WO2022047008A1 - Hsp90-binding conjugates and formulations thereof - Google Patents

Hsp90-binding conjugates and formulations thereof Download PDF

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WO2022047008A1
WO2022047008A1 PCT/US2021/047718 US2021047718W WO2022047008A1 WO 2022047008 A1 WO2022047008 A1 WO 2022047008A1 US 2021047718 W US2021047718 W US 2021047718W WO 2022047008 A1 WO2022047008 A1 WO 2022047008A1
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conjugate
acid
analogs
derivatives
active agent
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PCT/US2021/047718
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French (fr)
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Mark T. Bilodeau
Ashis K. Saha
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Tarveda Therapeutics, Inc.
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Publication of WO2022047008A1 publication Critical patent/WO2022047008A1/en

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    • 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
    • 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
    • A61K47/51—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 the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—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 the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/14—Ortho-condensed systems
    • 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
    • C07D487/12—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 three hetero rings
    • C07D487/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the disclosure generally relates to the field of therapeutic compounds. More particularly, the disclosure relates to the use of compounds targeting heat shock proteins including heat shock protein 90 (HSP90), e.g., for treating cancer.
  • HSP90 heat shock protein 90
  • HSP90 Heat shock protein 90
  • HSP90 is an intracellular chaperone protein that assists protein folding, stabilizes proteins against heat stress, and aids in protein degradation. It is upregulated in many types of cancer. Many Hsp90 client proteins are over-expressed in cancer, often in mutated forms, and are responsible for unrestricted cancer cell proliferation and survival. HSP90 is activated in cancer tissues and latent in normal tissues. HSP90 derived from tumour cells has higher binding affinity to HSP90 inhibitors than the latent form in normal cells, allowing specific targeting of HSP90 inhibitors to tumour cells with little inhibition of HSP90 function in normal cells. Further, HSP90 has also been recently identified as an important extracellular mediator for tumour invasion. Therefore, HSP90 is considered a major therapeutic target for anticancer drug development. There is a need in the art for developing therapeutic compounds that binds to HSP90.
  • the present application provides a conjugate comprising an active agent coupled to an HSP90 targeting moiety by a linker and a pharmaceutical composition comprising such a conjugate.
  • the conjugate inhibits of the activity or function of a target protein in cells.
  • the conjugates induces degradation of the target protein.
  • the active agent is a PI3K inhibitor, such as but not limited to PF-04979064, PQR514, BAY 80-6946 (Copanlisib), orfragments/derivatives/analogs thereof.
  • the present disclosure also provides methods of degrading a protein in a cell, reducing cell proliferation, and/or treating caner comprising administering a therapeutically effective amount of the conjugates of the present disclosure.
  • compositions comprising such conjugates and methods of making and using such conjugates are also provided.
  • FIG. 1 shows PI3K degradation 6 hours after receiving various doses of conjugates of the present disclosure.
  • FIG. 2 shows PI3K degradation 24 hours after receiving various doses of conjugates of the present disclosure.
  • FIG. 3 and FIG. 4 show tumor volume changes in LS174T xenograft model after receiving various doses of conjugates of the present disclosure.
  • FIG. 5 shows PI3K degradation in an in vivo study after treatment with conjugates of the present disclosure.
  • FIG. 6 shows the %PI3Ka degradations measured by western blot for Compound Cl, C50 and C48.
  • HSP90 targeting conjugates comprising an active agent and novel particles comprising such conjugates. Such targeting can, for example, improve the amount of active agent at a site and decrease active agent toxicity to the subject.
  • HSP90 targeting conjugates of the present disclosure have deep and rapid tumor penetration and do not require receptor internalization. High accumulation and long retention time of HSP90 targeting conjugates enable the use of cytotoxic and non-cytotoxic payloads, such as chemotherapeutic agents, kinase inhibitors, or immuno-oncology modulators.
  • toxicity refers to the capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment.
  • Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment.
  • Toxicity may further be measured relative to a subject’s weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity.
  • Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase.
  • Neutropenia or thrombopenia may also be metrics of toxicity.
  • Pharmacologic indicators of toxicity include elevated AST/ALT levels, neurotoxicity, kidney damage, GI damage and the like.
  • the conjugates are released after administration of the particles.
  • the targeted drug conjugates utilize active molecular targeting in combination with enhanced permeability and retention effect (EPR) and improved overall biodistribution of the particles to provide greater efficacy and tolerability as compared to administration of targeted particles or encapsulated untargeted drug.
  • EPR enhanced permeability and retention effect
  • the toxicity of a conjugate containing an HSP90 targeting moiety linked to an active agent for cells that do not overexpress HSP90 is predicted to be decreased compared to the toxicity of the active agent alone. Without committing to any particular theory, applicants believe that this feature is because the ability of the conjugated active agent to be retained in a normal cell is decreased relative to a tumor cell.
  • Conjugates include an active agent or prodrug thereof attached to a targeting moiety, e.g., a molecule that can bind to HSP90, by a linker.
  • the conjugates can be a conjugate between a single active agent and a single targeting moiety, e.g., a conjugate having the structure X-Y-Z where X is the targeting moiety, Y is the linker, and Z is the active agent.
  • the conjugate contains more than one targeting moiety, more than one linker, more than one active agent, or any combination thereof.
  • the conjugate can have any number of targeting moieties, linkers, and active agents.
  • the conjugate can have the structure X-Y-Z-Y-X, (X-Y)n-Z, X-(Y-Z)n, Xn-Y-Z, X-Y- Zn, (X-Y-Z)n, (X-Y-Z-Y)n-Z, where X is a targeting moiety, Y is a linker, Z is an active agent, and n is an integer between 1 and 50, between 2 and 20, for example, between 1 and 5.
  • Each occurrence of X, Y, and Z can be the same or different, e.g., the conjugate can contain more than one type of targeting moiety, more than one type of linker, and/or more than one type of active agent.
  • the conjugate can contain more than one targeting moiety attached to a single active agent.
  • the conjugate can include an active agent with multiple targeting moieties each attached via a different linker.
  • the conjugate can have the structure X-Y-Z-Y-X where each X is a targeting moiety that may be the same or different, each Y is a linker that may be the same or different, and Z is the active agent.
  • the conjugate can contain more than one active agent attached to a single targeting moiety.
  • the conjugate can include a targeting moiety with multiple active agents each attached via a different linker.
  • the conjugate can have the structure Z-Y-X-Y-Z where X is the targeting moiety, each Y is a linker that may be the same or different, and each Z is an active agent that may be the same or different.
  • a conjugate as described herein contains at least one active agent (a first active agent).
  • the conjugate can contain more than one active agent, that can be the same or different from the first active agent.
  • the active agent can be a therapeutic, prophylactic, diagnostic, or nutritional agent.
  • a variety of active agents are known in the art and may be used in the conjugates described herein.
  • the active agent can be a protein or peptide, small molecule, nucleic acid or nucleic acid molecule, lipid, sugar, glycolipid, glycoprotein, lipoprotein, or combination thereof.
  • the active agent is an antigen, an adjuvant, radioactive, an imaging agent (e.g., a fluorescent moiety) or a polynucleotide.
  • the active agent is an organometallic compound.
  • the active agent of the conjugate comprises a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%.
  • the amount of active agent(s) of the conjugate may also be expressed in terms of proportion to the targeting ligand(s).
  • the present teachings provide a ratio of active agent to ligand of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • the active agent can be a cancer therapeutic.
  • Cancer therapeutics include, for example, death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis.
  • TRAIL TNF-related apoptosis-inducing ligand
  • Suitable death receptors include, but are not limited to, TNFR1, Fas, DR3, DR4, DR5, DR6, LTpR and combinations thereof.
  • Cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy agents can be used as active agents.
  • Chemotherapeutic agents include, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Such agents typically affect cell division or DNA synthesis and function.
  • Additional examples of therapeutics that can be used as active agents include monoclonal antibodies and the tyrosine kinase inhibitors e.g. imatinib mesylate, which directly targets a molecular abnormality in certain types of cancer (e.g., chronic myelogenous leukemia, gastrointestinal stromal tumors).
  • Chemotherapeutic agents include, but are not limited to cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxol and derivatives thereof, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, epipodophyllotoxins, trastuzumab, cetuximab, and rituximab, bevacizumab, and combinations thereof. Any of these may be used as an active agent in a conjugate.
  • the small molecule active agents used in this disclosure include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PBK/m- TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors and proteasome inhibitors.
  • cytotoxic compounds e.g., broad spectrum
  • angiogenesis inhibitors e.g., cell cycle progression inhibitors, PBK/m- TOR/AKT pathway inhibitors
  • MAPK signaling pathway inhibitors e.g., kinase inhibitors
  • protein chaperones inhibitors e.g., HDAC inhibitors
  • PARP inhibitors e.g., RNA polymerase inhibitors
  • proteasome inhibitors e.g. antiproliferative (cytotoxic and cytostatic) agents
  • the small molecule active agents in
  • Broad spectrum cytotoxins include, but are not limited to, DNA-binding or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, and topoisomerase I or II inhibitors.
  • Exemplary DNA-binding or alkylating drugs include, CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and its analogs, alkylating agents, such as calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.
  • anthracyclines doxorubicin, epirubicin, idarubicin, daunorubicin
  • alkylating agents such as calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.
  • Exemplary doxorubicin analogs include nemorubicin metabolite or analog drug moiety disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.
  • Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin CI, duocarmycin C2, duocarmycin B2, DU-86, KW-2189, bizelesin, seco-adozelesin, and those described in U.S. Patent Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660;
  • Doxorubicin and its analogs include PNU- 159682 and those described in U.S. Patent No.6, 630, 579 and nemorubicin metabolite or analog drugs disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.
  • Calicheamicins include those described in U.S. Patent Nos. 5,714,586 and 5,739,116.
  • Duocarmycins include those described in U.S. Patent Nos.5, 070, 092;
  • Pyrrolobenzodiazepines include SG2057 and those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000), Anti-Cancer Agents in Medicinal Chemistry, 2009, 9, 1-31; WO 2011/130613 Al; EP 2 789 622 Al; Blood 2013, 122, 1455; J. Antimicrob. Chemother. 2012, 67, 1683-1696; Cancer Res. 2004, 64, 6693-6699; WO 2013041606; US 8481042; WO 2013177481; WO 2011130613; WO2011130598.
  • microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, cabazitaxel; maytansinoids, auristatins and analogs thereof, tubulysin A and B derivatives, vinca alkaloid derivatives, epothilones, PM060184 and cryptophy cins.
  • Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, maytansine or DM-1 and DM-4 are those described in U.S.
  • the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52: 127-131), maytansinoids or maytansinoid analogs.
  • suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Patent Nos.
  • Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F and dolastatin.
  • Suitable auristatins are also described in U.S. Publication Nos. 2003/0083263, 2011/0020343, and 2011/0070248; PCT Application Publication Nos. WO 09/117531, WO 2005/081711, WO 04/010957; W002/088172 and WOOl/24763, and U.S. Patent Nos.
  • Exemplary tubulysin compounds include compounds described in U.S. Patent Nos. 7,816,377; 7,776,814; 7,754,885; U.S. Publication Nos. 2011/0021568;
  • Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine).
  • Suitable Vinca alkaloids that can be used in the present disclosure are also disclosed in U.S. Publication Nos. 2002/0103136 and 2010/0305149, and in U.S. Patent No. 7,303,749 Bl, the disclosures of which are incorporated herein by reference in their entirety.
  • Exemplary epothilone compounds include epothilone A, B, C, D, E and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121,029;
  • Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOX ATINE®), iproplatin, ormaplatin, and tetraplatin.
  • Exemplary topoisomerase I inhibitors include camptothecin, camptothecin, derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11 (irinotecan), SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625.
  • Other camptothecin compounds that can be used in the present disclosure include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30: 1774 (1987).
  • topoisomerase II inhibitors include azonafide and etoposide.
  • Additional agents acting on DNA include Lurbinectedin (PM01183), Trabectedin (also known as ecteinascidin 743 or ET-743) and analogs as described in WO 200107711, WO 2003014127.
  • Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors.
  • Exemplary MetAP2 inhibitors include fumagillol analogs, meaning any compound that includes the fumagillin core structure, including fumagillamine, that inhibits the ability of MetAP-2 to remove NFL-terminal methionines from proteins as described in Rodeschini et al., I. Org. Chem., 69, 357-373, 2004 and Liu, et al., Science 282, 1324-1327, 1998.
  • Non limiting examples of "fumagillol analogs” are disclosed in /. Org. Chem., 69, 357, 2004; J. Org.
  • Exemplary cell cycle progression inhibitors include CDK inhibitors such as BMS-387032 and PD0332991; Rho-kinase inhibitors such as GSK429286; checkpoint kinase inhibitors such as AZD7762; aurora kinase inhibitors such as AZDI 152, MLN8054 and MLN8237; PLK inhibitors such as BI 2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors such as SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520.
  • CDK inhibitors such as BMS-387032 and PD0332991
  • Rho-kinase inhibitors such as GSK429286
  • checkpoint kinase inhibitors such as AZD7762
  • aurora kinase inhibitors such as AZDI 152,
  • Exemplary PI3K/m-T0R/AKT signaling pathway inhibitors include phosphoinositide 3 -kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors.
  • PI3K phosphoinositide 3 -kinase
  • Exemplary PI3 kinase inhibitors are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147, XL765, GSK2126458 (Omipalisib), GDC-0326, GDC-0032 (Taselisib, RG7604), PF-05212384 (Gedatolisib, PKI-587), BAY 80-6946 (copanlisib), PF- 04691502, PF-04989216, PF-04979064, PI-103, PKI-402 VS-5584 (SB2343), GDC- 09
  • Exemplary AKT inhibitors include, but are not limited to, AT7867, MK- 2206, Perifosine, GSK690693, Ipatasertib, AZD5363, TIC10, Afuresertib, SC79, AT13148, PHT-427, A-674563, and CCT128930.
  • Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.
  • Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD 8330 and GDC-0973.
  • Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.
  • Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB202190.
  • RTK Receptor tyrosine kinases
  • Exemplary inhibitors of ErbB2 receptor include but not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), and Gefitinib (Iressa).
  • Exemplary RTK inhibitors targeting more then one signaling pathway include AP24534 (Ponatinib) that targets FGFR, FLT-3, VEGFR-PDGFR and Bcr-Abl receptors; ABT-869 (Linifanib) that targets FLT-3 and VEGFR- PDGFR receptors; AZD2171 that targets VEGFR-PDGFR, Fit- 1 and VEGF receptors; CHR-258 (Dovitinib) that targets VEGFR-PDGFR, FGFR, Flt- 3, and c-Kit receptors.
  • AP24534 Panatinib
  • ABT-869 Liifanib
  • AZD2171 that targets VEGFR-PDGFR, Fit- 1 and VEGF receptors
  • CHR-258 Dovitinib
  • Exemplary kinase inhibtiors include inhibitors of the kinases ATM, ATR, CHK1, CHK2, WEE1, and RSK.
  • the active agents are ATR inhibitors, such as but not limited to berzosertib (VX-970, M6620, or VE-822), AZD6738, or BAY1895344.
  • Exemplary protein chaperon inhibitors include HSP90 inhibitors.
  • Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX- 5422, NVP-AUY-922, and KW-2478.
  • Exemplary HD AC inhibitors include Belinostat (PXD101), CUDC-101,
  • Doxinostat ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP- LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A, and Vorinostat (SAHA).
  • Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD- 2281), ABT-888 (Veliparib), AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3- aminobenzamide, A-966492, and AZD2461.
  • Exemplary Wnt/Hedgehog signaling pathway inhibitors include vismodegib (RG3616/GDC-0449), cyclopamine (11-deoxojervine) (Hedgehog pathway inhibitors), and XAV-939 (Wnt pathway inhibitor).
  • Exemplary RNA polymerase inhibitors include amatoxins.
  • Exemplary amatoxins include a- amanitins, P- amanitins, y- amanitins, s-amanitins, amanullin, amanullic acid, amaninamide, amanin, and proamanullin.
  • Exemplary proteasome inhibitors include bortezomib, carfilzomib, ONX 0912, CEP- 18770, and MLN9708.
  • the drug of the disclosure is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor (e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)), MEK inhibitor, KSP inhibitor, RNA polymerse inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, duocarmycin, tubulysin, auristatin or a platinum compound.
  • kinase inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • MEK inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • MEK inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • MEK inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • KSP inhibitor
  • the drug is a derivative of SN-38, vindesine, vinblastine, PI- 103, AZD 8330, auristatin E, auristatin F, a duocarmycin compound, tubulysin compound, or ARRY-520.
  • the drug used in the disclosure is a combination of two or more drugs, such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.
  • drugs such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.
  • the active agent can be a cancer therapeutic.
  • the cancer therapeutics may include death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis.
  • TRAIL TNF-related apoptosis-inducing ligand
  • Suitable death receptors include, but are not limited to, TNFR1, Fas, DR3, DR4, DR5, DR6, LTpR and combinations thereof.
  • the active agent can be a DNA minor groove binders such as lurbectidin and trabectidin.
  • the active agent can be E3 ubiquitin ligase inhibitors, adeubiquitinase inhibitors or an NFkB pathway inhibitor.
  • the active agent can be a phopsphatase inhibitors including inhibitors of PTP1B, SHP2, LYP, FAP-1, CD45, STEP, MKP-1, PRL, LMWPTP or CDC25.
  • the active agent can be an inhibitor of tumor metabolism, such as an inhibitor of GAPDH, GLUT1, HK II, PFK, GAPDH, PK, LDH orMCTs.
  • the active agent can target epigenetic targets including EZH2, MLL, DOTI -like protein (DOT IL), bromodomain-containing protein 4 (BRD4), BRD2, BRD3, NUT, ATAD2, or SMYD2.
  • the active agent can target the body's immune system to help fight cancer, including moecules targeting IDO1, IDO2, TDO, CD39, CD73, A2A antagonists, STING activators, TLR agonists (TLR 1-13), ALK5, CBP/EP300 bromodomain, ARG1, ARG2, iNOS, PDE5, P2X7, P2Y11, COX2, EP2 Receptor, or EP4 receptor.
  • the active agent can target Bcl-2, IAP, or fatty acid synthase.
  • the active agent can be 20-epi-l,25 dihydroxyvitamin D3, 4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizing morphogenetic protein- 1, antiestrogen, antineoplaston, antis
  • the active agent can be an inorganic or organometallic compound containing one or more metal centers.
  • the compound contains one metal center.
  • the active agent can be, for example, a platinum compound, a ruthenium compound (e.g., /raz/.s-fRuCh (DMSO)4], or Zraw -[RuC14(imidazole) 2, etc.), cobalt compound, copper compound, or iron compounds.
  • the active agent is a small molecule. In some embodiments, the active agent is a small molecule cytotoxin. In one embodiment, the active agent is cabazitaxel, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. In another embodiment, the active agent is mertansine (DM1) or DM4, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. DM1 or DM4 inhibits the assembly of microtubules by binding to tubulin. Structure of DM1 is shown below:
  • the active agent Z is Monomethyl auristatin E (MMAE), or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structure of MMAE is shown below:
  • the active agent Z is a sequence-selective DNA minor-groove binding crosslinking agent.
  • Z may be pyrrolobenzodiazepine (PBD), a PBD dimer, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structures of PBD and PBD dimer are shown below: dimer).
  • the active agent Z is a topoisomerase I inhibitor, such as camptothecin, irinotecan, SN-38, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.
  • SN-38 (7 -Ethyl- 10-hydroxy-camptothecin)
  • WO2015184246 the contents of each of which are incorporated herein by reference in their entirety, such as bendamustine, VDA, doxorubicin, pemetrexed, vorinostat, lenalidomide, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adozelesin, or derivatives/analogs thereof, may be used as an active agent in conjugates of the present disclosure.
  • the active agent is a topoisomerase inhibitor.
  • the topoisomerase inhibitor is a topoisomerase I inhibitor, including but not limited to irinotecan, topotecan, camptothecin, diflomotecan and lamellarin D, and fragments/derivatives/analogs thereof.
  • the topoisomerase inhibitor is a topoisomerase II inhibitor such as etoposide (VP- 16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, and HU-331, and fragments/derivatives/analogs thereof.
  • etoposide VP- 16
  • teniposide such as etoposide (VP- 16)
  • doxorubicin doxorubicin
  • daunorubicin daunorubicin
  • mitoxantrone amsacrine
  • ellipticines aurintricarboxylic acid
  • HU-331 fragments/derivatives/analogs thereof.
  • the PI3K/AKT/mTOR signaling network controls most hallmarks of cancer: cell cycle, survival, metabolism, motility and genomic stability.
  • the PI3K pathway is the most frequently altered pathway in human cancer. Activation of PI3K has been directly linked to cancer through mutations or amplifications of PIK3CA, and loss of function tumor suppressor PTEN.
  • PIK3CA gene is the 2 nd most frequently mutated oncogene. PTEN is among the most frequently mutated tumor suppressor genes.
  • Pathway inhibitors demonstrate antitumor efficacy in xenograft models, but toxicity limits clinical benefit in patients. Conjugating a PI3K inhibitor with a HSP90 targeting moiety provides a method to delivery PI3K inhibitors for sufficient PI3K inhibition in tumors with reduced toxicity.
  • Conjugates comprising PI3K inhibitors may be used to treat hematological malignancies and solid tumors.
  • conjugates comprising PI3K inhibitors are used to treat colorectal cancer, multiple myeloma, leukemia, lymphoma, colon cancer, gastric cancer, kidney cancer, lung cancer, or breast cancers including metastatic breast cancer.
  • conjugates comprising PI3K inhibitors are used to treat PIK3CA-altered cancers or HER2 positive cancers.
  • any PI3K inhibitor may be used as an active agent.
  • the PI3K inhibitor may be a small molecule.
  • Non-limiting examples include PQR514, PQR530, PF-04979064, BAY 80-6946 (Copanlisib), Omipalisib (GSK2126458, GSK458), , PF-04691502, PI-103, BGT226 (NVP-BGT226), Apitolisib (GDC-0980, RG7422), Duvelisib (IPI-145, INK1197), AZD8186, Pilaralisib (XL147), PIK-93, Idelalisib (GS-1101), MLN1117, VS-5584, SB2343, GDC-0941, BM120, NVP- BKM120, Buparlisib, AZD8835, XL765 (SAR245409), GS-9820 Acalisib,
  • the PI3K inhibitor may be an inhibitor of delta and gamma isoforms of PI3K. In some embodiments, the PI3K inhibitor is an inhibitor of alpha isoforms of PI3K. In other embodiments, the PI3K inhibitor is an inhibitor of one or more alpha, beta, delta and gamma isoforms of PI3K.
  • Non-limiting examples of PI3K inhibitors include compounds disclosed in US 9,546,180 (Infinity Pharmaceuticals), WO 2009088990 (Intellikine Inc.), WO 2011008302 (Intellikine Inc.), WO 2010036380 (Intellikine Inc.), WO 2010/006086 (Intellikine Inc.), WO 2005113556 (Icos Corp.), US 2011/0046165 (Intellikine Inc.), or US 20130315865 (Pfizer), the contents of each of which are incorporated herein by reference in their entirety.
  • the conjugates of the present disclosure comprises PQR514 or its fragments/derivatives/analogs, PF-04979064 or its fragments/derivatives/analogs, Omipalisib (GSK458) or its fragments/derivatives/analogs, BAY 80-6946 (Copanlisib) or its fragments/derivatives/analogs, PQR530 or its fragments/derivatives/analogs, PF- 04691502 or its fragments/derivatives/analogs, PI- 103 or its fragments/derivatives/analogs, BGT226 (NVP-BGT226) or its fragments/derivatives/analogs, Apitolisib (GDC-0980, RG7422) or its fragments/derivatives/analogs, Duvelisib (IPI-145, INK1197) or its fragments/de
  • Apitolisib (GDC-0980, RG7422) Duvelisib (IPI-145, INK1197) AZD8186
  • conjugates of the present disclosure may comprise an
  • HSP90 targeting moiety connected to PQR514 or its fragments/derivatives/analogs, PF-04979064 or its fragments/derivatives/analogs, BAY 80-6946 (Copanlisib) or its fragments/ derivatives/ analogs .
  • the conjugates of the present disclosure comprises PQR514 or its fragments/derivatives/analogs, such as r its fragments,
  • the conjugates of the present disclosure comprises a
  • the conjugates of the present disclosure comprises PF-04979064 or its fragments/derivatives/analogs, such as
  • the conjugates of the present disclosure comprises
  • Copanlisib or its fragments/derivatives/analogs such as
  • the conjugates of the present disclosure comprise a benzoxazepine group, such as GDC-0077 (Inavolisib), GDC-0326 or fragments/derivatives/analogs thereof, such as:
  • Targeting ligands include any molecule that can bind one or more HSP90 proteins.
  • targeting ligands can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates, or lipids.
  • the targeting moiety, X can be any HSP90 binding moiety such as, but not limited to, natural compounds (e.g., geldanamycin and radicicol), and synthetic compounds such as geldanamycin analogue 17-AAG (i.e., 17- allylaminogeldanamycin), a purine-scaffold HSP90 inhibitor series including PU24FC1 (He H., et al, J. Med. Chem., vol.49:381 (2006), the contents of which are incorporated herein by reference in their entirety), BIIB021 (Lundgren K., et al, Mol.
  • the targeting moiety may be a peptide, such as a bicyclic peptide or a modified bicyclic peptide.
  • the targeting moiety is a peptide having a sequence of (X)lC(X)mC(X)nC(X)o as disclosed in EP2474613, the contents of which are incorporated herein by reference in their entirety, wherein C is cysteine, X represents a random amino acid, m and n are numbers between 1 and 20 defining the length of intervening polypeptide segments and 1 and o are numbers between 0 and 20 defining the length of the flanking polypeptide segments.
  • the targeting moiety comprises at least 2 polypeptides covalently linked to tri s-(bromom ethyl) benzene (TBMB) at two or more amino acid residues as disclosed in US8680022 and US8778844, the contents of each of which are incorporated herein by reference in their entirety.
  • TBMB tri s-(bromom ethyl) benzene
  • the HSP90 binding moiety may be heterocyclic derivatives containing three heteroatoms.
  • W02009134110 to MATULIS et al. discloses 4,5- diaryl thiadiazoles which demonstrate good HSP90 binding affinity. Even though they have rather modest cell growth inhibition, they may be used as HSP90 binding moiety in conjugates of the present disclosure.
  • Another class of aza-heterocyclic adducts namely triazole derivatives or their analogs, may be used as HSP90 binding moiety in conjugates of the present disclosure.
  • the 1,2,4-triazole scaffold has been profusely documented as possessing HSP90 inhibiting properties.
  • WO2009139916 to BURLISON et al. discloses tricyclic 1,2,4-triazole derivatives inhibiting HSP90 at high micromolar concentrations. Any tricyclic 1,2,4- triazole derivatives disclosed in WO2009139916 or their derivatives/analogs may be used as HSP90 binding moiety in conjugates of the present disclosure.
  • Any tri substituted 1,2,4- triazole derivatives disclosed in WO 2010017479 and WO 2010017545 (Synta Pharmaceuticals Corp.) or their derivatives/analogs, the contents of which are incorporated herein by reference in their entirety, may be used as HSP90 binding moiety in conjugates of the present disclosure.
  • ganetespib a triazolone-containing HSP90 inhibitor named ganetespib (previously referred as to STA-9090, or as its highly soluble phosphate prodrug STA- 1474) disclosed in W02006055760 (Synta Pharmaceuticals Corp.), the contents of which are incorporated herein by reference in their entirety, or its derivatives/analogs may be used as HSP90 binding moiety in conjugates of the present disclosure.
  • ganetespib or its derivatives/analogs may be used a targeting moiety.
  • ganetespib derivatives/analogs are shown below.
  • Onalespib (AT13387) or its derivatives/analogs may be used as a targeting moiety in the conjugates of the present disclosure.
  • Onalespib and non-limiting examples of Onalespib derivatives/analogs are shown below.
  • the HSP90 targeting moiety comprises r its fragments/ derivatives/ analogs.
  • HSP90 binding moiety in the conjugates of the present disclosure, such as:
  • R1 may be alkyl, aryl, halide, carboxamide or sulfonamide
  • R2 may be alkyl, cycloalkyl, aryl or heteroaryl, wherein when R2 is a 6 membered aryl or heteroaryl, R2 is substituted at the 3- and 4- positions relative to the connection point on the triazole ring, through which a linker L is attached
  • R3 may be SH, OH, -CONHR4, aryl or heteroaryl, wherein when R3 is a 6 membered aryl or heteroaryl, R3 is substituted at the 3 or 4 position
  • Formula , wherein R1 may be alkyl, aryl, halo, carboxamido, sulfonamido
  • R2 may be optionally substituted alkyl, cycloalkyl, aryl or heteroaryl.
  • Examples of such compounds include 5-(2,4-dihydroxy-5- isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H- 1,2,4- triazole-3 -carboxamide and 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4- methylpiperazin- 1 -yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H- 1 ,2,4-triazole-3 - carboxamide;
  • R1 may be alkyl, aryl, halide, carboxamido or sulfonamido
  • R2 may be substituted alkyl, cycloalkyl, aryl or heteroaryl, where a linker L is connected directly or to the extended substitutions on these rings
  • R3 may be SH, OH, NR4R5 AND -CONHR6, to which an effector moiety may be connected
  • R4 and R5 may independently be H, alkyl, aryl, or heteroaryl
  • R6 may be alkyl, aryl, or heteroaryl, having a minimum of one functional group to which an effector moiety may be connected; or
  • R1 may be alkyl, aryl, halo, carboxamido or sulfonamido
  • R2 and R3 are independently C1-C5 hydrocarbyl groups optionally substituted with one or more of hydroxy, halogen, C1-C2 alkoxy, amino, mono- and di-Cl-C2 alkylamino; 5- to 12- membered aryl or heteroaryl groups; or, R2 and R3, taken together with the nitrogen atom to which they are attached, form a 4- to 8- membered monocyclic heterocyclic group, of which up to 5 ring members are selected from O, N and S. Examples of such compounds include AT-13387.
  • the HSP90 targeting moiety may be Ganetespib, Luminespib (AUY-922, NVP-AUY922), Debio-0932, MPC-3100, Onalespib (AT-13387), SNX-2112, 17- amino-geldanamycin hydroquinone, PU-H71, AT 13387, or derivatives/ analogs thereof.
  • the HSP90 targeting moiety may be SNX5422 (PF-04929113), or any other HSP90 inhibitors disclosed in US 8080556 (Pfizer), W02008096218 (Pfizer), W02006117669 (Pfizer), W02008059368 (Pfizer), W02008053319 (Pfizer), W02006117669 (Pfizer), EP1885701 (Novartis), EP1776110 (Novartis), EP2572709 (Novartis), WO2012131413 (Debiopharm), or WO2012131468 (Debiopharm), the contents of each of which are incorporated herein by reference in their entirety.
  • the HSP90 targeting moiety may also be PU-H71, an HSP90 inhibitor that is 124 I radiolabeled for PET imaging or its derivatives/analogs.
  • Conjugates comprising SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71, or ATI 3387 may have a structure of:
  • the HSP90 targeting moiety comprises a Sansalvamide A derivative.
  • Sansalvamide A (San A) is a cyclic pentapeptide isolated from a marine fungus and binds to HSP90. Any Di-Sansalvamide A derivative (dimerized San A molecules) disclosed in Alexander et al., J Med Chem., vol.52(24):7927 (2009), the contents of which are incorporated herein by reference in their entirety, for example, the Di-San A molecules in Figure 1 of Alexander, may be used as a targeting moiety of the conjugate of the current disclosure.
  • the HSP90 targeting moiety comprises alkynyl pyrrolo[2,3-d]pyrimidines and related analogs such as HSP90 inhibitors disclosed in US7544672 (CONFORMA THERAPEUTICS), the contents of which are incorporated herein by reference in their entirety.
  • the HSP90 targeting moiety compriese any HSP90 inhibitor disclosed in WO2010117425 (Biogen), the contents of which are incorporated herein by reference in their entirety.
  • Extracellular HSP90 eHSP90
  • HSP90 In normal cells, secretion of HSP90 occurs when cells are under environmental stress such as heat, drugs, cytokines, UV, and/or gamma rays.
  • the main function of the extracellular HSP90 (eHSP90) is to help tissue repair by promoting the cells at the edge of damaged tissue to migrate into the damaged area.
  • constitutively activated oncogenes trigger HSP90 secretion even without any environmental stress.
  • Secreted Hsp90 by tumors eHSP90a promotes both tumor and tumor stroma cell migration during invasion and metastasis.
  • HSP90a The extracellular promotility function of HSP90a depends on a 115-amino acid fragment (F-5) on the surface of HSP90 (Li et al., Ini Rev Cell Mol Biol., vol.303:203-235 (2013), the contents of which are incorporated herein by reference in their entirety).
  • F-5 115-amino acid fragment
  • eHSP90 has been shown to be present on the surface of tumor cells and to also be capable of being internalized (Crowe et al., ACS Chem. Biol., vol.12: 1047-1055 (2017)).
  • the surface expression of eHSP90 in tumor cells thus represents a target for directing therapies selectively to tumors over healthy cells. Therefore, eHSP90 (eHSP90a in particular) may be a good target for treating tumors.
  • eHSP90 can be measured using the method known in the art, such as ELISA, RIA, EIA, sandwich assay, Western Blot analysis, immunostaining, flow cytometry and immunohistological staining.
  • the targeting moiety selectively binds to eHSP90. In some embodiments, the targeting moiety binds to F-5 region of eHSP90.
  • the targeting moiety has low cell-permeability and prefers to bind to cell surface eHSP90. In some embodiments, the targeting moiety is cell-impermeable and binds exclusive to eHSP90. In some embodiments, the conjugates comprising the targeting moieties have a low cell permeability or is cell- impermeable.
  • HS- 131 (100) is a fluorescent specific small-molecule Hsp90 inhibitor. It is not cell permeable and it binds exclusively to eHsp90.
  • the five isogenic cell lines (67NR, 168FARN, 4T07, and 4T1) were isolated from a single spontaneous mammary tumor. They cells exhibit varying degrees of metastatic disease when injected into mice. 100 was internalized to a higher extent in 4T1 cells (the most aggressive of the five lines) over the less metastatic lines.
  • the targeting moieties comprise HS-23, HS-131, (disclosed in Crowe et al., ACS Chem. Biol., vol.12: 1047-1055 (2017), the contents of which are incorporated herein by reference in their entirety) or DMAG-N-oxide (a cell-impermeable for of 17-AAG disclosed in Tsutsumi et al., Oncogene, vol.27(17):2478-2487 (2008), the contents of which are incorporated herein by reference in their entirety), or analog/derivative thereof, the structures shown below.
  • the targeting moiety comprises BIIB021 (Dihydropyrrol opyrimidine), or any compound disclosed in WO2010117425, and fragments/analogs/derivatives thereof, such as the compounds of Formula I or Formula II or the compounds in Table 2 of WO2010117425, and the structures shown below:
  • the targeting moiety comprises an antibody or antibody fragment which binds to human extracellular Hsp90.
  • the targeting moiety may be a monoclonal antibody disclosed in US 7959915 (Tufts University), the contents of which are incorporated herein by reference in their entirety, such as m Ab 1.5.1.
  • the targeting moiety maybe an anti- HSP90 monoclonal antibody disclosed in US20110280881 (Riken Corp.), the contents of which are incorporated herein by reference in their entirety, wherein the antibody binds to an epitope comprising an amino acid sequence selected from the amino acid sequence VX1X2EX3PPLEGDX4 (wherein each of XI to X4, which may be identical to or different from each other, represents an arbitrary amino acid) (SEQ ID NO: 1 in US20110280881) or the amino acid sequence HX5IX6ETLRQKAE (wherein each of X5 to X6, which may be identical to or different from each other, represents an arbitrary amino acid) (SEQ ID NO:2 in US20110280881), and wherein the antibody recognizes cell surface HSP90.
  • VX1X2EX3PPLEGDX4 wherein each of XI to X4, which may be identical to or different from each other, represents an arbitrary amino acid
  • the targeting moiety or moieties of the conjugate are present at a predetermined molar weight percentage from about 0.1 % to about 10%, or about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%.
  • the amount of targeting moieties of the conjugate may also be expressed in terms of proportion to the active agent(s), for example, in a ratio of ligand to active agent of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5:1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • the conjugates contain one or more linkers attaching the active agents and targeting moieties.
  • the linker, Y is bound to one or more active agents and one or more targeting ligands to form a conjugate.
  • the linker Y is attached to the targeting moiety X and the active agent Z by functional groups independently selected from an ester bond, disulfide, amide, acylhydrazone, ether, carbamate, carbonate, and urea.
  • the linker can be attached to either the targeting ligand or the active drug by a non-cleavable group such as provided by the conjugation between a thiol and a maleimide, an azide and an alkyne.
  • the linker is independently selected from the group consisting alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally is substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl is optionally
  • the linker comprises a cleavable functionality that is cleavable.
  • the cleavable functionality may be hydrolyzed in vivo or may be designed to be hydrolyzed enzymatically, for example by Cathepsin B.
  • a “cleavable” linker refers to any linker which can be cleaved physically or chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by re- dox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes.
  • the cleavable functionality may be a disulfide bond or a carbamate bond.
  • the linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid.
  • the linker Y may be X’-RkY’-R 2 - ’ and the conjugate can be a compound according to Formula la:
  • V v Ia wherein X is a targeting moiety defined above; Z is an active agent; X’, R 1 , Y’, R 2 and Z’ are as defined herein.
  • X’ is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or more natural or unnatural amino acids, thio or succinimido;
  • R 1 and R 2 are either absent or comprised of alkyl, substituted alkyl, aryl, substituted aryl, polyethylene glycol (2-30 units);
  • Y’ is absent, substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol (2-30 units) or an amide;
  • Z’ is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, thio or succinimido.
  • the linker can allow one active agent molecule to be linked to two or more ligands, or one ligand to be linked to two or more active agent molecule.
  • a in Formula la is a spacer unit, either absent or independently selected from the following substituents.
  • the conjugate may be a compound according to Formula Ic:
  • C in Formula Ic is a branched unit containing three to six functionalities for covalently attaching spacer units, ligands, or active drugs, selected from amines, carboxylic acids, thiols, or succinimides, including amino acids such as lysine, 2,3- diaminopropanoic acid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, and cysteine.
  • active drugs selected from amines, carboxylic acids, thiols, or succinimides, including amino acids such as lysine, 2,3- diaminopropanoic acid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, and cysteine.
  • the active agent Z is a PI3K inhibitor and the HSP90 targeting moiety X is selected from the group consisting of TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM10, TM11, TM12 and their fragments/derivatives/analogs, wherein the active agent Z and the targeting moiety X are connected with a linker.
  • the linkers are cleavable. In some embodiments, the linkers are non-cleavable.
  • the conjugates of the present disclosure comprise Copanlisib or its fragments/derivatives/analogs, such as P7 or its fragments, P8 or its fragments, P9 or its fragments, P10 or its fragments, Pl 1 or its fragments, or P12 or its fragments.
  • Copanlisib or its fragments/derivatives/analogs such as P7 or its fragments, P8 or its fragments, P9 or its fragments, P10 or its fragments, Pl 1 or its fragments, or P12 or its fragments.
  • conjugates comprising HSP90-targeting moeities connected to Copanlisib or its fragments/derivatives/analogs include:
  • the conjugates of the present invention comprise copanlisib or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C53, C54, C62, C75, C76, and C77.
  • the conjugates of the present invention comprise copanlisib or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C48, C49, C50, C51, C52, C56, C57, C58, C59, C60, C61, C63, C64, C65, C66, C67, C68, C69, C70, C71, C72, C73, and C74.
  • the conjugates of the present disclosure comprise PF-04979064 or its fragments/derivatives/analogs, such as P4 or its fragments, P5 or its fragments, or P6 or its fragments.
  • PF-04979064 or its fragments/derivatives/analogs, such as P4 or its fragments, P5 or its fragments, or P6 or its fragments.
  • Non-limiting examples include:
  • conjugates comprising HSP90-targeting moeities connected to PF-04979064 or its fragments/derivatives/analogs include:
  • the conjugates of the present invention comprise PF-04979064 or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C81, C82, and C83.
  • the conjugates of the present invention comprise PF-04979064 or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C78, C79, C80, C84, C85, C86, and C87.
  • the active agent Z comprises PQR514 or its fragments/derivatives/analogs, such as Pl or its fragments, P2 or its fragments, or P3 or its fragments.
  • PQR514 or its fragments/derivatives/analogs, such as Pl or its fragments, P2 or its fragments, or P3 or its fragments.
  • Non-limiting examples include:
  • conjugates comprising HSP90-targeting moeities connected to PQR514 or its fragments/derivatives/analogs include:
  • the conjugates of the present invention comprise PQR514 or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C88, C89, C90, C91, C92, C93, C94, C95, C96, C98, C99, C100, C101, C104, C106, C107, C108, C109, C110, Cl 11, C112, C113, C114, C115, C116, and C120.
  • the conjugates of the present invention comprise PQR514 or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties.
  • PQR514 or its fragments/derivatives/analogs as active agents
  • ganetespib or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C102 and C103.
  • the active agent Z comprises GDC-0326 or its fragments/derivatives/analogs.
  • Non-limiting examples include:
  • the conjugates of the present invention comprise GDC-0326 or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C119.
  • the conjugates of the present invention comprise GDC-0326 or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties.
  • GDC-0326 or its fragments/derivatives/analogs as active agents
  • ganetespib or its fragments/derivatives/analogs as targeting moieties.
  • Non-limiting examples include C117 and C118.
  • the conjugates of the present disclosure may further comprise at least one external linker connected to a reacting group that reacts with a functional group on a protein or an engineered protein or derivatives/analogs/mimics thereof, or comprise at least one external linker connected to a pharmacokinetic modulating unit.
  • the external linkers connecting the conjugates and the reacting group or the pharmacokinetic modulating units may be cleavable linkers that allow release of the conjugates.
  • the conjugates may be separated from the protein or pharmacokinetic modulating units as needed.
  • Any reacting group or PMU (such as PMUs comprising polymers) disclosed in WO2017/197241, the contents of which are incorporated herein by reference in their entirety, may be attached to the conjugates of the present disclosure.
  • the conjugates of the present disclosure may further comprise at least one permeability modulating unit.
  • the permeability modulating unit is attached to the payload of the conjugate, wherein the permeablity modulating unit regulates the cell membrane permeability of the payload.
  • the permeability modulating unit reduces the permeability of the payload. Not willing to be bound by any theory, once the payload is released from the conjugate, the permability modulating unit that is attached to the payload reduces the cell membrane permability of the payload, increases the retention time of the payload in target cells, improves the intracellular accumulation of the payload, and improves its efficacy.
  • the permeability modulation unit does not adversely impact the permeability of the conjugate or the binding capability of the targeting moiety.
  • the permeability modulation unit is active only after the payload is released from the conjugate, e.g., after the cleavable linker between the payload and the targeting moiety is cleaved.
  • the permeability modulating unit is a functional group that is covalently attached to the payload of the conjugate. In some embodiments, the permeability modulating unit is an integral part of the payload. [0149] In some embodiments, the permeability modulating unit is attached to the payload via an external linker.
  • the external linker may be a non-cleavable linker.
  • the passive permeation of a payload through the biological cell membranes is strongly dependent on the molecule physicochemical properties. Important factors that influence cell memberane permeation include the acid-base character of the molecule (which influences the charge of the molecule at the specific pH), its lipophilicity (which affects its partition between aqueous and lipid environments), and its solubility. For a payload to be permeable, there should be an appropriate balance between the hydrophobicity and hydrophilicity.
  • the permeability moduclating unit is hydrophilic.
  • the permeability moduclating unit is hydrophobic.
  • the permeability moduclating unit is polar.
  • the permeability moduclating unit is charged at physiological pH.
  • the permeability modulating unit may be positively charged, negatively charged, or a combination of multiple charges.
  • Non-limiting examples of the permability modulating unit include a functional group that has at least one nitrogen, such as a piperazine functional group.
  • compositions are administered to humans, human patients or subjects.
  • active ingredient generally refers to the conjugate to be delivered as described herein.
  • compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • the conjugates of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) permit the sustained or delayed release (e.g., from a depot formulation of the monomaleimide); (3) alter the biodistribution (e.g., target the monomaleimide compounds to specific tissues or cell types); (4) alter the release profile of the monomaleimide compounds in vivo.
  • excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and preservatives.
  • Excipients of the present disclosure may also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the disclosure may include one or more excipients, each in an amount that together increases the stability of the monomaleimide compounds.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, crosslinked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone crosslinked poly(vinyl-pyrrolidone)
  • crospovidone
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g.
  • polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g.
  • acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMAB EN®II, NEOLONETM, KATHONTM, and/or EUXYL®.
  • Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, is
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • the conjugates the present disclosure may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (d
  • the formulations described herein contain an effective amount of conjugates in a pharmaceutical carrier appropriate for administration to an individual in need thereof.
  • the formulations may be administered parenterally (e.g., by injection or infusion).
  • the formulations or variations thereof may be administered in any manner including enterally, topically (e.g., to the eye), or via pulmonary administration. In some embodiments the formulations are administered topically.
  • the conjugates can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution, suspension or emulsion.
  • the formulation can be administered systemically, regionally or directly to the organ or tissue to be treated.
  • Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art.
  • compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in- water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • injectable formulations for example, solutions or suspensions
  • solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection emulsions, such as water-in-oil (w/o) emulsions, oil-in- water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)
  • 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/or by the use of surfactants.
  • an isotonic agent is included, for example, one or more sugars, sodium chloride, or other suitable agent known in the art.
  • Solutions and dispersions of the conjugates can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combinations thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4- oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Pol oxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N- dodecyl-P-alanine, sodium N-lauryl-P-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s).
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. If using 10% sucrose or 5% dextrose, a buffer may not be required.
  • Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the conjugates in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized conjugates into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • examples of methods of preparation include vacuum-drying and freeze-drying techniques that yield a powder of the particle plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are known in the art.
  • compositions for parenteral administration can be in the form of a sterile aqueous solution or suspension of conjugates formed from one or more polymer-drug conjugates.
  • Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution.
  • PBS phosphate buffered saline
  • the formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3 -butanediol.
  • the formulation is distributed or packaged in a liquid form.
  • formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation.
  • the solid can be reconstituted with an appropriate carrier or diluent prior to administration.
  • Solutions, suspensions, or emulsions for parenteral administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration.
  • Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.
  • Solutions, suspensions, or emulsions for parenteral administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, sucrose, dextrose, mannitol, sorbitol, sodium chloride, and other electrolytes. [0185] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations.
  • Suitable preservatives include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.
  • PHMB polyhexamethylenebiguanidine
  • BAK benzalkonium chloride
  • Purite® stabilized oxychloro complexes
  • phenylmercuric acetate chlorobutanol
  • sorbic acid chlorhexidine
  • chlorhexidine benzyl alcohol
  • parabens parabens
  • thimerosal and mixtures thereof.
  • Solutions, suspensions, or emulsions for parenteral administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.
  • the conjugates can be formulated for topical administration to a mucosal surface Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches.
  • the formulation may be formulated for transmucosal transepithelial, or transendothelial administration.
  • the compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.
  • the conjugates can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation.
  • the conjugates are formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, to the mucosa, such as the eye or vaginally or rectally.
  • Surfactants are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product.
  • Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof.
  • the non-ionic surfactant is stearyl alcohol.
  • Emmulsifiers are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds.
  • Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulf
  • Suitable classes of penetration enhancers include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.
  • the present disclosure provides methods comprising administering conjugates containing the conjugate as described herein to a subject in need thereof.
  • Conjugates containing the conjugates as described herein may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits).
  • a disease, disorder, and/or condition e.g., a disease, disorder, and/or condition relating to working memory deficits.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • compositions in accordance with the disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, from about 25 mg/kg to about 50 mg/kg, from about 50 mg/kg to about 100 mg/kg, from about 100 mg/kg to about 125 mg/kg, from about 125 mg/kg to about 150 mg/kg, from about 150 mg/ to about 175 mg/
  • the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • multiple administrations e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
  • split dosing regimens such as those described herein may be used.
  • the concentration of the conjugates of the present disclosure may be between about 0.01 mg/mL to about 50 mg/mL, about 0.1 mg/mL to about 25 mg/mL, about 0.5 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 5 mg/mL in the pharmaceutical composition.
  • a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose.
  • a “single unit dose” is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.
  • the monomal eimide compounds of the present disclosure are administed to a subject in split doses.
  • the monomaleimide compounds may be formulated in buffer only or in a formulation described herein.
  • a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous.
  • Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art including, but not limited to
  • compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3 -butanediol.
  • the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di glycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the rate of monomaleimide compound release can be controlled.
  • biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the monomaleimide compounds in liposomes or microemulsions which are compatible with body tissues.
  • Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
  • a formulation suitable for buccal administration may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • conjugates or formulations as described herein can be administered to treat any hyperproliferative disease, metabolic disease, infectious disease, or cancer, as appropriate.
  • Formulations may be administered by injection, orally, or topically, typically to a mucosal surface (lung, nasal, oral, buccal, sublingual, vaginally, rectally) or to the eye (intraocularly or transocularly).
  • cancer embraces any disease or malady characterized by uncontrolled cell proliferation, e.g., hyperproliferation. Cancers may be characterized by tumors, e.g., solid tumors or any neoplasm.
  • the cancer is a solid tumor.
  • Large drug molecules have limited penetration in solid tumors. The penetration of large drug molecules is slow.
  • small molecules such as conjugates of the present disclosure may penetrate solid tumors rapidly and more deeply. Regarding penetration depth of the drugs, larger molecules penetrate less, despite having more durable pharmacokinetics. Small molecules such as conjugates of the present disclosure penetrate deeper.
  • Dreher et al. (Dreher et al., JNCI, vol.98(5):335 (2006), the contents of which are incorporated herein by reference in their entirety) studied penetration of dextrans with different sizes into a tumor xenograft. As summarized in Fig.
  • conjugates of the present disclosure reach at least about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 75 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 pm, about 1400 pm or about 1500 pm into the solid tumor from the vascular surface of the tumor.
  • Zero distance is defined as the vascular surface of the tumor, and every distance greater than zero is defined as the distance measured in three dimensions to the nearest vascular surface.
  • conjugates of the present disclosure penetrate to the core of the tumor.
  • Core of the tumor refers to the central area of the tumor.
  • the distance from any part of the core area of the tumor to the vascular surface of the tumor is between about 30% to about 50% of the length or width of the tumor.
  • the distance from any part of the core area of the tumor to the center point of the tumor is less than about 20% of the length or width of the tumor.
  • the core area of the tumor is roughly the center 1/3 of the tumor.
  • conjugates of the present disclosure penetrate to the middle of the solid tumor.
  • “Middle” of the tumor refers to the middle area of the tumor.
  • the distance from any part of the middle area of the tumor to the vascular surface of the tumor is between about 15% and about 30% of the length or the width of the tumor.
  • the distance from any part of the middle area of the tumor to the center point of the tumor is between about 20% to about 35% of the length or width of the tumor.
  • the middle area of the tumor is roughly between the center 1/3 of the tumor and the outer 1/3 of the tumor.
  • the subject may be otherwise free of indications for treatment with the conjugates or formulations.
  • methods include use of cancer cells, including but not limited to mammalian cancer cells.
  • the mammalian cancer cells are human cancer cells.
  • the conjugates or formulations of the present teachings have been found to inhibit cancer and/or tumor growth. They may also reduce, including cell proliferation, invasiveness, and/or metastasis, thereby rendering them useful for the treatment of a cancer.
  • the conjugates or formulations of the present teachings may be used to prevent the growth of a tumor or cancer, and/or to prevent the metastasis of a tumor or cancer.
  • compositions of the present teachings may be used to shrink or destroy a cancer.
  • the conjugates or formulations provided herein are useful for inhibiting proliferation of a cancer cell.
  • the conjugates or formulations provided herein are useful for inhibiting cellular proliferation, e.g., inhibiting the rate of cellular proliferation, preventing cellular proliferation, and/or inducing cell death.
  • the conjugates or formulations as described herein can inhibit cellular proliferation of a cancer cell or both inhibiting proliferation and/or inducing cell death of a cancer cell.
  • cell proliferation is reduced by at least about 25%, about 50%, about 75%, or about 90% after treatment with conjugates or formulations of the present disclosure compared with cells with no treatment.
  • cell cycle arrest marker phospho histone H3 (PH3 or PHH3) is increased by at least about 50%, about 75%, about 100%, about 200%, about 400% or about 600% after treatment with conjugates or formulations of the present disclosure compared with cells with no treatment.
  • cell apoptosis marker cleaved caspase-3 (CC3) is increased by at least 50%, about 75%, about 100%, about 200%, about 400% or about 600% after treatment with conjugates or formulations of the present disclosure compared with cells with no treatment.
  • conjugates or particles of the present disclosure are effective for inhibiting tumor growth, whether measured as a net value of size (weight, surface area or volume) or as a rate over time, in multiple types of tumors.
  • the size of a tumor is reduced by about 60 % or more after treatment with conjugates or formulations of the present disclosure. In some embodiments, the size of a tumor is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, by a measure of weight, and/or area and/or volume.
  • the cancers treatable by methods of the present teachings generally occur in mammals.
  • Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs horses, pigs, sheep, goats, and cattle.
  • Cancers include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt’s lymphoma, dysproliferative changes
  • cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, kidney cancer, urothelium cancer, female genital tract cancer, uterine cancer, gestational trophoblastic disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary gland cancer, hemangioma, sarcoma arising from bone and soft tissues, Kaposi’s sarcoma, nerve cancer, ocular cancer, meningial cancer, glioblastomas, neuromas, neuroblastomas, Schwannomas, solid tumors arising from hematopoietic malignancies such as leukemias, metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer,
  • the conjugates or formulations as described herein are used to treat small cell lung cancer.
  • About 12%-15% of patients having lung cancer have small cell lung cancer. Survival in metastatic small cell lung cancer is poor. Survival rate is below 5% five years after diagnosis. US incidence of small cell lung cancer is about 26K-30K.
  • the conjugates or formulations as described herein are used to treat patients with tumors that express or over-express the HSP90.
  • a feature of conjugates or formulations of the present disclosure is relatively low toxicity to an organism while maintaining efficacy at inhibiting, e.g., slowing or stopping tumor growth.
  • toxicity refers to the capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment.
  • Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment.
  • Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment.
  • conjugates or formulations of the present disclosure may have lower toxicity than the active agent moiety Z administered alone.
  • DM1 their toxicity is lower than DM1 administered alone.
  • Toxicity may further be measured relative to a subject’s weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity.
  • Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase.
  • Neutropenia, thrombopenia, white blood cell (WBC) count, complete blood cell (CBC) count may also be metrics of toxicity.
  • Pharmacologic indicators of toxicity include elevated aminotransferases (AST/ALT) levels, neurotoxicity, kidney damage, GI damage and the like.
  • conjugates or formulations of the present disclosure do not cause a significant change of a subject’s body weight.
  • the body weight loss of a subject is less about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or formulations of the present disclosure.
  • conjugates or formulations of the present disclosure do not cause a significant increase of a subject’s AST/ALT levels.
  • the AST or ALT level of a subject is increased by less than about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or formulations of the present disclosure.
  • conjugates or formulations of the present disclosure do not cause a significant change of a subject’s CBC or WBC count after treatment with conjugates or formulations of the present disclosure.
  • conjugates or formulations of the present disclosure are combined with at least one additional active agent.
  • the active agent may be any suitable drug.
  • the conjugates and the at least one additional active agent may be administered simultaneously, sequentially, or at any order.
  • the conjugates and the at least one additional active agent may be administered at different dosages, with different dosing frequencies, or via different routes, whichever is suitable.
  • the additional active agent may be selected from any active agent described herein such as a drug for treating cancer. It may also be a cancer symptom relief drug.
  • Nonlimiting examples of symptom relief drugs include: octreotide or lanreotide; interferon, cypoheptadine or any other antihistamines.
  • conjugates or formulations of the present disclosure do not have drug-drug interference with the additional active agent.
  • conjugates or formulations of the present disclosure do not inhibit cytochrome P450 (CYP) isozymes.
  • CYP isozymes may include CYP3A4 Midazolam, CYP3A4 Testosterone, CYP2C9, CYP2D6, CYP1A2, CYP2C8, CYP2B6, and CYP2C19.
  • the additional active agent may be administered concomitantly with conjugates or formulations of the present disclosure.
  • conjugates or formulations of the present disclosure may be combined with a moderate dose of chemotherapy agents such as mitomycin C, vinblastine and cisplatin (see Ellis et al., Br J Cancer, vol.71(2): 366-370 (1995), the contents of which are incorporated herein by reference in their entirety).
  • chemotherapy agents such as mitomycin C, vinblastine and cisplatin
  • a patient may first receive a pharmaceutically effective dose of an unconjugated active agent, followed by a pharmaceutically effective dose of a conjugate comprising the same active agent.
  • conjugates or formulations as described herein or formulations containing the conjugates or formulations as described herein can be used for the selective tissue delivery of a therapeutic, prophylactic, or diagnostic agent to an individual or patient in need thereof.
  • conjugates or formulations of the present disclosure are used to deliver the payloads to selective tissues. These tissues may be tumor tissues.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic.
  • the conjugates of the present disclosure may further comprise at least one reactive functionality that covalently modifies the target protein.
  • the reactive functionality may be part of the linker, payload or HSP90 binding ligand. If part of the linker, the covalent modification step may be associated with the cleavage of the linker.
  • the conjugate of the present dislcoure covalently modifies the target protein.
  • the covalent modification of the target protein may be reversible or irreversible.
  • the covalent modification may occur at a nucleophilic amino acid in the target protein.
  • the amino acid that is modified may be a cysteine, serine, threonine, lysine, arginine, tyrosine, histidine, or tryptophan.
  • the amino acid that is covalently modified may be cysteine 862 in PI3Ka, cysteine 481 in BTK, cysteine 797 in EGFR or any member of the kinase cysteinome as described in Liu et al., Chemistry & Biology 20, February 21, 2013, the contents of which are incorporated herein by reference in their entirety.
  • Conjugates as Proteolysis Targeting Chimeras PROTACs
  • PROTACs Proteolysis targeting chimeras
  • POP ligand protein of interest
  • E3 ligand E3 ligase
  • the POI ligand and E3 ligand are connected through a crosslinker.
  • the PROTACs recuite POI to E3 ligase.
  • Ubiquitin (Ub) is added to the POI Lys residues.
  • the Ub-marked POI is degraded by the proteasome.
  • PROTACs induce the target protein to be degraded instead of (or in addition to) being inhibited. This opens up an alternate way to kill the activity of a protein to enhance the impact on the pathway or to target previously undruggable targets.
  • the challenges of PROTACs include finding tissue selective E3 ligases (only -600 members of the class).
  • conjugates of the present disclosure induces degradation of a target protein in the tumor cells, such as the PI3K protein in the tumor cells.
  • the target protein refers to the protein that is regulated (e.g., its functions or activities are up-regulated or inhibited) by the conjugates of the present disclosure.
  • the target protein of the conjugate is not a client protein of HSP90 and the degradation of the target protein is not driven by HSP90.
  • the target protein of the active agent is a client protein of HSP90 (such as but not limited to HER2) and the degradation of the target protein of the active agent is higher than the degradation achieved by the HSP90-binding targeting ligand alone or than the degradation achieved by the non-HSP90-binding binding control.
  • the conjugates of the present disclosure bind to HSP90, which associates with E3 ubiquitin ligases to degrade the target proteins of the payloads if folding is stalled.
  • HSP90-binding conjugates comprising PI3K inhibitors can ubiquitinate PI3K and induce the degradation of PI3K.
  • the conjugates of the present disclosure induce the degradation of the target protein of the active agent in cells. In some embodiments, the conjugates of the present disclosure induce the degradation of the target protein and inhibit the activity and/or function of the target protein of the active agent in cells. The degradation of the target protein and the inhibition of the target protein can be measured with any method known in the art. In some embodiments, the cells are tumor cells.
  • the level of degradation of target protein is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% higher than the degradation achieved by the HSP90-binding targeting ligand alone or than the degradation achieved by the non-HSP90-binding control.
  • the level of degradation of target protein is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% as compared to a vehicle or non-treated or other appropriate control.
  • the conjugates of the present disclosure are bifunctional. They induce the degradation of at least one HSP90 client protein, such as HER2, and they induce the degradatoin of the target protein of the active agent, wherein the target protein of the active agent is not an HSP90 client protein.
  • the conjguates of the present disclosure induce degradation of PI3K in cells.
  • conjugates of the presnet disclosure induce degradation of PI3K and inhibit the activity and/or function of PI3K in tumor cells.
  • the degradation of PI3K and the inhibition of PI3K can be measured in any method known in the art.
  • the cells are tumor cells.
  • the linkers of the conjugates are not cleavable.
  • the conjugates have low permeability.
  • kits and devices for conveniently and/or effectively carrying out methods of the present disclosure.
  • kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
  • kits for inhibiting tumor cell growth in vitro or in vivo comprising a conjugate and/or particle of the present disclosure or a combination of conjugates and/or formulations of the present disclosure, optionally in combination with any other active agents.
  • the kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition.
  • the delivery agent may comprise a saline, a buffered solution, or any delivery agent disclosed herein.
  • the amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations.
  • the components may also be varied in order to increase the stability of the conjugates and/or formulations in the buffer solution over a period of time and/or under a variety of conditions.
  • the present disclosure provides for devices which may incorporate conjugates and/or formulations of the present disclosure. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient. In some embodiments, the subject has cancer.
  • Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices.
  • the devices may be employed to deliver conjugates and/or formulations of the present disclosure according to single, multi- or split-dosing regiments.
  • the devices may be employed to deliver conjugates and/or formulations of the present disclosure across biological tissue, intradermal, subcutaneously, or intramuscularly.
  • conjugate is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.
  • Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium.
  • the compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • subject refers to any organism to which the conjugates may be administered, e.g., for experimental, therapeutic, diagnostic, and/or prophylactic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, guinea pigs, cattle, pigs, sheep, horses, dogs, cats, hamsters, lamas, non-human primates, and humans).
  • treating can include preventing a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having the disease, disorder or condition; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • a “target”, as used herein, shall mean a site to which targeted constructs bind.
  • a target may be either in vivo or in vitro.
  • a target may be cancer cells found in leukemias or tumors (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas).
  • a target may refer to a molecular structure to which a targeting moiety or ligand binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate or enzyme.
  • a target may be a type of tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.
  • the “target cells” that may serve as the target for the method or conjugates or formulations are generally animal cells, e.g., mammalian cells.
  • the present method may be used to modify cellular function of living cells in vitro, i.e., in cell culture, or in vivo, in which the cells form part of or otherwise exist in animal tissue.
  • the target cells may include, for example, the blood, lymph tissue, cells lining the alimentary canal, such as the oral and pharyngeal mucosa, cells forming the villi of the small intestine, cells lining the large intestine, cells lining the respiratory system (nasal passages/lungs) of an animal (which may be contacted by inhalation of the subject disclosure), dermal/epidermal cells, cells of the vagina and rectum, cells of internal organs including cells of the placenta and the so-called blood/brain barrier, etc.
  • a target cell expresses at least one type of HSP90.
  • a target cell can be a cell that expresses an HSP90 and is targeted by a conjugate described herein, and is near a cell that is affected by release of the active agent of the conjugate.
  • a blood vessel expressing an HSP90 that is in proximity to a tumor may be the target, while the active agent released at the site will affect the tumor.
  • therapeutic effect is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance.
  • the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease, disorder or condition in the enhancement of desirable physical or mental development and conditions in an animal, e.g., a human.
  • modulation is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
  • the modulation is generally compared to a baseline or reference that can be internal or external to the treated entity.
  • parenteral administration means administration by any method other than through the digestive tract (enteral) or non-invasive topical routes.
  • parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intraossiously, intracerebrally, intrathecally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.
  • Topical administration means the non-invasive administration to the skin, orifices, or mucosa. Topical administration can be delivered locally, i.e., the therapeutic can provide a local effect in the region of delivery without systemic exposure or with minimal systemic exposure. Some topical formulations can provide a systemic effect, e.g., via adsorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal administration.
  • Enteral administration means administration via absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.
  • “Pulmonary administration”, as used herein, means administration into the lungs by inhalation or endotracheal administration.
  • inhalation refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose.
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement or prevention of at least one symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life.
  • the therapeutically effective amount is thus dependent upon the specific biologically active molecule and the specific condition or disorder to be treated.
  • Therapeutically effective amounts of many active agents, such as antibodies, are known in the art.
  • bioactive agent and “active agent”, as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body.
  • a bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • prodrug refers to an agent, including a small organic molecule, peptide, nucleic acid or protein, that is converted into a biologically active form in vitro and/or in vivo.
  • Prodrugs can be useful because, in some situations, they may be easier to administer than the parent compound (the active compound). For example, a prodrug may be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. A prodrug may also be less toxic than the parent.
  • a prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962) Drug Latentiation in Jucker, ed.
  • biocompatible refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient.
  • biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.
  • biodegradable generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject.
  • the degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the U.S. Food and Drug Administration.
  • a “pharmaceutically acceptable carrier”, as used herein, refers to all components of a pharmaceutical formulation that facilitate the delivery of the composition in vivo.
  • Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • molecular weight generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (M w ) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.
  • small molecule generally refers to an organic molecule that is less than 2000 g/mol in molecular weight, less than 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than 500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.
  • hydrophilic refers to substances that have strongly polar groups that readily interact with water.
  • hydrophobic refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
  • lipophilic refers to compounds having an affinity for lipids.
  • amphiphilic refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties.
  • Amphiphilic material refers to a material containing a hydrophobic or more hydrophobic oligomer or polymer (e.g., biodegradable oligomer or polymer) and a hydrophilic or more hydrophilic oligomer or polymer.
  • targeting moiety refers to a moiety that binds to or localizes to a specific locale.
  • the moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule.
  • the locale may be a tissue, a particular cell type, or a subcellular compartment.
  • a targeting moiety can specifically bind to a selected molecule.
  • reactive coupling group refers to any chemical functional group capable of reacting with a second functional group to form a covalent bond.
  • the selection of reactive coupling groups is within the ability of those in the art.
  • Examples of reactive coupling groups can include primary amines (-NH2) and amine-reactive linking groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.
  • -NH2 primary amines
  • amine-reactive linking groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, ary
  • reactive coupling groups can include aldehydes (-COH) and aldehyde reactive linking groups such as hydrazides, alkoxyamines, and primary amines.
  • reactive coupling groups can include thiol groups (-SH) and sulfhydryl reactive groups such as maleimides, haloacetyls, and pyridyl disulfides.
  • reactive coupling groups can include photoreactive coupling groups such as aryl azides or diazirines.
  • the coupling reaction may include the use of a catalyst, heat, pH buffers, light, or a combination thereof.
  • protective group refers to a functional group that can be added to and/or substituted for another desired functional group to protect the desired functional group from certain reaction conditions and selectively removed and/or replaced to deprotect or expose the desired functional group.
  • Protective groups are known to the skilled artisan. Suitable protective groups may include those described in Greene and Wuts, Protective Groups in Organic Synthesis, (1991). Acid sensitive protective groups include dimethoxytrityl (DMT), tert- butylcarbamate (tBoc) and trifluoroacetyl (tFA).
  • Base sensitive protective groups include 9- fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) and phenoxyacetyl (pac).
  • Other protective groups include acetamidomethyl, acetyl, tertamyloxycarbonyl, benzyl, benzyloxy carbonyl, 2-(4-bi phenylyl )-2-propy!
  • activated ester refers to alkyl esters of carboxylic acids where the alkyl is a good leaving group rendering the carbonyl susceptible to nucleophilic attack by molecules bearing amino groups. Activated esters are therefore susceptible to aminolysis and react with amines to form amides. Activated esters contain a carboxylic acid ester group -CO2R where R is the leaving group.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl -substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer.
  • cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • carbonyl such as a carboxyl, alkoxycarbonyl, formyl, or an acyl
  • thiocarbonyl such as a thioester, a
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. In some embodiments, alkyl groups are lower alkyls. In some embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.
  • heteroalkyl refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, and ethylthio.
  • alkylthio also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined above for alkyl groups.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • alkoxyl or "alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy.
  • An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl.
  • Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below.
  • the alkoxy and aroxy groups can be substituted as described above for alkyl.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: wherein R9, Rio, and R'10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)m-Rs or R9 and Rio taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rs represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8.
  • R9 or Rio can be a carbonyl, e.g., R9, Rio and the nitrogen together do not form an imide.
  • the term “amine” does not encompass amides, e.g., wherein one of R9 and Rio represents a carbonyl.
  • R9 and Rio (and optionally R’10) each independently represent a hydrogen, an alkyl or cycloalkly, an alkenyl or cycloalkenyl, or alkynyl.
  • alkylamine as used herein means an amine group, as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R9 and Rio is an alkyl group.
  • amido is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: wherein R9 and Rio are as defined above.
  • Aryl refers to Cs-Cio-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring systems.
  • aryl includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN; and combinations thereof.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles.
  • heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2J/,6J/-l,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H- indazolyl, indolenyl, indolinyl, indoli
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • Carbocycle refers to an aromatic or nonaromatic ring in which each atom of the ring is carbon.
  • “Heterocycle” or “heterocyclic”, as used herein, refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, for example, from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (Ci-Cio) alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents.
  • heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4a77-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2J/,6J/-l,5,2-dithiazinyl, dihydrofuro[2,3-Z>]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 177-indazolyl, indolenyl, indolinyl,
  • Heterocyclic groups can optionally be substituted with one or more substituents at one or more positions as defined above for alkyl and aryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, and -CN.
  • substituents at one or more positions as defined above for alkyl and aryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imin
  • carbonyl is art-recognized and includes such moieties as can be represented by the general formula: wherein X is a bond or represents an oxygen or a sulfur, and Rn represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl, or an alkynyl, R'n represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl, or an alkynyl. Where X is an oxygen and Rn or R’n is not hydrogen, the formula represents an "ester".
  • X is an oxygen and Rn is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Rn is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen and R'n is hydrogen, the formula represents a "formate”. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group.
  • the term “monoester” as used herein refers to an analog of a dicarboxylic acid wherein one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or salt of a carboxylic acid.
  • monoesters include, but are not limited to, to monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Examples of heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. Other useful heteroatoms include silicon and arsenic.
  • nitro means -NO2; the term “halogen” designates -F, -Cl, -Br or -I; the term “sulfhydryl” means -SH; the term “hydroxyl” means -OH; and the term “sulfonyl” means -SO2-.
  • substituted refers to all permissible substituents of the compounds described herein.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example, 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, or elimination.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents.
  • the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfony
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, - CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, al
  • copolymer generally refers to a single polymeric material that is comprised of two or more different monomers.
  • the copolymer can be of any form, for example, random, block, or graft.
  • the copolymers can have any end- group, including capped or acid end groups.
  • polypeptide generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally-occurring amino acids or are unnatural amino acids.
  • protein refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and/or quaternary structure.
  • protein excludes small peptides by definition, the small peptides lacking the requisite higher- order structure necessary to be considered a protein.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. These terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • nucleic acid is a term of art that refers to a string of at least two base-sugar-phosphate monomeric units. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of a messenger RNA, antisense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acids refers to a string of at least two base-sugar- phosphate combinations. Natural nucleic acids have a phosphate backbone. Artificial nucleic acids may contain other types of backbones, but contain the same bases as natural nucleic acids. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
  • a "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains at least one function as the full-length protein, polypeptide or nucleic acid.
  • a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions.
  • the DNA binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis.
  • the ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, e.g., genetic or biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.
  • linker refers to a carbon chain that can contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long.
  • heteroatoms e.g., nitrogen, oxygen, sulfur, etc.
  • Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. Those of skill in the art will recognize that each of these groups may in turn be substituted.
  • linkers include, but are not limited to, pH-sensitive linkers, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
  • pH-sensitive linkers protease cleavable peptide linkers
  • nuclease sensitive nucleic acid linkers include lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
  • pharmaceutically acceptable salt(s) refers to salts of acidic or basic groups that may be present in compounds used in the present compositions.
  • Compounds included in the present compositions that are basic in nature are capable of forming a variety of salts with various inorganic and organic acids.
  • the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i
  • Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
  • Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations.
  • Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
  • the free base can be obtained by basifying a solution of the acid salt.
  • an addition salt particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds.
  • Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.
  • a pharmaceutically acceptable salt can be derived from an acid selected from l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2 -hydroxy ethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,
  • bioavailable is art-recognized and refers to a form of the subject disclosure that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.
  • the conjugates of the disclosure may be prepared using any convenient methodology.
  • the conjugates are constructed from their individual components, targeting moiety, in some cases a linker, and active agent moiety.
  • the components can be covalently bonded to one another through functional groups, as is known in the art, where such functional groups may be present on the components or introduced onto the components using one or more steps, e.g., oxidation reactions, reduction reactions, cleavage reactions and the like.
  • Functional groups that may be used in covalently bonding the components together to produce the pharmaceutical conjugate include: hydroxy, sulfhydryl, amino, and the like.
  • the particular portion of the different components that are modified to provide for covalent linkage will be chosen so as not to substantially adversely interfere with that components desired binding activity, e.g., for the active agent moiety, a region that does not affect the target binding activity will be modified, such that a sufficient amount of the desired drug activity is preserved.
  • certain moieties on the components may be protected using blocking groups, as is known in the art, see, e.g., Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).
  • the conjugates of the present disclosure may be synthesized by attaching a targeting moiety to the payload, optionally by a linker.
  • a targeting moiety to the payload, optionally by a linker.
  • a person skilled in the art can synthesize these conjugates by routine chemical steps.
  • the conjugate can be produced using known combinatorial methods to produce large libraries of potential conjugates which may then be screened for identification of a bifunctional, molecule with the pharmacokinetic profile.
  • the conjugates may be produced using medicinal chemistry and known structure-activity relationships for the targeting moiety and the active agent moiety. In particular, this approach will provide insight as to where to join the two moieties to the linker.
  • reaction mass quenched with ice-water (15 mL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (5 mL) dried under vacuum to afford crude compound.
  • reaction mixture stirred for 3h at 60°C. Progress of the reaction was monitor by TLC and LCMS. After completion of SM, reaction mixture quenched with water (10.0 mL), solid was filtered and washed with ice cold water, drier under reduced pressure to afford tert-butyl 4-(2-((5-(2-aminopyrimidine-5-carboxamido)-7-methoxy- 2,3-dihydroimidazo[l,2-c] quinazolin-8-yl) oxy) ethyl) piperazine- 1 -carboxylate (Int- 3) (130 mg, 81.21%) as off white solid.
  • reaction mixture was stirred for 2h at room temperature, after completion SM (monitored by TLC) to the reaction mixture a solution of 2-amino-N-(8-methoxy-9- (3 -(piperazin- 1 -yl)propoxy)-3 ,4-dihydro-2H-pyrimido[ 1 ,2-c]quinazolin-6- yl)pyrimidine-5-carboxamide (436 mg, 0.884 mmol, 1. eq) in DMF (1.0 mL) was added drop wise and stirred at room temperature for 16h. Progress of reaction was monitor by TLC and LCMS. After completion of SM, reaction mixture was quenched with water and extracted with ethyl acetate (3 x 100 mL).
  • reaction mass diluted with water (1 mL) and extracted with ethyl acetate (2 X 5 mL). Combined organic layers dried over sodium sulphate, filtered, evaporated the solvent under vaccue to afford (90 mg, crude).
  • the crude compound was purified with column chromatography by using 100-200 mesh size silica, eluting at 2-3% Methanol in DCM to afford tert-butyl 2-(4-(4-(3-(2,4- dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)acetate (Int-2) (75 mg, 80.2%) as an off white solid.
  • reaction mixture was stirred for 16 h at RT. Progress of the reaction mixture monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (100 mL) and extracted with DCM (2 x50 mL) washed with Brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure to get the crude compound.
  • reaction mixture was stirred for 16h at RT. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction mixture, the reaction mixture was quenched with ice water (50 mL) stirred for 20 min solid precipitated filtered and washed with ice water (20 mL) dried under vacuum to get solid crude 150 mg.
  • reaction mixture was stirred at such a rate as to maintain a gentle reflux of the solvent for 3h. Progress of the reaction was monitored by TLC and LC-MS. After completion of SM, the reaction was quenched in water (20 mL) and extracted with (2 x 50 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to obtain crude.
  • reaction mixture allowed room temperature for 18h. After completion of SM, reaction mass quenched with water (5 mL) and extracted with DCM (3 x 15 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude, the crude compound was purified by column chromatography using silica gel (100-200 mesh), compound eluted at 2- 5 % MeOH in DCM.
  • reaction mixture was heated to 80°C and stirred at for 4 h. After completion of starting material, reaction mass quenched with ice-water (20 mL), the resulting solid was filtered and washed with ice water dried to afford crude compound (200 mg).
  • the crude was purified by flash chromatography use silica gel 100-200 mesh compound eluted 5% in MeOH in DCM, determined pure fraction to get pure compound (4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)- 6-morpholino-l, 3, 5-triazin-2-yl) piperazin- l-yl)-4-oxobutanoic acid (Int-5) (0.25 g 77%) as a brown liquid.
  • reaction mixture warm to rt and stirred for 16 h at RT. Progress of reaction mixture monitored by LCMS. After completion of the SM, the reaction mixture was quenched with water (50.0 mL) and extracted with EtOAC (2 x 100 mL) organic layers combined washed ice water (2 x 100 mL) dried over sodium sulfate filtered concentrated under reduced pressure to afford crude (Int-7) (0.45 g, crude). The crude compound was purified by flash chromatography use silica gel (100-200 mesh), compound eluted 30% EtOAc in pet ether.
  • reaction mixture was stirred for 18 h at RT.
  • the progress of reaction was monitored by TLC and LCMS After completion of the reaction, the reaction was quenched with ice water (20 mL), solid principates and stirred for 10 min, filtered and washed with water (5 mL) dried well under vacuum to get crude product.
  • reaction mixture was quenched with ice water (50.0 mL) extracted with EtOAC (3 x 50.0 mL) the combined organic layers washed with ice water (2 x 100 mL) and brine soln (1 x 100 mL) dried over sodium sulphate filtered to afford crude compound.
  • the crude was stirred for 30 min in diethyl ether filtered to get crude compound.
  • reaction mixture was allowed RT and stirred for 4h at same temperature. After completion of SM, reaction mixture quenched with water (5 mL) and extracted with EtOAC (2 x 10 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude compound.
  • the crude compound was purified by column chromatography using silica gel (100-200 mesh) and compound eluted at 0-2% MeOH in DCM. All pure fractions were mixed and concentrated to afford tert-butyl N-[3-(prop-2-yn-l-yloxy) propyl] carbamate (Int-3) (280 mg, 73.6%) as a light yellow oil.
  • reaction mixture was degassed with argon for 20 min, then added Cui (0.0 64g, 0.33 mmol, 0.3 eq) and Pd (PPhs)4 (0.65g, 0.56 mmol, 0.05 eq) at RT.
  • the resulting reaction mixture stirred for 5h at 45°C. Progress of the reaction mixture monitored by LCMS. After completion of the reaction, volatiles evaporated under reduced pressure to get crude compound (600 mg crude).
  • BT474 (breast cancer) cells are plated at 12,000 cells per well and incubated for 20-24hrs at 37°C at 5% CO2. Post cell incubation, compounds were reconstituted in DMSO to a stock concentration of 5mM. A compound plate is then prepared containing a 10 point dilution in DMSO. 2uL of these dilutions are then added to the cells for a final working concentration of 5uM to 0.0003uM. Compounds and cells are incubated for 16hrs. Media is then removed, cells washed, lysed, and analyzed for human total EbB2/Her2 levels by ELISA. HSP90 Binding:
  • the bindings of the conjugates to HSP90 were studied wth the HSP90a Assay Kit.
  • the HSP90a Assay Kit was designed for identification of HSP90a inhibitors using fluorescence polarization.
  • the assay was based on the competition of fluorescently labeled geldanamycin (GM) for binding to purified recombinant HSP90a.
  • the key to the HSP90a Assay Kit was the fluorescently labeled geldanamycin.
  • the fluorescently labeled geldanamycin was incubated with a sample containing HSP90a enzyme to produce a change in fluorescent polarization that can then be measured using a fluorescence reader.
  • Some of the conjugates have low permeability.
  • PI3Ka mutations occur in about 15% to 30% of breast, endometrial and colon cancers.
  • LS174T cells colon cancer
  • PI3Ka mutations were plated at 500 cells per well and incubated for 20-24hrs at 37°C at 5% CO2.
  • Post cell incubation compounds were reconstituted in DMSO to a stock concentration of 200uM.
  • a compound plate was then prepared containing a 10 point dilution in RPMI +10%FBS + 0.25% DMSO. 5uL of the dilution was then added for a final working concentration range of lOuM to 0.0005uM.
  • Compounds and cells were then incubated for 72hrs.
  • TGI Tumor growth inhibition
  • the information for compounds preparation was listed below: [0459] The LS174T cells were seeded and 2pl of compounds were transferred to the 6-well plate containing 2ml medium. It is a 1000 x dilution in total. Cell plates were incubated with the compounds as well as vehicle control at 5% CO2, 37°C for 6 hours or 24 hours. The cell lysis was then prepared and Western Blot was carried out.
  • PI3Kalpha (pl lOalpha) and pAKT (S473) levels in LS 174T were shown in FIG. 1 and FIG. 2. All five conjugates tested lead to degradation. Cl caused slower degradation but highest level of inhibition. C32, having a PI3K/mTor dual inhibitor, caused very rapid degradation. At the highest concentration of 10 mM, there was less degradation. This is consistent with a distinctive feature of PROTACs called hook effect.
  • conjugates of the present disclosure were administered to mice bearing LS174T tumors and the degradation of the PI3K pl lOalpha protein was monitored.
  • Proteins were transferred from the gel to a nitrocellulose membrane using iBlot Transfer Stack, nitrocellulose, regular size (Thermo Fisher Scientific, IB301001). After the transfer, the membrane was blocked in TBS Blocking Buffer (LI-COR, 927-60001). This was followed by overnight incubation of PI3 Kinase pl 10a (C73F8) Rabbit mAb #4249 Rabbit mAb #4060 (1 :500 dilution) and GAPDH (D4C6R) Mouse mAb #97166 (1 : 1000 dilution) at 4°C.
  • the membrane was washed with Pierce 20X TBS Tween 20 Buffer (Thermo Fisher Scientific, 28360) which was diluted to IX and incubated further for one hour at room temperature with the secondary antibodies: 1 : 10,000 IRDye 680RD Goat anti-Rabbit IgG (LI-COR 926-68071) and 1 : 10,000 IRDye 800CW Goat anti -Mouse IgG (LI-COR, 926-32210). Then the membrane was imaged on the LI-COR Odyssey Imaging System. Bands were quantified on the basis of their size and brightness using LI-COR Odyssey imaging software.
  • PI3Kalpha protein levels were measured after treatments with Compound Cl (45 mg/kg (mgk) or 25 mgk) and C48 (30 mgk) upto 168 hours after dose. The reduction of PI3Kalpha protein is shown in FIG. 5.
  • PI3K Degradation - Western Blotting experiments were carried out following the steps of: Human colon carcinoma cell line LS174T were counted and diluted to 106 cell/mL, using 2 mL per well in a 6-well plate. Cells were incubated with 1% DMSO or compound in indicated concentrations for 6 or 24h or cells were left untreated. Lysates were prepared and samples measured for total protein quantification by bicinchonic acid (BCA) assay supplemented with 5X loading buffer. Western blot was then run using standard procedures.
  • BCA bicinchonic acid
  • PI3Kinase alpha CST# 4249, pAkt (S473): CST# 4060; Akt: CST# 4691; GAPDH: Millipore# MAB374.
  • Degradation concentraion (DC50), %PI3K loss, %pAkt inhibition, %PI3Ka loss PD, %pAkt inhibition, and %PI3Ka loss PD were calculated and are shown in Table 10 below.
  • the %PI3Ka degradations measured by western blot for Compound Cl, C50 and C48 are shown in FIG. 6.

Abstract

Conjugates of an active agent attached to a targeting moiety, such as an HSP90 binding moiety, via a linker, and formulations comprising such conjugates have been designed. Methods of making and using the conjugates and the formulations thereof are also provided.

Description

HSP90-BINDING CONJUGATES AND FORMULATIONS THEREOF
REFERENCED TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional Patent Application No. 63/070,418, filed August 26, 2020, entitled HSP90-B INDING CONJUGATES AND FORMULATIONS THEREOF, and U.S. Provisional Patent Application No. 63/184,348, filed May 5, 2021, entitled HSP90-BINDING CONJUGATES AND FORMULATIONS THEREOF, the contents of each of which are herein incorporated by reference in their entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure generally relates to the field of therapeutic compounds. More particularly, the disclosure relates to the use of compounds targeting heat shock proteins including heat shock protein 90 (HSP90), e.g., for treating cancer.
BACKGROUND
[0003] Heat shock protein 90 (HSP90) is an intracellular chaperone protein that assists protein folding, stabilizes proteins against heat stress, and aids in protein degradation. It is upregulated in many types of cancer. Many Hsp90 client proteins are over-expressed in cancer, often in mutated forms, and are responsible for unrestricted cancer cell proliferation and survival. HSP90 is activated in cancer tissues and latent in normal tissues. HSP90 derived from tumour cells has higher binding affinity to HSP90 inhibitors than the latent form in normal cells, allowing specific targeting of HSP90 inhibitors to tumour cells with little inhibition of HSP90 function in normal cells. Further, HSP90 has also been recently identified as an important extracellular mediator for tumour invasion. Therefore, HSP90 is considered a major therapeutic target for anticancer drug development. There is a need in the art for developing therapeutic compounds that binds to HSP90.
SUMMARY
[0004] The present application provides a conjugate comprising an active agent coupled to an HSP90 targeting moiety by a linker and a pharmaceutical composition comprising such a conjugate. In some embodiments, the conjugate inhibits of the activity or function of a target protein in cells. In some embodiments, the conjugates induces degradation of the target protein. In some embodiments, the active agent is a PI3K inhibitor, such as but not limited to PF-04979064, PQR514, BAY 80-6946 (Copanlisib), orfragments/derivatives/analogs thereof.
[0005] The present disclosure also provides methods of degrading a protein in a cell, reducing cell proliferation, and/or treating caner comprising administering a therapeutically effective amount of the conjugates of the present disclosure.
[0006] Pharmaceutical compositions comprising such conjugates and methods of making and using such conjugates are also provided.
BRIEF DESCRIPION OF DRAWINGS
[0007] FIG. 1 shows PI3K degradation 6 hours after receiving various doses of conjugates of the present disclosure.
[0008] FIG. 2 shows PI3K degradation 24 hours after receiving various doses of conjugates of the present disclosure.
[0009] FIG. 3 and FIG. 4 show tumor volume changes in LS174T xenograft model after receiving various doses of conjugates of the present disclosure.
[0010] FIG. 5 shows PI3K degradation in an in vivo study after treatment with conjugates of the present disclosure.
[0011] FIG. 6 shows the %PI3Ka degradations measured by western blot for Compound Cl, C50 and C48.
DETAILED DESCRIPTION
[0012] Applicants have designed HSP90 targeting conjugates comprising an active agent and novel particles comprising such conjugates. Such targeting can, for example, improve the amount of active agent at a site and decrease active agent toxicity to the subject. HSP90 targeting conjugates of the present disclosure have deep and rapid tumor penetration and do not require receptor internalization. High accumulation and long retention time of HSP90 targeting conjugates enable the use of cytotoxic and non-cytotoxic payloads, such as chemotherapeutic agents, kinase inhibitors, or immuno-oncology modulators.
[0013] As used herein, “toxicity” refers to the capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment.
[0014] Toxicity may further be measured relative to a subject’s weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity. Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase. Neutropenia or thrombopenia may also be metrics of toxicity.
[0015] Pharmacologic indicators of toxicity include elevated AST/ALT levels, neurotoxicity, kidney damage, GI damage and the like.
[0016] The conjugates are released after administration of the particles. The targeted drug conjugates utilize active molecular targeting in combination with enhanced permeability and retention effect (EPR) and improved overall biodistribution of the particles to provide greater efficacy and tolerability as compared to administration of targeted particles or encapsulated untargeted drug.
[0017] In addition, the toxicity of a conjugate containing an HSP90 targeting moiety linked to an active agent for cells that do not overexpress HSP90 is predicted to be decreased compared to the toxicity of the active agent alone. Without committing to any particular theory, applicants believe that this feature is because the ability of the conjugated active agent to be retained in a normal cell is decreased relative to a tumor cell.
[0018] It is an object of the disclosure to provide improved compounds, compositions, and formulations for temporospatial drug delivery.
[0019] It is further an object of the disclosure to provide methods of making improved compounds, compositions, and formulations for temporospatial drug delivery.
[0020] It is also an object of the disclosure to provide methods of administering the improved compounds, compositions, and formulations to individuals in need thereof.
I. Conjugates
[0021] Conjugates include an active agent or prodrug thereof attached to a targeting moiety, e.g., a molecule that can bind to HSP90, by a linker. The conjugates can be a conjugate between a single active agent and a single targeting moiety, e.g., a conjugate having the structure X-Y-Z where X is the targeting moiety, Y is the linker, and Z is the active agent.
[0022] In some embodiments the conjugate contains more than one targeting moiety, more than one linker, more than one active agent, or any combination thereof. The conjugate can have any number of targeting moieties, linkers, and active agents. The conjugate can have the structure X-Y-Z-Y-X, (X-Y)n-Z, X-(Y-Z)n, Xn-Y-Z, X-Y- Zn, (X-Y-Z)n, (X-Y-Z-Y)n-Z, where X is a targeting moiety, Y is a linker, Z is an active agent, and n is an integer between 1 and 50, between 2 and 20, for example, between 1 and 5. Each occurrence of X, Y, and Z can be the same or different, e.g., the conjugate can contain more than one type of targeting moiety, more than one type of linker, and/or more than one type of active agent.
[0023] The conjugate can contain more than one targeting moiety attached to a single active agent. For example, the conjugate can include an active agent with multiple targeting moieties each attached via a different linker. The conjugate can have the structure X-Y-Z-Y-X where each X is a targeting moiety that may be the same or different, each Y is a linker that may be the same or different, and Z is the active agent.
[0024] The conjugate can contain more than one active agent attached to a single targeting moiety. For example the conjugate can include a targeting moiety with multiple active agents each attached via a different linker. The conjugate can have the structure Z-Y-X-Y-Z where X is the targeting moiety, each Y is a linker that may be the same or different, and each Z is an active agent that may be the same or different.
A. Active Agents
[0025] A conjugate as described herein contains at least one active agent (a first active agent). The conjugate can contain more than one active agent, that can be the same or different from the first active agent. The active agent can be a therapeutic, prophylactic, diagnostic, or nutritional agent. A variety of active agents are known in the art and may be used in the conjugates described herein. The active agent can be a protein or peptide, small molecule, nucleic acid or nucleic acid molecule, lipid, sugar, glycolipid, glycoprotein, lipoprotein, or combination thereof. In some embodiments, the active agent is an antigen, an adjuvant, radioactive, an imaging agent (e.g., a fluorescent moiety) or a polynucleotide. In some embodiments the active agent is an organometallic compound. [0026] In certain embodiments, the active agent of the conjugate comprises a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of active agent(s) of the conjugate may also be expressed in terms of proportion to the targeting ligand(s). For example, the present teachings provide a ratio of active agent to ligand of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
[0027] In some embodiments, the active agent can be a cancer therapeutic. Cancer therapeutics include, for example, death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis. Suitable death receptors include, but are not limited to, TNFR1, Fas, DR3, DR4, DR5, DR6, LTpR and combinations thereof.
[0028] Cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy agents can be used as active agents. Chemotherapeutic agents include, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Such agents typically affect cell division or DNA synthesis and function. Additional examples of therapeutics that can be used as active agents include monoclonal antibodies and the tyrosine kinase inhibitors e.g. imatinib mesylate, which directly targets a molecular abnormality in certain types of cancer (e.g., chronic myelogenous leukemia, gastrointestinal stromal tumors).
[0029] Chemotherapeutic agents include, but are not limited to cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxol and derivatives thereof, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, epipodophyllotoxins, trastuzumab, cetuximab, and rituximab, bevacizumab, and combinations thereof. Any of these may be used as an active agent in a conjugate. [0030] The small molecule active agents used in this disclosure (e.g. antiproliferative (cytotoxic and cytostatic) agents) include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PBK/m- TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors and proteasome inhibitors. The small molecule active agents in some embodiments the active agent is an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.
[0031] Broad spectrum cytotoxins include, but are not limited to, DNA-binding or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, and topoisomerase I or II inhibitors.
[0032] Exemplary DNA-binding or alkylating drugs include, CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and its analogs, alkylating agents, such as calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.
[0033] Exemplary doxorubicin analogs include nemorubicin metabolite or analog drug moiety disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.
[0034] Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin CI, duocarmycin C2, duocarmycin B2, DU-86, KW-2189, bizelesin, seco-adozelesin, and those described in U.S. Patent Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660;
6,586,618; 6,756,397 and 7,049,316. Doxorubicin and its analogs include PNU- 159682 and those described in U.S. Patent No.6, 630, 579 and nemorubicin metabolite or analog drugs disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.
[0035] Calicheamicins include those described in U.S. Patent Nos. 5,714,586 and 5,739,116. Duocarmycins include those described in U.S. Patent Nos.5, 070, 092;
5,101,038; 5,187,186; 6,548,530; 6,660,742; and 7,553,816 B2; and Li et al., Tet Letts., 50:2932 - 2935 (2009). Pyrrolobenzodiazepines include SG2057 and those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000), Anti-Cancer Agents in Medicinal Chemistry, 2009, 9, 1-31; WO 2011/130613 Al; EP 2 789 622 Al; Blood 2013, 122, 1455; J. Antimicrob. Chemother. 2012, 67, 1683-1696; Cancer Res. 2004, 64, 6693-6699; WO 2013041606; US 8481042; WO 2013177481; WO 2011130613; WO2011130598.
[0036] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, cabazitaxel; maytansinoids, auristatins and analogs thereof, tubulysin A and B derivatives, vinca alkaloid derivatives, epothilones, PM060184 and cryptophy cins.
[0037] Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, maytansine or DM-1 and DM-4 are those described in U.S.
Patent Nos. 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821; RE39,151 and 7,276,497. In certain embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52: 127-131), maytansinoids or maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.
[0038] Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F and dolastatin. Suitable auristatins are also described in U.S. Publication Nos. 2003/0083263, 2011/0020343, and 2011/0070248; PCT Application Publication Nos. WO 09/117531, WO 2005/081711, WO 04/010957; W002/088172 and WOOl/24763, and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902;5,554,725; 5,530,097;
5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;
4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety.
[0039] Exemplary tubulysin compounds include compounds described in U.S. Patent Nos. 7,816,377; 7,776,814; 7,754,885; U.S. Publication Nos. 2011/0021568;
2010/004784; 2010/0048490; 2010/00240701; 2008/0176958; and PCT Application Nos. WO 98/13375; WO 2004/005269; WO 2008/138561; WO 2009/002993; WO 2009/055562; WO 2009/012958; WO 2009/026177; WO 2009/134279; WO 2010/033733; WO 2010/034724; WO 2011/017249; WO 2011/057805; the disclosures of which are incorporated by reference herein in their entirety.
[0040] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). Suitable Vinca alkaloids that can be used in the present disclosure are also disclosed in U.S. Publication Nos. 2002/0103136 and 2010/0305149, and in U.S. Patent No. 7,303,749 Bl, the disclosures of which are incorporated herein by reference in their entirety.
[0041] Exemplary epothilone compounds include epothilone A, B, C, D, E and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121,029;
6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO 97/19086; WO 98/08849; WO 98/22461; WO 98/25929; WO 98/38192; WO 99/01124; WO 99/02514; WO 99/03848; WO 99/07692; WO 99/27890; and WO 99/28324; the disclosures of which are incorporated herein by reference in their entirety.
[0042] Exemplary cryptophy cin compounds are described in U.S. Patent Nos. 6,680,311 and 6,747,021, the disclosures of which are incorporated herein by reference in their entirety.
[0043] Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOX ATINE®), iproplatin, ormaplatin, and tetraplatin.
[0044] Exemplary topoisomerase I inhibitors include camptothecin, camptothecin, derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11 (irinotecan), SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625. Other camptothecin compounds that can be used in the present disclosure include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30: 1774 (1987).
[0045] Exemplary topoisomerase II inhibitors include azonafide and etoposide. [0046] Additional agents acting on DNA include Lurbinectedin (PM01183), Trabectedin (also known as ecteinascidin 743 or ET-743) and analogs as described in WO 200107711, WO 2003014127.
[0047] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors. [0048] Exemplary MetAP2 inhibitors include fumagillol analogs, meaning any compound that includes the fumagillin core structure, including fumagillamine, that inhibits the ability of MetAP-2 to remove NFL-terminal methionines from proteins as described in Rodeschini et al., I. Org. Chem., 69, 357-373, 2004 and Liu, et al., Science 282, 1324-1327, 1998. Non limiting examples of "fumagillol analogs" are disclosed in /. Org. Chem., 69, 357, 2004; J. Org. Chem., 70, 6870, 2005; European Patent Application 0 354 787; / . Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999; W099/61432; U.S. Patent Nos. 6,603,812; 5,789,405; 5,767,293; 6,566,541; and 6,207,704.
[0049] Exemplary cell cycle progression inhibitors include CDK inhibitors such as BMS-387032 and PD0332991; Rho-kinase inhibitors such as GSK429286; checkpoint kinase inhibitors such as AZD7762; aurora kinase inhibitors such as AZDI 152, MLN8054 and MLN8237; PLK inhibitors such as BI 2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors such as SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520. [0050] Exemplary PI3K/m-T0R/AKT signaling pathway inhibitors include phosphoinositide 3 -kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors.
[0051] Exemplary PI3 kinase inhibitors are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147, XL765, GSK2126458 (Omipalisib), GDC-0326, GDC-0032 (Taselisib, RG7604), PF-05212384 (Gedatolisib, PKI-587), BAY 80-6946 (copanlisib), PF- 04691502, PF-04989216, PF-04979064, PI-103, PKI-402 VS-5584 (SB2343), GDC- 0941, NVP-BEZ235 (Dactoslisib), BGT226, NVP-BKM120 (Buparlisib), NVP- BYL719 (alpelisib), GSK2636771, AMG-319, GSK2269557, PQR309, PQR514, PQR530, PWT143, TGR-1202 (RP5264), PX-866, GDC-0980 (apitolisib), AZD8835, MLN1117, DS-7423, ZSTK474, CUDC-907, IPI-145 (INK-1197, Duvelisib), AZD8186, XL147 (SAR245408), XL765 (SAR245409), CAL-101 (Idelalisib, GS- 1101), GS-9820 (Acalisib) and KA2237.
[0052] Exemplary AKT inhibitors include, but are not limited to, AT7867, MK- 2206, Perifosine, GSK690693, Ipatasertib, AZD5363, TIC10, Afuresertib, SC79, AT13148, PHT-427, A-674563, and CCT128930.
[0053] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.
[0054] Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD 8330 and GDC-0973.
[0055] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885. [0056] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB202190.
[0057] Receptor tyrosine kinases (RTK) are cell surface receptors which are often associated with signaling pathways stimulating uncontrolled proliferation of cancer cells and neoangiogenesis. Many RTKs, which over express or have mutations leading to constitutive activation of the receptor, have been identified, including, but not limited to, VEGFR, EGFR, FGFR, PDGFR, EphR and RET receptor family receptors. Exemplary RTK specific targets include ErbB2, FLT-3, c-Kit, c-Met, and HIF.
[0058] Exemplary inhibitors of ErbB2 receptor (EGFR family) include but not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), and Gefitinib (Iressa).
[0059] Exemplary RTK inhibitors targeting more then one signaling pathway (multitargeted kinase inhibitors) include AP24534 (Ponatinib) that targets FGFR, FLT-3, VEGFR-PDGFR and Bcr-Abl receptors; ABT-869 (Linifanib) that targets FLT-3 and VEGFR- PDGFR receptors; AZD2171 that targets VEGFR-PDGFR, Fit- 1 and VEGF receptors; CHR-258 (Dovitinib) that targets VEGFR-PDGFR, FGFR, Flt- 3, and c-Kit receptors.
[0060] Exemplary kinase inhibtiors include inhibitors of the kinases ATM, ATR, CHK1, CHK2, WEE1, and RSK. In some embodiments, the active agents are ATR inhibitors, such as but not limited to berzosertib (VX-970, M6620, or VE-822), AZD6738, or BAY1895344.
[0061] Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX- 5422, NVP-AUY-922, and KW-2478.
[0062] Exemplary HD AC inhibitors include Belinostat (PXD101), CUDC-101,
Doxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP- LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A, and Vorinostat (SAHA).
[0063] Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD- 2281), ABT-888 (Veliparib), AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3- aminobenzamide, A-966492, and AZD2461. [0064] Exemplary Wnt/Hedgehog signaling pathway inhibitors include vismodegib (RG3616/GDC-0449), cyclopamine (11-deoxojervine) (Hedgehog pathway inhibitors), and XAV-939 (Wnt pathway inhibitor).
[0065] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include a- amanitins, P- amanitins, y- amanitins, s-amanitins, amanullin, amanullic acid, amaninamide, amanin, and proamanullin.
[0066] Exemplary proteasome inhibitors include bortezomib, carfilzomib, ONX 0912, CEP- 18770, and MLN9708.
[0067] In one embodiment, the drug of the disclosure is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor (e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)), MEK inhibitor, KSP inhibitor, RNA polymerse inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, duocarmycin, tubulysin, auristatin or a platinum compound. In specific embodiments, the drug is a derivative of SN-38, vindesine, vinblastine, PI- 103, AZD 8330, auristatin E, auristatin F, a duocarmycin compound, tubulysin compound, or ARRY-520.
[0068] In another embodiment, the drug used in the disclosure is a combination of two or more drugs, such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.
[0069] The active agent can be a cancer therapeutic. The cancer therapeutics may include death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis. Suitable death receptors include, but are not limited to, TNFR1, Fas, DR3, DR4, DR5, DR6, LTpR and combinations thereof.
[0070] The active agent can be a DNA minor groove binders such as lurbectidin and trabectidin.
[0071] The active agent can be E3 ubiquitin ligase inhibitors, adeubiquitinase inhibitors or an NFkB pathway inhibitor.
[0072] The active agent can be a phopsphatase inhibitors including inhibitors of PTP1B, SHP2, LYP, FAP-1, CD45, STEP, MKP-1, PRL, LMWPTP or CDC25. [0073] The active agent can be an inhibitor of tumor metabolism, such as an inhibitor of GAPDH, GLUT1, HK II, PFK, GAPDH, PK, LDH orMCTs. [0074] The active agent can target epigenetic targets including EZH2, MLL, DOTI -like protein (DOT IL), bromodomain-containing protein 4 (BRD4), BRD2, BRD3, NUT, ATAD2, or SMYD2.
[0075] The active agent can target the body's immune system to help fight cancer, including moecules targeting IDO1, IDO2, TDO, CD39, CD73, A2A antagonists, STING activators, TLR agonists (TLR 1-13), ALK5, CBP/EP300 bromodomain, ARG1, ARG2, iNOS, PDE5, P2X7, P2Y11, COX2, EP2 Receptor, or EP4 receptor. [0076] The active agent can target Bcl-2, IAP, or fatty acid synthase.
[0077] In some embodiments, the active agent can be 20-epi-l,25 dihydroxyvitamin D3, 4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizing morphogenetic protein- 1, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin, baccatin III derivatives, balanol, batimastat, benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride, bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene A, bizelesin, bleomycin, bleomycin sulfate, BRC/ ABL antagonists, breflate, brequinar sodium, bropirimine, budotitane, busulfan, buthionine sulfoximine, cabazitaxel, cactinomycin, calcipotriol, calphostin C, calusterone, camptothecin, camptothecin derivatives, canarypox IL-2, capecitabine, caracemide, carbetimer, carboplatin, carboxamide-amino-triazole, carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derived inhibitor, carubicin hydrochloride, carzelesin, casein kinase inhibitors, castano spermine, cecropin B, cedefingol, cetrorelix, chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam, cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, dronabinol, duazomycin, duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine, edrecolomab, eflornithine, eflornithine hydrochloride, elemene, elsamitrucin, emitefur, enloplatin, enpromate, epipropidine, epirubicin, epirubicin hydrochloride, epristeride, erbulozole, erythrocyte gene therapy vector system, esorubicin hydrochloride, estramustine, estramustine analog, estramustine phosphate sodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide, etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine, fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil, flurocitabine, forfenimex, formestane, fosquidone, fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabine hydrochloride, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride, idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor- 1 receptor inhibitor, interferon agonists, interferon alpha-2A, interferon alpha-2B, interferon alpha-Nl, interferon alpha-N3, interferon beta-IA, interferon gamma-IB, interferons, interleukins, iobenguane, iododoxorubicin, iproplatin, irinotecan, irinotecan hydrochloride, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, larotaxel, lanreotide acetate, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole, liarozole, liarozole hydrochloride, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantrone hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, maytansine, maytansinoid, mertansine (DM1), mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, merbarone, mercaptopurine, meterelin, methioninase, methotrexate, methotrexate sodium, metoclopramide, metoprine, meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1 - based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, mycophenolic acid, myriaporone, n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, nocodazole, nogalamycin, n-substituted benzamides, 06-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole, peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin, piritrexim, piroxantrone hydrochloride, placetin A, placetin B, plasminogen activator inhibitor, platinum(IV) complexes, platinum compounds, platinum-triamine complex, plicamycin, plomestane, porftmer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, puromycin, puromycin hydrochloride, purpurins, pyrazofurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxy ethylene conjugate, RAF antagonists, raltitrexed, ramosetron, RAS famesyl protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes, RII retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex, rubiginone Bl, ruboxyl, safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI 1 mimetics, semustine, senescence derived inhibitor 1 , sense oligonucleotides, siRNA, signal transduction inhibitors, signal transduction modulators, simtrazene, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin, spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide, teniposide, teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan hydrochloride, topsentin, toremifene, toremifene citrate, totipotent stem cell factor, translation inhibitors, trestolone acetate, tretinoin, triacetyluridine, triciribine, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tropisetron, tubulozole hydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, velaresol, veramine, verdins, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin stimalamer, or zorubicin hydrochloride. [0078] The active agent can be an inorganic or organometallic compound containing one or more metal centers. In some examples, the compound contains one metal center. The active agent can be, for example, a platinum compound, a ruthenium compound (e.g., /raz/.s-fRuCh (DMSO)4], or Zraw -[RuC14(imidazole) 2, etc.), cobalt compound, copper compound, or iron compounds.
[0079] In some embodiments, the active agent is a small molecule. In some embodiments, the active agent is a small molecule cytotoxin. In one embodiment, the active agent is cabazitaxel, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. In another embodiment, the active agent is mertansine (DM1) or DM4, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. DM1 or DM4 inhibits the assembly of microtubules by binding to tubulin. Structure of DM1 is shown below:
Figure imgf000018_0001
(DM1).
[0080] In some embodiments, the active agent Z is Monomethyl auristatin E (MMAE), or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structure of MMAE is shown below:
Figure imgf000018_0002
(MMAE).
[0081] In some embodiments, the active agent Z is a sequence-selective DNA minor-groove binding crosslinking agent. For example, Z may be pyrrolobenzodiazepine (PBD), a PBD dimer, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structures of PBD and PBD dimer are shown below:
Figure imgf000019_0001
dimer).
[0082] In some embodiments, the active agent Z is a topoisomerase I inhibitor, such as camptothecin, irinotecan, SN-38, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.
Figure imgf000019_0002
SN-38 (7 -Ethyl- 10-hydroxy-camptothecin)
[0083] Any cytotoxic moiety disclosed in WO2013158644, WO2015038649,
W02015066053, WO2015116774, WO2015134464, W02015143004,
WO2015184246, the contents of each of which are incorporated herein by reference in their entirety, such as bendamustine, VDA, doxorubicin, pemetrexed, vorinostat, lenalidomide, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adozelesin, or derivatives/analogs thereof, may be used as an active agent in conjugates of the present disclosure.
[0084] In some embodiments, the active agent is a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor, including but not limited to irinotecan, topotecan, camptothecin, diflomotecan and lamellarin D, and fragments/derivatives/analogs thereof. In some embodiments, the topoisomerase inhibitor is a topoisomerase II inhibitor such as etoposide (VP- 16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, and HU-331, and fragments/derivatives/analogs thereof. PI3K Inhibitors
[0085] The PI3K/AKT/mTOR signaling network (PI3K pathway) controls most hallmarks of cancer: cell cycle, survival, metabolism, motility and genomic stability. The PI3K pathway is the most frequently altered pathway in human cancer. Activation of PI3K has been directly linked to cancer through mutations or amplifications of PIK3CA, and loss of function tumor suppressor PTEN. PIK3CA gene is the 2nd most frequently mutated oncogene. PTEN is among the most frequently mutated tumor suppressor genes. Pathway inhibitors demonstrate antitumor efficacy in xenograft models, but toxicity limits clinical benefit in patients. Conjugating a PI3K inhibitor with a HSP90 targeting moiety provides a method to delivery PI3K inhibitors for sufficient PI3K inhibition in tumors with reduced toxicity.
[0086] Conjugates comprising PI3K inhibitors may be used to treat hematological malignancies and solid tumors. In some embodiments, conjugates comprising PI3K inhibitors are used to treat colorectal cancer, multiple myeloma, leukemia, lymphoma, colon cancer, gastric cancer, kidney cancer, lung cancer, or breast cancers including metastatic breast cancer. In some embodiments, conjugates comprising PI3K inhibitors are used to treat PIK3CA-altered cancers or HER2 positive cancers.
[0087] Any PI3K inhibitor may be used as an active agent. In some embodiments, the PI3K inhibitor may be a small molecule. Non-limiting examples include PQR514, PQR530, PF-04979064, BAY 80-6946 (Copanlisib), Omipalisib (GSK2126458, GSK458), , PF-04691502, PI-103, BGT226 (NVP-BGT226), Apitolisib (GDC-0980, RG7422), Duvelisib (IPI-145, INK1197), AZD8186, Pilaralisib (XL147), PIK-93, Idelalisib (GS-1101), MLN1117, VS-5584, SB2343, GDC-0941, BM120, NVP- BKM120, Buparlisib, AZD8835, XL765 (SAR245409), GS-9820 Acalisib, GSK2636771, AMG-319, IPI-549, Perifosine, Alpelisib, TGR 1202 (RP5264), PX- 866, AMG-319, GDC-0980, GDC-0941, Sanofi XL147, XL499, XL756, XL147, PF- 46915032, BKM 120, CAL 263, SF1126, PX-886, KA2237, a dual PI3K inhibitor (e.g., Novartis BEZ235), an isoquinolinone, or fragments/derivatives/analogs thereof. [0088] In some embodiments, the PI3K inhibitor may be an inhibitor of delta and gamma isoforms of PI3K. In some embodiments, the PI3K inhibitor is an inhibitor of alpha isoforms of PI3K. In other embodiments, the PI3K inhibitor is an inhibitor of one or more alpha, beta, delta and gamma isoforms of PI3K. Non-limiting examples of PI3K inhibitors include compounds disclosed in US 9,546,180 (Infinity Pharmaceuticals), WO 2009088990 (Intellikine Inc.), WO 2011008302 (Intellikine Inc.), WO 2010036380 (Intellikine Inc.), WO 2010/006086 (Intellikine Inc.), WO 2005113556 (Icos Corp.), US 2011/0046165 (Intellikine Inc.), or US 20130315865 (Pfizer), the contents of each of which are incorporated herein by reference in their entirety.
[0089] In some embodiments, the conjugates of the present disclosure comprises PQR514 or its fragments/derivatives/analogs, PF-04979064 or its fragments/derivatives/analogs, Omipalisib (GSK458) or its fragments/derivatives/analogs, BAY 80-6946 (Copanlisib) or its fragments/derivatives/analogs, PQR530 or its fragments/derivatives/analogs, PF- 04691502 or its fragments/derivatives/analogs, PI- 103 or its fragments/derivatives/analogs, BGT226 (NVP-BGT226) or its fragments/derivatives/analogs, Apitolisib (GDC-0980, RG7422) or its fragments/derivatives/analogs, Duvelisib (IPI-145, INK1197) or its fragments/derivatives/analogs, AZD8186 or its fragments/derivatives/analogs, Pilaralisib (XL147) or its fragments/derivatives/analogs, and PIK-93 or its fragments/ derivatives/ analogs .
Figure imgf000021_0001
PQR514
Figure imgf000021_0002
Figure imgf000022_0001
Apitolisib (GDC-0980, RG7422) Duvelisib (IPI-145, INK1197) AZD8186
Figure imgf000023_0001
Pilaralisib (XL147) PIK-93 Idelalisib (GS-1101)
[0090] In particular, the conjugates of the present disclosure may comprise an
HSP90 targeting moiety connected to PQR514 or its fragments/derivatives/analogs, PF-04979064 or its fragments/derivatives/analogs, BAY 80-6946 (Copanlisib) or its fragments/ derivatives/ analogs .
[0091] In some embodiments, the conjugates of the present disclosure comprises PQR514 or its fragments/derivatives/analogs, such as
Figure imgf000023_0002
r its fragments,
Figure imgf000024_0002
[0092] In some embodiments, the conjugates of the present disclosure comprises a
PI3K/mTOR dual inhibitor. In some embodiments, the conjugates of the present disclosure comprises PF-04979064 or its fragments/derivatives/analogs, such as
Figure imgf000024_0001
Figure imgf000025_0001
[0093] In some embodiments, the conjugates of the present disclosure comprises
Copanlisib or its fragments/derivatives/analogs, such as
Figure imgf000025_0002
P9 or its fragments,
Figure imgf000026_0001
[0094] In some embodiments, the conjugates of the present disclosure comprise a benzoxazepine group, such as GDC-0077
Figure imgf000026_0002
(Inavolisib), GDC-0326
Figure imgf000027_0001
or fragments/derivatives/analogs thereof, such as:
Figure imgf000027_0002
B. HSP90 Targeting Moieties
[0095] Targeting ligands (also referred to as targeting moieties) as described herein include any molecule that can bind one or more HSP90 proteins. Such targeting ligands can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates, or lipids.
[0096] The targeting moiety, X, can be any HSP90 binding moiety such as, but not limited to, natural compounds (e.g., geldanamycin and radicicol), and synthetic compounds such as geldanamycin analogue 17-AAG (i.e., 17- allylaminogeldanamycin), a purine-scaffold HSP90 inhibitor series including PU24FC1 (He H., et al, J. Med. Chem., vol.49:381 (2006), the contents of which are incorporated herein by reference in their entirety), BIIB021 (Lundgren K., et al, Mol. Cancer Ther., vol.8(4):921 (2009), the contents of which are incorporated herein by reference in their entirety), 4,5- diarylpyrazoles (Cheung K.M., et al, Bioorg. Med. Chem. Lett., vol.15:3338 (2005), the contents of which are incorporated herein by reference in their entirety), 3- aryl,4-carboxamide pyrazoles (Brough P.A., et al, Bioorg. Med. Chem. Lett., vol.15: 5197 (2005), the contents of which are incorporated herein by reference in their entirety), 4,5-diarylisoxazoles (Brough P.A., et al, J. Med. Chem., vol.51 : 196 (2008), the contents of which are incorporated herein by reference in their entirety), 3,4-diaryl pyrazole resorcinol derivative (Dymock B.W., et al, J. Med. Chem., vol.48:4212 (2005), the contents of which are incorporated herein by reference in their entirety), thieno[2,3- d]pyrimidine (W02005034950 to VERNALIS et al., the contents of which are incorporated herein by reference in their entirety), aryl triazole derivatives of Formula I in EP2655345 to Giannini et al., the contents of which are incorporated herein by reference in their entirety, or any other example of HSP90 binding ligands or their derivatives/analogs.
[0097] In some embodiments, the targeting moiety may be a peptide, such as a bicyclic peptide or a modified bicyclic peptide. In some embodiments, the targeting moiety is a peptide having a sequence of (X)lC(X)mC(X)nC(X)o as disclosed in EP2474613, the contents of which are incorporated herein by reference in their entirety, wherein C is cysteine, X represents a random amino acid, m and n are numbers between 1 and 20 defining the length of intervening polypeptide segments and 1 and o are numbers between 0 and 20 defining the length of the flanking polypeptide segments. In some embodiments, the targeting moiety comprises at least 2 polypeptides covalently linked to tri s-(bromom ethyl) benzene (TBMB) at two or more amino acid residues as disclosed in US8680022 and US8778844, the contents of each of which are incorporated herein by reference in their entirety.
[0098] In some embodiments, the HSP90 binding moiety may be heterocyclic derivatives containing three heteroatoms. W02009134110 to MATULIS et al., the contents of which are incorporated herein by reference in their entirety, discloses 4,5- diaryl thiadiazoles which demonstrate good HSP90 binding affinity. Even though they have rather modest cell growth inhibition, they may be used as HSP90 binding moiety in conjugates of the present disclosure. Another class of aza-heterocyclic adducts, namely triazole derivatives or their analogs, may be used as HSP90 binding moiety in conjugates of the present disclosure. For example, the 1,2,4-triazole scaffold has been profusely documented as possessing HSP90 inhibiting properties. WO2009139916 to BURLISON et al. (Synta Pharmaceuticals Corp.), the contents of which are incorporated herein by reference in their entirety, discloses tricyclic 1,2,4-triazole derivatives inhibiting HSP90 at high micromolar concentrations. Any tricyclic 1,2,4- triazole derivatives disclosed in WO2009139916 or their derivatives/analogs may be used as HSP90 binding moiety in conjugates of the present disclosure. Any tri substituted 1,2,4- triazole derivatives disclosed in WO 2010017479 and WO 2010017545 (Synta Pharmaceuticals Corp.) or their derivatives/analogs, the contents of which are incorporated herein by reference in their entirety, may be used as HSP90 binding moiety in conjugates of the present disclosure. In another example, a triazolone-containing HSP90 inhibitor named ganetespib (previously referred as to STA-9090, or as its highly soluble phosphate prodrug STA- 1474) disclosed in W02006055760 (Synta Pharmaceuticals Corp.), the contents of which are incorporated herein by reference in their entirety, or its derivatives/analogs may be used as HSP90 binding moiety in conjugates of the present disclosure.
Figure imgf000029_0001
Ganetespib
[0099] In some embodiments, ganetespib or its derivatives/analogs may be used a targeting moiety. Non-limiting examples of ganetespib derivatives/analogs are shown below.
Figure imgf000029_0002
Figure imgf000030_0001
Figure imgf000031_0001
[0100] In some embodiments, Onalespib (AT13387) or its derivatives/analogs may be used as a targeting moiety in the conjugates of the present disclosure. Onalespib and non-limiting examples of Onalespib derivatives/analogs are shown below.
Figure imgf000031_0002
Figure imgf000032_0001
[0101] In some embodiments, the HSP90 targeting moiety comprises
Figure imgf000032_0002
r its fragments/ derivatives/ analogs.
[0102] Any HSP90 ligand or HSP90 inhibitor disclosed in WO2013158644, WO2015038649, W02015066053, WO2015116774, WO2015134464,
W02015143004, WO2015184246, the contents of which are incorporated herein by reference in their entirety, or their derivatives/analogs may be used as HSP90 binding moiety in the conjugates of the present disclosure, such as:
Formula
Figure imgf000032_0003
, wherein R1 may be alkyl, aryl, halide, carboxamide or sulfonamide; R2 may be alkyl, cycloalkyl, aryl or heteroaryl, wherein when R2 is a 6 membered aryl or heteroaryl, R2 is substituted at the 3- and 4- positions relative to the connection point on the triazole ring, through which a linker L is attached; and R3 may be SH, OH, -CONHR4, aryl or heteroaryl, wherein when R3 is a 6 membered aryl or heteroaryl, R3 is substituted at the 3 or 4 position; Formula
Figure imgf000033_0001
, wherein R1 may be alkyl, aryl, halo, carboxamido, sulfonamido; and R2 may be optionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examples of such compounds include 5-(2,4-dihydroxy-5- isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H- 1,2,4- triazole-3 -carboxamide and 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4- methylpiperazin- 1 -yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H- 1 ,2,4-triazole-3 - carboxamide;
Formula I
Figure imgf000033_0002
, wherein X, Y, and Z may independently be CH,
N, O or S (with appropriate substitutions and satisfying the valency of the corresponding atoms and aromaticity of the ring); R1 may be alkyl, aryl, halide, carboxamido or sulfonamido; R2 may be substituted alkyl, cycloalkyl, aryl or heteroaryl, where a linker L is connected directly or to the extended substitutions on these rings; R3 may be SH, OH, NR4R5 AND -CONHR6, to which an effector moiety may be connected; R4 and R5 may independently be H, alkyl, aryl, or heteroaryl; and R6 may be alkyl, aryl, or heteroaryl, having a minimum of one functional group to which an effector moiety may be connected; or
Formula
Figure imgf000033_0003
, wherein R1 may be alkyl, aryl, halo, carboxamido or sulfonamido; R2 and R3 are independently C1-C5 hydrocarbyl groups optionally substituted with one or more of hydroxy, halogen, C1-C2 alkoxy, amino, mono- and di-Cl-C2 alkylamino; 5- to 12- membered aryl or heteroaryl groups; or, R2 and R3, taken together with the nitrogen atom to which they are attached, form a 4- to 8- membered monocyclic heterocyclic group, of which up to 5 ring members are selected from O, N and S. Examples of such compounds include AT-13387.
[0103] The HSP90 targeting moiety may be Ganetespib, Luminespib (AUY-922, NVP-AUY922), Debio-0932, MPC-3100, Onalespib (AT-13387), SNX-2112, 17- amino-geldanamycin hydroquinone, PU-H71, AT 13387, or derivatives/ analogs thereof.
Figure imgf000034_0001
AT-13387
Figure imgf000035_0001
[0104] The HSP90 targeting moiety may be SNX5422 (PF-04929113), or any other HSP90 inhibitors disclosed in US 8080556 (Pfizer), W02008096218 (Pfizer), W02006117669 (Pfizer), W02008059368 (Pfizer), W02008053319 (Pfizer), W02006117669 (Pfizer), EP1885701 (Novartis), EP1776110 (Novartis), EP2572709 (Novartis), WO2012131413 (Debiopharm), or WO2012131468 (Debiopharm), the contents of each of which are incorporated herein by reference in their entirety.
Figure imgf000035_0002
SNX5422
[0105] The HSP90 targeting moiety may also be PU-H71, an HSP90 inhibitor that is 124I radiolabeled for PET imaging or its derivatives/analogs.
[0106] Conjugates comprising SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71, or ATI 3387 may have a structure of:
Figure imgf000036_0001
17-amino-geldanamycin hydroquinone Conjugate
Figure imgf000036_0002
[0107] In some embodiments, the HSP90 targeting moiety comprises a Sansalvamide A derivative. Sansalvamide A (San A) is a cyclic pentapeptide isolated from a marine fungus and binds to HSP90. Any Di-Sansalvamide A derivative (dimerized San A molecules) disclosed in Alexander et al., J Med Chem., vol.52(24):7927 (2009), the contents of which are incorporated herein by reference in their entirety, for example, the Di-San A molecules in Figure 1 of Alexander, may be used as a targeting moiety of the conjugate of the current disclosure.
[0108] In some embodiments, the HSP90 targeting moiety comprises alkynyl pyrrolo[2,3-d]pyrimidines and related analogs such as HSP90 inhibitors disclosed in US7544672 (CONFORMA THERAPEUTICS), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the HSP90 targeting moiety compriese any HSP90 inhibitor disclosed in WO2010117425 (Biogen), the contents of which are incorporated herein by reference in their entirety. Extracellular HSP90 (eHSP90)
[0109] In normal cells, secretion of HSP90 occurs when cells are under environmental stress such as heat, drugs, cytokines, UV, and/or gamma rays. The main function of the extracellular HSP90 (eHSP90) is to help tissue repair by promoting the cells at the edge of damaged tissue to migrate into the damaged area. However, in tumors, constitutively activated oncogenes trigger HSP90 secretion even without any environmental stress. Secreted Hsp90 by tumors eHSP90a promotes both tumor and tumor stroma cell migration during invasion and metastasis. The extracellular promotility function of HSP90a depends on a 115-amino acid fragment (F-5) on the surface of HSP90 (Li et al., Ini Rev Cell Mol Biol., vol.303:203-235 (2013), the contents of which are incorporated herein by reference in their entirety). eHSP90 has been shown to be present on the surface of tumor cells and to also be capable of being internalized (Crowe et al., ACS Chem. Biol., vol.12: 1047-1055 (2017)). The surface expression of eHSP90 in tumor cells thus represents a target for directing therapies selectively to tumors over healthy cells. Therefore, eHSP90 (eHSP90a in particular) may be a good target for treating tumors.
[0110] The expression of eHSP90 can be measured using the method known in the art, such as ELISA, RIA, EIA, sandwich assay, Western Blot analysis, immunostaining, flow cytometry and immunohistological staining.
[OHl] In some embodiments, the targeting moiety selectively binds to eHSP90. In some embodiments, the targeting moiety binds to F-5 region of eHSP90.
[0112] In some embodiments, the targeting moiety has low cell-permeability and prefers to bind to cell surface eHSP90. In some embodiments, the targeting moiety is cell-impermeable and binds exclusive to eHSP90. In some embodiments, the conjugates comprising the targeting moieties have a low cell permeability or is cell- impermeable.
[0113] As disclosed in WO2017184956 to Haystead et al. (Duke University), HS- 131 (100) is a fluorescent specific small-molecule Hsp90 inhibitor. It is not cell permeable and it binds exclusively to eHsp90. To analyze the internalization of eHsp90 in transformed cells, the five isogenic cell lines (67NR, 168FARN, 4T07, and 4T1) were isolated from a single spontaneous mammary tumor. They cells exhibit varying degrees of metastatic disease when injected into mice. 100 was internalized to a higher extent in 4T1 cells (the most aggressive of the five lines) over the less metastatic lines.
[0114] High resolution confocal images of 100-treated cells revealed striking punctate formations both on the cell surface and within the cell itself, as well as diffuse fluorescence throughout the cell. Accumulation of the puncta intracellularly is time-dependent. Live imaging of cells treated with 100 with a lattice light sheet microscope shows puncta traveling throughout the cell, consistent with active trafficking. Puncta were not revealed using Hsp90 antibodies alone.
[0115] Studies in cells suggest that aggressive tumor cell lines, almost exclusively, express eHsp90. Further studies with 100 in tumor cells such as glioblastoma, nonsmall lung, prostate, and melanoma lines suggest that expression of eHsp90 is common to cells with an aggressive malignant cellular phenotype.
[0116] In some embodiments, the targeting moieties comprise HS-23, HS-131, (disclosed in Crowe et al., ACS Chem. Biol., vol.12: 1047-1055 (2017), the contents of which are incorporated herein by reference in their entirety) or DMAG-N-oxide (a cell-impermeable for of 17-AAG disclosed in Tsutsumi et al., Oncogene, vol.27(17):2478-2487 (2008), the contents of which are incorporated herein by reference in their entirety), or analog/derivative thereof, the structures shown below.
Figure imgf000038_0001
HS-131
Figure imgf000039_0001
»MA< -N-oxide
[0117] In some embodiments, the targeting moiety comprises BIIB021 (Dihydropyrrol opyrimidine), or any compound disclosed in WO2010117425, and fragments/analogs/derivatives thereof, such as the compounds of Formula I or Formula II or the compounds in Table 2 of WO2010117425, and the structures shown below:
Figure imgf000039_0002
[0118] In some embodiments, the targeting moiety comprises an antibody or antibody fragment which binds to human extracellular Hsp90. For example, the targeting moiety may be a monoclonal antibody disclosed in US 7959915 (Tufts University), the contents of which are incorporated herein by reference in their entirety, such as m Ab 1.5.1. In another example, the targeting moiety maybe an anti- HSP90 monoclonal antibody disclosed in US20110280881 (Riken Corp.), the contents of which are incorporated herein by reference in their entirety, wherein the antibody binds to an epitope comprising an amino acid sequence selected from the amino acid sequence VX1X2EX3PPLEGDX4 (wherein each of XI to X4, which may be identical to or different from each other, represents an arbitrary amino acid) (SEQ ID NO: 1 in US20110280881) or the amino acid sequence HX5IX6ETLRQKAE (wherein each of X5 to X6, which may be identical to or different from each other, represents an arbitrary amino acid) (SEQ ID NO:2 in US20110280881), and wherein the antibody recognizes cell surface HSP90.
[0119] In certain embodiments, the targeting moiety or moieties of the conjugate are present at a predetermined molar weight percentage from about 0.1 % to about 10%, or about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of targeting moieties of the conjugate may also be expressed in terms of proportion to the active agent(s), for example, in a ratio of ligand to active agent of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5:1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
C. Linkers
[0120] The conjugates contain one or more linkers attaching the active agents and targeting moieties. The linker, Y, is bound to one or more active agents and one or more targeting ligands to form a conjugate. The linker Y is attached to the targeting moiety X and the active agent Z by functional groups independently selected from an ester bond, disulfide, amide, acylhydrazone, ether, carbamate, carbonate, and urea. Alternatively the linker can be attached to either the targeting ligand or the active drug by a non-cleavable group such as provided by the conjugation between a thiol and a maleimide, an azide and an alkyne. The linker is independently selected from the group consisting alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally is substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl is optionally substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl.
[0121] In some embodiments, the linker comprises a cleavable functionality that is cleavable. The cleavable functionality may be hydrolyzed in vivo or may be designed to be hydrolyzed enzymatically, for example by Cathepsin B. A “cleavable” linker, as used herein, refers to any linker which can be cleaved physically or chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by re- dox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes. For example, the cleavable functionality may be a disulfide bond or a carbamate bond.
[0122] In some embodiments the alkyl chain of the linker may optionally be interrupted by one or more atoms or groups selected from -O-, -C(=O)-, -NR, -O- C(=O)-NR-, -S-, -S-S-. The linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid. In some embodiments, the linker Y may be X’-RkY’-R2- ’ and the conjugate can be a compound according to Formula la:
X R1 R2 Z
V v Ia wherein X is a targeting moiety defined above; Z is an active agent; X’, R1, Y’, R2 and Z’ are as defined herein.
[0123] X’ is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or more natural or unnatural amino acids, thio or succinimido; R1 and R2 are either absent or comprised of alkyl, substituted alkyl, aryl, substituted aryl, polyethylene glycol (2-30 units); Y’ is absent, substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol (2-30 units) or an amide; Z’ is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, thio or succinimido. In some embodiments, the linker can allow one active agent molecule to be linked to two or more ligands, or one ligand to be linked to two or more active agent molecule.
[0124] In some embodiments, the linker Y may be Am and the conjugate can be a compound according to Formula lb:
Figure imgf000041_0001
wherein A is defined herein, m=0-20. [0125] A in Formula la is a spacer unit, either absent or independently selected from the following substituents. For each substituent, the dashed lines represent substitution sites with X, Z or another independently selected unit of A wherein the X, Z, or A can be attached on either side of the substituent:
Figure imgf000042_0001
z = 0-40, R is H or an optionally substituted alkyl group, and R’ is any side chain found in either natural or unnatural amino acids.
[0126] In some embodiments, the conjugate may be a compound according to Formula Ic:
Figure imgf000042_0002
Ic wherein A is defined above, m=0-40, n=0-40, x=l-5, y=l-5, and C is a branching element defined herein.
[0127] C in Formula Ic is a branched unit containing three to six functionalities for covalently attaching spacer units, ligands, or active drugs, selected from amines, carboxylic acids, thiols, or succinimides, including amino acids such as lysine, 2,3- diaminopropanoic acid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, and cysteine. Non-Limiting Examples of Conjugates
PI3K Inhibitors as Active Agents
[0128] In some embodiments, the active agent Z is a PI3K inhibitor and the HSP90 targeting moiety X is selected from the group consisting of TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM10, TM11, TM12 and their fragments/derivatives/analogs, wherein the active agent Z and the targeting moiety X are connected with a linker. In some embodiments, the linkers are cleavable. In some embodiments, the linkers are non-cleavable.
[0129] In some embodiments, the conjugates of the present disclosure comprise Copanlisib or its fragments/derivatives/analogs, such as P7 or its fragments, P8 or its fragments, P9 or its fragments, P10 or its fragments, Pl 1 or its fragments, or P12 or its fragments. Non-limiting examples include:
Table 1: Non-Limiting Examples of Conjugates Comprising Copanlisib or its fragments/derivatives/analogs
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
[0130] Additional examples of conjugates comprising HSP90-targeting moeities connected to Copanlisib or its fragments/derivatives/analogs include:
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
[0131] In some embodiments, the conjugates of the present invention comprise copanlisib or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C53, C54, C62, C75, C76, and C77.
[0132] In some embodiments, the conjugates of the present invention comprise copanlisib or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C48, C49, C50, C51, C52, C56, C57, C58, C59, C60, C61, C63, C64, C65, C66, C67, C68, C69, C70, C71, C72, C73, and C74. [0133] In some embodiments, the conjugates of the present disclosure comprise PF-04979064 or its fragments/derivatives/analogs, such as P4 or its fragments, P5 or its fragments, or P6 or its fragments. Non-limiting examples include:
Table 2: Non-Limiting Examples of Conjugates Comprising PF-04979064 or its
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
[0134] Additional examples of conjugates comprising HSP90-targeting moeities connected to PF-04979064 or its fragments/derivatives/analogs include:
Figure imgf000063_0002
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0002
[0135] In some embodiments, the conjugates of the present invention comprise PF-04979064 or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C81, C82, and C83.
[0136] In some embodiments, the conjugates of the present invention comprise PF-04979064 or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C78, C79, C80, C84, C85, C86, and C87.
[0137] In some embodiments, the active agent Z comprises PQR514 or its fragments/derivatives/analogs, such as Pl or its fragments, P2 or its fragments, or P3 or its fragments. Non-limiting examples include:
Table 3: Non-Limiting Examples of Conjugates Comprising PQR514 or its fragments/derivatives/analogs
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
[0138] Additional examples of conjugates comprising HSP90-targeting moeities connected to PQR514 or its fragments/derivatives/analogs include:
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
[0139] In some embodiments, the conjugates of the present invention comprise PQR514 or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C88, C89, C90, C91, C92, C93, C94, C95, C96, C98, C99, C100, C101, C104, C106, C107, C108, C109, C110, Cl 11, C112, C113, C114, C115, C116, and C120.
[0140] In some embodiments, the conjugates of the present invention comprise PQR514 or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C102 and C103.
[0141] In some embodiments, the active agent Z comprises GDC-0326 or its fragments/derivatives/analogs. Non-limiting examples include:
Table 4: Non-Limiting Examples of Conjugates Comprising GDC-0326 or its
Figure imgf000076_0002
Figure imgf000077_0001
[0142] In some embodiments, the conjugates of the present invention comprise GDC-0326 or its fragments/derivatives/analogs as active agents, and BIIB021 or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C119.
[0143] In some embodiments, the conjugates of the present invention comprise GDC-0326 or its fragments/derivatives/analogs as active agents, and ganetespib or its fragments/derivatives/analogs as targeting moieties. Non-limiting examples include C117 and C118.
D. Pharmacokinetic Modulating Unit
[0144] The conjugates of the present disclosure may further comprise at least one external linker connected to a reacting group that reacts with a functional group on a protein or an engineered protein or derivatives/analogs/mimics thereof, or comprise at least one external linker connected to a pharmacokinetic modulating unit. The external linkers connecting the conjugates and the reacting group or the pharmacokinetic modulating units may be cleavable linkers that allow release of the conjugates. Hence, the conjugates may be separated from the protein or pharmacokinetic modulating units as needed.
[0145] Any reacting group or PMU (such as PMUs comprising polymers) disclosed in WO2017/197241, the contents of which are incorporated herein by reference in their entirety, may be attached to the conjugates of the present disclosure.
E. Permeability Modulating Unit
[0146] The conjugates of the present disclosure may further comprise at least one permeability modulating unit. In some embodiments, the permeability modulating unit is attached to the payload of the conjugate, wherein the permeablity modulating unit regulates the cell membrane permeability of the payload. In some embodiments, the permeability modulating unit reduces the permeability of the payload. Not willing to be bound by any theory, once the payload is released from the conjugate, the permability modulating unit that is attached to the payload reduces the cell membrane permability of the payload, increases the retention time of the payload in target cells, improves the intracellular accumulation of the payload, and improves its efficacy.
[0147] In some embodiments, the permeability modulation unit does not adversely impact the permeability of the conjugate or the binding capability of the targeting moiety. In some embodiments, the permeability modulation unit is active only after the payload is released from the conjugate, e.g., after the cleavable linker between the payload and the targeting moiety is cleaved.
[0148] In some embodiments, the permeability modulating unit is a functional group that is covalently attached to the payload of the conjugate. In some embodiments, the permeability modulating unit is an integral part of the payload. [0149] In some embodiments, the permeability modulating unit is attached to the payload via an external linker. The external linker may be a non-cleavable linker.
[0150] The passive permeation of a payload through the biological cell membranes is strongly dependent on the molecule physicochemical properties. Important factors that influence cell memberane permeation include the acid-base character of the molecule (which influences the charge of the molecule at the specific pH), its lipophilicity (which affects its partition between aqueous and lipid environments), and its solubility. For a payload to be permeable, there should be an appropriate balance between the hydrophobicity and hydrophilicity. In some embodiments, the permeability moduclating unit is hydrophilic. In some embodiments, the permeability moduclating unit is hydrophobic. In some embodiments, the permeability moduclating unit is polar. In some embodiments, the permeability moduclating unit is charged at physiological pH. For example, the permeability modulating unit may be positively charged, negatively charged, or a combination of multiple charges.
[0151] Non-limiting examples of the permability modulating unit include a functional group that has at least one nitrogen, such as a piperazine functional group.
II. Formulations
[0152] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the conjugate to be delivered as described herein. [0153] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
[0154] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
[0155] A pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. [0156] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
[0157] The conjugates of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) permit the sustained or delayed release (e.g., from a depot formulation of the monomaleimide); (3) alter the biodistribution (e.g., target the monomaleimide compounds to specific tissues or cell types); (4) alter the release profile of the monomaleimide compounds in vivo. Non-limiting examples of the excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and preservatives. Excipients of the present disclosure may also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the disclosure may include one or more excipients, each in an amount that together increases the stability of the monomaleimide compounds.
Excipients
[0158] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
[0159] In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[0160] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
[0161] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
[0162] Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, crosslinked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof. [0163] Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
[0164] Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof. [0165] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMAB EN®II, NEOLONE™, KATHON™, and/or EUXYL®. [0166] Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, etc., and/or combinations thereof.
[0167] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
[0168] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
[0169] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
Administration
[0170] The conjugates the present disclosure may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in a way which allows them to cross the bloodbrain barrier, vascular barrier, or other epithelial barrier.
[0171] The formulations described herein contain an effective amount of conjugates in a pharmaceutical carrier appropriate for administration to an individual in need thereof. The formulations may be administered parenterally (e.g., by injection or infusion). The formulations or variations thereof may be administered in any manner including enterally, topically (e.g., to the eye), or via pulmonary administration. In some embodiments the formulations are administered topically.
A. Parenteral Formulations
[0172] The conjugates can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution, suspension or emulsion. The formulation can be administered systemically, regionally or directly to the organ or tissue to be treated. [0173] Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in- water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
[0174] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations 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/or by the use of surfactants. In some cases, an isotonic agent is included, for example, one or more sugars, sodium chloride, or other suitable agent known in the art.
[0175] Solutions and dispersions of the conjugates can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combinations thereof.
[0176] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4- oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Pol oxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N- dodecyl-P-alanine, sodium N-lauryl-P-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0177] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).
[0178] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. If using 10% sucrose or 5% dextrose, a buffer may not be required. [0179] Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol. [0180] Sterile injectable solutions can be prepared by incorporating the conjugates in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized conjugates into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation include vacuum-drying and freeze-drying techniques that yield a powder of the particle plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are known in the art.
[0181] Pharmaceutical formulations for parenteral administration can be in the form of a sterile aqueous solution or suspension of conjugates formed from one or more polymer-drug conjugates. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3 -butanediol.
[0182] In some instances, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.
[0183] Solutions, suspensions, or emulsions for parenteral administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.
[0184] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, sucrose, dextrose, mannitol, sorbitol, sodium chloride, and other electrolytes. [0185] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.
[0186] Solutions, suspensions, or emulsions for parenteral administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.
B. Mucosal Topical Formulations
[0187] The conjugates can be formulated for topical administration to a mucosal surface Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulation may be formulated for transmucosal transepithelial, or transendothelial administration. The compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof. In some embodiments, the conjugates can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some embodiments, the conjugates are formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, to the mucosa, such as the eye or vaginally or rectally.
[0188] “ Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol.
[0189] “Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate.
[0190] Suitable classes of penetration enhancers are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.
Dosins
[0191] The present disclosure provides methods comprising administering conjugates containing the conjugate as described herein to a subject in need thereof. Conjugates containing the conjugates as described herein may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
[0192] Compositions in accordance with the disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[0193] In some embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, from about 25 mg/kg to about 50 mg/kg, from about 50 mg/kg to about 100 mg/kg, from about 100 mg/kg to about 125 mg/kg, from about 125 mg/kg to about 150 mg/kg, from about 150 mg/ to about 175 mg/kg, from about 175 mg/kg to about 200 mg/kg, from about 200 mg/kg to about 250 mg/kg of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.
[0194] The concentration of the conjugates of the present disclosure may be between about 0.01 mg/mL to about 50 mg/mL, about 0.1 mg/mL to about 25 mg/mL, about 0.5 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 5 mg/mL in the pharmaceutical composition. [0195] As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In one embodiment, the monomal eimide compounds of the present disclosure are administed to a subject in split doses. The monomaleimide compounds may be formulated in buffer only or in a formulation described herein.
Dosage Forms
[0196] A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous).
Liquid dosage forms
[0197] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Injectable
[0198] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di glycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
[0199] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0200] In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the monomaleimide compounds then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered monomaleimide compound may be accomplished by dissolving or suspending the monomalimide in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the monomaleimide compounds in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of monomaleimide compounds to polymer and the nature of the particular polymer employed, the rate of monomaleimide compound release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the monomaleimide compounds in liposomes or microemulsions which are compatible with body tissues.
Pulmonary
[0201] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose. [0202] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
[0203] General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
Coatings or Shells
[0204] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
III. Methods of Using the Conjugates and Formulations
[0205] The conjugates or formulations as described herein can be administered to treat any hyperproliferative disease, metabolic disease, infectious disease, or cancer, as appropriate. Formulations may be administered by injection, orally, or topically, typically to a mucosal surface (lung, nasal, oral, buccal, sublingual, vaginally, rectally) or to the eye (intraocularly or transocularly).
[0206] In various embodiments, methods for treating a subject having a cancer are provided, wherein the method comprises administering a therapeutically-effective amount of the conjugates, salt forms thereof, or formulations comprising such conjugates, as described herein, to a subject having a cancer, suspected of having cancer, or having a predisposition to a cancer. According to the present disclosure, cancer embraces any disease or malady characterized by uncontrolled cell proliferation, e.g., hyperproliferation. Cancers may be characterized by tumors, e.g., solid tumors or any neoplasm.
[0207] In some embodiments, the cancer is a solid tumor. Large drug molecules have limited penetration in solid tumors. The penetration of large drug molecules is slow. On the other hand, small molecules such as conjugates of the present disclosure may penetrate solid tumors rapidly and more deeply. Regarding penetration depth of the drugs, larger molecules penetrate less, despite having more durable pharmacokinetics. Small molecules such as conjugates of the present disclosure penetrate deeper. Dreher et al. (Dreher et al., JNCI, vol.98(5):335 (2006), the contents of which are incorporated herein by reference in their entirety) studied penetration of dextrans with different sizes into a tumor xenograft. As summarized in Fig. 6 and Table 1 of Dreher, Dextrans with a molecular weight of 3.3kDa or lOkDa showed rapid deep penetration into the tumor tissue (>35um from the vascular surface of the tumor). However, 40kDa, 70kDa or 2mDa sized dextrans penetrated much less than the 3.3kDa or lOkDa dextran. The 70kDa dextran reached only about 15um from the vascular surface of the tumor. Conjugates of the present disclosure have a molecule weight comparable to the 3.3kDa and lOkDa dextrans, while antibody drug conjugates have a molecule weight at least as big as the 70kDa dextran. Therefore, conjugates of the present disclosure may penetrate deep and rapidly into the core/center of the solid tumor.
[0208] In one embodiment, conjugates of the present disclosure reach at least about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 75 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 pm, about 1400 pm or about 1500 pm into the solid tumor from the vascular surface of the tumor. Zero distance is defined as the vascular surface of the tumor, and every distance greater than zero is defined as the distance measured in three dimensions to the nearest vascular surface.
[0209] In another embodiment, conjugates of the present disclosure penetrate to the core of the tumor. “Core” of the tumor, as used herein, refers to the central area of the tumor. The distance from any part of the core area of the tumor to the vascular surface of the tumor is between about 30% to about 50% of the length or width of the tumor. The distance from any part of the core area of the tumor to the center point of the tumor is less than about 20% of the length or width of the tumor. The core area of the tumor is roughly the center 1/3 of the tumor.
[0210] In another embodiment, conjugates of the present disclosure penetrate to the middle of the solid tumor. “Middle” of the tumor, as sued herein, refers to the middle area of the tumor. The distance from any part of the middle area of the tumor to the vascular surface of the tumor is between about 15% and about 30% of the length or the width of the tumor. The distance from any part of the middle area of the tumor to the center point of the tumor is between about 20% to about 35% of the length or width of the tumor. The middle area of the tumor is roughly between the center 1/3 of the tumor and the outer 1/3 of the tumor.
[0211] In some embodiments, the subject may be otherwise free of indications for treatment with the conjugates or formulations. In some embodiments, methods include use of cancer cells, including but not limited to mammalian cancer cells. In some instances, the mammalian cancer cells are human cancer cells.
[0212] In some embodiments, the conjugates or formulations of the present teachings have been found to inhibit cancer and/or tumor growth. They may also reduce, including cell proliferation, invasiveness, and/or metastasis, thereby rendering them useful for the treatment of a cancer.
[0213] In some embodiments, the conjugates or formulations of the present teachings may be used to prevent the growth of a tumor or cancer, and/or to prevent the metastasis of a tumor or cancer. In some embodiments, compositions of the present teachings may be used to shrink or destroy a cancer.
[0214] In some embodiments, the conjugates or formulations provided herein are useful for inhibiting proliferation of a cancer cell. In some embodiments, the conjugates or formulations provided herein are useful for inhibiting cellular proliferation, e.g., inhibiting the rate of cellular proliferation, preventing cellular proliferation, and/or inducing cell death. In general, the conjugates or formulations as described herein can inhibit cellular proliferation of a cancer cell or both inhibiting proliferation and/or inducing cell death of a cancer cell. In some embodiments, cell proliferation is reduced by at least about 25%, about 50%, about 75%, or about 90% after treatment with conjugates or formulations of the present disclosure compared with cells with no treatment. In some embodiments, cell cycle arrest marker phospho histone H3 (PH3 or PHH3) is increased by at least about 50%, about 75%, about 100%, about 200%, about 400% or about 600% after treatment with conjugates or formulations of the present disclosure compared with cells with no treatment. In some embodiments, cell apoptosis marker cleaved caspase-3 (CC3) is increased by at least 50%, about 75%, about 100%, about 200%, about 400% or about 600% after treatment with conjugates or formulations of the present disclosure compared with cells with no treatment.
[0215] Furthermore, in some embodiments, conjugates or particles of the present disclosure are effective for inhibiting tumor growth, whether measured as a net value of size (weight, surface area or volume) or as a rate over time, in multiple types of tumors.
[0216] In some embodiments the size of a tumor is reduced by about 60 % or more after treatment with conjugates or formulations of the present disclosure. In some embodiments, the size of a tumor is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, by a measure of weight, and/or area and/or volume.
[0217] The cancers treatable by methods of the present teachings generally occur in mammals. Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs horses, pigs, sheep, goats, and cattle. In various embodiments, Cancers include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt’s lymphoma, dysproliferative changes (dysplasias and metaplasias), embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen-receptor positive breast cancer, essential thrombocythemia, Ewing’s tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, hepatoma, hepatocellular cancer, hormone insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphagioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin’s and non-Hodgkin’s), malignancies and hyperproliferative disorders of the bladder, breast, colon, lung, ovaries, pancreas, prostate, skin, and uterus, lymphoid malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solid tumors (carcinomas and sarcomas), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenstrom’s macroglobulinemia, testicular tumors, uterine cancer, and Wilms’ tumor. Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, kidney cancer, urothelium cancer, female genital tract cancer, uterine cancer, gestational trophoblastic disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary gland cancer, hemangioma, sarcoma arising from bone and soft tissues, Kaposi’s sarcoma, nerve cancer, ocular cancer, meningial cancer, glioblastomas, neuromas, neuroblastomas, Schwannomas, solid tumors arising from hematopoietic malignancies such as leukemias, metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumors, colorectal cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small-cell lung cancer, malignant glioma, epithelial ovarian cancer, primary peritoneal serous cancer, metastatic liver cancer, neuroendocrine carcinoma, refractory malignancy, triple negative breast cancer, HER2- amplified breast cancer, nasopharageal cancer, oral cancer, biliary tract, hepatocellular carcinoma, squamous cell carcinomas of the head and neck (SCCHN), non-medullary thyroid carcinoma, recurrent glioblastoma multiforme, neurofibromatosis type 1, CNS cancer, liposarcoma, leiomyosarcoma, salivary gland cancer, mucosal melanoma, acral/ lentiginous melanoma, paraganglioma, pheochromocytoma, advanced metastatic cancer, solid tumor, triple negative breast cancer, colorectal cancer, sarcoma, melanoma, renal carcinoma, endometrial cancer, thyroid cancer, rhabdomysarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancy. [0218] In one embodiment, the conjugates or formulations as described herein are used to treat small cell lung cancer. About 12%-15% of patients having lung cancer have small cell lung cancer. Survival in metastatic small cell lung cancer is poor. Survival rate is below 5% five years after diagnosis. US incidence of small cell lung cancer is about 26K-30K.
[0219] In some embodiments, the conjugates or formulations as described herein are used to treat patients with tumors that express or over-express the HSP90.
[0220] A feature of conjugates or formulations of the present disclosure is relatively low toxicity to an organism while maintaining efficacy at inhibiting, e.g., slowing or stopping tumor growth. As used herein, “toxicity” refers to the capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment. For example, conjugates or formulations of the present disclosure may have lower toxicity than the active agent moiety Z administered alone. For conjugates comprising DM1, their toxicity is lower than DM1 administered alone.
[0221] Toxicity may further be measured relative to a subject’s weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity. Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase. Neutropenia, thrombopenia, white blood cell (WBC) count, complete blood cell (CBC) count may also be metrics of toxicity. Pharmacologic indicators of toxicity include elevated aminotransferases (AST/ALT) levels, neurotoxicity, kidney damage, GI damage and the like. In one embodiment, conjugates or formulations of the present disclosure do not cause a significant change of a subject’s body weight. The body weight loss of a subject is less about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or formulations of the present disclosure. In another embodiment, conjugates or formulations of the present disclosure do not cause a significant increase of a subject’s AST/ALT levels. The AST or ALT level of a subject is increased by less than about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or formulations of the present disclosure. In yet another embodiment, conjugates or formulations of the present disclosure do not cause a significant change of a subject’s CBC or WBC count after treatment with conjugates or formulations of the present disclosure. The CBC or WBC level of a subject is decreased by less than about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or formulations of the present disclosure. [0222] In some embodiments, conjugates or formulations of the present disclosure are combined with at least one additional active agent. The active agent may be any suitable drug. The conjugates and the at least one additional active agent may be administered simultaneously, sequentially, or at any order. The conjugates and the at least one additional active agent may be administered at different dosages, with different dosing frequencies, or via different routes, whichever is suitable. The additional active agent may be selected from any active agent described herein such as a drug for treating cancer. It may also be a cancer symptom relief drug. Nonlimiting examples of symptom relief drugs include: octreotide or lanreotide; interferon, cypoheptadine or any other antihistamines. In some embodiments, conjugates or formulations of the present disclosure do not have drug-drug interference with the additional active agent. In one embodiment, conjugates or formulations of the present disclosure do not inhibit cytochrome P450 (CYP) isozymes. CYP isozymes may include CYP3A4 Midazolam, CYP3A4 Testosterone, CYP2C9, CYP2D6, CYP1A2, CYP2C8, CYP2B6, and CYP2C19. The additional active agent may be administered concomitantly with conjugates or formulations of the present disclosure.
[0223] In another example, conjugates or formulations of the present disclosure may be combined with a moderate dose of chemotherapy agents such as mitomycin C, vinblastine and cisplatin (see Ellis et al., Br J Cancer, vol.71(2): 366-370 (1995), the contents of which are incorporated herein by reference in their entirety).
[0224] In yet another example, a patient may first receive a pharmaceutically effective dose of an unconjugated active agent, followed by a pharmaceutically effective dose of a conjugate comprising the same active agent.
[0225] The conjugates or formulations as described herein or formulations containing the conjugates or formulations as described herein can be used for the selective tissue delivery of a therapeutic, prophylactic, or diagnostic agent to an individual or patient in need thereof. For example, conjugates or formulations of the present disclosure are used to deliver the payloads to selective tissues. These tissues may be tumor tissues. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic.
[0226] In some embodiments, the conjugates of the present disclosure may further comprise at least one reactive functionality that covalently modifies the target protein. The reactive functionality may be part of the linker, payload or HSP90 binding ligand. If part of the linker, the covalent modification step may be associated with the cleavage of the linker. In some embodiments, the conjugate of the present dislcoure covalently modifies the target protein.
[0227] The covalent modification of the target protein may be reversible or irreversible. The covalent modification may occur at a nucleophilic amino acid in the target protein. The amino acid that is modified may be a cysteine, serine, threonine, lysine, arginine, tyrosine, histidine, or tryptophan. The amino acid that is covalently modified may be cysteine 862 in PI3Ka, cysteine 481 in BTK, cysteine 797 in EGFR or any member of the kinase cysteinome as described in Liu et al., Chemistry & Biology 20, February 21, 2013, the contents of which are incorporated herein by reference in their entirety. Conjugates as Proteolysis Targeting Chimeras (PROTACs)
[0228] Proteolysis targeting chimeras (PROTACs) have become a major focus of small biotech and large pharma as a new way to impact target proteins. These PROTAC molecules are usually conjugates comprising a ligand that binds to a protein of interest (POI ligand) and a ligand that binds to E3 ligase (E3 ligand). The POI ligand and E3 ligand are connected through a crosslinker. The PROTACs recuite POI to E3 ligase. Ubiquitin (Ub) is added to the POI Lys residues. The Ub-marked POI is degraded by the proteasome. Therefore, PROTACs induce the target protein to be degraded instead of (or in addition to) being inhibited. This opens up an alternate way to kill the activity of a protein to enhance the impact on the pathway or to target previously undruggable targets. However, the challenges of PROTACs include finding tissue selective E3 ligases (only -600 members of the class).
[0229] Surprisingly, conjugates of the present disclosure, including but not limited to HSP90-binding conjugates comprising PI3K inhibitors, induces degradation of a target protein in the tumor cells, such as the PI3K protein in the tumor cells. The target protein, as used herein, refers to the protein that is regulated (e.g., its functions or activities are up-regulated or inhibited) by the conjugates of the present disclosure. In certain instances, the target protein of the conjugate is not a client protein of HSP90 and the degradation of the target protein is not driven by HSP90. In certain instances, the target protein of the active agent is a client protein of HSP90 (such as but not limited to HER2) and the degradation of the target protein of the active agent is higher than the degradation achieved by the HSP90-binding targeting ligand alone or than the degradation achieved by the non-HSP90-binding binding control.
[0230] Not willing to be bound to any theory, the conjugates of the present disclosure bind to HSP90, which associates with E3 ubiquitin ligases to degrade the target proteins of the payloads if folding is stalled. For example, HSP90-binding conjugates comprising PI3K inhibitors can ubiquitinate PI3K and induce the degradation of PI3K.
[0231] In some embodiments, the conjugates of the present disclosure induce the degradation of the target protein of the active agent in cells. In some embodiments, the conjugates of the present disclosure induce the degradation of the target protein and inhibit the activity and/or function of the target protein of the active agent in cells. The degradation of the target protein and the inhibition of the target protein can be measured with any method known in the art. In some embodiments, the cells are tumor cells.
[0232] In some embodiments, when the target protein of the conjugate is an HSP90 client protein (such as but not limited to HER2), the level of degradation of target protein is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% higher than the degradation achieved by the HSP90-binding targeting ligand alone or than the degradation achieved by the non-HSP90-binding control. In other embodiments where the target protein of the conjugate in not the HSP90 client protein, the level of degradation of target protein is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% as compared to a vehicle or non-treated or other appropriate control.
[0233] In some embodiments, the conjugates of the present disclosure are bifunctional. They induce the degradation of at least one HSP90 client protein, such as HER2, and they induce the degradatoin of the target protein of the active agent, wherein the target protein of the active agent is not an HSP90 client protein.
[0234] In some embodiments, the conjguates of the present disclosure induce degradation of PI3K in cells. In some embodiments, conjugates of the presnet disclosure induce degradation of PI3K and inhibit the activity and/or function of PI3K in tumor cells. The degradation of PI3K and the inhibition of PI3K can be measured in any method known in the art. In some embodiments, the cells are tumor cells.
[0235] In some embodimentes, the linkers of the conjugates are not cleavable. [0236] In some embodiments, the conjugates have low permeability.
IV. Kits and Devices
[0237] The disclosure provides a variety of kits and devices for conveniently and/or effectively carrying out methods of the present disclosure. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
[0238] In one embodiment, the present disclosure provides kits for inhibiting tumor cell growth in vitro or in vivo, comprising a conjugate and/or particle of the present disclosure or a combination of conjugates and/or formulations of the present disclosure, optionally in combination with any other active agents.
[0239] The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, or any delivery agent disclosed herein. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of the conjugates and/or formulations in the buffer solution over a period of time and/or under a variety of conditions.
[0240] The present disclosure provides for devices which may incorporate conjugates and/or formulations of the present disclosure. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient. In some embodiments, the subject has cancer.
[0241] Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices. The devices may be employed to deliver conjugates and/or formulations of the present disclosure according to single, multi- or split-dosing regiments. The devices may be employed to deliver conjugates and/or formulations of the present disclosure across biological tissue, intradermal, subcutaneously, or intramuscularly.
V. Definitions
[0242] The term “compound”, as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. In the present application, compound is used interechangably with conjugate. Therefore, conjugate, as used herein, is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
[0243] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms. [0244] Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
[0245] Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium.
[0246] The compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
[0247] The terms "subject" or "patient", as used herein, refer to any organism to which the conjugates may be administered, e.g., for experimental, therapeutic, diagnostic, and/or prophylactic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, guinea pigs, cattle, pigs, sheep, horses, dogs, cats, hamsters, lamas, non-human primates, and humans).
[0248] The terms "treating" or “preventing”, as used herein, can include preventing a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having the disease, disorder or condition; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. [0249] A “target”, as used herein, shall mean a site to which targeted constructs bind. A target may be either in vivo or in vitro. In certain embodiments, a target may be cancer cells found in leukemias or tumors (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas). In still other embodiments, a target may refer to a molecular structure to which a targeting moiety or ligand binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate or enzyme. A target may be a type of tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.
[0250] The “target cells” that may serve as the target for the method or conjugates or formulations, are generally animal cells, e.g., mammalian cells. The present method may be used to modify cellular function of living cells in vitro, i.e., in cell culture, or in vivo, in which the cells form part of or otherwise exist in animal tissue. Thus, the target cells may include, for example, the blood, lymph tissue, cells lining the alimentary canal, such as the oral and pharyngeal mucosa, cells forming the villi of the small intestine, cells lining the large intestine, cells lining the respiratory system (nasal passages/lungs) of an animal (which may be contacted by inhalation of the subject disclosure), dermal/epidermal cells, cells of the vagina and rectum, cells of internal organs including cells of the placenta and the so-called blood/brain barrier, etc. In general, a target cell expresses at least one type of HSP90. In some embodiments, a target cell can be a cell that expresses an HSP90 and is targeted by a conjugate described herein, and is near a cell that is affected by release of the active agent of the conjugate. For example, a blood vessel expressing an HSP90 that is in proximity to a tumor may be the target, while the active agent released at the site will affect the tumor.
[0251] The term "therapeutic effect" is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease, disorder or condition in the enhancement of desirable physical or mental development and conditions in an animal, e.g., a human.
[0252] The term “modulation” is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart. The modulation is generally compared to a baseline or reference that can be internal or external to the treated entity.
[0253] “Parenteral administration”, as used herein, means administration by any method other than through the digestive tract (enteral) or non-invasive topical routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intraossiously, intracerebrally, intrathecally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.
[0254] “Topical administration”, as used herein, means the non-invasive administration to the skin, orifices, or mucosa. Topical administration can be delivered locally, i.e., the therapeutic can provide a local effect in the region of delivery without systemic exposure or with minimal systemic exposure. Some topical formulations can provide a systemic effect, e.g., via adsorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal administration.
[0255] “Enteral administration”, as used herein, means administration via absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.
[0256] “Pulmonary administration”, as used herein, means administration into the lungs by inhalation or endotracheal administration. As used herein, the term “inhalation” refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose.
[0257] The terms “sufficient” and “effective”, as used interchangeably herein, refer to an amount (e.g., mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s). A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement or prevention of at least one symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The therapeutically effective amount is thus dependent upon the specific biologically active molecule and the specific condition or disorder to be treated. Therapeutically effective amounts of many active agents, such as antibodies, are known in the art. The therapeutically effective amounts of compounds and compositions described herein, e.g., for treating specific disorders may be determined by techniques that are well within the craft of a skilled artisan, such as a physician. [0258] The terms “bioactive agent” and “active agent”, as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
[0259] The term "prodrug" refers to an agent, including a small organic molecule, peptide, nucleic acid or protein, that is converted into a biologically active form in vitro and/or in vivo. Prodrugs can be useful because, in some situations, they may be easier to administer than the parent compound (the active compound). For example, a prodrug may be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. A prodrug may also be less toxic than the parent. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962) Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977) Application of Physical Organic Principles to Prodrug Design in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA;
Acad. Pharm. Sci.; E. B. Roche, ed. (1977) Bior ever sible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches to the improved delivery of peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for Oral Delivery of P-Lactam antibiotics, Pharm. Biotech. 11 :345-365; Gaignault et al. (1996) Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs for the improvement of drug absorption via different routes of administration, Eur. J. DrugMetab. Pharmacokinet 15(2): 143-53; Balimane and Sinko (1999). Involvement of multiple transporters in the oral absorption of nucleoside analogues, Adv. Drug Delivery Rev., 39(l-3): 183-209; Browne (1997). Fosphenytoin (Cerebyx), Clin. Neuropharmacol . 20(1): 1-12; Bundgaard (1979). Bioreversible derivatization of drugs— principle and applicability to improve the therapeutic effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H. Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisher et al. (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130; Fleisher et al. (1985) Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting, Methods Enzymol. 112: 360-81; Farquhar D, et al. (1983) Biologically Reversible Phosphate-Protective Groups, J. Pharm. Sci., 72(3): 324-325; Han, H.K. et al. (2000) Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1): E6; Sadzuka Y. (2000) Effective prodrug liposome and conversion to active metabolite, Curr. Drug Metab., 1(1):31-48; D.M. Lambert (2000) Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm. Sci., 11 Suppl. 2: SI 5-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87.
[0260] The term “biocompatible”, as used herein, refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.
[0261] The term “biodegradable” as used herein, generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.
[0262] The term “pharmaceutically acceptable”, as used herein, refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the U.S. Food and Drug Administration. A “pharmaceutically acceptable carrier”, as used herein, refers to all components of a pharmaceutical formulation that facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
[0263] The term “molecular weight”, as used herein, generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.
[0264] The term “small molecule”, as used herein, generally refers to an organic molecule that is less than 2000 g/mol in molecular weight, less than 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than 500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.
[0265] The term “hydrophilic”, as used herein, refers to substances that have strongly polar groups that readily interact with water.
[0266] The term “hydrophobic”, as used herein, refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
[0267] The term “lipophilic”, as used herein, refers to compounds having an affinity for lipids.
[0268] The term “amphiphilic”, as used herein, refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties. “Amphiphilic material” as used herein refers to a material containing a hydrophobic or more hydrophobic oligomer or polymer (e.g., biodegradable oligomer or polymer) and a hydrophilic or more hydrophilic oligomer or polymer.
[0269] The term "targeting moiety", as used herein, refers to a moiety that binds to or localizes to a specific locale. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The locale may be a tissue, a particular cell type, or a subcellular compartment. In some embodiments, a targeting moiety can specifically bind to a selected molecule.
[0270] The term “reactive coupling group”, as used herein, refers to any chemical functional group capable of reacting with a second functional group to form a covalent bond. The selection of reactive coupling groups is within the ability of those in the art. Examples of reactive coupling groups can include primary amines (-NH2) and amine-reactive linking groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these conjugate to amines by either acylation or alkylation. Examples of reactive coupling groups can include aldehydes (-COH) and aldehyde reactive linking groups such as hydrazides, alkoxyamines, and primary amines. Examples of reactive coupling groups can include thiol groups (-SH) and sulfhydryl reactive groups such as maleimides, haloacetyls, and pyridyl disulfides. Examples of reactive coupling groups can include photoreactive coupling groups such as aryl azides or diazirines. The coupling reaction may include the use of a catalyst, heat, pH buffers, light, or a combination thereof.
[0271] The term “protective group”, as used herein, refers to a functional group that can be added to and/or substituted for another desired functional group to protect the desired functional group from certain reaction conditions and selectively removed and/or replaced to deprotect or expose the desired functional group. Protective groups are known to the skilled artisan. Suitable protective groups may include those described in Greene and Wuts, Protective Groups in Organic Synthesis, (1991). Acid sensitive protective groups include dimethoxytrityl (DMT), tert- butylcarbamate (tBoc) and trifluoroacetyl (tFA). Base sensitive protective groups include 9- fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) and phenoxyacetyl (pac). Other protective groups include acetamidomethyl, acetyl, tertamyloxycarbonyl, benzyl, benzyloxy carbonyl, 2-(4-bi phenylyl )-2-propy! oxy carbonyl, 2- bromobenzyloxycarbonyl, tert-butyb tert-butyloxycarbonyl, 1-carbobenzoxamido- 2,2.2- trifluoroethyl, 2,6-dichlorobenzyl, 2-(3,5-dimethoxyphenyl)-2- propyloxycarbonyl, 2,4- dinitrophenyl, dithiasuccinyl, formyl, 4- methoxybenzenesulfonyl, 4-methoxybenzyl, 4- methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl, a-2,4,5- tetramethylbenzyloxycarbonyl, p- toluenesulfonyl, xanthenyl, benzyl ester, N- hydroxysuccinimide ester, p-nitrobenzyl ester, p-nitrophenyl ester, phenyl ester, p- nitrocarbonate, p-nitrobenzylcarbonate, trimethyl silyl and pentachlorophenyl ester.
[0272] The term “activated ester”, as used herein, refers to alkyl esters of carboxylic acids where the alkyl is a good leaving group rendering the carbonyl susceptible to nucleophilic attack by molecules bearing amino groups. Activated esters are therefore susceptible to aminolysis and react with amines to form amides. Activated esters contain a carboxylic acid ester group -CO2R where R is the leaving group.
[0273] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl -substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
[0274] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
[0275] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. In some embodiments, alkyl groups are lower alkyls. In some embodiments, a substituent designated herein as alkyl is a lower alkyl.
[0276] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.
[0277] The term “heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.
[0278] The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, and ethylthio. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined above for alkyl groups.
[0279] The terms "alkenyl" and "alkynyl", refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
[0280] The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy and aroxy groups can be substituted as described above for alkyl.
[0281] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
Figure imgf000113_0001
wherein R9, Rio, and R'10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)m-Rs or R9 and Rio taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rs represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R9 or Rio can be a carbonyl, e.g., R9, Rio and the nitrogen together do not form an imide. In still other embodiments, the term “amine” does not encompass amides, e.g., wherein one of R9 and Rio represents a carbonyl. In additional embodiments, R9 and Rio (and optionally R’10) each independently represent a hydrogen, an alkyl or cycloalkly, an alkenyl or cycloalkenyl, or alkynyl. Thus, the term "alkylamine" as used herein means an amine group, as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R9 and Rio is an alkyl group.
[0282] The term "amido" is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
Figure imgf000113_0002
wherein R9 and Rio are as defined above.
[0283] “Aryl”, as used herein, refers to Cs-Cio-membered aromatic, heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ring systems. Broadly defined, “aryl”, as used herein, includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics”. The aromatic ring can be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN; and combinations thereof.
[0284] The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2J/,6J/-l,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H- indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3J/-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5- oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothi azole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4//-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6//-1 ,2,5-thiadiazinyl, 1,2,3- thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thi enothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or more of the rings can be substituted as defined above for “aryl”.
[0285] The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
[0286] The term "carbocycle", as used herein, refers to an aromatic or nonaromatic ring in which each atom of the ring is carbon. [0287] “Heterocycle” or “heterocyclic”, as used herein, refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, for example, from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (Ci-Cio) alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4a77-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2J/,6J/-l,5,2-dithiazinyl, dihydrofuro[2,3-Z>]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 177-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4- oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 477-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6/7-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5- thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thi enothiazolyl, thienooxazolyl, thi enoimidazolyl, thiophenyl and xanthenyl. Heterocyclic groups can optionally be substituted with one or more substituents at one or more positions as defined above for alkyl and aryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, and -CN. [0288] The term "carbonyl" is art-recognized and includes such moieties as can be represented by the general formula:
Figure imgf000116_0001
wherein X is a bond or represents an oxygen or a sulfur, and Rn represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl, or an alkynyl, R'n represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl, or an alkynyl. Where X is an oxygen and Rn or R’n is not hydrogen, the formula represents an "ester". Where X is an oxygen and Rn is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Rn is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen and R'n is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. Where X is a sulfur and Rn or R'n is not hydrogen, the formula represents a "thioester." Where X is a sulfur and Rn is hydrogen, the formula represents a "thiocarboxylic acid." Where X is a sulfur and R’n is hydrogen, the formula represents a "thioformate." On the other hand, where X is a bond, and Rn is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and Rn is hydrogen, the above formula represents an "aldehyde" group.
[0289] The term “monoester” as used herein refers to an analog of a dicarboxylic acid wherein one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or salt of a carboxylic acid. Examples of monoesters include, but are not limited to, to monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid. [0290] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Examples of heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. Other useful heteroatoms include silicon and arsenic.
[0291] As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-.
[0292] The term “substituted” as used herein, refers to all permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example, 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.
[0293] Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, or elimination.
[0294] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. The heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
[0295] In various embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.
[0296] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, - CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carb oxami doaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.
[0297] The term “copolymer” as used herein, generally refers to a single polymeric material that is comprised of two or more different monomers. The copolymer can be of any form, for example, random, block, or graft. The copolymers can have any end- group, including capped or acid end groups.
[0298] The terms "polypeptide," "peptide" and "protein" generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally-occurring amino acids or are unnatural amino acids. The term "protein", as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and/or quaternary structure. The term “protein” excludes small peptides by definition, the small peptides lacking the requisite higher- order structure necessary to be considered a protein.
[0299] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. These terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general and unless otherwise specified, an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T. The term “nucleic acid” is a term of art that refers to a string of at least two base-sugar-phosphate monomeric units. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of a messenger RNA, antisense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus. An antisense nucleic acid is a polynucleotide that interferes with the expression of a DNA and/or RNA sequence. The term nucleic acids refers to a string of at least two base-sugar- phosphate combinations. Natural nucleic acids have a phosphate backbone. Artificial nucleic acids may contain other types of backbones, but contain the same bases as natural nucleic acids. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
[0300] A "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains at least one function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, e.g., genetic or biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.
[0301] As used herein, the term “linker” refers to a carbon chain that can contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long. Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. Those of skill in the art will recognize that each of these groups may in turn be substituted. Examples of linkers include, but are not limited to, pH-sensitive linkers, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
[0302] The term “pharmaceutically acceptable salt(s)” refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
[0303] If the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.
[0304] A pharmaceutically acceptable salt can be derived from an acid selected from l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2 -hydroxy ethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isethionic, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic, naphthal ene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic, phosphoric acid, proprionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic, and undecylenic acid.
[0305] The term “bioavailable” is art-recognized and refers to a form of the subject disclosure that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.
[0306] It will be appreciated that the following examples are intended to illustrate but not to limit the present disclosure. Various other examples and modifications of the foregoing description and examples will be apparent to a person skilled in the art after reading the disclosure without departing from the spirit and scope of the disclosure, and it is intended that all such examples or modifications be included within the scope of the appended claims. All publications and patents referenced herein are hereby incorporated by reference in their entirety. EXAMPLES
EXAMPLE 1: Synthesis of the Conjugates
[0307] The conjugates of the disclosure may be prepared using any convenient methodology. In a rational approach, the conjugates are constructed from their individual components, targeting moiety, in some cases a linker, and active agent moiety. The components can be covalently bonded to one another through functional groups, as is known in the art, where such functional groups may be present on the components or introduced onto the components using one or more steps, e.g., oxidation reactions, reduction reactions, cleavage reactions and the like. Functional groups that may be used in covalently bonding the components together to produce the pharmaceutical conjugate include: hydroxy, sulfhydryl, amino, and the like. The particular portion of the different components that are modified to provide for covalent linkage will be chosen so as not to substantially adversely interfere with that components desired binding activity, e.g., for the active agent moiety, a region that does not affect the target binding activity will be modified, such that a sufficient amount of the desired drug activity is preserved. Where necessary and/or desired, certain moieties on the components may be protected using blocking groups, as is known in the art, see, e.g., Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).
[0308] In general, the conjugates of the present disclosure may be synthesized by attaching a targeting moiety to the payload, optionally by a linker. A person skilled in the art can synthesize these conjugates by routine chemical steps.
[0309] Alternatively, the conjugate can be produced using known combinatorial methods to produce large libraries of potential conjugates which may then be screened for identification of a bifunctional, molecule with the pharmacokinetic profile. Alternatively, the conjugates may be produced using medicinal chemistry and known structure-activity relationships for the targeting moiety and the active agent moiety. In particular, this approach will provide insight as to where to join the two moieties to the linker.
Conjugates C17-C86
[0310] Non-limiting examples of synthetic methods for Conjugates C17-C86 are included below. General procedure
Method A as follow:
Synthesis of route
Figure imgf000123_0001
1.1 (S)-benzyl 2-(chlorocarbonyloxy)propanoate
Figure imgf000123_0002
[0311] To an ice-cooled solution of bis(tri chloromethyl) carbonate (2.75 g, 9.27 mmol) in ether (60 mL) was added dropwise a solution of benzyl (2S)-2- hydroxypropanoate (5 g, 27.75 mmol) and pyridine (2.19 g, 27.75 mmol, 2.24 mL) in ether (40 mL) during 30 minutes, after addition the mixture was warmed gradually to rt and stirred at rt for 2 days, the resulting suspension was filtered, the cake was washed with ether (20 mL x 2), the combined filtrate and washing were concentrated under vacuum to leave the crude product as clear oil (6.0g, yield 90%).
LC-MS: (ESI) m/z: 252.2 [M+H]+ (note: product was treated with dimethylamine to get (S)-benzyl 2-(dimethylcarbamoyloxy)propanoate and then run LCMS)
1.2 3 -(((S)- 1 -(benzyloxy)- 1 -oxopropan-2-yloxy)carbonylamino)-2- fluoropropanoic acid
Figure imgf000123_0003
[0312] To an ice-cooled solution of 3-amino-2-fluoro-propanoic acid (214 mg, 2.00 mmol) and disodium;carbonate (424 mg, 4.00 mmol, 167.59 uL) in water (10 mL) was added dropwise the solution of benzyl (2S)-2-chlorocarbonyloxypropanoate (800 mg, 3.30 mmol) in THF (10 mL), the mixture was stirred at 0 °C for 10 minutes, LCMS showed almost full conversion of starting material. The mixture was quenched with a solution of NaHSO4 (1 g) in water (30 mL), stirred at 0 °C for 5 minutes and extracted with EtOAc (50 mL x 2), the combined extracts were dried over anhydrous Na2SO4, concentrated and purified by column chromatography (silica gel, EtOAc / petroleum ethers = 0:1 to 2:1) to afford the pure product as colorless oil (281 mg, yield 45%).
LC-MS: (ESI) m/z: 336.0 [M+Na]
1.3 (2S)-benzyl 2-(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-fluoro-3- oxopropy 1 carb amoy 1 oxy )propanoate
Figure imgf000124_0001
[0313] To a solution of 3-[[(lS)-2-benzyloxy-l-methyl-2-oxo- ethoxy]carbonylamino]-2-fluoro-propanoic acid (220 mg, 702.25 umol) in DMF (4 mL) was added DIEA (135.89 mg, 1.05 mmol) ,5-(2,4-dihydroxy-5-isopropyl- phenyl)-N-ethyl-4-[4-(piperazin-l-ylmethyl)phenyl]-l,2,4-triazole-3-carboxamide (114.18 mg, 245.79 umol) , phenol (66.09 mg, 702.25 umol, 61.77 uL) and then tripyrrolidin-l-yl(triazolo[4,5-b]pyridin-3-yloxy)phosphonium;hexafluorophosphate (256.30 mg, 491.58 umol) was added into the mixture and stirred for 1 hr at 25 °C The reaction mixture was purifed by combi-flash to get the product as a white solid (160 mg, yield 30%)
LC-MS: (ESI) m/z: 782.2 [M+Na]
1.4 (2S)-2-(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl)benzyl)piperazin- 1 -yl)-2-fluoro-3 - oxopropyl carb amoy 1 oxy )propanoi c aci d
Figure imgf000125_0001
[0314] To a solution of benzyl (2S)-2-[[3-[4-[[4-[3-(2,4-dihydroxy-5-isopropyl- phenyl)-5-(ethylcarbamoyl)-l,2,4-triazol-4-yl]phenyl]methyl]piperazin-l-yl]-2- fluoro-3-oxo-propyl]carbamoyloxy]propanoate (120 mg, 157.93 umol) in Methanol (15 mL) was added Palladiumon carbon 10% (20 mg, 157.93 umol) . The mixture was stirred for 1 hr at 25 °C under H2 atmosphere. After removed the cat. the residue was concentracted under vacuum to get the product as a white solid (lOOmg, yield 95%).
LC-MS: (ESI) m/z: 670.2 [M+Hp
1.5 (S)-l-(4-(3-methyl-8-(6-methylpyridin-3-yl)-2-oxo-2,3-dihydroimidazo[4,5- c] [ 1 ,5]naphthyridin- 1 -yl)piperidin- 1 -yl)- 1 -oxopropan-2-yl 3 -(4-(4-(3 -(2,4- dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)-2-fluoro-3 -oxopropylcarbamate
Figure imgf000125_0002
[0315] To a solution of(2S)-2-[[3-[4-[[4-[3-(2,4-dihydroxy-5-isopropyl-phenyl)-5- (ethylcarbamoyl)-l,2,4-triazol-4-yl]phenyl]methyl]piperazin-l-yl]-2-fluoro-3-oxo- propyl]carbamoyloxy]propanoic acid (100 mg, 149.32 umol) and 3-methyl-8-(6- methyl-3-pyridyl)-l-(4-piperidyl)imidazo[4,5-c][l,5]naphthyridin-2-one (67.09 mg, 179.19 umol) in N,N-Dimethylformamide (1 mL) and Tri chloromethane (25 mL) was added phenol (28.11 mg, 298.64 umol, 26.27 uL) ,N-ethyl-N-isopropyl- propan-2-amine (38.60 mg, 298.64 umol, 52.02 uL) and the reaction mixture was stirred for lOmin at 25°C, then tripyrrolidin-l-yl(triazolo[4,5-b]pyridin-3- yloxy)phosphonium;hexafluorophosphate (93.42 mg, 179.19 umol) was added into the mixture and stirred for more 2 hr at 26 °C . The reaction mixture was purified by Prep-HPLC (CF3COOH) to give the pure product as a off-white solid ( 1 mg, yield 26%).
C19
LC-MS: (ESI) m/z: 1026.2 [M+H]+
1H NMR (MeOD, 400 MHz): 5 9.40-9.28 (m, 1 H), 8.99-8.83 (m, 2 H), 8.54 (d, J = 7.6 Hz, 1 H), 8.40-8.29 (m, 1 H), 7.93-7.80 (m, 1 H), 7.55 (d, J = 6.8 Hz, 2 H), 7.43-7.34 (m, 2 H), 6.64 (d, J = 6.8 Hz, 1 H), 6.44-6.20 (m, 1 H), 6.15 (s, 1 H), 5.53-5.18 (m, 2 H), 4.75-4.60 (m, 3 H), 4.43-4.30 (s, 2 H), 4.28-4.09 (m, 1 H), 4.06-3.70 (m, 3 H), 3.65-3.24 (m, 11 H), 2.98-2.58 (m, 9 H), 2.09-1.90 (m, 2 H), 1.46-1.31 (m, 4 H), 1.10-0.99 (m, 3 H), 0.86-0.73 (m, 7 H).
[0316] A series of compounds can be synthesized by method A:
C17
LC-MS: (ESI) m/z: 359.5 [1/3 M+H], 538.7[1/2M+H], 1077.3 [M+H]+
1H NMR (MeOD, 500 MHz): 89.32 (s, 1 H), 8.93 (d, J= 5.0 Hz, 1 H), 8.87-8.75 (m, 1 H), 8.51 (d, J = 9.0 Hz, 1 H), 8.29 (d, J= 9.0 Hz, 1 H), 7.83-7.72 (m, 1 H), 7.52 (d, J= 8.0 Hz, 1 H), 7.45-7.36 (m, 2 H), 6.64 (s, 1 H), 6.17 (s, 1 H), 5.50-5.34 (m, 1 H), 4.72-4.63 (m, 2 H), 4.35-4.27 (m, 2 H), 4.21-4.10 (m, 1 H), 3.90-3.57 (m, 2 H), 3.51 (d, J = 19.0 Hz, 3 H), 3.39-3.23 (m, 4 H), 3.15-3.01 (m, 2 H), 2.98-2.52 (m, 13 H), 2.08-1.90 (m, 2 H), 1.43-1.14 (m, 6 H), 1.11-1.01 (m, 3 H), 0.93 (d, J= 7.0 Hz, 6 H).
C18
LC-MS: (ESI) m/z: 341.5[1/3M+H], 511.7[1/2M+H], 1023.3 [M+H]+
1H NMR (MeOD, 400 MHz): 89.32 (s, 1 H), 9.07-8.89 (m, 2 H), 8.58 (d, J= 9.6 Hz, 1 H), 8.44 (d, J= 9.2 Hz, 1 H), 8.03-7.93 (m, 1 H), 7.55 (d, J= 8.0 Hz, 1 H), 7.48-7.36 (m, 2 H), 6.65 (d, J= 8.4 Hz, 1 H), 6.48-6.23 (m, 1 H), 6.18 (s, 1 H), 5.45-5.18 (m, 1 H), 4.75-4.64 (m, 2 H), 4.43-4.30 (m, 2 H), 4.28-4.14 (m, 2 H), 4.12-3.62 (m, 3 H), 3.59-3.24 (m, 9 H), 2.98-2.33 (m, 11 H), 2.13-1.95 (m, 2 H), 1.46-1.28 (m, 3 H), 1.24-1.11 (m, 4 H), 1.10-0.99 (m, 3 H), 0.81 (d, J= 6.8 Hz, 6 H).
C33
LC-MS: (ESI) m/z: 362.2[1/3M+H], 542.8[1/2M+H], 1084.3 [M+H]+
1H NMR (MeOD, 400 MHz): 8 9.42-9.30 (m, 1 H), 9.11-8.94 (m, 2 H), 8.58 (d, J= 9.2 Hz, 1 H), 8.41 (d, J = 8.0 Hz, 1 H), 8.05-7.89 (m, 1 H), 7.45 (d, J= 8.0 Hz, 2 H), 7.40 (d, J = 8.0 Hz, 2 H), 7.31-7.13 (m, 5 H), 6.48-6.23 (m, 1 H), 6.18 (s, 1 H), 5.45-5.18 (m, 1 H), 4.75-4.64 (m, 2 H), 4.43- 4.30 (m, 2 H), 4.28-4.14 (m, 2 H), 4.12-3.62 (m, 3 H), 3.59-3.24 (m, 9 H), 2.98-2.33 (m, 11 H), 2.13-1.95 (m, 2 H), 1.46-1.28 (m, 3 H), 1.24-1.11 (m, 4 H), 1.10-0.99 (m, 3 H), 0.81 (d, J= 6.8 Hz, 6 H). C20
LC-MS: (ESI) m/z: 336.9[1/3M+H], 504.8[l/2M+H], 1008.2[M+H]+
1H NMR (MeOD, 500 MHz): 5 9.36-9.28 (m, 1 H), 8.92 (d, J= 10.0 Hz, 1 H), 8.88-8.80 (m, 1 H), 8.53 (d, J = 9.0 Hz, 1 H), 8.37-8.28 (m, 1 H), 7.87-7.74 (m, 1 H), 7.53 (d, J = 8.0 Hz, 2 H), 7.43- 7.34 (m, 2 H), 6.64 (d, J = 4.0 Hz, 1 H), 6.14 (s, 1 H), 5.35 (dd, J = 55.0, 6.5 Hz, 1 H), 4.85-4.78 (m, 2 H), 4.75-4.63 (m, 2 H), 4.42-4.31 (m, 2 H), 4.23-4.14 (m, 1 H), 3.50 (d, J = 15.0 Hz, 3 H), 3.42-3.24 (m, 6 H), 3.20-3.12 (m, 3 H), 2.93-2.75 (m, 2 H), 2.73-2.46 (m, 5 H), 2.08-1.92 (m, 1 H), 1.40-1.31 (m, 2 H), 1.10-0.98 (m, 2 H), 0.81 (d, J= 7.0 Hz, 6 H).
C21
LC-MS: (ESI) m/z: 352.9[1/3M+H], 528.8[1/2M+H], 1058.3 [M+H]+
1H NMR (MeOD, 400 MHz): 8 9.43-9.28 (m, 1 H), 8.99-8.80 (m, 2 H), 8.53 (d, J= 8.8 Hz, 1 H), 8.33 (d, J = 8.4 Hz, 1 H), 7.88-7.75 (m, 1 H), 7.58 (d, J= 3.6 Hz, 2 H), 7.46-7.27 (m, 5 H), 7.25- 7.15 (m, 1 H), 6.73-6.57 (m, 1 H), 6.47-6.23 (m, 1 H), 6.11 (s, 1 H), 5.55-5.35 (m, 1 H), 4.93-4.85 (m, 3 H), 4.75-4.65 (m, 1 H), 4.47-4.15 (m, 3 H), 3.83-3.25 (m, 11 H), 3.20-3.09 (m, 3 H), 2.96- 2.41 (m, 8 H), 2.12-1.90 (m, 2 H), 1.54-1.36 (m, 3 H), 1.25-1.12 (m, 1 H), 1.10-0.98 (m, 3 H), 0.76 (d, J = 6.4 Hz, 6 H).
C22
LC-MS: (ESI) m/z: 346.2[1/3M+H], 518.7[1/2M+H], 1037.3 [M+H]+
1H NMR (MeOD, 500 MHz): 8 9.40-9.21 (m, 1 H), 8.90 (d, J= 14.0 Hz, 1 H), 8.87-8.73 (m, 1 H), 8.51 (d, J= 9.0 Hz, 1 H), 8.36-8.26 (m, 1 H), 7.87-7.70 (m, 1 H), 7.54 (d, J= 7.5 Hz, 1 H), 7.39 (d, J = 8.0 Hz, 1 H), 6.63 (s, 1 H), 6.15 (s, 1 H), 5.45-5.25 (m, 1 H), 4.73-4.64 (m, 2 H), 4.35 (d, J = 6.0 Hz, 2 H), 4.23-4.11 (m, 1 H), 3.49 (d, J= 33.5 Hz, 3 H), 3.38-3.22 (m, 6 H), 3.17-2.98 (m, 5 H), 2.96-2.41 (m, 8 H), 2.09-1.90 (m, 2 H), 1.34 (d, J= 6.5 Hz, 3 H), 1.29-1.11 (m, 7 H), 1.11-0.99 (m, 3 H), 1.10-0.98 (m, 3 H), 0.78 (d, J= 6.5 Hz, 6 H).
C23
LC-MS: (ESI) m/z: 362.5 [1/3 M+H], 543.3[1/2M+H], 1086.3 [M+H]+
1H NMR (MeOD, 500 MHz): 8 9.35-9.20 (m, 1 H), 8.93-8.80 (m, 1 H), 8.74 (d, J= 8.0 Hz, 1 H), 8.53-8.43 (m, 2 H), 8.28 (d, J= 8.5 Hz, 1 H), 7.91-7.79 (m, 1 H), 7.76-7.69 (m, 1 H), 7.64-7.48 (m, 3 H), 7.43-7.28 (m, 3 H), 6.40 (d, J = 9.0 Hz, 1 H), 6.15 (d, J = 8.5 Hz, 1 H), 5.49-5.27 (m, 1 H), 5.21-5.12 (m, 1 H), 4.72-4.62 (m, 2 H), 4.47-4.15 (m, 2 H), 4.21-4.01 (m, 1 H), 3.56-3.43 (m, 3 H), 3.37-3.24 (m, 2 H), 3.20-3.09 (m, 8 H), 2.96-2.41 (m, 10 H), 2.06-1.90 (m, 2 H), 1.44-1.16 (m, 4 H), 1.11-0.99 (m, 3 H), 0.87-0.72 (m, 7 H). C25
LC-MS: (ESI) m/z: 518.8[1/2M+H], 1057.2 [M+Na]+
1H NMR (MeOD, 400 MHz): 5 9.46 (d, J= 6.4 Hz, 1 H), 9.06 (d, J = 11.2 Hz, 1 H), 9.01 (d, J = 8.8 Hz, 1 H), 8.66 (d, J = 9.2 Hz, 1 H), 8.47 (d, J = 8.4 Hz, 1 H), 7.96 (d, J = 8.0 Hz, 1 H), 7.69- 7.61 (m, 2 H), 7.50 (d, J= 8.4 Hz, 1 H), 7.42 (d, J= 8.0 Hz, 1 H), 6.84-6.70 (m, 1 H), 6.54-6.31 (m, 1 H), 6.25 (s, 1 H), 5.29-5.58 (m, 1 H), 4.79 (t, J= 14.0 Hz, 1 H), 4.48 (s, 2 H), 4.38-4.24 (m, 1 H), 4.17-3.73 (m, 2 H), 3.64 (d, J= 5.6 Hz, 3 H), 3.57-3.34 (m, 4 H), 3.32-3.09 (m, 4 H), 3.08-2.57 (m, 10 H), 2.22-2.03 (m, 2 H), 1.55-1.27 (m, 9 H), 1.20-1.04 (m, 3 H), 1.00-0.87 (m, 6 H).
C26
LC-MS: (ESI) m/z: 349.5 [1/3 M+H], 523.7[1/2M+H], 1068.3 [M+Na]+
1H NMR (MeOD, 400 MHz): 8 9.40-9.23 (m, 1 H), 8.98-8.85 (m, 2 H), 8.53 (d, J= 8.8 Hz, 1 H), 8.32 (d, J= 8.8 Hz, 1 H), 7.92-7.79 (m, 2 H), 6.17 (s, 1 H), 5.60-5.37 (m, 1 H), 4.77-4.66 (m, 3 H), 4.37 (s, 2 H), 4.27-4.13 (m, 1 H), 3.54 (s, 3 H), 3.24-3.22 (m, 5 H), 3.18-2.96 (m, 2 H), 2.95-2.78 (m, 3 H), 2.70 (d, J = 16.4 Hz, 4 H), 2.11-1.94 (m, 2 H), 1.48-1.33 (m, 3 H), 1.08 (t, J= 7.2 Hz, 3 H), 0.88-0.73 (m, 6 H).
C27
LC-MS: (ESI) m/z: 350.3 [1/3 M+H524.7[1/2M+H], 1049.3 [M+H]+
1H NMR (MeOD, 500 MHz): 89.32 (s, 1 H), 8.98-8.76 (m, 2 H), 8.53 (d, J= 8.0 Hz, 1 H), 8.33 (d, J = 6.5 Hz, 1 H), 7.94-7.69 (m, 1 H), 7.53 (d, J = 8.0 Hz, 2 H), 7.49-7.34 (m, 2 H), 6.65 (s, 1 H), 6.44-6.20 (m, 1 H), 6.16 (s, 1 H), 5.63-5.23 (m, 1 H), 4.72-4.62 (m, 2 H), 4.42-4.30 (m, 2 H), 4.27- 4.06 (m, 2 H), 4.05-3.79 (m, 3 H), 3.61-3.40 (m, 3 H), 3.39-3.24 (m, 3 H), 3.17-3.03 (m, 3 H), 3.03- 2.87 (m, 3 H), 2.84-2.61 (m, 8 H), 2.13-1.93 (m, 2 H), 1.90-1.80 (m, 1 H), 1.74-1.53 (m, 2 H), 1.52- 1.28 (m, 4 H), 1.13-0.97 (m, 3 H), 0.83 (d, J= 7.0 Hz, 6 H).
Method B as follow:
Synthesis of route:
Figure imgf000129_0001
2.1 (S)-benzyl2(4(hydroxymethyl)phenylcarbamoyloxy)propanoate
Figure imgf000129_0002
[0317] To a solution of (4-aminophenyl)methanol(246 mg, 2mmole) in THF (3ml) was added DIPEA(516mg, 4mmole) and then (S)-benzyl 2-
(chlorocarbonyloxy)propanoate(484 mg, 2mmole) was added into the mixture at OoC. After addition, the mixture was stirred for lOmin at r.t. LCMS showed almost full conversion of starting material. The mixture was quenched with a solution of NaHSO4 (1 g) in water (30 mL), stirred at 0 °C for 5 minutes and extracted with EtOAc (50 mL x 2), the combined extracts were dried over anhydrous Na2SO4, concentrated and purified by column chromatography (silica gel, EtOAc / petroleum ethers = 0: 1 to 1 : 1) to afford the pure product as colorless oil (430 mg yield 65%) LC-MS: (ESI) ni/z: 330.1 [M+H]+
2.2
(S)benzyl2(4(((4nitrophenoxy)carbonyloxy)methyl)phenylcarbamoyloxy)propanoate
Figure imgf000129_0003
[0318] To a solution of benzyl 2-[[4-
(hydroxymethyl)phenyl]carbamoyloxy]propanoate (200 mg, 607.26 umol) in THF (5 mL) was added DIEA (156.67 mg, 1.21 mmol) ,bis(4-nitrophenyl) carbonate (277.11 mg, 910.90 umol) . The mixture was stirred for 2 hr at 25 °C. After removed the solvent, the residue was dissolved in EA (30ml) and washed with brine (10ml*2) and then dried over Na2SO4, concentracted under reduced pressure to get the crude product (140 mg) which was used in the next step without further purificaiton.
LC- MS: (ESI) m/z: 495.1 [M+H]+
2.3 4-(((S)- 1 -(benzyloxy)- 1 -oxopropan-2-yloxy)carbonylamino)benzyl 4-(4-(3 -(2,4- dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazine-l -carboxylate
Figure imgf000130_0001
[0319] To a solution of b enzy 1 (2 S)2 [ [4 [(4nitrophenoxy )carb ony 1 oxy methyl ] phenyl ] carbamoyloxy]propanoate (100 mg, 202.25 umol) in DMF (4 mL) was added 5- (2,4-dihydroxy-5-isopropyl-phenyl)-N-ethyl-4-[4-(piperazin-l-ylmethyl)phenyl]- 1, 2, 4-triazole-3 -carboxamide (93.95 mg, 202.25 umol) ,DIEA (52.18 mg, 404.49 umol) . The mixture was stirred for 2 hr at 25 °C. The reaction mixture was purified by combi-flash to get the product (130mg, yield 74.8%)
LC -VIS- (ESI) /z: 820.3 [M+HJ+
2.4 (2S)-2-(4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl)benzyl)piperazine- 1 - carb ony 1 oxy )methy l)pheny 1 carb amoy 1 oxy )propanoi c aci d
Figure imgf000130_0002
[0320] To a solution of [4-[[(lS)-2-benzyloxy-l-methyl-2-oxo- ethoxy]carbonylamino]phenyl]methyl 4-[[4-[3-(2,4-dihydroxy-5-isopropyl-phenyl)-5- (ethylcarbamoyl)- 1, 2, 4-triazol-4-yl]phenyl]methyl]piperazine-l -carboxylate (110 mg, 134.16 umol) in Methanol (10 mL) was added Palladium on carbon, 10 wt.% 50% water (20 mg, 134.16 umol) . The mixture was stirred for 1 hr at 25 °C under H2 atmosphere. After removed the cat, the residue was concentracted under reduced pressure to get the product as a white solid (90mg, yield 91.2%)
LC-MS: (ESI) m/z: 730.3 [M+H]+
2.5 4-(((S)-l-(4-(3-methyl-8-(6-methylpyridin-3-yl)-2-oxo-2,3-dihydroimidazo[4,5- c] [ 1 ,5]naphthyridin- 1 -yl)piperidin- 1 -yl)- 1 -oxopropan-2-yloxy)carbonylamino)benzyl
4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazine-l -carboxylate
Figure imgf000131_0001
[0321] To a solution of (2S)-2-(4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl)benzyl)piperazine- 1 - carbonyloxy)methyl)phenylcarbamoyloxy)propanoic acid (90 mg, 123.50 umol) and 3-methyl-8-(6-methyl-3-pyridyl)-l-(4-piperidyl)imidazo[4,5-c][l,5]naphthyridin- 2-one (55.49 mg, 148.20 umol) in N,N-Dimethylformamide, ACS, 99.8+% (1 mL) and Trichloromethane (25 mL) was addedN-ethyl-N-isopropyl-propan-2-amine (31.92 mg, 246.99 umol, 43.02 uL) , phenol (23.24 mg, 246.99 umol, 21.72 uL) and the reaction mixture was stirred for lOmin at 25°C, thenbenzotriazol-1- yloxy(tripyrrolidin-l-yl)phosphonium;hexafluorophosphate (77.12 mg, 148.20 umol) was added into the mixture and stirred for more 2 hr at 25 °C. The reaction mixture was purification by Prep-HPLC (CF3COOH) to give the pure product as a white solid (49.7mg, yield 37%) C24
LC-MS: (ESI) m/z: 1086.3 [M+H]+ 1H NMR (MeOD, 400 MHz): 5 9.41-9.20 (m, 1 H), 9.03-8.90 (m, 2 H), 8.56 (d, J= 8.8 Hz, 1 H), 8.36 (d, J= 8.8 Hz, 1 H), 7.97-7.79 (m, 1 H), 7.52 (d, J= 8.4 Hz, 2 H), 7.39 (d, J = 8.0 Hz, 2 H), 7.33-7.13 (m, 4 H), 6.65 (d, J= 2.0 Hz, 1 H), 6.16 (d, J= 32 Hz, 1 H), 5.57-5.38 (m, 1 H), 4.99 (d, J= 21.5 Hz, 2 H), 4.91-4.85 (m, 2 H), 4.78-4.62 (m, 3 H), 4.36-4.19 (m, 3 H), 3.41 (s, 3 H), 3.46- 3.27 (m, 2 H), 3.26-3.21 (m, 3 H), 3.20-3.09 (m, 3 H), 2.96-2.79 (m, 3 H), 2.78-2.55 (m, 5 H), 2.13- 1.78 (m, 2 H), 1.49-1.35 (m, 3 H), 1.11-0.99 (q, J= 7.2 Hz, 3 H), 0.87-0.72 (m, 6 H). Method C as follow:
Synthesis of route:
Figure imgf000132_0001
3.1 (S)-benzyl 2-((4-nitrophenoxy)carbonyloxy)propanoate
Figure imgf000132_0002
[0322] To a solution of benzyl (2S)-2-hydroxypropanoate (3.4 g, 18.87 mmol) in DCM (50 mL) was added DIEA (7.30 g, 56.60 mmol) and a solution of (4- nitrophenyl) carbonochloridate (5.70 g, 28.30 mmol) in 5ml DCM was added into the mixture at 0°C. After addition, the mixture was stirred for more 16 hr at 25 °C. This crude product was purifed by combi-flash (PE/EA=9/1) to get the product as a colorless oil (4.2g yield 64.47%).
LC- S: (ESI) m/z: 368.0 [M+Na]+
3.2 (S)-l -(benzyloxy)- l-oxopropan-2-yl 4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-
(ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl)benzyl)piperazine- 1 -carboxylate
Figure imgf000132_0003
[0323] To a solution of 5-(2,4-dihydroxy-5-isopropyl-phenyl)-N-ethyl-4-[4- (piperazin-l-ylmethyl)phenyl]-l, 2, 4-triazole-3 -carboxamide (170 mg, 365.94 umol) in DMF (3 mL) was addded DIEA (94.41 mg, 731.88 umol), benzyl (2S)-2-(4- nitrophenoxy)carbonyloxypropanoate (139.00 mg, 402.53 umol) . the mixture was stirred for 2 hr at 25 °C. The crude product was purified by combi-flash to get the product as a white solid (160 mg, yield 65.19%).
LC-MS: (ESI) m/z: 671.2 [M+HJ+
3.3 (2S)-2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4- triazol-4-yl)benzyl)piperazine-l-carbonyloxy)propanoic acid
Figure imgf000133_0001
[0324] To a solution of [(1 S)-2-benzyloxy-l-methyl-2-oxo-ethyl] 4-[[4-[3-(2,4- dihydroxy-5-isopropyl-phenyl)-5-(ethylcarbamoyl)-l,2,4-triazol-4- yl]phenyl]methyl]piperidine-l -carboxylate (120.00 mg, 179.17 umol) in Methanol (10 mL) was added Palladium (19.07 mg, 179.17 umol) under N2 protection. The mxiture was stirred for 2 hr at 25 °C under H2 atmosphere, after removed the solid, the rusidue was concentracted to get the product as a white solid (75mg, yield 72.1%). LC-MS: (ESI) ni/z: 580.1 [M+H]+
3.4 (S)-l-(4-(3-methyl-8-(6-methylpyridin-3-yl)-2-oxo-2,3-dihydroimidazo[4,5- c][l,5]naphthyridin-l-yl)piperidin-l-yl)-l-oxopropan-2-yl 4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H- 1,2, 4-tri azol -4-yl)benzyl)piperazine-l- carb oxy late
Figure imgf000133_0002
[0325] To a solution of(2S)-2-[3-[4-[[4-[3-(2,4-dihydroxy-5-isopropyl-phenyl)-5- (ethylcarbamoyl)-l,2,4-triazol-4-yl]phenyl]methyl]piperazine-l-carbonyl]piperidine- l-carbonyl]oxypropanoic acid (70 mg, 101.19 umol) and 3-methyl-8-(6-methyl-3- pyridyl)-l-(4-piperidyl)imidazo[4,5-c][l,5]naphthyridin-2-one (45.47 mg, 121.43 umol) in N,N-Dimethylformamide (1 mL) and Trichloromethane (25 mL) was addedphenol (19.05 mg, 202.38 umol, 17.80 uL), N-ethyl-N-isopropyl-propan-2- amine (26.16 mg, 202.38 umol, 35.25 uL) and the reaction mixture was stirred for lOmin at 25°C, then tripyrrolidin-l-yl(triazolo[4,5-b]pyridin-3- yloxy)phosphonium;hexafluorophosphate (63.31 mg, 121.43 umol) was added into the mixture and stirred for more 2 hr at 25 °C. The reaction mixture was purification by Prep-HPLC (CF3COOH) to give the pure product as a white solid (70.5 mg, 64.9%). C28
LC-MS: (ESI) m/z: 1049.3 [M+H]+ 1H NMR (MeOD, 400 MHz): 5 10.27-9.95 (m, 1 H), 9.73 (s, 1 H), 9.35 (s, 1 H), 9.09-8.98 (m, 2 H), 8.50 (d, J= 8.4 Hz, 2 H), 8.36 (d, J= 9.2 Hz, 1 H), 7.68-7.37 (m, 4 H), 6.70 (s, 1 H), 6.30 (s, 1 H), 5.57-5.34 (m, 1 H), 4.73-4.58 (m, 2 H), 4.54-4.31 (m, 3 H), 4.30-4.08 (m, 7 H), 3.63-3.45 (m, 4 H), 3.41-3.26 (m, 2 H), 3.25-3.13 (m, 5 H), 3.12-2.88 (m, 2 H), 2.80-2.58 (m, 6 H), 2.08-1.96 (m, 2 H), 1.56 (d, J= 5.6 Hz, 2 H), 1.23 (s, 1 H), 0.97-0.80 (m, 6 H).
[0326] C29 was synthesized with the same method.
C29
LC-MS: (ESI) m/z: 313.2[1/3M+H], 469.2[1/2M+H], 938.1 [M+H]+ 1H NMR (MeOD, 400 MHz): 89.28 (s, 1 H), 8.88 (s, 1 H), 8.80-8.69 (m, 1 H), 8.54-8.43 (m, 1 H), 8.33-8.20 (m, 1 H), 7.78-7.67 (m, 4 H), 6.53 (s, 1 H), 6.24 (s, 1 H), 5.50-5.28 (m, 1 H), 4.28-3.89 (m, 3 H), 3.72-3.59 (m, 4 H), 3.17-2.99 (m, 12 H), 2.93-2.62 (m, 5 H), 2.63-2.47 (m, 1 H), 2.06- 1.86 (m, 2 H), 1.67-1.49 (m, 2 H), 1.21-0.93 (m, 5 H), 0.75 (s, 6 H).
Method D as follow:
Synthesis of route:
Figure imgf000135_0001
4.1 (S)-5-(l-(benzyloxy)-l-oxopropan-2-yloxy)-5-oxopentanoic acid
Figure imgf000135_0002
D1 D2
[0327] To a solution of benzyl (2S)-2-hydroxypropanoate (180 mg, 998.89 umol) in THF (5 mL) was added tetrahydropyran-2, 6-dione (227.94 mg, 2.00 mmol, 161.66 uL), DIEA (386.57 mg, 3.00 mmol). The mixture was stirred for 16 hr at 25 °C. After removed the solvent, the residue was purified by combi-flash to get the product as a oil (lOOmg, yield 34%) LC-MS: (ESI) m/z: 17.2 [M+Na]+
4.2 (S)-l -(benzyloxy)- l-oxopropan-2-yl 5-(4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-5- oxopentanoate
Figure imgf000135_0003
[0328] To a solution of 5-[(lS)-2-benzyloxy-l-methyl-2-oxo-ethoxy]-5-oxo- pentanoic acid (83 mg, 282.03 umol) inDMF (3 mL) was added [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl- ammonium;hexafhiorophosphate (160.85 mg, 423.04 umol) ,DIEA (109.14 mg, 846.08 umol) . The mixture was stirred for 10 min at 25°C, 5-(2,4-dihydroxy-5- isopropyl-phenyl)-N-ethyl-4-[4-(piperazin-l-ylmethyl)phenyl]-l,2,4-triazole-3- carboxamide (131.02 mg, 282.03 umol) was added and stirred for more 2 hr at 25 °C .The crude compound was purified by combi-flash to get the product as a white solid (146mg, yield 69.88%).
LC-MS: (ESI) m/z: 741.2
Figure imgf000136_0001
763.2 [M+Na]+
4.3(2S)-2-(5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H- l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-5-oxopentanoyloxy)propanoic acid
Figure imgf000136_0002
[0329] To a solution of [(lS)-2-benzyloxy-l-methyl-2-oxo-ethyl] 5-[4-[[4-[3-(2,4- dihydroxy-5-isopropyl-phenyl)-5-(ethylcarbamoyl)-l,2,4-triazol-4- yl]phenyl]methyl]piperazin-l-yl]-5-oxo-pentanoate (146 mg, 197.07 umol) in Methanol (10 mL) was added Palladium on carbon 10% (30 mg, 197.07 umol) and stirred for 1 hr at 25 °C under H2 atmosphere, after removed the solid, the residue was concentractedunder reduce pressure to get the product as a white solid (110 mg, yield 85.78%).
LC-MS: (ESI) m/z: 651.2 [M+H]+
4.4 (S)-l-(4-(3-methyl-8-(6-methylpyridin-3-yl)-2-oxo-2,3-dihydroimidazo[4,5- c] [ 1 ,5]naphthyridin- 1 -yl)piperidin- 1 -yl)- 1 -oxopropan-2-yl 5-(4-(4-(3 -(2,4-dihydroxy- 5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)- 5-oxopentanoate
Figure imgf000136_0003
[0330] To a solution of3-methyl-8-(6-methyl-3 -pyridyl)- 1 -(4- piperidyl)imidazo[4,5-c][l,5]naphthyridin-2-one (55.24 mg, 147.53 umol) and (2S)-2- [5-[4-[[4-[3-(2,4-dihydroxy-5-isopropyl-phenyl)-5-(ethylcarbamoyl)-l,2,4-triazol-4- yl]phenyl]methyl]piperazin-l-yl]-5-oxo-pentanoyl]oxypropanoic acid (80 mg, 122.94 umol) in N,N-Dimethylformamide (1 mL) and Tri chloromethane (10 mL) was addedphenol (23.14 mg, 245.88 umol, 21.63 uL), N-ethyl-N-isopropyl-propan-2- amine (31.78 mg, 245.88 umol, 42.83 uL) and the reaction mixture was stirred for lOmin at 25°C, then benzotriazol- l-yloxy(tri pyrrolidin-1- yl)phosphonium;hexafluorophosphate (76.77 mg, 147.53 umol) was added into the mixture and stirred for more 2 hr at 25 °C. The reaction mixture was purification by Prep-HPLC (CF3COOH) to give the pure product as a white solid (30 mg, yield 23.0%).
C30
LC-MS: (ESI) m/z: 1008.2 [M+H]+, 1030.3 [M+Na]+
1H NMR (MeOD, 400 MHz): 5 9.32 (s, 1 H), 8.93 (d, J= 9.2 Hz, 1 H), 8.86 (t, J = 6.8 Hz, 1 H), 8.53 (d, J= 8.8 Hz, 1 H), 8.37-8.29 (m, 1 H), 7.83 (d, J = 9.2 Hz, 1 H), 7.53 (d, 7 = 6.8 Hz, 2 H), 7.37 (t, J = 8.0 Hz, 2 H), 6.62 (d, J = 6.4 Hz, 1 H), 6.15 (s, 1 H), 5.45 (dq, J= 32.8 Hz, 6.4 Hz, 1 H), 4.73-4.62 (m, 2 H), 4.35 (d, J= 6.4 Hz, 2 H), 4.35 (d, J= 6.4 Hz, 2 H), 4.16 (d, J= 14.4 Hz, 1 H), 3.51 (d, J= 12.4 Hz, 3 H), 3.40-3.24 (m, 4 H), 3.19-3.05 (m, 3 H), 2.95-2.76 (m, 3 H), 2.56 (s, 4 H), 2.53-2.30 (m, 4 H), 2.11-1.73 (m, 4 H), 1.36 (d, J = 6.4 Hz, 3 H), 1.10-0.97 (m, 3 H), 0.88- 0.69 (m, 6 H).
Method E as follow:
Synthesis of route:
Figure imgf000137_0001
5.1 5-(4-(4-(3-(2, 4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H- 1,2,4- triazol-4-yl)benzyl)piperazin-l-yl)-2-methyl-5-oxopentanoic acid
Figure imgf000137_0002
[0331] To a solution of 2-methylpentanedioic acid (146 mg, 999.03 umol) inDMF (5 mL) was added 5-(2,4-dihydroxy-5-isopropyl-phenyl)-N-ethyl-4-[4-(piperazin-l- ylmethyl)phenyl]-l, 2, 4-triazole-3 -carboxamide (232.06 mg, 499.52 umol) ,DIEA (999.03 umol) and [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]- dimethyl-ammonium;hexafluorophosphate (227.92 mg, 599.42 umol) . The mixture was stirred for 1 hr at 25 °C. The reaction mixture was purified by combi-flash to get the product as a white solid (230 mg, yield 38.84%) i.C-VIS: (ESI) m/z: 593.3 [M+H]+
5.2 5-(4-(4-(3-(2,4-bis(tertbutyldimethylsilyloxy) 5isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-methyl-5- oxopentanoic acid
Figure imgf000138_0001
[0332] To the mixture of 5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-methyl-5- oxopentanoic acid (110 mg, 0.185 mmol), imidazole (102 mg, 1.49 mmol) and DMAP (10 mg) in DMF (1 mL) was added TBDSC1 (112 mg, 0.74 mmol), the mixture was stirred at rt overnight, LCMS showed full conversion of starting material. To the mixture was added MeOH (2 mL), the mixture was stirred at rt for 15 minutes, diluted with water (20 mL) and extracted with DCM (10 mL x 2), the combined organic was dried over anhydrous Na2SO4, concentrated and purified by column chromatography (silica gel, MeOH / DCM = 0: 1 to 1 :20) to afford the pure product as sticky oil (130 mg, yield 85%).
LC-MS: (ESI) m/z: 821.4 [M+H]+
5.3 (S)-l -(benzyloxy)- l-oxopropan-2-yl 5-(4-(4-(3-(2,4-bis(tert- butyldimethylsilyloxy)-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)-2-methyl-5oxopentanoate
Figure imgf000139_0001
[0333] The mixture of 5-(4-(4-(3-(2,4-bis(tert-butyldimethylsilyloxy)-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2- methyl-5-oxopentanoic acid (150 mg, 0.183 mmol), (S)-benzyl 2-hydroxypropanoate (66 mg, 0.366 mmol), DCC (206 mg, 1.0 mmol) and DMAP (10 mg) in DCM (3 mL) was stirred at rt overnight, LCMS showed full conversion of starting material. The mixture was filtered, the filtrate was diluted with water (20 mL) and extracted with DCM (10 mL x 2), the combined organic was dried over anhydrous Na2SO4, concentrated and purified by column chromatography (silica gel, EtOAc / petroleum ethers = 0: 1 to 2: 1) to afford the product as sticky oil (110 mg, yield 61%).
LC-MS: (ESI) m/z: 492.4 [1/2M+H]+
5.4 (2S)-2-(5-(4-(4-(3-(2,4-bis(tert-butyldimethylsilyloxy)-5-isopropylphenyl)-5-
(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-methyl-5- oxopentanoyloxy)propanoic acid
Figure imgf000139_0002
[0334] To a solution of [(lS)-2-benzyloxy-l-methyl-2-oxo-ethyl] 5-[4-[[4-[3-[2,4- bis[[tert-butyl(dimethyl)silyl]oxy]-5-isopropyl-phenyl]-5-(ethylcarbamoyl)-l,2,4- triazol-4-yl]phenyl]methyl]piperazin-l-yl]-2-methyl-5-oxo-pentanoate (110 mg, 111.86 umol) in Methanol (10 mL) was added Palladium on carbon, 10 wt.% 50% water (20 mg, 111.86 umol) . The result mixture was stirred for 1 hr at 25 °C under H2 atmosphere. After removed the solid, the residue was concentracted under reduced pressure to get the product as a white solid (80mg, yield 80.06%)
LC-MS: (ESI) m/z: 894.1 [M+H]+
5.5 (2S)-2-(5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H- l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-methyl-5-oxopentanoyloxy)propanoic acid
Figure imgf000140_0001
[0335] To a solution of (2S)-2-[5-[4-[[4-[3-[2,4-bis[[tert- butyl(dimethyl)silyl]oxy]-5-isopropyl-phenyl]-5-(ethylcarbamoyl)-l,2,4-triazol-4- yl]phenyl]methyl]piperazin-l-yl]-2-methyl-5-oxo-pentanoyl]oxypropanoic acid (80 mg, 89.56 umol) in THF (5 mL) was added tetrabutylammonium;fluoride (46.83 mg, 179.12 umol, 51.86 uL) . The mixture was stirred for 1 hr at 25 °C. removed the solvent to get the crude product which was used in the next step without further purification.
LC-MS: (ESI) m/z: 333.2 [1/2M+H]+ , 665.3 [M+H]+
5.6 (S)-l-(4-(3-methyl-8-(6-methylpyridin-3-yl)-2-oxo-2,3-dihydroimidazo[4,5- c] [ 1 ,5]naphthyridin- 1 -yl)piperidin- 1 -yl)- 1 -oxopropan-2-yl 5-(4-(4-(3 -(2,4-dihydroxy- 5-methylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2- methyl-5-oxopentanoate
Figure imgf000140_0002
[0336] To a solution of(2S)-2-[5-[4-[[4-[3-(2,4-dihydroxy-5-isopropyl-phenyl)-5- (ethylcarbamoyl)-l,2,4-triazol-4-yl]phenyl]methyl]piperazin-l-yl]-2-methyl-5-oxo- pentanoyl]oxypropanoic acid (99.26 mg, 149.32 umol) and 3-methyl-8-(6-methyl-3- pyridyl)-l-(4-piperidyl)imidazo[4,5-c][l,5]naphthyridin-2-one (67.09 mg, 179.19 umol) in N,N-Dimethylformamide, ACS, 99.8+% (1 mL) and Trichloromethane (25 mL) was addedN-ethyl-N-isopropyl-propan-2-amine (38.60 mg, 298.64 umol, 52.02 uL), phenol (28.11 mg, 298.64 umol, 26.27 uL) and the reaction mixture was stirred for lOmin at 25°C, thenbenzotriazol-l-yloxy(tripyrrolidin-l- yl)phosphonium;hexafluorophosphate (93.25 mg, 179.19 umol) was added into the mixture and stirred for more 2 hr at 25 °C. The reaction mixture was purification by Prep-HPLC (CF3COOH) to give the pure product as a white solid (10.5 mg, yield 6.8%) C31 LC-MS: (ESI) m/z: 341.3 [1/3M+H]+ , 511.2 [1/2M+H]+ ,1022.3 [M+H]+
1H NMR (MeOD, 400 MHz): 59.27-9.12 (M, 1 H), 8.78 (d, J= 18.8 Hz, 1 H), 8.47-8.29 (m, 2 H), 8.21-8.08 (m, 1 H), 7.47-7.26 (m, 3 H), 7.22-6.99 (m, 2 H), 6.54-6.40 (m, 1 H), 6.20-6.09 (m, 1 H), 5.56-5.33 (m, 1 H), 4.76-4.57 (m, 1 H), 4.24-4.09 (m, 1 H), 4.24-4.09 (m, 1 H), 3.72-3.27 (m, 10 H), 3.17-3.02 (m, 4 H), 2.93-2.64 (m, 3 H), 2.59-2.89 (m, 11 H), 2.09-1.68 (m, 4 H), 1.44-1.29 (m, 3 H), 1.25-0.91 (m, 8 H), 0.78-0.63 (m, 6 H).
Method F as follow:
Synthesis of route:
Figure imgf000141_0001
6.1 (S)-l -(benzyloxy)- l-oxopropan-2-yl 3 -formylbenzoate
Figure imgf000141_0002
[0337] To a suspension of 3 -formylbenzoic acid (0.2 g, 1.33 mmol) in Acetonitrile
(10 mL) was addedoxalyl dichloride (169.09 mg, 1.33 mmol, 115.81 uL) and DMF (0.01 mL). The mixture was stirred at 25 °C for 23 hr. Then benzyl (2S)-2- hydroxypropanoate (240.06 mg, 1.33 mmol) and N-ethyl-N-isopropyl-propan-2- amine (430.43 mg, 3.33 mmol, 580.10 uL) was added to the reation at 25 °C and the reaction mixture was sirred at 25 °C for 3h. This crude product was purifed by combi- flash (PE/EA=9/1) to get the product as a off-white solid (120 mg, yield 28.84%) LC-MS: (ESI) m/z: 335.1 [M+Na]+
6.2 (S)-l -(benzyloxy)- l-oxopropan-2-yl 3-((4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl)benzyl)piperazin- 1 - yl)methyl)benzoate
Figure imgf000142_0001
[0338] To a suspension of [(lS)-2 -benzyl oxy-1 -methyl-2-oxo-ethyl] 3- formylbenzoate (150 mg, 480.28 umol) and5-(2,4-dihydroxy-5-isopropyl-phenyl)-N- ethyl-4-[4-(piperazin- 1 -ylmethyl)phenyl]- 1 ,2,4-triazole-3 -carboxamide (223.12 mg, 480.28 umol) in Methanol (3 mL) was added 4A (15 mg, 480.28 umol). And the reaction was stirred at 26 °C for 12 hr. Then sodium;cyanoboranuide (30.18 mg, 480.28 umol) was added to the reation at 25 °C and the reaction mixture was sirred at 25 °C for Ih. The reaction mixture was quenched by adding a solution of NH4C1 and extracted with EA( 30ml*3) dried over Na2SO4, concentrated under vacuum to get the crude product. The crude product was purified by combi-flash to get the product as a white solid (120mg, 32.8%) LC-MS: (ESI) m/z: 761.7 [M+H]+
6.3 (S)-l -(benzyloxy)- l-oxopropan-2-yl 3-((4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl)benzyl)piperazin- 1 - yl)methyl)benzoate
Figure imgf000142_0002
[0339] To a solution of (S)-l -(benzyloxy)- l-oxopropan-2-yl 3-((4-(4-(3-(2,4- dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)methyl)benzoate (120 mg, 179.17 umol) in Methanol (15 mL) was added Palladium (19.07 mg) under N2 protection. The mxiture was stirred for 1 hr at 25 °C under H2 atmosphere. After removed the solid, the rusidue was concentrated to get the product as a white solid (95mg, 89%).
LC-MS: (ESI) m/z: 671.7 [M+H]+
6.4 (S)-l-(4-(3-methyl-8-(6-methylpyridin-3-yl)-2-oxo-2,3-dihydroimidazo[4,5- c] [ 1 ,5]naphthyridin- 1 -yl)piperidin- 1 -yl)- 1 -oxopropan -2 -yl 3 -((4-(4-(3 -(2,4- dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)methyl)benzoate
Figure imgf000143_0001
[0340] To a solution of(2S)-2-[3-[[4-[[4-[3-(2,4-dihydroxy-5-isopropyl-phenyl)-5- (ethylcarbamoyl)- 1 ,2,4-triazol-4-yl]phenyl]methyl]piperazin- 1 - yl]methyl]benzoyl]oxypropanoic acid (90 mg, 134.18 umol) and 3-methyl-8-(6- methyl-3-pyridyl)-l-(4-piperidyl)imidazo[4,5-c][l,5]naphthyridin-2-one (60.29 mg, 161.01 umol) in N,N-Dimethylformamide (1 mL) and Trichloromethane (25 mL) was addedphenol (25.26 mg, 268.36 umol, 23.60 uL), N-ethyl-N-isopropyl-propan-2- amine (34.68 mg, 268.36 umol, 46.74 uL) and the reaction mixture was stirred for lOmin at 25°C, then tripyrrolidin-l-yl(triazolo[4,5-b]pyridin-3- yloxy)phosphonium;hexafluorophosphate (83.95 mg, 161.01 umol) was added into the mixture and stirred for more 2 hr at 26 °C. The reaction mixture was purification by Prep-HPLC (CF3COOH) to give the pure product as a white solid (9.3 mg, 6.4%) C32
LC-MS: (ESI) m/z: 1050.2 [M+H]+, 514.3 [1/2M+H]+ 1H NMR (MeOD, 400 MHz): 5 9.37-9.25 (M, 1 H), 8.91 (d, J = 4.4 Hz, 1 H), 8.80 (d, J = 6.4 Hz, 1 H), 8.51 (d, J= 7.2 Hz, 1 H), 8.34-8.26 (m, 1 H), 8.09 (d, J = 9.2 Hz, 1 H), 8.02 (d, J= 5.2 Hz, 1 H), 7.81-7.70 (m, 1 H), 7.63 (d, J= 6.0 Hz, 1 H), 7.54-7.47 (m, 1 H), 7.45 (d, J= 6.8 Hz, 2 H), 7.34- 7.22 (m, 3 H), 6.59 (s, 1 H), 6.19 (s, 1 H), 5.81-5.64 (m, 1 H), 5.14-4.88 (m, 1 H), 4.33-4.20 (m, 1 H), 4.11-3.98 (m, 2 H), 3.92 (s, 2 H), 3.54 (s, 3 H), 3.43-3.30 (m, 1 H), 3.11-2.75 (m, 11 H), 2.72- 2.52 (m, 5 H), 2.14-1.95 (m, 3 H), 1.59-1.47 (m, 4 H), 1.45-1.35 (m, 1 H), 1.30-1.16 (m, 4 H), 1.07 (t, J= 5.6 Hz, 4 H), 0.78 (d, J= 5.6 Hz, 6 H).
Conjugates C1-C16 and C48-C77
[0341] Non-limiting examples of synthetic methods for Conjugates Cl -Cl 6, C48- C77 are included below.
C48:
Figure imgf000144_0001
[0342] Synthesis of methyl 2-(4-(benzyloxy)-2-fluorophenyl)acetate
Figure imgf000144_0002
Amixture of methyl 2-(2-fluoro-4-hydroxyphenyl)acetate (2 g, 10.9 mmol) and K2CO3 (4.5 g, 32.6 mmol) in DMF (30 mL) was added (bromomethyl)benzene (2.79 g, 16.29 mmol) and stirred at r t for 16 hr. The solution was added water(150 ml) and extracted with EA(80 ml), the organic phase was Concentrated in vacuo and purified by silica gel (EA:PE=1: 10) to give (2.8 g) iol. Yield: 94 %. LCMS (m/z): 275.1 M+ H+.
[0343] Synthesis of 2-(4-(benzyloxy)-2-fluorophenyl)acetic acid
Figure imgf000144_0003
Amixture of methyl 2-(4-(benzyloxy)-2-fluorophenyl)acetate (3.08 g, 11.23 mmol) in MeOH (30 mL) was added LiOH (807 mg, 33.69 mmol) and stirred at 40oC for 16 hr. The solution was acidated with 2 N HC1/H2O to PH=4, added water (150 ml) and fdtered to give (2.9 g) white solid. Yield: 99.23 %. LCMS (m/z): 283.0 M+ Na+.
[0344] Synthesis of tert-butyl 2-(4-(benzyloxy)-2-fluorophenyl)acetate
Figure imgf000144_0004
Amixture of 2-(4-(benzyloxy)-2-fluorophenyl)acetic acid (2.25 g, 8.65 mmol) in DCM (30 mL) was added 8% 2-methylprop-l-ene/DCM (20 mmol) and H2SO4 (0.2 eq) and stirred at r t for 48 hr. The solution was added water and extracted with EA (100 ml), Concentrated in vacuo and purified by silica gel (EA:PE=l:10) ) to give (2.5 g) solid. Yield: 91.41%. LCMS (m/z): 339.0 M+ H+.
[0345] Synthesis of tert-butyl 2-(2-fluoro-4-hydroxyphenyl)acetate
Figure imgf000145_0001
Amixture of 2-(4-(benzyloxy)-2-fluorophenyl)acetic acid (2.5 g, 0.411 mmol) ) in THF( 100 mL) was added Pd/C (0.2 eq) was stirred at r t for 16 hr. The solution was filtered, the filtrated was concentrated in vacuo to give (1.75 g) solid. Yield: 97.88 %. LCMS (m/z): 249.1 M+ Na+.
[0346] tert-butyl 2-(2-fluoro-4-((4-nitrophenoxy)carbonyloxy)phenyl)acetate
Figure imgf000145_0002
Amixture of tert-butyl 2-(2-fluoro-4-hydroxyphenyl)acetate (1.6 g, 7.07 mmol) and DIPEA (3.66 g, 28.29 mmol) in DMF (30 mL) was added (bromomethyl)benzene (4.3 g, 14.14 mmol) and stirred at r t for 2 hr. The solution was added water(100 ml) and extracted with EA(100 ml), the organic phase was Concentrated in vacuo and purified by silica gel (EA:PE=1 : 10) to give (2.3 g) iol. Yield: 83.10 %. LCMS (m/z): 413.9 M+ Na+.
[0347] 4-(2-tert-butoxy-2-oxoethyl)-3-fluorophenyl 4-(3-(5-(2-aminopyrimidine-5- carboxamido)-7-methoxy-2,3-dihydroimidazo[l,2-c]quinazolin-8- yloxy)propyl)piperazine- 1 -carboxylate
Figure imgf000145_0003
Amixture of tert-butyl 2-(2-fluoro-4-((4-nitrophenoxy)carbonyloxy)phenyl)acetate (2.4 g, 6.13 mmol), DIPEA (3.96 g, 30.66 mmol) in DMAC (30 mL) was added 2-amino-N-(7-methoxy-8-(3- (piperazin- 1 -yl)propoxy)-2,3 -dihydroimidazo [1 ,2-c]quinazolin-5 -yl)pyrimidine-5 -carboxamide (3.09 g, 6.44 mmol) and stirred at r t for 2 hr. The solution was added water (150 ml) and filtered to give (4 g) solid. Yield: 89.13 %. LCMS (m/z): 732.3 M+ H+. [0348] 2-(4-(4-(3-(5-(2-aminopyrimidine-5-carboxamido)-7-methoxy-2,3- dihydroimidazo[l,2-c]quinazolin-8-yloxy)propyl)piperazine-l-carbonyloxy)-2- fluorophenyl)acetic acid
Figure imgf000146_0001
A mixture of 4-(2-tert-butoxy-2-oxoethyl)-3-fluorophenyl 4-(3-(5-(2-aminopyrimidine-5- carboxamido)-7 -methoxy-2,3 -dihydroimidazo [ 1 ,2-c] quinazolin-8 -yloxy)propyl)piperazine- 1 - carboxylate (3.6 g, 4.92 mmol) in 50%TFA/DCM (40 mL) was stirred at r t for 2 hr. The solution was concentrated in vacuo, the crude purified by RP-Chrom (0.1% TFA MeCN) to give (0.74 g) white solid.. Yield: 22.26 %. LCMS (m/z): 676.1 M+ H+.
[0349] 4-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H- l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-oxoethyl)-3-fluorophenyl 4-(3-(5-(2- aminopyrimidine-5-carboxamido)-7-methoxy-2,3-dihydroimidazo[l,2-c]quinazolin-8- yloxy)propyl)piperazine- 1 -carboxylate
Figure imgf000146_0002
A mixture of 2-(4-(4-(3-(5-(2-aminopyrimidine-5-carboxamido)-7-methoxy-2,3- dihydroimidazo [ 1 ,2-c] quinazolin-8-yloxy)propyl)piperazine- 1 -carbonyloxy)-2- fluorophenyl)acetic acid (1.06 g, 1.57 mmol), DIPEA (1.01 g, 7.84 mmol) and HBTU (0.716 g, 1.88 mmol) in DMAC (20 mL) was added 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4- (piperazin-l-ylmethyl)phenyl)-4H-l, 2, 4-triazole-3 -carboxamide (0.875 mg, 1.88 mmol) and stirred at r t for 3 hr. The solution was added water (100 ml) and filtered, purified by RP-Chrom (0.1% FA MeCN) to give ( 0.9 g, FA salt) white solid. Yield: 51.12 %. LCMS (m/z): 1122.5M+ H+.
[0350] 1HNMR (400 MHz, DMSO): 8 13.44 (s, 1H), 11.78 (s, 2H), 9.89 (s, 1H), 9.06 (s, 1H), 8.99 (s, 2H), 8.41 (s, 1H), 7.74-7.69 (m, 3H), 7.45 (d, J=8.0 Hz, 3H), 7.27 (t, J=8.8 Hz, 1H), 7.13-7.09 (dd, J=2 Hz, J=2.4 Hz, 1H), 6.98 (d, J=9.6 Hz, 1H), 7.43 (d, J=9.2 Hz, 1H), 6.70 (s, 1H), 6.39 (m, 1H), 4.52-4.47 (m, 2H), 4.41 (m, 5H), 4.25-4.18 (m, 6H), 4.02 (s, 3H), 3.83 (d, J=7.0 Hz, 2H), 3.66-3.55 (m, 4H), 3.31 (m, 4H), 3.21-3.15 (m, 6H), 2.97-2.90 (m, 2H), 2.37 (s, 4H), 1.05 (t, J=7.2 Hz, 3H), 0.90 (d, J=6.8 Hz, 6H).
C50
Figure imgf000147_0001
[0351] Synthesis of tert-butyl 2-(4-hydroxyphenyl) acetate (Int-3):
To a stirred solution of 2-(4-hydroxyphenyl) acetic acid (Int-1) (500 mg, 3.29 mmol, 1.0 eq) in toluene (5.0 stirred for 5-10 min at 75-80°C.Then followed by [bis (tertbutoxy) methyl] dim ethylamine (2.74 g, 13.5 mmol, and 4.10 eq) was added at 75- 80°C. The resulting reaction mixture was stirred for 2h at 75-80°C. Progress of reaction monitor by TLC. After completion of the reaction mixture quenched with water and extracted with ethyl acetate (2 x 50 mL) and wash with brine soln (50 mL), dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure to get the crude compound. The crude compound was purified by flash chromatography, desired product eluded in 0- 5% ethyl acetate in Pet ether. All pure fraction determined to get tert-butyl 2-(4-hydroxyphenyl) acetate (Int-3) (360 mg, 52% yield), as pale yellow oil.
[0352] Synthesis of 4-(2-(tert-butoxy)-2-oxoethyl)phenyl 4-(2-((5-(2- aminopyrimidine-5-carboxamido)-7-methoxy-2,3-dihydroimidazo[l,2-c]quinazolin-8- yl)oxy)ethyl)piperazine-l -carboxylate (Int-4):
To a stirred solution of tert-butyl 2-(4-hydroxyphenyl) acetate (Int-3) (200 mg, 0.960 mmol, 1.0 eq) in DCM (6 mL), DIPEA (310 mg, 2.4 mmol, 2.5 eq) followed by 4- nitrophenyl carb onochlori date (194 mg, 0.960 mmol, 1.0 eq) were added at RT. The resulting reaction was stirred for 2h at RT. Progress of the reaction was monitored by TLC. After completion of the SM (monitor by TLC) a solution of 2-amino-N-{7- methoxy-8-[2-(piperazin- 1 -yl)ethoxy]-2H,3H-imidazo[ 1 ,2-c]quinazolin-5- yl}pyrimidine-5-carboxamide(666 mg, 0.966 mmol, 1.0 eq) in DMF (2.0 mL) was added at RT. The resulting reaction mixture was stirred for 16h at RT. Progress of the reaction monitored by TLC and LCMS. After completion of the SM, the reaction mixture was evaparatated under vacuum then quenched with water (100 mL) solid precipitated stirred for 10 min then filtered to get crude compound. The crude compound was stirred in minimum amount of DCM filtered to get pure compound Int-4 (250 mg, 37 % yield), as a gammy white solid.
[0353] Synthesis of 2-(4-((4-(2-((5-(2-aminopyrimidine-5-carboxamido)-7- m ethoxy-2, 3 -dihydroimidazof 1 ,2-c]quinazolin-8-yl)oxy)ethyl)piperazine- 1 - carbonyl)oxy)phenyl)acetic acid (Int- 5):
To stirred solution of 4-[2-(tert-butoxy)-2-oxoethyl]phenyl 4-(2-{[5-(2- aminopyrimidine-5-amido)-7-methoxy-2H,3H-imidazo[l,2-c]quinazolin-8- yl]oxy}ethyl)piperazine-l-carboxylate (Int- 4) (250 mg, 0.357 mmol, 1.0 eq) in DCM (10 mL), TFA (815 mg, 7.15 mmol, 20 eq) was added at RT. The resulting reaction mixture was stirred at RT for 6h. Progress of reaction mixture monitored by LCMS. After completion of the reaction, the reaction was volatile evaporated under reduced pressure to get crude compound. The crude compound was triturated with diethyl ether and followed by lyophilized to get 2-(4-((4-(2-((5-(2-aminopyrimidine-5- carboxamido)-7-methoxy-2,3-dihydroimidazo[l,2-c]quinazolin-8- yl)oxy)ethyl)piperazine-l-carbonyl)oxy)phenyl)acetic acid Int-5 (240 mg TFA salt, quantitative) as off white solid. This crude itself used for the next step without further purification.
[0354] XH NMR: (500 MHz, DMSO-a ) 8: 13.40 (s, 1H), 8.97 (s, 2H), 7.97 (d, J= 8.0 Hz, 2H),7.53 (s, 2H), 7.41 (d, J= 4.0 Hz, 1H), 7.28 (d, J= 8.0 Hz, 2H) 7.08 (d, J = 8.0Hz, 2H), 4.60 (bs, 2H), 4.46 (bs, 2H), 4.20 (t, J= 8.0 Hz, 2H), 4.01 (s, 3H), 3.38 (s, 9H, merged with water). LC-MS: 644.26 (M + H). C51
Figure imgf000149_0001
[0355] Synthesis of (Int-3): tert-butyl 2-(4-hydroxyphenyl) acetate:
To a suspension of 2-(4-hydroxyphenyl) acetic acid (500 mg, 3.29 mmol, 1.0 eq) in Toluene (5.0 mL), 1, 1-di-tert-butoxy-N, N-dimethylmethanamine (2.74 g, 13.50 mmol, 4.10 eq) was added at RT. The resulting reaction mass was slowly heated to 75-80°C for 1.5 h. Progress of the reaction was monitor by TLC. Reaction was quenched with water and extracted with ethyl acetate (3 x 150 mL), organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the crude. The crude obtained was purified by flash chromatography desired product was eluded in 0-5% Ethyl acetate in pet ether solvent system. All the pure fractions were collected and concentrated under reduced pressure to afford (Int-3) tert-butyl 2-(4-hydroxyphenyl)acetate (Int-2) (360 mg, 52.60% yield) as off white solid.
[0356] Synthesis of 4-(2-(tert-butoxy)-2-oxoethyl)phenyl 4-(2-((6-(2- aminopyrimidine-5-carboxamido)-8-methoxy-3,4-dihydro-2H-pyrimido[l,2- c]quinazolin-9-yl)oxy)ethyl)piperazine-l -carboxylate (Int-4): To a suspension of tert-butyl 2-(4-hydroxyphenyl)acetate (Int-2) (80 mg, 0.384 mmol, 1.0 eq) in DCM (2.5 mL), DIPEA (248 mg ,1.92 mmol, 5.0 eq) followed by 4- nitrophenyl chloroformate (77.40 mg, 0.384 mmol, 1.0 eq) were added at RT. The reaction mixture was stirred for 2h at room temperature, after completion of SM (monitored by TLC), a solution of 2-amino-N-(8-methoxy-9-(2-(piperazin-l- yl)ethoxy)-3,4-dihydro-2H-pyrimido[l,2-c]quinazolin-6-yl)pyrimidine-5- carboxamide (TVD-0004617) (184 mg, 0.384 mmol, 1. eq) in DMF (1.0 mL) was added at RT and the reaction mixture was stirred at room temperature for 16h , progress of reaction was monitor by TLC. After completion of the reaction, reaction mass quenched with water and extracted with ethyl acetate (3x 50 mL), organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the crude. The crude obtained was purified by flash chromatography desired product was eluded in 0-5% Methanol in DCM solvent system, all the pure fractions were collected and concentrated under reduced pressure to afford (Int-4) 4-(2-(tert- butoxy)-2-oxoethyl)phenyl 4-(2-((6-(2-aminopyrimidine-5-carboxamido)-8-methoxy- 3, 4-dihydro-2H-pyrimido[l,2-c]quinazolin-9-yl)oxy)ethyl)piperazine-l -carboxylate (TVD-0004617) (140 mg, 51.06% yield) as off white solid.
[0357] Synthesis of 2-(4-((4-(2-((6-(2-aminopyrimidine-5-carboxamido)-8- methoxy-3 ,4-dihydro-2H-pyrimido[ 1 ,2-c]quinazolin-9-yl)oxy)ethyl)piperazine- 1 - carbonyl)oxy)phenyl)acetic acid (Int-5):
To stirred solution of 4-(2-(tert-butoxy)-2-oxoethyl)phenyl 4-(2-((6-(2- aminopyrimidine-5-carboxamido)-8-methoxy-3,4-dihydro-2H-pyrimido[l,2- c]quinazolin-9-yl)oxy)ethyl)piperazine-l -carboxylate (Int-4) (140 mg ,0.196 mmol, 1.0 eq) in DCM (2.0 mL), TFA (0.45 mL, 5.88 mmol, 30.0 eq) was added at room temperature. The resulting reaction mixture was stirred for overnight at room temperature and progress of reaction monitored by TLC and LC-MS. After completion the reaction, volatilise was removed under reduced pressure and codistilled with toluene to get crude compound which was then lyophilized to afford 2- (4-((4-(2-((6-(2-aminopyrimidine-5-carboxamido)-8-methoxy-3,4-dihydro-2H- pyrimido[l,2-c]quinazolin-9-yl)oxy)ethyl)piperazine-l-carbonyl)oxy)phenyl)acetic acid (Int-5) (200 mg TFA salt, Crude) as off white solid.
[0358] Synthesis of 4-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-oxoethyl)phenyl 4- (2-( ( 6-(2-aminopyrimidine-5-carboxamido)-8-methoxy-3, 4-dihydro-2H-pyrimido[ 1, 2- c]quinazolin-9-yl)oxy)ethyl)piperazine-l -carboxylate (C51):
To a stirred solution of 2-(4-((4-(2-((6-(2-aminopyrimidine-5-carboxamido)-8- methoxy-3 ,4-dihydro-2H-pyrimido[ 1 ,2-c]quinazolin-9-yl)oxy)ethyl)piperazine- 1 - carbonyl)oxy)phenyl)acetic acid (Int-4) (150 mg, 0.228 mmol, 1.0 eq) in DMF (1.0 mL), DIPEA (0.202 mL, 1.14 mmol, 5.0 eq) followed by HOBT (61.60 mg, 0.456 mmol, 2.0 eq) and EDC.HC1 (87.40 mg, 0.456 mmol, 2.0 eq) and the reaction mass was stirred for 5-10 min at room temperature and then 5-(2,4-dihydroxy-5- isopropylphenyl)-N-ethyl-4-(4-(piperazin-l-ylmethyl)phenyl)-4H-l,2,4-triazole-3- carboxamide (TVD-0003510) (106 mg, 0.228 mmol, 1.0 eq) was added and the reaction mixture was stirred at room temperature for 18h. After completion of SM, as monitored by TLC. Then reaction quenched with ice-water (10 mL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (5 mL) dried under vacuum to afford crude compound .The crude was purified by Prep HPLC and lyophilized to afford 4-(2-(4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2- oxoethyl)phenyl 4-(2-((6-(2-aminopyrimidine-5-carboxamido)-8-methoxy-3,4- dihydro-2H-pyrimido[l,2-c]quinazolin-9-yl)oxy)ethyl)piperazine-l-carboxylate (C51) (50.90 mg, 20.21 % yield) as off white solid.
[0359] 'H NMR: (400 MHz, DMSO-tfe): 8: 14.18 (s, 1H), 11.21 (bs, 1H), 10.31 (bs, 1H), 9.70 (s, 1H), 9.01 (bs, 3H), 8.15 (d, J= 8.0 Hz, 1H), 7.23 (d, J= 12.0 Hz, 2H), 7.08 (d, J= 12.0 Hz, 2H), 6.97 (s, 1H), 6.31 (s, 1H), 4.60 (s, 2H), 4.40 (s, 4H), 4.02 (s, 3H), 3.84-3.34 (m, 24H), 3.21-3.14 (m, 6H), 2.95-2.92 (t, J = 6.8 Hz, 2H), 2.95-2.92 (m, 1H), 1.07-1.03 (t, J = 8.0 Hz, 3H), 0.87 (d, J = 8.0 Hz, 6H).
LC-MS: 1105.02 (M + H).
Figure imgf000152_0001
[0360] Synthesis of 2-(2-(tert-butoxy)-2-oxoethyl)phenyl 4-(3-((5-(2- aminopyrimidine-5-carboxamido)-7-methoxy-2,3-dihydroimidazo[l,2-c]quinazolin-8- yl)oxy)propyl)piperazine- 1 -carboxylate(Int-2):
To a suspension of tert-butyl 2-(2-hydroxyphenyl)acetate (Int-1) (150 mg, 0.720 mmol, 1.0 eq) in DCM (2.0 mL), DIPEA (233 mg, 1.80 mmol, 2.50 eq) followed by 4-nitrophenyl chloroformate (145 mg, 0.720 mmol, 1.0 eq) were added at RT. The reaction mixture was stirred for 2h at room temperature, after completion SM (monitored by TLC). A solution of 2-amino-N-(7-methoxy-8-(3-(piperazin-l- yl)propoxy)-2,3-dihydroimidazo[l,2-c]quinazolin-5-yl)pyrimidine-5-carboxamide (TVD-0003657) (345 mg, 0.720 mmol, 1.0 eq) in DMF (1.0 mL) was added and stirred at room temperature for 16. h. Progress of reaction was monitor by TLC. After completion of SM, reaction mixture was quenched with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the crude. The crude obtained was purified by flash chromatography desired product was eluded in 0-5% Methanol in DCM solvent system. All pure fractions were collected and concentrated under reduced pressure to afford 2-(2-(tert-butoxy)-2-oxoethyl)phenyl4- (3-((5-(2-aminopyrimidine-5-carboxamido)-7-methoxy-2,3-dihydroimidazo[l,2- c]quinazolin-8-yl)oxy)propyl)piperazine-l -carboxylate (Int-2) (250 mg, 48.63 % yield) as off white solid.
[0361] Synthesis of 2-{2-[4-(3-{[5-(2-aminopyrimidine-5-amido)-7-methoxy- 2H,3H-imidazo[l,2-c]quinazolin-8-yl]oxy}propyl)piperazine-l- carbonyloxy]phenyl} acetic acid (Int-3):
To a stirred solution of 2-[2-(tert-butoxy)-2-oxoethyl]phenyl 4-(3-{[5-(2- aminopyrimidine-5-amido)-7-methoxy-2H,3H-imidazo[l,2-c]quinazolin-8- yl]oxy}propyl)piperazine-l-carboxylate (Int-2) (230 mg, 0.322 mmol, 1.0 eq) in DCM (4.0 mL), TFA (0.73 mL, 9.67 mmol, 30.0 eq) was added at room temperature. The resulting reaction mixture was stirred at room temperature for 16h. Progress of the reaction was monitored by TLC and LC-MS. After completion the SM, all volatilise was removed under reduced pressure and co-distilled with toluene to get crude compound which was then lyophilized to afford 2-{2-[4-(3-{[5-(2- aminopyrimidine-5-amido)-7-methoxy-2H,3H-imidazo[l,2-c]quinazolin-8- yl]oxy}propyl)piperazine-l-carbonyloxy]phenyl}acetic acid (Int-3) (270 mg, quantitative) as off white solid.
[0362] Synthesis of 2-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-oxoethyl)phenyl 4- (3-((5-(2-aminopyrimidine-5-carboxamido)-7-methoxy-2, 3-dihydroimidazo[l,2- c]quinazolin-8-yl)oxy)propyl)piperazine-l-carboxylate (TVD-0004605):
To a stirred solution of 2-{2-[4-(3-{[5-(2-aminopyrimidine-5-amido)-7-methoxy- 2H,3H-imidazo[l,2-c]quinazolin-8-yl]oxy}propyl)piperazine-l- carbonyloxy]phenyl} acetic acid (Int-3) (270 mg, 0.411 mmol, l.Oeq) in DMF (5.0 mL), DIPEA (0.36 mL, 2.05 mmol, 5.0 eq), HOBt (111 mg, 0.821 mmol, 2.0 eq) followed by EDC.HC1 (157 mg, 0.821 mmol, 2.0 eq) were added at RT and the reaction mixture was stirred for 5-10 min at room temperature and then 5-(2,4- dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(piperazin-l-ylmethyl)phenyl)-4H- 1,2,4- triazole-3 -carboxamide (TVD-0003510) (191 mg, 0.411 mmol, 1.0 eq)was added and then reaction mixture was stirred at room temperature for 18h. After completion of SM, as monitored by TLC, reaction mass quenched with ice-water (15 mL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (5 mL) dried under vacuum to afford crude compound. The crude was purified by prep HPLC & lyophilized to afford 2-(2-(4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2- oxoethyl)phenyl 4-(3-( (5-(2-aminopyrimidine-5-carboxamido)-7-methoxy-2, 3- dihydroimidazo[l,2-c]quinazolin-8-yl)oxy)propyl)piperazine-l-carboxylateas (C52) (112 mg, 27.71% yield) as off white solid.
[0363] XH-NMR: (400 MHz, DMSO-a ) 8: 13.40 (s, 1H), 10.35-10.22 (bs, 2H), 9.75 (s, 1H), 9.02-8.97 (m, 3H), 7.98 (d, J= 8.0 Hz, 1H), 7.53-7.46 (m, 4H), 7.42- 7.38 (m, 3H), 7.34-7.30 (m, 2H), 7.24-7.21 (m, 1H), 7.15 (d, J= 8.0 Hz, 1H), 6.63 (s, 1H), 6.31 (s, 1H), 4.50-4.45 (m, 2H), 4.34 (bs, 2H), 4.21 (t, J= 8.0 Hz, 4H), 4.00 (s, 3H), 3.74 (s, 4H), 3.63-3.13 (m, 16H), 2.95-2.90 (m, 1H), 2.32-2.29 (m, 2H), 1.04 (t, J= 8.0 Hz, 3H), 0.84 (d, J = 6.8 Hz, 6H).
LC-MS: 1104.93 (M + H).
C53
Figure imgf000154_0001
[0364] Synthesis of (R)-2-amino-N-(8-(3-(4-(2-(2-amino-4-chloro-7-((4-methoxy- 3,5-dimethylpyridin-2-yl) methyl)-6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) ethyl) piperazin- 1-yl) propoxy)-7-methoxy-2,3-dihydroimidazo[l,2-c] quinazolin-5- yl) pyrimidine-5-carboxamide (TVD-0004600):
To stirred solution of 2-[(5R)-2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin- 2-yl) methyl]-5H, 6H, 7H-pyrrolo[2,3-d] pyrimidin-5-yl] acetaldehyde (Int-1) (198 mg, 0.547 mmol, 1.0 eq) in 10% Acetic acid in DMF (4.0 mL) was added 2-amino-N- {7-methoxy-8-[3-(piperazin-l-yl) propoxy]-2H,3H-imidazo[l,2-c] quinazolin-5-yl} pyrimidine-5-carboxamide (TVD-0003657) (175.0 mg, 0.365 mmol, 1.0 eq) at RT and the reaction mixture was stirred for 2h at RT. After 2h, Sodium triacetoxyborohydride (383 mg, 1.82 mmol, 5.0 eq) was added at RT. The resulting reaction mixture was stirred for 12h at RT. Progress of the reaction was monitor by TLC & LCMS. After completion of SM, reaction was quenched with water (12 mL) and extracted with ethyl acetate (3 x 20 mL). LCMS and TLC of aqueous layer indicated desired product. Then aqueous layer was lyophilized. Crude compound was purified by Prep.HPLC and lyophilized to afford (R)-2-amino-N-(8-(3-(4-(2-(2- amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl) methyl)-6,7-dihydro-5H- pyrrolo[2,3-d] pyrimidin-5-yl) ethyl) piperazin- 1-yl) propoxy)-7-methoxy-2,3- dihydroimidazo[l,2-c] quinazolin-5-yl) pyrimidine-5-carboxamide (39 mg, 12.95%) as an off white solid.
[0365] XH-NMR: (400 MHz, DMSO-a ) 8: 13.42 (s, 1H), 8.97 (s, 2H), 8.25 (s, 1H), 7.99 (d, J= 8.0 Hz, 1H), 7.54 (s, 2H), 7.40 (d, J= 8.0 Hz, 1H), 6.60 (bs, 2H), 4.63 (s, 2H), 4.51-4.47 (m, 2H), 4.31 (t, J= 4.0 Hz, 2H), 4.23-4.18 (m, 2H), 4.03 (s, 3H), 3.80 (s, 3H), 3.64 (t, J= 8.0 Hz, 1H), 3.34-3.24 (m, 4H), 2.95 (m, 10H), 2.24 (s, 3H), 2.20 (s, 3H), 2.12-2.05 (m, 3H), 1.86 (bs, 1H).
LC-MS: 825.08 (M + H).
C54
Figure imgf000155_0001
[0366] Synthesis of tert-butyl 4-(2-((5-(2-aminopyrimidine-5-carboxamido)-7- methoxy-2,3-dihydroimidazo[l,2-c] quinazolin-8-yl) oxy) ethyl) piperazine- 1- carboxylate (Int-3):
To a stirred solution of 2-amino-N- {8-hydroxy-7-methoxy-2H, 3 H-imidazo[l,2-c] quinazolin-5-yl] pyrimidine-5-carboxamide (100 mg, 0.238 mmol, 1.1 eq) in DMF (3.0 mL) was added K2CO3 (78 mg, 0.566 mmol, 2.0 eq) at ambient temperature and reaction mixture stirred for 10 min at RT. After 10 min, tert-butyl 4-(2 -bromoethyl) piperazine- 1 -carboxylate (99.6 mg, 0.340 mmol, 1.2 eq) was added at RT. The resulting reaction mixture stirred for 3h at 60°C. Progress of the reaction was monitor by TLC and LCMS. After completion of SM, reaction mixture quenched with water (10.0 mL), solid was filtered and washed with ice cold water, drier under reduced pressure to afford tert-butyl 4-(2-((5-(2-aminopyrimidine-5-carboxamido)-7-methoxy- 2,3-dihydroimidazo[l,2-c] quinazolin-8-yl) oxy) ethyl) piperazine- 1 -carboxylate (Int- 3) (130 mg, 81.21%) as off white solid.
[0367] Synthesis of 2-amino-N-(7-methoxy-8-(2-(piperazin-l-yl) ethoxy)-2,3- dihydroimidazo[l,2-c] quinazolin-5-yl) pyrimidine-5-carboxamide (Int-4):
To a solution of tert-butyl 4-(2-((5-(2-aminopyrimidine-5-carboxamido)-7-methoxy- 2,3-dihydroimidazo[l,2-c] quinazolin-8-yl) oxy) ethyl) piperazine- 1 -carboxylate (Int- 3) (120 mg, 0.212 mmol, 1.0 eq.) inDCM (1.5 ml) was added TFA (726 mg, 6.36 mmol, 30.0 eq) at 0°C. The resulting reaction mixture was stirred for 12h at RT. Progress of the reaction was monitor by TLC and LCMS. After completion of SM, all volatiles were evaporated under reduced pressure and the residue was triturated with DEE (10.0 mL) to afford 2-amino-N-(7-methoxy-8-(2-(piperazin-l-yl) ethoxy)-2,3- dihydroimidazo[l,2-c] quinazolin-5-yl) pyrimidine-5-carboxamide (Int-4) (150.0 mg, quantitative yield, TFA salt) as off white solid.
[0368] Synthesis of (R)-2-amino-N-(8-(2-(4-(2-(2-amino-4-chloro-7-((4-methoxy- 3,5-dimethylpyridin-2-yl) methyl)-6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) ethyl) piperazin- 1-yl) ethoxy)-7-methoxy-2,3-dihydroimidazo[l,2-c] quinazolin-5-yl) pyrimidine-5-carboxamide (TVD-0004599):
To stirred solution of 2-[(5R)-2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin- 2-yl) methyl]-5H, 6H, 7H-pyrrolo[2,3-d] pyrimidin-5-yl] acetaldehyde (Int-5) (204 mg, 0.564 mmol, 1.5 eq) in 10% Acetic acid in DMF (4.0 mL) was added 2-amino-N- {7-methoxy-8-[2-(piperazin-l-yl) ethoxy]-2H,3H-imidazo[l,2-c] quinazolin-5-yl} pyrimidine-5-carboxamide (Int-3) (175.0 mg, 0.376 mmol, 1.0 eq) at RT and the reaction mixture was stirred for 2h at RT. After 2h, Sodium triacetoxyborohydride (395 mg, 1.88 mmol, 5.0 eq) was added at RT. The resulting reaction mixture was stirred for 12h at RT. Progress of the reaction was monitor by TLC and LCMS. After completion of SM, reaction was quenched with water (12 mL) and extracted with ethyl acetate (3 x 20 mL). LCMS and TLC shows desired product was in aqueous layer. Then aqueous layer was lyophilized. Crude compound was purified by Prep.HPLC and lyophilized to afford (R)-2-amino-N-(8-(2-(4-(2-(2-amino-4-chloro- 7-((4-m ethoxy-3, 5 -dimethylpyri din-2 -yl) methyl)-6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) ethyl) piperazin- 1-yl) ethoxy)-7-methoxy-2,3-dihydroimidazo[l,2-c] quinazolin-5-yl) pyrimidine-5-carboxamide (19 mg, 6.23%) as an off white solid. [0369] 'H-NMR: (400 MHz, DMSO-a ) 8: 13.42 (s, 1H), 9.46 (bs, 1H), 8.97 (s, 1H), 8.25 (s, 1H), 7.98 (d, J= 8.0 Hz, 1H), 7.54 (s, 2H), 7.42 (d, J= 8.0 Hz, 1H), 6.58 (bs, 2H), 4.66 (s, 2H), 4.52-4.47 (m, 2H), 4.40 (bs, 2H), 4.23-4.18 (m, 2H), 4.04 (s, 3H), 3.81 (s, 3H), 3.64 (t, J= 8.0 Hz, 1H), 3.32-2.99 (m, 10H), 2.50 (s, 4H, merged with DMSO), 2.23 (s, 3H), 2.19 (s, 3H), 2.07 (bs, 1H), 1.92 (s, 1H).
LC-MS: 810.96 (M + H).
C55
Figure imgf000157_0001
[0370] Synthesis of tert-butyl 2-(2-fluoro-4-hydroxyphenyl) acetate (Int-2):
To a stirred solution of 2-(2-fluoro-4-hydroxyphenyl)acetic acid (250 mg, 1.47 mmol, 1.0 eq) in Toluene (8.0 mL), 1, l-di-tert-butoxy-N,N-dimethylmethanamine (1.22 g , 6.02 mmol, 4.10 eq) was at 70-80°C over a period of 5 min and the resulting reaction mass stirred at 70-80°C for 3h. Progress of the reaction was monitor by TLC and LCMS. After completion of SM, the reaction mixture was quenched with water and extracted with ethyl acetate (3 x 100 mL) the combined organic layers was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the crude. The crude obtained was purified by combi-flash, desired product was eluded in 0-5% Ethyl acetate in pet ether solvent system. All the pure fractions were collected and concentrated under reduced pressure to afford tert-butyl 2-(2-fluoro-4- hydroxyphenyl) acetate (Int-2) (230 mg, 69.18% yield) as pale yellow oil.
[0371] Synthesis of 4-(2-(tert-butoxy)-2-oxoethyl)-3-fluorophenyl 4-(3-((6-(2- aminopyrimidine-5-carboxamido)-8-methoxy-3,4-dihydro-2H-pyrimido[l,2- c]quinazolin-9-yl)oxy)propyl)piperazine-l-carboxylate(Int-3):
To a suspension of tert-butyl 2-(2-fluoro-4-hydroxyphenyl)acetate (Int-2) (80 mg, 0.384 mmol, 1.0 eq) in DCM (2.5 mL), DIPEA (570 mg, 4.42 mmol, 5.0 eq) followed by 4-nitrophenyl chloroformate (178 mg, 0.884 mmol, 1.0 eq) were added at RT. The reaction mixture was stirred for 2h at room temperature, after completion SM (monitored by TLC) to the reaction mixture a solution of 2-amino-N-(8-methoxy-9- (3 -(piperazin- 1 -yl)propoxy)-3 ,4-dihydro-2H-pyrimido[ 1 ,2-c]quinazolin-6- yl)pyrimidine-5-carboxamide (436 mg, 0.884 mmol, 1. eq) in DMF (1.0 mL) was added drop wise and stirred at room temperature for 16h. Progress of reaction was monitor by TLC and LCMS. After completion of SM, reaction mixture was quenched with water and extracted with ethyl acetate (3 x 100 mL). The combined organic layers was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain the crude. The crude obtained was purified by flash chromatography desired product was eluded in 0-5% methanol in DCM solvent system. All the pure fractions were collected and concentrated under reduced pressure to afford 4-(2-(tert-butoxy)-2-oxoethyl)-3 -fluorophenyl 4-(3-((6-(2-aminopyrimidine- 5-carboxamido)-8-methoxy-3,4-dihydro-2H-pyrimido[l,2-c]quinazolin-9- yl)oxy)propyl)piperazine-l-carboxylate(Int-3) (290 mg, 43.99 % yield) as off white solid.
[0372] Synthesis of 2-(4-((4-(3-((6-(2-aminopyrimidine-5-carboxamido)-8- methoxy-3,4-dihydro-2H-pyrimido[l,2-c]quinazolin-9-yl)oxy)propyl)piperazine-l- carbonyl)oxy)-2-fluorophenyl)acetic acid (Int-4):
To a stirred solution of 4-(2-(tert-butoxy)-2-oxoethyl)-3 -fluorophenyl 4-(3-((6-(2- aminopyrimidine 5-carboxamido)-8-methoxy-3,4-dihydro-2H-pyrimido[l,2- c]quinazolin-9yl)oxy)propyl) piperazine- 1 -carboxylate (Int-3) (290 mg, 0.389 mmol, 1.0 eq) in DCM (4.0 mL), TFA (0.89 mL, 11.70 mmol, 30.0 eq) was added at room temperature. The resulting reaction mixture was stirred for 16h at room temperature and progress of reaction monitored by TLC and LC-MS. After completion the SM, all volatilise were removed under reduced pressure and co-distilled with toluene to get crude compound which was then lyophilized to afford 2-(4-((4-(3-((6-(2- aminopyrimidine-5-carboxamido)-8-methoxy-3,4-dihydro-2H-pyrimido[l,2- c]quinazolin-9-yl)oxy)propyl)piperazine-l-carbonyl)oxy)-2-fluorophenyl)acetic acid (Int-4) (350 mg TFA salt, quantitative yield) as off white solid.
[0373] Synthesis of 4-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2-oxoethyl)-3- fluorophenyl 4-(3-((6-(2-aminopyrimidine-5-carboxamido)-8-methoxy-3,4-dihydro- 2H-pyrimido[ 1 ,2-c]quinazolin-9-yl)oxy)propyl)piperazine- 1 -carboxylate (C55) : To a stirred solution of 2-(4-((4-(3-((6-(2-aminopyrimidine-5-carboxamido)-8- methoxy-3,4-dihydro-2H-pyrimido[l,2-c]quinazolin-9-yl)oxy)propyl)piperazine-l- carbonyl)oxy)-2-fluorophenyl)acetic acid (Int-4) (300 mg, 0.435 mmol, 1.0 eq) in DMF (2.0 mL), DIPEA (0.38 mL, 2.17 mmol, 5.0 eq), HOBt (118 mg, 0.870 mmol, 2.0 eq) followed by EDC.HC1 (167 mg, 0.870 mmol, 2.0 eq) were added and the reaction mass stirred for 5-10 min at room temperature. After 10 min, 5-(2,4- dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(piperazin-l-ylmethyl)phenyl)-4H- 1,2,4- triazole-3 -carboxamide (TVD-0003510) (202 mg, 0.435 mmol, 1.0 eq) was added and then reaction mixture was stirred at room temperature for 18h. After completion of SM, as monitored by TLC. Then reaction quenched with ice-water (10 mL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (5 mL) dried under vacuum to afford crude compound .The crude was purified by prep HPLC & Lyophilized to afford 4-(2-(4-(4-(3-(2,4-dihydroxy-5- isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)-2- oxoethyl)- 3 -fluorophenyl 4-(3-((6-(2-aminopyrimidine-5-carboxamido)-8-methoxy- 3, 4-dihydro-2H-pyrimido[ 1, 2-c ]quinazolin-9-yl)oxy)propyl)piperazine-l-carboxylate (C55) (55.5mg, 11.23 % yield) as off white solid.
[0374] 'H-NMR: (400 MHz, DMSO-a ) 8: 14.16 (s, 1H), 11.44 (s, 1H), 10.95- 10.84 (m, 2H), 10.14 (bs, 1H), 9.74 (s, 1H), 9.01 (s, 3H), 8.31 (d, J = 8.0 Hz, 1H), 7.60-7.55 (m, 4H), 7.42 (d, J = 8.0 Hz, 3H), 7.29-7.24 (m, 1H), 7.11 -7.07 (m, 1H), 6.98(t, J = 4.0 Hz, 1H), 6.68 (s, 1H), 6.33 (s, 1H), 4.40-4.22 (m, 10H), 4.01 (s, 3H), 3.80 (bs, 3H), 3.65-3.50 (m, 6H), 3.21-2.92 (m, 9H), 2.32 (s, 2H), 2.18 (bs, 2H), 1.05 (t, J = 8.0 Hz, 3H), 0.88 (d, J = 8.0 Hz, 6H).
LC-MS: 1135.93 (M + H). C58
Figure imgf000160_0001
[0375] Synthesis of 5-(2, 4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-((4-(2- hydroxy ethyl) piperazin- 1-yl) methyl) phenyl)-4H-l, 2, 4-triazole-3 -carboxamide (Int-1):
To a stirred solution of 5-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-N-ethyl-4-{4- [(piperazin- 1 -yl)methyl]phenyl } -4H- 1 ,2,4-triazole-3 -carboxamide (TVD-0003510) (500 mg, 1.08 mmol, 1.0 eq) in anhydrous DMF (5.0 mL) was added 2-bromoethan-l- ol (1.34 g, 10.8 mmol, 10.0 eq), followed by dipotassium carbonate (595 mg, 4.31 mmol, 4.0 eq) at RT. The resulting reaction mixture was heated for 12h at 70° C. Progress of the reaction mixture monitored by TLC and LCMS. After completion of the SM, reaction mixture diluted with EtOAc and extracted with water. The combined organic layers washed with NaCl and dried over sodium sulphate, filtered, and concentrated to afford crude compound 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl- 4-(4-((4-(2-hydroxyethyl)piperazin-l-yl) methyl)phenyl)-4H-l,2,4-triazole-3- carboxamide (Int-1) (250 mg, 45.67% yield) as off white solid. The crude compound was used for next step without any further purification.
[0376] Synthesis of 4-(4-((4-(2-chloroethyl) piperazin- 1-yl) methyl) phenyl)-5-(2, 4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-l, 2, 4-triazole-3 -carboxamide (Int-2): To a stirred solution of 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-((4-(2- hydroxyethyl)piperazin- 1 -yl)methyl)phenyl)-4H- 1 ,2,4-triazole-3 -carboxamide (Int- 1 ) (500 mg 0.983 mmol, 1.0 eq) in DCM (4.0 mL) was added SOCh (1.17 g, 9.83 mmol, 10.0 eq) and DMF (7.19 mg, 0.098 mmol, 0.1 eq) at 0°C. The resulting reaction mixture was stirred for 18h at room temperature. Progress of the reaction was monitor by TLC and LC-MS. After completion of SM, all volatiles were evaporated and extorted with by using 20% NaHCOs and EtOAC (3 X 30 mL). The combined organic layers dried over Na2SO4 and filtered and concentrated to afford crude. Crude compound was purified by column chromatography 100-200 silica gel mesh, eluted with 20-40% Acetone in PE. All pure fractions collected and concentrated to afford 4- (4-((4-(2-chloroethyl) piperazin- l-yl)methyl)phenyl)-5-(2, 4-dihydroxy-5- isopropylphenyl)-N-ethyl-4H-l,2,4-triazole-3-carboxamide (Int-2) (200 mg, 38.6% yield) as off white solid.
[0377] Synthesis of 2-amino-N-(8-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)- 5-(ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)ethoxy)-7-methoxy- 2,3-dihydroimidazo[l,2-c]quinazolin-5-yl)pyrimidine-5-carboxamide (TVD- 0004567):
To a stirred solution of 2-amino-N-{8-hydroxy-7-methoxy-2H,3H-imidazo[l,2- c]quinazolin-5-yl}pyrimidine-5-carboxamide (T-1838) (200 mg, 0.566 mmol, 1.0 eq) in anhydrous DMF (5.0 mL) was added dipotassium carbonate (313 mg, 2.26 mmol, 4.0 eq) at RT. After 10 min, was added 4-(4-{[4-(2-chloroethyl)piperazin-l- yl]methyl}phenyl)-5-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-N-ethyl-4H-l,2,4- triazole-3 -carboxamide (Int-2) (298 mg, 0.566 mmol, 1.0 eq) added at RT. The resulting reaction mixture was heated for 12h at 50°C. Progress of the reaction mixture monitored by TLC and LCMS. After completion of SM, reaction mixture was quenched with ice water and resulting solid was collected by filtration. Crude was purified by prep HPLC followed by lyophilized to afford 2-amino-N-(8-(2-(4-(4-(3- (2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)ethoxy)-7-methoxy-2,3-dihydroimidazo[l,2-c]quinazolin-5- yl)pyrimidine-5-carboxamide (TVD-0004567) (80 mg, 16.75 % yield) as off white solid.
[0378] XH NMR: (400 MHz, DMSO-a ) 8: 13.45 (s, 1H), 12.50 (bs, 1H), 10.59 (s, 1H), 9.76 (s, 1H), 9.01-8.97 (m, 3H), 8.19 (d, J= 12.0 Hz, 2H), 7.55 (bs, 2H), 7.46- 7.39 (m, 4H), 6.63 (s, 1H), 6.34 (s, 1H), 4.50 (t, J= 8.0 Hz, 4H), 4.21 (t, J= 8.0 Hz, 4H), 4.00 (s, 3H), 3.64-2.91 (m, merged with water, 10H), 1.23 (s, 2H), 1.04 (t, J= 8.0 Hz, 3H), 0.85 (d, J= 8.0 Hz, 6H). LC-MS: 844.44 (M + H).
Conjugates C117, C118, C119
[0379] Non-limiting examples of synthetic methods for Conjugates Cl 17-C119 are included below.
C118
Figure imgf000162_0001
[0380] Synthesis of tert-butyl 2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)acetate (Int-2):
To a suspension of 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(piperazin-l- ylmethyl)phenyl)-4H-l, 2, 4-triazole-3 -carboxamide (TVD-0003510) (75 mg, 0.161 mmol, 1.0 eq) in THF (1.0 mL), TEA (0.022 mL, 0.161 mmol, 1.0 eq) followed by tert-butyl 2-bromoacetate (Int-1) (31.5 mg, 0.161 mmol, 1.0 eq) were added at RT. The reaction mixture was slowly heated to 75-80°C and stirred for 3h at the same temperature. Progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, reaction mass diluted with water (1 mL) and extracted with ethyl acetate (2 X 5 mL). Combined organic layers dried over sodium sulphate, filtered, evaporated the solvent under vaccue to afford (90 mg, crude). The crude compound was purified with column chromatography by using 100-200 mesh size silica, eluting at 2-3% Methanol in DCM to afford tert-butyl 2-(4-(4-(3-(2,4- dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl)benzyl)piperazin-l-yl)acetate (Int-2) (75 mg, 80.2%) as an off white solid. [0381] Synthesis of 2-(4-(4-(3-(2, 4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l, 2, 4-triazol-4-yl) benzyl) piperazin- 1-yl) acetic acid (Int-3): To a stirred solution of tert-butyl 2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l,2,4-triazol-4-yl)benzyl)piperazin-l-yl)acetate (Int-2) (75 mg, 0.130 mmol, 1.0 eq) in DCM (1.5 mL), TFA (0.14 mL, 1.94 mmol, 15.0 eq) was added at room temperature. The resulting reaction mixture was stirred for overnight at room temperature. Progress of the monitored by TLC and LC-MS. After completion the SM, DCM was removed under reduced pressure and co-distilled with toluene to get crude compound (80 mg crude). The crude compound was triturated with diethyl ether (2.0 mL) to afford (Int-3) 2-(4-(4-(3-(2, 4-dihydroxy-5-isopropylphenyl)-5- (ethylcarbamoyl)-4H-l, 2, 4-triazol-4-yl) benzyl) piperazin- 1-yl) acetic acid (70 mg, Quantitative) as an off white solid. This crude itself used for the next step without further purification.
[0382] Synthesis of (S)-4-(4-((4-(2-((6-amino-5-((2-(l-isopropyl-lH-l,2,4-triazol- 5-yl)-5,6-dihydrobenzo[f]imidazo[l,2-d][l,4]oxazepin-9-yl)amino)-6- oxohexyl)amino)-2-oxoethyl)piperazin-l-yl)methyl)phenyl)-5-(2,4-dihydroxy-5- isopropylphenyl)-N-ethyl-4H-l,2,4-triazole-3-carboxamide (Cl 18):
To a stirred solution of 2-{4-[(4-{3-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-5- (ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl }phenyl)methyl]piperazin- 1 -yl } acetic acid (Int-3) (40 mg, 0.076 mmol, l.Oeq) in DMF (0.5 mL), DIPEA (0.04 mL, 0.0230 mmol, 3.0 eq) followed by HATU (43.7 mg, 0.115 mmol, 1.5 eq) were added and the reaction stirred for 5-10 min at rt and then (R)-6-amino-2-((2-(l-isopropyl-lH-l,2,4- triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[l,2-d][l,4]oxazepin-9- yl)amino)hexanamide (TVD-0004440) (40.3 mg, 0.091 mmol, 1.2 eq) was added and the resulting reaction mixture was stirred at room temperature for 18h. After completion of SM (monitored by TLC) reaction mass quenched with ice-water (ImL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (2 mL) dried under vacuum to afford crude compound .The crude was purified by prep HPLC & Lyophilized to afford (Cl 18) (S)-4-(4-((4-(2-((6- amino-5-((2-(l-isopropyl-lH-l,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[l,2- d] [ 1 ,4]oxazepin-9-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)piperazin- 1 - yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-l,2,4-triazole-3- carboxamide (6.0 mg, 5.54%) as off white solid.
[0383] ’H-NMR: (500 MHz, DMSO-de) 6: 10.58 (s, 1H), 9.75 (s, 1H), 8.94 (s, 1H), 8.19 (bs, 1H), 8.09 (d, J = 10.0 Hz, 1H), 7.86 (s, 1H), 7.75 (s, 1H), 7.67 (t, J = 10.0 Hz , 1H), 7.40 (bs, 1H), 7.35 (d, J = 10.0 Hz, 2H), 7.28 (d, J = 10.0 Hz, 2H), 7.02 (s, 1H), 6.56 (s, 1H), 6.46 (dd, J = 10.0, 2.0 Hz, 1H) ,6.33 (s, 1H), 6.16- 6.15 (m, 2H), 5.90 (quint, J = 10.0 Hz, 1H), 4.41-4.39 (m, 4H), 3.69-3.68 (m, 1H), 3.47 (s, 2H), 3.31-3.07 (m, 4H), 2.90-2.86 (m, 3H), 2.43 (bs, 8H), 1.67-1.65 (m, 2H), 1.46-1.42 (m, 9H), 1.03 (t, J = 10.0 Hz, 3H), 0.79 (d, J = 10.0 Hz, 6H).
LC-MS: 943.65 (M + H).
C119
Figure imgf000164_0001
Figure imgf000164_0002
[0384] Synthesis of (S)-6-(2-((R)-2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)acetamido)-2-((2-(l-isopropyl-lH-l,2,4-triazol-5-yl)-5,6- dihydrobenzo[f]imidazo[ 1 ,2-d] [ 1 ,4]oxazepin-9-yl)amino)hexanamide(C 119): To a stirred solution of (R)-2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin- 2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetic acid (TVD- 0004473) (25.8 mg, 0.068 mmol, 1.5 eq) was dissolved in DMF (1.0 mL), DIPEA (23.6 mg, 0.182 mmol, 4.0 eq) followed by HATU (16.1 mg, 0.068 mmol, 1.5 eq) were added at RT. The resulting reaction mixture stirred for 20 min at RT. After 20 min, was added (S)-6-amino-2-((2-(l-isopropyl-lH-l,2,4-triazol-5-yl)-5,6- dihydrobenzo[f]imidazo[ 1 ,2-d] [ 1 ,4]oxazepin-9-yl)amino) hexanamide (TVD-
0004473) (20 mg, 0.045 mmol, 1.0 eq) at RT. Progress of the reaction was monitor by TLC & LCMS. The resulting reaction mixture was stirred at 80°C for 4h. After completion of SM, reaction mass quenched with ice-water (20 mL), the resulting precipitated was filtered and washed with ice water, then dried to afford crude. Crude compound was purified by prep .HPLC followed by lyophilization to afford (TVD- 0004527) (S)-6-(2-((R)-2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2- yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetamido)-2-((2-(l- isopropyl-lH-l,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[l,2-d][l,4]oxazepin-9- yl)amino)hexanamide (9.64 mg, 30.21%) as an off white solid.
[0385] ’H NMR: (500 MHz, DMSO-de) 6: 8.35 (bs, 1H), 8.06 (d, J = 5.0 Hz, 2H), 8.03-7.99 (m, 1H), 7.91 (s, 1H), 7.79 (s, 1H), 7.40 (bs, 1H), 7.04 (bs, 1H), 6.63 (bs, 2H), 6.46 (dd, J = 5.0 Hz, 1H), 6.15 (d, J = 5.0 Hz, 2H), 5.85 (quint, J = 5.0 Hz, 1H), 4.70 (bs, 2H), 4.43-4.40 (m, 4H), 4.03 (s, 3H), 3.89-3.55 (m, 2H), 3.55-3.51 (m, 1H), 3.30-3.27 (m, 1H), 3.03-3.01 (bs, 2H ), 2.73-2.69 (m, 1H), 2.36 (s, 3H), 2.21 (s, 3H), 1.64 (bs, 2H), 1.47 (d, J = 6.0 Hz, 6H), 1.38 (bs, 4H).
LC-MS: 798.45 (M + H).
Conjugates C39-C47, C87-C116 and C120
[0386] Non-limiting examples of synthetic methods for Conjugates C39-C47, C87-C116 and C120 are included below.
C90
Figure imgf000165_0001
[0387] Synthesis of tert-butyl (R)-2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl)piperazin- 1 -yl)acetate (Int-2) :
To a suspension of (R)-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-5- (2-(piperazin-l-yl)ethyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-amine (TVD- 0004553) (130 mg, 0.301 mmol, 1.0 eq) in THF (5.0 mL), TEA (30.50 mg, 0.301 mmol, 1.0 eq) followed by tert-butyl 2-bromoacetate (58.70 mg, 0.301 mmol, 1.0 eq) were added at RT. The reaction mixture was stirred at 75-80°C. Progress of the reaction was monitored by TLC and LC-MS. After completion of SM, volatiles were evaporated and extracted with ethyl acetate (80 mL) washed with water (30 mL). The combined organic layers dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude compound. The crude compound was purified by flash column chromatography, desired product eluting at 2-3% Methanol in DCM to afford tert-butyl (R)-2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyri din-2 - yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)piperazin-l-yl)acetate (Int-2) (120 mg, 73.01% yield) as light brown solid.
[0388] Synthesis of (R)-2-(4-(2-(2-amino-4-chloro-7-((4-m ethoxy-3, 5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl)piperazin-l-yl)acetic acid (Int-3):
To stirred solution of tert-butyl (R)-2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl)piperazin-l-yl)acetate (Int-2) (120 mg, 0.220 mmol, 1.0 eq) in DCM (4.0 mL) was added TFA (0.33 mL, 4.39 mmol, 20.0 eq) were added at room temperature. The resulting reaction mixture was stirred for overnight at room temperature and monitored by TLC and LC-MS. After completion of SM, all volatilise was removed under reduced pressure and co-distilled with toluene to get crude compound which was then lyophilized to afford (R)-2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl)piperazin-l-yl)acetic acid (Int-3) (150 mg, quantitative) as light brown solid. [0389] Synthesis of (R)-l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino- 1,3,5 -triazin-2-yl)piperazin- 1 -yl)-2-(4-(2-(2-amino-4-chloro-7-((4- methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 5-yl)ethyl)piperazin- 1 -yl)ethan- 1 -one (TVD-0004589) : To a stir solution of (R)-2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl)piperazin-l-yl)acetic acid (Int-3) (110 mg, 0.224 mmol, l.Oeq) in DMF (1.0 mL) was added DIPEA (0.19 mL, 1.12 mmol, 5.0 eq) followed by HOBT (60.70 mg, 0.449 mmol, 2.0 eq), EDC.HC1 (86.10 mg, 0.449 mmol) and the reaction was stirred for 5-10 min at room temperature. After 10 min, was added 4-(difluoromethyl)-5-(4- morpholino-6-(piperazin-l-yl)-l, 3, 5-triazin-2-yl) pyrimidin-2-amine (TVD- 0004425) (61.80 mg, 0.157 mmol, 0.70 eq) and then reaction stir at room temperature for 18h. After completion of SM, as monitored by TLC. Then reaction quenched with ice-water (5mL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (5 mL) dried under vacuum to afford crude compound .The crude was purified by prep HPLC & Lyophilized to afford (R)-l-(4- (4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-l, 3, 5-tri azin-2- yl)piperazin-l-yl)-2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyri din-2- yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)piperazin-l-yl)ethan- 1-one (C90) (62.0 mg, 31.91% yield) as off white solid.
[0390] XH-NMR: (400 MHz, DMSO-de) 6: 9.11 (s, 1H), 8.29 (s, 1H), 7.63 (t, J = 52.0 Hz, 1H), 7.57 (bs, 2H) 6.65 (bs, 2H), 4.65 (s, 2H), 4.0-3.77 (m, 13H), 3.65 (bs, 6H), 3.54 (bs, 5H), 3.42 (bs, 2H), 3.35-3.32 (m, 2H), 3.27-2.93 (m, 6H), 2.32 (s, 3H), 2.26 (s, 3H), 2.08 (bs, 1H), 1.91 (bs, 1H).
LC-MS: 865.23 (M + H).
C91
Figure imgf000167_0001
Figure imgf000167_0002
[0391] Synthesis of 5-[4-(4-{2-[(5S)-2-amino-4-chloro-7-[(4-methoxy-3,5- dimethylpyridin-2-yl)methyl]-5H,6H,7H-pyrrolo[2,3-d]pyrimidin-5- yl]ethyl}piperazin-l-yl)-6-(morpholin-4-yl)-l,3,5-triazin-2-yl]-4- (difluoromethyl)pyrimidin-2-amine (TVD-0004583):
To a stirred solution of (S)-2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin- 2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetaldehyde (Int-1) (200 mg , 0.553 mmol, 1.0 eq ) in DCE (5.0 mL) was added 4-(difluoromethyl)-5-(4- morpholino-6-(piperazin-l-yl)-l,3,5-triazin-2-yl)pyrimidin-2-amine (TVD-0004425) (174 mg, 0.442 mmol, 0.8 eq) and acetic acid (3.32 mg, 0.0553 mmol, 0.1 eq) and the reaction mixture was stirred at room temperature for Ih. After Ih, Sodium triacetoxyborohydride (363 mg, 1.66 mmol, 3.0 eq) was added and resulting reaction mixture allow to stir at room temperature for 16h. Progress of the reaction was monitor by TLC and LCMS. After completion of SM, reaction mixture was quenched with saturated solution of NaHCOs and extracted with DCM (3 x 50 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude, this crude was purified by Prep.HPLC followed by lyophilized to afford (S)-5-(2-(4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazin- 1 - yl)ethyl)-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2-amine (TVD-0004583) (91 mg, 22.27%) as off white solid. [0392] ’H NMR: ( DMSO, 400 MHz) 8: 9.79 (bs, IH), 9.13 (s, IH), 8.27 (s, IH), 7.61 (t, J = 52.0 Hz, IH), 7.60 (bs, 2H, merged with triplet), 6.63 (bs, 2H), 4.76 (bs, 2H), 4.64 (s, 2H), 3.81-3.78 (m, 8H), 3.65 (bs, 5H), 3.65 (bs, 2H), 3.38-3.32 (m, 6H), 3.06-3.04(m, 2H), 2.24 (s, 3H), 2.20 (s, 3H).
LC-MS: 739.26 (M+H)
C92
Figure imgf000169_0001
Figure imgf000169_0002
[0393] Synthesis of 4-(2-(tert-butoxy)-2-oxoethyl) phenyl4-(4-(2-amino-4- (difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperazine-1- carb oxy late (Int-2):
To a stirred solution of Methyl 2-(4-hydroxyphenyl) acetate (Int-1) (150 mg, 0.720 mmol, 1.0 eq) in DCM (2.0 mL), DIPEA (279 mg, 2.16 mmol, 3.0 eq) followed by 4- nitrophenyl carb onochlori date (160 mg, 0.792 mmol, 1.1 eq) were added at RT. The resulting reaction mixture was stirred for 2h at RT. after consumption of SM (conformed by TLC) a solution of (TVD-0004425) (255 mg, 0.648 mmol, 0.9 eq) dissolved in DMF (1.0 mL) was added at RT. The resulting reaction mixture was stirred for 16 h at RT. Progress of the reaction mixture monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was quenched with ice water (100 mL) and extracted with DCM (2 x50 mL) washed with Brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure to get the crude compound. To the crude compound MTBE (2 mL) added and stirred for 20 min, filtered to get pure 4-(2-(tert-butoxy)-2-oxoethyl) phenyl4-(4-(2-amino-4- (difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperazine-1- carboxylate (Int-2) (250 mg, 59.2%) off white solid.
[0394] Synthesis of 2-(4-((4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-1, 3, 5-triazin-2-yl) piperazine- 1 -carbonyl) oxy) phenyl) acetic acid (Int- 3):
To stirred solution of 4-(2-methoxy-2-oxoethyl)phenyl 4-{4-[2-amino-4- (difluoromethyl)pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-tri azin-2 -yl (piperazine- 1- carboxylate (Int-2) (250 mg, 0.4271 mmol, 1 eq) in DCM (4.0 mL), TFA (1.46g, 12.8 mmol, 30.0 eq) was added at 0°C. The resulting reaction mixture was stirred for 6h at RT. Progress of reaction mixture monitored by LCMS. After completion of the reaction, the reaction was volatile evaporated under reduced pressure to get crude compound. The crude compound was by lyophilisation to afford 2-(4-((4-(4-(2- amino-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperazine- 1 -carbonyl) oxy) phenyl) acetic acid (Int-3) (130 mg, 53.27% yield) off white solid. This crude itself used for the next step with out purification.
[0395] Synthesis of (R)-4-(2-(4-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl)piperazin- 1 -yl)-2-oxoethyl)phenyl4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazine- 1 - carboxylate (TVD-0004566):
To a stirred solution of 2-[4-(4-{4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6- (morpholin-4-yl)-l, 3, 5 -triazin-2 -yl( piperazine- 1 -carbonyloxy) phenyl] acetic acid (Int-3) (100 mg, 0.125 mmol, 1.0 eq) dissolved in DMF (1.0 mL), DIPEA (80.9 mg, 0.6 mmol, 5.0 eq) followed by EDC.HC1 (48 mg, 0.25 mmol, 2.0 eq) and HOBt (38.1 mg, 0.25 mmol, 2.0 eq) were added at RT. The resulting reaction mixture was stirred for 16h at RT. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction mixture, the reaction mixture was quenched with ice water (50 mL) stirred for 20 min solid precipitated filtered and washed with ice water (20 mL) dried under vacuum to get solid crude 150 mg. The crude compound was purified by Prep HPLC followed by lyophilized to afford (R)-4-(2-(4-(2-(2-amino-4- chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl)piperazin-l-yl)-2-oxoethyl)phenyl4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazine- 1 - carboxylate (TVD-0004566) (52 mg, 43.59 % yield) off white solid.
[0396] ’H NMR: (400 MHz, DMSO-de) 69.80 (bs, 1H), 9.12 (s, 1H), 8.28 (s, 1H), 7.64 (t, J = 52.0 Hz, 1H), 7.58 (bs, 2H), 7.22 (d, J = 12.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H) 6.64 (bs, 2H), 4.64 (s, 2H), 4.43 (bs, 2H), 3.89 (s, 4H), 3.81-3.77 (m, 10H), 3.67- 3.62 (m, 6H), 3.52 (bs, 4H), 3.35 (s, 2H), 3.26-3.22 (m, 5H), 2.26 (s, 3H), 2.21 (s, 3H), 2.10 (s, 1H), 2.0 (s, 1H).
LC-MS: 985.27 (M + H).
C93
Figure imgf000171_0001
[0397] Synthesis of tert-butyl (R)-2-(4-(3-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl)piperazin- 1 -yl)acetate (Int-2) :
To a suspension of (R)-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-5- (3-(piperazin-l-yl)propyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-amine (TVD- 0004554) (100 mg, 0.224 mmol, 1.0 eq) in THF (3.0 mL), TEA (22.70 mg, 0.224 mmol, 1.0 eq) was added at room temperature. After 30 min, tert-butyl 2- bromoacetate (Int-1) (43.70 mg, 0.224 mmol, 1.0 eq) was added at RT. The reaction mixture was stirred at such a rate as to maintain a gentle reflux of the solvent for 3h. Progress of the reaction was monitored by TLC and LC-MS. After completion of SM, the reaction was quenched in water (20 mL) and extracted with (2 x 50 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to obtain crude. The crude was purified by combi-flash, the desired product was eluted with 0-3% MeOH in DCM, all the pure fractions were collected and concentrated under reduced pressure to afford tert-butyl (R)-2-(4-(3-(2- amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-5-yl)propyl)piperazin-l-yl)acetate (Int-2) (75 mg, 59.72% yield) as a thick yellow oil.
[0398] Synthesis of (R)-2-(4-(3-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl)piperazin-l-yl)acetic acid (Int-3):
To a stirred solution of tert-butyl (R)-2-(4-(3-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl)piperazin-l-yl)acetate (Int-2) (50 mg ,0.089 mmol, 1.0 eq) in DCM (3.0 mL), TFA (0.137 mL, 1.79 mmol, 20.0 eq) was added at room temperature. The resulting, the reaction mixture was stirred overnight at room temperature. Progress of the reaction was monitored by TLC and LC-MS. After completion of SM, all volatiles were evaporated under reduced pressure and co-distilled with toluene to get desired product which was then lyophilized to afford (R)-2-(4-(3-(2-amino-4-chloro-7-((4- methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 5-yl)propyl)piperazin-l-yl)acetic acid (Int-3) (65 mg TFA salt, Quantitative) as a sticky light yellow oil.
[0399] Synthesis of (R)-l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino- 1,3,5 -triazin-2-yl)piperazin- 1 -yl)-2-(4-(3 -(2-amino-4-chloro-7-((4- methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 5 -yl)propyl)piperazin- 1 -yl)ethan- 1 -one (T VD-0004561 ) :
To a stirred solution of (R)-2-(4-(3-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl)piperazin-l-yl)acetic acid (Int-3) (50 mg, 0.099 mmol, l.Oeq) in DMF (0.5 mL) was added DIPEA (0.087 mL, 0.49 mmol, 5.0 eq) followed by HATU (47.10 mg, 0.124 mmol, 1.25 eq) and the reaction was stirred for 5-10 min at room temperature. After 10 min, was added 4-(difluoromethyl)-5-(4-morpholino-6- (piperazin-l-yl)-l, 3, 5-triazin-2-yl) pyrimidin-2-amine (TVD-0004425) (39.0 mg, 0.099 mmol, 1.0 eq) and stirred at room temperature for 18h. Progress of the reaction was monitored by TLC and LCMS. After completion of reaction, the reaction was quenched in ice-water (5.0 mL) stirred for 5 min, the resulting precipitated was filtered and washed with water followed by DEE (5.0 mL) dried under vacuum to afford crude compound . The crude compound was purified by prep HPLC & Lyophilized to afford (R)-l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino- 1,3,5 -triazin-2-yl)piperazin- 1 -yl)-2-(4-(3 -(2-amino-4-chloro-7-((4- methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 5-yl)propyl)piperazin-l-yl)ethan-l-one (TVD-0004561) (7.0 mg, 8.04%) as off white solid.
[0400] ’H-NMR: (400 MHz, DMSO-de) 6: 9.11 (s, 1H), 8.26 (s, 1H), 7.63 (t, J = 54.0 Hz, 1H), 7.57 (bs, 2H), 6.59 (bs, 2H), 4.63 (s, 2H), 3.96-3.77 (m, 14H), 3.66- 3.62 (m, 8H), 3.54 (s, 6H), 3.29-3.03 (m, 6H), 2.24 (s, 3H), 2.20 (s, 3H), 1.76 (bs, 1H), 1.56 (bs, 3H).
LC-MS: 879.26 (M + H).
C95
Figure imgf000173_0001
[0401] Synthesis of (R)-5-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino- 1,3,5 -triazin-2-yl)piperazin- 1 -yl)propyl)-4-chloro-7-((4-m ethoxy-3 , 5 - dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-amine (TVD-0004557): To a stirred solution of (R)-3-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin- 2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)propanal (Int-1) (90 mg, 0.239 mmol, 1.0 eq) in 1,2-di chloroethane (2.0 mL), 4-(difluoromethyl)-5-(4- morpholino-6-(piperazin-l-yl)-l,3,5-triazin-2-yl)pyrimidin-2-amine (TVD-0004425) (94.2 mg, 0.239 mmol, 1.0 eq) and acetic acid (1.44 mg, 0.23.9 mmol, 0.1 eq) were added at RT. After 30 min. sodium triacetoxybohydride (152 mg, 0.718 mmol, 3 eq) was added. The resulting reaction mixture was stirred at RT for 6h. Progress of reaction was monitored by LCMS and TLC. After completion of SM, reaction was quenched with water and resulting solid was collected by filtration. The crude was purified by prep. HPLC followed by lyophilized to afford (R)-5-(3-(4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazin- 1 - yl)propyl)-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2-amine (TVD-0004557) (56.0 mg, 31% yield) as an off white solid.
[0402] 'H NMR: (500 MHz, DMSO-de) 6: 9.65 (s, 1H), 9.13 (s, 1H), 8.28 (s, 1H), 7.61 (t, J = 54.0 Hz, 1H), 7.61 (bs, 2H), 6.61 (s, 2H), 4.74 (bs, 2H), 4.65 (s, 2H), 3.82- 3.78 (m, 6H), 3.68-3.66 (m, 5H), 3.51 (m, 2H), 3.30-3.21 (m, 4H), 3.16-3.02 (m, 4H), 2.25 (s, 3H), 2.21 (s, 3H), 1.71-1.59 (m, 4H).
LC-MS: 753.42 (M + H).
C98
Figure imgf000174_0001
[0403] Synthesis of tert-butyl (2-(2-hydroxyethoxy) ethyl) carbamate (Int-2):
To a stirred solution of tert-butyl (2-(2 -hydroxy ethoxy)ethyl)carbamate (Int-1) (1.50 g, 14.3 mmol, 1.0 eq) in DCM (15 mL) was added di-tert-butyl dicarbonate (3.11 g, 14.3 mmol, 1.0 eq) and stirred at room temperature for 18h. Progress of the reaction was monitor by TLC & LCMC. After completion of SM, the reaction mixture was concentrated under reduced pressure to get crude. The crude was purified by combi- flash, desired product eluted with 0-70% Ethyl acetate in pet ether solvent system, all pure fractions were collected and concentrated under reduced pressure to afford tertbutyl (2-(2-hydroxyethoxy)ethyl)carbamate (Int-2) (1.5 g, 51.22% yield) as color less oil.
[0404] Synthesis of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-(4-(2-amino- 4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-l,3,5-triazin-2-yl)piperazine-l- carb oxy 1 ate(Int-3 ) :
To a stirred solution of tert-butyl (2-(2 -hydroxy ethoxy)ethyl)carbamate (Int-2) (250 mg, 1.22 mmol, 1.0 eq) in DCM (4 mL), TEA (0.425 mL, 3.05 mmol, 2.5 eq) followed by 4-nitrophenyl carbonochloridate (246 mg, 1.22 mmol, 1.0 eq) were added at 0°C. The resulting reaction mixture was warm to RT and stirred for Ih. Progress of intermediate formation was monitored by TLC. After consumption of SM, 4- (difluoromethyl)-5-(4-morpholino-6-(piperazin-l-yl)-l, 3, 5-triazin-2-yl) pyrimidineamine (TVD-0004425) (479 mg, 1.22 mmol, 1.0 eq) in DMF (4.0 mL) was added and the resulting reaction mixture was stirred at RT for 2h. After consumption of SM, reaction mixture diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). Combined organic layers was washed with cold brine (100 mL). The organic layer was dried over sodium sulphate, filtered and evaporated. Crude compound was triturated with diethyl ether (10.0 mL), solid obtained was filtered dried well under vacuum to afford 2-(2-((tert-butoxycarbonyl) amino) ethoxy) ethyl 4-(4-(2-amino-4- (difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperazine-1- carboxylate (Int-3) (600 mg, 78.86% yield) as off white solid.
[0405] Synthesis of 2-(2-aminoethoxy) ethyl 4-(4-(2-amino-4-(difluorom ethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperazine- 1 -carboxylate (Int-4): To a stirred solution of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-(4-(2-amino- 4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-l,3,5-triazin-2-yl)piperazine-l- carboxylate (Int-3) (150 mg, 0.22 mmol, 1.0 eq) in DCM (8.0 mL), TFA (0.092 mL, 1.20 mmol, 5.0 eq) was added at 0°C. The resulting reaction mixture was warm to RT and stirred for 2h at same temperature. Progress of the reaction was monitor by TLC & LCMS. After consumption of SM, all volatiles were concentrated under reduced pressure to afford crude 2-(2-aminoethoxy)ethyl 4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazine- 1 - carboxylate (Int-4) . (190 mg, as TFA salt, quantitative yield) as a thick colourless oil. The crude compound was used for next step without any further purification.
[0406] Synthesis of (R)-2-(2-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)acetamido)ethoxy)ethyl 4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-l,3,5-triazin-2-yl)piperazine-l-carboxylate(TVD-0004537): To a stirred of (R)-2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2- yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetic acid (TVD-0004473) (100 mg, 0.265 mmol, l.Oeq) in DMF (2.0 mL), DIPEA (0.14 mL, 0.793 mmol, 3.0 eq) followed by HATU (151 mg, 0.397 mmol, 1.5 eq) were added at RT and the reaction mass was stirred for 5-10 min at room temperature and 2-(2- aminoethoxy)ethyl 4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- l,3,5-triazin-2-yl)piperazine-l-carboxylate (Int-4) (167 mg, 0.318 mmol, 1.2 eq) was added at RT and the reaction continue to stir at room temperature for 18h. After completion of SM, as monitored by TLC & LCMS. Reaction mixture was quenched in ice-water (10 mL) and stirred for 5 min, the resulting precipitated was filtered and washed with water followed by diethyl ether (10 mL), dried under vacuum to afford crude compound. The crude was purified by Prep.HPLC and lyophilized to afford (R)-2-(2-(2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7- dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetamido)ethoxy)ethyl 4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazine- 1 - carboxylate (C98) (74 mg, 31.62% yield) as off white solid.
[0407] ’H-NMR: (400 MHz, DMSO-de) 8: 9.10 (s, 1H), 8.13 (s, 1H), 7.98 (t, J = 8.0 Hz, 1H), 7.62 (t, J = 52.0 Hz, 1H), 7.57 (bs, 2H), 6.40 (s, 2H), 4.54 (s, 2H), 4.11 (d, J = 4.4 Hz, 2H), 3.75-3.56 (m, 16H), 3.44-3.39 (m, 8H), 3.18-3.12 (m, 4H), 2.66 (d, J = 2.8 Hz , 1H), 2.21-2.2 (m, 1H), 2.15 (d, J = 8.0 Hz, 6H).
LC-MS: 884.43 (M + H). C101
Figure imgf000177_0001
Figure imgf000177_0002
[0408] Synthesis of l-{4-[2-amino-4-(difluorom ethyl) pyrimidin-5-yl]-6- (morpholin-4-yl)-l, 3, 5-triazin-2-yl} piperidin-4-one (Int-1):
To a solution of l-{4-[2-amino-4-(difluorom ethyl) pyrimidin-5-yl]-6-(morpholin-4- yl)-l, 3, 5-triazin-2-yl} piperidin-4-ol (TVD-0004426) (200 mg, 0.48 mmol, 1.0 eq) in DCM (15 mL), Dess-Martin periodinane) (312 mg, 0.73 mmol, 1.5 eq) was added portion wise at 0°C. The resulting reaction mixture was slowly warm to RT and stirred for 18h at same temperature. Progress of the reaction was monitor by TLC. After complete conversion of SM, the reaction mixture quenched with water ( 10 mL) and adjusted pH- 8 with saturated NaHCOs and extracted DCM (3 x 15 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude. The crude compound was purified by column chromatography using silica gel (100-200 mesh). Compound eluted using 5-6% MeOH in DCM. All pure fractions were mixed and concentrated to afford l-{4-[2- amino-4-(difluoromethyl) pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-triazin-2-yl} piperidin-4-one (Int-1) (200 mg, 99%) as light-yellow solid.
[0409] Synthesis of 4-(difluoromethyl)-5-[4-(morpholin-4-yl)-6-[4-({2-[2-(prop-2- yn-l-yloxy)ethoxy] ethyl} amino) piperidin- 1 -yl]- 1 , 3, 5-triazin-2-yl] pyrimidineamine (Int-3): To a stirred solution of l-{4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6- (morpholin-4-yl)-l,3,5-triazin-2-yl}piperidin-4-one (200 mg , 0.49 mmol, 1.0 eq) in DCM ( 10.0 mL), 3-[2-(2-aminoethoxy)ethoxy]prop-l-yne (70.5 mg, 0.49 mmol, 1.0 eq) followed by Sodium triacetoxyborohydride (114 mg, 0.54 mmol , 1.1 eq) were added portion wise at 0°C. The resulting reaction mixture allowed room temperature for 18h. After completion of SM, reaction mass quenched with water (5 mL) and extracted with DCM (3 x 15 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude, the crude compound was purified by column chromatography using silica gel (100-200 mesh), compound eluted at 2- 5 % MeOH in DCM. All pure fraction were mixed and concentrated to afford 4-(difluoromethyl)-5-[4-(morpholin-4-yl)-6-[4-({2-[2-(prop-2- yn-l-yloxy)ethoxy]ethyl}amino)piperi din-1 -yl]-l , 3, 5-tri azin-2 -yl]pyrimidin-2-amine (Int-2) (160 mg, 60.93%) as a light yellow solid.
[0410] Synthesis of 5-(4-{4-[(2-{2-[(3-{2-amino-4-chloro-7-[(4-methoxy-3, 5- dimethylpyridin-2-yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}prop-2-yn-l- yl)oxy]ethoxy}ethyl)amino]piperi din- l-yl}-6-(morpholin-4-yl)-l, 3, 5-tri azin-2 -yl)-4- (difluoromethyl)pyrimidin-2-amine (C 101 ) :
To a stirred solution of 4-chloro-5-iodo-7-[(4-methoxy-3,5-dimethylpyridin-2- yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-2-amine (85 mg, 0.19 mmol, 1.0 eq) in DCM (15 mL), TEA (0.085 mL, 0.57 mmol, 3.0 eq) followed by (Int-3) 4-(difluoromethyl)- 5-[4-(morpholin-4-yl)-6-[4-({2-[2-(prop-2-yn-l-yloxy)ethoxy]ethyl}amino)piperidin- l-yl]-l,3,5-triazin-2-yl]pyrimidin-2-amine (153 mg, 0.28 mmol , 1.5 eq) were added at RT and reaction mass degassed for 10-20 min with argon, and then Cui (10.9 mg, 0.05 mmol, 0.3 eq) followed by Pd( PPhs)4 (11.1 mg, 0.009 mmol, 0.05 eq) were added and again degassed for 5 min. The resulting reaction mixture was heated to 45°C and stirred for 12h. After complete conversion of SM, reaction mixture was filtered through short celite bed and filtrate was concentrated under reduced pressure to afford crude .The crude compound was submitted to prep. HPLC to afford 5-(4-{4- [(2-{2-[(3-{2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-7H- pyrrolo[2,3-d]pyrimidin-5-yl}prop-2-yn-l-yl)oxy]ethoxy}ethyl)amino]piperidin-l- yl}-6-(morpholin-4-yl)- 1,3, 5-tri azin-2-yl)-4-(difluoromethyl)pyrimidin-2-amine (C101) (74 mg, 45.48%) as an off white solid. [0411] 'H NMR: (DMSO-de, 400 Hz) 8: 9.10 (s, 1H), 8.52 (bs, 2H), 8.04 (s, 1H), 7.62 (t, J = 52 Hz, 1H), 7.57 (bs, 2H), 7.39 (s, 1H), 6.74 (bs, 2H), 5.26 (s, 2H), 4.75 (bs, 2H), 4.41 (s, 2H), 3.73-3.58 (m, 18H), 3.18 (bs, 2H), 2.90 (bs, 2H), 2.24 (s, 3H), 2.15-2.07 (m, 5H), 1.45-1.39 (m, 1H).
LC-MS: 849.81 (M + H).
C102
Figure imgf000179_0001
[0412] Synthesis of (ethyl 4-{4-[(4-{3-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-5- (ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl }phenyl)methyl]piperazin- 1 -yl Jbutanoate ((Int-2):
To a stirred solution of 5-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-N-ethyl-4-{4- [ (piperazin- l-yl)methyl]phenyl}-4H-l, 2, 4-triazole-3 -carboxamide (200 mg, 0.43 mmol, 1.0 eq) in DMF (4.5 mL), NaHCOs (108 mg, 1.29 mmol, 3.0 eq) followed ethyl 4-bromobutanoate (126 mg, 0.64 mmol, 1.5 eq) were added at RT. The resulting reaction mixture was stirred at room temperature for 28h. Progress of the reaction was monitored by TLC. After consumption of SM, the reaction mixture was diluted with water (10 mL) and extracted with EtOAC (3 x 50 mL). The combined organic layers dried over sodium sulphate, filtered and evaporated to get crude. The crude compound was triturated with diethyl ether (50 mL) and filtered to afford (ethyl 4-{4-[(4-{3-[2,4- dihydroxy-5-(propan-2-yl)phenyl]-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl(phenyl)methyl]piperazin-l-yl(butanoate (Int-2) (200 mg, 80.28%) as light yellow solid.
[0413] Synthesis of 4-{4-[(4-{3-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-5- (ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl (phenyl)methyl]piperazin- 1 -yl (butanoic acid (Int-3):
To a stirred solution of ethyl 4-{4-[(4-{3-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-5- (ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl (phenyl)methyl]piperazin- 1 -yl (butanoate (Int- 2) (200 mg, 0.34 mmol, 1.0 eq) in a mixture of solvents THF:MeOH:H2O (5 mL, 3: 1 : 1), LiOH.HzO (16.0 mg, 0.34 mmol, 1.0 eq) was added at RT. The resulting reaction mixture was allowed to RT and stirred for 16 h. After completion of SM, volatiles were evaporated under reduced pressure, then the reaction mixture was diluted with water (5.0 mL) and neutralized pH - 6 with aq. HC1 extracted with 10% MeOH in DCM (16 x 10 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford 4-{4-[(4-{3-[2,4- dihydroxy-5-(propan-2-yl)phenyl]-5-(ethylcarbamoyl)-4H-l,2,4-triazol-4- yl(phenyl)methyl]piperazin-l-yl (butanoic acid (Int-3) (90 mg, 47.29%) as light yellow solid.
[0414] Synthesis of 4-[4-({4-[4-(4-{4-[2-amino-4-(difhioromethyl)pyrimidin-5- yl]-6-(morpholin-4-yl)- 1 ,3 , 5 -triazin-2 -yl (piperazin- 1 -yl)-4-oxobutyl]piperazin- 1 - yl(methyl)phenyl]-5-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-N-ethyl-4H-l,2,4- triazole-3 -carboxamide (Cl 02):
To a stirred solution of 4-{4-[(4-{3-[2,4-dihydroxy-5-(propan-2-yl)phenyl]-5- (ethylcarbamoyl)-4H- 1 ,2,4-triazol-4-yl (phenyl)methyl]piperazin- 1 -yl (butanoic acid (Int-3) (90 mg, 0.16 mmol, 1.0 eq) in DMF (3.5 mL), DIPEA (0.086 mL, 0.49 mmol, 3.0 eq) followed by HATU (93.2 mg, 0.24 mmol, 1.5 eq) was added at RT and stirred for 5-10 min. After that 4-(difluoromethyl)-5-[4-(morpholin-4-yl)-6-(piperazin-l-yl)- l,3,5-triazin-2-yl]pyrimidin-2-amine (TVD-0004425) (77.2 mg, 0.196 mmol, 1.2 eq) was added at room temperature. The resulting reaction mass was stirred for 18h at RT. Progress of the reaction was monitored by TLC and LCMS. After consumption of SM reaction mixture was diluted with water (8 mL) and the resulting solid was filtered and washed with diethyl ether (8 mL) and dried under vacuum to get crude. This crude was purified by prep.HPLC to afford 4-[4-({4-[4-(4-{4-[2-amino-4- (difluoromethyl)pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-tri azin-2 -yl (piperazin- 1- yl)-4-oxobutyl]piperazin-l-yl(methyl)phenyl]-5-[2,4-dihydroxy-5-(propan-2- yl)phenyl]-N-ethyl-4H-l, 2, 4-triazole-3 -carboxamide (C102) (20 mg, 13.21%) as an off white solid.
[0415] ’H NMR: (400 MHz, DMSO-de) 6: 9.74 (bs, 1H), 9.11 (s, 1H), 8.97 (bs, 1H), 7.63 (s, 1H), 7.49-7.40 (m, 1H), 7.63 (t, J = 56.0 Hz, 1H), 7.52 (bs, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 6.60 (s, 1H), 6.33 (s, 1H), 3.88-3.76 (m, 10H), 3.66-3.65 (m, 10H), 3.20-2.90 (m, 10H), 2.47 (s, 2H), 1.89 (bs, 2H), 1.04 (t, J = 8.0 Hz , 3H), 0.82 (d, J = 4.0 Hz, 6H).
LC-MS: 926.96 (M + H).
C103
Figure imgf000181_0001
[0416] Synthesis of ethyl 4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-1, 3, 5-triazin-2-yl) piperazin- 1-yl) butanoate (Int-2):
To a stirred solution of 4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-l-yl)-l, 3, 5- triazin-2-yl) pyrimidin-2-amine (0.2 g, 0.05 mol, 1.0 eq) and ethyl 4-bromobutanoate (0.119 g, 0.061 mol, 1.2 eq) were dissolved in DMF (8 mL). NaHCOs (0.085 g, 0.1 mol, 2.0 eq) was added at RT. The resulting reaction mixture was stirred for 24h at room temperature. After consumption of SM, reaction mass quenched with water (40 mL) and extracted with ethyl acetate (4 x 10 mL), combined organic layers was washed with brine, organic layer dried over Na2SO4, filtered and concentrated to afford crude 4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3,
5-triazin-2-yl) piperazin- 1-yl) butanoate (Int-2) (150 mg, 58.13%) as a colorless oil. [0417] Synthesis of 4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-1, 3, 5-triazin-2-yl) piperazin- 1-yl) butanoic acid (Int-3):
To a stirred solution of ethyl 4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-1, 3, 5-triazin-2-yl) piperazin- 1-yl) butanoate (Int-2) (0.15 g, 0.0296 mmol, 1.0 eq) in mixture of solvents THF/H2O (2:0.5 mL), LiOH.HzO (0.062 g, 0.14 mmol, 5.0 eq) was added at RT. The resulting reaction mixture was stirred at room temperature for 16h. After completion of SM, volatiles were evaporated under reduced pressure, the crude reaction mass was diluted with water (5 mL), adjusted pH~5-6 with IN HC1 and extracted with 10% methanol in DCM, combined organic layers were dried over NaSCU, filtered and concentrated to afford crude. Crude compound was triturated with n-hexane to afford 4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperazin- 1-yl) butanoic acid (Int- 3) (130 mg, 91.74%) as an off white solid.
[0418] Synthesis of 4-(4-((4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-
6-morpholino- 1,3,5 -triazin-2-yl)piperazin- 1 -yl)butanoyl)piperazin- 1 - yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-l,2,4-triazole-3- carboxamide (C103):
To a stirred solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-l,3,5-triazin-2-yl)piperazin-l-yl)butanoic acid (Int-3) (0.13 g, 0.027 mol, 1.0 eq) and 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(piperazin-l- ylmethyl)phenyl)-4H-l, 2, 4-triazole-3 -carboxamide (0.189 g, 0.04 mol, 1.5 eq) were in DMF (2.0 mL), DIPEA (0.140 g, 0.1 mmol, 4.0 eq) followed by HATU (0.155 g, 0.04 mmol, 1.5 eq) were added at RT. The resulting reaction mixture was heated to 80°C and stirred at for 4 h. After completion of starting material, reaction mass quenched with ice-water (20 mL), the resulting solid was filtered and washed with ice water dried to afford crude compound (200 mg). Crude compound purified by Prep.HPLC and lyophilized to afford 4-(4-((4-(4-(4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazin- 1 - yl)butanoyl)piperazin-l-yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N- ethyl-4H-l, 2, 4-triazole-3 -carboxamide (C103) (91 mg, 36.2%) as an off white solid. [0419] 'H NMR: (400 MHz, DMSO-de) 6: 10.24 (bs, 1H), 9.74 (s, 2H), 9.13 (s, 1H), 9.01 (bs, 1H), 7.75-7.42 (m, 7H), 6.66 (s, 1H), 6.31 (s, 1H), 4.76 (bs, 2H), 4.37- 4.01 (m, 2H), 3.78 (bs, 4H), 3.65 (bs, 4H), 3.30 (bs, 6H), 3.21-2.90 (m, 14H), 1.90 (bs, 2H), 1.05 (t, J = 7.2 Hz, 3H), 0.87 (d, J = 6.8 Hz, 6H).
LC-MS: 926.45 (M + H).
C104
Figure imgf000183_0001
[0420] Synthesis of 4-({[(tert-butoxy) carbonyl] amino} methyl) phenyl 4-{4-[2- amino-4-(difluoromethyl)pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-triazin-2-yl} piperazine- 1 -carboxylate (Int-2) :
To a stirred solution of tert-butyl N-[(4-hydroxyphenyl) methyl] carbamate (Int-1) (75 mg, 0.336 mmol, 1.0 eq) in DCM (4 mL) was added DIPEA (0.178 mL, 1.00 mmol, 3.0 eq) followed by 4-nitrophenyl carbonochloridate (67.7 mg, 0.336 mmol, 1.0 eq) at 0°C. The resulting reaction mixture was allowed to RT and stirred for 2h. Progress of intermediate formation was monitored by TLC. After consumption SM, 4- (difluoromethyl)-5-[4-(morpholin-4-yl)-6-(piperazin-l-yl)-l, 3, 5-triazin-2-yl] pyrimidin-2-amine (132 mg, 0.336 mmol, 1.0 eq) in DMF (2.5 mL) was added at RT. The resulting reaction mixture was stirred at RT for 2h. After consumption of SM, reaction mass diluted with water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layer was washed with sat. brine (1 x 50 mL). The organic layer was dried over sodium sulphate, filtered and evaporated to obtain crude compound. The crude compound was triturated with MTBE (5 mL) and filtered to afford pure 4-({[(tert-butoxy) carbonyl] amino} methyl) phenyl4-{4-[2-amino-4- (difluoromethyl)pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-triazin-2-yl} piperazine-1- carboxylate (Int-2) (130 mg, 62.22%) as an off white solid.
[0421] Synthesis of 4-(amino methyl) phenyl 4-{4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-triazin-2-yl} piperazine- 1 -carboxylate (Int- 3):
To a stirred solution of 4-({[(tert-butoxy)carbonyl]amino}methyl)phenyl 4-{4-[2- amino-4-(difluoromethyl)pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-tri azin-2- yl (piperazine- 1 -carboxylate (Int-2) (130 mg , 0.20 mmol, 1.0 eq) in DCM (5 mL), was added TFA (0.077 mL, 1.0 mmol, 5.0 eq) at 0°C. The resulting reaction mixture was warm to RT and stirred for 4h. Progress of the reaction was monitor by TLC. After consumption SM, volatiles were concentrated under reduced pressure to afford crude. The crude compound was triturating with MTBE (5.0 mL) and filtered to afford pure 4-(amino methyl)phenyl 4-{4-[2-amino-4-(difluoromethyl)pyrimidin-5- yl]-6-(morpholin-4-yl)-l,3,5-triazin-2-yl}piperazine-l-carboxylate (Int-3) (100 mg, as TFA salt, 91.12% yield) as an light brown solid.
[0422] Synthesis of 4-({2-[(5S)-2-amino-4-chloro-7-[(4-methoxy-3,5- dimethylpyridin-2-yl)methyl]-5H,6H,7H-pyrrolo[2,3-d]pyrimidin-5- yl]acetamido}methyl)phenyl4-{4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6- (morpholin-4-yl)- 1 ,3 , 5-triazin-2-yl (piperazine- 1 -carboxylate (C 104) :
To a stirred solution of 2-[(5S)-2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin- 2-yl)methyl]-5H,6H,7H-pyrrolo[2,3-d]pyrimidin-5-yl]acetic acid (TVD-0004512) (80 mg, 0.21 mmol, 1.0 eq) in DMF (3.0 mL) was added DIPEA (0.1 ImL, 0.63 mmol, 3.0 eq) followed by HATU (121 mg, 0.31 mmol, 1.5 eq) and the reaction was stirred for 5-10 min at room temperature. After 10 min, 4-(amino methyl) phenyl 4-{4-[2-amino- 4-(difluoromethyl) pyrimidin-5-yl]-6-(morpholin-4-yl)-l, 3, 5-triazin-2-yl( piperazine- 1 -carboxylate (138 mg, 0.25 mmol, 1.2 eq) was added at RT. The resulting reaction mixture was quenched with ice-water (10 mL) and stirred for 5 min, the resulting precipitated was filtered and washed with water followed by washed with diethyl ether (10 mL), dried under vacuum to afford crude compound. The crude was purified by prep. HPLC and Lyophilized to afford 4-({2-[(5S)-2-amino-4-chl oro-7- [(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-5H,6H,7H-pyrrolo[2,3-d]pyrimidin-5- yl]acetamido}methyl)phenyl 4-{4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6- (morpholin-4-yl)-l,3,5-triazin-2-yl}piperazine-l-carboxylate (C104) (60 mg, 31.4%) as an off white solid.
[0423] XH-NMR: (400 MHz, DMSO-de) 6: 9.12 (s, 1H), 8.58 (t, J = 8.0 Hz, 1H), 8.37 (s ,1H), 7.64 (t, J = 52.0 Hz, 1H), 7.58 (bs, 2H), 7.17 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.68 (bs, 2H), 4.73-4.65 (m, 2H), 4.34-4.22 (m, 2H), 3.91 (bs, 8H), 3.78 (bs, 4H), 3.75-3.65 (m, 5H), 3.61-3.54 (m, 3H), 3.51-3.37 (m, 1H), 2.77-2.76 (dd, J = 18.4, 3.2 Hz , 1H), 2.51-2.50 (m, 2H, merged with dmso-d6), 2.31 (s, 3H), 2.23 (s, 3H).
LC-MS: 902.63 (M + H).
C105
Figure imgf000185_0001
[0424] Synthesis of tert-butyl (4-hydroxy-4-methylpentyl) carbamate (Int-2): To a stirred solution of 5-amino-2-methylpentan-2-ol (Int-1) (1.0 g, 8.53 mmol, 1.0 eq) in tert-Butyl alcohol (15 mL) and IM NaOH aq solution (0.546 mL, 13.7 mmol, 1.6 eq) was added at 0°C and a solution of di-tert-butyl dicarbonate (2.79 g, 12.8 mmol, 1.5 eq) in tert-Butanol (5.0 mL) was added drop wise over a period of 10 min. The resulting reaction mixture was stirred for 12h at RT. Progress of the reaction mixture moniterd by TLC and LCMS. After completion of the SM, Reaction mixture diluted with IM ammonium chloride soln (50 mL) and pH adjusted (6-7) with IM HC1 soln, then extracted with EtOAC (2 x 100 mL) organic layers washed with brine solution (100 mL) and dried sodium sulphate evaporated to afford crude tert-butyl (4- hydroxy-4-methylpentyl) carbonate (Int-2) (1.2 g, 64.7 % yield) as a brown liquid. [0425] Synthesis of 1 -benzyl 4-(5-((tert-butoxy carbonyl) amino)-2-methylpentan- 2-yl) piperidine-l,4-dicarboxylate (Int-4):
To stirred solution of l-[(benzyloxy)carbonyl]piperidine-4-carboxylic acid (Int-2) (2.79 g, 10.6 mmol, 2.3 eq) in DCM (40 mL), N,N'-dicyclohexylmethanediimine (2.18 g, 10.6 mmol, 2.3eq), 4-(dimethylamino) pyridin-l-ium (1.12 g, 0.920 mmol, 0.2 eq) were added at RT. The resulting reaction mixture was stirred for 20 min, then followed by tert-butyl N-(4-hydroxy-4-methylpentyl) carbamate (1.0 g, 4.6 mmol, 1.0 eq) was added at RT. The resulting reaction mixture was stirred for 14 h at room temperature. Progress of the reaction mixture monitored by LCMS. After completion of the reaction mixture, the reaction mixture quenched with ice water (100 mL) extracted with EtOAC (3 x 50 mL) organic layers washed with ice water (3 x 100 mL) dried over sodium sulphate filtered to get afford crude compound. 1 -benzyl 4-(5- ((tert-butoxy carbonyl) amino)-2-methylpentan-2-yl) piperidine- 1, 4-dicarboxylate (Int-4) (0.82 g 38.52%) as a pale yellow solid.
[0426] Synthesis of (4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-1, 3, 5-triazin-2-yl) piperazin- l-yl)-4-oxobutanoic acid) (Int-5):
To a solution of 1-benzyl 4-(5-{[(tert-butoxy) carbonyl] amino }-2-methylpentan-2-yl) piperidine-1, 4-dicarboxylate (Int-4) (0.8 g, 1.73 mmol, 1 eq) in methanol (8 mL), 10% Pd/C (0.16 g) was added at RT, to the reaction mixture applied 50 PSI hydrogen gas for 2h in a parr-aparatus at RT. Progress of the reaction mixture monitored by TLC. After completion of the reaction, the reaction mixture was filtered on celite bed and washed with methanol (2 x 100 mL), filtrate mis concentrated under reduced pressure to afford crude compound. The crude was purified by flash chromatography use silica gel 100-200 mesh compound eluted 5% in MeOH in DCM, determined pure fraction to get pure compound (4-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)- 6-morpholino-l, 3, 5-triazin-2-yl) piperazin- l-yl)-4-oxobutanoic acid (Int-5) (0.25 g 77%) as a brown liquid. [0427] Synthesis of 5-((tert-butoxy carbonyl) amino)-2-methylpentan-2-yl l-(4-(2- (bis (tert-butoxycarbonyl) amino)-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2-yl) piperidine-4-carboxylate (Int-7):
To a stirred solution of tert-butyl N-f(tert-butoxy) carbonyl]-N-{5-[4-chloro-6- (morpholin-4-yl)-l,3,5-triazin-2-yl]-4-(difluoromethyl) pyrimidin-2-yl} carbamate (Int-6) (0.3g, 0.552 mmol, 1.0 eq) in DMF (6 mL), K2CO3 (0.091g, 0.662 mmol, 1.2 eq) followed by 5-{[(tert-butoxy) carbonyl] amino }-2-methylpentan-2-yl piperidine- 4-carboxylate (Int-5) (0.217g, 0.662 mmol, 1.2 eq) was added at 0°C. The resulting reaction mixture warm to rt and stirred for 16 h at RT. Progress of reaction mixture monitored by LCMS. After completion of the SM, the reaction mixture was quenched with water (50.0 mL) and extracted with EtOAC (2 x 100 mL) organic layers combined washed ice water (2 x 100 mL) dried over sodium sulfate filtered concentrated under reduced pressure to afford crude (Int-7) (0.45 g, crude). The crude compound was purified by flash chromatography use silica gel (100-200 mesh), compound eluted 30% EtOAc in pet ether. All pure fraction collected and concentrated to get pure 5-((tert-butoxy carbonyl) amino)-2-methylpentan-2-yl l-(4- (2-(bis (tert-butoxy carbonyl) amino)-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-1, 3, 5-triazin-2-yl) piperidine-4-carboxylate (Int-7) (0.35g, 75.9% yield) as a brown gammy solid.
[0428] Synthesis of 5 -amino-2-methylpentan-2-yl l-(4-(2-amino-4-
(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperidine-4- carboxylate (Int-8):
To a stirred solution of 5- {[(tert-butoxy) carbonyl] amino }-2-methylpentan-2-yl l-[4- (2-{bis[(tert-butoxy) carbonyl] amino }-4-(difluorom ethyl) pyrimidin-5-yl)-6- (morpholin-4-yl)-l,3,5-triazin-2-yl] piperidine-4-carboxylate (Int-7) (0.3 g, 0.359 mmol, 1.0 eq.) in DCM (3 mL), TFA (0.818 g, 7.18 mmol, 20.0 eq) was added drop wise at 0°C. The resulting reaction mixture was stirred for 16h at RT, Progress of the reaction mixture moniterd by TLC & LCMS. After completion of reaction, the reaction mixture was concentrated under vacuum to get crude 5-amino-2- methylpentan-2-yl l-(4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l, 3, 5-triazin-2-yl) piperidine-4-carboxylate (Int-8) (0.31g, crude 100% yield) as a brown liquid.
[0429] Synthesis of (R)-5-(2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl) methyl)-6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) acetamido)-2-methylpentan-2-yll-(4-(2-amino-4-(difluorom ethyl) pyrimidin-5-yl)-6- morpholino-l,3,5-triazin-2-yl) piperidine-4-carboxylate (C105):
To a stirred solution 2-[(5R)-2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin-2- yl)methyl]-5H,6H,7H-pyrrolo[2,3-d]pyrimidin-5-yl]acetic acid (Int-8) (0.212 g, 0.566 mmol,l eq) in DMF (3.0 mL), DIPEA ( 0.248 mL ,1.40 mmol, 2.5 eq), HATU ( 0.319 g,0.84 mmol,1.5eq) were added at RT, the reaction mixture stirred for 20 min, and then 5-amino-2-methylpentan-2-yl l-{4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]- 6-(morpholin-4-yl)-l,3,5-triazin-2-yl}piperidine-4-carboxylate ( 0.300 g, 0.56 mmol,l eq) was added at RT. The resulting reaction mixture was stirred for 18 h at RT. The progress of reaction was monitored by TLC and LCMS After completion of the reaction, the reaction was quenched with ice water (20 mL), solid principates and stirred for 10 min, filtered and washed with water (5 mL) dried well under vacuum to get crude product. The crude was purified by prep HPLC followed by lyophilized to afford (R)-5-(2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl) methyl)- 6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) acetamido)-2-methylpentan-2-yll-(4- (2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l,3,5-triazin-2-yl) piperidine-4-carboxylate (TVD-0004500) (88 mg, 17.5%) as an off white solid
C106
Figure imgf000188_0001
Figure imgf000188_0002
[0430] Synthesis of tert-butyl (5-(4-(4-(2-amino-4-(difluoromethyl) pyrimidin-5- yl)-6-morpholino-l,3,5-triazin-2-yl)piperazin-l-yl)-5-oxopentyl)carbamate (Int-2): To the stirred solution of 5-((tert-butoxycarbonyl)amino)pentanoic acid ( 108 mg, 0.496 mmol, 1.3 eq) in DMF( 5 ml) was added DIPEA ( 98.6 mg 0.763 mmol, 2 eq) and HATU ( 217 mg, 0.571 m mol, 1.5 eq) at RT. Reaction mixture was at RT for 20 minutes then 4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-l-yl)-l,3,5-triazin-2- yl)pyrimidin-2-amine (TVD-0004425) (150 mg, 0.381 mmol, 1 eq) was added and the reaction mixture was stirred at RT for 18h. Progress of reaction was monitored by TLC and LCMS. After completion of reaction, the reaction mixture was quenched with water and resulting solid was collected by filtration to afford tert-butyl (5-(4-(4- (2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-l, 3, 5-tri azin-2- yl)piperazin-l-yl)-5-oxopentyl)carbamate (Int-2) (160 mg, 70%) as a brown solid. [0431] Synthesis of 5-amino-l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)- 6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazin- 1 -yl)pentan- 1 -one (Int-3):
To the solution of tert-butyl (5-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-l,3,5-triazin-2-yl)piperazin-l-yl)-5-oxopentyl)carbamate (Int-2) (150 mg, 0.253 mmol, 1.0 eq) in DCM (5 mL) was added TFA (289 mg, 2.53 mmol, 10.0 eq) and reaction mixture was stirred at RT for 16h. Progress of reaction was monitored by TLC and LCMS. After completion of starting material, volatiles were removed under reduced pressure and crude was triturated with diethyl ether to afford 5-amino- l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3, 5-tri azin-2- yl)piperazin-l-yl)pentan-l-one (Int-3) (130 mg, TFA salt) as a brown solid.
[0432] Synthesis of (R)-N-(5-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)- 6-morpholino- 1,3, 5-tri azin-2-yl)piperazin-l-yl)-5-oxopentyl)-2-(2-amino-4-chl oro-7- ((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3- d]pyrimidin-5-yl)acetamide (C 106) :
To the stirred solution of (R)-2-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetic acid (TVD-0004473) (70 mg, 0.185 mmol, 1.0 eq) in DMF (2 mL) was added DIPEA (95.8 mg, 0.741 mmol, 4.0 eq) and HATU (106 mg, 0.278 mmol, 1.5 eq). Reaction was stirred at RT for 20 min. then 5-amino-l-(4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3 , 5-triazin-2-yl)piperazin- 1 - yl)pentan-l-one (Int-3) (110 mg, 0.222 mmol, 1.2 eq) was added and reaction mixture was stirred at 80°C for 4 h. Progress of reaction was monitored by TLC and LCMS. After completion of starting material, reaction was quenched with water and resulting solid was collected by filtration. The crude was purified by prep. HPLC followed by lyophilisation to afford (R)-N-(5-(4-(4-(2-amino-4-(difhioromethyl)pyrimidin-5-yl)-6- morpholino- 1,3,5 -tri azin-2 -yl)piperazin- 1 -y 1) - 5 -oxopentyl)-2-(2-amino-4-chl oro-7 - ((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3- d]pyrimidin-5-yl)acetamide (C106) (80 mg, 51%) as an off white solid.
C108
Figure imgf000190_0001
[0433] Synthesis of (4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6- morpholino-l,3,5-triazin-2-yl) piperidin-4-yl (R)-2-(2-amino-4-chloro-7-((4-methoxy- 3,5-dimethylpyridin-2-yl) methyl)-6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) acetate (Cl 08):
To a stirred solution of 2-[(5R)-2-amino-4-chloro-7-[(4-methoxy-3,5- dimethylpyridin-2-yl) methyl]-5H,6H,7H-pyrrolo[2,3-d] pyrimidin-5-yl] acetic acid (TVD-0004473) (278 mg, 0.73 mmol, 1.5 eq.) in DMF (4.0 mL), DCC (152 mg, 0.735 mmol, 1.5 eq) followed by DMAP (5.98 mg, 0.4 mmol, 0.1 eq) were added at RT. The resulting reaction mixture was stirred for 20 min. After 20 min, l-{4-[2- amino-4-(difluoromethyl) pyrimidin-5-yl]-6-(morphine- 4-yl)-l, 3, 5-triazin-2-yl} piperidin-4-ol (TVD-0004426) (200 mg, 0.49 mmol, 1.0 eq) was added at RT. The resulting reaction mixture was stirred for 72h at RT. Progress of the reaction was monitored by LCMS and TLC. After completion of the SM, the reaction mixture was quenched with ice water (50.0 mL) extracted with EtOAC (3 x 50.0 mL) the combined organic layers washed with ice water (2 x 100 mL) and brine soln (1 x 100 mL) dried over sodium sulphate filtered to afford crude compound. The crude was stirred for 30 min in diethyl ether filtered to get crude compound. The crude compound was purified by Prep HPLC followed by lyophilized to afford compound (4-(2-amino-4-(difluoromethyl) pyrimidin-5-yl)-6-morpholino-l,3,5-triazin-2-yl) piperidin-4-yl (R)-2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyri din-2 -yl) methyl)-6,7-dihydro-5H-pyrrolo[2,3-d] pyrimidin-5-yl) acetate (C108) (66 mg, 17.1% yield) as off white solid.
[0434] ’H NMR: (400 MHz, DMSO-de) 6 9.09 (s, 1H), 8.37 (bs, 1H), 7.62 (t, J = 52.0 Hz, 1H), 7.55 (bs, 2H), 6.67 (bs, 2H), 4.95 (s, 1H), 4.70 (s, 2H) 3.90-4.0 (m, 3H), 3.75-3.80 (m, 7H), 3.59-3.64 (m, 8H), 3.30 (dd, J = 8.0, 4.0 Hz, 1H), 2.84-2.89 (m, 1H), 2.68-2.61 (m, 1H), 2.30 (s, 3H), 2.24 (s, 3H), 1.83 (bs, 2H), 1.5 l(bs, 2H). LC-MS: 768.29 (M + H).
C109
Figure imgf000191_0001
TVD-0004473
Figure imgf000191_0002
[0435] Synthesis of (R)-l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-l,3,5-triazin-2-yl)piperazin-l-yl)-2-(2-amino-4-chloro-7-((4-methoxy- 3,5-dimethylpyridin-2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5- yl)ethan-l-one (Cl 09):
To a stirred solution of (R)-2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin- 2-yl)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)acetic acid (TVD- 0004473) (144 mg, 0.38 mmol, 1.5 eq) in DMF (5.0 mL) were added HATU (140 mg, 0.38 mmol, 1.5 eq) and DIPEA (130 mg, 1.02 mmol, 4.0 eq) at RT. Resulting reaction mixture was stirred at RT for 20 min. then 4-(difluoromethyl)-5-(4-morpholino-6- (piperazin- l-yl)-l, 3, 5-triazin-2-yl) pyrimidin-2-amine (C109) (100 mg, 0.25 mmol, 1.0 eq) was added and resulting reaction mixture was stirred at RT for 16h. Progress of reaction was monitored by LCMS and TLC. After completion of starting material, reaction was quenched with water and resulting solid was collected by filtration. The crude was purified by prep. HPLC followed by lyophilized to afford (R)-l-(4-(4-(2- arnino-4-(difhioromethyl)pyrimidin-5-yl)-6-morpholino-l, 3, 5-tri azin-2 -yl)piperazin- l-yl)-2-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-6,7- dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethan-l-one (TVD-0004496) (95 mg, 49%) as an off white solid.
[0436] ’H NMR: (400 MHz, DMSO-de) 8: 9.10 (s, 1H), 8.41 (s, 1H), 7.63 (t, J = 52.0 Hz, 1H), 7.58 (bs, 2H), 6.70 (bs, 2H), 4.72 (s, 2H), 3.92 (s, 3H), 3.80-3.77 (m, 2H), 3.66-3.58 (m, 10H), 3.54-3.53 (m, 6H), 3.23-3.19 (m, 1H), 2.96-2.91 (m, 1H), 2.77-2.73 (m, 1H), 2.32 (s, 3H), 2.24 (s, 3H).
LC-MS: 753.59 (M + H).
C112
Figure imgf000192_0001
[0437] Synthesis of tert-butyl N-[3-(prop-2-yn-l-yloxy) propyl] carbamate (Int-3): To a stirred solution of Prop-2-yn-l-ol (100 mg, 1.78 mmol, 1.0 eq) in DMF (4.0 mL), NaH (47.1mg, 1.96 mmol, 1.1 eq) was added portion wise at 0°C. The reaction mixture was allowed to RT and stirred for 30 min. then tert-butyl N-(3 -bromopropyl) carbamate (425 mg, 1.78 mmol, 1.0 eq) was added portion wise at 0°C. The resulting reaction mixture was allowed RT and stirred for 4h at same temperature. After completion of SM, reaction mixture quenched with water (5 mL) and extracted with EtOAC (2 x 10 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude compound. The crude compound was purified by column chromatography using silica gel (100-200 mesh) and compound eluted at 0-2% MeOH in DCM. All pure fractions were mixed and concentrated to afford tert-butyl N-[3-(prop-2-yn-l-yloxy) propyl] carbamate (Int-3) (280 mg, 73.6%) as a light yellow oil.
[0438] Synthesis of tert-butyl (3-((3-(2-amino-4-chloro-7-((4-methoxy-3,5- dimethylpyri din-2 -yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)prop-2-yn-l- yl)oxy)propyl)carbamate (Int-4):
To a stirred solution of 4-chloro-5-iodo-7-[(4-methoxy-3,5-dimethylpyridin-2- yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-2-amine (TVD-0004501) ( 230 mg , 0.5 mmol, 1.0 eq ) in DCM ( 20 mL), TEA ( 0.21 mL, 1.55 mmol, 3.0 eq) followed by tert-butyl N-[3-(prop-2-yn-l-yloxy)propyl]carbamate (276 mg, 1.4 mmol, 2.5 eq) were added at RT and degassed the reaction mass for 20 min with argon, and then Cui (29.5 mg, 0.15 mmol, 0.3 eq) followed by Pd( PPhs)4 (30 mg, 0.025 mmol, 0.05 eq) were added at same temperature again degassed for 5 min. The resulting reaction mixture was heated to 45°C and stirred for 2h. After completion of SM, volatiles were evaporated under reduced pressure to afford crude compound. The crude compound was purified by column chromatography using silica gel (100-200) and compound eluted at 1 to 1.5 % MeOH in DCM. All pure fraction were mixed and concentrated to afford tert-butyl N-{3-[(3-{2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin-2- yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}prop-2-yn-l-yl)oxy]propyl}carbamate (Int-4) (200 mg, 72.92%) as light yellow solid.
[0439] Synthesis of Int-5 5 -[3 -(3 -aminopropoxy) prop-l-yn-l-yl]-4-chloro-7-[(4- methoxy-3, 5-dimethylpyridin-2-yl) methyl]-7H-pyrrolo [2, 3-d] pyrimidin-2-amine: To a stirred solution of tert-butyl N-{3-[(3-{2-amino-4-chloro-7-[(4-methoxy-3,5- dimethylpyri din-2 -yl)methyl]-7H-pyrrolo[2, 3-d]pyrimidin-5-yl }prop-2-yn-l- yl)oxy]propyl} carbamate (Int-4) (160 mg, 0.30 mmol, 1.0 eq) in DCM ( 5.0 mL) was added TFA (0.116 mL, 1.5 mmol, 5.0 eq) at 0°C. The resulting reaction mixture was warm to room temperature and stirred for 2h at same temperature. Progress of reaction monitor by TLC, after complete conversion of SM, volatiles were concentrated under reduced pressure to afford crude 5-[3-(3-aminopropoxy)prop-l- yn-l-yl]-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-7H-pyrrolo[2,3- d]pyrimidin-2-amine (Int-5) (250 mg as TFA salt, quantitative yield). [0440] Synthesis of TVD-0004493 l-{4-[2-amino-4-(difluoromethyl)pyrimidin-5- yl]-6-(morpholin-4-yl)-l,3,5-triazin-2-yl}-N-{3-[(3-{2-amino-4-chloro-7-[(4- methoxy-3,5-dimethylpyridin-2-yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}prop-2- yn- 1 -yl)oxy]propyl }piperidine-4-carboxamide:
To a stirred solution of l-{4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6- (morpholin-4-yl)-l,3,5-triazin-2-yl}piperidine-4-carboxylic acid (Cl 12) (210 mg, 0.48 mmol, 1.0 eq.) in DMF (8 mL) was subsequently added DIPEA (0.25 mL, 1.4 mmol, 3.0 eq) and HATU (274 mg, 0.74 mmol, 1.5 eq) and stirred for 10 min. then (Int-5) 5 -[3 -(3 -aminopropoxy) prop-l-yn-l-yl]-4-chloro-7-[(4-methoxy-3, 5- dimethylpyridin-2-yl) methyl]-7H-pyrrolo [2, 3-d] pyrimidin-2-amine (248 mg, 0.57 mmol, 1.2 eq) was added and stirred for 18h at RT. After complete conversion of SM monitored by TLC. The reaction mixture was diluted with ice water (10 mL) and the resulting solid was collected by filtration to afford 250 mg crude, the crude product was purified by prep. HPLC to afford Cl 12, (58 mg, 14.23%) as an off white solid. [0441] 'H NMR: (400 MHz, DMSO-de) 6: 9.08 (s, 1H), 8.12 (s, 1H), 7.81 (t, J = 4.0 Hz, 1H), 7.62 (t, J = 56.0 Hz, 1H), 7.54 (bs, 2H), 7.41 (s, 1H), 6.75 (bs, 2H), 5.32 (s, 2H), 4.65 (bs, 2H), 4.34 (s, 2H), 3.65- 3.64 (m, 11H), 3.51 (t, J = 12.8 Hz, 2H), 3.09 (dd, J = 12.4, 6.8 Hz, 2H), 2.89 (bs, 2H), 2.42-2.32 (m, 1H), 2.25 (s, 3H), 2.19 (s, 3H), 1.73 (d, J = 12.0 Hz, 2H), 1.67 (d, J = 6.8 Hz, 2H), 1.47 (d, J = 9.2 Hz, 2H). LC-MS: 847.57 (M + H).
C114
Figure imgf000195_0001
[0442] Synthesis of 3-(prop-2-yn-l-yloxy) propan-l-ol (Int-2):
To a stirred solution of propane-1, 3-diol (Int-1) (5.0 g, 65.1 mmol, 1.0 eq) in dry THF (100 mL) potassium 2-methylpropan-2-olate (3.72 g, 32.9 mmol, 0.5 eq) was added at 0°C. The reaction mixture was stirred for 30 min at the same temperature and then 3 -bromoprop- 1-yne (3.91 g, 32.9 mmol, and 0.5 eq) in dry THF (100 mL) was added drop wise at RT for 30 min. The resulting reaction mixture stirred for 12h at RT. Progress of the reaction mixture monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under vacuum to get crude compound. The crude compound was purified flash chromatography using 100-200 mesh silica gel. Compound eluted 20-30% EtOAc/ Pet ether. All pure fraction was determined and concentrated to get pure 3-(prop-2-yn-l-yloxy) propan-l-ol (Int-3) (1.3g, 17.4%) brown liquid.
[0443] Synthesis of tert-butyl 3-(3-(prop-2-yn-l-yloxy) propoxy) propanoate (Int- 3):
To a stirred solution of 3 -(prop-2 -yn-l-yloxy) propan-l-ol (Int-2) (1.3g, 11.4 mmol, 1.0 eq) in THF (13 mL) potassium 2-methylpropan-2-olate (0.129 g, 1.14 mmol, 0.1 eq) was added at RT. the reaction mixture was stirred for 20 min, then followed by tert-butyl prop-2-enoate (1.9 g,14.8 mmol, 1.3 eq) drop wise added at RT. The resulting reaction mixture stirred for 16h at RT. Progress of reaction mixture monitored by TLC & LCMS. After completion of reaction. In this reaction mixture removed solvent under vacuum then diluted in water (50 mL) pH neutralize with IN HC1 extracted with EtAOc (2 x 100 mL) combined organic layers, dried sodium sulphate filtered mis was evaporated then to get crude compound (2 g). The crude compound was purified flash chromatography used silica gel 100-200 mesh compound eluted 20-30% EtAOc / Pet ether all pure fraction was determined to get pure compound. Tert-butyl 3-(3-(prop-2-yn-l-yloxy) propoxy) propanoate (Int-3) (1.32 g, 47.83 % yield) as brown liquid.
[0444] Synthesis of tert-butyl3-(3-((3-(2-amino-4-chloro-7-((4-methoxy-3, 5- dimethylpyridin-2-yl) methyl)-7H-pyrrolo [2, 3-d] pyrimidin-5-yl) prop-2-yn-l-yl) oxy) propoxy) propanoate (Int-4).
To stirred solution of 4-chloro-5-iodo-7-[(4-methoxy-3,5-dimethylpyridin-2- yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-2-amine (TVD-0004501) (0.50g, 1.13 mmol, 1.0 eq.) in DCM (15 mL) TEA (0.342 g, 3.38 mmol, 3.0 eq) and tert-butyl 3-[3-(prop- 2-yn-l-yloxy) propoxy]propionate (Int-3) (0.410 g, 1.69 mmol, 1.5 eq) were added at RT. The reaction mixture was degassed with argon for 20 min, then added Cui (0.0 64g, 0.33 mmol, 0.3 eq) and Pd (PPhs)4 (0.65g, 0.56 mmol, 0.05 eq) at RT. The resulting reaction mixture stirred for 5h at 45°C. Progress of the reaction mixture monitored by LCMS. After completion of the reaction, volatiles evaporated under reduced pressure to get crude compound (600 mg crude). The crude was purified by the reverse phase flash chromatography compound was eluted 60% ACN in water determined pure fractions concentrated followed by lyophilized to afford pure compound tert-butyl 3-(3-((3-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin- 2-yl) methyl)-7H-pyrrolo[2,3-d] pyrimidin-5-yl) prop-2-yn-l-yl) oxy) propoxy) propanoate (Int-4) (0.260 g, 41.37%) as a Brown liquid.
[0445] Synthesis of 3-(3-((3-(2-amino-4-chloro-7-((4-methoxy-3, 5- dimethylpyridin-2-yl) methyl)-7H-pyrrolo [2, 3-d] pyrimidin-5-yl) prop-2-yn-l-yl) oxy) propoxy) propionic acid (Int-5):
To a stirred solution of tert-butyl 3-{3-[(3-{2-amino-4-chloro-7-[(4-methoxy-3,5- dimethylpyridin-2-yl) methyl]-7H-pyrrolo[2,3-d] pyrimidin-5-yl} prop-2-yn-l-yl) oxy] propoxy} propanoate(Int-4) (0.25g, 0.448 mmol, 1.0 eq) in DCM (4 mL), TFA (1.45 g, 12.7 mmol, 20.0 eq) was added at 0°C. The resulting reaction mixture stirred for 6h at RT. Progress of the reaction mixture monitored by TLC. After completion of the reaction, volatiles the reaction mixture evaporated under reduced pressure at 0°C and to get crude compound Int-5 (0.180 g crude) as brown liquid. Crude compound was proceeded for the next step without purification.
[0446] Synthesis of l-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino- 1,3,5 -triazin-2-yl)piperazin- 1 -y 1 ) - 3 -(3 -((3 -(2-amino-4-chloro-7-((4- methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)prop-2- yn- 1 -yl)oxy)propoxy)propan- 1 -one(C 114):
To a stirred solution of 3-{3-[(3-{2-amino-4-chloro-7-[(4-methoxy-3,5- dimethylpyri din-2 -yl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl}prop-2-yn-l- yl)oxy]propoxy]propanoic acid (Int-5) (0.180 g, 0.359 mmol, 1.0 eq) in DMF (5.0 mL), HATU (0.127 g, 0.538 mmol, 1.5 eq), DIPEA (0.139 g, 1.08 mmol, 3.0 eq) followed by TVD-0004425 (0.141 g, 0.359 mmol, 1.0 eq) were added at RT. The resulting reaction mixture stirred for 12h at RT. Progress of the reaction mixture monitored by LCMS, after completion of SM. The reaction mixture quenched with ice water (5 mL) and stirred for 30 min solid precipitated. Solid was filtered and washed ice water (2 mL) dried under vacuum to afford crude compound (350 mg). The crude compound was purified by prep HPLC and lyophilized to afford Cl 14 (57 mg, 18.2 % yield) off white solid.
[0447] ’H-NMR: (400 MHz, DMSO-de) 8: 9.10 (s, 1H), 8.03 (s, 1H), 7.62 (t, J = 52.0 Hz, 1H), 7.56 (bs, 2H), 7.37 (s, 1H), 6.72 (bs, 2H), 5.27 (s, 2H), 4.34 (s, 2H), 3.75-3.71 (m, 10H), 3.65-3.51 (m, 12H), 3.44 (t, J = 4.0 HZ, 2H), 2.59 (t, J = 4.0 HZ, 2H), 2.24 (s, 3H), 2.14 (s, 3H), 1.75 (quint, J = 8.0, 4.0 Hz, 2H).
LC-MS: 877.4 (M + H)
EXAMPLE 2: In vitro Studies Using the Conjugates
HER2 Degradation Assay:
[0448] BT474 (breast cancer) cells are plated at 12,000 cells per well and incubated for 20-24hrs at 37°C at 5% CO2. Post cell incubation, compounds were reconstituted in DMSO to a stock concentration of 5mM. A compound plate is then prepared containing a 10 point dilution in DMSO. 2uL of these dilutions are then added to the cells for a final working concentration of 5uM to 0.0003uM. Compounds and cells are incubated for 16hrs. Media is then removed, cells washed, lysed, and analyzed for human total EbB2/Her2 levels by ELISA. HSP90 Binding:
[0449] The bindings of the conjugates to HSP90 were studied wth the HSP90a Assay Kit. The HSP90a Assay Kit was designed for identification of HSP90a inhibitors using fluorescence polarization. The assay was based on the competition of fluorescently labeled geldanamycin (GM) for binding to purified recombinant HSP90a. The key to the HSP90a Assay Kit was the fluorescently labeled geldanamycin. The fluorescently labeled geldanamycin was incubated with a sample containing HSP90a enzyme to produce a change in fluorescent polarization that can then be measured using a fluorescence reader.
[0450] In brief, compounds in DMSO were added into HSP solution by using Acoustic Technology and incubaged for 30 min. FITC-GM was added and incubated for 3 hours, fluorescent polarization (FP) was then measured and mP was calculated. Data pages included raw data (mP signal minus background), % Probe binding (relative to DMSO controls), and curve fits for control compound performed by GraphPad Prism software. Curve fits were performed where the enzyme activities at the highest concentration of compounds were less than 65%.
Table 5. HSP90a IC50 Data
Figure imgf000198_0001
Determining the Permeability of Payloads and Conjugates: [0451] In order to test the ability of the payloads and/or conjugates to enter cells, a cell monolayer assay was utilized employing Caco-2 cells (a human epithelial colorectal adenocarcinoma cell line).
[0452] Experimental Procedure: Caco-2 cells grown in tissue culture flasks were trypsinized, suspended in medium, and the suspensions were applied to wells of a Millipore 96 well Caco-2 plate. The cells were allowed to grow and differentiate for three weeks, feeding at 2-day intervals. For Apical to Basolateral (A— B) permeability, the test agent was added to the apical (A) side and amount of permeation was determined on the basolateral (B) side; for Basolateral to Apical (B— A) permeability, the test agent was added to the B side and the amount of permeation is determine on the A side.
[0453] Some of the conjugates have low permeability.
Table 6: Permeability Data
Figure imgf000199_0001
*: Papp values were expressed as “<” than the values that were calculated using the minimum concentration of the standards for receiver sides due to the fact that real concentration in the receivers were below quantitation limit (BQL).
Studies in LS174T Tumor Cell Assays:
[0454] PI3Ka mutations occur in about 15% to 30% of breast, endometrial and colon cancers. LS174T cells (colon cancer) with PI3Ka mutations were plated at 500 cells per well and incubated for 20-24hrs at 37°C at 5% CO2. Post cell incubation, compounds were reconstituted in DMSO to a stock concentration of 200uM. A compound plate was then prepared containing a 10 point dilution in RPMI +10%FBS + 0.25% DMSO. 5uL of the dilution was then added for a final working concentration range of lOuM to 0.0005uM. Compounds and cells were then incubated for 72hrs.
Cells were then analyzed by PI3K inhibiting and proliferation inhibition.
Figure imgf000200_0001
EXAMPLE 3: In vivo Studies Using the Conjugates
LS174t Colon Cancer Model
[0455] In this study using LS174t colon tumor xenograft model, accumulation and retention of the conjugates were tested. The conjugates were dosed at 25 mg/kg, 15 mg/kg or 7.5 mg/kg intravenously. Conjugate accumulation at 24hrs in tumor and plasma of the LS174t tumor-bearing mice were measured.
Figure imgf000201_0001
[0456] In further studies, efficacies of Compounds Cl, C48, C49 and C50 at various doses were evaluated in the LS174T xenograft model. Tumor volume changes are shown in FIG. 3 and FIG. 4. Tumor growth inhibition (TGI) data are shown in Table 7 and Table 8 below.
Table 7. TGI data of Cl and C48
Figure imgf000202_0001
Table 8. TGI data of Cl, C49 and C59
Figure imgf000202_0002
EXAMPLE 4: HSP90-PI3K Conjugates Induce Degradation of PI3Ka and
Inhibit Activation of Akt
[0457] In this study, the conjugates of the present disclosure were tested by
Western blot to determine PI3Kalpha (pl lOalpha) and pAKT (S473) levels in LS 174T cells after compounds treatment.
[0458] 5 compounds were tested in 6-well plate against LS 174T for 6 hours and
24 hours. The information for compounds preparation was listed below:
Figure imgf000202_0003
[0459] The LS174T cells were seeded and 2pl of compounds were transferred to the 6-well plate containing 2ml medium. It is a 1000 x dilution in total. Cell plates were incubated with the compounds as well as vehicle control at 5% CO2, 37°C for 6 hours or 24 hours. The cell lysis was then prepared and Western Blot was carried out.
Figure imgf000203_0001
[0460] The PI3Kalpha (pl lOalpha) and pAKT (S473) levels in LS 174T were shown in FIG. 1 and FIG. 2. All five conjugates tested lead to degradation. Cl caused slower degradation but highest level of inhibition. C32, having a PI3K/mTor dual inhibitor, caused very rapid degradation. At the highest concentration of 10 mM, there was less degradation. This is consistent with a distinctive feature of PROTACs called hook effect.
[0461] In a further in vivo study, the conjugates of the present disclosure were administered to mice bearing LS174T tumors and the degradation of the PI3K pl lOalpha protein was monitored.
[0462] Mice bearing LS174T tumors were treated with 45 mg/kg(mpk) or 25 mpk of Compound Cl or 30 mpk of Compound C48 (N = 3). Tumors were collected 6 hours and on each day from days 1-7 postdose. Xenograft tumors were processed for lysate using Cell Lysis Buffer 10X (Cell Signaling Technology, 9803) diluted to IX. Proteins were separated by SDS-PAGE on NuPAGE 4%-12% Bis-Tris Protein Gels, 1.5 mm, 15-well (Thermo Fisher Scientific, NP0336BOX). Proteins were transferred from the gel to a nitrocellulose membrane using iBlot Transfer Stack, nitrocellulose, regular size (Thermo Fisher Scientific, IB301001). After the transfer, the membrane was blocked in TBS Blocking Buffer (LI-COR, 927-60001). This was followed by overnight incubation of PI3 Kinase pl 10a (C73F8) Rabbit mAb #4249 Rabbit mAb #4060 (1 :500 dilution) and GAPDH (D4C6R) Mouse mAb #97166 (1 : 1000 dilution) at 4°C. After the primary incubation, the membrane was washed with Pierce 20X TBS Tween 20 Buffer (Thermo Fisher Scientific, 28360) which was diluted to IX and incubated further for one hour at room temperature with the secondary antibodies: 1 : 10,000 IRDye 680RD Goat anti-Rabbit IgG (LI-COR 926-68071) and 1 : 10,000 IRDye 800CW Goat anti -Mouse IgG (LI-COR, 926-32210). Then the membrane was imaged on the LI-COR Odyssey Imaging System. Bands were quantified on the basis of their size and brightness using LI-COR Odyssey imaging software.
[0463] PI3Kalpha protein levels were measured after treatments with Compound Cl (45 mg/kg (mgk) or 25 mgk) and C48 (30 mgk) upto 168 hours after dose. The reduction of PI3Kalpha protein is shown in FIG. 5.
[0464] In further studies, the effects of the compounds on PI3K degradation and cell proliferation (LS174T), and the concentration of the compounds in tumors were measured. The data are shown in Table 9 below.
Table 9. PI3K degradation, cell proliferation., and tumor concentration
Figure imgf000204_0001
Figure imgf000205_0001
[0465] In yet another study, the PI3K Degradation - Western Blotting experiments were carried out following the steps of: Human colon carcinoma cell line LS174T were counted and diluted to 106 cell/mL, using 2 mL per well in a 6-well plate. Cells were incubated with 1% DMSO or compound in indicated concentrations for 6 or 24h or cells were left untreated. Lysates were prepared and samples measured for total protein quantification by bicinchonic acid (BCA) assay supplemented with 5X loading buffer. Western blot was then run using standard procedures. Primary antibodies used were PI3Kinase alpha: CST# 4249, pAkt (S473): CST# 4060; Akt: CST# 4691; GAPDH: Millipore# MAB374. Degradation concentraion (DC50), %PI3K loss, %pAkt inhibition, %PI3Ka loss PD, %pAkt inhibition, and %PI3Ka loss PD were calculated and are shown in Table 10 below. The %PI3Ka degradations measured by western blot for Compound Cl, C50 and C48 are shown in FIG. 6.
Table 10. DC50, PI3K loss, and pAkt inhibition data
Figure imgf000206_0001

Claims

CLAIMS We claim:
1. A conjugate comprising an active agent coupled, via a linker, to an HSP90 targeting moiety, wherein the conjugate inhibits of the activity or function of the target protein of the active agent in cells.
2. The conjugate of claim 1, wherein the conjugate wherein the conjugate inhibits the activity or function of the target protein through degradation of the target protein.
3. The conjugate of claim 1 or claim 2, wherein the active agent is a PI3K inhibitor or a PI3K/mTor dual inhibitor.
4. The conjugate of claim 3, wherein the active agent comprises PQR514, PF- 04979064, BAY 80-6946 (Copanlisib), PQR530, Omipalisib (GSK2126458, GSK458), , PF-04691502, PI-103, BGT226 (NVP-BGT226), Apitolisib (GDC-0980, RG7422), Duvelisib (IPI-145, INK1197), AZD8186, Pilaralisib (XL147), PIK-93, Idelalisib (GS-1101), MLN1117, VS-5584, SB2343, GDC-0941, BM120, NVP- BKM120, Buparlisib, AZD8835, XL765 (SAR245409), GS-9820 Acalisib, GSK2636771, AMG-319, IPI-549, Perifosine, Alpelisib, TGR 1202 (RP5264), PX- 866, AMG-319, GDC-0980, GDC-0941, Sanofi XL147, XL499, XL756, XL147, PF- 46915032, BKM 120, CAL 263, SF1126, PX-886, KA2237, BEZ235, an isoquinolinone, or fragments/derivatives/analogs thereof.
5. The conjugate of claim 1, wherein the active agent is a PI3K/mT0R dual inhibitor.
6. The conjugate of claim 1, wehrein the active agent comprises Pl, P2, P3, P4, P5, P6, P7, P8, P9, P10, Pl 1, P12, or fragments/derivatives/analogs thereof.
7. The conjugate of claim 1, wherein the HSP90 targeting moiety comprises Ganetespib, Luminespib (AUY-922, NVP-AUY922), Debio-0932, MPC-3100, or Onalespib (AT-13387), SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71, AT13387, and fragments/derivatives/analogs thereof.
8. The conjugate of claim 7, wherein the HSP90 targeting moiety comprises TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM10, TM11, TM12, or fragments/derivatives/analogs thereof.
9. The conjugate of claim 1, wherein the linker is non-cleavable.
10. The conjugate of any of claims 1-9, wherein the cells are tumor cells.
11. A conjugate comprising an active agent coupled, via a linker, to an HSP90 targeting moiety, wherein the active agent is a PI3K inhibitor and wherein the PI3K inhibitor is PF-04979064, PQR514, BAY 80-6946 (Copanlisib), or fragments/derivatives/analogs thereof.
12. The conjugate of claim 11, wherein the HSP90 targeting moiety comprises Ganetespib, Luminespib (AUY-922, NVP-AUY922), Debio-0932, MPC-3100, or Onalespib (AT-13387), SNX-2112, 17-amino-geldanamycin hydroquinone, PU-H71, AT13387, or fragments/derivatives/analogs thereof.
13. The conjugate of claim 12, wherein the HSP90 targeting moiety comprises TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM10, TM11, TM12, or fragments/derivatives/analogs thereof.
14. The conjugate of claim 11, wherein the linker comprises an ester group, a disulfide group, an amide group, an acylhydrazone group, an ether group, a carbamate group, a carbonate group, or an urea group.
15. The conjugate of claim 11, wherein the linker is a cleavable linker.
16. The conjugate of claim 11, wherein the linker is a non-cleavable linker.
17. The conjugate of claim 11, wherein the conjugate has a molecular weight of less than about 50,000 Da, less than about 40,000 Da, less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, less than about 3,000 Da, less than 2000 Da, less than 1500 Da, less than 1000 Da, or less than 500 Da.
18. The conjugate of any of claims 11-17, wherein the active agent comprises Copanlisib or fragments/derivatives/analogs thereof.
19. The conjugate of claim 18, wherein the active agent comprises P7, P8, P9, P10, Pl 1, P12, or fragments/derivatives/analogs thereof.
20. The conjugate of claim 18, wherein the conjugate is Cl -Cl 6, C48-C77, or a pharmaceutically acceptable salt thereof.
21. The conjugate of any of claims 11-17, wherein the active agent comprises PF- 04979064 or fragments/derivatives/analogs thereof.
22. The conjugate of claim 21, wherein the active agent comprises P4, P5, P6, or fragments/derivatives/analogs thereof.
23. The conjugate of claim 21, wherein the conjugate is C17-C38, C78-87, or a pharmaceutically acceptable salt thereof.
24. The conjugate of any of claims 11-17, wherein the active agent comprises PQR530 or fragments/derivatives/analogs thereof.
25. The conjugate of claim 21, wherein the active agent comprises Pl, P2, P3, or fragments/derivatives/analogs thereof.
26. The conjugate of claim 24, wherein the conjugate is C39-C47, C88-C96, C98- C116, Cl 20, or a pharmaceutically acceptable salt thereof.
27. The conjugate of any of claims 11-17, wherein the active agent comprises GDC-0326 or fragments/derivatives/analogs thereof.
28. The conjugate of claim 27, wherein the conjugate is Cl 17, Cl 18, Cl 19, or a pharmaceutically acceptable salt thereof.
29. The conjugate of any of claims 1-28, further comprising a permeability modulating unit.
30. The conjugate of any of claims 1-28, further comprising a pharmacokinetic modulating unit.
31. A conjugating having structure selected from the group consisting of C1-C96, C98-C120, or a pharmaceutically acceptable salt thereof.
32. A pharmaceutical composition comprising the conjugate of any of claims 1-31 and at least one pharmaceutically acceptable excipient.
33. A method of degrading a protein in a cell comprising administering a therapeutically effective amount of the conjugate of any of claims 1-31 to the cell.
34. The method of claim 33, wherein the cell is a tumor cell.
35. The method of claim 33, wherein the protein is also inhibited.
36. The method of claim 33, wherein the protein is PI3K.
37. A method of reducing cell proliferation comprising administering a therapeutically effective amount of at least one conjugate of any of claims 1-31 to the cell.
38. The method of claim 37, wherein the cell is a cancer cell.
39. The method of claim 38, wherein the cancer cell is a small-cell lung cancer cell, a non-small-cell lung cancer cell, a sarcoma cell, a pancreatic cancer cell, a breast cancer cell, or a colon cancer cell.
40. A method of treating cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 32.
41. The method of claim 40, wherein the cancer is small-cell lung cancer cell, a non-small-cell lung cancer cell, a sarcoma cell, a pancreatic cancer cell, a breast cancer cell, or a colon cancer cell.
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