Verrucarin A derivatives and their antibody-drug conjugates

Abstract

Verrucarin A derivatives, linker-verrucarin A derivatives and antibody-drug conjugates thereof are provided herein. In one embodiment, the verrucarin A derivatives, linker-verrucarin A derivatives and antibody-drug conjugates are useful in treating viral diseases.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 299,824, filed January 14, 2022, and U.S. Provisional Patent Application No. 63 / 367,108, filed June 27, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Statement of Federally Funded Research This invention was made with United States Government support under OTA Contract No. HHSO100201700020C awarded by the Biomedical Advanced Research and Development Authority (BARDA). The United States Government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing, which is submitted herewith as an XML file entitled "10968WO01_SL.xml" created on January 9, 2023, having a size of 457,018 bytes, and which is hereby incorporated by reference in its entirety.

[0004] Field Verrucarin A derivatives and antibody-drug conjugates (ADCs) thereof are provided herein. The verrucarin A derivatives and ADCs provided herein are useful, inter alia, for the treatment of viral diseases. [Background technology]

[0005] Verrucarin A is a trichothecin toxin that has been studied in the treatment of various cancers. However, the toxicity of verrucarin A is too high to provide a therapeutic window for its use as a therapy. Therefore, ADCs of verrucarin A have been proposed to solve this problem (U.S. Pat. Nos. 4,744,981, 10,232,051 and 10,985,112; U.S. Patent Application Publication No. 2015 / 0250896). Verrucarin A has also been shown to have significant antiviral activity against, for example, vaccinia virus strain DII and Newcastle disease virus strain Miyadera (NDV) (Tamura et al. J. Anitbiot. (Tokyo) 1968, 21(2):160-161) and arenavirus Junin (JUNV) (Garcia et al. Planta Med. 2002, 68(3):209-212).

[0006] ADCs combine the power of antibody specificity with the ability to site-specifically target specific cell types or tissues with a payload. Such site specificity allows the use of toxic payloads that would otherwise lack a sufficient therapeutic window. Research in this area has attracted considerable interest and has led to marketed drugs, including ADCETRIS® (brentuximab vedotin) and KADCYLA™ (ado-trastuzumab emtansine).

[0007] With some exceptions, current antiviral therapies merely treat disease symptoms or inhibit viral replication. Such treatments can delay disease progression until either natural immune responses become effective or viral quiescence is reached. Furthermore, there are few, if any, effective broad-spectrum antiviral therapeutics. Thus, there is a large unmet need for the treatment and prevention of viral infections.

[0008] Furthermore, the development of antiviral drugs that utilize alternative mechanisms of action is important to reduce the risk of viruses developing resistance to existing therapeutics. The use of ADCs that contain broad-spectrum payloads can facilitate the use of broad-spectrum antiviral antibodies that often lack sufficient neutralizing activity to be effective on their own. Thus, there is a continuing need for effective and site-specific methods for treating viral infections. Summary of the Invention

[0009] Verrucarin A derivatives and ADCs thereof are provided herein. In one embodiment, the verrucarin A derivatives for use in the compositions and methods provided herein have the formula I: [ka] or a pharma- ceutically acceptable derivative thereof, where X, Y and Z are as defined elsewhere herein.

[0010] In another embodiment, the verrucarin A derivative for use in the compositions and methods provided herein has formula II: [ka] or a pharma- ceutically acceptable derivative thereof, wherein R 1 is as defined elsewhere herein.

[0011] In another embodiment, a compound of formula III: [ka] or a pharma- ceutical acceptable derivative thereof, wherein X 1 , D, L, Y and Z are as defined elsewhere herein.

[0012] In another embodiment, a compound of formula IV: [ka] or a pharma- ceutically acceptable derivative thereof, wherein L and n are as defined elsewhere herein.

[0013] In another embodiment, a compound of formula V: Z-(LX) v V or a pharma- ceutically acceptable derivative thereof, wherein: Z is an antiviral antigen-binding domain; L is a linking group as defined elsewhere herein; X is a verrucarin A derivative; v is an integer from 1 to 12.

[0014] In another embodiment, the verrucarin A derivatives and ADCs thereof provided herein are useful in methods of treating viral diseases. In one embodiment, the viral disease is influenza, COVID-19 or Ebola. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] i.Definition To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that there are multiple definitions for terms herein, the definitions in this section shall prevail unless otherwise stated.

[0017] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] As used herein, a "subject" is an animal, such as a mammal, including a human, such as a patient.

[0019] As used herein, biological activity refers to the in vivo activity of a compound or the physiological response that occurs upon in vivo administration of a compound, composition or other mixture. Thus, biological activity encompasses the therapeutic effects and pharmacokinetic behavior of such compounds, compositions and mixtures. Biological activity can be observed in in vitro systems designed to test such activity.

[0020] As used herein, "influenza" refers to influenza A, B, and C. "Influenza A" refers to a virus that includes 18 subtypes defined by its hemagglutinin protein (H1-H18) and 11 subtypes defined by its neuraminidase protein (N1-N11). All combinations of these subtypes are possible and are included within the scope of influenza A of this disclosure. Subtypes that affect humans include H1, H2, H3, H5, H6, H7, H9, and H10; and N1, N2, N6, N7, N8, and N9. Thus, in certain embodiments, influenza A has a combination of these subtypes. In certain embodiments, influenza A can be of subtype H1N1, H3N2, etc.

[0021] As used herein, "SARS-CoV-2" refers to the SARS-CoV-2 virus, including variants thereof (e.g., variants under surveillance, variants of interest, variants of concern, and variants of high importance). As of June 2022, variants under surveillance include alpha (B.1.1.7 and Q lineages), beta (B.1.351 and sublineages), gamma (P.1 and sublineages), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1), 1.617.3, mu (B.1.621 and B.1.621.1), and zeta (P.2). As of June 2022, variants of concern include Omicron (B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 and BA.5 lineages).

[0022] As used herein, "antigen-binding domain" refers to any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide-presenting molecule, or polypeptide-containing construct that can specifically bind to a particular antigen of interest. As used herein, "antigen-binding domain" includes antibodies and antigen-binding fragments of antibodies. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of the respective protein, polypeptide, or protein fragment, unless specified to be from a non-human species.

[0023] As used herein, the term "specifically binds" or cognate terms refers to an antigen-binding domain that binds to an antigen with a dissociation constant (K D ) means that the antibody forms a complex with a specific antigen, characterized by the fact that the antibody does not bind to other unrelated antigens under normal test conditions.

[0024] As used herein, an "unrelated antigen" is a protein, peptide, or polypeptide that shares less than 95% amino acid identity with each other.

[0025] The term "antibody," as used herein, refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., on or in an influenza virus particle or a SARS-CoV-2 virus particle). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or VVR). H The heavy chain constant region is made up of three domains: H 1. C H 2, and C H Each light chain comprises a light chain variable region (herein, LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0026] As used herein, the term "antigen-binding fragment" of an antibody means a naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0027] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0028] As used herein, the term "recombinant human antibody" means any human antibody prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving the joining of human immunoglobulin gene sequences to other DNA sequences.

[0029] As used herein in connection with amino acid sequences, the term "substantial identity" or "substantially identical" means that two amino acid sequences have at least 95%, 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weightings.

[0030] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time interactions by detection of changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0031] As used herein, "K D The term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise indicated, the K D Values ​​are K values ​​measured by surface plasmon resonance assay at 25 °C. D Points to a value.

[0032] As used herein, pharma- ceutically acceptable salts include amine salts, such as, but not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as, but not limited to, lithium, potassium, and sodium; alkaline earth metal salts, such as, but not limited to, barium, calcium, and magnesium; transition metal salts, such as, but not limited to, zinc; and inorganic salts, such as, but not limited to, sodium hydrogen phosphate and disodium phosphate, salts of mineral acids, such as, but not limited to, hydrochloride and sulfate; and salts of organic acids, such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, mesylate, and fumarate.

[0033] As used herein, treatment refers to any manner in which one or more of the symptoms of a disease or disorder are improved or otherwise beneficially altered. Treatment also includes any medical use of the compositions of the present invention, such as use to treat viral infections.

[0034] As used herein, amelioration of symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any relief, whether permanent or temporary, ongoing or transient, that may result from or be associated with administration of the compound or pharmaceutical composition.

[0035] As used herein, IC 50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures that response.

[0036] Where moieties are specified by their conventional chemical formula, written from left to right, they equally encompass the chemically identical moieties that would be obtained by writing the structure from right to left, e.g., -CH 2 O- is -OCH 2 -Equal to.

[0037] The term "alkyl" alone or as part of another substituent means, unless otherwise specified, a straight-chain (i.e., unbranched) or branched-chain saturated hydrocarbon radical. The term "alkylene" alone or as part of another substituent means a divalent radical derived from alkyl. Generally, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, and includes such groups having 10 or fewer carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 6 or fewer carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0038] The term "alkenyl," alone or as part of another substituent, means, unless otherwise specified, a straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical having one or more carbon-carbon double bonds. The term "alkenylene," alone or as part of another substituent, means a divalent radical derived from alkenyl. Generally, an alkenyl (or alkenylene) group will have from 1 to 24 carbon atoms, and includes those groups having 10 or fewer carbon atoms. A "lower alkenyl" or "lower alkenylene" is a shorter chain alkenyl or alkenylene group, generally having 6 or fewer carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., ethenyl), 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and the higher homologs and isomers.

[0039] The term “alkynyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical having one or more carbon-carbon triple bonds, which can be any number of carbon atoms as specified (i.e., C 1 ~C 10 (meaning 1 to 10 carbons) Examples of alkynyl groups include, but are not limited to, ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

[0040] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to such alkyl groups attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

[0041] The term "heteroalkyl", alone or in combination with another term, means, unless otherwise stated, a straight or branched chain hydrocarbon radical consisting of heteroatoms in the chain selected from the group consisting of O, N, P, Si and S, where the nitrogen and sulfur atoms may be optionally oxidized, the nitrogen atom may have alkyl substituents to fill the valence, and / or may be optionally quaternized. The heteroatom(s) O, N, P, Si and S may be placed at any interior position of the heteroalkyl group. Examples include -CH 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2 -S-CH 2 -CH 3 , -CH 2 -CH 2 -S(O)-CH 3 , -CH 2 -CH 2 -S(O) 2 -CH 3 , -CH=CH-O-CH 3 , -CH 2 -CH=N-OCH 3 , and -CH=CH-N(CH 3 )-CH 3 Up to two heteroatoms may be, for example, CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl, such as -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH2 Examples include, but are not limited to, -. For alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, a group of the formula -C(O) 2 R'- is -C(O) 2 R'- and -R'C(O) 2 - represents both.

[0042] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, represent cyclic forms of "alkyl" and "heteroalkyl", respectively, including bicyclic, tricyclic, and bridged bicyclic groups, unless otherwise specified. In addition, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. The terms "cycloalkylene" and "heterocycloalkylene", alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl or heterocycloalkyl. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornanyl, bicyclo(2.2.2)octanyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, 1- or 2-azabicyclo(2.2.2)octanyl, and the like.

[0043] The term "aryl", unless otherwise specified, means a polyunsaturated aromatic hydrocarbon substituent, which may be monocyclic or polycyclic (in one embodiment, 1 to 3 rings) fused together or covalently linked. The term "heteroaryl" refers to an aryl group containing from 1 to 4 heteroatoms selected from N, O and S in the ring(s), where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. The terms "arylene" and "heteroarylene", alone or as part of another substituent, mean a divalent radical derived from an aryl or heteroaryl. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4 Examples of heteroaryl groups include 1-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. The term "heteroarylium" refers to a heteroaryl group bearing a positive charge at one or more of the heteroatoms.

[0044] Each of the above terms is meant to include both substituted and unsubstituted forms of the indicated radical. Non-limiting examples of substituent moieties for each type of radical are provided below.

[0045] Substituent moieties for alkyl, heteroalkyl, alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups can in one embodiment be selected from deuterium, -OR', ═O, ═NR', ═N-OR', -NR'R", -SR', halo, -SiR'R"R'", -OC(O)R', -C(O)R', -CO 2 R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) 2 R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O) 2 R', -S(O) 2 NR'R", -NRSO 2 R', -CN and -NO 2 In one embodiment, substituent moieties for cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups also include substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl. R', R", R'", and R"" are each, in one embodiment, independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl groups. When the compounds provided herein include more than one R group, e.g., each R group is independently selected, as are each of the R', R", R"', and R"" groups, when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituent moieties, the term "alkyl" includes, for example, haloalkyl (e.g., -CF 3 and -CH2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 One of ordinary skill in the art will recognize that this is intended to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as aryl, aryloxy ...

[0046] Substituent moieties for aryl and heteroaryl groups, in one embodiment, include deuterium, halo, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO 2 R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) 2 R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O) 2 R', -S(O) 2 NR'R", -NRSO 2 R', -CN and -NO 2 , -R', -N 3 , -CH(Ph) 2 , Fluoro(C 1 ~C 4 ) alkoxy, and fluoro (C 1 ~C 4 ) alkyl, where R', R", R'', and R"" are, in one embodiment, independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound provided herein includes more than one R group, for example, each R group is independently selected, as are each of the R', R", R"', and R"" groups, when more than one of these groups is present.

[0047] Two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring are optionally of the formula -Q'-C(O)-(CRR') q Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -Q"-, where Q' and Q" are independently -NR-, -O-, -CRR', or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -A-(CH 2 ) r A and B are each independently selected from the group consisting of -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed may optionally be replaced with a double bond. Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -(CRR') s -X'-(CR”R”') d -, s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O) 2 - or -S(O) 2 The substituent moieties, R, R', R", and R"', in one embodiment, are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0048] The term "halo," by itself or as part of another substituent, means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, "halo(C 1 ~C 4 The term "alkyl" is meant to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0049] The term "oxo," as used herein, means an oxygen atom that is double bonded to a carbon atom.

[0050] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si).

[0051] Certain verrucarin A derivatives or ADCs provided herein have asymmetric carbon atoms (optical centers) or double bonds, and racemates, diastereomers, tautomers, geometric isomers, and individual isomers are encompassed within the scope of the present disclosure. The ADCs provided herein do not include those known in the art to be too unstable to synthesize and / or isolate.

[0052] II. Verrucarin A Derivatives In one embodiment, a compound of formula I: [ka] Provided herein is a verrucarin A derivative or a pharma- ceutically acceptable derivative thereof, wherein X is NR 1 R 2 , OR 3 or S.R. 4 and; R 1 and R 2 each independently represents H, alkyl, OR 5 Or COR 6 or together with the nitrogen atom to which they are attached form a heterocycloalkyl; R 3 is alkyl or COR 7 and; R 4 is H, alkyl, or COR 8 and; R 5 is H or alkyl; R 6 is R 9 , OR 10 or NR 11 R 12 and; R 7 ~R 9 each is independently alkyl or aralkyl; R 10 ~R 12 each is independently H, alkyl, or aralkyl; Y is H or OH; (i) Z is O; [ka] is a single bond; or (ii) Z is absent, [ka] is a double bond.

[0053] In another embodiment, R 1 and R 2 each independently represents H, lower alkyl, or COR 6 or together with the nitrogen atom to which they are attached form a heterocycloalkyl.

[0054] In another embodiment, R 1 and R 2 are each independently H, methyl, ethyl, and W is OR 13 Or NR 14 R 15 C(O)-alkylene-CO-W, or V is OR 16 Or NR 17 R 18 C(O)-CH(V)-CH 3 or together with the nitrogen atom to which they are attached form piperazinyl, piperidinyl, pyrrolidinyl, imidazolidinyl or azepinyl; R 13 and R 16are each independently H or alkyl; R 14 , R 15 , R 17 , and R 18 Each is independently H, alkyl, hydroxy, or alkoxy.

[0055] In another embodiment, R 1 and R 2 are each independently H, methyl, ethyl, and W is OR 13 Or NR 14 R 15 C(O)-(C 2~4 alkylene)-CO-W, or V is OR 16 Or NR 17 R 18 C(O)-CH(V)-CH 3 or together with the nitrogen atom to which they are attached form piperazinyl; R 13 and R 16 are each independently H or methyl; R 14 , R 15 , R 17 , and R 18 Each is independently H, methyl, hydroxy, or methoxy.

[0056] In another embodiment, R 1 is H, methyl, ethyl, C(O)CH 2 CH 2 COOH, C(O)CH 2 CH 2 CH 2 COOH, C(O)CH 2 CH 2 CH 2 CH 2 COOH, C(O)CH 2 CH 2 CH 2 COOMe, C(O)CH 2 CH 2 CH 2 CONHOH, C(O)CH 2 CH 2 CH 2 CONHOMe, C(O)-CH(OH)-CH 3, C(O)-CH(NH 2 )-CH 3 or C(O)-CH(NMe 2 )-CH 3 It is.

[0057] In another embodiment, R 2 is H or methyl.

[0058] In another embodiment, R 1 and R 2 together with the nitrogen atom to which they are attached form 4-methyl-1-piperazinyl.

[0059] In another embodiment, R 3 is methyl or C(O)-alkyl, where alkyl is optionally substituted. In another embodiment, R 3 is methyl or C(O)-CH(NR 19 R 20 )-CH 3 and R 19 and R 20 Each is independently H, alkyl, or CO-alkyl. 3 is methyl, C(O)-CH(NHMe)-CH 3 or C(O)-CH(NHAc)-CH 3 It is.

[0060] In another embodiment, R 4 COR 8 In another embodiment, R 4 is C(O)Me.

[0061] In another embodiment, R 5 is H or methyl.

[0062] In another embodiment, R 6 is R 9 and R 9 is alkylene-CO-W, W is OR 13 or NR 14 R 15 and R 13is H or methyl; R 14 and R 15 Each is independently H, methyl, hydroxy, or methoxy. 6 is CH 2 CH 2 COOH, CH 2 CH 2 CH 2 COOH, CH 2 CH 2 CH 2 CH 2 COOH, CH 2 CH 2 CH 2 COOMe, C.H. 2 CH 2 CH 2 CONHOH or CH 2 CH 2 CH 2 CONHOMe.

[0063] In another embodiment, R 7 and R 8 Each is independently alkyl. In another embodiment, R 7 and R 8 Each is independently methyl.

[0064] In another embodiment, R 10 ~R 12 Each is independently H or alkyl. In another embodiment, R 10 ~R 12 Each is independently H or methyl.

[0065] In another embodiment, Y is H and Z is absent. [ka] is a double bond. In another embodiment, Y is OH and Z is absent. [ka] is a double bond. In another embodiment, Y is H and Z is O; [ka] is a single bond.

[0066] In one embodiment, the verrucarin A derivative of formula I for use in the compositions and methods provided herein has formula Ia: [ka] or a pharma- ceutically acceptable derivative thereof, in which the variables are as defined elsewhere herein.

[0067] In another embodiment, the verrucarin A derivative of formula I for use in the compositions and methods provided herein has formula Ib: [ka] or a pharma- ceutically acceptable derivative thereof, in which the variables are as defined elsewhere herein.

[0068] In one embodiment, a compound of formula II: [ka] Provided herein is a verrucarin A derivative or a pharma- ceutically acceptable derivative thereof, wherein R 1 H or -COR 6 and; R 6 is -alkylene-COOH.

[0069] In another embodiment, R 1 is H. In another embodiment, R 1 -COR 6 It is.

[0070] In another embodiment, R 6 is -(CR 21 R 22 ) m COOH, where R 21 and R 22 is independently H or alkyl; and m is an integer from 0 to 6. 21 and R 22 Each is independently H or methyl. In another embodiment, R 21 and R 22 are H, respectively.

[0071] In another embodiment, m is 2, 3 or 4. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0072] In another embodiment, R is -COR 1 and R 1 is -(CH 2 ) m COOH and m is 2, 3, or 4. In another embodiment, R 1 is -CO-(CH 2 ) 2 In another embodiment, R 1 is -CO-(CH 2 ) 3 In another embodiment, R 1 is -CO-(CH 2 ) 4 It is COOH.

[0073] In another embodiment, the verrucarin A derivative of formula II for use in the compositions and methods provided herein has the formula IIa: [ka] or a pharma- ceutically acceptable derivative thereof, wherein R 1 is as defined elsewhere herein.

[0074] In another embodiment, the verrucarin A derivative of formula II for use in the compositions and methods provided herein has the formula IIb: [ka] or a pharma- ceutically acceptable derivative thereof, wherein R 1 is as defined elsewhere herein.

[0075] In one embodiment, the verrucarin A derivative for use in the compositions and methods provided herein is selected from the following: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0076] In another embodiment, the verrucarin A derivative for use in the compositions and methods provided herein is selected from the following: [Table 2]

[0077] III. Synthesis of Verrucarin A Derivatives The verrucarin A derivatives provided herein can be prepared by methods well known to those skilled in the art. For example, the free hydroxyl group of verrucarin A can be derivatized as a leaving group, e.g., triflate, using standard methods, e.g., trifluoromethanesulfonic anhydride and pyridine. The leaving group can be replaced by a nucleophile, e.g., an amine or a protected amine, an azide, etc. The resulting amino or azide derivative can then be deprotected or reduced, e.g., with triphenylphosphine, to obtain amino-verrucarin A, e.g., compound 4.

[0078] Amino-verrucarin A is then acylated under standard conditions, for example with an alkylene dicarboxylic anhydride and a tertiary amine, to give HOOC-alkylene-CO-verrucarin A amides, such as compounds 5, 6, and 7.

[0079] IV. Linker-Verrucarin A Derivatives In one embodiment, a compound of formula III: [ka] Provided herein is a linker-verrucarin A derivative of the formula: 1 is O or NH; D is absent or -C(O)-(CH 2 ) 2~5 -C(O)- or -O-NH-C(O)-(CH 2 ) 2~5 -C(O)-; L is a linking group; Y is H or OH; (i) Z is O, [ka] is a single bond; or (ii) Z is absent, [ka] is a double bond.

[0080] In one embodiment, Y is H and Z is absent. [ka] is a double bond. In another embodiment, Y is OH and Z is absent. [ka] is a double bond. In another embodiment, Y is H and Z is O; [ka] is a single bond.

[0081] In another embodiment, X is O. In another embodiment, X is NH.

[0082] In another embodiment, D is absent. In another embodiment, D is -C(O)-(CH 2 ) 2~5 In another embodiment, D is -O-NH-C(O)-(CH 2 ) 2~5 In another embodiment, D is -C(O)-(CH 2 ) 2 In another embodiment, D is -C(O)-(CH 2 ) 3 In another embodiment, D is -C(O)-(CH 2 ) 4 In another embodiment, D is -O-NH-C(O)-(CH 2 ) 3 -C(O)-.

[0083] In one embodiment, the linker-verrucarin A derivative of formula III has the formula IIIa: [ka] or a pharma- ceutically acceptable derivative thereof, in which the variables are as defined elsewhere herein.

[0084] In one embodiment, the linker-verrucarin A derivative of formula III has formula IIIb: [ka] or a pharma- ceutically acceptable derivative thereof, in which the variables are as defined elsewhere herein.

[0085] In one embodiment, the linker-verrucarin A derivative for use in the compositions and methods provided herein has formula IV: [ka] or a pharma- ceutically acceptable derivative thereof, wherein L is a linking group and n is an integer from 1 to 4.

[0086] In another embodiment, n is 1, 2 or 3. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3.

[0087] In one embodiment, the linker-verrucarin A derivative of formula IV has formula IVa: [ka] or a pharma- ceutically acceptable derivative thereof, wherein the variables are as defined herein.

[0088] In another embodiment, the linker-verrucarin A derivative of formula IV has formula IVb: [ka] or a pharma- ceutically acceptable derivative thereof, wherein the variables are as defined herein.

[0089] In certain embodiments, L is any group or moiety that links, connects, or binds selenium to a payload. Suitable linkers can be found, for example, in Antibody-Drug Conjugates and Immunotoxins, Phillips, GL, Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates, Wang, J., Shen, W.-C., and Zaro, JL, Eds.; Springer International Publishing, 2015. In certain embodiments, the L group of the verrucarin A derivatives and ADCs provided herein is stable enough to take advantage of the circulating half-life of the antigen-binding domain while at the same time being able to release its payload after internalization of the ADC with which the antigen is associated. The linker L may be cleavable or non-cleavable. Cleavable linkers for use as L herein include linkers that are cleaved by intracellular metabolism after internalization, such as hydrolysis, reduction, or enzymatic cleavage. Non-cleavable linkers for use as L herein include linkers that release the attached payload by lysosomal degradation of the antigen-binding domain after internalization. Suitable L linkers include, but are not limited to, acid labile linkers, hydrolytically unstable linkers, enzyme cleavable linkers, reduction labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable L linkers also include, but are not limited to, linkers that are or include peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyloxy (PAB) units.

[0090] Any linker molecule or linker technology known in the art can be used as L in the verrucarin A derivatives and ADCs provided herein. In certain embodiments, the L linker is a cleavable linker. In other embodiments, the L linker is a non-cleavable linker. In certain embodiments, L linkers that can be used in the ADCs provided herein include, for example, linkers that include or consist of MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide portion of a protease cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide portion of a protease cleavable linker, PAB (p-aminobenzyloxy), and variants and combinations thereof. Further examples of L linkers that can be used in the verrucarin A derivatives and ADCs provided herein are disclosed, for example, in U.S. Pat. No. 7,754,681, and Ducry, Bioconjugate Chem., 2010, 21:5-13 and references cited therein.

[0091] In certain embodiments, the L linker is stable under physiological conditions. In certain embodiments, the L linker is cleavable, e.g., in the presence of an enzyme or at a particular pH range or value, and can release at least the payload portion. In some embodiments, the L linker comprises an enzyme-cleavable moiety. In one embodiment, enzyme-cleavable L linkers include, but are not limited to, peptide bonds, ester bonds, and hydrazones. In some embodiments, the L linker comprises a cathepsin-cleavable linker.

[0092] In some embodiments, the L linker comprises a non-cleavable moiety.

[0093] In some embodiments, the L linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-α-amino acids. In some embodiments, the L linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of the amino acid are linked to the side chain groups shown below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the L linker comprises lysine, valine, and citrulline. In some embodiments, the L linker comprises lysine, valine, and alanine. In some embodiments, the L linker comprises valine and alanine. In some embodiments, the linker is a peptide comprising or consisting of the amino acids alanine and alanine, i.e., the divalent -AA-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid and alanine, i.e., -EA-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid and glycine, i.e., -EG-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glycine and glycine, i.e., -GG-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamine, valine, and citrulline, i.e., -QV-Cit- or -QVCit-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid, valine, and citrulline, i.e., -EV-Cit- or -EVCit-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids -GGGGS-. In some embodiments, the linker is a peptide comprising or consisting of the amino acids -GGGGG-.In some embodiments, the linker is a peptide that comprises or consists of the amino acids -GGGGK-. In some embodiments, the linker is a peptide that comprises or consists of the amino acids -GFGG-. In some embodiments, the linker is a peptide that comprises or consists of the amino acids lysine, valine, and citrulline, i.e., -KVCit-. In some embodiments, the linker is a peptide that comprises or consists of the amino acids -KVA-. In some embodiments, the linker is a peptide that comprises or consists of the amino acids -VA-.

[0094] In some embodiments, the L linker comprises a self-immolative group. The self-immolative group can be any such group known to one of skill in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC) and p-amino-α-methylbenzyl (MePAB). One of skill in the art will recognize that the self-immolative group can undergo a chemical reaction that releases the remaining atoms of the L linker from the payload.

[0095] In other embodiments, the L group may be modified with one or more enhancing groups. In certain embodiments, the enhancing group may be linked to the side chain of any amino acid of L. In one embodiment, the amino acids for linking the enhancing group include lysine, asparagine, aspartic acid, glutamine, glutamic acid, and citrulline. The link to the enhancing group may be a direct bond to the amino acid side chain, or the link may be indirect through a spacer and / or reactive group. In one embodiment, the spacer and reactive group include any of those described herein. In certain embodiments, the enhancing group may be any group that provides a beneficial effect to the payload, linker payload, or ADC, including, but not limited to, biological, biochemical, synthetic, solubilizing, imaging, detection, and reactive effects. In certain embodiments, the enhancing group is a hydrophilic group. In certain embodiments, the enhancing group is a cyclodextrin. In certain embodiments, the enhancing group is an alkyl, heteroalkyl, alkenyl, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, heteroalkenyl amine (e.g., a quaternary amine), or heteroalkenyl sugar. In certain embodiments, the sugar includes, but is not limited to, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like. In certain embodiments, the sugar includes sugar acids such as glucuronic acid, further including complex forms such as glucuronides (i.e., by glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like. The cyclodextrin may be any cyclodextrin known to one of skill in the art. In certain embodiments, the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or a mixture thereof, hi certain embodiments, the cyclodextrin is alpha cyclodextrin.In certain embodiments, the cyclodextrin is a beta cyclodextrin. In certain embodiments, the cyclodextrin is a gamma cyclodextrin. In certain embodiments, the enhancing group can improve the solubility of the remainder of the ADC. In certain embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or unsubstituted. In certain embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH. 2 ) 1~5 SO 3 H, -(CH 2 ) x -NH-(CH 2 ) 1~5 SO 3 H, -(CH 2 ) x -C(O)NH-(CH 2 ) 1~5 SO 3 H, -(CH 2 CH 2 O) y -C(O)NH-(CH 2 ) 1~5 SO 3 H, -(CH 2 ) x -N((CH 2 ) 1~5 C(O)NH(CH 2 ) 1~5 SO 3 H) 2 , -(CH 2 ) x -C(O)N((CH 2 ) 1~5 C(O)NH(CH 2 ) 1~5 SO 3 H) 2 , or -(CH 2 CH 2 O) y -C(O)N((CH 2 ) 1~5 C(O)NH(CH 2 ) 1~5 SO 3 H) 2where x is 1, 2, 3, 4, or 5 and y is 1, 2, 3, 4, or 5. In one embodiment, the alkyl or alkenyl sulfonic acid is -(CH 2 ) 1~5 SO 3 H. In another embodiment, the heteroalkyl or heteroalkenyl sulfonic acid is -(CH 2 ) x -NH-(CH 2 ) 1~5 SO 3 H, where x is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH 2 ) x -C(O)NH-(CH 2 ) 1~5 SO 3 H, where x is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH 2 CH 2 O) y -C(O)NH-(CH 2 ) 1~5 SO 3 H, where y is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH 2 ) x -N((CH 2 ) 1~5 C(O)NH(CH 2 ) 1~5 SO 3 H) 2 where x is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH 2 ) x -C(O)N((CH 2 ) 1~5 C(O)NH(CH 2 ) 1~5 SO 3 H) 2where x is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH 2 CH 2 O) y -C(O)N((CH 2 ) 1~5 C(O)NH(CH 2 ) 1~5 SO 3 H) 2 where y is 1, 2, 3, 4, or 5.

[0096] In some embodiments, the linker is [ka] where: SP 1 is a spacer; SP 2 is a spacer; [ka] is one or more bonds to antigen-binding domain Z; [ka] is one or more bonds to a verrucarin A derivative; each AA is an amino acid residue; n is an integer from 0 to 10.

[0097] SP 1 The spacer is (AA) n A moiety that links a moiety or residue to the antigen-binding domain Z or to a reactive group residue attached to Z. 1Spacers include, but are not limited to, those that contain alkylene or polyether, or both. The terminus of the spacer, e.g., the portion of the spacer that is linked to Z or AA, may be a moiety derived from a reactive moiety used to link the antibody or AA to the spacer during chemical synthesis of the conjugate. In certain embodiments, n is 0, 1, 2, 3, or 4 (i.e., when n is 0, AA is absent). In one embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4.

[0098] In some embodiments, the SP 1 The spacer comprises an alkylene. In some embodiments, SP 1 The spacer is C 5~7 In some embodiments, SP 1 The spacer comprises a polyether. In some embodiments, SP 1 The spacer comprises a polymer of ethylene oxide, such as polyethylene glycol (PEG). The polymer unit of polyethylene glycol generally consists of the group -(OCH 2 CH 2 ) p -, where p is an integer from 1 to 100. For example, -(OCH 2 CH 2 ) 2 -OCH 2 CH 2 -OCH 2 CH 2 -or PEG 2 In certain embodiments, the polyethylene glycol can be represented as PEG. 1 In certain embodiments, the polyethylene glycol is PEG 2 In certain embodiments, the polyethylene glycol is PEG 3 In certain embodiments, the polyethylene glycol is PEG 4 In certain embodiments, the polyethylene glycol is PEG 5 In certain embodiments, the polyethylene glycol is PEG 6In certain embodiments, the polyethylene glycol is PEG 7 In certain embodiments, the polyethylene glycol is PEG 8 In certain embodiments, the polyethylene glycol is PEG 9 In certain embodiments, the polyethylene glycol is PEG 10 In certain embodiments, the polyethylene glycol is PEG 11 In certain embodiments, the polyethylene glycol is PEG 12 In certain embodiments, the polyethylene glycol is PEG 13 In certain embodiments, the polyethylene glycol is PEG 14 In certain embodiments, the polyethylene glycol is PEG 15 In certain embodiments, the polyethylene glycol is PEG 16 In certain embodiments, the polyethylene glycol is PEG 17 In certain embodiments, the polyethylene glycol is PEG 18 In certain embodiments, the polyethylene glycol is PEG 19 In certain embodiments, the polyethylene glycol is PEG 20 In certain embodiments, the polyethylene glycol is PEG 21 In certain embodiments, the polyethylene glycol is PEG 22 In certain embodiments, the polyethylene glycol is PEG 23 In certain embodiments, the polyethylene glycol is PEG 24 In certain embodiments, the polyethylene glycol is PEG 25 In certain embodiments, the polyethylene glycol is PEG 26 In certain embodiments, the polyethylene glycol is PEG 27 In certain embodiments, the polyethylene glycol is PEG 28 In certain embodiments, the polyethylene glycol is PEG 29In certain embodiments, the polyethylene glycol is PEG 30 In certain embodiments, the polyethylene glycol is PEG 31 In certain embodiments, the polyethylene glycol is PEG 32 In certain embodiments, the polyethylene glycol is PEG 33 In certain embodiments, the polyethylene glycol is PEG 34 In certain embodiments, the polyethylene glycol is PEG 35 In certain embodiments, the polyethylene glycol is PEG 36 In certain embodiments, the polyethylene glycol is PEG 37 In certain embodiments, the polyethylene glycol is PEG 38 In certain embodiments, the polyethylene glycol is PEG 39 In certain embodiments, the polyethylene glycol is PEG 40 In certain embodiments, the polyethylene glycol is PEG 41 In certain embodiments, the polyethylene glycol is PEG 42 In certain embodiments, the polyethylene glycol is PEG 43 In certain embodiments, the polyethylene glycol is PEG 44 In certain embodiments, the polyethylene glycol is PEG 45 In certain embodiments, the polyethylene glycol is PEG 46 In certain embodiments, the polyethylene glycol is PEG 47 In certain embodiments, the polyethylene glycol is PEG 48 In certain embodiments, the polyethylene glycol is PEG 49 In certain embodiments, the polyethylene glycol is PEG 50 In certain embodiments, the polyethylene glycol is PEG 51 In certain embodiments, the polyethylene glycol is PEG 52In certain embodiments, the polyethylene glycol is PEG 53 In certain embodiments, the polyethylene glycol is PEG 54 In certain embodiments, the polyethylene glycol is PEG 55 In certain embodiments, the polyethylene glycol is PEG 56 In certain embodiments, the polyethylene glycol is PEG 57 In certain embodiments, the polyethylene glycol is PEG 58 In certain embodiments, the polyethylene glycol is PEG 59 In certain embodiments, the polyethylene glycol is PEG 60 In certain embodiments, the polyethylene glycol is PEG 61 In certain embodiments, the polyethylene glycol is PEG 62 In certain embodiments, the polyethylene glycol is PEG 63 In certain embodiments, the polyethylene glycol is PEG 64 In certain embodiments, the polyethylene glycol is PEG 65 In certain embodiments, the polyethylene glycol is PEG 66 In certain embodiments, the polyethylene glycol is PEG 67 In certain embodiments, the polyethylene glycol is PEG 68 In certain embodiments, the polyethylene glycol is PEG 69 In certain embodiments, the polyethylene glycol is PEG 70 In certain embodiments, the polyethylene glycol is PEG 71 In certain embodiments, the polyethylene glycol is PEG 72 In certain embodiments, the polyethylene glycol is PEG 73 In certain embodiments, the polyethylene glycol is PEG 74 In certain embodiments, the polyethylene glycol is PEG 75In certain embodiments, the polyethylene glycol is PEG 76 In certain embodiments, the polyethylene glycol is PEG 77 In certain embodiments, the polyethylene glycol is PEG 78 In certain embodiments, the polyethylene glycol is PEG 79 In certain embodiments, the polyethylene glycol is PEG 80 In certain embodiments, the polyethylene glycol is PEG 81 In certain embodiments, the polyethylene glycol is PEG 82 In certain embodiments, the polyethylene glycol is PEG 83 In certain embodiments, the polyethylene glycol is PEG 84 In certain embodiments, the polyethylene glycol is PEG 85 In certain embodiments, the polyethylene glycol is PEG 86 In certain embodiments, the polyethylene glycol is PEG 87 In certain embodiments, the polyethylene glycol is PEG 88 In certain embodiments, the polyethylene glycol is PEG 89 In certain embodiments, the polyethylene glycol is PEG 90 In certain embodiments, the polyethylene glycol is PEG 91 In certain embodiments, the polyethylene glycol is PEG 92 It is.

[0099] In some embodiments, the SP 1 The spacer is [ka] and During the ceremony, RG' is the reactive group residue following reaction of the reactive group RG with a binder; [ka] is binding to antigen-binding domain Z; [ka] is (AA) n is a combination with; n is an integer from 0 to 10; b is independently an integer from 1 to 92.

[0100] The reactive group RG may be any reactive group known to those skilled in the art to be capable of forming one or more bonds with the antigen-binding domain Z. The reactive group RG may have in its structure a moiety that reacts with a binding agent (e.g., with an antibody at a cysteine ​​or lysine residue, or with an antibody at an azide moiety, e.g., with a PEG-N at one or more glutamine residues). 3 Functionalized antibodies or antibodies at amino moieties, e.g., PEG-NH at one or more glutamine residues. 2 A moiety that includes a moiety that can react with a functionalized antibody to form an antibody-drug conjugate as described herein. After conjugation with an antigen-binding domain, the reactive group becomes a reactive group residue (RG'). Exemplary reactive groups include, but are not limited to, those that include a haloacetyl, isothiocyanate, succinimide, N-hydroxysuccinimide, or maleimide moiety that can react with a binding agent.

[0101] SP 2 The spacer, if present, is (AA) n A spacer is a moiety that links the moiety to the payload. Suitable spacers include SP 1 Spacers include, but are not limited to, those described above. Further suitable SPs 2 Spacers include, but are not limited to, those containing alkylene or polyether, or both. Spacer SP 2The terminus of, for example, the spacer portion directly linked to the verrucarin A derivative or AA, is used to link the verrucarin A derivative or AA to the SP during chemical synthesis of the conjugate. 2 It may be a moiety derived from the reactive moiety used to link the spacer. In some examples, the spacer SP 2 , for example, a SP that directly binds to a verrucarin A derivative or AA 2 The spacer portion may be a residue of a reactive moiety used to link the verrucarin A derivative or AA to the spacer during chemical synthesis of the conjugate.

[0102] In some embodiments, the SP 2 The spacer, when present, is -NH-(pC 6 H 4 )-CH 2 -, -NH-(pC 6 H 4 )-CH 2 OC(O)-, amino acids, dipeptides, tripeptides, oligopeptides, [ka] and any combination thereof. In certain embodiments, each [ka] is the payload and the binding, [ka] AA n or absent when n=0.

[0103] In the above formula, each (AA) n is an amino acid, or optionally a p-aminobenzyl-oxycarbonyl residue (PABC). n may be 0, in which case (AA) nIn one embodiment, if PABC is present, there is only one PABC. In another embodiment, if present, the PABC residue is adjacent to the payload and is not (AA) n The terminal AA of the group is attached. Suitable amino acids for each AA include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-α-amino acids. In some embodiments, AA includes alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or any combination thereof (e.g., dipeptides, tripeptides, oligopeptides, etc.). In certain embodiments, one or more side chains of the amino acid are linked to the side chain group shown below. In some embodiments, n is 2. In some embodiments, (AA) n is valine-citrulline. In some embodiments, (AA) n is citrulline-valine. In some embodiments, (AA) n is valine-alanine. In some embodiments, (AA) n is alanine-valine. In some embodiments, (AA) n is valine-glycine. In some embodiments, (AA) n is glycine-valine. In some embodiments, (AA) n is valine-citrulline-PABC. In some embodiments, (AA) n is citrulline-valine-PABC. In some embodiments, n is 3. In some embodiments, (AA) n is glutamic acid-valine-citrulline. In some embodiments, (AA) n is glutamine-valine-citrulline. In some embodiments, (AA) n is lysine-valine-alanine. In some embodiments, (AA) nis lysine-valine-citrulline. In some embodiments, n is 4. In some embodiments, (AA) n is glutamic acid-valine-citrulline-PAB. In some embodiments, (AA) n is glutamine-valine-citrulline-PABC.

[0104] In another embodiment, L is a cleavable linker.

[0105] In another embodiment, L is an acid labile linker, a hydrolytically unstable linker, an enzyme cleavable linker, a reduction labile linker, or a self-immolative linker.

[0106] In another embodiment, L comprises or is a peptide, a carbohydrate, an N-hydroxysuccinimidyl ester, a glucuronide, one or more polyethylene glycol units, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citrulline unit, or a para-aminobenzyl unit, or a combination thereof.

[0107] In another embodiment, L comprises or is a peptide, a carbohydrate, a glucuronide, one or more polyethylene glycol units, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citrulline unit, or a para-aminobenzyl unit, or a combination thereof.

[0108] In another embodiment, L comprises or is MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), isoleucine-phenylalanine-cutrulline, the dipeptide portion of a protease cleavable linker, ala-phe (alanine-phenylalanine), or PAB (p-aminobenzyloxy), or a combination thereof.

[0109] In another embodiment, L comprises or is MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), the dipeptide portion of a protease cleavable linker, ala-phe (alanine-phenylalanine), or PAB (p-aminobenzyloxy), or a combination thereof.

[0110] In another embodiment, L comprises val-cit. In another embodiment, L comprises or consists of val-cit-PAB. In another embodiment, L comprises or consists of val-cit-MePAB (val-cit-(p-amino-α-benzyloxy)). In another embodiment, L comprises or is AC(6-aminocaproyl). In another embodiment, L comprises or is MC(6-maleimidocaproyl). In another embodiment, L comprises or is MC-val-cit-PAB. In another embodiment, L comprises or is AC-val-cit-PAB. In another embodiment, L comprises or is MC-val-cit-MePAB. In another embodiment, L comprises or is AC-val-cit-MePAB. In another embodiment, L comprises or is AC-GGFG-CH 2 -Contains or is.

[0111] In certain embodiments, the linker-verrucarin A derivatives provided herein have the formula: [ka] [ka] where n is an integer from 1 to 4, or in another embodiment, n is 1, 2, or 3.

[0112] In one embodiment, the linker-verrucarin A derivative for use in the compositions and methods provided herein is selected from the following: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8]

[0113] In one embodiment, the linker-verrucarin A derivative for use in the compositions and methods provided herein is selected from the following: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0114] V. Synthesis of Linker-Verrucarin A Derivatives The linker-verrucarin A derivatives provided herein can be prepared according to methods well known to those skilled in the art. For example, the terminal carboxylic acid of the verrucarin A derivatives provided herein can then be esterified under standard conditions with, for example, p-aminobenzyl alcohol derivatives to obtain verrucarin A-linker compounds, such as compounds 10a-c, 12a-c, 19a-c, and 21a-c.

[0115] VI. ADCs for Use in Compositions and Methods In one embodiment, for use in the compositions and methods provided herein, there is provided a compound of formula V: Z-(LX) v V Provided herein is an ADC having the formula: Z is an anti-Influenza antigen-binding domain, an anti-SARS-CoV-2 antigen-binding domain, or an anti-Ebola antigen-binding domain; L is a linking group as defined herein; X is a verrucarin A derivative; v is an integer from 1 to 12.

[0116] In one embodiment, X is a verrucarin A derivative described herein. In another embodiment, v is an integer from 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In another embodiment, v is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, v is 1, 2, 3, 4, 5, 6, 7, or 8. In another embodiment, v is about 2 or is 2.

[0117] A. Antigen-binding domain Z Antigen-binding domains Z suitable for any ADC provided herein include, but are not limited to, antibodies, viral receptors, or any other cell-binding or peptide-binding molecules or substances. In other embodiments, antigen-binding domains usable in the ADCs provided herein include antibodies, antigen-binding fragments of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins that include the ligand-binding portion of a receptor that specifically binds to a particular antigen, antigen-binding scaffolds (e.g., scaffolds based on DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other naturally occurring repeat proteins (see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein), and aptamers or portions thereof. The full-length amino acid sequence of an exemplary influenza hemagglutinin (HA) is set forth in GenBank under the accession number ACP44150.1.

[0118] In one embodiment, the antigen-binding domain Z includes antibodies (e.g., fully human antibodies) and antigen-binding fragments thereof that specifically bind to influenza virus proteins, such as surface proteins hemagglutinin (HA), neuraminidase (NA), and matrix-2 (M2). In some embodiments, these antigen-binding domains Z modulate the interaction of influenza viruses with host cells. In some embodiments, the antibodies or antigen-binding fragments thereof bind to mature hemagglutinin. In some embodiments, the antibodies or antigen-binding fragments thereof bind to HA0 hemagglutinin precursor protein. The anti-influenza HA antibodies can bind to influenza virus HA with high affinity. In certain embodiments, the antibodies herein are blocking antibodies, where the antibodies can bind to influenza HA and block virus binding and / or entry into host cells. In some embodiments, the blocking antibodies herein can block influenza virus binding to cells and thus inhibit or neutralize virus infectivity of host cells. In some embodiments, the blocking antibodies can be useful for treating subjects suffering from influenza virus infection. The antibodies, when administered to a subject in need thereof, can reduce infection by a virus, such as influenza, in the subject. The antibody may be used to reduce viral load in a subject. The antibody may be used alone or as an adjunct therapy with other therapeutic moieties or modalities known in the art for treating viral infection. In certain embodiments, the antibody may bind to an epitope in the stem region of viral HA, the head region of viral HA, or both. Furthermore, the antibody may be used prophylactically (before infection) to protect a mammal from infection, or therapeutically (after infection is established) to improve an already established infection, or to improve at least one symptom associated with an infection.

[0119] In one embodiment, the antigen-binding domain Z includes antibodies (e.g., fully human antibodies) and antigen-binding fragments thereof that specifically bind to a SARS-CoV-2 protein, such as the spike glycoprotein (sometimes referred to as spike protein, or SARS-CoV-2-S). In some embodiments, these antigen-binding domains Z modulate the interaction of SARS-CoV-2 with a host cell. In some embodiments, the antibody or antigen-binding fragment thereof binds to the mature spike glycoprotein. In some embodiments, the antibody or antigen-binding fragment thereof binds to the spike precursor protein. The anti-SARS-CoV-2-S antibody can bind to the spike glycoprotein with high affinity. In certain embodiments, the antibodies herein are blocking antibodies, and can block viral binding and / or entry into a host cell by binding the antibody to SARS-CoV-2-S, e.g., by blocking the interaction between the spike glycoprotein and its receptor ACE2. In some embodiments, the blocking antibodies herein can block SARS-CoV-2 binding to a cell, and thus inhibit or neutralize viral infectivity of the host cell. In some embodiments, the blocking antibodies may be useful for treating subjects suffering from SARS-CoV-2 infection and / or experiencing COVID-19 symptoms. The antibodies, when administered to a subject in need thereof, can reduce infection by a virus such as SARS-CoV-2 in the subject. The antibodies may be used to reduce viral load in the subject. The antibodies may be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating viral infections. In certain embodiments, the antibodies may bind to an epitope of the receptor binding domain of the spike glycoprotein. Additionally, the antibodies may be used prophylactically (before infection) to protect a mammal from infection, or therapeutically (after infection has been established) to ameliorate an already established infection, or to ameliorate at least one symptom associated with an infection.

[0120] In certain embodiments, the antibodies are obtained from mice immunized with a first immunogen, such as full-length influenza HA, SARS-CoV-2-S, or Ebola virus GP, or a recombinant form of influenza HA, SARS-CoV-2-S, or Ebola virus GP, or a fragment thereof, followed by a second immunogen, or an immunogenic active fragment of influenza HA, SARS-CoV-2-S, or Ebola virus GP. In certain embodiments, the antibodies are obtained from mice immunized with an influenza, SARS-CoV-2, or Ebola vaccine composition, followed by a booster with one or more recombinantly produced influenza HA, SARS-CoV-2-S, or Ebola peptides, respectively. In certain embodiments, the antibodies are obtained from humans. In certain embodiments, the antibodies are obtained from mammals (e.g., non-human mammals). In certain embodiments, the antibodies are obtained from non-human primates.

[0121] The immunogen may be a biologically active and / or immunogenic fragment of influenza HA, SARS-CoV-2-S, or Ebola virus, or a DNA encoding the active fragment. In the case of influenza, the fragment may be derived from the stem region of the HA protein (see, e.g., Sui et al. Nature Struct. and Mol. Biol., published online on February 22, 2009; pp, 1-9), the head region of the HA protein, or a combination thereof. In the case of SARS-CoV-2, the fragment may be derived from the full-length SARS-CoV-2-S protein or the receptor binding domain (RBD). In the case of Ebola, the fragment may be derived from the full-length Ebola protein, or the Ebola virus GP, including the amino-terminal fragment (e.g., GP1), or the carboxy-terminal fragment (e.g., GP2).

[0122] The antigen-binding domain Z may be modified to include the addition or substitution of specific residues for tagging or for conjugation with a carrier molecule, such as keyhole limpet hemocyanin (KLH). For example, a cysteine ​​may be added to either the N-terminus or C-terminus of the peptide, or a linker sequence may be added to prepare the peptide for conjugation with, for example, KLH for immunization.

[0123] Certain anti-influenza antibodies, anti-influenza-HA antibodies, or ADCs provided herein have antiviral activity, such as the ability to bind to influenza-HA and neutralize its activity, as measured by in vitro or in vivo assays. Certain anti-influenza antibodies, anti-influenza-HA antibodies, or ADCs provided herein are capable of binding to HA but do not have neutralizing activity, as measured by in vitro or in vivo assays. The ability of an antibody or ADC herein to bind to influenza-HA and neutralize its activity, and thus viral infection subsequent to viral binding and / or entry into a host cell, can be measured using any standard method known to one of skill in the art, including the binding assays or activity assays described herein.

[0124] Certain anti-SARS-CoV-2 antibodies, anti-SARS-CoV-2-S antibodies, or ADCs provided herein have antiviral activity, such as the ability to bind to and neutralize the activity of SARS-CoV-2-S, as measured by in vitro or in vivo assays. Certain anti-SARS-CoV-2 antibodies, anti-SARS-CoV-2-S antibodies, or ADCs provided herein can bind to SARS-CoV-2-S but do not have neutralizing activity, as measured by in vitro or in vivo assays. The ability of an antibody or ADC herein to bind to SARS-CoV-2-S and neutralize its activity, and thus viral infection subsequent to viral binding and / or entry into a host cell, can be measured using any standard method known to one of skill in the art, including the binding assays described herein, or activity assays.

[0125] Certain anti-Ebola antibodies or ADCs provided herein have antiviral activity, such as the ability to bind to and neutralize the activity of Ebola virus, as measured by in vitro or in vivo assays. Certain anti-Ebola antibodies or ADCs provided herein can bind to Ebola but do not have neutralizing activity, as measured by in vitro or in vivo assays. The ability of an antibody or ADC herein to bind to Ebola virus and neutralize its activity, and thus viral infection subsequent to viral binding and / or entry into a host cell, can be measured using any standard method known to one of skill in the art, including the binding assays described herein, or activity assays.

[0126] The antigen-binding domain Z, such as an antibody specific for influenza-HA, SARS-CoV-2-S or Ebola virus, or an ADC may not include an additional label or moiety, or may include an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antibodies can be used in diagnostic assays, including imaging assays. In one embodiment, the additional moiety is a peptide tag. In one embodiment, the ADC includes an antibody heavy chain and further includes a peptide tag at the C-terminus of the antibody heavy chain. In one embodiment, an ADC comprises an antibody heavy chain and further comprises a peptide tag at the C-terminus of the antibody heavy chain, where the peptide tag is ELQRP, LLQG, LLQGG, LLQLLQG, LLQYQG, LLQGA, LLQGSG, SLLQG, LQG, LLQLQ, LLQLLQ, LLQGR, LLQYQGA, LQGG, LGQG, or LLQLLQGA. See, e.g., WO 2012 / 059882, U.S. Patent No. 9,676,871, and U.S. Patent Application Publication No. 2003 / 0138785. In one embodiment, an ADC provided herein comprises an antibody heavy chain and further comprises a peptide tag (e.g., a pentapeptide) at the C-terminus of the antibody heavy chain, where the peptide tag is the pentapeptide sequence LLQGA (e.g., for use in conjugating a linker payload with transglutaminase). In one embodiment, an ADC provided herein comprises an antibody heavy chain and further comprises a peptide tag (e.g., a pentapeptide) at the C-terminus of the antibody heavy chain, where the peptide tag is the pentapeptide sequence ELQGP (e.g., for use in conjugating a linker payload with transglutaminase). In one embodiment, an ADC comprises two antibody heavy chains and further comprises a peptide tag at the C-terminus of each antibody heavy chain.In one embodiment, an ADC comprises two antibody heavy chains and further comprises a peptide tag at the C-terminus of each antibody heavy chain, where the peptide tag is the pentapeptide sequence LLQGA. In one embodiment, an ADC comprises two antibody heavy chains and further comprises a peptide tag at the C-terminus of each antibody heavy chain, where the peptide tag is the pentapeptide sequence ELQGP.

[0127] In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a kappa light chain. In certain embodiments, the light chain is a lambda light chain. In certain embodiments, the antibody comprises a heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, the heavy chain is IgM. In some embodiments, the heavy chain is IgGY. In some embodiments, the heavy chain is any class, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or subclass. In some embodiments, the heavy chain is IgG1. In some embodiments, the heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, the heavy chain is IgA1. In some embodiments, the heavy chain is IgA2. In some embodiments, Z has a molecular weight of at least 500, 600, 700, 800, 900, 1000, 10000, 50000 or 100000 daltons.

[0128] In some embodiments, the antibody is an antibody fragment. Non-limiting examples of antigen-binding fragments for use in the ADCs provided herein include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs), e.g., CDR3 peptides) of an antibody, or the constraining FR3-CDR3-FR4 peptide. In other embodiments, antigen-binding fragments of antibodies include domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab') 2 In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment.

[0129] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain.

[0130] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

[0131] In certain embodiments, the antibody may be engineered to include a glutamine residue. Techniques for modifying antibody sequences to include glutamine residues are within the skill of the art (see, for example, Ausubel et al. Current Protoc. Mol. Biol.). In one embodiment, the antibody comprises an antibody heavy chain and further comprises a peptide tag at the C-terminus of the antibody heavy chain. In one embodiment, the antibody comprises an antibody heavy chain and further comprises a peptide tag, e.g., a transglutaminase recognition sequence or a pentapeptide tag, at the C-terminus of the antibody heavy chain, where the peptide tag is the pentapeptide sequence LLQGA or ELQGP.

[0132] B. Preparation of Human Antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present disclosure to generate human antibodies that specifically bind to influenza-HA, SARS-CoV-2-S or Ebola virus. An immunogen comprising any one of the following can be used to generate antibodies against influenza-HA, SARS-CoV-2-S or Ebola virus. In certain embodiments, the antibodies herein are obtained from mice immunized with full-length native influenza-HA (see, e.g., GenBank Accession No. FJ966082.1), full-length SARS-CoV-2-S, or SARS-CoV-2-S receptor binding domain, or live attenuated or inactivated virus, or DNA encoding the protein or fragment thereof. Alternatively, influenza-HA, SARS-CoV-2-S or Ebola protein or fragment thereof is generated and modified using standard biochemical techniques and used as an immunogen. In one embodiment, the immunogen is a recombinantly produced influenza-HA protein or SARS-CoV-2-S, or a fragment thereof. In certain embodiments herein, the immunogen may be an influenza virus vaccine or a SARS-CoV-2 vaccine. In certain embodiments, one or more booster injections of influenza, SARS-CoV-2, or Ebola may be administered. In certain embodiments, the influenza booster shot may include one or more influenza virus strains, or hemagglutinins from these strains, see, e.g., Protein Sciences H1 A / New Caledonia / 20 / 1999, H5 A / Indonesia / 05 / 2005, H3 A / Victoria / 361 / 2011, H7 A / Netherlands / 219 / 2003, or H9 A / Hong Kong / 1073 / 1988, or influenza B virus strains B / Victoria / 2 / 87, B / Nanchang / 3451 / 93, B / Singapore / 11 / 1994, B / Florida / 4 / 2006, or B / Yamagata / 16 / 88.In certain embodiments, the booster shot may contain a 1:1 mixture of influenza strains, or a 1:1 mixture of hemagglutinins from the strains. In certain embodiments, the immunogen may be a recombinant influenza-HA peptide or the influenza virus itself expressed in E. coli, or other eukaryotic or mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

[0133] Using VELOCIMMUNE® technology (see, e.g., U.S. Patent No. 6,596,541) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies against influenza-HA, SARS-CoV-2-S or Ebola are first isolated, having human variable regions and mouse constant regions. VELOCIMMUNE® technology involves the creation of transgenic mice with genomes that include human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies that include human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0134] Generally, VELOCIMMUNE® mice are exposed to an antigen of interest and lymphoid cells (such as B cells) are collected from the mice that express antibodies. The lymphoid cells may be fused with a myeloma cell line to create an immortalized hybridoma cell line, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains may be isolated and linked to constant regions of the desired isotypes of heavy and light chains. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0135] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desired properties, including affinity, selectivity, epitope, etc., as described in WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated by reference in its entirety. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody herein, e.g., wild-type or modified IgG1 or IgG4. The constant region selected may vary depending on the particular application, but the properties of high affinity antigen binding and target specificity reside in the variable region.

[0136] C. Biological equivalent Antigen-binding domain Z, including the anti-influenza-HA antibodies and antibody fragments, anti-SARS-CoV-2-S antibodies and antibody fragments, and anti-Ebola antibodies and antibody fragments provided for use in the ADCs herein, includes proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to influenza-HA, SARS-CoV-2-S, or Ebola, respectively. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit essentially equivalent biological activity to that of the described antibodies. Similarly, DNA sequences encoding the antibodies of the present disclosure include sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but are essentially biologically equivalent to the antibodies or antibody fragments herein. Other biologically equivalent anti-influenza-HA antibodies and antibody fragments are as described in WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated by reference in its entirety. Other bioequivalent anti-SARS-CoV-2-S antibodies and antibody fragments are described, for example, in U.S. Patent No. 10,787,501. Other bioequivalent anti-Ebola antibodies and antibody fragments are described, for example, in U.S. Patent Nos. 11,530,255 and 9,771,414.

[0137] D. Biological Characteristics of Antibodies Generally, the antigen-binding domain Z comprising the antibodies provided herein functions by binding to influenza-HA, SARS-CoV-2-S, or Ebola viruses, e.g., having a K of less than 10 nM (e.g., at 25° C. or 37° C.) as measured by a real-time biolayer interferometer-based biosensor (Octet HTX assay) or by surface plasmon resonance. D Provided herein are antibodies and antigen-binding fragments of antibodies that bind to influenza-HA, SARS-CoV-2-S, or Ebola virus at a K of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM, as measured by surface plasmon resonance, e.g., using an assay format as described in WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated by reference in its entirety, or a substantially similar assay. D and binds to influenza-HA, SARS-CoV-2-S or Ebola.

[0138] Non-limiting exemplary in vitro assays for measuring binding activity are shown in Example 3 of WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated herein by reference in its entirety. In Example 3 of WO2016 / 100807 or US2016 / 0176953A1, the binding affinity and dissociation constant of anti-influenza-HA antibodies to influenza-HA were measured by a real-time biolayer interferometer-based biosensor (Octet HTX assay). In Examples 4 and 5 of WO2016 / 100807 or US2016 / 0176953A1, a neutralization assay was used to determine the infectivity of various group 1 strains of influenza virus. Example 6 of WO2016 / 100807 or US2016 / 0176953A1 shows that certain antibodies mediate complement dependent cytotoxicity (CDC) of virus-infected cells in vitro. Examples 7 and 10 of WO2016 / 100807 or US2016 / 0176953A1 show that certain antibodies of the disclosure can neutralize influenza A infection in vivo when administered either prophylactically or therapeutically.

[0139] In one embodiment, the antigen-binding domain Z, including antibodies and antigen-binding fragments thereof for use in the ADCs provided herein, has a dissociation half-life (t t ) of greater than about 100 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined in WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated herein by reference in its entirety, or a substantially similar assay. 1 / 2) that bind to influenza-HA, SARS-CoV-2-S, or Ebola virus. In certain embodiments, an antibody or antigen-binding fragment for use in the ADCs provided herein has a t of greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, or greater than about 1000 minutes, as measured by surface plasmon resonance at 25° C. using, for example, an assay format (e.g., imAb-capture or antigen capture format) as defined in WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated herein by reference in its entirety, or a substantially similar assay. 1 / 2 In one embodiment, the antibodies and antigen-binding fragments herein bind to influenza-HA, SARS-CoV-2-S, or Ebola virus with a dissociation half-life (t 1 / 2 ) binds to influenza-HA, SARS-CoV-2-S or Ebola virus. In one embodiment, the antibodies herein provide an increase in dissociation half-life of about 1.5 to 2-fold when tested in monkeys and mice compared to a comparison antibody, referred to as Control I mAb.

[0140] In another embodiment, the antigen-binding domain Z, comprising an antibody or antigen-binding fragment thereof for use in the ADCs provided herein, neutralizes the infectivity of influenza virus, SARS-CoV-2 or Ebola virus to a host cell. In some embodiments, the antibody has an IC ranging from about 1.6 nM to about 130 nM in a microneutralization assay, e.g., as set forth in Examples 4 and 5 of WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated by reference in its entirety, or a substantially similar assay. 50and exhibits neutralizing activity against a variety of representative Group 1 influenza viruses (H1N1 A / Puerto Rico / 08 / 1934; H5N1 A / Vietnam / 1203 / 2004; H1N1 A California / 07 / 2009; H1N1 A / Wisconsin / 1933; H1N1 A / Brisbane / 59 / 1997, H9N2 A Hong Kong / 33982 / 2009, H13N6 A / gull / Maryland / 704 / 1977, and H16N3 A / shorebird / Delaware / 172 / 2006). In one embodiment, the antibody or antigen-binding fragment thereof that neutralizes influenza virus infectivity for host cells has an IC of less than 130 nM. 50 In some embodiments, the antibodies exhibit neutralizing power against any of the SARS-CoV-2 mutants described herein, including but not limited to, delta and omicron mutants. In some embodiments, the antibodies exhibit neutralizing power against any of the Ebola virus mutants.

[0141] In other embodiments, the antigen-binding domain Z, comprising an antibody or antigen-binding fragment thereof for use in the ADCs provided herein, has an EC 50 and mediates complement-dependent cytotoxicity of infected cells (see Example 6 of WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated by reference in its entirety). In one embodiment, the antibody or antigen-binding fragment thereof has an EC 50 mediates complement-dependent cytotoxicity of infected cells.

[0142] In another embodiment, an anti-influenza-A HA antibody for use in the ADCs provided herein exhibits increased protection or neutralization of influenza A infection in vivo compared to a control antibody. In another embodiment, an anti-SARS-CoV-2-S antibody for use in the ADCs provided herein exhibits increased protection or neutralization of SARS-CoV-2 infection in vivo compared to a control antibody. In another embodiment, an anti-Ebola antibody for use in the ADCs provided herein exhibits increased protection or neutralization of Ebola infection in vivo compared to a control antibody. Certain antibodies exhibit neutralization when administered either prophylactically (pre-infection) or therapeutically (post-infection). See Example 7 of WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated herein by reference in its entirety.

[0143] In one embodiment, the antigen-binding domain Z comprises an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza-HA, and the antibody or fragment thereof exhibits two or more of the following properties: (a) is a fully human monoclonal antibody; (b) has a specific binding affinity of 10 or more, as measured by a surface plasmon resonance assay. -9 Dissociation constant (K D ) binds influenza HA; (c) has a dissociation half-life (t 1 / 2 ) in the range of about 1.6 nM to about 130 nM; (d) IC 50 (e) neutralization of Group 1 influenza A viruses selected from H1N1, H5N1, H9N2, H13N6, and H16N3 at an EC 50 exhibits complement-mediated lysis of influenza virus-infected cells; or (f) exhibits protection as measured by enhanced survival in an animal model of influenza virus infection when administered either before or after virus exposure.

[0144] In one embodiment, the antigen-binding domain Z comprises an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 omicron mutant, and the antibody or fragment thereof exhibits two or more of the following properties: (a) is a fully human monoclonal antibody; (b) has a specific binding affinity of 10 or more to the SARS-CoV-2 omicron mutant as measured by a surface plasmon resonance assay; -9 Dissociation constant (K D ) to the SARS-CoV-2 omicron mutant; (c) dissociation half-lives (t 1 / 2 ) in the range of about 1.6 nM to about 130 nM; (d) IC 50 (e) neutralization of SARS-CoV-2 viruses selected from the Omicron B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 and BA.5 lineages with an EC of about 20 nM to about 66 nM; 50 (f) exhibits complement-mediated lysis of SARS-CoV-2 virus-infected cells; or (f) exhibits protection as measured by improved survival in an animal model of SARS-CoV-2 infection when administered either before or after virus exposure.

[0145] In one embodiment, the antigen-binding domain Z comprises an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to Ebola virus, and the antibody or fragment thereof exhibits two or more of the following properties: (a) is a fully human monoclonal antibody; (b) has a specific binding affinity of 10 or more, as measured by a surface plasmon resonance assay. -9 Dissociation constant (K D ) binds to Ebola virus; (c) has a dissociation half-life (t 1 / 2 ) in the range of about 1.6 nM to about 130 nM; (d) IC 50 (e) EC50 of about 20 nM to about 66 nM. 50 or (f) exhibits protection as measured by enhanced survival in an animal model of Ebola virus infection when administered either before or after virus exposure.

[0146] The antigen-binding domain Z, including antibodies and antigen-binding fragments thereof, for use in the ADCs herein may have two or more of the above biological properties, or any combination thereof. Other biological properties of the antigen-binding domain Z, including antibodies and antigen-binding fragments thereof, for use in the ADCs herein will be apparent to those of skill in the art from a review of the present disclosure, including the Examples herein.

[0147] E. Heavy and Light Chain Variable Region Amino Acid and Nucleotide Sequences In some embodiments, the antigen-binding domain Z conjugated to the linker-payload or payload may be an antibody targeting influenza-HA. Exemplary influenza-HA antibodies can be found, for example, in WO2016 / 100807 or US2016 / 0176953A1, each of which is incorporated herein by reference in its entirety. In some embodiments, the influenza-HA antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO:20; a HCDR2 comprising SEQ ID NO:22; a HCDR3 comprising SEQ ID NO:24; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO:28; a LCDR2 comprising SEQ ID NO:30; and a LCDR3 comprising SEQ ID NO:32. In some embodiments, the influenza-HA antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO:18 and a light chain variable region (LCVR) comprising SEQ ID NO:26. In any of the foregoing embodiments, the influenza-HA antibody can be prepared by site-directed mutagenesis to insert a glutamine residue at a site that does not abolish the function or binding of the antibody. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR, where the peptide tag is the pentapeptide sequence LLQGA. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR, where the peptide tag is the pentapeptide sequence ELQGP. In one embodiment, the antibody comprises two HCVRs and further comprises a peptide tag at the C-terminus of each HCVR. In one embodiment, the antibody comprises two HCVRs and further comprises a peptide tag at the C-terminus of the HCVR, where each peptide tag is independently the pentapeptide sequence LLQGA or the pentapeptide sequence ELQGP.

[0148] In some embodiments, the antigen-binding domain Z conjugated to the linker-payload or payload may be an antibody targeting SARS-CoV-2. Exemplary SARS-CoV-2 antibodies can be found, for example, in U.S. Pat. No. 10,787,501, which is incorporated herein by reference in its entirety. In some embodiments, the SARS-CoV-2 antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 315 or 335; a HCDR2 comprising SEQ ID NO: 317 or 337; a HCDR3 comprising SEQ ID NO: 319 or 339; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 323 or 343; a LCDR2 comprising SEQ ID NO: 325 or 345; and a LCDR3 comprising SEQ ID NO: 327 or 347. In some embodiments, the SARS-CoV-2 antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO: 313 or 333 and a light chain variable region (LCVR) comprising SEQ ID NO: 321 or 341. In any of the foregoing embodiments, the SARS-CoV-2 antibody can be prepared by site-directed mutagenesis to insert a glutamine residue at a site that does not abolish the function or binding of the antibody. In one embodiment, the antibody comprises an HCVR and further comprises a peptide tag at the C-terminus of the HCVR. In one embodiment, the antibody comprises an HCVR and further comprises a peptide tag at the C-terminus of the HCVR, where the peptide tag is the pentapeptide sequence LLQGA. In one embodiment, the antibody comprises an HCVR and further comprises a peptide tag at the C-terminus of the HCVR. In one embodiment, the antibody comprises an HCVR and further comprises a peptide tag at the C-terminus of the HCVR, where the peptide tag is the pentapeptide sequence ELQGP. In one embodiment, the antibody comprises two HCVRs and further comprises a peptide tag at the C-terminus of each HCVR. In one embodiment, the antibody comprises two HCVRs and further comprises a peptide tag at the C-terminus of the HCVR, where each peptide tag is independently the pentapeptide sequence LLQGA or the pentapeptide sequence ELQGP.

[0149] In some embodiments, the antigen-binding domain Z conjugated to the linker-payload or payload may be an antibody targeting Ebola. Exemplary Ebola antibodies can be found, for example, in U.S. Pat. Nos. 9,771,414 and 11,530,255, which are incorporated herein by reference in their entirety. In some embodiments, the Ebola antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 355; a HCDR2 comprising SEQ ID NO: 357; a HCDR3 comprising SEQ ID NO: 359; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 363; a LCDR2 comprising SEQ ID NO: WAS; and a LCDR3 comprising SEQ ID NO: 365. In some embodiments, the SARS-CoV-2 antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO: 353 and a light chain variable region (LCVR) comprising SEQ ID NO: 361. In any of the foregoing embodiments, the SARS-CoV-2 antibody can be prepared by site-directed mutagenesis to insert a glutamine residue into a site that does not abolish the function or binding of the antibody. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR, where the peptide tag is the pentapeptide sequence LLQGA. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR. In one embodiment, the antibody comprises a HCVR and further comprises a peptide tag at the C-terminus of the HCVR, where the peptide tag is the pentapeptide sequence ELQGP. In one embodiment, the antibody comprises two HCVRs and further comprises a peptide tag at the C-terminus of each HCVR. In one embodiment, the antibody comprises two HCVRs and further comprises a peptide tag at the C-terminus of the HCVR, where each peptide tag is independently the pentapeptide sequence LLQGA or the pentapeptide sequence ELQGP.

[0150] Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-Influenza-HA, anti-SARS-CoV-2 and Ebola antibodies. The corresponding nucleic acid sequence identifiers are shown in Table 2. [Table 5]

[0151] SEQ ID NO:14 is Lys Ala Ser; SEQ ID NO:30 is Ala Ala Ser; SEQ ID NO:46 is Lys Ala Ser; SEQ ID NO:62 is Ala Ala Ser; SEQ ID NO:70 is Ala Ala Ser; SEQ ID NO:86 is Thr Ala Ser; SEQ ID NO:102 is Gly Ala Ser; SEQ ID NO:118 is Lys Ile Ser; SEQ ID NO:134 is Ala Thr Ser; SEQ ID NO:150 is Ala Ala Ser; SEQ ID NO:166 is Ala Ala Ser; SEQ ID NO:182 is Ala Ala Ser; SEQ ID NO:198 is Ala Ala Ser; SEQ ID NO:214 is Lys Ala Ser; SEQ ID NO:230 is Gly Ala Ser; SEQ ID NO:304 is Gly Asn Ser; SEQ ID NO:325 is Gly Asn Ser; SEQ ID NO:345 is Asp Val Ser. The remaining SEQ ID NOs in Table 1 are set forth in the Sequence Listing XML, which is incorporated herein by reference. [Table 6]

[0152] SEQ ID NO:13 is aaggcgtct; SEQ ID NO:29 is gctgcgtcc; SEQ ID NO:45 is aaggcgtct; SEQ ID NO:61 is gctgcatcc; SEQ ID NO:69 is gctgcatcc; SEQ ID NO:85 is actgcatcc; SEQ ID NO:101 is ggtgcatcc; SEQ ID NO:117 is aagatttct; SEQ ID NO:133 is gctacatcc; SEQ ID NO:149 is gctgcatcc; SEQ ID NO:165 is gctgcatcc; SEQ ID NO:181 is gctgcatcc; SEQ ID NO:197 is gctgcatcc; SEQ ID NO:213 is aaggcgtct; SEQ ID NO:229 is ggtgcatcc; SEQ ID NO:324 is ggtaacagc; SEQ ID NO:344 is gatgtcagt. The remaining SEQ ID NOs in Table 2 are set forth in the Sequence Listing XML, which is incorporated herein by reference.

[0153] The 4A8 hIgG1 HC nucleic acid sequence is SEQ ID NO:374; the hIgG1-HC-C-term-LLQGA HC amino acid sequence is SEQ ID NO:375 and the nucleic acid sequence is SEQ ID NO:376; the hIgG1-HC-C-term-ELQRP HC amino acid sequence is SEQ ID NO:377 and the nucleic acid sequence is SEQ ID NO:378.

[0154] F. Linker-Verrucarin A Derivative-LX Conjugation with Antigen-Binding Domain Z The linker-verrucarin A derivative-LX may be conjugated to the antigen-binding domain Z, e.g., an antibody or antigen-binding fragment, by a bond at a specific amino acid in the antibody or antigen-binding fragment. In one embodiment, amino acid bonds that can be used herein include, for example, lysine (see, e.g., US 5,208,020; US 2010 / 0129314; Hollander et al., Bioconjugate Chem., 2008,19:358-361; WO2005 / 089808; US5,714,586; US2013 / 0101546; and US2012 / 0585592), cysteine ​​(see, e.g., US2007 / 0258987; WO2013 / 055993; WO2013 / 055990; WO2013 / 053873; WO2013 / 053872; WO2011 / 130598; US2013 / 0101546; and US7,750,116), selenocysteine ​​(see, e.g., WO2008 / 122039; and Hofer et al. al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formylglycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), unnatural amino acids (see, e.g., WO2013 / 068874, and WO2012 / 166559), and acidic amino acids (see, e.g., WO2012 / 05982). The linker L may also be conjugated to the antigen-binding domain Z by a carbohydrate bond (see, e.g., US2008 / 0305497, WO2014 / 065661, Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130, and Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997).

[0155] In some embodiments, the antigen-binding domain Z is an antibody or antigen-binding fragment that is linked to the L group of the linker-verrucarin A derivative-LX via a lysine residue. In some embodiments, the antigen-binding domain Z is an antibody or antigen-binding fragment that is linked to the L group of the linker-verrucarin A derivative-LX via a cysteine ​​residue.

[0156] In certain embodiments herein, the L group of the linker-verrucarin A derivative may be conjugated to one or more glutamine residues in the antigen-binding domain Z by transglutaminase-based chemoenzymatic conjugation (see, for example, Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997 and Dennler et al., Bioconjugate Chem. 2014, 25:569-578). For example, in the presence of transglutaminase, one or more glutamine residues of the antibody may be coupled to a primary amine compound. Primary amine compounds include, for example, verrucarin A derivative X and linker-verrucarin A derivative-LX, which directly results in an antibody-drug conjugate by transglutaminase-related coupling. Antibodies containing glutamine residues may be isolated from natural sources or engineered to contain one or more glutamine residues. Techniques for engineering the insertion of glutamine residues into antibody polypeptide chains (glutaminyl-modified antibodies or antigen-binding fragments) are within the skill of one in the art. In certain embodiments, the antibody is non-glycosylated. In certain embodiments, the antibody is glycosylated.

[0157] In certain embodiments, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises two heavy chain polypeptides, each having one Gln295 or Q295 residue. In yet another embodiment, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises one or more glutamine residues at a site other than heavy chain 295. Included herein are antibodies of this section having N297Q mutation(s) as described herein. In another embodiment, the antibody may also be conjugated in the presence of carbohydrate at N297 using the methods described in Dickdiesser, et al., Bioconjugate Chem. 2020, 31, 1070-1076.

[0158] In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises an antibody heavy chain and further comprises a peptide tag at the C-terminus of the antibody heavy chain. In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises an antibody heavy chain and further comprises a peptide tag at the C-terminus of the antibody heavy chain, where the peptide tag is the pentapeptide sequence LLQGA or ELQGP. In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises two antibody heavy chains and further comprises a peptide tag at the C-terminus of each antibody heavy chain. In one embodiment, the antibody or glutaminyl-modified antibody or antigen-binding fragment comprises two antibody heavy chains and further comprises a peptide tag at the C-terminus of each antibody heavy chain, where the peptide tag is the pentapeptide sequence LLQGA or ELQGP.

[0159] In another embodiment, the compound of the formula: [ka] [ka] where n, v, and Z are as defined herein.

[0160] VII. Synthesis of ADCs Methods for synthesizing the ADCs provided herein are also provided herein. The ADCs provided herein can be prepared according to standard methods well known to those of skill in the art.

[0161] For example, an antibody having a conjugation site engineered into its C-terminus, e.g., a polypeptide or "Q-tag," can be reacted with a linker-verrucarin A derivative bearing a primary amine in the presence of a transglutaminase, e.g., bacterial transglutaminase, to provide an ADC provided herein.

[0162] Alternatively, unengineered antibodies can be treated with excess TCEP (tris(2-carboxyethyl)phosphine) to reduce interchain disulfide bonds. The reduced antibody is then reacted with a linker-verrucarin A derivative bearing a maleimide group to yield the ADCs provided herein.

[0163] In either case, the ADC may be purified by standard techniques, such as size exclusion chromatography in PBS / 5% glycerol.

[0164] VIII. Pharmaceutical Compositions The pharmaceutical compositions provided herein comprise a therapeutically effective amount of one or more verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein and a pharma- ceutically acceptable carrier.

[0165] Verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs can be formulated into suitable pharmaceutical preparations. Typically, the above-mentioned verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).

[0166] In the compositions, an effective concentration of one or more verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs or pharma- ceutically acceptable salts are mixed with a suitable pharmaceutical carrier. In certain embodiments, the concentration of verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs in the compositions is effective for delivery of an amount that, upon administration, treats, prevents, or ameliorates one or more symptoms and / or progression of a disease or disorder disclosed herein.

[0167] Typically, the composition is formulated for administration in a single dosage. To formulate the composition, the weight fraction of verrucarin A derivative, linker-verrucarin A derivative and / or ADC is dissolved, suspended, dispersed or otherwise mixed in the selected carrier at an effective concentration to alleviate or improve the condition to be treated. Pharmaceutical carriers suitable for administration of verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein include any carrier known to those skilled in the art to be suitable for a particular mode of administration.

[0168] In some embodiments, the verrucarin A derivative, linker-verrucarin A derivative and / or ADC is included in a pharma- ceutically acceptable carrier in an amount sufficient to exert a therapeutically beneficial effect on the subject being treated in the absence of undesirable side effects. The therapeutically effective concentration is determined empirically by testing the compound in in vitro and in vivo systems described herein and known to those skilled in the art, and then human dosages can be extrapolated therefrom. In some embodiments, the verrucarin A derivative, linker-verrucarin A derivative and / or ADC is administered in a manner that achieves a therapeutically effective concentration of the payload. In some embodiments, a companion diagnostic (see, e.g., Olsen D and Jorgensen JT, Front. Oncol., 2014 May 16, 4:105, doi:10.3389 / fonC.2014.00105) is used to determine the therapeutic concentration and safety profile of the verrucarin A derivative, linker-verrucarin A derivative and / or ADC in a particular subject or subject population.

[0169] The concentration of verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs in the pharmaceutical composition will depend on the absorption, tissue distribution, inactivation and excretion rates of the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs, the physicochemical properties of the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs, the administration schedule, and the amount administered, as well as other factors known to those skilled in the art. For example, the amount delivered will be sufficient to ameliorate one or more symptoms of a disease or disorder disclosed herein.

[0170] The composition may be administered once or divided into several smaller doses and administered at intervals.Of course, the exact dosage and duration of treatment is a function of the disease to be treated and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data.It should be noted that concentration and dosage values ​​may also vary according to the severity of the condition to be alleviated.It should also be understood that for any particular subject, specific dosing regimes should be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the composition.

[0171] The compositions may contain other active compounds to obtain a desired combination of properties. The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein, or pharma- ceutical acceptable salts thereof described herein, may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the general art to be beneficial in treating one or more of the diseases or medical conditions referred to herein. It is to be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

[0172] IX. Medication The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein, and the pharmaceutical compositions provided herein, may be administered in specific therapeutically or prophylactically effective amounts, at specific time intervals, in specific dosage forms, and by specific dosage administration methods, as set forth below.

[0173] The methods provided herein encompass treating a subject regardless of the subject's age, although some diseases or disorders are more prevalent in certain age groups.

[0174] The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein, or pharma- ceutically acceptable salts thereof, may be administered repeatedly as necessary, for example, until the subject experiences stable disease or regression, or until the subject experiences disease progression or unacceptable toxicity.

[0175] The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs, or pharma- ceutically acceptable salts thereof, provided herein may be administered once a day (QD) or divided into multiple daily doses, such as twice a day (BID), three times a day (TID), and four times a day (QID). In addition, administration may be continuous (i.e., every day or every day for consecutive days), intermittent, e.g., in cycles (i.e., with a drug holiday of several days, weeks, or months). As used herein, the term "daily" is intended to mean that the therapeutic compound, such as the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs, or pharma- ceutically acceptable salts thereof, provided herein, is administered, for example, once or more than once each day of a period of time. The term "continuously" is intended to mean that a therapeutic compound, such as a verrucarin A derivative, linker-verrucarin A derivative and / or ADC, or a pharma- ceutically acceptable salt thereof, provided herein, is administered daily for a continuous period of at least 10 days to 52 weeks. As used herein, the term "intermittent" or "intermittently" is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of a verrucarin A derivative, linker-verrucarin A derivative and / or ADC, or a pharma- ceutically acceptable salt thereof, provided herein, can be administered 1-6 days per week, in cycles (e.g., daily administration for 2-8 consecutive weeks followed by a rest period of up to 1 week without administration), or every other day. The term "cycle", as used herein, is intended to mean that a therapeutic compound, such as a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein, or a pharma- ceutically acceptable salt thereof, is administered daily or consecutively, but with a rest period, in some such embodiments, once daily administration for 2-6 days, followed by a rest period of 5-7 days without administration.

[0176] X. Treatment Method In another embodiment, a method of treating a subject with a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein, or a pharma- ceutically acceptable salt thereof is provided. In another embodiment, a method of treating a subject with a pharmaceutical composition comprising a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein, or a pharma- ceutically acceptable salt thereof is provided. The pharmaceutical composition comprises any of the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs disclosed herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0177] In one embodiment, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are useful in treating viral diseases. In one embodiment, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are useful in treating COVID-19. SARS-CoV-2, the viral cause of COVID-19, has demonstrated a remarkable ability to mutate into additional pathogenic variants, e.g., beta, delta, omicron, etc. The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are useful in treating some or all of such SARS-CoV-2 variants. For example, the antibody portion of the ADCs provided herein can be generated or engineered to overcome viral resistance when SARS-CoV-2 variants develop resistance to existing antibody portions. In another embodiment, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are useful in treating Ebola.

[0178] In certain embodiments, the verrucarin A derivatives, linker-berucarin A derivatives and / or ADCs provided herein are useful in treating influenza, including influenza A, B and C. In another embodiment, a method of treating influenza in a subject by administering a verrucarin A derivative, linker-berucarin A derivative and / or ADC provided herein is provided. In another embodiment, a method of treating influenza A in a subject by administering a verrucarin A derivative, linker-berucarin A derivative and / or ADC provided herein is provided. In another embodiment, a method of treating influenza A in a subject by administering a verrucarin A derivative, linker-berucarin A derivative and / or ADC provided herein is provided, wherein the influenza A is any of subtypes H1, H2, H3, H5, H6, H7, H9 and H10; and N1, N2, N6, N7, N8 and N9. In another embodiment, there is provided a method of treating H1N1 influenza A in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein. In another embodiment, there is provided a method of treating H3N2 influenza A in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein.

[0179] In certain embodiments, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are selected from the group consisting of alpha (B.1.1.7 and Q lineages), beta (B.1.351 and sublineages), gamma (P.1 and sublineages), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​guanine (B.1.527 and B.1.529 ... In another embodiment, a method of treating COVID-19 in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein is provided. In another embodiment, a method of treating COVID-19 in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein is provided. In another embodiment, a method of treating COVID-19 in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein is provided. In another embodiment, a method is provided for treating COVID-19 caused by the omicron variant of SARS-CoV-2 in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein, where the COVID-19 caused by the omicron variant of SARS-CoV-2 is any of subtypes B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 and BA.5. In another embodiment, a method is provided for treating COVID-19 caused by the omicron BA.4 variant of SARS-CoV-2 in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein. In another embodiment, a method is provided for treating COVID-19 caused by the omicron BA.5 variant of SARS-CoV-2 in a subject by administering a verrucarin A derivative, linker-verrucarin A derivative and / or ADC provided herein.

[0180] In certain embodiments, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are useful in treating Ebola, including ameliorating or reducing the severity of at least one symptom of Ebola virus infection, including, but not limited to, fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration and unexplained bleeding. In another embodiment, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are useful prophylactically in subjects at risk of developing Ebola virus infection.

[0181] In the context of the methods of treatment provided herein, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs may be administered as monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0182] XI. Combination Therapy with a Second Active Agent Provided herein are compositions comprising any of the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject.

[0183] The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein may be formulated with and / or administered in combination with one or more additional therapeutically active ingredient(s) selected from oseltamivir, zanamivir, peramivir, baloxavir, amantadine and rimantadine. The verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein may also be formulated with and / or administered in combination with one or more additional therapeutically active ingredient(s) selected from molnupiravir, remdesivir, baricitinib, nilmatrevir, ritonavir, bebuterovimab, tocilizumab, casirivimab and imdevimab. In another embodiment, the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein may be formulated and / or administered in combination with one or more additional therapeutically active ingredient(s) selected from antiviral drugs, anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), anti-Ebola antibodies, vaccines against Ebola virus, TKM Ebola (small interfering RNA targeting viral RNA polymerase), brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomer targeting Ebola virus VP24 gene), interferon, or any other symptomatic therapy for treating Ebola virus infection.

[0184] The additional therapeutically active ingredient(s), for example, any of the drugs listed above or derivatives thereof, may be administered immediately prior to, simultaneously with, or shortly after administration of the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein. In another embodiment, a pharmaceutical composition is provided in which the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein are formulated with one or more additional therapeutically active ingredient(s) described herein.

[0185] As used herein, the term "in combination" includes the use of two or more therapies (e.g., one or more prophylactic and / or therapeutic agents), however, the use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or disorder. The first treatment (e.g., verrucarin A derivatives and / or ADCs provided herein) may be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second treatment (e.g., a prophylactic or therapeutic agent) to the subject. Triple therapies are also contemplated herein.

[0186] The administration of the verrucarin A derivatives, linker-verrucarin A derivatives and / or ADCs provided herein, or derivatives thereof, and one or more second active agents to a subject can be performed simultaneously or sequentially, by the same or different routes of administration. The suitability of a particular route of administration for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without being degraded before entering the bloodstream). EXAMPLES

[0187] XII. Examples The following examples are intended to illustrate certain embodiments provided herein, and are not intended to limit the scope of the disclosure.

[0188] The verrucarin A derivatives, linker-verrucarin A derivatives and ADCs provided herein were synthesized as shown below. All solvents used were used as received and purchased from either Sigma Aldrich or Fisher Scientific. 1H spectra were recorded on Varian Inova 300 MHz and 500 MHz NMR instruments. Chemical shifts (δ) are reported in ppm relative to the NMR solvent used for the analysis and are reported as s-singlet, d-doublet, t-triplet, q-quartet, dd-doublet, dt-doublet, dq-doublet, and m-multiplet. Coupling constants (J) are reported in Hertz (Hz). Chromatographic purity was determined on an Agilent 1100, 1260Infinity, or 1200 Series LC / MS system equipped with a 6130 Quadrupole LC / MS using a Chromolith® FastGradient RP-18e analytical column (50×2 mm, Merck KGaA, P / N 1.52007.0001) and the following analytical HPLC method: injection volume 2-10 μL; flow rate 1 mL / min; 5-95% acetonitrile in water over 4 min; Agilent diode array detector at λ=254 nm; room temperature. Low resolution mass spectrometry was performed on the Agilent system using an electrospray ionization source and analyzed with either a single quadrupole or ion trap mass detector.

[0189] Example 1 (R)-Amino-verrucarin A: Compound 4 [ka]

[0190] Compound 2: To a solution of verrucarin A (1, 5 mg, 0.01 mmol) in DCM (1.5 mL) at -20°C, pyridine (0.3 mL) was added, followed by the slow addition of trifluoromethanesulfonic anhydride (3.4 μL, 0.02 mmol). The reaction was stirred at the same temperature for 1 h, at which point it was complete by LCMS analysis. The reaction was diluted with water (2 mL) and extracted with DCM (3 x 2 mL). The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to give 2 as a colorless oil, which was used in the next step without purification. MS (ESI, pos.): C 28 H 33 F 3 O11 Calculated for S, 634.2; found 635.1 (M+H), 657.1 (M+Na).

[0191] Compound 3: To a solution of crude 2 (0.01 mmol) in anhydrous DMF (0.5 mL) at room temperature, sodium azide (2 mg, 0.03 mmol) was added and the mixture was stirred vigorously for 45 min, at which point the reaction was complete by LCMS analysis. The reaction was diluted with water (10 mL) and extracted with ethyl acetate (3×3 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. Residual solvent was removed under high vacuum to give 3, which was used in the next step without purification. MS (ESI, pos.): C 27 H 33 N 3 O 8 Calculated for, 527.2; found 528.3 (M+H), 550.2 (M+Na).

[0192] Compound 4: To a solution of 3 (0.01 mmol) in THF (1 mL) was added triphenylphosphine (6 mg, 0.02 mmol) at room temperature and the reaction was stirred for 16 h. DI water (0.2 mL) was added and the mixture was heated to 50 °C for 8 h. The reaction was concentrated in vacuo and the residue was dissolved in DMSO (0.6 mL) and injected into an ISCO (5.5 g C18Aq, both using 5-95% MeCN / water with 0.05% AcOH) for purification. Pure fractions were combined and lyophilized to give compound 4 as an off-white fluffy solid (3.8 mg, 77% over 3 steps). MS (ESI, pos.): C 27 H 35 NO 8 Calculated for, 501.2; Found 502.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.07 (dd, J = 15.7, 11.7 Hz,1H), 6.68 (t, J = 11.4 Hz, 1H), 6.14 (d, J = 11.1 Hz, 1H), 6.05 (d, J = 15.7 Hz,1H), 5.80 (dd, J = 8.1, 4.0 Hz, 1H), 5.45 (d, J = 4.6 Hz, 1H), 4.53-4.47 (m, 2H),4.27 (d, J = 12.3 Hz, 1H), 4.05 (td, J = 11.5, 3.2 Hz, 1H), 3.88 (d, J = 5.1 Hz,1H), 3.62 (d, J = 5.1 Hz, 1H), 3.18 (d, J = 9.1 Hz, 1H), 3.14 (d, J = 3.9 Hz, 1H),2.83 (d, J = 3.9 Hz, 1H), 2.53 (dd, J = 15.5, 8.2 Hz, 1H), 2.39-2.32 (m, 1H), 2.24-2.19(m, 1H), 2.09-1.95 (m, 5H), 1.81-1.76 (m, 2H), 1.76 (s, 3H), 1.35-1.30 (m, 1H),1.03 (d, J = 6.7 Hz, 3H), 0.91 (s, 3H).

[0193] Example 2 Compound 5, Compound 6, and Compound 7 [ka]

[0194] Compound 5: To a solution of (R)-amino-verrucarin A (4, 7 mg, 0.014 mmol) and succinic anhydride (2 mg, 0.02 mmol) in anhydrous THF (0.6 mL) at room temperature, N,N-diisopropylethylamine (8 µL, 0.042 mmol) was added and the reaction was stirred for 18 h. The volatiles were removed in vacuo and the residue was dissolved in DMF (0.5 mL) and purified by gradient elution 5-95% MeCN / H 2The mixture was purified by Teledyne ISCO EZ prep on a 30×150 mm Gemini column using 2×100 Hz and 100 Hz of 1000 Hz of 1H 2 O (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 5 as an off-white fluffy solid (7.8 mg, 93%). MS (ESI, pos.): 31 H 39 NO 11 Calculated for, 601.3; Found 602.3 (M+H). 1 H-NMR (500 MHz; CDCl 3 ): δ 8.15 (dd, J = 15.7, 11.7 Hz,1H), 6.70 (t, J = 11.4 Hz, 1H), 6.15 (d, J = 11.0 Hz, 1H), 6.07-6.04 (m, 2H), 5.80(dd, J = 7.9, 4.0 Hz, 1H), 5.41 (d, J = 4.5 Hz, 1H), 4.50 (dt, J = 10.8, 3.6 Hz,1H), 4.40 (d, J = 12.3 Hz, 1H), 4.33-4.28 (m, 2H), 4.01 (td, J = 11.2, 2.8 Hz, 1H),3.86 (d, J = 5.0 Hz, 1H), 3.59 (d, J = 5.0 Hz, 1H), 3.13 (d, J = 3.8 Hz, 1H), 2.83(d, J = 3.8 Hz, 1H), 2.75-2.64 (m, 2H), 2.56-2.42 (m, 2H), 2.21 (td, J = 9.9, 5.2Hz, 2H), 2.02-1.88 (m, 5H), 1.73 (s, 3H), 1.68-1.63 (m, 1H), 1.49-1.44 (m, 1H),1.08 (d, J = 6.7Hz, 3H), 0.85 (s, 3H).

[0195] Compound 6: Compound 6 was prepared using glutaric anhydride using the same method and scale as compound 5. The isolated yield was 8.0 mg (94% yield). MS (ESI, pos.): C 32 H 41 NO 11Calculated for, 615.3; found 616.3 (M+H). 1 H-NMR (500 MHz; CDCl 3 ): δ 8.14 (dd, J = 15.7, 11.7 Hz,1H), 6.70 (t, J = 11.3 Hz, 1H), 6.15 (d, J = 11.0 Hz, 1H), 6.06 (d, J = 15.8 Hz,1H), 5.85 (d, J = 8.2 Hz, 1H), 5.79 (dd, J = 7.7, 3.8 Hz, 1H), 5.41 (d, J = 4.6Hz, 1H), 4.49-4.46 (m, 1H), 4.39 (d, J = 12.4 Hz, 1H), 4.30 (q, J = 7.6 Hz, 2H),4.02 (td, J = 11.2, 2.0 Hz, 1H), 3.86 (d, J = 5.0 Hz, 1H), 3.58 (d, J = 5.0 Hz,1H), 3.12 (d, J = 3.8 Hz, 1H), 2.84 (d, J = 3.7 Hz, 1H), 2.53-2.40 (m, 3H), 2.29(t, J = 7.3 Hz, 2H), 2.21 (td, J = 9.9, 5.8 Hz, 2H), 1.99-1.88 (m, 6H), 1.73 (s,3H), 1.65 (d, J = 6.2 Hz, 1H), 1.49 (t, J = 12.6 Hz, 1H), 1.09 (d, J = 6.5 Hz, 3H), 0.84 (s, 3H).

[0196] Compound 7: Compound 7 was prepared using the same method and scale as compound 5, using adipic anhydride. The isolated yield was 5.6 mg (63% yield). MS (ESI, pos.): C 33 H 43 NO 11 Calculated for, 629.3; found 630.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.16 (dd, J = 15.6, 11.7 Hz,1H), 6.70 (t, J = 11.4 Hz, 1H), 6.15 (d, J = 11.1 Hz, 1H), 6.06 (d, J = 15.7 Hz,1H), 5.88 (d, J = 8.0 Hz, 1H), 5.80 (dd, J = 8.2, 4.0 Hz, 1H), 5.41 (d, J = 5.1Hz, 1H), 4.52 (dt, J = 11.0, 3.9 Hz, 1H), 4.40 (d, J = 12.3 Hz, 1H), 4.30 (t, J= 10.2 Hz, 2H), 4.03 (td, J = 11.4, 3.0 Hz, 1H), 3.86 (d, J = 5.0 Hz, 1H), 3.58 (d, J = 5.2 Hz, 1H), 3.13 (d, J = 3.8 Hz, 1H), 2.83 (d, J = 3.9 Hz, 1H), 2.51 (dd,J = 15.5, 8.1 Hz, 1H), 2.38-2.36 (m, 2H), 2.23-2.18 (m, 4H), 2.06-1.87 (m, 5H), 1.73 (s, 3H), 1.68-1.62 (m, 4H), 1.47 (dd, J = 14.0, 11.1 Hz, 1H), 1.10 (d, J =6.7 Hz, 3H), 0.83 (s, 3H).

[0197] Example 3 Compounds 10a, 10b and 10c

change

[0198] Compound 9a: Compound 8 (9mg, 0.011mmol) and Compound 5 (4.5mg, 0.007mmol) in THF / DCM (1+1mL) To the mixture, EDCI (2.3 mg, 0.011 mmol) and DMAP (0.5 mg, 0.004 mmol) were added and reactants were added and stirred for 18 hours. Volatile substances are removed under reduced pressure, and the residue is 5~95%MeCN / H 2The mixture was purified on an ISCO 30 g C18Aq column using 100 mL of 1000 sucrose and 100 mL of 0.05% AcOH (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 9b (7 mg, 77%) as a fluffy white solid. MS (ESI, pos.): 70 H 87 N 7 O 17 Calculated for, 1297.6; found 1298.5 (M+H).

[0199] Compound 9b: Compound 9b was prepared according to the general procedure using acid 6. Yield=76%. MS (ESI, pos.):C 71 H 89 N 7 O 17 Calculated for, 1311.6; found 1312.5 (M+H).

[0200] Compound 9c: Compound 9c was prepared according to the general procedure using acid 7. Yield=71%. MS (ESI, pos.):C 72 H 91 N 7 O 17 Calculated for, 1325.6; found 1326.5 (M+H).

[0201] Compound 10a: To a solution of compound 9a (7 mg .0053 mmol) in DMF (0.8 mL) was added 5% piperidine in DMF (0.5 mL) and the reaction was stirred for 60 min, then diluted with 5-95% MeCN / H 2 The mixture was purified on a 30×150 mm Gemini column using 100 ml of ethyl acetate and 20 ml of 2H2O (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 10a (4.8 mg, 84%) as a fluffy white solid. MS (ESI, pos.): 55 H 77 N 7 O 15 Calculated for, 1075.5; found 1076.4 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 10.01 (s, 1H), 8.22 (d, J = 5.8 Hz, 1H), 8.16 - 8.13 (m, 1H), 7.96 - 7.91 (m, 1H), 7.88 - 7.83 (m, 1H), 7.59 (d, J = 7.9 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 6.87 (t, J = 11.3 Hz, 1H), 6.24 (t, J = 12.3 Hz, 2H), 6.04 - 6.00 (m, 1H), 5.76 - 5.74 (m, 1H), 5.41 (s, 2H), 5.26 (d, J = 3.4 Hz, 1H), 5.00 (s, 2H), 4.40 - 4.35 (m, 2H), 4.18 - 4.11 (m, 2H), 4.02 - 3.99 (m, 1H), 3.95 - 3.90 (m, 1H), 3.74 - 3.71 (m, 1H), 3.68 - 3.65 (m, 2H), 3.03 - 2.94 (m, 5H), 2.74 (d, J = 3.3 Hz, 1H), 2.43 - 2.40 (m, 2H), 2.19 - 2.07 (m, 4H), 1.98 - 1.92 (m, 2H), 1.82 (s, 2H), 1.65 - 1.57 (m, 8H), 1.47 (dd, J = 4.2, 3.1 Hz, 3H), 1.36 - 1.34 (m, 3H), 1.24 - 1.23 (m, 4H), 0.91 (d, J = 6.4 Hz, 2H), 0.83 (dd, J = 11.9, 6.8 Hz, 7H), 0.74 (s, 3H).

[0202] Compound 10b was prepared from compound 9b according to the same procedure. Yield = 68%. MS (ESI, pos.): C 56 H 79 N 7 O 15 Calculated for, 1089.6; found 1090.6 (M + H). 1 H-NMR (500 MHz; DMSO-d 6): δ 10.01(s, 1H), 8.15 (d, J = 6.3 Hz, 1H), 7.94 (ddd, J = 15.6, 11.6, 0.9 Hz, 1H), 7.61(d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 6.87 (t, J = 11.7 Hz, 1H), 6.24 (dd,J = 13.3, 11.1 Hz, 2H), 5.76 (dd, J = 8.0, 3.7 Hz, 1H), 5.44-5.43 (m, 1H), 5.27(d, J = 2.3 Hz, 1H), 5.01 (s, 2H), 4.42-4.38 (m, 1H), 4.33-4.31 (m, 1H), 4.11 (d,J = 12.2 Hz, 2H), 4.02 (d, J = 12.3 Hz, 1H), 3.92 (td, J = 11.3, 4.2 Hz, 1H), 3.73(dd, J = 10.0, 6.1 Hz, 1H), 3.68 (d, J = 5.2 Hz, 1H), 3.65 (d, J = 5.3 Hz, 1H), 3.02 (d, J = 4.0 Hz, 1H), 2.99-2.93 (m, 3H), 2.74 (d, J = 4.1 Hz, 1H), 2.63 (dt,J = 3.7, 1.9 Hz, 1H), 2.60 (dd, J = 14.4, 7.1 Hz, 2H), 2.43-2.39 (m, 2H), 2.37-2.33(m, 4H), 2.21-2.16 (m, 5H), 2.01-1.92 (m, 2H), 0.84 (t, J = 6.4 Hz, 6H), 0.74 (s, 3H).

[0203] The same thing is done with compound 9c and compound 10c. Yield = 56%. MS (ESI, pos.): C 57 H 81 N 7 O15 Calculated value for it: 1103.6; measured value 1104.4 (M+H). 1 H-NMR (500 MHz; DMSO-d 6 ): δ 10.00 (s, 1H), 8.10 (d, J = 6.3 Hz, 1H), 7.96 - 7.90 (m, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 6.87 (t, J = 11.5 Hz, 1H), 6.61 - 6.59 (m, 1H), 6.24 (t, J = 12.6 Hz, 3H), 5.77 - 5.74 (m, 1H), 5.42 - 5.41 (m, 2H), 5.28 - 5.27 (m, 1H), 5.00 (s, 2H), 4.41 - 4.33 (m, 3H), 4.13 - 4.10 (m, 2H), 4.01 (d, J = 12.4 Hz, 1H), 3.95 - 3.90 (m, 2H), 3.74 (dd, J = 9.7, 6.3 Hz, 1H), 3.68 - 3.65 (m, 2H), 3.01 (d, J = 4.0 Hz, 2H), 2.99 - 2.94 (m, 2H), 2.73 (d, J = 3.9 Hz, 1H), 2.63 (t, J = 1.6 Hz, 1H), 2.45 - 2.40 (m, 1H), 2.35 - 2.32 (m, 3H), 2.20 - 2.14 (m, 5H), 1.99 - 1.92 (m, 2H), 1.84 (s, 1H), 1.78 - 1.72 (m, 1H), 1.66 - 1.61 (m, 4H), 1.50 - 1.46 (m, 4H), 1.39 - 1.34 (m, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.85 - 0.82 (m, 6H), 0.74 (s, 3H).

[0204] Example 4 Compounds 12a, 12b and 12c

Chemical Structure

[0205] Compound 12a: Compound 12a was prepared from acid 5 and alcohol 11 using the same procedure as for compound 9a. Yield=13% (after two purifications). MS (ESI, pos.): C 59 H 77 N 7 O 17 Calculated for, 1155.5; found 1156.3 (M+H). 1 H NMR (500 MHz; DMSO-d 6) δ 10.03 (s, 1H), 8.23-8.20(m, 1H), 8.15-8.12 (m, 1H), 7.93 (dd, J = 12.0, 15.5 Hz, 1H), 7.82 (d, J =9.0 Hz,1H), 7.58 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 7.16 (br m, 3H), 6.99 (s,2H), 6.86 (t, J = 11.5 Hz, 1H), 6.26-6.21 (m, 2H), 6.01-5.98 (br m, 1H), 5.76-5.74(m, 1H), 5.42-5.38 (m, 2H), 5.26 (d, J = 4.5 Hz, 1H), 5.02-4.97 (m, 2H), 4.42-4.34(m, 2H), 4.17 (dd,J= 8.5, 7.0 Hz, 1H), 4.12 (d, J = 12.5 Hz, 1H), 4.00 (d, J = 12.5,1H), 3.95-3.89 (m, 1H), 3.73 (dd, J = 6.5, 10.0 Hz, 1H), 3.67 (d, J = 5.0 Hz, 2H), 3.03 (d, J = 4.0 Hz, 2H), 3.00-2.91 (m, 1H) 2.73 (d, J = 4.0 Hz, 1H), 2.62 (dt,J = 3.5, 2.0 Hz, 1H), 2.45-2.38 (m, 1H), 2.35 (m, 2 H), 2.20-2.06 (m, 2H), 1.98-1.91(m, 2H), 0.82 (dd, J = 15.5, 7.0 Hz, 6H),0.74 (s, 3H).

[0206] Compound 12b: Compound 9a was prepared in the same manner as Compound 12b using Triacic Acid 6 and Trichosanthel 11. Yield = 41%. MS(ESI, pos.): C 60 H 79 N7 O 17 Calculated for, 1169.6; found 1170.3 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 9.98 (s, 1H), 8.15 (d, J = 6.2 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.94 (ddd,J = 15.6, 11.7, 0.9 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.6 Hz, 2H),7.27 (d, J = 8.6 Hz, 2H), 6.99 (s, 2H), 6.87 (t, J = 11.5 Hz, 1H), 6.54-6.48 (m,3H), 6.24 (t, J = 12.4 Hz, 2H), 5.95 (t, J = 5.6 Hz, 1H), 5.76 (dd, J = 8.1, 3.8Hz, 1H), 5.38 (dd, J = 1.6, 1.1 Hz, 2H), 5.27 (d, J = 4.7 Hz, 1H), 5.00 (s, 2H),4.42-4.34 (m, 2H), 4.18 (dd, J = 8.6, 6.9 Hz, 1H), 4.11 (d, J = 12.2 Hz, 1H), 4.02(d, J = 12.3 Hz, 1H), 3.92 (td, J = 11.4, 4.2 Hz, 1H), 3.73 (dd, J = 9.9, 6.2 Hz,1H), 3.67 (dd, J = 11.1, 5.1 Hz, 2H), 3.01 (dd, J = 12.7, 5.4 Hz, 2H), 2.93 (dt,J = 13.0, 6.5 Hz, 1H), 2.74 (d, J = 4.0 Hz, 1H), 2.63 (dt, J = 3.6, 1.8 Hz, 1H),2.42 (dd, J = 15.1, 8.2 Hz, 1H), 2.35 (td, J = 7.5, 2.8 Hz, 2H), 2.18 (dt, J = 11.4,7.1 Hz, 4H), 2.13-2.06 (m, 2H), 1.98-1.92 (m, 2H), 1.90 (d, J = 9.2 Hz, 1H), 1.73(dd, J = 14.1, 8.3 Hz, 3H), 1.67-1.56 (m, 6H), 1.47 (dq, J = 14.8, 7.4 Hz, 4H),1.27-1.23 (m, 2H), 1.17 (dt, J = 15.3, 7.7 Hz, 2H), 0.91 (d, J = 6.6 Hz, 3H), 0.82(dd, J = 15.5, 6.8 Hz, 6H), 0.74 (s, 3H). .

[0207] Compound 12c: Compound 12c was prepared from acid 7 and alcohol 11 using the same procedure as for compound 9a. Yield=23%. MS(ESI, pos.): C 61 H 81 N 7 O 17 Calculated for, 1183.6; found 1184.4 (M+H). 11H NMR (500 MHz; DMSO-d6) δ 10.00 (s, 1H), 8.12 - 8.07 (m, 2H), 7.93 (dd, J = 12.5, 16 Hz, 1H), 7.83 - 7.78 (br s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 6.98 (s, 2H), 6.86 (t, J = 11.5 Hz, 1H), 6.82 - 6.72 (br s, 1H), 6.26 - 6.20 (m, 2H), 6.00 - 5.96 (br s, 1H), 5.77 - 5.73 (m, 1H), 5.42 - 5.37 (m, 2H), 5.35 - 5.30 (br s, 1H), 5.29 - 5.26 (m, 1H), 5.01 - 4.97 (br s, 2H), 4.40 - 4.33 (m, 2H), 4.18 - 4.15 (m, 1H), 4.11 (d, J = 12.5 Hz, 1H), 4.03 - 3.98 (m, 1H), 3.95 - 3.89 (m, 1H), 3.74 (dd, J = 6.5, 10.0 Hz, 1H), 3.67 (d, J = 4.5 Hz, 1H), 3.66 - 3.63 (m, 1H), 3.05 - 2.87 (m, 4H), 2.73 (d, J = 3.5 Hz, 1H), 2.63 - 2.58 (m, 1H), 2.46 - 2.39 (m, 2H), 2.37 - 2.30 (m, 2H), 2.22 - 2.05 (m, 4H), 1.99 - 1.91 (m, 2H), 1.81 - 1.69 (m, 1H), 1.69 - 1.63 (m, 3H), 1.63 - 1.54 (m, 4H), 1.53 - 1.37 (m, 8H), 1.37 - 1.31 (m, 1H), 1.28 - 1.13 (m, 4H), 0.90 (d, J = 6.5 Hz, 3H), 0.82 (dd, J = 7, 15 Hz, 6H), 0.73 (s, 3H).

[0208] Example 5 Compounds 19a, 19b, 19c

Chemical Structure

[0209] Compound 15: To a solution of Fmoc-Val-Cit-OH (13, 497 mg, 1.0 mmol) and 1-(4-aminophenyl)ethan-1-ol (14, 274 mg, 2.0 mmol) in DCM (4.5 mL) and MeOH (2 mL) was added EEDQ (495 mg, 2.0 mmol) and the reaction was stirred at room temperature for 1 h. The reaction became a gum. Additional DCM (4.5 mL) and MeOH (2 mL) were added and the mixture was stirred overnight. The volatiles were removed under reduced pressure and the residue was washed successively with diethyl ether (5 mL), ethyl acetate (5 mL) and diethyl ether (5 mL). The residue was dried under high vacuum to give compound 15 (585 mg, 95%) as a pale yellow solid. MS (ESI, pos.): C 34 H 41 N 5 O 6 Calculated for, 615.3; found 616.3 (M+H).

[0210] Compound 16: To a solution of compound 15 (150 mg, 0.244 mmol) in DMF (1 mL) was added a 5% piperidine solution in DMF (1 mL) and the reaction was stirred at room temperature for 45 min. Gradient elution 5-95% MeCN / H 2 Purification on a Teledyne ISCO 50 g C18Aq column using 1,2-dichlorophenyl ether (COOH) and 2,3-dichlorophenyl ether (COOH) (both containing 0.05% AcOH) afforded compound 16 (103 mg, 94%) as the acetate salt. MS (ESI, pos.): 19 H 31 N 5 O 4 Calculated for, 393.2; found 394.3 (M+H).

[0211] Compound 17: To a solution of compound 16 (30 mg, 0.076 mmol) and Fmoc-N-amide-cap-NHS (34 mg, 0.076 mmol) in DMF, DIEA (20 μL, 0.114 mmol) was added and the reaction was stirred for 1.5 h. Gradient elution 5-95% MeCN / H 2Purification on a Teledyne ISCO 50 g C18Aq column using 1,2-dichlorophenyl ether (COOH) and 1,2-dichlorophenyl ether (COOH) (both containing 0.05% AcOH) gave compound 17 (20 mg, 36%). MS (ESI, pos.): 40 H 52 N 6 O 7 Calculated for, 728.4; found 729.3 (M+H).

[0212] Compound 18a: Compound 18a was prepared from acid 5 and alcohol 17 using the same procedure as for compound 9a. Yield=47%, MS (ESI, pos.): C 71 H 89 N 7 O 17 Calculated for, 1311.6; found 1312.5 (M+H).

[0213] Compound 18b: Compound 18b was prepared from acid 6 and alcohol 17 using the same procedure as for compound 9a. Yield=75%. MS (ESI, pos.): C 72 H 91 N 17 O 17 Calculated for, 1325.6; found 1326.6 (M+H).

[0214] Compound 18c: Compound 18c was prepared from acid 7 and alcohol 17 using the same procedure as for compound 9a. Yield=50%. MS (ESI, pos.): C 73 H 93 N 7 O 17 Calculated for, 1339.7; found 1340.5 (M+H).

[0215] Compound 19a: Compound 19a was prepared from compound 18a using the same procedure as compound 10a. Yield=60%. MS (ESI,pos.): C 56 H 79 N 7 O 15 Calculated for, 1089.6; found 1090.3 (M+H). 1 H-NMR (500 MHz; DMSO-d6 ): δ 9.98 (s, 1H), 8.21(s, 1H), 8.15 - 8.11 (m, 1H), 7.97 - 7.91 (m, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.27 (t, J = 7.0 Hz, 2H), 7.10 (d, J = 7.2 Hz, 1H), 6.86 (d, J = 11.4 Hz, 1H), 6.61 (s, 1H), 6.24 (t, J = 12.3 Hz, 2H), 6.02 (s, 1H), 5.75 - 5.73 (m, 2H), 5.40 (d, J = 6.3 Hz, 2H), 5.27 (t, J = 4.4 Hz, 1H), 4.40 - 4.35 (m, 2H), 4.18 - 4.11 (m, 2H), 4.03 - 3.98 (m, 1H), 3.94 - 3.90 (m, 1H), 3.73 - 3.71 (m, 1H), 3.68 - 3.64 (m, 2H), 3.03 (dd, J = 5.0, 4.4 Hz, 2H), 3.00 - 2.93 (m, 3H), 2.74 - 2.73 (m, 1H), 2.59 - 2.57 (m, 2H), 2.35 (s, 3H), 2.28 (s, 1H), 2.19 - 2.15 (m, 3H), 2.06 (s, 1H), 1.95 (td, J = 2.2, 0.9 Hz, 2H), 1.86 (s, 2H), 1.66 - 1.58 (m, 6H), 1.48 (s, 1H), 1.42 - 1.38 (m, 6H), 1.26 - 1.23 (m, 4H), 0.90 (d, J = 6.2 Hz, 3H), 0.83 (dd, J = 11.1, 6.9 Hz, 6H), 0.74 (s, 3H).

[0216] Compound 19b: Compound 19b was prepared from Compound 18b using the same procedure as Compound 10a. Yield = 33%. MS (ESI, pos.): C 57 H 81 N 7 O 15 Calculated for, 1103.6; Found 1104.3 (M + H). 1H-NMR (500 MHz; DMSO-d 6 ): δ 9.98-9.97 (m, 1H), 8.15-8.13 (m, 2H), 7.97-7.91 (m, 1H), 7.56 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.2Hz, 2H), 6.89-6.84 (m, 1H), 6.51 (s, 1H), 6.26-6.21 (m, 2H), 6.02 (s, 1H), 5.77-5.73(m, 2H), 5.41-5.39 (m, 2H), 5.28-5.26 (m, 1H), 4.83 (d, J = 5.2 Hz, 1H), 4.60 (t,J = 5.7 Hz, 1H), 4.40-4.35 (m, 2H), 4.19-4.16 (m, 1H), 4.12-4.09 (m, 1H), 4.02 (dd,J = 11.1, 4.1 Hz, 2H), 3.93-3.87 (m, 2H), 3.74-3.71 (m, 1H), 3.67 (ddd, J = 7.2,5.0, 3.1 Hz, 2H), 3.62-3.60 (m, 1H), 3.03-2.91 (m, 4H), 2.73 (d, J = 0.4 Hz, 1H), 2.66 (dd, J = 1.0, 0.5 Hz, 2H), 2.63 (t, J = 1.9 Hz, 1H), 2.36-2.30 (m, 3H), 2.27(t, J = 7.4 Hz, 1H), 2.20-2.15 (m, 4H), 1.98-1.93 (m, 2H), 1.89 (s, 1H), 1.72-1.70(m, 3H), 1.61-1.59 (m, 4H), 1.49 (d, J = 5.7 Hz, 4H), 1.43 (d, J = 6.4 Hz, 4H), 0.91-0.90 (m, 3H), 0.83 (m, 8H), 0.73 (d, J = 1.5 Hz, 3H).

[0217] Compound 19c: Compound 10a was prepared using the same method as compound 18c and compound 19c. Yield = 55%. MS (ESI,pos.): C 58 H 83 N 7 O15 Calculated value for it: 1117.6; Measured value 1118.4 (M+H). 1 H-NMR (500 MHz; DMSO-d 6 ): δ 9.98 (s, 1H), 8.10 (d, J = 6.1 Hz, 1H), 7.97 - 7.90 (m, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 7.2 Hz, 2H), 6.87 (t, J = 11.3 Hz, 1H), 6.24 (t, J = 12.9 Hz, 2H), 5.76 - 5.73 (m, 2H), 5.40 (ddd, J = 6.9, 2.1, 0.9 Hz, 2H), 5.28 - 5.27 (m, 1H), 4.40 - 4.34 (m, 2H), 4.16 - 4.10 (m, 2H), 4.02 - 3.99 (m, 1H), 3.99 - 3.90 (m, 1H), 3.76 - 3.72 (m, 1H), 3.69 - 3.65 (m, 2H), 3.06 - 2.91 (m, 6H), 2.73 (t, J = 0.6 Hz, 1H), 2.63 - 2.62 (m, 1H), 2.35 - 2.32 (m, 1H), 2.33 - 2.27 (m, 3H), 2.21 - 2.06 (m, 6H), 1.97 - 1.93 (m, 2H), 1.83 (s, 2H), 1.66 - 1.58 (m, 8H), 1.49 - 1.45 (m, 6H), 1.45 - 1.40 (m, 4H), 1.39 - 1.35 (m, 3H), 0.91 (d, J = 5.8 Hz, 3H), 0.85 - 0.82 (m, 6H), 0.74 (d, J = 0.3 Hz, 3H).

[0218] Example 6 Compounds 21a, 21b and 21c

Chemical Structure

[0219] Compound 20: To a solution of compound 16 (30 mg, 0.076 mmol) and mal-cap-NHS (23.5 mg, 0.076 mmol) in DMF (1 mL), DIEA (20 μL, 0.114 mmol) was added and the reaction was stirred for 1 h, after which it was purified by gradient elution 5–95% MeCN / H 2 The mixture was purified on a 50 g C18 Aq column using 100 mL of 1:1 hexanediaminetetraacetate (HCl) and 1:1 hexanediaminetetraacetate (HCl) (both containing 0.05% AcOH). The pure fractions were combined and lyophilized to give the title compound 20 (28 mg, 66%) as a fluffy off-white solid. MS (ESI, pos.): 29 H 42 N 6 O 7 Calculated for, 586.3; found 587.3 (M+H).

[0220] Compound 21a: Compound 21a was prepared from acid 5 and alcohol 20 using the same procedure as for compound 10a. Yield=13%. MS (ESI, pos.): C 60 H 79 N 7 O 17 Calculated for, 1169.6; found 1170.3 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 10.00 (s, 1H), 8.21-8.19(m, 1H), 8.13-8.11 (m, 1H), 7.96-7.90 (m, 1H), 7.83-7.80 (m, 1H), 7.56 (d, J = 8.5Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 6.99 (s, 2H), 6.87 (t, J = 11.5 Hz, 1H), 6.27-6.21(m, 2H), 6.00-5.97 (m, 1H), 5.77-5.71 (m, 2H), 5.39-5.38 (m, 2H), 5.28-5.26 (m,1H), 4.41-4.33 (m, 2H), 4.19-4.10 (m, 2H), 4.02-3.99 (m, 1H), 3.95-3.89 (m, 1H),3.73-3.71 (m, 1H), 3.71-3.65 (m, 3H), 3.04-3.03 (m, 2H), 3.00-2.92 (m, 3H), 2.74-2.73(m, 1H), 2.43-2.40 (m, 1H), 2.21-2.14 (m, 2H), 2.14-2.06 (m, 2H), 1.98-1.91 (m,2H), 1.80-1.76 (m, 1H), 1.66-1.57 (m, 9H), 1.48-1.45 (m, 4H), 1.42 (m, 5H), 1.23-1.15(m, 4H), 0.91-0.90 (m, 3H), 0.84-0.80 (m, 6H), 0.74 (s, 3H).

[0221] Compound 21b: Compound 21b was prepared from acid 6 and alcohol 20 using the same procedure as for compound 10a. Yield=15%. MS (ESI, pos.): C 61 H 81 N 17 O 17 Calculated for, 1183.6; found 1184.4 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 9.99 (s, 1H), 8.15-8.13(m, 1H), 8.11-8.09 (m, 1H), 7.94 (m, 2H), 7.82-7.79 (m, 1H), 7.57-7.55 (m, 2H),7.27-7.26 (m, 2H), 6.99 (s, 2H), 6.89-6.84 (m, 1H), 6.26-6.21 (m, 2H), 5.97 (m,1H), 5.77-5.73 (m, 2H), 5.39 (s, 2H), 5.28-5.26 (m, 1H), 4.41-4.33 (m, 2H), 4.17(dd, J = 8.3, 7.1 Hz, 1H), 4.12-4.09 (m, 1H), 4.03-4.01 (m, 1H), 3.94-3.89 (m, 1H), 3.73 (dd, J = 10.0, 6.1 Hz, 1H), 3.69-3.64 (m, 2H), 3.03 (d, J = 4.0 Hz, 1H), 3.01-2.99(m, 1H), 2.93 (t, J = 6.3 Hz, 2H), 2.73 (t, J = 3.6 Hz, 1H), 2.63 (t, J = 1.8 Hz,1H), 2.53 (t, J = 1.9 Hz, 1H), 2.45 (t, J = 1.8 Hz, 2H), 2.45-2.39 (m, 2H), 2.35(t, J = 1.8 Hz, 1H), 2.34-2.30 (m, 2H), 2.20-2.16 (m, 3H), 2.13-2.06 (m, 2H), 1.97-1.93(m, 2H), 1.72-1.69 (m, 3H), 1.66-1.57 (m, 6H), 1.50-1.42 (m, 5H), 1.24-1.23 (m,2H), 1.20-1.16 (m, 2H), 0.91-0.90 (m, 3H), 0.82 (dd, J = 14.7, 6.8 Hz, 6H), 0.73(d, J = 2.0 Hz, 3H).

[0222] Compound 21c: Compound 10a was prepared in the same manner as Compound 21c using Triacic Acid 7 and Trichosanthes 20. Yield = 11%. MS(ESI, pos.): C 62 H 83 N7 O 17 Calculated value for O, 1197.6; measured value 1198.3 (M+H). 1 H-NMR (500 MHz; DMSO-d 6 ): δ 9.98 (s, 1H), 8.10 - 8.08 (m, 2H), 7.93 - 7.90 (m, 1H), 7.58 - 7.55 (m, 2H), 7.27 - 7.25 (m, 2H), 6.99 (s, 2H), 6.85 (dd, J = 11.1, 0.6 Hz, 1H), 6.24 (t, J = 13.0 Hz, 2H), 5.77 - 5.72 (m, 2H), 5.39 - 5.38 (m, 2H), 5.28 (td, J = 2.2, 1.0 Hz, 1H), 4.39 - 4.35 (m, 2H), 4.18 - 4.10 (m, 2H), 4.00 (ddd, J = 11.7, 2.0, 0.8 Hz, 1H), 3.92 (s, 1H), 3.76 - 3.72 (m, 1H), 3.69 - 3.65 (m, 2H), 3.02 - 3.00 (m, 2H), 2.98 - 2.93 (m, 2H), 2.74 - 2.73 (m, 1H), 2.35 - 2.29 (m, 3H), 2.20 - 2.06 (m, 6H), 1.96 - 1.93 (m, 2H), 1.62 - 1.59 (m, 6H), 1.49 - 1.45 (m, 8H), 1.42 (d, J = 6.7 Hz, 4H), 0.91 (d, J = 6.6 Hz, 3H), 0.82 (dd, J = 14.7, 6.7 Hz, 6H), 0.73 (s, 3H).

[0223] Example 7 Compound 22

Chemical Structure

[0224] Compound 22: To a solution of methyl glutarate (2.4 mg, 0.016 mmol) in anhydrous DMF (0.8 mL) was added HATU (6.2 mg, 0.016 mmol), HOBt (2.2 mg, 0.016 mmol) and N,N-diisopropylylethylamine (5.7 μL, 0.033 mmol). The reaction mixture was stirred at room temperature for 5 min and then cooled to 0° C. (R)-amino-verrucarin A (4, 8.2 mg, 0.016 mmol) was added and stirring was continued at 0° C. for 20 min and then at room temperature for 1 h. The product was dissolved in 100 mL of H2O containing 0.05% AcOH. 2 Purification was performed on a 5.5 g C18Aq ISCO column eluted with 5-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 22 (7.5 mg, 73%) as a white fluffy solid. MS (ESI, pos.): C 33 H 43 NO 11 Calculated for, 629.28; found, 630.3 (M+H). 1 H NMR (500 MHz, DMSO-d 6 ): δ 8.27 (d, J = 6.5 Hz, 1H),7.98-7.92 (m, 1H), 6.88 (t, J = 11.5 Hz, 1H), 6.27-6.23 (m, 2H), 5.78-5.75 (m, 2H),5.30-5.29 (m, 1H), 4.43-4.39 (m, 1H), 4.12 (d, J = 12.0 Hz, 1H), 4.04 (d, J = 12.0Hz, 1H), 3.97-3.90 (m, 1H), 3.75 (dd, J = 11.0, 6.5 Hz, 1H), 3.70-3.66 (m, 2H),3.59 (s, 3H), 3.05 (d, J = 4.0 Hz, 1H), 2.76 (d, J = 4.0 Hz, 1H), 2.36-2.28 (m,3H), 2.23-2.17 (m, 3H), 2.12-2.08 (m, 1H), 1.98-1.93 (m, 1H), 1.81-1.68 (m, 2H), 1.67-1.59 (m, 5H), 1.29-1.22 (m, 2H), 0.92 (d, J = 6.5 Hz, 3H), 0.75 (s, 3H).

[0225] Example 8 compound 24 [ka]

[0226] Compound 23: To a solution of compound 6 (15 mg, 0.0244 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (3.4 mg, 0.029 mmol) and DMAP (0.3 mg, 0.002 mmol) in anhydrous DCM (0.24 mL) was added EDCI (7 mg, 0.037 mmol). The reaction mixture was stirred at room temperature for 2 h. The products were both purified by distillation with 0.05% AcOH in H 2 Purification was performed on a 5.5 g C18Aq ISCO column eluted with 5-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 23 (12 mg, 69%) as a white fluffy solid. MS (ESI, pos.): C 37 H 50 N 2 O 12 Calculated for, 714.34; found 715.64 (M+H).

[0227] Compound 24: MeCN and H 2 To a solution of compound 23 (6.0 mg, 0.0084 mmol) in a 1:1 mixture of 2H2O (0.16 mL) was added trifluoroacetic acid (13 μL, 0.1679 mmol). The reaction mixture was stirred at room temperature for 1 h. The products were both dissolved in 10 mM NH 4 H containing OAc 2 Purification was performed on a 5.5 g C18Aq ISCO column eluted with 5-95% MeCN in O. Pure fractions were combined and lyophilized to give compound 24 (4.0 mg, 76%) as a fluffy white solid. MS (ESI, pos.): C 32 H 42 N 2 O 11 Calculated for, 630.69; found 631.44 (M+H). 1 H NMR (500 MHz, DMSO-d 6): δ 10.3 (br, 1H), 8.65(br, 1H), 8.13 (dd, J = 6.0, 0.5 Hz, 1H), 7.95 (dd, J = 15.5, 12.5 Hz, 1H), 6.88(t, J = 11.5 Hz, 1H), 6.28-6.23 (m, 2H), 5.78-5.73 (m, 1H), 5.29-5.28 (m, 1H), 4.42-4.39(m, 1H), 4.12 (d, J = 12.0 Hz, 1H), 4.03 (d, J = 12.0 Hz, 1H), 3.96-3.91 (m, 1H),3.75 (dd, J = 10.0, 6.5 Hz, 1H), 3.69-3.66 (m, 2H), 3.03 (d, J = 1.5 Hz, 1H), 2.81(d, J = 4.0 Hz, 1H), 2.24-2.05 (m, 5H), 1.98-1.93 (m, 3H), 1.81-1.73 (m, 2H), 1.72-1.59(m, 5H), 1.28-1.23 (m, 2H), 0.92 (d, J = 6.5 Hz, 3H), 0.75 (s, 3H).

[0228] Example 9 compound 25 [ka]

[0229] Compound 25: Approximately 0.3M MeONH in THF 2 A solution of MeONH in THF (1.5 mL) 2 It was prepared from the HCl salt (42.5 mg, 0.5 mmol) and KOH (28 mg, 0.5 mmol). The suspension was filtered before use. A solution of compound 6 (5 mg, 0.00812 mmol) and DMAP (0.1 mg, 0.000818 mmol) in DCM was diluted with 0.3 M MeONH in THF (50 mL, 0.015 mmol). 2solution and EDC-HCl (3 mg, 0.0156 mmol) were added. The reaction was stirred at room temperature for 6 h, at which point LCMS showed the reaction was complete. The reaction was concentrated in vacuo. The products, both containing 0.05% HOAc, were purified by HCl distillation. 2 The product was purified by chromatography on a 5.5 g C18Aq ISCO column eluted with 5-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 25 (4.4 mg, 85%) as a white fluffy solid. MS (ESI, pos.): C 33 H 44 N 2 O 11 Calculated for, 644.29; found, 645.3 (M+H). 1 H-NMR (300 MHz; CDCl 3): δ 8.5 (br s, 1H), 8.11(dd, J = 15.7, 11.6 Hz, 1H), 6.69 (t, J = 11.4 Hz, 1H), 6.14 (d, J = 11.1 Hz, 1H),6.10 (d, J = 15.8 Hz, 1H) 5.97 (d, J = 8.2 Hz, 1H), 5.77 (dd, J = 8.1, 4.1 Hz, 1H),5.41 (br d, J = 5.5 Hz, 1H), 4.47 (ddd, J = 11.1, 4.7, 3.5 Hz, 1H), 4.38 (d, J =12.2 Hz, 1H), 4.30 (d, J = 12.9 Hz, 1H), 4.29 (d, J = 8.6 Hz, 1H), 4.07-3.98 (m,1H), 3.85 (d, J = 5.0 Hz, 1H), 3.75 (s, 3H), 3.58 (d, J = 5.0 Hz, 1H), 3.12 (d,J = 3.8 Hz, 1H), 2.88 (d, J = 3.9 Hz, 1H), 2.50 (dd, J = 15.5, 8.2 Hz, 1H), 2.31(t, J = 7.0 Hz, 2H), 2.26-2.15 (m, 3H), 2.09-1.85 (m, 6H), 1.73 (s, 3H), 1.68-1.61(m, 1H), 1.51-1.41 (m, 2H), 1.09 (d, J = 6.7 Hz, 3H), 0.86 (s, 3H).

[0230] Example 10 compound 37 [ka]

[0231] Compound 27: To a solution of compound 26 (100 mg, 0.36 mmol) and Fmoc-6-aminohexanoic acid N-hydroxysuccinimide ester (177.4 mg, 0.39 mmol) in anhydrous DMF (3.5 mL) was added N,N-diisopropylethylamine (0.19 mL, 1.07 mmol). The reaction was stirred at room temperature for 1 h. The products, both of which were purified by HCl distillation with 0.05% AcOH, were purified by HCl distillation with 0.05% AcOH.2 Purification was performed by chromatography on a 150 g C18Aq ISCO column eluting with 0-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 27 (200 mg, 91%) as a white fluffy solid. MS (ESI, pos.): C 34 H 38 N 4 O 7 Calculated for, 614.6; found 615.6 (M+H).

[0232] Compound 29: To a solution of compound 28 (2.00 g, 9.00 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (1.27 g, 10.80 mmol) and DMAP (0.11 g, 0.90 mmol) in anhydrous DCM (90 mL) was added EDCI (2.59 g, 13.50 mmol). The reaction mixture was stirred at room temperature for 2 h. The product was eluted with 50-100% EtOAc in hexanes onto 80 g SiO 2 The pure fractions were combined and concentrated to give compound 29 (2.8 g, 97%) as a colorless gel. MS (ESI, pos.): C 17 H 23 NO 5 Calculated for, 321.37; Found 344.39 (M+Na).

[0233] Compound 32: To a solution of compound 29 (576 mg, 1.79 mmol) in ethanol (6.0 mL) was added PPTS (225 mg, 0.90 mmol). The reaction mixture was stirred at 60° C. for 2 days until compound 30 was obtained as the major product. The reaction was concentrated in vacuo and the residue was dissolved in DCM (6.0 mL). Compound 31 (660 mg, 1.79 mmol) and PPTS (225 mg, 0.90 mmol) were added. The reaction mixture was stirred at 40° C. for 1 day. The reaction was diluted with EtOAc (20 mL) and washed with saturated NaHCO 3 (20 mL), water (20 mL), then brine (20 mL). The product was eluted with 0-100% EtOAc in DCM onto 40 g SiO 2 Column (0.1% Et 3The pure fractions were combined and concentrated to give compound 32 (293 mg, 30%) as a colorless gel. MS (ESI, pos.): 30 H 31 N 3 O 7 Calculated for, 545.59; found 546.55 (M+H).

[0234] Compound 33: To a solution of compound 32 (200 mg, 0.367 mmol) in DMF (1 mL) was added a solution of 5% piperidine in DMF (1 mL) and the reaction was stirred at room temperature for 2 h. The products were both purified by 10 mM NH 4 H containing OAc 2 The product was purified on a 50 g C18Aq ISCO column eluted with 0-40% MeCN in O. Pure fractions were combined and lyophilized to give compound 33 (80.0 mg, 67%) as a colorless gel. MS (ESI, pos.): C 15 H 21 N 3 O 5 Calculated for, 323.35; found 324.34 (M+H).

[0235] Compound 34: To a solution of compound 27 (50.0 mg, 0.081 mmol), compound 33 (34.2 mg, 0.106 mmol) and HATU (30.9 mg, 0.081 mmol) in anhydrous DMF (1 mL) was added N,N-diisopropylethylamine (28.3 μL, 0.163 mmol). The reaction mixture was stirred overnight at room temperature. The products were both dissolved in 10 mM NH 4 H containing OAc 2 Purification was performed on a 15.5 g C18Aq ISCO column eluted with 0-80% MeCN in O. Pure fractions were combined and lyophilized to give compound 34 (41 mg, 55%) as a white fluffy solid. MS (ESI, pos.): C 49 H 57 N 7 O 11 Calculated for, 920.03; found 921.13 (M+H).

[0236] Compound 35: A solution of compound 34 (35.0 mg, 0.038 mmol) and Pd / C (7 mg, 20 wt%) in absolute ethanol (760 μL) was prepared in an oven-dried round-bottom flask. The flask was evacuated and filled with H 2 The reaction was stirred at room temperature for 30 minutes. 2 The mixture was filtered through a 0.45 μm syringe filter and the filtrate was concentrated in vacuo. Compound 35 (16 mg, 51%) was obtained as a colorless gel, which was used without purification. MS (ESI, pos.): C 42 H 51 N 7 O 11 Calculated for, 829.91; found 830.93 (M+H).

[0237] Compound 36: To a solution of (R)-amino-verrucarin A (4, 9.0 mg, 0.018 mmol), compound 35 (16.0 mg, 0.019 mmol) and HATU (10.2 mg, 0.027 mmol) in anhydrous DMF (0.4 mL) was added N,N-diisopropylethylamine (6.3 μL, 0.036 mmol). The reaction mixture was stirred overnight at room temperature. The products were both dissolved in 10 mM NH 4 H containing OAc 2 Purification was performed on a 5.5 g C18Aq ISCO column eluted with 0-80% MeCN in O. Pure fractions were combined and lyophilized to give compound 36 (9 mg, 38%) as a white solid. MS (ESI, pos.): C 69 H 84 N 8 O 18 Calculated for, 1313.47; found 1314.50 (M+H).

[0238] Compound 37: To a solution of compound 36 (9 mg, 0.0069 mmol) in DMF (0.1 mL) was added a solution of 5% piperidine in DMF (0.3 mL) and the reaction was stirred at room temperature for 1 h. The products were both purified by 10 mM NH 4 H containing OAc 2Purification was performed on a 5.5 g C18Aq ISCO column eluted with 0-80% MeCN in O. Pure fractions were combined and lyophilized to give compound 37 (3.1 mg, 41%) as a white fluffy solid. MS (ESI, pos.): C 54 H 74 N 8 O 18 Calculated for, 1091.23; found 1092.4 (M+H). 1 H NMR (500 MHz, DMSO-d 6 ): δ 8.20-8.12 (m, 5H),8.08-8.04 (m, 1H), 7.95 (dd, J = 15.5, 12.5 Hz, 1H), 7.25-7.24 (m, 5H), 7.19-7.17(m, 1H), 6.87 (t, J = 11.5 Hz, 1H), 6.27-6.22 (m, 2H), 5.78-5.75 (m, 1H), 5.29-5.28(m, 1H), 4.76-4.75 (m, 2H), 4.53-4.49 (m, 1H), 4.42-4.38 (m, 1H), 4.12 (d, J = 12.0Hz, 1H), 4.03 (d, J = 12.0 Hz, 1H), 3.96-3.90 (m, 1H), 3.76-3.71 (m, 4H), 3.69-3.66(m, 5H), 3.60-3.55 (m, 1H), 3.07-3.02 (m, 3H), 2.81-2.80 (m, 1H), 2.59-2.56 (m,2H), 2.46-2.41 (m, 1H), 2.18-2.11 (m, 5H), 1.99-1.93 (m, 3H), 1.82-1.58 (m, 12H),1.50-1.46 (m, 2H), 1.40-1.35 (m, 2H), 1.29-1.22 (m, 4H), 0.92 (d, J = 7.0 Hz, 3H),0.75 (s, 3H).

[0239] Example 11 compound 46 [ka]

[0240] Compound 38 was prepared using the literature procedure in Bioconjugate Chemistry (2016), 27(10), 2549-2557.

[0241] Compound 39: Argon was bubbled through a solution of compound 38 (150 mg, 0.30 mmol) in THF (10 mL) for 10 min. Zinc powder (505 mg, 7.72 mmol) and ammonium formate (60 mg, 0.92 mmol) were added and the reaction was heated to 65° C. for 16 h. The reaction was filtered through a pad of Celite and the filtrate was concentrated to give compound 39 (141 mg, 99%), which was used without purification. MS (ESI, pos.): C 21 H 27 NO 11 Calculated for, 469.1; found, 470.2 (M+H).

[0242] Compound 42: To a solution of Fmoc-PEG8-amide-COOH (40, 240 mg, 0.36 mmol) in anhydrous DCM (10 mL) was added oxalyl chloride (62 μL, 0.72 mmol) and DMF (2 μL). After stirring for 30 min, the volatiles were removed in vacuo to give Fmoc-PEG8-amide-COCl, 41. In a separate vial, compound 39 (141 mg, 0.30 mmol) was dissolved in anhydrous THF (5 mL) and N,N-diisopropylylethylamine (125 μL, 0.72 mmol) was added followed by the acid chloride (41) in THF (5 mL). After 1 h, the solvent was removed under reduced pressure and the residue was purified by elution with 5-95% MeCN / H 2 The mixture was purified on an ISCO 100 g C18Aq column eluted with 0 (both containing 0.05% AcOH). Fractions containing the desired product were combined and lyophilized to give compound 42 (205 mg, 61%) as a fluffy off-white solid. MS (ESI, pos.): C 55 H 74 N 2 O 22 Calculated for, 1114.5; found 1115.4 (M+H).

[0243] Compound 43: To a solution of compound 42 (45 mg, 0.0404 mmol), glutaric acid (10 mg, 0.0757 mmol), and DMAP (9.0 mg, 0.0737 mmol) in DCM (0.7 mL) was added EDC-HCl (10 mg, 0.0522 mmol). The yellow solution was stirred at room temperature for 18 h. The reaction was shown to be complete by LCMS and was concentrated under vacuum. The product was purified by HPLC using H 2 The product was purified by chromatography on a 50 g C18Aq ISCO column eluted with 25-80% MeCN in O (both containing 0.05% HOAc). The pure fractions were combined and lyophilized to give compound 43 (40 mg, 81%) as a colorless viscous oil. MS (ESI, pos.): 60 H 80 N 2 O 25 Calculated for, 1228.5; Found, 1229.7 (M+H).

[0244] Compound 44: To a solution of compound 43 (32 mg, 0.0260 mmol), (R)-amino-verrucarin (4, 12 mg, 0.0239 mmol), and DMAP (3.2 mg, 0.0263 mmol) in DCM (1 mL) was added EDC-HCl (5.0 mg, 0.0263 mmol). The resulting solution was stirred at room temperature for 2.5 h, then diluted with DCM (4 mL) and washed with 0.5 N HCl (aq) (1 mL). The layers were separated and the aqueous layer was extracted with DCM (2 × 3 mL). The combined organic layers were washed with saturated NaHCO 3 (aqueous), then saturated brine, followed by Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo to give a viscous oil. The product was purified by chromatography on a 12 g silica ISCO column eluting with 0-30% methanol in DCM to give compound 44 (29 mg, 71%) as a colorless oil. MS (ESI, pos.): C 87 H 113 N 3 O 32 Calculated for, 1711.73; found 1713.27 (M+H).

[0245] Compound 45: To a -10 °C solution of compound 44 (10 mg, 0.00584 mmol) in methanol (0.9 mL) was added 0.1 M sodium methoxide solution in MeOH (117 μL, 0.0117 mmol) dropwise via syringe. The reaction was stirred in a cold bath for 1.25 h and then quenched by the addition of Dowex 50X8 resin (hydrogen form, 200-400 mesh, 30 mg). The mixture was stirred at room temperature for 2 min and then the solids were removed by filtration through a cotton plug, rinsing with additional methanol. The filtrate was concentrated in vacuo. The product was purified by HPLC using HPLC with HPLC. 2 Purification was performed by chromatography on a 5.5 g ISCO C18Aq column eluting with 10-100% MeCN in O (both containing 0.05% HOAc). Pure fractions were combined and lyophilized to give compound 45 (3.2 mg, 35%) as a white fluffy solid. MS (ESI, pos.): C 79 H 105 N 3 O 28 Calculated for, 1543.7; found 1545.2 (M+H).

[0246] Compound 46: To a solution of compound 45 (5.8 mg, 0.0038 mmol) in DMF (360 μL) was added 3.8 μL of a 10% solution of piperidine in DMF (0.38 μL, 0.0038 mmol). The reaction was stirred at room temperature for 6 h, at which point LCMS indicated the reaction was complete. The reaction solution was loaded onto an ISCO 5.5 g C18Aq column and purified by HPLC. 2 Elution was performed with 0-100% MeCN in O (both containing 0.05% HOAc). Pure fractions were combined and lyophilized to give compound 46 (2.4 mg, 48%) as a white fluffy solid. MS (ESI, pos.): 64 H 95 N 3 O 26 Calculated for, 1321.6; found 1323.1 (M+H). 1 H NMR (300 MHz; CD 3OD) δ 8.27-8.17 (m, 2H), 7.29 (d, J = 8.4 Hz, 1H), 7.12-7.08 (m, 1H), 6.85 (t,J = 11.3 Hz, 1H), 6.22 (d, J = 11.1 Hz, 1H), 6.14 (d, J = 15.6 Hz, 1H), 5.87-5.83(m, 1H), 5.41-5.39 (m, 1H), 5.16-5.06 (m, 2H), 4.79 (d, J = 7.8 Hz, 1H), 4.54-4.48(m, 1H), 4.37-4.24 (m, 2H), 4.10-4.01 (m, 2H), 3.95-3.75 (m, 10H), 3.69-3.58 (m,36H), 3.04 (d, J = 4.1 Hz, 1H), 2.96 (dd, J = 6.5, 3.9 Hz, 2H), 2.81 (d, J = 3.8Hz, 1H), 2.72 (t, J = 5.9 Hz, 2H), 2.56-2.43 (m, 3H), 2.43-2.26 (m, 4H), 2.19-2.11(m, 1H), 1.99-1.89 (m, 4H), 1.85-1.66 (m, 7H), 1.39-1.28 (m, 4H), 1.07 (d, J = 6.5Hz, 3H), 0.89 (s, 3H).

[0247] Example 12 Compound 53

change

[0248] Compound 47 is prepared in Bioconjugate Chemistry (2016), 27(10), 2549-2557.

[0249] Compound 48: Argon was bubbled through a solution of compound 47 (80 mg, 0.165 mmol) in THF (10 mL) for 10 min. Zinc powder (268 mg, 4.12 mmol) and ammonium formate (32 mg, 0.495 mmol) were then added and the reaction was heated to 65° C. for 16 h. The reaction was filtered through a pad of Celite and the filtrate was concentrated to give compound 48 (75 mg, 99%), which was used without purification. MS (ESI, pos.): C 20 H 25 NO 11 Calculated for, 455.1; found, 456.1 (M+H).

[0250] Compound 49: To a solution of Fmoc-PEG8-amide-COOH (40, 142 mg, 0.215 mmol) in anhydrous DCM (10 mL) was added oxalyl chloride (37 μL, 0.430 mmol) and DMF (2 μL). After stirring for 30 min, the volatiles were removed in vacuo to give Fmoc-PEG8-amide-COCl (41). In a separate vial, compound 48 (75 mg, 0.165 mmol) was dissolved in anhydrous THF (5 mL) and N,N-diisopropylylethylamine (73 μL, 0.430 mmol) was added followed by compound 41 in THF (5 mL). After 1 h, the volatiles were removed under reduced pressure and the residue was purified by elution with 5-95% MeCN / H 2 The product was purified on an ISCO 100 g C18Aq column eluted with 0 (both containing 0.05% AcOH). Fractions containing the desired product were combined and lyophilized to give compound 49 (47.1 mg, 25%) as a fluffy white solid. MS (ESI, pos.): C 54 H 72 N 2 O 22 Calculated for, 1100.5; found 1101.4 (M+H).

[0251] Compound 50: To a solution of compound 48 (56 mg, 0.051 mmol), glutaric acid (67 mg, 0.509 mmol) and DMAP (9.3 g, 0.076 mmol) in a 1:1 mixture of anhydrous DCM and THF (5 mL) was added EDCI (58.5 mg, 0.305 mmol). The reaction mixture was stirred at room temperature overnight. The products were both purified by distillation with 0.05% AcOH in H 2 Purification was performed on a 15.5 g C18Aq ISCO column eluted with 0-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 50 (37.5 mg, 61%) as a white solid. MS (ESI, pos.): C 59 H 78 N 2 O 25 Calculated for, 1215.26; found 1216.45 (M+H).

[0252] Compound 51: To a solution of compound 50 (17 mg, 0.014 mmol), (R)-amino-verrucarin A (4, 7.0 mg, 0.014 mmol) and DMAP (0.9 mg, 0.007 mmol) in anhydrous DCM (2.3 mL) was added EDCI (8.1 mg, 0.042 mmol). The reaction mixture was stirred at room temperature for 1 h. The products were both purified by distillation with 0.05% AcOH in H 2 Purification was performed on a 5.5 g C18Aq ISCO column eluted with 0-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 51 (20.5 mg, 86%) as a white solid. MS (ESI, pos.): C 86 H 111 N 3 O 32 Calculated for, 1698.82; found 1699.81 (M+H).

[0253] Compound 52: MeOH and H 2 A solution of compound 51 (20.5 mg, 0.012 mmol) in a 2:1 mixture of HO (4.5 mL) was cooled to 0 °C in an ice-water bath. 2A 0.1 M solution of LiOH in O (483 μL) was added dropwise and the reaction mixture was stirred for 30 min while the temperature was maintained at 0° C. The reaction was quenched by the dropwise addition of AcOH. Both products were purified by distillation in H2O containing 0.05% AcOH. 2 Purification was performed on a 15.5 g C18Aq ISCO column eluted with 0-100% MeCN in O. Pure fractions were combined and lyophilized to give compound 52 (6.8 mg, 30%) as a white solid. MS (ESI, pos.): C 79 H 103 N 3 O 29 Calculated for, 1558.69; found 1559.63 (M+H).

[0254] Compound 53: To a solution of compound 52 (6.8 mg, 0.004 mmol) in DMF (121 μL) was added a 5% solution of piperidine in DMF (24 μL) and the reaction was stirred at room temperature for 1 h. Both products were purified by HCl with 0.05% AcOH. 2 Purification was performed by Teledyne ISCO EZ prep on a 30 × 150 mm Gemini column eluted with 0–60% MeCN in O. Pure fractions were combined and lyophilized to give compound 53 (3.1 mg, 53%) as a white solid. MS (ESI, pos.): C 64 H 93 N 3 O 27 Calculated for, 1336.44; found 1337.4 (M+H). 1H NMR (500 MHz, MeOD): δ 8.22-8.17 (m, 2H), 7.35 (d, J= 8.5 Hz, 1H), 7.08 (dd, J = 8.5, 1.5 Hz, 1H), 6.82 (t, J = 11.0 Hz, 1H), 6.18 (d,J = 11.5 Hz, 1H), 6.11 (d, J = 15.5 Hz, 1H), 5.82 (dd, J = 8.0, 3.5 Hz, 1H), 5.38-5.37(m, 1H), 5.08 (br, 2H), 4.85 (br, 1H), 4.78 (d, J = 7.0 Hz, 1H), 4.49-4.46 (m, 1H), 4.32 (d, J = 12.5 Hz, 1H), 4.23 (d, J = 12.5 Hz, 1H), 4.03 (td, J = 11.5, 3.0 Hz, 1H), 3.89-3.83 (m, 3H), 3.76-3.70 (m, 5H), 3.67-3.59 (m, 34H), 3.53-3.51 (m, 3H), 3.10 (t, J = 10.5 Hz, 2H), 3.02 (d, J = 4.0 Hz, 1H), 2.81 (d, J = 3.5 Hz, 1H), 2.72(t, J = 5.0 Hz, 2H), 2.52-2.23 (m, 8H), 2.12 (dt, J = 15.5, 5.0 Hz, 1H), 1.94-1.89 (m, 3H), 1.81-1.73 (m, 3H), 1.67 (s, 3H), 1.35-1.29 (m, 1H), 1.05 (d, J = 6.5 Hz, 3H), 0.88 (s, 3H).

[0255] Example 13 Compound 54

change

[0256] Compound 54: To a solution of verrucarin A (1, 5 mg, 0.01 mmol) in anhydrous THF (1 mL) at 0° C. was added NaHMDS (11 μL, 1.0 M in THF, 0.011 mmol). The yellow solution was stirred for 5 min before methyl iodide (6.1 μL, 0.1 mmol) was added. The reaction was stirred at 0° C. for 30 min, at which point LC / MS indicated the reaction was complete. The reaction was diluted with saturated aqueous ammonium chloride (1 mL) and water (1 mL). The reaction was extracted with dichloromethane (3×3 mL). The combined organics were washed with brine before being dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by chromatography on an ISCO 4 g silica gold column using 35% ethyl acetate / hexane to give compound 54 (5 mg, 97%). MS (ESI, pos.): C 28 H 36 O 9 Calculated for, 516.2; Found 517.3 (M+H), 539.2 (M+Na). 1 H-NMR (500 MHz; CDCl 3): δ 8.02 (dd, J = 15.6,11.7 Hz, 1H), 6.67 (t, J = 11.3 Hz, 1H), 6.16 (d, J = 11.0 Hz, 1H), 6.05 (d, J =15.7 Hz, 1H), 5.80 (dd, J = 7.9, 3.8 Hz, 1H), 5.45-5.44 (m, 1H), 4.72 (d, J = 12.2Hz, 1H), 4.48-4.46 (m, 1H), 4.20 (d, J = 12.2 Hz, 1H), 4.02 (td, J = 11.4, 2.9 Hz,1H), 3.87 (d, J = 5.0 Hz, 1H), 3.69 (s, 1H), 3.59 (d, J = 5.0 Hz, 1H), 3.38 (s,3H), 3.13 (d, J = 3.8 Hz, 1H), 2.82 (d, J = 3.8 Hz, 1H), 2.50 (dd, J = 15.4, 8.2Hz, 1H), 2.35-2.33 (m, 1H), 2.23 (dt, J = 15.4, 4.5 Hz, 1H), 2.02-1.83 (m, 3H),1.77 (s, 3H), 1.56 (s, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.87 (s, 3H).

[0257] Example 14 compound 55 [ka]

[0258] Compound 55:1:1 v / v AcOH-Ac 2 Verrucarin A (1, 5 mg, 0.01 mmol) and SeO in O (1 mL) 2 The solution was refluxed for 1 h. The reaction was cooled to ambient temperature and concentrated to dryness. The residue was purified by dilution with 5-95% MeCN / H 2The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 1,2-dichlorophenyl ether (C1H2O) and 1,2-dichlorophenyl ether (C1H2O) (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 55 (3 mg, 58%) as a fluffy off-white solid. MS (ESI, pos.): 27 H 34 O 10 Calculated value for, 518.2; measured value 519.2M+H). 1 H-NMR (500 MHz; CDCl 3 ): δ 8.05 (dd, J = 15.2, 12.0 Hz,1H), 6.69 (t, J = 11.3 Hz, 1H), 6.16 (d, J = 11.1 Hz, 1H), 6.06 (d, J = 15.5 Hz,1H), 5.77 (dd, J = 8.1, 4.0 Hz, 1H), 5.53 (d, J = 4.9 Hz, 1H), 4.73 (d, J = 12.2Hz, 1H), 4.54-4.51 (m, 1H), 4.29 (d, J = 12.0 Hz, 1H), 4.18 (d, J = 1.1 Hz, 1H), 4.04-3.97 (m, 2H), 3.90 (d, J = 5.1 Hz, 1H), 3.59 (d, J = 4.8 Hz, 1H), 3.15 (d,J = 3.7 Hz, 1H), 2.85 (d, J = 3.7 Hz, 1H), 2.48 (dd, J = 15.3, 7.8 Hz, 1H), 2.39-2.35(m, 1H), 2.26 (m, 1H), 2.18 (dd, J = 8.7, 3.2 Hz, 1H), 2.11 (dd, J = 12.5, 6.4 Hz,1H), 1.99-1.93 (m, 1H), 1.89-1.86 (m, 4H), 1.85-1.77 (m, 2H), 0.89 (d, J = 6.7 Hz, 3H), 0.87 (s, 3H).

[0259] Example 15 compound 56 [ka]

[0260] Compound 56: To a solution of verrucarin A (1.5 mg, 0.01 mmol) in anhydrous chloroform (0.5 mL) was added m-CPBA (1.9 mg, 0.011 mmol). The reaction was stirred for 24 h, after which the volatiles were removed under reduced pressure. The residue was purified by elution with 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 1,2-dichlorophenyl ether (C1H2O) and 1,2-dichlorophenyl ether (C1H2O) (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 56 (3.5 mg, 68%) as a fluffy off-white solid. MS (ESI, pos.): 27 H 34 O 10 , calculated value for 518.2; measured value 519.2M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.06 (dd, J = 15.6, 11.8 Hz,1H), 6.69 (t, J = 11.3 Hz, 1H), 6.15 (d, J = 11.1 Hz, 1H), 6.05 (d, J = 15.7 Hz,1H), 5.78 (dd, J = 7.9, 4.0 Hz, 1H), 4.67 (d, J = 12.1 Hz, 1H), 4.54-4.51 (m, 1H),4.28 (d, J = 12.2 Hz, 1H), 4.16 (s, 1H), 4.00-3.96 (m, 1H), 3.96 (d, J = 4.8 Hz,1H), 3.55 (d, J = 4.2 Hz, 1H), 3.17 (d, J = 3.6 Hz, 1H), 3.12 (d, J = 5.2 Hz, 1H),2.76 (d, J = 3.5 Hz, 1H), 2.67-2.64 (m, 1H), 2.46 (dd, J = 15.4, 8.3 Hz, 1H), 2.36(t, J = 5.8 Hz, 1H), 2.24 (dt, J = 15.3, 4.4 Hz, 1H), 2.19 (s, 1H), 1.98-1.91 (m,2H), 1.83-1.70 (m, 2H), 1.59-1.52 (m, 1H), 1.41 (s, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.80 (s, 3H).

[0261] Example 16 compound 57 [ka]

[0262] Compound 57: To a solution of verrucarin A triflate (2, 5.0 mg, 0.0079 mmol) in DMF (0.1 mL) under argon was added potassium thioacetate (2.7 mg, 0.0236 mmol). The reaction was stirred at room temperature for 20 min, at which point LCMS indicated complete consumption of starting material. The reaction was diluted with EtOAc (1 mL) and washed with 1:1 brine / H 2The combined organic layers were washed with brine (1 mL) and then with NaCl. The aqueous layer was extracted with EtOAc (2×1 mL). 2 SO 4 The mixture was dried at 77° C., filtered and concentrated in vacuo. Chromatography on an ISCO 4 g silica gold column eluted with EtOAc / hexanes (0-100%) afforded compound 57 (4.4 mg, quantitative) as a white solid. MS (ESI, pos.): C 29 H 36 O 9 Calculated for S, 560.21; found 561.2 (M+H). 1 H-NMR (500 MHz; CDCl 3 ): δ 8.05-8.00 (m, 1H), 6.69-6.64(m, 1H), 6.15-6.13 (m, 1H), 6.03 (d, J = 15.5 Hz, 1H), 5.78-5.76 (m, 1H), 5.41 (d,J = 1.3 Hz, 1H), 4.55 (d, J = 11.8 Hz, 1H), 4.49-4.46 (m, 1H), 4.20 (d, J = 12.0Hz, 1H), 3.98-3.95 (m, 2H), 3.85 (s, 1H), 3.56 (t, J = 0.6 Hz, 1H), 3.13 (s, 1H),2.85 (s, 1H), 2.52-2.47 (m, 1H), 2.37 (s, 3H), 2.31-2.26 (m, 1H), 2.22-2.16 (m,2H), 1.98-1.83 (m, 3H), 1.80-1.74 (m, 4H), 1.33-1.25 (m, 1H), 1.13 (d, J = 5.4 Hz,3H), 0.89-0.86 (m, 3H).

[0263] Example 17 compound 58 [ka]

[0264] Compound 58: To a solution of verrucarin A triflate (2, 8 mg, 0.012 mmol) in DCM (2 mL) at room temperature was added methylamine (2 M in THF, 2 mL, 4 mmol). The reaction was stirred for 30 min, at which point LCMS analysis indicated approximately 40% conversion to the desired product. The reaction was concentrated in vacuo, redissolved in DCM (2 mL) and additional methylamine solution (2 mL) was added. The reaction was stirred for 2 h, after which the process was repeated, at which point LCMS indicated the reaction had reached completion. The volatiles were removed in vacuo. The residue was dissolved in DMF (0.5 mL) and injected onto an ISCO 5.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 58 (3.2 mg, 49%) as a white fluffy solid. MS (ESI, pos.): C 28 H 37 NO 8 Calculated for, 515.3; found 516.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.11 (dd, J = 15.5, 11.6 Hz,1H), 6.70 (t, J = 11.3 Hz, 1H), 6.15 (d, J = 11.1 Hz, 1H), 6.06 (d, J = 15.6 Hz,1H), 5.84-5.82 (m, 1H), 5.46 (dd, J = 3.1, 1.0 Hz, 1H), 4.65 (d, J = 12.2 Hz, 1H),4.55-4.52 (m, 1H), 4.17 (d, J = 12.3 Hz, 1H), 4.04-3.99 (m, 1H), 3.89 (d, J = 5.1Hz, 1H), 3.61 (d, J = 4.6 Hz, 1H), 3.15 (d, J = 3.8 Hz, 1H), 2.88-2.84 (m, 2H),2.54-2.49 (m, 3H), 2.42 (s, 3H), 2.27-2.22 (m, 1H), 2.00-1.95 (m, 4H), 1.77-1.75(m, 1H), 1.46 (s, 3H), 1.28-1.24 (m, 1H), 1.03 (d, J = 6.6 Hz, 3H), 0.90 (s, 3H).

[0265] Example 18 compound 59 [ka]

[0266] Compound 59: To a solution of verrucarin A triflate (2, 6.3 mg, 0.01 mmol) in THF (1 mL) at room temperature, ethylamine (2 M in THF, 1 mL) was added and the reaction was stirred for 18 h, at which point LCMS indicated complete consumption of the triflate. The volatiles were removed in vacuo. The residue was dissolved in DMF (0.5 mL) and injected onto an ISCO 5.5 g C18Aq column and purified by elution with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 59 (3.4 mg, 64%) as a white fluffy solid. MS (ESI, pos.): 29 H 39 NO8 Calculated value for it, 529.3; measured value 530.3 (M+H). 1 H-NMR (500 MHz; CDCl 3 ): δ 8.11 (dd, J = 15.7, 11.8 Hz, 1H), 6.70 (t, J = 11.3 Hz, 1H), 6.15 (d, J = 11.0 Hz, 1H), 6.06 (d, J = 15.7 Hz, 1H), 5.83 (dd, J = 7.9, 3.8 Hz, 1H), 5.46 (d, J = 4.1 Hz, 1H), 4.62 (d, J = 12.2 Hz, 1H), 4.55 - 4.52 (m, 1H), 4.17 (d, J = 12.3 Hz, 1H), 4.02 (d, J = 2.1 Hz, 1H), 3.89 (d, J = 5.0 Hz, 1H), 3.61 (d, J = 4.7 Hz, 1H), 3.15 (d, J = 3.9 Hz, 1H), 2.95 (d, J = 10.0 Hz, 1H), 2.84 (d, J = 3.9 Hz, 1H), 2.63 - 2.49 (m, 4H), 2.25 - 2.23 (m, 1H), 2.02 - 1.95 (m, 4H), 1.77 (s, 3H), 1.69 (d, J = 8.1 Hz, 1H), 1.26 - 1.23 (m, 1H), 1.12 (t, J = 7.1 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.90 (s, 3H).

[0267] Example 19 Compound 60

Chemical Structure

[0268] Compound 60: To a solution of (R)-amino-verrucarin A acetate (4.7 mg, 0.012 mmol) in DCE (1 mL) was added paraformaldehyde (3.6 mg, 0.12 mmol) and sodium triacetoxyborohydride (25 mg, 0.12 mmol). The reaction was heated to 60° C. for 5 h, at which point LCMS indicated the reaction was complete. The volatiles were removed in vacuo. The residue was dissolved in DMF (0.5 mL) and injected onto an ISCO 5.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 60 (2.0 mg, 29%) as a white fluffy solid. MS (ESI, pos.): 29 H 39 NO 8 Calculated for, 529.3; found 530.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.08 (ddd, J = 15.7, 11.6,1.0 Hz, 1H), 6.68 (t, J = 11.3 Hz, 1H), 6.15 (d, J = 11.1 Hz, 1H), 6.05 (d, J =15.7 Hz, 1H), 5.83 (dd, J = 8.1, 3.8 Hz, 1H), 5.44 (dd, J = 5.3, 1.2 Hz, 1H), 4.76(d, J = 12.3 Hz, 1H), 4.52 (ddd, J = 11.1, 6.1, 1.9 Hz, 1H), 4.09 (d, J = 12.3 Hz,1H), 4.03 (td, J = 11.6, 3.9 Hz, 1H), 3.87 (d, J = 5.2 Hz, 1H), 3.58 (d, J = 5.3Hz, 1H), 3.12 (d, J = 4.0 Hz, 1H), 2.99 (d, J = 11.0 Hz, 1H), 2.82 (d, J = 4.0 Hz,1H), 2.48 (dd, J = 15.5, 8.2 Hz, 1H), 2.34 (s, 6H), 2.31-2.21 (m, 3H), 2.01-1.95(m, 3H), 1.83-1.81 (m, 1H), 1.77 (s, 3H), 1.26-1.19 (m, 1H), 1.02 (d, J = 6.5 Hz,3H), 0.87 (s, 3H).

[0269] Example 20 compound 61 [ka]

[0270] Compound 61: To a solution of verrucarin A triflate (2, 10 mg, 0.0158 mmol) in DMF (100 mL) was added N-methylpiperazine (9 mL, 0.0811 mmol). The resulting solution was stirred for 3.5 days, at which point LCMS indicated the reaction was complete. The reaction solution was loaded onto an ISCO 5.5 g C18 column and purified by HPLC. 2Elution was performed with 0-100% MeCN (both containing 0.05% HOAc) in O. Fractions that gave a single peak by LCMS were combined and lyophilized. 1 H NMR analysis showed that the product was not pure. The impure product was separated by chromatography on an ISCO 4 g silica column eluted with a gradient of MeOH / DCM (0-10%). Fractions containing the pure product were combined and concentrated to give compound 61 as a white solid (1.8 mg, 20%). MS (ESI, pos.): C 32 H 44 N 2 O 8 Calculated for, 584.3; found 585.3 (M+H). 1 H NMR (300 MHz; CDCl 3 ) δ 8.09 (dd, J = 15.7, 11.6 Hz, 1H), 6.67 (t, J = 11.5 Hz, 1H), 6.14 (d, J =11.1 Hz, 1H), 6.04 (d, J = 15.7 Hz, 1H), 5.82 (dd, J = 8.1, 3.8 Hz, 1H), 5.42-5.41(m, 1H), 4.80 (d, J = 12.3 Hz, 1H), 4.53-4.48 (m, 1H), 4.08-3.96 (m, 2H), 3.85 (d,J = 5.0 Hz, 1H), 3.65-3.51 (m, 2H), 3.10 (d, J = 3.9 Hz, 1H), 2.99 (d, J = 11.2Hz, 1H), 2.83 (d, J = 3.8 Hz, 1H), 2.66-2.52 (m, 4H), 2.52-2.29 (m, 6H), 2.28-2.22(m, 4H), 2.22-2.11 (m, 2H), 2.05-1.77 (m, 6H), 1.75 (s, 3H), 0.99 (d, J = 6.4 Hz,3H), 0.85 (s, 3H).

[0271] Example 21 compound 64 [ka]

[0272] Compound 62: To a solution of verrucarin A triflate (2, 24 mg, 0.0378 mmol) in acetone (0.4 mL) was added sodium iodide (9 mg, 0.0600 mmol). The reaction was stirred at room temperature for 12.5 h, at which point LCMS indicated the reaction was complete. The reaction was diluted with EtOAc (2 mL) and diluted with H 2 The combined organic layers were washed with brine (2 mL) and then with NaCl. The aqueous layer was extracted with EtOAc (3×1 mL). 2 SO 4 The mixture was dried over H, filtered and concentrated in vacuo. 2 Chromatography on an ISCO 5.5 g C18Aq column eluted with 20-80% MeCN in O (both containing 0.05% HOAc) gave compound 62 (11 mg, 43%). MS (ESI, pos.): 27 H 33 IO 8 Calculated for, 612.12; Found 613.1 (M+H), 635.1 (M+Na).

[0273] Compound 63: To a solution of compound 62 (11 mg, 0.0178 mmol) in DMF (0.175 mL) was added sodium azide (3.5 mg, 0.0538 mmol). The reaction was stirred at room temperature for 4 h, then diluted with EtOAc (1 mL) and diluted with H 2 The combined organic layers were washed with brine (1 mL) and then with NaCl. The aqueous layer was extracted with EtOAc (3×1 mL). 2 SO 4 The mixture was dried at 77° C., filtered and concentrated in vacuo. Chromatography on an ISCO 4 g silica column eluting with EtOAc / hexane gave compound 63 (6 mg, 64%). MS (ESI, pos.): C 27 H 33 N 3 O 8 Calculated for, 527.23; found 528.3 (M+H).

[0274] Compound 64: To a solution of compound 63 (6 mg, 0.011 mmol) in THF (1 mL) was added triphenylphosphine (6 mg, 0.023 mmol). The reaction was stirred at room temperature for 18 h. DI water (0.2 mL) was added to the reaction and the mixture was heated in an aluminum block at 45° C. for 4.5 h. After cooling to room temperature, the reaction was concentrated in vacuo. The product was purified by HPLC using H 2 The product was purified by chromatography on an ISCO 5.5 g C18Aq column eluted with 5-100% MeCN in O (both containing 0.05% HOAc). Fractions containing the pure product were combined and lyophilized to give (S)-amino-verrucarin A (64, 2 mg, 36%) as a fluffy white solid. MS (ESI, pos.): C 27 H 35 NO 6 Calculated for, 501.24; found 502.2 (M+H). 1 H NMR (300 MHz; CDCl 3) δ 8.01(dd, J = 15.7, 11.6 Hz, 1H), 6.66 (t, J = 11.3 Hz, 1H), 6.16 (d, J = 10.6 Hz, 1H), 6.04 (d, J = 15.7 Hz, 1H), 5.79 (dd, J = 8.0, 3.9 Hz, 1H), 5.44-5.42 (m, 1H), 4.66(d, J = 12.1 Hz, 1H), 4.51-4.44 (m, 1H), 4.17 (d, J = 12.1 Hz, 1H), 4.04-3.95 (m,1H), 3.86 (d, J = 5.0 Hz, 1H), 3.65 (d, J = 4.3 Hz, 1H), 3.57 (d, J = 5.4 Hz, 1H), 3.44 (d, J = 2.1 Hz, 1H), 3.12 (d, J = 3.9 Hz, 1H), 2.81 (d, J = 4.0 Hz, 1H), 2.49(dd, J = 15.4, 8.2 Hz, 1H), 2.41-2.36 (m, 1H), 2.22 (dt, J = 15.6, 4.6 Hz, 1H), 1.99-1.74 (m, 7H), 1.74 (s, 3H), 0.88 (d, J = 6.8 Hz, 3H) Repeat 0.87 (s, 3H).

[0275] Example 22 Compound 65

change

[0276] Compound 65: Verrucarin A (1, 5 mg, 0.01 mmol) in a small vial was azeotropically dried with toluene (2 x 3 mL). To the vial containing verrucarin A in a glove box was added (S)-3,4-dimethyloxazolidine-2,5-dione (9 mg, 0.07 mmol), THF (0.6 mL) and DMF (0.2 mL). N,N-Diisopropylethylamine (11 μL, 0.06 mmol) was added followed by zinc triflate (11 mg, 0.03 mmol). The reaction vial was removed from the glove box and stirred under argon for 22 h, at which point the reaction was complete by LCMS. The reaction was quenched with water (0.3 mL) and stirred for 5 min before being injected onto an ISCO 15.5 g C18Aq column and purified using 5-40% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 65 (4.9 mg, 85%) as a fluffy off-white solid. MS (ESI, pos.): C 31 H 41 NO 10 Calculated for, 587.3; found 588.2 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.04 (dd, J = 15.4, 11.9 Hz,1H), 6.69 (t, J = 11.3 Hz, 1H), 6.19 (d, J = 11.1 Hz, 1H), 6.07 (d, J = 15.6 Hz,1H), 5.82 (dd, J = 7.8, 3.3 Hz, 1H), 5.42 (d, J = 3.9 Hz, 1H), 4.88 (s, 1H), 4.69(d, J = 12.0 Hz, 1H), 4.49 (d, J = 10.9 Hz, 1H), 4.23-4.20 (m, 1H), 4.03 (t, J =11.1 Hz, 1H), 3.88 (d, J = 5.0 Hz, 1H), 3.58 (d, J = 4.9 Hz, 1H), 3.41-3.36 (m,1H), 3.15-3.14 (m, 1H), 2.84-2.83 (m, 1H), 2.56-2.47 (m, 2H), 2.44 (s, 3H), 2.25(dt, J = 15.2, 4.6 Hz, 1H), 2.05-2.02 (m, 1H), 1.96-1.84 (m, 4H), 1.75 (d, J = 6.2Hz, 3H), 1.73-1.68 (m, 1H), 1.40 (d, J = 7.0 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 0.89 (s, 3H).

[0277] Example 23 compound 66 [ka]

[0278] Compound 66: To a solution of compound 65 (3.8 mg, 0.0065 mmol) in DCM (1 mL) at 0 °C was added pyridine (1 mL) and acetic anhydride (6.6 μL, 0.065 mmol). The reaction was stirred at 0 °C for 10 min, at which point LCMS indicated the reaction was complete. The volatiles were removed in vacuo. The residue was dissolved in DMF (0.5 mL) and purified with 5-95% MeCN / H 2The mixture was purified on an ISCO 5.5 g C18Aq column using 100 mL of 1000 sucrose / hexanes (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 66 (2.6 mg, 65%) as a fluffy off-white solid. MS (ESI, pos.): 33 H 43 NO 11 Calculated for, 629.3; Found 630.3 (M+H), 652.3 (M+Na). 1 H-NMR (500 MHz; CDCl 3 ): δ 8.01 (dd, J= 15.6, 11.7 Hz, 1H), 6.68 (t, J = 11.4 Hz, 1H), 6.18 (d, J = 11.1 Hz, 1H), 6.06(d, J = 15.8 Hz, 1H), 5.81 (dd, J = 7.1, 3.1 Hz, 1H), 5.43 (d, J = 3.0 Hz, 1H),5.20 (q, J = 7.3 Hz, 1H), 4.82 (s, 1H), 4.69 (d, J = 12.2 Hz, 1H), 4.47 (dd, J =11.4, 1.0 Hz, 1H), 4.22 (d, J = 11.9 Hz, 1H), 4.05-4.00 (m, 1H), 3.87 (d, J = 4.8Hz, 1H), 3.58 (d, J = 4.9 Hz, 1H), 3.14 (d, J = 3.5 Hz, 1H), 3.01 (s, 3H), 2.84(s, 2H), 2.63 (s, 1H), 2.49 (dd, J = 15.3, 8.2 Hz, 2H), 2.26-2.22 (m, 1H), 2.14(s, 3H), 2.05-2.01 (m, 1H), 1.95-1.83 (m, 2H), 1.75 (s, 3H), 1.70-1.63 (m, 1H),1.51 (d, J = 7.3 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H), 0.88 (s, 3H).

[0279] Example 24 Compound 70a and Compound 70b [ka]

[0280] Compound 68: A solution of Fmoc-6-aminohexanoic acid N-hydroxysuccinimide ester (150 mg, 0.33 mmol) and Val-Cit-OH TFA salt (67, 127 mg, 0.33 mmol) in acetonitrile (3 mL) and water (2 mL) was dissolved in saturated NaHCO 3 Aqueous solution (1 mL) was added at room temperature. After stirring for 18 h, the pH was adjusted to about 6 by adding acetic acid. The reaction was diluted with 5-95% MeCN / H 2 The mixture was purified on an ISCO 50 g C18Aq column using 100 mL of 1000 sucrose / hexanes (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 68 (133 mg, 66%) as a fluffy white solid. MS (ESI, pos.): C 32 H 43 N 5 O 7 Calculated for, 609.3; found 610.3 (M+H).

[0281] Compound 69: To a solution of compound 68 (39.6 mg, 0.065 mmol) and N,N-diisopropylethylamine (15.6 μL, 0.09 mmol) in DCM (1 mL) at 0 °C, 2,4,6-trichlorobenzoyl chloride (11 μL, 0.07 mmol) was added dropwise and the solution was stirred at 0 °C for 1 h. To the resulting solution was added a solution of DMAP (9.2 mg, 0.075 mmol) in DCM (1 mL) followed by a solution of verrucarin A (1, 25 mg, 0.05 mmol) in DCM (1 mL) at 0 °C. The reaction was allowed to warm slowly to room temperature and stirred for 2 h. The volatiles were removed in vacuo. The residue was dissolved in DMF (1 mL) and washed with 5-95% MeCN / H 2 The product was purified on an ISCO 50 g C18Aq column using 0 (both containing 0.05% AcOH). Fractions containing unreacted 1 were combined and lyophilized to recover 14 mg (56%) of verrucarin A. Fractions containing pure product were combined and lyophilized to give compound 69 (10 mg, 42% BRSM) as a fluffy off-white solid. MS (ESI, pos.): C 59 H75 N 5 O 15 Calculated for, 1093.5; found 1094.2 (M+H), 1116.0 (M+Na).

[0282] Compound 70a: To a solution of compound 69 (25 mg, 0.023 mmol) in DMF (1.5 mL) at room temperature was added 5% piperidine in DMF (0.5 mL) and the reaction was stirred for 30 min. Two isomers (29:71 at 254 nm) were observed by LC / MS. The reaction was injected directly onto an ISCO 15 g C18Aq column and purified by elution with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions containing the major isomer were combined and lyophilized to give compound 70a (10.1 mg, 51%) as a fluffy off-white solid. MS (ESI, pos.): C 44 H 65 N 5 O 13 Calculated for 871.5; Found 872.3 (M+H). 1H-NMR (500 MHz; CDCl 3): δ 8.02 (dd, J = 15.3, 11.5 Hz, 1H),7.83-7.79 (m, 1H), 7.19-7.14 (m, 1H), 6.71-6.66 (m, 1H), 6.18 (d, J = 11.0 Hz, 1H),6.07 (s, 1H), 5.99-5.96 (m, 1H), 5.81-5.78 (m, 1H), 5.42-5.41 (m, 2H), 4.87 (s,1H), 4.66-4.59 (m, 2H), 4.49-4.45 (m, 1H), 4.27 (t, J = 8.0 Hz, 1H), 4.22 (d, J= 12.8 Hz, 1H), 4.01 (t, J = 11.2 Hz, 1H), 3.87 (d, J = 4.6 Hz, 1H), 3.57 (d, J= 3.9 Hz, 1H), 3.18-3.12 (m, 7H), 2.83 (d, J = 3.2 Hz, 3H), 2.50 (td, J = 14.7,8.1 Hz, 2H), 2.23 (dt, J = 10.1, 4.9 Hz, 3H), 2.09-1.81 (m, 9H), 1.76 (s, 3H), 1.64-1.59(m, 5H), 1.37 (dd, J = 12.9, 6.6 Hz, 2H), 1.04 (d, J = 6.6 Hz, 3H), 0.98 (t, J =7.2 Hz, 6H), 0.87 (s, 3H).

[0283] Compound 70b: The mixed fractions from the RP purification were lyophilized and then purified with 5–95% MeCN / H 2 The product was repurified on an ISCO 15 g C18Aq column eluted with 0.05% AcOH. The pure fractions, including the by-product diastereomer, were combined and lyophilized to give compound 70b (6.1 mg, 31%). MS (ESI, pos.): C 44 H 65 N 5 O 13 Calculated for, 871.5; found, 872.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.00 (dd, J = 14.0,12.6 Hz, 1H), 7.84-7.81 (m, 1H), 6.99-6.93 (m, 1H), 6.68 (t, J = 11.3 Hz, 1H), 6.17(dd, J = 11.1, 0.8 Hz, 1H), 6.06-6.03 (m, 1H), 5.94-5.86 (m, 1H), 5.82-5.77 (m,1H), 5.41 (td, J = 1.9, 0.8 Hz, 1H), 5.21-5.09 (m, 1H), 4.85 (t, J = 1.7 Hz, 1H),4.66-4.62 (m, 1H), 4.59-4.56 (m, 1H), 4.46-4.42 (m, 1H), 4.29-4.19 (m, 2H), 4.00-3.98(m, 1H), 3.86 (td, J = 1.7, 0.8 Hz, 1H), 3.56 (ddd, J = 4.0, 1.5, 0.8 Hz, 1H), 3.24-3.22(m, 1H), 3.15-3.11 (m, 2H), 2.82 (m, 4H), 2.51-2.47 (m, 6H), 2.33-2.14 (m, 6H),1.94-1.85 (m, 8H), 1.74 (m, 4H), 1.42-1.38 (m, 3H), 1.27 (s, 2H), 1.03-0.96 (m, 6H), 0.86 (d, J = 0.4 Hz, 3H).

[0284] Example 25 Compound 74

change

[0285] Compound 72: To a mixture of Fmoc-Val-Ala-OH (71, 10.3 mg, 0.025 mmol), verrucarin A (1, 12.5 mg, 0.025 mmol), N,N-dicyclohexylcarbodiimide (10.3 mg, 0.05 mmol), 1-hydroxy-7-azabenzotriazole (3.5 mg, 0.025 mmol) and DMAP (3.1 mg, 0.025 mmol) was added anhydrous dichloromethane (1 mL). The reaction was stirred for 18 h, then concentrated in vacuo and purified with 5-100% MeCN / H 2 The mixture was purified on an ISCO 15.5 g C18Aq column using 100 mL of 1000 sucrose / hexanes (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 72 (15 mg, 48%) as a fluffy off-white solid. MS (ESI, pos.): C 50 H 58 N 2 O 13 Calculated for, 894.4; Found 895.2 (M+H), 917.3 (M+Na).

[0286] Compound 73: To a solution of compound 72 (14 mg, 0.015 mmol) in DMF (0.5 mL) was added a 5% piperidine solution in DMF (0.3 mL) and the reaction was stirred for 30 min. The reaction solution was injected directly onto an ISCO 15.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 73 (9 mg, 76%) as a fluffy off-white solid. MS (ESI, pos.): 35 H 48 N 2 O 11 Calculated for, 672.3; found, 873.2 (M+H).

[0287] Compound 74: To a solution of compound 73 (9 mg, 0.012 mmol) and 6-maleimidocaproic acid N-hydroxysuccinimide ester (8.7 mg, 0.027 mmol) in anhydrous DMF at 0 °C was added N,N-diisopropylethylamine. The reaction was allowed to warm to room temperature and stirred for 2 h. The reaction was directly injected onto an ISCO 15.5 g C18Aq column and eluted with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 74 (9 mg, 75%) as a fluffy off-white solid. MS (ESI, pos.): C 45 H 59 N 3 O 14 Calculated for, 865.4; found, 866.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.03 (dd, J = 15.7, 11.6 Hz, 1H), 6.69 (q, J = 7.1 Hz, 3H), 6.41 (d, J = 6.9 Hz, 1H), 6.18 (d, J = 11.0 Hz, 1H), 6.07 - 6.03 (m, 2H), 5.81 (dd, J = 7.8, 3.9 Hz, 1H), 5.42 (d, J = 4.9 Hz, 1H), 4.89 (d, J = 1.6 Hz, 1H), 4.75 - 4.66 (m, 2H), 4.49 - 4.46 (m, 1H), 4.31 (dd, J = 8.5, 6.7 Hz, 1H), 4.22 (dd, J = 12.3, 4.6 Hz, 1H), 4.03 - 3.98 (m, 1H), 3.88 (d, J = 5.1 Hz, 1H), 3.58 (d, J = 4.7 Hz, 1H), 3.54 (d, J = 7.1 Hz, 2H), 3.14 (d, J = 3.8 Hz, 1H), 2.83 (t, J = 4.1 Hz, 1H), 2.51 (td, J = 16.2, 7.5 Hz, 2H), 2.27 - 2.21 (m, 3H), 2.10 (dt, J = 13.0, 6.4 Hz, 1H), 2.06 - 2.01 (m, 1H), 1.93 (t, J = 10.7 Hz, 2H), 1.82 (d, J = 19.5 Hz, 1H), 1.77 (s, 3H), 1.69 (dt, J = 14.3, 7.3 Hz, 3H), 1.62 (t, J = 7.5 Hz, 4H), 1.49 (d, J = 7.1 Hz, 2H), 1.35 (q, J = 7.7 Hz, 2H), 1.05 (t, J = 5.6 Hz, 3H), 0.97 (q, J = 7.1 Hz, 6H), 0.87 (s, 3H).

[0288] Example 26 Compound 75 [Chemical formula]

[0289] Compound 75: Compound 73 (9 mg, 0.012 mmol), NaHCO in anhydrous DMF (1 mL) 3 A mixture of (3 mg, 0.036 mmol) and bis(2,5-dioxopyrrolidin-1-yl)adipate (77.4 mg, 0.24 mmol) was stirred at room temperature for 15 min. LCMS showed the reaction was complete. The reaction was injected onto an ISCO 15.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 75 (9.7 mg, 95%) as a fluffy off-white solid. MS (ESI, pos.): 45 H 59 N 3 O 16 Calculated for, 897.4; found, 898.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.03 (dd, J = 15.7,11.6 Hz, 1H), 6.68 (t, J = 11.4 Hz, 1H), 6.48 (d, J = 7.0 Hz, 1H), 6.17 (t, J =9.4 Hz, 2H), 6.06 (d, J = 15.7 Hz, 1H), 5.81 (dd, J = 8.1, 4.0 Hz, 1H), 5.43-5.42(m, 1H), 4.89 (d, J = 1.9 Hz, 1H), 4.74 (t, J = 7.1 Hz, 1H), 4.68 (dd, J = 11.8,3.7 Hz, 1H), 4.49-4.45 (m, 1H), 4.31 (dd, J = 8.5, 6.5 Hz, 1H), 4.25-4.20 (m, 1H), 4.01 (td, J = 11.7, 3.0 Hz, 1H), 3.88 (d, J = 5.1 Hz, 1H), 3.58 (d, J = 4.9 Hz, 1H), 3.14 (d, J = 3.9 Hz, 1H), 2.86-2.83 (m, 6H), 2.68-2.64 (m, 2H), 2.54-2.47 (m,2H), 2.33-2.30 (m, 2H), 2.27-2.22 (m, 1H), 2.19-2.11 (m, 1H), 2.05-2.00 (m, 1H),1.92 (d, J = 10.1 Hz, 2H), 1.84-1.81 (m, 6H), 1.77 (s, 3H), 1.48 (d, J = 7.1 Hz,3H), 1.04 (d, J = 6.8 Hz, 3H), 0.96 (dd, J = 8.7, 6.9 Hz, 6H), 0.87 (s, 3H).

[0290] Example 27 Compound 76

change

[0291] Compound 76: Compound 73 (7.4 mg, 0.0094 mmol), ビス-PEG-7-NHSエステル (87.4 mg, 0.14 mmol) and NaHCO in anhydrous DMF (2 mL)3 A mixture of (2.3 mg, 0.028 mmol) was stirred at room temperature for 30 min. The crude reaction mixture was loaded onto an ISCO 15.5 g C18Aq column and purified with 5–95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 76 (5 mg, 45%) as a fluffy off-white solid. MS (ESI, pos.): C 57 H 83 N 3 O 23 Calculated for, 1177.5; found 1178.5 (M+H). 1 H-NMR (300 MHz; CDCl 3 ): δ 8.05 (dd, J = 15.3,12.1 Hz, 1H), 6.92-6.89 (m, 1H), 6.70 (t, J = 11.3 Hz, 2H), 6.20 (d, J = 11.1 Hz,1H), 6.08 (d, J = 15.4 Hz, 1H), 5.84-5.80 (m, 1H), 5.45-5.43 (m, 1H), 4.91-4.88(m, 1H), 4.76-4.66 (m, 2H), 4.52-4.45 (m, 1H), 4.34-4.21 (m, 2H), 4.07-3.98 (m,1H), 3.88 (t, J = 6.2 Hz, 3H), 3.79 (t, J = 5.6 Hz, 3H), 3.74 (s, 24H), 3.59 (d,J = 5.1 Hz, 1H), 3.16 (d, J = 4.0 Hz, 1H), 2.96-2.85 (m, 5H), 2.58-2.47 (m, 4H),2.30 (d, J = 1.1 Hz, 2H), 2.30-2.20 (m, 3H), 2.09-1.81 (m, 4H), 1.81-1.76 (m, 3H),1.55-1.48 (m, 3H), 1.06 (d, J = 6.8 Hz, 3H), 0.99 (t, J = 6.3 Hz, 6H), 0.89 (s,3H).

[0292] Example 28 compound 78 [ka]

[0293] Compound 78: To a solution of compound 4 (5 mg, 0.01 mmol) and L-lactic acid (77, 0.9 mg, 0.01 mmol) in anhydrous THF (0.5 mL) at 0 °C was added EDCI (2.9 mg, 0.015 mmol), HOAt (2.0 mg, 0.015 mmol) and triethylamine (4.2 μL, 0.03 mmol). The reaction was stirred for 2 h, after which the volatiles were removed in vacuo. The residue was purified by elution with 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 100 mL of ethyl acetate (ethyl acetate, 0.05% acetic acid) and 100 mL of ethyl acetate (ethyl acetate, 0.05% acetic acid). Pure fractions were combined and lyophilized to give compound 78 as a fluffy white solid (4.9 mg, 86%). MS (ESI, pos.): 30 H 39 NO 10 Calculated for, 573.3; found 574.2 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.11-8.06 (m, 1H), 6.93 (d,J = 8.9 Hz, 1H), 6.69 (t, J = 10.9 Hz, 1H), 6.15 (d, J = 11.1 Hz, 1H), 6.05 (d,J = 15.7 Hz, 1H), 5.77 (dd, J = 7.8, 3.4 Hz, 1H), 5.43 (d, J = 4.2 Hz, 1H), 4.49-4.45(m, 1H), 4.39 (t, J = 10.4 Hz, 1H), 4.33 (m, 3H), 4.03 (td, J = 11.5, 2.5 Hz, 1H),3.87 (d, J = 5.2 Hz, 1H), 3.59 (d, J = 5.1 Hz, 1H), 3.13 (dd, J = 4.0, 1.3 Hz, 1H),2.82 (d, J = 3.3 Hz, 1H), 2.51 (dd, J = 15.1, 8.1 Hz, 1H), 2.22 (td, J = 9.6, 4.4Hz, 3H), 2.08-1.88 (m, 4H), 1.77-1.74 (m, 4H), 1.45 (m, 4H), 1.12 (d, J = 5.8 Hz,3H), 0.89 (s, 3H).

[0294] Example 29 compound 80 [ka]

[0295] Compound 80 (4.6 mg, 80%) was prepared using the procedure above on the same scale but starting from D-lactic acid (79). MS (ESI, pos.): C 30 H 39 NO 10 Calculated for, 573.3; found 574.2 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.09 (dd, J = 15.7, 11.6 Hz,1H), 6.97 (d, J = 8.8 Hz, 1H), 6.69 (t, J = 12.0 Hz, 1H), 6.15 (d, J = 11.0 Hz,1H), 6.05 (d, J = 15.7 Hz, 1H), 5.78 (dd, J = 7.9, 4.0 Hz, 1H), 5.42 (d, J = 4.9Hz, 1H), 4.48-4.45 (m, 1H), 4.40-4.32 (m, 3H), 4.28 (ddd, J = 6.6, 4.3, 2.1 Hz,1H), 4.06-4.00 (m, 1H), 3.86 (d, J = 5.0 Hz, 1H), 3.59 (d, J = 5.4 Hz, 1H), 3.13(d, J = 3.9 Hz, 1H), 2.81 (d, J = 4.0 Hz, 1H), 2.51 (dd, J = 15.5, 8.3 Hz, 1H),2.24-2.19 (m, 2H), 2.01-1.86 (m, 5H), 1.74 (s, 3H), 1.49 (d, J = 6.8 Hz, 3H), 1.48-1.44(m, 1H), 1.11 (d, J = 6.7 Hz, 3H), 0.88 (s, 3H).

[0296] Example 30 compound 82 [ka]

[0297] Compound 81: To a solution of (R)-amino-verrucarin A (4, 8 mg, 0.0142 mmol) and Fmoc-Ala-OSu (13 mg, 0.0318 mmol) in anhydrous DMF (280 μL) was added N,N-diisopropylethylamine (10 μL, 0.0574 mmol). The reaction was stirred at room temperature for 4 h, at which point LCMS indicated the reaction was complete. The reaction solution was loaded onto an ISCO 5.5 g C18 column and purified by HPLC. 2The mixture was eluted with 10-80% MeCN in O (both containing 0.05% HOAc). Fractions containing the pure product were combined and lyophilized to give compound 81 (8 mg, 73%) as a white solid. MSMS (ESI, pos.): 45 H 50 N 2 O 11 Calculated for, 794.3; found, 817.2 (M+Na).

[0298] Compound 82: Compound 81 (8.5 mg, 0.011 mmol) was treated with a 10% solution of piperidine in DMF (100 μL). The solution was stirred at room temperature for 40 min, at which point LCMS indicated the reaction was complete. The reaction solution was loaded onto an ISCO 5.5 g C18 column and purified by HPLC. 2 Elution was performed with 5-30% MeCN in O (both containing 0.05% HOAc). Fractions containing pure product were combined and lyophilized to give compound 82 (6 mg, 98%) as a fluffy white solid. MS (ESI, pos.): C 30 H 40 N 2 O 9 Calculated for, 572.3; found 573.3 (M+H). 1 H NMR (300 MHz; CDCl 3) δ 8.07 (dd, J = 15.6, 11.5 Hz, 1H), 7.88-7.85 (m, 1H), 6.68 (t, J = 11.4 Hz,1H), 6.14 (d, J = 11.1 Hz, 1H), 6.04 (d, J = 15.6 Hz, 1H), 5.78-5.74 (m, 1H), 5.43-5.41(m, 1H), 4.47-4.40 (m, 1H), 4.33 (dd, J = 10.4, 7.2 Hz, 3H), 4.05-4.01 (m, 1H),3.86 (d, J = 5.1 Hz, 1H), 3.57 (q, J = 5.7 Hz, 2H), 3.13 (d, J = 4.0 Hz, 1H), 2.80(d, J = 4.0 Hz, 1H), 2.51 (dd, J = 15.5, 8.1 Hz, 1H), 2.25-2.17 (m, 2H), 2.06-1.76(m, 6H), 1.76-1.70 (m, 3H), 1.49-1.39 (m, 2H), 1.34 (d,J = 7.0 Hz, 3H), 1.10 (d,J = 6.7 Hz, 3H), 0.88 (s, 3H).

[0299] Example 31 compound 84 [ka]

[0300] Compound 84: To a solution of N,N-dimethylalanine (83, 2.5 mg, 0.021 mmol), HOAt (2.7 mg, 0.020 mmol), and HATU (7.6 mg (0.020 mmol) in anhydrous DMF (75 mL) was added N,N-diisopropylethylamine (3.5 mL, 0.020 mmol). The resulting yellow solution was stirred for 5 min, after which a solution of (R)-amino-verrucarin A (4, 5 mg, 0.01 mmol) in DMF (175 mL) was added. The reaction was stirred at room temperature for 1 h, at which point LCMS indicated the reaction was complete. The reaction solution was loaded onto an ISCO 5.5 g C18 column and purified by HPLC. 2The mixture was eluted with 5-100% MeCN in O (both containing 0.05% HOAc). The fractions containing the product were lyophilized to give compound 84 (4.2 mg, 50%) as a fluffy white solid. MS (ESI, pos.): 32 H 44 N 2 O 9 Calculated for, 600.3; found 601.3 (M+H). 1 H NMR (300 MHz; CDCl 3 ) δ 8.09 (dd, J = 15,7, 11.7 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 6.69 (t, J =11.3 Hz, 1H), 6.14 (d, J = 10.8 Hz, 1H), 6.04 (d, J = 15.6 Hz, 1H), 5.76 (dd, J= 8.0, 4.0 Hz, 1H), 5.44-5.42 (m, 1H), 4.47-4.41 (m, 2H), 4.38-4.29 (m, 2H), 4.05-3.96(m, 1H), 3.87 (d, J = 5.1 Hz, 1H), 3.59 (d, J = 5.0 Hz, 1H), 3.15 (d, J = 3.9 Hz,1H), 3.03 (q, J = 7.0 Hz, 1H), 2.78 (d, J = 4.1 Hz, 1H), 2.51 (dd, J = 15.6, 8.1Hz, 1H), 2.25 (s, 6H), 2.25-2.18 (m, 1H), 2.00-1.76 (m, 6H), 1.73 (s, 3H), 1.46-1.37(m, 1H), 1.20 (d, J = 7.0 Hz, 3H), 1.09 (d, J = 6.6 Hz, 3H), 0.91 (s, 3H).

[0301] Example 32 compound 88 [ka]

[0302] Compound 85 (Fmoc-Ile-Phe-Arg-OH) was prepared using a peptide synthesizer.

[0303] Compound 86: To a solution of compound 85 (21 mg, 0.032 mmol) in DMF (1.3 mL) at room temperature was added HATU (15.2 mg, 0.04 mmol) and N,N-diisopropylethylamine (14 μL, 0.08 mmol), followed by (R)-amino-verrucarin A (4.9 mg, 0.016 mmol). The reaction was stirred for 6 h and then injected onto an ISCO 30 g C18Aq column and purified with 5-95% MeCN / H2O. 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 86 (12 mg, 59% yield) as a white fluffy solid. MS (ESI, pos.): 63 H 77 N 7 O 13 Calculated for, 1139.6; found 1140.5 (M+H).

[0304] Compound 87: To a solution of compound 86 (12 mg, 0.01 mmol) in DMF (0.8 mL) at room temperature was added a 5% solution of piperidine in DMF (0.4 mL) and the reaction was stirred for 20 min. The reaction solution was loaded onto an ISCO 30 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 87 (7.9 mg, 82%) as a fluffy white solid. MS (ESI, pos.): C 48 H 67 N 7 O 11 Calculated for, 917.5; found 918.2 (M+H).

[0305] Compound 88: To a solution of compound 87 (4.3 mg, 0.0046 mmol) in DMF at room temperature was added N-succinimidyl 6-maleimidohexanoate (2.1 mg, 0.0069 mmol), followed by diisopropylethylamine (2.4 µL, 0.013 mmol). The reaction was stirred for 1 h and then injected onto an ISCO 15.5 g C18Aq column and purified with 5–95% MeCN / H2O. 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 88 (2 mg, 38%) as a fluffy white solid. MS (ESI, pos.): C 58 H 78 N 8 O 14 Calculated for, 1110.6; found 1111.3 (M+H). 1 H-NMR (500 MHz; DMSO-d 6 ): δ 9.04-9.00 (m, 1H),8.22-8.20 (m, 1H), 7.98-7.86 (m, 3H), 7.79-7.75 (m, 1H), 7.21-7.15 (m, 5H), 6.99(s, 2H), 6.88-6.83 (m, 1H), 6.26-6.21 (m, 3H), 5.75-5.73 (m, 1H), 5.27-5.23 (m,2H), 4.54-4.48 (m, 2H), 4.47-4.40 (m, 1H), 4.14-4.03 (m, 3H), 3.93-3.87 (m, 1H),3.68-3.63 (m, 4H), 3.02-3.00 (m, 3H), 2.85-2.78 (m, 2H), 2.43-2.36 (m, 3H), 2.24-2.19(m, 3H), 2.15-2.08 (m, 3H), 2.05-2.00 (m, 2H), 1.95-1.91 (m, 2H), 1.74-1.69 (m,2H), 1.70-1.57 (m, 5H), 1.46-1.42 (m, 3H), 1.25-1.13 (m, 6H), 1.01-0.94 (m, 1H),0.91 (d, J = 7.7Hz, 3H), 0.77-0.68 (m, 9H).

[0306] Example 33 compound 91 [ka]

[0307] Compound 89: To a solution of compound 82 (14 mg, 0.0213 mmol), HATU (10 mg, 0.0262 mmol) and HOAt (3 mg, 0.0220 mmol) in anhydrous DMF (250 μL) was added compound 85 (6 mg, 0.0105 mmol) in DMF (250 μL). N,N-diisopropylethylamine (4 μL, 0.0230 mmol) was added and the reaction was stirred at room temperature for 1 h, at which point LCMS indicated the reaction was complete. The solution was loaded onto an ISCO 15.5 g C18Aq column and purified by HPLC. 2 Elution was performed with 0-100% MeCN in O (both containing 0.05% HOAc). Fractions containing pure product were combined and lyophilized to give compound 89 (5 mg, 38%) as a white fluffy solid. MS (ESI, pos.): C 66 H 82 N 8 O 14 Calculated for, 1210.6; Found, 1210.5 (M+).

[0308] Compound 90: Compound 89 (4.5 mg, 0.0037 mmol) was treated with 10% piperidine in DMF (150 μL). After 40 min, LCMS showed the reaction was complete. The reaction was loaded onto an ISCO 5.5 g C18 column and purified by H 2 Elution was performed with 5-40% MeCN in O (both containing 0.05% HOAc). Fractions containing >95% pure product were combined and lyophilized to give compound 90 (2.3 mg, 62%) as a white fluffy solid. MS (ESI, pos.): C 51 H 72 N 8 O 12 Calculated for, 988.5; found, 989.5 (M+H).

[0309] Compound 91: To a solution of compound 90 (4 mg, 0.00404 mmol) and N-succinimidyl 6-maleimidohexanoate (2 mg, 0.00649 mmol) in anhydrous DMF (100 μL) was added N,N-diisopropylethylamine (2 μL, 0.0115 mmol). After stirring at room temperature for 2.75 h, LCMS indicated the presence of unreacted amine, but the NHS ester had been consumed. Additional N-succinimidyl 6-maleimidohexanoate (1 mg, 0.00324 mmol) was added and the reaction was stirred for an additional 2 h, at which point LCMS indicated the reaction was complete. The reaction was loaded onto an ISCO 5.5 g C18Aq column and purified by HPLC. 2 The mixture was eluted with MeCN in O (both containing 0.05% HOAc). The pure fractions were combined and lyophilized to give compound 91 (1 mg, 17%) as a white fluffy solid. MS (ESI, pos.): 61 H 83 N 9 O 15 Calculated for, 1181.6; Found, 1182.5 (M+H). 1 H NMR (300 MHz; CDCl 3) δ 8.41 (br s, 1H), 8.10 (dd, J = 12.3, 16.6 Hz, 1H), 7.69 (br s, 1H), 7.49(br s, 1H), 7.18-7.15 (m, 5H), 7.03-7.00 (m, 1H), 6.78-6.73 (m, 2H), 6.55 (dd, J= 11.6, 10.8 Hz, 1H), 6.03 (d, J = 11.9 Hz, 1H), 5.95 (d, J = 15.5 Hz, 1H), 5.78-5.74(m, 1H), 5.43-5.41 (m, 1H), 4.67-4.57 (m, 1H), 4.47-3.91 (m, 6H), 3.90-3.85 (m,1H), 3.60-3.49 (m, 4H), 3.24-3.20 (m, 2H), 3.08-2.99 (m, 2H), 2.84-2.83 (m, 1H),2.65-2.54 (m, 2H), 2.37-2.16 (m, 4H), 2.05-2.04 (m, 3H), 1.46-1.27 (m, 9H), 1.10(d, J = 6.5 Hz, 3H), 0.99 (s, 3H), 0.82 (t, J = 7.8 Hz, 3H), 0.72 (d, J = 6.6 Hz,3H).

[0310] Example 34 compound 95 [ka]

[0311] Compound 8: To a solution of Fmoc-6-aminohexanoic acid n-hydroxysuccinimide ester (50 mg, 0.11 mmol) and Val-Cit-PAB-OH TFA salt (92, 50 mg, 0.1 mmol) in DMF (1.3 mL) at room temperature was added N,N-diisopropylethylamine (52 μL, 0.3 mmol). The reaction was stirred for 20 min and then injected onto an ISCO 30 g C18Aq column and purified with 5-95% MeCN / H 20 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 8 (58 mg, 81%) as a fluffy white solid. MS (ESI, pos.): C 39 H 50 N 6 O 7 Calculated for, 714.4; found 715.3 (M+H).

[0312] Compound 93: To a solution of compound 8 (40 mg, 0.056 mmol) in DMF (1.5 mL) at room temperature was added bis-(4-nitrophenyl)carbonate (20.4 mg, 0.067 mmol) and N,N-diisopropylethylamine (30 μL, 0.168 mmol). The reaction was stirred for 6 h and then loaded onto an ISCO 50 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 93 (29 mg, 57%) as a fluffy white solid. MS (ESI, pos.): 46 H 53 N 6 O 11 Calculated for, 879.4; found, 880.3 (M+H).

[0313] Compound 94: To a solution of compound 93 (30 mg, 0.034 mmol) and (R)-amino-verrucarin A (4, 17.1 mg, 0.031 mmol) in anhydrous DMF (1.3 mL) at room temperature was added diisopropylethylamine (16 μL, 0.093 mmol). The reaction was stirred for 36 h and then injected onto an ISCO EZ-Prep HPLC (30 × 150 mm Gemini column) and purified with 5–95% MeCN / H 2 0 (both containing 0.05% AcOH). Fractions containing unreacted starting amine were combined and lyophilized to recover (R)-aminoverrucarin A (4, 4 mg, 23% recovery). Fractions containing the desired product were combined and lyophilized to give compound 94 (10 mg, 32% BRSM yield) as a fluffy white solid. MS (ESI, pos.): C 67 H 83 N 7 O16 Calculated for, 1241.6; found 1242.3 (M+H).

[0314] Compound 95: To a solution of compound 94 (10 mg, 0.008 mmol) in DMF (0.5 mL) at room temperature was added a 5% solution of piperidine in DMF (0.5 mL) and the reaction was stirred for 20 min. The product was purified by elution with 5-95% MeCN / H 2 The product was purified by preparative HPLC chromatography on a 30×150 mm Gemini column eluted with 0.025 mL of 100% ethanol (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 95 (6.4 mg, 78%) as a fluffy white solid. MS (ESI, pos.): 52 H 73 N 7 O 14 Calculated for, 1019.5; found 1020.2 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 10.02 (s, 1H), 8.14-8.11(m, 1H), 7.92 (ddd, J = 15.5, 11.7, 0.9 Hz, 1H), 7.89-7.82 (m, 1H), 7.75 (d, J =6.5 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 6.85 (t, J = 11.4Hz, 1H), 6.23-6.18 (m, 2H), 6.01-5.98 (m, 1H), 5.75-5.73 (m, 1H), 5.40 (s, 2H),5.26 (d, J = 4.0 Hz, 1H), 5.03 (d, J = 12.4 Hz, 1H), 4.86 (d, J = 12.3 Hz, 1H), 4.45-4.41 (m, 1H), 4.39-4.35 (m, 1H), 4.17 (t, J = 7.8 Hz, 1H), 4.12-4.03 (m, 2H), 3.90-3.85 (m, 1H), 3.66-3.61 (m, 3H), 3.00-2.91 (m, 4H), 2.41 (dd, J = 15.2, 8.2Hz, 1H), 2.19-2.08 (m, 4H), 1.98-1.89 (m, 2H), 1.81 (s, 1H), 1.70-1.66 (m, 3H),1.62 (s, 3H), 1.50-1.41 (m, 6H), 1.37-1.32 (m, 3H), 1.26-1.22 (m, 6H), 0.91 (d,J = 6.6 Hz, 3H), 0.83 (dd, J = 12.4, 6.7 Hz, 6H), 0.65 (s, 3H).

[0315] Example 35 Compound 97

change

[0316] Compound 96 was prepared following the same procedure as compound 93, except starting with N-succinimidyl 6-maleimidohexanoate instead of Fmoc-6-aminohexanoic acid N-hydroxysuccinimide ester.

[0317] Compound 97: To a solution of (R)-amino-verrucarin A (4, 6.9 mg, 0.014 mmol) in DMF (1 mL) at room temperature was added compound 96 (11.4 mg, 0.015 mmol), followed by N,N-diisopropylethylamine (7 μL, 0.041 mmol). The resulting mixture was stirred for 18 h, at which point the reaction was judged complete by LCMS analysis. The reaction mixture was injected onto an ISCO 15.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 97 (9 mg, 60% yield) as a white fluffy solid. MS (ESI, pos.): 56 H 73 N 7 O 16 Calculated for, 1099.5; found 1100.5 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 10.06 (s, 1H), 8.17 (s, 1H), 7.93 (dd, J = 15.1, 12.1 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 6.5 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 7.00 (s, 2H), 6.86 (t, J = 11.4 Hz, 1H), 6.23 (m, 2H), 6.02 (s, 1H), 5.75 (dd, J = 7.0, 2.9 Hz, 1H), 5.41 (s, 2H), 5.27 (s, 1H), 5.04 (d, J = 12.3 Hz, 1H), 4.87 (d, J = 12.3 Hz, 1H), 4.46 - 4.43 (m, 1H), 4.38 (q, J = 6.5 Hz, 1H), 4.18 (t, J = 7.7 Hz, 1H), 4.08 (q, J = 14.7 Hz, 2H), 3.89 (td, J = 9.5, 3.3 Hz, 1H), 3.67 - 3.62 (m, 4H), 3.02 - 2.95 (m, 4H), 2.44 - 2.39 (m, 1H), 2.21 - 2.13 (m, 3H), 2.13 - 2.07 (m, 3H), 1.98 - 1.90 (m, 2H), 1.70 - 1.63 (m, 5H), 1.50 - 1.43 (m, 6H), 1.26 - 1.16 (m, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.83 (dd, J = 15.1, 6.7 Hz, 9H), 0.66 (s, 3H).

[0318] Example 36 Compound 101 [Chemical formula]

[0319] Compound 98: To a solution of valine-citrulline-PAB-OH TFA salt (92, 100 mg, 0.2 mmol) and Fmoc-amino-PEG8-NHS ester (167 mg, 0.22 mmol) in anhydrous DMF (2 mL) was added N,N-diisopropylethylamine (40 μL, 0.22 mmol) and the reaction was stirred for 45 min, at which point LC / MS indicated the reaction was complete. The product was purified by HPLC using 5-95% MeCN / H 2 The crude product was purified by chromatography on an ISCO 100 g C18Aq column eluted with 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 98 (145 mg, 71%) as an off-white fluffy solid. MS (ESI, pos.): 52 H 76 N 6 O 15 Calculated for, 1024.54; found 1025.5 (M+H).

[0320] Compound 99: To a solution of compound 98 (50 mg, 0.0488 mmol) and bis-(4-nitrophenyl)carbonate (18 mg, 0.0585 mmol) in anhydrous DMF (1 mL) at room temperature, N,N-diisopropylethylamine (19 μL, 0.146 mmol) was added and the reaction was stirred for 18 h. Additional bis-(4-nitrophenyl)carbonate (18 mg, 0.0585 mmol) was added and stirring was continued for 3 h, at which point LCMS indicated the reaction was complete. The reaction solution was injected onto an ISCO 50 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 99 (37 mg, 64%) as an off-white fluffy solid. MS (ESI, pos.): 59 H 79 N 7 O 19 Calculated for, 1189.5; found 1190.5 (M+H).

[0321] Compound 100: To a solution of compound 99 (30 mg, 0.025 mmol) and (R)-amino-verrucarin A (4, 14 mg, 0.025 mmol) in anhydrous DMF at room temperature, N,N-diisopropylethylamine (12 μL, 0.075 mmol) was added and the reaction was stirred for 24 h. The reaction mixture was injected onto an ISCO 30 g C18Aq column and purified with 5-95% MeCN / H 2 The mixture was eluted with 5-95% MeCN / HO (both containing 0.05% AcOH). Fractions containing the desired product were combined and lyophilized. The lyophilized solid was dissolved in DMF (1 mL) and purified by elution with 5-95% MeCN / HO. 2 The product was further purified by preparative HPLC on a Gemini 30×150 mm column eluted with 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 100 (8 mg, 21%) as an off-white fluffy solid. MS (ESI, pos.): 80 H 109 N 7 O 24 Calculated for, 1551.8; found 1552.7 (M+H).

[0322] Compound 101: To a solution of compound 100 (8 mg, 0.005 mmol) in DMF (0.6 mL) at room temperature was added a 5% solution of piperidine in DMF (0.3 mL) and the reaction was stirred for 45 min. The reaction solution was loaded onto an ISCO 30 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 101 (3.0 mg, 45%) as an off-white fluffy solid. MS (ESI, pos.): 65 H 99 N 7 O 22 Calculated for, 1329.7; found 1330.6 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 10.00 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.92 (dd, J = 15.5, 11.7 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.74 (d, J = 6.6 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 6.85 (t, J = 11.4 Hz, 1H), 6.21 (dd, J = 13.2, 10.3 Hz, 2H), 5.96 (t, J = 5.6 Hz, 1H), 5.74 (dd, J = 8.1, 4.1 Hz, 1H), 5.39 (s, 2H), 5.27 - 5.26 (m, 1H), 5.04 (d, J = 12.4 Hz, 1H), 4.86 (d, J = 12.3 Hz, 1H), 4.45 - 4.41 (m, 1H), 4.39 - 4.36 (m, 1H), 4.22 (dd, J = 8.6, 6.9 Hz, 1H), 4.10 (d, J = 12.1 Hz, 1H), 4.05 (d, J = 12.1 Hz, 1H), 3.90 - 3.85 (m, 1H), 3.66 - 3.56 (m, 5H), 3.46 (d, J = 16.6 Hz, 28H), 3.03 - 2.91 (m, 4H), 2.77 (t, J = 5.5 Hz, 2H), 2.41 - 2.35 (m, 3H), 2.18 - 2.16 (m, 1H), 2.09 - 2.06 (m, 1H), 1.94 (dt, J = 13.5, 5.6 Hz, 2H), 1.90 (s, 1H), 1.70 - 1.68 (m, 3H), 1.62 (s, 3H), 1.51 - 1.43 (m, 6H), 1.22 (s, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.83 (dd, J = 15.6, 6.7 Hz, 6H), 0.65 (s, 3H).

[0323] Example 37 Compound 106 [Chemical Structure] [ka]

[0324] Compound 102: To a solution of compound 49 (47.1 mg, 0.0428 mmol) in anhydrous DMF (1 mL) was added bis-(4-nitrophenyl)carbonate (20 mg, 0.064 mmol) and N,N-diisopropylethylamine (22 μL, 0.128 mmol). After stirring for 5 h, the reaction was diluted with 5-95% MeCN / H 2 The product was purified on an ISCO 50 g C18Aq column eluted with 0 (both containing 0.05% AcOH). Fractions containing the pure product were combined and lyophilized to give compound 102 (36 mg, 66%) as a yellowish fluffy solid. MS (ESI, pos.): C 61 H 75 N 3 O 26 Calculated for, 1265.5; found 1266.8 (M+H).

[0325] Compound 103: To a solution of compound 102 (18 mg, 0.014 mmol) and compound 4 (7 mg, 0.014 mmol) in anhydrous DMF (1 mL) was added N,N-diisopropylethylamine (7.3 μL, 0.042 mmol) and HOAt (1.0 mg, 0.007 mmol). After stirring for 24 h, the reaction was diluted with 5-95% MeCN / H 2 The mixture was purified on an ISCO 30 g C18Aq column eluted with 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 103 (12 mg, 53%) as a fluffy white solid. MS (ESI, pos.): 82 H 105 N 3 O 31 Calculated for, 1627.7; found 1628.6 (M+H).

[0326] Compound 105: To a solution of compound 103 (20 mg, 0.012 mmol) in anhydrous MeOH (1.5 mL) at 0° C. was added 0.05 M NaOMe in MeOH (0.48 mL, 0.024 mmol). After stirring for 10 min at 0° C., the reaction was neutralized with Dowex® resin (approximately 10 mg). The solids were filtered off and the filtrate was concentrated in vacuo to give crude compound 104, which was then dissolved in DMF (0.6 mL) and 5% piperidine in DMF (0.3 mL) was added. After stirring for 20 min, the reaction was neutralized with 5-95% MeCN / H 2 The mixture was purified on an ISCO 30 g C18Aq column eluted with 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 105 (4 mg, 26% over two steps) as a fluffy off-white solid. MS (ESI, pos.): 61 H 89 N 3 O 26 Calculated for, 1279.6; found 1280.5 (M+H).

[0327] Compound 106: To a solution of compound 105 (4 mg, 0.003 mmol) in THF (0.5 mL) and water (375 μL) was added 0.025 M aqueous LiOH (250 μL, 0.006 mmol) and the reaction was stirred for 2 h. The volatiles were removed under reduced pressure and the residue was purified by elution with 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 1,2-dichlorophenyl ether (C1H2O) and 1,2-dichlorophenyl ether (C1H2O) (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 106 (2.5 mg, 66%) as a fluffy off-white solid. MS (ESI, pos.): 60 H 87 N 3 O 26 Calculated for, 1265.6; found 1266.5 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 9.31 (s, 1H), 8.17 (dd, J = 1.3, 0.8 Hz, 1H), 7.90 (dd, J = 15.2, 11.1 Hz,1H), 7.77 (d, J = 6.5 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H),6.84 (t, J = 11.4 Hz, 1H), 6.21 (dd, J = 17.1, 13.2 Hz, 2H), 5.75-5.72 (m, 1H),5.69-5.68 (m, 1H), 5.26 (d, J = 4.5 Hz, 1H), 5.07-5.05 (m, 1H), 4.90 (q, J = 14.7Hz, 2H), 4.56 (d, J = 6.5 Hz, 1H), 4.44-4.41 (m, 1H), 4.11 (d, J = 10.8 Hz, 1H),4.02 (d, J = 11.7 Hz, 1H), 3.89-3.85 (m, 1H), 3.73-3.61 (m, 5H), 3.52-3.48 (m, 24H),3.09 (d, J = 5.4 Hz, 1H), 2.89 (t, J = 5.1 Hz, 4H), 2.58 (d, J = 4.6 Hz, 1H), 2.19-2.16(m, 2H), 2.08 (d, J = 2.9 Hz, 2H), 1.93-1.90 (m, 2H), 1.62 (m, 4H), 1.47 (s, 3H),1.25 (dd, J = 12.2, 10.5 Hz, 2H), 0.91 (d, J = 6.8 Hz, 3H), 0.67 (s, 3H).

[0328] Example 38 Compound 109 [Chemical Structure]

[0329] Compound 107: To a solution of compound 42 (190 mg, 0.17 mmol) in anhydrous DMF (2 mL) was added bis-(4-nitrophenyl)carbonate (104 mg, 0.34 mmol) and N,N-diisopropylethylamine (89 µL, 0.51 mmol). After stirring for 22 h, the reaction was loaded onto an ISCO 100 g C18Aq column and purified with 5–95% MeCN / H 2 0 (both containing 0.05% AcOH). Fractions containing the pure product were combined and lyophilized to give compound 107 (130 mg, 60%) as a fluffy solid. MS (ESI, pos.): C 62 H 77 N 3 O 26 Calculated for, 1279.5; found 1280.4 (M+H).

[0330] Compound 108: To a solution of compound 107 (45 mg, 0.035 mmol) and compound 4 (17.1 mg, 0.035 mmol) in anhydrous DMF (1.5 mL) was added N,N-diisopropylethylamine (18 μL, 0.105 mmol) and HOAt (2.5 mg, 0.018 mmol). After stirring for 9 h, the reaction was diluted with 5-95% MeCN / H 2 The mixture was purified on an ISCO 100 g C18Aq column, eluting with 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 108 (44 mg, 76%) as a fluffy off-white solid. MS (ESI, pos.): 83 H 105 N 3 O 31 Calculated for, 1641.7; found 1642.7 (M+H).

[0331] Compound 109: To a solution of compound 108 (30 mg, 0.018 mmol) in anhydrous MeOH (1.5 mL) at -10 °C was added 0.1 M NaOMe in MeOH (0.36 mL, 0.036 mmol). After stirring at -10 °C for 90 min, the reaction was neutralized with Dowex® resin (~100 mg). The solids were removed by filtration and the filtrate was concentrated in vacuo. The residue was purified by elution with 5-95% MeCN / H 2The mixture was purified by preparative HPLC on a 50×250 mm Luna column eluted with 100 mL of 1,000 sucrose (COOH) and 0.05% HCl (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 109 (5 mg, 18% over two steps) as a fluffy off-white solid. MS (ESI, pos.): 60 H 89 N 3 O 26 Calculated for, 1251.6; found 1252.5 (M+H). 1 H-NMR (500 MHz; DMSO-d 6 ): δ 9.16 (s, 1H), 8.23 ​​(d, J = 1.4 Hz, 1H), 7.91 (dd, J = 15.7, 11.8 Hz, 1H),7.77 (dd, J = 6.3, 4.2 Hz, 1H), 7.15 (dd, J = 8.3, 2.4 Hz, 1H), 7.02-6.99 (m, 1H),6.84 (t, J = 11.5 Hz, 1H), 6.20 (t, J = 13.3 Hz, 2H), 5.74 (dd, J = 8.1, 3.7 Hz,1H), 5.26 (dt, J = 4.1, 1.1 Hz, 1H), 4.98-4.85 (m, 3H), 4.56 (d, J = 7.7 Hz, 1H),4.45-4.41 (m, 1H), 4.19-3.98 (m, 3H), 3.90-3.84 (m, 2H), 3.71-3.63 (m, 8H), 3.52-3.47(m, 26H), 3.12-3.00 (m, 3H), 2.67-2.62 (m, 7H), 2.35 (t, J = 1.9 Hz, 1H), 2.20-2.06(m, 2H), 1.94-1.90 (m, 1H), 1.88 (d, J = 3.6 Hz, 2H), 1.67-1.62 (m, 5H), 1.55-1.46(m, 2H), 1.27-1.22 (m, 1H), 0.91 (d, J = 6.7 Hz, 3H), 0.66 (s, 3H).

[0332] Example 39 compound 114 [ka]

[0333] Compound 110: (2-(((allyloxy)carbonyl)amino)acetamido)methyl acetate (110) was prepared using the procedure from Tetrahedron, 2018, 74(15), 1951-1956.

[0334] Compound 111: To a solution of compound 110 (27.6 mg, 0.12 mmol) and verrucarin A (1, 30 mg, 0.06 mmol) in anhydrous THF (2.5 mL) at 0 °C was added LiHMDS (72 μL, 1.0 M solution in hexanes, 0.072 mmol). After 30 min, LCMS showed 26% desired product and 60% unreacted 1. Additional 110 (14 mg, 0.06 mmol) was added followed by LiHMDS (60 μL, 0.06 mmol) and the reaction was stirred for an additional 30 min. The reaction was quenched with saturated aqueous ammonium chloride (5 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The products were chromatographed on an ISCO 4 g silica gold column eluting with 50% ethyl acetate / hexane to recover unreacted verrucarin A (1, 10 mg, 29%), followed by 80% ethyl acetate / hexane to elute compound 111 (20.5 mg, 45%). MS (ESI, pos.): C 34 H 44 N 2 O 12 Calculated for, 672.3; found 673.3 (M+H).

[0335] Compound 112: A solution of compound 111 (15 mg, 0.022 mmol) in dichloromethane (2.5 mL) was added to Pd(PPh 3 ) 4(2.5 mg, 0.0022 mmol) and phenylsilane (4 μL, 0.033 mmol) were added. The reaction was stirred for 1 h, at which point LCMS indicated the reaction was complete. The reaction was filtered through a pad of Celite and washed with dichloromethane (3 mL). The filtrate was concentrated to give compound 112, which was used in the next step without purification. MS (ESI, pos.): C 30 H 40 N 2 O 10 Calculated for, 588.3; found 589.3 (M+H).

[0336] Compound 114: To a solution of compound 112 (0.022 mmol) in anhydrous DMF (1 mL) at room temperature, Mal-cap-Val-OH (113, 3.7 mg, 0.022 mmol) and HATU (12.6 mg, 0.033 mmol) were added followed by N,N-diisopropylethylamine (12 μL, 0.066 mmol). The reaction was complete within 30 min by LCMS. The product was purified by elution with 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a 30×150 mm Gemini column eluted with 0.05% AcOH (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 114 (5 mg, 25% over two steps). MS (ESI, pos.): 45 H 60 N 4 O 14 Calculated for, 880.4; found, 881.3 (M+H). 1 H-NMR (500 MHz; CDCl 3): δ 8.01 (dd, J = 15.0, 11.6 Hz, 1H), 6.71 (s, 2H), 6.67 (t, J = 11.4 Hz, 1H),6.46 (t, J = 5.2 Hz, 1H), 6.17 (d, J = 11.1 Hz, 1H), 6.04 (d, J = 15.6 Hz, 1H),5.99 (d, J = 6.9 Hz, 1H), 5.82 (dd, J = 7.9, 3.7 Hz, 1H), 5.44 (d, J = 3.3 Hz, 1H),4.91 (dd, J = 10.8, 8.5 Hz, 1H), 4.77 (d, J = 12.0 Hz, 1H), 4.63 (dd, J = 10.9,5.5 Hz, 1H), 4.47-4.44 (m, 1H), 4.14-4.08 (m, 3H), 4.01-3.96 (m, 2H), 3.87 (d, J= 5.0 Hz, 1H), 3.79 (dd, J = 16.9, 5.0 Hz, 1H), 3.58 (d, J = 5.0 Hz, 1H), 3.53 (t,J = 7.1 Hz, 2H), 3.14 (d, J = 3.9 Hz, 1H), 2.85 (d, J = 3.9 Hz, 1H), 2.49 (t, J= 7.5 Hz, 1H), 2.38-2.35 (m, 1H), 2.25-2.20 (m, 3H), 2.12-2.04 (m, 3H), 1.91-1.87(m, 2H), 1.77 (s, 3H), 1.69-1.62 (m, 3H), 1.36-1.27 (m, 6H), 0.99 (dd, J = 12.7,6.7 Hz, 6H), 0.93 (d, J = 6.8 Hz, 3H), 0.86 (s, 3H).

[0337] Example 40 Compound 118 [Chemical Structure]

[0338] Compound 116: To a solution of verrucarin A (1, 25 mg, 0.05 mmol) in anhydrous chloroform (0.25 mL) was added compound 115 (38 mg, 0.15 mmol) and N,N-diisopropylethylamine (35 μL, 0.2 mmol) and the reaction was heated to 40 °C for 22 h. The volatiles were removed under reduced pressure and the residue was purified by elution with 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 1,2-dichlorophenyl ether (C1H2O) and 1,2-dichlorophenyl ether (C1H2O) (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 116 (15 mg, 43%) as a fluffy white solid. MS (ESI, pos.): 33 H 44 N 4 O 12 Calculated for S, 720.3; found 721.3 (M+H) and 743.3 (M+Na).

[0339] Compound 117:10:1 THF / H 2 To a solution of compound 116 (15 mg, 0.02 mmol) in 0 (0.4 mL) was added triphenylphosphine (13.6 mg, 0.05 mmol). After stirring at room temperature for 24 h, the reaction was concentrated to dryness and used in the next step without purification. MS (ESI, pos.): C 33 H 46 N 2 O 12 Calculated for S, 694.3; found 695.3 M+H).

[0340] Compound 118: To a solution of compound 117 (0.02 mmol) and Mal-cap-Val-OH (113, 18.6 mg, 0.06 mmol) in anhydrous DMF (0.4 mL) was added HATU (19 mg, 0.05 mmol) and N,N-diisopropylethylamine (10.3 μL, 0.06 mmol). After stirring for 1 h, the reaction was diluted with 5-95% MeCN / H 2The mixture was chromatographed on an ISCO 30 g C18Aq column using 1,2-dichlorophenyl ether (C18Aq) and 1,2-dichlorophenyl ether (C18Aq) (both containing 0.05% AcOH). The pure fractions were combined and lyophilized to give compound 118 (5.2 mg, 27%) as a fluffy off-white solid. MS (ESI, pos.): C 48 H 66 N 4 O 16 Calculated for S, 986.4; found 987.4 M+H). 1 H-NMR (500 MHz; acetone-d 6 ): δ 8.11-8.05 (t, J -14.2 Hz, 1H), 7.39-7.37 (m, 1H), 7.10-7.08 (m, 1H), 6.87(d, J = 9.8 Hz, 2H), 6.26 (d, J = 11.2 Hz, 1H), 6.15 (d, J = 15.8 Hz, 1H), 5.90-5.89(m, 1H), 5.40-5.39 (m, 1H), 4.99 (d, J = 1.8 Hz, 2H), 4.73-4.71 (m, 1H), 4.46-4.43(m, 1H), 4.37-4.34 (m, 1H), 4.31 (s, 1H), 4.27-4.23 (m, 2H), 4.18-4.14 (m, 1H),4.03-3.92 (m, 2H), 3.85-3.84 (m, 1H), 3.73 (t, J = 6.5 Hz, 2H), 3.49-3.43 (m, 3H),3.40-3.38 (m, 1H), 3.06-3.03 (m, 2H), 3.00 (s, 3H), 2.54-2.48 (m, 2H), 2.30-2.27(m, 1H), 2.19-2.16 (m, 1H), 1.99-1.94 (m, 3H), 1.90-1.79 (m, 4H), 1.71 (s, 3H),1.67-1.64 (m, 2H), 1.60-1.56 (m, 2H), 1.33 (t, J = 7.6 Hz, 3H), 0.97-0.91 (m, 9H),0.89 (s, 3H).

[0341] Example 41 compound 121 [ka]

[0342] Compound 120: To a solution of crude 112 (20 mg, 0.03 mmol) in DMF (2 mL) at room temperature, HATU (17.2 mg, 0.045 mmol) and Fmoc-amino-cap-Val-OH (119, 15 mg, 0.033 mmol) were added, followed by N,N-diisopropylethylamine (16 μL, 0.09 mmol). The reaction was complete after 30 min by LCMS. The reaction solution was loaded onto an ISCO 15.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 120 (18.2 mg, 50% over two steps). MS (ESI, pos.): C 56 H 70 N 4 O 14 Calculated for, 1022.5; Found 1023.4 (M+H), 1045.4 (M+Na).

[0343] Compound 121: To a solution of compound 120 (18.2 mg, 0.0178 mmol) in DMF (1 mL) at room temperature was added a 5% piperidine in DMF solution (0.4 mL). The reaction was complete after 30 min by LCMS. The reaction was purified by 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a Gemini 30×150 mm column eluted with 0.05% AcOH (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 121 (12 mg, 83%) as a fluffy off-white solid. MS (ESI, pos.): 41 H 60 N 4 O 12 Calculated for, 800.4; found, 801.4 (M+H). 1 H-NMR (300 MHz; CDCl 3): δ 8.02 (m, 2H), 7.09 (d, J = 6.7 Hz, 1H), 7.01-6.97 (m, 1H), 6.68 (t, J = 11.3Hz, 1H), 6.19 (d, J = 11.2 Hz, 1H), 6.05 (d, J = 15.7 Hz, 1H), 5.85-5.81 (m, 1H),5.46 (dt, J = 2.6, 1.2 Hz, 1H), 4.79-4.72 (m, 3H), 4.48-4.44 (m, 1H), 4.20-4.12(m, 3H), 4.06-4.00 (m, 2H), 3.89 (d, J = 5.0 Hz, 1H), 3.76-3.69 (m, 1H), 3.61 (d,J = 5.3 Hz, 1H), 3.15 (d, J = 4.0 Hz, 1H), 2.86 (m, 1H), 2.56-2.48 (m, 2H), 2.37-2.23(m, 7H), 2.04 (d, J = 5.3 Hz, 5H), 1.92-1.88 (m, 2H), 1.78 (s, 3H), 1.75-1.61 (m,4H), 1.47-1.33 (m, 2H), 1.01 (dd, J = 11.9, 6.7 Hz, 6H), 0.93 (d, J = 6.8 Hz, 3H),0.88 (s, 3H).

[0344] Example 42 compound 124 [ka]

[0345] Compound 12To a solution of compound 27 (80 mg, 0.130 mmol) and N-hydroxysuccinimide (18 mg, 0.156 mmol) in 2:1:1 THF / DMF (0.8 mL) was added EDCI (37.4 mg, 0.195 mmol) and the reaction was stirred for 2 h. The volatiles were removed under reduced pressure and the product was purified by elution with 30-100% MeCN / H 2The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 1,2-dichlorophenyl ether (C1H2O) and 1,2-dichlorophenyl ether (C1H2O) (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 122 (66.2 mg, 72%) as a fluffy off-white solid. MS (ESI, pos.): 38 H 41 N 5 O 9 Calculated for, 711.3; found 712.3 (M+H).

[0346] Compound 123: To a solution of compound 112 (12.3 mg, 0.021 mmol) and compound 122 (16.5 mg, 0.023 mmol) in anhydrous DMF (1.5 mL) was added N,N-diisopropylethylamine. After stirring for 30 min, the reaction was loaded onto an ISCO 15.5 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 123 (14.2, 57%) as a fluffy off-white solid. MS (ESI, pos.): C 64 H 76 N 6 O 16 Calculated for, 1184.5; found 1185.5 (M+H).

[0347] Compound 124: To a solution of compound 123 (14.2 mg, 0.012 mmol) in DMF (1.0 mL) was added a solution of 5% piperidine in DMF (0.7 mL). After stirring for 40 min, the reaction was diluted with 5-95% MeCN / H 2 The mixture was purified by preparative HPLC on a 30×150 mm Gemini column using 1,2-dichlorophenyl ether (C1H2O) and 2,3-dichlorophenyl ether (C1H2O) (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 124 (9.2, 80%) as a fluffy off-white solid. MS (ESI, pos.): 49 H 66 N 6 O 14 Calculated for, 962.5; found 963.4 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 8.65 - 8.61 (m, 1H), 8.43 (m, 1H), 8.22 - 8.06 (m, 3H), 7.83 (dd, J = 15.5, 11.7Hz, 1H), 7.25 - 7.21 (m, 4H), 7.16 (t, J = 6.6 Hz, 1H), 6.84 (t, J = 11.4 Hz, 1H), 6.30 (d, J = 11.1 Hz, 1H), 6.21 (d, J = 15.5 Hz, 1H), 5.80 (dd, J = 7.9, 3.7 Hz, 1H), 5.29 (d, J = 4.4 Hz, 1H), 4.60 (dd, J = 10.3, 8.3 Hz, 1H), 4.52 - 4.45 (m, 3H), 4.30 (d, J = 10.0 Hz, 1H), 4.05 (t, J = 5.5 Hz, 2H), 3.91 (t, J = 5.8 Hz, 1H), 3.75 - 3.62 (m, 7H), 3.58 - 3.53 (m, 1H), 3.04 (dd, J = 13.5, 3.5 Hz, 2H), 2.94 - 2.88 (m, 3H), 2.80 (d, J = 13.1 Hz, 1H), 2.64 (t, J = 5.0 Hz, 1H), 2.26 - 2.21 (m, 1H), 2.12 (q, J = 7.2 Hz, 2H), 1.95 (dt, J = 14.9, 4.5 Hz, 1H), 1.88 - 1.85 (m, 2H), 1.81 (s, 2H), 1.76 (d, J = 13.1 Hz, 2H), 1.64 (s, 3H), 1.55 (t, J = 12.7 Hz, 1H), 1.48 (dt, J = 14.3, 7.2 Hz, 2H), 1.34 (q, J = 6.8 Hz, 2H), 1.24 (dt, J = 15.1, 7.4 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H), 0.69 (s, 3H).

[0348] Example 43 Compound 126 [Chemical formula]

[0349] Compound 125 was prepared using the same method as compound 27, except starting from 6-maleimidocaproic acid N-hydroxysuccinimide ester. MS (ESI, pos.): C 23 H 28 N 4 O 7 Calculated for, 472.2; found, 473.3 (M+H).

[0350] Compound 126: To a solution of compound 112 (10 mg, 0.017 mmol) and compound 125 (9.5 mg, 0.020 mmol) in anhydrous DMF (1.5 mL) was added HATU (9.7 mg, 0.026 mmol), HOAt (1.5 mg, 0.017 mmol) and N,N-diisopropylethylamine (6.0 µL, 0.034 mmol). After stirring for 30 min, the reaction was loaded onto an ISCO 30 g C18Aq column and purified with 5-95% MeCN / H 2 0 (both containing 0.05% AcOH). Pure fractions were combined and lyophilized to give compound 126 (5.6 mg, 33%) as a fluffy off-white solid. MS (ESI, pos.): C 53 H 66 N 6 O 16 Calculated for, 1042.5; found 1043.4 (M+H). 1 H-NMR (500 MHz; DMSO-d 6): δ 8.54 (t, J = 6.5 Hz, 1H), 8.34 (t, J = 8.2 Hz, 1H), 8.13-8.06 (m, 2H), 7.99(d, J = 3.8 Hz, 1H), 7.83 (ddd, J = 15.6, 11.6, 0.8 Hz, 1H), 7.25-7.20 (m, 4H), 7.18-7.15 (m, 1H), 6.98 (s, 2H), 6.83 (d, J = 11.6 Hz, 1H), 6.30 (d, J = 11.1 Hz,1H), 6.21 (d, J = 15.6 Hz, 1H), 5.80 (dd, J = 7.9, 3.9 Hz, 1H), 5.29 (d, J = 4.2Hz, 1H), 4.60 (dd, J = 10.7, 8.1 Hz, 1H), 4.52-4.45 (m, 3H), 4.32-4.28 (m, 1H), 4.06-4.04 (m, 2H), 3.92 (td, J = 11.5, 3.2 Hz, 1H), 3.72 (t, J = 11.3 Hz, 2H), 3.66(m, 4H), 3.58 (td, J = 18.4, 5.5 Hz, 2H), 3.04 (dd, J = 13.3, 4.2 Hz, 1H), 2.92(d, J = 3.9 Hz, 1H), 2.78 (dd, J = 13.6, 8.5 Hz, 1H), 2.63-2.61 (m, 1H), 2.41 (dd,J = 13.4, 6.6 Hz, 2H), 2.26-2.21 (m, 1H), 2.10 (t, J = 7.5 Hz, 2H), 1.95 (td, J= 9.7, 5.7 Hz, 1H), 1.89-1.85 (m, 4H), 1.81-1.71 (m, 2H), 1.62-1.61 (s, 3H), 1.58-1.53(m, 1H), 1.50-1.42 (m, 4H), 1.18 (dt, J = 15.5, 7.7 Hz, 2H), 0.76 (d, J = 6.9 Hz, 3H), 0.69 (s, 3H).

[0351] Example 44 Synthesis of Anti-Pyramid Antibody Complex The anti-hemagglutinin (anti-HA) monoclonal antibody mAb11729 was mutated to introduce a consensus LLQGA pentapeptide sequence at the C-terminus of the heavy chain. A non-HA binding mAb (derived from an immune antigen unrelated to infection) containing the same consensus sequence at the C-terminus of the heavy chain was used as a non-binding isotype control. This mutation allowed the antibody to be enzymatically conjugated to the heavy chain with up to two payloads (one on each heavy chain).

[0352] A second anti-hemagglutinin (H3N2) monoclonal antibody, F005-126, taken from the literature (Iba, et al., J. Virology 2014, vol 88, 7130-7144), was mutated to introduce a consensus ELQGP pentapeptide sequence at the C-terminus of the heavy chain. A non-HA binding mAb (derived from an immune antigen unrelated to infection) containing the same consensus sequence at the C-terminus of the heavy chain or the C-terminus of the light chain was used as a non-binding isotype control. This mutation allowed the antibody to be enzymatically conjugated to a maximum payload of two (one for each of the heavy or light chains).

[0353] Antibodies with conjugation sites at the C-terminus of the heavy chain were conjugated at 1 mg / mL in PBS, pH 7.4. Verrucarin A derivatives 10a, b, c; 19a, b, c; 46, 53, 101, 106, and 124 were added in 10-40 fold molar excess over the antibody, and the enzymatic reaction was initiated by adding 12 units of bacterial transglutaminase (Zedira or MilliporeSigma) per mg of antibody and incubated for 16 h at 37 °C. Conjugates were purified by size-exclusion chromatography (Superdex200) using PBS containing 5% glycerol and sterile filtered. Protein concentrations and drug-to-antibody ratios were determined by UV spectroscopy. All conjugates were confirmed to be >90% monomeric by size-exclusion HPLC. All conjugated antibodies were analyzed by mass spectrometry for linker-payload loading values. Drug-to-antibody ratios are reported in Table 3.

[0354] Native mAb11729 and isotype control antibodies (1-10 mg / ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5 were treated with 2.2 equivalents of tris(2-carboxyethyl)phosphine for 90 min at 37 °C to reduce interchain disulfide bonds. Maleimidoverrucarin A derivatives 12a, b, c, 21a, b, c, and 97 (1.2 equivalents / SH group) in DMSO (10 mg / ml) were added to the reduced antibodies and allowed to react for 1 h at 22 °C. Conjugates were purified by size-exclusion chromatography using PBS containing 5% glycerol and sterile filtered. Protein concentrations and drug-to-antibody ratios were determined by UV spectroscopy. Size-exclusion HPLC confirmed that all conjugates used were >95% monomeric. All conjugated antibodies were analyzed by mass spectrometry for linker-payload loading values. Drug-to-antibody ratios are reported in Table 3.

[0355] [Table 7-1] [Table 7-2]

[0356] Characterization of the complex by liquid chromatography-mass spectrometry To determine the linker-payload loading on the antibodies, the conjugates were reduced to heavy and light chains with 50 mM dithiothreitol, further deglycosylated with PNGase if the conjugate was glycosylated, and then analyzed by LC-MS. The resulting molecular ions, when weighted according to intensity, corresponded to the loading amounts listed in Table 3.

[0357] Conjugates were analyzed by ESI-MS to determine the drug:antibody ratio (DAR) using a Waters Acquity UPLC interfaced with a Xevo G2-S QTof mass spectrometer. Chromatographic separation was achieved on a C4 column (2.1 x 50 mm ACQUITY UPLC BEH Protein C4, 1.7 um, 300A) with a 10 min gradient (min: percentage of mobile phase B; 0:10%, 1:10%, 5:90%, 7:90%, 7.2:10%, 10:10%). Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was set at 0.3 mL / min. The detector TOF scan was set from m / z 500 to 4500 with the key parameters listed (capillary voltage 3.0 kV; sampling cone 80 V; source offset 100 V; source temperature 150 °C; desolvation temperature 450 °C; cone gas 0 L / h; desolvation gas 800 L / h). Spectra were deconvoluted using the MaxEnt function within MassLynx software. The resulting molecular ions, when weighted according to intensity, corresponded to the loadings listed in Table 3.

[0358] Alternatively, to determine the drug loading on the antibody, the conjugates were run on an Agilent 1260 using a TSK-NPR Butyl HIC (hydrophobic interaction chromatography) column using a linear gradient over 18 minutes from 25 mM sodium phosphate and 1.5 M ammonium sulfate pH 6.8 to 25 mM sodium phosphate pH 6.8. Payload loading was determined by integration of the peak areas corresponding to the conjugated and unconjugated antibody species.

[0359] Mass spectrometry spectra were deconvoluted using Masslynx software and the drug-to-antibody ratio (DAR) was calculated using the following equation: 1. Relative percentage (%) of drug (Dn) by peak intensity (PI) distribution: Dn%=PIn / Σ(PI0+PI1+PI2…….+PIi)×100 (n=0,1,2,3,…,i) 2. Average DAR calculation: DAR=Σ(1×D1%+2×D2%+3×D3%+……+i×Di%)

[0360] Example 45 background Antibodies that target the membrane-proximal stem domain of influenza HA generally show increased width compared to antibodies that target the globular head of this molecule. However, this increased width leads to a decrease in potency. It is therefore of interest whether the activity of these antibodies can be enhanced by conjugation with antiviral small molecules. mAb11729 is a monoclonal antibody that binds to the stem domain of influenza HA molecule of group 1 and shows antiviral activity against H1N1 in vitro. Verrucarin A is a broad-spectrum antiviral molecule that has been modified to be cell-impermeable in this case and conjugated to mAb11729.

[0361] Experimental Procedures, Results, and Conclusions [Table 8]

[0362] Antiviral activity To test for antiviral efficacy, mAb11729 and ADC10b, 19a, 19b and 19c were assayed for their ability to inhibit infection of cells by influenza virus. MDCK London cells were seeded at 20,000 cells / well in 100 μL of growth medium (DMEM containing 1% sodium pyruvate, 10% fetal bovine serum and 0.5% gentamicin) in 96-well plates. Cells were incubated at 37° C. and 5% CO. 2 The next day, all antibodies were diluted in trypsin infection medium (DMEM containing 1% sodium pyruvate, 0.21% low IgG BSA solution, 1 mg / mL trypsin, TPCK treatment and 0.5% gentamicin) to a starting concentration of 500 μg / mL and diluted with 8.5 × 10 -3The antibodies were titrated 1:3 to a final concentration of 1 μg / mL. H1N1 A / Puerto Rico / 08 / 1934 influenza virus engineered to express GFP in infected cells ("H1N1 A / Puerto Rico / 08 / 1934-GFP") was diluted to an MOI of 1 in trypsin infection medium (Life Technologies) and mixed 1:1 with the diluted antibodies or ADCs. Growth medium was removed from the seeded 96-well plates and the virus-antibody or virus-ADC mixtures were added to the cells at 100 μL per well. The plates were gently tapped and returned to 37°C, 5% CO2 for 20 hours. The plates were then washed once with PBS and overlaid with 50 μL of PBS. The GFP signal of the plates was immediately read on a Molecular Devices Spectramax i3x plate reader. The ADCs showed robustly increased antiviral potency against influenza A infection compared to unconjugated antibodies. [Table 9]

[0363] Example 46 To evaluate its potential as a broad-spectrum antiviral payload, cell-impermeable verrucarin A was conjugated to mAb10987, mAb10985, and clone 4A8, which are monoclonal antibodies that bind to different epitopes on the receptor-binding domain of SARS-CoV-2, and clone 4A8, which binds to the outside of the RBD on the SARS-CoV-2 spike protein.

[0364] To test the antiviral effect against SARS-CoV-2, mAb10987, mAb10985 and clone 4A8, as well as ADCs mAb10987-12b, mAb10985-12b, mAb10987-10b, mAb10985-10b and clone 4A8-10b were assayed for their ability to inhibit infection of cells by VSVΔG, which expresses the spike protein of SARS-CoV-2 on its surface. Vero cells were seeded at 20,000 cells / well in 100 μL of growth medium (DMEM with 10% fetal bovine serum and 1% Penn-Strep glutamine) in 96-well plates. Cells were incubated at 37 °C and 5% CO. 2 The next day, all antibodies were diluted in infection medium (DMEM containing 3% FBS and 1% PSG) to a starting concentration of 10 μg / mL and diluted with 1.69 × 10 -4 VSV-spike virus was titrated 1:3 to a final concentration of 4 × 10 4 The virus was diluted to PFU / mL and mixed 1:1 with the diluted antibody or ADC. The virus-antibody mixes were incubated together for 30 minutes. The growth medium was then removed from the seeded 96-well plates and the virus-antibody or virus-ADC mixes were added to the cells at 100 μL per well. The plates were gently tapped and incubated at 37°C, 5% CO 2The plates were then returned to RT for 20 hours. The plates were then removed from the incubator and fixed with 2% PFA for 30 minutes. The 2% PFA was then removed and blocking / permeabilization buffer (PBS with 3% BSA and 0.1% Triton-X100) was added to the cells for 1 hour at room temperature. The blocking buffer was then replaced with primary antibody solution (rabbit anti-VSV from Imanis diluted 1:100 in blocking buffer). The plates were incubated for 2 hours at room temperature. The plates were then washed 3 times with PBS. Secondary antibody solution (goat anti-rabbit AF488 diluted 1:100 in blocking buffer) was added to the cells for 1 hour at 37°C. The plates were then washed 3 times with PBS and overlaid with 100 μL of PBS. AF488 fluorescence was then read on a Molecular Devices Spectramax i3x plate reader. Anti-spike antibodies conjugated with a verrucarin-A payload demonstrated increased antiviral potency against VSV-spike infection compared to unconjugated antibodies (Table 6). [Table 10]

[0365] Example 47 To further evaluate its potential as a broad-spectrum antiviral payload, cell-impermeable verrucarin A was conjugated to mAb3471, a monoclonal antibody that binds to Ebola virus (EBOV) glycoprotein.

[0366] To test the antiviral effect against EBOV, mAb3471 and ADB mAb3471-10b were assayed for their ability to inhibit infection of cells by VSVΔG, which expresses the EBOV glycoprotein (GP) on its surface. Vero cells were seeded at 20,000 cells / well in 100 μL of growth medium (DMEM with 10% fetal bovine serum and 1% Penn-Strep glutamine) in 96-well plates. Cells were incubated at 37°C and 5% CO. 2The next day, all antibodies were diluted in infection medium (DMEM containing 3% FBS and 1% PSG) to a starting concentration of 200 μg / mL and diluted with 3.39 × 10 -3 VSV-EBOV-GP virus was titrated 1:3 to a final concentration of 4 × 10 4 The virus was diluted to PFU / mL and mixed 1:1 with the diluted antibody or ADC. The virus-antibody mixes were incubated together for 30 minutes. The growth medium was then removed from the seeded 96-well plates and the virus-antibody or virus-ADC mixes were added to the cells at 100 μL per well. The plates were gently tapped and incubated at 37°C, 5% CO 2 The plates were then removed from the incubator and fixed with 2% PFA for 30 minutes. The 2% PFA was then removed and blocking / permeabilization buffer (PBS with 3% BSA and 0.1% Triton-X100) was added to the cells for 1 hour at room temperature. The blocking buffer was then replaced with primary antibody solution (rabbit anti-VSV from Imanis diluted 1:100 in blocking buffer). The plates were incubated for 2 hours at room temperature. The plates were then washed 3 times with PBS. Secondary antibody solution (goat anti-rabbit AF488 diluted 1:100 in blocking buffer) was added to the cells for 1 hour at 37°C. The plates were then washed 3 times with PBS and overlaid with 100 μL of PBS. AF488 fluorescence was then read on a Molecular Devices Spectramax i3x plate reader. mAb3471 conjugated with a verrucarin-A payload demonstrated increased antiviral potency against VSV-EBOV-GP infection compared to the unconjugated antibody (Table 7). [Table 11]

[0367] The present disclosure should not be limited in scope by the embodiments disclosed in the examples, which are intended as single illustrations of individual aspects, and any equivalents are within the scope of the present disclosure. Various modifications will be apparent to those skilled in the art from the foregoing description, in addition to those shown and described herein. Such modifications are intended to fall within the scope of the appended claims.

[0368] Various references, such as patents, patent applications, and publications, are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. Formula I: 【Chemistry 1】 or a pharmaceutically acceptable derivative thereof, wherein X is NR １ R ２ , OR ３ or SR ４ and R １ and R ２ are each independently H, alkyl, OR ５ or COR ６ or together with the nitrogen atom to which they are attached form a heterocycloalkyl; R ３ is alkyl or COR ７ and R ４ is H, alkyl, or COR ８ and R ５ is H or alkyl; R ６ is R ９ , OR １０ or NR １１ R １２ and R ７ ~R ９ are each independently alkyl or aralkyl; R １０ ~R １２ are each independently H, alkyl, or aralkyl; Y is H or OH; (i) Z is O; 【Chemistry 2】 is a single bond; or (ii) Z is absent, 【Transformation 3】 is a double bond.

2. R １ and R ２ are each independently H, methyl, or ethyl; W is OR １３ Or NR １４ R １５ C(O)-(C ２～４ alkylene)-CO-W, or V is OR16 or NR １７ R １８ C(O)-CH(V)-CH ３ or together with the nitrogen atom to which they are attached form piperazinyl; R １３ and R １６ are each independently H or methyl; R １４ , R １５ , R １７ , and R １８ 10. The compound of claim 1, wherein each is independently H, methyl, hydroxy, or methoxy.

3. R １ is H, methyl, ethyl, C(O)CH ２ CH ２ COOH, C(O)CH ２ CH ２ CH ２ COOH, C(O)CH ２ CH ２ CH ２ CH ２ COOH, C(O)CH ２ CH ２ CH ２ COOMe, C(O)CH ２ CH ２ CH ２ CONHOH,C(O)CH ２ CH ２ CH ２ CONHOMe, C(O)-CH(OH)-CH ３ , C(O)-CH(NH ２ )-CH ３ or C(O)—CH(NMe ２ )-CH ３ and R 2 is H or methyl; or The compound of claim 1, wherein R 1 and R 2 together with the nitrogen atom to which they are attached form 4-methyl-1-piperazinyl.

4. R ３ The compound of claim 1, wherein is methyl or C(O)-alkyl, said alkyl being optionally substituted.

5. R ３ is methyl, C(O)—CH(NHMe)—CH ３ or C(O)—CH(NHAc)—CH ３ 2. The compound of claim 1, wherein:

6. R ４ 6. The compound of claim 1 or 5, wherein is C(O)Me and R<5> is H or methyl.

7. R ６ is CH ２ CH ２ COOH, CH ２ CH ２ CH ２ COOH, CH ２ CH ２ CH ２ CH ２ COOH, CH ２ CH ２ CH ２ COOMe, CH ２ CH ２ CH ２ CONHOH or CH ２ CH ２ CH ２ 2. The compound of claim 1, which is CONHOMe.

8. R ７ and R ８ The compound of claim 1 , wherein each is independently methyl.

9. R １０ ~R １２ 10. The compound of claim 1, wherein each is independently H or methyl.

10. Y is H and Z is absent; 【Chemistry 4】 The compound of claim 1 , wherein is a double bond.

11. Y is OH and Z is absent; 【Transformation 5】 The compound of claim 1 , wherein is a double bond.

12. Y is H and Z is O; 【Transformation 6】 The compound of claim 1 , wherein is a single bond.

13. Formula II: 【Transformation 7】 or a pharmaceutically acceptable derivative thereof, wherein R １ is H or -COR ６ and R ６ The compound of claim 1 , wherein is -alkylene-COOH.

14. R １ is -COR ６ 14. The compound of claim 13, wherein:

15. R ６ is -(CR ２１ R ２２ ) ｍ COOH, where R ２１ and R ２２ 14. The compound of claim 13, wherein each is independently H or alkyl; and m is an integer from 0 to 6.

16. 2. The compound of claim 1 selected from the following: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6

17. Formula III: 【Transformation 8】 or a pharmaceutically acceptable derivative thereof, wherein X １ is O or NH; D is absent or -C(O)-(CH ２ ) ２～５ —C(O)— or —O—NH—C(O)—(CH ２ ) ２～５ L is a linking group; Y is H or OH; (i) Z is O; 【Chemistry 9】 is a single bond; or (ii) Z is absent, 【Chemistry 10】 is a double bond.

18. Formula IV: 【Chemistry 11】 or a pharmaceutically acceptable derivative thereof, wherein L is a linking group and n is an integer from 1 to 4.

19. 19. The compound of claim 18, wherein n is 1, 2, or 3.

20. 18. The compound of claim 17, wherein L is a cleavable linker.

21. 18. The compound of claim 17, wherein L is an acid labile linker, a hydrolytically unstable linker, an enzyme cleavable linker, a reduction labile linker, or a self-immolative linker.

22. 18. The compound of claim 17, wherein L comprises or consists of MC-val-cit-PAB.

23. 18. The compound of claim 17, wherein L comprises or consists of AC-val-cit-PAB.

24. 18. The compound of claim 17, wherein L comprises or consists of MC-val-cit-MePAB.

25. 18. The compound of claim 17, wherein L comprises or consists of AC-val-cit-MePAB.

26. L is AC-GGFG-CH ２ 18. The compound of claim 17, comprising or consisting of:

27. 18. The compound of claim 17 having one of the following formulae: 【Chemistry 12-1】 【Chemistry 12-2】

28. 18. The compound of claim 17, wherein L is a non-cleavable linker.

29. 18. The compound of claim 17 selected from: Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9

30. Formula V: Z-(L-X) ｖ V or a pharmaceutically acceptable derivative thereof, wherein Z is an anti-influenza antigen-binding domain, an anti-SARS-CoV-2 antigen-binding domain, or an anti-Ebola virus antigen-binding domain; L is a linking group; X is a verrucarin A derivative; The compound wherein v is an integer of 1 to 12.

31. 31. The compound of claim 30, wherein -LX is a radical formed by removal of H from the compound of claim 17.

32. 31. The compound of claim 30, wherein v is 1, 2, 3, 4, 5, 6, 7, or 8.

33. 31. The compound of claim 30, wherein Z comprises an antibody heavy chain and further comprises a peptide tag at the C-terminus of the antibody heavy chain, wherein the peptide tag is ELQRP, LLQG, LLQGG, LLQLLQG, LLQYQG, LLQGA, LLQGSG, SLLQG, LQG, LLQLQ, LLQLLQ, LLQGR, LLQYQGA, LQGG, LGQG, or LLQLLQGA.

34. 31. The compound of claim 30, wherein Z comprises two antibody heavy chains and a peptide tag at the C-terminus of each antibody heavy chain.

35. 34. The compound of claim 33, wherein the peptide tag is the pentapeptide sequence LLQGA.

36. 34. The compound of claim 33, wherein the peptide tag is the pentapeptide sequence ELQGP.

37. 31. The compound of claim 30 having one of the following formulas: 【Chemistry 13-1】 【Chemistry 13-2】

38. 31. A pharmaceutical composition comprising a compound of any one of claims 1, 17 and 30 and a pharmaceutically acceptable carrier.

39. A compound according to any one of claims 1, 17 and 30 for use in treating influenza infection in a subject.

40. 40. The compound of claim 39, wherein the influenza is influenza A.

41. A compound according to any one of claims 1, 17 and 30 for use in treating SARS-CoV-2 infection in a subject.

42. 42. The compound of claim 41, wherein the SARS-CoV-2 is an omicron mutant.

43. A compound according to any one of claims 1, 17 and 30 for use in the treatment of an Ebola infection in a subject.

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