Patent Application
20250127918
The content of the electronic sequence listing (M065670540US01-SEQ-WWZ.xml; Size: 49,265 bytes; and Date of Creation: Sep. 30, 2024) is herein incorporated by reference in its entirety.
Peptides are highly selective biopolymers, usually able to bind to specific cell receptors, or ion channels, to trigger intracellular effects. They are also excellent vectors, capable of transporting cargos to specific targets, making them highly suitable for precision medicine as peptide-drug conjugates. Compared to other biologics, peptides are easier to produce, cheaper, and result in lower immunogenicity and enhanced tissue penetration (B. M. Cooper, J. Iegre, D. H. O′ Donovan, M. Ölwegård Halvarsson, D. R. Spring, Peptides as a platform for targeted therapeutics for cancer: peptide-drug conjugates (PDCs). Chem. Soc. Rev. 50, 1480-1494 (2021)). However, peptide therapeutics usually make poor drugs due to a low chemical and physical stability and very short plasmatic half-life, resulting in their renal elimination in a few minutes. Last decades efforts thereby focused on the extension of peptide pharmacokinetic (PK) and pharmacodynamic (PD) properties following chemical (or chemoenzymatic) modifications, to slow-down renal clearance (A. Rondon, S. Mahri, F. Morales-Yanez, M. Dumoulin, R. Vanbever, Protein engineering strategies for improved pharmacokinetics. Advanced Functional Materials. 31, 2101633 (2021)), increase chemical and physical stability (H. E. Blackwell, R. H. Grubbs, Highly efficient synthesis of covalently cross-linked peptide helices by ring-closing metathesis. Angewandte Chemie International Edition. 37, 3281-3284 (1998), C. Heinis, T. Rutherford, S. Freund, G. Winter, Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Nat Chem Biol. 5, 502-507 (2009)), and enhance bioavailability (D. J. Drucker, Advances in oral peptide therapeutics. Nat Rev Drug Discov. 19, 277-289 (2020)). Today, despite about 80 peptide drugs being already approved worldwide, and a hundred more currently in clinical trials, the drug market still faces a major challenge in designing and producing long-acting peptide therapeutics.
The present disclosure provides site-selective conjugation of a pharmaceutical agent to an antibody using an affinity peptide. In one aspect, the present disclosure provides a first modified affinity peptide, wherein n1 instances of the amino acid residues of the affinity peptide are independently modified with a moiety of Formula A′:
-L1-E2-L2-(E3)n3 (A′).
In certain embodiments, each instance of E2 is independently —O—C(═O)—, —O—S(═O)—, —O—S(═O)2—, —O—C(═O)—NRa—, —O—S(═O)—NRa—, —O—S(═O)2—NRa—. In certain embodiments, each instance of E3 is independently a first reactive moiety.
In another aspect, the present disclosure provides a second modified affinity peptide, wherein n1 instances of the amino acid residues of the affinity peptide are independently modified with a moiety of Formula B′:
-L1-E2-L2-(E34-L3-M)n3 (B′).
In certain embodiments, each instance of M is independently a radical of a pharmaceutical agent or absent.
In another aspect, the present disclosure provides an antibody-pharmaceutical agent conjugate, wherein n2 instances of the lysine residues of the antibody are independently modified with a moiety of Formula C′:
—(CH2)4-E12-L2-(E34-L3-M)n3 (C′).
In another aspect, the present disclosure provides a composition comprising:
In another aspect, the present disclosure provides a kit comprising:
The first modified affinity peptide may be useful in preparing the second modified affinity peptide. The second modified affinity peptide (e.g., the affinity peptide moiety of the second modified affinity peptide) may bind the antibody (e.g., the heavy chain of the antibody). E2 of the second modified affinity peptide may get close to, and as a result, react with, a lysine residue of the antibody. The reaction may occur under physiological conditions. The reaction may occur in a subject (e.g., a human). An antibody-pharmaceutical agent conjugate may form after the reaction. The antibody-pharmaceutical agent conjugate may not comprise the affinity peptide moiety.
The antibody-pharmaceutical agent conjugate may be useful for delivering the pharmaceutical agent to a subject, cell, tissue, or biological sample. In other aspects, the present disclosure provides a method comprising administering to a subject in need thereof an effective amount of a first modified affinity peptide, a second modified affinity peptide, an antibody-pharmaceutical agent conjugate, or a composition. In another aspect, the present disclosure provides a method comprising contacting a cell, tissue, or biological sample with a first modified affinity peptide, second modified affinity peptide, antibody-pharmaceutical agent conjugate, or a composition. In certain embodiments, the subject is in need of treatment, prevention, or diagnosis of a disease. The antibody-pharmaceutical agent conjugate may be useful for treating, preventing, or diagnosing a disease in a subject in need thereof. The antibody-pharmaceutical agent conjugate may be advantageous over the pharmaceutical agent because the former may show higher distribution, potency, efficacy, bioavailability, safety, and/or subject compliance; lower clearance; wider therapeutic window; fewer and/or less severe side effects; and/or lower toxicity and/or resistance to treatment than the latter.
In one embodiment of any one of the peptides or conjugates provided herein, the distance between an electrophile and a target of interest is about 10 Angstroms.
In one embodiment of any one of the peptides or conjugates provided herein, the administration is in vitro or in vivo. In one embodiment of any one of the peptides or conjugates provided herein, the “painting” or conjugation to a target occurs in vivo after the administration of the any one of the peptides or conjugates provided herein.
The details of one or more embodiments of the disclosure are set forth herein. Other features, objects, and advantages of the disclosure will be apparent from the Detailed Description, Examples, Figures, and Claims.
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March's Advanced Organic Chemistry, 7th Edition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
Compounds (e.g., the first modified affinity peptides, second modified affinity peptides, antibody-pharmaceutical agent conjugates) described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. For example, in some embodiments, the compounds described herein are in the form of an individual enantiomer, diastereomer or geometric isomer, or are in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. In some embodiments, isomers are isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers are prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The disclosure additionally encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
In a formula, the bond is a single bond, the dashed line
is a single bond or absent, and the bond
or
is a single or double bond.
Unless otherwise provided, formulae and structures depicted herein include compounds that do not include isotopically enriched atoms, and also include compounds that include isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 19F with 18F, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.
The term “isotopes” refers to variants of a particular chemical element such that, while all isotopes of a given element share the same number of protons in each atom of the element, those isotopes differ in the number of neutrons.
When a range of values (“range”) is listed, it encompasses each value and sub-range within the range. A range is inclusive of the values at the two ends of the range unless otherwise provided. For example “C1-6 alkyl” encompasses, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term “heteroaliphatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.
The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 200 carbon atoms (“C1-200 alkyl”). In some embodiments, an alkyl group has 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tert-amyl), and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), n-dodecyl (C12), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as fluorine). In certain embodiments, the alkyl group is an unsubstituted C1-12 alkyl (such as unsubstituted C1-6 alkyl, e.g., —CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C1-12 alkyl (such as substituted C1-6 alkyl, e.g., —CH2F, —CHF2, —CF3, —CH2CH2F, —CH2CHF2, —CH2CF3, or benzyl (Bn)).
The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, sulfur, and phosphorous within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 200 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-200 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-20 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-12 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 11 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-11 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-10 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-9 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-8 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-7 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC1-6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC1-5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC1-4 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC1-3 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC1-2 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC1 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC2-6 alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents (e.g., oxo, substituted or unsubstituted C1-6 alkyl (e.g., —CH3)). In certain embodiments, the heteroalkyl group is an unsubstituted heteroC1-12 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC1-12 alkyl. In some embodiments, unsubstituted heteroC1 alkyl is —OCH3 or —CH2OH. In some embodiments, substituted heteroC1 alkyl is —C(═O)NH2. In some embodiments, unsubstituted heteroC2 alkyl is —OCH2CH3, —CH2OCH3, or —CH2CH2OH. The terms “heteroCz1-z2 alkyl” and “Cz1-z2 heteroalkyl” are used interchangeably, wherein each of z1 and z2 is independently an integer.
The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 200 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 1 to 200 carbon atoms (“C1-200 alkenyl”). In some embodiments, an alkenyl group has at least 2 carbon atoms. In some embodiments, an alkenyl group has 1 to 20 carbon atoms (“C1-20 alkenyl”). In some embodiments, an alkenyl group has 1 to 12 carbon atoms (“C1-12 alkenyl”). In some embodiments, an alkenyl group has 1 to 11 carbon atoms (“C1-11 alkenyl”). In some embodiments, an alkenyl group has 1 to 10 carbon atoms (“C1-10 alkenyl”). In some embodiments, an alkenyl group has 1 to 9 carbon atoms (“C1-9 alkenyl”). In some embodiments, an alkenyl group has 1 to 8 carbon atoms (“C1-8 alkenyl”). In some embodiments, an alkenyl group has 1 to 7 carbon atoms (“C1-7 alkenyl”). In some embodiments, an alkenyl group has 1 to 6 carbon atoms (“C1-6 alkenyl”). In some embodiments, an alkenyl group has 1 to 5 carbon atoms (“C1-5 alkenyl”). In some embodiments, an alkenyl group has 1 to 4 carbon atoms (“C1-4 alkenyl”). In some 26 embodiments, an alkenyl group has 1 to 3 carbon atoms (“C1-3 alkenyl”). In some embodiments, an alkenyl group has 1 to 2 carbon atoms (“C1-2 alkenyl”). In some 28 embodiments, an alkenyl group has 1 carbon atom (“C1 alkenyl”). In certain embodiments, an alkenyl group is C2-3 alkenyl, C2-4 alkenyl, C2-5 alkenyl, C2-6 alkenyl, C2-7 alkenyl, C2-8 alkenyl, C2-9 alkenyl, C2-10 alkenyl, C2-12 alkenyl, C2-16 alkenyl, C2-20 alkenyl, C2-30 alkenyl, C2-40 alkenyl, C2-50 alkenyl, C2-60 alkenyl, C2-70 alkenyl, C2-80 alkenyl, C2-90 alkenyl, or C2-100 alkenyl. The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C1-4 alkenyl groups include methylidenyl (C1), ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C1-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C1-20 alkenyl. In certain embodiments, the alkenyl group is a substituted C1-20 alkenyl. In an alkenyl group, a C═C double bond for which the stereochemistry is not specified (e.g., —CH═CHCH3 or
may be in the (E)- or (Z)-configuration.
The term “heteroalkenyl” refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, sulfur, and phosphorous within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 200 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-200 alkenyl”). In some embodiments, a heteroalkenyl group has at least 2 carbon atoms. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-20 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 12 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-12 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 11 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-11 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-10 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-9 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-8 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-7 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC1-6 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-5 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-4 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC1-3 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 2 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC1-2 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-6 alkenyl”). In certain embodiments, a heteroalkenyl group is C2-3 heteroalkenyl, C2-4 heteroalkenyl, C2-5 heteroalkenyl, C2-6 heteroalkenyl, C2-7 heteroalkenyl, C2-8 heteroalkenyl, C2-9 heteroalkenyl, C2-10 heteroalkenyl, C2-12 heteroalkenyl, C2-16 heteroalkenyl, C2-20 heteroalkenyl, C2-30 heteroalkenyl, C2-40 heteroalkenyl, C2-50 heteroalkenyl, C2-60 heteroalkenyl, C2-70 heteroalkenyl, C2-80 heteroalkenyl, C2-90 heteroalkenyl, or C2-100 heteroalkenyl. Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents (e.g., oxo, substituted or unsubstituted C1-6 alkyl (e.g., —CH3)). In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC1-20 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC1-20 alkenyl. In some embodiments, unsubstituted heteroC1 alkenyl is —CH═NH or ═N—CH3. The terms “heteroCz1-z2 alkenyl” and “Cz1-z2 heteroalkenyl” are used interchangeably, wherein each of z1 and z2 is independently an integer.
The term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 200 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C1-200 alkynyl”). In some embodiments, an alkynyl group has 1 to 20 carbon atoms (“C1-20 alkynyl”). In some embodiments, an alkynyl group has at least 2 carbon atoms. In some embodiments, an alkynyl group has 1 to 10 carbon atoms (“C1-10 alkynyl”). In some embodiments, an alkynyl group has 1 to 9 carbon atoms (“C1-9 alkynyl”). In some embodiments, an alkynyl group has 1 to 8 carbon atoms (“C1-8 alkynyl”). In some embodiments, an alkynyl group has 1 to 7 carbon atoms (“C1-7 alkynyl”). In some embodiments, an alkynyl group has 1 to 6 carbon atoms (“C1-6 alkynyl”). In some embodiments, an alkynyl group has 1 to 5 carbon atoms (“C1-5 alkynyl”). In some embodiments, an alkynyl group has 1 to 4 carbon atoms (“C1-4 alkynyl”). In some embodiments, an alkynyl group has 1 to 3 carbon atoms (“C1-3 alkynyl”). In some embodiments, an alkynyl group has 1 to 2 carbon atoms (“C1-2 alkynyl”). In some embodiments, an alkynyl group has 1 carbon atom (“C1 alkynyl”). In certain embodiments, an alkynyl group is C2-3 alkynyl, C2-4 alkynyl, C2-5 alkynyl, C2-6 alkynyl, C2-7 alkynyl, C2-8 alkynyl, C2-9 alkynyl, C2-10 alkynyl, C2-12 alkynyl, C2-16 alkynyl, C2-20 alkynyl, C2-30 alkynyl, C2-40 alkynyl, C2-50 alkynyl, C2-60 alkynyl, C2-70 alkynyl, C2-80 alkynyl, C2-90 alkynyl, or C2-100 alkynyl. The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C1-4 alkynyl groups include, without limitation, methylidynyl (C1), ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C1-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C1-20 alkynyl. In certain embodiments, the alkynyl group is a substituted C1-20 alkynyl.
The term “heteroalkynyl” refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, sulfur, and phosphorous within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 200 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-200 alkynyl”). In some embodiments, a heteroalkynyl group has at least 2 carbon atoms. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-20 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-10 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-9 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-8 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-7 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC1-6 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-5 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-4 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC1-3 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 2 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC1-2 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC1-6 alkynyl”). In certain embodiments, a heteroalkynyl group is C2-3 heteroalkynyl, C2-4 heteroalkynyl, C2-5 heteroalkynyl, C2-6 heteroalkynyl, C2-7 heteroalkynyl, C2-8 heteroalkynyl, C2-9 heteroalkynyl, C2-10 heteroalkynyl, C2-12 heteroalkynyl, C2-16 heteroalkynyl, C2-20 heteroalkynyl, C2-30 heteroalkynyl, C2-40 heteroalkynyl, C2-50 heteroalkynyl, C2-60 heteroalkynyl, C2-70 heteroalkynyl, C2-80 heteroalkynyl, C2-90 heteroalkynyl, or C2-100 heteroalkynyl. Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents (e.g., oxo, substituted or unsubstituted C1-6 alkyl (e.g., —CH3)). In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC1-20 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC1-20 alkynyl. In some embodiments, unsubstituted heteroC1 alkynyl is —C═N. The terms “heteroCz1-z2 alkynyl” and “Cz1-z2 heteroalkynyl” are used interchangeably, wherein each of z1 and z2 is independently an integer.
The term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 13 ring carbon atoms (“C3-13 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 12 ring carbon atoms (“C3-12 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 11 ring carbon atoms (“C3-11 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. Exemplary C3-8 carbocyclyl groups include the aforementioned C3-10 carbocyclyl groups as well as cycloundecyl (C11), spiro[5.5]undecanyl (C11), cyclododecyl (C12), cyclododecenyl (C12), cyclotridecane (C13), cyclotetradecane (C14), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and is saturated or contains one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C3-14 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-14 carbocyclyl.
In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C3-14 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-14 cycloalkyl. In certain embodiments, the carbocyclyl includes 0, 1, or 2 C═C double bonds in the carbocyclic ring system, as valency permits.
The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In some embodiments, in heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment is a carbon or nitrogen atom, as valency permits. In some embodiments, a heterocyclyl group is either monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and is either saturated or contains one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits.
In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetra-hydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.
The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring 12 systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14 aryl.
“Aralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.
The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In some embodiments, in heteroaryl groups that contain one or more nitrogen atoms, the point of attachment is a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. In some embodiments, in polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment is on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In certain embodiments, the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In certain embodiments, the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.
In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each 24 instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.
Exemplary 5-membered heteroaryl groups containing 1 heteroatom include pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.
“Heteroaralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety.
The term “unsaturated bond” refers to a double or triple bond.
The term “unsaturated” or “partially unsaturated” refers to a moiety that includes at least one double or triple bond.
The term “saturated” or “fully saturated” refers to a moiety that does not contain a double or triple bond, e.g., the moiety only contains single bonds.
Affixing the suffix “-ene” to a group indicates the group is a polyvalent (e.g., divalent, trivalent, tetravalent) moiety as valency permits, e.g., alkylene is the polyvalent moiety of alkyl, alkenylene is the polyvalent moiety of alkenyl, alkynylene is the polyvalent moiety of alkynyl, heteroalkylene is the polyvalent moiety of heteroalkyl, heteroalkenylene is the polyvalent moiety of heteroalkenyl, heteroalkynylene is the polyvalent moiety of heteroalkynyl, carbocyclylene is the polyvalent moiety of carbocyclyl, heterocyclylene is the polyvalent moiety of heterocyclyl, arylene is the polyvalent moiety of aryl, and heteroarylene is the polyvalent moiety of heteroaryl. In certain embodiments, affixing the suffix “-ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl. In certain embodiments, the length of a polyvalent moiety is the smallest number of backbone atoms between any two attachment points.
A group is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. “Optionally substituted” refers to a group which is substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds and includes any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. The disclosure is not limited in any manner by the exemplary substituents described herein.
Exemplary carbon atom substituents include halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —ON(Rbb)2, —N(Rbb)2, —N(Rbb)3+X−, —N(ORcc)Rbb, —SH, —SRaa, —SSRcc, —C(═O)Raa, —CO2H, —CHO, —C(ORcc)2, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —OC(═NRbb)Raa, —OC(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —OC(═NRbb)N(Rbb)2, —NRbbC(═NRbb)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, —SO2ORaa, —OSO2Raa, —S(═O)Raa, —OS(═O)Raa, —Si(Raa)3, —OSi(Raa)3—C(═S)N(Rbb)2, —C(═O)SRaa, —C(═S)SRaa, —SC(═S)SRaa, —SC(═O)SRaa, —OC(═O)SRaa, —SC(═O)ORaa, —SC(═O)Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —OP(═O)(Raa)2, —OP(═O)(ORcc)2, —P(═O)(N(Rbb)2)2, —OP(═O)(N(Rbb)2)2, —NRbbP(═O)(Raa)2, —NRbbP(═O)(ORcc)2, —NRbbP(═O)(N(Rbb)2)2, —P(Rcc)2, —P(ORcc)2, —P(Rcc)3+X−, —P(ORcc)3+X−, —P(Rcc)4, —P(ORcc)4, —OP(Rcc)2, —OP(Rcc)3+X−, —OP(ORcc)2, —OP(ORcc)3+X−, —OP(Rcc)4, —OP(ORcc)4, —B(Raa)2, —B(ORcc)2, —BRaa(ORcc), C1-20 alkyl, C1-20 perhaloalkyl, C1-20 alkenyl, C1-20 alkynyl, heteroC1-20 alkyl, heteroC1-20 alkenyl, heteroC1-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, —ORaa, —SRaa, —N(Rbb)2, —CN, —SCN, —NO2, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —OC(═O)Raa, —OCO2Raa, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, or —NRbbC(═O)N(Rbb)2. In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, —ORaa, —SRaa, —N(Rbb)2, —CN, —SCN, —NO2, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —OC(═O)Raa, —OCO2Raa, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, or —NRbbC(═O)N(Rbb)2, wherein Raa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each Rbb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, —ORaa, —SRaa, —N(Rbb)2, —CN, —SCN, or —NO2. In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen moieties) or unsubstituted C1-10 alkyl, —ORaa, —SRaa, —N(Rbb)2, —CN, —SCN, or —NO2, wherein Raa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each Rbb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts).
The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).
The term “hydroxyl” or “hydroxy” refers to the group —OH. The term “substituted hydroxyl” or “substituted hydroxyl,” by extension, refers to a hydroxyl group wherein the oxygen atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from —ORaa, —ON(Rbb)2, —OC(═O)SRaa, —OC(═O)Raa, —OCO2Raa, —OC(═O)N(Rbb)2, —OC(═NRbb)Raa, —OC(═NRbb)ORaa, —OC(═NRbb)N(Rbb)2, —OS(═O)Raa, —OSO2Raa, —OSi(Raa)3, —OP(Rcc)2, —OP(Rcc)3+X−, —OP(ORcc)2, —OP(ORcc)3+X−, —OP(═O)(Raa)2, —OP(═O)(ORcc)2, and —OP(═O)(N(Rbb))2, wherein X−, Raa, Rbb, and Rcc are as defined herein.
The term “thiol” or “thio” refers to the group —SH. The term “substituted thiol” or “substituted thio,” by extension, refers to a thiol group wherein the sulfur atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from —SRaa, —S═SRcc, —SC(═S)SRaa, —SC(═S)ORaa, —SC(═S)N(Rbb)2, —SC(═O)SRaa, —SC(═O)ORaa, —SC(═O)N(Rbb)2, and —SC(═O)Raa, wherein Raa and Rcc are as defined herein.
The term “amino” refers to the group —NH2. The term “substituted amino,” by extension, refers to a monosubstituted amino, a disubstituted amino, or a trisubstituted amino. In certain embodiments, the “substituted amino” is a monosubstituted amino or a disubstituted amino group.
The term “monosubstituted amino” refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with one hydrogen and one group other than hydrogen, and includes groups selected from —NH(Rbb), —NHC(═O)Raa, —NHCO2Raa, —NHC(═O)N(Rbb)2, —NHC(═NRbb)N(Rbb)2, —NHSO2Raa, —NHP(═O)(OR)2, and —NHP(═O)(N(Rbb)2)2, wherein Raa, Rbb and Rcc are as defined herein, and wherein Rbb of the group —NH(Rbb) is not hydrogen.
The term “disubstituted amino” refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with two groups other than hydrogen, and includes groups selected from —N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —NRbbC(═NRbb)N(Rbb)2, —NRbbSO2Raa, —NRbbP(═O)(ORcc)2, and —NRbbP(═O)(N(Rbb)2)2, wherein Raa, Rbb, and Rec are as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not substituted with hydrogen.
The term “trisubstituted amino” refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with three groups, and includes groups selected from —N(Rbb)3 and —N(Rbb)3+X−, wherein Rbb and X− are as defined herein.
The term “sulfonyl” refers to a group selected from —SO2N(Rbb)2, —SO2Raa, and —SO2ORaa, wherein Raa and Rbb are as defined herein.
The term “sulfinyl” refers to the group —S(═O)Raa, wherein Raa is as defined herein.
The term “acyl” refers to a group having the general formula —C(═O)Raa, —C(═O)ORaa, —C(═O)—O—C(═O)Raa, —C(═O)SRaa, —C(═O)N(Rbb)2, —C(═S)Raa, —C(═S)N(Rbb)2, and —C(═S)S(Raa), —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)SRaa, and —C(═NRbb)N(Rbb)2, wherein Raa and Rbb are as defined herein. In some embodiments, the term “acyl” refers to a group having the general formula-C(═O)Raa, —C(═O)ORaa, —C(═O)—O—C(═O)Raa, —C(═O)SRaa, or —C(═O)N(Rbb)2.
The term “carbonyl” refers to a group wherein the carbon directly attached to the parent molecule is sp2 hybridized, and is substituted with an oxygen, nitrogen or sulfur atom, e.g., a group selected from ketones (—C(═O)Raa), carboxylic acids (—CO2H), aldehydes (—CHO), esters (—CO2Raa, —C(═O)SRaa, —C(═S)SRaa), amides (—C(═O)N(Rbb)2, —C(═O)NRbbSO2Raa, —C(═S)N(Rbb)2), and imines (—C(═NRbb)Raa, —C(═NRbb)ORaa), —C(═NRbb)N(Rbb)2), wherein Raa and Rbb are as defined herein.
The term “silyl” refers to the group —Si(Raa)3, wherein Raa is as defined herein.
Nitrogen atoms are substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)(ORcc)2, —P(═O)(Raa)2, —P(═O)(N(Rcc)2)2, C1-20 alkyl, C1-20 perhaloalkyl, C1-20 alkenyl, C1-20 alkynyl, hetero C1-20 alkyl, hetero C1-20 alkenyl, hetero C1-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.
In certain embodiments, each nitrogen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, or a nitrogen protecting group. In certain embodiments, each nitrogen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, or a nitrogen protecting group, wherein Raa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or an oxygen protecting group when attached to an oxygen atom; and each Rbb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group. In certain embodiments, each nitrogen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl or a nitrogen protecting group.
In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an “amino protecting group”). Nitrogen protecting groups include —OH, —ORaa, —N(Rcc)2, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRcc)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C1-20 alkenyl, C1-20 alkynyl, hetero C1-20 alkyl, hetero C1-20 alkenyl, hetero C1-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
For example, in certain embodiments, at least one nitrogen protecting group is an amide group (e.g., a moiety that include the nitrogen atom to which the nitrogen protecting groups (e.g., —C(═O)Raa) is directly attached). In certain such embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.
In certain embodiments, at least one nitrogen protecting group is a carbamate group (e.g., a moiety that include the nitrogen atom to which the nitrogen protecting groups (e.g., —C(═O)ORaa) is directly attached). In certain such embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.
In certain embodiments, at least one nitrogen protecting group is a sulfonamide group (e.g., a moiety that include the nitrogen atom to which the nitrogen protecting groups (e.g., —S(═O)2Raa) is directly attached). In certain such embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
In certain embodiments, each nitrogen protecting group, together with the nitrogen atom to which the nitrogen protecting group is attached, is independently selected from the group consisting of phenothiazinyl-(10)-acyl derivatives, N′-p-toluenesulfonylaminoacyl derivatives, N′-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys). In some embodiments, two instances of a nitrogen protecting group together with the nitrogen atoms to which the nitrogen protecting groups are attached are N,N′-isopropylidenediamine.
In certain embodiments, at least one nitrogen protecting group is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts.
In certain embodiments, each oxygen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, or an oxygen protecting group. In certain embodiments, each oxygen atom substituents is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, or an oxygen protecting group, wherein Raa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or an oxygen protecting group when attached to an oxygen atom; and each Rbb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group. In certain embodiments, each oxygen atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl or an oxygen protecting group.
In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”). Oxygen protecting groups include —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3+X−, —P(ORcc)2, —P(ORcc)3+X−, —P(═O)(Raa)2, —P(═O)(ORcc)2, and —P(═O)(N(Rbb)2)2, wherein X−, Raa, Rbb, and Rcc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
In certain embodiments, each oxygen protecting group, together with the oxygen atom to which the oxygen protecting group is attached, is selected from the group consisting of methoxy, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 4,4′-Dimethoxy-3″′-[N-(imidazolylmethyl)]trityl Ether (IDTr-OR), 4,4′-Dimethoxy-3″′-[N-(imidazolylethyl)carbamoyl]trityl Ether (IETr-OR), 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio) pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), 16 isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl carbonate (MTMEC-OR), 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl) phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
In certain embodiments, at least one oxygen protecting group is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl.
In certain embodiments, each sulfur atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, or a sulfur protecting group. In certain embodiments, each sulfur atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, or a sulfur protecting group, wherein Raa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or an oxygen protecting group when attached to an oxygen atom; and each Rbb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-10 alkyl, or a nitrogen protecting group. In certain embodiments, each sulfur atom substituent is independently substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl or a sulfur protecting group.
In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”). In some embodiments, each sulfur protecting group is selected from the group consisting of —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3+X−, —P(ORcc)2, —P(ORcc)3+X−, —P(═O)(Raa)2, —P(═O)(ORcc)2, and —P(═O)(N(Rbb)2)2, wherein Raa, Rbb, and Rcc are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
In certain embodiments, the molecular weight of a substituent is lower than 250, lower than 200, lower than 150, lower than 100, or lower than 50 g/mol. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, nitrogen, and/or silicon atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, and/or nitrogen atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, and/or iodine atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, and/or chlorine atoms. In certain embodiments, a substituent comprises 0, 1, 2, or 3 hydrogen bond donors. In certain embodiments, a substituent comprises 0, 1, 2, or 3 hydrogen bond acceptors.
A “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. In some embodiments, an anionic counterion is monovalent (e.g., including one formal negative charge). An anionic counterion may also be multivalent (e.g., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F−, Cl−, Br−, I−), NO3−, ClO4−, OH−, H2PO4−, HCO3−, HSO4−, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF4−, PF4−, PF6−, AsF6−, SbF6−, B[3,5-(CF3)2C6H3]4]−, B(C6F5)4−, BPh4−, Al(OC(CF3)3)4−, and carborane anions (e.g., CB11H12− or (HCB11Me5Br6)−). Exemplary counterions which may be multivalent include CO32−, HPO42−, PO43−, B4O72−, SO42−, S2O32−, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.
Use of the phrase “at least one instance” refers to 1, 2, 3, 4, or more instances, but also encompasses a range, as valency permits (e.g., from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive). In certain embodiments, “at least one instance” refers to each instance. When “at least one instance” refers to at least two instances, the at least two instances may be the same as, or different from, each other.
The term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts. Salts include ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge). Salts of the compounds of this disclosure include those derived from inorganic and organic acids and bases. Examples of acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, hippurate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C1-4 alkyl)4− salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
The term “leaving group” is given its ordinary meaning in the art of synthetic organic chemistry and refers to an atom or a group capable of being displaced by a nucleophile. Examples of suitable leaving groups include halogen (such as F, Cl, Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, and haloformates. In some cases, the leaving group is a sulfonic acid ester, such as toluenesulfonate (tosylate, —OTs), methanesulfonate (mesylate, —OMs), p-bromobenzenesulfonyloxy (brosylate, —OBs), —OS(═O)2(CF2)3CF3 (nonaflate, —ONf), or trifluoromethanesulfonate (triflate, —OTf). In some cases, the leaving group is a brosylate, such as p-bromobenzenesulfonyloxy. In some cases, the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy. In some embodiments, the leaving group is a sulfonate-containing group. In some embodiments, the leaving group is a tosylate group. The leaving group may also be a phosphineoxide (e.g., formed during a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate. Other examples of leaving groups are water, ammonia, alcohols, ether moieties, thioether moieties, zinc halides, magnesium moieties, diazonium salts, and copper moieties.
The term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (i.e., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of a small molecule is not more than 2,000 g/mol. In certain embodiments, the molecular weight of a small molecule is not more than 1,500 g/mol. In certain embodiments, the molecular weight of a small molecule is not more than 1,000 g/mol, not more than 900 g/mol, not more than 800 g/mol, not more than 700 g/mol, not more than 600 g/mol, not more than 500 g/mol, not more than 400 g/mol, not more than 300 g/mol, not more than 200 g/mol, or not more than 100 g/mol. In certain embodiments, the molecular weight of a small molecule is at least 100 g/mol, at least 200 g/mol, at least 300 g/mol, at least 400 g/mol, at least 500 g/mol, at least 600 g/mol, at least 700 g/mol, at least 800 g/mol, or at least 900 g/mol, or at least 1,000 g/mol. Combinations of the above ranges (e.g., at least 200 g/mol and not more than 500 g/mol) are also possible. In certain embodiments, the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (C.F.R.)). The small molecule may also be complexed with one or more metal atoms and/or metal ions. In this instance, the small molecule is also referred to as a “small organometallic molecule.” Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include radionuclides and imaging agents. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361, and 440 through 460; drugs for veterinary use are listed by the FDA under 21 C.F.R. §§ 500 through 589. All listed drugs are considered acceptable for use in accordance with the present disclosure.
The term “peptide,” “polypeptide,” or “protein” refers to an oligomer or polymer of amino acid residues covalently connected together by peptide bonds. A peptide, polypeptide, or protein may be of any size, structure, and function, and may be an individual peptide, polypeptide, or protein, or a collection (e.g., a complex) of peptides, polypeptides, and proteins, and optionally small molecules and/or metal ions. In certain embodiments, a peptide comprises between 2 and 10, between 11 and 20, between 21 and 30, between 31 and 40, or between 41 and 50, inclusive, amino acid residues. In certain embodiments, a polypeptide or protein comprises between 51 and 100, between 101 and 200, between 201 and 300, between 301 and 500, between 501 and 1,000, between 1,001 and 3,000, between 3,001 and 10,000, or between 10,001 and 30,000, inclusive, amino acid residues. A peptide, polypeptide, or protein may contain only natural amino acids but no non-natural amino acids; only non-natural amino acids but no natural amino acids; or both natural and non-natural amino acids. A peptide, polypeptide, or protein may contain amino acid analogs only or in addition to natural and/or non-natural amino acids. In certain embodiments, the amino acid residues of a peptide, polypeptide, or protein are residues of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and/or valine, in D and/or L form (e.g., in L form). One or more of the amino acid residues in a peptide, polypeptide, or protein may be alpha amino acid residues or homologs thereof (e.g., beta amino acid residues). One or more of the amino acid residues in a peptide, polypeptide, or protein May be protected or unprotected. One or more (e.g., two) of the termini of a peptide, polypeptide, or protein may be protected (e.g., to form an ester or amide) or unprotected (e.g., as —NH2, —NH3+, —C(═O) OH, or —C(═O)O−). One or more of the amino acid residues in a peptide, polypeptide, or protein may be modified or unmodified. A modification to an amino acid residue in a peptide, polypeptide, or protein may be an addition of a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, or a linker for conjugation or functionalization. A peptide, polypeptide, or protein may be naturally occurring, recombinant, synthetic, or a combination thereof. A peptide, polypeptide, or protein may be a fragment of a naturally occurring peptide, polypeptide, or protein.
The term “affinity peptide” refers to a peptide that binds preferentially to an antibody than to another protein. The affinity peptide may bind preferentially to the heavy chain of the antibody than to a light chain of the antibody. The preferential binding may be at least 2-fold, at least 5-fold, at least 10-fold, at least 30-fold, at least 100-fold, at least 1000-fold, or at least 10000-fold, inclusive, preferential binding. The binding may be under physiological conditions. The affinity peptide may be an unmodified peptide. The affinity peptide moiety of a modified affinity peptide is the affinity peptide radical without the modification.
The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. The antisense oligonuculeotide may comprise a modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, a thio-guanine, and 2,6-diaminopurine. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNAs) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing carbohydrate or lipids. Exemplary DNAs include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), plasmid DNA (pDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, and viral satellite RNA.
The terms “composition” and “formulation” are used interchangeably.
A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. In some embodiments, the non-human animal is a male or female at any stage of development. In some embodiments, the non-human animal is a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease.
The term “tissue” refers to any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and/or lymph vessels. In some embodiments, “tissue” is the object to which a compound, and/or composition of the disclosure is delivered. In some embodiments, a tissue is an abnormal or unhealthy tissue, which may need to be treated. A tissue may also be a normal or healthy tissue that is under a higher than normal risk of becoming abnormal or unhealthy, which may need to be prevented.
The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample.
The term “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound provided herein, a compound useful in a provided method, or a pharmaceutical composition provided herein, in or on a subject.
The terms “condition,” “disease,” and “disorder” are used interchangeably.
The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment is administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment is administered in the absence of signs or symptoms of the disease. For example, in some embodiments, treatment is administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.
An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular compound, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting the activity and/or production of a GSK3. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating a disease. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting the activity and/or production of a GSK3 and treating a disease.
A “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting the activity and/or production of a GSK3. In certain embodiments, a prophylactically effective amount is an amount sufficient for preventing a disease. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting the activity and/or production of a GSK3 and preventing a disease.
The term “genetic disease” refers to a disease caused by one or more abnormalities in the genome of a subject, such as a disease that is present from birth of the subject. Genetic diseases may be heritable and may be passed down from the parents' genes. A genetic disease may also be caused by mutations or changes of the DNAs and/or RNAs of the subject. In such cases, the genetic disease will be heritable if it occurs in the germline. Exemplary genetic diseases include Aarskog-Scott syndrome, Aase syndrome, achondroplasia, acrodysostosis, addiction, adreno-leukodystrophy, albinism, ablepharon-macrostomia syndrome, alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency, Alport's syndrome, Alzheimer's disease, asthma, autoimmune polyglandular syndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia telangiectasia, atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, baldness, Batten disease, Beckwith-Wiedemann syndrome, Best disease, bipolar disorder, brachydactyl), breast cancer, Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease, Crohn's disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer, congenital adrenal hyperplasia, Cornelia de Lange syndrome, Costello syndrome, Cowden syndrome, craniofrontonasal dysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob disease, cystic fibrosis, deafness, depression, diabetes, diastrophic dysplasia, DiGeorge syndrome, Down's syndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz syndrome, ectodermal dysplasia Ellis-van Creveld syndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Friedreich's ataxia, Gaucher's disease, glaucoma, glucose galactose malabsorption, glutaricaciduria, gyrate atrophy, Goldberg Shprintzen syndrome (velocardiofacial syndrome), Gorlin syndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis, hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditary non polyposis colorectal cancer (HNPCC), Huntington's disease, immunodeficiency with hyper-IgM, juvenile onset diabetes, Klinefelter's syndrome, Kabuki syndrome, Leigh's disease, long QT syndrome, lung cancer, malignant melanoma, manic depression, Marfan syndrome, Menkes syndrome, miscarriage, mucopolysaccharide disease, multiple endocrine neoplasia, multiple sclerosis, muscular dystrophy, myotrophic lateral sclerosis, myotonic dystrophy, neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Pendred syndrome, peroneal muscular atrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer, REAR syndrome, Refsum disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia, severe combined immunodeficiency, sickle cell anemia, spina bifida, spinal muscular atrophy, spinocerebellar atrophy, sudden adult death syndrome, Tangier disease, Tay-Sachs disease, thrombocytopenia absent radius syndrome, Townes-Brocks syndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, von Hippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome, Werner syndrome, Williams syndrome, Wilson's disease, xeroderma piginentosum, and Zellweger syndrome.
A “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.
The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and/or is associated with a disease.
The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is 10 located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.
The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. The cancer may be a solid tumor. The cancer may be a hematological malignancy. Exemplary cancers include acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenström's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous 16 panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).
The term “inflammatory disease” refers to a disease caused by, resulting from, or resulting in inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and/or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. Inflammatory diseases include atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, dermatitis (e.g., stasis dermatitis, allergic contact dermatitis, atopic dermatitis, irritant contact dermatitis, neurodermatitis perioral dermatitis, seborrheic dermatitis), hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hayfever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, necrotizing enterocolitis, inflammatory rosacea. An ocular inflammatory disease includes post-surgical inflammation.
An “autoimmune disease” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture's disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.
A “hematological disease” includes a disease which affects a hematopoietic cell or tissue. Hematological diseases include diseases associated with aberrant hematological content and/or function. Examples of hematological diseases include diseases resulting from bone marrow irradiation or chemotherapy treatments for cancer, diseases such as pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HTV, hepatitis virus or other viruses, myelophthisic anemias caused by marrow deficiencies, renal failure resulting from anemia, anemia, polycythemia, infectious mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, Wilm's tumor, Ewing's sarcoma, retinoblastoma, hemophilia, disorders associated with an increased risk of thrombosis, herpes, thalassemia, antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical trauma to red blood cells such as micro-angiopathic hemolytic anemias, thrombotic thrombocytopeniaurpura and disseminated intravascular coagulation, infections by parasites such as Plasmodium, chemical injuries from, e.g., lead poisoning, and hypersplenism.
The term “neurological disease” refers to any disease of the nervous system, including diseases that involve the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). Neurodegenerative diseases refer to a type of neurological disease marked by the loss of nerve cells, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), and Huntington's disease. Examples of neurological diseases include headache, stupor and coma, dementia, seizure, sleep disorders, trauma, infections, neoplasms, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular junctions. Addiction and mental illness, include bipolar disorder and schizophrenia, are also included in the definition of neurological diseases. Further examples of neurological diseases include acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers' disease; alternating hemiplegia; Alzheimer's disease; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia telangiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet's disease; Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome (CTS); causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's syndrome; cytomegalic inclusion body disease (CIBD); cytomegalovirus infection; dancing eyes-dancing feet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier's syndrome; Dejerine-Klumpke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia; frontotemporal dementia and other “tauopathies”; Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV-1 associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactica polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated dementia and neuropathy (see also neurological manifestations of AIDS); holoprosencephaly; Huntington's disease and other polyglutamine repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti; infantile; phytanic acid storage disease; Infantile Refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease; Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; lissencephaly; locked-in syndrome; Lou Gehrig's disease (aka motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; lyme disease-neurological sequelae; Machado-Joseph disease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motor neurone disease; moyamoya disease; mucopolysaccharidoses; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Parkinson's disease; paramyotonia congenita; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; Post-Polio syndrome; postherpetic neuralgia (PHN); postinfectious encephalomyelitis; postural hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive; hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (Type I and Type II); Rasmussen's Encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Saint Vitus Dance; Sandhoff disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; stiff-person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; tic douloureux; Todd's paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau Disease (VHL); Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wilson's disease; and Zellweger syndrome.
A “painful condition” includes neuropathic pain (e.g., peripheral neuropathic pain), central pain, deafferentiation pain, chronic pain (e.g., chronic nociceptive pain, and other forms of chronic pain such as post-operative pain, e.g., pain arising after hip, knee, or other replacement surgery), pre-operative pain, stimulus of nociceptive receptors (nociceptive pain), acute pain (e.g., phantom and transient acute pain), noninflammatory pain, inflammatory pain, pain associated with cancer, wound pain, burn pain, postoperative pain, pain associated with medical procedures, pain resulting from pruritus, painful bladder syndrome, pain associated with premenstrual dysphoric disorder and/or premenstrual syndrome, pain associated with chronic fatigue syndrome, pain associated with pre-term labor, pain associated with withdrawl symptoms from drug addiction, joint pain, arthritic pain (e.g., pain associated with crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis or Reiter's arthritis), lumbosacral pain, musculo-skeletal pain, headache, migraine, muscle ache, lower back pain, neck pain, toothache, dental/maxillofacial pain, visceral pain and the like. One or more of the painful conditions contemplated herein can comprise mixtures of various types of pain provided above and herein (e.g. nociceptive pain, inflammatory pain, neuropathic pain, etc.). In some embodiments, a particular pain can dominate. In other embodiments, the painful condition comprises two or more types of pains without one dominating. A skilled clinician can determine the dosage to achieve a therapeutically effective amount for a particular subject based on the painful condition.
The term “metabolic disease” refers to any disorder that involves an alteration in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination thereof. A metabolic disorder is associated with either a deficiency or excess in a metabolic pathway resulting in an imbalance in metabolism of nucleic acids, proteins, lipids, and/or carbohydrates. Factors affecting metabolism include the endocrine (hormonal) control system (e.g., the insulin pathway, the enteroendocrine hormones including GLP-1, PYY or the like), the neural control system (e.g., GLP-1 in the brain), or the like. Examples of metabolic disorders include diabetes (e.g., Type I diabetes, Type II diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and obesity.
The term “psychiatric disorder” refers to a condition or disorder relating to the functioning of the brain and the cognitive processes or behavior. Psychiatric disorders may be further classified based on the type of neurological disturbance affecting the mental faculties. Psychiatric disorders are expressed primarily in abnormalities of thought, feeling, emotion, and/or behavior producing either distress or impairment of function (for example, impairment of mental function such with dementia or senility). The term “psychiatric disorder” is, accordingly, sometimes used interchangeably with the term “mental disorder” or the term “mental illness”. A psychiatric disorder is often characterized by a psychological or behavioral pattern that occurs in an individual and is thought to cause distress or disability that is not expected as part of normal development or culture. Definitions, assessments, and classifications of mental disorders can vary, but guideline criteria listed in the International Classification of Diseases and Related Health Problems (ICD, published by the World Health Organization, WHO), or the Diagnostic and Statistical Manual of Mental Disorders (DSM, published by the American Psychiatric Association, APA) and other manuals are widely accepted by mental health professionals. Individuals may be evaluated for various psychiatric disorders using criteria set forth in these and other publications accepted by medical practitioners in the field and the manifestation and severity of a psychiatric disorder may be determined in an individual using these publications. Categories of diagnoses in these schemes may include dissociative disorders, mood disorders, anxiety disorders, psychotic disorders, eating disorders, developmental disorders, personality disorders, and other categories. There are different categories of mental disorder, and many different facets of human behavior and personality that can become disordered. One group of psychiatric disorders includes disorders of thinking and cognition, such as schizophrenia and delirium. A second group of psychiatric disorders includes disorders of mood, such as affective disorders and anxiety. A third group of psychiatric disorders includes disorders of social behavior, such as character defects and personality disorders. And a fourth group of psychiatric disorders includes disorders of learning, memory, and intelligence, such as mental retardation and dementia. Accordingly, psychiatric disorders encompass schizophrenia, delirium, attention deficit disorder (ADD), schizoaffective disorder, depression (e.g., lithium-resistant depression), mania, attention deficit disorders, drug addiction, dementia, agitation, apathy, anxiety, psychoses, personality disorders, bipolar disorders, unipolar affective disorder, obsessive-compulsive disorders, eating disorders, post-traumatic stress disorders, irritability, adolescent conduct disorder and disinhibition.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.
A recent success for chemical modifications leading to long-acting peptide therapeutics concerns the glucagon-like peptide-1 agonist receptors (GLP1 RAs). The incretin hormone GLP1, a 37-amino acid peptide, binds the GLP1 receptor (GLP1-R) that regulates glucogenesis by inducing glucose-dependent insulin secretion. In vivo, native GLP1 possess a very short plasmatic half-life, about few minutes, being quickly degraded through the kidneys by dipeptidyl peptidase-4 (DPP-4) and neutral endopeptidase 24.11 (NEP 24.11). However, the stability and pharmacological effects of synthetic GLP1 can be significantly improved after modifying its amino-acid sequence and conjugation to half-life extension moieties, leading to the development of several top-selling peptide drugs for the treatment of type II diabetes Mellitus (TDM) and obesity. For example, long-acting Semaglutide (Ozempic®, Wegovy®, Rybelsus®), FDA-approved in 2017, shows a 7-day half-life with a duration of efficacy of 3 days, allowing its administration once a week (refs), while Dulaglutide (Trulicity®) is covalently fused to the Fc fragment of an IgG1, with extended drug efficacy and stability. However, the first generations of synthetic GLP1 agonists require extensive chemical or biological engineering, Fc fusion requiring humanization to reducing the risks for immunogenicity. New technologies are thereby needed to manufacture the next generation of long-acting GLP1 or other drugs.
The present disclosure provides, in one aspect, a platform to “paint” antibodies (e.g., native antibodies) with pharmaceutical agents (payloads, e.g., therapeutic agents), after in vivo administration, to use the circulating antibodies as a vehicle to extend the PK/PD of payloads. First, IgG-binder electrophile peptides derived from the structure of Z33 (an affinity peptide) were synthesized and their affinity towards human IgG1,2,4 and mouse IgG1,2a,2b investigated. It was discovered that these electrophile peptides possess a high affinity and selectivity towards IgG in mouse sera.
Further investigations using the affinity peptides highlighted the IgG-binder electrophilic peptides are capable of “painting” native antibodies (IgG1,2,4) directly inside the body of a subject, through the covalent transfer of either a small molecule, a radionuclide, or a bioactive long peptide. The transfer reaction is biocompatible, occurs without catalyzers, and/or does not require biological engineering or production of antibody-pharmaceutical agent conjugates beforehand as the payload is attached on native circulating IgGs that are already naturally produced by the organism. The ability of the antibody-pharmaceutical agent conjugates to extend the PK/PD of the pharmaceutical agent (e.g., GLP1 agonists) was successfully demonstrated up to 6 days for blood glucose control along with a body weight loss sustained for about 10 days after one single injection (vs 3 days for Semaglutide). The results show promising outcomes for making the next generation pharmaceutical agents (e.g., antidiabetic agents and antiobesity agents), and highlight the technology versatility (e.g., by enabling the transfer of not only bioactive peptides, but also potent chemicals). The antibody-pharmaceutical agent conjugates may be useful in developing of long-acting drugs.
In one aspect, the present disclosure provides a first modified affinity peptide, wherein:
n1 instances of the amino acid residues of the affinity peptide are independently modified with a moiety of Formula A′:
-L1-E2-L2-(E3)n3 (A′);
In certain embodiments, the first modified affinity peptide is a first modified affinity peptide, wherein:
-L1-E2-L2-E3 (A);
In certain embodiments, Formula A′ is Formula A:
-L1-E2-L2-E3 (A).
In another aspect, the present disclosure provides a second modified affinity peptide, wherein:
-L1-E2-L2-(E34-L3-M)n3 (B′);
wherein:
In certain embodiments, the second modified affinity peptide is a second modified affinity peptide, wherein:
-L1-E2-L2-E34-L3-M (B);
wherein:
In certain embodiments, Formula B′ is Formula B:
-L1-E2-L2-E34-L3-M (B).
In another aspect, the present disclosure provides antibody-pharmaceutical agent conjugate, wherein:
—(CH2)4-E12-L2-(E34-L3-M)n3 (C′);
In certain embodiments, the antibody-pharmaceutical agent conjugate is an antibody-pharmaceutical agent conjugate, wherein:
—(CH2)4-E12-L2-E34-L3-M (C);
In certain embodiments, Formula C′ is Formula C:
—(CH2)4-E12-L2-E34-L3-M (C).
In certain embodiments, the affinity peptide has at least 80% identity to an amino acid sequence of SEQ ID NO.: 1. In certain embodiments, the affinity peptide has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO.: 1. In certain embodiments, Z33 is a peptide of an amino acid sequence of SEQ ID NO.: 1.
In certain embodiments, the affinity peptide has at least 80% identity to an amino acid sequence of SEQ ID NO.: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the affinity peptide has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to an amino acid sequence of SEQ ID NO.: 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In certain embodiments, the affinity peptide comprises at least one cysteine or homocysteine. In certain embodiments, the affinity peptide comprises at least one cysteine or homocysteine, wherein at least one cysteine or homocysteine is modified with a moiety of Formula A′ or B′.
In certain embodiments, n1 instances of the amino acid residues of the affinity peptide are independently replaced with a moiety of Formula A. In certain embodiments, n1 instances of the amino acid residues of the affinity peptide are independently substituted with a moiety of Formula A. In certain embodiments, n1 instances of the amino acid residues of the affinity peptide are independently replaced with a moiety of Formula B. In certain embodiments, n1 instances of the amino acid residues of the affinity peptide are independently substituted with a moiety of Formula B. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the N or C terminus of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of an amino acid at an internal position of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, or 36th position of the affinity peptide. In certain embodiments, the position is counted from the 5′ terminus, the amino acid at the 5′ terminus is at the 1st position, and as the total number of the amino acids of the affinity peptide permits. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 3rd position of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 17th position of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 20th position of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 20th position of the affinity peptide of SEQ ID NO.: 1. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 21st position of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 24th position of the affinity peptide. In certain embodiments, at least one residue that is modified (e.g., replaced or substituted) is the residue of the amino acid at the 31st position of the affinity peptide.
In certain embodiments, n1 is 1. In certain embodiments, n1 is 2. In certain embodiments, n1 is 3.
In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C1-18 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C1-18 heteroalkylene are independently replaced with substituted or unsubstituted arylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, or substituted or unsubstituted heteroarylene, as valency permits. In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C1-6 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C1-6 heteroalkylene are independently replaced with substituted or unsubstituted arylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, or substituted or unsubstituted heteroarylene, as valency permits. In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C7-12 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C7-12 heteroalkylene are independently replaced with substituted or unsubstituted arylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, or substituted or unsubstituted heteroarylene, as valency permits. In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C13-18 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C13-18 heteroalkylene are independently replaced with substituted or unsubstituted arylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, or substituted or unsubstituted heteroarylene, as valency permits.
The first modified affinity peptide or second modified affinity peptide, wherein at least one instance of L1 is substituted or unsubstituted, C1-18 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C1-18 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. The first modified affinity peptide or second modified affinity peptide, wherein at least one instance of L1 is substituted or unsubstituted, C1-6 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C1-6 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. The first modified affinity peptide or second modified affinity peptide, wherein at least one instance of L1 is substituted or unsubstituted, C7-12 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C7-12 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. The first modified affinity peptide or second modified affinity peptide, wherein at least one instance of L1 is substituted or unsubstituted, C13-18 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C13-18 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits.
In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C1-10 heteroalkylene, wherein one or two backbone atoms of the C1-10 heteroalkylene are independently replaced with substituted or unsubstituted phenylene. In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C1-3 heteroalkylene, wherein one or two backbone atoms of the C1-3 heteroalkylene are independently replaced with substituted or unsubstituted phenylene. In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C4-7 heteroalkylene, wherein one or two backbone atoms of the C4-7 heteroalkylene are independently replaced with substituted or unsubstituted phenylene. In certain embodiments, at least one instance of L1 is substituted or unsubstituted, C8-10 heteroalkylene, wherein one or two backbone atoms of the C8-10 heteroalkylene are independently replaced with substituted or unsubstituted phenylene.
In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)0-1-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)-(substituted or unsubstituted, C1-6 heteroalkylene)0-1-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)0-1-. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)0-1-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)-(substituted or unsubstituted, C1-6 heteroalkylene)0-1-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)0-1-*, wherein bond * is attached to E2. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)-. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)-*, wherein bond * is attached to E2. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)-(substituted or unsubstituted, C1-6 heteroalkylene)-(substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene)-*, wherein bond * is attached to E2.
In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)0-1-(substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene)-(substituted or unsubstituted, C1-6 heteroalkylene)0-1-(substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene)0-1-*, wherein bond * is attached to E2. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene)-. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)-(substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene)0-1-*, wherein bond * is attached to E2. In certain embodiments, at least one instance of L1 is -(substituted or unsubstituted, C1-6 heteroalkylene)-(substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene)-(substituted or unsubstituted, C1-6 heteroalkylene)-(substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene)-*, wherein bond * is attached to E2.
In certain embodiments, each atom in the backbone (e.g., shortest backbone) of at least one instance of L1 is independently C, O, or S.
In certain embodiments, each substituent in at least one instance of L1 is independently F; unsubstituted C1-6 alkyl; C1-6 alkyl substituted with one or more F; —O-(unsubstituted C1-6 alkyl); —O—(C1-6 alkyl substituted with one or more F); or oxo.
In certain embodiments, the shortest backbone length of at least one instance of L1 is between 1 and 3, between 3 and 5, between 5 and 7, between 7 and 10, between 10 and 12, between 12 and 15, between 15 and 20, between 20 and 25, or between 25 and 30, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L1 is between 5 and 15, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L1 is between 7 and 12, inclusive, atoms.
In certain embodiments, at least one instance of L1 is
wherein bond C1 is attached to E2.
In certain embodiments, at least one instance of L1 is
wherein bond C1 is attached to E2.
In certain embodiments, at least one instance of E2 is —O—C(═O)—. In certain embodiments, at least one instance of E2 is —O—C(═O)—NRa—. In certain embodiments, at least one instance of E2 is —O—C(═O)—NH—. In certain embodiments, at least one instance of E2 is —O—S(═O)—NRa— or —O—S(═O)2—NRa—. In certain embodiments, at least one instance of E2 is —O—S(═O)—NH— or —O—S(═O)2—NH—.
In certain embodiments, at least one instance of Ra is hydrogen. In certain embodiments, at least one instance of Ra is substituted or unsubstituted, C1-6 alkyl. In certain embodiments, at least one instance of Ra is —CH3. In certain embodiments, at least one instance of Ra is —C2H5, n-C3H7, or i-C3H7. In certain embodiments, at least one instance of Ra is C1-6 alkyl substituted with one or more F. In certain embodiments, at least one instance of Ra is —CF3. In certain embodiments, at least one instance of Ra is a nitrogen protecting group.
In certain embodiments, n2 is 1. In certain embodiments, n2 is 2. In certain embodiments, n2 is 3. In certain embodiments, n2 is 4, 5, or 6.
In certain embodiments, at least one instance of E12 is —NH—C(═O)—. In certain embodiments, at least one instance of E12 is —NH—C(═O)—NRa—. In certain embodiments, at least one instance of E12 is —NH—C(═O)—NH—. In certain embodiments, at least one instance of E12 is —NH—S(═O)—NRa— or —NH—S(═O)2—NRa—. In certain embodiments, at least one instance of E12 is —NH—S(═O)—NH— or —NH—S(═O)2—NH—.
In certain embodiments, the antibody is an immunoglobulin G (IgG). In certain embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the antibody is an immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), or immunoglobulin M (IgM). In certain embodiments, the antibody is IgA1 antibody or IgA2. In certain embodiments, the antibody is an anti-HER2 antibody (e.g., trastuzumab). In certain embodiments, the antibody is an anti-MUC1 antibody. In certain embodiments, the antibody is an anti-oncoprotein antibody, and the oncoprotein is an oncoprotein of the cancer described herein. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a peptide or protein. In certain embodiments, the antibody is a peptide or protein and comprises between 10 and 30, between 30 and 100, between 100 and 300, between 300 and 1,000, between 1,000 and 3,000, or between 3,000 and 10,000, inclusive, amino acids.
In certain embodiments, n2 instances of the lysine residues of the antibody are independently substituted with a moiety of Formula C. In certain embodiments, n2 instances of the lysine residues of the antibody are independently replaced with a moiety of Formula C. In certain embodiments, at least one instance of the lysine residues that are modified (e.g., substituted or replaced) is at the heavy chain of the antibody. In certain embodiments, at least one instance of the lysine residues that are modified (e.g., substituted or replaced) is at a surface domain of the antibody. In certain embodiments, the antibody is an unmodified antibody.
In certain embodiments, at least one instance of L2 is independently substituted or unsubstituted, C1-100 alkylene, substituted or unsubstituted, C2-100 alkenylene, substituted or unsubstituted, C2-100 alkynylene, substituted or unsubstituted, C1-100 heteroalkylene, substituted or unsubstituted, C2-100 heteroalkenylene, substituted or unsubstituted, C2-100 heteroalkynylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, optionally wherein one or more backbone atoms of the C1-100 alkylene, C2-100 alkenylene, C2-100 alkynylene, C1-100 heteroalkylene, C2-100 heteroalkenylene, or C2-100 heteroalkynylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits.
In certain embodiments, at least one instance of L2 is independently substituted or unsubstituted, C1-12 alkylene, substituted or unsubstituted, C2-12 alkenylene, substituted or unsubstituted, C2-12 alkynylene, substituted or unsubstituted, C1-12 heteroalkylene, substituted or unsubstituted, C2-12 heteroalkenylene, substituted or unsubstituted, C2-12 heteroalkynylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, optionally wherein one or more backbone atoms of the C1-12 alkylene, C2-12 alkenylene, C2-12 alkynylene, C1-12 heteroalkylene, C2-12 heteroalkenylene, or C2-12 heteroalkynylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits.
In certain embodiments, at least one instance of L2 is independently substituted or unsubstituted, C12-40 alkylene, substituted or unsubstituted, C12-40 alkenylene, substituted or unsubstituted, C12-40 alkynylene, substituted or unsubstituted, C12-40 heteroalkylene, substituted or unsubstituted, C12-40 heteroalkenylene, substituted or unsubstituted, C12-40 heteroalkynylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, optionally wherein one or more backbone atoms of the C12-40 alkylene, C12-40 alkenylene, C12-40 alkynylene, C12-40 heteroalkylene, C12-40 heteroalkenylene, or C12-40 heteroalkynylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits.
In certain embodiments, at least one instance of L2 is independently substituted or unsubstituted, C40-100 alkylene, substituted or unsubstituted, C40-100 alkenylene, substituted or unsubstituted, C40-100 alkynylene, substituted or unsubstituted, C40-100 heteroalkylene, substituted or unsubstituted, C40-100 heteroalkenylene, substituted or unsubstituted, C40-100 heteroalkynylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, optionally wherein one or more backbone atoms of the C40-100 alkylene, C40-100 alkenylene, C40-100 alkynylene, C40-100 heteroalkylene, C40-100 heteroalkenylene, or C40-100 heteroalkynylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits.
In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C1-100 alkylene or substituted or unsubstituted, C1-100 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C1-100 alkylene or C1-100 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C1-100 alkylene or substituted or unsubstituted, C1-100 heteroalkylene. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C1-12 alkylene or substituted or unsubstituted, C1-12 heteroalkylene. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C12-40 alkylene or substituted or unsubstituted, C12-40 heteroalkylene. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C40-100 alkylene or substituted or unsubstituted, C40-100 heteroalkylene.
In certain embodiments, each atom in the backbone (e.g., shortest backbone) of at least one instance of L2 is independently C, O, S, or N. In certain embodiments, each atom in the backbone (e.g., shortest backbone) of at least one instance of L2 is independently C, O, or S.
In certain embodiments, each substituent in at least one instance of L2 is independently F; unsubstituted C1-6 alkyl; C1-6 alkyl substituted with one or more F; —O— (unsubstituted C1-6 alkyl); —O—(C1-6 alkyl substituted with one or more F); or oxo.
In certain embodiments, the shortest backbone length of at least one instance of L2 is between 1 and 3, between 3 and 5, between 5 and 7, between 7 and 10, between 10 and 12, between 12 and 15, between 15 and 20, between 20 and 25, or between 25 and 30, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L2 is between 2 and 10, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L2 is between 2 and 8, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L2 is between 3 and 6, inclusive, atoms.
In certain embodiments, at least one instance of L2 is —(CH2)2-8—. In certain embodiments, at least one instance of L2 is —(CH2)3-6—.
In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C2-20 heteroalkylene, optionally wherein one or two backbone atoms of the C2-20 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C20-50 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C20-50 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C50-100 heteroalkylene, optionally wherein one, two, three, or four backbone atoms of the C50-100 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C2-20 heteroalkylene, optionally wherein one or two backbone atoms of the C2-20 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C20-50 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C20-50 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. In certain embodiments, at least one instance of L2 is substituted or unsubstituted, C50-100 heteroalkylene, optionally wherein one, two, three, or four backbone atoms of the C50-100 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits.
In certain embodiments, at least one instance of L2 is
In certain embodiments, at least one instance of L2 is
In certain embodiments, at least one instance of L2 is
In certain embodiments, at least one instance of
which is attached in any direction. In certain embodiments, at least one instance of
In certain embodiments, at least one of -L2A3-L2A4-, -L2A5-L2A6-, -L2A7-L2A8-, -L2A9-L2A10-, -L2A11-L2A12-, -L2A13-L2A14-, -L2A15-L2A16-, -L2A17-L2A18-, -L2A19-L2A20-, -L2A21-L2A22-, -L2A23-L2A24-, -L2A25-L2A26-, -L2A27-L2A28-, -L2A29-L2A30-, -L2A31-L2A32-, -L2A33-L2A34-, -L2A35-L2A36-, -L2A37-L2A38-, -LL2A39-L2A40-, and -L2A41-L2A42- is a single bond, —O—, —C(═O)NRb—, —NRbC(═O)—, —NRbC(═O)O—, —OC(═O)NRb—, —NRbC(═O)NRb—, or —C(═O)—. In certain embodiments, at least one of -L2A3-L2A4-, -L2A5-L2A6-, -L2A7-L2A8-, -L2A9-L2A10-, -L2A11-L2A12-, -L2A13-L2A14-, -L2A15-L2A16-, -L2A17-L2A18-, -L2A19-L2A20-, -L2A21-L2A22-, -L2A23-L2A24-, -L2A25-L2A26-, -L2A27-L2A28-, -L2A29-L2A30-, -L2A31-L2A32-, -L2A33-L2A34-, -L2A35-L2A36-, -L2A37-L2A38-, -L2A39-L2A40-, and -L2A41-L2A42- is a single bond, —O—, —C(═O)NRb—, —NRbC(═O)—, or —C(═O)—. In certain embodiments, at least one of -L2A3-L2A4-, -L2A5-L2A6-, -L2A7-L2A8-, -L2A9-L2A10-, -L2A11-L2A12-, -L2A13-L2A14-, -L2A15-L2A16-, -L2A17-L2A18-, -L2A19-L2A20-, -L2A21-L2A22-, -L2A23-L2A24-, -L2A25-L2A26-, -L2A27-L2A28-, -L2A29-L2A30-, -L2A31-L2A32-, -L2A33-L2A34-, -L2A35-L2A36-, -L2A37-L2A38-, -L2A39-L2A40-, and -L2A41-L2A42- is a single bond, —O—, —C(═O)NH—, —NHC(═O)—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, or —C(═O)—. In certain embodiments, at least one of -L2A3-L2A4-, -L2A5-L2A6-, -L2A7-L2A8-, -L2A9-L2A10-, -L2A11-L2A12, -L2A13-L2A14-, -L2A15-L2A16-, -L2A17-L2A18-, -L2A19-L2A20-, -L2A21-L2A22-, -L2A23-L2A24-, -L2A25-L2A26-, -L2A27-L2A28-, -L2A29-L2A30-, -L2A31-L2A32-, -L2A33-L2A34-, -L2A35-L2A36-, -LL2A37-L2A38-, -L2A39-L2A40- and -L2A41-L2A42- is a single bond, —O—, —C(═O)NH—, —NHC(═O)—, or —C(═O)—.
In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is a single bond, unsubstituted C1-100 alkylene, —(OCH2CH2)1-50—, —(CH2CH2O)1-50—, or —(CH2OCH2)1-50—.
In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is unsubstituted C1-12 alkylene. In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is unsubstituted C12-40 alkylene. In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is unsubstituted C12-40 alkylene. In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is unsubstituted C40-99 alkylene. In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is —(OCH2CH2)1-6—, —(CH2CH2O)1-6—, or —(CH2OCH2)1-6—. In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is —(OCH2CH2)6-20—, —(CH2CH2O)6-20—, or —(CH2OCH2)6-20—. In certain embodiments, at least one instance of L2B1, L2B2, L2B3, L2B4, L2B5, L2B6, L2B7, L2B8, L2B9, L2B10, L2B11, and L2B12 is —(OCH2CH2)20-49—, —(CH2CH2O)20-49—, or —(CH2OCH2)20-49—.
In certain embodiments, at least one instance of L2C1 and L2C2 is a single bond,
wherein bond C4 is attached to L2A4, L2A5, L2A10, or L2A11.
In certain embodiments, at least one instance of n3 is 1. In certain embodiments, at least one instance of n3 is 2. In certain embodiments, at least one instance of n3 is 3 or 4. When n3 is 2, 3, or 4, any 2 instances of E3 may be attached to the same atom (e.g., N or C) or different atoms of L2 as valency permits.
In certain embodiments, at least one instance of E3 is an electrophile. In certain embodiments, at least one instance of E3 is a first click-chemistry handle. Any “click chemistry” reaction known in the art can be used to this end. Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary coupling reactions (some of which may be classified as “click chemistry”) include formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., maleimide addition); and Diels-Alder reactions (e.g., tetrazine [4+2] cycloaddition). Examples of click chemistry reactions can be found in, e.g., Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004-2021. Kolb, H. C. and Shrapless, K. B. Drug Disc. Today, 2003, 8, 112-1137; Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Shrapless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596-2599; Tomoe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. 2002, 67, 3057-3064. Wang, Q. et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Lee, L. V. et al. J. Am. Chem. Soc. 2003 125, 9588-9589; Lewis, W. G. et al. Angew. Chem. Int. Ed. 2002, 41, 1053-41057; Manetsch, R. et al., J. Am. Chem. Soc. 2004, 126, 12809-12818; Mocharla, V. P. et al. Angew. Chem., Int. Ed. 2005, 44, 116-120. In certain embodiments, at least one instance of E3 is —N3 or substituted or unsubstituted 1,2,4,5-tetrazinyl. In certain embodiments, at least one instance of E3 is —N3. In certain embodiments, at least one instance of E3 is
In certain embodiments, at least one instance of E3 is —C(═O)-(a leaving group), —S(═O)-(a leaving group), —S(═O)2-(a leaving group), or —P(═O)(Rc)-(a leaving group), wherein Rc is substituted or unsubstituted, C1-6 alkyl, substituted or unsubstituted phenyl, —O-(substituted or unsubstituted, C1-6 alkyl), or —O-(substituted or unsubstituted phenyl).
In certain embodiments, at least one instance of E3 comprises non-aromatic, C≡C or C≡C. In certain embodiments, at least one instance of E3 is —C≡CH, substituted or unsubstituted cyclooctynyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropenyl, substituted or unsubstituted cyclobutenyl, substituted or unsubstituted trans-cyclooctenyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, or substituted or unsubstituted
In certain embodiments, at least one instance of E3 is substituted or unsubstituted
substituted or unsubstituted
substituted or unsubstituted trans-cyclooctenyl, or —CH≡CH. In certain embodiments, at least one instance of E3 is
In certain embodiments, at least one instance of E3 is —SH.
In certain embodiments, at least one instance of E3 is a nucleophile. In certain embodiments, at least one instance of E4 is a second click-chemistry handle. In certain embodiments, the second click-chemistry handle is orthogonal to the first click-chemistry handle (e.g., the second click-chemistry handle is capable of undergoing a click-chemistry reaction with the first click-chemistry handle (e.g., under suitable (e.g., ambient or physiological) conditions). In certain embodiments, at least one instance of E4 comprises non-aromatic, C≡C or C═C. In certain embodiments, at least one instance of E4 is —C≡CH, substituted or unsubstituted cyclooctynyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropenyl, substituted or unsubstituted cyclobutenyl, substituted or unsubstituted trans-cyclooctenyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, or substituted or unsubstituted
In certain embodiments, at least one instance of E4 is substituted or unsubstituted
substituted or unsubstituted
substituted or unsubstituted trans-cyclooctenyl, or —CH≡CH. In certain embodiments, at least one instance of E4 is
or —CH≡CH. In certain embodiments, at least one instance of E4 is —SH. In certain embodiments, at least one instance of E4 is —OH, —NH2, or —NH-(substituted or unsubstituted, C1-6 alkyl).
In certain embodiments, at least one instance of E4 is —N3 or substituted or unsubstituted 1,2,4,5-tetrazinyl. In certain embodiments, at least one instance of E4 is —N3. In certain embodiments, at least one instance of E4 is
In certain embodiments, at least one instance of E3 is —N3 or substituted or unsubstituted 1,2,4,5-tetrazinyl, and at least one instance of E4 is —C≡CH, substituted or unsubstituted cyclooctynyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropenyl, substituted or unsubstituted cyclobutenyl, substituted or unsubstituted trans-cyclooctenyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, or substituted or unsubstituted
In certain embodiments, at least one instance of E3 is —N3 or substituted or unsubstituted 1,2,4,5-tetrazinyl, and at least one instance of E4 is substituted or unsubstituted
substituted or unsubstituted
substituted or unsubstituted trans-cyclooctenyl, or —CH≡CH. In certain embodiments, at least one instance of E3 is
and at least one instance of E4 is —SH.
In certain embodiments, at least one instance of E4 is —N3 or substituted or unsubstituted 1,2,4,5-tetrazinyl, and at least one instance of E3 is —C≡CH, substituted or unsubstituted cyclooctynyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropenyl, substituted or unsubstituted cyclobutenyl, substituted or unsubstituted trans-cyclooctenyl optionally fused independently with one or more instances of substituted or unsubstituted phenyl, or substituted or unsubstituted
In certain embodiments, at least one instance of E4 is —N3 or substituted or unsubstituted 1,2,4,5-tetrazinyl, and at least one instance of E3 is substituted or unsubstituted
substituted or unsubstituted
substituted or unsubstituted trans-cyclooctenyl, or —CH≡CH. In certain embodiments, at least one instance of E4 is
and at least one instance of E3 is —SH.
When n3 is 2, 3, or 4, any 2 instances of E34 may be attached to the same atom (e.g., N or C) or different atoms of L2 as valency permits.
In certain embodiments, at least one instance of E34 is
wherein bond C2 is attached to L2 or L3. In certain embodiments, at least one instance of E34 is
wherein bond C2 is attached to L2 or L3. In certain embodiments, at least one instance of E34 is
In certain embodiments, at least one instance of L3 is
In certain embodiments, at least one instance of L3 is
In certain embodiments, at least one of -L3A1-L3A2-, -L3A3-L3A4-, -L3A5-L3A6-, -L3A7-L3A8-, -L3A9-L3A10-, -L3A11-L3A12-, and -L3A13-L3A14- is a single bond, —O—, —C(═O)NRb—, —NRbC(═O)—, —NRbC(═O)O—, —OC(═O)NRb—, —NRbC(═O)NRb—, or —C(═O)—. In certain embodiments, at least one of -L3A1-L3A2-, -L3A3-L3A4-, -L3A5-L3A6-, -L3A7-L3A8-, -L3A9-L3A10-, -L3A11-L3A12-, and -L3A13-L3A14- is a single bond, —O—, —C(═O)NRb—, —NRbC(═O)—, or —C(═O)—. In certain embodiments, at least one of -L3A1-L3A2-, -L3A3-L3A4-, -L3A5-L3A6-, -L3A7-L3A8-, -L3A9-L3A10-, -L3A11-L3A12- and -L3A13-L3A14- is a single bond, —O—, —C(═O)NH—, —NHC(═O)—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, or —C(═O)—. In certain embodiments, at least one of -L3A1-L3A2-, -L3A3-L3A4-, -L3A5-L3A6-, -L3A7-L3A8-, -L3A9-L3A10-, -L3A11-L3A12-, and -L3A13-L3A14- is a single bond, —O—, —C(═O)NH—, —NHC(═O)—, or —C(═O)—. In certain embodiments, each of -L3A1-L3A2-, -L3A3-L3A4-, -L3A5-L3A6-, -L3A7-L3A8-, -L3A9-L3A10-, -L3A11-L3A12- and -L3A13-L3A14- is independently a single bond, —O—, —NRb—, —C(═O)NRb—, or —NRbC(═O)—. In some embodiments, each of -L3A1-L3A2-, -L3A3-L3A4-, -L3A5-L3A6-, -L3A7-L3A8-, -L3A9-L3A10-, -L3A11-L3A12-, and -L3A13-L3A14- is independently a single bond, —O—, —NH—, —C(═O)NH—, or —NHC(═O)—.
In certain embodiments, at least one instance of Rb is hydrogen. In certain embodiments, at least one instance of Rb is substituted or unsubstituted, C1-6 alkyl. In certain embodiments, at least one instance of Rb is —CH3. In certain embodiments, at least one instance of Rb is —C2H5, n-C3H7, or i-C3H7. In certain embodiments, at least one instance of Rb is C1-6 alkyl substituted with one or more F. In certain embodiments, at least one instance of Rb is —CF3. In certain embodiments, at least one instance of Rb is a nitrogen protecting group.
In certain embodiments, each of L3B1, L3B2, L3B3, and L3B4 is independently a single bond, substituted or unsubstituted, C1-20 alkylene or substituted or unsubstituted, C1-20 heteroalkylene. In certain embodiments, each of L3B1, L3B2, L3B3, and L3B4 is independently substituted or unsubstituted, C1-10 alkylene or substituted or unsubstituted, C1-10 heteroalkylene. In certain embodiments, each L3B1, L3B2, L3B3, and L3B4 is independently unsubstituted C1-10 alkylene. In certain embodiments, each of L3B1, L3B2, L3B3, and L3B4 independently consists of one, two, three, four, five, six, seven, eight, nine, or ten PEG repeats. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is a single bond, unsubstituted C1-200 alkylene, —(OCH2CH2)1-100—, —(CH2CH2O)1-100—, or —(CH2OCH2)1-100—. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is unsubstituted C1-12 alkylene. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is unsubstituted C12-40 alkylene. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is unsubstituted C40-100 alkylene. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is unsubstituted C100-200 alkylene. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is —(OCH2CH2)1-6—, —(CH2CH2O)1-6—, or —(CH2OCH2)1-6—. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is —(OCH2CH2)6-20—, —(CH2CH2O)6-20—, or —(CH2OCH2)6-20—. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is —(OCH2CH2)20-50—, —(CH2CH2O)20-50—, or —(CH2OCH2)20-50—. In certain embodiments, at least one of L3B1, L3B2, L3B3, and L3B4 is —(OCH2CH2)50-100—, —(CH2CH2O)50-100—, or —(CH2OCH2)50-100—.
In certain embodiments, at least one instance of L3C1 and L3C2 is a single bond,
In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C12-200 alkylene or substituted or unsubstituted, C12-200 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C12-200 alkylene or C12-200 heteroalkylene are 16 independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C2-20 heteroalkylene, optionally wherein one or two backbone atoms of the C2-20 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C20-50 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C20-50 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C50-100 heteroalkylene, optionally wherein one, two, three, or four backbone atoms of the C50-100 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C100-200 heteroalkylene, optionally wherein one, two, three, four, or five backbone atoms of the C100-200 heteroalkylene are independently replaced with substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted carbocyclylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C2-20 heteroalkylene, optionally wherein one or two backbone atoms of the C2-20 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C20-50 heteroalkylene, optionally wherein one, two, or three backbone atoms of the C20-50 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C50-100 heteroalkylene, optionally wherein one, two, three, or four backbone atoms of the C50-100 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C100-200 heteroalkylene, optionally wherein one, two, three, four, or five backbone atoms of the C100-200 heteroalkylene are independently replaced with substituted or unsubstituted phenylene, substituted or unsubstituted naphthylene, substituted or unsubstituted, monocyclic, 5- or 6-membered heteroarylene, or substituted or unsubstituted, bicyclic, 8- to 10-membered heteroarylene, as valency permits.
In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C1-12 heteroalkylene. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C12-40 heteroalkylene. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C40-100 heteroalkylene. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C100-170 heteroalkylene. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C170-200 heteroalkylene. In certain embodiments, at least one instance of L3 is substituted or unsubstituted, C40-170 heteroalkylene.
The second modified affinity peptide or antibody-pharmaceutical agent conjugate, wherein at least one instance of L3 is —CH2—, —O—, —NH—, —N(CH3)—, —CH2CH2O—, —OCH2CH2—, —C(═O)—, —C(═O)NH—, —C(═O)N(CH3)—, —NHC(═O)—, or —N(CH3)C(═O)—, or a combination thereof, provided that:
In certain embodiments, each atom in the backbone (e.g., shortest backbone) of at least one instance of L3 is independently C, O, S, or N. In certain embodiments, each atom in the backbone (e.g., shortest backbone) of at least one instance of L3 is independently C, O, or N.
In certain embodiments, each substituent in at least one instance of L3 is independently F; unsubstituted C1-6 alkyl; C1-6 alkyl substituted with one or more F; —O— (unsubstituted C1-6 alkyl); —O—(C1-6 alkyl substituted with one or more F); or oxo.
In certain embodiments, the shortest backbone length of at least one instance of L3 is between 1 and 6, between 6 and 12, between 12 and 40, between 40 and 70, between 70 and 100, between 100 and 130, between 130 and 170, or between 170 and 200, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L3 is between 40 and 170, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L3 is between 50 and 150, inclusive, atoms. In certain embodiments, the shortest backbone length of at least one instance of L3 is between 60 and 130, inclusive, atoms.
In certain embodiments, at least one instance of M is a radical of a pharmaceutical agent. In certain embodiments, at least one instance of the pharmaceutical agent is a peptide, protein, polynucleotide, or small molecule. In certain embodiments, at least one instance of the pharmaceutical agent is a therapeutic agent, prophylactic agent, or diagnostic agent. In certain embodiments, at least one instance of the pharmaceutical agent is a therapeutic agent. In certain embodiments, at least one instance of the pharmaceutical agent is an actoprotector, Addison disease medication, alcohol deterrent, anti-aging agent, anti-hypoxic agent, anti-infective agent, anti-inflammatory agent, anti-neurodegenerative agent, antidiabetic agent, antidote, antifibrotic agent, antigout agent, antihistamine, antiobesity agent, antiosteoporotic agent, antitumor agent, antiulcer agent, antivenom, autoimmune disease medication, biopharmaceutical, COPD medication, calmidazolium, calmidazolium chloride, cardiovascular agent, Celiac disease medication, Crohn disease medication, cytoprotective agent, demulcent, dermatological agent, epignetic drug, G-quadruplex stabilizer, gastrointestinal agent, Graves disease medication, hematologic agent, hormone antagonist, lysosomotropic agent, Meniere disease medication, metabolic agent, natriuretic, nervous system agent, neuromuscular agent, ophthalmic agent, osmotherapy agent, oxytocic agent, pharmaceutical immune agent, pharmaceutical chemosensitizer, pharmaceutical photosensitizer, pharmacoenhancer, photothermal agent, pulmonary surfactant, radiomimetic, radiopharmaceutical, renal agent, reproductive control agent, respiratory system agent, secretagogue, sequestering agent, sialagogue, sodium-iodide symporter inhibitor, sonosensitizer, vilaprisan, or wound healing promoter. In certain embodiments, at least one instance of the pharmaceutical agent is an antidiabetic agent or antiobesity agent. In certain embodiments, at least one instance of the pharmaceutical agent is a glucagon-like peptide-1 receptor agonist, β-secretase 2 inhibitor, insulin mimetic, insulin secretagogue, or insulin sensitizer. In certain embodiments, at least one instance of the pharmaceutical agent is a glucagon-like peptide-1 receptor agonist. In certain embodiments, at least one instance of the pharmaceutical agent is semaglutide, tirzepatide, AC 163794, albiglutide, danuglipron, danuglipron tromethamine, dapiglutide, dulaglutide, ecnoglutide, efinopegdutide, efocipegtrutide, exenatide, liraglutide, lixisenatide, mazdutide, pemvidutide, sitagliptin, taspoglutide, utreglutide, or vurolenatide, or a pharmaceutically acceptable salt thereof. In certain embodiments, at least one instance of the pharmaceutical agent is semaglutide or tirzepatide. In certain embodiments, at least one instance of the pharmaceutical agent is liraglutide.
In certain embodiments, at least one instance of the pharmaceutical agent is an anti-cancer agent. In certain embodiments, at least one instance of the pharmaceutical agent is monomethyl auristatin E, abiraterone acetate, ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, ado-trastuzumab emtansine, afatinib dimaleate, aldesleukin, alemtuzumab, anastrozole, arsenic trioxide, asparaginase Erwinia chrysanthemi, axitinib, azacitidine, BEACOPP, belinostat, bendamustine hydrochloride, BEP, bevacizumab, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib-s-malate, CAF, capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomibcarmustine, carmustine implant, ceritinib, cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, clofarabine, CMF, COPP, COPP-ABV, crizotinib, CVP, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, Dinutuximab, docetaxel, doxorubicin hydrochloride, doxorubicin hydrochloride liposome, enzalutamide, epirubicin hydrochloride, EPOCH, erlotinib hydrochloride, etoposide, etoposide phosphate, everolimus, exemestane, FEC, fludarabine phosphate, fluorouracil, FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FU-LV, fulvestrant, gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, goserelin acetate, Hyper-CVAD, ibritumomab tiuxetan, ibrutinib, ICE, idelalisib, ifosfamide, imatinib mesylate, imiquimod, ipilimumab, irinotecan hydrochloride, ixabepilone, lanreotide acetate, lapatinib ditosylate, lenalidomide, lenvatinib, letrozole, leucovorin calcium, leuprolide acetate, liposomal cytarabine, lomustine, mechlorethamine hydrochloride, megestrol acetate, mercaptopurine, methotrexate, mitomycin c, mitoxantrone hydrochloride, MOPP, nelarabine, nilotinib, nivolumab, obinutuzumab, OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuccinate, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, pamidronate disodium, panitumumab, panobinostat, pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, pembrolizumab, pemetrexed disodium, pertuzumab, plerixafor, pomalidomide, ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, radium 223 dichloride, raloxifene hydrochloride, ramucirumab, R-CHOP, recombinant HPV bivalent vaccine, recombinant human papillomavirus, nonavalent vaccine, quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, rituximab, romidepsin, ruxolitinib phosphate, siltuximab, sipuleucel-t, sorafenib tosylate, STANFORD V, sunitinib malate, TAC, tamoxifen citrate, temozolomide, temsirolimus, thalidomide, thiotepa, topotecan hydrochloride, toremifene, tositumomab and iodine I 131, tositumomab, TPF, trametinib, trastuzumab, VAMP, vandetanib, VEIP, vemurafenib, vinblastine sulfate, vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, vismodegib, vorinostat, XELIRI, XELOX, ziv-aflibercept, or zoledronic acid, or a pharmaceutically acceptable salt thereof.
In certain embodiments, at least one instance of the pharmaceutical agent is a prophylactic agent. In certain embodiments, at least one instance of the pharmaceutical agent is an antibiotic, nutritional supplement, vaccine, interleukin, interferon, or cytokine. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts.
In certain embodiments, at least one instance of the pharmaceutical agent is a diagnostic agent. In certain embodiments, at least one instance of the pharmaceutical agent is selected from the group consisting of fluorescent molecules; gases; metals; imaging agents, such as commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials. In certain embodiments, at least one instance of the pharmaceutical agent is used in magnetic resonance imaging (MRI), such as iron oxide particles or gadolinium complexes. Gadolinium complexes that have been approved for clinical use include gadolinium chelates with DTPA, DTPA-BMA, DOTA and HP-DO3A which are reviewed in Aime, et al. (Chemical Society Reviews (1998), 27:19-29).
In certain embodiments, at least one instance of the pharmaceutical agent is a metal, inorganic compound, organometallic compound, organic compound, or salt thereof. In certain embodiments, the imaging agent contains a metal selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, rutherfordium, dubnium, seaborgium, bohrium, hassium, meitnerium, gadolinium, gallium, thallium, and barium. In certain embodiments, the imaging agent is a magnetic resonance imaging (MRI) agent. In certain embodiments, the MRI agent is gadolinium. In certain embodiments, the MRI agent is a nitroxide radical-containing compound. In certain embodiments, the imaging agent is a nuclear medicine imaging agent. In certain embodiments, the nuclear medicine imaging agent is selected from the group consisting of 64Cu diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ASTM), 18F-fluorodeoxyglucose (FDG), 18F-fluoride, 3′-deoxy-3′-[18F]fluorothymidine (FLT), 18F-fluoromisonidazole (FMISO), gallium, technetium-99m, and thallium. In certain embodiments, the imaging agent is radiographic imaging agent. In certain embodiments, the radiographic imaging agent is selected from the group consisting of barium, gastrografin, and iodine contrast agent. In certain embodiments, at least one instance of the pharmaceutical agent comprises a fluorescent molecule, a metal chelate, a contrast agent, a radionuclide, or a positron emission tomography (PET) imaging agent, an infrared imaging agent, a near-IR imaging agent, a computer assisted tomography (CAT) imaging agent, a photon emission computerized tomography imaging agent, an X-ray imaging agent, or a magnetic resonance imaging (MRI) agent. In certain embodiments, at least one instance of the pharmaceutical agent comprises a radionuclide. In some embodiments, at least one instance of the pharmaceutical agent is a fluorescent molecule. In some embodiments, the fluorescent molecule comprises an acridine dye, a cyanine dye, a rhodamine dye, a BODIPY dye, a fluorescein dye, a dansyl dye, an Alexa dye, an atto dye, a quantum dot, or a fluorescent protein. In some embodiments, the fluorescent molecule is a cyanine dye (e.g., Cy3, Cy 3.5, Cy5, Cy5.5, Cy7, or Cy7.5). In some embodiments, at least one instance of the pharmaceutical agent is an MRI agent (e.g., a contrast agent). Examples of suitable materials for use as MRI agents (e.g., contrast agents) include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. In some embodiments, at least one instance of the pharmaceutical agent is a CAT imaging agent or an X-ray imaging agent. Examples of materials useful for CAT and X-ray imaging include iodine-based materials. In some embodiments, at least one instance of the pharmaceutical agent is a PET imaging agent. Examples of suitable PET imaging agents include compounds and compositions comprising the positron emitting radioisotopoes 18F, 15O, 13N, 11C, 82Rb, 64Cu, and 68Ga, e.g., fludeoxyglucose (18F-FDG), 68Ga-DOTA-psuedopeptides (e.g., 68Ga-DOTA-TOC), 11C-metomidate, 11C-acetate, 11C-methionine, 11C-choline, 18F-fluciclovine, 18F-fluorocholine, 18F-fluorodeoxysorbitol, 18F-3′-fluoro-3′-deoxythymidine, 11C-raclopride, and 18F-desmethoxyfallypride. In some embodiments, at least one instance of the pharmaceutical agent is a near-IR imaging agent. Examples of near-IR imaging agents include Pz 247, DyLight 750, DyLight 800, cyanine dyes (e.g., Cy5, Cy5.5, Cy7), AlexaFluor 680, AlexaFluor 750, IRDye 680, IRDye 800CW, and Kodak X-SIGHT dyes. In certain embodiments, at least one instance of the pharmaceutical agent is a contrast agent. In certain embodiments, at least one instance of the contrast agent is a magnetic-resonance signal enhancing agent, X-ray attenuating agent, ultrasound scattering agent, or ultrasound frequency shifting agent.
In some embodiments, at least one instance of the pharmaceutical agent is a radionuclide. Among the radionuclides used, gamma-emitters, positron-emitters, and X-ray emitters may be suitable for diagnostic and/or therapy, while beta emitters and alpha-emitters may also be used for therapy. Suitable radionuclides include, but are not limited to, 123I, 125I, 130I, 131I, 133I, 135I, 47Sc, 72As, 72Sc, 90Y, 88Y, 97Ru, 100Pd, 101mRh, 119Sb, 128Ba, 197Hg, 211At, 212Bi, 212Pb, 109Pd, 111In, 67Ga, 68Ga, 67Cu, 75Br, 77Br, 99mTc, 14C, 13N, 15O, 32P, 33P, and 18F.
In certain embodiments, each instance of the pharmaceutical agent is a therapeutic agent. In certain embodiments, each instance of the pharmaceutical agent is a prophylactic agent. In certain embodiments, each instance of the pharmaceutical agent is a diagnostic agent. In certain embodiments, at least one instance of the pharmaceutical agent is a therapeutic agent, and at least one instance of the pharmaceutical agent is a diagnostic agent. In certain embodiments, each instance of the pharmaceutical agent is the same. In certain embodiments, at least two instances of the pharmaceutical agent are different from each other.
In another aspect, the present disclosure provides a composition comprising a first modified affinity peptide, second modified affinity peptide, or antibody-pharmaceutical agent conjugate; and optionally one or more excipients.
In certain embodiments, the composition comprises an effective amount of the antibody-pharmaceutical agent conjugate. In certain embodiments, the composition is a pharmaceutical composition. In certain embodiments, the excipients are pharmaceutically acceptable excipients.
In certain embodiments, the compositions are useful for delivering a pharmaceutical agent to a subject in need thereof, cell, tissue, or biological sample. In certain embodiments, the compositions are useful for treating a disease in a subject in need thereof. In certain embodiments, the compositions are useful for preventing a disease in a subject in need thereof. In certain embodiments, the compositions are useful for diagnosing a disease in a subject in need thereof.
In certain embodiments, the subject is an animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a human two-years and older. In certain embodiments, the subject is a human eighteen-years and older.
In certain embodiments, the cell, tissue, or biological sample is in vitro. In certain embodiments, the cell is in vivo.
Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the antibody-pharmaceutical agent conjugate (which includes a pharmaceutical agent (the “active ingredient”)) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly (vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan monostearate (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.
Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.
Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.
Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.
Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the antibody-pharmaceutical agent conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.
Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the antibody-pharmaceutical agent conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.
Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.
Dosage forms for topical and/or transdermal administration of an antibody-pharmaceutical agent conjugate described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the antibody-pharmaceutical agent conjugate in powder form through the outer layers of the skin to the dermis are suitable.
Formulations suitable for topical administration include liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops May further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
The antibody-pharmaceutical agent conjugates are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
The antibody-pharmaceutical agent conjugates and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the antibody-pharmaceutical agent conjugate or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.
The exact amount of an antibody-pharmaceutical agent conjugate required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular antibody-pharmaceutical agent conjugate, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of an antibody-pharmaceutical agent conjugate described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of an antibody-pharmaceutical agent conjugate described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of an antibody-pharmaceutical agent conjugate described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of an antibody-pharmaceutical agent conjugate described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of an antibody-pharmaceutical agent conjugate described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of an antibody-pharmaceutical agent conjugate described herein.
Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. In certain embodiments, a dose described herein is a dose to an adult human whose body weight is 70 kg.
In certain embodiments, the composition further comprises one or more additional pharmaceutical agents. The antibody-pharmaceutical agent conjugate or composition can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and/or in inhibiting the activity of a protein kinase in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including an antibody-pharmaceutical agent conjugate described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the antibody-pharmaceutical agent conjugate and the additional pharmaceutical agent, but not both.
The antibody-pharmaceutical agent conjugate or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which are different from the pharmaceutical agent included in the antibody-pharmaceutical agent conjugate or composition. This may be useful as, e.g., combination therapies. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent described in the “First modified affinity peptides, second modified affinity peptides, and antibody-pharmaceutical agent conjugates” section of the present disclosure. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the antibody-pharmaceutical agent conjugate or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the antibody-pharmaceutical agent conjugate described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
The additional pharmaceutical agents include anti-proliferative agents, anti-cancer agents, cytotoxic agents, anti-angiogenesis agents, anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, and pain-relieving agents. In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti-viral agent. In certain embodiments, the additional pharmaceutical agent is a binder or inhibitor of a protein kinase. In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the antibody-pharmaceutical agent conjugate described herein or pharmaceutical composition can be administered in combination with an anti-cancer therapy including surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.
In another aspect, the present disclosure provides a kit comprising:
In certain embodiments, the kit comprises a first container. In certain embodiments, the first container comprises the first modified affinity peptide, second modified affinity peptide, antibody-pharmaceutical agent conjugate, or composition. In some embodiments, the kit further comprises a second container. In certain embodiments, the second container comprises the instructions. In certain embodiments, the instructions comprise information required by a regulatory agency, such as the U.S. Food and Drug Administration (FDA) or European Medicines Agency (EMA). In certain embodiments, the instructions comprise prescribing information. In certain embodiments, the second container comprises the first container. In some embodiments, the kit further comprises a third container. In certain embodiments, the third container comprises the excipients. In certain embodiments, the third container comprises the additional pharmaceutical agents. In certain embodiments, the second container comprises the third container. In certain embodiments, each of the first, second, and third containers is independently a vial, ampule, bottle, syringe, dispenser package, tube, or box.
In another aspect, the present disclosure provides a method comprising administering to a subject in need thereof an effective amount of a first modified affinity peptide, a second modified affinity peptide, an antibody-pharmaceutical agent conjugate, or a composition.
In another aspect, the present disclosure provides a method of delivering a pharmaceutical agent to a cell comprising contacting the cell with an effective amount of: the antibody-pharmaceutical agent conjugate, or a tautomer, isotopically labeled conjugate, or salt thereof; or the composition.
In another aspect, the present disclosure provides a method comprising contacting a cell, tissue, or biological sample with a first modified affinity peptide, second modified affinity peptide, antibody-pharmaceutical agent conjugate, or a composition.
In certain embodiments, the subject is in need of treatment of a disease. In certain embodiments, the subject is in need of prevention of a disease. In certain embodiments, the subject is in need of diagnosis of a disease.
In certain embodiments, the subject is in need of treatment of a disease, and at least one instance of the pharmaceutical agent is a therapeutic agent. In certain embodiments, the subject is in need of prevention of a disease, and at least one instance of the pharmaceutical agent is a prophylactic agent. In certain embodiments, the subject is in need of diagnosis of a disease, and at least one instance of the pharmaceutical agent is a diagnostic agent.
In certain embodiments, the disease is a metabolic disease, cancer, benign neoplasm, pathologic angiogenesis, inflammatory disease, autoimmune disease, hematological disease, genetic disease, neurological disease, painful condition, or psychiatric disease.
In certain embodiments, the disease is a metabolic disease. In certain embodiments, the disease is diabetes. In certain embodiments, the disease is Type I diabetes. In certain embodiments, the disease is Type II diabetes. In certain embodiments, the disease is gestational diabetes. In certain embodiments, the disease is obesity. In certain embodiments, the disease is hyperglycemia. In certain embodiments, the disease is hyperinsulinemia. In certain embodiments, the disease is insulin resistance.
In certain embodiments, the disease is cancer. In certain embodiments, the disease is a solid tumor. In certain embodiments, the disease is a hematological malignancy. In certain embodiments, the disease is leukemia. In certain embodiments, the disease is lymphoma.
In certain embodiments, the administration is parenteral administration. In certain embodiments, the administration is intramuscular, subcutaneous, or intracerebroventricular administration. In certain embodiments, the administration is intrathecal administration. In certain embodiments, the administration is intravenous administration. In certain embodiments, the administration is intracerebroventricular administration. In certain embodiments, the administration is oral administration. In certain embodiments, the administration is topical administration.
In certain embodiments, the method further comprises administering to or implanting in the subject in need thereof an effective amount of an additional therapy. In certain embodiments, the additional therapy is an additional pharmaceutical agent. In certain embodiments, the additional therapy is surgery, radiation, or transplantation.
In order that the embodiments described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the oligonucleotides, pharmaceutical compositions, uses, and methods provided herein and are not to be construed in any way as limiting their scope.
A platform to “paint” native antibodies with therapeutic payloads was developed and tested for the ability to use the circulating antibodies as a vehicle to extend the PK/PD of peptide drugs after in vivo administration in mice (
The structures of the IgG-binder peptides Z33-P16p, Z33-E20Hcy, PEG8-Z33-E20Hcy and their reactivity towards human IgG1, IgG2, and IgG3 and mouse IgG1, IgG2a, and IgG2b was first evaluated in vitro. The IgG-binder peptides were also incubated in mouse sera for 2-, 6-, and 24-hours to evaluate the percentage of in vitro azido transfer to mouse IgG (
To evaluate the in vivo azido transfer in mice, two groups of mice were injected subcutaneously or intraperitoneally with the IgG-binder peptides. Mice were sacrificed at 24 hours post-injection, and the blood was collected for ELISA analysis. Sandwich ELISA assays. ELISA analysis of the azido transfer to mouse IgG following the subcutaneous injection of 30 mg/kg of Z33-E20Hcy or pegylated-Z33-E20Hcy indicated that both performed similarly (mean 30-40% azido transfer), with the pegylated-Z33-E20Hcy performing moderately better (
The ability of the IgG-binder electrophilic peptides to extend the PK/PD of GLP1 drugs was then evaluated. A cell experiment was first step up to evaluate GLP1 analog binding to the GLP1 receptor and evaluate the functionality of GLP1 after conjugation to IgG. After in vitro confirmation, an in vivo experiment was designed to evaluate GLP1 transfer to mouse IgG in wile-type Swiss mice. Mice were injected intraperitoneally with PBS (n=5), semaglutide (n=15), GLP1 peptide (n=15), Z33-E20Hcy-GLP1 (n=15), or pegylated-Z33-E20Hcy-GLP1 (n=15). An intraperitoneal glucose tolerance test (IP-GTT) was performed 24-, 72-, and 144 hours post-injection (
The same study was repeated in obese mice models, which are deficient in leptin (Lepob) and present spontaneous type II diabetes and cardiometabolic diseases. Obese mice were injected peritoneally with PBS (n=6), semaglutide (n=12), GLP1 peptide (n=12), Z33-E20Hcy-GLP1 (n=12), or pegylated-Z33-E20Hcy-GLP1 (n=12). An intraperitoneal glucose tolerance test (IP-GTT) was performed 24-, 72-, and 144 hours post-injection (
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Mouse IgG Painting with Radionuclides
Further investigations using the most efficient electrophilic peptides highlighted that the IgG-binder electrophilic peptides could be capable of “painting” native antibodies (IgG1, IgG2, and IgG3) directly inside the body through the covalent transfer of either a small molecule, a radionuclide, or a bioactive long peptide. To evaluate this, wile-type Swiss mice were injected with about 1 MBq of Zr-89 labelled Z33-P16p, Z33-E20Hcy, or pegylated-Z33-E20Hcy intravenously, intraperitoneally, or subcutaneously (
The whole blood biodistribution of the radiolabeled electrophilic peptides was evaluated at 144 hours post-intravenous injection (
PET imaging and uptake in the spleen at 144 hours post-intravenous injection was also evaluated (
Finally, a comparison between Z33-P16p and Z33-E20Hcy was performed. Whole-body biodistribution at 2-, 24-, 72-, and 144-hours post-injection showed markedly different biodistributions between the two electrophilic peptides (
The transfer reaction is fully biocompatible, occurs without catalyzers, and does not require any biological engineering or production of antibody-drug conjugates beforehand as the payload is attached on native circulating IgGs that are already naturally produced by the organism. Taken together, these results show promising outcomes for making the next generation of weight loss and TDM drugs, and highlight the technology versatility, by enabling the transfer of not only bioactive peptides, but also potent chemicals. The IgG peptide-transfer technology represents a novel and powerful alternative for the manufacture of long-acting drugs.
Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp-(Ot-Bu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(Ot-Bu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(But)-OH, Fmoc-L-Thr(t-Bu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(t-Bu)-OH, and Fmoc-L-Val-OH were purchased from Novabiochem, Millipore Sigma, or Chem-Impex Inc. Fmoc-alpha-methylalanine (Fmoc-Aib-OH) and Fmoc-L-HomoCys(Trt)-OH (Fmoc-Hcy-OH) were purchased from Combi-Blocks Inc. 5-azidopentanoic acid was purchased from ChemPep®. 1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oic acid (Fmoc-PEG8-CO2H) was purchased by AmBeed. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and (7-azabenzotriazol-lyloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were purchased from P3 Biosystems. H-Rink Amide-ChemMatrix resin was purchased from PCAS BioMatrix Inc. Polyethylene filter paper for peptide synthesis (0.60 mm thick, pore size 7-12 μm) was purchased from Interstate Specialty Products. N,N-dimethylformamide (OmniSolv® for Biosynthesis, DMF, stored with an AldraAmine trapping packet), diethyl ether (≥99.0% stabilized by BHT, Et2O), acetonitrile (≥99.9%, CH3CN), dichloromethane (≥99.8% stabilized by amylene, CH2Cl2), 1,4-dioxan (≥99.5% stabilized by BHT), 2-propanol (99.9%), 2-methyl-tetrahydrofuran (≥99.5% stabilized by BHT, 2-Me-THF), dimethylsulfide (≥99.5%, BioUltra for molecular biology, DMSO), and pentane (98% reagent grade) were obtained from Millipore-Sigma. Ethyl acetate (≥99.5%), hexane (≥98.5%, mixture of isomers), Methanol (≥99.8%), and acetone (≥99.5%) were purchased from VWR. The water used in all reactions on proteins, in the preparation of buffers, and in the preparation of mobile phases for purification was obtained via filtration of deionized water through a Millipore Sigma Milli-Q™ Ultrapure Water System. AldraAmine trapping packets and piperidine were purchased from Millipore Sigma. Amine-free DMF refers to DMF stored over AldraAmine trapping packets for a minimum of 24 hours prior to use. LC/MS grade water, acetonitrile, and formic acid were purchased from Thermo Fisher Scientific Inc and were used for liquid chromatography-mass spectrometry (LC-MS). All small compounds were purchased from TCI America, Millipore Sigma, AmBeed, Matrix Scientific, or Combi-Blocks in purities ≥95%, and were used without further purification (unless otherwise noted). PBS buffer (1× and 10×) were purchased from CORNING®. Reactions monitored by analytical thin-layer chromatography (TLC) were carried out using glass-backed plates pre-coated with silica gel impregnated with a fluorescent indicator (254 nm). All deuterium solvents were purchased from Cambridge Isotope Laboratories, Inc. C18-ZipTips (0.6 μL) were purchased from Millipore Sigma. Zeba spin desalting columns were purchased from Thermo Fisher Scientific Inc. Trastuzumab and its biosimilar were purchased from Syd Labs Inc., MedChemExpress LLC., and Bio X Cell.
NMR spectra were recorded on Bruker Avance-III HD 400 MHz spectrometer. All 1H NMR chemical shifts are expressed in parts per million (ppm, δ scale) and are referenced to the residual proton in the NMR solvent (CDCl3-d3: 7.26, DMF-d7: 8.03, DMSO-d6: 2.50). All 13C spectra recorded are proton decoupled. The 13C NMR chemical shifts are expressed in part per million (ppm, δ scale) and are referenced to the carbon resonance of the NMR solvent (CDCl3-d3: 77.16, DMF-d7: 163.15, DMSO-d6: 39.52). All 31P chemical shifts are expressed in parts per million (ppm, δ scale). 1H NMR spectroscopic data are reported as follows: a chemical shift in ppm (multiplicity, coupling constants J (Hz), integration intensity, assigned number of protons in molecule). The multiplicities are abbreviated with s (singlet), br. s (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). In the case of combined multiplicities, the multiplicity with the larger coupling constant is stated first. The chemical shift of all signals is reported as the center of the resonance range, except in the case of multiplets, which are reported as ranges in chemical shift. All raw fid files were processed, and the spectra analyzed using the program MestReNova 14.2.3 from Mestrelab Research S. L.
LC-MS chromatograms and associated mass spectra were acquired using an Agilent Technologies 6550 Q-ToF LC-MS system. Solvent compositions are 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in acetonitrile (solvent B). For methods A, B, and C, a calibration solution containing mass 922.009798 constantly flowed through the system. The following LC-MS methods were used:
Q-ToF LC-MS Method A: LC conditions: Zorbax 300SB C3 column: 2.1×150 mm, 5 μm, column temperature: 40° C., gradient: 0-1 minutes 1% B, 1-6 minutes 1-61% B, flow rate: 0.8 mL/minute. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300-3000 m/z, temperature of drying gas=350° C., flow rate of drying gas=11 L/minute, pressure of nebulizer gas=60 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 175, and 750 V, respectively.
Q-ToF LC-MS Method B: LC conditions: Zorbax 300SB C3 column: 2.1×150 mm, 5 μm, column temperature: 40° C., gradient: 0-1 minutes 1% B, 1-7 minutes 1-91% B, flow rate: 0.7 mL/minute. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300-3000 m/z, temperature of drying gas=350° C., flow rate of drying gas=11 L/minute, pressure of nebulizer gas=60 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 175, and 750 V, respectively.
Q-ToF LC-MS Method C: LC conditions: Zorbax 300SB C3 column: 2.1×150 mm, 5 μm, column temperature: 40° C., gradient: 0-1 minutes 1% B, 1-6 minutes 1-41% B, flow rate: 0.8 mL/minute. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300-3000 m/z, temperature of drying gas=350° C., flow rate of drying gas=11 L/minute, pressure of nebulizer gas=60 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 175, and 750 V, respectively.
LC-MS Method D: LC conditions: Aeris WIDEPORE C4 200 column: 2.1×150 mm, 3.6 μm, column temperature: 40° C., gradient: 0-2 minutes 1% B, 2-8 minutes 1-91% B, 8-10 minutes 91-95% B, flow rate: 0.3 mL/minute. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 100-1700 m/z, temperature of drying gas=200° C., flow rate of drying gas=14 L/minute, pressure of nebulizer gas=55 psi, the capillary, fragmentor, and octapole voltages were set at 3500, 175, and 750 V, respectively.
Data were processed using Agilent MassHunter Workstation Qualitative Analysis Version B.06.00 Software or Agilent MassHunter BioConfirm Software B.10.00. Deconvoluted masses of proteins were obtained using a maximum entropy algorithm. Unless otherwise depicted, the following parameters were used for deconvolution: Mass range is given in the experimental section for each peptide; Mass step was set to 1.00 Daltons; Baseline was set to Subtract baseline, Baseline factor as 7.00. The y-axis of all chromatograms shown in the
Mass spectra were obtained on an Agilent 6125B mass spectrometer attached to an Agilent 1260 Infinity LC. Solvent compositions are 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The following LC-MS method was used:
LC conditions: Poroshell 120 SB C18: 2.1×50 mm, 2.7 μm, column temperature: 40° C., gradient: 0-1 minutes 10% B, 1-5 minutes 10-100% B, 5-6 minutes 100% B, 6-7 minutes 100-10% B, flow rate: 0.4 mL/minute. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 100-1500 m/z, temperature of drying gas=350° C., flow rate of drying gas=13 L/minute, pressure of nebulizer gas=35 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 70, and 650 V, respectively.
Nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS)
Analysis was performed on an EASY-nLC 1200 nano-liquid chromatography system connected to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer or to an Orbitrap Fusion Eclipse Tribrid Mass Spectrometer (Thermo Fisher Scientific). Samples were run on a PepMap RSLC C18 column (C18, 50 um×15 cm, 2 um, 100 Å, Thermo Fisher Scientific, P/N ES901). An Acclaim PepMap 100 Trap column (C18, 75 um×2 cm, 3 um, 100 Å, Thermo Fisher Scientific, P/N 164946) was used for desalting. Solvent compositions are 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in 80% acetonitrile and 19.9% H2O (solvent B). The following conditions were used for each sample measurement:
nLC-MS/MS Method A: LC conditions: column temperature: 40° C., gradient: 0-30 minutes 30-95% B, 30-40 minutes 95% B, flow rate: 300 nL/minute. MS conditions: positive ion spray voltage was set to 2200 V. Orbitrap detection was used for primary MS, with the following parameters: Application mode=peptide, cycle time=3 s, resolution=120000, mass range=normal, scan range=250-2000 m/z, RF Lens=30%, AGC target=standard, maximum injection time mode=auto, 1 microscan, data type-profile, polarity=positive, The following filters were applied for MS2 precursor selection: monoisotopic peak determination=peptide, filter type=threshold, Intensity threshold=5.0e5, include charge state=2-6, mass list type=m/z, target mass*, mass tolerance=25 ppm. Fragmentation was induced by higher-energy collisional dissociation (HCD) and electron-transfer dissociation with higher-energy collision (EThcD). Specifications HCD: isolation mode=quadrupole, isolation window=1.2 m/z, isolation offset=0.6 m/z, activation type=HCD, collision energy mode=fixed (30%), detection type=orbitrap, resolution=30000, mass range=normal, first mass=120 m/z, AGC target=standard, maximum injection time mode=dynamic, 1 microscan, data type=profile. Specifications EThcD: isolation mode=quadrupole, isolation window=1.2 m/z, isolation offset=0.6 m/z, activation type=ETD, use calibrated charge-dependent ETD parameters, ETD supplemental activation=EThcD, SA collision energy=30%, detection=orbitrap, resolution=30000, mass range=normal, first mass=120 m/z, normalized AGC target=standard, maximum injection time=dynamic, 1 microscans, data type=profile. A list of target masses was created by calculating the intrinsic masses of the measured samples.
nLC-MS/MS Method B: LC conditions: column temperature: 40° C., gradient: 0-30 minutes 1-10% B, 30-120 minutes 10-81% B, 120-125 minutes 81-90% B, 125-135 minutes 90% B, flow rate: 300 nL/minute. MS conditions: positive ion spray voltage was set to 2200 V, and negative ion spray voltage was set to 600 V. Orbitrap detection was used for primary MS, with the following parameters: Application mode=peptide, cycle time=3 s, resolution=120000, mass range=normal, scan range=200-1400 m/z, RF Lens=30%, AGC target=custom, normalized AGC target (%)=250, maximum injection time mode=auto, 1 microscan, data type=profile, polarity=positive, The following filters were applied for MS2 precursor selection: monoisotopic peak determination=peptide, exclude after 4 times, If occurs within 30 seconds, exclusion duration=30 seconds, mass tolerance=10 ppm, exclude isotope, include charge state=2-10, filter type=threshold, Intensity threshold=5.0e4, precursor selection range=200-1400, Fragmentation was induced by collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation with higher-energy collision (EThcD). Specifications CID: isolation mode=quadrupole, isolation window=1.3 m/z, activation type=CID, collision energy mode=fixed (30%), CID activation time=10 milliseconds, activation Q=0.25, detection type=orbitrap, resolution=30000, mass range=normal, AGC target=custom, normalized AGC target=40%, maximum injection time mode=auto, 1 microscan, data type=centroid. Specifications HCD: isolation mode=quadrupole, isolation window=1.3 m/z, activation type=HCD, detection type=orbitrap, resolution=30000, mass range=normal, scan range mode=auto, AGC target=custom, normalized AGC target=40%, maximum injection time mode=auto, 1 microscan, data type=centroid. Specifications EThcD: isolation mode=quadrupole, isolation window=1.3 m/z, activation type=ETD, use calibrated charge-dependent ETD parameters, ETD supplemental activation=EThcD, SA collision energy=25%, detection=orbitrap, resolution=30000, mass range=normal, scan range mode=auto, normalized AGC target=40%, normalized AGC target=custom, maximum injection time=auto, 1 microscans, data type=profile.
The samples were analyzed using an Agilent Technologies 1290 Infinity II LC system which was computer-controlled through Agilent ChemStation software. The following methods were used:
UHPLC Method A (for peptide/protein analysis excluding IgG): Solvent compositions used in the UHPLC are 0.1% TFA in H2O (solvent A) and 0.1% TFA in acetonitrile (solvent B). ACQUITY UPLC Protein BEH C4 column: 2.1×50 mm, 1.7 μm (Waters, P/N 186004495), ACQUITY UPLC Protein BEH C4 VanGurde pre-column: 2.1×5 mm, 1.7 μm (Waters, P/N 186004623), column temperature: 27° C., gradient: 0-3 minutes 1% B, 3-13 minutes 1-61% B, flow rate: 0.5 mL/minute.
UHPLC Method B (For IgGs analysis): Solvent compositions used in the UHPLC are 0.1% TFA in 98% H2O and 2% 2-propanol (solvent A) and 70% 2-propanol, 20% acetonitrile, and 10% solvent A (solvent B). AdvanceBio RP-mAb Diphenyl USP L11 column: 2.1×150 mm, 3.5 μm (Agilent, P/N 793775-944), column temperature: 80° C., gradient: 0-1 minutes 15% B, 1-3 minutes 15-30% B, 3-15 minutes 30-40% B, 15-16 minutes 40-60% B, 16-20 minutes 60-15% B, flow rate: 0.5 mL/minute.
A Biotage® Selekt automated flash chromatography system was used for the purification of small molecules and peptides. The columns, solvents, and solvent gradients employed for the purification of individual compounds are written in the synthesis section of each compound. Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE)
Bolt 4-12% Bis-Tris Plus 1.0 mm×15 well or 1.0 mm×10 well plates (Invitrogen), Mini Gel Tank (Invitrogen) along with the PowerPac HC (BIO-RAD) were used for SDS-PAGE analysis. SeeBlue® Plus2 standard (Invitrogen) was used as the molecular weight standard. Gels were run using Bolt MOPS SDS Running Buffer (1×, Invitrogen) under the conditions of 135V for 55 minutes. After electrophoresis, the running buffer was discarded, and the gel was placed in deionized water. Subsequently, the water with gel was heated in a microwave for 1 minute and seconds. The heated water was replaced, and the gel was thoroughly rinsed. Gel staining was performed for 10 minutes using SimpleBlue SafeStain (Invitrogen). The stain was removed following staining, and the gel was immersed in deionized water. The tray with gel and deionized water was placed on a shaker overnight to remove the stain from the gels. Then, ChemDoc MP Imaging System (BIO-RAD) was used to analyze the stained gels.
The conversion rate for each reaction was calculated by one of the following methods. Determination by LC-MS: Reported yields based on LC-MS spectra were determined by extracting the total ion current (TIC) spectra of all protein-containing species in the chromatogram utilizing Agilent MassHunter 6.0 software with BioConfirm package. These extracted chromatograms were deconvoluted utilizing a maximum entropy algorithm and abundance of each species determined using total ion count.
Determination by SDS-PAGE: Band densitometry was calculated using ImageJ software. Densitometry-based yields were calculated based on the product: starting material protein ratio in lanes and standardized based on molecular weight. An example is shown in
All animal experiments were performed under an MIT institute approved IACUC protocol following federal, state, and local guidelines for the care and use of animals (protocol number #0821-058-24). The studies were performed on 30-35 g (5-8 weeks-old) healthy female Swiss mice, purchased from The Jackson Laboratory (MA, USA). Mice were housed with free access to food and water ad libitum unless stated otherwise for the need of the experiment. For subcutaneous (SC) injections, the mice were shortly anesthetized beforehand with 2-3% isoflurane along with O2, then shaved on the right flank. For intravenous injections (IV), the mice were anesthetized with 2-3% isoflurane along with O2, then a catheter was inserted in the lateral tail vein to ensure the injection was properly done. Intraperitoneal injections (IP) were performed on vigil mice, in the lower right abdominal quadrant. For blood collection, mice were either punctured in the heart (terminal procedure), or gently pressed at the tail following tail pricks (vigil). For multiple blood collections following the injection of radionuclides, the mice were first anesthetized with 2-3% isoflurane along with O2, before performing tail pricks, for radiation safety matter. The animals were sacrificed by CO2 inhalation followed by cervical dislocation. All compounds injected in mice were either USP grade, sterile, or filtered using 0.22 μm.
The synthesis of the IgG binding peptide Z33 (Proc. Natl. Acad. Sci. U.S.A. 93, 5688-5692 (1996)) analogs were performed using the Automated-flow solid phase peptide synthesis (AFPS) system (Science, 368, 980-987 (2020)). The synthesis conditions are summarized in
A plastic fritted syringe (6 mL) was equipped with polyethylene filter paper, and ChemMatrix® H-Rink Amide resin (0.49 mmol/g, 150 mg) was loaded onto it. The syringe assembly was then placed on a manifold. The resin was swollen in DMF, and subsequently, a homogeneous resin slurry was prepared by repeatedly drawing back 500 μL of the slurry using a 1 mL pipette. Next, the syringe was set to the AFPS system for peptide synthesis. After completion of the synthesis, the syringe was transferred from the AFPS system to the manifold and washed three times with CH2C12. To minimize methionine oxidation in the peptide sequence, drying of the resin was not conducted using air flow on the manifold but rather using nitrogen flow. Specifically, the syringe was capped with a rubber septum, and a needle connected to a nitrogen flow line was inserted into the septum for 15 minutes. The resulting peptide-bound dry resin was subjected to subsequent steps of cleavage and purification.
The peptide was synthesized using the method described in the “Synthesis of Z33 Variants Composed of Natural Amino Acids” section from the C-terminus of the sequence to one unnatural amino acid before the sequence. The resin in a plastic fritted syringe was transferred from the AFPS system to a manifold. Fmoc-protected unnatural amino acid (10 equivalents for the peptide on the resin) in HATU/DMF (0.38 M, 9.5 equivalents) solution and DIEA (15 equivalents) were added to the resin and allowed to react for 50 minutes at room temperature.
The reaction solution was stirred with a spatula every 10 minutes. After the reaction, the reaction solution was removed, and the resin in the syringe was washed three times with 5 mL of DMF. Then, 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added and allowed to react for 10 minutes. The reaction solution was removed, and 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added again and allowed to react for 10 minutes. The reaction solution was removed, and the resin was washed three times with 4 mL of DMF. Subsequent synthesis of the sequence using natural amino acids was again performed according to the procedure previously described.
After synthesis of the peptide by AFPS, the fritted syringe with resin was placed on a manifold. A HATU/DMF solution (0.38 M, 9.5 equivalents) of the carboxylic acid corresponding to the acyl group (10 equivalents for the peptide on the resin) and DIEA (15 equivalents) were added and allowed to react for 50 minutes at room temperature. During that time, the reaction solution was stirred with a spatula every 10 minutes. After the reaction, the reaction solution was removed, and the resin in the syringe was washed three times with 10 mL of DMF. If the introduced acyl group contained the Fmoc protecting group, 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added and allowed to react for 10 minutes. The reaction solution was removed, and 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added again and allowed to react for 10 minutes. The reaction solution was removed and washed three times with 4 mL of DMF and three times with CH2Cl2. To minimize methionine oxidation in the peptide sequence, the resin was not dried using airflow on the manifold but rather using nitrogen flow. Specifically, the syringe was capped with a rubber septum, and a needle connected to a nitrogen flow line was inserted into the septum for 15 minutes. The resulting peptide-bound dry resin was subjected to subsequent steps of cleavage and purification.
Cleavage of Z33 Variants from Resin
The peptide-bound resin was transferred to a 15 mL centrifuge tube. Subsequently, a 7.5 mL peptide cleavage solution (a mixture of TFA, water, EDT, and TIPS in a ratio of 94:2.5:2.5:1) was added to the centrifuge tube, and the reaction was allowed to proceed at room temperature for 2 hours on the shaker. Afterward, 5 mL of a plastic fritted syringe was placed on top of a 50 mL centrifuge tube, and the reaction solution containing the resin was filtered. The resin inside the syringe was washed twice with 3 mL of TFA. To the resulting solution, diethyl ether pre-cooled at −80° C. was added until a total volume of 45 mL was reached. The centrifuge tube was then capped, and the reaction solution was vortexed for 5 seconds, followed by centrifugation at 3220 rcf (relative centrifugal force) for 4 minutes. Subsequently, the supernatant was removed by decantation. The obtained crude peptide was dissolved in 10 mL of 50:50 water and acetonitrile solution (+0.1% TFA), followed by freezing the solution with liquid nitrogen and subjecting it to lyophilization. When the peptide contained the azido moiety, another peptide cleavage solution (a mixture of TFA, water, thioanisole, and TIPS in a ratio of 94:2.5:2.5:1) was used instead of the solution described above.
Purification of the Z33 Variants from Resin
The crude Z33 analog was dissolved in a 95:5 water-acetonitrile solution and loaded onto 12 g of Biotage® Sfär C18 column. The gradient used for purification was as follows: 3 CV (column volume) 5% B, 1 CV 5-10% B, 30 CV 10-40% B. Fractions containing the desired product were collected in 50 mL centrifuge tubes, and the solution was frozen using liquid nitrogen. The target Z33 variant was obtained as white solids by removing the solvent through lyophilization. The purified Z33 analogs are characterized in Table 3
aaverage molecular mass,
bafter deconvolution
Synthesis of Electrophiles with Allyl Halides
A 50 mL round-bottom flask equipped with a stir bar was charged with 4-bromoaniline (1000 mg, 5.81 mmol) followed by the addition of 20 mL of dichloromethane to provide a clear solution. The flask was cooled in an ice bath, and then phenyl chloroformate (803 μL, 6.40 mmol) and pyridine (562 μL, 6.98 mmol) were added, and the resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was quenched with NaHCO3 (100 mL) and partitioned between ethyl acetate (300 mL) and water (200 mL). The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (100 mL) twice. The combined organic extract was washed with 1M HCl aq. (100 mL) and brine (50 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure. 500 mg of the 1.61 g residue obtained was then treated with a mixture of hexane and diethyl ether (10:1, 15 mL) and subjected to sonication for approximately 30 seconds. The reaction solution was filtered using a Büchner funnel, and the resulting solid was washed with hexane and diethyl ether (10:1, 5 mL). Finally, the solid was dried under reduced pressure, yielding the desired compound as a white solid (460 mg, 87%). The 1H and mass spectra of the obtained material were identical to those reported in the patent (WO2017205709).
A 50 mL round-bottom flask equipped with a stir bar was charged with 4-bromoaniline (250 mg, 1.45 mmol) followed by the addition of 4 mL of dioxane to provide a clear solution. The flask was cooled in an ice bath, and then 4-fluorophenyl chloroformate (279 mg, 1.60 mmol) and NaHCO3 (244 mg, 2.91 mmol) were added, and the resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was quenched with water (100 mL) and partitioned between ethyl acetate (300 mL) and water (200 mL). The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (100 mL) twice. The combined organic extract was washed with brine (50 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was then treated with a mixture of hexane and diethyl ether (10:1, 15 mL) and subjected to sonication for approximately 30 seconds. The reaction solution was filtered using a Büchner funnel, and the resulting solid was washed with a solution of hexane and diethyl ether (10:1, 5 mL). Finally, the solid was dried under reduced pressure, yielding the desired compound as a white solid (183 mg, 41%). 1H NMR (400 MHZ, CDCl3): δ 7.46 (d, J=8.8 Hz, 2H), 7.34 (d, J=8.4 Hz, 2H), 7.15 (dd, J=9.1, 4.5 Hz, 2H), 7.08 (dd, J=9.1, 8.0 Hz, 2H), 6.90 (brs, 1H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 4′-Aminoacetophenone (110 mg, 0.815 mmol), followed by the addition of 4 mL of dioxane to provide a clear solution. The flask was cooled in an ice bath, and then 4-bromophenyl chloroformate (230 mg, 0.978 mmol) and NaHCO3 (137 mg, 1.63 mmol) were added, and the resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was quenched with water (100 mL) and partitioned between ethyl acetate (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (100 mL) twice. The combined organic extract was washed with brine (50 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was then treated with a mixture of hexane and diethyl ether (10:1, 15 mL) and subjected to sonication for approximately 30 seconds. The reaction solution was filtered using a Büchner funnel, and the resulting solid was washed with a solution of hexane and diethyl ether (10:1, 5 mL). Finally, the solid was dried under reduced pressure, yielding the desired compound as a white solid (183 mg, 67%).
1H NMR (400 MHZ, DMSO): δ 10.68 (s, 1H), 7.95 (d, J=8.8 Hz, 2H), 7.69-7.59 (m, 4H), 7.25 (d, J=8.8 Hz, 2H), 2.53 (s, 3H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 3-iodophenol (324 mg, 1.47 mmol), followed by the addition of 7 mL of CH2Cl2 to provide a clear solution. Next, 4-Acetylphenyl Isocyanate (250 mg, 1.55 mmol) and DIEA (330 μL, 1.86 mmol) were added, and the resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was then treated with diethyl ether (4 mL) and subjected to sonication for approximately seconds. The reaction solution was filtered using a Büchner funnel, and the resulting solid was washed with dichloromethane (1 mL). Finally, the solid was dried under reduced pressure, yielding the desired compound as a white solid (241 mg, 43%).
1H NMR (400 MHZ, DMSO): δ 10.68 (s, 1H), 7.95 (d, J=8.8 Hz, 2H), 7.72-7.59 (m, 4H), 7.34-7.20 (m, 2H), 2.53 (s, 3H).
A 100 mL round-bottom flask equipped with a stir bar was charged with 4-bromophenol (314 mg, 1.82 mmol), followed by the addition of 8 mL of dioxane to provide a clear solution. Next, 5-azidopentanoic acid (200 mg, 1.40 mmol), DIEA (0.973 μL, 5.59 mmol), and HATU (797 mg, 2.10 mmol) were added, and the resulting mixture was stirred at room temperature for hours. The resulting mixture was quenched with saturated NaHCO3 solution (100 mL) and partitioned between CH2Cl2 (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with CH2Cl2 (100 mL) twice. The combined organic extract was washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a clear oil (338 mg, 81%).
1H NMR (400 MHZ, CDCl3): δ 7.49 (d, J=8.8 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H), 3.35 (t, J=6.6 Hz, 2H), 2.60 (t, J=7.3 Hz, 2H), 1.90-1.78 (m, 2H), 1.77-1.67 (m, 2H).
A 100 mL round-bottom flask equipped with a stir bar was charged with 3-iodophenol (400 mg, 1.82 mmol), followed by the addition of 8 mL of CH2Cl2 to provide a clear solution. Next, 5-azidopentanoic acid (200 mg, 1.40 mmol), DIEA (0.973 μL, 5.59 mmol), and HATU (797 mg, 2.10 mmol) were added, and the resulting mixture was stirred at room temperature for hours. The resulting mixture was quenched with saturated NaHCO3 solution (100 mL) and partitioned between CH2Cl2 (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with CH2Cl2 (100 mL) twice. The combined organic extract was washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a clear oil (269 mg, 78%).
1H NMR (400 MHZ, CDCl3): δ 7.57 (dt, J=7.5, 1.5 Hz, 1H), 7.46 (t, J=1.8 Hz, 1H), 7.15-7.03 (m, 2H), 3.35 (t, J=6.6 Hz, 2H), 2.60 (t, J=7.3 Hz, 2H), 1.90-1.78 (m, 2H), 1.79-1.66 (m, 2H).
The INT-01 was synthesized as previously described (WO2014065860). The 1H NMR spectra of the obtained material were identical to those reported in the literature.
A 20 mL scintillation glass vial equipped with a stir bar was charged with 3-iodophenol (45.4 mg, 0.309 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, INT-01 (40.0 mg, 0.238 mmol), DIEA (104 μL, 0.952 mmol), and HATU (90.5 mg, 0.357 mmol) were added, and the resulting mixture was stirred at room temperature for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a pink oil (50 mg, 57%).
1H NMR (400 MHZ, CDCl3): δ 7.57 (ddd, J=6.0, 3.1, 1.6 Hz, 1H), 7.50-7.44 (m, 1H), 7.14-7.05 (m, 2H), 3.75 (t, J=6.9 Hz, 2H), 3.31 (t, J=6.9 Hz, 2H), 3.06 (s, 3H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 1-bromo-4-(2-bromoethoxy)benzene (600 mg, 2.14 mmol), followed by the addition of 10 mL of acetone to provide a clear solution. Next, resorcinol (1.18 g, 10.7 mmol) and K2CO3 (1.48 g, 10.7 mmol) were added, and the resulting mixture was stirred at 45° C. for 12 hours. The resulting mixture was cooled to room temperature, followed by filtration using a Büchner funnel. The collected solid was then washed once with acetone (5 mL), and the resulting solution was concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a clear oil (415 mg, 63%).
1H NMR (400 MHz, CDCl3): δ 7.39 (d, J=9.0 Hz, 2H), 7.14 (t, J=8.5 Hz, 1H), 6.83 (d, J=9.0 Hz, 2H), 6.57-6.49 (m, 1H), 6.47-6.43 (m, 2H), 4.71 (s, 1H), 4.28 (s, 4H).
A 20 mL scintillation glass vial equipped with a stir bar was charged with INT-02 (150 mg, 0.485 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, 5-azidopentanoic acid (63.1 mg, 0.441 mmol), DIEA (300 μL, 1.76 mmol), and HATU (252 mg, 0.662 mmol) were added, and the resulting mixture was stirred at room temperature for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a pale pink solid (116 mg, 61%).
1H NMR (400 MHZ, CDCl3): δ 7.39 (d, J=9.0 Hz, 2H), 7.31-7.26 (m, 1H), 6.87-6.78 (m, 3H), 6.74-6.66 (m, 2H), 4.29 (s, 4H), 3.35 (t, J=6.7 Hz, 2H), 2.60 (t, J=7.2 Hz, 2H), 1.91-1.79 (m, 2H), 1.78-1.67 (m, 2H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 1-bromo-3-(2-bromoethoxy)benzene (600 mg, 2.14 mmol), followed by the addition of 10 mL of acetone to provide a clear solution. Next, resorcinol (1.18 g, 10.7 mmol) and K2CO3 (1.48 g, 10.7 mmol) were added, and the resulting mixture was stirred at 45° C. for 12 hours. The resulting mixture was cooled to room temperature, followed by filtration using a Büchner funnel. The collected solid was then washed once with acetone (5 mL), and the resulting solution was concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a clear oil (415 mg, 63%).
1H NMR (400 MHZ, CDCl3): δ 7.20-7.07 (m, 4H), 6.91-6.86 (m, 1H), 6.53 (ddd, J=8.3, 2.3, 1.0 Hz, 1H), 6.48-6.42 (m, 2H), 4.72 (s, 1H), 4.29 (s, 4H).
A 20 mL scintillation glass vial equipped with a stir bar was charged with INT-03 (75 mg, 0.242 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, 5-azidopentanoic acid (52.0 mg, 0.363 mmol), DIEA (158 μL, 0.968 mmol), and HATU (184 mg, 0.484 mmol) were added, and the resulting mixture was stirred at room temperature for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a pale pink solid (74 mg, 70%).
1H NMR (400 MHZ, CDCl3): δ 7.29 (t, J=8.1 Hz, 1H), 7.20-7.09 (m, 3H), 6.92-6.79 (m, 2H), 6.75-6.66 (m, 2H), 3.35 (t, J=6.7 Hz, 2H), 2.60 (t, J=7.2 Hz, 2H), 1.91-1.79 (m, 2H), 1.78-1.68 (m, 2H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 7-amino-heptanoic acid t-butyl ester (346 mg, 1.72 mmol), followed by the addition of 9 mL of DMF to provide a clear solution. Next, Fmoc-L-azidolysine (746 mg, 1.89 mmol), 2,4,6-colidine (0.909 μL, 6.84 mmol), and HATU (781 mg, 2.06 mmol) were added, and the resulting mixture was stirred at room temperature for 12 hours. The resulting mixture was quenched with saturated NaHCO3 solution (100 mL) and partitioned between ethyl acetate (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (100 mL) twice. The combined organic extract was washed with saturated ammonium chloride solution (100 mL) twice and brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 2:1 hexane:ethyl acetate) and dried under reduced pressure, yielding INT-04 as a clear oil (868 mg, 87%).
1H NMR (400 MHZ, CDCl3): δ 7.77 (d, J=7.5 Hz, 2H), 7.58 (d, J=7.5 Hz, 2H), 7.41 (t, J=7.5 Hz, 2H), 7.32 (t, J=7.4 Hz, 2H), 5.90 (s, 1H), 5.32 (d, J=8.3 Hz, 1H), 4.42 (s, 2H), 4.21 (t, J=6.7 Hz, 1H), 3.34-3.17 (m, 4H), 2.19 (t, J=7.4 Hz, 2H), 1.57 (s, 8H), 1.43 (s, 9H), 1.43-1.40 (m, 2H), 1.31 (s, 4H).
A 50 mL round-bottom flask equipped with a stir bar was charged with INT-04 (400 mg, 0.69 mmol), followed by the addition of piperidine in DMF (3.4 mL, 20 v/v %) to provide a clear solution. The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was diluted with 20 mL of toluene and concentrated under reduced pressure. To remove DMF, 20 mL of toluene was added to the reaction mixture, and the solvent was subsequently evaporated under reduced pressure three times. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 100:0 to 95:5 CH2Cl2+0.5 v/v % trimethylamine: MeOH) and dried under reduced pressure, yielding INT-05 as a clear oil (162 mg, 66%).
1H NMR (400 MHZ, CDCl3): δ 7.30 (s, 1H), 3.43 (dd, J=7.7, 4.8 Hz, 1H), 3.34-3.19 (m, 4H), 2.20 (t, J=7.5 Hz, 2H), 2.11-1.69 (m, 4H), 1.69-1.44 (m, 8H), 1.44 (s, 9H), 1.33 (p, J=3.5 Hz, 4H).
A 20 mL scintillation glass vial equipped with a stir bar was charged with INT-05 (93 mg, 0.261 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, Methyltetrazine acid (50 mg, 0.217 mmol), DIEA (154 μL, 0.868 mmol), and HATU (124 mg, 0.326 mmol) were added, and the resulting mixture was stirred at room temperature for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding INT-06 as a pale pink solid (101 mg, 82%).
1H NMR (400 MHZ, CDCl3): δ 8.58 (d, J=8.4 Hz, 2H), 7.50 (d, J=8.2 Hz, 2H), 6.19 (d, J=8.0 Hz, 1H), 5.98 (s, 1H), 4.36 (q, J=7.2 Hz, 1H), 3.68 (s, 2H), 3.23 (dq, J=7.3, 4.9 Hz, 4H), 3.10 (s, 3H), 2.19 (t, J=7.4 Hz, 2H), 1.90-1.77 (m, 1H), 1.66-1.41 (m, 7H), 1.44 (s, 9H), 1.40-1.24 (m, 6H).
A 10 mL round-bottom flask equipped with a stir bar was charged with INT-06 (30 mg, 0.053 mmol), followed by the addition of dichloromethane (2 mL) to provide a clear solution. Next, TFA (0.4 mL) was added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was used in the next step without further purification.
Dichloromethane (1 mL) was added to a 10 mL round-bottom flask containing the residue obtained from the previous reaction, resulting in a clear solution. Next, 3-iodophenol (17.5 mg, 0.080 mmol), DIEA (55.0 μL, 0.318 mmol), and HATU (34.3 mg, 0.090 mmol) were added, and the resulting mixture was stirred at room temperature for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding AHL-10 as a pale pink solid (35.9 mg, 95%).
MS (m/z): C30H37IN9O4+ (M+H+) 714.2, found 714.0′
Synthesis of [(cod)Pd(CH2TMS)2] (cod=1,5 cyclooctadiene)
This compound was synthesized as previously described (Nature 526, 687-691 (2015)). The 1H and 13C NMR spectra of the obtained material were identical to those reported in the literature.
Synthesis of Sodium 2′-dicyclohexylphosphino-2,6-dimethoxy-1,1′-biphenyl-3-sulfonate hydrate (sSPhos)
This compound was synthesized as previously described (Org. Lett. 23, 777-780 (2021)). The 1H and 13C NMR spectra of the obtained material were identical to those reported in the literature.
A 5 mL scintillation glass vial equipped with a stir bar was charged with 4-bromobenzenesulfonyl fluoride (20.4 mg, 0.085 mmol) and sSPhos (43.4 mg, 0.085 mmol), and 2-methyl tetrahydrofuran (0.51 mL). The reaction solution was sonicated for 1 minute until it became completely clear, and then the [(cod)Pd(CH2TMS)2] (30.0 mg, 0.077 mmol) was added, and the resulting mixture was stirred at room temperature for 2 hours. After the reaction time, pentane (1 mL) was added to the reaction mixture. The vial containing the white suspension was placed in a centrifuge and centrifuged at 3220 ref for 2 minutes. The cap was removed, and the supernatant was decanted. The stir bar was removed, and the solid was resuspended in 2-methyltetrahydrofuran (1.0 mL) and pentane (1.0 mL). The vial was capped and sonicated for 1 minute to obtain a homogeneous suspension. The vial was then centrifuged again at 3220 ref for 2 minutes. The cap was removed, and the supernatant was decanted. This sonication/centrifugation/decanting procedure was repeated twice. The resulting beige solid was dried under reduced pressure, yielding the desired compound as a pale brown solid (51 mg, 77%).
MS (m/z): C32H39FO7PPdS2+ (M−Br−Na+H+) 755.1, found 755.0.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. AHL-01 (16.6 mg, 0.057 mmol), sSPhos (28.9 mg, 0.056 mmol), [(cod)Pd(CH2TMS)2] (20.0 mg, 0.052 mmol), and 2-methyltetrahydrofuran (0.34 mL) were used, and the desired compound was obtained as a light brown solid (11 mg, 25%).
MS (m/z): C39H45NO7PPdS+ (M−Br−Na+H+) 808.2, found 808.1.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. 3-bromobenzenesulfonyl fluoride (6.8 mg, 0.029 mmol), sSPhos (14.5 mg, 0.028 mmol), [(cod)Pd(CH2TMS)2] (10.0 mg, 0.026 mmol), and 2-methyltetrahydrofuran (0.34 mL) were used, and OAC-03 was obtained as a light brown solid (21 mg, 95%).
MS (m/z): C32H39FO7PPdS2+ (M−Br−Na+H+) 755.1, found 755.0.
This compound was synthesized as previously described (J. Am. Chem. Soc., 142, 21237-21242 (2020)). The 1H, 13C and 31P NMR spectra of the obtained material were identical to those reported in the literature.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. AHL-02 (17.7 mg, 0.057 mmol), sSPhos (28.9 mg, 0.056 mmol), [(cod)Pd(CH2TMS)2] (20.0 mg, 0.052 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and the desired compound was obtained as a light brown solid (41 mg, 87%).
MS (m/z): C39H44FNO7PPdS+ (M−Br−Na+H+) 826.2, found 826.1.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. AHL-03 (9.5 mg, 0.028 mmol), sSPhos (14.5 mg, 0.028 mmol), [(cod)Pd(CH2TMS)2] (10.0 mg, 0.026 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and OAC-06 was obtained as a light brown solid (12 mg, 50%).
MS (m/z): C41H47NO8PPdS+ (M−Br−Na+H+) 850.2, found 850.1.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. AHL-04 (16.3 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and the desired compound was obtained as a light brown solid (39 mg, quantitative).
MS (m/z): C41H47NO8PPdS+ (M−I−Na+H+) 850.2, found 850.1.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. AHL-05 (12.7 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and OAC-08 was obtained as a light brown solid (34 mg, 96%).
MS (m/z): C37H47NO7PPdS+ (M−N2−Br−Na+H+) 786.2, found 786.1.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. ALH-06 (58.8 mg, 0.171 mmol), sSPhos (82.8 mg, 0.162 mmol), [(cod)Pd(CH2TMS)2] (60.0 mg, 0.154 mmol), and 2-methyltetrahydrofuran (1.0 mL) were used, and OAC-09 was obtained as a light brown solid (111 mg, 75%).
MS (m/z): C37H47N3O7PPdS+ (M−I−Na+H+) 814.2, found 814.1.
This compound was synthesized following the general procedure described in the section on the synthesis of OAC-01. ALH-07 (15.8 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and OAC-10 was obtained as a light brown solid (21 mg, 55%).
MS (m/z): C38H46N4O7PPdS+ (M−I−Na+H+) 839.2, found 839.1.
This compound was synthesized following the general procedure described previously. ALH-08 (18.5 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and OAC-11 was obtained as a light brown solid (32 mg, 80%).
MS (m/z): C45H55N3O9PPdS+ (M−Br−Na+H+) 950.0, found 950.1.
This compound was synthesized following the general procedure described previously. ALH-09 (17.0 mg, 0.039 mmol), sSPhos (19.0 mg, 0.037 mmol), [(cod)Pd(CH2TMS)2] (13.8 mg, 0.035 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and OAC-12 was obtained as a light brown solid (26 mg, 71%).
MS (m/z): C45H55N3O9PPdS+ (M−Br−Na+H+) 950.0, found 950.1.
This compound was synthesized following the general procedure described previously ALH-10 (15.0 mg, 0.021 mmol), sSPhos (10.3 mg, 0.020 mmol), [(cod)Pd(CH2TMS)2] (7.5 mg, 0.019 mmol), and 2-methyltetrahydrofuran (0.4 mL) were used, and OAC-12 was obtained as a light brown solid (22 mg, 85%).
MS (m/z): C56H71N9O9PPdS+ (M−I−Na+H+) 1182.0, found 1182.0.
Synthesized electrophile-attached Z33 reagents are listed in Table 4 The ADC mass analysis results for the synthesized electrophile-attached Z33 reagents are shown in
CFNMQQQRRFYEALH DPNLNEEQRNAKIKS IRDD
aElectrophile-attached amino acids are highlighted in bold.,
baverage molecular mass,
cafter deconvolution
PEP-04 (5.0 mg) was weighed into a 50 mL centrifuge tube, and Histidine buffer (500 mM, pH 5.8, 6 mL) was added. The mixture was then stirred by hand to obtain a homogeneous solution. Additionally, OAC-01 (2.3 mg) was weighed into a 0.6 mL microcentrifuge tube, and DMF (0.18 mL) was added to prepare a separate brown solution using sonication. The OAC-01 solution was added to the PEP-04 solution, and the mixture was vortexed briefly and allowed to stand at room temperature for 2 hours. Subsequently, the reaction solution was directly loaded onto a Biotage® Sfär C18 D column (12 g) and purified using the following conditions: 5 CV 5% B, 1 CV 5-10% B, 20 CV 10-35% B. 1 μL of a 20-fold diluted solution of the obtained fractions was analyzed by LC-MS, and the fractions containing only the target product were collected and lyophilized to obtain PEL-01 (1.3 mg) as a white solid.
PEP-04 (5.0 mg) and OAC-02 (2.2 mg) were used to synthesize PEL-02 following the same procedure as for PEL-01. PEL-02 (0.82 mg) was obtained as a white solid.
PEP-04 (5.0 mg) and OAC-03 (2.1 mg) were used to synthesize PEL-03 following the same procedure as for PEL-01. PEL-03 (2.2 mg) was obtained as a white solid.
PEP-04 (5.0 mg), OAC-04 (2.3 mg), and phosphate buffer (20 mM, pH 7.2) were used to synthesize PEL-04 following the same procedure as for PEL-01. PEL-04 (0.41 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-01 (2.1 mg) were used to synthesize PEL-05 following the same procedure as for PEL-01. PEL-05 (2.8 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-03 (2.1 mg) were used to synthesize PEL-06 following the same procedure as for PEL-01. PEL-06 (3.3 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-02 (2.2 mg) were used to synthesize PEL-07 following the same procedure as for PEL-01. PEL-07 (2.1 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-05 (2.3 mg) were used to synthesize PEL-08 following the same procedure as for PEL-01. PEL-08 (2.1 mg) was obtained as a white solid.
PEP-11 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-09 following the same procedure as for PEL-01. PEL-09 (2.1 mg) was obtained as a white solid.
PEP-01 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-10 following the same procedure as for PEL-01. PEL-10 (1.3 mg) was obtained as a white solid.
PEP-02 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-11 following the same procedure as for PEL-01. PEL-11 (0.45 mg) was obtained as a white solid.
PEP-03 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-12 following the same procedure as for PEL-01. PEL-12 (4.9 mg) was obtained as a white solid.
PEP-05 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-13 following the same procedure as for PEL-01. PEL-13 (2.8 mg) was obtained as a white solid.
PEP-06 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-14 following the same procedure as for PEL-01. PEL-14 (1.3 mg) was obtained as a white solid.
PEP-07 (5.0 mg) and OAC-01 (2.3 mg) were used to synthesize PEL-15 following the same procedure as for PEL-01. PEL-15 (2.7 mg) was obtained as a white solid.
PEP-04 (5.0 mg) and OAC-06 (2.3 mg) were used to synthesize PEL-16 following the same procedure as for PEL-01. PEL-16 (1.6 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-06 (2.3 mg) were used to synthesize PEL-17 following the same procedure as for PEL-01. PEL-17 (3.0 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-07 (2.5 mg) were used to synthesize PEL-18 following the same procedure as for PEL-01. PEL-18 (2.5 mg) was obtained as a white solid.
PEP-04 (5.0 mg) was weighed into a 1.5 mL microcentrifuge tube, and DMF (200 μL) was added. Additionally, OAC-09 (2.3 mg) was weighed into a 0.6 mL microcentrifuge tube, and DMF (0.18 mL) was added to prepare a separate brown solution using sonication. The OAC-solution was added to the PEP-04 solution, and the mixture was vortexed briefly and allowed to stand at room temperature for 30 minutes. Then the reaction mixture was transferred to a 50 mL centrifuge tube and diluted to 5 mL with water. Subsequently, the reaction solution was directly loaded onto a Biotage® Sfär C18 D column (12 g) and purified using the following conditions: 5 CV 5% B, 1 CV 5-10% B, 20 CV 10-35% B. 1 μL of a 20-fold diluted solution of the obtained fractions was analyzed by LC-MS, and the fractions containing only the target product were collected and lyophilized to obtain PEL-019 (2.8 mg) as a white solid.
PEP-09 (5.0 mg) and OAC-08 (2.2 mg) were used to synthesize PEL-20 following the same procedure as for PEL-01. PEL-20 (3.4 mg) was obtained as a white solid.
PEP-09 (15 mg) and OAC-09 (7.1 mg) were used to synthesize PEL-21 following the same procedure as for PEL-19. PEL-21 (10 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-10 (2.4 mg) were used to synthesize PEL-22 following the same procedure as for PEL-19. PEL-22 (1.2 mg) was obtained as a white solid.
PEP-02 (5.0 mg) and OAC-11 (2.6 mg) were used to synthesize PEL-23 following the same procedure as for PEL-01. PEL-23 (3.2 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-11 (2.6 mg) were used to synthesize PEL-24 following the same procedure as for PEL-01. PEL-24 (3.1 mg) was obtained as a white solid.
PEP-02 (5.0 mg) and OAC-12 (2.6 mg) were used to synthesize PEL-25 following the same procedure as for PEL-01. PEL-25 (3.2 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-12 (2.6 mg) were used to synthesize PEL-26 following the same procedure as for PEL-01. PEL-26 (4.5 mg) was obtained as a white solid.
PEP-07 (5.0 mg) and OAC-12 (2.6 mg) were used to synthesize PEL-27 following the same procedure as for PEL-01. PEL-27 (3.5 mg) was obtained as a white solid.
PEP-08 (5.0 mg) and OAC-12 (2.6 mg) were used to synthesize PEL-28 following the same procedure as for PEL-01. PEL-28 (0.5 mg) was obtained as a white solid.
PEP-13 (5.0 mg) and OAC-12 (2.6 mg) were used to synthesize PEL-29 following the same procedure as for PEL-19. PEL-29 (0.48 mg) was obtained as a white solid.
PEP-11 (15.7 mg) and OAC-09 (6.7 mg) were used to synthesize PEL-30 following the same procedure as for PEL-19. PEL-30 (9.5 mg) was obtained as a white solid.
PEP-12 (15.0 mg) and OAC-09 (7.1 mg) were used to synthesize PEL-31 following the same procedure as for PEL-19. PEL-31 (6.7 mg) was obtained as a white solid.
PEP-09 (5.0 mg) and OAC-13 (3.3 mg) were used to synthesize PEL-32 following the same procedure as for PEL-19. PEL-32 (0.32 mg) was obtained as a white solid.
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes (
The reaction was performed on a 210 μL scale using a 1.5 mL microcentrifuge tube. First, PEL-09 was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (Bio X Cell, 9.2 mg/mL, 32.2 μL), PEL-09 (2 mg/mL, 43.8 μL), HEPES buffer (161 mM, 124 μL, pH 8.7), and NaCl solution (4M, 10 μL) were added and mixed using a 20 μL pipette, followed by incubation at room temperature in the dark for 24 hours. The reaction mixture was diluted to 400 μL with citrate buffer (100 mM, pH 2.7) and filtered through an Amicon® filter (0.5 mL, 50KMWCO) at 14000 ref for 7 minutes. After filtration, 400 μL of citrate buffer (100 mM, pH 2.68) was added and centrifuged again, followed by centrifugation with 400 μL of Tris (100 mM, pH 8.1) buffer (total of 3 centrifugations). Finally, the remaining solution (roughly 20 μL) on the Amicon® filter was transferred to a microcentrifuge tube, and the Amicon® filter was washed twice with 40 μL of Tris buffer (100 mM, pH 8.1). The obtained solution was analyzed by LC-MS (
The reaction solution corresponding to 3.5 μg of IgG was taken into a PCR tube and diluted with Tris buffer (100 mM, pH 8.1) to a final volume of 3 μL. Then, Laemmli sample buffer (BIO-RAD, 2×+5v/v % 2-mercaptoethanol, 3 μL) was added, and the mixture was heated at 70° C. for 5 minutes. After returning the reaction solution to room temperature, 2 μL was loaded onto an SDS-Gel. Subsequently, electrophoresis, washing, staining, and analysis were performed using the methods described in General Information Section.
A reaction solution corresponding to 9.0 μg of IgG was taken into a PCR tube and diluted with Tris buffer (100 mM, pH 8.1) to a final volume of 6 μL. Separately, a mixed solution of PNGase F (New England BioLabs, 63 units) and Glycoprotein Denaturing Buffer (New England Biolabs, 1×, 4 μL) was prepared and added to the reaction solution, followed by incubation at 37° C. for 4 to 12 hours. Afterward, DTT (200 mM in water, 2 μL) was added, and the mixture was further incubated at 37° C. for 1 hour. Finally, the solution was diluted to a volume of 40 μL with a mixture of water and acetonitrile (95:5), and 8 μL of the diluted solution was injected into LC-MS.
aReaction was conducted at 37° C. using the 8 equivalents of the reagent.
b200 mM NaCl was added to the reaction.
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes (
The reaction was performed on a 2.1 mL scale using a 1.5 mL microcentrifuge tube. First, PEL-21 was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (Bio X Cell, 9.2 mg/mL, 322 μL), PEL-21 (2 mg/mL, 429 μL), HEPES buffer (160 mM, 1249 μL, pH 8.5), and NaCl solution (4M, 100 μL) were added and mixed using a 200 μL pipette, followed by incubation at room temperature on the shaker for 24 hours. The reaction mixture was diluted to 4 mL with citrate buffer (100 mM, pH 2.7) and filtered through an Amicon® filter (4 mL, 50KMWCO) at 3220 rcf for 10 minutes. After filtration, 2 mL of citrate buffer (100 mM, pH 2.7) was added and centrifuged twice, followed by centrifugation with 2 mL of Tris buffer (50 mM, pH 8.0) three times (total of 6 centrifugations). The remaining solution on the Amicon® filter was transferred to a microcentrifuge tube, and the Amicon® filter was washed twice with 40 μL of Tris buffer (100 mM, pH 8.1). Finally, modified trastuzumab (1.79 mg/mL) was obtained in 75% isolation yield.
a10 equivalents of the reagent were added at the beginning and after 4 hours of reaction, respectively.
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes (
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes (
Reactions were performed on a 10 μL scale using 0.2 mL PCR tubes (
The modified trastuzumab (30 μg) from entry 6 of 5.2.1 was diluted to 36 μL using Tris buffer (50 mM, pH 8.0). PNGase F (New England BioLabs, 200 units) was added to the solution and incubated at 37° C. for 4 hours. The reaction solution was then brought to room temperature, and urea solution (6M in 50 mM Tris pH 8.0, 14 μL) and DTT solution (200 mM, 1.5 μL) were added and incubated at 37° C. for 1 hour. After that, iodoacetamide (800 mM, 1 μL) was added and incubated at room temperature for 30 minutes. The resulting solution was diluted 2-fold using Tris buffer (50 mM, pH 8.0), and then Trypsin/Lys-C mix (Promega, 0.2 μg/μL in solution, 6 μL) was added and incubated at 37° C. for 18 hours. The reaction solution was then purified by pipetting with Ziptip, lyophilized, and analyzed by nLC-MS/MS (using nLC-MS/MS method B). Obtained raw data were analyzed using Thermo Scientific FreeStyle™ 1.6. De novo sequencing was performed with PEAKS 8.5 with the following search parameters: Parent Mass Error Tolerance=15.0 ppm; Fragment Mass Error Tolerance=0.02 Da, Enzyme=None; modification setting=table below; Max Variable PTM Per Peptide=10; Report #Peptides=10. Sequencing results were exported as .csv reports for all de novo candidates. Finally, fragments corresponding to trastuzumab sequences were manually searched from the .csv file, and the main modification sites were estimated using each fragment's peak area (Table 11). Since the mass of the modified lysine is the same as the mass of Pro-Arg (271.1644), both possibilities were considered in the fragment search (
Reactions were performed on a 10 μL scale using 0.2 mL PCR tubes. Modified trastuzumab (7.26 μg) obtained from entry 6 of 5.2.1 was mixed with DBCO-PEG4-VC-PAB-MMAE (MedChemExpress, 50 mM in DMSO), Tris buffer (pH 8.0), and NaCl solution to achieve final concentrations of 5 uM, 100 μM, 50 mM, and 200 mM, respectively. The reaction mixture was then incubated at room temperature for 24 hours. After incubation, the reaction solution was divided into two portions: 2 μL for analysis by LC-MS and 8 μL for analysis by HPLC. The 2 μL sample was prepared for LC-MS analysis in a similar manner as described in the “Mouse IgG Painting with Azido Moieties” section. The 8 μL sample was diluted to 40 μL using a 95:5 water-acetonitrile solution (+0.1% TFA), and 20 μL of this diluted sample was injected into the HPLC system for analysis (
Two sandwich ELISA assays have been developed in-house, one for the detection of the azido moiety conjugated mouse IgG (mIgG, E1), the other one for the detection of the whole mIgGs (modified and unmodified) in the sample (E2). Both E1 and E2 were run simultaneously on a same plate, using the same initial stock sample, and were processed in parallel at the same time for every steps. The same azido-free capture goat anti-mouse polyclonal antibody (Creative Diagnostics) was used for both E1-E2 assays at 1 μg/mL (100 μL per well) for incubation overnight at 4° C. Washing steps were performed thoroughly between each incubation by using 0.02% Tween 20-PBS. Saturation step (1 h, ROOM TEMPERATURE) and sample dilutions were all carried in 5% milk-PBS. For E1, the detection was performed using 1:150 dilution of DBCO-PEG4-Biotin (Jena Bioscience) (in 5% milk-PBS) while for E2 the detection was made using 1:2000 azido-free biotin-conjugated donkey anti-mouse polyclonal antibody (Creative Diagnostics) in 5% milk-PBS, both for 150 minutes at ROOM TEMPERATURE, in the dark. A dilution of 1:500 HRP-conjugated streptavidin (Thermo Fisher Scientific Inc) in 5% milk-PBS was then added for 1 h, ROOM TEMPERATURE, in the dark. TMB substrate (Thermo Fisher Scientific Inc) was added (100 μL per well, ROOM TEMPERATURE) simultaneously in E1 and E2 wells, and the reaction was stopped with 2N H2SO4 stop solution (Thermo Fisher Scientific Inc) after 5 to 10 minutes. Absorbances were measured at 450 nm and 540 nm (A0=A450 nm-A540 nm) using a Spark microplate reader (Tecan, USA). The background noise (Ablank) from the serum was subtracted to get the final absorbance of the sample (Asample=A0-Ablank). The ratio Asample (E1)/Asample (E2) was calculated to estimate the percentage of azido transfer on the total amount of IgG in the sample.
Reactions were performed on a 40 μL scale using 0.2 mL PCR tubes. First, the electrophile-attached Z33 peptide reagent PEL-21, PEL-30 or PEL-31 was dissolved in water to prepare a 2 mg/mL solution. Mouse serum (Invitrogen, S/N 24-5544-94, 31.2 μL) and electrophile-attached Z33 peptide reagent (2 mg/mL, 8.8 μL) were added to the PCR tubes to a final concentration of 100 mM of electrophile-attached Z33 reagent. The contents were mixed using a 10 μL pipette, followed by incubation at 37° C. in the dark for 2, 6, or 24 hours. To remove the excess amount of the remaining electrophile-attached Z33 reagent, the reaction mixture was filtered using an Amicon® filter. The reaction mixture was diluted to 200 μL with citrate buffer (100 mM, pH 2.7) and filtered through an Amicon® filter (0.5 mL, 30KMWCO) at rcf for 7 minutes. After filtration, 400 μL of PBS buffer was added and centrifuged again. Finally, the remaining solution (roughly 20 μL) on the Amicon® filter was transferred to a microcentrifuge tube. Obtained samples were then diluted 1:100 to 1:2000 in 5% milk-PBS for dosage using E1/E2 assays.
Preparation of Deferoxamine B-Attached Z33 Reagents and Radiolabeling with Zirconium-89
First, PEL-21 or PEL-31 were dissolved in HEPES buffer (100 mM in ultra-trace elemental water; Fischer scientific, pH 6.7) to prepare a 2 mg/mL solution of each. Deferoxamine-DBCO (Macrocycles™, 1 mM in DMSO, 11.1 μL) and PEL-21 or PEL-31 (360 μL) solutions were then added to the tube, stirred gently by hand, and the tube was placed on ice for 1 hour.
The obtained samples were incubated with 37 MBq of [89Zr]Zr-oxalate (dilution in 0.5M HEPES, pH 6.7) for 1 hour in the wet ice under mild agitation. Purification was then performed using a Zeba spin column (0.5 mL, 7KMWCO) to remove the non-complexed [89Zr]Zr-oxalate, for 5 min at 1,200 rcf. Instant-layer thin chromatography (iTLC) was performed to verify the success of the radiolabeling with elution in 0.1 M citrate buffer.
Pharmacokinetic of Radionuclide Transfer to mIgGs and Whole-Body Biodistribution
For injection in animals, samples were incubated with 55.5 MBq of [89Zr]Zr-oxalate (dilution in 0.5M HEPES, pH 6.7) for 1.5 hours in wet ice under mild agitation. No purification step was performed as a previous iTLC analysis showed no big purity difference between the pre and post purification of samples.
Swiss mice (n=4 per group) were IP injected with 1.2 to 1.5 MBq of [89Zr]Zr-PEL-21 or [89Zr]Zr-PEL-31. After 2 h, 24 h, 72 h, and 144 hours post injection, the mice were sacrificed to collect the following fluids and organs: blood, urine, feces, heart, lungs, liver, spleen, stomach, small intestines, colon, kidneys, bladder, pancreas, caecum, uterus+ovaries, gastrocnemius muscle, bone, skin, brain, and eyes. Each sample was weighted and count in a gamma counter (Wizard2, Perkin Elmer LLC, USA) to get the counts per minutes (cpm) for determining the percentage of injected activity per gram (% IA/g) in each organ. Cpm were decay-corrected by using the following formula: A=A0 e((−(ln2)Δt)/T) with A=decay-corrected cpm, A0=raw cpm, Δt=time difference between the injection and the cpm measurement, and T=half-life of the radionuclide (i.e. 78.41 h for Zr-89).
Preparation of the GLP1 analog-attached Z33 transfer reagents
GLP1 analog GLP-01 was synthesized according to the previously described method, except for the following conditions: ChemMatrix® H-Rink Amide resin (0.19 mmol/g, 150 mg) was used, and the noncanonical amino acid Fmoc-Aib-OH was prepared as a 0.40 M DMF solution and incorporated into the sequence by AFPS. For the introduction of Aib and N-terminal H, the condensation reagent was changed to PyAOP, and pump stroke number was changed to 35. A mixture of TFA, water, thioanisole, phenol, and TIPS in a ratio of 33:2:2:1 was used as the cleavage solution from the resin. Additionally, if CO2 adducts were observed in the crude product, the crude product was dissolved in a 95:5 water-acetonitrile solution (5 mL), and 200 μL of acetic acid was added before purification. The mixture was incubated at 37° C. for 1 hour and then loaded into a Biotage for purification (Table 12).
amonoisotopic mass
The reaction was performed using a 1.5 mL microcentrifuge tube (
The modification site of GLP-02 was inferred from the measurement results using nLC-MS/MS (method A). GLP-02 (100 nmol, 1 μL) was injected into the nLC-MS/MS system, and nLC-MS/MS method B was used for this experiment (Table 13).
aaverage mass, bafter deconvolution, *modified lysine
The reaction was performed using a 15 mL centrifuge tube. First, PEL-21 (4 mg) and GLP-(4.9 mg) were dissolved in HEPES buffer (100 mM, pH 6.7) to prepare a 2 mg/mL solution of each. The PEL-21 and GLP-02 solutions were then added to the centrifuge tube, stirred gently by hand, and the tube was placed on ice for 30 minutes. Then, the reaction solution was directly loaded onto a Biotage® Sfär C18 D column (12 g) and purified using the following conditions: 5 CV 5% B, 1CV 5-20% B, 25CV 20-60% B. 1 μL of a 5-fold diluted solution of the obtained fractions was analyzed by LC-MS, and the fractions containing only the target product were collected and lyophilized to obtain GLP-03 (6.22 mg) as a white solid (
PEL-30 (7.0 mg) and GLP-02 (7.8 mg) were used to synthesize GLP-04 following the same procedure as GLP-03 (
Reactions were performed on a 20 μL scale using 1.5 mL microcentrifuge tubes (
Pharmacodynamic Studies of Native IgG Painting with GLP1 Analogs in Living Mice
Swiss mice were SC injected, with either PBS (n=5), or with 10 mg/kg of semaglutide Ozempic® (Adipogen Inc.) (n=15), GLP-01 (n=15), GLP-03 (n=15), or GLP-04 (n=15). Body weight was monitored over 21 days for the entire cohort. At 24 h, 72 h, or 144 hours post injection, an IP-glucose tolerance test (Ip-GTT) was performed on the mice. Six hours before the Ip-GTT, the mice were fasted by removing the food, the enrichment, and by placing them in a new cage to avoid coprophagy. Access to water was, however kept free and ad libitum for the entire experiment. Five minutes before the Ip-GTT, basal glycemia was measured by taking a small drop of blood using tail pricks. Then, 2 g/kg of a 20% solution of dextrose (Sigma Aldrich Inc.) was IP injected followed by glycemia measurement over 120 min using a glucometer (Contour®). NB: At the 24 hours time-point, the test was stopped after 30-45 minutes as the glycemia of the mice was severely dropping and started showing moderate to severe hypoglycemia. The results are shown in
A tirzepatide-transfer reagent was synthesized as described previously (
The purified tirzepatide-transfer reagent was then evaluated for its reactivity against human IgG trastuzumab. Briefly, 5 μM of trastuzumab was reacted with 100 μM (20 equivalents) of the purified tirzepatide-transfer reagent under one of three conditions: 1) 100 mM HEPES pH 8.5 at room temperature for 24 hours; 2) 100 mM HEPES pH 8.5 at 37° C. for 24 hours; and, 3) PBS at 37° C. for 24 hours (
Synthesis of IgG binding peptide Z33 analogs were performed as described in Example 3. A co-crystal structure of mAB-FC and Z33 was used to determine lysine residues in the active site (
Each of the Z33 analogs containing cysteine were designed with the suitable distance for each lysine in mind. At the same time, palladium oxidative addition complex with finely designed electrophilic positions were prepared. The electrophile-attached IgG binding peptides were prepared by C-S arylation for selective IgG modification, as described previously (
The first target was K317 modification using the Z33 peptide as the IgG binding peptide (
The reaction was set up using trastuzumab as the IgG under basic pH conditions (
To evaluate whether quantitative modification could be achieved by changing the electrophile, a homocysteine was introduced in place of cysteine to optimize the distance between IgG and Z33 (
To demonstrate the generality of this reaction, other IgGs were used to determine reactivity with Z33. WN1 was used as another IgG1 (
Samples were then evaluated using Orbitrap LC-MS/MS to confirm the modification site and determine which lysine reacted with the reagent. A number of fragments were detected (
Next, other bioconjugation handles were tested to further demonstrate the generality of this reaction. Tetrazine was reacted as described above. LC-MS analysis of the reaction at three different conditions demonstrated that the reagent with tetrazine had a similar reactivity to that with azido under similar conditions (
One of the purposes of this technology is to create selective ADCs. The reaction was optimized and demonstrated that selective modification worked well even at the 1 mg scale (
A second lysine modification was then performed to evaluate whether the electrophile with a longer linker could react with lysine 248. The second lysine targeted was K248, which is located close to the M3 in Z33 (
The next step was to achieve double modification of the K317 and K248 on the same IgG. When the previous reagents were reacted sequentially with IgG, the double modified IgG was obtained as the main product and each modification reactivity was similar to the reactivity in the single modification (
Finally, a third lysine modification K288 was evaluated using a long linker. The R31 and D32 were shown to be located close to the K288 (
To modify IgG in vivo, the reagents need to react in neutral pH conditions. The reactivity of the reagents was evaluated using PBS and found that more than about 70% of Tmab was modified (
The nucleophilic Z33 variants were each prepared in a single step (
Next, a one-pot procedure was tested to determine whether it was necessary to isolate and purify the electrophilic Z33 (
Small compound conjugation via the azide group was evaluated for its reactivity with DBCO (
Thus, successful IgG modification was demonstrated in vitro and showed that most of the heavy chain could be modified by 5-azido-penatnoic acid-3-phenylester reagent (
An in-house sandwich ELISA was used to evaluate whether Z33 peptides react to mouse IgG when incubated in mouse serum (
To evaluate the whole-body biodistribution in mice, Z33 peptides were radiolabeled. Z33-P16p and Z33-E20Hcy were both successfully conjugated to DFO and used for radiolabeling (
For both peptides, quick renal clearance was discovered. The control Z33-P16p was found to be cleared in about 2 hours, while the Z33-H20Hcy peptide was found to remain in specific organs for up to 144 hours post-injection (
Finally, a proof-of-concept was developed to evaluate whether the PK/PD of GLP1 peptides using the Z33 transfer technology could be increased. An in vivo mouse model was set up (
An in vivo experiment was set up, wherein mice were administered PBS, semaglutide, GLP1 peptide, Z33-E20Hcy-GLP1, or pegylated Z33-E20Hcy-GLP1 subcutaneously. At 24, 72, and 144 hours post-injection, an intraperitoneal glucose tolerance test (Ip-GTT) was performed with 20% dextrose (
The pharmacodynamics of blood glucose management were also evaluated to determine for how long Z33-GLP1 peptide transfer reagents could regulate glucogenesis. Both Z33-GLP1 peptides were found to have an extended effect of around 6 days, whereas GLP1 alone and semaglutide were found to induce hypoglycemia (
Thus, the results demonstrate a biotechnology platform for in vivo biorthogonal site-selective modification of IgGs.
Peptide drugs are advantageous due to their high target selectivity, high efficacy, safety, and low cost (1-3). However, they also suffer from poor in vivo stability, short plasma half-life (a few minutes), and poor oral availability. Recent efforts in biotechnology focused on improving peptide pharmacokinetic (PK) and pharmacodynamic (PD) profiles using chemical modifications, to diminish renal clearance (4), increase chemical stability (5, 6), and enhance bioavailability (7).
One of the remarkable recent successes in the development of long-acting peptide drugs is exemplified by glucagon-like peptide-1 receptor agonists (GLP-1 RAs) (8-10). While native GLP-1 has a very short plasma half-life, of a few minutes, long-acting GLP-1 RAs have been proven efficient for the treatment of type II diabetes mellitus (T2DM) and obesity (11-16). GLP-RAs conjugation to neonatal Fc receptor (FcRn) binders, such as serum albumin or IgGs, leads to endosomal internalization, transportation and recycling to the blood (17, 18). Despite great achievements, the first generations of long-acting GLP-1 RAs are associated with high costs for IgG production and purification. New technologies that leverage native antibodies, obviate in vitro IgG production, and enable the assembly of the IgG-GLP-1 conjugate in vivo would greatly streamline the manufacturing process and reduce costs of next-generation long-acting GLP-1 RAs.
Here, a novel drug delivery platform to attach therapeutic payloads specifically to IgGs directly in vivo, aiming to extend the PK/PD of both existing and candidate drugs via FcRn binding, is described. The technology is at times referred to as “in vivo antibody painting” as different lysine (Lys) residues from the Fc domain of native IgGs can be subsequently modified site-selectively to incorporate multiple drugs (
Fc-binder affinity peptides were synthesized by Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS) using previously reported automated fast-flow peptide synthesis (AFPS) instrumentation (20). Their safety and duration of efficacy was assessed in two different mice models, WT lean Swiss and Lepob/ob, the latter having spontaneous type II diabetes mellitus (T2DM) related obesity. The results show promising outcomes for advancing the next generation of weight loss and T2DM drugs. Furthermore, the applicability of IgG painting to a variety of payloads (small molecules, peptides, and radionuclides) is demonstrated, highlighting its versatility and potential for numerous applications. This work thereby represents a novel impactful insight for designing long-acting drugs and antibody-drug conjugates.
The minimal Z-domain of protein A, a 33-mer peptide (Z33), was shown to bind the Fc fragment of IgG1 with an apparent dissociation constant (KD) of 43 nM (21). In a previous study, it was determined that specific amino acids in the sequence of the Z33 peptide have little to no influence on its binding to IgGs while other residues play a crucial role (22). The interaction between Glu-20 (E20) of protein A and Lys-317 (K317, (23, 24)) of the Fc domain of IgG1 was previously confirmed using co-crystal structure (
First several variants of Z33 were designed and synthesized using previously reported AFPS technology (
In vitro selective transfer of azido moieties onto human and mouse IgGs
Several electrophiles (I, II, III, IV, V) were synthesized, and their reactivity after 24 h was assessed in vitro, using hIgG1 trastuzumab (Tmab; 1 eq.) (
a10 equivalents of the reagent were added at the beginning and after 4 hours of reaction, respectively.
Peptide N3-2-I (20 eq.) incubated with a mixture of Tmab (1 eq.) and RNase A (1 eq.) showed that only Tmab ended being decorated with azido moieties (DAR 1-2) (
In Vitro Selective Transfer of GLP-1 Conjugates onto Mouse IgGs
Motivated by the encouraging results obtained with azido transfer, the IgG painting technology was applied to larger biomolecular cargos, such as GLP-1 RAs for extending their PK. Three different GLP-1 backbones were synthesized using AFPS (
Three different doses of the commercial semaglutide (0.5 mg/kg (˜5 nmol), 3 mg/kg (˜30 nmol) or 10 mg/kg (˜100 nmol)) were first SC injected in WT mice to verify the tolerability for GLP-1 RAs (Table 20). In the other cohorts, 2a,3a was SC administered following either 10 mg/kg (25 nmol), 30 mg/kg (75 nmol), or three injections of 10 mg/kg, 1 per week for 3 weeks (30 mg/kg total, 75 nmol total). As expected, hypoglycemia was observed starting 0.5 mg/kg of semaglutide and became critical for the highest dose suggesting 10 mg/kg as the maximum tolerated dose. None of the mice receiving SC injections of 2a,3a showed hypoglycemia or behavioral change, confirming the safety of IgG painting with GLP-1 RAs.
In Lepob/ob obese mice, two routes were assessed for the 10 mg/kg dose: SC and IP (Table 21). For Semaglutide, both routes did significantly impact the mice behavior and induce hypoglycemia. In the 2a,3a cohorts, the SC route was better tolerated with no significant toxicity while IP induced some behavioral changes. Investigations about the PD of 2a,3a following SC injection of 10 mg/kg were pursued in both mice models.
The Pharmacodynamic Profile of GLP-1 is Significantly Improved after In Vivo IgG Painting in Lean Mice
A dose response using the commercial semaglutide (0.5 mg/kg (˜5 nmol), 3 mg/kg (˜30 nmol), or 10 mg/kg (˜100 nmol), SC) allowed us to validate the WT Swiss mice model as suitable for measuring blood glucose change in addition to body weight loss (
Intraperitoneal glucose tolerance tests (Ip-GTT) demonstrated blood glucose decrease for all the GLP-1 analogs compared to the naïve mice, after 24 h of injection (
In vivo IgG painting with GLP-1a sustains body weight loss and improves blood glucose management in obese Lepob/ob mice for at least 10 days after SC injection.
Both males and females were used for this efficacy study to avoid sex-related biases. One single injection of 10 mg/kg (˜170 nmol) of Semaglutide, or 10 mg/kg (˜45 nmol) of 2a,3a was SC in Lepob/ob mice (
Lepob/ob are insulin resistant as confirmed by the blood glucose levels measured for the naïve, with 440±9 mg/dL vs 178±14 mg/dL for the non-obese C57BL/6J mice, both sexes combined (
In Vivo IgG Painting with GLP-1a Improved Metabolic Health in Lepob/ob Mice after IP Administration as Well
The IP route was assessed in male and female Lepob/ob mice (
IgG Painting with Radionuclides Highlights the Payload Versatility
It was hypothesized that the in vivo IgG painting approach can be used as a general drug delivery platform suitable for numerous kinds of payloads. Radionuclides are among the most challenging payloads but are particularly useful for in vivo detection and quantification. N3-1-3-I was radiolabeled using zirconium-89 and the fate of [89Zr]Zr-1-3 was assessed in female WT mice, after either intravenous (IV), SC, or IP injection of 1.2-1.5 MBq (
Longitudinal PET-CT imaging and ex vivo quantifications in organs confirmed the uptake of IV injected [89Zr]Zr-1-3 in organs rich in FcRn, for up to 6 days p.i. (
Altogether, these results indicate that in vivo IgG painting using electrophile peptide 2,3-I may be impactful for the development of long-acting drugs by proposing a broad platform freed from any cumbersome in vitro antibody engineering.
To enable the conjugation of two or more different drugs on one single IgG, two different strategies have been explored: (i) the site-specific modification of two different lysine side chains using different electrophilic warheads, (ii) the use of a bivalent electrophile. First, 5 novel Z33 variants (Table 16) were synthesized. For peptides 7,8, the L-Cys or L-Hcy residue substituted Met-3, then were conjugated to azido electrophiles VI and VII (
Multi-drug conjugation was then achieved through the linkage of peptides 2,3 to VIII, a dendronized electrophile bearing both an azido and a Tz moiety (
It is important to note that the drug delivery system can also be used in vitro, for engineering traditional antibody-drug conjugates (ADC), in a fast, site-selective, and highly reproducible manner (
Fusion to FcRn binder molecules for PK extension purposes was explored in the past two decades and has already led to the approval of several drugs (18, 35). Applied to GLP-1 analogs, such approaches have provided T2DM patients an efficient therapeutic option and recently, FDA approval was obtained for weight loss applications (36, 37). A recent report on AMG-133 indicated that the fusion of two GLP-1 moieties on an anti-GIP IgG enabled body-weight loss in db/db mice after 24 h and persistent up to 216 h post IP injection of 2 mg/kg. In the same study, blood glucose was reduced up to 144 h post injection (15). In addition, in humans, AMG-133 was safe and tolerable besides an increase of amylase and lipase, possessed a half-life of 14-16 days with peak of duration reached 4-7 days post SC injection, and maintained body weight loss up to 120 days after the last dose. These data suggest that an antibody-GLP-1 conjugate may be effective at a twice monthly dosing regimen rather than the once-weekly semaglutide, with a significant lower toxicity. The manufacturing of antibodies is however time- and resource-intensive, and among the most costly steps in the entire production pipeline. Therefore, the in vivo antibody painting technology developed proposes an efficient, straightforward, and cost efficient alternative.
The synthesis of 13 different Fc-binder peptides, derived from the structure of Z33 is reported (21). Every compound was obtained with high purity and good yield, as a result of AFPS synthesis technology (20). Seven electrophilic groups were synthesized, conjugated to the Fc-binder peptides to evaluate their transfer reactivity towards the heavy chain Fc domain of Tmab, enabling us to determine the best Fc-binder peptide/electrophile pair for moving forward to in vivo experiments. The ability to transfer a cargo from a peptide to a native IgG while circulating in blood, in two hours has been successfully demonstrated, which is believed to have not been reported before.
The proof-of-concept of in vivo IgG painting with GLP-1 analogs was performed in WT mice and Lepob/ob (38) to evaluate the safety, the PK/PD extension, and the efficacy on obesity and TD2M. In both models a sustained body weight loss for 10 to 15 days, associated with blood glucose decrease for 144 h post SC injection, was successfully demonstrated. Moreover, in the obesity model, a better response to insulin was also measured after 10 days following either SC or IP injection. While the outcome was similar, slight sex-related differences were observed for the treatment response among the male and female obese mice, a pattern observed also for GLP-1 agonists in humans (39). Overall, in vivo IgG painting with GLP-1 achieved either similar or better results than the commercial semaglutide, at a dose 4 times lower (40, 41).
Finally, in vivo IgG painting can achieve site-selective modification of either one or two lysine side chains, from both sides of the heavy chain Fc domain depending on the electrophile used, making the conjugation of two different drugs possible on the same antibody. The development of long-acting GLP-1 analogs is currently moving towards the combination of two or more incretin co-agonists to boost the effect on T2DM and weight loss (16, 42). While the engineering of bispecific antibodies is challenging, the IgG painting platform, which can be performed either in vitro or in vivo, introduces a facile modality to access multi-drug antibody conjugates in the body.
Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp-(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, and Fmoc-L-Val-OH were purchased from Novabiochem, Millipore Sigma, or Chem-Impex Inc. Fmoc-alpha-methylalanine (Fmoc-Aib-OH) and Fmoc-L-HomoCys(Trt)-OH (Fmoc-Hcy-OH) were purchased from Combi-Blocks Inc. 5-Azidopentanoic acid was purchased from ChemPep®. 1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oic acid (Fmoc-PEG8-CO2H) was purchased from AmBeed. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and (7-azabenzotriazol-lyloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) were purchased from P3 Biosystems. H-Rink Amide-ChemMatrix resin was purchased from PCAS BioMatrix Inc. Polyethylene filter paper for peptide synthesis (0.60 mm thick, pore size 7-12 μm) was purchased from Interstate Specialty Products. N,N-Dimethylformamide (OmniSolv® for Biosynthesis, DMF, stored with an AldraAmine trapping packet), diethyl ether (≥99.0% stabilized with BHT, Et2O), acetonitrile (≥99.9%, CH3CN), dichloromethane (≥99.8% stabilized with amylene, CH2Cl2), 1,4-dioxan (≥99.5% stabilized by BHT), 2-propanol (99.9%), 2-methyl-tetrahydrofuran (≥99.5% stabilized with BHT, 2-Me-THF), dimethylsulfide (≥99.5%, BioUltra for molecular biology, DMSO), and pentane (98% reagent grade) were obtained from Millipore-Sigma. Ethyl acetate (≥99.5%), hexane (≥98.5%, mixture of isomers), methanol (≥99.8%), and acetone (≥99.5%) were purchased from VWR. The water used in all reactions involving proteins, in the preparation of buffers, and in the preparation of mobile phases for purification was obtained via filtration of deionized water through a Millipore Sigma Milli-Q Ultrapure Water System. AldraAmine trapping packets and piperidine were purchased from Millipore Sigma. Amine-free DMF refers to DMF stored over AldraAmine trapping packets for a minimum of 24 hours prior to use. LC/MS grade water, acetonitrile, and formic acid were purchased from Thermo Fisher Scientific Inc and were used for liquid chromatography-mass spectrometry (LC-MS). All small compounds were purchased from TCI America, Millipore Sigma, AmBeed, Matrix Scientific, or Combi-Blocks in purities ≥95%, and were used without further purification (unless otherwise noted). PBS buffer (1× and 10×) were purchased from CORNING®. Reactions monitored by analytical thin-layer chromatography (TLC) were carried out using glass-backed plates pre-coated with silica gel impregnated with a fluorescent indicator (254 nm). All deuterium solvents were purchased from Cambridge Isotope Laboratories, Inc. C18-ZipTips (0.6 μL) were purchased from Millipore Sigma. Zeba spin desalting columns were purchased from Thermo Fisher Scientific Inc. Trastuzumab and its biosimilar were purchased from Syd Labs Inc., MedChemExpress LLC., and Bio X Cell.
NMR spectra were recorded on a Bruker Avance III HD 400 MHz spectrometer. All 1H NMR chemical shifts are expressed in parts per million (ppm, δ scale) and are referenced to the residual proton in the NMR solvent (CDCl3-d3: 7.26, DMSO-d6: 2.50). All 13C spectra recorded are proton decoupled. The 13C NMR chemical shifts are expressed in part per million (ppm, δ scale) and are referenced to the carbon resonance of the NMR solvent (CDCl3-d3: 77.16, DMSO-d6: 39.52). 1H NMR spectroscopic data are reported as follows: a chemical shift in ppm (multiplicity, coupling constants J (Hz), integration intensity, assigned number of protons in molecule). The multiplicities are abbreviated with s (singlet), br. s (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). In the case of combined multiplicities, the multiplicity with the larger coupling constant is stated first. The chemical shift of all signals is reported as the center of the resonance range, except in the case of multiplets, which are reported as ranges in chemical shift. All raw fid files were processed, and the spectra analyzed using the program MestReNova 14.2.3 from Mestrelab Research S. L. Copies of the 1H and 13C spectra can be found at the end of this document (unless otherwise noted).
LC-MS chromatograms and associated mass spectra were acquired using an Agilent Technologies 6550 Q-ToF LC-MS system. Solvent compositions are 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in acetonitrile (solvent B). For methods A, B, and C, a calibration solution containing m/z at 922.0098 constantly flowed through the system. The following LC-MS methods were used:
LC conditions: Zorbax 300SB C3 column: 2.1×150 mm, 5 μm, column temperature: 40° C., gradient: 0-1 min 1% B, 1-6 min 1-61% B, flow rate: 0.8 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300-3000 m/z, temperature of drying gas=350° C., flow rate of drying gas=11 L/min, pressure of nebulizer gas=60 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 175, and 750 V, respectively.
LC conditions: Zorbax 300SB C3 column: 2.1×150 mm, 5 μm, column temperature: 40° C., gradient: 0-1 min 1% B, 1-7 min 1-91% B, flow rate: 0.7 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300-3000 m/z, temperature of drying gas=350° C., flow rate of drying gas=11 L/min, pressure of nebulizer gas=60 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 175, and 750 V, respectively.
LC conditions: Zorbax 300SB C3 column: 2.1×150 mm, 5 μm, column temperature: 40° C., gradient: 0-1 min 1% B, 1-6 min 1-41% B, flow rate: 0.8 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300-3000 m/z, temperature of drying gas=350° C., flow rate of drying gas=11 L/min, pressure of nebulizer gas=60 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 175, and 750 V, respectively.
LC conditions: Aeris WIDEPORE C4 200 column: 2.1×150 mm, 3.6 μm, column temperature: 40° C., gradient: 0-2 min 1% B, 2-8 min 1-91% B, 8-10 min 91-95% B, flow rate: 0.3 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 100-1700 m/z, temperature of drying gas=200° C., flow rate of drying gas=14 L/min, pressure of nebulizer gas=55 psi, the capillary, fragmentor, and octapole voltages were set at 3500, 175, and 750 V, respectively.
LC conditions: Aeris WIDEPORE C4 200 column: 2.1×150 mm, 3.6 μm, column temperature: 40° C., gradient: 0-2 min 1% B, 2-7 min 1-61% B, 7-8 min 61-95% B, flow rate: 0.3 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 100-1700 m/z, temperature of drying gas=200° C., flow rate of drying gas=14 L/min, pressure of nebulizer gas=55 psi, the capillary, fragmentor, and octapole voltages were set at 3500, 175, and 750 V, respectively.
Data were processed using Agilent MassHunter Workstation Qualitative Analysis Version B.06.00 Software or Agilent MassHunter BioConfirm Software B.10.00. Deconvoluted masses of proteins were obtained using a maximum entropy algorithm. Unless otherwise depicted, the following parameters were used for deconvolution: Mass range is given in the experimental section for each peptide; Mass step was set to 1.00 Daltons; Baseline was set to Subtract baseline, Baseline factor as 7.00. The y-axis of all chromatograms shown in the
Mass spectra were obtained on an Agilent 6125B mass spectrometer attached to an Agilent 1260 Infinity LC. Solvent compositions are 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The following LC-MS method was used:
LC conditions: Poroshell 120 SB C18: 2.1×50 mm, 2.7 μm, column temperature: 40° C., gradient: 0-1 min 10% B, 1-5 min 10-100% B, 5-6 min 100% B, 6-7 min 100-10% B, flow rate: 0.4 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 100-1500 m/z, temperature of drying gas=350° C., flow rate of drying gas=13 L/min, pressure of nebulizer gas=35 psi, the capillary, fragmentor, and octapole voltages were set at 4000, 70, and 650 V, respectively.
Nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS)
Analysis was performed on an EASY-nLC 1200 nano-liquid chromatography system connected to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer or to an Orbitrap Fusion Eclipse Tribrid Mass Spectrometer (Thermo Fisher Scientific). Samples were run on a PepMap RSLC C18 column (C18, 50 μm×15 cm, 2 μm, 100 Å, Thermo Fisher Scientific, P/N ES901). An Acclaim PepMap 100 Trap column (C18, 75 μm×2 cm, 3 μm, 100 Å, Thermo Fisher Scientific, P/N 164946) was used for desalting. Solvent compositions are 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in 80% acetonitrile and 19.9% H2O (solvent B). The following conditions were used for each sample measurement:
nLC-MS/MS Method A
LC conditions: column temperature: 40° C., gradient: 0-30 min 30-95% B, 30-40 min 95% B, flow rate: 300 nL/min. MS conditions: positive ion spray voltage was set to 2200 V. Orbitrap detection was used for primary MS, with the following parameters: Application mode=peptide, cycle time=3 s, resolution=120000, mass range=normal, scan range=250-2000 m/z, RF Lens=30%, AGC target=standard, maximum injection time mode=auto, 1 microscan, data type=profile, polarity=positive. The following filters were applied for MS2 precursor selection: monoisotopic peak determination=peptide, filter type=threshold, Intensity threshold=5.0e5, include charge state=2-6, mass list type=m/z, target mass*, mass tolerance=25 ppm. Fragmentation was induced by higher-energy collisional dissociation (HCD) and electron-transfer dissociation with higher-energy collision (EThcD). Specifications HCD: isolation mode=quadrupole, isolation window=1.2 m/z, isolation offset=0.6 m/z, activation type=HCD, collision energy mode=fixed (30%), detection type=orbitrap, resolution=30000, mass range=normal, first mass=120 m/z, AGC target=standard, maximum injection time mode=dynamic, microscan, data type=profile. Specifications EThcD: isolation mode=quadrupole, isolation window=1.2 m/z, isolation offset=0.6 m/z, activation type=ETD, use calibrated charge-dependent ETD parameters, ETD supplemental activation=EThcD, SA collision energy=30%, detection=orbitrap, resolution=30000, mass range=normal, first mass=120 m/z, normalized AGC target=standard, maximum injection time=dynamic, 1 microscans, data type=profile.
LC conditions: column temperature: 40° C., gradient: 0-30 min 1-10% B, 30-120 min 10-81% B, 120-125 min 81-90% B, 125-135 min 90% B, flow rate: 300 nL/min. MS conditions: positive ion spray voltage was set to 2200 V, and negative ion spray voltage was set to 600 V. Orbitrap detection was used for primary MS, with the following parameters: Application mode=peptide, cycle time=3 s, resolution=120000, mass range=normal, scan range=200-1400 m/z, RF Lens=30%, AGC target=custom, normalized AGC target (%)=250, maximum injection time mode=auto, 1 microscan, data type=profile, polarity=positive. The following filters were applied for MS2 precursor selection: monoisotopic peak determination=peptide, exclude after 4 times, If occurs within 30 s, exclusion duration=30 s, mass tolerance=10 ppm, exclude isotope, include charge state=2-10, filter type=threshold, Intensity threshold=5.0e4, precursor selection range=200-1400, Fragmentation was induced by collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation with higher-energy collision (EThcD). Specifications CID: isolation mode=quadrupole, isolation window=1.3 m/z, activation type=CID, collision energy mode=fixed (30%), CID activation time=10 ms, activation Q=0.25, detection type=orbitrap, resolution=30000, mass range=normal, AGC target=custom, normalized AGC target=40%, maximum injection time mode=auto, 1 microscan, data type=centroid. Specifications HCD: isolation mode=quadrupole, isolation window=1.3 m/z, activation type=HCD, detection type=orbitrap, resolution=30000, mass range=normal, scan range mode=auto, AGC target=custom, normalized AGC target=40%, maximum injection time mode=auto, 1 microscan, data type=centroid. Specifications EThcD: isolation mode=quadrupole, isolation window=1.3 m/z, activation type=ETD, use calibrated charge-dependent ETD parameters, ETD supplemental activation=EThcD, SA collision energy=25%, detection=orbitrap, resolution=30000, mass range=normal, scan range mode=auto, normalized AGC target=40%, normalized AGC target=custom, maximum injection time=auto, 1 microscans, data type=profile.
The samples were analyzed using an Agilent Technologies 1290 Infinity II LC system which was computer-controlled through Agilent ChemStation software. The following methods were used:
Solvent compositions used in the UHPLC are 0.1% TFA in H2O (solvent A) and 0.1% TFA in acetonitrile (solvent B). ACQUITY UPLC Protein BEH C4 column: 2.1×50 mm, 1.7 μm (Waters, P/N 186004495), ACQUITY UPLC Protein BEH C4 VanGurde pre-column: 2.1×5 mm, 1.7 μm (Waters, P/N 186004623), column temperature: 27° C., gradient: 0-3 min 1% B, 3-13 min 1-61% B, flow rate: 0.5 mL/min.
A Biotage® Selekt automated flash chromatography system was used for the purification of small molecules and peptides. The columns, solvents, and solvent gradients employed for the purification of individual compounds are written in the synthesis section of each compound.
Bolt 4-12% Bis-Tris Plus 1.0 mm×15 well or 1.0 mm×10 well plates (Invitrogen), Mini Gel Tank (Invitrogen) along with the PowerPac HC (BIO-RAD) were used for SDS-PAGE analysis. SeeBlue® Plus2 standard (Invitrogen) was used as the molecular weight standard. Gels were run using Bolt MOPS SDS Running Buffer (1×, Invitrogen) under the conditions of 135 V for 55 min. After electrophoresis, the running buffer was discarded, and the gel was placed in deionized water. Subsequently, the water with gel was heated in a microwave for 1 min and 30 s. The heated water was replaced, and the gel was thoroughly rinsed. Gel staining was performed for 10 min using SimpleBlue SafeStain (Invitrogen). The stain was removed following staining, and the gel was immersed in deionized water. The tray with gel and deionized water was placed on a shaker overnight to remove the stain from the gels. Then, ChemDoc MP Imaging System (BIO-RAD) was used to analyze the stained gels.
The conversion rate for each reaction was calculated by one of the following methods.
Reported yields based on LC-MS spectra were determined by extracting the total ion current (TIC) spectra of all protein-containing species in the chromatogram utilizing Agilent MassHunter 6.0 software with the BioConfirm package. These extracted chromatograms were deconvoluted utilizing a maximum entropy algorithm and abundance of each species determined using total ion count.
Band densitometry was calculated using ImageJ software (imagej.nih.gov/ij/).
Densitometry-based yields were calculated based on the product: starting material protein ratio in lanes and standardized based on molecular weight.
After plotting bands and integration using ImageJ software, the value of each band was obtained as 7122.93 and 10408.80, respectively.
Starting material: right peak standardization (MW 49151) 10408.80/49151=0.21177
Modified product: left peak standardization (MW 53381) 7122.93/53381=0.13344
Thus, the determined conversion (%) corresponds to Standardized Product/(Standardized Total): 0.13344/(0.21177+0.13344)*100=39%
Chemical Synthesis of IgG Binding Peptide Z33 Analogs Automated Flow-based Peptide Synthesis System
The synthesis of the IgG binding peptide Z33 analogs were performed using the Automated fast-flow solid phase peptide synthesis (AFPS) system assembled in the Pentelute lab (20, 21). The synthesis conditions are summarized in the following table.
A plastic fritted syringe (6 mL) was equipped with polyethylene filter paper, and ChemMatrix® H-Rink Amide resin (0.49 mmol/g, 150 mg) was loaded onto it. The syringe assembly was then placed on a manifold. The resin was swollen in DMF, and subsequently, a homogeneous resin slurry was prepared by repeatedly drawing back 500 μL of the slurry using a 1 mL pipette. Next, the syringe was set to the AFPS system for peptide synthesis. After completion of the synthesis, the syringe was transferred from the AFPS system to the manifold and washed three times with CH2Cl2. To minimize methionine oxidation in the peptide sequence, drying of the resin was not conducted using air flow on the manifold but rather using nitrogen flow. Specifically, the syringe was capped with a rubber septum, and a needle connected to a nitrogen flow line was inserted into the septum for 15 min. The resulting peptide-bound dry resin was subjected to subsequent steps of cleavage and purification.
The peptide was synthesized using the method described in the “Synthesis of Z33 Variants Composed of Natural Amino Acids” section from the C-terminus of the sequence to the position of the unnatural amino acid. The resin in a plastic fritted syringe was transferred from the AFPS system to a manifold. Fmoc-protected unnatural amino acid (10 equivalents for the peptide on the resin) in HATU/DMF (0.38 M, 9.5 equivalents) solution and DIEA (15 equivalents) were added to the resin and allowed to react for 50 min at room temperature. The reaction solution was stirred with a spatula every 10 min. After the reaction, the reaction solution was removed, and the resin in the syringe was washed three times with 5 mL of DMF. Then, 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added and allowed to react for 10 min. The reaction solution was removed, and 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added again and allowed to react for 10 min. The reaction solution was removed, and the resin was washed three times with 4 mL of DMF. Addition of the subsequent natural amino acids to the peptide chain was again performed according to the procedure described in the “Synthesis of Z33 Variants Composed of Natural Amino Acids” section.
After synthesis of the peptide by AFPS, the fritted syringe with resin was placed on a manifold. A HATU/DMF solution (0.38 M, 9.5 equivalents) of the carboxylic acid corresponding to the acyl group (10 equivalents for the peptide on the resin) and DIEA (15 equivalents) were added and allowed to react for 50 min at room temperature. During that time, the reaction solution was stirred with a spatula every 10 min. After the reaction, the reaction solution was removed, and the resin in the syringe was washed three times with 10 mL of DMF. If the introduced acyl group contained the Fmoc protecting group, 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added and allowed to react for 10 min. The reaction solution was removed, and 3 mL of 40% piperidine/DMF solution (+2% formic acid) was added again and allowed to react for 10 min. The reaction solution was removed and washed three times with 4 mL of DMF and three times with CH2Cl2. To minimize methionine oxidation in the peptide sequence, the resin was not dried using airflow on the manifold but rather using nitrogen flow. Specifically, the syringe was capped with a rubber septum, and a needle connected to a nitrogen flow line was inserted into the septum for 15 min. The resulting peptide-bound dry resin was subjected to subsequent steps of cleavage and purification.
Cleavage of Z33 Variants from Resin
The peptide-bound resin was transferred to a 15 mL centrifuge tube. Subsequently, a 7.5 mL peptide cleavage solution (a mixture of TFA, water, EDT, and TIPS in a ratio of 94:2.5:2.5:1 by volume) was added to the centrifuge tube, and the reaction was allowed to proceed at room temperature for 2 hours on the shaker. Afterward, 5 mL of a plastic fritted syringe was placed on top of a 50 mL centrifuge tube, and the reaction solution containing the resin was filtered. The resin inside the syringe was washed twice with 3 mL of TFA. To the resulting solution, diethyl ether pre-cooled at −80° C. was added until a total volume of 45 mL was reached. The centrifuge tube was then capped, and the reaction solution was vortexed for 5 s, followed by centrifugation at 3220 rcf (relative centrifugal force) for 4 min. Subsequently, the supernatant was removed by decanting. The obtained crude peptide was dissolved in 10 mL of 50:50 water and acetonitrile solution (+0.1% TFA), followed by freezing the solution with liquid nitrogen and subjecting it to lyophilization. When the peptide contained the azido moiety, another peptide cleavage solution (a mixture of TFA, water, thioanisole, and TIPS in a ratio of 94:2.5:2.5:1 by volume) was used instead of the solution described above.
Purification of the Z33 Variants from Resin
The crude Z33 analog was dissolved in a 95:5 water-acetonitrile solution and loaded onto 12 g of Biotage® Sfär C18 column. The gradient used for purification was as follows: 3 CV (column volume) 5% B, 1 CV 5-10% B, 30 CV 10-40% B. Fractions containing the desired product were collected in 50 mL centrifuge tubes, and the solution was frozen using liquid nitrogen. The target Z33 variant was obtained as white solids by removing the solvent through lyophilization.
Synthesis of Electrophiles with Allyl Halides
A 100 mL round-bottom flask equipped with a stir bar was charged with 3-iodophenol (400 mg, 1.82 mmol), followed by the addition of 8 mL of CH2Cl2 to provide a clear solution. Next, 5-azidopentanoic acid (200 mg, 1.40 mmol), DIEA (0.973 μL, 5.59 mmol), and HATU (797 mg, 2.10 mmol) were added, and the resulting mixture was stirred at rt for 48 hours. The resulting mixture was quenched with saturated NaHCO3 solution (100 mL) and partitioned between CH2Cl2 (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with CH2Cl2 (100 mL) twice. The combined organic extract was washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate v/v) and dried under reduced pressure, yielding the desired compound as a clear oil (269 mg, 78%).
1H NMR (400 MHZ, CDCl3): δ 7.57 (dt, J=7.5, 1.5 Hz, 1H), 7.46 (t, J=1.8 Hz, 1H), 7.15-7.03 (m, 2H), 3.35 (t, J=6.6 Hz, 2H), 2.60 (t, J=7.3 Hz, 2H), 1.90-1.78 (m, 2H), 1.79-1.66 (m, 2H).
13C NMR (101 MHz, CDCl3): δ 171.27, 150.95, 135.04, 130.81, 130.79, 121.19, 93.62, 51.08, 33.69, 2.
HRMS (DART) m/z calcd for C11H13O2N3I ([M+H]+) 346.0047, found 346.0052.
A 50 mL round-bottom flask equipped with a stir bar was charged with 3-iodophenol (324 mg, 1.47 mmol), followed by the addition of 7 mL of CH2Cl2 to provide a clear solution. Next, 4-acetylphenyl isocyanate (250 mg, 1.55 mmol) and DIEA (330 μL, 1.86 mmol) were added, and the resulting mixture was stirred at rt for 1 hour. The resulting mixture was then treated with diethyl ether (4 mL) and subjected to sonication for approximately 30 s. The reaction solution was filtered using a Büchner funnel, and the resulting solid was washed with dichloromethane (1 mL). Finally, the solid was dried under reduced pressure, yielding the desired compound as a white solid (241 mg, 43%).
1H NMR (400 MHZ, DMSO): δ 10.68 (s, 1H), 7.95 (d, J=8.8 Hz, 2H), 7.72-7.59 (m, 4H), 7.34-7.20 (m, 2H), 2.53 (s, 3H).
A 100 mL round-bottom flask equipped with a stir bar was charged with 4-bromophenol (314 mg, 1.82 mmol), followed by the addition of 8 mL of dioxane to provide a clear solution. Next, 5-azidopentanoic acid (200 mg, 1.40 mmol), DIEA (0.973 μL, 5.59 mmol), and HATU (797 mg, 2.10 mmol) were added, and the resulting mixture was stirred at rt for 48 hours. The resulting mixture was quenched with saturated NaHCO3 solution (100 mL) and partitioned between CH2Cl2 (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with CH2Cl2 (100 mL) twice. The combined organic extract was washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a clear oil (338 mg, 81%).
1H NMR (400 MHz, CDCl3): δ 7.49 (d, J=8.8 Hz, 2H), 6.97 (d, J=8.8 Hz, 2H), 3.35 (t, J=6.6 Hz, 2H), 2.60 (t, J=7.3 Hz, 2H), 1.90-1.78 (m, 2H), 1.77-1.67 (m, 2H).
13C NMR (101 MHz, CDCl3): δ 171.41, 149.74, 132.59, 123.44, 119.02, 51.13, 33.78, 28.34, 22.13.
HRMS (DART) m/z calcd for C11H13O2N3Br ([M+H]+) 298.0186, found 298.0183.
The BB-10-intermediate was synthesized as previously described (WO2014065860). The 1H NMR spectra of the obtained material were identical to those reported in the literature.
A 20 mL scintillation glass vial equipped with a stir bar was charged with 3-iodophenol (45.4 mg, 0.309 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, BB-10-intermediate (40.0 mg, 0.238 mmol), DIEA (104 μL, 0.952 mmol), and HATU (90.5 mg, 0.357 mmol) were added, and the resulting mixture was stirred at rt for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a pink oil (50 mg, 57%).
1H NMR (400 MHZ, CDCl3): δ 7.57 (ddd, J=6.0, 3.1, 1.6 Hz, 1H), 7.50-7.44 (m, 1H), 7.14-7.05 (m, 2H), 3.75 (t, J=6.9 Hz, 2H), 3.31 (t, J=6.9 Hz, 2H), 3.06 (s, 3H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 1-bromo-4-(2-bromoethoxy)benzene (600 mg, 2.14 mmol), followed by the addition of 10 mL of acetone to provide a clear solution. Next, resorcinol (1.18 g, 10.7 mmol) and K2CO3 (1.48 g, 10.7 mmol) were added, and the resulting mixture was stirred at 45° C. for 12 hours. The resulting mixture was cooled to room temperature, followed by filtration using a Büchner funnel. The collected solid was then washed once with acetone (5 mL), and the resulting solution was concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the BB-11-intermediate as a clear oil (415 mg, 63%).
1H NMR (400 MHZ, CDCl3): δ 7.39 (d, J=9.0 Hz, 2H), 7.14 (t, J=8.5 Hz, 1H), 6.83 (d, J=9.0 Hz, 2H), 6.57-6.49 (m, 1H), 6.47-6.43 (m, 2H), 4.71 (s, 1H), 4.28 (s, 4H).
13C NMR (101 MHZ, CDCl3): δ 160.05, 157.90, 156.85, 132.46, 130.38, 129.66, 116.68, 113.49, 108.43, 108.06, 107.22, 102.49, 66.89, 66.57.
A 20 mL scintillation glass vial equipped with a stir bar was charged with BB-11-intermediate (150 mg, 0.485 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, 5-azidopentanoic acid (63.1 mg, 0.441 mmol), DIEA (300 μL, 1.76 mmol), and HATU (252 mg, 0.662 mmol) were added, and the resulting mixture was stirred at rt for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a pale pink solid (116 mg, 61%).
1H NMR (400 MHZ, CDCl3): δ 7.39 (d, J=9.0 Hz, 2H), 7.31-7.26 (m, 1H), 6.87-6.78 (m, 3H), 6.74-6.66 (m, 2H), 4.29 (s, 4H), 3.35 (t, J=6.7 Hz, 2H), 2.60 (t, J=7.2 Hz, 2H), 1.91-1.79 (m, 2H), 1.78-1.67 (m, 2H).
A 50 mL round-bottom flask equipped with a stir bar was charged with 1-bromo-3-(2-bromoethoxy)benzene (600 mg, 2.14 mmol), followed by the addition of 10 mL of acetone to provide a clear solution. Next, resorcinol (1.18 g, 10.7 mmol) and K2CO3 (1.48 g, 10.7 mmol) were added, and the resulting mixture was stirred at 45° C. for 12 hours. The resulting mixture was cooled to room temperature, followed by filtration using a Büchner funnel. The collected solid was then washed once with acetone (5 mL), and the resulting solution was concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 3:1 hexane:ethyl acetate) and dried under reduced pressure, yielding BB-12-intermediate as a clear oil (415 mg, 63%).
1H NMR (400 MHZ, CDCl3): δ 7.20-7.07 (m, 4H), 6.91-6.86 (m, 1H), 6.53 (ddd, J=8.3, 2.3, 1.0 Hz, 1H), 6.48-6.42 (m, 2H), 4.72 (s, 1H), 4.29 (s, 4H).
13C NMR (101 MHZ, CDCl3): δ 160.01, 159.52, 156.86, 130.72, 130.37, 124.38, 122.95, 118.13, 113.89, 108.44, 107.18, 102.49, 66.85, 66.50.
HRMS (ESI) m/z calcd for C14H14O3Br ([M+H]+) 309.0126, found 309.0133.
A 20 mL scintillation glass vial equipped with a stir bar was charged with BB-12-intermediate (75 mg, 0.242 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, 5-azidopentanoic acid (52.0 mg, 0.363 mmol), DIEA (158 μL, 0.968 mmol), and HATU (184 mg, 0.484 mmol) were added, and the resulting mixture was stirred at rt for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding the desired compound as a pale pink solid (74 mg, 70%).
1H NMR (400 MHZ, CDCl3): δ 7.29 (t, J=8.1 Hz, 1H), 7.20-7.09 (m, 3H), 6.92-6.79 (m, 2H), 6.75-6.66 (m, 2H), 3.35 (t, J=6.7 Hz, 2H), 2.60 (t, J=7.2 Hz, 2H), 1.91-1.79 (m, 2H), 1.78-1.68 (m, 2H).
13C NMR (101 MHz, CDCl3): δ 171.51, 159.40, 159.35, 151.58, 130.62, 129.95, 124.28, 122.83, 117.98, 114.29, 113.73, 112.28, 108.44, 66.64, 66.57, 51.06, 33.75, 28.26, 22.10.
HRMS (ESI) m/z calcd for C19H21O4N3Br ([M+H]+) 434.0715, found 434.0673.
A 50 mL round-bottom flask equipped with a stir bar was charged with 7-amino-heptanoic acid t-butyl ester (346 mg, 1.72 mmol), followed by the addition of 9 mL of DMF to provide a clear solution. Next, Fmoc-L-azidolysine (746 mg, 1.89 mmol), 2,4,6-colidine (0.909 μL, 6.84 mmol), and HATU (781 mg, 2.06 mmol) were added, and the resulting mixture was stirred at rt for 12 hours. The resulting mixture was quenched with saturated NaHCO3 solution (100 mL) and partitioned between ethyl acetate (200 mL) and water (100 mL). The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (100 mL) twice. The combined organic extract was washed with saturated ammonium chloride solution (100 mL) twice and brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 95:5 to 2:1 hexane:ethyl acetate) and dried under reduced pressure, yielding BB-13-intermediate-1 as a clear oil (868 mg, 87%).
1H NMR (400 MHZ, CDCl3): δ 7.77 (d, J=7.5 Hz, 2H), 7.58 (d, J=7.5 Hz, 2H), 7.41 (t, J=7.5 Hz, 2H), 7.32 (t, J=7.4 Hz, 2H), 5.90 (s, 1H), 5.32 (d, J=8.3 Hz, 1H), 4.42 (s, 2H), 4.21 (t, J=6.7 Hz, 1H), 3.34-3.17 (m, 4H), 2.19 (t, J=7.4 Hz, 2H), 1.57 (s, 8H), 1.43 (s, 9H), 1.43-1.40 (m, 2H), 1.31 (s, 4H).
13C NMR (101 MHZ, CDCl3): δ 173.28, 171.35, 156.33, 143.85, 141.45, 127.89, 127.21, 125.13, 120.14, 80.19, 67.13, 54.94, 51.26, 47.28, 39.62, 35.52, 32.38, 29.38, 28.69, 28.63, 28.24, 26.60, 24.99, 22.80.
HRMS (ESI) m/z calcd for C32H44O5N5 ([M+H]+) 578.3342, found 578.3315.
A 50 mL round-bottom flask equipped with a stir bar was charged with BB-13-intermediate-1 (400 mg, 0.69 mmol), followed by the addition of piperidine in DMF (3.4 mL, 20 v/v %) to provide a clear solution. The resulting mixture was stirred at rt for 2 hours. The resulting mixture was diluted with 20 mL of toluene and concentrated under reduced pressure. To remove DMF, 20 mL of toluene was added to the reaction mixture, and the solvent was subsequently evaporated under reduced pressure three times. The obtained residue was purified by column chromatography (using Biotage® Sfär Silica 60 μm 25 g column, 100:0 to 95:5 CH2Cl2+0.5 v/v % trimethylamine: MeOH) and dried under reduced pressure, yielding BB-13-intermediate-2 as a clear oil (162 mg, 66%).
1H NMR (400 MHZ, CDCl3): δ 7.30 (s, 1H), 3.43 (dd, J=7.7, 4.8 Hz, 1H), 3.34-3.19 (m, 4H), 2.20 (t, J=7.5 Hz, 2H), 2.11-1.69 (m, 4H), 1.69-1.44 (m, 8H), 1.44 (s, 9H), 1.33 (p, J=3.5 Hz, 4H).
13C NMR (101 MHZ, CDCl3): δ 173.68, 173.41, 80.22, 54.89, 51.30, 39.22, 35.58, 34.12, 29.46, 28.79, 28.75, 28.24, 26.71, 25.05, 22.94.
HRMS (ESI) m/z calcd for C17H34O3N5 ([M+H]+) 356.2662, found 356.2644.
A 20 mL scintillation glass vial equipped with a stir bar was charged with BB-13-intermediate-2 (93 mg, 0.261 mmol), followed by the addition of 2 mL of CH2Cl2 to provide a clear solution. Next, methyltetrazine acid (50 mg, 0.217 mmol), DIEA (154 μL, 0.868 mmol), and HATU (124 mg, 0.326 mmol) were added, and the resulting mixture was stirred at rt for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate) and dried under reduced pressure, yielding BB-13-intermediate-3 as a pale pink solid (101 mg, 82%).
1H NMR (400 MHZ, CDCl3): δ 8.58 (d, J=8.4 Hz, 2H), 7.50 (d, J=8.2 Hz, 2H), 6.19 (d, J=8.0 Hz, 1H), 5.98 (s, 1H), 4.36 (q, J=7.2 Hz, 1H), 3.68 (s, 2H), 3.23 (dq, J=7.3, 4.9 Hz, 4H), 3.10 (s, 3H), 2.19 (t, J=7.4 Hz, 2H), 1.90-1.77 (m, 1H), 1.66-1.41 (m, 7H), 1.44 (s, 9H), 1.40-1.24 (m, 6H).
13C NMR (101 MHz, CDCl3): δ 173.24, 171.27, 170.37, 167.39, 163.89, 139.63, 130.95, 130.26, 128.43, 80.18, 53.21, 51.21, 43.46, 39.62, 35.49, 32.22, 29.32, 28.70, 28.63, 28.24, 26.62, 24.98, 22.76, 21.28.
HRMS (ESI) m/z calcd for C28H42O4N9 ([M+H]+) 568.3360, found 568.3334.
A 10 mL round-bottom flask equipped with a stir bar was charged with BB-13-intermediate-3 (30 mg, 0.053 mmol), followed by the addition of dichloromethane (2 mL) to provide a clear solution. Next, TFA (0.4 mL) was added, and the resulting mixture was stirred at rt for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was used in the next step without further purification.
Dichloromethane (1 mL) was added to a 10 mL round-bottom flask containing the residue obtained from the previous reaction, resulting in a clear solution. Next, 3-iodophenol (17.5 mg, 0.080 mmol), DIEA (55.0 μL, 0.318 mmol), and HATU (34.3 mg, 0.090 mmol) were added, and the resulting mixture was stirred at rt for 12 hours. The resulting mixture was directly purified by column chromatography (using Biotage® Sfär Silica 60 μm 10 g column, 80:20 to 1:1 hexane:ethyl acetate v/v) and dried under reduced pressure, yielding BB-13 as a pale pink solid (35.9 mg, 95%).
1H NMR (400 MHZ, CDCl3): δ 8.58 (d, J=8.1 Hz, 2H), 7.56 (d, J=7.5 Hz, 1H), 7.53-7.42 (m, 3H), 7.14-7.02 (m, 2H), 6.15 (d, J=8.0 Hz, 1H), 5.97 (t, J=6.0 Hz, 1H), 4.35 (q, J=7.2 Hz, 1H), 3.67 (s, 2H), 3.23 (dq, J=7.0, 4.5 Hz, 4H), 3.09 (s, 3H), 2.80 (s, 1H), 2.53 (t, J=7.4 Hz, 2H), 1.84 (dq, J=14.2, 7.3 Hz, 1H), 1.72 (p, J=7.3 Hz, 2H), 1.65-1.55 (m, 2H), 1.56-1.45 (m, 3H), 1.47-1.23 (m, 6H).
13C NMR (101 MHz, CDCl3): δ 171.84, 171.25, 170.38, 167.43, 163.90, 151.08, 139.55, 135.04, 131.03, 130.90, 130.82, 130.27, 128.50, 121.30, 93.65, 53.26, 51.22, 43.53, 39.57, 38.75, 34.18, 32.11, 29.79, 29.34, 28.70, 28.62, 26.56, 24.73, 22.79, 21.30.
HRMS (ESI) m/z calcd for C30H37O4N9I ([M+H]+) 714.2013, found 714.2008.
Synthesis of [(cod)Pd(CH2TMS)2] (cod=1,5 cyclooctadiene)
This compound was synthesized as previously described (28). The 1H and 13C NMR spectra of the obtained material were identical to those reported in the literature.
Synthesis of sodium 2′-dicyclohexylphosphino-2,6-dimethoxy-1,1′-biphenyl-3-sulfonate hydrate (sSPhos)
This compound was synthesized as previously described (43). The 1H and 13C NMR spectra of the obtained material were identical to those reported in the literature.
A 5 mL scintillation glass vial equipped with a stir bar was charged with BB-7 (58.8 mg, 0.171 mmol) and sSPhos (82.8 mg, 0.162 mmol), and 2-methyltetrahydrofuran (0.51 mL). The reaction solution was sonicated for 1 min until it became completely clear, and then the [(cod)Pd(CH2TMS)2] (60.0 mg, 0.154 mmol) was added, and the resulting mixture was stirred at rt for 2 hours. After the reaction time, pentane (1 mL) was added to the reaction mixture. The vial containing the white suspension was placed in a centrifuge and centrifuged at 3220 rcf for 2 min. The cap was removed, and the supernatant was decanted. The stir bar was removed, and the solid was resuspended in 2-methyltetrahydrofuran (1.0 mL) and pentane (1.0 mL). The vial was capped and sonicated for 1 min to obtain a homogeneous suspension. The vial was then centrifuged again at 3220 rcf for 2 min. The cap was removed, and the supernatant was decanted. This sonication/centrifugation/decanting procedure was repeated twice. The resulting beige solid was dried under reduced pressure to give the desired crude product as a light brown solid (111 mg, 75%). The obtained crude product was used in the following reaction without further purification.
MS (m/z): C37H47N3O7PPdS+ ([M−I−Na+H]+) 814.2, found 814.1.
This compound was synthesized following the general procedure described in the section on the synthesis of BB-14. BB-8 (16.3 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and BB-15 was obtained as a light brown solid (39 mg, quantitative).
MS (m/z): C41H47NO8PPdS+ ([M−I−Na+H]+) 850.2, found 850.1.
This compound was synthesized following the general procedure described in the section on the synthesis of BB-14. BB-9 (12.7 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and BB-16 was obtained as a light brown solid (34 mg, 96%).
MS (m/z): C37H47NO7PPdS+ ([M−N2−Br−Na+H]+) 786.2, found 786.1.
This compound was synthesized following the general procedure described in the section on the synthesis of BB-14. BB-10 (15.8 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and BB-17 was obtained as a light brown solid (21 mg, 55%).
MS (m/z): C38H46N4O7PPdS+ ([M−I−Na+H]+) 839.2, found 839.1.
This compound was synthesized following the general procedure described in the section on the synthesis of BB-14. BB-11 (18.5 mg, 0.043 mmol), sSPhos (20.7 mg, 0.040 mmol), [(cod)Pd(CH2TMS)2] (15.0 mg, 0.039 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and BB-18 was obtained as a light brown solid (32 mg, 80%).
MS (m/z): C45H55N3O9PPdS+ ([M−Br−Na+H]+) 950.0, found 950.1.
This compound was synthesized following the general procedure described in the section on the synthesis of BB-14. BB-12 (17.0 mg, 0.039 mmol), sSPhos (19.0 mg, 0.037 mmol), [(cod)Pd(CH2TMS)2] (13.8 mg, 0.035 mmol), and 2-methyltetrahydrofuran (0.5 mL) were used, and BB-19 was obtained as a light brown solid (26 mg, 71%).
MS (m/z): C45H55N3O9PPdS+ ([M−Br−Na+H]+) 950.0, found 950.1.
This compound was synthesized following the general procedure described in the section on the synthesis of BB-14. BB-13 (15.0 mg, 0.021 mmol), sSPhos (10.3 mg, 0.020 mmol), [(cod)Pd(CH2TMS)2] (7.5 mg, 0.019 mmol), and 2-methyltetrahydrofuran (0.4 mL) were used, and BB-20 was obtained as a light brown solid (22 mg, 85%).
MS (m/z): C56H71N9O9PPdS+ ([M−I−Na+H]+) 1182.0, found 1182.0.
Synthesized electrophile-attached Z33 reagents are listed in Table 16.
Each modified peptide was synthesized as follows:
1 (15 mg) was weighed into a 1.5 mL microcentrifuge tube, and DMF (200 μL) was added. Additionally, BB-14 (7.1 mg) was weighed into a 0.6 mL microcentrifuge tube, and DMF (0.18 mL) was added to prepare a separate brown solution using sonication. The BB-14 solution was added to the solution of 1, and the mixture was vortexed briefly and allowed to stand at room temperature for 30 min. Then the reaction mixture was transferred to a 50 mL centrifuge tube and diluted to 5 mL with water. Subsequently, the reaction solution was directly loaded onto a Biotage® Sfär C18 D column (12 g) and purified using the following conditions: 5 CV 5% B, 1 CV 5-10% B, 20 CV 10-35% B. 1 μL of a 20-fold diluted solution of the obtained fractions was analyzed by LC-MS, and the fractions containing only the target product were collected and lyophilized to obtain N3-1-I (6.7 mg, 45%) as a white solid.
2 (15.0 mg) and BB-14 (7.1 mg) were used to synthesize N3-2-I following the same procedure as for N3-1-I. N3-2-I (6.7 mg, 42%) was obtained as a white solid.
3 (15.7 mg) and BB-14 (6.7 mg) were used to synthesize N3-3-I following the same procedure as for N3-1-I. N3-3-I (9.5 mg, 58%) was obtained as a white solid.
4 (5.0 mg) and BB-14 (2.3 mg) were used to synthesize N3-4-I following the same procedure as for N3-1-I. N3-4-I was obtained as a white solid.
5 (5.0 mg) and BB-14 (2.3 mg) were used to synthesize N3-5-I following the same procedure as for N3-1-I. N3-5-I was obtained as a white solid.
6 (5.0 mg) and BB-14 (2.3 mg) were used to synthesize N3-6-I following the same procedure as for N3-1-I. N3-6-I was obtained as a white solid.
2 (5.0 mg) and BB-15 (2.5 mg) were used to synthesize BB-21 following the same procedure as for N3-1-I. BB-21 (2.5 mg, 47%) was obtained as a white solid.
2 (5.0 mg) and BB-16 (2.2 mg) were used to synthesize BB-22 following the same procedure as for N3-1-I. BB-22 (3.4 mg, 65%) was obtained as a white solid.
2 (5.0 mg) and BB-17 (2.4 mg) were used to synthesize BB-23 following the same procedure as for N3-1-I. BB-23 (1.2 mg, 23%) was obtained as a white solid.
2 (5.0 mg) and BB-20 (3.3 mg) were used to synthesize N3-12-VIII following the same procedure as for N3-1-I. N3-12-VIII (0.32 mg, 6%) was obtained as a white solid.
BB-2 (5.0 mg) and BB-18 (2.6 mg) were used to synthesize N3-7-VI following the same procedure as for N3-1-I. N3-7-VI (3.2 mg, 59%) was obtained as a white solid.
BB-3 (5.0 mg) and BB-18 (2.6 mg) were used to synthesize N3-8-VI following the same procedure as for N3-1-I. N3-8-VI (3.1 mg, 57%) was obtained as a white solid.
BB-2 (5.0 mg) and BB-19 (2.6 mg) were used to synthesize N3-7-VII following the same procedure as for N3-1-I. N3-7-VII (3.2 mg, 59%) was obtained as a white solid.
BB-3 (5.0 mg) and BB-19 (2.6 mg) were used to synthesize N3-8-VII following the same procedure as for N3-1-I. N3-8-VII (4.5 mg, 83%) was obtained as a white solid.
BB-4 (5.0 mg) and BB-19 (2.6 mg) were used to synthesize N3-9-VII following the same procedure as for N3-1-I. N3-9-VII (3.5 mg, 64%) was obtained as a white solid.
BB-5 (5.0 mg) and BB-19 (2.6 mg) were used to synthesize N3-10-VII following the same procedure as for N3-1-I. N3-10-VII (0.5 mg, 9%) was obtained as a white solid.
BB-6 (5.0 mg) and BB-19 (2.6 mg) were used to synthesize N3-11-VII following the same procedure as for N3-1-I. N3-11-VII (0.48 mg, 9%) was obtained as a white solid.
Click handle transfer to IgGs using electrophile-Z33 variants.
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes. First, the electrophile-attached Z33 peptide reagent was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (MedChemExpress LLC. or Bio X Cell), electrophile-attached Z33 peptide reagent (2 mg/mL), HEPES (pH 8.5), and NaCl were added to achieve final concentrations of 10 μM, 100 μM, 100 mM, and 200 mM respectively. The contents were mixed using a 10 μL pipette, followed by incubation at room temperature in the dark for 24 hours. The resulting reaction solution was quenched with a 100 mM glycine buffer or 100 mM Tris buffer and analyzed by LC-MS.
A reaction solution corresponding to 9.0 μg of IgG was taken into a PCR tube and diluted with Tris buffer (100 mM, pH 8.1) to a final volume of 6 μL. Separately, a mixed solution of PNGase F (New England BioLabs, 63 units) and Glycoprotein Denaturing Buffer (New England Biolabs, 1×, 4 μL) was prepared and added to the reaction solution, followed by incubation at 37° C. for 4 to 12 hours. DTT (200 mM in water, 2 μL) was added, and the mixture was further incubated at 37° C. for 1 hour. Finally, the solution was diluted to a volume of 40 μL with a mixture of water and acetonitrile (95:5), and 8 μL of the diluted solution was injected into LC-MS.
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes. First, the electrophile-attached Z33 peptide reagent was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (Bio X Cell), electrophile-attached Z33 peptide reagent (2 mg/mL), HEPES (pH 8.5), and NaCl were added to achieve final concentrations of 10 μM, 100 μM, 100 mM, and 200 mM respectively. The contents were mixed using a 10 μL pipette, followed by incubation at 37° C. in the dark for 24 hours. 4 hours after the start of incubation, an additional 10 equivalent of electrophile-attached Z33 peptide was added (200 μM in total). The resulting reaction solution was quenched with a 100 mM glycine buffer or 100 mM Tris buffer and analyzed by LC-MS. Bioconjugation to other IgGs (human IgG1,2,4 & mouse IgG1) (See
Reactions were performed on a 10 μL scale using 0.2 mL PCR tubes. First, the electrophile-attached Z33 peptide reagent was dissolved in water to prepare a 2 mg/mL solution. Denosumab (Invitrogen), dupilumab (Invitrogen), or mouse IgG1 (Invitrogen, subtype controlled)), electrophile-attached Z33 peptide reagent (2 mg/mL), HEPES buffer (pH 8.5), and NaCl solution were added to achieve final concentrations of 5 μM, 100 μM, 100 mM, and 200 mM. The contents were mixed using a 5 μL pipette and incubated at room temperature in the dark for 24 hours. The resulting reaction solution was quenched with a 100 mM Tris buffer and analyzed by LC-MS. Sample preparation for LC-MS analysis was performed in the same manner as described in the “Bioconjugation to Trastuzumab (IgG1) (See
The modified trastuzumab (30 μg) from entry 6 of 5.2.1 was diluted to 36 μL using Tris buffer (50 mM, pH 8.0). PNGase F (New England BioLabs, 200 units) was added to the solution and incubated at 37° C. for 4 hours. The reaction solution was then brought to room temperature, and urea solution (6 M in 50 mM Tris pH 8.0, 14 μL) and DTT solution (200 mM, 1.5 L) were added and incubated at 37° C. for 1 hour. After that, iodoacetamide (800 mM, 1 μL) was added and incubated at room temperature for 30 min. The resulting solution was diluted 2-fold using Tris buffer (50 mM, pH 8.0), and then Trypsin/Lys-C mix (Promega, 0.2 μg/μL in solution, 6 μL) was added and incubated at 37° C. for 18 hours. The reaction solution was then purified by pipetting with Ziptip, lyophilized, and analyzed by nLC-MS/MS (using nLC-MS/MS method B). Obtained raw data were analyzed using Thermo Scientific FreeStyle™ 1.6. De novo sequencing was performed with PEAKS 8.5 with the following search parameters: Parent Mass Error Tolerance=15.0 ppm; Fragment Mass Error Tolerance=0.02 Da, Enzyme=None; modification setting are listed in the table below; Max Variable PTM Per Peptide=10; Report #Peptides=10. Sequencing results were exported as .csv reports for all de novo candidates. Finally, fragments corresponding to trastuzumab sequences were manually searched from the .csv file, and the main modification sites were estimated using each fragment's peak area.
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes. First, the electrophile-attached Z33 peptide reagent was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (Bio X Cell), electrophile-attached Z33 peptide reagent (2 mg/mL), HEPES buffer (pH 8.5), and NaCl solution were added to achieve final concentrations of 10 μM, 200 μM, 100 mM, and 200 mM. The contents were mixed using a 10 μL pipette, followed by incubation at room temperature or 37° C. in the dark for 24 hours. The resulting reaction solution was quenched with a 100 mM Tris buffer and analyzed by LC-MS. Sample preparation for LC-MS analysis was performed in the same manner as described in the “Bioconjugation to Trastuzumab (IgG1) (See
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes. First, the electrophile-attached Z33 peptide reagent was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (Bio X Cell), electrophile-attached Z33 peptide reagent (2 mg/mL), HEPES buffer (pH 8.5), and NaCl solution were added to achieve final concentrations of 10 μM, 200 μM, 100 mM, and 200 mM. The contents were mixed using a 10 μL pipette, followed by incubation at room temperature or 37° C. in the dark for 24 hours. The resulting reaction solution was quenched with a 100 mM Tris buffer and analyzed by LC-MS. Sample preparation for LC-MS analysis was performed in the same manner as described in the “Bioconjugation to Trastuzumab (IgG1) (See
First Tmab modification reaction was performed on a 200 μL scale using 1.5 mL microcentrifuge tube. N3-2-I was dissolved in water to prepare a 2 mg/mL solution. Trastuzumab (Bio X Cell), N3-2-I (2 mg/mL), HEPES buffer (pH 8.5), NaCl solution were added to achieve final concentrations of 10 μM, 100 μM, 100 mM, and 200 mM. The contents were mixed using a 200 μL pipette and incubated at room temperature for 24 hours. The resulting reaction solution was transferred to an Amicon 30K MWCO filter, and 200 μL of a 100 mM citric acid (pH 2.7) was added. The solvent was removed by centrifugation, and 200 μL of a 50 mM Tris buffer (pH 8.0) was added. Another round of centrifugation, 30 μL of a reaction mixture was obtained. Sample preparation for LC-MS analysis was performed in the same manner as described in the “Bioconjugation to Trastuzumab (IgG1) (See
Second Tmab modification was performed on a 20 μL scale using 0.2 mL PCR tubes. N3-8-VI was dissolved in water to prepare a 2 mg/mL solution. Modified Tmab above, N3-8-VI (2 mg/mL), HEPES buffer (pH 8.5), NaCl solution were added to achieve final concentrations of 10 μM, 100 μM, 100 mM, and 200 mM. The contents were mixed using a 20 μL pipette and incubated at 37° C. for 24 hours. The resulting reaction solution was quenched with a 100 mM Tris buffer and analyzed by LC-MS. Sample preparation for LC-MS analysis was performed in the same manner as described in the “Bioconjugation to Trastuzumab (IgG1) (See
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes. First, N3-2-I was dissolved in water to prepare a 2 mg/mL solution. Tmab (Bio X Cell), RNAse A (VWR), N3-2-I (2 mg/mL), and PBS were added to achieve final concentrations of 10 μM, 10 μM, 200 μM, and 1×. The contents were mixed using a 5 μL pipette and incubated at 37° C. for 24 hours. The resulting reaction solution was transferred to an Amicon 10K MWCO filter, and 200 μL of a 100 mM citric acid (pH 2.7) was added. The solvent was removed by centrifugation, and 200 μL of a 100 mM Tris buffer (pH 8.1) was added. Another round of centrifugation, 30 μL of a reaction mixture was obtained. Sample preparation for LC-MS analysis was performed in the same manner as described in the “Bioconjugation to Trastuzumab (IgG1) (See
Antibody-Drug Conjugate (ADC) Preparation with MMAE Transfer Reagent
Reactions were performed on a 20 μL scale using 0.2 mL PCR tubes. First, N3-2-I and DBCO-PEG4-Val-Cit-PAB-MMAE (BROADPHARM) were dissolved to prepare 2 mg/mL (in water) and 2 mM (in DMSO) solutions, respectively. A mixture of HEPES buffer and NaCl solution or PBS was added to the tubes, followed by N3-2-I (2 mg/mL, 4.29 μL) and DBCO-PEG4-Val-Cit-PAB-MMAE (2 mM, 1.1 μL) were added, mixed using a 10 μL pipette, and incubated at 4° C. for 30 min. The final concentrations of HEPES buffer (pH 8.5), NaCl solution, and PBS buffer were 100 mM, 200 mM and 1×, respectively. Tmab (Bio X Cell, 9.2 mg/mL, 1.61 μL) was then added to the reaction solution and incubated at room temperature or 37° C. for hours. The resulting reaction solution was quenched with 100 mM Tris buffer and analyzed by SDS-PAGE.
The reaction mixture corresponding to 3.5 μg of IgG was taken to a PCR tube, and diluted with Tris buffer (100 mM, pH 8.1) to a final volume of 3 μL. Then, Laemmli sample buffer (BIO-RAD, 2×+5% 2-mercaptoethanol, 3 μL) was added, and the mixture was heated at 70° C. for 5 min. After returning the reaction solution to room temperature, 2 μL was loaded onto an SDS-Gel. Subsequently, electrophoresis, washing, staining, and analysis were performed using the methods described in General Information Section.
All animal experiments were performed under an MIT institute approved IACUC protocol following federal, state, and local guidelines for the care and use of animals (protocol number #0821-058-24). The studies were performed either on 35-45 g (12-16 weeks-old) wild-type female Swiss mice, 25-30 g (14-20 weeks-old) male and female C57BL/6J mice, or 55-70 g (16 weeks-old) male and female obese Lepob/ob mice, all purchased from The Jackson Laboratory (MA, USA). Mice were housed with free access to normal food diet and water ad libitum unless stated otherwise for the need of the experiment. For subcutaneous (SC) injections, the mice were shortly anesthetized beforehand with 2-3% isoflurane along with O2, then shaved on the right flank. Intravenous injections (IV) were performed through IV catheterization; the mice were anesthetized with 2-3% isoflurane along with O2, then a catheter was inserted in the lateral tail vein to ensure the injection was properly done. Intraperitoneal injections (IP) were performed on non-anesthetized mice, in the lower right abdominal quadrant. Blood collection for the ELISA assays and biodistributions was performed as a terminal procedure by heart puncture immediately after the sacrifice. For Ip-GTT and Ip-ITT mice were restrained and gently pressed at the tail following tail pricks. The animals were sacrificed by CO2 inhalation followed by cervical dislocation. All compounds injected in mice were either USP grade, sterile, or filtered using 0.22 μm.
Mouse IgG Painting with Azido Moieties
Two sandwich ELISA assays have been developed in-house, one for the detection of the azido moiety conjugated mouse IgG (mIgG, E1), the other one for the detection of the whole mIgGs (modified and unmodified) in the sample (E2). Both E1 and E2 were run simultaneously on the same plate, using the same initial stock sample, and were processed in parallel at the same time for every step. The same azido-free capture goat anti-mouse polyclonal antibody (Creative Diagnostics) was used for E1-E2 assays at 1 μg/mL (100 μL per well) for incubation overnight at 4° C. Washing steps were performed thoroughly between each incubation by using 0.02% Tween 20-PBS. Saturation step (1 h, RT) and sample dilutions were all carried in 5% milk-PBS. For E1, the detection was performed using 1:150 dilution of DBCO-PEG4-Biotin (Jena Bioscience) (in 5% milk-PBS) while for E2 the detection was made using 1:2000 azido-free biotin-conjugated donkey anti-mouse polyclonal antibody (Creative Diagnostics) in 5% milk-PBS, both for 150 min at RT, in the dark. A dilution of 1:500 HRP-conjugated streptavidin (Thermo Fisher Scientific Inc.) in 5% milk-PBS was then added for 1 h, RT, in the dark. TMB substrate (Thermo Fisher Scientific Inc.) was added (100 μL per well, RT) simultaneously in E1 and E2 wells, and the reaction was stopped with 2N H2SO4 stop solution (Thermo Fisher Scientific Inc.) after 5 to 10 min. Absorbances were measured at 450 nm and 540 nm (A0=A450nm−A540nm) using a Spark microplate reader (Tecan, USA). The background noise (Ablank) from the serum was subtracted to get the final absorbance of the sample (Asample=A0−Ablank). The ratio Asample (E1)/Asample (E2) was calculated to estimate the percentage of azido transfer on the total amount of IgG in the sample.
Reactions were performed on a 40 μL scale using 0.2 mL PCR tubes. First, the azido electrophile-attached Z33 peptide reagent 1, 2 or 3 was dissolved in water to prepare a 2 mg/ml solution. Mouse serum (Invitrogen, S/N 24-5544-94, 31.2 μL) and electrophile-attached Z33 peptide reagent (2 mg/mL, 8.8 μL) were added to the PCR tubes to a final concentration of 100 mM of electrophile-attached Z33 reagent. The contents were mixed using a 10 μL pipette, followed by incubation at 37° C. in the dark for 2, 6, or 24 hours. To remove the excess amount of the remaining electrophile-attached Z33 reagent, the reaction mixture was filtered using an Amicon® filter. The reaction mixture was diluted to 200 μL with citrate buffer (100 mM, pH 2.7) and filtered through an Amicon® filter (0.5 mL, 30KMWCO) at 14000 rcf for 7 min. After filtration, 400 μL of PBS buffer was added and centrifuged again. Finally, the remaining solution (roughly 20 μL) on the Amicon® filter was transferred to a microcentrifuge tube. Obtained samples were then diluted 1:100 to 1:2000 in 5% milk-PBS for dosage using E1/E2 assays.
Female Swiss mice (n=3 per group) were either IP or SC injected with 10 mg/kg (˜90 nmol) to 30 mg/kg (˜280 nmol) of N3-1, N3-2, or N3-3 (diluted in 1×PBS). Blood was recovered by intracardiac puncture (terminal procedure) at 24 hours post injection, then was centrifuged 10 min at 15,000 rcf to get serum. Obtained samples were then diluted 1:2 in 5% milk-PBS for dosage using E1/E2 assays.
Mouse IgG Painting with Radionuclides
Preparation of Deferoxamine B-Attached Z33 Reagents and Radiolabeling with Zirconium-89
First, 1, 2, 3 were dissolved in HEPES buffer (100 mM in ultra-trace elemental water; Fisher Scientific, pH 6.7) to prepare a 2 mg/mL solution of each. Deferoxamine-DBCO (obtained from Macrocycles, 1 mM in DMSO, 11.1 μL) and 1, 2, 3 (360 μL) solutions were then added to the tube, stirred gently by hand, and the tube was placed on ice for 1 hour.
The obtained samples were incubated with 37 MBq of [89Zr]Zr-oxalate (obtained from Mallinckrodt Institute of Radiology, Nuclear Pharmacy Cyclotron; dilution in 0.5 M HEPES, pH 6.7) for 1 hour in the wet ice under mild agitation. Purification was then performed using a Zeba spin column (0.5 mL, 7K MWCO) to remove the non-complexed [89Zr]Zr-oxalate, for 5 min at 1,200 rcf. Instant-thin layer chromatography (iTLC) was performed to verify the success of the radiolabeling with elution in 0.1 M citrate buffer.
In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
The present application claims the benefit of priority under 35 U.S.C. § 119 (e) to U.S. Provisional Application No. 63/587,137, filed Oct. 1, 2023, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63587137 | Oct 2023 | US |
