Patent Application
20250082768
The present invention relates to antibody conjugates, and methods of manufacturing the same. More especially, the present invention relates to providing an antibody, linker and an active agent, for example a drug or labelling moiety, to produce a conjugate. Additionally, the present invention provides an improved linker for use in conjugates and methods of introducing said linker into said conjugates. More specifically, the conjugate may be an antibody drug conjugate (ADC).
The selective delivery of drugs to malignant cells without undesired toxicity towards healthy tissues is one of the primary goals in the development of modern cancer therapeutics. Antibody-drug conjugates (ADCs) are a promising class of targeted therapeutics which aim to address this need (Walsh 2021, Beck 2014, Teicher 2012). By harnessing the exquisite specificity of antibodies for cell-surface antigens overexpressed by cancer cells, ADCs enable the selective delivery of highly cytotoxic payloads to tumour sites. The utility of this therapeutic strategy has been showcased by the recent Food and Drug Administration (FDA) approval of enfortumab vedotin (Padcev™), trastuzumab deruxtecan (Enhertu™) sacituzumab govitecan (Trodelvy™), and belantamab mafodotin (Blenrep™) increasing the number of ADCs on the market to ten (Challita-Eid 2016, Modi 2020, Kaplon 2020, Syed 2020 and Hamadani (2021)).
However, current methods for the production of ADCs still suffer from various shortcomings. One common strategy for ADCs formation involves the conjugation of the payload to surface-exposed lysine residues via N-hydroxysuccinimide (NHS) ester motifs. Another approach involves conjugation to antibody cysteine residues. Human IgG1 antibodies contain four interchain disulfides, which can be reduced to reveal eight nucleophilic cysteine residues that can be conjugated to linkers containing electrophilic motifs such as maleimides. Due to the high abundance of reactive lysine (>50) and cysteine (8) residues in antibodies, modifications at these residues suffer from an inherent lack of site-selectivity and result in highly heterogeneous ADCs which vary in terms of conjugation site and the number of drug molecules attached to each antibody (drug-to-antibody ratio, DAR) (Kim 2014, Jackson 2016, Freedy 2016, Chudasama 2016). Conjugation site and DAR have been shown to have a significant effect on both the safety and efficacy of ADCs (Hamblett 2004, Shen 2012, Strop 2013). All of the currently FDA-approved ADCs are synthesised via lysine or cysteine conjugation and exist as heterogenous mixtures of conjugates with a DAR distribution of 0-8 and up to 70 different conjugation sites; this makes their pharmacokinetic profiles unnecessarily complex and difficult to predict (Kim 2014, Agarwal 2015, Godwin 2017, Bross 2001, Hedrich 2018).
Furthermore, one of the most commonly employed cysteine modification strategies—maleimide bioconjugation—has been shown to yield unstable linkages which are prone to undergo retro-Michael additions under physiological conditions (Alley 2008, Lyon 2014). This instability may lead to premature dissociation of the payload from the antibody and result in undesired toxicity in healthy tissues.
Several strategies to circumvent these issues through the incorporation of unnatural amino acids with bioorthogonal functionality or modification of antibody glycans have been reported (Fang 2014, Amant 2019, Amant 2019*, Walker 2019, Axup 2012, Zimmerman 2014). While these approaches have indeed produced ADCs with high stability, defined attachment points and precise DAR, the generation of such non-native antibody formats is generally laborious, low-yielding and may present an immunogenic risk. Therefore, the establishment of site-selective conjugation strategies using native antibodies is preferable. Disulfide bridging linkers have emerged as an attractive class of reagents that enable the synthesis of homogenous ADCs from native antibodies (Forte 2018). These linkers contain two cysteine-reactive groups that may undergo reaction with reduced interchain disulfides in an IgG molecule leading to covalent rebridging of the antibody chains. Dibromomaleimides (Behrens 2015, Smith 2010), pyridazinediones (Robinson 2017), bissulfones (Badescu 2014) and arylene-dipropiolonitrile (Koniev 2018) reagents have all been used in this context to successfully generate ADCs with demonstrated in vitro and in vivo activity. Also recently reported is the development of disulfide rebridging divinylpyrimidine (DVP) linkers (Walsh 2019, Walsh 2020). The rebridging approach yields immense improvements in homogeneity when compared to stochastic cysteine modifications and results in a defined DAR of 4 in every synthetic batch. However, a major limitation of this approach is the formation of “half-antibody” species during bioconjugation, which is the result of non-native intrachain cross-linking of the cysteine residues in the hinge region of the antibody. Half-antibody formation has been observed with all kinds of disulfide bridging linkers (Forte 2018). The loss of covalent linkages between the antibody heavy chains effected by this non-native rebridging is associated with reduced antibody stability; therefore the development of methods which abrogate half-antibody formation is highly desirable (Bahou 2019).
The present invention aims to provide a disulfide bridging linker platform which addresses the reported stability issues yet retains the advantages of a precise ratio between the active agent and the antibody, and ability to distribute the active agent. Furthermore, the present invention aims to provide a disulfide bridging linker platform which addresses the problem of half-antibody formation, and provides greater control to the number of drug molecules added to an antibody.
Accordingly, in a first aspect of the present invention, there is provided a conjugate comprising an antibody, a linker and at least one active agent, wherein:
The present invention provides a linker for use in conjugates, with utility in linking antibodies and active agents, for example antibodies and cytotoxins to provide antibody-drug conjugate (ADC) molecules. The linker provides an improved method for controlling loading of active agents on the antibody. Thus, the present invention provides a linker for use in providing homogeneous ADCs with controlled drug-to-antibody ratios.
The linker also provides an improved targeted payload of the active agent, and thus may improve the activity of the conjugate where the active agent exerts a biological activity, such as cytotoxicity. Additionally or alternatively, the linker provides the conjugate with increased stability as compared to currently known linker molecules for use in conjugates. This may improve the tolerability of such conjugates.
The linker may be directly bound to five to eight thiol groups (sulfur atoms) of cysteine residues in the antibody. Thus, the linker connects to four reduced interchain disulfide bonds via one or both of the sulfur atoms associated with the four reduced interchain disulfide bonds. Accordingly, the linker serves to re-bridges between one and four reduced interchain disulfide bonds in the antibody. Thus, the linker may be connected to the antibody (e.g. human IgG1 antibodies) via 5 to 8 covalent bonds to the eight thiol groups of the cysteine residues involved in the interchain disulfide bonds. Specifically, the linker may be connected to the antibody (e.g. human IgG1 antibodies) via 5 to 8 covalent bonds to the eight thiol groups of the cysteine residues involved in the interchain disulfide bonds at the hinge region of the antibody.
Preferably, the linker is directly bound to six to eight, or more preferably seven to eight, thiol groups (sulfur atoms) of cysteine residues in the antibody.
Thus, the linker may re-bridge one to four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 5 to 8 covalent bonds to the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bond. Preferably, the linker is directly bound to six to eight, or more preferably seven to eight, thiol groups (sulfur atoms) of cysteine residues in the antibody. Thus, the linker preferably re-bridges two to four, or more preferably three to four, of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 6 to 8 (or more preferably 7 to 8) covalent bonds to the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. Most preferably, the linker is directly bound to eight thiol groups (sulfur atoms) of cysteine residues in the antibody. Thus, the linker re-bridges all four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds.
In a second aspect of the present invention, there is provided a conjugating reagent capable of reaction with an antibody, said conjugating reagent comprising a linker, at least one active agent and 8 functional groups capable of reaction with between 5 and 8 sulfur atoms on the antibody.
Thus, suitably, the conjugating reagent of the invention is a compound of Formula (II), shown below:
(D-L1-FG)n-L-(Z)8 (Formula II)
This conjugating reagent may be reacted with an antibody to form the conjugate of the first aspect of the invention.
Preferably, the conjugating reagent comprises a linker, at least one active agent and 8 functional groups capable of reaction with between 6 and 8, more preferably 7 to 8, and most preferably 8, sulfur atoms on the antibody.
In a third aspect of the invention, there is provided an intermediate conjugate comprising an antibody, a linker and at least one functional group capable reacting with another moiety to form a functional linking moiety, wherein:
Thus, the intermediate conjugate may be reacted with at least one active agent to form the conjugate of the first aspect of the invention. This reaction between the intermediate conjugate and the active agent(s) can take place via reaction of the at least one functional group on the intermediate conjugate and another functional moiety on the active agent. As such, a functional linking moiety linking the intermediate conjugate to the active agent(s) is formed.
In a fourth aspect of the present invention, there is provided a compound capable of reaction with both an antibody and at least one active agent, said compound comprising a linker, 8 functional groups capable of reaction with between 5 and 8 sulfur atoms on the antibody and at least one functional group capable reacting with another moiety to form a functional linking moiety. The functional linking moiety serving as the means by which the at least one active agent may be attached to the compound.
Thus, suitably, the compound of the invention is a compound of Formula (I), shown below:
(FG)n-L-(Z)8 (Formula I)
The compound of the fourth aspect is a precursor of the conjugating reagent of the second aspect of the invention. The compound of the fourth aspect is also a precursor of the intermediate conjugate of the third aspect of the invention. The conjugating reagent can be formed by reacting the compound of Formula (I) with an active agent, which may have a linker group already attached. The intermediary conjugate can be formed by reacting the compound of Formula (I) with an antibody (an antibody that has had its interchain disulfide bonds reduced).
In a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising, a conjugate of the first aspect of the invention, where the active agent is a drug, and a carrier, excipient or diluent. The fifth aspect also provides a conjugate of the first aspect of the invention, where the active agent is a drug, for use in a method of treatment.
It will be understood that the pharmaceutical composition according to fifth aspect of the present invention may comprise a mixture of conjugates of the first aspect of the invention. That is, it will be understood that the pharmaceutical composition may comprise a mixture of conjugates wherein the linker is attached to the antibody through either 5, 6, 7 or 8 independent covalent bonds. It will also be understood that pharmaceutical composition may also comprise conjugates wherein the linker is attached to the antibody through 1 to 4 independent covalent bonds.
Suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 65% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. More suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 70% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. Even more suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 75% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. Yet more suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 80% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. Still more suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 85% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. Still more suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 90% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. Still more suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 95% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds. Most suitably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 98% of the conjugates comprise a linker which is attached to the antibody through 8 independent covalent bonds.
Thus, suitably, at least 65% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. More suitably, at least 70% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. Yet more suitably, at least 75% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. Even more suitably, at least 85% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. Even more suitably, at least 90% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. Even more suitably, at least 95% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds. Most suitably, at least 98% of the conjugate present in the pharmaceutical composition is a conjugate of the first aspect of the invention wherein the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g. human IgG1 antibodies), via 8 covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine residues involved in the interchain disulfide bonds.
In a sixth aspect of the present invention there is provided the use of a conjugate of the first aspect of the invention, where the active agent is a cytotoxic drug, in the manufacture of a medicament for treating a proliferative disease. The sixth aspect also provides a conjugate of the first aspect of the invention, where the active agent is a cytotoxic drug, for use in the treatment of a proliferative disease. The sixth aspect also provides a method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate of the first aspect of the invention, where the active agent is a cytotoxic drug, to a patient in need thereof.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
The terms “specifically binds” and “specific binding” refer to the binding of an antibody to a predetermined molecule (e.g., an antigen). Typically, the antibody binds with an affinity of at least about 1×107 M−1, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
“Antibody fragments” comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).
The monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies.
Examples of cell binding agents include those agents described for use in WO 2007/085930, which is incorporated herein.
Tumour-associate antigens and cognate antibodies for use in embodiments of the present invention are listed below, and are described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein.
The active agent may be a drug, a labelling moiety or a targeting moiety. Preferably, the active agent is a drug or a labelling agent. The active agent needs to comprise a functional group that can be linked to the linker. Such a group may include, for example, an amino group, an imine group or a hydroxy group. In situations where there exists more than one active agent, it is to be understood that the two or more active agents may either be the same or different. For example, in situations where there exist two or more active agents, the two or more active agents may independently be selected from a drug, a labelling moiety or a targeting moiety. It will also be understood that in situations where there exist more than one drug, a labelling moiety or a targeting moiety, each drug, labelling moiety and targeting moiety may be the same or different.
The drug may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the drug is preferably a cytotoxin.
Preferably the cytotoxin is a biologically active cytotoxic material. The cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camptothecin analogues and doxorubicin.
However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials, although such materials are less commonly utilised in the field of the art.
The labelling moiety may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).
The labelling moiety may also be a biotin tag, derived from biotin.
The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11C]acetate, [11C]methionine, [11C] choline, copper 64Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F]fluorocholine, [18F]fluoroethyl-L-tyrosine, [18F]fluoromisonidazole, [18F]fluorothymidine F-18, [64Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).
The targeting moiety is a moiety that helps to direct to the conjugate to a particular location within the body. It will be appreciated that any suitable targeting moiety may be used. Suitably, the targeting moiety is a carbohydrate, such as alpha-D-galacto-hexopyranosyl-(1->3)-2-acetamido-2-deoxy-D-galacto-hexopyranose (Gal-alpha 1,3-GalNAc).
According to one aspect of the invention there is provided a conjugate comprising an antibody, a linker and at least one active agent, wherein:
Suitably, each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom on the antibody and an electrophilic functional group on the linker.
In certain embodiments, the conjugate comprises between one and four re-bridging moieties of Formula (III), shown below:
It will be understood that the functional linking group ZA can be any functional group that is capable of linking the antibody to the linker. The functional linking group ZA is thus a functional grouping that is capable of forming a covalent attachment to sulfur atoms present on the antibody. Suitably, the sulfur atoms are sulfur atoms from cysteine residues on the antibody, and more suitably, sulfur atoms from cysteine residues from reduced interchain disulfide bonds of the antibody.
Functional linking groups capable of linking the antibody to the linker in this way are well known in the art. Thus, the skilled person will be able to select suitable functional linking groups to use with the invention. Examples of suitable functional linking groups, ZA, include, for instance, those described in, for example, Xu 2021, Stieger 2021, Walsh 2019, Walsh 2020, Badescu 2014, Koniev 2018, Robinson 2017, Behrens 2015 and WO2019/011078.
In certain embodiments, ZA is selected from one of the functional linking groups shown below, and thus the conjugate comprises between one and four (preferably between two and four, more preferably between three and four, and most preferably four) re-bridging moieties independently selected from one of the following groups:
In certain embodiments, two of A1, A2 and A3 are N (e.g. A1 and A2 are N) and the other of A1, A2 and A3 is CH (e.g. A3 is CH). Suitably, in this embodiment, integers a and b are 1.
In other embodiments, all three of A1, A2 and A3 are N. Suitably, in this embodiment, integers a and b are 1.
In some embodiments, one of A1, A2 and A3 is N (e.g. A3 is N) and the other two of A1, A2 and A3 are CH (e.g. A1 and A2 are CH). Suitably, in this embodiment, integers a and b are 0.
Suitably, X is NH or O. Most suitably, X is NH.
In certain embodiments, R1 and R2 are independently selected from C1-C6 alkylene. Suitably, R1 and R2 are independently selected from C1-C4 alkylene.
In some embodiments, R3 is a C1-C4 alkyl (e.g. a methyl group).
In some embodiments, Y1 and Y2 are independently absent or selected from O, NR4 and C(═O). Suitably, Y1 and Y2 are independently absent.
Suitably, Q is CR6 or N. In some embodiments, Q is CR6. In other embodiments, Q is N.
In some embodiments, R4, R5 and R6 are independently selected from hydrogen and methyl. Suitably, R4, R5 and R6 are each hydrogen.
In certain embodiments, the conjugate comprises between one and four (preferably between two and four, more preferably between three and four, and most preferably four) re-bridging moieties independently selected from the following groups:
In other embodiments, the conjugate comprises between one and four (preferably between two and four, more preferably between three and four, and most preferably four) re-bridging moieties of general formula (IIIa), shown below:
wherein each of A1, A2, A3, X, Pep and
are as defined herein.
In some embodiments, A1 and A2 are N.
In other embodiments, A1 and A3 are N.
In some embodiments, X is selected from NRN, O and S, where RN is H or C1-2 alkyl. In these embodiments, only a single active agent can be attached is to X via a linker.
In some of these embodiments, X is NRN. X may be NH, NCH3 or NCH2CH3.
In others of these embodiments, X is O.
In others of these embodiments, X is S.
In some embodiments, X is N. In these embodiments, X can be attached to the linker via two covalently bonds, as shown below:
wherein each of A1, A2, A3, Pep and
are as defined herein.
The linker is a group (functional grouping) that joins the antibody to the active agent(s). The linker attaches to the antibody via 5 to 8 (suitably via 6 to 8, more suitably via 7 to 8, and most suitably via 8) covalent bonds formed between sulfur atoms on the antibody and functional groups (e.g. electrophilic functional groups) on the linker. The sulfur atoms on the antibody may be sulfur atoms on cysteine residues on the antibody, such as sulfur atoms from the cysteine residues on the antibody that are involved in the interchain disulfide linkages of the antibody (i.e. sulfur atoms from cysteine residues made available following the reduction of antibody's interchain disulfide linkages).
The linker may therefore attach to the antibody via the functional linking groups (ZA). The linker may therefore connect the functional linking groups (ZA) to the at least one active agent. The linker can connect to the at least one active agent(s) via a functional linking moiety (FG′) as defined hereinbelow.
It will be understood that the linker may be any group (functional grouping) that is capable of connecting the at least one active agent to the antibody.
In certain embodiments, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (III)) are separated by between 10 and 60 atoms. Suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (III)) are separated by between 10 and 50 atoms. More suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (III)) are separated by between 10 and 40 atoms. Even more suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (III)) are separated by between 12 and 40 atoms. Still more suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (III)) are separated by between 15 and 40 atoms. Most suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (III)) are separated by between 16 and 38 atoms.
In some embodiments, the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue, an arylene-containing moiety, a heteroarylene-containing moiety, a heterocyclyl-containing moiety, a cycloalkyl-containing moiety and combinations thereof. Suitably, the linker comprises between 1 and 20 of the above-mentioned groups. More suitably, the linker comprises between 1 and 15 of the above-mentioned groups. Even more suitably, the linker comprises between 1 and 10 of the above-mentioned groups.
Suitably, the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue and combinations thereof.
The alkylenediamine moiety will be understood to be a functional grouping that contains or is derived from an alkylenediamine moiety, such as, an ethylenediamine.
The polyethylene glycol moiety will be understood to be a functional grouping that comprises one or more alkylene glycol moieties, such as, one or more ethylene glycol moieties. In certain embodiments, the linker comprises between 1 and 10 alkylene glycol moieties (e.g. ethylene glycol moieties).
The amino acid residues will be understood to constitute both natural and unnatural amino acid residues. Suitably, the amino acid residue will be a natural amino acid residue. The amino acid residue may also comprise two or more amino acids, such as, for example, dipeptide and tripeptide moieties. Suitable amino acid residues include Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp residues.
The arylene-containing moiety will be understood to be a functional grouping that comprises one or more arylene groups. Suitably, the arylene will be a 6-membered arylene such as phenylene. In certain embodiments, the arylene-containing moiety is a functional grouping that includes a 1,3,5-benzyl group.
The heteroarylene-containing moiety will be understood to be a functional grouping that comprises one or more heteroarylene groups. Suitably, the heteroarylene will be a functional grouping that includes a 6-membered heteroaryl such as triazine. In certain embodiments, the arylene-containing moiety is a functional grouping that includes a 1,3,5 triazine group.
The heterocyclyl-containing moiety will be understood to be a functional grouping that comprises one or more heterocycle groups. Suitably, the heterocycle is a 6-membered heterocycle such as triazinane. In certain embodiments, the heterocyclyl-containing moiety is a functional grouping that includes a 1,3,5 triazinane group.
The cycloalkyl-containing moiety will be understood to be a functional grouping that comprises one or more cycloalkyl groups. Suitably, the cycloalkyl is a 6-membered cycloalkyl such as cyclohexane. In certain embodiments, the cycloalkyl-containing moiety is a functional grouping that includes a 1,3,5 cyclohexane.
In certain embodiments, the conjugate is a compound of Formula (IIA), shown below:
It will be understood that when integer v is 2 the two
groups may be the same or different.
It will also be understood that when integer m is 2 the two WB and two R24 groups may be the same or different. Suitably, the two WB and two R24 groups are the same.
Suitably, m is 1.
Suitably, Ring A is selected from a phenyl, 1,3,5-triazine or 1,3,5 triazinane.
Suitably, W1 is a group of Formula IVc.
In certain embodiments, W1 is a group of Formula IVc and W1C is a group of Formula WC1.
In other embodiments, W1 is a group of Formula IVc and W1C is a group of Formula WC2.
In certain embodiments, LW is absent or selected from a C1-C6alkylene and a C1-C6alkylene containing one or more of an O or an N in the backbone.
When W1 is a group of Formula IVc and W1C is a group of Formula WC2, suitably t is an integer selected from 1 to 6, most suitably t is an integer selected from 1 to 4.
Thus, it will be understood that the linker defined hereinabove in connection with the first aspect of the invention may take the form of Formula LX1 or Formula LX2 shown below:
wherein each of Z1, Q1, Q2, Q3, W1A W1B, W1C, W2A, W2B, W2C, WQ1, WQ2, R23, R24, R25, R26, R27, FG′, L1, LW, L2W, LQ1, m, t, r and s are as defined herein; and
indicates the position where the linker is attached to the active agent(s).
Thus, in certain embodiments, there is provided a conjugate comprising an antibody, a linker and at least one active agent, wherein:
Suitably, each Z1 of Formula LX1 and Formula LX2 is a re-bridging moiety independently selected from a group consisting of Formula IIIa, Formula IIIb, Formula IIIc, Formula IIId, Formula IIIe, Formula IIIf and Formula IIIg, as defined hereinabove. More suitably, each Z1 is a re-bridging moiety independently selected from the group consisting of Formula IIIa, Formula IIIb and Formula IIIe, as defined hereinabove. Most suitably, each Z1 is a re-bridging moiety of Formula IIIa, as defined hereinabove.
It will also be understood that when conjugate is attached to the antibody through 5 to 7 independent covalent bonds, the ZA functional linking group of each re-bridging moiety of Formula III) may be attached to the antibody, either directly or indirectly, via one or both of the
bonds shown hereinabove.
Suitably, r and s are selected such that the total number of active agents per conjugate is between 1 and 10, more suitably between 1 and 8, and most suitably between 1 and 4.
Suitably, r and s are selected such that there are 1, 2, 3, 4, 5, 6, 7 or 8 active agents per conjugate.
In some embodiments Q1 is a bond.
In other embodiments Q1 is a group of Formula IVa:
Suitably, Q1 is a group of Formula IVa wherein:
More suitably, Q1 is a group of Formula IVa wherein:
In some embodiments, Q2 is N. In other embodiments, Q2 is CR11, wherein R11 is selected from hydrogen and methyl.
In some embodiment, Q3 is a group of Formula IVb:
In some embodiments, Q3 is a group of Formula IVb1:
In some embodiments, Q3 is a group of Formula IVb1, wherein:
In some embodiments, W1 is a group of Formula IVc, wherein:
In other embodiments, W1 is a group of Formula IVc, wherein:
In other embodiments, W1 is a group of Formula IVc, wherein:
In further embodiments, W1 is a group of Formula IVc, wherein:
When W1C is a group of Formula WC2, suitably LQ1 is a group of Formula LQ2;
In some embodiments, W2 is a group of Formula IVd, wherein:
In other embodiments, W2 is a group of Formula IVd, wherein:
In certain embodiments, FG′ is a bond or a functional group formed from the reaction between: i) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; ii) an azide and an alkyne; iii) a amine and an acyl halide or carboxylic acid; iv) electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine); and iii) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine. Suitably, FG′ is a bond or a functional group formed from the reaction between an azide and an alkyne or an amine and an acyl halide or carboxylic acid. Most suitably, FG′ is a functional group formed from the reaction between an azide and an alkyne.
Thus, in some embodiments FG′ is a functional group selected from a bond,
In other embodiments FG′ is a functional group selected from a bond,
In some embodiments, FG′ is or
Suitably, FG′ is
Most suitably, FG′ is
wherein N is bound to L1.
Linker L1 is a linker that attaches the functional linking moiety, FG′, to the active agent(s), D.
It will be appreciated that any suitable linker may be used connect the functional linking moiety, FG′, to the active agent, D.
In certain embodiments, the linker L1 comprises one or more groups that are susceptible to enzymatic cleavage (e.g. proteolytic or peptidase cleavage, sulfatase cleavage or galactosidase cleavage). Thus, in some embodiments, the linker L1 comprises one or more amino-acid residues, dipeptide residues or tripeptide residues, one or more aryl sulfates, one or more aryl galactosides or combinations thereof. Suitably, the linker L1 comprises one or more amino-acid residues, dipeptide residues or tripeptide residues, wherein the amino-acid residues, dipeptide residues or tripeptide residues are as defined hereinbelow.
In certain embodiments, the linker L1 comprises one or more groups that are susceptible to chemical cleavage (e.g. a disulfide or a hydrazone).
In some embodiments, the linker L1 comprises one or more groups selected from a C1-C20alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereof.
In certain embodiments, L1 is selected from a bond, a C1-C10alkylene, a C1-C10alkylene containing O in the backbone and a group of formula Va:
Suitably, LQ is a selected from a bond, a C1-C10alkylene and a C1-C10alkylene containing O in the backbone.
In some embodiments, LQ is a selected from a bond. In other embodiments, LQ is a C1-C10alkylene or a C1-C10alkylene containing O in the backbone. Suitably, LQ is a C1-C10alkylene containing O in the backbone.
In certain embodiments, L1 is selected from a bond, a C1-C10alkylene, a C1-C10alkylene containing O in the backbone and a group of formula Va1:
In certain embodiments, L1 is selected from a C1-C10alkylene and a C1-C10alkylene containing O in the backbone.
In other embodiments, L1 is a group of Formula Va or Formula Va1.
In some embodiments, Q4 is a single bond.
In other embodiments, Q4 is
In one embodiment, Qx is an amino acid residue. The amino acid may be a natural amino acid or a non-natural amino acid.
In one embodiment, QX is selected from: Phe, Lys, Val, Ala, Cit, Leu, IIe, Arg, and Trp, where Cit is citrulline.
In one embodiment, Qx comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, Qx is selected from:
Preferably, Qx is selected from:
Most preferably, Qx is selected from NH-Phe-Lys-C═O, NH-Val-Cit-C═O or NH-Val-Ala-C═O.
Other dipeptide combinations of interest include:
Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
In some embodiments, Qx is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin.
In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.
In some embodiments, LD is selected from:
In some of these embodiments, LD is a single bond. If Q4 is also a single bond, then FG′ is attached directly to the active agent.
In other of these embodiments, LD is —C(═O)— or —NH—.
In other of these embodiments, LD is
This group can act as a self-immolative group in conjunction with a cleavable linking group.
In some embodiments, the conjugate is a compound of Formula (IIIA), shown below:
In other embodiments, the conjugate is a compound of Formula (IIIA), shown below:
In certain embodiments, the conjugate is selected from one of the following:
wherein:
In some embodiments, the conjugate is selected from one of the following:
wherein:
In some embodiments, the conjugate is selected from one of the following:
wherein:
In some embodiments, the conjugate is selected from ADC 1 to ADC 20 described below.
According to another aspect, the present invention provides a conjugating reagent of Formula (II), shown below:
(D-L1-FG′)n-L-(Z)3 (Formula II)
Suitably, Z is an electrophilic functional group capable of reaction with a sulfur atom of an antibody.
Suitably, n is an integer selected from 1 to 12, more suitably from 1 to 10, even more suitably, from 1 to 8, and most suitably from 1 to 4.
In certain embodiments, Z is a Michael Acceptor (e.g. alkene or alkyne typically with an electron withdrawing group) that is capable of reaction with a sulfur atom of an antibody via a conjugate addition (Michael reaction). Conjugate addition reactions and the Michael reaction are well known in the field of chemistry. Thus, a skilled person will be able to select suitable Michael Acceptors for use in the conjugating reagents of the invention.
In certain embodiments, Z is a functional group that reacts with the sulfur atoms on the antibody selected from an alkene, an alkyne, a vinylarene (e.g. divinylpyrimidine), a maleimide, a halo-maleimide, a sulfone, an arylene-propiolonitrile, a pyridazinedione, a halo-(hetro)arene, a benzyl halide, a group comprising a β-unsaturated ketone, a group capable of an SNAr reaction or a group capable of alkylating sulfur.
In certain embodiments, Z is a functional group that reacts with the sulfur atoms on the antibody selected from an alkene, an alkyne, a vinylarene (e.g. divinylpyrimidine), a maleimide, a halo-maleimide, a sulfone, an arylene-propiolonitrile, a pyridazinedione or a group comprising a β-unsaturated ketone.
In certain embodiments, the conjugating reagent is of Formula (IIa), shown below:
(D-L1-FG′)n-L-(Z2)4 (Formula IIa)
Preferred and suitable substituent groups for each of D, L, L1, FG′ and n will be understood to be analogous to the preferred and suitable substituent groups for each of D, L, L1, FG′ and n for the conjugates of the invention described hereinabove.
In certain embodiments, each Z2 is a re-bridging linking group independently selected from one of the following groups:
In certain embodiments, each Z2 is a re-bridging linking group independently selected from a group of Formula IIZA, Formula IIZB, Formula IIZE and Formula IIZH. Suitably, each Z2 is a re-bridging linking group independently selected from a group of Formula IIZA, Formula IIZB and Formula IIZE. More suitably, each Z2 is a re-bridging linking group of Formula IIZA.
In certain embodiments, there is provided a conjugating reagent selected from one of the following:
wherein each of D, L, L1, FG′, A1, A2, A3, X, R1, R2, R3, Q, Y1, Y2, p, q and n are as defined hereinabove.
In other embodiments, the conjugating reagent is of Formula (IIB), shown below:
Preferred and suitable substituent groups for each of D, Q1, Q2, Q3, W1, W2, r and s will be understood to be analogous to the preferred and suitable substituent groups for each of D, Q1, Q2, Q3, W1, W2, r and s for the conjugates of the invention described hereinabove. Preferred and suitable Z2 groups will be understood to those described hereinabove.
In certain embodiments, the conjugating reagent is a compound of Formula (IIB), shown below:
In certain embodiments, the conjugating reagent is a compound of Formula (IIB), shown below:
In other embodiments, the conjugating reagent is selected from one of the following:
In some embodiments, the conjugating reagent is selected from one of the following:
According to another aspect of the invention, there is provided compound of Formula (I), shown below:
(FG)n-L-(Z)8 (Formula I)
Preferred and suitable substituent groups for each of FG, n, L and Z will be understood to be analogous to the preferred and suitable substituent groups for each of FG, n, L and Z for the conjugates and conjugating reagents of the invention described hereinabove.
Thus, in certain embodiments, FG is a functional group selected from an alkene, an alkyne, an azide, a hydroxyl, an amine, a carboxylic acid, an aldehyde, an acyl halide, a tetrazine, an alkoxyamine (e.g. hydroxylamine), a hydrazine, an electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine), nitrone, an isocyanate and an isothiocyanate.
Suitably, FG is a functional group selected from an alkene, an alkyne, an azide, a hydroxyl, an amine, a carboxylic acid, an aldehyde, an acyl halide, a tetrazine, an alkoxyamine (e.g. hydroxylamine), and a hydrazine. More suitably, FG is a functional group selected from an alkyne, an azide, hydroxyl, amine, carboxylic acid, aldehyde and acyl halide. Most suitably, FG is a functional group selected from an alkyne and an azide.
In certain embodiments, the compound is of Formula (Ia), shown below:
(FG)n-L-(Z2)4 (Formula Ia)
wherein Z2, L, FG and n are each as defined hereinabove.
In some embodiments, the compound is selected from one of the following groups:
wherein each of A1, A2, A3, X, L, FG, n, Q, Y1, Y2, R1, R2, R3, q and p are as defined hereinabove.
In some embodiments, the compound is of Formula (Ic), shown below
wherein:
In other embodiments, the compound is of Formula (Ic), shown below:
wherein:
In other embodiments, the compound is selected from one of the following:
In some embodiments, the compound is selected from one of the following:
In some embodiments, the compound is selected from one of the following:
According to another aspect of the invention there is provided an intermediate conjugate comprising an antibody, a linker and at least one functional group capable reacting with another moiety to form a functional linking moiety, wherein:
In certain embodiments, the intermediate conjugate comprises between one and four (preferably between two and four, more preferably between three and four, and most preferably four) re-bridging moieties of Formula (III), shown below:
Preferred and suitable ZA groups will be understood to be analogous to the preferred and suitable ZA groups for the conjugates of the invention described hereinabove.
In other embodiments, the intermediate conjugate is a compound of Formula (VI), shown below:
wherein, Z1, Q1, Q2, Q3, W1′, W2′, r and s are as defined hereinabove.
In other embodiments, the intermediate conjugate is selected from one of the following:
wherein:
In other embodiments, the intermediate conjugate is selected from one of the following:
wherein:
In other embodiments, the intermediate conjugate is selected from ALC 1 to 12 described below.
The conjugates of the present invention where the active agent is a drug may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate according to the first aspect of the invention, where the active agent is a drug. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a conjugate according to the first aspect of the invention, where the active agent is a drug, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
The conjugates can be used to treat proliferative disease and autoimmune disease. The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin. Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.
In certain embodiments, the conjugates can be used to treat microbial diseases. Suitably, the conjugates can be used to treat bacterial diseases, such tuberculosis (TB) and methicillin-resistant Staphylococcus aureus (MRSA).
It will also be understood that features, including optional, suitable, and preferred features in relation to any one of the aspects of the present invention detailed above (e.g. conjugates of the present invention) may also be features, including optional, suitable and preferred features in relation to any other aspects of the invention (e.g. the uses of the conjugates of the present invention).
All solvents and reagents were used as received unless otherwise stated. Ethyl acetate, methanol, dichloromethane, acetonitrile and toluene were distilled from calcium hydride. Diethyl ether was distilled from a mixture of lithium aluminium hydride and calcium hydride. Petroleum ether (PE) refers to the fraction between 40-60° C. upon distillation. Tetrahydrofuran was dried using Na wire and distilled from a mixture of lithium aluminium hydride and calcium hydride with triphenylmethane as indicator.
Non-aqueous reactions were conducted under a stream of dry nitrogen using oven-dried glassware. Temperatures of 0° C. were maintained using an ice-water bath. Room temperature (rt) refers to ambient temperature.
Yields refer to spectroscopically and chromatographically pure compounds unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) or liquid chromatography mass spectroscopy (LC-MS). TLC was performed using glass plates pre-coated with Merck silica gel 60 F254 and visualized by quenching of UV fluorescence (λmax=254 nm) or by staining with potassium permanganate. Retention factors (Rf) are quoted to 0.01. LC-MS was carried out using a Waters ACQUITY H-Class UPLC with an ESCi Multi-Mode Ionisation Waters SQ Detector 2 spectrometer using MassLynx 4.1 software; ESI refers to the electrospray ionisation technique; LC system: solvent A: 2 mM NH4OAc in H2O/MeCN (95:5); solvent B: MeCN; solvent C: 2% formic acid; column: ACQUITY UPLC® CSH C18 (2.1 mm×50 mm, 1.7 μm, 130 Å) at 40° C.; gradient: 5-95% B with constant 5% C over 1 min at flow rate of 0.6 mL/min; detector: PDA eλ Detector 220-800 nm, interval 1.2 nm.
Flash column chromatography was carried out using slurry-packed Merck 9385 Kieselgel 60 SiO2 (230-400 mesh) or Combiflash Rf200 automated chromatography system with Redisep® reverse-phase C18-silica flash columns (20-40 μm).
HPLC purification was carried out via semi-preparative reverse phase HPLC on an Agilent 1260 Infinity using a SupeIcosil ABZ+PLUS column (250 mm×21.2 mm, 5 μm) eluting with a linear gradient system (solvent A: 0.1% (v/v) TFA in water, solvent B: 0.05% (v/v) TFA in acetonitrile) over 20 minutes at a flow rate of 20 mL/min.
Analytical high performance liquid chromatography (HPLC) was performed on Agilent 1260 Infinity machine, using a SupeIcosil™ ABZ+PLUS column (150 mm×4.6 mm, 3 μm) with a linear gradient system (solvent A: 0.05% (v/v) TFA in H2O; solvent B: 0.05% (v/v) TFA in MeCN) over 20 min at a flow rate of 1 mL/min, and UV detection (λmax=220-254 nm).
Infrared (IR) spectra were recorded neat on a Perkin-Elmer Spectrum One spectrometer with internal referencing. Selected absorption maxima (vmax) are reported in wavenumbers (cm−1).
1H and 13C nuclear magnetic resonance (NMR) were recorded using an internal deuterium lock on Bruker DPX-400 (400 MHz, 101 MHz), Bruker Avance 400 QNP (400 MHz, 101 MHz) and Bruker Avance 500 Cryo Ultrashield (500 MHz, 126 MHz). Tetramethylsilane was used as an internal standard. In 1H NMR, chemical shifts (δH) are reported in parts per million (ppm), to the nearest 0.01 ppm and are referenced to the residual non-deuterated solvent peak (CDCl3: 7.26, DMSO-d6: 2.50, CD3OD: 3.31, D2O: 4.79). Coupling constants (J) are reported in Hertz (Hz) to the nearest 0.1 Hz. Data are reported as follows: chemical shift, multiplicity (s=singlet; d=doublet; t=triplet; q=quartet; quint=quintet; m=multiplet; or as a combination of these, e.g. dd, dt etc.), integration and coupling constant(s). In 13C NMR, chemical shifts (δC) are quoted in ppm, to the nearest 0.1 ppm, and are referenced to the residual non-deuterated solvent peak (CDCl3: 77.16, DMSO-d6, 39.52, CD3OD: 49.00).
Unless, otherwise specified, high resolution mass spectrometry (HRMS) measurements were recorded with a Micromass Q-TOF mass spectrometer or a Waters LCT Premier Time of Flight mass spectrometer. Mass values are reported within the error limits of ±5 ppm mass units. ESI refers to the electrospray ionisation technique.
UV-Vis spectroscopy was used to determine protein concentrations and fluorophore-to-antibody ratios (FAR) using a nanodrop ND-1000 spectrophotometer. Sample buffer was used as blank for baseline correction with extinction coefficients ε280=215,380 M−1 cm−1 for trastuzumab and ε495=71,000 M−1 cm−1 for AlexaFluor 488™ (AF488). The correction factor for AF488 absorption at 280 nm is 0.11. FAR was calculated using the following formula:
Protein LC-MS was performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1×50 mm). H2O with 0.1% formic acid (solvent A) and 95% MeCN and 5% H2O with 0.1% formic acid (solvent B) were used as the mobile phase at a flow rate of 0.2 mL/min. The gradient was programmed as follows: 95% A for 0.93 min, then a gradient to 100% B over 4.28 min, then 100% B for 1.04 minutes, then a gradient to 95% A over 1.04 min. The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 190 V. Nitrogen was used as the desolvation gas at a total flow of 850 L/h. Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v4.2 from Waters) according to the manufacturer's instructions. Trastuzumab samples were deglycosylated with PNGase F (New England Biolabs) prior to LC-MS analysis.
4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) was synthesised as previously described (Bargh et al, Chem. Sci., 2020, 11, 2375-2380).
To a solution of diethylenetriamine (2.16 mL, 20.0 mmol) in THF (25 mL) at 0° C. was slowly added a solution of Boc-ON (9.92 g, 40.0 mmol) in THF (25 mL). The reaction was stirred at 0° C. under nitrogen for 1 h and then concentrated in vacuo. The crude yellow oil was dissolved in CH2Cl2 and sequentially washed with 10% aq. NaOH, water and brine. The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by column chromatography (10% MeOH/CH2Cl2) yielded the product as a colourless oil (5.61 g, 18.5 mmol, 92% yield). Rf 0.08 (SiO2, 10% MeOH/CH2Cl2); vmax (neat/cm1) 3336 (m, N—H), 2975 (m, C—H), 1687 (s, C═0); δH (400 MHz, CDCl3) 4.93 (br s, 2H), 3.24-3.19 (m, 4H), 2.73 (t, 4H, J=5.7 Hz), 1.44 (s, 18H); δC (101 MHz, CDCl3) 156.3, 79.4, 49.0, 40.4, 28.6; HRMS (ESI) m/z found [M+H]+ 304.2221, C14H30O4N3+ required 304.2231.
To a solution of di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (3) (5.61 g, 18.5 mmol) in DMF (80 mL) were added DIPEA (3.86 mL, 22.2 mmol) and methyl bromoacetate (2.61 mL, 27.8 mmol). The reaction was stirred at room temperature for 6 h and then concentrated under a stream of nitrogen. The crude residue was purified by column chromatography (40-50% EtOAc/PE) yielding the product as a colourless oil (6.56 g, 17.5 mmol, 94% yield). Rf 0.28 (SiO2, 50% EtOAc/PE); vmax (neat/cm−1) 3345 (m, N—H), 2979 (m, C—H), 1740 (s, C═O), 1688 (s, C═O); δH (400 MHz, CDCl3) 5.13 (br s, 2H, NH), 3.70 (s, 3H), 3.37 (s, 2H), 3.15 (q, 4H, J=5.6 Hz), 2.72 (t, 4H, J=5.9 Hz), 1.44 (s, 18H); δC (101 MHz, CDCl3) 172.3, 156.3, 79.3, 55.1, 54.3, 51.8, 38.7, 28.6; LRMS (ESI) m/z found [M+H]+ 376.5.
A solution of propargylamine (1.16 mL, 18.2 mmol) in CH2Cl2 (33 mL) and sat. NaHCO3 (33 mL) at maximum stirring was cooled to −10° C. and 2-bromoacetyl bromide (2.42 mL, 27.2 mmol) was added dropwise over 15 min. The reaction mixture was allowed to slowly reach rt, and upon completion the reaction mixture was concentrated. Following addition of water (30 mL), the aqueous solution was extracted with EtOAc (2×80 mL) and the combined organic phases were washed with sat. NaHCO3 (30 mL), 5% HCl (30 mL) and brine (30 mL). Combined organic phases were dried over Na2SO4 and concentrated in vacuo to give the title compound as a light yellow solid (2.97 g, 16.9 mmol, 93%). Rf 0.69 (SiO2, 20% MeOH/CH2Cl2); vmax (neat/cm−1) 3289 (m, N—H), 3071 (m, C—H), 2120 (w, C≡C), 1645 (s, C═O); δH (400 MHz, CDCl3) 6.68 (s, 1H), 4.09 (dd, J=5.3, 2.6 Hz, 2H), 3.90 (s, 2H), 2.28 (t, J=2.6 Hz, 1H); δC (100 MHz, CDCl3) 165.1, 78.5, 72.3, 30.0, 28.7; LRMS (ESI) m/z found [M+H]+ 178.0.
In a pre-dried microwave vial containing methyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4) (200 mg, 0.53 mmol) dissolved in dry MeOH (0.3 mL) was added diethylenetriamine (10.7 μL, 0.098 mmol), the vial was capped, flushed with nitrogen, and stirred over night at 110° C. The reaction mixture was concentrated and purified by column chromatography (0-10% MeOH/CH2Cl2) to give the desired compound as a colourless oil (60.6 mg, 0.077 mmol, 78%). Rf 0.14 (SiO2, 10% MeOH/CH2Cl2); vmax (neat/cm−1) 3316 (m, N—H), 2974 (m, C—H), 1687 (s, C═O); δH (400 MHz, CD3OD) 3.48 (t, 4H, J=5.8 Hz), 3.21 (s, 4H), 3.14 (t, 8H, J=6.3 Hz), 3.06 (t, 4H, J=5.7 Hz), 2.61 (t, 8H, J=6.3 Hz), 1.45 (s, 36H); δC (101 MHz, CD3OD) 175.6, 158.6, 80.2, 59.9, 56.4, 49.5, 39.7, 38.4, 28.9; HRMS (ESI) m/z found [M+H]+ 790.5425, C3H72O10N9+ required 790.5402.
In a pre-dried microwave vial, a suspension of compound 6 (59.3 mg, 0.075 mmol) and K2CO3 (20.7 mg, 0.15 mmol) in anhydrous MeCN (0.15 mL) was cooled to 0° C. by means of an ice-bath. A solution of compound 5 (16.5 mg, 0.094 mmol) in MeCN (0.5 mL) was slowly added to the stirring suspension, after which the reaction was brought to rt and stirred overnight under nitrogen. The reaction mixture was concentrated and purified by flash chromatography (0-10% MeOH/CH2Cl2) to give the title compound as a white solid (35.9 mg, 0.041 mmol, 54%). Rf0.30 (SiO2, 10% MeOH/CH2Cl2); vmax (neat/cm−1) 3359 (m, N—H), 2985 (m, C—H), 2101 (m, C≡C), 1724 (s, C═O); δH (600 MHz, CDCl3) 7.79 (s, 2H), 5.70 (s, 4H), 4.04 (dd, 2H, J=2.4, 5.4 Hz), 3.37-3.32 (m, 4H), 3.24 (s, 2H), 3.20-3.14 (m, 8H), 3.14 (s, 4H), 2.75-2.70 (m, 4H), 2.60-2.55 (m, 8H), 2.25-2.22 (m, 1H), 2.10 (s, 1H), 1.42 (s, 36H); δC (101 MHz, CDCl3) 172.2, 171.5, 156.7, 80.0, 79.4, 71.5, 59.5 (two peaks), 55.8 (two peaks), 38.8, 37.9, 29.0, 28.6; HRMS (ESI) m/z found [M+H]+ 885.5760, C41H77O11N10 + required 885.5768.
To a solution of compound 7 (69.0 mg, 0.078 mmol) in CH2Cl2 (0.5 mL) at 0° C. was added HCl (4M in dioxane, 1.0 mL). The reaction was stirred under nitrogen for 6 h and then concentrated in vacuo to yield the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt was re-dissolved in CH2Cl2 (1 mL) and cooled to 0° C. To this solution, a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) (72.8 mg, 0.312 mmol), triethylamine (217 μL, 1.56 mmol) and HBTU (118 mg, 0.312 mmol) in CH2Cl2 (2 mL) at 0° C. was added. The reaction was stirred under nitrogen for 18 h and then concentrated in vacuo. Purification by column chromatography (2.5-10% MeOH/CH2Cl2) yielded the product as a colourless oil (29.6 mg, 0.022 mmol, 28% over 2 steps). Rf 0.15 (SiO2, 10% MeOH/CH2Cl2); vmax (neat/cm−1) 3290 (m, N—H), 2944 (m, C—H), 2187 (m, C≡C), 1633 (s, C═O); δH (600 MHz, CD3OD) 6.67 (s, 4H), 6.59 (dd, 8H, J=10.7, 17.4 Hz), 6.35 (d, 8H, J=17.4 Hz), 5.55 (dd, 8H, J=1.5, 10.7 Hz), 3.97 (d, 2H, J=2.5), 3.45 (t, 8H, J=6.8 Hz), 3.27 (t, 4H, J=6.5 Hz), 3.24 (t, 8H, J=6.3 Hz), 3.21 (s, 2H), 3.18 (s, 4H), 2.65 (t, 4H, J=6.4 Hz), 2.62-2.59 (m, 1H), 2.60 (t, 8H, J=6.2 Hz), 2.28 (t, 8H, J=7.5 Hz), 1.90 (quint, 8H, J=7.2 Hz); δC (101 MHz, CD3OD) 175.9, 174.1, 173.7, 165.3, 164.0, 137.1, 122.1, 105.8, 80.8, 72.5, 59.7, 59.3, 55.9, 55.8, 44.0, 38.7, 38.5, 34.7, 29.4, 27.0; HRMS (ESI) m/z found [M+H]+ 1345.7930, C69H97O7N22+ required 1345.7905.
In a pre-dried microwave vial containing methyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4) (203 mg, 0.54 mmol) dissolved in dry MeOH (0.3 mL) was added triethylenetetramine (14.9 μL, 0.100 mmol), the vial was capped, flushed with nitrogen, and stirred over night at 110° C. The reaction mixture was concentrated and purified by column chromatography (0-30% MeOH/CH2Cl2) to give the desired compound as a colourless oil (40.8 mg, 0.049 mmol, 49%). Rf0.38 (SiO2, 20% MeOH/CH2Cl2); vmax (neat/cm−1) 3335 (m, N—H), 2980 (m, C—H), 1690 (s, C═O); δH (600 MHz, CD3OD) 3.38 (t, 4H, J=6.3 Hz), 3.17 (s, 4H), 3.13 (t, 8H, J=6.1 Hz), 2.80-2.77 (m, 4H), 2.76 (s, 4H), 2.60 (t, 8H, J=5.7 Hz), 1.45 (s, 36H); δC (101 MHz, CD3OD) 174.5, 158.6, 80.2, 60.1, 56.4, 49.7, 49.4, 39.7, 39.7, 28.9; HRMS (ESI) m/z found [M+H]+ 833.5843, C38H77O10N10+ required 833.5824.
In a pre-dried microwave vial, a suspension of compound 9 (39.2 mg, 0.047 mmol) and K2CO3 (26.0 mg, 0.188 mmol) in anhydrous MeCN (0.1 mL) was cooled to 0° C. by means of an ice-bath. A solution of compound 5 (20.8 mg, 0.118 mmol) in MeCN (0.4 mL) was slowly added to the stirring suspension, after which the reaction was brought to rt and stirred overnight under nitrogen. The reaction mixture was concentrated and purified by flash chromatography (0-10% MeOH/CH2Cl2) to give the title compound as a white solid (30.0 mg, 0.029 mmol, 62%). Rf 0.18 (SiO2, 10% MeOH/CH2Cl2); vmax (neat/cm−1) 3296 (m, N—H), 2978 (m, C—H), 2157 (m, C≡C), 1654 (s, C═O); δH (600 MHz, CD3OD) 4.20 (s, 4H), 4.08 (d, 4H, J=2.5 Hz), 3.80 (s, 4H), 3.57 (t, 4H, J=6.0 Hz), 3.51-3.44 (m, 16H), 3.27 (s, 4H), 3.21-3.17 (m, 4H), 2.72 (t, 2H, J=2.5 Hz), 1.47 (s, 36H); δC (101 MHz, CD3OD) 169.8, 166.3, 159.1, 81.3, 80.3, 73.2, 57.0, 56.1, 56.0, 55.9, 53.3, 37.0, 36.7, 30.0, 28.8; HRMS (ESI) m/z found [M+H]+ 1023.6547, C48H87O12N12+ required 1023.6566.
To a solution of compound 10 (79.8 mg, 0.078 mmol) in CH2Cl2 (0.5 mL) at 0° C. was added HCl (4M in dioxane, 1.0 mL). The reaction was stirred under nitrogen for 6 h and then concentrated in vacuo to yield the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt was re-dissolved in CH2Cl2 (1 mL) and cooled to 0° C. To this solution was added a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) (72.8 mg, 0.312 mmol), triethylamine (217 μL, 1.56 mmol) and HBTU (118 mg, 0.312 mmol) in CH2Cl2 (2 mL) at 0° C. The reaction was stirred under nitrogen for 18 h and then concentrated in vacuo. Purification by reverse phase flash column chromatography (35-70% solvent B in solvent A. Solvent A: 100 mM NH4OH (aq). Solvent B: MeCN) and lyophilisation yielded the product as a colourless oil (46.1 mg, 0.031 mmol, 40% over 2 steps). vmax (neat/cm−1) 3289 (m, N—H), 2938 (m, C—H), 2186 (m, C≡C), 1637 (s, C═O); δH (600 MHz, CD3OD) 6.67 (s, 4H), 6.59 (dd, 8H, J=10.6, 17.4 Hz), 6.35 (d, 8H, J=17.3 Hz), 5.56 (dd, 8H, J=1.2, 10.7 Hz), 3.98 (d, 4H, J=2.3), 3.45 (t, 8H, J=6.7 Hz), 3.27 (t, 4H, J=6.8 Hz), 3.24 (t, 8H, J=6.4 Hz), 3.18 (s, 4H), 3.17 (s, 4H), 2.64-2.57 (m, 4H), 2.64-2.57 (m, 2H), 2.64-2.57 (m, 8H), 2.60 (s, 4H), 2.29 (t, 8H, J=7.5 Hz), 1.91 (quint, 8H, J=7.1 Hz); δC (101 MHz, CD3OD) 175.9, 174.1, 173.7, 165.3, 164.0, 137.1, 122.1, 105.8, 80.9, 72.4, 59.7, 59.3, 55.8 (two peaks), 54.4, 41.7, 38.7, 38.4, 34.7, 29.4, 27.0; HRMS (ESI) m/z found [M+H]+ 1483.8687, C76H107O8N24+ required 1483.8698.
In a pre-dried microwave vial containing methyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4) (964 mg, 2.57 mmol) dissolved in dry MeOH (1 mL) was added tetraethylenepentamine (90 μL, 0.475 mmol), the vial was capped, flushed with nitrogen, and stirred over night at 110° C. The reaction mixture was concentrated and purified by column chromatography (0-20% MeOH/CH2Cl2) to give the desired compound as a white foam (184 mg, 0.21 mmol, 44%). Rf 0.06 (BuOH:H2O:MeCN, 0.8/0.1/0.1); vmax (neat/cm−1) 3330 (m, N—H), 2980 (m, C—H), 1689 (s, C═O); δH (400 MHz, CDCl3) 3.40-3.30 (m, 4H), 3.16-3.09 (m, 8H), 3.07 (s, 4H), 2.83-2.66 (m, 12H), 2.60-2.52 (m, 8H), 1.40 (s, 36H); δC (100 MHz, CDCl3) 171.5, 156.6, 79.3, 59.2, 55.4, 48.6 (three peaks), 38.7, 38.6, 28.6; HRMS (ESI) m/z found [M+H]+ 876.6284, C40H82N11NaO10+ required 876.6246.
In a pre-dried microwave vial, a suspension of compound 12 (184 mg, 0.21 mmol) and K2CO3 (174 mg, 1.26 mmol) in anhydrous MeCN (0.26 mL) was cooled to 0° C. by means of an ice-bath. A solution of compound 5 (anh. MeCN, 0.5 M, 1.58 mL) was slowly added to the stirring suspension, after which the reaction was brought to rt and stirred overnight under nitrogen. The reaction mixture was concentrated, re-dissolved in CH2Cl2 (40 mL), the organic phase washed with water (2×10 mL), brine (1×20 mL), dried over Na2SO4, and purified by flash chromatography (5-10% MeOH/CH2Cl2) to give the title compound as a white foam (119 mg, 0.102 mmol, 49%). Rf 0.5 (SiO2, 20% MeOH/CH2Cl2); vmax (neat/cm−1) 3309 (m, N—H), 2928 (m, C—H), 2158 (m, C≡C), 1658 (s, C═O); δH (400 MHz, CD3OD) 4.05 (d, J=2.6 Hz, 4H), 4.03 (d, J=2.5 Hz, 2H), 3.39-3.32 (m, 7H), 3.27 (s, 4H), 3.25 (s, 2H), 3.20 (s, 4H), 3.17-3.13 (m, 8H), 2.77-2.67 (m, 8H), 2.66-2.58 (m, 8H), 1.47 (s, 36H); δC (100 MHz, CD3OD) 174.3, 173.7, 173.6, 158.4, 80.9, 80.8, 80.1, 72.6, 72.3, 60.2, 59.3, 59.2, 56.5, 55.7, 54.4, 54.3, 39.7, 38.5, 29.5, 29.3, 28.9; HRMS (ESI) m/z found [M+H]+ 1161.7365, C55H97O13N14+ required 1161.7360.
To a solution of compound 13 (25 mg, 22 μmol) in CH2Cl2 (150 μL) at 0° C. was added HCl (4 M in dioxane, 270 uL). The reaction was brought to rt and stirred for 1 h. The reaction mixture was concentrated to give the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt (23.0 mg, 22.0 μmol) was suspended in CH2Cl2 (150 μL), cooled to 0° C. by means of an ice-bath, and added first DIPEA (74.0 μL, 0.42 mmol) and then 2 (25.0 mg, 0.11 mmol). The solution was stirred at rt for 10 min, after which a solution of BTFFH in CH2Cl2 (0.6 M, 205 μL) was added dropwise over 3 h during maximum stirring. After complete addition of BTFFH the reaction was stirred for an additional 12 h at rt, concentrated, and purified by reverse phase flash column chromatography (35-70% solvent B in solvent A. Solvent A: 100 mM NH4OH (aq). Solvent B: MeCN) to yield the desired product after lyophilization as a pale yellow solid (6.70 mg, 41.4 μmol, 20% over 2 steps). vmax (neat/cm1) 3305 (m, N—H), 2970 (m, C—H), 2156 (m, C≡C), 1637 (s, C═O); δH (400 MHz, CD3OD) 6.68 (s, 4H), 6.60 (dd, J=17.4, 10.7 Hz, 8H), 6.36 (d, J=17.3 Hz, 8H), 5.56 (dd, J=10.6, 1.6 Hz, 8H), 4.02-3.96 (m, 6H), 3.45 (t, J=6.8 Hz, 8H), 3.35 (s, 4H), 3.29-3.15 (m, 21H), 2.67-2.56 (m, 20H), 2.29 (t, J=7.5 Hz, 8H), 1.91 (quint, J=7.0 Hz, 8H); δC (126 MHz, CD3OD) 175.9, 174.1, 173.8, 173.7, 165.3, 164.0, 137.1, 122.1, 105.8, 81.0 (two peaks), 72.5, 72.4, 59.8, 59.4, 59.3, 55.9, 55.8, 54.5 (two peaks), 54.3 (two peaks), 41.7, 38.7, 38.4, 34.7, 29.4, 29.3, 27.0; HRMS (ESI) m/z found [M+H]+ 1621.9491, C83H117N26O9+required 1621.9497.
A solution of diethanolamine (2.44 g, 23.0 mmol) in CH2Cl2 (25 mL) was cooled to 0° C. by means of an ice-bath and a solution of di-tert-butyl dicarbonate (6.33 g, 29.0 mmol) in anh. CH2Cl2 (5 mL) as slowly added. The reaction was stirred for 12 h at rt, diluted in CH2Cl2 (40 mL), added water (40 mL), and the aqueous phase was extracted twice with CH2Cl2 (2×20 mL). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated to give tert-butyl bis(2-hydroxyethyl)carbamate as a clear oil, which was used directly in the next step without further purification. To a 50% NaOH(aq) solution (11 mL) was sequentially added tert-butyl bis(2-hydroxyethyl)carbamate in toluene (11 mL), tetrabutylammonium bisulfate (16.0 mg, 47.0 μmol), and propargyl bromide (80 w % in toluene, 8.7 mL, 78.0 mmol), and the reaction was stirred at rt for 48 h under nitrogen atmosphere. The organic layer was isolated, concentrated and the desired compound was purified by flash chromatography (50% EtOAc/hexane) to give the title compound as a yellow viscous oil (2.30 g, 7.57 mmol, 35% over 2 steps). Rf 0.60 (SiO2, 50% EtOAc/hexane); vmax (neat/cm−1) 2975 (m, C—H), 2117 (m, C≡C), 1686 (s, C═O); δH (400 MHz, CDCl3) 4.13 (d, J=2.4 Hz, 4H), 3.66-3.57 (m, 4H), 3.52-3.38 (m, 4H), 2.41 (t, J=2.4 Hz, 2H), 1.45 (s, 9H); δC (100 MHz, CDCl3) 155.6, 79.9, 79.8, 74.5, 68.7, 58.3, 47.9, 28.6; LRMS (ESI) m/z found [M+Na]+ 304.2.
A solution of compound 15 (281 mg, 1.00 mmol) in CH2Cl2 (1.5 mL) was cooled to 0° C., followed by addition of HCl (4 M in dioxane, 3 mL). The reaction was brought to rt and stirred for 2 h. The reaction mixture was concentrated to give the desired amine hydrochloride salt as a white solid. The solid was re-suspended in MeCN (10 mL). Sodium carbonate (1.06 g, 10.0 mmol) was added. After stirring for 5 min, a solution of tert-Butyl (5-bromopentyl)carbamate (399 mg, 1.50 mmol) in MeCN (2 mL) was added. The reaction mixture was then refluxed at 70° C. for 48 h. Subsequently, the reaction mixture was diluted with brine and extracted with EtOAc (3×). The combined organic layers were dried over Na2SO4, concentrated, and purified by flash chromatography (30-50% EtOAc/hexane) to give the title compound as a pale yellow oil (242 mg, 66% over 2 steps). Rf 0.21 (SiO2, 80% EtOAc/PE); vmax (neat/cm−1) 3342 (m, N—H), 2938 (m, C—H), 2111 (m, C≡C), 1676 (s, C═O); δH(400 MHz, CDCl3) 4.54 (br s, 1H), 4.16 (d, J=2.3 Hz, 4H), 3.60 (t, J=6.0 Hz, 4H), 3.14-3.06 (m, 2H), 2.73 (t, J=6.0 Hz, 4H), 2.55-2.49 (m, 2H), 2.42 (t, J=2.2 Hz, 2H), 1.51-1.42 (m, 4H), 1.44 (s, 9H), 1.30 (quint, J=7.6 Hz, 2H); Sc (100 MHz, CDCl3) 156.0, 79.9, 79.0, 74.3, 68.3, 58.2, 55.0, 53.8, 40.5, 29.9, 28.4, 26.7, 24.5; HRMS (ESI) m/z found [M+H]+ 367.2587, C20H34O4N2+ required 367.2597.
To a solution of compound 16 (100 mg, 0.27 mmol) in CH2Cl2 (0.5 mL) at 0° C. was added HCl (4M in dioxane, 1 mL). The reaction was stirred under nitrogen for 2 h and then concentrated in vacuo to yield the desired amine hydrochloride salt as a white solid. The hydrochloride salt was re-dissolved in a mixture of CH2Cl2 (1 mL) and sat. NaHCO3 (1 mL) and cooled to −10° C., followed by dropwise addition of 2-bromoacetyl bromide (35.3 μL, 0.405 mmol). The reaction mixture was stirred for 1 h, and then diluted with brine and extracted with CH2Cl2 (×3). Combined organic phases were dried over Na2SO4 and concentrated in vacuo. Purification by flash chromatography (0-5% MeOH/CH2Cl2) yielded the title compound as a pale yellow oil (57.3 mg, 0.148 mmol, 55% over 2 steps). Rf 0.41 (SiO2, 10% MeOH/CH2Cl2); vmax (neat/cm−1) 3219 (m, N—H), 2928 (m, C—H), 2111 (m, C≡C), 1672 (s, C═O); δH (400 MHz, CDCl3) 6.55 (br s, 1H), 4.15 (d, J=2.4 Hz, 4H), 3.87 (s, 2H), 3.62 (t, J=5.5 Hz, 4H), 3.27 (q, 2H, J=6.7 Hz), 2.78-2.75 (m, 4H), 2.58-2.54 (m, 2H), 2.43 (t, J=2.4 Hz, 2H), 1.55 (quint, J=7.4 Hz, 2H), 1.50 (quint, J=7.4 Hz, 2H), 1.33 (quint, J=7.6 Hz, 2H); δC (100 MHz, CDCl3) 165.5, 79.7, 74.8, 67.8, 58.4, 55.0, 53.9, 40.2, 29.8, 29.5, 29.1, 24.5; HRMS (ESI) m/z found [M+H]+ 387.1273, C17H28O3N2Br+ required 387.1283.
To a solution of compound 9 (49.3 mg, 59.2 μmol) in MeCN (1 mL) was added anh. K2CO3 (32.7 mg, 237 μmol), followed by a solution of N-(5-(bis(2-(prop-2-yn-1-yloxy)ethyl)amino)pentyl)-2-bromoacetamide (17) (57.3 mg, 148 μmol) in MeCN (0.5 mL). The reaction was stirred at room temperature for 48 h. The reaction mixture was concentrated, and the desired compound purified by flash chromatography (0%-15% MeOH/CH2Cl2) to give the title compound as a clear oil (38.8 mg, 26.6 μmol, 45%). Rf 0.35 (15% MeOH/CH2Cl2); vmax (neat/cm−1) 3283 (m, N—H), 2936 (m, C—H), 2155 (m, C≡C), 1654 (s, C═O); δH (400 MHz, CD3OD) 4.17 (s, 4H), 3.66 (t, J=5.5 Hz, 8H), 3.32-3.29 (m, 8H), 3.26-3.21 (m, 4H), 3.20 (s, 4H), 3.16 (s, 4H), 3.13 (t, J=5.8 Hz, 8H), 2.89-2.84 (m, 8H), 2.70-2.65 (m, 12H), 2.59 (t, J=5.7 Hz, 8H), 1.61-1.50 (m, 8H), 1.44 (s, 36H), 1.38-1.30 (m, 4H); δC (126 MHz, CD3OD) 174.3, 173.8, 158.5, 80.7, 80.2, 76.2, 68.3, 60.2, 59.6, 59.0, 56.5 (two peaks), 56.0 (two peaks), 54.6, 40.3, 39.7, 38.6, 30.5, 29.0, 26.8, 25.8; HRMS (ESI) m/z found [M+H]+ 1445.9692, C72H129N14O16+ required 1445.9711.
To a solution of compound 18 (13.0 mg, 8.99 μmol) in CH2Cl2 (0.25 mL) at 0° C. was added HCl (4M in dioxane, 0.5 mL). The reaction was stirred under nitrogen for 2 h and then concentrated in vacuo to yield the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt was re-dissolved in CH2Cl2 (0.25 mL) and cooled to 0° C. To this solution was added a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) (12.6 mg, 53.9 μmol), triethylamine (25 μL, 180 μmol) and HBTU (20.5 mg, 53.9 μmol) in CH2Cl2 (0.25 mL) at 0° C. The reaction was stirred under nitrogen for 18 h and then concentrated in vacuo. Purification by reverse phase flash column chromatography (35-70% solvent B in solvent A. Solvent A: 100 mM NH4OH (aq). Solvent B: MeCN) and lyophilisation yielded the product as a white solid (1.80 mg, 0.94 μmol, 10% over 2 steps). vmax (neat/cm−1) 3299 (m, N—H), 2928 (m, C—H), 2153 (m, C≡C), 1645 (s, C═O); δH (400 MHz, CD3OD) 6.69 (s, 4H), 6.60 (dd, J=17.4, 10.7 Hz, 8H), 6.36 (d, J=17.3 Hz, 8H), 5.57 (dd, J=10.7, 1.5 Hz, 8H), 4.23 (d, J=2.4 Hz, 8H), 3.83 (t, J=4.9 Hz, 8H), 3.45 (t, J=6.8 Hz, 8H), 3.30-3.15 (m, 32H), 3.00 (t, J=2.3 Hz, 4H), 2.65-2.60 (m, 24H), 2.29 (t, J=7.5 Hz, 8H), 1.91 (quint, J=7.1 Hz, 8H), 1.56 (quint, J=7.5 Hz, 4H), 1.39-1.30 (m, 8H); δC (126 MHz, CD3OD) 175.9, 174.2 (two peaks), 165.3, 164.0, 137.2, 122.2, 105.9, 89.9, 77.2, 68.8, 59.9 (two peaks), 59.2, 56.1, 56.0, 55.3, 54.0, 47.1, 41.8, 40.4, 38.8 (two peaks), 34.7, 30.2, 26.8, 27.1, 25.0; HRMS (ESI) m/z found [M+H]+ 1906.1851, C100H149N26O12+ required 1906.1848.
Dibromopyridazinedione (20) was synthesised as previously described (Bahou et al, Org. Biomol. Chem., 2018, 16, 1359-1366).
Compound 7 (3.90 mg, 4.40 μmol) was dissolved in CH2Cl2 (0.25 mL) and 4M HCl in 1,4-dioxane (0.25 mL) and the solution stirred at rt for 1 h. The reaction mixture then was concentrated under a stream of N2. To the crude residue was added dibromopyridazinedione 20 (10.0 mg, 22.0 μmol) in CH2Cl2DMF (1:1, 1 mL) followed by K2CO3 (3.00 mg, 22.0 μmol) and the reaction mixture stirred at rt for 3 h. Upon completion, the reaction was concentrated under a stream of N2 and purified by preparative RP-HPLC (5-95% B) to yield the title compound (2.50 mg, 1.36 mol, 31%) as a white solid. HPLC (5-95% MeCN/H2O over 20 min) retention time 7.433 min; LRMS (ESI) m/z found [M+H]+ 1837.0, C53H69Br8N18O15+ required 1837.5, m/z found [M+HCOO]− 1881.1, C54H69Br8N18O17− required 1881.5.
Compound 10 (10.0 mg, 9.80 μmol) was dissolved in CH2Cl2 (0.25 mL) and 4M HCl in 1,4-dioxane (0.25 mL) and the solution stirred at rt for 1 h. The reaction mixture then was concentrated under a stream of N2. To the crude residue was added dibromopyridazinedione 20 (22.0 mg, 48.9 μmol) in CH2Cl2DMF (1:1, 1 mL) followed by K2CO3 (6.80 mg, 48.9 μmol) and the reaction mixture stirred at rt for 3 h. Upon completion, the reaction was concentrated under a stream of N2 and purified by preparative RP-HPLC (5-95% B) to yield the title compound (4.20 mg, 2.13 μmol, 22%) as a white solid. HPLC (5-95% MeCN/H2O over 20 min) retention time 7.507 min; LRMS (ESI) m/z found [M+H]+ 1976.7, C60H79Br8N20O16+ required 1975.7, m/z found [M+HCOO]− 2018.8, C61H79Br8N20O18− required 2019.7.
Divinyltriazine (23) was synthesised as previously described (Counsell et al, Org. Biomol. Chem., 2020, 18, 4739-4743).
A solution of compound 10 (20.0 mg, 19.5 μmol) in HCl (4 M in 1,4-dioxane, 0.5 mL) and CH2Cl2 (0.5 mL) was stirred at rt for 1 h. Upon completion, the reaction mixture was concentrated in vacuo. To this deprotected residue was added HBTU (41.7 mg, 110 μmol), dinvinyltriazine 23 (24.2 mg, 110 μmol) and DMF (1 mL) followed by DIPEA (76.6 μL, 440 μmol) and the mixture stirred at rt for 45 min. Upon completion, the reaction mixture was purified by automated reverse phase FCC (10-60% solvent B in solvent A; Solvent A: 0.1 M NH4OH/H2O, Solvent B: MeCN) to yield the title compound (12.2 mg, 8.52 μmol, 44%) as a white solid. HPLC (5-95% solvent B in solvent A) retention time 8.257 min; LRMS (ESI) m/z found [M+H]+ 1432.9, C68H95N28O8+ required 1431.8, m/z found [M+HCOO]− 1477.1, C69H95N28O10− required 1475.8.
To a solution of Fmoc-hexamethylenediamine hydrochloride (200 mg, 0.533 mmol) and bromoacetyl bromide (116 μL, 1.33 mmol) in CH2Cl2 (2 mL) was added saturated aqueous NaHCO3 (2 mL) and the mixture stirred at rt for 24 h. The reaction mixture was then diluted with H2O and extracted with CH2Cl2 (3×50 mL). The combined organic fractions were washed with 1M HCl (2×50 mL), dried (MgSO4) and concentrated in vacuo. The crude residue was purified via FCC (50-100% EtOAc/PE) to give the title compound (107 mg, 0.233 mmol, 44%) as a white solid. Rf 0.16 (SiO2; 50% EtOAc/PE); δH (400 MHz, DMSO-d6) 8.24 (t, 1H, J=5.2 Hz), 7.89 (d, 2H, J=7.2 Hz), 7.68 (d, 2H, J=7.4 Hz), 7.41 (t, 2H, J=7.4 Hz), 7.31 (td, 2H, J=11.1, 0.7 Hz), 7.25 (t, 1H, J=5.5 Hz), 4.29 (d, 2H, J=7.5 Hz), 4.22-4.18 (m, 1H), 3.82 (s, 2H), 3.05 (q, 2H, J=6.6 Hz), 2.96 (q, 2H, J=6.6 Hz), 1.41-1.36 (m, 4H), 1.25-1.23 (m, 4H); δC (101 MHz, DMSO-d6) 165.8, 156.1, 143.9, 140.7, 127.6, 127.0, 125.1, 120.1, 65.1, 46.8, 29.6, 29.3, 28.8, 26.0, 25.9. LRMS (ESI) m/z found [M+H]+ 459.3, C23H28Br1N2O3+ required 459.1.
A solution of compound 6 (30.0 mg, 38.0 μmol), compound 25 (29.0 mg, 63.0 μmol) and DIPEA (8.60 μL, 49.4 μmol) in DMF (0.5 mL) was stirred at rt for 21 h. Upon completion, the reaction mixture was concentrated under a stream of N2. The crude residue was purified by FCC (0-8% MeOH/CH2Cl2) to yield the title compound (40.0 mg, 34.0 μmol, 90%) as a white solid. HPLC (5-95% solvent B in solvent A) retention time 10.824 min; LRMS (ESI) m/z found [M+H]+ 1169.1, C59H98N11O13+ required 1168.7, m/z found [M+HCOO]− 1213.1, C60H98N11O15− required 1212.7.
A solution of compound 26 (44.0 mg, 38.0 μmol) and piperidine (7.50 μL, 76.0 μmol) in DMF (1 mL) was stirred at rt for 2 h. Upon completion, the reaction mixture was concentrated under a stream of N2. To this crude residue was added HBTU (28.8 mg, 76.0 μmol) and DMF (1 mL) followed by N3-PEG4-COOH (114 μL, 57.0 μmol, 0.5 M in TBME) and DIPEA (13.2 μL, 76.0 μmol) and the mixture stirred at rt for 20 h. Upon completion, the reaction mixture was concentrated under a stream of N2 and the crude purified by FCC (0-10% MeOH/CH2Cl2) to yield the title compound (38.0 mg, 31.5 μmol, 83%) as a clear oil. HPLC (5-95% solvent B in solvent A) retention time 10.471 min; LRMS (ESI) m/z found [M+H]+ 1205.8, C54H105N14O16+ required 1205.8, m/z found [M+HCOO]− 1250.7, C55H105N14O18− required 1249.8.
A solution of compound 27 (29.0 mg, 24.0 μmol) in HCl (4 M in 1,4-dioxane, 0.5 mL) and CH2Cl2 (0.5 mL) was stirred at rt for 1 h. Upon completion, the reaction mixture was concentrated in vacuo. To this deprotected residue was added HBTU (45.5 mg, 120 μmol), compound 2 (28.0 mg, 120 μmol) and DMF (1 mL) followed by DIPEA (83.8 μL, 480 μmol) and the mixture stirred at rt for 1 h. Upon completion, the reaction mixture was purified by automated reverse phase FCC (10-100% solvent B in solvent A; Solvent A: 0.1 M NH4OH/H2O, Solvent B: MeCN) to yield the title compound (12.0 mg, 7.20 μmol, 30%) as a pale yellow solid. HPLC (5-95% solvent B in solvent A) retention time 8.401 min; LRMS (ESI) m/z found [M+Na]+ 1689.2, C82H124N2623Na1O12+ required 1689.0, m/z found [M+HCOO]− 1711.1, C3H125N26O14− required 1711.0.
Fmoc-Lys-OH·HCl (500 mg, 1.23 mmol) was suspended in DMF/CH2Cl2 (1:1, 3 mL) and a solution of N3-dPEG4-NHS ester (480 mg, 1.23 mmol) in DMF/CH2Cl2 (1:1, 3 mL) was added dropwise with stirring. K2CO3 (324 mg, 2.35 mmol) was added and the mixture was stirred for 23 hours. The reaction mixture was diluted with CH2Cl2 and washed with aqueous LiCl (3M, 2×15 mL). The combined aqueous layer was extracted with CH2Cl2 (15 mL) and the combined organic extracts concentrated in vacuo. The crude organic fraction was purified by automated reverse phase FCC (10-60% solvent B in solvent A; Solvent A: 0.5% formic acid/H2O, Solvent B: MeCN) to yield the product, Fmoc-Lys(COdPEG4-N3)—OH (560 mg, 0.810 mmol, 66%) as a colourless oil. δH (400 MHz, CDCl3) 7.72 (d, 2H, J=7.5 Hz), 7.58 (t, 2H, J=7.3 Hz), 7.36 (t, 2H, J=7.4 Hz), 7.27 (t, 2H, J=7.4 Hz), 6.90 (br s, 1H), 6.23 (br s, 1H), 5.90 (d, 1H, J=7.1 Hz), 4.44-4.30 (m, 3H), 4.18 (t, 1H, J=6.9 Hz), 3.72-3.63 (m, 2H), 3.60 (s, 10H), 3.56 (s, 4H), 3.34 (t, 2H, J=4.9 Hz), 3.23 (qt, 2H, J=6.1 Hz), 2.45 (t, 2H J=5.5 Hz), 1.94-1.82 (m, 1H), 1.81-1.72 (m, 1H), 1.58-1.30 (m, 4H); Sc (101 MHz, CDCl3) 174.5, 172.6, 171.0, 156.3, 144.0, 143.8, 141.3, 127.7, 127.1, 125.2, 125.2, 120.0, 80.6, 70.6, 70.5, 70.5, 70.3, 70.2, 70.1, 70.0, 67.2, 66.9, 53.8, 50.6, 47.2, 38.9, 36.7, 36.3, 31.7, 28.8, 28.1, 22.1; LRMS (ESI) m/z found [M+H]+ 642.5, C32H43N5O9+ required 642.3.
The peptide was synthesised on a 0.1 mmol scale using a general Fmoc solid phase peptide synthesis procedure, producing the desired peptide 30 (40.5 mg, 30.0 μmol, 27%) as a colourless, viscous oil. HPLC (5-95% solvent B in solvent A) retention time 4.313 min; LRMS (ESI) m/z found [M+H]+ 1031.3, C45H87N15O12+ required 1030.7.
Compound 30 (35.0 mg, 24.0 μmol) was suspended in DMF/CH2Cl2 (1:1, 0.4 mL)) and cooled to 0° C. A suspension of compound 2 (27.5 mg, 120 μmol), HBTU (44.7 mg, 120 μmol) and NEt3 (66.0 uL, 470 μmol) in DMF/CH2Cl2 (1:1, 0.4 mL) was added and the mixture stirred for 3 hours. The mixture was purified by automated reverse phase FCC (10-60% solvent B in solvent A; Solvent A: H2O, Solvent B: MeCN) to yield the title compound (18.3 mg, 9.70 μmol, 40%) as a colourless viscous oil. HPLC (5-95% solvent B in solvent A) retention time 8.084 min; LRMS (ESI) m/z found [M+Na]+ 1914.2, C93H13923Na1N27O16+ required 1914.1, m/z found [M+HCOO−]− 1936.3, C94H140N27O18− required 1936.1.
The peptide was synthesised on a 0.1 mmol scale using the general peptide synthesis procedure, producing the product (30.6 mg, 16.0 μmol, 5%) as a colourless, viscous oil. HPLC (5-95% solvent B in solvent A) retention time 5.942 min; LRMS (ESI) m/z found [M+H]+ 1489.8, C64H122N21O19+ required 1488.9, m/z found [M+HCOO−]− 1532.9, C65H122N21O21− required 1533.7.
Compound 32 (23.3 mg, 12.0 μmol) was suspended in DMF (0.5 mL) and cooled to 0° C. A suspension of compound 2 (14.0 mg, 60.0 μmol), HBTU (22.7 mg, 60.0 μmol) and NEt3 (33.4 uL, 240 μmol) in DMF (0.5 mL) was added and the mixture stirred for 5 hours. The mixture was purified by automated reverse phase FCC (40-55% MeCN in H2O) to yield the title compound (1.81 mg, 0.770 μmol, 5%) as a colourless viscous oil. HPLC (5-95% solvent B in solvent A) retention time 8.376 min; LRMS (ESI) m/z found [M+2H]2+ 1176.3, C112H175N33O432+ required 1175.7.
The peptide was synthesised on a 0.3 mmol scale using the general peptide synthesis procedure, producing the desired product (44.1 mg, 60.0 μmol, 60%) as a colourless, viscous oil. HPLC (5-95% solvent B in solvent A) retention time 5.985 min; LRMS (ESI) m/z found [M+H]735.5, C30H54N8O13+ required 735.4.
Compound 26 (20.0 mg, 17.0 μmol) was dissolved in DMF (0.5 mL) and piperidine (3.40 uL, 34.0 μmol) was added. The mixture was stirred for 90 minutes, then concentrated under N2. The crude mixture was redissolved in DMF (0.5 mL) and compound 34 (18.9 mg, 26.0 μmol), HBTU (13.0 mg, 34.0 μmol) and DIPEA (6.00 μL, 34.0 μmol) were added. The reaction mixture was stirred for 3 hours. The mixture was purified by automated reverse phase FCC (10-100% solvent B in solvent A; Solvent A: 0.1 M NH4OH/H2O, Solvent B: MeCN) to yield the title compound (11.4 mg, 6.90 μmol, 40%) as a pale yellow powder. HPLC (5-95% solvent B in solvent A) retention time 8.538 min; LRMS (ESI) m/z found [M+2H]2+ 832.4, C74H139N19O23+ required 832.0.
Compound 35 (7.00 mg, 4.20 μmol) was dissolved in CH2Cl2 (0.3 mL) and HCl (2M in Et2O, 0.3 mL) added. The mixture was stirred for 1 hour and then concentrated. The crude mixture was redissolved in DMF (0.5 mL) and compound 2 (7.00 mg, 30.0 μmol), HBTU (11.4 mg, 30.0 μmol) and DIPEA (10.5 μL, 60.0 μmol) were added. The mixture was stirred for 2 hours and then purified twice via automated reverse phase FCC (40-70% MeCN in H2O) to yield the desired product (0.120 mg, 0.600 μmol, 1.3%) as a yellow residue. HPLC (5-95% solvent B in solvent A) retention time 7.66 min; LRMS (ESI) m/z found [M+Na]+ 2124.4, C102H160N31O19+ required 2124.3, m/z found [M+HCOO−]− 2169.0, C103H160N31O21− required 2168.2.
To a solution of trastuzumab (198 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37° C. for 1 h. A solution of conjugating reagent 1, 8, 11 or 14 (10 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37° C. for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). Samples were stored at 4° C. until analysis. LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the corresponding bridged conjugates 36, 37, 38 and 39.
To a solution of trastuzumab (50 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37° C. for 1 h. A solution of conjugating reagent 19, 21, 22, 24, 28, 31 or 33 (5 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37° C. for 2 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific). Samples were either stored at 4° C. or flash frozen and stored at −20° C. until analysis. LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the corresponding bridged conjugates 40, 41, 42, 43, 44, 45 and 46.
To a solution of brentuximab (50 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37° C. for 1 h. A solution of conjugating reagent 1, 8, 19 or 21 (5 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37° C. for 2 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific). Samples were either stored at 4° C. or flash frozen and stored at −20° C. until analysis. LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the corresponding bridged conjugates 47, 48, 49 and 50.
Size-exclusion chromatography (SEC) was carried out using a Superdex 200 10/300 GL column. Samples were injected at a concentration of 1 mg/mL and eluted with TBS buffer (25 mM Tris HCl pH 8, 200 mM NaCl, 0.5 mM EDTA) at a flow rate of 0.5 mL/min.
Size exclusion chromatograms for conjugates 36, 37, 38 and 39 are shown in
A 96-well plate was coated with 100 μL of a 0.25 μg/mL solution of HER2 (Sino Biological, His-tagged) overnight at 4° C. Coating solutions were removed and each well washed with PBS (2×200 μL). Each well was then blocked with 1% BSA in PBS (200 μL) for 1 h at room temperature. The blocking solution was then removed and each well washed with PBS (3×200 μL). Wells were treated with a serial dilution of trastuzumab and trastuzumab conjugates 36, 37, 38 and 39 in PBS (100 μL of 90 nM, 30 nM, 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0 nM) and incubated at room temperature for 2 h. The conjugate solutions were removed, and each well was washed with 0.1% Tween 20 in PBS (2×200 μL) followed by PBS (3×200 μL). Next, 100 μL of detection antibody (1:500 dilution of a mouse anti-human IgG-HRP, ThermoFisher) in PBS was added to each well and incubated at room temperature for 1 h. Each well was washed with 0.1% Tween 20 in PBS (2×200 μL) followed by PBS (3×200 μL). Finally, an OPD solution (100 μL of a solution prepared by dissolving 1 capsule in 9 mL H2O and 1 mL stable peroxide substrate buffer (10×), ThermoFisher) was added to each well. After 15 minutes, 4M HCl (aq.) (50 μL) was added to each well to quench the reaction. Absorbance at 490 nm and 590 nm was measured using a CLARIOstar Microplate Reader. Measurements were performed in quadruplicate and three independent repeats were performed.
ELISA results for conjugates 36, 37, 38 and 39 are shown in
To a solution of trastuzumab conjugates 36, 37, 38 or 39 in PBS was added CuSO4·5H2O (20 equiv. per alkyne), THPTA (100 equiv. per alkyne), sodium ascorbate (150 equiv. per alkyne) and AlexaFluor™ 488 Azide (Thermo Fisher Scientific) (5 mM in DMSO, 12 equiv. per alkyne). The mixture was vortexed and incubated at 37° C. for 4 h (for 36 and 37) or 6 h (for 38 and 39). The excess reagents were removed by use of a Zeba™ Spin Desalting Column (7,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). UV-vis analysis revealed conversion of 36, 37, 38 and 39 to antibody-fluorophore conjugates 51, 52, 53 and 54 with average fluorophore-to-antibody ratios (FAR) of 1.0, 2.0, 2.9 and 4.0, respectively. Each reaction was repeated at least three times and gave consistent results with FAR values within ±0.1 units of those reported above.
To a solution of trastuzumab-AlexaFluor488 conjugates 51, 52, 53 and 54 (138 μL, 3.75 μM) in PBS were added 12 μL of reconstituted human plasma (Sigma). The mixture was incubated at 37° C. for 14 days. Aliquots were removed every 2 days, flash frozen, and stored at −80° C. until analysis. SDS-PAGE was followed by in-gel fluorescence and coomassie brilliant blue staining.
Payload 55 was synthesised as previously described (Walsh et al, Chem. Sci., 2019, 10 (3), 694-700).
A solution of Fmoc-Val-Cit-PABC-PNP (21.6 mg, 30.1 μmol), MMAE (30.0 mg, 39.1 μmol), HOBt·H2O (10.1 mg, 60.0 μmol), DIPEA (15.7 μL, 90.0 μmol) and pyridine (24.3 μL, 300 μmol) in DMF (1 mL) was stirred at rt for 24 h. Upon completion, the reaction mixture was purified by automated reverse phase FCC (10-70% solvent B in solvent A. Solvent A: 0.1 M NH4OH (aq). Solvent B: MeCN) and lyophilised to yield the desired compound (39.0 mg, 29.0 μmol, 96%) as a white solid. HPLC (5-95% solvent B in solvent A) retention time 13.189 min; LRMS (ESI) m/z found [M+Na]+ 1368.6, C73H104N1023Na1O14+ required 1367.8.
To a solution of Fmoc-Val-Cit-PABC-MMAE (57) (20.0 mg, 14.9 μmol) in DMF (2 mL) was added piperidine (7.30 μL, 74.3 μmol) and the mixture stirred at rt for 2 h. Upon completion, the reaction was concentrated under a stream of N2. To the free amine was added HBTU (11.3 mg, 29.8 μmol) and DMF (2 mL) followed by N3-PEG4-CO2H (50 μL of a 0.5 M solution in TBME, 22.4 μmol) and DIPEA (7.80 μL, 44.7 μmol) and the reaction mixture stirred at rt for 18 h. Upon completion, the reaction was concentrated under a stream of N2 and purified by automated reverse phase FCC (10-100% solvent B in solvent A. Solvent A: 0.1 M NH4OH (aq). Solvent B: MeCN) and lyophilised to yield the title compound (13.6 mg, 9.80 μmol, 66%) as a white solid. HPLC (5-95% solvent B in solvent A) retention time 11.319 min; LRMS (ESI) m/z found [M+H]+ 1282.8, C68H112N13O17+ required 1382.8, m/z found [M+HCOO]− 1428.0, C69H112N13O19 − required 1426.8.
To a solution of trastuzumab conjugates 36, 37, 38 or 39 in PBS was added CuSO4·5H2O (150 equiv.), THPTA (600 equiv.), sodium ascorbate (1000 equiv.) and compound 55 (20 mM in DMSO, 100 equiv.). The mixture was vortexed and incubated at 37° C. for 6 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). Hydrophobic interaction chromatography (HIC) revealed conversion of 36, 37, 38 and 39 to antibody-drug conjugates 58, 59, 60 and 61 with average drug-to-antibody ratios (DAR) of 0.8, 1.4, 2.1 and 2.8, respectively.
Hydrophobic interaction chromatograms for conjugates 58, 59, 60 and 61 are shown in
To a solution of trastuzumab conjugates 36, 37, 38 or 39 in PBS was added CuSO4·5H2O (150 equiv.), THPTA (600 equiv.), sodium ascorbate (1000 equiv.) and compound 56 (20 mM in DMSO, 100 equiv.). The mixture was vortexed and incubated at 37° C. for 6 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). Hydrophobic interaction chromatography (HIC) revealed conversion of 36, 37, 38 and 39 to antibody-drug conjugates 62, 63, 64 and 65 with average drug-to-antibody ratios (DAR) of 0.5, 1.0, 1.6 and 2.4, respectively.
Hydrophobic interaction chromatograms for conjugates 62, 63, 64 and 65 are shown in
HER2-positive SKBR3 and BT474 cells were obtained from the American Type Culture Collection (ATCC) and HER2-negative MCF7 and MDA-MB-468 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC) and ATCC, respectively. SKBR3 cells were maintained in high glucose McCoy's 5A medium, supplemented with 10% heat-inactivated foetal-bovine serum (FBS), GlutaMAX™, 50 U/mL penicillin and 50 μg/mL streptomycin. MCF7 and MDA-MB-468 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal-bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. BT474 cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal-bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. All cell lines were incubated at 37° C. with 5% CO2.
Cells were seeded in 96-well plates for 24 h at 37° C. with 5% CO2. SKBR3 cells were seeded at 15,000 cells/well, BT474 cells were seeded at 20,000 cells/well, MCF7 cells were seeded at 7,500 cells/well and MDA-MB-468 cells were seeded at 10,000 cells/well. Serial dilutions of 62, 63, 64, 65 and trastuzumab were added to the cells in complete growth medium and incubated at 37° C. with 5% CO2 for 96 h. Cell viability was measured using CellTiter-Glo viability assay (Promega) according to the manufacturer's instructions. Cell viability was plotted as a percentage of untreated cells. Each measurement was taken in triplicate and three independent repeats were performed.
Each of the conjugates displayed a significant increase in cytotoxicity in HER2-positive cells compared to trastuzumab alone. In contrast, the proliferation of HER2-negative cells was not affected compared to vehicle control, thus confirming the selectivity for HER2-positive cells. Also, higher DAR conjugates (conjugates 64 and 65) displayed an increase in cytotoxicity relative to lower DAR conjugates (conjugates 62 and 63). This showcases the value of DAR tunability offered by the present invention.
SKBR3 or MCF7 cells were seeded at 40,000 cells/well in 8-well chambered μ-slide (Ibidi, 80826) for 48 h at 37° C. with 5% CO2. Slides were then placed on ice and washed with Ham's F12 Nutrient Mix media containing 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin (3×200 μL). Antibody conjugates 51 and 52 (50 nM), trastuzumab (50 nM) or vehicle (PBS) were added to the cells in complete F12 growth medium and incubated at 4° C. in the dark for 1 h. Cells were placed back on ice and washed with complete F12 growth medium (3×200 μL). Complete F12 growth medium (200 μL) was added and the cells incubated for 3.5 h at 37° C. with 5% CO2. After 3 h incubation, Hoechst 33342 trihydrochloride trihydrate (1 μg/mL, Invitrogen, H3570) was added. Live cell microscopy was performed on an Operetta CLS confocal microscope (Perkin Elmer) with a 40× water objective. Cells were maintained in a humidified atmosphere at 37° C. and 5% CO2 throughout analysis. Data analysis was performed using ImageJ (Fiji).
Microscopy images are shown in
Compound 3 (4.43 g, 14.6 mmol) was dissolved in DMF (49 mL) and benzyl bromoacetate (3.47 mL, 21.9 mmol) and DIPEA (3.05 mL, 17.5 mmol) were added dropwise. The reaction mixture was stirred for 16 hours. The mixture was diluted with water (850 mL) and extracted with EtOAc (3×300 mL). The combined organic extracts were washed with brine (5×350 mL) and aqueous LiCl (3M, 2×350 mL), dried (MgSO4), filtered and concentrated in vacuo. The crude mixture was submitted to flash column chromatography (0-50% EtOAc/Pet. Ethers 40-60) to yield the product, benzyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4.99 g, 11.1 mmol, 76%) as a colourless oil. Rf: 0.39 (50% EtOAc/Pet. Ethers 40-60); 1H NMR (500 MHz, CDCl3, 25° C.): δ (ppm)=7.39-7.31 (m, 5H), 5.16 (s, 2H), 3.49 (br s, 2H), 3.20 (br s, 4H), 2.81 (br s, 4H), 1.44 (s, 18H); HRMS (ESI) C22H35N3O6 m/z: [M+Na]+ 474.2576 (calc. 474.2574).
Benzyl ester 202 (7.78 g, 17.2 mmol) was dissolved in MeOH (170 mL) and degassed with N2 for 15 minutes. Palladium on carbon (10 wt % Pd, 340 mg) was added and the suspension flushed with H2 for 10 minutes. The vent needle was removed, and a fresh H2 balloon applied, and the suspension stirred for 20 hours. The mixture was filtered through Super-Cel®, washed with MeOH (2×125 mL) and concentrated in vacuo to yield the product, bis(2-((tert-butoxycarbonyl)amino)ethyl)glycine (5.81 g, 16.1 mmol, 93%) as a white solid. 1H NMR (500 MHz, DMSO-d6, 25° C.): δ (ppm)=6.64 (t, J=5.0 Hz, 2H), 3.27 (s, 2H), 2.95 (q, J=6.0 Hz, 4H), 2.60 (t, J=6.7 Hz, 4H), 1.37 (s, 18H); 13C NMR (126 MHz, DMSO-d6): δ (ppm)=172.6, 155.6, 77.5, 54.9, 53.4, 38.4, 28.2; HRMS (ESI) C16H31N3O6 m/z: [M+H]+ 362.2271 (calc. 362.2286).
Acid 203 (100 mg, 0.28 mmol) was dissolved in DMF (1 mL). 11-Azido-3,6,9-trioxaundecanamine (55 μL, 0.28 mmol), HBTU (105 mg, 0.28 mmol) and DIPEA (96 μL, 0.55 mmol) were added, and the reaction mixture stirred for 71 hours. The mixture was diluted with aqueous HCl (1M, 25 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (25 mL), dried (Na2SO4), filtered and concentrated in vacuo. The crude residue was submitted to flash column chromatography (0-6% MeOH/CH2Cl2) to yield the product, 204 (94 mg, 0.17 mmol, 60%) as a colourless residue. Rf: 0.27 (6% MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3, 25° C.): δ (ppm)=7.47 (br s, 1H), 5.68 (br s, 2H), 3.68-3.57 (m, 12H), 3.46 (q, J=3.2 Hz, 2H), 3.37 (t, J=4.9 Hz, 2H), 3.26-2.99 (m, 5H), 2.58 (br s, 3H), 1.44 (s, 18H); 13C NMR (101 MHz, CDCl3): δ (ppm)=171.0, 156.7, 79.5, 70.8, 70.7, 70.6, 70.2, 70.1, 69.6, 55.3, 50.8, 39.2, 38.3, 28.6; HRMS (ESI) C24H47N7O8 m/z: [M+Na]+ 584.3401 (calc. 584.3378).
N-Boc amine 204 (64 mg, 0.11 mmol) was suspended in HCl/dioxane (4M, 1 mL) and stirred for 1.5 hours. The solvent was removed under a stream of nitrogen to leave a white solid, which was used without further purification. The intermediate was suspended in DMF (0.5 mL) and DIPEA (119 μL, 0.68 mmol), acid 203 (103 mg, 0.28 mmol) and HBTU (108 mg, 0.28 mmol) were added. The reaction mixture was stirred for 22 hours. The mixture was diluted with water (20 mL) and extracted with EtOAc (2×20 mL). The combined organic extracts were washed with brine (4×50 mL), dried (Na2SO4), filtered and concentrated. The crude product was submitted to flash column chromatography (0-8% MeOH/CH2Cl2) to yield the product, 205 (53.5 mg, 0.051 mmol, 46%) as a colourless residue. Rf: 0.15 (8% MeOH/CH2Cl2); 1H NMR (500 MHz, CDCl3, 25° C.): δ (ppm)=6.77 (s, 3H), 3.61 (t, J=5.0 Hz, 3H), 3.58-3.51 (m, 8H), 3.47 (t, J=6.0 Hz, 2H), 3.39 (t, J=5.0 Hz, 2H), 3.28 (q, J=5.8 Hz, 2H), 3.24 (s, 1H), 3.12 (s, 1H), 1.39 (s, 36H); 13C NMR (125 MHz, CDCl3): δ (ppm)=155.7, 78.2, 69.8, 69.8, 69.7, 69.6, 69.3, 68.9, 54.1, 50.0, 28.2; LRMS (ESI) C46H89N13O14 m/z: [M+H]+ 1048.7 (calc. 1048.7).
Tetra-N-Boc amine 205 (20 mg, 0.019 mmol) was dissolved in CH2Cl2 (1.5 mL) and HCl in dioxane (4M, 0.5 mL) was added. The reaction mixture was stirred for 1.5 hours and then the solvent was removed in vacuo. The intermediate was redissolved in DMF (1 mL) and compound 2 (22.3 mg, 0.095 mmol), HBTU (36.2 mg, 0.095 mmol) and DIPEA (33 μL, 0.191 mmol) were added. The reaction mixture was stirred for 3.5 hours. LCMS indicated incomplete conversion, so further compound 2 (8.9 mg, 0.038 mmol), HBTU (14.5 mg, 0.038 mmol) and DIPEA (13 μL, 0.076 mmol) were added. The mixture was stirred for a further 1.5 hours and then submitted to reverse-phase Combiflash chromatography (10-70% MeCN in 0.1M aqueous NH4OH) to yield the product, 206 (9.05 mg, 0.0060 mmol, 32%) as a pale-yellow solid. 1H NMR (400 MHz, CDCl3, 25° C.): δ (ppm)=7.73 (br s, 2H; H), 6.55 (dd, J=16.9, 10.1 Hz, 14H), 6.39 (d, J=17.3 Hz, 8H), 5.62 (d, J=10.5 Hz, 8H), 2.38 (t, J=7.1 Hz, 9H), 1.95 (qu, J=6.6 Hz, 9H); 13CNMR (125 MHz, CDCl3): δ (ppm)=174.3, 163.4, 161.6, 136.4, 131.3, 121.3, 107.9, 105.2, 70.7, 70.7, 70.6, 70.6, 70.6, 70.2, 70.1, 70.1, 69.4, 59.0, 55.9, 53.8, 50.8, 48.2, 46.9, 40.9, 39.3, 37.3, 33.9, 26.3, 25.7, 24.8, 24.4; LRMS (ESI) C74H109N25O10 m/z: [M+formate]− 1553.0 (calc. 1552.7).
Tetra N-Boc amine 205 (35 mg, 0.033 mmol) was suspended in HCl/dioxane (4M, 1 mL) and stirred for 1.5 hours. The solvent was removed under a stream of nitrogen to leave a white solid, which was used without further purification. The intermediate was suspended in DMF (0.3 mL) and Fmoc-8-amino-3,6-dioxaoctanoic acid (51 mg, 0.13 mmol), HBTU (50 mg, 0.13 mmol) and DIPEA (70 μL, 0.40 mmol) were added. The reaction mixture was stirred for 18 hours. The mixture was diluted with water (20 mL) and extracted with EtOAc (2×20 mL). The combined organic extracts were washed with aqueous LiCl (5 wt %, 30 mL), dried (Na2SO4), filtered and concentrated. The crude product was submitted to flash column chromatography (4-10% MeOH/CH2Cl2) to yield the product, 207 (13 mg, 0.0059 mmol, 18%) as a white residue. Rf: 0.10 (10% MeOH/CH2Cl2); 1H NMR (400 MHz, MeOD, 25° C.): δ (ppm)=7.76 (d, J=7.5 Hz, 8H), 7.61 (d, J=7.4 Hz, 8H), 7.36 (t, J=7.4 Hz, 8H), 7.27 (t, J=7.4 Hz, 8H), 4.58 (s, 2H), 4.46 (s, 1H), 4.32 (d, J=6.8 Hz, 7H), 4.16 (t, J=6.7 Hz, 4H), 3.94 (s, 8H), 3.64-3.57 (m, 17H), 3.56-3.53 (m, 8H), 3.53-3.43 (m, 10H), 3.36 (t, J=5.4 Hz, 2H), 3.27 (t, J=5.9 Hz, 18H), 3.20 (s, 2H), 3.18 (s, 4H), 3.05 (br s, 1H), 2.66 (t, J=6.5 Hz, 4H), 2.61 (t, J=6.2 Hz, 8H); 13C NMR (101 MHz, MeOD): δ (ppm)=174.2, 172.7, 158.8, 145.3, 142.6, 128.8, 128.2, 126.2, 121.0, 72.0, 71.6, 71.5, 71.4, 71.2, 71.1, 71.0, 67.7, 59.7, 55.9, 55.6, 48.5, 41.7, 40.0, 38.3; LRMS (ESI) C110H141N17O26 m/z: [M+formate]− 2161.4 (calc. 2161.0).
Tetra-N-Fmoc amine 207 (10 mg, 0.0047 mmol) was dissolved in DMF (0.3 mL) and piperidine (4.6 μL, 0.047 mmol) was added. The mixture was stirred for 2 hours. The solvent was removed under a stream of nitrogen to leave a white solid, which was used without further purification. The intermediate was suspended in DMF (0.3 mL) and compound 2 (5 mg, 0.021 mmol), HBTU (8 mg, 0.021 mmol) and DIPEA (7.4 μL, 0.043 mmol) were added. The reaction mixture was stirred for 2 hours and then submitted directly to reverse-phase Combiflash chromatography (10-80% MeCN in water). Fractions containing product were combined and lyophilised to yield the product, 208 (1.9 mg, 0.00089 mmol, 19%) as an off-white solid residue. 1H NMR (400 MHz, DMSO-d6, 25° C.): δ (ppm)=7.85 (t, J=5.5 Hz, 4H), 7.81-7.74 (m, 3H), 7.69 (t, J=5.6 Hz, 4H), 7.04 (t, J=5.5 Hz, 4H), 6.76 (s, 4H), 6.57 (dd, J=10.7, 17.3 Hz, 8H), 6.34 (d, J=16.1 Hz, 8H), 5.57 (dd, J=1.6, 10.5 Hz, 8H), 3.85 (s, 8H), 3.59-3.46 (m, 29H), 3.40 (t, J=5.7 Hz, 8H), 3.23-3.11 (m, 23H), 3.07 (br s, 6H), 2.67 (t, J=1.8 Hz, 3H), 2.59-2.53 (m, 7H), 2.33 (m, 5H), 2.13 (t, J=7.6 Hz, 11H), 1.74 (t, J=7.4 Hz, 11H); HRMS (ESI) C98H153O22N29 m/z: [M+H]+ 2089.1782 (calc. 2089.1818).
Compound 204 (385 mg, 0.69 mmol) was dissolved in HCl solution (4 M in dioxanes, 4 mL) and CH2Cl2 (2 mL) and stirred for 3 hours. The solvent was removed under a stream of nitrogen, and the resulting residue was redissolved in DMF (2.5 mL). Fmoc-8-Amino3,6-dioxaoctonoic acid (532 mg, 1.38 mmol), HBTU (523 mg, 1.38 mmol) and DIPEA (721 μL, 4.14 mmol) were added, and the reaction mixture stirred for 17 hours. Some acid starting material remained, so further HBTU (261 mg, 0.69 mmol) and DIPEA (120 μL, 0.69 mmol) were added and the mixture stirred for a further 1 hour. The mixture was submitted directly to reverse-phase column chromatography (10-100% MeCN in H2O) to yield the product, 209 (378 mg, 0.34 mmol, 50%) as a viscous orange residue. Rf: 0.18 (7% MeOH in CH2Cl2); IR(neat): vmax(cm−1)=3313 (N—H, medium), 2928 (C—H, medium), 2870 (C—H, medium), 2107 (N3, medium), 1714 (C═O, strong), 1658 (C═O, strong), 1529 (N—H, strong), 1448 (C—H, strong), 1247 (C—N, strong), 1102 (CO, strong); 1H NMR (500 MHz, CDCl3, 25° C.): δ (ppm)=7.75 (d, J=7.5 Hz, 4H), 7.60 (d, J=7.2 Hz, 4H), 7.39 (t, J=7.5 Hz, 4H), 7.30 (t, J=7.4 Hz, 4H), 7.18 (br s, 2H), 5.71 (br s, 2H), 4.40 (d, J=6.4 Hz, 4H), 4.20 (t, J=6.7 Hz, 2H), 3.99 (s, 4H), 3.68-3.51 (m, 24H), 3.43 (q, J=5.6 Hz, 2H), 3.40-3.30 (m, 10H), 3.15 (s, 2H), 2.64 (t, J=6.0 Hz, 4H); 13C NMR (125 MHz, CDCl3): δ (ppm)=171.4, 170.5, 156.8, 144.1, 141.5, 127.8, 127.2, 125.2, 120.1, 77.4, 71.1, 70.8, 70.7, 70.6, 70.6, 70.4, 70.2, 70.1, 70.1, 69.7, 66.7, 59.2, 54.8, 50.8, 47.4, 41.0, 39.0, 37.2; HRMS (ESI) C56H73O14N9 m/z: [M+H]+ 1096.5346 (calc. 1096.5350).
N-Fmoc amine 209 (161 mg, 0.15 mmol) was dissolved in DMF (2 mL) and piperidine (116 μL, 1.18 mmol) was added. The mixture was stirred for 1 hour. The solvent was removed under a stream of nitrogen to leave a white solid. The residue was suspended in DMF (1.5 mL) and acid 4 (133 mg, 0.37 mmol), HBTU (139 mg, 0.37 mmol) and DIPEA (128 μL, 0.73 mmol) were added. The reaction mixture was stirred for 1.5 hours. The crude mixture was submitted to reverse-phase column chromatography (10-100% MeCN/H2O) to yield the product, 210 (118 mg, 0.088 mmol, 59%) as a pale-yellow residue. 1H NMR (500 MHz, CDCl3, 25° C.): δ (ppm)=7.49 (br s, 2H), 7.22 (br s, 1H), 5.60 (s, 2H), 4.09-3.93 (m, 4H), 3.70-3.59 (m, 25H), 3.57 (t, J=4.8 Hz, 4H), 3.50-3.41 (m, 7H), 3.38 (t, J=5.0 Hz, 6H), 3.27-3.03 (m, 10H), 2.82-2.48 (m, 8H), 2.16 (br s, 2H), 1.44 (s, 36H); 13C NMR (125 MHz, CDCl3): δ (ppm)=171.0, 163.2, 156.6, 152.0, 82.9, 79.7, 71.0, 70.8, 70.7, 70.6, 70.3, 70.1, 70.1, 69.6, 69.5, 63.1, 55.9, 55.6, 55.1, 54.8, 53.7, 50.8, 47.2, 41.0, 39.2, 28.6; HRMS (ESI) C58H111N15O20 m/z: [M+2H]2+ 669.9153 (calc. 669.9138).
Tetra-N-Boc amine 210 (25 mg, 0.019 mmol) was dissolved in HCl/dioxane (4 M, 1 mL) and stirred for 1 hour. The solvent was removed under a stream of nitrogen. Compound 2 (26.1 mg, 0.112 mmol), HBTU (42.5 mg, 0.112 mmol) were added, and the combined solids suspended in DMF (0.75 mL). DIPEA (59 μL, 0.336 mmol) was added and the reaction mixture stirred for 1 hour. The mixture was submitted to reverse-phase column chromatography (10-100% MeCN in 0.1M NH4OH(aq)) to yield the product, 211 (9.71 mg, 0.0054 mmol, 28%) as a brown solid. 1H NMR (500 MHz, DMSO-d6, 25° C.): δ (ppm)=7.80-7.72 (m, 7H), 7.68 (t, J=5.7 Hz, 2H), 7.04 (t, J=5.6 Hz, 4H), 6.76 (s, 4H), 6.58 (dd, J=10.6, 17.3 Hz, 8H), 6.35 (d, J=16.6 Hz, 8H), 5.58 (dd, J=1.4, 10.3 Hz, 8H), 3.85 (s, 4H), 3.61-3.57 (m, 2H), 3.56-3.45 (m, 16H), 3.44-3.36 (m, 9H), 3.29 (q, J=6.6 Hz, 9H), 3.24 (q, J=5.9 Hz, 6H), 3.17 (q, J=6.3 Hz, 4H), 3.11 (q, J=6.2 Hz, 8H), 3.08-3.03 (m, 6H), 2.56 (t, J=6.8 Hz, 4H), 2.14 (t, J=7.7 Hz, 8H), 1.76 (m, J=7.2 Hz, 9H), 0.95 (d, J=6.5 Hz, 4H); 13C NMR (125 MHz, DMSO-d6): δ (ppm)=172.1, 170.6, 170.5, 169.2, 163.1, 162.4, 136.1, 121.4, 104.5, 70.1, 70.0, 69.8, 69.7, 69.7, 69.5, 69.3, 69.2, 69.0, 70.0, 58.3, 58.0, 54.2, 54.1, 50.0, 40.3, 40.1, 39.9, 39.8, 39.6, 38.1, 36.8, 36.4, 33.1; HRMS (ESI) C86H132O16N27 m/z: [M+H]+ 1799.0355 (calc. 1799.0340).
Tetra-N-Boc amine 210 (102 mg, 0.076 mmol) was dissolved in HCl solution (4 M in dioxanes, 2 mL) and stirred for 1 hour. The solvent was removed under a stream of nitrogen, and the resulting white solid redissolved in DMF (2 mL). Fmoc-8-amino-3,6-dioxaoctonoic acid (176 mg, 0.46 mmol), HBTU (173 mg, 0.46 mmol) and DIPEA (239 μL, 1.37 mmol) were added and the reaction mixture stirred for 19.5 hours. The mixture was submitted to reverse-phase column chromatography (10-100% MeCN in H2O) to yield the product, 212 (50.6 mg, 0.021 mmol, 28%) as a white solid. 1H NMR (500 MHz, DMSO-d6, 25° C.): δ (ppm)=7.87 (d, J=7.5 Hz, 8H), 7.77-7.71 (m, 3H), 7.68 (m incl. d, J=7.5 Hz, 13H), 7.40 (t, J=7.4 Hz, 8H), 7.31 (m incl. t, J=7.4 Hz, 11H), 4.28 (d, J=6.9 Hz, 8H), 4.20 (t, J=6.8 Hz, 4H), 3.87-3.84 (m, 11H), 3.58-3.47 (m, 32H), 3.44-3.39 (m incl. t, J=5.2 Hz, 13H), 3.36 (t, J=4.9 Hz, 2H), 3.24 (q, J=5.6 Hz, 6H), 3.15 (m, J=6.4 Hz, 19H), 3.07 (s, 6H), 2.56 (t, J=6.7 Hz, 11H); 13C NMR (100 MHz, DMSO-d6): δ (ppm)=170.6, 169.8, 169.3, 169.2, 142.6, 139.4, 137.4, 128.9, 127.3, 121.4, 120.0, 109.8, 70.2, 70.2, 70.0, 69.8, 69.7, 69.7, 69.5, 69.3, 69.0, 58.0, 54.1, 50.0, 40.3, 40.1, 39.8, 38.1, 36.4; HRMS (ESI) C122H164O32N19 m/z: [M+H]+ 2407.1743 (calc. 2407.1784).
Compound 212 (24 mg, 0.01 mmol) was dissolved in DMF (1 mL) and piperidine (9.9 μL, 0.10 mmol) was added. The mixture was stirred for 30 minutes, and the solvent was removed under a stream of nitrogen. The crude residue was redissolved in DMF (0.75 mL). Acid 2 (14 mg, 0.06 mmol), HBTU (23 mg, 0.06 mmol) and DIPEA (31 μL, 0.18 mmol) were added and the reaction mixture was stirred for 2 hours. The crude mixture was submitted to reverse-phase flash column chromatography (0-100% MeCN in 0.1M NH4OH(aq)) to yield the product, 213 (8.6 mg, 0.0036 mmol, 36%) as a pale brown solid. 1H NMR (500 MHz, DMSO-d6, 25° C.): δ (ppm)=7.86 (t, J=5.5 Hz, 4H), 7.78-7.72 (m, 3H), 7.70-7.63 (m, 6H), 7.04 (t, J=5.7 Hz, 4H), 6.76 (s, 4H), 6.57 (dd, J=10.7, 17.2 Hz, 8H), 6.35 (d, J=16.8 Hz, 8H), 5.57 (dd, J=1.4, 10.7 Hz, 8H), 3.86 (s, 12H), 3.60-3.47 (m, 36H), 3.45-3.36 (m, 17H), 3.30-3.22 (m, 18H), 3.22-3.13 (m, 21H), 3.07 (s, 6H), 2.56 (t, J=6.4 Hz, 10H), 2.13 (t, J=7.5 Hz, 8H), 1.74 (app. qu, J=7.2 Hz, 9H); 13C NMR (125 MHz, DMSO-d6): δ (ppm)=172.1, 170.6, 170.5, 169.2, 169.2, 163.2, 162.4, 136.1, 121.4, 104.5, 70.2, 70.0, 69.8, 69.7, 69.7, 69.5, 69.3, 69.3, 69.2, 69.1, 69.0, 58.0, 54.1, 50.0, 40.3, 40.1, 39.9, 39.8, 39.6, 38.4, 38.1, 38.1, 36.4, 33.0, 28.3, 25.3; HRMS (ESI) C110H175O28N31 m/z: [M+H]+ 2379.3252 (calc. 2379.3296).
To a mixture of compound 214 (10 g, 45.61 mmol) and compound 215 (4.9 g, 38.23 mmol) in DCM (100 mL) was added Bu4NBr (2.4 g, 7.44 mmol) and NaOH (9.85 g, 246.31 mmol). The reaction mixture was stirred at 15° C. for 3 hours. The reaction mixture was poured into water (200 mL), extracted with DCM (200 mL*2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, concentrated in vacuum to give the residue, which was purified by column (SiO2, 30% to 40% of EtOAc in Petroleum) to afford compound 216 (5.2 g, 32.8% yield) as yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 3.73-3.69 (m, 2H), 3.68-3.64 (m, 10H), 3.64-3.59 (m, 4H), 2.04 (s, 2H), 1.44 (s, 9H).
A mixture of compound 216 (5.2 g, 14.97 mmol) in HCl/dioxane (4 M, 50 mL) was stirred at 20° C. for 2 hours. LCMS showed starting material was consumed and desired mass was detected as major product. The reaction mixture was concentrated in vacuum to afford compound 217 (4.3 g, 98.6% yield) as yellow oil. LCMS: rt=0.502 min, (292.2 [M+H]+), 74.6% purity.
To a mixture of compound 217 (4.3 g, 14.76 mmol) and compound 218 (2.8 g, 16.24 mmol) in DCM (60 mL) was added HATU (5.6 g, 14.76 mmol) and DPIEA (9.5 g, 73.81 mmol). The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to give the residue, which was purified by reversed phase Combiflash (water (0.1% FA)-ACN; B %: 30%-50%). The mixture was adjusted to pH=8 by aq. NaHCO3 solution and concentrated in vacuum to remove CH3CN. Then the mixture was extracted with EtOAc (200 mL*2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, concentrated in vacuum to afford compound 219 (5 g, 75.7% yield) as light-yellow oil. LCMS: rt=0.740 min, (448.3 [M+H]+), 100.00% purity. 1H NMR (400 MHz, METHANOL-d4) δ 3.72 (t, J=6.0 Hz, 2H), 3.69-3.61 (m, 14H), 3.39-3.36 (m, 2H), 3.21 (t, J=6.8 Hz, 2H), 3.07 (t, J=6.8 Hz, 2H), 2.43 (t, J=6.0 Hz, 2H), 1.66-1.58 (m, 2H), 1.44 (s, 9H).
To a mixture of compound 219 (5 g, 11.17 mmol) in dioxane (30 mL) was added HCl/dioxane (4 M, 30 mL). The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 220 (4.6 g, 97.9% yield) as light-yellow oil. LCMS: rt=0.610 min, (348.2 [M+H]+), 100.00% purity.
To a mixture of compound 220 (2.3 g, 5.99 mmol) and compound 203 (2.17 g, 5.99 mmol) in DCM (50 mL) was added DIPEA (2.3 g, 17.97 mmol) and HATU (2.28 g, 5.99 mmol). The reaction mixture was stirred at 20° C. for 8 hours. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuum to give the residue, which was purified by reversed phase Combiflash (water (0.1% HCl)-ACN; B % 10%-54%). The mixture was adjusted to pH=8 by aq. NaHCO3 solution and concentrated in vacuum to remove CH3CN. Then the mixture was extracted with EtOAc (100 mL*2). The combined organic layers were washed with brine, dried over Na2SO4, concentrated in vacuum to afford compound 221 (3.8 g, 88.5% yield) as yellow oil. m/z 691.4 (M+H)+(ES+). LCMS: rt=0.530 min, (691.4 [M+H]+), 96.41% purity. 1H NMR (400 MHz, METHANOL-d4) δ3.76 (t, J=6.0 Hz, 2H), 3.69-3.61 (m, 14H), 3.37 (t, J=5.2 Hz, 2H), 3.29-3.24 (m, 4H), 3.19-3.11 (m, 6H), 2.60 (t, J=6.0 Hz, 2H), 2.48 (t, J=6.0 Hz, 2H), 1.68 (q, J=6.4 Hz, 2H), 1.44 (s, 18H).
To a mixture of compound 221 (3.8 g, 5.50 mmol) in dioxane (20 mL) was added HCl/dioxane (4 M, 20 mL). The reaction mixture was stirred at 20° C. for 16 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 222 (3.2 g, 96.9% yield) as light-yellow oil. LCMS: rt=0.404 min, (491.2 [M+H]+), 82.75% purity.
To a mixture of compound 222 (1.65 g, 2.75 mmol) and compound 203 (2.09 g, 5.78 mmol) in DCM (20 mL) was added DIPEA (2.13 g, 16.50 mmol) and HATU (2.20 g, 5.78 mmol). The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuum to give the residue, which was purified by reversed phase Combiflash (water (0.1% FA)-ACN; B % 50%-70%) and then concentrated in vacuum to afford compound 223 (2.7 g, 79.8% yield) as a light-yellow solid. LCMS: rt=0.739 min, (1178.0 [M+H]+), 95.74% purity. 1H NMR (400 MHz, METHANOL-d4) δ3.73 (t, J=6.0 Hz, 2H), 3.70-3.67 (m, 2H), 3.66-3.65 (m, 4H), 3.64-3.63 (m, 4H), 3.62-3.58 (m, 4H), 3.39-3.33 (m, 6H), 3.29-3.22 (m, 6H), 3.17 (s, 4H), 3.13 (t, J=6.0 Hz, 8H), 2.74 (t, J=6.4 Hz, 4H), 2.60 (t, J=6.0 Hz, 8H), 2.46 (t, J=6.0 Hz, 2H), 1.75-1.67 (m, 2H), 1.44 (s, 36H).
To a mixture of compound 223 (600 mg, 0.51 mmol) in dioxane (4 mL) was added HCl/dioxane (4 M, 2 mL). The reaction mixture was stirred at 20° C. for 8 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 224 (525 mg, 99.8% yield) as a light-yellow solid. LCMS: rt=0.390 min, (777.3 [M+H]+), 93.39% purity.
To a mixture of compound 2 (593.2 mg, 2.54 mmol) and compound 224 (525 mg, 0.51 mmol) in DCM (8 mL) was added DIPEA (1.31 g, 10.17 mmol). Then, HATU (773.58 mg, 2.03 mmol) was slowly added into the above solution. The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuum to give the residue, which was purified by prep-HPLC (column: Waters Xbridge 150 mm*25 mm*5 um; mobile phase: [water (ammonia hydroxide v/v)-ACN]; B %: 37%-67%, 9 min) and lyophilized to afford 225, Scaffold E (236 mg, 25.7% yield) as yellow oil. Then the product was dissolved in DMSO (13 mL) immediately. LCMS: rt=1.022 min, (1638.9 [M+H]+), 93.22% purity.
To a mixture of compound 202 (10 g, 22.15 mmol) in dioxane (50 mL) was added HCl/dioxane (4 M, 50 mL). The reaction mixture was stirred at 20° C. for 8 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 226 (7.9 g, 98.9% yield) as a white solid. LCMS: rt=0.435 min, (252.3 [M+H]+), 100.00% purity.
To a mixture of compound 226 (5 g, 12.59 mmol) and compound 227 (6.96 g, 26.44 mmol) in DCM (100 mL) was added DIPEA (11.39 g, 88.12 mmol) and HATU (10.05 g, 26.44 mmol). The reaction mixture was stirred at 20° C. for 2 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to give a residue, which was purified by reversed phase combiflash (water (0.1% FA)-ACN; B % 70%-90%). The mixture was adjusted to pH=8 by aq. NaHCO3 solution and concentrated in vacuum to remove CH3CN. Then the mixture was extracted with EtOAc (100 mL*2). The combined organic layer was washed with brine (100 mL), dried over sodium sulfate, concentrated in vacuum to afford compound 228 (6.1 g, 57.4% yield) as yellow oil. LCMS: rt=0.555 min, (742.6 [M+H]+), 87.94% purity. 1H NMR (400 MHz, METHANOL-d4) δ 7.39-7.31 (m, 5H), 5.16 (s, 2H), 3.99 (s, 4H), 3.63-3.69 (m, 8H), 3.55 (s, 2H) 3.51 (t, J=5.6 Hz, 4H), 3.34-3.31 (m, 4H), 3.22 (t, J=5.6 Hz, 4H), 2.79 (t, J=6.0 Hz, 4H), 1.43 (s, 18H).
To a mixture of compound 228 (6.1 g, 8.22 mmol) in MeOH (200 mL) was added Pd/C (800 mg, 10% purity) under N2. The reaction mixture was stirred at 20° C. for 3 hours under H2 (15 psi). LCMS showed the reaction was completed. The catalyst was filtered. The filtrate was concentrated in vacuum to afford compound 229 (5.3 g, 98.9% yield) as colourless oil. LCMS: rt=0.823 min, (652.5 [M+H]+), 100.00% purity. 1H NMR (400 MHz, METHANOL-d4) δ4.04 (s, 4H), 3.74-3.68 (m, 4H), 3.68-3.62 (m, 6H), 3.55 (t, J=6.0 Hz, 8H), 3.26-3.17 (m, 8H), 1.44 (s, 18H).
To a mixture of compound 229 (700 mg, 1.17 mmol) and compound 221 (1.52 g, 2.33 mmol) in DCM (20 mL) was added DIPEA (904.73 mg, 7.00 mmol) and HATU (887.23 mg, 2.33 mmol). The reaction mixture was stirred at 20° C. for 2 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to give a residue, which was purified by reversed phase Combiflash (water (0.1% FA)-ACN; B % 50%-70%) and lyophilized to afford compound 230 (800 mg, 39.0% yield) as yellow oil. 1H NMR (400 MHz, METHANOL-d4) δ 4.02 (s, 8H), 3.73 (t, J=6.0 Hz, 2H), 3.71-3.68 (m, 10H), 3.67-3.66 (m, 10H), 3.65-3.61 (m, 10H), 3.53 (t, J=6.0 Hz, 8H), 3.43-3.40 (m, 2H), 3.35 (t, J=6.0 Hz, 12H), 3.27-3.22 (m, 18H), 2.75-2.67 (m, 12H), 2.47 (t, J=6.0 Hz, 2H), 1.74-1.67 (m, 2H), 1.44 (s, 36H).
To a mixture of compound 230 (800 mg, 0.46 mmol) in dioxane (5 mL) was added HCl/dioxane (4 M, 5 mL). The reaction mixture was stirred at 20° C. for 4 hours. LCMS starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 231 (700 mg, 95.4% yield) as yellow oil. LCMS: rt=0.471 min, (1357.7 [M+H]+), 59.49% purity.
To a mixture of compound 2 (343.6 mg, 1.47 mmol) and compound 231 (400 mg, 294.64 umol) in DCM (20 mL) was added DIPEA (571.2 mg, 4.42 mmol). Then HATU (448.1 mg, 1.18 mmol) was slowly added into the above solution. The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to give a residue, which was purified by prep-HPLC (column: Waters Xbridge 150 mm*25 mm*5 um; mobile phase: [water (ammonia hydroxide v/v)-ACN]; B %: 30%-60%, 9 min) and lyophilized to afford 232, Scaffold F (155 mg, 22.7% yield) as yellow oil. Then the product was dissolved in DMSO (7 mL) immediately. LCMS: rt=0.961 min, (1110.3 [[M+H]/2]+), 97.65% purity.
To a mixture of compound 229 (3 g, 4.60 mmol) and compound 219 (1.93 g, 4.60 mmol) in DCM (30 mL) was added DIPEA (2.97 g, 23.02 mmol) and HATU (1.75 g, 4.60 mmol). The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to give a residue, which was purified by reversed phase Combiflash (water (0.1% FA)-ACN; B % 40%-60%). The mixture was adjusted to pH=8 by aq. NaHCO3 solution and concentrated in vacuum to remove CH3CN. Then the mixture was extracted with EtOAc (200 mL*2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, concentrated in vacuum to afford compound 233 (2.0 g, 44.3% yield) as yellow oil. LCMS: rt=0.533 min, (981.4 [M+H]+), 94.19% purity. 1H NMR (400 MHz, METHANOL-d4) δ 4.00 (s, 4H), 3.74 (t, J=6.0 Hz, 2H), 3.69-3.61 (m, 22H), 3.53 (t, J=6.0 Hz, 4H), 3.40-3.34 (m, 6H), 3.28-3.22 (m, 10H), 2.71 (t, J=6.8 Hz, 4H), 2.46 (t, J=6.0 Hz, 2H), 1.70 (q, J=6.4 Hz, 2H) 1.44 (s, 18H).
To a mixture of compound 233 (2 g, 2.04 mmol) in dioxane (10 mL) was added HCl/dioxane (4 M, 9.1 mL). The reaction mixture was stirred at 20° C. for 4 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 234 (1.81 g, 100% yield) as yellow oil. LCMS: rt=0.354 min, (781.5 [M+H]+), 55.4% purity.
To a mixture of compound 203 (487.1 mg, 1.35 mmol) in DCM (5 mL) was added HATU (512.5 mg, 1.35 mmol) and DIPEA (348.4 mg, 2.70 mmol). The reaction mixture was stirred at 20° C. for 0.5 hour. Then, a solution of compound 234 (600 mg, 0.67 mmol) and DIPEA (522.6 mg, 4.04 mmol) in DCM (5 mL) was added dropwise into the above solution. The reaction mixture was stirred at 20° C. for another 0.5 hour. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuum to give a residue, which was purified by reversed phase Combiflash (water (0.1% FA)-ACN; B % 40%-60%). The mixture was adjusted to pH=8 by aq. NaHCO3 solution and concentrated in vacuum to remove CH3CN. Then the mixture was extracted with EtOAc (100 mL*2). The combined organic layer was washed with brine (100 mL), dried over sodium sulfate, concentrated in vacuum to afford compound 235 (360 mg, 34.5% yield) as yellow oil. LCMS: rt=0.739 min, (1468.1 [M+H]+), 100.00% purity. 1H NMR (400 MHz, METHANOL-d4) δ 4.01 (s, 4H), 3.76-3.70 (m, 4H), 3.70-3.59 (m, 28H), 3.45 (t, J=5.6 Hz, 4H), 3.40-3.34 (m, 6H), 3.29-3.22 (m, 6H), 3.18-3.10 (m, 14H), 2.71 (t, J=6.6 Hz, 4H), 2.60 (br t, J=6.0 Hz, 8H), 2.46 (t, J=6.0 Hz, 2H), 1.74-1.66 (m, 2H), 1.45 (s, 36H).
To a mixture of compound 235 (250 mg, 0.17 mmol) in dioxane (4 mL) was added HCl/dioxane (4 M, 1 mL). The reaction mixture was stirred at 20° C. for 2 hours. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to afford compound 236 (225 mg, 99% yield) as yellow oil. LCMS: rt=0.354 min, (1167.8 [M+H]+), 75.55% purity.
To a mixture of compound 236 (225 mg, 0.17 mmol) in DCM (10 mL) was added DIPEA (439.7 mg, 3.40 mmol) and compound 2 (198.4 mg, 0.85 mmol). Then HATU (323.5 mg, 0.85 mmol) was slowly added into the above solution in potions. The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed starting material was consumed and desired mass was detected. The reaction mixture was concentrated in vacuum to give a residue, which was purified by prep-HPLC (column: Waters Xbridge 150 mm*25 mm*5 μm; mobile phase: [water (ammonia hydroxide v/v)-ACN]; B %: 36%-66%, 9 min) and lyophilized to afford 237, Scaffold G (278 mg, 78.2% yield) as yellow oil. Then the product was dissolved in DMSO (13 mL) immediately. LCMS: rt=0.958 min, (1929.4 [M+H]+), 95.17% purity.
To a mixture of compound 229 (878.5 mg, 1.35 mmol) in DCM (10 mL) was added DIEA (174.2 mg, 1.35 mmol) and HATU (512.5 mg, 1.35 mmol). The reaction mixture was stirred at 20° C. for 0.5 hour. Then a solution of compound 234 (600 mg, 0.67 mmol) and DIEA (522.61 mg, 4.04 mmol) in DCM (5 mL) was added into the above solution. The reaction mixture was stirred at 20° C. for another 0.5 hour. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuum to give a residue, which was purified by reversed phase Combiflash (water (0.1% FA)-ACN; B % 60%-80%). The mixture was adjusted to pH=8 by aq. NaHCO3 solution and concentrated in vacuum to remove CH3CN. Then the mixture was extracted with EtOAc (100 mL*2). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, concentrated in vacuum to afford compound 238 (360 mg, 26.1% yield) as yellow oil. LCMS: rt=0.740 min, (1025.2 [[M+H]/2]+), 100.00% purity. 1H NM R (400 MHz, METHANOL-d4) δ 4.02 (s, 12H), 3.76-3.71 (m, 4H), 3.70-3.64 (m, 32H), 3.63-3.58 (m, 8H), 3.53 (t. J=5.6 Hz, 8H), 3.47-3.42 (m, 4H), 3.41-3.34 (m, 14H), 3.28-3.22 (m, 18H), 2.71 (t, J=6.4 Hz, 12H), 2.47 (t, J=6.0 Hz, 2H), 1.74-1.67 (m, J=6.4 Hz, 2H), 1.44 (s, 36H).
To a mixture of compound 238 (300 mg, 0.15 mmol) in dioxane (5 mL) was added HCl/dioxane (4 M, 5 mL). The reaction mixture was stirred at 20° C. for 4 hours. The reaction mixture was concentrated in vacuum to afford compound 239 (270 mg, 96.9% yield) as yellow oil. LCMS: rt=0.414 min, (1647.9 [M+H]+), 44.49% purity.
To a mixture of compound 239 (270 mg, 0.14 mmol) and compound 2 (165.5 mg, 0.71 mmol) in DCM (5 mL) was added DIEA (366.7 mg, 2.84 mmol). Then HATU (215.8 mg, 0.57 mmol) was added into the above solution. The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuum to give a residue, which was purified by prep-HPLC (column: Waters Xbridge 150 mm*25 mm*5 um; mobile phase: [water (ammonia hydroxide v/v)-ACN]; B %: 34%-64%, 9 min) and lyophilized to afford to afford 240, Scaffold H (131 mg, 34.4% yield) as yellow oil. Then the product was dissolved in DMSO (4.7 mL) immediately. LCMS: rt=0.946 min, (1255.3 [[M+H]/2]+), 100.00% purity.
L-citrulline (85 mg, 0.486 mmol) in DME (1.5 mL) was added to a solution of Fmoc-Val-OSu (202 mg, 0.463 mmol) and NaHCO3 (42.8 mg, 0.509 mmol) in H2O (3 mL) and THF (4 mL) at 0° C. The reaction warmed to rt and stirred for 48 h. Upon completion, the reaction was adjusted to pH 10 with sat. aq. K2CO3 and washed with EtOAc (2×20 mL). The aqueous layer was acidified to pH 4 with 10% aq. citric acid and the formed gelatinous mixture was filtered and dried in vacuo to yield Fmoc-Val-Cit-OH 241 (141 mg, 0.284 mmol, 61%) as an off-white solid. 1H NMR (700 MHz, DMSO-d6) δ ppm: 7.89 (d, 2H, J=7.6 Hz), 7.75 (dd, 2H, J=11.7, 7.8 Hz), 7.41 (t, 2H, J=7.4 Hz), 7.34-7.30 (m, 2H), 6.00 (s, 1H), 4.31-4.26 (m, 1H), 4.27-4.19 (m, 2H), 4.17-4.13 (m, 1H), 3.94-3.87 (m, 1H), 2.99-2.92 (m, 2H), 1.98 (app sx, 1H, J=6.8 Hz), 1.74-1.66 (m, 1H), 1.61-1.53 (m, 1H), 1.46-1.35 (m, 2H), 0.89 (d, 3H, J=6.8 Hz), 0.86 (d, 3H, J=6.8 Hz). 13C NMR (176 MHz, DMSO-d6) δ ppm: 173.4, 171.3, 158.8, 156.1, 143.9, 143.9, 140.7, 127.7, 127.1, 125.4, 120.1, 65.7, 59.8, 51.9, 46.7, 38.8, 30.6, 28.4, 26.6, 19.2, 18.2. HRMS (ESI) m/z found [M+H]+ 497.2394, C26H33N4O6 required 497.2395.
A solution of Fmoc-Val-Cit-OH 241 (104 mg, 0.210 mmol), 4-aminobenzyl alcohol (PABA, 52.0 mg, 0.419 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 104 mg, 0.419 mmol) in DCM (2.5 mL) and MeOH (1.5 mL) was stirred at rt for 22 h. Upon completion, the mixture was diluted with Et2O (10 mL), filtered and washed with Et2O (2×4 mL) to yield Fmoc-Val-Cit-PABA 242 (66.4 mg, 0.110 mmol, 53%) as a white solid. 1H NMR (700 MHz, DMSO-d6) δ ppm: 9.98 (s, 1H), 8.12 (d, 1H, J=7.5 Hz), 7.89 (d, 2H, J=7.6 Hz), 7.74 (t, 2H, J=8.1 Hz), 7.54 (d, 2H, J=8.4 Hz), 7.46-7.39 (m, 3H), 7.34-7.30 (m, 2H), 7.23 (d, 2H, J=8.4 Hz), 6.00 (t, 1H, J=5.8 Hz), 5.41 (s, 2H), 5.09 (t, 1H, J=5.5 Hz), 4.43-4.39 (m, 3H), 4.33-4.20 (m, 3H), 3.95-3.91 (m, 1H), 3.06-2.89 (m, 2H), 2.04-1.95 (m, 1H), 1.74-1.55 (m, 2H), 1.50-1.32 (m, 2H), 0.89-0.84 (m, 6H); 13C NMR (176 MHz, DMSO-d6) δ ppm: 171.3, 170.4, 158.9, 156.1, 143.9, 140.7, 137.5, 137.4, 127.7, 127.1, 126.9, 125.4, 120.1, 118.9, 65.7, 62.6, 60.1, 53.1, 46.7, 38.6, 30.5, 29.5, 26.8, 19.2, 18.3; HRMS (ESI) m/z found [M+H]+ 602.2968, C33H40N5O6+ required 602.2973.
A solution of Fmoc-Val-Cit-PABA 242 (100 mg, 0.166 mmol) and diethylamine (343 μL, 3.33 mmol) in DMF (1.5 mL) was stirred at rt for 19 h. Upon completion, the solvent was evaporated under reduced pressure, the product was crashed out with DCM, filtered, and washed with EtOAc and Et2O to give H-Val-Cit-PABA (52.3 mg, 0.138 mmol, 83%) as a white solid, which was carried through without further purification. A solution of H-Val-Cit-PABA (52.3 mg, 0.138 mmol), Boc-PEG4-COOH (50.0 mg, 0.138 mmol), HBTU (52.5 mg, 0.138 μmol) and DIPEA (48.0 μL, 0.276 μmol) in DMF (3 mL) was stirred at rt for 2 h. Upon completion, the reaction was concentrated under a stream of N2 and the crude residue purified by reverse-phase flash column chromatography (5-100% solvent B in solvent A; Solvent A: 0.1 M NH4OH (aq), Solvent B: MeCN) and lyophilised to yield Boc-PEG4-Val-Cit-PABA 243 (50.5 mg, 69.4 μmol, 51%) as a white solid. 1H NMR (700 MHz, DMSO-d6) δ ppm: 9.87 (s, 1H), 8.07 (d, 1H, J=7.6 Hz), 7.86 (d, 1H, J=8.7 Hz), 7.53 (d, 2H, J=8.5 Hz), 7.21 (d, 2H, J=8.6 Hz), 6.73 (t, 1H, J=5.0 Hz), 6.00 (t, 1H, J=5.8 Hz), 5.39 (s, 2H), 5.09 (t, 1H, J=5.7 Hz), 4.41 (d, 2H, J=5.5 Hz), 4.39-4.31 (m, 1H), 4.23-4.17 (m, 1H), 3.63-3.53 (m, 2H), 3.50-3.43 (m, 12H), 3.07-2.96 (m, 3H), 2.96-2.87 (m, 1H), 2.47-2.42 (m, 1H), 2.40-2.31 (m, 1H), 1.95 (h, 1H, J=6.8 Hz), 1.76-1.63 (m, 1H), 1.62-1.52 (m, 1H), 1.45-1.38 (m, 2H), 1.37-1.35 (s, 9H), 0.82 (dd, 6H, J=6.8 Hz); 13C NMR (176 MHz, DMSO-d6) δ ppm: 171.6, 170.8, 159.3, 156.0, 137.9, 127.4, 119.3, 78.1, 70.2, 70.2, 70.1, 69.9, 69.9 69.6, 67.4, 63.0, 58.0, 53.5, 39.0, 36.4, 31.0, 29.8, 28.7, 27.3, 19.6, 18.5; LRMS (ESI) m/z found [M+H]+ 726.8, C34H59N59O11+ required 727.4273.
A solution of Boc-PEG4-Val-Cit-PABA 243 (60.0 mg, 82.5 μmol), bis(4-nitrophenyl) carbonate (50.2 mg, 0.165 mmol) and DIPEA (43.0 μL, 0.248 mmol) in DMF (2 mL) was stirred at rt for 24 h. Upon completion, the mixture was diluted with Na2CO3 (10 mL) and extracted with DCM (10 mL). The aqueous layer was washed with DCM (4×20 mL), the organic layers combined and washed with Na2CO3 (2×10 mL). The combined organic fractions were dried (MgSO4), concentrated in vacuo to yield Boc-PEG4-Val-Cit-PABC-PNP as a brown oil, which was carried through without further purification. A solution of MMAE (25.0 mg, 34.8 μmol), Boc-PEG4-Val-Cit-PABC-PNP (25.9 mg, 29.0 μmol), HOBt·H2O (4.70 mg, 34.8 μmol) and DIPEA (12.1 μL, 69.6 μmol) in DMF (2 mL) was stirred at rt for 21 h. Upon completion, the reaction mixture was concentrated under a stream of N2. The crude product was purified by reverse-phase flash column chromatography (5-100% solvent B in solvent A; Solvent A: 0.1 M NH4OH (aq), Solvent B: MeCN) and lyophilised to yield Boc-PEG4-Val-Cit-PABC-MMAE 244 (45.6 mg, 31.0 μmol, 89%) as a light-yellow solid. HPLC (5-95% MeCN/H2O over 20 min) retention time 10.370 min; HRMS (ESI) m/z found [M+H]+ 1470.9042, C74H24N11O19+ required 1470.9075.
A solution of Boc-PEG4-Val-Cit-PABC-M MAE 244 (40.0 mg, 27.2 μmol), TIPS (100 μL), TFA (500 μL) and DCM (950 μL) was stirred at rt for 20 min. Upon completion, the reaction mixture was concentrated under a stream of N2 and carried forward without further purification. A solution of H-PEG4-Val-Cit-PABC-M MAE (37.3 mg, 27.2 μmol), Fmoc-PEG2-Glu-PEG2-Glu-NH2 (8.57 mg, 10.9 μmol), HATU (18.7 mg, 49.1 μmol) and DIPEA (12.3 μL, 70.9 μmol) in DMF (2 mL) was stirred at rt for 48 h. Upon completion, the reaction was concentrated under a stream of N2, and the crude product purified by preparative HPLC and lyophilised to yield Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-M MAE)-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE) 245 (1.10 mg, 0.315 μmol, 3%) as a white solid. HPLC (5-95% MeCN/H2O over 20 min) retention time 12.721 min; LRMS (ESI) m/z found [M+2H]+ 1165.3, [M+3H]+ 1747.7, [M+4H]+ 873.9, C175H276N27O46+ required 3492.0073.
A solution of Boc-PEG4-Val-Cit-PABC-MMAE 244 (38.0 mg, 25.8 μmol), TIPS (100 μL), TFA (500 μL) and DCM (950 μL) was stirred at rt for 20 min. Upon completion, the reaction mixture was concentrated under a stream of N2, dissolved in H2O (5 mL), lyophilised, and carried forward without further purification. A solution of Fmoc-PEG2-Glu-PEG2-PEG2-Glu-NH2 (10.9 mg, 11.7 μmol), BTTFH (7.77 mg, 24.6 μmol) and DIPEA (8.55 μL, 49.2 μmol) in DMF (0.5 mL) was stirred at 0° C. for 1.5 h. To the solution, TFA.HN-PEG4-Val-Cit-PABC-MMAE (33.7 mg, 23.0 μmol) and DIPEA (6.09 μL, 35.0 μmol) in DMF (0.5 mL) were added and the reaction mixture stirred at rt for 24 h. Upon completion, the reaction was concentrated under a stream of N2, and the crude product purified by preparative HPLC and lyophilised to yield Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2 246 (6.39 mg, 1.76 μmol, 15%) as a white solid. HPLC (5-95% MeCN/H2O over 20 min) retention time 12.720 min; HRMS (ESI) m/z found [M+H]+ 3637.140, C181H387N28O49+ required 3637.0773.
Polymer-bound piperazine (30 mg, mmol, 200-400 mesh, 1.0-2.0 mmol/g loading, 2% cross-linked with divinylbenzene) was added to a solution of Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2 246 (3.60 mg, 0.989 μmol) in DMF (200 μL), followed by the addition of 0.1% DBU. The resulting mixture was stirred for 2 h at room temperature. Upon completion, the solvent was evaporated under N2 stream, and the residue taken forward without further purification. A solution of H-PEG2-Glu(-PEG4-Val-Cit-PABC-M MAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-M MAE) (3.38 mg, 0.989 μmol), DBCO-PEG5-COOH (1.60 mg, 2.68 μmol), HATU (1.02 mg, 2.68 μmol) and DIPEA (0.932 μL, 5.36 μmol) in DMF (100 μL) was stirred at rt for 24 h. Upon completion, the reaction was concentrated under a stream of N2, and the crude product purified by preparative HPLC to yield DBCO-PEG5-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2 247 (0.60 mg, 0.150 μmol, 16%) as a white solid. HPLC (5-95% MeCN/H2O over 20 min) retention time 13.208 min; H RMS (ESI) m/z found [M+H]+ 3995.4700, [M+2H]+ 1997.1381, [M+3H]+ 1331.7620 and [M+4H]+ 999.0757, C198H315N30O55+ required 3993.2773.
Manual peptide synthesis: Manual peptide synthesis was carried out on Merck LL MHBA low-loading Rink amide resin (0.308 mmol/g, 100-200 mesh, 1 eq) to afford C-terminal amides. Amino acid coupling was performed using Fmoc-protected amino acids (3 eq), HATU (3 eq) and DIPEA (6 eq) in DMF for 1-3 h. Fmoc deprotection was carried out using 20% piperidine in DMF for 10 min.
Resin cleavage: Side chain deprotection and cleavage from resin were achieved using a TFA cleavage cocktail containing TFA/TIPS/H2O (95:2.5:2.5) at rt for 3 h. Upon filtration and evaporation under a stream of N2, the peptides were precipitated in ice-cold Et2O. The crude peptides were lyophilised, the mass analysed by LCMS and the purity determined by analytical HPLC. The crude peptides were carried through without further purification.
The peptide sequence, mass observed LCMS, peptide purity and yield, together with the retention time determined by analytical HPLC are shown in Table 1 and 2 below.
To a solution of trastuzumab in TBS buffer (1×, pH 8, [Tras]=2.5 mgml−1, 200 μL, 3.4 nmol) was added TCEP (5 mM stock in TBS, 6.8 μL, 34 nmol, 10 equiv.) and the mixture was incubated at 37° C. for 1 hour on a thermal shaker at 400 rpm. DMSO was added to ensure a final organic solvent concentration of 5-10%. Linker/scaffold (1-10 mM stock in DMSO, 2-10 equiv.) was added, and the mixture incubated at 37° C. for 4 hours on a thermal shaker at 400 rpm. The conjugate was purified by Zeba™ Spin Desalting Column (MW cut-off 40,000 Da, Thermo Fisher Scientific) which had been equilibrated into PBS (3×300 μL). Organic solvent was reduced to <0.01% by PBS diafiltration using an Amicon-Ultra centrifugal filter (MW cut-off 10,000 Da, Merck Millipore).
To a solution of trastuzumab-linker conjugate in PBS ([Tras]=1.0 mgml−1, 60 μL, 0.4 nmol) was added a solution of DBCO click reagent in DMSO (10 mM stock concentration, 0.4 μL, 4 nmol, 10 equiv.) and the mixture was incubated at 37° C. for 4-24 hours on a thermal shaker at 400 rpm. The conjugate was purified by Zeba™ Spin Desalting Column (MW cut-off 40,000 Da, Thermo Fisher Scientific) which had been equilibrated into PBS (3×300 μL). Organic solvent was reduced to <0.01% by PBS diafiltration using an Amicon-Ultra centrifugal filter (MW cut-off 10,000 Da, Merck Millipore).
Bioconjugation was carried out using scaffold A (206). SDS-PAGE (12% polyacrylamide gel, run at 200V over 50 minutes, visualised with Coomassie Brilliant Blue stain) indicated that the fully re-bridged antibody was the major product. HRMS (ESI) [M+H]+ 146,687 Da (calc. 146,687 Da).
Bioconjugation was carried out using scaffold B (208). SDS-PAGE (12% polyacrylamide gel, run at 200V over 50 minutes, visualised with Coomassie Brilliant Blue stain) indicated that the fully re-bridged antibody was the major product. HRMS (ESI) [M+H]+ 147,269 Da (calc. 147,267 Da).
Bioconjugation was carried out using scaffold C (211). SDS-PAGE (12% polyacrylamide gel, run at 200V over 50 minutes, visualised with Coomassie Brilliant Blue stain) indicated that the fully re-bridged antibody was the major product. HRMS (ESI) [M+H]+ 146,979 Da (calc. 146,978 Da).
Bioconjugation was carried out using linker scaffold D (213). SDS-PAGE (12% polyacrylamide gel, run at 200V over 50 minutes, visualised with Coomassie Brilliant Blue stain) indicated that the fully re-bridged antibody was the major product. HRMS (ESI) [M+H]+ 147,559 Da (calc. 147,557 Da).
Click reaction was carried out using ALC 1, ALC 2, ALC 3 and ALC 4 respectively. DBCO-PEG5-Val-Cit-PAB-MMAE (248) as the click partner in each reaction, and the mixture was incubated for 24 hours to produce the desired ADC.
A solution of brentuximab (5.7 mg/mL) in PBS was pH adjusted with a 5% v/v addition of 500 mM Tris and 25 mM EDTA (pH8.5). The mixture was reduced by the addition of TCEP (10 eq) for 90 minutes at room temperature. The solution was then further diluted to 2.5 mg/mL with 50 mM Tris (pH 8.0). 5 eq of a 10 mM solution of conjugating reagent (Scaffolds E, F, G and H) in DMSO was added as well as additional solvent to a final concentration of 5% v/v. The reaction was allowed to proceed at room temperature for 24 hours. The mixture was then desalted into PBS (pH 7.4) using NAP 25 desalting columns to remove excess reagents, and re-bridging confirmed by RP-HPLC (PLRP) and HRMS to yield ALC 5, ALC 6, ALC 7 and ALC 8 respectively.
Each re-bridged mAb sample (ALC 5, ALC 6, ALC 7 and ALC 8) was conjugated with commercially available DBCO-VC-MMAE using 10 eq of the DBCO-linker-warhead and incubated at room temperature for 24 hours. Conjugation was confirmed by RP-HPLC (PLRP) and HRMS to yield ADC 5-8 respectively.
Each re-bridged mAb sample (ALC 5, ALC 6, ALC 7 and ALC 8) was conjugated with commercially available DBCO-PBD using 10 eq of the DBCO-linker-warhead and incubated at room temperature for 24 hours. Conjugation was confirmed RP-HPLC (PLRP) and HRMS to yield ADC 9-12 respectively.
A solution of trastuzumab (25.6 mg/mL) in PBS was pH adjusted with a 5% v/v addition of 500 mM Tris and 25 mM EDTA (pH8.5). The mixture was reduced by the addition of TCEP (6 eq) for 90 minutes at room temperature. The solution was then further diluted to 2.5 mg/mL with 50 mM Tris (pH8.0). 5 eq of a 10 mM solution of conjugating reagent (Scaffolds E, F, G and H) in DMSO was added as well as additional solvent to a final concentration of 5% v/v. The reaction was allowed to proceed at room temperature for 24 hours. The mixture was then desalted into PBS (pH7.4) using NAP 25 desalting columns to remove excess reagents, and re-bridging confirmed by RP-HPLC (PLRP) and HRMS to yield ALC 9, ALC 10, ALC 11 and ALC 12 respectively.
Each re-bridged mAb sample (ALC 9, ALC 10, ALC 11 and ALC 12) was conjugated with commercially available DBCO-VC-MMAE using 10 eq of the DBCO-linker-warhead and incubated at room temperature for 24 hours. Conjugation was confirmed by SEC, HIC, PLRP and MS to yield ADC 13-16 respectively.
Each re-bridged mAb sample (ALC 9, ALC 10, ALC 11 and ALC 2) was conjugated with commercially available DBCO-PBD using 10 eq of the DBCO-linker-warhead and incubated at room temperature for 24 hours. Conjugation was confirmed by SEC, HIC, PLRP and MS to yield ADC 17-20 respectively.
DBCO-VC-MMAE was purchased from Syntabio LLC.
DBCO-PBD purchased from Levena biopharma, catalogue code SET0317.
For each of Examples 42 to 61 the following reverse phase-HPLC (PLRP) conditions were used:
For each of Examples 42 to 61 the following HRMS conditions were used:
The measured mass of the major glycoform for each antibody was also used to calculate the theoretical (calculated) molecular weights.
Brentuximab at 5.7 mg/ml was pH adjusted with a 5% v/v addition of 500 mM Tris, 25 mM EDTA pH 8.5 then reduced with 10 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 90 minutes at room temperature. RP-HPLC analysis showed complete reduction achieved. Reduced Brentuximab diluted to 2.5 mg/ml with 50 mM Tris pH 8.0 was then re-bridged by the addition of 5 equivs of 10 mM of scaffold (Scaffold E, F, G and H respectively) in DMSO plus additional solvent to 5% v/v total. The re-bridging reaction was allowed to proceed at room temperature for 24 hours. Th re-bridged antibody samples were then desalted into PBS pH 7.4 using NAP 25 desalting columns to remove solvent and residual scaffold. UV analysis showed all samples to have an approx. [P]=2 mg/ml post desalting. Analysis was performed using SEC, HIC, PLRP (intact) and MS (intact). Each re-bridged antibody sample was split and conjugated with the following click compounds: DBCO-Sulfo-Gly3, DBCO-VC-MMAE and DBCO-PBD, using 10 equivalents of the DBCO-compounds in 10% v/v DMA. Conjugation reactions were incubated at room temperature overnight and SEC, HIC, PLRP (intact) and MS (intact) analysis was repeated.
The results are summarised in Table 1 below.
In the tables below:
Trastuzumab at 25.6 mg/ml was pH adjusted with a 5% v/v addition of 500 mM Tris, 25 mM EDTA pH 8.5 then reduced with 6 equivalents of TCEP for 90 minutes at room temperature. RP-HPLC analysis showed complete reduction achieved. The reduced trastuzumab was then diluted to 2.5 mg/ml with 50 mM Tris pH 8.0 and then re-bridged by the addition of 5 equivs of 10 mM scaffold (Scaffold E, F, G and H respectively) in DMSO plus additional solvent to 5% v/v total. The re-bridging reaction was allowed to proceed at RT for 23 hours. Re-bridged antibody samples were then desalted into PBS pH 7.4 using NAP 25 desalting columns to remove solvent and residual scaffold. UV analysis showed all samples to have a [P]=1.8 mg/mi post desalting. Analysis was performed using SEC, HIC, PLRP (intact) and MS (intact). Each re-bridged antibody sample was split and conjugated with the following click compounds: DBCO-Sulfo-Gly3, DBCO-vcE and DBCO-PBD, using 10 equivalents of DECO-compounds in 10% v/v DMA. Conjugation reactions were incubated at room temperature overnight and SEC, HIC, PLRP (intact) and MS (intact) analysis was repeated.
The results are summarised in Table 2 below:
While specific embodiments of the invention have been described for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims
The following numbered paragraphs describe certain aspects and embodiments of the invention:
(FG)n-L-(Z)8 (Formula I)
(FG)n-L-(Z2)4 (Formula Ia)
(D-L1-FG′)n-L-(Z)8 (Formula II)
(D-L1-FG′)n-L-(Z2)4 (Formula IIa)
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2110726.3 | Jul 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/071003 | 7/26/2022 | WO |
