WO2018178277A1

WO2018178277A1 - New targeted cytotoxic isocombretaquinoline derivatives and conjugates thereof

Info

Publication number: WO2018178277A1
Application number: PCT/EP2018/058168
Authority: WO
Inventors: Zaki SELLAM; Gary BRANDAM; Romain BLANC; Alami MOUAD; Abdallah Hamze; Olivier Provot; Ilhelm KHÉLIFI
Original assignee: Avicenna Oncology Gmbh; Centre National De La Recherche Scientifique (Cnrs); Universite Paris-Sud
Priority date: 2017-03-29
Filing date: 2018-03-29
Publication date: 2018-10-04

Abstract

The present invention is directed to novel natural product-derived combretastatin- based compounds useful as payloads in drug-conjugates constructs with cell target binding moieties (CTBM) and payload-linker compounds useful in connection with drug conjugates. The present invention further relates to new isoNH2CombretaQuinoline compositions including the aforementioned payloads, payload-linkers and drug conjugates, and methods for using these payloads, payload-linkers and drug conjugates, to treat pathological conditions including cancer.

Description

NEW TARGETED CYTOTOXIC ISOCOMBRETAQUINOLINE DERIVATIVES

AND CONJUGATES THEREOF

FIELD OF THE INVENTION:

[0001 ] The present invention is directed to novel natural product-derived combretastatin-based compounds useful as payloads (or toxins) in drug-conjugates constructs with cell target binding moieties (CTBM) and payload-linker compounds useful in connection with drug conjugates. The present invention further relates to new isoNH₂CombretaQuinoline compositions including the aforementioned payloads, payload-linkers and drug conjugates, and methods for using these payloads, payload-linkers and drug conjugates, to treat pathological conditions including cancer, inflammatory and infectious diseases.

BACKGROUND OF THE INVENTION

[0002] For many types of cancer classic chemotherapy is still the only effective form of treatment. Chemotherapy functions on the basis of a cytotoxic effect: a toxin kills cancer cells, thus halting tumor growth. Chemotherapeutic agents primarily damage and destroy cells with a high level of cell-division activity. However, these therapeutics have long struggled with the need to target and destroy malignant cells while minimizing undesired collateral toxicity to normal tissue. Since such drugs also damage healthy cells, patients suffer severe side effects. Numerous highly cytotoxic drugs are of limited clinical utility because they are equally aggressive against both normal and malignant tumoral cells. Healthy tissue can be heavily affected by cytotoxins. Since these drugs do not explicitly discriminate between tumor cells and normal cells, leading to side effects, drugs are often dosed at minimum levels, which may be not effective. This is the reason why it is important to find a way to target specifically cells tumor.

[0003] Improving the delivery of drugs and other agents to target cells, tissues and tumors to achieve maximal efficacy and minimal toxicity has been the focus, of considerable research for many years. Modern cancer therapies now target the cancer more precisely using large molecules such as antibodies. Antibodies are an important, naturally occurring part of the immune system, large molecules that can specifically bind to the cell surface of an 'intruder' (e.g. a virus) and in this way eliminate it. However, often not curative, antibodies need to be combined with chemotherapeutic agents.

[0004] Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient.

[0005] Drug Conjugate constructs seek to overcome these limitations of both nonspecific cytotoxic drugs and specific CTBM (Antibody, small molecule, antibody fragment, antibody mimic, etc.). For instance, ADCs (Antibody Drug Conjugates) are composed of 3 key elements: an antibody (designed to selectively target the tumor of interest), a toxic payload (a cytotoxic compound that will kill the tumor) and the linker (used to conjugate the toxic payload to the antibody). The benefits with such constructs are the significant improvement of the therapeutic window: increased half life and specificity of the toxic payload, reducing off target effects and toxicity. The use of ADCs has been extensively investigated for the last three decades (Moolten et al. (1972), J Natl. Cancer Inst. 49(4):1057-62, Chari et al., (2014) Angew. Chem. Int. Ed., 53:3796-3827; Jackson, (2016) Org. Process Res. Dev. 2016, 20:852-866) Two are already approved and commercialized (Kadcyla from Roche and Adcetris from Seattle Genetics).

[0006] Payloads (or toxins) used in ADCs include bacterial toxins such as diphtheria toxin (Levy et al. (1975) Cancer Res. 35(5):1 182-6), plant toxins such as ricin, small molecule toxins such as maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode et al. (1998) Cancer Res. 58:2925-2928; Upeslacis et al., (1993) Cancer Res. 53, 3336-3342), auristatins (Sanderson et al. (2005) Clin. Cancer Res. 1 1 :843-52), SN-38 or irinotecan analogs (Govidan et al. (2013) Mol. Cancer. Ther. 12:968-78, US 2014/0227180A1 , Goldenberg et al. (2007), Clin. Cancer Res. 13, 5556s-5563s), pyrrolobenzodiazepins (US 201 1 /0256157A1 , Kung Sutherland et al. Blood (2013) 122:1455-1463, Chari et al., (2009) ol. Cancer Ther. 8, B126.) or cryptophycines (WO 201 1/001052A1 , Verma et al. (20 5) Bioorg. Med. Chem. Lett. 25:864-8, US 2012/0225089). Currently, more than 60% of all ADCs currently in clinical evaluation carry toxins related to monomethyl Auristatin E and F (MMAE, MMAF tubulin inhibitor). MMAE could be considered by the skilled person as the standard payload to compare with.

[0007] Conjugation of drugs to CTBM, either directly or via linkers, involves a consideration of a variety of factors, including the identity and location of the chemical group for conjugation of the drug, the mechanism of drug release, the structural elements providing drug release, and the structural modification to the released free drug. In addition, if the drug has to be released after antibody internalization, the mechanism of drug release must be consonant with the intracellular trafficking of the conjugate. Therefore, while a number of different drug classes have been tried as payloads, only a few drug classes have proved efficacious as antibody drug conjugates, because of limited effectiveness, selectivity and/or stability (Tolcher et al. (2000) J Clin. Oncol. 18:4000, Laguzza et al (1989) J. Med. Chem. 32:548-555, Uadia P, (1984). Cancer Res. 44:4263-4266). It turns out that compounds, in order to successfully be eligible as conjugates, should present a level of cytotoxicity below the nanomolar IC₅o level. (Casi and Neri, (2012) J. Control Release. 20;161 (2):422-8.; Wu and Senter (2005) Nat. Biotechnol. 23(9):1 137-46). Ideal payloads should escape from the multi-drug resistance mechanism (MDR). In the case of MMAE, which is subject to MDR, some tumors can develop a mechanism of resistance (Chen et al., 2015). The few payloads accessible are not effective against the large spectrum of cancer indications. Because of these limitations, there is a clinical demand of new payloads with differentiated mode of actions, improved selectivity and toxicity profile and not subject to MDR. For these reasons, combretastatins have been considered as potential toxic payload.

[0008] Indeed, Combretastatin was isolated from the native African tree Combretum caffrum and the like in the 1980s, and was verified to have tubulin polymerization inhibitory activity (Pettit GR (1987). J Nat Prod 50:1 19-131 ). The compound has blood flow inhibitory activity by causing morphological changes of the vascular endothelial cells. Therefore, it can be used as a therapeutic agent for diseases associated with neovascularization, such as cancer and inflammatory indications. Combretastatin A-4 (or CA-4, see figure 1 A) was initially found to compete with combretastatin A-1 , another compound isolated from Combretum caffrum, as an inhibitor of colchicine binding to tubulin. CA-4 and analogs are cytotoxic and selectively disrupt tumoral vasculature or prevent its neoformation (so- called antivascular or anti-angiogenic effect, respectively). They also bind to tubulin and inhibit its polymerization thus impeding the cell proliferation (antimitotic effect). In combination, these effects lead to an inhibition of tumor cell proliferation and of the growth and spread of solid tumors (invasion, metastasis). Several studies have demonstrated that CA-4 has deleterious effects on tumour vasculature, causing rapid vascular shutdown, leading to central tumour necrosis (Dark et al. (1997) Cancer Res 57, 1829- 1834, Grosios K, et al. (1999) Br J Cancer 81 :1318-1327).

[0009] However, this structurally very simple stilbene CA-4 include several drawbacks such as a low water-solubility and a chemical instability of Z-configured double bond, which isomerizes during storage, administration and metabolism. This characteristic has significantly interfered with the development of pharmaceutical compositions comprising CA-4. In order to increase its solubility as well as its efficacy, efforts have been made to create prodrug derivatives of CA-4 to regenerate combretastatin A-4 in physiological conditions (Pettit GR et al. (2000) Anticancer Drug Des 15:203-216, Ohsumi K et al. (1998) J Med Chem 41 :3022-3032). Another drawback of CA-4 is its insufficient cytotoxicity. Treatment with CA-4 alone often led to the persistence of peripheral cancer cells and hence to tumor relapses (Tron et al., (2006) J. Med. Chem., 49:3033-3044; Lippert, (2007) Bioorg. Med. Chem. 15:605-615). The problem of insufficient cytotoxicity of CA-4 in vivo was overcome by developing combretastatin A-1 diphosphate with a direct impact on cancer cells. However, like CA-4 this compound is prone to isomerization and thus deactivation. In addition the 3,4,5-trimethoxyphenyl group, considered as responsible of neurotoxicity or cardiotoxicity effects, has led to new derivatives using N containing heterocycles like quinazolines (see Figure 1 B) and quinolines (see Figure 1 C & D) (Soussi MA et al. (2015) Chem. Med. Chem.10(8):1392-402; I. Khelifi, et al., (2017), European Journal of Medicinal Chemistry 127:1025-1034). [0010] In parallel to that, research has also been conducted on the use of a macromolecule as a carrier for the purpose of imparting water-solubility to poorly water-soluble anticancer agents, or accumulating the anticancer agents in the affected areas. Various investigations to develop derivatives of these compounds have been made, but compounds, which are effective on a conjugate format, are still unknown. Combretastatins were cited to be potentially used as payloads within few patents but with no exemplifications and supportive data: (EP 1 912 677 B1 "PSMA antibody-drug conjugates"); WO 2014/164534 ("Site-specific antibody-drug conjugation through glycoengineering"); US 8,535,678 B2 ("Anti-CD70 antibody-drug conjugates and their use for the treatment of cancer and immune disorders"). On the other hand, combretastatins were evaluated as potential payloads within a couple of studies but with no clinical applications so far because of limited potency, stability issues and unfavorable metabolization profiles acknowledged by the authors themselves (Toki et al. (2002) J. Org. Chem. 67, 1866-1872, Bolu et al. (2016) Mol. Pharmaceutics, 13, 1482-1490, R. Nani et al. (2015) Angew. Chem. Int. Ed. Engl. 9; 54(46): 13635-13638).

[001 1 ] As a conclusion, on one hand, in spite of significant improvements (cardiotoxicity, stability and metabolization profile) resulting from this new generation of quinazoline and quinoline combretastatin derivatives, none of them are eligible for a conjugation to a cell target binding moiety (relevant potency and structure). On the other hand, most conjugation attempts have failed so far because of ineffective combretastatin being used. Last but not least, new derivatives with improved profile compared to MMAE could result in a potential commercial success. These and other matters are addressed by the present invention.

SUMMARY OF THE INVENTION

[0012] As a result of intensive studies for solving the problem described above, the present inventors have found iso-amino-combretaquinolines (ICQO) with improved efficacy against cancer, in particular against resistant tumor cells, and a structure allowing proper efficient conjugation. Furthermore, they have an improved chemical stability and thus reduced tendency to inactivation. This approach has so far neither been taught nor suggested by the prior art. [0013] Thus, in first aspect, the present invention relates to a compound according to Formula I

I

wherein:

R¹ is selected from CH₃, CN, CONH₂, CONHR^*, CH_xX_y, OR^*, NH₂, NHR^*, NR^* ₂ and H,

wherein x is selected from 0, 1 , 2, and 3, X is a halogen selected from F, CI and

Br, and y is 3-x, and wherein R^* is selected from Ci_-5-alkyl and CF₃;

R² is selected from H, CH₃, CN, F, CI and OR^**, wherein R^** is a Ci_-5-alkyl

R³ is selected from H, CH₃, CN, F, CI and OR^*, wherein R^* is selected from Ci-

5-alkyl and CF₃;

R⁴ and R⁵ are either both independently selected from H and F; are H and CN; or are jointly a keto group;

R⁶ is H;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH_3, SCH₃, SCH₂CH₃ and OCHF₂.

[0014] In a second aspect, the present invention relates to a compound according to Formula II

II wherein:

R¹ is selected from CH₃, CN, CONH₂, CONHR^*, CH_xX_y, OR^*, NH₂, NHR^*, NR^* ₂, and H,

wherein x is selected from 0, 1 , 2, and 3, X is a halogen selected from F, CI and Br, and y is 3-x, and wherein R^* is selected from Ci_-5-alkyl and CF₃;

R² is selected from H, CH₃, CN, F, CI and OR^**, wherein R^** is a Ci_-5-alkyl; R³ is selected from H, CH₃, CN, F, CI and OR^*, wherein R^* is selected from Ci- 5-alkyl and CF₃:

R⁶ is L-RM^*, wherein L is a linker, particularly a self-immolative linker, RM^* is selected from RM and RM', wherein RM is a reactive moiety being able to form a covalent bond with a targeting moiety, particularly a target-binding antibody or functional antigen-binding fragment thereof, and wherein RM' is a moiety RM carrying a protecting group;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH_3, SCH₃, SCH₂CH₃ and OCHF₂.

[0015] In a third aspect, the present invention relates to a compound according to Formula III

III

wherein:

Br, and y is 3-x, and wherein R^* is selected from Ci_-5-alkyl and CF₃;

R² is selected from H, CH₃, CN, F, CI and OR^**, wherein R^** is a Ci_-5-alkyl; R³ is selected from H, CH₃, CN, F, CI and OR^*, wherein R^* is selected from Ci- 5-alkyl and CF₃;

R⁶ is L^*-TM, wherein L^* is a linker, particularly a self-immolative linker, and TM is a targeting moiety, particularly a target-binding antibody or functional antigen- binding fragment thereof;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH₃, SCH₃, SCH₂CH₃ and OCHF₂.

[0016] In a fourth aspect, the present invention relates to a method of synthesizing a toxic compound-linker-reactive moiety compound of the present invention, comprising the step of reacting a free toxic compound of the present invention via the amino group attached to the phenyl ring with a compound X-L'-RM^*, wherein

X is a group that is (i) able to react with an amine, or (ii) can be replaced by an amine; and

L' is a linker;

wherein the reaction of said amino group with the moiety X-L' results in the formation of the moiety -NR⁶-L-RM^* or -NR⁶-L-RM^*.

[0017] In a fifth aspect, the present invention relates to a method of synthesizing a toxic compound-linker-targeting moiety compound of the present invention, comprising the step of reacting a toxic compound-linker-reactive moiety compound of the present invention with a targeting moiety.

[0018] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising the toxic compound-linker-targeting moiety compound of the present invention.

[0019] In a seventh aspect, the present invention relates to a pharmaceutical composition of the present invention for use in the treatment of cancer.

[0020] In an eight aspect, the present invention relates to a method for the treatment of cancer comprising the step of administering a toxic compound-linker- targeting moiety compound of the present invention or the pharmaceutical composition of the present invention to a patient in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021 ] Fig. 1 shows the chemical structure of combretastatin A-4 (1A), quinazoline (1 C) and quinolones analogues (1 B and 1 D) as closest prior art.

[0022] Fig. 2 shows the chemical synthesis of Maleimide-Valine-Citrulline-PAB- ICQO-1 as utilized for the bioconjugation with monoclonal antibodies. Step 1 : phosgene/CH₂Cl2; extraction with aq. NaHCOs; room temperature, 2 h. Step 2: CH2CI2, room temperature, overnight. Step 3: Et₂NH, DMF, room temperature, 2 h. Step 4: Hunig's Base, DMF, room temperature, overnight.

[0023] Fig. 3 represents the mass analysis of the compound ICQO-1 by mass spectrometry.

[0024] Fig. 4 represents the mass analysis of the compound ICQO-2 by mass spectrometry.

[0025] Fig. 5 represents the mass analysis of the compound ICQO-3 by mass spectrometry.

[0026] Fig. 6 represents the retention time of the compound ICQO-4 by High Performance Liquid Chromatography.

[0027] Fig. 7 represents the mass analysis of the compound ICQO-5 by mass spectrometry.

[0028] Fig. 8 represents the retention time of the compound ICQO-6 by High Performance Liquid Chromatography.

[0029] Fig. 9 represents the mass analysis of the compound ICQO-7 by mass spectrometry.

[0030] Fig. 10 represents the mass analysis of the compound ICQO-8 by mass spectrometry. [0031 ] Fig. 11 represents the mass analysis of the compound ICQO-9 by mass spectrometry.

[0032] Fig. 12 represents the retention time of the compound ICQO-10 by High Performance Liquid Chromatography.

[0033] Fig. 13 represents the retention time of the compound ICQO-1 1 by High Performance Liquid Chromatography.

[0034] Fig. 14 represents the mass analysis of the compound ICQO-12 by mass spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0036] Particularly, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0037] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer, composition or step or group of integers or steps, while any additional integer, composition or step or group of integers, compositions or steps may optionally be present as well, including embodiments, where no additional integer, composition or step or group of integers, compositions or steps are present. In such latter embodiments, the term "comprising" is used coterminous with "consisting of.

[0038] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety to the extent possible under the respective patent law.

[0039] The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being of particular relevance or advantageous may be combined with any other feature or features indicated as being of particular relevance or advantageous.

[0040] The present invention is based on a combination of different advantageous elements and features and in particular on the unexpected observation that conjugates of iso-amino-combretaquinolines are particularly stable, while simultaneously being highly toxic in target cells.

[0041 ] Thus, in first aspect, the present invention relates to a compound according to Formula I

wherein: R¹ is selected from CH₃, CN, CONH₂, CONHR^*, CH_xX_y, OR^*, NH₂, NHR^*, NR^* ₂ and H,

R⁶ is H;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH_3, SCH₃, SCH₂CH₃ and OCHF₂. ] In a second aspect, the present invention relates to a compound accordingrmula II

II

wherein:

R⁴ and R⁵ are either both independently selected from H and F; are H and CN; or are jointly a keto group; R⁶ is L-RM^*, wherein L is a linker, particularly a self-immolative linker, RM^* is selected from RM and RM', wherein RM is a reactive moiety being able to form a covalent bond with a targeting moiety, particularly a target-binding antibody or functional antigen-binding fragment thereof, and wherein RM' is a moiety RM carrying a protecting group;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH₃, SCH₃, SCH₂CH₃ and OCHF₂.

[0043] The terms "targeting moiety" or "target-binding moiety", as used herein, refer to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. Preferred target-binding moieties in the context of the present application are (i) antibodies or antigen-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers. "Target-binding moieties" suitable for use in the present invention typically have a molecular mass of 40 000 Da (40 kDa) or more.

[0044] As used herein, a first compound (e.g. an antibody) is considered to "specifically bind" to a second compound (e.g. an antigen, such as a target protein), if it has a dissociation constant KD to said second compound of 100 μΜ or less, particularly 50 μΜ or less, particularly 30 μΜ or less, particularly 20 μΜ or less, particularly 10 μΜ or less, particularly 5 μΜ or less, more particularly 1 μΜ or less, more particularly 900 nM or less, more particularly 800 nM or less, more particularly 700 nM or less, more particularly 600 nM or less, more particularly 500 nM or less, more particularly 400 nM or less, more particularly 300 nM or less, more particularly 200 nM or less, even more particularly 100 nM or less, even more particularly 90 nM or less, even more particularly 80 nM or less, even more particularly 70 nM or less, even more particularly 60 nM or less, even more particularly 50 nM or less, even more particularly 40 nM or less, even more particularly 30 nM or less, even more particularly 20 nM or less, and even more particularly 10 nM or less.

[0045] In the context of the present application the terms "target molecule" and "target epitope", respectively, refers to an antigen and an epitope of an antigen, respectively, that is specifically bound by a target-binding moiety. Particularly the target molecule is a tumour-associated antigen, in particular an antigen or an epitope which is present on the surface of one or more tumour cell types in an increased concentration and/or in a different steric configuration as compared to the surface of non-tumour cells. Particularly, said antigen or epitope is present on the surface of one or more tumour cell types, but not on the surface of non-tumour cells.

[0046] The term "antibody or functional antigen-binding fragment thereof, as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen-binding site that immunospecifically binds an antigen. Thus, the term "functional antigen-binding fragments thereof refers to a fragment of an antibody comprising at least a functional antigen-binding domain, i.e. a domain that is able to specifically interact with its target antigen. In particular embodiments, immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to a target molecule are also comprised by that term. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule. "Antibodies and functional antigen-binding fragments thereof suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (in particular CDR-grafted), deimmunized, or chimeric antibodies, single chain antibodies (e.g. scFv), Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, diabodies or tetrabodies (Holliger P. et ai, Proc Natl Acad Sci USA. 90 (1993) 6444-8), nanobodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.

[0047] In some embodiments the functional antigen-binding fragments are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable domain(s) alone or in combination with the entirety or a portion of the following: hinge region, CL, CH1 , CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable domain(s) with a hinge region, CL, CH1 , CH2, and CH3 domains.

[0048] Antibodies usable in the invention may be from any animal origin including birds and mammals. Particularly, the antibodies are from human, rodent (e.g. mouse, rat, guinea pig, or rabbit), chicken, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog origin. It is particularly preferred that the antibodies are of human or murine origin. As used herein, "human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described for example in U.S. Patent No. 5,939,598 by Kucherlapati & Jakobovits.

[0049] The term "antibody-like protein" refers to a protein that has been engineered (e.g. by mutagenesis of loops) to specifically bind to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the antibody-like protein to levels comparable to that of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein may be any protein having good solubility properties. Particularly, the scaffold protein is a small globular protein. Antibody-like proteins include without limitation affibodies, anticalins, and designed ankyrin repeat proteins (for review see: Binz et al. (2005) Nat Biotechnol, 1257-68). Antibody-like proteins can be derived from large libraries of mutants, e.g. be panned from large phage display libraries and can be isolated in analogy to regular antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

[0050] The term "nucleic acid aptamer" refers to a nucleic acid molecule that has been engineered through repeated rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a target molecule (for a review see: Brody and Gold, (2000) J Biotechnol. 74:5-13). The nucleic acid aptamer may be a DNA or RNA molecule. The aptamers may contain modifications, e.g. modified nucleotides such as 2'-fluorine-substituted pyrimidines. [0051 ] A "linker" in the context of the present invention refers to a structure that is connecting two components, each being attached to one end of the linker. In the case of the linker being a bond, a direct linkage of the toxic compound to the antibody may decrease the ability of the toxic compound to interact with its molecular target inside the cell. In particular embodiments, the linker increases the distance between two components and alleviates steric interference between these components, such as in the present case between the antibody and the toxic compound. In particular embodiments, the linker has a continuous chain of between 1 and 30 atoms (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 atoms) in its backbone, i.e. the length of the linker is defined as the shortest connection as measured by the number of atoms or bonds between the toxic compound moiety and the antibody, wherein one side of the linker backbone has been reacted with the toxic compound and, the other side is available for reaction, or has been reacted, with an antibody. In the context of the present invention, a linker particularly is a Ci-20-alkylene, Ci-20-heteroalkylene, C2-20- alkenylene, C2-2o-heteroalkenylene, C2-2o-alkynylene, C2-2o-heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or a heteroaralkylene group, optionally substituted. The linker may contain one or more structural elements such as carboxamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties and the like. The linker may also contain combinations of two or more of these structural elements. Each one of these structural elements may be present in the linker more than once, e.g. twice, three times, four times, five times, or six times. In some embodiments the linker may comprise a disulfide bond. It is understood that the linker has to be attached either in a single step or in two or more subsequent steps to the toxic compound and the antibody. To that end the linker to be will carry two groups, particularly at a proximal and distal end, which can (i) form a covalent bond to a group present in one of the components to be linked, particularly an activated group on an toxic compound or the target binding-peptide or (ii) which is or can be activated to form a covalent bond with a group on an toxic compound. Accordingly, it is preferred that chemical groups are at the distal and proximal end of the linker, which are the result of such a coupling reaction, e.g. an ester, an ether, a urethane, a peptide bond etc. [0052] In particular embodiments, the linker L is a linear chain of between 1 and 20 atoms independently selected from C, O, N and S, particularly between 2 and 18 atoms, more particularly between 5 and 16 atoms, and even more particularly between 6 and 15 atoms. In particular embodiments, at least 60% of the atoms in the linear chain are C atoms. In particular embodiments, the atoms in the linear chain are linked by single bonds.

[0053] In particular embodiments, the linker L is an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or a heteroaralkylene group, comprising from 1 to 4 heteroatoms selected from N, O, and S, wherein said linker is optionally substituted.

[0054] The term "alkylene" refers to a bivalent straight chain saturated hydrocarbon groups having from 1 to 20 carbon atoms, including groups having from 1 to 10 carbon atoms. In certain embodiments, alkylene groups may be lower alkylene groups. The term "lower alkylene" refers to alkylene groups having from 1 to 6 carbon atoms, and in certain embodiments from 1 to 5 or 1 to 4 carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH₂-), ethylene (-CH₂-CH₂-), n-propylene, n-butylene, n-pentylene, and n-hexylene.

[0055] The term "alkenylene" refers to bivalent straight chain groups having 2 to 20 carbon atoms, wherein at least one of the carbon-carbon bonds is a double bond, while other bonds may be single bonds or further double bonds. The term "alkynylene" herein refers to groups having 2 to 20 carbon atoms, wherein at least one of the carbon-carbon bonds is a triple bond, while other bonds may be single, double or further triple bonds. Examples of alkenylene groups include ethenylene (- CH=CH-), 1 -propenylene, 2-propenylene, 1 -butenylene, 2-butenylene, 3-butenylene, and the like. Examples of alkynylene groups include ethynylene, 1 -propynylene, 2- propynylene, and so forth.

[0056] As used herein, "cycloalkylene" is intended to refer to a bivalent ring being part of any stable monocyclic or polycyclic system, where such ring has between 3 and 12 carbon atoms, but no heteroatom, and where such ring is fully saturated, and the term "cydoalkenylene" is intended to refer to a bivalent ring being part of any stable monocyclic or polycyclic system, where such ring has between 3 and 12 carbon atoms, but no heteroatom, and where such ring is at least partially unsaturated (but excluding any arylene ring). Examples of cycloalkylenes include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Examples of cycloalkenylenes include, but are not limited to, cyclopentenylene and cyclohexenylene.

[0057] As used herein, the terms "heterocycloalkylene" and "heterocycloalkenylene" are intended to refer to a bivalent ring being part of any stable monocyclic or polycyclic ring system, where such ring has between 3 and about 12 atoms, and where such ring consists of carbon atoms and at least one heteroatom, particularly at least one heteroatom independently selected from the group consisting of N, O and S, with heterocycloalkylene referring to such a ring that is fully saturated, and heterocycloalkenylene referring to a ring that is at least partially unsaturated (but excluding any arylene or heteroarylene ring).

[0058] The term "arylene" is intended to mean a bivalent ring or ring system being part of any stable monocyclic or polycyclic system, where such ring or ring system has between 3 and 20 carbon atoms, but has no heteroatom, which ring or ring system consists of an aromatic moiety as defined by the "4n+2" π electron rule, including phenylene.

[0059] As used herein, the term "heteroarylene" refers to a bivalent ring or ring system being part of any stable mono- or polycyclic system, where such ring or ring system has between 3 and 20 atoms, which ring or ring system consists of an aromatic moiety as defined by the "4n+2" π electron rule and contains carbon atoms and one or more nitrogen, sulfur, and/or oxygen heteroatoms.

[0060] In the context of the present invention, the term "substituted" is intended to indicate that one or more hydrogens present in the backbone of a linker is replaced with a selection from the indicated group(s), provided that the indicated atom's normal valency, or that of the appropriate atom of the group that is substituted, is not exceeded, and that the substitution results in a stable compound. The term "optionally substituted" is intended to mean that the linker is either unsubstituted or substituted, as defined herein, with one or more substituents, as defined herein. When a substituent is a keto (or oxo, i.e. =O) group, a thio or imino group or the like, then two hydrogens on the linker backbone atom are replaced. Exemplary substituents include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, aroyl, heteroaroyl, carboxyl, alkoxy, aryloxy, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, halogen, (thio)ester, cyano, phosphoryl, amino, imino, (thio)amido, sulfhydryl, alkylthio, acylthio, sulfonyl, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, nitro, azido, haloalkyi, including perfluoroalkyl (such as trifluoromethyl), haloalkoxy, alkylsulfanyl, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino, arylsulfonoamino, phosphoryl, phosphate, phosphonate, phosphinate, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino (optionally mono- or di-substituted, e.g. by alkyl, aryl, or heteroaryl), imino, carboxamide, carbamoyl (optionally mono- or di-substituted, e.g. by alkyl, aryl, or heteroaryl), amidino, aminosulfonyl, acylamino, aroylamino, (thio)ureido, (arylthio)ureido, alkyl(thio)ureido, cycloalkyl(thio)ureido, aryloxy, aralkoxy, or - O(CH₂)_n-OH, -O(CH₂)_n-NH₂, -O(CH₂)_nCOOH, -(CH₂)_nCOOH, -C(O)O(CH₂)_nR, - (CH₂)_nN(H)C(O)OR, or -N(R)S(O)₂R wherein n is an integer selected from 1 to 4 and R is independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, - cycloalkenyl, -(C-linked-heterocycloalkyl), -(C-linked-heterocycloalkenyl), -aryl, and -heteroaryl, with multiple degrees of substitution being allowed. It will be understood by those skilled in the art that substituents, such as heterocycloalkyl, aryl, heteroaryl, alkyl, etc., or functional groups such as -OH, -NHR etc., can themselves be substituted, if appropriate. It will also be understood by those skilled in the art that the substituted moieties themselves can be substituted as well when appropriate.

[0061 ] In particular embodiments, the linker L comprises a moiety selected from one of the following moieties: a disulfide (-S-S-), an ether (-O-), a thioether (-S-), an amine (-NH-), an ester (-O-C(=O)- or -C(=O)-O-), a carboxamide (-NH-C(=O)- or - C(=O)-NH-), a urethane (-NH-C(=0)-0- or -O-C(=O)-NH-), and a urea moiety (-NH- C(=0)-NH-).

[0062] In particular embodiments of the present invention, the linker L comprises a number of m groups selected from the list of: alkylene, alkenylene, alkynylene, cycloalkylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, and a heteroaralkylene group, wherein each group may optionally be independently substituted, the linker further comprises a number of n moieties independently selected from one of the following moieties: a disulfide (-S-S-), an ether (-O-), a thioether (-S-), an amine (- NH-), an ester (-O-C(=O)- or -C(=O)-O-), a carboxamide (-NH-C(=O)- or -C(=O)- NH-), a urethane (-NH-C(=O)-O- or -O-C(=O)-NH-), and a urea moiety (-NH-C(=O)- NH-), wherein m = n+1 . In particular embodiments, m is 2 and n is 1 , or m is 3 and n is 2. In particular embodiments, the linker comprises 2 or 3 unsubstituted alkylene groups, and 1 or 2, respectively, disulfide, ether, thioether, amine, ester, carboxamide, urethane or urea moieties linking the unsubstituted alkylene groups.

[0063] In particular embodiments, the C atoms in the linear chain are independently part of optionally substituted methylene groups (-CH₂-). In particular such embodiments, the optional substituents are independently selected from halogen and Ci-6-alkyl, particularly methyl.

[0064] In particular embodiments, the linker L is a stable linker.

[0065] In the context of the present invention, the term "stable linker" refers to a linker that is stable (i) in the presence of enzymes, and (ii) in an intracellular reducing environment.

[0066] In particular embodiments, the stable linker does not contain (i) an enzyme- cleavable substructure, and/or (ii) a disulfide group. In particular such embodiments, the linker has a length of up to 12 atoms, particularly from 2 to 10, more particularly from 4 to 9, and most particularly from 6 to 8 atoms.

[0067] In certain embodiments the linker is a cleavable linker.

[0068] In the context of the present invention, the term "cleavable linker" refers to a linker that is (i) cleavable by chemical cleavage, or (ii) a reducible linker. [0069] In certain such embodiments, the linker is cleavable by reduction. In the context of the present invention, the term "cleavable by reduction" refers to a linker that can be cleaved in the intracellular reducing environment, particularly a linker that contains a disulfide groups, resulting in the intracellular release of the toxin cargo conjugated to the target-binding moiety after internalization by the intracellular reducing environment (see Shen et ai, (1985) J. Biol. Chem. 260:10905-10908).

[0070] In certain such embodiments, the linker comprises a disulfide bond, particularly a -CMe₂-S-S-CMe₂- moiety. In certain other embodiments, the linker is attached to the thiol group of the targeting moiety via a disulfide bond.

[0071 ] In certain other such embodiments, the linker is cleavable by chemical cleavage, particularly by hydrolysis or proteolysis, particularly wherein such chemical cleavage is catalyzed by an enzyme.

[0072] In the context of the present invention, the term "chemical cleavage is catalyzed by an enzyme" refers to a linker that can be cleaved by an enzyme, particularly by a lysosomal peptidase, such as Cathepsin B, resulting in the intracellular release of the toxin cargo conjugated to the targeting antibody after internalization (see Dubowchik et ai, (2002) Bioconjug Chem. 13:855-69). In particular embodiments, the cleavable linker comprises a dipeptide selected from: Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit, particularly wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptides and the toxic payload.

[0073] In certain such embodiments, the linker comprises a hydrazone group. In particular such embodiments, cleavage occurs by hydrolysis in the lysosome.

[0074] In certain embodiments, the linker is a self-immolative linker.

[0075] In the context of the present invention, the term "self-immolative linker" refers to a linker that comprises a cleavable bond, wherein after cleavage a fragmentation takes place that removes that part of the linker that is still attached to the toxin after said cleavage. Thus, a self-immolative linker is a linker that can completely be cleaved off a compound according to Formula II resulting in the corresponding free toxin of Formula I (R⁶ = L-RM^* in Formula II is converted to R⁶ = H in Formula I). It is well understood by one of ordinary skill in the art, particularly in the art of antibody-drug conjugates, that a variety of chemical structures and fragmentation processes may be employed in the design and application of self- immolative linkers (see, for example, Tranoy-Opalinski et al., Anticancer Agents Med Chem. 8 (2008) 618-37; Blencowe et al., Polym. Chem. 2 (201 1 ) 773-790; Roth, et al., Chemical Reviews 1 16 (2016) 1309-1352; Walther et al., Advanced Drug Delivery Reviews 1 18 (2017) 65-77).

[0076] In certain embodiments, the linker comprises a group -(cleavable bond)-X- phenyl-CH₂-O-C(=O)-, wherein the carbonyl group is attached to the amino group attached to the phenyl ring in the compounds of the present invention, wherein after cleavage of the cleavable bond, the compound of the present invention with a free amino group -NR⁶H is released.

[0077] In the context of the present invention, the term "reactive moiety" relates to a chemical group present in a molecule A that can specifically react with a second moiety present in a molecule B by forming a covalent bond, thus connecting the two molecules, under conditions that leave the remaining moieties and bonds present in A and B unchanged. It is well understood by one of ordinary skill in the art, particularly in the art of antibody-drug conjugates, that a variety of reactive groups, and of second moieties that may react with such reactive groups, may be employed (see, for example, Wu and Senter, Nat Biotechnol. 23 (2005) 1 137-46; Chari, Acc Chem Res. 41 (2008) 98-107; Carter and Senter,. Cancer J. 14 (2008) 154-69).

[0078] In a third aspect, the present invention relates to a compound according to Formula III

III

wherein:

R¹ is selected from CH₃, CN, CONH₂, CONHR^*, CH_xX_y, OR^*, NH₂, NHR^*, NR^* ₂ and H, wherein x is selected from 0, 1 , 2, and 3, X is a halogen selected from F, CI and Br, and y is 3-x, and wherein R^* is selected from Ci_-5-alkyl and CF₃; R² is selected from H, CH₃, CN, F, CI and OR^**, wherein R^** is a Ci_-5-alkyl; R³ is selected from H, CH₃, CN, F, CI and OR^*, wherein R^* is selected from Ci- 5-alkyl and CF₃;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH_3, SCH₃, SCH₂CH₃ and OCHF₂.

[0079] In a fourth aspect, the present invention relates to a method of synthesizing a toxic compound-linker-reactive moiety compound of the present invention according to Formula II, comprising the step of reacting a free toxic compound of the present invention according to Formula I via the amino group attached to the phenyl ring with a compound X-L'-RM', wherein

X is a group that is (i) able to react with an amine, or (ii) can be replaced by an amine;

L' is a linker, and RM' is an optionally protected reactive moiety being able, in the optional case after deprotection, to form a covalent bond with a targeting moiety, particularly a target-binding antibody or functional antigen-binding fragment thereof;

wherein the reaction of said amino group with the moiety X-L' results in the formation of the moiety -NR⁶-L-RM^* or -NR⁶'-L-RM^*.

[0080] In the context of the present invention, a group that is able to react with an amine refers to a moiety of a molecule that can react with the nitrogen of an amino group by forming a covalent bond. It is well understood by one of ordinary skill in the art, particularly in the art of antibody-drug conjugates, that a variety of reactive groups may be employed, including, for example and without limitation, carboxylic acid derivatives such carboxylic acid halides and carboxylic acid esters, alkyl groups with leaving groups (i. e. groups that can be replaced by an amine) for nucleophilic substitutions (see, for example, Hartshorn, Aliphatic Nucleophilic Substitution, Cambridge University Press, London, 1973), such as halides, trifluoromethanesulfonat.es, methanesulfonates, toluenesulfonates, or fluorosulfonates, Michael acceptors, such as a,b-unsaturated ketones or carboxylic acid derivatives.

[0081 ] In a particular embodiment, wherein RM^* is RM', the method further comprises the step of deprotecting the moiety RM' to result in RM.

[0082] In a fifth aspect, the present invention relates to a method of synthesizing a toxic compound-linker-targeting moiety compound of the present invention according to Formula III, comprising the step of reacting a toxic compound-linker-reactive moiety compound of the present invention according to Formula II with a targeting moiety.

[0083] In a particular embodiment, wherein RM^* in Formula II is RM', the method further comprises the step of first deprotecting the moiety RM' to result in RM.

[0084] Conjugation to the targeting moiety may be achieved via coupling of the toxin-L-RM construct to free amino groups present in the targeting moiety. In such case, the group RM may be selected from an activated carboxylic acid derivative, such as an N-hydroxy succinimide ester; or an activated carbonic acid derivative, such as an isothiocyanate.

[0085] Conjugation to the targeting moiety may further be achieved via coupling of the toxin-L-RM construct to free thiol groups present in the targeting moiety. In such case, the group RM may be selected from a haloacetyl group; an RM group comprising an acceptor-substituted alkene (Michael system), particularly a maleimide group or propenoyl group (see Badescu et al., (2014) Bioconjugate Chem. 25:460-469); a maleimide group substituted in 3-position or disubstituted in 3,4-positions with a leaving group X, particularly wherein X is selected from CI, Br, and aryl-S-, particularly Ph-S-.

[0086] In certain such embodiments, the thiol group is part of a single, uncoupled cysteine residue present in the wildtype targeting moiety. In certain other such embodiments, the thiol group is part of a single, uncoupled cysteine residue that has been generated from a wildtype targeting moiety, particularly by recombinant genetic engineering, for example by insertion into the wildtype sequence, by removing a second cysteine that is forming a disulfide bridge with the first cysteine residue in the wildtype targeting moiety, or by replacing a non-cysteine residue. In certain other such embodiments, the thiol group is generated by reduction of a disulfide linkage between two cysteines present in the wildtype targeting moiety.

[0087] Conjugation to the targeting moiety may further be achieved via coupling of the toxin-L-RM construct to two free thiol groups present in the targeting moiety. In such case, the group RM may be a maleimide group disubstituted in 3,4-positions with leaving groups X, particularly wherein X is selected from CI, Br, and aryl-S-, particularly Ph-S-.

[0088] In certain such embodiments, the two thiol groups are each part of a single, uncoupled cysteine residue present in the wildtype targeting moiety. In certain other such embodiments, the thiol groups are part of two single, uncoupled cysteine residues that have been generated from a wildtype targeting moiety, particularly by recombinant genetic engineering, for example by insertion into the wildtype sequence, by removing a second cysteine that is forming a disulfide bridge with the first cysteine residue in the wildtype targeting moiety, or by replacing a non-cysteine residue. In particular embodiments, the two thiol groups are generated by reduction of a disulfide linkage between two cysteines present in the wildtype targeting moiety. By reaction of such two thiol groups with a disubstituted maleimide, the thiol groups are bridged, thus mimicking the originally present disulfide bridge.

[0089] In certain other embodiments, a free thiol group may be generated by thiolation of free amino groups present in the targeting moiety, particularly by reaction of such free amino groups with a thiolation reagent selected from 2- iminothiolane (see Still et al., (1984) Can. J. Org. Chem. 62:586) and an activated acylthioacetic acid derivative (X-C(=O)-CH₂-SAcyl), such as an N-hydroxy succinimide ester of acetylthioacetic acid.

[0090] Conjugation to the targeting moiety may further be achieved by coupling to unnatural amino acids introduced by genetic engineering, for example by introducing p-acetyl phenylalanine and subsequent oxime ligation (see Kazane et al., (2012) Proc. Natl. Acad. Sci. U.S.A, 109:3731-3736).

[0091 ] Conjugation to the targeting moiety may further be achieved by coupling of cyclic diazodicarboxamides to the phenyl ring of tyrosine residues in the targeting moiety (see Ban et al., (2010) J Am. Chem. Soc. 13:1523-5).

[0092] Conjugation to the targeting moiety may be achieved via 1 ,3-dipolar cycloaddition (click chemistry).

[0093] In certain such embodiment the targeting moiety comprises a double or triple bond and the toxin-L-RM construct comprises a 1 ,3-dipole, particularly an azide group. In certain such embodiments, the targeting moiety is first reacted with dibenzocyclooctyne-N-hydroxysuccinimide ester or azadibenzocyclooctyne-N- hydroxysuccinimide ester (see, for example, Zhou et al., (2013) J Am Chem Soc. 135:12994-7).

[0094] In certain other such embodiment the targeting moiety comprises a 1 ,3- dipole, particularly an azide group and the toxin-L-RM construct comprises a double or triple bond. In certain such embodiments, the targeting moiety is a glycosylated antibody that is first coupled to an azide-containing molecule by an enzyme- catalyzed reaction (tradename SiteClick; see Zeglis et al., (2013) Bioconjug Chem. 24:1057-67). In certain other embodiments an azido group is incorporated via the unnatural amino acid p-azido-phenylalanine (see Kazane et al., (2012) Proc. Natl. Acad. Sci. U.S.A, 109:3731-3736).

[0095] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising the toxic compound-linker-targeting moiety compound of the present invention according to Formula III.

[0096] In a seventh aspect, the present invention relates to a pharmaceutical composition of the present invention for use in the treatment of cancer.

[0097] As used herein, "treat", "treating" or "treatment" of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

[0098] As used herein, the treatment may comprise administering a conjugate or a pharmaceutical composition according to the present invention to a patient, wherein "administering" includes in vivo administration, as well as administration directly to tissue ex vivo, such as vein grafts.

[0099] In particular embodiments, a therapeutically effective amount of the conjugate of the present invention is used.

[00100] A "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.

[00101 ] And further comprising one or more pharmaceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents; and/or preservatives.

[00102] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[00103] In particular embodiments, the pharmaceutical composition is used in the form of a systemically administered medicament. This includes parenterals, which comprise among others injectables and infusions. Injectables are formulated either in the form of ampoules or as so called ready-for-use injectables, e.g. ready-to-use syringes or single-use syringes and aside from this in puncturable flasks for multiple withdrawal. The administration of injectables can be in the form of subcutaneous (s.c), intramuscular (i.m.), intravenous (i.v.) or intracutaneous (i.e.) application. In particular, it is possible to produce the respectively suitable injection formulations as a suspension of crystals, solutions, nanoparticular or a colloid dispersed systems like, e.g. hydrosols.

[00104] Injectable formulations can further be produced as concentrates, which can be dissolved or dispersed with aqueous isotonic diluents. The infusion can also be prepared in form of isotonic solutions, fatty emulsions, liposomal formulations and micro-emulsions. Similar to injectables, infusion formulations can also be prepared in the form of concentrates for dilution. Injectable formulations can also be applied in the form of permanent infusions both in in-patient and ambulant therapy, e.g. by way of mini-pumps. [00105] It is possible to add to parenteral drug formulations, for example, albumin, plasma, expander, surface-active substances, organic diluents, pH-influencing substances, complexing substances or polymeric substances, in particular as substances to influence the adsorption of the target-binding moiety toxic compound conjugates of the invention to proteins or polymers or they can also be added with the aim to reduce the adsorption of the target-binding moiety toxic compound conjugates of the invention to materials like injection instruments or packaging- materials, for example, plastic or glass.

[00106] The toxic compounds (payloads) of the present invention comprising a target-binding moiety can be bound to microcarriers or nanoparticles in parenterals like, for example, to finely dispersed particles based on poly(meth)acrylates, polylactates, polyglycolates, polyamino acids or polyether urethanes. Parenteral formulations can also be modified as depot preparations, e.g. based on the "multiple unit principle", if the conjugates of the present invention are introduced in finely dispersed, dispersed and suspended form, respectively, or as a suspension of crystals in the medicament or based on the "single unit principle" if the conjugate of the invention is enclosed in a formulation, e.g. in a tablet or a rod which is subsequently implanted. These implants or depot medicaments in single unit and multiple unit formulations often consist of so called biodegradable polymers like e.g. polyesters of lactic acid and glycolic acid, polyether urethanes, polyamino acids, poly(meth)acrylates or polysaccharides.

[00107] Adjuvants and carriers added during the production of the pharmaceutical compositions of the present invention formulated as parenterals are particularly aqua sterilisata (sterilized water), pH value influencing substances like, e.g. organic or inorganic acids or bases as well as salts thereof, buffering substances for adjusting pH values, substances for isotonization like e.g. sodium chloride, sodium hydrogen carbonate, glucose and fructose, tensides and surfactants, respectively, and emulsifiers like, e.g. partial esters of fatty acids of polyoxyethylene sorbitans (for example, Tween^®) or, e.g. fatty acid esters of polyoxyethylenes (for example, Cremophor^®), fatty oils like, e.g. peanut oil, soybean oil or castor oil, synthetic esters of fatty acids like, e.g. ethyl oleate, isopropyl myristate and neutral oil (for example, Miglyol^®) as well as polymeric adjuvants like, e.g. gelatine, dextran, polyvinylpyrrolidone, additives which increase the solubility of organic solvents like, e.g. propylene glycol, ethanol, N,N-dimethylacetamide, propylene glycol or complex forming substances like, e.g. citrate and urea, preservatives like, e.g. benzoic acid hydroxypropyl ester and methyl ester, benzyl alcohol, antioxidants like e.g. sodium sulfite and stabilizers like e.g. EDTA.

[00108] When formulating the pharmaceutical compositions of the present invention as suspensions in a preferred embodiment thickening agents to prevent the setting of the conjugates of the invention or, tensides and polyelectrolytes to assure the resuspendability of sediments and/or complex forming agents like, for example, EDTA are added. It is also possible to achieve complexes of the active ingredient with various polymers. Examples of such polymers are polyethylene glycol, polystyrene, carboxymethyl cellulose, Pluronics^® or polyethylene glycol sorbit fatty acid ester. The conjugates of the invention can also be incorporated in liquid formulations in the form of inclusion compounds e.g. with cyclodextrins. In particular embodiments dispersing agents can be added as further adjuvants. For the production of lyophilisates scaffolding agents like mannite, dextran, saccharose, human albumin, lactose, PVP or varieties of gelatine can be used.

[00109] In an eight aspect, the present invention relates to a method for the treatment of cancer comprising the step of administering a toxic compound-linker- targeting moiety compound of the present invention or the pharmaceutical composition of the present invention to a patient in need of such treatment.

EXAMPLES

[001 10] In order to illustrate the invention, the following examples are included. However, it is to be understood that these examples do not limit the invention and are only meant to suggest a method of practicing the invention.

Example 1 : Generation and synthesis of the toxin unit ICQO-1

[001 1 1 ] Melting points (mp) were recorded on a Buchi B-450 apparatus and are uncorrected. NMR spectra were performed on a Bruker AMX 200 (1 H, 200 MHz; 13C, 50 MHz), Bruker AVANCE 300. Yields refer to isolated yields of compounds estimated to be >95% pure [1 H NMR (25°C) and capillary GC]. Chemical shifts are reported as δ values in ppm relative to the solvent peak. NMR spectra were recorded in CDCI₃ solution (residual CDCI3: δ = 7.26 ppm for 1 H NMR and δ = 77.16 ppm for 13C NMR), residual acetone-d6 (δ = 2.05 ppm for 1 H NMR and δ = 206.26 and 29.84 ppm for 13C NMR), residual dimethylsulfoxide-d6 (δ = 2.50 ppm for 1 H NMR and δ = 39.52 ppm for 13C NMR). Chemical shifts δ are reported in ppm, and the following abbreviations are used: singlet (s), doublet (d), triplet (t), multiplet (m), broad (br). Mass spectra were obtained using a Bruker Esquire electrospray ionization apparatus. Thin-layer chromatography (TLC) was performed on silica gel 60 plates with a fluorescent indicator and visualized under a UVP Mineralight UVGL- 58 lamp (δ 254 nm) and with a 7% solution of phosphomolybdic acid in EtOH. Flash chromatography was performed on silica gel 60 (40-63 mm, 230-400 mesh ASTM) at medium pressure (200 mbar). All solvents were distilled and stored over 4 A molecular sieves before use. All reagents were obtained from commercial suppliers unless otherwise stated. Organic extracts were, in general, dried over MgSO₄ or Na₂SO4.

[001 12] The toxin unit ICQO-1 was generated using the following process. First, to a round necked bottom flask equipped with a reflux apparatus (Argon, stirrer) were added polyphosphoric acid (40 g), aniline (4.67 g, 4.58 ml_, 50 mmol, 1 eq.) and ethyl acetoacetate (6.52 g, 6.34 ml_, 50 mmol, 1 eq.). The reaction mixture was stirred at 130°C for 2 h. Reaction completion was monitored by TLC. The reaction mixture was poured into ice water (170 ml_) slowly with vigorous stirring. The solution was neutralized by an addition of NaHCO₃. Then a precipitated solid appears and the solution was filtered and the residue was dried under reduce pressure oven for 12 h to afford the crude mixture of 2-methylquinolin-4-ol (4.4 g, 55% yield) as white solid.

[001 13] The crude mixture was taken as such for the next step without further purification.

¹H NMR (300 MHz, DMSO-D₆): δ [ppm] = 1 1 .58 (br s, 1 H, OH), 8.02 (d, 1 H, J = 8.3 Hz), 7.60 (t, 1 H, J = 8.1 Hz), 7.49 (d, 1 H, J = 8.5 Hz), 7.26 (1 H, J = 7.9 Hz), 5.90 (s, 1 H), 2.33 (s, 3H). [001 14] Then, to a round necked bottom flask equipped with a reflux apparatus (Argon, stirrer) were added 2-methylquinolin-4-ol (2 g, 12.56 mmol, 1 eq.) and freshly distilled POCI3 (7.4 mL). The mixture was heated at 120°C for 2 h. The reaction was monitored by TLC (DCM: 100%). After completion of the reaction and cooling to room temperature the reaction mixture was poured into a saturated solution of sodium carbonate, and neutralized to pH = 7 with NaHCO3. Then, DCM was added, and the organic phase was separated. The aqueous phase was extracted with DCM twice. The combined organic phase was dried over MgSO4, filtered and concentrated under reduce pressure to afford the desired product 4-chloro-2- methylquinoline (3.157 g, 69%) as a pale yellow oil.

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.17 (d, 1 H, J = 8.3 Hz), 8.03 (d, 1 H, J = 8.5 Hz), 7.73 (t, 1 H, J = 8.3 Hz), 7.57 (t, 1 H, J = 8.3 Hz), 7.39 (s, 1 H), 2.72 (s, 3H).

1³C NMR (75 MHz, CDCI₃): δ [ppm] = 159.0, 148.8, 142.7, 130.5, 129.1 , 126.8, 124.9, 124.1 , 122.1 , 25.3.

[001 15] Into a microwave tube dried and flushed with argon (Stirrer) were placed 4- chloro-2- methylquinoline (204 mg, 1 .14 mmol, 1 eq.), Pd₂dba₃CHCI₃ (30 mg, 0.028 mmol, 0.025 eq.), X-Phos (54 mg, 0.1 14 mmol, 0.1 eq.), (E)-N'-(1 -(4-methoxy-3- nitrophenyl)ethylidene)-4-methylbenzenesulfonohydrazide (498 mg, 1 .378 mmol, 1 .2 eq.), LiOfBu (228 mg, 2.85 mmol, 2.5 eq.) in dioxane (4 mL). Then, the mixture is heated at 100°C and stirred at the same temperature for 3 h. Then, the solution was cooled at RT, degassed (warning: formation of N₂), filtered over Celite, washed with EtOAc (3 x 10 mL) and concentrated under reduce pressure. The crude mixture was purified by column chromatography on silica gel using a Compagnon apparatus and DCM/EtOAc 7/3 as eluents to afford the desired product (4-(1 -(4-methoxy-3- nitrophenyl)vinyl)-2-methylquinoline (240 mg, 66%) as yellow solid.

1 H NMR (300 MHz, CDCI₃): δ [ppm] = 8.10 (d, 1 H, J = 8.5 Hz), 7.90 (d, 1 H, J = 2.5 Hz), 7.72-7.65 (m, 2H), 7.427.30 eq(m, 2H), 6.98 (d, 1 H, J = 8.9 Hz), 6.04 (s, 1 H), 5.49 (s, 1 H), 3.96 (s, 3H), 2.81 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 159.0, 152.7, 148.3, 147.3, 143.8, 132.5, 132.4, 129.7, 129.2, 126.1 , 125.6, 125.0, 123.4, 122.7, 1 17.8, 1 13.7, 56.7, 25.4. [001 16] To a round necked bottom flask equipped with a reflux apparatus (25 mL) were added 4- (1 -(4-methoxy-3-nitrophenyl)vinyl)-2-methylquinoline (209 mg, 0.72 mmol, 1 eq.) in EtOH (7.2 mL) and water (1 .8 mL). Iron powder (402 mg, 7.2 mmol, 10 equiv.) and HCI (12N) (100 μί) were added. The mixture was heated under reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through Celite. The pad of Celite was washed with EtOAc (3 x 15 mL). The organic layer was neutralized with a solution of NaHCO₃. The organic layer was separated, dried over MgSO4, filtered and concentrated under reduce pressure. The crude mixture was purified by column chromatography on silica gel to afford the 2- methoxy-5-(1 -(2-methylquinolin-4-yl)vinyl)aniline as a yellow solid (120 mg, 64% yield).

¹H NMR (300 MHz, CDCI₃) δ [ppm] = 8.04 (d, 1 H, J = 8.7 Hz), 7.75 (d, 1 H, J = 8.3 Hz), 7.62 (t, 1 H, J = 8.5 Hz), 7.32 (t, 1 H, J = 7.2 Hz), 7.21 (s, 1 H), 6.70-6.59 (m, 3H), 5.86 (s, 1 H), 5.24 (s, 1 H), 3.82 (s, 3H), 3.75 (br s, 2H), 2.75 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 158.8, 149.2, 148.2, 147.6, 146.1 , 136.2, 133.1 , 129.3, 128.8, 126.3, 125.7, 125.6, 122.5, 1 17.3, 1 15.0, 1 13.2, 1 10.2, 55.6, 25.4.

Product Name: ICQO-1

Overall Yield: 16%

Molecular formula: C19H18N20

Molecular weight: 290.37 g/mol

Example 2: Generation and synthesis of the toxin unit ICQO-2 [001 17] The toxin unit ICQO-2 was generated using the following process. First, in a round bottom flask, dried and flushed with argon, equipped with a reflux apparatus were dissolved 4-methoxy-3-nitrophenylacetophenone (5 g, 39.04 mmol, 1 eq.) in EtOH (350 ml_). Tosyl hydrazide (8.72 g, 46.86 mmol, 1 .2 eq.) was added to the solution in one portion. The reaction mixture was warmed under reflux. The reaction was stirred for 4 h and followed by TLC (cyclohexane/EtOAc: 2/8). The mixture was cooled at RT and filtrated. The residue was recrystallized in EtOH to afford the desired product (E)-N'-(1 -(4-methoxy-3-nitrophenyl)ethylidene)-4-methylbenzene- sulfonohydrazide (8.96 g, 63% yield) as yellow solid.

¹H NMR (300 MHz, DMSO-D₆): δ [ppm] = 10.52 (br s, 1 H, NH), 8.05 (s, 1 H), 7.91 - 7.80 (m, 3H), 7.41 -7.34 (m, 3H), 3.93 (s, 3H), 2.36 (s, 3H), 2.17 (s, 3H).

1³H NMR (75 MHz, DMSO-D₆): δ [ppm] = 152.6, 150.9, 143.4, 139.0, 136.1 , 131 .5, 129.8, 129.4 (2C), 127.6 (2C), 122.3, 1 14.3, 56.9, 21 .0, 14.0.

[001 18] In a round bottom flask, dried and flushed with argon, was placed 2,4- dimethoxyaniline (3.48 g, 3.57 ml_, 22.72 mmol, 1 eq.), ethylacetoacetate (5.01 g, 4.88 ml_, 38.5 mmol, 1 .7 eq.), anhydrous magnesium sulfate (5.05 g, 42 mmol, 1 .8 eq.) and catalytic amount of acetic acid (1 ml_) in ethanol (EtOH) (250 ml_). The mixture was refluxed for 7 h and TLC (cyclohexane/EtOAc: 9/1 ) analysis indicated that the reaction was complete. The reaction mass was filtered and evaporated under reduced pressure to afford a brown liquid which was further purified by flash column chromatography on silica gel (cyclohexane/EtOAc: 95/5) as eluents to afford the desired product in good yield (3.95 g, 65%).

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 9.93 (br s, 1 H), 7.00 (d, J = 8.6 Hz, 1 H), 6.49 (d, J = 2.6 Hz, 1 H), 6.42 (dd, J = 8.6 and 2.6 Hz, 1 H), 4.66 (br s, 1 H), 4.14 (q, J = 7.2 Hz, 2H), 3.81 (s, 6H), 1 .86 (s, 3H), 1 .28 (t, J = 7.2 Hz, 3H).

[001 19] In a round bottom flushed flask, dried and flushed with argon, were added polyphosphoric acid (PPA) (40 g) to the ethyl (Z)-3-((2,4- dimethoxyphenyl)imino)butanoate (3.95 g, 14.88 mmol, 1 eq.) and phosphorous oxychloride (POCI₃) ( mmol, 0.28 eq.). The mixture was heated to 75°C for 8 h, and TLC analysis indicated that the reaction was complete. The reaction mixture was quenched slowly with 10% sodium hydroxide solution by keeping it in ice bath adjusted to pH 7, and the solid was filtered then dried and washed with diethyl ether (3 x 20 mL) and then acetone. The solid was dried under reduce pressure to afford the desired product as a beige solid (714 mg, 20%).

¹H NMR (200 MHz, DMSO-D₆): δ [ppm] = 10.98 (br s, 1 H), 7.04 (d, J = 2.4 Hz, 1 H), 6.83 (J = 2.4 Hz, 1 H), 5.87 (s, 1 H), 3.96 (s, 3H), 3.81 (s, 3H), 2.34 (s, 3H).

[00120] In a round bottom flushed flask, POCI₃ (1 .6 mL, 16.90 mmol, 5 eq.) was added slowly to the 6,8-dimethoxy-2-methylquinolin-4-ol (714 mg, 3.38 mmol, 1 eq.) and refluxed for 3 h at 105°C. TLC analysis indicated that the reaction was completed. Excess POCI₃ was removed under reduced pressure and the crude mixture was quenched into crushed ice then neutralized with saturated sodium bicarbonate solution (100 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layer was dried under anhydrous magnesium sulfate filtered and evaporated under reduced pressure to get corresponding desired product (665 mg, 83% yield). ¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.37 (s, 1 H), 6.97 (d, J = 2.6 Hz, 1 H), 6.71 (d, J = 2.6 Hz, 1 H), 4.02 (s, 3H), 3.93 (s, 3H), 2.70 (s, 3H).

[00121 ] In a microwave vial dried and flushed with argon, was placed 4-chloro-6,8- dimethoxy-2-methylquinoline (1 18.5 mg, 0.5 mmol, 1 eq.), PdCI₂(MeCN)₂ (13 mg, 0.05 mmol, 0.1 eq.), dppf (55 mg, 0.1 mmol, 0.2 eq.), (E)-N'-(1 -(4-methoxy-3- nitrophenyl)ethylidene)-4-methylbenzenesulfonohydrazide (273 mg, 0.75 mmol, 1 .5 eq.), LiOtBu (100 mg, 1 .25 mmol, 2.5 eq.) in dioxane (4 mL). (The order of the addition is essential). Then, the mixture is heated at 100°C and stirred at the same temperature for 3 h. (Followed by TLC DCM/EtOAc: 8/2). Then, the solution was cooled at RT, degassed (warning: formation of N₂), filtered through celite, washed with EtOAc (3 x 10 mL) and concentrated under reduce pressure. The crude mixture was purified by flash column chromatography on silica gel using DCM/EtOAc (9/1 to 8/2) as eluents to afford the desired product (95 mg, 50% yield) as brown solid.

1H NMR (300 MHz, CDCI₃): δ [ppm] = 7.90 (d, J = 2.6 Hz, 1 H), 7.26 (dd, J = 8.6 and 2.5 Hz, 2H), 7.20 (s, 1 H), 6.95 (d, J = 8.6 Hz, 1 H), 6.67 (d, J = 2.5 Hz, 1 H), 6.45 (d, J = 2.5 Hz, 1 H), 5.97, (s, 1 H), 5.47 (s, 1 H), 4.05 (s, 3H), 3.93 (s, 3H), 3.65 (s, 3H), 2.78 (s, 3H). [00122] To a round necked bottom flask, dried and flushed with argon, equipped with a reflux apparatus (25 mL) was placed 6,8-dimethoxy-4-(1 -(4-methoxy-3- nitrophenyl)vinyl)-2-methylquinoline (79 mg, 0.206 mmol, 1 eq.) in EtOH (2 mL) and water (0.5 mL). Iron powder (1 16 mg, 2.06 mmol, 10 eq.) and concentrated hydrochloric acid (20 μί) were added. The mixture was heated under reflux for 30 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through celite. The pad of celite was washed with EtOAc (3 x 15 mL). The mixture was cooled at RT and filtered through celite. The pad of celite was washed with EtOAc (3 x 15 mL) and the organic layer was concentrated under reduce pressure. Then the crude mixture was purified by flash column chromatography on silica gel using (DCM/EtOAc/NEt₃: 7/3/0.1 ) to afford the product as a pale solid (45 mg, 61 % yield).

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.20 (s, 1 H), 6.69-6.59 (m, 5H), 5.83 (s, 1 H), 5.26 (s, 1 H), 4.04 (s, 3H), 3.83 (s, 3H), 3.65 (s, 3H), 2.76 (s, 3H). (absence of the NH₂ signal).

¹³C NMR (75 MHz, CDCI₃): δ = 157.2, 155.9, 155.2, 147.5, 146.6, 136.1 , 127.2, 123.6, 1 17.3, 1 14.8, 1 13.2, 1 10.2, 100.9, 96.1 , 56.3, 55.7, 55.4, 25.4. (absence of 3 carbons).

Product Name: ICQO-2

Overall Yield: 5.5%

Molecular formula: C21 H22N2O3

Molecular weight: 350.42 g/mol

Example 3: Generation and synthesis of the toxin unit ICQO-3

[00123] A mixture of 2,4-dichloroquinoline (0.3 g, 1 .5 mmol) and n-butylamine (3 mL) was heated at 75°C in a sealed vial for 1 .5 h. The mixture was then concentrated under reduced pressure, dissolved in ethyl acetate (40 mL) and washed with water (20 mL) and with brine (20 mL). The organic layer was dried over MgSO₄, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using ethyl acetate in hexane (5/95 to 1/9) to give a mixture of 2 isomers (105 mg, 30%) and (1 13 mg, 32%). First regioisomers:

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.99 (d, J = 8.3 Hz, 1 H), 7.66 (d, J = 8.3 Hz, 1 H), 7.57 (t, J = 7.5 Hz, 1 H), 7.27 (t, J = 7.2 Hz, 1 H), 6.76 (s, 1 H), 4.72 (br s, 1 H, NH), 3.45 (q, J = 6.0 Hz, 2H), 1 .64 (quint, J = 7.4 Hz, 2H), 1 .44 (quint, J = 7.6 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] =156.8, 149.0, 143.0, 130.6, 126.5, 124.1 , 122.7, 121 .7, 1 10.7, 41 .7, 31 .9, 20.3, 14.0.

[00124] In a sealed tube and under an argon atmosphere were added successively, ArCI (0.5 mmol, 1 eq.), hydrazone (0.75 mmol, 1 .5 eq.) PdCI₂(CH₃CN)₂ (10 mol%), dppf (20 ιτιο /ο) in dry dioxane (4 mL) and the mixture was stirred for 5 min. at RT. Then dry LiOfBu (1 .25 mmol, 2.5 eq.) was added and the mixture was stirred at 120° C for 3 h. The resulting suspension was cooled to room temperature, filtered through a pad of Celite eluting with ethyl acetate and the inorganic salts were removed. The filtrate was concentrated and the crude was purified by flash column chromatography on silica gel to afford the desired product.

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.92 (d, J = 2.5 Hz, 1 H), 7.70 (d, J = 8.3 Hz, 1 H), 7.48 (t, J = 8.1 Hz, 1 H), 7.38 (d, J = 7.7 Hz, 1 H), 7.34 (dd, J = 8.1 and 2.3 Hz, 1 H), 7.05 (t, J = 8.1 Hz, 1 H), 6.94 (d, J = 8.9 Hz, 1 H), 6.58 (s, 1 H), 5.93 (s, 1 H), 5.44 (s, 1 H), 4.74 (br s, NH), 3.93 (s, 3H), 3.51 (q, J = 6.5 Hz, 2H), 1 .72-1 .62 (m, 4H), 0.99 (t, J = 7.2 Hz, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 156.9, 148.8, 148.2, 144.4, 144.3, 139.9, 132.5, 132.4, 129.8, 126.7, 125.7, 123.3, 122.2, 122.1 , 1 17.1 , 1 13.6, 1 1 1 .7, 56.8, 41 .7, 32.1 , 20.4, 14.1 .

[00125] To a round-necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 mL) and water (0.8 mL). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μί) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of Celite was washed with EtOAc (3 x 20 ml_). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O: 8/2) to afford the desired product.

1H NMR (300 MHz, CDCI₃): δ [ppm] = 7.67 (d, J = 9.2 Hz, 1 H), 7.56-7.48 (m, 2H), 7.08-7.02 (m, 1 H), 6.80-6.66 (m, 3H), 6.57 (s, 1 H), 5.80 (s, 1 H), 5.24 (s, 1 H), 4.80 (br s, NH), 3.82 (s, 3H), 3.75 (br s, NH2), 3.59-3.45 (m, 2H), 1 .75-1 .62 (m, 2H), 1 .56- 1 .41 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 129.5, 126.4, 122.0, 1 17.3, 1 13.2, 1 1 1 .3, 1 10.3, 55.7, 41 .7, 32.1 , 20.4, 14.0

Product Name: ICQO-3

Overall Yield: 4.3%

Molecular formula: C22H25N30

Molecular weight: 347.46 g/mol

Example 4: Generation and synthesis of the toxin unit ICQO-4

[00126] The toxic unit ICQO-4 was synthesized according the following process. In a microwave vial, dried and flushed with argon, was charged 2,4-chloroquinoline (407 mg, 2.055 mmol, 1 eq.) in DMF (4.6 ml_). Then, Zn(CN)₂ (123 mg, 1 .048 mmol, 0.51 eq.) was added followed by Pd(PPh₃)₄ (237.5 mg, 0.206 mmol, 0.1 eq.). The reaction mixture was warmed at 120°C and stirred at this temperature for 1 h. After complete consumption of the starting material (TLC: cyclohexane/EtOAc: 9/1 ), the reaction was hydrolyzed by addition of saturated solution of NH₄CI (50 ml_). The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 30 ml_). The combined organic layers were dried over MgSO₄, filtered and concentrated under reduce pressure. The crude mixture was purified by flash column chromatography on silica gel (cyclohexane/EtOAc: 9/1 ) to afford the desired product (310 mg, 80%).

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.28 (d, J = 8.6 Hz, 1 H), 8.19 (d, J = 8.4 Hz, 1 H), 7.92 (t, J = 7.5 Hz, 1 H), 7.82 (t, J = 7.8 Hz, 1 H), 7.80 (s, 1 H).

[00127] In a sealed tube and under an argon atmosphere were added successively, ArCI (0.5 mmol, 1 eq.), hydrazone (0.75 mmol, 1 .5 eq.) PdCI₂(CH₃CN)₂ (10 mol%), dppf (20 mol%) in dry dioxane (4 ml_) and the mixture was stirred for 5 min. at RT. Then dry LiOfBu (1 .25 mmol, 2.5 eq.) was added and the mixture was stirred at 120° C for 3 h. The resulting suspension was cooled to room temperature, filtered through a pad of Celite eluting with ethyl acetate and the inorganic salts were removed. The filtrate was concentrated and the crude was purified by flash column chromatography on silica gel to afford the desired product.

[00128] To a round-necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 ml_) and water (0.8 ml_). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μΙ_) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of Celite was washed with EtOAc (3 x 20 ml_). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

[00129] ¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.24 (s, 1 H), 8.1 1 (d, J = 8.5 Hz, 1 H), 7.85 (t, J = 8.5 Hz, 1 H), 7.70 (d, J = 6.7 Hz, 1 H), 7.46 (t, J = 8.3 Hz, 1 H), 6.67-6.65 (m, 2H), 6.58 (dd, J = 8.3 and 2.1 Hz, 1 H), 5.89 (s, 1 H), 5.72 (br s, NH2), 5.30 (s, 1 H), 3.82 (s, 3H), 3.74 (br s, NH2).

[00130] ¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 167.2, 150.8, 149.2, 147.1 , 146.0, 145.1 , 136.3, 133.0, 130.1 , 130.0, 128.6, 128.0, 126.6, 1 19.5, 1 17.5, 1 15.9, 1 13.2, 1 10.3, 55.7.

Product Name: ICQO-4 Overall Yield: 8.5%

Molecular formula: C19H17N302

Molecular weight: 319.36 g/mol

Example 5: Generation and synthesis of the toxin unit ICQO-5

[00131 ] The synthesis of the toxic unit ICQO-5 was performed according to the following process. First, to a round-necked bottom flask equipped with a reflux apparatus (Argon, stirrer) was placed polyphosphoric acid (20 g), aniline (25 mmol, 1 eq.) and ethyl acetoacetate (25 mmol, 1 eq.). The reaction mixture was stirred at 130°C for 2 h. Reaction completion was monitored by TLC. The reaction mixture was poured into ice water (170 ml_) slowly with vigorous stirring. The solution was neutralized by addition of NaHCO3 or KOH. Then a precipitated solid appears and the solution was filtered and the residue was dried under reduce pressure oven for 12 h to afford the crude mixture. The crude mixture was taken as such for the next step without further purification.

¹H NMR (300 MHz, DMSO-D₆): δ [ppm] = 1 1 .70 (br s, 1 H), 7.67 (dd, J = 9.4 and 2.6 Hz, 1 H), 7.59-7.48 (m, 2H), 5.91 (br s, 1 H), 2.34 (s, 3H).

¹³C NMR (75 MHz, DMSO-D₆): δ [ppm] = 175.8, 159.6, 156.5, 149.9, 136.8, 125.5 (d, J = 6.0 Hz), 120.3 (dd, J = 28.5 and 1 1 .5 Hz), 108.7 (d, J = 21 .9 Hz), 107.7, 19.4. ¹⁹F NMR (300 MHz, DMSO-D₆): δ [ppm] = -1 18.8

[00132] To a round necked bottom flask equipped with a reflux apparatus (Argon, stirrer) was placed 2-methylquinolin-4-ol derivative (6.69 mmol, 1 eq.) and freshly distilled POCI3 (2 ml_). The mixture was heated at 120°C for 2 h. The reaction was monitored by TLC (DCM: 100%). After completion of the reaction and cooling to room temperature the reaction mixture was poured into a saturated solution of sodium carbonate or KOH, and neutralized to the pH value 7. Then, DCM was added, and the organic phase was separated. The aqueous phase was extracted with DCM (2 x). The combined organic phase was dried over MgSO₄, filtered and concentrated under reduce pressure. The crude was purified by flash column chromatography on silica gel to afford the desired product.

[00133] ¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.97 (dd, J = 9.2 and 5.7 Hz, 1 H), 7.73 (td, J = 9.4 and 2.6 Hz, 1 H), 7.46 (td, J = 9.2 and 2.6 Hz, 1 H), 7.36 (s, 1 H), 2.68 (s, 3H).

[00134] ¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 160.5, 158.3 (d, J = 2.7 Hz), 145.8, 141 .8 (d, J = 5.5 Hz), 131 .7 (d, J = 9.3 Hz), 125.7 (d, J = 10.4 Hz), 122.6, 120.5 (d, J = 25.8 Hz), 107.8 (d, J = 24.7 Hz), 25.1 .

[00135] ¹⁹F NMR (300 MHz, CDCI₃): δ [ppm] = -1 12.4

[00136] In a sealed tube and under an argon atmosphere were added successively, ArCI (0.5 mmol, 1 eq.), hydrazone (0.75 mmol, 1 .5 eq.) PdCI₂(CH₃CN)₂ (10 mol%), dppf (20 mol%) in dry dioxane (4 ml_) and the mixture was stirred for 5 min. at RT. Then dry LiOfBu (1 .25 mmol, 2.5 eq.) was added and the mixture was stirred at 120° C for 3 h. The resulting suspension was cooled to room temperature, filtered through a pad of Celite eluting with ethyl acetate and the inorganic salts were removed. The filtrate was concentrated and the crude was purified by flash column chromatography on silica gel to afford the desired product.

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.02 (dd, J = 9.2 and 5.5 Hz, 1 H), 7.80 (d, J = 2.5 Hz, 1 H), 7.41 (ddd, J = 9.2, 8.1 and 2.8 Hz, 1 H), 7.34 (dd, J = 8.9 and 2.5 Hz, 1 H), 7.25-7.21 (m, 1 H), 7.23 (s, 1 H), 6.98 (d, J = 8.9 Hz, 1 H), 6.00 (s, 1 H), 5.44 (s, 1 H), 3.94 (s, 3H), 2.75 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 161 .8, 158.4 (d, J = 14.3 Hz), 152.8, 146.6 (d, J = 5.5 Hz), 145.5, 143.4, 139.8, 132.2, 132.1 , 131 .8 (d, J = 8.8 Hz), 125.8 (d, J = 9.3 Hz), 123.5, 123.3, 1 19.8 (d, J = 25.8 Hz), 1 18.0, 1 13.8, 109.1 (d, J = 22.5 Hz), 56.8, 25.3.

¹⁹F NMR (300 MHz, CDCI₃): δ [ppm] = -1 13.3

[00137] To a round necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 ml_) and water (0.8 ml_). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μΙ_) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of Celite was washed with EtOAc (3 x 20 mL). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

1H NMR (300 MHz, CDCI₃): δ [ppm] = 8.02 (dd, J = 9.6 and 6.2 Hz, 1 H), 7.37 (ddd, J = 1 1 .5, 8.3 and 2.8 Hz, 1 H), 7.35 (d, J = 9.8 Hz, 1 H), 7.22 (s, 1 H), 6.69-6.64 (m, 2H), 6.59 (dd, J = 8.3 and 2.1 Hz, 1 H), 5.85 (s, 1 H), 5.24 (s, 1 H), 3.83 (s, 3H), 3.77 (br s, NH2), 2.74 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 161 .6, 158.3, 158.1 (d, J = 2.7 Hz), 148.6 (d, J = 5.5 Hz), 147.7, 145.4 (d, J = 1 .1 Hz), 136.3, 132.6, 131 .2, 126.4 (d, J = 9.9 Hz), 123.1 , 1 19.4 (d, J = 25.8 Hz), 1 17.3, 1 15.2, 1 13.1 , 1 10.3, 109.7 (d, J = 23.0 Hz), 55.6, 25.3.

¹⁹F NMR (300 MHz, CDCI₃): δ [ppm] = -1 12.3

Product Name: ICQO-5

Overall Yield: 17.7%

Molecular formula: C19H17FN20

Molecular weight: 308.36 g/mol

Example 6: Generation and synthesis of the toxin unit ICQO-6

[00138] The synthesis of the toxic unit ICQO-6 was done as following. First, to a round necked bottom flask equipped with a reflux apparatus (Argon, stirrer) was placed polyphosphoric acid (20 g), aniline (25 mmol, 1 eq.) and ethyl acetoacetate (25 mmol, 1 eq.). The reaction mixture was stirred at 130°C for 2 h. Reaction completion was monitored by TLC. The reaction mixture was poured into ice water (170 mL) slowly with vigorous stirring. The solution was neutralized by addition of NaHCOs or KOH. Then a precipitated solid appears and the solution was filtered and the residue was dried under reduce pressure oven for 12 h to afford the crude mixture. The crude mixture was taken as such for the next step without further purification.

¹H NMR (300 MHz, DMSO-D₆): δ [ppm] = 1 1 .73 (br s, 1 H), 8.60 (br s, 1 H), 8.06 (d, J = 8.6 Hz, 1 H), 7.50 (m, J = 8.6 Hz, 1 H), 5.96 (br s, 1 H), 2.35 (s, 3H).

[00139] To a stirred solution of diethylcyanomethylphosphonate (83 μί, 0.52 mmol, 1 .1 eq.) in THF (3.1 mL) was added a 1 M solution of LiHMDS in THF (520 μΙ_, 0.52 mmol, 1 .1 eq.) at 0°C. The solution was allowed to warm to room temperature and was stirred for 30 min, and then a mixture of (4-methoxy-3-nitrophenyl)(2- methylquinolin-4-yl)methanone (150 mg, 0.465 mmol, 1 eq.) in THF (4 mL) was added. The reaction mixture was refluxed overnight and was then cooled and quenched with water (10 mL). The resulting mixture was extracted with methylene chloride (2 x 5 mL). The combined extracts were washed with water (10 mL), dried over MgSO₄, and concentrated under reduce pressure. This crude mixture was purified by flash column chromatography on silica gel (cyclohexane/EtOAc: 1/1 ) to give a mixture of the product and the SM. Et₂O was added in the mixture, the solution was filtered. The residue collected was the desired product (64 mg, 40% yield) as slightly yellow solid.

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.07 (d, J = 8.7 Hz, 1 H), 7.90 (d, J = 8.9 Hz, 1 H), 7.87 (s, 1 H), 7.70 (t, J = 7.0 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.41 (t, J = 7.5 Hz, 1 H), 7.20 (s, 1 H), 7.16 (d, J = 8.7 Hz, 1 H), 5.72 (s, 1 H), 4.01 (s, 3H), 2.79 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 158.9, 156.9, 154.5, 148.5, 139.7, 144.7, 134.2, 130.3, 129.7, 128.6, 127.0, 126.2, 124.7, 123.8, 122.3, 1 16.7, 1 14.1 , 99.4, 57.0, 25.5.

[00140] To a round necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 mL) and water (0.8 mL). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μί) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of Celite was washed with EtOAc (3 x 20 ml_). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

1H NMR (300 MHz, CDCI₃) δ [ppm] = 8.05 (d, J = 8.5 Hz, 1 H), 7.68-7.59 (m, 2H), 7.37 (t, J = 8.3 Hz, 1 H), 7.18 (s, 1 H), 6.89 (dd, J = 8.3 and 2.3 Hz, 1 H), 6.89 (d, J = 2.3 Hz, 1 H), 6.75 (d, J = 8.5 Hz, 1 H), 5.49 (s, 1 H), 3.87 (s, 3H), 3.85 (br s, 2H), 2.77 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 159.5, 158.7, 149.4, 148.3, 146.6, 136.6, 134.5, 129.9, 129.3, 126.5, 125.4, 124.5, 122.1 , 120.1 , 1 17.7, 1 14.6, 1 10.2, 96.0, 55.7, 25.5.

[00141 ] To a stirred solution of diethylcyanomethylphosphonate (83 μΙ_, 0.52 mmol, 1 .1 eq.) in THF (3.1 ml_) was added a 1 M solution of LiHMDS in THF (520 μΙ_, 0.52 mmol, 1 .1 eq.) at 0°C. The solution was allowed to warm to room temperature and was stirred for 30 min, and then a mixture of (4-methoxy-3-nitrophenyl)(2- methylquinolin-4-yl)methanone (150 mg, 0.465 mmol, 1 eq.) in THF (4 ml_) was added. The reaction mixture was refluxed overnight and was then cooled and quenched with water (10 ml_). The resulting mixture was extracted with methylene chloride (2 x 5 ml_). The combined extracts were washed with water (10 ml_), dried over MgSO₄, and concentrated under reduce pressure. This crude mixture was purified by flash column chromatography on silica gel (cyclohexane/EtOAc: 1/1 ) to give a mixture of the product and the SM. Et₂O was added in the mixture, the solution was filtered. The residue collected was the desired product (64 mg, 40% yield) as slightly yellow solid.

[00142] To a round necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 ml_) and water (0.8 ml_). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μΙ_) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of celite was washed with EtOAc (3 x 20 mL). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

Product Name: ICQO-6

Overall Yield: 6%

Molecular formula: C20H17N3O

Molecular weight: 315.37 g/mol

Example 7: Generation and synthesis of the toxin unit ICQO-9

[00143] The toxic unit ICQO-9 was synthesised according the following procedure. In a dried round bottom flask was placed 4-Hydroxyacetophenone (5.5 g, 40 mmol, 1 eq.) in dry DMF (80 ml). K₂CO₃ (18 g, 130 mmol, 3.25 eq.) was added and the suspension was stirred for 10 min. Ethyliodide (4.8 ml, 60 mmol, 1 .5 eq.) was added drop wise. After stirring for 16 h, water (100 ml) and ethyl acetate (500 ml) were added. The aqueous phase was separated from the organic layer, which was washed with water (12 x 250 ml), dried over MgSO₄ and concentrated to afford the desired product (5.31 g, 81 %).

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.92 (d, J = 9.8 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.09 (q, J = 7.0 Hz, 2H), 2.54 (s, 3H), 1 .43 (t, J = 7.0 Hz, 3H).

[00144] In to a round bottom flask was placed 4-ethoxyacetophenone (5.31 g, 32.3 mmol) in acetic anhydride (1 1 mL). 80% fuming HNO3 (7.9 mL) was added drop wise at 0°C. After stirring for 1 h, the reaction mixture was poured into ice water (250 mL). The organic compound was extracted with EtOAc (2 x 100 mL). The combined organic layer was washed with brine and dried over MgSO₄, filtrated and concentrated under reduce pressure. The residue was purified by flash column chromatography on silica gel (cyclohexane/EtOAc: 9/1 ) to afford the desired product (3.95 g, 58%).

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.4 (d, J = 2.1 Hz, 1 H), 8.12 (dd, J = 8.7 and 2.3 Hz, 2H) 7.13 (d, J = 8.9 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 2.59 (s, 3H), 1 .50 (t, J = 7.0 Hz, 3H).

[00145] In a round bottom flask (Argon, Stirrer) was placed 1 -(2-methylquinolin-4- yl)ethan-1 -one (432 mg, 2.33 mmol, 1 eq.) in MeOH (75 mL). Tosyl hydrazide (520 mg, 2.79 mmol, 1 .2 eq.) was added in one portion. The reaction mixture was stirred at RT (overnight or more) and followed by TLC. The mixture concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (DCM/EtOAc: 7/3 or cyclohexane/EtOAc: 1/1 ) on silica gel to afford the desired compound (1 .6 g, 67%) as slightly orange solid.

¹H NMR (200 MHz, CDCI₃): δ [ppm] = 8.17 (br s, NH), 8.01 (d, J =9.2, 1 H), 7.90 (d, J = 8.1 , 2H), 7.80 (d, J = 7.2 Hz, 1 H), 7.66 (t, J = 6.8 Hz, 1 H), 7.39-7.31 (m, 3H), 7.14 (s, 1 H), 2.72 (s, 3H), 2.45 (s, 3H), 2.30 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 158.4, 151 .7, 144.6, 144.5, 138.5, 135.4, 129.9 (2C), 129.6, 128.9, 128.4 (2C), 126.4, 125.5, 123.4, 121 .1 , 25.2, 21 .8, 17.6.

[00146] In a round bottom flask (Argon, stirrer) equipped with a reflux apparatus was placed 1 -(4-ethoxy-3-nitrophenyl)ethan-1 -one (3.91 g, 18.69 mmol, 1 eq.) in EtOH (200 mL). Tosyl hydrazide (3.828 g, 20.56 mmol, 1 .1 eq.) was added in one portion. The reaction mixture was warmed at reflux (1 10°C) (yellow solution). The solution was stirred for 6.5 h and followed by TLC (reaction not completed = equilibrium). The mixture was cooled at RT and filtered using a funnel apparatus. The residue was collected as a slightly yellow solid and was recrystallized in EtOH to afford the desired product. The filtrate was concentrated and was purified by flash column chromatography on silica gel (100% DCM) to afford the desired product (m = 3.64 g, 52%).

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.04 (d, , J = 2.13 Hz, 1 H), 7.90 (d, J = 8.32 Hz, 1 H), 7.82 (dd, J = 8.8 and 2.3 Hz, 2H), 7.71 (s, 1 H), 7.34 (d, J = 7.8 Hz, 2H), 7.03 (d, J = 8.3 Hz, 2H), 4.20 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H), 2.14 (s, 3H), 1 .48 (t, J = 7.1 Hz, 3H).

[00147] The palladium-catalyzed cross coupling reaction between (E)-4-methyl-N'- (1 -(2-methylquinolin-4-yl)ethylidene)benzenesulfonohydrazide 4 and 4- chloromethylquinoline afforded the coupling product in 69% yield.

1H NMR (300 MHz, CDCI₃) δ [ppm] = 8.07 (d, J = 8.5 Hz, 1 H), 7.85 (d, J = 2.5 Hz, 1 H), 7.62 (dt, J = 9.8 and 1 .7 Hz, 2H), 7.36 (t, J = 8.3 Hz, 1 H), 7.28 (dd, J = 8.7 and 2.1 Hz, 1 H), 7.23 (s, 1 H), 6.93 (d, J = 8.7 Hz, 1 H), 5.99 (s, 1 H), 5.45 (s, 1 H), 4.15 (t, J = 7.0 Hz, 2H), 2.78 (s, 3H), 1 .45 (t, J = 7.0 Hz, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 159.0, 148.4, 144.0, 132.4, 132.2, 129.7, 129.3, 128.1 , 125.7, 125.1 , 123.2, 122.7, 1 17.6, 1 14.6, 65.7, 25.5, 14.7. (3 carbons missing).

[00148] To a round necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 mL) and water (0.8 mL). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μί) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of Celite was washed with EtOAc (3 x 20 mL). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

1H NMR (300 MHz, CDCI₃) δ [ppm] = 8.02 (d, J = 8.5 Hz, 1 H), 7.75 (dt, J = 6.8 and 1 .5 Hz, 1 H), 7.62 (t, J = 8.5 Hz, 1 H), 7.32 (dt, J = 6.8 and 1 .5 Hz, 1 H), 7.21 (s, 1 H), 6.70-6.57 (m, 3H), 5.85 (s, 1 H), 5.24 (s, 1 H), 4.01 (t, J = 7.0 Hz, 1 H), 3.76 (br s, 2H), 2.75 (s, 3H), 1 .41 (t, J = 7.0 Hz, 1 H). ¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 158.8, 149.2, 148.2, 146.9, 146.1 , 136.3, 132.9, 129.4, 128.8, 126.3, 125.7, 125.6, 122.5, 1 17.3, 1 14.9, 1 13.2, 1 1 1 .1 , 55.6, 25.4, 15.0.

Product Name: ICQO-9

Overall Yield: 13%

Molecular formula: C20H20N2O

Molecular weight: 304.39 g/mol

Example 8: Generation and synthesis of the toxin unit ICQO-11

[00149] The synthesis of the toxic unit ICQO-11 was performed according to the following process. In a sealed tube and under an argon atmosphere were added successively, ArCI (0.5 mmol, 1 eq.), hydrazone (0.75 mmol, 1 .5 eq.) PdCl2(CH₃CN)₂ (10 mol%), dppf (20 mol%) in dry dioxane (4 mL) and the mixture was stirred for 5 min. at RT. Then dry LiOfBu (1 .25 mmol, 2.5 eq.) was added and the mixture was stirred at 120° C for 3 h. The resulting suspension was cooled to room temperature, filtered through a pad of Celite eluting with ethyl acetate and the inorganic salts were removed. The filtrate was concentrated and the crude was purified by flash column chromatography on silica gel to afford the desired product.

¹H NMR (300 MHz, CDCI₃): δ [ppm] = 7.86 (s, 1 H), 7.51 -7.47 (m, 2H), 7.32 (d, J = 8.7 Hz, 1 H), 7.25 (d, J = 8.3 Hz, 1 H), 7.20 (s, 1 H), 6.95 (d, J = 9.6 Hz, 1 H), 5.98 (s, 1 H), 5.43 (s, 1 H), 3.92 (s, 3H), 2.83 (s, 3H), 2.78 (s, 3H).

¹³C NMR (75 MHz, CDCI3): δ [ppm] = 157.7, 152.6, 147.5, 147.1 , 144.2, 140.0, 137.2, 132.8, 132.3, 129.7, 125.6, 124.9, 123.6, 123.3, 122.3, 1 17.4, 1 13.6, 56.7, 25.8, 18.5. [00150] To a round necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 mL) and water (0.8 mL). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μί) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of celite was washed with EtOAc (3 x 20 mL). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

1H NMR (300 MHz, CDCI₃): δ [ppm] = 7.61 (d, J = 8.3 Hz, 1 H), 7.47 (d, J = 7.2 Hz, 1 H), 7.21 (t, J = 7.5 Hz, 1 H), 7.19 (s, 1 H), 6.68-6.60 (m, 3H), 5.85 (s, 1 H), 5.22 (s, 1 H), 3.82 (s, 3H), 3.72 (br s, 2H, NH2), 2.82 (s, 3H), 2.76 (s, 3H).

1³C NMR (75 MHz, CDCI₃): δ [ppm] = 157.6, 149.1 , 147.5, 147.4, 146.5, 136.6, 136.1 , 133.3, 129.4, 125.6, 125.2, 124.3, 122.2, 1 17.4, 1 14.7, 1 13.2, 1 10.2, 56.6, 25.8, 18.5.

Product Name: ICQO-11

Overall Yield: 26%

Molecular formula: C20H20N2O

Molecular weight: 304.39 g/mol

Example 8: Generation and synthesis of the toxin unit ICQO-12

[00151 ] The toxic unit ICQO-12 was synthesised as follows. First, in round bottom flask, dried and flushed with argon, was placed 4-chloro-2-methylquinoline (2.329 g, 13.1 1 mmol, 1 eq.) in 23 mL of THF was added 4 M HCI in 1 ,4-dioxane (3.6 mL, 14.42 mmol, 1 .1 eq.). After 5 min, the solvent was removed and the precipitate was dried under reduced pressure. The hydrochloride salt and Nal previously dried at 120°C under reduce pressure (9.82 g, 65.55 mmol, 5 eq.) were suspended in 70 mL of anhydrous acetonitrile and refluxed for 24 h. After this mixture was cooled to room temperature, 100 mL of an aqueous solution of 10% K2CO3 and 5% NaHSO3 was added. After the mixture was extracted with CH2CI2 twice, the combined organic layers were dried over MgSO₄, filtered and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (100:20:1 cyclohexane/EtOAc/triethylamine) to afford the desired product (3.21 g, 91 %) as white crystals.

¹H NMR (300 MHz, CDCI₃) δ [ppm] = 7.98 (dd, J = 8.2 and 1 .4 Hz, 1 H), 7.94 (dd, J = 8.4 and 1 .4 Hz, 1 H), 7.91 (s, 1 H), 7.70 (ddd, J = 16.9, 8.4, and 1 .4 Hz, 1 H), 7.55 (ddd, J = 8.2 6.9, and 1 .4 Hz, 1 H), 2.69 (s, 3H).

[00152] In round bottom flask, dried and flushed with argon, was placed 4-iodo-2- methylquinoline (296 mg, 1 .1 mmol, 1 .1 eq.) in 5 mL of anhydrous THF was cooled to -78°C under nitrogen. To the solution was added 1 .3 M iPrMgCI LiCI in THF (0.92 mL, 1 .2 mmol, 1 .2 eq.) dropwise. After 30 min, the magnesium solution was added to a solution of 4-methoxy-3-nitrobenzaldehyde (232 mg, 1 mmol, 1 eq.) in THF (5 mL) at -78°C. The resulting mixture was allowed to come to room temperature, stirred for 5 h, quenched with NH CI, and extracted with EtOAc. The combined organic layers were dried over MgSO₄, filtered and concentrated under reduce pressure. The crude mixture was purified by flash column chromatography on silica gel (DCM/EtOAc/NEts: 6/4/0.1 ) to afford the desired product (483 mg, 75%).

1H NMR (300 MHz, CDCI₃): δ [ppm] = 8.00 (d, J = 8.7 Hz, 1 H), 7.90 (d, J = 2.4 Hz, 1 H), 7.80 (d, J = 9.0 Hz, 1 H), 7.63 (d, J = 7.6 Hz, 1 H), 7.51 (s, 1 H), 7.47-7.36 (m, 2H), 6.98 (d, J = 8.9 Hz, 1 H), 6.43 (s, 1 H), 3.91 (s, 3H), 3.56 (br s, OH), 2.72 (s, 3H). ¹³C NMR (75 MHz, CDCI3): δ [ppm] = 159.2, 152.8, 148.3, 147.4, 141 .7, 134.6, 132.8, 129.6, 129.5, 126.2, 124.6, 123.7, 123.4, 1 19.6, 1 13.9, 71 .5, 56.8, 25.6.

[00153] The alcohol (440 mg, 1 .356 mmol, 1 eq.) was diluted in dried DCM (30 mL). Then, PCC (671 mg, 3.12 mmol, 2.3 eq.) was added at 0°C and stirred at RT for 2 h. The reaction was filtered through a pad of Celite, and the crude was concentrated under reduce pressure. The crude mixture was purified by flash column chromatography on silica gel (DCM/EtOAc: 6/4) to afford the desired product (284 mg, 65%). ¹H NMR (300 MHz, CDCI₃): δ [ppm] = 8.31 (s, 1 H), 8.10 (d, J = 8.1 Hz, 1 H), 8.01 (d, J = 8.3 Hz, 1 H), 7.71 (d, J = 7.9 Hz, 2H), 7.46 (t, J = 7.6 Hz, 1 H), 7.26 (s, 1 H), 7.14 (d, J = 8.9 Hz, 1 H), 4.03 (s, 3H), 2.79 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 193.2, 158.5, 157.0, 148.4, 143.6, 139.8,

136.0, 130.4, 129.5, 129.2, 127.8, 127.1 , 123.0, 120.3, 1 13.6, 71 .5, 57.2, 25.6.

[00154] To a round necked bottom flask equipped with a reflux apparatus was placed nitro derivative (0.326 mmol, 1 eq.) in EtOH (3.2 mL) and water (0.8 mL). Iron powder (3.26 mmol, 10 eq.) and concentrated hydrochloric acid (50 μί) were added. The mixture was heated at reflux for 90 min and followed by TLC DCM/EtOAc (7/3). The mixture was cooled at RT and filtered through a pad of Celite. The pad of celite was washed with EtOAc (3 x 20 mL). The crude mixture was purified by flash column chromatography on silica gel (DCM/Et₂O (8/2) to afford the desired product.

1H NMR (300 MHz, CDCI₃): δ [ppm] = 8.07 (d, J = 8.5 Hz, 1 H), 7.75 (d, J = 8.3 Hz, 1 H), 7.70 (td, J = 8.1 and 1 .1 Hz, 1 H), 7.43 (t, J = 7.6 Hz, 1 H), 7.31 (d, J = 2.1 Hz, 1 H), 7.26 (s, 1 H), 7.14 (dd, J = 8.3 and 2.3 Hz, 1 H), 6.75 (d, J = 8.5 Hz, 1 H), 3.96 (br s, NH2), 3.91 (s, 3H), 2.78 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ [ppm] = 195.4, 158.4, 152.3, 148.2, 145.9, 136.7,

130.1 , 130.0, 129.1 , 126.6, 125.5, 123.6, 123.5, 120.1 , 1 15.0, 109.5, 55.9, 25.5.

Product Name: ICQO-12

Overall Yield: 10%

Molecular formula: C18H16N202

Molecular weight: 292.34 g/mol

Example 9: Generation of maleimide-valine-citrulline-PAB-ICQO-1 payload conjugated monoclonal antibody trastuzumab [00155] A two phase of mixture of ICQO-1 (30 mg, 103.3 μιτιοΙ) in dichloromethane (10.1 mL) and saturated aqueous sodium hydrogen carbonate (10.1 mL) was cooled to 0°C. A solution of 20 % phosgene in toluene (1 .5 mol eq.) was quickly added to the organic layer under N₂ and the reaction mixture was stirred vigorously for 1 h at room temperature. The layers were separated and the aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic layers were dried (MgSO₄) and concentrated to five the isocyanate as oil. This oil was dissolved in anhydrous DMF (2.25 mL), Fmoc-Val-Cit-PAB (1 .3 mol eq.) was added and the mixture was stirred overnight at room temperature under N₂. The resulting mixture was dry- loaded onto silica and the purified using automated normal phase chromatography eluting with a gradient of 4 to 32 % methanol in dichloromethane gave 28 mg (30 %) of Fmoc-Val- Cit-PAB-ICQO-1 .

[00156] Fmoc removal was achieved using diethylamine (67 mol eq.) to give 21 .5 mg of NH₂-Val-Cit- PAB-ICQO-1 in quantitative yield along with the fluorenyl residue at 8.6 min.

[00157] This material was coupled to Mal-Hex-OSu (1 .5 mol eq.). Purification using automated normal phase chromatography eluting with a gradient of 2 to 40 % methanol in dichloromethane gave 1 1 mg (40 %) of Mai -Hex- Val-Cit-PAB- ICQO-1 .

[00158] The conditions used for Mal-Val-Cit-PAB-ICQO-1 conjugation to Herceptin (trastuzumab): Partially reduced Herceptin (55 μΜ), Mal-Val-Cit- PAB-ICQO-1 (0.55 mM, 10 eq.) and DMSO (30 % v/v) at 25 ^°C for 18 hours. ADC was purified using 3 sequential desalting steps (PD-10 columns). ADCs were analyzed by HIC to estimate DAR: Herceptin-Mal-Val-Cit-PAB-ICQO-1 has a DAR 2.0.

Example 10: In cellulo cytotoxicity assay with ICQO derivatives on a free format against cancer cell lines

[00159] Cytotoxicity of ICQO derivatives was investigated using cell lines representing a broad spectrum of indications where there is still a high unmet medical need and where ADCs could be developed with ICQO payloads.

K562 - Chronic myelogenous leukemia cell line (Blood cancer) HCT1 16 - Colorectal carcinoma

A549 - Adenocarcinomic human alveolar basal epithelial cells (Non Small Cell Lung Cancer)

PC3 - Prostatic adenocarcinoma cell lines (Prostate Cancer)

MDA-MB-231 - Human metastatic adenocarcinoma (Breast Cancer)

[00160] For these studies, the cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cell viability was determined by an MTS assay according to the manufacturer's instructions (Promega, Madison, Wl, USA). The cells were counted using a Vi-cell XR (Beckman Coulter) and their viability assessed by 0,25% trypan blue dye exclusion. For IC₅o determination, the cells were seeded in 96-well plates (3 10³ cells/well) containing 100 μΙ_ of growth medium. After 24 h of culture, the cells were treated with the tested compounds at 10 different final concentrations (10^"7, 5x10^"8, 10^"8, 5x10^"9, 10^"9, 5x10^"10, 10^"10, 5x10^"11, 10^"11, 5x10^"12 M). Each concentration was obtained from serial dilutions in culture medium starting from the stock solution. Control cells were treated with the vehicle. Experiments were performed in triplicate. After 72 h of incubation, 20 μί of CellTiter 96^® AQueous One Solution Reagent was added for 2 h before recording absorbance at 490nm with a spectrophotometric plate reader Softmax 384 plus (Molecular devices Sunnyvale, California). The dose-response curves were plotted with Graph Prism software and the IC₅o values were calculated using the Graph Prism software from polynomial curves (four or five-parameter logistic equations).

IC₅0 in pM (picomolar)

[00161 ] Results indicate subnanomolar cytotoxicity against different type of cancer cell lines (solid and liquid tumors). This is a key criterion for a toxic payload. Example 11 : Comparison of IC₅₀ values of ICQO derivatives vs IC₅₀ value of MMAE on a free format against cancer cell lines

[00162] Approach: Ratio between the IC₅o value of MMAE and the IC₅o value of ICQO. If the ratio is above 1 then it means that the level of cytotoxicity of the respective ICQO derivatives is higher than for MMAE. The higher the ratio, the stronger is the therapeutic improvement and potential for commercial success.

A549 Adenocarcinomic human alveolar basal epithelial cells (Non Small Cell Lung Cancer)

PC3 - Prostatic adenocarcinoma cell lines (Prostate Cancer)

Mia-Paca2 - Human pancreatic adenocarcinoma (Pancreatic Cancer)

[00163] Results indicate that ICQO derivatives are showing a significant advantage compared to MMAE in terms of potency. In the case of PC3 for instance ICQO-10 is 16.9 times more potent than MMAE.

Example 12: Comparison of IC₅₀ ICQO derivatives vs closest prior art on a free format against cancer cell lines

[00164] Approach: Ratio between the IC₅o value of 1 D (or 1 B) and the IC₅o value of ICQO. If the ratio is above 1 then it means that the level of cytotoxicity of ICQO derivatives is higher than for the respective prior art reference compound. The higher the ratio, the stronger is the therapeutic improvement.

K562 - Chronic myelogenous leukemia cell line (Blood cancer)

PC3 - Prostatic adenocarcinoma cell lines (Prostate Cancer)

HCT1 16 - Colorectal carcinoma

A549 Adenocarcinomic human alveolar basal epithelial cells (Non Small Cell Lung Cancer) Ratio vs Compound 1 D K562 PC3

ICQO-1 4 9

ICQO-7 2 16

ICQO-9 15 2

ICQO-10 ND 32

[00165] The skilled person in the art would appreciate that for such level of cytotoxicity, improving the cytotoxicity from the nanomolar level down to the subnanomolar level is extremely cumbersome. Considering the structure similarity between compound 1 D and ICQO derivatives, this improvement ranging from a factor 2 to 32 (ICQO-10 in PC3) could be considered by the skilled person as completely unexpected and surprising.

[00166] All in all, ratios indicate that ICQO derivatives are showing a significant advantage compared to compound 1 D in terms of potency.

[00167] The skilled person in the art would appreciate that for such level of cytotoxicity, improving the cytotoxicity from the nanomolar level down to the subnanomolar level is extremely cumbersome. Considering the structure similarity between compound 1 B and ICQO derivatives, this improvement ranging from a factor 5 to 98 (ICQO-10 in PC3) could be considered by the skilled person as completely unexpected and surprising.

[00168] All in all, ratios indicate that ICQO derivatives are showing a significant advantage compared to compound 1 B in terms of potency against other type of cell lines. In addition, ICQO structures bearing a primary amine would also largely facilitate the conjugation process compared to compound 1 B and 1 D. Example 13: Comparison of the IC₅₀ values of ICQO derivatives vs MMAE and closest prior art [1 D] on a free format against resistant cancer cell line (MDR effect)

[00169] K562 R - Chronic myelogenous leukemia cell line with overexpressed MDR profile (resistant to various chemotherapeutics and payloads including MMAE)

[00170] A skilled person in the field of payloads and drug conjugates would acknowledge that an IC₅o of 35 nM for MMAE is not anymore sufficient to be used as a payload. A minimum acceptable level would be subnanomolar level of cytotoxicity which is the case for ICQO-8. In addition, considering the structure similarity between compound 1 D and ICQO derivatives, the 3.15 fold cytotoxicity improvement from 1 D to ICQO-8 could be considered by the skilled person as completely unexpected and surprising.

Example 14: In cellulo cytotoxicity assay with Trastuzumab-Mal-VC-PAB-ICQO- 1

[00171 ] Cytotoxicity of Trastuzumab-Mal-VC-PAB-ICQO-1 was investigated using SKBR3, human breast cancer cell line that overexpresses the Her2. Trastuzumab (Herceptin, Roche) has been used a positive control for the experiment. For this, the cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cell viability was determined by an MTS assay according to the manufacturer's instructions (Promega, Madison, Wl, USA). The cells were counted using a Vi-cell XR (Beckman Coulter) and their viability assessed by 0.25% trypan blue dye exclusion. For IC₅o determination, the cells were seeded in 96-well plates (3 10³ cells/well) containing 100 μΙ_ of growth medium. After 24 h of culture, the cells were treated with the tested compounds at 10 different final concentrations (5x10^"1, 10^"1, 5x10^"2, 10^"2, 5x10^"3, 10^"3, 5x10^"4, 10^"4, 5x10^"5, 10^"5 pg/ml). Each concentration was obtained from serial dilutions in culture medium starting from the stock solution. Control cells were treated with the vehicle. Experiments were performed in triplicate. After 72 h of incubation, 20 μΙ_ of CellTiter 96® AQueous One Solution Reagent was added for 2 h before recording absorbance at 490nm with a spectrophotometric plate reader Softmax 384 plus (Molecular devices Sunnyvale, California). The dose- response curves were plotted with Graph Prism software and the IC₅o values were calculated using the Graph Prism software from polynomial curves (four or five- parameter logistic equations). Trastuzumab-VC-PAB-ICQO-1 resulted in an IC₅o of 27 pg/mL against SKBR3 cell line.

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Claims

1 . A compound according to Formula I

I wherein:

R¹ is selected from CH₃, CN, CONH₂, CONHR^*, CH_xX_y, OR^*, NH₂, NHR^*, NR*2 and H,

R² is selected from H, CH₃, CN, F, CI and OR^**, wherein R^** is a Ci_-5-alkyl; R³ is selected from H, CH₃, CN, F, CI and OR^*, wherein R^* is selected from Ci-5-alkyl and CF₃;

R⁶ is H;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH₃, SCH₃, SCH₂CH₃ and OCHF₂.

2. A compound according to Formula II

II wherein:

R² is selected from H, CH₃, CN, F, CI and OR^**, wherein R^** is a Ci_-5-alkyl R³ is selected from H, CH₃, CN, F, CI and OR^*, wherein R^* is selected from Ci-5-alkyl and CF₃;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH₃, SCH₃, SCH₂CH₃ and OCHF₂.

3. A compound according to Formula III

III wherein:

R⁶ is L^*-TM, wherein L^* is a linker, particularly a self-immolative linker, and

TM is a targeting moiety, particularly a target-binding antibody or functional antigen-binding fragment thereof;

R^6' is selected from H, COR^* and COOR^*; and

R⁷ is selected from OCH₃, OCH₂CH₃, SCH₃, SCH₂CH₃ and OCHF₂.

4. A method of synthesizing a compound according to claim 2, comprising the step of reacting a compound according to claim 1 via the amino group attached to the phenyl ring with a compound X-L'-RM^*, wherein

L' is a linker;

5. The method according to claim 4, wherein RM^* is RM', further comprising the deprotection of the moiety RM' to result in RM.

6. A method of synthesizing a compound according to claim 3, comprising the step of reacting a compound according to claim 2 with a targeting moiety.

7. A pharmaceutical composition comprising the compound according to claim 3.

8. The pharmaceutical composition according to claim 7 for use in the treatment of cancer.

9. A method for the treatment of cancer comprising the step of administering the compound according to claim 3, or the pharmaceutical composition according to claim 7 or 8, to a patient in need of such treatment.