WO2018146166A1 - Heterobifunctional linkers for modifying thiols - Google Patents

Abstract

The invention relates to certain compounds of formula (I) (I) wherein Y is an electron-withdrawing group; Q is (formula) Z is a leaving group; J is NR5, with R5 being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group. R1 to R4 optional substituents and m is an integer between 1 and 12; n is an integer between 0 and 4; o is between 0 and 4. These compounds are useful as linkers for realisably joining a targeting moiety such as an antibody to a "cargo" compound such as a drug, particularly an anti-cancer drug. The invention also relates to conjugates of a targeting moiety and a drug utilising the linker, to methods of synthesis of the linker and to processes for the production of the conjugate. The use of the conjugate in the treatment of disease such as cancer is also disclosed.

Description

HETEROBIFUNCTIONAL LINKERS FOR MODIFYING THIOLS

Field of the invention

The invention generally relates to medical and/or diagnostic conjugates, their synthesis and use. The invention comprises of a set of heterobifunctional linkers that can be used in the synthesis of conjugates where the linkage between a cargo molecule and a carrier molecule is required to be transient of nature.

Background of the invention

Cancer is a group of diseases that will affect one in three people in their lifetime. Many cancer treatments are currently available and they range from local (surgery, radiation, etc.) to systemic (chemotherapy, etc.) treatment, each with their own advantages and disadvantages.

Cytotoxic drugs used in the treatment of solid tumors and hematological malignancies have, over the last 50 years, seen much improvement. However, the non-specificity and the extreme toxicity of some of these drugs limit their usability, and this has led to discontinuation of some of these drugs.

Great emphasis has therefore been put on increasing the therapeutic index of anticancer drugs. The therapeutic index of a drug can be defined as the ratio between the toxic dose and the therapeutic dose of a drug, used as a measure of the relative safety of the drug for a particular treatment. It is well-known that the therapeutic index of anticancer drugs can be improved by temporary chemical modifications. It has been shown that the cytotoxicity of these drugs can be reduced or eliminated by attaching special chemical entities (protective groups) to the functional groups causing toxicity. These temporarily modified and inactivated drugs are often referred to as prodrugs.

A prodrug is a medication or compound that, after administration, is metabolized (i.e., converted within the body) into a pharmacologically active drug. Prodrugs contain specialized nontoxic protective groups, known as promoieties, used in a transient manner to alter or eliminate undesirable properties in the parent molecule, see Figure 1.

After parenteral administration (e.g. intravenous injection), the inactive prodrug will be distributed or transported throughout the body without killing healthy cells. However, once the prodrug reaches its target, the temporary modifications are removed by biochemical transformations within the tumor, thus fully restoring the toxicity of the anticancer drug and resulting in the selective killing of cancer cells. This strategy has already been validated, and currently there are several anticancer prodrugs in clinical use. (1)

Several different anticancer prodrug strategies based on tumor-selective activation exist. These include selective enzyme expression in tumor cells, tumor hypoxia, therapeutic radiation, pH differences, tumor-specific antigens, etc. The design of the prodrug will greatly depend on the targeting strategy that is chosen. When targeting pH differences and antigens associated with tumors, a tripartite prodrug approach is often taken. A tripartite prodrug usually comprises of a carrier connected to a drug (cargo) through a linker, see Figure 2.

A carrier can be defined as a chemical entity (e.g. antibody, nanoparticle, peptide) that can be used to transport another compound to its target to improve its bioavailability and kinetics. The two types of carriers which have received most attention, are monoclonal antibodies and tumor-homing peptides. Monoclonal antibodies (mAbs) bind selectively to overexpressed tumor-antigens (e.g. CD20). Numerous tumor-homing peptides have been discovered in recent years by using phage display. These peptides are very often ligands for overexpressed receptors on cancer cells (e.g. α_νβ₃ receptor).

A linker can be defined as a chemical entity that covalently joins two separate compounds (e.g. a carrier molecule and a cargo molecule) in a permanent or non-permanent manner.

Permanent linkers are stable towards enzymatic, oxidation, reduction, hydrolysis and other biochemical reactions and ensure that the carrier and the cargo remains conjugated under physiological conditions (see Figure 3A). Examples include maleimide/amide crosslinkers.

In contrast, cleavable linkers are sensitive to enzymatic, oxidation, reduction, hydrolysis or other biochemical transformations. Conditions associated with cancer or tumor cells, such as low pH, hypoxia, or overexpression of proteolytic enzymes facilitate catalysis or trigger events that lead to dissociation of the carrier and drug entities (see Figure 3B) at target sites. These linkers are normally sensitive to low pH (e.g. hydrazone, czs-aconityl, acetal), enzymatic cleavage (e.g. Val-Cit sequence), or thiols. Examples of linkers sensitive to thiols include disulfide linkers.

Self-eliminating linkers constitute a subgroup of the cleavable linkers that are designed to yield the native cargo molecule following a trigger event. As with normal cleavable linkers, the trigger event can be low pH or enzymatic cleavage by thiolysis, but the initial trigger event is followed by a subsequent intramolecular reaction which then yields the free native, unmodified cargo, see Figure 3C. Examples include electronic cascades and cyclization.

This type of linker would be essential for conjugates where the desired activity of the cargo depends on the cargo molecule being completely unmodified. Examples of cargo molecules that require complete liberation from chemical modifications include the anticancer drugs camptothecin, paclitaxel, and doxorubicin. Luciferin is an example of a reporter cargo that requires full liberation.

The cargo can be defined as a chemical entity (e.g. a bioactive agent, a diagnostic agent, a visualization agent) that is to be delivered at a specific target site (e.g. cells, neoplasm).

An example of tripartite prodrug is antibody-drug conjugates (ADC). Such prodrugs combine the unique targeting capabilities of mAbs with the specific cancer-killing ability of cytotoxic drugs. By attaching biologically active chemotherapeutic drugs or cytotoxins via chemical linkers with labile bonds to a monoclonal antibody directed to overexpressed antigens in tumor cells, ADCs significantly improve the sensitive discrimination between healthy and diseased tissue. Several studies have shown that linking cytotoxic drugs to tumor- homing peptides improves the selectivity of the drug (2).

An example of FDA approved ADC with permanent linkers is ado-trastuzumab emtansine. In this conjugate the linker is connected with the mAb and the drug via an amide and a thioether bond, respectively (3).

An example of FDA approved ADC with cleavable linker is brentuximab vedotin. this conjugate the linker between the mAb and the drug is cleavable by cathepsin (4).

In ADCs, the cytotoxicity of the cargo can be modulated by masking certain functional groups (i.e. amine or hydroxyl). For example, when the hydroxyl group of camptothecin is masked nearly all cytotoxic activity is lost (5). However, once the ADC with self-eliminating linker reaches its target the temporary modifications are removed by triggering reactions and the toxicity of the anticancer drug can be fully restored.

The invention discloses a series of heterobifunctional cleavable linkers that are self- eliminating, and their synthesis, but not restricted to, conjugates with biomedical applications.

The linkers consist of three components; (i) a self-eliminating linker compound activated with (ii) a thiol- specific activation group, preferably in the carrier molecule, and (iii) a hydroxy 1/amine specific activation group, preferably in the cargo molecule (see Figure 4). It is envisaged that a conjugate will be formed through the reaction of the activated linker with a thiol-containing carrier and a hydroxyl or amine group of a cargo molecule.

Four similar heterobifunctional releasable linker molecules believed used in various applications have been identified in the prior art (see below).

Compound 1 is described in a document (Genentech WO2016/040724, WO2013/55987) where the preparation and characterization of monoclonal antibodies that binds to human B7 homo log 4 protein (B7-H4/VTCN1) is described. The linker is synthesized as a component of ADC, but it is not isolated pure and not fully characterized. The synthesis is performed using a surplus of triphosgene, but the resulting chloroformate is not purified before it is used to link to a drug. Also, unreacted triphosgene, which causes severe skin burns and eye damage, may cause respiratory irritation, is fatal if inhaled, and form deadly phosgene upon decomposition (Acros data sheet for Triphosgene), is not accounted for. Furthermore, without purification and chemical characterization it is impossible to know if the desired compound is the main product of the synthesis. A side reaction yielding a by-product (i.e. a trichloromethyl carbonate) has also been found, (see below).

This by-product is capable of reacting with hydroxyl and amino groups to form a carbonate and carbamate, respectively, in the same way as the chloroformate. It is therefore unclear the extent to which compound 1 is actually provided by the disclosure of WO2016/040724/ WO2013/55987. In one embodiment, applicable to all aspects of the invention, the compounds of and for use in the present invention are preferably not Compound 1.

Compound 2 is described in a document (Spirogen Sari and Genentech Inc., WO2014/159981) related to the preparation of dimeric pyrrolobenzodiazepine immunoconjugates for treatment of proliferative diseases. The compound has not been isolated pure and has not been fully characterized. Again, the extent to which this compound is in fact made available by this document is therefore uncertain.

Compound 3 is described in several documents. With n = 1 it is used in the synthesis of polyglyoxylates and oxoacetate (The University of Western Ontario, WO2015/168809), the preparation of folate receptor binding ligand-drug conjugates (Brightgene Bio-Medical Technology Co. Ltd, WO2015/106599), and the synthesis of epothilone compounds and analogs (Bristol-Myers Squibb Company, WO2007/140298 and WO2008/147941). With n = 1 - 3 it has been applied in DNA sequencing-by-synthesis (Helicos Biosciences Corporation, WO2008/144544), synthesis of conjugates for intracellular delivery (Stanford University, WO2008/069824), and the synthesis of a glutathione-sensitive nanoplatform for monitored intracellular delivery and controlled release of Camptothecin (UPV Instituto de Tecnologia Quimica, RSC Adv., 2013, 3, 15121-15131). The compound has not been isolated pure and has not been fully characterized.

Compound 4 is described in a document related to DNA sequencing-by-synthesis (Helicos Biosciences Corporation, WO2008/144544). The compound has not been isolated pure and has not been fully characterized.

In view of the above, it would be of considerable advantage to provide self-eliminating linkers with a greater molecular diversity that allows for the modulation of drug release kinetics and/or a modulation of the way in which it is activated towards thiol (SH), hydroxyl (OH) and amino (N¾) groups. It would be an advantage if the structure of the linkers enabled the modulation of stability towards cleavage of the linker, whether thio lytic cleavage or amino lysis. It would be a further advantage to provide linkers which are prepared as purified and fully characterized reagents. It would be a further advantage to provide linkers which are more reactive towards thiols and offer superior thiol conjugation. It would be a further advantage to provide linkers of which the leaving groups may be chosen so that they act as reporting compounds that can be used to monitor the progression of the conjugation reactions.

Furthermore, it would be of considerable advantage to provide conjugates which are more stable than some known carbonate-based conjugates (i.e. conjugates formed from a 'mercaptoalcohoP linker and an OH-containing drug). In some cases, conjugates featuring a carbonate linkage release the bioactive agent before its intended destination, thereby reducing the targeting efficiency and/or risking side-effects in some cases. In particular, it would be useful to provide improved linkers for the preparation of conjugates with OH-containing bioactive agents.

Overall it would be an advantage to provide new compounds and technology that can be used to improve the therapeutic index of drugs already approved clinically, in particular anticancer drugs, previously discontinued drugs, or newly discovered cytotoxic agents that hitherto have been considered too toxic for clinical use. SUMMARY OF INVENTION

The present inventors have now established that certain linkers can address some or all of the above issues.

In a first aspect, the invention provides a compound of formula (I)

wherein

Y is an electron- withdrawing group;

Z is a leaving group;

J is O or NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group;

R¹ and R² are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, halogen (e.g. F, CI, Br, I), alkoxy, acyloxy, ester, carboxylic acid, amino, amido, and substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl groups;

R³ and R⁴, where present, are each independently selected for each carbon (i.e. for each value of o) from the group consisting of H, halogen (e.g. F, CI, Br, I), alkoxy, acyloxy, ester, carboxylic acid, amino, amido, and substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl groups;

m is an integer between 1 and 12; n is an integer between 0 and 4; o is between 0 and 4. In a particular embodiment, the compound is not (i?)-2-((5-nitropyridin-2-yl)disulfanyl)propyl chloroformate.

In a further particular embodiment, the invention provides a compound of formula II

wherein Y is one or more nitro groups at the para and/or ortho positions;

R¹ and R² are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, halogens (e.g. F, CI, Br, I), Ci- C₆ alkoxy, Ci- C₆ alkyl, C₂- C₆ alkenyl groups, and phenyl optionally substituted by one or more fluorine atoms; and

Q is as defined for formula I; and m is an integer from 1 to 10.

In a further particular embodiment, the invention provides a compound of formula III

wherein Y, R¹, R² and Q are as defined above, m is and integer of 0 to 10 and each group R^s and R° are independently selected from the group consisting of H and Ci-C₆ alkyl groups, wherein at least one group R^s is a Ci- C₆ alkyl group and/or at least one group R° is a Ci- C₆ alkyl group. In a further particular embodiment, the invention provides a compound selected from the group consisting of:

2- (2-(5-Nitropyridin-2-yl)disulfanyl)ethyl chloroformate

3- (2-(5-Nitropyridin-2-yl)disulfanyl)propyl chloroformate

1 -Methyl-2-(2-(5 -nitropyridin-2-yl)disulfanyl)propyl chloroformate

1 -Methyl-2-(2-(5 -nitropyridin-2-yl)disulfanyl)ethyl chloroformate

4- (2-(5-Nitropyridin-2-yl)disulfanyl)butyl chloroformate

3-(2-(5-Nitropyridin-2-yl)disulfanyl)hexyl chloroformate

3 -Methyl-3 -(2-(5 -nitropyridin-2-yl)disulfanyl)butyl chloroformate

1 ,3 -Dimethyl-3 -(2-(5 -nitropyridin-2-yl)disulfanyl)butyl chloro formate

6-(2-(5-Nitropyridin-2-yl)disulfanyl)hexyl chloroformate

8- (2-(5-Nitropyridin-2-yl)disulfanyl)octyl chloroformate

9- (2-(5-Nitropyridin-2-yl)disulfanyl)nonyl chloroformate

1 l-(2-(5-Nitropyridin-2-yl)disulfanyl)undecyl chloroformate

In a further aspect, the invention provides a process for producing a compound comprising the

reacting a compound of formula (IV)

with a compound of formula (V):

to obtain a compound of formula (VI):

ii) reacting the compound of formula (VI) with phosgene, triphosgene or a compound of formula (VII)

(VII) to obtain a compound of formula (I), (II) or (III) as defined above, wherein m, n and o and the groups Y, Z and R^!-R⁴ are as defined in any of formulae (I), (II) or (III); J is O or NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group; wherein Z=Z' or Z≠Z', and Z' is a leaving group selected from halides, (such as fluoride, chloride, bromide, iodide), alkoxides, 2,5-pyrrolidinedione 1 -oxide, tosylate, mesylate, nitrate, phosphate, phenoxide, /?-nitrophenoxide, o-nitrophenoxide benzotriazoloxide and 2,5-pyrrolidinedione 1 -oxide.

In a further aspect, the invention provides a conjugate of formula (VIII):

wherein R'-R⁴, m, n and o are as defined in formulae (I), (II) or (III); wherein J is O or NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group; wherein A is such that A-SH is a carrier entity such as an antibody, antibody fragment, nanoparticle, protein, protein fragment or peptide; wherein B and W are such that B-WH is a bioactive agent, a diagnostic agent or a visualization agent; and

W = NH or O; or a pharmaceutically acceptable salt, hydrate or solvate thereof .

In a further aspect, the invention provides a process for producing the conjugate of formula (VIII) comprising the steps of: a) reacting a compound of formula as defined above with a bioactive agent, a diagnostic agent or a visualization agent of formula B-WH to obtain a compound of formula IX):

reacting said compound of formula (IX) with the carrier entity of formula A-SH; wherein R¹- R⁴, m, n, o, Y, A, B, W are as defined for the conjugate above. In a further aspect, the invention provides a conjugate as described above for use in the treatment of cancer, preferably wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer and adrenal cancer.

In a further aspect, the invention provides a method of treating cancer comprising administering to an animal, preferably a mammal, e.g. human, an effective amount of a conjugate as described herein, preferably wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer and adrenal cancer.

In a further aspect, the invention provides the use of a conjugate as described herein in the manufacture of a medicament for the treatment of cancer, preferably wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer and adrenal cancer.

The features of the aspects and/or embodiments indicated herein are usable individually and in combination in all aspects and embodiments of the invention where technically viable, unless otherwise indicated.

DETAILED DESCRIPTION OF INVENTION

The present invention discloses a series of pre-synthesized, purified, and fully characterized linker compounds that provide a way of facile conjugation of a carrier to a cargo molecule. The invention offers several advantages over the prior art in terms of ease of conjugate synthesis and drug release kinetics. Enabling technology for the facile synthesis of tripartite conjugates wherein the linker moiety is fully releasable resulting in the liberation of the cargo in its native (i.e. unmodified) form is provided.

Due to an inherent propensity to react with itself, self-eliminating linkers present certain synthetic challenges. In conventional synthesis of ADC prodrugs the challenges are normally circumvented by following laborious, multistep syntheses resulting in low yields, long preparation times, and high production costs. Heterobifunctional and self-eliminating linkers offer an alternative to this approach. By modifying both ends of a linker with compatible activating groups it is possible to fully control the conjugation chemistry, as well as prevent intramolecular cyclization (self-elimination). By simplifying the conjugation chemistry, this approach allows for a higher throughput in tripartite ADC/prodrug synthesis and eventual testing.

Linkers

The linkers of the present invention and their synthesis can be used for conjugating thiol-containing targeting moieties, such as an antibody, nanoparticle, peptide (e.g. a tumor- homing peptide), or peptide fragment to an amine and/or hydroxyl-containing cargo, such as a cytotoxic drug or reporter molecule. The disclosed linkers of formula I are designed to selectively and orthogonally react with molecules containing SH groups and with molecules containing OH or NH₂ groups.

In formula (I) Y is an electron- withdrawing group;

Z is a leaving group;

J is O or NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group.

R¹ and R² are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, halogen (e.g. F, CI, Br, I), alkoxy, ester, carboxylic acid, amino, amido, and substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl groups;

R³ and R⁴, where present, are each independently selected for each carbon (i.e. for each value of o) from the group consisting of H, halogen (e.g. F, CI, Br, I), alkoxy, ester, carboxylic acid, amino, amido, and substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl groups; m is an integer between 1 and 12; n is an integer between 0 and 4; and o is between 0 and 4.

Preferably J is NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group. Preferred linkers and conjugates of the invention are therefore 'mercaptoamine' linkers and conjugates. Typically, R⁵ is hydrogen, a Ci - C₆ alkyl, e.g. methyl or ethyl, or a substituted or unsubstituted aryl group, such as a substituted or unsubsituted phenyl group, such as phenyl. Preferably, R⁵ is a Ci - C₆ alkyl, e.g. methyl or ethyl, or a substituted or unsubstituted aryl group, such as a substituted or unsubsituted phenyl group (e.g. phenyl). It will be clear to the skilled reader that these considerations also apply to the conjugates of the invention and to all technically compatible aspects of the invention.

Preferably n is zero. Typically o will be zero when n is zero. In a particular embodiment, 2 < m + n + o < 12; preferably 2 < m + n + o < 6, more preferably 2 < m + n + o < 4. In a further particular embodiment, 3 < m + n + o < 12; preferably 3 < m + n + o < 6, more preferably 3 < m + n + o < 4. In a particular embodiment, therefore, the linker comprises three or more carbon atoms between the disulfide unit and the Q group. It will be clear to the skilled reader that these considerations also apply to the conjugates of the invention and to all technically compatible aspects of the invention.

The term 'substituted' covers substitution at any position within the relevant group (for example alkyl, alkenyl, alkynyl or aryl) with one or more alkyl groups, such as a Ci-C₆ alkyl group, or one or more halogen atoms, such as F, CI, Br or I, especially F. Preferably, the alkyl, alkenyl or alkynyl groups, if substituted, are substituted with at least one Ci-C₆ alkyl group, and preferably the aryl group, if substituted, is substituted with at least one F atom. A preferred unsubstituted or substituted aryl group is a phenyl group or the same substituted with one or more fluorine atoms respectively.

In the present invention the term 'Hydrogen' or the symbol Ή' encompasses all isotopes of hydrogen such as protium, deuterium and tritium, in particular protium and deuterium. Most typically, hydrogen will comprise, consist essentially of or consist of protium.

Preferably the linker is not (i?)-2-((5-nitropyridin-2-yl)disulfanyl)propyl chloro formate. Preferably the linker is not the compound of formula:

Preferably the electron withdrawing group Y is selected from N0₂, CN, COOH, ester, CONH₂, amide, CX₃ (wherein X is a halogen atom such as F, CI, Br, I), acyl, S0₃H. By ester, acyloxy, amide or acyl groups is meant groups COOR^a, OCOR^a, CONHR^a/CONR^a ₂ or COR^a respectively, wherein R^a is for example a Ci - C₆ alkyl or phenyl optionally substituted by one or more halogen atoms (F, CI, Br, I). Preferably the leaving group is in the ortho or para position, preferably para position. Preferred electron-withdrawing groups are /?ara-N0₂, para-COOH and ort/zo-N0₂, preferably /?ara-N0₂.

The presence of the electron- withdrawing groups is important for thiol conjugation. Pyridine rings without electron-withdrawings groups are not as effective. For example, the following compound:

which appears in WO 2014/086952, is unsuitable to achieve the results of the present invention.

Preferably the leaving group Z is a leaving group selected from halides (such as fluoride, chloride, bromide, iodide), alkoxides, acyloxy, tosylate, mesylate, nitrate, phosphate, phenoxide, /?-nitrophenoxide, o-nitrophenoxide, benzotriazoloxide and 2,5-pyrrolidinedione 1 -oxide. More preferably the leaving group Z is selected from halide (fluoride, chloride, bromide, iodide), /?ara-nitrophenoxide and 2,5-pyrrolidinedione 1 -oxide. Z=C1 constitutes a particularly preferred embodiment of the invention. The leaving group may be chosen such that it acts as a reporting compound that can be used to monitor the progression of the conjugation reactions. In this case, Z is preferably such that Z and/or Z-H contains a chromophore, or a fluorophore, or is a phosphorescent compound, preferably a chromophore. A suitable leaving group containing a chromophore may be /?ara-nitrophenoxide.

R¹ and R² are preferably independently selected from the group consisting of H, F, OR^a, NHR, OCOR^a, COOR^a, COOH, Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, and phenyl optionally substituted by one or more fluorine atoms (R^a is a Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, or phenyl optionally substituted by one or more halogen atoms, preferably R^a is a Ci - C₆ alkyl). In the case of R¹ and/or R² being alkoxy (OR), preferably the alkoxy group is a Ci-C₄ alkoxy group (by C₁-C₄ alkoxy is meant OMe, OEt, 0^;Pr, OⁿPr, OⁿBu, 0¾u, O^sBu, 0¾u). In a preferred embodiment, R¹ and R² are independently H or Ci - C₆ alkyl.

R³ and R⁴ are preferably independently selected from the group consisting of H, F, OR^a, NHR, OCOR^a, COOR^a, COOH, Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, and phenyl optionally substituted by one or more fluorine atoms (R^a is a Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, or phenyl optionally substituted by one or more halogen atoms, preferably R^a is a Ci - C₆ alkyl). In the case of R³ and/or R⁴ being alkoxy (OR), preferably the alkoxy group is a C₁-C₄ alkoxy group (By C₁-C₄ alkoxy is meant OMe, OEt, 0^;Pr, OⁿPr, OⁿBu, 0¾u, O^sBu, 0¾u). In a preferred embodiment, R³ and R⁴ are independently H or Ci - C₆ alkyl.

In the linkers of the present invention, R⁵ is preferably hydrogen, a Ci - C₆ alkyl, or a substituted or unsubstituted aryl group, such as phenyl. In a particular embodiment, R⁵ is not hydrogen.

By Ci-C₆ alkyl is meant methyl (Ci), ethyl (C₂) or any linear or branched C₃-C₆ alkyl group. For example, for C₃ the group may be /? -propyl or isopropyl, for C₄ the group may be n-butyl, isobutyl, sec-butyl or tert-butyl, for C₅ the group may be n-pentyl, 2-methylbutan-2- yl, 2,2-dimethylpropyl, 3-methylbutyl, pentan-2-yl, pentan-3-yl etc. By C₂-C₆ alkenyl group is meant any C=C unit-containing group made up of 2-6 carbon atoms and H atoms. By C₂-C₆ alkynyl group is meant any C≡C unit-containing group made up of 2-6 carbon atoms and H atoms.

The linker of the present invention may be provided as a salt, e.g. a pyridinium salt, a solvate, or a hydrate.

It will be appreciated from the above formula (I) that the linker may be provided as a 'mercaptoalcohol linker', a 'mercaptoamine' linker or a 'mercaptoisocyanate' linker (sometimes shorted herein to isocyanate linker). The 'mercaptoamine' linker and 'mercaptoisocyanate' linker (sometimes shorted herein to isocyanate linker) are particularly preferred. Both the mercaptoalcohol linker and the mercaptoamine linker have the residue Q as

The mercaptoalcohol linker is one in which J = O, and the mercaptoamine linker has J (with R⁵ as defined above). The mercaptoisocyanate linker (or 'isocyanate' linker) is which Q is -NCO. Overall, it is preferred if J = NR⁵.

In one particular embodiment the linker may be of formula (II):

In formula II, Y is preferably a nitro group at the para and/or ortho positions (preferably one nitro group at the para position); R¹ and R² are preferably each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, halogen (e.g. F, CI, Br, I), Ci-C₆ alkoxy, Ci-C₆ alkyl, C₂-C₆ alkenyl groups, and phenyl optionally substituted by one or more fluorine atoms. Q, and the corresponding groups J and Z if appropriate, are as defined for formula I; and m is and integer from 1 to 10, preferably 2-10 (such as 2-6 or 2-4, e.g. 2).

A particular embodiment of the compound of formula (II) is the following compound:

wherein R , R , m, R and Z are as defined above or below. Typically, R and R are, independently and for each value of m, H or a Ci-C₆ alkyl. The value of m is preferably 2-10 (such as 2-6 or 2-4, e.g. 2), Z is preferably CI, and R⁵ is preferably H, a Ci-C₆ alkyl or phenyl.

In a further embodiment of the invention, the linker is of formula (III):

Y, R¹, R², Z and Q are as defined above, m is 0 to 10 and each group R^s and R° are independently selected from the group consisting of H and Ci-C₆ alkyl groups, wherein at least one group R^s is a Ci-C₆ alkyl group and at least one group R° is a Ci-C₆ alkyl group. Substitutions on the carbon adjacent to the sulfur atom allows for the modulation of stability towards thio lytic cleavage. Substitutions on the carbon adjacent to the oxygen atom allow for the modulation of stability towards, for example, hydrolysis and amino lysis of the linker.

Particularly suitable linkers are compounds of formula:

wherein Z and R⁵ are as defined hereinbefore or hereinafter. In particular, the linker could be butyl(2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl)carbamic chloride, or the compound of formula:

In a further embodiment, the linker is preferably selected from the group consisting of:

2- (2-(5-Nitropyridin-2-yl)disulfanyl)ethyl chloro formate

3- (2-(5-Nitropyridin-2-yl)disulfanyl)propyl chloroformate

1 -Methyl-2-(2-(5 -nitropyridin-2-yl)disulfanyl)propyl chloroformate 1 -Methyl-2-(2-(5 -nitropyridin-2-yl)disulfanyl)ethyl chloroformate

4- (2-(5-Nitropyridin-2-yl)disulfanyl)butyl chloroformate 3-(2-(5-Nitropyridin-2-yl)disulfanyl)hexyl chloroformate

3 -Methyl-3 -(2-(5 -nitropyridin-2-yl)disulfanyl)butyl chloroformate 1 ,3 -Dimethyl-3 -(2-(5 -nitropyridin-2-yl)disulfanyl)butyl chloro formate 6-(2-(5-Nitropyridin-2-yl)disulfanyl)hexyl chloroformate

8- (2-(5-Nitropyridin-2-yl)disulfanyl)octyl chloroformate

9- (2-(5-Nitropyridin-2-yl)disulfanyl)nonyl chloroformate

1 l-(2-(5-Nitropyridin-2-yl)disulfanyl)undecyl chloroformate

Typically, the linker units are reacted directly with the drug, without further modification or introduction of a spacer unit. The conjugates of interest in the invention are therefore ones which have been obtained without prior modification of the drug or linker. The linker moiety in the conjugates of the invention are therefore based on the compounds of formula (I), preferably with no additional spacers.

Isomers

All compounds and formulae indicated herein represent all possible stereoisomers, where chiral centres exist and where a specific isomeric configuration is not specified.

Thiol substitution

In the majority of cases, the linkers in the prior art use a pyridinesulfenyl-group for thiol protection/activation. The linkers according to the present invention preferably contain a pyridinesulfanyl-group substituted with at least one electron withdrawing group Y as defined above. Such substitution is preferably at the para position and/or at one or both ortho positions. Nitropyridinesulfanyl groups are highly appropriate, more preferably ortho- or /?ara-nitropyridinesulfanyl groups (i.e. Y = N0₂), even more preferable para- nitropyridinesulfanyl. Nitropyridinesulfanyl groups have been demonstrated to be superior in terms of thiol conjugation.

The rate of the self- elimination process is important for efficient drug release. However, seeing that there is a great level of structural variance between drug molecules, it is envisaged that there exists no "one-size-fits-all" linker solution. In order to accommodate for the ability to modulate linker stability and drug release rates of the resultant prodrug, substitutions have been introduced in the structure of the linkers.

Examples of linkers are given below. Most preferred linkers will have alkyl substitution adjacent to the disulfide unit and/or adjacent to the O or N atom of group Q.

Process for the preparation of linkers

The linkers of the present invention can be made by a process comprising the following steps. The first step comprises reacting compounds of formulae (IV) and (V) to form compound (VI).

In this process,

R², R³ and R⁴ are as defined above, J is O or NR⁵, preferably NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group. In this process, preferably R⁵ is hydrogen, a Ci - C₆ alkyl, or a substituted or unsubstituted aryl group, such as phenyl. It will be appreciated that for the formation of the isocyanate linker (i.e. one in which Q is -NCO), R⁵ must be H (and hence J must be NH) in the above scheme. Conversion to the isocyanate linker is discussed further below.

In a second step, compound (VI) may be reacted with phosgene, triphosgene or a compound of formula (VII)

(VII) to obtain a linker of formula (I), (II) or (III) as defined above wherein m, n and o and the groups Y, J, Z and R^!-R⁴ are as defined above; wherein Z=Z' or Z≠Z', and Z' is a leaving groups selected from halides (such as fluoride, chloride, bromide, iodide), alkoxides, tosylate, mesylate, nitrate, phosphate, phenoxide, p- nitrophenoxide, o-nitrophenoxide benzotriazoloxide and 2,5-pyrrolidinedione 1 -oxide;

If triphosgene is used in the second step, a compound of formula (I), (II) or (III) with Q being (-J(C=0)Z) and Z = CI may be produced, or Q being -NCO. Preferably, at least one of Z and Z' is CI. In particular, it is preferable if in the above process if compound (VI) is reacted with phosgene (i.e. compound (VII) with Z = Z' = CI) or triphosgene.

It is postulated that the isocyanate linkers (i.e. the linkers of formula (I), (II) or (III) in which Q is -NCO) are obtained upon decomposition of intermediate compounds of the formula shown below:

when R⁵ is H and Z in particular is CI. Whilst linkers with carbamoyl chloride groups (-NH(C=0)C1) tend to decompose to the isocyanate, linkers of the above with R⁵ being H (i.e. with the -NH(C=0)Z group still intact) are also within the scope of the invention, in particular when Z is not chloride.

Conjugates

The linkers of the present invention are of particular use in the preparation of conjugates, i.e. compounds in which a 'carrier' moiety is linked to a 'cargo' moiety via the linker. The conjugates of the present invention are of the formula (VIII):

R*-R⁴, m, n and o are as defined herein (e.g. previously). In the conjugates of formula (VIII), J is O or NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group. Preferably, J is NR⁵. Preferably, in the conjugates of formula (VIII), R⁵ is a hydrogen, a Ci-C₆ alkyl group or a substituted or unsubstituted aryl group, such as phenyl. In a particular embodiment of the conjugate of formula (VIII), R⁵ is not hydrogen. It will be appreciated by the skilled worker that conjugates with J = NH (i.e. R⁵ = H) can be made from the addition of the bioactive agent to the mercaptoamine linker with R⁵ = H or to an isocyanate linker. A is such that A-SH is a carrier entity such as an antibody, antibody fragment, nanoparticle, protein, protein fragment or peptide; B and W are such that B-WH is a bioactive agent, a diagnostic agent or a visualization agent; and W = NH or O. Preferably, when J = NPv⁵, W = O. The present invention also encompasses any pharmaceutically acceptable salt, hydrate or solvate of the conjugate.

In a particular embodiment, the conjugate of the invention comprises a single linker moiety and a single bioactive agent, diagnostic agent or visualization agent B. Conjugates which comprise multiple linker-drug units attached to a single carrier unit are therefore preferably excluded. Preferably, therefore, the carrier unit A does not contain any further linker and/or drug units attached thereto. Thus, in a corresponding embodiment, the conjugate may consist of one carrier (e.g. antibody), one linker moiety (as described in any embodiment herein) and one moiety "B", such as a bioactive agent, diagnostic agent or visualization agent (e.g. as described herein). In a related embodiment, the ratio of moiety "A" to linker moiety to moiety "B" will be around 1 : 1 : 1 (e.g. ±20%, preferably ±10% or ±5%).

In a further embodiment of the invention, the conjugate may be based on a linker of formula (II) or (III), providing corresponding conjugates i.e. compounds of formula:

Wherein R¹, R², R^s, R°, J and m are as defined herein, and particularly as defined for the linkers of formula (I), (II) and/or (III). Again, it will be appreciated by the skilled worker that conjugates with J = NH (i.e. R⁵ = H) can be made from the addition of the bioactive agent to the mercaptoamine linker with R⁵ = H or to an isocyanate linker. Suitable conjugates include, for example:

R¹ R² o

R⁵

wherein A, B, W, R¹, R², m, R⁵ and Z are as defined above or below; typically, R¹ and R² are, independently and for each value of m, H or a Ci-C₆ alkyl. The value of m is preferably 2-10 (such as 2-6 or 2-4, e.g. 2), Z is preferably CI, and R⁵ is preferably H, a Ci-C₆ alkyl or phenyl.

By 'carrier' is meant the unit 'A-S-' (e.g. in formula (VIII)) either attached to the conjugate (e.g. as A-S-linker-cargo") or free as the free carrier A-SH. The skilled worker understands that the free carrier A-SH and the attached unit A will possess the same or similar targeting properties and therefore the term carrier may apply to any of Ά', 'A-S-' and Ά- SH'. The 'SH' in Ά-SH' is the SH group used to conjugate the carrier to the linker. In the case of cysteine-engineered antibodies for example, this SH group originates from the cysteine unit. In the case of a carrier with more than one thiol moiety, the A-SH may not represent a single specific -SH group but rather represents the thiol moieties available for conjugation. The degree of that conjugation may be optimized by the skilled worker depending upon the desired properties of the conjugate.

Similar consideration apply for the cargo moiety B. The cargo is attached to the linker via an -NH₂ or -OH group represented by -WH (W = NH or O). The naked, unprotected cargo is therefore B-WH but the term 'cargo' may apply to B, B-W or B-WH and may contain one or more "W" groups, of which one, some or all may be available for conjugation. Typically it will be desirable to conjugate the cargo moiety B through a specific W moiety and so other amine or hydroxyl groups may be kinetically unfavorable or may be protected by any of the well know protective moieties.

It has been found during drug release studies with glutathione in aqueous buffer solution at pH 7.6 that carbamate conjugates are more stable than carbonate conjugates. When mercaptoalcohol linkers (i.e. linkers with J = O) are used for the preparation of conjugates with a hydroxyl-containing bioactive agent, it is possible in some circumstances that the corresponding conjugate with a carbonate linkage is insufficiently stable. It is possible, for example, for the bioactive agent to be released prior to its targeted destination, thereby reducing the targeting efficiency and/or risking side-effects in some cases. For bioactive agents having an -OH functionality (i.e. B-O- / B-OH bioactive agents), mercaptoamine linkers (i.e. linkers with J = NR⁵) or isocyanate linkers (i.e. linkers with Q = -NCO) provide a useful alternative. The resulting carbamate conjugate formed can have improved stability compared to the carbonate conjugate. It is preferable for the mercaptoamine linkers (i.e. those with J = NR⁵) and isocyanate linkers (i.e. linkers with Q = -NCO) to be used with hydroxyl-containing bioactive agents, as the use of amine-containing bioactive agents would result in urea derivatives. Such linkages may be too stable to be effectively releasable under the relevant physiological conditions.

Amine-containing bioactive agents are therefore preferably used with mercaptoalcohol linkers. In some cases, it has been found during release studies that the drug is released as a carbamic acid derivative. In cases in which the drug does not need to be released in its native form, this can be of use, and thus the release of carbamic acid derivatives of bioactive agents constitute a further embodiment of the invention. a particular embodiment, the conjugate is not the compound of formula:

or any sa t or cat on t ereo , suc as ts + or + a cat ons.

Carrier

The 'carrier' A-SH is envisaged to preferably be a thiol-containing antibody, antibody fragment, protein, protein fragment, peptide, or nanoparticle.

The linkers of the present invention are of particular use in the preparation of antibody drug conjugates (ADC's), and thus the carrier unit is preferably an antibody. The carrier unit A-SH acts as a targeting moiety and is preferably a cysteine-containing or cysteine- engineered antibody. The antibody may be any antibody known to be effective in targeting cancer cells, including polyclonal and monoclonal antibodies (including IgG and IgM type antibodies), fragments and/or constructs thereof. Preferably the antibody is a monoclonal antibody. Antibodies, antibody constructs, fragments of antibodies (e.g. FAB fragments, interchain thiol or any fragment comprising at least one antigen binding region(s)), constructs of fragments (e.g. single chain antibodies) or a mixture thereof may also be used.

Preferably, the carrier is not a Shiga toxin.

The carrier is preferably tumor-homing, i.e. it targets cancer cells. Such cancer cell targeting is typically the result of the carrier moiety "A" targeting a tumor-associated antigen. In one embodiment, therefore, the carrier (A) may bind to a tumor-associated antigen. Many such tumor associated antigens are known in the art, including "Cluster of Differentiation (CD)" antigens (e.g. CD20, CD22, CD30, CD32, CD33 and/or CD52), glycoprotein antigens (e.g. EpCAM, CEA, Mucins, TAG-72m Carbonic anhydrase IX, PSMA and/or folate binding protein), Glycolipid antigens (e.g. Gangliosides such as GD2, GD3, amd/or GM2), Carbohydrate antigens (e.g. Lewis- Y), Vascular antigens (e.g. VEGF, VEGFR, α,νβ3, α5β1), Growth factor antigens (e.g. ErbBl, EGFR, ErbB2, HER2, ErbB3, c-MET, IGF1R, EphA3, TRAIL-R!, TRAIL-R2, RANKL), extracellular matrix antigens (e.g. FAP, Tenascin), and/or overexpressed receptors (e.g α_νβ₃).

The antibody may be an antibody (e.g. a monoclonal antibody) which is in itself an immunotherapeutic agent which binds to certain cells or proteins and then stimulates the patient's immune system to attack those cells. In this case, the cargo species of the conjugate acts in tandem with the immunotherapeutic effects of the antibody. Alternatively, the carrier agent may be a mere targeting agent and does not provoke any immunotherapeutic effects by itself. In this case, it is solely the cargo unit which acts as the active, cell-destroying agent.

In one particular embodiment, the carrier is the antibody RS7-3G 1 1 . In another embodiment. engineered antibodies of the invention comprise an antibody that comprises an epitope binding domain (for example, but not limited to, an antibody variable region having all 6 CDRs, or an equivalent region that is at least 90% identical to an antibody variable region) chosen from: abagovomab, abatacept (also known as ORENCIA®), abciximab (also known as REOPRO®, c7E3 Fab), adaiimumab (also known as HUMIRA®), adecatumumab, alemtuzumab (also known as CAMPATH®, MabCampath or Campath- 1 1 1), aitumomab, afelimomab, anatumomab mafenatox, anetumiimab. anrukizumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab, bapineuzumab, basiliximab (also known as

SIMULECT®), bavituximab, bectumomab (also known as LYMPHOSCAN®), belimumab (also known as LYMPFIO-STAT-B®), bertilimumab, besilesomab, bevacizumab (also known as AVASTIN®), biciromab brailobarbital, bivatuzumab mertansine, campath, canakinumab (also known as ACZ885), cantuzumab mertansine, capromab (also known as

PROSTASCINT®), catumaxomab (also known as REMOVAB®), cedelizumab (also known as CIMZIA®), certoiizumab pegol. cetuximab (also known as ERBITUX®), clenoliximab, dacetuzumab, dacliximab, daclizumab (also known as ZENAPAX.®), denosumab (also known as AMG 162), detumomab, dorlimomab aritox, doriixizumab, diintiimumab, durimuiumab, durmulumab, ecromeximab, eculizumab (also known as SOLIRIS®), cdobacomab, edrecolomab (also known as Mabl7-1A, PANOREX®), efalizumab (also known as RAPTIVA®), efungumab (also known as MYCOGRAB®), elsilimomab, cnlimomab pegol, epitumomab cituxctan, efalizumab, epitumomab, epratuzumab, erlizumab, crtuma.xomab (also known as REXOMU ©), etanercept (also known as ENBREL®), etaracizumab (also known as etaratuziimab, VITAXLN®, ABEGRIN™), e.xbivirumab.

fanolesomab (also known as NEUTROSPEC®), faralimomab, felvizumab, fontoliziimab (also known as HUZAF®), ga!iximab, gantenerumab, gavilimomab (also known as ABX-CBL®), gemtuzumab ozogamicin (also known as MYLOTARG®), golimumab (also known as CNTO 148), gomiiiximab, ibalizumab (also known as TNX-355), ibritumomab tiuxetan (also known as ZEVALIN®), igov o truth, imciromab, infliximab (also known as REM1CADE®), inolimomab, inotuzumab ozogamicin, ipilimumab (also known as MDX-010, MDX-101), iratumumab, keliximab, labetuzumab, lemaiesomab, iebriiiziimab, lerdel imumab,

lexatumumab (also known as, HGS-ETR2, ETR2-ST01), lexitumumab, libivirumab,

lintuzumab, lucatumiimab, liimiliximab, mapatumiimab (also known as HGS-ETRl , TRM-1), masiimomab, matuzumab (also known as EMD72000), mepolizumab (also known as

BOSATRIA®), metclimumab, milatuzumab, minretumomab, mitumomab, moroiimumab, motavizumab (also known as NUMAX™), muromonab (also known as OKT3), nacolomab tafenatox, naptumomab estafenatox, natalizumab (also known as TYSABRI®,

ANTEGREN®), nebacumab, nerelimomab, nimotuzumab (also known as THERACIM hR3®, THERA-CIM-hR3®, THERALOC®), nofetumomab merpentan (also known as VERLUMA®), ocrelizumab, odulimomab, ofatumumab, omalizumab (also known as XOLAIR®), oregovomab (also known as OVA REX* ). otelixizumab, pagibaximab, palivizumab (also known as SYNAGIS®), panitumumab (also known as ABX-EGF,

VECTIBIX®), pascoiizumab, pemtumomab (also known as THERAGYN®), pertuzumab (also known as 2C4, OMNITARG®), pexelizumab, pintumomab, priiiximab, pritumumab, ranibizumab (also known as LUCENTIS®), raxibacumab, regavirumab, resiizumab, rituximab (also known as R1TUXAN®, MabTHERA®), roveiizumab, ruplizumab, satumomab, sevirumab, sibrotuzumab, siplizumab (also known as MEDI-507), sontuzumab, stamulumab (also known as MYO-029), sulesomab (also known as LEUKOSCA ®), tacatuzumab tetraxetan, tadocizumab, taiizumab, taplitumomab paptox, tefibazumab (also known as AUREXIS®), teiimomab aritox, teneiiximab, teplizumab, ticilimiimab, tociliziimab (also known as ACTEMRA®), toraiizumab, tositumomab, trastuzumab (also known as HERCEPTIN®), tremelimumab (also known as CP-675,206), tucotuzumab ceimoleukin, tuvirumab, urtoxazumab, ustekinumab (also known as CNTO 1275), vapaiiximab, veituzumab, vepaiimomab, visilizumab (also known as NUVION®), volociximab (also known as M200), votumumab (also known as HUMASPECT®), zalutumumab, zano!imumab (also known as HuMAX-CD4), ziralimumab, or zolimomab aritox.

Whilst antibodies constitute a preferred embodiment of the invention, the carrier unit may also be a single type of protein, protein fragment or construct of protein, or a mixture of proteins, fragments or constructs of protein, especially those that are known to localize to diseased cells such as cancer/tumor cells.

The carrier may also be a peptide, preferably a cysteine-containing cell-penetrating peptide such as Tat-peptide, penetratin, MPG and Pep-1. Protein fragments, such as histidine- rich glycoprotein fragments, for example HRGP-335 also constitute an embodiment of the invention. Tumor-homing peptides such as the NGR- and cRGD peptides constitute a further embodiment. Suitable moieties also include other poly- and oligo-peptides.

It is also envisaged that aptamers, DNA or RNA fragments may be used as carrier moieties in the present invention when suitably modified with at least one -SH moiety.

Nanoparticles that include, but are not limited to, liposomes, nano worms, and dendrimers that contain at least one surface -SH group may also be used as the carrier and thus constitute a further embodiment of the invention. Similarly, colloidal (e.g. silicon) nanoparticles, particularly colloidal porous silicon nanoparticles may be used as the carrier moiety in the various aspects of the present invention. These may utilize the inherent properties of such particles (e.g. by loss through "leaky" tumour capillaries) or may use "active" targeting using other targeting moieties such as those described herein (antibodies and their derivatives for example). In such cases, the "cargo" may be within or on the surface of such colloidal nanoparticles. The cargo may thus be joined to the nanoparticles by means of the linker moieties described herein and thus the nanoparticles may be functionalized with a suitable group, particularly a thiol group, for such purpose. Reactions for attachment and release with colloidal particles will be analogous to those for other targeting moieties, as discussed herein.

Cargo

In all compatible aspects of the present invention, preferably the 'cargo' B-WH is a hydroxyl- and/or amine-containing molecule (i.e. W = NH or O). Preferably, W = O. The cargo molecules could be cytotoxic compounds, substrate, diagnostic compounds, bioactive compounds. Preferably the cargo molecule is a bioactive agent, a diagnostic agent and/or a visualization agent. More preferably the cargo is an anti-cancer drug. In the case of cytotoxic or bioactive compounds, the cargo may for example be an alkylating agent, an antimetabolite, an anti-microtubule agent, a topoisomerase inhibitor or a cytotoxic antibiotic. The cargo may also be a cell division inhibitor, a DNA replication inhibitor or an angiogenesis inhibitor. Further categories of cargo include nucleotide analogues, radioisotopes, folate analogues.

In a preferred embodiment the hydroxyl-containing cargo is selected from the group consisting of doxorubicin, everolimus, becalutamide, cabazitaxel, irinotecan, bicalutamide, dasatinib, degarelix, dexamethasone, docetaxel, raloxifene, fulvestrant, goserilin, topotecan, ixabepilone and leuprolide. Hydroxyl-containing cargos represent a preferred embodiment, applicable to all technically compatible aspects of the invention.

In another preferred embodiment the amine- containing cargo is selected from the group consisting of methotrexate, imiquimod, melphalane, pemetrexed disodium, aminolevulinic acid, dactinomycine, crizotinib, dafrafenib, pralatrexate, ibrutinib, lenalidomide, leucovorin, mitomycin C, pomalidomide, and tioguanine.

Preparation of conjugate

The first step in the conjugation reaction is between the cargo and linker. The linker of formula (I) is reacted with the carrier B-WH to form cargo-linker intermediate (IX):

(I) + HW B

In a second stage, the obtained carrier-linker intermediate (IX) is reacted further with the carrier molecule to form the carrier-cargo conjugate (VIII) described above.

(VIII)

In the above schemes for the preparation of the conjugates (VIII) and intermediate compounds (IX), it will be appreciated that in the case of a mercaptoalcohol or mercaptoamine linker, J is O or NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group. In the case of an 'isocyanate' linker, R⁵ will be H and hence J will be NH.

In a preferable embodiment of the invention the preparation of the conjugate is carried out in the order described above in order to ensure the desired conjugate is obtained in good yield. Carbonates and carbamates are reactive towards nucleophiles such as SH-groups, thus the OH- or NH₂-containing cargo, which do not react with disulfide bonds, should be conjugated first. Failing to do so may lead to excessive generation of byproducts (e.g. ' 'carrier- linker-carrier' ') .

Self- elimination and release of cargo

It is a feature of the invention that the backbone of the linker, and therefore of the conjugate, be self-eliminating (self-immolative) under physiological conditions.

Once the targeting moiety reaches and adheres to the target cell, the conjugate is taken up via endocytosis. Endocytosis is envisaged to involve the intact ADC. Endocytosis could also involve only the linker-cargo entity, since it has been shown that exofacial protein thiols can serve as a route of entry into the cell. In this case, thiol-disulfide reactions between membrane proteins and the conjugate could displace the carrier molecule leaving the linker- cargo entity covalently bond to the surface of the target cell, followed by internalization into the cytoplasm. This route of internalization would be advantageous in the cases where non- internalized mAbs are used as carriers.

Upon cytosolic release form the endosome/lysosome, the disulfide bond between the targeting moiety, or exofacial protein, and cargo (drug) is cleaved by thiol-containing compounds, most notably glutathione, which are present in high concentrations within the cytoplasm. This initial cleavage is followed by a spontaneous intramolecular self-elimination reaction resulting in the release of the cargo (drug) in its free form (B-WH). The drug release is a two-step process, starting with the thiol-disulfide exchange with intracellular thiol- containing compounds as seen in the scheme below.

(i) Thiol-disulfide exchan e - - — -

(ii) Intramolecular cycl

HW B 'naked', unmodified drug

In the above Scheme, G-SH represents a thiol-containing compound in the cytopli as glutathione. The second step, whereby the cargo (drug) is released, comprises an intramolecular cyclization. The rate of this reaction can be adjusted by introduction of substituents as described herein.

Substitutions on the carbon adjacent to the sulfur atom allows for the modulation of stability towards thio lytic cleavage. In nearly all prior art cases there is no substitution on the carbon adjacent to sulfur. This disulfide bond formed between the thiol-containing carrier and the linker will be too sensitive towards thiolytic cleavage to be relevant for in vivo applications. Thus, many of the linkers according to the present invention contain one or two substituent groups (e.g. alkyl, such as methyl groups) on the adjacent carbon to provide increased stability through steric hindrance (e.g. at position R^s in formula III). It is preferred if the steric hindrance is provided by substitution on the carbon which the disulfide moiety is attached, however it is also conceivable that protection may be obtained via substitution at carbon atoms which are further away. Where the substitution is present at such carbon positions this may be in addition to substitution on the adjacent carbon or instead of that. In either case, to be effective, such substitution at a carbon not adjacent to the sulfur will preferably comprise at least one branched substituent (e.g. an isoalkyl, sec-alkyl or tert-alkyl group) and/or may comprise at least two substitutions at the same or neighboring carbon atoms.

Substitutions on the carbon adjacent to the oxygen or nitrogen atom (i.e. adjacent to J, such as at R° in Formula III) allow for the modulation of stability towards aminolysis and/or hydrolysis of the linker. If the carrier moiety of the conjugate contains at least one amino group (e.g. the side chain of lysine) intramolecular attack may take place at physiological pH and result in premature drug release in the plasma. The conjugate's stability towards this side reaction can be improved by incorporating substitution on the carbon atom adjacent to the oxygen/nitrogen atom (group J in formulae such as I-IV) of the carbonate/carbamate group (Figure 5). Such substitution may be any described herein, such as alkyl substitution (e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-bvXy\ substitution). As with the substitution neighboring the disulfide group, substitution on or in the vicinity of the carbon to which group "J" is attached may be present analogously.

Treatment

The conjugates of the present invention are preferably for use in the treatment of cancer. All types of cancer are deemed to be within the scope of the invention, but in particular the conjugates are for use in the treatment of breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer or adrenal cancer.

EXAMPLES

The example linkers shown below have been synthesized.

Structure Name

Y 2- (2-(5-Nitropyridin-2- yl)disulfanyl)ethyl chloroformate

3- (2-(5-Nitropyridin-2- y l)disulfany l)propy 1 chloro formate

1 -Methyl-2-(2-(5-nitropyridin-2- y l)disulfany l)propy 1 chloro formate

1 -Methyl-2-(2-(5-nitropyridin-2- yl)disulfanyl)ethyl chloroformate

4- (2-(5-Nitropyridin-2- yl)disulfanyl)butyl chloroformate

3-(2-(5-Nitropyridin-2- yl)disulfanyl)hexyl chloroformate

3 -Methyl-3 -(2-(5 -nitropyridin-2- yl)disulfanyl)butyl chloroformate

1 ,3 -Dimethyl-3 -(2-(5 -nitropyridin- 2-yl)disulfanylbutyl chloroformate

6-(2-(5-Nitropyridin-2- yl)disulfanyl)hexyl chloroformate

8- (2-(5-Nitropyridin-2- yl)disulfanyl)octyl chloroformate

9- (2-(5-Nitropyridin-2- yl)disulfanyl)nonyl chloroformate

1 l-(2-(5-Nitropyridin-2- yl)disulfanyl)undecyl

chloroformate butyl(2-(2-(5 -nitropyridin-2- yl)disulfanyl)ethyl)carbamic chloride

General procedure for the preparation of linkers

Step 1.

A solution of mercaptoalcohol (1.0 mmol) in dichloromethane (5.0 mL) was added to a solution of 2,2'-dithiobis(5-nitropyridine) (1.0 mmol) in dichloromethane (20 mL) containing 5% methanol (v/v), and the resulting mixture was stirred at room temperature for 24 hr . The solvent was removed and the remaining crude product was added by hexane containing 10% ethyl acetate (20 mL) and the resulting suspension was filtered on a glass filter. The filtrate was concentrated on a rotary evaporator and the residue was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions were collected and lyophilized to give (5-nitropyridin-2-yl) disulfanylalkyl alcohol (62-78%).

Step 2.

Method 1. A 15%> solution of phosgene (0.25 mmol) in toluene was added to a solution of (5-nitropyridin-2-yl)disulfanylalkyl alcohol (0.20 mmol) in toluene (1.0 mL). The reaction mixture was stirred at room temperature for 8 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the fractions were lyophilized to get the final product (90-95%).

Method 2. Triphosgene (0.2 mmol) was dissolved in toluene (2 mL) at 0 °C a solution of (5-nitropyridin-2-yl)disulfanylalkyl alcohol (0.2 mmol) in toluene (1 mL) was added. The reaction mixture was stirred at 0 °C for 2 hr and then at room temperature for 24 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent to get the final product (90-95%). Chromatographic and mass spectrometric analyses have shown that product obtained by this method contained in some cases trichloromethyl carbonate as a byproduct along with the corresponding chloroformate as a main product. Trichloromethyl carbonate is capable of reacting with hydroxyl and amino groups to form a carbonate and carbamate, respectively, in the same way as the chloroformate. However, this reaction involves a release of phosgene. Therefore, method 1 is preferred to method 2 if method 2 gives the by-product.

In the following four representative examples are described.

Example 1

Step 1.

A solution of 2-mercapto-l-ethanol (0.078 g, 1.0 mmol) in dichloromethane (5 mL) was added to a solution of 2,2'-dithiobis(5-nitropyridine) (0.31 g, 1.0 mmol) in dichloromethane (20 mL) containing 5% methanol (v/v), and the resulting mixture was stirred at room temperature for 24 hr . The solvent was removed and the remaining crude product was added by hexane containing 10% ethyl acetate (20 mL) and the resulting suspension was filtered on a glass filter. The filtrate was concentrated on a rotary evaporator and the residue was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions were collected and lyophilized to give 2-(2-(5- nitropyridin-2-yl)disulfanyl)ethanol (0.167 g, 72%) as a yellowish powder.

1H NMR (400 MHz, CDC1₃) δ 9.28 (d, J = 2.5 Hz, 1H), 8.41 (dd, J = 9.0, 2.5 Hz, 1H), 7.89 (d, J= 9.0 Hz, 1H), 3.79 (t, J= 7.0 Hz, 2H), 3.02 (t, J= 7.0 Hz, 2H)

¹³C NMR (100 MHz, CDC1₃) δ 167.6, 145.4, 140.6, 131.5, 120.9, 58.7, 42.6

Step 2.

Method 1. A 15% solution of phosgene (0.18 mL, d= 0.924 g/mL, 0.25 mmol) in toluene was added to a solution of 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethanol (0.046 g, 0.20 mmol) in toluene (1.0 mL). The reaction mixture was stirred at room temperature for 8 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the collected fractions were lyophilized to get 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl chloroformate (0.054 g, 93%>) as an off-white powder. Method 2. Triphosgene (0.059 g, 0.2 mmol) was dissolved in toluene (2 mL) at 0 °C before solution of 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethanol (0.046 g, 0.20 mmol) in toluene (1 mL) was added.. The reaction mixture was stirred at 0 °C for 2 hr and then at room temperature for 24 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the collected fractions were lyophilized to get 2-(2-(5-nitropyridin-2- yl)disulfanyl)ethyl chloroformate (0.053 g, 90%>) as an off-white powder.

1H NMR (400 MHz, CDC1₃) δ 9.28 (d, J = 2.5 Hz, 1H), 8.35 (dd, J = 9.0, 2.5 Hz, 1H), 7.79 (d, J= 9.0 Hz, 1H), 4.60 (t, J= 7.0 Hz, 2H), 3.21 (t, J= 7.0 Hz, 2H)

¹³C NMR (100 MHz, CDC1₃) δ 167.2, 150.6, 145.4, 142.4, 131.8, 119.7, 68.7, 36.5

Example 2

Step 1.

A solution of 3-mercapto-l-propanol (0.092 g, 1.0 mmol) in dichloromethane (5 mL) was added to a solution of 2,2'-dithiobis(5-nitropyridine) (0.31 g, 1.0 mmol) in dichloromethane (20 mL) containing 5% methanol (v/v), and the resulting mixture was stirred at room temperature for 24 hr . The solvent was removed and the remaining crude product was added by hexane containing 10% ethyl acetate (20 mL) and the resulting suspension was filtered on a glass filter. The filtrate was concentrated on a rotary evaporator and the residue was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions were collected and lyophilized to give 3-(2-(5-nitropyridin-2-yl)disulfanyl)propan-l-ol (0.172 g, 70%>) as a yellowish powder.

1H NMR (400 MHz, CDC1₃) δ 9.28 (d, J = 2.5 Hz, 1H), 8.41 (dd, J = 9.0, 2.5 Hz, 1H), 7.89 (d, J= 9.0 Hz, 1H), 3.80 (t, J= 7.0 Hz, 2H), 2.99 (t, J= 7.0 Hz, 2H), 1.97 (m, 2H).

¹³C NMR (100 MHz, CDC1₃) δ 168.7, 145.2, 141.1, 131.6, 119.4, 60.8, 35.5, 31.5

Step 2.

Method 1. A 15% solution of phosgene (0.18 mL, d= 0.924 g/mL, 0.25 mmol) in toluene was added to a solution of 3-(2-(5-nitropyridin-2-yl)disulfanyl)propan-l-ol (0.049 g, 0.2 mmol) in toluene (1.0 mL). The reaction mixture was stirred at room temperature for 8 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the collected fractions were lyophilized to give 3-(2-(5-nitropyridin-2-yl)disulfanyl)propyl chloro formate (0.057 g, 92%) as an off- white powder.

1H NMR (400 MHz, CDC1₃) δ 9.28 (d, J = 2.5 Hz, 1H), 8.32 (dd, J = 9.0, 2.5 Hz, 1H), 7.79 (d, J= 9.0 Hz, 1H), 4.39 (t, J= 7.0 Hz, 2H), 2.87 (t, J= 7.0 Hz, 2H), 2.09 (m, 2H).

¹³C NMR (100 MHz, CDC1₃) δ 167.8, 150.5, 145.2, 142.1, 131.7, 119.4, 69.7, 34.6, 27.6

Example 3

Step 1.

A solution of l-mercapto-2-propanol (0.092 g, 1.0 mmol) in dichloromethane (5 mL) was added to a solution of 2,2'-dithiobis(5-nitropyridine) (0.310 g, 1.0 mmol) in dichloromethane (20 mL) containing 5% methanol (v/v), and the resulting mixture was stirred at room temperature for 24 hr . The solvent was removed and the remaining crude product was added by hexane containing 10% ethyl acetate (20 mL) and the resulting suspension was filtered on a glass filter. The filtrate was concentrated on a rotary evaporator and the residue was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions were collected and lyophilized to give l-(2-(5-nitropyridin-2-yl)disulfanyl)propan-2-ol (0.169 g, 69%>) as a yellowish powder.

1H NMR (400 MHz, CDC1₃) δ 9.33 (d, J = 2.2 Hz, 1H), 8.36 (dd, J = 9.0, 2.2 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 4.32 (s, 1H), 3.89-3.82 (m, 1H), 3.03 (dd, J = 14.0, 2.8 Hz, 1H), 2.71(dd, J= 14.0, 9.5 Hz, 1H) 1.27 (d, J= 6.5 Hz, 3H).

¹³C NMR (100 MHz, CDC1₃) δ 167.5, 145.4, 135.8, 131.4, 120.7, 64.5, 48.6, 21.6 Step 2.

Method 1. A 15% solution of phosgene (0.18 mL, d= 0.924 g/mL, 0.25 mmol) in toluene was added to a solution of l-(2-(5-nitropyridin-2-yl)disulfanyl)propan-2-ol (0.049 g, 0.2 mmol) in toluene (1.0 mL). The reaction mixture was stirred at room temperature for 8 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the collected fractions were lyophilized to give l-methyl-2-(2-(5-nitropyridin-2-yl)disulfanyl)- ethyl chloroformate (0.056 g, 90%>) as an off-white powder.

1H NMR (400 MHz, CDC1₃) δ 9.29 (d, J = 2.2 Hz, 1H), 8.41 (dd, J = 9.0, 2.2 Hz, 1H), 7.80 (d, J= 9.0 Hz, 1H), 5.21-5.13 (m, 1H), 3.17-3.02 (m, 2H), 1.49 (d, J= 6.5 Hz, 3H). ¹³C NMR (100 MHz, CDC1₃) δ 167.2, 150.0, 145.4, 142.4, 131.7, 119.7, 77.9, 43.7, 19.0

Example 4

Step 1.

A solution of 3-mercapto-3-methylbutan-l-ol (0.120 g, 1.0 mmol) in dichloromethane (5 mL) was added to a solution of 2,2'-dithiobis(5-nitropyridine) (0.310 g, 1.0 mmol) in dichloromethane (20 mL) containing 5% methanol (v/v), and the resulting mixture was stirred at room temperature for 24 hr . The solvent was removed and the remaining crude product was added by hexane containing 10% ethyl acetate (20 mL) and the resulting suspension was filtered on a glass filter. The filtrate was concentrated on a rotary evaporator and the residue was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions were collected and lyophilized to give 3-methyl-3-(2-(5-nitropyridin-2-yl)disulfanyl)butan-l-ol (0.186 g, 68%>) as a yellowish powder.

1H NMR (400 MHz, CDC1₃) δ 9.24 (d, J = 2.7 Hz, 1H), 8.37 (dd, J = 9.0, 2.7 Hz, 1H), 7.92 (d, J= 9.0 Hz, 1H), 2.17 (t, J= 6.8 Hz, 2H), 1.92 (t, J= 6.8 Hz, 2H) 1.37 (s, 6H).

¹³C NMR (100 MHz, CDC1₃) δ 169.6, 144.8, 142.0, 131.4, 119.6, 59.5, 52.2, 43.5, 27.9

Step 2.

Method 1. A 15% solution of phosgene (0.18 mL, d= 0.924 g/mL, 0.25 mmol) in toluene was added to a solution of 3-methyl-3-(2-(5-nitropyridin-2-yl)disulfanyl)butan-l-ol (0.055 g, 0.2 mmol) in toluene (1.0 mL). The reaction mixture was stirred at room temperature for 8 hr. The solvent was removed under reduced pressure and the crude product was purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the collected fractions were lyophilized to give 3-methyl-3-(2-(5-nitropyridin-2- yl)disulfanyl)butyl chloroformate (0.063 g, 94%>) as an off-white powder.

1H NMR (400 MHz, CDCI3) δ 9.19 (d, J = 2.7 Hz, 1H), 8.31 (dd, J = 9.0, 2.7 Hz, 1H), 7.87 (d, J= 9.0 Hz, 1H), 4.42 (t, J= 6.8 Hz, 2H), 1.97 (t, J= 6.8 Hz, 2H) 1.31 (s, 6H).

¹³C NMR (100 MHz, CDCI3) δ 168.8, 150.6, 145.0, 142.2, 131.5, 119.5, 68.7, 51.4, 39.0, 27.8, 27.7 General procedure for the preparation of conjugates Step 1. Cargo-linker conjugate

Conjugation with cargo containing hydroxyl group.

A solution of cargo (0.1 mmol) in acetonitrile and a solution of triethylamine (0.02 mmol) are added to a solution of linker (0.1 mmol) in acetonitrile and the reaction mixture is stirred at room temperature. When reaction is complete, the mixture is concentrated under reduced pressure and the remaining crude is purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give the cargo-linker conjugate after lyophilization.

Example 5

A 0.1 M solution of paracetamol (1.0 mL) in acetonitrile and 0.1 M solution of triethylamine (0.25 mL) were added to 0.1 M solution of 2-(2-(5-nitropyridin-2- yl)disulfanyl)ethyl chloro formate (1 mL) in acetonitrile and the reaction mixture is stirred at room temperature. When reaction was complete, the reaction mixture was concentrated under reduced pressure and the remaining crude was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give 4-acetamidophenyl 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl carbonate (0.035 g, 86%>) as a white powder after lyophilization.

Conjugation with cargo containing amine group

A solution of cargo (0.1 mmol) in acetonitrile is added to a solution of linker (0.1 mmol) in acetonitrile and the reaction mixture is stirred at room temperature. When reaction is complete, the mixture is concentrated under reduced pressure and the remaining crude is purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give the cargo-linker conjugate after lyophilization.

Example 6

A 0.05 M solution of 4-acetamidoaniline (2 mL) in acetonitrile was added to 0.1 M solution of 2-(2-(5-Nitropyridin-2-yl)disulfanyl)ethyl chloroformate (1 mL) in acetonitrile and the reaction mixture is stirred at room temperature. When reaction was complete, the mixture was concentrated under reduced pressure and the remaining crude was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl 4-acetamidophenylcarbamate (0.033 g, 81%) as a white powder after lyophilization.

Step 2. Cargo-Linker-Carrier conjugate

A solution of cargo-linker conjugate (0.1 mmol) in acetonitrile or mixture of acetonitrile and water is added to a solution of carrier (0.05 mmol) in water or mixture of acetonitrile and water and the reaction mixture is stirred at room temperature. The color of the reaction mixture turns to more intense yellow due to releasing pyridinethiol. When reaction is complete, the mixture is directly purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give the cargo-linker- carrier conjugate after lyophilization.

Example 7

A 0.1 M solution of 4-acetamidophenyl 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl carbonate (1 mL) in acetonitrile was added to 0.1 M solution of peptide with formula X (0.5 mL) in water and the reaction mixture is stirred at room temperature for 24 hr. The crude mixture was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give the final conjugate with formula XI (0.068 g, 82%>) as a white powder after lyophilization.

Ac-CysAhxGlyHisHisProHisGlyHisHisProHis-CONH₂

(X)

Example 8

A 0.1 M solution of 2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl 4- acetamidophenylcarbamate (1 mL) in acetonitrile was added to 0.1 M solution of peptide with formula X (0.5 mL) in water and the reaction mixture is stirred at room temperature for 24 hr. The crude mixture was purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent to give the final conjugate with formula XII (0.070 g, 84%) as a white powder after lyophilization.

Example 9 - Proposed synthesis of linkers from aminothiols.

Step 1.

A solution of an aminothiol (1.0 mmol) in dichloromethane (5.0 mL) is added to a solution of 2,2'-dithiobis(5-nitropyridine) (1.0 mmol) in dichloromethane (20 mL) containing 5% methanol (v/v), and the resulting mixture is stirred at room temperature for 1 fir. The solvent is removed and to the remaining crude product is added ethyl acetate (20 mL) and the resulting suspension is filtered on a glass filter. The filtrate is concentrated on a rotary evaporator and the residue is purified by performing reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions are collected and lyophilized to give (5-nitropyridin-2-yl)disulfanylalkylamine partly in the form of TFA salt.

Step 2.

(5-Nitropyridin-2-yl)disulfanylalkylamine (0.20 mmol) is dissolved in a 1 : 1 (v/v) mixture of saturated aqueous sodium bicarbonate and acetonitrile (1 mL) and is added a 15% solution of phosgene (1.0 mmol) in toluene. The reaction mixture is stirred at 0 °C for 15 min and then is treated with water and dichloromethane, and the organic layer is separated. The aqueous layer is washed by dichloromethane and the combined organic layer is dried (MgSC^), filtered and concentrated on a rotary evaporator. The crude product is purified by performing reverse phase preparative HPLC with acetonitrile 0.1% TFA as eluent and the fractions are lyophilized to get the final product. Example 10 - Synthesis of linkers from aminothiols.

Step 1.

A solution of 2-(butylamino)ethanethiol (0.118 g, 1.0 mmol) in dichloromethane (5.0 mL) was added over 15 min to a solution of 2,2'-dithiobis(5-nitropyridine) (0.620 g, 2.0 mmol) in dichloromethane (40 mL) containing 5% methanol (v/v), and the resulting mixture is stirred at room temperature for 1 hr. The dichloromethane was removed under reduced pressure and the remaining methanol solution of the crude product was cooled to room temperature. The resulting precipitate was removed and the filtrate was purified by reverse phase preparative HPLC with a gradient of acetonitrile and water containing 0.1% TFA as eluent. The fractions were collected and lyophilized to give (N-(2-(2-(5-nitropyridin-2- yl)disulfanyl)ethyl)butylamine (0.181 g, 63%) as a white powder.

1H NMR (400 MHz, CDC1₃) δ 9.87 (bs, 1H), 9.29 (d, J= 2.6 Hz, 1H), 8.40 (dd, J= 8.8, 2.6 Hz, 1H), 7.70 (d, J= 8.8 Hz, 1H), 3.32 (t, J= 6.8 Hz, 2H), 3.20 (t, J= 6.7 Hz, 2H), 2.99 (t, J = 7.9 Hz, 2H), 1.83 - 1.54 (m, 2H), 1.42 (sext, J= 7.4 Hz, 2H), 0.96 (t, J= 7.4 Hz, 3H).

¹³C NMR (100 MHz, CDC1₃) δ 166.2, 145.4, 142.4, 131.9, 120.7, 47.6, 45.9, 33.9, 28.2, 19.8, 13.5.

Step 2.

A 15%) solution of phosgene (0.18 mL, d= 0.924 g/mL, 0.25 mmol) in toluene was added to a solution of (N-(2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl)butylamine (0.057 g, 0.20 mmol) and triethylamine (0.020 g, 0.20 mmol) in toluene (3 mL). The reaction mixture was stirred at room temperature for 4 hr. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel using 9: 1 mixture of hexane and ethyl acetate as eluent to give butyl(2-(2-(5-nitropyridin-2-yl)disulfanyl)ethyl)carbamic chloride (0.049 g, 70%>) as a colorless oil.

1H NMR (400 MHz, CDC1₃) δ 9.31 (d, J = 2.6 Hz, 1H), 8.42 (dd, J = 9.0, 2.6 Hz, 1H), 7.83 (d, J= 9.0 Hz, 1H), 3.67 (t, J= 7.0 Hz, 2H), 3.45 (t, J= 7.2 Hz, 2H), 3.07 (t, J= 7.8 Hz, 2H), 1.65-1.54 (m, 2H), 1.36-1.25 (m, 1H), 0.92 (t, J= 7.4 Hz, 3H).

¹³C NMR (100 MHz, CDC1₃) δ 166.7, 149.0, 145.3, 142.4, 131.7, 119.7, 52.2, 49.0, 35.4, 29.7, 19.8, 13.7. REFERENCES

1. Engel, J.; Emons, G.; Pinski, J.; Schally, A. V. Expert Opin. Investig. Drugs, 2012, 21, 891

2. (a) Zitzmann, S., Ehemann, V. and Schwab, M. Cancer Res. 2002, 62, 5139; (b) Pasqualini, R. et al. Cancer Res 2000, 60, 722; (c) Ruoslahti, E., Bhatia, S. N. and Sailor, M. J. J. Cell Biol. 2010, 188, 759; (d) Ruoslahti, E. Adv. Mater. 2012, 24, 3747

3. Niculescu-Duvaz I. Curr. Opin. Mol. Ther. 2010, 12(3), 350

4. Phillips, G.L., Antibody-Drug Conjugates and Immunotoxins From Pre-Clinical Development to Therapeutic Applications, Humana Press, Springer, 2013, pl61-175.

5. Zhang, W.; Song, J.; Zhang, B.; Liu, L.; Wang, K.; Wang, R. Bioconjugate Chem. 2011, 22, 1410

Claims

Claims

1. A compound of formula (I)

wherein

Y is an electron- withdrawing group;

Z is a leaving group;

J is NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group.

R¹ and R² are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, halogen (e.g. F, CI, Br, I), alkoxy, acyloxy, ester, carboxylic acid, amino, amido, and substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl groups;

R³ and R⁴, where present, are each independently selected for each carbon (i.e. for each value of o) from the group consisting of H, halogen (e.g. F, CI, Br, I), alkoxy, acyloxy, ester, carboxylic acid, amino, amido, and substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl groups;

m is an integer between 1 and 12; n is an integer between 0 and 4; o is between 0 and 4.

2. A compound as claimed in claim 1 wherein 2 < m + n + o < 12; preferably 2 < m + n + o < 6, more preferably 2 < m + n + o < 4.

3. A compound as claimed in claim 1 or 2 wherein 3 < m + n + o < 12; preferably 3 < m + n + o < 6, more preferably 3 < m + n + o < 4

4. A compound as claimed in any preceding claim, wherein n is 0.

5. A compound as claimed in any preceding claim wherein

- Y is an electron-withdrawing group selected from N0₂, CN, COOH, ester, CONH₂, amide, CX₃ (wherein X is a halogen such as F, CI, Br, I), acyl, S0₃H; and/or

- Z is a leaving group selected from halides (such as fluoride, chloride, bromide, iodide), alkoxides, acyloxy, tosylate, mesylate, nitrate, phosphate, phenoxide, p- nitrophenoxide, o-nitrophenoxide, benzotriazoloxide and 2,5-pyrrolidinedione 1 -oxide; and/or

- R¹ and R² are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, F, OR^a, NHR^a, COOR^a, COOH, Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, and phenyl optionally substituted by one or more fluorine atoms, preferably R^J-R² are each independently H or optionally substituted Ci - C₆ alkyl; and/or

- R³ and R⁴ are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, F, OR^a, NHR^a, COOR^a, COOH, Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, and phenyl optionally substituted by one or more fluorine atoms, preferably R³-R⁴ are each independently H or optionally substituted Ci - C₆ alkyl; and/or -R⁵ is hydrogen, a Ci - C₆ alkyl, or a substituted or unsubstituted aryl group, such as phenyl; wherein R^a is a Ci - C₆ alkyl, C₂ - C₆ alkenyl, C₂ - C₆ alkynyl, C₃ - C₆ cycloalkyl, C₄ - C₆ cycloalkenyl, or phenyl optionally substituted by one or more fluorine atoms, preferably Ci-C₆ alkyl or phenyl optionally substituted by one or more fluorine atoms;

6. A compound as claimed in any preceding claim wherein at least one of R¹ and R² is not H, preferably wherein at least one o f R¹ and R² is Ci - C₆ alkyl, more preferably Ci - C₃ alkyl, even more preferably CH₃.

7. A compound as claimed in any preceding claim, wherein Z is such that Z and/or Z-H contains a chromophore, or a fluorophore, or is a phosphorescent compound, preferably a chromophore.

8. A compound as claimed in any preceding claim, wherein Z is selected from halide (fluoride, chloride, bromide, iodide), /?ara-nitrophenoxide, and 2,5-pyrrolidinedione 1 -oxide, preferably chloride.

9. A compound as claimed in any preceding claim wherein the group Y is in the para or ortho position, preferably para position.

10. A compound as claimed in any preceding claim wherein Y is para -N0₂ or para- COOH, preferably para -N0₂.

11. A compound as claimed in any preceding claim wherein at least one of R³ and R⁴ is not H, preferably wherein at least one o f R³ and R⁴ is Ci - C₆ alkyl, more preferably Ci - C₃ alkyl, even more preferably CH₃.

12. A compound as claimed in any of claims 1 to 10 of formula II

wherein Y is one or more nitro groups at the para and/or ortho positions;

R¹ and R² are each independently selected for each carbon (i.e. for each value of m) from the group consisting of H, halogens (e.g. F, CI, Br, I), Ci- C₆ alkoxy, Ci- C₆ alkyl, C₂- C₆ alkenyl groups, and phenyl optionally substituted by one or more fluorine atoms; and

Q is as defined for formula I; and m is an integer from 1 to 10, preferably 2-10.

13. A compound of any of claims 1 to 10 having formula III

wherein Y, R¹, R² and Q are as defined in any preceding claim, m is and integer of 0 to 10 and each group R^s and R° are independently selected from the group consisting of H and Ci- C₆ alkyl groups, wherein at least one group R^s is a Ci- C₆ alkyl group and/or at least one group R° is a Ci- C₆ alkyl group.

14. A process for producing a compound according to any of claims 1 to 13 comprising the steps of: i) reacting a compound of formula (IV):

with a compound of formula (V):

to obtain a compound of formula (VI):

ii) reacting the compound of formula (VI) with triphosgene or a compound of formula (VII)

to obtain a compound of formula (I), (II) or (III) as defined in any of claims 1 to 13, wherein m, n and o and the groups Y, Z and R^]-R⁴ are as defined in any of the preceding claims; J is NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group; wherein Z=Z' or Z≠Z', and Z' is a leaving group selected from halides, (such as fluoride, chloride, bromide, iodide), alkoxides, 2,5-pyrrolidinedione 1 -oxide, tosylate, mesylate, nitrate, phosphate, phenoxide, /?-nitrophenoxide, o-nitrophenoxide benzotriazoloxide and 2,5-pyrrolidinedione 1 -oxide.

A conjugate of formula (VIII)

wherein R'-R⁴, m, n and o are as defined in any of claims 1 to 13; wherein J is NR⁵, with R⁵ being hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl or aryl group; wherein A is such that A-SH is a carrier entity such as an antibody, antibody fragment, nanoparticle, protein, protein fragment or peptide; wherein B and W are such that B-WH is a bioactive agent, a diagnostic agent or a visualization agent; and

W = NH or O; or a pharmaceutically acceptable salt, hydrate or solvate thereof

16. A conjugate as claimed in claim 15 wherein A has affinity for a tumor specific antigen.

17. A conjugate as claimed in claim 15 or claim 16 wherein A is an antibody, antibody fragment, antibody construct, construct of antibody fragments or a tumor-homing peptide, preferably an antibody, more preferably a monoclonal antibody.

18. A conjugate as claimed in claims 15 to 17, wherein the conjugate is an Antibody Drug Conjugate (ADC).

19. A conjugate as claimed in claims 15 to 18 wherein the conjugate is self-immolative (i.e. self-eliminating).

20. A conjugate as claimed in claims 15 to 19, wherein B-WH is: - a hydroxyl-containing bioactive agent such that W = O, such as doxorubicin everolimus, becalutamide, cabazitaxel, irinotecan, bicalutamide, dasatinib, degarelix, dexamethasone, docetaxel, raloxifene, fulvestrant, goserilin, topotecan, ixabepilone and leuprolide. or

- an amine-containing bioactive agent such that W = NH, such as methotrexate, imiquimod, melphalane, pemetrexed disodium, aminolevulinic acid, dactinomycine, crizotinib, dafrafenib, pralatrexate, ibrutinib, lenalidomide, leucovorin, mitomycin C, pomalidomide, and tio guanine.

21. A process for producing the conjugate of claims 15 to 20 comprising the steps of: a) reacting a compound as claimed in claims 1 to 13 with a bioactive agent, a diagnostic agent or a visualization agent of formula B-WH to obtain a compound of formula (IX):

b) reacting said compound of formula (IX) with the carrier entity of formula A- SH; wherein R^!-R⁴, m, n, o, Y, A, B, W and J are as defined in any preceding claim.

22. A conjugate as claimed in claims 15 to 20 for use in the treatment of cancer, preferably wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer and adrenal cancer.

23. A method of treating cancer comprising administering to an animal, preferably a mammal, e.g. human, an effective amount of a conjugate as claimed in claims 15 to 20, preferably wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer and adrenal cancer.

24. Use of a conjugate as claimed in any one of claims 15 to 20 in the manufacture of a medicament for the treatment of cancer, preferably wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, liver cancer and adrenal cancer.