Tubulysin compounds, methods of making and use

BACKGROUND OF THE INVENTION

The present invention relates to compounds structurally similar to tubioules, their ligand conjugates, methods of making and using such compounds and conjugates, and compositions containing such compounds and conjugates.

Tubulisins are cytotoxins originally isolated from myxobacteria Archangium gephyra or Angiococcus disciformis, each of which produces a different mixture of tubiques (Sasse et al. 2000, Reichenbach et al. 1998). Their crystalline structure and path of biosynthesis were explained (Steinmetz et al., 2004) and their biosynthetic genes were sequenced (Hoefle et al. 2006b). Preitulizine, a biosynthetic precursor of tubioules, has also been shown to show some activity (Ullrich et al. 2009). (A full list of documents cited here is given by the first author or inventor and the year is given at the end of this specification).

Tubulisins belong to a group of naturally occurring antimitotic polypeptides and depsipeptides, which include fomopsins, dolastatins and cryptophycins (Hamel 2002). There are also antimitotic agents other than polypeptides or depsipeptides, e.g. paclitaxel, majtansins and epothilones. During mitosis, cellular microtubules reorganize to form a mitotic spindle, which requires fast assembly and disassembly of the proteins forming the α- and β-tubulin microtubules. Antimitotic agents block this process and prevent the process of mitosis in the cell. At the molecular level, the exact mechanism of action of one agent may vary the effects of another agent. Tubulins prevent the assembly of tubulins in the microtubules, which causes the cells they are affected to remain in the G2 / M phase and undergo apoptosis (Khalil et al. 2006).

Tubulisines have a tetrapeptidyl backbone built of one proteinogenic amino acid subunit and three non-proteinaceous amino acid subunits as shown in formula (A): N-methylpipecolic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), and either tubufenylalanine (Tup, R 'means H) or tubutyrosine (Tut, R is OH). Between the better-known naturally occurring tubiisins (named A, B, etc.), the structural site varieties occur at residues R ', R "and R"' (A) as in formula (I) and are shown in Table 1:

<img file="PL2956173T3_D0001.tif" />

<td colspan="4">Table 1 - Naturally occurring tubulins</td>

<td>Tubulizyna</td><td>R '</td><td>R "</td><td>R " '</td>

<td>AND</td><td>OH</td><td>OC (= O) Me</td><td>CH₂OC (= O) / - Bu</td>

<td>B</td><td>OH</td><td>OC (= O) Me</td><td>CH₂OC (= O) n-Pr</td>

<td>C</td><td>OH</td><td>OC (= O) Me</td><td>CH₂OC (= O) Et</td>

<td>D</td><td>H</td><td>OC (= O) Me</td><td>CH₂OC (= O) / - Bu</td>

<td>E</td><td>H</td><td>OC (= O) Me</td><td>CH₂OC (= O) n-Pr</td>

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<td>F</td><td>H</td><td>OC (= O) Me</td><td>CH₂OC (= O) Et</td>

<td>G</td><td>OH</td><td>OC (= O) Me</td><td>CH₂OC (= O) CH = CH₂</td>

<td>H</td><td>H</td><td>OC (= O) Me</td><td>CH₂OC (= O) Me</td>

<td>AND</td><td>OH</td><td>OC (= O) Me</td><td>CH₂OC (= O) Me</td>

<td>AT</td><td>H</td><td>OC (= O) Me</td><td>H</td>

<td>V</td><td>H</td><td>OH</td><td>H</td>

<td>Y</td><td>OH</td><td>OC (= O) Me</td><td>H</td>

<td>WITH</td><td>OH</td><td>OH</td><td>H</td>

<td>Pretubulizyna</td><td>H</td><td>H</td><td>Me</td>

In addition, other naturally occurring tubulins have been designated (Chai et al., 2010).

Kaur et al. 2006 studied the anti-proliferative properties of tubulisin A and found that it was much stronger than other antimitotic agents such as paclitaxel and vinblastine, and was active in the study of xenografts of various tumor cell lines. In addition, tubulisin A induces apoptosis in tumor cells, whereas it has no such activity in normal cells, and has demonstrated significant potential anti-angiogenic properties in in vitro assays. Antimitotic properties of other tubulin were also evaluated and, in general, were found to be favorable for non-cytosine antimitotic agents (see, e.g., Balasubramanian et al., 2009, Steinmetz et al., 2004, Wipf et al., 2004). For these reasons, there is considerable interest in tubiizols as anti-cancer agents (see, e.g., Domling et al. 2005c, Hamel 2002).

Many publications describe efforts focused on the synthesis of tubiisins, including: Balasubramanian et al. 2009; Domling et al. 2006; Hoefle et al. 2003; Neri et al. 2006; Peltier et al. 2006; Sani et al. 2007; Sasse et al. 2007; Shankar et al. 2009; Shibue et al. 2009 and 2010 and Wipf et al. 2004. Other publications describe the study structure-activity relationship (SAR), through the development and evaluation of tubulinizine analogues or derivatives: Balasubramanian et al. 2008 and 2009; Chai et al. 2011; Domling 2006; Domling et al. 2005a; Ellman et al. 2013; Hoefle et al. 2001 and 2006a; Pando et al. 2011; Patterson et al. 2007 and 2008; Richter 2012a, 2012b and 2012c; Shankar et al. 2013; Shibue et al. 2011; Sreejith et al. 2011; Vlahov et al. 2010a; Wang et al. 2007; Wipf et al. 2007 and 2010 and Zanda et al. 2013.

Domling et al. 2005 disclosed tubioules conjugates with a partner molecule described generically as a polymer or biomolecule, but the actual examples were limited to polyethylene glycol (PEG) as a partner molecule. Also Cheng et al. 2011 revealed tubulinizine analogs adapted for use in conjugates. Other documents disclosing tubiouin conjugates were articles by Loyd et al. 2008 and 2010; Jackson et al. 2013; Vlahov et al. 2008a, 2008b and 2010b; Leamon et al. 2008 and 2010; Reddy et al. 2009 and Low et al. 2010. Leung et al. 2002 revealed polyanionic polypeptides that can be conjugated with drugs (including tubiisins) to improve their bioactivity and solubility in water.

Davis et al. 2008 and Schluep et al. 2009 disclosed formulations based on cyclodextrin in which tubulins were covalently attached to the cyclodextrin through the hydrazide disulfide linker molecule linked to the Tup / Tut carboxyl group. EP2292639 and WO2001 / 069116 disclose tubiysin derivatives and their synthesis.

Deacetylation of the Tuv subunit (i.e., R &amp; quot; in formula (A) is a hydroxyl group instead of acetyl) supposedly leads to loss of biological activity (Domling et al., 2006). In studies of U and V tubulises, the first being acetylated and the other deacetylated , it was reported that VV showed less potency of about 200X to 600X, depending on

- 3 EP 2956173 from the study (Balasubramanian et al. 2009). Since the acetyl group is sensitive to hydrolysis, deacetylation at the R "position is a problem, since the potential unstable medium leads to loss of activity in the development of tubuline analogs for pharmaceutical applications.

BRIEF DESCRIPTION OF THE INVENTION

We have detected that it is possible to protect against the loss of biological activity associated with deacetylation, as discussed above, by exchanging the acetyl group at the R "position on the carbamate group. The carbamate group as described herein does not cause significant loss of biological activity but is more stable.

Accordingly, in one aspect, the present invention provides a compound with the structural structure depicted in formula (I)

<img file="PL2956173T3_D0002.tif" />

where _R1 is H, unsubstituted or substituted C 1 -C 10 alkyl, unsubstituted or substituted C 2 -C 10 alkenyl, unsubstituted or substituted C 2 -C 10 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted (CH 2) 1-2 O (C 1 -C 10 alkyl), unsubstituted or substituted (CH 2) 1-2 O (C 2 -C 10 alkenyl), unsubstituted or substituted (CH 2) 1-2 O (C 2 -C 10 alkynyl), (CH 2) 1-2OC (= O) (C1-C10 alkyl), unsubstituted or substituted (CH2) 1-2OC (= O) (C2-C10 alkenyl), unsubstituted or substituted (CH2) 1-2OC (= O) (C2-C10) alkynyl), unsubstituted or substituted C (= O) (C1-C10 alkyl), unsubstituted or substituted C (= O) (C2-C10 alkenyl), unsubstituted or substituted by C (= O) (C2-C10 alkynyl) .unsubstituted or substituted cycloaliphatic, unsubstituted or substituted heterocycloaliphatic, unsubstituted or substituted arylalkyl, or unsubstituted or substituted alkylaryl;

_R2 is H, unsubstituted or substituted C 1 -C 10 alkyl, unsubstituted or substituted C 2 -C 10 alkenyl, unsubstituted or substituted C 2 -C 10 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted (CH 2) 1-2 O (C 1 -C 10 alkyl), unsubstituted or substituted (CH 2) 1-2 O (C 2 -C 10 alkenyl), unsubstituted or substituted (CH 2) 1-2 O (C 2 -C 10 alkynyl), (CH 2) 1-2OC (= O) (C1-C10 alkyl), unsubstituted or substituted (CH2) 1-2OC (= O) (C2-C10 alkenyl), unsubstituted or substituted (CH2) 1-2OC (= O) (C2-C10) alkynyl), unsubstituted or substituted C (= O) (C1-C10 alkyl), unsubstituted or substituted C (= O) (C2-C10 alkenyl), unsubstituted or substituted by C (= O) (C2-C10 alkynyl) .unsubstituted or substituted cycloaliphatic, unsubstituted or substituted heterocycloaliphatic, unsubstituted or substituted arylalkyl, or unsubstituted or substituted alkylaryl, or_t / ^ (R^2a)₂ | - (CH₂)and-₄-;

where every R^2a is independently H, NH 2, NHMe, Cl, F, Me, Et, or CN;

_R^3a_{and R}^3b

Are each independently H, C 1 -C 5 alkyl, CH 2 (C 5 -C 6 cycloalkyl), CH 2 C 6 H 5, C 6 H 5, or CH 2 CH 2 OH;

R⁴ is

<img file="PL2956173T3_D0003.tif" />

where R^4a is H or a C1-C3 alkyl group; and Y is H, OH, Cl, F, CN, Me, Et, NO2, or NH2;

R⁵ is H, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, CO (C1-C5 alkyl), CO (C2-C5 alkenyl), or CO (C2-C5 alkynyl);

W is O or S (preferably O); and

N is 0, 1, or 2;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a conjugate comprising a compound of formula (I) bound covalently to a target molecule that is specifically or preferentially associated with a chemical molecule on a target cell, which target cell is preferably a cancer cell. Preferably, the targeting molecule is an antibody - more preferably a monoclonal antibody and even more preferably a human monoclonal antibody - or an antigen binding portion thereof and the chemical molecule is a tumor-coupled antigen. The tumor-associated antigen may be one of the tumor cells or one secreted by the tumor cell into the surrounding extracellular space.

In another embodiment, there is provided a substance composition comprising a compound of the present invention and a linker molecule having a reactive functional group suitable for coupling to a target molecule.

In another embodiment, the present invention provides a compound for use in a method of treating cancer in a patient suffering from such a cancer, comprising administering to the patient a therapeutically effective amount of a compound of the present invention or a conjugate thereof with a target molecule (particularly an antibody). In another embodiment, a compound of the present invention or a conjugate thereof with a target molecule (particularly an antibody) is provided for use in the preparation of a medicament for the treatment of cancer in a patient suffering from such a cancer. The cancer may cause kidney, stomach, lung or ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1, 2a-2b, and 3 show, in combination, the synthesis scheme of compound (III-1). Fig. 4 shows a synthesis scheme of the compound (III-2).

Fig. 5 shows a synthesis scheme of compounds (I-2) and (I-3).

Figs. 6a-6c show, in combination, a synthetic scheme of compounds (III-4) and (III-5).

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Fig. 7 shows a synthesis scheme of the compound (I-1).

Fig. 8 shows a synthesis scheme of the compound (1-4).

Figs. 9a and 9b show, in combination, a synthesis scheme of compound (III-6).

Figures 10 and 11 show synthetic schemes for the intermediates useful to form the compounds of the present invention.

Figure 12 shows synthetic schemes for additional compounds of the present invention.

Figs. 13a-13d show the biological activity of some of the compounds of the present invention.

Fig. 14 shows the in vitro activity of the conjugate of the present invention.

Fig. 15 shows the in vivo activity of the conjugate of the present invention.

Figures 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b and 20 show additional in vivo data regarding the activity of the conjugates of the present invention.

Figures 21 and 22 show synthetic schemes for additional compounds of the present invention, showing structural changes in the carbamate group.

Figure 23 shows a synthesis scheme of the compounds of the present invention suitable for coupling in reactions of the "click" type.

Figure 24 shows a synthesis scheme of the compounds of the present invention suitable for coupling via an aliphatic amine group.

DETAILED DESCRIPTION OF THE INVENTION

Each VH and VL contains three CDRs and four FRs arranged from the amino to carboxylic terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions contain a binding domain that interacts with the antigen. The constant regions may participate in binding the antibody to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The antibody is said to "bind specifically" to the antigen X if the antibody binds to the X antigen with a K D of 5 x 10 The constant regions may participate in binding the antibody to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The antibody is said to "bind specifically" to the antigen X if the antibody binds to the X antigen with a K D of 5 x 10 The constant regions may participate in binding the antibody to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The antibody is said to "bind specifically" to the antigen X if the antibody binds to the X antigen with a K D of 5 x 10^-8 M or less, more preferably 1 x 10^-8M or less, more preferably 6 x 10^-9 M or less, more preferably 3 x 10^-9 M or less, even more preferably 2 x 10^-9 M or less. The antibody may be chimeric, humanized, or, preferably, human. The heavy chain constant region may be engineered to affect the type or size of glycosylation, extend the half-life of the antibody, increase or decrease interactions with effector cells or the complement system, or modulate certain other features. The structure can be obtained by replacing, adding, or deleting one or more amino acids or by replacing the domain with a domain of another type of immunoglobulin, or a combination of the above.

- "Antigen-binding fragment of an antigen" and "antigen-binding portion" of an antibody (or simply "part of an antibody" or "antibody fragment") are one or more antibody fragments that retain the ability to specifically bind to an antigen. the function of binding to the antigen may have antibody full-length antibody fragments, such as (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) the F (ab ') fragment²a divalent fragment comprising two Fab fragments linked by a disulphide bridge in the hinge region; (iii) a Fab 'fragment, which is essentially a Fab fragment with part of the hinge region (see, e.g., Abbas et al., Cellular and Molecular Immunology, 6th edition, Saunders Elsevier 2007); (iv) a Fd fragment consisting of the VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of a single antibody arm, (vi) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of the VH domain; vii) an isolated complementarity determining region (CDR); and (viii) a nanobase, a heavy chain variable region comprising one variable domain and two constant domains. Preferred antigen binding fragments are Fab, F (ab ') 2, Fab', Fv, and Fd fragments. Furthermore, although the two domains of the Fv, VL and VH fragment, encoded by separate genes, they can be joined using recombination methods with a synthetic linker, so they can be made from one protein chain in which the VL and VH regions pair to form monovalent molecules (known as one Fv chain, or scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also encompassed by the term "antigen-binding portion" of the antibody. 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also encompassed by the term "antigen-binding portion" of the antibody. 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also encompassed by the term "antigen-binding portion" of the antibody.

"Isolated antibody" means an antibody essentially free of other antibodies having different antigenic characteristics (e.g., an isolated antibody that specifically binds to the X antigen does not essentially contain antibodies that specifically bind antigens other than the X antigen). An isolated antibody that specifically binds to the X antigen may, however, be cross-reactive with other antigens, such as X-antigen molecules of other species. In certain example embodiments, the isolated antibody binds specifically to the human X antigen and does not cross-react with other (non-human) X antigens. Furthermore, the isolated antibody may be substantially free of other cellular material and / or chemical compounds.

A "monoclonal antibody" or "monoclonal antibody composition" means a preparation of antibody molecules with one molecular composition that exhibits one specific binding specificity and affinity to a particular epitope.

"Human antibody" means an antibody having variable regions in which both the framework and CDR regions (and the constant region, if present) are derived from human germline immunoglobulin sequences. Human antibodies may contain subsequent modifications, including natural or synthetic modifications. The human antibodies can contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis or somatic mutation in vivo). However, a "human antibody" does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences.

"Human monoclonal antibody" means an antibody having a single binding specificity comprising variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that comprises a B cell derived from a non-human transgenic animal, e.g. a transgenic mouse having a human heavy chain transgene genome and a light chain transgene linked to the immortalized cell.

"Aliphatic" means a linear or branched, saturated or unsaturated, non-aromatic group containing the specified number of carbon atoms (e.g., as in "C3 aliphatic,"

- "C1-C5 aliphatic," or "C1 to C5 aliphatic," the last two terms are synonyms of an aliphatic group having 1 to 5 carbon atoms) or, where the number of carbon atoms is not clearly defined, from 1 to 4 atoms carbon (2 to 4 carbon atoms in the case of unsaturated aliphatic groups).

"Alkyl" means a saturated aliphatic group using the same convention to determine the number of carbon atoms. For example, C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl and the like. "Alkylene" means a divalent analog of an alkyl group, such as CH 2 CH 2, CH 2 CH 2 CH 2 and CH 2 CH 2 CH 2 CH 2.

"Alkenyl" means an aliphatic group having at least one carbon-carbon double bond using the same convention to determine the number of carbon atoms. For example, C2-C4 alkenyl groups include, but are not limited to, ethenyl (vinyl), 2-propenyl (allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E (i.e. or Z-) 2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl) and the like.

"Alkynyl" means an aliphatic group having at least one carbon-carbon triple bond, using the same convention to determine the number of carbon atoms. For example, C2-C4 alkynyl groups include ethynyl (acetylenyl), propargyl (prop-2-ynyl), 1-propynyl, but-2-ynyl and the like.

"Cycloaliphatic" means a saturated or unsaturated non-aromatic hydrocarbon group having from 1 to 3 rings, with each ring having from 3 to 8 (preferably from 3 to 6) carbon atoms. "Cycloalkyl" means a cycloaliphatic group in which each ring is saturated. "Cycloalkenyl" means a cycloaliphatic group in which at least one ring has at least one carbon-carbon double bond ". "Cycloalkynyl" means a cycloaliphatic group wherein at least one ring has at least one carbon-carbon triple bond. For example, cycloaliphatic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl. The preferred cycloaliphatic groups are cycloalkyl groups, especially cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. "Cyclyleneilene" means the divalent counterpart of a cycloalkyl group.

"Heterocycloaliphatic" means a cycloaliphatic group in which at least one of its rings has been replaced by up to three (preferably 1 to 2) carbon atoms a heteroatom independently selected from N, O, or S, where N and S optionally can be oxidized and N optionally can be quaternized . Similarly, "heterocycloalkyl", "heterocycloalkenyl" and "heterocycloalkynyl" mean a cycloalkyl, cycloalkenyl or cycloalkynyl group, respectively, in which at least one ring has been modified as above. Exemplary heterocycloaliphatic groups include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulphoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl, tetrahydro-1,1-dioxotienyl, 1,4-dioxanyl, tietanyl, and the like. "Heterocycloalkylene" means the divalent counterpart of a heterocycloalkyl group.

"Alkoxy", "aryloxy", "alkylthio" and "arylthio" are -O (alkyl), -O (aryl), -S (alkyl), and -S (aryl), respectively. Examples are methoxy, phenoxy, methylthio and phenylthio, respectively.

"Halogen" or "halo" means fluorine, chlorine, bromine or iodine.

"Aryl" means a hydrocarbon group with a mono-, bi- or tricyclic ring system in which each ring has from 3 to 7 carbon atoms and at least one ring is aromatic. Rings in the ring system can be condensed with one another (as in naphthyl) or joined together (as in biphenyl) and can be condensed or joined to non-aromatic rings (as in indanyl or cyclohexylphenyl). Further, for example, aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracenyl and phenyl.

- acenaphthyl. "Arylene" means the divalent equivalent of an aryl group, for example 1,2-phenylene, 1,3-phenylene or 1,4-phenylene.

"Heteroaryl" means a group having a mono-, bi-, or tricyclic ring system in which each ring has from 3 to 7 carbon atoms and at least one ring is an aromatic ring containing from 1 to 4 heteroatoms independently selected from N, O, or S, where N and S optionally can be oxidized and N optionally can be quaternized. Such an aromatic ring containing at least one heteroatom may be condensed with other types of rings (as in benzofuranyl or tetrahydroisoquinolyl) or directly attached to other types of rings (as in phenyl pyridyl or 2-cyclopentylpyridyl). Further, for example, heteroaryl groups include pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, quinazalinyl, naphthyridinyl, benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl, phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl, carbazolyl, dibenzothiophenyl, acridinyl, and the like. "Heteroarylene" means the divalent equivalent of an aryl group.

Where it is indicated that a group may be substituted, such as the use of the phrases "substituted or unsubstituted" or "optionally substituted" as in "substituted or unsubstituted C1-C5 alkyl" or "optionally substituted heteroaryl" indicates that such a group may have one or more independently selected substituents, preferably one to five, more preferably one or two. Substituents and substitution patterns may be selected by one of skill in the art, including the group to which a substituent is attached, to provide chemically stable compounds that can be synthesized by techniques known in the art as well as the methods described herein.

"Arylalkyl", "(heterocycloaliphatic) alkyl", "arylalkenyl", "arylalkynyl", "biarylalkyl" and the like mean alkyl, alkenyl, or alkynyl groups as the case may be, substituted with an aryl, heterocycloaliphatic, biaryl group, etc., depending from the case, with an open (unsaturated) valency of the alkyl, alkenyl or alkynyl group, for example as in benzyl, phenethyl, N-imidazoleethyl, N-morpholinoethyl, and the like, in contrast, "alkylaryl", "alkenylcycloalkyl" and the like mean aryl groups, cycloalkyl groups, etc., as the case may be, substituted with an alkyl, alkenyl group, etc., as the case may be for example methyl phenyl (tolyl) or allylcyclohexyl. "Hydroxyalkyl", "haloalkyl", "alkylaryl","cyanoaryl" and the like means an alkyl, aryl group, etc., as the case may be, substituted with one or more identified substituents (hydroxyl, halo, etc., as the case may be).

For example, acceptable substituents include, but are not limited to, alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl, aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especially fluoro), haloalkyl (especially trifluoromethyl), hydroxy, hydroxyalkyl (especially hydroxyethyl), cyano, nitro, alkoxy, -O (hydroxyalkyl), -O (haloalkyl) (especially -OCF3), -O (cycloalkyl), -O (heterocycloalkyl), -O (aryl), alkylthio, arylthio, = O, = NH, = N (alkyl), = NOH, = NO (alkyl), -C (= O) (alkyl), -C (= O) H, -CO2H, -C (= O) NHOH, -C (= O) O (alkyl), C (= O) O (hydroxyalkyl), -C (= O) NH2, -C (= O) NH (alkyl), -C (= O) N (alkyl) 2, -OC (= O) (alkyl), OC (= O) (hydroxyalkyl), -OC (= O) O (alkyl), -OC (= O) O (hydroxyalkyl), -OC (= O) NH2 , -OC (= O) NH (alkyl), OC (= O) N (alkyl) 2, azido, -NH 2, -NH (alkyl), -N (alkyl) 2, -NH (aryl), -NH (alkyl) hydroxyalkyl), -NHC (= O) (alkyl), NHC (= O) H,-NHC (= O) NH2, -NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2, -NHC (= NH) NH2, -OSO2 (alkyl), -SH, -S (alkyl), -S (aryl), -S (cycloalkyl), -S (= O) alkyl, -SO2 (alkyl), -SO2NH2, -SO2NH (alkyl), -SO2N (alkyl) 2, and the like.

Where the substituent group is an aliphatic group, the preferred substituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo, hydroxy, cyano, nitro, alkoxy, O (hydroxyalkyl), -O (haloalkyl), -O (cycloalkyl), -O (heterocycloalkyl) , -O (aryl), alkylthio, arylthio, = O, = NH, = N (alkyl), = NOH, = NO (alkyl), -CO2H, -C (= O) NHOH, -C (= O) O (alkyl), -C (= O) O (hydroxyalkyl), - 295-2173

C (= O) NH 2, -C (= O) NH (alkyl), -C (= O) N (alkyl) 2, -OC (= O) (alkyl), -OC (= O) (hydroxyalkyl), -OC (= O) O (alkyl), -OC (= O) O (hydroxyalkyl), -OC (= O) NH2, -OC (= O) NH (alkyl), -OC (= O) N (alkyl) ) 2, azido, -NH 2, -NH (alkyl), -N (alkyl) 2, -NH (aryl), -NH (hydroxyalkyl), -NHC (= O) (alkyl), -NHC (= O) H , -NHC (= O) NH2, NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2, -NHC (= NH) NH2, -OSO2 (alkyl), -SH, -S (alkyl), -S (aryl), S (= O) alkyl, -S (cycloalkyl), -SO2 (alkyl), -SO2NH2, -SO2NH (alkyl), and -SO2N (alkyl) 2. More preferred substituents are halo, hydroxyl, cyano, nitro, alkoxy, -O (aryl), = O, = NOH, = NO (alkyl), -OC (= O) (alkyl), -OC (= O) O ( alkyl), -OC (= O) NH 2, -OC (= O) NH (alkyl), -OC (= O) N (alkyl) 2, azido, -NH 2, -NH (alkyl), -N (alkyl) 2, -NH (aryl), NHC (= O) (alkyl), -NHC (= O) H, -NHC (= O) NH2, NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2 and -NHC (= NH) NH2. Particularly preferred substituents are phenyl, halo,

and -SO2N (alkyl) 2. More preferred substituents are alkyl, alkenyl, halo, haloalkyl, hydroxy, hydroxyalkyl, cyano, nitro, alkoxy, -O (hydroxyalkyl), -C (= O) (alkyl), -C (= O) H, -CO2H, - C (= O) NHOH, C (= O) O (alkyl), -C (= O) O (hydroxyalkyl), -C (= O) NH 2, -C (= O) NH (alkyl), -C ( = O) N (alkyl) 2, -OC (= O) (alkyl), OC (= O) (hydroxyalkyl), -OC (= O) O (alkyl), -OC (= O) O (hydroxyalkyl), -OC (= O) NH2, -OC (= O) NH (alkyl), OC (= O) N (alkyl) 2, -NH2, -NH (alkyl), -N (alkyl) 2, -NH (aryl) ), -NHC (= O) (alkyl), -NHC (= O) H, -NHC (= O) NH2, NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2, and -NHC (= NH) NH2. Particularly preferred substituents are C 1 -C 4 alkyl, cyano, nitro, halo and C 1 -C 4 alkoxy. -C (= O) O (hydroxyalkyl), -C (= O) NH 2, -C (= O) NH (alkyl), -C (= O) N (alkyl) 2, -OC (= O) (alkyl ), OC (= O) (hydroxyalkyl), -OC (= O) O (alkyl), -OC (= O) O (hydroxyalkyl), -OC (= O) NH2, -OC (= O) NH (alkyl) ), OC (= O) N (alkyl) 2, -NH2, -NH (alkyl), -N (alkyl) 2, -NH (aryl), -NHC (= O) (alkyl), -NHC (= O) ) H, -NHC (= O) NH 2, NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2, and -NHC (= NH) NH 2. Particularly preferred substituents are C 1 -C 4 alkyl, cyano, nitro, halo and C 1 -C 4 alkoxy. -C (= O) O (hydroxyalkyl), -C (= O) NH 2, -C (= O) NH (alkyl), -C (= O) N (alkyl) 2, -OC (= O) (alkyl ), OC (= O) (hydroxyalkyl), -OC (= O) O (alkyl), -OC (= O) O (hydroxyalkyl), -OC (= O) NH2, -OC (= O) NH (alkyl) ), OC (= O) N (alkyl) 2, -NH2, -NH (alkyl), -N (alkyl) 2, -NH (aryl), -NHC (= O) (alkyl), -NHC (= O) ) H, -NHC (= O) NH 2, NHC (= O) NH (alkyl), -NHC (= O) N (alkyl) 2, and -NHC (= NH) NH 2. Particularly preferred substituents are C 1 -C 4 alkyl, cyano, nitro, halo and C 1 -C 4 alkoxy.

Where the range is given, as in "C1-C5 alkyl" or "5 to 10%", this range includes end points of the range, as in C1 and C5 in the first case and 5% and 10% in the second case.

If specific stereoisomers are not clearly labeled (e.g., as a thickened or intermittent bond on a suitable stereocentre in a structural formula, by displaying a double bond having E or Z configuration in the structural formula, or by using a nomenclature specifying stereochemistry), all stereoisomers are included in the scope of the invention as pure compounds as well as mixtures thereof. Unless otherwise stated, individual enantiomers, diastereomers, geometric isomers, and combinations and mixtures thereof are all included in the present invention.

Those skilled in the art will recognize that the compounds may have tautomeric forms (e.g., ketic and enol forms), resonance forms, and zwitterionic forms that are equivalent to those shown in the structural formulas used herein, and that the structural patterns include such tautomeric forms, resonant forms. or zwitterions.

"Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (e.g. in the human body) to produce a parent compound or a salt thereof or has activity similar to that of the parent compound that is per se. Suitable esters include C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl esters, especially methyl, ethyl or npropyl esters.

"Pharmaceutically acceptable salt" means a salt of the compound suitable for use in a pharmaceutical preparation When the compound contains one or more basic groups, the salt may be an acid addition salt such as sulphate, hydrogen bromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamonate (benzoate), hydroiodide, nitrate, hydrochloride, lactate, methylsulphate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, etc. Where the compound has one or more acidic groups, the salt may be a salt

Such as calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like. Polymorphic crystalline forms and solvates are also within the scope of the present invention.

COMPOSITIONS

In formula (I), repeated below for ease,

<img file="PL2956173T3_D0004.tif" />

group R¹ preferably Me, Et, n-Pr, i-Pr, or

<img file="PL2956173T3_D0005.tif" />

more preferably the last one.

Also in formula (I), the R group² preferably C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, CH2OC (= O) C1-C5 alkyl, CH2OC (= O) C1-C5 alkenyl, CH2OC (= O) C1-C5 alkynyl, [~ ( CH₂) I-2 ~ ^ ^or | - (CH₂)and.₂-fi

Also in formula (I), preferred N (R) groups^3a) (R^3b) are:

<img file="PL2956173T3_D0006.tif" />

with an especially preferred H for R^3a and R^3b and Me for others. In other preferred embodiments, both Rs^3a and R^3b they are H or both are Me, or one of R^3a and R^3b is H and the other C₆H₅.

In another preferred embodiment of R^3a and R^3b are independently H, a C1-C5 alkyl group, CH2 (C5-C6 cycloalkyl), CH2C6H5 or CH2CH2OH;

In the definitions of R¹ and R² in formula (I) wherein the group is defined as being substituted or unsubstituted, it is preferably unsubstituted.

In the formula of this specification, the bond passes through a phenyl ring between two phenyl carbons meaning that the group attached to the bond may be located in the ortho, meta or para position of the phenyl ring. For example, a pattern

<img file="PL2956173T3_D0007.tif" />

shows

<img file="PL2956173T3_D0008.tif" />

Synthesis of the equivalents of Tuv and Tup tubulizine subunits with different R groups² and R⁴ was clarified by Cheng et al. , 2011.

- EP 2956173

In a preferred embodiment of the compounds according to formula (I), R¹ is

<img file="PL2956173T3_D0009.tif" />

R² is C1-C5 alkyl (especially Me or n-Pr) or

<img file="PL2956173T3_D0010.tif" />

one of R^3a and R^3b is H and the other is Me; R⁴ is

<img file="PL2956173T3_D0011.tif" />

where Y is H or NO2 and R^4a is H, Me, or Et; R⁵ is Me; W is O, in is 1.

In another preferred embodiment of the compounds according to formula (I), n is 1, W is 0, Y in R⁴ is H or NO2 (preferably H), and R² is

<img file="PL2956173T3_D0012.tif" />

A compound according to this preferred embodiment is represented by formula (Ia)

<img file="PL2956173T3_D0013.tif" />

where Y is H or NO2; R^4a is H, Me, or Et; and R^3a and R^3b are independently H, C 6 H 5, Me, or Et; or a pharmaceutically acceptable salt thereof.

Even more preferably, the compound has the structure shown in formula (Ia '):

<img file="PL2956173T3_D0014.tif" />

where R^4a is H, Me, or Et; and R^3a and R^3b are independently H, C 6 H 5, Me, or Et; or a pharmaceutically acceptable salt thereof.

- EP 2956173

And still in another preferred embodiment W is O, Y is NH2, n is 1, and both R groups^2a in R² they are different from NH2. The compound according to this embodiment is represented by formula (Ib):

<img file="PL2956173T3_D0015.tif" />

where R^4a is H, Me, or Et; R^3a and R^3b are independently H, C 6 H 5, Me, or Et; and R⁶ is C1-C5 alkyl, CH2OC (= O) C1-C5 alkyl, or (CH2) 1-2C6H5; or a pharmaceutically acceptable salt thereof.

Even in an even more preferred embodiment, the compound has the structure shown in formula (Ib '):

<img file="PL2956173T3_D0016.tif" />

where R^4a is H, Me, or Et (preferably H) and R⁶ is Me or n-Pr; or a pharmaceutically acceptable salt thereof.

Preferably, in formula (Ia), (Ia '), and (Ib), one of R^3a and R^3b is H and the other is Me. In other preferred embodiments, both Rs^3a and R^3b they are H or both are Me, or one of R^3a and R^3b is H and the other is C6H5. In still other preferred embodiments, R^3a and R^3b are independently H, Me, or Et.

Specific examples of the compounds of the present invention include the compounds set forth immediately below, together with their pharmaceutically acceptable salts:

<img file="PL2956173T3_D0017.tif" />

- EP 2956173

<img file="PL2956173T3_D0018.tif" />

- EP 2956173

<img file="PL2956173T3_D0019.tif" />

<img file="PL2956173T3_D0020.tif" />

Compounds (I-2), (I-7), (I-8) and (I-9) are preferred.

conjugates

Optionally, the compounds of the present invention may be conjugated to a targeting group that specifically or preferentially binds to a chemical entity on a cancer cell. Preferably, the targeting molecule is an antibody or antigen binding portion thereof and the chemical molecule is a tumor-coupled antigen. Preferably, the conjugation is made by chemical linkage to a functional group in the Tuv or Tup subunit, such as an amino group.

In another aspect, a conjugate is provided comprising a cytotoxic compound of the present invention and a ligand represented by formula (II) [D (X)^D) C (X^WITH) b] mZ (II) where Z is a ligand; D is a cytotoxic compound of the present invention (e.g., a compound according to formula (I), (Ia), (Ia '), or (Ib)); and - (X^D) C (X^WITHb) together is referred to as a "linker" or "linker" because it links Z and D. In the linker, C is a cut-off group to be cleaved at the site of the intended biological action of compound D;^D and X^WITH they are defined as a spacer group (or "walks") because they separate D and C and C and Z, respectively; the subscripts a and b are independently 0 or 1 (i.e., the occurrence of X^D and / or X^WITH it is optional); and the subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 1, 2, 3, or 4). D, X^D, C, X^WITH and Z are more precisely defined below.

The Z ligand - for example, an antibody - has a targeting (directional) function. The ligand Z, by binding to the target tissue or cell, where its antigen or receptor is located, directs the conjugate there. Preferably, the target tissue or cell is a tumor tissue or cell, and the antigen or receptor is a tumor-associated antigen, i.e. an antigen, which is unique in cancer cells or over-expressed by tumor cells compared to non-cancer cells. Cleavage of the C group in the target tissue or cell releases compound D which allows local cytotoxic effects. In some cases, the conjugate is internalized to the target cell as a result of endocytosis and the cleavage takes place in the target cell. In this way, the compound is delivered exactly at the place of intended action,

- EP 2956173

Also, compound D is usually biologically inactive (or much less active) in a conjugated form, thereby reducing undesirable toxicity to the non-target tissue or cells. This is an important issue because anti-cancer drugs are often generally highly toxic to cells.

As shown in the subscript m, each Z ligand molecule may be conjugated with more than one compound D, depending on the number of available Z ligand sites for conjugation and the experimental conditions used. It will be appreciated by those skilled in the art that when each individual Z-ligand molecule is conjugated to the total number of Compounds D, conjugate production can be analyzed for the incomplete proportion of Compounds D to the Z-ligand, which is a statistical average. The ratio is called the substitution index (SR) or, alternatively, in the case of antibody-drug conjugates, a drug-antibody indicator (DAR).

L / gand Z / his accreditation

Preferably, the ligand Z is an antibody. For convenience and shortening, not limitation, the following detailed discussion of Z ligand conjugation is written in the context of being an antibody, but those skilled in the art understand that other types of Z ligand may be conjugated, taking into account existing differences (mutat / s mutand / s). For example, conjugates with folic acid as a ligand may target cells having a folate receptor on the surface (Vlahov et al. 2008, Leamon et al. 2008). For the same reason, a detailed description below is written with regard to the 1: 1 ratio of antibody Z to compound D (m = 1).

Preferably, the ligand Z is an antibody against a tumor-associated antigen, which allows the conjugate containing such a ligand Z to selectively target target tumor cells. Examples of such antigens include: mesothelin, prostate-specific membrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H4 (also known as O8E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, CTLA-4 and CD44. The antibody may be animal (e.g., mouse), chimeric, humanized or, preferably, human. The antibody is preferably a monoclonal antibody, especially a human monoclonal antibody. The production of human monoclonal antibodies against some of the above-mentioned antigens is disclosed in Korman et al., US 2009/0074660 A1 (B7H4); RaoNaik et al., 8,097,703 B2 (CD19); King et al., US 2010/0143368 A1 (CD22); Keler et al., US 7, 387,776 B2 (2008) (CD30); Terrett et al., US 8,124,738 B2 (CD70); Korman et al., US 6,984,720 B1 (2006) (CTLA-4); Korman et al., US 8,008,449 B2 (2011) (PD-1); Huang et al., US 2009/0297438 A1 and Cardarelli et al., US 7,875,278 B2 (PSMA); Terrett et al., US 2010/0034826 A1 (PTK7); Terrett et al., US 2010/0209432 (A1) (glypican-3); Harkins et al., US 7,335,748 B2 (2008) (RG1); Terrett et al., US 8,268,970 B2 (2012) (mesothelin) and Xu et al., US 2010/0092484 A1 (CD44).

The Z ligand may also be an antibody fragment or mimetic of an antibody, such as an affibody molecule, a domain antibody (dAb), a nanobody, a conjugate, a DARPin, an antiallergine, a version, a duocalin, a lipocalin or an avimer.

Any of the many different reactive groups on the Z ligand can be a conjugation site, including ε-amino groups in lysine residues, side carbohydrate molecules, carboxylic acid groups, disulfide groups and thiol groups. Each type of reactive group represents a compromise because it has certain advantages and some disadvantages. An overview of the reactive groups of antibodies suitable for conjugation is given, for example, in

Garnett, Adv. Drug Delivery Rev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology &amp; Therapeutics 83 (1999), 67-123.

In yet another embodiment, the Z ligand may be conjugated through the ε-amino group of lysine. Most antibodies have many exposed ε-amino lysine groups that can be conjugated via amide, urea, thiourea or carbamate linkages using techniques known in the art, including modifications with a heterobifunctional agent (as further described below). However, the control of which and how many ameamine groups reacts is difficult, which leads to potential variability between series

- conjugates preparations. Also, conjugation can cause neutralization of the protonated ε-amino group important for maintaining the native conformation of the antibody, or can take place in lysine alongside or at the antigen-binding site, none of which is desirable.

In another embodiment, the Z ligand may be conjugated through the carbohydrate side chain, as many antibodies are glycosylated. The side carbohydrate chain may be oxidized with periodate to form aldehyde groups that in turn may react with amines to form an imino group, such as semicarbazone, oxime or hydrazone. If necessary, the imine group can be converted into a more stable amino group by reduction with a cyanoborohydride. For additional disclosure of conjugation by side carbohydrate chains see, e.g., Rodwell et al., Proc. Nat'l Acad. Sci. USA 83, 2632-2636 (1986); the disclosure is incorporated herein by reference. As in the case of ε-amino groups of lysine,

In yet another embodiment, the Z ligand may be conjugated via a carboxylic acid group. In one embodiment, the carboxylic acid end group is functionalized to produce a carbohydroazide, which is then reacted with a carrier molecule conjugated to the aldehyde. See Fisch et al., Bioconjugate

Chemistry 1992, 3, 147-153.

In yet another embodiment, the Z antibody may be conjugated through a disulfide group that forms a bridge between the cysteine ​​residue on the Z antibody and the sulfur on the other part of the conjugate. Certain antibodies lack the free sulfhydryl groups but have disulphide groups, e.g. in the hinge region. In this case, free thiol groups can be produced by reduction of native disulfide groups. The thiol groups so generated can be used for conjugation. See Packard et al., Biochemistry 1986, 25, 3548-3552; King et al., Cancer Res. 54, 6176-6185 (1994) and Doronina et al., Nature Biotechnol. 21 (7), 778-784 (2003). Again, there are doubts regarding the location of the site and the stoichiometry of the conjugation and the possible disruption of the native conformation of the antibody.

There are many known methods for introducing free thiol groups into antibodies without disrupting native disulfide bonds, which methods can be practiced with the Z ligand of the present invention. Depending on the method used, it may be possible to introduce a predictable number of free sulfhydryl at predetermined locations. In one approach, mutant antibodies are produced in which the cysteine ​​is substituted for another amino acid. See, for example, Eigenbrot et al., US 7,521,541 B2 (2009); Chilkoti et al., Bioconjugate Chem. 1994, 5, 504-507; Urnovitz et al., US 4,698,420 (1987); Stimmel et al., J. Biol. Chem., 275 (39), 30445-30450 (2000); Bam et al., US 7,311,902 B2 (2007); Kuan et al., J. Biol. Chem., 269 (10), 7610-7618 (1994); Poon et al., J. Biol. Chem., 270 (15), 85718577 (1995). In a different approach, an additional cysteine ​​is added to the C-terminal. See, for example, Cumber et al., J. Immunol. 149, 120-126 (1992); King et al, Cancer Res., 54, 6176-6185 (1994); Li et al., Bioconjugate Chem., 13, 985-995 (2002); Yang et al., Protein Engineering, 16, 761-770 (2003) and Olafson et al., Protein Engineering Design &amp; Selection, 17, 21-27 (2004). A preferred method of introducing free cysteines is explained in Liu et al., WO 2009/026274 A1, in which the amino acid sequence bearing cysteine ​​is added to the C-terminus of the heavy chain of the antibody. The given method introduces a known number of cysteine ​​residues (one per heavy chain) to a known site away from the antigen-binding site. Bioconjugate Chem., 13, 985-995 (2002); Yang et al., Protein Engineering, 16, 761-770 (2003) and Olafson et al., Protein Engineering Design &amp; Selection, 17, 21-27 (2004). A preferred method of introducing free cysteines is explained in Liu et al., WO 2009/026274 A1, in which the amino acid sequence bearing cysteine ​​is added to the C-terminus of the heavy chain of the antibody. The given method introduces a known number of cysteine ​​residues (one per heavy chain) to a known site away from the antigen-binding site. Bioconjugate Chem., 13, 985-995 (2002); Yang et al., Protein Engineering, 16, 761-770 (2003) and Olafson et al., Protein Engineering Design &amp; Selection, 17, 21-27 (2004). A preferred method of introducing free cysteines is explained in Liu et al., WO 2009/026274 A1, in which the amino acid sequence bearing cysteine ​​is added to the C-terminus of the heavy chain of the antibody. The given method introduces a known number of cysteine ​​residues (one per heavy chain) to a known site away from the antigen-binding site. wherein the cysteine ​​bearing amino acid sequence is added to the C-terminus of the antibody heavy chain. The given method introduces a known number of cysteine ​​residues (one per heavy chain) to a known site away from the antigen-binding site. wherein the cysteine ​​bearing amino acid sequence is added to the C-terminus of the antibody heavy chain. The given method introduces a known number of cysteine ​​residues (one per heavy chain) to a known site away from the antigen-binding site.

In yet another embodiment, the ε-amino groups of lysine can be modified with heterobifunctional reagents such as 2-iminothiolate or N-succinimidyl-3- (2-pyridyliodithio) propionate (SPDP), converting the ε-amino group to a thiol or disulphide group - forming in a way, a cysteine ​​surrogate. However, the described method has the same limitations on the place and stoichiometry of conjugation associated with the appropriate εamino groups.

- EP 2956173

In yet another preferred embodiment, the Z ligand is conjugated by the product of the nucleophilic addition of the thiol group to the acceptor group. A preferred receptor group is a maleimide group whose reaction with the thiol group of the antibody is generically shown below. The thiol group may be native or introduced as described above.

<img file="PL2956173T3_D0021.tif" />

The Z ligand can also be conjugated via a functional group adapted for use in "click" reactions, as discussed below.

Linker - (X^D) C (X^WITH) As noted above, the linker portion of the conjugate of the present invention contains up to three members: the C-group being removed and the optional X-spacer.^WITH and X^D.

Cut off group C is a group cut off under physiological conditions, preferably one that is relatively stable when the conjugate is in general circulation in the blood plasma, but is easily cut off when the conjugate reaches the site of intended action, i.e., close to, , or in the target cell. Preferably, the conjugate is internalized by endocytosis by the target cell after binding of the Z antibody to the antigen on the surface of the target cell. Then, the C-group is cleaved in the alveolar cell of the target cell (early endosome, late endosome, or especially lysosomes).

In one embodiment, the C group is a pH sensitive group. The pH of the blood plasma is slightly above neutral, whereas the pH inside the lysosom is acidic, about 5. Thus, the C group, which is acid catalyzed, will cut at a speed several times faster in the lysosome interior than in the blood plasma. Examples of suitable acid-sensitive groups include cisacononamides and hydrazones as described in Shen et al., US 4,631,190 (1986); Shen et al., US 5,144,011 (1992); Shen et al., Biochem. Biophys. Res. Commun. 102, 1048-1054 (1981) and Yang et al., Proc. Natl Acad. Sci (USA), 85, 1189-1193 (1988).

In another embodiment, the C group is a disulphide. Disulfides may be cut by a thiol-disulphide exchange mechanism at a rate depending on the thiol concentration in the environment. Since the intracellular concentration of glutathione and other thiols is higher than their serum concentration, the disulfide cleavage rate will be higher inside the cell. Further, the thiol-disulphide exchange rate can be modulated by adjusting the spatial and electronic characteristics of the disulphide (e.g., alkyl-aryl disulphide with alkylalkyl disulphide, substitution on the aryl ring, etc.), allowing a disulfide bond pattern with increased serum stability or a specific speed. cut. Additional disclosures of disulfide groups cleaved in conjugates are given, for example, in Thorpe et al., Cancer Res. 48, 6396-6403 (1988); Santi et al., US 7,541,530 B2 (2009); Ng et al., US 6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1; Boyd et al., US 7,691,962 B2 and Sufi et al., US 2010/0145036 A1.

The preferred C group has a peptide bond that is cleaved, preferably by a protease, at the intended site of action, in contrast to the serum protease. Typically, the C group contains from 1 to 20 amino acids, preferably from 1 to 6 amino acids, more preferably from 1 to 3 amino acids. The amino acid (s) may be natural and / or unnatural α-amino acids. The natural amino acids are encoded with the genetic code, as well as derivatives of these amino acids, e.g. hydroxyproline, γ-carboxyglutamate, citrulline and O-phosphoserine. The term amino acid also includes amino acid analogs and mimetics. Analogs are compounds having the same general structure H2N (R) CHCO2H of a natural amino acid, except that the R group does not exist among natural amino acids. Examples of analogs include homoserine, norleucine, methionine sulfoxide and methylsulfonium methionine. The amino acid mimetic is a compound having a structure different from the general chemical structure of an amino acid but operating in a similar manner to it. The term "unnatural amino acid" is to have the stereochemical form "D", the natural amino acids have the form "L".

Preferably, the C group contains an amino acid sequence which is a protease cleavage recognition sequence. Many recognition recognition sequences are known in the art. See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995).

For conjugates that are not intended to be internalized by the cell, a C group can be selected which is cleaved by a protease present in the extracellular matrix near the target tissue, e.g., a protease released by nearby dying cells or a tumor associated protease. Exemplary extracellular proteases associated with a tumor tumor are matrix metalloproteases (MMPs), thimet oligopeptidases (TOP) and CD10.

In the case of conjugates to be internalized by a cell, the C group preferably comprises an amino acid sequence selected for cleavage by an endosomal or lysosomal protease, especially the latter. Non-limiting examples of such proteases include cathepsins B, C, D, H, L and S, especially cathepsin B. Cathepsin B preferably cuts peptides in the sequence -AA²-AA¹- where AA¹ is a basic or strongly hydrogen bond of an amino acid (such as lysine, arginine, or citrulline), and AA² is a hydrophobic amino acid (such as phenylalanine, valine, alanine, leucine, or isoleucine), for example Val-Cit (where Cit is citrulline) or Val-Lys. (Here, the amino acid sequences are written in the N-to-C direction, as in H2N-AA²-AA¹-CO2H, unless the context clearly indicates otherwise.) For additional information on cathepsin cleaved groups, see, e.g., Dubowchik et al., Biorg. Med. Chem. Lett. 8, 3341-3346 (1998); Dubowchik et al., Bioorg. Med. Chem. Lett., 8, 3347-3352 (1998) and Dubowchik et al., Bioconjugate Chem. 13, 855-869 (2002). Another enzyme that can be used to cut peptidyl linkers is legumine, a lysosomal cysteine ​​protease preferentially cleaving in Ala-Ala-Asn.

In another embodiment, the C group is a peptide containing the amino acid sequence -AA²-AA¹- where AA¹ is lysine, arginine, or citrulline and AA² is phenylalanine, valine, alanine, leucine or isoleucine. In another embodiment, C consists of a sequence of one to five amino acids selected from the group consisting of Val-Cit, Ala-Val, Val-AlaVal, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit -Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser and Glu.

The production and design of cleavable C groups consisting of one amino acid is disclosed in Chen et al., US 2010/0113476 A1.

Group C can also be a photo-cleavable one such as a nitrobenzyl ether that is cleaved when exposed to light.

Group C may be directly attached to the antibody Z or compound D; I mean, X walks^WITH and X^D, depending on the case, may not be present. For example, if group C is a disulphide, one of the two sakires may be a cysteine ​​residue or a surrogate of it on the Z antibody. Or, the C group may be a hydrazone linked to the aldehyde on the carbohydrate side chain of the antibody. Or the C group may be a bond formed with the ε-amino group of the antibody Z. Lysine. In a preferred embodiment, the compound D is directly attached to the group C via a peptidyl bond to the carboxyl or amino group of the compound D.

When present, walk X^WITH provides a spatial separation between the C group and the Z antibody, so that the first spatially does not interfere with the binding of the antigen by the latter or that the latter does not interfere spatially with the first. Next, walk X^WITH may be used to confer upon the conjugates the features of increased solubility or reduced aggregation. Walk X^WITH may contain one or more modular segments,

2956173 that can be assembled in any number of combinations. Examples of suitable segments for the X spacer^WITH are:

&gt; Η τ. ° iN- (CH₂)₂.₆- (NH)_q-l ξ- (CH₂)₂.₆-C-ξ h (CH₂)₂.₆- (NH)_q-and

9 9

Ο&gt; ¹¹ 5,,? ^c_(CH₂)₂6- (NH)_q-Ξ j- (CH₂CH₂ABOUT)_rCH₂CH₂- |

9

O&gt; n S - (NH) q- (CH₂CH₂ABOUT)_rCH₂CH₂C- | and combinations thereof, wherein the subscript q is 0 or 1 and the subscript r is 1 to 24, preferably 2 to 4. These segments can be combined as shown below:

HCH₂)₃-CN- (CH₂CH₂ABOUT)₄'CH₂CH₂-CN- (CH₂)₂- (NH)_q about _H

THEIR₂)₃^-CN- (CH₂)₂- (NH)_q-And / or

H ° C (0132) 2-6 - ^ - O- (O ^) ^ 6 (NH)_qWalk X^Dif present, provides a spatial separation between the group C and the compound D so that the latter does not interrupt spatially or electronically in cutting the first group. Walk X^D it may also serve to introduce additional molecular weight and chemical functionality into the conjugate. In general, additional mass and functionality will affect the serum half-life and other properties of the conjugate. Thus, a reasonable selection of spacer groups can provide modulation of the serum half-life of the conjugate. Walk X^Dit can also be assembled from modular segments as described in the context of the X spacer^WITH.

X Walks^WITH and / or X^Dwhere present, preferably provides a linear separation of 4 to 25 atoms, more preferably from 4 to 20 atoms, between Z and C or D and C, respectively.

Any walk X^WITH or X^D, or both, may contain self-metabolizing moiety. The self-degrading group is a group, (1) bound to group C and either the Z antibody or cytotoxin D and (2) has a structure such that cleavage from group C initiates a sequence of reactions resulting in detachment of the self-degrading group from the Z antibody or cytotoxin D, as the case may be. . In other words, a reaction at a distal site from the Z antibody or cytotoxin D (cleavage from group C) also results in the X binding being interrupted^WITH-Z or X^D-D. The occurrence of a self-degrading group is required for the X spacer^Dbecause if, after cutting the conjugate, walk X^D or part of it would remain attached to cytotoxin D, then the biological activity of the latter could be weakened. The use of a self-degrading group is especially required when the cleavage group C is a polypeptide.

Exemplary self-degrading groups (i) - (v) linked to a hydroxyl or amino group on a partner D molecule are shown below:

<img file="PL2956173T3_D0022.tif" />

- EP 2956173

The self-degrading group is the structure between the dashed lines a and b, with the neighboring structures shown to show the context. The self-degrading groups (i) and (v) are bound to the compound D-NH2 (i.e., the compound D is conjugated via an amino group) while the self-degrading groups (ii), (iii) and (iv) are bound to the compound D-OH. (i.e., Compound D is conjugated via a hydroxyl or carboxyl group). The cleavage of the amide bond on dashed line b releases the amide nitrogen as the amine nitrogen, initiating the sequence of reactions resulting in cleavage of the bond on the dashed line a followed by the release of D-OH or D-NH2 as the case may be. Additional disclosures regarding self-degrading groups are provided in Carl et al., J. Med. Chem., 24 (3), 479-480 (1981); Carl et al., WO 81/01145 (1981); Dubowchik et al., Pharmacology &amp; Therapeutics, 83, 67-123 (1999); Firestone et al., US 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 67, 1866-1872 (2002); Doronina et al., Nature Biotechnology 21 (7), 778-784 (2003) (erratum, p. 941); Boyd et al., US 7,691,962 B2; Boyd et al., US 2008/0279868 A1; Sufi et al., WO 2008/083312 A2; Feng, US 7,375,078 B2 and Senter et al., US 2003/0096743 A1.

In another preferred embodiment, the targeting group of the antibody and the cytotoxic compound D are conjugated by a non-cut linker. Degradation of the antibody optionally reduces the linker to a small added molecule that does not interfere with the biological activity of the cytotoxic compound D.

Compositions Compound D - linker

The conjugates of the present invention are preferably produced by first combining compound D and a linker (X^D) C (X^WITH) b (where X^D, C, X^WITH, a, ib are as given for formula (II)) to form a drug-linker composition represented by the formula (III):

D (X^D) C (X^WITH) bR³¹ (III) where R³¹ is a functional group suitable for reacting with a functional group on the Z antibody to form a conjugate. Examples of suitable R groups³¹ include amine, azide, cyclooctyne,

OO

<img file="PL2956173T3_D0023.tif" />

where R³² is Cl, Br, F, mesylate or tosylate and R³³ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O- (4-nitrophenyl), -O-pentafluorophenyl or -O-tetrafluorophenyl. In general, reactions useful for the preparation of appropriate D- (X^D) C (X^WITH) bR³¹ disclosed in Ng et al., US 7,087,600 B2 (2006); Ng et al., US 6,989,452 B2 (2006); Ng et al., US 7,129,261 B2 (2006); Ng et al., WO 02/096910 A1; Boyd et al., US 7,691,962 B2; Chen et al., US 7,517, 903 B2 (2009); Gangwar et al., US 7,714,016 B2 (2010); Boyd et al., US 2008/0279868 A1; Gangwar et al., US 7,847,105 B2 (2010); Gangwar et al., US 7,968, 886 B2 (2011); Sufi et al., US 2010/0145036 A1; and Chen et al., US 2010/0113476 A1.

Preferably, the functional group is -R³¹ is -NH2, -OH, -CO2H, -SH is a maleimide, cyclooctyne, azido (-N3), hydroxylamino (-ONH2) or N-hydroxysuccinimide group.

Particularly preferably, the -R functional groups³¹ are selected from the group containing:

- EP 2956173

<img file="PL2956173T3_D0024.tif" />

The -OH group can be esterified with a carboxyl group on the antibody, for example, on the side chain of aspartic and glutamic acid.

The -CO 2 H group can be esterified with an -OH group or amidated with an amine group (e.g. on the lysine side chain) on the antibody.

The N-hydroxysuccinimide group is functionally activated with a carboxyl group and can conveniently be amidated by reaction with an amino group (e.g., from lysine).

The maleimide group may be conjugated to the -SH group on the antibody (e.g., from cysteine ​​or from a chemical modification of the antibody to introduce a sulfhydryl function) in the Michael addition reaction.

The -SH group is particularly useful for conjugation, where the antibody is to be modified by introducing the maleimide group into it, in the Michael addition reaction it is the "mirror image" described above. Antibodies may be modified to have maleimide groups with N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate (SMCC) or its sulfonated sulfo-SMCC variant, both reagents available from Sigma-Aldrich.

Azide and cyclooctane are complementary functional groups that can affect conjugation through so-called "click" reactions without copper, in which azide is added through the tethered cyclooctyne alkyne linkage to form a 1,2,3-thiazole ring. See, e.g., Agard et al., J. Amer. Chem. Soc. 2004, 126, 15046-15047; Best, Biochemistry 2009, 48, 6571-6584. Azide may be a reactive functional group of R³¹ in formula (III) and the cyclooctin can be placed on its antibody or antigen binding portion or vice versa. The cyclooctin group may be provided by a DIBO reagent (available from Invitrogen / Molecular Probes, Eugene, Oregon).

Techniques for introducing unnatural amino acids into antibodies can be used with unnatural amino acids that provide conjugation functionality with a reactive functional group. For example, the unnatural amino acid p-acetylphenylalanine may be introduced into an antibody or other polypeptide as explained in Tian et al., WO 2008/030612 A2 (2008). The ketone group in p-acetylphenylalanine can be a conjugation site by oxime formation with a hydroxylamine reactive functional group. Alternatively, the unnatural amino acid p-acetylphenylalanine can be introduced into the antibody to provide an azide function group for conjugation in a "click" chemical reaction. Unnatural amino acids can also be introduced into an antibody or other polypeptide using non-cell-based methods as explained in Goerke et al. US 2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009, 102 (2), 400-416.

The amino group (NH2) can be used for conjugation using the transglutaminase enzyme as explained in Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995 -9997.

The conjugation may also be influenced by the enzyme sortase A, as explained in Levary et al., PLoS One 2011, 6 (4), e18342; Proft, Biotechnol. Lett. 2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010) and Mao et al., WO 2005/051976 A2 (2005). The sort A recognizer motif (usually LPXTG, where X is any natural amino acid) may be on the Z ligand and the nucleophilic acceptor motif (usually GGG) may be the R group³¹ in formula (III) or vice versa.

Group D in the formula [D (X^D) C (X^WITH) b] mZ and D- (X^D) C (X^WITH) bR³¹ preferably it has a structure according to formula (Da)

- EP 2956173

<img file="PL2956173T3_D0025.tif" />

or a formula (Db)

<img file="PL2956173T3_D0026.tif" />

where Y is H or NO₂ ; R^4a is H, Me, or E_t; R^3a and R^3b are independently H, Me, or Et; and R⁶ is C1-C5 alkyl, CH2OC (= O) C1-C5 alkyl or (CH2) 1-2C6H5.

Examples of such D groups include:

<img file="PL2956173T3_D0027.tif" />

<img file="PL2956173T3_D0028.tif" />

where R⁷ is H, Me or Et;

Examples of compositions according to formula D- (X^D) C (X^WITH) bR³¹ include those given immediately below; together with their pharmaceutically acceptable salts:

- EP 2956173

<img file="PL2956173T3_D0029.tif" />

- EP 2956173

<img file="PL2956173T3_D0030.tif" />

- EP 2956173

<img file="PL2956173T3_D0031.tif" />

A preferred drug-linker compound has the structure shown in formula (III-a):

<img file="PL2956173T3_D0032.tif" />

R

3a _{and R}^3b are independently H, Me, or Et;

R⁶ is Me, Et or n-Pr;

AA^and and every AA^b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;

- p is 1, 2, 3, or 4;

q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 2, 3 or 4); r is 1, 2, 3, 4 or 5; s is 0 or 1; and

R³¹ is selected from the group consisting of

<img file="PL2956173T3_D0033.tif" />

or a pharmaceutically acceptable salt thereof.

In the formula (III-a), -AA^and- [AA^b] p- represents a polypeptide whose length is determined by the p-value (e.g., dipeptide if p is 1, tetrapeptide, when p is 3, etc.). AA^and it is the carboxyl terminus of a polypeptide and its carboxyl group forms a peptide (amide) bond with aniline nitrogen or drug. On the contrary, the last AA^b is the amino terminus of a polypeptide and its alpha-amino group forms a peptide bond with s if s is and z

<img file="PL2956173T3_D0034.tif" />

if s is 0.

A more preferred drug-linker compound has the structure represented by formula (III-b):

<img file="PL2956173T3_D0035.tif" />

R⁶ is Me or n-Pr;

Q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 2, 3 or 4);

r is 1, 2, 3, 4 or 5;

s is 0 or 1; and

R³¹ is selected from the group consisting of

- EP 2956173 ο

<img file="PL2956173T3_D0036.tif" />

ο ο

<img file="PL2956173T3_D0037.tif" />

and

<img file="PL2956173T3_D0038.tif" />

or a pharmaceutically acceptable salt thereof.

Preparation of conjugates

The following is an illustrative procedure based on the introduction of free thiol groups into the antibody by reaction of ε-amino groups of lysine with 2-iminothiolane followed by reaction with a maleimide-containing drug-linker moiety as described above.

Initially, the antibody undergoes buffer exchange to 0.1 M phosphate buffer (pH 8.0) containing 50 mM NaCl and 2 mM diethylenetriaminepentaacetic acid (DTPA) and concentrated to 5-10 mg / ml. Thiolation was achieved by the addition of 2-iminothiolate to the antibody. The amount of added 2-iminothiolate can be determined in the initial experiment and is different depending on the antibody. In the preliminary experiment, the increasing amount of 2-iminothiolan added to the antibody is titrated and after incubation with the antibody for 1 hour. at room temperature (RT, about 25 ° C) the antibody is desalted into 50 mM HEPES buffer pH 6.0 using a SEPHADEX ™ G-25 column, and the number of thiol groups introduced is quickly determined in reaction with dithiodipyridine (DTDP). The reaction of thiol groups with the DTDP results in the release of thiopyridine, which can be monitored spectroscopically at 324 nm. Samples with a protein concentration of 0.5-1.0 mg / ml are usually used. Absorbance at 280 nm can be used to accurately determine the protein concentration in the samples, and then a submersion of each sample (0.9 mL) is incubated with 0.1 mL of DTDP (5 mM stock in ethanol) for 10 min at room temperature. At the same time, blind samples of the buffer plus DTDP are also incubated. After 10 min, the absorbance at 324 nm was measured and the number of thiol groups was counted using a disappearance factor of thiopyridine of 19 800 M 1 ml DTDP (5 mM stock solution in ethanol) for 10 min at room temperature. At the same time, blind samples of the buffer plus DTDP are also incubated. After 10 min, the absorbance at 324 nm was measured and the number of thiol groups was counted using a disappearance factor of thiopyridine of 19 800 M 1 ml DTDP (5 mM stock solution in ethanol) for 10 min at room temperature. At the same time, blind samples of the buffer plus DTDP are also incubated. After 10 min, the absorbance at 324 nm was measured and the number of thiol groups was counted using a disappearance factor of thiopyridine of 19 800 M^-1.

Typically a thiolation level of about three thiol groups per antibody is required. For example, for some antibodies this can be achieved by adding a 15-fold molar excess of 2-iminothiolane and then incubating at room temperature for 1 hour. The antibody is then incubated with 2-iminothiolane at the required molar ratio and then desalted into a conjugation buffer (50 mM HEPES buffer pH 6.0 containing 5 mM glycine and 2 mM DTPA). The thiolated material is kept on ice while the number of thiol groups introduced is calculated as above.

After verification of the number of thiol groups introduced, the drug-linker group was added at a 3-fold molar excess per thiol. The conjugation reaction may further proceed in a conjugation buffer containing a final concentration of 5% dimethylsulfoxide (DMSO) or a similar alternative solvent. In general, the drug-linker stock solution is dissolved in 100% DMSO. The stock solution is added directly to the thiolated antibody, which contains a sufficient amount of DMSO added to raise the final concentration to 10%, or prediluted in a conjugation buffer at 10% DMSO final concentration, and then added to an equal volume of the thiolated antibody.

The conjugation reaction mixture is incubated at room temperature for 2 hours. with mixing. After incubation, the conjugation reaction mixture is centrifuged and filtered through a 0.2 μm filter. Purification of the conjugate can be achieved using a variety of chromatographic methods. In one method, the conjugate is purified by a size exclusion chromatography on a SEPHACRYL ™ S200 column of pre-equilibrated 50 mM HEPES buffer pH 7.2 containing 5 mM glycine and 150 mM NaCl. Chromatography is performed at a linear flow rate of 28 cm / h. The fractions containing the conjugate were collected, pooled and concentrated. In an alternative method, purification was achieved using chromatography

- ion exchange. Conditions vary depending on the antibody and should in each case be optimized. For example, the antibody-antibody conjugate reaction mixture was used in a SP SEPHAROSE ™ column pre-equilibrated in 50 mM HEPES buffer pH 5.5 containing 5mM glycine. The antibody conjugate was eluted with a 0-1 M NaCl gradient in equilibration buffer at pH 5.5. The appropriate fractions containing the conjugate are pooled and dialyzed against the formulation buffer (50 mM HEPES buffer pH 7.2 containing 5 mM glycine and 100 mM NaCl).

Those skilled in the art will understand that the conditions and methodologies described above are illustrative and non-limiting and that other conjugation approaches known in the art may be used in the present invention.

The conjugate prepared in the procedure described above is shown in formula (II-1). It is a conjugate of compound (III-1) and anti-isotonic antibody 6A4 (Terrett et al. 2012):

<img file="PL2956173T3_D0039.tif" />

Those skilled in the art will understand that such conjugate preparation may result in groups with 15 different substitution ratios, usually ranging from 1 to 5, and that the given preparation may be represented by formula (II-1 '):

<img file="PL2956173T3_D0040.tif" />

Where R⁶ is Me or n-Pr and Ab is an antibody. The antibody is preferably an anti-CD70 antibody, anti-isothelin or anti-glypican-3 antibody.

PHARMACEUTICAL COMPOSITIONS

In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present invention, or a conjugate thereof, prepared together with a pharmaceutically acceptable excipient. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as an antibody or other drug. The pharmaceutical compositions may be administered in combination therapy with another therapeutic agent, especially another anti-cancer agent.

The pharmaceutical composition may contain one or more excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, binders, dispersions or suspending agents, solubilizers, dyes, flavors, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents , and their combinations. Choice and

- use of suitable excipients is discussed in Gennaro, ed., Remington: The Science and Practice of Pharmacy, eds. 20 (Lippincott Williams &amp; Wilkins 2003).

Preferably, the pharmaceutical composition should be suitable for intravenous, intramuscular, subcutaneous, parenteral, paraspinal or skin application (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated with material to protect against acids or other natural conditions, which may lead to its deactivation. The term "parenteral administration" means modes of administration other than intestinal and local, usually injectable, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcuticular, intraarticular delivery , subcapsular, subarachnoid, intraspinal, epidural and trichomoniasis as well as infusion. Alternatively,

The pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They can also be prepared in a microemulsion, liposome or other ordered structure suitable to achieve high drug concentration. The compositions may also be provided in the form of lyophilates, for reconstitution in water prior to administration.

The amount of active ingredient that can be combined with a carrier material to obtain a unit dosage form varies depending upon the host treated and the particular mode of administration and will generally be the amount of the composition that causes the therapeutic effect. Generally, one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of the active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of the active ingredient in combination with a pharmaceutically acceptable carrier.

Dose regimens are set to provide a therapeutic response. For example, one bolus can be given, several divided doses can be administered over time, or the dose can be proportionately reduced or increased depending on the requirements (situation). It is especially preferred to formulate the parenteral composition in a unit dosage form that provides easy administration and unification of the dose. "Unit dosage form" refers to physically discrete units envisaged as unitary doses for treated patients; each unit containing a predetermined amount of active compound calculated to produce the required therapeutic response in association with the required pharmaceutical carrier.

The dose is from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of the host's body weight. For example, doses may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or lie within the range of 1-10 mg / kg. Exemplary treatment regimes include administering once a week, every two weeks, every three weeks, every four weeks, once a month, every three months, or every three to six months. Preferred dosing regimens include 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, using one of the following dosing schedules: (i) every four weeks to six doses, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once and then 1 mg / kg body weight every three weeks. In some methods, the dosage is adjusted,

A "therapeutically effective amount" of a compound of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevents the deterioration of the condition or disability caused by disease. For example, in the treatment of patients with a tumor, a "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably by about

At least about 40%, more preferably at least about 60%, and even more preferably at least about 80%, for untreated patients. A therapeutically effective amount of the therapeutic compound can reduce the size of the tumor, or otherwise alleviate the symptoms in a patient who is usually a human, but may be another mammal.

The pharmaceutical composition may be a controlled- or sustained-release formulation, which includes implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as vinyl-ethylene acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, e.g., Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices such as (1) needless hypodermic devices (e.g., US 5,399,163; 5,383,851; 5,312,335;

5,064,413; 4,941,880; 4.790.824 and 4.596,556); (2) microinfusion pumps (US 4,487,603); (3) percutaneous devices (US 4,486,194); (4) infusion devices (US 4,447,233 and 4,447,224) and (5) osmotic devices (US 4,439,196 and 4,475,196).

In certain embodiments, a pharmaceutical composition may be prepared to ensure proper distribution in vivo. For example, to ensure the passage of the therapeutic compounds of the invention across the blood-brain barrier, they can be prepared in liposomes that can additionally contain targeting groups to increase selective transport to specific cells or organs. See, e.g., US 4,522,811; 5,374,548; 5,416,016 and 5,399,331; VV Ranade (1989) J. Clin. Pharmacol. 29: 685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe et al. (1995) Am. J. Physiol. 1233: 134; Schreier et al. (1994) J. Biol. Chem. 269: 9090; Keinanen and Laukkanen (1994) FEBS Lett. 346: 123 and Killion and Fidler (1994) Immunomethods 4: 273.

USAGE

The compounds of the present invention or conjugates thereof can be used to treat diseases such as, but not limited to, hyperproliferative diseases, including: head and neck cancers that include head, neck, nasal cavity, paranasal sinus, nasopharyngeal cancer, mouth, oropharynx, larynx, throat larynx, salivary gland and paragangoma; liver and biliary tract cancer, especially hepatocellular carcinoma; bowel cancer, especially colon cancer; ovarian cancer; small cell and non-small cell lung cancer (SCLC and NSCLC); breast sarcomas, such as fibrosarcomas, malignant fibrous histiocytoma, rhabdomyosarcoma, leiomyosarcoma, neurofibromasarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and soft tissue sarcoma; leukemias such as acute promyelocytic leukemia (APL), acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML); tumors of the central nervous system, in particular brain cancer; multiple myeloma (MM), lymphomas such as Hodgkin's lymphoma (Hodgkin's lymphoma), lymphoplasmacytic lymphoma, follicular lymphoma, mucosal lymphoma associated with mucous membrane, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and large anaplastic lymphoma T cells. Clinically, in practice, the methods and use of the compositions described herein will reduce the size or number of cancer growth outbreaks and / or reduce associated symptoms (where applicable). In pathology, in practice, the methods and use of the compositions described herein will provide an adequate pathological response, such as inhibiting the proliferation of tumor cells, reducing the size of the cancer or tumor, preventing further metastasis, and inhibiting tumor angiogenesis. A method of treating such diseases comprises administering to a patient a therapeutically effective amount of a combination according to the invention. The method can be repeated as required. Especially, cancer can be cancer of the kidneys, lungs, stomach or ovaries. The method can be repeated as required. Especially, cancer can be cancer of the kidneys, lungs, stomach or ovaries. The method can be repeated as required. Especially, cancer can be cancer of the kidneys, lungs, stomach or ovaries.

- 29 EP 2956173

The compounds of the present invention or conjugates thereof may be administered in combination with other therapeutic agents including antibodies, alkylating agents, angiogenesis inhibitors, antimetabolites, DNA cutting agents, DNA crosslinking agents, DNA intercalators, small DNA groin binding agents, enediins, heat shock protein inhibitors, histone deacetylase inhibitors, immunomodulators, microtubule stabilizers, nucleoside analogs (purines or pyrimidines), inhibitors of nuclear export, proteasome inhibitors, topoisomerase inhibitors (I or II), tyrosine kinase inhibitors, and serine-threonine kinase inhibitors. Specific antitumor agents include adalimumab, P3 ansamitocyte, auristatin, bendamustine, bevacizumab, bicalutamide, bleomycin, bortezomib, busulfan, calistatin A, camptothecin, capecitabine,

EXAMPLES

The practice of the present invention may be further understood by reference to the following examples.

Example 1 - Compound (III-1)

The example describes the synthesis of compound (III-1), the corresponding scheme is shown in the combined figures 1, 2a-2b and 3.

Compound 2. A mixture of compound 1 (6 g, 16.6 mmol, prepared according to Peltier et al. 2006) and paraformaldehyde (9.94 g, 331 mmol) in toluene (150 mL) was heated in a sealed vessel at 70 ° C for 24 hours. . Thin layer chromatography (TLC) showed the reaction was complete. The reaction mixture was filtered through CELITE ™ filtration media, and the filter cake was washed thoroughly with toluene. After evaporation of the solvent, the crude product was purified by flash chromatography eluting with silica gel with a 0-70% gradient of ethylene acetate (EtOAc) in dichloromethane (DCM) to give 4.76 g of compound 2 as a light yellow oil. MS: (+) m / z 375.2 (M + 1).

Compound 3. Hydrochloric acid (4.0 M in 1,4-dioxane, 12.24 mL, 50.8 mmol) was added dropwise to a solution of compound 2 (4.76 g, 12.7 mmol) in acetonitrile (62 mL) and methanol (6.8 mL) in the presence of compound cyanoborohydride resin (MP-BH3CN) (4.85 g, 12.7 mmol). The reaction mixture was stirred at room temperature (RT) for 3 hours. LCMS indicated the reaction was complete. The resin was filtered off and washed with a mixture of acetonitrile-methanol. After evaporation of the solvent, the crude product was purified by flash chromatography eluting with silica gel with a 0-10% methanol gradient in DCM containing 1% NH4OH to give crude compound 3.

The product fractions were concentrated, diluted with EtOAc, and washed once with saturated NaHCO 3 to remove excess ammonium salts. The aqueous fraction was re-extracted once with EtOAc. The combined organic phases were dried and concentrated to give 2.82 g of compound 3 as a foam solid. MS: (+) m / z 273.2 (M + 1).

Compound 4. N-benzyl-N-cyclohexylcarbodiimide bound to the polymer (Aldrich, 4.5 g,

5.21 mmol) was added to a solution of compound 3 (1.42 g, 5.21 mmol), t-butanol (0.72 g, 5.32 mmol) and Boc protected isoleucine 3a (1.27 g, 5.47 mmol) in DCM (48 mL) at 0 ° C. . The reaction mixture was stirred overnight at room temperature. The resin was filtered off and washed with DCM.

- EP 2956173

The filtrate was concentrated, diluted with EtOAc, and washed once with saturated NaHCO 3. The aqueous solution was extracted twice with EtOAc. The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a 0-10% gradient of methanol in DCM containing 1% NH4OH to give crude 3b.

The product containing fractions were combined, concentrated, diluted with EtOAc, and washed with saturated NaHCO 3 to remove excess ammonium salts. The aqueous fraction was back-extracted once with EtOAc. The combined organic phases are dried and concentrated to yield intermediate 3b as a white solid.

Intermediate 3b in toluene (50 ml) was heated to 90 ° C in a sealed vessel, stirring overnight. LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-100% EtOAc in hexanes to give 1.3 g of compound 4 as a light yellow solid. MS: (+) m / z 486.3 (M + 1).

Compound 5. Trifluoroacetic acid (TFA, 26 mL) was added to a mixture of compound 4 in DCM (26 mL). After stirring for 30 min at room temperature, LCMS indicated complete reaction. The solution was concentrated, diluted with EtOAc, and once washed with saturated NaHCO 3. The aqueous solution was extracted twice with EtOAc. The combined organic phases were dried and concentrated to give 1.03 g of compound 5 as a white solid. MS: (+) m / z 386.3 (M + 1).

Compound 6. DCC (0.664 g, 3.22 mmol) was added to a mixture of compound 5 (1.03 g, 2.68 mmol), (R) -1-methylpiperidine-2-carboxylic acid 5a (0.4 g, 2.81 mmol; prepared according to Peltier et al. 2006), and t-butanol (0.369 g, 2.73 mmol) in DCM at 0 ° C. The reaction mixture was allowed to warm to room temperature and was stirred overnight at room temperature. The solid was filtered off and the filtrate was concentrated. The residue was dissolved in EtOAc, and once washed with saturated NaHCO 3. The aqueous solution was extracted twice with EtOAc. The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a 0-20% gradient of methanol in DCM to yield 1.23 g of compound 6 as a light yellow solid. MS: (+) m / z 511.4 (M + 1).

Compound 7. A mixture of N, N-diisopropylethylamine (DIEA, also called DIPEA, 0.972 ml, 5.58 mmol), bis (4-nitrophenyl) carbonate (BNPC, 1.698 g, 5.58 mmol) and compound 6 (0.57 g, 1.116 mmol) in N, N-dimethylformamide (DMF, 10 ml) was stirred at room temperature overnight. LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography on silica gel with a 0-20% methanol in DCM gradient to give 0.68 g of compound 7 as a light yellow oil. MS: (+) m / z 676.4 (M + 1).

Compound 8. Methylamine in methanol (2.0 M, 0.089 ml, 0.178 mmol) was added to compound 7 (0.1 g, 0.148 mmol) in methanol (1 ml). The reaction mixture was then stirred at room temperature for 10 min, LCMS indicated complete reaction. The diluent was evaporated to give 0.084 g of compound 8. MS: (+) m / z 568.4 (M + 1).

Compound 9. Lithium hydroxide (7.09 mg, 0.296 mmol) was added in water (0.5 mL) to a solution of compound 8 (0.084 g, 0.148 mmol) in 1,4-dioxane (0.5 mL) at room temperature. The reaction mixture was then stirred at room temperature for 2 hours, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a 0-30% methanol in DCM gradient to give 0.075 g of compound 9 as a white solid. MS: (+) m / z 554.4 (M + 1).

Compound 10. Triethylamine (11.73 mL, 84 mmol) was added to a mixture of ditbutyldicarbonate (BOC2O, 10.57 mL, 46.0 mmol) and (S) -methyl 2-amino-3- (4-nitrophenyl) propanoate hydrochloride 9a (10 g, 38.4 mmol) in acetonitrile (300 ml) at 0 ° C. The reaction mixture was allowed to warm to room temperature and was stirred overnight at room temperature. LCMS indicated the reaction was complete. The reaction mixture was concentrated and the product was redissolved in 200 mL of diethyl ether. The solid was filtered off and the filtrate was filtered off

Was concentrated. The crude product was purified by flash chromatography eluting with silica gel with 0-50% EtOAc in hexanes gradient to give 11.3 g of the Boc-protected intermediate as a white solid.

The Pd / C catalyst (10 wt%, 0.85 g, 7.99 mmol) was added to a solution of the Boc protected intermediate (15 g, 46.2 mmol) in MeOH (200 mL). The reaction mixture was stirred under a hydrogen atmosphere overnight. The Pd / C catalyst was filtered off and the filtrate was concentrated to give 13.6 g of compound 10 as a white solid. MS: (+) m / z 195.2 (M + 1-Boc).

Compound 11. Pyridine (5.77 ml, 71.3 mmol) was added to a solution of benzyl chloroformate (10.18 ml, 71.3 mmol) and compound 10 (17.5 g, 59.5 mmol) in DCM (185 ml) at 0 ° C. The reaction mixture was allowed to warm to room temperature and was stirred overnight at room temperature. The reaction was quenched by addition of saturated aqueous NaHCO 3 solution, and washed with brine. The organic layer was dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with 0-50% EtOAc in hexanes to afford 22.6 g of compound 11 as a colorless oil. MS: (+) m / z 329.2 (M + 1-Boc).

Compound 12. Diisobutylaluminium hydride (DIBAL-H) ​​in hexanes (1M, 26.5 mL, 26.5 mmol) was added to a solution of compound 11 (5.17 g, 12.07 mmol) in DCM (39 mL) at -78 ° C. The reaction mixture was stirred at -78 ° C for 2 hours. Acetic acid (24 ml) and toluene (36 ml) were added at -78 ° C. The reaction mixture was warmed to room temperature. To the reaction mixture was added tartaric acid (10% aq., 69 ml). The aqueous solution was extracted with a mixture of hexanes and EtOAc (v / v 1: 1). The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-50% EtOAc in hexanes to yield 3.12 g of compound 12 as a white solid. MS: (+) m / z 299.2 (M + 1-Boc).

Compound 13. Dibutyl (((trifluoromethyl) sulfonyl) oxy) borate (Bu2BOTf, 1M in DCM, 8.61 ml, 8.61 mmol) and DIEA (1.637 ml, 9.40 mmol) was added to the solution of (S) -4-isopropyl-3-propionyloxazolidine-2. -on 12a (1.450 g, 7.83 mmol) in DCM (7.8 ml) at 0 ° C.

The reaction mixture was stirred at 0 ° C for 45 min. A solution of compound 12 (3.12 g, 7.83 mmol) in DCM (7.8 mL) was added to the reaction mixture at -78 ° C. The reaction mixture was allowed to warm to rt overnight. Sodium phosphate buffer (pH 7, 29 ml) was added. The aqueous solution was extracted with DCM. The combined organic phases are washed with brine, dried, filtered and concentrated.

The residue was redissolved in methanol (130 ml) and cooled to 0 ° C. Water H 2 O 2 (30% aq., 39.7 ml) was added to the reaction mixture at 0 ° C. The reaction mixture was stirred at 0 ° C for 4 hours. Water (39 ml) was added. Part of the solvent (MeOH) was evaporated. The aqueous solution was extracted with EtOAc. The combined organic phases were washed with a 5% NaHCO3 solution and brine, dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a 0-50% gradient of EtOAc in hexanes to give 4.23 g of compound 13 as a colorless oil. MS: (+) m / z 484.3 (M + 1-Boc).

Compound 14. Di (1H-imidazol-1-yl) methanethiolane (1.5 g, 8.42 mmol) was added to a solution of compound 13 (2.46 g, 4.21 mmol) in THF (20 mL). The reaction mixture was kept under reflux overnight. LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with 0-50% EtOAc in hexanes to afford 1.25 g of compound 14 as a white solid. MS: (+) m / z 694.3 (M + 1).

Compound 15. (E) -2,2 '- (diazene-1,2-diyl) bis (2-methylpropanenitrile) (AIBN, 0.016 g, 0.095 mmol) was added to a solution of compound 14 (1.78 g, 2.57 mmol) and tributylstyannane (Bu3SnH, 1.380 ml, 5.13 mmol). The reaction mixture was kept under reflux for 30 min (water bath at 142 ° C). The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-33% EtOAc in

Hexanes to give 0.84 g of compound 15 as a light yellow oil. MS: (+) m / z 468.3 (M + 1-Boc).

Compound 16. LiOH (0.071 g, 2.96 mmol) in water (3.7 ml) was added to a solution of compound 15 (0.84 g, 1.480 mmol) in tetrahydrofuran (THF, 11.4 ml) followed by addition of 30% aqueous H2O2 (0.271 ml, 8.88). mmol) at 0 ° C. The reaction mixture was then stirred at 0 ° C for 4 h, 20 mL of 1.33 M aqueous Na 2 SO 3 was added to complete the reaction. Hydrochloric acid (1M) was added to adjust the pH to 2-3. The resulting aqueous solution was extracted with DCM. The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-75% EtOAc in hexanes to give 0.53 g of compound 16 as a colorless oil. MS: (+) m / z 357.3 (M + 1-Boc).

Compound 17. Concentrated hydrochloric acid (4 drops) was added to a solution of 2,2-dimethoxypropane (3.53 mL, 28.7 mmol) and compound 16 (0.53 g, 1.161 mmol) in methanol (17.7 mL). The reaction mixture was stirred overnight at room temperature. LCMS indicated the reaction was complete. LCMS also showed the formation of some unprotected by-products 16a. The diluent was evaporated.

Triethylamine (2.2 eq., 0.36 ml) was added to a solution of the above residue and BOC2O (1.2 eq., 304.3 mg) in acetonitrile at room temperature to again protect the by-product 16a. The reaction mixture was stirred at room temperature for 2 hours. LCMS indicated the reaction was complete. The diluent was evaporated. Water (7 ml) was added, the aqueous solution was extracted with EtOAc. The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-50% EtOAc in hexanes to give 0.3 g of compound 17 as a colorless oil. MS: (+) m / z 371.3 (M + 1-Boc).

Compound 18. A mixture of compound 17 (0.223 g, 0.474 mmol) and Pd / C 10 wt. (20 mg, 0.474 mmol) in methanol (6 mL) was stirred under H2 atmosphere overnight. The Pd / C catalyst was filtered off and the filtrate was concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-50% EtOAc in hexanes to yield 0.112 g of compound 18 as a white solid. MS: (+) m / z 237.2 (M + 1-Boc).

Compound 19. A mixture of compound 18 (0.204 g, 0.606 mmol), N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.174 g, 0.910 mmol) and Fmoc protected citrulline 18a (0.361 g, 0.910 mmol) in DMF ( 12.4 ml) was stirred at room temperature overnight. NH 4 Cl saturated solution (20 mL) was added to complete the reaction. The aqueous solution was extracted with EtOAc. The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash silica gel chromatography with a 0-30% MeOH in DCM gradient to yield 0.25 g of compound 19 as a white solid. MS: (+) m / z 716.4 (M + 1).

Compound 20. Piperidine (0.5 mL, 5.06 mmol) was added to a solution of compound 19 (0.25 g, 0.349 mmol) in DMF (5 mL). The reaction mixture was then stirred at room temperature for 20 min, the solvent was evaporated to obtain an intermediate with the removed Fmoc protection as residue

DIEA was added to a solution of (S) -2 - ((((9H-fluorene-9-yl) methoxy) carbonyl) -amino) 3-methylbutanoic acid 19a (0.142 g, 0.418 mmol) and N, N, N 'hexafluorophosphate, N'-tetramethyl-O- (7-azabenzotriazol-1-yl) uronium (HATU, 0.146 g, 0.383 mmol) in DMF (2 ml), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 5 min. The above residue in DMF (1ml) and DIEA was added to the reaction mixture, pH was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 15 min and 20 mL of water containing 8 mL of an aqueous solution of 0.1% TFA was added. The aqueous solution was extracted with EtOAc. The combined organic phases are dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a 0-20% gradient of MeOH in DCM to afford 0. 24 g of compound 20 in the form of a white solid. MS: (+) m / z 815.4 (M + 1).

- EP 2956173

Compound 21. Piperidine (0.3 ml) was added to a solution of compound 20 in DMF (3 ml). The reaction mixture was stirred at room temperature (RT) for 1 hour. LCMS indicated the reaction was complete. The diluent was evaporated.

Lithium hydroxide (0.028 mg, 1.176 mmol) in water (2 mL) was added to a solution of the above residue in THF (4 mL). The reaction mixture was stirred at room temperature for 4 hours. and then aqueous HCl (0.1N) was added to acidify the reaction mixture (pH 23). The solution was partially evaporated and lyophilized to afford compound 21 as a white solid. MS: (+) m / z 579.4 (M + 1).

Compound 22. DIEA was added to the mixture of ε-maleimidocaprone 21-N-hydroxysuccinimide ester (Tokyo Chemical Industry, 64.7 mg, 0.210 mmol) and compound 21 (81 mg, 0.14 mmol) in DMF (3 mL), adjusted to pH 8-9. After mixing before 2 hours of the reaction mixture at room temperature, 10 ml of a 1: 1 mixture of acetonitrile in water containing 0.1% TFA are added. The product 22 was purified by preparative high performance liquid chromatography (HPLC). MS: (+) m / z 772.5 (M + 1).

Compound 23. 2,2,2-trifluoroacetic acid (0.7 mL, 0.013 mmol) was added to a mixture of compound 22 (30 mg, 0.039 mmol) in DCM (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 10 min, LCMS indicated complete reaction. The diluent was evaporated to yield compound 23. MS: (+) m / z 672.4 (M + 1).

Compound (III-1). DIEA was added to a solution of compound 9 (23.66 mg, 0.043 mmol) and HATU (14.77 mg, 0.039 mmol) in DMF (1 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, compound 23 (26.1 mg, 0.039 mmol) in DMF (1ml) and DIEA was added. The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, LCMS indicated complete reaction. The reaction was terminated by the addition of 10 ml of 1: 1 (v / v) of a water mixture containing 0.1% TFA and acetonitrile. Compound (III-1) was purified by preparative HPLC. MS: (+) m / z 1207.7 (M + 1).

Compounds such as (III-1) containing a maleimide group can be used to prepare conjugates by reaction with a sulfhydryl group on an antibody or other ligand. The sulfidryl group may be derived from a cysteine ​​residue or may be obtained by derivatization with a 2-iminothiolane of a lysine residue.

Example 2 - Compound (III 2)

The example describes the synthesis of compound (III-2), the corresponding scheme is shown in figure 4.

Compound 24. N-Ethyl-N-isopropylpropan-2-amine (0.556 ml, 3.19 mmol) was added to a solution of glycine-tert-butyl ester hydrochloride 23a (0.209 g, 1.596 mmol), Fmocaminoxyacetic acid 23b (0.5g, 1.596 mmol) and HATU (0.607 g, 1.596 mmol) in DMF (5 ml), at temp.

room temperature. The reaction mixture was then stirred at room temperature (RT) for 1 hour, 0.1% aqueous TFA (20 ml) was added. The aqueous layer was extracted with EtOAc and the combined organic layers were dried, filtered and concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-70% EtOAc in hexanes to give 0.45 g of compound 24 as a colorless oil. MS: (+) m / z 449.2 (M + 23).

Compound 25. TFA (3 mL, 1.437 mmol) was added to a solution of compound 24 (0.45 g, 1.055 mmol) in DMF (0.5 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-30% MeOH in DCM to give 0.39 g of compound 25 as a white solid. MS: (+) m / z 371.1 (M + 1).

Compound 26. N, N'-methanediylenedene cyclohexanamine (DCC, 0.261 g, 1.267 mmol) was added to a solution of compound 25 and 1-hydroxypyrrolidine-2,5-dione (0.146 g, 1.267 mmol) in DCM (6 mL) at room temperature. The reaction mixture was then stirred overnight at temperature

- room temperature, and the solid was filtered off. The filtrate was then concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-100% EtOAc in hexanes to yield 0.43 g of compound 26 as a colorless oil.

Compound 27. DIEA was added to a solution of compound 21 (50 mg, 0.086 mmol) and compound 26 (60.6 mg, 0.130 mmol) in DMF at room temperature, pH adjusted to 8-9. The reaction mixture was then stirred at room temperature (RT) for 4 h, 10 ml of a 1: 1 mixture of aqueous 0.1% TFA and acetonitrile were added. Purified by preparative HPLC to afford 55 mg of compound 27 as a white solid, MS: (+) m / z 931.4 (M + 1).

Compound 28. TFA (1 mL, 0.059 mmol) was added to a mixture of compound 27 (55 g, 0.059 mmol) in DCM (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 10 min. The diluent was evaporated.

DIEA was added to a solution of compound 9 (32.7 mg, 0.059 mmol) and HATU (22.47 mg, 0.059 mmol) in DMF (1 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, DMF (2ml) and DIEA were added. The pH of the reaction solution was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, LCMS indicated complete reaction. The reaction was terminated by the addition of 20 ml of 1: 1 (v / v) of a water mixture containing 0.1% TFA and acetonitrile. Purified by preparative HPLC to afford 70 mg of compound 28 as a white solid. MS: (+) m / z 684.1 (M / 2 + 1).

Compound (III-2). The piperidine was added to a mixture of compound 28 (70 mg, 0.051 mmol) in DCM (4 ml). The reaction mixture was then stirred at room temperature for 20 min, a 1: 1 mixture of acetonitrile and 0.1% aqueous TFA (40 ml) was added. Purified by preparative HPLC to afford 46 mg of compound (III-2) as a white solid. MS: (+) m / z 1144.6 (M + 1).

Compounds such as (III-2) having a hydroxylamine group can be used to form conjugates with antibodies or other ligands having the functionality of aldehydes or ketones, e.g. by incorporating an unnatural amino acid 4-acetylphenylalanine.

Example 3 - Compound (I-2) and (I-3)

The synthesis of compounds (I-2) and (I-3) is shown schematically in Figure 5.

Compound (I-3). DIEA was added to a solution of compound 9 (10 mg, 0.018 mmol) and HATU (6.87 mg,

0.018 mmol) in DMF (0.3 ml), adjusting the pH to 8-9. The reaction mixture was then stirred for 10 min at room temperature, compound 29 (prepared according to Cheng et al. 2011, Example 17, 4.56 mg, 0.018 mmol) in DMF (0.5 ml) and DIEA, adjusted to pH 8-9. Then the reaction mixture is for 20 min. After stirring at room temperature, the reaction was quenched with 4 mL of a 1: 1 mixture of acetonitrile and 0.1% aqueous TFA. Purified by preparative HPLC to afford 12 mg of compound (I-3) (I-2) as a white solid. MS: (+) m / z 788.4 (M + 1).

Compound (I-2). A mixture of compound (I-3) (12 mg, 0.015 mmol) and Pd / C 10% wt. (4 mg, 0.015 mmol) in methanol (0.5 mL) was stirred under H2 atmosphere overnight. The catalyst was filtered off and the filtrate was concentrated. Purified by preparative HPLC to give 8.1 mg of compound (I-2) as a white solid. MS: (+) m / z 758.4 (M + 1).

Compound (I-1) can be prepared analogously by replacing compound 29 with a compound having a mixed stereochemistry at the alpha-methyl position (Cheng et al. 2011).

Example 4 - Compound (III-4)

In FIGS. 6a to 6c, the synthesis of compound (III-4) is shown schematically.

- 29 EP 2956173

Compound 32. Compound 30 (Aldrich, 3.5 g, 13.9 mmol) was dissolved in 50 mL of DCM. To this solution was added Dess-Martin periodinane (11.8 g, 27.9 mmol) at 5 ° C. After 10 minutes the reaction was warmed to room temperature. After another hour, the reaction was quenched with saturated aqueous NaHCO3 and saturated aqueous NaS2O3. After extracting with ether, the ether extract was washed with aqueous NaHCO 3 and then brine, dried and evaporated to give a viscous oil. The oil was dissolved in 50 mL of DCM, to which compound 31 (5.05 g, 13.93 mmol) was commercially available. After 10 minutes, the reaction mixture was dissolved in EtOAc, washed with aqueous NaHCO 3 and then brine and dried, filtered and the solvent was evaporated. Column chromatography (EtOAc: hexane, 0-20% gradient) afforded compound 32 (2.3 g, 6.90 mmol, 49 yield. 5%) as a white solid. Its NMR spectrum was consistent with literature (Wipf et al. 2004a).

Compound 33. Hydrochloric acid (7.80 ml, 31.2 mmol, 4M in dioxane) was added at 5 ° C to a solution of compound 32 (5.2 g, 15.60 mmol) in DCM. After completion of the deprotection reaction, the reaction mixture was evaporated to give compound 33 (4.21 g, 15.60 mmol, 100% yield, hydrochloride) as a white solid which was used in the next step without further purification.

Compound 35. HATU (4.61 g, 12.12 mmol) and DIPEA (6 mL, 34.4 mmol) were added to a solution of compound 34 (prepared by Sani et al. 2007, 4.00 g, 11.6 mmol) in 20 mL DMF at 5 ° C. . After 10 minutes Compound 33 (2.71 g, 11.60 mmol) was added. After another 30 min. the mixture was dissolved in EtOAc, then rinsed with 10% aqueous citric acid, saturated aqueous NaHCO 3 and brine. After drying and filtration, the organic phase was evaporated to yield compound 35 (6.49 g, 11.60 mmol, 100% yield, [M + Na]⁺, calculated 582.3, determined 582.3) as an oil which was used in the next step without further purification.

Compound 36. NaBH4 (4.66 g, 123 mmol) was added in portions to 100 mL of a solution of methanol of compound 35 (6.49 g, 11.6 mmol) and NiSO4 (H2O) 6 (6.48 g, 24.66 mmol) at 5 ° C. (Note: Hydrogen formed.) After 30 min, saturated aqueous NaHCO 3 was added followed by EtOAc. After filtration over CELITE ™, the organic phase was separated from the aqueous phase, washed with brine, dried, filtered and evaporated to yield compound 36 (5.6 g, 9.97 mmol, 81% yield, [M + 1]⁺, 562.3, found 562.4), which was used in the next step without further purification.

Compound 37. Compound 36 (5.6 g, 9.97 mmol) was dissolved in 30 ml of pyridine at 5 ° C. Acetic anhydride (4 g, 39.2 mmol) was added to the above solution. After 10 minutes the reaction was warmed to room temperature. After about one hour, the reaction mixture was concentrated. The resulting residue was dissolved in EtOAc, and the organic phase was washed successively with 10% aqueous citric acid, saturated aqueous NaHCO 3, and brine. The organic phase was dried, filtered and concentrated to give compound 37 (5.8 g, 9.61 mmol, 100% yield, [M + 1]⁺, 604.3, found 604.4), which was used in the next step without further purification.

Compounds 38a and 38b. Compound 37 (0.8 g, 1.3 mmol) was dissolved in 5 mL of methanol at -78 ° C. NaOMe (331 μl, 1.33 mmol, 4M in MeOH) was added to this solution. The mixture was allowed to warm to temperature over 1 hour. The mixture was dissolved in EtOAc, then rinsed with 10% aqueous citric acid, saturated aqueous NaHCO 3 and brine. The separated organic phase was dried, filtered off and evaporated to yield a mixture of ethyl and methyl esters (compounds 38a and 38b, respectively). The ester mixture was not separated during the next few steps until hydrolysis of both esters to the carboxylic acid in the last step.

Compounds 40a and 40b. A mixture of compounds 38a and 38b from the above reaction was dissolved in 20 ml of DCM. To this solution was added 4-nitrophenylchloroformate 39 (524 mg, 2.6 mmol) and pyridine (210 μL, 2.6 mmol) at 5 ° C. The temperature was allowed to rise to room temperature, then after 1 hour. methylamine (1.950 ml, 3.9 mmol, 2M w

THF). After 10 minutes, the solvent was evaporated and the residue was passed through the column

- chromatographic to obtain a mixture of compounds 40a and 40b (ethyl and methyl esters, respectively, 420 mg, 40a / 40b, 3: 1 ratio from HPLC, yield about 53% in two steps, [M + 1]⁺: calculated at 603.3, 603.4 for 40a were obtained; calculated on 589.3, 589.4 were obtained for 40b).

Compounds 41a and 41b. A mixture of compounds 40a and 49b (420 mg, about 0.68 mmol) was dissolved in 3 ml of DCM to which HCl (4.8 mmol, 1.2 ml, 4N in dioxane) was added. After 1 hour at 5 ° C, the solvent was evaporated and the mixture of compounds 41a (ethyl ester) and 41b (methyl ester), in a 3: 1 ratio, was used in the next step without further purification.

Compounds 43a and 43b. A mixture of compounds 41a and 41b (400 mg, = 0.72 mmol), compound 42 (commercially available from Anichem, 170 mg, 0.721 mmol) and acetic acid (0.041 mL, 0.721 mmol) were mixed in DCM at 5 ° C. Triacetoxy borohydride (306 mg, 1.44 mmol) was added. After 30 min. the mixture was dissolved in EtOAc. After washing with 7% aqueous K 2 CO 3 and brine, the organic phase was dried, filtered off and evaporated to give a residue. Purification by column chromatography (MeOH: DCM, 0-7% gradient) afforded compounds 43a (ethyl ester) and 43b (methyl ester) (310 mg, approximately 0.42 mmol, yield about 58.3%, proportion 43a: 43b about 3: 1, [M + 1]⁺: calculated at 738.4, 738 for 43a were obtained; 724.4 were calculated, 724 for 43b were obtained).

Compounds 45a and 45b. A mixture of compounds 43a and 43b (310 mg, about 0.42 mmol) was dissolved in 5 ml of DCM at room temperature. To the resulting solution was added 2,6-ditert-butylpyridine (161 mg, 0.840 mmol) and 2 mL of a solution in compound 44 (prepared according to Peltier et al. 2006, 73.8 mg, 0.420 mmol) in DCM. After half an hour Et was added₃N (58.6 μΐ, 0.420 mmol). The mixture was dissolved in EtOAc, then rinsed with 10% aqueous citric acid, saturated aqueous NaHCO 3 and brine. The organic phase was dried, filtered off and evaporated to obtain a residue. Purification on column chromatography afforded compounds 45a and 45b (294 mg, approximately 0.334 mmol, yield 80%, 45a: 45b in a 3: 1 ratio, [M + 1]⁺: calculated 877.5, 877 for 45a were obtained; calculated 863.5, 863 obtained for 45b) as a viscous oil.

Compounds 46a and 46b. A mixture of compounds 45a and 45b (100 mg, about 0.114 mmol) was added to a Pd / C suspension (65 mg, 10%) in 20 ml MeOH. HCl (28.5 μΐ, 0.114 mmol, 4M in dioxane) was added. The atmosphere was removed from the flask and the H2 filled, the process repeated three times. After 2 hours the suspension was filtered off and the solvent was evaporated to give a residue. A suspension of compound 5a (19.59 mg, 0.137 mmol) was added together in 500 μl DMF, HATU (43.4 mg, 0.114 mmol) and DIPEA (49.8 μ (0.285 mmol) at 5 ° C. After obtaining a homogeneous suspension, the above residue was added in solution DMF (1 ml) .More added DIPEA to adjust the pH to about 12. After 10 min, the mixture was dissolved in EtOAc, then rinsed with 10% aqueous citric acid, saturated aqueous NaHCO 3 and brine, The separated organic phase was dried, filtered off and evaporated.⁺: calculated 976.5, 976.5 for 46a were obtained; calculated 962.5, obtained 962.5 for 46b).

Compounds 47a and 47b. HCl (256 μ (1.024 mmol, 4M in dioxane) was added to 2 mL of a solution of compounds 46a and 46b (200 mg, 0.205 mmol) in MeOH at 5 ° C. After 1 h the solution was evaporated and dried under high vacuum for 5 h. The resulting viscous oil, the Fmoc 18a protected citrulline (81 mg, 0.205 mmol) and DIPEA (179 μ (1.024 mmol) were dissolved in 2 mL of DMA at room temperature. Propylphosphonate acid anhydride (T3P, 178 μL) was added. 0.410 mmol, 2.3 M in EtOAc.) After 1 h, the reaction mixture is dissolved in EtOAc, then washed with saturated aqueous NaHCO 3 and brine. After separation, drying and evaporation, the resulting residue is passed through column chromatography (MeOH: DCM, 0-10% gradient) to obtain a mixture of compounds 47a and 47b (ethyl and methyl esters, respectively, 150 mg, about 0.119 mmol,⁺: for 47a, 1255.6 were calculated, 1255.6 were obtained; for 47b, 1241.6 were calculated, 1241.6 were obtained).

- EP 2956173

Compound 49. A mixture of compounds 47a and 47b (200 mg, about 0.165 mmol) was dissolved in 5 ml DMF (with 5% piperidine) at room temperature. After 30 min the solution was evaporated to dryness. The resulting residue was mixed with the N-hydroxysuccinimide ester with Boc 48 protected valine (61.9 mg, 0.198 mmol), 5 ml DMF and DIPEA (87 μΐ, 0.496 mmol). After allowing the reaction to proceed overnight, the reaction mixture was evaporated to dryness. The resulting mixture was dissolved in 5 mL of a mixture of MeOH, THF and water (1: 1: 1). NaOH was added, the pH of the final solution was 14. After allowing the reaction to run overnight at room temperature, the mixture was acidified with HCl to pH 3 and evaporated under high vacuum. The obtained solid was treated with TFA and after 10 minutes the mixture was evaporated to yield compound 49 (80 mg, 0.072 mmol, 43.8% yield, [M + 1]⁺, calculated 1104.6, found 1104.6) after purification in preparative HPLC.

Compound (III-4) Compound 49 (80 mg, 0.072 mmol), commercially available compound 21a (Aldrich, 26.6 mg, 0.087 mmol), and DIPEA (38.0 μL 0.217 mmol) were dissolved in 2 mL of DMF. After leaving the reaction overnight, the mixture was evaporated and the residue was purified by preparative HPLC to obtain the major isomer (15 mg, 16% yield, 1/2 [M + 2]²⁺, 649.3 was calculated, designated 649.5) and the secondary isomer (3.7 mg, 4% yield, 1/2 [M + 2]²⁺, 649.3 was calculated, found 649.5)). The main isomer pre-labeled as (III-4), had the stereochemical structure of the natural tubulinis in the alpha-methyl group of the Tup subunit, and the secondary isomer designated compound (III-5) had an inverted stereochemical structure at this point.

Example 5 - Compounds (I-5), (I-6), and (I-7)

A small portion of the viscous oil described above resulting from treatment of compounds 46a and 46b of HCl was not conjugated to compound 18a, but instead was dissolved in the mixture

THF, MeOH and water (1: 1: 1). The pH of the reaction mixture was adjusted to 14. After leaving the reaction overnight, half of the mixture was evaporated and purified by preparative chromatography to obtain compounds (I-5) (1 m ;, M + 1, 876.6), (1-6) (1 mg, M + 1, 862.5) and (I-7) (1 mg, M + 1, 848.5).

Example 6 - Compound (I-1)

The synthesis scheme of compound (I-1) is shown in Figure 7.

Compound 51.HCl (6N, 0.2 mmol) was added to a solution of compound 50 (Cheng et al 2011, 50 g, 0.198 mmol) and 2,2-dimethoxypropane (0.244 mL, 1.982 mmol) in MeOH (1 mL). The reaction mixture was then stirred at room temperature overnight. The solvent was evaporated to give 52.8 mg of compound 51. MS: (+) m / z 267.2 (M + 1).

Compound 52. A mixture of compound 51 (52.8 mg, 0.198 mmol) and palladium on carbon (10% wt, 8 mg) in MeOH (1 mL) was stirred under H2 atmosphere overnight. The crystallizer was then filtered off and the solvent was evaporated to give 46.9 mg of compound 52. MS: (+) m / z 237.3 (M + 1).

Compound (I-1). A mixture of pentafluorophenol (2.493 mg, 0.014 mmol), 1,3-dicyclohexylcarbodiimide (2.049 mg, 9.93 μmol), and compound 9 (5 mg, 9.03 μmol) in DCM (0.5 mL) was stirred at room temperature overnight. The diluent was then evaporated.

To a solution of the resulting residue (6.50 mg, 9.03 μmol) and compound 52 (4.27 mg, 18.06 μmol) in DMF (0.2 ml) was added DIEA (1 drop). Then the reaction mixture is for 10 min. After stirring at room temperature, the reaction was quenched by the addition of a 1: 1 mixture of acetonitrile and 0.1% aqueous TFA (4 mL). Purification by preparative HPLC gave 2.5 mg of compound (I-1) as a white solid. MS: (+) m / z 772.5 (M + 1)

Example 6 - Compound (I-4)

Figure 8 shows a synthesis scheme of compound (I-4).

- EP 2956173

DIEA was added to a solution of compound 9 (10.69 g, 0.019 mmol) and HATU (7.34 mg, 0.019 mmol) in DMF (0.3 mL), adjusting the pH to 8-9. The reaction mixture was then stirred at room temperature for 10 min, compound 53 (4 mg, 0.019 mmol, prepared according to Sani et al., 2007) in DMF (0.5 ml) and DIEA, pH adjusted to 8-9. Then the reaction mixture is for 20 min. After stirring at room temperature (RT), the reaction was quenched by the addition of a 1: 1 mixture of acetonitrile and 0.1% aqueous TFA (4 ml). Purification by preparative HPLC gave 12.5 mg of compound (I-4) as a white solid. MS: (+) m / z 743.4 (M + 1).

Example 7 - Tiocarbamates

Compounds according to formula (I) wherein W is S (i.e., thiocarbamates) can be obtained by treating a suitable precursor such as compound 6 (Fig. 1) with sodium hydride followed by thioisocyanate as follows:

NHMe

<img file="PL2956173T3_D0041.tif" />

(partial construction)

Example 8 - Compound (I-4)

The synthesis scheme of compound 56 is shown in Figs. 9a and 9b.

Compound 56. 1,3-Dicyclohexylcarbodiimide (DCC, 0.160 g, 0.778 mmol) was added to a solution of tert-butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-niate 54 (Quanta Biosciences, 0.25 g, 0.778 mmol ) and 2 - (((((9H-fluorene-9-yl) methoxy) carbonyl) amino) oxy) acetic acid (Chem-Impex, 0.244 g, 0.778 mmol) in DCM (5 ml) at room temperature. The reaction mixture was then stirred at room temperature overnight and the precipitate was filtered off. The filtrate was then concentrated. The crude product was purified by flash chromatography eluting with silica gel with a 0-10% methanol in DCM gradient to give 0.30 g of 56 as a colorless oil. MS: (+) m / z 617.4 (M + 1).

Compound 57. A solution of compound 56 (0.303 g, 0.491 mmol) in TFA (2 mL, 0.662 mmol) was stirred at rt for 2 h. The solution was then concentrated, the residue was rinsed with hexanes to give 0.28 g of compound 57. MS: (+) m / z 561.3 (M + 1).

Compound 58. DCC (0.199 g, 0.963 mmol) was added to a solution of compound 57 (0.27 g, 0.482 mmol) and 1-hydroxypyrrolidine-2,5-dione (also known as N-hydroxysuccinimide or NHS, 0.111 g, 0.963 mmol) in DCM (5 ml) at room temperature. The reaction mixture was then stirred at room temperature overnight, and the solid was filtered off. The filtrate was then concentrated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-100% ethyl acetate in hexanes to give 0.12 g of compound 58 as a colorless oil. MS: (+) m / z 658.3 (M + 1).

Compound 59. DIEA (2 drops) was added to a solution of compound 58 (0.12 g, 0.182 mmol) and compound 21 (0.106 g, 0.182 mmol) in DMF (2 mL) at room temperature. The reaction mixture was then stirred at room temperature for 1 hour, the reaction was terminated by the addition of a mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified in preparative HPLC to give 0.12 g of compound 59 as a white solid. MS: (+) m / z 1121.6 (M + 1).

Compound 60. TFA (0.5 mL) was added to a solution of compound 59 (20 g, 0.018 mmol) in DCM (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 20 min, the solution was concentrated to give 18.2 mg of compound 60. MS: (+) m / z 1021.6 (M + 1).

- EP 2956173

Compound 61. DIEA was added to a solution of compound 9 (9.87 mg, 0.018 mmol) and HATU (6.78 mg, 0.018 mmol) in DMF (0.4 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, compound 60 (18.2 mg, 0.018 mmol) in DMF (1ml) and DIEA was added. The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, the reaction was terminated by the addition of 10 ml of 1: 1 (v / v) of a water mixture containing 0.1% TFA and acetonitrile. The crude product was purified by preparative HPLC to give 25 g of compound 61 as a white solid. MS: (+) m / z 779.0 (M / 2 + 1).

Compound (III-6). One drop of piperidine was added to a solution of compound 61 (25 mg, 0.016 mmol) in DMF (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 1 hour, the reaction was terminated by the addition of a mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified by preparative HPLC to give 20 g of compound (III-6) as a white solid. MS: (+) m / z 668.0 (M / 2 + 1). Compound (III-6) has a hydroxylamine group that can be used for conjugation by the formation of an oxime, for example with the keto group of the p-acetylphenylalanin residue, which has been introduced into the protein as outlined above.

The conjugate of compound (III-6) and the anti-mesothelin antibody modified to contain a p-acetyl phenylalanine residue exhibited an EC50 value of 0.14 against gastric N87 gastric cells, in an introduction study ³H thymidine.

In one embodiment, the present invention provides a conjugate of compound (III-6) and an anti-anothelin antibody, modified to contain a keto group. Preferably, the modification is effected by introducing the p-acetylphenylalanin residue to the antibody polypeptide chain. Also, preferably, the antibody so modified is 6A4.

Example 9 - Intermediate 69

Figure 10 shows a synthesis scheme of compound 69 that can be used as an intermediate for the synthesis of compounds of the present invention.

Compound 63. Compound 62 (Chem-Impex, 5 g, 23.8 mmol) was added to a solution of SOCl 2 (3.47 ml, 47.6 mmol) in 20 ml MeOH at 5 ° C. After allowing the reaction to run overnight, the reaction mixture was heated for half an hour at 45 ° C. The volatile materials were evaporated and the residue was dissolved in 20 ml of DCM. Boc2O (7.8 g, 35.7 mmol) was added. Et3N was used to adjust the pH of the solution to 9 (tested with wet pH indicator paper). After several hours, the mixture was dissolved in EtOAc. The EtOAc mixture was then rinsed with a 10% citric acid solution, a saturated NaHCO3 solution and brine. The organic phase was dried, separated and evaporated. The final residue after evaporation of the dried organic phase was passed through the column to give compound 63 (5 g, 65% yield, [M + 1Boc]⁺, calculated 225.1, found 225.2).

Compound 65. To 20 ml of a solution of compound 63 (2 g, 6.2 mmol) in DCM was added DIBAL-H (12.4 ml, 12.4 mmol, 1M in DCM) at -78 ° C. After half an hour, the reaction was quenched with MeOH. HCl was used to adjust the pH of the solution to about 2. The mixture was dissolved in EtOAc, then rinsed with 10% citric acid, brine, separated and evaporated. The residue was dissolved in 30 mL of DCM. Compound 64 (2.4 g, 6.2 mmol, US 4.894,386) was added at 0 ° C. The mixture was evaporated after 1 hour. at room temperature, the residue was passed through the column to give compound 65 (2 g, 79% yield, [M + 1-Boc]⁺, calculated 307.2, found 307.1).

Compound 66. Compound 65 (600 mg, 1.48 mmol) was dissolved in 75 mL of EtOAc at room temp. The solution was transferred to a flask with N2 and Pd / C (600 mg, 10%). The atmosphere was removed from the flask and filled with H2; the cycle was repeated three times. After 1 hour the solution was filtered off and evaporated to give compound 66 (560 mg, 100% yield, [M + 1]⁺, calculated 379.3, designated 379.3) as a mixture of epimers. The mixture was separated later.

- EP 2956173

Compound 67. Compound 66 (1.30 g, 3.43 mmol), protected with Fmooc citrulline 18a (1.6 g, 4.12 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 1.06 g, 4.3 mmol) was dissolved in the mixture. DCM and MeOH (22 mL, 10: 1). After leaving the reaction overnight, the mixture was evaporated and the residue was passed through a column (MeOH: DCM, 0-5% gradient) to give compound 67 [M + 1]⁺, 758.4 was calculated, 758.4 determined) as a mixture of epimers (= 4: 1 from HPLC analysis). The resulting compound was transferred to the next step without additional cleaning.

Compound 68a. A mixture of compound 67 from the above reaction was dissolved in 10 ml of DMF (with 5% piperidine) at room temperature. After 30 min. the reaction mixture was evaporated and dried with a high vacuum pump overnight. The residue was mixed with compound 48 (1.5 g, 3.45 mmol) in 5 mL DMF. Et3N was used to adjust the pH of the solution to about 12. After 1 hour. the reaction mixture was dissolved in EtOAc, then rinsed with 10% citric acid, saturated aqueous NaHCO 3 and brine. The separated organic phase was dried, filtered off and evaporated to dryness. The residue was deprotected with 5% piperidine in DMF in the same way as the deprotection of compound 67 was carried out. The amine obtained in this step and compound 21a (1 g, 3.27 mmol) were mixed in 10 ml of DMF. Et3N was used to adjust the pH of the solution to about 12 at room temperature. After leaving the reaction for the night, the reaction mixture was dissolved in EtOAc, then rinsed with 10% citric acid, saturated aqueous NaHCO 3 and brine. The organic phase was dried, filtered off and evaporated to obtain a residue. The residue was passed through a standard silica gel column to obtain a mixture of compounds 68a and 68b (1g, 35% yield from compound 66, [M + 1]⁺, calculated 828.5, found 828.5). After separation in the HPLC column, 600 mg of the main epimer was obtained (the structural structure was theoretically established as the compound 68a with the compound 68b as the secondary epimer).

Compound 69. Compound 68a (600 mg, 0.72 mmol) was dissolved in 5 mL of DCM and DCM

TFA (1: 1). After 2 h, the reaction mixture was evaporated to yield compound 69 (quantitative yield, [M + 1]⁺, calculated on 672.4, found 672.4).

Example 10 - Intermediate 75

Figure 11 shows a synthesis scheme of intermediate 75 that can be used to synthesize compounds of the present invention.

Compound 71. Compound 70 (Cheng et al 2011, 25 mg, 0.044 mmol, N, N'-diisopropylcarbodiimide (DIC, 0.014 ml, 0.088 mmol), 4- (dimethylamino) pyridine (DMAP, 10.78 mg, 0.088 mmol) and MeOH. (0.036 ml, 0.882 mmol) was stirred at room temp. The mixture was evaporated after 1 hour and passed through the column to give compound 71 (10 mg, 39% yield, M + 1, 581.4).

Compound 72. Compound 71 (122 mg, 0.210 mmol from another synthetic batch) was dissolved in MeOH (2 ml) at 5 ° C. NaOMe (0.441 ml, 0.221 mmol) was added. After 0.5 hours the mixture was neutralized with HCl (4M in dioxane) and evaporated to give compound 72 (m + 1, 539.4) which was used in the next step without purification.

Compound 73. Compound 72 (60 mg, 0.111 mmol) was dissolved in DCM (1.5 ml) at 5 ° C. Pyridine (0.045 ml, 0.557 mmol). 4-Nitrophenylchloroformate 72a (67.3 mg, 0.334 mmol, Aldrich) in 0.5 mL of DCM was slowly added. After allowing the reaction to proceed overnight, the mixture was evaporated and purified by column chromatography to obtain compound 73 (42 mg, 0.060 mmol, 53.6% yield) (m + 1, 704.4).

Compound 74. Compound 73 (42 mg, 0.060 mmol) was dissolved in DCM (1 mL) at 5 ° C.

Methylamine (1.853 mg, 0.060 mmol) was added. After 0.5 hours the mixture was evaporated and the residue was passed through a chromatographic column (MeOH: DCM, 0-15% gradient, product elution at 7-10%) to give compound 74 (35 mg, 0.059 mmol, 98% yield) (m + 1,

596.4).

- EP 2956173

Compound 75. Compound 74 (93 mg, 0.156 mmol) was dissolved in THF (1 mL) at 5 ° C. LiOH (7.47 mg, 0.94 mmol) was added in 0.34 ml of water. After completion of the reaction, the mixture was neutralized with HCl (4M in dioxane) and evaporated under high vacuum to yield compound 75 (m + 1, 582.3) which was used in the next step without purification.

Example 11 - Chemical compounds (III-7) and (III-8)

Figure 12 shows a synthesis scheme of compounds (III-7) and (III-8).

Compound (III-7 '). Compound 75 (11 mg, 0.019 mmol), was activated in DMF (0.5 ml) with HATU (6.83 mg, 0.018 mmol) and DIEA (14.86 μΙ, 0.085 mmol). Compound 69 (15.24 mg, 0.023 mmol) was added. After 10 minutes the reaction mixture was dissolved in DMSO and purified by preparative chromatography to obtain compound (111-7) (12 mg, 9.71 μmol, 51.4% yield) (M + 1, 1235.7).

Compound (III-8). Compound 75 (10 mg, 0.017 mmol), was activated in DMF (0.5 mL) with HATU (6.21 mg, 0.016 mmol) and DIEA (0.014 mL, 0.077 mmol). Compound 23 (13.86 mg, 0.021 mmol) was added. After 10 minutes the reaction mixture was dissolved in DMSO and purified by preparative chromatography to obtain compound (111-8) (13 mg, 10.52 μmol, 61.2% yield) (M + 1, 1235.7).

Example 12 - Chemical compounds (I-8) and (I-9)

In principle, compounds (I-8) and (I-9) can be obtained by treating compounds (III-7) and (III-8) with cathepsin protease B, wherein the motif is dipeptide Val-Cit Compound (III). -7) with its ortho-amino group, it may have a more spatial protection against cutting.

Relationship (111-7)

Relationship (111-8)

<img file="PL2956173T3_D0042.tif" />

Example 13 - Biological activity of compounds

Figures 13a and 13b show the biological activity of compounds (I-4) and (I-2) of the present invention against lung cancer H226 and kidney cancer 786-O, respectively, in a luminescent ATP study. As a control, the analog of doxorubicin and tubulinizine of compound A, which contains the acetic group instead of the carbamate group in the Tuv subunit. Compound A can be prepared in accordance with the explanations given by Cheng et al. , 2011.

<img file="PL2956173T3_D0043.tif" />

- EP 2956173

The EC50 values ​​against the H226 cells were: doxorubicin, 115.4 nM; Compound A, 2.4 nM; and compound (I-4), 12.1 nM. The EC 50 values ​​against cells of 786-O were: doxorubicin, 68.9 nM; Compound A, 1.2 nM; and compound (I-4), 7.1 nM.

Figures 13c and 13d show similar data for compounds (I-5), (I-6) and (I-7). The comparative compounds were doxorubicin and tubulisin D. Against the H226 cells, the EC50 values ​​were: doxorubicin, 115.4 nM; tubulisin D, 0.05 nM; compound (I-5) 19.5 nM; compound (I-6), 9.9 nM; and compound (I-7), 15.4 nM. The EC 50 values ​​against cells of 786-O were: doxorubicin, 68.9 nM; tubulisin D, 0.02 nM; compound (I-5), 22.9 nM; compound (I-6), 22.8 nM; and compound (I-7), 12.5 nM.

Human tumor cell lines were obtained from the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108, USA, and expanded as instructed by the ATCC. The cells were seeded at 1.0 x 10³ cells / well in 96-well plates for 3 h for the ATP assay. Serial dilutions of 1: 3 compounds were added to the wells. The plates were incubated for 24 to 72 h. ATP levels on ATP plates were measured with a luminescence kit for determining the viability of CELLTITER-GLO® cells according to the manufacturer's instructions and read on a GLOMAX 20/20 luminometer (both devices manufactured by Promega, Madison, WI, USA). EC values₅₀ - concentration at which the agent inhibits or decreases cell proliferation by 50% - determined using the PRISM ™ software version 4.0 (GraphPad Software, La Jolla, CA, USA).

Example 14 - In vitro conjugate activity

Fig. 14 shows the in vitro activity of the conjugate (II-1) against N87 gastric cancer cells (American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108, USA).

The cells were sown at 1.0 x 10⁴ cells / well in 96-well plates for 3 h for the test ³H thymidine. Serial dilutions (1: 3) of the conjugate (II-1) were added to the wells. The plates were incubated for 120 hours. The plates were pulsed with 1.0 pCi³H-thymidine per well over the last 24 hours of the entire incubation period was collected and read on a Top Count scintillation counter (Packard Instruments, Meriden, CT). EC 50 values ​​- concentration at which the agent inhibits or reduces cell proliferation by 50% maximal inhibition - was determined using PRISM ™ software version 4.0 (GraphPad Software, La Jolla, CA, USA) as 0.2 nM.

Comparison of the EC50 values ​​of Figures 13a-13d and Fig. 14 indicates two points. The first is to strengthen the strength associated with the target delivery of the cytotoxin through the conjugate and then release the mechanism of active internalization by binding the antibody portion of the conjugate to its antigen (Schrama et al., 2006). Second, unconjugated toxins are relatively polar compounds, and when there is no mechanism for active internalisation, diffusion through the cell membrane is hindered, resulting in higher (lower strength) EC50 values ​​measured.

Example 15 - In vivo conjugate activity

In this example, the in vivo activity of the conjugate (II-1) was compared to the activity of conjugate B (Cheng et al 2011) whose structural structure is identical to the conjugate (II1), except that it has acetate in the Tuv subunit instead of carbamate:

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<img file="PL2956173T3_D0044.tif" />

Five million OVCAR3 ovarian carcinoma cells, suspended in 0.1 ml phosphate buffered saline ("PBS") plus 0.1 ml matrigel, were implanted subcutaneously in the side region of SCID mice. Tumor measurements were started 28 days later, mice were randomized in groups of 7 mice each, with an average tumor size of 60 mm³ estimated by LWH / 2 tumor. 29 days after tumor implantation, mice were given one intraperitoneal dose of the test compound. Fig. 15 shows that the conjugate (II-1) used against OVCAR3 xenografts suppressed tumor growth more efficiently than conjugate B. The difference is particularly marked after 20 days.

Figures 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b and 20 provide additional in vivo efficacy data for the conjugates of the present invention prepared according to the protocol described above, mutatis mutandis.

Fig. 16a shows OVCAR3 tumor volume data (ovarian cancer) with respect to a series of conjugates of compound (III-1) with anti-CD70 1F4 antibody or anti-anothelinic antibody 6A4. The legend is given in order of the drug-antibody indicator DAR (e.g., "2.7") and the dose in μmol / kg (e.g., "0.1"). In each case, the administration schedule was intraperitoneal in a single dose (SD), with the exception of the last data set (♦) in which the conjugate was administered Q7Dx3. Fig. 16b shows the change in body weight for the same experiment.

The preparation and characterization of the human monoclonal antibody 6A4 is described in Terrett et al. 2012, which disclosure is incorporated herein by reference. The VH CDR1, CDR2 and CDR3 and VK CDR1, CDR2 and CDR3 sequences of the 6A4 antibody are set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The VH and VK variable region sequences of the 6A4 antibody are set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The preparation and characterization of human 1F4 monoclonal antibody is described in Coccia et al. 2010, which disclosure is incorporated herein by reference. The VH CDR1, CDR2 and CDR3 and VK CDR1, CDR2 and CDR3 sequences of the 1F4 antibody are set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The VH and VK variable region sequences of antibody 1F4 are given in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

Figures 17a and 17b show the results of a similar experiment, but with conjugates of compound (III-8) and anti-mesothelin antibody 6A4. In each case, a single intraperitoneal dose was administered.

The efficacy of the conjugate of compound (III-1) and anti-mesothelin antibody 6A4 against H226 tumors (lung cancer) is shown in Figures 18a and 18b, with information on DAR and dose given again in legends. In each case, the conjugate was administered as a single intraperitoneal dose.

Figures 19a and 19b show H226 results for the conjugate of compound (III-8) and anti-mesothelin antibody 6A4. The first set of data (•) was given as a single dose, whereas in the last two data sets (▼ and ♦) the administration schedule was Q7Dx3.

Fig. 20 shows efficacy data of conjugates of compound (III-1) with anti-mesothelin or anti-CD70 6A4 antibody against N87 gastric carcinoma.

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Antibodies, particularly used in conjugates, may have a natural fixed or engineered (modified) region designed to reduce or eliminate the effector function such as ADCC. Examples of such modified constant antibody regions provide the polypeptides of SEQ ID NO: 25 and SEQ ID NO: 26. SEQ ID NO: 25 is the constant region of the IgG4 isotype modified by substitution of the specified amino acid. SEQ ID NO: 26 is a hybrid of the IgG1 / IgG4 constant region. In both SEQ ID NO: 25 and SEQ ID NO: 26, the occurrence of C-terminal lysine is optional.

Example 16 - Stability tests

In this example, the mouse plasma stability of the conjugate (II-1) and conjugate B, with their respective carbamate and acetic groups, was compared.

The conjugate was injected to the mice at a dose of 0.1 μmol / kg. For the conjugate (II-1), the concentration was 3.4 mg / ml and the conjugate was characterized by a substitution index (SR) of 4.2. For B-conjugate, the concentration was 1.2 mg / ml and SR was 2.3. Approximately 100 μl plasma was collected from each of 3 animals at each time point for analysis.

Plasma samples from different animals were collected, giving 200-300 μl at each time point. The collected volume was centrifuged to remove solids and the supernatant was used for analysis. The conjugate was isolated from plasma by uptake of an immunoaffinity using an anti-idiotype monoclonal antibody conjugated to SEPHAROSE ™ beads. After uptake, the conjugate was washed out by exposure to a low pH and then neutralized with the Tris base. The cytotoxin found on the conjugate was released by adding activated cathepsin to cut off the Cit-Val peptide linker. Cathepsin B digestion was performed at 37 ° C for 3 hours, and then 1 volume of cold methanol was added. The cytoxine extracted from the solution was analyzed in LC-MS using ESI-TOF MS followed by UPLC with reverse phase chromatography (Acquity HSS T3 2.1 X 50mm).

In the case of the conjugate (II-1), the occurrence of the hydroxyl compound from the hydrolysis of the carbamate group was not detected at any time, although time points were set to 240 hours. Only the carbamate compound was detected.

Conversely, in the case of conjugate B, the hydrolysis product was detected after 6 hours, and about 50 percent of the hydrolysis occurred up to 72 hours. Table 2 lists the relative amounts of acetate and hydroxyls based on the intensity of the mass spectrum for the corresponding double charged ions (M [H²⁺] 372.2 Da and 351.2 Da):

<img file="PL2956173T3_D0045.tif" />

<td colspan="4">Table 2</td>

<td rowspan="2">Time (h)</td><td colspan="2">Relative intensity peak</td><td rowspan="2">Hydroxyl ion (% of total)</td>

<td>Acetate ion</td><td>Hydroxyl ion</td>

<td>0.25</td><td>360.587</td><td>-</td><td>0</td>

<td>2</td><td>649.982</td><td>-</td><td>0</td>

<td>6</td><td>165.680</td><td>11.779</td><td>7</td>

<td>24</td><td>77.110</td><td>14.619</td><td>16</td>

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<td>48</td><td>26.760</td><td>16.312</td><td>38</td>

<td>72</td><td>18.243</td><td>17.124</td><td>48</td>

<td>168</td><td>7.978</td><td>18.330</td><td>70</td>

<td>240</td><td>4.268</td><td>15.654</td><td>79</td>

The obtained results indicate that the exchange of the acetic group in the Tuv subunit leads to a more stable compound, which, however, retains the basic biological activity.

Example 17 - Compounds 72a, 72b and 72c.

This example describes the preparation and properties of the compounds of the present invention that have structural changes in the carbamate group. Refer to FIG. 21 for a synthesis scheme.

Compound 70a. Ammonia in methanol (2M, 0.089 ml, 0.178 mmol) was added to compound 7 (0.1 g, 0.148 mmol) in MeOH (2 ml). The reaction mixture was then stirred at room temperature for 20 min, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-15% MeOH in DCM to give 55 g of compound 70a as a white solid. MS: (+) m / z 554.3 (M + 1).

Compound 71a. Lithium hydroxide (4.41 mg, 0.184 mmol) in water (0.5 mL) was added to a solution of 70a (51 mg, 0.092 mmol) in THF (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 2 hours, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-30% MeOH in DCM to give 47 g of compound 71a as a white solid. MS: (+) m / z 540.3 (M + 1).

Compound 72a. DIEA (6.45 μL, 0.037 mmol) was added to a solution of compound 71a (20 mg, 0.037 mmol) and HATU (14.09 mg, 0.037 mmol) in DMF (0.5 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, compound 23 (24.9 mg, 0.037 mmol) in DMF (1 mL) and DIEA (6.45 μL, 0.037 mmol) was added. The pH of the reaction solution was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, LCMS indicated the reaction was complete. The reaction was terminated by the addition of 10 mL of 1: 1 (v / v) of a water mixture containing 0.1% TFA and acetonitrile containing 0.1% TFA. The crude product was purified in preparative HPLC to give 35.2 g of 72a as a white solid. MS: (+) m / z 1193.6 (M + 1). Compound 72a above is also referred to as compound (III-9).

The conjugate of compound 72a and anti-CD70 antibody 1F4 showed an EC50 at 0.19 nM against 786-O kidney cancer cells. The 72a conjugate and anti-anothelin antibody 6A4 showed an EC 50 at 0.16 nM against N87 gastric cancer cells. In both cases, inclusion testing³H-thymidine.

Compound 70b. A solution of compound 7 (0.1 g, 0.148 mmol) and aniline (0.041 mL, 0.444 mmol) in DMF (1.8 mL) was heated at 50 ° C overnight. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-15% MeOH in DCM to give 58 g of compound 5 as a white solid.

MS: (+) m / z 630.4 (M + 1).

Compound 71b. LiOH (4.03 mg, 0.168 mmol) was added in water (0.5 mL) to a solution of 70b (53 mg, 0.084 mmol) in THF (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 2 hours, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by chromatography

Flash eluting with silica gel with a gradient of 0-30% MeOH in DCM to give 43 g of compound 71b as a white solid. MS: (+) m / z 616.3 (M + 1).

Compound 72b. DIEA (5.66 μL 0.032 mmol) was added to a solution of compound 71b (20 mg, 0.032 mmol) and HATU (12.35 mg, 0.032 mmol) in DMF (0.5 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, compound 23 (21.82 mg, 0.032 mmol) in DMF (1 mL) and DIEA (5.66 μL 0.032 mmol) was added. The pH of the reaction solution was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, LCMS indicated the reaction was complete. The reaction was terminated by the addition of 10 mL of 1: 1 (v / v) of a water mixture containing 0.1% TFA and acetonitrile containing 0.1% TFA. The crude product was purified in preparative HPLC to give 35 g of compound 72b as a white solid. MS: (+) m / z 1269.7 (M + 1). Compound 72b above is also referred to as compound (III-10).

The 72b conjugate and anti-CD70 antibody 1F4 showed an EC50 at 0.58 nM against 786-O kidney cancer cells. The conjugate of compound 72b and the anti-anothelinic 6A4 antibody showed an EC50 at 0.47 against gastric N87 stomach cancer cells. An inclusion test was used in both cases³H-thymidine.

Compound 70c. Dimethylamine in DMF (1 ml, 0.148 mmol) was added dropwise to a solution of compound 7 (0.1 g, 0.148 mmol) in DMF (1 ml) at room temperature until the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-15% MeOH in DCM to give 56 g of compound 70c as a white solid. MS: (+) m / z 582.3 (M + 1).

Compound 71c. LiOH (8.23 mg, 0.344 mmol) was added in water (0.5 mL) to a solution of 70c (0.1 g, 0.172 mmol) in THF (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 2 hours, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a gradient of 0-30% MeOH in DCM to give 50.8 g of compound 71c as a white solid. MS: (+) m / z 568.3 (M + 1).

Compound 72c. DIEA (6.14 μ (0.035 mmol) was added to a solution of compound 71c (20 mg, 0.035 mmol) and HATU (13.39 mg, 0.035 mmol) in DMF (0.5 mL) .The pH of the reaction mixture was adjusted to 8-9. At room temperature for 20 min, compound 23 (23.67 mg, 0.035 mmol) in DMF (1 ml) and DIEA (6.14 μ (0.035 mmol) was added, the pH of the reaction solution was adjusted to 8- 9. The reaction mixture was then stirred at room temperature for 20 min LCMS showed completion of the reaction The reaction was terminated by the addition of 10 ml of 1: 1 (v / v) of a water mixture containing 0.1% TFA and acetonitrile containing 0.1% TFA The crude product was purified by preparative HPLC to give 23.8 g of Compound 72c as a white solid. MS: (+) m / z 1221.6 (M + 1) The compound 72c above is also referred to as compound (III-11).

The conjugate of compound 72c and antibody 1F4 anti-CD70 showed EC50 at 0.32 nM against 786-O kidney cancer cells. The conjugate of Compound 72c and Antibemotine Antibody 6A4 showed an EC50 at 0.34 nM against N87 gastric cancer cells. An inclusion test was used in both cases³H-thymidine.

Example 18 - Compounds 75a, 75b and 75c.

This example describes the preparation of tubulinizine analogs from the previous examples, but without an attached linker group. Refer to FIG. 22 for a synthesis scheme.

Compound 73. LiOH (0.071 g, 2.97 mmol) was added in water (5 mL) to a solution of 18 (0.25 g, 0.743 mmol) in THF (5 mL) at room temperature. The reaction mixture was stirred at room temperature (RT) for 2 hours. The crude product was purified by means of chromatography

Flash eluted from a silica gel with a gradient of 0-20% MeOH in DCM to give 0.22 g of compound 73 as a white solid. MS: (+) m / z 223.3 (M + 1-Boc).

Compound 74. A mixture of compound 73 (0.2 g, 0.620 mmol) and 4N HCl in 1,4-dioxane (4 mL, 0.620 mmol) was stirred at rt for 1 h. The diluent was evaporated. The white solid 74 was washed twice with hexanes. MS: (+) m / z 223.3 (M + 1).

Compound 75a. DIEA (3.23 μL 0.019 mmol) was added to a solution of compound 71a (10 mg, 0.019 mmol) and HATU (7.05 mg, 0.019 mmol) in DMF (0.5 mL), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, DIEA (3.23 μL 0.019 mmol) was added and compound 74 (5.35 mg, 0.024 mmol) in DMF (0.5 mL), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 1 h, LCMS indicated the reaction was complete. The reaction was terminated by the addition of 10 ml of 1: 1 (v / v) of a mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified by preparative HPLC to give 6.0 g of compound 75a as a white solid. MS: (+) m / z 744.4 (M + 1). Compound 75a above is also referred to as compound (I-10).

Compound 75a has an EC50 at 75.8 nM against N87 gastric cancer cells using a luminescent ATP assay.

Compound 75b. DIEA (2.83 μL 0.016 mmol) was added to a solution of compound 71b (10 mg, 0.016 mmol) and HATU (6.17 mg, 0.016 mmol) in DMF (0.5 mL), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, DIEA (2.83 μL 0.016 mmol) was added and compound 74 (4.69 mg, 0.021 mmol) in DMF (0.5 mL), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 1 h, LCMS indicated the reaction was complete. The reaction was terminated by the addition of 10 ml of 1: 1 (v / v) of a mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified by preparative HPLC to afford 7.6 g of compound 75b as a white solid. MS: (+) m / z 820.4 (M + 1). Compound 75b above is also referred to as compound (I-11).

EC 50 at 0.39 nM was determined for compound 75b against N87 gastric cancer cells using a luminescent ATP assay.

Compound 75c. DIEA (3.07 μL 0.018 mmol) was added to a solution of compound 71c (10 mg, 0.018 mmol) and HATU (6.70 mg, 0.018 mmol) in DMF (0.5 mL), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, DIEA (3.07 μL 0.018 mmol) was added and compound 74 (5.09 mg, 0.023 mmol) in DMF (0.5 mL), pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 1 h, LCMS indicated the reaction was complete. The reaction was terminated by the addition of 10 ml of 1: 1 (v / v) of a mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified in preparative HPLC to give 7.6 g of compound 75c as a white solid. MS: (+) m / z 772.5 (M + 1) Compound 75c above is also called compound (I-12).

The EC 50 at 5.4 nM was determined for compound 75c against N87 gastric cancer cells using the ATP luminescence assay.

Example 19 - Compound 80

The example describes the synthesis of compound 80 suitable for conjugation in a "click" reaction having a cyclooctyne group capable of reacting with a group of azide on a partner molecule.

A suitable synthesis scheme is given in Figure 23.

Compound 77. DCC (22.40 mg, 0.109 mmol) was added to a solution of DBCO-PEG4-Acid 76 (50 mg, 0.090 mmol, provided by Click Chemistry Tools, Scottsdale, AZ) and N-hydroxysuccinimide (NHS, 20.83 mg, 0.181 mmol) in DCM. (1 ml) at room temperature.

The reaction mixture was stirred overnight at room temperature. The solid was filtered off and the filtrate was concentrated to give compound 77. MS: (+) m / z 650.3 (M + 1).

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Compound 78. DIEA was added to a solution of compound 77 (58.8 mg, 0.091 mmol) and compound 21 (57.6 mg, 0.100 mmol) in DMF (1 mL) at room temperature, pH adjusted to 8-9. The reaction mixture was then stirred at room temperature for 1 h, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was purified by preparative HPLC to give 20 g of compound 78 as a white solid. MS: (+) m / z 1113.6 (M + 1).

Compound 79. TFA (0.5 mL, 6.53 mmol) was added to a mixture of compound 78 (17.2 mg, 0.015 mmol) in DCM (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 1 h, LCMS indicated the reaction was complete. The diluent was evaporated to yield compound 79. MS: (+) m / z 1013.5 (M + 1).

Compound 80. DIEA (2.96 μΙ, 0.017 mmol) was added to a solution of compound 9 (8.55 mg, 0.015 mmol) and HATU (5.87 mg, 0.015 mmol) in DMF (0.4 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, compound 79 (15.65 mg, 0.015 mmol) in DMF (1ml) and DIEA (2.96 μΙ, 0.017 mmol) was added. The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 20 min, LCMS indicated the reaction was complete. The diluent was evaporated. The crude product was re-dissolved in 1 mL of DMSO, and purified by preparative HPLC to provide compound 80 as a white solid. MS: (+) m / z 775.0 (M / 2 + 1). Compound 80 above is also referred to as compound (III-12).

The conjugate of compound 80 and seven variants of the 4A6 antigipican 3 antibody modified to have an azide group at different sites showed EC50 for 1.4, 0.17, 0.13, 0.22, 0.14, 0.064, and 0.25 nM against N87 gastric cancer cells, using the inclusion study. ³H thymidine.

The preparation and characterization of the human monoclonal antibody 4A6 is described in Terrett et al. 2010, which disclosure is incorporated herein by reference. The VH CDR1, CDR2, and CDR3 and VK CDR1, CDR2, and CDR3 sequences of the 4A6 antibody are set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively. VH and VK variable region sequences are given in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

In one embodiment, the present invention provides a conjugate of compound 80 and a polypeptide, preferably an antibody, modified to contain an azide group.

Example 20 - Compound 88

This example describes the synthesis of compound 88 which has the functionality of a primary alkyl amine that can be used for conjugation. Please refer to FIG. 24 for a synthesis scheme.

Compound 82. A mixture of tert-butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-niate 54 (0.285 g, 0.888 mmol, obtained from VWR; see also Example 8) and 2,5-dioxo-pyrrolidine 1-yl 6 ((((9H-fluorene-9-yl) methoxy) carbonyl) amino) hexanoate 81 (0.4 g, 0.888 mmol, obtained from Chem-Impex) in DMF (3 ml) was stirred at room temperature for 2 minutes. h. The diluent was evaporated. The crude product was purified by flash chromatography eluting with silica gel with a 0-10% MeOH in DCM gradient to give 0.4 g of compound 82 as a white solid. MS: (+) m / z 657.4 (M + 1).

Compound 83. A mixture of compound 82 (0.258 g, 0.393 mmol) in TFA (2 mL, 0.393 mmol) was stirred at rt for 1 h. The diluent was evaporated. Compound 83 (white solid) was washed twice with hexanes and used in the next reaction without further purification.

Compound 84. DCC (0.162 g, 0.786 mmol) was added to a mixture of compound 83 (0.236 g, 0.393 mmol) and NHS (0.090 g, 0.786 mmol) in DCM (5 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. of room temperature, and the solid was filtered off and the filtrate was concentrated. The crude product was purified by flash chromatography eluting from the gel

Silica with 0-100% EtOAc in hexanes to give 0.195 g of compound 84 as a colorless oil. MS: (+) m / z 698.3 (M + 1).

Compound 85. DIEA was added to a solution of compound 21 (0.162 g, 0.279 mmol) and compound 84 (0.195 g, 0.279 mmol) in DMF (1.5 mL) at room temperature. The reaction mixture was then stirred at room temperature (RT) for 1 hour, the reaction was terminated by the addition of 6 ml of a 1: 1 mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified in preparative HPLC to give 0.292 g of compound 85 as a white solid. MS: (+) m / z 1161.6 (M + 1).

Compound 86. TFA (0.5 mL) was added to a mixture of compound 85 (22.1 mg, 0.019 mmol) in DCM (1 mL) at room temperature. The reaction mixture was then stirred at room temperature for 20 min, the solvent was evaporated to give compound 86. MS: (+) m / z 1061.6 (M + 1).

Compound 87. DIEA was added to a solution of compound 9 (10.53 mg, 0.019 mmol) and HATU (7.23 mg, 0.019 mmol) in DMF (0.4 mL). The pH of the reaction mixture was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, compound 86 (20.19 mg, 0.019 mmol) in DMF (1ml) and DIEA was added. The pH of the reaction solution was adjusted to 8-9. The reaction mixture was then stirred at room temperature for 10 min, LCMS indicated complete reaction. The reaction was quenched with 10 mL of 1: 1 (v / v) water (0.1% TFA) and acetonitrile. The crude product was purified by preparative HPLC to give 27.3 g of compound 87 as a white solid. MS: (+) m / z 799.1 (M / 2 + 1).

Compound 88. The piperidine was added to a solution of compound 87 (27.3 mg, 0.017 mmol) in DMF (2 ml) at room temperature, the pH adjusted to 9-10. The reaction mixture was then stirred at room temperature for 1 hour, the reaction was terminated by the addition of 6 ml of a 1: 1 mixture of acetonitrile and water containing 0.1% TFA. The crude product was purified in preparative HPLC to give 22.5 g of compound 88 as a white solid. MS: (+) m / z 688.1 (M / 2 + 1). Compound 88 above is also referred to as compound (III-13).

In one embodiment, a conjugate is provided wherein the compound 88 is conjugated to a polypeptide that is preferably an antibody, by forming an amide over the primary alkyl amine of compound 88 and a carboxyl group on the amino acid residue of the side chain - preferably glutamic acid - in the polypeptide. The present invention also provides a method of making such a conjugate, comprising combining a compound 88 and a polypeptide in the presence of a transglutaminase enzyme.

REFERENCE

A full list of cited documents in abbreviated form with the first author (or inventor) and earlier date in this specification is given below.

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SEQUENCE LIST

Included here, based on the references in its entirety, is the Sequence Listing "SEQT_12026WOPCT.txt", comprising SEQ ID NO: 1 to SEQ ID NO: 26, which includes the nucleic acid and / or amino acid sequences disclosed herein. The Sequence List has been included in the ASCII text format via EFS-Web, and thus it is both its paper and digital form (readable on a computer). The Sequence List was first created using PatentIn 3.5 on January 4, 2014, and has approximately 15 KB.

The following table 3 summarizes the description of the sequences contained in the present patent application.

<td colspan="2">Table 3 - Sequence Summary</td>

<td>SEQ ID NO:</td><td>SEQUENCE DESCRIPTION</td>

<td>1</td><td>amino acid 6A4 V_H CDR1</td>

<td>2</td><td>amino acid 6A4 V_H CDR2</td>

<td>3</td><td>amino acid 6A4 V_H CDR3</td>

<td>4</td><td>amino acid 6A4 V_K CDR1 a</td>

<td>5</td><td>amino acid 6A4 V_K CDR2</td>

<td>6</td><td>amino acid 6A4 V_K CDR3</td>

<td>7</td><td>amino acid 6A4 V_H</td>

<td>8</td><td>amino acid 6A4 V_K</td>

<td>9</td><td>amino acid 1F4 V_H CDR1</td>

<td>10</td><td>amino acid 1F4 V_H CDR2</td>

<td>11</td><td>amino acid 1F4 V_H CDR3</td>

<td>12</td><td>amino acid 1F4 V_K CDR1</td>

<td>13</td><td>amino acid 1F4 V_K CDR2</td>

<td>14</td><td>amino acid 1F4 V_K CDR3</td>

<td>15</td><td>amino acid 1F4 V_H</td>

<td>16</td><td>amino acid 1F4 V_K</td>

<td>17</td><td>amino acid 4A6 V_H CDR1</td>

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<td>18</td><td>amino acid 4A6 V_H CDR2</td>

<td>19</td><td>amino acid 4A6 V_H CDR3</td>

<td>20</td><td>amino acid 4A6 V_K CDR1</td>

<td>21</td><td>amino acid 4A6 V_K CDR2</td>

<td>22</td><td>amino acid 4A6 V_K CDR3</td>

<td>23</td><td>amino acid 4A6 V_H</td>

<td>24</td><td>amino acid 4A6 V_K</td>

<td>25</td><td>modified constant region</td>

<td>26</td><td>modified constant region</td>

SEQUENCE LISTING &lt;110&gt; Bristol-Myers Squibb Company &lt;120&gt; TUBULYSIN COMPOUNDS, METHODS OF MAKING AND USE &lt;130&gt; 12026-WO-PCT &lt;150&gt; US 61/764825 &lt;151&gt; 2013-02-14 &lt;160&gt; 26 &lt;170 &gt; PatentIn version 3.5 &lt;210&gt; 1 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 1

How much Tyr Gly Met His 1 5 &lt;210&gt; 2 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 2

Val Leu Trp Tyr Asp Gly Cheese His Glu Tyr Tyr Ala Asp Ser Val Lys 15 10 15

Gly &lt;210&gt; 3 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 3

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Asp Gly Asp Tyr Tyr Asp Ser Gly Pro Pro Leu Asp Tyr 15 10 &lt;210&gt; 4 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 4

Arg Ala Ser Gin Cheese Val Ser Ser Tyr Leu Ala 15 10 &lt;210&gt; 5 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 5

Asp Ala Ser Asn Arg Ala Thr 1 5 &lt;210&gt; 6 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 6

Gin Gin Arg Ser Asn Trp Pro Leu Thr 1 5 &lt;210&gt; 7 &lt;211&gt; 122 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 7

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Gin Val His Leu Val Glu Gly Gly Gly Val Val Gin Pro Gly Arg 15 10 15

Leu Arg Leu Ser Ser Cys Val Ala Ser Gly Ile Thr Phe Arg Ile Tyr 20 25 30

Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Val Leu Trp Tyr Asp Gly Cheese His Glu Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Cheese Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gin Met Asn Leu Ser Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95

Ala Arg Asp Gly Asp. Tyr Tyr Asp. Ser Gly Pro Pro Leu Asp Tyr Trp 100 105 110

Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 &lt;210&gt; 8 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 8

Glu Ile Val Leu Thr Gin Cheese Pro Ala Thr Leu Cheese Leu Cheese Pro Gly 15 10 15

Glu Arg Ala Thr Leu Cheese Cys Arg Ala Cheese Gin Cheese Val Ser Cheese Tyr 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60

Cheese Gly Cheese Gly Thr Asp Phe Thr Leu Thr Ile Cheese Cheese Leu Glu Pro 65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu 85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 &lt;210&gt; 9 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 9

- EP 2956173

How much Tyr Ala Met Ser 1 5 &lt;210&gt; 10 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 10

Ala Ile Cheese Asp Ser Gly Gly Arg Thr Tyr Phe Ala Asp Ser Val Arg 15 10 15

Gly &lt;210&gt; 11 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 11

Val Asp Tyr Ser Asn Tyr Leu Phe Phe Asp Tyr 15 10 &lt;210&gt; 12 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 12

Arg Ala Ser Gin Ser Ile Ser Ser Ser Tyr Leu Ala 15 10 &lt;210&gt; 13 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 13

Gly Ala Ser Ser Arg Ala Thr 1 5 &lt;210&gt; 14 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 14

Gin Gin Tyr Gly Cheese Ser Pro Tyr Thr 1 5 &lt;210&gt; 15 &lt;211&gt; 120 &lt;212&gt; PRT &lt;213&gt; Homo sapiens

- &lt;40&gt; 2856173 &lt;400&gt; 15

Glu Val Gin Leu Leu Glu Cheese Gly Gly Gly Leu Val Gin Pro Gly Gly 15 10 15

Leu Arg Leu Cheese Ser Cys Ala Ala Cheese Gly Phe Thr Phe Ser Ile Tyr 20 25 30

Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Ala Ile Cheese Asp Ser Gly Gly Arg Thr Tyr Phe Ala Asp Ser Val 50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Cheese Lys Asn Thr Leu Cheese

70 75 80

Leu Gin Met Asn Leu Ser Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

90 95

Ala Lys Val Asp Tyr Cheese Asn Tyr Leu Phe Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 &lt;210&gt; 16 &lt;211&gt; 108 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 16

Glu Ile Val Leu Thr Gin Cheese Pro Gly Thr Leu Cheese Leu Cheese Pro Gly 15 10 15

Glu Arg Ala Thr Leu Cheese Cys Arg Ala Cheese Gin Cheese Ile Cheese Cheese Cheese 20 25 30

Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40 45

How much Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60

Gly Cheese Gly Cheese Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Cheese Pro Pro 85 90 95

Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 &lt;210&gt; 17 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 17

- EP 2956173

Cheese Tyr Trp Ile Ala 1 5 &lt;210&gt; 18 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 18

How Much Ile Phe Pro Gly Asp Ser Asp Thr Arg Tyr Cheese Pro Phe Gin 15 15 15

Gly &lt;210&gt; 19 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 19

Thr Arg Glu Gly Tyr Phe Asp Tyr 1 5 &lt;210&gt; 20 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 20

Arg Ala Val Gin Cheese Val Ser Ser Ser Tyr Leu Ala 15 10 &lt;210&gt; 21 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 21

Gly Ala Ser Ser Arg Ala Thr 1 5 &lt;210&gt; 22 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 22

Gin Gin Tyr Gly Cheese Ser Pro Thr 1 5 &lt;210&gt; 23 &lt;211&gt; 117 &lt;212&gt; PRT

- EP 2956173 &lt;213&gt; Homo sapiens &lt;400&gt; 23

<img file="PL2956173T3_D0046.tif" />

Glu

Tyr

Underworld

phe

Tyr

Cys

Leu &lt;210&gt; 24 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 24

<img file="PL2956173T3_D0047.tif" />

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105

Gly

Cheese

Leu

Cheese

Glu

Pro &lt;210&gt; 25 &lt;211&gt; 327 &lt;212&gt; PRT

- 61 EP 2956173 &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Modified Antibody Constant Region &lt;400&gt; 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 15 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Cheese Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gin Cheese Gly Cheese Leu Tyr Cheese 50 55 60

Leu Cheese Cheese Val Val Thr Val Pro Cheese Cheese Cheese Leu Gly Thr Lys Thr 65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Arg Val Glu Cheese Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110

Glu Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140

Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp 145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe 165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 180 185 190

- 62 EP 2956173

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro 210 215 220

Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr 225 230 235

Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Cheese Phe Phe Leu Tyr 275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe 290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 305 310 315

Leu

Arg

lys

240

Asp

lys

Cheese

Cheese

Cheese

320

Leu Ser Leu Ser Leu Gly Lys 325 &lt;210&gt; 26 &lt;211&gt; 330 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Modified Antibody Constant Region &lt;400&gt; 26

Ala Ser Thr Lys Gly Pro Cheese Val Phe Pro Leu Ala Pro Ser Ser 15 10 15

lys

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30

Tyr

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45

Cheese

Gly Val His Thr Phe Pro Ala Val Leu Gin Cheese Ser Gly Leu Tyr 50 55 60

Cheese

- EP 2956173

<img file="PL2956173T3_D0048.tif" />

<img file="PL2956173T3_D0049.tif" />

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- EP 2956173

1. A compound with the structural structure depicted in formula (I)