Conjugate of an ERK5 inhibitor

Abstract

The present invention describes a conjugate of an ERK5 inhibitor and use thereof as an antitumor agent, in particular for treating malignant mesothelioma.

Description

[0001] PATENT APPLICATION FOR THE INDUSTRIAL INVENTION ENTITLED:

[0002] CONJUGATE OF AN ERK5 INHIBITOR

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of antitumor drugs, in particular to an ERK5 inhibitor Antibody Drug Conjugate (ADC).

[0005] BACKGROUND ART ERK5 (Extracellular-regulated protein kinase 5) is a MAP kinase which is part of a growth factor-stimulated signaling pathway which has been shown to be involved in tumorigenesis and resistance to treatments of many tumor types, including malignant mesothelioma (MM). ERK5 is activated by different extracellular stimuli, such as stress stimuli and growth factors, and has a key role in different cellular responses, such as cellular proliferation and differentiation. ERK5 inhibition suppresses proliferation and induces cell death in various tumor types.

[0006] XMD8-92 (CAS No. 1234480-50-2) and JWG-071 (CAS No. 2250323-50-1) are ERK5 inhibitor molecules active as antitumor agents, especially in certain particular forms such as malignant mesothelioma (MM).

[0007]

[0008] XMD8-92 JWG-071

[0009] Patent application W02020219606A1 discloses methods for treating glioma in pediatric patients by administering ERK5 inhibitors, including XMD8-92. Joyce T.K. et al “Extracellular signal regulated kinase 5 and inflammasome in progression of mesothelioma”, Oncotarget, 2018, 9, 1, 293-305 uses XMD8-92 in in-vitro and in-vivo models to demonstrate that ERK5 inhibition can slow mesothelioma tumorigenesis.However, selective and effective targeting of the ERK5 pathway still remains a chimera and the side effects of ERK5 inhibitors together with the development of innate or acquired resistance by the target tissue, as often occurs for other kinases, limit the therapeutic success thereof.

[0010] An ADC (antibody drug conjugate) consists of a molecule conjugated by means of a covalent bond to a monoclonal antibody (mAb) that interacts with a receptor expressed on target cells and following such interaction are internalized into such cells carrying therewith the molecule to target a key process in the growth of the tumor. This molecule is released through lysosomal digestion and acts exclusively in the tumor compartment where it has been delivered by the antibody. To date about 15 ADCs have been approved by FDA and EMA and about a hundred are in the clinical development step.

[0011] Cetuximab, commonly known under the trade name Erbitux®, is an mAb which belongs to the class of antitumor drugs for treating patients suffering from metastatic colorectal carcinoma or for treating patients affected by squamous cell carcinoma of the head and neck. Several clinical studies are currently underway aimed at verifying the efficacy of cetuximab for treating other tumor types.

[0012] Trastuzumab, commonly known by the trade name Herceptin®, is an mAb which belongs to the class of antitumor drugs for treating adult patients with HER2 positive metastatic breast cancer (MBC), for treating adult patients with early HER2 positive breast cancer (EBC); in combination with other chemotherapeutic agents it is indicated for treating adult patients with HER2 positive metastatic adenocarcinoma of the stomach or gastroesophageal junction.

[0013] Bevacizumab (Avastin® by Genentech / Roche) selectively binds to "human vascular endothelial growth factor" (VEGF). Avastin® is a medicinal product used for treating patients affected by advanced carcinoma of the large intestine i.e. of the colon or rectum, metastatic carcinoma of the breast, advanced non-small cell lung carcinoma, advanced carcinoma of the kidney, epithelial ovarian carcinoma, carcinoma of the Fallopian tubes or advanced primary peritoneal carcinoma. Avastin® is administered in association with a chemotherapeutic treatment.

[0014] Panitumumab (Vectibix®) is indicated for treating patients with metastatic colorectal carcinoma, a tumor of the large intestine which has spread to other parts of thebody. Vectibix® is used in monotherapy (alone) in patients the tumor cells of whom present, on the surface, a protein called epidermal growth factor receptor (EGFR) and contain a non-mutated "KRAS" gene. KRAS is a gene which, if mutated in tumor cells, stimulates the growth of the tumor. Vectibix® is used when therapeutic regimens with combinations of antitumor drugs comprising a "fluoropyrimidine" (e.g. , 5-fluorouracil), oxaliplatin and irinotecan are no longer effective.

[0015] Emactuzumab (RG 7155) is a humanized monoclonal antibody directed against the colony-stimulating factor 1 receptor (CSF-1R) expressed on macrophages and has demonstrated a profound antitumor effect through interference with the CSF-1 / CSF-1 R axis, together with a manageable safety profile in patients with tenosynovial giant cell tumors of diffuse type.

[0016] Anti-mouse CD115 (clone AFS98) is another example of an anti CSF-1 R mAb. It is an object of the present invention to provide a product which is capable of inhibiting ERK5 selectively in tumor cells and therefore potentially useful as an antitumor agent, in particular for use in treating MM.

[0017] DEFINITIONS AND ABBREVIATIONS

[0018] Ab - antibody

[0019] ADC - Antibody Drug Conjugate

[0020] mAb - monoclonal antibody

[0021] MM - Malignant Mesothelioma

[0022] ERK5 - Extracellular-Regulated protein Kinase 5

[0023] MDSCs - Myeloid-Derived Suppressor Cells

[0024] DAR - Drug to Antibody Ratio

[0025] TAMs - Tumor-Associated Macrophages

[0026] CSF-1 R - Colony Stimulating Factor 1 Receptor

[0027] MAP - Mitogen-Activated Protein

[0028] EP - Petroleum Ether

[0029] SUMMARY OF THE INVENTION

[0030] The present invention solves the aforesaid problems by means of a conjugate of formula (I)

[0031] A-(L-D)r (l)wherein

[0032] A is a targeting agent capable of specifically binding to a cell surface protein expressed on target tumor cells;

[0033] L is a linker,

[0034] D is a payload consisting of an ERK5 inhibitor molecule;

[0035] r is a number from 1 to 10 and indicates the average D / A ratio (DAR); wherein A is covalently conjugated to L through at least one s-amino group of a Lys residue or through at least one thiol group of a Cys residue; and

[0036] wherein D is of formula (D-l) or (D-ll)

[0037]

[0038] wherein

[0039] Ri is H, (Ci-Cs)alkyl;

[0040] R2 is H, (Ci-Csjalkyl; (Cs-Cejcycloalkyl, CH3SO2-, p-toluenesulfonyl;

[0041] R3is H, (Ci-Cs)alkyl;

[0042] R4 and Rs are independently from each other H, (C1-C5) alkyl:

[0043] Het is

[0044]

[0045] where * indicates the bonding site with L.

[0046] It has surprisingly been found that a conjugate according to the present invention is capable of binding specifically to the surface of tumor cells and the microenvironment thereof and of being then internalized in the lysosome, thus suppressing the activation of ERK5 and reducing cell proliferation, both of tumor cells, and tumor-associated macrophages and immunosuppressive myeloid cells. The present inventors have surprisingly found that the conjugate according to the present invention is capable of selectively inhibiting cell proliferation, not only of mesothelioma cells but also of TAMs and MDSCs, thereby reducing theimmunosuppressive tumor microenvironment and paving the way for a more effective immune response.

[0047] Through the use of a conjugate of the present invention the difficulties associated with the overall side effects of ERK5 inhibitors are overcome, since the amounts of chemotherapeutic used are much lower compared to the systemic use. Indeed, the active substance is released only within the tumor cell, TAM and MDSC, where it has been delivered by the antibody.

[0048] As will be apparent from the experimental section (Figure 1 B), the present inventors have unexpectedly found that the conjugate according to the present invention has advantageously obtained, at an effective concentration 25 times lower, a double cell proliferation inhibition rate with respect to the inhibition rate obtained with the payload alone.

[0049] Furthermore, the active substance being covalently bound to the antibody, there are no “off-target” release problems, a phenomenon which often occurs for nanotechnological complexes. Bioconjugation with the hydrophilic targeting agent ensures low distribution in the peripheral tissues and the high molecular weight of the targeting agent limits the “clearance” ensuring a high conjugate level in the blood for long times. The elimination of a conjugate generally occurs by slow proteolytic degradation and renal excretion of the formed polypeptides. The invention therefore envisages a preclinical candidate for conjugated immunotherapy based on dual selectivity ensured by the monoclonal antibody and by the specificity of the ERK5 inhibitor.

[0050] For an aspect, the present invention relates to a conjugate as described above for antitumor use, in particular for use in the treatment of MM.

[0051] For an aspect, the present invention relates to a linker-payload molecule of formula (II)

[0052] L’-D (II)

[0053] wherein D is a payload as described above, and L’ is a precursor of linker L selected from the group consisting of:

[0054] L’a-COOH, which has a -COOH group as a terminal group (hereinafter also referred to as L’a);L’b-=, which has an alkyne as terminal group or forming part of a cycloalkyl (hereinafter also referred to as L’b);

[0055] o

[0056]

[0057] 0, which has a maleimide group as a terminal group (hereinafter also referred to as L’c);

[0058] L’d-Ns, which has an -Ns group as a terminal group (hereinafter also referred to as L’d).

[0059] For an aspect, the present invention relates to the use of a molecule of formula (II) as an intermediate for the preparation of a conjugate of formula (I) according to the present invention.

[0060] For a further aspect, the present invention relates to a process for preparing a conjugate of formula (I) as described above, said process comprising contacting a compound of formula (II) as described above with a targeting agent A as described above, appropriately activated if required.

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] Preferably the targeting agent (A) is an antigen-binding protein or a small molecule which selectively binds to cellular receptors overexpressed in tumor cells, e.g. folic acid as a targeting agent for folate receptor Alpha (FRa) overexpressed in many tumor cells and folate receptor Beta (FR|3) often overexpressed in tumor-associated macrophages (TAMs). An antigen-binding protein describes a protein capable of specifically binding to an antigen expressed on the surface of target tumor cells. An antigen-binding protein can comprise an antibody, a fragment of an antibody (e.g. scFv, Fab, etc.), an antibody-like molecule (e.g. a bispecific T-cell engager), a chimeric antigen receptor (CAR) and a ligand (e.g. a natural ligand, a synthetic ligand).

[0063] Preferably, the targeting agent comprises an antibody.

[0064] Preferably, A is a monoclonal antibody (mAb) selected from the group consisting of an anti-CSF-1R, cetuximab (ctx), trastuzumab, bevacizumab and panitumumab. Preferably, anti-CSF-1R mAb is selected from the group consisting of anti-CD115, Emactuzumab (RG 7155) and a fragment thereof (i.e. scFv, Fab).Preferably, A is Ctx(Lys), Ctx(Cys), anti-CD115(Lys), anti-CD115(Cys), RG7155(Lys) or RG7155(Cys) where (Lys) indicates that A is covalently conjugated to L through at least one s-amino group of a Lys residue and (Cys) indicates that A is covalently conjugated to L through at least one thiol group of a Cys residue. D is a small-molecule ERK5 inhibitor covalently linked to L, preferably through a carbonate or carbamate group, preferably by reaction between a hydroxyl group of D activated as a carbonate and a hydroxyl or amino group of L, or by means of an alkylamino bond, preferably by reaction between an amino group of D and an alkyl halide of L (or vice versa). According to the present invention, D is of formula (D-l) or (D-ll)

[0065]

[0066] wherein

[0067] Ri is H, (Ci-Cs)alkyl;

[0068] R2 is H, (Ci-C5)alkyl; (C5-C6)cycloalkyl, CH3SO2-, p-toluenesulfonyl;

[0069] R3is H, (Ci-Cs)alkyl;

[0070] R4 and Rs are independently from each other H, (C1-C5) alkyl:

[0071] Het is

[0072]

[0073] where * indicates the bonding site with L.

[0074] Preferably,

[0075] R1 is Me; R2 is Me, sec-Bu, cyclopentyl; R3 is Me, Et; and R4 and Rs, equal to each other, are H or Me.

[0076] Preferably D is selected from the group consisting of

[0077]

[0078] L is a linker which covalently connects A and D and is preferably of formula (L-l) or

[0079]

[0080] wherein

[0081] * indicates the bonding site with D and ** indicates the bonding site with A;

[0082] n is an integer between 1 and 12, preferably 5;

[0083] m is an integer between 1 and 8, preferably 4;

[0084] Y isH N

[0085] o , where p is an integer between 1 and 4, preferably 2;

[0086] X is

[0087]

[0088] where

[0089] W is -(CH2)q-, -(OCH2CH2)S- with q integer between 0 and 12 and s integer between 1 and 6, v is 0, 1 and Z is

[0090]

[0091] L is preferably selected from the group consisting of

[0092]

[0093] 0(Ls);

[0094]

[0095] o

[0096]

[0097] where * indicates the bonding site with D and ** indicates the bonding site with A. Preferably when D is Di then L is Li, L2, l_3, l_4, Ls or L7; when L is Lethen D is D2, Ds or D4 .

[0098] Preferably r=1.0-6.5.

[0099] Preferably, the conjugate of the present invention is of formula:

[0100] A L D description

[0101] Ctx(Lys) Li Di Ctx(Lys)-Li-XMD8-92

[0102] Anti-CD115 (Lys) Li Di Anti-CD115(Lys)-Li-XMD8-92 RG7155(Lys) Li Di RG7155 (Lys)-Li-XMD8-92

[0103] Ctx(Lys)) L2 Di Ctx(Lys)-L2-XMD8-92

[0104] Anti-CD115 (Lys) L2 Di Anti-CD115 (Lys)-L2-XMD8-92 RG7155(Lys) L2 Di RG7155 (Lys)-L2-XMD8-92

[0105] Ctx(Lys) L3 Di Ctx(Lys)-Ls-XMD8-92

[0106] Anti-CD115 (Lys) L3 Di Anti-CD115 (Lys)-L3-XMD8-92 RG7155(Lys) L3 Di RG7155 (Lys)-Ls-XMD8-92

[0107] Ctx(Lys) L4Di Ctx(Lys)-L4-XMD8-92

[0108] Anti-CD115 (Lys) L4Di Anti-CD115 (Lys)-L4-XMD8-92 RG7155(Lys) L4Di RG7155 (Lys)-L4-XMD8-92

[0109] Ctx(Cys) L5 Di Ctx(Cys)-L5-XMD8-92

[0110] Anti-CD115 (Cys) L5 Di Anti-CD115 (Cys)-L5-XMD8-92 RG7155(Cys) L5 Di RG7155(Cys)-L5-XMD8-92

[0111] Ctx(Lys) Le D2 Ctx(Lys)-L6- JWG-071

[0112] Anti-CD115 (Lys) Le D2Anti-CD115 (Lys)-Le- JWG-071 RG7155(Lys) Le D2RG7155 (Lys)-Le- JWG-071

[0113] Ctx(Lys) L7 DI Ctx(Lys)-L7-XMD8-92

[0114]

[0115] CD115(Lys) l_7 Di Anti-CD115 (Lys)-L7-XMD8-92 RG7155(Lys) l_7 Di RG7155 (Lys)-L7-XMD8-92

[0116]

[0117] Preferably, the linker-payload compounds of formula (II) are those in which D is as described above and

[0118] L’ is a precursor of the linker preferably of formula (L’-l) or (L’-ll)

[0119]

[0120] wherein

[0121] * indicates the bonding site with D;

[0122] n is an integer between 1 and 12, preferably 5;

[0123] m is an integer between 1 and 8, preferably 4;

[0124] Y is

[0125]

[0126] O X where p is an integer between 1 and 4, preferably 2;

[0127] X’ is

[0128]

[0129] whereW is -(CH2)q-, -(OCH2CH2)S- with q integer between 0 and 12, and s integer between

[0130]

[0131] L’ is preferably selected from the group consisting of

[0132]

[0133]

[0134]

[0135] where * indicates the bonding site with D.

[0136] The conjugates of the present invention can preferably be prepared by means of one of the following strategies for conjugating A with an L’-D of formula (II): a) conjugation to the s-amino group of at least one lysine of A using a D-L’a-NHS containing N-hydroxysuccinimide

[0137] A

[0138]

[0139] (Lys)-(CH2)4-NH2 + D-L’a-NHSS A(Lys)-NHCO-L-D;

[0140] b) conjugation to the s-amino group of at least one lysine of A using a D-L’ahaving the carboxyl terminus activated in situ with (N-hydroxysulfosuccinimide, NHSS)

[0141]

[0142] NH

[0143] A(Lys2D-L'a-COOH A(Lys)-NHCO-L-D

[0144] c) conjugation, through pre-activation of the s-amino group of at least one lysine of A with an azido-polyethylene glycol chain, with a D-Lb-dibenzocyclooctyne (DBCO)

[0145]

[0146] wherein L’b is as described above preferably of formula

[0147]

[0148] o (L’-l) wherein

[0149] * indicates the bonding site with D;

[0150] n is an integer between 1 and 12, preferably 5;

[0151] Y is

[0152] O , where p is an integer between 1 and 4, preferably 2;

[0153]

[0154] d) conjugation to the thiol group of at least one cysteine of A using a D-L’c-maleimide i o

[0155] D A

[0156] A(Cys) SH X?N-L;CA(Cys) S D

[0157]

[0158] O e) conjugation with a D-L’d-Ns, through pre-activation of the s-amino group of at least one lysine of A with a DBCO-polyethylene glycol chain

[0159]

[0160] Wherein L’d is as described above, preferably of formula

[0161]

[0162] 0(L’-l)

[0163] wherein

[0164] * indicates the bonding site with D;

[0165] n is an integer between 1 and 12, preferably 5;

[0166] Y is

[0167] X’ is

[0168]

[0169] where

[0170] Z’ is

[0171]

[0172] In the conjugation strategies described above, the different types of bonds that can be formed, in a variable number in accordance with the definition of formula (I), between the targeting agent A and the linker-payload of formula (II) have been specified.The present invention will now be further described with reference to the following examples. These examples are merely for illustrative purposes and must not be interpreted as limiting the scope of the present invention

[0173] BRIEF DESCRIPTION OF THE DRAWINGS

[0174] Figure 1: ADC 32 (anti CSF-1R-XMD8-92) inhibits the growth of mesothelioma cells:

[0175] A) Colony formation assay of AB1 cells showing the inhibitory effect of anti-CSF-1 R (alone), ADC (conjugate 32), XMD8-92 (ERK5 inhibitor) or GW2580 (CSF-1R inhibitor) at the indicated concentration for 7 days.

[0176] B) Quantification of the inhibitory effect of anti-CSF-1 R, ADC 32 conjugate, XMD8-92, or GW2580 as described in B. The data are the mean of triplicate experiments; p < 0,05 with respect to control cells.

[0177] C) Representative images of AB1 treated with anti-CSF-1 R (Ctrl) or conjugate 32 (30 pg / ml) for 48 hours which show proliferating Edu+ cells. The nuclei of the cells were stained with DAPI. The quantification of Edu+ cells, as mean ± SD, indicates that the inhibition of ERK5 mediated by ADC significantly reduced the proliferation of AB1 cells.

[0178] Figure 2. Internalization of ADC 32 and inhibition of pERK5:

[0179] A) Immunofluorescence images of ADC (conjugate 32) and LysoTracker to localize anti CSF-1 R-XMD8-92 and lysosomes in AB1 cells (upper panel) and in tumor-associated macrophages TAMs (lower panel), after incubation with conjugate 32 at the indicated times. The arrows show the colocalization of ADC with the lysosomes in the cells. The nuclei of the cells were stained with DAPI.

[0180] B) Protein analysis through Western blot showing the suppression of phospho-ERK5 (pERK5) after treatment of AB1 cells with anti-CSF-1 R-XMD8-92 (ADC 32, 30 pg / ml) with respect to that induced with anti-CSF-1 R alone (Ctrl, 30 pg / ml). Figure 3. Suppression of ERK5 activation and tumor-associated macrophages proliferation after treatment with ADC:

[0181] A) Protein analysis by immunoblot which shows the suppression of pERK5 in TAMs after treatment with anti-CSF-1 R-XMD8-92 (ADC 32, 30 pg / ml) with respect to anti-CSF1R alone (Ctrl, 30 pg / ml), for 20 hours.B) Representative images of TAMs treated with ADC 32 or anti-CSF-1 R alone for 48 hours which show proliferating Edu+ cells. The nuclei of the cells were stained with DAPI.

[0182] C) Percentages of the means of Edu+ cells ± SD of three independent experiments as in B indicate that the proliferation of TAMs was significantly reduced by ADC-induced ERK5 inhibition, p < 0.05 with respect to control cells.

[0183] Figure 4. Reduction of ERK5 activation and MDSC proliferation after treatment with ADC:

[0184] A) Flow cytometric analysis of CSF-1R (CD115) expression in bone marrow precursors (day 0) and in MDSCs after incubation with tumor factors enriched with GM-CSF (50 ng / ml) and IL-6 (25 ng / ml) for 7 days. Ly6-C indicates the marker of the myeloid cells of the monocytic type used to identify the M-MDSC (monocytic-MDSC) population.

[0185] B) Protein analysis of ERK5 expression and ERK5 phosphorylation (pERK5-TEY) in bone marrow-derived MDSCs at the indicated time.

[0186] C-D) Flow cytometric analysis and quantification of the percentage of proliferating Click-iT Alexa Fluor Edu+ (C) or apoptotic (D) MDSCs after treatment for 3 days with control isotype, anti-CD115 or ADC 36 (30 mg / ml, DAR 4). * p<0.05 with respect to the control condition. In Figure 4-D, the level of Annexin V, i.e. the marker used for apoptotic cells, positivity is expressed on the x-axis, while the level of Propidium iodide, i.e. the marker used for dead cells due to necrosis, positivity is expressed on the y-axis.

[0187] E) Protein analysis of the expression of pERK5-TEY and ERK1 / 2 in the MDSCs derived from bone marrow after treatment as in C.EXPERIMENTAL SECTION

[0188] EXAMPLE 1 - SYNTHESIS AND CHARACTERIZATION OF LINKER-PAYLOADS EXAMPLE 1.1 - Synthesis of linker-payload 8

[0189] Scheme 1. Synthesis scheme of linker-payload 8

[0190]

[0191] Synthesis of intermediate 3

[0192] Under nitrogen atmosphere, compound 1 (500 mg, 2.02 mmol) and 2-(Boc-amino)ethanethiol (0.85 mL, 5.04 mmol) were dissolved in CH3CN (3 mL) and the solution was stirred at room temperature for 16 hours. The solvent was removed under vacuum and the mixture was purified by flash chromatography on silica gel with a gradient from 0 to 100% of EtOAc in PE to obtain the product 2 in the form of a yellow oil (380 mg, 71%).

[0193] 1H NMR: (400 MHz, MeOD) 53.91 (d, J = 2.6 Hz, 2H), 3.46 (t, J = 7.1 Hz, 2H), 3.34 - 3.31 (m, 2H), 3.15 (dd, J = 12.0, 8.0 Hz, 1H), 3.03 - 2.93 (m, 1H), 2.79 (s, 1H), 2.77 - 2.70 (m, 1 H), 2.54 (t, J = 2.6 Hz, 3H), 2.48 - 2.42 (m, 1 H), 2.16 (t, J = 7.5 Hz, 2H), 1.67 - 1.50 (m, 4H), 1.41 (s, 9H), 1.36 - 1.26 (m, 2H).

[0194] MS (ESI): m / z 426 [M+H]+, 448 [M+Na]+, 464 [M+K]+Synthesis of intermediate 4

[0195] After dissolving compound 3 (220 mg, 0.52 mmol) in CH2CI2 (3 mL) TFA (796 pL, 10.40 mmol) was added. The solution was stirred at room temperature for 16 hours. The solvent was removed under vacuum and the product was obtained as trifluoroacetate salt. (The reaction was considered quantitative and the product was used for the next step without further purification)

[0196] 1H NMR: (600 MHz, MeOD) 5 3.99 (dd, J = 9.2, 4.2 Hz, 1H), 3.96 (d, J = 2.5 Hz, 2H), 3.52 (t, J = 7.2 Hz, 2H), 3.31 (d, J = 6.4 Hz, 3H), 3.24 (ddd, J = 16.5, 11.5, 7.2 Hz, 4H), 3.06 (dt, J = 14.2, 6.5 Hz, 2H), 2.83 (s, 2H), 2.59 (t, J = 2.5 Hz, 1H), 2.53 (dd, J = 12.0, 4.0 Hz, 1 H), 2.21 (t, J = 7.4 Hz, 2H), 1.68 - 1.58 (m, 4H), 1.37 - 1.30 (m, 2H).

[0197] MS (ESI): m / z 326 [M+H]+

[0198] Synthesis of intermediate 5

[0199] XMD8-92 (30 mg, 0.06 mmol) is dissolved in CH2CI2 (2 mL) under an argon flow, then EtsN (22 pL, 0.16 mmol) and p-nitrophenyl chloroformate (20 mg, 0.10 mmol) are added allowing the reaction to proceed under magnetic stirring for 16 hours at room temperature. After evaporating the solvent under reduced pressure, the reaction mixture was purified by flash chromatography (eluent mixture from 7:3 EP / EtOAc up to a gradient of 8:2 EtOAc / EP). Given the instability of the obtained compound, it was used immediately for the next reaction confirming the formation thereof solely through mass spectrometry.

[0200] MS (ESI): m / z 640 [M+H]+, 662 [M+Na]+

[0201] Synthesis of intermediate 6

[0202] Under a nitrogen atmosphere, XMD8-92 carbonate 5 (28 mg, 0.04 mmol) was dissolved in CH2CI2 (1 mL), and compound 4 (20 mg, 0.04 mmol), previously dissolved in DMF (0.5 mL), and EtsN (31 pL, 0.22 mmol) were added. The resulting solution was stirred at room temperature for 16 hours. The solvent was removed under vacuum and the mixture was purified by flash chromatography on silica gel with a gradient of MeOH in CH2CI2 from 0 to 5%. The product was obtained as a light yellow oil (23 mg, 63%).1H NMR: (600 MHz, CDCI3) 6 8.26 (s, 1 H), 8.12 (s, 1H), 7.87 - 7.82 (m, 1H), 7.48 (s, 1H), 7.42 (t, J = 7.3 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.65 - 6.51 (m, 1H), 5.72 (s, 1H), 4.90 - 4.83 (m, 1H), 4.17 - 4.07 (m, 2H), 4.08 -4.02 (m, 2H), 3.82 - 3.72 (m, 1 H), 3.52 (t, J = 7.0 Hz, 3H), 3.49 (s, 3H), 3.42 (s, 3H), 3.21 - 3.10 (m, 3H), 3.01 (s, 1H), 2.91 - 2.83 (m, 1H), 2.51 (d, J = 18.6 Hz, 1H), 2.22 (s, 1 H), 2.19 (t, J = 7.4 Hz, 2H), 2.10 - 2.03 (m, 2H), 1.86 (s, 3H), 1.70 - 1.57 (m, 7H), 1.47 (t, J = 6.8 Hz, 3H), 1.38 - 1.29 (m, 3H).

[0203] ES-MS: m / z 826 [M+H]+, 848 [M+Na]+, 864 [M+K]+

[0204] Synthesis of compound 8

[0205] Under an argon atmosphere, compound 6 (7 mg, 0.01 mmol) and 6-azidohexanoic acid 7 (1 mg, 0.01 mmol) were dissolved in DMF (300 pL) and the solution was degassed with three vacuum / argon cycles. To this solution the aqueous mixture (150 pL) of CU(OAC)2 (0.4 mg, 0.30 mmol) and sodium ascorbate (0.8 mg, 0.60 mmol), previously degassed under argon, was added. The reaction mixture was stirred at room temperature for 24 hours. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography on silica gel with a gradient from 0 to 10% MeOH in CH2CI2, obtaining 6 mg (95%) of the desired product 8.

[0206] 1H NMR (600 MHz, MeOD) 58.32 - 8.21 (m, 1 H), 8.11 (d, J = 8.5 Hz, 1 H), 7.84 (s, 1H), 7.76 - 7.71 (m, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.65 (d, J = 36.1 Hz, 2H), 4.45 - 4.29 (m, 5H), 4.13 (s, 2H), 3.48 (s, 3H), 3.41 (s, 3H), 3.24 - 3.15 (m, 3H), 3.09 - 2.98 (m, 3H), 2.49 (d, J = 18.4 Hz, 2H), 2.32 - 2.26 (m, 2H), 2.21 (t, J = 6.9 Hz, 2H), 2.11 - 2.00 (m, 2H), 1.94 - 1.86 (m, 3H), 1.85 - 1.74 (m, 2H), 1.66 - 1.55 (m, 8H), 1.43 (t, J = 6.9 Hz, 3H), 1.33 -1.30 (m, 9H).

[0207] MS (ESI): m / z 981 [M+Hp

[0208] EXAMPLE 1.2 - Synthesis of linker payload 10

[0209] Scheme 2 Synthesis scheme of linker payload 10

[0210]

[0211] Following the procedure described above, the linker-payload 6 system (23 mg, 0.03 mmol) was reacted with the 14-azido-3,6,9,12-tetraoxatetradecanoic acid 90.5 M in MTBE (42 pL, 0.02 mmol). Compound 10 was obtained with a 90% yield (23 mg).

[0212] 1H NMR (600 MHz, CDCIs) 58.23 (d, J = 8.3 Hz, 1 H), 8.11 (s, 1 H), 7.87 - 7.76 (m, 1H), 7.48 (s, 1H), 7.42 (t, J = 7.4 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.57 (d, J = 12.7 Hz, 2H), 5.49 (s, 1H), 4.81 (s, 1H), 4.52 (d, J = 40.2 Hz, 4H), 4.11 (d, J = 6.7 Hz, 2H), 3.91 (d, J = 32.3 Hz, 4H), 3.66 (s, 10H), 3.49 (s, 6H), 3.41 (s, 6H), 3.22 - 3.06 (m, 4H), 3.03 - 2.94 (m, 3H), 2.93 - 2.81 (m, 3H), 2.27 -2.14 (m, 3H), 2.13 - 2.00 (m, 2H), 1.87 - 1.77 (m, 2H), 1.69 - 1.50 (m, J = 34.3 Hz, 6H), 1.46 (t, J = 6.5 Hz, 3H).

[0213] MS (ESI): m / z 1103 [M+H]+, 1125 [M+Na]+.

[0214] EXAMPLE 1.3 - Synthesis of linker payload 14

[0215] Scheme 3 Synthesis scheme of linker payload 14

[0216]

[0217] is of intermediate 134-(N-maleimidomethyl)cyclohexane-1 -carboxylic acid 11 (0.154 mmol) was solubilized in anhydrous acetonitrile (2 mL). 2(Boc-amino)ethanethiol 12 (0.308 mmol) was then added to the solution. The reaction was maintained at room temperature under magnetic stirring for 16 hours. The solvent was then removed by evaporation at reduced pressure and the crude reaction mixture was purified by means of flash chromatography on silica gel with isocratic elution EP:EtOAc (1:1). Obtaining 310 mg (49% yield) of Michael addition product. MS (ESI): m / z 415 [M+H]+; 437 [M+Na]+. Such product (0.75 mmol) was solubilized in CH2CI2 (2 mL) at room temperature. The solution was brought to 0 °C and trifluoroacetic acid (15 mmol) was added dropwise. The reaction was left at room temperature under magnetic stirring with the flask hermetically sealed for 16 hours. The solvent and excess TFA were then evaporated under reduced pressure and the obtained product was used as such for the next step.

[0218] 1H NMR (400 MHz, MeOD, 5 ppm, J Hz): 53.97 (dd, J = 9.2, 4.1 Hz, 1H), 3.34 (s, 2H), 3.28 - 3.24 (m, J = 1.5 Hz, 2H), 3.24 - 3.17 (m, 2H), 3.07 - 2.95 (m, 2H), 2.50 (dd, J = 18.6, 4.1 Hz, 1H), 2.19 (tt, J = 12.2, 3.5 Hz, 1H), 1.96 - 1.91 (m, 2H), 1.72 (d, J = 13.7 Hz, 2H), 1.39 - 1.26 (m, 2H), 1.01 (qd, J = 13.0, 3.1 Hz, 2H).

[0219] MS (ESI): m / z 315 [M]+.

[0220] Synthesis of compound 14

[0221] Compound 13 (26 mg, 0.060 mmol) was dissolved in CH2CI2 / DMF (100 / 1, 2 mL), and triethylamine (42 pL, 0.300 mmol) was added. To the solution, after being left under magnetic stirring for 15 minutes, XMD8-92 carbonate 5 (42 mg) was added. The reaction was brought to 40 °C and left under magnetic stirring for 24 hours. The solvent was then removed by evaporation at reduced pressure and the crude reaction product was purified through chromatography on silica gel with a mediumpressure system (MPLC) eluting a 0-3 % gradient of MeOH in CH2CI2, the product was obtained as a light yellow solid (29 mg, 0.035 mmol) with a 58% yield.

[0222] 1H NMR (600 MHz, CDCI3, 5 ppm, J Hz) 5 12.14 (s, 1 H), 8.21 (d, J = 8.8 Hz, 1 H), 8.12 (s, 1 H), 7.85 (dd, J = 7.8, 1.6 Hz, 1 H), 7.60 (s, 1 H), 7.45 - 7.40 (m, 1 H), 7.14 (t, J = 7.5 Hz, 1 H), 7.09 (d, J = 8.3 Hz, 1 H), 6.62 (d, J = 7.3 Hz, 2H), 4.85 (s, 1 H), 4.11 (q, J = 7.0 Hz, 2H), 3.82 - 3.78 (m, 1 H), 3.50 (s, 2H), 3.42 (s, 3H), 3.39 (d, J = 6.9 Hz, 2H), 3.18 - 3.12 (m, 6H), 3.07 - 3.02 (m, 2H), 2.90 - 2.84 (m, 1H), 2.24 (t,J = 12.2 Hz, 1 H), 2.03 (d, J = 12.6 Hz, 2H), 1.90 - 1.83 (m, 1 H), 1.78 - 1.67 (m, 2H), 1.47 (t, J = 7.0 Hz, 3H), 1.42 - 1.37 (m, 2H), 1.34 (t, J = 7.3 Hz, 3H), 1.27 (s, 3H), 1.08 - 0.98 (m, 2H), 0.89 (t, J = 6.9 Hz, 1 H).

[0223] MS (ESI): m / z 815 [M+H]+.

[0224] EXAMPLE 1.4 - Synthesis of linker payload 15

[0225] Scheme 4. Synthesis scheme of linker payload 15

[0226]

[0227] Compound 14 (14 mg, 0.018 mmol), NHS (4 mg, 0.031 mmol) and EDC- HCI (6 mg, 0.031 mmol) were dissolved in anhydrous CH2CI2 (3 mL). The reaction was left at room temperature under magnetic stirring under an N2 atmosphere for 24 hours. The solvent was then removed by evaporation under reduced pressure and the crude reaction product was purified by means of flash chromatography on silica gel with elution from 0.5% to 2% of MeOH in CH2CI2 as a white solid (7 mg, 0.008 mmol) with a 44% yield.

[0228] 1H NMR (600 MHz, CDCI3, 5 ppm, J Hz) 5 8.30 (d, J = 8.5 Hz, 1H), 8.13 (s, 1H), 7.86 (dd, J = 7.8, 1.7 Hz, 1H), 7.61 (s, 1H), 7.46 - 7.42 (m, 1H), 7.15 (dd, J = 10.9, 4.1 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.67 (d, J = 7.3 Hz, 2H), 5.21 (s, 1H), 4.87 (s, 1 H), 4.13 (q, J = 7.0 Hz, 2H), 3.81 (dd, J = 8.8, 3.0 Hz, 1 H), 3.51 (s, J = 6.9 Hz, 3H), 3.44 (s, J = 13.5 Hz, 3H), 3.42 (d, J = 6.9 Hz, 2H), 3.23 - 3.14 (m, 2H), 3.09 (s, 2H), 2.92 - 2.86 (m, 1 H), 2.82 (s, 4H), 2.64 (s, 1 H), 2.63 - 2.57 (m, 1 H), 2.57 - 2.51 (m, 1 H), 1.93 (s, 2H), 1.85 - 1.75 (m, 4H), 1.57 (dd, J = 25.8, 13.0 Hz, 4H), 1.49 (t, J = 7.0 Hz, 3H), 1.28 (s, 4H), 1.10 (d, J = 12.6 Hz, 2H).

[0229] MS (ESI): m / z 912 [M+H]+.EXAMPLE 1.5 - Synthesis of linker payload 17

[0230] Scheme 5. Synthesis scheme of linker payload 17

[0231]

[0232] The activated acid 15 (6 mg, 0.007 mmol) was solubilized in anhydrous DMF / CH2CI2 (6 / 1 , 700 pL) and then triethylamine (1.5 pL, 0.011 mmol) was added. To the freshly formed solution dibenzocyclooctyne-amine (2 mg, 0.007 mmol) was added and the reaction was then left at room temperature under magnetic stirring under N2 atmosphere for 16 hours. The solvent was then removed by evaporation at reduced pressure with heptane and the crude reaction product was purified by means of flash chromatography on silica gel with elution from 1% to 3% of MeOH in CH2CI2 as a white solid (2 mg, 0.002 mmol), with a 27% yield.

[0233] 1H NMR (400 MHz, CDCI3, 5 ppm, J Hz) 5 8.21 (d, J = 8.6 Hz, 1H), 8.09 (s, 1H), 7.82 (dd, J = 7.8, 1.6 Hz, 1 H), 7.67 (d, J = 7.2 Hz, 1 H), 7.44 (s, 1 H), 7.43 - 7.29 (m, 6H), 7.26 (s, 1 H), 7.22 (s, 1 H), 7.10 (t, J = 7.5 Hz, 2H), 7.05 (d, J = 7.9 Hz, 2H), 6.54 (d, J = 2.1 Hz, 1 H), 5.88 (t, J = 5.8 Hz, 1 H), 5.14 (s, 1 H), 5.11 (s, 1 H), 4.80 (s, 1 H), 4.08 (q, J = 7.0 Hz, 2H), 3.47 (s, 3H), 3.39 (s, 3H), 3.34 (d, J = 6.7 Hz, 2H), 3.25 (dd, J = 11.5, 5.7 Hz, 2H), 3.16 (dt, J = 14.1 , 7.4 Hz, 2H), 3.02 - 2.93 (m, 2H), 2.85 (dt, J = 13.6, 7.0 Hz, 2H), 2.42 - 2.30 (m, 2H), 2.02 (s, 4H), 1.66 (s, 5H), 1.44 (t, J = 7.0 Hz, 3H), 1.31 (s, 6H), 0.86 (t, J = 6.4 Hz, 4H).

[0234] MS (ESI): m / z 1073 [M+H]+.

[0235] EXAMPLE 1.6 - Synthesis of linker payload 19

[0236] Scheme 6. Synthesis of the linker payload 19

[0237]

[0238] Compound 14 (9 mg, 0.010 mmol) was solubilized in THF / DMF (3 / 1, 700 pL). To the resulting solution 1-(2-aminoethyl)maleimide (2 mg, 0.010 mmol), HOBt (2 mg, 0.015 mmol) and HBtu (6 mg, 0.015 mmol) were added. The solution was then brought to 0 °C and then DIPEA (4 pL, 0.025 mmol) was added. The solvent was then removed by evaporation under reduced pressure with heptane and the crude reaction product was purified by means of flash chromatography on silica gel with elution from 1% to 3% MeOH in CH2CI2 as a white solid (8 mg, 0.009 mmol) with a 90% yield.

[0239] 1H NMR (600 MHz, DMSO, 5 ppm, J Hz): 57.99 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.80 (s, J = 9.4 Hz, 1 H), 7.72 (d, J = 8.3 Hz, 1 H), 7.67 (d, J = 7.7 Hz, 1 H), 7.55 (t, J = 7.6 Hz, 1 H), 7.50 (t, J = 7.7 Hz, 1 H), 7.42 (t, J = 7.6 Hz, 1 H), 7.23 (d, J = 8.4 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.98 (s, 1H), 6.63 (s, 1H), 6.55 -6.50 (m, 1H), 4.07 (q, J = 6.8 Hz, 2H), 3.28 (s, 4H), 3.22 (d, J = 6.6 Hz, 6H), 3.19 - 3.15 (m, 3H), 3.15 -3.07 (m, 2H), 2.97 - 2.85 (m, 3H), 2.77 - 2.71 (m, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.99 - 1.87 (m, 4H), 1.64 (d, J = 13.7 Hz, 6H), 1.30 (t, J = 6.7 Hz, 3H), 1.21 - 1.15 (m, 4H), 0.91 - 0.83 (m, 2H).

[0240] MS (ESI): m / z 937 [M+H]+.EXAMPLE 1.7 - Synthesis of linker payload 27

[0241] Scheme 7. Synthesis scheme of linker payload 27

[0242]

[0243] Synthesis of intermediate 22

[0244] 4-(N-maleimidomethyl)cyclohexane-1 -carboxylic acid (300 mg, 1.264 mmol) was dissolved in THF / DMF (3 / 1, 40 mL). To the formed solution 4-azidobenzylamine 21 (187 mg, 1.264 mmol), HOBt (256 mg, 1.896 mmol) and HBtu (719 mg, 1.896 mmol) were added. The solution was then brought to 0 °C and then DIPEA (550 pL, 3.160 mmol) was added. The reaction was left at room temperature under magnetic stirring under N2 flow for 3 hours. The solvent was then removed by evaporation at reduced pressure with heptane and the crude reaction product was purified by means of flash chromatography on silica gel with elution from 0% to 15% EtOAc in CHCI3 as a white solid (390 mg, 1.062 mmol) with an 84% yield.

[0245] 1H NMR (600 MHz, CDCI3, 5 ppm, J Hz): 57.21 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 6.67 (s, 2H), 5.75 (t, J = 5.7 Hz, 1H), 4.36 (d, J = 5.8 Hz, 2H), 3.34 (d, J = 7.1 Hz, 2H), 2.02 (tt, J = 12.2, 3.5 Hz, 1H), 1.92 - 1.86 (m, 2H), 1.76 - 1.69 (m, 2H), 1.69 - 1.63 (m, 1H), 1.44 (qd, J = 13.1, 3.3 Hz, 2H), 0.97 (qd, J = 13.1 , 3.5 Hz, 2H).

[0246] MS (ESI): m / z 368 [M+H]+.Synthesis of intermediate 24

[0247] 4-mercaptobenzyl alcohol 23 (36 mg, 0.254 mmol) was dissolved in MeCN (710 pL) forming a solution at a concentration of 50 mg / mL. To this solution compound 22 (93 mg, 0.254 mmol) and a catalytic quantity of triethylamine (5 pL) were added. The reaction was left at room temperature under magnetic stirring for 2 hours. The solvent was then removed by evaporation at reduced pressure and the crude reaction product was purified by means of silica gel chromatography with a medium -pressure system (MPLC) eluting with a 0-3% gradient of MeOH in CHCh, as a transparent and colorless oil (58 mg, 0.114 mmol) with a 45% yield.

[0248] 1H NMR (400 MHz, CDCI3, 5 ppm, J Hz): 57.46 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.19 - 7.16 (m, 2H), 6.92 (d, J = 8.4 Hz, 2H), 6.16 (t, J = 5.7 Hz, 1 H), 4.64 (s, 2H), 4.31 (d, J = 5.6 Hz, 2H), 3.91 (dd, J = 9.2, 4.3 Hz, 1 H), 3.27 - 3.06 (m, 4H), 2.75 (dd, J = 14.5, 4.3 Hz, 1 H), 1.90 (m, 1 H), 1.81 - 1.74 (m, 2H), 1.65 (m, 1 H), 1.60 - 1.51 (m, 2H), 1.42 - 1.08 (m, 2H), 0.84 - 0.65 (m, 2H).

[0249] MS (ESI): m / z 508 [M+H]+; 530 [M+Na]+.

[0250] Synthesis of intermediate 25

[0251] Compound 24 (100 mg, 0.197 mmol) was dissolved in anhydrous CH2CI2 (15 mL) then the solution was cooled to 0 °C and SOCI2 (29 pL, 0.394 mmol) was added. The reaction was left at 0 °C under magnetic stirring under N2 atmosphere for 1 hour. The solvent and excess SOCI2 were removed by evaporation at reduced pressure and the crude reaction mixture was purified by means of silica gel chromatography with with a medium-pressure system (MPLC) with isocratic elution of CHCh / EtOAc (6 / 1), as a transparent and slightly yellow-colored oil (82 mg, 0.156 mmol) with a 79% yield.

[0252] 1H NMR (400 MHz, CDCI3, 5 ppm, J Hz): 57.46 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.93 (d, J = 8.1 Hz, 2H), 6.05 (t, J = 5.1 Hz, 1 H), 4.52 (s, 2H), 4.05 - 4.02 (m, 2H), 3.66 (s, 2H), 3.25 (d, J = 6.9 Hz, 2H), 3.15 (m, 2H), 2.64 (dd, J = 15.0, 3.7 Hz, 1H), 2.01 (s, 1H), 1.84 (s, 2H), 1.55 (m, 2H), 1.22 (t, J = 7.1 Hz, 1 H), 0.99 - 0.83 (m, 2H).

[0253] 1.22 (t, J = 7.1 Hz, 1 H), 0.99 - 0.83 (m, 2H).

[0254] MS (ESI): m / z 526 [M+H]+; 548 [M+Na]+; 564 [M+K]+.Synthesis of compound 27

[0255] To a solution of chloride 25 (82 mg, 0.156 mmol) in anhydrous CH2CI2 (3 mL) tetrabutylammonium iodide (53 mg, 0.144 mmol) and compound JWG-071 26 (88 mg, 0.144 mmol) were added under an N2 flow. The solution was brought to 0 °C and DIPEA (63 pL, 0.360 mmol) was added. The reaction was left at room temperature under magnetic stirring under N2 flow for 6 days. The solvent was then removed by evaporation at reduced pressure and the crude reaction product was purified by means of semi-preparative HPLC (Luna® Omega 5 mm C18 100 A; 250x21.2 mm) elution from 50% to 95% MeCN in H2O as a white solid (74 mg, 0.060 mmol) with a 42% yield.

[0256] 1H NMR (400 MHz, CDCI3-MeOD2:i) 58.43 (dd, J = 18.8, 8.1 Hz, 1H), 8.20 (s, 1H), 7.65 (s, 1H), 7.60 (d, J = 7.7 Hz, 2H), 7.47 (d, J = 7.8 Hz, 2H), 7.41 (d, J = 2.9 Hz, 4H), 7.21 (d, J = 8.1 Hz, 2H), 7.13 (s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.98 -6.89 (m, 3H), 4.50 (s, 2H), 4.29 (s, 2H), 3.92 (s, 3H), 3.48 (s, 3H), 3.33 - 3.27 (m, 4H), 3.17 - 3.10 (m, 5H), 2.99 (s, J = 17.3 Hz, 3H), 2.88 - 2.79 (m, 2H), 2.70 - 2.61 (m, 2H), 2.07 (t, J = 11.4 Hz, 1 H), 1.86 - 1.76 (m, 3H), 1.63 - 1.54 (m, 8H), 1.43 - 1.35 (m, 9H), 1.23 (s, 4H), 0.99 (t, J = 7.2 Hz, 3H).

[0257] MS (ESI): m / z 1102.5458 [Mcation]+;

[0258] EXAMPLE 1.8 - Synthesis of linker payload 30

[0259] Scheme 8. Synthesis of linker payload 30

[0260]

[0261] Synthesis of intermediate 29

[0262] Under an inert N2 atmosphere XMD8-92 carbonate 5 (35 mg, 0.05 mmol,) CH2CI2 (3 mL) is dissolved and orthoester 28 (13 mg, 0.05 mmol,) and triethylamine (50 pL, 0.13 mmol) are added. The solution is left under magnetic stirring at room temperature for 16 h. After evaporating the solvent under vacuum, the crude reaction product was purified by flash chromatography on silica gel eluting with a gradient of MeOH in CH2Cl2from 0 to 5%. The product 29 was isolated with a 67% yield (25 mg).

[0263] 1H NMR (400 MHz, MeOD) 5 8.20 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.71 (dd, J = 7.8, 1.7 Hz, 1 H), 7.56 - 7.41 (m, 1 H), 7.29 - 7.05 (m, 2H), 6.92 (s, 1 H), 6.72 (d, J = 1.4 Hz, 1H), 6.65 (d, J = 2.2 Hz, 2H), 6.58 (dd, J = 8.9, 2.6 Hz, 1H), 5.47 (s, 1H), 5.04 (s, 2H), 4.82 - 4.80 (m, 2H), 4.79 - 4.73 (m, 1 H), 4.09 (q, J = 7.0 Hz, 2H), 3.75 - 3.66 (m, 2H), 3.62 (d, J = 2.5 Hz, 1 H), 3.45 (s, 3H), 3.38 (s, 3H), 3.04 - 2.96 (m, 2H), 2.94 (t, J = 2.4 Hz, 1 H), 2.10 - 1.99 (m, 2H), 1.89 - 1.78 (m, 2H), 1.40 (t, J = 7.0 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H).

[0264] MS (ESI): 751 [M+H]+.

[0265] Synthesis of compound 30

[0266] In a flask under magnetic stirring and in an inert atmosphere compound 29 (13 mg, 0.02 mmol,) and 14-azido-3,6,9,12-tetraoxatetradecanoic acid 90.5 M in MTBE (26 pL 0.01 mmol), were solubilized in DMF (1 mL) and 3 vacuum-Ar cycles were carried out. Separately, a mixture of Cu(OAc)2 (1 mg, 0.01 mmol,) and sodium ascorbate (2 mg, 0.02 mmol,) was prepared in H2O (0.5 mL). The latter was degassed under argon and was added to the solution of alkyne and TEG-azide in DMF. The reaction was maintained under magnetic stirring at room temperature for 24 h. The solvent was evaporated under vacuum and the reaction mixture was purified by flash chromatography using a gradient of MeOH in CH2CI2 from 5% to 50%). The product 30 was obtained with a 55% yield (9 mg).

[0267] 1H NMR (600 MHz, MeOD) 5 8.22 (s, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.73 (dd, J = 7.8, 1.6 Hz, 1H), 7.58 - 7.43 (m, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1 H), 6.94 (d, J = 5.4 Hz, 1 H), 6.78 (s, 1 H), 6.67 (d, J = 2.4 Hz, 1 H), 6.65 (d, J = 0.9 Hz, 1 H), 6.60 (dd, J = 8.8, 2.4 Hz, 1 H), 5.30 (s, 2H), 5.05 (s, 2H), 4.83 - 4.76 (m, 1 H), 4.71 - 4.66 (m, 2H), 4.11 (q, J = 7.0 Hz, 2H), 3.97 - 3.84 (m, 4H), 3.72 - 3.68(m, 2H), 3.68 - 3.65 (m, 7H), 3.62 - 3.60 (m, 2H), 3.59 - 3.54 (m, 3H), 3.54 - 3.50 (m, 2H), 3.50 - 3.47 (m, 2H), 3.48 - 3.46 (m, 2H), 3.41 - 3.34 (m, 4H), 2.11 - 2.02 (m, 2H), 1.92 - 1.80 (m, 2H), 1.42 (t, J = 7.0 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H).

[0268] MS (ESI): 1026 [M-H]’ ; 1028 [M+H]+; 1050 [M+Na]+.

[0269] EXAMPLE 2 - SYNTHESIS AND CHARACTERIZATION OF ADCs

[0270] By way of example, the ADCs according to the present invention were prepared with different monoclonal antibodies selected from the group consisting of Trastuzumab (Herceptin® by Roche), Cetuximab (Erbitux® by Merck), Bevacizumab (Avastin® by Genentech / Roche), Panitumumab (Vectibix®), anti-mouse CD115 and Emactuzumab (RG 7155). Among these, the antibodies which have already obtained authorization from the regulatory authorities for placement on the market are Trastuzumab which recognizes ErbB2, Cetuximab and Panitumumab which recognize ErbB1, Bevacizumab which recognizes VEGFR.

[0271] The exemplary ADCs of the present invention were prepared primarily via 4 routes: 1) conjugation to the £ -amino groups of the lysines using linker-payload containing N-hydroxysuccinimide, 2) conjugation to the £ -amino groups of the lysines using linker-payload activated in situ with (N-hydroxysulfosuccinimide, NHSS), 3) conjugation through pre-activation of the s-amino groups of the lysines with azidopolyethylene glycol chains 4) conjugation to the thiol group of the cysteines using linker payload containing a maleimide 5) conjugation through pre-activation of the s-amino groups of the antibody with dibenzocyclooctyne (DBCO). The average DAR was calculated by MALDI mass spectrometry using an Ultraflex III mass spectrometer (Broker, GmbH).

[0272] EXAMPLE 2.1 - Method of preparing an ADC through conjugation to the £-amino groups of Ab lysines using a linker-payload containing N-hydroxysuccinimide A 10 mM solution in DMSO of the linker-payload system 15 (28 pL) were added to 500 pL of anti-CD115 (0,025 eg, 2.0 mg / mL, PBS pH 8,0) or RG7155 (0.25 eg, 2.5 mg / mL, PBS pH 7.8) and the mixture was gently mixed at room temperature for 1 hour. An agueous solution of glycine (5.6 pL, 20 mM) was then added, and the mixture was incubated at room temperature for 10 min. The resulting bioconjugate 33 or 38 was purified using Cytiva PD SpinTrap G25 5K MWCO size-exclusiondialysis columns (10 kDa cutoff), as described by the manufacturer. The concentration after dialysis was determined by measuring the OD280. The conjugate is stored at 4 °C.

[0273] EXAMPLE 2.2 - Method of preparing an ADC through conjugation to the s-amino groups of Ab lysines using a linker-payload activated in situ with (N-hydroxysulfosuccinimide, NHSS)

[0274] A 10 mM solution of linker-payload 1040 eguivalents (calculated with respect to the antibody) were initially incubated with Sulfo-NHS and EDC HCI to activate the carboxylic acid to the corresponding Sulfo-NHS derivative, the reaction was conducted at room temperature for 16 hours. After activation a solution of the antibody cetuximab (PBS, pH 7.4) oranti-CD115 (EPPS, pH 8) was added. After 1 hour, a 20 mM solution of glycine in water was added. The resulting bioconjugate 31 or 32 was purified using Cytiva PD SpinTrap G25 5K MWCO size-exclusion dialysis columns (10 kDa of cutoff), as described by the manufacturer. The concentration after dialysis was determined by measuring the OD280. The conjugate is stored at 4 °C.

[0275] EXAMPLE 2.3 - Method of preparing an ADC through pre-activation of the s-amino groups of Ab lysines with azido-polyethylene glycol chains

[0276] 2.3.1 Pre-functionalization of anti-CD115

[0277] To 200 pL of anti-CD115 (1 eg, 2.0 mg / mL, PBS pH 7,4) a solution of the azido-PEG3-NHS ester (2.8 pL, 10 eg, 10 mM DMSO) was added and the mixture was stirred at room temperature for 1 hour. An agueous solution of glycine (2.8 pL, 20 eg, 20 mM in water) was then added and the mixture was incubated at room temperature for 10 minutes. The resulting bioconjugate was purified using Cytiva PD SpinTrap G25 5K MWCO size exclusion dialysis columns (10 kDa cutoff), as described by the manufacturer.

[0278] 2.3.2 Click chemistry reaction

[0279] To 100 pL of a 0.20 mg / mL agueous solution of anti-CD115 functionalized as in example 2.3.1 a DMSO solution of the linker-payload-DBCO system 17 (5.6 pL, 40 eg, 10 mM DMSO) was added and the mixture was stirred gently at 4 °C for 16 h.The resulting bioconjugate 34 was purified using Cytiva PD SpinTrap G25 5K MWCO size-exclusion dialysis columns (10 kDa cutoff), as described by the manufacturer. The concentration after dialysis was determined by measuring the OD280. The conjugate is stored at 4 °C.

[0280] EXAMPLE 2.4 - Method of preparing an ADC through conjugation to the thiol group of cysteines using linker payloads containing a maleimide

[0281] The solution of compound 19 (3.4 pL, 10 mM in DMSO) is added to a solution of cetuximab antibody, anti-CD115, or RG7155 (50 pL, 10 mg / mL) in PBS (pH 7.4) after which TCEP HCI (41.4 pL of a 1 mM solution in water) is added and the solution is incubated at room temperature for one hour. At the end the solution is purified by means of centrifugation on PD-SpinTrap (5000 MWCO, Cytiva®). The resulting bioconjugate 35, 36 or 39 was purified using Cytiva PD SpinTrap G25 5K MWCO size-exclusion dialysis columns (10 kDa cutoff), as described by the manufacturer. The concentration after dialysis was determined by measuring the OD280. The conjugate is stored at 4 °C.

[0282] EXAMPLE 2.5 - Method of preparing an ADC through pre-activation of the s-amino groups of Ab with dibenzocyclooctyne (DBCO)

[0283] 5.1 Pre-functionalization of anti-CD115 or RG7155

[0284] To 200 pL of anti-CD115 or RG7155 (1 eg, 2.0 mg / mL, PBS pH 7,4) a solution of DBCO-PEG4-NHS (2.8 pL, 10 eg, 10 mM DMSO) was added and the mixture was stirred at room temperature for 1 hour. An agueous solution of glycine (2.8 pL, 20 eg, 20 mM in water) was then added and the mixture was incubated at room temperature for 10 minutes. The resulting pre-functionalized antibody was purified using Cytiva PD SpinTrap G255K MWCO size-exclusion dialysis columns (10 kDa cutoff), as described by the manufacturer. The concentration after dialysis was determined by measuring the OD280.

[0285] 5.2 Click chemistry reaction

[0286] To 100 pL of anti-CD115 or RG7155 pre-functionalized as in example 5.1 (0.20 mg / mL in water) a DMSO solution of the linker-payload-Ns 27 system (5.6 pL, 40 eg, 10 mM DMSO) was added and the mixture was gently stirred at 4 °C for 16 hours. The resulting bioconjugate 37 or 40 was purified using Cytiva PD SpinTrapG25 5K MWCO size-exclusion dialysis columns (10 kDa cutoff), as described by the manufacturer. The resulting bioconjugate was purified using Cytiva PD SpinTrap G25 5K MWCO size exclusion dialysis columns (10 kDa cutoff), as described by the manufacturer. The concentration after dialysis was determined by measuring the OD280. The conjugate is stored at 4 °C.

[0287] EXAMPLE 3 - Functional assays in vitro

[0288] Table 1. Inhibition of cell proliferation

[0289] ADC

[0290] Code Brief description DAR Linker- Inhibition of Inhibition of Payload tumor cell TAM and / or proliferation MDSC proliferation 31 Ctx(Lys)-L2- 1.5-1.6 10a+++ ND

[0291] XMD8-92

[0292] 32 anti-CD115(Lys)- 1.2-1.7 10 b ++

[0293] L2-XMD8-92

[0294] 33 anti-CD115(Lys)- 3.0-6.3 15 b++ d

[0295] L3-XMD8-92 (14

[0296] activated)

[0297] 34 anti-CD115(Lys)- 3.0-4.7 17 b+d+

[0298] L4-XMD8-92

[0299] 35 Ctx(Cys)-Ls- 4.0-6.0 19a++ ND

[0300] XMD8-92

[0301] 36 anti-CD115(Cys)- 2.0-4.0 19 b ++ d

[0302] L5-XMD8-92 f++37 anti-CD115(Lys)- 2.0-5.2 27 b+d +

[0303] Le-JWG-071

[0304] 38 RG7155 (Lys)-Ls- 1.0-2.0 15CND ND

[0305] XMD8-92 (14

[0306] activated)

[0307] 39 RG7155(Cys)-L5- 2.0-4.0 19c++e++

[0308] XMD8-92

[0309] 40 RG7155 (Lys)-Le- 2.0-3.0 27CND ND

[0310] JWG-071

[0311] 41 Ctx(Lys)L7-XMD8- 0.5-0.9 30 ND ND

[0312] 92

[0313] 42 Anti-CD115(Lys)- 0.3-0.7 8 ND ND

[0314]

[0315] L1-XMD8-92

[0316] Ctx indicates Cetuximab; Cys indicates bond with the cysteines, Lys indicates the bond with the lysines, NCL indicates a non-cleavable linker. The inhibitory effect of the individual ADCs is compared to that induced by the control antibodies.

[0317] aHela adenocarcinoma of the cervix.bAB1 murine mesothelioma.

[0318] cH-2052 human mesothelioma.

[0319] dTAMs: murine tumor-associated macrophages.

[0320] eTAMs: human tumor-associated macrophages.

[0321] fMDSCs: myeloid immunosuppressive cells.

[0322] EXAMPLE 3.1 - Inhibition of mesothelioma cell proliferation

[0323] The inhibitory capacity of the ADCs was tested in a first analysis on murine mesothelioma cells and on tumor-associated macrophages, both expressing CSF-1R on the membrane. Regarding the mesothelioma cells, the AB1 cells were incubated with conjugate 32, or with anti-CSF-1R (unconjugated antibody), XMD8-92 (ERK5 inhibitor), or GW2580 (CSF-1R inhibitor) at the indicated concentrations for 7 days (Figure 1 A). Using the colony formation assay, it was observed that the ADC 32 conjugate had an inhibitory effect on AB1 cell growth greater than that induced by anti-CSF-1 R alone (Figure 1 A).

[0324] To test the efficacy of the conjugate ADC 32, the growth of tumor cells after treatment with the inhibitors XMD8-92 or GW2580 (alone) was compared. In particular, the growth inhibition rates induced by 200 nM of ADC 32 are significantly higher than those induced by XMD8-92 or anti-CSF-1 R used at the same concentration (Figure 1B). Observing Figure 1B, it can be noted that the ADC according to the present invention obtained a cell growth inhibition rate, at a concentration corresponding to 200 nM, equal to about twice the cell growth inhibition rate induced by only the payload XMD8-92 at a concentration equal to 5 pM, namely 25 times higher.

[0325] Using the Click-iT Edu Alexa Fluor proliferation-specific assay the inhibitory effect induced by ADC 32 on the proliferation of tumor cells was observed (Figure 1 C) demonstrating that the conjugate ADC 32 reduces the progression of the G1 / S phase of AB1 cells. Therefore, it has been demonstrated that the ADC according to the present invention significantly reduces the proliferation of mesothelioma cells, obtaining a cell quantification equal to almost half with respect to cells treated with only anti-CSF1 R.

[0326] EXAMPLE 3.2 - Internalization analysis of ADC complexes in tumor cells and in tumor-associated macrophagesTo explore the mechanism of action of anti-CSF-1 R-XMD8-92 according to the invention the binding thereof with CSF-1R_and internalization in AB1 tumor cells were first analyzed. It was observed that ADC 32 binds to its target to form an ADC-CSF-1R complex, which leads to endocytosis of the complex. The internalized complex undergoes lysosomal processing and the cytotoxic inhibitor XMD8-92 is released within the cells. The binding of anti-CSF-1 R-XMD8-92 to CSF-1R on the surface of tumor cells was first examined and then whether it is efficiently internalized. Using an anti-Rat IgG conjugated to 488 which binds anti-CSF-1 R-XMD8-92 and the lysosomal marker LysoTracker to show the lysosome in live cells, the binding and internalization of ADC 32 were tracked after it was incubated with AB1 cells for 24 hours or with macrophages cultured with AB1 -conditioned medium (tumor-associated macrophages) for 1 hour and 24 hours, and then washed. Immunofluorescence analysis showed that the anti-CSF-1 R-XMD8-92 conjugate is present in the plasma membrane and cytoplasm, and co-localizes with LysoTracker in AB1 cells (Figure 2A, upper panel) and in macrophages (Figure 2A, lower panel), indicating that anti-CSF-1 R-XMD8-92 specifically binds to the surface of CSF-1R-expressing cells and is then internalized into the lysosome.

[0327] EXAMPLE 3.3 - ADCs inhibit the activation of ERK5 in tumor cells and TAMs To verify that the ADC, once internalized, is capable of inactivating ERK5, AB1 tumor cells were incubated with anti-CSF1R alone or anti-CSF-1 R-XMD8-92 (ADC 32) for 24 hours. As shown in Figure 2B, with respect to cells treated with anti-CSF-1R alone, the activation of ERK5 (pERK5) is inhibited in cells treated with ADC 32, indicating that anti-CSF-1 R-XMD8-92 efficiently targets ERK5 of the tumor cells. Furthermore, the phosphorylation of ERK1 / 2 (pERK1 / 2) is not affected by treatment with ADC 32 (Figure 2B), indicating that anti-CSF-1 R-XMD8-92 has a specific inhibitory activity against ERK5.

[0328] Similar results were obtained in tumor-associated macrophages which were treated with anti-CSF1R alone or ADC 32 (anti-CSF-1 R-XMD8-92). ERK5 phosphorylation is strongly suppressed (Figure 3A) and macrophage proliferation is significantly reduced (Figure 3B-C) after treatment with ADC 32 with respect to cells treated with anti-CSF1 R alone, obtaining a quantification of cells equal to about half with respect to the cells treated with anti-CSF1 R alone.These results indicate that the anti-CSF-1 R-XMD8-92 conjugates according to the present invention suppress the activation of ERK5 and reduce the cell proliferation, both of AB1 cells and of pro-tumor macrophages.

[0329] EXAMPLE 3.4. - ADCs inhibit the activation of ERK5 in MDSCs

[0330] To demonstrate the ability of ADC molecules to also target the immunosuppressive myeloid cells of the tumor microenvironment expressing CSF-1R (Figure 4A), the expression of ERK5 was analyzed in MDSCs generated from bone marrow precursors (day 0) following exposure to tumor factors enriched with GM-CSF and IL-6 (day 4 and day 7). While, on one hand, the expression and activation of ERK5 is not quantifiable in bone marrow precursors, said expression and activation of ERK5 is induced after incubation with tumor factors (Figure 4-B), indicating that ERK5 plays a key role during the expansion of the MDSCs generated from bone marrow precursors.

[0331] The inhibitory capacity of ADCs was tested following the incubation of MDSCs with the conjugate ADC 36, or with anti-CSF-1 R (unconjugated antibody), or Rat IgG isotype antibody, each at a concentration equal to 30 pg / mL. Using the specific Click-iT Edu Alexa Fluor proliferation assay, the inhibitory effect induced by ADC 36 on the proliferation of MDSCs (Figure 4C) was observed, which is greater than that induced by anti-CSF-1 R alone, without inducing particular apoptotic effects (Figure 4D). Furthermore, with respect to the effect of CSF-1 R alone, the activation of ERK5 (pERK5) is inhibited to a greater extent in the cells treated with ADC 36, demonstrating that, as in the tumor cells and tumor-associated macrophages, anti-CSF-1 R-XMD8-92 according to the present invention efficiently targets the phosphorylation of ERK5 of MDSCs and is capable of inhibiting the proliferation thereof.

Claims

CLAIMS1. A conjugate of formula (I)A-(L-D)r(I)whereinA is a targeting agent capable of specifically binding to a cell surface protein expressed on target tumor cells;L is a linker;D is a payload consisting of an ERK5 inhibitor molecule;r is a number from 1 to 10 and indicates the average D / A ratio (DAR); wherein A is covalently conjugated to L through at least one s-amino group of a Lys residue or through at least one thiol group of a Cys residue; and wherein D is of formula (D-l) or (D-l I)whereinRi is H, (Ci-Cs)alkyl;R2 is H, (Ci-Csjalkyl, (Cs-Cejcycloalkyl, CH3SO2-, p-toluenesulfonyl;R3is H, (Ci-Cs)alkyl;R4 and Rs are independently from each other H, (Ci-Cs)alkyl; andHet iswhere * indicates the bonding site with L.

2. The conjugate according to claim 1 , wherein A is a protein binding an antigen expressed on the surface of target tumor cells or a small molecule selectively binding to cell receptors overexpressed in target tumor cells.

3. The conjugate according to claim 1 or 2, wherein A is a monoclonal antibody (mAb) selected from the group consisting of anti-CSF-1R, cetuximab (ctx), trastuzumab, bevacizumab, and panitumumab or Fabs or scFvs thereof.

4. The conjugate according to any one of claims 1 -3, wherein A is an anti-CSF-1R monoclonal antibody selected from the group consisting of anti-CD115, Emactuzumab (RG 7155) and Fabs or scFvs thereof.

5. The conjugate according to claim 1 or 2, wherein A is folic acid as a targeting agent for folate receptor Alpha (FRa) overexpressed in many tumor cells or folate receptor Beta (FR|3) often overexpressed in tumor-associated macrophages (TAMs).

6. The conjugate according to claim 5, wherein Ri is Me; R2 is Me, sec-Bu, cyclopentyl; R3 is Me, Et; R4 and Rs equal to each other are H, Me.

7. The conjugate according to claim 6 wherein D is selected from the group consisting ofo o8. The conjugate according to any one of claims 1 -7 wherein L is of formula (L-I) or (L-ll)wherein* indicates the bonding site with D and ** indicates the bonding site with A;n is an integer between 1 and 12, preferably 5;m is an integer between 1 and 8, preferably 4;Y isO X where p is an integer between 1 and 4, preferably 2;X is **whereinw is -(CH2)q-, -(OCH2CH2)S- with q integer between 0 and 12 and s integer between 1 and 6; v is 0, 1 and Z is9. The conjugate according to claim 8 wherein L is selected from the group 5 consisting ofwhere * indicates the bonding site with D and ** indicates the bonding site with A.

10. A conjugate according to any one of claims 1 -9 for use as a medicament.

11. A conjugate according to any one of claims 1 -9 for use in the treatment of at least one tumor, preferably of malignant mesothelioma.

12. A linker-payload molecule of formula (II)L’-D (II)whereinD is a payload as defined in claims 1 or 6-7, andL’ is a precursor of linker L, selected from the group consisting of:L’a-COOH, which has a -COOH group as a terminal group;L’b-=, which has an alkyne as a terminal group or as part of a cycloalkyl;o° , which has a maleimide group as a terminal group;L’d-Ns, which has an -Ns group as a terminal group.

13. The molecule of formula (II) according to claim 12 whereinL’ is of formula (L’-l) or (L’-l I)0(L’-l)wherein* indicates the bonding site with D;n is an integer between 1 and 12;m is an integer between 1 and 8;Y isoX’ iswhereW is -(CH2)q-, -(OCH2CH2)S- with q integer between 0 and 12, and s integer between14. Use of a molecule of formula (II) according to claim 12 or 13 as an intermediate for the preparation of a conjugate of formula (I) according to any one of claims 1-9.

15. A process for preparing a conjugate of formula (I) according to any one of claims 1-9, said process comprising contacting a compound of formula (II) according to claim 12 or 13 with a targeting agent A as defined in any one of claims 1-4, if required appropriately activated.

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