Antibody-drug conjugates involving small molecule ATR / CHK1 inhibitors

Abstract

The present invention relates to antibody-drug conjugate, comprising an antibody or antigen-binding fragment thereof, and an ATR / CHK1 inhibitor. The present invention further relates to the method of making inventive conjugates and their metabolites inside the cell, as well as to pharmaceutical compositions comprising said antibody-drug conjugate. Said conjugate is particularly useful in the treatment of cancer.

Description

[0001] Antibody-drug conjugates involving small molecule ATR / CHK1 inhibitors

[0002] Field of the invention

[0003] The present invention relates to antibody-drug conjugate, comprising an antibody or antigen-binding fragment thereof, and an ATR / CHK1 inhibitor. The present invention further relates to the method of making inventive conjugates and their metabolites inside the cell, as well as to pharmaceutical compositions comprising said antibody-drug conjugate. Said conjugate is particularly useful in the treatment of cancer.

[0004] Background of the invention

[0005] Antibody-drug conjugates (ADCs) are a class of anticancer drugs composed of an antibody scaffold, a chemical linker and a payload. Through their antibody moiety, ADCs bind to cancer cells, become internalized and their cytotoxic payload is released intracellularly resulting in cell death. The current payloads have no selectivity for cancer cells over normal cells. Nevertheless, ADCs exhibit cancer selectivity, because their antibody component recognizes antigens that are expressed preferentially on cancer cells. Improvements in ADC technology, especially in the linker, have resulted in ADCs becoming superior to current standard of care therapies (doi: 10.1056 / nejmoa2115022) and, as of September 2022, the FDA had approved twelve ADCs for clinical use (doi: 10.1016 / j.trecan.2023.01.003).

[0006] Current ADCs use antibody-targeting as the way to curtail the very high, non-selective toxicity of their payloads. However, treatment with even the best ADCs is associated with toxicity in a significant fraction of patients (doi:10.1186 / s13045-022-01397-y). For example, up to 26% of patients treated with Trastuzumab Deruxtecan may exhibit interstitial lung pneumonitis and fibrosis and fatal cases have been reported (doi / 10.1056 / NEJMoa2112431). Further, emergence of resistance mechanisms represents also a major challenge (doi.org / 10.1038 / s41571 -023-00783-w; doi.org / 10.1208 / s12248-022-00717-x; doi.org / 10.1158 / 2159-8290.CD-21 -0702; doi.org / 10.3390 / cancers 15041278).

[0007] Developing ADCs with payloads that, upon release, have a wider therapeutic window could potentially reduce the toxicity associated with ADC treatments. Consequently, the introduction of new payloads becomes pivotal in the advancement of ADCs. Furthermore, developing ADCs with payloads that have a novel mechanism of action could potentially overcome the development of resistance to earlier-line ADCs.

[0008] There is continued need for further improved anticancer therapies based on antibody-drug conjugates.

[0009] Summary of the invention

[0010] Accordingly, it was an objective technical problem of the present invention to provide novel cancer- selective ADCs with improved properties. The problem is solved by the embodiments disclosed herein and as characterized by the claims.

[0011] The present invention provides novel, cancer-selective ADC payloads, by exploiting known differences in DNA replication and DNA repair between cancer and normal cells. Most cancers have oncogene-induced DNA replication stress (doi:10.1038 / nature03485), which renders them sensitive to inhibition of the replication stress checkpoint kinases ATR and CHK1 (doi:10.1158 / 2159-8290.cd-20-0868; doi.org / 10.1038 / s41573-022-00558-5). The most potent clinical ATR and CHK1 inhibitors have picomolar efficiency. Therefore, we propose to develop these inhibitors as ADC payloads with the intent to significantly improve the therapeutic window of ADCs in the clinic.

[0012] In the present application, novel ADCs according to the claims and including an ATR inhibitor or CHK1 inhibitor are demonstrated to show particular anti-cancer activity. In particular, it has been demonstrated in Example 16 that antibody-drug conjugates based on trastuzumab or its derivatives (i.e., moieties targeting the conjugate to HER2+ cancer cells) and including CHK1 specific inhibitors selectively kill HER2+ cancer cells, without causing any toxicity to cells that are not HER2+. Accordingly, it has been demonstrated that according to the present invention the efficacy of ATR / CHK1- specific inhibitors can be targeted to specific cells, and thereby their toxicity can be mitigated.

[0013] The invention is summarized in the following embodiments.

[0014] In a first embodiment, the present invention relates to an antibody-drug conjugate, comprising an antibody or antigen-binding fragment thereof, and an ATR / CHK1 inhibitor.

[0015] In a second embodiment, the present invention relates to a compound of formula (II):

[0016] or a pharmaceutically acceptable salt thereof, wherein: R3and R4are each independently hydrogen or methyl, and n is an integer from 1 to 5, preferably n is 3.

[0017] In a third embodiment, the present invention relates to a compound of formula: wherein R1is selected from

[0018] R3and R4are each independently hydrogen or methyl, and n is an integer from 1 to 5, preferably from 2 to 4, more preferably n is 3. Preferably, it is to be understood that the salts of the compounds are also encompassed. In a fourth embodiment, the present invention relates to a pharmaceutical composition comprising the antibody-drug conjugate of the first embodiment of the present invention, and a pharmaceutically acceptable carrier. In a fifth embodiment, the present invention relates to the antibody-drug conjugate of the first embodiment of the present invention or the pharmaceutical composition of the fourth embodiment of the present invention for use as a medicament.

[0019] In a sixth embodiment, the present invention relates to the antibody-drug conjugate ofthe first embodiment of the present invention or the pharmaceutical composition of the fourth embodiment of the present invention for use in the treatment of cancer.

[0020] In a seventh embodiment, the present invention relates to use of the antibody-drug conjugate of the first embodiment of the present invention or the pharmaceutical composition of the fourth embodiment of the present invention in the manufacture of the medicament for the treatment of cancer.

[0021] In an eight embodiment, the present invention relates to a method of treating a cancer in a subject, the method comprising the step of administering the antibody-drug conjugate of the first embodiment of the present invention or the pharmaceutical composition of the fourth embodiment of the present invention to a subject in need thereof. It is to be understood that, preferably, a therapeutically effective amount is to be administered.

[0022] In a further embodiment, the present invention relates to a compound selected from:

[0023]

[0024] 

[0025] is to be understood that the salts of the compounds are also encompassed. Brief description of figures

[0026] The invention is further presented in the appended figures. These are not meant to be considered as limiting the scope of protection in any way, which is defined by the hereto appended claims. Fig.1 presents A. Chemical structure of the ATR inhibitor BAY1895344 and synthesized derivatives; B. Activity of BAY1895344 and selective derivatives in the ATR / ATRIP in vitro kinase assay. Dose- response curves and IC50 values for BAY1895344 and derivatives, examined over a 2 μM - 200 pM range (3.33-x dilution steps); C-E. Activity of BAY1895344 and selective derivatives in the cell-based yH2AX assay: C. Top: Experimental timeline for monitoring yH2AX accumulation upon ATR inhibition in cells (top). Bottom: Representative flow cytometry profiles showing DNA content

[0027] (x-axis; DNA content to indicate cells in the G1 , S and G2 phases of the cell cycle) versus yH2AX levels (y-axis; No yH2AX, black - bottom of the Y-axis; Low yH2AX, gray - middle of the Y-axis; High yH2AX, light-gray - top of the Y-axis) of U2OS osteosarcoma cells treated with the indicated compounds (400 nM). D. Graphs showing the percentage of U2OS cells assigned to the Low (Lo) and High (Hi) yH2AX gates for each of the compounds shown in panel C. E. Graph showing the EC50 values calculated for each compound tested in the cell-based yH2AX assay. For the last 3 compounds, EC50 values could not be determined, as the compounds were inactive or had very weak activity.

[0028] Fig. 2 shows A. Purification profiles of the antibodies. Non-reduced PAGE with Coomassie blue staining. Note that the mutant V205C is referred to as V225C, because the numbering includes the length of the N-terminal signal peptide. IN, input; FT, flow through; E, eluted fractions; MW, Molecular weight marker (kDa). B. Final sample QC. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. 1 , Trastuzumab; 2, Trastuzumab-m_V225C. 2pg loaded per lane. MW, Molecular weight marker (kDa).

[0029] Fig. 3 presents A. Final sample QC for Trastuzumab and Trastuzumab-mV205C conjugated to HALA- 009 with DAR 2 or 4. B. Additional QC Trastuzumab unconjugated or conjugated to HALA-009 with DAR 2 or 4. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. Reduced and Non-Reduced PAGE analysis. MW. Molecular weight marker. 2pg loaded per lane.

[0030] Fig. 4 shows SEC-HPLC profiles of purified Trastuzumab, Trastuzumab-mV205C and conjugates.

[0031] Fig. 5 depicts A. OD values obtained with Trastuzumab, Trastuzumab-HALA-009-DAR2 and Trastuzumab-HALA-009-DAR4 against CD340 (HER2); B. Superimposition of ELISA titration curves of samples from A.

[0032] Fig. 6 presents synthetic schemes for the compounds prepared in Example 9.

[0033] Fig. 7 shows A. Purification profiles of the antibodies. Non-reduced PAGE with Coomassie blue staining. IN, input; FT, flow through; E, eluted fractions; MW, Molecular weight marker (kDa). B. Final sample QC. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. WT, wild-type trastuzumab; 1, Antibody-1; 2, Antibody-2; 3, Antibody- 3; 4, Antibody-4. 2pg loaded per lane. MW, Molecular weight marker (kDa).

[0034] Fig. 8 depicts A. OD values obtained with Trastuzumab, Antibody-1 , Antibody-2 and Antibody-3 against CD340 (HER2). B. Superimposition of ELISA titration curves of samples from A. C. OD values obtained with Trastuzumab and Antibody-4 against CD240 (HER2). D. Superimposition of ELISA titration curves of samples from C.

[0035] Fig. 9 presents final sample QC. A. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. 1 , wild-type trastuzumab; 2, B24-05_HALA-015. 5pg loaded per lane. MW, Molecular weight marker (kDa). B. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. 1, wild-type trastuzumab; 2, B24-031_HG-036. 5pg loaded per lane. MW, Molecular weight marker (kDa). C. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. 1, wild-type trastuzumab; 2, Antibody-5; 3, proteo_WT; 4, proteo_V205C. 2pg loaded per lane. MW, Molecular weight marker (kDa). D. OD values obtained with wilde-type trastuzumab, Antibody-5 (trastuzumab V205C mutant), ADC proteo_WT and ADC proteo_V205C against CD340 (HER2). E. Superimposition of ELISA titration curves of samples from B

[0036] Fig. 10 presents final sample QC. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. A A. 1 , Antibody-5; 2, B24-06_HALA-015 B. 1, Antibody-1; 2, B24-013_HALA-015; 3, Antibody-3; 4, B24-014_HALA-015. C. 1, Antibody-2; 2, B24-037_HALA-015; 3, B24-037_HG-028. D. 1 , Antibody-2; 2, B24-025_HG-016. E. 1, Antibody-2; B24-032_HG-036. F. 1, Antibody-2; 2, B25- 01_HG-043; 3, B25-01_HG-044; 4, B25-01_HALA-018; 5, B25-01_HALA-017; 6, B25-01.HALA- 016.

[0037] Fig. 11 presents Final sample QC. Reduced / non-reduced SDS-PAGE with Coomassie blue staining. 5pg loaded per lane. MW, Molecular weight marker (kDa). A. 1, Antibody-4; 2, B24-041_HALA-015. B. 1 , Antibody-4; 2, B24-041 JHALA-015; 3, B24-038_HG-036; 4, B24-038_HG-028. C. 1, Antibody-4; 2, B25_02_UGTH-HG-043; 3, B25_02_HALA-018; 4, B25_02_HALA-017; 4, B25_02_HALA-016.

[0038] Fig. 12 shows quantification of yH2AX mean intensity in HCC1569 (HER2-positive) and HUH7 (HER2- negative) cell lines treated with IgG, wild-type trastuzumab and the ADC proteo_V205C.

[0039] Detailed description of the invention

[0040] As already mentioned, in one embodiment the present invention relates to an antibody-drug conjugate, comprising an antibody or antigen-binding fragment thereof, and an ATR / CHK1 inhibitor. As mentioned before, the antibody-drug conjugate comprises the antibody (or antigen-binding fragment thereof), and a payload, which may also be referred to as a drug. The skilled person will appreciate that said antibody and said payload are connected to each other by the means of chemical linkage. The skilled person is capable of devising a linker connecting said payload and said antibody. Exemplary and suitable payloads are disclosed herein.

[0041] Said antibody-drug conjugate of the present invention comprises an antibody or antigen-binding fragment thereof.

[0042] In general, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully-human antibodies and antibody fragments so long as they exhibit the desired antigen-binding activity. Unless explicitly indicated to the contrary, when discussing the antibody or the properties thereof, whenever a reference is made to an antibody, an antibody of an antigen-binding fragment thereof is meant.

[0043] As preferably referred to herein, an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0044] Preferably, the antibody or the antigen binding fragment thereof is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a single chain antibody, a humanized antibody, a bispecific antibody, a multi-specific antibody, or an antibody displayed upon the surface of a phage or displayed upon the surface of a chimeric antigen receptor (CAR) T cell.

[0045] The term “monoclonal antibody” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, / .e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modified "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495.

[0046] The term “chimeric antibodies”, refers to an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, human or non- human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

[0047] The term “recombinant antibody” includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Accordingly, the term antibody also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0048] A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.

[0049] The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.

[0050] The term antibody also relates to humanized antibodies. "Humanized" forms of non-human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see: JonesNature 321 (1986), 522-525; Reichmann Nature 332 (1998), 323-327 and Presta Curr Op Struct Biol 2 (1992), 593-596.

[0051] Accordingly, in context of the present invention, the term “antibody” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., “antigen-binding fragment thereof”). Furthermore, the term relates, as discussed above, to modified and / or altered antibody molecules. The term also relates to recombinantly or synthetically generated / synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fv, Fab’, Fab’-SH, F(ab’)2. The term antibody also comprises but is not limited to fully-human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.

[0052] A single chain antibody, i.e., “single-chain Fv” or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies are described, e.g., in Pliickthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, N.Y. (1994), 269- 315. A bispecific antibody, as referred to herein, is an antibody that can simultaneously bind to two different types of antigen, or to two different epitopes of the same antigen. Upon development, bispecific antibodies can be manufactured in several structural formats, which are known to the skilled person. Within the invention, at least one of antigens relates to L1-CAM, as defined herein.

[0053] A multi-specific antibody, as referred to herein, is an antibody that can simultaneously bind to more than two different types of antigen, or to more than two different epitopes of the same antigen.

[0054] Preferably, the antibody or the antigen binding fragment thereof is a monoclonal antibody.

[0055] The antibody as referred to herein may be an lgG1, lgG2a or lgG2b, lgG3, lgG4, IgM, lgA1, lgA2, IgAsec, IgD, IgE. As used herein, "isotype" refers to the antibody class (e.g., IgM or lgG1 ) that is encoded by heavy chain constant region genes. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG 1 , 1 gG2, lgG3, 1 gG4, lgA1, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, £, y, and p, respectively.

[0056] Preferably, the monoclonal antibody as described herein is an lgG1 antibody.

[0057] Accordingly, the antibodies of the present invention can be full length or can include only an antigen- binding fragment such as the antibody constant and / or variable domain of lgG1, lgG2, lgG3, lgG4, IgM, lgA1, lgA2, IgAsec, IgD or IgE or could consist of a Fab fragment, a F(ab')2 fragment and a Fv fragment.

[0058] A ‘‘Fab fragment” as used herein is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0059] An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0060] A "Fab1fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the C H1 domain and also the region between the CH1 and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0061] A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[0062] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0063] In the antibody-drug conjugates of the present invention, it is particularly preferred that the antibody or antigen-binding fragment thereof is capable of targeting the antibody-drug conjugate to cancer cells.

[0064] As preferably it is to be understood herein, the ability of targeting the antibody-drug conjugate to cancer cells is to be understood as the ability of colocalizing said antibody-drug conjugate with the cancer cells. This may be achieved, for example, by binding of the antibody of said antibody-drug conjugate to a receptor on a surface of a cancer cell. To that extent, the antibody is to bind an antigen that is specifically present on the surface of the cancer cells. By binding, an interaction of sufficient affinity so that the two binding partners can effectively colocalize, is understood. Preferably, the binding as described herein is specific. That means, the of antibody to its antigen is at least 10 fold, preferably at least 100 fold stronger than binding to any further binding partner.

[0065] The skilled person appreciates that antibody can in generic terms be defined by a specific antigen its binds to. Accordingly, it is preferred that the antibody, or antigen-binding fragment thereof, present in the antibody-drug conjugate of the present invention, specifically binds to an antigen selected from HER2, CD30, CD33, CD22, CD79b, Nectin-4, Trop2, BCMA, EGFR, CD19, Fra and B7H4. The skilled person will further appreciate that only a fragment of sequence of any of these proteins is the actual antigen said antibody its binding to.

[0066] The skilled person further appreciates that antibody can also be defined in generic terms by defining its epitope through another antibody, or antigen binding fragment thereof, that binds to the same epitope. Accordingly, within the scope of the present invention, it is preferred that the antibody or antigen-binding fragment thereof specifically binds to the same epitope as an antibody selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, Mirvetuximab, Datopotamab and Telisotuzumab, Preferably, the antibody or antigen-binding fragment thereof specifically binds to the same epitope as an antibody selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab, loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, and Mirvetuximab. More preferably, the antibody or antigen-binding fragment thereof specifically binds to the same epitope as an antibody selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab and loncastuximab. Even more preferably, the antigen-binding fragment thereof specifically binds to the same epitope as an antibody selected from trastuzumab, and Sacituzumab. Still more preferably, the antigen-binding fragment thereof specifically binds to the same epitope as trastuzumab. In one embodiment, the antigen-binding fragment thereof specifically binds to the same epitope as an antibody Sacituzumab.

[0067] Accordingly, in the antibody-drug conjugate of the present invention, the antibody or antigen-binding fragment thereof may be selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab, loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, Mirvetuximab, Datopotamab and Telisotuzumab. Preferably, the antibody or antigen-binding fragment thereof may be selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab, loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, and Mirvetuximab. More preferably, the antibody or antigen-binding fragment thereof may be selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab, and loncastuximab. Even more preferably, the antibody or antigen-binding fragment thereof may be selected from trastuzumab and Sacituzumab. Still more preferably, the antibody or antigen-binding fragment thereof may be trastuzumab. However, alternatively the antibody or antigen-binding fragment thereof may be Sacituzumab.

[0068] According to the present invention, the trastuzumab may be an antibody comprising (preferably consisting of) a heavy chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 1 , and a light chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID N0.:2. It is to be understood that, preferably, the sequences of the light chain and the heavy chain, as referred to herein, are such that the resulting antibody binds to the same epitope as trastuzumab. Accordingly, as understood herein, trastuzumab preferably comprises (i.e., preferably consists of) a heavy chain characterized by a sequence identical to a sequence according to SEQ ID NO.: 1 , and a light chain characterized by a sequence identical to a sequence according to SEQ ID NO.: 2. However, also encompassed by the present invention are variants of trastuzumab deviating from these sequences, preferably engineered specifically for payload attachment, wherein preferably the resulting antibody binds to the same epitope as trastuzumab.

[0069] Accordingly, light chain of the antibody in the antibody-drug conjugate of the present invention may be characterized by the presence of V205C mutation. Preferably, the light chain of the antibody in the antibody-drug conjugate of the present invention is characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 3. It is preferred that the sequence, when compared to the sequence according to SEQ ID NO.: 2, is characterized by the presence of V205C mutation, and that the antibody binds to the same epitope as trastuzumab.

[0070] The light chain of the antibody in the antibody-drug conjugate of the present invention may also be characterized by the presence of V205C mutation and of Q155C mutation. Preferably, the light chain of the antibody in the antibody-drug conjugate of the present invention is characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 6. It is preferred that the sequence, when compared to the sequence according to SEQ ID NO.: 2, is characterized by the presence of V205C mutation and the presence of Q155C mutation, and that the antibody binds to the same epitope as trastuzumab.

[0071] The heavy chain of the antibody in the antibody-drug conjugate of the present invention may be characterized by the presence of V170C mutation. Preferably, the heavy chain of the antibody in the antibody-drug conjugate of the present invention is characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 5. It is preferred that the sequence, when compared to the sequence according to SEQ ID NO.: 1, is characterized by the presence of V170C mutation, and that the antibody binds to the same epitope as trastuzumab. The heavy chain of the antibody in the antibody-drug conjugate of the present invention may also be characterized by the presence of V170C mutation, the presence of S340C mutation, the presence of E391 C mutation and the presence of Q441C mutation. Preferably, the heavy chain of the antibody in the antibody-drug conjugate of the present invention is characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 7. It is preferred that the sequence, when compared to the sequence according to SEQ ID NO.: 1, is characterized by the presence of V170C mutation, the presence of S340C mutation, the presence of E391 C mutation and the presence of Q441 C mutation, and that the antibody binds to the same epitope as trastuzumab.

[0072] Accordingly, in one embodiment of the present invention, the trastuzumab may be an antibody comprising (preferably consisting of) a heavy chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 1, and a light chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID N0.:3. It is to be understood that, preferably, the sequences of the light chain and the heavy chain, as referred to herein, are such that the resulting antibody binds to the same epitope as trastuzumab.

[0073] In one embodiment of the present invention, the trastuzumab may be an antibody comprising (preferably consisting of) a heavy chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 5, and a light chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID N0.:3. It is to be understood that, preferably, the sequences of the light chain and the heavy chain, as referred to herein, are such that the resulting antibody binds to the same epitope as trastuzumab.

[0074] In one embodiment of the present invention, the trastuzumab may be an antibody comprising (preferably consisting of) a heavy chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 1 , and a light chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID N0.:6. It is to be understood that, preferably, the sequences of the light chain and the heavy chain, as referred to herein, are such that the resulting antibody binds to the same epitope as trastuzumab.

[0075] In one embodiment of the present invention, the trastuzumab may be an antibody comprising (preferably consisting of) a heavy chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID NO.: 7, and a light chain characterized by sequence at least 95% identical to, preferably a least 97% identical to, more preferably at least 99% identical to, even more preferably identical to a sequence according to SEQ ID N0.:6. It is to be understood that, preferably, the sequences of the light chain and the heavy chain, as referred to herein, are such that the resulting antibody binds to the same epitope as trastuzumab.

[0076] The antibody-drug conjugate of the present invention further comprises an ATR / CHK1 inhibitor. Accordingly, the payload or the drug in the antibody-drug conjugate of the present invention is necessarily an ATR / CHK1 inhibitor.

[0077] As it is to be understood herein, the ATR / CHK1 inhibitor is a compound that has downregulating effect on the ATR / CHK1 signaling pathway. ATR / CHK1 inhibitor may be ATR inhibitor. ATR is ATR kinase or ATR, is also referred to as ataxia telangiectasia and RAD3 related. It is a kinase belonging to phosphatidylinositol 3-kinase-related kinase protein family, which is activated in response to single strand breaks, and functions to ensure genome integrity. Alternatively or additionally, ATR / CHK1 inhibitor may be CHK1 inhibitor. CHK1 is Checkpoint kinase 1, commonly referred to as Chk1. It is a serine / threonine- specific protein kinase that, in humans, is encoded by the CHEK1 gene. Chk1 coordinates the DNA damage response (DDR) and cell cycle checkpoint response. Activation of Chk1 results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle. As it is to be understood herein, an inhibitor of an enzyme is a compound that causes, at a concentration of 20 micromolar, at least 50% reduction in activity of the enzyme. The particular mechanism of inhibition is not meant to be limited in any way and inhibitors may be both competitive, non-competitive or allosteric. Preferably, a compound can be considered to be an inhibitor of a particular enzyme if it has Ki against this enzyme not higher than 10 micromolar. The compound is considered an ATR / CHK1 pathway inhibitor if it is at least one of the following: an ATR inhibitor, a CHK1 inhibitor. Preferably, the ATR / CHK1 inhibitor is an ATR inhibitor, as defined herein. It is preferred that said ATR / CHK1 inhibitor is a small molecule inhibitor. Small molecule is defined as a chemical compound made of C, H, 0, N, and S atoms with a molecular weight of up to 1500 g / mol, preferably with a molecular weight of up to 1000 g / mol, more preferably with a molecular weight of up to 500 g / mol, wherein said small molecule is not a peptide.

[0078] Preferably, the ATR / CHK1 inhibitor is an ATR inhibitor.

[0079] The ATR inhibitor may be Elimusertib or its derivative. Elimusertib is a compound according to formula:

[0080] Elimusertib (BAY-1895344) is a potent, orally active and selective ATR inhibitor with an IC50 of 7 nM. Elimusertib has shown anti-tumor activity and can be used for the treatment, as well as the research, of solid tumors and lymphomas.

[0081] As it is to be understood herein, preferably Elimusertib or its derivative is Elimusertib. Alternatively, derivative of Elimusertib is as disclosed in any of embodiments disclosed in the following.

[0082] As encompassed by the present invention, the ATR inhibitor can be derivative of Elimusertib. In one embodiment, said derivative of Elimusertib being ATR inhibitor may be a compound of formula (I):

[0083] ,

[0084] In formula (I), R2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -0-(C1-6 alkyl), -0- heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-( C1-6 alkyl), -N(C1-6 alkyl)-( C1-6 alkyl), -COO-( C1-6 alkyl), -C0NH-( C1-6 alkyl), -CON(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -SO-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHSO2-(C1-6 alkyl), and -N(C1-6 alkyl)-SO2-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0085] Preferably, R2is selected from C1-6 alkyl, C2-6 alkenyl, -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, and heteroaryl, wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0086] More preferably, R2is selected from -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, and heteroaryl, wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl. Even more preferably, R2is selected from phenyl, and heteroaryl, wherein said said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0087] Still more preferably, R2is heteroaryl, optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl. Particularly suitable heteroaryl are 5- and 6-membered heteroaryls comprising at least one N atom, preferably, 5-membered heteroaryls comprising at least one N atom. Thus, R2may be, for example, imidazolyl or pyrazolyl, preferably pyrazolyl, in particular pyrazol-5-yl, optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0088] In a particularly preferred embodiment,

[0089] In formula (I), R3and R4are each independently hydrogen or methyl. It is particularly preferred that R3is hydrogen and R4is methyl. It is particularly preferred that the compound of formula (I) comprises a moiety of formula:

[0090] As it is to be understood herein, and as encompassed by the present invention, the ATR inhibitor may be according to formula (I), as defined hereinabove, or in any preferred definition or any preferred embodiment of the compound of formula (I).

[0091] As encompassed by the present invention, the compound of formula (I) may be a compound of formula (la):

[0092]

[0093] (la).

[0094] Accordingly, the ATR inhibitor, or more generally defined ATR / CHK1 inhibitor may be defined herein as a complete (small) molecule, without any defined attachment points and without any empty valences. It is apparent to the skilled person that for the attachment of said molecule to the antibody or antigen-binding fragment thereof, it is necessary to generate / define the attachment point in said molecule. Said attachment point may be made by removal of an -H from the molecule, thereby creating an empty valence for the attachment of antibody (or antigen-binding fragment thereof), optionally through a linker moiety, as recognizable to the skilled person.

[0095] As referred to herein, a workable attachment is herein understood as an attachment that maintains, or substantially maintains, activity of the compound as ATR / CHK1 inhibitor. In other words, the attachment of a compound to antibody (or antigen-binding fragment thereof) can be considered to be a workable attachment if said compound, despite being chemically modified for the attachment, is still capable of showing activity towards as ATR / CHK1 inhibitor. As it is to be understood herein, maintaining (or substantially maintaining) the activity of the compound as ATR / CHK1 inhibitor means that the effect of the compound as ATR / CHK1 inhibitor upon chemical modification necessary for attachment is not diminished by more than 50%, preferably by not more than 40%, more preferably by not more than 30%, even more preferably by not more than 40%, still more preferably by not more than 50%.

[0096] In the following, alternative definitions of ATR inhibitors, in particular based on Elimuserti b or derivatives thereof, which explicitly recite possible and preferred attachment points, will be provided. However, it is to be noted that these definitions are not meant to be considered limiting, and any workable attachment of said molecule to the antibody (or its antigen-binding fragment) will be considered encompassed by the invention. Accordingly, in one embodiment of the antibody-drug conjugate of the present invention, the antibody- drug conjugate comprises a moiety of formula (lb):

[0097] In formula (lb), it is to be understood that the empty valence is connected to the antibody or antigen- binding fragment thereof. Optionally, said empty valence may be connecting to the antibody or antigen- binding fragment thereof through a linker moiety.

[0098] In formula (lb), R1is selected from

[0099] Preferably, R1is selected from , In formula (lb), R3and R4are each independently hydrogen or methyl. It is particularly preferred that R3is hydrogen and R4is methyl. It is particularly preferred that in the moiety of formula (lb) the following moiety is comprised:

[0100] In view of the foregoing, it is particularly preferred that the antibody-drug conjugate of the present invention comprises a moiety according to formula (lb-1):

[0101] (lb-1). In other words, the moiety of formula (lb) may be a moiety of formula (lb-1), as defined hereinabove.

[0102] In one embodiment of the antibody drug conjugate of the present invention, the antibody-drug conjugate comprises a moiety according to formula (Ic): In formula (Ic), it is to be understood that the empty valence is connected to the antibody or antigen- binding fragment thereof. Optionally, said empty valence may be connecting to the antibody or antigen- binding fragment thereof through a linker moiety.

[0103] In formula (Ic), R2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -0-(C1-6 alkyl), -0- heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-(C1-6 alkyl), -N(CI-B alkyl)-( C1-6 alkyl), -C00-(C1-6 alkyl), -C0NH-(C1-6 alkyl), -C0N(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -S0-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHSO2-(C1-6 alkyl), and -N(C1-6 alkyl)-SO2-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0104] Preferably, R2is selected from C1-6 alkyl, C2-6 alkenyl, -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, and heteroaryl, wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0105] More preferably, R2is selected from -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, and heteroaryl, wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0106] Even more preferably, R2is selected from phenyl, and heteroaryl, wherein said said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0107] Still more preferably, R2is heteroaryl, optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl. Particularly suitable heteroaryl are 5- and 6-membered heteroaryls comprising at least one N atom, preferably, 5-membered heteroaryls comprising at least one N atom. Thus, R2may be, for example, imidazolyl or pyrazolyl, preferably pyrazolyl, in particular pyrazol-5-yl, optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0108] In a particularly preferred embodiment, In formula (Ic), R3and R4are each independently hydrogen or methyl. It is particularly preferred that R3is hydrogen and R4is methyl. It is particularly preferred that in the moiety of formula (Ic), the following moiety is comprised:

[0109] Accordingly, in view of the foregoing, it is particularly preferred that the antibody-drug conjugate of the present invention comprises a moiety according to formula (lc-1):

[0110] (lc-1)

[0111] In other words, as encompassed by the present invention, the moiety of formula (Ic) may be a moiety of formula (lc-1) as defined hereinabove.

[0112] In one embodiment of the present invention, relating to the antibody-drug conjugate of the present invention, the ATR inhibitor is selected from Elimusertib, Camonsertib, Ceralasertib, Gartisertib, Tuvusertib and Berzosertib.

[0113] Elimusertib is as defined hereinabove.

[0114] Camonsertib is a compound of formula:

[0115]

[0116] Camonsertib is an orally active, selective ATR kinase inhibitor (ATRi) with an IC50 of 1.00 nM in biochemical assays. Camonsertib shows 30-fold selectivity for ATR over mTOR (IC5o=120 nM) and >2, 000-fold selectivity over ATM, DNA-PK, and PI3Ka kinases. The safety and tolerability profile of

[0117] Camonsertib was consistent with a highly selective and potent ATR inhibitor, and preliminary anti-tumor activity was demonstrated in heavily pre-treated tumors across a range of histologic types and enrollment gene alterations. Ceralasertib is a compound of formula:

[0118] Ceralasertib is an orally active, and selective ATR kinase inhibitor with IC50 of 1 nM. It is currently undergoing phase 1 / 2 clinical trials. Gartisertib is a compound of formula: Gartisertib is an ATP-competitive, orally active, and selective ATR inhibitor, with a Ki of <150 pM. Gartisertib potently inhibits ATR-driven phosphorylated checkpoint kinase- 1 (Chk1) phosphorylation with an I C50 of 8 nM. Gartisertib shows antitumor activity.

[0119] Tuvusertib is a compound of formula:

[0120] Tuvusertib is a potent ATR inhibitor, which exhibits anti-proliferative and anti-tumor effects in cancer cell lines.

[0121] Berzosertib is a compound of formula:

[0122] Berzosertib is a highly potent and selective, first-in-class inhibitor of ataxia telangiectasia and Rad3- related protein kinase (ATR).

[0123] In one embodiment of the present invention, relating to the antibody-drug conjugate of the present invention, the ATR inhibitor is selected from Elimusertib and Camonsertib.

[0124] In one preferred embodiment of the present invention, relating to the antibody-drug conjugate of claim the present invention, the ATR inhibitor is Camonsertib. It is to be understood that Camonsertib is to be attached to the antibody or the antigen-binding fragment thereof as described hereinabove. Preferred attachment of Camonsertib to the antibody or the antigen-binding fragment thereof is described in the following. Accordingly, the antibody-drug conjugate of the present invention further relates to an embodiment, wherein the antibody-drug conjugate comprises a moiety according to formula:

[0125] It is to be understood that the empty valence in the moiety derived from Camonsertib compound is connected to the antibody or antigen-binding fragment thereof. Optionally, said empty valence may be connecting to the antibody or antigen-binding fragment thereof through a linker moiety. In one embodiment, the ATR inhibitor is Berzosertib. As discussed herein, Berzosertib can be attached to the antibody or the antigen-binding fragment thereof in any workable way, as defined herein. Exemplary workable way of attaching Berzosertib to said antibody (or its fragment) will be disclosed in the following.

[0126] Accordingly, in one embodiment, the antibody-drug conjugate of the present invention comprises a moiety according to formula: Alternatively, in the antibody-drug conjugate of the present invention, the ATR / CHK1 inhibitor may be a CHK1 inhibitor. According to the present invention, the antibody-drug conjugates, wherein the ATR / CHK1 inhibitor is a CHK1 inhibitor, are particularly preferred.

[0127] In one embodiment, the CHK1 inhibitor is Prexasertib. Prexasertib is a compound of formula:

[0128] In one embodiment, the CHK1 inhibitor is according to formula:

[0129] In this formula, X’ is CH or N. Preferably, X’ is CH.

[0130] In this formula, R1is selected from hydrogen, C1-5 alkyl, halogen, cycloalkyl and heterocycloalkyl, wherein said cycloalkyl and said heterocycloalkyl are each optionally substituted with one or more groups selected from C1-5 alkyl and halogen.

[0131] Preferably, R1is selected from C1-5 alkyl (such as methyl), halogen (such as -F), and heterocycloalkyl, wherein said heterocycloalkyl is optionally substituted with one or more groups selected from C1-5 alkyl and halogen. More preferably, R1’ is selected from methyl, -F Even more preferably, R1is selected from methyl, and -F.

[0132] In this formula, R2 1is -CN and R2 2is hydrogen, or R2 1is hydrogen

[0133] Preferably, R2 1’ is -CN and R2 2’ is hydrogen.

[0134] Further in this formula, R3is — (C1-5 alkylene)-NH2, — (C1-5 alkylene)-NH-CO-(C1-5 alkylene)-NH2, - (cycloalkylene)-NH2 or -(cycloalkylene)-NH-CO-(C1-5 alkylene)-NH2. Preferably, R3is — (C1-5 alkylene)- NH2, — (C1-5 alkylene)-NH-CO-(C1-5 alkylene)-NH2, or -(cycloalkylene)-NH2. More preferably, R3’ is -(C1-5 alkylene)-NH2, or -(cycloalkylene)-NH2. Particularly suitable — (C1-5 alkylene)-NH2, is -(CH2CH2CH2)-NH2, and particular suitable — (cycloalkylene)

[0135] In one embodiment, the CHK1 inhibitor is according to formula:

[0136] In this formula, R1' is selected from hydrogen, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, halogen, -0-(C1-5 alkyl), -0-(C1-5 alkenyl), and -0-(C1-5 alkynyl). Preferably, R1is selected from C1-5 alkyl, halogen, and -0-(C1-5 alkynyl). More preferably, R1is selected from -F and -0-(CH2CCH).

[0137] In this formula, R5is selected from hydrogen and -CN, Preferably, R5is -CN. Further in this formula, R6’ is — (C1-5 alkylene)-NH2, — (C1-5 alkylene)-NH-CO-(C1-5 alkylene)-NH2, - (cycloalkylene)-NH2 or -(cycloalkylene)-NH-CO-(C1-5 alkylene)-NH2. Preferably, R6is — (C1-5 alkylene)- NH2, — (C1-5 alkylene)-NH— CO-(C1-5 alkylene)-NH2, or -(cycloalkylene)-NH2. More preferably, R6is -(C1-5 alkylene)-NH2, or — (cycloalkylene)-NH2. Particularly suitable — (C1-5 alkylene)-NH2, is -(CH2CH2CH2)-NH2, and particular / suitable — (cycloalkylene)-

[0138] In one embodiment, the antibody-drug conjugate of the present invention comprises a moiety according to formula:

[0139] In one preferred embodiment, the antibody-drug conjugate of the present invention comprises a moiety according to formula:

[0140] In one embodiment, the CHK1 inhibitor is according to formula:

[0141]

[0142] In one embodiment, the CHK1 inhibitor is according to formula: In one embodiment, the ATR inhibitor in the antibody-drug conjugate of the present invention is according to formula:

[0143] In this formula, R1is selected from More preferably, R1is selected from

[0144] Even more preferably,

[0145] In this formula, R2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -0-(C1-6 alkyl), -0- heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-(C1-6 alkyl), -N(CI-B alkyl)-(C1-6 alkyl), -C00-(C1-6 alkyl), -C0NH-(C1-6 alkyl), -CON(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -SO-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHSO2-(C1-6 alkyl), and -N(C1-6 alkyl)-SO2-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0146] Preferably, R2is selected from C1-6 alkyl, C2-6 alkenyl, -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, and heteroaryl, wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0147] More preferably, R2is selected from -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, and heteroaryl, wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0148] Even more preferably, R2is selected from phenyl, and heteroaryl, wherein said said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl.

[0149] Still more preferably, R2is heteroaryl, optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl. Particularly suitable heteroaryl are 5- and 6-membered heteroaryls comprising at least one N atom, preferably, 5-membered heteroaryls comprising at least one N atom. Thus, R2may be, for example, imidazolyl or pyrazolyl, preferably pyrazolyl, in particular pyrazol-5-yl, optionally substituted with one or more groups selected from -OH, halogen and Ci -6 alkyl. In a particularly preferred embodiment,

[0150] In this formula, R3and R4are each independently hydrogen or methyl. It is particularly preferred that R3is hydrogen and R4is methyl. It is particularly preferable that the compound comprises a moiety of formula:

[0151] As mentioned above, the antibody drug conjugate of the invention optionally includes a linker that connects the antibody (or antigen-binding fragment thereof), and the drug, i.e. ATR / CHK1 inhibitor. Accordingly, said linker is a bivalent moiety, wherein one empty valence of said linker is attached to the ATR / CHK1 inhibitor (including any specific examples and preferred embodiments of said ATR / CHK1 inhibitors, including attachment to any specific preferred points of attachment, as discussed herein), and wherein the second empty valence of said linker is attached to the antibody (or antigen-binding fragment thereof).

[0152] As referred to herein, linkers are not to be particularly limited and any linker conceivable to the skilled person as usable in the antibody-drug conjugate, can be used within the scope of the present invention.

[0153] An exemplary embodiment of an antibody-drug conjugate of the present invention comprises an antibody or an antigen-binding fragment thereof (Ab), an ATR / CHK1 inhibitor (D), and a linker moiety (L) that attaches Ab to D. In some embodiments, the antibody or its fragment as defined herein is attached to the linker moiety (L) through one or more amino acid residues, such as lysine and / or cysteine.

[0154] Accordingly, the linker may comprise one or more amino acid residue. Such a linker is thus referred to as peptide linker. Said peptide linker is understood to include two attachment points, also referred to as empty valences of the linker. It is highly preferred that said attachment points are to be formed through -COOH group on the C-terminus on the peptide and through -NH2 amino group on the N-terminus of the peptide. However, this is not meant to be limiting and the peptide linker may be attached to the antibody or the antigen-binding fragment thereof as well as to the ATR / CHK1 inhibitor in any way conceivable to the skilled person. Particularly preferred peptide linker comprises -GlyGlyPheGly- sequence (also referred to in a single letter sequence code as -GGFG- sequence). Insofar this embodiment to of the linker is concerned, preferably the left empty valence of the moiety is connected, directly or indirectly, to the antibody or the antigen- binding fragment thereof, and wherein the right empty valence is connected, directly or indirectly, to the ATR / CHK1 inhibitor. The direct attachment necessitates lack on any further chemical moieties between the parts of the conjugate to be connected, while indirect attachment allows for further chemical moieties to be present. The indirect attachment is thus consistent with the peptide linker comprising the amino acid sequence as defined above, which does not exclude the presence of further structural elements of said linker.

[0155] Thus, in one embodiment, the antibody-drug conjugate of the present invention comprises a moiety of formula:

[0156] According to the present invention, as it relates to the embodiment including the moiety of the aforementioned formula, it is particularly preferred that the left empty valence of the moiety is connected (directly or indirectly) to the antibody or the antigen-binding frag me nt thereof, and wherein the right empty valence is connected (directly or indirectly) to the ATR / CHK1 inhibitor.

[0157] The ATR / CHK1 inhibitor may preferably be a CHK1 inhibitor, as described herein. Thus, in one embodiment, the antibody-drug conjugate comprises moiety of formula: In accordance with the foregoing, the linker may further comprise a PEG moiety, which may be defined as - (O-CH2CH2)n- wherein n is an integer, preferably n is an integer from 1 to 10, more preferably n is an integer from 1 to 5, more preferably n is an integer from 2 to 4.

[0158] In one particularly preferred embodiment of the present invention, the antibody-drug conjugate comprises a moiety according to formula: wherein n is an integer from 1 to 5, preferably wherein n is 2 to 4, more preferably wherein n is 3. Accordingly, in this particularly preferred embodiment, the linker comprises -GGFG- amino acid sequence, and a PEG moiety, as defined hereinabove. It is particularly preferred that the left empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof, and wherein the right empty valence is connected to the ATR / CHK1 inhibitor. Thus, the ATR / CHK1 inhibitor is in this embodiment connected to the peptide part of the linker through the PEG moiety.

[0159] The ATR / CHK1 inhibitor may preferably be a CHK1 inhibitor, as described herein. Thus, in one embodiment, the antibody-drug conjugate comprises moiety of formula:

[0160] Further preferred peptide linkers include the following preferred amino acid sequences including GGNN (SEQ ID NO.: 9), GPVK (SEQ ID NO.: 10) GNLK (SEQ ID NO.: 11), FK, VC and VA. In accordance with the foregoing, the linker may further comprise a PEG moiety, which may be defined as -(O-CFhCFbjn- wherein n is an integer, preferably n is an integer from 1 to 10, more preferably n is an integer from 1 to 5, more preferably n is an integer from 2 to 4.

[0161] Further preferred peptide linkers include the following preferred amino acid sequences including GGGF (SEQ ID NO.: 12), GPVR (SEQ ID NO.: 13) GPLK (SEQ ID NO.: 14), and GLVK (SEQ ID NO.: 15). In accordance with the foregoing, the linker may further comprise a PEG moiety, which may be defined as - (O-CH2CH2)n- wherein n is an integer, preferably n is an integer from 1 to 10, more preferably n is an integer from 1 to 5, more preferably n is an integer from 2 to 4.

[0162] The linker in the antibody-drug conjugate of the present invention can be a cleavable linker. It is to be understood that said linker is configured to be cleaved after successful delivery of the antibody-drug conjugate into its destination, e.g. in the vicinity of the cancer cell, to the surface of the cancer cell or, in a particularly preferred embodiment, inside the cancer cell. In one embodiment, one may refer to the linker comprised in the antibody-drug conjugate of the present invention as self-immolative linker.

[0163] Particularly preferred self-immolative linker is described in the following. Accordingly, it is preferred that the antibody-drug conjugate of the present invention comprises a moiety according to formula:

[0164] Preferably, in the formula shown above the left empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof, and the right empty valence is connected to the ATR / CHK1 inhibitor.

[0165] More preferably, the antibody-drug conjugate comprises a moiety according to formula:

[0166] Preferably, in the formula shown above the left empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof, and the right empty valence is connected to the ATR / CHK1 inhibitor. Further according to the present invention, the antibody-drug conjugate may comprise a moiety selected from the following moieties:

[0167] It is to be understood that, preferably, the left empty valence is connected to the ATR / CHK1 inhibitor, and the right empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof.

[0168] In one embodiment, more than one linker moiety is attached to the antibody or antigen-binding fragment thereof. Accordingly, in such embodiment, the immunoconjugate can be described according to the formula A-(L-D)i, wherein i is an integer number, wherein i > 1. In one embodiment, i = 8, as in Enhertu, trastuzumab-based antibody-drug conjugate of deruxtecan.

[0169] The linker, as defined herein, may be attached to the antibody or antigen-binding fragment thereof in any way that is conceivable to the skilled person. One particularly preferred way is relying on the presence of surface-exposed cysteine residues in the antibody (or its fragment) and reacting the -SH moiety of said cysteine residue(s) with e.g. a Michael acceptor introduced into the precursor of linker, or a precursor of linker-ATR / CHK1 inhibitor. One particularly preferred Michael acceptor is based on N-succinimide moiety, which reacts with a cysteine residue according to the following reaction scheme:

[0170] It is thus preferred that the conjugate of the present invention is formed according to said reaction scheme. In addition, or alternatively, it is preferred that the conjugate of the present invention comprises a moiety of formula:

[0171] It is to be understood that the left empty valence of the moiety depicted above is connected to the antibody or antigen-binding fragment thereof, whereby the S atom originates from the cysteine that is preferably surface exposed in said antibody or antigen-binding fragment thereof.

[0172] In accordance with the foregoing, the antibody-drug conjugate of the present invention comprises in one embodiment the following moiety:

[0173] It is to be understood that the left empty valence of the moiety depicted above is connected to the antibody or antigen-binding fragment thereof, preferably to the S atom that originates from the cysteine that is preferably surface exposed in said antibody or antigen-binding fragment thereof. It is further to be understood that the right empty valence of the moiety depicted above is to be connected, directly or indirectly, to the ATR / CHK1 inhibitor.

[0174] Further in accordance with the foregoing, the antibody-drug conjugate of the present invention may comprise the following moiety:

[0175] It is to be understood that the left empty valence of the moiety depicted above is connected to the antibody or antigen-binding fragment thereof, preferably to the S atom that originates from the cysteine that is preferably surface exposed in said antibody or antigen-binding fragment thereof. It is further to be understood that the right empty valence of the moiety depicted above is to be connected, directly or indirectly, to the ATR / CHK1 inhibitor.

[0176] The antibody-drug conjugate of the present invention may also be defined through the process of obtaining the same. Accordingly, as mentioned before, the antibody-drug conjugate of the present invention is obtainable in the reaction of a compound of formula (III):

[0177] with a cysteine residue of the antibody or the antigen-binding fragment thereof.

[0178] More preferably, R1is selected from

[0179] Even more preferably,

[0180] In formula (III), R3and R4are each independently hydrogen or methyl. Preferably, R3is hydrogen and R4is methyl.

[0181] Preferably, the compound of formula (III) comprises a moiety of formula:

[0182] In formula (III), n is an integer from 1 to 5, preferably from 2 to 4, more preferably n is 3. Preferred compound of formula (III) is according to formula (Illa): The present invention further refers explicitly to the compound of formula (III), as defined hereinabove, or its salt, including any preferred definition of the compound of formula (III) or any specific embodiment of the compound of formula (III).

[0183] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound selected from:

[0184] with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0185] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0186] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula:

[0187] with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0188] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention. In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula:

[0189] with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0190] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0191] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula:

[0192] with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0193] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0194] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula:

[0195] with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0196] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0197] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0198] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula:

[0199] Phe with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0200] In another embodiment of the present invention, the antibody-drug conjugate is obtainable in a reaction of a compound according to formula: with a cysteine residue of the antibody or the antigen-binding fragment thereof. It is to be understood that the present invention also refers to any one of the compounds disclosed herein (as well as their salts), useful in obtaining the antibody-drug conjugate of the present invention.

[0201] As mentioned before, encompassed by the present invention are embodiments of the antibody-drug conjugate of the present invention wherein the ATR / CHK1 inhibitor is released inside the cell upon delivery through said conjugate. In one embodiment, the compound capable of being an ATR / CHK1 inhibitor is a compound of formula (II): or a pharmaceutically acceptable salt thereof.

[0202] In formula (II), R1is selected from

[0203] Preferably, R1is selected from

[0204] More preferably, R1is selected from Even more preferably,

[0205] In formula (II), R3and R4are each independently hydrogen or methyl.

[0206] Preferably, R3is hydrogen and R4is methyl.

[0207] Preferably, the compound of formula (II) comprises a moiety of formula:

[0208] In formula (II), n is an integer from 1 to 5, preferably from 2 to 4, more preferably n is 3.

[0209] In a particularly preferred embodiment, the compound of formula (II) is a compound of formula (Ila): or a pharmaceutically acceptable salt thereof.

[0210] In formula (Ila), n is an integer from 1 to 5 (accordingly, n can be 1, 2, 3, 4, or 5), preferably from 2 to 4 (thus, n can be 2, 3 or 4), more preferably n is 3.

[0211] The following definitions apply throughout the present specification and the claims, unless specifically indicated otherwise. The term “hydrogen” is herein used to refer to protium, deuterium and / or tritium, preferably to protium. Accordingly, the term “non-hydrogen atom” refers to any atoms that is not hydrogen, i.e. that is not protium, deuterium or tritium.

[0212] The term “hydrocarbon group” refers to a group consisting of carbon atoms and hydrogen atoms.

[0213] The term “alicyclic” is used in connection with cyclic groups and denotes that the corresponding cyclic group is non-aromatic.

[0214] As used herein, the term “alkyl” refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to- carbon double bond or any carbon-to-carbon triple bond. A “C1-5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term “alkyl” preferably refers to C1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.

[0215] As used herein, the term “alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term “C2-5 alkenyl” denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-

[0216] 1-en-1-yl, prop-1 -en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1 ,3-dien-

[0217] 2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term “alkenyl” preferably refers to C2-4 alkenyl.

[0218] As used herein, the term “alkynyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term “C2-5 alkynyl” denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term “alkynyl” preferably refers to C2- 4 alkynyl.

[0219] As used herein, the term “alkylene” refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A “Ci -5 alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the term “C0-3 alkylene” indicates that a covalent bond (corresponding to the option “Co alkylene”) or a C1-3 alkylene is present. Preferred exemplary alkylene groups are methylene (- CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH3)-), propylene (e g., -CH2-CH2-CH2-, -CH(-CH2-CH3)-, -CH2- CH(-CH3)-, or -CH(-CH3)-CH2-), or butylene (e.g., -CH2-CH2-CH2-CH2-). Unless defined otherwise, the term “alkylene” preferably refers to C1.4 alkylene (including, in particular, linear Ci.4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.

[0220] As used herein, the term “alkenylene” refers to an alkenediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon- to-carbon double bonds while it does not comprise any carbo n-to-carbon triple bond. A “C2-5 alkenylene” denotes an alkenylene group having 2 to 5 carbon atoms. Unless defined otherwise, the term “alkenylene” preferably refers to C2-4 alkenylene (including, in particular, linear C2-4 alkenylene).

[0221] As used herein, the term “alkynylene” refers to an alkynediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon- to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. A “C2-5 alkynylene” denotes an alkynylene group having 2 to 5 carbon atoms. Unless defined otherwise, the term “alkynylene” preferably refers to C2-4 alkynylene (including, in particular, linear C2-4 alkynylene).

[0222] As used herein, the term “carbocyclyl” refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, “carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.

[0223] As used herein, the term “heterocyclyl” refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise, “heterocyclyl” preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.

[0224] As used herein, the term “aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and / or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). “Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1 ,2-dihydronaphthyl), tetralinyl (i.e., 1,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H- fluorenyl, or azulenyl. Unless defined otherwise, an “aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.

[0225] As used herein, the term “heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and / or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. “Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1- benzopyranyl or 4H-1 -benzopyranyl), isochromenyl (e.g., 1 H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1 H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3- pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, p-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [1 ,10]phenanthrolinyl, [1 ,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, or 1 ,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[1 ,5-a]pyrimidinyl (e.g., pyrazolo[1,5-a]pyrimidin-3-yl), 1,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., 1 H-1 ,2,3-triazolyl, 2H-1,2,3-triazolyl, 1 H-1 ,2,4-triazolyl, or 4H-1 ,2,4-triazolyl), benzotriazolyl, 1 H-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1,2,3-triazinyl, 1 ,2,4-triazinyl, or 1 ,3,5-triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or 1 ,3-dihydrofuro[3,4- cjpyridinyl), imidazopyridinyl (e.g., imidazo[1 ,2-a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1,3-benzodioxolyl, benzodioxanyl (e.g., 1 ,3-benzodioxanyl or 1 ,4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term “heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.

[0226] As used herein, the term “cycloalkyl” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). “Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, “cycloalkyl” preferably refers to a Ca n cycloalkyl, and more preferably refers to a C3-7 cycloalkyl. A particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members (e.g., cyclopropyl or cyclohexyl).

[0227] As used herein, the term “heterocycloalkyl” refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom- containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. “Heterocycloalkyl” may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl (e.g., 1 ,4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholine- yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 1,3-dioxolanyl, tetrahydropyranyl, 1 ,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl), 1 ,3-dithiolanyl, thianyl, 1,1-dioxothianyl, thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl. Unless defined otherwise, “heterocycloalkyl” preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, “heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.

[0228] As used herein, the term “cycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to- carbon double bonds and does not comprise any carbon-to-carbon triple bond. “Cycloalkenyl” may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, “cycloalkenyl” preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3.7 cycloalkenyl. A particularly preferred “cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.

[0229] As used herein, the term “halogen” refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-1). As used herein, the term “haloalkyl” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. “Haloalkyl” may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. A particularly preferred “haloalkyl” group is -CF3.

[0230] The terms “bond” and “covalent bond” are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.

[0231] As used herein, the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent. Whenever the term “optional”, “optionally” or “may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression “X is optionally substituted with Y” (or “X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be “optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.

[0232] Various groups are referred to as being “optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the “optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.

[0233] A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples. As used herein, unless explicitly indicated otherwise or contradicted by context, the terms “a”, “an” and “the” are used interchangeably with “one or more” and “at least one”. Thus, for example, a composition comprising “a” compound of formula (I) can be interpreted as referring to a composition comprising “one or more” compounds of formula (I).

[0234] It is to be understood that wherever numerical ranges are provided / disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, as well as each subrange encompassed by a numerical range disclosed herein.

[0235] As used herein, the term “about” preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term “about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint.

[0236] As used herein, the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e., “containing, among further optional elements, ...”. In addition thereto, this term also includes the narrower meanings of “consisting essentially of’ and “consisting of’. For example, the term “A comprising B and C” has the meaning of “A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., “A containing B, C and D” would also be encompassed), but this term also includes the meaning of “A consisting essentially of B and C” and the meaning of “A consisting of B and C” (i.e., no other components than B and C are comprised in A).

[0237] The term “amino acid” refers, in particular, to any one of the 20 standard proteinogenic o-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) but also to non-proteinogenic and / or non-standard a-amino acids (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group such as, e.g., cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine, or phenylglycine) as well as p-amino acids (e.g., P-alanine), y-amino acids (e.g., y-aminobutyric acid, isoglutamine, or statine) and / or b-amino acids as well as any other compound comprising at least one carboxylic acid group and at least one amino group. Unless defined otherwise, an “amino acid” preferably refers to an a-amino acid, more preferably to any one of the 20 standard proteinogenic a-amino acids (which can be present as the L-isomer or the D- isomer, and are preferably present as the L-isomer). The term “amino acid” may also refer to a monovalent or divalent radical derived from an “amino acid” as described herein above, wherein preferably an amino group and / or a carboxylic acid group may serve as point(s) of attachment, preferably through an amide bond. Thus, the term “amino acid” may refer to an amino acyl moiety attached to the rest of the molecule, e.g., through a -CO- group formed from its carboxylic acid group.

[0238] The term “peptide” refers to a polymer of two or more amino acids linked via amide bonds that are formed between an amino group of one amino acid and a carboxylic acid group of another amino acid. The amino acids comprised in the peptide or protein, which are also referred to as amino acid residues, may be selected from the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) but also from non-proteinogenic and / or non- standard a-amino acids (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group such as, e.g., cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine, or phenylglycine) as well as 0-amino acids (e.g., p-alanine), y-amino acids (e.g., y- aminobutyric acid, isoglutamine, or statine) and b-amino acids. Preferably, the amino acid residues comprised in the peptide or protein are selected from a-amino acids, more preferably from the 20 standard proteinogenic a-amino acids (which can be present as the L-isomer or the D-isomer, and are preferably all present as the L-isomer). The peptide may be unmodified or may be modified, e.g., at its N-terminus, at its C-terminus and / or at a functional group in the side chain of any of its amino acid residues (particularly at the side chain functional group of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and / or Arg residues). Such modifications may include, e.g., the attachment of any of the protecting groups described for the corresponding functional groups in: Wuts PG & Greene TW, Greene’s protective groups in organic synthesis, John Wiley & Sons, 2006. Such modifications may also include the covalent attachment of one or more polyethylene glycol (PEG) chains (forming a PEGylated peptide), the glycosylation and / or the acylation with one or more fatty acids (e.g., one or more Cs-3o alkanoic or alkenoic acids; forming a fatty acid acylated peptide or protein). Moreover, such modified peptides or proteins may also include peptidomimetics, provided that they contain at least two amino acids that are linked via an amide bond (formed between an amino group of one amino acid and a carboxyl group of another amino acid). The amino acid residues comprised in the peptide or protein may, e.g. , be present as a linear molecular chain (forming a linear peptide) or may form one or more rings (corresponding to a cyclic peptide). The peptide may also form oligomers consisting of two or more identical or different molecules. The term “peptide” may also refer to a monovalent radical derived from a “peptide” as described herein above, wherein preferably an amino group or a carboxylic acid group, in particular the N-terminal amino group or the C- terminal carboxylic acid group, serves as point of attachment, preferably through an amide bond. Thus, the term “peptide” may refer to a peptidyl moiety attached to the rest of the molecule through a -CO- group (formed from a carboxylic acid group, e.g., from its C-terminal carboxylic acid group).

[0239] The scope of the invention embraces all salts, in particular pharmaceutically acceptable salt forms of the dislosed compounds which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2- hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2 naphthalenesulfonate (napsylate), 3 phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. Preferred pharmaceutically acceptable salts of the compounds of the invention include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically acceptable salt of the compounds of the invention is a hydrochloride salt. Accordingly, it is preferred that the compound disclosed herein, including any one of the specific compounds described herein, is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that the compound of the invention is in the form of a hydrochloride salt. The present invention also specifically relates to any of the compounds described herein, in its non- salt form.

[0240] Throughout the present description, when a reference to a specific compound is made, this reference encompasses said compound, as well as its salts, in particular pharmaceutically acceptable salts thereof. In other words, any reference to a compound is meant to refer to “a compound or its salt”, preferably “a compound or its pharmaceutically acceptable salt’.

[0241] Pharmaceutical compositions

[0242] The present invention further relates to a pharmaceutical composition comprising the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier.

[0243] Pharmaceutical formulations of antibody-drug conjugate of the present invention as described herein are prepared by mixing such antibody-drug conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA or GLDA; sugars such as sucrose, mannitol, trehalose or sorbitol; osmo-protectants like ectoin; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0244] The term "pharmaceutical formulation" or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0245] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0246] Exemplary lyophilized antibody-drug conjugate formulations are described in US Patent No. 6,267,958. Aqueous antibody-drug conjugate formulations include those described in US Patent No. 6,171,586 and W02006 / 044908, the latter formulations including a histidine-acetate buffer.

[0247] The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

[0248] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0249] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody-drug conjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

[0250] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0251] In one embodiment, the present invention relates to the antibody-drug conjugate of the present invention or pharmaceutical composition of the present invention for use as a medicament. In other words, the present invention relates to the antibody-drug conjugate of the present invention or pharmaceutical composition of the present invention for use in therapy. It is to be understood that the antibody-drug conjugates or the pharmaceutical compositions of the present invention can be used in the treatment of a disease or a disorder.

[0252] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0253] An antibody-drug conjugate of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0254] Antibody-drug conjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody-drug conjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody-drug conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0255] For the prevention or treatment of disease, the appropriate dosage of an antibody-drug conjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or immunoconjugate, the severity and course of the disease, whether the antibody or immunoconjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody or antibody-drug conjugate, and the discretion of the attending physician. The antibody-drug conjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg-10 mg / kg) of antibody-drug conjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody-drug conjugate would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0256] In a particular embodiment of the present invention, the antibody-drug conjugate of the present invention is provided for use in the treatment of cancer. Preferably, the cancer is HER2-positive cancer, CD30-positive cancer, CD33-positive cancer, CD22- positive cancer, CD79b-positive cancer, Nectin-4-positive cancer, Trop2-positive cancer, BCMA-positive cancer, EGFR-positive cancer, CD 19-positive cancer, Fra-positive cancer, or B7H4-positive cancer. It is further preferred, in addition or in alternative, that the cancer is characterized by DNA-repair deficiency.

[0257] Preferably, the cancer is selected from breast cancer, gastric cancer, lung cancer (e.g. non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, and pancreatic cancer.

[0258] The present invention further relates to use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention in the manufacture of the medicament for the treatment of cancer. The cancer is preferably as described hereinabove.

[0259] The present invention also relates to a method of treating a cancer in a subject, the method comprising the step of administering the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention to a subject in need thereof. It is to be understood that, preferably, a therapeutically effective amount is to be administered.

[0260] Antibody-drug conjugates of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody-drug conjugate of the invention may be co-administered with at least one additional therapeutic agent.

[0261] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody or immunoconjugate of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant. Antibody-drug conjugates of the invention can also be used in combination with radiation therapy.

[0262] Preferably, said antibody-drug conjugates to be administered to a subject with an additional therapeutic agent, selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP and PARG inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, immune checkpoint inhibitors, immune checkpoint therapies and bisphosphonate therapy agent, preferably selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP and PARG inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.

[0263] Preferably, said antibody-drug conjugateof the invention is to be administered to a subject with an additional therapeutic agent, wherein said additional therapeutic agent may be an agent sensitizing the cells to radiotherapy, for example selected from protein kinase inhibitor and DNA intercalating agent. Preferably, protein kinase inhibitor is selected from Alisertib, MK1775, MK2206, Saracatinib, Temsirolimus, Crizotinib, Ceritinib, Alectinib, Brigatinib, Bosutinib, Dasatinib, Imatinib, Nilotinib, Ponatinib, Vemurafenib, Dabrafenib, Ibrutinib, Ibrutinib, Palbociclib, Sorafenib, Ribociclib, Crizotinib, Cabozantinib, Gefitinib, Erlotinib, Lapatinib, Vandetanib, Afatinib, Osimertinib, Ruxolitinib, Tofacitinib, Trametinib, Axitinib, Gefitinib, Imatinib, Lenvatinib, Nintedanib, Pazopanib, Regorafenib, Sorafenib, Sunitinib, Vandetanib, Bosutinib, Dasatinib, Ponatinib, Vandetanib, Axitinib, Lenvatinib, Nintedanib, Regorafenib, Pazopanib, Sorafenib, and Sunitinib, more preferably selected from Alisertib, MK1775, MK2206, Saracatinib, and Temsirolimus. Preferably, the DNA intercalating agent is selected from Doxorubicin and Nemorubicin.

[0264] Further, particularly preferred embodiments of the present invention are disclosed in the following numbered items.

[0265] 1 . An antibody-drug conjugate, comprising an antibody or antigen-binding fragment thereof, and an ATR / CHK1 inhibitor.

[0266] 2. The antibody-drug conjugate of item 1 , wherein the antibody or antigen-binding fragment thereof is capable of targeting the antibody-drug conjugate to cancer cells.

[0267] 3. The antibody-drug conjugate of item 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds to the same epitope as trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, Mirvetuximab, Datopotamab and Telisotuzumab preferably wherein the antibody or antigen-binding fragment thereof is selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, Mirvetuximab, Datopotamab and Telisotuzumab. The antibody-drug conjugate of any one of items 1 to 3, wherein the antibody-drug conjugate comprises trastuzumab or sacituzumab. The antibody-drug conjugate of any one of items 1 to 4, wherein the antibody-drug conjugate comprises trastuzumab. The antibody-drug conjugate of any one of items 1 to 5, wherein the ATR / CHK1 inhibitor is an ATR inhibitor. The antibody-drug conjugate of item 6, wherein the ATR inhibitor is Elimusertib or its derivative. The antibody-drug conjugate of item 6 or 7, wherein the ATR inhibitor is according to formula (I):

[0268] R2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -O-(C1-6 alkyl), -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-(C1-6 alkyl), -N(C1-6 alkyl)-(C1-6 alkyl), - C00-(C1-6 alkyl), -C0NH-(C1-6 alkyl), -C0N(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -S0-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHS02-(CI-B alkyl), and -N(C1-6 alkyl)-S02-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl; and R3and R4are each independently hydrogen or methyl. The antibody-drug conjugate of item 8, wherein

[0269]

[0270] R3is hydrogen and R4is methyl. 10. The antibody-drug conjugate of item 9, wherein the compound of formula (I) is a compound of formula (la):

[0271] (la).

[0272] 11. The antibody-drug conjugate of any one of items 1 to 10, wherein the antibody-drug conjugate comprises a moiety of formula (lb): or the antibody-drug conjugate comprises a moiety according to formula (Ic):

[0273] wherein: the empty valence is connected to the antibody or antigen-binding fragment thereof;

[0274] R1is selected from

[0275] R2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -0-(C1-6 alkyl), -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-(C1-6 alkyl), -N(C1-6 alkyl)-(C1-6 alkyl), - C00-(C1-6 alkyl), -C0NH-(C1-6 alkyl), -C0N(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -S0-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHS02-(C1-6 alkyl), and -N(C1-6 alkyl)-SO2-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl; and R3and R4are each independently hydrogen or methyl. The antibody-drug conjugate of item 11, wherein the antibody-drug conjugate comprises a moiety according to formula (lb), wherein

[0276] R3is hydrogen and R4is methyl. The antibody-drug conjugate of item 12, wherein the antibody-drug conjugate comprises a moiety according to formula (I b-1 ):

[0277]

[0278] (lb-1). The antibody-drug conjugate of item 11, wherein the antibody-drug conjugate comprises a moiety according to formula (Ic), wherein:

[0279] R3is hydrogen and R4is methyl. The antibody-drug conjugate of item 14, wherein the antibody-drug conjugate comprises a moiety according to formula (I c- 1 ): The antibody-drug conjugate of item 6, wherein the ATR inhibitor is selected from Elimusertib, Camonsertib, Ceralasertib, Gartisertib, Tuvusertib and Berzosertib. The antibody-drug conjugate of item 16, wherein the ATR inhibitor is selected from Elimusertib and Camonsertib. The antibody-drug conjugate of item 16 or 17, wherein the ATR inhibitor is Camonsertib. The antibody-drug conjugate of item 18, wherein the antibody-drug conjugate comprises a moiety according to formula: The antibody-drug conjugate of item 16, wherein the ATR inhibitor is Berzosertib. The antibody-drug conjugate of item 16, wherein the antibody-drug conjugate comprises a moiety according to formula: The antibody-drug conjugate of any one of items 1 to 5, wherein the ATR / CHK1 inhibitor is a

[0280] CHK1 inhibitor. The antibody-drug conjugate of item 22, wherein the CHK1 inhibitor is: The antibody-drug conjugate of any one of items 1 to 23, wherein the antibody-drug conjugate comprises a moiety according to formula: wherein n is an integer from 1 to 5, preferably wherein n is 2 to 4, more preferably wherein n is 3, preferably wherein the left empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof, and wherein the right empty valence is connected to the ATR / CHK1 inhibitor. The antibody-drug conjugate of any one of items 1 to 23, wherein the antibody-drug conjugate comprises a moiety according to formula: preferably wherein the antibody-drug conjugate comprises a moiety according to formula:

[0281] preferably wherein the left empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof, and wherein the right empty valence is connected to the ATR / CHK1 inhibitor. The antibody-drug conjugate of any one of items 1 to 23, wherein the antibody-drug conjugate is obtainable in a reaction of a compound selected from: with a cysteine residue of the antibody or the antigen-binding fragment thereof. A compound of formula (III): or a pharmaceutically acceptable salt thereof, wherein:

[0282] R3and R4are each independently hydrogen or methyl, and n is an integer from 1 to 5, preferably n is 3.

[0283] The compound of item 27, of formula (Illa): or a pharmaceutically acceptable salt thereof. A compound of formula (II):

[0284]

[0285] R3and R4are each independently hydrogen or methyl, and n is an integer from 1 to 5, preferably from 2 to 4, more preferably n is 3. The compound of item 29, wherein the compound is according to formula: A pharmaceutical composition comprising the antibody-drug conjugate of any one of items 1 to

[0286] 26, and a pharmaceutically acceptable carrier. The antibody-drug conjugate of any one of items 1 to 26 or the pharmaceutical composition of item 31 for use as a medicament. The antibody-drug conjugate of any one of items 1 to 26 or the pharmaceutical composition of item 31 for use in the treatment of cancer. 34. The antibody-drug conjugate for use of item 33 or the pharmaceutical composition for use of item 30, wherein the cancer is HER2-positive cancer, CD30-positive cancer, CD33-positive cancer, CD22-positive cancer, CD79b-positive cancer, Nectin-4-positive cancer, Trop2-positive cancer, BCMA-positive cancer, EGFR-positive cancer, CD 19-positive cancer, Fra-positive cancer or B7H4-positive cancer.

[0287] 35. The antibody-drug conjugate for use of item 33 or 34, or the pharmaceutical composition for use of item 33 or 34, wherein the cancer is characterized by DNA-repair deficiency.

[0288] 36. The antibody-drug conjugate for use of any one of items 33 to 35, wherein the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, and pancreatic cancer.

[0289] Examples

[0290] The invention will be illustrated in the following Examples which, however, are not meant to be construed as limiting. It is noted that, unless explicitly indicated to the contrary, the present invention may relate to each and every compound and every antibody-drug conjugate, as well as every intermediate as disclosed in the Examples.

[0291] Example 1 : Methods to assay the activity of ATR and CHK1 inhibitors suitable as ADC payloads.

[0292] The present invention focuses on the protein kinases ATR and CHK1 that function as checkpoints in cells experiencing DNA replication stress. The activities of compounds that will serve as ADC payloads are examined in biochemical and cell-based assays. These assays are described below. Example 2 shows data generated using these assays.

[0293] Biochemical assays

[0294] A biochemical assay to monitor the kinase activity of ATR is performed using recombinant, purified human ATR / ATRIP protein complex incubated in buffer containing GST-cMyc-p53 and Mg / ATP (cat# 14-953KP Eurofins). Phosphorylation of a p53 substrate is monitored by homogeneous time-resolved fluorescence. A biochemical assay to monitor the kinase activity of CHK1 is performed using recombinant, purified human CHK1 protein incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 pM KKKVSRSGLYRSPSMPENLNRPR (SEQ ID NO.: 4), 10 mM Mg Acetate and [gamma-33P]-ATP. An aliquot of the reaction is then spotted onto a filter and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting (cat# 14-346KP Eurofins).

[0295] Dose-response curves for the tested compounds are obtained and IC50 values calculated.

[0296] The compounds are tested in duplicates at final concentrations ranging from 0.0001 pM to 1 pM using three-fold dilution steps. Averaged values of the samples are normalized to control. Curves were fit as % of the control vs. log of the compound concentration.

[0297] Cellular assays

[0298] Inhibition of ATR and CHK1 activity is measured in ATR-CHK1 inhibition-sensitive U2OS osteosarcoma cells overexpressing cyclin E upon treatment with ATR or CHK1 inhibitors. Cells are incubated with drugs for a given time (between 4 - 24 h; preferably for 16 h) at 37°C in a cell culture incubator, before being fixed with a fixative agent. After incubating the cells with anti-phospho-Histone H2A.X antibody (Millipore / FlowCellect™ Histone H2A.X Phosphorylation Assay Kit; according to the instructions of the manufacturer), histone H2AX phosphorylation is quantified by flow cytometry.

[0299] The cytotoxicity of ATR and CHK1 inhibitors is assessed in a cellular viability assay. SKOV3, ovarian cancer cells, which are sensitive to ATR or CHK1 -inhibition, are plated in 96-well black plates with a clear flat bottom. After allowing the cells to adhere to the plates for 24 h, inhibitors are dispensed in triplicates. After 96 h of incubation, the number of viable cells is determined using Cell Titer-Gio (cat#G9243 Promega) and cellular viability EC50 values of the inhibitors are calculated.

[0300] Cellular Viability Assay for CHK1 inhibitors

[0301] Inhibition of CHK1 activity is measured in CHK1 inhibition-sensitive HCC1569 breast cancer cells upon treatment with CHK1 inhibitors. The cell line is cultured according to the manufacturer specifications. HCC1569 cells were plated at 2500 cells / well, in 96-well white plates with clear flat bottom. The outer wells of the plate are excluded and filled with PBS (ThermoFisher Scientific) to compensate evaporation mediated effects in the plate periphery. After 24 hours, the compounds are added manually, in duplicates, af final concentrations ranging from 0.015 nM to 1000 nM using four-fold dilution steps. Cells were treated for 120 hrs. At the end of the treatment, 150 pl of the growth medium are removed and 50 pl of Cell Titer- Glo (Promega) are added per well. Following an incubation of 10 minutes, luminescence is read using a plate reader (Tecan). Averaged values of the samples are normalized to DMSO treated control samples. Curves are fit as % of the control vs. log of the compound concentration using a 4-parameter log-logistic function:

[0302] Example 2: Identification of exit vectors suitable for development of the ATR inhibitor E lim usertib as an Antibody-Drug Conjugate (ADC) payload.

[0303] Variants of Elimusertib (BAY1895344) were synthesized to identify suitable derivatives, which can be used as ADC payloads (Fig. 1A). Specifically, linkers were attached at various positions, called “exit vectors”, of BAY1895344. The generated compounds were tested for activity in biochemical enzymatic activity assays and in cell-based assays. Compounds that maintain activity define the exit vectors that are compatible with development of these compounds as ADC payloads.

[0304] A biochemical assay to monitor the kinase activity of ATR was performed using recombinant, purified human ATR / ATRIP protein complex; phosphorylation of a p53 substrate was monitored by homogeneous time-resolved fluorescence. A small number of ATR compounds were tested to demonstrate feasibility of this approach; dose-response curves for the tested compound were obtained and IC50 values were calculated. Two of the three BAY1895344 derivative compounds tested maintained inhibitory activity (Fig. 1B). In this assay, due to the high ATP concentrations used, the calculated I C50 values are higher than those reported in the literature; however, the assay is still suitable for comparing the activities of the derivative compounds to the original BAY1895344 compound.

[0305] Next, the activity of derivative BAY1895344 inhibitors was tested in a cell-based assay. This assay involves exposing cells experiencing oncogene-induced DNA replication stress to an ATR inhibitor and monitoring the induction of a DNA damage response. Specifically, DNA replication stress was induced in U2OS osteosarcoma cells by overexpressing cyclin E; these cells were treated for 16 h with either unmodified BAY1895344 or BAY1895344-derivative compounds over a compound concentration range of 10-400 nM. The cells were fixed, immunostained for the DNA damage response marker phosphorylated H2AX (yH2AX), counterstained with propidium iodide to monitor genomic DNA content, and analyzed by flow cytometry. In this assay, inhibition of ATR leads to high levels of yH2AX; therefore, the fraction of cells scoring positive for yH2AX can be used as a surrogate for ATR inhibition (Fig. 1C). As compared to unmodified BAY1895344, three out of six BAY1895344 derivatives tested, retained activity in the nM range (Fig. 1 D,E). A structure-activity relationship analysis of the compounds retaining activity in the biochemical and cell-based assays, identified exit vectors suitable for development of BAY1895344 as ADC payloads (Fig. 1A).

[0306] The same strategy described here for Elimusertib can be applied to other ATR inhibitors, as well as to Chk1 inhibitors.

[0307] Example 3: Synthesis of ATR inhibitors serving as ADC payloads.

[0308] Derivatives of the ATR inhibitor Elimusertib (BAY1895344), which can be used as ADC payloads, were synthesized.

[0309] Synthetic scheme for ATR inhibitor HALA-003

[0310] Experimental Procedure:

[0311] Step-1 : (R)-1-(3-methylmorpholino)ethan-1-one (2):

[0312] AcCI, K2CO3, DCM, RT Step-1 To a solution of (R)-3-methylmorpholine (6.0 g, 59.3 mmol), in DCM (80 ml) was added Potassium carbonate (16.37 g, 118.6 mmol) at 0 °C, after 15 min was added Acetyl chloride (6.98 g, 88.9 mmol) at same temperature. The suspension was stirred at room temperature for 16 h. After completion of reaction, the reaction mixture was diluted with water and extracted with DCM (30 mL x 2 times). The organic phase was dried with sodium sulfate and concentrated under reduced pressure. The crude product was used next step without purification. Finally 8.2 g of crude product (R)-1-(3-methylmorpholino)ethan-1-one (2) (8.2 g) was obtained as a thick brown oil.

[0313] Step-2: methyl(R,E)-2-chloro-3-((1 -(3-methylmorpholino)ethylidene)amino)isonicotinate (4):

[0314] To a stirred solution of compound-2 (8.2 g, 57.2 mmol) in a 1 ,2-dichloroethane (100 ml) was added POCI3 (26.34 g, 171 mmol) at O°C. After 10 min was added compound-3 (10 g, 57.2 mmol) in DCM for 10 min and the progress of the reaction mixture was warmed to 80 °C and stirred for Overnight. After completion of reaction the suspension was quenched with saturated sodium bicarbonate solution (basify PH-8) then extracted with DCM (30 mL x 3 times). The combined organics were dried with sodium sulfate and concentrated under reduced pressure. Finally 19.5 g of crude product methyl(R,E)-2-chloro-3-((1-(3- methylmorpholino)ethylidene)amino)isonicotinate (4) (19.5 g, 68 %) was obtained as a brown oil.

[0315] Step 3: (R)-8-chloro-2-(3-methylmorpholino)-1,7-naphthyridin-4-ol (5):

[0316] To a stirred solution of compound 4 (19.5 g, 62.6mmol) in Dry THF was allowed to cool to 0 °C, then added LiHMDS (31.42 g, 187mmol) dropwise for 20 min. The reaction mixture was warmed to room temperature and stirred for 4 h. After completion of reaction the suspension was diluted with water and removed THF from the reaction then added 100 ml saturate NH4CI solution and then extracted with 20% IPA in DCM (40 ml_ x 4 times). Separated organic layer was dried with sodium sulfate and concentrated at 20 °C. The combined organic part was concentrated and purified by combi column to afford the desired product. Accordingly, 5.6 g of pure product (R)-8-chloro-2-(3-methylmorpholino)-1,7-naphthyridin-4- ol (5) (5.6 g, 47 %) was obtained as a brown solid.

[0317] Step 4: 2-((R)-3-methylmorpholino)-8-(1 -(tetrahvdro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7- naphthyridin-4-ol (7)

[0318] To a stirred solution of compound-5 (5.6 g, 20 mmol) in 1 ,4-Dioxane (60 ml) was added compound-6 (7.24 g, 26 mmol) and cesium carbonate (26.1 g, 80.1 mmol) then degassed with nitrogen for 15 min then added palladium complex (Pd(dppf)Cl2) (1.636 g, 2 mmol), Then again degassed with nitrogen for 15 min then refluxed at 80 °C for 3h. After completion of reaction the RM was filtered through celite and washed with 10% MeOH in DCM (10 mL x 3 times). The organic layer dried with sodium sulfate and concentrated. The crude product was purified through combi-flash column. Finally, 5.6 g of pure product 2-((R)-3- methylmorpholino)-8-(1 -(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1,7-naphthyridin-4-ol (7) (5.6 g, 70.7 %) was obtained as a yellow solid.

[0319] Step-5: 2-((R)-3-methylmorpholino)-8-(1 -(tetrahvdro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7- naphthyridin-4-yl trifluoromethanesulfonate (9):

[0320] To a stirred solution of compound- 7 (5.6 g, 14.17 mmol) in THF (100 ml) was added DI PEA (4.59 g, 35.42 mmol) and Intermediate-8 (7.08 g, 19.83 mmol) at room temperature then heated to 60 °C and stirred for 2.5 h. After completion of reaction, the RM was cooled to RT then added 50 ml water and extracted with DCM (30 mL x 3 times). The organic layer was dried with sodium sulfate and concentrated. The crude product was purified through combi-flash column. Finally, 4.8 g of pure product 2-((R)-3- methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)-1,7-naphthyridin-4-yl trifluoromethanesulfonate (9) (4.8 g, 64.2 %) was obtained as a yellow solid.

[0321] Step-6: 2-((tert-butoxycarbonyl)(methyl)amino)ethyl methanesulfonate (11):

[0322] To a stirred solution of compound- 10 (6 g, 34.7mmol) in Dry DCM (80 ml) was added DIPEA (9.9 g, 76.52mmol) at 0°C then stirred for 15 min, was added Methane sulfonyl chloride (4.9 g, 43.4 mmol) at 0°C and stirred for 2.5 h. After completion of reaction the RM was quenched with saturated NaHCOa solution and extracted with DCM (50 ml_ x 3 times). The organic was dried with sodium sulfate and concentrated. The crude product was directly used for the next step without purification. Finally, 8.2 g of pure product 2- ((tert-butoxycarbonyl)(methyl)amino)ethyl methanesulfonate (11) (8.2 g, 93 %) was obtained as a colorless oil.

[0323] Step-7: tert-butyl (2-(1H-pyrazol-1-yl)ethyl)(methyl)carbamate (13):

[0324] To a stirred solution of compound-12 (2.84 g, 41.7mmol) in Dry THF (50 ml) was added NaH (0.83 g, 34.7mmol) at 0 °C then stirred for 15 min, was added Compound-11 (8.2 g, 32.4 mmol) at 0 °C. The reaction was heated to 60 °C and stirred for 16 h. After completion of reaction the RM was quenched with saturated NH4CI solution and extracted with DCM (10 mL x 3 times). The organic was dried with sodium sulfate and concentrated. The crude product was purified through column (neutral Alumina). Finally 6 g of pure product tert-butyl (2-(1H-pyrazol-1-yl)ethyl)(methyl)carbamate (13) (6 g, 82.3 %) was obtained as a colorless oil. Step-8: tert-butyl methyl(2-(5-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazol-1 ■ yl)ethyl)carbamate (15):

[0325] 13 Compound-15

[0326] To a stirred solution of compound-13 (2.6 g, 1.10mmol) in Dry THF (15 ml) was added n-Butyl Lithium (1 .5 g, 2.20mmol) at -78 °C dropwise for 10 min then stirred for 30 min, was added Compound-14 (4.28 g, 2.20 mmol) at -78 °C. The reaction mixture was stirred for 1 h at -78 °C. After completion of reaction the RM was quenched with saturated NH4CI solution at -50 °C and extracted with EtOAc (10 mL X 3 times). The organic was dried with sodium sulfate and concentrated. The crude product was used to next step without purification. 3.2 g of crude product tert-butyl methyl(2-(5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1 H-pyrazol-1-yl)ethyl)carbamate (15) (3.2 g) was obtained as a yellow oil.

[0327] Step 9: tert-butyl methyl(2-(5-(2-((R)-3-methylmorpholino)-8-(1-(tetrahvdro-2H-pyran-2-yl)-1H- Pyrazol-5-yl)-1,7-naphthyridin-4-yl)-1H-pyrazol-1-yl)ethyl)carbamate (16):

[0328] To a stirred solution of compound-9 (1.2 g, 2.2 mmol) in 1 ,4-Dioxane (30 ml) was added compound-15 (3.6 g, 10.2 mmol) and cesium carbonate (2.96 g, 9.1 mmol) then degassed with nitrogen for 15 min then added palladium complex Pd(dppf)Cl2 (120 mg, 0.14 mmol), Then again degassed with nitrogen for 15 min then stirred at room temperature for 4h. After completion of reaction to the reaction mixture added water and extracted with DCM (10 mL x 3 times). The organic was dried with sodium sulfate and concentrated. The crude product was purified through C-18 reverse phase column. The product was eluted with 60% ACN in 1 % ammonium acetate solvent system & eluted product was extracted with DCM and dried with sodium sulfate and concentrated. Finally, 250 mg of pure product tert-butyl methyl(2-(5- (2-((R)-3-methylmorpholino)-8-(1 -(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4- yl)-1H-pyrazol-1-yl)ethyl)carbamate (16) (0.25 g) was obtained as a brown gummy liquid.

[0329] Step-10: (R)-N-methyl-2-(5-(2-(3-methylmorpholino)-8-(1H-pyrazol-5-yl)-U-naphthyridin-4-yl)-1H-

[0330] To a stirred solution of compound- 16 (250 mg, 0.41) in Dry THF (10 ml) was added PTSA.H2O (475 mg, 2.4mmol) at 0 °C. The progress of reaction mixture was monitored by LCMS at 0 °C to 20 °C for 48 h. After completion of reaction the RM was quenched with water and basified with saturated NaHCCh solution and extracted with DCM (5 mL x 3 times). The organic was dried with sodium sulfate and concentrated. The crude product was purified through preparative HPLC method. 35 mg of pure product (R)-N-methyl-2-(5-(2-(3-methylmorpholino)-8-(1 H-pyrazol-5-yl)-1,7-naphthyridin-4-yl)-1H-pyrazol- 1 -yl)ethan-1 -amine ( HALA-003) (35 mg, 20 %) was obtained as a yellow solid. Synthetic scheme for ATR inhibitor HALA-008

[0331] Synthesis of lnt-10:

[0332]

[0333] Experimental Procedure:

[0334] To a stirred solution of 2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7- naphthyridin-4-yl trifluoromethanesulfonate (lnt-9) (0.5 g, 0.948 mmol) in Tetrahydrofuran (10 ml) was added Triethylamine (0.396 mL, 2.843 mmol), ethynyltrimethylsilane (0.140 g, 1.422 mmol) and Copper(l) iodide (0.036 g, 0.190 mmol) at 25°C. The reaction mixture was degassed with argon balloon for 15 min. Then Bis(triphenylphosphine)palladium(ll) dichloride (0.067 g, 0.095 mmol) was added at room temperature. The reaction mixture was stirred at 70°C for 6h. Progress of the reaction was monitored by LCMS and TLC [(TLC silica gel plate), 50% EtOAc:Petether (visualizing the spot under uv)] Rfvalue of the product was 0.5. The reaction mixture was filtered through a pad of celite bed washed with ethyl acetate. Filtrate was concentrated under vacuum. Crude was dissolved with EtOAc (100 mL) and washed with water (50 mL) followed by brine (30 mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure to afford crude lnt-10. The crude compound was purified by column chromatography by using silicagel (100-200 mesh), pure compound was eluted at 30% EtOAc: Petether to afford (3R)-3-methyl-4-(8-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)-4-((trimethylsilyl)ethynyl)-1,7- naphthyridin-2-yl)morpholine (lnt-10) (290 mg, 64.32%) as a yellow gummy.

[0335] Synthesis of lnt-13:

[0336] Experimental Procedure:

[0337] To a stirred solution of (3R)-3-methyl-4-(8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-4- ((trimethylsilyl)ethynyl)-1 ,7-naphthyridin-2-yl)morpholine (lnt-10) (0.160 g, 0.336 mmol) in methanol (1.6 mL) was added Potassium carbonate (0.093 g, 0.673 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2h. Progress of the reaction was monitored by LCMS and TLC, [(TLC silica gel plate), 50% EtOAc:Petether (visualizing the spot under uv)] Rf value of the product was 0.33. The reaction mixture was distilled out the solvent and diluted with ethyl acetate (80 mL), washed with water (40 mL), brine solution (30 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to obtained crude. Crude was purified by column chromatography by using silicagel (100-200 mesh) and pure compound was eluted at 40% EtOAc:Petether to afford (3R)- 4-(4-ethynyl-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-2-yl)-3-methylmorpholine (lnt.13) (100 mg, 73.68%) of as glassy yellow solid.

[0338] Synthesis of lnt-14:

[0339] Experimental Procedure:

[0340] Chloro(pentamethylcyclopentadienyl)(cyclooctadiene)ruthenium(ll) (Cp*RuCI(COD)) (0.008 g, 0.020 mmol, 0.1 equiv.) was added to a glass tube with a septa cap. The glass tube was sealed, then evacuated, and filled with nitrogen three times. Toluene (1.6 mL, degassed for 1 h with nitrogen purge) was added followed by (3R)-4-(4-ethy nyl-8- ( 1 -(tetrahyd ro-2H-pyran-2-yl)- 1 H-pyrazol-5-yl)- 1 , 7-n ap hthyri d i n-2-yl)-3- methylmorpholine (lnt-13) (0.080 g, 0.198 mmol) and tert-butyl (2-(2-azidoethoxy)ethyl)carbamate (Int- 12) (0.055 g, 0.238 mmol). The reaction mixture was stirred at room temperature for 1 h. Reaction was monitored by LCMS and TLC [(TLC silica gel plate), 5% MeOH:DCM (visualize the spot under uv)] Rf value of the product was 0.25. Reaction mixture was evaporated under reduce pressure to get crude lnt- 14. Crude was purified by column chromatography by using silicagel (100-200 mesh) and pure compound was eluted at 80% EtOAc:Petether and evaporated under reduced pressure to afford tert-butyl (2-(2-(5- (2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1,7-naphthyridin-4-yl)-1 H- 1 ,2,3-triazol-1 -yl)ethoxy)ethyl)carbamate (lnt-14) (70 mg, 55.71 %) as dark yellow solid.

[0341] Synthesis of HALA-008:

[0342] Experimental Procedure:

[0343] To a stirrred solution of tert-butyl (2-(2-(5-(2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)- 1 H-pyrazol-5-yl)-1,7-naphthyridin-4-yl)-1 H-1 ,2,3-triazol-1-yl)ethoxy)ethyl)carbamate (lnt-14) (0.150 g, 0.237 mmol) in tetrahydrofuran (3.0 mL) was added 6N aqueous HCI (0.30 mL) at room temperature. Reaction mixture was stirred at room temperature for 2h. Reaction was monitored by LCMS and TLC [(TLC silica gel plate), 10% MeOH:DCM (visualizing the spot under uv)] Rf value of the product was 0.15. Reaction mixture was concentrated under reduce pressure to afford crude. Crude residue was triturated with THF and dried under reduce pressure to get 150 mg crude. Crude was purified by prep HPLC to obtain the HALA-008 as formate salt.

[0344] Prep-HPLC condition: Column :Sunfire C18 (4.6x150) mm, 3.5|jm Mobile Phase-A : 0.1% FA in water Mobile Phase-B :Acetonitrile Gradient (A / B) :0 / 5, 2 / 5, 10 / 45, 11 / 45, 11.01 / 100, 13 / 100, 15 / 5 Flow Rate : ImL / min Column oven Temp : 35°C. The pure fractions were concentrated under reduced pressure and lyophilized to afford (R)-2-(2-(5-(2-(3-methylmorp holino)-8-(1 H-pyrazol-5-yl)-1 , 7-naphthyridi n-4-yl)-1 H- 1 ,2,3-triazol-1 -yl)ethoxy)ethan-1 -amine (HALA-008) (34 mg, 32%) as pale yellow solid.

[0345] Example 4: Synthesis of maleimide peptide-based cleavable linkers

[0346] Two maleimide peptide-based cleavable linkers were synthesized.

[0347] 1) a maleimide linker with a valine-citrulline (VC) peptide and a self-immolative pami nobenzyloxycarbamoyl (pABC) linker (HALA005).

[0348] 2) a maleimide linker with a glycine-glycine-phenylalani ne-glycine (GGFG) peptide and a self-immolative amino methylene (AM) spacer (HALA007).

[0349] Synthetic scheme for maleimide linker HALA-005

[0350]

[0351] Step-1 : (9H-fluoren-9-yl)methyl (R)-(1-((4-(hvdroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2- vDcarbamate (3):

[0352] To a stirred solution of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid (1 g, 2.5 mmol), in DCM (50 ml) and methanol(3ml) was added 4-aminobenyl alcohol-2 (0.368 g, 2.9 mmol) and EEDQ (N-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline) (1.24 g, 5.0 mmol) added at room temperature then stirred for 16 h. After completion of reaction the RM directly concentrated to get solid crude RM, it was washed with MTBE (10 ml_ x 3 times) and filtered washed with MTBE then dried under vacuum.

[0353] Finally, 1.1 g of pure product (9H-fluoren-9-yl)methyl(R)-(1-((4-(hydroxymethyl)phenyl)amino)-1 -oxo- 5-ureidopentan-2-yl)carbamate (3) (1.1 g, 87 %) was obtained as an off-white solid. [Mass: 502.5]

[0354] Step-2: (R)-2-amino-N-(4-(hvdroxymethyl)phenyl)-5-ureidopentanamide (4):

[0355] To a stirred solution of Compound 3 (1.05 g, 2.03 mmol) in DMF (20 ml) was added piperidine (0.739 g, 8.68 mmol) then stirred at rt for 2.5 h. After completion of reaction the suspension was concentrated. The crude product was washed with MTBE (10 mL x 3 times). Finally, 0.45 g of pure product (R)-2- amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide-(4) (0.45 g, 76.7 %) was obtained as a thick brown oil. (0.45 g, 76%) [Mass: 280.5]

[0356] Step 3: (9H-fluoren-9-yl)methyl ((S)-1-(((R)-1-((4-(hvdroxymethyl)phenyl)amino)-1-oxo-5- ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (5):

[0357] To a stirred solution of compound 4 (0.25 g, 0.85 mmol) in dry DMF (5 ml) was added Fmoc-L-Valine (0.317 g, 0.93 mmol) followed by addition of EDC.HCI (0.195 g, 1.00 mmol) and HOBt (0.137 g, 1.00 mmol). The reaction mixture was stirred at rt for 4h. After completion of reaction the suspension was poured into ice cold water stirred for 10 min. Then filtered the precipitated solid and washed 2 times with water and dried under vacuum. Finally we got 0.36 g of pure product (9H-fluoren-9-yl)methyl ((S)-1- (((R)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl) amino)-3-methyl-1- oxobutan-2-yl) carbamate-5 (0.36 g, 67 %) as Off-white solid (0.36 g, 67%) as Off-white solid. [Mass: 601.29]

[0358] Step-4:(R)-2-((S)-2-amino-3-methylbiitanamido)-N-(4-(hvdroxymethyl)phenyl)-5- ureidopentanamide (6):

[0359] To a stirred solution of compound 5 (0.35 g, 0.56 mmol) in DMF (20 ml) was added piperidine (0.1 g, 1.13 mmol) then stirring at rt for 2.5 h. After completion of reaction the suspension was concentrated under vacuum. The crude product was washed with MTBE (10 mL X 3 times). Finally, 0.25 g of crude product (R)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl) phenyl)-5-ureidopentanamide (6) (0.25 g, 98 %) was obtained as a brown gummy oil. [Mass: 379.2], This crude product was used as such in the next step without any further purification.

[0360] Step-5: 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-N-((S)-1 -(((R)-1 ■((4-(hvdroxymethyl)phenyl)amino)-

[0361] 1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(HALA-005):

[0362] HALA-005

[0363] To a stirred solution of 6 (1.54 g, 3.9 mmol) in DMF was added N-Succinimidyl-6-malimidohexanoate 7 (2.6 g, 8.5mmol) at room temperature then stirred at RT for 4.5 h. After completion of reaction the reaction mass was concentrated. The crude product was washed with MTBE (10 mL x 3 times) to get solid crude RM. Then the product was recrystallized with 20 ml mixture of MTBE (5 vol), DCM (2 vol), Methanol (0.5 vol) and ethyl acetate (1 vol) added and stirred for 1 h then filtered and washed 2 times with same solvent then dried under vacuum. Finally 0.950 g of pure product 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-N- ((S)-1-(((R)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1- oxobutan-2-yl)hexanamide (HALA-005) (0.95 g, 42%) was obtained as an off-white solid [Mass: 572.6].

[0364] Synthetic scheme for maleimide linker HALA-007

[0365] Step-1 : tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanylglvcinate (3):

[0366] To a stirred solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine-2 (2.0 g, 5.16 mmol) in DCM (70 ml) and Methanol (10 ml) was added tert-butyl glycinate 2 (0.949 g, 7.23 mmol), and EEDQ (2.55 g, 10.31 mmol) added at room temperature then stirred for 16 h. After completion of reaction the RM directly concentrated to get solid crude, it was washed with MTBE (30 mL X 3 times) and filtered washed with MTBE then dried under vacuum. Finally, we got 2.2 g of pure product tert-butyl(((9H-fluoren-9- yl)methoxy)carbonyl)-L-phenylalanyl glycinate (3) (2.2 g, 85 %) as Off-white solid. [Mass: 500.5], Step-2: tert-butyl L-phenylalanylqlvcinate (4):

[0367] To a stirred solution of Compound-3 (2.2 g, 4.39mmol) in DMF (20 ml) was added piperidine (0.748 g, 8.79 mmol) then stirred at RT for 2.5 h. After completion of reaction the suspension was concentrated. The crude product was washed with MTBE (30 mL X 3 times). Finally, we got 1 .05 g of rude product tert- butyl L-phenylalanylglycinate (4) as a thick brown oil. (1.05 g, 86%) as brown oil. [Mass: 278.35]. This crude product was used as such in the next step without any further purification.

[0368] Step 3: tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)qlycyl-L-phenylalanylqlycinate (6): o pc

[0369] To a stirred solution of compound-4 (1.058 g, 3.7 mmol) in DCM (40 ml) and Methanol (6 ml) was added (9H-fluoren-9-yl)methoxy)carbonyl)glycine-5 (1 .12 g, 3.7 mmol) followed by addition of EEDQ (1 .87 g, 7.5 mmol). The reaction mixture was stirred at RT for 5h. After completion of reaction the RM directly concentrated to get solid crude RM, it was washed with MTBE (10 mL x 3 times) and filtered washed with MTBE then dried under vacuum. Finally, 1.5 g of pure product tert-butyl (9H-fluoren-9- yl)methoxy)carbonyl)glycyl-L-phenylalanylglycinate (6) (1.5 g, 71 %) was obtained as an off-white solid [Mass: 557.65].

[0370] Step-4: tert-butyl qlvcvI-L-phenylalanylqlvcinate (7):

[0371] To a stirred solution of compound 6 (1.5 g, 2.68 mmol) in DMF (20 ml) was added piperidine (0.472 g, 5.37 mmol) then stirred at RT for 2.5 h. After completion of reaction the suspension was concentrated. The crude product was washed with (10 mL X 3 times). Finally, 0.75 g of pure product tert-butyl glycyl- L-phenylalanylglycinate (7) (0.75 g, 98 %) was obtained as a brown gummy oil. [Mass: 335.2] Step-5: tert-butyl qlycvIqlvcvI-L-phenvIalanylqlycinate (8):

[0372] Step-5

[0373] To a stirred solution of compound 7 (0.75 g, 2.23 mmol) in DCM (50 ml) and methanol (10 ml) was added (9H-fluoren-9-yl)methoxy)carbonyl)glycine-5 (0.66 g, 2.23 mmol) followed by addition of EEDQ (1.87 g, 7.5 mmol). The reaction mixture was stirred at RT for 5h. After completion of reaction the RM was directly concentrated to get solid crude RM, it was washed with MTBE (10 mL x 3 times) and filtered washed with MTBE then dried under vacuum. We got 1 .3 g of crude product. This crude product was used as such in the next step without any further purification. To the crude product in DMF (20 ml) was added piperidine (0.372 g, 4.23 mmol) then stirred at RT for 2.5 h. After completion of reaction the suspension was concentrated. The crude product was washed with MTBE (10 mL x 3 times). Finally, 0.75 g of crude amine product tert-butyl glycylglycyl-L-phenylalanylglycinate (8) (0.75 g) was obtained as a brown gummy oil. [Mass: 392.2], This crude product was used as such in the next step without any further purification.

[0374] Step-6:(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl)qlvcylqlvcyl-L-phenylalanyl glycine

[0375] (HALA-007):

[0376] To a stirred solution of Compound 8 (0.75 g, 2.1 mmol) in DMF was added N-Succinimidyl-6- malimidohexanoate 9 (1 .32 g, 4.2 mmol) at room temperature then stirred at RT for 4.5 h. After completion of reaction the reaction mass was directly concentrated. To the reaction mass in dry DCM was added Trifluoroacetic acid (0.368 g, 3.22 mmol) at RT and stirred for 2 h. After completion of reaction the reaction mass was concentrated. The crude product was washed with MTBE (10 mL x 3 times) to get solid crude RM. Then the crude product was recrystallized with mixture of MTBE (5 vol), DCM (2 vol), washed 2 times with same solvent then dried under vacuum. Finally, 121 mg of pure product (6-(2,5-dioxo-2, 5-dihydro- 1 H-pyrrol-1 -yl) hexanoyl)glycylglycyl-L-phenylalanyl glycine (HALA-007) (0.121 g, 12 %) was obtained as an off-white solid. [Mass: 529.2] Example 5: Coupling of ATR and CHK1 inhibitors as ADC payloads to maleimide peptide-based cleavable linkers.

[0377] Synthetic Scheme for compound: HALA-009.

[0378] HALA-009, as ATR inhibitor HALA-008 coupled to maleimide-VC-pABC linker HALA-005.

[0379] Synthetic Scheme for compound (lnt-2):

[0380] Experimental Procedure:

[0381] To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((R)-1-((4- (hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (HALA-005) (0.025 g, 0.044 mmol, 1 equiv.) in DMF (1.25 mL) at RT and were added bis(4-nitrophenyl) carbonate (lnt-1) (0.027 g, 0.087 mmol, 2 equiv.) and N,N-Diisopropylethylamine (0.017 g, 0.131 mmol, 3.0 equiv.) at RT. The reaction mixture was stirred at RT for 16h. The progress of the reaction was monitored by LCMS. The reaction mixture was quenched with ice water (20 mL) and extracted with ethylacetate (2x20 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude lnt-2. The crude lnt-2 was purified by FCC using 10% methanol in DCM to afford 4-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (lnt-2) (12 mg, 37.26%) as an off-white solid.

[0382] Synthetic Scheme for HALA-009: Experimental Procedure:

[0383] To a stirred solution of 4-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (lnt-2) (0.012 g, 0.016 mmol, 1 .0 equiv.) and (R)-N-methyl-2-(5-(2-(3-methylmorpholino)-8-(1 H-pyrazol-5-yl)- 1 , 7-nap hthyridi n-4-yl)-1 H- pyrazol-1-yl)ethan-1-amine (0.007 g, 0.016 mmol, 1 equiv.) in DMF (1.2 mL) at RT and were added 1- Hydroxybenzotriazole (0.004 g, 0.033 mmol, 2.0 equiv.) and pyridine (0.001 g, 0.016 mmol, 1 equiv.) at RT. The reaction mixture was stirred at RT for 16h. The progress of the reaction was monitored by LCMS. The reaction mixture was quenched with ice water (10 mL) and extracted with ethylacetate (3x20 mL) and the organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude HALA-009. The crude was purified by prep-HPLC.

[0384] Prep-HPLC condition: Column: SUNFIRE-C18 (150*19), 5u, Instument : MCL-PREP-COL-2021-081, Mobile phase: 10mM Ammonium Bi Carbonate in H2O: ACETONITRILE, GRADIENT: (T%B) :- 0 / 5,2 / 5,10 / 40,13.7 / 40,13.8 / 98,15 / 98, Flow Rate : 8 ml / min, Diluent: ACN +H2O.

[0385] The pure fractions were lyophilised to afford 4-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl methyl (2-(5-(2-((R)-3- methylmorpholino)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl)-1 H-pyrazol-1 -yl)ethyl)carbamate (HALA- 009) (4.5 mg, 27.20%) as a pale yellow solid.

[0386] ARG-ROI-1015-24 (2.5 mg) and ARG-ROI-1015-25 (4.5 mg) batches were dissolvedin acetonitrile and water, lyophilised to afford 4-((R)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl methyl(2-(5-(2-((R)-3-methylmorpholino)-8-(1 H- pyrazol-5-yl)-1,7-naphthyridin-4-yl)-1 H-pyrazol-1 -yl)ethyl)carbamate (HALA-009) (7.0 mg) as a pale yellow solid.

[0387] Synthetic Scheme for compound: HALA-010

[0388] HALA-010, as ATR inhibitor HALA-008 coupled to maleimide-GGFG-AM linker HALA-007. Experimental Procedure:

[0389] To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl)glycylglycyl-L- phenylalanylglycine (HALA-007) (0.015 g, 0.028 mmol, 1 equiv.) and (R)-2-(2-(5-(2-(3-methylmorpholino)- 8-(1 H-pyrazol-5-y I)- 1 , 7-n ap hthyri di n-4-y I)- 1 H-1 ,2, 3-triazol- 1 -y l)ethoxy)ethan-1 -amine (HALA-008) (0.013 g, 0.028 mmol, 1 equiv.) in DMF (1.5 mL) and was added HATU (0.022 g, 0.057 mmol, 2.0 equiv.) at RT. Then N,N-Diisopropylethylamine (0.011 g, 0.085 mmol, 3.0 equiv.) was added dropwise to the reaction mixture at RT. The reaction mixture was stirred at RT for 3h. The progress of the reaction was monitored by LCMS. The reaction mixture was quenched with ice-water (5 mL) and the compound was extracted with ethylacetate (3x10 mL). The organic layer was dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude compound. Crude was purified by prep-HPLC.

[0390] Prep-HPLC condition: Column Name: X-BRIDGE-OBD C18 (250*10mm), 5u, Column No# MCL-PREP- COL-2023-045, Mobile Phase-A: 10mM Ammonium Bicarbonate in water Mobile Phase-B: Acetonitrile, Gradient program (T / %B) 0 / 5,3 / 5,12 / 50,12.8 / 50,12.9 / 98,16 / 98,16.1 / 5,19 / 5, Flow Rate (mL / minute) 8ml_.

[0391] The pure fractions were concentrated under reduced pressure to afford N-((S)-7-benzyl-17-(5-(2-((R)-3- methylmorpholino)-8-(1 H-pyrazol-5-y I)- 1 ,7-nap hthyridi n-4-yl)- 1 H-1 , 2, 3-triazol- 1 -y l)-2, 5, 8, 11 -tetraoxo-15- oxa-3,6,9,12-tetraazaheptadecyl)-6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamide (HALA-010) (6 mg, 22.04%) as an off-white solid.

[0392] ARG-FFS-1094-35 (4 mg) and ARG-FFS-1094-36 (6 mg) batches were dissolved in acetonitrile and water, lyophilized to afford N-((S)-7-benzyl-17-(5-(2-((R)-3-methylmorpholino)-8-(1 H-pyrazol-5-yl)-1 ,7- naphthyridin-4-yl)-1 H-1 ,2,3-triazol-1 -yl)-2,5,8,11-tetraoxo-15-oxa-3,6,9,12-tetraazaheptadecyl)-6-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamide (HALA-010) (9.7 mg) as a pale yellow solid.

[0393] Synthetic Scheme for compound: HALA-011

[0394] HALA-010, as ATR inhibitor Berzosertib coupled to maleimide-VC-pABC linker HALA-005.

[0395] Experimental Procedure: To a stirred solution of 5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((methylamino)methyl)phenyl)isoxazol-5- yl)pyrazin-2-amine (berzosertib) (0.010 g, 0.022 mmol, 1 equiv.) and N-((S)-1-(((R)-1-((4- (chloromethyl)phenyl)amino)-1 -oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo- 2, 5-dihydro-1 H-pyrrol-1 -yl)hexanamide (HALA-006) (0.013 g, 0.022 mmol, 1 equiv.) in DMF (0.8 mL) at RT and were added sodium iodide (0.003 g, 0.022 mmol, 1 equiv.), potassium carbonate anhydrous (0.009 g, 0.065 mmol, 3 equiv.) at RT. The reaction was stirred at RT for 3h. The progress of the reaction was monitored by LCMS. The reaction mixture was directly submitted to prep-HPLC without any workup.

[0396] Prep-HPLC condition: Column: YMC TRIART 10*150, Mobile phase: A: 0.1% HCI buffer B: Acetonitrile, Gradient: (% of B): 0 / 30,1 / 30,11 / 75,16 / 75,16.01 / 100,18 / 100,18.01 / 30,20 / 30, flow rate- 06 mL.

[0397] The pure fractions were concentrated under reduced pressure to afford N-((S)-1-(((R)-1-((4-(((4-(5-(3- amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3- yl)benzyl)(methyl)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan- 2-yl)-6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamide (HALA-011) (5.9 mg, 26.86%) as an off-white solid.

[0398] Synthetic Scheme for HALA-015:

[0399] HALA-015, as CHK1 inhibitor Prexasertib coupled to maleimide-GGFG-AM linker HALA-007.

[0400] Experimental Procedure:

[0401] To a stirred solution of 5-((5-(2-(3-aminopropoxy)-6-methoxyphenyl)-1H-pyrazol-3-yl)amino)pyrazine-2- carbonitrile (prexasertib) (0.010 g, 0.027 mmol, 1 equiv.) and ((6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)hexanoyl)glycylglycyl-L-phenylalanylglycine (HALA-007) (0.014 g, 0.027 mmol, 1 equiv.) in DMF (0.8 mL) and were added EDC.HCI (0.010 g, 0.055 mmol, 2.0 equiv.) and 1 -Hydroxybenzotriazole, anhydrous (0.007 g, 0.055 mmol, 2.0 equiv.) at at 0°C. Then N,N-Diisopropylethylamine (0.011 g, 0.082 mmol, 3.0 equiv.) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was stirred at RT for 3h. The progress of the reaction was monitored by LCMS. The reaction mixture was quenched with ice water and extracted with ethyl acetate (2x20 mL). The organic layer was dried over sodium sulphate, filtered and concentrated under reduced pressure to afford crude compound.

[0402] Prep-HPLC condition: Column: YMC TRIART , Mobile phase: 0.1 % HCI in CAN, Flow rate: 06 ML / MIN, Gradient: (% of B): 0 / 10,1 / 10,10 / 58,15 / 70,15.01 / 100,17 / 0100,17.01 / 10,19 / 10.

[0403] The pure fractions were lyophilized to afford (S)-N-(7-benzyl-15-(2-(3-((5-cyanopyrazin-2-yl)amino)-1 H- pyrazol-5-yl)-3-methoxyphenoxy)-2,5,8,11-tetraoxo-3,6,9,12-tetraazapentadecyl)-6-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)hexanamide (HALA-015) (4 mg, 16.6%) as an off-white solid.

[0404] Example 6: Production of Trastuzumab and Trastuzumab-mV205C Antibodies.

[0405] The wild-type form of the anti-HER2 antibody, human monoclonal lgG1, was produced with reference to the amino acid sequence of Trastuzumab [Ohri, Rachana et al. Bioconjugate chemistry vol. 29,2 (2018): 473-485], appended as SEQ ID NO.: 1 (heavy chain) and SEQ ID NO.: 2 (light chain). Engineered trastuzumab with a V205C substitution in its light chain, is appended as SEQ ID NO: 3. The native trastuzumab (wild-type trastuzumab) antibody and the modified trastuzumab antibody with the V205C substitution in the light chain (Antibody-5) were produced and purified, according to the following steps: gene design with codons optimized for protein expression; gene synthesis; subcloning into expression vector; plasmid amplification and preparation; XtenCHO Transient expression protocol; one-step affinity purification.

[0406] Quality control SDS-PAGE analysis of purified Trastuzumab and Trastuzumab V205C was performed (Fig. 2). Please note that in this figure the Trastuzumab V205C antibody is referred to as Trastuzumab-m V225C, as the numbering of the amino acids includes the signal peptide of 20 amino acids, which is removed upon maturation of the antibody.

[0407] Example 7: ADC Development: Conjugation of Trastuzumab and Trastuzumab-mV205C Antibodies to HALA-009.

[0408] Trastuzumab and Trastuzumab-mV205C were conjugated to HALA-009 with specific Drug-Antibody Ratios (DAR) of 2 and 4 according to the following protocol.

[0409] ADC development- Brief protocol

[0410] 1. Disulfide bonds reduction by TECP

[0411] - Antibody concentration: C1 (pM) in PBS, PH7.4 - Addition of a 10-fold molar excess of a reducing agent TCEP

[0412] - Incubation: 37°C for 1 h.

[0413] 2. Conjugation with maleimide Drug-Linker

[0414] - Addition of drug-linker and NEM into the reaction system of step 1 :

[0415] - Final concentration of drug-linker 3 x C1

[0416] - Incubation: 0°C for 1 h.

[0417] Drug-linker and NEM ratio [Lyon RP et al. Methods Enzymol. 2012;502:123-138]:

[0418] DAR 2: Mole fraction of drug-linker in a mixture of drug-linker and NEM = 0.31 => Concentration of NEM added = 6.67 x C1.

[0419] DAR 4: Mole fraction of drug-linker in a mixture of drug-linker and NEM = 0.54 -> Concentration of NEM added = 2.56 x C1.

[0420] - Buffer exchange vs PBS, pH7.5 by dialysis method for at least 3 times

[0421] - Filtration by 0.22 pm filter.

[0422] Quality control of Trastuzumab-HALA-009-DAR2, Trastuzumab-HALA-009-DAR4, Trastuzumab- mV205C-HALA-009-DAR2 and Trastuzumab-mV205C-HALA-009-DAR4 was performed by SDS-PAGE analysis and SEC-HPLC analysis. Unconjugated Trastuzumab and Trastuzumab-mV205C were used as controls.

[0423] SDS-PAGE analysis was performed to control antibody integrity and drug loading after conjugation (Fig. 3A-B).

[0424] SEC-HPLC analysis was performed to confirm antibody integrity (Fig. 4). % of monomer observed: Trastuzumab (91.5% monomer with retention time of 8.266 min); Trastuzumab-HALA-009-DAR2 (77.48% with retention time of 8.213 min); Trastuzumab-HALA-009-DAR4 (79.06% with retention time of 8.216 min); Trastuzumab-mV205C (88.17% with retention time of 8.286 min); Trastuzumab-mV205C-HALA- 009-DAR2 (82.33% with retention time of 8.269 min); Trastuzumab-mV205C-HALA-009-DAR24 (79.48% with retention time of 8.272 min).

[0425] ELISA analysis was performed to evaluate the binding capacity of the conjugated antibodies of Trastuzumab-HALA-009-DAR2 and Trastuzumab-HALA-009-DAR4 to CD340 (HER2) antigen (Fig. 5A- B).

[0426] ELISA- Brief protocol

[0427] 1. Coating antigen: 5pg / mL in PBS pH7.4 10OpL / well - 4°C - overnight 2. Blocking: Protein-free blocking buffer 300pL / well - 37C - 1.5H

[0428] 3. Washing: 0.05%PBST 300pl / well 3 times

[0429] 4. Incubation primary antibody: dilutions 1 pg / mL to 0.016pg / mL in PBS pH 7.4 - 37°C - 1H

[0430] 5. Washing: 0.05%PBST 300pL / well 3 times

[0431] 6. Incubation secondary antibody (@human IgG-HRP): 10OpL / well - 37°C - 30min

[0432] 7. Washing: 0.05%PBST 300pL / well 3 times

[0433] 8. Incubation TMB substrate: 1 OOpL / well - 37C - 10min

[0434] 9. Stop: 2M HCI - 50pL / well

[0435] 10. Reading: OD450

[0436] Increasing OD was observed for increased concentration of Trastuzumab, Trastuzumab-HALA- 009(DAR2) and Trastuzumab-HALA-009(DAR4) against CD340 (HER2) protein. The signal measured for all three antibodies were similar, meaning that binding capacity of antibodies Trastuzumab DAR2 and Trastuzumab DAR4 is maintained when compared to Trastuzumab (Fig. 5A-B).

[0437] Example 8: Method for the Assessment of Target-specific Growth Inhibition by the Developed HER2 ADCs in vitro.

[0438] Developed HER2 ADCs are examined for their ability to inhibit growth of cancer cell lines in a target- selective manner, in a cellular viability assay. SKOV3, HER2 positive and ATR-CHK1 -inhibition sensitive ovarian cancer cell line is plated in 96-well black plates with clear flat bottom. After 24 hours, ADCs bearing ATR or CHK1 inhibitor payloads are dispensed in triplicates. Cells are incubated with the ADCs for a given time (96-144 h) at 37°C in a cell culture incubator. After incubation, the number of viable cells is determined using Cell Titer-Gio (cat#G9243 Promega) and cellular viability EC50 values of the ADCs are calculated. MCF7, HER2 negative invasive breast carcinoma cell line, is similarly treated with HER2 ADCs which are expected not to inhibit cell growth.

[0439] Bystander effect of developed HER2 ADCs, bearing ATR or CHK1 inhibitor payloads, is studied by exposing coculture of HER2 positive SKOV3 cells and HER2-negative MCF7 cells, according to the assays described by [Wu S, Shah DK. Methods Mol Biol. 2020;2078:329-340],

[0440] SEQ ID N01: Amino acid sequence of “Trastuzumab” antibody heavy chain.

[0441] 1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR

[0442] 51 IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG 101 GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT

[0443] 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP

[0444] 251 KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN

[0445] 301 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

[0446] 351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

[0447] 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[0448] SEQ ID NO 2: Amino acid sequence of “Trastuzumab” antibody light chain.

[0449] 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS

[0450] 51 ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ

[0451] 101 GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

[0452] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

[0453] 201 LSSPVTKSFN RGEC

[0454] SEQ ID NO 3: Amino acid sequence of “Trastuzumab” antibody light chain with V205C amino acid substitution.

[0455] 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS

[0456] 51 ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ

[0457] 101 GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

[0458] 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 LSSPcTKSFN RGEC

[0459] Example 9 - Synthesis of further derivatives and intermediates of the present invention

[0460] Synthetic schemes for the compounds prepared herein are shown in Figure 6.

[0461] Methyl 3-amino-2-chloroisonicotinate (2) (WJ013)

[0462] Methyl 3-aminoisonicotinate (1, 10.0 g, 65.7 mmol, 1.0 eq.) was dissolved in cone. HCI (100 mL) and hydrogen peroxide (10 mL, 35% cone.) was added at 0 °C dropwise and then stirred for 1 h at rt. The reaction solution was quenched with the addition of a NaOH solution (10 mL, 1 M). The precipitate that forms was filtered off and the filtrate was continued stirring. The second precipitate that formed was filtered off. The second precipitate was then dissolved in dichloromethane and filtered again. The filtrate was the died over sodium sulphate and concentrated in vacuo to yield (2, 4.38 g, 36% yield) as a beige solid.

[0463] 1H NMR (500 MHz, CDCI3) δ 7.70 (d, J = 5.1 Hz, 1 H), 7.59 (d, J = 5.1 Hz, 1 H), 6.21 (br s, 2H), 3.92 (s, 3H).

[0464] 13C NMR (126 MHz, CDCh) δ 166.79, 141.91, 139.09, 134.81, 122.83, 116.62, 52.29.

[0465] (R)-1-(3-methylmorpholino)ethan-1-one (4) (WJ008)

[0466] A three-neck flask, equipped with a stirring bar and a thermometer, was charged with (R)-3-methylmorpholine (3, 2.22 mL, 19.7 mmol, 1.0 eq.) and potassium carbonate (8.23 g) in dichloromethane (50 mL). The mixture was stirred at it for 30 min when acetyl chloride (2.83 mL, 39.6 mmol, 2.0 eq.) was added. After stirring for 18h at rt. the precipitate was filtered off and washed with dichloromethane (30 mL). The mother liquor was concentrated to dryness to obtain 1-((R)-3- methylmorpholin-4-yl)ethenone (4, 2.70 g, 18.9 mmol, 95%) as a yellow oil.

[0467] 1H-NMR (500 MHz, 298 K, CDCI3 δ / ppm): 4.55 (dd, J = 7.3, 3.7 Hz, 1 H), 4.26 - 4.19 (m, 1 H), 3.92 - 3.83 (m, 2H), 3.81 - 3.75 (m, 1H), 3.66 (d, J = 11 .4 Hz, 2H), 3.55 (ddd, J = 24.8, 11 .6, 3.2 Hz, 2H), 3.49 - 3.34 (m, 4H), 2.97 (d, J = 4.1 Hz, 1 H), 2.03 (d, J = 6.5 Hz, 3H), 1.33 (d, J = 6.9 Hz, 3H), 1.22 (d, J = 6.8 Hz, 3H).

[0468] 13C{1H}-NMR (126 MHz, CDCI35 / ppm): 169.17, 71.04, 70.65, 67.06, 66.72, 49.77, 44.44, 41.80, 36.52, 21.52, 20.84, 15.73, 14.60.

[0469] Methyl (R,E)-2-chloro-3-((1-(3-methylmorpholino)ethylidene)amino)isonicotinate (5) (WJ012)

[0470] Under inert atmosphere a two necked flask was charged with 1-((R)-3-methylmorpholin-4-yl)ethenone (4, 0.33 mL, 2.70 mmol, 1.0 eq.) in dichloroethane (20 mL). The solution was cooled to 0 °C when phosphorous oxychloride (0.73 mL) was added. After stirring the solution for 30 min at rt. methyl 3-amino- 2-chloroisonicotinate (2, 0.50 g, 2.70 mmol, 1 .0 eq.) was added and the mixture was stirred at 80 °C for 2h. After allowing to cool down the mixture, it was stirred overnight at rt. and heated for another 3h the next day. The progress of the reaction was monitored with UPLC-MS. The solvent was removed under reduced pressure and dichloromethane (10 mL) and water (5.0 mL) were added. Under vigorous stirring a saturated sodium carbonate solution was added until the pH of the solution was basic (pH=9) and the mixture was extracted three times with dichloromethane (3x25 mL). The combined organic phases were dried over sodium sulfate and the solvent was removed under reduced pressure. The brown oil was the purified with isolera RP chromatography (H2O +0.1% TFA / MeCN + 0.1 % TFA) to obtain methyl 2-chloro- 3-[1-((R)-3-methylmorpholin-4-yl)eth-(E)-ylideneamino]isonicotinate (5, 850 mg, 2.70 mmol, 100%) as a brown oil.

[0471] 1H-NMR (500 MHz, 298 K, CDCI3 δ / ppm): 8.39 (dd, J = 5.0, 2.8 Hz, 1H), 7.66 (dd, J = 5.0, 3.9 Hz, 1 H), 4.39 (s, 1 H), 4.05 - 3.89 (m, 2H), 3.84 (d, J = 1 .9 Hz, 3H), 3.76 (d, J = 2.8 Hz, 2H), 3.61 (dd, J = 10.2, 7.4 Hz, 2H), 1.99 (s, 3H), 1.46 (dd, J = 10.2, 6.8 Hz, 3H).

[0472] 13C{1H}-NMR (126 MHz, CDCb b / ppm): 163.88, 163.82, 162.71 , 160.52, 160.22, 150.39, 150.36, 148.27, 148.20, 136.90, 132.87, 123.19, 123.17, 116.88, 114.58, 70.20, 70.15, 66.20, 66.11 , 53.31, 51.32, 42.93, 16.66, 16.60, 15.21.

[0473] 8-Chloro-2-((R)-3-methylmorpholin-4-yl)-[1,7]naphthyridin-4-ol (6) (WJ014)

[0474] Under inert atmosphere at 0°C a solution of lithium bis(trimethylsilyl)amide (1M in THF, 8.5 mL) was added dropwise over 30 min to a solution of methyl 2-chloro-3-[1 -((R)-3-methylmorpholin-4-yl)eth-(E)- ylideneamino]isonicotinate (5, 850 mg, 2.70 mmol, 1.0 eq.) in 50 mL of THF. The mixture was stirred for 3 h at rt. Water (10 mL) was carefully added and the solvent was removed under reduced pressure. The residue was taken up in saturated ammonium chloride solution and extracted with dichloromethane / isopropanol (4:1, 4x20 mL) The combined organic phases were dried over sodium sulfate, filtered and concentrated under reduced pressure, acetonitrile (12 mL) was added and the solid was filtered off. The precipitate was purified with isolera RP chromatography (H2O + 0.1 % TFA / MeCN + 0.1 % TFA) to obtain 8-chloro-2-((R)-3-methylmorpholin-4-yl)-[1 ,7]naphthyridin-4-ol (6, 450 mg, 1.61 mmol, 59%) as a yellow solid.

[0475] 1H-NMR (500 MHz, 298 K, CDCI3 δ / ppm): 11.62 (s, 1 H), 7.98 (dd, J = 5.4, 2.0 Hz, 1 H), 7.72 (dd, J = 5.3, 2.0 Hz, 1 H), 6.60 (d, J = 2.0 Hz, 1 H), 4.41 (d, J = 7.4 Hz, 1H), 4.16 (s, 1 H), 3.97 (d, J = 3.7 Hz, 1 H), 3.77 (d, J = 11 .2 Hz, 1 H), 3.65 (dd, J = 11.4, 3.1 Hz, 1 H), 3.49 (td, J = 11 .8, 3.0 Hz, 1 H), 3.18 (td, J = 12.8, 3.9 Hz, 1 H), 1.21 (dd, J = 6.7, 1.9 Hz, 3H). 2-[((R)-3-methylmorpholin-4-yl)-8-[2-(tetrahydropyran-2-yl)-2H-pyrazol-3yl]-[1,7]naphthyridin-4-ol

[0476] (7) (WJ015)

[0477] Under inert atmosphere a 10 mL pressure tube was charged with 8-chloro-2-((R)-3-methylmorpholin-4- yl)-[1 ,7] naphthyridin-4-ol (6, 80.0 mg, 0.29 mmol, 1 .0 eq.), 1 -(tetrahydropyran-2-yl)-5-(4,4, 5, 5-tetramethyl- [1 ,3,2]dioxaborolane-2-yl)-1 H-pyrazole (130 mg, 0.46 mmol, 1.6 eq.), [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium(l I) (30.0 mg) and caesium carbonate (370 mg) in Dioxane (3.0 mL). The suspension was stirred at 90 °C overnight. The progress of the reaction was monitored with UPLC-MS. After completion of the reaction, water (5.0 mL) and dichloromethane (5.0 mL) were added and the reaction suspension was extracted with dichloromethane until the organic phase was transparent. The solvent was removed under reduced pressure and the crude product was purified with Preparative HPLC SHIMADZU® to obtain (R)-2-(3-methylmorpholino)-8-(1 H-pyrazol-5-yl)-1 ,7- naphthyridin-4-ol (7, 30.0 mg, 0.096 mmol, 33%) as a yellow oil.

[0478] LCMS: m / z: [M+H] = 396.3.

[0479] Methyl ( / ?)-5-(4-hydroxy-2-(3-methylmorpholino)-1,7-naphthyridin-8-yl)picolinate (9) (WJ022)

[0480] (R)-8-chloro-2-(3-methylmorpholino)-1 ,7-naphthyridin-4-ol (6, 80.0 mg, 286 μmol, 1.0 eq.) was dissolved under inert conditions in dioxane (3.0 mL). PdCl2(dppf) (30.0 mg, 41.0 pimol, 0.14 eq.), CS2CO3 (370 mg, 1.14 mmol, 4.0 eq.) and methyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinate (121 mg, 460 μmol, 1.6 eq.) were added and the suspension was stirred at 90 °C for 36 h. The reaction mixture was filtered over Celite®, purified by preparative HPLC (H2O / MeCN) with 0.1 % TFA to yield the desired product (9, 32.0 mg, 29% yield) as a yellow solid. 2-[(3R)-3-methylmorpholin-4-yl]-8-[1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl]-1,7naphthyridin-

[0481] 4-yl trifluoromethanesulfonate (10) (WJ010)

[0482] Under inert atmosphere a solution of (R)-2-(3-methylmorpholino)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4- ol (7, 30 mg, 0.096 mmol, 1.0 eq.), N-Phenylbis(trifluoromethanesulfonimide) (40 mg, 0.11 mmol, 1.1 eq.) and DIPEA (26.0 ul) in DMF (1 mL) was stirred for 0.5 h at rt. The solvent was removed under reduced pressure and the crude product was purified with Preparative HPLC SHIMADZU® to obtain (R)-2-(3- methylmorpholino)-8-(1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl trifluoromethanesulfonate (10, 10 mg, 0.023 mmol, 23%, lit.yield: 42%).

[0483] LCMS: m / z: [M+H] = 528.2.

[0484] Methyl ( / ?)-5-(2-(3-methylmorpholino)-4-(((trifluoromethyl)sulfonyl)oxy)-1 ,7-naphthyridin-8- yl)picolinate (12) (WJ024)

[0485] Methyl (R)-5-(4-hydroxy-2-(3-methylmorpholino)-1 ,7-naphthyridin-8-yl)picolinate (9, 32.0 mg, 84.0 μmol, 1.0 eq.) was dissolved in DMF (0.80 mL) under inert conditions. Phenyl triflimide (33.0 mg, 92.4 μmol, 1.1 eq.) and DIPEA (23.3 μL, 134 μmol, 1 .6 eq.) were added and the solution was stirred at it for 30min. The solution was concentrated and the crude was used for the next step of the synthesis.

[0486] (3R)-3-methyl-4-(8-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)-4-((trimethylsilyl)ethynyl)-1,7- naphthyridin-2-yl)morpholine (14.1) (WJ027)

[0487] 2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl trifluoromethanesulfonate (10, 20.0 mg, 37.9 μmol, 1.0 eq.) was dissolved under inert conditions in MeCN (0.5 mL) and PdCl2(PPh3)2 (2.67 mg, 3.80 pimol, 0.1 eq.), Cui (0.724 mg, 3.80 pimol, 0.1 eq.), ethynyltrimethylsilane (8.10 μL, 56.9 μmol, 1.5 eq.) and diisopropylamine (84.9 piL, 606 pimol, 16 eq.) were added. The solution was stirred for 2 h at 70 °C. The reaction mixture was filtered over Celite®, purified by preparative HPLC (PfeO / MeCN) and concentrated by sublimation in vacuo. The desired product (14.1, 9.80 mg, 54% yield) was obtained as a yellow solid.

[0488] 2_(3 / ?)-4-(4-(1H-pyrazol-5-yl)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1,7-naphthyridin-2- yl)-3-methylmorpholine (17) (WJ017)

[0489] 2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl trifluoromethanesulfonate (10, 20.0 mg, 38.0 μmol, 1 .0 eq.) was added under N2-flowto a microwave tube and dissolved in MeCNWD (1 :1 , 1.0 mL). PdCl2(PPhs)2 (2.67 mg, 3.80 μmol, 0.10 eq.), K2CO3 (13.1 mg, 95.0 pimol, 2.5 eq.) and 5-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazole (14.7 mg, 76.0 pimol, 2.0 eq.) were added and the suspension was stirred on microwave installation for 15 min at 130 °C. The reaction mixture was filtered over Celite® and purified by preparative HPLC (H2O / MeCN) without 0.1% TFA to yield the desired product (17, 3.1 mg, 18% yield) as a yellow solid.

[0490] (R)-4-(4-(1 -(2-(2-azidoethoxy)ethyl)-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridin-2-yl)-3- methylmorpholine (18) (WJ019)

[0491] (3R)-4-(4-(1 H-pyrazol-5-y l)-8- (1 - (tetrahy dro-2 / 7-pyran-2-yl)- 1 H-pyrazol-5-yl)-1 ,7-nap hthyridi n-2-yl)-3- methylmorpholine (17, 3.00 mg, 6.75 μmol, 1.0 eq.), powdered NaOH (0.540 mg, 13.5 μmol, 2.0), TBAB (0.11 mg, 0.341 pimol, 0.05 eq.), 1-azido-2-(2-bromoethoxy)ethane (WJ004, 9.6 μL, 119 μmol, 18 eq.) were added to an Eppendorf vial and the reaction was shaken at rt for 24 h. The reaction mixture was dissolved in DCM / TFA (2:1) and shaken for another 30 min. The solution was purified by preparative HPLC (H2O / MeCN) without 0.1% TFA to yield the desired product (18, 2.47 mg, 77% yield) as a yellow solid.

[0492] (R)-5-(2-(3-methylmorpholino)-8-(1H-pyrazol-5-yl)-1,7-naphthyridin-4-yl)-A / -propylthiophene-2- carboxamide (19) (WJ020a)

[0493] (R)-5- (2- (3-methylmorp hoi i no)-8- ( 1 H-pyrazol-5-yl)-1 , 7- nap hthyridi n-4-yl)thiop hene-2-carboxylic acid (23, 5.00 mg, 11.0 μmol, 1.0 eq.) was dissolved under inert conditions in DMF (0.10 mL). DIPEA (3.83 μL, 22.0 μmol, 2.0 eq.), HOBt (1.99 mg, 13.0 μmol, 1.2 eq.), EDOHCI (2.49 mg, 13.0 μmol, 1.2 eq.) and propylamine (0.90 μL, 11.0 μmol, 1.0 eq.) were added and the suspension was stirred at rt for 20 h. The reaction mixture was filtered over Celite®, purified by preparative HPLC (H2O / MeCN) with 0.1% TFA to yield the desired product (19, 2.1 mg, 41 % yield) as a yellow solid.

[0494] (R)-4-(2-(3-methylmorpholino)-8-(1H-pyrazol-5-yl)-1,7-naphthyridin-4-yl)benzaldehyde (20)

[0495] (WJ011)

[0496] 2-(( / ?)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl trifluoromethanesulfonate (10, 35.0 mg, 66.0 μmol, 1 .0 eq.) was added under N2-flow to a microwave tube and dissolved in MeCN / H2O (1 : 1 , 1.0 mL). PdCl2(PPh3)2 (4.67 mg, 13.0 |jmol, 0.20 eq.), K2CO3 (22.9 mg, 165 μmol, 2.5 eq.) and 4- Formyl p he ny Iboron ic acid pinacol ester (33.5 mg, 132 pi mol, 2.0 eq.) were added and the suspension was stirred on microwave installation for 15 min at 130 °C. The reaction mixture was filtered over Celite®, purified and THP-deprotected by preparative HPLC (FW / MeCN) with 0.1 % TFA to yield the desired product (20, 10.2 mg, 37% yield) as a yellow solid.

[0497] (R)-4-(4-ethynyl-8-(1 H-pyrazol-5-yl)-1,7-naphthyridin-2-yl)-3-methylmorpholine (22) (WJ027a deprot)

[0498] (3R)-3-methyl-4-(8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-4-((trimethylsilyl)ethynyl)-1 ,7- naphthyridin-2-yl)morpholine (14, 9.80 mg, 21.0 μmol, 1.0 eq.) was dissolved in (0.32 mL) in an ice bath at 0 °C under inert conditions. A solution of TBAF in THF (32 piL, 1.0 M) was added dropwise and then the solution was stirred at rt. for 30 min. The reaction mixture was purified by preparative HPLC (H2O / MeCN) and concentrated by sublimation in vacuo. The desired product (22, 7.90 mg, 93% yield) was obtained as a yellow solid.

[0499] (R)-5-(2-(3-methylmorpholino)-8-(1H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl)thiophene-2-carboxylic acid (23) (WJ018)

[0500]

[0501] 2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl trifluoromethanesulfonate (10, 35.0 mg, 66.0 μmol, 1 .0 eq.) was added under N2-flowto a microwave tube and dissolved in MeCN / FhO (1 :1 , 1.0 mL). PdCl2(PPh3)2 (4.67 mg, 13.0 |jmol, 0.20 eq.), K2CO3 (22.9 mg, 165 pimol, 2.5 eq.) and 5-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)thiophene-2-carboxylic acid (33.5 mg, 132 μmol, 2.0 eq.) were added and the suspension was stirred on microwave installation for 15 min at 130 °C. The reaction mixture was filtered over Celite®, purified and THP-deprotected by preparative HPLC (H2O / MeCN) with 0.1 % TFA to yield the desired product (23, 10.2 mg, 37% yield) as a yellow solid.

[0502] ( / ?)-3-methyl-4-(4-(1-methyl-1H-pyrazol-5-yl)-8-(1 H-pyrazol-5-yl)-1,7-naphthyridin-2-yl)morpholine

[0503] (28) (WJ026, BAY1895344)

[0504] 2-((R)-3-methylmorpholino)-8-(1-(tetrahydro-2H-pyran-2-yl)-1 H-pyrazol-5-yl)-1 ,7-naphthyridin-4-yl trifluoromethanesulfonate (10, 50.0 mg, 95.0 pimol, 1 .0 eq.) was added under N2-flowto a microwave tube and dissolved in MeCN / PhO (1 :1 , 2.0 mL). PdCb(PPh3)2 (6.67 mg, 9.50 μmol, 0.10 eq.), K2CO3 (32.9 mg, 238 μmol, 2.5 eq.) and 1-methyl-5-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazole (39.5, 190 pimol, 2.0 eq.) were added and the suspension was stirred on microwave installation for 15 min at 130 °C. The reaction mixture was filtered over Celite®, purified and THP-deprotected by preparative HPLC (H2O / MeCN) with 0.1 % TFA to yield the desired product (28, 20.0 mg, 56% yield) as a yellow solid.

[0505] Example 10: Production of wild-type and cysteine mutants of trastuzumab

[0506] The wild-type form of the anti-HER2 antibody, human monoclonal lgG1, was produced with reference to the amino acid sequence of Trastuzumab [Ohri, Rachana et al. Bioconjugate chemistry vol. 29,2 (2018): 473-485], appended as SEQ ID NO.:1 (heavy chain) and SEQ ID NO.: 2 (light chain). Engineered trastuzumab with a V205C substitution in its light chain, and a V170C substitution in its heavy chain, is identified as Antibody-1 and appended as SEQ ID NO.: 3 and SEQ ID NO.: 5, respectively.

[0507] Engineered trastuzumab with Q155C and V205C substitutions in its light chain, and a V170C substitution in its heavy chain, is identified as Antibody-2 and appended as SEQ ID NO.: 6 and SEQ ID NO.: 5, respectively.

[0508] Engineered trastuzumab with Q155C and V205C substitutions in its light chain, and wild-type heavy chain, is identified as Antibody-3 and appended as SEQ ID NO.: 6 and SEQ ID NO.: 1, respectively.

[0509] Engineered trastuzumab with Q155C and V205C substitutions in its light chain, and V170C-S340C- E391 C-Q441 C substitutions in its heavy chain, is identified as Antibody-4 and appended as SEQ ID NO.:

[0510] 6 and SEQ ID NO.: 7, respectively.

[0511] Engineered trastuzumab with a V205C substitution in its light chain, is identified as Antibody-5 and appended as SEQ ID NO.: 3.

[0512] The native trastuzumab antibody and the modified trastuzumab antibodies were produced and purified, according to the following steps: gene design with codons optimized for protein expression; gene synthesis; subcloning into expression vector; plasmid amplification and preparation; XtenCHO Transient expression protocol; one-step affinity purification.

[0513] Quality control SDS-PAGE analysis of purified trastuzumab and cysteine mutants of trastuzumab was performed (Fig. 7).

[0514] ELISA analysis was performed to evaluate the binding capacity of the produced antibodies to CD340 (HER2) antigen (Fig. 8).

[0515] ELISA- Brief protocol

[0516] 1. Coating antigen: 5pg / mL in PBS pH7.4 10OpL / well - 4°C - overnight

[0517] 2. Blocking: Protein-free blocking buffer 300pL / well - 37C - 1.5H

[0518] 3. Washing: 0.05%PBST 300pl / well 3 times

[0519] 4. Incubation primary antibody: dilutions 1 pg / mL to 0.016pg / mL in PBS pH 7.4 - 37°C - 1 H

[0520] 5. Washing: 0.05%PBST 300pL / well 3 times 6. Incubation secondary antibody (@human IgG-HRP): 1 OOpL / well - 37°C - 30min

[0521] 7. Washing: 0.05%PBST 300pL / well 3 times

[0522] 8. Incubation TMB substrate: 1 OOpL / well - 37C - 10min

[0523] 9. Stop: 2M HCI - 50pL / well

[0524] 10. Reading: OD450

[0525] Increasing OD was observed for increased concentration of Trastuzumab and cysteine mutants of trastuzumab against CD340 (HER2) protein. The signal measured for all three six antibodies were similar, meaning that binding capacity of antibodies Trastuzumab DAR2 and Trastuzumab DAR4 is maintained when compared to Trastuzumab (Fig. 7).

[0526] SEQ ID NO 5: Amino acid sequence of “Trastuzumab” antibody heavy chain with V170C amino acid substitution.

[0527] 1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR

[0528] 51 IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG

[0529] 101 GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK

[0530] 151 DYFPEPVTVS WNSGALTSGc HTFPAVLQSS GLYSLSSWT VPSSSLGTQT

[0531] 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP

[0532] 251 KDTLMISRTP EVTCWVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN

[0533] 301 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

[0534] 351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

[0535] 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[0536] SEQ ID NO 6: Amino acid sequence of “Trastuzumab” antibody light chain with Q155C and V205C amino acid substitutions.

[0537] 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS

[0538] 51 ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ

[0539] 101 GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

[0540] 151 DNALcSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

[0541] 201 LSSPcTKSFN RGEC

[0542] SEQ ID NO 7: Amino acid sequence of “Trastuzumab” antibody heavy chain with V170C, S340C, E391C and Q441C amino acid substitutions.

[0543] 1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR Ill

[0544] 51 IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG

[0545] 101 GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK

[0546] 151 DYFPEPVTVS WNSGALTSGc HTFPAVLQSS GLYSLSSWT VPSSSLGTQT

[0547] 201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP

[0548] 251 KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN

[0549] 301 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIc KAKGQPREPQ

[0550] 351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP cNNYKTTPPV

[0551] 401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT cKSLSLSPGK

[0552] Example 11 : Synthesis of CHK1 inhibitors serving as ADC payloads.

[0553] CHK1 inhibitors, which can be used as ADC payloads, were synthesized.

[0554] Experimental procedures for CAN-001:

[0555] Synthesis of compound lnt-1 :

[0556] Int 1 fert-Butyl (R)-3-((6-chloropyrazin-2-yl)oxy)piperidine-1-carboxylate: To a stirred solution of sodium hydride (4.865 g, 2.02 mol) in tetrahydrofuran (60 mL) at O - 5 °C was added 3-hydroxy-1-Boc piperidine (37.348 g, 1 .85 mol ) in tetrahydrofuran (60 mL) and the reaction mixture was stirred at 0-5 °C for 1 h. 2,6- Dichloropyrazine (25 g, 1.68 mol) as a solution in tetrahydrofuran (60 mL) was added dropwise for 1.5 hours at same temperature and the reaction mixture was stirred at room temperature for 2h.The progress of the reaction was monitored by TLC and LCMS. After completion of reaction, the reaction mixture was quenched with ice-cold water and extracted with ethyl acetate twice. The combined organic layer was dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residual oil is triturated with 5 % dichloromethane in hexane and filtered under vacuum to give crude. The crude product was triturated with 5 % dichloromethane in hexanes to afford lnt-1 (15 g) as a white solid.1H NMR (500 MHz, CHLOROFORM-d) δ 1.45-1.32 (m, 9H), 1.97-1.88 (m, 4H), 3.75- 3.55 (m, 4H), 4.13 (q, J = 7.00 Hz, 1 H), 8.09 (s, 1 H), 8.14 (s, 1 H). LC-MS (M+H): 314.30.

[0557] Synthesis of compound lnt-2:

[0558] Int 2 fert-Butyl (R)-3-(6-((5-bromo-1H-Pyrazol-3-yl amino) pyrazin-2-yloxy) piperidine-1 -carboxylate: To a stirred solution of tert-butyl (R)-3-((6-chloropyrazin-2-yl) oxy) piperidine-1 -carboxylate (500 mg, 1.593 mmol, 1 .0 eq.) in terf-Butanol (2 mL) were added sodium tert-Butoxi de (306.264 mg, 3.187 mmol, 2.0 eq.), 5-bromo-1 H-pyrazol-3-amine (387.188 mg, 2.390 mmol, 1 .5 equiv.) and XPhosPdG2 (116.482 mg, 0.159 mmol, 0.1 eq.) at room temperature and the reaction mixture was stirred at 80 °C for 5h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was filtered through celite pad and washed with methanol: DCM, filtrate was concentrated under reduced pressure to give a residue. The residue was diluted with brine solution, extracted with ethyl acetate twice. The combined organic layer was dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography, using silica gel (230-400 mesh), eluted with 80% ethyl acetate in petroleum ether to afford lnt-2 (200 mg, Y: 28%) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 1.23-1.05 (m, 9H), 1.47-1.38 (m, 2H), 1.83-1.75 (m, 2H), 1.95 (s, 1 H), 3.16-3.08 (m, 1 H), 3.63-3.50 (m, 1 H), 3.92 (s, 1 H), 4.91 (br s, 1H), 6.31 (br s, 1 H), 7.55 (s, 1 H), 7.85 (s, 1 H), 9.97 (s, 1 H), 12.70 (s, 1 H). LC-MS (M+H): 439.27 & (M+H+2): 441.26.

[0559] Synthesis of compound lnt-3: tert-Butyl (R)-3-( (6-( (5-bromo-1-(tert-butoxy carbonyl)-1 H-pyrazol-3-yl) (tert-butoxy carbonyl) amino) pyrazin-2-yl) oxy) piperidine-1 -carboxylate: To a stirred solution of methyl tert-butyl (R)-3-((6- ((5-bromo-1 H-pyrazol-3-yl) amino) pyrazin-2-yl) oxy) piperidine-1 -carboxylate (2.0 g, 0.45 mmol, 1 eq.) in dichloromethane (20 mL) was added di-tert-butyl di carbonate (10 mL, 5 vol.) and / V-Ethyl di isopropyl amine (3.96 ml, 0.22 mmol, 5 eq.) at 00C and the reaction mixture was stirred at 70 °C for 16h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was quenched with ice cold water, extracted with dichloromethane twice. The combined organic layer was washed with brine and dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by column chromatography using silica gel (230-400 mesh), eluted with 30% ethyl acetate in petroleum ether to afford lnt-3 (1.2 g, Y: 41.21%) as a yellow solid. LCMS (M+H): 639.64 & (M+H+2): 641.65.

[0560] Synthesis of compound Int-A: tert-Butyl (R)-3-( (6-( (tert-butoxy carbonyl) (1 -(tert-butoxy carbonyl)-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-Pyrazol-3-yl) amino) pyrazin-2-yl)oxy) piperidine-1 -carboxylate: To a stirred solution of tert-butyl tert-butyl (R)-3-( (6-( (5-bromo-1 -(tert-butoxy carbonyl)-1 H-pyrazol-3-yl) (tert-butoxy carbonyl) amino) pyrazin-2-yl) oxy)piperidine-1 -carboxylate (500 mg, 0.78 mmol, 1.0 eq.) in 1 ,4-dioxane (5 mL) were added bis(pinacolato) di boron (277.94 mg, 1.09 mmol, 1.4 eq.), potassium acetate (153.23 mg, 1.56 mmol, 2.0 eq.), and [1 ,1 '-bis(diphenyl phosphino) ferrocene] di chloro palladium(ll) (114.41 mg, 0.15 mmol, 0.2 eq.) at room temperature and the reaction mixture was stirred at 110 °C for 12h under nitrogen. After completion of reaction, the reaction mixture was filtered through celite pad and filtrate was concentrated under reduced pressure to give crude compound to afford Int-A (220 mg, Y: 40.98) as a brown solid. LC-MS (M+H): 605.78.

[0561] Synthesis of compound lnt-4: tert-Butyl (3-(2-bromo-3-methoxy phenoxy) propyl) carbamate: To the stirred solution of 2-bromo-3- methoxy phenol (1 .0 g, 4.92 mmol, 1 .0 eq.) and tert-butyl (3-bromo propyl) carbamate (1 .75 g, 7.38 mmol, 1.5 eq.) in N,N-Dimethylformamide (10 mL) was added cesium carbonate (3.21 g, 9.85 mmol, 2.0 eq.) at room temperature and the reaction mixture was stirred at same temperature for 12h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was diluted with water and extracted with ethyl acetate twice. The combined organic layer dried over anhydrous NasSCU, filtered and filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica gel (100-200 mesh), eluted with 20% ethyl acetate in pert ether to afford lnt-4 (1.4 g, Y: 78.9%) as a white solid.1H NMR (500 MHz, CHLOROFORM-d) δ 1 .43 (s, 9H), 2.04 (t, J = 5.50 Hz, 2H), 3.45-3.37 (m, 2H), 3.90 (s, 3H), 4.11 (t, J= 5.50 Hz, 2H), 6.59-6.54 (m, 2H), 7.26-7.20 (m, 1 H). LC-MS (M+H-100): 260.34. NH proton was not observed in proton NMR.

[0562] Synthesis of compound lnt-5: tert-Butyl (R)-3- ( (6-((tert-butoxy carbonyl) (1 -(tert-butoxy carbonyl)-5-(2-(3-( (tert-butoxy carbonyl)amino)propoxy)-6-methoxy phenyl)-1H-pyrazol-3-yl) amino) pyrazin-2-yl) oxy) piperidine-1 -carboxylate: To the stirred solution of tert-butyl (R)-3-( (6-( (tert-butoxy carbonyl)(1-(tert- butoxy carbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1 H-pyrazol-3-yl)amino) pyrazin-2-yl)oxy) piperidine-1 -carboxylate (200 mg, 0.29 mmol, 1.0 eq.) in toluene (2 mL) and water (0.2 ml) were added tert-butyl (3-(2-bromo-3-methoxy phenoxy) propyl)carbamate (125.92 mg, 0.35 mmol, 1.2 eq.) and potassium phosphate (123 mg, 0.58 mmol, 2.0 eq.) and after degassed with nitrogen for 5 min, then SPhos (11 .95 mg, 0.02 mmol, 0.10 eq.) and palladium acetate (3.27 mg, 0.01 mmol, 0.05 eq.) were added and the reaction mixture was stirred at 110 °C for 12h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was filtered through celite pad, filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica gel (230-400 mesh), eluted with 30-80% ethyl acetate in 1 % triethylamine in petroleum ether to afford lnt-5 (150 mg, Y: 61.31%) as a yellow solid. LC-MS (M+H-100): 740.92 & (M+H+2): 741.02.

[0563] Synthesis of compound CAN-001 :

[0564] CAN-001

[0565] (R)-N-(5-(2-(3-Aminopropoxy)-6-methoxyphenyl)-1 H-pyrazol-3-yl)-6-(piperidin-3-yloxy)pyrazin-2- amine: To a stirred solution of tert-butyl (R)-3-((6-((tert-butoxycarbonyl)(1-(tert-butoxycarbonyl)-5-(2- ((1r,3R)-3-((tert-butoxycarbonyl)amino)cyclobutoxy)-6-methoxyphenyl)-1 H-pyrazol-3-yl)amino)pyrazin-2- yl)oxy)pi peridi ne-1 -carboxylate (100 mg, 0.11 mmol, 1.0 eq.) in dichloromethane (1 ml) formic acid (0.5 mL) was added at room temperature and the reaction mixture was stirred at 50°C for 18h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure to give crude compound. The crude compound was purified by Prep-HPLC to afford CAN-001 (16 mg, Y: 30.58%) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 1 .33-1 .44 (m, 2H), 1.47-1.57 (m, 2H), 1.59-1.71 (m, 2H), 1.86-1.96 (m, 2H), 2.05-2.16 (m, 2H), 2.65-2.68 (m, 2H), 2.71-2.78 (m, 2H), 3.08-3.13 (m, 2H), 3.80 (s, 1 H), 4.09 (br t, J = 5.88 Hz, 2H), 6.62 (s, 1 H), 6.76 (d, J = 8.50 Hz, 2H), 7.29-7.34 (m, 1 H), 7.46 (s, 1 H), 8.14 (s, 1 H), 8.39 (s, 2H), 9.64 (s, 1 H). LCMS (M+H): 440.55. NH Proton was not observed in proton NMR.

[0566] Prep-HPLC Condition: Mobile Phase: A- 0.1% FA in Water: B-Acetonitrile Column: XSELECT C18 19X150mm, 5pm Gradient: Time / %B of 0 / 5; 7 / 32; 7.1 / 100; 13 / 100; 13.1 / 5; 15 / 5 Flow Rate: 12ml / min Synthetic scheme for CAN-002:

[0567] Experimental procedures for CAN-002:

[0568] Synthesis of compound lnt-1 : lnt-1

[0569] 2-Bromo-5-methylbenzene-1,3-diol: 5-methylbenzene-1 ,3-diol (500 mg, 4.02 mmol, 1 eq.) in chloroform (8 mL) was added bromine (686 mL, 13.29 mmol, 3.3 eq.) at room temperature and the reaction mixture was stirred at 70 °C for 1 h. The progress of the reaction was monitored by TLC and LCMS. After completion of reaction, the reaction mixture was concentrated under reduced pressure to give residue. The residue was dissolved in methanol (2 mL) and water (5 mL). The solution of sodium sulphate anhydrous (1.015 g, 8.05 mmol, 2 eq.) and sodium hydroxide (322.22 mg, 8.05 mmol, 2 eq.) was added at room temperature and the reaction mixture was stirred at same temperature for 1 h. Acidified with a 5% HCI, and extracted with ethyl acetate thrice. The combined organic layer washed with brine and dried over anhydrous Na2S O4, filtered and filtrate was concentrated under reduced pressure to give crude compound to afford lnt-1 (190 mg, Y; 38%), as a white semi-solid.

[0570] 1H NMR (400 MHz, CHLOROFORM-D) δ 2.24 (s, 3H), 5.28 (s, 2H), 6.43 (s, 2H). LC-MS (M+H): 203.07 & (M+H+2): 205.05. Synthesis of compound lnt-2: lnt-2

[0571] 2-Bromo-3-methoxy-5-methylphenol: To the stirred solution of 2-bromo-5-methylbenzene-1,3-diol (180 mg, 0.88 mmol, 1 eq.) in acetone (2 mL) were added dimethyl sulfate (167.72 mg, 1.33 mmol, 1.5 eq.) and potassium carbonate (245.04 mg, 1.77 mmol, 2.0 eq.) at 0 °C, and the reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure to give residue. The residue was diluted with ethyl acetate and washed with water, the combined organic layer was dried over anhydrous Na2SC>4, filtered and filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica gel (100-200 mesh), eluted with 20% ethyl acetate in petroleum ether to afford lnt-2 (110 mg, Y: 57.16%) as a white solid. LC-MS (M+H+2): 219.16.

[0572] Synthesis of compound lnt-3: tert-Butyl (3-(2-bromo-3-methoxy-5-methylphenoxy) propyl) carbamate: To the stirred solution of 2- bromo-3-methoxy-5-methylphenol (500 mg, 2.30 mmol, 1 .0 eq.) and tert-butyl (3-bromopropyl) carbamate (822.77 mg, 3.45 mmol, 1.5 eq.) in / V, / V-Dimethylformamide (5 mL) was added cesium carbonate (1.50 g, 4.60 mmol, 2.0 eq.) at 0 °C and the reaction mixture was stirred room temperature for 5h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was quenched with ice cold water and extracted with ethyl acetate three times. The combined organic layer was washed with brine and dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica gel (100-200 mesh), eluted with 20-30% ethyl acetate in petroleum ether to afford lnt-3 (680 mg, Y: 78.87%) as a brown semi-solid. LC-MS (M+H): 374.37. Synthesis of compound lnt-4: tert-Butyl (R)-3-( (6-((tert-butoxy carbonyl) (1 -(tert-butoxy carbonyl)-5-(2-(3-( (tert-butoxy carbonyl) amino) propoxy)-6-methoxy-4-methyl phenyl )-1 H-pyrazo l-3-y I) amino) pyrazin-2-yl)oxy) piperidine- 1 -carboxylate: To the stirred solution of tert-butyl (R)-3-( (6-( (tert-butoxy carbonyl) (1 -(tert-butoxy carbonyl)-5-(4, 4, 5, 5-tetramethyl- 1 ,3, 2-dioxaborolan-2-yl)-1 H-pyrazol-3-yl)amino) pyrazin-2-yl) oxy) piperidine-1 -carboxylate (200 mg, 0.29 mmol, 1.0 eq.) in toluene (2 ml_) and water (0.2 mL) were added tert-butyl (3-(2-bromo-3-methoxy-5-methyl phenoxy) propyl) carbamate (163.53 mg, 0.43 mmol, 1.5 eq.) and tri potassium phosphate (61.75 mg, 0.29 mmol, 2.0 eq.) and after degassed with nitrogen for 5 min, followed by addition of SPhos (11.95 mg, 0.02 mmol, 0.10 eq.) and palladium acetate (3.27 mg, 0.01 mmol, 0.05 eq.) and the reaction mixture was stirred at 120 0°C for 12h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was filtered through celite pad, filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica gel (230-400 mesh), eluted with 50% ethyl acetate in petroleum ether to afford lnt-4 (70 mg, Y: 28.14%) as a brown semi- solid. LCMS (M+H-100): 755.01 & (M-H-100): 753.00.

[0573] Synthesis of compound CAN-002:

[0574] (R)-N-(5-(2-(3-Amino propoxy)-6-methoxy-4-methyl phenyl)-1 H-pyrazol-3-yl)-6-(piperidin-3-yloxy) pyrazin-2-amine: To the stirred solution of tert-butyl (R)-3-( (6-((tert-butoxy carbonyl) (1 -(tert-butoxy carbonyl)-5-(2-(3-( (tert-butoxy carbonyl) amino) propoxy)-6-methoxy-4-methyl phenyl)-1 H-pyrazol-3- yl)amino) pyrazin-2-yl) oxy) piperidine-1 -carboxylate (50 mg, 0.05 mmol, 1 eq.) in formic acid (1.0 mL, 0.58 mmol) at room temperature and the reaction mixture was stirred at 50 °C for 16h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure to give crude compound. The crude compound was purified by reverse phase using C18 column, eluted with 1 % formic acid H2O: Acetonitrile and further purified by Prep HPLC to afford CAN-002 (7 mg, Y: 26.36%) as a pale brown semi-solid.1H NMR (400 MHz, DMSO-d6) δ 1.35-1.46 (m, 1 H), 1.48-1.59 (m, 1 H), 1.63-1.71 (m, 1 H), 1.88-1.98 (m, 2H), 2.05-2.13 (m, 1 H), 2.32-2.35 (m, 2H), 2.40- 2.45 (m, 1 H), 2.65-2.68 (m, 1 H), 2.72-2.79 (m, 1 H), 2.81-2.88 (m, 2H), 3.08-3.14 (m, 1 H), 3.79 (s, 4H), 4.08 (br t, J = 5.75 Hz, 2H), 4.82-4.90 (m, 2H), 6.59 (d, J = 2.25 Hz, 3H), 7.46 (s, 1 H), 8.17 (s, 1 H), 8.37 (br s, 3H), 9.65 (s, 1 H). LCMS (M+H): 454.54.

[0575] Prep-HPLC Condition: Mobile Phase:A- 0.1% FA in Water: B-Acetonitrile Column: XSELECT C18 19X150mm, 5|jm Gradient: Time / %B of 0 / 5; 7 / 32; 7.1 / 100; 13 / 100; 13.1 / 5; 15 / 5 Flow Rate: 12ml / min

[0576] Synthetic scheme for CAN-003:

[0577] Experimental procedures for CAN-003:

[0578] Synthesis of compound lnt-1 : ferf-Butyl ((1r,3r)-3-(2-bromo-3-methoxyphenoxy) cyclo butyl) carbamate: To the stirred solution of 2-bromo-3-methoxyphenol (300 mg, 1.47 mmol, 1 eq.) and tert-butyl ((1s,3s)-3-hydroxycyclobutyl) carbamate (414.47 mg, 2.21 mmol, 1.5 eq.) in tetrahydrofuran (3.0 mL) were added triphenyl phosphine, pure (775.09 mg, 2.95 mmol, 2.0 eq.), diethyl azodicarboxylate (0.46 mL, 2.95 mmol, 2.0 eq.) at 0 °C and the reaction mixture was stirred at 50 °C for 12h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was diluted with water and extracted with ethyl acetate twice. The combined organic layer was dried over anhydrous Na2SC>4, filtered and filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by sepa bean using (100-200 silica), eluted with 25% ethyl acetate in petroleum ether to afford lnt-1 (100 mg, Y: 18.24%) as a white solid.1H NMR (400 MHz, CHLOROFORM-d) δ 1.60-1.45 (s, 9H), 2.41-2.40 (m, 2H), 2.66-2.60 (m, 2H), 3.89 (s, 3H), 4.31 (s, 1 H), 4.86-4.75 (m, 2H), 6.32 (d, J = 8.40 Hz, 1 H), 6.55 (d, J = 8.40 Hz, 1 H), 7.17 (t, J = 8.40 Hz, 1 H).

[0579] Synthesis of compound lnt-2: terf-Butyl(R)-3-((6-((tert-butoxycarbonyl)(1-(tert-butoxycarbonyl)-5-(2-((1r,3R)-3-((tert- butoxycarbonyl)amino)cyclobutoxy)-6-methoxyphenyl)-1 H-pyrazol-3-yl)amino)pyrazin-2- yl)oxy)piperidine-1-carboxylate: To a stirred solution of tert-butyl (R)-3-((6-((tert-butoxycarbonyl)(1 - (tert-butoxycarbonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1 H-pyrazol-3-yl)amino)pyrazin-2- yl)oxy)piperidine-1 -carboxylate (0.15 mg, 0.21 mmol, 1.0 eq.) in toluene (1.5 ml_) and water (0.5 mL) were added tert-butyl ((1 r,3r)-3-(2-bromo-3-methoxyphenoxy) cyclo butyl)carbamate (0.09 mg, 0.26 mmol, 1.2 eq.), potassium phosphate (0.09 mg, 0.43 mmol, 2.0 eq.) in Sphos (0.009 mg, 0.021 mmol, 0.10 eq.), palladium acetate (0.002 mg, 0.010 mmol, 0.05 eq.) at room temperature and the reaction mixture was stirred at 110° C for 12 h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was filtered through celite pad, filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica (230-400 mesh), eluted with 50%-80% ethyl acetate and petroleum ether in 1 % triethylamine to afford I nt-2 (90 mg, Y: 50.56%) as a yellow solid. LCMS (M+H-100): 753.00 Synthesis of compound CAN-003:

[0580] (R)-N-(5-(2-(3-Aminopropoxy)-6-methoxy-4-methylphenyl)-1H-Pyrazol-3-yl)-6-(piperidin-3-yloxy) pyrazin-2-amine: To a stirred solution of tert-butyl (R)-3-((6-( (tert-butoxy carbonyl) (1-(tert-butoxy carbonyl)-5-(2-( (1 r,3R)-3-( (tert-butoxy carbonyl) amino) cyclo butoxy)-6-methoxy phenyl)-1 H-pyrazol-3- yl) amino) pyrazin-2-yl) oxy) piperidi ne-1 -carboxylate (70 mg, 0.082 mmol, 1 .0 eq.) was added formic acid (1mL) at 0 °C and the reaction mixture was stirred at 50 °C for 16h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure to give crude compound. The crude compound was purified by Prep-HPLC to afford CAN-003 (10 mg, Y: 40.43%) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 1.35-1.48 (m, 2H), 1.53-1.74 (m, 2H), 2.03-2.13 (m, 2H), 2.36 (br t, J = 5.88 Hz, 2H), 2.59 (br d, J= 9.01 Hz, 2H), 2.68-2.81 (m, 2H), 3.13 (br d, J = 10.26 Hz, 1 H), 3.65-3.71 (m, 2H), 3.80 (s, 4H), 6.48 (d, J = 8.50 Hz, 1 H), 6.66 (s, 1 H), 6.75 (d, J = 8.50 Hz, 2H), 7.29 (t, J = 8.38 Hz, 1 H), 7.47 (s, 2H), 8.15 (s, 1 H), 9.60 (s, 1 H), 11.99-12.12 (m, 1 H). LCMS (M+H): 452.52.

[0581] Prep-HPLC Condition: Mobile Phase: A- 0.1% FA in Water: B-Acetonitrile Column: XSELECT C18 19X150mm, 5|jm Gradient: Time / %B of 0 / 5; 7 / 32; 7.1 / 100; 13 / 100; 13.1 / 5; 15 / 5 Flow Rate: 12ml / min.

[0582] Synthetic scheme for CAN-005:

[0583]

[0584] Synthesis of compound lnt-3: tert-Butyl (3-((3-bromo-2-methoxy-6-methylpyridin-4-yl) oxy) propyl) carbamate: To the stirred solution of 3-bromo-2-methoxy-6-methylpyridin-4-ol (300 mg, 1.37 mmol, 1 eq.) in N,N- Dimethylformamide (3.0 mL) were added tert-butyl (3-bromopropyl) carbamate (393.14 mg, 1.65 mmol, 1.2 eq.) and cesium carbonate (896.54 mg, 2.75 mmol, 2.0 eq.) at 0 °C and the reaction mixture was stirred at room temperature for 8 h. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was quenched with water and extracted with ethyl acetate twice. The combined organic layer was washed with brine and dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure to give crude. The crude compound was purified by column chromatography using silica (100-200 mesh), eluted with 10-30% ethyl acetate in petroleum ether to afford lnt-3 (320 mg, Y: 62.18%) as a yellow semi-solid.1H NMR (500 MHz, CHLOROFOM-D) δ 1.43 (s, 9H), 2.05 (t, J = 6.00 Hz, 2H), 2.39 (s, 3H), 3.38 (q, J = 6.00 Hz, 2H), 3.98 (s, 3H), 4.13 (t, J = 5.50 Hz, 2H), 5.18 (s, 1 H), 6.34 (s, 1 H). LC-MS (M+H): 375.40 & (M+H+2): 377.38.

[0585] Synthesis of compound lnt-4:

[0586] tert-Butyl (R)-3-( (6-((tert-butoxy carbonyl)(1 -(tert-butoxy carbonyl)-5-(4-(3-( (tert-butoxy carbonyl) amino) propoxy)-2-methoxy-6-methyl pyridin-3-yl)-1H-pyrazol-3-yl)amino) pyrazin-2-yl)oxy) piperidine-1 -carboxylate: To the stirred solution of tert-butyl (R)-3-( (6-( (tert-butoxy carbonyl) (1-(tert- butoxy carbonyl)-5-(4,4, 5, 5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazol-3-yl) amino) pyrazin-2-yl) oxy) piperidine-1 -carboxylate (500 mg, 0.72 mmol, 1.0 eq.) and tert-butyl (3-( (3-bromo-2-methoxy-6- methyl pyridin-4-yl)oxy) propyl)carbamate (409.90 mg, 1.09 mmol, 1.5 eq.) in toluene (5.0 mL) and water (1.0 mL), tri potassium phosphate (308.76 mg, 1.45 mmol, 2.0 eq.), was added and the reaction mixture was degassed with nitrogen for 5 minutes, followed by addition of SPhos (14.94 mg, 0.03 mmol, 0.05 eq.) and palladium acetate (16.34 mg, 0.07 mmol, 0.10 eq.) and the reaction mixture was stirred at 120 °C for 12 h. The progress of the reaction was monitored by TLC. After completion of reaction, reaction mixture was filtered through celite pad and washed with ethyl acetate. Filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by flash chromatography using silica gel (234-400 mesh), eluted with 50%-80% ethylacetate: 1 % triethylamine in petroleum ether to afford Int- 4 (220 mg, Y: 35.33%) as a brown semi-solid. LC-MS (M+H-100): 755.81 & (M-H-100): 753.77.

[0587] Synthesis of compound CAN-005:

[0588] (R)-N-(5-(4-(3-Amino propoxy)-2-methoxy-6-methyl pyridin-3-yl)-1H-Pyrazol-3-yl)-6-(piperidin-3- yloxy) pyrazin-2-amine: To the stirred solution of tert-butyl (R)-3-( (6-( (tert-butoxy carbonyl) (1 -(tert- butoxy carbonyl)-5-(2-((1r,3R)-3-( (tert-butoxy carbonyl) amino) cyclo butoxy)-6-methoxy phenyl)-1 H- pyrazol-3-yl) amino) pyrazin-2-yl) oxy) piperidine-1-carboxylate (200 mg, 0.23 mmol, 1.0 eq.) in dichloromethane (1 .0 mL) was added formic acid (1.0 ml_) at rt and the reaction mixture was stirred at 50 °C for 12h. The progress of the reaction was monitored by TLC and LCMS. After completion of reaction, the reaction mixture was concentrated under reduced pressure to give crude product. The crude compound was purified by reverse phase purification using C18 column, eluted with 1% formic acid in water and acetonitrile to give CAN-005. Further purified by Prep-HPLC, to afford CAN-005 (10 mg, Y: 12.54%) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 1.20-1.27 (m, 1 H), 1.64-1.80 (m, 3H), 1.88- 2.12 (m, 3H), 2.28-2.35 (m, 3H), 2.42 (d, J = 2.75 Hz, 1H), 2.67 (dt, J = 3.69, 1.78 Hz, 1 H), 2.85-3.00 (m, 1 H), 3.09-3.21 (m, 2H), 3.28-3.33 (m, 1 H), 3.90 (d, J = 1.50 Hz, 2H), 4.20-4.29 (m, 2H), 4.33-4.39 (m, 1 H), 5.18-5.42 (m, 1 H), 6.62 (s, 1 H), 6.78-6.82 (m, 1 H), 7.56 (d, J= 2.75 Hz, 1 H), 7.68-7.85 (m, 2H), 8.17- 8.22 (m, 1 H), 9.63 (br d, J = 16.76 Hz, 1 H), 12.01 (br s, 1 H). LCMS (M+H): 455.54.

[0589] Prep-HPLC Condition: Mobile Phase: A- 0.1% FA in Water: B-Aceto nitrile Column: YMC ACTUS C18 150*20mm 5um, GRADIANT- Time / %B 0 / 5;3 / 5; 12 / 34; 12.1 / 98; 15 / 98; 15.1 / 5; 17 / 5, Flow Rate: 15ml / min.

[0590] Synthetic scheme for CAN-010: 4-amino-N-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]butanamide

[0591] 3-(dimethylamino)-1-(2-hydroxy-6-methoxy-phenyl)prop-2-en-1-one To a mixture of 1-(2-hydroxy-6-methoxy-phenyl) ethanone (10 g, 60.18 mmol, 1 eq) (Bide) in toluene (100 mL) was added DMF-DMA (25 g, 209.80 mmol, 27.87 mL, 3.49 eq), then the mixture was stirred at 80 °C for 12 h. The reaction mixture was concentrated to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 120 mL / min) to afford 3-(dimethylamino) -1-(2-hydroxy-6-methoxy- phenyl) prop-2-en-1-one (13.1 g, 41.45 mmol, 68.87% yield, 70% purity) as yellow oil.

[0592] MS m / z: 222.2 [M+H]+tert-butyl N-[3-[2-[3-(dimethylamino)prop-2-enoyl]-3-methoxy-phenoxy]propyl]carbamate

[0593] 2 4

[0594] To a mixture of 3-(dimethylamino) -1-(2-hydroxy-6-methoxy-phenyl) prop-2-en-1-one (13.1 g, 59.21 mmol, 1 eq) in DMF (120 mL) was added tert-butyl N-(3-bromopropyl) carbamate (17 g, 71.39 mmol, 1.21 eq) (Bide) and K3PO4 (16.4 g, 77.26 mmol, 1.3 eq), then the mixture was stirred at 25 °C for 12 h. The residue was partitioned between ethyl acetate (300 mL) and brine (200 mL). The aqueous layer was extracted with ethyl acetate (100 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product tert-butyl N-[3-[2-[3-(dimethylamino) prop-2- enoyl]-3-methoxy-phenoxy]propyl]carbamate (26.4 g, 27.90 mmol, 47.13% yield, 40% purity) as yellow oil was used in next step without purification.

[0595] MS m / z: 379.3 [M+H]+tert-butyl N-[3-(2-isoxazol-5-yl-3-methoxy-phenoxy)propyl]carbamate

[0596] 4 5

[0597] To a mixture of tert-butyl N-[3-[2-[ 3-(dimethylamino) prop-2-enoyl]-3-methoxy-phenoxy]propyl]carbamate (26.4 g, 27.90 mmol, 1 eq) in EtOH (100 mL) was added NH2OH. HCI (1.94 g, 27.90 mmol, 1 eq), then the mixture was stirred at 35 °C for 12 h. The reaction mixture was concentrated then partitioned between ethyl acetate (50 mL) and water (30 mL). The aqueous layer was extracted with ethyl acetate (30 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 100 mL / min) to afford tert-butyl N-[3- (2-isoxazol-5-yl-3-methoxy-phenoxy) propyl]carbamate (6.3 g, 18.08 mmol, 64.81% yield, 100% purity) as yellow oil.

[0598] 1H NMR (400 MHz, CHLOROFORM-d) δ = 8.33 (d, J = 1.0 Hz, 1 H), 7.36 (t, J= 8.4 Hz, 1 H), 6.65 (dd, J= 3.9, 8.4 Hz, 2H), 6.51 - 6.48 (m, 1 H), 4.06 (t, J = 5.8 Hz, 2H), 3.84 (s, 3H), 3.29 - 3.22 (m, 2H), 1 .95 (quin, J = 5.9 Hz, 2H), 1.43 (s, 9H).

[0599] MS m / z: 249.1 [M-100+H]+tert-butyl N-[3-[2-(2-cyanoacetyl)-3-methoxy-phenoxy]propyl]carbamate

[0600] To a mixture of tert-butyl N-[3-(2-isoxazol-5-yl-3-methoxy-phenoxy) propyl]carbamate (6.3 g, 18.08 mmol, 1 eq) in MeOH (30 mL) was added KOH (3.5 M, 6.72 mL, 1.30 eq), then the mixture was stirred at 40 °C for 12 h. The reaction mixture was added AcOH to adjust pH=7 then was partitioned between ethyl acetate (80 mL) and brine (40 mL). The aqueous layer was extracted with ethyl acetate (50 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product was triturated with MTBE at 25°C for 30 min then collected the tert-butyl N-[3-[2-(2-cyanoacetyl) -3-methoxy-phenoxy]propyl]carbamate (7.2 g, 15.50 mmol, 85.71% yield, 75% purity) as yellow solid. MS m / z: 249.2 [M- 100+H]+tert-butyl N-[3-[2-(3-amino-1H-pyrazol-5-yl)-3-methoxy-phenoxy]propyl]carbamate

[0601] To a mixture of tert-butyl N-[3-[2-(2-cyanoacetyl) -3-methoxy-phenoxy]propyl]carbamate (1 g, 2.15 mmol, 1 eq) in EtOH (15 mL) / n-BuOH (5 mL) was added AcOH (130 mg, 2.16 mmol, 123.93 μL, 1.01 eq), the N2H4’H2O (210 mg, 3.57 mmol, 203.49 85% purity, 1.6 μ6L e,q) was added into the mixture at 0 °C, then the mixture was stirred at 80°C for 12 h. The reaction mixture was partitioned between ethyl acetate (30 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ether gradient @ 20 mL / min) to afford tert-butyl N-[3-[2-(3-amino-1 H-pyrazol-5- yl) -3-methoxy-phenoxy]propyl]carbamate (600 mg, 1.64 mmol, 76.13% yield, 99% purity) as yellow oil. MS m / z: 363.3 [M+H]+tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxyjpropyljcarbamate

[0602] To a mixture of tert-butyl N-[3-[2-(3-amino-1 H-pyrazol-5-yl) -3-methoxy-phenoxy]propyl]carbamate (600 mg, 1.66 mmol, 1 eq) in DMSO (10 mL) was added 5-chloropyrazine-2-carbonitrile (270 mg, 1.93 mmol, 1.17 eq) (Bide) and 4-ethylmorpholine (230 mg, 2.00 mmol, 252.75 1.21 eq), then the mixture μL w,as stirred at 80 °C for 3 h. The reaction mixture was partitioned between ethyl acetate (40 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product was triturated with MTBE at 25°C for 30 min then collected the tert-butyl N-[3-[2-[3- [(5-cyanopyrazi n-2-yl) ami no]- 1 H-pyrazol- 5-yl]-3-methoxy-phenoxy]propyl]carbamate (516 mg, 1.11 mmol, 66.96% yield, 100% purity) as yellow solid.

[0603] MS m / z: 466.2 [M+H]+

[0604] 5-[[5-[2-(3-aminopropoxy)-6-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile To a mixture of tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]carbamate (516 mg, 1.11 mmol, 1 eq) in dioxane (1 mL) was added HCI / dioxane (2 M, 1 mL, 1 .80 eq), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was filtered and collected the residue. The crude product 5-[[5-[2-(3-aminopropoxy) -6-methoxy-phenyl]-1 H-pyrazol-3- yl]amino]pyrazine-2-carbonitrile (500 mg, crude) as yellow solid was used in next step without purification.1H NMR (400 MHz, DMSO-d6) δ = 10.79 (s, 1 H), 8.73 - 8.67 (m, 1 H), 8.61 - 8.49 (m, 1 H), 8.19 - 8.04 (m, 3H), 7.33 (t, J = 8.4 Hz, 1 H), 6.91 - 6.83 (m, 1 H), 6.80 - 6.76 (m, 2H), 4.11 - 4.10 (m, 2H), 3.81 (s, 3H), 3.00 - 2.90 (m, 2H), 2.09 - 2.01 (m, 2H) tert-butyl N-[4-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propylamino]-4-oxo-butyl]carbamate

[0605] To a mixture of 5-[[5-[2-(3-aminopropoxy) -6-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile (50 mg, 136.84 μmol, 1 eq) in DMF (1 mL) was added 4-(tert-butoxycarbonylamino) butanoic acid (30 mg, 147.61 μmol, 1.08 eq) (Bide), HATU (80 mg, 210.40 μmol, 1.54 eq) and DIEA (60 mg, 464.24 μmol, 80.86 3 μ.3L9, eq), then the mixture was stirred at 25 °C for 2 h. The residue was partitioned between ethyl acetate (5 mL) and brine (5 mL). The aqueous layer was extracted with ethyl acetate (5 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product tert-butyl N-[4-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3- methoxy-phenoxy]propylamino]-4-oxo-butyl]carbamate (60 mg, crude) as yellow oil was used in next step directly without purification.

[0606] MS m / z: 551.4 [M+H]+

[0607] 4-amino-N-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]butanamide

[0608] To a mixture of tert-butyl N-[4-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propylamino]-4-oxo-butyl]carbamate (60 mg, 108.97 μmol, 1 eq) in DCM (1 mL) was added TFA (153.50 mg, 1.35 mmol, 0.1 mL, 12.35 eq), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Phenomenex luna Cis 150*25 mm* 10um;mobile phase: [water (TFA) -ACN];gradient:15%-45% B over 12 min). The elute was directly lyophilized to give product then wash resin with 10 mL of deionized water then wash resin with 4-amino-N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]butanamide in MeOH (target was adsorbed to the resin), wash resin with deionized water until the pH of the solution is neutral, wash resin with NH3H2O / MeOH=1 :20 (21 mL), target was cleaved from the resin, concentration and lyophilization to give 4-amino-N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]- 1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propyl]butanamide (5.4 mg, 11.27 μmol, 10.34% yield, 94% purity) as yellow solid.

[0609] 1H NMR (400 MHz, METHANOL-d4) δ = 8.49 (s, 2H), 7.35 - 7.28 (m, 1 H), 6.97 (s, 1 H), 6.76 (dd, J= 5.8, 8.2 Hz, 2H), 4.10 (t, J= 5.8 Hz, 2H), 3.89 (s, 3H), 3.41 (t, J = 6.7 Hz, 2H), 2.70 (t, J= 7.3 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H), 2.01 (quin, J= 6.3 Hz, 2H), 1.80 - 1.71 (m, 2H) MS m / z: 451.3 [M+H]+

[0610] Synthetic scheme for CAN-011 :

[0611] 5-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxyjpropylaminojpentanamide

[0612] methyl 5-[tert-butoxycarbonyl-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]amino]pentanoate To a mixture of 5-[[5-[2-(3-aminopropoxy) -6-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile (300 mg, 746.55 μmol, 1 eq, HCI) in DMF (4 mL) was added methyl 5-oxopentanoate (80 mg, 614.72 μmol, 8.23e-1 eq), NaBHaCN (120 mg, 1.91 mmol, 2.56 eq) and TEA (240 mg, 2.37 mmol, 3.18 eq), then the mixture was stirred at 0°C for 6 h. The BOC2O (360 mg, 1 .65 mmol, 2.21 eq) and TEA (240 mg, 2.37 mmol, 3.18 eq) was added into the mixture and stirred at 25°C for 12h. The reaction mixture was partitioned between ethyl acetate (20 mL) and brine (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL) twice, The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-60% Ethyl acetate / Petroleum ether gradient @ 20 mL / min) to afford methyl 5-[tert-butoxycarbonyl-[3-[2-[3-[(5-cyanopyrazi n-2-yl) amino]- 1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]amino]pentanoate (150 mg, 173.38 μmol, 23.22% yield, 67% purity) as yellow oil.

[0613] MS m / z: 580.4 [M+H]+ 5-[tert-butoxycarbonyl-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]amino]pentanoic acid

[0614] To a mixture of methyl 5-[tert-butoxycarbonyl-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3- methoxy-phenoxy]propyl]amino]pentanoate (100 mg, 172.52 μmol, 1 eq) in dioxane (2 mL) was added hydroxy (trimethyl) stannane (150 mg, 829.55 μmol, 4.81 eq), then the mixture was stirred at 80 °C for 12 h. The residue was partitioned between ethyl acetate (10 mL) and aq. KF (5 mL). The aqueous layer was extracted with ethyl acetate (5 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product 5-[tert-butoxycarbonyl-[3-[2-[3-[(5-cyanopyrazin- 2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propyl]amino]pentanoic acid (130 mg, crude) as yellow oil was used in next step without purification.

[0615] MS m / z: 566.4 [M+H]+tert-butyl N-(5-amino-5-oxo-pentyl)-N-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3- methoxy -phenoxy]propyl]carbamate

[0616] To a mixture of 5-[tert-butoxycarbonyl-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]amino]pentanoic acid (130 mg, 229.84 μmol, 1 eq) in DMF (2 mL) was added HATU (130 mg, 341.90 μmol, 1.49 eq) and stirred at 25 °C for 0.5 h, the NH4CI (40 mg, 747.79 μmol, 3.25 eq) and DIEA (90 mg, 696.37 μmol, 121.29 3.03 eq) w μaLs, added into the mixture and stirred at 25°C for 2h. The reaction mixture was partitioned between ethyl acetate (5 mL) and brine (5 mL). The aqueous layer was extracted with ethyl acetate (5 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product was purified by prep-TLC with EtOAc to give tert- butyl N-(5-amino-5-oxo-pentyl) -N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]- 1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]carbamate (50 mg, 88.55 μmol, 38.53% yield, 100% purity) as yellow oil. MS m / z: 565.3[M+H]+

[0617] 5-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxyjpropylaminojpentanamide

[0618] To a mixture of tert-butyl N-(5-amino-5-oxo-pentyl) -N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol- 5-yl]-3-methoxy-phenoxy]propyl]carbamate (50 mg, 88.55 μmol, 1 eq) in DCM (0.5 mL) was added TFA (153.50 mg, 1.35 mmol, 0.1 mL, 15.20 eq), then the mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated to give a residue. The residue was purified by Prep-HPLC (column: Waters Xbridge 150*25 mm* 5pm;mobile phase: [water (FA) -ACN]; gradient: 12%-42% B over 10 min). The eluate was directly lyophilized to give 5-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propylamino]pentanamide (10.4 mg, 22.39 μmol, 25.28% yield, 100% purity) as yellow solid.

[0619] MS m / z: 465.3 [M+H]+

[0620] 1H NMR (400 MHz, DMSO-d6) δ = 12.55 - 12.14 (m, 1 H), 10.83 - 10.68 (m, 1 H), 8.65 (d, J = 1.2 Hz, 1 H), 8.63 - 8.46 (m, 1 H), 8.26 (s, 1 H), 7.34 (t, J = 8.4 Hz, 1 H), 7.26 (br s, 1 H), 6.88 - 6.70 (m, 4H), 4.09 (br t, J = 5.7 Hz, 2H), 3.80 (s, 3H), 2.92 (br t, J = 7.0 Hz, 2H), 2.73 (br s, 2H), 2.08 - 1 .96 (m, 4H), 1 .47 (br s, 4H)

[0621] Synthetic scheme for CAN-012:

[0622] 5-[[5-[2-methoxy-6-[3-(pent-4-ynylamino)propoxy]phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1H-pyrazol-5-yl]-3-methoxy-phenoxy]propyl]-N- pent-4-ynyl-carbamate

[0623] To a mixture of 5-[[5-[2-(3-aminopropoxy) -6-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile (300 mg, 746.55 μmol, 1 eq, HCI) in DMF (4 mL) was added pent-4-ynal (50 mg, 609.01 μmol, 8.16e-1 eq), NaBHsCN (120 mg, 1.91 mmol, 2.56 eq) and TEA (240 mg, 2.37 mmol, 330.12 μL, 3.18 eq), then the mixture was stirred at 25 °C for 6 h. The BOC2O (360 mg, 1.65 mmol, 378.95 μL, 2.21 eq) and TEA (240 mg, 2.37 mmol, 330.12 3.18 eq) w μaLs, added into the mixture and stirred at 25°C for 12h. The reaction mixture was partitioned between ethyl acetate (20 mL) and brine (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL) twice, The combined organic layers were dried over(Na2SO4) and evaporated to give a crude material. The crude product was purified by prep-TLC with PE: EtOAc=1 :1 to give tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propyl]-N- pent-4-ynyl-carbamate (60 mg, 74.49 μmol, 9.98% yield, 66% purity) as yellow oil.

[0624] MS m / z: 532.3 [M+H]+

[0625] 5-[[5-[2-methoxy-6-[3-(pent-4-ynylamino)propoxy]phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile

[0626] To a mixture of tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]-N-pent-4-ynyl-carbamate (60 mg, 112.87 μmol, 1 eq) in dioxane (0.5 mL) was added HCI / dioxane (2 M, 0.1 mL, 1.77 eq), then the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Welch Xtimate Cis 150*25 mm*5pm;mobile phase: [water (FA) -ACN];gradient: 10%-40% B over 10 min). The eluate was directly lyophilized to give 5-[[5-[2-methoxy-6-[3-(pent-4-ynylamino) propoxy]phenyl]-1 H-pyrazol-3- yl]amino]pyrazine-2-carbonitrile (8.4 mg, 19.47 μmol, 17.25% yield, 100% purity) as yellow solid. MS m / z: 432.2 [M+H]+

[0627] 1 H NMR (400 MHz, DMSO-d6) δ = 10.83 - 10.68 (m, 1 H), 8.65 (d, J = 1 .3 Hz, 1 H), 8.61 - 8.46 (m, 1 H), 8.21 (s, 1 H), 7.32 (t, J = 8.4 Hz, 1 H), 6.92 - 6.82 (m, 1 H), 6.77 (d, J = 8.4 Hz, 2H), 4.14 - 4.04 (m, 2H), 3.81 (s, 3H), 2.80 (br t, J = 6.5 Hz, 2H), 2.75 (t, J = 2.6 Hz, 1 H), 2.71 - 2.63 (m, 2H), 2.22 - 2.13 (m, 2H), 1.99 - 1.89 (m, 2H), 1.67 - 1.55 (m, 2H)

[0628] Synthetic scheme for CAN-014:

[0629] 3-(dimethylamino) -1 -(2-hydroxy-6-methoxy -phenyl) prop-2-en-1 -one

[0630] 1 2

[0631] To a mixture of 1-(2-hydroxy-6-methoxy-phenyl) ethanone (10 g, 60.18 mmol, 1 eq) in toluene (100 mL) was added DMF-DMA (25 g, 209.80 mmol, 27.87 mL, 3.49 eq), then the mixture was stirred at 80°C for 12 h. The reaction mixture was concentrated to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 120 mL / min) to afford 3-(dimethylamino) -1-(2-hydroxy-6-methoxy- phenyl) prop-2-en-1-one (11.1 g, 25.08 mmol, 41 .68% yield, 50% purity) as yellow oil.

[0632] MS m / z: 222.2 [M+H]+ 3-(dimethylamino) -1-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]prop-2-en-1-one

[0633] 2 3

[0634] To a mixture of 3-(dimethylamino) -1-(2-hydroxy-6-methoxy-phenyl) prop-2-en-1-one (1 g, 2.26 mmol, 1 eq) in DMF (10 mL) was added K3PO4 (1.44 g, 6.78 mmol, 3 eq), the PMB-CI (346.20 mg, 2.21 mmol, 0.3 mL, 9.78e-1 eq) was added into the mixture at 0 °C, then the mixture was stirred at 25°C for 12 h. The residue was partitioned between ethyl acetate (40 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (30 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product 3-(dimethylamino) -1-[2-methoxy-6-[(4- methoxyphenyl) methoxy]phenyl]prop-2-en-1-one (1.8 g, crude) as yellow oil.

[0635] MS m / z: 342.2 [M+H]+

[0636] 5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]isoxazole

[0637] 3 4

[0638] To a mixture of 3-(dimethylamino) -1 -[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]prop-2-en-1-one (1.8 g, 5.27 mmol, 1 eq) in EtOH (20 mL) was added hydroxylamine;hydrochloride (190 mg, 2.73 mmol, 5.19e-1 eq), then the mixture was stirred at 35 °C for 12 h. The residue was partitioned between ethyl acetate (50 mL) and brine (30 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to afford 5-[2-methoxy-6-[(4- methoxyphenyl) methoxy]phenyl]isoxazole (790 mg, 2.49 mmol, 47.17% yield, 98% purity) as yellow oil. MS m / z: 312.0 [M+H]+

[0639] 1H NMR (400 MHz, CHLOROFORM-d) δ = 8.31 (d, J= 1.8 Hz, 1 H), 7.37 - 7.28 (m, 3H), 6.91 - 6.87 (m, 2H), 6.68 (dd, J = 8.3, 18.0 Hz, 2H), 6.47 (d, J = 1.7 Hz, 1 H), 5.06 (s, 2H), 3.85 (s, 3H), 3.82 (s, 3H)

[0640] 3-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-3-oxo-propanenitrile

[0641] To a mixture of 5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]isoxazole (590 mg, 1.90 mmol, 1 eq) in MeOH (3 mL) was added KOH (3.5 M, 0.7 mL, 1 .29 eq), then the mixture was stirred at 40°C for 12 h. The reaction mixture was added AcOH to adjust pH=7, then partitioned between ethyl acetate (10 mL) and water (5 mL). The aqueous layer was extracted with ethyl acetate (10 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product was triturated with MTBE at 25°C for 30 min then collected the 3-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-3-oxo-propanenitrile (560 mg, crude) as yellow solid.

[0642] 5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-1H-pyrazol-3-amine

[0643] To a mixture of 3-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-3-oxo-propanenitrile (560 mg, 1.80 mmol, 1 eq) in EtOH (6 mL) / butan-1-ol (1 mL) was added AcOH (160 mg, 2.66 mmol, 152.53 μL, 1.48 eq), the lOrHsO (0.860 g, 14.60 mmol, 85wt%, 8.12 eq) was added into the mixture at 0 °C, then the mixture was stirred at 80°C for 12 h. The residue was partitioned between ethyl acetate (30 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ether gradient @ 18 mL / min) to afford 5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-1 H-pyrazol-3-amine (480 mg, 1.45 mmol, 80.38% yield, 98% purity) as off-white oil.

[0644] MS m / z: 326.3 [M+H]+

[0645] 5-[[5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2- carbomtnle

[0646] To a mixture of 5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-1 H-pyrazol-3-amine (480 mg, 1.48 mmol, 1 eq) in DMSO (6 mL) was added 5-chloropyrazine-2-carbonitrile (270 mg, 1.93 mmol, 1.31 eq) and 4-ethylmorpholine (220 mg, 1.91 mmol, 1.29 eq), then the mixture was stirred at 80°C for 3 h. The residue was partitioned between ethyl acetate (30 mL) and brine (30 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product was triturated with MTBE at 25°C for 10 min then filtered and collected the 5-[[5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-1 H-pyrazol-3- yl]amino]pyrazine-2-carbonitrile (440 mg, 1.03 mmol, 69.61 % yield, 100% purity) as yellow solid.

[0647] MS m / z: 429.2 [M+H]+

[0648] 5-[[5-(2-hydroxy-6-methoxy-phenyl) -1 H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile

[0649] To a mixture of 5-[[5-[2-methoxy-6-[(4-methoxyphenyl) methoxy]phenyl]-1 H-pyrazol-3-yl]amino]pyrazine- 2-carbo nitrile (510 mg, 1 .19 mmol, 1 eq) in HCI / dioxane (2 M, 5 mL, 8.40 eq), then the mixture was stirred at 80 °C for 12 hr. The reaction mixture was filtered and collected the residue. The crude product was triturated with MTBE at 25 °C for 10 min then was filtered and collected the 5-[[5-(2-hydroxy-6-methoxy- phenyl) -1 H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile (360 mg, 1.11 mmol, 93.19% yield, 95% purity) as yellow solid.

[0650] MS m / z: 309.2 [M+H]+tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]cyclobutyl]carbamate

[0651] To a solution of 5-[[5-(2-hydroxy-6-methoxy-phenyl) -1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile (100 mg, 324.37 μmol, 1 eq), tert-butyl N-(3-hydroxycyclobutyl) carbamate (70 mg, 373.86 μmol, 1.15 eq), PPhs (100.00 mg, 381.26 μmol, 1.18 eq) in THF (2 mL) was added DIAD (75.00 mg, 370.91 μmol, 1.14 eq) dropwsied at 0 °C, then the mixture was stirred at 50°C for 1 h. The mixture was concentrate to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0~60% Ethyl acetate / Petroleum ethergradient @ 30 mL / min) to give tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]- 1 H-pyrazol-5-yl]-3-methoxy- phenoxy]cyclobutyl]carbamate (200 mg, crude) as a white solid.

[0652] MS m / z: 478.3 [M+H]+tert-butyl N-[2-[5-[6-[(3R) -3-methylmorpholin-4-yl]-1-(1H-pyrazol-3-yl) pyrazolo[3, 4-b]pyridin-4- y I] py razo I - 1 -yl]ethyl]carbamate

[0653] To a solution of tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]cyclobutyl]carbamate (200 mg, 418.84 μmol, 1 eq) in DCM (1 mL) was add TFA (307.00 mg, 2.69 mmol, 0.2 mL, 6.43 eq), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex luna Cis 150*25 mm* 10pm;mobile phase: [water (FA) -ACN];gradient:4%-34% B over 10 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give 5-[[5-[2-(3-aminocyclobutoxy) -6-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile (12.3 mg, 32.59 μmol, 7.78% yield) was obtained as a white solid. MS m / z: 378.1 [M+H]+

[0654] 1H NMR (400 MHz, DMSO-ofe) δ ppm: 12.37 (s, 1 H), 10.77 (s, 1H), 8.64 (d, J = 1.2 Hz, 1 H), 8.55 (s, 1 H), 7.31 (t, J = 8.4 Hz, 1 H), 6.98 (s, 1 H), 6.78 (d, J = 8.0 Hz, 1 H), 6.51 (d, J = 8.4 Hz, 1 H), 5.0-4.91 (m, 1 H), 3.83 (s, 3H), 3.79 - 3.75 (m, 1 H), 2.41 (t, J = 5.6 Hz, 4H)

[0655] Synthetic scheme for CAN-029: tert-butyl N-[3-(2-nitrophenoxy)propyl]carbamate: To a mixture of 2-nitrophenol (2 g, 14.38 mmol, 1 eq) in THF (30 mL) was added tert-butyl N-(3-bromopropyl) carbamate (4.1 g, 17.22 mmol, 1.2 eq), KI (600 mg, 3.61 mmol, 2.51 e-1 eq) and CS2CO3 (7.03 g, 21.57 mmol, 1.5 eq), then the mixture was stirred at 70 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (40 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford tert-butyl N-[3-(2-nitrophenoxy) propyl]carbamate (3.4 g, 11.47 mmol, 79.81% yield) as yellow solid.

[0656] 1H NMR (400 MHz, DMSO-d6) δ = 7.86 (dd, J = 1.6, 8.1 Hz, 1 H), 7.68 - 7.60 (m, 1 H), 7.33 (d, J= 8.4 Hz, 1 H), 7.14 - 7.05 (m, 1 H), 6.92 - 6.83 (m, 1 H), 4.15 (t, J = 6.1 Hz, 2H), 3.08 (q, J = 6.6 Hz, 2H), 1 .83 (quin, J = 6.4 Hz, 2H), 1.36 (s, 9H) tert-butyl N-[3-(2-aminophenoxy)propyl]carbamate: To a mixture of tert-butyl N-[3-(2-nitrophenoxy) propyl]carbamate (3.4 g, 11.47 mmol, 1 eq) in MeOH (30 mL) / H2O (3 mL) was added Fe (3.20 g, 57.37 mmol, 5 eq) and NH4CI (3.07 g, 57.37 mmol, 5 eq), then the mixture was stirred at 80 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (50 mL) and water (30 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford tert-butyl N-[3-(2-aminophenoxy) propyl]carbamate (2.77 g, 10.09 mmol, 87.92% yield, 97% purity) as yellow solid.

[0657] MS m / z: 267.4 [M+H]+

[0658] Phenyl N-pyrazin-2-ylcarbamate: To a mixture of pyrazin-2-amine (2 g, 21.03 mmol, 1 eq) in Py (9.80 g, 123.89 mmol, 10 mL, 5.89 eq), the phenyl carbonochloridate (3.49 g, 22.32 mmol, 2.8 mL, 1.06 eq) was added into the mixture at 0 °C and stirred at 25°C for 12 h. The reaction mixture was added HCI (1 N) to adjust pH<7 then was partitioned between ethyl acetate (30 mL) and water (20 mL). The aqueous layer was extracted with ethyl acetate (10 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford phenyl N-pyrazin-2-ylcarbamate (600 mg, 2.79 mmol, 13.26% yield, N / A purity) was yellow solid.

[0659] 1H NMR (400 MHz, DMSO-d6) δ = 11.09 (s, 1H), 9.09 (d, J = 1.4 Hz, 1 H), 8.43 - 8.34 (m, 2H), 7.49 - 7.41 (m, 2H), 7.33 - 7.22 (m, 3H)

[0660]

[0661] Tert-butyl N-[3-[2-(pyrazin-2-ylcarbamoylamino)phenoxy]propyl]carbamate: To a mixture of phenyl N-pyrazin-2-ylcarbamate (90 mg, 418.20 μmol, 1.11 eq) in DMF (1 mL) was added tert-butyl N-[3-(2- aminophenoxy) propyl]carbamate (100 mg, 375.47 μmol, 1 eq) and DIEA (148.40 mg, 1.15 mmol, 0.2 mL, 3.06 eq), then the mixture was stirred at 70 °C for 12 h. The reaction mixture was added into H2O, a lot of precipitate was formed, filtered, collected the residue and washed with H2O (10 mL) *2. The crude residue tert-butyl N-[3-[2-(pyrazin-2-ylcarbamoylamino) phenoxy]propyl]carbamate (140 mg, crude) as yellow solid was used in next step directly without purification.

[0662] MS m / z: 388.2 [M+H]+

[0663] 1-[2-(3-aminopropoxy)phenyl]-3-pyrazin-2-yl-urea: To a mixture of tert-butyl N-[3-[2-(pyrazin-2- ylcarbamoylamino) phenoxy]propyl]carbamate (140 mg, 361.35 μmol, 1 eq) in DCM (1 mL) was added TFA (153.50 mg, 1 .35 mmol, 0.1 mL, 3.73 eq), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Phenomenex Luna Cis 150*25 mm*10um;mobile phase: [H2O (0.1 %TFA) -ACN];gradient:0%-25% B over 15.0 min). The elute was directly lyophilized to give the crude product, the crude product was purified by Prep-HPLC (column: Phenomenex Luna Cis 150*25mm*10pm;mobile phase: [H2O (0.1 %TFA) - ACN];gradient: 0%-25% B over 15.0 min). The elute was directly lyophilized to give 1-[2-(3-aminopropoxy) phenyl]-3-pyrazin-2-yl-urea (28.3 mg, 95.54 μmol, 26.44% yield, 97% purity) as white solid. MS m / z: 288.2 [M+H]+1H NMR (400 MHz, DMSO-ds) δ = 10.61 - 10.22 (m, 1 H), 8.71 (s, 1 H), 8.29 (dd, J= 1 .4, 2.7 Hz, 1 H), 8.26 - 8.19 (m, 2H), 7.07 - 6.96 (m, 2H), 6.94 - 6.87 (m, 1 H), 4.13 (t, J= 6.3 Hz, 2H), 2.78 (t, J= 6.8 Hz, 2H), 1.91 (quin, J = 6.5 Hz, 2H) Synthetic scheme for CAN-030:

[0664] Phenyl N-(5-cyanopyrazin-2-yl)carbamate: To a mixture of 5-aminopyrazine-2-carbonitrile (2 g, 16.65 mmol, 1 eq) in THF (10 mL) was added Py (1.47 g, 18.58 mmol, 1.5 mL, 1.12 eq), the phenyl carbonochloridate (3.74 g, 23.91 mmol, 3 mL, 1.44 eq) was added into the mixture and stirred at 25 °C for 12 h. The reaction mixture was added HCI (1 N) to adjust pH<7, then partitioned between ethyl acetate (30 mL) and water (20 mL). The aqueous layer was extracted with ethyl acetate (10 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford phenyl N-(5-cyanopyrazin-2-yl) carbamate (1.85 g, 7.70 mmol, 46.25% yield) as yellow solid.

[0665] 1H NMR (400 MHz, DMSO-d6) δ = 11.76 (s, 1 H), 9.20 (d, J= 1.4 Hz, 1H), 8.97 (d, J = 1.5 Hz, 1 H), 7.49 - 7.44 (m, 2H), 7.34 - 7.25 (m, 3H) Tert-butyl N-[3-[2-[(5-cyanopyrazin-2-yl)carbamoylamino]phenoxy]propyl]carbamate: To a mixture of phenyl N-(5-cyanopyrazin-2-yl) carbamate (100 mg, 416.29 μmol, 1.11 eq) in DMF (2 mL) was added tert-butyl N-[3-(2-aminophenoxy) propyl]carbamate (100 mg, 375.47 μmol, 1 eq) and DIEA (148.40 mg, 1.15 mmol, 0.2 mL, 3.06 eq), then the mixture was stirred at 70 °C for 12 h. The reaction mixture was added into H2O, a lot of precipitate was formed, filtered, collected the residue and washed with H2O (10 mL) *2. The crude residue tert-butyl N-[3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]phenoxy]propyl]carbamate (150 mg, crude) as yellow solid which was used in next step directly without purification.

[0666] MS m / z: 313.1 [M-100+H]+

[0667] 1-[2-(3-aminopropoxy)phenyl]-3-(5-cyanopyrazin-2-yl)urea: To a mixture of tert-butyl N-[3-[2-[(5- cyanopyrazin-2-yl) carbamoylamino]phenoxy]propyl]carbamate (150 mg, 363.69 μmol, 1 eq) in DCM (1 mL) was added TFA (0.1 mL), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Phenomenex Luna Cis 150*25 mm*10pm;mobile phase: [H2O (0.1 %TFA) -ACN]; gradient:0%-28% B over 15.0 min). The elute was directly lyophilized to give 1-[2-(3-aminopropoxy) phenyl]-3-(5-cyanopyrazin-2-yl) urea (50.7 mg, 118.92 μmol, 32.70% yield, 100% purity, TFA) as white solid.

[0668] MS m / z: 313.1 [M+H]+

[0669] 1H NMR (400 MHz, DMSO-d6) δ = 10.98 - 10.74 (m, 1 H), 9.94 (br s, 1 H), 8.92 (s, 1 H), 8.84 (s, 1 H), 8.16 (d, J = 7.9 Hz, 1 H), 8.03 - 7.60 (m, 3H), 7.11 - 7.02 (m, 2H), 7.00 - 6.92 (m, 1 H), 4.18 (t, J = 6.2 Hz, 2H), 3.05 (t, J = 7.3 Hz, 2H), 2.11 (quin, J = 6.7 Hz, 2H)

[0670] Synthetic scheme for CAN-031 :

[0671]

[0672] 1 3

[0673] Tert-butyl N-[3-(5-fluoro-2-nitro-phenoxy)propyl]carbamate: To a mixture of 5-fluoro-2-nitro-phenol (1 g, 6.37 mmol, 1 eq) in DMF (10 mL) was added tert-butyl N-(3-bromopropyl) carbamate (1.82 g, 7.64 mmol, 1.2 eq) and K2CO3 (1.50 g, 10.82 mmol, 1.7 eq), then the mixture was stirred at 60 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (40 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (IS^SCU) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-40% Ethyl acetate / Petroleum ether gradient @ 20 mL / min) to afford tert-butyl N-[3-(5-fluoro-2-nitro-phenoxy) propyl]carbamate (1.9 g, 5.44 mmol, 85.47% yield, 90% purity) as yellow solid.

[0674] MS m / z: 215.2 [M-Boc+H]+

[0675] 1H NMR (400 MHz, DMSO-d6) δ = 8.02 (dd, J = 6.1, 9.1 Hz, 1 H), 7.29 (dd, J = 2.3, 11.1 Hz, 1 H), 6.96 (ddd, J = 2.5, 7.9, 9.0 Hz, 1 H), 6.87 (br s, 1 H), 4.17 (t, J= 6.0 Hz, 2H), 3.08 (q, J= 6.6 Hz, 2H), 1.83 (quin, J = 6.4 Hz, 2H), 1.36 (s, 9H)

[0676] 3 4

[0677] Tert-butyl N-[3-(2-amino-5-fluoro-phenoxy)propyl]carbamate: To a mixture of tert-butyl N-[3-(5-fluoro- 2-nitro-phenoxy) propyl]carbamate (900 mg, 2.86 mmol, 1 eq) in EtOH (10 mL) / H2O (1 mL) was added Fe (1 .60 g, 28.63 mmol, 10 eq) and NH4CI (1 .53 g, 28.63 mmol, 10 eq), then the mixture was stirred at 80 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (20 mL) and water (20 mL). The aqueous layer was extracted with ethyl acetate (10 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to afford tert-butyl N-[3-(2-amino-5-fluoro-phenoxy) propyl]carbamate (300 mg, 949.62 μmol, 33.16% yield, 90% purity) as yellow solid.

[0678] MS m / z: 185.0 [M-100+H]+

[0679] (4-nitrophenyl) N-[2-[3-(tert-butoxycarbonylamino)propoxy]-4-fluoro-phenyl]carbamate: To a mixture of tert-butyl N-[3-(2-amino-5-fluoro-phenoxy) propyl]carbamate (150 mg, 527.56 μmol, 1 eq) in DCM (1 mL) was added Py (60 mg, 758.54 μmol, 61 .22 1 .44 eq), the (4-nitroph μeLn,yl) carbonochloridate (130 mg, 644.96 μmol, 1.22 eq) was added into the mixture and stirred at 25 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (5 mL) and water (2 mL), then washed by HCI (1 N) till pH=7, The aqueous layer was extracted with ethyl acetate (3 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product (4-nitrophenyl) N- [2-[3-(tert-butoxycarbonylamino) propoxy]-4-fluoro-phenyl]carbamate (230 mg, crude) as yellow oil was used in next step directly without purification.

[0680] MS m / z: 350.0 [M-Boc+H]+

[0681] tert-butyl N-[3-[2-[(5-cyanopyrazin-2-yl)carbamoylamino]-5-fluoro-phenoxy]propyl]carbamate: To a mixture of (4-nitrophenyl) N-[2-[3-(tert-butoxycarbonylamino) propoxy]-4-fluoro-phenyl]carbamate (230 mg, 511.76 μmol, 1 eq) in DMF (3 mL) was added 5-aminopyrazine-2-carbonitrile (70 mg, 582.79 μmol, 1.14 eq) and DIEA (200 mg, 1.55 mmol, 269.54 3.02 eq), then th μeL m, ixture was stirred at 80 °C for 1 h. The reaction mixture was partitioned between ethyl acetate (5 mL) and brine (2 mL). The aqueous layer was extracted with ethyl acetate (3 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-50% Ethyl acetate / Petroleum ether gradient @ 20 mL / min) to afford tert-butyl N-[3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]-5-fluoro- phenoxy]propyl]carbamate (80 mg, 130.10 μmol, 25.42% yield, 70% purity) as yellow oil.

[0682] MS m / z: 331.1 [M-Boc+H]+

[0683] 1-[2-(3-aminopropoxy)-4-fluoro-phenyl]-3-(5-cyanopyrazin-2-yl)urea: To a mixture of tert-butyl N-[3- [2-[(5-cyanopyrazin-2-yl) carbamoylamino]-5-fluoro-phenoxy]propyl]carbamate (80 mg, 185.86 μmol, 1 eq) in DCM (1 mL) was added TFA (153.50 mg, 1 .35 mmol, 0.1 mL, 7.24 eq), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Phenomenex Luna Cis 150*25 mm*10pm;mobile phase: [H2O (0.1%TFA) - ACN];gradient: 0%-30% B over 15.0 min). The elute was directly lyophilized to give 1-[2-(3-aminopropoxy) -4-fluoro-phenyl]-3-(5-cyanopyrazin-2-yl) urea (7.5 mg, 16.88 μmol, 9.08% yield, 100% purity, TFA) as white solid.

[0684] MS m / z: 331.1 [M+H]+ 1 H NMR (400 MHz, DMSO-d6) 3 = 10.83 (br s, 1 H), 9.90 (br s, 1 H), 8.94 - 8.80 (m, 2H), 8.12 (dd, J = 6.3, 9.1 Hz, 1 H), 7.85 - 7.65 (m, 3H), 7.03 (dd, J= 2.7, 10.6 Hz, 1 H), 6.81 (dt, J= 2.8, 8.7 Hz, 1 H), 4.20 (t, J= 6.3 Hz, 2H), 3.08 - 2.98 (m, 2H), 2.09 (quin, J = 6.6 Hz, 2H) Synthetic scheme for CAN-032:

[0685] 1 3

[0686] Tert-butyl N-[(1S,3R)-3-(2-nitrophenoxy)cyclopentyl]carbamate: To a mixture of 2-nitrophenol (650 mg, 4.67 mmol, 1 eq) in THF (20 mL) was added tert-butyl N-[(1 S, 3S) -3-hydroxycyclopentyl]carbamate (1 g, 4.97 mmol, 1.06 eq) and PPh3 (2.45 g, 9.35 mmol, 2 eq), the mixture was stirred at 0 °C for 0.5 h. The DIAD (1.88 g, 9.28 mmol, 1.99 eq) was dropwise into the mixture and stirred at 25°C for 12h. The reaction mixture was partitioned between ethyl acetate (30 mL) and water (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to afford tert-butyl N-[(1 S, 3R) -3-(2-nitrophenoxy) cyclopentyl]carbamate (2 g, crude) as yellow solid.

[0687] 1H NMR (400 MHz, DMSO-d6) δ = 7.84 (dd, J = 1.2, 8.0 Hz, 1 H), 7.65 - 7.58 (m, 1 H), 7.29 (d, J = 8.4 Hz, 1 H), 7.08 (t, J = 7.6 Hz, 1 H), 6.77 (br d, J = 7.3 Hz, 1 H), 3.88 - 3.76 (m, 1 H), 3.41 - 3.39 (m, 1 H), 2.39 (td, J = 7.0, 13.7 Hz, 1 H), 1.97 - 1.75 (m, 3H), 1.64 - 1.54 (m, 2H), 1.36 (s, 9H)

[0688] 3 4

[0689] Tert-butyl N-[(1S,3R)-3-(2-aminophenoxy)cyclopentyl]carbamate: To a mixture of tert-butyl N-[(1S, 3R) -3-(2-nitrophenoxy) cyclopentyl]carbamate (1 g, 3.10 mmol, 1 eq) in MeOH (10 mL) / H2O (1 mL) was added Fe (870 mg, 15.58 mmol, 5.02 eq) and NH4CI (830 mg, 15.52 mmol, 5 eq), then the mixture was stirred at 80 °C for 3 h. The reaction mixture was partitioned between ethyl acetate (30 mL) and water (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 20 mL / min) to afford tert-butyl N-[(1 S, 3R) -3-(2-aminophenoxy) cyclopentyl]carbamate (200 mg, 684.06 μmol, 22.05% yield, N / A purity) as yellow solid.

[0690] 1H NMR (400 MHz, DMSO-d6) δ = 6.92 (br d, J = 6.9 Hz, 1 H), 6.71 (d, J = 7.8 Hz, 1 H), 6.66 - 6.58 (m, 2H), 6.51 - 6.43 (m, 1 H), 4.67 (br s, 3H), 3.88 - 3.76 (m, 1 H), 3.47 (br d, J = 4.9 Hz, 1 H), 2.36 - 2.26 (m, 1 H), 1 .88 - 1 .75 (m, 3H), 1 .68 - 1 .54 (m, 2H), 1 .37 (s, 9H) Tert-butyl N-[(1S,3R)-3-[2-[(5-cyanopyrazin-2- yl)carbamoylamino]phenoxy]cyclopentyl]carbamate: To a mixture of tert-butyl N-[(1 S, 3R) -3-(2- aminophenoxy) cyclopentyl]carbamate (100 mg, 342.03 μmol, 1 eq) in DMF (2 mL) was added phenyl N- (5-cyanopyrazin-2-yl) carbamate (90 mg, 374.66 μmol, 1.1 eq) and DIEA (130 mg, 1.01 mmol, 2.94 eq), then the mixture was stirred at 70 °C for 3 h. The reaction mixture was partitioned between ethyl acetate (10 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0-60% Ethyl acetate / Petroleum ether gradient @ 18 mL / min) to afford tert-butyl N-[(1S, 3R) -3-[2-[(5- cyanopyrazin-2-yl) carbamoylamino]phenoxy]cyclopentyl]carbamate (75 mg, 164.20 μmol, 48.01% yield, 96% purity) as yellow solid.

[0691] MS m / z: 339.3 [M-100+H]+

[0692] 1-[2-[(1 R,3S)-3-aminocyclopentoxy]phenyl]-3-(5-cyanopyrazin-2-yl)urea: To a mixture of tert-butyl N- [(1S, 3R) -3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]phenoxy]cyclopentyl]carbamate (75 mg, 171.05 μmol, 1 eq) in DCM (1 mL) was added TFA (0.1 mL), then the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Phenomenex Luna Cis 150*25 mm*10pm;mobile phase: [H2O (0.1 %TFA) -ACN];gradient:8%-38% B over 15.0 min). The elute was directly lyophilized to give 1-[2-[(1 R, 3S) -3-aminocyclopentoxy]phenyl]-3-(5- cyanopyrazin-2-yl) urea (24.5 mg, 72.41 μmol, 42.33% yield, 100% purity) as white solid.

[0693] MS m / z: 339.2 [M+H]+

[0694] 1H NMR (400 MHz, DMSO-d6) δ = 9.85 (br s, 1 H), 8.95 (s, 1 H), 8.82 (d, J = 1.2 Hz, 1 H), 8.17 (dd, J = 1.2, 8.0 Hz, 1 H), 7.96 (br s, 2H), 7.07 - 6.98 (m, 2H), 6.97 - 6.91 (m, 1 H), 4.86 (br d, J = 4.8 Hz, 1 H), 3.61 - 3.52 (m, 1 H), 2.61 (td, J = 7.2, 14.2 Hz, 1 H), 2.11 - 2.02 (m, 3H), 1.92 - 1.81 (m, 1 H), 1.79 - 1.69 (m, 1 H)

[0695] Synthetic scheme for CAN-037:

[0696] 5-[[5-[2-(3-aminopropoxy)-4-fluoro-6-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile

[0697]

[0698] 1-(4-fluoro-2,6-dimethoxy-phenyl)ethanone

[0699] 1 3 To a mixture of AICI3 (10 g, 75.00 mmol, 4.10 mL, 1.17 eq) in DCM (100 mL) was added 1 -fluoro-3, 5- dimethoxy-benzene (10 g, 64.04 mmol, 1 eq) in DCM (100 mL), the acetyl chloride (5.52 g, 70.32 mmol, 5 mL, 1.10 eq) was added into the mixture then the mixture was stirred at 25 °C for 12 h. The reaction mixture was partitioned between DCM (300 mL) and water (200 mL). The aqueous layer was extracted with DCM (200 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-10% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to afford 1- (4-fluoro-2, 6-dimethoxy-phenyl) ethanone (2.6 g, 13.12 mmol, 20.49% yield, N / A purity) as white solid.1H NMR (400 MHz, DMSO-d6) δ = 6.63 (td, J = 2.9, 11.2 Hz, 2H), 3.81 - 3.72 (m, 6H), 2.37 - 2.31 (m, 3H) 1-(4-fluoro-2-hydroxy-6-methoxy-phenyl)ethanone

[0700] To a mixture of 1 -(4-fluoro-2, 6-dimethoxy-phenyl) ethanone (2.6 g, 13.12 mmol, 1 eq) in DCM (50 mL) was added BBrs (2 M, 7.2 mL, 1.10 eq) at -20 °C, then the mixture was stirred at -20°C for 1 h. The reaction mixture was added H2O to quenched then was partitioned between DCM (50 mL) and water (30 mL). The aqueous layer was extracted with DCM (30 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford 1-(4-fluoro-2-hydroxy-6-methoxy-phenyl) ethanone (2.2 g, 11 .95 mmol, 91 .06% yield, N / A purity) as white solid.

[0701] 1H NMR (400 MHz, CHLOROFORM-d) δ = 13.77 (d, J= 1.3 Hz, 1 H), 6.27 (dd, J= 2.4, 10.1 Hz, 1 H), 6.13 (dd, J = 2.4, 11.1 Hz, 1 H), 3.90 (s, 3H), 2.65 (s, 3H)

[0702] (Z)-3-(dimethylamino)-1-(4-fluoro-2-hydroxy-6-methoxy-phenyl)prop-2-en-1-one

[0703] To a mixture of 1-(4-fluoro-2-hydroxy-6-methoxy-phenyl) ethanone (2 g, 10.86 mmol, 1 eq) in toluene (15 mL) was added DMF-DMA (4.49 g, 37.64 mmol, 5 mL, 3.47 eq), then the mixture was stirred at 80 °C for 12 h. The reaction mixture was concentrated to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford (Z) -3-(dimethylamino) -1-(4-fluoro-2-hydroxy-6- methoxy-phenyl) prop-2-en-1-one (2.1 g, 8.78 mmol, 80.83% yield, n / a purity) as yellow oil.

[0704] MS m / z: 240.2 [M+H]+tert-butyl N-[3-[2-[(Z)-3-(dimethylamino)prop-2-enoyl]-5-fluoro-3-methoxy- phenoxyjpropyljcarbamate

[0705] To a mixture of (Z) -3-(dimethylamino) -1-(4-fluoro-2-hydroxy-6-methoxy-phenyl) prop-2-en-1-one (2.1 g, 8.78 mmol, 1 eq) in DMF (30 mL) was added tert-butyl N-(3-bromopropyl) carbamate (2.4 g, 10.08 mmol, 1.15 eq) and K3PO4 (2.4 g, 11.31 mmol, 1.29 eq), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (50 mL) and brine (50 mL). The aqueous layer was extracted with ethyl acetate (40 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product tert-butyl N-[3-[2-[(Z) -3-(dimethylamino) prop- 2-enoyl]-5-fluoro-3-methoxy-phenoxy]propyl]carbamate (4.5 g, crude) as yellow oil was used in next step directly without purification.

[0706] MS m / z: 397.2 [M+H]+tert-butyl N-[3-(5-fluoro-2-isoxazol-5-yl-3-methoxy-phenoxy)propyl]carbamate

[0707] To a mixture of tert-butyl N-[3-[2-[(Z) -3-(dimethylamino) prop-2-enoyl]-5-fluoro-3-methoxy- phenoxy]propyl]carbamate (4.5 g, 11 .35 mmol, 1 eq) in EtOH (30 mL) was added NH2OH. HCI (800 mg, 11 .51 mmol, 1.01 eq), then the mixture was stirred at 35 °C for 3 h. The reaction mixture was partitioned between ethyl acetate (50 mL) and water (30 mL). The aqueous layer was extracted with ethyl acetate (30 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to afford tert-butyl N-[3-(5-fluoro-2-isoxazol-5-yl-3-methoxy-phenoxy) propyl]carbamate (310 mg, 829.19 μmol, 7.31% yield, 98% purity) as off-white oil and 5-fluoro-2-isoxazol-5-yl-3-methoxy-phenol (2.13 g, 10.18 mmol, 89.71% yield, N / A purity) as yellow oil.

[0708] MS m / z: 267.1 [M-100+H]+tert-butyl N-[3-[2-(2-cyanoacetyl)-5-fluoro-3-methoxy-phenoxy]propyl]carbamate

[0709] 8 9

[0710] To a mixture of tert-butyl N-[3-(5-fluoro-2-isoxazol-5-yl-3-methoxy-phenoxy) propyl]carbamate (310 mg, 846.11 μmol, 1 eq) in MeOH (5 mL) was added KOH (3.5 M, 0.3 mL, 1.24 eq), then the mixture was stirred at 40 °C for 12 h. The reaction mixture was added AcOH to adjust pH=7 then was partitioned between ethyl acetate (30 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 20 mL / min) to afford tert-butyl N-[3- [2-(2-cyanoacetyl) -5-fluoro-3-methoxy-phenoxy]propyl]carbamate (285 mg, 777.87 μmol, 91.94% yield, 100% purity) as off-white oil.

[0711] 1H NMR (400 MHz, DMSO-d6) δ = 6.92 - 6.76 (m, 1 H), 6.71 - 6.57 (m, 2H), 4.02 - 3.95 (m, 2H), 3.78 (s, 3H), 3.30 (br s, 2H), 3.04 (br s, 2H), 1.83 - 1.74 (m, 2H), 1.36 (s, 9H).

[0712] MS m / z: 267.0 [M-100+H]+tert-butyl N-[3-[2-(3-amino-1H-pyrazol-5-yl)-5-fluoro-3-methoxy-phenoxy]propyl]carbamate

[0713] To a mixture of tert-butyl N-[3-[2-(2-cyanoacetyl) -5-fluoro-3-methoxy-phenoxy]propyl]carbamate (285 mg, 777.87 μmol, 1 eq) in EtOH (9 mL) / n-BuOH (3 mL) was added AcOH (50 mg, 832.61 μmol, 47.66 μL, 1.07 eq), the (0.370 g, 6.28 mmol, 85wt%, 8.08 eq) was added into the mixture at 0 °C, then the mixture was stirred at 80°C for 12 h. The reaction mixture was partitioned between ethyl acetate (30 mL) and brine (20 mL). The aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 80-100% Ethyl acetate / Petroleum ether gradient @ 18 mL / min) to afford tert-butyl N-[3-[2-(3-amino-1 H-pyrazol-5- yl) -5-fluoro-3-methoxy-phenoxy]propyl]carbamate (170 mg, 446.88 μmol, 57.45% yield, 100% purity) as yellow oil.

[0714] MS m / z: 381.3 [M+H]+ tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl)amino]-1 H-pyrazol-5-yl]-5-fluoro-3-methoxy- phenoxy]propyl]carbamate phenoxy]propyl]carbamate (170 mg, 446.88 μmol, 1 eq) in DMSO (3 mL) was added 5-chloropyrazine-2- carbonitrile (70 mg, 501.64 μmol, 1.12 eq) and 4-ethylmorpholine (60 mg, 520.97 μmol, 65.93 μL, 1.17 eq), then the mixture was stirred at 80 °C for 12 h. The reaction mixture was partitioned between ethyl acetate (10 mL) and brine (8 mL). The aqueous layer was extracted with ethyl acetate (5 mL) twice. The combined organic layers were dried over (Na2SO4) and evaporated to give a crude material. The crude product was triturated with MTBE at 25°C for 30 min then collected the tert-butyl N-[3-[2-[3-[(5- cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-5-fluoro-3-methoxy-phenoxy]propyl]carbamate (200 mg, 359.88 μmol, 80.53% yield, 87% purity) as yellow solid.

[0715] MS m / z: 484.2 [M+H]+

[0716] 5-[[5-[2-(3-aminopropoxy)-4-fluoro-6-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile

[0717] To a mixture of tert-butyl N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-5-fluoro-3-methoxy- phenoxy]propyl]carbamate (100 mg, 206.83 μmol, 1 eq) in DCM (1 mL) was added TFA (307.00 mg, 2.69 mmol, 0.2 mL, 13.02 eq), then the mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated to give a residue. The filter liquor was purified by Prep-HPLC (column: Unisil 3-100 Cis Ultra 150*50 mm*3 pm;mobile phase: [water (FA) -ACN];B%: 1%-30%, 10min). The elute was directly lyophilized to give 5-[[5-[2-(3-aminopropoxy) -4-fluoro-6-methoxy-phenyl]-1 H-pyrazol-3- yl]amino]pyrazine-2-carbonitrile (25.2 mg, 65.73 μmol, 31.78% yield, 100% purity) as white solid.1H NMR (400 MHz, DMSO-d6) δ = 12.35 (br s, 1 H), 10.73 (br s, 1 H), 8.65 (d, J = 1.2 Hz, 1H), 8.61 - 8.35 (m, 1 H), 7.73 (br s, 3H), 6.81 (br s, 1 H), 6.73 - 6.67 (m, 2H), 4.11 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H), 2.99 - 2.90 (m, 2H), 1.99 (quin, J= 6.7 Hz, 2H) MS m / z: 384.1 [M+H]+

[0718] Synthetic Scheme for CAN-038:

[0719] 5-[(5-bromo-1 H-py razol-3-y I ) amino]pyrazine-2-carbonitrile

[0720] A mixture of 5-bromo-1 H-pyrazol-3-amine (1.22 g, 7.53 mmol, 1 .05 eq), 5-chloropyrazine-2-carbonitrile (1 g, 7.17 mmol, 1 eq), CS2CO3 (7.00 g, 21.48 mmol, 3.00 eq) in DMSO (20 mL) was stirred at 80°C for 12 h. The mixture was diluted in EtOAc (100 mL) and water (100 mL). The mixture was filtered and the cake was dried under vacuum to give the product. The mixture was filtered and the cake was dried under vacuum to give 5-[(5-bromo-1 H-pyrazol-3-yl) amino]pyrazine-2-carbonitrile (1 g, 2.79 mmol, 38.96% yield, 74% purity) as yellow solid.1H NMR (400 MHz, DMSO-d6) δ ppm: 12.91 (s, 1 H), 10.63 (s, 1 H), 8.61 (s, 1 H), 8.37 (s, 1 H), 6.45 (s, 1 H) tert-butyl 4-(4-chloro-3-methoxy-phenyl) -4-hy droxy -pi peri di ne-1 -carboxy late

[0721] To a solution of Mg (600 mg, 24.69 mmol, 1.82 eq) in THF (20 mL) was added b (50 mg, 197.00 μmol, 39.68 μ 1L.,45e-2 eq) at 50°C under N2. A solution of 4-bromo-1-chloro-2-methoxy-benzene (3 g, 13.55 mmol, 1.84 mL, 1 eq) in THF (10 mL) was added dropwise at 50°C under N2. Then the mixture was stirred at 50°C under N2 for 1 h. Then the mixture was cooled to 0°C. A solution of tert-butyl 4-oxopiperidine-1 - carboxylate (3.00 g, 15.06 mmol, 1.11 eq) in THF (10 mL) was added 0°C under N2. Then mixture was stirred at 20°C for 1 h. TLC (Petroluem / EtOAc=5:1) indicated the starting material was consumed completely and a major spot observed. The mixture was poured in sat. NH4CI (50 mL) and extracted with EtOAc (30 mL*3). The organic layers were dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient ® 60 mL / min) to give tert-butyl 4-(4-chloro-3-methoxy-phenyl) -4-hydroxy-piperidine-1 -carboxylate (3 g, 8.78 mmol, 64.79% yield) was obtained as colourless oil.

[0722] 1H NMR (400 MHz, CHLOROFORM-d) δ = 7.34 (d, J = 8.0 Hz, 1 H), 7.11 (d, J = 2.0 Hz, 1H), 6.96 (dd, J = 2.0, 8.4 Hz, 1 H), 4.09-4.00 (m, 2H), 3.93 (s, 3H), 3.27 - 3.19 (m, 2H), 2.03-1.91 (m, 2H), 1.76-1.70 (m, 2H), 1.49 (s, 9H). tert-butyl 4-(4-chloro-3-methoxy-phenyl) -4-flu oro-pi peri din e- 1 -carboxylate

[0723] 3 4

[0724] To a solution of tert-butyl 4-(4-chloro-3-methoxy-phenyl) -4-hydroxy-piperidine-1 -carboxylate (2.5 g, 7.31 mmol, 1 eq), N, N-diethylethanamine;trihydrofluoride (1.50 g, 9.30 mmol, 1.52 mL, 1.27 eq) in DCM (20 mL) was added (difluoro-sulfanylidene) -diethyl-ammonium;tetrafluoroborate (2.00 g, 8.73 mmol, 1 .19 eq) at 0°C, then the mixture was stirred at 20°C for 1 h. TLC (Petroluem / EtOAc=5: 1 ) indicated the starting material was consumed completely and a major spot observed. The mixture was poured into sat. Na2CO3 (50 mL) and extracted with DCM (50 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-10% Ethyl acetate / Petroleum ether gradient @ 50 mL / min) to give tert-butyl 4-(4-chloro-3-methoxy-phenyl) -4-fluoro-piperidine-1-carboxylate (1.5 g, 4.36 mmol, 59.65% yield) as colourless oil.

[0725] 1H NMR (400 MHz, CHLOROFORM-d) δ = 7.35 (d, J = 8.0 Hz, 1 H), 6.99 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 2.0, 8.4 Hz, 1 H), 4.20-4.10 (m, 2H), 3.93 (s, 3H), 3.17 (t, J = 10.0 Hz, 2H), 2.05 - 1.83 (m, 4H), 1.50 (s, 9H) tert-butyl 4-fluoro-4-[3-methoxy-4-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) ph eny I] piperi di ne-1 -carboxylate

[0726] A mixture of tert-butyl 4-(4-chloro-3-methoxy-phenyl) -4-fluoro-piperidine-1 -carboxylate (1 .5 g, 4.36 mmol, 1 eq), 4, 4, 5, 5-tetramethyl-2-(4, 4, 5, 5-tetramethyl-1 , 3, 2-dioxaborolan-2-yl) -1, 3, 2-dioxaborolane (2.79 g, 10.97 mmol, 2.51 eq), Pd2 (dba)3 (400 mg, 436.82 μmol, 0.1 eq), XPhos (400 mg, 839.07 μmol, 1.92e- 1 eq), KOAc (1 .3 g, 13.25 mmol, 3.04 eq) in dioxane (20 mL) was stirred at 100°C under N2 for 12 h. The mixture was filtered and the filtrate was concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~5% Ethyl acetate / Petroleum ether gradient @ 50 mL / min) to give tert-butyl 4-fluoro-4-[3-methoxy-4-(4, 4, 5, 5-tetramethyl-1 , 3, 2-dioxaborolan-2-yl) phenyl]piperidine-1 -carboxylate (1 g, 1.61 mmol, 36.86% yield, 70% purity) as colorless oil.

[0727] MS m / z: 380.1 [M+H-56]+tert-butyl 4-[4-[3-[(5-cyanopyrazi n-2-y I) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenyl]-4-hydroxy- piperidine-1 -carboxylate

[0728] A mixture of tert-butyl 4-fluoro-4-[3-methoxy-4-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl]piperidine-1 -carboxylate (300 mg, 689.12 μmol, 1 eq), 5-[(5-bromo-1 H-pyrazol-3-yl) amino]pyrazine-2-carbonitrile (200 mg, 754.52 μmol, 1.09 eq), K2CO3 (300.01 mg, 2.17 mmol, 3.15 eq), Pd(dppf) CI2 (100 mg, 136.67 μmol, 1.98e-1 eq) in dioxane (3 mL) and H2O (1 mL) was stirred at 100°C for 12 h. The mixture was filtered and the filtrate was concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-50% THF / Petroleum ether gradient @ 30 mL / min) to give tert-butyl 4-[4-[3-[(5-cyanopyrazi n-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenyl]-4-hydroxy-piperidine-1-carboxylate (180 mg, 270.98 μmol, 39.32% yield, 74% purity) as a white solid.

[0729] MS m / z: 492.1 [M+H]+tert-butyl 4-[4-[3-[(5-cyanopyrazi n-2-y I) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenyl]-4-fluoro- piperidine-1 -carboxylate

[0730] To a solution of tert-butyl 4-[4-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenyl]-4- hydroxy-piperidine-1 -carboxylate (150 mg, 305.16 μmol, 1 eq), N, N-diethylethanamine;trihydrofluoride (60 mg, 372.30 μmol, 60.67 1.22 e μq)L, in DCM (2 mL) was added (difluoro-sulfanylidene)-diethyl- ammonium;tetrafluoroborate (90.00 mg, 393.02 μmol, 1.29 eq) at 0°C, then the mixture was stirred at 20°C for 1 h. The mixture was poured into sat. Na2COs (30 mL) and extracted with DCM (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0-40% THF / Petroleum ether gradient @ 20 mL / min) to give tert-butyl 4-[4-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenyl]-4-fluoro-piperidine-1 -carboxylate (100 mg, 202.62 μmol, 66.40% yield) as a white solid.

[0731] 1H NMR (400 MHz, DMSO-of6) δ ppm: 12.65 (s, 1 H), 10.75 (s, 1 H), 8.67 (d, J = 0.8 Hz, 1 H), 8.61 - 8.45 (m, 1 H), 7.68 (d, J = 8.4 Hz, 1 H), 7.15 (s, 1 H), 7.11 (d, J = 8.0 Hz, 1 H), 4.05 - 3.97 (m, 2H), 3.94 (s, 3H), 3.16 - 2.94 (m, 2H), 2.17 - 1.84 (m, 4H), 1.44 (s, 9H).

[0732] 5-[[5-[4-(4-fluoro-4-piperidyl) -2-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile To a soluton of tert-butyl 4-[4-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenyl]-4- fluoro-piperidine-1 -carboxylate (100 mg, 202.62 μmol, 1 eq) in DCM (1 mL) was added TFA (307.00 mg, 2.69 mmol, 0.2 mL, 13.29 eq), then the mixture was stirred at 20°C for 12 h. The mixture was concentrated to 5-[[5-[4-(4-fl uoro-4-piperidyl) -2-methoxy-phenyl]-1 H-pyrazol-3-yl]ami no] pyrazi ne-2-carbonitrile (100 mg, 197.07 μmol, 97.26% yield, TFA) as yellow solid.

[0733] MS m / z: 394.1 [M+H]+

[0734] 5-[[5-[4-(4-fluoro-1 -methyl-4-piperidyl) -2-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile

[0735] To a solution of 5-[[5-[4-(4-fluoro-4-piperidyl) -2-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2- carbonitrile (90 mg, 228.77 μmol, 1 eq) in EtOH (1 mL) was added HCHO (27.00 mg, 332.71 μmol, 1.45 eq) at 20 °C, then NaBH (OAc)s (63.00 mg, 297.25 μmol, 1.30 eq) was added and the mixture was stirred at 20°C for another 1 h. The mixture was filtered and the filtrate was concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex Luna Cis 150*25 mm*10um;mobile phase: [H2O (0.225% FA) -ACN];gradient: 13%-33% B over 10.0 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give 5-[[5-[4-(4-fluoro-1-methyl-4-piperidyl) -2-methoxy-phenyl]-1 H-pyrazol-3- yl]amino]pyrazine-2-carbonitrile (29.0 mg, 69.25 μmol, 30.27% yield, 97.3% purity) as a white solid.

[0736] MS m / z: 408.3 [M+H]+

[0737] 1H NMR (400 MHz, DMSO-d6) δ ppm: 12.64 (s, 1 H), 10.76 (s, 1 H), 8.67 (s, 1 H), 8.53 (s, 1 H), 8.14 (s, 1 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.13 (s, 1 H), 7.08 (d, J = 8.4 Hz, 1 H), 6.96 (s, 1 H), 3.94 (s, 3H), 3.01-2.94 (m, 2H), 2.62-2.55 (m, 2H), 2.46 (s, 3H), 2.33 - 2.24 (m, 1 H), 2.24 - 2.13 (m, 1 H), 2.08 - 1.98 (m, 2H).

[0738] Synthetic scheme for CAN-039:

[0739]

[0740] Tert-butyl N-[(1 S, 3R) -3-(5-fluoro-2-nitro-phenoxy) cyclopentyljcarbamate: To a mixture of 5-fluoro- 2-nitro-phenol (500 mg, 3.18 mmol, 1 eq), tert-butyl N-[(1S, 3S) -3-hydroxycyclopentyl]carbamate (750 mg, 3.73 mmol, 1.17 eq), PPhs (1.00 g, 3.82 mmol, 1.2 eq) in THF (10 mL) was added DIAD (770 mg, 3.81 mmol, 1.2 eq) dropwise at 0 °C, then the mixture was stirred at 20°C for 12 h. TLC (Petroleum / EtOAc=5:1) indicated the starting material was consumed completely and a major spot observed. The mixture was concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-10% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to give tert-butyl N-[(1S, 3R) -3-(5-fluoro-2-nitro- phenoxy) cyclopentyljcarbamate (500 mg, 1.47 mmol, 46.16% yield, 100% purity) as colourless oil.

[0741] MS m / z: 285.1 [M+H-t-Bu]+ Tert-butyl N-[(1 S, 3R) -3-(2-nitro-5-prop-2-ynoxy-phenoxy) cyclopentyljcarbamate: To a mixture of tert-butyl N-[(1S, 3R) -3-(5-fluoro-2-nitro-phenoxy) cyclopentyljcarbamate (300 mg, 881.46 μmol, 1 eq), K2CO3 (300 mg, 2.17 mmol, 2.46 eq) in DMF (5 mL) was added prop-2-yn-1-ol (340 mg, 6.06 mmol, 358.27 μ 6L.8, 8 eq), then the mixture was stirred at 70 °C for 3 h. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (30 mL*3). The resulting mixture was washed with brine (30 mL*3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0~20% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to give tert- butyl N-[(1 S, 3R) -3-(2-nitro-5-prop-2-ynoxy-phenoxy) cyclopentyljcarbamate (300 mg, 781.08 μmol, 88.61% yield, 98% purity) as a white solid.

[0742] MS m / z: 277.1 [M+H-Boc]+

[0743] Tert-butyl N-[(1S, 3R) -3-(2-amino-5-prop-2-ynoxy-phenoxy) cyclopentyljcarbamate: A mixture of tert-butyl N-[(1S, 3R) -3-(2-nitro-5-prop-2-ynoxy-phenoxy) cyclopentyljcarbamate (300 mg, 797.02 μmol, 1 eq), Fe (250 mg, 4.48 mmol, 5.62 eq), NH4CI (500 mg, 9.35 mmol, 11.73 eq) in MeOH (10 mL) and H2O (1 mL) stirred at 80 °C for 5 h. The mixture was diluted in THF (100 mL) and filtered. The filtrate was concentrated to give tert-butyl N-[(1 S, 3R) -3-(2-amino-5-prop-2-ynoxy-phenoxy) cyclopentyljcarbamate (200 mg, crude) was obtained as yellow solid. MS m / z: 347.1 [M+H]+

[0744] Tert-butyl N-[(1S, 3R) -3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]-5-prop-2-ynoxy- phenoxyjcyclopentyljcarbamate: To a mixture of tert-butyl N-[(1S, 3R) -3-(2-amino-5-prop-2-ynoxy- phenoxy) cyclopentyljcarbamate (200 mg, 577.33 μmol, 1 eq), DIEA (222.60 mg, 1.72 mmol, 2.98 eq) in DMF (2 mL) was added phenyl N-(5-cyanopyrazin-2-yl) carbamate (140 mg, 582.81 μmol, 1.01 eq), then the mixture was stirred at 70 °C for 2 h. The mixture was duilted in EtOAc (40 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-80% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to give tert-butyl N-[(1 S, 3R) -3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]-5-prop-2-ynoxy- phenoxy]cyclopentyl]carbamate (200 mg, 406.07 μmol, 70.34% yield) as a white solid.

[0745] MS m / z: 393.0 [M+H-Boc]+

[0746] 1-[2-[(1 R, 3S) -3-aminocyclopentoxy]-4-prop-2-ynoxy-phenyl]-3-(5-cyanopyrazin-2-yl) urea: To a solution of tert-butyl N-[(1 S, 3R) -3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]-5-prop-2-ynoxy- phenoxy]cyclopentyl]carbamate (200 mg, 406.07 μmol, 1 eq) in DCM (5 mL) was added TFA (1 mL), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex Luna Cie 150*25 mm*10um;mobile phase: [H2O (0.1 %TFA) -ACN];gradient:10%-40% B over 15.0 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give 1-[2-[(1 R, 3S) -3-aminocyclopentoxy]-4-prop-2-ynoxy-phenyl]-3-(5-cyanopyrazin-2-yl) urea (60.2 mg, 114.94 μmol, 28.30% yield, 96.69% purity, TFA salt) as a white solid.

[0747] MS m / z: 393.0 [M+H]+

[0748] 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.80 (s, 1 H), 9.69 (s, 1 H), 8.93 (d, J = 1.2 Hz, 1 H), 8.80 (d, J = 1.2 Hz, 1 H), 8.00 (d, J = 8.8 Hz, 1 H), 7.95 (s, 2H), 6.65 (d, J = 2.4 Hz, 1 H), 6.59 (dd, J = 2.4, 8.8 Hz, 1 H), 4.92-4.84 (m, 1 H), 4.78 (d, J = 2.4 Hz, 2H), 3.60 - 3.51 (m, 2H), 2.66-2.56 (m, 1 H), 2.12 - 1.98 (m, 3H), 1.90 - 1.79 (m, 1 H), 1.77 - 1.66 (m, 1 H)

[0749] Table CHK1 : Inhibition of CHK1 and cellular activity of compounds according to the present invention

[0750] Example 12: Synthesis of peptide-based cleavable linkers and coupling with CHK1 inhibitors.

[0751] Either maleimide or bromoacetamide peptide-based cleavable linkers were synthesized, as presented in the following Table.

[0752] All presented precursors include Prexasertib, except for UGTH-HG-044 (marked with * in the Table), which includes CAN-030. Synthesis of a GGFG-PEG maleimide linker with Prexasertib (UGTH-HG-043)

[0753] Synthetic scheme for UGTH-HG-043 tert-butyl N-[2-[[2-[[(1S) -1-benzyl-2-[[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3- methoxy -phenoxy]propylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2- oxo-ethyl]carbamate

[0754] To a mixture of 2-[[(2S) -2-[[2-[[2-(tert-butoxycarbonylamino) acetyl]amino]acetyl]amino]-3-phenyl- propanoyl]amino]acetic acid (140 mg, 320 μmol, 9.92e-1 eq), DIEA (148 mg, 1.15 mmol, 3.55 eq) in DMF (1 mL) was added HATU (150 mg, 394 μmol, 1 .22 eq) at 0 °C, then the mixture was stirred at 0°C for 0.5 h. 5-[[5-[2-(3-aminopropoxy) -6-methoxy-phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile (130 mg, 323.50 μmol, 1 eq, HCI salt) was added and the mixture was stirred at 20°C for 2 h. The mixture was diluted in EtOAc (30 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0-10% MeOH / DCM gradient @ 20 mL / min) to give tert-butyl N-[2-[[2-[[(1S) -1-benzyl-2-[[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo- ethyl]amino]-2-oxo-ethyl]carbamate (120 mg, 145.44 μmol, 44.96% yield, 95% purity) as a white solid. MS m / z: 784.3 [M+H]+ (2S) -2-[[2-[(2-ami noacetyl) amino]acetyl]amino]-N-[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H- pyrazol-5-yl]-3-methoxy-phenoxy]propylamino]-2-oxo-ethyl]-3-phenyl-propanamide

[0755] To a solution of tert-butyl N-[2-[[2-[[(1S) -1 -benzyl-2-[[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol- 5-yl]-3-methoxy-phenoxy]propylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo- ethylcarbamate (120 mg, 153.09 μmol, 1 eq) in DCM (3 mL) was added TFA (0.5 mL), then the mixture was stirred at 20°C for 1 h. The mixture was concentrated to give (2S) -2-[[2-[(2-aminoacetyl) amino]acetyl]amino]-N-[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propylamino]-2-oxo-ethyl]-3-phenyl-propanamide (120 mg, 150.43 μmol, 98.26% yield, TFA) as yellow solid. MS m / z: 684.5 [M+H]+

[0756] (2S) -N -[2-[3-[2- [3-[(5-cya nopy razi n-2-yl) aminoJ-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propylamino]-2-oxo-ethyl]-2-[[2-[[2-[[2-[2-[2-[2-[2-(2, 5-dioxopyrrol-1-yl) ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]acetyl]amino]acetyl]amino]-3-phenyl-propanamide -

[0757] To a mixture of (2S) -2-[[2-[(2-aminoacetyl) amino]acetyl]amino]-N-[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propylamino]-2-oxo-ethyl]-3-phenyl-propanamide (100 mg, 125 μmol, 1 eq, TFA salt), 2-[2-[2-[2-[2-(2, 5-dioxopyrrol-1-yl) ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (50.0 mg, 150 μmol, 1.2 eq), DIEA (50.00 mg, 386.87 μmol, 3.09 eq) in DMF (2 mL) was added T4P (110 mg, 152.67 μmol, 50wt%, 1.22 eq), then the mixture was stirred at 20°C for 1 h. The mixture was filtered and concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex Luna C18 150*25 mm*10pm;mobile phase: [H2O (0.225% FA) -ACN];gradient:34%-54% B over 10.0 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give (2S) -N-[2-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]- 1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propylamino]-2-oxo-ethyl]-2-[[2-[[2-[[2-[2-[2-[2-[2-(2, 5-dioxopyrrol- 1-yl) ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]acetyl]amino]acetyl]amino]-3-phenyl-propanamide (81.9 mg, 78.95 μmol, 62.98% yield, 96.11 % purity) as a white solid.

[0758] MS m / z: 997.4 [M+H]+ 1 H NMR (400 MHz, DMSO-d6) 5 = 12.34 ( s, 1 H), 10.67 ( s, 1 H), 8.62 (s, 1 H), 8.59 - 8.46 (m, 1 H), 8.29 - 8.20 (m, 1 H), 8.14 - 8.03 (m, 2H), 7.89 (t, J = 5.2 Hz, 1 H), 7.74 (t, J = 4.8 Hz, 1 H), 7.34 - 7.27 (m, 1 H), 7.25 - 7.15 (m, 5H), 7.01 (s, 2H), 6.88 (s, 1 H), 6.75 (d, J = 8.8 Hz, 2H), 4.49 - 4.41 (m, 1 H), 4.02 (t, J = 5.6 Hz, 2H), 3.92 (s, 2H), 3.80 (s, 3H), 3.78 - 3.70 (m, 4H), 3.68-3.63 (m, 2H), 3.61-3.52 (m, 8H), 3.52 - 3.44 (m, 10H), 3.28 - 3.23 (m, 2H), 3.04-2.99 (m, 1H), 2.80 - 2.73 (m, 1 H), 1.91 - 1.82 (m, 2H).

[0759] Synthesis of a GGFG maleimide linker with CAN-030 compound (UGTH-HG-044)

[0760] Synthetic scheme for UGTH-HG-044

[0761] UGTH-HG-044 tert-butyl N-[3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]phenoxy]propyl]carbamate

[0762] A mixture of tert-butyl N-[3-(2-aminophenoxy) propyl]carbamate (200 mg, 750 μmol, 1 eq), phenyl N-(5- cyanopyrazin-2-yl) carbamate (200 mg, 832 μmol, 1.11 eq), DIEA (194.10 mg, 1.50 mmol, 2 eq) in DMF (1 mL) was stirred at 60°C for 2 h. The mixture was poured into ethyl acetate (20 mL) and water (20 mL).

[0763] The mixture was filtered and the cake was dried under vacuum to give tert-butyl N-[3-[2-[(5-cyanopyrazin- 2-yl) carbamoylamino]phenoxy]propyl]carbamate (200 mg, 387.93 μmol, 51.66% yield, 80% purity) as yellow solid. MS m / z: 413.1 [M+H]+ 1-[2-(3-aminopropoxy) phenyl]-3-(5-cyanopyrazin-2-yl) urea

[0764] To a solution of tert-butyl N-[3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]phenoxy]propyl]carbamate (150 mg, 363 μmol, 1 eq) in DCM (2 mL) was added TFA (0.2 mL), then the mixture was stirred at 20°C for 0.5 h. The mixture was concentrated to give 1-[2-(3-aminopropoxy) phenyl]-3-(5-cyanopyrazin-2-yl) urea (150 mg, crude, TFA salt) as yellow solid.

[0765] MS m / z: 313.0 [M+H]+

[0766] N-[2-[[2-[[(1S) -1-benzyl-2-[[2-[3-[2-[(5-cyanopyrazin-2-yl) carbamoylaminojphenoxyjpropylamino]- 2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]-6-(2, 5-dioxopyrrol-1 -yl) hexanamide

[0767] UGTH-HG-OM

[0768] To a solution of 2-[[(2S) -2-[[2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetyl]amino]-3- phenyl-propanoyl]amino]acetic acid (70 mg, 132 μmol, 1.02 eq), T4P (110.00 mg, 152 μmol, 50wt%, 1.18 eq) in DMF (1 mL) was added DIEA (55.00 mg, 425 μmol, 3.30 eq) at 0°C, then the mixture was stirred at 20°C for 0.5 h. 1-[2-(3-aminopropoxy) phenyl]-3-(5-cyanopyrazin-2-yl) urea (55 mg, 129.00 μmol, 1 eq, TFA salt) was added and the mixture was stirred at 20°C for 1 h. The crude product was purified by prep- HPLC (column: Phenomenex Luna Cis 150*25 mm*10 pm;mobile phase: [H2O (0.225% FA) - ACN];gradient:34%-54% B over 10.0 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give N-[2-[[2-[[(1 S) -1-benzyl- 2-[[2-[3-[2-[(5-cyanopyrazin-2-yl) carbamoylamino]phenoxy]propylamino]-2-oxo-ethyl]amino]-2-oxo- ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]-6-(2, 5-dioxopyrrol-1-yl) hexanamide (30.6 mg, 36.40 μmol, 28.21% yield, 97.99% purity) as a white solid.

[0769] MS m / z: 824.3 [M+H]+

[0770] 1H NMR (400 MHz, DMSO-d6) δ ppm: 5 = 10.87 (s, 1H), 9.89 (s, 1H), 8.95 (s, 1H), 8.84 (d, J = 1.2 Hz, 1H), 8.26 (t, J = 6.0 Hz, 1H), 8.19 - 8.15 (m, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.07 (t, J = 6.0 Hz, 1H), 8.02 (t,

[0771] J = 5.6 Hz, 1H), 7.79 (t, J = 5.2 Hz, 1H), 7.25 - 7.20 (m, 4H), 7.19-7.15 (m, 1H), 7.06 - 7.00 (m, 2H), 6.99 (s, 2H), 6.95 - 6.90 (m, 1H), 4.49 - 4.40 (m, 1H), 4.09 (t, J = 6.0 Hz, 2H), 3.77 - 3.68 (m, 2H), 3.67 - 3.54 (m, 4H), 3.39 - 3.34 (m, 2H), 3.32 - 3.27 (m, 2H), 3.07-3.00 (m, 1H), 2.84-2.76 (m, 1H), 2.10 (t, J = 7.2 Hz, 2H), 2.02- 1.95 (m, 2H), 1.50-1.41 (m, 4H), 1.22- 1.14 (m, 2H)

[0772] Synthesis of a legumain maleimide linker with Prexasertib (UGTH-HG-028)

[0773] Synthetic scheme for UGTH-HG-028

[0774] tert-butyl 2-[6-(2, 5-dioxopyrrol-1 -yl) hexanoylaminojacetate

[0775] To a solution of 6-(2, 5-dioxopyrrol-1-yl) hexanoic acid (2 g, 9.47 mmol, 1 eq), DIEA (3.67 g, 28.41 mmol, 4.95 mL, 3 eq) in DMF (20 ml_) was added HATU (4.32 g, 11.36 mmol, 1.2 eq) at 0°C, then the mixture was stirred at 0°C for 0.5 h. Tert-butyl 2-ami noacetate (1.7 g, 10.14 mmol, 1.07 eq, HCI) was added and the mixture was stirred at 20°C for another 1 h. The mixture was diluted in EtOAc (50 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO® ; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH / DCM @ 50 mL / min) to give tert-butyl 2-[6- (2, 5-d ioxopyrrol- 1 -yl) hexanoylamino]acetate (2 g, 5.06 mmol, 53.39% yield, 82% purity) as a white solid. MS m / z: 269.0 [M-t-Bu-HH]+

[0776] 2-[6-(2, 5-dioxopyrrol-1 -yl) hexanoylaminojacetic acid

[0777] 3 4

[0778] To a solution of tert-butyl 2-[6-(2, 5-dioxopyrrol-1 -yl) hexanoylamino]acetate (2 g, 6.17 mmol, 1 eq) in DCM (20 mL) was added TFA (2 mL), then the mixture was stirred at 20°C for 1 h. The mixture was concentrated to give 2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetic acid (1.6 g, crude) as yellow oil. MS m / z: 269.1 [M+H]+tert-butyl 2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetate

[0779] 6

[0780] To a solution of 2-[6-(2, 5-dioxopyrrol-1 -yl) hexanoylamino]acetic acid (1.6 g, 5.96 mmol, 1 eq), DIEA (2.31 g, 17.89 mmol, 3 eq) in DMF (10 mL) DMF (10 mL) was added HATU (2.7 g, 7.10 mmol, 1.19 eq) at 0 °C, then the mixture was stirred at 0°C for 0.5 h. Tert-butyl 2-ami noacetate (1 g, 5.97 mmol, 1 .00 eq, HCI salt) was added and the mixture was stirred at 20°C for another 1 h. The mixture was diluted in EtOAc (50 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over IsfeSCU, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0~10% MeOH / DCM @ 50 mL / min) to give tert-butyl 2-[[2-[6-(2, 5-dioxopyrrol-1 -yl) hexanoylamino]acetyl]amino]acetate (2 g, 3.41 mmol, 57.15% yield, 65% purity) as yellow solid.

[0781] MS m / z: 326.1 [M-t-Bu+H]+

[0782] 2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetic acid To a mixture of tert-butyl 2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetate (2 g, 5.24 mmol, 1 eq) in DCM (10 mL) was added TFA (4.00 mL), then the mixture was stirred at 20°C for 1 h. The mixture concentrated to give the crude product. The crude product was triturated with EA (50 mL) and filtered to give 2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetic acid (1.5 g, 4.61 mmol, 87.93% yield) as a white solid.

[0783] 1H NMR (400 MHz, DMSO-ob) δ ppm: 8.08 (t, J = 5.6 Hz, 1 H), 8.03 (t, J = 5.6 Hz, 1 H), 7.00 (s, 2H), 3.75 (d, J = 6.0 Hz, 2H), 3.69 (d, J = 6.0 Hz, 2H), 3.37-3.36 (m, 2H), 2.10 (t, J = 7.6 Hz, 2H), 1.52 - 1.43 (m, 4H), 1.24 - 1.16 (m, 2H).

[0784] 9H-fluoren-9-ylmethyl N-[(1S) -1 -[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3- methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyljcarbamate

[0785] To a solution of (2S) -2-(9H-fluoren-9-ylmethoxycarbonylamino) -4-oxo-4-(tritylamino) butanoic acid (300 mg, 502 μmol, 1 eq), DIEA (195 mg, 1.51 mmol, 3 eq) in DMF (5 mL) was added HATU (210mg, 552 μmol, 1.1 eq), then the mixture was stirred at 20 °C for 0.5 h. 5-[[5-[2-(3-aminopropoxy) -6-methoxy- phenyl]-1 H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile (200 mg, 497.70 μmol, 0.99 eq, HCI salt) was added and the mixture was stirred at 20 °C for 1 h. The mixture was diluted in EtOAc (60 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-60% Ethyl acetate / Petroleum ether gradient @ 30 mL / min) to give 9H-fluoren-9-ylmethyl N-[(1 S) -1 -[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]- 3-methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyljcarbamate (300 mg, 292.36 μmol, 58.15% yield, 92% purity) as a white solid.

[0786] MS m / z: 945.8 [M+H]+

[0787] (2S) -2-amino-N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propyl]-N'-trityl-butanediamide

[0788]

[0789] To a solution of 9H-fluoren-9-ylmethyl N-[(1S) -1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]- 3-methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]carbamate (250 mg, 264.82 μmol, 1 eq) in DCM (20 mL) was added DBU (404 mg, 2.65 mmol, 10.02 eq), then the mixture was stirred at 20°C for 15 mins. The mixture was diluted in DCM (100 mL). The mixture was washed with brine (50 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was triturated with MTBE (10 mL) and filtered. The cake was dried under vacuum to give (2S) -2- amino-N-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propyl]-N'-trityl- butanediamide (150 mg, 193.27 μmol, 72.98% yield, 93% purity) a white solid.

[0790] MS m / z: 722.5 [M+H]+

[0791] 9H-fluoren-9-ylmethyl N-[(1 S) -1-[[(1 S) -1 -[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3- methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]carbamoyl]-3-oxo-3-

[0792] (tritylamino) propyl]carbamate

[0793] To a mixture of (2S) -2-(9H-fluoren-9-ylmethoxycarbonylamino) -4-oxo-4-(tritylamino) butanoic acid (130 mg, 218 μmol, 1.05 eq), DIEA (80 mg, 623 μmol, 3 eq) in DMF (3 mL) was added HATU (100 mg, 263 μmol, 1.27 eq) at 0 °C, then the mixture was stirred at 0°C for 0.5 h. (2S) -2-amino-N-[3-[2-[3-[(5- cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy] propyl]-N'-trityl-butanediamide (150 mg, 208 μmol, 1 eq) was added and the mixture was stirred at 20°C for 1 h. The mixture was diluted in EtOAc (60 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was triturated with MTBE (10 mL) and filtered. The cake was dried under vacuum to give 9H-fluoren-9-ylmethyl N-[(1 S) -1 -[[(1 S) -1 -[3-[2-[3-[(5- Yin cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]carbamoyl]-3-oxo-3-(tritylamino) propyl]carbamate (200 mg, 154 μmol, 74.01% yield) as a white solid.

[0794] MS m / z: 1301.5 [M+H]+

[0795] (2S) -2-amino-N-[(1S) -1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]-N'-trityl-butanediamide

[0796] To a solution of 9H-fluoren-9-ylmethyl N-[(1S) -1-[[(1S) -1 -[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H- pyrazol-5-yl]-3-methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]carbamoyl]-3-oxo-3- (tritylamino) propyl]carbamate (200 mg, 154 μmol, 1 eq) in DCM (5 mL) was added DBU (404 mg, 2.65 mmol, 17.26 eq), then the mixture was stirred at 20 °C for 15 mins. The mixture was diluted in DCM (20 mL). The mixture was washed with brine (10 mL*3). The organic layer was dried over Na2SC>4, filtered and concentrated to give the crude product. The crude product was triturated with MTBE (10 mL) and filtered. The cake was dried under vacuum to give (2S) -2-amino-N-[(1 S) -1 -[3-[2-[3-[(5-cyanopyrazin-2- yl) amino]-1 H-pyrazol-5-yl]-3-methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]-N'-trityl- butanediamide (120 mg, 92.37 μmol, 60.06% yield, 83% purity) as a white solid.

[0797] MS m / z: 1078.3 [M+H]+

[0798] (2S) -N-[(1S) -1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]-2-[[2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetyl]amino]-N'-trityl-butanediamide To a mixture of (2S) -2-amino-N-[(1 S) -1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3- methoxy-phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) propyl]-N'-trityl-butanediamide (130 mg, 121 μmol, 1 eq), 2-[[2-[6-(2, 5-dioxopyrrol-1 -yl) hexanoylamino]acetyl]amino]acetic acid (50 mg, 154 μmol, 1.27 eq), DIEA (46 mg, 362 μmol, 3 eq) in DMF (1 mL) was added T4P (100 mg, 139 μmol, 50wt%, 1.15 eq), then the mixture was stirred at 20°C for 1 h. The mixture was diluted in EtOAc (50 mL). The mixture was washed with brine (30 mL*3). The organic layer was dried over Na2SO4, filtered and concentrated to give (2S) -N-[(1 S) -1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- p henoxy] p ropy lcarbamoyl]-3-oxo-3- (trityl ami no) p ropy l]-2- [[2- [[2- [6- (2, 5-d ioxopy rrol- 1 -yl) hexanoylamino]acetyl]amino]acetyl]amino]-N'-trityl-butanediamide (130 mg, crude) as a white solid.

[0799] MS m / z: 1386.4 [M+H]+

[0800] (2S) -N-[(1S) -3-amino-1 -[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1H-pyrazol-5-yl]-3-methoxy- phenoxy]propylcarbamoyl]-3-oxo-propyl]-2-[[2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetyl]amino]butanediamide

[0801] To a solution of (2S) -N-[(1S) -1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propylcarbamoyl]-3-oxo-3-(tritylamino) p ropy l]-2- [[2- [[2- [6- (2, 5-d ioxopy rrol- 1 -yl) hexanoylamino]acetyl]amino]acetyl]amino]-N'-trityl-butanediamide (100 mg, 72.17 μmol, 1 eq) in DCM (5 mL) was added TFA (1.54 g, 13.46 mmol, 1.00 mL, 186.52 eq) and triisopropylsilane (231 mg, 1.46 mmol, 20.24 eq), then the mixture was stirred at 20°C for 1 h. The mixture was concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex Luna Cis 150*25 mm*10pm;mobile phase: [H2O (0.1%TFA) -ACN];gradient:18%-48% B over 15.0 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to (2S) -N-[(1 S) -3-amino-1-[3-[2-[3-[(5-cyanopyrazin-2-yl) amino]-1 H-pyrazol-5-yl]-3-methoxy- phenoxy]propylcarbamoyl]-3-oxo-propyl]-2-[[2-[[2-[6-(2, 5-dioxopyrrol-1-yl) hexanoylamino]acetyl]amino]acetyl]amino]butanediamide (24.1 mg, 26.75 μmol, 37.06% yield, 100% purity) as a white solid.

[0802] MS m / z: 901.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ ppm: 12.35 (s, 1 H), 10.68 (s, 1 H), 8.63 (s, 1 H), 8.56 (s, 1 H), 8.23 - 8.14 (m, 2H), 8.12 - 8.03 (m, 2H), 7.85 (t, J = 5.6 Hz, 1 H), 7.50 (s, 1 H), 7.35 - 7.30 (m, 1 H), 7.29 (s, 1 H), 7.00 (s, 3H), 6.86 (s, 2H), 6.78-6.72 (m, 2H), 4.55 - 4.48 (m, 1 H), 4.48 - 4.42 (m, 1H), 4.00 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H), 3.72 - 3.68 (m, 4H), 3.37 (t, J = 6.8 Hz, 2H), 3.29 - 3.18 (m, 2H), 2.59 - 2.55 (m, 2H), 2.48 - 2.42 (m, 2H), 2.11 (t, J = 7.2 Hz, 2H), 1.91 - 1.82 (m, 2H), 1.54 - 1.44 (m, 4H), 1.23 - 1.14 (m, 2H)

[0803] 1H NMR (400 MHz, DMSO+D2O-d6) δ ppm: 8.59 (s, 1 H), 8.51 (s, 1 H), 7.30 (t, J = 8.4 Hz, 1 H), 6.92 (s, 2H), 6.84 (s, 1 H), 6.77-6.68 (m, 2H), 4.47 (t, J = 6.8 Hz, 1 H), 4.43-4.38 (m, 1 H), 3.97 (t, J = 5.6 Hz, 2H), 3.78 (s, 3H), 3.68 (s, 4H), 3.34 (t, J = 6.8 Hz, 2H), 3.28 - 3.13 (m, 2H), 2.60 - 2.52 (m, 2H), 2.48 - 2.40 (m, 2H), 2.09 (t, J = 7.6 Hz, 2H), 1.89 - 1.77 (m, 2H), 1.50-1.39 (m, 4H), 1.20 - 1.11 (m, 2H)

[0804] Synthesis of a GPVK maleimide linker with Prexasertib (H ALA-016)

[0805] Experimental Procedure: Step-1 :

[0806] Experimental procedure: 2-CI CTC Resin (2 g, 39.61 mmol, 1.1 eq) was swelled in DMF (50 mL) for 15 min at RT, filtered and washed with DMF (3 x 50 mL). Fmoc-Lys(Boc)-0H: A solution of Fmoc-Lys(Boc)-OH (55.68 g, 118.84 mmol, 3.0 eq) and DIPEA in DMF (50 mL) was added to the resin and shaken at RT for 4 h. The resin was filtered and washed with DMF (3 x 50 mL) and DCM (3 x 50 mL) washed with diethyl ether (3x25 mL), dried under vacuum.

[0807] Loading factor: 0.66 mmol / g was observed.

[0808] Resin was taken in peptide flask and added capping solution (10 vol) (DMF: DIPEA: MeOH = 80: 10: 10). The resin was agitated under nitrogen bubbling at 25-28 °C for 30 min. The solvents were drained. The resin was washed with DMF (10 Vol; 3 x 5 min) and DCM (10 Vol; 3 x 5 min).

[0809] Fmoc-Deprotection: A mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated under nitrogen bubbling at RT for 15 min. The solvents were drained. A second lot of a mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated at RT for 15 min. The solvents were drained. The resin was washed with DMF (10 Vol; 3 x 5 min) and DCM (10 Vol; 3 x 5 min). Fmoc-Val-OH: A solution of Fmoc-Val-OH (40.335 g, 118.84 mmol, 3.0 equiv.) and Oxyma (3.0 eq) and DIC (5.0 eq) in DMF (50 mL) was added to the resin and the resin was shaken at RT for 1 h. The resin was filtered and washed with DMF (3 x 50 mL) followed by DCM (3 x 50 mL).

[0810] Fmoc-Deprotection: A mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated under nitrogen bubbling at RT for 15 min. The solvents were drained. A second lot of a mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated at RT for 15 min. The solvents were drained. The resin was washed with DMF (10 Vol; 3 x 5 min) and DCM (10 Vol; 3 x 5 min). Fmoc-Pro-OH: A solution of Fmoc-Pro-OH (40.095 g, 118.84 mmol, 3.0 eq) and Oxyma (3 eq) and DIC (5 eq) in DMF (50 mL) was added to the resin and the resin was shaken at RT for 1 h. The resin was filtered and washed with DMF (3 x 50 mL) followed by DCM (3 x 50 mL).

[0811] Fmoc-Deprotection: A mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated under nitrogen bubbling at RT for 15 min. The solvents were drained. A second lot of a mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated at RT for 15 min. The solvents were drained. The resin was washed with DMF (10 Vol; 3 x 5 min) and DCM (10 Vol; 3 x 5 min). Fmoc-Gly-OH: A solution of Fmoc-Gly-OH (35.334 g, 118.847 mmol, 3.0 eq) and Oxyma(3.0 eq) and DIC (5.0 eq) in DMF (50 mL) was added to the resin and the resin was shaken at RT for 1 h. The resin was filtered and washed with DMF (3 x 50 mL) followed by DCM (3 x 50 mL) to get N2-(((9H-fl uoren-9-yl) methoxy) carbonyl) glycyl-L-prolyl-L-valyl-N6-(tert-butoxycarbonyl)-L-lysine.

[0812] Analytical data: LCMS (ES) m / z = 722.32 [M]+

[0813] Step-2:

[0814] Experimental procedure: A solution of methyl N2-(((9H-fluoren-9-yl) methoxy) carbonyl) glycyl-L-prolyl- L-valyl-N6-(tert-butoxycarbonyl)-L-lysinate (2 g, 2.718 mmol, 1.0 eq) was swelled in DMF (20 mL) for 15 min at RT, filtered and washed with DMF (3 x 30 mL) mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated under nitrogen bubbling at RT for 15 min. The solvents were drained.

[0815] Fmoc-Deprotection: A second lot of a mixture of 20 % piperidine in DMF (10 Vol) was added to the resin and gently agitated at RT for 15 min. The solvents were drained. The resin was washed with DMF (10 Vol; 3 x 5 min) and DCM (10 Vol; 3 x 5 min). A solution of 1 ,T-(1-oxohexane-1 ,6-diyl)bis(1 H-pyrrole-2,5- dione) (1.578 g, 5.436 mmol, 2.0 eq) in N,N-dimethylformamide (20 mL) was added to the resin and shaken at RT for 2 h. The resin was filtered and washed with DMF (3 x 50 mL) and DCM (3 x 50 mL) washed with diethylether (3x25 mL),

[0816] Fmoc-Deprotection: A mixture of 30 % HFIPA in DCM (10 Vol) was added to the resin and gently agitated under nitrogen bubbling at RT for 15 min. The solvents were collected and distilled under vaccum. A second lot of a mixture of 30 % HFIPA in DCM (10 Vol) was added to the resin and gently agitated at RT for 15 min. The solvents were collected and distilled under vacuum to afford N6- (tert- butoxycarbo nyl)-N2- (6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl) hexanoyl) glycyl-L-prolyl-L-valyl-L-lysine (0.5 g).

[0817] Nature of the compound: Off-White Solid.

[0818] Step-3: perfluorophenyl A / 3-(tert-butoxycarbonyl)- / V2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 ■ yl)hexanoyl)glvcyl-L-prolyl-L-valyl-L-lvsinate (3A):

[0819] To a solution of / V3-(tert-butoxycarbonyl)- / V2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl)glycyl-Z_- prolyl-L-valyl-L-lysine (190 mg, 0.27 mmol), in Dry 1 ,4-Dioxane (2 ml) was added A / , / V- Dicyclohexylcarbod iimide (70.73 mg, 0.34 mmol), followed addition of Pentafluorophenol SM-A (62.5 mg, 0....

Claims

New PCT-Patent Application based on EP 24 158 292.3Uni versite de Geneve, Universitat BaselVossius Ref.: AG3349 PCT BSCLAIMS1 . An antibody-drug conjugate, comprising an antibody or antigen-binding fragment thereof, and an ATR / CHK1 inhibitor.

2. The antibody-drug conjugate of claim 1 , wherein the antibody or antigen-binding fragment thereof is capable of targeting the antibody-drug conjugate to cancer cells.

3. The antibody-drug conjugate of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds to the same epitope as trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, Mirvetuximab, Datopotamab and Telisotuzumab.

4. The antibody-drug conjugate of claim 3, wherein the antibody or antigen-binding fragment thereof is selected from trastuzumab, sacituzumab, gemtuzumab, brentuximab, inotuzumab, polatuzumab, enfortumab, belantamab loncastuximab, Moxetumomab, Cetuximab, Disitamab, Tisotumab, Mirvetuximab, Datopotamab and Telisotuzumab5. The antibody-drug conjugate of claim 4, wherein the antibody-drug conjugate comprises trastuzumab or sacituzumab.

6. The antibody-drug conjugate of claim 4, wherein the antibody-drug conjugate comprises trastuzumab.

7. The antibody-drug conjugate of any one of claims 1 to 6, wherein the antibody comprises a heavy chain characterized by sequence at least 95% identical to a sequence selected from sequences according to SEQ ID NO.: 1, 5 and 7, and a light chain characterized by sequence at least 95% identical to a sequence selected from sequences according to SEQ ID NO.:

2. 3 and 6, wherein the antibody binds to the same epitope as trastuzumab.

8. The antibody-drug conjugate of any one of claims 1 to 6, wherein the antibody comprises a heavy chain characterized by sequence identical to a sequence selected from sequences according to SEQ ID NO.: 1 , 5 and 7, and a light chain characterized by sequence identical to a sequence selected from sequences according to SEQ ID NO.:

2. 3 and 6.

9. The antibody-drug conjugate of any one of claims 1 to 8, wherein the ATR / CHK1 inhibitor is an ATR inhibitor, preferably wherein the ATR inhibitor is Elim userti b or its derivative, more preferably wherein the ATR inhibitor is Elimuserti b.

10. The antibody-drug conjugate of claim 9, wherein the ATR inhibitor is according to formula (I):R2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -O-(C1-6 alkyl), -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-(C1-6 alkyl), -N(C1-6 alkyl)-(C1-6 alkyl), - COO-(C1-6 alkyl), -CONH-(C1-6 alkyl), -CON(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -SO-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHSO2-(C1-6 alkyl), and -N(C1-6 alkyl)-SO2-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl; and R3and R4are each independently hydrogen or methyl.

11. The antibody-drug conjugate of claim 10, wherein the compound of formula (I) is a compound of formula (la):

12. The antibody-drug conjugate of any one of claims 1 to 11, wherein the antibody-drug conjugate comprises a moiety of formula (lb):or the antibody-drug conjugate comprises a moiety according to formula (Ic):wherein: the empty valence is connected to the antibody or antigen-binding fragment thereof;R1is selected fromR2is selected from H, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, -O-(C1-6 alkyl), -O-heterocycloalkyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, -NH2, -NH-(C1-6 alkyl), -N(C1-6 alkyl)-(C1-6 alkyl), - COO-(C1-6 alkyl), -CONH-(C1-6 alkyl), -CON(C1-6 alkyl)-(C1-6 alkyl), -S-(C1-6 alkyl), -SO-(C1-6 alkyl), -SO2-(C1-6 alkyl), -NHSO2-(C1-6 alkyl), and -N(C1-6 alkyl)-SO2-(C1-6 alkyl); wherein said heterocycloalkyl, said cycloalkyl, said cycloalkenyl, said phenyl and said heteroaryl are each optionally substituted with one or more groups selected from -OH, halogen and C1-6 alkyl; and R3and R4are each independently hydrogen or methyl.

13. The antibody-drug conjugate of any one of claims 1 to 11, wherein the antibody-drug conjugate comprises a moiety according to formula (lb-1):(lb-1). or the antibody-drug conjugate comprises a moiety according to formula (lc-1):

14. The antibody-drug conjugate of claim 9, wherein the ATR inhibitor is selected from Elimusertib, Camonsertib, Ceralasertib, Gartisertib, Tuvusertib and Berzosertib.

15. The antibody-drug conjugate of claim 9, wherein the ATR inhibitor is Camonsertib.

16. The antibody-drug conjugate of claim 15, wherein the antibody-drug conjugate comprises a moiety according to formula:

17. The antibody-drug conjugate of any one of claims 1 to 8, wherein the ATR / CHK1 inhibitor is a CHK1 inhibitor.

18. The antibody-drug conjugate of claim 17, wherein the CHK1 inhibitor is according to formula:wherein:X’ is CH or N;R1is selected from hydrogen, C1-5 alkyl, halogen, cycloalkyl and heterocycloalkyl, wherein said cycloalkyl and said heterocycloalkyl are each optionally substituted with one or more groups selected from C1-5 alkyl and halogen;R2 1is -CN and R2 2is hydrogen, or R2 1is hydrogenR3is — (C1-5 alkylene)-NH2, - (C1-5 alkylene)-NH— CO-(C1-6 alkylene)-NH2, - (cycloalkylene)-NH2 or -(cycloalkylene)-NH-CO-(C1-5 alkylene)-NH2.

19. The antibody-drug conjugate of claim 17, wherein the CHK1 inhibitor is:

20. The antibody drug conjugate of claim 17, wherein the antibody-drug conjugate comprises a moiety according to formula:21 . The antibody drug conjugate of claim 17, whereinthe antibody-drug conjugate comprises a moiety according to formula:

22. The antibody-drug conjugate of claim 17, wherein the CHK1 inhibitor is23. The antibody-drug conjugate of any one of claims 1 to 22, wherein the antibody-drug conjugate comprises a moiety according to formula:wherein n is an integer from 1 to 5, preferably wherein n is 2 to 4, more preferably wherein n is 3, preferably wherein the left empty valence of the moiety is connected to the antibody or the antigen-binding fragment thereof, and wherein the right empty valence is connected to the ATR / CHK1 inhibitor.

24. The antibody-drug conjugate of any one of claims 1 to 22, wherein the antibody-drug conjugate comprises a moiety selected from the following moieties:preferably wherein the left empty valence of the moiety is connected to the ATR / CHK1 inhibitor, and wherein the right empty valence is connected to the antibody or the antigen-binding fragment thereof.

25. The antibody-drug conjugate of any one of claims 1 to 22, wherein the antibody-drug conjugate is obtainable in a reaction of a compound selected from:with a cysteine residue of the antibody or the antigen-binding fragment thereof.

26. The antibody-drug conjugate of any one of claims 1 to 22, wherein the antibody-drug conjugate is obtainable in a reaction of a compound selected from:5 with a cysteine residue of the antibody or the antigen-binding fragment thereof.

27. A compound of formula (III):or a pharmaceutically acceptable salt thereof, wherein:R3and R4are each independently hydrogen or methyl, and n is an integer from 1 to 5, preferably n is 3,28. The compound of claim 27, wherein the compound is a compound of formula (Illa):or a pharmaceutically acceptable salt thereof.

29. A compound of formula (II):R3and R4are each independently hydrogen or methyl, and n is an integer from 1 to 5, preferably from 2 to 4, more preferably n is 3.

30. The compound of claim 29, wherein the compound is according to formula:31 . A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to26, and a pharmaceutically acceptable carrier.

32. The antibody-drug conjugate of any one of claims 1 to 26 or the pharmaceutical composition of claim 31 for use as a medicament.

33. The antibody-drug conjugate of any one of claims 1 to 26 or the pharmaceutical composition of claim 31 for use in the treatment of cancer.

34. The antibody-drug conjugate for use of claim 33 or the pharmaceutical composition for use of claim 33, wherein the cancer is HER2-positive cancer, CD30-positive cancer, CD33-positive cancer, CD22-positive cancer, CD79b-positive cancer, Nectin-4-positive cancer, Trop2-positive cancer, BCMA-positive cancer, EGFR-positive cancer, CD19-positive cancer, Fra-positive cancer or B7H4-positive cancer.

35. The antibody-drug conjugate for use of claim 33 or the pharmaceutical composition for use of claim 33, wherein the cancer is characterized by DNA-repair deficiency.

36. The antibody-drug conjugate for use of claim 33 or the pharmaceutical composition for use of claim 33, wherein the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, and pancreatic cancer.

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