Novel linkers and its preparation methods thereof

Abstract

The present invention relates to a novel linker for Antibody-drug conjugates (ADCs), Peptide- drug conjugates (PDCs) and a preparation method for it, wherein the linker's one end can selectively and covalently link a targeting substance, and its other end can covalently link to a small molecule compound or a cytotoxic payload. The linkers of the present invention can be used to prepare an antibody drug conjugate, and it can be used for various applications including anticancer activity and other medicinal uses.

Description

[0001] NOVEL LINKERS AND ITS PREPARATION METHODS THEREOF Field of invention

[0002] The present invention relates to novel linkers utilized in Antibody-drug conjugates (ADCs), Peptide-drug conjugates (PDCs) and a preparation method for it. The novel stable linkers described herewith are of Formula (I), these novel linkers at one end can selectively conjugated with suitable small molecule compounds or a cytotoxic payload through a spacer linker and at another end it can covalently link to suitable monoclonal antibody or peptides. The linkers of the present invention can be used to prepare ADCs / PDCs, and it can be used for various applications including cytotoxic activity and other medicinal uses. In some aspect, the present disclosure provides compounds of the Formula (I)

[0003] Pm1-Z1-Z2-Z3-Z4-Xp1

[0004] Formula (I)

[0005] The linkers presented herein are utilized to generate cell-specific delivery agents and medications that target tumour cells.

[0006] Background of the invention

[0007] The linkers are biochemical compounds which is attached to the payload at one end and at another end it attached to the agent which can give us a targeted drug delivery. This strategy aims to improve the therapeutic index by minimizing off-target effects associated with traditional chemotherapeutic agents. The most critical aspect influencing the success of targeted therapy is the nature of the linker that connects the cytotoxic payload and the agents which regulates the drug's targeting release, and the cytotoxins are substances that either cause cell death, promote apoptosis, or impair cell viability. For linkers to be an effective, it should have the best stability in the bloodstream as well as it should allow an efficient cleavage upon internalization into target cells. Despite the great potential and many years of research and clinical experiments, only few targeted drug delivery agents are approved for clinical use. One of the lessons learned from the previous discoveries was that we require highly stable and potentially releasable linkers. There is, therefore, a compelling need in the art for novel linkers that overcome these limitations and offer enhanced stability, controlled and targeted release, and improved selectivity. The present invention will focus on our efforts to develop a novel stable linker and the application of these linkers for conjugation for the treatment of cancer and other diseases. Here we design a novel stable peptide-based linker, that exclusively impact the linker mechanism on both efficacy and toxicity. Another key factor is the design of the proper coupling strategy that is coupling of linkers to the conjugating agents, as it is critical for the drug targeting. The heterogeneity of conjugates seriously affects the pharmacokinetics, drug stability and reproducibility. Therefore, site specific, highly homogenous coupling is the important aspect in the linkers design. The innovation described here has the potential to advance the field of conjugation therapy, by providing new opportunities for more precise and effective treatment for a variety of diseases, including cancers.

[0008] Antibody drug conjugates (ADC’s), composed of an antibody, linker, and cytotoxic agent (drug), are one of the most complex drug platforms in the oncology armamentarium[1]. They are a payload delivery system with major variables influencing their success including: (i) the rate of internalization of the payload; (ii) the expression of the target antigen on the tumor and normal tissues with the implications for both patient selection as well as therapeutic index; (iii) the linker chemistry and, implicit within the choice of chemistry, the extracellular as well as intracellular stability; and (iv) the selection of payload for the tumor indication[2]. The mechanisms of action of ADCs include direct binding between the antibody and its targeted cell surface antigen, and intracellular or extracellular release of cytotoxic drugs[3]. Present invention provides Trastuzumab derived antibody drug conjugates to enable specific delivery of drug (Exatecan and / or its analogues) to target antigen on cancer cells.

[0009] In summary, the present invention introduces novel linkers that overcomes existing limitations, thereby contributing to the development of more effective and targeted therapeutic interventions for various diseases.

[0010] Summary of the invention

[0011] The present invention relates to a novel linker for Antibody-drug conjugates (ADCs), Peptide-drug conjugates (PDCs) and a preparation method for it, wherein the linker's one end can selectively and covalently link a targeting substance, and its other end can covalently link to a small molecule compound or a cytotoxic payload. The linkers of the present invention can be used to prepare an antibody drug conjugate, and it can be used for various applications including anticancer activity and other medicinal uses.

[0012] Embodiment(s) of the invention

[0013] An embodiment of the present invention is to provide novel linkers of general Formula (I), novel intermediates involved in their synthesis, their pharmaceutically acceptable salts, its stereoisomeric forms, or mixtures thereof, wherein at least one of the amino acids is unnatural amino acid,

[0014] Pm1-Z1-Z2-Z3-Z4-Xp1

[0015] Formula (I)

[0016] when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent.

[0017] In a further embodiment is provided a process for the preparation of novel linkers of general Formula (I), novel intermediates involved in their synthesis, their pharmaceutically acceptable salts, its stereoisomeric forms, or mixtures thereof.

[0018] In another embodiment is provided use of the linkers of general Formula (I), novel intermediates involved in their synthesis, their pharmaceutically acceptable salts, its stereoisomeric forms, or mixtures for the preparation of ADCs / PDCs.

[0019] In an embodiment of the present invention is to provide the novel compounds of general formula (II), novel intermediates involved in their synthesis, their pharmaceutically acceptable salts, its stereoisomeric forms, or mixtures thereof, wherein at least one of the amino acid is unnatural amino acid,

[0020] Pm1-Z1-Z2-Z3-Z4-Xp1-D

[0021] Formula (II)

[0022] when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent.

[0023] In an embodiment, the present invention provides antibody-linker-drug conjugates (ADC) of Formula (A),

[0024] Ab-Pm1-Z1-Z2-Z3-Z4-Xp1-D

[0025] Formula (A)

[0026] when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent.

[0027] In a further embodiment is provided use of the present invention for the treatment of cancer, specifically breast cancer. Description of the figure

[0028] Figure 1: JIMT-1 breast carcinoma xenograft study of ADC- 1 in Nude mice

[0029] Detailed description of the invention

[0030] Definitions

[0031] The term ‘natural amino acids’ indicates all those twenty amino acids, which are present in nature. The term ‘unnatural amino acids’ or ‘non-natural amino acids’ preferably represents either replacement of L-amino acids with corresponding D-amino acids such as replacement of L-Ala with D-Ala and the like or suitable modifications of the L or D amino acids, amino alkyl acids, either by

[0032] α-alkylation such as substitution of Ala with α-methyl Ala (Aib), replacement of Leu with α-methyl Leu;

[0033] substitution on the side chain of amino acid such as substitution of aromatic amino acid side chain with halogen, (C1-C3) alkyl, aryl groups, more specifically the replacement of Phe with halo Phe;

[0034] β amino acids such as β alanine;

[0035] The various groups, radicals and substituents used anywhere in the specification are described in the following paragraphs.

[0036] The term “alkyl” used herein, either alone or in combination with other radicals, denotes a linear or branched radical containing one to eighteen carbons, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, amyl, t-amyl, n-pentyl, n-hexyl, iso-hexyl, heptyl, octyl, decyl, tetradecyl, octadecyl and the like.

[0037] The term “cycloalkyl” used herein, either alone or in combination with other radicals, denotes a radical containing three to seven carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

[0038] Unless otherwise indicated, the term ‘amino acid’ as employed herein alone or as part of another group includes, without limitation, an amino group and a carboxyl group linked to the same carbon, referred to as ‘a’ carbon.

[0039] The absolute ‘S’ configuration at the ‘a’ carbon is commonly referred to as the ‘L’ or natural configuration. The ‘R’ configuration at the ‘a’ carbon is commonly referred to as the ‘D’ amino acid. In the case where both the ‘a-substituents’ is equal, such as hydrogen or methyl, the amino acids are Gly or Aib and are not chiral.

[0040] The term ‘derivatives’ mentioned anywhere in document indicates any substituted or homologous non-natural amino acid of that particular amino acid.

[0041] The term ‘mAb’ mentioned anywhere in the specification represents monoclonal antibody or antibody.

[0042] In accordance with the present invention, the various novel stable linkers of formula (I) which at one end can selectively conjugated with suitable cytotoxic drugs (D) through a spacer linker (S) and at another end can covalently link to suitable antibody (A). The linkers of the present invention can be used to prepare ADCs and it can be used for various applications including anticancer activity and other medicinal uses. The linkers of the present invention can be used to prepare PDCs also.

[0043] In an embodiment, the present disclosure provides novel linkers of general Formula (I)

[0044] Pm1-Z1-Z2-Z3-Z4-Xp1

[0045] Formula (I)

[0046] wherein,

[0047] P is selected from maleimide containing functional residue which is unsubstituted or substituted linear or branched (Ci-s)alkyl chain wherein (Ci-s)alkyl chain is unsubstituted or substituted by (C1-6)alkoxy, (C3-C6)cycloalkyl, aryl, heteroaryl or arylalkyl or polyethylene glycol (PEG) with more than one monomer, carboxylic acyl unit or its derivatives, unsubstituted or substituted amino acids or its derivatives; m1 is 0-8;

[0048] ‘Zi’ group may independently present or absent and is group selected from the group of uncharged amino acid residues, preferably selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl-isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl), etc;

[0049] ‘Z2’ group may independently present or absent and is group selected from the group of uncharged amino acid residues, preferably selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl-isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl), etc;

[0050] ‘Z3’ is an amino acid residue selected from the group of aromatic amino acid and aryl group wherein aromatic amino acid is selected from phenylalanine and their derivatives, α-methyl phenylalanine, 2-fluorophenylalanine, α-methyl-2-fluorophenylalanine, α-methyl-2,6-diflurophenylalanine, 2-Amino-5 -phenylpentanoic acid; the aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl, etc;

[0051] ‘Z4’ is an amino acid residue selected from the group of uncharged amino acid residues and their derivatives, preferably selected from the group of glycine, sarcosine, alanine, isoleucine, leucine, norleucine, homoleucine, [3-alanine, AIB and their derivatives such as N-methyl-isoleucine, N-methyl-leucine, [3-alanine, 1 -amino-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc; with the proviso that at least one of Zi to Z4 always represents an unnatural amino acid;

[0052] when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent;

[0053] X is selected from but not limited to 2-(aminomethoxy)acetic acid and their derivatives like 2-(aminomethoxy)propionic acid or 2-(aminomethoxy)butanoic acid, or it may be paraaminobenzylmethylcarbamate (PBMC) and its derivatives, aromatic compounds that are electronically similar to PAB group such as 2- aminoimidazol-5 -methanol derivatives; ortho or para-aminobenzylacetals; pl is 0-3.

[0054] In a preferred embodiment, P is selected from maleimide containing carboxylic acyl unit, Zi is absent or amino acid residues comprising branch or liner Ci-Cs alkyl, Z2 is absent or amino acid residues comprising branch or liner Ci-Cs alkyl, Z3 is phenylalanine and their derivatives, Z4 is glycin, [3-alanine, AIB, X is selected from 2-(aminomethoxy)acetic acid and their derivatives, ml is 1, 2 and pl is 1, 2. Linkers in ADC synthesis are crucial for connecting the antibody to the cytotoxic drug, influencing stability, release mechanism, and therapeutic efficacy.

[0055] Role and Importance of Linkers in ADCs

[0056] Linkers are the chemical bridges that tether the cytotoxic payload to the monoclonal antibody. Their design directly affects:

[0057] • Stability in circulation: Prevents premature drug release

[0058] • Controlled release: Ensures drug is released only in target cells

[0059] • Pharmacokinetics and toxicity: Influences how the ADC behaves in the body Linker Design Considerations

[0060] • Hydrophilicity: Impacts solubility and reduces aggregation

[0061] • Bioconjugation chemistry: Determines how the linker attaches to the antibody and drug (e.g., maleimide-thiol, click chemistry)

[0062] The invention also relates to methods for production of these medicaments and pharmaceutical composition thereof and their use in medicine. The conjugation techniques and linkers presented herein are utilized to generate cell-specific delivery agents and medications that target tumor cells.

[0063] While the invention has been primarily exemplified in relation to novel linkers containing amino acids, it will also be understood that the amide linkage between the residues may be replaced by a non-amide bond provided that the therapeutic potential is retained. The person skilled in the art will be aware of suitable modifications, such as thioamide bond formation, N-methylation of amide bonds and the like.

[0064] Sequences encompassing conservative substitutions of amino acids are also within the scope of the invention, provided that the biological activity is retained.

[0065]

[0066] Figure 1: Examples of some protected non-natural amino acids used in synthesis of novel linkers.

[0067] It is to be clearly understood that the compounds of the invention include amides and nonamides analogues, including but not limited to the following:

[0068] a) Compounds in which one or more amino acids is replaced by its corresponding D-amino acid. The skilled person will be aware that retro-inverso amino acid sequences can be synthesized by standard methods; see for example, Chorev M., Acc. Chem. Res., 26, 1993, 266-273;

[0069] b) Compounds, in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example, Olson G. L., et al., J. Med. Chem., 36(21), 1993, 3039-3049 and

[0070] c) Compounds in which individual amino acids are replaced by analogous structures for example Gly with Aib or [3-Ala; or Phe with α-Me-Phe etc;

[0071] Throughout the description the conventional one-letter and three-letter code for natural amino acids are used as well as generally acceptable three-letter codes for other unnatural amino acids such as Aib (α-amino isobutanoic acid), [3-Ala [beta alanine] are used. [Figure 1] Table 1: List of Novel linkers prepared

[0072]

[0073]

[0074]

[0075] Abbreviations used

[0076] The following abbreviations are employed in the examples and elsewhere herein: Ac = Acetyl

[0077] AIB = a-Amino-isobutyric acid,

[0078] a-Me-APPA = alpha-methyl- 2-aminophenyl pentanoic acid,

[0079] a-Me-L = alpha-methyl-leucine,

[0080] a-Me-Phe = alpha-methyl-phenylalanine,

[0081] a-Me-2F-Phe = alpha-methyl-2-fluorophenylalanine,

[0082] a-Me-2,6-diF-Phe = alpha-methyl-2,6-diflurophenylalanine,

[0083] AC3C= 1-amino-cyclopropanecarboxylic acid,

[0084] AC4C= 1-amino-cyclobutanecarboxylic acid,

[0085] AC5C= 1-amino-cyclopentanecarboxylic acid,

[0086] AC6C= 1-amino-cyclohexanecarboxylic acid,

[0087] ACN = Acetonitrile,

[0088] APPA = 2-Aminophenyl pentanoic acid,

[0089] p-Ala = beta alanine,

[0090] Boc = tert-Butoxycarbonyl,

[0091] But=O-tert-butyl group,

[0092] DBU = 1,8-Diazabicyclo[5.4.0]undec-7-ene

[0093] DCM = Dichloromethane,

[0094] DMF = N, N-Dimethylformamide,

[0095] DIPEA= Diisopropylethylamine,

[0096] Et = Ethyl,

[0097] Et2O = Diethyl ether,

[0098] 2F-Phe = 2-fluorophenylalanine,

[0099] Fmoc = Fluorenylmethoxycarbonyl,

[0100] g = Gram (s),

[0101] h = Hour (s),

[0102] HoLeu = homoleucine, HATU = Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium

[0103] HOBt = 1 -Hydroxybenzotriazole,

[0104] HOAt= 7-Aza-hydroxybenzotriazole,

[0105] HBTU = 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl aminium hexafluorophosphate, HPLC = High Performance Liquid Chromatography,

[0106] L = Liter,

[0107] LC / MS = Liquid Chromatography / Mass Spectrometry,

[0108] Me = Methyl,

[0109] Min = minute (s),

[0110] mL = milliliter,

[0111] μl = microliter,

[0112] mg = milligram (s),

[0113] mmol = millimole (s),

[0114] MS= Mass Spectrometry,

[0115] Nle = Norleucine,

[0116] NMe-Ile = N-methyl-isoleucine

[0117] NMe-Leu = N-methyl-leucine

[0118] NMe-Nle = N-methyl-norleucine

[0119] PyBOP = Benzotriazole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, Pd / C = Palladium on carbon

[0120] Sar = Sarcosine,

[0121] SPPS = Solid Phase Peptide Synthesis,

[0122] TATU = O-(7-Azabenzotriazol-l-yl)- N, N, N', N'-tetramethyluronium tetrafluoroborate TBTU= 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate,

[0123] Trt= Trityl group.

[0124] The novel compounds of the present invention were prepared using the reactions and techniques described above, together with conventional techniques known to those skilled in the art of organic synthesis, or variations thereon as appreciated by those skilled in the art. The reactions can be performed in solvents appropriate to the reagents and materials employed and are suitable for the transformations being affected. Preferred methods include, but not limited to those described below, where all symbols are as defined earlier unless and otherwise defined below.

[0125] The compounds of the formula (I) can be prepared as described in schemes above along with suitable modifications / variations which are well within the scope of a person skilled in the art.

[0126] Preparation of the novel linkers:

[0127] Several synthetic routes can be employed to prepare the novel linkers of the present invention well known to one skilled in the art of organic synthesis. The novel linkers of formula (I), where all symbols are as defined earlier can be synthesized using the methods described below, together with conventional techniques known to those skilled in the art of synthesis, or variations thereon as appreciated by those skilled in the art. Referred methods include, but not limited to those described below.

[0128] The novel linkers thereof described herein may be produced by chemical synthesis using suitable variations of both the solution-phase (preferably, using Boc-chemistry or Cbz-Chemistry; M. Bodansky, A. Bodansky, “The practice of peptide synthesis”, Springer-Verlag, Berlim, 1984; E. Gross, J. Meinhofer, “The peptide synthesis, analysis, biology”, Vol. 1, Academic Press, London, 1979).

[0129] The preferred strategy for preparing the novel linkers of this invention is based on the use of solution phase approach, wherein Cbz (benzyloxycarbonyl) group is used for temporary protection of the a-amino group, in combination with the acid labile protecting groups, such as tert-butoxycarbonyl (Boc), tert-butyl (But). Trityl (Trt) groups whenever it needed a temporary protection of the amino acid side chains, if present (see for example E. Atherton & R. C. Sheppard, "The Fluorenylmethoxycarbonyl amino protecting group", in "The peptides: Analysis, synthesis, Biology"; Volume 9 - "Special methods in peptide synthesis, Part C", pp.

[0130] 1-38, S. Undenfriend & J. Meienhofer, Eds., Academic Press, San Diego, 1987).

[0131] The novel linkers can be synthesized in a stepwise manner in solution phase as described below: The coupling of an amino acid is performed by activation of its carboxyl group as an active ester and reaction thereof with unprotected a-amino group of the N-terminal amino acid. After coupling, the deprotection of Cbz group was achieved by Pd / C and Hydrogen to get free amine which is again coupled with Cbz protected acids to get desired linker. The sequence of a-amino group deprotection and coupling is repeated until the desired linker sequence is assembled (Scheme 1). The resulting linker is finally purified by column chromatography or reverse phase HPLC.

[0132]

[0133] Scheme 1: General scheme for preparation of novel linkers in solution phase

[0134] Compound 1 of above scheme is protected amino acid.

[0135] R is -H, substituted or unsubstituted alkyl or aryl; q is 0-12.

[0136] Step G Step H

[0137]

[0138] O

[0139] Step I H2 / Pd / C Et / EA

[0140]

[0141] Step J

[0142]

[0143] DBU / DCM Intermediate 1

[0144]

[0145] Scheme 2: General Scheme for intermediate (1): 2-(aminomethoxy)acetic acid (XVII) The coupling of the subsequent amino acids can be accomplished using HOBt or HO AT or active esters produced from EDC / HOBt or EDC / HOAT or HATU / DIPEA or O-Hydroxy succinimide. In case of some difficult coupling, especially coupling of those amino acids, which are hydrophobic or amino acids with bulky side chain protection; complete coupling can be achieved using a combination of highly efficient coupling agents such as HBTU, PyBOP or TATU, with additives such as DIPEA.

[0146] The non-natural non-commercial amino acids present at different position were incorporated into the linker chain, using one or more methods known in the art. In one approach, Cbz-protected non-natural amino acid or Boc-protected non-natural amino acid was prepared in solution, using appropriate literature procedures. For example, the Boc-protected APPA analogs, described above, were prepared from L-pyroglutamic acid, in good enantiomeric purity, using modified literature procedure (Betsbrugge J. V., et al., Tetrahedron, 54, 1988, 1753-1762)

[0147] In another method the novel linkers of the present invention can be chemically synthesized by methods which are well known in the art. It is also possible to produce the linkers of the present invention using solid phase peptide synthesis [SPPS] methods using Boc or Fmoc Chemistry. Also, methods for isolating and purifying the novel linkers prepared by SPPS are well known in the literature and includes e.g. gel filtration, semi preparative HPLC etc.

[0148] The Cbz-protected a-methylated amino acids were prepared using asymmetric Strecker synthesis (Boesten, W. H. J., et al., Org. Lett., 3(8), 2001, 1121-1124; Cativiela C., Diaz-de-villegas M. D., Tetrahedran Asymmetry, 9, 1988, 3517-3599). The resulting derivative was then used in the step-wise synthesis of the linkers. Alternatively, the required non-natural amino acid was synthesized directly using synthetic organic chemistry procedures and a linear linker chain were prepared.

[0149] The linker precursors for their respective novel linkers may be cleaved and deprotected using suitable variations of any of the standard cleavage procedures described in the literature (King D. S., et al., Int. J. Peptide Protein Res., 36, 1990, 255-266).

[0150] Column Chromatography purification of the novel linker:

[0151] The novel linkers with the desired purity can be purified by using the conventional column chromatography or by using the automated column chromatography instruments like using flash chromatography for purification [Teledyne Combiflash]. Preparative HPLC purification of the novel linker:

[0152] The novel linkers with the desired purity can be obtained by purification using preparative HPLC. The solution of crude linker is injected into a semi-Prep column (Luna 10μ; C18; 100 Å°), dimension 250 X 50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1 % TFA or 0.1% Acetic acid or ammonium acetate, using a flow rate of 40 ml / min with effluent monitoring by PDA detector at 220 nm. The linker after purification from semi-Prep HPLC was filtered through a membrane filter (0.2 μm) and subsequently lyophilized to yield the desired compounds.

[0153] The structures of the purified novel linkers can be confirmed by Electrospray Mass Spectroscopy (ES-MS) analysis.

[0154] HPLC analysis of the purified novel linkers:

[0155] After purification by preparative HPLC as described above, each novel linker attached with spacer and payloads was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLC system. For analytical HPLC analysis of novel linkers attached with spacer and payloads, Luna 5μ; C18; 100 Å°, dimension 250 X 4.6 mm column was used, with a linear gradient of 0.1% TFA and ACN buffer and the acquisition of chromatogram was carried out at 220 nm, using a PDA detector.

[0156] The compounds can be purified whereever required, by recrystallization, trituration, precipitation, preparative thin layer chromatography, flash chromatography or by preparative HPLC method.

[0157] 'H NMR spectral data given in the examples (vide infra) are recorded using a 400 MHz spectrometer (Bruker AVANCE-400) and reported in 8 scale. Until and otherwise mentioned the solvent used for NMR is CDCl3using TMS as the internal standard.

[0158] Column Chromatography purification of the novel linker attached with spacer and payloads:

[0159] The novel linkers with the desired purity can be purified by using the conventional column chromatography or by using the automated column chromatography instruments like using flash chromatography for purification [Teledyne Combiflash]. Preparative HPLC purification of the novel linker attached with spacer and payloads:

[0160] The novel linkers atached with spacer and payloads having desired purity can be obtained by purification using preparative HPLC. The solution of crude novel linkers atached with spacer and payloads is injected into a semi-Prep column (Luna 10μ; C18; 100 Å°), dimension 250 X 50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1 % TFA, using a flow rate of 40 ml / min with effluent monitoring by PDA detector at 220 nm. The novel linkers attached with spacer and payloads after purification from semi-Prep HPLC was filtered through a membrane filter (0.2 μm) and subsequently lyophilized to yield the white to off-white compounds. The some of the novel linkers atached with spacer and payloads may be purified using column chromatography by using the conventional columns or automated column chromatography techniques like using flash chromatography for purification [eg. Teledyne combiflash]. The structures of the purified novel linkers attached with spacer and payloads can be confirmed by Electrospray Mass Spectroscopy (ES-MS) analysis.

[0161] HPLC analysis of the purified novel linkers attached with spacer and payloads

[0162] After purification by preparative HPLC as described above, each novel linker atached with spacer and payloads was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLC system. For analytical HPLC analysis of novel linkers atached with spacer and payloads, Luna 5μ; C18; 100 Å°, dimension 250 X 4.6 mm column was used, with a linear gradient of 0.1% TFA and ACN buffer and the acquisition of chromatogram was carried out at 220 nm, using a PDA detector.

[0163] Characterization by Mass Spectrometry

[0164] Each novel linker atached with spacer and payloads was characterized by electrospray ionization mass spectrometry (ESI-MS), either in flow injection or LC / MS mode. Triple quadrupole mass spectrometers (API-3000 (MDS-SCIES, Canada) was used in all analyses in positive and negative ion electrospray mode. Full scan data was acquired over the mass range of quadrupole, operated at unit resolution. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotopic molecular weight. Quantification of the mass chromatogram was done using Analyst 1.4.1 software.

[0165] The invention is further illustrated by the following non-limiting examples which describe the preferred way of carrying out the present invention. These are provided without limiting the scope of the present invention in any way. The novel compounds of general formula (II), novel intermediates involved in their synthesis, their pharmaceutically acceptable salts, its stereoisomeric forms, or mixtures thereof.

[0166] Pm1-Z1-Z2-Z3-Z4-Xp1-D

[0167] Formula (II)

[0168] Pm1, Z1, Z2, Z3, Z4, Xp1are described as above;

[0169] when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent.

[0170] D is selected from Exatecan mesylate.

[0171] Table 2: List of Novel linkers with payload

[0172]

[0173]

[0174] Procedure for conjugation of novel linkers with payload

[0175]

[0176] Scheme 3: General scheme for preparation of novel linkers with payload To the solution of novel linker (10) (200 mg, 0.349 mmol) and Exatecan Mesylate [DX8951f] (15) (185 mg, 0.349 mmol) and in DMF (2.0 mL) was added HATU (133 mg, 0.349 mmol) and DIPEA (0.142 ml, 0.762 mmol) at 0°C, the reaction mixture was stirred 18h at room temperature. Then DMF was evaporated and residue was diluted with ethyl acetate. The organic layer was washed sequentially with aqueous saturated sodium bicarbonate solution, then 10% aqueous citric acid solution and brine (20 ml each) and the solvent was concentrated under reduced pressure. The crude mixture was purified by RP HPLC to obtain conjugated product of novel linkers with payload.

[0177] The present invention provides antibody-linker-drug conjugates of Formula (A),

[0178] Ab-Pmi -Z i -Z2-Z3 -Z4-Xpi-D

[0179] Formula (A)

[0180] wherein “Ab” is selected from Trastuzumab; “D” is selected from Exatecan mesylate; Pmi is a stretcher unit that links the antibody to linker; Z1-Z2-Z3-Z4 is a linker unit; Xpi is a spacer unit that links the drug unit to linker; when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent.

[0181] Antibody (Ab):

[0182] The antibody of the antibody drug conjugates (ADC) of the present invention is Trastuzumab or any posttranslational modification of Trastuzumab thereof. The posttranslational modification of the antibody includes, but not limited to, deamidated, acidic, basic or oxidized variants or any other variants of an antibody that do not impact the biological function or efficacy of the antibody. In one embodiment, the antibody of the antibody drug conjugates (ADC) of the present invention is Trastuzumab variants. In preferred embodiment, the Trastuzumab antibody according to the present invention is an antibody that comprises heavy chain SEQ ID NO. 1: (EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG) and light chain sequence SEQ ID NO. 2: (DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GIKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC) that represent Trastuzumab antibody. The said sequences have been disclosed in WHO Drug Information, Vol.

[0183] 37, No. 2, 2023. In one embodiment, the antibody of the antibody drug conjugates (ADC) of the present invention is Trastuzumab or its variants that can improve binding affinity of ADC to the HER-2 antigen. In one more embodiment, the antibody of the antibody drug conjugates (ADC) of the present invention is Trastuzumab or its variants that can improve binding affinity of ADC to the HER-2 antigen at suitable pH. The preferred pH can be pH 6.0 or pH 7.0. In another embodiment, the antibody of the present invention is Trastuzumab or its variants that can be used to increase the half-life of ADC. The half-life of an ADC of the present invention can be modified in blood by mutation in the Fc domain of the antibody. The mutation in Fc domain includes but not limited to one or more amino acid substitution, insertion, and / or deletion in amino acid sequence of the antibody.

[0184] In an embodiment, the present invention provides antibody drug conjugate (ADC), wherein average number of units of the selected one drug-linker structure conjugated per antibody molecule is in the range of 1 to 12 e.g., one drug-linker structure conjugated per antibody molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; in the range of 3 to 10; in the range of 4 to 9; in the range of 6 to 9; in the range of 7 to 8.5 e.g., 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5.

[0185] Pmi-Zi-Z2-Z3-Z4-Xpidescribed above and D is selected from Exatecan mesylate.

[0186] when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent.

[0187] In a further embodiment, present invention provides use of the antibody drug conjugate (ADC) for the treatment of cancer, specifically breast cancer.

[0188] Preparation of novel ADCs link with novel linkers

[0189] Preparation of novel ADCs was carried out through covalent conjugation of the drug-linkers to suitable monoclonal antibody, wherein the drug-linker molecules comprise of a novel linker that is covalently attached to the payload though spacer. The conjugation reaction is conducted in a controlled manner, under specific reaction conditions, to achieve the desired antibody-drug-conjugated molecules. Reagents / chemicals, process aids that are required for such reactions to conduct, and isolate the conjugated complex, include typical antibody, linker molecule, spacer molecule, payload molecule, coupling groups like maleimide, buffer solution (PBS, succinate, citrate, MES, borate, acetate or similar), reducing agent, salt, anti-oxidant, surfactant, sugar, organic solvent etc., desalting columns or dialysis membranes or tangential flow filtration, centrifugation, chromatography etc.

[0190] Preparation of antibody:

[0191] Purified monoclonal antibody preparation was subjected to covalent conjugation with the druglinker, in the presence or absence of reducing agent. Prior to conjugation reaction, the antibody preparation can be reconditioned to tune up with the reaction conditions, either by using desalting column, dialysis membrane or tangential flow filtration. The drug -linker molecule can be attached to the protein directly or upon controlled reduction of the side-chains of certain amino acid. Prior to conjugation reaction, the drug-linker was dissolved in a suitable solvent and added to the protein solution for conjugation, under controlled conditions.

[0192] The drug-linker was added to the protein solution in molar ratio to control the drug-antibody-ratio (DAR). The drug -linker conjugation reaction to antibody was carried out about 25 °C for about 2 hours, under stirring condition.

[0193] Purification: At the end of reaction, the reaction mixture comprising the antibody-drug-conjugate, free / unreacted drug-linker etc was subjected to desalting or dialysis or tangential flow filtration for the removal of the excess, unreacted drug-linker while recovering the antibody-drug -conjugate (ADC). The conjugation efficiency and purity of ADCs were analysed using various analytical techniques, e.g., SDS-PAGE, HPLC, or mass spectrometry, and protein concentration in the preparation was measured. The conjugated antibody preparation was stored in suitable buffer in the presence or absence of sugar or other additives, as necessary, between 2-8 °C and at or below -20 °C, in suitable container-closure system. Synthetic scheme for representative Example 1

[0194]

[0195] Example 1

[0196] Scheme 1A: Preparation of (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazahenicosanoic acid (10)

[0197] Step VII

[0198] Step VI 9 Tris (pentafluorophenyl)

[0199] Fmoc^

[0200]

[0201] -L jorane _ N

[0202] Copper (ll)acetate H

[0203]

[0204]

[0205] Lead tetracetate 13

[0206] Acetic acid

[0207]

[0208] Benzoic 2-hydroxyacetic anhydride Step VIII H2 / Pd / C Et / EA

[0209]

[0210] Step IX

[0211] FmocHN

[0212]

[0213] DBU / DCM

[0214]

[0215] Intermediate 1 14 Scheme 2A: Preparation of intermediate (1): 2-(aminomethoxy)acetic acid (9) Procedure for representative Example 1

[0216] To the solution of Cbz-hexanoic acid (1) and 1 -hydroxypyrrolidine-2,5 -dione in dry DCM was added EDC and the reaction mixture was stirred for 2 h, to this solution was added L-phenylalanine (2) and reaction mixture was stirred at RT for overnight. The compound was purified by flash chromatography to afford (6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanine(3). This compound was reacted with 1 -hydroxypyrrolidine-2, 5-dione in dry DCM and to this was added EDC and the reaction mixture was stirred for 2 h, after to this solution was added L-glycine (4) and reaction mixture was stirred at RT for overnight. The compound (6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanylglycine (5) was purified by flash or can be prepared by the method reported in literature along with their suitable modifications as may be necessary. In compound (5) the Cbz group was deprotected by H₂ / Pd / C in Methanol to give (6-aminohexanoyl)-L-phenylalanylglycine (6). The compound (6) was then dissolved in DMF and to this was added l-(3-(2,5-dioxopyrrolidin-l-yl)-3-oxopropyl)-lH-pyrrole-2, 5-dione (7) and was stirred for overnight to get ethyl (6-(3-(2,5-dioxo-2,5-dihydro- lH-pyrrol-l-yl)propanamido)hexanoyl)-L-phenylalanylglycine (8). Then to compound (8) the 1 -hydroxypyrrolidine-2, 5-dione in dry DCM was added EDC and the reaction mixture was added 2-(aminomethoxy) acetic acid (Intermediate 1) and was stirred for 2h to afford (S)-12-benzyl-3,10,13,16-tetraoxo-l-phenyl-2,19-dioxa-4,l l,14,17-tetraazahenicosan-21-oic acid (10).

[0217] Procedure for synthesis of intermediate 1[9]

[0218] To a mixture containing N-9-fluorenylmethoxycarbonylglycylglycine (11) in tetrahydrofiiran was added copper acetate, lead tetraacetate and acetic acid and heated under reflux for 30 mins. After workup and purification it yielded ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl acetate (12). This compound is then it is treated with benzoic 2-hydroxyacetic anhydride with in PTSA to afford 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic benzoic anhydride (13). The benzyl group was removed by the H₂ / PdC to give 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic acid (14). The Fmoc group was removed by DBU in DCM to afford compound 2-(aminomethoxy)acetic acid (9). Synthetic procedure of linker Example 1

[0219] Scheme 1A:

[0220] Step I: Synthesis of ethyl ((6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanine (3) To the solution of 6-(((benzyloxy)carbonyl)amino)hexanoic acid (1) (5.0 g, 18.85 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (2.169 g, 18.85 mmol) in dry DCM (50 mL) was added EDC (3.61 g, 18.85 mmol) and stirred for 2 h. To this solution added L-phenylalanine (2) (3.11 g, 18.85 mmol), and the reaction mixture was stirred at RT for overnight. After completion of reaction, the mixture was poured into distilled water (50 mL). The mixture was extracted with DCM (3 x 50 mL). The organic layer was washed with 10% NaHCO₃ (50 mL), and brine (50 mL), and dried over MgSO. The organic solvent was evaporated in vacuum. The crude product was purified by flash silica chromatography (DCM: Methanol, 10: 1 v / v) to obtain the (6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanine (5.46 g, 13.23 mmol, 70.2 % yield) as white solid.

[0221] ¹H NMR: (DMSO, 400 MHz): δ: 12.890 (bs, 1H), 8.084 (s, 1H), 7.388-7.292 (m, 5H), 7.272-7.201 (m, 5H), 7.194 (s, 1H), 5.007 (s, 2H), 4. 431-4. 380 (m, 1H), 3.065-3.031 (t, 2H), 2.957-2.908 (d, 2H), 2.048-2.011 (t, 2H), 1.382-1.319 (m, 4H), 1.126-1.109 (m, 2H); ESI-MS: (+ve mode) 413.18 (M+H)+(100 %).

[0222] Step II: Synthesis of (6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanylglycine (5)

[0223] To the solution of ethyl ((6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanine (3) (4.0 g, 9.70 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (1.116 g, 9.70 mmol) in dry DCM (50 mL) was added EDC (1.859 g, 9.70 mmol) and stirred for 2 h. To this solution added L-glycine (4) (0.728 g, 9.70 mmol), and the reaction mixture was stirred at RT for overnight. After completion of reaction, the mixture was poured into distilled water (50 mL). The mixture was extracted with DCM (3 x 50 mL). The organic layer was washed with 10% NaHCO₃ (50 mL), and brine (50 mL), and dried over MgSO. The organic solvent was evaporated in vacuum. The crude product was purified by flash silica chromatography (DCM: Methanol, 10:1 v / v) to obtain the (6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanylglycine (5) (2.96 g, 6.30 mmol, 65 % yield)) as white solid.

[0224] ‘H NMR: (DMSO, 400 MHz): 8: 12.351 (bs, 1H), 8.340 (s, 1H), 8.016 (s, 1H), 7.368-7.325 (m, 5H), 7.247-7.203 (m, 5H), 7.196 (s, 1H), 5.006 (s, 2H), 4.434-4.385 (m, 1H), 3.353-3.233 (d, 2H), 3.067-3.032 (t, 2H), 2.956-2.910 (d, 2H), 2.046-2.013 (t, 2H), 1.302-1.213 (m, 4H), 1.106-1.082 (m, 2H); ESI-MS: (+ve mode) 470.22 (M+H)+(100 %).

[0225] Step III: Synthesis of (6-aminohexanoyl)-L-phenylalanylglycine (6)

[0226] To the solution of (6-(((benzyloxy)carbonyl)amino)hexanoyl)-L-phenylalanylglycine (5) (4.0 g, 8.52 mmol) in methanol (20 mL) was added Pd / C (10%, wet 50% water) (0.4 g, 0.441 mmol) in Parr apparatus at 40 psi. The reaction mixture was stirred 2h at room temperature. Then reaction mixture was filtered through hyflow and the solvent was concentrated under reduced pressure to afford (6-aminohexanoyl)-L-phenylalanylglycine (6) as oily compound (2.71 g, 8.09 mmol, 95 % yield)). Product was used as such in next reaction step without any purification. ¹H NMR: (DMSO, 400 MHz): δ: 12.251 (bs, 1H), 8.346 (s, 1H), 8.026 (s, 1H), 7.287-7.201 (m, 5H), 4.245-4.216 (m, 1H), 3.353-3.233 (d, 2H), 3.249-3.213 (br s, 2H), 3.067-3.032 (t, 2H), 2.956-2.910 (d, 2H), 2.046-2.013 (t, 2H), 1.302-1.213 (m, 4H), 1.106-1.082 (m, 2H); ESI-MS: (+ve mode) 336.42 (M+H)+(100 %).

[0227] Step IV: Synthesis of (6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)hexanoyl)-L-phenylalanylglycine (8)

[0228] To a solution of (6-aminohexanoyl)-L-phenylalanylglycine (6) (500 mg, 1.491 mmol) in DMF (5 mL) was added 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (476 mg, 1.789 mmol). The reaction mixture was stirred for 18 h at room temperature. After completion of reaction the DMF was evaporated under reduced pressure. The crude mixture was purified by RP HPLC to obtain (6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)hexanoyl)-L-phenylalanylglycine (330 mg, 0.667 mmol, 44.7 % yield) ¹H NMR: (DMSO, 400 MHz): δ: 12.556 (bs, 1H), 8.301 (s, 1H), 8.024 (s, 1H), 7.859 (s, 1H), 7.256-7.173 (m, 5H), 7.006 (s, 2H), 4.548-4.616 (m, 1H), 3.787-3.769 (q, 2H), 3.614-3.578 (d, 2H), 3.354-3.333 (t, 1H), 3.052-3.018 (t, 2H), 2.918-2.901 (t, 1H), 2.372-2.90 (t, 2H), 2.021-1.984 (t, 2H), 1.338-1.301 (m, 4H), 1.042-1.024 (m, 2H); ESI-MS: (+ve mode) 487.21 (M+H)+(100 %).

[0229] Step V: Synthesis of (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazahenicosanoic acid (10) [Example 1]

[0230] To the solution of (6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)hexanoyl)-L-phenylalanylglycine (8) (500 mg, 1.028 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (124 mg, 1.079 mmol) in dry DCM (50 mL) was added EDC (236 mg, 1.233 mmol) and stirred for 2 h. To this solution added 2-(aminomethoxy)acetic acid (108 mg, 1.028 mmol), and the reaction mixture was stirred at RT for 2h. After completion of reaction, the mixture was poured into distilled water (50 mL). The mixture was extracted with DCM (3 x 50 mL). The organic layer was washed with 10% NaHCCh (50 mL), and brine (50 mL), and dried over MgSO. The organic solvent was evaporated in vacuum. The crude product was purified by flash silica chromatography (DCM: Methanol, 10: 1 v / v) to obtain (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,l 1,18-tetraazahenicosanoic acid (14) (271 mg, 0.473 mmol, 46 % yield)) as white solid.

[0231] ¹H NMR: (DMSO, 400 MHz): δ: 12.556 (bs, 1H), 8.301 (s, 1H), 8.024 (s, 1H), 7.859 (s, 1H), 7.256-7.173 (m, 5H), 7.006 (s, 2H), 6.987 (s, 1H), 4.618 (s, 2H), 4.548-4.616 (m, 1H), 3.787-3.769 (q, 2H), 3.741 (s, 2H)3.614-3.578 (d, 2H), 3.354-3.333 (t, 1H), 3.052-3.018 (t, 2H), 2.918-2.901 (t, 1H), 2.372-2.90 (t, 2H), 2.021-1.984 (t, 2H), 1.338-1.301 (m, 4H), 1.042-1.024 (m, 2H); ESI-MS: (+ve mode) 574.6 (M+H)+(100 %).

[0232] Scheme 2A: Preparation of Intermediate 1

[0233] Step VI: Synthesis of ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl acetate (12 ) The compound (((9H-fluoren-9-yl)methoxy)carbonyl)glycine (11) (2.5 g, 8.41 mmol) was dissolved in anhydrous DMF (40 mL) and magnetically stirred in a 100 mL flask as COPPER (II) ACETATE (0.573 g, 3.15 mmol), ACETIC ACID (1.088 mL, 19.00 mmol) and LEAD TETRAACETATE (4.25 g, 9.59 mmol) were added. The flask was heated in a 60 °C for 15 min. The heating was removed and the reaction was allowed to cool to room temperature. The mixture was poured in water and extracted with ethyl acetate. The crude mixture was purified by Flash silica column chromatography [Hexane: Ethylacetate 5:1 v / v] to give 1.2 g of white semi-solid ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl acetate (12) (1.6 g, 5.14 mmol, 61 % yield).

[0234] ¹H NMR: (DMSO, 400 MHz): δ: 7.787-7.258 (m, 8H), 5.983 (s, 1H), 5.247 (s, 2H), 4.483-4.466 (d, 2H), 4.265-4.232 (t, 1H), 2.109 (s, 3H); ESI-MS: (+ve mode) 312.31 (M+H)+(100 %). Step-VII: Synthesis of 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic benzoic anhydride (13)

[0235] To a suspension of({N-[(9H-fluoren-9-ylmethoxy) carbonyl]glycyl}amino)methyl acetate (1.5 g, 4.82 mmol) in anhydrous 1,2-dimethoxyethane (20 mL), benzyl 2-hydroxyacetate (1.367 mL, 9.64 mmol)was added under a nitrogen atmosphere, and the resulting mixture was cooled to about 0° C. Tris (pentafluorophenyl)borane (1.42 g, 0.27 mol) was added thereto, and the resulting mixture was stirred at the same temperature as the above for 3 hours. Then, ethyl acetate (30 mL) and a 10% aqueous potassium bicarbonate solution were added thereto, and the resulting mixture was heated to room temperature and separated into organic and aqueous layers. The organic layer was washed by the addition of 10% saline (20.1 L). The organic layer was concentrated under reduced pressure until the amount of the liquid. The thick residue was purified by flash silica chromatography (Hexane: Ethylacetate, 10:2 v / v) to obtain the solid compound 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic benzoic anhydride (13) (1.609 g, 3.85 mmol, 80 % yield)

[0236] ¹H NMR: (DMSO, 400 MHz): δ: 8.231 (s, 1H), 7.906-7.305 (m, 13H), 5.142 (s, 2H), 4.574-4.557 (d, 2H), 4.374-4.357 (d, 2H), 4.254-4.220 (t, 1H), 4.139 (s, 2H); ESI-MS: (+ve mode) 418.65 (M+H)+(100 %).

[0237] Step VIII: 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic acid (14) To the solution of benzyl 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetate (1.0 g, 2.395 mmol) in mixture of ethanol (10 mL) and Ethylacetate (10 mL) was added Pd / C (0.255 g, 0.240 mmol)(10%, wet 50% water) in Parr apparatus at 40 psi. The reaction mixture was stirred 2h at room temperature. Then mixture was filtered through hyflow and the solvent was concentrated under reduced pressure to afford 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic acid (0.706 g, 2.156 mmol, 90 % yield). Product was used as such in next reaction step without any purification.

[0238] ¹H NMR: (DMSO, 400 MHz): δ: 12.306 (bs, 1H), 8.256 (s, 1H), 7.907-7.318 (m, 8H), 4.548-4.534 (d, 2H), 4.347-4.330 (d, 2H), 4.252-4.239 (t, 1H), 3.939 (s, 1H), 3.747 (s, 1H); ESI-MS: (+ve mode) 328.56 (M+H)+(100 %). Step IX:2-(aminomethoxy)acetic acid (9) [Intermediate 1]

[0239] To the solution of 2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methoxy)acetic acid (500 mg, 1.527 mmol) in DCM (20 mL) was added DBU (0.276 ml, 1.833 mmol). The reaction mixture was stirred 2h at room temperature. After completion of reaction, solvent was concentrated under reduced pressure to afford 2-(aminomethoxy)acetic acid (159 mg, 1.512 mmol, 99 % yield). ESI-MS: (+ve mode) 106.40 (M+H)+(100 %).

[0240] Product was used as such in next reaction step without any purification [To prepare a linker mentioned in Example 1.

[0241] By using the similar procedure following linkers were prepared,

[0242] Example 2

[0243] Synthesis of (S)-ll-benzyl-22-(2, 5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6, 10,13, 20-tetraoxo-3-oxa-5,9,12,19-tetraazadocosanoic acid

[0244]

[0245] To the solution of (6-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)hexanoyl)-L-phenylalanylglycine (500 mg, 0.999 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (115 mg, 0.999 mmol) in dry DCM (50 mL) was added EDC (239 mg, 1.249 mmol) and stirred for 2 h. To this solution added 2-(aminomethoxy)acetic acid (115 mg, 1.099 mmol), and the reaction mixture was stirred at RT for 2h. After completion of reaction, the mixture was poured into distilled water (50 mL). The mixture was extracted with DCM (3 x 50 mL). The organic layer was washed with 10% NaHCO₃ (50 mL), and brine (50 mL), and dried over MgSO. The organic solvent was evaporated in vacuum. The crude product was purified by flash silica chromatography (DCM: Methanol, 10: 1 v / v) to obtain (S)-ll-benzyl-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,10,13,20-tetraoxo-3-oxa-5,9,12,19-tetraazadocosanoic acid (247 mg, 0.420 mmol, 42 % yield)) as white solid.

[0246] ¹H NMR: (DMSO, 400 MHz): δ: 12.446 (bs, 1H), 8.015-7.987 (d, 1H), 7.966-7.873 (d, 1H), 7.992 (s, 1H), 7.247-7.171 (m, 5H), 6.997 (s, 2H), 6.987 (s, 1H), 4.620 (s, 2H), 4.618-4.606 (m, 1H), 3.784-3.765 (q, 2H), 3.742 (s, 2H), 3.621-3.574 (d, 2H), 3.344-3.330 (t, 1H), 3.046-3.023 (t, 2H), 2.924-2.912 (t, 1H), 2.364-2.320 (m, 4H), 2.024-1.989 (m, 2H), 1.338-1.280 (m, 6H), 1.042-1.024 (m, 2H); ESI-MS: (+ve mode) 588.6 (M+H)+(100 %).

[0247] Example 3

[0248] Synthesis of (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7,7-dimethyl-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazahenicosanoic acid

[0249]

[0250] To the solution of (S)-2-(2-(6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)hexanamido)-3-phenylpropanamido)-2-methylpropanoic acid (500 mg, 0.972 mmol) and 1 -hydroxypyrrolidine-2,5 -dione (112 mg, 0.972 mmol) in dry DCM (50 mL) was added EDC (233 mg, 1.215 mmol) and stirred for 2 h. To this solution added 2-(aminomethoxy)acetic acid (112 mg, 1.069 mmol), and the reaction mixture was stirred at RT for 2h. After completion of reaction, the mixture was poured into distilled water (50 mL). The mixture was extracted with DCM (3 x 50 mL). The organic layer was washed with 10% NaHCO₃ (50 mL), and brine (50 mL), and dried over MgSO. The organic solvent was evaporated in vacuum. The crude product was purified by flash silica chromatography (DCM: Methanol, 10: 1 v / v) to obtain (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7,7-dimethyl-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazahenicosanoic acid (228 mg, 0.379 mmol, 39 % yield)) as white solid.

[0251] ¹H NMR: (DMSO, 400 MHz): δ: 12.528 (bs, 1H), 8.138-7.939 (d, 1H), 7.917-7.862 (d, 1H), 7.859 (s, 1H), 7.249-7.170 (m, 5H), 7.007 (s, 2H), 6.987 (s, 1H), 4.554 (s, 2H), 4.538-4.524 (m, 1H), 3.613-3.595 (q, 2H), 3.741 (s, 2H), 3.614-3.578 (d, 2H), 3.354-3.333 (t, 1H), 2.971-2.937 (t, 2H), 2.921-2.905 (t, 1H), 2.362-2.290 (t, 2H), 2.016-1.979 (t, 2H), 1.323 (s, 6H), 1.318-1.301 (m, 4H), 1.042-1.024 (m, 2H); ESI-MS: (+ve mode) 602.6 (M+H)+(100 %).

[0252] Example 4

[0253] Synthesis of (S)-10-benzyl-24-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazatetracosanoic acid

[0254]

[0255] To the solution of (6-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)hexanoyl)-L-phenylalanylglycine (500 mg, 0.946 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (109 mg, 0.946 mmol) in dry DCM (50 mL) was added EDC (227 mg, 1.182 mmol) and stirred for 2 h. To this solution added 2-(aminomethoxy)acetic acid (109 mg, 1.040 mmol), and the reaction mixture was stirred at RT for 2h. After completion of reaction, the mixture was poured into distilled water (50 mL). The mixture was extracted with DCM (3 x 50 mL). The organic layer was washed with 10% NaHCO₃ (50 mL), and brine (50 mL), and dried over MgSO. The organic solvent was evaporated in vacuum. The crude product was purified by flash silica chromatography (DCM: Methanol, 10: 1 v / v) to obtain (S)-10-benzyl-24-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,l 1,18-tetraazatetracosanoic acid (180 mg, 0.292 mmol, 31 % yield)) as white solid.

[0256] ¹H NMR: (DMSO, 400 MHz): δ: 12.546 (bs, 1H), 8.293 (s, 1H), 8.014 (s, 1H), 7.992 (s, 1H), 7.165-7.283 (m, 5H), 7.000 (s, 2H), 6.987 (s, 1H), 4.620 (s, 2H), 4.548-4.616 (m, 1H), 3.784-3.765 (q, 2H), 3.742 (s, 2H), 3.621-3.574 (d, 2H), 3.344-3.330 (t, 1H), 3.046-3.023 (t, 2H), 2.924-2.912 (t, 1H), 2.364-2.320 (t, 2H), 2.024-1.989 (m, 2H), 1.338-1.280 (m, 10H), 1.042-1.024 (m, 2H); ESI-MS: (+ve mode) 616.5 (M+H)+(100 %).

[0257] Furthermore, these novel linkers linked with spacer were attached with cytotoxic payloads, which were synthesized separately by using the following protocol. Representative examples are given below,

[0258]

[0259] Scheme 3A: Coupling of compound (10) with payload [eg. Exatecan mesylate] Example 24

[0260] Scheme 3A: Coupling of compound (10) [Example 1] with Exatecan Mesylate

[0015] Step X: Synthesis of 6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((2-(((2-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)amino)-l-oxo-3-phenylpropan-2-yl)hexanamide (16)

[0261] To the solution of (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,l 1,18-tetraazahenicosanoic acid (10) (200 mg, 0.349 mmol) and Exatecan Mesylate [DX8951f] (15) (185 mg, 0.349 mmol) and in DMF (2.0 mL) was added HATU (133 mg, 0.349 mmol) and DIPEA (0.142 ml, 0.762 mmol) at 0°C, the reaction mixture was stirred 18h at room temperature. Then DMF was evaporated and residue was diluted with ethyl acetate. The organic layer was washed sequentially with aqueous saturated sodium bicarbonate solution, then 10% aqueous citric acid solution and brine (20 ml each) and the solvent was concentrated under reduced pressure. The crude mixture was purified by RP HPLC to obtain 6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((2-(((2-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo [de]pyrano [3 ',4': 6,7]indolizino [ 1,2-b]quinolin- 1 -yl)amino)-2-oxoethoxy)methyl)amino)- 2-oxoethyl)amino)-l -oxo-3 -phenylpropan-2-yl)hexanamide (138 mg, 0.139 mmol, 40 % yield)).

[0262] ESI-MS: (+ve mode) 991.31 (M)+(100 %), 992.31 (M+H)+(80 %).

[0263] Example 25

[0264] Coupling of linker [Example 2] with Exatecan Mesylate

[0015]

[0265] Synthesis of 6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((3-(((2-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropyl)amino)-l-oxo-3-phenylpropan-2-yl)hexanamide To the solution of (S)-ll-benzyl-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6, 10, 13,20-tetraoxo-3-oxa-5,9,12,19-tetraazadocosanoic acid (200 mg, 0.340 mmol) and Exatecan Mesylate [DX8951f] (15) (175 mg, 0.340 mmol) and in DMF (2.0 mL) was added HATU (129 mg, 0.340 mmol) and DIPEA (0.149 ml, 0.851 mmol) at OoC, the reaction mixture was stirred 18h at room temperature. Then DMF was evaporated and residue was diluted with ethyl acetate. The organic layer was washed sequentially with aqueous saturated sodium bicarbonate solution, then 10% aqueous citric acid solution and brine (20 ml each) and the solvent was concentrated under reduced pressure. The crude mixture was purified by RP HPLC to obtain 6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((3-(((2-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo [de]pyrano [3 ',4': 6,7]indolizino [ 1,2-b]quinolin- 1 -yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropyl)amino)-l-oxo-3-phenylpropan-2-yl)hexanamide (140 mg, 0.140 mmol, 41 % yield)).

[0266] ESI-MS: (+ve mode) 1005.09 (M)+ (100 %), 1006.09 (M+H)+ (80 %).

[0267] Example 26

[0268] Coupling of linker [Example 3] with Exatecan Mesylate

[0015]

[0269] Synthesis of 6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((l-(((2-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-2-oxoethoxy)methyl)amino)-2-methyl-l-oxopropan-2-yl)amino)-l-oxo-3-phenylpropan-2-yl)hexanamide To the solution of (S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7,7-dimethyl-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazahenicosanoic acid (200 mg, 0.332 mmol) and Exatecan Mesylate [DX8951f] (15) (171 mg, 0.332 mmol) and in DMF (2.0 mL) was added HATU (126 mg, 0.332 mmol) and DIPEA (0.145 ml, 0.762 mmol) at OoC, the reaction mixture was stirred 18h at room temperature. Then DMF was evaporated and residue was diluted with ethyl acetate. The organic layer was washed sequentially with aqueous saturated sodium bicarbonate solution, then 10% aqueous citric acid solution and brine (20 ml each) and the solvent was concentrated under reduced pressure. The crude mixture was purified by RP HPLC to obtain 6-(3 -(2,5 -dioxo-2, 5 -dihydro- IH-pyrrol- 1 -yl)propanamido)-N-((S)- 1 -(( 1 -(((2- (((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de]pyrano [3 ',4': 6,7] indolizino [ 1,2-b]quinolin- 1 -yl)amino)-2-oxoethoxy)methyl)amino)-2 -methyl- 1 -oxopropan-2-yl)amino)- 1 -oxo-3 -phenylpropan-2-yl)hexanamide (132 mg, 0.130 mmol, 39 % yield)).

[0270] ESI-MS: (+ve mode) 1019.13 (M)+ (100 %), 1020.16 (M+H)+ (79 %).

[0271] Example 27

[0272] Coupling of linker [Example 4] with Exatecan Mesylate

[0015]

[0273] Synthesis of N-((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12-tetraoxo-3-oxa-5,8,ll-triazaheptadecan-17-yl)-6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamide

[0274] To the solution of (S)-10-benzyl-24-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa-5,8,ll,18-tetraazatetracosanoic acid (200 mg, 0.325 mmol) and Exatecan Mesylate [DX895H] (15) (167 mg, 0.325 mmol) and in DMF (2.0 mL) was added HATU (124 mg, 0.325 mmol) and DIPEA (0.142 ml, 0.812 mmol) at 0°C, the reaction mixture was stirred 18h at room temperature. Then DMF was evaporated and residue was diluted with ethyl acetate. The organic layer was washed sequentially with aqueous saturated sodium bicarbonate solution, then 10% aqueous citric acid solution and brine (20 ml each) and the solvent was concentrated under reduced pressure. The crude mixture was purified by RP HPLC to obtain N-((S)-10-benzyl- 1 -((( 1 S,9S)-9-ethyl-5 -fluoro-9-hydroxy-4-methyl- 10,13 -dioxo-2, 3,9, 10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-l,6,9,12-tetraoxo-3 -oxa-5, 8, 11 -triazaheptadecan- 17-yl)-6-(2, 5 -dioxo-2, 5 -dihydro- IH-pyrrol- 1 -yl)hexanamide (117 mg, 0.114 mmol, 35 % yield)). ESI-MS: (+ve mode) 1033.39 (M)+(100 %), 1034.33 (M+H)+(80 %).

[0275] Example 28: Preparation of ADC-1

[0276] Trastuzumab was expressed in CHO cells and purified using conventional column chromatography techniques, including r-Protein A affinity chromatography, Ion exchange chromatography and hydrophobic interaction chromatography. Post purification, trastuzumab material was subjected to the buffer exchange in 10 mM phosphate pH 7.4 + 137 mM NaCl + 5 mM EDTA. Trastuzumab, was subjected to partial reduction (inter-chain S-S cross-links) by 2.0 to 7.0 molar excess amount of TCEP [tris(carboxyethyl)phosphine] for a period of 2 h, while maintaining the reaction temperature between 4 °C to 37 °C, at pH between 5.0 and pH 8.5. under stirring condition (200 rpm). At the end of reaction, the drug -linker molecule was added to the protein-TCEP reaction mixture for conjugation. Partially reduced trastuzumab was conjugated by < 20 molar excess amount of ADC-1. Incubation was performed between 4 °C to 37 °C, at pH between 5.0 and 8.5 for a maximum period of 3 h, with gentle stirring (200 rpm). The linker-drug complex was dissolved in 5 - 20 % DMSO before mixing to the protein solution. The linker-drug complex can be also dissolved in other organic solvents like dimethyl acetamide (DMAc) and acetonitrile (ACN). Conjugation takes place with the formation of covalent thioether bond between the free -SH groups of inter-chain Cys residues (reduced) of the monoclonal antibody and the maleimide group of the drug-linker. Post-conjugation, the antibody drug conjugate (ADC-1) was buffer exchanged, formulated in appropriate buffer of pH 5.5 and filtered through a 0.2 µm filter and stored under frozen condition at -25 ± 5 °C. ADC-1 was subjected to the quality analysis for pH, Protein concentration, Purity by HP-SEC, DAR profile by mass spectrometry (MS). Results are summarized in Table 1A.

[0277] Table 1A: Results obtained with ADC- 1 drug conjugate

[0278]

[0279] The present invention introduces a novel linker designed for the preparation of antibody-drug conjugates (ADCs). In present study, one end of the linker, referred to as the stretcher unit is conjugated to the antibody, while the opposite end, the spacer unit is linked to the payload. The efficacy data for the resulting ADC has been evaluated in comparison with Enhertu, a commercially available ADC which consists of Trastuzumab as an Antibody and Exatecan mesylate as a payload.

[0280] Efficacy ADC-1 in breast carcinoma xenograft disease model in Nude mice

[0281] In present study, the therapeutic potential of ADC- 1 has been evaluated in comparison to market leader ADC molecule (named Enhertu which consists of Trastuzumab as an Antibody and Exatecan mesylate as a payload). Specifically, to induce breast carcinoma xenograft, Immunodeficient Nude mice were injected with JIMT-1 cells (10 million cells / animal / 200 pL volume, In 1:1 ratio of cell suspension & matrigel) in right flank region of animals. Animals were observed for palpable tumor development & drug administration was done on the day when mean tumor volume of animals reached to approximately 150-200 mm3, which has been considered as day 0 of the study. In this study, total 2 dosage have been administered, i.e. On day 0 & Day 32. ADC-1 & Enhertu were compared head to head at dose level of 3 mg / kg (I. V.) to evaluate whether ADC- 1 demostrated either similarity or any superiority over commercially available Enhertu.

[0282] Figure 1 demonstrates efficacy of ADC- 1 in comparison to Enhertu in JIMT-1 breast carcinoma xenograft disease model. Here, Enhertu as well as ADC-1 at a tested dose were able to delay the tumor progression in comparison to placebo control group, but it was observed that the tumor progression inhibition potential of ADC- 1 is much better than the Enhertu, positioning it as a promising candidate for further development in cancer treatment.

[0283] In addition to tumor volume inhibition, clinical sign observation was also carried out visually, as in preclinical studies, clinical sign observation plays a crucial role in identifying potential toxic effects of drug candidates. Clinical sign observation plays a major role in study of safety profile of ADCs when ADC is delivered to the subject. Here in this study, subject was observed till 32 days after 1stinjection for various clinical sign observations where it was found that body weight and food consumption observed normal across the study group. The details findings of the clinical signs observed are as below:

[0284] No abnormal clinical signs were noted in any of the study groups. All animals exhibited normal grooming behaviour, activity levels, and posture.

[0285] • No signs of distress, toxicity, or adverse reactions were observed.

[0286] • Body weight and food consumption were closely monitored up to day 32 after 1stinjection & observed to be normal across study groups.

[0287] • Skin, fur, eyes, and mucous membranes appeared normal throughout the study duration. Above clinical sign observations suggests that the ADC-1 molecule exhibits similar safety profile as Enhertu in animal model.

[0288] References incorporated in current patent application:

[0289] 1. Diamantis N, Banerji U. Antibody-drug conjugate — an emerging class of cancer treatment.

[0290] Br J Cancer 2015; 114: 362-367.

[0291] 2. Bouchard H, Viskov C, Garcia-Echeverria C. Antibody-drug conjugates — a new wave of cancer drugs. Bioorganic Med. Chem Lett 2014; 24: 5357-5363.

[0292] 3. Tolcher AW, Ann Oncol. 2016; 27: 2168-2172. 10.1093 / annonc / mdw424. [PubMed:

[0293] 27733376]

Claims

We claim:

1. A novel linker of general formula (I),Pm1-Z1-Z2-Z3-Z4-Xp1Formula (I)wherein,P is selected from maleimide containing functional residue which is unsubstituted or substituted linear or branched (C1-8)alkyl chain wherein (C1-8)alkyl chain is unsubstituted or substituted by (C1-6)alkoxy, (C3-C6)cycloalkyl, aryl, heteroaryl or arylalkyl or polyethylene glycol (PEG) with more than one monomer, carboxylic acyl unit or its derivatives, unsubstituted or substituted amino acids or its derivatives;Zi is present or absent and is selected from the group of uncharged amino acid residues, selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl -isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl); Z2is present or absent and is selected from the group of uncharged amino acid residues, selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl -isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl); Z3is an amino acid residue selected from aromatic amino acid and aryl group wherein aromatic amino acid is selected from phenylalanine and their derivatives, oc-methyl phenylalanine, 2-fluorophenylalanine, a-methyl-2-fluorophenylalanine, a-methyl-2,6- diflurophenylalanine, 2-Amino-5 -phenylpentanoic acid; the aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl;Z4 is an amino acid residue selected from the group of uncharged amino acid residues and their derivatives selected from the group of glycine, sarcosine, alanine, isoleucine, leucine, norleucine, homoleucine, [3-alanine, AIB and their derivatives selected from N-methyl-isoleucine, N-methyl-leucine, [3-alanine, 1 -amino-cyclopropanecarboxylic acid, 1 -amino-cyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid;when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent;X is selected from 2-(aminomethoxy)acetic acid and their derivatives, paraaminobenzylmethylcarbamate (PBMC) and its derivatives wherein derivatives of 2- (aminomethoxy)acetic acid selected from 2-(aminomethoxy)propionic acid, 2- (aminomethoxy)butanoic acid;pl is 0-3;ml is 0-8.

2. The linker as claimed in claim 1 wherein P is selected from maleimide containing carboxylic acyl unit, Zi is absent or amino acid residues comprising branch or liner Ci- Cs alkyl, Z2 is absent or amino acid residues comprising branch or liner Ci-Cs alkyl, Z3 is phenylalanine and their derivatives, Z4 is glycine, [3-alanine, AIB, X is selected from 2-(aminomethoxy)acetic acid and their derivatives, ml is 1, 2 and pl is 1, 2.

3. The linker as claimed in claim 1 is selected from:(S)-10-benzyl-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa- 5.8.11.18-tetraazahenicosanoic acid;(S)-l l-benzyl-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6, 10, 13,20-tetraoxo-3-oxa- 5.9.12.19-tetraazadocosanoic acid;(S)- 10-benzyl-21 -(2,5 -dioxo-2,5-dihydro- IH-pyrrol- 1 -yl)-7,7-dimethyl-6,9, 12,19- tetraoxo-3-oxa-5,8, 11,18-tetraazahenicosanoic acid;(S)-10-benzyl-24-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa- 5.8.11.18-tetraazatetracosanoic acid;(S)- 10-benzyl-24-(2,5 -dioxo-2,5-dihydro- IH-pyrrol- 1 -yl)-7,7-dimethyl-6,9, 12,19- tetraoxo-3-oxa-5,8, 11,18-tetraazatetracosanoic acid;(S)-ll-benzyl-25-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,10,13,20-tetraoxo-3-oxa- 5.9.12.19-tetraazapentacosanoic acid;(S)-10-benzyl-20-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,18-tetraoxo-3-oxa- 5,8,11,17-tetraazaicosanoic acid;(S)-10-benzyl-19-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,17-tetraoxo-3-oxa-5,8,11,16-tetraazanonadecanoic acid;(R)- 10-benzyl- 18-(2,5 -dioxo-2, 5 -dihydro- IH-pyrrol- 1 -yl)-6,9, 12,16-tetraoxo-3 -oxa-5,8,11,15 -tetraazaoctadecanoic acid;(S)- 11 -benzyl -24-(2, 5 -dioxo-2, 5 -dihydro- IH-pyrrol- 1 -yl)- 14, 14-dimethyl- 6,10,13, 16, 19-pentaoxo-3-oxa-5,9, 12, 15, 18-pentaazatetracosanoic acid;(S)-21-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-10-phenethyl-3-oxa-5,8,11, 18-tetraazahenicosanoic acid;(S)-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,10,13,17-tetraoxo-ll-(3-phenylpropyl)-3-oxa-5,9,12,16-tetraazadocosanoic acid;(S)-10-benzyl-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa- 5.8.11.18-tetraazadocosanoic acid;(S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,9,12,19-tetraoxo-3-oxa- 5.8.11.18-tetraazatricosanoic acid;(S)- 11 -benzyl -24-(2, 5 -dioxo-2, 5 -dihydro- IH-pyrrol- 1 -yl)- 17,17 -dimethyl- 6.10.13.16.19-pentaoxo-3-oxa-5, 9, 12, 15, 18-pentaazatetracosanoic acid;(S)-ll-benzyl-25-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-18,18-dimethyl- 6.10.13.17.20-pentaoxo-3-oxa-5,9, 12, 16, 19-pentaazapentacosanoic acid;(S)-l l-benzyl-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol- 1-yl)- ll-methyl-6, 10, 13,20-tetraoxo-3-oxa-5,9,12,19-tetraazadocosanoic acid;(S)-3 -benzyl- 1 -( 1 -( 6 -(3 -(2,5 -dioxo-2, 5 -dihydro- IH-pyrrol- 1 -yl)propanamido)hexanamido)cyclopentyl)- 1,4,8-trioxo- 11 -oxa-2,5,9-triazatridecan- 13-oic acid;(S)-2-((l-(2-(6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)hexanamido)-3-phenylpropanamido)cyclobutane-l-carboxamido)methoxy)acetic acid;(S)-ll-benzyl-26-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-6,10,13,17,24-pentaoxo-3-oxa-5,9, 12, 16,23-pentaazahexacosanoic acid;(S)-l l-benzyl-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7, 10, 13,20-tetraoxo-4-oxa- 6,9,12,19-tetraazadocosanoic acid;(S)-12-benzyl-l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3,10,13,16-tetraoxo-19-oxa- 4,11, 14, 17-tetraazatricosan-23-oic acid;(S)-6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-(l-((2-((4- (hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)- 1 -oxo-3 -phenylpropan-2- yl)hexanamide.

4. The novel compounds of general formula (II), novel intermediates,Pm1-Z1-Z2-Z3-Z4-Xp1-DFormula (II)wherein,P is selected from maleimide containing functional residue which is unsubstituted or substituted linear or branched (C1-8)alkyl chain wherein (C1-8)alkyl chain is unsubstituted or substituted by (C1-6)alkoxy, (C3-C6)cycloalkyl, aryl, heteroaryl or arylalkyl or polyethylene glycol (PEG) with more than one monomer, carboxylic acyl unit or its derivatives, unsubstituted or substituted amino acids or its derivatives;Zi is present or absent and is selected from the group of uncharged amino acid residues, selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl -isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl); Z2is present or absent and is selected from the group of uncharged amino acid residues, selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl -isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl); Z3is an amino acid residue selected from aromatic amino acid and aryl group wherein aromatic amino acid is selected from phenylalanine and their derivatives, oc-methyl phenylalanine, 2-fluorophenylalanine, a-methyl-2-fluorophenylalanine, a-methyl-2,6- diflurophenylalanine, 2-Amino-5 -phenylpentanoic acid; the aryl group is selected fromphenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl;Z4 is an amino acid residue selected from the group of uncharged amino acid residues and their derivatives selected from the group of glycine, sarcosine, alanine, isoleucine, leucine, norleucine, homoleucine, [3-alanine, AIB and their derivatives selected from N- methyl-isoleucine, N-methyl-leucine, [3-alanine, 1 -amino-cyclopropanecarboxylic acid, 1 -amino-cyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid;when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent;X is selected from 2-(aminomethoxy)acetic acid and their derivatives, paraaminobenzylmethylcarbamate (PBMC) and its derivatives wherein derivatives of 2- (aminomethoxy)acetic acid selected from 2-(aminomethoxy)propionic acid, 2- (aminomethoxy)butanoic acid;D is selected from Exatecan mesylate;pl is 0-3;ml is 0-8.

5. The compound as claimed in claim 4 wherein P is selected from maleimide containing carboxylic acyl unit, Zi is absent or amino acid residues comprising branch or liner Ci- Cs alkyl, Z2 is absent or amino acid residues comprising branch or liner Ci-Cs alkyl, Z3 is phenylalanine and their derivatives, Z4 is glycine, [3-alanine, AIB, X is selected from 2-(aminomethoxy)acetic acid and their derivatives, D is selected from Exatecan mesylate, ml is 1, 2 and pl is 1, 2.

6. The compound as claimed in claim 4 selected from:6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((2-(((2- (((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)- 2-oxoethoxy)methyl)amino)-2-oxoethyl)amino)- 1 -oxo-3 -phenylpropan-2- yl)hexanamide;6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((3-(((2- (((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-2-oxoethoxy)methyl)amino)-3-oxopropyl)amino)-l-oxo-3-phenylpropan-2- yl)hexanamide;6-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-((S)-l-((l-(((2- (((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)- 2-oxoethoxy)methyl)amino)-2-methyl- 1 -oxopropan-2-yl)amino)- 1 -oxo-3 - phenylpropan-2-yl)hexanamide;N-((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9, 10, 13, 15 -hexahydro- 1H, 12H-benzo [de]pyrano[3 ',4 ': 6,7]indolizino [1,2- b]quinolin-l-yl)amino)-l,6,9,12-tetraoxo-3-oxa-5,8,ll-triazaheptadecan-17-yl)-6-(2,5- dioxo-2, 5 -dihydro- IH-pyrrol- l-yl)hexanamide.

7. An antibody drug conjugates (ADC) of Formula (A),Ab-Pml-Zl-Z2-Z3-Z4-Xpl-DFormula (A)wherein,Ab is Trastuzumab;P is selected from maleimide containing functional residue which is unsubstituted or substituted linear or branched (C1-8)alkyl chain wherein (C1-8)alkyl chain is unsubstituted or substituted by (C1-6)alkoxy, (C3-C6)cycloalkyl, aryl, heteroaryl or arylalkyl or polyethylene glycol (PEG) with more than one monomer, carboxylic acyl unit or its derivatives, unsubstituted or substituted amino acids or its derivatives;Zi is present or absent and is selected from the group of uncharged amino acid residues, selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl -isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl); Z2is present or absent and is selected from the group of uncharged amino acid residues, selected from the group of glycine, sarcosine, isoleucine, leucine, norleucine, homoleucine, alanine, [3-alanine, AIB or their derivatives such as N-methyl -isoleucine, N-methyl-leucine, 1 -amino-cyclopropanecarboxylic acid, 1 -aminocyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -amino-cyclohexanecarboxylic acid, etc. or amino fatty acid or polyethylene glycol (PEG) with various length of (C1-C8), linear or branched alkyl chain comprising (C1-C8alkyl); Z3is an amino acid residue selected from aromatic amino acid and aryl group wherein aromatic amino acid is selected from phenylalanine and their derivatives, oc-methyl phenylalanine, 2-fluorophenylalanine, a-methyl-2-fluorophenylalanine, a-methyl-2,6- diflurophenylalanine, 2-Amino-5 -phenylpentanoic acid; the aryl group is selected from phenyl, naphthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl;Z4 is an amino acid residue selected from the group of uncharged amino acid residues and their derivatives selected from the group of glycine, sarcosine, alanine, isoleucine, leucine, norleucine, homoleucine, [3-alanine, AIB and their derivatives selected from N- methyl-isoleucine, N-methyl-leucine, [3-alanine, 1 -amino-cyclopropanecarboxylic acid, 1 -amino-cyclobutanecarboxylic acid, 1 -amino-cyclopentanecarboxylic acid, 1 -aminocyclohexanecarboxylic acid;when either Zi or Z2 is an unnatural amino acid, at least one of Zi or Z2 is present; both Zi and Z2 are not simultaneously absent;X is selected from 2-(aminomethoxy)acetic acid and their derivatives, paraaminobenzylmethylcarbamate (PBMC) and its derivatives wherein derivatives of 2- (aminomethoxy)acetic acid selected from 2-(aminomethoxy)propionic acid, 2- (aminomethoxy)butanoic acid;D is selected from Exatecan mesylate;pl is 0-3;ml is 0-8;8. The antibody drug conjugates as claimed in claim 7 wherein P is selected from maleimide containing carboxylic acyl unit, Zi is absent or amino acid residues comprising branch or liner Ci-Cs alkyl, Z2 is absent or amino acid residues comprising branch or liner Ci-Cs alkyl, Z3 is phenylalanine and their derivatives, Z4 is glycine, [3- alanine, AIB, X is selected from 2-(aminomethoxy)acetic acid and their derivatives, D is selected from Exatecan mesylate, ml is 1, 2 and pl is 1, 2.

9. The antibody drug conjugates as claimed in claim 7 wherein antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2.

10. The antibody drug conjugates as claimed in claim 7 wherein average number of units of the selected one drug-linker structure conjugated per antibody molecule is in the rangeof 1 to 12; average number of units of the selected one drug-linker structure conjugated per antibody molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; average number of units of the selected one drug -linker structure conjugated per antibody molecule is in the range of 7 to 8.5; average number of units of the selected one drug-linker structure conjugated per antibody molecule is 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, average number of units of the selected one drug-linker structure conjugated per antibody molecule is in the range of 6 to 9.

11. The antibody drug conjugates as claimed in claim 7 wherein antibody drug conjugate is ADC-1.

12. The antibody drug conjugates as claimed in claim 7 wherein antibody drug conjugates is useful for the treatment of cancer, specifically breast cancer.

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