EA046150B1 - CONJUGATES OF QUATERNIZED TUBULISIN COMPOUNDS - Google Patents

Description

Cross reference to related applications

This application claims priority under US Application Ser. No. 62/263578, filed December 4, 2015, US Application Ser. No. 62/263587, filed December 4, 2015, US Application Ser. No. 62/309448, filed March 16, 2016, and US Application Ser. No. 62/309462, filed March 17, 2016, all of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The invention relates to drug-ligand conjugates (LDCs) for targeted delivery of tubulisin compounds to pathological cells associated with a given disease state or in close proximity to such cells. The targeting ligand in such an LDC selectively targets pathological cells with the tubulisin compound, but not normal cells distant from the pathological cells. This selective action is achieved by concentrating the compound at the desired site of action as a result of binding of the LDC targeting ligand to or near the pathological cells. As a result, the exposure of the removed normal cells to the tubulisin compound is reduced, thereby reducing the undesirable side effects due to the cytotoxicity of the tubulisin compound, while reducing the contribution of pathological cells to the disease state resulting from this cytotoxicity.

Typically, the design of an LDC involves consideration of multiple factors, including the requirement that the drug compound has an attachment site on a linker moiety that binds the drug compound to a targeting ligand and is capable of releasing the drug compound at the target site. In one approach, tubulysin compounds have previously been introduced into LDCs by covalently attaching a linker moiety to the C-terminal component of the tubulysin compound, which is typically tubuphenylalanine (Tup) or tubutyrosine (Tut), either through its carboxylic acid moiety converted to a hydrazide functional group, or through the phenyl part of the C-terminal component by means of an amino substituent introduced into this fragment. In another approach, the attachment of the linker moiety is carried out to the N-terminal component, which in natural tubulysins is D-N-methylpipecolic acid (D-Mep), after removal of its methyl substituent. In both approaches, the modified tubulisin compound is released with a significant loss of cytotoxicity compared to the parent compound, reducing its effectiveness as a therapeutic compound.

Due to the difficulty of introducing a tubulisin compound into an LDC that will conditionally release the compound in an unchanged form that retains its cytotoxic activity, there is a need in the art for conjugates that utilize the tertiary amine nitrogen in the N-terminal component of tubulisin as a conjugation site for releasing the fully active tubulisin compound at the target site of its action. There is also a need to suppress the hydrophobicity of the hydrophobic tubulisin compound in the LDC to provide increased compound loading at the LDC targeting ligand unit so as to increase the amount of tubulisin compound delivered to the desired site of action while reducing aggregation leading to LDC elimination and eliminating acetate loss. parts in the tubuvalin (Tuv) component in vivo. Either alone or in combination, this reduces the effectiveness of the administered LDC.

The essence of the invention

Primary embodiments of the invention are ligand drug conjugate (LDC) compositions, wherein the LDC composition is represented by the structure of Formula 1A

(Formula 1A) where L represents a ligand unit (L); LB is a covalently binding ligand unit; LP is a parallel connecting link; PEG is a polyethylene glycol unit; subscript a denotes 0 or 1; subscript b denotes 0 or 1; A represents the first optional extension unit, such that when the index a is 0, A is absent, or when the index a is 1, A is present and optionally consists of two, three or four independently selected subunits (A1, _A2 , A ₃ , A4); B represents a branching unit or a second optional extension unit (Ao), such that when the subscript b is 0, B is absent, or when the subscript b is 1, B is present and optionally consists of two, three or four subunits regardless of A; the index n denotes 1, 2, 3 or 4, provided that the subscript b denotes 1, and B represents a branch when the subscript n denotes 2, 3 or 4 and the subscript b denotes 0 or 1, so that B represents Ao, when the subscript b denotes 1 and the subscript n denotes 1; Su

- 1 046150 is a carbohydrate fragment; -O'- represents the oxygen atom of the O-glycosidic bond cleaved by the glycosidase; -J'- represents a heteroatom, optionally substituted if nitrogen; V, Z ¹ , Z ² and Z ³ represent =N-hjh =C(R ²⁴ )-, where R ²⁴ represents hydrogen or alkyl, alkenyl or alkynyl, optionally substituted, or halogen, -NO2, -CN or other an electron-withdrawing group, or -OCH3 or another electron-donating group, -O'-Su or -C(R ⁸ )(R ⁹ )-D ⁺ , where at least two of V, Z ¹ , Z ² and Z ³ are = C(R ²⁴ )-, provided that one and only one R ²⁴ is -C(R ⁸ )(R ⁹ )-D ⁺ , so that -C(R ⁸ )(R ⁹ )-D ⁺ is attached to one of V, Z ¹ , Z ² , Z ³ when that variable group is =C(R ²⁴ )-, and one and only one other R ²⁴ is -O'-Su, so that -O'-Su attached to another one of V, Z ¹ , Z ² , Z ³ when the variable group is =C(R ²⁴ )-, and the substituents are -O'-Su and -C(R ⁸ )(R ⁹ )-D ⁺ are ortho or para in relation to each other; R ⁸ and R ⁹ are independently hydrogen, alkyl, alkenyl or alkynyl, optionally substituted, or aryl or heteroaryl, optionally substituted; R' represents hydrogen or represents a halogen, -NO2, -CN or other electron-withdrawing group; D ⁺ represents a quaternized tubulisin drug unit; the subscript p denotes a number in the range from 1 to 24; and wherein said glycosidase cleavage initiates the release of the therapeutic tubulisin compound (D) from the drug-ligand conjugate compound in the composition.

In some aspects, the ligand unit is an antibody, thereby providing an antibody drug conjugate (ADC) having the antibody ligand unit, and the target fragment to which the antibody ligand unit is capable of binding is a cell surface antigen of the pathological target cells, which is capable of cellular internalization of the bound ADC.

Other major embodiments of the invention are drug-linker compounds having the structure of Formula IA

Su

(Formula IA) wherein LB' represents the precursor unit of the covalently binding ligand and the remaining variable groups are as specified for Formula 1A.

In other general embodiments, the invention provides drug-ligand conjugate and drug-linker conjugate compositions having structures of Formula 1B and Formula IB, respectively

(Formula 1B)

(Formula IB) where V, Z ¹ , Z ² and Z ³ represent =N- or =C(R ²⁴ )-, where R ²⁴ represents hydrogen or alkyl, alkenyl or alkynyl, optionally substituted, or halogen, -NO2 , -CN or other electron-withdrawing group, or -OCH3 or other electron-withdrawing group, or -C(R ⁸ )(R ⁹ )-D ⁺ , where at least one of V, Z ¹ and Z ³ is =C(R ²⁴ )-, provided that one and only one R ²⁴ is -C(R ⁸ )(R ⁹ )-D ⁺ , so that -C(R ⁸ )(R ⁹ )-D ⁺ is attached to one of the V , Z ¹ and Z ³ when the variable group is =C(R ²⁴ )-, J is a heteroatom, optionally substituted if it is a nitrogen atom; R' is hydrogen or -OCH3 or other electron-donating group, W is a peptide consisting of an amino acid sequence covalently attached to J by an amide bond, where the amide bond can be cleaved by a protease, and other variable groups are as specified for the formula 1A and formulas IB; and wherein said protease cleavage initiates release of the therapeutic tubulisin compound (D) from the drug-ligand conjugate compound in the composition.

- 2 046150

In some aspects, the invention relates to LDC conjugate compositions prepared by contacting a compound of formula IA or a compound of formula IB with a targeting moiety having a reactive sulfhydryl, amino or aldehyde group, under suitable conditions to effect condensation of the reactive moiety with the L _B ' moiety of the compound of formula I, where LB' is converted to LB covalently linked to a ligand unit which, as a result of this contact, corresponds to or incorporates the targeting agent into the structure.

In some aspects, LB' has one of the following structures:

where R represents hydrogen or C ₁ -C ₆ optionally substituted alkyl; R' represents hydrogen or halogen, or R and R' are independently selected from halogen; T represents -Cl, -Br, -I, -O-mesyl or -O-tosyl or other sulfonate leaving group; U is -F, -Cl, -Br, -I, -ON-succinimide, -O-(4-nitrophenyl), -O-pentafluorophenyl, -O-tetrafluorophenyl or -OC(=O)-OR ⁵⁷ ; X ² represents Cj-yualkylene, ^-^-carbocycle, -O-(C ₁ -C ₆ -alkyl), -arylene-, C ₁ -C ₁₀ -alkylene-arylene, -arylene-C ₁ -C ₁₀ - αlkylene, -C ₁ -C ₁₀ -alkylene-(C ₃ -C ₆ -carbocycle)-, -(^-^-carbocycle) -C ₁ -C ₁₀ -alkylene-, C ₃ -C ₈ heterocycle, -C ₁ -C ₁₀ -alkylene-(C ₃ -C ₈ -heterocyclo)-, -C ₃ -C ₈ -heterocyclo)-C ₁ -C ₁₀ -alkylene, -(CH ₂ CH ₂ O)u or -CH ₂ CH ₂ O)u-CH ₂ - where u is an integer ranging from 1 to 10 and R ⁵⁷ is C1-C6 alkyl or aryl.

Brief description of the figures

Fig. 1 - average tumor volume (mm ³ ) depending on the time (in days) subsequent post-implantation treatment of Hodgkin lymphoma xenograft CD30+ L540cy with DAR for 4 antibody-tubulysin drug conjugates linked to a quaternary amine, which contain units of the drug compound quaternized tubulisin M , conjugated via a Val-Ala protease-cleavable dipeptide unit (cAC10-15), versus conjugation via a β-glucuronidase-cleavable glucuronide unit (cAC10-82), and comparison of antibody-drug conjugates that contain quaternized tubulisin M drug compound units ( cAC10-82) and tubulisin ethyl ester (cAC10-57), both linked via β-glucuronide-cleavable glucuronide linkers, which all have non-PEGylated linker units.

Fig. 2 - average tumor volume (mm ³ ) depending on the time (days) of subsequent post-implantation treatment with DAR for 8 glucuronide conjugates of an antibody-tubulysin drug compound associated with a quaternary amine, which contain units of the drug compound ethyl ester of quaternized tubulisin with (cAC10- 66) and without (cAC10-57) PEGylation of their linker units, and quaternized tubulisin propyl ester drug units with (cAC10-67) and without (cAC10-58) PEGylation of their linker units.

Fig. 3 - average tumor volume (mm ³ ) depending on the time (in days) of subsequent post-implantation treatment of Hodgkin's lymphoma xenograft CD30+ L540cy with DAR for 8 glucuronide conjugates antibody-tubulisin drug compound M, coupled to a quaternary amine, (cAC10-99) with PEGylation of their linker units.

Fig. 4 - average tumor volume (mm ³ ) depending on the time (days) subsequent post-implantation treatment of a xenograft consisting of a mixed population of CD30+ Karpas and CD30-negative KarpasBVR tumor cells, to assess bystander activity, in mice with xe

- 3 046150 the nograft tumor was treated with a single dose of 0.5 mg/kg of glucuronide antibody-drug conjugates tubulisins bound to a quaternary amine, in which the quaternized drug compound unit is tubulisin M (cAC10-99), tubulisin ethyl ester (cAC10-66) or tubulisin methyl (propen-methyl)2-yl) ester (cAC10-185).

Fig. 5 - pharmacokinetic profiles, presented as the total amount of antibodies (μg/ml) depending on time (days), antibody-drug conjugates administered intravenously to rats at 1 mg/kg, having a DAR with 4 or 8 quaternized tubulisin M conjugated to humanized IgG via a protease-cleavable linker from Val-Ala (hIgG-15) or val-glu (hIgG-91) dipeptide quaternary amine without or with PEGylation of the linker unit (hIgG-95) versus a β-glucuronidase-cleavable glucuronide antibody-drug conjugate compound quaternized tubulisin M (hIgG-82) having a DAR for 4 without PEGylation of the linker unit.

Fig. 6 - Pharmacokinetic profiles presented as total amount of antibody (μg/ml) versus time (days) for antibody-drug conjugates administered intravenously to rats at 1 mg/kg with DAR for 8 loads per humanized IgG antibody with tubulisin glucuronide propyl ester , linked to a quaternary amine, with (hIgG-67) and without (hIgG-58) PEGylation of the linker unit.

Fig. 7 - pharmacokinetic profiles, presented as total amount of antibodies (μg/ml) as a function of time (days), for antibody-drug conjugates administered intravenously to rats at 1 mg/kg with DAR for 8 substitutions on the humanized IgG antibody with glucuronide tubulisin M , linked to a quaternary amine, (hIgG-99) and tubulisin ethyl ester (hIgG-66), both with a PEGylated linker unit.

Fig. 8 - average tumor volume (mm ³ ) depending on the time (days) subsequent after implantation of treatment of the Hodgkin lymphoma xenograft CD30+ L540cy dosed intraperitoneally with 0.15 mg/kg or 0.3 mg/kg glucuronide conjugate antibody-tubulisin drug compound M, bound with a quaternary amine, with (cAC10-99) or without (cAC10-82) PEGylation of the linker, with DAR for 8 and 4, respectively, or treatment with a glucuronide conjugate antibody-drug compound tubulisin ethyl ester linked to a quaternary amine, with (cAC10-66) or without (cAC10-57) PEGylation of the linker, with DARs for 8 and 4, respectively, compared to the 0.3 mg/kg intraperitoneal dose treatment with DARs for 4 quaternary amine-linked antibody-tubulisin drug conjugates M, which linked via a Val-Ala protease-cleavable dipeptide unit (cAC10-15) without PEGylation of the linker unit.

Fig. 9 - average tumor volume (mm ³ ) depending on the time (in days) subsequent post-implantation treatment of drug-resistant renal cell carcinoma xenograft CD70' 786-O (MDR+) intraperitoneally at a dose of 0.5 mg/kg or 1, 5 mg/kg PEGylated glucuronide conjugate antibody-drug compound tubulisin ethyl ester bound to a quaternary amine (h1F6-66) or PEGylated glucuronide methyl-(propen-2-yl)tubulysin ester bound to a quaternary amine (hF16- 185), both with a DAR for 8, compared to treatment with a quaternary amine-coupled antibody-tubulisin drug glucuronide conjugate M (h1F6-99), with a DAR for 8.

Fig. 10 - mean tumor volume (mm ³ ) depending on the time (in days) of subsequent post-implant treatment of drug-resistant anaplastic large cell lymphoma xenograft CD30 ⁺ cAC10-mc-Val-Cit-MMAE (DELBVR ALCL) with DAR for 4 non-PEGylated glucuronide antibody-drug conjugates tubulisin ethyl ester coupled to a quaternary amine (cAC10-57) or non-PEGylated glucuronide conjugate antibody-tubulysin drug compound M coupled to a quaternary amine, compared with their respective DARs for 8 PEGylated conjugates (cAC10-66 and cAC10-99) administered intraperitoneally at a dose of 0.3 mg/kg or 1 mg/kg, and with DAR for 4 antibody-tubulisin drug compound M conjugates coupled to a quaternary amine, which are linked through a Val-Ala protease-cleavable dipeptide without PEGylation of the linker unit (cAC10-15), administered intraperitoneally at a dose of 1 mg/kg.

Detailed Description of the Invention

Definitions.

As used herein and unless otherwise stated or implied by context, terms as used herein have the meanings defined below. Unless otherwise contradicted or implied, for example by the inclusion in these definitions and throughout this description of mutually exclusive elements or options, the singular terms mean one or more and the term or means and/or, where the context permits. Thus, as used in the specification and the accompanying claims, the singular forms include references to the plural unless the context clearly dictates otherwise.

In various parts of the description of the present invention, for example in any described embodiments or in the claims, reference is made to compounds, compositions or methods,

- 4 046150 which include one or more of the specified components, elements or steps. Embodiments of the invention also specifically include those compounds, compositions, compositions or methods that consist of or that essentially consist of the specified components, elements or steps. The term consists is used interchangeably with the term including, and they are considered equivalent terms. For example, the description of a composition, device, article or methods that includes a component or step is open, and it includes or covers these compositions or methods plus additional components or steps. However, these terms do not cover unspecified elements that may interfere with the functionality of the described compositions, devices, articles or methods for their intended purpose. Likewise, compositions, devices, articles, or methods described that consist of a component or steps are proprietary, and they will not include or indicate such compositions or methods that have additional component(s) or additional step(s). In addition, the use of the term including, as well as other forms such as include, includes and included, is not limiting. Finally, the term consisting essentially of allows for the inclusion of unspecified elements that do not significantly affect the functionality of the described compositions, devices, articles or methods for their intended purpose and are specified further herein. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. Unless otherwise noted, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used.

The term about, as used herein, when used in connection with a numerical value or range of values provided to describe a particular property of a compound or composition, indicates that the value or range of values may vary to an extent considered reasonable by anyone of ordinary skill in the art. in the art as part of the description of this particular property. Reasonable deviations include deviations that are within the accuracy or precision of the instrument(s) used to measure, determine, or obtain a particular property. In particular, the term about, as used in this context, means that a numerical value or range of values may differ by 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or 0.01% of the specified value or range of values, usually by 10 to 0.5%, more typically by 5 to 1%, within the description of that particular property.

The term essentially retains, essentially retains, and similar terms as used herein refer to a property, characteristic, or activity of a compound, or composition, or fragment that has not been appreciably altered or is within the experimental error of determining the same activity, characteristic, or property of another the compound, or composition, or fragment from which they were obtained.

The term negligible or insignificant as used herein is an amount of impurity below the level of quantitation by HPLC analysis and, if present, represents from about 0.5% to about 0.1% w/w of the composition which it pollutes. Depending on the context, these terms can also mean that there is no statistically significant difference between the measured values or results, or is within the experimental error of the instruments used to obtain those values. Minor differences in the values of a parameter determined experimentally do not mean that the impurity characterized by this parameter is present in an insignificant amount.

As used herein, the term substantially preserve refers to a measured value of a physical property of a compound or composition or fragment thereof that is statistically different from the determination of the same physical property of another compound or composition or fragment from which it was derived, but where such difference is not limited to a statistically significant difference in biological activity in a suitable biological test system for assessing that activity (that is, the biological activity is substantially preserved) or which has no biological consequence. Thus, the phrase essentially retains is made with reference to the effect that a physical property of a compound or composition has on the biological activity that is clearly associated with that property.

The term predominantly containing, predominantly having and similar terms refer to the main component of the mixture. When a mixture consists of two components, then the main component constitutes more than 50% by weight of the mixture. When using a mixture of three or more components, the predominant component is the one present in the greatest amount in the mixture and may or may not constitute the majority of the mixture's mass.

The term electron-withdrawing group as used herein refers to a functional group or electronegative atom that withdraws electron density from the atom to which it is bonded inductively and/or by resonance, whichever is more dominant (i.e., functional group or atom can be an electron donor through resonance, but in general can inductively withdraw electrons) and have a ten

- 5 046150 tendency to stabilize anions or fragments enriched with electrons. The electron-withdrawing action is usually transferred inductively, albeit in a weakened form, to other atoms attached to the bonded atom, which creates an electron deficiency via the electron-withdrawing group (EWG), thus affecting the electrophilicity of the more distant reactive site. Typical electron-withdrawing groups include, but are not limited to, -C(=O), -CN, -NO2, -CX ₃ , -X, -C(=O)OR', -C(=O)NH ₂ , -C (=O)N(R')R ^op , -C(=O)R', -C(=O)X, -S(=O)2R ^op , -S(=O)2OR', -SO3H2, -S(=O) ₂ NH ₂ , -S(=O) ₂ N(R')R ^op , -PO3H2, -P(=O)(OR')(OR ^op )2, -NO, -NH2, -NH(R')(R ^op ), -N(R ^op )3 ⁺ , and salts thereof, where X is -F, -Br, -Cl or -I, and R ^op is in each case independently selected from the group , previously described for optional substituents, and is sometimes selected from the group consisting of C ₁ -C ₆ -alkyl and phenyl, and R' is selected from the group previously described for optional substituents, and is sometimes ^-^-alkyl. Typical EWGs may also include aryl groups (eg, phenyl) depending on substitution, and some heteroaryl groups (eg, pyridine). Thus, the term electron-withdrawing group also includes aryls or heteroaryls that are further substituted with electron-withdrawing groups. Common electron withdrawing groups are -C(=O), -CN, -NO2, -CX3 and -X, where X is a halogen. Depending on its substituents, the unsaturated alkyl moiety can also be an electron-withdrawing group.

The term electron-donating group refers to a functional group or electropositive atom that increases the electron density of the atom to which it is bonded, inductively and/or through resonance, whichever is more dominant (that is, the functional group or atom may be inductively electron-withdrawing, but in general can be electron-donating through resonance) and tend to stabilize cations or electron-poor systems. The electron-donating effect is usually transferred via resonance to other atoms attached to a bonded atom that has been enriched with electrons by an electron-donating group (EDG), thus influencing the nucleophilicity of the more distant reactive site. Typical electron-donating groups include, but are not limited to, -OH, -OR', -NH ₂ , -NHR' and N(R') ₂ , where each R' is an independently selected alkyl, typically ^-^-alkyl. Depending on their substituents, an aryl, heteroaryl, or unsaturated alkyl moiety can also be an electron-donating group.

The term fragment as used herein means a specified segment, fragment or functional group of a molecule or compound. Chemical moieties are sometimes referred to as chemical entities that are embedded or attached (that is, a substituent or variable group) to a molecule, compound, or chemical formula.

For any group of substituents or any fragment described herein, defined by a range of carbon atoms, the specified range means that any single number of carbon atoms is described. Thus, reference is, for example, to optionally substituted Cj-C-halkyl. optionally substituted C ₂ -C ₆ -alkenyl, optionally substituted C ₃ -C ₈ -heterocycle, in particular means that an optionally substituted alkyl moiety with 1, 2, 3 or 4 carbon atoms is represented, as defined herein, or optionally a substituted alkenyl moiety having 2, 3, 4, 5 or 6 carbon atoms, or a 3, 4, 5, 6, 7 or 8 membered heterocycle as defined herein. All such numerical designations are specifically intended to describe all individual groups of carbon atoms; and thus, optionally substituted C'1-Cgalkyl includes methyl, ethyl, C3 alkyls and C4 alkyls, including all positional isomers thereof, whether substituted or unsubstituted. Thus, when an alkyl moiety is substituted, the numerals refer to the unsubstituted backbone moiety and are not intended to include carbon atoms that may be present on substituents on that backbone moiety. For esters, carbonates, carbamates and ureas as defined herein, which are identified by a given range of carbon atoms, the range includes the carbonyl carbon of the corresponding functional group. Thus, a C1 ester is a formate ester, a _C2 ester is an acetate ester, and an unsubstituted _C1 urea is a _NH2 (C=O) _NH2 .

Organic substituents, moieties and groups described herein, and for any other moieties described herein, generally exclude unstable moieties, unless such unstable moieties are transition components that can be used in the preparation of a compound with sufficient chemical stability for one or more of the applications described herein. Substituents, moieties, or groups are necessarily excluded when, by the definitions provided herein, this results in substituents containing a pentavalent carbon.

The term alkyl, as used herein, by itself or as part of another term refers to a methyl or collection of carbon atoms where one or more carbon atoms are saturated (that is, composed of one or more sp ³ carbon atoms) that are covalently bonded together in a normal, secondary, tertiary or cyclic arrangement, that is, in a linear, branched, cyclic arrangement or some combination thereof. When adjacent

- 6 046150 saturated carbon atoms are in a cyclic arrangement, such alkyl groups are sometimes called cycloalkyl as defined herein. Saturated alkyl substituents contain saturated carbon atoms (ie sp ³ carbon atoms) and do not contain aromatic, sp ² or sp carbon atoms (ie not substituted by unsaturated, aromatic and heteroaromatic moieties). Unsaturated alkyl substituents are alkyl moieties or groups that contain moieties as described herein for alkenyl, alkynyl, aryl and heteroaryl moieties.

Thus, unless otherwise specified, the term alkyl will mean a saturated non-cyclic hydrocarbon radical, optionally substituted with one or more cycloalkyl or unsaturated, aromatic or heteroaromatic moieties, or some combination thereof, wherein the saturated hydrocarbon radical has the specified number of covalently bonded saturated carbon atoms (e.g. ''QC^-alkyl'' or C ₁ -C ₆ -alkyl means an alkyl moiety or group that contains 1, 2, 3, 4, 5 or 6 contiguous non-cyclic saturated carbon atoms, and C 1 -C ₈ -alkyl refers to an alkyl moiety or group having 1, 2, 3, 4, 5, 6, 7 or 8 contiguous saturated non-cyclic carbon atoms). The number of saturated carbon atoms in an alkyl moiety or group can vary and is typically 1-50, 1-30 or 1-20, and more typically 1-8 or 1-6. Generally, alkyl refers to a saturated C1- _C8 alkyl moiety or, more typically, a C1- _C6 or _C1 - _C4 alkyl moiety, where the latter is sometimes called lower alkyl. When the number of carbon atoms is not specified, the alkyl moiety or group contains from 1 to 8 carbon atoms.

When referring to an alkyl moiety or group as an alkyl substituent, the alkyl substituent in the Markush structure or other organic moiety to which it is associated is a chain of contiguous saturated carbon atoms covalently attached to the structure or moiety through the sp ³ carbon of the alkyl substituent. Therefore, an alkyl substituent, as used herein, contains at least one saturated moiety and may also contain or optionally be substituted with cycloalkyl, unsaturated alkyl, aromatic or heteroaromatic moieties or groups. Thus, the alkyl substituent may further contain one, two, three or more independently selected double bonds, triple bonds or cycloalkyl, aromatic or heteroaromatic moieties or some combination thereof, typically one double bond, one triple bond, or be substituted by one cycloalkyl aromatic or heteroaromatic fragment. When an alkyl substituent, moiety, or group is indicated, examples include those resulting from the removal of a hydrogen atom from the parent alkane (i.e., is monovalent) and may include methyl, ethyl, 1propyl (n-propyl), 2-propyl (isopropyl , -CH(CH ₃ ) ₂ ), 1-butyl (n-butyl), 2-methyl-1-propyl (iso-butyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (sec-butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (tert-butyl, -C(CH ₃ ) ₃ ), amyl, isoamyl, sec-amyl and other alkyl fragments with linear, cyclic and branched chain.

The term alkylene, as used herein, by itself or as part of another term refers to a saturated, branched, cyclic or linear hydrocarbon diradical, substituted or unsubstituted, where one or more carbon atoms are unsaturated (that is, include one or more sp ³ carbon atoms) of a specified number of carbon atoms, usually 1-10 carbon atoms, and having two radical centers (i.e. divalent) formed by removing two hydrogen atoms from one or two different saturated (i.e. sp ³ ) carbon atoms of the parent alkane. Alkylene moieties also include alkyl radicals, as described herein, in which a hydrogen atom is removed from a saturated moiety or from a carbon of the alkyl radical to form a diradical. Typically, alkylene moieties include, but are not limited to, divalent moieties resulting from the removal of a hydrogen atom from a saturated carbon atom of the parent alkyl moiety and are represented, for example, by (-CH2-), 1,2-ethylene (-CH2CH2- ), 1,3-propylene (-CH ₂ CH ₂ CH ₂ -), 1,4-butylene (-CH ₂ CH ₂ CH ₂ CH ₂ -) and similar diradicals. Typically, an alkylene is a branched or straight chain hydrocarbon, typically containing only sp ³ carbon atoms (that is, fully saturated regardless of radical carbon atoms).

Cycloalkyl, as used herein, is a monocyclic, bicyclic, or tricyclic ring system radical, where each of the atoms forming the ring system (i.e., the backbone atoms) is a carbon atom, and where one or more of these carbon atoms in each ring cyclic ring system are saturated (that is, composed of one or more sp ³ carbon atoms). Thus, cycloalkyl represents cyclic saturated carbon atoms, but may also contain unsaturated carbon atom(s), and therefore its carbocyclic ring may be saturated or partially unsaturated, or may be fused with an aromatic ring, where the condensation points of the cycloalkyl with aromatic ring refers to adjacent unsaturated carbon atoms of a cycloalkyl moiety, group, or substituent and to adjacent aromatic carbon atoms of an aromatic ring.

- 7 046150

Unless otherwise indicated, a cycloalkyl moiety, group, or substituent may be substituted with moieties described for alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, and the like, or may be substituted by other cycloalkyl moieties. Cycloalkyl moieties, groups or substituents include cyclopropyl, cyclopentyl, cyclohexyl, adamantyl or other cyclic moieties containing only carbon atoms. Cycloalkyls further include cyclobutyl, cyclopentenyl, cyclohexenyl, cycloheptyl and cyclooctyl. Depending on its structure, the cycloalkyl substituent may be a monoradical, as described above for cycloalkyl moieties or groups, or a diradical (i.e., cycloalkylene or, alternatively, carbocyclo), such as, but not limited to, cyclopropane-1,1-diyl, cyclobutane -1,1-diyl, cyclopentane-1,1-diyl, cyclohexane-1,1-diyl, cyclohexane-1,4-diyl, cycloheptane-1,1-diyl and the like).

When a cycloalkyl is used as a Markush group (i.e., a substituent), the cycloalkyl is attached to a Markush formula or other organic moiety to which it is linked via a carbon that is included in the carbocyclic ring system of the cycloalkyl group, provided that the carbon is not an aromatic fused carbon ring system. When an unsaturated carbon of an alkene moiety containing a cycloalkyl substituent is attached to the Markush formula to which it is associated, the cycloalkyl is sometimes called a cycloalkenyl substituent. The number of carbon atoms in the cycloalkyl substituent is determined by the total number of backbone atoms of the ring system. This number can vary and typically ranges from 3 to 50, 1-30 or 1-20 and more typically 3-8 or 3-6 unless otherwise specified, for example C _3-8 -cycloalkyl means cycloalkyl substituent , a fragment or group containing 3, 4, 5, 6, 7 or 8 carbocyclic carbon atoms, and O^cycloalkyl means a cycloalkyl substituent, fragment or group containing 3, 4, 5 or 6 carbocyclic carbon atoms. Therefore, cycloalkyl substituents, moieties or groups typically have 3, 4, 5, 6, 7, 8 carbon atoms in their carbocyclic ring system and may contain exo- or endocyclic double bonds, or endocyclic triple bonds, or a combination of both, where endocyclic double or triple bonds or their combination do not form a cyclically conjugated system of 4n+2 electrons. A bicyclic ring system may have one (that is, a spiro ring system) or two carbon atoms, and a tricyclic ring system may have a total of 2, 3, or 4 carbon atoms, usually 2 or 3.

The term alkenyl, as used herein, means a substituent, moiety, or group that contains one or more double bond groups (e.g., -CH=CH- functional group) or 1, 2, 3, 4, 5, or 6 or more , typically 1, 2 or 3 such moieties, and may be replaced by an aryl moiety or group such as benzene, or linked by normal, secondary, tertiary or cyclic carbon atoms, i.e. linear, branched, cyclic or any combination thereof, except when the alkenyl substituent, fragment or group is a vinyl group (for example, the -CH=CH ₂ functional group). An alkenyl moiety, group or substituent having multiple double bonds may have the double bonds arranged continuously (i.e., 1,3-butadienyl moiety) or non-continuously with one or more intermediate saturated carbon atoms or a combination thereof, provided that the cyclic continuum the arrangement of double bonds does not form a cyclically conjugated system of 4n+2 electrons (that is, it is not aromatic).

When an alkenyl moiety, group or substituent is indicated, species include, by way of example and not limitation, any of the alkyl or cycloalkyl groups, moieties or substituents described herein that have one or more endo double bonds and monovalent moieties formed as a result of removing a hydrogen atom from the sp ² carbon of the parent alkene compound. Such monovalent groups typically include vinyl (-CH=CH2), allyl, 1-methylvinyl, butenyl, isobutenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methylcyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl and other linear, cyclic and branched chains, all carbon-containing units containing at least one double bond. When alkenyl is used as a Markush group (ie, a substituent), the alkenyl is attached to the Markush formula or other organic moiety to which it is linked via the double bond carbon (ie, sp ² carbon) of the alkenyl moiety of the group. The number of carbon atoms in an alkenyl substituent is determined by the number of sp ² carbon atoms in the alkene functional group that defines it as an alkenyl substituent, and the total number of adjacent saturated carbon atoms attached to each of these sp ² carbon atoms. This number can vary and, unless otherwise stated, ranges from 1 to 50, for example typically 1-30 or 1-20, more typically 1-8 or 1-6 when the double bond functional group is in the exo position in a Markush structure, or may vary and range from 2 to 50, typically 2-30 or 2-20, more typically 2 to 8 or 2-6 when the double bond functional group is endo to the Markush structure . For example, O^-alkenyl or O^-alkenyl means an alkenyl group containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms, in which at least two are sp ² carbon atoms in conjugation with each other, and O^-alkenyl or O^-alkenyl means an alkenyl group containing 2, 3, 4, 5 or 6 carbon atoms, in which at least two are sp ²

- 8 046150 carbon atoms that are in conjugation with each other. Typically, the alkenyl substituent is a C ₂ -C ₆ -or ^-^-alkenyl moiety having two sp ² carbons that are in conjugation with each other.

The term alkenylene, as used herein, by itself or as part of another term, refers to a substituent, moiety, or group that contains one or more double bond groups as previously described for alkenyl, with a specified number of carbon atoms, typically 1 -10 carbon atoms when the double bond functional group is exo to the larger moiety, or 2-10 when the double bond functional group is endo in the alkenylene moiety, and have two radical centers formed by removing two hydrogen atoms from one or two different sp ² carbon atoms in the double bond group of the parent alkene. Alkenylene moieties further include alkenyl radicals, as described herein, in which a hydrogen atom has been removed from the same or another sp ² carbon atom in the double bond moiety of the alkenyl radical to form a diradical or from an sp ² carbon in another moiety with double bond to provide another radical carbon. Typically, alkenylene moieties include diradicals having the structure -C=C- or -C=CX ¹ -C=C-, where X ¹ is absent or is an alkylene as defined herein.

The term aryl as used herein means an organic moiety, substituent or group that defines an aromatic ring system or a fused ring system without ring heteroatoms, containing 1, 2, 3 or 4 to 6 rings, typically 1 to 3 rings, where the rings consist only of carbon atoms that participate in a cyclically conjugated system of 4n+2 electrons (Hückel's rule), typically 6, 10 or 14 electrons, some of which may additionally participate in exocyclic conjugation with a heteroatom (cross-conjugated with e.g. quinone). Aryl substituents, moieties or groups are usually formed of six, eight, ten or more aromatic carbon atoms. Aryl substituents, moieties or groups are optionally substituted. Examples of aryls include C ₆ -C ₁₀ aryls such as phenyl and naphthalenyl and phenanthryl. Since aromaticity in a neutral aryl moiety requires an even number of electrons, it should be understood that this range for this moiety will not cover species with an odd number of aromatic carbons. When aryl is used as a Markush group (i.e., substituent), the aryl is attached to the Markush formula or other organic moiety to which it is linked through the aromatic carbon of the aryl group. Depending on the structure, the aryl group can be monoradical (ie, monovalent) or diradical (ie, the arylene group described herein, which is divalent).

The term arylene or heteroarylene, as used herein, by itself or as part of another term, is an aryl or heteroaryl moiety, group, or substituent, as defined herein, that forms two covalent bonds (i.e., is divalent) within a larger moiety, which may be in the ortho, meta, or para configuration, or an aromatic diradical moiety. Examples of arylenes include, but are not limited to, phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene, as shown by the following structures:

The term arylalkyl, as used herein, means a substituent, moiety or group wherein the aryl moiety is linked to an alkyl group, i.e. -alkyl-aryl, where alkyl and aryl groups are as described above, for example CH2-C6H5, - CH ₂ CH(CH ₃ )-C ₆ H ₅ or -CH(CH ₂ CH ₂ CH ₃ )-CH ₂ -C ₆ H ₅ . When an arylalkyl is used as a Markush group (i.e., a substituent), the alkyl moiety of the arylalkyl is added to the Markush formula to which it is bonded via the sp ³ carbon of the alkyl group.

The term alkylaryl as used herein means a substituent, moiety or group wherein the alkyl moiety is linked to an aryl group, i.e. -aryl-alkyl, where the aryl and alkyl groups are as described above, for example -C6H4-CH3 or -C6H4-CH2CH(CH3). When an alkylaryl is used as a Markush group (i.e., a substituent), the aryl moiety of the alkylaryl is added to the Markush formula to which it is linked via the aromatic carbon of the aryl moiety.

Optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkylaryl, optionally substituted arylalkyl, optionally substituted heterocycle, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkylheteroaryl, optionally substituted heteroarylalkyl and similar terms refer to alkyl, alkenyl, alkynyl , alkylaryl, arylalkyl, heterocyclic, aryl, heteroaryl, alkylheteroaryl, heteroaryl

- 9 046150 to a lalkyl or other substituent, moiety or group as defined or described herein, wherein the hydrogen atom(s) of the substituent, moiety or group are optionally replaced by other moiety(s) or group(s), or where the alicyclic carbon chain, which includes one of these substituents, fragment or group, is interrupted by the replacement of the carbon atom(s) of that chain with other fragment(s) or group(s).

Optional substituent(s) replacing hydrogen(s) in any of the above substituents, moieties or groups includes substituents independently selected from the group consisting of halogen, -CN, -NH2, -OH, -N(CH3)2, alkyl, fluoroalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone and arylsulfone, or those selected from the group consisting of halogen, -CN, -NH2, -OH , -NH(CH ₃ ), -N(CH ₃ b, -C(=O)OH (that is, CO2H), -C(=O)O-alkyl (that is, ^-alkyl), C(=O) NH2, -C(=O)NH(alkyl), -C(=O)N^k^) ₂ , -S(=O) ₂ NH ₂ , S(=O) ₂ NH^km), ^(^ ^(alkyl^, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, -S-alkyl and S(=O) ₂ alkyl.

Typically, the optional substituent(s) replacing the hydrogen(s) in any of the above substituents, moieties or groups is independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroaliphatic ring, hydroxyl, alkoxy, aryloxy, cyano, halogen , nitro, haloalkyl, fluoroalkyl, fluoroalkoxy and amino, including mono-, di- and tri-substituted amino groups and their protected derivatives, or selected from the group consisting of halogen, -CN, -NH2, -OH, NH(CH ₃ ), - N(CH ₃ ) ₂ , -CH ₃ , -CH2CH3 , -CF ₃ , -OCH3 , and -OCF3 . Typically, any of the above substituents, moieties or groups that are optionally substituted by replacing one or more of its hydrogens has the hydrogen(s) replaced by one or two of the preceding optional substituents or, more typically, one of the preceding optional substituents. An optional substituent on a saturated aliphatic carbon atom in an acyclic or cyclic ring system further includes oxo (=O). For a phenyl or 6-membered heteroaryl moiety, the arrangement of any two substituents present on the aromatic or heteroaromatic ring may be ortho (o), meta (m) or para (p).

Typically, the optional substituent replacing a carbon in an acyclic carbon chain is selected from the group consisting of -O-, -C(=O)-, -C(=O)O-, -S-, -S(=O)-, -S(=O) ₂ -, -NH-, -NHC(=O)-, -C(=O)NH-, S(=O)2NH-, -NHS(=O)2, -OC(= O)NH- and -NHC(=O)O-.

Typically, any of the above substituents, moieties or groups that are optionally substituted by substitution of one or more hydrogens has the hydrogen(s) replaced by one or two of the preceding optional substituents or, more typically, by one of the preceding optional substituents.

It is understood that an optional alkyl or alkylene substituent, moiety, or group excludes alkyl, and that an optional alkene or alkenylene substituent, moiety, or group excludes alkenyl, since such substitutions provide moieties that fall within the definition of basic moieties, thus the substituted and optional substituent alkyl or alkylene further excludes alkylene or alkenylene, since such substitution provides moieties that fall within the definition of unsaturated alkyl and unsaturated alkylene, respectively.

The term heterocycle as used herein means a carbocycle in which one or more, but not all, of the backbone carbon atoms in the carbocyclic ring system are independently replaced by a heteroatom, optionally substituted where permitted, including N, O, S, Se, B Si, P , where two or more heteroatoms may be adjacent to each other or separated by one or more carbon atoms in the same ring system, typically 1-3 atoms. These heteroatoms typically include N, O, or S. A heterocycle typically contains a total of one to ten heteroatoms in a heterocyclic ring system, provided that not all of the backbone atoms of any one ring in a heterocyclic ring system are heteroatoms, wherein each heteroatom in the ring(s) ), optionally substituted, where permitted, is independently selected from the group consisting of O, S and N, provided that any one ring does not contain two adjacent O or S atoms. When non-aromatic, heterocycles have at least 3 atoms in their ring system, and when they are aromatic, heterocycles have at least 5 atoms in their ring system. Typical heterocycles are suggested by Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7 and 9; The Chemistry of Heterocyclic Compounds, A series of Monographs (John Wiley & Sons, New York, 1950 to date), particularly volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. 1960, 82:5545-5473, in particular 5566-5573).

When a heterocycle is used as a Markush group (i.e., a substituent), the heterocycle is attached to a Markush formula or larger moiety to which it is linked via a carbon or heteroatom of the heterocycle, where such addition does not result in an unstable or formal oxidation state of that carbon or heteroatom . Thus, heterocycles in this context are monovalent units, sometimes called heterocyclyls, which

- 10 046150 these include heteroaryls which have a heteroaromatic ring system, or heterocycloalkyl in which the ring system is non-aromatic, any of which can be fused with a carbocyclic aryl or heteroaryl moiety and includes phenyl- (i.e. benzo) fused heterocycloalkyl and heteroaryl moieties, provided that, when a heteroaryl moiety is fused to a heterocycloalkyl or carbocyclic moiety (i.e., when the heterocyclic portion of the fused ring system is monovalent), the resulting fused ring system is classified as heteroaryl, and when a heterocycloalkyl moiety is fused to a carbocyclic moiety (i.e., when the carbocyclic part of the fused ring system is monovalent), the resulting fused ring system is classified as heterocycloalkyl.

Generally, heterocycloalkyl is a cycloalkyl group, fragment or substituent wherein 1, 2 or 3 carbon atoms of the cycloalkyl chain are replaced by a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and is a C ₃ -C ₁₀ -heterocycloalkyl, more typically , C5C ₁₀ -heterocycloalkyl, in which the subscript indicates the total number of backbone atoms (including carbon atoms and heteroatoms) in the heterocycloalkyl ring system. Non-limiting heterocycloalkyls may contain 0-2 N atoms, 0-2 O atoms or 0-1 S atoms, or some combination thereof, provided that at least one of these heteroatoms is present in the cyclic ring system and can be replaced by one or two oxo (=O) fragments, as in pyrrolidin-2-one. More typically, heterocycloalkyls include pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl.

Heteroaryls generally contain from one to four heteroatoms in the ring(s) of the heteroaryl ring system, provided that not all of the backbone atoms of any one ring system in a heteroaryl are heteroatoms, optionally substituted where permitted, and have 0-3 N atoms , 1-3 N atoms or 0-3 N atoms with 0-1 O atom or 0-1 S atom, provided that at least one heteroatom is present. Heteroaryl may be monocyclic, bicyclic or polycyclic. Monocyclic heteroaryls include C ₅ -C ₂₄ heteroaryls, typically C5-C12 or C5-C6 heteroaryls, in which the subscript indicates the total number of backbone atoms (including its carbon atoms and heteroatoms) of the aromatic ring system of the heteroaryl. A more typical heteroaryl is an aryl moiety where one of the 1, 2, or 3 carbon atoms of the aromatic ring(s) of the original aryl moiety is replaced by a heteroatom, optionally substituted where permitted, including N, O, and S, provided that not all backbone atoms are which -either one aromatic ring system in the aryl moiety is replaced by heteroatoms and is typically replaced by oxygen (-O-), sulfur (-S-), nitrogen (=N-) or -NR-, where R is -H, protective group or alkyl, aryl, or is nitrogen substituted by another organic moiety in a manner that maintains a cyclic conjugated system where a heteroatom of nitrogen, sulfur, or oxygen participates in the conjugated system either through a pi bond to an adjacent atom in the ring system or through a lone a pair of electrons on a heteroatom.

In other aspects, heteroaryl is monocyclic and is typically a heteroaryl with a 5-membered or 6-membered heteroaromatic ring system. A 5-membered heteroaryl is a monocyclic C5 heteroaryl containing from 1 to 4 carbon atoms and the required number of heteroatoms in its heteroaromatic ring system. A 6-membered heteroaryl is a monocyclic C ₆ heteroaryl containing from 1 to 5 carbon atoms and the required number of heteroatoms in its heteroaromatic ring system. Heteroaryls that are 5-membered have four, three, two or one aromatic heteroatom(s), and heteroaryls that are 6-membered include heteroaryls having four, three, two or one aromatic heteroatom(s). C5 heteroaryls are monovalent moieties formed by the removal of a hydrogen atom from an aromatic carbon or an electron from an aromatic heteroatom, where possible, from the parent heterocyclic compound, including pyrrole, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, triazole and tetrazole. Examples of C ₆ heteroaryls that are 6-membered are monovalent moieties formed by the removal of a hydrogen atom from an aromatic carbon or an electron from an aromatic heteroatom, where allowed, in the following heterocyclic parent compounds: pyridine, pyridazine, pyrimidine and triazine.

5-membered nitrogen-containing heteroaryl, 5-membered nitrogen-containing heteroaryl and similar terms refer to a 5-membered heteroaromatic moiety containing at least one nitrogen atom in its aromatic ring system, and is a monocyclic heteroaryl or is fused to an aryl or other heteroaryl ring system and may contain one or more other independently selected heteroatoms, such as N, O or S. Examples of 5-membered nitrogen heteroaryls include thiazole, imidazole, oxazole and triazole, and are typically a thiazole or oxazole, more typically a thiazole.

Heteroarylalkyl, as used herein, means a substituent, moiety, or group wherein a heteroaryl group is linked to an alkyl group, that is, -alkylheteroaryl, wherein the alkyl and heteroaryl groups are as described above. When arylalkyl is used in ca

- 11 046150 As a Markush group (that is, a substituent), the alkyl moiety of the arylalkyl is attached to the Markush formula to which it is linked through the sp ³ carbon of the alkyl group.

Alkylheteroaryl, as used herein, means a substituent, moiety, or group wherein a heteroaryl group is linked to an alkyl group, that is, heteroarylalkyl, wherein the heteroaryl and alkyl groups are as described above. When heteroarylalkyl is used as a Markush group (i.e., substituent), the heteroaryl moiety of the heteroarylalkyl is attached to the Markush formula to which it is linked via the sp ² carbon or heteroatom of the alkyl moiety.

O-linked moiety, O-linked substituent, and similar terms as used herein refer to a group or substituent that is attached to a moiety directly through an oxygen atom of the group or substituent. The O-linked group may be monovalent, including groups such as -OH, acetoxy (ie -OC(=O)CH ₃ ), acyloxy (ie -OC(=O)R ^a where R ^a is -H , optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocycle) and further includes monovalent groups such as optionally substituted alkyloxy, where the alkyl moiety is saturated or unsaturated, and other ethers including aryloxy (aryl-O-), phenoxy (Ph-O-), heteroaryloxy (heteroaryl-O-), optionally substituted, and silyloxy, (i.e. R3SiO-, where each R independently represents is alkyl or aryl, optionally substituted), and -OR ^pr , where R ^pr is a protecting group as defined above, or the O-linked group may be divalent, i.e. =O or -X-(CH2) _n -Y-, where X and Y are independently S and O, and n is 2 to 3, to form a spiro ring system with the carbon to which X and Y are attached. Typically, the O-linked substituent is a monovalent moiety selected from the group consisting of -OH, -OC(=O)CH ₃ , -OC(=O)R ^a , C1-C6 saturated alkyl ether and C3-C6 unsaturated ether, where R ^a is C1-C6 saturated alkyl or C3-C ₆ unsaturated alkyl or ^-^-alkenyl, or selected from this group, excluding -OH. Other typical O-linked substituents are provided in the definitions for carbamate, ether and carbonate described herein.

The term halogen as used herein means fluorine, chlorine, bromine or iodine and is typically -F or -Cl.

The term protecting group, as used herein, means a moiety that prevents or reduces the ability of the atom or functional group to which it is bonded to participate in unwanted reactions. Typical protecting groups for atoms or functional groups are reviewed by Greene (1999), Protective groups in organic synthesis, ^3rd ed., Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur and nitrogen are sometimes used to minimize or prevent unwanted reactions with electrophilic compounds. In other cases, a protecting group is used to reduce or eliminate the nucleophilicity and/or basicity of the unprotected heteroatom. Non-limiting examples of protected oxygen are given as OR ^pr , where R ^pr is a hydroxyl protecting group, where the hydroxyl is typically protected as an ester (eg, acetate, propionate, or benzoate). Other hydroxyl protecting groups do not affect the nucleophilicity of organometallic reagents or other highly basic reagents where the hydroxyl is typically protected as an ether, including alkyl or heterocycloalkyl ethers (e.g., methyl or tetrahydropyranyl ethers), alkoxymethyl ethers (e.g., methoxymethio or ethoxymethyl ethers), optionally substituted aryl ethers and silyl ethers (e.g. trimethylsilyl (TMS), triethylsilyl (TES), t-butyldiphenylsilyl (TBDPS), t-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS) and [2-(trimethylsilyl)ethoxy] methylsilyl (SEM)). Nitrogen protecting groups include groups for primary or secondary amines, such as in -NHR ^pr or -N(R ^pr ) ₂ -, where at least one of the R ^pr represents a protecting group for a nitrogen atom, or both R ^pr together form protective group.

A protecting group is a suitable protection when it is capable of preventing or avoiding unwanted side reactions or premature loss of the protecting group under reaction conditions necessary to effect a desired chemical transformation elsewhere in the molecule, and during purification of the newly formed molecule when desired and can be removed under conditions that do not adversely affect the structure or stereochemical integrity of the newly formed molecule. By way of example, and not limitation, a suitable protecting group may include those previously described for protecting functional groups. In some aspects, a suitable protecting group is typically a protecting group used in peptide condensation reactions. For example, a suitable nitrogen protecting group is an acid-sensitive carbamate protecting group such as BOC.

The term ester, as used herein, means a substituent, moiety, or group that contains the structure -C(=O)-O- (i.e., ester functionality), in

- 12 046150 in which a carbon atom in the structure is not directly bonded to another heteroatom and is directly bonded to -H or another carbon atom of an organic moiety, and a monovalent oxygen atom is attached to the same organic moiety to form a lactone or other organic moiety. Typically, esters include or consist of organic moieties containing 1-50 carbon atoms, typically 1-20 carbon atoms, or more typically 1-8 carbon atoms, and 0-10 independently selected heteroatoms (e.g., O, S, N, P, Si, but usually O, S and N), usually 0-2, where the organic moieties are linked through the -C(O)-O- structure (that is, through the ester functional group). When an ester is a substituent or variable group of a Markush structure, the substituent is linked to the structure through the monovalent oxygen atom of the ester functional group. In these cases, the organic moiety attached to the carbonyl carbon of the ester functionality includes any of the organic groups described herein, for example, Cj-C.'2-alkyl moieties, C ₂ -C ₂₀ -alkenyl moieties, C ₂ -C ₂₀ -alkynyl moieties, C ₆ -C ₂₄ -aryl moieties, C ₅ -C ₂₄ -heterocycles or substituted derivatives of any of them, for example containing 1, 2, 3, 4 or more substituents, where each substituent is independently selected. Typical esters include, by way of example, but are not limited to, acetate, propionate, isopropionate, isobutyrate, butyrate, valerate, isovalerate, caproate, isocaproate, hexanoate, heptanoate, octanoate, phenylacetate esters or benzoate esters, or have the structure -OC(=O )R ^a where R ^a is as defined for the acyloxy O-linked substituent and is typically selected from the group consisting of methyl, ethyl, propyl, isopropyl, 3-methylprop-1-yl, 3,3-dimethylprop-1 -silt and vinyl. The ester substituents described herein are examples of monovalent O-linked substituents.

The term ether, as used herein, means an organic moiety, group, or substituent that contains 1, 2, 3, 4 or more -O- (i.e., oxy) moieties that are not associated with carbonyl moiety(s), typically 1 or 2, where no two fragments -O- are directly adjacent (that is, directly related) to each other. Typically, the ether structure is formed by or consists of the formula -O-organic moiety, where the organic moiety is as described for an organic moiety associated with an ester functionality. More typically, the ether moiety, group, or substituent has the formula -O-organic moiety, where the organic moiety is as described herein for an optionally substituted alkyl group. When an ether (that is, an ether substituent) is used as the Markush group, the oxygen of the ether functional group is added to the Markush formula to which it is associated. When an ether is used as a substituent on a Markush group, it is sometimes referred to as an alkoxy group, which is an example of an O-linked substituent. Alkoxy includes C1-^-ether substituents such as, by way of example and not limitation, methoxy, ethoxy, propoxy, isopropoxy, butoxy and allyloxy.

The term amide or carboxamide, as used herein, means a group that contains the structure R-C(=O)N(R)- or -C(=O)N(R)2 (i.e., amide or carboxamide functionality, respectively) without any other heteroatom directly bonded to the carbonyl carbon of the structure, and wherein R, independently selected, is hydrogen, a protecting group, or an organic moiety as described herein for an organic moiety linked to an ester functionality, and is typically optional substituted alkyl group. Typically, hydrogen or an organic moiety independently selected from R is associated with a carboxamide or amide functionality, which organic moiety is also as described herein for an organic moiety associated with an ester functionality. When bound to an organic fragment, the resulting structure is represented by an R-C(=O)N(R)-organic fragment or an organic fragment -C(=O)N(R)2. When an amide is specified as a variable for a Markush structure, the amide nitrogen is associated with that structure. For carboxamide substituents, the carbonyl carbon of the amide functional group is associated with a Markush structure. Amides and carboxamides are typically prepared by condensing an acid halide, such as an acid chloride, with a compound containing a primary or secondary amine. Alternatively, amide condensation reactions are used, well known in the art of peptide synthesis, which often proceed through an activated ester of a carboxylic acid-containing compound. Examples of the formation of amide bonds using peptide condensation methods are presented in Benoiton (2006) Chemistry of peptide synthesis CRC Press, Bodansky Peptide synthesis: A practical textbook (1988) Springer-Verlag; Frinkin, M. et al. Peptide Synthesis Ann. Rev. Biochem. (1974) 43:419-443. Reagents used in the preparation of activated carboxylic acids are presented in Han, et al. Recent development of peptide coupling agents in organic synthesis Tet. (2004) 60:2447–2476.

The term carbonate, as used herein, means a substituent, moiety or group that contains the structure -O-C(=O)-O- (ie, a carbonate functional group). Typically, carbonate groups, as used herein, are formed or consist of an organic moiety, where the organic moiety is as described herein for an organic moiety linked to an ester functionality linked through

- 13 046150 structure -O-C(=O)-O-, for example, organic fragment -O-C(=O)-O-. When a carbonate is used as a Markush group (i.e., a substituent), one of the single-bonded oxygen atoms of the carbonate functional group is attached to the Markush formula to which it is linked, and the other is linked to the carbon atom of the organic moiety, as described previously for the organic moiety linked with an ester functional group. In such cases, the carbonate is an example of an O-linked substituent.

The term carbamate or urethane as used herein means a substituent, moiety or group that contains a carbamate functional group represented by -OC(=O)N(R ^a )- or -OC(=O)N(R ^a )2 , and include OC(=O)NH (optionally substituted alkyl) or OC(=O)N(optionally substituted alkyl)2, which are typical carbamate substituents, where R ^a and optionally substituted alkyl are independently selected, where R ^a , independently selected is hydrogen, a protecting group, or an organic moiety, wherein the organic moiety is as described herein for an organic moiety attached to an ester functionality, and is typically an optionally substituted alkyl. Typically, the carbamate groups used herein are formed by or consist of an organic moiety independently selected from R ^a , wherein the organic moiety is as described herein for an organic moiety linked to an ester functional group linked through the structure -OC(= O)-N(R ^a )-, in which the formed group has the formula of an organic fragment -OC(=O)-N(R ^a )- or an organic fragment -OC(=O)-N(R ^a )-. When a carbamate is used as a Markush group (i.e., a substituent), the single-bonded oxygen (O-linked) or nitrogen (N-linked) of the carbamate functional group is added to the Markush formula to which it is linked. The linkage of the carbamate substituent is indicated either explicitly (N- or O-linked) or implicitly in the context to which the substituent relates. The O-linked carbamates described herein are examples of monovalent O-linked substituents.

The term antibody, as used herein, is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity, provided that the fragment the antibody has the required number of attachment sites for the drug linker. The native form of the antibody is a tetramer and consists of two identical pairs of immunoglobulin chains, each having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) together are primarily responsible for binding to the antigen. The light chain and heavy chain variable domains consist of a framework region interrupted by three hypervariable regions, also called complementarity determining regions or CDRs. Constant regions can be recognized by and interact with the immune system (see, for example, Janeway et al., 2001, Immunol. Biology, 5th Ed., Garland Publishing, New York). The antibody can be of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibody can be obtained from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody may be, for example, human, humanized or chimeric. An antibody or antibody fragment thereof is a typical targeting agent that corresponds to or is included in the LDC of the present invention as a ligand unit from the antibody.

In some aspects, the antibody selectively and specifically binds to an epitope on hyperproliferating cells or hyperstimulated mammalian cells (i.e., pathological cells), where the epitope is preferentially displayed or more characteristic of pathological cells as opposed to normal cells, or is preferentially displayed or more characteristic of normal cells near pathological cells, in contrast to normal cells, which are not located in pathological cells. In these aspects, mammalian cells are typically human cells. Other aspects of antibodies included in ligand units are described in embodiments for drug-ligand conjugates.

The term monoclonal antibody, as used herein, refers to an antibody derived from a population of substantially hydrogen antibodies, that is, the individual antibodies within the population are identical except for naturally occurring mutations that may be present in small quantities. Monoclonal antibodies are highly specific because they are directed against a single region of the determinant. The definition of monoclonal indicates that the antibody is derived from a substantially homogeneous population of antibodies; and should not be construed as requiring antibody production in a particular manner.

Cytotoxic activity, as used herein, refers to the cytotoxic effect of a drug compound, a drug-ligand conjugate, or an intracellular metabolite of a drug-ligand conjugate. Cytotoxic activity can be expressed as an IC ₅₀ value, which is the concentration (molar or mass) per unit volume at which half the cells survive.

- 14 046150

The expression cytostatic activity, as used herein, refers to the antiproliferative effect of a drug compound, a drug-ligand conjugate, or an intracellular metabolite of a drug-ligand conjugate, which is independent of cell death, but the effect of which is due to the inhibition of cell division of hyperproliferating cells, hyperstimulated immune cells or other abnormal or unwanted cells.

The terms specific binding and specifically bind mean that the antibody or ligand unit of an antibody in an LDC as a targeting moiety is capable of binding in a selective or highly selective manner to a corresponding target antigen rather than to a variety of other antigens. Typically, the antibody or antibody derivative binds with an affinity of at least about 1 x 10 ^-7 M, and preferably from 10 ^-8 M to 10 ^-9 M, 10 ^-10 M, 10 ^-11 M or 10 ^-12 , and binds to a predetermined antigen with an affinity that is at least two times greater than its binding affinity to a nonspecific antigen (eg, BSA, casein) other than a closely related antigen.

The term ligand drug conjugate or LDC, as used herein, refers to a construct consisting of a ligand unit of a targeting agent and a drug unit containing a quaternized tertiary amine (D'). structurally corresponding to a drug compound containing a tertiary amine, which are linked to each other through a linker unit, where the LDC selectively binds to the target moiety through its targeting ligand unit. In some cases, the term LDC represents multiple (i.e., composition) individual LDC compounds, differing primarily in the number of D+ units associated with each ligand unit and/or the location on the ligand unit to which the D+ units are associated. In other cases, the term LDC is applied to a single member or compound of a composition.

The term targeting agent as used herein is a group corresponding to or included as a ligand unit in a drug-ligand conjugate such that the ligand unit is a targeting moiety of the conjugate that is capable of selectively binding to a commonly present target moiety on , in or near hyperproliferating cells, hyperstimulated immune cells, or other pathological or unwanted cells, compared to other fragments present on, in or near normal cells, where these pathological or unwanted cells are not normally present. Sometimes the target fragment is present on, in or near the pathological ones in greater numbers compared to normal cells or the environment of normal cells, where pathological cells are not usually present. In some cases, the targeting agent is an antibody that specifically binds to an available antigen that is characteristic of the pathological cell, or is an available antigen that is specific to the environment in which the cells reside. In other cases, the targeting agent is a ligand that specifically binds to or is abundantly present on an accessible receptor specific to pathological cells or other unwanted cells, or is an accessible receptor specific to cells in the environment in which the pathological cells are found. Typically, the targeting agent is an antibody, as defined herein, that selectively binds to a target fragment of a pathological or unwanted mammalian cell, more typically to a target fragment of a pathological or unwanted human cell.

The terms target cells or target cells as used herein are putative cells (ie, pathological or other unwanted cells) that are intended to interact with LDC to inhibit proliferation or other unwanted activity of the intended cells. In some cases, the target cells are hyperproliferating cells or hyperactivated immune cells, which are examples of pathological cells. Typically, these pathological cells are mammalian cells and, more typically, human cells. In other cases, the target cells are in close proximity to pathological or unwanted cells, such that exposure of neighboring cells to LDC has the expected effect on the pathological or unwanted cells. For example, the adjacent cells may be epithelial cells, which are characteristic of the abnormal vasculature of the tumor. Targeting these vascular cells with LDC will have a cytotoxic or cytostatic effect on these cells by inhibiting the delivery of nutrients to pathological tumor cells, or indirectly have a cytotoxic or cytostatic effect on pathological cells and/or have a direct cytotoxic or cytostatic effect on pathological cells by releasing D+ in the form of tubulisin drug compound (D) in close proximity to these cells.

A target fragment, as used herein, is a fragment predominantly recognized by a targeting agent or ligand unit of a drug-ligand conjugate, corresponding to or including that targeting agent.

- 15 046150 agent (that is, selectively bound by a ligand unit) and present on, within or near the target cells. Sometimes the target fragment is an antigen that is available for selective binding by an antibody, which is a typical targeting agent that matches or is included in the LDC as an antibody ligand unit. In these cases, such antigen is a cell surface protein present on pathological cells or other unwanted cells, or is present on cells that are specific to the environment in which pathological or unwanted cells are found, such as vascular cells that are characteristic of a hyperproliferating environment cells in the tumor. More typically, an antigen is a cell surface protein of a pathological cell or other unwanted cell that is capable of internalization upon binding to its cognate targeting agent or fragment. In other cases, the targeting agent is a ligand for an extracellularly accessible cell membrane receptor that can be internalized upon binding of a targeting moiety or is capable of passive or facilitated transport of LDCs by targeting a cell surface receptor such that the receptor is a targeting moiety. In some aspects, the target moiety is present on pathological mammalian cells or on mammalian cells characteristic of the environment of such pathological cells.

Antibody-drug conjugate or ADC as the term used herein refers to a drug-ligand conjugate wherein the targeting agent corresponding to or included in its ligand unit is an antibody, thereby defining the ligand unit of the antibody, wherein the ligand the antibody unit is covalently attached to the quaternized drug compound (D+) unit, usually through a linker intermediate. Often the term refers to a population (i.e., population or plurality) of conjugate compounds containing the same antibody ligand units, quaternized drug units, and linker units, but having a variable loading and/or distribution of linker-drug moieties for each antibody (such as when the number of quaternized drug units (D+) of any two ADC compounds in many such compounds is the same, but the location of their attachment sites on the targeting moiety is different). In such cases, the ADC is described by the average drug loading of the conjugate compounds. An ADC prepared by the methods described herein has the general structure Ab-LB-LO-D ⁺ , where -LB-LO- defines a linker unit, in which LB is a covalently binding ligand moiety or a covalently binding ligand unit, sometimes referred to as a major linker (LR), so named because this fragment or unit must be present in the ADC linker unit, and LO is a secondary linker susceptible to enzymatic (eg, protease or glycosidase) or non-enzymatic (eg, reductive or hydrolytic) cleavage. In some cases, such cleavage is enhanced in the environment of pathological cells or occurs after intracellular internalization of the ADC following binding of the ligand arm of the targeting antibody of the ADC to its cognate antigen; D ⁺ is a quaternized drug unit and is typically formed by the quaternization of a drug compound containing a tertiary amine (D), or corresponds to the quaternized of D, where D ⁺ is released as a tertiary amine drug compound as a result of this enzymatic or non-enzymatic effects on L _O .

The average number of quaternized drug units per ligand unit from an antibody or fragment thereof in an ADC composition (i.e., the average number for a population of ADC conjugate compounds that differ primarily in the number of conjugated quaternized drug units per ligand unit from an antibody in each of the ADC compounds, that are present in this population, and/or by their location), when the linker units are not branched, is denoted by p, or, when the linkers are branched, p is the average number of linker drug moieties attached to a ligand unit from the antibody. In any context, p is a number ranging from about 2 to about 24, or from about 2 to about 20, and is typically about 2, about 4, or about 8. In other contexts, p is the number of units of the quaternized drug compound when the linker units are unbranched, or the number of linker moieties of a quaternized drug compound, when the linker units are branched, which are covalently linked to a single ligand unit from an antibody in an ADC in a population of compounds of antibody-drug conjugates, in which the compounds of this population may primarily vary in number and /or the arrangement of conjugated units of the quaternized drug compound or linker fragments of the quaternized drug compound in each of the ADC compounds. In this context, p is denoted p' and denotes an integer ranging from 1 to 24 or 1 to 20, typically 1 to 12 or 1 to 10, and more typically 1 to 8.

The average number of quaternized drug units per ligand unit produced by the conjugation reaction can be characterized by conventional means, for example, mass spectroscopy, ELISA, HIC and/or HPLC analysis. The quantitative distribution of conjugate compounds in terms of p' can also be determined. In some cases, separation, purification and character

- 16 046150 The differentiation of homogeneous compounds of drug-ligand conjugates in which p' is a specific value in the drug-ligand conjugate composition from those having other drug loads can be achieved by methods such as reversed-phase HPLC or electrophoresis.

An antigen is an entity that is capable of selectively binding to an unconjugated antibody or fragment thereof or to an ADC containing an antibody ligand unit corresponding to or including the antibody or fragment thereof. In some aspects, an antigen is an extracellularly accessible cell surface protein, glycoprotein, or carbohydrate that is preferentially cleared by pathological or other unwanted cells over normal cells. In some cases, the unwanted cells bearing the antigen are hyperproliferating mammalian cells. In other cases, the unwanted cells bearing the antigen are hyperactivated immune cells of the mammal. In other aspects, the specifically bound antigen is present in a particular environment of hyperproliferating cells or hyperactivated immune cells of a mammal, different from the environment that normally affects normal cells in the absence of such pathological cells. In other aspects, a cell surface antigen is capable of internalization upon selective binding of an ADC compound and is associated with cells that are specific to the environment in which hyperproliferating or hyperstimulated immune cells are found in the absence of such pathological cells. An antigen is a typical LDC target fragment, wherein its targeting ligand unit is or includes an antibody to the target antigen and is capable of preferentially recognizing that antigen through selective binding.

Antigens associated with hyperproliferating cells accessible to cell surface ADCs include, by way of example, but not limitation, CD19, CD70, CD30, CD33, NTBA, ave6, and CD123.

A ligand unit is a moiety comprising a drug-ligand conjugate that is capable of selectively binding to its cognate targeting moiety and is sometimes referred to as an LDC targeting moiety. The ligand unit includes, but is not limited to, targeting agents such as receptor ligands, antibodies to cell surface antigens, and transporter substrates. Sometimes the receptor, antigen, or transporter that is supposed to bind to LDC is present in greater numbers on pathological cells as opposed to normal cells. In other cases, the receptor, antigen or transporter that should bind to LDC is present in greater abundance in normal cells that are intrinsic to the environment of pathological cells as opposed to normal cells in the periphery. Various aspects of the ligand units are further described by embodiments of the invention.

A covalently binding ligand moiety or covalently binding ligand is a fragment or linker unit (LU) component of an LDC that is covalently attached to a ligand unit that targets pathological or unwanted cells or their environment, as well as to the remainder of the linker unit and is formed as a result interaction of the corresponding LB' fragment or component in the linker precursor with the targeting agent. For example, when L _B ' consists of a maleimide moiety, interaction of this moiety with a reactive sulfhydryl group of the targeting agent converts L _B ' to L _B which consists of a thio-substituted succinimide moiety linked to a ligand unit that represents or includes the targeting agent. In another example, when L _B ' consists of an activated carboxylic acid functional group, interaction of this functional group with the epsilon-amino group of lysine in the targeting agent converts the functional group into an amide, where this amide includes the LB moiety covalently attached to the ligand unit, which is or includes this targeting agent. Other L _B fragments or units and their conversion from LB'-containing fragments or units are described in embodiments of the invention. In some cases, the targeting agent is derivatized with a bifunctional molecule to produce an intermediate that is fused to a covalently binding ligand (L _B ') moiety or precursor unit. As a result of this condensation, the L _B fragment or unit thus formed contains atoms belonging to the bifunctional molecule and L _B '.

A covalently binding ligand moiety precursor or a covalently binding ligand moiety precursor is a fragment or component of a linker unit or a substructure thereof used in the preparation of a linker unit that is capable of covalently binding to a targeting agent in the process of preparing an LDC, followed by a ligand binding (LB') moiety or the precursor unit is converted into a covalently ligand binding (LB) moiety or unit covalently attached to a ligand unit corresponding to or including the targeting agent. In some aspects, the LB' moiety typically has a functional group capable of reacting with a nucleophile or electrophile native to the antibody or fragment thereof, or being introduced into the antibody through chemical transformation or genetic engineering. In some aspect, the nucleophile is the N-terminal amino group of a peptide-containing antibody or epsilon

- 17 046150 lone-amino group of the lysine residue of the antibody. In other aspects, the nucleophile is a sulfhydryl group of a cysteine residue of an antibody introduced by genetic engineering or chemical reduction of an interchain disulfide of the antibody. In some aspects, the electrophile is an aldehyde introduced by selective oxidation of a carbohydrate moiety of an antibody, or is a non-natural amino acid ketone introduced into an antibody using a genetically engineered tRNA/tRNA synthetase pair. These and other methods are reviewed by Behrens and Liu in Methods for site-specific drug conjugation to antibodies mAB (2014) 6(1): 46-53.

A linker unit, as that term is used herein, refers to an organic moiety in a drug-ligand conjugate (LDC) that is inserted and covalently attached between a quaternized drug unit (D+) and a ligand unit, or an organic moiety connecting a drug compound with a linker embedded and covalently attached between a quaternized drug compound unit (D+) and a fragment or precursor unit of a covalently binding ligand (LB'). Typically, the linker unit (LU) of an LDC or drug-linker compound consists of a covalently binding ligand moiety or unit ( _LB ) or a covalently binding ligand precursor moiety or unit (LB'), respectively, and a secondary linker (LO) as described herein document. In some aspects, the precursor or covalently binding ligand unit contains a maleimide (M ¹ ) group. The attachment of the targeting agent through M ¹ , leading to the covalent attachment of the corresponding ligand unit to the linker unit, occurs through the sulfhydryl group of the cysteine of the targeting agent by Michael addition of the sulfur atom of the sulfhydryl group to the maleimide ring system of M ¹ . This addition results in a succinimide (M ² ) moiety having a sulfur-substituted succinimide ring system. Subsequent hydrolysis of this ring system, either spontaneously or under controlled conditions, such as when the system is part of a self-stabilizing linker (LSS) moiety, results in the formation of a succinic acid-amide moiety ( ^M3 ), which is an example of a self-stabilizing moiety (LS), described below. Also covalently linked to LB or LB', which is the main linker (LR fragments), is a secondary linker moiety (LO), which is further inserted between the ligand unit and the quaternized drug unit (D+) in the LDC or between LB' and D+ in the compound a drug compound with a linker, wherein the covalent binding to the LR occurs through an intermediate ether, ester, carbonate, urea, disulfide, amide or carbamate functional group, more typically through an ether, amide or carbamate functional group.

A parallel link unit, as used herein, refers to a branched linker unit component that connects the PEG unit in a parallel orientation to the quaternized drug compound (D+) unit. As used herein, parallel orientation, parallel arrangement, parallel connection, and similar terms refer to a configuration in which parallel, or parallel-oriented, or parallel-connected components are connected to a parallel link ( _LP ) such that each component has one end tied to L _P and one free end. Typically, L _P connects a quaternized drug unit (D+) through one or more linker unit components, such as AO-WY- or AO-Y(W')-, where AO _-Y (W')- is optionally present, and a PEG unit, so that the quaternized drug compound and PEG units are arranged in parallel, as a result of which the hydrophobicity of the quaternized drug compound unit is effectively masked by the PEG unit. Only those PEG units that are necessary to mask hydrophobicity for a given LU-D+ moiety (i.e., the quaternized drug moiety with the linker) need to be oriented parallel to the quaternized drug unit, which does not necessarily require that all quaternized drug units and polyethylene glycol ( PEG) links connected to LP must be parallel to each other. Thus, one PEG unit can effectively mask the hydrophobicity of 1, 2, 3, 4 or more units of a quaternized drug compound, typically 1 to 4 D+ and more typically 1 or 2 D+.

The term parallel is used herein to denote the branching of two components of a drug-ligand conjugate (LDC) or drug-linker conjugate from L _P that contains the LDC or drug-linker conjugate, and is not used to indicate that the two components are located side by side in space or have the same distance between them over some or all of their length. An LDC or drug-linker compound having a PEG unit that is in parallel orientation with respect to the quaternized drug unit or drug-linker linker refers to an LDC or drug-linker linker containing a PEG unit, one end of which is connected to a component of a linker unit (ie, a parallel connecting unit) and one or more free unlinked ends (or ends). The free unbonded end of the PEG unit when attached to L _P can, when

- 18 046150 take the form, for example, of an unreacted functional group, for example an alkoxy, carboxylic acid, alkylene carboxylic acid, alcohol or other functional group. In cases where the parallel oriented PEG component is itself branched and therefore has multiple ends, it still only has one end connected to L _P . The parallel orientation of the PEG unit with respect to the D+ also minimizes the number of atoms between the ligand unit and the drug unit, since the PEG unit atoms are not located between the D+ and the ligand unit.

An example of a graphical representation of an LDC having a PEG unit that is in a parallel (i.e., branched) orientation with respect to the quaternized drug compound unit is the following:

C(O)CH ₂ CH ₂ (OSN ₂ CH ₂ ) _p PSN ₃

Ligand------Linker----D ⁺ _f where the subscript n' ranges from 1 to 24.

The expression main linker, as used, is a covalently binding ligand (LB) moiety or unit, or a precursor of a covalently binding (LB') ligand moiety or unit, and is present as a component of a linker unit in an LDC or as a component of an LB'-containing moiety , such as LB'-LO- or LB'-LO-D ⁺ , in connecting the drug compound to the linker. The main linker LB' consists of a reactive functional group that can interact with an electrophilic or nucleophilic functional group of the targeting agent. As a result of this interaction, the targeting agent becomes covalently bound as a ligand unit to the LB of the main linker through a functional group derived from the reactive functional group of LB'.

A secondary linker moiety, as used herein, refers to an organic moiety in a linker unit where the secondary linker (LO) is covalently attached to the LB or L _B ' moiety (i.e., the main linker unit) by interposing a functional group between the L _B moiety or L _B ' and the remainder of a linker unit to which a quaternized drug compound unit can be covalently attached. In an LDC, a secondary linker is also covalently attached to a quaternized drug unit (D ⁺ ) through the benzyl position of a self-disrupting PAB moiety or a PAB-type that contains a self-disrupting spacer unit. In addition to such a spacer unit (Y), secondary linkers consist of a cleavable unit (W or W'), where W, Y and D ⁺ or W', Y and D ⁺ are arranged in either a linear or orthogonal relationship, respectively, and additionally consist of a fragment -LP(PEG)- and a first optional extension unit (A) and/or a branching unit, in which the latter can be replaced by a second optional extension unit (AO) when the LU is attached to only one unit of the quaternized drug compound (D ⁺ ). When present, A binds LB' or an LB derived from it to a secondary linker residue via -LP(PEG)- or via AO, or B, when either present or binds D ⁺ and -L _P (PEG)-via -WY - or -Y(W')- when B and AO are absent.

In LDC, LO consists of a self-destructive spacer unit (Y) that contains a self-destructive moiety and is covalently attached to a cleavable unit (W or W'), such that cleavage of W or W', under the conditions to which pathological cells are more likely to be exposed, results in self-destruction of the self-degrading fragment with concomitant release of D ⁺ as a drug compound (D). Alternatively, this shedding may occur in close proximity to these pathological cells compared to normal cells in their normal environment. Typically, this self-destruction occurs through 1,6-elimination in the self-destruction fragment, as described herein. In these cases, the self-destructive fragment of the self-destructive spacer unit is attached to a drug compound containing a tertiary amine by quaternizing the tertiary amine nitrogen of the drug compound.

The secondary linker ( _LO ), when linked to a D ⁺ in a linker unit attached to only one D ⁺ , is usually represented by structure (1) or (2)

P.E.G. I.

-ξ-Aa—Lp—Ao W _w --Y _y D

P.E.G.W.

-§-A _a --L _P -A ₀ ---Y _y -D where the variable groups are as defined herein. In some aspects of the invention, the Y in structure (1) is formed or consists of a self-interrupting moiety (SI) described herein, substituted with W and D ⁺ . In other aspects of the invention, Y in structure (2) is formed or consists of a self-destructive moiety as described herein, substituted with D ⁺ through the quaternary nitrogen of the amino group of the tubulisin compound, and additionally for

- 19 046150 placed W' and ligand -L _B -A _a -LP(PEG)-A _O - or L _B '-A _a -L _P (PEG)-AO- in a conjugate of a drug compound with a ligand or a connection of a drug compound with a linker, respectively, where AO is optionally present (that is, AO is linked to the self-destructive moiety Y when AO is present), or is further substituted by L _B -A _a -L _P (PEG)- or L _B ' -A _a -L _P (PEG ) - in a drug-ligand conjugate or a drug-linker conjugate, respectively, when AO is absent.

Typically, secondary linkers with structure (1) are represented by

R'

Y(=S1) and secondary linkers with structure (2) are presented

R' where Y is a self-destructive fragment of the PAB or PAB type, and E, J/J', V, Z ¹ , Z ² , Z ³ , R', R ⁸ and R ⁹ are as defined in the embodiments for self-destructive fragments of type PAB or PAB.

A maleimide moiety, as used herein, is a covalently binding ligand precursor moiety having a maleimide ring system. The maleimide moiety (M ¹ ) as LB' is capable of participating in Michael addition (i.e., 1,4-conjugate addition) of the thiol functionality of the targeting agent to produce the thio-substituted succinimide (M ² ) moiety described herein, which becomes component in the linker unit in LDC. The M ¹ moiety is attached to the linker moiety of the drug-linker compound via its imide nitrogen, converting it to a thio-substituted succinimide moiety. Apart from the imide nitrogen, the M ¹ moiety is usually unsubstituted, but can be asymmetrically substituted at the cyclic double bond of its maleimide ring system. Such substitution typically results in a regiochemically preferential addition of the sulfhydryl group sulfur atom to the less hindered or more electron-deficient double bond carbon (depending on the more dominant contribution) of the maleimide ring system. Controlled hydrolysis of the succinimide ring system of a thio-substituted succinimide moiety M ² derived from such a substituted M ¹ moiety, if present in the LDC self-stabilizing linker (LSS) moiety, is expected to provide or may provide regiochemical isomers of succinic acid-amide (M ³ ) moieties in form LB in a self-stabilized linker (L _S ) fragment, the relative amount of which is due to differences in the reactivity of the two carbonyl carbon atoms M ² related to the substituent(s) present in the precursor M ¹ .

A succinimide moiety, as used herein, is an organic moiety of the linker unit of a drug-ligand conjugate and results from the Michael addition of a thiol functional group of a targeting agent to the maleimide ring system of the maleimide moiety (M ¹ ) as LB', where M ¹ is typically is the connection of a drug compound with a linker. In some aspects, the drug-ligand conjugate is a drug-antibody conjugate, and the thiol functional group is derived from a cysteine residue of the antibody or a fragment thereof. Thus, the succinimide (M ² ) moiety as LB consists of a thio-substituted succinimide ring system and contains an imide nitrogen substituted by a linker moiety and is optionally substituted by the substituent(s) present in the M ¹ precursor. Typically, when A is present (ie, the subscript a for Aa is 1), the imide nitrogen is covalently attached to the extender unit (A) or a subunit thereof (ie, A1), as described herein. Sometimes M ² -A (or M2-A1) refers to the self-stabilizing linker (L _SS ) moiety described herein.

A succinic acid-amide moiety, as used herein, refers to a succinic acid having an amide substituent that is formed from the thio-substituted succinimide ring system of the M ² succinimide moiety as LB that has undergone cleavage of one of its carbonyl nitrogen bonds by hydrolysis. When hydrolysis of an ADC results in the formation of a succinic acid-amide (M ³ ) moiety, the linker unit is less susceptible to premature loss of its ligand unit from an antibody having this M ³ moiety by eliminating the thio substituent of the antibody. When present in a self-stabilizing linker (LSS) moiety, controlled hydrolysis of the succinimide ring system of the thio-substituted succinimide moiety ^M2 derived from the substituted ^M1 moiety as _LB ' is expected to provide regiochemical isomers of the ^M3 moieties (from

- 20 046150 specifically designated as M ^3A and M ^3B ), which are due to differences in the reactivity of the two carbonyl carbon atoms of M ² related to the substituent(s) that was present in the precursor M ¹ and the thio substituent of the ligand unit, which is formed from antibody or corresponds to a targeting agent from an antibody.

A self-stabilizing linker, as used herein, is an LB-containing fragment of an LDC linker unit or a precursor thereof (i.e., an LB'-containing fragment) that, under controlled conditions, is capable of undergoing chemical conversion to a self-stabilizing linker (L _S ), such that LDC , initially composed of a self-stabilizing protein (LSS), becomes more resistant to premature loss of its targeting ligand unit. Typically, the LSS moiety, in addition to the L _B or L _B ' moiety, consists of a first extension unit (A) to which the LSS and -L _P (PEG)- are covalently attached. However, sometimes intermediate A is missing and LSS is covalently attached directly to -L _P (PEG)-. In some aspects, the LSS, prior to its incorporation into the LDC, contains a maleimide moiety (M ¹ ) as its L _B ' moiety (through which the targeting agent is to be attached as a ligand unit) and a first extender unit (A) or subunit thereof (i.e., A1 ) and is represented by the formula M1-A- or M1-A1-. After incorporation into the LDC (that is, after attachment of the targeting moiety as a ligand unit via Michael addition to the maleimide moiety), the M ¹ -A- (or M ¹ -A1 -) moiety in the LSS is converted to its corresponding thio-substituted succinimide moiety M2-A- (or M2-A1-). Typically, the LSS also consists of a base unit (BU), as described herein, and is typically a substitute for an extender unit associated with M ² or its precursor M ¹ . In these aspects, BU promotes the hydrolysis of the succinimide moiety M ² to its corresponding ring-open form M ³ [i.e., M ² -A(BU)- or M ² -A1(BU)- becomes M ³ -A(BU) or M ³ -A1(BU)].

A self-stabilized linker is an organic moiety formed from an LSS moiety, both of which are LB-containing moieties, into an LDC that has been hydrolyzed, usually under controlled conditions, providing a new LB-containing moiety that is less likely to reverse the targeting condensation reaction agent with an LB'-containing fragment, which would give the original LB-containing fragment. Typically, the self-stabilizing linker (L _S ) consists of an extender unit or subunit thereof covalently attached to a moiety resulting from the conversion of a succinimide moiety (M ² ) by hydrolysis of its succinimide ring system. In these cases, the M ² precursor of this moiety has a thio-substituted succinimide ring system resulting from the Michael addition of the targeting agent's thiol functional group to the M ¹ maleimide ring system, such that the moiety derived from M ² (M ³ ) has reduced elimination reactivity its thio substituent compared to the corresponding substituent in M ² . In these aspects, the moiety derived from M ² has the structure of the succinic acid-amide moiety (M ³ ) corresponding to M ² , where M ² has undergone hydrolysis of one of its carbonyl-nitrogen bonds in its succinimide ring system. This hydrolysis can occur spontaneously or is typically catalyzed by the BU core functional group. For this purpose, BU is covalently attached to the M ² -linked extender unit such that BU is in appropriate proximity as a result of this attachment to promote carbonyl-nitrogen cleavage. Thus, the product of this hydrolysis has a carboxylic acid functional group and an amide functional group substituted on the amide nitrogen atom, where this nitrogen atom corresponds to the imide nitrogen atom in the M ² -containing LSS precursor in the structure of the above extender unit. Typically this major functional group is an amino group whose effectiveness in increasing the rate of hydrolysis to convert M ² to M ³ is controlled by pH. Thus, a self-stabilizing linker (LS) typically has an M ³ structure covalently linked to an extension unit or subunit thereof, which in turn is covalently linked to the remainder of the secondary linker LO (LO') in a linear arrangement and has a base unit covalently associated with the expanding link (A) in an orthogonal arrangement relative to A and LO'. LS with M ³ , A, BU and LO arranged as indicated, is represented by the formula M ³ -A(BU)-LO- or M ³ -A ₁ (BU)-L _O- .

After hydrolysis, the resulting self-stabilized linker ( _LS ) will typically have an M ³ structure covalently linked to a BU-substituted extender unit (eg, M ³ -A(Bu) or M ³ A1(BU)-). This first extender unit is in turn covalently linked to the LO moiety in a linear arrangement with the main unit orthogonal to M ³ and the other units of the LO component. Examples of LSS and LS fragment structures with M ² or M ³ , A(BU) [or A ₁ (BU)| and LO'-, where LO'- represents the residue LO-, arranged in the manner indicated, are shown by way of example and not limitation:

- 21 046150

where the fragment -CH(CH2NH2)C(=O)- shown is the structure of the first extender unit (A) or subunit thereof covalently linked to the imide or amide nitrogen M2 or M ³ , respectively, where the fragment -CH2NH2 is the BU substituent of this extender link The remainder of the LSS and LS structures is a succinimide fragment M2 and a succinic acid-amide fragment ^M3 resulting from hydrolysis of the succinimide ring of M2, respectively, where M2 and ^M3 are replaced at the imide and corresponding amide nitrogen, respectively, by the sp ³ carbon of the extender unit. The wavy line indicates the point of covalent attachment to the ligand unit as a result of Michael addition of the thiol functionality of the targeting agent to the maleimide ring system M ¹ , and the asterisk indicates the point of covalent attachment to D ⁺ . Since the succinimide ring system ^M2 is asymmetrically substituted due to its thio-substitution by the ligand unit, regiochemical isomers of succinic acid-amide fragments ( ^M3 ) can be obtained, which differ in the position of the ligand unit relative to the carboxylic acid group released by hydrolysis of M2. In the above structures, the carbonyl functionality of the shown extender unit is an example of a hydrolysis enhancer (HE) as defined herein that is included in the structure of such unit.

M ³ -A(BU)- are examples of self-stabilized linker (LS) structures because these structures are less likely to eliminate the thio substituent of the targeting ligand moiety and thus cause loss of the targeting moiety from the drug-ligand conjugate compared to the corresponding M2 structure -A(BU)- LSS fragment. Without being limited by theory, it is believed that the increased stability results from greater conformational flexibility in M ³ compared to M ² , which no longer restricts the thio substituent to a conformation favorable for E ² elimination.

The backbone unit, as used herein, is an organic moiety in the self-stabilizing linker (L _SS ) described herein that can be transferred to the corresponding _LS by hydrolyzing the succinimide ring system in the M ² moiety including the LSS (i.e. catalyzed by the addition of water, the addition of a water molecule to one of the succinimide carbonyl-nitrogen bonds) and can be initiated or enhanced under controlled conditions suitable for the targeting ligand unit attached to the LSS. To this end, the backbone functional group (BU) and its relative position in the LSS relative to its M ² component are in some aspects selected for its ability to form a hydrogen bond with the M ² carbonyl group, which effectively increases its electrophilicity and hence sensitivity to exposure to water. In another aspect, this choice is made in such a way that the water molecule, the nucleophilicity of which is increased by hydrogen bonding with the main functional group BU, is directed towards the carbonyl group of M ² . In the third aspect, this choice is made in such a way that the basic nitrogen, when protonated, increases the electrophilicity of the succinimide carbonyls due to the induced electron-withdrawing ability. In the latter aspect, some combination of these mechanisms promotes catalysis in the hydrolysis of LSS to L _S .

To increase the electrophilicity of carbonyl M2 through hydrogen bonding, BU must have a primary or secondary amine as its main functional group, since increasing the nucleophilicity of water or the electrophilicity of a carbonyl in the manner described above can be done with a primary, secondary or tertiary amine as the main functional group. In order for the basic amine to be in the required proximity to promote hydrolysis of the succinimide moiety M ² to the corresponding carboxylic acid amide M ³ corresponding to its open ring by either of these mechanisms, the amine-containing carbon chain BU is typically attached to the extender unit LSS at the alpha carbon of this fragment relative to the point of attachment of A (or A1) to the succinimide nitrogen of M2 (and therefore to the maleimide nitrogen of the structure of its corresponding precursor M ¹ -A or M ¹ -A1). In some aspects, the alpha carbon has the (S) stereochemical configuration or configuration corresponding to the alpha carbon of L-amino acids.

The term hydrolysis enhancer unit as used herein is an electron-withdrawing group or moiety and is an optional substituent of an extender unit containing an LSS moiety. When present, a hydrolysis enhancer (HE) unit is typically included in an extender unit that is bonded to the imide nitrogen of the ^M2 moiety, such that its electron-withdrawing effects increase the electrophilicity of the succinimide carbonyl groups in this moiety

- 22 046150 mente. When the extending unit also has a BU substituent, the action of HE on carbonyl groups in combination with the action of BU, which depends on the basicity of the main functional group BU and the distance between this functional group and this carbonyl, is balanced such that premature hydrolysis of M ¹ or M ² to M ³ does not occur to a significant extent, which would require an excessive excess of coupling of the drug compound to the linker to produce LDC from the LSS precursor having the structure M'-A(BU)-. but allows hydrolysis (ie, conversion of the LDC-containing moiety -M ² - to its corresponding moiety -M ³ -A(BU)-) under controlled conditions (eg, when the pH is deliberately elevated) that is permissible for the attached targeting ligand unit. Typically, the HE unit is a carbonyl or carbonyl-containing functional group located remote from the end of an extender unit that is linked to M ² or an M ³ derived therefrom such that HE is covalently attached to the extender unit and to the secondary linker residue. Carbonyl-containing functional groups other than ketone (ie HE is -C(=O)-) include esters, carbamates, carbonates and ureas. When HE is a carbonyl-containing functional group other than a ketone, the carbonyl moiety of that functional group is typically bonded to A. In some aspects, the HE unit may be sufficiently distant in the extender unit from the imide nitrogen to which the extender unit is covalently bonded such that there is no noticeable effect on The hydrolytic sensitivity of the succinimide carbonyl-nitrogen bonds in the ^M2 -containing fragment is not observed.

A extender unit, as the term is used herein, refers to an organic moiety in a secondary linker that physically separates the targeting moiety from other intermediate components of the linker that are located distal to the extender unit, such as the cleavage unit and/or or spacer link. A first extension unit (A) and/or a second extension unit (AO) may be required when L _B and/or the -L _P (PEG) moiety provide insufficient steric relief to allow effective processing of the linker unit at W or W' for release of D+ as a tubulisin drug compound, and/or are sometimes included for ease of synthesis when constructing the linker unit of the present invention. The first or second expansion link may comprise one or more expansion links described herein. Before inclusion in the LDC, A has functional groups capable of covalently linking LB' to -L _P (PEG)-, and AO has functional groups capable of covalently linking together -L _P (PEG)- and the cleavage unit (W) in some linker constructs (as with A, W and Y in a linear arrangement, i.e. -AYW-), or AO has functional groups capable of covalently linking together -L _P (PEG)- and the spacer unit (Y) in other linker constructs (as with W ' orthogonal to A and Y, that is -AY(W')-). In some aspects of the invention, the secondary linker is attached to the LB or LB' moiety through a first extension unit while another of its subunits is covalently linked to -LP(PEG)-.

The first extending unit (A), when present in an LDC or in a drug-linker combination, is typically found in linker units having the formula -AL _P (PEG)-WY-, -AL _P (PEG)-A _O -WY- , AL _p (PEG)-Y(W')- or AL _P (PEG)-AO-Y(W')-, in which A is attached to a covalently binding ligand unit or a precursor of a covalently binding ligand unit. The first or second expansion unit may contain or consist of two, three or more subunits. Typically, A is one single unit or has 2 to 4 additional individual subunits, designated A ₂ , A _{3 ,} and A4 . In these, Aa is -A1-A2-, -A1-A2-A3- and -A1-A2-A3-A4-, collectively referred to as -A1-A2-4-. Typically, the first extender unit or subunit thereof has from one to six contiguous carbon atoms that are located between the covalently linking ligand unit or precursor of the covalently linking ligand unit and the functional group that covalently attaches A to LP(PEG)- or to another subunit A. B In some aspects, this functional group can also serve as a hydrolysis enhancing (HE) unit.

The term branching unit, as used herein, refers to a trifunctional organic moiety that is an optional component of a linker unit (LU). A branching unit (B) is present when more than one quaternary tubulisin drug unit (D+), typically 2, 3 or 4, is attached to a linker unit (LU) of a drug-ligand conjugate or drug-linker compound. In a conjugate of a drug compound with a ligand of formula 1A, 1B, 1C or 1D or in a conjugate of a drug compound with a linker of formula IA, IB or 1D, the presence of a branching unit is indicated when the index b in B _b is 1, and occurs when the index n is greater than 1 in any of these structural formulas. The branching unit is trifunctional so that it can be included in a secondary linker unit (LO). In aspects where n is 1 in any of these structural formulas, the branch unit is either absent, as indicated when the subscript b is 0, or the subscript b is 1, and B is replaced by a second optional branch unit, referred to as AO. A compound of a drug compound with a linker or a conjugate of a drug compound with a ligand having a branching unit due to a number of D+ units on the LU have a linker

- 23 046150 core units consisting of fragments such as -A _a -L _P (PEG)-BWY- or -A _a -L _P (PEG)-BY(W')-.

In some aspects, a natural or non-natural amino acid or other amino acid compound having a functionalized side chain serves as a branching unit. In some aspects, B is a lysine, glutamic acid, or aspartic acid moiety in an L- or D-configuration in which an epsilon-amino, gammacarboxylic acid, or beta-carboxylic acid functional group, respectively, connects B to the LU residue.

A cleavage unit is defined to provide a reactive site in which the reactivity at that site is greater in or around a hyperproliferating cell or a hyperstimulated immune cell (i.e., a pathological cell) compared to a normal cell, such that action at that site results in a preferential effect on pathological cell tubulisin drug compound (D). This effect results from the possible release of the quaternized tubulisin drug compound (D+) unit as D from the LDC having such a cleavable unit. In some aspects of the invention, the cleavage unit, W or W', includes a reactive site cleavable by an enzyme (i.e., W or W' consists of an enzyme substrate) whose activity or location is greater in or around a hyperproliferating, immunostimulatory, or other pathological or unwanted cell. In other aspects, the cleavable unit consists of a reactive site that is cleaved by other mechanisms (ie, non-enzymatic) more likely to be operative in the environment in or around the pathological cells of the target site compared to the environment of normal cells in which pathological cells are typically absent. In other aspects of the invention, the reactive site is more likely to act upon cellular internalization of the LDC into the pathological cell. This internalization is more likely to occur in these cells compared to normal cells due to the greater presence of the target moiety that is recognized by the target ligand moiety in the LDC on the cell membrane of pathological or unwanted cells. Therefore, target cells are more likely to be intracellularly exposed to the active drug moiety upon release from the LDC. The cleavage unit may contain one or more sites sensitive to cleavage under these conditions for the target site, but typically there is only one such site.

In some aspects of the invention, the cleavable unit is a substrate for a regulatory protease, hydrolase, or glycosidase located intracellularly in target cells (i.e., the reactive site W or W' is a peptide bond or glycosidic bond, respectively, cleaved by a regulatory protease, hydrolase, or glycosidase). In these aspects, the peptide or glycosidic bond is capable of being selectively cleaved by a regulatory protease, hydrolase, or glycosidase over serum proteases, hydrolases, or glycosidases. These regulatory proteases, hydrolases or glycosidases may be more specific to pathological or other unwanted target cells compared to normal cells, or W or W' have the ability to be selectively degraded by a protease, hydrolase or glycosidase secreted in large quantities by pathological or other unwanted target cells by compared to normal cells or normal target cells, which are characteristic of the environment of pathological cells compared to normal cells in the periphery. Alternatively, W provides a functional group that, when incorporated into LDC, is susceptible to the acidic environment of lysozymes where LDC is preferentially internalized into the abnormal cell, or to the more reducing environment in or around these cells compared to the environment of normal cells where abnormal cells are typically absent, such that the release of D+ as a tubulisin drug compound preferentially exposes pathological cells to the drug compound compared to normal cells distant from the location of the pathological cells.

A cleavable unit (W or W') at the junction of a drug compound with a linker or upon its incorporation into an LDC provides a cleavable bond (i.e., a reactive site) that in some aspects, upon the action of an enzyme present in hyperproliferating cells or hyperactivated immune cells, releases the drug compound , containing a tertiary amine, from D+. In other aspects, the releasing enzyme is specific to the immediate environment of these abnormal or unwanted cells. In other aspects, the non-enzymatic action on W, due to conditions more likely to be experienced by hyperproliferating cells compared to normal cells, releases the free tubulisin drug compound from D+. Typically, W or W' provides a cleavable bond that is likely to act intracellularly in a hyperproliferating cell or hyperactivated immune cell due to its preferential entry into such cells over normal cells. Typically, W or W' in an LDC connection or in a drug-linker connection is covalently attached to a spacer unit (Y) having a self-disruption moiety such that enzymatic action on W or W' causes self-destruction of this moiety within -Y-D+ of -WY -D+ or -Y(W')-D ⁺ , respectively, to release D ⁺ as D.

- 24 046150

Functional groups that provide cleavable bonds include, by way of example and not limitation, (a) sulfhydryl groups, forming a disulfide bond that is more susceptible to the more reducing environments of pathological cells compared to normal cells or to excess glutathione produced in hypoxic conditions experienced by such cells, (b) aldehyde, ketone or hydrazine groups, which form Schiff base functional groups, or hydrazone groups, which are more sensitive to the acidic conditions of lysozymes upon selective internalization of LDC having a linker unit with such a cleavable bond, the pathological cell by compared with its internalization by normal cells, (c) carboxyl or amino groups that form an amide bond, as in peptide bonds, more sensitive to enzymatic cleavage by proteases produced or secreted preferentially by pathological cells compared with normal cells, or preferentially secreted by normal cells, which are inherent in the environment of pathological cells, compared with normal cells in the periphery, or by a regulatory protease in the target cell, (d) amino or hydroxyl groups that form the corresponding urea or carbamate groups, or carbonic or hydroxyl groups that form ester or carbonate groups that are more susceptible to enzymatic degradation by hydrolase or esterase produced or secreted preferentially by pathological cells as compared to normal cells, or secreted preferentially by normal cells inherent in the environment of pathological cells as compared to normal cells in the periphery.

Still other functional groups that provide cleavable bonds are found in sugars or carbohydrates that have a glycosidic bond, which are substrates for glycosides that can sometimes be produced preferentially by pathological cells compared to normal cells. Alternatively, the protease, hydrolase or glycosidase enzyme required to process the linker unit to release D' as the active tubulisin drug compound should not be produced preferentially by the pathological cells over normal cells, provided that the effecting enzyme is not secreted by normal cells in those limits that would cause undesirable side effects from premature release of the free drug compound. In other cases, the desired protease, hydrolase or glycosidase enzyme may be excreted from the body, but to avoid undesirable premature release of the drug compound, it is preferable that the enzyme involved is secreted in the vicinity of the pathological cells and remains localized in this environment, whether produced by the pathological cells or by neighboring normal cells. cells in response to an abnormal environment caused by abnormal cells. In this regard, W or W' are selected so that they are preferentially affected by a protease, hydrolase or glycosidase in or within the environment of the pathological cells, as opposed to freely circulating enzymes. In these cases, the LDC is less likely to release the tubulisin drug compound in the vicinity of normal cells distant from the desired site of action, nor is it internalized into normal cells that produce but do not secrete the enzyme of choice, since such cells are less likely to display the target moiety. required for entry into LDCs through selective binding via a targeting ligand moiety.

In some aspects, W is a peptide cleavable unit formed from an amino acid, or formed from or consisting of one or more amino acid sequences that provide a substrate for a protease present in or localized to the pathological cells. Thus, W may be formed by or consist of a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide included in the linker unit through an amide bond with a self-cleaving Y, where this peptide fragment is the sequence recognition for this protease. In some aspects, W is the only naturally occurring L-amino acid, such as L-glutamate.

In some aspects, the W' of a glucuronide unit of formula -Y(W')- is formed or consists of a carbohydrate moiety attached to a self-destructive moiety Y by a glycosidic linkage that is cleaved by a glycosidase preferentially produced by or found in pathological cells in such cells in which LDC, which consists of these self-destructive and carbohydrate fragments, has selective entry due to the presence of the target fragment on pathological cells.

The term spacer unit, as used herein, is an organic moiety in a secondary linker (LO) in a linker unit that is covalently linked to D+ and a second optional extender unit (AO) or branching unit (B), if either is present, or with -Lp(PEG)- if AO and B are absent, and/or with a cleavage unit (W or W'), depending on the configuration of the cleavage unit to the spacer unit relative to each other. Typically in one configuration, the quaternized drug unit (D+) and W' are both covalently linked to Y, which in turn is also linked to A _O or B when present, or to L _P (PEG),

- 25 046150 when both are missing so that W' is positioned orthogonal to the LO residue, whereas in the other configuration W, Y, D ⁺ are arranged in a linear configuration with D+ bound to Y. In either configuration, Y may also serve to separate the cleavage site W or W' from D ⁺ to avoid steric interactions with this unit, which would interfere with W/W' cleavage whenever this cleavage occurs by enzymatic action.

Typically, a spacer unit (Y) having a self-destruction moiety as defined herein has that moiety covalently linked to a cleavage unit (W/W') such that in vivo treatment of the cleavage unit activates the self-destruction of Y, thereby releasing the tubulisin drug compound from D+. In some aspects, the sacrificial moiety Y is linked to W via an amide (or anilide) functional group, with Y also covalently bonded to the quaternary nitrogen of the amine D ⁺ such that spontaneous degradation of the sacrificial moiety occurs upon enzymatic action on this functional group, resulting in the release of free tubulisin drug compound from D+. In other aspects, the self-cleaving moiety Y is linked to W' via a glycosidic bond such that cleavage of this bond releases the free tubulisin drug compound from D+.

The term self-destructive moiety, as used herein, refers to a bifunctional moiety in a spacer unit (Y) having an organic moiety inserted between the first and second functional group moieties and covalently incorporating these moieties into an normally stable three-part molecule if is not activated. Upon activation, when the covalent bond to the first functional group moiety is cleaved, the second functional group moiety spontaneously separates from the three-part molecule by self-destruction of the remainder of the self-destructive moiety. This self-destruction, when activated, releases the free tubulisin drug compound (D). In some aspects, this self-destruction occurs following cellular internalization of an LDC containing D+ and a linker unit having a self-destructive spacer unit. The intermediate organic moiety between the moieties of the functional groups of the self-destructive moiety is sometimes an arylene or heteroarylene moiety capable of undergoing fragmentation to form a quinone methide or related structure by 1,4 or 1,6 elimination, concomitantly releasing the free tubulisin drug compound. Examples of such self-destructive moieties are optionally substituted p-aminobenzyl alcohol (PAB) moieties, ortho- or para-aminobenzylacetals, or aromatic compounds that are electronically similar to the PAB group (i.e., PAB type), such as 2-aminoimidazole-5-methanol derivatives (see ., for example, Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and other heteroaryls described herein.

In one aspect, the aromatic carbon of the arylene or heteroarylene group of a self-degrading moiety of the PAB or PAB type, when incorporated into a linker unit, is replaced by an electron-donating (EDG) heteroatom attached to the W cleavage site through a first heteroatom-containing functional group, wherein the heteroatom is functionalized such that its electron-donating the ability is weakened (that is, the EDG is masked by the inclusion of a self-destructive moiety Y in the linker unit). Another substituent that provides a second functional group is a benzyl carbon, which is also attached to another aromatic carbon atom of the central arylene or heteroarylene group and has a quaternary amine substituent, wherein the quaternary amine is or includes a tubulisin drug compound attached through the benzyl carbon, where the aromatic carbon bearing the weakened electron-donating heteroatom is adjacent (i.e., 1,2-arrangement) or two additional positions (i.e., 1,4-arrangement) away from this benzylic carbon atom. The EDG is selected such that targeting the W cleavage site restores the electron donating capacity of the masked EDG, thereby causing 1,4- or 1,6-elimination to displace the tubulisin drug compound from the benzyl quaternary amine substituent.

Examples of -LO-D+ moieties having PAB or PAB-linked self-destructive moieties containing a central arylene or heteroarylene with the required 1,2- or 1,4-substitution variants that allow 1,4- or 1,6-fragmentation to release D from the quaternized unit of the medicinal compound are represented by the following:

where D+ in -C(R ⁸ )(R ⁹ )-D ⁺ is covalently attached to the above benzylic carbon through the quaternary amine nitrogen, which corresponds to the tertiary amine nitrogen of the tubulisin drug compound (D) to be released from D ⁺ , and J represents heteroatom, which is bonded

- 26 046150 occupied with W through the above weakened functional group containing J, and is optionally substituted if nitrogen (i.e. J is optionally substituted -NH), where J is -O-, -N(R ³³ )- or -S-, and R ⁸ , R ⁹ , R ³³ , R', V, Z ¹ , Z ² , Z ³ are defined in embodiments of self-destructive spacer units containing PAB or PAB type moieties. These variables are selected so that the reactivity of J when released upon exposure to W at the target site is appropriately balanced with the reactivity of the tertiary amine of the tubulisin drug compound eliminated from the self-degrading moiety of Y and with the stability of the quinone methide type intermediate formed as a result of this elimination, to effectively release D+ as D.

In some aspects of the -LO-D+ fragment structure (2), the LO spacer unit containing a self-damaging PAB type fragment or PAB linked to D+ has the following structure:

where the wavy line to J' indicates stable covalent binding (i.e. not affected at the target site) to the ligand-LB-AL _P (PEG)-A _O - in a drug-ligand conjugate, or to L _B' -AL _P (PEG)-A _O - in the connection of a drug compound with a linker when a is 1 and optionally AO is present, or with a ligand-L _B -A _a -L _P (PEG)-B- or L _B '-A _a -L _P (PEG)-B-, where a is 0 or 1 and where J' is -O-, N(R ³³ )- or -S- linked directly to A _O , B or a subunit thereof, or to a ligand- L _B -L _P (PEG)- or L _B '-L _P (PEG)-, when there is none of A, B and AO, through a functional group containing J', and where R', R ⁸ , R ⁹ , V, Z ¹ , Z ² and Z ³ have the meanings defined in Formula 1A or Formula IA, and E', independently selected from J', represents an electron-donating group in W' such as -O-, -N(R ³³ )- or -S-, wherein R ³³ is -H, optionally substituted alkyl, or optionally substituted aralkyl, and the electron-donating ability of E' is weakened by its bond to the carbohydrate moiety W', where the W'-E' bond provides a cleavage site for the glycosidase , and E' and the benzyl carbon of the -C(R ⁸ )(R ⁹ )-D ⁺ fragment are linked to the above central arylene or heteroarylene at positions defined by V, Z ¹ , Z ² or Z ³ , so that E' and the fragment -C(R ⁸ )(R ⁹ )-D ⁺ are in a 1,2- or 1,4-reciprocal arrangement to allow 1,4- or 1,6-fragmentation, which results in the release of D ⁺ as the tubulisin drug compound.

In some aspects of the -LO _-D ⁺ fragment having structure (1), the spacer unit L _O containing a self-damaging PAB type fragment or a PAB linked to D ⁺ has the following structure:

v=/ ²

4-ν—ζ UD _R 33 /1^ [Г where R', R ⁸ , R ⁹ , V, Z ¹ and Z ² are as defined in Formula 1D or Formula ID, R ³³ represents -H, optional substituted alkyl or optionally substituted aralkyl, and a wavy line adjacent to an optionally substituted nitrogen heteroatom indicates the site of covalent binding of W to the LO residue, where W is bound to the ligand-L _B -AL _P (PEG)-A _O - in the drug compound conjugate with ligand or with L _B '-AL _P (PEG)-A _O - in connection of a drug compound with a linker when the subscript a is 1 and AO is optionally present, or with ligand-L _B -A _a -L _P (PEG)-B - or L _B '-A _a -L _P (PEG)-B-, where the subscript a denotes 0 or 1, or c ligand -L _B -L _P (PEG)- or L _B '-L _P (PEG)- , when none of A, B and A _O are present, where the selective action of the protease on this covalent bond initiates self-disruption of the spacer unit to release D ⁺ as D.

In some aspects of the _-LO -D ⁺ fragment having structure (2), the spacer unit LO containing a self-damaging PAB type fragment or a PAB linked to D ⁺ has the following structure:

where E', J', R', R ⁸ , R ⁹ , V and Z ³ have the meanings defined in Formula 1A or Formula IA. Other definitions of structures and variable groups thereof, including the self-disruptive moiety in the secondary linker moiety of the D ⁺ structure (2), are provided in the embodiments.

The arylene or heteroarylene self-destructive moiety Y may be further substituted to influence the 1,2- or 1,4-elimination kinetics to modulate the release of D ⁺ as D or improve the physicochemical properties of the drug-ligand conjugate (e.g., reduced hydrophobicity) , in which it is included. For example, the non-hydrogen group R' may be an electron-withdrawing group such as chlorine, fluorine or -NO2, for example when E' is

- 27 046150 represents the oxygen atom of the glycosidic bond with the carbohydrate fragment W'.

Other illustrative and non-limiting examples of self-breaking structures that can be modified to accommodate benzylic quaternary amine substituents are presented in the works of Blencowe et al. Self-immolative linkers in polymeric delivery systems Polym. Chem. (2011) 2: 773-790; Greenwald et al. Drug delivery systems employing 1,4- or 1,6-elimination: poly(ethylene glycol) products of amine-containing compounds J. Med. Chem. (1999) 42: 3657-3667; and US Patent Nos. 7,091,186, 7,754,681, 7,553,816, and 7,989,434, all of which are incorporated herein by reference in their entirety, with the structures and variable groups set forth therein specifically incorporated by reference.

A cytotoxic drug compound, as used herein, refers to a compound or metabolite derived from LDCs that has an anti-proliferative effect on hyperproliferating cells, hyperactivated immune cells, or other pathological or unwanted cells. In some aspects, the cytotoxic drug compound acts directly on these cells or acts indirectly by affecting abnormal vasculature that supports the survival and/or growth of hyperproliferating or other pathological or unwanted cells, or the cytotoxic drug compound acts at sites of infiltration of hyperactivated immune cells. Typically, the pathological or unwanted cells that are affected by the cytotoxic drug compound are mammalian cells, more typically human cells. The cytotoxic activity of a cytotoxic drug compound can be expressed as an IC ₅₀ value, which is the effective concentration, usually the molar amount per unit volume, at which half of the cancer cells in an in vitro cell model system survive exposure to the cytotoxic agent. Thus, the value of IC5o depends on the model. Typically, a cytotoxic agent included in an LDC will have an IC ₅₀ value in an in vitro cell model of hyperproliferating cells in the range of 100 nM to 0.1 pM or more, typically 10 nM to 1 pM. A highly toxic cytotoxic drug compound typically has an IC ₅₀ value in such models of about 100 pM or lower. Although a number of drug-resistant inhibitors that reverse resistance to cytotoxic drugs are not themselves cytotoxic, they are sometimes included as cytotoxic drugs. In some cases, the cytotoxic drug compound for use in the present invention is a cytotoxic tubulisin compound containing a tertiary amine nitrogen that can be quaternized to be included as D+ in a structure representing a drug-ligand conjugate composition. In other cases, a cytotoxic tubulisin drug compound having a tertiary amine nitrogen is obtained when D+ is released as D from the drug-ligand conjugate compound in the composition of the present invention.

A cytotoxic drug compound, as used herein, refers to a compound or metabolite derived from LDCs that has an inhibitory effect on the growth and proliferation of hyperproliferating cells, hyperactivated immune cells, or other pathological or unwanted cells. In some aspects, the cytotoxic drug compound acts directly on these cells or acts indirectly by affecting abnormal vasculature that supports the survival and/or growth of hyperproliferating or other pathological or unwanted cells, or the cytotoxic drug compound acts at sites of infiltration of hyperactivated immune cells. Typically, the pathological or unwanted cells that are affected by the cytotoxic drug compound are mammalian cells, more typically human cells. Although a number of drug-resistant inhibitors that reverse resistance to cytotoxic drugs are not themselves cytotoxic, they are sometimes included as cytotoxic drugs. In some cases, the cytotoxic drug compound for use in the present invention is a cytotoxic tubulisin compound containing a tertiary amine nitrogen that can be quaternized to be included as D+ in a structure representing a drug-ligand conjugate composition. In other cases, a tubulisin compound, which is cytostatic and has a tertiary amine nitrogen, is formed when D+ is released as D from the drug-ligand conjugate compound in the composition of the present invention.

Hemoblastosis as used herein refers to a tumor of blood cells that arises from cells of lymphoid or myeloid origin and is synonymous with the term liquid tissue tumor. Hemoblastoses can be classified as indolent, moderately aggressive or highly aggressive.

Lymphoma, as used herein, is a hematologic malignancy that typically develops from hyperproliferating cells of lymphoid origin. Lymphomas are sometimes divided into two main types: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Lymphomas can also be classified according to type

- 28 046150 normal cells that are most similar to cancer cells according to phenotypic, molecular or cytogenic markers. Subtypes of lymphoma according to this classification include, but are not limited to, mature B-cell neoplasms, mature T-cell and natural killer (NK) cell neoplasms, Hodgkin lymphoma, and immunodeficiency-related lymphoproliferative disorders. Subtypes of lymphoma include T-cell progenitor lymphoma (sometimes called lymphoblastic leukemia because T-cell lymphoblasts are produced in the bone marrow), follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic lymphoma (sometimes called leukemia due to peripheral blood involvement), MALT lymphoma, Burkitt's lymphoma, mycosis fungoides and its more aggressive variant Sézary's disease, peripheral T-cell lymphoma not otherwise specified, nodular sclerosis Hodgkin's lymphoma, and a subtype of mixed cell Hodgkin's lymphoma.

Leukemia, as used herein, is a hematologic malignancy that typically develops from hyperproliferating cells of myeloid origin and includes, but is not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and acute monocytic leukemia (AML). Other leukemias include hairy cell leukemia (HCL), T-cell lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia.

The term quaternized drug unit or quaternized tubulisin drug unit, as used herein, is an attached tubulisin compound containing a tertiary amine, (D) or corresponds to such a compound in which the tertiary amine nitrogen is present in the structure of the compound as a quaternary amine salt , and exhibits cytotoxic, cytostatic, immunosuppressive or anti-inflammatory properties, typically against mammalian cells, upon release from the drug-ligand conjugate compound. In some aspects, a quaternized tubulisin drug compound (D+) unit is prepared by condensing the tertiary amine nitrogen of the C-terminal component of the tubulisin compound with a secondary linker LO precursor having a suitable leaving group. In some aspects, a tubulisin compound containing a tertiary amine is converted to its quaternized form when incorporated into an L _B or L _B ' containing moiety. In other aspects, the C-terminal component is first quaternized with a tubulisin residue and then added to complete the D+ unit. Therefore, structures such as L-LB-LO-D+ and L _B' -LO-D ⁺ do not imply any particular method by which D ⁺ is formed, and the reagent used in their formation does not require that is a drug containing a tertiary amine, but only requires D ⁺ to incorporate or match the structure of the compound containing a tertiary amine intended to release the drug-ligand conjugate from the compound. The class of tertiary amine containing drug compounds released from the LDCs of the present invention includes the tubulisin compounds described herein, which have a cytotoxic or cytostatic effect on pathological or other unwanted cells.

The term hyperproliferating cells, as used herein, refers to cells that are characterized by unwanted cellular proliferation or an abnormally high rate or persistent state of cell division that is unrelated or inconsistent with that of surrounding normal tissues. Typically the hyperproliferating cells are mammalian cells. In some aspects, hyperproliferating cells are hyperstimulated immune cells, as defined herein, in which a persistent state of cell division occurs after the cessation of the stimulus that may have initially caused a change in their cell division. In other aspects, hyperproliferating cells are transformed normal cells or cancer cells, and their uncontrolled and progressive state of cellular proliferation can result in a tumor that is benign, potentially malignant (precancerous), or clearly malignant. Conditions of hyperproliferation arising from transformed normal cells or cancer cells include, but are not limited to, conditions characterized as precancerous condition, hyperplasia, dysplasia, adenoma, sarcoma, blastoma, cancer, lymphoma, leukemia or papilloma. Precancerous lesions are generally defined as lesions that show histological changes associated with an increased risk of developing malignancy and sometimes have some, but not all, of the molecular and phenotypic properties that characterize malignancy. Hormone-related or hormone-sensitive precancerous conditions include prostatic intraepithelial neoplasia (PIN), especially high-grade PIN (HGPIN), atypical small acinar proliferation (ASAP), cervical dysplasia, and ductal carcinoma in situ. Hyperplasia generally refers to the proliferation of cells within an organ or tissue beyond what is normally observed, which can result in significant enlargement of the organ or the formation of a benign tumor or growth. Hyperplasia includes, but is not limited to, endometrial hyperplasia

- 29 046150 (endometriosis), benign prostatic hyperplasia and ductal hyperplasia.

Normal cells, as used herein, refer to cells undergoing concerted cell division associated with maintaining the integrity of normal tissue cells or replenishing circulating lymph or blood cells, which is necessary for regulated cell regeneration or for tissue repair required by injury, or for a regulated immune or inflammatory response resulting from exposure to a pathogen or other cell-damaging factor, where the induced cell division or immune response ceases after the necessary maintenance, replenishment, or clearance of the pathogen has been completed. Normal cells include normally proliferating cells, normal quiescent cells, and normally activated immune cells.

Normal quiescent cells are non-cancerous cells at rest and are not stimulated by stress or mitogen, or are immune cells that are normally inactive or have not been activated by exposure to pro-inflammatory cytokines.

Hyperstimulated immune cells as a term used herein refer to cells involved in innate or adaptive immunity characterized by abnormally persistent proliferation or an inappropriate state of stimulation that occurs after the cessation of the stimulus that may have initially caused the change in proliferation or stimulation, or that occurs in absence of any external damage. Often the persistent spread or inappropriate state of stimulation results in a chronic state of inflammation characteristic of a disease or disease state. In some cases, the stimulus that may initially cause the change in proliferation or stimulation is not due to an external lesion, but is caused internally, as in an autoimmune disease. In some aspects, hyperstimulated immune cells are proinflammatory immune cells that have been hyperactivated as a result of chronic exposure to proinflammatory cytokines.

In some aspects of the invention, the LDC binds to an antigen predominantly detected by pro-inflammatory immune cells that are abnormally proliferating or inappropriately activated. These immune cells include classically activated macrophages or T helper type 1 (Th1) cells, which produce interferon-gamma (INF-γ), interleukin-2 (IL-2), interleukin-10 (IL-10), and tumor necrosis factor-beta (TNF-β), which are cytokines involved in the activation of macrophages and CD8+ T cells.

Glycosidase as used herein refers to a protein capable of enzymatic cleavage of a glycosidic bond. Typically, the glycosidic bond to be cleaved is present in the cleavable unit (W') in the LDC. Sometimes the glycosidase acting on LDC is present intracellularly in hyperproliferating cells, hyperactivated immune cells, or other pathological or unwanted cells to which LDC has preferential access compared to normal cells due to the targeting ability of the ligand binding moiety (i.e., the ligand unit) . Sometimes the glycoside is more specific to pathological or unwanted cells, or is preferentially secreted by pathological or unwanted cells compared to normal cells, or is present in greater quantities near pathological or unwanted cells compared to the amount in serum. Often, the glycosidic bond in W', which is acted upon by a glycosidase, attaches the anomeric carbon of the carbohydrate moiety (Su) to the phenolic oxygen of the self-disrupting (SI) moiety, which contains the self-disrupting spacer unit (Y), so that glycosidic cleavage of this bond triggers the 1,4- or 1,6-elimination of a tubulisin drug compound containing a tertiary amine from a quaternary amine moiety linked to the benzyl position of the SI.

In some aspects, drug-linker compounds or drug-ligand conjugates consist of groups such as -A _a -L _P (PEG)-Bb-Y(W')-D ⁺ or -A _a -L _P (PEG) -AO-Y(W')-D ⁺ , in which the subscripts a and b independently denote 0 or 1, where the fragment -Y(W')- is usually a Su-O' fragment associated with the self-destructive fragment Y defined in herein containing AO or -LP(PEG)-, W' and D ⁺ attached to a self-destructive moiety in a manner that provides self-destructive release of a free tubulisin compound containing a tertiary amine by the action of a glycosidase. Such -Y(W')- moieties are sometimes referred to as a glucuronide unit, where Su is a carbohydrate moiety which is not limited to such glucuronic acid.

Typically, the Su-O'- moiety (where -O'- is the oxygen of the glycosidic bond and Su is the carbohydrate moiety) associated with a self-disruptive moiety Y is represented by the structure described for self-disruptive moieties, where E', associated with a central arylene or heteroarylene self-destructive moiety is oxygen, and where this heteroatom is replaced by a carbohydrate moiety through its anomeric carbon atom. More typically, Su-O'-YD ⁺ has the structure

- 30 046150

wherein R ^24A , R ^24B and R ^24C have the meanings defined for R ²⁴ in the summary of the invention, and are selected such that the electron donating ability of the phenolic -OH released from the glycosidic bond is sensitive to selective cleavage by the desired glycosidase of the glycosidase bond with the carbohydrate moiety Su and the fragmentation stability of the quinone methide intermediate were balanced by the ability to remove the tertiary amine-containing tubulisin drug compound so that D was efficiently released from D+ via 1,4- or 1,6-elimination. Such Su-O'-Y- structures having a PAB or PAB type moiety for self-destruction are typical glucuronide units. When a glycosidic bond is linked to the carbohydrate moiety of a glucuronic acid, the glycosidase capable of enzymatically cleaving the glycosidic bond is a glucuronidase.

A carbohydrate moiety, as used herein, refers to a monosaccharide having the empirical formula Cm(H ₂ O)n, where n is m, containing an aldehyde moiety in its hemiacetal form or a derivative thereof, where the -CH2OH moiety of this formula has been oxidized to a carboxylic moiety acid (for example, to glucuronic acid as a result of oxidation of the CH2OH group in glucose). Typically the carbohydrate moiety (Su) is a cyclic hexose such as pyranose or a cyclic pentose such as furanose. Typically, pyranose is a glucuronide or hexose in the eD conformation. In some cases, the pyranose is the eD-glucuronide moiety (i.e., the eD-glucuronic acid linked to the self-degrading part of Y via a glycosidic bond, which is cleaved by β-glucuronidase). Often the carbohydrate moiety is unsubstituted (eg, a natural cyclic hexose or cyclic pentose). In other cases, the carbohydrate moiety may be a cyclic hexose or cyclic pentose in which the hydroxyl group has been removed or replaced by a halogen or lower alkyl, or alkylated with a lower alkyl.

A protease, as defined herein, refers to a protein capable of enzymatic cleavage of a carbonyl-nitrogen bond, such as an amide bond, typically present in a peptide. Proteases are divided into six main classes: serine proteases, threonine proteases, cysteine proteases, glutamic acid proteases, aspartic acid proteases, and metalloproteases, named for the catalytic residue in the active site that is primarily responsible for breaking the carbonyl and nitrogen bonds of the substrate. Proteases are characterized by different specificities, which depend on the identity of the residues on the N-terminal and/or C-terminal side of the carbonyl-nitrogen bond and different distributions.

When W consists of an amide or other functional group containing a carbonyl nitrogen bond that is cleavable by a protease, the cleavage site is often limited to those recognized by proteases present in hyperproliferating cells or hyperstimulated immune cells, or in cells specific to the environment in which hyperproliferating cells are present. cells or hyperstimulated immune cells. In these cases, the protease need not be predominantly present or found in greater abundance in the cells targeted by the LDC, since the LDC will have less access to those cells that do not have a preferential presence of the target fragment. In other cases, the protease is preferentially secreted by pathological cells or normal cells inherent in the environment in which the pathological cells are found, compared with normal cells in the environment distant from the site of pathological cells. Thus, in cases where protease is released, it is imperative that the protease is present predominantly or in greater abundance in the vicinity of LDC target cells compared to normal cells not in the vicinity of pathological cells.

When incorporated into an LDC, the W-containing peptide will present a recognition sequence to the protease, which cleaves the carbonyl-nitrogen bond in W to initiate fragmentation of the linker unit to cause the release of the tertiary amine-containing drug compound from D+. Sometimes the recognition sequence is selectively recognized by an intracellular protease present in pathological cells to which LDC has preferential access compared to normal cells due to targeting of pathological cells, or is preferentially produced by pathological cells compared to normal cells in order to properly deliver the drug compound to desired location. Typically, the peptide is resistant to circulating proteases to minimize premature release of the tubulisin drug compound and thus minimize unwanted systemic exposure to the compound. Typically a peptide will have one or more unnatural or unclassified

- 31,046,150 ical amino acids in its sequence to have such stability. Often, the amide bond that is specifically cleaved by the protease produced by the pathological cell is an anilide, in which the nitrogen of the anilide is an electron-donating heteroatom (ie J) arising from a self-degrading moiety having the structures defined above. Thus, the action of the protease on such a peptide sequence in W results in the release of D+ as D from the linker unit through 1,4- or 1,6-elimination occurring through the central arylene or heteroarylene moiety of the self-degrading moiety.

Regulatory proteases are typically located intracellularly and are required to regulate cellular activities that sometimes become aberrant or dysregulated in pathological or other undesirable cells. In some cases, when W targets a protease that has a predominant intracellular distribution, that protease is a regulatory protease involved in cell maintenance or proliferation. In some cases, these proteases include cathepsins. Cathepsins include serine proteases, cathepsin A, cathepsin G, aspartic acid proteases, cathepsin D, cathepsin E and cysteine proteases, cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W and cathepsin Z.

In other cases, when W is directed to a protease that is preferentially distributed extracellularly in the vicinity of hyperproliferating or hyperstimulated immune cells due to preferential secretion by such cells or neighboring cells secreted by the environment of hyperproliferating or hyperstimulated immune cells, the protease is typically a metalloprotease. Typically, such proteases are involved in tissue remodeling, which promotes the invasiveness of hyperproliferating cells, or are the result of the unwanted accumulation of hyperactivated immune cells, which leads to further recruitment of such cells.

The term tubulisin drug compound or tubulisin compound as used is a peptide-based tubulin-disrupting agent having cytotoxic activity, cytostatic or anti-inflammatory activity, and consists of one natural or unnatural amino acid component and three other natural amino acid components, where one of these components is characterized by a central 5-membered or 6-membered heteroarylene moiety, and the other component provides a tertiary amine for inclusion in the quaternized drug compound unit.

Tubulisin compounds have a DG or DH structure

where a straight dotted line indicates an optional double bond, a curved dotted bond indicates an optional cyclization, circled Ar denotes an arylene or heteroarylene that has a 1,3-substitution on the tubulisin carbon skeleton and is optionally substituted anywhere, where arylene or heteroarylene and others the variable groups are as specified in the embodiments of the invention.

Natural tubulisin compounds have the structure DG _-6

and are conventionally divided into four amino acid subunits, as shown by the dotted vertical lines, called N-methyl-pipecolic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), and either tubuphenylalanine (Tup, when R ^7A is hydrogen) or tubutyrosine (Tut when R ^7A is -OH). There are about a dozen naturally occurring tubulysins, currently known as tubulysin AI, tubulysin U, tubulysin V and tubulysin Z, the structures of which are designated by variable groups for the D structure _{of G-6} , defined in variants

- 32 046150 implementation in units of a quaternized medicinal substance based on tubulisin.

Pretubulisins have the structure DG or DH, where R ³ is -CH3 and R ² is hydrogen, and desmethyl tubulisins have the structure DG, D _G-1 , D _G-6 , DH or D _H-1 , in which R ³ is hydrogen, and other tubulisin structures are variants of tubulisin-based quaternized drug units wherein R ³ is hydrogen and where the other variable groups are as described for tubulisins. Pretubulysins and desmethyl tubulysins are not necessarily included in the definitions of tubulysins.

In the structures _DG , DG _-1 , _DG-6 , DH, DH- _i and other tubulisin structures described herein, in embodiments of tubulisin-based quaternized drug compound units, said (f) nitrogen atom is quaternization site, when such structures correspond to or are included in the LDC or its precursor in the form of a quaternized tubulisin drug compound unit. When incorporated into LDC or its precursor, this nitrogen is quaternized by covalent binding to LO or to LO in an LB or LB'-containing moiety containing LO. Typically, this quaternized D+ moiety results from the covalent addition of a nitrogen atom of a tertiary amine moiety to the benzyl carbon of a PAB or PAB moiety in the self-breaking Y spacer unit in LO. The structures of other typical tertiary amine-containing tubulisins and pretubulesins and the method of their incorporation into LDCs as D+ are presented in embodiments of tubulisin-based caternized drug units.

Examples of methods for preparing tubulisin drug compounds and their structurally active bonds are proposed in the works of Shankar et al. Synthesis and structure-activity relationship studies of novel tubulysin U analogs-effect on cytotoxicity of structural variations in the tubuvaline fragment Org. Biomol. Chem. (2013) 11: 2273-2287; Xiangming et al. Recent advances in the synthesis of tubulysins Mini-Rev. Med. Chem. (2013) 13: 1572-8; Shankar et al. Synthesis and cytotoxic evaluation of diastereomers and Nterminal analogs of Tubulysin-U Tet. Lett. (2013) 54: 6137–6141; Shankar et al. Total synthesis and cytotoxicity evaluation of an oxazole analogue of Tubulysin U Synlett (2011) 2011(12): 1673-6; Raghavan et al. J. Med. Chem. (2008) 51: 1530-3; Balasubramanian, R. et al. Tubulysin analogs including desmethyl and dimethyl tubuphenylalanine derivatives Bioorg. Med. Chem. Lett. (2008) 18:2996-9; and Raghavan et al. Cytotoxic simplified tubulysin analogues J. Med. Chem. (2008) 51: 1530-3.

The terms intracellularly cleaved, intracellular cleavage, and similar terms as used herein refer to a metabolic process or reaction within a cell to LDC or the like, whereby the covalent bond, e.g., by a linker, between the quaternary amine of the quaternized tubulisin compound and the antibody is disrupted, resulting in to the formation of a free drug compound containing a tertiary amine, or another metabolite of the conjugate, dissociated from the targeting moiety within the cell. Thus, cleaved LDC fragments are intracellular metabolites.

Bioavailability, as used herein, refers to the systemic availability (ie, blood/plasma levels) of a given amount of a drug compound administered to a patient. Bioavailability is an absolute term that refers to parameters of both the time (rate) and the total amount (extent) of a drug compound that reaches the general circulation from the administered dosage form.

The term subject as used herein refers to a human, non-human primate, or mammal having or susceptible to a hyperproliferative, inflammatory or immune disorder that will benefit from administration of an effective amount of LDC. Non-limiting examples of the subject include human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and poultry. Typically, the subject is a human, non-human primate, rat, mouse or dog.

The term inhibit or inhibit means to reduce by a measurable amount or completely prevent. Inhibition of hyperproliferating cell proliferation by ADCs is typically determined against untreated cells (mimicking drug carrier media) in a suitable test system such as a cell culture (in vitro) or a xenograft model (in vivo). Typically, an LDC consisting of an antigen targeting moiety that is not present on the hyperproliferating cells or activated immunostimulatory cells of interest is used as a negative control.

The term therapeutically effective amount refers to an amount of an antibody or other drug compound effective to treat a disease or disorder in a subject or mammal. In the case of malignancy, a therapeutically effective amount of the drug compound can reduce the number of cancer cells; reduce the primary size of the tumor; inhibit (ie, slow to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie, slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate to some extent one or more symptoms associated with the malignancy

- 33 046150 new growth. To some extent, the drug compound can inhibit the growth and/or destroy existing cancer cells, and it may be cytostatic and/or cytotoxic. In cancer treatment, efficacy can be measured, for example, by assessing time to progression (TTP) and/or rate of response (RR).

In the case of immune disorders arising from hyperstimulated immune cells, a therapeutically effective amount of a drug compound may reduce the number of hyperstimulated immune cells, the extent of their stimulation and/or infiltration into other normal tissues and/or alleviate to some extent one or more symptoms associated with dysregulation of the immune system due to overstimulated immune cells. For immune disorders caused by hyperstimulated immune cells, effectiveness can be measured, for example, by assessing one or more surrogate inflammatory endpoints, including one or more cytokine levels, such as levels for IL-1β, TNFa, INFy, and MCP-1, or the amount classically activated macrophages.

In some aspects of the invention, the drug-ligand conjugate binds to an antigen on the surface of a target cell (ie, a hyperproliferating cell or hyperstimulated immune cell), and the drug-ligand conjugate is then placed inside the target cell via receptor-mediated endocytosis. Once inside the cell, one or more cleavage units in the linker unit are cleaved, resulting in the release of the drug compound containing the tertiary amine. The released tubulisin drug compound containing the tertiary amine can then migrate freely in the cytosol and cause cytotoxic or cytostatic activity, or in the case of hyperstimulated immune cells, can alternatively inhibit inflammatory signal transduction. In another aspect of the invention, the quaternized drug compound unit is cleaved from the drug-ligand conjugate outside the target cell, but in close proximity to the target cell, such that the released tubulisin compound containing the tertiary amine subsequently enters the cell rather than being released at distant sites .

The term carrier as used herein refers to the diluent, adjuvant or excipient with which the compound is administered. Pharmaceutical excipients can be liquids such as water and oils, including those derived from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. Carriers can be, for example, saline solution, gum acacia, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea. In addition, auxiliary, stabilizing, thickening, lubricating agents and dyes can be used. In one embodiment, when administered to a patient, the compound or compositions and pharmaceutically acceptable carriers are sterile. Water is the typical vehicle when compounds are administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, glycol , water and ethanol. The pharmaceutical compositions, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffering agents.

The terms treat, treatment and the like, unless otherwise clear from the context, refer to therapeutic treatments and preventive measures to prevent relapse, where the goal is to inhibit or slow (reduce) unwanted physiological changes or disorders, such as the development or spread of cancer or tissue damage from chronic inflammation. Generally, favorable or desirable clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in the severity of the disease, stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, alleviation or temporary relief of the disease state, and remission (partial or complete) , both detectable and undetectable. Treatment may also mean prolongation of survival or improvement of quality of life and the like, compared to the expected survival or quality of life if there is no treatment. Those in need of treatment include those who already have a condition or disorder, as well as those who are likely to have that condition or disorder.

In the context of a malignancy or disease state associated with chronic inflammation, the term treatment includes any or all options for inhibiting the growth of tumor cells, cancer cells or tumors; inhibiting the replication of tumor cells or cancer cells, inhibiting the spread of tumor cells or cancer cells, reducing the overall tumor burden or reducing the number of cancer cells, inhibiting the replication or stimulation of pro-inflammatory immune cells, inhibiting or reducing the chronic inflammatory state of a dysregulated immune system or reducing the frequency and/or intensity sudden exacerbations experienced by subjects with an autoimmune condition or disease,

- 34 046150 or relief of one or more symptoms associated with cancer or a hyperimmune-stimulated disease or condition.

The phrase pharmaceutically acceptable salt, as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound described herein. The compound typically contains at least one amino group and accordingly acid addition salts can be formed with this amino group. Other examples of salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e. 1,1'-methylene-bis-(2- hydroxy-3-naphthoate)).

The pharmaceutically acceptable salt may have the inclusion of another molecule, such as an acetate ion, succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, they may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions. Typically, the quaternized tubulisin drug unit is in the form of a pharmaceutically acceptable salt.

Typically, the pharmaceutically acceptable salt is selected from those reviewed by P. H. Stahl and C.G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zurich:Wiley-VCH/VHCA, 2002. The choice of salt depends on the properties that the drug product must exhibit, including adequate solubility in water at various pH values, in depending on the intended route(s) of administration, crystallinity with characteristics of flow and low hygroscopicity (i.e. water absorption depending on relative humidity), suitable processing and the required shelf life determined by chemical and solid-state stability under conditions of accelerated degradation (i.e. determination of degradation or changes in solid state when stored at 40°C and 75% relative humidity).

The terms load, drug load, payload load, or similar terms denote or refer to the average number of payloads (payload and payloads are used interchangeably herein with drug compound and drug compounds) in a population of LDCs (i.e., a composition of LDCs differing in number attached D' units having the same or different attachment sites, but otherwise being substantially identical in structure). The drug load can range from 1 to 24 drug compounds per targeting moiety. This is sometimes referred to as DAR or drug-to-targeting moiety ratio. The LDC compositions described herein typically have a DAR in the range of 1-24 or 1-20, and in some aspects in the range of 1-8, 2-8, 2-6, 2-5 and 2-4. Typical DAR values are about 2, about 4, about 6 and about 8. The average amount of drug per antibody or DAR value can be characterized by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA analysis and HPLC. The quantitative value of DAR can also be determined. In some cases, separation, purification, and characterization of homogeneous LDCs having a specific DAR value can be achieved by means such as reversed-phase HPLC or electrophoresis. The DAR may be limited by the number of attachment sites on the targeting moiety.

For example, when the targeting moiety is an antibody and the attachment site is a cysteine thiol, the antibody may have only one or more sufficiently reactive thiol groups that react with the LB'-containing moiety. Sometimes a cysteine thiol is a thiol group derived from a cysteine residue that participates in an interchain disulfide bond. In other cases, a cysteine thiol is a thiol group of a cysteine residue that was not involved in the interchain disulfide bond but was introduced through genetic engineering. Typically, fewer than the theoretical maximum of D+ fragments are conjugated to the antibody during the conjugation reaction. For example, an antibody may contain many lysine residues that do not react with the LB'-containing moiety because only the most reactive lysine groups can react with the moiety.

I. Embodiments.

Disclosed herein are ligand drug conjugates (LDCs) capable of preferentially delivering a tubulisin compound containing a tertiary amine into hyperproliferative cells or hyperactivated immune cells or into the vicinity of such pathological cells as compared to normal cells or the environment of normal cells where the pathological cells are not usually present and thus can be used to treat diseases and conditions characterized by these abnormal cells.

- 35 046150

1.1. General provisions.

LDC has three main components: (1) a ligand unit corresponding to or including a targeting agent that selectively binds to a target moiety present in or near pathological cells or other unwanted cells over other moieties present on, within, or in the vicinity of normal cells, where these pathological or unwanted cells are usually absent or present in or near pathological or other unwanted cells in greater numbers compared to normal cells or the environment around normal cells in which pathological or unwanted cells are usually absent, (2) a quaternized tubulisin drug compound unit (D'). comprising or corresponding to the structure of a tubulisin compound containing a tertiary amine by quaternizing the tertiary nitrogen atom of the amine, and (3) a linker unit connecting D+ to a ligand unit and capable of conditionally releasing the free tubulisin drug compound containing a tertiary amine in or near pathological or unwanted cells , which are targeted by the ligand unit.

The tubulisin compound for use in the present invention is a compound that primarily or selectively exerts its effects (eg, cytotoxic, cytostatic effect) on mammalian cells as compared to prokaryotic cells. In some aspects, the target fragment is an epitope of an extracellular membrane protein that is preferentially found on abnormal or unwanted cells compared to normal cells. Specificity for pathological or unwanted cells (i.e., target cells) is due to the ligand unit (L) of the LDC, which includes D. In addition to L and D ⁺ , an LDC targeting pathological or unwanted cells has a linker unit, which covalently connects the quaternized tubulisin drug compound unit to the ligand unit. In some aspects, the ligand unit consists of an antibody that is a typical targeting agent that recognizes pathological mammalian cells.

In some aspects, the target fragment recognized by the ligand unit is an epitope of an extracellular membrane protein that is preferentially found on pathological or unwanted cells compared to normal cells. In some of these aspects, and as an additional requirement, the membrane protein targeted by the ligand moiety must have sufficient copy number and be internalized upon LDC binding to intracellularly deliver an effective amount of the cytotoxic, cytostatic, immunosuppressive, or anti-inflammatory tubulisin compound, preferably , to pathological cells.

The tertiary amine containing tubulisin compound generally exhibits adverse peripheral effects when administered in an unconjugated form. Therefore, this compound must be selectively delivered by its LDC so that the LDC linker block is not simply a passive structure that serves as a bridge between the target ligand block and the D+ from which the tubulisin compound is released, but must be carefully designed to be stable from the LDC injection site before it is delivered to the target site, and then must effectively release the active tubulisin compound. To accomplish this task, the targeting agent is reacted with an LB'-containing moiety to form an LB-containing moiety in a drug-linker conjugate. When an LB-containing moiety is formed in this manner, the resulting drug-ligand conjugate typically consists of a targeting moiety (as a ligand unit), a covalently binding ligand unit (LB), also called a major linker (LR), a quaternized tubulisin compound (D+) and a secondary linker (LO), located between LB and D ⁺ .

1.1. Main linker (LR).

A major linker (LR) is a covalently binding ligand unit (LB) or a precursor to a covalently binding ligand unit ( _LB ') and is typically present as a component of the linker unit in an LDC or LB'-containing fragment such as LB'-LO in a compound a drug compound with a linker having the formula LB'-LO-D ⁺ . The main linker of the LB'-containing fragment consists of a functional group capable of interacting with the electrophilic or nucleophilic functional group of the targeting agent. As a result of this interaction, the targeting agent becomes covalently linked as a ligand unit to the main linker through LB, where the main linker is now LB having a functional group derived from LB'.

In some embodiments, the LB' in the LB'-containing moiety has one of the following structures:

- 36 046150

where R represents hydrogen or ^-^-optionally substituted alkyl; T represents -Cl, -Br, -I, -O-mesyl, -O-tosyl or other sulfonate leaving group; U is -F, -Cl, -Br, -I, -ON-succinimide, -O-(4-nitrophenyl), -O-pentafluorophenyl, tetrafluorophenyl or -OC(=O)-OR ⁵⁷ ; X ² is C1-10alkylene, C3-C8 carbocycle, -O-(C1-C6-alkyl), -arylene-, C1-C10alkylene-arylene, -arylene-C1-C10-alkylene, -C1-C10-alkylene -(C3-C6-carbocycle)-, -(C3-C8-carbocycle)-C1-C10alkylene-, ^-^-heterocycle, -C1-C10-alkylene-(C3-C8-heterocyclo)-, -C3-C8 -heterocyclo)-C1-C10-alkylene, -(CH2CH2O)u or -CH2CH2O)u-CH2-, where u is an integer ranging from 1 to 10 and R ⁵⁷ is ^-^-alkyl or aryl; and where the wavy line denotes covalent attachment to the LO subunit.

When reacted with an electrophile or nucleophile, the targeting agent L _B ' is converted into a ligand-LB moiety, as illustrated below, where one such interaction has occurred:

O

where the atom marked (#) is derived from the reactive ligand moiety and X ² has the meanings defined above.

1.2. Secondary linkers ( _LO ).

Secondary linkers in an LDC or its LB'-containing precursor are an organic moiety located between the main linker (LR) and the quaternized drug (D+) that allows processing of the cleavable unit (W or W') in the linker unit of the LDC after selective targeting binding. an LDC fragment with its cognate target fragment present on, within or near hyperproliferating cells, hyperactivated immune cells or other pathological or unwanted cells targeted by the LDC. In some embodiments, W provides a substrate for a protease that is present in hyperproliferating cells or hyperactivated immune cells. Preferred cleavage units are those that are not recognized by proteases secreted by normal cells that are not typically present in hyperproliferating cells or hyperactivated immune cells, or are substrates for proteases in the systemic circulation, to minimize inappropriate release of the drug compound or systemic exposure to the drug compound containing tertiary amine, due to its premature release

- 37 046150 denier from LDC. More preferred are those proteases that are regulatory proteases or proteases found in lysosomes, which are the cellular compartments into which LDC is delivered upon internalization of a membrane surface receptor to which LDC specifically binds. Regulatory and lysosomal proteases are typical intracellular proteases.

In one embodiment, W in the second linker includes or consists of a dipeptide moiety having the structure

where R ²⁹ represents benzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH(OH)CH ₃ , or has the following structure:

and R ³⁰ is methyl, -(CH ₂ ) ₄ -NH ₂ , -(CH ₂ ) ₃ NH(C=O)NH ₂ , -(CH ₂ ) ₃ NH(C=NH)NH ₂ or -(CH ₂ ) ₂ CO ₂ H, where the dipeptide fragment refers to the recognition site of a regulatory or lysosomal protease.

In preferred embodiments, the dipeptide is valine-alanine (Val-Ala). In another preferred embodiment, W contains or consists of the dipeptide valine-citrulline (Val-Cit). In another preferred embodiment, W is formed or consists of the dipeptide threonine glutamic acid (Thr-Glu). In other embodiments, W is one naturally occurring L-amino acid, preferably L-glutamate or L-lysine. In some of these embodiments, the dipeptide moiety or L-amino acid is covalently attached to the self-interruptible moiety (SI) of Y through an amide bond formed between an alanine or citrulline carboxylic acid functional group or a glutamate alpha-carboxylic acid functional group and an aryl or heteroaryl amino group of SI. Thus, in these embodiments, the SI consists of an arylamine or heteroarylamine moiety, and the above-mentioned carboxylic acid functional group of the dipeptide moiety forms an anilide bond to the amine nitrogen of the arylamine moiety.

In another embodiment, the W' in the secondary linker consists of a carbohydrate moiety linked to a glycoside having a recognition site for an intracellularly located glycosidase. In such embodiments, W' is a carbohydrate moiety linked to E' via a glycosidic bond, where W'-E' provides a recognition site for the cleavage of W' from E', and where E' is an optionally substituted heteroatom that is included in self-destructive spacer unit to which W' is attached in the glucuronide unit of the formula -Y(W')-. In such embodiments, W'-E'- typically has the following structure:

HE

BUT, 1 .OH

R ⁴⁵ O ^e'|where R ⁴⁵ is -CH2OH or -CO ₂ H and E' is a heteroatom moiety such as -O-, -S- or -NH- associated with a carbohydrate moiety and a self-destructive moiety of Y (as indicated by the wavy line), where the linkage to the carbohydrate moiety provides a recognition site for the glycosidase. Preferably, this site is recognized by lysosomal glycosidase. In some embodiments, the glycosidase is a glucuronidase when R ⁴⁵ is -CO2H.

The secondary linker unit (LO), in addition to W or W', also consists of a spacer unit (Y) and a -LP(PEG)- moiety and may further include a second extender ( _AO ) or branching unit (B) located relative to W/ W' in a linear relationship represented by the L _O -D ⁺ structures of (la) and (lb), or in an orthogonal relationship represented by the L _O -D ⁺ structures of (2a) and (2b), respectively

- 38 046150

where A _a represents the first optional extension link, AO represents the second optional extension link; W _w and W' _w ' represent detachable units; and Y _y represents a spacer unit, where subscripts a and b independently denote 0 or 1, subscript w or w' denotes 1, and subscript y denotes 1. When subscript a denotes 1, a wavy line in front of A _a indicates covalent binding of that LO subunit with LB' or LB (formed from LB' after its inclusion in the LDC). When subscript a is 0, the wavy line indicates covalent binding of LB' or LB to the -LP(PEG)- moiety in structure (1a), (1b) (2a) or (2b).

In preferred embodiments, the subscript a is 1. In other preferred embodiments, -LO-D+ has the following structure (2a) or 2(b), more preferably when A _O is present, or the subscript b is 0. In particular preferred embodiments - LO-D+ has the following structure (2a), where AO is present and the subscript a denotes 1, so A is also present.

The structures of some examples of the A/AO, W and Y moieties in LO and their substituents are described in WO 2004/010957, WO 2007/038658, US Patent Nos. 6214345, 7498298, 7968687 and 8163888, and US Patent Publications No. 2009-0111756, 2009 - 0018086 and 2009-0274713, which are incorporated herein by reference.

In some embodiments, AO, A, or subunits thereof have the structure

(4) where the wavy lines indicate covalent binding at the L _O residue, and where for A _O the wavy line to the carbonyl moiety in any structure in (1a) represents the point of attachment to the amino terminus of the dipeptide moiety or natural L-amino acid containing W when Y is located linearly with respect to Y and D ⁺ , or in (2b) represents the point of attachment to a self-destructive fragment of Y described herein, in which W' is associated with Y, and is located orthogonal with respect to Y and D ⁺ , and wherein the wavy line to the amine moiety of either structure represents for (1a) or (2a) the point of attachment to the carbonyl-containing functional group L _P in -L _P (PEG);

where K and L are independently C, N, O or S, provided that when K or L is O or S, R ⁴¹ and R42-K or R ⁴³ and R44-L are absent, and when K or L is N, one of ^R41 , R42-K or one of ^R42 , ^R43 -L is absent, and provided that two adjacent Ls are not independently selected as N, O or S;

where the subscripts e and f are independently chosen integers that range from 0 to 12, and the subscript g denotes an integer ranging from 1 to 12:

where G represents hydrogen, optionally substituted with C1-C ₆ -alkyl, -OH, -OR ^pr , -CO2H, CO2RPR, where R ^pr represents an appropriate protecting group, -N(R ^pr )(R ^pr ), where RPR is independent represent a protecting group, or R ^pr together form a corresponding protecting group, or -N(R ⁴⁵ )(R ⁴⁶ ), where one of R ⁴⁵ , R ⁴⁶ represents hydrogen or R ^pr , where R ^pr represents a corresponding protecting group, and the other is hydrogen or optionally substituted C1- _C6 alkyl;

where R ³⁸ represents hydrogen or optionally substituted C1-C6 alkyl; R ³⁹ -R ⁴⁴ are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, or both R ³⁹ and R ⁴⁰ together with the carbon atom to which they are attached form C ₃ -C ₆ -cycloalkyl, or R ⁴¹ , R ⁴² together with the K to which they are attached,

- 39 046150 when K represents C or R ⁴³ , R ⁴⁴ together with L to which they are attached, when L represents C, form C ₃ -C ₆ -cycloalkyl, or R ⁴⁰ and R ⁴¹ , or R ⁴⁰ and R ⁴³ , or R ⁴¹ and R ⁴³ together with the carbon atom or heteroatom to which they are attached and the atoms intermediate between these carbon atoms and/or heteroatoms form a 5- or 6-membered cycloalkyl or heterocycloalkyl, provided that, when K is O or S, R ⁴¹ and R ⁴² are absent, when K is N, one of R ⁴¹ is absent, R ⁴² is absent, when L is O or S, R ⁴³ and R ⁴⁴ are absent, and when L is N, one of R ⁴³ , R ⁴⁴ is missing.

In some embodiments, R ³⁸ is hydrogen. In other embodiments, -K(R ⁴¹ )(R ⁴² ) is -(CH ₂ )-. In other embodiments, whenever the subscript e is not O, R ³⁹ and R ⁴⁰ are hydrogen. In other embodiments, whenever the subscript f is not O, L(R ⁴³ )(R ⁴⁴ )- is CH2-.

In preferred embodiments, G is -CO ₂ H. In other preferred embodiments, K and/or L is C. In other preferred embodiments, the subscript e or f is 0. In still other preferred embodiments, e+f is an integer in the range from 1 to 4.

In some embodiments, _AO , A, or a subunit thereof has the following structure -NHC ₁ -C ₁₀ -alkylene-C(=O)-, -NH-C ₁ -C ₁₀ -alkylene-NH-C(=O)-C ₁ -C ₁₀ -αalkylene-C(=O)-, -NH-Ci-Cu-alkyleneC(=O)-NI-C ₁ -C ₁₀ -alkylene (C=O)-, -NH-(CH ₂ CH ₂ O) _s -CH ₂ (C=O)-, ^-(C ₃ -C ₈ -carbocyclo)(C=O)-, -NH(arylene-)-C(=O)- and -NI-( C ₃ -C ₈ -heterocyclo-)C(=O).

In other embodiments, A _O , A, or a sub-unit thereof have the following structure:

O —NH—R ¹³ —C— where R ¹³ represents -C1-C10-alkylene-, -C3-C8-carbocyclo-, -arylene-, -C1-C30-heteroalkylene-, C3-C8-heterocyclo-, - C1-C10-alkylene-arylene-, arylene-C1-C10-alkylene-, -C1-C10-alkylene-(C3-C8-carbocyclo)-, - (C3-C8-carbocyclo)-C1-C10-alkylene-, -C1-C10-alkylene- (C3-C8-heterocyclo)-, - (C3-C8-heterocyclo)C1-C10-alkylene-, - (CH2CH2O)1-10(-CH2)1-3- or -(CH2CH2NH )1-10(-CH2)1-3-. In some embodiments, R ¹³ is -C1-C ₁₀ -alkylene- or -C ₁ -C ₃₀ -heteroalkylene-. In some embodiments, R ¹³ is -C1-C10-alkylene-, -(CH2CH2O)1-10(-CH2)1-3-, or -(CH2CH2NH)1-10(-CH2)1-3-. In some embodiments, R ¹³ is -C1-C ₁₀ -alkylene-polyethylene glycol or polyethyleneimine.

In more preferred embodiments, _AO , A, or a subunit thereof is structurally matched or a residue of an alpha-amino acid moiety, beta-amino acid moiety, or other amine-containing acid. Other embodiments for A as a single unit and A subunits _1-4 for A are described in embodiments for linker units having the formula LR-LO.

In some embodiments, the spacer units are capable of undergoing a 1,4- or 1,6-elimination reaction following enzymatic treatment of W covalently linked to Y (ie, Y includes a self-destructive spacer unit). In some embodiments, Y-D+, positioned in line with W at LO, has the following structure:

where V, Z ¹ , Z ² and Z ³ are, independently, -C(R ²⁴ ) = or -N=; U represents -O-, -S or -N(R ²⁵ )-; ^R24 is independently hydrogen, halogen, -NO2, -CN, ^OR25 , ^-SR26 , -N( ^R27 )( ^R28 ), optionally substituted C1-C6 alkyl, or -C( ^R29 )= C(R ³⁰ )-R ³¹ where R ²⁵ is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, R ²⁶ is optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, R ²⁷ and R ²⁸ are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, or both R ²⁷ and R ²⁸ together with the nitrogen atom to which they are attached form 5 - or a 6-membered heterocycle, R ²⁹ and R ³⁰ are independently hydrogen or optionally substituted C1-C6 alkyl, and R ³¹ is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)OR ³² or -C(=O)NR ³² , where R ³² represents hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, R ⁸ and R ⁹ independently represent is hydrogen or optionally substituted C1- _C6 alkyl; and R' is hydrogen or is a halogen, -NO ₂ , -CN or other electron-withdrawing group or is an electron-donating group, provided that no more than two R ²⁴ are other than hydrogen; where J represents -O-, S-or

- 40 046150

-N(R ³³ )-, where R ³³ represents hydrogen or methyl;

where the wavy line to J represents covalent binding of J containing a functional group W that inhibits the electron donating ability of J sufficiently to stabilize the (hetero)arylene moiety of SI prior to 1,4- or 1,6-elimination, and where enzymatic treatment of W results in to inhibit this ability to cause elimination to release D+ bound to Y as a tubulisin drug compound containing a tertiary amine (eg, when J is linked to the carbonyl moiety of a carbonyl-containing functional group W);

In other embodiments, W' and Y are orthogonal in L _O (i.e., represent -Y(W')- in the linker unit), wherein the SI in Y is linked to a glycosidic bonded carbohydrate moiety having a recognition site for a glycosidase, wherein the orthogonal arrangement involving SI in Y is usually represented by the structural formula

where E' is attached to one of V, Z ¹ , Z ³ , provided that the other V, Z1, Z ² (that is, not connected to E') is defined as =C(R ²⁴ )- or =N-. In preferred embodiments, the orthogonal arrangement involving SI in Y is represented by the following structure:

where V, Z ¹ and Z ³ are independently -C(R ²⁴ )= or -N=; R ²⁴ are independently hydrogen, halogen, -NO2, -CN, -OR ²⁵ , -SR ²⁶ , -N(R ²⁷ )(r ²⁸ ), -C(R ²⁹ )=C(R ³⁰ )-R ³¹ or optionally substituted C1- _C6 ;

where R ²⁵ represents hydrogen, optionally substituted ^-^-alkyl, optionally substituted aryl or optionally substituted heteroaryl; R ²⁶ is optionally substituted QQ^kuk, optionally substituted aryl or optionally substituted heteroaryl, and R ²⁷ and R ²⁸ are independently hydrogen, optionally substituted ^-^-alkyl, optionally substituted aryl or optionally substituted heteroaryl, or both R ²⁷ and R ²⁸ together with the nitrogen atom to which they are attached form or represent a 5- or 6-membered heterocycle, R ²⁹ and R ³⁰ are independently hydrogen or optionally substituted ^-^-alkyl, and R ³¹ is hydrogen, optionally substituted ^-^-alkyl, optionally substituted aryl, optionally substituted heteroaryl, -CN, C(=O)OR ³² or -C(=O)NR ³² ; where R ³² represents hydrogen, optionally substituted ^-^-alkyl, optionally substituted aryl or optionally substituted heteroaryl;

R ⁸ and R ⁹ are independently hydrogen or optionally substituted ^-^-alkyl; R' is hydrogen or is a halogen, -NO2, -CN or other electron-withdrawing group or is an electron-withdrawing group; R ⁴⁵ represents -CH2OH, -CO ₂ H; E' represents -O- or -NH-; J' represents -NH-; and D+ has the meanings defined in the embodiments described for the quaternized drug units.

In more preferred embodiments, the orthogonal arrangement involving SI in Y has the following structure:

In preferred embodiments, -E'- is -O- or -NH- and V or Z ³ is =C(R ²⁴ ), where R ²⁴ is hydrogen or an electron-withdrawing group. In other preferred embodiments, R ⁸ and R ⁹ are hydrogen and V, Z ¹ or Z ² is =CH-B in other preferred embodiments -J'- is

-NH, V, Z ¹ or Z ² represents =CH- and R' represents hydrogen or electron-withdrawing

- 41 046150 group, preferably -Cl, -F or -NO2.

1.3. L _R -L _O as linker units.

A quaternized drug compound (D ⁺ ) unit attached to any of the above self-destructive moieties described herein is a quaternized tubulisin compound in which the tertiary amine of the C-terminal component of the tubulisin compound is quaternary (i.e., D ⁺ is a tubulisin compound containing a quaternized tertiary amine), where the quaternary nitrogen is attached to the benzyl position of the SI moiety in the self-breaking spacer unit.

In some embodiments, -LB-LO-D+ or LB'-L _O -D ⁺ has the following structure:

wherein V, Z ¹ and Z2 are independently =N- or =C(R ²⁴ )-, where R ²⁴ is independently selected to represent hydrogen, an optionally substituted alkyl or electron-donating group, and R ⁸ and R ⁹ are independently selected to represent is hydrogen or optionally substituted alkyl, and J is -O- or -N(R ³³ ), where R ³³ is hydrogen or lower alkyl.

In preferred embodiments where A, W and Y are in a linear configuration, -LBL _O -D ⁺ or LB'-L _O -D ⁺ has the following structure:

In more preferred embodiments, where A, W and Y have a linear configuration, the drug-ligand conjugate or drug-linker compound of formula LR-LO-D+ has the following structure:

where W is a naturally occurring L-amino acid residue or represents successive amino acid subunits such that W includes or consists of a dipeptide, where the dipeptide is at the distal end of W, where the dipeptide and said bond is an amide bond specifically cleavable by an intracellular protease versus freely circulating serum proteases. In preferred embodiments, the amino acid subunit attached to the J/NH in Y is a naturally occurring L-amino acid or a non-natural amino acid in which the amine-bearing carbon atom has the same stereochemical configuration.

In any of the above embodiments, where W consists of a dipeptide that is recognized by an intracellular protease, preferably a cathepsin protease, preferred dipeptides have the structure

where R ³⁴ represents benzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH(OH)CH ₃ or has the following structure

- 42 046150

and R ³⁵ is methyl, -(CH ₂ ) ₄ -NH2, -(CH ₂ ) ₃ NH(C=O)NH ₂ , (CH ₂ ) ₃ NH(C=NH)NH ₂ or -(CH ₂ ) ₂ CO ₂ H, where the wavy line at the dipeptide N-terminus indicates covalent attachment to A or to L _B or L _B ', and the wavy line at the dipeptide C-terminus indicates covalent attachment to J.

In preferred embodiments, -L _B and L _B ' are succinimide (M ² ) or maleimide (M ¹ ) moieties, respectively. In such embodiments, -L _B -A- and -L _B -A ₁ -A ₂ are referred to as succinimide-containing moieties, which are typical LSS moieties when A or A ₁ includes a backbone unit, and L _B '-A and L _B '-A ₁ - are indicated as maleimide-containing moieties that are precursors to L _SS moieties when A or A ₁ includes a backbone unit.

Preferably, A _O , when A _O is present, in any of the above L _R -L _O structures in which W, Y and D+ are in a linear configuration, corresponds in structure to an amine acid, or is an amine acid containing residue where the carboxy termini of the acids are containing an amine are linked to W as an ester or amide, preferably as an amide, and their N-termini are linked to LP in -LP(PEG)- via a carbonyl-containing functional group.

In other preferred embodiments, -L _B -L _O -D ⁺ or L _B '-L _O -D ⁺ has the following structure:

where V and Z ³ are, independently, =N- or =C(R ²⁴ )-, where R ²⁴ , independently selected, is hydrogen, an optionally substituted alkyl or electron-donating group, R ⁸ and R ⁹ , independently, are hydrogen or optionally substituted alkyl, and J' represents -O- or -N(R ³³ ), where R ³³ represents hydrogen or lower alkyl, and R' represents hydrogen or an electron-withdrawing group.

In other more preferred embodiments, L _R -L _O -D ⁺ (i.e. -LB-LO-D ⁺ or LB'L _O -D ⁺ ) has the following structure:

- 43 046150 where V, Z ¹ or Z ³ represents =N- or =C(R ²⁴ )-, where R ²⁴ , independently selected, represents hydrogen, an optionally substituted alkyl or electron-donating group, R ⁸ and R ⁹ , independently , represent hydrogen or an optionally substituted alkyl, and J' represents -O- or -N(R ³³ ), where R ³³ represents hydrogen or lower alkyl, R' represents hydrogen or an electron-withdrawing group, and O' represents a bonded glycosidic bond oxygen, where the bond can be cleaved by glycosidase. In preferred embodiments, J' is -NH-.

Preferably, _AO , when _AO is present, in any of the above structures, LR-Lo- corresponds in structure to the amine acid, where the carboxyl terminus of the amine acid is linked to J/J' as an ester or amide, preferably as amide, and its N-terminus is linked to LP via a carbonyl-containing functional group.

In specific preferred embodiments, -LB-A- or LB'-A in any of the above embodiments -LB-LO-D+, LB'-L _O -D ⁺ or LR-LO-D+ has the following structure M1-A, M1-A1-A2-, M2-A or M2-A1-A2-, provided

or

ABOUT

AnA

R^A [C(R ^b1 )(R ^b1 )] _q -[HE]-A ₂ -A k _y J

Ai

I where A1 and A ₂ are subunits of A, LB is a succinimide moiety (M ² ) and LB' is a maleimide moiety (M ¹ ), where -[C(R ^b1 )(R ^b1 )]q-[HE] - represents A or a subunit (A1) in A; R and Ra ² independently represent hydrogen or optionally substituted alkyl; R ^a1 represents hydrogen, lower alkyl or BU; HE is an optional hydrolysis enhancer (HE) unit; subscript q denotes an integer in the range from 0 to 6; each R ^b1 independently represents hydrogen, optionally substituted C1- _C6 alkyl, optionally substituted aryl or optionally substituted heteroaryl, or two R ^b1 together with the carbon atom(s) to which they are attached form or represent C3-C6 cycloalkyl , or one R ^b1 and HE together with the carbon atom to which they are attached form or represent a 5- or 6-membered cycloalkyl or a 5- or 6-membered heterocycloalkyl, and the other R ^b1 is hydrogen, optionally substituted with C1-C ₆ -alkyl, optionally substituted aryl or optionally substituted heteroaryl; BU is a main unit having the structure -[C(R ¹ )(R ¹ )]-[C(R ² )(R ² )]nN(R ²² )(R ²³ ), where the subscript n denotes 0, 1, 2 or 3, R ¹ is, independently, hydrogen or lower alkyl, or two R ¹ together with the carbon atom to which they are attached form or represent C3-C6 cycloalkyl, R ² , independently, is hydrogen, optionally substituted C1- _C6 -alkyl, optionally substituted aryl or optionally substituted heteroaryl, or two ^R2s together with the carbon atom(s) to which they are attached and intermediate carbon atoms are C3-C6cycloalkyl, or one ^R1 and one ^R2 together with the carbon atoms to which they are attached and intermediate carbon atoms form or denote a 5- or 6-membered cycloalkyl, and the remaining R ¹ and R ² are as indicated; R ²² and R ²³ , independently, represent hydrogen or an optionally substituted ^-^-alkyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached form or represent a 5- or 6-membered heterocycloalkyl, or one of R ²² , R ²³ represents hydrogen, and the other represents an acid-sensitive protecting group; and wherein the sulfhydryl moiety of the targeting agent is linked to M ² as a targeting ligand unit as indicated by the wavy line to the succinimide moiety, and where the wavy line to HE (or to [C(R ^b1 )(R ^b1 )]q when HE is not present ) denotes covalent attachment to another subunit A or to -LP(PEG)-.

In other specific preferred embodiments, -LB-A- or -LB-A1-A2- in any of the above embodiments containing _LB has the following structure -M ³ -A- or

-M ³ -A ₁ -A ₂ -, represented by

- 44 046150

where A1 and A2 are subunits of A, and M ^3A and M ^3B are regioisomers of M ³ , and where the variable groups and binding to the sulfhydryl group of the target moiety and HE (or [C(R ^b1 )(R ^b1 )]q) are such as indicated for the corresponding succinimide-containing fragments shown immediately above. In the illustrated embodiments, LB refers to the succinic acid-amide (M ³ ) moiety, and -LB-A-, L _B' -A-, -L _B -A ₁ - or Lg'-A ₁ - are indicated as moieties containing succinic acid amide, which are representative fragments of L _S .

In these and any of the above embodiments involving HE, HE is preferably -C(=O)-.

In any of such embodiments, -L _B -A-, L _B '-A-, -LB-A1- and L _B '-A1- or M1-A, M ¹ -A1-A2-, M2-A-, M2-A1-A2-, M ³ -A- and M ³ -ArA ₂ - each R ^b independently is preferably hydrogen or lower alkyl, and the subscript m is 0 or 1, R ^a1 is preferably hydrogen, lower alkyl or BU or R ^a 2 is preferably hydrogen.

In any of the above embodiments where W, Y and D are in a linear configuration and include A or A1-A2, preferred embodiments are those wherein W is linked to A or _A2 via an amide functionality. In such embodiments, preferably, A, A ₁ and A ₂ have independently selected structures corresponding to or including amine containing acids as described herein with respect to embodiments of the extender unit. In any of the above embodiments, -L _B -A-, L _B '-A-, -LB-A1- and L _B '-A].- include A or A1, A and A1 preferably having structures corresponding to or including amine-containing acids, or are amine-containing acid residues, as described herein with respect to embodiments of the extender unit, wherein A is linked to W, or A ₁ is linked to A ₂ via an amide functionality. In any of the above embodiments, M1-A-, M1-A1-A2-, M2-A-, M2-A1-A2-, M ³ -A- and M3-A1-A2-, preferably A, A ₁ and A ₂ have independently selected structures corresponding to or including amine-containing acids, or are independently selected amino acid residues, as described herein with respect to embodiments of the first extension unit A and the second extension unit A _o . In any of the above embodiments, the L _SS or _LS that are part of the -A(Bi)- or -A1(Bi)- moieties, A and A1 preferably have structures corresponding to or including acids containing an amine substituted with BU , and are therefore diamino-containing acids, as described herein with respect to embodiments of the extender unit and the base unit. In fragments containing M ¹ , M2 and M ³ having fragments A or A1 corresponding to the structure of the amine-containing acid or the residue of the amine-containing acid, the amine nitrogen of the amine-containing acid is included as the imine nitrogen of the M ¹ or M2 ring system or the amide nitrogen of the ^M3 fragment. In fragments containing L _SS or _LS , the N-terminal amine nitrogen of the diamino-containing acid is included as the imine nitrogen of the M ¹ or M ² ring system or the amide nitrogen of the M ³ fragment. Preferably, for any of the above fragments containing M ¹ -, M2

- 45 046150 and M ³ -, or fragments containing L _SS - or L _S -, the carboxylic acid group of the amine-containing acid or diamino-containing acid is included in the amide functional group attached to A2 for those fragments that contain A1-A2-, or attached to W, for those fragments that contain A when A is a single unit.

In more preferred embodiments, A or A ₁ and A2 in -A1-A2- are independently represented by structures (3) or (4) wherein L is absent (i.e., the subscript e is 0) and G is hydrogen, BU, -CO2H, or -NH2, or a side chain of a natural amino acid such as aspartic acid, glutamic acid or lysine, and other variable group has the meanings defined above. In other preferred embodiments, L and K are carbon, and R ⁴¹ , R ⁴² , R ⁴³ and R ⁴⁴ are each hydrogen, and R ³⁸ - R ⁴⁰ and the subscripts e, f and g are as defined above. In other preferred embodiments, R ³⁸ -R ⁴⁴ are in each case hydrogen, and K, L and the subscripts e, f and g are as defined above. Other preferred embodiments have structure (3) wherein K is nitrogen and one of R ⁴¹ , R ⁴² is absent and the other is hydrogen, and L, subscripts e, f and g and the remaining variable groups R ³⁹ -R ⁴² have the meanings defined above. Other preferred embodiments have structure (4) where the subscript g is 1, K is nitrogen, and one of R ⁴¹ , R ⁴² is absent and the other is hydrogen, L, subscripts e and f and the remaining variable groups R ³⁹ - R ⁴² have the meanings defined above. In other preferred embodiments, the subscripts e and f in structure (3) are each 0, or the subscripts f and g in structure (4) are each 0, where K, L and the remaining variable groups R ³⁸ -R ⁴⁴ have the meanings defined above. Other preferred embodiments have structure (3) wherein the subscripts e and f are both 0 and K together with R ⁴¹ and R ⁴² is -C(=O)- and the remaining variable groups R ³⁸ -R ⁴⁰ are , defined above. Other preferred embodiments have structure (4) wherein the subscript f is 1 and L together with R ⁴³ and R ⁴⁴ is -C(=O)-, and K, L, subscript g and R ³⁸ -R ⁴² are , defined above.

In more preferred embodiments, A or A1 and A2 in -A1-A2- are independently of structure (3a) or (4a) wherein the subscript e or f is 0 or 1, and G and ^R39 - ^R44 are defined above.

When the fragment L _SS or L _S includes A or A1, the preferred structures of A or A1 are those shown for (3), (3a), (4) and (4a), where R ³⁸ is absent, G is the main unit (BU), and the N-terminal nitrogen is included in the M ¹ or M ² fragment as the imine nitrogen of this fragment ring system or is included in the M ³ fragment as the amide nitrogen of the succinic acid amide.

In other more preferred embodiments, A or A1 and A _O in A correspond independently in structure to, or are independently selected alpha-amino, beta-amino, or other amine-containing acid residues. When the L _SS or L _S fragment consists of A or A1, the preferred A or A ₁ fragments correspond in structure to or are residues of an alpha-amino, beta-amino, or other amine-containing acid substituted with BU (i.e., a diamino-containing acid) wherein the N-terminal nitrogen of a BU-substituted alpha-amino, beta-amino or other amine-containing acid that is A(BU) or A1(BU) is included in the ^M1 or ^M2 moiety as the imino nitrogen of that fragment ring system or included in M ³ as the amide nitrogen of the succinic acid amide moiety.

In such embodiments, particularly preferred A(BU) or A1(BU) have structure (3) or (3a), where the subscript e denotes 0 and G represents BU, or have structure (4) or (4a), where the subscript f stands for 1 and G represents BU. In embodiments where W, Y and D+ are in a linear arrangement where _AO is present, particularly preferred amine-containing acids included as _AO have the structure NH2-X1-CO2H, where X ¹ is an optionally substituted C1-C _6- alkylene, including s-amino-caproic acid and β-amino-propionic acid.

Preferred embodiments wherein A, W and Y being in an orthogonal configuration in -L _B -L _O -D ⁺ or L _B '-L _O -D ⁺ have the structure

- 46 046150

and other preferred embodiments wherein A, W and Y have a linear configuration in -LB-L _O -D ⁺ or LB'-L _O -D ⁺ have the structure

where A represents a unit link and A _O is present.

In more preferred embodiments, E' is O', J/J' is -NH-, R ³⁴ is methyl, isopropyl or -CH(OH)CH3, and R ³⁵ is methyl, -(CH2)3NH( C=O)NH2 or -(CH2)2CO2H, R ⁴⁵ is -CO2H, and R', R ⁸ , R ⁹ , J, V, Z ¹ , Z ² and Z ³ are as defined above or in Eqs. 1A, 1B, 1D, or in formulas IA, IB or ID. In more preferred embodiments, R ⁸ and R ⁹ are each hydrogen. In yet other more preferred embodiments, E is O', J/J' is -NH-, V, Z ¹ , Z ² and Z ³ are each -CH=. Also more preferred are embodiments wherein LB' has the structure of a maleimide moiety (M ¹ ), or wherein LB has the structure of a succinimide moiety (M ² ) or a succinic acid-amide moiety (M ³ ).

More preferred are those embodiments in which L _B '-A has the structure given above for any of the M1-A-, and more preferably, the -LB-A- fragments have the structure given above for any of the M ² -A- fragments. or M3-A1-. In any such embodiments, J/J' is preferably -NH-.

In preferred embodiments in which W', Y and D ⁺ are orthogonally bonded, A _O is present and has the structure previously defined for (3), (3a), (4) or (4a), where the wavy line to the carbonyl moiety of either structure represents the point of attachment of AO to J', preferably via an amide functionality, and wherein the wavy line to the amine moiety of either structure represents the point of attachment to a carbonyl-containing functionality in LP or -LP(PEG)-, preferably , forming an amide functional group; wherein the variable groups have the meanings defined above for structures representing A or A ₁ and A ₂ in -A1-A2-. In preferred embodiments, L is absent (ie, the subscript q is 0) and G is hydrogen, -CO ₂ H or -NH ₂ or a side chain of a naturally occurring amino acid such as aspartic acid, glutamic acid or lysine. In other preferred embodiments, L and K are carbon, and R ⁴¹ , R ⁴² , R ⁴³ and R ⁴⁴ are each hydrogen. In other preferred embodiments, R ³⁸ -R ⁴⁴ is hydrogen in each case. Other preferred embodiments have structure (3) wherein K is nitrogen and one of ^R41 , ^R42 is absent and the other is hydrogen. Other preferred embodiments have structure (4) wherein r is 1, K is nitrogen and one of ^R41 , ^R42 is absent and the other is hydrogen. In other preferred embodiments, the subscripts p and q in structure (3) are 0, or the subscripts q and r in structure (4)

- 47 046150 denotes 0. Other preferred embodiments have structure (3), where the subscripts p and q both denote 0, and K together with R ⁴¹ and R ⁴² represents -C(=O)-. Other preferred embodiments have structure (4) wherein the subscript q is 1 and L together with R ⁴³ and R ⁴⁴ is -C(=O)-.

In more preferred embodiments, A or A1 and AO in A in the structure independently correspond to or represent an alpha-amino, beta-amino, or other amine-containing acid moiety. In other more preferred embodiments having an L _SS or L _S moiety, the moiety consists of A or A1 with preferred structures corresponding to those shown for (3), (3a), (4) and (4a), where R ³⁸ is missing, G is a backbone unit (BU), and the N-terminal nitrogen is included in the M ¹ or M ² moiety as the imine nitrogen of these ring system moieties or is included in the M ³ moiety as the amide nitrogen of the succinic acid amide. Other preferred A or A1 moieties for LSS or LS correspond in structure to or include an alpha-amino, beta-amino, or other BU-substituted amine acid (i.e., a diamino-containing acid), wherein the N-terminal nitrogen is BU-substituted alpha -amino, beta-amino or other amine-containing acid, which is A(BU) or A ₁ (BU), is included in the M ¹ or M ² moiety as the imine nitrogen of these ring system moieties or is included in M ³ as the amide nitrogen succinic acid amide fragment.

In embodiments where A, W' and Y have an orthogonal configuration in -L _B -L _O -D ⁺ or LB'-LO-D ⁺ where AO is present, amine-containing acids that correspond to the AO include the NH2 structure are particularly preferred -X'-CO ₂ H. where X ¹ represents an optionally substituted C ₁ -C ₆ -alkylene, including s-aminocaproic acid and β-amino-propionic acid.

Particularly preferred are any of the above-described embodiments containing LB, wherein the target moiety associated with the LB is an antibody.

1.3.1. Ligand unit.

In some embodiments, a ligand moiety is present. The ligand unit (L-) is a targeting moiety that specifically binds to the target moiety. The ligand unit can specifically bind to a cell component (cell-binding agent) or other target molecules of interest. The ligand unit acts to target and deliver quaternized tubulisin drug compound units to specific target cell populations with which the ligand unit interacts to selectively release D ⁺ as the free tubulisin compound. Targeting agents include, but are not limited to, proteins, polypeptides and peptides. Suitable ligand units include targeting agent units such as antibodies, e.g., full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules (such as, but not limited to, transferrin) or any other cell-binding molecule or substance. The ligand unit may, for example, be from a non-targeting agent targeting the antibody protein. Alternatively, the targeting agent may be, for example, an antibody. Preferred targeting agents are higher molecular weight proteins, for example, cell binding agents having a molecular weight of at least about 80 kDa.

The targeting agent reacts with the covalent binding unit of the ligand precursor L _B ' to form LL _B -, where L is the ligand unit and L _B is the covalent binding ligand unit. The targeting agent must have the required number of attachment sites to accommodate linker drug moieties, each containing an LB, whether natural or non-natural (eg, engineered). For example, for a subscript p value to be between 6 and 14, the targeting agent must be able to bind 6 to 14 moieties of the drug compound to the linker. The targeting agent may form a bond with LB' in the linker unit of the drug compound-linker connection via a reactive or heteroatom-activated or heteroatom-containing functional group of the targeting agent. Reactive or heteroatom-activated or heteroatom-containing functional groups that may be present on the targeting agent include sulfur (in one embodiment from the sulfhydryl group of the targeting agent), C=O or (in one embodiment from the carbonyl, carboxyl or hydroxyl group of the targeting agent) and nitrogen (in one embodiment, from the primary or secondary amino group of the targeting agent). These heteroatoms may be present in the targeting agent in the natural state of the targeting agent, for example, in a natural antibody, or may be introduced into the targeting agent through chemical modification or biological engineering.

In one embodiment, the targeting agent has a sulfhydryl group and its ligand unit is attached to the linker unit through the sulfur atom of the sulfhydryl group.

In another embodiment of the invention, the targeting agent has lysine residues that can react with activated esters (such as esters

- 48 046150 include, but are not limited to, N-hydroxysuccinimide, pentafluorophenyl and p-nitrophenyl esters) of the LB' linker unit connecting the drug compound with the linker and thereby forming an amide bond between the nitrogen atom of the ligand unit and the C=O group of the linker unit.

In yet another aspect, the targeting agent has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The ligand unit from such a targeting agent is attached to the linker unit through the sulfur atom of the included sulfhydryl group. Reagents that can be used to modify lysines in this manner include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Trot's reagent).

In another embodiment of the invention, the targeting agent may have one or more carbohydrate groups, which may be chemically modified to have one or more sulfhydryl groups. The ligand unit from such a targeting agent is attached to the linker unit through the sulfur atom of the included sulfhydryl group.

In yet another embodiment, the targeting agent may have one or more carbohydrate groups that can be oxidized to produce an aldehyde (-CHO) group (see, e.g., Laguzza, et al., 1989, J. Med. Chem. 32( 3):548-55). The corresponding aldehyde can then react with L _B ', which has a nucleophilic nitrogen. Reaction sites on L _B ' that can react with the carbonyl group on the targeting agent include, but are not limited to, hydrazine and hydroxylamine. Other protocols for modifying proteins to attach drug linker moieties are described by Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002) (incorporated herein by reference).

The targeting agent forms a bond with a reactive group on LB' in the drug-linker connection to form an LDC in which the drug-linker moiety is included in the LB-containing moiety. Various reactive groups can be used and will depend on the nature of the desired ligand unit. The reactive group may be a maleimide that is present on L _B ' (prior to attachment to L), and covalent attachment of L to L _B is achieved through the sulfhydryl group of the targeting agent to form a thio-substituted succinimide. The sulfhydryl group may be present on the targeting agent in the natural state of the targeting agent, for example, as a natural moiety, or may be introduced into the targeting agent through chemical modification.

In yet another embodiment of the invention, the targeting agent is an antibody and the sulfhydryl group is formed by reduction of an interchain disulfide. Accordingly, in some embodiments, the linker unit is conjugated to a cysteine residue of reduced interchain disulfides of the ligand unit.

In yet another embodiment of the invention, the targeting agent is an antibody and the sulfhydryl group is chemically incorporated into the antibody, for example, by introducing a cysteine residue. Accordingly, in some embodiments, the linker unit is conjugated to the introduced cysteine residue.

For bioconjugates, it has been observed that the site of conjugation of a drug compound can influence a number of parameters, including ease of conjugation, stability of the drug compound with the linker, influence on the biophysical properties of the resulting bioconjugates, and in vitro cytotoxicity. With respect to drug-linker stability, the site of conjugation of the drug-linker moiety to the ligand unit may affect the ability of the drug-linker conjugate moiety to undergo an elimination reaction and transfer the drug-linker moiety from the ligand unit of the bioconjugate to an alternative reactive thiol present in the bioconjugate environment. , such as, for example, reactive thiol in albumin, free cysteine or glutathione in plasma. Such regions include, for example, interchain disulfides, as well as individual cysteine-constructed regions. The drug-ligand conjugates described herein can be conjugated to thiol residues at sites that are less susceptible to elimination reactions (eg, EU index position 239 as reported in Kabat) among other sites.

When the conjugates contain ligands from a non-immunoreactive protein, polypeptide or peptide instead of an antibody, the ligands from the non-immunoreactive protein, polypeptide or peptide used include, but are not limited to, transferrin, epidermal growth factors (EGF), bombesin, gastrin, gastrin releasing peptide, platelet-derived growth factor , IL-2, IL-6, transforming growth factors (TGFs) such as TGF-α and TGF-β, vaccinia virus growth factor (VGF), insulin and insulin-like growth factors I and II, somatostatin, lectins and apoproteins from low density lipoproteins.

Particularly preferred targeting agents are antibodies, including intact antibodies. In fact, in any of the embodiments described herein, whether

- 49 046150 the gangnd unit may be an antibody. The polyclonal antibodies used are homogeneous populations of antibody molecules obtained from the sera of immunized animals. The monoclonal antibodies used are homogeneous populations of antibodies to a specific antigenic determinant (eg, cancer cell antigen, viral antigen, microbial antigen, protein, peptide, carbohydrate, chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen of interest can be produced using any method known in the art that produces antibody molecules from continuous cell lines in culture.

Suitable monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be prepared by any of numerous methods known in the art (eg, Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

An antibody may be a functionally active fragment, derivative, or analogue of an antibody that immunospecifically binds to target cells (eg, cancer cell antigens, viral antigens, or microbial antigens) or other antibodies associated with tumor cells or matrix. In this regard, functionally active means that the fragment, derivative or analogue is capable of immunospecifically binding to target cells. To determine which CDR sequences bind an antigen, synthetic peptides containing the CDR sequences can be used in antigen binding assays by any binding assay known in the art (e.g., BIAcore assay) (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. Immunology 125(3):961-969).

Other antibodies used include antibody fragments such as, but not limited to, F(ab') ₂ fragments, Fab fragments, Fvs, single chain antibodies, diabodies, tribodies, tetrabodies, scFv, scFv-FV or any other molecule with the same specificity like an antibody.

In addition, the antibodies used are recombinant antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human parts, which can be produced using standard recombinant DNA techniques. A chimeric antibody is a molecule in which different parts are derived from different animal species, for example, those that have a variable region derived from the constant regions of a murine monoclonal and human immunoglobulin (see, for example, US Pat. No. 4,816,567; and US Pat. No. 4,816,397 , which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules of a non-human species having one or more complementarity determining regions (CDRs) from a non-human species and a framework region of a human immunoglobulin molecule (see, for example, US Pat. No. 5,585,089, which is incorporated herein by reference in its entirety ). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA methods known in the art, for example, using the methods described in international publication No. WO 87/02671; European Patent Publication No. 0184187; European Patent Publication No. 0171496; European Patent Publication No. 0173494; International publication No. WO 86/01533; US Patent No. 4816567; European Patent Publication No. 0012023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; US Patent No. 5225539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.

Fully human antibodies are particularly desirable and can be produced using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.

Antibodies include analogs and derivatives that are either modified, that is, by covalent attachment of any type of molecule, provided that such covalent attachment allows the antibody to substantially retain its antigen-binding immunospecificity. For example, but not by way of limitation, antibody derivatives and analogs include those that have been further modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody moiety, or other squirrel, etc. Any of numerous chemical modifications can be carried out by known methods, including, but not limited to

- 50 046150 limited to this, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. The analogue or derivative may contain one or more unnatural amino acids.

Antibodies may have modifications (eg, substitutions, deletions or modifications) at amino acid residues that interact with Fc receptors. In particular, antibodies may have modifications to amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, for example, International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety).

Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one skilled in the art, such as, for example, chemical synthesis or recombinant expression methods. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or a database like this, literature publications, or by conventional cloning and sequencing.

In a specific embodiment, known antibodies for the treatment of cancer can be used. Antibodies that are immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one skilled in the art, for example, by recombinant expression methods. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or a database like this, literature publications, or by conventional cloning and sequencing.

In another specific embodiment, antibodies for treating an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies that are immunospecific for a cell antigen responsible for the production of autoimmune antibodies can be obtained from any organization (for example, a university scientist or company) or produced by any method known to one skilled in the art, such as, for example, chemical synthesis or recombinant expression methods .

In some embodiments, the antibodies used may bind to a receptor or receptor complex expressed on an activated lymphocyte. The receptor or receptor complex may comprise a member of the immunoglobulin gene superfamily, a member of the TNF receptor superfamily, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.

In some aspects, the antibody will specifically bind to CD19, CD20, CD30, CD33, CD70, alpha-y-beta-6, or Lewis Y antigen.

The antibody may be a humanized anti-CD33 antibody (US 2013/0309223, incorporated herein by reference in its entirety for all purposes), a humanized anti-Beta6 antibody (see, for example, WO 2013/123152, incorporated herein by reference in its entirety and for all purposes), a humanized anti-Liv-1 antibody (see, for example, US 2013/0259860, incorporated herein by reference in its entirety for all purposes), or a humanized AC10 antibody (see, for example, US 8,257,706, incorporated herein by reference). herein by reference and for all purposes).

An example of attachment to a ligand is through thioether bonds. Thioether bonds can be through interchain disulfide bonds, introduced cysteine residues, and combinations thereof.

1.3.2. Parallel connecting link.

The drug-ligand conjugate and its precursor drug-linker compound of the present invention include a PEG unit that is in parallel orientation with the quaternized tubulisin drug unit to influence the pharmacokinetics of the resulting LDC. Parallel orientation of the PEG link is achieved using a parallel connecting link (L _P ). For this purpose, the parallel connecting unit arranges the ligand, PEG and drug units in a branched configuration. Accordingly, the parallel connecting link can be considered as a scaffold component having attachment sites for other components of the drug-ligand conjugate and the drug-linker conjugate.

To act as a parallel connector, the LP unit is attached through three attachment sites in the linker unit. One of the attachment sections connects the L _P link to the PEG link. In one embodiment, the second attachment site connects the LP unit to a protease-sensitive cleavable unit (W), which is connected to a self-destructive spacer unit (Y). In another embodiment, the second LP attachment site is coupled directly to a self-cleaving spacer unit (Y) to which a glycosidase-sensitive cleavable unit (W') is also attached, or in such cases is indirectly coupled to Y via an extender intermediate (Ao). The third attachment site attaches the L _P unit to the remainder of the linker unit,

- 51 046150 which in LDC is usually connected to the ligand unit through another extender unit (A). A parallel connecting link is a link that is different from the PEG link and is attached to it through the PEG component of the connecting link in the PEG link. In other words, the parallel connecting link is not a sub-link of the PEG link.

For drug-ligand conjugates and intermediates thereof having more than one drug per PEG unit, the attachment of the parallel connecting unit to W or Y may be via a branching unit (B) instead of AO. In all of these embodiments, the L _P unit can be considered a trifunctional chemical moiety that is capable of covalently linking together three spaced apart chemical moieties. It is understood that for selected intermediates, the precursor to LP is represented as LP' and is not yet fully incorporated into the linker unit (eg, not yet attached to A, AO, B, W or Y, but has an optionally protected functional group for this attachment). It is also understood that the term trifunctional is used to refer to the three attachment sites and not the number of functional groups present in any LP, LP' or subunit thereof.

The parallel connecting unit may be formed from one or more (typically 1 to 5, or 1 to 4, or 1 to 3, or 1 or 2) natural or unnatural amino acids, amino alcohols, amino aldehydes or polyamines, or some combination thereof.

It is understood that when referring to a natural or unnatural amino acid, amino alcohol, aminoaldehyde or polyamine present in the linker LDC or drug compound of the present invention (whether it is part of the L _P unit or another component of the LDC or drug compound with the linker described herein), the amino acid, amino alcohol, amino aldehyde or polyamines will exist in residual form. For example, in embodiments where the parallel connecting unit is two amino acids, the two amino acids will exist as residues with a peptide bond between them. In embodiments where the parallel linker consists of an amino alcohol, the amino alcohol will exist as a residue where, for example, its amino group is linked to another moiety of the parallel linker or another component of the LDC compound or drug compound with a linker through the carbonyl-containing functional group of that other residue. component while its hydroxyl group is ether-linked or via a carbonyl-containing functional group to another residue of a parallel connecting unit or other component of the LDC compound or drug compound with the linker. In embodiments where the parallel linker unit consists of an amino aldehyde, the amino aldehyde will exist as a residue where, for example, its amino group is linked to another moiety of the parallel linker unit or another component of the LDC compound or drug linker compound through a carbonyl-containing functional group of that other residue /component, wherein its aldehyde functional group is converted to an imino functional group or, by subsequent reduction, forms a nitrogen-carbon bond when it is linked to the amino group of another residue of the parallel connecting unit or another component of the LDC compound or drug compound with a linker. An amino alcohol or amino aldehyde can be produced from a natural or non-natural amino acid by reducing the carboxylic acid functionality to an aldehyde or hydroxyl functionality.

In some embodiments, the parallel linking unit or subunit thereof having the required trifunctionality is provided by an amino acid or other amine-containing acid residue that has or can be substituted with a functionalized side chain to provide the required three points of attachment. For example, the series has three functional groups, that is, acid, amino and hydroxyl functional groups, and it can be considered as a combined amino acid and amino alcohol residue for its inclusion in a parallel connecting unit. Tyrosine also contains a hydroxyl group, in this case on its phenolic side chain, and can also be considered similarly to serine for its inclusion as a trifunctional component of the parallel connecting unit.

In another example, when the three attachment sites of a parallel linker or subunit thereof are provided by cysteine, its amino and carboxylic acid groups will exist in residual form in a manner as discussed previously for amino acids or amino acids to provide two of the three required attachment points, while while its thiol group will exist in a residual form to provide another necessary point of attachment. In some cases, the residual thiol group is in its oxidized form (ie -S(=O)- or -S(=O) ₂ -) when it is linked to another subunit of the parallel linking unit or to another component of the linker unit. In yet another example, the alpha-amino and carboxyl groups of lysine will exist in residual form to provide two of the three required attachment points for the parallel linking unit, while its epsilon-amino group in its residual form provides the remaining attachment point. Histidine can also be thought of as an amino acid with two amino groups, where the second amino group is NH containing

- 52 046150 cabbage soup imidazole side chain.

In another example, when the three attachment sites of a parallel linker are provided by aspartic or glutamic acid, the alpha amino and C-terminal carboxyl functional groups of the amino acid in their residual forms provide two of the three required attachment points, while its beta or gamma the carboxyl functional group in its residual form provides the remaining attachment. Where a naturally occurring amino acid is specified as a parallel linker or subunit thereof, but does not contain a naturally functionalized amino acid side chain, although it is required to be a trifunctional L _P component, it is understood that the structure of the amino acid is modified to have an additional functional group in addition to its amino and carboxylic acid functional groups in residual form to provide the necessary third point of attachment. For example, an amino acid having an aliphatic side chain may be substituted on a carbon atom of that side chain with a hydroxyl, amino, aldehyde, thiol, carboxylic acid or other functional group or other moiety (e.g., aryl or arylalkyl substituted with any of these functional groups) to provide a non-natural amino acid having the required three points of attachment. Such unnatural amino acids are included in the parallel connecting link, as described above for amino acids and residual forms of introduced functional groups.

Likewise, when an amino aldehyde or amino alcohol is included in a parallel linking unit, the amino aldehyde or amino alcohol will have a third functional group to provide, along with its amino and aldehyde functional groups, the required three points of attachment. In these cases, the amino aldehyde or amino alcohol may correspond in structure to a naturally occurring amino acid that has a functionalized side chain, or to an unnatural amino acid having a functional group that has been introduced into the side chain of the natural amino acid, as described above, wherein the carboxylic acid group of the natural or unnatural amino acid is reduced to hydroxyl or aldehyde functional group.

The amino acid residue L _P may be an alpha, beta or gamma amino acid residue or other acidic amine-containing compound and may be present as its D or L isomer if it contains a chiral carbon to which a natural or natural side chain is linked. unnatural amino acid providing the remaining necessary point of attachment. When the parallel connecting unit consists of more than one natural or non-natural amino acid, amino alcohol, amino aldehyde or polyamine, the amino acids, amino alcohols, amino aldehydes, polyamines or combinations thereof are linked to each other through covalent bonds to form the parallel connecting link.

The amino acid, amino alcohol, or amino aldehyde may be non-natural and may be modified to have a functionalized side chain for attachment to components of a drug-ligand conjugate or drug-linker conjugate (as described above for the parallel linker moiety), as appropriate. Examples of functionalized amino acids, amino alcohols, or amino aldehydes include, for example, azido or alkyne functionalized amino acids, amino alcohols, or amino aldehydes (e.g., an amino acid, amino alcohol, or amino aldehyde modified to have an azide group or alkyne group for attachment using click chemistry).

The attachment within the parallel connecting unit or with other components of the conjugate (or linker) can be, for example, through amino, carboxy or other functional groups. Methods for independently activating and interacting functional groups present on an amino acid - e.g., an amino site, a carboxylic acid site, and a side chain site (e.g., an amine moiety, a hydroxyl group, another carboxylic acid group, a thiol, an azide, or an alkyne) involve a suitable type of chemistry peptides.

The parallel linking unit may contain 1 or more (usually 1-5 or 1-4 or 13 or 1 or 2) amino acids, optionally substituted Ci-20-heteroalkylenes (preferably optionally substituted Ci-12-heteroalkylene), optionally substituted C _3-8 heterocycles, optionally substituted C _6-14 arylenes, optionally substituted O-O carbocycles, or combinations thereof. In some embodiments, the parallel connecting unit comprises no more than 2 or no more than one optionally substituted C _1-20 -heteroalkylene, optionally substituted O^ heterocycle, optionally substituted O^-arylene, or optionally substituted O-O-carbocycle. Optional substituents include (=O), -X, -R, -OR, -SR, -NR ₂ , -NR ³ , =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 ^- , -SO3H, -S(= O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO=3, -PO3H2, -ASO2H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2 or -C(=NR)NR2, where each X is independently a halogen: -F , -Cl, -Br or -I; and each R independently represents -H, -^^(halkyl, ^-C^-aryl, -C3-C ₁₄ -heterocycle, protecting group or prodrug moiety. Preferred optional substituents are (=O), -X, - R, -OR, -SR, and -NR ₂ .

The parallel connecting link can be represented by the formula AA

- 53 046150

Formula AA, where AA ¹ is a subunit of LP independently selected from an amino acid optionally substituted with C ₁ . _{a 20} -heteroalkylene (preferably an optionally substituted C _1-12 heteroalkylene), an optionally substituted C _3-8 heterocycle, an optionally substituted C _6-14 arylene or an optionally substituted C ₃ -C8 carbocycle;

and index h is independently selected from 0 to 4; and the wavy line indicates the sites of covalent attachment in the ligand-drug conjugate or intermediate thereof. The optionally substituted heteroalkylene, heterocycle, arylene or carbocyclo must have functional groups to form a connection between subunits and in a ligand-drug conjugate or intermediates thereof.

In some embodiments, in at least one instance, AA ¹ is an amino acid. The subscript h may be 0, 1, 2, 3, or 4. In some embodiments, AA ¹ is an amino acid and h is 0. In some embodiments, the parallel connecting unit consists of no more than 2 optionally substituted C _1-20 heteroalkylenes, optionally substituted C _3-8 heterocycles, optionally substituted C _6-14 arylenes or optionally substituted C ₃ -C ₈ carbocycles. In some embodiments of Formula AA, the parallel connecting unit consists of no more than 1 optionally substituted C _1-20 heteroalkylene, optionally substituted C _3-8 heterocyclo, optionally substituted C _6-14 arylene, or optionally substituted C ₃ -C ₈ carbocycle.

The parallel connecting unit or amino acid subunit thereof may be independently selected from a thiol-containing amino acid. The thiol-containing amino acid may be, for example, cysteine, homocysteine or penicillamine in a D- or L-stereochemical configuration.

The parallel connecting unit or amino acid subunit thereof may be independently selected from the group consisting of L- or D-isomers of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine , phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, β-alanine, aminoalkynoic acid, aminoalkandioic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, and their derivatives.

Preferred amino acids include cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine and alanine.

Examples of LP or its sub-units AA ¹ include _R 100 _R 100

- 54 046150 where R ¹¹⁰ represents

*-ch ₂ o-£· *—CH ₂ CH ₂ COO-£- ₅ *--(CH ₂ ) ₄ NHC(=N-NH)CH ₃ , *—ССОНз *—ch ₂ conh-|— *— CH ₂ COO-<>*—CH ₂ CH ₂ CONH-^*—(CH ₂ ) ₄ NHC(=NH)NH-£>, *—(CH ₂ ) ₃ NHC(=NH)NH-£- ₅ < *---(CH ₂ ) ₃ NHC(=N-NH)CH ₃ , > *---(CH ₂ ) ₃ NHC(=NO)CH ₃ , ' *----(CH ₂ ) ₃ NHCH= N-NH-^- ₅ , *—(CH ₂ ) _m NH-^-, *---(CH ₃ ) ₄ NHC(=NO)CH ₃ , * (CH ₂ ) ₃ NHCONH-<>-, * --CH ₂ CH ₂ CH(OH)CH ₂ NH-^— *--CH ₂ CH ₂ OCH ₂ CH ₂ NH--?— *---{CH ₂ ) ₃ NHCH=NO-^- , *- -(CH ₂ ) ₄ NHCONH-£-

_R 111

-CH2OH, independently selected from hydrogen, p-hydroxybenzyl, methyl, isopropyl, isobutyl, sec-butyl,

-CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH,

-(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC (=NH)NH2, -(CH2)4NH2, -(CH2) 4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -С11X11X11(( )11)С1 h\l l·. 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-,

where the asterisk indicates the attachment to the carbon atom marked x;

R ¹⁰⁰ is independently selected from hydrogen or -C ₁ -C ₃ -alkyl (preferably hydrogen or CH3),

R ¹³ is independently selected from the group consisting of -C ₁ -C ₆ -alkylene-, -C ₃ -C8-carbocyclo-, -arylene-, -C ₁ -C ₁₀ -heteroalkylene-, -C ₃ -C8-heterocyclo-, -C ₁ -C ₁₀ -alkylene-arylene-, -arylene-C ₁ -C ₁₀ -alkylene-, C ₁ -C ₁₀ alkylene-(C ₃ -C8-carbocyclo)-, -(C ₃ -C8-carbocyclo) -C ₁ -C ₁₀ -alkylene-, -C ₁ -C ₁₀ -alkylene-(C ₃ -C8-heterocyclo) and -(C ₃ -C8-heterocyclo)-C ₁ -C ₁₀ -alkylene- (preferably - OT^S^-);

Y represents

Y' represents -C(=O)-, -O-, -S-, -NH-, or -N(OT ₃ )-, and the subscripts p, q, and d are integers independently selected from 0 up to 5; and the wavy line indicates covalent addition of an OH or C _1-3 hydrogen of an unsubstituted alkyl group to the compound, provided that at least one of the wavy lines indicates covalent addition to the compound. In some aspects, all wavy lines indicate covalent attachment in the compound (eg, when the LP does not contain any subunits).

In one set of embodiments, LP is a heterocyclic ring having functional groups that can independently form covalent bonds with the noted components (eg, a triazole heterocyclic ring formed from cyanuric acid chloride). In another set of embodiments, LP is an alkane having functional groups attached as described above. In still other embodiments, LP may represent a nitrogen atom.

In some embodiments, the LP in -LP(PEG)-, when assembled, has the formula set forth below:

- 55 046150

wherein the wavy line indicates attachment sites in a ligand-drug conjugate or intermediate thereof, R ¹⁰⁰ is as defined above, an asterisk indicates attachment to the carbon atom marked x, and the wavy line indicates one of three attachment sites; Y is independently selected from N or CH, Y' is independently selected from NH, O or S, and each subscript c represents an integer independently selected from 1 to 10, and preferably 1, 2 or 3.

In preferred embodiments, R ¹¹⁰ is not *— CH ₂ CH ₂ CH(O)CH ₂ NH ₂ .

In some embodiments, LP or a subunit thereof is an aminoalkanedioic acid, a diaminoalkanoic acid, a sulfur-substituted alkanedioic acid, a sulfur-substituted aminoalkanoic acid, a diaminoalkanol, an aminoalkandinol, a hydroxyl-substituted alkanedioic acid, a hydroxyl-substituted aminoalkanoic acid, or a substituted with a sulfur-containing substituent, an aminoalkanol residue, optionally substituted, where the sulfur-containing substituent is in a reduced or oxidized form.

In some embodiments, the parallel linking unit or amino acid subunit thereof has formula A or formula B ⁺ Y --n -g (Formula A) (Formula B) wherein the subscript v denotes an integer in the range of 1 to 4; subscript v' denotes an integer ranging from 0 to 4; XLP is a side chain of a natural or unnatural amino acid or is selected from the group consisting of -O-, -NRLP-, -S-, -S(=O)-, S(=O) ₂ -, -C(=O)-, -C(=O)N(R ^LP )-, -N(R ^LP )C(=O)N(R ^LP )- and -N(R ^lp )C(=NR ^lp )N(R ^lp )-, wherein each ^RLP is independently selected from the group consisting of hydrogen and optionally substituted alkyl, or the two ^RLPs together with their intermediate atoms define heterocycloalkyl, and the remaining RLPs are as defined above; Ar represents arylene or heteroarylene, optionally substituted; each RE and ^RF are independently selected from the group consisting of -H, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl, or RE and ^RF together with the same carbon atom to which they are attached, or RE and ^RF of adjacent atmospheres carbon together with these carbon atoms denote optionally substituted cycloalkyl, the remaining substituents RE and ^RF being defined above; and where the wavy lines indicate covalent attachment in the formula A structure or formula B in the LDC structure.

In some embodiments, -LP(PEG)- has a structure of the formula A1 or A2 / V^ ^LP -PEG / _R E

Ί ^-_Fh ;^ ^peg (Formula Al) (Formula A2) where the variable groups are as specified in Formula A.

In some embodiments, L _P has the structure of the formula wherein X ^P is a side chain of a natural or unnatural amino acid.

In preferred embodiments, Formula A, Formula A1, Formula A2, or Form

- 56 046150 ly B, R ^e and R ^f are independently selected from the group consisting of -H and -Cj-C halkyl. In preferred embodiments of Formula A, Formula A1, or Formula A2, X ^LP is selected from the group consisting of -O-, -NH, -S-, and -C(=O)-.

In some embodiments, L _P or a subunit thereof is selected from the group consisting of lysine, glutamic acid, aspartic acid, cysteine, penicillamine, serine, or threonine in the D or L stereochemical configuration.

In other embodiments, L _P or a subunit thereof is selected from the group consisting of lysine, glutamic acid, aspartic acid, cysteine, or penicillamine in a D- or L-stereochemical configuration.

In other embodiments, L _P or a subunit thereof is a thiol-containing amino acid in a D- or L-stereochemical configuration. The thiol-containing amino acid is preferably cysteine, homocysteine or penicillamine.

In other embodiments, L _P or a subunit thereof is selected from the group consisting of the following amino acids or amine-containing acids: arginine, aspartic acid, asparagine, cysteine, histidine, glutamic acid, glutamine, phenylalanine, series, tyrosine, threonine, tryptophan, ornithine, penicillamine , aminoalkandioic acid, heterocyclocarboxylic acid, citrulline, diaminoalkanoic acid and their derivatives in D- or L-stereochemical configuration.

In other embodiments, L _P or a subunit thereof is selected from the group consisting of cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glutamine, aspartic acid, glutamic acid, and selenocysteine.

In other embodiments, L _P or L _P ' or a subunit thereof is selected from the group consisting of arginine and arginine derivatives. Representative examples of arginine and their derivatives include, but are not limited to: arginine (Arg), N-alkyl-arginine, H-Arg(Me)-OH, H-Arg(NH ₂ )-OH, H-Arg(NO ₂ ) -OH, H-Arg(Ac) ₂ -OH, H-Arg(Me) ₂ -OH (asymmetric), H-Arg(Me) ₂ -OH (symmetric), 2-amino-4-(2'-hydroxyguanidino )butanoic acid (Nm-hydroxy-nor-arginine) and homoarginine.

In other embodiments, LP or LP' or a subunit thereof is selected from the group consisting of aspartic acid and derivatives thereof. Representative examples of aspartic acid and its derivatives include, but are not limited to: aspartic acid (Asp), N-alkyl-aspartic acid, and H-Asp(O-tBu)-OH.

In other embodiments, L _P or a subunit thereof is selected from the group consisting of asparagine and derivatives thereof. Representative examples of asparagine and its derivatives include, but are not limited to: asparagine (Asn), N-alkyl-asparagine and iso-asparagine (H-Asp-NH ₂ ).

In other embodiments, L _P or L _P ' or a subunit thereof is selected from the group consisting of glutamic acid and derivatives thereof. Representative examples of glutamic acid and its derivatives include, but are not limited to: glutamic acid (Glu), N-alkyl-glutamic acid, H-Glu(OtBu)-OH, H-γ-hydroxy-Glu-OH, H-γ- methylene-Glu-OH, H-γ-carboxy-[Glu(O-tBu)] ₂ -OH and pyroglutamic acid.

In other embodiments, LP or LP' or a subunit thereof is selected from the group consisting of glutamine and derivatives thereof. Representative examples of glutamine and its derivatives include, but are not limited to: glutamine (Gln), N-alkyl-glutamine, isoglutamine (H-Glu-NH ₂ ), H-Gln(Trt)-OH and HGln(isonropyl)-OH.

In other embodiments, L _P or L _P ' or a subunit thereof is selected from the group consisting of lysine and derivatives thereof. Representative examples of lysine and its derivatives include, but are not limited to: lysine (Lys), N-alkyl-lysine, H-Lys(Boc)-OH, H-Lys(Ac)-OH, H-Lys^opM^)- OH, H-Lys(Me) ₂ -OH, H-Lys(nicotinoyl)-OH, H-Lys(Me) ₃ -OH, H-trans-4,5-dehydro-Lys-OH, H-Lys(Alloc )-OH, HH-δ-hydroxy-Lys-OH, H-δ-hydroxy-Lys(Boc)-OH, H-Lys(acetamidoyl)-OH and H-Lys(isopropyl)-OH.

In other embodiments, L _P or L _P ' or a subunit thereof is selected from the group consisting of serine and derivatives thereof. Representative examples of serine and its derivatives include, but are not limited to: serine (Ser), N-alkylserine, H-Ser(O-Ac)-OH, H-Ser(Ot-Bu)-OH, H-Ser(O- Bzl)-OH, H-Ser(pχλορ-Ο-Βζ1)-ΟΗ, Hp-(3,4-dihydroxyphenyl)-Ser-OH, H-β-(2-thienyl)-Ser-OH, isoserine N-alkylisoserine , and 3-phenylisoserine.

In other embodiments, LP or LP' or a subunit thereof is selected from the group consisting of tyrosine and derivatives thereof. Representative examples of tyrosine and its derivatives include, but are not limited to: tyrosine (Tyr), N-alkyltyrosine, H-3,5-dinitro-Tyr-OH, H-3-amino-Tyr-OH, H-3,5-dibromo- Tyr-OH, H-3,5-diiodo-Tyg-OH, H-Tyr(OMe)-OH, H-Tyr(O-t-Bu)-OH, H-Tyr(O-Boc)-OH, H-Tyr (OBzl)-OH, H-Tyr(O-Et)-OH, H-3-iodine-Tyr-OH and H-3-nitro-Tyr-OH.

In other embodiments, L _P , L _P ' or a subunit thereof is selected from the group consisting of threonine and derivatives thereof. Representative examples of threonine and its derivatives include, but are not limited to: threonine (Thr), N-alkyl-threonine, allo-threonine, H-Thr(OAc)-OH, H-Thr(Ot-Bu)-OH and HThr( OBzl)-OH.

In other embodiments, LP, LP', or a subunit thereof is selected from the group consisting of tryp-57 046150 and derivatives thereof. Representative examples of tryptophan and its derivatives include, but are not limited to: tryptophan (Trp), N-alkyl-tryptophan, H-5-Me-Trp-OH, H-5-hydroxy-Trp-OH, H-4-MeTrp- OH, H-a-Me-Trp-OH, H-Trp(Boc)-OH, H-Trp(Formyl)-OH and H-Trp(Mesitylene-2-sulfonyl)-OH.

In other embodiments, _LP , LP', or a subunit thereof is selected from the group consisting of ornithine and derivatives thereof. Representative examples of ornithine and its derivatives include, but are not limited to: ornithine (Orn), N-alkylornithine, H-Orn(Boc)-OH, H-Orn(Z)-OH, H-α-difluoro-Me-Orn- OH (eflornithine) and H-Orn(Alloc)-OH.

In other embodiments, LP, LP', or a subunit thereof is selected from the group consisting of penicillamine and derivatives thereof. Representative examples of penicillamine and its derivatives include, but are not limited to: penicillamine, H-penicillamine(Acm)-OH (H-β,β-dimethylcys(Acm)-OH) and N-alkylpenicillamine.

In other embodiments, LP or a subunit thereof is selected from the group consisting of aminoalkanedioic acid and derivatives thereof. Representative examples of aminoalkanedioic acid and its derivatives include, but are not limited to: N-alkylaminoalkanedioic acid, 2-aminohexanedioic acid, 2-aminoheptanedioic acid, 2-aminooctanedioic acid (H-Asu-OH).

In other embodiments, L _P or a subunit thereof is selected from the group consisting of citrulline and its derivatives. Representative examples of citrulline and its derivatives include, but are not limited to: citrulline (cit), N-alkylcitrulline, thiocitrulline, S-methylthiocitrulline, and homocitrulline.

In other embodiments, LP, LP', or a subunit thereof is selected from the group consisting of diaminoalkanoic acid and derivatives thereof. Representative examples of diaminoalkanoic acid (Dab) and its derivatives include, but are not limited to: N-alkyldiaminoalkanoic acids, \,\-dial<ylaminoalkanoic acids, α,γ-diaminobutanoic acid (H-Dab-OH), H-Dab(Alloc )-OH, H-Dab(Boc)OH, H-Dab(Z)-OH, α,β-diaminopropionic acid and its side chain protected variants.

An example of an LP unit or subunit thereof, lysine or cysteine or penicillamine is shown below. The wavy line indicates the sites of attachment to PEG and LP in LP(PEG)- in the linker unit. L- and D-isomers of amino acids are suitable for use herein

An example of a ligand-drug conjugate having lysine as the LP unit is shown below, where L _B , A, AO, L, W, W', Y, D ⁺ , PEG, subscripts a and p, and PEG have the meanings described in this document. L- and D-isomers of amino acids are suitable for use herein

- 58 046150

An example of a ligand-drug conjugate having cysteine or penicillamine as the LP unit is shown below, where

LB, A, AO, L, W, W', Y, D ⁺ , PEG, the subscripts a and p and PEG have the meanings described herein. L- and D-isomers are amino acid isomers suitable for use herein

1.3.3. PEG link.

The PEG units discussed herein are intended to impart an appropriate level of hydrophobicity masking to the hydrophobic units of the quaternized tubulisin drug compound and other hydrophobic components of the quaternized drug compound moiety to the drug-ligand conjugate linker. For this reason, inclusion of a PEG moiety as described herein is particularly suitable for quaternized tubulisin linker couplings that might otherwise impart sufficient hydrophobicity to negatively impact the pharmacokinetics of the resulting conjugate compared to an unconjugated targeting agent of the ligand moiety. These degraded pharmacokinetic parameters include greater plasma clearance, which may be due to the hydrophobicity of the tubulisin compound that is quaternized in the drug-ligand conjugate. Thus, drug-ligand conjugates having a quaternized tubulisin drug moiety that exhibit significantly greater plasma clearance and therefore lower plasma exposure compared to an unconjugated targeting agent ligand moiety will be advantageous according to the present invention. The drug-ligand conjugates of the present invention have these more favorable pharmacokinetic properties due to the parallel orientation within the hydrophobic moiety of the drug compound with the linker of the quaternized tubulisin drug compound unit and the PEG unit, as a result of which the negative effect of the hydrophobicity of the quaternized drug compound unit can be further aggravated by other hydrophobic components fragment of a quaternized tubulisin drug compound with a linker, with a sufficient reduction or elimination of plasma clearance (that is, the hydrophobicity of the drug compound fragment with a linker is masked).

Polydisperse PEGs, monodisperse PEGs and discrete PEGs can be used to prepare the compounds of the present invention. Polydisperse PEGs are a heterogeneous mixture in size and molecular weight, whereas monodisperse PEGs are typically purified from heterogeneous mixtures and therefore provide a single chain length and molecular weight. Preferred PEG units are discrete PEGs, compounds that are synthesized stepwise rather than through a polymerization process. Discrete PEGs provide one molecule with a specific and

- 59 046150 specific chain length.

The PEG unit provided herein contains one or more polyethylene glycol chains. Polyethylene glycol chains can be linked together, for example, in a linear, branched or star configuration. Typically at least one of the PEG chains is derivatized at one end to be covalently attached to a parallel connecting link. Examples of joining to a parallel connecting link are through bonds that are not cleavable under certain conditions or through bonds that are cleavable under certain conditions. Examples of attachments are amide bonds, ester bonds, ester bonds, hydrazone bonds, oxime bonds, disulfide bonds, peptide bonds or triazole bonds. In some aspects, the attachment to L _P is accomplished through a bond that is non-cleavable under certain conditions. In some aspects, the attachment to L _P occurs through an ester bond, a hydrazone bond, an oxime bond, or a disulfide bond. In some aspects, attachment to the LP does not occur through the hydrazone bond.

A bond that is cleavable under certain conditions refers to a bond that is substantially insensitive to cleavage when circulating in plasma, but is sensitive to cleavage in the intracellular or intratumoral environment. A bond that is not cleavable under certain conditions is a bond that is substantially insensitive to cleavage in any biological environment. Chemical hydrolysis of hydrazone, disulfide reduction, and enzymatic cleavage of a peptide bond or glycosidic bond are examples of bonds that can be cleaved under certain conditions.

The PEG unit will be directly attached to the drug-ligand conjugate (or intermediate thereof) in a parallel link. The other end (or ends) of the PEG unit will be free and unbound, and may take the form of a methoxy, carboxylic acid, alcohol or other suitable functional group. A methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a plug for the terminal PEG subunit in the PEG unit. By unbound, it is meant that at this unlinked site the PEG unit will not be attached to a drug unit, ligand unit, or binding moiety linking the drug unit and/or ligand unit. For those embodiments in which the PEG element contains more than one PEG chain, the plurality of PEG chains may be the same or different chemical moieties (eg, PEGs with different molecular weights or numbers of subunits). Multiple PEG chains are attached to a parallel connecting link at a single attachment site. One skilled in the art will appreciate that the PEG unit, in addition to the repeating polyethylene glycol subunits, may also contain a non-PEG material (eg, to facilitate connection of multiple PEG chains to each other or to facilitate connection to a parallel link unit). Non-PEG refers to atoms in a PEG unit that are not part of the -CH2CH2O- repeating subunits. In embodiments presented herein, the PEG unit may comprise two PEG monomer chains linked to each other through non-PEG elements. In other embodiments presented herein, a PEG link may comprise two linear PEG chains attached to a central core that is connected to a parallel connecting link (ie, the PEG link itself is branched).

There are a number of methods for attaching PEGs available to those skilled in the art [see, for example, Goodson, et al. (1990) Bio/Technology 8:343 (PEGylation of interleukin-2 at its glycosylation site after site-directed mutagenesis); EP 0401384 (PEG binding to G-CSF); Malik, et al., (1992) Exp. Hematol. 20:1028-1035 (PEGylation of GM-CSF using tresyl chloride); ACT Pub. No. WO 90/12874 (PEGylation of erythropoietin containing a recombinantly introduced cysteine residue using a cysteine-specific mPEG derivative); US Patent No. 5,757,078 (PEGylation of EPO peptides); US Patent No. 5,672,662 (Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acid and functional derivatives thereof for biotechnical applications); US Patent No. 6,077,939 (PEGylation of the N-terminal alpha carbon of a peptide); Veronese et al., (1985) Appl. Biochem. Bioechnol 11:141–142 (PEGylation of the N-terminal α-carbon of a peptide with PEG-nitrophenyl carbonate (PEG-NPC) or PEG-trichlorophenyl carbonate); and Veronese (2001) Biomaterials 22: 405-417 (review article on PEGylation of peptides and proteins)].

For example, PEG may be covalently linked to amino acid residues through a reactive group. Reactive groups are those to which the activated PEG molecule can be bound (eg, a free amino or carboxyl group). For example, N-terminal amino acid residues and lysine (K) residues have a free amino group; and the C-terminal amino acid residues have a free carboxyl group. Sulfhydryl groups (eg, as found on cysteine residues) can also be used as a reactive group for PEG attachment. In addition, enzymatic methods for introducing activated groups (eg, hydrazide, aldehyde, and aromatic amine groups) specifically at the C-terminus of a polypeptide have been described (See, Schwarz, et al. (1990) Methods Enzymol. 184:160; Rose, et al. al (1991) Bioconjugate Chem 2:154 and Gaertner, et al (1994) J Biol Chem 269:7224].

- 60 046150

In some embodiments, PEG molecules can be attached to amino groups using methoxylated PEG (mPEG) having various reactive moieties. Non-limiting examples of such reactive groups include succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA) and cyanuric acid chloride. Non-limiting examples of such mPEGs include mPEG-succinimidyl succinate (mPEG-SS), mPEG ₂ -succinimidyl succinate (mPEG2-SS); mPEG-succinimidyl carbonate (mPEG-SC), mPEG ₂ -succinimidyl carbonate (mPEG2-SC); mPEG-imidate, mPEG-p-nitrophenyl carbonate (mPEG-NPC), mPEG-imidate; mPEG ₂ -nitrophenyl carbonate (mPEG ₂ NPC); mPEG-succinimidyl propionate (mPEG-SPA); mPEG ₂ -succinimidyl propionate (mPEG, -SPA); mPEG-N-hydroxysuccinimide (mPEG-NHS); mPEG ₂ -N-hydroxysuccinimide (mPEG ₂ -NHS); mPEGcyanurchloride; mPEG ₂ - cyanuric chloride; mPEG2-Lysinol-NPC b mPEG2-Lys-NHS.

Typically, at least one of the PEG chains making up the PEG link is functionalized such that it can be connected to a parallel connecting link. Functionalization may be through, for example, an amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional group. The PEG unit may further comprise a non-PEG material (ie, a material not containing -CH2CH2O-) to facilitate connection to a parallel connecting link or to facilitate connection of two or more PEG chains.

A wide variety of polyethylene glycol (PEG) types can be used, and essentially any suitable PEG reactive reagent can be used. In some embodiments, the PEG reactive reagent will form a carbamate or amide bond when attached to L _P . The following PEG reagents are used in various embodiments: mPEG2-NHS, mPEG ₂ -ALD, multi-Arm PEG, mPEG(MAL)2, mPEG2(MAL), mPEG-NH2, mPEGSPA, mPEG-SBA, mPEG thioesters, mPEG- double esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET, heterofunctional PEG (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEGVS, NHS-PEG-MAL), PEG- acrylates (ACRL-PEG-NHS), PEG phospholipids (e.g. mPEG-DSPE), multi-armed SUNBRITE™ series PEGs, including glycerol-based GL series, chemically activated, selected by those skilled in the art, any SUNBRITE-activated PEG (including, but not limited to, carboxyl-PEG, p-NP-PEG, tresyl-PEG, aldehyde PEG, acetal-PEG, aminoPEG, thiol-PEG, maleimido-PEG, hydroxyl-PEG-amine, amino-PEG-COOK-hydroxy- PEG-aldehyde, PEG-carboxyl anhydride, functionalized PEG-phospholipid and other similar and/or suitable reactive PEGs selected by those skilled in the art for their particular application and use.

The addition of a PEG moiety may have two potential effects on the pharmacokinetics of the resulting drug-linker conjugate. The desired effect is a decrease in clearance (and consequent increase in exposure), which occurs due to a decrease in nonspecific interactions caused by the interfering hydrophobic elements of the drug compound with the linker. The second effect is an undesirable effect, and is a decrease in volume and rate of distribution that may result from an increase in the molecular weight of the drug-ligand conjugate. Increasing the number of PEG subunits increases the hydrodynamic radius of the conjugate, which leads to a decrease in diffusivity. In turn, reduced diffusivity may reduce the ability of the drug-ligand conjugate to penetrate the tumor (Schmidt and Wittrup, Mol. Cancer Ther. 2009;8:2861-2871). Because of these two competing pharmacokinetic effects, it is desirable to use a PEG that is large enough to reduce the clearance of the LDC, thereby increasing plasma exposure, but not so large as to significantly reduce its diffusivity, which may reduce the drug-ligand conjugate ability reach target cell populations.

In one set of embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits , at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, according to at least 21 sub-units, at least 22 sub-units, at least 23 sub-units or at least 24 sub-units. As used herein, a subunit, when referred to in relation to a PEG unit, refers to a polyethylene glycol subunit having the formula

In another set of embodiments, a PEG unit comprises one or more straight PEG chains, each having at least 2 subunits, at least 3 subunits, at least 4 subunits, at least 5 subunits, at least 6 subunits, at least

- 61 046150 at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, according to at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 sub-units or at least 24 sub-units. In preferred embodiments, the PEG unit contains a total of at least 6 subunits, at least 8, at least 10 subunits, or at least 12 subunits. In some such embodiments, the PEG unit contains a total of no more than about 72 subunits, preferably a total of no more than about 36 subunits.

In another set of embodiments, the PEG unit is derived from a linear PEG chain having 2 to 72, 2 to 60, 2 to 48, 2 to 36, or 2 to 24 subunits, 2 to 72, 2 to 60 , from 2 to 48, from 2 to 36 or from 2 to 24 subunits, from 3 to 72, from 3 to 60, from 3 to 48, from 3 to 36 or from 3 to 24 subunits, from 3 to 72, from 3 up to 60, from 3 to 48, from 3 to 36 or from 3 to 24 subunits, from 4 to 72, from 4 to 60, from 4 to 48, from 4 to 36 or from 4 to 24 subunits, from 5 to 72, from 5 to 60, from 5 to 48, from 5 to 36 or from 5 to 24 subunits.

Examples of linear PEG units that can be used in any of the embodiments presented herein are as follows:

-^-P ^PE1 -(CH ₂ CH ₂ 0) _p ---R ^PEG2

R ^ΡΕΘ1 -(CH2CHgO)rf —R ^REez —(CH2CH ₂ 0)—R ^ΡΕΘ2

-|_R ^PE,s 'i_l:i:n2CH2Q)r|1 J^R ^pe<: ' ³ —(CH2CH ₂ O) _rf ^R ^PES2 where the wavy line shows the point of attachment to the parallel connecting link, r ^PEG1 represents the PEG linking unit, R ^PEG2 is a PEG restricting unit; R ^PEG3 is a PEG linker (ie, for linking together multiple chains of PEG subunits), the index n is independently selected from 2 to 72 (preferably 4 to 72, more preferably 6 to 72, 8 to 72, 10 to 72, 12 to 72 or 6 to 24); the index e denotes 2 to 5, each index n' is independently selected from 1 to 72.

In preferred embodiments, there are at least 6, preferably at least 8, at least 10, or at least 12 PEG subunits in a PEG unit. In some embodiments, there are no more than 72 or 36 PEG subunits in a PEG unit.

In other preferred embodiments, the subscript n is 8 or about 8, 12 or about 12, 24 or about 24.

The PEG connection link is part of the PEG link and serves to connect the PEG link to the parallel connecting link. In this regard, the parallel connecting link has a functional group that forms a bond with the PEG link. Functional groups for attaching a PEG unit to a parallel linking unit include sulfhydryl groups to form disulfide bonds or thioether bonds, aldehyde, ketone or hydrazine groups to form hydrazone bonds, hydroxylamine to form oxime bonds, carboxyl or amino groups to form peptide bonds, carbonic or hydroxy groups to form ester bonds, sulfonic acids to form sulfonamide bonds, alcohols to form carbamate bonds, and amines to form sulfonamide bonds or carbamate bonds or amide bonds. Accordingly, the PEG unit can be attached to the parallel connecting unit, for example, through disulfide, thioether, hydrazone, oxime, peptide, ester, sulfonamide, carbamate or amide bonds. Typically, a PEG addition unit is the product of a cycloaddition, addition, addition/elimination, or displacement reaction that occurs when a PEG unit is attached to a parallel link unit.

A PEG linker is part of a PEG unit and is a non-PEG substance that functions to connect two or more chains of CH ₂ CH ₂ O- repeating subunits. In exemplary embodiments, the PEG linker R ²² is -C ₁ . ₁₀ αlkyl-C(O)-NH-, -C ₁ . ₁₀ alkyl-NH-C(O)-, -C ₂ . ₁₀ alkyl-NH-, -C^^ku^-O-, -C _1-10 alkyl-8- or -C ₂ . ₁₀ alkyl-NH-.

In exemplary embodiments, the PEG linker R ²⁰ is -C(O)-, -O-, -S-, -S(O)-, -NH-, -C(O)O-, -C(O) C1. _yu alkyl, -C(O)C _M 0alkyl-O-, -C(O)C1.10alkyl-CO ₂ -, -C(O)C _M 0alkylNH-, -C(O)C ₁ . ₁₀ alkyl-B-, -QO^M^Kua-QOyNH-, -QO^^^Kua-NH-QO)-, -C1. ₁₀ alkyl, -C _1-10 alkyl-O, -C1. ₁₀ alkyl-CO ₂ -, -Ср^alkyl-KI-, -C1. ₁₀ alkyl-B-, -Srualkyl-C^-KI-, -Srmalkyl-KI-OR)-, -CH ₂ CH ₂ SO ₂ -C ₁ . ₁₀ alkyl-, -CH ₂ C(O)-C ₁ . ₁₀ alkyl-, =N-(O or IO-C _1-10 alkyl-O-, =N-(O or N)-C ₁ . ₁₀ alkyl-NH-, =N-(O or N)-C ₁ . ₁₀ alkyl-CO ₂ -, =N-(O or Yu-C _1-10 alkyl-8-,

- 62 046150

where each R ²¹ independently represents -C1-10alkyl, -C2-10alkyl-CO2H, -C2-10alkyl-OH, -C2-10alkyl-KH2, C2-10alkyl-UN(C1-3 alkyl) or C2-10alkyl-N (C1-3alkyl)2; and each R ²² independently represents -C1-10 alkyl-C(O)-UN-, -C _1-10 alkyl-NH-C(O)-, -C _2-10 alkyl-CN-, -C _2-10 alkyl-O-, -C _1-10 alkylS- or -C _2-10 alkyl-CNB in some embodiments, R ²⁰ is -NH-, -C(=O)-, triazole-linked groups or -S- or maleimido -related groups such as

where the wavy line indicates the attachment portion of the parallel connecting link, and the asterisk indicates the attachment portion of the PEG link. In some such aspects, R ²¹ is C _1-10 alkyl, -C _2-10 alkyl-CO ₂ H, -C _2-10 alkyl-OH, or -C _2-10 alkyl-UN ₂ .

Exemplary linear PEG units that may be used in any of the embodiments presented herein are as follows:

-^NH-(CH ₂ CH ₂ 0) _n -CH ₂ CH ₂ C0 ₂ H

-|-NH-(CH ₂ CH ₂ 0) _n -CH ₂ CH ₂ C(=0)NH-(CH ₂ CH ₂ 0)-CH ₂ CH ₂ C0 ₂ H o s and |-C—(CH ₂ CH ₂ 0) _p -CH ₃ -^-NH-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ NH - (CH ₂ CH ₂ o) -CH ₂ CH ₂ with ₂ N where the wavy line shows the place of attachment to parallel connecting link, and each sublink n is independently selected from 4 to 72, 6 to 72, 8 to 72, 10 to 72, 12 to 72, 6 to 24, or 8 to 24. In some aspects, n means about 8, about 12 or about 24.

As described herein, the PEG unit is selected so that it improves the clearance of the resulting drug-ligand conjugate, but does not significantly affect the ability of the conjugate to penetrate the tumor. In embodiments wherein the quaternized drug compound unit and -W-Y- or -Y(W')- in the linker unit of the drug-ligand conjugate has a hydrophobicity comparable to that of the maleimido glucuronide MMAE drug compound with the linker (as shown in the Examples), The PEG unit that will be selected for use will consist of from 8 subunits to about 24 subunits, more preferably about 12 subunits. In embodiments where the quaternized drug compound and -W-Y- or -Y(W')- unit in the linker unit of the drug-ligand conjugate has a hydrophobicity greater than that of the maleimido glucuronide MMAE drug compound with the linker, a PEG unit with a higher amount of subunits. The methodology shown in the examples section can be used to determine the ideal number of subunits for a particular linker-drug compound.

It will be understood that when referring to PEG subunits, and depending on the context, the number of subunits may be an average number, for example, when referring to a collection of drug-ligand conjugates or intermediates (eg, drug-linker conjugates), and using polydisperse PEG.

1.3.4. Quaternized tubulisins.

In one set of embodiments, the quaternized tubulisin drug compound unit comprises or corresponds in structure to tubulisin having a tertiary amine at the N-terminus, wherein the nitrogen atom of the tertiary amine is quaternized.

In some embodiments, the quaternized drug unit is a tubulisin that is represented by a structure of the formula DG, _DH , or _DH ', wherein said nitrogen (f) represents the quaternization site when such compounds are included in an LDC or in a drug compound linked to a linker in the form of a quaternized drug unit (D+)

- 63 046150

wherein said nitrogen (f) represents a quaternization site when such a tubulisin compound is included in an LDC or a drug compound linked to a linker in the form of a quaternized tubulisin drug unit (D+); the ring is a 5-membered or 6-membered nitrogen-containing heteroaryl, wherein said required substituents of this heteroaryl are in 1,3- or meta-linkages to each other with optional substitution at the remaining positions; a curved dotted line denotes optional cyclization; the straight dotted line to R ² denotes an optional double bond, or optionally two occurrences of R ² independently selected, or a divalent O-linked moiety; R ² represents X ^A -R ^2A , where R ^2A represents hydrogen, optionally substituted alkyl, saturated or unsaturated, or -C(=O)R ^B , where R ^B represents hydrogen, optionally substituted alkyl, saturated or unsaturated, optionally substituted alkenyl or optionally substituted aryl; X ^A represents -O-, -S-, -N(R ^2C )-, -CH ₂ -, -C(=O)-, (C=O)N(R ^2C )- or -O (C= O)N(R ^2C )-, where R ^2C represents hydrogen or optionally substituted alkyl, or R ² represents a monovalent O-linked substituent and there is no double bond to R ² , or R ² represents O and a double bond to R ² is present; R ³ represents hydrogen or optionally substituted alkyl; R ⁴ , R ^4A , R ^4B , R ⁵ and R ⁶ represent optionally substituted alkyl, independently selected, or R ^4A and R ^4B , together with the atoms to which they are attached, define optionally substituted heterocycloalkyl, as indicated by the curved dotted line between R ^4A and R ^4B and R ⁴ , R ⁵ and R ⁶ have the meanings defined above; one R ⁷ is hydrogen or optionally substituted alkyl, and the other R ⁷ is optionally substituted arylalkyl or optionally substituted heteroarylalkyl.

In other embodiments, the quaternized drug substance is tubulisin, represented by the structure of formula DG, where one R ⁷ is hydrogen or an optionally substituted alkyl, preferably hydrogen or C1-C4, and the other R ⁷ is an independently selected optionally substituted alkyl, preferably ^-^-alkyl, optionally substituted with phenyl or -CO2H or an ester prodrug thereof; R ^4A and R ^4B , together with the atoms to which they are attached, define optionally substituted C5-C ₆ heterocycloalkyl; and other variable groups have the meanings defined above.

In some embodiments of formula D _G R ² is X ^A -R ^2A where X ^A is -O- and R ^2A is -C(=O) ^RC where R ^C is hydrogen, optionally substituted alkyl, preferably methyl, ethyl, vinyl or branched alkyl, or R ² is a monovalent O-linked substituent selected from the group consisting of an ester.

In another embodiment of formula D _G R ² is X ^A -R ^2A where X ^A is -O-; and R ^2A is hydrogen or optionally substituted alkyl, saturated or unsaturated, or R ² is a monovalent O-linked substituent selected from the group consisting of ethers.

In preferred embodiments, the quaternized drug unit is tubulisin, represented by the structure of formula DG'

wherein the subscript m is 0 or 1, one R ⁷ is hydrogen, and the other R ⁷ is an optionally substituted arylalkyl, where the alkyl moiety is substituted by -CO ₂ H or an ester thereof and the remaining variable groups are as specified for formula D _G .

In other preferred embodiments, in Formula D, _G -N(R ⁷ )(R ⁷ ) is replaced by -N(R ⁷ )-CH(R ¹⁰ )(CH2R ¹¹ ) to define the quaternized tubulisin drug substances of Formula DH'

- 64 046150

where R ¹⁰ represents a C1-C6 alkyl substituted by -CO2H or an ester thereof, and R ⁷ represents hydrogen or a C1-C6 alkyl independently selected from R ¹⁰ , or R ⁷ and R ¹⁰ together with the atoms to which they attached, denotes a 5 or 6 membered heterocycle; and R ¹¹ is aryl or 5- or 6-membered heteroaryl, optionally substituted with one or more, preferably 1 or 2, more preferably 1, substituents independently selected from the group consisting of halogen, lower alkyl, -OH and -O -C1-C ₆ -alkyl, preferably -F, -CH3, and -OCH3; and the remaining variable groups are as specified for formula _DH .

In still other aspects, one R ⁷ in -N(R ⁷ )(R ⁷ ) in formula DG, formula DG', or formula DH is hydrogen or C1-C6 alkyl, and the other R ⁷ is independently selected from C1-C6 alkyl , optionally substituted by -CO ₂ H or an ester thereof, or optionally substituted by phenyl.

In some embodiments of Formula DG, Formula DG', or Formula DH, one R ⁷ is hydrogen and the other R ⁷ is an optionally substituted arylalkyl having the structure

where R ^7B represents hydrogen or an O-linked substituent, preferably hydrogen or -OH in the para position, and R ^8A represents hydrogen or lower alkyl, preferably methyl; and where the wavy line indicates the point of attachment to a D _G , D _G ', or D _H residue.

In preferred embodiments of Formula DG, Formula DG', or Formula DH, one R ⁷ is hydrogen and the other R ⁷ is an optionally substituted arylalkyl having the structure wherein R ^7B is -H or -OH; And

where the wavy line indicates the point of attachment to the DG or DG' residue.

In other embodiments of the structure of Formula DG, Formula DG', or Formula DH, one R ⁷ is hydrogen or C1-C ₄ alkyl, preferably hydrogen or methyl, more preferably hydrogen, and the other R ⁷ is an optionally substituted arylalkyl having one of the structures

where Z is an optionally substituted alkylene or an optionally substituted alkenylene, R ^7B is hydrogen or an O-linked substituent, preferably hydrogen or OH in the para position, R ^8A is hydrogen or lower alkyl, preferably methyl, and the subscript n is 0 , 1 or 2, preferably 0 or 1; and where the wavy line indicates the point of attachment to the D _G or D _H residue.

In some embodiments, the structures of _DH ' R ⁷ and R ¹⁰ , together with the atoms to which they are attached, define an optionally substituted 5 or 6 membered heterocycle, wherein -N(R ⁷ )CH(R ¹⁰ )(CH2R ¹¹ ) has the following structure

where the wavy line indicates the point of attachment to the DH' residue.

- 65 046150

Some preferred quaternized drug units are tubulisin, represented by the formula D _H-1 , wherein said nitrogen (f) represents the quaternization site when such tubulisin compound is included in an LDC or drug compound linker quaternized drug unit (D+)

⁰ D _H -i, where the ring is a 5-membered or 6-membered nitrogen-containing heteroaryl, wherein said required substituents on that heteroaryl are in the 1,3- or meta-position to each other with optional substitution at the remaining positions, R ^2A is hydrogen or optionally substituted alkyl, or R ^2A together with the oxygen atom to which it is attached denotes an O-linked substituent other than -OH; R ³ represents hydrogen or optionally substituted alkyl; R ⁴ , R ^4A , R ^4B , R ⁵ and R ⁶ are optionally substituted alkyl, independently selected; R ^7A is optionally substituted aryl or optionally substituted heteroaryl, R ^8A is hydrogen or optionally substituted alkyl, and the subscript m is 0 or 1.

In some preferred embodiments of formula DG, DG', _DH , DH', or _DH-1 , R ⁴ is methyl or ethyl, R ³ is optionally substituted alkyl, and R ⁵ and R ⁶ are independently selected side chain moieties natural hydrophobic amino acids and the remaining variable groups are as indicated.

In other preferred embodiments of Formula D _H-1 R ^7A is optionally substituted phenyl. In another preferred embodiment, R ^8A is methyl in the (S) configuration. In other preferred embodiments, _DH , _DH ' or _DH-1 R ^2A , together with the oxygen atom to which it is attached, represents an O-linked substituent other than -OH, more preferably an ester, ether or O- bound carbamate. In more preferred embodiments, the ring is a 5-membered nitrogen-containing heteroarylene with a divalent oxazole or thiazole moiety particularly preferred. In other preferred embodiments, R ⁴ is methyl, or R ^4A and R ^4B are methyl. In other preferred embodiments, R ⁷ is optionally substituted arylalkyl, where aryl is phenyl, and R ^7A is optionally substituted phenyl.

In other embodiments of the formula, ring DG, DG', _DH , _DH ', or _DH-1 is a 5-membered heteroarylene nitrogen, preferably represented by the structure

where X ^B represents O, S or NR ^B , where R ^B represents hydrogen or lower alkyl. Preferably, the quaternized drug substance is a tubulisin represented by the structure of the formula DG', _DH , _DH ' or _DH-1 , where the subscript m is 1. More preferably, tubulisins are represented by the structure of the formula _DG ', _DH , D _H ' or D _H-1 , where the subscript m is 1 and the ring is an optionally substituted divalent thiazole moiety.

Other quaternized drug units are tubulisin, represented by the structure of the formula DI

wherein said nitrogen (f) represents a quaternization site when such a tubulisin compound corresponds to or is included in an LDC or a drug compound linked to a quaternized drug unit (D+) linker; curved dashed lines indicate optional cyclization; R ^2A is hydrogen or an optionally substituted alkyl, or R ^2A together with the oxygen atom to which it is attached is an O-linked substituent other than -OH, or R ^2A is absent when R ⁶ is bonded to that oxygen atom as shown a curved dotted line between R ⁶ and an oxygen atom, denoting an oxygen-containing heterocycloalkyl; circled Ar is a 5-membered nitrogen-containing heteroarylene, wherein said required substituents on the heteroarylene are in the 1,3 position relative to each other with optional substitution at the remaining positions, R ³ is hydrogen or optionally substituted alkyl; R ⁴ , R ⁵ and R ⁶ are optionally substituted alkyl, independently selected, or R ⁶ is attached to an oxygen atom of an -OR ^2A moiety in which R ^2A is absent, and R ⁴

- 66 046150 and R ⁵ have the meanings defined above; R ^4a represents hydrogen or optionally substituted alkyl, and R ^4B represents optionally substituted alkyl, or both, together with the nitrogen atom to which they are attached, as indicated by the curved dotted line between R ^4A and R ^4B , represent heterocycloalkyl with a quaternized nitrogen atom, optionally substituted; one R ⁷ is hydrogen or optionally substituted alkyl, and the other R ⁷ is optionally substituted aralkyl or heteroaralkyl; where the wavy line indicates covalent binding of the D ⁺ structure to the residue of the LDC structure.

In these embodiments, the tubulisin compound preferably has the structure of formula DI-1

where index m denotes 0 or 1; Z represents optionally substituted alkylene or optionally substituted alkenylene; R ^7A is optionally substituted aryl or optionally substituted heteroaryl; and other variable groups have the meanings defined above for the DI formula.

In preferred embodiments of Formula D _I, the tubulisin compound has the structure of Formula DI-2

where R ^7A represents optionally substituted phenyl; R ^8A represents hydrogen or methyl; and other variable groups have the meanings defined above for the formula D _I .

In other preferred embodiments of Formula DI, the tubulisin compound has the structure of Formula DI-3

⁰ Dj-з, where R ⁵ and R ⁶ represent alkyl side chain residues of natural or unnatural hydrophobic amino acids, independently selected; the index u indicating the number of substituents R ^7B means 0, 1, 2 or 3; each R ^7B , when present, is an independently selected O-linked substituent; R ^8A represents hydrogen or optionally substituted alkyl; and other variable groups have the meanings defined above for the formula D _I .

In more preferred embodiments of Formula DI, the tubulisin compound has the structure of Formula DI-4

wherein said nitrogen (f) represents a quaternization site when such a tubulisin compound corresponds to or is included in an LDC or a drug compound linked to a quaternized drug unit (D ⁺ ) linker; R ⁴ represents methyl; the subscript u denotes 0, 1 or 2; R ³ represents H, methyl, ethyl, propyl, -CH2-OC(O)R ^3A , -CH2CH(R ^3B )C(O)R ^3A or -CH(R ^3B )C(O)NHR ^3A , where R ^3A is C1-C6 alkyl, and R ^3B is H or C1-C6 alkyl independently selected from R ^3A ; R ^2A , together with the oxygen atom to which it is attached, represents an O-linked substituent selected from the group consisting of OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -CH2OC(O)R ^2B , -OC( O)N(R ^2B )(R ² c) and -OCH2C(O)N(R ^2B )(R ² c), where R ^2B and R ^2C are independently selected from the group consisting of H, C1-C6 alkyl and C2 -C6-alkenyl; and each R ^7B , when present, is independently -OH or -OCH3.

In other more preferred embodiments of Formula D _I , the tubulisin compound has the structure of Formula D _I-5

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wherein said nitrogen (f) represents a quaternization site when such a tubulisin compound corresponds to or is included in an LDC or a drug compound linked to a quaternized drug unit (D+) linker; R ^2A represents hydrogen, optionally substituted alkyl, saturated or unsaturated, or R ² together with the oxygen atom to which it is attached represents an O-linked substituent other than -OH; R ³ represents optionally substituted C|-C6 alkyl; R ⁴ represents methyl; R ⁵ and R ⁶ represent alkyl side chain residues of natural hydrophobic amino acids; and the fragment -N(R ⁷ )(R ⁷ ) is -KH(C1-C ₆ -alkyl) or -KH-K(C1-C6-alkyl) ₂ , where one and only one C^C ₆ -alkyl is optional substituted with a -CO ₂ H group or its ester or optionally substituted phenyl with the moiety -N(R ⁷ )(R ⁷ ) preferably selected from the group consisting of -NI 1 (CH ₃ ), -UNCH ₂ CH ₂ PK and -NI1CH ₂ CO ₂ H, YANSN ₂ CH ₂ CO ₂ H and -KNSN ₂ CH ₂ CH ₂ CO ₂ H.

In any one formula 1)ts, _DH ', _Dh-1 , DI, DI _{- 1} , DI-2, DI _-3 , _DI-4 and _DI-5 , preferably R ^2A represents - CH ₂ CH ₃ , -CH ₂ -CH = CH ₂ or -CH ₂ -C(CH ₃ ) = CH ₂ .

In specific preferred embodiments of Formula D _I, the tubulisin compound has the following structure:

where R ^2B represents -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH2, -CH2CH(CH3)2, -CH ₂ C(CHe)3; and the indicated nitrogen (f) represents the quaternization site when such a tubulisin compound corresponds to or is included in an LDC or connection of a drug compound with a linker in the form of a quaternized drug unit (D ⁺ ).

In other specific preferred embodiments of Formula DI, the tubulisin compound has the following structure:

or

where R ^2B represents hydrogen, methyl or -OCH3 (i.e. -OCH^ ^2B represents a methyl, ethyl or methoxymethyl ether substituent), or -OCH2I ^2B represents OCH ₂ CH=CH ₂ or OCH ₂ C(CH ₃ )= CH ₂ ; and said nitrogen (f) represents a quaternization site when such a tubulisin compound corresponds to or is included in an LDC or a drug compound linked to a quaternized drug unit (D ⁺ ) linker.

In other preferred embodiments, any of the formulas DI _-1 , DI _-2 , DI _-2 , DI-4 or DI _-5 : V ^N yi- 'W heterocyclic thiazole ring $ ' is replaced by θ ^or .

In some preferred embodiments, any of the formulas _DH , DH', DH _-1 , DI, DI _-1 , DI _-2 , DI _-3 , DI-4, and DI _-5 R ³ is methyl or is -CH ₂ OC(=O^ ^3A where R ^3A is an optionally substituted alkyl. In other preferred embodiments, any of these structures R ³ is -C^ ^3A )^ ^3A )C(=O)-X ^C where X ^C is is -ОЯ ^3В or -Н(К ^3С )(К ^3С ), where each R ^3A , R ^3B and R^ independently represents hydrogen, optionally

- 68 046150 substituted alkyl or optionally substituted cycloalkyl. Preferably, R ³ is -C(R ^3A )(R ^3A )C(=O)-N(R ^3C )(R ³ c), where each R ^3A is hydrogen, one R ^3C is hydrogen and the other R ^3C is n-butyl or, more preferably, isopropyl.

In other preferred embodiments, the tubulysin corresponding to or included as D+ in the LDC is a naturally occurring tubulysin, including tubulysin A, tubulysin B, tubulysin C, tubulysin D, tubulysin E, tubulysin F, tubulysin G, tubulysin H, tubulysin I, tubulisin U, tubulisin V, tubulisin W, tubulisin X or tubulisin Z, the structures of which are represented by the descriptions of the following structures and variable groups, wherein said nitrogen (f) represents the quaternization site when such compound is included in the LDC as a quaternized drug unit (D+ )

Table 1. Some natural tubulisins

Tubulisin R ^7b R ^2a R ³

A He c(=0)CH ₃ CH ₂ O(0=0)i-Bu IN He C(=0)CH ₃ CH ₂ O(0=0)p-Rg WITH He c(=0)CH ₃ CH ₂ O (0=0) Et D n C(=0)CH ₃ CH ₂ O(0=0)i-Bu E n c(=0)CH ₃ CH ₂ O(0=0)p-Rg F n C(=0)CH ₃ CH ₂ O (0=0) Et G He c(=0)CH ₃ CH ₂ O(0=0)CH= _CH2 N n c(=0)CH ₃ CH ₂ O (0=0) Me I He c(=0)CH ₃ CH ₂ O (0=0) Me And n C(=0)CH ₃ N V n He N Z He He N

In specific preferred embodiments, the quaternized tubulysin is such tubulysin M.

1.4.1. Connections of a drug compound with a linker L _B '-Lo-D ⁺ .

In other preferred embodiments, L _B '-L _O -D ⁺ or LB-LO-D + has the following structure:

R

- 69 046150

free carcinogenic substances in the structure DG', DH, DH', D _H-1 , DI, DI-1, DI-2, DI _-3 , DI-4 and DI-5, and LB, LA', LP, PEG, A, Ao, V and Z ³ and the p indices are as described above for the LB- and LB'-containing fragments described herein; E' and J' independently represent -O-, -S- or -N(R ³³ ), where R ³³ represents hydrogen or optionally substituted alkyl; and R ⁴⁵ is CO2H or CH2OH. In more preferred embodiments, J' is -NH-. In other preferred embodiments, E' is -O-.

More preferred embodiments are those wherein LB' is a maleimide (M ¹ ) moiety or LB is a succinimide (M2) or amide acid (M ³ ) moiety.

In other more preferred embodiments, LB'-L _O -D ⁺ has the following structure:

oh oh he

or where A, R2A, R ³ , R ⁴⁵ , R ⁷ B, R ⁴⁵ , and the subscript u have the meanings defined above. In more preferred embodiments, one or both of V, Z ³ are =CH-.

In more preferred embodiments, the LB'-L _O -D ⁺ or -LB-L _O -D ⁺ moiety comprising the quaternized tubulisin drug unit has the following structure:

- 70 046150

where the wavy line indicates covalent binding to the sulfhydryl group of the ligand unit. In more preferred embodiments, the carbohydrate moiety covalently attached to the self-destructive moiety Y via a glycosidic bond has its anomeric carbon in the β configuration. In other more preferred embodiments, R ⁴⁵ is -CH2OH or -CO2H.

In any of the above embodiments, for LB-, LB'-, M ¹ -, M2- or M ³ -containing moieties comprising a quaternized tubulisin drug compound, R ³ is preferably methyl, or R 2 is preferably acetate , or the subscript m is preferably 1. Also preferred for such LB-, LB'-, M ¹ -, M2- or M ³ containing moieties are those wherein R ³ is methyl, ethyl or propyl and -OR2 ^A is -OC(O)CH3, -OCH3, -OCH2CH3, -OCH2CH2CH3, OCH2CH=CH2 or -OCH2C(CH ₃ )=CH2. In any such embodiments, the subscript u is 0 or is 1 and R7B is -OH.

1.5.1. Treatment of hyperproliferative conditions.

Conjugates of drug compounds with a ligand can be used to inhibit the proliferation of a tumor or cancer cell by inducing apoptosis in the tumor or cancer cell, or to treat a malignancy in a patient. Conjugates of drug compounds with a ligand can suitably be used in various clinical settings for the treatment of cancer. Conjugates of drug compounds with a ligand can be used to deliver a drug compound to a tumor cell or cancer cell. Without being limited to any theory, in one embodiment, the ligand unit of the drug-ligand conjugate binds or associates with the cell surface of the cancer cell or an antigen or receptor associated with the tumor cell, and upon binding of the drug-ligand conjugate can be taken up (internalized) within a tumor or cancer cell through antigen- or receptor-mediated endocytosis or other internalization mechanism. The antigen may be attached to a tumor cell or cancer cell, or may be an extracellular matrix protein associated with a tumor cell or cancer cell. Once inside the cell through an enzymatic or non-enzymatic cleavage mechanism, depending on the components of the linker system, the drug compound is released inside the cell. In an alternative embodiment, the drug compound or drug compound unit is cleaved from the drug-ligand conjugate in close proximity to the tumor cell or cancer cell, and the drug compound or drug compound unit subsequently enters the cell.

- 71 046150

Conjugates of drug compounds with a ligand can provide specific conjugative targeting of a drug compound to a tumor or cancer, thereby reducing the overall toxicity of the drug compound.

In some embodiments, the linker units stabilize conjugates of drug compounds with a ligand in the blood, but are capable of releasing the drug compound once inside the cell.

In one embodiment, the ligand unit binds to a tumor cell or cancer cell.

In another embodiment of the invention, the ligand unit binds to a tumor cell or cancer cell antigen that is located on the surface of the tumor cell or cancer cell.

In another embodiment of the invention, the ligand unit binds to a tumor cell or cancer cell antigen, which is an extracellular matrix protein associated with a tumor cell or cancer cell.

The specificity of the ligand unit for a particular tumor cell or cancer cell may be important in determining which tumors or cancers can be treated most effectively. For example, a drug-ligand conjugate having a BR96 ligand unit can be used to treat antigen-positive carcinomas, including lung, breast, colon, ovarian and pancreatic cancers. Drug-ligand conjugates having an anti-CD30 or anti-CD70 binding ligand moiety can be used to treat hematological malignancies.

Other specific types of malignancies that can be treated with ligand drug conjugates include, but are not limited to, solid tumors, blood cancers, acute and chronic leukemias and lymphomas.

Solid tumors include, but are not limited to, fibrosarcoma, malignant myxoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, cancer kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous cancer glands, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer , lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma and retinoblastoma.

Blood-borne malignancies include, but are not limited to, acute lymphoblastic leukemia ALL, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia AML, acute promyelocytic leukemia APL, acute monoblastic leukemia, acute erythroleukemic leukemia , acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute undifferentiated leukemia, chronic lymphocytic leukemia CLL, hairy cell leukemia and multiple myeloma.

Acute and chronic leukemias include, but are not limited to, lymphoblastic, myelogenous, lymphocytic and myelocytic leukemias.

Lymphomas include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, and polycythemia vera.

Malignancies, including, but not limited to, tumor, metastasis, or other diseases or disorders characterized by hyperproliferating cells, can be treated or their progression inhibited by administration of an ADC composition.

In other embodiments, methods of treating cancer are provided, comprising administering to a patient in need thereof an effective amount of an LDC composition and a chemotherapeutic agent. In one embodiment, the cancer being treated with a chemotherapeutic agent in combination with an LDC has not been found to be refractory to the chemotherapeutic agent. In another embodiment, the cancer being treated with a chemotherapeutic agent in combination with an ADC is refractory to the chemotherapeutic agent. LDC compositions may be administered to a patient who has also undergone surgery for treatment of malignancy.

In some embodiments, the patient also receives additional treatment such

- 72 046150 something like radiation therapy. In a specific embodiment, the drug-ligand conjugate is administered simultaneously with a chemotherapeutic agent or radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the drug-ligand conjugate.

The chemotherapy agent may be administered over a series of sessions. Any one or combination of chemotherapeutic agents, such as standard therapeutic chemotherapeutic agent(s), may be administered.

In addition, methods of treating a malignancy using a drug-ligand conjugate are provided as an alternative to chemotherapy or radiation therapy when chemotherapy or radiation therapy has proven itself or may be too toxic, such as resulting in unacceptable or intolerable side effects for the subject being treated . The patient being treated may optionally be treated with another cancer treatment, such as surgery, radiation therapy, or chemotherapy, depending on which treatment is considered acceptable or tolerable.

1.6.1. Pharmaceutical compositions.

The present invention relates to pharmaceutical compositions containing a composition of LDC described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients or one to four pharmaceutically acceptable excipients, which in some embodiments include a pharmaceutically acceptable carrier. The pharmaceutical compositions can be in any form that allows the drug-antibody conjugate to be administered as an LDC to a patient to treat a disorder associated with the expression of an antigen to which the antibody from the ADC binds. For example, the pharmaceutical compositions may be in the form of a liquid or a lyophilized solid. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular and intrasternal injections or infusions. In preferred embodiments, the pharmaceutical composition containing the ADC is administered intravenously as a liquid solution. Preferably, the liquid solution is prepared by reconstituting the solid preformulation obtained by lyophilizing the liquid preformulation containing the ADC using a suitable pharmaceutically acceptable carrier.

Pharmaceutical compositions can be formulated to ensure bioavailability of the compound when the composition is administered to a patient. Such compositions may take the form of one or more dosage units, where, for example, a lyophilized solid may provide a single dosage unit when reconstituted as a solution or suspension upon addition of a suitable liquid carrier.

The substances used in the preparation of pharmaceutical compositions are preferably non-toxic in the quantities used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient(s) in a pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (eg, human), the specific form of the pharmaceutical composition, the route of administration, and the LDC composition used.

The pharmaceutical composition may be, for example, in the form of a liquid. The liquid can be used for delivery by injection. One or more of the following may also be included in the composition for administration by injection: a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

Liquid compositions, whether solutions, suspensions or other similar forms, may also include one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fatty oils such as synthetic mono- or diglycerides, which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates or phosphates; detergents such as nonionic surfactants, polyols; and tonicity agents such as sodium chloride or dextrose. The parenteral composition may be contained in an ampoule, disposable syringe, or multiple dose vial made of glass, plastic, or other material. Physiological saline solution is an example of an adjuvant. The injectable pharmaceutical composition is preferably sterile.

The amount of conjugate that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may not necessarily be used.

- 73 046150 to determine optimal dose ranges. The exact dosage to be used in the compositions will also depend on the route of administration and the severity of the disease or disorder, and should be selected in accordance with the judgment of the practitioner and the circumstances of each patient.

The pharmaceutical composition contains an effective amount of the LDC composition so that a suitable dosage is obtained for administration to a subject in need thereof. Typically this amount is at least about 0.01% by weight of the pharmaceutical composition.

For intravenous administration, the pharmaceutical composition may contain from about 0.01 to about 100 mg of the LDC composition per kg of animal body weight. In one aspect, the pharmaceutical composition may include from about 1 to about 100 mg of the ADC composition per kg of animal body weight. In another aspect, the amount administered will range from about 0.1 to about 25 mg/kg body weight of the ADC composition.

Typically, the dose of the LDC composition administered to a patient is typically from about 0.01 mg/kg to about 100 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is from about 0.01 mg/kg to about 15 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is from about 0.1 mg/kg to about 15 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is from about 0.1 mg/kg to about 20 mg/kg of the subject's body weight. In other embodiments, the dose administered is from about 0.1 mg/kg to about 5 mg/kg, or from about 0.1 mg/kg to about 10 mg/kg of the subject's body weight. In some embodiments, the dose administered is from about 1 mg/kg to about 15 mg/kg of the subject's body weight. In other embodiments, the dose administered is from about 1 mg/kg to about 10 mg/kg of the subject's body weight. In some embodiments, the dose administered is in the range of about 0.1 to 4 mg/kg, preferably 0.1 to 3.2 mg/kg or more, preferably 0.1 to 2.7 mg/kg body weight the subject's body during the treatment cycle.

LDC can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous integuments (eg, oral mucosa, rectal and intestinal mucosa). Administration may be systemic or local. Various delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, capsules, and can be used to administer the compound. In certain embodiments, more than one compound or composition is administered to a patient.

In one embodiment, the conjugates are formulated according to conventional procedures into a pharmaceutical composition adapted for intravenous administration to animals, especially humans. Typically, vehicles or carrier media for intravenous administration are sterile isotonic aqueous buffer solutions. If necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally contain a local anesthetic, such as lignocaine, to relieve pain at the injection site. Typically, the ingredients are supplied either individually or mixed in a unit dosage form, such as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. If the conjugate is to be administered by infusion, it can be dispensed, for example, into an infusion bottle containing sterile water or pharmaceutical grade saline. When the conjugate is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and fully compliant with all US Food and Drug Administration Good Manufacturing Practices (GMP).

1.7.1. Numbered options.

The following numbered embodiments further describe the invention without limiting it.

1. Drug-ligand conjugate, represented by the structure of one of the formulas 2A2F

- 74 046150

(Formula 2F) or its salt,

L represents a ligand unit from an antibody;

LB is the unit that covalently binds the ligand;

LP represents a parallel connecting unit, where LP represents an aminoalkanedioic acid, a diaminoalkanoic acid, a sulfur-alkanedioic acid, a sulfur-substituted aminoalkanoic acid, a diaminoalkanol, an aminoalkanedinol, a hydroxyl-alkanedioic acid, a hydroxyl-substituted aminoalkanoic acid, or a sulfur-substituted aminoalkanol moiety, optionally placed, where the substituent containing sulfur, is in reduced or oxidized form or LP is an amino acid residue of lysine, arginine, asparagine, glutamine, ornithine, citrulline, cysteine, homocysteine, penicillamine, threonine, serine, glutamic acid, aspartic acid, tyrosine, histidine or tryptophan, where the amino acid has D - or L-configuration;

PEG is a polyethylene glycol unit;

subscript a denotes 0 or 1;

A represents the first optional extension link, so when index a

- 75 046150 is 0, A is absent, or, when the subscript a is 1, A is present and optionally includes two, three or four independently selected subunits (A ₁ , A2, A3, A4);

AO represents a second optional extension unit and optionally includes two, three or four subunits independent of A;

Su represents a carbohydrate moiety;

-O'- represents the oxygen atom of the O-glycosidic bond cleaved by the glycosidase;

-J'- represents -O-, -N(R33)- or -S- associated directly with AO or a subunit thereof, or with LL _B -LP(PEG)- or LB'-LP(PEG)-, when none of A and AO, via a functional group containing J', where R33 represents -H, optionally substituted C1-C8 alkyl or optionally substituted aralkyl;

V, Z ¹ and Z3 are =N- or =C(R ²⁴ )-, where R ²⁴ is hydrogen or C1-C6 alkyl, C ₂ -C ₆ alkenyl or C ₂ -C8 alkynyl, optionally substituted, or halogen, -NO2, -CN or other electron-withdrawing group, or -OCH3 or other electron-donating group,

R8 and ^R9 are, independently, hydrogen, C1- _C6 -alkyl, _C2 - _C6 -alkenyl or _C2 - _C6 -alkynyl, optionally substituted, or _C6 - _C14 -aryl or _C5 -C ₁₄ -heteroaryl, optionally substituted;

R' represents hydrogen or represents a halogen, -NO ₂ , -CN or other electron-withdrawing group;

D+ is a quaternized tubulisin drug unit having the structure

wherein the Het ring is a 5-membered nitrogen-containing heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, and wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with optional substitution for other provisions;

subscript m denotes 0 or 1;

R2A represents hydrogen or optionally substituted C1-C8 alkyl, or R2A together with the oxygen atom to which it is attached represents an O-linked substituent;

R3 represents hydrogen or optionally substituted C1- _C6 alkyl;

R ⁴ , R ⁵ and R ⁶ represent optionally substituted C1-C ₆ alkyl;

one R ⁷ is optionally substituted C1-C6 alkyl, optionally substituted C6-C18 arylalkyl, optionally substituted C6-C18 heteroarylalkyl, and the other R ⁷ is hydrogen or optionally substituted C1-C6 alkyl;

subscript p' denotes an integer in the range from 1 to 24;

wherein the wavy line indicates covalent binding of D+ to the compound residue of the drug-ligand conjugate structure, and wherein each optionally substituted alkyl is independently selected;

wherein cleavage of said O-glycosidic bond by a glycosidase results in the release of the tubulisin compound (D) from the drug-ligand conjugate compound, wherein the term optionally substituted refers to a moiety that may be unsubstituted or substituted by a group selected from one or more atoms selected from the group , consisting of halogen, -CN, -OH, -NH2, -NH(CH3) -N(CH3) ₂ , C1-C ₆ -alkyl, C1-C ₆ -fluoroalkyl, C1-C ₆ -fluoroalkoxy, C1-C ₆ -heteroalkyl, C3-C ₈ -cycloalkyl, C3-C ₈ -heterocycloalkyl, C ₅ -C ₁₄ -aryl, C ₅ -C ₁₄ -heteroaryl, C1-C ₆ -alkoxy, C ₅ -C ₁₄ -aryloxy, C1 -C ₆ -alkylthio, C ₅ -C ₁₄ -arylthio, C1-C ₆ -alkyl sulfoxide, C ₅ -C ₁₄ -aryl sulfoxide, C^-alkyl sulfone, -C(=O)OH, -C(=O)O- C1-C6-αalkyl, -C(=O)NH2, -C(=O)NH(C1-C ₆ alkyl) and -C(=O)N(C ₁ -C ₆ -αalkyl) ₂ ;

where the term electron-withdrawing group refers to a species selected from the group consisting of optionally substituted C ₂ -C ₆ -alkenyl, optionally substituted C ₂ -C ₆ -kynyl, -C(=O), -CN, -NO2, -CX3, -X, -C(=O)OR', -C(=O)NH ₂ , -C(=O)N(R')R ^op , -C(=O)R', -C(=O) X, -S(=O)2R ^op , -S(=O)2OR', -SO3H2, -S(=O)2NH2, -S(=O)2N(R')R ^op , -PO3H2, -P (=O)(OR')(OR ^op )2, -NO, -NH2, -NH(R ^op ), N(R')(R ^op ) and -N(R ^op )3 ⁺ , where X represents -F, -Cl, -Br or -I, and each of R and R ^op is independently selected from the group of optional substituents;

where the term electron-donating group refers to a species selected from the group consisting of OH, -OR', -NH2, -NHR', N(R') ₂ , optionally substituted _C5 - _C14 aryl, optionally substituted C5- _C14 -heteroaryl, optionally substituted C ₂ -C ₆ -alkenyl and optionally substituted C2 -C 6 -alkynyl, where each R' independently represents C 1 -C ₆ -alkyl; and wherein the term O-linked substituent refers to a substituent selected from the group consisting of -OH, -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -CH2OC(O)R ^2B , -OC(O)N (R ^2B )(R ^2C ) and OCH2C(O)N(R ^2B )(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, C1-C6 alkyl and

- 76 046150 ^-^-alkenyl.

2. The drug-ligand conjugate of Embodiment 1, wherein the antibody ligand moiety is capable of selectively binding to an available cell surface antigen of abnormal cells, wherein the antigen is capable of cellular internalization of the bound ADC and is preferentially present on abnormal or other undesirable cells as compared to normal cells .

3. A drug-ligand conjugate according to any one of embodiments 1 or 2, represented by the structure of formula 4

in the form of a pharmaceutically acceptable salt, where

Ab is a ligand unit from an antibody;

J' represents -N(R ³³ )-, where R ³³ represents hydrogen or methyl;

V and Z ³ are independently =CH- or =N-;

R' represents hydrogen or an electron-withdrawing group;

R ⁸ represents hydrogen;

R ⁹ is hydrogen, optionally substituted with C ₁ -C ₆ -alkyl or optionally substituted phenyl;

R ⁴⁵ represents -CO2H; and the subscript p' denotes a number in the range from 1 to 24.

4. The drug-ligand conjugate according to any one of embodiment 13, represented by the structure of formula 6

[Formula 6) wherein S represents a sulfur atom of a ligand unit from an antibody (Ab);

an asterisk (*) denotes chirality or lack thereof at the specified carbon atom;

A _2-4 represent independently selected optional subunits of A, where -[C(R ^b1 )(R ^b1 )] _q [HE]- represents A1 when one or more such subunits are present;

R represents hydrogen;

R' represents hydrogen or an electron-withdrawing group;

R ^a1 represents hydrogen or a basic unit (BU), where BU represents a basic unit having the structure -CH2-N(R ²² )(R ²³ ), or its acid addition salt, where R ²² and R ²³ , independently, represent is hydrogen, methyl or ethyl, or both together with the nitrogen atom to which they are attached represent a 5- or 6-membered heterocycloalkyl, or one of R ²² and R ²³ is hydrogen and the other is an acid-sensitive carbamate protecting group ;

R ^a2 represents hydrogen;

subscript q denotes an integer ranging from 0 to 5 when HE is present, or from 1 to 5 when HE is absent;

each R ^b1 independently represents hydrogen or optionally substituted ^-^-alkyl;

HE is absent or represents -C(=O)-;

R ⁴⁵ represents -CO2H;

J' represents -NH-;

V and Z ³ represent =CH-;

R ⁸ represents hydrogen;

R ⁹ represents hydrogen or methyl;

subscript p' denotes an integer in the range from 1 to 16; and where the remaining group variables have the values defined for formula 4, or

- 77 046150 where the conjugate has the structure of formula 9A or formula 9B

where S represents the sulfur atom of the ligand unit from the antibody (Ab);

an asterisk (*) denotes chirality or lack thereof at the specified carbon atom;

A2-4 represent independently selected optional subunits of A, where -[C(R ^b1 )(R ^b1 )]q[HE]- represents A ₁ when one or more such subunits are present;

R represents hydrogen;

R' represents hydrogen or an electron-withdrawing group;

R ^a1 is -H or BU, where BU is a backbone having the structure -CH ₂ -N(R ²² )(R ²³ ), or an acid addition salt thereof, where R ²² and R ²³ are independently hydrogen either methyl, or both together with the nitrogen atom to which they are attached represent a basic nitrogen-containing 5- or 6-membered heterocycloalkyl, or one of R ²² and R ²³ is hydrogen and the other is an acid-sensitive protecting group;

R ^a2 represents hydrogen;

subscript q denotes an integer ranging from 0 to 5 when HE is present, or from 1 to 5 when HE is absent;

each R ^b1 independently represents hydrogen or optionally substituted C ₁ -C ₆ -αalkyl;

HE is absent or represents -C(=O)-;

J' represents -O- or -NH-;

R ⁸ and R ⁹ independently represent -H or optionally substituted C ₁ -C ₆ -alkyl, or both, together with the carbon atom to which they are attached, represent ^-^-cycloalkyl;

subscript p' denotes an integer in the range from 1 to 24; and where the remaining group variables have the values defined for formula 4.

5. The drug-ligand conjugate of any one of embodiment 14 represented by the structure of Formula 10A or Formula 10B

- 78 046150 where R represents hydrogen;

R' is hydrogen, -NO2, -Cl or -F;

HE represents -C(=O)-;

R ⁴⁵ represents -CO2H;

J' represents -NH-;

V and Z ³ are each =CH2-;

R ⁸ represents hydrogen;

R ⁹ represents hydrogen or methyl;

p' is an integer in the range from 1 to 12; and where the remaining group variables have the values defined for formula 4.

6. The drug-ligand conjugate of Embodiment 4, wherein said carbon marked with an asterisk (*) predominantly has the same absolute configuration as the alpha carbon of the L-amino acid when said carbon is chiral, and/or wherein

A and AO, independently, have the structure of formula 7 or formula 8 _: 43 _R 44\ _{r41 r} 42 O (Formula 7) (Formula 8) where the wavy lines indicate covalent bonding in the conjugate structure, where K and L, independently, represent C, N, O or S, provided that when K or L is O or S, R ⁴¹ and R42-K or R ⁴³ and R44-L are absent, and when K or L is N, one of R ⁴¹ and R42-K or one of R ⁴² and R ⁴³ -L are absent, and provided that two adjacent Ls are not independently selected as N, O or S;

where the subscripts e and f are independently selected integers that range from 0 to 12, and the subscript g denotes an integer ranging from 1 to 12, where G represents hydrogen, optionally substituted with C^C^alkyl, -OH, -OR ^pr , -CO2H, CO2RPR, where R ^pr is an appropriate protecting group, or G is -N(R ^pr )(R ^pr ), where each R ^pr is a protecting group, or G is -N(R ⁴⁵ )( ^R46 ), wherein one of ^R45 and ^R46 is hydrogen or ^Rpr , wherein ^Rpr is an appropriate protecting group, and the other is hydrogen or optionally substituted C1- _C6 alkyl;

where R ³⁸ represents hydrogen or optionally substituted C1-C6 alkyl; R ³⁹ -R ⁴⁴ are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C ₆ -C ₁₄ aryl or optionally substituted C ₅ -C ₁₄ heteroaryl, or both R ³⁹ and R ⁴⁰ together with an atom carbon to which they are attached include C ₃ -C ₆ -cycloalkyl, or R ⁴¹ and R ⁴² together with the K to which they are attached, when K is C, or R ⁴³ and R ⁴⁴ together with L to which they are attached attached when L represents a carbon atom, include C ₃ -C ₆ -cycloalkyl, or R ⁴⁰ and R ⁴¹ , or R ⁴⁰ and R ⁴³ , or R ⁴¹ and R ⁴³ together with the carbon atom or heteroatom to which they are attached, and the atoms located between these carbon atoms and/or heteroatoms form a 5- or 6-membered cycloalkyl or heterocycloalkyl, provided that when K is O or S, R ⁴¹ and R ⁴² are absent when K is N, one of R ⁴¹ and R ⁴² is absent when L is O or S, R ⁴³ and R ⁴⁴ are absent, and when L is N, one of R ⁴³ and R ⁴⁴ is absent, or where AO has a structure corresponding to alpha amino, beta-amino or other amino acid.

7. The drug-ligand conjugate according to embodiment 1, wherein the quaternized tubulisin drug unit (-D+) has the following structure:

where index m denotes 0 or 1;

Z represents an optionally substituted C1- _C6 alkylene or an optionally substituted _C2 - _C8 alkenylene; And

- 79 046150

R ^7A is optionally substituted C ₆ -C ₁₄ -aryl or optionally substituted C6 -C ₁₄ -heteroaryl.

8. The drug-ligand conjugate of Embodiment 1, wherein the quaternized tubulisin drug (-D+) unit has the following structure:

where R ^7A represents optionally substituted phenyl and R ⁸ represents hydrogen or methyl.

9. The drug-ligand conjugate according to any one of embodiments 7 or 8, wherein the quaternized tubulisin drug unit (-D+) has the structure:

where R ⁴ represents methyl; the subscript u denotes 0, 1 or 2;

R ³ represents H, methyl, ethyl, propyl, -CH2-OC(O)R ^3A , -CH2CH(R ^3B )C(O)R ^3A or -CH(R ^3B )C(O)NHR ^3A , where R ^3A is AA-alkyl and R ^3B is H or C1-C6 alkyl independently selected from R ^3A ;

R ^2A , together with the oxygen atom to which it is attached, represents an O-linked substituent selected from the group consisting of -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -OCH2OC (0) R ^2B , -OC (O)N(R ^2B )(R ^2C ) and -OCH ₂ C(O)N(R ^2B )(R ² c), where R ^2B and R ^2C are independently selected from the group consisting of H, CpC^-alkyl and A-A-alkenyl; and each R ^7B , when present, is independently -OH or -OCH ₃ .

10. The drug-ligand conjugate of Embodiment 9, wherein the quaternized tubulisin drug unit (-D+) has the structure

where R ^2A is -CH ₂ CH ₃ , -CH ₂ -CH=CH ₂ or CH ₂ C(CH ₃ )=CH ₂ , R ^2B is CH3 , R ³ is -CH3 , subscript u is 0 or 1 and R ^7B is -OH.

11. The drug-ligand conjugate according to embodiment 9, wherein the quaternized tubulisin drug unit (-D+) has the structure

where R ^2A represents -CH ₂ CH ₃ or -CH ₂ -CH=CH ₂ .

12. The drug-ligand conjugate of Embodiment 9, wherein the quaternized tubulisin drug unit (-D+) has the structure

- 80 046150 where R ^2B represents -CH ₃ , -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH2C(CH3)3, or where a quaternized tubulisin drug unit (-D+) has a structure

or where ^R2B is hydrogen, methyl or -OCH3, or

-OCH2R ^2B represents -OC l.-CI ICI,- or -OC Ι.<(Ο 1 _: ) =СШ

13. The drug-ligand conjugate of Embodiment 12, wherein the quaternized tubulisin drug -D+ unit is such tubulisin

M, which has the following structure:

14. The drug-ligand conjugate of Embodiment 1, wherein LP is an aminoalkanedioic acid, a diaminoalkanoic acid, a sulfur-alkanedioic acid, a sulfur-aminoalkanoic acid, a diaminoalkanol, an aminoalkandinol, a hydroxyl-alkanedioic acid, a hydroxyl-aminoalkanoic acid, or a sulfur-substituted ny residue of aminoalkanol, aminoalkanedioic acid, diaminoalkanoic acid, sulfur-substituted aminoalkanoic acid or hydroxyl-substituted aminoalkanoic acid residue has the structure of formula A or formula B

(Formula A) (Formula B) where the index v denotes an integer in the range from 1 to 4;

subscript v' denotes an integer ranging from 0 to 4;

XLP is selected from the group consisting of -O-, -NRLP-, -S-, -S(=O)-, -S(=O) ₂ -, -C(=O)-, -C(=O)N (R ^LP )-, -N(R ^lp )C(=O)N(R ^lp )- and -N(R ^lp )C(=NR ^lp )N(R ^lp )-, where each R ^LP is independently selected from a group comprising hydrogen and an optionally substituted C ₁ -C ₆ -alkyl, or two RLPs together with their intermediate atoms define an optionally substituted C ₅ -C ₁₀ -heterocycloalkyl, and the remaining RLPs have the meanings defined above;

Ar represents arylene or heteroarylene, optionally substituted;

each RE and R ^f are independently selected from the group consisting of -H, optionally substituted C ₁ -C ₆ -alkyl, optionally substituted C ₆ -C ₁ 4-aryl and optionally substituted C ₅ -C ₁ 4-heteroaryl, or R ^e and R ^f together with the same carbon atom to which they are attached, or R ^E and R ^F of adjacent carbon atoms together with these carbon atoms denote optionally substituted cycloalkyl, the remaining substituents R ^E and R ^F having the meanings defined above; and where the wavy lines indicate covalent attachment in the structure of formula A or formula B in the structure of the conjugate, or where -LP(PEG)- has a structure of formula A1 or A2:

(Formula A1) (Formula A2) where X ^LP is selected from the group consisting of -O-, -NH, -S- and -C(=O)-;

- 81 046150

R ^e and R ^f are independently selected from the group consisting of -H and -C1-C ₄ -alkyl; and where the wavy line indicates covalent addition in Formula A1 or Formula A2 in the conjugate structure.

15. The drug-ligand conjugate of Embodiment 1, represented by the following structure:

where Ab represents a ligand unit from an antibody;

S represents the sulfur atom of the ligand unit from the antibody;

R ^2A is C1-C4 alkyl, C2-C4 alkenyl, SE-Sralkynyl or -C(=O)R ^2B , where R ^2B is C1-C4 alkyl;

AO is absent or is an amino acid residue;

The subscript p' denotes an integer in the range from 1 to 8.

subscript q denotes an integer in the range from 1 to 4;

index u is 0 or 1;

subscript v denotes an integer in the range from 1 to 4;

R ^7B , when present, represents -OH;

X ^LP is selected from the group consisting of -O-, -NH, -S- and -C(=O)-; And

R ^e and R ^f are independently selected from the group consisting of -H and C1-C ₄ -alkyl, or wherein the conjugate is represented by the structure

where Ab represents a ligand unit from an antibody;

S represents the sulfur atom of the ligand unit from the antibody;

the Ab-S- fragment is connected to the α or β carbon of the specified carboxylic acid M ³ ;

R2A is C1- _C4 alkyl, _C2 - _C4 alkenyl, _C2 - _C4 alkynyl or -C(=O)R ^2B , where ^R2B is C1- _C4 alkyl;

the subscript p' denotes an integer in the range from 1 to 8.

subscript q denotes an integer in the range from 1 to 4;

the subscript u denotes 0 or 1;

subscript v denotes an integer in the range from 1 to 4;

R ^7B , when present, represents -OH;

X ^LP is selected from the group consisting of -O-, -NH, -S- and -C(=O)-;

R ^e and R ^f are independently selected from the group consisting of -H and C1- _C4 alkyl; And

PEG is a polyethylene glycol unit.

16. The drug-ligand conjugate of Embodiment 15, wherein the PEG has a structure selected from the group consisting of

- 82 046150

-^-R ^PEG1 -(CH ₂ CH ₂ O) _n ---R ^PEe2

R ^PEG1 -(CH2CH£i)rt—R ^PE03 —(CH2CH ₂ O) _rf —R ^PE&2

-£-R ^PEe1 —(ij:H2CH2Ci:ir|1J^R ^PE,:?3 —(CE2CE ₂ Oj _r .^R ^PE * ² where the wavy line shows the place of connection to X ^LP of the parallel connecting link (Lp),

R ^peg1 is an optional PEG joining unit,

RPEG2 is a PEG restriction unit;

R ^PEG3 is a PEG linker;

index n varies from 2 to 72;

each index n' is independently selected from 1 to 72; and the index e varies from 2 to 5.

17. The drug-ligand conjugate of any one of embodiments 15 or 16, wherein R ^2A is ^-^-alkyl or ^-^-alkenyl, wherein ^-^-alkyl is -CH3, -CH2CH3, -CH2CH2CH3 and C3-C4-alkenyl is -CIRCII Cll _; or -CH(CH3)CH=CH2, and -XLP-PEG has the structure:

-^-C(0) — (CH ₂ CH ₂ 0) _p ----R ^PE $ ²

18. The drug-ligand conjugate of Embodiment 15, wherein R ^2A is -C(O)CH3, -CH2CH3 or -CH2CH=CH2; n is 12 and r ^peG2 is hydrogen or -CH3.

19. The drug-ligand conjugate of Embodiment 1, represented by the structure

where Ab represents a ligand unit from an antibody;

S represents the sulfur atom of the ligand unit from the antibody;

the Ab-S- fragment is connected to the α or β carbon of the specified carboxylic acid M ³ ;

the subscript p' denotes an integer in the range from 1 to 8;

the subscript u denotes 0 or 1;

R ^7B , when present, represents -OH; And

R ^2A together with the oxygen atom to which it is attached is -OC(O)CH ₃ , -OCH2CH3 or -OCH2CH=CH2.

20. The drug-ligand conjugate of embodiment 19, represented by the structure

- 83 046150

21. A drug compound with a linker having the structure of one of the formulas IIA-IIF R ⁸ R ⁹

PEG R(Formula HA)

- 84 046150

(Formula IIF) or a pharmaceutically acceptable salt thereof, wherein

LB' is a precursor unit of a covalently binding ligand;

LP represents a parallel connecting unit, where LP represents an aminoalkanedioic acid, a diaminoalkanoic acid, a sulfur-alkanedioic acid, a sulfur-aminoalkanic acid, a diaminoalkanol, an aminoalkanedinol, a hydroxyl-alkanedic acid, a hydroxyl-aminoalkanoic acid, or a sulfur-substituted aminoalkanol moiety, optionally placed, where the substituent containing sulfur , is in reduced or oxidized form, or LP is an amino acid residue of lysine, arginine, asparagine, glutamine, ornithine, citrulline, cysteine, homocysteine, penicillamine, threonine, serine, glutamic acid, aspartic acid, tyrosine, histidine or tryptophan, where the amino acid has D- or L-configuration;

PEG is a polyethylene glycol unit;

subscript a denotes 0 or 1;

A represents the first optional extension unit, such that when the subscript a is O, A is absent, or when the subscript a is 1, A is present and optionally includes two, three, or four independently selected subunits (A1, A2, A ₃ , A4);

A _O represents a second optional extension unit, and optionally includes two, three or four subunits independent of A;

Su represents a carbohydrate moiety;

- O'- represents the oxygen atom of the O-glycosidic bond, cleaved by glycosidase;

- J'- represents -O-, -N(R ³³ )- or -S- associated directly with A _O or a subunit thereof, or with L-LB-LP(PEG)- or LB'-LP(PEG) -, when there is none of A and AO, via a functional group containing J', where R ³³ represents -H, optionally substituted ^-^-alkyl or optionally substituted aralkyl;

V, Z ¹ and Z ³ represent =N- or =C(R ²⁴ )-, where R ²⁴ represents hydrogen or C1-C6 alkyl, ^-^-alkenyl or ^-^-alkynyl, optionally substituted, or halogen, -NO2, -CN or other

- 85 046150 electron-withdrawing group or electron-donating group,

R ⁸ and R ⁹ are, independently, hydrogen, C 1 -C ₆ -alkyl, C 2 -C ₈ -alkenyl or C 2 -C ₈ -kynyl, optionally substituted, or C ₆ -C ₁₄ -aryl or C ₅ -C ₁₄ -heteroaryl, optionally substituted;

R' represents hydrogen or represents a halogen, -NO2, -CN or other electron-withdrawing group;

D+ is a quaternized tubulisin drug unit having the structure

wherein the Het ring is a 5-membered nitrogen-containing heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, and wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with optional substitution for other provisions, the index m denotes 0 or 1;

R ^2A represents hydrogen or optionally substituted C1-C ₈ -alkyl, or R ^2A together with the oxygen atom to which it is attached denotes an O-linked substituent other than -OH;

R ³ represents hydrogen or optionally substituted C1-C ₆ -alkyl;

R ⁴ , R ⁵ and R ⁶ represent optionally substituted C1-C ₆ alkyl;

one R ⁷ is optionally substituted C1-C6 alkyl, optionally substituted C6-C18 arylalkyl, optionally substituted C6-C18 heteroarylalkyl, and the other R ⁷ is hydrogen or optionally substituted C1-C6 alkyl;

R ^8A represents hydrogen or optionally substituted C1- _C6 alkyl, wherein the wavy line indicates covalent bonding of D+ to the residue of the drug compound structure with the linker, and wherein the optionally substituted C1- _C6 alkyl is independently selected;

wherein cleavage of said O-glycosidic bond by a glycosidase results in the release of a tubulisin compound (D) from a drug-linker compound or a drug-ligand conjugate compound derived from a linker-drug compound, wherein the drug-ligand conjugate compound has the structure of Formula 1A according to claim 1, where the index p' denotes an integer in the range from 1 to 24;

wherein the term optionally substituted refers to a species that may be unsubstituted or substituted by a group selected from one or more atoms selected from the group consisting of halogen, -CN, -OH, -NH ₂ , -NH(CH ₃ ) -N( CH ₃ )2, C1-C ₆ -alkyl, C1-C ₆ -fluoroalkyl, C1-C ₆ -fluoroalkoxy, C1-C6 heteroalkyl, C ₃ -C ₈ -cycloalkyl, C ₃ -C ₈ -heterocycloalkyl, C ₅ -C ₁₄ -aryl, C ₅ -C ₁₄ -heteroaryl, C1- _C6 alkoxy, C ₅ -C 14 -aryloxy, C1-C ₆ -alkylthio, C ₅ -C ₁₄ -arylthio, C1-C ₆ -alkyl sulfoxide, C ₅ -C ₁₄ -aryl sulfoxide, C1-C ₆ -alkylsulfone, -C(=O)OH, -C(=O)OC ₁ -C ₆ -alkyl, -C(=O)NH ₂ , -C(=O)NH( C ₁ -C ₆ -alkyl) and -C(=O)N(Ci-C6^ku3)2;

where the term electron-withdrawing group refers to a species selected from the group consisting of optionally substituted C ₂ -C ₆ -alkenyl, optionally substituted C ₂ -C ₆ -kynyl, -C(=O), -CN, -NO2, -CX3, -X, -C(=O)OR', -C(=O)NH2, -C(=O)N(R')R ^op , -C(=O)R', -C(=O)X , -S(=O)2R ^op , -S(=O)2OR', -SO3H2, -S(=O)2NH2, -S(=O)2N(R')R ^op , -PO3H2, -P( =O)(OR')(OR ^op )2, -NO, -NH2, -NH(R ^op ), -N(R')(R ^op ) and -N(R ^op ) ₃ ⁺ , where X represents -F, -Cl, -Br or -I, and each of R and R ^op is independently selected from the group of optional substituents;

where the term electron-donating group refers to a species selected from the group consisting of -OH, -OR', -NH2, -NHR', N(R') ₂ , optionally substituted C ₅ -C ₁₄ -aryl, optionally substituted C ₅ - C ₁₄ -heteroaryl, optionally substituted C ₂ -C ₆ -alkenyl and optionally substituted C 2 -C 6 -alkynyl, where each R' independently represents C 1 -C ₆ -alkyl; and wherein the term O-linked substituent refers to a substituent selected from the group consisting of -OH, -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -CH2OC(O)R ^2B , -OC(O)N (R ^2B )(R ^2C ) and -OCH2C(O)N(R ^2B )(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, C1-C6 alkyl and C ₂ -C ₈ -alkenyl.

22. The drug-linker compound of Embodiment 21, wherein L _B ' has a structure selected from the group consisting of

- 86 046150

where the wavy line indicates covalent binding to A or LP, depending on the presence or absence, respectively, of A;

R represents hydrogen or optionally substituted Q-C^-alkyl;

R is hydrogen or halogen, or R and R' are independently selected from halogen;

T represents -Cl, -Br, -I, -O-mesyl or -O-tosyl or other sulfonate leaving group;

U represents -F, -Cl, -Br, -I, -ON-succinimide, 10-nitrophen^-O-pentafluorophenyl, -Otetrafluorophenyl or -OC(=O)-OR ⁵⁷ ; And

X ² represents C _1-10 αalkylene, C ₃ -C ₈ -carbocycle, -O-(C ₁ -C ₆ -αalkyl), -arylene-, C ₁ -C ₁₀ -alkylenarylene, -arylene-C ₁ -C ₁₀ -alkylene, -C ₁ -C ₁₀ -αalkylene-(C ₃ -C ₆ -carbocycle)-, -(C ₃ -C ₈ carbocycle)-C ₁ -C ₁₀ -αalkylene-, ^-^-heterocycle, - C ₁ -C ₁₀ -alkylene-(C ₃ -C ₈ -heterocyclo)-, -C ₃ -C ₈ -heterocyclo)-C ₁ -C ₁₀ -alkylene,

-(CH ₂ CH ₂ O)u or -(CH ₂ CH ₂ O)u-CH ₂ -, where the subscript u denotes an integer ranging from 1 to 10 and R ⁵⁷ represents ^-^-alkyl or C ₆ - C ₁₄ -aryl.

23. The drug-linker compound of any one of embodiments 21 or 22, wherein -O'-Su has the structure of formula 3

HE

O O'-|— , ha * (Formula 3) where the wavy line represents the covalent binding of O' to the residue of the drug-linker compound structure; and R ⁴⁵ is -CH2OH or -CO ₂ H.

24. The drug-linker compound of any one of embodiments 21-23 having the structure of Formula IV

where J' represents -N(R ³³ )-, where R ³³ represents hydrogen or methyl;

V and Z ³ are independently =CH- or =N-;

R' represents hydrogen or an electron-withdrawing group;

R ⁸ represents hydrogen;

R ⁹ represents hydrogen, optionally substituted ^-^-alkyl or optionally substituted phenyl; And

R ⁴⁵ is -CO ₂ H.

25. The connection of a drug with a linker according to any one of embodiments 21-23, where the index a denotes 1; and Lb'-A- of formula IA has the structure of formula V

- 87 046150

ABOUT

R'4J R ^a1

JN—(—R ^a2

[C(R ^b1 )(R ^b1 )] _q [HE]-(A ₂ . ₄ ) _γ ________J

Lr a or Ai ^D (Formula V) where the fragment -[C(R ^b1 )(R ^b1 )] q- [HE]- represents A or A1, where A ₁ represents a subunit of A;

A _2-4 are optional A subunits; R represents hydrogen, chlorine or CrClkyl; R represents hydrogen or chlorine;

R ^a1 represents hydrogen, optionally substituted CrCg-alkyl or backbone (BU), optionally protected; And

R ^a2 represents hydrogen or optionally substituted ^-^-alkyl, or

R ^a1 and R ^a2 together with the carbon atom to which they are attached represent nitrogen-containing C ₃ Cw-heterocycloalkyl;

HE is an optional hydrolysis enhancer (HE) unit;

subscript q denotes an integer in the range from 0 to 6;

each R ^b1 independently represents hydrogen, optionally substituted ^-^-alkyl, optionally substituted ^-C^-aryl or optionally substituted C5-C10-heteroaryl, or two R ^b1 together with the carbon atom(s) to which they are attached, is ^-^-cycloalkyl, or one R ^b1 and HE together with the carbon atom to which they are attached is a 5 or 6 membered cycloalkyl or a 5 or 6 membered heterocycloalkyl, and the other R ^b1 is hydrogen, optionally substituted with ^ -^-alkyl, optionally substituted C6-C10-aryl or optionally substituted C5-C ₁₀ -heteroaryl;

BU, optionally protected, has the structure -[C(R ¹ )(R ¹ )]-[C(R ² )(R ² )] _r -N(R ²² )(R ²³ ) or its acid addition salt, where subscript r represents 0, 1, 2 or 3;

each R ¹ is independently hydrogen or ^-^-alkyl, or two R ¹ together with the carbon atom to which they are attached form a ^-^-cycloalkyl, and each R ² is independently hydrogen, optionally substituted with ^-^- alkyl, optionally substituted ^-C^-aryl or optionally substituted ^-^-heteroaryl, or two R ² together with the carbon atom(s) to which they are attached and intermediate carbon atoms are ^-^-cycloalkyl, or one R ¹ and one R ² together with the carbon atoms to which they are attached and intermediate carbon atoms represents a 5- or 6-membered cycloalkyl, and the remaining R ¹ and R ² are as indicated; And

R ²² and R ²³ independently represent hydrogen, an optionally substituted ^-^-alkyl or an acid-sensitive protecting group, or together with the nitrogen atom to which they are attached, a 5- or 6-membered heterocycloalkyl, or one of R ²² and R ²³ represents hydrogen, and the other represents an easily removable protecting group; and where the wavy line indicates the site of covalent attachment to the remainder of the drug-linker structure.

26. The drug-linker compound of Embodiment 25 having the structure VA

where the subscript q denotes an integer ranging from 0 to 4, or having the structure of a VB formula

where one of R ²² and R ²³ is hydrogen and the other is an acid-sensitive carbamate protecting group, and the subscript q is an integer ranging from 0 to 4.

27. Connecting a drug to a linker according to any of the embodiments

- 88 046150 or 26, where formula VA or formula VB has the following structure:

respectively.

28. The drug-linker compound of one of embodiments 25 or 26 having the structure of formula VI

Р _r8 R ^{9 R} 'M Г ¹ PEG Г° ⁺

T n^-r ^{32 reg} У=\

[C(R ^b1 )(R ^b1 )] _q —[HE]-(A ₂ . ₄ )-L _P -A _O -J'—d/ ³

HO O'R' ^H °^

HO r45, (Formula VI) where the asterisk (*) denotes the chirality or lack thereof of the specified carbon atom;

A _2-4 represent independently selected optional subunits of A, where -[C(R ^b1 )(R ^b1 )] _q [HE]- represents A1 when one or more such subunits are present;

one of R and R is hydrogen and the other is hydrogen or chlorine;

R' represents hydrogen or an electron-withdrawing group;

R ^a1 represents hydrogen or a base unit (BU), optionally protected, having the structure -CH ₂ -N(R ²² )(R ²³ ) or an acid addition salt thereof, where R ²² and R ²³ are independently hydrogen, methyl either ethyl or both together with the nitrogen atom to which they are attached represent a 5- or 6-membered heterocycloalkyl, or one of R ²² and R ²³ is hydrogen and the other is an acid-sensitive carbamate protecting group;

R ^a2 represents hydrogen;

subscript q denotes an integer ranging from 0 to 5 when HE is present, or from 1 to 5 when HE is absent;

each R ^b1 independently represents hydrogen or optionally substituted CrClkyl;

HE is absent or represents -C(=O)-;

R ⁴⁵ represents -CO ₂ H;

J' represents -NH-;

V and Z ³ represent =CH-;

R ⁸ represents hydrogen; And

R ⁹ represents hydrogen or methyl.

29. The drug-linker compound of Embodiment 28, wherein said carbon marked with an asterisk (*) predominantly has the same absolute configuration as the alpha carbon of the L-amino acid when said carbon is chiral, or wherein A and AO are independently have the structure of formula 7 or formula 8:

wherein the wavy lines indicate covalent attachment to a moiety of the drug compound structure with a linker wherein K and L are independently C, N, O or S, with the proviso that when K or L is O or S, R is ⁴¹ and R ⁴² -K or R ⁴³ and R ⁴⁴ -L are absent, and, when K or L is N, one of R ⁴¹ and R ⁴² -K or one of R ⁴² and R ⁴³ -L is absent, and provided that two adjacent Ls are not independently selected as N, O or S;

where the subscripts e and f are independently selected integers that range from 0 to 12, and the subscript g denotes an integer ranging from 1 to 12;

where G is hydrogen, optionally substituted with Q^-alkyl, -OH, -OR ^pr , -CO ₂ H, CO ₂ R ^pr , where R ^pr is an appropriate protecting group, or G is -N(R ^pr ) R ^pr ), where R ^pr independently represent a protecting group or R ^pr together form a corresponding protecting group,

- 89 046150 or G is -N(R ⁴⁵ )(R ⁴⁶ ), where one of R ⁴⁵ , R ⁴⁶ is hydrogen or RPR, where RPR is an appropriate protecting group, and the other is hydrogen or an optionally substituted C1- C ₆ -alkyl;

where R ³⁸ represents hydrogen or optionally substituted C1-C6 alkyl; R ³⁹ -R ⁴⁴ are independently hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C ₆ -C ₁₄ aryl or optionally substituted C ₅ -C ₁₄ heteroaryl, or both R ³⁹ and R ⁴⁰ together with an atom carbon to which they are attached include C ₃ -C ₆ -cycloalkyl, or R ⁴¹ and R ⁴² together with the K to which they are attached, when K is C, or R ⁴³ and R ⁴⁴ together with L to which they are attached attached when L represents a carbon atom, form C ₃ -C ₆ -cycloalkyl, or R ⁴⁰ and R ⁴¹ , or R ⁴⁰ and R ⁴³ , or R ⁴¹ and R ⁴³ together with the carbon atom or heteroatom to which they are attached , and the atoms located between these carbon atoms and/or heteroatoms include 5- or 6-membered cycloalkyl or heterocycloalkyl, with the proviso that when K is O or S, R ⁴¹ and R ⁴² are absent when K is N , one of R ⁴¹ and R ⁴² is absent when L is O or S, R ⁴³ and R ⁴⁴ are absent, and, when L is N, one of R ⁴³ and R ⁴⁴ is absent, or where A _O is an alpha residue -amino, beta-amino or other amine-containing acid.

30. The drug-linker compound of any one of embodiments 28 or 29, wherein the quaternized tubulisin drug unit (-D+) has the structure

where the index m denotes 0 or 1;

Z represents an optionally substituted C1- _C6 alkylene or an optionally substituted _C2 - _C8 alkenylene; And

R ^7A is optionally substituted C ₆ -C ₁₀ -aryl or optionally substituted C6C10-heteroaryl.

31. The drug-linker compound of Embodiment 30, wherein the quaternized tubulisin drug (-D) unit has the structure

where R ^7A represents optionally substituted phenyl and R ⁸ represents hydrogen or methyl.

32. The drug-linker compound of any one of embodiments 30 or 31, wherein the quaternized tubulisin drug unit (-D+) has the structure

where R ⁴ represents methyl;

the subscript u denotes 0, 1 or 2;

R ³ represents H, methyl, ethyl, propyl, -CH2-OC(O)R ^3A , -CH2CH(R ^3B )C(O)R ^3A or -CH(R ^3B )C(O)NHR ^3A , where R ^3A is C1-C6 alkyl and R ^3B is H or C1-C6 alkyl independently selected from R ^3A ;

R ^2A , together with the oxygen atom to which it is attached, represents an O-linked substituent selected from the group consisting of -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -OCH2OC (O)R ^2B , -OC (O)N(R ^2B )(R ^2C ) and -OCH2C(O)N(R ^2B )(R ² c), where R ^2B and R ^2C are independently selected from the group consisting of H, C1-C ₆ -alkyl and C ₂ -C ₆ -alkenyl; and each R ^7B , when present, is independently -OH or -OCH ₃ .

33. The drug-linker connection of Embodiment 32, wherein the quaternized tubulisin drug unit (-D+) has the structure

- 90 046150

where R ^2A represents -CH ₂ CH ₃ or -CH ₂ -CH=CH ₂ .

34. Combining a drug with a linker according to any one of embodiments 32 or 33, wherein

R ^2A is -CH ₂ CH ₃ , -CH ₂ -CH=CH ₂ or -CH ₂ C(CH ₃ )=CH ₂ , R ^2B is

-CH ₃ , R ³ is -CH ₃ , and the subscript u is 0, or

R ^2A is -CH ₂ CH ₃ or -CH ₂ -CH=CH ₂ , or CH ₂ C(CH ₃ )=CH ₂ , R ^2B is

-CH ₃ , R ³ is -CH ₃ , and the subscript u is 1, where R ^7B is -OH, or where the quaternized tubulisin drug unit (-D+) has the structure

where R ^2B represents -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -CH ₂ CH( _CH3 ) ₂ or -CH2C(CH3)3, or where the quaternized tubulisin drug unit (-D+) has the structure

where R ^2B represents hydrogen, methyl or -OCH ₃ , or

-OCH2R ^2B is -OCH2CH=CH2 or -OCH2C(CH3)=CH2, or wherein the quaternized tubulisin drug unit -D+ is such tubulisin M, the structure of which is

35. The drug-linker compound of Embodiment 21, wherein LP is an aminoalkandioic acid, a diaminoalkanoic acid, a sulfur-alkanedioic acid, a sulfur-aminoalkanoic acid, or a hydroxyl-substituted aminoalkanoic acid residue, wherein the aminoalkandioic acid, a diaminoalkanoic acid, a sulfur-substituted aminoalkanoic acid, or a hydroxy l-substituted aminoalkanoic acid residue has the structure of formula A or formula B

- 91 046150

where the index v denotes an integer in the range from 1 to 4;

subscript ν' denotes an integer in the range from 0 to 4;

X ^LP is selected from the group consisting of -O-, -NR ^LP -, -S-, -S(=O)-, -S(=O) ₂ -, -C(=O)-, -C(=O )N(R ^LP )-, -N(R ^lp )C(=O)N(R ^lp ) - and

-N(R ^lp )C(=NR ^lp )N(R ^lp )-, wherein each R ^LP is independently selected from the group consisting of hydrogen and an optionally substituted C1-C6 alkyl, or the two R ^LPs together with their intermediate atoms are optionally defined substituted C5-C ₁₀ -heterocycloalkyl, and the remaining ^RLP are as defined above;

Ar is C ₆ -C ₁₄ -arylene or C ₅ -C ₁₄ -heteroarylene, optionally substituted;

each RE and R ^f are independently selected from the group consisting of -H, optionally substituted C6-C14-aryl, optionally substituted C6-C14-aryl, and optionally substituted C5-C14-heteroaryl, or R ^e and R ^f together with the same carbon atom, to to which they are attached, or R ^E and R ^F of adjacent carbon atoms, together these carbon atoms denote an optionally substituted C3-C ₈ -cycloalkyl, the remaining substituents R ^E and R ^F being defined above; and where the wavy lines indicate covalent attachment in a structure of Formula A or a formula in a drug-linker compound structure, or where -L _P (PEG)- has a structure of Formula A1 or A2

(Formula A1) (Formula A2) where X ^LP is selected from the group consisting of -O-, -NH, -S- and -C(=O)-;

R ^e and R ^f are independently selected from the group consisting of -H and -C1-C ₄ -alkyl; and wherein the wavy line indicates a covalent attachment in Formula A1 or Formula A2 in the drug-linker compound structure.

36. The drug-linker compound of any one of embodiments 21-23, represented by the following structure:

x ^LP

A PEG where R ^2A represents C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or -C(=O)R ^2B where R ^2B represents C1-C4 alkyl;

A _O is absent or is an amino-containing acid residue;

subscript q denotes an integer in the range from 1 to 4;

the subscript u denotes 0 or 1;

subscript v denotes an integer in the range from 1 to 4;

R ^7B , when present, represents -OH;

- 92 046150

X ^LP is selected from the group consisting of -O-, -NH, -S- and -C(=O)-;

R ^e and R ^f are independently selected from the group consisting of -H and C1- _C4 alkyl;

one of R ²² , R ²³ is hydrogen, and the other is an easily removable protecting group, or R ²² and R ²³ are each hydrogen on the nitrogen atom to which they are attached, optionally protonated as an acid addition salt.

37. The drug-linker connection of Embodiment 21, wherein ^R2A is C1- _C4alkyl or _C3 - _C4alkenyl , wherein C1- _C4alkyl is _-CH3 , _{CH2CH3 ,} -CH ₂ CH ₂ CH ₃ and C ₃ -C ₄ -alkenyl is -CH ₂ CH=CH ₂ or -CH (CH ₃ ) CH=CH ₂ , or

R ^2a is -C(O)CH ₃ , -CH2CH3 or -CH2CH=CH2.

38. The connection of a drug with a linker according to any one of embodiments 21-23, wherein the PEG has a structure selected from the group consisting of

4-R ^ΡΕΘ1 - (CH ₂ CH ₂ 0) _p ---R ^PEe2

R ^PEei -(CH2CH^))rt—R ^PE<53 —(CH2CH ₂ 0)ri—R ^PEe2

-|-R ^PE ' ⁵¹ —i:CH2CH2Ci)nl ^ ^|ΡΕ,5:Ξ; —(CH2CH ₂ O) _rf -^-R ^PE02 where the wavy line shows the point of connection to X ^LP of the parallel connecting link (Lp),

R ^peg1 is an optional PEG joining unit,

R ^PEG2 is a PEG restriction unit;

R ^PEG3 is a PEG linker;

index n varies from 2 to 72;

each index n' is an integer independently selected from the range 1 to 72; and the index e is an integer from 2 to 5.

39. The drug-linker connection of Embodiment 36, wherein -X ^LP PEG has the structure

-|-C(O) - (CH ₂ CH ₂ 0) _p ----ρ ^ρΕΘ2

40. Connecting a drug with a linker according to embodiment 37, wherein the subscript n is 12 and R ^PEG2 is hydrogen or -CH3.

41. A drug-linker compound according to any one of embodiments 36 or 37, wherein the compound has the structure

where the index u denotes 0 or 1;

R ^7B , when present, represents -OH; And

R ^2A together with the oxygen atom to which it is attached is -OC(O)CH ₃ , -OCH2CH3 or -OCH2CH=CH2.

42. The drug-linker compound of Embodiment 41, wherein the compound has the structure

- 93 046150

43. Drug-ligand conjugate represented by the structure of formula 1D

or a pharmaceutically acceptable salt thereof, wherein L represents a ligand unit from an antibody, thereby defining an antibody-drug conjugate;

LB is the unit that covalently binds the ligand;

LP represents a parallel connecting unit, where LP represents an aminoalkanedioic acid, a diaminoalkanoic acid, a sulfur-alkanedioic acid, a sulfur-substituted aminoalkanoic acid, a diaminoalkanol, an aminoalkanedinol, a hydroxyl-alkanedioic acid, a hydroxyl-substituted aminoalkanoic acid, or a sulfur-substituted aminoalkanol moiety, optionally placed, where the substituent containing sulfur, is in reduced or oxidized form or L _P is an amino acid residue of lysine, arginine, asparagine, glutamine, ornithine, citrulline, cysteine, homocysteine, penicillamine, threonine, serine, glutamic acid, aspartic acid, tyrosine, histidine or tryptophan, where the amino acid has D- or L-configuration;

PEG is a polyethylene glycol unit;

subscript a denotes 0 or 1;

subscript b denotes 0 or 1;

A represents a first optional extension unit, such that when the subscript a is 0, A is absent, or when the subscript a is 1, A is present and optionally includes two, three, or four independently selected subunits (A ₁ , A 2 , A ₃ , A ₄ );

B represents a branching unit or a second optional extension unit (AO), such that when the subscript b is 0, B is absent, or when the subscript b is 1, B is present and optionally includes two, three or four subunits independent of A;

the index n denotes 1, 2, 3 or 4, provided that the subscript b denotes 1 and B represents a branch when the subscript n denotes 2, 3 or 4, and the proviso that B represents AO or is absent when the subscript n stands for 1;

V, Z ¹ , Z ² and Z ³ represent =N- or =C(R ²⁴ )-, where R ²⁴ represents hydrogen or C ₁ -C ₆ alkyl, ^-^-alkenyl or ^-^-alkynyl, optionally substituted, or halogen, -NO2, -CN or other electron-withdrawing group, or -OCH ₃ or other electron-withdrawing group, or -C(R8)(R ⁹ )-D ⁺ , where one of V, Z ¹ and Z ³ is is =C(R ²⁴ )-, where R ²⁴ is -C(R8)(R ⁹ )-D ⁺ ;

R' represents hydrogen or -OCH ₃ or other electron-donating group;

subscript p' denotes an integer in the range from 1 to 24;

- 94 046150

D ⁺ is a quaternized tubulisin drug unit, preferably having the structure

wherein the Het ring is a 5-membered nitrogen-containing heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, and wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with optional substitution for other provisions;

subscript m denotes 0 or 1;

R ^2A represents hydrogen or optionally substituted ^-^-alkyl, or R ^2A together with the oxygen atom to which it is attached denotes an O-linked substituent;

R ³ represents hydrogen or optionally substituted Ci-C^-alkyl;

R ⁴ , R ⁵ and R ⁶ represent optionally substituted ^-^-alkyl;

one R ⁷ is optionally substituted ^-^-alkyl, optionally substituted C ₆ -C ₁₈ -arylalkyl, optionally substituted C ₆ -C ₁₈ -heteroarylalkyl, and the other R ⁷ is hydrogen or optionally substituted ^-^-alkyl;

J represents a heteroatom, optionally substituted in the case of nitrogen, preferably J represents -N(R ³³ )-, where R ³³ represents hydrogen or methyl;

W is a peptide comprising an amino acid sequence covalently attached to J via an amide bond, wherein the amide bond can be cleaved by a protease, wherein cleavage of said amide bond by a protease initiates the release of the tubulisin compound (D) from the drug-ligand conjugate compound;

wherein the term optionally substituted refers to a species that may be unsubstituted or substituted by a group selected from one or more atoms selected from the group consisting of halogen, -CN, -OH, -NH ₂ , -NH(CH ₃ ) -N( CH ₃ ) ₂ , ^-^-alkyl, C ₁ -C ₆ -fluoroalkyl, C ₁ -C ₆ -fluoroalkoxy, ^-^-heteroalkyl, ^-^-cycloalkyl, C ₃ -C ₈ -heterocycloalkyl, C ₅ - C ₁₄ -aryl, C ₅ -C ₁₄ -heteroaryl, ^-^-alkoxy, C ₅ -C ₁₄ -aryloxy, C ₁ -C ₆ -αalkylthio, C ₅ -C ₁₄ -arylthio, C ₁ -C ₆ -alkyl sulfoxide , C5-C14 arylsulfoxide, C ₁ -C ₆ -αalkyl sulfone, -C(=O)OH, -C(=O)OC ₁ -C ₆ -αalkyl, -C(=O)NH ₂ , -C(=O) NH(C ₁ -C ₆ alkyl) and -C(=O)N(C ₁ -C ₆ ^kuh) ₂ ;

where the term electron-withdrawing group refers to a species selected from the group consisting of optionally substituted ^-^-alkenyl, optionally substituted ^-^-alkynyl, -C(=O), -CN, -NO2, -CX3, -X, - C(=O)OR', -C(=O)NH2, -C(=O)N(R')R ^op , -C(=O)R', -C(=O)X, -S( =O)2R ^op , -S(=O)2OR', -SO3H2, -S(=O)2NH2, -S(=O)2N(R')R ^op , -PO3H ₂ , -P(=O) (OR')(OR ^op )2, -NO, -NH2, -NH(R ^op ), -N(R')(R ^op ) and -N(R ^op )3 ⁺ , where X is -F, -Cl, -Br or -I, and each of R and R ^op is independently selected from the group of optional substituents;

where the term electron-donating group refers to a species selected from the group consisting of -OH, -OR', -NH ₂ , -NHR', N(R') ₂ , optionally substituted N-aryl, optionally substituted C ₅ -C ₁₄ -heteroaryl, optionally substituted ^-^-alkenyl and optionally substituted C2-C6alkynyl, wherein each R' is independently CpQ^kuh.

44. The drug-ligand conjugate of Embodiment 43, represented by the structure

wherein W includes a dipeptide, wherein the dipeptide is at the distal end of W, and wherein said bond is an amide bond specifically cleaved by an intracellular protease compared to free circulating serum proteases, or wherein the dipeptide has the structure

where R ³⁴ represents benzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH(OH)CH ₃ or has the following structure:

- 95 046150

R ³⁵ is methyl, -(CH ₂ ) ₄ -NH2, -(CH ₂ ) ₃ NH(C=O)NH ₂ , (CH2) ₃ NH(C=NH)NH2 or

-(CH ₂ ) ₂ CO ₂ H, where the wavy line at the dipeptide N-terminus denotes covalent attachment to A _O or LP, depending on the presence or absence of A _O , respectively, and the wavy line at the dipeptide C-terminus denotes covalent attachment to J.

45. The drug-ligand conjugate of any one of embodiments 43 or 44, wherein the quaternized tubulisin drug unit (-D+) has the structure

where the curved dotted lines indicate optional cyclization;

R ^2A represents hydrogen or an optionally substituted C1-C6 alkyl, or R ^2A together with the oxygen atom to which it is attached represents a substituent selected from the group consisting of -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -CH2OC(O)R ^2B , -OC(O)N(R ^2B )(R ^2C ) and -OCH2C(O)N(R ^2B )(R ^2C ), or R ^2A is absent when R ⁶ is bound to this oxygen atom, as shown by the curved dotted line between R ⁶ and the oxygen atom, denoting an oxygen-containing C5-C ₁₀ -heterocycloalkyl;

circled Ar is a 5-membered nitrogen-heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with optional substitution for other provisions

R ³ represents hydrogen or optionally substituted C1-C ₆ -alkyl;

R ⁴ , R ⁵ and R ⁶ are optionally substituted C 1 -C ₆ -alkyl, independently selected, or R ⁶ is attached to an oxygen atom of the -OR ^2A moiety in which R ^2A is absent, and R ⁴ and R ⁵ are as defined higher;

^R4a is hydrogen or optionally substituted CpCg-alkyl, and ^R4B is optionally substituted C1- _C6 -alkyl, or both together with the nitrogen atom to which they are attached, as shown by the curved dotted line between ^R4A and ^R4B . denotes heterocycloalkyl with a quaternized nitrogen atom, optionally substituted;

one R ⁷ is hydrogen or optionally substituted C1-C6 alkyl, and the other R ⁷ is optionally substituted C6-C ₁₄ aralkyl or C ₅ -C ₁₄ heteroaralkyl;

where the wavy line indicates covalent binding of the D ⁺ structure to the remainder of the conjugate structure.

46. The drug-ligand conjugate of Embodiment 43, wherein the quaternized tubulisin drug (-D ⁺ ) unit has the structure

where index m denotes 0 or 1;

Z represents an optionally substituted C1-C ₆ -alkylene or an optionally substituted C ₂ -C ₈ -alkenylene; And

R ^7A is optionally substituted C ₆ -C ₁₂ -aryl or optionally substituted C ₅ C ₁₂ -heteroaryl.

47. The drug-ligand conjugate of any one of embodiments 45 or 46, wherein the quaternized tubulisin drug unit -D ⁺ has the structure

where R ⁴ represents methyl;

the subscript u denotes 0, 1 or 2;

R ³ is H, methyl, ethyl, propyl, -CH2-OC(O)R ^3A , -CH2CH(R ^3B )C(O)R ^3A or

- 96 046150

-CH(R ³ b)C(O)NHR ^3A , where R ^3A represents ^-^-alkyl and R3B represents H or C ₁ -C ₆ alkyl independently selected from R3A;

R ^2A , together with the oxygen atom to which it is attached, represents an O-linked substituent selected from the group consisting of -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ² b, -CH2OC(O)R ² b, -OC(O)N(R ^2B )(R ² c) and -OCH2C(O)N(R ² b)(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, ^-^- alkyl and C2-C6-alkenyl; and each R ^7B , when present, is independently -OH or -OCH3.

48. The drug-ligand conjugate of Embodiment 45, wherein the quaternized tubulisin drug unit has the structure

where R ^2B represents -CH3, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , -CH ₂ C(CH ₃ ) ₃ , or the quaternized tubulisin drug unit has the structure

where R2B represents hydrogen, methyl or -OCH3, or -OCH ₂ R ^2B represents OCH ₂ CH=CH ₂ or -OCH2C(CH3) =CH2.

49. The drug-ligand conjugate of Embodiment 45, represented by the structure

50. A drug-linker compound represented by the following structure

or a pharmaceutically acceptable salt thereof, wherein LB' is a precursor unit of a covalently binding ligand;

LP represents a parallel connecting link, where LP represents aminoalkanedioic acid, diaminoalkanoic acid, sulfur-alkanedioic acid, sulfur-aminoalkanic acid, diaminoalkanol, aminoalkandinol, hydroxylalkanedic acid

- 97 046150 slot, hydroxyl-substituted aminoalkanoic acid or sulfur-substituted aminoalkanol residue, optionally substituted, where the sulfur-containing substituent is in a reduced or oxidized form or LP is an amino acid residue of lysine, arginine, asparagine, glutamine, ornithine, citrulline, cysteine, homocysteine, penicillamine, threonine, serine, glutamic acid, aspartic acid, tyrosine, histidine or tryptophan, where the amino acid has a D or L configuration;

PEG is a polyethylene glycol unit;

the indices a and b are independently 0 or 1;

subscript n denotes 1, 2, 3 or 4;

A represents a first optional extension unit such that when the index a is 0, A is absent, or when the index a is 1, A is present and optionally includes two, three, or four independently selected subunits (A ₁ , A ₂ , A ₃ , A4);

B represents a branch unit or a second optional extension unit (AO), such that when the subscript b is 0, B is absent, or when the subscript b is 1, B is present and optionally includes two, three or four subunits independent of A, where the index b is 1 and B is a branch when the index n is 2, 3 or 4 or b is 0 or 1, so that B is AO when the index n is 1;

V, Z ¹ , Z ² and Z ³ represent =N- or =C(R ²⁴ )-, where R ²⁴ represents hydrogen or C ₁ -C ₆ alkyl, ^-^-alkenyl or ^-^-alkynyl, optionally substituted, or halogen, -NO2, -CN or other electron-withdrawing group, or -OCH ₃ or other electron-withdrawing group, or -C(R8)( ^R9 )-D ⁺ , where one of V, Z ¹ and Z ³ is is =C(R ²⁴ )-, where R ²⁴ is -C(R8)(R ⁹ )-D ⁺ ;

R' represents hydrogen or -OCH ₃ or other electron-donating group;

D ⁺ is a quaternized tubulisin drug unit, preferably having the structure

wherein the Het ring is a 5-membered nitrogen-containing heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, and wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with optional substitution for other provisions;

subscript m denotes 0 or 1;

R ^2A represents hydrogen or optionally substituted ^-^-alkyl, or R ^2A together with the oxygen atom to which it is attached denotes an O-linked substituent;

R ³ represents hydrogen or optionally substituted ^-^-alkyl;

R ⁴ , R ⁵ and R ⁶ represent optionally substituted ^-^-alkyl;

one R ⁷ is optionally substituted ^-^-alkyl, optionally substituted C ₆ -C ₁₈ -arylalkyl, optionally substituted C 6-C _{1 8} heteroarylalkyl, and the other R ⁷ is hydrogen or optionally substituted ^-^-alkyl;

J is a heteroatom, optionally substituted with nitrogen, preferably J is -N(R ³³ )-, where R ³³ is hydrogen or methyl;

W is a peptide comprising an amino acid sequence covalently attached to J via an amide bond, wherein cleavage of the amide bond by said protease initiates the release of a tubulisin compound (D) from a drug-linker compound or a drug-ligand conjugate compound derived from a linker-linker compound. a drug, wherein the drug-ligand conjugate compound has the structure of formula 1D according to claim 43, in which the subscript p is replaced by the subscript p', where the subscript p' denotes an integer in the range from 1 to 24;

wherein the term optionally substituted refers to a species that may be unsubstituted or substituted by a group selected from one or more atoms selected from the group consisting of halogen, -CN, -OH, -NH2, -NH(CH ₃ ) -N(CH ₃ )2, ^-^-alkyl, C ₁ -C ₆ -fluoroalkyl, C ₁ -C ₆ -fluoroalkoxy, C ₁ -C ₆ -heteroalkyl, ^-^-cycloalkyl, ^-^-heterocycloalkyl, C ₅ -C ₁₄ -aryl, C ₅ -C ₁₄ -heteroaryl, ^-^-alkoxy, C ₅ -C ₁₄ -aryloxy, ^-^-alkylthio, C ₅ -C ₁₄ -arylthio, ^-Oz-alkyl sulfoxide, C ₅ -C ₁₄ arylsulfoxide, C ₁ -C ₆ -alkylsulfone, -C(=O)OH, -C(=O)O-C ₁ -C ₆ -alkyl, -C(=O)NH ₂ , -C(=O) NH(C ₁ -C ₆ alkyl) and -C(=O)N(C ₁ -C ₆ -alkyl) ₂ ;

where the term electron-withdrawing group refers to a species selected from the group consisting of optionally substituted ^-^-alkenyl, optionally substituted ^-^-alkynyl, -C(=O), -CN, -NO2, -CX3, -X, - C(=O)OR', -C(=O)NH2, -C(=O)N(R')R ^op , -C(=O)R', -C(=O)X, -S( =O)2R ^op , -S(=O)2OR', -SO3H2, -S(=O)2NH2, -S(=O)2N(R')R ^op , -PO3H2, -P(=O)( OR')(OR ^op )2, -NO, -NH2, -NH(R ^op ), -N(R')(R ^op ) and -N(R ^op ) ₃ ⁺ where X represents -F, - Cl, -Br or -I, and each of R and R ^op is independently selected from the group

- 98 046150 optional deputies;

where the term electron-donating group refers to a species selected from the group consisting of -OH, -OR', -NH ₂ , -NHR', N(R') ₂ , optionally substituted 05-0¼^^^ optionally substituted C ₅ -C ₁₄ -heteroaryl, optionally substituted C ₂ -C ₆ -alkenyl and optionally substituted C2 -C 6 -alkynyl, where each R' independently represents C ₁ -C ₆ -alkyl; and wherein the term O-linked substituent refers to a substituent selected from the group consisting of -OH, -OCH2OCH2R ²¹⁵ , -OCH2R ^2B , -OC(O)R ^2B , -CH2OC(O)R ^2B , -OC(O)N (R ^2B )(R ² c) and -OCH2C (O)N(R ^2B )(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, C1-C ₆ alkyl and C ₂ - C ₈ alkenyl.

51. The drug-linker compound of Embodiment 50, represented by the following structure:

wherein W includes a dipeptide, wherein the dipeptide is at the distal end of W, and wherein said bond is an amide bond specifically cleaved by an intracellular protease compared to free circulating serum proteases, and wherein the dipeptide has the structure

where R ³⁴ represents benzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH(OH)CH ₃ or has the following structure:

R ³⁵ is methyl, -(CH ₂ ) ₄ -NH ₂ , -(CH ₂ ) ₃ NH(C=O)NH ₂ , (CH ₂ ) ₃ NH(C=NH)NH ₂ or -(CH ₂ ) ₂ CO _2H where the wavy line at the dipeptide N-terminus denotes covalent attachment to AO or to _LB , depending on the presence or absence of AO, respectively, and the wavy line at the dipeptide C-terminus denotes covalent attachment to the nitrogen atom of said amide bond.

52. The drug-linker compound of Embodiment 51, wherein D ⁺ is a quaternized tubulisin drug unit having the structure

where the curved dotted lines indicate optional cyclization; R ^2A is hydrogen or optionally substituted ^-^-alkyl, or R ^2A together with the oxygen atom to which it is attached is an O-linked substituent other than -OH, or R ^2A is absent when R ⁶ is bonded to that atom oxygen, as shown by the curved dotted line between R ⁶ and the oxygen atom, denoting an oxygen-containing C ₅ -C ₁₀ -heterocycloalkyl;

circled Ar is a 5-membered nitrogen-heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with an optional substitution in the remaining positions;

R ³ represents hydrogen or optionally substituted ^-^-alkyl;

R ⁴ , R ⁵ and R ⁶ are optionally substituted ^-^-alkyl, independently selected, or R ⁶ is attached to an oxygen atom of the -OR ^2A moiety in which R ^2A is absent, and R ⁴ and R ⁵ are as defined above ;

R ^4a is hydrogen or optionally substituted ^-^-alkyl, and R ^4B is optionally substituted ^-^-alkyl, or both together with the nitrogen atom to which they are attached, as shown by the curved dotted line between R ^4A and R ^4B , denote heterocycloalkyl with a quaternized nitrogen atom, optionally substituted;

one R ⁷ is hydrogen or optionally substituted ^-^-alkyl, and the other R ⁷ is optionally substituted ^-^-aralkyl or C ₅ -C ₁₄ -heteroaralkyl;

where the wavy line indicates covalent binding of the D ⁺ structure to the residue of the LDC structure.

- 99 046150

53. The drug-linker compound of Embodiment 52, wherein the quaternized tubulisin drug unit (-D+) has the structure

subscript m denotes 0 or 1;

Z represents optionally substituted ^-^-alkylene or optionally substituted C ₂ -C ₈ -alkenylene; And

R ^7A is optionally substituted 06-0^^^ or optionally substituted C ₅ -C ₁₂ -heteroaryl.

54. The drug-linker compound of any one of embodiments or 53, wherein the quaternized tubulisin drug unit (-D+) has the structure

where R ^7A represents optionally substituted phenyl and R ^8A represents hydrogen or methyl.

55. The drug-linker compound of Embodiment 52, wherein the quaternized tubulisin drug unit (-D+) has the structure

where R ⁴ represents methyl;

the subscript u denotes 0, 1 or 2;

R ³ represents H, methyl, ethyl, propyl, -CH2-OC(O)R ^3A , -CH2CH(R ^3B )C(O)R ^3A or -CH(R ^3B )C(O)NHR ^3A , where R ^3A is C1- _C6alkyl and ^R3B is H or C1-C6alkyl independently selected from ^R3A ;

R ^2A , together with the oxygen atom to which it is attached, represents an O-linked substituent selected from the group consisting of -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -OCH2OC(O)R ^2B , OC( O)N(R ^2B )(R ^2C ) and -OCH2C(O)N(R ^2B )(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl and ^ -C^alkenyl; and each R ^7B , when present, is independently -OH or -OCH ₃ .

56. The drug-linker compound of any one of embodiments 54 or 55, wherein the quaternized tubulisin drug unit (-D+) has the structure

where R ^2B represents -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , -CH ₂ C(CH ₃ ) ₃ , or the quaternized tubulisin drug unit (-D+) has the structure

- 100 046150

where R ^2B represents hydrogen, methyl or -OCH3, or -OCH2R ^2B represents -OCH2CH=CH ₂ or -OCH ₂ C(CH3) =CH ₂ .

57. The drug-linker compound of Embodiment 51, having the structure

wherein one of R ²² and R ²³ is hydrogen and the other is an easily removable protecting group, or R ²² and R ²³ are each hydrogen on the nitrogen atom to which they are attached, optionally protonated as an acid addition salt.

58. A pharmaceutical composition containing a drug-ligand conjugate according to embodiment 1 and one or more excipients.

59. The composition of Embodiment 58, wherein the precursor of the pharmaceutically acceptable formulation is a solid suitable for dilution into a solution for intravenous injection into a subject, or wherein the pharmaceutically acceptable formulation is a liquid suitable for intravenous injection into a subject;

and wherein the drug-ligand conjugate is present in a pharmaceutically acceptable composition in an effective amount for treating the hyperproliferative condition.

60. Use of a drug-ligand conjugate according to any of embodiments 1 or 43 for the manufacture of a drug for treating a hyperproliferative disease or condition.

61. Use according to embodiment 60, wherein the hyperproliferative disease or condition is a malignant neoplasm.

62. Use according to embodiment 61, wherein the hyperproliferative disease or condition is leukemia or lymphoma.

63. A method of inhibiting the proliferation of tumor cells or cancer cells or inducing apoptosis in tumor or cancer cells by exposing said cells in vitro to an effective amount of a drug-ligand conjugate according to any of Embodiments 1 or 43.

Examples

General information.

All commercially available anhydrous solvents were used without further purification. Analytical thin layer chromatography was performed on aluminum plates with silica gel 60 F254 (EMD Chemicals, Gibbstown, NJ). Radial chromatography was performed on a Chromatotron instrument (Harris Research, Palo Alto, CA). Column chromatography was performed on a Biotage Isolera One flash purification system (Charlotte, NC). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system equipped with a Varian ProStar 330 PDA detector. Samples were eluted on a Phenomenex Synergi 2.0 x 150 mm, 4 μm, 80 A C12 reverse phase column. The acidic mobile phase consisted of acetonitrile and water, both containing 0.05% trifluoroacetic acid or 0.1% formic acid (indicated for each compound ). Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min after injection to 95% at 11 min, and then an isocratic 95% acetonitrile until 15 min (flow rate=1.0 ml/min). LC-MS was performed on two different systems. LC-MS system 1 consisted of a ZMD Micromass mass spectrometer coupled to an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0 x 150 mm, 4 μm, 80 A reverse phase column. Ki

- 101 046150 slot eluent consisted of a linear gradient of 5% to 95% acetonitrile in 0.1% aqueous formic acid over 10 min, followed by an isocratic 95% acetonitrile over 5 min (flow rate = 0.4 ml/min) . LC-MS System 2 consisted of a Waters Xevo G2 ToF mass spectrometer coupled to a Waters 2695 separation module via a Waters 2996 photodiode array detector; column, mobile phases, gradient and flow rate were the same as for LC-MS system 1. UPLC-MS system 1 consisted of a Waters SQ mass detector connected to an Acquity Ultra Performance liquid chromatograph equipped with an Acquity UPLC BEH C18 2.1 column x50 mm, 1.7 µm reverse phase. The acidic mobile phase (0.1% formic acid) consisted of a gradient from 3% acetonitrile/97% water to 100% acetonitrile (flow rate=0.5 ml/min). System 2 UPLC-MS consisted of a Waters Xevo G2 ToF mass spectrometer coupled to a Waters Acquity H-Class Ultra Performance LC equipped with an Acquity UPLC BEH C18 2.1 x 50mm reverse phase 1.7 µm column. The acidic mobile phase (0.1% formic acid) consisted of a gradient from 3% acetonitrile/97% water to 100% acetonitrile (flow rate=0.7 ml/min). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system equipped with a Varian ProStar 330 PDA detector. Products were purified on a Phenomenex Synergi 10.0 x 250 mm, 4 μm, 80 A C12 reverse phase column, eluting with 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). Purification methods typically consisted of linear gradients of solvent A to solvent B, gradually increasing from 90% aqueous solvent A to 10% solvent A. The flow rate was 4.6 ml/min when monitored at 254 nm. NMR spectrum data were collected on a Varian Mercury 400 MHz spectrometer. Coupling constants (J) are given in hertz.

Scheme 1

^)-2-(^)-2-((tert-Butoxycarbonyl)amino)-3-methylbutanamido)-propanoic acid (3): Boc-Val-OSu (1, 1.0 g, 3.18) was placed in the flask mmol) and H-Ala-OH (2.312 mg, 3.5 mmol) in anhydrous dimethylformamide (10.6 ml). N,N-diisopropylethylamine (1.1 mL, 6.4 mmol) was added and the solution was stirred under N2 at 50°C for 12 h. The reaction mixture was taken up in DMSO and purified by preparative HPLC to give 3 (808 mg, 88%). Analytical UHPLC-MS (system 1): t _r =1.38 min, m/z (ES+) found 289.60.

tert-Butyl ((S)-1 -(((S)-1 -((4-(hydroxymethyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 oxobutan-2- yl)carbamate (5). Dipeptide 3 (808 mg, 2.8 mmol) and 4-aminobenzyl alcohol 4 (345 mg, 2.8 mmol) in anhydrous dichloromethane (14 ml) were placed in a flame-dried flask. EEDQ (762 mg, 3.1 mmol) was added as a solid and stirred under nitrogen at room temperature for 12 hours. The reaction mixture was then concentrated and purified on silica gel using a Biotage column (CH ₂ Cl ₂ /MeOH, 0% - 10%) to obtain 5 (660 mg, 60%). Analytical UHPLC-MS (system 1): t _r =1.51 min, m/z (ES+) found 394.51.

tert-butyl ((S)-1 -(((S)-1 -((4-(Bromomethyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3-methyl-1 -oxobutan-2 -yl)carbamate (6). A flask containing Boc-Val-Ala-PABA-OH (5.100 mg, 254 µmol), Nbromosuccinimide (68 mg, 381 µmol) and triphenylphosphine (100 mg, 381 µmol) was purged with nitrogen. The reaction mixture was placed in THF (4 ml) and stirred for 12 hours. The reaction mixture was concentrated and purified on silica gel on a Biotage column (hexane/EtOAc, 10%-100%) to give 6 (94 mg, 81%). Analytical UHPLC-MS (system 1): t _r =2.09 min, m/z (ES+) found 456.10.

(2S,4R)-tert-Butyl 4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine -2-carboxamido)pentanamido)-4-methylpentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (8). Tubulisin analogue (7.10 mg, 14 µmol) in anhydrous DCM (0.7 ml) and tert-butanol (0.7 ml) were placed in a flame-dried flask. Diisopropylcarbodiimide (3.2 μL, 21 μmol) and DMAP (0.08 mg, 0.7 μmol) were added and the reaction mixture was stirred at room temperature for 48 h. Reaction

- 102 046150 The mixture was concentrated, placed in DMSO and purified by preparative HPLC to give 8 (3.5 mg, 32%). Analytical UHPLC-MS (system 1): t _r =1.35 min, m/z (ES+) found 784.56.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-5-(tert-butoxy)-4 -methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl) carbamoyl)-1-(4-(^)-2-(^)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperidin-1-ium (9). Boc-Val-Ala-PAB-Br (6, 3.5 mg, 7.7 µmol) and protected tubulisin 8 (4.0 mg, 5.1 µmol) in anhydrous butanone (0.765 ml) were placed in a pressure vessel. N,Ndiisopropylethylamine (1.8 µl, 10 µmol) was added and the reaction mixture was purged with nitrogen. The vessel was sealed and left to stir at 80°C for 12 hours. The reaction mixture was concentrated, placed in DMSO and purified by preparative HPLC to give 9 (3.5 mg, 58%). Analytical UHPLC-MS (system 1): t _r =1.51 min, m/z (ES+) found 1159.58.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1 -oxopentan-2-yl)carbamoyl)-1 -(4-((S) -2(^)-2-amino-3-methylbutanamido)propanamido)benzyl)-1-methylpiperidin-1-ium (10). The flask containing Boc-Val-Ala-PAB-TubM-OtBu (9, 3.5 mg, 3 µmol) was cooled to 0°C under nitrogen atmosphere. A solution of 10% TFA in CH ₂ Cl ₂ (0.3 ml) was added dropwise and stirred for 4 hours. The reaction mixture was concentrated, placed in DMSO and purified by preparative HPLC to give 10 (1.9 mg, 63%). Analytical UHPLC-MS (system 1): t _r =1.05 min, m/z (ES+) found 1003.60.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1 -oxopentan-2-yl)carbamoyl)-1 -(4-((S) -2(^)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperidin-1- y (12). MC-OSu (11, 0.6 mg, 2 µmol) was placed in anhydrous dimethylformamide (0.2 ml) and added to the flask containing Val-Ala-PAB-Tub (10, 1.9 mg, 2 µmol). N,Ndiisopropylethylamine (1.0 mg, 8 µmol) was added and the reaction mixture was stirred under nitrogen for 3 hours. The reaction mixture was taken up in DMSO and purified by preparative HPLC to yield tubulisin quaternary amine linker 12 (1.2 mg, 53 %). Analytical UHPLC-MS (system 1): t _r =1.25 min, m/z (ES+) found 1196.45.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1-oxopentan-2-yl)carbamoyl)-1 -(4((7S,10S ,13S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-isopropyl-2,2,13-trimethyl-4,8,11-trioxo3-oxa- 5,9,12-triazatetradecanamido)benzyl)-1-methylpiperidin-1-ium (14). MDPR(Boc)-OSu (13, 1.3 mg, 3.5 μmol) was placed in anhydrous dimethylformamide (0.3 ml) and added to the flask containing ValAla-PAB-TubM (10, 3.2 mg, 3. 2 µmol). NN-Diisopropyleth'ylamine (1.6 mg, 13 µmol) was added and the reaction mixture was stirred under nitrogen for 3 hours. The reaction mixture was taken up in DMSO and purified by preparative HPLC to give 14 (2.0 mg, 49%) . Analytical UHPLC-MS (system 2): t _r =1.35 min, m/z (ES+) found 1269.76.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1-oxopentan-2-yl)carbamoyl)-1 -(4-((S) -2(^)-2-(^)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl) -1-methylpiperidin-1-ium (15). The flask containing MDPR(Boc)-Val-Ala-PAB-TubM (14.2 mg, 1.6 µmol) was cooled to 0°C under nitrogen atmosphere. A solution of 10% TFA in CH ₂ Cl ₂ (1.6 ml) was added dropwise and stirred for 4 hours. The reaction mixture was concentrated, placed in DMSO and purified by preparative HPLC to give 15 (1.0 mg, 54%). Analytical UHPLC-MS (system 2): t _r =1.02 min, m/z (ES+) found 1169.72.

Tubulysin analogues, in which tubuvalin acetate was replaced by an alkyl ester, were prepared as shown in Scheme 3.

- 103 046150

Scheme 3

General method for esterification of tubuvalin. Boc-protected known tubuvalin (J. Org. Chem., 2008, 73, 4362-4369) intermediate 17 in anhydrous tetrahydrofuran (50 mM) was placed in a flame-dried flask, to which 18-crown-6 (2.0 equivalents) was added. and cooled to -78°C. Potassium hexamethyldisilazide (1.5 equivalents) was added dropwise as a 1M solution in tetrahydrofuran and the reaction mixture was then stirred for 1 hour at -78°C under a nitrogen atmosphere. Iodoalkane (2-5 equivalents) was then added and the reaction mixture was slowly warmed to room temperature and then subjected to UHPLC/MS. After the starting material was consumed, the reaction mixture was cooled on ice and quenched with saturated ammonium chloride and diluted with dichloromethane (10 volumes). The organic layer was washed with 0.1 M HCl and the resulting aqueous phase was extracted twice with dichloromethane. The combined organics were then dried over sodium sulfate, filtered and concentrated to dryness. Purification of crude O-alkylated products was achieved by flash chromatography on silica gel or preparative HPLC.

Ethyl 2-(( 1 R,3R)-3 -((tert-butoxycarbonyl)(methyl)amino)-1-methoxy-4-methylpentyl)thiazole-4-carboxylate (18). Intermediate 17 tubuvalin (170 mg, 440 μmol) was O-methylated as above with iodomethane (89 μL, 880 μmol) to give 170 mg (97%) of the title compound after purification on silica gel, eluting with mixtures of methanol and dichloromethane. UHPLC-MS(system 2): tr=1.62 min, m/z (ES+) calculated 401.21, found 401.28.

Ethyl 2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-ethoxy-4-methylpentyl)thiazole-4-carboxylate (19). Intermediate 17 tubuvalin (392 mg, 1.01 mmol) was O-ethylated as above with iodoethane (791 mg, 5.05 mmol) to give 407 mg (97%) of the title compound after purification on silica gel eluting with methanol mixtures and dichloromethane. UHPLC-MS (system 2): tr=1.66 min, m/z (ES+) calculated 415.23 (M+H) ⁺ , found 415.29.

Ethyl 2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-propoxypentyl)thiazole-4-carboxylate (20). Intermediate 17 tubuvalin (22 mg, 57 μmol) was O-propylated as above with 1-iodopropane (28 μL, 285 μmol) to give 9 mg (37%) of the title compound after purification by preparative HPLC. UHPLC-MS (system 2): tr=1.77 min, m/z (ES+) calculated 428.23 (M+H) ⁺ , found 451.30 (M+Na) ⁺ . 1H NMR (1:1 mixture of rotamers, CDCl ₃ ) δ (ppm) 0.91 (m, 9H), 1.40 (t, J=7.0 Hz, 3H), 1.47 (two s from rotamers, 9H), 1.64 (m, 3H), 1.87 (m, 2H), 2.74 (m, 3H), 3.42 (m, 2H), 4.10 (m, 1H) , 4.42 (kv, J=7.0 Hz, 2H), 4.50 (m, 1H), 8.14 (two s from rotamers, 1H).

General method for saponification of O-alkylated tubuvalin esters. Saponification reactions were carried out at a reaction concentration of 20 mM using a 1:1:1 solvent mixture of tetrahydrofuran:methanol:water. O-alkylated tubuvalin intermediates 18-20 were dissolved in 1 volume each of tetrahydrofuran and methanol. The mixture was then cooled in an ice bath at 0°C. Lithium hydroxide monohydrate (2-3 equivalents) was dissolved in 1 volume of distilled water and added dropwise to the reaction flask, with stirring at 0°C. The reaction mixture was then allowed to warm to room temperature and the progress of the reaction was monitored using UHPLC/MS data. Once the starting material has been converted to the free acid,

- 104 046150 the reaction mixture was quenched with glacial acetic acid (2-3 equivalents) and concentrated by evaporation on a rotor. The crude carboxylic acids were then purified by preparative HPLC.

Ethyl 2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-methoxy-4-methylpentyl)thiazole-4-carboxylate (21). Intermediate 18 tubuvalin methyl ester (170 mg, 425 μmol) was saponified as above with lithium hydroxide monohydrate (19 mg, 1.28 mmol) to give 140 mg (89%) of the title compound. UHPLC-MS (system 1): t _r =1.47 min, m/z (ES+) calculated 373.18, found 373.41. ¹ H NMR (1:1 mixture of rotamers, CDCl ₃ ) δ (ppm) 0.87 (dd, J=6.7, 2.0 Hz, 3H), 0.96 (dd, J=6, 7, 1.2 Hz, 3H), 1.49 (two s from rotamers, 9H), 1.67 (m, 1H), 1.85 (m, 1H), 2.01 (m, 1H), 2 .70 (m, 3H), 3.41 (s, 3H), 4.12 (m, 1H), 4.36 (first rotamer, dd, J=10.5, 2.3 Hz, 0.5H) , 4.48 (second rotamer, d, J=8.6 Hz, 0.5H), 8.28 (two s from rotamers, 1H).

Ethyl 2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-ethoxy-4-methylpentyl)thiazole-4-carboxylate (22). Intermediate 19 tubuvalin ethyl ester (170 mg, 425 μmol) was saponified as above with lithium hydroxide monohydrate (19 mg, 1.28 mmol) to give 140 mg (89%) of the title compound. UHPLC-^(system 2): t _r =1.48 min, m/z (ES+) calculated 387.20 (M+H) ⁺ , found 387.26. 1H NMR (CDCl ₃ ) δ (ppm) 0.88 (dd, J=6.7, 2.0 Hz, 3H), 0.96 (d, J=6.6 Hz, 3H), 1 .49 (two s from rotamers, 9H), 1.68 (m, 1H), 1.86 (m, 1H), 2.00 (m, 1H), 2.69 (m, 3H), 3.53 (m, 2H), 4.09 (m, 1H), 4.43 (first rotamer, dd, J=10.2, 2.7 Hz, 0.5H), 4.54 (second rotamer, d, J =7.0 Hz, 0.5H), 8.24 (two s from rotamers, 1H).

Ethyl 2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-propoxypentyl)thiazole-4-carboxylate (23). Intermediate 20 Tubuvalin propyl ester (9 mg, 20 µmol) was saponified as above with lithium hydroxide monohydrate (1.7 mg, 40 µmol) to give 7.6 mg (95%) of the title compound. UHPLC-MS (system 2): t _r =1.58 min, m/z (ES+) calculated 401.21 (M+H)+, found 401.28 (M+Na)+.

General method for the amide condensation of the O-alkylated free acids of tubuvalin and the allyl ester of tubuphenylalanine. The O-alkylated free acids of tubuvalin 21-23 were pre-activated by dissolving in anhydrous dimethylformamide (25-50 mM) and adding HATU (2.4 equivalents) and DIPEA (5 equivalents); the mixture was then stirred under a nitrogen atmosphere at room temperature for 10 minutes. The activated acid was then added to the known (Org. Lett., 2007, 9, 1605-1607) tubuphenylalanine allylic ester 16 and then the reaction mixture was stirred at ambient temperature under nitrogen atmosphere, this reaction was monitored using UHPLC/MS. After completion of the reaction, glacial acetic acid (14 equivalents) was then added and the product was purified by preparative HPLC.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-methoxy-4-methylpentyl)thiazol-4-carboxamido)-2- methyl 5-phenylpentanoate (24). Intermediate 21 tubuvalin methyl ester (TuvOMe) (140 mg, 380 μmol) was coupled to tubuphenylalanine allylic ester (Tup) 16 (188 mg, 760 μmol) to give 164 mg (72%) of the title compound. UHPLC-MS (system 1): t _r =1.96 min, m/z (ES+) calculated 602.33 (M+H)+, found 602.26.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-ethoxy-4-methylpentyl)thiazol-4-carboxamido)-2- methyl 5-phenylpentanoate (25). Intermediate 22 tubuvalin ethyl ester (TuvOEt) (140 mg, 380 μmol) was coupled to tubuphenylalanine allylic ester (Tup) 16 (188 mg, 760 μmol) to give 164 mg (72%) of the title compound. UHPLC-MS (system 2): t _r =1.84 min, m/z (ES+) calculated 616.34 (M+H)+, found 616.43.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-propoxypentyl)thiazol-4-carboxamido)-2- methyl 5-phenylpentanoate (26). Intermediate 23 tubuvalin propyl ester (TuvOPr) (7.6 mg, 19 μmol) was coupled to tubuphenylalanine allyl ester (Tup) 16 (9.4 mg, 38 μmol) to give 8 mg (67%) of the title compound. UHPLC-MS (system 2): t _r =2.00 min, m/z (ES+) calculated 630.36 (M+H)+, found 630.45. 1H NMR (CDCl ₃ ) δ (ppm) 0.94 (m, 9H), 1.19 (d, J=7.4 Hz, 3H), 1.49 (s, 9H), 1.64 (m, 5H), 1.84 (m, 1H), 2.03 (m, 2H), 2.63 (m, 1H), 2.73 (m, 3H), 2.93 (m, 2H) , 3.41 (m, 2H), 4.07 (m, 2H), 4.29 (m, 1H), 4.41 (m, 2H), 4.55 (m, 2H), 5.25 ( m, 2H), 5.88 (m, 1H), 7.24 (m, 5H), 8.05 (two s from rotamers, 1H).

General method for removing the Boc protecting group from Tuv(O-Alk)-Tup intermediates. Intermediates 24-26 O-alkylated tubuvaline-tubuphenylalanine were deprotected to expose the secondary amine functionality under acidic conditions with 10% TFA in dichloromethane (25 mM). Specifically, the starting material was dissolved in anhydrous dichloromethane (9 volumes) and stirred under a nitrogen atmosphere at 0°C. Then trifluoroacetic acid (1 volume) was added dropwise to the stirred solution. The reaction mixture was slowly warmed to room temperature and the progress of the reaction was monitored using UHPLC/MS data. After completion of the reaction, the reaction mixture was concentrated by evaporation on a rotor and pumped out in a vacuum line overnight. Free amines 27-29 were further used without additional purification.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-methoxy-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamido)-2methyl-5-phenylpentanoate (27). Intermediate 24 Boc-protected TuvOMe-Tup (160 mg,

- 105 046150

267 μmol) was deprotected as described above to give 133 mg (99%) of the title compound. UHPLC-MS (system 1): t _r =1.17 min, m/z (ES+) calculated 524.2 6 (M+Na)+, found 524.27.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-ethoxy-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamido)-2methyl-5-phenylpentanoate (28). Intermediate 25 Boc-protected TuvOEt-Tup (160 mg, 267 μmol) was deprotected as described above to give 133 mg (99%) of the title compound. UHPLC-MS(system 2): tr=1.11 min, m/z (ES+) calculated 516.29 (M+H) ⁺ found 516.37.

(2S,4R)-Allyl 2-methyl-4-(2-((1R,3R)-4-methyl-3-(methylamino)-1-propoxypentyl)thiazole-4-carboxamido)-5-phenylpentanoate (29) . Intermediate 2 Boc-protected TuvOEt-Tup 6 (8 mg, 13 μmol) was deprotected as described above to give 7 mg (quantit) of the title compound. UHPLC-MS (system 2): t _r =1.16 min, m/z (ES+) calculated 530.31 (M+H)+, found 530.40.

General method for amide condensation of O-alkylated tubuvaline-tubuphenylalanine dipeptides with Fmoc-protected L-isoleucine. Commercially available Fmoc-L-isoleucine (1.3-2 equivalents) was dissolved in anhydrous dimethylformamide (50-200 mM) and preactivated with HATU (1.5-2 equivalents) and DIPEA (2 equivalents); the mixture was stirred for 10 minutes at room temperature under a nitrogen atmosphere. Activated acid was then added to Tuv(O-3(|)hp)-Tup dipeptides 27-29; the reaction mixture was stirred at room temperature under a nitrogen atmosphere and the reaction progress was monitored using UHPLC/MS data. After the reaction stopped or was completely completed, glacial acetic acid (13 equivalents) was added and the reaction mixture was purified using rep. HPLC.

(2S,4R)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 -methyl-3,6dioxo-2,11-dioxa-4,7-diazadodecan-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (30). Intermediate 27 TuvOMe-Tup (160 mg, 265 μmol) was coupled to Fmoc-L-Ile as described above to give 67 mg (30%) of the title compound. UHPLC-MS (system 1): t _r =2.07 min, m/z (ES+) calculated 837.43 (M+H)+, found 837.20.

(28.4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl- 7-methyl-3,6dioxo-2,11-dioxa-4,7-diazatridecan-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (31). Intermediate 28 TuvOEt-Tup (160 mg, 265 μmol) was coupled to Fmoc-L-Ile as described above to give 133 mg (99%) of the title compound. UHPLC-MS (system 2): t _r =1.95 min, m/z (ES+) calculated 851.44 (M+H)+, found 851.54.

(2S,4R)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 -methyl-3,6dioxo-2,11-dioxa-4,7-diazatetradecane-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (32). Intermediate 29 TuvOPr-Tup (7 mg, 13 μmol) was coupled to Fmoc-L-Ile as described above to give 9 mg (82%) of the title compound. UHPLC-MS (system 2): t _r =2.25 min, m/z (ES+) calculated 865.46 (M+H)+, found 865.65.

A general method for deprotecting Fmoc isoleucine-O-alkylated tubuvaline-tubuphenylalanine tripeptides. Fmoc-Πe-Tuv(O-ester)-Tup allyl ether (30-32) was treated with 20% piperidine in dimethylformamide (20 mM), with stirring under nitrogen at room temperature. Once complete deprotection was achieved, as monitored by UHPLC/MS, the reaction mixture was concentrated by rotary evaporation. The crude product was then purified by preparative HPLC to give tripeptides with free amines 33-35.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-1-methoxy-4-methylpentyl)thiazole -4-Carboxamido)-2-methyl-5-phenylpentanoate (33): Fmoc-Ile-TuvOMe-Tup intermediate 30 (67 mg, 80 μmol) was deprotected as described above to give 30 mg (61% ) specified in the connection header. UHPLC-MS (system 1): t _r =1.30 min, m/z (ES+) calculated 637.34 (M+Na)+, found 637.57.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethyl-pentanamido)-1-ethoxy-4-methylpentyl)thiazole -4-Carboxamido)-2-methyl-5-phenylpentanoate (34): Fmoc-Ile-TuvOEt-Tup intermediate 31 (67 mg, 80 μmol) was deprotected as described above to give 30 mg (61% ) specified in the connection header. UHPLC-MS (system 2): t _r =1.18 min, m/z (ES+) calculated 629.38 (M+H)+, found 629.45.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethyl-pentanamido)-4-methyl-1-propoxypentyl)thiazole -4-carboxamido)-2-methyl-5-phenylpentanoate (35). Fmoc-Ile-TuvOPr-Tup intermediate 32 (9 mg, 10 μmol) was deprotected as described above to give 7 mg (quantit) of the title compound. UHPLC-MS (system 2): t _r =1.29 min, m/z (ES+) calculated 643.39 (M+H)+, found 643.55.

General method for amide condensation of isoleucine-tubuvaline(ester)tubuphenylalanine tripeptides with (L)-N-methyl-pipecolic acid. Commercially available (L)-N-methyl-pipecolic acid (DMep) 36 (1.5-2 equivalents) was dissolved in anhydrous dimethylformamide (25-50 mM) and pre-activated with HATU (2 equivalents) and DIPEA (4 equivalents) ; the mixture was stirred

- 106 046150 for 10 minutes at room temperature in a nitrogen atmosphere. The activated acid was then added to the tripeptides Ile-Tuv(O-ester)-Tup 33-35; the reaction mixture was stirred at room temperature under a nitrogen atmosphere and the reaction progress was monitored using UHPLC/MS data. After completion of the reaction, glacial acetic acid (14 equivalents) was then added and the product was purified by preparative HPLC.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-1-methoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (37). Intermediate 33 Ile-TuvOMe-Tup (20 mg, 33 μmol) was coupled to D-Mep 36 as described above to give 17 mg (71%) of the title compound. UHPLC-MS (system 1): t _r =1.29 min, m/z (ES+) calculated 762.42 (M+Na)+, found 762.32.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (38). Intermediate 34 Ile-TuvOEt-Tup (20 mg, 33 μmol) was coupled to D-Mep 36 as described above to give 17 mg (71%) of the title compound. UHPLC-MS (system 2): t _r =1.25 min, m/z (ES+) calculated 754.46 (m+H) ⁺ , found 754.55.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-4-methyl-1-propoxypentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (39). Intermediate 35 Ile-TuvOPr-Tup (7 mg, 11 μmol) was coupled to D-Mep 36 as described above to give 4.5 mg (53%) of the title compound. UHPLC-MS (system 2): t _r =1.36 min, m/z (ES+) calculated 768.48 (M+H) ⁺ , found 768.55.

General method for removing allyl ester from D-methylpipecolic acid-isoleucine-tubuvaline(ester)-tubuphenylalanine tubulysine intermediates. Intermediates (37-39) allyl ether-protected tubulisin ester were dissolved in anhydrous dichloromethane (20 mM) treated with tetrakis(triphenylphosphine) palladium (0.1 equiv.), triphenylphosphine (0.2 equivalents) and anhydrous pyrrolidine (8 equivalents), and the reaction mixture was stirred at ambient temperature under a nitrogen atmosphere. After UHPLC/MS data indicated conversion of the free acid to the product, the reaction mixture was quenched with glacial acetic acid (22 equivalents), diluted with acetonitrile and dimethylformamide, and then concentrated by rotary evaporation. The crude tubulisin ester was then purified by preparative HPLC.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido) -1-methoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (40). Allyl ether-protected tubulisin methyl ester (TubOMe) intermediate 37 (2.9 mg, 4 μmol) was deprotected as described above to yield 2.5 mg (93%) tubulisin methyl ester 40 (TubOMe). SVeLC-MS (system 2): tr=1.05 min, m/z (ES+) calculated 700.41 (M+H)+, found 700.50.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido) -1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (41). Allyl ester-protected tubulysin ethyl ester (TubOEt) intermediate 38 (2.9 mg, 4 μmol) was deprotected as described above to yield 2.5 mg (93%) tubulisin ethyl ester 41 (TubOEt). UHPLC-MS (system 2): t _r =1.09 min, m/z (ES+) calculated 714.43 (M+H)+, found 714.51.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido) -4-methyl-1-propoxypentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (42). Allyl ether-protected tubulysin propyl ester (TubOPr) intermediate 39 (6 mg, 8 μmol) was deprotected as described above to yield 6 mg (qty.) tubulisin propyl ester 42 (TubOPr). UHPLC-MS (system 2): t _r =1.19 min, m/z (ES+) calculated 728.44 (M+H)+, found 728.54.

Scheme 4

A)-Perfluorophenyl 3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-III-pyrrol-1yl)propanoate (44). mDPR(Boc)-OH (Nature Biotech, 2014, 32, 1059-1062) 43 (500 mg, 1.76 mmol) was placed in the flask, and a solution of PFP-OH (324 mg, 1.7 6 mmol) in DMF (8.8 ml) then 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (371 mg, 1.93 mmol) as a solid. The reaction mixture was stirred for 1 hour at room temperature, then quenched with 50 ml saturated NH ₄ Cl in H2O and 50 ml H2O. The aqueous layer was extracted twice with DCM, the organics were then washed with brine, dried over NaSO ₄ and condensed under reduced pressure to give 44 (589 mg, 74%). Analytical UHPLC-MS (System 2): t _r =1.51 min, m/z (ES+) calculated

- 107 046150

473.07 (M+Na)+, found 473.14.

Scheme 5

X=Cd

NB3 ΡΡΊ.. f 37¾

S.C. Me i< 46 Χ=Βι

(28,3^48,58,68)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(bromomethyl)phenoxy)-6 -(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (46). A known fragment (Bioconjugate Chem. 2006, 17, 831-840) glucuronide linker (45, 210 mg, 281 μmol) in 4.5 ml of anhydrous THF was placed in a flame-dried flask. The solution was stirred at room temperature under N ₂ atmosphere. Triphenylphosphine (111 mg, 421.5 µmol) and Nbromosuccinimide (75 mg, 421.5 µmol) were added sequentially and the solution was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure and purified on silica gel using a Biotage column (hexane/EtOAc, 30 %-50%-70%) to obtain 46 (222 mg, 97%). Analytical UHPLC-MS (system 1): tr=2.36 min, m/z (ES+) found 811.34.

General method for quaternizing Tub(OR)-OAlyl to Fmoc-Gluc-Br: Place Tub(OR)-OAlyl (37-39, 1 equivalent) and brominated glucuronide linker moiety (46, 1.5 equivalents) in anhydrous 2-butanone (50 mM). The reaction vessel was purged with N2 and hermetically sealed. The reaction mixture was then stirred and heated at 60°C for 18 hours. The resulting mixture was cooled, condensed to a residue under reduced pressure, then used crude or placed in a minimal amount of DMSO for purification by preparative HPLC.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)3, 4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1(4- (((2R,4S)-5-(allyloxy)-4-methyl-5-oxo-1 -phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1 -methoxy-4methylpentan-3 -yl)( methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (47).

Tub(OMe)-OAlyl 37 (13 mg, 18 μmol) was quaternized as described above with Gluc-Br 46 (17 mg, 28 μmol) and used further without further purification. Analytical UHPLC-MS (system 1): tr=1.61 min, m/z (ES+) calculated 1470.68 (M) ⁺ found 1471.68.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)3, 4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1(4- (((2R,4S)-5-(allyloxy)-4-methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-ethoxy-4methylpentan-3-yl)( methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (48).

Tnb(OE1:)-OA.p.pi.p 38 (14 8 mg, 196 μmol) was quaternized as described above with Gluc-Br 46 (175 mg, 216 μmol) and used further without additional purification. Analytical UHPLC-MS (system 2): tr=1.49 min, m/z (ES+) calculated 1484.69 (M) ⁺ , found 1484.84.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)3, 4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1(4- (((2K.^)-5-(allyloxy)-4-methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1propoxypentan-3-yl)( methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (49). Tub(()Pr)-()A.LiiLi 39 (43 mg, 56 μmol) was quaternized as above with Gluc-Br 46 (50 mg, 62 μmol) to give 49 (68%) after preparative LC. Analytical UHPLC-MS (System 2):

- 108 046150 t _r =1.47 min, m/z (ES+) calculated 1498.71 (M) ⁺ , found 1498.85.

General method for complete deprotection of Fmoc-GlucQ-Tub(OR)-OAlyl. Fmoc-GlucQ-Tub(OR)-OAlyl (47-49) in THF and MeOH was placed in the flask and cooled to 0°C. LiOH-H2O (6.0 equivalents) in H2O (1:1:1 THF:MeOH: _H2O , 50 mM final concentration) was added dropwise and the reaction mixture was allowed to warm to room temperature and stirred overnight. THF and MeOH were removed under reduced pressure, the resulting precipitate was redissolved using a minimal amount of DMSO, and the mixture was purified by preparative HPLC.

(2R)-1-(3-(3-Aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2Hpyran-2-yl)oxy )benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl )thiazol-2-yl)-1-methoxy-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)1-methylpiperidin-1-ium (50) . Fmoc-GlucQ-Tub(OMe)-OAlyl 47 (17 mg, 12 μmol) was deprotected as described above to give 50 (4.3 mg, 34%). Analytical UHPLC-MS (system 1): t _r =1.08 min, m/z (ES+) calculated 1068.53 (M) ⁺ , found 1068.66.

(2R)-1-(3-(3-Aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2Hpyran-2-yl)oxy )benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl )thiazol-2-yl)-1-ethoxy-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1-oxopentan-2-yl)carbamoyl)-1 methylpiperidin-1-ium (51) . Fmoc-GlucQ-Tub(OEt)-OAlyl 48 (292 mg, 197 μmol) was deprotected as described above to give 51 (116 mg, 54%). Analytical UHPLC-MS (system 2): t _r =0.95 min, m/z (ES+) calculated 1082.55 (M) ⁺ , found 1082.68.

(2R)-1-(3-(3-Aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2Hpyran-2-yl)oxy )benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl )thiazol-2-yl)-4-methyl-1-propoxypentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)1-methylpiperidin-1-ium (52) . Fmoc-GlucQ-Tub(OPr)-OAlyl 49 (57 mg, 38 μmol) was deprotected as described above to give 52 (34 mg, 41%). Analytical UHPLC-MS (system 2): t _r =0.98 min, m/z (ES+) calculated 1096.56 (M) ⁺ , found 1096.67.

General method for condensation of H-GlucQ-Tub(OR) with mDPR-OPFP. H-GlucTub(OR)(50-52) was placed in the flask, to which mDPR(Boc)-OPFP (44, 1.2 equivalents) was added as a solution in DMF (10 mM). N.N-Diisopropylethylamine (4.0 equivalents) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with AcOH (4.0 equivalents), then diluted with DMSO (1 volume) and purified by preparative HPLC.

(2R)-1-(3-(3-((S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )propanamido)propanamido)-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2yl)oxy)benzyl)-2-(((2S,3S)- 1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-methoxy-4- methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (53). H-GlucQ-Tub(OMe) 50 (4.3 mg, 4 μmol) was coupled to mDPR-OPFP 44 (2.2 mg, 4.8 μmol) as described above to give 53 (4 mg, 75%) . Analytical UHPLC-MS (system 1): t _r =1.22 min, m/z (ES+) calculated 1334.62 (M) ⁺ , found 1334.68.

(2R)-1-(3-(3-((S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-digcdro-1H-pyrrol-1-yl )propanamcdo)propanamido)-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2yl)oxy)benzyl)-2-(((2S,3S)- 1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-ethoxy-4- methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (54). H-GlucQ-Tub(OEt) 51 (29 mg, 27 μmol) was coupled to mDPR-OPFP 44 (14 mg, 32 μmol) as described above to give 54 (26 mg, 72%). Analytical UHPLC-MS (system 2): t _r =1.19 min, m/z (ES+) calculated 1348.64 (M) ⁺ , found 1348.79.

(2R)-1-(3-(3-((S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-digcdro-1H-pyrrol-1-yl )propanamcdo)npropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2yl)oxy)benzyl)-2-((( 2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4 -methyl-1-propoxypentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (55). H-GlucQ-Tub(OPr) 52 (6 mg, 5 μmol) was coupled to mDPR-OPFP 44 (3 mg, 6 μmol) as described above to give 55 (6 mg, 84%). Analytical UHPLC-MS (system 2): t _r =1.24 min, m/z (ES+) calculated 1362.65 (M) ⁺ , found 1362.78.

General deprotection method mDPR(Boc)-GlucQ-Tub(OR). mDPR(Boc)-GlucQ-Tub(OR) (53-55) was placed in the flask and cooled to 0°C. A 10% solution of TFA in DCM (50 mM) was added and the reaction mixture was allowed to warm to room temperature with stirring for 1 hour. The reaction mixture was then diluted with DMSO (1 volume), DCM was removed under reduced pressure, then purified by preparative HPLC.

(2R)-1-(3-(3-((S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)4- ((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-((^^)-1(((1R,3R) -1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-methoxy-4-methylpentan-3-yl)(methyl) amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (56).

mDPR(Boc)-GlucQ-Tub(OMe) 53 (4 mg, 3 μmol) was deprotected as described above to give 56 (2 mg, 54%). Analytical UHPLC-MS (system 2): t _r =1.09 min, m/z (ES+) you

- 109 046150 numerical 1234.57 (M) ⁺ , found 1234.65.

(2R)-1 -(3-(3-((S)-3 -Amino-2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)propanamido)4 -(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)- 1(((1R,3R)-1 -(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-ethoxy-4-methylpentane -3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (57).

mDPR(Boc)-GlucQ-Tub(OEt) 54 (26 mg, 19 μmol) was deprotected as described above to give 57 (24 mg, 99%). Analytical UHPLC-MS (system 2): tr=0.95 min, m/z (ES+) calculated 1248.59 (M) ⁺ , found 1248.72.

(2R)-1 -(3-(3-((S)-3 -Amino-2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)propanamido)4 -(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)- 1(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1-propoxypentane -3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (58).

mDPR(Boc)-GlucQ-Tub(OPr) 55 (6 mg, 4 μmol) was deprotected as described above to give 58 (4 mg, 75%). Analytical UHPLC-MS (system 2): tr=1.03 min, m/z (ES+) calculated 1262.60 (M) ⁺ found 1262.73.

Scheme 6

General method for condensation of H-GlucQ-Tub(OR) with Fmoc-Lys(PEG12)-OSu: HGlucQ-Tub(OR) (51 or 52) was placed in a flask, to which Fmoc-Lys(PEG12)-OSu was added (WO 2015057699 ) (1.2 equivalents) solution in DMF (20 mM), then HC-diisopropylethylamine (4.0 equivalents). The reaction mixture was stirred at room temperature for 4 h, then quenched with AcOH (4.0 equivalents), diluted with DMSO (1 volume) and purified by preparative HPLC.

(2R)-1 -(3 -(^)-44-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3 8,45-dioxo-2,5,8,11,14 ,17,20,23, 26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3 S)-1 -(((1 R,3R)-1 -(4-(((2R,4S)- 4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-ethoxy-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan2-yl)carbamoyl )-1-methylpiperidin-1-ium (60). H-GlucQ-Tub(OEt) 51 (87 mg, 80 μmol) was coupled to Fmoc-Lys(PEG12)-OSu 59 (100 mg, 96 μmol) as described above to give 60 (108 mg, 67%). Analytical UHPLC-MS (system 2): tr=1.29 min, m/z (ES+) calculated 2003.04 (M) ⁺ found 2003.24.

(2R)-1 -(3 -(^)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3 8,45-dioxo-2,5,8,11,14 ,17,20,23, 26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4- carboxy-1phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1 -propoxypentan-3 -yl)(methyl)amino)-3 -methyl-1 oxopentan-2-yl)carbamoyl)-1 -methylpiperidin-1-ium (61). H-GlucQ-Tub(OPr) 52 (20 mg, 18 μmol) was coupled to Fmoc-Lys(PEG12)-OSu 59 (23 mg, 22 μmol) as described above to give 61 (27 mg, 73%). Analytical UHPLC-MS (system 2): tr=1.31 min, m/z (ES+) calculated 2017.05 (M) ⁺ , found 2017.22.

- 110 046150

General method for deprotection of Fmoc-Lys(PEG12)-GlucQ-Tub(OR): Fmoc-Lys(PEG12)-GlucQ-Tub(OR)(60 or 61) was placed in a flask, to which a 20% solution of piperidine in DMF was added (20 mM). The reaction mixture was stirred for 30 min, then diluted with DMSO (1 volume) and purified by preparative HPLC.

(2R)-1-(3-((8)-44-Amvno-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa- 39,46-diazanonatetracontanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2- (((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiαzol-2yl)- 1-ethoxy-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (62). Fmoc-Lys(PEG12)-GlucQ-Tub(OEt) 60 (108 mg, 54 μmol) was deprotected as described above to give 62 (83 mg, 86%). Analytical UHPLC-MS (system 2): t _r =0.99 min, m/z (ES+) calculated 1780.97 (M) ⁺ , found 1781.14.

(2R)-1-(3-((8)-44-Amvno-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa- 39,46-diazanonatetracontanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2- (((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2yl)- 4-methyl-1-propoxypentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (63). Fmoc-Lys(PEG12)-GlucQ-Tub(OPr) 61 (27 mg, 13 μmol) was deprotected as described above to give 63 (17 mg, 71%). Analytical UHPLC-MS (system 2): t _r =1.03 min, m/z (ES+) calculated 1794.98 (M) ⁺ , found 1795.14.

General method for condensation of H-Lys(PEG12)-GlucQ-Tub(OR) with mDPR-OPFP: HLys(PEG12)-GlucQ-Tub(OR) (62 or 63) was placed in a flask, to which mDPR-OPFP (44, 1.2 equivalents) as a solution in DMF (10 mM), then N.N-diisopropylethylamine (4.0 equivalents). The reaction mixture was stirred at room temperature for 4 hours, then quenched with AcOH (4.0 equivalents), diluted with DMSO (1 volume) and purified by preparative HPLC.

(2R)-1-(3-((S)-44-((S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1yl)propanamido)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-((^ ^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2(((2S,3S)-1-(((1R,3R)- 1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-ethoxy-4methylpentan-3-yl)(methyl)amino)- 3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (64). H-Lys(PEG12)-GlucQ-Tub(OEt) (83 mg, 46 μmol) was coupled to mDPR-OPFP 44 (25 mg, 56 μmol) as described above to give 64 (43 mg, 45%). Analytical UHPLC-MS (system 2): t _r =1.22 min, m/z (ES+) calculated 2047.06 (M) ⁺ , found 2047.25.

(2R)-1-(3-((S)-44-((S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1yl)propanamido)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-((^ ^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2(((2S,3S)-1-(((1R,3R)- 1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1propoxypentan-3 -yl)(methyl)amino)- 3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (65). H-Lys(PEG12)-GlucQ-Tub(OPr) (17 mg, 9 μmol) was coupled to mDPR-OPFP 44 (5 mg, 11 μmol) as described above to give 65 (14 mg, 74%). Analytical UHPLC-MS (system 2): t _r =1.22 min, m/z (ES+) calculated 2061.07 (M) ⁺ , found 2 0 61.26.

General method for deprotection of mDPR(Boc)-Lys(PEG12)-GlucQ-Tub(OR): Place mDPR(Boc)-Lys(PEG12)-GlucQ-Tub(OR) (64 or 65) in a flask and cool to temperature 0°C. A 10% solution of TFA in DCM (50 mM) was added and the reaction mixture was allowed to warm to room temperature with stirring for 1 hour. The reaction mixture was then diluted with DMSO (1 volume), DCM was removed under reduced pressure, then purified by preparative HPLC.

(2R)-1-(3-((S)-44-((S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propαnamido) -38,45dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4((^^^^^)-6- carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-((^^)-1(((1R,3R)-1-(4-(((2R, 4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-ethoxy-4methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentane-2- yl)carbamoyl)-1-methylpiperidin-1-ium (66). mDPR(Boc)-Lys(PEG12)-GlucQ-Tub(OEt) 64 (43 mg, 21 μmol) was deprotected as described above to give 66 (34 mg, 83%). Analytical UHPLC-MS (System 2): t _r =0.96 min, m/z (ES+) calculated 1947.01 (M) ⁺ , found 1947.22.

(2R)-1-(3-((S)-44-((S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido) -38,45dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4((^^^^^)-6- carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-((^^)-1(((1R,3R)-1-(4-(((2R, 4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1-npropoxypentan-3-yl)(methyl)amino)-3-methyl-1-oxopentane- 2-yl)carbamoyl)-1-methylpiperidin-1-ium (67).

mDPR(Boc)-Lys(PEG12)-GlucQ-Tub(OPr) 65 (14 mg, 7 μmol) was deprotected as described above to give 67 (12 mg, 92%). Analytical UHPLC-MS (system 2): t _r =1.05 min, m/z (ES+) calculated 1961.02 (M) ⁺ found 1961.20.

- 111 046150

Scheme 7

(2S,4R)-4-(2-((1R,3R)-3-((tert-Butoxycarbonyl)(methyl)amino)-1-hydroxy-4-methylpentyl)thiazole4-carboxamido)-2-methyl-5 -phenylpentanoic acid (69). The flask was filled with commercially available Boc-Tuv(OAc)-Tup-OEt (68.50 mg, 81 µmol) in THF (1.35 ml) and MeOH (1.35 ml) and cooled to 0°C. LiOH-H ₂ O (27 mg, 64 7 µmol) was dissolved in H2O (1.35 ml), then added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction mixture was then quenched with acetic acid (37 μl, 647 μmol) and condensed under reduced pressure. The residue was taken up in minimal DMSO and purified by preparative HPLC to give 69 (44 mg qt). Analytical UHPLC-MS (system 2): tr=1.53 min, m/z (ES+) calculated 548.28 (M+H) ⁺ found 548.24.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-acetoxy-3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazol-4-carboxamido)-2- methyl 5-phenylpentanoate (70). Boc-Tuv(OH)-Tup-OH (69.44 mg, 81 µmol) was dissolved in anhydrous pyridine (1.6 ml) and stirred at room temperature under N ₂ atmosphere. Acetic anhydride (15.4 µl, 162 µmol) was added dropwise. After stirring for 1 hour, 1.0 additional equivalent of acetic anhydride was added and the reaction was complete by LCMS after 2 hours. The reaction mixture was concentrated to dryness under reduced pressure, then redissolved in anhydrous allyl alcohol (1.6 ml). Diallyl pyrocarbonate (54 µl, 325 µmol) was added followed by solid DMAP (3.0 mg, 24 µmol). The reaction mixture was allowed to stir at room temperature and monitored by LCMS, with additional diallyl pyrocarbonate (4.0 additional equivalents) added as needed to push the reaction to completion. The reaction mixture was then taken into DMSO, condensed under reduced pressure and purified by preparative HPLC to give 70 (33 mg, 65%). Analytical UHPLC-MS (system 2): tr=1.75 min, m/z (ES+) calculated 630.32 (M+H) ⁺ , found 630.42.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-acetoxy-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamido)-2methyl-5-phenylpentanoate (71). The flask containing Boc-Tuv(OAc)-Tup-OEt (70.33 mg, 21 µmol) was cooled to 0°C under N2 atmosphere. A solution of 10% TFA in CH ₂ Cl ₂ (0.52 ml) was added dropwise and stirred for 4 hours. The reaction mixture was concentrated under reduced pressure, redissolved in DCM, and condensed 3 times to remove TFA, then used further without additional cleaning. Analytical UHPLC-MS (system 2): tr=1.03 min, m/z (ES+) calculated 530.27 (M+H) ⁺ , found 530.36.

(2S,4R)-Allyl 4-(2-(^,9^1^)-6-(^)-sec-butyl)-9-isopropyl-2,2,8-trimethyl-4,7,13- trioxo3,12-dioxa-5,8-diazatetradecane-11-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (72). H-Tuv(OAc)-Tup-OAlyl (71.28 mg, 53 µmol) was placed in the flask, Boc-L-Ile-OH (15 mg, 63 µmol) and HATU (40 mg, 106 µmol) were added as solids. substances, then DMF (1.0 ml). N,Ndiisopropylethylamine (37 µL, 211 µmol) was added and the reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was then taken up in DMSO, condensed under reduced pressure and purified by preparative HPLC to give 72 (19 mg, 49%) . Analytical UHPLC-MS (system 2): tr=1.72 min, m/z (ES+) calculated 743.41 (M+H) ⁺ found 743.51.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-4-methylpentyl)thiazol-4 -carboxamido)-2-methyl-5-phenylpentanoate (73). The flask containing Boc-Ile-Tuv(OAc)-TupOEt (72.19 mg, 26 µmol) was cooled to 0°C under N2. A solution of 10% TFA in CH ₂ Cl ₂ (0.52 ml) was added dropwise and stirred for 4 hours. The reaction mixture was concentrated under reduced pressure, redissolved in DCM and condensed 3 times to remove TFA, then used without further purification . Analytical UHPLC-MS (system 2): t _r =1.18 min, m/z (ES+) calculated 643.36 (M+H) ⁺ , found 643.42.

- 112 046150

Scheme 8

(X)-tert-Butyl 1-methylpiperidine-2-carboxylate (75). Commercially available H-Pip-OtBu (74.500 mg, 2.70 mmol) was placed in MeOH (4.50 mL), AcOH (4.50 mL) and 37% CH ₂ O in H ₂ O (4.50 mL ) and stirred for 20 minutes. NaBH ₃ CN (509 mg, 8.10 mmol) was added slowly as a solid while bubbling vigorously and stirred for 30 min. The reaction mixture was then poured into 200 ml saturated NaHCO ₃ solution and extracted with 3 x 200 ml DCM. The organic layers were washed with brine, dried over NaSO ₄ and condensed under reduced pressure to give 75 (516 mg, 96%) and used without further purification. Analytical UHPLC-MS (System 2): t=0.53 min, m/z (ES+) calculated 200.17 (M+H)+, found 200.21.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)3, 4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(tert-butoxycarbonyl)-1-methylpiperidin-1-ium (76). The brominated glucuronide linker moiety (46.104 mg, 128 µmol) and Mep-OtBu (75.34 mg, 171 µmol) in anhydrous 2butanone (1.71 ml) were placed in a pressure vessel. The reaction vessel was purged with N2 and hermetically sealed. The reaction mixture was then stirred and heated at 60°C for 12 hours. The resulting mixture was cooled, condensed under reduced pressure, placed in a minimal amount of DMSO and purified by preparative HPLC to give 76 (97 mg, 82%). Analytical UHPLC-MS (system 2): tr=1.32 min, m/z (ES+) calculated 930.40 (M) ⁺ found 930.49.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)-6 ((allyloxy)carbonyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(tert-butoxycarbonyl)-1-methylpiperidin-1-ium (77). Fmoc-GlucQ-Mep-OtBu (76.97 mg, 104 µmol) in anhydrous allyl alcohol (2.09 ml) under N2 was placed in a flame-dried flask. Ti(OC ₂ H ₅ ) ₄ (87 µl, 417 µmol) was added and the reaction mixture was heated at 80°C with stirring for 2 hours. The reaction mixture was then cooled to room temperature and poured into 50 ml of 1M HCl. After standing for 45 min, HCl was extracted with 3x 50 ml DCM. The resulting organics were washed with brine, dried over NaSO ₄ , condensed and purified by preparative HPLC to give 77 (42 mg, 48%). Analytical UHPLC-MS (system 2): t _r =1.18 min, m/z (ES+) calculated 830.39 (M) ⁺ , found 830.49.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)-6 - 113 046150 ((allyloxy)carbonyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-carboxy-1-methylpiperidin-1-ium (78). The flask containing Fmoc-Gluc(Allyl)Q-Mep-OtBu (77.42 mg, 50 µmol) was cooled to 0°C under N ₂ atmosphere. A solution of 30% TFA in CH ₂ Cl ₂ (2.5 ml) was added dropwise and stirred for 18 hours. The reaction mixture was concentrated under reduced pressure, taken up in a minimal amount of DMSO and purified by preparative HPLC to give 78 (25 mg, 64 %). Analytical UHPLC-MS (system 2): t _r =1.05 min, m/z (ES+) calculated 774.32 (M) ⁺ found 774.42.

(2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)-6 ((allyloxy)carbonyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)1-acetoxy -1-(4-(((2R,4S)-5-(allyloxy)-4-methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4methylpentan-3-yl )(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (79). H-Πe-Tuv(OAc)-Tup-OAlyl (73.23 mg, 36 µmol) was placed in the flask, Fmoc-Gluc(Allyl)QMep-OH (78.28 mg, 36 µmol) and HATU (27 mg, 72 µmol) as solids, then DMF (0.714 ml). NN-diisopropylethylamine (25 μL, 143 μmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then taken into DMSO and purified by preparative LC to give 79 (23 mg, 46%). Analytical UHPLC-MS (system 2): t _r =1.39 min, m/z (ES+) calculated 1398.66 (M) ⁺ , found 1398.81.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1-oxopentan-2-yl)carbamoyl)-1 -(3 -(3 -aminopropanamido )-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-1-methylpiperidin-1-ium (80). Fmoc-Gluc(Allyl)Q-TubM-OAlyl (79.21 mg, 15 µmol) was placed in DCM (1.5 ml) with stirring under N2 atmosphere. Pd(PPh ₃ ) ₄ (3.5 mg, 3.1 µmol) and PPh ₃ (1.6 mg, 6.1 µmol) were added as solids, followed by pyrrolidine (20.1 µl, 245 µmol). The reaction mixture was stirred for 2 h at room temperature, then taken into 1 ml DMSO, condensed under reduced pressure and purified by preparative LC to give 80 (13 mg, 79%). Analytical UHPLC-MS (system 2): t _r =0.94 min, m/z (ES+) calculated 1096.53 (M) ⁺ , found 1096.65.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1-oxopentan-2-yl)carbamoyl)-1 -(3 -(3 -( (S)3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)-4-((^^^^ ^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-1-methylpiperidin-1-ium (81). H-GlucQ-TubM (80, 13.1 mg, 11.9 µmol) in anhydrous DMF (0.595 ml) was placed into the flask, to which mDPR(Boc)-OSu (13, 4.6 mg, 11) was added under N2 atmosphere. .9 µmol). N,N-diisopropylethylamine (8.3 µl, 47.8 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid (8.3 µl) and purified by preparative HPLC to give 81 (5.2 mg, 33%). Analytical UHPLC-MS (system 2): t _r =1.20 min, m/z (ES+) calculated 1362.62 (M) ⁺ , found 1362.75.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3 -yl)(methyl)amino)-3 -methyl-1-oxopentan-2-yl)carbamoyl)-1 -(3 -(3 -( (S)3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamcdo)nropanamido)-4-(((2S,3R,4S,5S,6S) -6carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-1-methylpiperidin-1-ium (82). The flask containing mDPR(Boc)-GlucQ-TubM (81, 5.2 mg, 3.8 µmol) was cooled to 0°C under N ₂ atmosphere. A solution of 10% TFA in CH ₂ Cl ₂ (0.84 ml) was added dropwise and stirred for 4 hours. The reaction mixture was then taken up in DMSO, condensed under reduced pressure and purified by preparative HPLC to give 82 (4.8 mg, 81%). Analytical UHPLC-MS (system 2): t _r =0.95 min, m/z (ES+) calculated 1262.56 (M) ⁺ , found 1262.68.

- 114 046150

Scheme 9

(3)-tert-Butyl 4-amino-5-((4-(hydroxymethyl)phenyl)amino)-5-oxopentanoate (84). A flask containing Fmoc-Glu(OtBu)-OH (83, 2.0 g, 4.7 mmol), H-PABA (4, 579 mg, 4.7 mmol) and Cl ₂ CH ₂ (25 ml) was stirred at room temperature. EEDQ (1.40 g, 5.6 mmol) was added as a solid and the mixture was stirred overnight. The product was eluted on a 4 mm EtOAc chromatotron plate and the product containing fractions were condensed. The resulting residue was taken into 20% piperidine in DCM, stirred for 15 minutes, then condensed to an oil. The oil was dissolved in DCM and eluted on a 2 mm chromatotron plate using a gradient of 10%-20% MeOH in DCM to give 84 (860 mg, 60%). Analytical UHPLC-MS (system 2): t _r =0.65 min, m/z (ES+) calculated 309.18 (M+H) ⁺ , found 309.24.

^)-tert-Butyl 4-(^)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-((4-(hydroxymethyl)phenyl)amino)-5-oxopentanoate (85). H-Glu(OtBu)-PABA (84, 860 mg, 2.78 mmol) was placed in the flask in DMF (10 ml), Boc-Val-OSu 1 (1.13 g, 3.60 mmol) and DIPEA (0) were added .75 ml). After 30 min, the reaction mixture was poured into 100 ml EtOAc and washed with H2O 3x, brine 1x and dried over _NaSO4 . The solution was dried under reduced pressure, dissolved in 50 ml EtOAc and precipitated with 10% EtOAc in hexane (50 ml). The solids were collected and dried to provide 85 as a white solid (0.97 g, 70%). Analytical UHPLC-MS (system 2): t=1.37 min, m/z (ES+) calculated 508.30 (M+H) ⁺ , found 508.38.

^)-tert-Butyl 5-((4-(bromomethyl)phenyl)amino)-4-(^)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-oxopentanoate (86). The flask containing Boc-Val-Glu(OtBu)-PABA-OH (85, 200 mg, 394 µmol), N-bromosuccinimide (105 mg, 591 µmol) and triphenylphosphine (155 mg, 591 µmol) was purged with N2. The reaction mixture was placed in THF (4 ml) and stirred for 12 hours. The reaction mixture was concentrated and purified on silica gel on a Biotage column (hexane/EtOAc, 10%-100%) to give 86 (210 mg, 93%). Analytical UHPLC-MS (system 2): tr=1.56 min, m/z (ES+) calculated 570.22 (M+H) ⁺ , found 570.30.

(2R)-2-(((2S,3 S)-1 -(((1R,3R)-1 -Acetoxy-1 -(4-(((2R,4S)-5-(allyloxy)-4- methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl )-1-(4-(^)-5-(tert-butoxy)-2-(^)-2-((tert-butoxycarbonyl) amino)-3-methylbutanamido)-5-oxopentanamido)benzyl)-1- methylpiperidin-1-ium (88). Flask containing Boc-Val-Glu(OtBu)-PABA-Br (86.40 mg, 70 µmol) and Tub-OAlyl (Org. Lett., 2007, 9, 1605-1607) (87.45 mg, 59 µmol ), purged with N2. Butanone (1.17 ml) was added and the reaction mixture was heated at 60°C with stirring. After 18 hours, the reaction mixture was concentrated to dryness, placed in minimal DCM and purified with Biotage (0-20% DCM/MeOH) to give 88 (62 mg, 85%). Analytical UHPLC-MS (system 2): t _r =1.47 min, m/z (ES+) calculated 1257.72 (M) ⁺ , found 1257.85.

(2R)-2-(((2S,3 S)-1 -(((1 R,3R)-1 -Acetoxy-1 -(4-(((2R,4S)-4-carboxy-1-phenylpentane -2-yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-(4-(^ )-2(^)-2-amino-3-methylbutanamido)-4-carboxybutanamido)benzyl)-1-methylpiperidin-1-ium (89). The flask containing Boc-Val-Glu(OtBu)-PABQ-Tub-OAlyl (88.42 mg, 50 µmol) was cooled to 0°C under N2 atmosphere. A solution of 30% TFA in CH ₂ Cl ₂ (0.99 ml) was added dropwise and stirred

- 115 046150 18 h. The reaction mixture was concentrated under reduced pressure, placed in DCM and condensed again 3 times. The residue was then taken up in DCM (0.98 ml) to which Pd(PPh ₃ ) ₄ (5.7 mg, 4.9 µmol) and PPh ₃ (2.6 mg, 9.8 µmol) were added as solids , then pyrrolidine (32 µl, 392 µmol). After 1 hour, the reaction mixture was taken up in minimal DMSO, condensed, and purified by preparative HPLC to give 89 (47 mg, 90%). Analytical UHPLC-MS (system 2): t _r =0.95 min, m/z (ES+) calculated 1061.57 (M) ⁺ , found 1061.69.

Scheme 10

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-(4((7S,10S ,13S)-13-(2-carboxyethyl)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-isopropyl-2,2-dimethyl-4,8, 11-trioxo-3-oxa-5,9,12-triazatetradecanamido)benzyl)-1-methylpiperidin-1-ium (90). H-ValGluPABQ-Tub (89, 22.5 mg, 21.2 µmol) in anhydrous DMF (0.420 ml) was placed in the flask, to which mDPR(Boc)-OSu (13, 8) was added as a solid under N ₂ atmosphere .9 mg, 23.3 µmol). N,Ndiisopropylethylamine (14.8 µl, 84.7 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid (14.8 µl) and purified by preparative HPLC to give 90 (11 .5 mg, 40%). Analytical UHPLC-MS (system 2): t _r =1.31 min, m/z (ES+) calculated 1327.66 (M) ⁺ , found 1327.94.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-(4-(^)- 2((S)-2-((S)-3 -amino-2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)-3-methylbutanamido)-4carboxybutanamido )benzyl)-1-methylpiperidin-1-ium (91). The flask containing mDPR(Boc)ValGluPABQ-Tub (90, 11.5 mg, 8.6 µmol) was cooled to 0°C under N ₂ atmosphere. A solution of 10% TFA in CH ₂ Cl ₂ (0.86 ml) was added dropwise and stirred for 2 hours. The reaction mixture was then taken into DMSO, condensed under reduced pressure and purified by preparative HPLC to give 91 (9.9 mg, 93%). Analytical UHPLC-MS (system 2): t _r =0.99 min, m/z (ES+) calculated 1227.61 (M) ⁺ , found 1227.83.

- 116 046150

Scheme 11

(2R)-1-(4-((44S,47S,50S)-44-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-50-(2-carboxyethyl)-47isopropyl-38 ,45,48-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46,4 9-triazagenpentaconeTaHaMugo)6eH3ug)-2-((( 2S,3S)-1-(((1R,3R)-1-a^TOKCu-1-(4-(((2R,4S)-4-Kap6oKCu-1^eHugneHTaH-2-ug)Kapbamoyl)thiazol- 2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (92). Fmoc-Lys(PEG12)-OSu (59.26 mg, 25 µmol) was added to the flask containing H-ValGluPABQ-Tub (89.24 mg, 23 µmol) as a solution in anhydrous DMF (0.457 ml) in an atmosphere N ₂ . Diisopropylethylamine (16 µl, 91 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid (16 µl) and purified by preparative HPLC to give 92 (34 mg, 75%). Analytical UHPLC-MS (system 2): tr=1.35 min, m/z (ES+) calculated 1982.06 (M) ⁺ found 1982.37.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1 -oxopentan-2-yl)carbamoyl)-1 -(4((44S,47S ,50S)-44-amino-50-(2-carboxyethyl)-47-isopropyl-38,45,48-trioxo-2,5,8,11,14,17,20,23,26,29, 32, 35-dodecaoxa-39,46,49-triazagenpentacontanamido)benzyl)-1-methylpiperidin-1-ium (93). 20% piperidine in DMF (1.7 ml) was added to the flask containing Fmoc-Lys(PEG12)-ValGluPABQ-Tub (92.34 mg, 17 µmol). The reaction mixture was stirred under N ₂ atmosphere at room temperature for 30 minutes. The reaction mixture was then diluted with DMSO/H ₂ O and purified by preparative HPLC to give 93 (26 mg, 86%). Analytical UHPLC-MS (system 2): tr=1.01 min, m/z (ES+) calculated 1759.99 (M) ⁺ , found 1760.26.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1 -oxopentan-2-yl)carbamoyl)-1 -(4((44S,47S ,50S)-44-((S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-50- (2-carboxyethyl)-47-isopropyl-38,45,48-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46,49- triazagenpentacontanamido)benzyl)-1-methylpiperidin-1-ium (94). The flask was filled with HLys(PEG12)-ValGluPABQ-Tub (93.26 mg, 15 µmol) in anhydrous DMF (0.735 ml), to which mDPR(Boc)-OSu (13.6 mg, 16 µmol). N,Ndiisopropylethylamine (10 µl, 59 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid (10 µl), diluted with DMSO and purified by preparative HPLC to give 94 (16 mg, 46 %). Analytical UHPLC-MS (system 2): tr=1.26 min, m/z (ES+) calculated 2026.08 (M) ⁺ , found 2026.38.

(2R)-2-(((2S,3S)-1-(((1R,3R)-1-Acetoxy-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1 -oxopentan-2-yl)carbamoyl)-1 -(4((44S,47S ,50S)-44-((S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-50-(2-carboxy

- 117 046150 ethyl)-47-I3opropyl-38,45,48-trioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46,49- triazagenpentacontanamido)benzyl)-1-methylpiperidin-1-ium (95). The flask containing mDPR(Boc)-Lys(PEG12)ValGluPABQ-Tub (94.14 mg, 7 µmol) was cooled to 0°C under N2 atmosphere. A solution of 10% TFA in CH ₂ Cl ₂ (1.03 ml) was added dropwise and stirred for 2 hours. The reaction mixture was then taken into DMSO, condensed under reduced pressure, and purified by preparative HPLC to give 95 (9 mg, 70 %). Analytical UHPLC-MS (system 2): tr=1.03 min, m/z (ES+) calculated 1926.03 (M) ⁺ , found 1926.32.

Scheme 12

(2R)-1 -(3 -((S)-44-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-38,45-dioxo-2,5,8,11,14 ,17,20,23, 26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-acetoxy-1-(4-(((2R,4S )-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl )-1-methylpiperidin-1-ium (96). Fmoc-Lys(PEG12)-OSu (59, 4.4 mg, 4.3 µmol) was added to the flask containing H-GlucQ-Tub (80, 3.9 mg, 3.6 µmol) as a solution in anhydrous DMF (0.355 ml) under N2 atmosphere. Na-diisopropylethylamine (1.9 µl, 10.7 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid (1.9 µl) and purified by preparative HPLC to give 96 (5 .0 mg, 70%). Analytical UHPLC-MS (system 2): tr=1.29 min, m/z (ES+) calculated 2017.02 (M) ⁺ , found 2017.21.

(2R)-2-(((2S,3 S)-1 -(((1 R,3R)-1 -Acetoxy-1 -(4-(((2R,4S)-4-carboxy-1-phenylpentane -2-yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-(3-(^ )-44amino-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4(((2S,3R ,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-1-methylpiperidin-1-ium (97). 20% piperidine in DMF (0.248 ml) was added to a flask containing Fmoc-Lys(PEG12)-GlucQ-Tub (96, 5.0 mg, 2.5 µmol). The reaction mixture was stirred under N2 atmosphere at room temperature for 30 min. The reaction mixture was then diluted with DMSO/H ₂ O and purified by preparative HPLC to give 97 (3.6 mg, 82%). Analytical UHPLC-MS (system 2): t _r =0.99 min, m/z (ES+) calculated 1794.95 (M) ⁺ found 1795.12.

(2R)-2-(((2S,3 S)-1 -(((1 R,3R)-1 -Acetoxy-1 -(4-(((2R,4S)-4-carboxy-1-phenylpentane -2-yl)carbamoyl)thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-(3-(^ )-44(^)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-III-pyrrol-1-yl)propanamido)-38,45dioxo-2, 5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diasanonetetracontanamido)-4-(((2S,3R,4S,5S, 6S)-6-carboxy -3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-1-methylpiperidin-1-ium (98). The flask was filled with H-Lys(PEG12)-GlucQ-Tub (97, 3.8 mg, 2.14 µmol) in anhydrous DMF (0.212 ml), to which mDPR(Boc)- was added as a solid under N ₂ atmosphere. OPFP (44, 1.4 mg, 3.2 µmol). Na-diisopropylethylamine (0.74 µl, 4.2 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid (0.74 µl), diluted with DMSO and purified by preparative HPLC to give 98 (2.7 mg, 61%). Analytical UHPLC-MS (system 2): t _r =1.20 min, m/z (ES+) calculated 2061.04 (M) ⁺ , found 2061.20.

- 118 046150 (2I)-2-(((2838M-(((1H-3NM-AcetoxA1-1-(4-(((2I,48)-4-carboxy-1-phenyl11enta11-2-yl)carbamoyl) thiazol-2-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-(3-((8)-44((8 )-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-38,45-dioxo-2,5,8,11,14,17, 20,23, 26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-(((28,3^48,58,68)-6-carboxy-3,4,5-trihydroxytetrahydro-2H -pyran-2-yl)oxy)benzyl)-1-methylpiperidin-1-ium (99) Flask containing mDPR(Boc)-Lys(PEG12)-GlucQ-Tub (98, 2.7 mg, 1.3 µmol), cooled to 0°C under N2. A solution of 10% TFA in CH ₂ Cl ₂ (0.26 ml) was added dropwise and stirred for 2 hours. The reaction mixture was then placed in DMSO, condensed under reduced pressure and purified by preparative HPLC to give 99 (2.5 mg, 97%). Analytical UHPLC-MS (System 2): t _r =1.00 min, m/z (E8+) calculated 1960.99 (M) ⁺ , found 1961.17.

Scheme 13

112, R=CH ₃

2-((1R,3R)-3-((tert-Butoxycarbonyl)(methyl)amino)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylic acid (101). Tubuvalin acetate (Org. Lett., 2007, 9, 1605-1607) 100 (225 mg, 560 µmol), dissolved in methanol (5 ml) and tetrahydrofuran (5 ml), was placed in the flask, then cooled in an ice bath under a nitrogen atmosphere. to a temperature of 0°C. Lithium hydroxide monohydrate (71 mg, 1680 µmol) (5 ml) was dissolved in water and the solution was added dropwise to the reaction flask. The reaction mixture was then stirred at room temperature until UHPLC/MS data indicated complete conversion to product. The substance was diluted with dichloromethane and washed with 0.1 M HCl. The aqueous layer was extracted twice with dichloromethane, then the combined organics were dried over sodium sulfate, filtered and concentrated to give free acid 101 (200 mg quant). Analytical UHPLC-MS (system 1): tr=1.33 min, m/z (E8+) calculated 359.17 (M+H) ⁺ , found 359.14.

(28,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-hydroxy-4-methylpentyl)thiazol-4-carboxamido)-2- methyl 5-phenylpentanoate (102). Tubuvalin free acid 101 (200 mg, 560 µmol) was pre-activated by dissolving in anhydrous dimethylformamide (5.4 ml, 100 mmol) and adding HATU (250 mg, 670 µmol) and DIPEA (0.59 ml, 3.36 mmol) ; the mixture was then stirred under a nitrogen atmosphere at room temperature for 10 minutes. The activated acid was then added to the known (Org. Lett., 2007, 9, 1605-1607) tubuphenylalanine allylic ester 16 and then the reaction mixture was stirred at ambient temperature under nitrogen atmosphere, the progress of the reaction was monitored using UHPLC/MS data. After completion of the reaction, glacial acetic acid (14 equivalents) was then added and the product was purified by preparative HPLC to give Thiu(OH)-Tyr-O-allyl dipeptide 102 (272 mg, 83%). Analytical UHPLC-MS (system 1): t=1.84 min, m/z (E8+) calculated 588.31 (M+H) ⁺ , found 588.29.

(28,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-(propionyloxy)pentyl)thiazole-4-carboxamido) -2-methyl-5-phenylpentanoate (105). Thiu(OH)-Tyr-O-allyl dipeptide 102 (26 mg, 44 μmol) was dissolved in anhydrous pyridine (1.8 ml, 25 mmol) and stirred under nitrogen atmosphere at room temperature. Propionic anhydride 103 (113 μL, 20 equivalents) was added dropwise and the reaction mixture was then monitored by UHPLC/MS. Additional propionic anhydride (20 equivalents) was added to achieve conversion to product. The substance was diluted with dichloromethane and washed with 0.1 M HCl. The aqueous layer was extracted twice with dichloromethane, then the combined organic products were dried over sodium sulfate, filtered and condensed.

- 119 046150 was centrated to give the crude product, which was then purified by preparative HPLC to give the esterified product 105 (17 mg, 61%). Analytical UHPLC-MS (system 1): t _r =1.99 min, m/z (ES+) calculated 644.34 (M+H) ⁺ , found 644.26.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-(butyryloxy)-4-methylpentyl)thiazol-4-carboxamido)- 2-methyl-5-phenylpentanoate (106). Tuv(OH)-Tup-O-allyl dipeptide 102 (27 mg, 46 μmol) was dissolved in anhydrous pyridine (0.9 ml, 50 mmol) and stirred under nitrogen at room temperature. Butanoic anhydride 104 (225 μL, 30 equivalents) was added dropwise and the reaction mixture was then monitored by UHPLC/MS. Additional butanoic anhydride (40 equivalents) was added in three parts to achieve conversion to the product. The substance was diluted with dichloromethane and washed with 0.1 M HCl. The aqueous layer was extracted twice with dichloromethane, then the combined organics were dried over sodium sulfate, filtered and concentrated to give the crude product, which was then purified by preparative HPLC to give esterified product 106 (24 mg, 80%). Analytical UHPLC-MS (system 1): t _r =2.13 min, m/z (ES+) calculated 658.35 (M+H)+, found 658.23.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-(isobuturyloxy)-4-methylpentyl)thiazol-4-carboxamido)- 2-methyl-5-phenylpentanoate (108). Tuv(OH)-Tup-O-allyl dipeptide 102 (26 mg, 44 μmol) was dissolved in anhydrous pyridine (1.8 ml, 25 mmol) and stirred under nitrogen at room temperature. Isobutyryl chloride 107 (93 µl, 20 equivalents) was added dropwise and the reaction mixture was then monitored by UHPLC/MS. Once converted to product, the material was then diluted with dichloromethane and washed with 0.1M HCl. The aqueous layer was extracted twice with dichloromethane, then the combined organics were dried over sodium sulfate, filtered and concentrated to give the crude product, which was then purified by preparative HPLC to give esterified product 108 (29 mg, qty). Analytical UHPLC-MS (system 1): t _r =2.13 min, m/z (ES+) calculated 658.35 (M+H)+, found 658.33.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-((3-methylbutanoyl)oxy)pentyl)thiazole -4-carboxamido)-2-methyl-5-phenylpentanoate (111). Isovaleric acid 109 (94 µl, 851 µmol) dissolved in anhydrous dichloromethane (5.6 ml, 15 mmol) was placed in the flask and the solution was stirred at 0°C under a nitrogen atmosphere. DMAP (10 mg, 85 µmol) was then added followed by DCC (88 mg, 425 µmol) and the reaction mixture was allowed to warm to room temperature over 2 hours. The resulting activated acid was then added to Tuv(OH)-Tup-Oallyl dipeptide 102 (50 mg, 85 µmol) and the reaction mixture was stirred overnight, at which time UHPLC/MS data indicated conversion to product. The reaction mixture was then diluted with dichloromethane and washed with 0.1 M HCl. The aqueous layer was extracted twice with dichloromethane, then the combined organics were dried over sodium sulfate, filtered and concentrated to give the crude product, which was then purified by preparative HPLC to give esterified product 111 (52 mg, 91%). Analytical UHPLC-MS (system 2): t _r =1.91 min, m/z (ES+) calculated 672.37 (M+H)+, found 672.46.

(2S,4R)-Allyl 4-(2-((1R,3R)-3 -((tert-butoxycarbonyl)(methyl)amino)-1 -((3,3-dimethylbutanoyl)oxy)-4-methylpentyl) thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (112). Gemdimethylbutanoic acid 110 (98 μL, 766 μmol) dissolved in anhydrous dichloromethane (5.1 mL, 15 mmol) was placed in the flask and the solution was stirred at 0°C under a nitrogen atmosphere. DMAP (9 mg, 77 µmol) was then added followed by DCC (79 mg, 383 µmol) and the reaction mixture was allowed to warm to room temperature over 2 hours. The resulting activated acid was then added to Tuv(OH)-Tup-Oallyl dipeptide 102 (45 mg, 77 µmol) and the reaction mixture was stirred overnight, at which time UHPLC/MS data indicated conversion to product. The reaction mixture was then diluted with dichloromethane and washed with 0.1 M HCl. The aqueous layer was extracted twice with dichloromethane, then the combined organics were dried over sodium sulfate, filtered and concentrated to give the crude product, which was then purified by preparative HPLC to give esterified product 112 (49 mg, 93%). Analytical UHPLC-MS (system 2): t _r =1.88 min, m/z (ES+) calculated 686.39 (M+H)+, found 686.47.

- 120 046150

Scheme 14

Vos

TFA/DCM

105, K=CH ₂ CH ₃ O

106, R=CH ₂ CH ₂ CH ₃

108, R=CH(CH ₃ ) ₂

111, R=CH ₂ CH(CH ₃ ) ₂

112, R=CH ₂ C(CH ₃ ) ₃

_H2N .

124, R=CH ₂ CH ₂ CH ₃

125, R=CH(CH ₃ ) ₂

126, R=CH ₂ CH(CH ₃ ) ₂

127, R=CH ₂ C(CH ₃ ) ₃

113, r=ch ₂ ch ₃ o

114, R=CH ₂ CH ₂ CH ₃

115, R=CH(CH ₃ ) ₂

116, R=CH ₂ CH(CH ₃ ) ₂

117, R-CH ₂ C(CH ₃ ) ₃

Fmoc-lle, HATU, DIPEA, DMF

H„ ^-N '.

Fmoc ¹

119, R-CH ₂ CH ₂ CH ₃

120, R=CH(CH ₃ ) ₂

121, R=CH ₂ CH(CH ₃ ) ₂

122, R=CH ₂ C(CH ₃ ) ₃

129, R=CH ₂ CH ₂ CH ₃

130, R=CH(CH ₃ ) ₂

131, R=CH ₂ CH(CH ₃ ) ₂

132, R=CH ₂ C(CH ₃ ) ₃

134, R=CH ₂ CH ₂ CH ₃

135, R=CH(CH ₃ ) ₂

136, R=CH ₂ CH(CH ₃ ) ₂

137, R=CH ₂ C(CH ₃ ) ₃

General method for deprotection of Boc T^(O-ester)-Tyr intermediates. Esterified tubuvalin-tubuphenylalanine intermediates 105, 106, 108, 111, or 112 were deprotected to expose the secondary amine functionality under acidic conditions with 10% TFA in dichloromethane (25 mM). Specifically, the starting material was dissolved in anhydrous dichloromethane (9 volumes) and stirred under a nitrogen atmosphere at 0°C. Then trifluoroacetic acid (1 volume) was added dropwise to the stirred solution. The reaction mixture was slowly warmed to room temperature and the reaction progress was monitored using UHPLC/MS data. After completion of the reaction, the reaction mixture was concentrated by evaporation on a rotor and pumped out in a vacuum line overnight. Free amines 135-139 were further used without further purification.

(2S,4R)-Allyl 2-methyl-4-(2-((1R,3R)-4-methyl-3-(methylamino)-1-(propionyloxy)pentyl)thiazol-4carboxamido)-5-phenylpentanoate (113 ). Intermediate 105 Boc-protected Tuv-Tup (17 mg, 26 μmol) was deprotected as described above to give 14 mg (quantit) of the title compound. UHPLC-MS (system 1): t _r =1.24 min, m/z (ES+) calculated 544.29 (M+H) ⁺ , found 544.25.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(butyryloxy)-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (114). Intermediate 106 Boc-protected Tuv-Tup (24 mg, 37 μmol) was deprotected as described above to give 21 mg (quantit) of the title compound. UHPLC-MS (system 2): tr=1.20 min, m/z (ES+) calculated 558.30 (M+H) ⁺ , found 558.38.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(isobutyryloxy)-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (115). Intermediate 108 Boc-protected Tuv-Tup (29 mg, 44 μmol) was deprotected as described above to give 25 mg (quantit) of the title compound. UHPLC-MS (system 1): tr=1.30 min, m/z (ES+) calculated 558.30 (M+H) ⁺ , found 557.93.

(2S,4R)-Allyl 2-methyl-4-(2-((1R,3R)-4-methyl-3-(methylamino)-1-((3-methylbutanoyl)oxy)pentyl)thiazole-4-carboxamido )-5-phenylpentanoate (116). Intermediate 111 Boc-protected Tuv-Tup (52 mg, 78 μmol) was deprotected as described above to give 45 mg (quantit) of the title compound. UHPLC-MS (system 2): t _r =1.23 min, m/z (ES+) calculated 572.32 (M+H) ⁺ , found 572.40.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-((3,3-dimethylbutanoyl)oxy)-4-methyl-3-(methylamino)pentyl)thiazole4-carboxamido)-2- methyl 5-phenylpentanoate (117). Intermediate 112 Boc-protected Tuv-Tup (49 mg, 71 μmol) was deprotected as described above to give 46

- 121 046 150 mg (quant.) of the title compound. UHPLC-MS (system 2): t _r =1.27 min, m/z (ES+) calculated 586.33 (M+H) ⁺ , found 586.42.

General method for amide condensation of O-esterified tubuvaline-tubuphenylalanine dipeptides with Fmoc-protected L-isoleucine. Commercially available Fmoc-L-isoleucine (4 equivalents) was dissolved in anhydrous dimethylformamide (50 mM) and preactivated with HATU (4 equivalents) and DIPEA (8 equivalents); the mixture was stirred for 10 minutes at room temperature under a nitrogen atmosphere. The activated acid was then added to TuviO-ester)-Tch dipeptides 113-117; the reaction mixture was stirred at room temperature under a nitrogen atmosphere and the reaction progress was monitored using UHPLC/MS data. Once the reaction was stopped or completely completed, glacial acetic acid (13 equivalents) was added and the reaction mixture was purified using Rev. HPLC.

(2S,4R)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 -methyl3,6,12-trioxo-2,11-dioxa-4,7-diazatetradecane-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (118). Intermediate 113 Tuv-Tup (14 mg, 25 μmol) was coupled to Fmoc-L-Ile as described above to give 17 mg (77%) of the title compound. UHPLC-MS (system 1): t _r =2.11 min, m/z (ES+) calculated 879.44 (M+H)+, found 879.60.

DO,4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 -methyl3,6,12-trioxo-2,11-dioxa-4,7-diazapentadecan-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (119). Intermediate 114 Tuv-Tup (21 mg, 37 μmol) was coupled to Fmoc-L-Ile as described above to give 24 mg (73%) of the title compound. UHPLC-MS (system 2): t _r =1.94 min, m/z (ES+) calculated 893.45 (M+H)+, found 893.56.

DO,4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 ,13-dimethyl-3,6,12-trioxo-2,11-dioxa-4,7-diazatetradecane-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (120). Intermediate 115 Tuv-Tup (25 mg, 44 μmol) was coupled to Fmoc-L-Ile as described above to give 26 mg (67%) of the title compound. UHPLC-MS (system 2): t _r =1.85 min, m/z (ES+) calculated 893.45 (M+H)+, found 893.56.

(28.4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl- 7,14-dimethyl-3,6,12-trioxo-2,11-dioxa-4,7-diazapentadecan-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (121). Intermediate 116 Tuv-Tup (45 mg, 79 μmol) was coupled to Fmoc-L-Ile as described above to give 54 mg (75%) of the title compound. UHPLC-MS (system 2): t _r =2.06 min, m/z (ES+) calculated 907.47 (M+H)+, found 907.58.

(28.4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl- 7,14,14-trimethyl-3,6,12-trioxo-2,11-dioxa-4,7-diazapentadecan-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (122). Intermediate 117 Tuv-Tup (46 mg, 79 μmol) was coupled to Fmoc-L-Ile as described above to give 55 mg (75%) of the title compound. UHPLC-MS (system 2): t _r =2.50 min, m/z (ES+) calculated 921.49 (M+H)+, found 921.59.

A general method for deprotecting Fmoc isoleucine-O-esterified tubuvalintubuphenylalanine tripeptides. Fmoc-Πe-Tuv(O-ester)-Tcr allyl ester (118-122) was treated with 20% piperidine in dimethylformamide (20 mM) with stirring under nitrogen at room temperature. Once complete deprotection was achieved, as monitored by UHPLC/MS, the reaction mixture was concentrated by rotary evaporation. The crude product was then purified by preparative HPLC to give tripeptides with free amines 145-149.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-4-methyl-1-(propionyloxy)pentyl) thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (123): Fmoc-Ile-Tuv-Tup intermediate 118 (17 mg, 19 μmol) was deprotected as described above to give 15 mg (qty. .) specified in the connection header. UHPLC-MS (system 1): t _r =1.29 min, m/z (ES+) calculated 657.37 (M+H)+, found 658.04.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-1-(butyryloxy)-4-methylpentyl)thiazole -4-carboxamido)-2-methyl-5-phenylpentanoate (124). Intermediate 119 Fmoc-Ile-Tuv-Tup (23 mg, 26 μmol) was deprotected as described above to give 15 mg (86%) of the title compound. UHPLC-MS (system 2): t _r =1.24 min, m/z (ES+) calculated 671.39 (M+H)+, found 671.48.

(28,4I.)-Allyl 4-(2-((^^)-3-(^^)-2-amino-N,3-dimethylpentanamido)-1-(isobutyryloxy)-4methylpentyl)thiazole-4-carboxamido )-2-methyl-5-phenylpentanoate (125). Intermediate 120 Fmoc-Ile-Tuv-Tup (26 mg, 29 μmol) was deprotected as described above to give 20 mg (quantit) of the title compound. UHPLC-MS (system 1): t _r =1.33 min, m/z (ES+) calculated 671.39 (M+H)+, found 671.33.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-4-methyl-1-((3-methylbutanoyl )oxy)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (126). Intermediate 121 Fmoc-Ile-Tuv-Tup (54 mg, 60 μmol) was deprotected as described above to give 41 mg (quantit) of the title compound. UHPLC-MS (system 2): t _r =1.31 min, m/z (ES+) calculated 685.40 (M+H)+, found 685.49.

- 122 046150 (2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-1-((3,3- dimethylbutanoyl)oxy)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (127). Intermediate 122 Fmoc-Ile-Tuv-Tup (55 mg, 60 μmol) was deprotected as described above to give 36 mg (86%) of the title compound. UHPLC-MS (system 2): t _r =1.30 min, m/z (ES+) calculated 699.42 (M+H) ⁺ , found 699.51.

General method for the amide condensation of tripeptides isoleucine-tubuvaline-ester)tubuphenylalanine with N-methyl-pipecolic acid. Commercially available D-N-methylpipecolic acid (D-Mep) 36 (2 equivalents) was dissolved in anhydrous dimethylformamide (20-25 mM) and preactivated with HATU (2 equivalents) and DIPEA (4 equivalents); the mixture was stirred for 10 minutes at room temperature under a nitrogen atmosphere. The activated acid was then added to the Πe-Tuv(O-ester) tripeptides 123-127; the reaction mixture was stirred at room temperature under nitrogen and the reaction progress was monitored using UHPLC/MS data. Glacial acetic acid was then added after completion of the reaction (14 equivalents) and the product was purified by preparative HPLC.

(28.4I.)-Allyl 4-(2-((^^)-3-(^^)-Н,3-dimethyl-2-(^)-1-methylpiperidine-2-carboxamido)pentanamido)-4 -methyl-1-(propionyloxy)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (128). Intermediate 123 Ile-Tuv-Tup (5 mg, 8 μmol) was coupled to D-Mep 36 as described above to give 6 mg (97%) of the title compound. UHPLC-MS (system 1): t _r =1.33 min, m/z (ES+) calculated 782.45 (M+H)+, found 7 81.82.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(butyryloxy)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine -2-carboxamido)pentanamido)-4-methylpentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (129). Intermediate 124 Ile-Tuv-Tup (6 mg, 9 μmol) was coupled to D-Mep 36 as described above to give 7 mg (98%) of the title compound. UHPLC-MS (system 2): t _r =1.31 min, m/z (ES+) calculated 796.47 (M+H)+, found 796.57.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-1-(isobutyryloxy)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (130). Intermediate 125 Ile-Tuv-Tup (5 mg, 8 μmol) was coupled to D-Mep 36 as described above to give 6 mg (94%) of the title compound. UHPLC-MS (system 1): t _r =1.37 min, m/z (ES+) calculated 796.47 (M+H)+, found 795.78.

(28.4I.)-Allyl 4-(2-((^^)-3-(^^)-Н,3-dimethyl-2-(^)-1-methylpiperidine-2-carboxamido)pentanamido)-4 -methyl-1-((3-methylbutanoyl)oxy)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (131). Intermediate 126 Ile-Tuv-Tup (7 mg, 10 μmol) was coupled to D-Mep 36 as described above to give 6 mg (94%) of the title compound. UHPLC-MS (system 2): t _r =1.35 min, m/z (ES+) calculated 810.49 (M+H)+, found 810.59.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-1-((3,3-dimethylbutanoyl)oxy)-4-methylpentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (132). Intermediate 127 Ile-Tuv-Tup (7 mg, 10 μmol) was coupled to D-Mep 36 as described above to give 6 mg (94%) of the title compound. UHPLC-MS (system 2): t _r =1.38 min, m/z (ES+) calculated 824.50 (M+H)+, found 824.60.

General method for removing allyl ester from D-methylpipecolic acid-isoleucine-tubuvaline^-ester)-tubuphenylalanine intermediates. Intermediate (128132) allylic ether-protected tubulisin was dissolved in anhydrous dichloromethane (20 mM), treated with tetrakis(triphenylphosphine) palladium (0.1 equiv.), triphenylphosphine (0.2 equivalents) and anhydrous pyrrolidine (8 equivalents), and the reaction mixture stirred at ambient temperature under nitrogen atmosphere. After UHPLC/MS data indicated conversion of the free acid to the product, the reaction mixture was quenched with glacial acetic acid (22 equivalents), diluted with acetonitrile and dimethylformamide, and then concentrated by rotary evaporation. The crude tubulisin ester was then purified by preparative HPLC.

^^)-4-(2-((^^)-3-(^^)-Н,3-Dimethyl-2-(^)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methyl-1 -(propionyloxy)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (133). Allyl ether-protected tubulisin intermediate 128 (6 mg, 8 μmol) was deprotected as described above to yield 3.8 mg (67%) tubulisin 133 (Tub propionate). UHPLC-MS (system 2): t _r =1.11 min, m/z (ES+) calculated 742.42 (M+H)+, found 742.51.

^^)-4-(2-((^^)-1-(Butyryloxy)-3-(^^)-Н,3-dimethyl-2-(^)-1-methylpiperidine-2carboxamido)pentanamido)-4 -methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (134). Allyl ether-protected tubulisin intermediate 129 (7 mg, 9 μmol) was deprotected as described above to yield 6 mg (88%) of tubulisin 134 (Tub butyrate). UHPLC-MS (system 2): t _r =1.16 min, m/z (ES+) calculated 756.44 (M+H)+, found 756.54.

(2S,4R)-4-(2-((1R,3R)-3 -(^^)-Н,3-Dimethyl-2-(^)-1-methylpiperidine-2-carboxamido)pentanamido)-1- (isobutyryloxy)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (135). Allyl ether-protected tubulisin intermediate 130 (6 mg, 8 μmol) was deprotected as described above to yield 3 mg (50%) tubulisin 135 (Tub isobu

- 123 046150 tirate). UHPLC-MS (system 1): ^=1.23 min, m/z (ES+) calculated 756.44 (M+H)+, found 756.82.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-guMeTun-2-((R)-1-MeTunnunepuguH-2-Kap6oKcaMugo)neHTaHaMudo) -4-methyl-1-((3-methylbutanoyl)oxy)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (136). Allyl ether-protected tubulisin intermediate 131 (7 mg, 9 μmol) was deprotected as described above to yield 7 mg (quantit) of tubulisin 136 (Tub isovalerate). UHPLC-MS (system 2): tr=1.22 min, m/z (ES+) calculated 770.45 (M+H)+, found 770.55.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido) -1 -((3,3-dimethylbutanoyl)oxy)-4-methylpentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (137). Allyl ether-protected tubulisin intermediate 132 (7 mg, 8.5 μmol) was deprotected as described above to yield 8 mg (quantit) of tubulisin 137 (Tub gem-dimethylbutyrate). UHPLC-MS (system 2): tr=1.23 min, m/z (ES+) calculated 784.47 (M+H)+, found 784.57.

Scheme 15

142, R=BOC

TFYUSM (

143, RH (2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S )-6((allyloxy)carbonyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R) 1-(4-(((2R,4S)-5-(allyloxy)-4-methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-(isobutyryloxy)- 4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin1-ium (138). To a flask containing H-Ile-Tuv(O-isobutyrate)-Tup-OAlyl (125.15 mg, 22 µmol), Fmoc-Gluc(Allyl)Q-Mep-OH (78.17 mg, 22 µmol) was added and HATU (9 mg, 22 µmol) as solids, then DMF (0.9 ml). N,N-Diisopropylethylamine (15 µl, 8 8 µmol) was added and the reaction mixture was stirred at room temperature and the reaction progress was monitored using UHPLC/MS data. The reaction mixture was then taken up in DMSO and purified by preparative HPLC to give 138 (4 mg, 13%). Analytical UHPLC-MS (system 1): tr=1.53 min, m/z (ES+) calculated 1426.69 (M) ⁺ , found 1426.17.

(2R)-1-(3-(3-Aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2Hpyran-2-yl)oxy )benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl )thiazol-2-yl)-1-(isobutyryloxy)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (139). Fmoc-Gluc(Allyl)Q-Tub(iso-O-Allyl) (138.4 mg, 3 µmol) was placed in DCM (0.3 ml) with stirring under N ₂ atmosphere. Pd(PPh ₃ ) ₄ (0.4 mg, 0.3 µmol) and PPh ₃ (0.2 mg, 0.6

- 124 046 150 µmol) were added as solids, then pyrrolidine (2 µl, 2 4 µmol). The reaction mixture was stirred for 2 hours at room temperature, then taken into DMSO, condensed under reduced pressure, and purified by preparative HPLC to give 139 (3 mg, 8-8%). Analytical UHPLC-MS (system 1): t _r =1.04 min, m/z (ES+) calculated 1124.56 (M) ⁺ , found 1124.69.

(2R)-1-(3-((S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38,45-dioxo-2,5,8,11,14 ,17,20, 23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran -2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentane -2-yl)carbamoyl)thiazol-2-yl)-1-(isobutyryloxy)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1 -methylpiperidin-1-ium (140). Fmoc-Lys(PEG12)-OSu (59.10 mg, 9 µmol) was added to a flask containing H-GlucQ-Tub(iso-O-allyl) (139.3 mg, 3 µmol) as a solution in anhydrous DMF ( 0.3 ml) in N2 atmosphere. NN-diisopropylethylamine (2 µl, 9 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid and purified by preparative HPLC to give 140 (0.8 mg, 13%). Analytical UHPLC-MS (system 2): t _r =1.35 min, m/z (ES+) calculated 1023.03 (M+H) ²⁺ , found 1023.15.

(2R)-1-(3-((8)-44-Amino-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa- 39,46-diazanonatetracontanamido)-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S, 3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2yl)-1 -(isobutyryloxy) -4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1 methylpiperidin-1-ium (141). 20% piperidine in DMF (0.2 ml) was added to a flask containing Fmoc-Lys(PEG12)-GlucQ-Tub-(O-iso-allyl) (140, 0.8 mg, 0.4 µmol). The reaction mixture was stirred under N2 at room temperature for 2 hours. The reaction mixture was then diluted with DMSO/H2O and purified by preparative HPLC to give 141 (3.6 mg, 82%). Analytical UHPLC-MS (system 2): t _r =1.07 min, m/z (ES+) calculated 1822.98 (M) ⁺ , found 1823.15.

(2R)-1-(3-((S)-44-((S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1yl)propanamido)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-((^ ^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2(((2S,3S)-1-(((1R,3R)- 1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-(isobutyryloxy)-4-methylpentan-3-yl)(methyl )amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidinium (142). H-Lys(PEG12)-GlucQ-Tub-(O-iso-allyl) (141, 0.5 mg, 0.4 µmol) in anhydrous DMF (0.212 ml) was placed in the flask, to which it was added as a solid in an atmosphere N ₂ mDPR(Boc)OPFP (44, 1.4 mg, 3.2 µmol) was added. N,N-diisopropylethylamine (0.74 µl, 4.2 µmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was then quenched with acetic acid, diluted with DMSO and purified by preparative HPLC to give 142 (0. 5 mg, 61%). Analytical UHPLC-MS (system 2): t _r =1.25 min, m/z (ES+) calculated 1045.04 (M+H) ²⁺ , found 1045.16.

(2R)-1-(3-((S)-44-((S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido) -38,45dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4((^^^^^)-6- carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-((^^)-1(((1R,3R)-1-(4-(((2R, 4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-(isobutyryloxy)-4methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentane- 2-yl)carbamoyl)-1-methylpiperidin-1-ium (143). The flask containing mDPR(Boc)-Lys(PEG12)-GlucQ-Tub(O-iso-allyl)(142, 0.5 mg, 0.24 µmol) was cooled to 0°C under N2. A solution of 10% TFA in CH ₂ Cl ₂ (0.26 ml) was added dropwise and stirred for 2 hours. The reaction mixture was then taken up in DMSO, condensed under reduced pressure and purified by preparative HPLC to give 143 (0.7 mg, quantity). Analytical UHPLC-MS (system 2): t _r =1.06 min, m/z (ES+) calculated 1989.02 (M) ⁺ , found 1989.20.

- 125 046150

Scheme 16

General method for esterification of tubuvalin. In a flame-dried flask, intermediate 17 (or 186) Boc-protected known tubuvalin (J. Dept. Chem., 2008, 13, 4362-4369) was placed in anhydrous tetrahydrofuran (50 mM), to which 18-crown-6 was added (2.0 equivalents) and cooled to -7 8°C. Potassium hexamethyldisilazide (1.5 equivalents) was added dropwise as a 1M solution in tetrahydrofuran, and the reaction mixture was then stirred for 1 hour at -78°C under a nitrogen atmosphere. Unsaturated bromoalkane (2 equivalents) was then added and the reaction mixture was slowly warmed to room temperature and the reaction progress was monitored using UHPLC/MS data. After the starting material was consumed, the reaction mixture was cooled on ice and quenched with saturated ammonium chloride and diluted with dichloromethane (10 volumes). The organic layer was washed with 0.1M HC1 and the resulting aqueous phase was extracted twice with dichloromethane. The combined organics were then dried over sodium sulfate, filtered and concentrated to dryness. Purification of crude O-alkylated products was achieved by flash chromatography on silica gel or preparative HPLC.

Ethyl 2-((1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-((2-methylallyl)oxy)pentyl)thiazole-4-carboxylate (148). Intermediate 186 protected tubuvalin propyl ester (299 mg, 750 µmol) was O-alkylated as described above with 3-bromo-2-methylprop-1-ene (151 µl, 1.5 mmol) to give, after purification on silica gel, eluting with mixtures of methanol and dichloromethane, 243 mg (71%) of the title compound. UHPLC-MS (system 2): tr=1.77MHH, m/z (ES+) calculated 455.26 (M+H) ⁺ found 455.32.

Ethyl 2-((1R,3R)-1-(allyloxy)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-4carboxylate (149). Intermediate 17 tubuvalin (84 mg, 218 µmol) was O-allylated as above with allyl bromide (40 µl, 43 6 µmol) to give, after purification on silica gel eluting with mixtures of methanol and dichloromethane, 77 mg (83%) of the title connections. UHPLC-MS (system 2): tr=1.69 min, m/z (ES+) calculated 427.23 (M+H)+, found 427.30.

Ethyl 2-((1R,3R)-3-((Ίret-butoxycarbonyl)(methyl)amino)-4-methyl-1-(nrop-2-yn-1-yloxy)pentyl)thiazol-4-carboxylate (150 ). Intermediate 17 tubuvalin (101 mg, 262 μmol) was propargylated as above with propargyl bromide (56 μL, 524 μmol) to give 76 mg (68%) of the title compound after purification. UHPLC-MS (system 2): tr=1.63 min, m/z (ES+) calculated 425.21 (M+H)+, found 425.28.

Ethyl 2-((1R,3R)-1-(benzyloxy)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-4carboxylate (151). Intermediate 17 tubuvalin (90 mg, 230 µmol) was O-benzylated as above with benzyl bromide (55 µl, 460 µmol) to give after purification 21 mg (19%) as indicated

- 126 046150 in the connection header. UHPLC-MS (system 1): t _r =1.95 min, m/z (ES+) calculated 477.24 (M+H) ⁺ , found 477.22.

General method for saponification of O-alkylated tubuvaline esters. Saponification reactions were carried out at a reaction mixture concentration of 20 mM using a 1:1:1 mixture of tetrahydrofuran:methanol:water solvents. O-Alkylated tubuvalin intermediate 148-151 was dissolved in tetrahydrofuran and methanol, 1 volume each. The mixture was then cooled in an ice bath at 0°C. Lithium hydroxide monohydrate (2-3 equivalents) was dissolved in 1 volume of distilled water and added dropwise to the reaction flask with stirring at 0°C. The reaction mixture was then allowed to warm to room temperature and the progress of the reaction was monitored using UHPLC/MS data. Once the starting material was converted to the free acid, the reaction mixture was quenched with glacial acetic acid (2-3 equivalents) and concentrated by rotary evaporation. The crude carboxylic acids were then purified by preparative HPLC.

2-((1R,3R)-3-((tert-Butoxycarbonyl)(methyl)amino)-4-methyl-1-((2-methylallyl)oxy)pentyl)thiazole4-carboxylic acid (152). Intermediate 148 tubuvalin ester (143 mg, 315 µmol) was saponified as above with lithium hydroxide monohydrate (27 mg, 63 0 µmol) to give 114 mg (88%) of the title compound. UHPLC-MS (system 1): tr=1.57 min, m/z (ES+) calculated 413.21, found 413.28.

2-((1R,3R)-1-(Allyloxy)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-4-carboxylic acid (153). Intermediate 149 tubuvalin allyl ester (77 mg, 181 µmol) was saponified as above with lithium hydroxide monohydrate (15 mg, 3-60 µmol) to give 51 mg (71%) of the title compound. UHPLC-MS (system 2): t _r =1.51 min, m/z (ES+) calculated 399.20 (M+H) ⁺ , found 399.26.

2-((1R,3R)-3 -((tert-Butoxycarbonyl)(methyl)amino)-4-methyl-1-(prop-2-yn-1-yloxy)pentyl)thiazole4-carboxylic acid (154). Intermediate 150 tubuvalin propargyl ester (76 mg, 180 μmol) was saponified as described above with lithium hydroxide monohydrate (15 mg, 360 μmol) to give 50 mg (69%) of the title compound. UHPLC-MS (system 2): t _r =1.47 min, m/z (ES+) calculated 397.18 (M+H)+, found 397.25.

2-((1R,3R)-1-(Benzyloxy)-3-((m-tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-4-carboxylic acid (155). Intermediate 151 Tubuvalin benzyl ester (21 mg, 44 μmol) was saponified as described above with lithium hydroxide monohydrate (5.5 mg, 132 μmol) to give the title compound. UHPLC-MS (system 1): t _r =1.72 min, m/z (ES+) calculated 449.21 (M+H)+, found 449.18.

General method for amide condensation of allyl esters of the free acids of O-alkylated tubuvaline and tubuphenylalanine. O-alkylated tubuvalin free acids 152-155 were pre-activated by dissolving in anhydrous dimethylformamide (25-50 mM) and adding HATU (2.4 equivalents) and DIPEA (5 equivalents); the mixture was then stirred under a nitrogen atmosphere at room temperature for 10 minutes. The activated acid was then added to the known (Org. Lett., 2007, 9, 1605-1607) tubuphenylalanine allylic ester 16 and the reaction mixture was then stirred at ambient temperature under a nitrogen atmosphere and the reaction progress was monitored by UHPLC/MS. Glacial acetic acid (14 equivalents) was then added after completion of the reaction and the product was purified by preparative HPLC.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((mret-butoxycarbonyl)(methyl)amino)-4-methyl-1-((2-methylallyl)oxy)pentyl)thiazole -4-carboxamido)-2-methyl-5-phenylpentanoate (156). Intermediate 152 tubuvalin ester (114 mg, 277 μmol) was added to tubuphenylalanine allylic ester (Tup) 16 (137 mg, 554 μmol) to give 159 mg (90%) of the title compound. UHPLC-MS (system 1): t _r =1.97 min, m/z (ES+) calculated 642.36 (M+H)+, found 642.44.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(allyloxy)-3-((mret-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazol-4-carboxamido)- 2-Methyl-5-phenylpentanoate (157): Intermediate 153 tubuvalin allyl ester (44 mg, 100 µmol) was added to tubuphenylalanine (Tup) allyl ester 16 (37 mg, 150 µmol) to give 60 mg (95%) of connection header. UHPLC-MS (system 1): t _r =2.06 min, m/z (ES+) calculated 628.34 (M+H)+, found 628.26.

(2S,4R)-Allyl 4-(2-((1R,3R)-3 -((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1 -(prop-2-yn-1 -yloxy) pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (158): intermediate 154 tubuvalin propargyl ester (42 mg, 106 μmol) was coupled to tubuphenylalanine allyl ester (Tup) 16 (52 mg, 212 μmol) with obtaining 48 mg (73%) of the title compound. UHPLC-MS (system 2): t _r =1.73 min, m/z (ES+) calculated 626.33 (M+H)+, found 626.41.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(benzyloxy)-3-((mret-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazol-4-carboxamido)- 2-Methyl-5-phenylpentanoate (159): Intermediate 155 Tubuvalin benzyl ester (50 mg, 110 µmol) was added to tubuphenylalanine allyl ester (Tup) 16 (60 mg, 165 µmol) to give 43 mg (58%) of connection header. UHPLC-MS (system 2): t _r =1.93 min, m/z (ES+) calculated 678.36 (M+H)+, found 678.45.

- 127 046150

General method for removing the Boc Tuv(OAlk)-Tup protecting group from intermediates. Intermediates 156-159 O-alkylated tubuvaline-tubuphenylalanine were deprotected to expose the secondary amine functionality under acidic conditions with 10% TFA in dichloromethane (25 mM). Specifically, the starting material was dissolved in anhydrous dichloromethane (9 volumes) and stirred under a nitrogen atmosphere at 0°C. Then trifluoroacetic acid (1 volume) was added dropwise to the stirred solution. The reaction mixture was slowly warmed to room temperature and the reaction progress was monitored using UHPLC/MS data. After completion of the reaction, the reaction mixture was concentrated by evaporation on a rotor and pumped out in a vacuum line overnight. Free amines 160-163 were further used without additional purification.

(2S,4R)-Allyl 2-methyl-4-(2-((1R,3R)-4-methyl-1-((2-methylallyl)oxy)-3-(metulamino)pentyl)thiazole-4-carboxamido )-5-phenylpentanoate (160): Intermediate 156 Boc-protected Tuv-Tup (159 mg, 248 μmol) was deprotected as described above to give 127 mg (95%) of the title compound. UHPLC-MS (system 1): t _r =1.18 min, m/z (ES+) calculated 542.31 (M+H) ⁺ , found 542.38.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(allyloxy)-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamudo)2-methyl-5-phenylpentanoate ( 161): Intermediate 157 Boc-protected Tuv(O-allyl)-Tup (60 mg, 96 μmol) was deprotected as described above to give 52 mg (quantit) of the title compound. UHPLC-MS (system 1): t _r =1.16 min, m/z (ES+) calculated 528.29 (M+H)+, found 528.05.

(2S,4R)-Allyl 2-methyl-4-(2-((1R,3R)-4-methyl-3 -(methylamino)-1 -(prop-2-yn-1 -yloxy)pentyl)thiazol- 4-Carboxamido)-5-phenylpentanoate (162): Intermediate 158 Boc-protected Tuv(Opropargyl^^ (39 mg, 62 μmol) was deprotected as described above to give 45 mg (quantit) of the title compounds. UHPLC-MS (system 2): t _r =1.08 min, m/z (ES+) calculated 526.28 (M+H)+, found 526.35.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(benzyloxy)-4-methyl-3-(methylamino)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (163): Intermediate 159 Boc-protected Tuv^^m^)Tup (38 mg, 56 µmol) was deprotected as described above to give the title compound in quantitative yield. UHPLC-MS (system 2): t _r =1.20 min, m/z (ES+) calculated 578.31 (M+H)+, found 578.38.

General method for amide condensation of O-alkylated tubuvaline-tubuphenylalanine dipeptides with Fmoc-protected L-isoleucine. Commercially available Fmoc-L-isoleucine (1.3-2 equivalents) was dissolved in anhydrous dimethylformamide (50-200 mM) and preactivated with HATU (1.5-2 equivalents) and DIPEA (2 equivalents); the mixture was stirred for 10 minutes at room temperature under a nitrogen atmosphere. The activated acid was then added to the Tuv(O-3(|ihp)-Tup 160-163 dipeptides; the reaction mixture was stirred at room temperature under nitrogen and the reaction progress was monitored using UHPLC/MS data. Once the reaction had stopped or completely When complete, glacial acetic acid (13 equivalents) was added and the reaction mixture was purified by pre-HPLC.

(28.4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl- 7,13-dimethyl-3,6-dioxo-2,11-dioxa-4,7-diazatetradec-13-en-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (164): Tuv-Tup intermediate 160 (127 mg, 235 μmol) was coupled to Fmoc-L-Ile as described above to give 90 mg (44%) of the title compound. UHPLC-MS (system 1): t _r =2.14 min, m/z (ES+) calculated 877.46 (M+H)+, found 877.56.

(2S,4R)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 -methyl-3,6dioxo-2,11-dioxa-4,7-diazatetradec-13-en-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (165). Intermediate 161 Tuv(O-allyl)-Tup (52 mg, 100 μmol) was coupled to Fmoc-L-Ile as described above to give 38 mg (44%) of the title compound. UHPLC-MS (system 2): t _r =1.94 min, m/z (ES+) calculated 863.44 (M+H)+, found 863.54.

(28.4I.)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl- 7-methyl-3,6dioxo-2,11-dioxa-4,7-diazatetradec-13-yn-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (166). Intermediate 162 Tuv(O-nropargyl)-Tup (62 μmol) was coupled to Fmoc-L-Ile as described above to give 33 mg (62%) of the title compound. UHPLC-MS (system 2): t _r =1.83 min, m/z (ES+) calculated 861.43 (M+H)+, found 861.53.

(2S,4R)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7 -methyl-3,6dioxo-12-phenyl-2,11-dioxa-4,7-diazadodecan-10-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (167). Intermediate 163 Tuv^^^^^T^ (56 µmol) was coupled to Fmoc-L-Ile as described above to give 23 mg (45%) of the title compound. UHPLC-MS (system 2): t _r =2.04 min, m/z (ES+) calculated 913.46 (M+H)+, found 913.56.

A general method for deprotecting Fmoc tripeptides isoleucine-O-alkylated tubuvaline-tubuphenylalanine: Fmoc-Ile-Tuv(O-ester)-Tup allyl ether (164-167) was treated with 20% piperidine in dimethylformamide (164-167) while stirring under nitrogen at room temperature. 20 mM).

- 128 046150

Once complete deprotection was achieved, as monitored by UHPLC/MS, the reaction mixture was concentrated by rotary evaporation. The crude product was then purified by preparative HPLC to give tripeptides with free amines 168-171.

(28.4I.)-Allyl 4-(2-((^^)-3-(^^)-2-amino-H,3-dimethylpentanamido)-4-methyl-1-((2-methylallyl)oxy )pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (168). Intermediate 164 Fmoc-Ile-Tuv-Tup (90 mg, 103 μmol) was deprotected as described above to give 29 mg (43%) of the title compound. UHPLC-MS (system 1): t _r =1.29 min, m/z (ES+) calculated 655.39 (M+H)+, found 655.48.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(allyloxy)-3-((2S,3S)-2-amino-N,3-dimethylpentanamdo)-4-methylpentyl)thiazole -4-carboxamido)-2-methyl-5-phenylpentanoate (169). Intermediate 165 FmocΠe-Tuv(O-allyl)-Tup (38 mg, 44 μmol) was deprotected as described above to give 25 mg (89%) of the title compound. UHPLC-MS (system 2): t _r =1.20 min, m/z (ES+) calculated 641.38 (M+H) ⁺ , found 641.46.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-4-methyl-1-(prop-2- in-1yloxy)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (170). Intermediate 166 Fmoc-Ile-Tuv(O-propargyl)-Tup (33 mg, 38 μmol) was deprotected as described above to give 25 mg (quantit) of the title compound. UHPLC-MS (system 2): t _r =1.15 min, m/z (ES+) calculated 639.36 (M+H)+, found 639.44.

(28,4I.)-Allyl 4-(2-((^^)-3-(^^)-2-amino-N,3-dimethylpentanamido)-1-(benzyloxy)-4-methylpentyl)thiazol-4 -carboxamido)-2-methyl-5-phenylpentanoate (171). Intermediate 167 Fmoc11e-Tiu(O-benzyl)-Tyr (23 mg, 25 μmol) was deprotected as described above to give the title compound in quantitative yield. UHPLC-MS (system 2): t _r =1.30 min, m/z (ES+) calculated 691.39 (M+H)+, found 691.48.

General method for amide condensation of isoleucine-tubuvaline(ester)-tubuphenylalanine tripeptides with (L)-N-methylpipecolic acid. Commercially available (L)-H-methylpipecolic acid (DMep) 36 (1.5-2 equivalents) was dissolved in anhydrous dimethylformamide (25-50 mM) and pre-activated with HATU (2 equivalents) and DIPEA (4 equivalents); the mixture was stirred for 10 minutes at room temperature under a nitrogen atmosphere. The activated acid was then added to 11e-Tiu(O-e(|)yr)-Tyr tripeptides 168-171; the reaction mixture was stirred at room temperature under a nitrogen atmosphere, and the progress of the reaction was monitored using UHPLC/MS data. Glacial acetic acid (14 equivalents) was then added after completion of the reaction and the product was purified by preparative HPLC.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-4-methyl-1 -((2-methylallyl)oxy)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (172). Intermediate 168 Ile-Tuv-Tup (55 mg, 84 μmol) was coupled to D-Mep 36 as described above to give 40 mg (62%) of the title compound. UHPLC-MS (system 1): t _r =1.27 min, m/z (ES+) calculated 780.48 (M+H)+, found 780.58.

DODU-Allyl 4-(2-((1R,3R)-1-(allyloxy)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido )pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (173). Intermediate 169 11e-Tiu(O-allyl)-Tyr (5 mg, 7.8 μmol) was coupled to D-Mep 36 as described above to give 6 mg (quantit) of the title compound. UHPLC-MS (system 2): t _r =1.28 min, m/z (ES+) calculated 766.46 (M+H)+, found 766.54.

(2S,4R)-Allyl 4-(2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido )-4-methyl-1-(prop-2-yn-1-yloxy)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (174). Intermediate 170 I1e-Tuv(O-propargyl)-Tup (18 mg, 28 μmol) was coupled to DMep 36 as described above to give 20 mg (93%) of the title compound. UHPLC-MS (system 2): t _r =1.23 min, m/z (ES+) calculated 764.44 (M+H)+, found 764.53.

(2S,4R)-Allyl 4-(2-((1R,3R)-1-(benzyloxy)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine -2-carboxamido)pentanamido)-4-methylpentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoate (175). Intermediate 171 Ι^-Τυν^^^^^Τ^ (5 mg, 7 µmol) was coupled to D-Mep 36 as described above to give 4.4 mg (75%) of the title compound. UHPLC-MS (system 1): t _r =1.42 min, m/z (ES+) calculated 816.48 (M+H)+, found 816.64.

General method for removing allyl ester from D-methylpipecolic acid-isoleucine-tubuvaline(ester)-tubuphenylalanine tubulysine intermediates. Intermediate (172-175) allyl ether-protected tubulisin ester was dissolved in anhydrous dichloromethane (20 mM) treated with tetrakis(triphenylphosphine)palladium (0.1 equiv.), triphenylphosphine (0.2 equivalents) and anhydrous pyrrolidine (8 equivalents) and the reaction mixture was stirred at ambient temperature under a nitrogen atmosphere. After UHPLC/MS data indicated conversion of the free acid to the product, the reaction mixture was quenched with glacial acetic acid (22 equivalents), diluted with acetonitrile and dimethylformamide, and then concentrated by rotary evaporation. The crude tubulisin ester was then purified by preparative HPLC.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanes

- 129 046150 to)-4-methyl-1-((2-methylallyl)oxy)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (176). Intermediate 172 allyl ether-protected tubulisin methyl allyl ether (19 mg, 24 μmol) was deprotected as described above to give 14 mg (93%) of tubulisin ester 176. UHPLC-MS(System 2): ^.=1 ,16 min, m/z (ES+) calculated 740.44 (M+H)+, found 740.54.

(2S,4R)-4-(2-((1R,3R)-1-(Allyloxy)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidin- 2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (177). Intermediate 173 allyl ether-protected tubulisin allyl ester (7 mg, 9 μmol) was deprotected as described above to give 6 mg (92%) tubulisin allyl ester 177. UHPLC-MS (System 1): tr=1 ,20 min, m/z (ES+) calculated 726.43 (M+H)+, found 726.33.

(2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido) -4-methyl-1-(prop-2-yn-1-yloxy)pentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (178). Allyl ether-protected tubulisin propargyl ester intermediate 174 (5.2 mg, 6.8 μmol) was deprotected as described above to yield 2.7 mg (55%) tubulisin propargyl ester 178. UHPLC-MS (system 2): t=1.09 min, m/z (ES+) calculated 724.41 (M+H)+, found 724.50.

(2S,4R)-4-(2-((1R,3R)-1-(Benzyloxy)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidin- 2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (179). Intermediate 175 allyl ether-protected tubulisin benzyl ester (5 mg, 6 μmol) was deprotected as described above to give 4.5 mg (96%) tubulisin benzyl ester 179. UHPLC-MS (System 1): tr =1.26 min, m/z (ES+) calculated 776.44 (M+H)+, found 776.58.

Scheme 17

ΒΟύ (2R)-1-(3-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((2S,3R,4S,5S,6S)- 6((allyloxy)carbonyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)1- (4-(((2R,4S)-5-(allyloxy)-4-methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1-(( 2methylallyl)oxy)pentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin1-ium (180). Into a flask containing H-Ile-Tuv(O-methylpropene)-Tup-OAlyl (168.24 mg, 31 µmol) as

- 130 046150 solids Fmoc-Gluc (Allyl) Q-Mep-OH (78.28 mg, 31 µmol) and HATU (18 mg, 45 µmol) were added, followed by THF (1.2 ml). NN-diisopropylethylamine (19 μL, 109 μmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then taken into DMSO and purified by preparative LC to give 180 (21 mg, 49%). Analytical UHPLC-MS (system 2): t _r =1.46 min, m/z (ES+) calculated 1410.69 (M) ⁺ , found 1410.84.

(2R)-1-(3-(3-Aminopropanamido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2Hpyran-2-yl)oxy )benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl )thiazol-2-yl)-4-methyl-1-((2-methylallyl)oxy)pentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2yl)carbamoyl)-1-methylpiperidine -1st (181). Fmoc-GlucQ-Tub(O-methylpropene)-OAlyl 180 (12 mg, 8 µmol) was deprotected as described above (see: General method for complete deprotection of Fmoc-GlucQ-Tub(OR)-OAlyl) yielding 181 (8.6 mg, 97%). Analytical UHPLC-MS (system 1): t _r =1.02 min, m/z (ES+) calculated 1108.56 (M) ⁺ , found 1108.69.

(2R)-1-(3-((S)-44-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-38,45-dioxo-2,5,8,11,14 ,17,20,23, 26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran -2-yl)oxy)benzyl)-2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1phenylpentane-2 -yl)carbamoyl)thiazol-2-yl)-4-methyl-1-((2-methylallyl)oxy)pentan-3-yl)(methyl)amino)-3methyl-1-oxopentan-2-yl)carbamoyl) -1-methylpiperidin-1-ium (182). H-GlucQ-Tub(O-methylpropene) 181 (12.5 mg, 11 μmol) was coupled to Fmoc-Lys(PEG12)-OSu 59 (14 mg, 13.5 μmol) as described above (see: General Method condensation of H-GlucQ-Tub(OR) with Fmoc-Lys(PEG12)-OSu) to give 182 (13 mg, 56%). Analytical UHPLC-MS (system 2): tr=1.32 min, m/z (ES+) calculated 2029.05 (M) ⁺ found 2029.24.

(2R)-1-(3-((8)-44-Amvno-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa- 39,46-diazanonatetracontanamido)-4-((^^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-(((2S, 3S)-1-(((1R,3R)-1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2yl)-4-methyl-1 -((2-methylallyl)oxy)pentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)1-methylpiperidin-1-ium (183). Fmoc-Lys(PEG12)-GlucQ-Tub(O-methylpropene) 182 (13 mg, 6 μmol) was deprotected as described above (see: General Method for Deprotecting Fmoc-Lys(PEG12)-GlucQ-Tub (OR)) to obtain 183 (11 mg, quant.). Analytical UHPLC-MS (system 2): t _r =1.04 min, m/z (ES+) calculated 1806.98 (M) ⁺ , found 1807.16.

(2R)-1-(3-((S)-44-((S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1yl)propanamido)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4-((^ ^^^^)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2(((2S,3S)-1-(((1R,3R)- 1-(4-(((2R,4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1((2-methylallyl)oxy)pentane-3 -yl)(methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (184). H-Lys(PEG12)-GlucQ-Tub(O-methylpropene) 183 (11.5 mg, 6.4 μmol) was coupled to mDPR-OPFP 44 (4.3 mg, 9.5 μmol) as described above, ( see: General method for condensing HLys(PEG12)-GlucQ-Tub(OR) with mDPR-OPFP) to give 184 (8.3 mg, 63%). Analytical UHPLC-MS (system 2): t _r =1.24 min, m/z (ES+) calculated 2073.07 (M) ⁺ , found 2073.25.

(2R)-1-(3-((S)-44-((S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido) -38,45dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamido)-4((^^^^^)-6- carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)-2-((^^)-1(((1R,3R)-1-(4-(((2R, 4S)-4-carboxy-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methyl-1-((2-methylallyl)oxy)pentan-3 -yl)(methyl)amino)- 3-methyl-1-oxopentan-2-yl)carbamoyl)-1-methylpiperidin-1-ium (185). mDPR(Boc)-Lys(PEG12)-GlucQ-Tub(O-methylpropene) 184 (8.3 mg, 4 μmol) was deprotected as described above (see: General Method for Deprotecting mDPR(Boc)Lys (PEG12)-GlucQ-Tub(OR)) to obtain 185 (7.4 mg, 94%). Analytical UHPLC-MS (system 2): t _r =1.05 min, m/z (ES+) calculated 1973.02 (M) ⁺ , found 1973.21.

Solid-phase synthesis of TubOEt analogues by replacing Tubulisin residues with Ile or D-Mep.

- 131 046150

Scheme 18

2-((1R,3R)-3-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-1-ethoxy-4-methylpentyl)thiazole-4-carboxylic acid (200) : Boc-MeN-TuvOEt-COOH (22.153 mg, 0.40 mmol) was placed in a vessel and dissolved in anhydrous DCM (4 ml). The solution was cooled to 0°C, then TFA (0.40 mL, 5.22 mmol) was added dropwise. After adding TFA, the reaction mixture was allowed to slowly warm to room temperature. After 4 h, complete removal of the Boc protecting group was observed. The reaction mixture was diluted with 5 ml of toluene and concentrated to dryness. The intermediate amine was used without further purification. Analytical UHPLC-MS (system 1): tr=0.86 min, m/z (ES+) calculated 287.1 (M+H) found 287.2. The trifluoroacetic acid amine salt intermediate was dissolved in 1,4-dioxane (2.0 mL), then water (2.0 mL) and DIPEA (0.41 mL, 2.4 mmol) were added. A solution of FmocCl (122 mg, 0.47 mmol) in 1,4-dioxane was then added dropwise. After 8 h, the reaction mixture was diluted with 10% DMSO in MeCN and purified by preparative HPLC to give FmocMeN-TuvOEt-COOH (200.88 mg, 44%). Analytical UHPLC-MS (system 1): tr=2.35 min, m/z (ES+) calculated 509.2 (M+H) ⁺ found 509.2; calculated 531.2 (M+Na)+, found 531.1.

(2S,4R)-4-(2-((1R,3R)-3-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)-amino)-1-ethoxy-4-methylpentyl )thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (202): FmocMeN-TuvOEt-COOH (200, 80 mg, 0.16 mmol) was dissolved in anhydrous DMF (0.8 ml) and then the solution was added HATU (60 mg, 0.16 mmol) in anhydrous DMF (0.8 ml), then DIPEA (0.21 ml, 1.2 mmol). After 5 min, (2S,4R)-4-amino-2-methyl-5-phenylpentanoic acid hydrochloride salt (201, HCl-TupCOOH, 96 mg, 0.39 mmol) was added in one portion. The reaction mixture was stirred for 5 h at room temperature, then diluted with 10% DMSO in MeCN and purified by preparative HPLC to give FmocMeNTuvOEt-Tup-COOH (202.95 mg, 87%). Analytical UHPLC-MS (system 1): t _r =2.40 min, m/z (ES+) calculated 698.3(M+H)+, found 698.3.

General Chlorotrityl Resin Loading Procedure: Six SynPhase™ Series D polyacrylamide resins functionalized with trityl alcohol linker and nominal loading of 8 µM (Mimotopes, Victoria, Australia) were placed in a dry vial and converted to trityl chloride linker by treatment with 3 mL of 10% AcCl solution. in anhydrous DCM solution. After 3 hours of gentle stirring at room temperature, the solution was removed and the resins were washed 3 times with 5 ml of anhydrous DCM. A solution of 202 (57 mg, 81.7 µmol) and DIPEA (57 µl, 327 µmol) in 3 ml anhydrous DCM was then added to the resins. After 5 hours of gentle stirring at room temperature, the solution was separated from the resins and the resins were washed 3 times with 5 ml DMF, 3 times with 5 ml DCM, then dried overnight under a stream of argon to obtain FmocMeN-TuvOEt-Tup loaded resins.

General procedure for obtaining Ile replacement analogues TubOEt (203-215): step 1: removal of Fmoc. The resin loaded with FmocMeN-TuvOEt-Tup as prepared above was treated with 1 ml of 20% piperidine in DMF for 30 min. The solution was removed and then the resin was washed 3 times with 1.5 ml DMF. Stage 2. Amino acid addition. In a separate vial, combine 0.5 mL of a 240 mM solution of the appropriate Fmoc-protected amino acid (FmocHN-Xxx-COOH) in DMF and 0.5 mL of a 200 mM solution of HATU in DMF, and add DIPEA (45 μL, 0.24 mmol). After 2 minutes, the activated amino acid solution was added to the resin and stirred gently for 2 hours. The resin was treated a second time with an activated Fmoc amino acid solution for 2 hours. The solution was then removed and the resin was washed 3 times

- 132 046150

1.5 ml DMF. Stage 3. Removal of Fmoc. The resin loaded with FmocHNXxx-TuvOEt-Tup was treated with 1 ml of 20% piperidine in DMF for 30 min. The solution was removed and then the resin was washed 3 times with 1.5 ml of DMF. Stage 4. Attachment of Mep. In a separate vial, combine 0.5 ml of a 240 mM solution of N)-1-methylpiperidine-2-carboxylic acid (Mep) in DMF and 0.5 ml of a 200 mM solution of HATU in DMF and add DIPEA (45 μl, 0.24 mmol) . After 2 minutes, the activated Mep solution was added to the resin and stirred gently for 1.5 hours. The solution was removed and the resin was then washed with 3 times 1.5 ml DMF and 3 times 1.5 ml DCM. Stage 5. Cleavage from resin. Each resin was treated for 90 min with a solution of 20% HFIP in DCM. The solution was removed and the resin was washed with an additional 0.5 ml of 20% HFIP in DCM. The combined solutions were filtered through a 45 micron syringe filter and concentrated to dryness to give analogues 203-215 (as shown immediately below). Analogues were obtained with purity &gt; 85% based on UPLC-MS analysis and chromatography with diode array detection.

(2S,4R)-4-(2-(( 1R,3R)-1 -Ethoxy-3 -((S)-3 -ethyl-K-methyl-2-((K)-1 -methylpiperidine-2- carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (203): The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-ethylpentanoic acid in step 2 to give 203 (1.17 mg). Analytical UHPLC-MS (system 1): tr=1.77 min, m/z (ES+) calculated 728.4 (M+H) ⁺ found 728.4.

(2S,4R)-4-(2-((1R,3R)-3-((S)-2-Cyclopentyl-N-methyl-2-((R)-1-methylpiperidine-2-carboxamido)acetamido) -1-ethoxy-4-methylpentyl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (204): The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopentylacetic acid in step 2 to give 204 (1.71 mg). Analytical UHPLC-MS (system 1): tr=1.72 min, m/z (ES+) calculated 726.4 (M+H)+, found 726.4.

(2S,4R)-4-(2-((1R,3R)-3-((S)-2-Cyclohexyl-N-methyl-2-((R)-1-methylpiperidine-2-carboxamido)acetamido) -1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (205): The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclohexylacetic acid in step 2 to give 205 (1.59 mg). Analytical UHPLC-MS (system 1): tr=1.77 min, m/z (ES+) calculated 740.4 (M+H)+, found 740.4.

(2S,4R)-4-(2-(( 1R,3R)-1 -Ethoxy-3 -((2S,3 S)-3-methoxy-N-methyl-2-(^)-1 -methylpiperidin- 2-Carboxamido)butanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (206): The TubOEt analogue at the Ile position was prepared according to the general method described above using N-(( (9H-fluoren-9-yl)methoxy)carbonyl)-O-methyl-L-allotreonine in step 2 to give 206 (1.50 mg). Analytical UHPLC-MS (system 1): tr=1.58 min, m/z (ES+) calculated 716.4 (M+H)+, found 716.4.

(2S,4R)-4-(2-((1R,3R)-1-Ethoxy-3-((2S,3R)-3-methoxy-N-methyl-2-((R)-1-methylpiperidin- 2-Carboxamido)butanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (207): The TubOEt analogue at the Ile position was prepared according to the general method described above, using

- 133 046150 Zuya No. (((9H-fluoren-9-yl)methoxy)carbonyl)-O-methyl-E-threonine in step 2, giving 207 (1.44 mg). Analytical UHPLC-MS (system 1): t _r =1.59 min, m/z (ES+) calculated 716.4 (M+H) ⁺ found 716.4. (2S,4R)-4-(2-((1R,3R)-3-((S)-2-(2,3-dihydro-1H-inden-2-yl)-N-methyl-2-( (R)-1-methylpiperidine-2carboxamido)acetamido)-1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (208). The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(2,3-dihydro- 1H-inden-2yl)acetic acid in step 2 to give 208 (1.15 mg). Analytical UHPLC-MS (system 1): t _r =1.84 min, m/z (ES+) calculated 774.4 (M+H)+, found 774.4.

(2S,4R)-4-(2-((1R,3R)-3 -((2S,3R)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido) -1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (209). The Ile analogue of TubOEt was prepared according to the general method described above using (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alloisoleucine in step 2 to give 209 (1.33 mg). Analytical UHPLC-MS (system 1): tr=1.78 min, m/z (ES+) calculated 714.4 (M+H)+, found 714.4.

(2S,4R)-4-(2-((1R,3R)-1-Ethoxy-4-methyl-3-(^)-N-methyl-2-(N)-1-methylpiperidine-2-carboxamido) 2-(tetrahydro-2H-pyran-4-yl)acetamido)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (210): The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(tetrahydro-2H-pyran-4yl)acetic acid in step 2 to give 210 (1. 07 mg). Analytical UHPLC-MS (system 1): t _r =1.20 min, m/z (ES+) calculated 742.4 (M+H)+, found 742.4.

(2S,4R)-4-(2-((1R,3R)-1-Ethoxy-3-(2-(3-fluorobicyclo[1,1,1]pentan-1-yl)-N-methyl-2 -((R)-1-methylpiperidine-2-carboxamido)acetamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (211): The TubOEt analogue at the Ile position was prepared according to the general in the manner described above using racemic 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(3-fluorobicyclo[1,1,1]pentan-1-yl)acetic acid on step 2 to obtain 211 (2.69 mg) as a mixture of epimers. Analytical UHPLC-MS (system 1): Epimer 1: tr=1.67 min, m/z (ES+) calculated 742.4 (M+H)+, found 742.3; Epimer 2: t _r =1.83 min, m/z (ES+) calculated 742.4 (M+H)+, found 742.3.

(2S,4R)-4-(2-((1R,3R)-3 -(^)-3-Cyclopropyl-N-methyl-2-(N)-1-methylpiperidine-2-carboxamido)propanamido)-1 -ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (212): The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2-(( ((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-cyclopropylpropanoic acid in step 2 to give 212 (3.21 mg). Analytical UHPLC-MS (system 1): t _r =1.66 min, m/z (ES+) calculated 712.4 (M+H)+, found 712.3.

(2S,4R)-4-(2-((1R,3R)-3 -(^)-3-Cyclohexyl-N-methyl-2-(N)-1-methylpiperidine-2-carboxamido)propanamido)-1 -ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (213): The TubOEt analogue at the Ile position was prepared according to the general method described above using (S)-2-(( ((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-cyclohexylpropanoic acid in step 2 to give 213 (1.72 mg). Analytical UHPLC-MS (system 1): t _r =1.87 min, m/z (ES+) calculated 754.5 (M+H)+, found 754.4.

(2S,4R)-4-(2-((1R,3R)-3 -(^^)-3-(tert-Butoxy)-N-methyl-2-((K)-1-methylpiperidin-2- carboxamido)butanamido)-1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (214): The TubOEt analogue at the Ile position was prepared according to the general method described above using N- (((9H-fluoren-9-yl)methoxy)carbonyl)-O-(tert-butyl)-L-allothreonine in step 2 to give 214 (2.31 mg). Analytical UHPLC-MS (system 1): t _r =1.77 min, m/z (ES+) calculated 758.5 (M+H)+, found 758.4.

(2S,4R)-4-(2-((1R,3R)-3 -((2S,3R)-3-(tert-Butoxy)-N-methyl-2-((R)-1-methylpiperidin- 2-Carboxamido)butanamido)-1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (215): The TubOEt analogue at the Ile position was prepared according to the general method described above using N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(tert-butyl)-L-mthreonine in step 2 to give 215 (2.35 mg). Analytical UHPLC-MS (system 1): t _r =1.76 min, m/z (ES+) calculated 758.5 (M+H)+, found 758.4.

General procedure for preparing D-Mep replacement T-Mer analogues: Step 1: Fmoc removal. The resin loaded with Fmoc-MeN-TuvOEt-Tup as prepared above was treated with 1 ml of 20% piperidine in DMF for 30 min. The solution was removed and then the resin was washed 3 times with 1.5 ml DMF. Stage 2. Amino acid addition. In a separate vial, 0.5 ml of a 240 mM solution of FmocHN-IleCOOH in DMF and 0.5 ml of a 200 mM solution of HATU in DMF were combined and DIPEA (45 μL, 0.24 mmol) was added. After 2 minutes, the activated Fmoc-Ile solution was added to the resin and stirred gently for 2 hours. The resin was treated a second time with a solution of the activated Fmoc-Ile amino acid for 2 hours. The solution was then removed and the resin was washed 3 times with 1.5 ml of DMF. Stage 3. Removal of Fmoc. The resin loaded with FmocHN-Ile-TuvOEt-Tup was treated with 1 ml of 20% piperidine in DMF for 30 min. The solution was removed and then the resin was washed 3 times with 1.5 ml of DMF. Stage 4. Attaching analogue measures. In a separate vial, 0.5 ml of a 240 mM solution of Mer analog in DMF and 0.5 ml of a 200 mM solution of HATU in DMF were combined and DIPEA (45 μl, 0.24 mmol) was added. After 2 minutes, activated Mer with analogue

- 134 046150 the solution was added to the resin and stirred gently for 2 hours. The resin was treated a second time with an activated Mer analogue solution for 2 hours. The solution was removed and the resin was washed 3 times with 1.5 ml of DMF, and then washed 3 times with 1 .5 ml DCM. Stage 5. Cleavage from resin. Each resin was treated for 90 min with a 20% solution of HFIP in DCM. The solution was removed and the resin was washed with an additional 0.5 ml of 20% HFIP in DCM. The combined solutions were filtered through a 45 micron syringe filter and concentrated to dryness to give analogues 216-221 (as shown immediately below). The analogues were obtained with a purity of >85% based on LC-MS analysis and chromatography with diode array detection.

General procedure for N-methylation of D-Mep analogues (224-227). Commercially available analogues of pipecolic acid (ref. 222, 0.30 mmol) were dissolved in a mixture of 0.4 ml of methanol and a solution of 37% formaldehyde in water (0.23 ml, 3.0 mmol). Pd/C (10 wt%, 10 mg) was added, the flask was equipped with a balloon filled with hydrogen, and the reaction mixture was stirred overnight. Pd was removed by filtration through a celite pad and concentrated by rotary evaporation. The resulting residue was concentrated from 2.0 ml of methanol 5 more times to obtain the desired N-methylated pipecolic acid analogues 224-227.

! ] Formalin >' ] >>y ^oh ----- sA/* ¹ n n n ₂ , MeOH I Η

224 225 226 227

5,5-Difluoro-1-methylpiperidine-2-carboxylic acid (224): 5,5-difluoropiperidine-2-carboxylic acid (50 mg, 0.30 mmol) was methylated using the method described above to give 5,5 -difluoro-1-methylpiperidine-2-carboxylic acid (224.47 mg, 87%). Analytical UHPLC-MS (system 1): t _r =0.32 min, m/z (ES+) calculated 18 0.1 (M+H) ⁺ , found 180.0.

2-Methyl-2-azabicyclo[3.1.1]heptane-1-carboxylic acid (225): 2-Azabicyclo[3.1.1]heptane-1-carboxylic acid hydrochloride (53 mg, 0.30 mmol) was methylated using the method described above to give 2-methyl-2-azabicyclo[3.1.1]heptane-1-carboxylic acid (225, 60 mg, quantitative yield). Analytical UHPLC-MS (system 1): t _r =0.39 min, m/z (ES+) calculated 156.1 (M+H) ⁺ , found 156.1.

(1S,3R,4S)-2-Methyl-2-azabicyclo[2,2,2]octane-3-carboxylic acid (226): (1S,3R,4S)-2-azabicyclo[2,2,2] octane-3-carboxylic acid (47 mg, 0.30 mmol) was methylated using the method described above to give (1S,3R,4S)-2-methyl-2-azabicyclo[2,2,2]octane-3 -carboxylic acid (226.53 mg, quantitative yield). Analytical UHPLC-MS (system 1): t _r =0.40 min, m/z (ES+) calculated 170.1 (M+H) ⁺ , found 170.0.

(1S,3R,4R)-2-Methyl-2-azabicyclo[2,2,1]heptane-3-carboxylic acid (227): (^,3E,4K)-2-(tert-butoxycarbonyl)-2-azabicyclo[ 2,2,1]heptane-3-carboxylic acid (73 mg, 0.30 mmol) was dissolved in 1.0 ml DCM, then 0.30 ml TFA was added and the reaction mixture was stirred for 1.5 h to remove the protecting group Boc. The reaction mixture was diluted with 3 ml of toluene and concentrated to dryness to give (1S,3R,4R)-2-azabicyclo[2,2,1]heptane-3-carboxylic acid. Analytical UHPLC-MS (system 1): tr=0.85 min, m/z (ES+) calculated 142.1 (M+H) ⁺ found 142.1. The intermediate (1S,3R,4R)-2-azabicyclo[2,2,1]heptane-3-carboxylic acid was then methylated using the method described above to give the trifluoroacetic acid salt (1S,3R,4R)-2- methyl-2azabicyclo[2,2,1]heptane-3-carboxylic acid (227.66 mg, 83%). Analytical UHPLC-MS (system 1): t _r =0.89 min, m/z (ES+) calculated 156.1 (M+H) ⁺ found 156.1.

^AA)-4-(2-((^^)-3-(^^)-2-(5,5-Difluoro-1-methylpiperidine-2-carboxamido)-H3-dimethylpentanamido)-1-ethoxy-4 -methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (216). The Mer analogue of TubOEt was prepared according to the general method described above using racemic 5,5-difluoro-1-methylpiperidine-2-carboxylic acid (224, prepared above)

- 135 046150 in step 4, yielding 216 (3.05 mg) as a mixture of epimers. Analytical UHPLC-MS (system 1): Epimer 1: t _r =2.06 min, m/z (ES+) calculated 750.4 (M+H) ⁺ found 750.3; Epimer 2: t _r =2.10 min, m/z (ES+) calculated 750.4 (M+H)+, found 750.4.

(I.4P)-4-(2-((1P.3P)-3-((II)-K3-Dimethyl-2-(2-methyl-2-azabicyclo[3.1.1|heptane-1-carboxamido) pentanamido)-1-ethoxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (217): The Mer analogue of TubOEt was prepared according to the general method described above using 2-methyl- 2-azabicyclo[3.1.1]heptane-1-carboxylic acid (225, obtained above) in step 4 to give 217 (4.44 mg). Analytical UHPLC-MS (system 1): tr=1.69 min, m/z (ES+) calculated 726.4 (M+H) ⁺ found 726.3.

^^)-4-(2-((^^)-3-(^^)-Н,3-Dimethyl-2-((^^^)-2-methyl-2-azabicyclo[2,2,2 ]octane-3-carboxamido)pentanamido)-1-ethoxy-4-methyl-pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (218. The TubOEt analogue at the Mer position was prepared according to the general method described above using (1S,3R,4S)-2-methyl-2-azabicyclo[2,2,2]octane-3-carboxylic acid (226, obtained above) in step 4 to give 218 (3.35 mg). Analytical UHPLC-MS (system 1): t _r =1.74 min, m/z (ES+) calculated 740.4 (M+H)+, found 740.6.

^^)-4-(2-((^^)-3-(^^)-H3-Dimethyl-2-((^^^)-2-methyl-2-azabicyclo[2,2,1]heptane -3carboxamido)pentanamido)-1-ethoxy-4-methylpentyl)-thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (219). The Mer analogue of TubOEt was prepared according to the general method described above using (1S,3R,4R)-2-methyl-2-azabicyclo[2,2,1]heptane-3-carboxylic acid (227, prepared above) in step 4 to obtain 219 (2.82 mg). Analytical UHPLC-MS (system 1): t _r =1.70 min, m/z (ES+) calculated 726.4 (M+H)+, found 726.4.

(2S,4R)-4-(2-((3R,6S,9R,11R)-6-((S)-Secon-butyl)-3,9-diisopropyl-2,8-dimethyl-4,7- dioxo-12-oxa2,5,8-triazatetradecane-11-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (220). The Mer analogue of TubOEt was prepared according to the general method described above using N,Ndimethyl-O-valine in step 4 to give 220 (3.51 mg). Analytical UHPLC-MS (system 1): t _r =1.72 min, m/z (ES+) calculated 716.4 (M+H)+, found 716.3.

^^)-4-(2-((^^)-3-(^^)-2-(^)-1,2-Dimethylpyrrolidine-2-carboxamido)-N,3-dimethylpentanamido)-1-ethoxy- 4-Methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (221): The Mer analogue of TubOEt was prepared according to the general method described above using ^)-1,2-dimethylpyrrolidine-2- carboxylic acid in step 4 to give 221 (2.90 mg). Analytical UHPLC-MS (system 1): t _r =1.67 min, m/z (ES+) calculated 714.4 (M+H)+, found 714.4.

In vitro assays. Cells cultured in log growth phase were seeded for 24 h in 96-well plates containing 150 μl of RPMI 1640 supplemented with 20% FBS. Serial dilutions of drug-antibody conjugates in cell culture medium were prepared at 4 working concentrations; 50 μl of each dilution was added to 96-well plates. After addition of ADC, cells were incubated with test drugs for 4 days at 37°C. After 96 h, growth inhibition was assessed using CellTiter-Glo® (Promega, Madison, WI) and luminescence was measured on a plate reader. The IC ₅₀ value, determined in triplicate, is defined herein as the concentration that results in a 50% reduction in cell growth compared to untreated controls.

In vivo xenograft models. All experiments were conducted in accordance with the requirements of the Institutional Animal Care and Use Committee, which is fully supervised by the Association for the Assessment and Accreditation of Laboratory Animal Care. Efficacy experiments were conducted in L540cy Hodgkin lymphoma, Karpas:KarpasBVR anaplastic large cell lymphoma, MDR+DELBVR anaplastic large cell lymphoma, and MDR+786-O xenograft model of renal cell carcinoma. Tumor cells were implanted subcutaneously in SCID mice with immunodeficiency. Tumor cells in the form of a cell suspension were implanted subcutaneously into SCID mice with immunodeficiency. After tumor engraftment, mice were randomly assigned to study groups when the average tumor volume reached approximately 100 mm ³ . ADCs or controls were administered once via intraperitoneal injection. Tumor volume as a function of time was determined using the formula (LxW2)/2. Animals were sacrificed when tumor volumes reached 1000 mm ³ . Mice showing long-term regression were sacrificed approximately 100 days after implantation.

ADC Pharmacokinetic (PK) experiments. Pharmacokinetic (PK) experiments were performed using radiolabeled antibody or ADC. Preparations tested for PK were labeled with a radioactive isotope using the following procedure. To a solution of the antibody or ADC in PBS supplemented with a further 50 mM potassium phosphate (pH 8.0) and 50 mM sodium chloride, was added 55 μCi of N10 succinimidyl propionate, [propionate^^^^- (Moravek Biochemicals, Cat. No: MT 919, 80 Ci/mmol, 1 mCi/ml, 9:1 hexane:ethyl acetate solution) per mg of antibody or ADC. The resulting mixture was shaken and left at room temperature for 2 hours. The mixture was centrifuged at 4000 g for 5 minutes and the lower aqueous layer was removed and separated on Amicon Ultra-15 centrifugal filter units (Milli

- 136 046150 pore, Cat. No: UFC903024, 30 kDa, MWCO). Unconjugated radioactivity was removed by 4 rounds of dilution and centrifugation at 4000xg. The resulting products were filtered through sterile Ultrafree-MC 0.22 μm centrifugal filter units (Millipore, Cat. No: UFC30GV0S) and the final concentration of antibody or ADC was measured spectrophotometrically. The specific activity (μCi/mg) of each product was determined using liquid scintillation detection.

The pharmacokinetic properties of the unconjugated antibody or ADC have been studied in several rodent models. In each experiment, 1–3 mg of radiolabeled antibody or ADC per kg of animal weight was administered via the tail vein. Each study drug was dosed once to the animals and the experiment was repeated. Blood was collected into K2EDTA tubes via saphenous vein or cardiac puncture for terminal blood at various time points. Plasma was isolated by centrifugation for 10 min at 10,000xg. 10-20 μl of plasma sample from each time point was added to 4 ml of Ecoscint-A liquid scintillation cocktail (National Diagnostics) and total radioactivity was measured by 5 liquid scintillation counts. The resulting disintegration per minute values were converted to μCi, and the specific activity of the radiolabeled study drugs was used to calculate the concentration of antibody or ADC remaining in plasma at each time point.

In vitro assays - drug compounds without tubulisin. Cells were treated for 96 h with tubulysin ester analogs (40-42) or tubulisin M (7) and then assessed for viability as described in Methods.

_____________________________________________________________Table 1

connection medicinal Karpas299, L540cy, L428, HL60, HL60/RV, substance ALCL H.L. MDR+HL AML MDR+ ML 7 TubM 0.17 0.14 0, 11 0.3 2.2 40 TubOMe 0.19 0.12 0.5 0.13 29 41 TubOEt 0.05 0.05 0.14 0.05 5 42 TubOPr 0.15 0.13 0.4 0.17 14

IC ₅₀ values (nM) in table. 1 show that all four study drugs were highly effective for each cell line. Tubulysin ethyl ester (41) tends to be more active than the methium (40) and propyl (42) analogues. Ethyl ester and tubulisin M maintained the highest level of activity against MDR+L428 and HL60/RV cell lines.

table 2

connection medicinal substance Karpas299, ALCL L540cy,HL L428, MDR+HL HL60, AML HL60/RV, MDR+AML 7 TubM 0.04 0.08 0.04 0.15 1 133 Tubpropionate 0.02 0.06 0.04 0.1 1 135 Tubizobutyrate 0.02 0, 1 0, 12 0.1 6 134 Tub-butyrate 0.011 0.04 0.05 0.08 3 136 Tubisovalerate 0.02 0.06 0, 11 0.04 6 137 Tub-3,3-dimethylbutyra T 0.18 0.7 0.93 0.7 47 176 Tub-methyl(propen-2yl) 0, 12 0.06 0, 12 0.07 6 177 Tub allyl ether 0.02 0, 12 0.15 0.1 9 178 Tub propargyl ester 0.05 0.17 0.41 0.2 27 179 Tub benzyl ether 0.08 0.4 0.57 0.4 24

- 137 046150

Tubulysin ester analogues (133-137) and unsaturated ether analogues (176-179) were tested in vitro for activity against tubulisin M (7) after 96 hours of exposure. As shown by the IC50 values (nM) in the table. 2, tubulisin esters 133-136 acted comparable to tubulisin M with similar efficacy in cell lines. The most hindered, bulky analog of tubulisin (137) showed reduced activity compared to tubulisin M, with potency losses ranging from 5- to 47-fold compared to tubulisin M.

The unsaturated ethers of tubulisin 176 and 177 showed comparable potency compared to the parent tubulisin 7. In contrast, propargyl (178) and benzyl (179) esters of tubulisin were weaker in potency compared to tubulisin 7.

A series of analogues of tubulisin ethyl ester compounds TubOEt (41) have been synthesized on solid-phase resins that support ethyl ester displacement by changing the residue at either the Ile (203-215) or Mer (216-221) position. After separation from the solid phase, analogues with a purity of >85% were obtained and were studied without further purification. The compounds were tested in vitro for activity against a TubOEt sample synthesized in parallel on resin (41-resin), after 96 hours of exposure, with IC ₅₀ values expressed in nM (Table 3). Although attenuation of activity relative to the 41-resin was observed in many cases, some well-tolerated replacements were found. At the Ile position, the cyclic substituents R ⁵ cyclopentyl (204) and cyclohexyl (205) give effective compounds; however, the tetrahydropyranyl analogue (210) was inactive against all cell lines tested. In addition, when replacing Ile with allo-Ile (209), a 25-40-fold loss of efficiency is observed. Although this stereochemical preference is not observed when Ile is replaced by Omethylthreonine (207) or O -methylallotreonine (206), bulkier substituents result in greater differentiation. In particular, substitution with O-tert-butylthreonine (215) provides an analogue that is significantly more potent than the corresponding O-tert-butylallotreonine compound 216. Substitution of the Mer residue is generally less well tolerated. The most effective compounds obtained in this series (217 and 221) have substitution at the 4- and 2-position of the piperidine or pyrrolidine-2-carboxylic acid core, respectively. Substitution at the 5-position (216) or 6-position (218 and 219) resulted in a decrease in the observed cytotoxicity, especially in the HL60/RV cell line.

Table 3

connect tion Replaced remainder ok HepG2, nss L540cy,HL Ramos, NHL U266 MM HL60, AML HL60/RV, MDR+AML 41lantern N.A. 2 0.3 0.3 0.3 4 94 203 Not 8 1 0.4 0.5 8 604 204 Not 2 0.4 0.2 0, 1 1 40 205 Not 7 1 0.5 0.5 2 177 206 Not 41 6 2 3 16 >1000 207 Not 46 6 2 2 9 >1000 208 Not 250 58 44 40 221 >1000 209 Not 50 12 7 8 15 >1000 210 Not >1000 >1000 >1000 >1000 >1000 >1000 211 Not 14 2 1 2 5 224 212 Not 19 2 1 1 8 810 213 Not 70 20 10 9 42 >1000 214 Not 91 thirty 20 22 190 >1000 215 Not 1 0.2 0.2 0, 1 1 33 216 Mer 58 12 10 9 61 >1000 217 Mer 2 1 0.4 0.4 4 244 218 Mer 9 5 6 7 24 >1000 219 Mer 19 6 8 8 25 >1000 220 Mer 41 13 10 10 299 >1000 221 Mer 4 1 1 2 12 522

In vitro assays - tubulisin ADC. ADCs were prepared by complete reduction of interchain disulfides to identify 8-conjugated cysteines/antibodies that were alkylated via Michael addition with maleimide-containing drug compounds with a linker. Anti-CD30 conjugates containing quaternized tubulisin ethers with and without a PEG12 side chain were compared with their tubulisin M analogues. Cells were treated with cAC10 (anti-CD30) conjugates loaded at a concentration of 8 drug/Ab for 96 h and then assessed for viability. IC ₅₀ values (ng/ml) are given in table. 4.

Table 4

SASY ADC DAR 8 lec. vvolinker description Karpas299, ALCL L540cy,HL L428, MDR+HL DEL, ALCL DEL/BVR, MDR+ALCL 82 glucQ-TubM 0.6 4 0.5 2 3 56 glucQTubOMe 3 10 >1000 2 20 57 glucQTubOEt 1 5 4 2 5 58 glucQTubOPr 2 6 117 2 10 99 PEG12glucQ-TubM 0.3 2 0.5 1 2 66 PEG12glucQTubOEt 1 5 6 2 6 67 PEG12glucQTubOPr 4 10 >1000 2 12

The conjugate containing tubulisin ethyl ester linker 57 was consistently more effective than the methyl (56) or propyl (58) analogues. With the exception of L428, tubulisin ethyl ester linker 57 acted similarly to tubulisin M analogue 82. The presence of a PEG12 side chain in the linker had minimal effect on the activity of the conjugate. All ADCs were inactive (no effect at 1000 ng/ml) in the CD30-negative Ramos NHL cell line, indicating a high degree of immunological specificity.

Table 5

sACYA ADCs 8 drug substance/mAb drug substance linker MDPR- Linker- TubM L540cy,HL L428 MDR+ H.L. DEL, ALCL DEL/B VR, MDR+ ALCL NerZV CD30- HCC 15 ValAlaPABQ 2 1 0.3 4 >1000 82 glucQ 1 0.4 0.3 4 >1000 91 ValGluPABQ 1 0.3 0, 1 2 >1000 95 PEG12- ValGluPABQ 0.8 0.2 0, 1 2 >1000

Several hydrophilic linker constructs incorporating quaternized tubulisin M have been prepared and evaluated in vitro; the results are shown in table. 5. Data (IC ₅₀ values in ng/ml) indicate that conjugates derived from drug-linker compounds having quaternized tubulisin M linked through the hydrophilic ValGlu dipeptide with (95) or without (91) a PEG12 side chain or bound via a hydrophilic glucuronide (82), provide ADCs that are equally effective compared to ValAla (15). All conjugates demonstrated a high degree of immunological specificity: IC ₅₀ s &gt; 1000 ng/ml on antigen-negative Hep3B hepatocellular carcinoma cells.

In vivo xenograft models—comparison of dipeptide- and glucuronide-linked tubulisins. Quaternary amine-linked tubulysin M conjugated to a dipeptide linker ValAla

- 139 046150 (15) and glucuronide (82), were compared in a CD30+L540cy Hodgkin lymphoma xenograft model with tubulisin ethyl ester bound to glucuronide (57). A glucuronide drug compound with a linker has been shown to have improved physicochemical and pharmacokinetic conjugate properties compared to dipeptide linkers for ADC payloads (Bioconjugate Chem., 2006, 17, 831-840; Nature Biotech, 2014, 32, 1059 -1062). Conjugates were loaded at 4 drug/mAb to minimize the effects of ADC PK. The research results are presented in Fig. 1. Tumor-bearing mice were administered a single dose of study drug intraperitoneally once the average tumor volume reached 100 mm ³ on day 7. Significantly greater conjugate activity was observed when tubulisin M (7) was conjugated in quaternized form as a glucuronide to cAC10-82. In mice treated with a single dose of 0.6 mg/kg cAC10-82, 5/5 achieved long-term complete regression. In contrast, in the study group treated with a single dose of 0.6 mg/kg ValAla dipeptide conjugate, cAC10-15, there was one cure, while the remaining mice showed a temporary delay in tumor growth. The higher dose of 2 mg/kg dipeptide conjugate was also inferior to the glucuronide, with only 2/5 mice cured at day 78. Thus, the glucuronide conjugate bearing tubulisin 7 was more than 3-fold more active than the corresponding Val-Ala dipeptide control. Likewise, tubulisin ethyl ester bound to glucuronide (41) in the form of cAC10-57 conjugate was also highly active. A single dose of 0.6 mg/kg cAC10-57 caused 5/5 long-term complete regression.

In Vivo Xenograft Models - Effect of PEGylation with DAR 8 Conjugates We recently reported that the addition of a PEG side chain to the drug compound glucuronide monomethyl auristatin E with a linker provides improved pharmacological properties of ADCs with 8-drug/mAb-loaded ADCs (Nature Biotech, 2014, 32 , 1059-1062). The side chain of PEG12 was incorporated into a drug moiety based on the quaternary amine ethyl ester and propyl ester of tubulisin with a linker to produce the conjugates AC10-66 and c-AC10-67, respectively. Anti-CD30 cAC10 conjugates loaded at 8 drug/mAb concentrations were prepared and evaluated against non-PEGylated ethyl (cAC10-57) and propyl (cAC10-58) ester analogues in the L540cy xenograft model. The results are shown in Fig. 2. For ethyl and propyl ester glucuronide constructs, inclusion of PEG12 resulted in enhanced antitumor activity. For ethyl ester, the non-PEGylated cAC10-57 conjugate delayed tumor growth in mice treated with a single dose of 0.5 mg/kg; whereas the PEGylated variant cAC10-66 provided cure in 5/5 mice at the same dose of antibody. For propyl ether, non-PEGylated cAC10-58 conjugate delayed tumor growth with growth at approximately day 40 in mice treated with a single dose of 0.5 mg/kg; whereas the PEGylated variant cAC10-67 further delayed growth until around day 60.

A PEGylated version of glucuronide-tubulisin M, referred to as linker 99, was also studied in the CD30 + L540cy xenograft as a DAR 8 cAC10 conjugate. As stated above, tumor-bearing mice were administered a single dose of 0.5 mg/kg study drug intraperitoneally once the average tumor volume reached 100 mm ³ on day 8. Similar to the PEGylated ethyl ester construct (cAC10-66), the PEGylated tubulisin M conjugate cAC10-99 caused a 5/5 cure in mice treated with 0.5 mg/kg.

A subset of drug-antibody conjugates were evaluated at lower doses in the L540cy xenograft model. Anti-CD30 conjugates carrying PEGylated drug moieties with a linker containing quaternized tubulisin M (cAC10-99) or quaternized tubulisin ethyl ester (cAC10-66) were conjugated at a concentration of 8 drugs/mAb. Similarly, anti-CD30 conjugates carrying non-PEGylated linker glucuronide drug moieties containing quaternized tubulisin M (cAC10-82) or quaternized tubulisin ethyl ester (cAC10-57), or a Val-Ala dipeptide variant containing quaternized tubulisin M, have been prepared. (cAC10-15) in 4drug/mAb. Tumor-bearing mice were administered a single dose of each conjugate at 0.15 or 0.3 mg/kg after tumor size reached approximately 100 mm ³ . The results are shown in Fig. 8. At a lower dose of 0.15 mg/kg, a delay in tumor growth was observed for all conjugates, with the longest delay observed in mice treated with a conjugate (cAC10-66) carrying PEGylated drug moieties with a linker containing quaternized tubulisin ethyl ester in quaternized medicinal units. At a dose of 0.3 mg/kg, long-term complete regression was observed in 2/6 mice treated with an ADC carrying non-PEGylated glucuronide drug moieties with a linker containing quaternized tubulisin ethyl ester (cAC10-57) in the drug units, and in 3/ 6 mice treated with conjugates carrying either PEGylated glucuronide drug linker moieties containing quaternized tubulisin M (cAC10-99) or quaternized tubulisin ethyl ester (cAC1066) in the drug units.

In vivo xenograft models - demonstration of the bystander effect. PEGylated constipates

- 140 046150 hands were studied in the xenograft model Karpas:KarpasBVR bystander activity. Equal numbers of CD30+ Karpas299 and CD30 ⁻ KarpasBVR cells were injected subcutaneously to establish a tumor mass with a heterogeneous population of antigen-positive and negative cells. Conjugates carrying charges that are unable to diffuse freely across plasma membranes exhibit minimal activity. Anti-CD30 cAC10 conjugates loaded at a concentration of 8 drugs/mAbs with PEGylated glucuronide drug moieties with a linker carrying quaternized tubulisin M (cAC-10-99), quaternized tubulisin ethyl ester (cAC10-66) and methyl-( propen-2-yl) ester (cAC10-185) of quaternized tubulisin. As stated above, tumor-bearing mice were administered a single dose of 0.5 mg/kg study drug intraperitoneally once the average tumor volume reached 100 mm ³ on day 6. The results are shown in Fig. 4. All mice treated with conjugates containing quaternized tubulisin ethyl ester (cAC10-66 and cAC10-185) achieved complete tumor regression on day 41 of the study. The quaternized tubulisin M conjugate (cAC10-99) showed more variable activity: 2/5 mice achieved complete regression by day 41, and the remaining 3/5 mice showed temporary tumor regression.

In vivo xenograft models - demonstration of activity in MDR+ models. Conjugates bearing PEGylated constructs were studied in MDR+ 786-O renal cell carcinoma models and DELBVR xenograft models of anaplastic large cell lymphoma. Anti-CD70 conjugates (mAb=h1F6) were loaded at a concentration of 8 drugs/mAb with PEGylated glucuronide drug moieties with a linker containing quaternized tubulisin M (cAC10-99), quaternized tubulisin ethyl ester (cAC10-66) or methylpropene ester of quaternized tubulisin (cAC10-66). cAC10-185) in medicinal units. Mice bearing CD70 ⁺ 786-O renal cell carcinoma tumors were administered a single dose of 0.5 or 1 mg/kg of study drugs after the tumor had reached approximately 100 mm ³ . The results are shown in Fig. 9. Delayed tumor growth was observed in mice treated with ADC conjugated to quaternized tubulisin M (cAC10-99) at a dose of 0.5 mg/kg, or with ADC conjugated to quaternized tubulisin ethyl ester (cAC10-66) at a dose of 1. 5 mg/kg. A higher dose of 1.5 mg/kg quaternized tubulisin M conjugate (cAC10-99) caused 5/5 long-term complete regression. ADCs having a subset of linkers have also been studied in the MDR+, CD30-expressing xenograft model of anaplastic large cell lymphoma DELBVR. Anti-CD30 conjugates (mAb=cAC10) carrying PEGylated drug moieties with a linker containing quaternized tubulisin M (cAC10-99) and quaternized tubulisin ethyl ester (cAC10-66) were conjugated at 8-drug/mAb. Similarly, anti-CD30 conjugates carrying non-PEGylated glucuronide drug moieties with a linker containing quaternized tubulisin M (cAC10-82) and quaternized tubulisin ethyl ester (cAC10-57) and the Val-Ala dipeptide version of quaternized tubulisin M (cAC10-15) were conjugated at a dose of 4 drug compounds/mAb. Tumor-bearing mice were administered a single dose of study drugs at 0.3 or 1 mg/kg after tumor size had reached approximately 100 mm ³ . The results are shown in Fig. 10. At a dose of 0.3 mg/kg, conjugates having PEGylated fragments of glucuronide drug compounds with a linker, containing ethyl ester of quaternized tubulisin (cAC10-66), gave 2/5 long-term complete regression, and having PEGylated fragments of glucuronide drug compounds with a linker, containing quaternized tubulisin M (cAC10-99), provided 5/5 long-term complete regression. At the higher dose of 1 mg/kg, long-term complete regression was observed with non-PEGylated glucuronide ethyl ester conjugates of tubulisin (1/5, cAC10-57) and tubulisin M (5/5 mice cured, cAC10-82). In the PEGylated series, a dose of 1 mg/kg provided 4/5 and 5/5 long-term complete regression rates for tubulisin glucuronide-ethyl ester conjugates (cAC10-66) and tubulisin M (cAC10-99), respectively.

Pharmacokinetic evaluation in rats. In fig. Figures 5-7 show the clearance profiles of various tubulisin-quaternary amine linker designs. In all experiments, rats were administered a single intravenous dose of 1 mg/kg at time zero with radiolabeled ADCs. Plasma samples were collected at various time points and analyzed as described to quantify total antibody as a function of time. In fig. Figure 5 shows the exposure profiles of humanized IgG conjugates derived from the drug compound linker MDPRVal-Ala-PABQ-TubM (15) and the hydrophilic drug compound linker MDPRGlucQ-TubM (82) MDPR-val-glu-TubM (91) and MDPR-val-glu (PEG12)-TubM (9). A humanized IgG carrying four copies of quaternized TubM bound to al-Ala dipeptide (hIgG 15) had a clearance profile identical to the unmodified antibody; however, at DAR 8, ADC was cleared from circulation much more quickly. Replacing the alanine residue of hIgG-15 with the glutamate residue that provides IgG-91 did not lead to a noticeable increase in exposure. The addition of a PEG12 side chain to the val-glu linker in hIgG-91, resulting in hIgG-95, provided increased exposure to the ADC. Likewise, replacing the Val-Ala dipeptide in IgG-15 with a glucuronide unit providing hIgG-82 did result in increased conjugate potency on DAR 8 conjugates.

- 141 -

Claims (12)

In fig. Figure 6 shows the effects of PK on DAR 8 conjugates with humanized IgG carrying fragments of the drug compounds glucuronide propyl ester of tubulizide linked to a quaternary amine with a linker in the absence (hIgG-58) and presence (hIgG-67) of the PEG12 side chain. A conjugate carrying 8 copies of quaternized drug linker fragments from the drug linker compound MDPR-glucQ-TubOPr (58) was cleared from circulation much more rapidly than the unmodified antibody. The addition of the PEG12 side chain providing hIgG-67 significantly improved exposure, more closely approximating the PK properties of the naked antibody. The PK effects of DAR 8 conjugates with humanized IgG containing PEGylated fragments of glucuronide drug compounds with a linker having drug units of quaternized tubulisin M (hIgG-99) and quaternized tubulisin ethyl ester (hIgG-66) are shown in Fig. 7. Both conjugates exhibited long-lasting exposures that closely approximated the line representing the literature average exposure for the parent antibody. CLAIM 1. Drug-ligand conjugate, represented by the structure of one of the formulas 2A2F - 142 046150 or its salt, L represents a ligand unit from an antibody; LB is the unit that covalently binds the ligand; LP represents a parallel connecting unit, where LP represents an aminoalkanedioic acid, a diaminoalkanoic acid, a sulfur-alkanedioic acid, a sulfur-substituted aminoalkanoic acid, a diaminoalkanol, an aminoalkanedinol, a hydroxyl-alkanedioic acid, a hydroxyl-substituted aminoalkanoic acid, or a sulfur-substituted aminoalkanol moiety, optionally placed, where the substituent containing sulfur, is in reduced or oxidized form or L _P is an amino acid residue of lysine, arginine, asparagine, glutamine, ornithine, citrulline, cysteine, homocysteine, penicillamine, threonine, serine, glutamic acid, aspartic acid, tyrosine, histidine or tryptophan, where the amino acid has D- or L-configuration; PEG is a polyethylene glycol unit; subscript a denotes 0 or 1; A represents a first optional extension unit such that when the index a is 0, A is absent, or when the index a is 1, A is present and optionally includes two, three, or four independently selected subunits (A1, A2, _A3 , A ₄ ); AO represents a second optional extension unit and optionally includes two, three or four subunits independent of A; Su represents a carbohydrate moiety; -O'- represents the oxygen atom of the O-glycosidic bond cleaved by the glycosidase; -J'- represents -O-, -N(R ³³ )- or -S- associated directly with AO or a subunit thereof, or with L-LB-LP(PEG)- or LB'-LP(PEG)- when there is none of A and AO, via a functional group containing J', where R ³³ represents -H, optionally substituted C1-C8 alkyl or optionally substituted aralkyl; V, Z ¹ and Z ³ represent =N- or =C(R ²⁴ )-, where R ²⁴ represents hydrogen or C1-C6 alkyl, C ₂ -C ₆ alkenyl or C ₂ -C ₆ alkynyl, optionally substituted , or halogen, -NO2, -CN or other electron-withdrawing group, or -OCH ₃ or other electron-donating group, R8 and ^R9 are, independently, hydrogen, C1- _C6 -alkyl, _C2 - _C6 -alkenyl or _C2 - _C6 -alkynyl, optionally substituted, or _C6 - _C14 -aryl or _C5 -C ₁₄ -heteroaryl, optionally substituted; R' represents hydrogen or represents a halogen, -NO2, -CN or other electron-withdrawing group; D ⁺ is a quaternized tubulisin drug unit having the structure wherein the Het ring is a 5-membered nitrogen-containing heteroarylene containing at least one heteroatom selected from the group consisting of N, O and S, and wherein said obligatory substituents on the heteroarylene are in the 1,3-position relative to each other with optional substitution for other provisions; subscript m denotes 0 or 1; R2A represents hydrogen or optionally substituted C1-C8 alkyl, or R2A together with atom - 143 046150 mΩ of oxygen to which it is attached denotes an O-linked substituent; R ³ represents hydrogen or optionally substituted C1-C ₆ -alkyl; R ⁴ , R ⁵ and R ⁶ represent optionally substituted C1-C ₆ alkyl; one R ⁷ is optionally substituted C1-C6 alkyl, optionally substituted C6-C18 arylalkyl, optionally substituted C6-C18 heteroarylalkyl and the other R ⁷ is hydrogen or optionally substituted C1-C6 alkyl; subscript p' denotes an integer in the range from 1 to 24; wherein the wavy line indicates covalent binding of D+ to the compound residue of the drug-ligand conjugate structure and wherein each optionally substituted alkyl is independently selected; wherein cleavage of said O-glycosidic bond by a glycosidase results in the release of the tubulisin compound (D) from the drug-ligand conjugate compound, wherein the term optionally substituted refers to a moiety that may be unsubstituted or substituted by a group selected from one or more atoms selected from the group , consisting of halogen, -CN, -OH, -NH2, -NH(CH ₃ ) -N(CH ₃ ) ₂ , C1-C ₆ -alkyl, C1-C ₆ -fluoroalkyl, C1-C ₆ -fluoroalkoxy, C1 -C ₆ -heteroalkyl, C ₃ -C ₈ -cycloalkyl, C ₃ -C ₈ -heterocycloalkyl, C ₅ -C ₁₄ -aryl, C ₅ -C ₁₄ -heteroaryl, C1-C ₆ -alkoxy, C ₅ -C ₁₄ -aryloxy, C1-C ₆ -alkylthio, C ₅ -C ₁₄ -arylthio, C1-C ₆ -alkyl sulfoxide, C ₅ -C ₁₄ arylsulfoxide, C1-C ₆ -alkyl sulfone, -C(=O)OH, -C( =O)O-C ₁ -C ₆ -alkyl, -C(=O)NH ₂ , -C(=O)NH(C1-C ₆ alkyl) and -C(=O)^C1-C ₆ -alkyl ) ₂ ; where the term electron-withdrawing group refers to a species selected from the group consisting of optionally substituted C ₂ -C ₆ -alkenyl, optionally substituted C ₂ -C ₆ -kynyl, -C(=O), -CN, -NO2, -CX3, -X, -C(=O)OR', -C(=O)NH2, -C(=O)N(R')R ^op , -C(=O)R', -C(=O)X , -S(=O)2R ^op , -S(=OhOR', -SO3H2, -S(=O)2NH2, -S(=O)2N(R')R ^op , -PO3H2, -P(=O )(OR')(0R ^op )2, -NO, -NH2, -NH(R ^op ), -N(R')(R ^op ) and -N(R ^op ) ₃ ⁺ where X is -F , -Cl, -Br or -I, and each of R and R ^op is independently selected from the group of optional substituents; where the term electron-donating group refers to a species selected from the group consisting of -OH, -OR', -NH2, -NHR', N(R') ₂ , optionally substituted C ₅ -C ₁₄ -aryl, optionally substituted C ₅ - C ₁₄ -heteroaryl, optionally substituted C ₂ -C ₆ -alkenyl and optionally substituted C 2 -C 6 -alkynyl, where each R' independently represents C 1 -C ₆ -alkyl; and wherein the term O-linked substituent refers to a substituent selected from the group consisting of -OH, -OCH2OCH2R ^2B , -OCH2R ^2B , -OC(O)R ^2B , -CH2OC(O)R ^2B , -OC(O)N (R ^2B )(R ² c) and -OCH2C (O)N(R ^2B )(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, C1-C ₆ alkyl and C ₂ - C ₈ alkenyl. 2. The drug-ligand conjugate of claim 1, wherein the antibody ligand moiety is capable of selectively binding to an available cell surface antigen of abnormal cells, wherein the antigen is capable of cellular internalization of the bound ADC and is preferentially present on abnormal or other undesirable cells compared to normal cells . 3. Drug-ligand conjugate according to claim 1 or 2, represented by the structure of formula 4 in the form of a pharmaceutically acceptable salt, where Ab is a ligand unit from an antibody; J' represents -N(R ³³ )-, where R ³³ represents hydrogen or methyl; V and Z ³ are independently =CH- or =N-; R' represents hydrogen or an electron-withdrawing group; R ⁸ represents hydrogen; R ⁹ is hydrogen, optionally substituted with C ₁ -C ₆ -alkyl or optionally substituted phenyl; R ⁴⁵ represents -CO2H; and the subscript p' denotes a number in the range from 1 to 24. 4. Drug-ligand conjugate according to any one of claims 1-3, represented by the structure of formula 6 - 144 046150 (Formula 6) where S represents the sulfur atom of the ligand unit from the antibody (Ab); an asterisk (*) denotes chirality or lack thereof at the specified carbon atom; A2-4 represent independently selected optional subunits of A, where -[C(R ^b1 )(R ^b1 )] _q [HE]- represents A ₁ when one or more such subunits are present; R represents hydrogen; R' represents hydrogen or an electron-withdrawing group; R ^a1 represents hydrogen or a basic unit (BU), where BU represents a basic unit having the structure -CH2-N(R ²² )(R ²³ ), or its acid addition salt, where R ²² and R ²³ , independently, represent is hydrogen, methyl or ethyl, or both together with the nitrogen atom to which they are attached represent a 5- or 6-membered heterocycloalkyl, or one of R ²² and R ²³ is hydrogen and the other is an acid-sensitive carbamate protecting group ; R ^a2 represents hydrogen; subscript q denotes an integer ranging from 0 to 5 when HE is present, or from 1 to 5 when HE is absent; each R ^b1 independently represents hydrogen or optionally substituted C ₁ -C ₆ -αalkyl; HE is absent or represents -C(=O)-; R ⁴⁵ represents -CO2H; J' represents -NH-; V and Z ³ represent =CH-; R ⁸ represents hydrogen; R ⁹ represents hydrogen or methyl; subscript p' denotes an integer in the range from 1 to 16; and where the remaining group variables have the meanings defined for Formula 4, or where the conjugate has the structure of Formula 9A or Formula 9B Lb (Formula 9A) Lb (Formula 9B) wherein S represents a sulfur atom of a ligand unit from an antibody (Ab); an asterisk (*) denotes chirality or lack thereof at the specified carbon atom; A2-4 represent independently selected optional subunits of A, where -[C(R ^b1 )(R ^b1 )]q[HE]- represents A ₁ when one or more such subunits are present; R represents hydrogen; R' represents hydrogen or an electron-withdrawing group; R ^a1 is -H or BU, where BU is a backbone having the structure -CH ₂ -N(R ²² )(R ²³ ), or an acid addition salt thereof, where R ²² and R ²³ are independently - 145 046150 hydrogen or methyl, or both, together with the nitrogen atom to which they are attached, denote a basic nitrogen-containing 5- or 6-membered heterocycloalkyl, or one of R ²² and R ²³ is hydrogen and the other is an acid-sensitive protective group; R ^a2 represents hydrogen; subscript q denotes an integer ranging from 0 to 5 when HE is present, or from 1 to 5 when HE is absent; each R ^b1 independently represents hydrogen or optionally substituted C ₁ -C ₆ -alkyl; HE is absent or represents -C(=O)-; J' represents -O- or -NH-; R ⁸ and R ⁹ independently represent -H or optionally substituted C ₁ -C ₆ -αalkyl, or both, together with the carbon atom to which they are attached, represent ^-^-cycloalkyl; subscript p' denotes an integer in the range from 1 to 24; and where the remaining group variables have the values defined for formula 4. 5. A drug-ligand conjugate according to any one of claims 1 to 4, represented by the structure of formula 10A or formula 10B (Formula 10A) ^L B (Formula 10B) where R represents hydrogen; R' represents hydrogen, -NO ₂ , -Cl or -F; HE represents -C(=O)-; R ⁴⁵ represents -CO ₂ H; J' represents -NH-; V and Z ³ are each =CH ₂ -; R ⁸ represents hydrogen; R ⁹ represents hydrogen or methyl; p' is an integer in the range from 1 to 12; and where the remaining group variables have the values defined for formula 4. 6. The drug-ligand conjugate of claim 4, wherein said carbon marked with an asterisk (*) predominantly has the same absolute configuration as the alpha carbon of the L-amino acid when said carbon is chiral, and/or wherein A and AO, independently, have the structure of formula 7 or formula 8 g (Formula 7) (Formula 8) - 146 046150 where the wavy lines indicate covalent bonding in the conjugate structure, where K and L are independently C, N, O or S, provided that when K or L is O or S, R ⁴¹ and R ⁴² -K or R ⁴³ and R ⁴⁴ -L are absent, and, when K or L is N, one of R ⁴¹ and R ⁴² -K or one of R ⁴² and R ⁴³ -L is absent, and provided that two neighboring Ls are not independently selected as N, O or S; where the subscripts e and f are independently selected integers that range from 0 to 12, and the subscript g denotes an integer ranging from 1 to 12, where G represents hydrogen, optionally substituted with ^-^-alkyl, -OH, -ORPR, -CO ₂ H, CO2RPR, where RPR is an appropriate protecting group, or G is -N(R ^pr )(R ^pr ), where each RPR is a protecting group, or G is -N(R ⁴⁵ )(R ⁴⁶ ), where one of R ⁴⁵ and R ⁴⁶ is hydrogen or RPR, where RPR is an appropriate protecting group, and the other is hydrogen or optionally substituted ^-^-alkyl; where R ³⁸ represents hydrogen or optionally substituted C1-C6-alkyl; R ³⁹ -R ⁴⁴ are, independently, hydrogen, optionally substituted ^-^-alkyl, optionally substituted ^-^4^^ or optionally substituted C5-C ₁₄ -heteroaryl, or both R ³⁹ and R ⁴⁰ together with a carbon atom, to which they are attached include ^-^-cycloalkyl, or R ⁴¹ and R ⁴² together with the K to which they are attached when K is C, or R ⁴³ and R ⁴⁴ together with L to which they are attached when L represents a carbon atom, include C ₃ -C ₆ -cycloalkyl, or R ⁴⁰ and R ⁴¹ , or R ⁴⁰ and R ⁴³ , or R ⁴¹ and R ⁴³ , together with the carbon atom or heteroatom to which they are attached, and the atoms located between these carbon atoms and/or heteroatoms form a 5- or 6-membered cycloalkyl or heterocycloalkyl, provided that when K is O or S, R ⁴¹ and R ⁴² are absent, when K is N, one of R ⁴¹ and R ⁴² are absent, when L is O or S, R ⁴³ and R ⁴⁴ are absent, and, when L is N, one of R ⁴³ and R ⁴⁴ is absent, or where A _O has an alpha-amino structure, beta-amino or other amino acid. 7. The drug-ligand conjugate according to claim 1, where the quaternized tubulisin drug unit (-D+) has the following structure: where index m denotes 0 or 1; Z represents optionally substituted ^-^-alkylene or optionally substituted ^-^-alkenylene; And R ^7A is an optionally substituted ^-^4^^ or optionally substituted C ₆ -C ₁₄ -heteroaryl. 8. The drug-ligand conjugate according to claim 1, where the quaternized tubulisin drug unit (-D+) has the following structure: where R ^7A represents optionally substituted phenyl and R ^8A represents hydrogen or methyl. 9. The drug-ligand conjugate according to claim 7 or 8, wherein the quaternized tubulisin drug unit (-D+) has the structure where R ⁴ represents methyl; the subscript u denotes 0, 1 or 2; R ³ represents H, methyl, ethyl, propyl, -CH2-OC(O)R ^3A , -CH2CH(R ^3B )C(O)R ^3A or -CH(R ^3B )C(O)NHR ^3A , where R ^3A is C1-C6-alkyl and R ^3B is H or C1- _C6 alkyl independently selected from R ^3A ; R ^2A , together with the oxygen atom to which it is attached, represents an O-bonded - 147 046150 substitute selected from the group consisting of -OCH ₂ OCH ₂ R ^2B , -OCH2R2B, -OC(O)R ^2B , -OCH2OC(O)R ^2B , OC(O)N(R ^2B )(R ^2C ) and -OCH2C(O)N(R ^2B )(R ^2C ), where R ^2B and R ^2C are independently selected from the group consisting of H, Q^-alkyl and ^-C^-alkenyl; and each R ^7B , when present, is independently -OH or -OCH ₃ . 10. The drug-ligand conjugate according to claim 9, wherein the quaternized tubulisin drug unit (-D+) has the structure where R ^2A is -CH ₂ CH ₃ , -CH ₂ -CH=CH ₂ or -CH ₃ , the u subscript is 0 or 1, and R ^7B is -OH. -CH2C(CH ₃ )=CH2, R ³ represents 11. The drug-ligand conjugate according to claim 9, wherein the quaternized tubulisin drug unit (-D+) has the structure where R ^2A represents -CH2CH3 or -CH ₂ -CH=CH ₂ . 12. The drug-ligand conjugate according to claim 9, wherein the quaternized tubulisin drug unit (-D+) has the structure where R ^2B is -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH2C(CH3)3, or where the quaternized tubulisin drug unit (-D+) has the structure where R