WO2016025752A1 - Reagents for thiol conjugation and conjugates formed therefrom - Google Patents

Abstract

Reagents for preparing stabilized thioethers for in vivo administration are disclosed, along with said thioethers thus prepared.

Description

REAGENTS FOR THIOL CONJUGATION AND

CONJUGATES FORMED THEREFROM

Related Application

[0001] This application claims priority from U.S. 62/037,465 filed 14 August 2014, the contents of which are incorporated herein by reference in its entirety.

Technical Field

[0002] The invention relates to reagents for complexing to thiols and to the products of said complexing.

Background Art

[0003] Maleimides react rapidly and selectively with thiol groups to form succinimidyl thioethers; these thioethers may be unstable towards hydrolytic ring-opening, which provides a mi

hydrolytic ring-opening

[0004] Due to the high selectivity of maleimides towards thiols and the rapid rate of reaction to form the succinimidyl thioethers, the reaction of a thiol group with a maleimide is a commonly used method for conjugating molecules. For example, it is a common method to conjugate toxins to antibodies or antibody fragments to generate antibody-drug conjugates (ADCs), albumin-drug conjugates, and poly(ethylene glycol)-drug conjugates. It is being increasingly recognized that this conjugation chemistry may be unsuitable for generating highly stable conjugates due to the reversibility of the thiol-maleimide addition reaction which leads to thiol exchange. The equilibrium constant for thiol addition to maleimides is quite large, such that reversal of the reaction is unobservable under normal in vitro conditions; in vivo, however, where competing thiols such as glutathione or thiol-containing proteins such as albumin are present, the exchange of a first thiol (RSH) with a second thiol (R'SH) on the conjugate has been observed leading to loss of the RSH from the conjugate (see, for example, Shen, et ah , "Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates," Nature Biotech (2012) 30: 184-189;

Baldwin & Kiick, Bioconj. Chem. (2011) 22: 1946-1953.

[0005] In contrast to the succinimidyl thioethers, the hydrolytic ring-opening products appear to be relatively stable towards thiol exchange. Thus, if the rate of hydrolytic ring- opening can be increased such that it is feasible to prepare fully ring-opened conjugate prior to in vivo administration, the problem of thiol-maleimide conjugate instability may be ameliorated.

[0006] It has been previously noted that the rate of hydrolytic ring-opening of succinimidyl thioethers is strongly dependent on environment, with a positively-charged environment leading to more rapid ring-opening (Shen, et ah). It has also been shown that ring-opening can be catalyzed in some instances using anions such as molybdate (Kalia & Raines, Bioorg. Med. Chem. Lett. (2007) 17:6286-6289). It is further known that the rate of hydrolytic ring-opening in maleimides is influenced by neighboring electron-withdrawing groups. U.S. Patent 8,106,131 B2 discloses maleimides that are more resistant to ring- opening hydrolysis by virtue of having a hydrocarbon spacer of at least 4 contiguous saturated carbon atoms between the maleimide ring and the functional group through which the maleimide is attached to a polymer. While maleimido-ethyl-carboxamido-PEG showed a hydrolysis half-life of 8.1 h, increasing the length of the alkylene spacer between the maleimide and the carboxamido group increased the half-life to as much as 64.6 h.

[0007] U.S. Patent Application 2013/0309256 Al (filed 13 March 2013) discloses self- stabilizing linkers comprising a pendant base and an electron- withdrawing group operably linked to stabilize the conjugate in plasma by increasing the rate of succinimide ring hydrolysis. These linkers were shown to slow, although not eliminate, the in vivo loss of maleimide-linked drug from an antibody-drug conjugate after administration to rats relative to unstabilized linkers. This resulted in improved performance in a tumor xenograft model.

[0008] The instability of thiol-maleimide conjugates towards thiol exchange may interfere with the controlled release of drugs from conjugates, especially when long-term therapy involving controlled slow release of the drug from the conjugate is desired. There is thus a need for improved maleimides showing reduced thiol exchange for use in both stable and releasable conjugates.

Disclosure of the Invention

[0009] The present invention provides improved reagents for and methods of conjugating to thiols. These reagents are designed to provide conjugates that are stabilized towards thiol exchange, and that allow for the preparation of thiol-linked conjugates that are sufficiently stable for use in long-term therapies.

[0010] In one aspect, the invention is directed to a maleimide reagent of the formula (A):

wherein p = 0-2;

each R¹⁰ is independently H, CF₃, (CH₂)_p(C=0)R¹², (CH₂)_pC(=0)ORⁿ,

(CH₂)_pC(=0)NR¹² ₂, CN, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, (CH₂)_pS0₂Rⁿ, (CH₂)_pSRⁿ, (CH₂)_pS0₂Rⁿ, CH₂NR¹³ ₂ or CH₂N⁺R¹³ ₃;

Y is C=0, C(=0)NR¹², alkynyl, S0₂, S0₂NR¹², (CF₂)_q, NR¹³, NR¹³ ₂, N⁺R¹³ ₂, N⁺R¹³ ₃, or is absent;

q = l-4;

each R¹¹ is independently C1-C15 alkyl, aryl, heteroalkyl (1-15 joined members) or heteroaryl;

each R¹² is independently H, C1-C15 alkyl, aryl, heteroalkyl (1-15 joined members) or heteroaryl;

each R¹³ is independently C1-C15 alkyl or heteroalkyl (1-15 joined members) or in NR¹³ ₂ or N⁺R¹³3 two R¹³ can independently be C1-C15 alkylene or heteroalkylene (1-15 joined members) and optionally form a ring with 3-8 members and the third R¹³ on N⁺R¹³3 is C1-C15 alkyl or heteroalkyl (1-15 joined members);

at least one of R¹⁰ or Y is an electron withdrawing group;

m = 0 or 1 ;

linker is C1-C15 alkylene or heteroalkylene (1-15 joined members); Z is a functional group for attachment to another molecule which Z is optionally included in Y or in R¹ ';

with the proviso that when Y is C(=0)NR " or is absent at least one of Rⁱ⁰ is other than H.

[0011] In another aspect, the invention is directed to reagents of formula (A) wherein each R¹² is independently H, C1-C15 alkyl or heteroalkyl (1-15 joined members),

linker is heteroalkylene (1-15 joined members); and

Z is an amine, carboxylic acid, active ester, alcohol, or activated carbonate, optionally coupled to a protecting group.

[0012] In still another aspect the invention provides maleimide conjugation reagents having the formula (I):

p = 0-2;

n = 0-6;

each R¹⁰ is independently H, CF₃, C(=0)R^{! 1}, CN, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, S0₂R^{1 !}, CH₂SRⁿ, CH₂S0₂R^{1 !}, CH₂NR¹³ ₂ or CH₂N^÷R¹³ ₃;

Y is C=0, C(=0)NR¹², alkynyl, S0₂, S0₂NR¹², (CF₂)_q, NRⁱ³, NRⁱ³ ₂, N⁺R¹³ ₂, N⁺R¹³ ₃ or is absent;

q = 1-4;

R^u is CrQ alkyl or aryl;

R¹² is H, Q-Ce alkyl, or aryl;

R¹³ is Ci-C₆ alkyl in NRⁱ³ and N⁺R⁵ ₂ or two R¹³ may be Ci-C₆ alkylene in NR⁵ ₂ and two R ^~ may be C1-C6 alkylene and one R¹³ Ci-Ce alkyl in N⁺R¹³ ₃, and

Z is an amine, carboxylic acid, active ester, alcohol, or activated carbonate which may be protected or further derivatized,

with the proviso that when Y is C(=0)NR¹² or is absent, at least one of Rⁱ⁰ is other than H. [0013] In a second aspect the invention provides maleimide-drug conjugates having the formula (B):

wherein m, p, R¹⁰, Y and linker are as defined in formula (A);

X is NHCO, CONH, NHC(0)0, or OC(0)NH; and

D is the residue of a drug.

[0014] In some embodiments, in formula (B) each R¹² is independently H, Q-Q 5 alkyl or heteroalkyl (1-15 joined members),

linker is heteroalkylene (1-15 joined members); and

Z is an amine, carboxylic acid, active ester, alcohol, or activated carbonate, optionally coupled to a protecting group.

[0015] In still other embodiments, the drug conjugate is of formula (II)

wherein Rⁱ⁰, p, Y and n are as defined for formula (I); wherein X is NH-CO, CO-NH, NH- CO-O, O-CO-NH; D is the residue of a drug; and other groups are as defined above. The invention further provides conjugates of the compounds of formula (A), (I), (B) or (II) with macromolecular carriers or other thiol containing moieties, formed by reaction of (A), (I), (B) or (II) with a moiety comprising one or more thiol groups.

[0016] In a third aspect, the invention provides releasable maleimide conjugation reagents having formula (C):

wherein m, p, R , Y and linker are as defined in formula (A); t is 0 or 1 ;

at least one or both R¹ and R² is independently CN; N0₂;

optionally substituted aryl;

optionally substituted heteroaryl;

optionally substituted alkenyl;

optionally substituted alkynyl;

COR³ or SOR³ or S0₂R³ wherein

R³ is H or optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted;

heteroaryl or heteroarylalkyl, each optionally substituted; or

OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H or optionally substituted alkyl, or both R^{9 s}roups taken together with the nitrogen to which they are attached form a heterocyclic ring;

SR⁴ wherein

R⁴ is optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted;

wherein R¹ and R² may be joined to form a 3-8 membered ring; and

wherein one and only one of R¹ and R² may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and

each R⁵ is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (CH₂CH₂0)_p wherein p = 1-1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; wherein at least one of R¹, R², or R⁵ further comprises a functional group for coupling to a macromolecular carrier or is coupled to a macromolecular carrier.

[0017] In some embodiments, the releasable reagent is of formula (III):

wherein

P, n, R¹⁰ and Y are as defined in formula (I);

t is 0 or 1 ; at least one or both R¹ and R² is independently CN; N0₂;

optionally substituted aryl;

optionally substituted heteroaryl;

optionally substituted alkenyl;

optionally substituted alkynyl;

COR³ or SOR³ or S0₂R³ wherein

R³ is H or optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted;

heteroaryl or heteroarylalkyl, each optionally substituted; or

OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H or optionally substituted alkyl, or both R^{9 s}roups taken together with the nitrogen to which they are attached form a heterocyclic ring;

SR⁴ wherein

R⁴ is optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted;

wherein R¹ and R² may be joined to form a 3-8 membered ring; and

wherein one and only one of R¹ and R² may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and

each R⁵ is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (CH₂CH₂0)_p wherein p=l-1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and

wherein at least one of R¹, R², or R⁵ further comprises a functional group for coupling to a macromolecular carrier or is coupled to a macromolecular carrier.

The invention is also directed to conjugates of formula (C) or (III) with thiols.

Brief Description of the Drawings

[0018] Figure 1 illustrates the competition between thiol exchange and ring-opening hydrolysis of thiol-maleimide conjugates. While ring-opening hydrolysis provides a product that is more stable towards thiol exchange, the initial conjugate is susceptible to loss of conjugated thiol R-SH through exchange in the presence of competing thiols R*-SH. [0019] Figure 2 illustrates one method for the formation of conjugates using the compounds of the invention. In this illustration, the potent cytotoxin monomethyl auristatin E (MMAE) is first connected to a maleimide of formula (I) to produce a maleimide-drug conjugate of formula (II). This is subsequently reacted with a

macromolecular carrier comprising at least one thiol group to produce a macromolecular drug conjugate.

[0020] Figure 3 illustrates formation of compounds of formula (III) and their use in producing macromolecule-maleimide conjugates connected by a releasable linker. A maleimide of formula (I) is reacted with a suitable β-eliminative linker to form a compound of formula (III). In this illustration, one R^{5 s}roup in (III) comprises a BOC-protected amine which after deprotection is used to connect (III) to a macromolecular carrier comprising at least one reactive carboxylate or active ester group. Group R¹ is chosen to provide the appropriate release rate.

[0021] Figure 4 illustrates the use of the macromolecule-maleimide conjugates of Figure 3 in producing macromolecule-drug conjugates wherein the drug undergoes controlled release by a β-eliminative mechanism. The conjugate of Figure 3 is reacted with a thiol- containing drug D-SH to produce the releasable drug-macromolecule conjugate initially as a succinimidyl-thioether. Subsequent hydrolysis produces a drug-macromolecule conjugate that is stabilized towards thiol exchange. Subsequent β-elimination releases the drug with a small remnant from the hydrolyzed maleimide.

Modes of Carrying out the Invention

[0022] The present invention provides improved reagents for and methods of conjugating to thiols. These reagents are designed to provide conjugates that are stabilized towards thiol exchange, and that allow for the preparation of thiol-linked conjugates that are sufficiently stable for use in long-term therapies. In one aspect the invention thus provides maleimide conjugation reagents of formula (A) or (I) as well as compounds of the formulas (B), (II), (C), and (III) as described above.

[0023] The ability of R¹⁰ and Y in formulas (A), (B), (C), (I), (II) and (III) to promote hydrolytic ring-opening may be correlated with their electron-withdrawing ability, with more highly electron-withdrawing groups increasing the sensitivity of the ring carbonyl groups towards addition of water and subsequent ring-opening. The enhanced rate of ring opening is provided by ensuring that at least one of R and Y contains an electron-withdrawing group. Therefore, if Y is absent R¹⁰ cannot be simply hydrogen. On the other hand, if Y presents an effective electron-withdrawing moiety, this possibility for R¹⁰ is available. Thus, R¹⁰ and/or Y are typically electron-withdrawing groups. While these may be amines, it is considered that under physiologically relevant conditions (pH ~ 7.4) such amines will be predominantly in their protonated forms and thus more highly electron-withdrawing than their free base forms; this protonation under physiological conditions makes it less probable that amines may effectively act as general bases to catalyze ring-opening. As illustrated in the working examples below, this is consistent with the similar effects on hydrolysis rates observed for cognate trialkylamines and tetraalkylammonium salts at pH 7.4. The R¹⁰ and Y groups may act synergistically to provide greater rates of hydrolysis than might be observed with either group alone.

[0024] When the reagents of formulas (A) or (I) or the compounds of formulas (B), (II), (C) or (III) are coupled to a thiol-containing moiety, either a thiol-containing drug such as an antibody or a protein or peptide or to a macromolecule such as a polyethylene glycol that has been derivatized to react with the maleimide double bond, the exchange rate of the derivatized moiety is greatly decreased once the maleimide ring is opened. While in some circumstances, this ring opening can occur after administration in vivo, it is generally preferred to carry out ring opening in vitro prior to administration. This can be conducted as illustrated in Example 17 below, which provides rates of hydrolysis of the ring system for various exemplified reagents after coupling to DNP-PEG₄-cys— i.e. , analogous to the product of the specified reagent and the thioethers set forth in Example 16. Other conditions for hydrolysis in vitro can also be used.

[0025] Z is needed in reagent A or I to provide a means of connecting the maleimide to another molecule. Thus, Z can be any functional group that is compatible with a maleimide for which such coupling methods are known. In one embodiment of the invention, Z is an amine and may be coupled with molecules comprising carboxylic acids, active esters such as nitrophenyl or succinimidyl esters, or active carbonates such as chloroformates, succinimidyl carbonates, or nitrophenyl carbonates to provide amide or carbamate linkages. In another embodiment of the invention, Z is a carboxylic acid or active ester, which may be analogously coupled to molecules comprising amine groups to provide amide linkages. In another embodiment of the invention, Z is an alcohol or activated carbonate, which may be analogously coupled to molecules comprising amine groups to provide carbamate linkages. Z may be included in Y or R¹³.

[0026] The maleimides of formula (A) or (I) may be prepared using synthetic methodology generally known in the art. For example, maleimides may be prepared using a two-step process in which an amine is first reacted with maleic anhydride to provide an intermediate maleamic acid. This intermediate is then cyclized to the maleimide under dehydrating conditions, for example using acid or base catalysis and/or heat. Alternately, maleimides may be prepared by the substitution of an alcohol group using the Mitsunobu process (maleimide, triphenylphosphine, azodicarboxylate diester). Specific methods for synthesis are illustrated in the working examples given below.

[0027] Alternative intermediate compounds are those of formula (B) or (II) where Z is replaced by X = D wherein X is an amide or carbamate; and D is the residue of a drug. The compounds of formula (B) or (II) may be prepared using methods known in the art. For example, an amine-containing drug D-NH₂ may be conjugated to a maleimide of formula (A) or (I) wherein Z is a carboxylic acid using a condensing agent such as a carbodiimide or uranium salt to form a compound of formula (B) or (II) wherein X = CO-NH. Alternately, a maleimide of formula (A) or (I) wherein Z is an active ester of a carboxylic acid, for example a nitrophenyl or succinimidyl ester, or wherein Z is an acid chloride, may be used with D- NH₂ directly. Similarly, if Z is an activated carbonate such as a chloroformate, nitrophenyl carbonate, or succinimidyl carbonate, reaction with D-NH₂ will provide (B) or (II) wherein X is O-CO-NH. Analogous methods may be used for carboxylate drugs, D-COOH, or alcohol drugs, D-OH, using the cognate (I) when Z is an amine.

[0028] Drugs useful in the invention include small molecules, peptides, proteins, oligonucleotides, aptamers, and the like. In certain embodiments, D is a potent cytotoxin useful in preparing antibody-drug conjugates, including maytansines such as mertansine and DM1, enediynes such as calicheamicin, and auristatins such as monomethyl auristatin E (MMAE) and its analogues.

[0029] Coupling to certain carriers through thiol linkages may be performed prior to obtaining the compounds of formula (B) or (II) where the carrier does not contain competing groups for reaction with the drug. Typically, proteins such as antibodies should be coupled to these reagents subsequent to formation of the compounds of formula (B) or (II) in order to avoid cross-reaction of the drug with the protein. However, certain carriers which do not contain competing groups, such as polyethylene glycol may be used first to form the thioether followed by coupling of the Z group ultimately to the drug to obtain the compounds of formula (B) or (II).

[0030] The invention further provides macromolecular conjugates of the compounds of formula (B) or (II) with macromolecular carriers, formed by reaction of (B) or (II) with a macromolecular carrier comprising one or more thiol groups. The macromolecular carrier can be any thiol-containing macromolecule, including proteins and oligosaccharides and synthetic polymers such as poly(ethylene glycols), and may be soluble or insoluble, for example as hydrogels. In certain embodiments of the invention, the macromolecular carrier is a protein, for example an antibody, an albumin, or a designed-sequence protein such as those disclosed in PCT Publication WO2013/130683 A2. Such macromolecular conjugates may be useful in the targeted delivery of therapeutic agents, for example as antibody-drug conjugates. In other embodiments of the invention, the macromolecular carrier is a synthetic polymer such as a poly (ethylene glycol).

[0031] The invention also provides releasable maleimide reagents having

formula (C) or (III) as described above.

[0032] The reagents of formula (C) or (III) may be prepared by reaction of a compound of formula (IV) wherein X is a displaceable group such as CI, F, O-succinimidyl, O-phenyl, O-nitrophenyl, O-dinitrophenyl, imidazolyl, triazolyl, tetrazolyl, or the like, with the appropriate amino-maleimide.

R\ R², or R⁵ and m are as defined in formula (C) or (III).

[0033] Compounds of formula (IV) and methods for their preparation have been described, for example in U.S. Patents 8,680,315 and 8,754,190; PCT publication

WO2013/036857; Santi, et al., "Predictable and tunable half-life extension of therapeutic reagents by controlled chemical release from macromolecular conjugates," Proc. Natl. Acad. Sci. USA (2012) 109:6211-6216; and Schneider, et al., "β-eliminative linkers adapted for bioconjugation of macromolecules to phenols," Bioconjugate Chem. (2013) 24: 1990-1997, each of which is incorporated herein by reference in its entirety. As described in the above- cited publications, R¹ and R² are electron-withdrawing groups that activate the adjacent C-H for ionization, thus controlling the rate at which the 0-(C=0)-X group is eliminated under physiological conditions; subsequent decarboxylation of 0-(C=0)-X then releases the group X. Reactive functional groups for coupling to macromolecular carriers may be amines, protected amines, carboxylic acids, protected carboxylates, aldehyde, ketone, alkynes, cycloalkynes, irans-cyclooctenes, norbornenes, or 1,2,4,5-tetrazines. In certain embodiments of the invention, the reactive functional group is an alkyne, cycloalkyne, irans-cyclooctene, norbornene, or 1,2,4,5-tetrazine. In a preferred embodiment, the reactive functional group is a cyclooctyne, irans-cyclooctene, norbornene, or 1,2,4,5-tetrazine. In certain other embodiments of the invention, the reactive functional group is a protected amine, for example a carbamate-protected amine such as ieri-butoxycarbonylamino.

[0034] The compounds of formula (C) or (III) may serve to couple, releasably, a macromolecular carrier to a thiol-containing drug which will be linked to the maleimide moiety through a thioether linkage. Thus, intermediates in the formation of such conjugates would include the compound of formula (C) or (III) coupled simply to a thiol-containing drug such as an antibody or a peptide through a thioether linkage and alternatively, the compound of formula (C) or (III) coupled through the reactive functional group on R¹, R² or R⁵ to a macromolecular carrier. The final product conjugate would thus contain both the thiol- containing drug coupled through the maleimide moiety and the macromolecular carrier coupled to R¹, R² or R⁵.

[0035] Suitable macromolecular carriers are similar to those described above but which do not themselves comprise free thiol groups. Thus proteins, oligosaccharides or synthetic polymers are suitable. Proteins include antibodies, albumins, and designed-sequence proteins such as those disclosed in U.S. Patent Publication 2011/0171687 Al. Oligosaccharides include dextrans and hyaluronic acids, and synthetic polymers include poly(ethylene glycol). The macromolecular carrier may be either soluble or insoluble, for example as in an insoluble hydrogel. In one embodiment of the invention, the macromolecular carrier is a biodegradable hydrogel such as those disclosed in PCT Patent Publication WO2013/036847 Al.

[0036] The macromolecular carrier comprises a cognate reactive functional group to that in formula (C) or (III) to allow for connection to (C) or (III). Thus, when (C) or (III) comprises a protected amine the macromolecular carrier comprises a group that is reactive with the amine group that is liberated from deprotection of the protected amine, for example a carboxylic acid or activated carboxylate group such as a succinimidyl or substituted phenyl ester or carbonate or a thioester in which case (C) or (III) is connected to the macromolecular carrier through an amide or carbamate linkage. Similarly, when (C) or (III) comprises a protected carboxylate group the macromolecular carrier comprises an amine group, in which case (C) or (III) is connected to the macromolecular carrier through an amide or carbamate linkage after deprotection of the carboxylate group. Alternatively, when (C) or (III)

comprises a protected amine the amine may be deprotected then elaborated into a carboxylate group, for example by reaction with succinic or glutaric anhydride. The resulting carboxylate may be couple with a macromolecular carrier comprising an amine group either by prior activation, for example to the succinimidyl or substituted phenyl ester, or by use of a coupling reagent such as a carbodiimide or uranium salt. When (C) or (III) comprises an alkyne or cycloalkyne, the macromolecular carrier comprises an azide group, in which case (C) or (III) is connected to the macromolecular carrier through a triazole linkage.

When (C) or (III) comprises a irans-cyclooctene or norbornene, the macromolecular carrier comprises a 1,2,4,5-tetrazine group, in which case (C) or (III) is connected to the

macromolecular carrier through a diazine or pyridazine linkage. When (C) or (III) comprises a 1,2,4,5-tetrazine group, the macromolecular carrier comprises a irans-cyclooctene or norbornene, in which case (C) or (III) is similarly connected to the macromolecular carrier through a diazine or pyridazine linkage. When (C) or (III) comprises an aldehyde or ketone, the macromolecular carrier comprises an amino-ether in which case (C) or (III) is connected to the macromolecular carrier through an oxime linkage.

[0037] Reaction of (C) or (III) with the thiol-containing drug and with the

macromolecular carrier may be performed either simultaneously or sequentially, and if sequentially in either order.

[0038] "Alkyl" means linear, branched, or cyclic saturated hydrocarbon groups of 1-15 carbons, 1-8 carbons, or in some embodiments 1-6 or 1-4 carbons. "Alkylene" is similarly defined but is bivalent.

[0039] "Alkoxy" means alkyl groups bonded to oxygen.

[0040] "Alkenyl" means non-aromatic unsaturated hydrocarbons which may be linear, branched or cyclic and contain 2-15 C with carbon-carbon double bonds. "Alkenyl" may be mono-, di-, tri- or tetra-substituted carbon-carbon double bonds of any geometric

configuration. [0041] "Alkynyl" means non-aromatic unsaturated hydrocarbons which may be linear, branched or cyclic and contain 3-15 C and contain carbon-carbon triple bonds. "Alkynyl" may have one or two carbon-carbon triple bonds.

[0042] "Aryl" means aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl and anthracenyl. "Heteroaryl" includes aromatic rings comprising 3-15 carbons containing at least one N, O or S atom,

preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.

[0043] In some instances, alkenyl, alkynyl, aryl or heteroaryl moieties may be coupled to the remainder of the molecule through an alkyl linkage. Under those circumstances, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.

[0044] "Heteroalkyl" means linear, branched, or cyclic saturated hydrocarbon groups which further include one or more O, N or S atoms as joined members - i.e. C or these heteroatoms. "Heteroalkyl" includes moieties that contain C = O or C = NH as well.

"Heteroalkylene" is similarly defined but is bivalent. Heteroalkyl and heteroalkylene contain 1-15 joined members counting both carbons and heteroatoms in the backbone of the moiety. The oxygen and N in C = O and C = NH are not included in this count, however hydrogen atoms are not. Thus "member" does not include H. It will also be evident that heteroalkyl and heteroalkylene may include one or more functional groups for example including Z that will couple the compound of formula (A) or (I) to moieties to give compounds of formulas (B), (II), (C) or (III).

[0045] The following examples are intended to illustrate and not limit the invention.

PREPARATION A

7-(ieri-butoxycarbonylamino - 1 -(phenylsulfonyl)-2-heptyl succinimidyl carbonate

[0046] (1 ) 7-(fe? -butoxycarbonylamino)- 1 -phenylsulfonyl-2-heptanol: A 1.0 M solution of trimethylphosphine in THF (2.2 mL, 2.2 mmol) was added dropwise to a stirred solution of 7-azido-l-phenylsulfonyl-2-heptanol (600 mg, 2.0 mmol; Santi, et ah , PNAS (2012) 109:6211-6216) in 1.8 mL of THF, resulting in vigorous gas evolution. After 10 min, the gas evolution had subsided and water (0.2 mL) was added, followed by di-ieri-butyl dicarbonate (500 mg, 2.3 mmol). After 15 min, the mixture was diluted with ethyl acetate and washed with sat. aq. NaHCC>₃. The organic phase was washed sequentially with 0.1 N HC1, water, sat. aq. NaHCC>₃, and brine, then dried over MgS0₄, filtered, and evaporated to a colorless oil. Chromatography on Si0₂ using a step gradient of hexane and 60:40 hexane/ethyl acetate provided the product (592 mg, 80%) as a colorless oil. ^]H-NMR (400 MHz, d₆-DMSO): d 7.89 (2H, m), 7.72 (1H, m), 7.63 (2H, m), 6.75 (1H, t, J = 5.2 Hz), 4.82 (1H, d, J = 6 Hz), 3.83 (1H, m), 3.34 (2H, m), 2.85 (2H, q, J = 6.8 Hz), 1.36 (9H, s), 1.6 (4H, m), 1.15 (4H, m).

[0047] (2) 7-(ferZ-butoxycarbonylamino)- 1 -phenylsulfonyl-2-heptyl succinimidyl carbonate: Pyridine (0.16 mL, 2.0 mmol) was added dropwise to a stirred solution of l-(tert- butoxycarbonylamino)-l-phenylsulfonyl-2-heptanol (372 mg, 1.0 mmol) and triphosgene (500 mg, 1.7 mmol) in 15 mL of anhydrous THF. A white precipitate formed. After 10 minutes, the mixture was concentrated to dryness. The residue was dissolved in 15 mL of anhydrous THF and treated with N-hydroxysuccinimide (350 mg, 3.0 mmol) and pyridine (0.25 mL, 3.0 mmol) for 15 minutes. The mixture was evaporated to dryness, and the residue was dissolved in ethyl acetate, filtered, and washed sequentially with water, 5% KHS0₄, and brine, then dried over MgS0₄, filtered, and evaporated. Chromatography on Si0₂ using a step gradient of 0, 20, 40, and 60% ethyl acetate in hexane provided the product (300 mg, 59%) as a foam which crystallized from 1: 1 ethyl acetate/hexane. ^]H-NMR (400 MHz, d₆-DMSO): d 7.89 (2H, m), 7.75 (1H, m), 7.65 (2H, m), 6.74 (1H, t, J = 5.2 Hz),

5.11 (1H, m), 3.95 (1H, dd, J = 8.8, 15.2 Hz), 3.83 (1H, dd, J = 2.8, 15.2 Hz), 2.85 (2H, q, J = 6.8 Hz), 2.81 (4H, s), 1.65 (2H, m), 1.36 (9H, s), 1.30 (2H, m), 1.15 (4H, m).

PREPARATION B

-(15-(2,4,-dinitrophenylamino)-4,7,10,13-tetraoxap

[0048] 1. N-(15-(2,4,-dinitrophenylamino)-4,7, 10,13-tetraoxapentadecanoyl) succinimide (DNP-PEGa-HSE). To a stirred solution of 15-(2,4,-dinitrophenylamino)- 4,7,10, 13-tetraoxapenta-decanoic acid (DNP-PEG₄-C0₂H, 0.060 g; 0.139 mmol) in anhydrous CH₃CN (1 mL) was added disuccinimidyl carbonate (0.041 g; 0.180 mmol) and 4- (dimethylamino) -pyridine (2.5 mg; 20.5 mmol). The reaction solution was stirred at ambient temperature for 1 hr at which time HPLC analysis showed that the starting material had been consumed and replaced a new less polar product. The solvent was evaporated and the residue taken up in CH₂C1₂ (10 mL). This solution was washed with water and brine (10 mL each) then dried over MgS0₄ and concentrated to a yellow oil (0.075 g). Purification using a RediSep^® (Teledyne) silica gel column (4 g) and eluting with 50% ethyl acetate/50% hexanes followed by 70% ethyl acetate/30% hexanes and finally 100% ethyl acetate furnished the succinimidyl ester (DNP-PEG4-HSE) as a yellow oil (0.056 g; 0.108 mmol; 77%). ^]H nmR δ (CDC1₃) 2.84 (4H, br. s), 2.88 (2H, t, J = 6.5 Hz), 3.58 (2H, q, J = 5.3 Hz), 3.65 (8H, m), 3.70 (4H, m), 3.83 (4H, m), 6.95 (1H, d, J = 9.6 Hz), 8.26 (1H, dd, J = 2.9 Hz, J = 9.6 Hz), 8.61 (1H, br. m), 9.15 (1H, d, J = 2.5 Hz). Purity (HPLC): 99% at 254 nm, 98% at 355 nm. _max = 355 nm.

[0049] 2. N,N' -bis(15-(2,4,-dinitrophenylamino)-4,7, 10, 13-tetraoxapentadecanoyl)-L- cystine. A solution of DNP-PEG4-HSE (0.054 g; 0.102 mmol) in CH₃CN (400 uL) was added to L-cystine (0.012 g; 0.050 mmol) dissolved in a mixture of 100 uL water, 100 uL 1 M NaOH, and 100 uL 1 M NaHCC>3. The reaction mixture was stirred at ambient temperature for 45 min at which time HPLC analysis (355 nm) indicated an 80.6% conversion to product. Also present was a polar impurity (16.4%) and DNP-PEG₄-C0₂H (3%). The reaction mixture was partitioned between CH₂C1₂ and 5% KHSO4 and the phases separated (the polar impurity remained in the aqueous phase). The aqueous phase was extracted with CH₂CI₂ (2 x 5 mL) and the combined organic extracts washed with water (5 mL) and dried over magnesium sulfate. The organic solution was concentrated to a yellow oil (0.039 g; 0.037 mmol; 73%). ^]H nmR δ (CDC1₃) 2.55 (4H, t, J = 5.4 Hz), 3.20 (4H, m), 3.60 (32H, m), 3.82 (4H, t, J = 5.4 Hz), 4.84 (2H, m), 6.96 (2H, d, J = 9.7 Hz), 7.48 (2H, d, J = 7.4 Hz), 8.26 (2H, dd, J = 2.6 Hz, J = 9.5 Hz), 8.80 (2H, br. m), 9.13 (2H, d, J = 2.4 Hz). Purity (HPLC): 99% at 254 nm, 98% at 355 nm. max = 355 nm.

[0050] 3. N-(15-(2,4,-dinitrophenylamino)-4,7,10,13-tetraoxapentadecanoyl)-L- cysteine. The disulfide from Step 2 (0.011 g; 10.3 mol) was dissolved in 2 mL of N₂-sparged 50 mM HEPES, 5 mM EDTA, pH 7.4. Tris(2-carboxyethyl)phosphine hydrochloride (0.005 g; 17.4 mmol) was added and the solution stirred at ambient temperature for 24 hr. HPLC analysis of the reaction solution showed complete conversion of the starting material to a new more polar product. The reaction solution was loaded onto a 1 g Cis BondElut™ column. The column was washed with 10 mL water + 0.1% TFA, followed by 10%, 20% and 50% CH₃CN in water + 0.1% TFA. The product was eluted with CH₃CN + 0.1% TFA to afford the thiol as a yellow oil (0.011 g; 20.6 mmol; 100%). Purity (HPLC): 99% at 254 nm, 98% at 355 nm. max = 355 nm. MS [M+H]⁺ 535.1.

Example 1

3-maleimido-4,4,4-trifluorobutanoic acid

Compound of Formula (I) wherein R¹⁰ = CF₃; R¹⁰ = H; p = 1 ; Y is absent; n = 0;

and Z is a carboxylic acid

[0051] 1. N-r3-hydroxycarbonyl-l-trifluoromethyllmaleamic acid: 3-Amino-4,4,4,- trifluoromethyl butanoic acid (0.217 g; 1.38 mmol) was partially dissolved in glacial acetic acid (1.4 mL) and maleic anhydride (0.135 g; 1.38 mmol) was added. The reaction mixture was stirred at room temperature for 5 hr. TLC analysis (100% ethyl acetate) showed consumption of the ninhydrin reactive baseline spot and the appearance of a KMn0₄ reactive streak (R_f ~ 0.28). The reaction mixture was concentrated and CH2CI2 (14 mL) was added to precipitate the product. The solid was collected by filtration, washed with CH2CI2 and dried under high vacuum to give the maleamic acid as a white sold (0.262 g; 1.02 mmol; 74%). ^]H nmR δ (J6-DMSO) 2.54 (1H, dd, J = 9.0 Hz, J = 16.5 Hz), 2.75 (1H, dd, J = 5.0 Hz, J = 16.6 Hz), 4.89 (1H, m), 6.22 (1H, d, J = 12.1 Hz), 6.28 (1H. d, J = 12.1 Hz), 8.97 (1H, d, J = 8.9 Hz), 12.2 (2H, br. m).

[0052] 2. 3-maleimido-4,4,4-trifluorobutanoic acid. The maleimide (0.108 g;

0.423 mmol) was suspended in anhydrous toluene (4.4 mL) and triethylamine (294 mL; 2.11 mmol) and 4 A molecular sieves (0.35 g) added. The reaction mixture was heated at gentle reflux for 6 hr (substrate dissolves on heating) at which time TLC analysis (20: 1 CHCVMeOH + 1 % AcOH) showed that the starting material had been consumed and replaced by a less polar product. After cooling to room temperature the reaction solution was concentrated to a pale yellow oil (0.108 g). Purification using a SiliCycle^® 4 g silica gel column and eluting with 20: 1 CHC^/MeOH + 1% AcOH gave the product as a white solid (0.061 g; 0.259 mmol; 61%). ^]H nmR (J6-DMSO) δ 2.98 (1H, dd, J = 5.3 Hz, J = 17.2 Hz), 3.32 (2H, m), 4.99 (1H, m)7.18 (2H, s), 12.83 (1H, s). TLC R_f = 0.63 (20/1 CHCl₃/MeOH + 1% AcOH; KMn0₄).

Example 2

Compound of Formula (I) wherein R¹⁰ = CF^; R¹⁰ = H; p = 1 ; Y is CO-NH; n = 2;

and Z is a protected amine, subsequently deprotected

[0053] To a stirred solution of the maleimide of Example 1 (0.025 g; 0.105 mmol), N- Boc-ethylenediamine (0.017 g; 0.106 mmol) and triethylamine (29.5 mL; 0.211 mmol) in anhydrous acetonitrile (0.5 mL) was added HBTU (0.040 g; 0.106 mmol). The reaction mixture was stirred at room temperature for 2 hr. TLC analysis (70% ethyl acetate/30% hexanes; KMn0₄) showed that the starting material had been consumed and replaced by a less polar product. The solution was concentrated to a dark oil and purified using a 4 g SiliCycle silica gel column and eluting with 50% ethyl acetate/50% hexanes to furnish the product as a colorless oil (0.025 g; 0.068 mmol; 64%). 1H nmR (c/6-OMSO): δ 1.36 (9H, s), 2.91 (4H, m), 3.16 (2H, m), 5.04 (1H, m), 6.74 (1H, br. m), 7.14 (2H, s), 8.16 (1H, br. m). TLC: R_f = 0.57 (70% EtOAc/30% hexanes; KMn0₄).

[0054] The Boc protecting group was removed by treatment with 1 : 1 CH₂C1₂ to provide the amine as the trifluoroacetate salt.

Example 3

Compound of Formula (I) w^rherein both R¹⁰ = H; p = 2; Y is NR¹³?; each R¹³ is the alkylene

-CH7CH7-; and Z is a protected amine, subsequently deprotected

[0055] Prepared by the method described in Example 1 starting from 1-tert- butyloxycarbonyl-4-(3-aminopropyl)piperazine. The intermediate did not precipitate from CH₂C1₂ so it was concentrated from 1 : 1 CH₂Cl₂/heptane (3 x 5 mL) to a sticky solid. The maleimide was purified using a 4 g SiliCycle*^' silica gel column and eluting with 50% ethyl acetate/50% hexanes to give a yellow oil (0.121 g; 0.372 mmol; 88%). 1H nmR (CDC1₃): δ 1.45 (9H, s), 1.76 (2H, m), 2.34 (6H, br. m), 3.38 (4H, br. m), 3.58 (2H, t, J = 5.0 Hz), 6.69 (2H, s). TLC: R_f = 0.62 (100% EtOAc; Mn0₄).

[0056] The Boc protecting group was removed by treatment with 1 : 1 CH₂C1₂ to provide the amine as the trifluoroacetate salt.

Example 4

Compound of Formula (I) wherein both R^so = H; p = 1 ; Y is NR¹³?: each R¹³ is -CPLCFL-;

and Z is a protected amine, subsequently deprotected

[0057] Prepared by the method described in Example 1 starting from 1-tert- butyloxycarbonyl-4-(3-aminoethyl)piperazine. The Boc protecting group was removed by treatment with 1 : 1 CH₂C1₂ to provide the amine as the trifluoroacetate salt.

Example 5

Compound of Formula (I) wherein both R^so = H; p = 1; Y is NR¹³ _Z; each R¹³ is -CH₂CH ;

and Z is a derivatized amine

[0058] The amine salt of Example 4 is derivatized by dissolving in acetonitrile, neutralized with N,N-diisopropylethylamine, and reacted with glutaric anhydride. After acidification, the crude product is isolated by chromatography on silica gel.

Example 6

Compound of Formula (I) wherein both R^so = H; p = 2; Y is N^"R^L\; one R¹³ is C¾ and the other two are -CH?CH?-; n = 2; and Z is a protected amine, subsequently deprotected

[0059] l-(Tert-butoxycarbonyl)-4-(3-maleimidopropyl)piperazine (Example 3; 0.050 g; 0.155 mmol) was dissolved in anhydrous CH₃CN (1 niL) and iodomethane (1 mL;

16.1 mmol) added. The solution was stirred at ambient temperature for 24 hr at which time TLC analysis (1 : 1 ethyl acetate/ hexanes; ΚΜη0₄) showed that the starting material had been consumed and replaced by a baseline product spot. The solvent was evaporated and the product dried under high vacuum to produce the quaternary ammonium salt as an orange solid (0.066 g; 0.142 mmol; 92%). ⁵H nmR (dtf-DMSO): δ 1.41 (9H, s), 1.90 (2H, br. m), 3.04 (3H, s), 3.34-3.53 (8H, br. m), 3.72 (2H, br. m).

[0060] The Boc protecting group is removed by treatment with 1 : 1 CH₂C1₂ to provide the amine as the trifluoroacetate salt. Example 7

Compound of Formula (I) wherein both R¹⁰ = H; p = 1 ; Y is NR¹³: R¹³ is C¾; n = 2;

and Z is a protected amine, subsequently deprotected

[0061] Prepared by the method described in Example 1 starting from {2-[(2-aminoethyl)- methylamino]ethyl}carbamic acid, t-butyl ester. The intermediate did not precipitate from CH₂CI₂ so it was concentrated from 1 : 1 Cl^C^/heptane (3 x 5 mL) to a sticky solid. The maleimide was purified using a 4 g SiliCycle^® silica gel column and eluting with 50% ethyl acetate/50% hexanes to give a yellow oil (0.090 g; 0.303 mmol; 72%). ^]H nmR (CDC1₃): δ 1.44 (9H, s), 2.25 (3H, s), 2.47 (2H, t, J = 5.9 Hz), 2.57 (2H, t, J = 6.4 Hz), 3.14 (2H, br. m), 3.61 (2H, t, J = 6.4 Hz), 4.90 (1H, br. s), 7.00 (2H, s). TLC: R_f = 0.50 (50% EtOAc/50% hexanes; KMn0₄).

[0062] The Boc protecting group is removed by treatment with 1 : 1 CH₂CI₂ to provide the amine as the trifluoroacetate salt.

Example 8

Compound of Formula (I) wherein both R¹⁰ = H; p = 1 ; Y is N⁺R¹³ ₂; each R¹³ is CT¾; n = 2; and Z is a protected amine, subsequently deprotected, subsequently deprotected

[0063] Prepared as described in Example 6 on a 0.138 mmol scale to yield the quaternary ammonium salt as an orange solid (0.044 g; 0.100 mmol; 72%). ^]H nmR d (J6-DMSO) 1.39 (9H, s), 3.12 (6H, s), 3.38 (4H, br. m), 3.49 (2H, t, J = 7.1 Hz), 3.83 (2H, t, J = 7.0 Hz), 7.13 (2H, s), 7.19 (1H, br. m).

[0064] The Boc protecting group is removed by treatment with 1 : 1 CH₂CI₂ to provide the amine as the trifluoroacetate salt. Example 9

Compound of Formula (I) wherein R¹⁰ = CO7H; R¹⁰ = H; p = 0; Y is absent; n = 2;

and Z is a protected amine, subsequently deprotected

[0065] Prepared by the method described in Example 1 starting from 2-amino-4-(tert- butoxycarbonylamino)butyric acid. The maleimide was purified using a SiliCycle^® 4 g silica gel column and eluting with 20: 1 CHCl₃/MeOH + 1% AcOH to give pale yellow oil (0.070 g; 0.237 mmol; 56%). ^]H nmR (J6-DMSO): δ 1.35 ((H, s), 2.09 (2H, m), 2.89 (2H, br. m), 4.55 (1H. dd, J = 5.4 Hz, J = 10.3 Hz), 6.79 (1H, br. t, J = 5.6 Hz), 7.09 (2H, s), 13.1 (1H, br. s). TLC: R_f = 0.60 (20/1 CHCl₃/MeOH + 1% AcOH; KMn0₄).

[0066] The Boc protecting group is removed by treatment with 1 : 1 CH₂CI₂ to provide the amine as the trifluoroacetate salt.

Example 10

3-maleimidopropionic (2-(tert-butoxycarbonylamino)ethyl)amide wherein both R¹⁰ are H, p = 1, Y = CONH, n = 2, Z = protected amine, subsequently deprotected

[0067] To a stirred suspension of 3-maleimidopropionic acid NHS ester (0.100 g;

0.376 mmol) in anhydrous CH₃CN (2 mL) was added N-Boc-ethylenediamine (59 uL;

0.373 mmol). The resulting solution was stirred at room temperature for 1 hr at which time TLC analysis (70% ethyl acetate/30% hexanes; KMn0₄) showed conversion of the starting material to a new more polar product. The solvent was evaporated and the residue purified using a 4 g SiliCycle^® silica column and eluting with 50% ethyl acetate/50% hexanes followed by 70% ethyl acetate/30% hexanes to furnish the amide as a white solid (0.088 g; 0.282 mmol; 75%). ^]H nmR (CDC1₃): δ 1.44 (9H, s), 2.49 (2H, t, J = 7.2 Hz), 3.24 (2H, m), 3.32 (2H, m), 3.82 (2H, t, J = 7.0 Hz), 4.95 (1H, br. s), 6.34 (1H, br. s), 6.70 (2H, s). TLC: R_f = 0.61 (70% EtO Ac/30% hexanes; KMn0₄). Example 11

3-maleimidoacetic (2-(tert-butoxycarbonylamino)ethyl)amide wherein R¹⁰ is H, p = O, Y = CONH, n = 2, Z is a protected amine, subsequently deprotected

[0068] To a stirred suspension of maleimidoacetic acid NHS ester (10 mg; 39.7 umol) in anhydrous CH₃CN (0.25 mL) was added N-Boc-ethylenediamine (6.3 uL; 39.8 umol). The resulting solution was stirred at room temperature for 1 hr at which time TLC analysis (70% ethyl acetate/30% hexanes; KMn0₄) showed conversion of the starting material to a new more polar product. The solvent was evaporated and the residue redissolved in ethyl acetate and washed with water, 5% KHS0₄, water, and brine to furnish the amide as a white solid (13 mg). ^]H nmR (CDC1₃): δ 1.44 (9H, s), 3.26 (2H, m), 3.36 (2H, m), 4.17 (2H, s), 4.88 (1H, br. s), 6.79 (2H, s), 6.83 (1H, br s). TLC: R_f = 0.52 (70% EtOAc/30% hexanes;

KMn0₄).

Example 12

3-amino-2-maleimido-propionic ac iidd,, wwhheerreeiinn oo:ne R is COOH, one R is H, p is 1,

Y is absent, n is 0 and Z is NH^

[0069] Equal molar quantities of 2-amino-3-(Boc-amino)propionic acid and maleic anhydride are stirred in acetic acid at ambient temperature. After concentration under reduced pressure, the product is precipitated by addition of dichloromethane and dried. This is heated with triethylamine to close the maleimide ring. The Boc group is removed by treatment with 1 : 1 dichloromethane/trifluoroacetic acid, and the product salt is precipitated and washed with ethyl ether.

Example 13

Compound of Formula (II)

[0070] As an illustration, an amide-linked drug-maleimide conjugate is prepared by coupling of monomethyl auristatin E (MMAE) with a maleimide of formula (I) wherein Z is an active ester. Thus, the compound of Example 5 is first converted into the succinimidyl ester by treatment with Ν,Ν'-disuccinimidyl carbonate and 4-(dimethylamino)pyridine, or alternately by treatment with N-hydroxysuccinimide and dicyclohexylcarbodiimide. The succinimidyl ester is then reacted with MMAE to form the compound of formula (II).

Example 14

Preparation of Antibody-Drug Conjugates

[0071] The generation of antibody-drug conjugates entails the partial reduction of the mAb disulfide bonds followed by reaction of the resulting thiols with a maleimide-drug conjugate of formula (II), for example a compound as illustrated in Example 13. In a typical procedure, the antibody (-10 mg/mL) is partially reduced by addition of 3 equivalents of DTT at 37°C for 2 hours. The reduction reaction is then applied to a PD-10 desalting column to remove excess DTT. The thiol content in the partially reduced mAb is determined using Ellman's reagent. The compound of formula (II) is added to the reduced mAb at -1.2 equivalents per free thiol, and the conjugation reaction allowed to proceed at 4°C for approximately 1 h. The reaction mixture is then applied to a PD-10 desalting column to isolate the antibody-drug conjugate.

Example 15

Compound of Formula (III) wherein R¹ is PhSO?; ² is H; m = 0; one R⁵ is H and the other is

(CHTkNHBoc; both R¹⁰ are H; p = 0; Y is NHCO; and n = 2

[0072] The preparation of compounds of formula (III) is illustrated. Triethylamine (15 uL, 0.11 mmol) is added to a stirred mixture of 7-(ieri-butoxycarbonylamino)- l-phenylsulfonyl-2-heptyl succinimidyl carbonate (55 mg, 0.11 mmol) and N- ((2-aminoethyl)aminocarbonyl) methylmaleimide trifluoroacetate salt (40 mg, 0.13 mmol) in 2 mL of THF. After 1 h, the mixture is diluted with ethyl acetate and washed sequentially with 0.5 M citric acid, water, sat. aq. NaHCC>3, water, and brine, then dried over MgS0₄, filtered, and evaporated to provide the product.

Example 16

Synthesis of thioethers

[0073] 3-Maleimidoacetic (2-(tert-butoxycarbonylamino)ethyl)amide (1.5 mg, 5.1 umol; Example 12) was added to a solution of N-(15-(2,4,-dinitrophenylamino)-4,7, 10,13- tetraoxapentadecanoyl)-L-cysteine (4.23 umol; Preparation B) dissolved in 1 mL of

N₂-sparged 50 mM HEPES, 5 mM EDTA, pH 7.4. After 2 h, HPLC showed 95% conversion to product, and the mixture was loaded onto a 1 g Cis BondElut™ extraction column. The column was washed with 10 mL of water/0.1% TFA followed by 20% and 50%

CH₃CN/H₂O/0.1% TFA. The product was then eluted with 100% CH₃CN/0.1% TFA and evaporated to yield the product as an orange oil (4.8 mg). Purity (HPLC): 95% (254 nm), 93% (355 nm). Example 17

Kinetics of thioether exchange and hydrolysis

[0074] A solution of oxidized glutathione (GSSG, 5.0 mM) in 50 mM HEPES, 0.5 mM EDTA, was adjusted to pH 7.61 at ambient temperature. Given the temperature coefficient of HEPES, this gives a solution buffered at pH 7.4 at 37 °C. A solution of the thioether

(Example 16) in acetonitrile (5.3 mM, 28 uL) was added to 1500 uL of the GSSG solution, and the mixture was divided into 3 equal aliquots that were incubated in a 37°C water bath and analyzed periodically by HPLC using a C₁₈ column and a water/acetonitrile/0.1 % TFA gradient. Ring-opened hydrolysis products eluted at 8.75 (major) and 8.95 (minor) min, while the starting thioether eluted at 9.55 min. The mixed disulfide of glutathione and DNP- PEG₄-Cys was observed at 6.35 min. Kinetic data were fit for ki (pseudo-first order rate constant for ring-opening hydrolysis) and k₂ (pseudo-first order rate constant for thiol exchange) to the kinetic scheme of Baldwin & Kiick (Bioconjugate Chemistry 22(10)- 1946-53 (2011). Fractional conversions X ([species]/[Mal-SR]o) were calculated as the ratios of HPLC peak areas for each species to the sum of the peak areas of the three species, such that the final equations fit were:

X(Mal-SR) = e^"at

X(ring-open) = Qi · (1 - e^"at)

X (total exchanged) = (1 - Qj) · (1 - e^"at),

where a = ki + k₂

Q₂ = k₂/(k₃ - a)

[0075] The final equation yields the total fraction of thiol exchange in the limit where t ->∞:

Total exchange = 1 - Qi = k₂/(k] + k₂).

[0076] Kinetic parameters from the three aliquots were averaged to give the final results, with half-lives calculated as ln(2)/k. Similar experiments were performed using 2.5 mM GSSG or 5.0 mM GSH. Table 1

Kinetics of Hydrolytic Ring-Opening

Maleimide k_RO (b/¹) tl/2 (h)

N-ethyl 0.003 220

2-aminoethyl 0.691 1.0

Example 2 0.1 7.6

Example 3 0.064 10.8

Example 4 0.4 1.7

Example 6 0.11 6.4

Example 7 1.7 0.4

Example 8 0.424 1.6

Example 9 0.0036 193

Example 10 0.0233 30

Example 11 0.16 4.5

[0077] In subsequent determinations using the same methods as set forth above but further including the compounds of subsequent Examples 18-20, the following results were obtained. Larger values of Qi in column 4 of Table 2 indicate better ratios of ring opening hydrolysis to file exchange.

Table 2

Maleimide ti/2 (RO) ti 2 (exchange) Qi

N-ethyl 220 400 0.65

2-aminoethyl 0.42 12 0.97

Example 2 4.1 69 0.77

Example 3 12 180 0.93

Example 4 1.6 61 0.97

Example 6 6.2 67 0.92

Example 7 0.37 36 0.99

Example 8 1.6 29 0.95

Example 9 220 690 0.77

Example 10 30 220 0.88

Example 11 4.5 87 0.92 Maleimide ti/2 (RO) ti/2 (exchange) Qi

Example 18 4.1 69 0.94

Example 19 1 1 150 0.93

Example 20 14.5 nd

Example 18

(2i? -2-(2.5-Dioxo-2.5-dihvdro- 1 H-pyrrol- 1 -yl)-N-(2-methoxyethyl)- 3 -(methylsulfanyl)propanamide

[0078] (2i?)-2-(2,5-Dioxo-2,5-dihydro- lH-pyrrol- l-yl)-3-(methylsiilfanyl)propanic acid and (2i?)-2-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N-(2-methoxyethyl)-3-(methylsulfanyl)- propanamide: The acid was prepared according to the procedure of Example 1, starting with S-methyl cysteine. Maleamic diacid: 1H NMR (300 MHz, DMSO-d6) δ 13.41 (br. s, 2 H), 9.26 (d, J=7.9 Hz, 1 H), 6.41 (d, 5=12 A Hz, 1 H), 6.31 (d, J=12.3 Hz, 1 H), 4.51 (td, J=8.1, 5.0 Hz, 1 H), 2.92 (dd, J=13.8, 4.9 Hz, 1 H), 2.78 (dd, J=13.8, 8.2 Hz, 1 H), 2.09 (s, 3 H). Maleimide acid: ^!H NMR (300 MHz, DMSO-d6) δ 7.15 (s, 2 H), 4.78 (dd, J=l 1.5, 4.7 Hz, 1 H), 3.15 (dd, J=14.1, 4.9 Hz, 1 H), 3.03 (dd, J=14.1 , 1 1.3 Hz, 1 H), 2.01 (s, 3 H).

[0079] The acid was converted to the amide as follows. To a cold (0°C) solution of the acid (0.150 g, 0.697 mmol, 1 equiv) prepared above in CH₂C1₂ (3 mL) was added

2-methoxyethylamine (64 μΐ,, 0.73 mmol, 1.1 equiv), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (0.543 g, 2.80 mmol, 4 equiv), N-hydroxysuccinimide (0.160 g, 1.39 mmol, 2 equiv). The reaction mixture was allowed to warm to ambient temperature and was stirred at ambient temperature for 16 h. Additional methoxyethylamine (32 μί, 0.366 mmol, 0.55 equiv) was added and the reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was diluted with CH₂C1₂ (20 mL) and 5% aq KHSO₄ (30 mL, resulting aqueous phase pH 1-2). The aqueous phase was separated and extracted with C¾C1₂ (3 x 20 mL) and 10% MeOH/CH₂Cl₂ (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over MgS0₄, filtered, and

concentrated to afford an orange semi-solid. Purification via column chromatography (12 g silica gel cartridge; stepwise gradient elution 50%, 75%, 100% EtOAc) afforded the desired amide (0.130 g, 68%) as a pale yellow semi-solid. Ή NMR (300 MHz, DMSO-t ₆) δ ppm 8.22 (t, J=5.3 Hz, 1 H), 7.11 (s, 2 H), 4.63 (dd, J=l 1.7, 4.7 Hz, 1 H), 3.27 - 3.34 (m, 3 H), 3.21 - 3.24 (m, 3 H), 3.14 - 3.21 (m, 2 H), 2.98 - 3.10 (m, 1 H), 1.99 (s, 3 H).

Example 19

4-[2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethanesu^

[0080] Step 1 : To a solution of 10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (0.250 g, 1.51 mmol, 1 equiv) in MeCN (10 mL) was added ₂C0₃ (1.04 g, 7.55 mmol, 5 equiv), and 4-(2-chlorethylsulfonyl)butyric acid (0.357 g, 1.67 mmol, 1.1 equiv). The reaction mixture was stirred at ambient temperature for 16 h, diluted with CH₂C1₂ (20 mL) and 5% aq KHS0₄ (20 mL). The aqueous phase was separated and extracted with CH₂C1₂ (2 x 10 mL), extracted with 10% MeOH/CH₂Cl₂ (2 x 10 mL). The combined organic phases were washed with brine (20 mL), dried over MgS0₄, filtered, and concentrated to afford the desired sulfone (0.212 g, 41% yield, 63:37 mixture of endo.exo isomers) as a white solid. ⁵H NMR (300 MHz, DMSO-i/6) δ 12.27 (br. s., 1 H), 6.41 (t, J = 0.8 Hz, 2 H), 5.29 - 5.34 (m, 2 H), 3.76 (dd, J = 8.3, 6.4 Hz, 1 H), 3.57 - 3.63 (m, 1 H), 3.55 (dd, J = 3.8, 1.7 Hz, 2 H), 3.26 - 3.36 (m, 2 H), 3.14 - 3.23 (m, 2 H), , 2.38 (td, J = 7.3, 2.8 Hz, 2 H), 1.87 (m, 2 H).

Resonances corresponding to the minor isomer were observed". 6.55 (t, J= 0.9 Hz, 2 H), 5.14 (s, 2 H), 2.95 (s, 2 H). This material was used in the next step without further purification.

[0081] Step 2: To a cooled solution (0°C) of the carboxylic acid prepared above (0.100 g, 0.291 mmol, 1 equiv) and 2-methoxyethyl amine (28 iL, 24 mg, 0.320 mmol, 1.1 equiv) in CH₂C1₂ (1 mL) was added Et₃N (0.25 mL, 0.183 g, 1.75 mol„ 6 equiv), N- (3-dimethylaminopiOpyl)-N'-ethylcarbodiimide hydrochloride (0.223 g, 1.16 mmol, 4 equiv) and N-hydroxysuccinimide (0.134 g, 0.582 mmol, 2 equiv). The reaction mixture was allowed to warm to ambient temperature and was stirred at ambient temperature for 20 h. Additional 2-methoxyethyl amine (28 μί, 24 mg, 0.320 mmol, 1.1 equiv) was added and the reaction mixture was stirred at ambient temperature for 3 h. The reaction mixture was then diluted with CH₂C1₂ (20 mL) and 5% aq KHSO₄ (20 mL). The aqueous layer was separated and extracted with 10% MeOH/CH₂Cl₂ (5 x 15 mL). The combined organic phases washed with brine (20 mL), dried over MgS0₄, filtered, and concentrated to afford a light pink oil. Purification via column chromatography (4 g silica gel cartridge; gradient elution 5-10% MeOH/CH₂Cl₂) afforded the desired amide (0.070 g, 60%) as a white semi-solid. ^SH NMR (300 MHz, DMSO-*¼) δ 7.95 (br. s., 1 H), 6.45 (s, 2 H), 5.32 (m, J = 3.6 Hz, 2 H), 3.75 (t, J = 7.3 Hz, 1 H), 3.59 - 3.62 (m, 1 H), 3.55 (dd, J = 3.7, 1.6 Hz, 2 H), 3.26 - 3.36 (m, 2 H), 3.23 (s, 3 H), 3.10 - 3.22 (m, 6 H), 2.18 - 2.26 (m, 2 H), 1.86 (ap. quin, J = 7.8 Hz, 2 H). Resonances corresponding to the minor isomer were observed: 6.55 (s, 2 H), 5.14 (s, 2 H), 2.95 (s, 2 H).

[0082] Step 3: A solution of the amide (0.070 g, 0.174 mmol, 1 equiv) prepared above in 1 : 1 MeCN:toluene (6 mL) was heated at reflux for 18 h and then concentrated. The resulting oil was suspended and concentrated twice from Et₂0 (10 mL) to afford the desired maleimide (0.055 g, 95%) as an off-white solid. 1H NMR (300 MHz, Acetone-d₆) δ 7.15 (br. s., 1 H), 6.91 (s, 2 H), 3.98 (t, J = 6.9 Hz, 2 H), 3.30 - 3.44 (m, 6 H), 3.27 (s, 3 H), 3.14 - 3.23 (m, 2 H), 2.77 - 2.87 (m, 2 H), 2.37 (t, J = 7.0 Hz, 2 H).

[0083] Step 1. tert-Butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 4-(diethylamino)-4-oxobutanoate. A 20-mL vial was charged with Fmoc-Asp(O^tBu)-OH (500 mg, 1.22 mmol, 1 equiv), DMF (3 mL), iPr₂NEt (0.45 mL 2.4 mmol, 2 equiv), and HATU (456 mg, 1.22 mmol, 1 equiv). The reaction mixture was stirred at ambient temperature for 15 min then Et₂NH (0.13 mL, 1.22 mmol, 1.0 equiv) was added dropwise over 5 min. The reaction mixture was stirred at ambient temperature for 30 min then diluted with EtOAc (100 mL) and H20 (30 mL). The organic phase was separated and washed with H20 (3x30 mL) and brine (30 mL), dried over MgS0₄, filtered, and concentrated to afford a yellow oil. Purification via column chromatography (12 g silica gel cartridge; stepwise gradient elution, 20%, 30%, 40% EtOAc/hexanes) afforded 512 mg (90%) of a colorless viscous oil. ^]H NMR (CDC1₃, 300 MHz) δ 7.77 (d, J=7.5 Hz, 2 H), 7.56 - 7.64 (m, 2 H), 7.41 (t, J=7.3 Hz, 2 H), 7.31 (t, J=7.3 Hz, 2 H), 5.63 (d, J=9.6 Hz, 1 H), 4.92 - 5.06 (m, 1 H), 4.31 - 4.46 (m, 2 H), 4.19 - 4.28 (m, 1 H), 3.46 - 3.64 (m, 2 H), 3.19 - 3.46 (m, 2 H), 2.71 (dd, J=15.4, 6.4 Hz, 1 H), 2.54 (dd, J=15.4, 6.6 Hz, 1 H), 1.44 (s, 9 H), 1.22 - 1.30 (m, 3 H), 1.14 (t, J=7.2 Hz, 3 H). LRMS (ESI) m/z [M+Na]⁺ calcd for C27H₃4N2O5: 489.2; found: 489.8. C18 HPLC was monitored at 265 nm: >98% (0-100% B; R_T = 10.7 min).

[0084] Step 2. tert-Butyl 3-amino-4-(diethylamino)-4-oxobutanoate. A 20-mL vial was charged with tert-Butyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(diethylamino)- 4-oxobutanoate (512 mg, 1.10 mmol, 1 equiv), DMF (6 mL), and 4-methyl piperidine (1.8 mL, 15 mmol, 14 equiv). The reaction mixture was stirred at ambient temperature for 30 min and diluted with 10% MeOH/DCM (100 mL) and H₂0 (30 mL). The aqueous layer was separated and extracted with 10% MeOH/DCM (3 x 50 mL). The combined organic phases were dried over MgS0₄, filtered, and concentrated to afford a yellow oil. Purification via column chromatography (12 g silica gel cartridge, stepwise gradient elution with 5% MeOH/DCM, 10% MeOH/DCM; 30% acetone/DCM) afford 189 mg (70%) of a pale-yellow semi-solid. ^]H NMR (CDC1₃, 300 MHz) δ 3.97 - 4.17 (m, 1 H), 3.19 - 3.56 (m, 4 H), 2.36 - 2.63 (m, 2 H), 1.38 - 1.52 (m, 9 H), 1.18 - 1.30 (m, 3 H), 1.06 - 1.17 (m, 3 H). LRMS (ESI) m/z [M+Na]⁺ calcd for C₁₂H24N₂0₃: 267.2; found: 267.6.

[0085] Step 3. (Z)-4-((4-(tert-butoxy)-l-(diethylamino)-l,4-dioxobutan-2-yl)amino)- 4-oxobut-2-enoic acid. A 20-mL vial was charged with tert-butyl 3-amino-4-(diethylamino)- 4-oxobutanoate (189 mg, 0.774 mmol, 1 equiv), maleic anhydride (76 mg, 0.77 mmol, 1 equiv), DCM (2 mL), and iPr₂NEt (0.27 mL, 1.6 mmol, 2 equiv). The reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was diluted with DCM (20 mL) and 2.5% aq KHSO₄ (20 mL). The aqueous layer was separated and extracted with DCM (3 x 20-mL). The combined organic phases were washed with brine (30 mL), dried over MgS0₄, filtered, and concentrated to afford 215 mg (81%) of an orange oil which was used in the next step without further purification. ^]H NMR (CDC13, 300 MHz) δ 6.40 (s, 2 H), 5.21 - 5.32 (m, 1 H), 3.48 - 3.63 (m, 2 H), 3.35 - 3.48 (m, 1 H), 3.20 - 3.34 (m, 1 H), 2.73 (dd, J=15.6, 5.8 Hz, 1 H), 2.60 (dd, J=15.4, 6.6 Hz, 1 H), 1.45 (s, 9 H), 1.28 (d, J=0.6 Hz, 3 H), 1.14 (t, J=6.2 Hz, 3 H); LRMS (ESI) m z [M+H]⁺ calcd for C₁₆H₂₆N₂0₆: 343.2; found 343.7. C18 HPLC was monitored at 254 nm: >98% (0-100%B, R_T = 8.94 min).

[0086] Step 4. Tert-butyl 3-(N,N-diethylcarboxamido)-3-maleimidopropionate. A 20- niL vial was charged with (Z)-4-((4-(tert-butoxy)-l-(diethylamino)-l,4-dioxobutan- 2-yl)amino)-4-oxobut-2-enoic acid (215 mg, 0.628 mmol, 1 equiv), DCE (1 mL), acetic anhydride (0.29 mL, 3.1 mmol, 5 equiv), and sodium acetate (63 mg, 0.77 mmol, 1.3 equiv). The reaction mixture was heated at 80°C for 2 h, allowed to cool to ambient temperature, and diluted with DCM (20 mL) and H20 (20 mL). The aqueous layer was separated and extracted with DCM (3x20 mL). The combined organic phases were washed with brine (20 mL), dried over MgS0₄, filtered, and concentrated to afford a brown oil. Purification via column chromatography (12 g silica gel cartridge; stepwise gradient elution 20%, 30%, 40%, 50% EtOAc/hexanes) afforded 141 mg (56%) of an yellow oil. 1H NMR (CDC1₃, 300 MHz) δ 6.73 (s, 2 H), 5.25 (dd, J=8.9, 6.2 Hz, 1 H), 3.18 - 3.49 (m, 4 H), 3.13 (dd, J=16.4, 8.9 Hz, 1 H), 2.95 (dd, J=16.2, 6.0 Hz, 1 H), 1.42 (s, 9 H), 1.15 (t, J=7.5 Hz, 3 H), 1.10 (t, J=7.2 Hz, 3 H). LRMS (ESI) m/z [M+Na]⁺ calcd for ^Η₂₄Ν₂0₅: 347.2; found: 347.6. C 18 HPLC monitored at 295 nm: 98% (0-100%B, R_T = 9.50 min).

Example 21

di-/er/-butyl 2-maleimidosuccinate

[0087] Step 1. Maleamic acid: This compound was prepared according to the procedure of Example 20 to afford 503 mg (82%) of a colorless foamy oil. 1H NMR (CDC1₃, 300 MHz) δ 6.43 (d, J=12.8 Hz, 1 H), 6.29 (d, J=12.8 Hz, 1 H), 4.69 (dt, J=7.7, 4.3 Hz, 1 H), 2.99 (m, J=17.3, 4.1 Hz, 1 H), 2.81 (dd, J=17.3, 4.1 Hz, 1 H), 1.49 (s, 9 H), 1.45 - 1.47 (m, 9 H). LRMS (ESI) m/z [M+Na]⁺ calcd for ^Η₂₅Ν0₇: 366.2; found: 366.6. C18 monitored at 250 nm: >98% (0-100%B; R_T = 8.53 min).

[0088] Step 2. Di-tert-butyl 2-maleimidosuccinate: This compound was prepared according to the procedure above (Ac₂0 NaOAc). Purification via column chromatography (12 g silica gel column; stepwise gradient elution 10%, 15%, 20% EtOAc/hexanes) afforded 205 mg (44%) of an orange semi-solid. 1H NMR (CDC1₃, 300 MHz) δ 6.74 (s, 2 H), 5.05 (dd, J=9.4, 5.7 Hz, 1 H), 3.12 (dd, J=16.2, 5.7 Hz, 1 H), 2.93 (dd, J=16.4, 9.4 Hz, 1 H), 1.42 (s, 9 H), 1.41 (s, 9 H). LRMS (ESI) m/z [M+Naf calcd for C₁₆H₂₃N0₆: 348.2; found: 348.6. C18 monitored at 292 nm: 97% (0-100%B, R_T = 10.83 min).

Example 22

2-maleimidosuccinic bis-[N-metbyl-N-3,6,9-trioxa-9-oxononyllamide

[0089] A solution of di-/<?r/-butyl 2-maleimidosuccinate (Example 21) in CH₂C1₂ is treated with an equal volume of trifluoroacetic acid then concentrated to yield the

2-maleimidosuccinic acid. This is coupled with 2 equivalents of tert-butyl 9-(methylamino)- 4,7-dioxanonanoate in the presence of a coupling reagent such as HATU

((l-[Bis(dimethylamino)methylene]-lH-l ,2,3-triazolo[4,5-b]pyridinium 3- oxid

hexafluorophosphate). The resulting di-/er/-butyl ester is deblocked by treatment with 1 : 1

Example 23

N-methyl N-3,6,9-trioxa-9-oxononyl 3-(N,N-diethylcarboxamido)-3-maleimidopropionamide

[0090] A solution of tert-Butyl 3-(diethylcarboxamido)-3-maleimidopropionate (Example 20) in CH₂C1₂ is treated with an equal volume of trifluoroacetic acid then concentrated to yield the amido-acid. This is coupled with tert-butyl 9-(methylamino)-4,7-dioxanonanoate in the presence of a coupling reagent such as HATU ((l-[Bis(dimethylamino)methylene]-lH- l ,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate). The resulting terZ-butyl ester is deblocked by treatment with 1 : 1 CF3CO2H/CH2CI2.

Claims

Claims

1. A maleimide reagent of the formula

wherein p = 0-2;

each R¹⁰ is independently H, CF₃, (CH₂)_p(C=0)R¹², (CH₂)_pC(=0)ORⁿ,

(CH₂)_pC(=0)NR¹² ₂, CN, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, (CH₂)_pS0₂Rⁿ, (CH₂)_pSRⁿ, (CH₂)_pS0₂Rⁿ, CH₂NR¹³ ₂ or CH₂N⁺R¹³ ₃;

Y is C=0, C(=0)NR¹², alkynyl, S0₂, S0₂NR¹², (CF₂)_q, NR¹³, NR¹³ ₂, N⁺R¹³ ₂, N⁺R^1: or is absent;

q = l-4;

each R¹¹ is independently C1-C15 alkyl, aryl, heteroalkyl (1-15 joined members) or heteroaryl;

each R¹² is independently H, C1-C15 alkyl, aryl, heteroalkyl (1-15 joined members) heteroaryl;

each R¹³ is independently C1-C15 alkyl or heteroalkyl (1-15 joined members) or in NR¹³ ₂ or N⁺R¹³3 two R¹³ can independently be C1-C15 alkylene or heteroalkylene (1-15 joined members) and optionally form a ring with 3-8 members and the third R¹³ on N⁺R¹³3 C1-C15 alkyl or heteroalkyl (1-15 joined members);

at least one of R¹⁰ or Y is an electron withdrawing group;

m = 0 or 1 ;

linker is C1-C15 alkylene or heteroalkylene (1-15 joined members);

Z is a functional group for attachment to another molecule which Z is optionally included in Y or R¹³ ;

with the proviso that when Y is C(=0)NR or is absent at least one of R¹⁰ is other than H.

2. The maleimide reagent of claim 1 wherein each R¹² is independently H, C1-C15 alkyl or heteroalkyl (1-15 joined members), linker is heteroalkylene (1-15 joined members); and

Z is an amine, carboxylic acid, active ester, alcohol, or activated carbonate, optionally coupled to a protecting group.

3. The maleimide reagent of claim 1 having the formula (I)

wherein

P = 0-2;

n = 0-6;

each R¹⁰ is independently H, CF₃, C(=0)Rⁿ, CN, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, S0₂Rⁿ, CH₂SRⁿ, CH₂S0₂Rⁿ, CH₂NR¹³ ₂ or CH₂N⁺R¹³ ₃;

Y is C=0, C(=0)NR¹², alkynyl, S0₂, S0₂NR¹², (CF₂)_q, NR¹³, NR¹³ ₂, N⁺R¹³ ₂, N⁺R¹³ ₃ or is absent;

q = l-4;

R¹¹ is C C₆ alkyl or aryl;

R¹² is H, Ci-C₆ alkyl, or aryl;

R¹³ is Ci-Ce alkyl in NR¹³ and N⁺R¹³ ₂ or two R¹³ may be C C₆ alkylene in NR¹³ ₂ and two R¹³ may be C C₆ alkylene and one R¹³ C C₆ alkyl in N⁺R¹³ ₃, and

Z is an amine, carboxylic acid, active ester, alcohol, or activated carbonate which may be protected or further derivatized,

with the proviso that when Y is C(=0)NR or is absent, at least one of R¹⁰ is other than H.

4. The maleimide reagent of any of claims 1-3 wherein Z is an amine or a carboxylic acid or ester; and/or

wherein Y is S0₂ or S0₂NR¹².

5. The maleimide reagent of claim 3

wherein Y-(CH₂)_n-Z is N(CH₂CH₂)₂N or N^÷Rⁱ³(CH₂CH₂)₂N^÷R^L\ or wherein Y is NR¹³ or N⁺R¹³ ₂, wherein each R^S3 independently is Ci-C₆ alkyl.

6. The maleimide reagent of claim 1 selected from the group consisting of

The maleimide reagent of claim 1 selected from the group consisting of

8. A conjugate comprising the maleimide reagent of any of claims 1-7 formed as a thioether with a thiol-containing moiety.

9. The conjugate of claim 8 wherein the thiol-containing moiety is a protein, an oligosaccharide or a synthetic polymer.

10. The conjugate of claim 8 or 9 wherein said thioether has been hydro lytically ring-opened.

1 1. A compound of the formula

wherein p, R , Y and linker are as defined in claim 1 ;

X is NHCO, CONH, NHC(0)0, or OC(0)NH; and

D is the residue of a drug.

12. The compound of claim 11 wherein each R is independently H, Ci-C₁₅ alkyl or hetero alkyl (1-15 joined members),

linker is heteroalkylene (1-15 joined members); and Z is an amine, carboxylic acid, active ester, alcohol, or activated carbonate, optionally coupled to a protecting group.

13. The compound of claim 11 which is of formula (II)

wherein

P, n, R¹⁰ and Y are as defined in claim 3;

X is NHCO, CONH, NHC(0)0, or OC(0)NH; and

D is the residue of a drug.

14. A conjugate comprising the compound of any of claims 11-13 coupled through a thioether to a thiol-containing moiety.

15. The conjugate of claim 14 wherein the thiol is a protein, oligosaccharide, or synthetic polymer.

16. The conjugate of claim 15 wherein the thiol-containing moiety is an antibody, an albumin, or a designed-sequence protein.

17. The conjugate of any of claims 14-16 wherein said thioether has been hydrolytically ring-opened.

18. A compound of the formula

wherein m, p, R¹⁰, Y and linker are as defined in claims 1 or 2;

t is 0 or 1 ; at least one or both R¹ and R² is independently CN; N0₂;

optionally substituted aryl;

optionally substituted heteroaryl;

optionally substituted alkenyl;

optionally substituted alkynyl;

COR³ or SOR³ or S0₂R³ wherein

R³ is H or optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted;

heteroaryl or heteroarylalkyl, each optionally substituted; or

OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H or optionally substituted alkyl, or both R^{9 s}roups taken together with the nitrogen to which they are attached form a heterocyclic ring;

SR⁴ wherein

R⁴ is optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted;

wherein R¹ and R² may be joined to form a 3-8 membered ring; and

wherein one and only one of R¹ and R² may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and

each R⁵ is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (CH₂CH₂0)_p wherein p=l-1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; wherein at least one of R¹, R², or R⁵ further comprises a functional group for coupling to a macromolecular carrier or is coupled to a macromolecular carrier.

19. The compound of claim 18 which is of formula (III)

wherein

p, n, R and Y are as defined in claim 3;

t is 0 or 1 ; at least one or both R¹ and R² is independently CN; N0₂;

optionally substituted aryl;

optionally substituted heteroaryl;

optionally substituted alkenyl;

optionally substituted alkynyl;

COR³ or SOR³ or S0₂R³ wherein

R³ is H or optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted;

heteroaryl or heteroarylalkyl, each optionally substituted; or

OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H or optionally substituted alkyl, or both R^{9 s}roups taken together with the nitrogen to which they are attached form a heterocyclic ring;

SR⁴ wherein

R⁴ is optionally substituted alkyl;

aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted;

wherein R¹ and R² may be joined to form a 3-8 membered ring; and

wherein one and only one of R¹ and R² may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and

each R⁵ is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (CH₂CH₂0)_p wherein p=l-1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; wherein at least one of R¹, R², or R⁵ further comprises a functional group for coupling to a macromolecular carrier or is coupled to a macromolecular carrier.

20. The compound of claim 18 or 19 wherein t = 0.

21. The compound of any of claims 18-20 wherein the functional group for coupling to a macromolecular carrier is an amine, protected amine, carboxylic acid, active ester, protected carboxylate, aldehyde, ketone, alkyne, cycloalkyne, irans-cyclooctene, norbornene, or 1,2,4,5-tetrazine.

22. A conjugate thioether formed by the reaction of a thiol-containing drug with the compound of any of claims 18-21.

23. The conjugate of claim 22 wherein the thioether has been hydrolytically ring-opened.

24. The conjugate of claim 23 wherein the macromolecular carrier is a protein, oligosaccharide, synthetic polymer, or hydrogel.

25. The conjugate of claim 24 wherein the macromolecular carrier is a hydrogel.

26. A method to administer a drug to a subject which comprises administering the conjugate of any of claims 14-17 or 23-25 to said subject.