Releasable Linkers in Targeted Conjugates

In this section as well as in other sections throughout this book, (tumor-activated) prodrugs in which a releasable (self-elimination) linker (or spacer) is incorporated between a specifier (targeting unit part of prodrug or conjugate that is (enzymatically) removed) and the parent drug are discussed. Two main reasons for application of releasable linker systems in prodrugs and bioconjugates are facilitation of (enzymatic) activation, and incorporation of appropriate linkage chemistry. In a prodrug designed for enzymatic activation, steric burden caused by a bulky specifier or a bulky parent drug (or both) can (partially) block efficient enzymatic cleavage. In Figure 2, the principle of drug release from a self-elimination spacer-containing conjugate is schematically depicted.

Two types of self-elimination spacers can be distinguished. The first concerns self-elimination spacers that eliminate as a consequence of shifting conjugated electron pairs, ultimately leading to expulsion of the leaving group (the drug). We call this spacer type 'electronic cascade linker.' The second releasable spacer type, the 'cyclization linker.' involves self-elimination spacers that release the drug following an intramolecular cyclization reaction.

The most prominent example of an electronic cascade spacer is the 1,6-elimination linker that was developed in the early 1980s by Katzenellenbogen and colleagues (Figure 3) (Carl et al., 1981). Following enzymatic hydrolysis of the

Figure 3. First application of the 1,6-elimination electronic cascade linker in prodrug design.

Figure 3. First application of the 1,6-elimination electronic cascade linker in prodrug design.

lysine residue by trypsin, the resulting strongly electron-donating 4-aminobenzyl group triggers a 1,6-elimination to release the model compound 4-nitroaniline. This 1,6-elimination spacer can be considered as one of the most versatile self-immolative connectors that can be incorporated in drug conjugates.

When the electron-donating group and the leaving group on the benzene ring are positioned ortho with respect to one another, 1,4-elimination can take place in a similar fashion. Other 1,4-elimination linkers have also been reported (Rivault et al., 2004). The 1,4- and 1,6-elimination processes can occur when an electron-donating amino or hydroxyl group is generated from a masked amino or hydroxyl functionality.

The second self-elimination spacer type concerns the cyclization spacer, of which several variations exist. One prominent example is the ethylene diamine spacer that, after removal of the specifier R1, intramolecularly cyclizes to yield a cyclic urea derivative and the liberated parent drug HOR2 (Figure 4, A) (Saari et al., 1990). Because some cyclization spacers possess long half-lives of cyclization, bulky substituents have been introduced on the spacer with the goal of conforma-tionally pre-orientating the functional groups involved in the cyclization reaction and achieving a favorable enthalpy and entropy effect for ring closure (ThorpeIngold effect) (Eliel, 1962). According to this principle, the trimethyl lock spacer is a frequently used cyclization spacer (Figure 4, B) (Nicolaou et al., 1996; Wang et al., 1997). Following activation, an intramolecular cyclization leads to the formation of a lactone and release of free drug R2H.

°yor! <Vor2 ft ilH Nation, H2N^H cyclizali0n. HN^NH + HOr2

°yor! <Vor2 ft ilH Nation, H2N^H cyclizali0n. HN^NH + HOr2

The need for incorporation of a linker in between the parent drug and the specifier seems to be a common theme for conjugates designed for enzymatic activation. Several structurally different enzymes cleaved spacer-containing prodrugs much more readily than the corresponding prodrugs lacking a spacer. Evidence for this recurring theme was obtained, for example, with substrates for the enzyme P-glucuronidase. A glucuronide directly coupled to epirubicin was inert toward ^-glucuronidase cleavage, whereas a 1,6-elimination spacer-containing doxorubicin analog did show substrate behavior for the enzyme (Desbene et al., 1998; Papot et al., 1998; Madec-Lougerstay et al., 1999). Another P-glucuronide carbamate prodrug of daunorubicin was only a moderate substrate for this enzyme (Leenders et al., 1995b), whereas incorporation of a spacer drastically enhanced the affinity of the enzyme for the prodrug (Leenders et al., 1995a; Houba et al., 1996). Incorporation of a spacer between 5-fluorouracil (5-FU) and the glucuronide specifier dramatically enhanced the rate of hydrolysis by glucuronidase (Madec-Lougerstay et al., 1999). Doxorubicin and Mmc prodrugs designed for cleavage by cathepsins and lacking a linker were resistant to enzymatic activation, whereas incorporation of a linker led to drug release for both parent drugs (Dubowchik and Firestone, 1998; Dubowchik et al., 1998).

We and others have reported novel releasable linker chemistries and concepts. For example, we have developed releasable linkers for which both the length and the chemistry are tunable (de Groot et al., 2001b). Advantages are that efficiency of drug release can be improved and that different linker chemistries are available for different drug molecules. Depending on the drug's functional group used for linkage, the linkers enable conjugation via appropriate chemistry, preferably through stable carbamate linkages. Senter et al. have reported another interesting electronic cascade releasable linker system, which is applicable for conjugation via the drug's aromatic hydroxy group (Toki et al., 2002).

A novel releasable linker concept has been recently reported simultaneously by two groups, among which is our own group (de Groot et al., 2003; Amir et al., 2003). In the same year, a third group reported their work on the same concept (Li et al., 2003). Branched self-elimination linker systems release multiple leaving groups upon a single activation event. Double release and triple release spacers were developed for which proof of principle has been delivered. Multiple generations of multiple release spacers can be coupled to one another to yield dendrimeric multiple release conjugates, which we termed 'cascade-release dendrimers' (Figure 5).

The multiple release spacers in these exploding dendrimers fall apart into the corresponding separate monomers and release all drug molecules upon a single

Figure Type Dendrimer
Figure 5. A 'cascade-release dendrimer.' containing two generations of double-release linker monomers, releases all end groups (drug molecules) following a single activation event.

activation (enzymatic cleavage). Proof of this has been reported for complete release of four paclitaxel molecules as end groups from a cascade-release dendrimer prodrug containing two generations of double release self-elimination linkers. This type of branched releasable linker system may prove useful in construction of single-activation/multiple release prodrugs (Meijer and van Genderen, 2003).

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