Daunorubicin substituted at N-3' with a benzyloxycarbonyl group (self-immolative spacer) linked to an α-D-galactosyl residue such as 7a and 7b have been prepared as prodrugs. Conversion of 7a and 7b to daunorubicin will be mediated by an immunoconjugate consisting of an α-D-galactosidase enzyme covalently attached to a tumor-specific monoclonal antibody. Anthracycline antibiotics are widely used in cancer chemotherapy but their clinical efficacy is limited by severe dose-cumulative cardiotoxicity and the appearance of acquired resistance. Rational design attempts to avoid these side effects can be represented either by drug targeting or prodrug synthesis. A combination of both should be optimal. In this regard, the combination of prodrugs and tumor-specific antibody-enzyme conjugates for use as therapeutic agents has been recently reported in the literature. This entails monoclonal antibodies directed against a particular tumor and covalently bonded to a prodrug-cleaving enzyme. Therefore, the antibody-enzyme conjugate localizes on the tumor cell surface antigen, and the non-cytotoxic prodrug is converted into the active cytotoxic species at the tumor cell surface. To avoid non-specific liberation of the active compound and the disadvantage of an immune response, glycosidases—preferably human enzymes such as human α-D-galactosidase or human β-D-glucuronidase, which are non-immunogenic or of low immunogenicity—have been chosen for our studies. Preliminary experiments showed that enzymatic hydrolysis was ineffective when galactose or glucuronic acid was directly bonded to the 14-OH of anthracycline via a classical glycosidic linkage. Therefore, linking these sugars to the primary amino group of daunosamine (the carbohydrate constituent of daunorubicin and doxorubicin) was considered. However, N-acylation or N-carbamylation of amines to form amide or carbamate prodrugs has been used only to a limited extent due to the relative stability of these derivatives in vivo. In contrast, o- and p-substituted (OH or NH2) benzylcarbamate derivatives or (acyloxy)alkyl carbamates can be chemically or enzymatically activated to release the free amino function, with concomitant formation of quinone methide and CO2 or via the intermediate formation of an unstable carbamic acid, respectively. Based on these observations, our initial goal was to prepare potentially enzymatically cleavable prodrugs of daunorubicin 5 (general formula A) with a sugar residue (galactose or glucuronic acid) linked to daunorubicin via a self-immolative spacer. In this letter, we report the initial work using galactose as the sugar residue. The general route for preparing such compounds involved initial glycosidation of o- or p-cresol with peracetyl-α-D-galactose, benzylic hydroxylation of the resulting o- or p-hydroxymethyl phenyl glycosides, and activation of the OH group with a peptide coupling reagent prior to condensation with daunorubicin. As an illustration (Scheme 2), condensation of penta-O-acetyl-D-galactose with p-cresol in the presence of ZnCl2 (160°C, 10 min) stereoselectively afforded the α-glycoside 1a (mp 163–165°C, [α]D +164, 30% yield), which was purified by column chromatography (hexane-EtOAc, 9:1). Benzylic bromination of 1a was performed using N-bromosuccinimide in CCl4, either under irradiation (1000 W, 15 min) or in the presence of a radical initiator (benzoyl peroxide), yielding bromoderivative 2a (82% yield). This was subsequently converted to alcohol 3a (AgNO3, acetone-H2O, 56% yield). Activation of alcohol 3a with N-hydroxysuccinimidocarbonate gave 4a (96% yield), which was condensed with daunorubicin 5 (DMF, Et3N, room temperature) to yield 6a (32% yield). Finally, N-[4-(α-D-galactopyranosyl)benzyloxycarbonyl]-daunorubicin 7a (amorphous solid, [α]D20 +243) was obtained in 89% yield by transesterification of 6a with MeONa-MeOH. Following the same reaction sequence, the corresponding N-[2-(α-D-galactopyranosyl)benzyloxycarbonyl]-daunorubicin 7b was prepared from 2-methylphenyl 2,3,4,6-tetra-O-acetyl-α-D-galactopyranoside 1b. Bromination of 1b (NBS, CCl4) afforded 2b (80% yield), which was converted (AgNO3) to a mixture of the desired alcohol 3b (21% yield) and the corresponding aldehyde (52%). The aldehyde was easily reduced (NaBH4) to 3b (80% yield). Activation of 3b with succinimido chloroformate (72% yield), condensation with daunorubicin 5 (85% yield), and deprotection of 6b yielded the desired compound 7b (83% yield). The cytotoxicity of compounds 7a and 7b was investigated against L1210 murine leukemia. As expected, both exhibited strongly reduced cytotoxicity (>1 µg/mL) compared to daunorubicin 5 (0.02 µg/mL). The stability of these prodrugs in buffer and plasma, as well as their cleavability by α-D-galactosidase, is currently under investigation.