We previously reported the synthesis of a series of doxorubicin analogue prodrugs that give rise to intensely cytotoxic metabolites in the presence of carboxylate esterases. We now report studies on structurally related beta-glucuronide prodrugs that are converted to similar potent metabolites in the presence of beta-glucuronidases. These prodrugs were prepared by reductive condensation of daunomycin or doxorubicin with methyl 1-O-[(1'RS)-1'-ethoxy-4'-oxobutyl]-2,3,4-tri-O-acetyl-beta-D- glucopyranosyluronate in the presence of sodium cyanoborohydride followed by base-mediated cleavage of the glucuronate protective groups. The doxorubicin derivatives were isolated in very low yield, most likely because of the inherent base lability of the parent aglycone. By contrast, fairly good yields of the more base-stable daunomycin analogues were obtained. The target daunomycin glucuronide, N-[(4"RS)-4"-ethoxy-4"-(sodium 1"'-O-beta-D-glucopyranuronate)butyl]daunorubicin (6a), had a half-life of 30 h when incubated at a concentration of 12 microM in aqueous 0.05 M phosphate buffer, pH 7.4, at 37 degrees C. Under identical conditions in the presence of 197 units/mumol of Escherichia coli beta-glucuronidase, 6a was hydrolyzed with a half-life of 1.7 h. The single metabolite observed was chromatographically identical with that formed from the hydrolysis of N-(4,4-diacetoxybut-1-yl)daunomycin by carboxylate esterases. 6a was approximately 10,000-fold more toxic to human A375 melanoma cells in the presence of E. coli beta-glucuronidase than in the absence of the enzyme. These findings indicate the therapeutic potential of anthracycline glucuronide prodrugs as independent entities or four use in conjunction with enzyme tissue-targeting strategies such as antibody-directed enzyme prodrug therapy (ADEPT) or gene-directed enzyme prodrug therapy (GDEPT).

N-(5,5-Diacetoxypent-1-yl)doxorubicin (1b) is an intensely cytotoxic doxorubicin analogue that retains full potency against tumor cells that express elevated levels of P-glycoprotein and are resistant to doxorubicin. 1b was designed to be hydrolyzed in the presence of carboxylate esterases to N-(5-oxypent-1-yl)doxorubicin, an aldehyde capable of existing in equilibrium with a cyclic carbinolamine. To investigate the structural determinants of potency for 1b, we have prepared a series of chemically related compounds in which various omega-[bis(acetoxy)]alkyl or omega-[bis(acetoxy)]alkoxyalkyl groups are substituted at the 3'-amino position of the daunosamine sugar. These groups were selected to assess the effect of chain length, oxygen substitution, and carbinolamine ring size on analogue potency. The compounds were evaluated for their ability to inhibit the in vitro growth of the following cell lines: (a) Chinese hamster ovary (CHO) cells, (b) a CHO cell mutant 100-fold resistant to doxorubicin that expresses elevated levels of P-glycoprotein, (c) a murine ductal cell pancreatic adenocarcinoma (Panc 02), and (d) a murine mammary carcinoma (CA 755). The most potent members of the series were those that could form a straight chain aldehyde intermediate after esterase-mediated hydrolysis of the omega-bis(acetoxy) groups and give rise to 5- or 6-membered ring carbinolamines. Analogues capable of forming 7-, 8-, or 9-membered carbinolamines were markedly less active. The N-methyl derivative of 1b, which cannot give rise to a cyclic carbinolamine, was 2 orders of magnitude less potent than 1b. A branched chain analogue, 1f, which contained a tertiary carbon atom adjacent to the omega-bis(acetoxy) groups, was also substantially less active than its nonbranched counterpart, 1a. These findings suggest that the chain length of the 3'-amino substituents and the ability of the derived aldehydes to form 5- or 6-membered carbinolamines are critical determinants of biologic potency.

A series of phenylthiourea and ethylthiourea derivatives of daunorubicin and its congeners was prepared by reaction of the 3'-amino group of the antibiotic with phenylisothiocyanate or ethylisothiocyanate.S-Methylation yielded 5-methylisothiouronium salts which when reacted with amines resulted in an intramolecular cyclization with the participation of the neighboring 4'-OH group. The structures and predominant conformations of the thiourea derivatives andwere determined by *H and 13C NMR.Cytostatic activities of the thiourea and oxazoline derivatives were compared with the cytostatic activities of TV-methylurea and A'-methyl-TV-nitrosourea containing daunorubicin and its congeners. Carminomycin derivatives were endowedwith the highest cytostatic activity.In the course of a screening program for novel second generation antitumor anthracycline antibiotics of dauftorabicin (1) series, derivatives containing in 3' -position N-methylurea (2) or 7V-methyl-iV-nitrosourea moieties (3) were synthesized1}. In alkaline conditions, 3/-(7V-methyl-ALnitrosoaminocarbonyl)daunorubicin (3a) or related compounds produce 3'-JVr,4'-0-carbonyl derivatives by intramolecular cyclization of intermediate 3-deamino-3'-isocyanato derivativesX).In this paper we report on the preparation of thiourea-containing derivatives of daunorubicin and its analogs which are susceptible to transformations with neighboring 4'-hydroxy group participation. Also, the structure-cytostatic activity relationship among urea and thiourea derivatives of anthracycline antibiotics was investigated. ChemistryDaunorubicin (la), carminomycin (lb), doxorubicin (1c) and 14-pimeloyloxydaunorubicin (ld)1}, upon interaction with phenylisothiocyanate or ethylisothiocyanate in pyridine afforded the corresponding phenylthiourea (5a~5d) of ethylthiourea (6a and 6c) derivatives in 77~99% yield. Previously, 5a was obtained from la and phenylisothiocyanate in chloroform-methanol mixture in a yield of 64%2). S-Methylisothiouronium salts of these compounds7a, 7b and 8a were obtained by alkylation with CH3I in methanol. Usually S-alkylisothiouronium salts easily produce guanidines by the action of amines. Compounds 7 and 8 were selected for their transformation to substituted guanidine derivatives (9). Upon interaction with primary amines (methylamine, n-pentylamine or tris(hydroxymethyl)aminomethane) 7a, 7b and 8a yielded daunorubicino(3',4'-d)oxazoline derivatives (10a, 10b or lla), respectively. The formation of a 5-membered cycle is facilitated as in the case of transformations of Af-methyl-iV-nitrosoaminocarbonylderivatives of daunorubicin (4)1}.We could demonstrate by HPLCthat storage of 5-methylisothiouronium salt 7a during 1-day in