Marion
Grinda
a,
Jonathan
Clarhaut
bc,
Brigitte
Renoux
a,
Isabelle
Tranoy-Opalinski
a and
Sébastien
Papot
*a
aUniversité de Poitiers, UMR-CNRS 6514, 4 rue Michel Brunet, BP 633, 86022, Poitiers, France. E-mail: sebastien.papot@univ-poitiers.fr; Fax: +33 549 453 501; Tel: +33 549 453 862
bUniversité de Poitiers, UMR-CNRS 6187, 1 rue Georges Bonnet, BP 633, 86022, Poitiers, France
cINSERM CIC 0802, 2 rue de la Milétrie, CHU de Poitiers, 86021, Poitiers, France
First published on 19th October 2011
The first self-immolative dendritic glucuronide prodrug of COMPOUND LINKS
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Download mol file of compounddoxorubicin was studied with the aim to target β-glucuronidase overexpressed in the microenvironment of numerous tumors. This compound includes a chemical amplifier programmed to release two molecules of COMPOUND LINKS
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Download mol file of compounddoxorubicin after a single enzymatic activation step. Upon β-glucuronidase activation, the dendritic prodrug was twice more toxic than its monomeric counterpart against H661 lung cancer cells.
β-Glucuronidase has been detected at high concentration in a wide range of malignancies including breast, lung, colon and ovarian carcinomas.3 In 1988, Tietze4 was the first who proposed to target this tumor specificity by means of enzyme-responsive prodrugs in the course of a prodrug monotherapy (PMT3b). Ever since then, the validity of this targeting strategy was demonstrated with several glucuronide prodrugs.5 Within this framework, one of us has been involved in the development of a glucuronide prodrug of COMPOUND LINKS
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Download mol file of compounddoxorubicin, HMR 1826,6 which led to superior therapeutic efficacy compared to standard treatment in various xenograft models.7 However, the efficiency of HMR 1826 was limited by the poor turnover of β-glucuronidase in the tumor microenvironment. Indeed, the optimal pH for activity of β-glucuronidase is approximately 4 whereas that of the tumor extracellular medium is 6–7. Thus, above a certain threshold dose of prodrug the enzyme is saturated and consequently the amount of drug liberated in targeted tissues is not sufficient to induce total and lasting remission of the tumor.
As proposed earlier by Shabat et al.,8 de Groot et al.9 and McGrath et al.,10 this problem could be overcome by the use of self-immolative dendrimers allowing the release of several drug units after a single enzymatic activation step. Thus, we decided to study the dendritic glucuronide prodrug 1 designed for the selective targeting of two doxorubicins (Fig. 1). This targeting system is composed of five units comprising an enzymatic trigger, a self-immolative linker11 and two drug molecules articulated around a chemical amplifier. With this design, enzymatic hydrolysis of the glycosidic bond should result in the formation of the phenol intermediate 2 which will induce the release of the aniline 3via an 1,6 elimination. The amplifier unit will then conduct to the expulsion of two doxorubicins through successive 1,4 and 1,6 elimination processes as depicted in Fig. 1.12 Therefore, in the presence of the same quantity of β-glucuronidase, the dendritic glucuronide prodrug 1 should be twice more toxic than its monomeric counterpart HMR 1826.
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Fig. 1 β-Glucuronidase-catalysed drug release mechanism. |
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Scheme 1
Reagents and conditions: (a) 5, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundHOBt, DMF, 50 °C, 3 h, 70%; (b) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundp-nitrophenyl chloroformate, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundpyridine, CH2Cl2, 0 °C to RT, 1 h, 98%; (c) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounddoxorubicin (HCl), Et3N, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundHOBt, DMF, RT, 3 h, 57%; (d) MeONa/COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundMeOH, −10 °C, 3 h, 81%; and (e) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundNaOH, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundH2O/THF, −5 °C or −15 °C, 15 min or 2 h. |
The cleavage of the methyl ester was undertaken with COMPOUND LINKS
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Download mol file of compoundNaOH in COMPOUND LINKS
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Download mol file of compoundH2O/THF (1:
1) at −5 °C for 2 hours. In this case, the synthesis of prodrug 1 was confirmed by HPLC/MS analysis of the crude mixture. However, this was accompanied by the formation of several side products making impossible the purification of compound 1 (see the ESI†). Other attempts at a lower temperature (−15 °C) or with a shorter reaction time (15 min) gave similar results. At this stage, although the reasons of this failure remain unclear, we considered it preferable to turn our attention to other protecting groups for the carbohydrate moiety. Thus, we decided to start again the synthesis of prodrug 1 from the fully allyl protected bis-carbonate 10 recently prepared in our laboratory (Scheme 2).15 Indeed, both allyl ester and carbonates are compatible with the presence of either alkali- or acid-sensitive anticancer drugs.16 Furthermore, the entire deprotection of the glucuronide can be achieved in a one step procedure under mild conditions in the presence of Pd(0) as a catalyst.
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Scheme 2
Reagents and conditions: (a) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounddoxorubicin (HCl), Et3N, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundHOBt, DMF, RT, 3 h, 58%; and (b) Pd(PPh3)4, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundaniline, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundMeOH/CH2Cl2, RT, 24 h, 80%. |
Compound 10 reacted with COMPOUND LINKS
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Download mol file of compounddoxorubicin to yield the bis-carbamate 11 (58%). No difference of reactivity was recorded between the bis-carbonates 7 and 10 for this coupling reaction. Finally, the dendritic prodrug 1 was obtained by the cleavage of protecting groups performed in the presence of a catalytic amount of Pd(PPh3)4 and two equivalents of COMPOUND LINKS
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Download mol file of compoundaniline (80%). As expected, under such conditions the full deprotection of the glucuronide moiety proceeded cleanly avoiding the formation of the side products observed previously.
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Fig. 2
E. coli β-glucuronidase-catalysed release of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounddoxorubicin from prodrug 1 (phosphate buffer, pH 7, 37 °C). |
As soon as incubated with the enzyme, prodrug 1 rapidly disappeared concomitantly with the appearance of COMPOUND LINKS
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Download mol file of compounddoxorubicin. Thanks to the presence of the nitrobenzylphenoxy carbamate linker, the glucuronide trigger is substantially far away from the two bulky drug units to allow an easy recognition of 1 by β-glucuronidase. Only six hours after the addition of the enzyme, the liberation of COMPOUND LINKS
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Download mol file of compounddoxorubicin was completed. Thus, in spite of the complexity of the double drug release mechanism from prodrug 1, it appeared that the whole process occurred relatively rapidly.
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Fig. 3 Cell viability of H661 cells treated 3 days with indicated compounds from 10 to 1000 nM. Graphs represent mean +/− SEM of four independent experiments performed in triplicate. |
When incubated alone compound 1 did not exhibit significant toxicity until the highest tested dose of 1000 nM. This result indicated that the derivatisation of COMPOUND LINKS
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Download mol file of compounddoxorubicin in the form of the prodrug 1 markedly reduced its anti-proliferative activity. The reduced toxicity could be attributed to the hydrophilicity imparted by the glucuronide trigger which probably prevented passive cellular uptake and further intracellular activation of prodrug 1 by lysosomal β-glucuronidase. On the other hand, addition of the enzyme in the culture medium produced a dramatic anti-proliferative effect with an IC50 value of 110 nM. In contrast, the recorded cytotoxicity for HMR 1826 in the presence of the same quantity of β-glucuronidase was approximately 2-fold lower and similar to that obtained for COMPOUND LINKS
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Download mol file of compounddoxorubicin (IC50 = 280 nM and 250 nM respectively). All together, these results confirm the β-glucuronidase-catalysed double drug release from prodrug 1 in the cell culture medium.
Footnote |
† Electronic supplementary information (ESI) available: General experimental procedures, chemical structure of HMR 1826, synthesis and NMR spectra of the compounds, cell culture and cell proliferation assays. See DOI: 10.1039/c1md00193k |
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