A series of alpha-amino acid ester prodrugs of camptothecin: in vitro hydrolysis and A549 human lung carcinoma cell cytotoxicity

Abstract

The objective of the present study was to identify a camptothecin (CPT) prodrug with optimal release and cytotoxicity properties for immobilization on a passively targeted microparticle delivery system. A series of alpha-amino acid ester prodrugs of CPT were synthesized, characterized, and evaluated. Four CPT prodrugs were synthesized with increasing aliphatic chain length (glycine (Gly) (2a), alanine (Ala) (2b), aminobutyric acid (Abu) (2c), and norvaline (Nva) (2d)). Prodrug reconversion was studied at pH 6.6, 7.0, and 7.4 corresponding to tumor, lung, and extracellular/physiological pH, respectively. Cytotoxicity was evaluated in A549 human lung carcinoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hydrolytic reconversion rate to parent CPT increased with decreasing side chain length as well as increasing pH. The Hill slope of 2d was significantly less than CPT and the other prodrugs tested, indicating a higher cell death rate at lower concentrations. These results suggest that 2d is the best candidate for a passively targeted sustained release lung delivery system.

Figure 1

Schematic representation of prodrugs. Hydrolysis site is shown by the red zig-zag line.

Figure 2

¹H NMR spectrum of all four prodrugs in DMSO-d₆ at 37 °C after 6.5 h of incubation in PB pH7.4: (A) section of ¹H NMR spectrum showing changes in the H-5 and H-17 signals of 2a-d, I-1, and I0-1; (B) section of ¹H NMR spectrum showing changes in the H-23 signals of 2a-d, and I-1; (C) section of ¹H NMR spectrum showing changes in the H-7 and H-14 signals of 2a-d, I-1, and I0-1. Similar spectra were observed at pH 7.0 and 6.6.

Figure 3

LC-MS spectra of the intermediates obtained during the hydrolysis studies of 2a-d: (A) 2a (I-1); (B) 2a (I-2); (C) 2b (I-1); (D) 2b (I-2); (E) 2c (I-1); (F) 2c (I-2); (G) 2d (I-1); (H) 2d (I-2).

Figure 4

Release of CPT in PB at three pH values at 37 °C: (A) pH 7.4; (B) pH 7.0; (C) pH 6.6.

Figure 5

Hydrolysis of 2a-d in PB at three pH values at 37°C: (A) pH 7.4; (B) pH 7.0; (C) pH 6.6.

Figure 6

Correlation of 2a-d hydrolysis half-life (see steps b and d, Scheme 2) and hydrophobicity constants (π) of amino acid. The hydrolysis half-lives of 2a-d in PB (pH 6.6, 7.0, and 7.4) at 37°C are reported as the mean ± SD (n=3). Slopes: pH 7.4 = 0.7788 (r²= 0.8493), pH 7.0 = 1.465 (r²= 0.9731), pH 6.6 = 1.322 (r²= 0.7161). The slopes at pH 6.6 and 7.0 are approximately twice as large as the slope at pH 7.4 suggesting that the prodrug reconversion in cancerous tissues or in the lung (the target organ) would be expected to be twice as fast.

Figure 7

Correlation of CPT formation half-life (see step e, Scheme 2) at three pH values and hydrophobicity constant (π) of amino acid. The reported half-life of CPT formation in PB in three pH values at 37°C represents the mean ± SD (n=3). Even though the rate of prodrug hydrolysis is dependent upon pH (Figure 6), once prodrug hydrolysis occurs the rate of CPT formation is independent of pH. Slopes: pH 7.4 = 14.89 (r²= 0.9837), pH 7.0 = 17.26 (r²= 0.9783), pH 6.6 = 17.42 (r²= 0.9954).

Figure 8

Cytotoxicity of CPT and 2a-d in A549 human lung carcinoma cells. The data were fit using the Hill equation.

Scheme 1

Synthesis of 2a-d: Reagents and conditions: (a) DIPC, DMAP, DCM, RT, 4 h; (b) 30% TFA in DCM, RT, 1 h.

Scheme 2

The proposed degradation pathway of 2a-d in three pH values: pH 6.6, 7.0 and 7.4. Prodrug hydrolysis involves steps b and d whereas CPT formation is possible via pathways a, e, or f. However, results in this study demonstrate that only pathway e is responsible for CPT formation.