Selective water-soluble gelatinase inhibitor prodrugs - PubMed (original) (raw)

. 2011 Oct 13;54(19):6676-90.

doi: 10.1021/jm200566e. Epub 2011 Sep 6.

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Selective water-soluble gelatinase inhibitor prodrugs

Major Gooyit et al. J Med Chem. 2011.

Abstract

SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were nonmutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than those of 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in the treatment of acute gelatinase-dependent diseases.

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Figures

Figure 1

Figure 1

Ex vivo hydrolysis of 5d in human blood generates 3.

Figure 2

Figure 2

Reconstructed ion chromatogram following 60-min incubation of 5d with rat S9 liver microsomes.

Figure 3

Figure 3

Product ion mass spectra of (A) metabolite M1 (MH+, m/z 406) (B) M2 (MH+, m/z 250) and (C) M3 (MH+, m/z 364).

Figure 4

Figure 4

Product ion mass spectra of (A) metabolite M4 and (B) M5 (Inset: Reconstructed ion chromatogram of m/z 338 (MH + 16)+, following 30-min incubation of 3 with rat liver microsomes).

Figure 5

Figure 5

Identification of M4 by comparison of HPLC retention time to synthetic standards. (A) Reconstructed ion chromatogram of m/z 338 following 30-min incubation of 3 with rat liver microsomes and (B) HPLC chromatogram of a mixture of synthetic standards 13 and 14 using UV absorbance at 245 nm.

Figure 6

Figure 6

Product ion mass spectra of (A) metabolite M4, (B) synthetic standard 13, and (C) synthetic standard 14.

Figure 7

Figure 7

Metabolism pathway of 5d. Active gelatinase inhibitors are boxed; the major pathway is represented by a thick arrow, minor pathways are depicted by thin arrows, minute pathways are represented by broken thin arrows. The crossed red arrow indicates the absence of 14.

Figure 8

Figure 8

Plasma concentration-time curves of 3 after a single bolus intravenous dose of 5d and 3 to mice.

Scheme 1

Scheme 1

Synthesis of 3_a_ a Reagents and condition: (a) (i) Cs2CO3, room temperature, 2 h; (ii) Zn, AcOH, 0 °C to room temperature, 2 h, 79%. (b) Boc2O, Et3N, MeOH, 60 °C, 2 h, 82%. (c) _m_-CPBA, CH2Cl2, room temperature, 72 h, 81%. (d) thiourea, MeOH/CH2Cl2, room temperature, 18 h, 72%. (e) 4 N HCl in 1,4-dioxane, ethyl acetate/CH2Cl2, 0 °C to room temperature, 48 h, 82%.

Scheme 2

Scheme 2

Syntheses of the Ester and Amide Prodrugs_a_ a Reagents and condition: (a) DMAP, _i_Pr2EtN, CH2Cl2, room temperature, 3 h, 72 - 81%. (b) 4 N HCl in 1,4-dioxane, ethyl acetate/CH2Cl2, 0 °C to room temperature, 48 h, 71-79%. (c) THF, −20 °C to room temperature, 1 h, 61-79%.

Scheme 3

Scheme 3

Syntheses of 13 and the _N_-Hydroxylamine 14 Derivative.a a Reagents and condition: (a) Cs2CO3, DMF, room temperature, 24 h, 88%. (b) Fe, NH4Cl, MeOH/H2O, reflux, 2 h, 83%. (c) Boc2O, Et3N, MeOH, room temperature, 24 h, 76%. (d) KOH, MeOH, reflux, 4 h; then epichlorohydrin, room temperature, 10 min, 68%. (e) _m_-CPBA, CH2Cl2, 0 °C to room temperature, 10 min, 89%. (f) thiourea, MeOH/CH2Cl2, room temperature, 24 h, 83%. (g) HCl, MeOH, reflux, 1 h, 95%. (h) Zn, NH4Cl, CH2Cl2/H2O, room temperature, 0.5 h, 69%.

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