Towards aspirin-inspired self-immolating molecules which target the cyclooxygenases

Abstract

Cyclooxygenases (COXs) are enzymes that play a vital role in the inflammatory cascade through the generation of prostaglandins. Their over-expression has been implicated in numerous diseases. In particular, over-expression of COX-2 has been shown to be a predictive biomarker for progression of pre-malignant lesions towards invasive cancer in various tissues. This makes the early detection of COX-2 expressing lesions of high clinical relevance. Herein we describe the development of the first self-immolating trigger which targets COXs. We incorporated our trigger design into 2 activatable fluorogenic probes and demonstrated COX-specific activation in vitro. Experimental data revealed probe activation was likely caused by solvent-exposed amino acids on the surface of the COXs. Overall, the probes reported here mark the first step towards developing self-immolating imaging/therapeutic agents targeted to specific COXs.

Figure 1

Molecular Design of COX-Sensitive Fluorogenic Probes. (A) Overall Design Strategy. (B) Structure of CP-1 and CP-2.

Figure 2

Mechanism of Probe Activation by COXs. The probe is optically silent until deacetylation by a COX reveals a phenol group, resulting in a 1,6-quinone-methide elimination and the release of fluorescent 6-aminoquinoline.

Figure 3

Emission spectra for (A) CP-1 and (B) CP-2. Spectra were measured at 0.1 mgmL⁻¹ in water and deprotection solution (2:1:1, v:v:v, methanol:water:saturated NaHCO₃). Release of 6-aminoquinoline via removal of an acetyl moiety by the deprotection solution is clearly documented by the appearance of a new peak around 540 nm.

Figure 4

Activation of A) CP-1 B) CP-2 by COX-1 and COX-2 over a time-course of 180 mins. Purified COX-1 and COX-2 (0.125 mgmL⁻¹) were incubated with either CP-1 or CP-2 (100 μM) in Tris buffer (80 mM, pH = 8.0, 0.1% Tween 20, 300 μM DDC) at 37 °C for 180 mins and fluorescent readings (λ_ex = 355 nm, λ_em = 535 nm) were taken at 0, 30, 60, 120 and 180 mins.

Figure 5

Activation of CP-1 and CP-2 by Purified COX and Porcine Liver Esterase Enzymes in Vitro. Either COX-1, COX-2, albumin, carbonic anhydrase (all 0.125 mgmL⁻¹) or porcine liver esterase (0.309 mgmL⁻¹) were incubated with either CP-1 or CP-2 (100 μM) in Tris buffer (80 mM, pH = 8.0, 0.1% Tween 20, 300 μM DDC) at 37 °C for 60 minutes. Pre-treatment with the COX-2 inhibitor celecoxib (100 mM) was performed at 0 °C for 10 minutes prior to addition of probe. Albumin and carbonic anhydrase were tested as non-enzymatic protein controls. Fluorescence readings (λ_ex = 355 nm, λ_em = 535 nm) were then taken. Results are reported as fold increase in fluorescent signal compared to a blank solution with no protein.

Scheme 1

Synthesis of CP-1 and CP-2. ia) PPh₃, DIAD, THF; 0 °C to r.t.; 16 h b) NaBH₄, MeOH; 0 °C to r.t.; 3 h; 28% over 2 steps ii) K₂CO₃, Ac₂O, DMF; 0 °C; 2 h; 58% iiia) 6-aminoquinoline, pyridine, triphosgene, DCM/toluene; 0 °C to reflux; 16 h b) TBAF, THF, acetic acid; r.t.; 15 h; 34% over 2 steps iv) HSO₃Cl; r.t.; 15 h; 53% va) PPh₃, THF/toluene b) H₂SO₄, MeOH; reflux; 2 days c) LiAlH₄, THF; 0 °C to reflux; 15 h d) 1-chloro-2-octyne, KHCO₃, DMF; r.t.; 15 h; 39% over 4 steps vi) K₂CO₃, Ac₂O, DMF; 0 °C; 1 h; 79% viia) Na₂CO₃, triphosgene, toluene; 0 °C to r.t.; 4 h b) 6-aminoquinoline, THF; r.t.; 1 h; 26% over 2 steps. DIAD = diisopropyl azodicarboxylate; TBAF = tetrabutylammonium fluoride; TBDMS = tert-butyldimethylsilyl.