Synthesis and chemical and biological comparison of nitroxyl- and nitric oxide-releasing diazeniumdiolate-based aspirin derivatives

Abstract

Structural modifications of nonsteroidal anti-inflammatory drugs (NSAIDs) have successfully reduced the side effect of gastrointestinal ulceration without affecting anti-inflammatory activity, but they may increase the risk of myocardial infarction with chronic use. The fact that nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction, and enhances contractility led us to synthesize a diazeniumdiolate-based HNO-releasing aspirin and to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described for these compounds. In addition to protection against stomach ulceration, these prodrugs exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent diazeniumdiolate toward nonsmall cell lung carcinoma cells (A549), but they were not appreciably toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2 and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere shortening on murine ventricular myocytes compared to control. Together, these anti-inflammatory, antineoplasic, and contractile properties suggest the potential of HNO-NSAIDs in the treatment of inflammation, cancer, or heart failure.

Figure 1

Spontaneous hydrolysis of (A) IPA/NO-aspirin, (B) DEA/NO-aspirin or (C) aspirin at pH 7.4 and (D, E) DEA/NO-aspirin at pH 10 in PBS containing 50 μM DTPA at 37°C. Scans are plotted at 0, 90, 210, 390, 570, 930, 1410 min in (A), 60, 570, 930, 1290, 1770, 2370, 2970, 3690 min in (B), 60, 210, 570, 1170, 1770, 2970, 5730 min in (C), 0, 10, 20, 30, 40, 50 min in (D) and 80, 110, 150, 190, 230, 270, 320, 360, 420, 460, 540, 660, 820 min in (E). The respective first-order rate constants of IPA/NO-aspirin and DEA/NO-aspirin decomposition at 241 nm are (2.6 ± 0.2) × 10⁻⁵ s⁻¹ (n > 3) and 5.3 × 10⁻⁶ s⁻¹ (n = 1 given the slow rate) while that of aspirin at 295 nm is 9.8 × 10⁻⁶ s⁻¹ (n = 1 given the slow rate); all R² > 0.997.

Figure 1

Spontaneous hydrolysis of (A) IPA/NO-aspirin, (B) DEA/NO-aspirin or (C) aspirin at pH 7.4 and (D, E) DEA/NO-aspirin at pH 10 in PBS containing 50 μM DTPA at 37°C. Scans are plotted at 0, 90, 210, 390, 570, 930, 1410 min in (A), 60, 570, 930, 1290, 1770, 2370, 2970, 3690 min in (B), 60, 210, 570, 1170, 1770, 2970, 5730 min in (C), 0, 10, 20, 30, 40, 50 min in (D) and 80, 110, 150, 190, 230, 270, 320, 360, 420, 460, 540, 660, 820 min in (E). The respective first-order rate constants of IPA/NO-aspirin and DEA/NO-aspirin decomposition at 241 nm are (2.6 ± 0.2) × 10⁻⁵ s⁻¹ (n > 3) and 5.3 × 10⁻⁶ s⁻¹ (n = 1 given the slow rate) while that of aspirin at 295 nm is 9.8 × 10⁻⁶ s⁻¹ (n = 1 given the slow rate); all R² > 0.997.

Figure 1

Spontaneous hydrolysis of (A) IPA/NO-aspirin, (B) DEA/NO-aspirin or (C) aspirin at pH 7.4 and (D, E) DEA/NO-aspirin at pH 10 in PBS containing 50 μM DTPA at 37°C. Scans are plotted at 0, 90, 210, 390, 570, 930, 1410 min in (A), 60, 570, 930, 1290, 1770, 2370, 2970, 3690 min in (B), 60, 210, 570, 1170, 1770, 2970, 5730 min in (C), 0, 10, 20, 30, 40, 50 min in (D) and 80, 110, 150, 190, 230, 270, 320, 360, 420, 460, 540, 660, 820 min in (E). The respective first-order rate constants of IPA/NO-aspirin and DEA/NO-aspirin decomposition at 241 nm are (2.6 ± 0.2) × 10⁻⁵ s⁻¹ (n > 3) and 5.3 × 10⁻⁶ s⁻¹ (n = 1 given the slow rate) while that of aspirin at 295 nm is 9.8 × 10⁻⁶ s⁻¹ (n = 1 given the slow rate); all R² > 0.997.

Figure 2

Guinea pig serum induced hydrolysis of (A, C) IPA/NO-aspirin, (B, D) DEA/NO-aspirin, or (E) aspirin in PBS containing 50 μM DTPA at pH 7.4 and 37°C. Scans are plotted at 0, 5, 10, 15, 20, 25, 40 s in (A), 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 s in (B), 2, 14, 26, 38, 62 min in (C), 3, 6, 9, 12, 15, 18, 39 min in (D) and 1, 7, 19, 31, 43, 59, 79, 99, 119 min in (E). Panel (F) is as in (A) with the substitution of fetal bovine serum; scans are plotted at 0, 15, 30, 60, 150, 255, 555 and 885 min. All kinetic data fit to a first order equation (n ≥ 3 except for n = 1 for F) with R² > 0.983. The presence of serum proteins impacts the blank-corrected spectra in the low 200 nm region in Figure 2 compared to Figure 1.

Figure 2

Guinea pig serum induced hydrolysis of (A, C) IPA/NO-aspirin, (B, D) DEA/NO-aspirin, or (E) aspirin in PBS containing 50 μM DTPA at pH 7.4 and 37°C. Scans are plotted at 0, 5, 10, 15, 20, 25, 40 s in (A), 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 s in (B), 2, 14, 26, 38, 62 min in (C), 3, 6, 9, 12, 15, 18, 39 min in (D) and 1, 7, 19, 31, 43, 59, 79, 99, 119 min in (E). Panel (F) is as in (A) with the substitution of fetal bovine serum; scans are plotted at 0, 15, 30, 60, 150, 255, 555 and 885 min. All kinetic data fit to a first order equation (n ≥ 3 except for n = 1 for F) with R² > 0.983. The presence of serum proteins impacts the blank-corrected spectra in the low 200 nm region in Figure 2 compared to Figure 1.

Figure 2

Guinea pig serum induced hydrolysis of (A, C) IPA/NO-aspirin, (B, D) DEA/NO-aspirin, or (E) aspirin in PBS containing 50 μM DTPA at pH 7.4 and 37°C. Scans are plotted at 0, 5, 10, 15, 20, 25, 40 s in (A), 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 s in (B), 2, 14, 26, 38, 62 min in (C), 3, 6, 9, 12, 15, 18, 39 min in (D) and 1, 7, 19, 31, 43, 59, 79, 99, 119 min in (E). Panel (F) is as in (A) with the substitution of fetal bovine serum; scans are plotted at 0, 15, 30, 60, 150, 255, 555 and 885 min. All kinetic data fit to a first order equation (n ≥ 3 except for n = 1 for F) with R² > 0.983. The presence of serum proteins impacts the blank-corrected spectra in the low 200 nm region in Figure 2 compared to Figure 1.

Figure 3

HPLC analysis of guinea pig serum induced hydrolysis of (A) DEA/NO-aspirin or (B) IPA/NO-aspirin (100 μM) in 100 mM phosphate buffer containing 50 μM DTPA at pH 7.4 and 37°C. Authentic standards eluted from a Phenomenex Luna C18 column, 3 μm, 150 × 2.1 mm, using a water and acetonitrile gradient containing 0.1% formic acid as follows: aspirin (19.6 min), salicylate (20.5 min), IPA/NO-aspirin (23.8 min), IPA/NO-salicylate (24.6 min), DEA/NO-aspirin (24.6 min) and DEA/NO-salicylate (25.7 min).

Figure 4

Representative spectral changes (n ≥ 3) indicating trapping of NO and HNO by MbO₂ (50 μM) during decomposition at 37°C of IPA/NO-aspirin or DEA/NO-aspirin in assay buffer ± GSH (1 mM): (A) 250 μM IPA/NO-aspirin, (B) 250 μM DEA/NO-aspirin, (C) 100 μM IPA/NO-aspirin + 2% guinea pig serum and (D) 50 μM DEA/NO-aspirin (given production of 2 eqs of NO) + 2% guinea pig serum. Initial scans are in red (MbO₂), final scans without GSH are in blue (155 min for A; 350 min for B; 1170 s for C; and 600 s for D) and final scans with GSH are in green (145 min for A; 210 min for B; 1170 s for C; and 690 s for D).

Figure 4

Representative spectral changes (n ≥ 3) indicating trapping of NO and HNO by MbO₂ (50 μM) during decomposition at 37°C of IPA/NO-aspirin or DEA/NO-aspirin in assay buffer ± GSH (1 mM): (A) 250 μM IPA/NO-aspirin, (B) 250 μM DEA/NO-aspirin, (C) 100 μM IPA/NO-aspirin + 2% guinea pig serum and (D) 50 μM DEA/NO-aspirin (given production of 2 eqs of NO) + 2% guinea pig serum. Initial scans are in red (MbO₂), final scans without GSH are in blue (155 min for A; 350 min for B; 1170 s for C; and 600 s for D) and final scans with GSH are in green (145 min for A; 210 min for B; 1170 s for C; and 690 s for D).

Figure 5

Maximum current intensity from an NO-specific electrode as a measure of NO and HNO release from 100 μM of (A) IPA/NO-aspirin or (B) DEA/NO-aspirin in assay buffer containing 2% guinea pig serum (in A, blue bars in assay buffer alone and green bars with addition of 1 mM ferricyanide. The data are expressed as mean ± SD (n ≥ 3).

Figure 6

NO and HNO release measured in A549 cells. The cells were exposed 100 μL of 10 μM DAF-2DA in PBS pH 7.4 for 75 min at 37°C and washed three times with PBS pH 7.4 to remove excess dye. Upon addition of 10 μM of DEA/NO-aspirin, IPA/NO-aspirin or aspirin in DMSO (<0.1%), 10 μM IPA/NO or DEA/NO in 10 mM NaOH or DMSO (n = 2, six replicates per plate), the increase in fluorescence intensity at 535 nm was measured as a function of time at 37°C following excitation at 485 nm. The data are expressed as mean ± SD.

Figure 7

Examination of the stomach ulcerogenicity of IPA/NO-aspirin and DEA/NO-aspirin (1.38 mmol/kg). Ulcerogenicity was not significantly increased compared to control.

Figure 8

Effect of IPA/NO-aspirin, DEA/NO-aspirin, IPA/NO, DEA/NO and aspirin (25 and 50 μM) on PGE₂ levels after 24 h exposure to A549 cells (experiment in triplicate; *, p < 0.001 vs. control) at 37°C.

Figure 9

The effect of NONO-aspirin prodrugs and appropriate controls (10-200 and 25-100 μM, respectively) on cell viability of (A) A549 and (B) HUVEC cells. Cells were treated for 48 h at 37°C and then analyzed by the spectrophotometric MTT assay (n = 3 for A549s and n = 1 for HUVECs in at least triplicate per plate).

Figure 10

Effect of IPA/NO-aspirin, DEA/NO-aspirin, IPA/NO, DEA/NO and aspirin (100 μM) on GAPDH activity in A549 cells at 1 h (experiment in triplicate) at 37°C. The data are expressed as mean ± SD (experiment in triplicate), and one-way ANOVA was applied to determine the significance of the difference between control and treatment groups (*, p < 0.01).

Figure 11

The effect of IPA/NO-aspirin (500 μm) on contractility and relaxation in isolated ventricular myocytes (IPA/NO-aspirin in blue compared to control in red): (A) representative trace of sarcomere length (μm), (B) representative trace of calcium transient (ratio 365 nm/380 nm), (C) absolute changes in sarcomere shortening (n = 12 cells), (D) calcium transients and (E) myocyte relaxation (time of half relaxation from peak shortening (t₅₀). The data are expressed as mean ± SEM (*, p < 0.005; **, p < 0.001; ***, p < 0.0001).

Figure 11

The effect of IPA/NO-aspirin (500 μm) on contractility and relaxation in isolated ventricular myocytes (IPA/NO-aspirin in blue compared to control in red): (A) representative trace of sarcomere length (μm), (B) representative trace of calcium transient (ratio 365 nm/380 nm), (C) absolute changes in sarcomere shortening (n = 12 cells), (D) calcium transients and (E) myocyte relaxation (time of half relaxation from peak shortening (t₅₀). The data are expressed as mean ± SEM (*, p < 0.005; **, p < 0.001; ***, p < 0.0001).

Figure 11

The effect of IPA/NO-aspirin (500 μm) on contractility and relaxation in isolated ventricular myocytes (IPA/NO-aspirin in blue compared to control in red): (A) representative trace of sarcomere length (μm), (B) representative trace of calcium transient (ratio 365 nm/380 nm), (C) absolute changes in sarcomere shortening (n = 12 cells), (D) calcium transients and (E) myocyte relaxation (time of half relaxation from peak shortening (t₅₀). The data are expressed as mean ± SEM (*, p < 0.005; **, p < 0.001; ***, p < 0.0001).

Scheme 1

Dual decomposition mechanisms available for primary amine diazeniumdiolates leading to release of HNO or NO.

Scheme 2

Synthesis of NO or HNO releasing aspirin prodrugs.

Scheme 3

Possible pathways leading to HNO and salicylate production from spontaneous decomposition of IPA/NO-aspirin via initial amine deprotonation.

Scheme 4

Possible pathways leading to salicylate and free diazeniumdiolate production from spontaneous decomposition of NONO-aspirin prodrugs via initial ester hydrolysis.