Novel R-roscovitine NO-donor hybrid compounds as potential pro-resolution of inflammation agents

Abstract

Neutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and possess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution derivatives, here we describe the design, synthesis and investigation of the biological potential of a small series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties (compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an increased pro-apoptotic activity when compared with either R-roscovitine or structurally related compounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apoptosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi-NO-donor hybrids may have additive pro-resolution of inflammation effects.

Figure 1

Induction of neutrophil apoptosis by R-roscovitine and NO-donor R-roscovitine compounds. Representative flow cytometric plot showing forward and side scatter with neutrophils gated (a), and representative flow cytometric plots of annexin V (x axis)/propidium iodide (y axis) staining after 6 h in culture for control untreated neutrophils (b), neutrophils cultured with R-roscovitine 3 μM (c) and neutrophils cultured with compound 9a 3 μM (d). For annexin V/PI plots lower left quadrant contains viable cells (annexin V negative, PI negative), lower right quadrant contains apoptotic cells (annexin V positive, PI negative), upper right quadrant contains necrotic cells (PI positive). Neutrophil morphology. Representative cyto-centrifuge preparations after staining with Diff-Quick™ (400× light microscopy) in freshly isolated neutrophils (e), untreated neutrophils aged for 6 h (f), neutrophils treated with R-roscovitine 3 μM for 6 h (g) and neutrophils treated with compound 9a 3 μM for 6 h (h). Black arrows indicate apoptotic cells, recognizable by cellular shrinkage and nuclear pyknosis. Contaminating eosinophils (<4%) are also visible, identified by the pink/red staining of their cytoplasm.

Figure 2

R-Roscovitine and NO-donor R-roscovitine compounds induce neutrophil apoptosis in a time-dependent manner. Time course demonstrating the percentage (%) of viable (solid line, ■), apoptotic (dotted line, ●) and necrotic (dashed line, ▴) neutrophils induced by treatment with 10 μM R-roscovitine (1) (panel a), compounds 2 (panel b), 9a (panel c) and 9c (panel d). Results expressed as mean ± SEM, n = 3.

Figure 3

R-Roscovitine and NO-donor R-roscovitine compounds induce neutrophil apoptosis in a concentration-dependent manner. Percentage of neutrophil death induced by treatment with R-roscovitine (1), compounds 2 and 9a,b (panel a) and compounds 9c,d (panel b), normalized with respect to the control cells after 6 h of culture. All non-viable, annexin V positive neutrophils are included as at 6 h the percentage of necrotic cells are negligible (Fig. 2). Results expressed as mean ± SEM, n = 6–35, analysed by one-way ANOVA with Newman–Keuls post hoc test with. ^∗p <0.05, ^∗∗p <0.01,^∗∗∗p <0.001 with respect to R-roscovitine and ^#p <0.05, ^##p <0.01, ^###p <0.001 with respect to the corresponding des-NO derivative.

Figure 4

(a and b) Neutrophil apoptosis induced by NO-donor R-roscovitine compounds is not affected by inhibition of NO-synthase nor soluble guanylate cyclase. Percentage of neutrophil apoptosis after 6 h of culture using different concentrations of l-NAME (panel a) and 10 μM ODQ (panel b), in the presence of R-roscovitine (1) and compound 2, 9a and 9c (3 μM and 10 μM). Results expressed as mean ± SEM, n = 3. (c) PTIO and NO scavenging. Percentage of neutrophil apoptosis after 6 h of culture using different concentrations of PTIO, in the presence of gliotoxin (1 μg/mL), R-roscovitine (1), compound 2 and 9a (3 μM). Results as mean ± SEM, n = 5–6, analysed by one-way ANOVA with Newman–Keuls post hoc test ^∗p <0.05, ^∗∗p <0.01, ^∗∗∗p <0.001, and ^#p <0.05, ^##p <0.01, ^###p <0.001 with respect to R-roscovitine in the same conditions.

Scheme 1

Preparation of derivative 2. Reagents: (i) 4-nitrooxybutirric acid, EDC, DMAP, dry Pyr, 4 h, rt.

Scheme 2

Preparation of derivatives 9a–d. Reagents and conditions: (i) H₂SO₄, H₂O/MeOH, 3 h, Δ; (ii) benzylmercaptane, DIPEA, EtOH, 4 h, Δ; (iii) 2-bromopropane, K₂CO₃, DMSO, 12 h, 50 °C; (iv) R-(2)-amino-1-butanol, nBuOH, 5 days, Δ; (v) mCPBA, CH₂Cl₂, 3 h, rt; (vi) 12, 14, 16 or 18, Et₃N, MeOH, 5 days, 40 °C.

Scheme 3

Preparation of amino derivatives 12, 14, 16, 18. Reagents and conditions: (i) K₂CO₃, KI, DMF, CH₃CN, 20 h, 50 °C; (ii) TFA 10% CH₂Cl₂, 3 h, rt; (iii) tBuO⁻K⁺, dry THF, 4 h, rt → Δ; (iv) PPh₃, DIAD, dry THF, 12 h, 0 °C → rt; (v) DBU, CH₂Cl₂, 12 h, rt.