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beyond reason
nitro oxide는 gas signal molecul로서 치료용으로 사용할 수 있음
Abstract
The biological effects attributed to nitric oxide (•NO) and nitroxyl (HNO) have been extensively studied, propelling their array of putative clinical applications beyond cardiovascular disorders toward other age-related diseases, like cancer and neurodegenerative diseases. In this context, the unique properties and reactivity of the N- O bond enabled the development of several classes of compounds with potential clinical interest, among which •NO and HNO donors, nitrones, and nitroxides are of particular importance. Although primarily studied for their application as cardioprotective agents and/or molecular probes for radical detection, continuous efforts have unveiled a wide range of pharmacological activities and, ultimately, therapeutic applications. These efforts are of particular significance for diseases in which oxidative stress plays a key pathogenic role, as shown by a growing volume of in vitro and in vivo preclinical data. Although in its early stages, these efforts may provide valuable guidelines for the development of new and effective N-O-based drugs for age-related disorders. In this report, we review recent advances in the chemistry of NO and HNO donors, nitrones, and nitroxides and discuss its pharmacological significance and potential therapeutic application.
2.1.1.2 Dual-acting antithrombotic and anti-inflammatory agents
The development and rupture of atheroma plaques and subsequent thrombus formation in coronary arteries leads to
vessel obstruction and inflammatory damage, which are triggering events in unstable coronary syndromes. Diseased
vessels have impaired •NO release, which contributes to thrombus formation and enhances the risk of thrombotic
events. Local endothelial release of •NOcan prevent platelet adherence and activation, thus having an antithrombotic
and anti-inflammatory outcome. In this sense, controlled •NO release with •NO donors can be an effective approach
to treat thrombotic events, owing to their antiplatelet, antithrombotic, anti-inflammatory, and blood pressure lowering
activities.60,61
The antiplatelet agent 3-n-butylphthalide (NBP, Fig. 3) was recently recognized as a potential antithrombotic drug
that can improve microcirculation and reduce brain infarct volume, providing clinical benefits to patients with ischemic
stroke,62 being recently approved in China for the treatment of ischemic stroke.63 In this context, NBP has been used
as a scaffold for the development of •NO donors (compounds 10–11, Table 1, Fig. 4).64,65 Compound 10 (Table 1,
Fig. 4), bearing a four-carbon linker and a diethylamino side chain,was the most potent of the developed series,with an
inhibition of platelet aggregation of 88.5% (IC50 = 0.24 mM), a remarkable •NO release (0.3 𝜇g/mL) and an enhanced
antithrombotic activity overNBP and aspirin, in a rat extra-corporeal circulation of arteriovenous (A-V) cannulamodel.
The thrombusweights in the rats treated with compound 10 (27.95°æ7.32 mg)were slightly lower than that in the NBPtreated
rats (29.97 °æ 7.01 mg) and with aspirin (30.73 °æ 6.08 mg). The antithrombotic effect was also evaluated on a
mouse model of thromboembolism with collagen- and adrenalin-induced thrombosis. Treatment with compound 10
significantly reduced the onset of hemiplegia and death inmice and its protective effect was similar to that of NBP and
aspirin (72.2, 70.6, and 80.0 %, respectively).64 The isosteric substitution of the ester function by an amide (compound
11, Table 1) led to a similar derivative that inhibited ADP-induced platelet aggregation by 83.0% (IC50 = 54.44 𝜇M)
and maintained sustained •NO release (0.057 𝜇g/mL).65 Moreover, the in vivo hydrolysis of compounds 10 and 11
released NPB and ferulic acid (Fig. 4), a hydroxycinnamic acid (HCA) with potent antioxidant and cardioprotective
properties,66,67 improving their antithrombotic potential.
Salicylic acid (Fig. 3) was used as a template for the development of nitrooxyacyl derivatives targeting
cyclooxygenase-1 (COX-1), as a strategy to obtain vasoactive anti-inflammatory agents.68 Compound 12a (Table 1)
stood out as a potent and irreversible COX-1 inhibitor, blocking collagen-induced platelet aggregation in human platelet-rich plasma (IC50 COX-1 = 0.74 °æ 0.17 𝜇M; IC50 COX-1/IC50 COX-2 = 0.39). Interestingly, themodification of the
carbon chain length to a higher homologous sequence (compound 12b) switched the pharmacological profile toward
preferential COX-2 inhibition (IC50 COX-2 = 1.1 °æ 0.2 𝜇M; IC50 COX-2/IC50 COX-1 = 0.13).68
2.1.1.3 •NOdonors as neuroprotective agents
Neurodegeneration is a multifactorial and complex process, involving several pathogenic mechanisms, namely oxidative/
nitrosative stress, mitochondrial dysfunction, and neuroinflammation. Thus, recent drug design strategies for
the development of neuroprotective agents encompassed the use of scaffolds with established antioxidant and antiinflammatory
activities.
Owing to its plethora of in vitro biological activities, ferulic acid (Fig. 3)was also explored as a scaffold for the development
of drugs to tackle neuroinflammation.69 Compound 13 (NCX 2057, Table 1), a 4-nitrooxybutyl ester of ferulic
acid, elicited neuroprotection in an animalmodel for chronic neuroinflammation and Alzheimer’s disease.69 NCX 2057
(13) inhibited iNOS mRNA and protein expression (IC50 = 6.2 °æ 1.0 𝜇M) without altering iNOS protein degradation
rate, decreased the levels of LPS/IFN-𝛾-induced nitrite accumulation (IC50 = 4.3 °æ 0.7 𝜇M) in RAW 264.7 cells, and
inhibited LPS-induced translocation/activation of the nuclear factor 𝜅B (NF-𝜅B).70 These effects were dependent on
•NOrelease, since its denitratedmetabolite (NCX2059) was onlyweakly effective and ferulic acid displayed no activity
whatsoever.70 NCX 2057 also showed a favorable outcome in chronic inflammatory and neuropathic pain models,71,72
and again the activitywas •NOdependent. Finally,NCX2057 competitively inhibitedCOX-1 andCOX-2 in whole RAW
macrophages (IC50 = 14.7 °æ 7.4 and 21.6 °æ 7.5 𝜇M, respectively71), further improving its anti-inflammatory outline.
Flurbiprofen (Fig. 3), a widely used nonsteroid anti-inflammatory drug (NSAID) and potent COX inhibitor, was also
used as a model for the development of •NOreleasing neuroprotective derivatives. HCT 1026 (compound 14, Table 1)
was thus developed as a prodrug of flurbiprofen by derivatizing the carboxylic acid function with a 4-(nitrooxy)butyl
ester as a •NO releasing moiety. Compounds 13 and 14 (Table 1) can undergo rapid hydrolysis (≥ 80%) yielding the
parent compounds ferulic acid and flurbiprofen, respectively, and the corresponding nitrooxybutyl alcohol. The nitrate
moiety is subsequently metabolized by glutathione-S-transferase (GST) to the corresponding nitrogen oxides.36 The
involvement of erythrocytes in bioactivation was also proposed, since sustained •NO release was observed in deoxygenated
whole blood.36 The nitrosylated flurbiprofen derivativeHCT1026 (14)was also shown to inhibit bone resorption,
both in vivo and in vitro, in an •NO-independent fashion.73,74 Moreover, HCT 1026 (14) was also found to inhibit
cytokine-induced signaling by inhibiting the effects of receptor activator of nuclear factor kappa B ligand (RANKL),
TNF, IL-1, and LPS in both osteoclast and macrophage cultures.75 Considering the role of RANKL, TNF, and IL1 as mediators
of inflammation,HCT1026and structurally related derivatives could be of pharmacological interest as novel class
of anti-inflammatory agents.
2.1.1.4 •NOdonors as pulmonary vasodilators
Amine-based diazeniumdiolates, also calledNONOates, were originally synthesized byDrago and colleagues and have
come into wide use as •NO donors.76 NONOates hold advantages over organic nitrates, namely spontaneous release
•NOin physiologicalmedia, which mitigates the risk of metabolic tolerance observed for organic nitrates.77 NONOate
derivative DETA/NO (compound 15, Table 1) was studied as a pulmonary vasodilator in patients with acute respiratory
distress syndrome (ARDS).78 Lam et al. concluded that inhalation of DETA/NO reduced pulmonary vascular resistance
and mean pulmonary arterial pressure without affecting the systemic blood pressure or cardiac output in ARDS
patients.
2.1.1.5 Cancer and chemoprevention
Excessive and unregulated •NO production has been implicated as a causal or contributing factor on several types of
cancer, due to its tumor growth and proliferation properties.79 Accordingly, controlled and sustained •NO production
and release may have the opposite outcome,80–82 providing the rationale for the use of •NO donors in cancer. In this
context, several research groups developed •NOdonors using scaffolds with described anticancer activity.
Cai et al. synthesized 5-fluorouracil (5-FU)-NONOate hybrids 16a-b (Table 1), which displayed enhanced cytotoxicity
on human cancer cells lines HeLa and DU145 (16a: LD50 HeLa = 112 𝜇Mand LED50 DU145 = 98 𝜇M; 16b: LD50
HeLa = 50 𝜇M and LD50 DU145 = 129 𝜇M).83 These derivatives outperformed 5-FU under the same experimental
conditions (LD50 HeLa = 278 𝜇M and LD50 DU145 = 204 𝜇M). These observations are in accordance with previous
reports from Saavedra et al. concerning in vitro antileukemic activity of structurally simpler NONOate derivatives.84
Lu et al. developed •NO-releasing derivatives based on the HCA scaffold and evaluated their in vitro cytotoxicity
against human hepatocellular carcinoma cells (SMMC-7721 andHepG2) and human breast cancer cells (MCF-7), using
5-FU and adriamycin drugs as standards.85 Compound 17 (Table 1) displayed potent cytotoxic activity (LD50 SMMC-7721
= 6.1 𝜇M; LD50 HepG2 = 7.3 𝜇M; LD50 MCF-7 = 3.8 𝜇M), enhanced over that of the standard anticancer drug 5-FU (LD50
SMMC-7721 = 39.0 𝜇M; LD50 HepG2 = 43.3 𝜇M; LD50 MCF-7 = 16.7 𝜇M). Compound 17 inhibited HepG2 cell proliferation
in a concentration-dependent manner and did not present cytotoxicity toward nontumor LO2 hepatic cell line, which is
indicative of a selective cytotoxicity. The cytotoxic effectwas associatedwith the levels of •NOreleased in cancer cells
and to the antioxidant properties of the HCA unit.85 Using a similar approach, Li et al. developed structurally related
•NO donors, and assessed their in vitro cytotoxic activity in a panel of human cancer cell lines (lung cancer,melanoma,
cervical, neck and head, and breast).86 The hybrids based on ferulic acid containing a phenylsulfonylfuroxan moiety
(18a–c, Table 1) were the most potent compounds, displaying IC50 values below 10 𝜇M against all the tested human
cancer cell lines. TheHCAunit and the •NOreleasing propertieswere identified as effective contributors to anticancer
activity under the current experimental conditions.86
Naturally occurring curcumin (Fig. 3) was also used in the development of •NO donors.87,88 Ahmed et al. developed
curcumin-based •NOdonors 19a–d (Table 1) and their anticancer and anti-inflammatory activities were screened
in human monocytic leukemia (THP-1) cells. Curcumin and derivatives 19a–d were not cytotoxic when incubated at
10 𝜇Mand significantly increased nitrite production in the same cellularmodel.87
The tetracyclic diterpenoid isosteviol (Fig. 3) has attracted considerable interest due to its broad spectrum of biological
activities,89–91 andwas thus used as scaffold for the development of •NOdonors.92 Biological screening identified
compound 20 (Table 1) as a potent antiproliferative compound on B16F10 cells (ED50 = 0.016 °æ 0.004 𝜇M), a significant
increase over isosteviol (ED50 ˃ 100 𝜇M).92
The concept of chemopreventionwas also explored using the salicylic acid scaffold.93 A series of aspirin derivatives
bearing •NOand hydrogen sulfide (H2S) releasing moieties was thus developed, and compound 21 (NOSH-1, Table 1)
was identified as the most potent derivative.93 NOSH-1 effectively inhibited cell proliferation in a panel of human
cancer cell lines: adenomatous (ED50 = 48–60 nM), pancreatic (ED50 = 47–57 nM), lung (ED50 = 50 nM), prostate
(ED50 = 88 nM), epithelial (ED50 = 75–280 nM), and lymphocytic leukemia (ED50 = 100 nM) cell lines. Moreover,
NOSH-1 displayed anti-inflammatory activity similar to that of aspirin in different experimental models, including the
carrageenan rat paw edema model and COX-dependent production of PGE2. Rats treated with compound 21 (0.52
mmol/kg), showed a plateaued change in paw volume of ΔV = 0.45 mL after 1−2 hr, which then deceased steadily over
the next 4 hr to ΔV = 0.35 mL, an effect comparable to that of aspirin (0.56 mmol/kg). Significant COX inhibition was
also observed: compound 21 reduced PGE2 levels to 42°æ3and21°æ4 pg/mgprotein at 0.21 and 0.52 mmol/kg, respectively
(control group = 82 °æ 2 pg/mg), albeit less active than aspirin (12 °æ 3 pg/mg protein at 0.21 mmol/kg).93
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