nitric oxide: state of the art in drug design
TRANSCRIPT
386 Current Medicinal Chemistry, 2012, 19, 386-405
0929-8673/12 $58.00+.00 © 2012 Bentham Science Publishers
Nitric Oxide: State of the Art in Drug Design
R.A.M. Serafim, M.C. Primi, G.H.G. Trossini and E.I. Ferreira*
LAPEN: Laboratory of Design and Synthesis of Chemotherapeutic Potentially Active against Neglected Diseases, Department of
Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo – FCF/USP, Brazil
Abstract: Since the great discovery of Furchgott, Ignarro and Murad in the late 90´s, nitric oxide (NO) is considered one of the most
versatile endogenous molecules, which is involved in important signaling biochemistry pathways of the human body. Thus, it is directly
related to pathological processes and its over- or low-production is able to cause damage in systems that are involved. By using certain
functional groups present in molecules that already have potential therapeutic value, hybrid compounds, by means of inclusion of NO-
donors (e.g., ester nitrates, furoxans, benzofuroxans, NONOates, S-nitrosothiols, metal complexes), can be generated that have a NO
release benefit along with maintaining the activity of the native drug. This approach has proved to be useful in many spheres of
Medicinal Chemistry, such as cardiovascular, inflammatory, bacterial, fungal, viral, parasitic, ocular diseases and cancer. Potent and
selective nitric oxide synthase inhibitors are being designed, mainly through enzyme structure based process, however, due to high
homology between the isoforms, these studies have proved to be very difficult. The objective of the research is to achieve a balance
between the release of therapeutic amounts of NO, especially in specific site of action, and maintaining the native drug activity. The
search for new and effective NO-donor hybrid drugs, as well as selective nitric oxide synthase inhibitors, is an important focus in modern
drug design in order to manipulate biochemical pathways involving NO that influence many dysfunctions of the human organism.
Keywords: Drug design, nitric oxide, NO-releasing agents, nitric oxide synthase inhibitors, immune system, hybrid compounds, mutual prodrugs, medicinal chemistry, therapeutic potential, biochemical pathways.
1. INTRODUCTION
Nitric Oxide (NO) is an uncharged diatomic molecule containing 11 electrons in its valence shell, one of which is unpaired [1,2]. The molecule is produced by bacteria, plants and animals and notably, the majority of the chemical interactions involving NO in biological systems entail the stabilization of the unpaired electron. NO solubility is higher in nonpolar media and therefore tends to dissolve in cell membrane and lipid phases [1,3]. In its pure state, and under ambient temperature and pressure conditions, NO remains in gaseous form. However, within organisms it acts in the form of a dissolved non-electrolyte, except at the lung, where NO can also be found in its gaseous state [1].
The importance of research in NO has been recognized by the scientific community at several junctures. In 1992, Science magazine elected NO as the molecule of the year [4], and in 1998 the Nobel Prize in Physiology and Medicine was awarded to the researchers R. Furchgott, L. Ignarro and F. Murad, whose work led to the discovery of NO as a biological mediator produced by mammalian cells [5,6]. Further, in 1999, Salvador Moncada was the most cited scientist in the biomedical field for his group of work on mammalian NO [5]. The Nitric Oxide Society was founded in 1996, publishers of the official journal on NO, “Nitric Oxide: Biology and Chemistry” [7]. An online journal about NO is also available called “The Open Nitric Oxide Journal”, which seeks to fast-track the publication of quality articles and provides ease of access to researchers worldwide [8].
1.1. Historical Aspects
NO was originally regarded as an environmental pollutant but in the late 1970s Robert Furchgott, upon analyzing vascular relaxation by acetylcholine in blood vessels, noted the obligatory role of endothelial cells to enable vasodilation [9,10,11]. Thus, Furchgott identified the interference of a factor needed in vasodilation which he termed endothelium-derived relaxing factor (EDRF) [9,10]. Furchgott later suggested that EDRF could in fact be NO, and in 1986 found several similar properties shared by both EDRF and NO. In 1987, the research groups of Ignarro and Moncada demonstrated that EDRF and NO were indeed the same molecule [9,12,13].
*Address correspondence to this author at the Av. Prof. Lineu Prestes, 580 05508-900 Sao Paulo – SP, Brazil; Tel: 55 11 3091-3793; Fax: 55 11 3815-4418;
E-mail: [email protected]
1.2. NO Generation and Nitric Oxide Synthases
NO is generated from L-arginine by a family of enzymes denominated nitric oxide synthases (NOSs) [14,15,16]. These enzymes were originally identified and described in 1989 and the first crystallographic structures of their domains were determined in 1998 and published in 1999 [5]. The NOSs present as three distinct isoforms (Fig. 1), two of which are defined as constitutive and the other induced [14,16]. The constitutive isoforms include: neuronal (nNOS), present in nerve cells, skeletal muscle and heart muscle; and endothelial (eNOS), present in the cells coating blood vessels [14,3]. The inducible isoform (iNOS) occurs in many types of cells as an immune system response to inflammatory processes [14,3].
Fig. (1). Isoforms of NOSs.
The isoforms of NOS are dimeric, where each monomer has two domains, namely the reductase and oxygenase domains. The structure of the reductase domain comprises FAD, FMN and NADPH whereas the oxygenase domain comprises the heme group and BH4. These domains are linked by calmodulin, a calcium sensing portion, which can act as the enzyme activator [14]. The need for calcium in order to activate the enzyme is a factor differentiating the two types of NOS: the constitutive synthases (eNOS and nNOS) activated during high intracellular calcium, and the induced synthase (iNOS) which is calcium independent [17].
1.3. Biological Properties
NO can regulate a wide array of biological processes, in many cases through activation of guanylyl cyclase and increase of cyclic guanosine monophosphate (cGMP) synthesis from cyclic guanosine triphosphate (cGTP) [18,19,6]. However, there are many independent effects from cGMP, such as NO interactions with
nitric oxide synthases (NOSs)
constitutive inducible(iNOS)
neuronal(nNOS)
endothelial(eNOS)
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transition metals, thiol groups, free radicals, oxygen, superoxide anion, unsaturated fatty acids and other molecules. These effects, dependent or independent of cGMP, can regulate and alter a variety of biochemical and physiological events important in cell regulation and function [18]. NO is a simple molecule which possesses different signaling functions and is capable of acting as an intracellular messenger, autacoid, paracrine substance, as well as a neurotransmitter or hormone [18,20].
NO has a number of actions within organisms (Fig. 2), such as within the cardiovascular system, where NO is continuously produced by endothelial cells coating the lumen, and helps to maintain micro- and macro-vascular homeostasis through vascular relaxation and inhibition of platelet aggregation [15,19]. With regard to the nervous system, NO regulates neurotransmission. In the central nervous system, NO plays a key role in learning and in the formation of memory, whilst in the peripheral nervous system it regulates different reflexes such as gastrointestinal, respiratory and genitourinary [15,21,20]. These physiological activities take place by stimulating soluble guanylyl cyclase or NO-sensitive guanylyl cyclase, generating intracellular cGMP which causes relaxation in cells of smooth muscle [15,10,22].
At high concentrations, NO also performs the function of stimulating the host’s immune response against pathogens and tumor cells, and is able to invade and destroy pathogens [15,19,23,20]. At low concentrations however, NO can induce activation of inflammatory cells, particularly monocytes [15,19]. Moreover, NO has been shown to play a role in gastric mucosa protection, either independently or by modulating the effects of other substances [20,16].
Fig. (2). Systems in which NO is involved.
1.4. Therapeutic Potential
Owing to the properties outlined, NO has huge therapeutic potential. In its gaseous form, NO can be administered via the inhalatory route particularly in cases of pulmonary hypertension and for treating neonates. Along with the fact that the respiratory route of administration prevents gaseous NO from reaching all potential target sites of action, it also is difficult to deploy since NO oxidizes into NO2 in the presence of oxygen. NO transporting molecules, therefore, are needed that are able to stabilize its radical until time of release [19].
Currently, there is great interest in a class of molecules called nitric oxide donors (NO-donors) [15]. NO-donors are substances able to transport and release NO under physiological conditions to different areas of the organism [15,2]. Consequently, NO-donors are able to provide different types of therapeutic benefits [15]. The NO-donors most commonly used in clinical practice include organic nitrates, but there is also extensive use of sodium nitroprusside (SNP) from the class of NO-metal complexes [24,19,25]. In addition, a number of other promising NO-donors are being studied such as diazeniumdiolates (NONOates), S-nitrosothiols, furoxans and benzofuroxans [19,25].
The most extensively studied organic nitrate is glyceryl trinitrate (GTN) (Fig. (3) – compound 1), also known as nitroglycerine [19]. This is the prototype of the class and is used mainly for relief of angina-associated acute pain [19,25,26]. Slow release preparations are also widely employed, such as mononitrate isosorbide (ISMN) (Fig. 3 – compound 2), frequently used in cases of chronic angina [19]. SNP (Fig. 3 – compound 3) is a complex between NO and iron, and is widely used in hospitals via the intravenous route for rapid reduction of blood pressure in severe hypertension episodes [19,25].
The NO-donors cited are currently the focus of research and, although not yet in routine clinical use, have known biological activities [19]. NONOates (Fig. 4 – compound 4), one of the most studied classes of NO-donors to date, exhibit vasodilatation, platelet aggregation, inhibition of blood coagulation and inhibition of proliferation of cells of the vascular smooth muscle [24,19]. With regard to the S-nitrosothiols (Fig. 4 – compound 5), endogenous S-nitrosothiols have been identified, and some advantages over other NO-releasers have been established such as tissue selectivity in some cases, and potent platelet anti-aggregant action [19,27]. NO-releasing furoxans (Fig. 4 – compound 6) and some benzofuroxan derivatives (Fig. 4 – compound 7) are characterized by well-known vasodilatory and platelet anti-aggregant activities [28,25].
1.5. NO in Drug Design
A number of pathologic states often require action in more than one biochemical pathway, i.e. the use of supplementary pharmacodynamic mechanisms [29]. Great advances in knowledge on biochemical pathways and about pharmacological properties involving NO have sparked interest in devising hybrid molecules containing NO-donors (Fig. 5) that allow synergism of action between NO and the broad chemical diversity of ¨native¨ drugs [30].
Thus, owing to NO-donors groups already mentioned, releasing NO under different conditions, and pathologies where exogenous supplementation of NO is required, the design of hybrid molecules containing those groups may be beneficial for a variety of disorders, such as: cardiovascular, inflammatory, bacterial, fungal, viral, parasitic, ocular diseases and cancer. Likewise, the design of molecules that act selectively inhibiting NOS isoforms may be useful in diseases involving high concentration of NO, e.g.: arthritis, asthma, cerebral ischemia, Parkinson’s disease, neurodegenerative diseases and seizures [31].
O
O2NO O
NO2
O
NO2
O
OH
O NO21 - glyceryl trinitrate
2 - mononitrate isosorbide
Fe
NC NO
CN
CN
NC
NC
2
3 - sodium nitroprusside
2 Na
Fig. (3). Some NO-donors used in practice clinical.
NO
CARDIOVASCULAR SYSTEM
CENTRAL AND PERIPHERALNERVOUS SYSTEM
GASTRIC MUCOSA PROTECTION
IMMUNE RESPONSE(PATHOGENS AND TUMOR CELLS)
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N
N
NO O
HO NH
HN
OH
O
O
SNO
O
NH2
O
45
NNO O
6
N
O
N
O
7
Fig. (4). Structure of NO-donors: NONOate (diazeniumdiolate, DEA/NO)
(4), S-nitrosothiol (S-nitroso-glutathione, GSNO) (5) and general structure
of furoxans (6) and benzofuroxans (7).
In this review the design of potential NO-donors and selective NOS isoforms inhibitors for those diseases will be addressed.
DRUG NO-RELEASING MOIETY
DRUG NO-RELEASING MOIETY
NEW MOLECULE
LINKER
Fig. (5). NO-donor moiety linked to native drug generates a new hybrid
molecule. Adapted from [36].
2. NITRIC OXIDE IN MEDICINAL CHEMISTRY
2.1. NO-Releasing Agents
2.1.1. Cardiovascular
NO is strongly implicated in essential signalling of the cardiovascular system and is responsible for maintaining its homeostasis. NO mediates relaxation of smooth muscle, mainly by increasing production of cytosolic cGMP (cGMP-dependent mechanism). This occurs due to affinity of NO for metals such as Fe from the heme group present in guanylyl cyclase, and thus hemoglobin can serve as a group transporting NO throughout the organism [32,33].
The quantity of NO released into veins and flow regulation, especially the presence of eNOS, defines the physiological and pathophysiological profile of NO. A range of different pathologies such as arteriosclerosis, congestive heart failure, thrombosis and hypercholesterolemia are related to dysfunction in the release, to inadequate bioavailability at the action site, or to inactivation, of NO [32,33].
Mice submitted to eNOS knockout developed abnormalities in vascular relaxation, blood pressure regulation and heart contractility, showing a rise in blood pressure of around 30% compared to wild-type animals. NOS inhibitors also exhibit vasoconstrictor effects [33,34].
NO has also been shown to be an important regulator in the excitation-contraction of the cardiac system, influencing for instance, the pathway and signalling of -adrenergic receptors, with
nNOS and eNOS playing key roles in this modulation [35]. Other studies point to the role of iNOS in the pathogenesis of cardiac dysfunctions in mouse models of cytokine-induced cardiomyopathy [36]. Despite this finding and the other evidence available, future studies are needed to shed further light on action of NO in the heart.
By virtue of these properties, NO-donors have in recent years broadened the outlook for use of NO in the treatment of cardiovascular dysfunctions. Organic nitrates (e.g. trinitroglycerine and dinitrate isosorbide) have been used for this purpose from the 19
th century to the present day [37]. NO-donors in the furoxan class
(1,2,5-oxadiazole-2-oxides, furazan-N-oxides) are considered prodrugs since they exert their biological activity through NO release, created by sulfhydryl groups which mediate this release. The NO released activates soluble guanylyl cyclase, increasing intracellular levels of cGMP, thereby possessing vasodilatory potential. In vitro studies in rat heart, and in vivo studies in rabbit arteries, have shown concentration-dependent increase in blood flow [38].
Another pathway of action in the cardiovascular system is modulation of the renin-angiotensin system (RAS). In this system, the angiotensin converting enzyme (ACE) transforms angiotensin I (AT1) into angiotensin II (AT2), a peptide with vasoconstrictor potential. The use of ACE inhibitors (e.g. captopril, enalapril) is one of the main approaches for managing hypertension. In order to boost the effects of ACE inhibitors, an S-NO bond is introduced in the sulfhydryl group of captopril (S-nitroso-captopril) (Fig. 6), which is released by plasma esterases to promote its anti-hypertensive, platelet anti-aggregant, anti-thrombotic activity while potentializing the effect of captopril, and exert angiogenic inhibition activity [39,40]. However, derivative of enalapril containing the ester nitrate grouping (NCX-899) showed stronger vascular effects than the prototype and also prevention of cardiac remodeling [37].
N COOH
SH
O
N COOH
S
O
NO
captopril S-nitroso-captopril
Fig. (6). Captopril and it S-NO-donor analogue.
ACE inhibitors also have beneficial effects resulting from increased bradykinin concentration and NO production. However, bradykinin is responsible for coughing - the main side-effect of ACE inhibitors. In addition, AT2 can be produced through non-ACE pathways such as chymases [41]. Drugs of the antagonist class of AT2 receptors, referred to as “sartans”, have poorer clinical efficacy compared to ACE inhibitors but cause no side-effects since they do not inhibit bradykinin catabolism. Hence, they do not induce the cough and angioedema observed in ACE inhibitors [42].
Based on “sartans” and exploiting the activity of NO-donor groups, these two structures were hybridized by means of linker groups in order to increase the therapeutic potential of the class. Therefore, they have the potential to antagonize the AT2 receptor (more specifically, type I receptors) and exhibit vasodilatory properties independent of bradykinin derived from NO [43]. The structure of the linkers between NO-donor and sartan is chosen so as to modulate NO release in a therapeutically beneficial manner. Fig. (7) shows two of the NO-sartans (8 and 9) synthesized by Breschi and co-workers (2006) [44], which release NO slower than SNP (known rapid NO-donor drug).
Some NO-sartans have prodrug properties since the antagonist activity of type I AT2 receptors only manifest after release of the
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 389
respective sartan by esterases. The 9 compound has shown anti-hypertrophic effects similar to those observed in losartan and captopril. The additional vasorelaxant effects of 9 are thought to stem from NO release since this effect is inhibited upon addition of a guanylyl cyclase inhibitor. Moreover, 9 was also found to influence platelet aggregation, denoting the action of the released NO. It is noteworthy that the effects of adenosine diphosphate - ADP (an important platelet aggregant) were inhibited 50% by the 9 compound, an effect seen to a lesser degree in losartan [44].
Another important group of drugs used both for hypertension as well as arrhythmias and vascular problems are the L-type voltage-dependent calcium channel blockers, represented by the 1,4-dihydropyridines (DHPs) [45]. A series of 4-phenyl-DHPs linked to furazan and furoxan rings was synthesized (Fig. 8 – compounds 10 and 11, respectively), with the former exhibiting only antagonistic properties on calcium channels and predictably, devoid of NO-donor activity. The latter however, showed NO-dependent vasodilatory activity in studies with guanylyl cyclase inhibitors [46].
Therefore, the hybridization of DHPs with NO-donors, especially furoxans, indicates the possibility of achieving a balanced vasodilator effect between the NO-dependent mechanism and calcium channel blocking [46]. In 1999, Dhein and co-workers had previously showed that DHPs are capable of activating an endogenous NO release.
Another class of prodrugs which act on the cardiovascular system, the 1-antagonists, was also the target of molecular hybridization. Prazosin ( 1-antagonist) was linked to furoxan to potentialize vasodilatory action. The compound 12 (Fig. 9) had similar activity values to both prazosin and SNP. The molecular hybrid showed dual activity (NO-donor and 1-antagonism) at approximately the same concentration interval. NO-donor potential was again confirmed since, in the presence of oxyhemoglobin (HbO2), a known NO scavenger, the compounds appeared as typical
1-antagonists. Additionally, in vasodilation trials with K+-
depolarized aortic strips, an increased IC50 value was obtained in the presence of HbO2. It is important to stress that, overall, the ring system of furoxans is characterized by its high flexibility, which
N
NHO
N
N
HN N
losartan
N
NO
N
N
HN N
O
N
NO
N
N
HN N
O
(n)O2NO
O2NO
8
9
Fig. (7). Losartan and NO-sartans.
NH
R
COOCH3H3COOC
NH
COOCH3H3COOC
OCH2
N O
N
R
NH
COOCH3H3COOC
OCH2
N O
N
CN
O
4-phenyl-DHP scaffold
10 11
Fig. (8). 4-Phenyl-DHPs scaffold and its furoxan derivatives.
390 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
enables its inclusion in diverse active molecules in order to potentialize activity [47].
Drawing on this feature, a series of furoxans was linked to a propranolol-like skeleton and the resulting compounds assessed according to their 1-adrenoceptor blocking and NO-dependent vasodilating activity (Fig. 10 – compound 13) [48]. A reduction in the potency of the compounds was seen when the experiments were carried out in the presence of HbO2. Furoxanic derivatives evidenced the potential for thiol-induced NO release through the formation of nitrites (NO metabolite). Generally, hybrids of the series had lower affinity to -receptors, more specifically 2, compared to propranolol. However, compound 14 showed similar potency to propranolol and 21-fold greater affinity for the 1 than the 2 receptor. The same compound also has a high level of NO-dependent vasodilation due to the furoxanic substructure introduced during molecular hybridization. Both properties were attained at the same concentration range thus demonstrating the obtention of a molecule with highly desirable pharmacological characteristics.
Currently, the use of acetyl salicylic acid (ASA) (Fig. 11) in the prophylaxis of cardiovascular diseases such as thrombosis and myocardial infarct is routine clinical practice. This prophylactic effect is due to ability of ASA to irreversibly inhibit thromboxane production by platelets thereby reducing platelet aggregation. Doses of around 80 mg/day of ASA for long periods of time inhibit the synthesis of thromboxanes while having little or no activity on cyclooxygenases in many tissues [49,37]. However, ASA blocks only one pathway of platelet activation, a process that may occur through alternative pathways [37]. In addition, it is worth recalling that, even at low doses, the non-steroidal anti-inflammatories (NSAID) can cause ulcers and hemorrhages with prolonged use.
It should be noted that cGMP independent pathways may exist, despite NO showing anti-aggregant platelet activity through guanylyl cyclase activation and intracellular calcium increases diminishing the binding of elements to fibronectin (responsible for platelet activity) [37,49,50]. Thus, the coupling of NSAIDS with NO-donor moieties represents an important advance in the design of molecules with anti-thrombotic and anti-inflammatory potential. Moreover, owing to the gastrosparing potential of NO (via cGMP
dependent and independent pathways), the hybridization of these molecules does not lead to gastrointestinal damage [51,49,37]. The administration of NCX-4016 (Fig. 11) for example, produced lower blood pressure (around 10-20 mm Hg) in comparison to animals treated with ASA alone where, besides the cardioprotective effect, the derivative showed marked vasodilation related to the released NO [52].
O
O
OH
O
O
O
O
OONO2
NCX-4016ASA
Fig. (11). Acetyl salicylic acid and NCX-4016.
In the field of hypocholesterolemics, the prodrugs known as “statins” are widely used due to their potential for inhibiting the enzyme 3-hydroxy-3-methylglutaryl-Coa reductase (HMGCoA-reductase). Pathologies such as arteriosclerosis and diabetes can be conducive to cardiovascular risk, reducing endogenous production of NO. Thus, NO-statins such as NCX-6550 and NCX-6553 (Fig. 12) represent an important supplementary source of NO, reducing inflammatory effects, inhibiting in vitro platelet aggregation, improving endothelial dysfunction in animals, and maintaining inhibitory activity of HMGCoA reductase [37,53].
2.1.2. Inflammation and Gastro-Sparing
NSAIDs are widely used for inflammatory diseases such as arthritis, rheumatism and atherosclerosis. Their mechanism of action is by the inhibitory activity of the enzyme cyclooxygenase in both isoforms (Cox-1 and Cox-2), thereby inhibiting the formation of prostaglandins (PGs) [54]. PGs produced by Cox-1 are important for sparing gastric mucosa since they regulate the secretion of
N
N N NH3CO
H3CO
NH2
O
ON
N N NH3CO
H3CO
NH2
S
NO
N
O
O
O
prazosin 12
Fig. (9). Prazosin and the NO-donor hybrid with dual activity.
O NH
X
OH NO
N
R
OO N
H
O
OH NO
N
S
O
OO
1314
Fig. (10). Propranolol-like skeleton with furoxan group and the active compound.
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 391
cytoprotective mucosa [54]. Consequently, NSAIDs cause ulcers in around 20% of patients in long term use of these drugs [56].
After prolonged use of ASA and consequent reduction in gastric PG synthesis, increased NO synthesis by eNOS in the mucosa takes place. NO-donors increase the blood flow of gastric mucosa, effectively reducing the gastric damage caused by NSAIDs. Excessive amounts of NO, however, can directly damage mucosa [16].
The introduction of NO-releasing groups into the structure of NSAIDs led to the emergence of a new class of drugs, the NO-NSAIDS. Given their release of NO, these drugs confer marked reduction in the gastric damage secondary to NSAIDs. This effect may be attributed to an increase in the blood flow of gastric mucosa, stimulation of cytoprotective mucosa production, among other mechanisms, in addition to other NO-dependent anti-inflammatory activities [57,16,58,59].
ASA have become one of the best selling drugs worldwide, thus serving as a potential prototype for creating NO-aspirins [60]. In recent years, a number of NO-aspirin derivatives have been synthesized using several different linkers (aliphatic, aromatic and
heterocyclic) between the NO-donor moiety (Fig. 13 lists some of these: NONOates – 15, furoxans – 16, nitrate ester – 17 and 18) and the carboxylic acid of ASA. The linkers used depend on the potential release of NO in molecules and their pharmacokinetic properties [37,61,62].
Studies have shown that the NCX-4016 compound (Fig. 11) is well absorbed by the oral route, releasing NO only after release of the spacer group by the hepatic esterases or those present in plasma [49]. Compound 16 however, showed reduction in gastric lesions compared to equimolar doses of ASA. This furoxan derivative has also revealed great NO-dependent platelet anti-aggregant potential (around 100-fold greater than ASA) in studies of collagen-induced aggregation, producing high levels of NO in plasma. In fact, this is potentialized owing to the break in furoxan-moiety by elements from plasma which have yet to be fully established. On the other hand, its Cox-inhibiting activity is likely more related with ASA-moiety than to NO-dependent activity [63,64].
Among several activities, compound 16 was able to reduce TNF- (tumor necrosis factor-alpha) cytokine release by monocytes and macrophages by 72% and 64%, respectively, even though the underlying mechanism remains unclear [65]. Studies have shown
HO
HO
O
COOH
OH OH
HO
HO
O
OH OH
O
OONO2
N
F
COOH
OH OH
N
F
OH OH O
OONO2
NCX-6550
NCX-6553pravastatin
fluvastatin
Fig. (12). Pravastatin and fluvastatin with its respective NO-statins.
O
O
O
O
NO
N
CN
O
O
O
O
O
ON
NN
O
O
O
O
O
OONO2 O
O
O
O
N ONO2
15 16
17
18
Fig. (13). NO-aspirin derivatives with different linkers and NO-donor groups.
392 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
that the NCX-4016 compound inhibits the formation of pro-inflammatory cytokines, such as interleukin-1 and interleukin-18 (IL-1 and IL-18) through the inhibition of the proteolythic enzyme caspase-1 [66]. Dimmeler and co-workers (1997) [67] showed that this cysteine-protease can be subject to S-nitrosylation in two cysteine subunits by NO-donors, thus leading to reduced formation of pro-inflammatory cytokines that require its activation.
The insertion of nitrooxy-acyl moieties in the phenolic hydroxyl group of ASA (Fig. 14) was another approach used, yielding compounds with anti-inflammatory activity as potent as the native drug, stable at physiologic pH (>90% stability), yet labile when tested in plasma-containing medium. Gastrosparing behavior was also maintained for these compounds [68].
Based on the novel nitrooxy-acyl esters of ASA developed by Lazzarato and co-workers (2009) [69], aromatic derivatives have showed enhanced results. Benzoyl derivatives with the NO-donor chain in the meta position was found to exhibit greater activity in a hydrolysis study involving numerous molecular variations.
Other NSAIDS linked to NONOates (Fig. 15) have shown benefits over NO-free NSAIDS. The compounds 19 (NONO-aspirin) and 20 (NONO-indomethacin) increased potency of analgesic activity 4- and 1.7-fold, respectively, compared with the parent compounds (ASA and indomethacin). With regard to toxicity to gastric mucosa in the experimental animals, NONO-aspirin produced few detectable lesions whereas NONO-indomethacin led to no apparent lesions. Notably, indomethacin caused considerable damage to mucosa. On the NO release test performed in phosphate
buffer at pH 7.4, the compounds of this class showed an interesting release profile. As expected, the profile was one of slow release [24], requiring the elapsing of 43-45 hours before detection of 1 mmol of NO/mol of compound [70].
Diclofenac derivatives containing S-nitrosothiol moiety as the NO-donor (S-NO-diclofenac – Fig. 16), were synthesized and assessed in vivo. These compounds showed good bioavailability by the oral route, depending on the volume of the spacing group used for molecule linking. These act as prodrugs, releasing significant amounts of diclofenac into plasma within 15 minutes and also exhibit anti-inflammatory and analgesic activities in equimolar doses with doclofenac. In addition, few gastric lesions were produced compared with diclofenac, causing only negligible stomach lesions compared to the NSAID prototype which caused lesions of >40 mm [71].
Other diclofenac derivatives were obtained via linking with benzofuroxan (Fig. 16 – compound 21). These derivatives also appeared promising in in vivo trials, where they retained the pharmacological characteristics of diclofenac after insertion of the NO-donor moiety, including analgesic and anti-inflammatory activity. Akin to the previous S-nitrosothiols, the damage to mucosa was reduced compared to the prototype. This reduction was likely due to NO, whose release by benzofuroxan-moiety was confirmed in pH 7.4 by nitrite formation which, as expected, did not occur with diclofenac [72].
Varying the groups linked to the NO-donor enabled the synthesis of ASA and diclofenac derivatives (Fig. 17 – compounds
O OH
O ONO2
O
R R
n
O OH
O
O
R R
ONO2
ONO2
n
Fig. (14). ASA with nitrooxy-acyl moieties in phenolic hydroxyl group.
O
O
O O
O O
NN
NO
O
O
O
19
N
OCl
H3CO
O O
O O
N
N N
OO
O
O
20
Fig. (15). ASA (19) and indomethacin (20) linked to NONOates.
Cl Cl
NH
O
O
SNO
RR`
Cl Cl
NH
O
O
N
O
N
O
S-NO-diclofenac 21
Fig. (16). NO-diclofenac compounds.
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 393
22 and 23, respectively). Groups containing disulfide at the position linked to nitrate esters were used for latentiation of these NSAIDs. The use of these spacers was based on the metabolism of the nitrate ester by sulfhydryl groups. SS-NO-aspirin and SS-NO-diclofenac derivatives act like prodrugs of salicylic acid and diclofenac, respectively. On the other hand, compounds in which the NO-donor was linked to the NSAID through amide function (Fig. 17 – compound 22c) failed to release the prototypes, perhaps because release occurs through enzymatic hydrolysis. Nonetheless, further studies investigating this release are required. It is important to point out that the compounds with greater bioavailability also had greater anti-inflammatory activity as well as gastric-sparing effect, due to the release of NO and the protection of the acid group of the NSAIDs, the main cause of their toxicity [73].
Cl ClNH
O
RO
O
R
O
22 23
22a ; 23a =
22b ; 23b =
22c =
OS
SO
NO2
OO
SS
O
ONO2
HNS
SO
NO2
Fig. (17). NO-aspirin and NO-diclofenac derivatives containing disulfide
bond.
In addition to these derivatives, a number of other NO-NSAIDs have been synthesized such as NO-ketoprofen, and NO-acetaminophen among others. All showed therapeutic advantages over the native drugs including (besides those already mentioned) protection against hepatotoxic effects due to the formation of NO in the organism [51,74,75].
The anti-inflammatory drugs of the coxib class were also targets of hybridization with NO-donor groups. The NO-rofecoxib prodrug was synthesized some years ago [76,77], while more recently, a series of NO-celecoxib derivatives containing the diazeniumdiolate as the NO-donor directly linked to the aromatic ring of this anti-inflammatory drug, has shown promise in terms of release and pharmacological properties [78].
2.1.3. Cancer
The exact role of NO in the physiopathology of tumors is a matter of heated debate and consensus on this question has yet to be
reached [79,80]. NO is known to play a role in numerous signaling pathways essential in the malignant aspects of cancer [79].
Experimental evidence has shown that the constitutive expression of NO by eNOS and iNOS has contrasting effects. On one hand, a
protumor effect has been observed while on the other, antitumor effects. This disparity is most likely associated to the concentrations
of NO involved [79,81].
Tumor tissue typically has high levels of endogenous NO compared to normal tissue, suggesting that NO exerts a protumor
effect [79,80]. NO can promote tumor growth through a variety of actions, such as regulating blood flow, maintaining vasodilatory
tonus, promoting metastases by increasing vascular permeability, affecting matrix metalloproteinases, and stimulating angiogenesis
[80,81]. The presence of NOSs has been confirmed in many different types of tumor including those of the breast, head,
prostate, bladder, colon and central nervous system. Many studies have also correlated high iNOS expression with a high incidence of
metastases and poor prognosis in gastric and colorectal carcinomas [79,80,82].
Studies involving specific NOS inhibitors have shown that such
inhibition reduces tumor blood flow and slows tumor growth, demonstrating an anti-tumor effect [79,83]. In addition, the
expression and exogenous presence of NO at high levels also creates an antitumor effect by triggering cell demise through
exposure to high levels of oxidative and nitrosative stress promoting cellular apoptosis and necrosis. However, despite the
apoptosis observed, this does not signify a halt in the development of clear tumors [79,80,81,84]. Moreover, studies have shown that
NO expression may exert indirect antitumor action by reducing metastatic potential of tumor cells, inhibiting platelet aggregation,
which in turn reduces adhesion of emboli of tumor cells to capillary beds [79].
More recently, the dose-dependent nature of NO has been demonstrated through the NO-donor S-nitro-N-acetyl-penicillamine
(SNAP). Low SNAP levels significantly increase angiogenesis yet when SNAP concentrations progressively raise, pro-angiogenic
effects start to wane with 80% inhibition of angiogenesis at a concentration of 4 mM [79]. Also notable in this case, is the fact
that the cell death caused by NO is related to its concentration, being capable of causing apoptosis or even of antagonizing cell
death [80,81].
These features seen in the overexpression of NO have been exploited therapeutically, with these approaches holding great
promise for novel anticancer strategies [79,83].
N
NHR
O
OO
HO
ONO2
24a, R= H
24b, R= Me
24c, R= F
24d, R= I
N
N
OO
R
NH2
HO
ONO2
25a, R= H
25b, R= Me
25c, R= I
N
NHR
O
OO
O2NO
OH
26a, R= H
26b, R= Me
26c, R= I
Fig. (18). Nucleoside analogues with NO-donor activity.
394 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
One such promising strategy in the development of anti-neoplastic drugs is the design of nucleoside analogues with NO-releasing potential [85,86]. To this end, 3`-O-nitro derivatives of 2`-deoxyuridine (Fig. 18 – compounds 24a-d), 2`-deoxycytidine (Fig. 18 – compounds 25a-c), as well as 5`-O-nitro derivatives of 2`-deoxyuridine (Fig. 18 – compounds 26a-c), were synthesized and assessed in a number of neoplastic cell lines. These compounds show potential for NO release in the presence of L-cysteine (1.5-5.4% range at 1 h, and 17.1-59.9% range at 16 h), a value greater than or equal to the reference drug isosorbide dinitrate (3.5 and 24% release at 1 h and 16 h). In vitro cytotoxic activity in a variety of tumor cell lines of these 3`- and 5`-O-nitrate ester derivatives was assessed, exhibiting activity comparable (CC50 from 10
-3 to
10-6
) to 5-iodo-2`-deoxyuridine, but lower than 5-fluoro-2`-deoxyuridine [85].
Furoxans can produce high concentrations of NO and exhibit strong anti-cancer activity but owing to their broad biological effects, furoxan-based anti-neoplastic compounds can result in serious adverse effects [87,88]. In this regard, furoxan/glycyrr-hetinic acid (GA) hybrids (Fig. 19) have attracted interest since GA, besides having antineoplastic activity, also has an affinity for hepatocytes. Hence, these hybrids with furoxans are able to be more selective for hepatic tissue and consequently generate potent cytotoxic action against hepatic tumor cells while minimizing collateral damage to other organs as well as healthy hepatocytes. Comparison of some NO-donors with the proposed hybrids showed that the former inhibit growth both in tumor cells and healthy liver cells at a concentration of 10 M, whereas the latter reveal a high inhibition of tumor cells of between 83.52–96.50% and a lesser effect on healthy cells of less than 18% inhibition, hence offering a safer profile. Furoxans were also found to produce similar concentrations of NO both in the presence of tumor cells and healthy hepatocytes. By contrast, the proposed hybrid created higher NO concentrations in tumor cells compared to healthy hepatic cells, resulting in action selectivity [87].
Given that inflammation is currently regarded as a risk factor for the development of cancer, and that chronic inflammation contributes to 1 in 4 cancer cases worldwide, the regular use of ASAs or other NSAIDs is attributed to reducing the risk of developing several types of cancer [84,89]. Studies show that chronic use of ASA for ten years lowers the risk of developing colorectal cancer [90]. Despite the growing body of evidence pointing to a link between regular NSAID use and reduced risk of developing cancer, some studies show conflicting results regarding this protective effect, with some findings associating the use of NSAIDs with increased risk for cancer of the prostate and pancreas [86].
Although controversy remains over the protective role of NSAIDs against cancer, the chemical entity referred to as “NO-NSAID” does appear to hold promise, exhibiting greater cytostatic and cytocidal activity than the native molecule in both in vivo and in vitro cancer models. In the case of the compound NCX 4040 (Fig. 20), a NO-releasing aspirin derivative, greater cytotoxic
activity compared to the original NO-donor and other NO-donors has been found, producing 50% inhibition of cell growth at 5.4 M. A cytocidal effect has also been observed in 50% of cells at 9.6
M, and 90% of apoptotic cells at 10 M, detected after 24h. This finding may be explained by the high capacity for NO-molecule release exhibited by the spacer. Tests of NCX 4040, along with its denitrated molecule, confirmed the inactivity of the latter, giving evidence that the antitumor property of NO-NSAID does not stem from the spacer but that its presence is important for facilitating the NO release. Furthermore, the study confirmed the importance of the presence of ASA and showed that NCX 4040 is a promising molecule in the management of cancer, possessing greater anti-proliferative, cytocidal and pro-apoptotic activities than either ASA or NO donors [90].
C
OCOCH3
O
CH2ONO2O
NCX 4040
Fig. (20). Promising NO-NSAID with anti-cancer activity.
On the other hand, some studies ascribe the anti-tumor activity of NCX 4040 to formation of a quinone methide intermediate (QM) which can be attacked by a nucleophile (e.g. glutathione) triggering cell events that culminate in cytotoxic and cytostatic effects. The study affirmed that nitrate ester is only an electron-withdrawing group which helps the formation of the intermediate, and that the ASA skeleton plays no relevant role in the activity [91].
2.1.4. Antiviral, Antibacterial, Antifungal
NO is a necessary component of the non-specific defense mechanism against many pathogens, including bacteria, viruses and fungi [92]. NO has a wide spectrum anti-microbial activity produced by macrophages and other inflammatory cells for destroying microorganisms [93,92,94]. It is acknowledged that the production of NO by iNOS is of great importance in the intracellular destruction of microorganisms such as Mycobacterium tuberculosis, Candida, among others [93,95]. The mechanism through which NO exerts its antimicrobial effect is based on interaction with the Fe-prosthetic groups of mitochondrial enzymes or its reaction with the superoxide anion (O2) to produce peroxynitrite [92]. Peroxynitrite has multiple molecular targets including intracellular targets. In vivo studies have shown that the NO derivative of eNOS can reproduce the mycobacterial action of the NO derivative of iNOS. However, future studies confirming the viability of its use are needed [93].
Antimycotic activity by NO has been observed and verified, for instance through inhibition of the Candida species by NONOates [96].
H
CONHCH2CO(CH2)4OO
NO
NO
SO2Ph
O
H
HHO
Fig. (19). NO-glycyrrhetinic acid.
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 395
The antiviral effect of NO occurs in several families of virus DNA and RNA whereby S-nitrosylation mediated by NO of (macro) viral and host molecules constitutes an interesting mechanism for general control of the viral life cycle [97,98,99]. S-nitrosylation normally takes place in one or a few Cys residues in target proteins, where local hydrophobicity can be a determining factor for this to occur [100].
Given the bactericidal effect of NO outlined, NO donors have
been used to achieve an antibacterial effect, such as in the case of SNAP. Virulent samples of Photobacterium damselae subsp.
Piscicida treated with SNAP have shown reduced viability at doses
upward of 10 M [92,101,102]. However, the majority of NO-
donors used for bactericidal action do not have adequate specificity,
creating a host of side effects. In a bid to control the intracellular
release of NO, which in this case occurs only in the presence of –
galactosidase, a novel NO-releasing compound was synthesized,
namely, -galactosylpyrrolidinyl NONOates (Fig. 21 – -Gal-
NONOate). In bacteria induced with the –galactosidase-producing
gene, treatment with the compound led to lower survival (22.4%)
compared to treatment with NONOates (87.3%) [92].
O
HO
HOOH
OH
ON
NN
O
Fig. (21). -Gal-NONOate.
NO is also known to cause conversion of the replicating form of
the Helicobacter pylori into its non-replicating form, besides having
a bactericidal effect on the replicant form when used at sufficient
doses [103]. Thus, the NO-Donor/metronidazole (Fig. 22) hybrids
were synthesized in order to combine the reported properties of NO
with the anti-microbial effects of metronidazol. The cited hybrids
were found to be potential therapeutic instruments, since they
offered greater than or equal potency to metronidazol against H.
pylori. The majority of the minimum inhibitory concentrations of
hybrids was found to be less than 32 g/mL, equivalent to that of
metronidazol. In addition, all hybrids showed good-to-high
potential against metronidazol-resistant H.pylori strains [104].
ONN
N
N
O2N
O R
O
Fig. (22). NO-metronidazole.
The use of nanoparticles as drug transporters represents a new
paradigm in the development of anti-bacterial therapies, and can
even reduce the production of biofilms [94,105]. The incorporation
of traditional antibiotics within particles or annexing them to outer
layers has also been employed, in many cases leading to improved
efficacy over antibiotics alone. Nanoparticles that release NO, known as NO-releasing silica nanoparticles, by using NONOates as
NO-donors (Fig. 23) produce a bactericidal effect at concentrations
of between 800 and 3200 g/mL, compared to the NO donor alone,
whose bactericidal effect occurs at 12 mg. This difference in
concentration is due to the highly localized NO release achieved
using nanoparticles. Additionally, in vitro studies have also shown
that NO-releasing silica nanoparticles have no cytotoxicity to
mammalian fibroblasts at bactericidal concentrations [94]. Also, the
size of NO-releasing silica nanoparticles have influence on
bactericidal activity of those compounds, as small NO-releasing
silica nanoparticles showed to be more effective at bacteria killing
when evaluated against Pseudomonas aeruginosa [106].
Fig. (23). Scheme of nanoparticles containing NO-releasing agents. Adapted
from [93].
Also noteworthy is the role of phagocytes in the defense against microorganisms, where two microbicide processes occur during phagocytosis [107]. The processes in question are oxidation and degranulation. In the former, there is production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), where RNI result from the production of NO [107,101]. Observations revealed that the use of NO-donors causes inhibition of adhesion and aggregation of neutrophils. Thus, it was verified that administration of SNP and 3-morpholinosydnonimine (SIN-1) (Fig. 24) significantly increased the bactericidal activity of the neutrophils. Use of SNP showed an increase in bactericidal activity of neutrophils against P. vulgaris at concentrations of 100 and 1000
M and also against E. coli at 1000 M. SIN-1, however, induced neutrophils against P. vulgaris and S. Anatum at 1000 M and also against E. coli at both 100 and 1000 M [108].
N OO
N
N
NH
Fig. (24). SIN-1.
With regards to antifungal activity of NO, besides inhibiting Candida species, NO-donors also produced a synergic effect when used in conjunction with antimycotic azoles [96,109]. Based on these effects, hybrid compounds of ketoconazole with NO-donors NONOates and nitrate ester were synthesized to achieve synergic action against Candida sp. This action stems from the inhibitory activity of NO against Candida species associated with the known antifungal activity of ketoconazol. These hybrids can be considered a useful approach for treating antifungal mycosis, since these compounds proved more active than fixed doses of cetoconazol and NO-donor. The in vivo activity of these hybrids is presumed to be greater given the regulatory role of NO in the immunologic system [96].
Antiviral activity of NO has also been found, for instance against the coronavirus and dengue virus, in tests determining
Si N
OMe
MeO
OMe NNO
O
NH2
Si
OEt
OEt
OEt
EtO+
AHAP3/NO TEOS
+-
-Na+
R´N N
N O
R O+-
-
NO-releasing silica nanoparticle
=
396 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
SNAP activity against these viruses [98,99]. Concerning the coronavirus, SNAP (222 M) was found to inhibit the viral concentrations to non-toxic levels [98]. In the case of the dengue virus, an inhibitory effect by NO was noted at a concentration of 150 M, with the maximum effect observed in the treatment of contaminated cells 10-14 h after infection [99]. It is also noteworthy that the synthesized series 3`-O-nitro derivatives of 2`-deoxyuridine, 2`-deoxycytidine and also 5`-O-nitro derivatives of 2`-deoxyuridine, intended as novel anti-neoplastic drugs, was assessed for activity against the poliovirus, picornavirus and rhinovirus but exhibited no antiviral activity [83].
2.1.5. Anti-Parasitics
The notion that NO is merely an inspecific cytostatic agent has changed over the years, following recent discoveries of a wide variety of effects on leukocyte activity. Currently, NO is considered an important molecule with cytotoxic and cytostatic properties for many parasitic organisms, evidencing host protection against T. cruzi, Toxoplasma gondii, Leishmania major, L. donovani, Plasmodium sp and Schistosoma mansoni. A number of parasitic antigens are recognized by macrophage receptors, which activate NO production through inducible NOS [4,110].
From a physiopathological standpoint, induced NO production by macrophages during the acute phase of Chagas´ disease can result in lysis of the etiological agent. These cells have the induced form of NO synthase (iNOS) whose activation occurs by induction of cytokines (interferon- , TNF- , among others) released by some cells of the immunologic system. This release is prolonged for several hours at high concentrations, with the aim of provoking toxicity for parasitic cells. Therefore, macrophages constitute a source of NO in the organism, where its functions are dependent on the release of this compound [110].
Nevertheless, the prolonged activation of iNOS and consequent excessive NO release can have negative repercussions on host cells. Studies associate this phenomenon to the cardiac dysfunction in infections by T. cruzi, seen in patients and animal models with Chagas´ disease [111].
The effects of NO on the immune system are due to the formation of reactive oxygen and nitrogen species such as peroxynitrite. NO also regulate metalloproteinases or chemically alter parasitic cysteine-proteases, mediators of important metabolic processes and macromolecular targets of NO. In the case of the latter, NO and NO-donors (such as SNP and SNAP) inhibit the catalytic activity of these enzymes through the formation of S-nitrosothiol in the cysteine of the active site, as shown in studies on cruzipain of T. cruzi and falcipain of Plamodium falciparum [4,112,113,114,115].
Therapies against T. cruzi based on NO-releasing compounds represent an alternative approach for the synthesis of new trypanocidal agents [116,117,110].
Metal complexes for treating and creating new active compounds in Chagas´ disease have been in use for many years
[116]. More specifically, the NO-releasing ruthenium complexes have recently emerged as a promising alternative for treating this disease. These compounds (Fig. 25) have low toxicity and show high survival rates in mouse infected by T. cruzi treated with them, attaining rates of 40% parasitological cure, rising to over 80% when used in combination with benznidazole [117,118].
Results of studies suggest that S-nitrosylation in catalytic Cys166 of glyceraldehyde 3-phosphate dehydrogenase (GADPH) by NO-releasing ruthenium complexes is an important event for inhibition of the glycolytic metabolic pathway of the parasite, achieving from 85 to 97% enzymatic inhibition using 200 mM [119].
The ability of parasites to survive within macrophages is at least in part due to the inability of activated macrophages to produce sufficient NO [120]. The entire surface of macrophages is covered by mannose receptors (MR) which also recognize other types of saccharides such as: fucose and N-acetylglucosamine [121]. Thus, there is the possibility of exploiting MRs for the design of directed drugs, creating compounds able to selectively release NO within intracellular compartments [122].
Based on this strategy, monosaccharides covalently bonded to diazeniumdiolates were planned and synthesized to selectively enter, via MR, macrophages infected by Leishmania major. In this process they are cleaved, releasing the NO molecule, concentrating this cytotoxic agent within the cell. L. major is susceptible to the toxic effects of NO but has the ability to survive and proliferate within macrophages when these are devoid of induction of NO by iNOS. Therefore, in order to become active, compounds require sufficient stability to pass through the bloodstream without being cleaved, possible only within macrophages [122].
Analysis of the potential NO-donor was carried out through in vitro studies using macrophages of iNOS-deficient rats, thus excluding any influence of endogenous NO. Without iNOS, stimulus with the cytokines TNF and IFN- has low anti- L. major activity. Derivatives of synthesized fucose and mannose exhibited no relevant activity against the parasitic load of cells tested. By contrast, macrophages, due to the low pH (4-5.5) environment and/or the presence of N-acetylglucosaminase within them, were able to cleave the derivatives 27a and 27b (Fig. 26), subsequently releasing NO, which exerted its antiparasitic activity. The derivatives N-acetylglucosamine 27a and 27b showed significant reduction in the number of intracellular parasites, with the 27a molecule being the most active. In addition, both were devoid of toxicity at the concentrations tested (125 and 250 μM). Studies have also identified a directly proportional relationship between the quantity of nitrite formed (due to auto-oxidation of NO) and leishmanicidal activity [122].
Also, the leishmanicidal activity shown by GTN is of interest (Fig. 3 – compound 1), a known NO-donor and the first drug used for angina. The drug reduced the size of cutaneous leishmaniasis lesions from the eighth week of treatment in infected mice. It also
Ru
NO
R
H3N NH3
H3NNH3
n
3+
R = N-heterocyclic, H2O, SO32-, P(OEt)3
Ru
NON
NN
N
L
3+
L = imN, miN, SO32-
Fig. (25). General structure of NO-releasing ruthenium complexes of first and second generations.
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 397
showed a 63% difference compared to controls, a lowering of the number of amastigotes in macrophages of mice spleens, besides a 100% animal survival rate at week 12 of the study [123].
Malaria is one of the most devastating diseases globally, causing an estimated 800,000 deaths per year, and is considered endemic in Saharan Africa, South Asia and parts of South America [124]. Although the therapeutic arsenal consists of different classes of anti-malarials, the problem of plasmid resistance to currently used drugs gives rise to the need for new chemotherapics against the parasitosis [125,126]. Furoxan sulfones (Fig. 27) were assessed for their activity against P. falciparum in vitro, where preliminary results in sensitive and chloroquine-resistant strains showed that furoxan sulfones possessing the R-SO2 group at the 3-position had an IC50 of between 3.03 μM (compound 28a) and 5.36 μM (compound 28c). These have shown to be potent NO-releasing compounds compared to furazan derivatives which exhibited no NO-releasing potential and low activity. The results suggest that NO makes an important contribution to the antiparasitic potential [127].
Activity against cerebral malaria (CM) and in vitro P. falciparum was also observed in NO-donor amodiaquine derivatives (NO-AQ) containing either furoxan or nitrate ester as NO-donor. Piperidine and piperazine derivatives (Fig. 28 – compounds 29a, 29b and 29c) showed to be the most active antiplasmodial compounds. However, the contribution of NO to the in vitro activity of NO-AQ derivatives against strains of P.
falciparum seems to be secondary when compared with AQ activity. Nevertheless, compound 29d increased the survival of infected mice with late stage CM to 23%, almost three times less than the 8% survival showed by AQ, what makes NO an important factor to the activity of that compound [128].
Cysteine-proteases are enzymes fundamental for parasitic development and penetration into host cells, being considered validated targets for developing new molecules with antiparasitic activity [129].
Studies have shown that the water soluble compound 4-(phenylsulfonyl)-3-((2-dimethylamino)ethyl)thio)-furoxan oxalate (SNO-102 – Fig. 29) exhibits dose- and time-dependent inhibitory activity in cruzipain, falcipain and cysteine-protease of L. infantum owing to its NO-releasing potential. Studies of the fluorogenic substrate Z-Phe-Arg-AMC show its ability to protect the enzyme from being inactivated by SNO-102, thus suggesting that the action of this NO-donor involves chemical modification (S-nitrosylation) in Cys25 catalysis of cysteine-proteases [130].
This valuable data highlights the promising aspect of this approach and justifies the search for novel NO-donor compounds with antiparasitic activities [117,119,118,114].
A study is currently underway at our laboratory involving a mutual prodrug containing NO-donor groups and nitro-heterocyclic derivatives which have activity against cruzipain and T. cruzi in vitro. Nitro-heterocycles and drugs releasing NO in the form of
O
HO
HO
OH
ON
NNEt2
O
NHAc
27a
O
HO
HO
OH
ON
NN
O
NHAc
27b
Fig. (26). N-acetylglucosamine linked to NONOate.
O
NN
SO2Ph
O
Ph
O
NN O
PhS
OH
O
O
O
NN O
PhS
O
O
O
O
NN O
PhS
O
O
S
O
NN O
PhS
O
O
NH
O
28a 28b 28c
28d 28e
Fig. (27). Anti-malarial furoxan sulfones derivatives.
398 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
nitrate esters are being synthesized and subsequently shall undergo tests in cruzain and cultures of cells infected with T. cruzi. In addition to these studies, molecular modeling has been used as a tool for elucidating the behavior at both electronic and molecular levels of these new NO-releasing chemical entities.
SO2
NO
NO
S N
Fig. (29). SNO-102.
2.1.6. Ocular Disease
Ocular tissue was one of the first environments where NOS were found and nowadays it is observed in all compartments of the eyes. The presence of NOS in retina shows the important role of NO in maintaining basal resting tone in its circulation, through cGMP-dependent pathways. Ocular blood vessels exposure to NOS inhibitors increases smooth muscle contraction. Isolated strips of bovine ciliary muscle also contract in response to those compounds and relax when treated with organic nitro vasodilators. Nitroglycerin, for instance, decreases intraocular pressure (IOP) in experiments with rabbits and monkeys, showing the important role of NO in solving the problem of glaucoma [131,132].
NCl
HN
OH
NO N
O
N
SO
O
O
NCl
HN
OH
N
ONO2
NCl
HN
OH
N
ONO2
NCl
HN
OH
N
N
ONO2
29a29b
29c29d
Fig. (28). Anti-malarial NO-donors amodiaquine derivatives.
O
HO
F H
OOH
O
O
O
HO
F H
O
O
O
O
HN
ONO2O
O
Triamcinolone
acetonide
NCX-434
O
HO
F
O
OH
OH
Dexamethasone
O
HO
F
O
O
OH
O
ONO2
NCX-1021
Fig. (30). NO-donors containing steroid nucleus.
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 399
The glucocorticoid triamcinolone acetonide (TA – Fig. 30) is used intravitreally to treat patients with diabetic macular edema (DME). However, this compound has been reported to enhance the incident of hypertensive glaucoma and cataract lesions, as well as IOP. The prodrug NCX-434 (Fig. 30) corresponds to TA linked to a spacer group containing NO-donor nitrate ester, which releases NO in vivo by esterases. Therefore, besides TA anti-inflammatory effect, observed in animal studies, IOP is unaffected due to NO properties showed by NO-releasing groups [133].
The same rational design was used to generate NCX-1021 (Fig. 30) from a steroid nucleus. IOP levels did not modify significantly during the treatment with NO-donor dexamethasone derivative. Nevertheless, the treatment with dexamethasone alone carried out the highest levels of IOP in two weeks. Thus, based on those and on other data is possible to suggest that NCX-1021 avoid the IOP increasing [134].
Latanoprost acid (Fig. 31), a synthetic prostaglandin-F2 analog, is widely used to treat ocular hypertension and the design of derivatives linked to NO-donor, such as NCX-125 and NCX-139 (Fig. 31), has proven beneficial to decrease IOP [135,136]. Latanoprost showed a decrease of 26% in IOP compared with 36% of NCX-125 in glaucomatous dogs. A decrease also was observed in IOP studies in non-human primates [135]. NCX-139 was able to bind to recombinant human prostaglandin F (FP) receptors and effectively lowered IOP in ocular hypertensive rabbits, while in the analogue without NO the anti-hypertensive activity is absent at equimolar dose, suggesting two distinct mechanism of action that contribute, concomitantly, to the NCX-139 activity [136].
Carbonic anhydrase (CA) inhibitors are other class of drugs that has been used to reduce IOP. Then, sulfonamide CA inhibitors (CAI) linked to NO-donor can be an interesting approach in cases of glaucoma. Some sulfonamides linked to NO-donor presented inhibitory activity against human CA (hCA), such as compound 30 (Fig. 32). Isoforms of CA, associated to tumor activity, were also inhibited by those compounds [137]. Other study reported new NO-donating CAIs Dorzolamide analogues that also presented inhibitory activity against hCA, such as compounds NCX-278 and NCX-201 (Fig. 32). Crystallographic analysis of NCX-201 showed some hydrogen bonds important to the activity. Those bonds are observed between secondary nitrate and carboxamide hydrogens of Asn62; between ester oxygen and also with those carboxamide hydrogens and between primary nitrate oxygen with the imidazole moiety of His64 present in CA [138].
2.2. NOSs Inhibitors
As previously outlined, NO is involved in diverse physiological events of the human organism. However, NO overproduction, predominantly by nNOS and iNOS, secondary to dysfunctions is strongly associated with some diseases such as arthritis, asthma, cerebral ischemia, Parkinson’s disease, neurodegeneration, and seizures. Blocking this target enzyme is an important strategy to obtain inhibitor derivatives for use in the treatment of these diseases [139,140,141,142,143].
Against this background, rational planning of inhibitors of iNOS and nNOS isoforms is an approach gaining increasing support in the scientific milieu. Selectivity for eNOS represents the
HO
HO
O
ONO2
O
ONO2
OH
HO
HO
OH
O
OH
HO
HO
HN
O
OH
ONO2
Latanoprost acidNCX-125
NCX-139
Fig. (31). Latanoprost acid and its NO-donors analogues.
400 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
major challenge in this approach because the isoforms are highly similar [144,145,146].
In a bid to identify possible selective inhibitors of the iNOS enzyme, studies investigating the structure-activity relationship of a series of substituted 2-aminopyridines have been conducted. The substituted 4,6-dialkyl-substituted analogues (Fig. 33 – compounds 31 and 32) were found to be the most potent inhibitors, presenting a significant degree of selectivity for this isoform. On the other hand, the larger volume compound containing the phenylpropylic group showed lower activity [147].
NH2N NH2N
31 32
Fig. (33). 4,6-dialkyl-substituted analogues from 2-aminopyridine.
Subsequently, synthesis of the derivatives N-4-piperidinyl-2-aminopyridine led to compound 33 (Fig. 34), with the 4-methoxy substitute, yielding a 4-fold increase in iNOS potency compared to the 4-methyl compound. After optimization, the derivative 4-cyanobenzamide (AR-C133057XX – Fig. 34) was synthesized, showing an IC50 of 0.071 M and selectivity of over 1400-fold and approximately 100-fold for endothelial and neuronal isoforms, respectively. X-ray crystallography of AR-C133057XX revealed important interactions of the pyridine ring with the heme grouping of the iNOS, and of exocyclic groups with other cavities [148].
Another class of selective inhibitors of iNOS comprises the 1,2-dihydro-4-quinazolinamines, principally the derivative of spirocyclic amides AR-C102222 (Fig. 35), which showed dose-dependent inhibition of NO production induced by lipopolysaccharide (LPS). In chronic inflammatory arthritis induced in rats, the highest dose of AR-C102222 assessed (100 mol/kg) led to complete block of the pathology, thus confirming the in vivo efficacy of the class [149].
Garcin and co-workers (2008) [150] showed that, in addition to the cis-amidine nucleus of quinazoline and aminopyridine derivatives promoting interactions of hydrogen with Glu present at the binding site, and with the heme grouping (thus mimicking the L-arginine substrate), the greater affinity for iNOS of the inhibitors containing bulky groups was due to the interaction of these groups with a new pocket. This occurs when the amino acid Gln rotates on its own axis, taking on an open conformation, which in turn creates conformational changes in cascades with other amino acids.
The compounds 34 and 35 with values of 0.2 and 0.3 μM, respectively (Fig. 36), were also selective for nNOS, exhibiting 500- and 1166-fold selectivity over eNOS and 50 and 100-fold over iNOS, respectively. Molecular modeling showed this was due to the greater cavity size of the heme group of nNOS compared to the other isoforms, thus adequately accommodating the more bulky groups [151].
Aminopyridine compounds 36 and 37 (Fig. 37) were found to be active and selective against the nNOS isoform, although they proved unable to penetrate the blood-brain-barrier in studies in vivo. The two compounds exhibit aminopyridine moiety as a possible pharmacophore, capable of interaction with different regions of the enzyme [152,153,154]. Double-headed aminopyridine derivatives were designed where 38 (Fig. 37) with Ki of 25 nM and 107-fold
H2NO2S
O
O O
O
OMe
(CH2)4ONO2
S
O O
S
S
NH2
O
O
NO
O
O2NO
S
O O
S
S
NH2
O
O
NO
O
ONO2
O2NO
30
NCX-278 NCX-201
Fig. (32). NO-sulfonamides derivatives with potential activity in ocular diseases.
N
N NH
OMe
N
N NH
OMe
CN
33 AR-C133057XX
O
O O
Fig. (34). N-4-piperidinyl-2-aminopyridine and 4-cyanobenzamide derivatives, respectively.
Nitric Oxide in Drug Design Current Medicinal Chemistry, 2012 Vol. 19, No. 3 401
selectivity for nNOS over eNOS, showed greater potency and degree of selectivity. The first aminopyridine part of the 38 molecule interacts with Glu592 and the second, with the heme group. However, in the nNOS isoform, X-ray crystallography revealed that a second molecule of 38 also interacts with the enzyme, more specifically, at the ligation site of the H4B cofactor, with the pyridine region coordinated with the Zn atom. The second 38 molecule does not bond to the eNOS isoform, where only the first unit of the compound interacts. This most likely explains the greater selectivity of this compound for nNOS. Given the greater lipophilicity, the 38 derivative has better permeability in cell-based tests (5 M) compared to that of compound 37. Therefore, the alternative cavity found in the target, the data on activity and permeability, coupled with the structural simplicity of 38, render this a good candidate as a lead-compound for designing selective inhibitors of nNOS [155,156].
In addition to the design strategies mentioned, the ultra HTS (High-Throughput Screening) technique warrants mention since it has been used to identify new leads for iNOS inhibitors and is responsible for the discovery of ligand 39 (Fig. 38). After optimization, this compound generated the potent human iNOS inhibitor (human iNOS = 0.011 μM) quinoline amide derivative 40 (Fig. 38), > 2000-fold selective over human eNOS. This compound had oral activity in mouse, albeit with a short action due to its high clearance value (Clp >100 mL/min/kg) [157].
Based on these results, efforts to improve the pharmacokinetic characteristics of the prototype entailing assessment of various analogues led to the dual iNOS/nNOS inhibitor 4,7-imidazopyrazine 41 (Fig. 38). This showed high potency in human iNOS (0.091 μM) and also activity over nNOS (0.30 μM), while maintaining the desired selectivity over eNOS (180-fold). This derivative was shown to be effective in in vivo models of neuropathic pain, and showed no tolerance after use of repeated doses. Moreover, the derivative also had a low clearance value (4-9 mL/min/kg) and good bioavailability conferring useful properties to this new ligand. Studies have confirmed that the activity of 41 is based on its ability to inhibit the dimerization of iNOS [158].
2.3. Other Activities
In addition to the activities outlined earlier, NO is active in various pathologies such as type II diabetes mellitus (DM-II), schizophrenia, obsessive-compulsive behavior, depression, migraine, among others [159,160,161,162,163].
Notably, the NO-donor hybrids are considered a rational strategy in the treatment of DM-II, since they confer vasodilatory properties to the hypoglycemics [159]. The properties cited for these hybrids are of great importance, given that cardiovascular complications are amongst the most serious in type II diabetes mellitus [164,159]. In addition, NO is also indicated as an inactivator of insulin-degrading enzyme (IDE), possibly inhibiting it in a competitive manner. Thus, the NO-donors are in use for the management of DM-II, reducing the degradation of insulin through S-nitrosylation of the IDE [165].
NO and its metabolites are high in erythrocytes of schizophrenics, affecting a number of processes known to be changed in this pathology such as cellular migration, synapse
N
N
N
HN
F
F NH2
O
CN
AR-C102222
Fig. (35). 1,2-dihydro-4-quinazolinamines derivative.
NH
NH
NH
NH
34 35
Fig. (36). Selective nNOS inhibitors.
N
HN
NH
H2N
HN F
N
HN
NH
H2N
HN F
36
37
N
N
N
NH2
H2N
38
Fig. (37). Ligands containing double-headed aminopyridine are interesting prototypes as selective nNOS inhibitors.
402 Current Medicinal Chemistry, 2012 Vol. 19, No. 3 Serafim et al.
formation and so forth [166,160]. It has been observed that the administration of NOS inhibitors to rats treated with phencyclidine, an anesthetic causing hallucinations, halts this hallucinogenic behavior, implying that the NOS system in the brain needs be intact in order for the hallucinations to take place [160].
The search for new agents to manipulate the biochemical pathways involving NO is important, thereby attenuating many disease dysfunctions.
3. CONCLUSION AND PERSPECTIVES
After the discovery of the numerous benefits of the multifunctional NO molecule, compounds that acquire NO-releasing potential by the inclusion of certain functionalizations have become highly useful in many spheres of medicinal chemistry, particularly in pathologies involving dysfunction in the production of the molecule. However, striking a balance between the release of therapeutic amounts of NO and maintaining the native drug activity, as well as the release selectivity at specific sites, still poses a challenge to researchers. Potent inhibitors able to selectively inhibit certain isoforms of NOS are also the focus of current research, selectivity which has proven hard to achieve given the high homology among the isoforms.
The search for compounds, which in some way modulate the amount of NO in the human organism, represents the latest, most effective strategy for combating a wide range of dysfunctions and pathogens.
It is hoped that further elucidation of the role of NO in various diseases will lead to the rational design of NO derivatives, paving the way for innovation and novel approaches in the management of disease. This applies especially to neglected diseases which afflict millions of people, but have current availability to a limited therapeutic arsenal.
ACKNOWLEDGEMENTS
The authors thank CAPES, CNPq and FAPESP for the scholarships and financial support.
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NH
O
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F
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Received: September 29, 2011 Revised: November 15, 2011 Accepted: November 16, 2011