Novel analgesic/anti-inflammatory agents: 1,5-Diarylpyrrole nitrooxyethyl sulfides and related compounds as Cyclooxygenase-2 inhibitors containing a nitric oxide donor moiety endowed with vasorelaxant properties

Highlights

• Novel analgesic/anti-inflammatory agents 13–16 as dual selective COX-2 inhibitors containing a NO donor moiety were proposed.

• SARs of target compounds 13–16 are carefully explored.

• The isosteric O-to-S replacement led to selective and highly potent COX-2 inhibitors 13a-c and 15a-c.

• Hydroxyethyl sulfides 14a-c also showed an efficacious and selective COX-2 inhibitory activity.

• Compounds 13a-c and 15a-c, containing a NO donor moiety, showed intriguing vasorelaxant properties.

Abstract

The design of compounds able to combine the selective inhibition of cyclooxygenase-2 (COX-2) with the release of nitric oxide (NO) is a promising strategy to achieve potent anti-inflammatory agents endowed with an overall safer profile and reduced toxicity upon gastrointestinal and cardiovascular systems. With the aim of generating novel and selective COX-2 inhibiting NO-donors (CINOD) and encouraged by the promising results obtained with our nitrooxy- and hydroxyethyl ethers 11 and 12 reported in previous works, we shifted our attention on the synthesis of isosteric thioanalogs nitrooxy- and hydroxy ethyl sulfides 13a-c and 14a-c, respectively, along with their oxidation products nitrooxy- and hydroxyethyl sulfoxides 15a-c and 16a-c, respectively, also referred to as thio-CINOD. Preliminary data and metabolic analysis highlighted how the isosteric substitution of the ethereal oxygen atom of 11a-c with sulfur in compounds 13a-c, independently from the presence and the number of fluorine atoms in N₁-phenyl ring, leads to new selective and highly potent COX-2 inhibitors, capable to induce vasorelaxant responses in vivo. The same behavior is observed with their oxidized counterparts nitrooxyethyl sulfoxides 15a-c, in which the oxidation state of the sulfur atom and the presence of the additional oxygen atom play a substantial role in enhancing compounds activity and vasorelaxation. In addition, the screened compounds proved significantly efficacious in mouse models of inflammation and nociception at the dose of 20 mg/kg.

Introduction

The traditional non-steroidal anti-inflammatory drugs (tNSAIDs) represent a class of therapeutic agents widely used in the treatment of various pathologies [1]. Up to now, treatment with tNSAIDs is the best therapy for pain caused by rheumatoid arthritis (RA) and osteoarthritis (OA) [2]. Other examples of common applications are in the treatment of intestinal bowel disease (IBD) inflammation [3], urogenital tract [4] and respiratory system [5] inflammation, dysmenorrhea [6], lupus erythematosus [7] and fibromyalgia [8]. Although the analgesic action of tNSAIDs does not match in potency that of the opiates, their co-administration with usual narcotics has found wide application for the treatment of both post-operative pain and of chronic pain induced by various pathologies, including cancer pathologies [9,10]. tNSAIDs perform their anti-inflammatory and analgesic action via inhibition of cyclooxygenases (COX) [11]. At least two isoforms of COX are known: COX-1, expressed constitutively, and COX-2, which is absent from most tissues in physiological conditions and is expressed as a result of pro-inflammatory stimuli (e.g.: cytokines) [11]. As tNSAIDs are not selective, they inhibit both isoforms often with a preference for COX-1 [12]. This poor selectivity leads, with concomitant inhibition of COX-1, to inhibition of the synthesis of prostanoids that are essential for maintenance of the functions of the gastric mucosa and of renal homeostasis, giving rise, especially in prolonged use, to severe gastrointestinal (GI) complications (mucosal damage, bleeding) and renal damage [12]. Clinical use of selective COX-2 inhibitors also referred to as Coxib has recently shown that the gastric toxicity associated with the use of tNSAIDs can be reduced considerably [13]. Several recent clinical studies have shown that selective COX-2 inhibition, as well as giving rise to anti-inflammatories and analgesics with a safer GI profile, prove effective in the treatment of various pre-cancerous and cancerous forms [14]. In fact, COX-2 is overexpressed in gastric, hepatic, pancreatic, esophageal, colon, breast, bladder, and lung tumors [15]. However, various clinical and epidemiological studies have shown that long-term use of selective COX-2 inhibitors is associated with a higher incidence of adverse effects relating to the cardiovascular system, and in particular with an increased incidence of myocardial infarction, angina pectoris and transient ischemic attacks [16,17]. The cause of this toxicity for cardiovascular system, which is also found with some tNSAIDs that are rather selective in inhibiting COX-2, arises from the fact that this isoform, constitutively expressed in the vascular epithelium, is fundamental to the synthesis of prostaglandin (PG)-I, a potent vasodilator [18,19]. Thus, high selectivity in inhibition of COX-2 leads, in the cardiovascular system, to prevalence of the pro-aggregative and vasoconstrictive stimulus exerted by thromboxane (TxA₂) no longer counterbalanced by the vasodilator effect of prostacyclin (PGI₂) [18]. Such a pharmacological effect caused well-known dramatic consequences: following the results of clinical studies (VIGOR and APPROVe) [19], demonstrating increased incidence of myocardial infarction and thrombotic events, two vicinal diaryl-substituted heterocycles (VDHs), namely rofecoxib and valdecoxib (Fig. 1) were withdrawn from the global pharmaceutical market in 2004 and 2005, respectively [20].

The GI side effects associated with the use of tNSAIDs and those relating to the use of selective COX-2 inhibitors created the need for new analgesics and anti-inflammatories agents that have a better profile of tolerability.

Similarly to PGI₂, nitric oxide (NO) at low concentrations has an important role in maintaining appropriate functionality of the cardiovascular system [21]. Through the activation of guanylate cyclase (sGC), NO gives rise to an increase in cGMP, which leads to vasodilation in smooth muscles, inhibits adhesion of leukocytes to the vessel wall and inhibits platelet aggregation, eliciting an overall anti-thrombotic action [22]. Furthermore, NO is now widely recognized as a critical mediator of gastrointestinal mucosal defense, exerting many of the same actions as PGs in the GI tract [23]. It was also demonstrated that NO was able to reduce the severity of gastric injury in experimental models [24,25]. As a consequence, it was proposed that linking of a NO-releasing moiety to a tNSAID might reduce the toxicity of the latter [26]. NO-releasing drugs mimic the well-known physiological process of endothelium-dependent relaxation of blood vessels, resulting in NO-mediated activation of sGC and accumulation of cGMP in adjacent vascular smooth muscle cells. In different animal studies, NO-releasing derivatives of a wide array of tNSAIDs (Fig. 2), including NO-aspirin (1), NO-naproxen (2), NO-flurbiprofen (3), NO-diclofenac (4), and NO-indomethacin (5) have shown to spare the GI tract, even though they suppressed PG synthesis as effectively as the parent drug [27,28]. The synthesis of this kind of hybrid molecules capable of selectively inhibiting COX-2 and at the same time of releasing NO (or NO-donors) appropriately, also referred to as CINOD [29], boosted the search of new anti-inflammatory and analgesic drugs, devoid of the cardiovascular and renal side effects, especially those associated with the use of VDHs rofecoxib and valdecoxib [20], (Fig. 1) cited above and known as Viooxx® and Bextra®, respectively.

VDHs represent a family of privileged scaffolds, bearing two phenyl rings on adjacent atoms of a five- or six-membered heterocyclic system, that in the last few decades elicited enormous interest for their clinical applications [30].

Among VDHs, vicinal diarylpyrroles came out as promising building blocks for the discovery of novel drug molecules. In fact, highly substituted diarylpyrrole, including 1,2-, 2,3-, 3,4-, 4,5-, and 1,5-diarylpyrrole heterocycles, have been reported along with their various and promising biological profiles [31]. In particular, new chemical entities based on the 1,5-diarylpyrrole scaffold have been developed as anti-inflammatory, anticancer, antimycobacterial, analgesic, anticoccidial, antinociceptive, antimicrobial, antihyperlipidemic agents, and EP1-receptor antagonist [31].

As only a few number of drugs with vicinal diarylpyrrole scaffold were available on the market, since 2005 extensive research work has been reported by our research group on the development of 3-substituted 1,5-diarylpyrrole derivatives having carboxylic acids and esters (6) [32] alkoxy ethers (7a,b) [33], alkyl sulfides (8a,b) and sulfoxides (9a,b) [34] along with nitric oxide-releasing moieties as nitrooxyalkyl esters (10a,b) [35], and ethers (11a-c) [36] for their anti-inflammatory and selective COX-2 inhibitory activities [37]. Most of these compounds exhibited potent biological activities, which were comparable to the standard selective COX-2 inhibitors (e.g. celecoxib) in in vitro, in vivo, and ex vivo studies [[32], [33], [34], [35], [36]]. The discovery of nitrooxy analogs (10a and 11a-c) has laid the foundation for the development of novel dual COX-2 inhibitors/NO-donors with NO-dependent vasorelaxant property [38], thus facilitating gastrointestinal and cardiovascular safety. Both nitrooxyalkyl inverse ethers (11a-c) and their metabolites, hydroxyethyl derivatives (12a-c), displayed very potent COX-2 inhibitory activity in the nanomolar range [36] (Fig. 3).

As an extension of our previous work focused on novel COX-2 inhibiting NO-donors and encouraged by the promising results of previously reported nitrooxy- and hydroxyethyl ethers 11 and 12 [36], we shifted our attention on the synthesis of nitrooxy- and hydroxy ethyl sulfides 13a-c and 14a-c respectively, as thio-analogs of 11a-c and 12a-c along with their oxidation products nitrooxy- and hydroxyethyl sulfoxides 15a-c and 16a-c, respectively (Fig. 4).

In fact, as recently reported by some of us, the transformation of 1,5-diarylpyrrole-3-alkoxyethyl ethers (7) into the corresponding alkyl sulfides (thioethers) (8a,b) and respective alkyl sulfoxides (9a,b), still lead to selective and active compounds endowed with COX-2 inhibitory activity in the low nanomolar range [34]. The synthetic procedure and the biological evaluation of target compounds 13a-c and 15a-c, along with that of their metabolites 14a-c and 16a-c, as highly selective COX-2 inhibitors and efficacious vasorelaxant agents is the object of the present study. Our findings also suggested that our best compounds caused vasorelaxation through NO-generation and activation of the sCG/cGMP/PKG pathway.