molecules Review
Inhibitors of the Hydrolytic Enzyme Dimethylarginine Dimethylaminohydrolase (DDAH): Discovery, Synthesis and Development Rhys B. Murphy † , Sara Tommasi † , Benjamin C. Lewis and Arduino A. Mangoni * Department of Clinical Pharmacology, School of Medicine, Flinders University, Bedford Park, Adelaide 5042, Australia;
[email protected] (R.B.M.);
[email protected] (S.T.);
[email protected] (B.C.L.) * Correspondence:
[email protected]; Tel.: +61-8-8204-7495; Fax: +61-8-8204-5114 † These authors contributed equally to this work. Academic Editors: Jean Jacques Vanden Eynde and Sylvain Rault Received: 9 February 2016; Accepted: 4 May 2016; Published: 11 May 2016
Abstract: Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), with ADMA proposed to be a putative marker of cardiovascular disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders, and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors as potential drug candidates represent a growing field of interest. This review article summarizes the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the main DDAH inhibitors reported in the literature. Furthermore, current methods of development and chemical synthetic pathways are discussed. Keywords: dimethylarginine dimethylaminohydrolase; arginine; nitric oxide; asymmetric dimethylarginine; monomethyl arginine; enzyme inhibitors; organic synthesis
1. Introduction Dimethylarginine dimethylaminohydrolase (DDAH) is a modulator of the nitric oxide (NO) pathway and is responsible for the metabolism of methylated arginines [1]. DDAH is conserved throughout many species and has been demonstrated to pre-date evolution of the Nitric Oxide Synthase (NOS) family [2], suggesting an alternative function for methylated arginines independent to their role as NOS regulatory molecules. Two different isoforms of human DDAH have been identified, with DDAH-1 and DDAH-2 sharing 62% homology. DDAH-1 is primarily expressed in different areas of the brain [3], the dorsal root ganglia and the spinal dorsal horn [4]. DDAH-2 appears prevalent in pancreatic islet cells [5] and infected tissues [6]. In addition, Altman et al. reported a study showing high expression of DDAH-2 in porcine immune tissues [7]. The two isoforms are both expressed in kidney [8–10], heart [11,12], adipose tissue [13], placenta and trophoblasts [14], and the liver [15–18]. Phylogenetic analyses indicate DDAH-1 was generated by duplication of the genomic segment located on chromosome 6 comprising DDAH-2 [2,19]. Although hydrolysis of asymmetrically methylated arginines appears to be the primary function of DDAH-1, alternate roles for both DDAH isoforms are evident when they independently interact with other enzymes [20,21]. For example, DDAH-1 is
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reported to bind the tumour suppressor protein neurofibromin, resulting in increased phosphorylation of neurofibromin by levels of Molecules 2016, 2016, 21, 21, 615protein kinase A (PKA) [20] and is additionally reported to regulate the of 31 Molecules 615 22 of 31 phosphorylated protein kinase B (p-Akt) by increasing Ras activity via a mechanism independent to of neurofibromin neurofibromin by Similarly, protein kinase kinase A (PKA) (PKA) [20] and and to is additionally additionally reported to regulate regulate the levels of of by protein A is reported to levels the NOS pathway [22]. DDAH-2 is [20] known interact with PKA causing thethe induction of phosphorylated protein kinase B (p-Akt) by increasing Ras activity via a mechanism independent of phosphorylated proteinfactor kinase(VEGF) B (p-Akt)via byphosphorylation increasing Ras activity viaSp1 a mechanism independent vascular endothelial growth of the transcription factor [21]. to the the NOS NOS pathway pathway [22]. [22]. Similarly, Similarly, DDAH-2 DDAH-2 is is known known to to interact interact with with PKA PKA causing causing the the induction induction of of to Asymmetric dimethylarginine (ADMA, 1), monomethylarginine (L-NMMA, 2), and symmetric vascular endothelial growth factor (VEGF) via phosphorylation of the Sp1 transcription factor [21]. vascular endothelial growth factor (VEGF) via phosphorylation of the Sp1 transcription factor [21]. dimethylarginine (SDMA, 3) are endogenous methylated arginines (Figure 1).2),They are formed by Asymmetric dimethylarginine dimethylarginine (ADMA, (ADMA, 1), 1), monomethylarginine monomethylarginine ((LL-NMMA, -NMMA, 2), and symmetric symmetric Asymmetric and two biochemical processes: post-translational methylation of1). protein arginine dimethylarginine (SDMA,(1) 3)the are initial endogenous methylated arginines arginines (Figure 1). They are are formedresidues by dimethylarginine (SDMA, 3) are endogenous methylated (Figure They formed by which is catalyzed by the protein arginine methyltransferases (PRMT’s); and (2) the subsequent two biochemical processes: (1) the initial post-translational methylation of protein arginine residues two biochemical processes: (1) the initial post-translational methylation of protein arginine residues release from their respective methylated protein(s) by proteolysis [23]. Once released into the which is catalyzed catalyzed by the the protein protein arginine methyltransferases methyltransferases (PRMT’s); and and (2) the the subsequent release which is by arginine (PRMT’s); (2) subsequent release + fromtransport their respective respective methylated protein(s) by proteolysis proteolysis [23]. Once released released into the cytosol, cytosol, and efflux of theprotein(s) methylated arginines is mediated by theinto y the cationic amino from their methylated by [23]. Once cytosol, ++ cationic transport and and efflux efflux of of the the methylated methylated arginines is mediated mediated by the the cationic amino amino acid (CAT-1) is by (CAT-1) acid transport (CAT-1) transporter [24]. ADMA arginines and L-NMMA inhibit allyymembers of theacid NOS family of transporter [24]. ADMA and Lcompetes L-NMMA -NMMA inhibit inhibit all members aof ofNOS the NOS NOS family of of enzymes enzymes [25–30], transporter [24]. ADMA and members the family [25–30], enzymes [25–30], while SDMA with Lall -arginine, substrate, for transport by the y+ while SDMA SDMA competes competes with with LL-arginine, -arginine, aa NOS NOS substrate, substrate, for for transport transport by by the the yy++ cation cation transporter transporter [31]. [31]. while cation transporter [31]. Different enzymes play a role in the metabolism of methylated arginines, Different enzymes enzymes play play aa role role in in the the metabolism metabolism of of methylated methylated arginines, arginines, particularly particularly alanine-glyoxylate alanine-glyoxylate Different particularly alanine-glyoxylate aminotransferase 2 (AGXT2) [32–34]. However, ADMA and L-NMMA, aminotransferase 22 (AGXT2) (AGXT2) [32–34]. [32–34]. However, However, ADMA ADMA and and LL-NMMA, -NMMA, but but not not SDMA, SDMA, are are primarily primarily aminotransferase but not SDMA, primarily converted by dimethylamine DDAH-1 to L(5) -citrulline (4) and dimethylamine converted byare DDAH-1 to LL-citrulline -citrulline (4) and and or monomethylamine monomethylamine (6), respectively respectively(5) or converted by DDAH-1 to (4) dimethylamine (5) or (6), monomethylamine [35] (Scheme (Scheme 1). 1). (6), respectively [35] (Scheme 1). [35]
Figure 1. The endogenous methylated arginines, asymmetric dimethylarginine (ADMA 1),
Figure Theendogenous endogenous methylated methylated arginines, dimethylarginine (ADMA 1), 1), Figure 1. 1.The arginines, asymmetric asymmetric dimethylarginine (ADMA -NMMA 2), 2), and and symmetric symmetric dimethylarginine dimethylarginine (SDMA (SDMA 3) 3) act act as as direct direct or or monomethylarginine ((LL-NMMA monomethylarginine monomethylarginine (L-NMMA 2), and symmetric dimethylarginine (SDMA 3) act as direct or indirect L -NMMA (2) are substrates for indirect inhibitors of the NOS family of enzymes. ADMA (1) and indirect inhibitors of the NOS family of enzymes. ADMA (1) and L-NMMA (2) are substrates for inhibitors of the NOS family of enzymes. ADMA (1) and L-NMMA (2) are substrates for human DDAH-1. human DDAH-1. human DDAH-1.
Scheme 1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and
Scheme 1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and Scheme 1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and an amine amine species species 55 or or 6. 6. an an amine species 5 or 6.
The role role of of ADMA ADMA accumulation accumulation and and impaired impaired DDAH DDAH expression expression and/or and/or activity activity in in the the The pathophysiology of several several diseases is is well well documented [36–44]. Increased plasma or serum serum ADMA The role of ADMA accumulation anddocumented impaired [36–44]. DDAHIncreased expression and/or activity in the pathophysiology of diseases plasma or ADMA concentrations have been associated with coronary events [45–47], renal disease [29,37], atherosclerosis concentrations have been associated with coronary events [45–47], renal disease [29,37], atherosclerosis pathophysiology of several diseases is well documented [36–44]. Increased plasma or serum [48],concentrations and cerebrovascular cerebrovascular disease [49,50]. Up-regulation Up-regulation of DDAH DDAH expression and activity might, [48], and [49,50]. of expression activity might, ADMA havedisease been associated with coronary events [45–47],and renal disease [29,37], therefore, represent a good strategy in the treatment of the aforementioned pathologies to increase therefore, represent a good strategy in the treatment of the Up-regulation aforementioned of pathologies to increase and atherosclerosis [48], and cerebrovascular disease [49,50]. DDAH expression ADMA metabolism metabolism and lower lower its plasma concentrations. concentrations. ADMA activity might, therefore,and representitsaplasma good strategy in the treatment of the aforementioned pathologies In contrast, reduced ADMA concentrations have been been demonstrated demonstrated in in other other disease disease states, states, such such as as In contrast, reduced ADMA concentrations have to increase ADMA metabolism and lower its sclerosis plasma [43], concentrations. amyotrophic lateral sclerosis [51], multiple Alzheimer’s disease and dementia [52,53]. amyotrophic lateral sclerosis [51], multiple sclerosis [43], Alzheimer’s disease and dementia [52,53]. In contrast, reduced ADMA have been demonstrated in other disease states, ADMA has also also been shown shown to toconcentrations have neuroprotective neuroprotective effects in aa model model of of Parkinson’s Parkinson’s disease [54]. such ADMA has been have effects in disease [54]. as amyotrophic lateral sclerosis [51], multiple sclerosis [43], Alzheimer’s disease and dementia [52,53].
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ADMA has also been shown to have neuroprotective effects in a model of Parkinson’s disease [54]. Furthermore, DDAH overexpression in tumours enhances their metastatic potential [42,55,56]. Several reports also show that inhibition of NO synthesis results in significant anti-angiogenic effects both in vitro and in vivo, with potential beneficial effects in cancer [57–61]. Moreover, excessive NO concentrations play a key role in the pathophysiology of septic shock [62–65] (an early therapeutic approach for the treatment of septic shock involved the administration of 546C88 (L-NMMA) to inhibit NOS and reduce NO synthesis, however the increase in mortality in patients receiving L-NMMA caused the termination of the clinical trial in phase III [66]. Further analysis of the data revealed that mortality was associated with elevated L-NMMA concentrations; conversely, lower L-NMMA concentrations were associated with improved survival [36]. In this context a more controlled, indirect modulation of methylated arginine concentrations through pharmacological DDAH-1 inhibition (DDAH-2 knockout affects outcome in models of polymicrobial sepsis [44]) might represent an alternative strategy, and appears particularly promising in a rat model of septic shock [67]). In these circumstances, DDAH inhibition could exert a controlled inhibition of the NO synthesis by increasing ADMA concentrations and offering a new approach in the treatment of these diseases. Therefore, alterations in DDAH and NOS activity might exert either beneficial or toxic effects, according to specific experimental models and disease conditions. The potential of DDAH as a molecular target in the treatment of these pathological conditions has led several groups to study the endogenous mechanisms involved in the modulation of the expression and activity of human DDAH-1 and DDAH-2 [68]. The elucidation of these mechanisms may identify new strategies for the pharmacological activation or inhibition of these enzymes to restore physiological homeostasis after pathological imbalance. Since the discovery of the DDAH family of enzymes in 1987 [35], a number of research groups have identified inhibitors with the aim of developing such therapeutics. While no ‘selective’ DDAH inhibitor has entered clinical trials, numerous compounds have been synthesized that act as prototypic in vitro inhibitors. Herein, we discuss all published DDAH inhibitors according to their chemical structure (‘substrate-like’ and ‘non-substrate-like’), and summarize the details of their synthetic pathways as well as how they alter the pharmacokinetic parameters for L-citrulline formation by DDAH. 2. Endogenous Modulation of DDAH Activity The production of specific gene products, both in terms of RNA and proteins, is physiologically regulated by a plethora of mechanisms and can be triggered or silenced by various stimuli and factors. Of particular note, DDAH activity is reported to be inhibited by divalent transition metals [69,70] and it is known that elevated zinc(II) concentrations are associated with oxidative stress in numerous pathological conditions [71–73]. S-Nitrosylation of the enzyme’s cysteine residues is a further important post-translational regulatory mechanism [74] and is generally associated with increased expression of inducible NOS (iNOS). The induction of iNOS generates excessive NO that can bind covalently (but reversibly) to the thiol (SH) moiety of each DDAH cysteine [74]. The apparent S-nitrosylation of the catalytic DDAH cysteine residue results in DDAH inhibition. This in turn causes the subsequent accumulation of DDAH substrates, ADMA and L-NMMA, which eventually reach concentrations that reversibly inhibit the NOS enzymes [75]. Oxidative stress additionally modulates DDAH activity. Factors associated with oxidative stress, including shear stress [76], tumour necrosis factor α (TNF-α), oxidized low density lipoprotein (LDL) [76], and homocysteine [77] each suppresses DDAH expression and activity. In contrast, antioxidant mediators such as probucol [78], estradiol [79], and interleukin-1β (IL-1β) [80] up-regulate DDAH expression. Moreover, nicotine and cigarette smoking have been shown to decrease DDAH expression and activity in endothelial cells, with consequential accumulation of ADMA, NOS inhibition, and endothelial damage [81,82]. Furthermore, it is known that selective down-regulation of DDAH-1 and up-regulation of DDAH-2 occurs when angiotensin II binds to the type 1 angiotensin receptor (AT1 -R), linking DDAH transcription to angiotensin-induced inflammation [8]. In addition, epigenetic modifications are known to alter DDAH-2 regulation at the promoter. For example, DNA
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hypermethylation has the ability to suppress DDAH-2 transcription, while histone acetylation has the ability to up-regulate DDAH-2 transcription [83]. Molecules 2016, 21, 615
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3. Pharmacological Induction of DDAH
hypermethylation has the ability to suppress DDAH-2 transcription, while histone acetylation has
As resulttoofup-regulate diminishedDDAH-2 DDAH transcription activity and/or theaability [83].accumulation of ADMA and L-NMMA in various disease states, several studies have investigated the effects of restoring or increasing DDAH expression. Pharmacological Induction of DDAH Statin3.treatment is associated with a significant reduction in ADMA concentrations [84–86], and an increase in DDAH-1 and DDAH-2 expression and activity [87–89]. class of compounds Asboth a result of diminished DDAH activity and/or accumulation of Another ADMA and L-NMMA in various disease states, several studies have investigated the effects of restoring or increasing DDAH enhancing ADMA metabolism via DDAH up-regulation is represented by the farnesoid X receptor treatment is associated with a significant in ADMA concentrations (FXR)expression. agonists.Statin GW4064 upregulates both DDAH-1 andreduction the cationic transporter CAT-1,[84–86], leading to and an increase in both DDAH-1 DDAH-2 and expression and serum activityADMA [87–89].concentrations Another class of increased ADMA cellular uptake and and metabolism, decreased [15,90]. compounds enhancing ADMA metabolism via DDAH up-regulation is represented by the farnesoid Another FXR agonist, INT-747, can increase DDAH-1 expression [91,92], however this is not associated X receptor (FXR) agonists. GW4064 upregulates both DDAH-1 and the cationic transporter CAT-1, with significant changes in ADMA and NO concentrations [91]. The lipid lowering drug probucol leading to increased ADMA cellular uptake and metabolism, and decreased serum ADMA also lowers ADMA concentrations [93] via up-regulation of both DDAH expression and activity, and concentrations [15,90]. Another FXR agonist, INT-747, can increase DDAH-1 expression [91,92], down-regulation ofnot PRMT-1 expression [78,94]. changes Anotherinlipid lowering fenofibrate, however this is associated with significant ADMA and NOdrug, concentrations [91].decreases The ADMA concentrations by restoring DDAH activity in conditions of oxidative stress [95]. lipid lowering drug probucol also lowers ADMA concentrations [93] via up-regulation of bothAdditionally, DDAH the oral antidiabetic drug pioglitazone and theofβPRMT-1 blocker[78,94]. nebivolol both decrease serum expression and activity, and down-regulation expression Another lipid lowering 1 receptor drug, fenofibrate, decreases ADMA concentrations by restoring ADMA concentrations by increasing DDAH-2 expression [96–99]. DDAH activity in conditions of oxidative stress [95]. Additionally, the oral antidiabetic drug pioglitazone and the β1 receptor blocker
4. Pharmacological Inhibition DDAH nebivolol both decrease serumofADMA concentrations by increasing DDAH-2 expression [96–99]. Contrary to the comments inof Section 4. Pharmacological Inhibition DDAH3 above, there is a growing body of evidence to suggest that the modulation of DDAH enzyme activity may be utilized in conditions characterized by excessive Contrary to the comments in Section 3 above, there is a growing body of evidence to suggest NO production [36,67]. The remainder of this review will focus on highlighting the current knowledge that the modulation of DDAH enzyme activity may be utilized in conditions characterized by associated with the main DDAH inhibitors reported in the literature to date. excessive NO production [36,67]. The remainder of this review will focus on highlighting the current knowledge associated with the main DDAH inhibitors reported in the literature to date.
4.1. Substrate-Like Inhibitors
4.1. Substrate-Like Inhibitors
4.1.1. S-Nitroso-L-Homocysteine (HcyNO) 4.1.1. S-NitrosoL-Homocysteine (HcyNO) Since the identification of S-nitrosylation as an important mechanism of DDAH regulation, the discovery ofidentification L -homocysteine’s ability as toaninhibit DDAH-1 ledof to the regulation, development Since the of S-nitrosylation important mechanism DDAH the of S-nitrosoL -homocysteine (HcyNO, 9), an endogenous S-nitrosothiol and a source of endogenous discovery of L-homocysteine’s ability to inhibit DDAH-1 led to the development of S-nitrosoLhomocysteine (HcyNO, 9), an endogenous S-nitrosothiol source ofDDAH endogenous NO [100–102]. NO [100–102]. Synthesis and characterisation of HcyNOand as aa bovine inhibitor was reported Synthesis characterisation of HcyNO as a bovineinDDAH inhibitor wasyield reported Knipp etreaction al. by Knipp et al.and (2005) [102]. HcyNO was synthesized near quantitative in aby two-step (2005) [102]. HcyNO was synthesized in near quantitative yield in a two-step reaction (Scheme 2) (Scheme 2) via alkaline hydrolysis of commercially available L-homocysteine thiolactone hydrochloride via alkaline hydrolysis of commercially available L-homocysteine thiolactone hydrochloride (7) to (7) to L-homocysteine (8) [103], followed by S-nitrosylation of L-homocysteine with sodium nitrite L-homocysteine (8) [103], followed by S-nitrosylation of L-homocysteine with sodium nitrite (NaNO2) (NaNO2 ) and acidification to afford HcyNO (9) [104]. Alternatively, S-nitrosylation can be undertaken and acidification to afford HcyNO (9) [104]. Alternatively, S-nitrosylation can be undertaken directly directly on commercially available L -homocysteine (8). Mild reaction conditions are required, and on commercially available L-homocysteine (8). Mild reaction conditions are required, and excellent excellent yields HcyNO to prepare a large scale. yields makemake HcyNO simple simple to prepare on a largeon scale.
Scheme 2. Synthetic scheme for generating HcyNO (9). Reagents and Conditions: (i) 5 M NaOH, 37 °C, ˝ Scheme 2. Synthetic scheme for generating HcyNO (9). Reagents and Conditions: (i) 5 M NaOH, 37 C, 5 min, 96%; (ii) NaNO2, 2 M HCl, 37˝°C, 15 min, >99%. 5 min, 96%; (ii) NaNO2 , 2 M HCl, 37 C, 15 min, >99%.
HcyNO irreversibly inhibits DDAH by covalently binding to the putative catalytic cysteine
HcyNO irreversibly inhibits by[105]. covalently binding to the putative catalytic with an inhibitory constant (Ki) DDAH of 690 μM Mass spectrometry analyses using differentcysteine isotopic with 15NO and HcyN18O) and oxygen-18 labelled water (H218O) forms of S-nitroso(Hcy an inhibitory constantL-homocysteine (Ki ) of 690 µM [105]. Mass spectrometry analyses using different isotopic 15 NO and HcyN 18 O) with HcyNO forms (Hcy a N-thiosulfoximide adduct cysteine 273labelled (Cys273)water of bovine formsdemonstrated of S-nitroso-that L -homocysteine and oxygen-18 (H2 18 O) DDAH-1. The authors’ proposed mechanism of inhibition requires the lone pair of electrons of the demonstrated that HcyNO forms a N-thiosulfoximide adduct with cysteine 273 (Cys273) of bovine
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Molecules 2016,authors’ 21, 615 5 of 31 DDAH-1. The proposed mechanism of inhibition requires the lone pair of electrons of the Cys273 SH to attack the S-nitroso group in HcyNO to eliminate a hydroxyl anion (Scheme 3). This Cys273 the S-nitroso group in HcyNO to eliminate a hydroxyl anion (Scheme 3).5This Molecules SH 2016,to 21,attack 615 of 31 mechanism is likely stabilized by neighbouring amino acids, namely His172. A further hydroxyl anion mechanism is likely stabilized by neighbouring amino acids, namely His172. A further hydroxyl anion originating from water attacks the cationic sulfur of the conjugated intermediate, and subsequently originating water the group cationic of to theeliminate conjugated intermediate, subsequently Cys273 SH from to attack theattacks S-nitroso insulfur HcyNO a hydroxyl anionand (Scheme 3). This rearranges to give the the covalent thiosulfoximide adduct [102]. rearranges to covalent adduct [102]. mechanism is give likely stabilized bythiosulfoximide neighbouring amino acids, namely His172. A further hydroxyl anion
originating from water attacks the cationic sulfur of the conjugated intermediate, and subsequently rearranges to give the covalent thiosulfoximide adduct [102].
Scheme 3. Schematic of the reaction mechanism proposed by Knipp et al. [102] for the covalent
Scheme 3. Schematic of the reaction mechanism proposed by Knipp et al. [102] for the covalent inhibition of DDAH-1 by HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005 inhibition of DDAH-1 by HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005 American Society. Scheme 3.Chemical Schematic of the reaction mechanism proposed by Knipp et al. [102] for the covalent American Chemical Society. inhibition of DDAH-1 by HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005
4.1.2.American 2-Chloroacetamidine and N-But-3-ynyl-2-chloroacetamidine Chemical Society.
4.1.2. 2-Chloroacetamidine and N-But-3-ynyl-2-chloroacetamidine Stone et al. [106] identified 2-chloroacetamidine (11) as a nonspecific inhibitor of two enzymes of 4.1.2. 2-Chloroacetamidine and N-But-3-ynyl-2-chloroacetamidine
the amidinotransferases superfamily, Pseudomonas (11) aeruginosa DDAH (PaDDAH) human Stone et al. [106] identified 2-chloroacetamidine as a nonspecific inhibitor and of two enzymes peptydilarginine deaminase (PAD). 2-Chloroacetamidine (11, SchemeDDAH 4) inhibitor is a commercially available Stone et al. [106] identified 2-chloroacetamidine (11) as a nonspecific of two enzymes of of the amidinotransferases superfamily, Pseudomonas aeruginosa (PaDDAH) and human compound bearing a 2-chloromethylene chain and an aeruginosa amidinium DDAH group, which partially resembles the amidinotransferases superfamily, Pseudomonas (PaDDAH) and human peptydilarginine deaminase (PAD). 2-Chloroacetamidine (11, Scheme 4) is a commercially available arginine. It can bedeaminase synthesized(PAD). via reaction of chloroacetonitrile (10) with4)sodium methoxide available followed peptydilarginine 2-Chloroacetamidine (11, Scheme is a commercially compound bearing a 2-chloromethylene chain and an amidinium group, which partially resembles by treatment with ammonium chloride [107,108]. as apartially weak irreversible compound bearing a 2-chloromethylene chain and 2-Chloroacetamidine an amidinium group,acts which resembles arginine. It can be synthesized reaction chloroacetonitrile (10) with to sodium methoxide followed amidinotransferase inhibitorvia with greaterof affinity for PaDDAH, PAD4 (Table followed 1). Data arginine. It can be synthesized via reaction of chloroacetonitrile (10)relative with sodium methoxide by treatment with ammonium chloride [107,108]. 2-Chloroacetamidine acts as a weak irreversible acquired by electrospray ionization mass spectrometry revealed the formation an irreversible by treatment with ammonium chloride [107,108]. 2-Chloroacetamidine acts as a of weak irreversible amidinotransferase inhibitor with affinity for PaDDAH, PaDDAH, relative PAD4 (Table 1). Data thiouronium-enzyme adduct whereby the loss of chlorine from therelative inhibitor observed [106] amidinotransferase inhibitor withgreater greater affinity for to isto PAD4 (Table 1).. Data acquired by electrospray ionization revealedthe theformation formation of irreversible an irreversible acquired by electrospray ionizationmass massspectrometry spectrometry revealed of an thiouronium-enzyme adduct whereby fromthe theinhibitor inhibitor is observed [106]. . thiouronium-enzyme adduct wherebythe theloss lossof ofchlorine chlorine from is observed [106]
Scheme 4. Synthetic scheme for generating 2-chloroacetamidine (11) (the conditions described afford the hydrochloride salt). Reagents and Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4Cl, reflux, h, Synthetic 89%. Scheme6 4. scheme for generating 2-chloroacetamidine (11) (the conditions described afford
Scheme 4. Synthetic scheme for generating 2-chloroacetamidine (11) (the conditions described afford the hydrochloride salt). Reagents and Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4Cl, the hydrochlorideTable salt).1.Reagents Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4 Cl, Reportedand inhibitory constants for 2-chloroacetamidine (11) [106]. reflux, 6 h, 89%. reflux, 6 h, 89%. Enzyme Ki (mM) Kinact (min−1) Table 1. Reported inhibitory constants for 2-chloroacetamidine (11) [106]. PaDDAH 3.1 ± 0.8 1.2 ± 0.1 Table 1. Reported inhibitory constants for 2-chloroacetamidine (11) [106]. PAD4 ±5 0.7 (min ± 0.1 −1) Enzyme K20 i (mM) Kinact PaDDAH 3.1 (mM) ± 0.8 1.2 ± 0.1(min´1 ) Enzyme K Kinact i The addition of an N-but-3-ynyl PAD4 substituent 20 ±to5 the acetamidine 0.7 ± 0.1 group of 2-chloroacetamidine PaDDAH 3.1 ˘ 0.8 1.2 ˘ 0.1 led to formation of the human DDAH-1 probe, N-but-3-ynyl-2-chloro-acetamidine (18, Scheme 5) PAD4 20 ˘ 5 0.7 ˘ 0.1 [109].The N-But-3-ynyl-2-chloro-acetamidine binds within putative active-site, while the N-alkynyl addition of an N-but-3-ynyl substituent to thethe acetamidine group of 2-chloroacetamidine substituent is reacted with biotin-PEO 3-azide under copper catalysis via click chemistry. led to formation of the human DDAH-1 probe, N-but-3-ynyl-2-chloro-acetamidine (18, Successful Scheme 5) conjugation can detected in immunoblots using anti-biotin antibody [109].the Synthesis of led [109].addition N-But-3-ynyl-2-chloro-acetamidine binds to within theanputative active-site, while N-alkynyl The ofbe aneasily N-but-3-ynyl substituent the acetamidine group of 2-chloroacetamidine the N-but-3-ynyl-2-chloro-acetamidine probe was achieved in five steps via using twochemistry. modules (Scheme 5).[109]. substituent is reacted with biotin-PEO 3-azide under copper catalysis click Successful to formation of the human DDAH-1 probe, N-but-3-ynyl-2-chloro-acetamidine (18, Scheme 5) Amine module 15 easily was prepared inimmunoblots threewithin steps: using activation of the terminal group of conjugation can be detected in an anti-biotin antibodyhydroxyl [109]. Synthesis N-But-3-ynyl-2-chloro-acetamidine binds the putative active-site, while the N-alkynyl 3-butyn-1-ol (12) through mesylation probe to afford 13 in 62% in yield, followed bytwo conversion to amine 15 the N-but-3-ynyl-2-chloro-acetamidine was achieved five steps using modules (Scheme 5). substituent is reacted with biotin-PEO3 -azide under copper catalysis via click chemistry. Successful Amine module 15 was prepared in three steps: activation of the terminal hydroxyl group of conjugation can be easily detected in immunoblots using an anti-biotin antibody [109]. Synthesis of the 3-butyn-1-ol (12) through mesylation to afford 13 in 62% yield, followed by conversion to amine 15
N-but-3-ynyl-2-chloro-acetamidine probe was achieved in five steps using two modules (Scheme 5).
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Amine module 15 was prepared in three steps: activation of the terminal hydroxyl group of 3-butyn-1-ol Molecules 2016, 21, 615 of 31azide (12) through mesylation to afford 13 in 62% yield, followed by conversion to amine 15 6via intermediate 14 in 49% yield across two steps. The imidic ester module 17 was obtained in moderate via azide2016, intermediate 14 in 49% yield across two steps. The imidic ester module 17 was obtained 21, 615 48%) 6 of in 31 yieldsMolecules (approximately by reacting chloroacetonitrile (10) with anhydrous ethanol (16) in the moderate yields (approximately 48%) by reacting chloroacetonitrile (10) with anhydrous ethanol (16) presence of dry hydrochloric acid gas. Final coupling between amine 15 and the imidic ester 17 in presence of dry hydrochloric acid gas. two Finalsteps. coupling between amine 15 and the imidic ester viathe azide intermediate 14 in 49% yield across The imidic ester module 17 was obtained in proceeds under basic aqueous conditions to afford probe N-but-3-ynyl-2-chloro-acetamidine (18) in 17 proceeds under basic aqueous conditions to afford probe N-but-3-ynyl-2-chloro-acetamidine moderate yields (approximately 48%) by reacting chloroacetonitrile (10) with anhydrous ethanol (18) (16) reasonable yields (approximately 63%). To date, no data are available regarding the selectivity of in reasonable yields (approximately 63%).gas. To Final date, no data are available regarding of the presence of dry hydrochloric acid coupling between amine 15 andthe theselectivity imidic ester N-but-3-ynyl-2-chloro-acetamidine human DDAH-1 and the potential N-but-3-ynyl-2-chloro-acetamidine for human DDAH-1 and the potentialcross crossreactivity reactivityofofthis thisprobe 17 proceeds under basic aqueousfor conditions to afford probe N-but-3-ynyl-2-chloro-acetamidine (18) with PaDDAH. with probe PaDDAH. in reasonable yields (approximately 63%). To date, no data are available regarding the selectivity of N-but-3-ynyl-2-chloro-acetamidine for human DDAH-1 and the potential cross reactivity of this NH.HCl probe with PaDDAH. (iv) Cl Cl
N + HO 16
10 Cl
(iv)
N + HO 16
10
O 17 NH.HCl
Cl
O
HCl.H2N
HO
17
Cl
N H NH
18
N H 18
HCl.H2N
(i) 12
MsO
(v)
NH Cl
15
12 HO
(v)
(i) 13
15(iii)
(ii) (iii)
N3 (ii)
14
Scheme 5. SyntheticMsO scheme for generating Nthe DDAH-1 biochemical probe N-but-3-ynyl-2-chloro3 Scheme 5. Synthetic scheme for generating the DDAH-1 biochemical probe N-but-3-ynyl-2-chloro13 Conditions: (i) DCM,14 acetamidine (18). Reagents and TEA, DMAP, MsCl, 0 °C for 2 h then 25 °C for 3 h, acetamidine (18). Reagents and Conditions: (i) DCM, TEA, DMAP, MsCl, 0 ˝ C for 2 h then 25 ˝ C for 3 h, °C, 3.5 h; the (iii) Et2O, Ph3biochemical P, 0 °C for 2 h, addition of H2O, then 25 °C ˝ 62%; (ii) anhydrous NaN3, 70 5. SyntheticDMF, N-but-3-ynyl-2-chloro˝ C, 62%; Scheme (ii) anhydrous DMF,scheme NaN3 ,for 70 generating 3.5 h; (iii) DDAH-1 Et2 O, Ph3 P, 0 ˝ C for 2probe h, addition of H2 O, then 25 C 0 °C for 1TEA, h, stop HCl(g)MsCl, , then 00 °C °C for for 24 h h then and RT overnight for 20 h, 49%(18). (twoReagents steps); (iv) HCl(g) at(i) acetamidine anddry Conditions: DCM, DMAP, 25 °C for 3 h, ˝ C for 1 h, stop HCl , then 0 ˝ C for 4 h and for 2048%–76%; h, 49% (two steps); (iv) dry HCl at 0 RT overnight (g)3 h, 25 °C for 2 h, neutralized, (g) RT for 1.5 days, 62%. Adapted 2 O, pH = 10, 0 °C for (v) H 3, 70 °C, 3.5 h; (iii) Et2O, Ph3P, 0 °C for 2 h, addition of H2O, then 25 °C 62%; (ii) anhydrous DMF, NaN ˝ C for 2 h, neutralized, RT for 1.5 days, 62%. Adapted 48%–76%; (v) H O, from pH =[109]. 10, 0 ˝ C for 3 h, 25American with Chemical at 0 °C for 1 h, stop HCl(g)Society. , then 0 °C for 4 h and RT overnight for 20permission h, 49%2(two steps); (iv)Copyright dry HCl(g)2009
with 48%–76%; permission Copyright American Society. pH = 10, 0 °C for 2009 3 h, 25 °C for 2 h,Chemical neutralized, RT for 1.5 days, 62%. Adapted (v)from H2O,[109]. 4.1.3.with (S)-2-Amino-4-(3-Methylguanidino)butanoic Acid Analogues permission from [109]. Copyright 2009 American Chemical Society.
4.1.3. (S)-2-Amino-4-(3-Methylguanidino)butanoic Acid Screening experiments with a library comprising 26Analogues analogues of ADMA (1) and L-NMMA (2) 4.1.3. (S)-2-Amino-4-(3-Methylguanidino)butanoic Acid Analogues
identified (S)-2-amino-4-(3-methylguanidino)butanoic (4124W, 21),ofasADMA a mammalian Screening experiments with a library comprisingacid 26 analogues (1) andDDAH L -NMMA inhibitor [110]. The original synthesis of 4124W (21), first described by Ueda et al. inL-NMMA 2003 [80](2) is Screening experiments with a library comprising 26 analogues of ADMA (1) and (2) identified (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W, 21), as a mammalian DDAH shown in Scheme 6, and was achieved by coupling N,S-dimethylthiopseudouronium iodide (19) identified (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W, 21), as a mammalian DDAH inhibitor [110]. The original synthesis of 4124W (21), first described by Ueda et al. in 2003 [80] is shown with the copper complex of synthesis 2,4-diamino-n-butyric acid dihydrochloride (20) [32,111]. 4124W (21) inhibitor [110]. The original of 4124W (21), first described by Ueda et al. in 2003 [80] is in Scheme 6, and was achieved by coupling N,S-dimethylthiopseudouronium iodide (19) with the was finally crystallized as the salt. shown in Scheme 6, and washydrochloride achieved by coupling N,S-dimethylthiopseudouronium iodide (19) copper complex of 2,4-diamino-n-butyric acid dihydrochloride (20) [32,111]. 4124W (21) was finally with the copper complex of 2,4-diamino-n-butyric acid dihydrochloride (20) [32,111]. 4124W (21) crystallized as the hydrochloride salt. was finally crystallized as the hydrochloride salt.
Scheme 6. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W), as reported by Ueda et al. [80] using the methods of Ogawa et al. [32] and Corbin et al. [111]. Reagents and Conditions: (i) (1) 2,4-diamino-n-butyric acid dihydrochloride (20), copper acetate, 1:1 NH(4124W), 4OH/H2O; Scheme 6. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid as Scheme 6. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W), 4+ [111]. form),Reagents 4124W (21) (2) N,S-dimethylthiopseudouronium iodide (19) 24 h;et(3) reported by Ueda et al. [80] using the methods of RT, Ogawa al.Dowex [32] and50WX4 Corbin(NH et al. and as reported by Ueda as et al. using the methods of Ogawa et al. [32] and Corbin et al. [111]. Reagents and was crystallized the[80] hydrochloride salt. Conditions: (i) (1) 2,4-diamino-n-butyric acid dihydrochloride (20), copper acetate, 1:1 NH4OH/H2O;
Conditions: (i) (1) 2,4-diamino-n-butyric acid dihydrochloride (20), copper acetate, 1:1 NH4 OH/H2 O; (21) (2) N,S-dimethylthiopseudouronium iodide (19) RT, 24 h; (3) Dowex 50WX4 (NH4+ form), 4124W + form), 4124W (21) (2) N,S-dimethylthiopseudouronium iodide (19) RT, 24 h; (3) Dowex 50WX4 (NH High chemical and structural similarity between 4124W and the DDAH substrate L-NMMA exists 4 was crystallized as the hydrochloride salt. (compound 21, Scheme 6, and compound was crystallized as the hydrochloride salt.2, Figure 1), and although exhibiting weak DDAH inhibition (IC50 High = 416 chemical μM) [110], this prompted Rossiterbetween et al. [112] to synthesize a further 34 compounds and structural similarity 4124W and the DDAH substrate L-NMMAbased exists on the 4124W structure. Furthermore, Rossiter et al. [112] reported an alternative synthetic approach, (compound 21, Scheme 6, and compound 2, Figure 1), and although exhibiting weak DDAH inhibition High chemical and structural similarity between 4124W and the DDAH substrate L-NMMA exists which although requiring three steps, is synthetically versatile and awas applied to developbased their (IC50 = 416 [110], Rossiter et al. 1), [112] to although synthesize further 34weak compounds (compound 21, μM) Scheme 6,this andprompted compound 2, Figure and exhibiting DDAH inhibition library of 4124W analogues. Rossiter’s strategy protected intermediates to avoid complications on the 4124W structure. Furthermore, Rossiterutilized et al. [112] reported an alternative synthetic approach, (IC50 associated = 416 µM)with [110], this prompted Rossiter et al. [112] to synthesize a further 34 compounds based the purification polarisamino acids. Intermediate 24 was (Scheme 7) istogenerated which although requiring threeofsteps, synthetically versatile and applied develop from their on the 4124W structure. Furthermore, Rossiter et al. [112] reported an alternative approach, commercially available N,N′-bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine (22)synthetic by Mitsunobu library of 4124W analogues. Rossiter’s strategy utilized protected intermediates to avoid complications
which although requiring three steps, is amino synthetically versatile and was applied to develop associated with the purification of polar acids. Intermediate 24 (Scheme 7) is generated from their library of 4124W analogues. Rossiter’s strategy utilized protected intermediates to avoid complications commercially available N,N′-bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine (22) by Mitsunobu associated with the purification of polar amino acids. Intermediate 24 (Scheme 7) is generated from
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Molecules 2016, 21, 615 7 of 31 commercially available N,N1 -bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine (22) by Mitsunobu reaction with alcohol 23 containing the desired R1 group, followed by reaction with (S)-tert-butyl reaction with21,alcohol 23 containing the desired R1 group, followed by reaction with (S)-tert-butyl Molecules 2016, 615 7 of 31 4-amino-2-((tert-butoxycarbonyl)amino)butanoate (25) to afford the protected amino acid analogue 26 4-amino-2-((tert-butoxycarbonyl)amino)butanoate (25) to afford the protected amino acid analogue (Scheme 7). Cleavage of the protecting isR1achieved with a 4by M solutionwith of HCl in 1,4-dioxane reaction with 23Boc containing the groups desiredgroups group, followed (S)-tert-butyl 26 (Scheme 7).alcohol Cleavage of the Boc protecting is achieved withreaction a 4 M solution of HCl in and produced 4124W (21) in moderate yields (approximately 44%) or analogues 27 in moderate 4-amino-2-((tert-butoxycarbonyl)amino)butanoate to afford the protected amino acid analogue 1,4-dioxane and produced 4124W (21) in moderate(25) yields (approximately 44%) or analogues 27 in to goodmoderate yields (12%–67%). 26 (Scheme 7). Cleavage of the Boc protecting groups is achieved with a 4 M solution of HCl in to good yields (12%–67%).
1,4-dioxane and produced 4124W (21) in moderate yields (approximately 44%) or analogues 27 in moderate to good yields (12%–67%).
Scheme 7. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid analogues.
Scheme 7. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid analogues. Reagents and Conditions: (i) DEAD, PPh3, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH3CN, RT, 24 h; (iii) 4M Reagents and Conditions: (i) DEAD, PPh3 , THF, 0 ˝ C-RT, 3–16 h; (ii) DIPEA, CH3 CN, RT, 24 h; (iii) Scheme 7. Synthetic generating (S)-2-amino-4-(3-methylguanidino)butanoic analogues. HCl/1,4-dioxane, RT, scheme 24–72 h,for 12%–67% overall; (iv) SOCl2, 0 °C for 30 min, reflux for 1 h,acid RT overnight, ˝ C for 30 min, reflux for 1 h, RT 4M HCl/1,4-dioxane, RT, 24–72 h, 12%–67% overall; (iv) SOCl , 0 2 3 , THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH 3 CN, RT, 24 h; (iii) 4M Reagents and Conditions: (i) DEAD, PPh 44%–84%. Adapted with permission from [112]. Copyright 2005 American Chemical Society. overnight, 44%–84%. RT, Adapted with permission [112]. Copyright 2005 American Chemical Society. HCl/1,4-dioxane, 24–72 h, 12%–67% overall;from (iv) SOCl 2, 0 °C for 30 min, reflux for 1 h, RT overnight,
Additionally, Rossiter et al. from developed a synthetic method for Society. N,N-disubstituted 44%–84%. Adapted with permission [112]. Copyright 2005 American Chemical alkylguanidinobutanoic acids and N-arylguanidines (Scheme 8). This involved N,N’-bisAdditionally, Rossiter et al. developed a synthetic method forreacting N,N-disubstituted Additionally, Rossiter et al. developed a synthetic method for ester N,N-disubstituted tert-butoxycarbonylpyrazole-1H-carboxamidine (22) with Boc-homoserine-tert-butyl (30), whereby alkylguanidinobutanoic acids and N-arylguanidines (Scheme 8). This involved reacting N,N’-bisalkylguanidinobutanoic and N-arylguanidines 8). Thiswith involved N,N’-bisthe pyrazole group in theacids intermediate 31 is displaced (Scheme by the reaction amine reacting 32 containing the tert-butoxycarbonylpyrazole-1H-carboxamidine (22) with Boc-homoserine-tert-butyl ester (30), tert-butoxycarbonylpyrazole-1H-carboxamidine (22) with Boc-homoserine-tert-butyl ester (30), whereby desired R1 and R2 groups. whereby the pyrazole group the intermediate 31 is displaced the reaction with32 amine 32 containing the pyrazole group in theinintermediate 31 is displaced by the by reaction with amine containing the 1 2 the desired R2 groups. groups. desiredRR1 and and R
Scheme 8. Synthetic scheme for generating N,N-disubstituted alkylguanidinobutanoic acid and N-arylguanidine analogues. Reagents and Conditions: (i) DEAD, Ph3P, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, Scheme RT, 8. Synthetic for generating N,N-disubstituted alkylguanidinobutanoic acid and CH 24 h; (iii) 4scheme M HCl/1,4-dioxane, h, 0.29%–41% overall. Adapted with permission Scheme3CN, 8. Synthetic scheme for generating24–72 N,N-disubstituted alkylguanidinobutanoic acid and 3–16 h; (ii) DIPEA, N-arylguanidine analogues. Reagents and Conditions: (i) DEAD, Ph3P, THF, 0 °C-RT, from [112]. Copyright 2005 American Chemical Society. N-arylguanidine analogues. Reagents and Conditions: (i) DEAD, Ph3 P, THF, 0 ˝ C-RT, 3–16 h; (ii) DIPEA, CH3CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 0.29%–41% overall. Adapted with permission CH3 CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 0.29%–41% overall. Adapted with permission from [112]. Copyright 2005 American Chemical Society.
from [112]. Copyright 2005 American Chemical Society.
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Deprotection of the amino acid analogues 33 is carried out using the same acidic conditions described in the synthesis of Rossiter’s 4124W analogues in Scheme 7 and provides analogues 34 Molecules 2016, 21, 615 8 of 31 in low to moderate yields (0.29%–41%). Each analogue’s inhibitory potential was assessed based Deprotection of the of amino acid analogues is carried out the same acidic conditions on the reduced conversion [14 C]-NMMA to [1433 C]-citrulline byusing rat DDAH. Screening experiments described in the synthesis of to Rossiter’s 4124W analogues Scheme 7 and provides analogues 34 in revealed N-monosubstitution be more effective thanindisubstitution, and that a R1 substituent low to moderate yields (0.29%–41%). Each analogue’s inhibitory potential was assessed based on the 27a; comprising a N-2-methoxyethyl group increased inhibitor binding affinity (viz. compound reduced conversion of [14C]-NMMA to [14C]-citrulline by rat DDAH. Screening experiments revealed IC50 = 189 µM; Scheme 7). Furthermore, a series of N-2-methoxyethyl guanidinobutanoate esters [112] N-monosubstitution to be more effective than disubstitution, and that a R1 substituent comprising a was synthesized via thionyl chloride promoted esterification with alcohol containing the desired R3 N-2-methoxyethyl group increased inhibitor binding affinity (viz. compound 27a; IC50 = 189 μM; group (Scheme 7). The generated benzyl ester 29a resulted in substantial improvement in DDAH-1 Scheme 7). Furthermore, a series of N-2-methoxyethyl guanidinobutanoate esters [112] was synthesized inhibition (IC50chloride = 27 µM). A series of amidewith analogues based on the structures (structures 3 group (Schemeomitted) 7). via thionyl promoted esterification alcohol containing desired R29 exhibited poor DDAH-1 potentials. The generated benzylinhibitory ester 29a resulted in substantial improvement in DDAH-1 inhibition (IC50 = 27 μM). A series of amide analogues based on structures 29 (structures omitted) exhibited poor
4.1.4.DDAH-1 NG -SubstitutedinhibitoryL-Arginine potentials. Analogues
Rossiter et al. [112] also synthesized a library of N-substituted arginine analogues. A significant 4.1.4. NG-Substituted-L-Arginine Analogues increase in inhibitor binding affinity was obtained for NG -(2-methoxyethyl)-L-arginine (commonly et al. [112] also39; synthesized library of N-substituted analogues. A significant known asRossiter L-257 (compound IC50 = 22aµM; Scheme 9) and itsarginine corresponding methyl ester (also G increase in inhibitor binding affinity was obtained for N -(2-methoxyethyl)-L-arginine (commonly known as L-291 (compound 40; IC50 = 20 µM; Scheme 9), relative to compound 27a (Scheme 7) that known as L-257 (compound 39; IC50 = 22 μM; Scheme 9) and its corresponding methyl ester (also known contains one less carbon within the main-chain. Interestingly, the benzyl ester of L-257 did not increase as L-291 (compound 40; IC50 = 20 μM; Scheme 9), relative to compound 27a (Scheme 7) that contains one DDAH-1 inhibition as the wasmain-chain. observed with compound 29a. These suggest that arginine analogues less carbon within Interestingly, the benzyl ester data of L-257 did not increase DDAH-1 may inhibition bind differently within the putative DDAH-1 active-site. Moreover, neither the corresponding as was observed with compound 29a. These data suggest that arginine analogues may bind methyl amide ofwithin L-257,the norputative the (R)-isomer L-257 (structures omitted) in DDAH-1methyl inhibition. differently DDAH-1ofactive-site. Moreover, neitherresulted the corresponding amidesynthesis of L-257, nor (R)-isomer L-257 (structures DDAH-1 inhibition. The of the L-257 (39) isofrepresented in omitted) Scheme resulted 9. Thein reaction sequence follows a The synthesis of described L-257 (39) is in Scheme 9. The reaction sequence follows a similar similar approach to that inrepresented the synthesis of the (S)-2-amino-4-(3-methylguanidino)butanoic to that described in the synthesis the (S)-2-amino-4-(3-methylguanidino)butanoic acid acid approach analogues, using Boc protected aminoof acid analogues. Firstly, 2-methoxyethyl pyrazole analogues, using Boc protected amino acid analogues. Firstly, 2-methoxyethyl pyrazole carboxamidine (36) is prepared under Mitsunobu conditions [113] from 2-methoxyethanol (35) and carboxamidine (36) is prepared under Mitsunobu conditions [113] from 2-methoxyethanol (35) and N,N1 -bis-tert-butoxycarbonylpyrazole-1H-carboxamidine (22). This intermediate 36 is reacted with N,N′-bis-tert-butoxycarbonylpyrazole-1H-carboxamidine (22). This intermediate 36 is reacted with Boc-Orn-OBut (37) to introduce the N-alkyl-guanidino group to compound 38. Final deprotection of Boc-Orn-OBut (37) to introduce the N-alkyl-guanidino group to compound 38. Final deprotection of 38 with 4 M HCl in afforded in 44% 44%overall overallyield. yield. L-257 be subsequently 38 with 4 M HCl1,4-dioxane in 1,4-dioxane affordedL-257 L-257 (39) (39) in L-257 cancan be subsequently esterified to afford L-291 (40) in yields of approximately 80%. esterified to afford L-291 (40) in yields of approximately 80%.
Scheme 9. Synthetic scheme for generating L-257 (39) and L-291 (40). Reagents and Conditions: (i) Scheme 9. Synthetic scheme for generating L-257 (39) and L-291 (40). Reagents and Conditions: (i) DEAD, DEAD, Ph˝3P, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH3CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, Ph3 P, THF, 0 C-RT, 3–16 h; (ii) DIPEA, CH3 CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 44% 44% overall; (iv) SOCl2, 0 °C for 30 min, reflux for 1 h, RT overnight, 80%. Adapted with permission overall; (iv) SOCl2 , 0 ˝ C for 30 min, reflux for 1 h, RT overnight, 80%. Adapted with permission from [112]. Copyright 2005 American Chemical Society. from [112]. Copyright 2005 American Chemical Society.
Both L-257 (39) and L-291 (40) inhibit DDAH-1 in vivo [112]. They are non-cytotoxic and selective for DDAH-1 against the three isoforms of in NOS and are arginase [115], an and enzyme Both L-257 (39) and L-291 (40) main inhibit DDAH-1 vivo[112,114] [112]. They non-cytotoxic selective responsible for the metabolism of L -arginine into urea and ornithine [112,114,115]. L-257 (39) was for DDAH-1 against the three main isoforms of NOS [112,114] and arginase [115], an enzyme responsible utilized by Kotthaus al. [114] as a validated active ‘parent’[112,114,115]. compound for L-257 determining viable lead by for the metabolism of Let -arginine into urea and ornithine (39) was utilized compounds, and to obtain initial structure-activity relationships (SARs) for those leads. In
Kotthaus et al. [114] as a validated active ‘parent’ compound for determining viable lead compounds,
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9 of 31 and to obtain initial structure-activity relationships (SARs) for those leads. In particular, the role of the N-substituent in DDAH inhibition was investigated as the guanidino moiety is considered to particular, the role of the N-substituent in DDAH inhibition was investigated as the guanidino moiety be important in the proposed catalytic mechanism and binding to the enzyme [116]. In the work is considered to be important in theGproposed catalytic mechanism and binding to the enzyme [116]. undertaken by Kotthaus et al., the N -(2-methoxyethyl) side-chain was varied along with the degree of In the work undertaken by Kotthaus et al., the NG-(2-methoxyethyl) side-chain was varied along with guanidine substitution. Similarly, Tommasi et al. synthesized further L-257 derivatives with differing the degree of guanidine substitution. Similarly, Tommasi et al. synthesized further L-257 derivatives side-chains [117]. In both studies, the NG -(2-methoxyethyl) side-chain conferred inhibitors with activity with differing side-chains [117]. In both studies, the NG-(2-methoxyethyl) side-chain conferred toward DDAH-1. N-monosubstituted examples are represented in Table 2. inhibitors with activity toward DDAH-1. N-monosubstituted examples are represented in Table 2.
Table 2. Inhibitory parameters for the N-monosubstituted L-arginine analogues with varied Table 2. Inhibitory parameters for the N-monosubstituted L-arginine analogues with varied N-substituents (R groups) [114,117]. N-substituents (R groups) [114,117].
Entry Inhibition at IC50 (µM) Kii (μM) (µM) 50 (μM) Entry R–R– Inhibition at11mM mM(%) (%) IC K * CH 83 31 13 1 11 1 * CH 3OCH 2CH 2– 83 31 13 3 OCH 2 CH 2– 1 CH NHCH CH – 29 2 1 3 2 2– 2 2 1 CH3NHCH 2CH 29 -(CH ) NHCH CH – 14 3 3 2 2 2 31 1 (CH3)2NHCH2CH2– 14 CH3 SHCH2 CH2 – 80 408 4 4 15 1 CH3SHCH 2CH2– 80 408 -FCH2 CH 67 379 2– 5 16 1 FCH 2 CH 2 – 67 379 -BnCH2 – 55 866 2 1 CH CH CH – 60 283 90 7 6 BnCH 55 866 3 2– 2 2 CH 70 207 58 2 CHCH 2– 7 28 CH 3CH 2CH2– 60 283 90 2 CH2 CHCH2 CH2 – 71 189 57 8 29 2 CH2CHCH2– 70 207 58 CHCCH2 – 83 55 17 10 9 211 2 CH2CHCH 2CH2– 71 189 57 F3 CCH 41 2– 2 2 10 12 CHCCH 83 55 17 O2 N–2– 6 2 2 11 13 F23CCH 2–2 CH2 – 41 -NH COCH 43 12 2 L-257 (39) (Scheme O2N–9); 1 Measured by Ultra Performance 6 * Denotes Liquid Chromatography-Mass Spectrometry; 2 Measured by colorimetric assay. 13 2 NH2COCH2CH2– 43 -
* Denotes L-257 (39) (Scheme 9);
1
Measured by Ultra Performance Liquid Chromatography-Mass
4.1.5.Spectrometry; NG -(2-Methoxyethyl)-Arginine Carboxylate Bioisosteres 2 MeasuredLby colorimetric assay.
The use of bioisosteric replacements is a well-known strategy adopted by medicinal 4.1.5. NG-(2-Methoxyethyl)L-Arginine Carboxylate Bioisosteres chemists to improve the pharmacokinetic and pharmacodynamic properties of certain compounds, including butreplacements not limited istoa inhibition potency, pKa , andchemists metabolic The use of bioisosteric well-known strategylipophilicity, adopted by medicinal to stability [118]. Tommasi et al. [117] reported the synthesis and pharmacological evaluation improve the pharmacokinetic and pharmacodynamic properties of certain compounds, including of five carboxylate bioisosteres of L-257 (compounds 44a–c; Scheme 10, [118]. 49a–b;Tommasi Schemeet11). but not limited to inhibition potency, lipophilicity, pKa, and metabolic stability al. In compounds 44a–c (Scheme the carboxylicevaluation acid moiety is modified by bioisosteres introducingof anL-257 acyl [117] reported the synthesis and10), pharmacological of five carboxylate methyl sulfonamide 44a (2-amino-5-(3-(2-methoxyethyl)guanidino)-N-(methylsulfonyl)-pentanamide; (compounds 44a–c; Scheme 10, 49a–b; Scheme 11). In compounds 44a–c (Scheme 10), the carboxylic acid ZST316), O-methyl-hydroxamate a hydroxamate The replacement is introduced moiety is an modified by introducing an44b acyland methyl sulfonamide44c. 44a (2-amino-5-(3-(2-methoxyethyl) on Z-ornithine(Boc)-OH (41) in moderate to good yieldsan (51%–86%) to afford 42, followed by the guanidino)-N-(methylsulfonyl)-pentanamide; ZST316), O-methyl-hydroxamate 44b and a quantitative cleavage of Boc protected 42 with 4 M HCl in 1,4-dioxane. Guanidination of 42 with hydroxamate 44c. The replacement is introduced on Z-ornithine(Boc)-OH (41) in moderate to good 2-methoxyethylpyrazole carboxamidine undertaken as described et al. [112] (see yields (51%–86%) to afford 42, followed(36) by isthe quantitative cleavage by of Rossiter Boc protected 42 with Sections 4.1.3 and 4.1.4 Schemes 7 and 9). Deprotection of the Bocand Z-appended arginine derivatives 4 M HCl in 1,4-dioxane. Guanidination of 42 with 2-methoxyethylpyrazole carboxamidine (36) is 43a–c is achieved in a single usingetTFMSA and(see TFASections [117,119]4.1.3 andand produces undertaken as described bystep Rossiter al. [112] 4.1.4, compounds Schemes 7 44a–c. and 9). Bioisosteric replacement the carboxylic acid moiety in L-257 withisO-methyl 44b Deprotection of the Boc- andofZ-appended arginine derivatives 43a–c achievedhydroxamate in a single step and hydroxamate 44c resulted in a decrease in DDAH-1 inhibition (IC = 230 and 131 µM, respectively). using TFMSA and TFA [117,119] and produces compounds 44a–c. 50 By contrast, introduction of anofacyl sulfonamidic moiety in ZST316 (44a) led hydroxamate to significant Bioisosteric replacement the methyl carboxylic acid moiety in L-257 with O-methyl improvements in pharmacokinetic (98%ininhibition 1 mM, IC50 Ki 131 = 1 µM). 44b and hydroxamate 44c resultedparameters in a decrease DDAH-1atinhibition (IC=503 =µM 230and and μM, Compound ZST316 (44a) is the most potent human DDAH-1 inhibitor with ‘substrate-like’ structure respectively). By contrast, introduction of an acyl methyl sulfonamidic moiety in ZST316 (44a) led to reported inimprovements the literature toindate. significant pharmacokinetic parameters (98% inhibition at 1 mM, IC50 = 3 μM and Ki = 1 μM). Compound ZST316 (44a) is the most potent human DDAH-1 inhibitor with ‘substrate-like’ structure reported in the literature to date.
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Scheme 10. Synthetic scheme for generating NG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres.
Scheme 10. Synthetic scheme for generating NG -(2-methoxyethyl)-L-arginine carboxylate bioisosteres. Reagents and Conditions: (i) CDI, DBU, methanesulfonamide, THF, RT, 7 h, 51%; (ii) HATU, DIPEA, Scheme Synthetic scheme for generating NG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres. Reagents and10. Conditions: (i) CDI, DBU, methanesulfonamide, THF, RT, 7 h, 51%; (ii) HATU, O-methylhydroxylamine hydrochloride, THF, 0 °C-RT, 16 h, 86%; (iii) HATU, DIPEA, Reagents and Conditions: (i) CDI, DBU, methanesulfonamide, THF, RT, 7 h, 51%; (ii) HATU, DIPEA, ˝ DIPEA, O-methylhydroxylamine hydrochloride, THF, 0 C-RT, 16 h, 86%; (iii) HATU, DIPEA, O-benzylhydroxylamine, THF, 0 °C-RT, 16 h, 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; O-methylhydroxylamine hydrochloride, 0 °C-RT, 16 h, 86%; (iii) HATU, DIPEA, ˝ C-RT, 16 h,THF, O-benzylhydroxylamine, THF, 0 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, (v) DIPEA, DCM, RT, 24 h, 43a 42%, 43b 22%, 43c 56%; (vi) TFMSA/TFA, RT, 1 h, 99%. Reproduced 99%; O-benzylhydroxylamine, THF, 0 °C-RT, 16 h, 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (v) DIPEA, RT, 24 h,permission 43a 42%, 43b 22%, 43cSociety 56%; (vi) TFMSA/TFA, RT, 1 h, 99%. Reproduced in in partDCM, from [117] with of the Royal of Chemistry. (v) DIPEA, DCM, RT, 24 h, 43a 42%, 43b 22%, 43c 56%; (vi) TFMSA/TFA, RT, 1 h, 99%. Reproduced part from [117] with permission of the Royal Society of Chemistry. in part from [117] with permission of the Royal Society of Chemistry.
Scheme 11. Synthetic scheme for generating NG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres. Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH4OH (30%), THF, −10 °C-RT, 3 h, Scheme 11. Synthetic scheme for generating NGG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres. Scheme scheme generating N (iii) -(2-methoxyethyl)L-arginine bioisosteres. 99%;11. (ii)Synthetic TFAA, TEA, THF, for 0 °C-RT, 16 h, 47%; 4 M HCl/1,4-dioxane, DCM, carboxylate RT, 30 min, 99%; (iv) Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH4OH (30%), THF, −10 ˝°C-RT, 3 h, Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH OH (30%), THF, ´10 C-RT, 3 h, 99%; 16 h, 30%; (vi) (1) NH2OH·HCl, DIPEA, DCM, RT, 24 h, 40%; (v) NaN3, ZnBr2, H2O/2-propanol, reflux, 4 99%; (ii) TFAA, TEA, THF, 0 °C-RT, 16 h, 47%; (iii) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (iv) ˝ C-RT, (ii) TFAA, TEA, THF, 0reflux, 4 THF, M HCl/1,4-dioxane, DCM, 30 min, 99%; (iv)RT, DIPEA, NaHCO 3, DMSO, 1616 h; h, (2)47%; CDI, (iii) DBU, reflux, 30 min, 73%; (vii)RT, TFMSA/TFA, DCM, DIPEA, DCM, RT, 24 h, 40%; (v) NaN3, ZnBr2, H2O/2-propanol, reflux, 16 h, 30%; (vi) (1) NH2OH·HCl, 30RT, min,2499%. Reproduced in part from2 ,[117] with permission reflux, of the Royal Society of Chemistry. DCM,NaHCO h, 40%; (v) NaN , ZnBr H O/2-propanol, 16 h, 30%; (vi) (1) NH OH¨ HCl, 3 2 2 3, DMSO, reflux, 16 h; (2) CDI, DBU, THF, reflux, 30 min, 73%; (vii) TFMSA/TFA, DCM, RT, NaHCO , DMSO, reflux, 16 h; (2) CDI, DBU, THF, reflux, 30 min, 73%; (vii) TFMSA/TFA, DCM, RT, 3 30 min, 99%. Reproduced in part from [117] with permission of the Royal Society of Chemistry.
Other inhibitors in this series the tetrazole 1-[4-amino-4-(1H-tetrazol-5-yl) 30 min, 99%.noteworthy Reproduced in part from [117] withinclude permission of the Royal Society of Chemistry. butyl]-3-(2-methoxyethyl)guanidine (ZST086, 49a, Scheme 11) and the 5-oxo-1,2,4-oxadiazole Other noteworthy inhibitors in this series include the tetrazole 1-[4-amino-4-(1H-tetrazol-5-yl) 1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine (ZST152, 49b, butyl]-3-(2-methoxyethyl)guanidine (ZST086, 49a, Scheme 11) and the 5-oxo-1,2,4-oxadiazole Other noteworthy inhibitors inofthis series include theazole tetrazole 1-[4-amino-4-(1H-tetrazol-5-yl) Scheme 11) [117]. The synthesis these inhibitors with bioisosteric groups is achieved49b, via 1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine (ZST152, butyl]-3-(2-methoxyethyl)guanidine (ZST086, 49a, Schemeamide 11) 45 and the 5-oxo-1,2,4-oxadiazole conversion of the terminal carboxylic acid in 41 to primary in quantitative yields using Scheme 11) [117]. The synthesis of these inhibitors with azole bioisosteric groups is achieved via 1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine (ZST152, isobutylchloroformate, N-methylmorpholine, andto aqueous amideyields 45 isusing then conversion of the terminal carboxylic acid in 41 primary ammonia. amide 45 inPrimary quantitative converted11) to nitrile using trifluoroacetic anhydride and with triethylamine in 47% yield. Compound 46 49b, Scheme [117]. 46 The synthesis of these inhibitors azole bioisosteric groups isobutylchloroformate, N-methylmorpholine, and aqueous ammonia. Primary amide 45 is is achieved then is guanylated [112] (see Sections 4.1.3 and 4.1.4, Schemes 7 and 9) to afford compound 47, from via conversion of the terminal carboxylic acid in 41 to primary amide 45 in quantitative yields converted to nitrile 46 using trifluoroacetic anhydride and triethylamine in 47% yield. Compound 46using which the heterocycle is constructed from the4.1.4, nitrile. The tetrazole is9)introduced by treating nitrile 47 then isobutylchloroformate, N-methylmorpholine, andSchemes aqueous ammonia. Primary amide is is guanylated [112] (see Sections 4.1.3 and 7 and to afford compound 47,45 from with sodium azide in the presence of zinc(II) bromide to afford compound 48a, using the click which to thenitrile heterocycle is constructed from the nitrile. Theand tetrazole is introduced by treating 47 converted 46 using trifluoroacetic anhydride triethylamine in 47% yield.nitrile Compound chemistry procedure by Demko and Sharpless [120]. Although the use48a, of zinc(II) bromide sodium azide (see inreported the presence of zinc(II) bromide to afford using the click 46 is with guanylated [112] Sections 4.1.3 and 4.1.4 Schemes 7 andcompound 9) to afford compound 47, from in the synthesis of Boc-amino tetrazoles derived from α-amino acids has been reported [121],bromide the use procedure reported by Demko and nitrile. Sharpless [120]. Although the use of zinc(II) whichchemistry the heterocycle isresult constructed from the The tetrazole is introduced byAmmonium treating nitrile of a Lewis acid may in partial deprotection of compound 48a during the reaction. in the synthesis of Boc-amino tetrazoles derived from α-amino acids has been reported [121], the use 47 with sodium azide in the presence of zinc(II) bromide to afford compound 48a, usingfrom the click chloride represents alternative reagent [122]. The 5-oxo-1,2,4-oxadiazole 48b is synthesized of a Lewis acid may an result in partial deprotection of compound 48a during the reaction. Ammonium chemistry procedure reported by Demko and Sharpless [120]. Although the use of zinc(II) bromide nitrile 47represents using hydroxylamine and5-oxo-1,2,4-oxadiazole sodium bicarbonate in followed by in chloride an alternative hydrochloride reagent [122]. The 48bDMSO, is synthesized from the synthesis ofusing Boc-amino tetrazoles derived fromand α-amino acids has been [121], thebyuse of treatment CDI and DBU [123]. Acidic deprotection of 48a–b is achieved asreported described previously nitrile 47 with hydroxylamine hydrochloride sodium bicarbonate in DMSO, followed [117,119]. Despite obtaining lower yields for heterocyclic adducts 49a–b (7%–13%) relative to a Lewis acid may result in partial deprotection of compound 48a during the reaction. Ammonium treatment with CDI and DBU [123]. Acidic deprotection of 48a–b is achieved as described previously bioisosteres 44a–c (19%–32%), the inhibitory potential of 5-oxo-1,2,4-oxadiazole ZST152 (49b) was chloride represents an alternative reagent [122]. The 5-oxo-1,2,4-oxadiazole 48b is synthesized [117,119]. Despite obtaining lower yields for heterocyclic adducts 49a–b (7%–13%) relative tofrom nitrilebioisosteres 47 using hydroxylamine hydrochloride and sodiumofbicarbonate in DMSO, followed by treatment 44a–c (19%–32%), the inhibitory potential 5-oxo-1,2,4-oxadiazole ZST152 (49b) was
with CDI and DBU [123]. Acidic deprotection of 48a–b is achieved as described previously [117,119]. Despite obtaining lower yields for heterocyclic adducts 49a–b (7%–13%) relative to bioisosteres 44a–c
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(19%–32%), the inhibitory potential of 5-oxo-1,2,4-oxadiazole ZST152 (49b) was significant (95% at 21, 615K = 7 µM), while tetrazole ZST086 (49a) displayed 91% inhibition at 111 of 31 with 1 mM,Molecules IC50 =2016, 18 µM, mM, i Molecules 2016, 21,K 615 11 of 31 IC50 = 34 µM and i = 14 µM [117]. significant (95% at 1 mM, IC50 = 18 μM, Ki = 7 μM), while tetrazole ZST086 (49a) displayed 91% 50 = 34 μM and Ki = 14 μM [117]. inhibition at 1 mM, IC (95% at 1with mM, IC 50 = 18 μM, Ki = 7 μM), while tetrazole ZST086 (49a) displayed 91% 4.1.6.significant N5 -(1-Iminoalk(en)yl)L -Ornithine Derivatives
inhibition at 1 mM, with IC50 = 34 μM and Ki = 14 μM [117]. 4.1.6. N5-(1-Iminoalk(en)yl)L-Ornithine Derivatives Amidines are commonly known bioisosteres of the guanidine moiety [124]. N5 -(1-iminoalk(en)yl)-L5 -(1-imino-3-butenyl)- L -ornithine (L-VNIO, 54a, Figure 2), are reported to 5-(1-Iminoalk(en)yl)ornithines asareNcommonly 4.1.6. Amidines Nsuch L-Ornithine Derivatives known bioisosteres of the guanidine moiety [124]. N5-(1-iminoalk(en)yl)-L5 inhibit NOS by competing with L -arginine for binding at moiety the Figure enzyme catalytic-site [125–127]. ornithines such ascommonly N -(1-imino-3-butenyl)L-ornithine (L-VNIO, 54a, reported to inhibit Amidines are known bioisosteres of the guanidine [124].2), N5are -(1-iminoalk(en)yl)LTherefore, of this class of have been extensively studied as DDAH L-arginine for compounds binding at the enzyme catalytic-site [125–127]. Therefore, NOS byderivatives competing with ornithines such as N5-(1-imino-3-butenyl)L-ornithine (L-VNIO, 54a, Figure 2), are reported to inhibit derivatives of synthetic this class compounds have extensively asL-ornithine DDAH inhibitors. The 54 inhibitors. approach to binding affordbeen N5the -(1-iminoalk(en)yl)derivatives L-arginine for at enzyme studied catalytic-site [125–127]. Therefore, NOS by The competing with of 5 synthetic approach to afford N -(1-iminoalk(en)yl)-ornithine derivatives (Scheme 12) [127,128], (Scheme 12) [127,128], similar approach to Lthe N-but-3-ynyl-2-chloro-acetamidine probe derivatives of this utilized class of acompounds have been extensively studied as54DDAH inhibitors. The (18) utilized4.1.2, aapproach similar approach to the N-but-3-ynyl-2-chloro-acetamidine probe (18) Section 4.1.2,ethyl synthetic to afford N5-(1-iminoalk(en)yl)54 to (Scheme 12) [127,128], (see Section Scheme 5) [109]. The R1 furnishedL-ornithine nitrile 50 derivatives is converted its(see corresponding Scheme a5)similar [109]. The R1 furnished nitrile 50 is converted to its corresponding ethyl imidic ester 4.1.2, 51 by utilized approach to the N-but-3-ynyl-2-chloro-acetamidine probe (18) (see Section imidic ester 51 by bubbling dry hydrochloric acid gas through a solution of anhydrous ethanol (16). bubbling hydrochloric acid gas through a solution to of its anhydrous ethanol (16).imidic The ethyl 1 Scheme 5) dry [109]. The nitrile 50 is converted corresponding ethyl esterimidic 51 by 53. The ethyl imidic ester 51Ris furnished then reacted with Nα -Boc-L-ornithine (52) to afford the protected adduct α-Boc-L-ornithine (52) to afford the protected adduct 53. Deprotection ester 51 is then reacted with N bubbling dry hydrochloric acid gas through a solution of anhydrous ethanol (16). The ethyl imidic 5 Deprotection is obtained by treatment with M achieve HCl to achieve the N -(1-iminoalk(en)yl)L-ornithine 5-(1-iminoalk(en)yl)is obtained byreacted treatment 6 M HCl6 to the Nthe -ornithine 54 in ester 51 is then withwith Nα-BocL-ornithine (52) to afford protected adductL53. Deprotection 54 in excellent excellentoverall overall yields (84%–94%). yields (84%–94%). 5
is obtained by treatment with 6 M HCl to achieve the N -(1-iminoalk(en)yl)-L-ornithine 54 in excellent overall yields (84%–94%).
Scheme 12. Synthetic scheme for generating N5-(1-iminoalk(en)yl)-L-ornithine derivatives. Reagents
Scheme 12. Synthetic scheme for generating N5 -(1-iminoalk(en)yl)-L-ornithine derivatives. Reagents and Conditions: (i) dry scheme HCl(g) atfor 0 °C for 1 h, stop HCl(g), then 0 °C for-ornithine 4 h and RT overnight; Reagents (ii) H2O, Scheme 12.(i) Synthetic N5-(1-iminoalk(en)yl)and Conditions: dry HCl 0 ˝ Cgenerating for 1 h, stop HCl(g) , then 0 ˝ CLfor 4 h andderivatives. RT overnight; (ii) H2 O, (g) at 5 °C, pH = 10, 1.5 h, adjust pH = °C 7, overnight, Dowex 50WX8-400 (H24Ohthen 10% aqueous pyridine); and Conditions: (i) dry HCl (g) at 0 for 1 h, stop HCl (g) , then 0 °C for and RT overnight; (ii) H2O, 5 ˝ C, (iii) pH 6= M 10,HCl, 1.5 h, adjust pH = 7, overnight, Dowex 50WX8-400 (H O then 10% aqueous pyridine); 2 with permission from [127]. EtOAc, 84%–94% from Nα-Boc-Dowex L-ornithine (52). Adapted 5 °C, pH = 10, 1.5 h, adjust pH = 7, overnight, 50WX8-400 (H2O then 10% aqueous pyridine); α -Boc- L (iii) 6Copyright M HCl, EtOAc, 84%–94% from N -ornithine (52). Adapted with permission from [127]. 1999 American Chemical Society. (iii) 6 M HCl, EtOAc, 84%–94% from Nα-Boc-L-ornithine (52). Adapted with permission from [127]. Copyright 1999 American Chemical Society. Copyright 1999 American Chemical Society.
Figure 2. N5-(1-Iminoalk(en)yl)-L-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c), as synthesized by Kotthaus et al. [114] and Wang et al. [128,129]. Figure 2. N5-(1-Iminoalk(en)yl)L-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c),
Figure 2. N5 -(1-Iminoalk(en)yl)-L-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c), as synthesized by Kotthaus et al. [114] and Wang et al. [128,129]. 5-(1-iminoalk(en)yl)as synthesized by Kotthaus et al. similarity [114] and Wang [128,129]. The significant structural of theetNal. L-ornithine derivatives with
DDAH (1) and L-NMMA groups to study this class of compounds Thesubstrates, significantADMA structural similarity of (2) theled N5different -(1-iminoalk(en)yl)L-ornithine derivatives with 5-(1-iminoalk(en)yl)-L5 as potential DDAH inhibitors [114,128]. Kotthaus et al. [114] examined five N DDAH substrates,structural ADMA (1) similarity and L-NMMA (2) led groups to study this class ofderivatives compounds with The significant of the N different -(1-iminoalk(en)yl)L -ornithine ornithines with varying side-chain and bond saturation for theirfive ability to inhibit L-citrulline as potential DDAH inhibitors Kotthaus et al. [114]different examined N5-(1-iminoalk(en)yl)LDDAH substrates, ADMA (1) [114,128]. andlengths L-NMMA (2) led groups to study this class of formation by human DDAH-1 using both high performance liquid chromatography and colorimetry. ornithines with varying side-chain lengths and bond saturation for their ability to inhibit L -citrulline compounds as potential DDAH inhibitors [114,128]. Kotthaus et al. [114] examined five L-VNIO (54a, FigureDDAH-1 2) emerged the high best performance DDAH-1 inhibitor of this series of derivatives (97% formation by human usingasboth liquid chromatography and colorimetry. N5 -(1-iminoalk(en)yl)L -ornithines with varying side-chain lengths and bond saturation for their inhibition at 1 mM, IC 50 = 13 μM, Ki = 2 μM). However, increasing the main-chain length by one L-VNIO (54a, Figure 2) emerged as the best DDAH-1 inhibitor of this series of derivatives (97% ability to inhibit L -citrulline formation by human DDAH-1 using both high performance liquid carbon atom lysine DDAH inhibitory potential. inhibition at 1from mM,ornithine IC50 = 13toμM, Ki reduced = 2 μM).itsHowever, increasing the main-chain length by one
chromatography and colorimetry. L-VNIO (54a, Figure 2) emerged as the best DDAH-1 inhibitor of carbon atom from ornithine to lysine reduced its DDAH inhibitory potential. this series of derivatives (97% inhibition at 1 mM, IC50 = 13 µM, Ki = 2 µM). However, increasing the main-chain length by one carbon atom from ornithine to lysine reduced its DDAH inhibitory potential.
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Wang et al. [128] investigated this class of compounds as dual DDAH-1 and NOS 5 inhibitors, Moleculesreporting 2016, 21, 615 the synthesis and kinetic parameters for four N -(1-iminoalkyl)- L -ornithines. 12 of 31 5 N -(1-Iminopropyl)-L-ornithine (L-IPO 54b, Figure 2) was identified as the best compound exhibiting et human al. [128]DDAH-1 investigated compounds as dual DDAH-1 and NOS inhibitors, inhibitionWang of both (Ki this = 52class µM) of and rat nNOS (K i = 3 µM). Wang et al. [129] additionally reporting the synthesis and kinetic parameters5 for four N5-(1-iminoalkyl)-L-ornithines. developed an irreversible human DDAH-1 inhibitor, N -(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO, N5-(1-Iminopropyl)-L-ornithine (L-IPO 54b, Figure 2) was identified as the best compound 54c, Figure 2), whose chemical structure reflects a combination of both L-IPO (54b) and exhibiting inhibition of both human DDAH-1 (Ki = 52 μM) and rat nNOS (Ki = 3 μM). Wang et al. 2-chloroacetamidine (11). Theansynthesis Cl-NIO (54c) inhibitor, is similar to that described for the [129] additionally developed irreversibleofhuman DDAH-1 N5-(1-imino-2-chloroethyl)5 α N -(1-iminoalk(en)yl)L -ornithine derivatives (Scheme 12). reflects Here, aNcombination -Boc-L-ornithine is reacted L-ornithine (Cl-NIO, 54c, Figure 2), whose chemical structure of both L-IPO with (54b) 2-chloro-1-ethoxyethanimine, intermediate is deprotected with 4.6 M HCl and 2-chloroacetamidine (11).and Thethe synthesis of Cl-NIOobtained (54c) is similar to that described for the L-ornithine (Scheme 12). Here, NαThe -Boc-inhibition L-ornithine potency is reactedofwith N5-(1-iminoalk(en)yl)in dioxane to generate Cl-NIO (54c,derivatives Figure 2) in low yields (10%). Cl-NIO and assay the intermediate obtained deprotected with 4.6 M HCl in (54c) 2-chloro-1-ethoxyethanimine, was assessed via colorimetric and exhibited Ki =is1.3 µM. In summary, several of the 5 dioxane to generate Cl-NIO (54c, Figure 2) in low yields (10%). The inhibition potency of Cl-NIO N -(1-iminoalk(en)yl)-L-ornithine derivatives (54a–c, Figure 2) are potent human DDAH-1 inhibitors. (54c) was assessed via colorimetric assay and exhibited Ki = 1.3 μM. In summary, several of the Off-target effects on the broader NO pathway (NOSs, arginases, AGTX-2) may either represent an N5-(1-iminoalk(en)yl)-L-ornithine derivatives (54a–c, Figure 2) are potent human DDAH-1 inhibitors. advantage (strong NO suppression), or limit their application (loss of NO physiological functions) as Off-target effects on the broader NO pathway (NOSs, arginases, AGTX-2) may either represent an potential therapeutics. advantage (strong NO suppression), or limit their application (loss of NO physiological functions) as potential therapeutics.
4.2. Non-Substrate-Like Inhibitors 4.2. Non-Substrate-Like Inhibitors
4.2.1. Pentafluorophenyl (PFP) Sulfonate Esters 4.2.1. Pentafluorophenyl (PFP) Sulfonate Esters Vallance et al. [130] identified pentafluorophenyl (PFP) sulfonate esters 61 as nonspecific PaDDAH and bacterial arginine (ADI)pentafluorophenyl inhibitors, after highlighting their structural similarity with Vallance et al.deaminase [130] identified (PFP) sulfonate esters 61 as nonspecific PaDDAH and bacterial deaminase (ADI) afterbehighlighting structural the cysteine protease familyarginine of enzymes. This class of inhibitors, inhibitor can obtained intheir moderate to good similarity with the cysteine protease family of enzymes. This class of inhibitor can be obtained in yields (44%–75%) via the 1,3-dipolar cycloaddition between nitrone 60 and the electron deficient moderate to good yields (44%–75%) via the 1,3-dipolar cycloaddition between nitrone 60 and the vinyl-appended PFP sulfonate 57 (Scheme 13) [131]. The shelf-stable PFP vinylsulfonate 57 is electron in deficient vinyl-appended 57 (Scheme (55) 13) and [131].2-chloroethane-1-sulfonyl The shelf-stable PFP synthesized moderate yield (59%) PFP fromsulfonate pentafluorophenol vinylsulfonate 57 is synthesized in moderate yield (59%) from pentafluorophenol (55) and chloride (56) [132], while various nitrones 60 can be generated from their corresponding aldehydes 58 2-chloroethane-1-sulfonyl chloride (56) [132], while various nitrones 60 can be generated from their by reaction with N-methyl hydroxylamine (59) under mild conditions and in good yield (for examples, corresponding aldehydes 58 by reaction with N-methyl hydroxylamine (59) under mild conditions pleaseand seein[133,134]). good yield (for examples, please see [133,134]).
Scheme 13. Synthetic scheme for generating PFP sulfonate esters 61. Reagents and Conditions: (i) DCM,
Scheme 13. Synthetic scheme for generating PFP sulfonate esters 61. Reagents and Conditions: (i) DCM, TEA, 0 °C, 59%; (ii) Method A: NaHCO3, anhydrous MgSO4, DCM, reflux, 24 h, 73%–90% [133]; TEA, 0 ˝ C, 59%; (ii) Method A: NaHCO3 , anhydrous MgSO4 , DCM, reflux, 24 h, 73%–90% [133]; Method B: NaOAc, EtOH, RT, 90% [134]; (iii) dry toluene, reflux, 1–24 h, 44%–75%. Reproduced in part Method B: NaOAc, EtOH, RT, 90% [134]; (iii) dry toluene, reflux, 1–24 h, 44%–75%. Reproduced in part from [130–133] with permission of the Royal Society of Chemistry and the American Chemical Society. from [130–133] with permission of the Royal Society of Chemistry and the American Chemical Society.
The electron deficiency of the vinyl PFP sulfonate dipolarophile 57 affords the reversed regioselective of the in compound 57 61,affords contrarytheto reversed the The electron 4C-substituted deficiency of adduct the vinyl PFPisoxazolidine sulfonate dipolarophile 5C-substituted adduct typically observed with isoxazolidine olefins. Although was previously observed for regioselective 4C-substituted adduct of the inthis compound 61, contrary to the electron deficient dipolarophiles [135–138], Caddick et al. [131] reported the 4C-substituted 5C-substituted adduct typically observed with olefins. Although this was previously observed isoxazolidine 61 is increasingly favoured at higher temperatures. Each of the nitrones 60 reported by for electron deficient dipolarophiles [135–138], Caddick et al. [131] reported the 4C-substituted Caddick et al. [131] participates in cycloaddition with the vinyl PFP sulfonate 57, including C-arylisoxazolidine 61 is increasingly higher temperatures. Each of the nitrones 60 reported and C-alkyl-N-methyl speciesfavoured with both at electron-donating and electron-withdrawing substituents in by Caddick et al. [131] participates in cycloaddition with the vinyl PFP sulfonate 57, including C-aryladdition to poly- and hetero-aromatic substituents. This robust synthetic methodology provides the and C-alkyl-N-methyl with both electron-donating andesters electron-withdrawing substituents ability to generate a species structurally diverse library of PFP sulfonate 61 with moderate ease and high in yields PFP sulfonate esters 61 have beenThis screened forsynthetic their activity as PaDDAH provides and ADI the addition to[130]. poly-Several and hetero-aromatic substituents. robust methodology
ability to generate a structurally diverse library of PFP sulfonate esters 61 with moderate ease and high yields [130]. Several PFP sulfonate esters 61 have been screened for their activity as PaDDAH and
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ADI inhibitors at two concentrations (500 µM and 50 µM), with five compounds emerging as13relatively Molecules 2016, 21, 615 of 31 potent PaDDAH inhibitors (IC50 = 16–58 µM, see [130] for structures). Vallance et al. revealed the modeinhibitors of inhibition competitive and reversibility experiments at twowas concentrations (500utilising μM andtime-dependence 50 μM), with five compounds emerging as relatively[130]. potent PaDDAH 50 = 61 16–58 μM, seeproposed [130] for structures). et al. revealed the Interestingly, the PFP inhibitors sulfonate (IC esters have been as potentialVallance antibacterial agents, however mode inhibition was identifying competitive utilising time-dependence and reversibility experiments [130]. no data hasofbeen reported if PaDDAH inhibition modulates bacterial growth or viability. Interestingly, theutilizing PFP sulfonate 61 have have been been reported proposedidentifying as potential antibacterial agents, Likewise, no studies humanesters DDAH whether the PFP sulfonate however no data has been reported identifying if PaDDAH inhibition modulates bacterial growth or esters exhibit specificity for bacterial PaDDAH. Non-specific PFP sulfonate ester binding to both human viability. Likewise, no studies utilizing human DDAH have been reported identifying whether the DDAH and PaDDAH would be counterproductive for their proposed application as antibiotics. PFP sulfonate esters exhibit specificity for bacterial PaDDAH. Non-specific PFP sulfonate ester binding to both human DDAH and PaDDAH would be counterproductive for their proposed application as 4.2.2. Indolylthiobarbituric Acid Derivatives antibiotics.
The indolylthiobarbituric acid derivatives 68 [139] were identified as PaDDAH inhibitors by 4.2.2. Indolylthiobarbituric Acid Derivatives a combination of virtual screening using a compound library comprising 308,000 structures and subsequent screening. acid Among the 10968highest scoring compounds, 90 were commercially Thebiological indolylthiobarbituric derivatives [139] were identified as PaDDAH inhibitors by a combination screening usingactivity a compound comprising 308,000 structures and available and usedofinvirtual colorimetric enzyme assays.library Three compounds consisting of two chemical subsequent biological screening. Among the 109 highest scoring compounds, 90 were commercially scaffolds were identified as potential PaDDAH inhibitors. One of these scaffolds, indolylthiobarbituric available and used in colorimetric activity 68b–c assays. identified Three compounds of two chemical acid (68a), together with additionalenzyme compounds from aconsisting similarity search, emerged scaffolds were identified as potential PaDDAH inhibitors. One of these scaffolds, indolylthiobarbituric as promising inhibitors of PaDDAH (IC50 = 2–16.9 µM). The most potent inhibitor of this series was acid (68a), together with additional compounds 68b–c identified from a similarity search, emerged SR445 (68c, IC50 = 2 µM), and no member of this class of compounds showed any human DDAH as promising inhibitors of PaDDAH (IC50 = 2–16.9 μM). The most potent inhibitor of this series was inhibition [114]. SR445 (68c, IC50 = 2 μM), and no member of this class of compounds showed any human DDAH This class[114]. of inhibitors is comprised of thiobarbituric acid module 64 and indolyl module 67 inhibition (Scheme 14). The moduleof64thiobarbituric is synthesized using a modified versionmodule of the method This classthiobarbituric of inhibitors isacid comprised acid module 64 and indolyl 67 described by Fisher and Mering in 1903, by reaction of N-substituted 2-thiourea 63 diethyl (Scheme 14). The thiobarbituric acid module 64 is synthesized using a modified version of thewith method described by Fisher The and indolyl Mering module in 1903, 67 by is reaction of N-substituted 2-thiourea 63 with diethyl malonate (62) [140,141]. alkylated by deprotonation of indole-3-carbaldehyde malonate (62)with [140,141]. The indolyl module 67 is alkylated by deprotonation of indole-3-carbaldehyde (65) and reaction 2-chloro-N-furan-2-ylmethyl-acetimide (66) using a method previously reported and[142]. reaction with 2-chloro-N-furan-2-ylmethyl-acetimide (66) using a67method previously reported (yield(65) 83%) The aldehyde substituent of the indolyl derivative is subsequently reacted with (yield 83%) [142]. The aldehyde substituent of the indolyl derivative 67 is subsequently reacted with the thiobarbituric acid derivative 64 under acidic conditions to afford 68 (yields not reported) [139,143]. the thiobarbituric acid derivative 64 under acidic conditions to afford 68 (yields not reported) This produced a mixture of (E)- and (Z)-isomers about the linking double bond. [139,143]. This produced a mixture of (E)- and (Z)-isomers about the linking double bond.
Scheme 14. Synthetic scheme for generating indolylthiobarbituric acid derivatives 68. Reagents and
Scheme 14. Synthetic scheme for generating indolylthiobarbituric acid derivatives 68. Reagents and Conditions: (i) NaOMe, MeOH, 60 °C, 6 h, yields not reported; (ii) NaH, DMF, RT, overnight, 83%; Conditions: (i) NaOMe, MeOH, 60 ˝ C, 6 h, yields not reported; (ii) NaH, DMF, RT, overnight, 83%; (iii) 6 M HCl, EtOH, RT, 2–3 h, yields not reported. (iii) 6 M HCl, EtOH, RT, 2–3 h, yields not reported.
4.2.3. Ebselen
4.2.3. Ebselen
Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, 73), a selenazole heterocyclic compound with well-known antioxidant properties [144], was recently as a potent DDAH-1 inhibitor. with Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, 73),identified a selenazole heterocyclic compound This was achieved by utilizing a high throughput screening (HTS) assay of two commercial libraries well-known antioxidant properties [144], was recently identified as a potent DDAH-1 inhibitor. comprising 2446 compounds [145]. The HTS identified each compound’s ability to inhibit the This was achieved by utilizing a high throughput screening (HTS) assay of two commercial libraries conversion of the synthetic substrate, S-methyl-L-thiocitrulline (SMTC) to citrulline and methanethiol comprising 2446 compounds [145]. The HTS identified each compound’s ability to inhibit the by PaDDAH. Compounds exhibiting PaDDAH inhibitory potentials were validated for their ability conversion of the synthetic substrate, S-methyl-L-thiocitrulline (SMTC) to citrulline and methanethiol to inhibit human DDAH-1 using ADMA as the substrate. The quantification of inhibition was
by PaDDAH. Compounds exhibiting PaDDAH inhibitory potentials were validated for their ability to inhibit human DDAH-1 using ADMA as the substrate. The quantification of inhibition was measured using derivatized citrulline formation and colorimetric detection. Characterisation of PaDDAH and
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human DDAH-1 inhibition by ebselen was subsequently determined by dose-response, reversibility, measured using derivatized citrulline formation and colorimetric detection. Characterisation of and ESI-MS experiments. PaDDAH and human DDAH-1 inhibition by ebselen was subsequently determined by dose-response, reversibility, and ESI-MS experiments.available, it can be prepared via two main methods [146–148]. While ebselen (73) is commercially While ebselen (73) is commercially available, is it can be prepared main methods [146–148]. The method described in Scheme 15 [147,148] preferred due via to two its shorter reaction times and The method described in Scheme 15 [147,148] is preferred due to its shorter reaction times elementary design. This method involves the treatment of benzoic acid (69) with thionyl and chloride elementary design. This method involves the treatment of benzoic acid (69) with thionyl chloride and aniline to afford benzanilide (70) followed by ortho-lithiation to dianion 71 using n-butyl lithium. and aniline to afford benzanilide (70) followed by ortho-lithiation to dianion 71 using n-butyl lithium. Selenium is then inserted into the carbon-lithium bond of 71, forming dianion intermediate 72, whose Selenium is then inserted into the carbon-lithium bond of 71, forming dianion intermediate 72, cyclization to ebselen to (73) is promoted by copper(II) bromide oxidant. whose cyclization ebselen (73) is promoted by copper(II) bromide oxidant.
Scheme 15. Synthetic scheme for generating ebselen (73). Reagents and Conditions: (i) (1) DCM, Scheme 15. Synthetic scheme for generating ebselen (73). Reagents and Conditions: (i) (1) DCM, pyridine, pyridine, SOCl2, reflux, 2 h; (2) toluene, aniline, reflux, 3 h, 80%; (ii) dry THF, n-BuLi, nitrogen SOCl2 , reflux, 2 h; (2) toluene, aniline, reflux, 3 h, 80%; (ii) dry THF, n-BuLi, nitrogen atmosphere, atmosphere, 0 °C, 30 min, 76% (determined via methylation of 72); (iii) same solution as step (ii), Se, 0 ˝ C, 30 min, 76% (determined via methylation of 72); (iii) same solution as step (ii), Se, 0 ˝ C, 30 min; 0 °C, 30 min; (iv) same solution as step (iii), CuBr2, 30 min at −78 °C, then 2 h at RT, 63%. Adapted (iv) same solution as step (iii), CuBr2 , 30 min at ´78 ˝ C, then 2 h at RT, 63%. Adapted with permission with permission from [147], copyright 1989 American Chemical Society, and [148], copyright 2003 from The [147], copyright 1989 American Chemical Society, and [148], copyright 2003 The Pharmaceutical Pharmaceutical Society of Japan. Society of Japan. Ebselen is reported to irreversibly inhibit both PaDDAH (IC50 = 96 ± 2 nM) and human DDAH-1 (IC50 = 330 30 nM), forming a covalent selenosulfide bond with each enzyme’s catalytic Ebselen is ±reported to irreversibly inhibit both PaDDAH (IC50 = 96 ˘ 2 nM)respective and human DDAH-1 cysteine. In silico docking experiments suggest inhibitor binding is supported by π-stacking (IC50 = 330 ˘ 30 nM), forming a covalent selenosulfide bond with each enzyme’s respective catalytic interactions with Phe76 in addition to other polar interactions involving His162. In vitro experiments cysteine. In silico docking experiments suggest inhibitor binding is supported by π-stacking interactions demonstrated ebselen exhibits no effect on human DDAH-1 expression [145]. with Phe76 in addition to other polar interactions His162. In vitrostructure experiments demonstrated Ebselen is the most potent DDAH inhibitorinvolving with ‘non-substrate-like’ reported in the ebselen exhibits no effect on human DDAH-1 expression [145]. literature to date and demonstrates an anti-inflammatory response [149]. However, its application as Ebselen is the most potent with ‘non-substrate-like’ structure a DDAH inhibitor is limited due toDDAH its poor inhibitor DDAH selectivity. A wide range of molecular targetsreported are modulated by ebselen NADPH oxidase [150,151], H+/K+ ATPase transporter [152], However, Ca2+in the literature to dateincluding: and demonstrates an 2anti-inflammatory response [149]. and phospholipid-dependent protein [151], [153],selectivity. thioredoxin A reductase its application as a DDAH inhibitor is kinase limitedC due to lipoxygenase its poor DDAH wide range + + ATPase [154], and metallothionein [155]). Moreover, cellular toxicity in leukocytes and hepatocytes has of molecular targets are modulated by ebselen including: NADPH oxidase 2 [150,151], H /Kbeen reported, further reducing the likelihood of ebselen’s use in clinical practice [156,157]. 2+
transporter [152], Ca - and phospholipid-dependent protein kinase C [151], lipoxygenase [153], thioredoxin reductase [154], and metallothionein [155]). Moreover, cellular toxicity in leukocytes 4.2.4. 4-Hydroxy-2-Nonenal and hepatocytes has been reported, further reducing the likelihood of ebselen’s use in clinical 4-Hydroxy-2-nonenal (4-HNE, 76) is a cytotoxic aldehyde generated from lipid peroxidation practice [156,157].
during inflammation [158,159] and is abundant in the body. 4-HNE is highly reactive toward nucleophilic groups, such as thiol and imidazole moieties. Nucleophilic attack affords 4.2.4.proteinaceous 4-Hydroxy-2-Nonenal Michael adducts, whilst Schiff bases are formed with primary amines (e.g., lysine residues) [160–163]. 4-Hydroxy-2-nonenal (4-HNE, 76) for is athe cytotoxic generated from lipid[164–166]. peroxidation Different methods have been reported synthesisaldehyde of 4-HNE (76) and its derivatives Theinflammation method reported by Soulérand et al. is [166] is described to its4-HNE simplicity, versatility during [158,159] abundant in here the due body. is speed, highlyand reactive toward (Scheme 16). The synthesis is completed in a single cross-metathesis reaction between octen-3-ol (75) proteinaceous nucleophilic groups, such as thiol and imidazole moieties. Nucleophilic attack affords and adducts, 2-propenal (74) inSchiff the presence of Hoveyda-Grubbs 2nd generation ruthenium under a Michael whilst bases are formed with primary amines (e.g., lysine catalyst residues) [160–163]. nitrogen atmosphere and in dry oxygen-free solvent (Scheme 16). The reaction proceeds at room Different methods have been reported for the synthesis of 4-HNE (76) and its derivatives [164–166]. temperature for 25 h affording the (E)-isomer of 4-HNE (76) in 75% yield.
The method reported by Soulér et al. [166] is described here due to its simplicity, speed, and versatility (Scheme 16). The synthesis is completed in a single cross-metathesis reaction between octen-3-ol (75) and 2-propenal (74) in the presence of Hoveyda-Grubbs 2nd generation ruthenium catalyst under a nitrogen atmosphere and in dry oxygen-free solvent (Scheme 16). The reaction proceeds at room temperature for 25 h affording the (E)-isomer of 4-HNE (76) in 75% yield.
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Scheme 16. Synthetic scheme for generating 4-hydroxy-2-nonenal (4-HNE, 76). Reagents and Scheme 16. scheme for generating 4-hydroxy-2-nonenal (4-HNE,(4-HNE, 76). Reagents Conditions: Scheme 16.Synthetic Synthetic scheme for generating 4-hydroxy-2-nonenal 76). and Reagents and Conditions: (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.). (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.). Conditions: (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.).
4-HNE is reported to react with cysteine and histidine residues and it was this reactivity that
4-HNE is reported to react with andDDAH-1 histidineinhibitor residues andInitForbes’ was this reactivity that led Forbes et al. to investigate its usecysteine as a human [167]. experiments, DDAHetactivity was measured and radioisotope assays, withexperiments, 4-HNE its via useboth led Forbes al. to investigate investigate as acolorimetric human DDAH-1 inhibitor activity [167]. In Forbes’ showing dose-dependent inhibition (ICcolorimetric 50 = 50 μM) and near complete inhibition 500 μM. Mass both colorimetric and radioisotope 4-HNE DDAH activity was measured via both and radioisotope activity at assays, with 4-HNE spectrometry experiments elucidated the mechanism of inhibition, whereby the formation of μM.aMass showing dose-dependent inhibition (IC50 50 μM) µM) and and near near complete complete inhibition inhibition at 500 µM. 50 == 50 Michael adduct between 4-HNE and His173 of the DDAH-1 active-site occurs. spectrometryexperiments experimentselucidated elucidated mechanism of inhibition, whereby the formation of a spectrometry thethe mechanism of inhibition, whereby the formation of a Michael Michael adduct between 4-HNE and His173 of the DDAH-1 active-site occurs. adduct between 4-HNE and His173 of the DDAH-1 active-site occurs. 4.2.5. PD 404182 (PD 404182, 79), is a 4.2.5. PD 6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine 404182
commercially available heterocyclic pyrimidobenzothiazine and a suitable lead compound for SAR
6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine (PD 404182, 404182, 79), is a 6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine studies due to its simple synthetic methodology [168]. PD 404182 (79) is a (PD compound listed 79), in theis commercially available heterocyclic pyrimidobenzothiazine and a suitable lead compound for heterocyclic pyrimidobenzothiazine and a suitable lead compound for SAR Library ofavailable Pharmacologically Active Compounds (LOPAC) and is reported to have antibacterial SAR studies due to its simple methodology PD (79) is aassay compound [168,169] [170,171]synthetic activity. Ghebremariam et[168]. al. [61] utilized toin the studies due toand its antiviral simple synthetic methodology [168]. PD 404182 (79)404182 is aa colorimetric compound listed screen a Library library ofofover 1200Active compounds for theirCompounds ability to inhibit conversion of ADMA listed in of thePharmacologically Pharmacologically Active andtois have reported toto have Library Compounds (LOPAC) and(LOPAC) is the reported antibacterial L -citrulline and dimethylamine by recombinant human DDAH-1. Compounds with reasonable antibacterial antiviral [170,171] activity. Ghebremariam et al. [61]a utilized a colorimetric [168,169] and[168,169] antiviraland [170,171] activity. Ghebremariam et al. [61] utilized colorimetric assay to inhibitory potentials were subjected kinetic characterisation via fluorimetric using the synthetic assay toascreen a library of1200 over 1200 to compounds for their ability to inhibit the conversion of ADMA screen library of over compounds for their ability to inhibit theassay conversion of ADMA to substrate, S-methylthiocitrulline. Reversibility and ESI-MS studies were additionally undertaken. to L -citrulline and dimethylaminebybyrecombinant recombinanthuman human DDAH-1. DDAH-1. Compounds Compounds with with reasonable L-citrulline and dimethylamine reasonable PD 404182 was subsequently evaluated for its ability to reduce angiogenesis and to protect against inhibitory potentials were subjected to kinetic characterisation via fluorimetric assay using the synthetic subjected to kinetic characterisation synthetic lipopolysaccharide-induced NO production. PD 404182 (79) was found to be a potent irreversible substrate, S-methylthiocitrulline. Reversibility and ESI-MS studies were additionally undertaken. S-methylthiocitrulline. Reversibility studies wereasadditionally undertaken. competitive human DDAH-1 inhibitor (IC50 = 9and μM)ESI-MS and showed promise an anti-inflammatory PD 404182 was subsequently against and antiangiogenic agent. evaluated for its ability to reduce angiogenesis and to protect against lipopolysaccharide-induced NO production. production. PDvia 404182 (79) was was found irreversible lipopolysaccharide-induced NO PD 404182 (79) found to approaches be a potent potentgenerate irreversible The synthesis of PD404182 can be achieved two routes [172,173]. Both competitive human DDAH-1 inhibitor over (IC50 µM) and showed promise as an anti-inflammatory reasonable to high yields (61%–88%) synthetic steps. The first approach involves the 50several == 99 μM) anti-inflammatory reaction of 2-(2′-haloaryl)-tetrahydropyrimidine 77 with carbon disulfide in the presence of sodium and antiangiogenic agent. hydride, followed byPD404182 hydrolysis ofbethe derivative 78 [172,173]. andBoth subsequent The synthesis becarbamodithioate achieved via routes two routes Bothtreatment approaches synthesis ofofPD404182 cancan achieved via two [172,173]. approaches generate with cyanogen bromide to afford 79 (Scheme 17; Route A). Alternatively, the 2-(2′-haloaryl)generate reasonable to high yields (61%–88%) over severalsteps. synthetic steps. The first approach reasonable to high yields (61%–88%) over several synthetic The first approach involves the tetrahydropyrimidine 77 can1 be reacted with tert-butylisothiocyanate (80) in the presence of sodium involves reaction of 2-(2 -haloaryl)-tetrahydropyrimidine 77 with incarbon disulfide in the reaction ofthe 2-(2′-haloaryl)-tetrahydropyrimidine 77 with carbon disulfide the presence of sodium hydride to afford intermediate 81, followed by treatment with trifluoroacetic acid to afford 79 presence of sodiumbyhydride, followed hydrolysis of the carbamodithioate derivative 78 and hydride, followed hydrolysis of the by carbamodithioate derivative 78 and subsequent treatment (Scheme 17; Route B). subsequent treatment with afford 79 A). (Scheme 17; Routethe A).2-(2′-haloaryl)Alternatively, with cyanogen bromide to cyanogen afford 79bromide (Schemeto17; Route Alternatively, the 2-(21 -haloaryl)-tetrahydropyrimidine 77 can be reacted with tert-butylisothiocyanate in the tetrahydropyrimidine 77 can be reacted with tert-butylisothiocyanate (80) in the presence (80) of sodium presence of sodium hydride to afford intermediate 81, followed by treatment with trifluoroacetic hydride to afford intermediate 81, followed by treatment with trifluoroacetic acid to affordacid 79 to afford 17; 79 (Scheme (Scheme Route B).17; Route B).
Scheme 17. Synthetic scheme for generating PD 404182 (79). Reagents and Conditions: Route A: (i) DMF, NaH, CS2, argon atmosphere, 80 °C, 12 h, 88%; (ii) (1) 0.1 M NaOH, MeOH/H2O (9:1), reflux, 12 h; (2) anhydrous EtOH, BrCN, argon atmosphere, reflux, 2 h, 61% (two steps). Route B: (iii) DMF, NaH, argon atmosphere, 80 °C, 2 h, 62%; (iv) TFA, CHCl3, molecular sieves 4 Å, reflux, 1 h, 85%. Adapted with permission from [172,173]. Copyright 1975, 2010 American Chemical Society.
Scheme 17. 17. Synthetic Syntheticscheme schemeforfor generating 404182 Reagents and Conditions: (i) Scheme generating PDPD 404182 (79).(79). Reagents and Conditions: RouteRoute A: (i) A: DMF, ˝ 2 , argon atmosphere, 80 °C, 12 h, 88%; (ii) (1) 0.1 M NaOH, MeOH/H 2 O (9:1), reflux, 12 DMF, NaH, CS NaH, CS2 , argon atmosphere, 80 C, 12 h, 88%; (ii) (1) 0.1 M NaOH, MeOH/H2 O (9:1), reflux, 12 h; h; (2) anhydrous EtOH, BrCN, argon atmosphere, reflux, 2 h, 61% steps). (iii) DMF, (2) anhydrous EtOH, BrCN, argon atmosphere, reflux, 2 h, 61% (two (two steps). RouteRoute B: (iii)B:DMF, NaH, 3, molecular sieves 4 Å, reflux, 1 h, 85%. NaH, argon atmosphere, 2 h, 62%; (iv) TFA, CHCl argon atmosphere, 80 ˝ C, 280h,°C, 62%; (iv) TFA, CHCl , molecular sieves 4 Å, reflux, 1 h, 85%. Adapted 3 Adapted with permission from [172,173]. 2010 American with permission from [172,173]. CopyrightCopyright 1975, 20101975, American Chemical Chemical Society. Society.
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4.2.6. Proton Pump Inhibitors
4.2.6. Proton Pump Inhibitors
+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric Molecules 2016, 21, 615 16 of 31 acid Proton H+ /K Proton H+/K+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric acid secretion and are characterized by a 2-pyridylmethylsulfinylbenzimidazole scaffold. The chemical secretion and are characterized by a 2-pyridylmethylsulfinylbenzimidazole scaffold. The chemical 4.2.6. Proton Pump Inhibitors structures of the prescribed inFigure Figure3.3. structures of commonly the commonly prescribedPPIs PPIs82a–f 82a–f are are shown shown in
Proton H+/K+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric acid secretion and are characterized by a 2-pyridylmethylsulfinylbenzimidazole scaffold. The chemical structures of the commonly prescribed PPIs 82a–f are shown in Figure 3.
Figure 3. Chemical structures of commonly prescribed proton pump inhibitors (PPIs) 82a–f.
Figure 3. Chemical structures of commonly prescribed proton pump inhibitors (PPIs) 82a–f.
HTS experiments utilising a library of 130,000 compounds and recombinant enzyme identified
HTS experiments utilising a library of 130,000 compounds and recombinant identified Figure 3.DDAH-1 Chemical structures of commonly prescribed proton pump inhibitors using (PPIs)enzyme 82a–f. PPIs as human inhibitors [174,175]. Initial screening was undertaken a colorimetric assay with ADMA as the substrate. Further characterization of compounds of interest was achieved PPIs as human DDAH-1 inhibitors [174,175]. Initial screening was undertaken using a colorimetric experiments a library of 130,000 compounds and recombinant enzyme (SPR) identified aHTS fluorimetric using S-methylthiocitrulline [174,175]. of Surface plasmonof resonance was assayvia with ADMA asassay the utilising substrate. Further characterization compounds interest was achieved PPIs as human DDAH-1 inhibitors [174,175].ofInitial screening was undertaken using a sensor colorimetric additionally utilized to detect the binding PPIs to amine-coupled DDAH-1 (CM5 chip) via a fluorimetric assay using S-methylthiocitrulline [174,175]. Surface plasmon resonance (SPR) was assay ADMA as theavailable substrate. Further of compounds of interest achieved [175]. with All commercially PPIs werecharacterization found to reversibly inhibit DDAH-1 (IC50 was = 51–63 μM). additionally utilized to detect the binding of PPIs to amine-coupled DDAH-1 (CM5 sensor chip) [175]. via a fluorimetric assay using S-methylthiocitrulline [174,175]. Surface plasmon resonance (SPR) was Interestingly, in vitro experiments using human endothelial cells and in vivo experiments in mice All commercially available PPIsthewere found to to reversibly inhibit DDAH-1 (IC = 51–63 µM). 50 additionally utilized to detect binding of PPIs amine-coupled DDAH-1 (CM5 sensor chip) demonstrated that PPIs increase ADMA concentrations [175] further supporting the evidence that Interestingly, inuse vitro experiments using human cells and indisease vivo (IC experiments in mice [175]. All commercially PPIs were foundendothelial to risk reversibly inhibit DDAH-1 50 = 51–63 μM). prolonged of PPIs isavailable associated with an increased of cardiovascular [176,177]. demonstrated that PPIs increase ADMA concentrations [175] further supporting the evidence Interestingly, in vitro experiments using human endothelial cells and in vivo experiments in mice PPIs are comprised of pyridine and benzimidazole moieties with different substituents. Their that demonstrated that is PPIs increase ADMA [175] further supporting the evidence that prolonged use are of PPIs associated with anconcentrations increased of cardiovascular disease [176,177]. syntheses protected by current patents (with therisk exception of omeprazole (82a)). The original prolonged use of PPIs is associated with an increased risk of cardiovascular disease [176,177]. PPIs pyridine and benzimidazole moieties with different substituents. patentare forcomprised omeprazoleof (82a) [178] details preparation of the 2-(chloromethyl)pyridine derivative 84.Their PPIs comprised of pyridine andwith benzimidazole moieties of with different substituents. Their Condensation [179] of 84 2-mercaptobenzimidazole derivative 83 affords the syntheses are are protected bycompound current patents (with the exception omeprazole (82a)). The original syntheses are protected by current patents (with the exception of omeprazole (82a)). The original thioether precursor (82a) 85, which subsequently oxidized sulfoxide in omeprazole (82a) using 84. patent for omeprazole [178]isdetails preparation of to thethe 2-(chloromethyl)pyridine derivative patent for omeprazole (82a) [178] details preparation ofcatalyst the 2-(chloromethyl)pyridine derivative 84. conditions such of as compound hydrogen peroxide a vanadium [180] derivative (Scheme 18;83 Route A). the thioether Condensation [179] 84 withand 2-mercaptobenzimidazole affords Condensation [179] of compound 84 with 2-mercaptobenzimidazole derivative 83 affords the precursor 85, which is subsequently oxidized to the sulfoxide in omeprazole (82a) using conditions thioether precursor 85, which is subsequently oxidized to the sulfoxide in omeprazole (82a) using such conditions as hydrogen andperoxide a vanadium [180] (Scheme Route18; A). suchperoxide as hydrogen and acatalyst vanadium catalyst [180] 18; (Scheme Route A).
Scheme 18. Synthetic scheme for generating omeprazole (82a). Reagents and Conditions: Route A: (i) 1 M NaOH (2 eq.), EtOH, RT, 8 h; (ii) catalyst: V2O5, NaVO3, NH4VO3, [(CH3COCH2COCH3)2VO] (0.0001–0.1 eq.), H2O2: 10%–70% aqueous or organic solution (1–3 eq.), solvent: halogenated Scheme Synthetic forketones, generating omeprazole Reagents0and Route of A:the (i) temperature: °C Conditions: to boiling point hydrocarbons, ethers, scheme alcohols, nitriles, or H2O, (82a). Scheme 18. 18. Synthetic scheme for generating omeprazole (82a). Reagents and Conditions: Route A: 1solvent, M NaOH (2 eq.), EtOH, RT,Route 8 h; (ii) V2O5, NaVO 3, NH4VOnot 3, [(CH 3COCHin 2COCH 3)2VO] 0.5–24 h, 89%–93%. B: catalyst: (iii) condensation, conditions specified patent; (iv) (i) 1 M NaOH (2 eq.), EtOH, RT, 8 h; (ii) catalyst: V2 O5 , NaVO3 , NH4 VO3 , [(CH3 COCH2 COCH3 )2 VO] (0.0001–0.1 H23O : 10%–70% aqueous solvent: halogenated [(CH3COCHeq.), 2COCH )22VO] (0.006 eq.), 30% H2or O2, organic acetone, solution 0–5 °C for(1–3 1 h eq.), then 20–22 °C for 1 h, 90%; (0.0001–0.1 eq.), H2 O2 : 10%–70% aqueous or organic solution (1–3 eq.), solvent: halogenated temperature: °Cv/v to isopropanol/toluene, boiling point of the hydrocarbons, alcohols, ketones, nitriles, or CO H2O, (v) 10% NaOH,ethers, nitrogen atmosphere, 50 °C, 3 h; (vi) 2, extraction into01:1 hydrocarbons, ethers, alcohols, ketones, nitriles, or H2 O,conditions temperature: 0 ˝ C toinboiling of solvent, 0.5–24 h, 42% 89%–93%. B: and (iii) condensation, not specified patent; point (iv) reflux, 20–30 min, (acrossRoute steps (v) (vi)). the solvent, 0.5–24 h, 89%–93%. Route B: (iii) condensation, conditions not specified in patent; [(CH3COCH2COCH3)2VO] (0.006 eq.), 30% H2O2, acetone, 0–5 °C for 1 h then 20–22 °C for 1 h, 90%; (iv) [(CH (0.006 eq.), 30% H2(vi) O2 CO , acetone, 0–5 ˝into C for1:11 h 20–22 ˝ C for 1 h, 90%; (v) 10% NaOH, nitrogen atmosphere, 50 °C, 3 h; 2, extraction v/vthen isopropanol/toluene, 3 COCH 2 COCH 3 )2 VO] ˝ (v) 10% NaOH, C, 3(vi)). h; (vi) CO2 , extraction into 1:1 v/v isopropanol/toluene, reflux, 20–30nitrogen min, 42%atmosphere, (across steps 50 (v) and
reflux, 20–30 min, 42% (across steps (v) and (vi)).
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Further development of the synthetic method to yield omeprazole (82a) [180] identified that the Molecules 2016, 21, the 615 pyridine and benzimidazole moieties can be modified to simplify purification. 17 of 31 coupling between Principally, this method avoids the formation of the thioether intermediate 85. Furthermore, the crude Further development of the synthetic method to yield omeprazole (82a) [180] identified that the product containing omeprazole (82a) produced from the original synthesis is prone to discolouration coupling between the pyridine and benzimidazole moieties can be modified to simplify purification. during the oxidation step [180]. The revised synthesis (Scheme 18; Route B) proceeds via condensation Principally, this method avoids the formation of the thioether intermediate 85. Furthermore, the of 2-chloromercaptobenzimidazole derivative 86 with afrom primary amide pyridine crude product containing omeprazole (82a) produced the original synthesis derivative is prone to87 to afford the acetamide thioether intermediate 88, which is oxidized to the acetamide sulfoxide 89. discolouration during the oxidation step [180]. The revised synthesis (Scheme 18; Route B) proceeds Compound 89 is subsequently hydrolysed to the carboxylic acid salt 90 under alkaline conditions, via condensation of 2-chloromercaptobenzimidazole derivative 86 with a primary amide pyridine with derivative 87yielding to affordomeprazole the acetamide thioether intermediate(82c), 88, which is oxidized to the acetamide 86 decarboxylation (82a) or lansoprazole depending on the benzimidazole sulfoxide 89. Compound 89 isused subsequently hydrolysed to the carboxylic acid salt 90 under alkaline and the pyridine 87 derivatives as the starting materials. conditions, with decarboxylation yielding omeprazole (82a) or lansoprazole (82c), depending on the 86 and the pyridine 87 derivatives used as the starting materials. 4.2.7.benzimidazole 1,2,3-Triazoles
The of 1,2,3-triazoles 95 (Scheme 19) comprised of 4-halopyridine and benzimidazole 4.2.7.synthesis 1,2,3-Triazoles units have been explored as ‘non-substrate-like’ DDAH-1 inhibitors [117]. The synthesis of these The synthesis of 1,2,3-triazoles 95 (Scheme 19) comprised of 4-halopyridine and benzimidazole derivatives was undertaken based on reports that identified binding of both the 4-halopyridine and units have been explored as ‘non-substrate-like’ DDAH-1 inhibitors [117]. The synthesis of these benzimidazole fragments to human DDAH-1 [181]. A triazole linker wasthe selected to connect derivatives was undertaken based on reports that identified binding of both 4-halopyridine andthese fragments via copper(I)-catalysed ‘click’ cycloaddition between thewas terminal 92 and benzimidazole fragments to human DDAH-1 [181]. A triazole linker selectedalkyne to connect theseazide 94 (Scheme 19). alkyne 92 is synthesized by Sonogashira of the92alkyne derivative fragments via The copper(I)-catalysed ‘click’ cycloaddition between thecoupling terminal alkyne and azide 94 with (Scheme the required species 91by[182,183]. The azideof94theisalkyne synthesized bywith reaction of 19). Thehalogenated alkyne 92 is synthesized Sonogashira coupling derivative the required halogenated species 91 [182,183]. The azide 94 is synthesized by reaction of diphenylphosphoryl azide with the required primary alcohol 93 [184]. diphenylphosphoryl azide with albeit the required primary alcohol 93 [184]. Human DDAH-1 inhibition, low (IC 50 = 422 µM, 57%, inhibition at 1 mM), was reported Human DDAH-1 inhibition, albeit low (IC 50 = 422 μM, 57%, inhibition at 1 mM), was reported with triazole 95a. In general, 95a–b failed to show significant inhibitory potential and may be due to with triazole 95a. In general, 95a–b failed to show significant inhibitory potential and may be due to the triazole linker not facilitating a productive inhibitory binding conformation of the fragments in the the triazole linker not facilitating a productive inhibitory binding conformation of the fragments in enzyme active-site. the enzyme active-site.
Scheme 19. Synthetic scheme for generating 1,2,3-triazoles 95. Reagents and Conditions: (i) 92a
Scheme 19. Synthetic scheme for generating 1,2,3-triazoles 95. Reagents and Conditions: (i) 92a (Ph3P)2PdCl2, CuI, TEA, TIPS-acetylene, DMF, 80 °C, 72 h, 30%, 92b (Ph3P)2PdCl2, CuI, TEA, ethynyl-TMS, (Ph3 P)2 PdCl2 , CuI, TEA, TIPS-acetylene, DMF, 80 ˝ C, 72 h, 30%, 92b (Ph3 P)2 PdCl2 , CuI, TEA, 80 °C, 20 h, 48%; (ii) DPPA, DBU, toluene, RT, 16 h, 94a 55%, 94b 83%; (iii) CuI, sodium ascorbate, ethynyl-TMS, 80 ˝ C, 20 h, 48%; (ii) DPPA, DBU, toluene, RT, 16 h, 94a 55%, 94b 83%; (iii) CuI, sodium TEA, DCM, RT, 24 h, 95a 65%, 95b 30%. Reproduced in part from [117] with permission of the Royal ascorbate, DCM, RT, 24 h, 95a 65%, 95b 30%. Reproduced in part from [117] with permission of SocietyTEA, of Chemistry. the Royal Society of Chemistry. 5. Conclusions
5. Conclusions Current evidence suggest that inhibiting the DDAH family of enzymes may be used as a therapeutic tool to indirectly the NOS enzymes via increasing of endogenous Current evidence suggestinhibit that inhibiting the DDAH family concentrations of enzymes may be used as a NOS inhibitors, such as ADMA and L-NMMA. Therefore the development and synthesis of DDAH therapeutic tool to indirectly inhibit the NOS enzymes via increasing concentrations of endogenous inhibitors is emerging as a promising field of clinical research. Furthermore, engineering compounds NOS inhibitors, such as ADMA and L-NMMA. Therefore the development and synthesis of DDAH that target bacterial DDAH may represent a suitable strategy for the development of new inhibitors is emerging as a promising field of clinical research. Furthermore, engineering compounds antimicrobial agents; however further investigations are required. This has led to different classes of that target DDAH represent a suitable strategycharacterized for the development of new antimicrobial DDAHbacterial inhibitors beingmay reported within the last decade by alternative features in agents; however further investigations are required. This has led to different classes of DDAH terms of chemical structure, potency, enzyme selectivity, and mechanism of action. The importance inhibitors reported within depends the last on decade characterized features in terms of placedbeing on each of these features the end application of by the alternative inhibitor. Table 3 summarizes the most potent DDAH inhibitors themechanism literature to date. Some compounds, namely chemical structure, potency, enzymedocumented selectivity,inand of action. The importance placed PFPof sulfonate esters 61depends or indolylthiobarbituric acid derivatives exhibit Table significant PaDDAH the on each these features on the end application of the 68, inhibitor. 3 summarizes most potent DDAH inhibitors documented in the literature to date. Some compounds, namely PFP
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sulfonate esters 61 or indolylthiobarbituric acid derivatives 68, exhibit significant PaDDAH inhibition. In particular, selectivity experiments using indolylthiobarbiturates failed to exhibit inhibitory effects on the human isoenzyme, therefore derivatives of this structural class are well positioned for further investigation in antibiotic applications. Among classes of molecules which are active on the human isoenzyme (amino acid derivatives 27, 29, 39, 40, 44, 49, 54, ebselen (73), 4-HNE (76), PD 404182 (79), PPIs 82, 1,2,3-triazoles 95), L-257 (39) and L-291 (40) are highly selective for DDAH-1, while ornithine derivatives such as L-VNIO (54a) or L-IPO (54b) are known to inhibit not only DDAH, but also arginase and the NOS family of enzymes. Furthermore, compounds such as ebselen (73) and 4-HNE (76), although very potent DDAH inhibitors, are well known to exhibit a range of off-target effects, thus are more suitable for experiments in vitro rather than in vivo. In terms of the varied approaches to chemical synthesis of the DDAH inhibitors discussed here, many can be synthesized in moderate to excellent overall yields and use reaction sequences that can be easily modified to enable the design of a large number of analogues. The commercial availability of several of the compounds determined to be DDAH inhibitors (2-chloroacetamidine (11), ebselen (73) 4-HNE (76) and the PPIs 82) demonstrates the versatility of some of the synthetic routes for large scale production. Based on the in vitro pharmacokinetic and/or enzyme selectivity data compiled in this review, there appear to be three promising human DDAH-1 inhibitors that have the potential to progress to clinical trial: ZST316 (Ki = 1 µM), Cl-NIO (Ki = 1.3 µM), and L-257 (Ki = 13 µM) (Table 3). Despite exhibiting the most potent inhibitory response in vitro, a paucity of data exists with respect to the in vivo pharmacokinetic and pharmacodynamic profiling of ZST316. Literature reports involving the enzyme selectivity of Cl-NIO shows considerable promise, with no measurable cross-reactivity in experiments specifically involving eNOS [129]. However, data are not available regarding this inhibitor‘s interactions with iNOS, nNOS, or the arginase family of enzymes. Contrary to this, data reported for L-257 by Kotthaus et al. [115] demonstrates that L-257 is highly selective for DDAH-1. The administration of L-257 in a rodent model of septic shock not only improved survival, but additionally improved haemodynamic and organ function [67], indicating a potential clinical role for L-257. Interestingly, little is understood about the regulation and pharmacological modulation of human DDAH-2. As such, the development of improved metabolomic approaches for the sensitive measurement of DDAH-2 activity in vitro is an important step forward in profiling the endogenous substrates and biochemical role(s) of this important enzyme in humans. The continued exploration of DDAH inhibition and the synthesis of new chemical entities with improved pharmacokinetic parameters are therefore at an exciting junction in the development of novel medicines.
Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Molecules 2016, 21, 615 Chemical Chemical Molecules 2016,Chemical 21, 615 Name
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Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.
Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. Chemical Enzyme IC5050(μM) (µM) Table 3. Summary of theStructure main DDAH inhibitors and their kinetic parameters. Chemical Name Chemical Structure Enzyme IC Chemical Number Table 3. Summary of theStructure main DDAH inhibitors and their kinetic parameters. Chemical Number Chemical Name Chemical Enzyme IC50 (μM) Table 3. Summary of theStructure main DDAH inhibitors and their kinetic parameters. Chemical Chemical Name Chemical Enzyme IC 50 (μM) Number Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. Chemical Chemical Structure Enzyme IC50 (μM) Number Chemical Name Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. Chemical Chemical Structure Enzyme IC50 (μM) Chemical Number L-homocysteine S-Nitroso-Name Chemical Number Chemical Name Chemical Structure Enzyme IC50 (μM) (9) Bovine DDAH-1 75 L -homocysteine S-Nitroso(9) Chemical S-NitrosoL -homocysteine (HcyNO) Bovine DDAH-1 75 Chemical Name Chemical Structure Enzyme IC50 (μM) Number L -homocysteine S-Nitroso(HcyNO) Name (9) Bovine DDAH-1 Chemical Chemical Chemical Structure Enzyme IC5075 (μM) Number LName -homocysteine S-Nitroso(9) Bovine DDAH-1 (HcyNO) Chemical Chemical Structure Enzyme IC 5075 (μM) Number S-Nitroso(9) Bovine DDAH-1 75 (HcyNO) L-homocysteine Number (9) Bovine DDAH-1 75 S-Nitroso-L-homocysteine (HcyNO) S-Nitroso-L-homocysteine (HcyNO) (9) Bovine DDAH-1 75 1 L-homocysteine S-Nitroso(9) Bovine DDAH-1 75(HcyNO) (11) 2-Chloroacetamidine PaDDAH 11 S-Nitroso-L-homocysteine (9) 2-Chloroacetamidine Bovine DDAH-1 75 (HcyNO) -2-Chloroacetamidine PaDDAH (11) (11) 1 (9) Bovine DDAH-1 75 (HcyNO) (11) 2-Chloroacetamidine PaDDAH1 (HcyNO) (11) 2-Chloroacetamidine PaDDAH 1 (11) 2-Chloroacetamidine PaDDAH 1 N-But-3-ynyl-2-chloro-acetamid (11) 2-Chloroacetamidine PaDDAH (18) Human DDAH-1 350 N-But-3-ynyl-2-chloro-acetamid 1 (11) 2-Chloroacetamidine PaDDAH N-But-3-ynyl-2-chloro-acetamid 1 ine (18) Human DDAH-1 350 2-Chloroacetamidine PaDDAH N-But-3-ynyl-2-chloro-acetamid 1 (18) N-But-3-ynyl-2-chloro-acetamidine DDAH-1 350 (18) (11) Human DDAH-1 350 ine (11) 2-Chloroacetamidine PaDDAH N-But-3-ynyl-2-chloro-acetamid (18) Human DDAH-1 350 ine NH NH2 (18) Human DDAH-1 350 N-But-3-ynyl-2-chloro-acetamid ine NH NH 2 N-But-3-ynyl-2-chloro-acetamid ine (18) Human DDAH-1 350 (S)-2-Amino-4-(3-methylguanid NH NH2 OH N-But-3-ynyl-2-chloro-acetamid (18) Human DDAH-1 350 ine (21) Rat DDAH-1 416 (S)-2-Amino-4-(3-methylguanid NH2 OH N NH N N-But-3-ynyl-2-chloro-acetamid (18) Human DDAH-1 350 ine (S)-2-Amino-4-(3-methylguanid ino)butanoic acid (4124W) (21) Rat DDAH-1 416 NH NH NH2 H OH N (18) Human DDAH-1 350 ine (S)-2-Amino-4-(3-methylguanid (21) (S)-2-Amino-4-(3-methylguanidino) Rat DDAH-1 416 ino)butanoic acid (4124W) NH NH OH N N O H H 2 ine (S)-2-Amino-4-(3-methylguanid Rat 416 ino)butanoic acid (4124W) (21) (21) RatDDAH-1 DDAH-1 416 NH2O OH NH NHNH (21) butanoic Rat DDAH-1 416 (S)-2-Amino-4-(3-methylguanid ino)butanoic acid (4124W) acid (4124W) NH2O OH N NHH N H (S)-2-Amino-4-(3-methylguanid ino)butanoic acid (4124W) (21) Rat DDAH-1 416 NH NH H H OH G N N O 2 NG -(2-Methoxyethyl)L-arginine (S)-2-Amino-4-(3-methylguanid (21) Rat DDAH-1 416 2 ino)butanoic acid (4124W) N N O OH H H (S)-2-Amino-4-(3-methylguanid (39) Human DDAH-1 31 N -(2-Methoxyethyl)L-arginine (21) Rat DDAH-1 416 ino)butanoic acid (4124W) N N H H G-(2-Methoxyethyl)O OH N L-arginine (21) Rat DDAH-1 41622 (L-257) (39) Human DDAH-1 31 ino)butanoic acid (4124W) N N H H G-(2-Methoxyethyl)O N L -arginine (39) G (L-257) Human DDAH-1 312 ino)butanoic acid (4124W) H H G O N(L-257) -(2-Methoxyethyl)L-arginine (39) N -(2-Methoxyethyl)Human DDAH-1 31 2 2 L -arginine O Human 31312 NGG-(2-Methoxyethyl)-L-arginine (L-257) (39) (39) HumanDDAH-1 DDAH-1 N GG-(2-Methoxyethyl)-L-arginine (L-257) (39) (L-257) Human DDAH-1 31 2 N -(2-Methoxyethyl)LL-arginine (39) Human DDAH-1 31 2 (L-257) N -(2-Methoxyethyl)-arginine G NG-(2-Methoxyethyl)-(2-Methoxyethyl)-LL-arginine -arginine (39) Human DDAH-1 31 (L-257) (40) Rat DDAH-1 20 N G-(2-Methoxyethyl)-L-arginine (39) Human DDAH-1 31 (L-257) N methyl ester (L-291) (40) Rat DDAH-1 20 2 G (L-257) Nmethyl -(2-Methoxyethyl)L-arginine (40) Rat DDAH-1 20 ester (L-291) G Nmethyl -(2-Methoxyethyl)(40) Rat DDAH-1 20 ester (L-291) L-arginine (40) NG -(2-Methoxyethyl)Rat DDAH-1 20 NGG-(2-Methoxyethyl)L-arginine methyl ester (L-291) L -arginine N2-Amino-5-(3-(2-methoxyethyl) -(2-Methoxyethyl)methyl ester (L-291) L-arginine Rat DDAH-1 2020 (40) (40) Rat DDAH-1 G 2-Amino-5-(3-(2-methoxyethyl) N Gester -(2-Methoxyethyl)L-arginine (40) methyl Rat DDAH-1 20 methyl ester (L-291) (L-291) 2-Amino-5-(3-(2-methoxyethyl) N -(2-Methoxyethyl)(44a) guanidino)-N-(methylsulfonyl)Human DDAH-1 3 (40) Rat DDAH-1 20 methyl ester (L-291) L-arginine 2-Amino-5-(3-(2-methoxyethyl) (40) Rat DDAH-1 20 (44a) guanidino)-N-(methylsulfonyl)Human DDAH-1 3 methyl ester (L-291) 2-Amino-5-(3-(2-methoxyethyl) (44a) guanidino)-N-(methylsulfonyl)Human DDAH-1 3 methyl ester (L-291) pentanamide (ZST316) 2-Amino-5-(3-(2-methoxyethyl) (44a) guanidino)-N-(methylsulfonyl)Human DDAH-1 3 pentanamide (ZST316) 2-Amino-5-(3-(2-methoxyethyl) (44a) 2-Amino-5-(3-(2-methoxyethyl) guanidino)-N-(methylsulfonyl)Human DDAH-1 3 pentanamide (ZST316) 2-Amino-5-(3-(2-methoxyethyl) (44a) guanidino)-N-(methylsulfonyl)Human DDAH-1 3 pentanamide (ZST316) 5 2-Amino-5-(3-(2-methoxyethyl) N5 -(1-Imino-3-butenyl)guanidino)-N-(methylsulfonyl)Human DDAH-1 33 pentanamide (ZST316) L-ornith (44a) (44a) guanidino)-N-(methylsulfonyl)Human DDAH-1 (54a) Human DDAH-1 DDAH-1 13 -(1-Imino-3-butenyl)N (44a) guanidino)-N-(methylsulfonyl)Human 3 pentanamide (ZST316) LL-ornith 5-(1-Imino-3-butenyl)-ornith N (44a) pentanamide guanidino)-N-(methylsulfonyl)Human DDAH-1 3 ine (L-VNIO) (54a) Human DDAH-1 13 (ZST316) pentanamide (ZST316) L-ornith 5-(1-Imino-3-butenyl)N (54a) Human DDAH-1 13 ine (L-VNIO) pentanamide (ZST316) 5 -(1-Imino-3-butenyl)L-ornith Npentanamide (54a) Human DDAH-1 13 ine (L-VNIO) (ZST316) (54a) Human DDAH-1 13 L-ornith N55-(1-Imino-3-butenyl)ine (L-VNIO) L-ornith N 5-(1-Imino-3-butenyl)(L-VNIO) (54a) 5 ine Human DDAH-1 13 5-(1-Imino-3-butenyl)L-ornith N (54a) N -(1-Imino-3-butenyl)Human DDAH-1 13 L -ornithine ine (L-VNIO) N -(1-Iminopropyl)L-ornithine 5 -(1-Imino-3-butenyl)L-ornith N5-(1-Iminopropyl)Human DDAH-1 13 (54a) (54a) 13 ine (L-VNIO) (54b) Human DDAH-1 N L -ornithine 5-(1-Iminopropyl)(54a) (L-VNIO) Human DDAH-1 DDAH-1 13 ine (L-VNIO) N L-ornithine (L-IPO) (54b) Human 5 ine (L-VNIO) N(L-IPO) -(1-Iminopropyl)L-ornithine (54b) Human DDAH-1 5 N(L-IPO) -(1-Iminopropyl)-L-ornithine (54b) Human DDAH-1 (54b) Human DDAH-1 N55-(1-Iminopropyl)-L-ornithine (L-IPO) N 5-(1-Iminopropyl)L-ornithine (L-IPO) (54b) Human DDAH-1 5-(1-Iminopropyl)N L-ornithine (54b) 5 (L-IPO) Human DDAH-1 N -(1-Imino-2-chloroethyl)L-or 5 N5-(1-Imino-2-chloroethyl)-(1-Iminopropyl)L-ornithine (54b) Human -(L-IPO) L -ornithine (54c)N -(1-Iminopropyl)Human DDAH-1 DDAH-1 N L-or 5 (54b) (54b) (54c) Human DDAH-1 (L-IPO) N -(1-Imino-2-chloroethyl)L-or nithine (Cl-NIO) Human -- 5 (L-IPO) Nnithine -(1-Imino-2-chloroethyl)L -or (54c)(L-IPO) Human DDAH-1 (Cl-NIO) 5-(1-Imino-2-chloroethyl)-L-or Nnithine (54c) Human DDAH-1 (Cl-NIO) (54c) Human DDAH-1 N55-(1-Imino-2-chloroethyl)L-or nithine (Cl-NIO) N 5-(1-Imino-2-chloroethyl)L-or nithine (Cl-NIO) (54c) Human DDAH-1 N 5-(1-Imino-2-chloroethyl)L-or (54c) Human DDAH-1 nithine (Cl-NIO) N -(1-Imino-2-chloroethyl)L-or (54c) Human DDAH-1 nithine (Cl-NIO) (54c) Human DDAH-1 nithine (Cl-NIO) nithine (Cl-NIO)
Ki (µM) Ki (μM) Ki (μM) Ki (μM) Ki (μM) Ki (μM) Ki (μM) 690 690 Ki (μM) Ki690 (μM) Ki 690 (μM) 690 690 690 690 3100 6903100 3100 690 3100 3100 3100 3100 3100 3100 - 3100 ---- 2 13 13- 22 132 13 2 13 2 13 2 13 2 13 2 13- 2 13-1 11 1 1 1 1 1 12 21 2 2 2 2 2 2 2 52 2 52 52 52 52 52 52 52 1.3 52 52 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3
19 of 31 19 of 31 19 of 31 19 of 31 19 of 31 19 of 31 19 of 31 19 of 31 19 ofReference 31
´1 ) Kinact Reference Kinact−1Kinact (min Kinact ) (min Reference Kinact−1−1) Reference (min Kinact Reference ) (min −1) Reference Kinact (min −1) Kinact (min Reference −1) [105] 0.38 K0.38 inact Reference (min −1) 0.38 [105] K inact Reference (min −1 0.38−1) [105] Reference (min 0.38 ) [105] (min 0.38 [105] 0.38 [105] 0.38 [105] 1.2 [106] 0.38 [105] 1.2 1.2 [106] 0.38 [105] 1.2 [106] 1.2 [106] 1.2 [106] 1.2[106] [109] 1.2 [106] [109] 1.2 [109] - [106] 1.2 [106] [109] [109] [109] -[109] [110] [109] --[110] [109] [110] - [110] [110] [110] [110] [112] -[110] [110] -[112] [112] [112] - [112] [112] [112] -[112] [112] [112] -[112] [112] [112] [112] - [112] [112] [117] -[112] [112] -[117] [117] [117] [117] [117] [117] [114] -[117] [117] -[114] [114] [114] [114] [114] [114] -- [114] [128] [114] -[128] [128] [128] [128] [128] [128] [128] 0.34 [129] [128] 0.34 [129] 0.34 [129] 0.34 [129] 0.34 [129] 0.34 [129] 0.34 [129] 0.34 [129] 0.34 [129]
[105]
[106]
[109]
[110]
[112]
[112]
[117]
[114]
[128]
(40)
NG-(2-Methoxyethyl)-L-arginine methyl ester (L-291)
Rat DDAH-1
20
-
-
[112]
(44a)
2-Amino-5-(3-(2-methoxyethyl) guanidino)-N-(methylsulfonyl)pentanamide (ZST316)
Human DDAH-1
3
1
-
[117]
Molecules 2016, 21, 615
(54a)
20 of 32
N5-(1-Imino-3-butenyl)-L-ornith ine (L-VNIO)
N5-(1-Iminopropyl)-L-ornithine Chemical(54b) Chemical Name (L-IPO) Number Molecules 2016, 21, 615 Molecules 2016, 21,5 615 5 N -(1-Imino-2-chloroethyl)L-or L-ornithine Molecules 2016, N 21,-(1-Imino-2-chloroethyl)615 (54c) 21, 615 (54c) 2016, Molecules (Cl-NIO) Molecules 2016, 21, 615nithine (Cl-NIO) Molecules 2016, 21, 615
Human DDAH-1
13
2
-
Chemical Structure
Human DDAH-1 Enzyme
IC50- (µM)
52K (µM) i
- K [128] ´1 inact (min )
Human Human DDAH-1
--
1.3 1.3
PaDDAH PaDDAH 11 PaDDAH 11 PaDDAH1 PaDDAH 1 PaDDAH PaDDAH 1
16–58 16–58 16–58 16–58 16–58 16–58 16–58
-
Indolylthiobarbituric acid Indolylthiobarbituric acid 68a–c Indolylthiobarbituric acid derivatives 68a–c 68a–c Indolylthiobarbituric acid derivatives 68a–c Indolylthiobarbituric acid derivatives Indolylthiobarbituric acid derivatives 68a–c Indolylthiobarbituric acid 68a–c derivatives 68a–c derivatives derivatives
PaDDAH 111 PaDDAH PaDDAH PaDDAH 1 PaDDAH 11 PaDDAH 1 PaDDAH
2–17 2–17 2–17 2–17 2–17 2–17 2–17
-
Ebselen Human DDAH-1 0.33 Ebselen Human DDAH-1 0.33 Ebselen Human 0.33 Ebselen Human DDAH-1 DDAH-1 0.33 Ebselen Human DDAH-1 0.33 Ebselen Human DDAH-1 0.33 Ebselen Human DDAH-1 0.33 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 4-Hydroxy-2-nonenal (4-HNE) Human 5050 4-Hydroxy-2-nonenal (4-HNE) HumanDDAH-1 DDAH-1 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 6H-6-Imino-(2,3,4,5-tetrahydropy 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 6H-6-Imino-(2,3,4,5-tetrahydropy 6H-6-Imino-(2,3,4,5-tetrahydropy rimido)[1,2-c]-[1,3]benzothiazine Human DDAH-1 9 6H-6-Imino-(2,3,4,5-tetrahydropy rimido)[1,2-c]-[1,3]benzothiazine Human DDAH-1 9 6H-6-Imino-(2,3,4,5-tetrahydropy rimido)[1,2-c]-[1,3]benzothiazine Human DDAH-1 9 (PD 404182) 6H-6-Imino-(2,3,4,5-tetrahydropy rimido)[1,2-c]-[1,3]benzothiazine Human DDAH-1 9 6H-6-Imino-(2,3,4,5-tetrahydropyrimido) (PD 404182) rimido)[1,2-c]-[1,3]benzothiazine Human 99 HumanDDAH-1 DDAH-1 (PD 404182) rimido)[1,2-c]-[1,3]benzothiazine Human DDAH-1 9 [1,2-c]-[1,3]benzothiazine (PD 404182) (PD 404182) (PD 404182) (PD 404182) Proton H+/K+ ATPase pump Proton H++/K++ ATPase pump 82a–f Human DDAH-1 51–63 ATPase pump Proton H+ /K 82a–f Human DDAH-1 51–63 inhibitors (PPIs) + ATPase pump 82a–f Human DDAH-1 51–63 Proton H +/K inhibitors (PPIs) + ATPase pump /K Proton H inhibitors (PPIs) 82a–f Human DDAH-1 51–63 + ATPase pump Proton H++/K 82a–f Proton Human DDAH-1 51–63 H+ /K ATPase pump inhibitors (PPIs) Human 51–63 82a–f 82a–f HumanDDAH-1 DDAH-1 51–63 inhibitors (PPIs) 1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117]. inhibitors (PPIs) inhibitors (PPIs) 1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117]. 1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117]. 1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117]. 1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117]. 1 Pa: 2 as 2determined by Tommasi et al. [117]. aeruginosa; 1 Pseudomonas Pa: Pseudomonas aeruginosa; as determined by Tommasi et al. [117].
-
(73) (73) (73) (73) (73) (73) (76) (76) (76) (76) (76) (76) (76) (79) (79) (79) (79) (79) (79) (79) (73)
0.34
-
-
-
-
-
-
-
Reference 20 of 31 20 of 31 20 of 31 20 of 31 20 of 31
0.34 [129] 20 of 31 [129]
Table 3. Cont. Table 3. Cont. Table 3. Cont. Table 3. Cont. Table 3. Cont. Table 3. Cont.
Pentafluorophenyl (PFP) Pentafluorophenyl (PFP)(PFP) Pentafluorophenyl 61 Pentafluorophenyl (PFP) 61 sulfonate sulfonate estersesters 61 Pentafluorophenyl (PFP) sulfonate esters Pentafluorophenyl sulfonate esters (PFP) 61 Pentafluorophenyl (PFP) 61 sulfonate esters 61 sulfonate esters sulfonate esters
61
[114]
Table 3. Cont.
-
- [130] [130] [130] [130] [130] [130]
-
[139] - [139] [139] [139] [139] [139]
-
[145] [145] - [145] [145] [145] [145] [167] [167] [167] - [167] [167] [167] [61] [61] [61] [61] - [61] [61]
-
[175] [175] [175] [175] [175] - [175]
[130]
[139]
[145]
[167]
[61]
[175]
Molecules 2016, 21, 615
21 of 32
Acknowledgments: The authors thank Flinders Medical Centre Foundation and Flinders Faculty of Health Sciences, the Scottish Universities Life Sciences Alliance (SULSA), and the NHS Grampian Endowment Fund for providing financial support which assisted with reading available evidence and conducting experimental work. Author Contributions: The manuscript was conceived by A.A.M., R.B.M. and S.T. collected the primary data and compiled draft manuscripts. B.C.L. and A.A.M. supervised development of the manuscript, and assisted in data interpretation, manuscript evaluation and editing. Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations The following abbreviations are used in this manuscript: DDAH ADMA L-NMMA NOS NO SDMA PRMTs CAT AGXT2 PKA Akt VEGF iNOS TNF-α LDL IL-1β AT1 -R DNA FXR HcyNO NaNO2 NaOH HCl Min Ki µM H2 O Cys His PaDDAH PAD NaOMe MeOH AcOH NH4 Cl PEO DCM TEA DMAP MsCl Et2 O RT NaN3
Dimethylarginine dimethylamino hydrolase Asymmetric dimethylarginine Monomethyl arginine Nitric oxide synthase Nitric oxide Symmetric dimethylarginine Protein arginine methyl transferases Cationic amino acid transporter Alanine-glyoxylate amino transferase 2 Protein kinase A Protein kinase B Vascular endothelial growth factor Inducible nitric oxide synthase Tumour necrosis factor α Low density lipoprotein Interleukin-1β Type 1 angiotensin receptor Deoxyribonucleic acid Farnesoid X receptor S-Nitroso-L-homocysteine Sodium nitrite Sodium hydroxide Hydrochloric acid Minutes Inhibition constant Micromolar Water Cysteine Histidine Pseudomonas aeruginosa dimethylarginine dimethylamino hydrolase Peptydilarginine deaminase Sodium methoxide Methanol Acetic acid Ammonium chloride Poly(ethylene oxide) Dichloromethane Triethylamine 4-Dimethylaminopyridine Methanesulfonyl chloride Diethyl ether Room temperature Sodium azide
Molecules 2016, 21, 615
DMF Ph3 P 4124W Boc M DEAD THF DIPEA CH3 CN SOCl2 SAR IC50 L-257 L-291 Orn Bu Z TFMSA TFA CDI DBU h HATU mM DMSO i-BuCOCl NH4 OH TFAA ZnBr2 NH2 OH.HCl NaHCO3 L-VNIO EtOH EtOAc L-IPO Cl-NIO PFP NaOAc ADI HTS SMTC ESI-MS BuLi Se CuBr2 NADPH H+/K+ ATPase Ca2+ 4-HNE LOPAC NaH
22 of 32
N,N-Dimethylformamide Triphenylphosphine (S)-2-Amino-4-(3-methylguanidino)butanoic acid tert-Butyloxycarbonyl Molar Diethyl azodicarboxylate Tetrahydrofuran N,N-Diisopropylethylamine Acetonitrile Thionyl chloride Structure activity relationship Inhibition constant NG -(2-Methoxyethyl)-L-arginine NG -(2-Methoxyethyl)-L-arginine methyl ester Ornithine Butyl Carboxybenzyl Trifluoromethanesulfonic acid Trifluoroacetic acid 1,11 -Carbonyldiimidazole 1,8-Diazabicyclo[5.4.0]undec-7-ene Hours 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate Millimolar Dimethyl sulfoxide Isobutyl chloroformate Ammonium hydroxide Trifluoroacetic anhydride Zinc bromide Hydroxylamine hydrochloride Sodium bicarbonate N5 -(1-Imino-3-butenyl)-L-ornithine Ethanol Ethyl acetate N5 -(1-Iminopropyl)-L-ornithine N5 -(1-Imino-2-chloroethyl)-L-ornithine Pentafluorophenyl Sodium acetate Arginine deaminase High throughput screening S-methyl-L-thiocitrulline Electrospray ionization mass spectrometry Butyl lithium Selenium Copper(II) bromide Nicotinamide adenine dinucleotide phosphate Proton/potassium ATPase Calcium 2+ cation 4-Hydroxy-2-nonenal Library of Pharmacologically Active Compounds Sodium hydride
Molecules 2016, 21, 615
CS2 BrCN Å PPIs SPR CM (Ph3 P)2 PdCl2 CuI TIPS TMS DPPA
23 of 32
Carbon disulfide Cyanogen bromide Angstrom Proton pump inhibitors Surface plasmon resonance Carboxymethylated Bis(triphenylphosphine)palladium chloride Copper(I) iodide Triisopropylsilyl Trimethylsilane Diphenylphosphoryl azide
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50.
51.
52.
53.
54.
55.
56. 57.
58.
59.
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61.
62. 63. 64. 65. 66.
67.
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