molecules

Review Inhibitors of the Hydrolytic 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; rhys.murphy@flinders.edu.au (R.B.M.); sara.tommasi@flinders.edu.au (S.T.); ben.lewis@flinders.edu.au (B.C.L.) * Correspondence: arduino.mangoni@flinders.edu.au; 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 . Current literature reports identify the DDAH family of as a potential therapeutic target in the regulation of (NO) production, mediated via its biochemical interaction with the (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 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 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

Molecules 2016, 21, 615; doi:10.3390/molecules21050615 www.mdpi.com/journal/molecules Molecules 2016, 21, 615 2 of 32 reported to bind the tumour suppressor protein neurofibromin, resulting in increased phosphorylation of neurofibrominMoleculesMolecules 20162016,, 2121 by,, 615615 protein kinase A (PKA) [20] and is additionally reported to regulate the22 ofof levels 3131 of phosphorylated protein kinase B (p-Akt) by increasing Ras activity via a mechanism independent to the NOSofof neurofibrominneurofibromin pathway [22 byby]. Similarly,proteinprotein kinasekinase DDAH-2 AA (PKA)(PKA) is [20] known[20] andand toisis additionallyadditionally interact with reportedreported PKA causing toto regulateregulate the thethe induction levelslevels of vascularofof phosphorylatedphosphorylated endothelial growth proteinprotein factor kinasekinase (VEGF) BB (p-Akt)(p-Akt) via byby phosphorylation increasingincreasing RasRas activityactivity of the viavia Sp1 aa mechanismmechanism transcription independentindependent factor [21 ]. toto thethe NOSNOS pathwaypathway [22].[22]. Similarly,Similarly, DDAH-2DDAH-2 isis knowknownn toto interactinteract withwith PKAPKA causingcausing thethe inductioninduction ofof Asymmetric dimethylarginine (ADMA, 1), monomethylarginine (L-NMMA, 2), and symmetric vascularvascular endothelialendothelial growthgrowth factorfactor (VEGF)(VEGF) viavia phosphorylationphosphorylation ofof thethe Sp1Sp1 transcriptiontranscription factorfactor [21].[21]. dimethylarginine (SDMA, 3) are endogenous methylated arginines (Figure1). They are formed by AsymmetricAsymmetric dimethylargininedimethylarginine (ADMA,(ADMA, 11),), monomethylargininemonomethylarginine ((LL-NMMA,-NMMA, 22),), andand symmetricsymmetric two biochemicaldimethylargininedimethylarginine processes: (SDMA,(SDMA, (1) 33)) the areare initialendogenousendogenous post-translational methylatedmethylated argininesarginines methylation (Figure(Figure of 1).1). protein TheyThey areare arginine formedformed residues byby whichtwotwo is biochemicalbiochemical catalyzed byprocesses:processes: the protein (1)(1) thethearginine initialinitial post-transpost-trans methyltransferaseslationallational methylationmethylation (PRMT’s); ofof proteinprotein and argininearginine (2) the residuesresidues subsequent releasewhichwhich from isis catalyzedcatalyzed their respective byby thethe proteinprotein methylated argininearginine methyltrmethyltr protein(s)ansferasesansferases by proteolysis (PRMT’s);(PRMT’s); andand [23 (2)(2)]. thethe Once subsequentsubsequent released releaserelease into the cytosol,fromfrom transport theirtheir respectiverespective and efflux methylatedmethylated of the protein(s)protein(s) methylated byby prpr argininesoteolysisoteolysis is[23].[23]. mediated OnceOnce releasedreleased by the intointo y+ thethecationic cytosol,cytosol, amino acidtransport (CAT-1)transport and transporterand effluxefflux ofof the [the24 ].methylatmethylat ADMAeded argininesarginines and L-NMMA isis mediatedmediated inhibit byby thethe all yy members++ cationiccationic aminoamino of the acidacid NOS (CAT-1)(CAT-1) family of enzymestransportertransporter [25–30 [24].[24].], while ADMAADMA SDMA andand L competesL-NMMA-NMMA inhibitinhibit with Lallall-arginine, membersmembers aofof NOS thethe NOSNOS , familyfamily of forof enzymesenzymes transport [25–30],[25–30], by the y+ ++ cationwhilewhile transporter SDMASDMA competescompetes [31]. Different withwith LL-arginine,-arginine, enzymes aa NOSNOS play substrate,substrate, a role forfor in transporttransport the metabolism byby thethe yy cation ofcation methylated transportertransporter arginines, [31].[31]. DifferentDifferent enzymesenzymes playplay aa rolerole inin thethe metabolismmetabolism ofof methylatedmethylated arginines,arginines, particularlyparticularly alanine-glyoxylatealanine-glyoxylate particularly alanine-glyoxylate aminotransferase 2 (AGXT2) [32–34]. However, ADMA and L-NMMA, aminotransferaseaminotransferase 22 (AGXT2)(AGXT2) [32–34].[32–34]. However,However, ADMAADMA andand LL-NMMA,-NMMA, butbut notnot SDMA,SDMA, areare primarilyprimarily but not SDMA, are primarily converted by DDAH-1 to L- (4) and dimethylamine (5) or convertedconverted byby DDAH-1DDAH-1 toto LL-citrulline-citrulline ((44)) andand dimethylaminedimethylamine ((55)) oror monomethylaminemonomethylamine ((66),), respectivelyrespectively monomethylamine[35][35] (Scheme(Scheme 1).1). (6), respectively [35] (Scheme1).

Figure 1. The endogenous methylated arginines, asymmetric dimethylarginine (ADMA 1), FigureFigure 1. 1.The The endogenous endogenous methylatedmethylated arginine arginines,s, asymmetric asymmetric dimethylarginine dimethylarginine (ADMA (ADMA 1), 1), monomethylargininemonomethylarginine ((LL-NMMA-NMMA 22),), andand symmetricsymmetric dimethylargininedimethylarginine (SDMA(SDMA 33)) actact asas directdirect oror monomethylarginine (L-NMMA 2), and symmetric dimethylarginine (SDMA 3) act as direct or indirect indirectindirect inhibitorsinhibitors ofof thethe NOSNOS familyfamily ofof enzymes.enzymes. ADMAADMA ((11)) andand LL-NMMA-NMMA ((22)) areare substratessubstrates forfor inhibitors of the NOS family of enzymes. ADMA (1) and L-NMMA (2) are substrates for human DDAH-1. humanhuman DDAH-1.DDAH-1.

SchemeScheme 1.1. ADMAADMA andand LL-NMMA-NMMA areare substratessubstrates forfor DDDDAH-1AH-1 andand areare convertedconverted toto LL-citrulline-citrulline ((44)) andand Scheme 1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and anan amineamine speciesspecies 55 oror 66.. an amine species 5 or 6. TheThe rolerole ofof ADMAADMA accumulationaccumulation andand impairedimpaired DDAHDDAH expressionexpression and/orand/or activityactivity inin thethe Thepathophysiologypathophysiology role of ADMA ofof severalseveral accumulation diseasesdiseases isis wellwell and documedocume impairedntednted [36–44].[36–44]. DDAH IncreasedIncreased expression plasmaplasma and/or oror serumserum activity ADMAADMA in the pathophysiologyconcentrationsconcentrations ofhavehave several beenbeen associatedassociated diseases withwith is coronary wellcoronary documented eventsevents [45–47],[45–47], [36 renalrenal–44]. diseasedisease Increased [29,37],[29,37], atherosclerosis plasmaatherosclerosis or serum ADMA[48],[48], concentrations andand cerebrovascularcerebrovascular have diseasedisease been associated [49,50].[49,50]. Up-regulationUp-regulation with coronary ofof DDAHDDAH events expressionexpression [45–47], andand renal activityactivity disease might,might, [29 ,37], atherosclerosistherefore,therefore, representrepresent [48], and aa goodgood cerebrovascular strategystrategy inin thethe disease treatmentreatmen [49tt of,of50 thethe]. Up-regulation aforementionedaforementioned ofpathologiespathologies DDAH expression toto increaseincrease and ADMA metabolism and lower its plasma concentrations. activityADMA might, metabolism therefore, and represent lower its a plasma good strategyconcentrations. in the treatment of the aforementioned pathologies InIn contrast,contrast, reducedreduced ADMAADMA concentrationsconcentrations havehave beenbeen demonstrateddemonstrated inin otherother diseasedisease states,states, suchsuch asas to increase ADMA metabolism and lower its plasma concentrations. amyotrophicamyotrophic laterallateral sclerosissclerosis [51],[51], multiplemultiple sclerosissclerosis [43],[43], Alzheimer’sAlzheimer’s diseasedisease andand dementiadementia [52,53].[52,53]. InADMAADMA contrast, hashas alsoalso reduced beenbeen shown ADMAshown toto concentrations havehave neuroprotectivneuroprotectiv haveee been effectseffects demonstrated inin aa modelmodel ofof Parkinson’s inParkinson’s other disease diseasedisease states, [54].[54]. such 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 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 (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 [77] each suppresses DDAH expression and activity. In contrast, 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 Molecules 2016, 21, 615 4 of 32 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 4 of 31 3. Pharmacological Induction of DDAH hypermethylation has the ability to suppress DDAH-2 transcription, while histone acetylation has Asthe aability result to of up-regulate diminished DDAH-2 DDAH transcription activity and/or [83]. accumulation of ADMA and L-NMMA in various disease states, several studies have investigated the effects of restoring or increasing DDAH expression. Statin3. treatmentPharmacological is associated Induction with of DDAH a significant reduction in ADMA concentrations [84–86], and an increase inAs both a result DDAH-1 of diminished and DDAH-2 DDAH expression activity and/or and activityaccumulation [87–89 of]. AnotherADMA and class L-NMMA of compounds in enhancingvarious ADMA disease metabolismstates, several viastudies DDAH have up-regulation investigated the is effects represented of restoring by the or increasing farnesoid DDAH X receptor (FXR)expression. agonists. Statin GW4064 treatment upregulates is associated both with DDAH-1 a significant and reduction the cationic in AD transporterMA concentrationsCAT-1 [84–86],, leading to increasedand an ADMA increase cellular in both uptake DDAH-1 and and metabolism, DDAH-2 andexpr decreasedession and serumactivity ADMA [87–89]. concentrations Another class of [15 ,90]. Anothercompounds FXR agonist, enhancing INT-747, ADMA can metabo increaselism DDAH-1 via DDAH expression up-regulation [91,92 is ],represented however thisby the is notfarnesoid 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-regulationhowever this is of not PRMT-1 associated expression with significant [78,94]. changes Another in lipid ADMA lowering and NO drug, concentrations fenofibrate, [91]. decreases The ADMAlipid concentrations lowering drug byprobucol restoring also DDAHlowers ADMA activity co inncentrations conditions [93] of via oxidative up-regulation stress of [95 both]. Additionally, DDAH the oralexpression antidiabetic and activity, drug pioglitazoneand down-regulation and the of βPR1 receptorMT-1 expression blocker [78,94]. nebivolol Another both lipid decrease lowering serum ADMAdrug, concentrations fenofibrate, decreases by increasing ADMA DDAH-2 concentrations expression by restoring [96–99]. DDAH activity in conditions of oxidative stress [95]. Additionally, the oral antidiabetic drug pioglitazone and the β1 receptor blocker 4. Pharmacologicalnebivolol both decrease Inhibition serum of ADMA DDAH concentrations by increasing DDAH-2 expression [96–99].

Contrary4. Pharmacological to the comments Inhibition in of Section 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) Since4.1.1. S the-Nitroso- identificationL-Homocysteine of S-nitrosylation (HcyNO) as an important mechanism of DDAH regulation, the discoverySince the of identificationL-homocysteine’s of S-nitrosylation ability as to an inhibit important DDAH-1 mechanism led of toDDAH the regulation, development the of S-nitroso-discoveryL-homocysteine of L-homocysteine’s (HcyNO, ability9), anto inhibit endogenous DDAH-1S-nitrosothiol led to the development and a source of S of-nitroso- endogenousL- NO [100homocysteine–102]. Synthesis (HcyNO, and 9), an characterisation endogenous S-nitrosothiol of HcyNO and as aa bovinesource of DDAH endogenous inhibitor NO [100–102]. was reported by KnippSynthesiset al. and(2005) characterisation [102]. HcyNO of HcyNO was synthesized as a bovine in DDAH near quantitativeinhibitor was yieldreported in a by two-step Knipp et reaction al. (Scheme(2005)2) via[102]. alkaline HcyNO hydrolysis was synthesized of commercially in near quanti availabletative Lyield-homocysteine in a two-step thiolactone reaction (Scheme hydrochloride 2) 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) (NaNO ) and acidification to afford HcyNO (9)[104]. Alternatively, S-nitrosylation can be undertaken and2 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 excellentyields yields make makeHcyNO HcyNO simple simpleto prepare to prepareon a large on scale. a large scale.

SchemeScheme 2. Synthetic 2. Synthetic scheme scheme for for generating generating HcyNOHcyNO ( 9).). ReagentsReagents and and Conditions Conditions: (i) :(5 iM) 5NaOH, M NaOH, 37 °C, 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 HcyNOwith an inhibitory irreversibly constant inhibits (Ki) DDAHof 690 μM by [105]. covalently Mass spectrometry binding to theanal putativeyses using catalytic different cysteine isotopic with 15 18 18 an inhibitoryforms of S constant-nitroso-L-homocysteine (Ki) of 690 µ M[(Hcy105NO]. Massand HcyN spectrometryO) and oxygen-18 analyses labelled using differentwater (H2 isotopicO) demonstrated that HcyNO forms a N-thiosulfoximide15 adduct18 with cysteine 273 (Cys273) of bovine 18 forms of S-nitroso-L-homocysteine (Hcy NO and HcyN O) and oxygen-18 labelled water (H2 O) demonstratedDDAH-1. The that authors’ HcyNO proposed forms a mechN-thiosulfoximideanism of inhibition adduct requires with the cysteine lone pair 273 of (Cys273)electrons of of the bovine

Molecules 2016, 21, 615 5 of 32

DDAH-1.Molecules The 2016 authors’, 21, 615 proposed mechanism of inhibition requires the lone pair of electrons5 of 31 of the Cys273 SH to attack the S-nitroso group in HcyNO to eliminate a hydroxyl anion (Scheme3). This Cys273 SH to attack the S-nitroso group in HcyNO to eliminate a hydroxyl anion (Scheme 3). This mechanismMolecules is2016 likely, 21, 615 stabilized by neighbouring amino acids, namely His172. A further hydroxyl5 of 31 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 originatingCys273 SH fromto attack water the attacks S-nitroso the groupcationic in sulfurHcyNO of tothe eliminat conjugatede a hydroxyl intermediate, anion and (Scheme subsequently 3). This rearrangesrearrangesmechanism to give to is givelikely the the covalent stabilized covalent thiosulfoximide by thiosulfoximide neighbouring amin adductadducto acids, [102]. [102 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 AmericanScheme 3. Chemical Schematic Society. 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 Chemical Society. and N-But-3-ynyl-2-chloroacetamidine 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 Stonethe amidinotransferaseset al. [106] identified superfamily, 2-chloroacetamidine Pseudomonas (11aeruginosa) as a nonspecific DDAH (Pa inhibitorDDAH) and of two human enzymes of thepeptydilarginine amidinotransferasesStone et al. [106] deaminase identified superfamily, (PAD). 2-chloroacetamidine 2-ChloroacetamidinePseudomonas (11) as aeruginosa ( 11a nonspecific, SchemeDDAH 4) inhibitoris a commercially (Pa ofDDAH) two enzymes available andhuman of peptydilargininecompoundthe amidinotransferases bearing deaminase a 2-chloromethylene (PAD).superfamily, 2-Chloroacetamidine Pseudomonaschain and an aeruginosaamidinium (11, Scheme DDAHgroup,4) which is(Pa aDDAH) commercially partially and resembles human available arginine.peptydilarginine It can be deaminase synthesized (PAD). via reaction 2-Chloroacetamidine of chloroacetonitrile (11, Scheme(10) with 4) sodium is a commercially methoxide availablefollowed compound bearing a 2-chloromethylene chain and an amidinium group, which partially resembles bycompound treatment bearing with ammoniuma 2-chloromethylene chloride [107,108].chain and 2-Chloroacetamidinean amidinium group, acts which as apartially weak irreversible resembles arginine. It can be synthesized via reaction of chloroacetonitrile (10) with sodium methoxide followed amidinotransferasearginine. It can be synthesized inhibitor with via reactiongreater ofaffinity chloroacetonitrile for PaDDAH, (10 )relative with sodium to PAD4 methoxide (Table followed1). Data by treatmentacquiredby treatment withby electrospraywith ammonium ammonium ionization chloride chloride mass [ 107[107,108]. spectrom,108]. 2-Chloroacetamidineetry revealed the formation acts acts as asa ofweak a an weak irreversibleirreversible irreversible amidinotransferasethiouronium-enzymeamidinotransferase inhibitor inhibitor adduct with wherebywith greater greater the loss affinityaffinity of chlorine for for PaPaDDAH, fromDDAH, the relative inhibitor relative to is toPAD4 observed PAD4 (Table (Table[106] 1).. 1Data). Data acquiredacquired by electrosprayby electrospray ionization ionization mass mass spectrometryspectrometry revealed revealed the the formation formation of an of irreversible an irreversible thiouronium-enzymethiouronium-enzyme adduct adduct whereby whereby the the loss loss of of chlorinechlorine from from the the inhibitor inhibitor is observed is observed [106] [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, 6 h, 89%. SchemeScheme 4. Synthetic 4. Synthetic scheme scheme for for generating generating2-chloroacetamidine 2-chloroacetamidine (11 (11) (the) (the conditions conditions described described afford afford the hydrochloride salt). Reagents and Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4Cl, the hydrochloride salt). Reagents and Conditions:(i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4Cl, reflux, 6 h, 89%.Table 1. Reported inhibitory constants for 2-chloroacetamidine (11) [106]. reflux, 6 h, 89%. Enzyme Ki (mM) Kinact (min−1) Table 1. Reported inhibitory constants for 2-chloroacetamidine (11) [106]. Table 1. ReportedPa inhibitoryDDAH constants 3.1 ± 0.8 for 2-chloroacetamidine 1.2 ± 0.1 (11)[106]. EnzymePAD4 K 20i (mM) ± 5 Kinact 0.7 (min± 0.1 −1) PaDDAH 3.1 ± 0.8 1.2 ± 0.1 ´1 Enzyme Ki (mM) Kinact (min ) The addition of an N-but-3-ynylPAD4 substituent 20 ± to 5 the acetam 0.7 ±idine 0.1 group of 2-chloroacetamidine led to formation of the humanPaDDAH DDAH-1 probe, 3.1 ˘N-but-3-ynyl-2-chloro-acetamidine0.8 1.2 ˘ 0.1 (18, Scheme 5) PAD4 20 ˘ 5 0.7 ˘ 0.1 [109].The N-But-3-ynyl-2-chloro-acetamidine addition of an N-but-3-ynyl substituent binds with to inthe the acetam putativeidine active-site, group of 2-chloroacetamidinewhile the N-alkynyl substituentled to formation is reacted of the with human biotin-PEO DDAH-13-azide probe, under N-but-3-ynyl-2-chloro-acetamidine copper via click chemistry. (18, SuccessfulScheme 5) Theconjugation[109]. addition N-But-3-ynyl-2-chloro-acetamidine can of be an easilyN-but-3-ynyl detected in substituent immunoblots binds towith theusingin acetamidinethe an putative anti-biotin active-site, group antibody of 2-chloroacetamidinewhile [109]. the Synthesis N-alkynyl of led to formationthesubstituent N-but-3-ynyl-2-chloro-acetamidine of theis reacted human with DDAH-1 biotin-PEO probe, probe3-azide Nwas-but-3-ynyl-2-chloro-acetamidine under achieved copper in fivecatalysis steps viausing click two chemistry. modules (18, Scheme (Scheme Successful5 5).)[ 109]. N-But-3-ynyl-2-chloro-acetamidineAmineconjugation module can be15 easilywas prepareddetected in bindsin immunoblots three within steps: using theactivation putative an anti-biotin of the active-site, terminal antibody hydroxyl while[109]. Synthesis the groupN-alkynyl of 3-butyn-1-olthe N-but-3-ynyl-2-chloro-acetamidine (12) through mesylation probeto afford was 13 achieved in 62% inyield, five followedsteps using by two conversion modules to (Scheme amine 155). 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 (Scheme5).

Molecules 2016, 21, 615 6 of 32

Amine module 15 was prepared in three steps: activation of the terminal hydroxyl group of 3-butyn-1-ol (12) throughMolecules 2016 mesylation, 21, 615 to afford 13 in 62% yield, followed by conversion to amine 15 6via of 31 azide intermediate 14 in 49% yield across two steps. The imidic ester module 17 was obtained in moderate via azide intermediate 14 in 49% yield across two steps. The imidic ester module 17 was obtained in yieldsMolecules (approximately 2016, 21, 615 48%) by reacting chloroacetonitrile (10) with anhydrous ethanol (166 of) 31 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 invia the azide presence intermediate of dry hydrochloric14 in 49% yield acid across gas. twoFinal steps. coupling The imidicbetween ester amine module 15 and 17 wasthe imidicobtained ester in proceeds under basic aqueous conditions to afford probe N-but-3-ynyl-2-chloro-acetamidine (18) in 17moderate proceeds yields under (approximately basic aqueous 48%) conditions by reacting to afford chloroacetonitrile probe N-but-3-ynyl-2-chloro-acetamidine (10) with anhydrous ethanol (1816) reasonablein reasonablethe presence yields yields (approximately of dry (approximately hydrochloric 63%). acid63%). Togas. To date, Finaldate, no nocoupling datadata are between available available amine regarding regarding 15 and the the theselectivity imidic selectivity ester of of N-but-3-ynyl-2-chloro-acetamidineN17-but-3-ynyl-2-chloro-acetamidine proceeds under basic aqueous forconditions for human human to DDAH-1 affordDDAH-1 probe and and theN- thebut-3-ynyl-2-chloro-acetamidine potential potential cross cross reactivity reactivity of of this (this18) probe with probeinPa reasonableDDAH. with Pa DDAH.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.Cl (iv) Cl N + HO O NH 10 16 17NH.HCl (v) (iv) Cl Cl N Cl H N + HO O NH 10 16 17 (v) 18 HCl H N Cl HO . 2 N 12 15 H 18 HO (i) HCl.H2N (iii) 12 15 (ii) (i) MsO N3 (iii) 13 14 (ii) MsO N SchemeScheme 5. Synthetic 5. Synthetic scheme scheme for for generating generating thethe3 DDAH-1 biochemical biochemical probe probe N-but-3-ynyl-2-chloro-N-but-3-ynyl-2-chloro- acetamidine (18). Reagents and13 Conditions: (i) DCM, 14 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, 62%;Scheme (ii) anhydrous5. Synthetic DMF, scheme NaN for3, 70generating˝ °C, 3.5 h; the(iii )DDAH-1 Et2O, Ph 3biochemicalP, 0 °C˝ for 2 h,probe addition N-but-3-ynyl-2-chloro- of H2O, then 25 °C ˝ 62%; (ii) anhydrous DMF, NaN3, 70 C, 3.5 h; (iii) Et2O, Ph3P, 0 C for 2 h, addition of H2O, then 25 C foracetamidine 20 h, 49% ((two18). Reagents steps); ( ivand) dry Conditions HCl(g) at: ( i0) °CDCM, for 1TEA, h, stop DMAP, HCl(g) MsCl,, then 0 °C°C forfor 24 hh thenand RT25 °Covernight for 3 h, for 20 h, 49% (two steps); (iv) dry HCl at 0 ˝C for 1 h, stop HCl , then 0 ˝C for 4 h and RT overnight 48%–76%; (v) H2O, pH = 10, 0 °C for(g) 3 h, 25 °C for 2 h, neutralized,(g) RT for 1.5 days, 62%. Adapted 62%; (ii) anhydrous DMF, NaN˝ 3, 70 °C, 3.5 h; ˝(iii) Et2O, Ph3P, 0 °C for 2 h, addition of H2O, then 25 °C 48%–76%; (v)H2O, pH = 10, 0 C for 3 h, 25 C for 2 h, neutralized, RT for 1.5 days, 62%. Adapted withfor 20 permission h, 49% (two from steps); [109]. (iv )Copyri dry HClght(g) 2009 at 0 °CAmerican for 1 h, stopChemical HCl(g) Society., then 0 °C for 4 h and RT overnight with permission from [109]. Copyright 2009 American Chemical Society. 48%–76%; (v) H2O, pH = 10, 0 °C for 3 h, 25 °C for 2 h, neutralized, RT for 1.5 days, 62%. Adapted 4.1.3.with (S)-2-Amino-4-(3-Methylguanidino)butanoic permission from [109]. Copyright 2009 American Acid Chemical Analogues Society. S 4.1.3. ( )-2-Amino-4-(3-Methylguanidino)butanoicScreening experiments with a library comprising Acid 26 Analoguesanalogues of ADMA (1) and L-NMMA (2) 4.1.3. (S)-2-Amino-4-(3-Methylguanidino)butanoic Acid Analogues Screeningidentified (S experiments)-2-amino-4-(3-methylguanidino)butanoic with a library comprising acid 26 analogues(4124W, 21), of as ADMA a mammalian (1) and DDAHL-NMMA (2) identifiedinhibitorScreening ([110].S)-2-amino-4-(3-methylguanidino)butanoic experimentsThe original with synthesis a library of 4124Wcomprising (21), 26first analogues aciddescribed (4124W, of byADMA Ueda21), as( 1et) a al.and mammalian in L -NMMA2003 [80] ( DDAH2is) shown in Scheme 6, and was achieved by coupling N,S-dimethylthiopseudouronium iodide (19) inhibitoridentified [110]. ( TheS)-2-amino-4-(3-methylguanidino)butanoic original synthesis of 4124W (21), first acid described (4124W, by 21 Ueda), as eta al.mammalianin 2003 [ 80DDAH] is shown withinhibitor the copper[110]. The complex origin alof synthesis2,4-diamino- of 4124Wn-butyric (21 acid), first dihydrochloride described by Ueda(20) [32,111].et al. in 20034124W [80] (21 is) in Scheme6, and was achieved by coupling N,S-dimethylthiopseudouronium iodide (19) with the wasshown finally in Scheme crystallized 6, and as thewas hydrochloride achieved by couplingsalt. N,S-dimethylthiopseudouronium iodide (19) n 20 21 copperwith complex the copper of 2,4-diamino- complex of 2,4-diamino--butyric acidn-butyric dihydrochloride acid dihydrochloride ( )[32, 111(20)]. [32,111]. 4124W (4124W) was (21 finally) crystallizedwas finally as thecrystallized hydrochloride as the hydrochloride salt. 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 ConditionsScheme 6.: Synthetic(i) (1) 2,4-diamino- scheme forn generating-butyric acid (S)-2-amino-4-(3-methylguanidino)butanoic dihydrochloride (20), copper acetate, 1:1 acid NH (4124W),4OH/H2 O;as Scheme 6. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic+ acid (4124W), (2)reported N,S-dimethylthiopseudouronium by Ueda et al. [80] using the methodsiodide (19 of) RT,Ogawa 24 h; et (3) al. Dowex [32] and 50WX4 Corbin (NH et al.4 [111].form), Reagents 4124W (and21) as reported by Ueda et al. [80] using the methods of Ogawa et al. [32] and Corbin et al. [111]. Reagents and wasConditions crystallized: (i) (1) as 2,4-diamino- the hydrochloriden-butyric salt. acid dihydrochloride (20), copper acetate, 1:1 NH4OH/H2O; Conditions:(i) (1) 2,4-diamino-n-butyric acid dihydrochloride (20), copper acetate, 1:1 NH4OH/H2O; (2) N,S-dimethylthiopseudouronium iodide (19) RT, 24 h; (3) Dowex 50WX4 (NH4+ form), 4124W (21) + (2) N,HighwasS-dimethylthiopseudouronium crystallized chemical andas the structural hydrochloride similari iodide salt.ty between (19) RT, 244124W h; (3) and Dowex the DDAH 50WX4 substrate (NH4 form), L-NMMA 4124W exists (21) was(compound crystallized 21, Scheme as the hydrochloride6, and compound salt. 2, Figure 1), and although exhibiting weak DDAH inhibition (IC50 High= 416 chemicalμM) [110], and this structural prompted similari Rossiterty between et al. [112] 4124W to synthesize and the DDAH a further substrate 34 compounds L-NMMA basedexists on the 4124W structure. Furthermore, Rossiter et al. [112] reported an alternative synthetic approach, High(compound chemical 21, Scheme and structural 6, and compound similarity 2, betweenFigure 1), 4124Wand although and the exhi DDAHbiting weak substrate DDAHL-NMMA inhibition exists which although requiring three steps, is synthetically versatile and was applied to develop their (compound(IC50 = 41621, μ SchemeM) [110],6, this and prompted compound Rossiter2, Figure et al.1 [112]), and to although synthesize exhibiting a further 34 weak compounds DDAH based inhibition libraryon the 4124Wof 4124W structure. analogues. Furthermore, Rossiter’s strategy Rossiter utilized et al. [112] protected reported intermediates an alternative to avoidsynthetic complications approach, (IC = 416 µM) [110], this prompted Rossiter et al. [112] to synthesize a further 34 compounds based 50associatedwhich although with therequiring purification three ofsteps, polar is amino syntheti acids.cally Intermediate versatile and 24 was(Scheme applied 7) is to generated develop fromtheir on the 4124W structure. Furthermore, Rossiter et al. [112] reported an alternative synthetic approach, commerciallylibrary of 4124W available analogues. N,N ′Rossiter’s-bis-tert-butoxycarbonylpyrazole-1 strategy utilized protectedH intermediates-carbox-amidine to avoid (22) by complications Mitsunobu which associated although with requiring the purification three steps, of polar is amino synthetically acids. Intermediate versatile and24 (Scheme was applied 7) is generated to develop from their librarycommercially of 4124W analogues. available N Rossiter’s,N′-bis-tert-butoxycarbonylpyrazole-1 strategy utilized protectedH-carbox-amidine intermediates to(22 avoid) by Mitsunobu complications associated with the purification of polar amino acids. Intermediate 24 (Scheme7) is generated from Molecules 2016, 21, 615 7 of 32 commerciallyMolecules 2016 available, 21, 615 N,N1-bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine (22) by Mitsunobu7 of 31 reaction with alcohol 23 containing the desired R1 group, followed by reaction with (S)-tert-butyl reactionMolecules 2016with, 21 alcohol, 615 23 containing the desired R1 group, followed by reaction with (S)-tert-butyl7 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 (Scheme7). Cleavage of the Boc protecting groups is achieved with a 4 M solution of HCl in 1,4-dioxane 26reaction (Scheme with 7). alcohol Cleavage 23 ofcontaining the Boc protectingthe desired groups R1 group, is achieved followed with by reaction a 4 M solution with (S )-oftert HCl-butyl in and produced1,4-dioxane4-amino-2-(( 4124W andtert-butoxycarbonyl)amino)butanoate produced (21) in moderate4124W (21) yieldsin moderate (approximately ( 25yields) to afford (approximately 44%)the protected or analogues 44%) amino or analogues 27acidin analogue moderate 27 in to goodmoderate26 yields (Scheme (12%–67%). to good7). Cleavage yields (12%–67%). of the Boc protecting groups is achieved with a 4 M solution of HCl in 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, CH3CN, RT, 24 h; (iii) HCl/1,4-dioxane,Scheme 7. Synthetic RT, scheme24–72 h, for 12%–67% generating overall; (S)-2-amino-4-(3-methylguanidino)butanoic (iv) SOCl2, 0 °C for 30 min, reflux for 1 h, acid RT overnight,analogues. 4M HCl/1,4-dioxane, RT, 24–72 h, 12%–67% overall; (iv) SOCl , 0 ˝C for 30 min, reflux for 1 h, RT 44%–84%.Reagents and Adapted Conditions with: ( permissioni) DEAD, PPh from3, THF, [112]. 0 Copyright°C-RT, 3–16 2005 h; ( iiAmerican2) DIPEA, ChemicalCH3CN, RT, Society. 24 h; (iii) 4M overnight,HCl/1,4-dioxane, 44%–84%. RT, Adapted 24–72 h, with 12%–67% permission overall; from (iv) SOCl [1122]., 0 Copyright °C for 30 min, 2005 reflux American for 1 h, ChemicalRT overnight, Society. Additionally,44%–84%. Adapted Rossiter with permission et al. fromdeveloped [112]. Copyright a synthetic 2005 American method Chemical for Society.N,N-disubstituted Additionally,alkylguanidinobuta Rossiternoic acidset and al. N-arylguanidinesdeveloped a(Scheme synthetic 8). This method involved for reactingN,N-disubstituted N,N’-bis- Additionally, Rossiter et al. developed a synthetic method for N,N-disubstituted alkylguanidinobutanoictert-butoxycarbonylpyrazole-1 acids andH-carboxamidineN-arylguanidines (22) with (Scheme Boc-homoserine-8). Thistert involved-butyl ester reacting (30), wherebyN,N’ -bis- thealkylguanidinobuta pyrazole group innoic the acids intermediate and N-arylguanidines 31 is displaced (Schemeby the reaction 8). This with involved amine reacting32 containing N,N’-bis- the tert-butoxycarbonylpyrazole-1H-carboxamidine (22) with Boc-homoserine-tert-butyl ester (30), desiredtert-butoxycarbonylpyrazole-1 R1 and R2 groups. H-carboxamidine (22) with Boc-homoserine-tert-butyl ester (30), whereby wherebythe pyrazole the pyrazole group group in the in intermediate the intermediate 31 is displaced31 is displaced by the byreaction the reaction with amine with 32 amine containing32 containing the 1 2 the desireddesired R R1 andand R 2 groups.groups.

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,

Scheme3 8. Synthetic scheme for generating N,N-disubstituted alkylguanidinobutanoic acid and SchemeCH CN, 8. SyntheticRT, 24 h; (iii scheme) 4 M HCl/1,4-dioxane, for generating 24–72N,N -disubstitutedh, 0.29%–41% overall. alkylguanidinobutanoic Adapted with permission acid and fromN-arylguanidine [112]. Copyright analogues. 2005 Amer Reagentsican and Chemical Conditions Society.: (i) DEAD, Ph3P, THF, 0 °C-RT,˝ 3–16 h; (ii) DIPEA, N-arylguanidine analogues. Reagents and Conditions:(i) DEAD, Ph3P, 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 CH CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 0.29%–41% overall. Adapted with permission 3 from [112]. Copyright 2005 American Chemical Society. from [112]. Copyright 2005 American Chemical Society.

Molecules 2016, 21, 615 8 of 32

Deprotection of the amino acid analogues 33 is carried out using the same acidic conditions describedMolecules in 2016 the, 21 synthesis, 615 of Rossiter’s 4124W analogues in Scheme7 and provides analogues8 of 31 34 in low to moderate yields (0.29%–41%). Each analogue’s inhibitory potential was assessed based on the reducedDeprotection conversion of the ofamino [14C]-NMMA acid analogues to [14 33C]-citrulline is carried out by using rat DDAH. the same Screening acidic conditions experiments revealeddescribedN-monosubstitution in the synthesis of toRossiter’s be more 4124W effective analogues than in disubstitution, Scheme 7 and provides and that analogues a R1 substituent 34 in comprisinglow to moderate a N-2-methoxyethyl yields (0.29%–41%). group Each increased analogue’s inhibitor inhibitory binding potential affinity was assessed (viz. based compound on the 27a; reduced conversion of [14C]-NMMA to [14C]-citrulline by rat DDAH. Screening experiments revealed IC50 = 189 µM; Scheme7). 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 (Scheme7). 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 inhibitionvia thionyl (IC50 chloride= 27 µM promoted). A series esterificati of amideon with analogues alcohol containing based on the structures desired R293 group(structures (Scheme omitted) 7). exhibitedThe generated poor DDAH-1 benzyl inhibitoryester 29a resulted potentials. in substantial improvement in DDAH-1 inhibition (IC50 = 27 μM). A series of amide analogues based on structures 29 (structures omitted) exhibited poor G 4.1.4.DDAH-1N -Substituted- inhibitoryL -Argininepotentials. 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 Rossiter et al. [112] also synthesized a library of N-substituted arginine analogues. A significant known as L-257 (compound 39; IC50 = 22 µM; Scheme9) and its corresponding methyl ester (also increase in inhibitor binding affinity was obtained for NG-(2-methoxyethyl)-L-arginine (commonly known as L-291 (compound 40; IC50 = 20 µM; Scheme9), relative to compound 27a (Scheme7) 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-1less carbon inhibition within as the was main-chain. observed withInterestingly, compound the benzyl29a. These ester dataof L-257 suggest did not that increase arginine DDAH-1 analogues mayinhibition bind differently as was obse withinrved thewith putative compound DDAH-1 29a. These active-site. data suggest Moreover, that arginine neither analogues the corresponding may bind methyldifferently amide ofwithin L-257, the nor putative the (R)-isomer DDAH-1 of active-sit L-257 (structurese. Moreover, omitted) neither resultedthe corresponding in DDAH-1 methyl inhibition. Theamide synthesis of L-257, nor of the L-257 (R)-isomer (39) is of represented L-257 (structures in omitted) Scheme 9resulted. The in reaction DDAH-1 sequence inhibition. follows a similar approachThe synthesis to that of describedL-257 (39) is in represented the synthesis in Scheme of the (9.S)-2-amino-4-(3-methylguanidino)butanoic The reaction sequence follows a similar acidapproach analogues, to that using described Boc protected in the synthesis amino of acidthe (S analogues.)-2-amino-4-(3-methylguanidino)butanoic Firstly, 2-methoxyethyl pyrazoleacid carboxamidineanalogues, (using36) is preparedBoc protected under amino Mitsunobu acid conditionsanalogues. [113Firstly,] from 2-methoxyethyl 2-methoxyethanol pyrazole (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 with38 with 4 M 4 HCl M HCl in 1,4-dioxane in 1,4-dioxane afforded afforded L-257 L-257 ((3939) in 44% 44% overall overall yield. yield. L-257 L-257 can can be subsequently be subsequently esterifiedesterified to afford to afford L-291 L-291 (40 ()40 in) in yields yields of of approximately approximately 80%. 80%.

SchemeScheme 9. Synthetic 9. Synthetic scheme scheme for generatingfor generating L-257 L-257 (39 )( and39) and L-291 L-291 (40 ).(40Reagents). Reagents and and Conditions Conditions:(i:) ( DEAD,i) 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, Ph3P, THF, 0 C-RT, 3–16 h; (ii) DIPEA, CH3CN, 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) SOCl , 0 ˝C for 30 min, reflux for 1 h, RT overnight, 80%. Adapted with permission from [112]. Copyright2 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 Bothfor DDAH-1 L-257 (39 against) and L-291the three (40) main inhibit isoforms DDAH-1 of inNOS vivo [112,114][112]. They and arearginase non-cytotoxic [115], an andenzyme selective for DDAH-1responsible against for the the metabolism three main isoformsof L-arginine of NOS into [urea112, 114and] andornithine [112,114,115]. [115], an enzymeL-257 (39 responsible) was utilized by Kotthaus et al. [114] as a validated active ‘parent’ compound for determining viable lead for the metabolism of L-arginine into urea and ornithine [112,114,115]. L-257 (39) was utilized by 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,

Molecules 2016, 21, 615 9 of 32

Moleculesand to obtain2016, 21, initial 615 structure-activity relationships (SARs) for those leads. In particular, the role9 of of31 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 the proposed catalytic mechanism and binding to the enzyme [116]. undertaken by Kotthaus et al., the NG-(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 Table2. 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 R– Inhibition at 1 mM (%) IC (µM) K (µM) Entry R– Inhibition at 1 mM (%) IC5050(μM) Kii (μM) 1 1 1 1 * CH* CH3OCH3OCH2CH2CH2–2 – 8383 31 31 13 2 1 CH NHCH CH – 29 - - 2 1 CH3NHCH3 2CH2 2–2 29 - - 1 1 3 (CH3)2NHCH2CH2– 14 - - 3 1 (CH3)2NHCH2CH2– 14 - - 4 CH3SHCH2CH2– 80 408 - 4 1 1 CH3SHCH2CH2– 80 408 - 5 FCH2CH2– 67 379 - 1 1 5 6 FCHBnCH2CH22– 5567 379 866 - 2 6 1 7 CHBnCH3CH2–CH 2– 6055 866 283 90- 2 7 2 8 CHCH3CH2CHCH2CH2–2– 7060 283 207 90 58 2 9 CH2CHCH2CH2– 71 189 57 8 2 CH2CHCH2– 70 207 58 10 2 CHCCH – 83 55 17 2 2 9 2 CH2CHCH2CH2– 71 189 57 11 F3CCH2– 41 - - 2 2 2 10 12 CHCCHO2N–– 83 6 55 - 17 - 2 2 11 13 NHF23CCHCOCH2–2 CH2– 4341 - - - * Denotes12 2 L-257 (39) (SchemeO2N–9 ); 1 Measured by Ultra Performance6 Liquid Chromatography-Mass- Spectrometry;- 2 Measured13 2 byNH colorimetric2COCH assay.2CH2– 43 - - * Denotes L-257 (39) (Scheme 9); 1 Measured by Ultra Performance Liquid Chromatography-Mass G 4.1.5.Spectrometry;N -(2-Methoxyethyl)- 2 MeasuredL by-Arginine colorimetric Carboxylate assay. Bioisosteres The use of bioisosteric replacements is a well-known strategy adopted by medicinal G 4.1.5.chemists N -(2-Methoxyethyl)- to improve theL-Arginine pharmacokinetic Carboxylate and Bioisosteres pharmacodynamic properties of certain compounds,The use of including bioisosteric but replacements not limited is to a inhibitionwell-known potency, strategy lipophilicity, adopted by medicinal pKa, and chemists metabolic to improvestability the [118 pharmacokinetic]. Tommasi et and al. [ 117pharmacodynamic] reported the synthesisproperties andof certain pharmacological compounds, evaluationincluding butof fivenot limited carboxylate to inhibition bioisosteres potency, of L-257lipophilicity, (compounds pKa, and44a metabolic–c; Scheme stability 10, [118].49a–b ;Tommasi Scheme et11 al.). [117]In compounds reported the44a synthesis–c (Scheme and 10 pharmacological), the carboxylic eval aciduation moiety of five is modified carboxylate by bioisosteres introducing of an L-257 acyl (compoundsmethyl sulfonamide 44a–c; Scheme44a (2-amino-5-(3-(2-methoxyethyl)guanidino)- 10, 49a–b; Scheme 11). In compounds 44a–Nc -(methylsulfonyl)-pentanamide;(Scheme 10), the carboxylic acid moietyZST316), is anmodifiedO-methyl-hydroxamate by introducing an 44bacyland methyl a hydroxamate sulfonamide44c 44a. (2-amino-5-(3-(2-methoxyethyl) The replacement is introduced guanidino)-on Z-ornithine(Boc)-OHN-(methylsulfonyl)-pentanamide; (41) in moderate to goodZST316), yields an (51%–86%) O-methyl-hydroxamate to afford 42, followed 44b and by thea hydroxamatequantitative cleavage 44c. The ofreplacement Boc protected is introduced42 with 4 on M HClZ-ornithine(Boc)-OH in 1,4-dioxane. Guanidination(41) in moderate of to42 goodwith yields2-methoxyethylpyrazole (51%–86%) to afford carboxamidine 42, followed ( 36by) isthe undertaken quantitative as describedcleavage byof RossiterBoc protectedet al. [ 11242 ]with (see 4Sections M HCl 4.1.3 in 1,4-dioxane. and 4.1.4 Schemes Guanidination7 and9). Deprotection of 42 with of2-methoxyethylpyrazole the Boc- and Z-appended carboxamidine arginine derivatives (36) is undertaken43a–c is achieved as described in a single by step Rossiter using et TFMSA al. [112] and (see TFA Sections [117,119 4.1.3] and and produces 4.1.4, compoundsSchemes 7 44aand– c9).. DeprotectionBioisosteric of the replacement Boc- and of Z- theappended carboxylic arginine acid moiety derivatives in L-257 43a with–c isO achieved-methyl hydroxamate in a single step44b usingand hydroxamate TFMSA and44c TFAresulted [117,119] in a and decrease produces in DDAH-1 compounds inhibition 44a– (ICc. 50 = 230 and 131 µM, respectively). By contrast,Bioisosteric introduction replacement of an of acylthe methylcarboxylic sulfonamidic acid moiety moiety in L-257 in ZST316 with O (-methyl44a) led hydroxamate to significant 44bimprovements and hydroxamate in pharmacokinetic 44c resulted parameters in a decrease (98% in inhibition DDAH-1 at inhibition 1 mM, IC 50(IC=50 3 =µ M230 and andK i131= 1 µμM).M, respectively).Compound ZST316 By contrast, (44a) isintroduction the most potent of an human acyl methyl DDAH-1 sulfonamidic inhibitor withmoiety ‘substrate-like’ in ZST316 (44a structure) led to significantreported in improvements the literature to in date. 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.

Molecules 2016, 21, 615 10 of 32 Molecules 2016, 21, 615 10 of 31

Molecules 2016, 21, 615 10 of 31

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, ReagentsScheme and 10. Conditions Synthetic :(schemei) CDI, for generating DBU, methanesulfonamide, NG-(2-methoxyethyl)-L-arginine THF, RT, carboxylate 7 h, 51%; bioisosteres. (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-benzylhydroxylamine,O-methylhydroxylamine THF, 0 °C-RT, hydrochloride, 16 h, 57%; ( THF,iv) 4 M 0 HCl/1,4-dioxane,C-RT, 16 h, 86%; DCM,( RT,iii) 30 HATU, min, 99%; DIPEA, O-methylhydroxylamine hydrochloride,˝ THF, 0 °C-RT, 16 h, 86%; (iii) HATU, DIPEA, O-benzylhydroxylamine,(v) DIPEA, DCM, RT, 24 THF, h, 43a 0 42%,C-RT, 43b 16 22%, h, 57%; 43c (56%;iv) 4 (vi M) HCl/1,4-dioxane,TFMSA/TFA, RT, 1 h, DCM, 99%. RT,Reproduced 30 min, 99%; O-benzylhydroxylamine, THF, 0 °C-RT, 16 h, 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (v) DIPEA,in part DCM,from [117] RT, 24with h, permission43a 42%, 43b of the22%, Royal43c Society56%; (ofvi )Chemistry. TFMSA/TFA, RT, 1 h, 99%. Reproduced in (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. Scheme99%; 11. (ii)Synthetic TFAA, TEA, scheme THF, for0 °C-RT, generating 16 h, 47%;N (-(2-methoxyethyl)-iii) 4 M HCl/1,4-dioxane,L-arginine DCM, carboxylateRT, 30 min, 99%; bioisosteres. (iv) Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH4OH (30%), THF, −10 ˝°C-RT, 3 h, ReagentsDIPEA, and DCM, Conditions RT, 24:( ih,) i40%;-BuCOCl, (v) NaNN3-methylmorpholine,, ZnBr2, H2O/2-propanol, NH reflux,4OH 16 (30%), h, 30%; THF, (vi)´ (1)10 NHC-RT,2OH·HCl, 3 h, 99%; 99%; (ii) TFAA, TEA,˝ THF, 0 °C-RT, 16 h, 47%; (iii) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (iv) (ii) TFAA,NaHCO TEA,3, DMSO, THF, 0reflux,C-RT, 1616 h; h,(2)47%; CDI, (DBU,iii) 4 THF, M HCl/1,4-dioxane, reflux, 30 min, 73%; DCM, (vii) RT, TFMSA/TFA, 30 min, 99%; DCM, (iv )RT, DIPEA, DIPEA, DCM, RT, 24 h, 40%; (v) NaN3, ZnBr2, H2O/2-propanol, reflux, 16 h, 30%; (vi) (1) NH2OH·HCl, DCM,30 RT, min, 24 99%. h, 40%;Reproduced (v) NaN in part, ZnBr from ,H[117]O/2-propanol, with permission reflux,of the Royal 16 h, Society 30%; of (vi Chemistry.) (1) NH OH¨ HCl, NaHCO3, DMSO, reflux, 16 h;3 (2) CDI,2 DBU,2 THF, reflux, 30 min, 73%; (vii) TFMSA/TFA, DCM,2 RT, NaHCO30 min,3, DMSO, 99%. Reproduced reflux, 16 h;in (2)part CDI, from DBU,[117] with THF, permission reflux, 30 of min, the Royal 73%; (Societyvii) TFMSA/TFA, of Chemistry. DCM, RT, 30 min,Other 99%. noteworthy Reproduced inhibitors in part from in this [117 seri] withes include permission the tetraz of theole Royal 1-[4-amino-4-(1 Society of Chemistry.H-tetrazol-5-yl) butyl]-3-(2-methoxyethyl)guanidineOther noteworthy inhibitors in this(ZST086, series include49a, Scheme the tetraz 11)ole and 1-[4-amino-4-(1 the 5-oxo-1,2,4-oxadiazoleH-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, Otherbutyl]-3-(2-methoxyethyl)guanidine noteworthy inhibitors in this (ZST086, series include49a, Scheme the tetrazole 11) and 1-[4-amino-4-(1 the 5-oxo-1,2,4-oxadiazoleH-tetrazol-5-yl) 1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazoScheme 11) [117]. The synthesis of these inhibitorsl-3-yl)butyl]-3-(2-methoxyethyl)guanidine with azole bioisosteric groups is (ZST152,achieved 49b via, butyl]-3-(2-methoxyethyl)guanidine (ZST086, 49a, Scheme 11) and the 5-oxo-1,2,4-oxadiazole Schemeconversion 11) of[117]. the terminalThe synthesis carboxylic of these acid inhibitors in 41 to primarywith azole amide bioisosteric 45 in quantitative groups is achievedyields using via 1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine (ZST152, conversionisobutylchloroformate, of the terminal N-methylmorpholine, carboxylic acid in 41and to aqueousprimary ammonia.amide 45 inPrimary quantitative amide yields 45 is using then 49b, Schemeisobutylchloroformate,converted 11 to)[ nitrile117]. 46 The using N synthesis-methylmorpholine, trifluoroacetic of these anhydride inhibitorsand aqueous and withtriethylamine ammonia. azole bioisosteric inPrimary 47% yield. amide groups Compound 45 is achievedthen 46 via conversionconvertedis guanylated to of nitrile the[112] terminal 46 (see using Sections carboxylictrifluoroacetic 4.1.3 and acid anhydride 4.1.4, in 41 Schemesto and primary triethylamine 7 and amide 9) to 45 inafford 47%in quantitative yield.compound Compound 47 yields, from 46 using isobutylchloroformate,iswhich guanylated the heterocycle [112] (seeN is-methylmorpholine, constructed Sections 4.1.3 from and the 4.1.4, nitrile. and Schemes aqueousThe tetrazole 7 and ammonia. is9) introduced to afford Primary compoundby treating amide nitrile47,45 from is47 then convertedwhichwith sodium tothe nitrile heterocycle azide46 usingin is the constructed trifluoroaceticpresence fromof zinc(II) the anhydride nitrile. bromide The and tetrazoleto triethylamineafford is compoundintroduced in 47%by48a treating, using yield. nitrilethe Compound click 47 chemistry procedure reported by Demko and Sharpless [120]. Although the use of zinc(II) bromide 46 iswith guanylated sodium [azide112] (seein the Sections presence 4.1.3 of zinc(II)and 4.1.4 bromide Schemes to afford7 and 9compound) to afford 48a compound, using the click47, from chemistryin the synthesis procedure of Boc-amin reportedo tetrazoles by Demko derived and Sharple from αss-amino [120]. acidsAlthough has been the us reportede of zinc(II) [121], bromide the use whichof thea Lewis heterocycle acid may is result constructed in partial fromdeprotection the nitrile. of compound The tetrazole 48a during is introduced the reaction. by Ammonium treating nitrile 47 within the sodium synthesis azide of Boc-amin in the presenceo tetrazoles of derived zinc(II) from bromide α-amino to acids afford has compound been reported48a [121],, using the theuse click ofchloride a Lewis represents acid may anresult alternative in partial reagent deprotection [122]. The of compound 5-oxo-1,2,4-oxadiazole 48a during the 48b reaction. is synthesized Ammonium from chemistry procedure reported by Demko and Sharpless [120]. Although the use of zinc(II) bromide in chloridenitrile 47 represents using hydroxylamine an alternative hydrochloridereagent [122]. Theand 5-oxo-1,2,4-oxadiazole sodium bicarbonate in48b DMSO, is synthesized followed from by α the synthesisnitriletreatment 47 of withusing Boc-amino CDI hydroxylamine and DBU tetrazoles [123]. hydrochloride Acidic derived deprotection from and-amino sodium of 48a acids –bicarbonateb is achieved has been in as reported DMSO,described followed [ 121previously], the by use of a Lewistreatment[117,119]. acid may withDespite resultCDI obtainingand in DBU partial [123].lower deprotection Acidic yields deprotection for ofheterocyclic compound of 48a –adductsb48a is achievedduring 49a–b as the (7%–13%) described reaction. previouslyrelative Ammonium to chloride[117,119].bioisosteres represents Despite 44a– anc (19%–32%),obtaining alternative lower the reagent inhibitory yields [122 for ].potential Theheterocyclic 5-oxo-1,2,4-oxadiazole of 5-oxo-1,2,4-oxadiazole adducts 49a–b (7%–13%)48b ZST152is synthesized relative (49b) was to from nitrilebioisosteres47 using hydroxylamine 44a–c (19%–32%), hydrochloride the inhibitory and potential sodium of bicarbonate 5-oxo-1,2,4-oxadiazole in DMSO, followedZST152 (49b by) treatment 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 Molecules 2016, 21, 615 11 of 32

(19%–32%), the inhibitory potential of 5-oxo-1,2,4-oxadiazole ZST152 (49b) was significant (95% at Molecules 2016, 21, 615 11 of 31 1 mM, IC50 = 18 µM, Ki = 7 µM), while tetrazole ZST086 (49a) displayed 91% inhibition at 1 mM, with IC50 =Molecules 34 µM 2016 and, 21, K615i = 14 µM[117]. 11 of 31 significant (95% at 1 mM, IC50 = 18 μM, Ki = 7 μM), while tetrazole ZST086 (49a) displayed 91% inhibition5 at 1 mM, with IC50 = 34 μM and Ki = 14 μM [117]. 4.1.6.significantN -(1-Iminoalk(en)yl)- (95% at 1 mM,L -OrnithineIC50 = 18 μM, Derivatives Ki = 7 μM), while tetrazole ZST086 (49a) displayed 91% inhibition at 1 mM, with IC50 = 34 μM and Ki = 14 μM [117]. Amidines4.1.6. N5-(1-Iminoalk(en)yl)- are commonly knownL-Ornithine bioisosteres Derivatives of the guanidine moiety [124]. N5-(1-iminoalk(en)yl)-L- 5 5 ornithines4.1.6. AmidinesN such-(1-Iminoalk(en)yl)- as areN commonly-(1-imino-3-butenyl)-L-Ornithine known bioisoster DerivativesL-ornithinees of the guanidine (L-VNIO, moiety54a [124]., Figure N5-(1-iminoalk(en)yl)-2), are reportedL- to inhibit NOS by competing5 with L-arginine for binding at the enzyme catalytic-site [125–127]. ornithinesAmidines such are as commonly N -(1-imino-3-butenyl)- known bioisosterL-ornithinees of the guanidine(L-VNIO, moiety54a, Figure [124]. 2), N 5are-(1-iminoalk(en)yl)- reported to inhibitL- Therefore,ornithinesNOS by derivatives competing such as N 5-(1-imino-3-butenyl)-with of this L-arginine class offor compoundsbindingL-ornithine at the(L-VNIO,have enzyme been 54a, catalytic-site Figure extensively 2), are[125–127]. reported studied Therefore, to inhibit as DDAH 5 inhibitors.NOSderivatives by Thecompeting of syntheticthis classwith of approachL-arginine compounds for to bindinghave afford been atN the-(1-iminoalk(en)yl)-extensively enzyme studiedcatalytic-site asL -ornithineDDAH [125–127]. inhibitors. derivativesTherefore, The 54 (Schemederivativessynthetic 12)[127 approach of,128 this], utilized classto afford of acompounds N similar5-(1-iminoalk(en)yl)- approach have been toL theextensively-ornithineN-but-3-ynyl-2-chloro-acetamidine derivatives studied as 54 DDAH (Scheme inhibitors. 12) [127,128], probeThe (18) (see Sectionsyntheticutilized 4.1.2a approach similar, Scheme approach to afford5)[ 109 toN]. 5-(1-iminoalk(en)yl)-the The N-but-3-ynyl-2-chloro-acetamidine R1 furnishedL-ornithine nitrile 50 derivativesis converted probe 54 to(Scheme(18 its) (see corresponding 12)Section [127,128], 4.1.2, ethyl 1 imidicutilizedScheme ester 51a5) similar [109].by bubbling The approach R furnished dry to hydrochloricthe nitrileN-but-3-ynyl-2-chloro-acetamidine 50 is acidconverted gas through to its corresponding a solution probe ofethyl(18 anhydrous) (see imidic Section ester ethanol 4.1.2,51 by (16). bubbling dry hydrochloric1 acid gas through αa solution of anhydrous ethanol (16). The ethyl imidic The ethylScheme imidic 5) [109]. ester The51 Ris furnished then reacted nitrile with 50 isN converted-Boc-L-ornithine to its corresponding (52) to afford ethyl the imidic protected ester 51 adduct by 53. ester 51 is then reacted with Nα-Boc-L-ornithine (52) to afford the protected adduct 53. Deprotection bubbling dry hydrochloric acid gas through a solution of anhydrous ethanol5 (16). The ethyl imidic Deprotection is obtained by treatment with 6 M HCl to achieve5 the N -(1-iminoalk(en)yl)-L-ornithine esteris obtained 51 is then by reacted treatment with with Nα-Boc- 6 ML-ornithine HCl to achieve(52) to afford the N the-(1-iminoalk(en)yl)- protected adductL 53-ornithine. Deprotection 54 in 54 in excellent overall yields (84%–94%). isexcellent obtained overall by treatment yields (84%–94%). with 6 M HCl to achieve the N5-(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 Schemeand Conditions 12. Synthetic: (i) dry schemeHCl(g) at for ˝0 °Cgenerating for 1 h, stop N5-(1-iminoalk(en)yl)- HCl(g), then 0 °C˝ forL-ornithine 4 h and RT derivatives. overnight; Reagents(ii) H2O, and Conditions:(i) dry HCl(g) at 0 C for 1 h, stop HCl(g), then 0 C for 4 h and RT overnight; (ii)H2O, 5 °C, pH = 10, 1.5 h, adjust pH = 7, overnight, Dowex 50WX8-400 (H2O then 10% aqueous pyridine); 5 ˝C,and pH Conditions = 10, 1.5: h,(i) adjustdry HCl pH(g) at = 0 7, °C overnight, for 1 h, stop Dowex HCl(g), 50WX8-400 then 0 °C for (H 4 hO and then RT 10%overnight; aqueous (ii) pyridine);H2O, (iii) 6 M HCl, EtOAc, 84%–94% from Nα-Boc-L-ornithine (52). Adapted2 with permission from [127]. 5 °C, pH = 10, 1.5 h, adjust pH = 7, overnight,α Dowex 50WX8-400 (H2O then 10% aqueous pyridine); (iii) 6 M HCl, EtOAc, 84%–94% from N -Boc-L-ornithine (52). Adapted with permission from [127]. (Copyrightiii) 6 M HCl, 1999 EtOAc, American 84%–94% Chemical from Society. 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),

Figureas synthesized 2. N5-(1-Iminoalk(en)yl)- by Kotthaus et al.L-ornithine [114] and derivatives,Wang et al. [128,129]. 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]. as synthesizedThe significant by Kotthaus structuralet al. similarity[114] and Wangof theet N al.5-(1-iminoalk(en)yl)-[128,129]. L-ornithine derivatives with DDAHThe substrates, significant ADMA structural (1) and similarity L-NMMA of (the2) led N 5different-(1-iminoalk(en)yl)- groups to studyL-ornithine this class derivatives of compounds with 5 DDAHTheas potential significant substrates, DDAH structural ADMA inhibitors (1) similarityand [114,128]. L-NMMA ofKotthaus the(2) ledN 5 etdifferent-(1-iminoalk(en)yl)- al. [114] groups examined to study fiveL-ornithine thisN -(1-iminoalk(en)yl)- class of derivatives compoundsL- with DDAHasornithines potential substrates, with DDAH varying ADMA inhibitors side-chain (1) [114,128]. and lengths L-NMMA Kotthaus and bond (et2 )al. saturation led [114] different examined for their groups five ability N5-(1-iminoalk(en)yl)- to inhibit study L-citrulline this classL- of formation by human DDAH-1 using both high performance liquid chromatography and colorimetry. compoundsornithines as with potential varying side-chain DDAH lengths inhibitors and bond [114, 128saturation]. Kotthaus for their abilityet al. to [inhibit114] examinedL-citrulline five formationL-VNIO ( 54aby human, Figure DDAH-1 2) emerged using as both the highbest perfDDAH-1ormance inhi liquidbitor chromatographyof this series of derivativesand colorimetry. (97% N5-(1-iminoalk(en)yl)-L-ornithines with varying side-chain lengths and bond saturation for their L-VNIOinhibition (54a at , 1Figure mM, IC2)50 emerged = 13 μM, as K ithe = 2 best μM). DDAH-1 However, inhi increasingbitor of this the seriesmain-chain of derivatives length by (97% one ability to inhibit L-citrulline formation by human DDAH-1 using both high performance liquid inhibitioncarbon atom at 1from mM, ornithine IC50 = 13 to μ lysineM, Ki reduced= 2 μM). its However, DDAH inhibitory increasing potential. the main-chain length by one chromatography and colorimetry. L-VNIO (54a, Figure2) 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, IC = 13 µM, K = 2 µM). However, increasing the 50 i main-chain length by one carbon atom from ornithine to lysine reduced its DDAH inhibitory potential.

Molecules 2016, 21, 615 12 of 32

Wang et al. [128] investigated this class of compounds as dual DDAH-1 and NOS 5 inhibitors,Molecules reporting 2016, 21, 615 the synthesis and kinetic parameters for four N -(1-iminoalkyl)-L-ornithines.12 of 31 N5-(1-Iminopropyl)-L-ornithine (L-IPO 54b, Figure2) was identified as the best compound exhibiting Wang et al. [128] investigated this class of compounds as dual DDAH-1 and NOS inhibitors, inhibition of both human DDAH-1 (Ki = 52 µM) and rat nNOS (Ki = 3 µM). Wang et al. [129] additionally reporting the synthesis and kinetic parameters for four N5-(1-iminoalkyl)-L-ornithines. developed an irreversible human DDAH-1 inhibitor, N5-(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, Figure2), 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[129] additionally developed (11). The an synthesis irreversible of human Cl-NIO DDAH-1 (54c) inhibitor, is similar N5-(1-imino-2-chloroethyl)- to that described for the 5 α N -(1-iminoalk(en)yl)-L-ornithine (Cl-NIO,L -ornithine54c, Figure 2), derivatives whose chemical (Scheme structure 12). reflects Here, aN combination-Boc-L-ornithine of both L-IPO is reacted with( 2-chloro-1-ethoxyethanimine,54b) and 2-chloroacetamidine (11). and Thethe synthesis intermediate of Cl-NIO obtained (54c) is similar is deprotected to that described with 4.6for the M HCl in dioxaneN5-(1-iminoalk(en)yl)- to generate Cl-NIOL-ornithine (54c ,derivatives Figure2) in (Scheme low yields 12). Here, (10%). Nα The-Boc- inhibitionL-ornithine potency is reacted of with Cl-NIO (54c)2-chloro-1-ethoxyethanimine, was assessed via colorimetric and assaythe intermediate and exhibited obtainedKi = is 1.3 deprotectedµM. In summary, with 4.6 M several HCl in of the N5-(1-iminoalk(en)yl)-dioxane to generateL -ornithineCl-NIO (54c derivatives, Figure 2) in (54a low–c ,yields Figure (10%).2) are The potent inhibition human potency DDAH-1 of Cl-NIO inhibitors. Off-target(54c) was effects assessed on the via broader colorimetric NO pathwayassay and(NOSs, exhibited arginases, Ki = 1.3 μ AGTX-2)M. In summary, may either several represent of the 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 Vallance4.2.1. Pentafluorophenylet al. [130] identified (PFP) Sulfonate pentafluorophenyl Esters (PFP) sulfonate esters 61 as nonspecific PaDDAH and bacterialVallance arginine et al.deaminase [130] identified (ADI) pentafluorop inhibitors,henyl after highlighting(PFP) sulfonate their esters structural 61 as nonspecific similarity with the cysteinePaDDAH protease and bacterial family arginine of enzymes. deaminase This class(ADI) of inhi inhibitorbitors, canafter be highlighting obtained in their moderate structural to good yieldssimilarity (44%–75%) with viathe thecysteine 1,3-dipolar protease cycloadditionfamily of enzymes. between This class nitrone of inhibitor60 and can the be electron obtained deficient in vinyl-appendedmoderate to good PFP sulfonateyields (44%–75%)57 (Scheme via the 131,3-dipolar)[131]. cycloaddition The shelf-stable between PFP nitrone vinylsulfonate 60 and the57 is synthesizedelectron indeficient moderate vinyl-appended yield (59%) PFP from sulfonate pentafluorophenol 57 (Scheme (13)55) and[131]. 2-chloroethane-1-sulfonyl The shelf-stable PFP 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 see in [ 133good,134 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: NaHCO , anhydrous MgSO , DCM, reflux, 24 h, 73%–90% [133]; Method B: NaOAc, EtOH, RT, 90% [134]; (3iii) dry toluene, reflux, 41–24 h, 44%–75%. Reproduced in part iii Methodfrom B: [130–133] NaOAc, with EtOH, permission RT, 90% of [134 the]; Royal ( ) drySociet toluene,y of Chemistry reflux, and 1–24 the h, American 44%–75%. Chemical Reproduced Society. in part 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 Theregioselective electron 4C-substituted deficiency of adduct the vinyl of the PFP isoxazolidine sulfonate dipolarophile in compound 5761,affords contrary the to reversedthe regioselective5C-substituted 4C-substituted adduct typically adduct observed of thewith isoxazolidineolefins. Although in this compound was previously61, contraryobserved for 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-aryl- isoxazolidineand C-alkyl-61Nis-methyl increasingly species favoured with both atelectron-donating higher temperatures. and electron-withdrawing Each of the nitrones substituents60 reported in by Caddickadditionet al. to[ 131poly-] participates and hetero-aromatic in cycloaddition substituents with. This the robust vinyl synthetic PFP sulfonate methodology57, including provides theC-aryl- and Cability-alkyl- toN generate-methyl a speciesstructurally with di bothverse electron-donatinglibrary of PFP sulfonate and esters electron-withdrawing 61 with moderate ease substituents and high in additionyields to [130]. poly- Several and hetero-aromatic PFP sulfonate esters substituents. 61 have been This screened robust for synthetic their activity methodology as PaDDAH providesand ADI the 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 Molecules 2016, 21, 615 13 of 32

ADI inhibitors at two concentrations (500 µM and 50 µM), with five compounds emerging as relatively Molecules 2016, 21, 615 13 of 31 potent PaDDAH inhibitors (IC50 = 16–58 µM, see [130] for structures). Vallance et al. revealed the modeinhibitors of inhibition at two was concentrations competitive (500 utilising μM and time-dependence50 μM), with five compounds and reversibility emerging experiments as relatively [130]. Interestingly,potent Pa theDDAH PFP inhibitors sulfonate (IC esters50 = 6116–58have μM, been see proposed[130] for structures). as potential Vallance antibacterial et al. revealed agents, howeverthe no datamode has of been inhibition reported was identifyingcompetitive utilising if PaDDAH time-dependence inhibition modulates and reversibility bacterial experiments growth or [130]. viability. Likewise,Interestingly, no studies the utilizing PFP sulfonate human esters DDAH 61 havehave beenbeen reportedproposed identifying as potential whether antibacterial the PFP agents, sulfonate estershowever exhibit specificityno data has forbeen bacterial reportedPa identifyingDDAH. Non-specific if PaDDAH inhibition PFP sulfonate modulates ester bindingbacterial togrowth both or 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 a combination4.2.2. Indolylthiobarbituric of virtual screening Acid Derivatives using a compound library comprising 308,000 structures and subsequentThe biological indolylthiobarbituric screening. acid Among derivatives the 109 68 highest [139] were scoring identified compounds, as PaDDAH 90 wereinhibitors commercially by a availablecombination and used of in virtual colorimetric screening enzyme using activity a compou assays.nd library Three compoundscomprising 308,000 consisting structures of two chemicaland scaffoldssubsequent were identified biological asscreening. potential AmongPaDDAH the 109 inhibitors. highest scoring One of compounds, these scaffolds, 90 were indolylthiobarbituric commercially acid (available68a), together and used with in colorimetric additional enzyme compounds activity 68bassays.–c identifiedThree compounds from aconsisting similarity of two search, chemical 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 Thisinhibition class [114]. of inhibitors is comprised of thiobarbituric acid module 64 and indolyl module 67 (Scheme 14This). Theclass thiobarbituric of inhibitors is acid comprised module of64 thiobarbituricis synthesized acid using module a modified 64 and indolyl version module of the method67 described(Scheme by 14). Fisher The thiobarbituric and Mering acid in 1903,module by 64reaction is synthesized of N using-substituted a modified 2-thiourea version of63 thewith method diethyl malonatedescribed (62)[ by140 Fisher,141]. Theand indolylMering modulein 1903, 67by isreaction alkylated of N by-substituted deprotonation 2-thiourea of indole-3-carbaldehyde 63 with diethyl (65) andmalonate reaction (62) with[140,141]. 2-chloro- The indolylN-furan-2-ylmethyl-acetimide module 67 is alkylated by deprotonation (66) using a methodof indole-3-carbaldehyde previously reported (yield(65 83%)) and [ reaction142]. The with aldehyde 2-chloro- substituentN-furan-2-ylmethyl-acetimide of the indolyl derivative (66) using a67 methodis subsequently previously reported reacted with the thiobarbituric(yield 83%) [142]. acid The derivative aldehyde64 substituentunder acidic of the conditions indolyl derivative to afford 6768 is(yields subsequently not reported) reacted with [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 Ebselenwith well-known (2-phenyl-1,2-benzisoselenazol-3(2 antioxidant properties [144], wasH)-one, recently73), identified a selenazole as a potent heterocyclic DDAH-1 compound inhibitor. with well-knownThis was antioxidantachieved by utilizing properties a high [144 throughput], was recently screening identified (HTS) assay as of a two potent commercial DDAH-1 libraries inhibitor. This wascomprising achieved 2446 by compounds utilizing a high[145]. throughputThe HTS identifi screeninged each (HTS) compound’s assay of twoability commercial to inhibit librariesthe 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 S L conversionto inhibit of thehuman synthetic DDAH-1 substrate, using AD-methyl-MA as the-thiocitrulline substrate. The (SMTC) quantification to citrulline of inhibition and methanethiol was by Pa DDAH. 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 Molecules 2016, 21, 615 14 of 32 Molecules 2016, 21, 615 14 of 31 humanmeasured DDAH-1 using inhibition derivatized by ebselen citrulline was formation subsequently and colorimetric determined detection. by dose-response, Characterisation reversibility, of and ESI-MSPaDDAH experiments. and human DDAH-1 inhibition by ebselen was subsequently determined by dose-response, Whilereversibility, ebselen and (73 ESI-MS) is commercially experiments. available, it can be prepared via two main methods [146–148]. The methodWhile described ebselen (73 in) is Scheme commercially 15[ 147available,,148] isit can preferred be prepared due via to two its shortermain methods reaction [146–148]. times and elementaryThe method design. described This methodin Scheme involves 15 [147,148] the treatment is preferred of due benzoic to its acid shorter (69 )reaction with thionyl times 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, cyclizationwhose cyclization to ebselen to (73 ebselen) is promoted (73) is promoted by copper(II) by copper(II) bromide bromide oxidant. oxidant.

SchemeScheme 15. Synthetic 15. Synthetic scheme scheme forgenerating for generating ebselen ebselen (73). (Reagents73). Reagents and Conditionsand Conditions:(i): (1) (i)DCM, (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) samewith solutionpermission as from step ([147],iii), CuBr copyright2, 30 min1989 atAmerican78 C, Chemical then 2 h atSo RT,ciety, 63%. and Adapted[148], copyright with permission 2003 fromThe [147 Pharmaceutical], copyright 1989 Society American of Japan. Chemical Society, and [148], copyright 2003 The Pharmaceutical 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 respective catalytic Ebselen is reported to irreversibly inhibit both PaDDAH (IC50 = 96 ˘ 2 nM) and human DDAH-1 cysteine. In silico docking experiments suggest inhibitor binding is supported by π-stacking (IC = 330 ˘ 30 nM), forming a covalent selenosulfide bond with each enzyme’s respective catalytic 50interactions 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 Phe76Ebselen in addition is the most to other potent polar DDAH interactions inhibitor involving with ‘non-substrate-like’ His162. In vitro structureexperiments reported demonstrated in the ebselenliterature exhibits to date no effect and demonstrates on human DDAH-1 an anti-inflammatory expression response [145]. [149]. However, its application as Ebselena DDAH inhibitor is the mostis limited potent due to DDAH its poor inhibitorDDAH selectivity. with ‘non-substrate-like’ A wide range of molecular structure targets reported are in themodulated literature by ebselen to date including: and demonstrates NADPH oxidase an 2 anti-inflammatory[150,151], H+/K+ ATPase response transporter [149 [152],]. However,Ca2+- its applicationand phospholipid-dependent as a DDAH inhibitor protein is kinase limited C due[151], to lipoxygenase its poor DDAH [153], selectivity.thioredoxin Areductase wide range of molecular[154], and targets metallothionein are modulated [155]). byMoreover, ebselen cellular including: toxicity NADPH in leukocytes oxidase and 2[ 150hepatocytes,151], H +has/K been+ ATPase transporterreported, [152 further], Ca reducing2+- and thephospholipid-dependent likelihood of ebselen’s use protein in clinical kinase practice C [156,157]. [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 practice [1564-Hydroxy-2-nonenal,157]. (4-HNE, 76) is a cytotoxic aldehyde generated from lipid peroxidation during [158,159] and is abundant in the body. 4-HNE is highly reactive toward 4.2.4.proteinaceous 4-Hydroxy-2-Nonenal nucleophilic groups, such as thiol and imidazole moieties. Nucleophilic attack affords Michael adducts, whilst Schiff bases are formed with primary amines (e.g., lysine residues) [160–163]. 4-Hydroxy-2-nonenalDifferent methods have been (4-HNE, reported76) for is athe cytotoxic synthesis aldehydeof 4-HNE ( generated76) and its derivatives from lipid [164–166]. peroxidation duringThe inflammation method reported [158 by ,Soulér159]and et al. is[166] abundant is described in here the due body. to its 4-HNE simplicity, is speed, highly and reactive versatility toward proteinaceous(Scheme 16). nucleophilic The synthesis groups, is completed suchas in thiola single and cross-metathesis imidazole moieties. reaction Nucleophilic between octen-3-ol attack (75 affords) Michaeland adducts,2-propenal whilst (74) in Schiff the presence bases are of Hoveyda-Grubbs formed with primary 2nd generation amines (e.g., ruthenium lysine catalyst residues) under [160 a– 163]. Differentnitrogen methods atmosphere have beenand in reported dry oxygen-free for the synthesissolvent (Scheme of 4-HNE 16). The (76) reaction and its derivativesproceeds at room [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. Molecules 2016, 21, 615 15 of 32 MoleculesMolecules 2016, 2016 21, 615, 21, 615 15 of 3115 of 31

Scheme 16. Synthetic scheme for generating 4-hydroxy-2-nonenal (4-HNE, 76). Reagents and Scheme 16.16.Synthetic Synthetic scheme scheme for generatingfor generating 4-hydroxy-2-nonenal 4-hydroxy-2-nonenal (4-HNE, (4-HNE,76). Reagents 76). andReagents Conditions and: Conditions: (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.). (Conditionsi) dry oxygen-free: (i) dry oxygen-free DCM, RT, 25 DCM, h, 75%, RT, Hoveyda-Grubbs 25 h, 75%, Hoveyd 2nda-Grubbs generation 2nd (0.025generation eq.). (0.025 eq.). 4-HNE is reported to react with cysteine and histidine residues and it was this reactivity that 4-HNEled Forbes is reportedet al. to investigate to react withits use cysteine as a human and DDAH-1 histidinehistidine inhibitor residues [167]. and In it Forbes’ was this experiments, reactivity that led ForbesForbesDDAH et activity al. to investigate was measured its viause both as acolorimetric humanhuman DDAH-1DDAH-1 and radioisotope inhibitorinhibitor activity [[167].167]. In assays, Forbes’ with experiments, 4-HNE DDAHshowing activity dose-dependent was measured inhibition via bothboth (IC colorimetriccolorimetric50 = 50 μM) and andand near radioisotoperadioisotope complete inhibition activity at assays, 500 μ M. with Mass 4-HNE4-HNE spectrometry experiments elucidated the mechanism of inhibition, whereby the formation of a showing dose-dependent inhibition (IC 50 == 50 50 μµM)M) and and near complete inhibition at 500 µμM. Mass spectrometryMichael adduct experimentsexperiments between elucidated 4-HNEelucidated and the His173the mechanism mechan of the DDAH-1ism of inhibition, of inhibition,active-site whereby occurs. whereby the formation the formation of a Michael of a Michael adduct between 4-HNE and His173 of the DDAH-1 active-site occurs. adduct4.2.5. between PD 404182 4-HNE and His173 of the DDAH-1 active-site occurs.

4.2.5. PD 6 404182H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2- c]-[1,3]benzothiazine (PD 404182, 79), is a commercially available heterocyclic pyrimidobenzothiazine and a suitable lead compound for SAR 6studiesH-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2- due to its simple synthetic methodology [168].cc]-[1,3]benzothiazine]-[1,3]benzothiazine PD 404182 (79) is a (PDcompound (PD 404182, 404182, listed 7979in), ),the is is a commerciallyLibrary of available availablePharmacologically heterocyclic heterocyclic Active pyrimidobenzothiazine pyrimidobenzothiazine Compounds (LOPAC) and and isa suitablereported a suitable leadto have lead compound antibacterial compound for SAR for SARstudies[168,169] studies due to and due its antiviralsimple to its simplesynthetic[170,171] synthetic activity.methodology Ghebremariam methodology [168]. PD et [ 168al.404182 [61]]. utilized PD(79) 404182 is aa colorimetriccompound (79) isa assaylisted compound to in the listedLibraryscreen in of the Pharmacologicallya Librarylibrary of of over Pharmacologically 1200 Active compounds Compounds for Active their Compounds(LOPAC)ability to inhibitand (LOPAC) is thereported conversion and to is have reportedof ADMA antibacterial toto have antibacterial[168,169]L-citrulline and [168 antiviral and,169 dimethylamine] and [170,171] antiviral activity. by [170 recombinant,171 Ghebremariam] activity. human Ghebremariam DDAH-1.et al. [61] Compoundsutilizedet al. [61 ]a utilizedcolorimetric with reasonable a colorimetric assay to assayscreeninhibitory to a screenlibrary potentials a libraryof over were of1200 over subjected compounds 1200 to compounds kinetic for characterisation their for theirability ability via to fluorimetricinhibit to inhibit the assay theconversion conversionusing the ofsynthetic ADMA of ADMA to substrate, S-methylthiocitrulline. Reversibility and ESI-MS studies were additionally undertaken. toL-citrullineL-citrulline and and dimethylamine dimethylamine by by recombinant recombinant human human DDAH-1. DDAH-1. Compounds Compounds with reasonablereasonable PD 404182 was subsequently evaluated for its ability to reduce and to protect against inhibitory potentials were subjected to kinetic characterisation via fluorimetric assay using the synthetic inhibitorylipopolysaccharide-induced potentials were subjected NO production.to kinetic characterisation PD 404182 (79) viawas fluorimetric found to be assaya potent using irreversible the synthetic substrate, S-methylthiocitrulline. Reversibility and ESI-MS studies were additionally undertaken. substrate,competitive S-methylthiocitrulline. human DDAH-1 inhibitor Reversibility (IC50 = 9and μM) ESI-MSand showed studies promise were as additionally an anti-inflammatory undertaken. PD 404182and antiangiogenic was subsequently agent. evaluated for its ability to reduce angiogenesis and to protect againstagainst lipopolysaccharide-inducedThe synthesis of PD404182 NO NO production. can be achieved PD PD via 404182 404182 two routes ( (7979)) was was[172,173]. found found Both to approachesbe a potentpotent generate irreversibleirreversible competitivereasonable human to high DDAH-1 yields (61%–88%) inhibitor over (IC50 several== 9 9 μµM) synthetic and showed steps. The promise first approach as an anti-inflammatoryanti-inflammatory involves the and antiangiogenicreaction of 2-(2′-haloaryl)-tetrahydropyrimidine agent. 77 with carbon in the presence of sodium Thehydride, synthesis synthesis followed of of PD404182by PD404182 hydrolysis can canof be the achieved be carbamodithioate achieved via tw viao routes twoderivative routes [172,173]. 78 [and172 Both ,173subsequent]. approaches Both treatment approaches generate generatereasonablewith reasonablecyanogen to high yieldsbromide to high (61%–88%) to yields afford (61%–88%) 79over (Scheme several over17; synthetic Route several A). steps.synthetic Alternatively, The first steps. approachthe The2-(2′-haloaryl)- first involves approach the tetrahydropyrimidine 77 can be reacted with tert-butylisothiocyanate (80) in the presence of sodium involvesreaction of the 2-(2 reaction′-haloaryl)-tetrahydropyrimidine of 2-(21-haloaryl)-tetrahydropyrimidine 77 with carbon disulfide77 with in carbon the presence disulfide of sodium in the hydride to afford intermediate 81, followed by treatment with trifluoroacetic acid to afford 79 presence of sodium hydride, followed by hydrolysis of the carbamodithioate derivative 78 and hydride,(Scheme followed 17; Route by B).hydrolysis of the carbamodithioate derivative 78 and subsequent treatment subsequentwith cyanogen treatment bromide with to cyanogenafford 79 bromide(Scheme to17; afford Route79 A).(Scheme Alternatively, 17; Route the A). 2-(2 Alternatively,′-haloaryl)- thetetrahydropyrimidine 2-(21-haloaryl)-tetrahydropyrimidine 77 can be reacted with77 can tert be-butylisothiocyanate reacted with tert-butylisothiocyanate (80) in the presence (of80 sodium) in the presencehydride ofto sodiumafford intermediate hydride to afford 81, followed intermediate by 81treatment, followed with by treatmenttrifluoroacetic with trifluoroaceticacid to afford acid 79 to(Scheme afford 17;79 (SchemeRoute 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. SyntheticSynthetic scheme scheme for for generating generating PD PD 404182 404182 (79 ).(79Reagents). Reagents and Conditionsand Conditions: Route: Route A: (i) A: DMF, (i)

2 ˝ 2 NaH,DMF, CSNaH,2, argon CS , argon atmosphere, atmosphere, 80 C, 80 12 °C, h, 12 88%; h, 88%; (ii) (1) (ii) 0.1 (1) M 0.1 NaOH, M NaOH, MeOH/H MeOH/H2O (9:1),O (9:1), reflux, reflux, 12 12 h; (2)h; (2) anhydrous anhydrous EtOH, EtOH, BrCN, BrCN, argon argon atmosphere, atmosphere reflux,, reflux, 2 h, 61%2 h, 61% (two (two steps). steps). Route Route B: (iii B:) DMF, (iii) DMF, NaH, ˝ argonNaH, argon atmosphere, atmosphere, 80 C, 280 h, °C, 62%; 2 h, (iv 62%;) TFA, (iv CHCl) TFA,3, molecularCHCl3, molecular sieves 4 Å,sieves reflux, 4 Å, 1 h,reflux, 85%. 1 Adapted h, 85%. withAdapted permission with permission from [172 ,from173]. [172,173]. Copyright Copy 1975,right 2010 1975, American 2010 American Chemical Chemical Society. Society.

Molecules 2016, 21, 615 16 of 32

Molecules 2016, 21, 615 16 of 31 4.2.6. Proton Pump Inhibitors 4.2.6. Proton Pump Inhibitors ProtonMolecules 2016 H+,/K 21, 615+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric16 of 31 acid Proton H+/K+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric acid secretionsecretion and and are characterizedare characterized by by a 2-pyridylmethylsulfinylbenzimidazolea 2-pyridylmethylsulfinylbenzimidazole scaffold. scaffold. The Thechemical chemical 4.2.6. Proton Pump Inhibitors structuresstructures of the of commonlythe commonly prescribed prescribed PPIs PPIs82a 82a––ff areare shown in in Figure Figure 3.3 . 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 HTSPPIs as experiments humanFigure 3.DDAH-1 Chemical utilising inhibitors structures a library of[174,175]. commonly of 130,000 Initial prescribed compounds screening proton was pump and undertaken recombinantinhibitors using(PPIs) enzyme82aa colorimetric–f. identified PPIsassay as human with ADMA DDAH-1 as the inhibitors substrate. [174 Further,175]. characterization Initial screening of compounds was undertaken of interest using was a achieved colorimetric assayvia with aHTS fluorimetric ADMA experiments as assay the utilising substrate. using S -methylthiocitrullinea library Further of 130,000 characterization compounds [174,175]. ofSurface and compounds recombinant plasmonof resonance interestenzyme (SPR)identified was achievedwas PPIs as human DDAH-1 inhibitors [174,175]. Initial screening was undertaken using a colorimetric via aadditionally fluorimetric utilized assay usingto detectS-methylthiocitrulline the binding of PPIs to [174 amine-coupled,175]. Surface DDAH-1 plasmon (CM5 resonance sensor (SPR)chip) was assay with ADMA as the substrate. Further characterization of compounds of interest was achieved additionally[175]. All utilized commercially to detect available the binding PPIs were of PPIs found to to amine-coupled reversibly inhibit DDAH-1 DDAH-1 (CM5 (IC50sensor = 51–63 chip) μM). [175]. viaInterestingly, a fluorimetric in vitro assay experiments using S-methylthiocitrulline using human endothelial [174,175]. cellsSurface and plasmon in vivo experimentsresonance (SPR) in mice was All commercially available PPIs were found to reversibly inhibit DDAH-1 (IC = 51–63 µM). additionallydemonstrated utilized that PPIs to detectincrease the ADMA binding concentrat of PPIs ionsto amine-coupled [175] further supportingDDAH-1 (CM5 the evidence sensor50 chip) that in vitro in vivo Interestingly,[175].prolonged All commercially use of PPIsexperiments is available associated usingPPIs with were humanan incr foundeased endothelial to riskreversibly of cardiovascular cells inhibit and DDAH-1 disease (ICexperiments [176,177].50 = 51–63 μM). in mice demonstratedInterestingly,PPIs are that comprisedin PPIsvitro increaseexperiments of pyridine ADMA using and concentrations humanbenzimidazol endotheliale moieties [175 cells] further withand indiffer supporting vivoent experiments substituents. the evidence in Theirmice that prolongeddemonstratedsyntheses use are of PPIsprotectedthat isPPIs associated increaseby current ADMA with patent an concentrats increased (with theions riskexception [175] of cardiovascular further of omeprazole supporting disease (82a the)). evidence [The176 ,177original ].that PPIsprolongedpatent are for comprised omeprazoleuse of PPIs ofis(82a associated pyridine) [178] details with and benzimidazolean preparation increased riskof the moietiesof 2cardiovascular-(chloromethyl)pyridine with different disease [176,177]. substituents. derivative 84. Their synthesesCondensationPPIs are are protected comprised [179] of by compoundof current pyridine patents 84and with benzimidazol (with 2-mercaptobenzimidazole the exceptione moieties ofwith omeprazole differ derivativeent substituents. ( 82a83 )).affords The Their originalthe patentsynthesesthioether for omeprazole precursorare protected (8582a, which)[by178 current ]is details subsequently patent preparations (with oxidized the ofexception to the the 2-(chloromethyl)pyridine sulfoxide of omeprazole in omeprazole (82a)). The (82a derivative original) using 84. patent for omeprazole (82a) [178] details preparation of the 2-(chloromethyl)pyridine derivative 84. Condensationconditions [ 179such] ofas compoundhydrogen peroxide84 with and 2-mercaptobenzimidazole a vanadium catalyst [180] derivative(Scheme 18;83 Routeaffords A). the thioether 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 suchconditions as hydrogen such peroxide as hydrogen and peroxide a vanadium and a catalyst vanadium [180 catalyst] (Scheme [180] 18(Scheme; Route 18; A). 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 SchemeSchemehydrocarbons, 18. 18.Synthetic Synthetic ethers, scheme schemealcohols for for, ketones, generatinggenerating nitriles, omeprazole omeprazole or H2O, (temperature:82a (82a). Reagents). Reagents 0 and °C Conditionsto and boiling Conditions: pointRoute : ofA: Route the (i) A: 1solvent, M NaOH 0.5–24 (2 eq.), h, 89%–93%. EtOH, RT, Route 8 h; (iiB:) catalyst:(iii) condensation, V2O5, NaVO conditions3, NH4VO not3, [(CH specified3COCH in2COCH patent;3)2 VO](iv) (i) 1 M NaOH (2 eq.), EtOH, RT, 8 h; (ii) catalyst: V2O5, NaVO3, NH4VO3, [(CH3COCH2COCH3)2VO] (0.0001–0.1[(CH3COCH eq.),2COCH H23O)22VO]: 10%–70% (0.006 eq.), aqueous 30% H 2orO 2, organicacetone, solution0–5 °C for (1–3 1 h eq.),then 20–22solvent: °C forhalogenated 1 h, 90%; (0.0001–0.1 eq.), H2O2: 10%–70% aqueous or organic solution (1–3 eq.), solvent: halogenated hydrocarbons,(v) 10% NaOH, ethers, nitrogen alcohols atmosphere,, ketones, 50 °C,nitriles, 3 h; ( vior) COH2O,2, extractiontemperature: into 0 1:1 °C v /tov isopropanol/toluene,boiling point of the ˝ hydrocarbons,solvent,reflux, 20–30 0.5–24 ethers, min, h, 42%89%–93%. alcohols, (across Route steps ketones, (B:v) and(iii nitriles,) (vicondensation,)). or H2O, conditions temperature: not specified 0 C toin boilingpatent; point(iv) of the solvent,[(CH3COCH 0.5–242COCH h,3 89%–93%.)2VO] (0.006 eq.), Route 30% B: H (2iiiO2), acetone, condensation, 0–5 °C for conditions 1 h then 20–22 not specified°C for 1 h, in90%; patent; ˝ ˝ (iv) [(CH(v) 10%3COCH NaOH,2COCH nitrogen3)2 VO]atmosphere, (0.006 eq.), 50 °C, 30% 3 h; H 2(viO)2 CO, acetone,2, extraction 0–5 intoC for 1:1 1 hv/v then isopropanol/toluene, 20–22 C for 1 h, 90%; ˝ (v) 10%reflux, NaOH, 20–30 nitrogen min, 42% atmosphere, (across steps 50(v) andC, 3 (vi h;)). (vi ) CO2, extraction into 1:1 v/v isopropanol/toluene, reflux, 20–30 min, 42% (across steps (v) and (vi)).

Molecules 2016, 21, 615 17 of 32

Further development of the synthetic method to yield omeprazole (82a)[180] identified that the couplingMolecules between 2016, 21, the615 pyridine and benzimidazole moieties can be modified to simplify purification.17 of 31 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 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-chloromercaptobenzimidazolecrude product containing omeprazole derivative (82a) produced86 with afrom primary the original amide pyridinesynthesis derivativeis prone to 87 to afforddiscolouration the acetamide during thioether the oxidation intermediate step [180].88 The, which revised is synthesis oxidized (Scheme to the 18; acetamide Route B) proceeds sulfoxide 89. Compoundvia condensation89 is subsequently of 2-chloromercaptobenzimidazole hydrolysed to the carboxylic derivative acid salt86 with90 under a primary alkaline amide conditions, pyridine with decarboxylationderivative 87 yielding to afford omeprazolethe acetamide (82a thioether) or lansoprazole intermediate ( 82c88, ),which depending is oxidized on the to the benzimidazole acetamide 86 and thesulfoxide pyridine 89. Compound87 derivatives 89 isused subsequently as the starting hydrolysed materials. to the carboxylic acid salt 90 under alkaline conditions, with decarboxylation yielding omeprazole (82a) or lansoprazole (82c), depending on the 4.2.7.benzimidazole 1,2,3-Triazoles 86 and the pyridine 87 derivatives used as the starting materials.

The4.2.7. synthesis 1,2,3-Triazoles of 1,2,3-triazoles 95 (Scheme 19) comprised of 4-halopyridine and benzimidazole 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 benzimidazolederivatives was fragments undertaken to human based on DDAH-1 reports that [181 id].entified A triazole binding linker of both was the selected 4-halopyridine to connect and these fragmentsbenzimidazole via copper(I)-catalysed fragments to human ‘click’ DDAH-1 cycloaddition [181]. A triazole between linker the was terminal selected alkyne to connect92 andthese azide 94 (Schemefragments 19 via). Thecopper(I)-catalysed alkyne 92 is synthesized‘click’ cycloaddition by Sonogashira between the coupling terminal alkyne of the 92 alkyne and azide derivative 94 with(Scheme the required 19). The halogenated alkyne 92 is synthesized species 91 by[182 Sonogashira,183]. The coupli azideng of94 theis alkyne synthesized derivative by with reaction the of diphenylphosphorylrequired halogenated azide withspecies the 91 required [182,183]. primary The alcoholazide 9493 [is184 synthesized]. by reaction of diphenylphosphoryl azide with the required primary alcohol 93 [184]. Human DDAH-1 inhibition, albeit low (IC50 = 422 µM, 57%, inhibition at 1 mM), was reported with triazoleHuman95a .DDAH-1 In general, inhibition,95a–b albeitfailed low to show(IC50 = significant 422 μM, 57%, inhibitory inhibition potential at 1 mM), and was may reported 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, (Ph P) PdCl , CuI, TEA, TIPS-acetylene, DMF, 80 ˝C, 72 h, 30%, 92b (Ph P) PdCl , CuI, TEA, 3 802 °C, 202 h, 48%; (ii) DPPA, DBU, toluene, RT, 16 h, 94a 55%, 94b 83%; (iii) CuI,3 sodium2 ascorbate,2 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,Society TEA, of Chemistry. DCM, RT, 24 h, 95a 65%, 95b 30%. Reproduced in part from [117] with permission of the Royal Society of Chemistry. 5. Conclusions 5. ConclusionsCurrent evidence suggest that inhibiting the DDAH family of enzymes may be used as a Currenttherapeutic evidence tool to indirectly suggest inhibit that inhibitingthe NOS enzy themes DDAH via increasing family concentrations of enzymes mayof endogenous be used as a therapeuticNOS inhibitors, tool to indirectlysuch as ADMA inhibit and the L-NMMA. NOS enzymes Therefore via the increasing development concentrations and synthesis of of endogenous DDAH 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 targetDDAH bacterial inhibitors DDAH being mayreported represent within a suitablethe last decade strategy characterized for the development by alternative of new features antimicrobial in agents;terms however of chemical further structure, investigations potency, enzyme are required. selectivity, This and mechanism has led to of different action. The classes importance of DDAH inhibitorsplaced being on each reported of these features within depends the last on decade the end characterized application of bythe alternativeinhibitor. Table features 3 summarizes in terms of chemicalthe most structure, potent DDAH potency, inhibitors enzyme documented selectivity, in and the mechanismliterature to date. of action. Some compounds, The importance namely placed on eachPFP ofsulfonate these features esters 61 depends or indolylthiobarbituric on the end application acid derivatives of the 68 inhibitor., exhibit Tablesignificant3 summarizes PaDDAH the most potent DDAH inhibitors documented in the literature to date. Some compounds, namely PFP Molecules 2016, 21, 615 18 of 32 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) (Table3). 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 19 of 32

Molecules 2016, 21, 615 19 of 31 Molecules 2016, 21, 615 19 of 31 Molecules 2016, 21, 615 19 of 31 Molecules 2016, 21, 615 Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. 19 of 31 Molecules 2016, 21, 615 19 of 31 Molecules 2016, 21, 615 Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. 19 of 31 Molecules 2016, 21, 615 Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. 19 of 31 Molecules 2016, 21, 615 Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. 19 of 31 ChemicalChemical Table 3. Summary of the main DDAH inhibitors and their kinetic parameters. inact ´1 Molecules 2016,Chemical 21, 615 NameTable 3. Summary Chemical of theStructure main DDAH inhibitors and Enzyme their kinetic parameters. IC50 (µM) K (µM) K K (min ) 19 ofReference 31 NumberChemical Chemical Name Chemical Structure Enzyme IC50 (μM) Ki (μM)i Kinact inact Reference ChemicalNumber Chemical Name Table 3. Summary Chemical of the Structure main DDAH inhibitors and Enzyme their kinetic parameters. IC50 (μM) Ki (μM) (minKinact−1 ) Reference ChemicalNumber Chemical Name Table 3. Summary Chemical of the Structure main DDAH inhibitors and Enzyme their kinetic parameters. IC50 (μM) Ki (μM) (minKinact− 1) Reference ChemicalNumber Chemical Name Table 3. Summary Chemical of the Structure main DDAH inhibitors and Enzyme their kinetic parameters. IC50 (μM) Ki (μM) (minKinact− 1) Reference ChemicalNumber Chemical Name Table 3. Summary Chemical of the Structure main DDAH inhibitors and Enzyme their kinetic parameters. IC50 (μM) Ki (μM) (minKinact−1) Reference ChemicalNumber ChemicalS-Nitroso- NameL-homocysteine Chemical Structure Enzyme IC50 (μM) Ki (μM) (minKinact−1 ) Reference (9) ChemicalNumber(9) S -Nitroso- ChemicalS-Nitroso-L-homocysteine NameL-homocysteine (HcyNO) Chemical Structure BovineBovine Enzyme DDAH-1DDAH-1 IC50 (75 75μM) Ki (690μM) 690 (minK0.38inact−1) 0.38Reference[105] [105] ChemicalNumber(9) ChemicalS(HcyNO)-Nitroso- NameL-homocysteine Chemical Structure Bovine Enzyme DDAH-1 IC5075 (μ M) Ki 690(μM) (minK0.38inact−1 ) Reference[105] Number(9) S(HcyNO)Chemical-Nitroso- L Name-homocysteine Chemical Structure Bovine Enzyme DDAH-1 IC50 75(μ M) Ki 690(μM) (min0.38−1 ) Reference[105] Number(9) S(HcyNO)-Nitroso- L-homocysteine Bovine DDAH-1 75 690 (min0.38 −1) [105] (9) (HcyNO)S-Nitroso- L-homocysteine Bovine DDAH-1 75 690 0.38 [105] (9) (HcyNO)S-Nitroso- L-homocysteine Bovine DDAH-1 75 690 0.38 [105] ((911) ) (HcyNO)S2-Chloroacetamidine-Nitroso- L-homocysteine BovinePaDDAH DDAH-1 1 75- 6903100 0.381.2 [105][106] (11)((119)) 2-Chloroacetamidine(HcyNO)2-ChloroacetamidineS-Nitroso- L-homocysteine BovinePaDDAH DDAH-1 1 75- - 3100690 3100 0.381.2 1.2[106][105] [106] ((119)) (HcyNO)2-Chloroacetamidine BovinePaDDAH DDAH-1 1 75- 3100690 0.381.2 [105][106] (11) 2-Chloroacetamidine(HcyNO) PaDDAH 1 - 3100 1.2 [106] (11) 2-Chloroacetamidine PaDDAH 1 - 3100 1.2 [106] (11) 2-ChloroacetamidineN-But-3-ynyl-2-chloro-acetamid PaDDAH 1 - 3100 1.2 [106] ((1118)) 2-ChloroacetamidineN-But-3-ynyl-2-chloro-acetamid HumanPaDDAH DDAH-1 1 350- 3100- 1.2- [106][109] ((1811)) 2-ChloroacetamidineNine-But-3-ynyl-2-chloro-acetamid HumanPaDDAH DDAH-1 1 350- 3100- 1.2- [109][106] (18) ((1118)) N-But-3-ynyl-2-chloro-acetamidineNine2-Chloroacetamidine-But-3-ynyl-2-chloro-acetamid HumanPaDDAH DDAH-1 1 350 350- 3100- -1.2- -[106][109] [109] (18) Nine-But-3-ynyl-2-chloro-acetamid NH NH Human DDAH-1 350 - - [109] (18) ineN-But-3-ynyl-2-chloro-acetamid 2 Human DDAH-1 350 - - [109] (18) ineN(S-But-3-ynyl-2-chloro-acetamid)-2-Amino-4-(3-methylguanid NH NH2 Human DDAH-1 350 - - [109] ((1821)) ine(NS-But-3-ynyl-2-chloro-acetamid)-2-Amino-4-(3-methylguanid NH NH2 OH HumanRat DDAH-1 DDAH-1 350416 -- -- [109][110] ((2118)) ineN(ino)butanoicS-But-3-ynyl-2-chloro-acetamid)-2-Amino-4-(3-methylguanid acid (4124W) NNH N NH2 OH HumanRat DDAH-1 DDAH-1 416350 -- -- [110][109] ((1821)) (Sine)-2-Amino-4-(3-methylguanid NH NH NH NH2 OH HumanRat DDAH-1 DDAH-1 350416 -- -- [109][110] (S)-2-Amino-4-(3-methylguanidino)ino)butanoicine acid (4124W) HN NH HN NH2OOH (21) (21) (Sino)butanoic)-2-Amino-4-(3-methylguanid acid (4124W) NH NHNH NH2O OH RatRat DDAH-1 DDAH-1 416 416 - -- -[110] [110] (21) butanoicino)butanoic(S)-2-Amino-4-(3-methylguanid acid (4124W) acid (4124W) HN NHHN NH2O OH Rat DDAH-1 416 - - [110] (21) ino)butanoic(S)-2-Amino-4-(3-methylguanid acid (4124W) NH NH Rat DDAH-1 416 - - [110] G L HN HN O2 OH (21) ino)butanoic(NSG)-2-Amino-4-(3-methylguanid-(2-Methoxyethyl)- acid (4124W)-arginine HN HN O OH Rat DDAH-1 416 2 - 2 - [110] ((2139)) ino)butanoicN(S)-2-Amino-4-(3-methylguanid-(2-Methoxyethyl)- acid (4124W)L-arginine OH HumanRat DDAH-1 DDAH-1 41631 13- -- [110][112] ((3921)) N(L-257)G-(2-Methoxyethyl)- L-arginine HN HN O Rat DDAH-1 4162 - 2 - [110] ino)butanoicG acid (4124W) HN HN O Human DDAH-1 31 2 13 2 - [112] (39) N(L-257)ino)butanoicG-(2-Methoxyethyl)- acid (4124W)L-arginine H H O Human DDAH-1 31 13 - [112] (39) NG-(2-Methoxyethyl)-N(L-257)-(2-Methoxyethyl)- L-arginineL-arginine Human DDAH-1 31 2 13 2 - [112] (39) (39) (L-257)NG-(2-Methoxyethyl)- L-arginine O HumanHuman DDAH-1 DDAH-1 3131 2 2 13 2 13 2 - -[[112] 112] (39) (L-257)NG-(2-Methoxyethyl)- L-arginine Human DDAH-1 31 2 13 2 - [112] (L-257)GG 2 2 (39) (L-257)NNG-(2-Methoxyethyl)--(2-Methoxyethyl)- LL-arginine-arginine Human DDAH-1 31 13 - [112] ((3940)) (L-257)NG-(2-Methoxyethyl)--(2-Methoxyethyl)- LL-arginine-arginine HumanRat DDAH-1 DDAH-1 3120 2 13- 2 -- [112][112] ((4039)) (L-257)NmethylG-(2-Methoxyethyl)- ester (L-291) L-arginine HumanRat DDAH-1 DDAH-1 3120 2 13- 2 -- [112][112] (40) Nmethyl(L-257)G-(2-Methoxyethyl)- ester (L-291) L-arginine Rat DDAH-1 20 - - [112] (40) NmethylG-(2-Methoxyethyl)- ester (L-291) L-arginine Rat DDAH-1 20 - - [112] (40) NG-(2-Methoxyethyl)-methylNG-(2-Methoxyethyl)- ester (L-291)L-arginine L-arginine Rat DDAH-1 20 - - [112] (40) (40) methylN2-Amino-5-(3-(2-methoxyethyl)G-(2-Methoxyethyl)- ester (L-291) L-arginine RatRat DDAH-1 DDAH-1 20 20 - -- -[112] [112] (40) methylmethyl2-Amino-5-(3-(2-methoxyethyl)NG ester-(2-Methoxyethyl)- ester (L-291) (L-291) L-arginine Rat DDAH-1 20 - - [112] ((44a40) ) methylN2-Amino-5-(3-(2-methoxyethyl)guanidino)-G-(2-Methoxyethyl)- ester N(L-291)-(methylsulfonyl)- L-arginine HumanRat DDAH-1 DDAH-1 203 -1 -- [112][117] (44a(40)) 2-Amino-5-(3-(2-methoxyethyl)guanidino)-methyl esterN (L-291)-(methylsulfonyl)- HumanRat DDAH-1 DDAH-1 203 1- -- [117][112] (44a) 2-Amino-5-(3-(2-methoxyethyl)methylguanidino)-pentanamide esterN (L-291) -(methylsulfonyl)-(ZST316) Human DDAH-1 3 1 - [117] (44a) guanidino)-2-Amino-5-(3-(2-methoxyethyl)pentanamideN-(methylsulfonyl)- (ZST316) Human DDAH-1 3 1 - [117] (44a) 2-Amino-5-(3-(2-methoxyethyl)guanidino)-2-Amino-5-(3-(2-methoxyethyl)pentanamideN-(methylsulfonyl)- (ZST316) Human DDAH-1 3 1 - [117] (44a) pentanamideguanidino)-2-Amino-5-(3-(2-methoxyethyl)N (ZST316)-(methylsulfonyl)- Human DDAH-1 3 1 - [117] (44a) (44a) guanidino)-pentanamideguanidino)-2-Amino-5-(3-(2-methoxyethyl)N5-(1-Imino-3-butenyl)-N-(methylsulfonyl)-N (ZST316)-(methylsulfonyl)- L-ornith HumanHuman DDAH-1 DDAH-1 3 31 1- -[117] [117] ((44a54a)) pentanamideNguanidino)-5-(1-Imino-3-butenyl)-N (ZST316)-(methylsulfonyl)- L-ornith HumanHuman DDAH-1DDAH-1 133 12 -- [117][114] ((54a44a)) pentanamidepentanamideguanidino)-Nine5-(1-Imino-3-butenyl)- (L-VNIO) (ZST316)N (ZST316)-(methylsulfonyl)- L-ornith Human DDAH-1 133 21 -- [114][117] (54a) Ninepentanamide5-(1-Imino-3-butenyl)- (L-VNIO) (ZST316) L-ornith Human DDAH-1 13 2 - [114] (54a) Npentanamideine5-(1-Imino-3-butenyl)- (L-VNIO) (ZST316) L-ornith Human DDAH-1 13 2 - [114] (54a) ineN5-(1-Imino-3-butenyl)- (L-VNIO) L-ornith Human DDAH-1 13 2 - [114] (54a) ineN5-(1-Imino-3-butenyl)- (L-VNIO) L-ornith Human DDAH-1 13 2 - [114] 5 55 (54a) N -(1-Imino-3-butenyl)-ineNN5-(1-Imino-3-butenyl)- -(1-Iminopropyl)-(L-VNIO) L-ornithineL-ornithineL-ornith Human DDAH-1 13 2 - [114] (54a) ((54a54b)) ineN5-(1-Iminopropyl)--(1-Imino-3-butenyl)- (L-VNIO) L-ornithineL-ornith HumanHuman DDAH-1DDAH-1 13 13- 522 2-- -[114][128] [114] ((54b54a) (L-VNIO)ineN(L-IPO)5-(1-Iminopropyl)- (L-VNIO) L-ornithine Human DDAH-1 13- 522 -- [128][114] (54b) N(L-IPO)ine5-(1-Iminopropyl)- (L-VNIO) L-ornithine Human DDAH-1 - 52 - [128] (54b) N(L-IPO)5-(1-Iminopropyl)- L-ornithine Human DDAH-1 - 52 - [128] (54b) (L-IPO)N5-(1-Iminopropyl)- L-ornithine Human DDAH-1 - 52 - [128] (54b) (L-IPO)N5-(1-Iminopropyl)- L-ornithine Human DDAH-1 - 52 - [128] 55 L (54b) 5 (L-IPO)NN5-(1-Iminopropyl)--(1-Imino-2-chloroethyl)- -ornithineL-or Human DDAH-1 - 52 - [128] ((54b54c)) N -(1-Iminopropyl)-(L-IPO)N5-(1-Imino-2-chloroethyl)--(1-Iminopropyl)- L-ornithineL-ornithineL-or HumanHuman DDAH-1DDAH-1 -- 521.3 0.34- [128][129] (54b) (54c54b)) (L-IPO)Nnithine5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 -- -1.352 520.34- -[129][128] [128] (54c) (L-IPO)Nnithine(L-IPO)5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 - 1.3 0.34 [129] (54c) Nnithine5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 - 1.3 0.34 [129] (54c) nithineN5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 - 1.3 0.34 [129] (54c) nithineN5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 - 1.3 0.34 [129] (54c) nithineN5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 - 1.3 0.34 [129] (54c) nithineN5-(1-Imino-2-chloroethyl)- (Cl-NIO) L-or Human DDAH-1 - 1.3 0.34 [129] (54c) nithine (Cl-NIO) Human DDAH-1 - 1.3 0.34 [129] nithine (Cl-NIO)

Molecules 2016, 21, 615 19 of 31

Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.

Chemical Kinact Chemical Name Chemical Structure Enzyme IC50 (μM) Ki (μM) Reference Number (min−1)

S-Nitroso-L-homocysteine (9) Bovine DDAH-1 75 690 0.38 [105] (HcyNO)

(11) 2-Chloroacetamidine PaDDAH 1 - 3100 1.2 [106]

N-But-3-ynyl-2-chloro-acetamid (18) Human DDAH-1 350 - - [109] ine

NH NH2 (S)-2-Amino-4-(3-methylguanid OH (21) N N Rat DDAH-1 416 - - [110] ino)butanoic acid (4124W) H H O

NG-(2-Methoxyethyl)-L-arginine (39) Human DDAH-1 31 2 13 2 - [112] (L-257)

NG-(2-Methoxyethyl)-L-arginine (40) Rat DDAH-1 20 - - [112] methyl ester (L-291)

2-Amino-5-(3-(2-methoxyethyl) (44a) guanidino)-N-(methylsulfonyl)- Human DDAH-1 3 1 - [117] Molecules 2016, 21, 615 pentanamide (ZST316) 20 of 32

N5-(1-Imino-3-butenyl)-L-ornith (54a) Human DDAH-1 13 2 - [114] ine (L-VNIO) Table 3. Cont.

N5-(1-Iminopropyl)-L-ornithine Chemical (54b) Chemical Name Chemical StructureHuman Enzyme DDAH-1 IC - (µM) 52K (µM) - K (min[128]´1) Reference Number (L-IPO) 50 i inact Molecules 2016, 21, 615 20 of 31 Molecules 2016, 21, 615 20 of 31 Molecules 2016, N215,-(1-Imino-2-chloroethyl)- 615N 5-(1-Imino-2-chloroethyl)-L-ornithineL-or 20 of 31 Molecules(54c) 2016(54c,) 21, 615 Human DDAH-1 - -1.3 1.30.34 0.34[129] 20 of 31 [129] Molecules 2016, 21(Cl-NIO), 615nithine (Cl-NIO) 20 of 31 Molecules 2016, 21, 615 Table 3. Cont. 20 of 31 Table 3. Cont. Table 3. Cont. Table 3. Cont. Table 3. Cont. Table 3. Cont.

PentafluorophenylPentafluorophenyl (PFP) (PFP) 61 61 Pentafluorophenyl (PFP) PaPaDDAHDDAH 11 16–5816–58 - -- -[130] [130] 61 sulfonatesulfonatePentafluorophenyl esters esters (PFP) PaDDAH 1 16–58 - - [130] 61 Pentafluorophenylsulfonate esters (PFP) PaDDAH 1 16–58 - - [130] 61 Pentafluorophenylsulfonate esters (PFP) PaDDAH 1 16–58 - - [130] 61 sulfonatePentafluorophenyl esters (PFP) PaDDAH 1 16–58 - - [130] 61 sulfonate esters PaDDAH 1 16–58 - - [130] sulfonate esters

Indolylthiobarbituric acid 68a–c Indolylthiobarbituric acid PaDDAH 1 2–17 - - [139] 68a–c 68a–c IndolylthiobarbituricderivativesIndolylthiobarbituric acid derivativesacid PaPaDDAHDDAH 11 2–172–17 - -- -[139] [139] 68a–c Indolylthiobarbituricderivatives acid PaDDAH 1 2–17 - - [139] 68a–c Indolylthiobarbituricderivatives acid PaDDAH 1 2–17 - - [139] 68a–c derivativesIndolylthiobarbituric acid PaDDAH 1 2–17 - - [139] 68a–c derivatives PaDDAH 1 2–17 - - [139] derivatives

(73) Ebselen Human DDAH-1 0.33 - - [145] (73) Ebselen Human DDAH-1 0.33 - - [145] (73)(73) EbselenEbselen HumanHuman DDAH-1 DDAH-1 0.33 0.33 - -- -[145] [145] (73) Ebselen Human DDAH-1 0.33 - - [145] (73) Ebselen Human DDAH-1 0.33 - - [145] (73) Ebselen Human DDAH-1 0.33 - - [145] (76) 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 - - [167] (76) 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 - - [167] (76) 4-Hydroxy-2-nonenal (4-HNE) Human DDAH-1 50 - - [167] (76)(76) 4-Hydroxy-2-nonenal4-Hydroxy-2-nonenal (4-HNE) (4-HNE) HumanHuman DDAH-1 DDAH-1 5050 - -- -[167] [167] (76) 4-Hydroxy-2-nonenal6H-6-Imino-(2,3,4,5-tetrahydropy (4-HNE) Human DDAH-1 50 - - [167] (76) 4-Hydroxy-2-nonenal6H-6-Imino-(2,3,4,5-tetrahydropy (4-HNE) Human DDAH-1 50 - - [167] (79) rimido)[1,2-6H-6-Imino-(2,3,4,5-tetrahydropyc]-[1,3]benzothiazine Human DDAH-1 9 - - [61] (79) 6rimido)[1,2-H-6-Imino-(2,3,4,5-tetrahydropyc]-[1,3]benzothiazine Human DDAH-1 9 - - [61] (79) 6(PDrimido)[1,2-H-6-Imino-(2,3,4,5-tetrahydropy 404182) c]-[1,3]benzothiazine Human DDAH-1 9 - - [61] (79) 6H-6-Imino-(2,3,4,5-tetrahydropyrimido)6rimido)[1,2-(PDH-6-Imino-(2,3,4,5-tetrahydropy 404182)c ]-[1,3]benzothiazine Human DDAH-1 9 - - [61] (79) (79) rimido)[1,2-(PD 404182)c ]-[1,3]benzothiazine HumanHuman DDAH-1 DDAH-1 9 9- -- -[61] [61] (79) [1,2-(PDrimido)[1,2-c]-[1,3]benzothiazine 404182)c ]-[1,3]benzothiazine (PD 404182) Human DDAH-1 9 - - [61] (PD 404182) (PD 404182) Proton H+/K+ ATPase pump 82a–f Proton H+/K+ ATPase pump Human DDAH-1 51–63 - - [175] 82a–f inhibitorsProton H+ /K(PPIs)+ ATPase pump Human DDAH-1 51–63 - - [175] 82a–f Protoninhibitors H+ /K(PPIs)+ ATPase pump Human DDAH-1 51–63 - - [175] 82a–f Protoninhibitors H+/K (PPIs)+ ATPase pump Human DDAH-1 51–63 - - [175] 82a–f ProtoninhibitorsProton H+/K H+ /K(PPIs)ATPase+ ATPase pump pump Human DDAH-1 51–63 - - [175] 82a–f 82a–f inhibitors (PPIs) HumanHuman DDAH-1 DDAH-1 51–63 51–63 - -- -[175] [175] inhibitorsinhibitors (PPIs) (PPIs) 1 2 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: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117]. Pa1: PseudomonasPa: Pseudomonas aeruginosa aeruginosa; as; 2determinedas determined by Tommasi by Tommasi et alet. [117]. al.[117 ].

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 Dimethylarginine dimethylamino ADMA Asymmetric dimethylarginine L-NMMA Monomethyl arginine NOS Nitric oxide synthase NO Nitric oxide SDMA Symmetric dimethylarginine PRMTs Protein arginine methyl CAT Cationic amino acid transporter AGXT2 Alanine-glyoxylate amino 2 PKA Protein kinase A Akt Protein kinase B VEGF Vascular endothelial growth factor iNOS Inducible nitric oxide synthase TNF-α Tumour necrosis factor α LDL Low density lipoprotein IL-1β Interleukin-1β AT1-R Type 1 angiotensin receptor DNA Deoxyribonucleic acid FXR Farnesoid X receptor HcyNO S-Nitroso-L-homocysteine NaNO2 Sodium nitrite NaOH Sodium hydroxide HCl Hydrochloric acid Min Minutes Ki Inhibition constant µM Micromolar H2O Water Cys Cysteine His Histidine PaDDAH Pseudomonas aeruginosa dimethylarginine dimethylamino hydrolase PAD Peptydilarginine deaminase NaOMe Sodium methoxide MeOH Methanol AcOH Acetic acid NH4Cl Ammonium chloride PEO Poly(ethylene oxide) DCM Dichloromethane TEA Triethylamine DMAP 4-Dimethylaminopyridine MsCl Methanesulfonyl chloride Et2O Diethyl ether RT Room temperature NaN3 Sodium azide Molecules 2016, 21, 615 22 of 32

DMF N,N-Dimethylformamide Ph3P Triphenylphosphine 4124W (S)-2-Amino-4-(3-methylguanidino)butanoic acid Boc tert-Butyloxycarbonyl M Molar DEAD Diethyl azodicarboxylate THF Tetrahydrofuran DIPEA N,N-Diisopropylethylamine CH3CN Acetonitrile SOCl2 Thionyl chloride SAR Structure activity relationship IC50 Inhibition constant L-257 NG-(2-Methoxyethyl)-L-arginine L-291 NG-(2-Methoxyethyl)-L-arginine methyl ester Orn Ornithine Bu Butyl Z Carboxybenzyl TFMSA Trifluoromethanesulfonic acid TFA Trifluoroacetic acid CDI 1,11-Carbonyldiimidazole DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene h Hours 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide HATU hexafluorophosphate mM Millimolar DMSO Dimethyl sulfoxide i-BuCOCl Isobutyl chloroformate NH4OH Ammonium hydroxide TFAA Trifluoroacetic anhydride ZnBr2 Zinc bromide NH2OH.HCl Hydroxylamine hydrochloride NaHCO3 Sodium bicarbonate L-VNIO N5-(1-Imino-3-butenyl)-L-ornithine EtOH Ethanol EtOAc Ethyl acetate L-IPO N5-(1-Iminopropyl)-L-ornithine Cl-NIO N5-(1-Imino-2-chloroethyl)-L-ornithine PFP Pentafluorophenyl NaOAc Sodium acetate ADI Arginine deaminase HTS High throughput screening SMTC S-methyl-L-thiocitrulline ESI-MS Electrospray ionization mass spectrometry BuLi Butyl lithium Se Selenium CuBr2 Copper(II) bromide NADPH Nicotinamide adenine dinucleotide phosphate H+/K+ ATPase Proton/potassium ATPase Ca2+ Calcium 2+ cation 4-HNE 4-Hydroxy-2-nonenal LOPAC Library of Pharmacologically Active Compounds NaH Sodium hydride Molecules 2016, 21, 615 23 of 32

CS2 Carbon disulfide BrCN Cyanogen bromide Å Angstrom PPIs Proton pump inhibitors SPR Surface plasmon resonance CM Carboxymethylated (Ph3P)2PdCl2 Bis(triphenylphosphine)palladium chloride CuI Copper(I) iodide TIPS Triisopropylsilyl TMS Trimethylsilane DPPA Diphenylphosphoryl azide

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