The Biochemical Pathways of Nicotinamide-Derived Pyridones

International Journal of Molecular Sciences

Article The Biochemical Pathways of Nicotinamide-Derived Pyridones

Faisal Hayat 1,2,†, Manoj Sonavane 1,2,3,†, Mikhail V. Makarov 2 , Samuel A. J. Trammell 4 , Pamela McPherson 2, Natalie R. Gassman 2,3 and Marie E. Migaud 1,2,*

1 Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA; [email protected] (F.H.); [email protected] (M.S.) 2 Mitchell Cancer Institute, College of Medicine, University of South Alabama, Mobile, AL 36604, USA; [email protected] (M.V.M.); [email protected] (P.M.); [email protected] (N.R.G.) 3 Department of Physiology & Cell Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA 4 Novo Nordisk Foundation, Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen, Denmark; [email protected] * Correspondence: [email protected] † Shared ﬁrst authorship.

Abstract: As catabolites of nicotinamide possess physiological relevance, pyridones are often included in metabolomics measurements and associated with pathological outcomes in acute kidney injury (AKI). Pyridones are oxidation products of nicotinamide, its methylated form, and its ribosylated form. While they are viewed as markers of over-oxidation, they are often wrongly reported or mislabeled. To address this, we provide a comprehensive characterization of these catabolites of vitamin B3, justify their nomenclature, and differentiate between the biochemical pathways that lead to their generation. Furthermore, we identify an enzymatic and a chemical process that accounts for the formation of the ribosylated form of these pyridones, known to be cytotoxic. Finally, we Citation: Hayat, F.; Sonavane, M.; demonstrate that the ribosylated form of one of the pyridones, the 4-pyridone-3-carboxamide riboside Makarov, M.V.; Trammell, S.A.J.; (4PYR), causes HepG3 cells to die by autophagy; a process that occurs at concentrations that are McPherson, P.; Gassman, N.R.; Migaud, M.E. The Biochemical comparable to physiological concentrations of this species in the plasma in AKI patients. Pathways of Nicotinamide-Derived Pyridones. Int. J. Mol. Sci. 2021, 22, Keywords: NAD; redox cofactor; nicotinamide; pyridones 1145. https://doi.org/10.3390/ijms 22031145

Academic Editor: Maurizio Battino 1. Introduction Received: 28 December 2020 Through multiple yet convergent biosynthetic pathways, all components of vitamin Accepted: 19 January 2021 B3 are precursors to the nicotinamide-derived redox cofactor, nicotinamide adenine dinu- Published: 24 January 2021 cleotide (NAD), its reduced form NADH, its phosphate parent NADP, and the reduced form NADPH, collectively referred to as NAD(P)(H) [1]. The water-soluble components Publisher’s Note: MDPI stays neutral that constitute vitamin B3 are obtained through the diet and/or supplementation. Its with regard to jurisdictional claims in regular intake must compensate for the daily losses stemming from the degradation of published maps and institutional afﬁl- the cofactors [2]. Some of the NAD(P)(H) degradation products (Figure1) include N- iations. methyl-4-pyridone-3-carboxamide (N-Me-4PY) and N-methyl-6-pyridone-3-carboxamide, often reported as N-methyl-2-pyridone-5-carboxamide (N-Me-6PY). Both pyridones are detected in plasma and urine as major vitamin B3 degradation products and are oxidized forms of methyl-nicotinamide (Me-Nam) [3,4]. The ribosylated form of the pyridone car- Copyright: © 2021 by the authors. boxamides, 4-pyridone-3-carboxamide riboside (4PYR), and 6-pyridone-3-carboxamide Licensee MDPI, Basel, Switzerland. riboside also known as 2-pyridone-5-carboxamide riboside (6PYR), are also detected in This article is an open access article urine [5]. In blood, these ribosylated pyridones are mostly present intracellularly in distributed under the terms and their triphosphorylated (4PYR-TP and 6PYR-TP) [6,7] or adenine dinucleotidic forms [8]. conditions of the Creative Commons N-Methyl-2-pyridone-3-carboxamide (Me-2PY) and 2-pyridone-3-carboxamide riboside Attribution (CC BY) license (https:// (2PYR) are the least prominent pyridones in the literature, as they are much less often creativecommons.org/licenses/by/ detected in biological samples [9]. 4.0/).

Int. J. Mol. Sci. 2021, 22, 1145. https://doi.org/10.3390/ijms22031145 https://www.mdpi.com/journal/ijms Int. J. J. Mol. Mol. Sci. Sci. 20212021,, 2222,, x 1145 2 of 27 28

N-Me-Nam NAD(P)H NAD(P)

2NAD(P)O 6NAD(P)O 4NAD(P)O N-Me-2PY N-Me-6PY N-Me-4PY

NQO2 NQO2

2PYR 6PYR 4PYR NR NRH

2PYR-TP 6PYR-TP 4PYR-TP Figure 1. NAD(P)(H)-derivedNAD(P)(H)-derived pyridone pyridone catabolites catabolites.. An An additional additional route route to tothe the tripho triphosphatesphate from from the the monophosphate monophosphate ri- bosylatedribosylated pyridone pyridone generated generated from from the the dinucleotides dinucleotides can can also also be envisaged. be envisaged. NAD(P)(H): NAD(P)(H): nicotinamide nicotinamide adenine adenine dinucleo- dinu- tidecleotide (phosphate)(reduced (phosphate)(reduced form); form); NAD(P)O: NAD(P)O: hyper- hyper- oxidized oxidized forms forms of NAD(P) of NAD(P) containing containing pyridone pyridone moieties; moieties; PYR-TP: PYR-TP: pyr- idonepyridone riboside riboside triphosphate; triphosphate; PYR: PYR: pyridone pyridone riboside; riboside; The The 2, 2,4, 4,and and 6 6positions positions of of the the carbonyl carbonyl unit unit are are indicated indicated in the abbreviation; N-Me-PY: N-methyl pyridone. abbreviation; N-Me-PY: N-methyl pyridone.

AsAs waste products, thesethese by-productsby-products of of NAD(P)(H) NAD(P)(H) metabolism metabolism associate associate with with aging ag- ingand and nephrotic nephrotic dysfunction dysfunction as their as their levels levels associate associate with advancedwith advanced kidney kidney injuries injuries (AKI) (AKI)and kidney and kidney failure failure [4,6,10 –[4,6,10–12].12]. Liquid Liquid chromatography chromatography combined combined with mass with spectrometry mass spec- trometryis the most is commonthe most methodcommon used method to detect used nicotinamide-derivedto detect nicotinamide-derived pyridones pyridones in biological in biologicalsamples [13 samples–15]. However, [13–15]. muchHowever, confusion much arisesconfusion from arises their detection,from their identification, detection, identi- and fication,reporting and as additionalreporting as nicotinamide additional nicotina metabolitesmide that metabolites are the non-methylated that are the non-methylated or ribosylated orpyridones ribosylated are alsopyridones often mistaken are also foroften their mistak methylateden for speciestheir methylated in reports [species5,9,11,15 in– 18reports]. The [5,9,11,15–18].misperception The stems misperception from confusing stems nomenclature, from confusing compounded nomenclature, by poor compounded quantification by poordue toquantification facile fragmentation due to facile and adduct fragmentation formation and under adduct mass formation spectrometry under conditions. mass spec- trometryCritically, conditions. the chemical nature of each form of the pyridone informs on the type of catabolicCritically, pathway the thechemical nicotinamide-derived nature of each form species of the have pyridone undertaken. informs For on instance, the type only of catabolicthe methylated pathway form the of nicotinamide-derived nicotinamide (N-Me-Nam) species can have be the undertaken. precursor ofFor the instance, three methy- only thelated methylated pyridones form (N-Me-2PY, of nicotinamideN-Me-4PY, (N-Me-Nam) and N-Me-6PY, can be Figure the precursor1)[ 19–22 of]. the Similarly, three meth- only ylatedthe ribosylated pyridones form (N-Me-2PY, of nicotinamide N-Me-4PY, can beand the N-Me-6PY, source of theFigure pyridone 1) [19–22]. ribosides Similarly, [17]. Eachonly theof the ribosylated catabolic form routes of indicates nicotinamide dysregulation can be th ine source NAD-dependent of the pyridonemetabolism ribosides [23 [17].,24]. Each Pyri- ofdone the ribosides, catabolic unlikeroutes the indicates methylated dysregulation species, can in be NAD-dependent converted to the adeninemetabolism dinucleotide [23,24]. Pyridoneor the triphosphate ribosides, unlike form [the17, 25methylated]. The 4PYR species, isomer can not be onlyconverted circulates to the in adenine plasma dinu- once cleotidegenerated or butthe istriphosphate also internalized form and[17,25]. metabolized The 4PYR to isomer species not that only interfere circulates with enzymaticin plasma onceprocesses generated [26–28 but]. Possessing is also internalized a systematic, and reliablemetabolized account to species of the pyridones’ that interfere distribution with en- zymaticwould greatly processes help [26–28]. identify Possessing whether these a systemat speciesic, are reliable pathophysiologically account of the pyridones’ significant. dis- tributionHere, would we present greatly a help comprehensive identify whethe comparativer these species characterization are pathophysiologically of each species andsig- nificant.explore the mechanisms by which their under-detection and misidentification can arise.

Int. J. Mol. Sci. 2021, 22, 1145 3 of 28

We also propose a mechanism for the wide-spread production of the ribosylated pyridone 4PYR and rationalize selective cytotoxicity by comparing how exogenous exposure to ribosylated pyridones affects cell survival.

2. Results The N-methylated 6-pyridone were easily generated from their respective chloronicotinic acid precursors as were the ribosylated 6-pyridone according to published syntheses [6,29–32]. However, N-Me-2PY, 2PYR, N-Me-4PY, and 4PYR converted readily to the methylated ester instead of the amide under aqueous methanolic conditions. Alterations to the published synthetic sequences were deemed necessary to ensure sample authen- ticity and are described in detail in the experimental and methods section (Schemes1–8 in Methods). All synthetic compounds were puriﬁed by separation by ﬂash column, and their chemical structures were fully characterized by both NMR and mass spectrometry (Supplementary Material Table S1 and Spectra). More details on structural characterization can be found in the supplementary material section. In Tables1–3, we have compiled the 1H NMR experimental data for the pyridones (Table1), the methylated pyridones ( Table2 ), and the ribosylated pyridones (Table3), while a comprehensive mass spectrometry report can be found in the supplementary material section. The 1H NMR of each one of the pyridone is very characteristic of each species according to the position of the carbonyl moiety as well as the type of substitution present on the heterocycle. The simple 4PY was synthesized in a three-step process as reported by Slominska et al. [25] First, 4-chloronicotinic acid was hydrolyzed then converted to the acyl chloride with thionyl chloride in MeOH solution. The 4-pyridone-formylchloride was treated with aq.NH4OH in a sealed tube to generate 4PY. For the 6PY, 5-hydroxy-nicotinic was activated with HOBt in DMF and subsequently treated with aq.NH4OH in a sealed tube at rt to afford 6PY in 70% yield. Finally, 2PY was generated from 2-hydroxy-nicotinic acid, which following its conversion to the acyl halide using SOCl2 reagent was converted to the amide using aq.NH4OH in a sealed tube at rt. When stirred with aq. MeOH, the acyl chloride hydrolyzes to its nicotinate salt that exists in both keto and enol forms. The synthesis of ribosylated forms of 2PY, 4PY, and 6PY (2PYR, 4PYR, and 6PYR, respectively) is based on Vorbrüggen glycosylation reaction [31,32]. Following silylation of the nucleobase using either HMDS (2-PY and 4-PY) or BSTFA (6-PY), the silylated pyridones were mixed with beta-D-riboside tetraacetate in the presence of a Lewis acid. For the ribosylation of 2PY and 4PY, TMSOTf in 1,2-DCE/acetonitrile was applied, while SnCl4 was used for the ribosylation of 6PY. While the deacetylation should have been straightforward, it had to be optimized for 2PYR and 4PYR to prevent the loss of the amine moiety and generation of the corresponding carboxylic acid derivatives, materials that we have also fully characterized for reasons described below. Finally, the N-methyl derivatives of 4PY and 6PY (N-Me-2PY, N-Me-4PY, and N-Me-6PY, respectively) were prepared by methylation with methyl iodide. N-methyl-2-pyridone, N-Me-2PY was obtained by oxidation of N-methyl nicotinamide with K3Fe(CN)6 under basic conditions [33,34]. Int. J. Mol. Sci. 2021, 22, 1145 4 of 28

Table 1. Tables of 1H NMR chemical shift for the non-substituted pyridones.

Compound Chemical Shift (Multiplicity, J Values in Hz) (Solvent) NH H2 H3 H4 H5 H6 2PY 12.3 (s), 9.05 (s), 7.51 (s) 7.66 (d, J = 4.32 Hz) 6.41 (t, J = 6.66 Hz) 8.29 (d, J = 5.28 Hz) (DMSO-d6) 2PY 11.80 (s), 9.02 (s), 7.57 (s) 7.69 (q, 1H) 6.44 (t) 8.31 (q) (DMSO-d6)[25] 4PY 8.54 (s) 6.56 (d, J = 6.53 Hz) 7.79 (d, J = 5.56 Hz) (CD3OD) 4PY 8.56 (d, J = 1.7 Hz) 6.58 (d, J = 7.2 Hz) 7.80 (dd, J = 7.2, 1.7 Hz) (CD3OD) [25] 6PY 11.92(s), 7.72 (s) 7.18 (s) 8.00 (s) 7.85 (d, J = 9.6 Hz) 6.32 (d, J = 9.56 Hz) (DMSO-d6) 6PY 11.84 (s), 7.71 (s), 7.18 (s) 7.99 (d, J = 2.6 Hz) 7.82–7.87 (dd, J = 2.6 & 9.5 Hz) 6.32 (d, J = 9.5 Hz) (DMSO-d6)[31] Int. J. Mol. Sci. 2021, 22, 1145 5 of 28

Table 2. Tables of 1H NMR chemical shift for the methylated pyridones.

Compound Chemical Shift (Multiplicity, J Values in Hz) (Solvent) NH H2 H3 H4 H5 H6 CH3 N-Me-2PY 8.01 (dd, J = 2.16 & 9.06 (s) 7.54 (s) 6.44 (t, J = 6.88 Hz) 8.27 (dd, J = 2.20; 2.16 Hz) 3.08 (s) (DMSO-d6) 2.16 Hz) N-Me-2PY * 9.09 (brs, 1H, H-Nb), 8.31 (dd, J = 7.21, 2.21 Hz) 6.47 (dd, J = 7.10, 7.10 Hz) 8.04 (dd, J = 6.55, 2.21 Hz) 3.56 (s) (DMSO-d6)[29] 7.57 (brs, 1H, H-Na) N-Me-4PY 9.36 (s), 7.54 (s) 8.54 (s) 6.53 (d, J = 7.4 Hz) 7.87 (d, J = 5.08 Hz) 3.82 (s) (DMSO-d6) N-Me-4PY * 7.74 (dd, J = 2.44, 2 9.56 (brs), 7.41 (brs) 6.38 (d, J = 7.49 Hz) 8.44 (d, J = 2.44 Hz) 3.75 (s) (DMSO-d6) [29] 7.49 Hz) N- Me-6PY 8.38 (s) 7.95 (d, 1H, J = 6.69 Hz) 6.53 (d, J = 9.44 Hz) 3.62 (s) (D2O) N- Me-6PY * 7.23 (brs), 7.69 (brs) 6.38 (d, J = 9.49 Hz) 7.85 (dd, J = 9.49, 2.54 Hz) 8.36 (d, J = 2.54 Hz) 3.46 (s) (DMSO-d6)[29] *: non-IUPAC atom numbering. Int. J. Mol. Sci. 2021, 22, 1145 6 of 28

Table 3. Tables of 1H NMR chemical shift for the ribosylated pyridones.

Compound Chemical Shift (Multiplicity, J Values in Hz) (Solvent) NH H2 H3 H4 H5 H6 H1’ H2’ H3’ H4’ H5’ CH3 2PYR 8.46 (dd, J = 5.4, 7.04 Hz, 2H, H4 & H6), 3.97 (d, J = 12.9 Hz), 3.80 (d, 6.14 (s), 4.12 (bs, 3H, C H) (D O) 2’,3’,4’ J = 11.56 Hz) 2 6.55 (t, J = 6.96 Hz) 5.55 (d, 1H 5.10 (d, 1H, 2PYR 7.63 and 6.08 (d, 5.23 (t, 1H, C5’OH), 3.64 8.32 (m) 6.55 (t) 8.42 (m) (C2’OH) C3’OH) (DMSO-d6)[32] 8.96 (2 brs, J = 1.17 Hz) (m) CONH2) 3.79–3.97 (m, 3H) 7.94 (dd, 3.81 and 3.73 (ABX, 2H, 4PYR 8.67 (d, 6.65 (d, 5.55 (d, 4.23 (t, J = 2.24 Hz, 4.19–4.13 (m) JAB = 12.7 Hz, JAX = (D O) J = 2.24 Hz) J = 7.6 Hz) J = 5.5 Hz) J = 5.28 Hz) 2 2.24 Hz) 3.04 Hz, JBX = 4.0 Hz) 7.93 (dd, 4.14 (ddd, 4PYR 8.66 (d, 6.58 (d, 5.53 (d, 4.22 (t, 4.16 (dd, 3.72 (dd, J = 12.6, 3.0 Hz), J = 7.8, J = 4.0, 3.5, (D O) [25] J = 2.4 Hz) J = 7.8) J = 5.9) J = 5.4) J = 5.1, 3.5) 3.79 (dd, J = 12.6, 3.0 Hz) 2 2.4 Hz 3.0 Hz) 6PYR 7.84 (d, 6.54 (d, 3.97 (d, J = 12.92 Hz), 3.80 8.60 (s) 6.01 (s) 4.19–4.13 (m) (D2O) J = 9.2 Hz) J = 9.44 Hz) (d, J = 12.88 Hz) 5.45 (d, 5.07 (d, 6PYR 7.54(1 H, 8.52 (d, 7.81–7.86 (dd, 6.40 (d, 6.01 (d, J = 4.6 Hz,1H, J = 4.9 Hz, 1 5.17 (t, J = 4.5 Hz, C5’OH, (DMSO-d6)[31] Amid-NH), J = 1.9 Hz) J = 1.9 Hz, J = 9.5 Hz) J = 3.5 Hz) C2’ OH) H, C3’ OH) 3.57–3.76 (mb) 7.30 (s) 9.5 Hz) 3.92–4.08 (m, 3H, C2’,3’,4’H) 7.89 (dd, 4.26 (dd, 4.20 (dd, 4.15 (dd, 4PYR ACID 8.21 (d, 6.52 (d, 5.51 (d, 3.82 (dd, J = 12.8, 4.5 Hz), J = 7.6, J = 5.6 and J = 5.3 and J = 8.3, (D O) J = 2.4 Hz) J = 7.6 Hz) J = 5.6 Hz) 3.74 (dd, J = 12.8, 4.5 Hz) 2 2.4 Hz) 5.3 Hz) 3.6 Hz) 3.6 Hz) Int. J. Mol. Sci. 2021, 22, x 6 of 27 Int. J. Mol. Sci. 2021, 22, 1145 7 of 28

As described above, N-methyl-nicotinamideN-methyl-nicotinamide oxidizes to three possible forms, N-Me--Me- 2PY, N-Me-4PY, andand NN-Me-6PY.-Me-6PY. ByBy massmass spectrometryspectrometry (ESI-MS,(ESI-MS, SupplementarySupplementary Material Table S1),S1), itit was was found found that that the the three three isomers isomer fragments fragment were were similar similar to each to othereach other but differ but differin the in relative the relative abundance abundance of the of predominant the predominant fragmentation fragmentation products products (Supplementary (Supplemen- taryMaterial Material Table Table S1). TheS1). fragmentationThe fragmentation pattern patte ofrn each of each pyridone pyridone derivative derivative is a ﬁngerprintis a finger- printof that of pyridone that pyridone and canand be can used be used to identify to identify these these species species unambiguously unambiguously (Supplemen- (Supple- mentarytary Material Material Spectra). Spectra). Crucially, Crucially, the functionalthe functional group group exchange exchange between between the amidethe amide and andthe carboxylicthe carboxylic acid acid described described above above is is observed observed under under simple simple electrospray electrospray conditionsconditions (Supplementary Material Material Table Table S1 S1 and and Spectr Spectra).a). All All three three methylated methylated pyridone pyridone forms forms (N- (Me-2PY,N-Me-2PY, N-Me-4PY,N-Me-4PY, and and N-Me-6PY)N-Me-6PY) are aredeamidated deamidated on the on thecarboxamide carboxamide to form to form an ion an ation m/z at m/z136. 136.However, However, this fragment this fragment is the is predominant the predominant product product at low at energy low energy when when frag- mentingfragmenting N-Me-2PY,N-Me-2PY, and andN-Me-4PYN-Me-4PY but more but more minor minor when when fragmenting fragmenting N-Me-6PY.N-Me-6PY. The Thesame same fragmentation fragmentation profile proﬁle is observed is observed for 2PYR, for 2PYR, 4PYR, 4PYR, and and6PYR 6PYR (Supplementary (Supplementary Ma- terialMaterial Table Table S1). S1). Critically, Critically, when when the the mass mass spectrometry spectrometry analyses analyses are are conducted conducted in the presence of water and/orand/or methanol, methanol, the the ionized carbonium fragment (Figure 22)) cancan reactreact with inin situsitu solventssolvents to to generate generate adducts adducts that that in in turn turn get get ionized ionized and and thus thus get get detected. detected. We Wehave have provided provided a comparative a comparative characterization characterizati ofon theseof these materials materials to addressto address the the possible possi- blemischaracterization mischaracterization of these of these metabolites metabolites by MS. by MS. While While not endogenouslynot endogenously generated, generated, they theycan be can major be major contributors contributors to the to pyridone the pyridone pool theypool originatedthey originated from. from.

ESI [M1+H]+

in source fragmentation

in source adduct formation

ESI [M2+H]+

R= Me or C H O ; R1= OH or OCH 5 9 4 3

Figure 2. Pyridone carboxamides, their ESI-induced fragment, and the subsequent in source gen- gener- eratedated adduct. adduct. The The contribution contribution of of in-source in-source fragmentation fragmentation of theof the N-Me-6PY N-Me-6PY is thought is thought to beto minimal.be minimal. While the origins of the methylated pyridones (N-Me-2PY, N-Me-4PY, and N-Me-6PY) haveWhile been attributedthe origins to of the the oxidation methylated of Npy-methylridones nicotinamide(N-Me-2PY, N-Me-4PY, by aldehyde and oxidases N-Me- 6PY)and xanthinehave been oxidases attributed [35 –to37 the], the oxidation formation of N-methyl of the pyridone nicotinamide ribosides by remainsaldehyde more oxi- daseselusive. and Interestingly, xanthine oxidases side reactions [35–37], between the formation water and of th NADPe pyridone in the bindingribosides pocket remains of moreadrenodoxin elusive. Interestingly, reductase (FDXR) side reactions have been between reported water [38 –and40]. NADP The nucleophilic in the binding addition pocket of water on the ribosylated nicotinamide moiety of NADP was thought to promote the of adrenodoxin reductase (FDXR) have been reported [38–40]. The nucleophilic addition formation of 4NADPO. We explored the possibility that nicotinamide riboside, NR, and its of water on the ribosylated nicotinamide moiety of NADP was thought to promote the reduced form, NRH are oxidized by the FAD-dependent reductase, dihydronicotinamide formation of 4NADPO. We explored the possibility that nicotinamide riboside, NR, and riboside: quinone reductase 2 via a similar mechanism, using LC-MS combined to mass its reduced form, NRH are oxidized by the FAD-dependent reductase, dihydronicotina- spectrometry. The oxidation of NRH by the FAD-dependent NQO2 was monitored at mide riboside: quinone reductase 2 via a similar mechanism, using LC-MS combined to 340 nM by LC-UV-spectroscopy, while the appearance of NR was conﬁrmed by monitoring mass spectrometry. The oxidation of NRH by the FAD-dependent NQO2 was monitored at 254 nm. Similarly, when seeking to oxidize NR, the reaction was monitored at 254 nm at 340 nM by LC-UV-spectroscopy, while the appearance of NR was confirmed by moni- to follow NR consumption. We observed not only that NRH was oxidized to NR in the toring at 254 nm. Similarly, when seeking to oxidize NR, the reaction was monitored at presence of FAD and menadione, but that the pyridone riboside, 4PYR, was also generated 254 nm to follow NR consumption. We observed not only that NRH was oxidized to NR over time, as conﬁrmed by LC-MS-MS. This chemistry was not observed when NR was used in the presence of FAD and menadione, but that the pyridone riboside, 4PYR, was also as the starting material. This indicates that the mechanism responsible for the oxidation of generated over time, as confirmed by LC-MS-MS. This chemistry was not observed when

Int. J. Mol. Sci. 2021, 22, x 7 of 27

NR was used as the starting material. This indicates that the mechanism responsible for the oxidation of NRH to 4PYR by NQO2 is not the same as the mechanism observed and characterized for the oxidation of NADP by adrenodoxin reductase. Seeking to establish the mechanism of 4PYR formation by NQO2, we turned to Fen- ton Chemistry. In the presence of K3(Fe(CN)6) in 1 M NaOH, methyl-nicotinamide is readily oxidized, and N-Me-2PY and N-Me-6PY were detected. A carboxylic acid [C7H7NO3+H+] was also detected by direct ESI-MS. This outcome could be consistent with the synthetic challenges we experienced in the synthesis of N-Me-4PY. When we applied this chemistry to nicotinamide riboside (NR), we detected 2PYR [41] and 6PYR [42,43], the two isomers of 4PYR (Figure 3), along with nicotinamide, the product formed from NR hydrolysis. These outcomes indicate that the NQO2-catalyzed over-oxidation of NRH favors one isomer, 4PYR, and occurs via a mechanism that differs from Fenton chemistry as it is more regioselective. It is also different from adrenodoxin reductase (FDXR) oxidation of NADP, as the over-oxidation of NRH by NQO2 requires the initial release of a hydride for superoxide formation via FAD.

Int. J. Mol. Sci. 2021, 22, 1145 Acetaminophen, also known as paracetamol is oxidized to a quinone8 by of 28 the FDXR- dependent cytochrome CYP450, CYP2E1, for which acetaminophen administration stim- ulates the overexpression [44]. acetaminophen exposure also promotes the overexpression of NADPH:NRH to 4PYR quinone by NQO2 oxide-reductase is not the same as1 (NQO1) the mechanism [45], observedan homolog and characterized of NQO2, forwhich cata- lyzesthe the oxidation reduction of NADP of the by quinone adrenodoxin back reductase. to acetaminophen. Catabolites of vitamin B3 found in urine includeSeeking tonicotinuric establish the acid, mechanism trigonellin of 4PYRe, formationmethyl-nicotinamide, by NQO2, we turned Me-2PY, to Fenton Me-4PY, and Chemistry. In the presence of K3(Fe(CN)6) in 1 M NaOH, methyl-nicotinamide is readily 4PYR. In a human preliminary study, we measured the levels of Me-Nam, N-Me-4PY,+ and oxidized, and N-Me-2PY and N-Me-6PY were detected. A carboxylic acid [C7H7NO3+H ] 4PYRwas in urine also detected over a by 24 direct h period ESI-MS. and This compared outcome couldthese be levels consistent with withthose the obtained synthetic following the ingestionchallenges weof experienced1 gm of acetaminophen in the synthesis of (SuN-Me-4PY.pplementary When we Figure applied S3). this chemistryWe observed that acetaminophento nicotinamide consumption riboside (NR), increases we detected the 2PYR urinary [41] and levels 6PYR of [42 4PYR,43], the by two more isomers than 40-fold but doesof 4PYR not (Figurechange3), the along levels with of nicotinamide, the non-ribosylated the product species. formed from This NR observation hydrolysis. would in- dicateThese that outcomes acetaminophen indicate thatonly the promotes NQO2-catalyzed the oxidation over-oxidation of the ofribosylated NRH favors forms one of nico- isomer, 4PYR, and occurs via a mechanism that differs from Fenton chemistry as it is tinamide,more regioselective.i.e., NR, NMN, It is alsoor NAD(P) different fromand adrenodoxinthis via a mechanism reductase (FDXR) which oxidation is independent of of the aldehydeNADP, as theoxidase, over-oxidation the enzyme of NRH responsible by NQO2 requires for the the generation initial release of of N a-Me-2PY hydride for and N-Me- 4PY fromsuperoxide methyl-nicotinamide formation via FAD. in vivo.

Figure 3. FigureFenton 3. chemistryFenton chemistry facilitates facilitates the the formation formation ofof 2PYR 2PYR and an 6PYRd 6PYR from NR.from Overlay NR. 1OverlayH NMR of 1H nicotinamide NMR of ribosidenicotinamide riboside (bottom(bottom spectrum) spectrum) andand crude crude reaction reaction mixture mixture (above). (above). NR decomposes NR decomposes to nicotinamide to nicotinamide under basic conditionsunder basic (3rd conditions (3rd spectrumspectrum up). up). The The crude crude reaction reaction mixture mixture was was spiked spiked with eachwith pyridone each pyridone ribosides (4th–6thribosides spectra). (4th–6th Pure spectra). PYR 1H NMR Pure PYR 1H NMR spectraspectra (7th–9th (7th–9th spectra). spectra). Only Only 2PYR 2PYR and and 6PYR 6PYR are present are present in the reaction in the mixture. reaction• 2PYR; mixture. x 6PYR; ● T2PYR 4PYR; ; Fx Nicotinamide6PYR; T 4PYR ;★Nic- otinamideRiboside; Riboside Nicotinamide.; ♦ Nicotinamide. Acetaminophen, also known as paracetamol is oxidized to a quinone by the FDXR- dependent cytochrome CYP450, CYP2E1, for which acetaminophen administration stimu- lates the overexpression [44]. acetaminophen exposure also promotes the overexpression of NADPH: quinone oxide-reductase 1 (NQO1) [45], an homolog of NQO2, which catalyzes the reduction of the quinone back to acetaminophen. Catabolites of vitamin B3 found in urine include nicotinuric acid, trigonelline, methyl-nicotinamide, Me-2PY, Me-4PY, and 4PYR. In a human preliminary study, we measured the levels of Me-Nam, N-Me-4PY, and 4PYR in urine over a 24 h period and compared these levels with those obtained following the ingestion of 1 gm of acetaminophen (Supplementary Figure S3). We observed that acetaminophen consumption increases the urinary levels of 4PYR by more than 40-fold but Int. J. Mol. Sci. 2021, 22, x 8 of 27

The studies evaluating the PYR-family cytotoxicity are somewhat confusing and im- precise, as not all cell-lines appear to be sensitive to exposure to these nucleosides [26,46]. Given the literature, we decided to explore which one if not all the pyridone ribosides were cytotoxic to two very metabolically different cell lines, the human embryonic kidney HEK293T cell line and the human hepatocarcinoma HepG3 cell line. Compared to Int. J. Mol. Sci. 2021HEK293T, 22, 1145 that is more glycolytic, HepG3 is more reliant on mitochondrial respiration for 9 of 28 energy production, processes that are NAD(P) dependent and therefore potentially sensitive to the presence of PYR-derived dinucleotides. Using a CellTiter-FluorTM viability assay as a reporter [47], cytotoxicity assays on HEK293T and HepG3 cell cultures indicated does not change the levels of the non-ribosylated species. This observation would indicate that none of the PYR-derivates were cytotoxic to HEK293T cells but that the 4PYR reduced that acetaminophen only promotes the oxidation of the ribosylated forms of nicotinamide, the cell viability of HepG3 cells by 62% after 24 h of treatment at 100 µM followed by 48 h i.e., NR, NMN, or NAD(P) and this via a mechanism which is independent of the aldehyde recovery in cell medium and by 45% after 72 h of continuous exposure (Figure 4). oxidase, the enzyme responsible for the generation of N-Me-2PY and N-Me-4PY from A dose-dependent sensitivity assay of HepG3 cells exposed to continuous 4PYR methyl-nicotinamide in vivo. treatment showed that 4PYR was cytotoxic to HepG3 in a dose-dependent manner with The studies evaluating the PYR-family cytotoxicity are somewhat confusing and an IC50 at 50 µMimprecise, (Figure as5). notCrucially, all cell-lines even appearat higher to beconcentrations sensitive to exposure in this cell to theseline, sur- nucleosides [26,46]. viving cells remain,Given and the the literature, loss of cell we viability decided appears to explore to plateau. which oneWe also if not characterized all the pyridone ribosides the effects of 4PYRwere on cytotoxic HepG3 cells to two in verya clonogenic metabolically survival different assay, cellwith lines, HepG3 the cell human density embryonic kidney less than 50% afterHEK293T 6 days cell at line 25 andµM theof human4PYR (Figure hepatocarcinoma 6). Together, HepG3 these cell data line. provided Compared to HEK293T some indicationsthat of isthe more possible glycolytic, mode of HepG3 action isof more4PYR reliantin HepG3 on mitochondrialcells, which likely respiration pro- for energy motes senescenceproduction, in the remaining processes viable that cells. are NAD(P) Therefore, dependent we sought and to thereforeestablish whether potentially sensitive to markers of autophagythe presence could of PYR-derivedbe detected dinucleotides.in 4PYR-treated Using HepG3. a CellTiter-Fluor ImmunoblottingTM viability assay as a showed an increasereporter in protein [47], cytotoxicity expression assays levels on of HEK293Tthe autophagy and HepG3 marker cell LC3BII, cultures Beclin- indicated that none 1, and a decreaseof thein phospho-mTOR PYR-derivates werecompared cytotoxic to vehicle-treated to HEK293T cells control but thatcells the(Figure 4PYR 7). reduced the cell Furthermore, weviability confirmed of HepG3 that apoptosis cells by 62% wa afters not 24observed h of treatment over the at 100sameµM period followed (data by 48 h recovery not shown). in cell medium and by 45% after 72 h of continuous exposure (Figure4).

Figure 4. 4PYR is cytotoxic to HepG3 cells but not HEK293T. Cell viability after 72 h was assessed for HEK293T (A) and Figure 4. 4PYR is cytotoxic to HepG3 cells but not HEK293T. Cell viability after 72 h was assessed HepG3 (B) cells. Cells were exposed to 100 µM of 2PYR, 4PYR, or 6PYR for 24 h with pyridones removed and fresh medium for HEK293T (A) and HepG3 (B) cells. Cells were exposed to 100 µM of 2PYR, 4PYR, or 6PYR for TM replaced for24 an h with additional pyridones 48 h removed or continuously and fresh exposed medium to replaced the pyridone for an isomers additional for 7248 h.h or After continuously 72 h, CellTiter-Fluor viability assayexposed was performedto the pyridone with resultsisomers expressed for 72 h. After as the 72 mean h, CellTiter-Fluor fluorescence intensityTM viability relative assay to was control, per- vehicle-treated cells (% Viability)formed± withstandard results error expressed of the meanas the (SEM).mean fluorescence Statistical significance: intensity relative **** p < to 0.0001. control, vehicle- treated cells (% Viability) ± standard error of the mean (SEM). Statistical significance: **** p < 0.0001. A dose-dependent sensitivity assay of HepG3 cells exposed to continuous 4PYR treatment showed that 4PYR was cytotoxic to HepG3 in a dose-dependent manner with an IC50 at 50 µM (Figure5). Crucially, even at higher concentrations in this cell line, surviving cells remain, and the loss of cell viability appears to plateau. We also characterized the effects of 4PYR on HepG3 cells in a clonogenic survival assay, with HepG3 cell density less than 50% after 6 days at 25 µM of 4PYR (Figure6). Together, these data provided some indications of the possible mode of action of 4PYR in HepG3 cells, which likely promotes senescence in the remaining viable cells. Therefore, we sought to establish whether markers of autophagy could be detected in 4PYR-treated HepG3. Immunoblotting showed an increase in protein expression levels of the autophagy marker LC3BII, Beclin-1, and a decrease in phospho-mTOR compared to vehicle-treated control cells (Figure7). Furthermore, we confirmed that apoptosis was not observed over the same period (data not shown). Int. J. Mol. Sci. 2021, 22, x 9 of 27

Int. J.Int. Mol. J. Mol. Sci. Sci. 20212021, 22, 22, x, 1145 10 of 28 9 of 27

Figure 5. Dose-dependent sensitivity of HepG3 cells to continuous 4PYR treatment. HEK239T (A) Figure 5. Dose-dependent sensitivity of HepG3 cells to continuous 4PYR treatment. HEK239T (A) and HepG3 (B) cellsFigure were 5. Dose-dependentexposed to 25, 50, sensitivity 75, 100, andof HepG3 150 µM cells continuously to continuous for 4PYR 72 h. treatment.CellTiter- HEK239T (A) and HepG3 (B) cells were exposed to 25, 50, 75, 100, and 150 µM continuously for 72 h. CellTiter- FluorTM Viability andassay HepG3 was performed (B) cells were with exposed results to expressed 25, 50, 75, as100, the and mean 150 fluorescenceµM continuously intensity for 72 h. CellTiter- FluorTM Viability assay was performed with results expressed as the mean ﬂuorescence intensity relative to control,Fluor vehicle-treatedTM Viability assay cells (%was viability) performed ± SEM. with resultsStatistical expressed significance: as the mean** p < fluorescence0.005, **** intensity relative to control, vehicle-treated cells (% viability) ± SEM. Statistical signiﬁcance: ** p < 0.005, p < 0.0001. relative to control, vehicle-treated cells (% viability) ± SEM. Statistical significance: ** p < 0.005, **** **** p < 0.0001. p < 0.0001.

Figure 6. Clonogenic survival of HepG3 cells continuously exposed to 4PYR. HepG3 cells were exposed to 25, 50, and 100 µMFigureof 4PYR 6. for Clonogenic six daysFigure then survival stained 6. Clonogenic with of HepG3 crystal survival violet cells ( Acontinuouslyof). HepG3 Cell density cells wasexposed continuously assessed to 4PYR. by measuringexposed HepG3 to the 4PYR.cells area were fraction HepG3 of cells were the crystalexposed violet-stained to 25, 50, cellsexposed and after 100 4PYR to µM 25, exposure of50, 4PYR and (B 100 ).for Results µMsix daysof are 4PYR presented then for stained si asx days percentage with then crystal stained cell density violet with to (crystalA control). Cell violet± den-SEM (A of). Cell den- three independentsity was assessed experiments.sity by measuringwas Statistical assessed signiﬁcance: the by area measuring fraction *** p < the 0.001, of areathe **** crystal fractionp < 0.0001. viol of theet-stained crystal violcellset-stained after 4PYR cells expo- after 4PYR exposure (B). Results aresure presented (B). Results as are percentage presented cell as percentagedensity to controlcell density ± SEM to controlof three ± independentSEM of three independent experiments. Statisticalexperiments.Finally, significance: weStatistical exposed *** significance:p < HEK293T 0.001, **** and*** p p< HepG3 <0.0001. 0.001, cells **** top < 100 0.0001.µM NRH, the reduced form of nicotinamide riboside, for 1, 4, and 24 h and measured the cell extract for the presence of the over-oxidized form of NAD, NADO by LC-MS. While HEK293T did not produce NADO under such conditions, HepG3 cells produced 4NADO intracellularly, and its abundance increased with the length of exposure (Figure8).

Int. J. Mol. Sci. 2021, 22, x 10 of 27

Int. J.Int. Mol. J. Mol. Sci. Sci. 20212021, 22, 22, x, 1145 11 of 28 10 of 27

Figure 7. 4PYR induced autophagy in HepG3 cells. HepG3 cells were continuously dosed with 100 µM of 4PYR for 48, 72, and 96 h. (A) Increased protein expression levels of the autophagy marker LC3BII, Beclin-1, and decrease in phospho-mTOR were assessed using immunoblot in 4PYR treated HepG3 cells compared to vehicle-treated control cells. α-tubulin was used as a loading control for immunoblotting. The graph shows quantified protein expression levels relative to controls for (B) LC3B-II, (C) Beclin-1 and (D) phosphor-mTOR in HepG3 cells. Results are expressed as relative abundance (a.u.) ± SEM of two biological replicates. Statistical significance: * p < 0.05. FigureFigure 7. 4PYR4PYR induced autophagyautophagy in in HepG3 HepG3 cells. cells. HepG3 HepG3 cells cells were were continuously continuously dosed dosed with with 100 µ µM100 ofM 4PYRof 4PYR for for48, 48, 72, 72, and and 96 96 h. h. ( (AA)) Increased Increased protein protein expression expression levels levels of the of autophagy the autophagy marker marker Finally, we exposed HEK293T and HepG3 cells to 100 µM NRH, the reduced form of LC3BII,LC3BII, Beclin-1, and and decrease decrease in phospho-mTORin phospho-mTOR were were assessed assessed using immunoblotusing immunoblot in 4PYR in treated 4PYR α nicotinamide riboside,treatedHepG3 forHepG3 cells 1, compared 4, cells and compared 24 to vehicle-treatedh and tomeasured vehicle-treated control the cells. cell control extract-tubulin cells. for was α -tubulinthe used presence as awas loading used of control as a loading for the over-oxidizedcontrolimmunoblotting. form for of immunoblotting. NAD, The NADO graph shows Theby LC-MS.graph quantiﬁed shows While protein quantifi HEK293T expressioned protein levelsdid expression not relative produce to levels controls relative for (B) to con- NADO under suchtrolsLC3B-II, conditions,for ( (BC)) LC3B-II, Beclin-1 HepG3 and(C) Beclin-1 (D )cells phosphor-mTOR producedand (D) phosphor-mTOR in4NADO HepG3 cells.intracellularly, in Results HepG3 are ce expressed lls.and Results its as are relative expressed abundance increasedasabundance relative with abundance (a.u.)the length± SEM (a.u.) of twoexposure ± SEM biological of (Figuretwo replicates. biol 8).ogical Statistical replicates. signiﬁcance: Statistical * p

Finally, we exposed HEK293T and HepG3 cells to 100 µM NRH, the reduced form of nicotinamide riboside, for 1, 4, and 24 h and measured the cell extract for the presence of the over-oxidized form of NAD, NADO by LC-MS. While HEK293T did not produce NADO under such conditions, HepG3 cells produced 4NADO intracellularly, and its abundance increased with the length of exposure (Figure 8).

Figure 8. A B FigureNRH 8. induced NRH induced 4NADO production4NADO production in HepG3 cellsin HepG but not3 cells in HEK293T. but not HEK293Tin HEK293T. ( ) andHEK293T HepG3 ( A)) cells were µ continuouslyand HepG3 dosed ( withB) cells 100 wereM of continuously 4PYR for 1, 4, anddosed 24 h.with The 100 crude µM cell of extract 4PYR wasfor 1, measured 4, and 24 by h. LC-MS The crude for its NADO ± content.cell The extract NADO was detected measured matches by the LC-MS 4NADO for standard. its NADO Results content. are presentedThe NADO as relative detected abundance matches to the control SEM of three4NADO independent standard. experiments. Results Statistical are presented signiﬁcance: as relati ****vep abundance< 0.0001. N.D: to Notcontrol Detected. ± SEM of three independent experiments. Statistical significance: **** p < 0.0001. N.D: Not Detected.

Figure 8. NRH induced 4NADO production in HepG3 cells but not in HEK293T. HEK293T (A) and HepG3 (B) cells were continuously dosed with 100 µM of 4PYR for 1, 4, and 24 h. The crude cell extract was measured by LC-MS for its NADO content. The NADO detected matches the 4NADO standard. Results are presented as relative abundance to control ± SEM of three independent experiments. Statistical significance: **** p < 0.0001. N.D: Not Detected.

Int. J. Mol. Sci. 2021, 22, 1145 12 of 28

3. Discussion Pyridones and methyl-nicotinamide are measured in clinical settings to identify vitamin B3 deficiencies [48,49] and in metabolomics as they associate with increased NAD consumption [50,51] and cellular dysfunctions in tissues [51–55]. Unfortunately, over the years the nomenclature used to describe the pyridone’s nature thus characterized has been somewhat unsystematic and exquisitely confusing (e.g., [5,11,56]). For instance, the term “pyridone” can describe both the methylated or non-methylated forms, abbreviated PY. Similarly, PY and PYR have been used to describe the ribosylated or non-ribosylated species indiscriminately (e.g., [28,57]). Surely, there is a need for consistency, as these species come from very distinct pathways and are sought systematically during physiological and pathophysiological investigations [11,55]. We proposed that by implementing the IUPAC nomenclature, inconsistencies can be readily rectified. We have named, characterized, and compared each of the pyridone species to facilitate this transition to provide clarity to the field. The abundance of urinary pyridones derived from nicotinamide has been shown to correlate with metabolic syndromes and aging [4,58]. While N-Me-2PY, N-Me-6PY, 4PYR, and 6PYR are detected in human urine, N-Me-2PY and 2PYR could go undetected because of their rapid conversion to the free acid or an ester during acidic or basic extraction protocols [59–61]. When the amide bond can share hydrogen bond interactions with the neighboring carbonyl, as is the case in N-Me-4PY, 4PYR, N-Me-2PY, and 2PYR, the loss of ammonia occurs readily, as we observed in solution chemistry, a process that could readily occur during sample processing. Mass spectrometry detects all metabolites in ionic forms. For the NAD metabolome, in some cases, such as for NR, the metabolite exists in an ionic form before reaching the detector; however, in most cases, the metabolite requires protonation/deprotonation or adduction, usually with sodium, potassium, chloride, ammonium. Many metabolites included in the NAD metabolome react to the most common ionization source, electrospray ionization (ESI) [62,63]. ESI employs a steady stream of nitrogen and heat to produce ions. N-Me-4PY, 4PYR, N-Me-2PY, and 2PYR react within the ESI chamber in a potentially confounding way because of an MS/MS fragmentation and formation of an acylium ion. In the ESI-MS instrument, such fragments, if stabilized or produced at a high enough rate, can react with water or methanol to form the acid or methyl ester analogs of N-Me-4PY, 4PYR, N-Me-2PY, and 2PYR (Figure2). Here, the in-source reaction can lead to a loss of signal and potentially the false identification of metabolites that likely do not exist in the biological sample. Furthermore, metabolite derivatives, artifacts of ESI, could go unmeasured. When a low-resolution instrument is used, the carboxylic acids may be non- resolved from the carboxamide form (Supplementary Materials). However, high-resolution mass spectrometers are more than capable of resolving the amide form from the carboxylic acid [64]. In quantitative metabolomics, internal isotopically labeled standards are used to identify and quantify specific metabolites of interest [63]. In a more encompassing but less accurate, semi-quantitative metabolomics, identifying a species often relies upon the m/z ratio and fragmentation. As such, the materials can go undetected or underrepresented unless dedicated internal standards are used for their quantification. It is with this in mind that one can rationalize the often-contradictory reports of pyridones’ quantifications. Crucially, the ribosylated pyridones derived from nicotinamide can only be generated from the ribosylated forms of nicotinamide [17]. Such forms include nicotinamide riboside (NR), its mononucleotide parent (NMN) or NAD(P), and their reduced forms [1]. Circulat- ing endogenous and exogenous 4PYR is readily intracellularized and metabolized by blood and tissues where it is phosphorylated and converted to either 4PYR-triphosphate (4PYR- TP) or the NAD derivative 4NADO, all inhibitors of intracellular enzymes [25,27,46,65]. The cells’ ability to convert exogenous pyridone ribosides to NAD-like species might select for 4PYR’s capacity to promote cell death in a cell-specific manner, as it has been observed here and by others [26,28]. Crucially, we observed similar cell line selectivity regarding cell survival when the adenine dinucleotide form of 4PYR, 4NADO, is generated Int. J. Mol. Sci. 2021, 22, 1145 13 of 28

endogenously following exposure to the reduced form of NR, NRH (Figure8)[ 47]. Still, the causality of cell death observed in HepG3 cells exposed to NRH, and the formation of 4NADO remain to be explored. Finally, exogenous 2PYR and 6PYR do not affect HEK293T and HepG3 cells’ viability (Figure6). Yet, their function, if generated endogenously, remains unknown. Therefore, the physiological properties of endogenously produced toxins derived from the oxidation of NAD, NADP, and NR warrant further investigations. Finally, aldehyde oxidases are responsible for the formation of the methylated pyridones (N-Me-6PY, and N-Me-4PY) from methyl-nicotinamide [36]. Yet, the generation of the three ribosylated pyridone derivatives, 2PYR, 4PYR, and 6PYR, remains unexplained. A hint as to the source of the ribosylated pyridones was obtained when the FAD (flavin adenine dinucleotide)-dependent FDXR was shown to generate the oxidized form of NADP, NADPO, in a side-reaction. In the proposed mechanism, NADP, instead of the normal substrate NADPH, binds FAD-bound FDXR [39]. The 1,4-nucleophilic addition of an active site water molecule on the enzyme-bound NADP leads to a 4-hydroxylated dihydropyri- dine derivative that can then enter in hydride-transfer type catalysis with the bound FAD to generate the 4-pyridone form of NADP (i.e., 4NADPO). At first sight and given the abundance of the urinary ribosylated pyridones, the kinetics of this side-reaction enzymatic process would preclude it to be the major source of 4PYR. Furthermore, this enzymatic process only accounts for the generation of the dinucleotide form of 4PYR and cannot explain the formation of 2PYR, known to also be in circulation [66]. NQO1 and NQO2 are FAD-dependent redox enzymes that bind NAD(P)H and NRH, respectively, and could potentially generate 4NAD(P)O and 4PYR via a mechanism like that of FDXR. Recent work on NQO2 and acetaminophen in the presence of NRH has identified superoxide HOO- as a by-product of the NQO2-catalyzed turn-over, especially in the presence of O2 [45]. We envisage that the detection of 4PYR in the NQO2-catalyzed oxidation of NRH is promoted by the NQO2-catalyzed production of superoxide upon the reduction of NRH, with O2 as a co-oxidant and that the production of superoxide within the vicinity of a still FAD-NQO2-bound NR, favors the addition of superoxide onto the electrophilic NR. Superoxide is likely to react within the constraints of the enzyme binding pocket and lead to a favored isomer. As such, this type of side reaction or a variation thereof could apply to other NAD(P)H binding FAD-dependent oxidoreductases and explain the generation of a substantial amount of ribosylated pyridone metabolites in vivo. It is important to note that CYP450 and FAD-dependent aryl hydroxylation has long been recognized as a source of hydroxylated catabolites of aryl amino acids and riboflavin, e.g., 2/3-hydroxylated tyrosine and 7/8-hydroxyriboflavins, respectively [67,68]. In such a process, the transient formation of the ROS species is responsible for the outcomes of the conversion. We, therefore, explored whether Fenton chemistry [69], often used to emulate ROS-chemistry in these systems, could promote PYR formation from NR and found that only 2PYR and 6PYR but not 4PYR were readily detected. We have therefore identified two related oxidative mechanisms that could be wide-spread and responsible for the formation of functionalized ribosylated pyridones.

4. Methods 4.1. Syntheses 4.1.1. Synthesis of N-methyl-4-pyridone 3-carboxamide (1) The synthesis of N-methyl-4-pyridone 3-carboxamide was conducted according to Scheme1. Int. J. Mol. Sci. 2021, 22, x 13 of 27

4. Methods 4.1. Syntheses 4.1.1. Synthesis of N-Methyl-4-Pyridone 3-Carboxamide (1) Int. J. Mol. Sci. 2021, 22, 1145 The synthesis of N-methyl-4-pyridone 3-carboxamide was conducted14 of 28 according to Scheme 1.

Scheme 1. Synthesis of N-methyl-4-pyridone 3-carboxamide. Scheme 1. Synthesis of N-methyl-4-pyridone 3-carboxamide. Synthesis of O-methyl 4-methoxypyridine-3-carboxylate (9) (Step-1): To a solution of 4- chloronicotinic acid (8) (1.0 g, 6.34 mmol) in anhydrous methanol (18.5 mL) was added acetylSynthesis chloride (1.42 of mL)O-methyl at 0 ◦C. 4-methoxypyridine-3-carboxylate The reaction mixture was stirred under (9 nitrogen) (Step-1) overnight: To a solution of 4- atchloronicotinic 65–70 ◦C. Upon acid completion (8) (1.0 of g, the 6.34 reaction, mmol) the in reaction anhydrous mixture methanol was concentrated (18.5 mL) was added underacetyl reduced chloride pressure. (1.42 mL) The crude at 0 was°C. dissolvedThe reaction in saturated mixture NaHCO was 3stirred(pH = 8–9) under and nitrogen over- thennight extracted at 65–70 with °C. CHCl Upon3 (2 completion× 15 mL). The of combined the reaction, organics the were reaction washed mixture with brine was concentrated (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford theunder desired reduced product pressure. as a white The solid crude (87%). was1H NMR dissolved (MeOD), inδ saturated, ppm: 8.74 NaHCO (s, 1H, H1),3 (pH = 8–9) and 8.51then (d, extracted J = 6.04 Hz, with 1H, H6),CHCl 7.183 (d,(2 J× =15 6.04 mL). Hz, 1H,The H5), combined 3.96 (s, 3H, organics COOMe), were 3.87 (s, washed 3H, with brine 13 –OMe).(30 mL),C dried NMR (MeOD),over Naδ,2SO ppm:4, filtered, 165.58 (C4), and 164.92 concentrated (C7), 153.52 (C2), under 151.37 reduced (C6), 116.45 pressure to afford (C3),the desired 107.93 (C5), product 55.33 (C9), as a 51.31 white (C8) solid [29]. (87%). 1H NMR (MeOD), δ, ppm: 8.74 (s, 1H, H1), 8.51 Synthesis of 4-methoxypyridine-3-carboxamide (10) (Step-2): O-Methyl-4-methoxypyridine- (d, J = 6.04 Hz, 1H, H6), 7.18 (d, J = 6.04 Hz, 1H, H5), 3.96 (s, 3H, COOMe), 3.87 (s, 3H, – 3-carboxylate (9) (0.5 g, 2.99 mmol) in NH4OH (27%, 20 mL) was stirred at rt for 15 in a 13 50OMe). mL sealed C NMR tube and(MeOD), the reaction δ, ppm: was 165.58 monitored (C4), by 164.921H NMR. (C7), After 153.52 the reaction (C2), 151.37 had (C6), 116.45 gone(C3), to 107.93 completion, (C5), the 55.33 solvent (C9), was 51.31 evaporated (C8). [29] under reduced pressure and the obtained whiteSynthesis solid was usedof 4-methoxypyridine-3-carboxamide for the next step without purification (10 (96%).) (Step-2)1H NMR: O-Methyl-4-methoxypyri- (MeOD), δ, ppm: 8.87 (s, 1H, H1), 8.51 (d, J = 5.96 Hz, 1H, H6), 7.21 (d, J = 6.0 Hz, 1H, H5), 4.04 (s, 3H, dine-3-carboxylate (9) (0.5 g, 2.99 mmol) in NH4OH (27%, 20 mL) was stirred at rt for 15 –OMe). 13C NMR (MeOD), δ, ppm: 166.50 (C4), 164.35 (C7), 153.02 (C2), 151.37 (C6), 118.16 1 (C3),in a 50 107.51 mL (C5), sealed 55.55 tube (C8) and [29]. the reaction was monitored by H NMR. After the reaction had goneSynthesis to completion, of 1-N-methyl-4-oxo-pyridine-3-carboxamide the solvent was evaporated (under1) (Step-3) reduced: To a solutionpressure of and 4- the obtained methoxypyridine-3-carboxamidewhite solid was used for the (10 )next (438 mg,step 2.88 without mmol) in purification methanol:water (96%). (9:1) mixture1H NMR (MeOD), δ, (10 mL) was added iodomethane (1.74 mL, 28 mmol). The reaction was stirred under ppm: 8.87 (s, 1H, H1),◦ 8.51 (d, J = 5.96 Hz, 1H, H6),◦ 7.21 (d, J = 6.0 Hz, 1H, H5), 4.04 (s, 3H, nitrogen for13 24 h at 60 C. After stirring for 24 h at 60 C with an oil bath, the mixture was–OMe). cooled C in NMR an iced (MeOD), bath for 1δ h, ppm: and the 166.50 resulting (C4), yellow 164.35 precipitate (C7), 153.02 was filtered (C2), 151.37 off, (C6), 118.16 washed(C3), 107.51 with methanol, (C5), 55.55 and (C8) dried [29]. under high vacuum. The final product did not require purification.Synthesis Yield: of 65%.1-N-methyl-4-oxo-pyridine-3-carboxamide1H NMR (DMSO, d6), δ, ppm: 9.36 (s, 1H, NH),(1) (Step-3) 8.54 (s, 1H,: To H2), a solution of 4- 7.87methoxypyridine-3-carboxamide (d, J = 5.08 Hz, 1H, H6), 7.54 (s, 1H, NH),(10) 6.53(438 (d, mg, J = 7.42.88 Hz, mmol) 1H, H5), in 3.82 methanol:water (s, 3H, Me). (9:1) mix- 13 δ tureC NMR (10 mL) (MeOD), was ,added ppm: 175.66 iodomethane (C4), 165.51 (1.74 (C7), mL, 146.51 28 (C2), mmol). 143.27 The (C6), reaction 119.66 (C3), was stirred under 118.97 (C5), 44.37 (C8), HRMS calcd for C H N O [M + H]+ 153.0664 found 153.0652 [29]. nitrogen for 24 h at 60 °C. After stirring7 9 2 for2 24 h at 60 °C with an oil bath, the mixture was Int. J. Mol. Sci. 2021, 22, x 4.1.2.cooled Synthesis in an iced of O-methyl, bath for 1- 1N -methyl-4-oxo-pyridine-3-carboxylateh and the resulting yellow precipitate (12) was filtered14 off,of 27 washed withThe methanol, synthesis ofandO-methyl, dried under 1-N-methyl-4-oxo-pyridine-3-carboxylate high vacuum. The final product was did conducted not require purifica- accordingtion. Yield: to Scheme 65%.1H2. NMR (DMSO, d6), δ, ppm: 9.36 (s, 1H, NH), 8.54 (s, 1H, H2), 7.87 (d, J = 5.08 Hz, 1H, H6), 7.54 (s, 1H, NH), 6.53 (d, J = 7.4 Hz, 1H, H5), 3.82 (s, 3H, Me). 13C NMR (MeOD), δ, ppm: 175.66 (C4), 165.51 (C7), 146.51 (C2), 143.27 (C6), 119.66 (C3), 118.97 (C5), 44.37 (C8), HRMS calcd for C7H9N2O2 [M + H]+ 153.0664 found 153.0652 [29].

4.1.2. Synthesis of O-Methyl, 1-N-Methyl-4-Oxo-Pyridine-3-Carboxylate (12) SchemeScheme 2.2. SynthesisSynthesis of of the theO -methyl,O-methyl, 1-N 1--methyl-4-oxo-pyridine-3-carboxylateN-methyl-4-oxo-pyridine-3-carboxylate (12). (12). The synthesis of O-methyl, 1-N-methyl-4-oxo-pyridine-3-carboxylate was conducted accordingSynthesis to ofScheme O-methyl 2. 4-pyridone-3-carboxylate (11) (Step-1): To a solution of the 4-chloronicotinic acid (8) (5 g, 31.7 mmol) in H2O (50 mL) was refluxed for 3 h and then concentrated under reduced pressure. The obtained yellow solid was azeotroped with toluene and then dissolved in anhydrous MeOH (50 mL). To the resulting mixture was added SOCl2 (4.99 mL, 8.19 g, 68.9 mmol) at room temperature, and then the reaction mixture was heated to 75 °C. After stirring at 75 °C for 15 h, the mixture was concentrated under reduced pressure, and the residual product was treated with saturated NaHCO3. As the remaining amount of thionyl chloride got neutralized by NaHCO3, the desired product precipitated. The precipitate was filtered, washed twice with water, and used directly for the next step without further purification. (e.g., Scheme Yield 62%. 1H NMR (D2O), δ, ppm: 8.44 (s, 1H, H2), 7.76 (d, J = 7.2 Hz, 1H, H6), 6.53 (d, J = 6.53 Hz, 1H, H5), 3.77 (s, 3H, OMe). 13C NMR (CDCl3), δ, ppm: 178.34 (C4), 166.77 (COOCH3), 144.41 (C2), 139.15 (C6), 120.11 (C5), 117.13 (C3), 52.26 (Me), HRMS calcd for C7H8NO3 [M + H]+ 154.0504 found 154.0490. Spectral data are consistent with published literature. [25] Synthesis of O-methyl, 1-N-methyl 4-pyridone-3-carboxylate (12) (Step-2): In a 50-mL round bottom flask, equipped with a magnetic stirring bar, NaH (15.30 mg, 0.65 mmol) was added to 2 mL of anhydrous methanol in a portion-wise manner, and then methyl 4- pyridone-3-carboxylate (11) (50 mg, 0.33 mmol) was added. The resulting reaction mixture was stirred at rt for 10 min. Then, CH3I (81.17 µL, 1.30 mmol) was added, and the resulting mixture was stirred again at the same temperature for 36 h. The reaction progress was monitored by TLC. Upon completion of the reaction, the resulting mixture was concentrated under reduced pressure, and the desired product was precipitated by adding EtOAc (2 mL). (Yield = 70%).1H NMR (MeOD), δ, ppm: 8.47 (s, 1H, H2), 7.72 (dd, J = 2.28 & 2.28 Hz, 1H, H6), 6.49 (d, J = 7.52 Hz, 1H, H5), 3.83 (s, 3H, OMe), 3.80 (s, 3H, -NMe). 13C NMR (CDCl3), δ, ppm: 176.38 (C4), 164.88 (C7), 147.58 (C2), 142.16 (C6), 120.79 (C3), 117.26 (C5), 50.86 (–OCH3), 43.23 (–CH3). HRMS calcd for C8H10NO3 [M + H]+ 168.0661 found 168.0648. Spectral data are consistent with published [M+ + H].

4.1.3. Synthesis of N-Methyl-6-Pyridone 3-Carboxamide (2) The synthesis of N-methyl-6-pyridone 3-carboxamide was conducted according to Scheme 3.

Scheme 3. Synthesis of N-methyl-6-pyridone 3-carboxamide.

Synthesis of O-ethyl 6-oxo-1H-pyridine-3-carboxylate (14) (Step-1): To a stirred solution of 6-hydroxynicotinic acid (13) (5 g, 3.59 mmol) in absolute ethanol (25 mL) was added sulfuric acid (0.2 mL) at room temperature. The mixture was heated to reflux for 48 h. After cooling down to room temperature, water (2.5 mL) was added, and the reaction mixture was neutralized to pH = 6–7 by portion-wise addition of sodium hydrogen carbonate (482 mg). Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was extracted with ethyl acetate (3 × 10 mL). The

Int. J. Mol. Sci. 2021, 22, x 14 of 27

Int. J. Mol. Sci. 2021, 22, 1145 15 of 28 Scheme 2. Synthesis of the O-methyl, 1-N-methyl-4-oxo-pyridine-3-carboxylate (12).

Synthesis of O-methyl 4-pyridone-3-carboxylate (11) (Step-1): To a solution of the 4-chlo- ronicotinicSynthesis acid of (8 O-methyl) (5 g, 31.7 4-pyridone-3-carboxylate mmol) in H2O (50 mL) (was11) (Step-1)refluxed: for To 3 a h solution and then of concen- the 4- chloronicotinictrated under reduced acid (8 pressure.) (5 g, 31.7 The mmol) obtained in H 2yellowO (50 mL)solid was was refluxed azeotroped for 3with h and toluene then concentratedand then dissolved under in reduced anhydrous pressure. MeOH The (50 obtained mL). To yellow the resulting solid was mixture azeotroped was added with tolueneSOCl2 (4.99 and mL, then 8.19 dissolved g, 68.9 inmmol) anhydrous at room MeOH temperature, (50 mL). and To thethen resulting the reaction mixture mixture was addedwas heated SOCl to2 (4.9975 °C. mL, After 8.19 stirring g, 68.9 at mmol) 75 °C for at room15 h, the temperature, mixture was and concentrated then the reaction under mixture was heated to 75 ◦C. After stirring at 75 ◦C for 15 h, the mixture was concentrated reduced pressure, and the residual product was treated with saturated NaHCO3. As the under reduced pressure, and the residual product was treated with saturated NaHCO . As remaining amount of thionyl chloride got neutralized by NaHCO3, the desired product3 theprecipitated. remaining The amount precipitate of thionyl was chloride filtered, got wash neutralizeded twice with by NaHCO water,3 and, the used desired directly product for precipitated. The precipitate was filtered, washed twice with water, and used directly for the next step without further purification. (e.g., Scheme Yield 62%. 1H NMR (D2O), δ, ppm: 1 δ the8.44 next (s, 1H, step H2), without 7.76 (d, further J = 7.2 purification. Hz, 1H, H6), E.g., 6.53 Scheme (d, J = 6.53 Yield Hz, 62%. 1H, HH5), NMR 3.77 (D (s,2O), 3H, ,OMe). ppm: 8.4413 (s, 1H, H2), 7.76 (d, J = 7.2 Hz, 1H, H6), 6.53 (d, J = 6.53 Hz, 1H, H5), 3.77 (s, 3H, OMe). 13C NMR (CDCl3), δ, ppm: 178.34 (C4), 166.77 (COOCH3), 144.41 (C2), 139.15 (C6), 120.11 C NMR (CDCl3), δ, ppm: 178.34 (C4), 166.77 (COOCH3), 144.41 (C2), 139.15 (C6), 120.11 (C5), 117.13 (C3), 52.26 (Me), HRMS calcd for C7H8NO3 [M + H]+ 154.0504 found 154.0490. (C5), 117.13 (C3), 52.26 (Me), HRMS calcd for C H NO [M + H]+ 154.0504 found 154.0490. Spectral data are consistent with published literature.7 8 3 [25] Spectral data are consistent with published literature [25]. Synthesis of O-methyl, 1-N-methyl 4-pyridone-3-carboxylate (12) (Step-2): In a 50-mL Synthesis of O-methyl, 1-N-methyl 4-pyridone-3-carboxylate (12) (Step-2): In a 50-mL round round bottom flask, equipped with a magnetic stirring bar, NaH (15.30 mg, 0.65 mmol) bottom flask, equipped with a magnetic stirring bar, NaH (15.30 mg, 0.65 mmol) was added was added to 2 mL of anhydrous methanol in a portion-wise manner, and then methyl 4- to 2 mL of anhydrous methanol in a portion-wise manner, and then methyl 4-pyridone-3- pyridone-3-carboxylate (11) (50 mg, 0.33 mmol) was added. The resulting reaction mixture carboxylate (11) (50 mg, 0.33 mmol) was added. The resulting reaction mixture was stirred was stirred at rt for 10 min. Then, CH3I (81.17 µL, 1.30 mmol) was added, and the resulting at rt for 10 min. Then, CH I (81.17 µL, 1.30 mmol) was added, and the resulting mixture mixture was stirred again3 at the same temperature for 36 h. The reaction progress was was stirred again at the same temperature for 36 h. The reaction progress was monitored monitored by TLC. Upon completion of the reaction, the resulting mixture was concen- by TLC. Upon completion of the reaction, the resulting mixture was concentrated under reducedtrated under pressure, reduced and pressure, the desired and product the desir wased precipitated product was by precipitated adding EtOAc by (2adding mL). 1 (YieldEtOAc = (2 70%). mL).1 (YieldH NMR = 70%). (MeOD),H NMRδ, ppm: (MeOD), 8.47 (s, δ 1H,, ppm: H2), 8.47 7.72 (s, (dd, 1H, J H2), = 2.28 7.72 & 2.28(dd, Hz, J = 2.28 1H, & 2.28 Hz, 1H, H6), 6.49 (d, J = 7.52 Hz, 1H, H5), 3.83 (s, 3H, OMe), 3.8013 (s, 3H, -NMe). 13C H6), 6.49 (d, J = 7.52 Hz, 1H, H5), 3.83 (s, 3H, OMe), 3.80 (s, 3H, –NMe). C NMR (CDCl3), δNMR, ppm: (CDCl 176.383), δ (C4),, ppm: 164.88 176.38 (C7), (C4), 147.58 164.88 (C2), (C7), 142.16 147.58 (C6), (C2), 120.79142.16 (C3),(C6), 120.79 117.26 (C3), (C5), 117.26 50.86 (C5), 50.86 (–OCH3), 43.23 (–CH3). HRMS calcd for C8H10NO+3 [M + H]+ 168.0661 found (–OCH3), 43.23 (–CH3). HRMS calcd for C8H10NO3 [M + H] 168.0661 found 168.0648. + Spectral168.0648. data Spectral are consistent data are consistent with published with published [M+ + H]. [M + H].

4.1.3.4.1.3. Synthesis of N-methyl-6-pyridone-Methyl-6-Pyridone 3-carboxamide3-Carboxamide ( 2(2) ) TheThe synthesissynthesis ofof NN-methyl-6-pyridone-methyl-6-pyridone 3-carboxamide3-carboxamide waswas conductedconducted accordingaccording toto SchemeScheme3 3..

SchemeScheme 3.3. Synthesis of N-methyl-6-pyridone-methyl-6-pyridone 3-carboxamide.3-carboxamide.

SynthesisSynthesis ofof O-ethylO-ethyl 6-oxo-1H-pyridine-3-carboxylate6-oxo-1H-pyridine-3-carboxylate (14) (Step-1)(Step-1): To aa stirredstirred solutionsolution ofof 6-hydroxynicotinic6-hydroxynicotinic acid (13)) (5(5 g,g, 3.593.59 mmol)mmol) inin absoluteabsolute ethanolethanol (25(25 mL)mL) waswas addedadded sulfuricsulfuric acidacid (0.2 (0.2 mL) mL) at at room room temperature. temperature. The The mixture mixture was was heated heated to reﬂux to reflux for 48 for h. After48 h. coolingAfter cooling down todown room to temperature,room temperature, water (2.5 water mL) (2.5 was mL) added, was andadded, the reactionand the mixturereaction wasmixture neutralized was neutralized to pH = to 6–7 pH by = portion-wise6–7 by portion-wise addition addition of sodium of sodium hydrogen hydrogen carbonate car- (482bonate mg). (482 Upon mg). completion Upon completion of the reaction, of the reaction, the reaction the reaction mixture mixture was concentrated was concentrated under reducedunder reduced pressure, pressure, and the and residue the residue was was extracted extracted with with ethyl ethyl acetate acetate (3 (3× ×10 10 mL). mL). TheThe combined organic layers were washed with brine, dried over Na2SO4, and evaporated under reduced pressure leading to the pure ethyl 6-hydroxynicotinate (14) (yield = 82%). 1 H NMR (CDCl3), δ, ppm: 8.16 (s, 1H, H6), 7.95 (dd, J = 2.44 & 2.44 Hz, 1H, H4), 6.51 (d, J = 9.56 Hz, 1H, H3), 4.25 (q, J = 7.14 Hz, 2H, -CH2), 1.28 (t, J = 7.12 Hz, 3H, Me). 13C NMR

(CDCl3), δ, ppm: 165.61 (COCH3), 164.00 (C2), 141.11 (C6), 139.69 (C4), 119.36 (C3), 111.44 (C5), 60.68 (–CH2), 14.23 (Me) [30]. Synthesis of O-ethyl-1-methyl-6-oxo-pyridine-3-carboxylate (15) (Step-2): In a 50-mL round bottom ﬂask, equipped with a magnetic stirring bar, NaH (56.11, 2.39 mmol) was added to Int. J. Mol. Sci. 2021, 22, x 15 of 27

combined organic layers were washed with brine, dried over Na2SO4, and evaporated under reduced pressure leading to the pure ethyl 6-hydroxynicotinate (14) (yield = 82%).1H NMR (CDCl3), δ, ppm: 8.16 (s, 1H, H6), 7.95 (dd, J = 2.44 & 2.44 Hz, 1H, H4), 6.51 (d, J = 9.56 Hz, 1H, H3), 4.25 (q, J = 7.14 Hz, 2H, -CH2), 1.28 (t, J = 7.12 Hz, 3H, Me). 13C NMR (CDCl3), δ, ppm: 165.61 (COCH3), 164.00 (C2), 141.11 (C6), 139.69 (C4), 119.36 (C3), 111.44 Int. J. Mol. Sci. 2021, 22, 1145 (C5), 60.68 (–CH2), 14.23 (Me) [30]. 16 of 28 Synthesis of O-ethyl-1-methyl-6-oxo-pyridine-3-carboxylate (15) (Step-2): In a 50-mL round bottom flask, equipped with a magnetic stirring bar, NaH (56.11, 2.39 mmol) was added to 3 mL of anhydrous methanol in a portion-wise manner, and then ethyl 6-hy- 3droxynicotinate mL of anhydrous (14 methanol) (0.2 g, 1.19 in a portion-wisemmol) was added, manner, th ande resulting then ethyl reaction 6-hydroxynicotinate mixture was (stirred14) (0.2 at g, rt 1.19for 10 mmol) min, and was then added, CH3I the (0.297 resulting mL, 4.78 reaction mmol) mixture was added, was and stirred the resulting at rt for 10mixture min, andwas then stirred CH 3againI (0.297 at mL,the same 4.78 mmol)temperature was added, for 36 and h. The the resultingreaction progress mixture waswas stirredmonitored again by at TLC the analysis. same temperature Upon completion for 36 h. of The the reaction reaction, progress the resulting was monitored mixture was by TLCconcentrated analysis. under Upon completionvacuum and of dissolved the reaction, in EtOAc the resulting (15 mL). mixture The organic was concentrated layer was underwashed vacuum with water and dissolved(3 × 10 mL), in dried EtOAc over (15 mL).Na2SO The4, and organic concentrated layer was under washed reduced with water pres- (3 × 10 mL), dried over Na SO , and concentrated under reduced1 pressure to afford the sure to afford the product (215) as4 white solid (Yield = 80%). H NMR (CDCl3), δ, ppm: 8.12 1 product(s, 1H, H6), (15) 7.77 as white (dd, J solid = 2.52 (Yield & 2.52 = 80%).Hz, 1H,H H4), NMR 6.46 (CDCl (d, J3 ),= 9.52δ, ppm: Hz, 8.121H, (s,H3), 1H, 3.79 H6), (s, 7.773H, (dd, J = 2.52 & 2.52 Hz, 1H, H4), 6.46 (d, J = 9.52 Hz, 1H, H3), 3.79 (s, 3H, OMe), 3.52 (s, OMe), 3.52 (s, 3H, Me). 13C NMR (CDCl3), δ, ppm: 165.61 (COCH3), 162.89 (C2), 143.55 13 δ 3H,(C6), Me). 138.63C NMR(C4), 119.40 (CDCl 3(C3),), , ppm: 108.58 165.61 (C5), (COCH 52.04 3(OMe),), 162.89 38.20 (C2), 143.55(Me). HRMS (C6), 138.63 calcd (C4), for 119.40 (C3), 108.58 (C5), 52.04 (OMe), 38.20 (Me). HRMS calcd for C H NO [M + H]+ C8H10NO3 [M + H]+ 168.0661 found 168.0647. 8 10 3 168.0661Synthesis found of 168.0647. 1-N-methyl-6-pyridone-3-carboxamide (2) (Step-3): A sealed tube equipped Synthesis of 1-N-methyl-6-pyridone-3-carboxamide (2) (Step-3): A sealed tube equipped with a Teflon-coated magnetic stirring bar was charged with O-methyl, 1-N-methyl-6-oxo- with a Teﬂon-coated magnetic stirring bar was charged with O-methyl, 1-N-methyl-6-oxo- pyridine-3-carboxylic acid (15) (2 g, 13.0 mmol) and 10 mL mixture of NH4OH solution pyridine-3-carboxylic acid (15) (2 g, 13.0 mmol) and 10 mL mixture of NH OH solution (28–30%) and methanol (1:1). The resulting mixture was stirred at room temperature4 for (28–30%) and methanol (1:1). The resulting mixture was stirred at room temperature for 24 h. After 24 h, reaction progress was monitored by TLC. Upon completion of the reac- 24 h. After 24 h, reaction progress was monitored by TLC. Upon completion of the reaction, tion, the solvent was evaporated under reduced pressure, and the resulting crude (2) was the solvent was evaporated under reduced pressure, and the resulting crude (2) was ﬁltered filtered off and washed with ethyl acetate. Yield 66%. 1H NMR (D2O), δ, ppm: 8.38 (s, 1H, off and washed with ethyl acetate. Yield 66%. 1H NMR (D O), δ, ppm: 8.38 (s, 1H, H2), H2), 7.95 (d, 1H, J = 6.69 Hz, 1H, H4), 6.53 (d, 1H, J = 9.44 Hz,2 H5), 3.62 (s, 3H, OMe). 13C 7.95 (d, 1H, J = 6.69 Hz, 1H, H4), 6.53 (d, 1H, J = 9.44 Hz, H5), 3.62 (s, 3H, OMe). 13C NMR NMR (CDCl3), δ, ppm: 167.17 (CONH2), 163.69 (C2), 142.03 (C6), 138.61 (C4), 117.89 (C3), (CDCl3), δ, ppm: 167.17 (CONH2), 163.69 (C2), 142.03 (C6), 138.61 (C4), 117.89 (C3), 113.52 113.52 (C5), 37.27 (Me). HRMS calcd for C7H9N2O2 [M+ + H]+ 153.0664 found 153.0663. (C5), 37.27 (Me). HRMS calcd for C7H9N2O2 [M + H] 153.0664 found 153.0663.

4.1.4. Synthesis of N-methyl-6-pyridone-Methyl-6-Pyridone 3-carboxamide3-Carboxamide ( 2(2)) The synthesis of 1-N-methyl-2-pyridone-3-carboxamide-methyl-2-pyridone-3-carboxamide waswas conductedconducted accordingaccording toto Scheme4 4..

Scheme 4. 4. SynthesisSynthesis of 1- ofN 1--methyl-2-pyridone-3-carboxamideN-methyl-2-pyridone-3-carboxamide and 1- andN-methyl-6-pyridone-3-car- 1-N-methyl-6-pyridone-3- carboxamide.boxamide.

N-Methylnicotinamide ( (77)) (0.5 (0.5 g, g, 1.89 1.89 mmol) mmol) was was dissolved dissolved in 10 in mL 10 mLNaOH NaOH (1M, (1M, aq.) aq.)and andthen then commercially commercially available available K3[Fe(CN) K3[Fe(CN)6] (1.876] (1.87 g, 5.68 g, 5.68mmol), mmol), dissolved dissolved in 2 inmL 2 mLde- deionizedionized water water was was added. added. The The resulting resulting mixture mixture wa wass stirred stirred at at rt rt for for 1 h. After 1 h, 25 mLmL MeOH was addedadded toto precipitateprecipitate KK44[Fe(CN)[Fe(CN)6]] and then the resulting mixture waswas filteredfiltered off. The filtratefiltrate was concentrated on rotavaprotavap and the crude was purifiedpurified by using hexane, ethyl acetate and methanol as an eluent to afford 1-methyl-2-oxo-pyridine-3-carboxamide 1 ((55)) (15%)(15%) andand 1-methyl-6-oxo-pyridine-3-carboxamide1-methyl-6-oxo-pyridine-3-carboxamide ( (22)) (25%). (25%).1H HNMR NMR (DMSO- (DMSO-d6),d 6δ),, δ, ppm: 9.06 (s, 1H, NH), 8.27 (dd, J = 2.2 Hz & 2.16 Hz, 1H, H6), 8.01 (dd, J = 2.16 & 2.16 Hz, 1H, H4), 7.54 (s, 1H, NH), 6.44 (t, J = 6.88 Hz, 1H, H5), 3.08 (s, 3H, Me). 13C NMR (DMSO-d6), δ, ppm: 165.08 (CONH2), 162.22 (CO), 144.43 (C6), 143.70 (C4), 120.45 (C3), + 106.10 (C5), 38.16 (CH3) HRMS calcd for C7H9N2O2 [M + H] 153.0664 found 153.0648 [29]. Int. J. Mol. Sci. 2021, 22, x 16 of 27

ppm: 9.06 (s, 1H, NH), 8.27 (dd, J = 2.2 Hz & 2.16 Hz, 1H, H6), 8.01 (dd, J = 2.16 &2.16 Hz, 1H, H4), 7.54 (s, 1H, NH), 6.44 (t, J = 6.88 Hz, 1H, H5), 3.08 (s, 3H, Me). 13C NMR (DMSO- d6), δ, ppm: 165.08 (CONH2), 162.22 (CO), 144.43 (C6), 143.70 (C4), 120.45 (C3), 106.10 (C5), 38.16 (CH3) HRMS calcd for C7H9N2O2 [M + H]+ 153.0664 found 153.0648. [29] Int. J. Mol. Sci. 2021, 22, 1145 17 of 28 4.1.5. Synthesis of the Ribosylated Pyridone Carboxamides

Synthesis of 4-Pyridone-3-Carboxamide4.1.5. Synthesis of the Ribosylated Riboside Pyridone (3) Carboxamides The synthesis Synthesisof 4-pyridone-3-carboxamide of 4-pyridone-3-carboxamide riboside riboside (was3) conducted according to Scheme 5. The synthesis of 4-pyridone-3-carboxamide riboside was conducted according to Scheme5.

Scheme 5. Synthesis Schemeof 4-pyridone-3 5. Synthesis-carboxamide of 4-pyridone-3-carboxamide riboside. riboside. Synthesis of 4-pyridone-3-carboxamide (16) (Step-1): A sealed tube equipped with a Synthesis of 4-pyridone-3-carboxamideTeflon-coated magnetic stirring (16) (Step-1) bar was: chargedA sealed with tube methyl equipped 4-pyridone-3-carboxylate with a Tef- lon-coated magnetic(11 stirring) (2 g, 13.0 bar mmol) was and charge 50 mLd ofwith NH methyl4OH methanolic 4-pyridone-3-carboxylate solution (28–30%). The ( resulting11) (2 g, 13.0 mmol) andmixture 50 mL was of stirredNH4OH at room methanolic temperature solution for 15 h.(28–30%). Upon completion The resulting of the reaction, mix- the ture was stirred at roomsolvent temperature was evaporated for at15 reduced h. Upon pressure, completion and the of residue the reaction, was triturated the solvent with water. The solid (16) was filtered off and washed with water. Yield 66%. 1H NMR (MeOD), δ, was evaporated at reducedppm: 8.54 pressure, (s, 1H, H2), and 7.79 (d,the J residue = 5.56 Hz, was 1H, H6),triturated 6.56 (d, with J = 6.53 water. Hz, 1H, The H5). solid13C NMR 1 (16) was filtered off(CDCl and washed3), δ, ppm: with 179.04 water. (C4), 168.48 Yield (CONH2), 66%. H 142.95NMR (C2), (MeOD), 139.32 (C6),δ, ppm: 119.45 8.54 (C5), (s, 117.69 + 1H, H2), 7.79 (d, J =(C3); 5.56 HRMS Hz, 1H, calcd H6), for C 66.56H7N (d,2O2 J[M = +6.53 H] Hz,139.0508 1H, foundH5). 139.049513C NMR [25 (CDCl]. 3), δ, 0 0 0 ppm: 179.04 (C4), 168.48Synthesis (CONH2), of 1-(2 ,3 142.95,5 -tri-O -acetyl-(C2), β139.32-D-ribofuranosyl)-4-pyridone-3-carboxamide (C6), 119.45 (C5), 117.69 (C3);(18) (Step- 2): Silylation of 4-pyridone-3-carboxamide (17): 4-pyridone-3-carboxamide (16) (0.5 g, HRMS calcd for C6H7N2O2[M + H]+ 139.0508 found 139.0495. [25] 3.62 mmol): Hexamethyldisilazane (10 mL) and trimethylchlorosilane (1.5 mL, 1.30 g, Synthesis of 121-(2 mmol)′,3′,5 were′-tri-O-acetyl- added sequentiallyβ-D-ribofuranosyl)-4-pyridone-3-carboxamide in a 50 mL round bottom flask under nitrogen. (18) The (Step-2): Silylation ofresulting 4-pyridone-3-carboxamide mixture was stirred at 120 (17◦):C 4-pyridone-3-carboxamide for 2 h. Upon completion of the (16 reaction,) (0.5 the g, 3.62 mmol): Hexamethyldisilazanesolution was cooled to (10 room mL) temperature and trimethylchlorosilane and concentrated under (1.5 reduced mL, pressure.1.30 g, The 12 mmol) were addedresidue sequentially was co-evaporated in a 50 with mL anhydrous round bottom toluene (2flask to 3 times)under to nitrogen. afford the mixtureThe of mono and bis-silylated 4-pyridone-3-carboxamide (17), which was used directly for the resulting mixture wasnext stirred step. at 120 °C for 2 h. Upon completion of the reaction, the solution was cooled to roomVorbrüggen temperature glycosylation and concentrated(Step-3): The under crude reduced mono and pressure. bis-silylated The 4-pyridone- res- idue was co-evaporated3-carboxamide with anhydrous (17) mixture toluene was dissolved (2 to 3 in times) 6 mL ofto anhydrousafford the 1,2- mixture dichloroethane. of mono and bis-silylatedThen, 4-pyridone-3-carboxamide a solution of 1,2,3,5-tetra-O-acetyl- (17β),- Dwhich-ribofuranoside was used (0.6 directly g, 2 mmol) for in the 2 mL of 1,2-dichloroethane was added, followed by the addition of trimethylsilyl triflate (0.4 mL, next step. 2.3 mmol). The resulting mixture was stirred at 40–45 ◦C for overnight. The reaction was Vorbrüggen glycosylationmonitored by 1(Step-3)H NMR: analysis The crude of the mono crude mixture.and bis-silylated After completion 4-pyridone-3- of the reaction, carboxamide (17) mixturethe resulting was solutiondissolved was in cooled 6 mL down of anhydrous and poured into1,2- a dichloroethane. mixture of ice-cooled Then, saturated a solution of 1,2,3,5-tetra-aq. NaHCOO-acetyl-3 solution.β-D-ribofuranoside The mixture was extracted(0.6 g, 2 with mmol) DCM in (3 2× mL10 mL). of 1,2-di- The organic chloroethane was added,phases were followed collected, by washed the addition with a saturated of trimethylsilyl NaCl solution, triflate and dried (0.4 over mL, anhydrous 2.3 Na2SO4. Then, the filtrate was concentrated under reduced pressure and the residue was mmol). The resultingpurified mixture by flash was column stirred chromatography at 40–45 °C for using overnight. DCM: Acetone The reaction (7:3) as an was eluent mon- to afford itored by 1H NMR theanalysis pure product of the (18 crude) (72%). mixture.1H NMR After (MeOD), completionδ, ppm: 8.75 of (d, the J = 2.4reaction, Hz, 1H, H2),the 7.87 resulting solution was cooled down and poured into a mixture of ice-cooled saturated aq. NaHCO3 solution. The mixture was extracted with DCM (3 × 10 mL). The organic phases were collected, washed with a saturated NaCl solution, and dried over anhydrous Na2SO4.

Int. J. Mol. Sci. 2021, 22, x 17 of 27

Then, the filtrate was concentrated under reduced pressure and the residue was purified by flash column chromatography using DCM: Acetone (7:3) as an eluent to afford the pure product (18) (72%). 1H NMR (MeOD), δ, ppm: 8.75 (d, J = 2.4 Hz, 1H, H2), 7.87 (dd, J = 2.4 Hz, 2.4 Hz, 1H, H6), 6.51 (d, J = 7.6 Hz, 1H, H5), 5.80 (d, J = 5.6 Hz, 1H, H1′), 5.38–5.25 (m, 2H, H2′ and H3′), 4.45 (q, J = 3.07 Hz,1H, H4′), 4.36 and 4.27 (AB part of ABX system, 2H, JAB = 12.56 Hz, JAX = 3.12 Hz, JBX = 2.76 Hz, H5′), 2.08 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.98 (s, 3H, Ac).13C NMR (MeOD), δ, ppm: 178.69 (C4), 170.73 (CO), 169.88 (CO), 169.69 (CO), Int. J. Mol.166.32 Sci. 2021 ,(CONH2),22, 1145 141.78 (C2), 138.23 (C6), 119.91 (C5), 119.04 (C3), 94.59 (C1′), 81.48 (C4′18), of 28 74.48 (C2′), 70.43 (C3′), 62.83 (C5′), 19.28 (Me), 18.96 (Me), 18.73 (Me); HRMS calcd for C17H21N2O9 [M + H]+ 397.1249 found 397.1229. (dd, J = 2.4 Hz, 2.4 Hz, 1H, H6), 6.51 (d, J = 7.6 Hz, 1H, H5), 5.80 (d, J = 5.6 Hz, 1H, H10), Synthesis of 4-Pyridone-3-carboxamide-1-β-D-ribofuranoside (3) (Step-4): Method A: A 5.38–5.25 (m, 2H, H20 and H30), 4.45 (q, J = 3.07 Hz,1H, H40), 4.36 and 4.27 (AB part of ABX mixture of compoundsystem, (18 2H,) (0.078 JAB = g, 12.56 0.196 Hz, mmol), JAX = 3.12 1 mL Hz, NH JBX =4OH 2.76 (27–30%) Hz, H50), 2.08 and (s, cat. 3H, K Ac),2CO 2.043 (s, in methanol (10 mL)3H, was Ac), stirred 1.98 (s, 3H, at rt Ac). overni13C NMRght, (MeOD),and theδ ,reaction ppm: 178.69 progress (C4), 170.73 was (CO), monitored 169.88 (CO), by 1H NMR analysis169.69 of the (CO), crude 166.32 reaction. (CONH2), Upon 141.78 (C2),completion 138.23 (C6), of 119.91the reaction, (C5), 119.04 the (C3), reaction 94.59 (C1 0), 0 0 0 0 mixture was filtered81.48 and (C4 evaporated), 74.48 (C2 ),in 70.43 vacuo (C3 to), 62.83afford (C5 the), 19.28 desired (Me), product 18.96 (Me), 3 as 18.73 semisolid. (Me); HRMS calcd for C H N O [M + H]+ 397.1249 found 397.1229. Yield 50%. 17 21 2 9 Synthesis of 4-Pyridone-3-carboxamide-1-β-D-ribofuranoside (3) (Step-4): Method A: A Method B: A sealedmixture tube of compound was char (18ged) (0.078 with g, a 0.196 mixture mmol), of 1 compound mL NH4OH (27–30%)(18) (0.408 and g, cat. 1.02 K2CO 3 mmol), 2 mL 1,4-dioxanein methanol (saturated (10 mL) was with stirred 0.5N at NH rt overnight,3), and 15 and mL the methanol. reaction progress The wasresulting monitored mixture was stirredby at1H rt NMR overnight, analysis ofand the crudethe reaction reaction. Uponprogress completion was monitored of the reaction, by theNMR reaction mixture was ﬁltered and evaporated in vacuo to afford the desired product 3 as semisolid. analysis of the crude mixture. After overnight stirring at rt, the 1H NMR of the crude Yield 50%. showed complete removalMethod of the B: A acetates. sealed tube The was resulting charged withmixture a mixture was concentrated of compound (18 under) (0.408 g, reduced pressure to1.02 afford mmol), the 2 mLdesired 1,4-dioxane product (saturated 3. Yield: with 62%. 0.5N NH3), and 15 mL methanol. The Method C: A resultingPTFE jar mixture was wascharged stirred with at rt overnight, pure compound and the reaction (18) progress(0.2 g, 0.50 was monitoredmmol), by NMR analysis of the crude mixture. After overnight stirring at rt, the 1H NMR of the crude K2CO3 (6.91 mg, 0.1 mmol) and 20 microliter MeOH and was vibrated at a rate of 1800 showed complete removal of the acetates. The resulting mixture was concentrated under 1 rpm (30 Hz) at roomreduced temperature pressure to for afford 30 min. the desired The reaction product 3. progress Yield: 62%. was monitored by H NMR analysis. Yield: 85%.Method 1H C:NMR A PTFE (D2 jarO), was δ, ppm: charged 8.67 with (d, pure J = 2.24 compound Hz, 1H, (18 )H2), (0.2 g,7.94 0.50 (dd, mmol), J = 2.24 Hz, 2.24 Hz,K2 1H,CO3 (6.91H6), mg,6.65 0.1 (d, mmol) J = 7.6 and Hz, 20 microliter 1H, H5), MeOH 5.55 and(d, wasJ = 5.5 vibrated Hz, 1H, at a rateH1′ of), 18004.23 rpm 1 (t, 1H, J = 5.28 Hz,(30 H2 Hz)′), at4.19–4.13 room temperature (m, 2H, for H4 30′ min.and TheH3 reaction′), 3.81 progressand 3.73 was (AB monitored part of by ABXH NMR 1 analysis. Yield: 85%. H NMR (D2O), δ, ppm: 8.67 (d, J = 2.24 Hz, 1H, H2), 7.94 (dd, system, 2H, JAB = 12.7 Hz, JAX = 3.04 Hz, JBX = 4.0 Hz, H5′).13C NMR (D2O), δ, ppm: 179.13 J = 2.24 Hz, 2.24 Hz, 1H, H6), 6.65 (d, J = 7.6 Hz, 1H, H5), 5.55 (d, J = 5.5 Hz, 1H, H10), 4.23 (t, (C4), 167.75 (CONH1H,2), J =142.72 5.28 Hz, (C2), H20), 138.73 4.19–4.13 (C6), (m, 2H,120.35 H40 and (C5), H3 0118.25), 3.81 and (C3), 3.73 96.96 (AB part (C1 of′), ABX 86.03 system, 0 13 + (C4′), 75.61 (C2′), 70.022H, JAB (C3 = 12.7′), 60.89 Hz, JAX (C5 =′ 3.04); HRMS Hz, JBX calcd = 4.0 Hz, for H5 C11).H15CN NMR2O6 (D[M2O), + δH], ppm: 271.0930 179.13 (C4), 0 0 found 271.0919. 167.75 (CONH2), 142.72 (C2), 138.73 (C6), 120.35 (C5), 118.25 (C3), 96.96 (C1 ), 86.03 (C4 ), 0 0 0 + 75.61 (C2 ), 70.02 (C3 ), 60.89 (C5 ); HRMS calcd for C11H15N2O6 [M + H] 271.0930 found 271.0919. Synthesis of 6-Pyridone 3-Carboxamide Riboside (4) The synthesisSynthesis of 6-pyridone of 6-pyridone 3-carboxamid 3-carboxamidee riboside riboside ( 4was) conducted according to Scheme 6. The synthesis of 6-pyridone 3-carboxamide riboside was conducted according to Scheme6.

Scheme 6. Synthesis of Scheme6-pyridone 6. Synthesis 3-carboxamide of 6-pyridone riboside. 3-carboxamide riboside. Synthesis of 6-pyridone 3-carboxamide (6PY) (20) (Step-1): To an oven-dried 50-mL Synthesis of 6-pyridoneround bottom 3-carboxamide ﬂask equipped (6PY) with a( magnetic20) (Step-1) stir bar,: To 6-hydroxynicotinic an oven-dried acid 50-mL (13) (5 g, round bottom flask3.59 equipped mmol), HOBt with (0.540 a magnetic g, 4.0 mmol), stir bar, DCC 6-hydroxynicotinic (0.928 g, 4.5 mmol), and acid anhydrous (13) (5 g, DMF

Int. J. Mol. Sci. 2021, 22, 1145 19 of 28

(10 mL) were added. The RBF was placed under nitrogen sealed with a septum. The reaction mixture was stirred at rt for 2 h under nitrogen. The reaction progress was monitored by 1H NMR of the crude reaction mixture. Upon completion of the reaction, the solid residue (19) was filtered off and washed with fresh DMF (10 mL). The organic layer was concentrated under reduced pressure, and the residue was dissolved in 10 mL NH4OH (28–30%) and stirred at rt for 1 h. The reaction progress was monitored by 1H NMR of the crude reaction mixture. After completion of the reaction, the solvent was evaporated under high vacuum to afford the 6-pyridone-3-carboxamide (20) (6PY) as a brown solid. Yield 70%. 1H NMR (DMSO, d6), δ, ppm: 11.92 (s, 1H, NH), 8.00 (s, 1H, H2), 7.85 (d, J = 9.6 Hz, 1H, H4), 7.72 (s, 1H, NH), 7.18 (s, 1H, NH), 6.32 (d, J = 9.56 Hz, 1H, H5). HRMS calcd for + C12H16NO7 [M + H] 139.0508 found 139.0499 [31]. Silylation of 6-pyridone-3-carboxamide (21) (Step-2): 6PY (20) (0.3 g, 2.17 mmol) and N,O-Bis (trimethylsilyl) trifluoroacetamide (2.3 mL, 2.23 g, 8.69 mmol) were dissolved in anhydrous acetonitrile (5 mL) at rt, and the reaction mixture was stirred at rt for 24 h under nitrogen. After 24 h, the reaction mixture was concentrated under reduced pressure, and the crude product was used directly in the next step. 0 0 0 Synthesis of 1-(2 ,3 ,5 -tri-O-acetyl-β-D-ribofuranosyl)-6-pyridone-3-carboxamide (22) (Step- 3): To a mixture of pre-silylated 6-pyridone-3-carboxamide (21) (0.5 g, 1.6 mmol) and 1, 2, 3, 5-tetra-O-acetyl-β-ribofuranoside (0.767 g, 2.4 mmol) in anhydrous 1,2-dichloroethane (20 mL) was added SnCl4 (0.754 mL, 1.67 g, 6.4 mmol) and stirred at rt for 16 h under nitrogen gas environment. Once the reaction was complete, the reaction mixture was poured into water and extracted with CHCl3 (10 mL × 3), washed with saturated NaHCO3 and dried over anhydrous Na2SO4. The organic layer was concentrated, and the oily 1 product was purified by flash column chromatography. Yield 78%. H NMR (CDCl3), δ, ppm: 8.30 (br.s., 1H, H6), 7.69 (d, J = 9.04 Hz, 1H, H4), 6.45 (d, J = 9.44 Hz, 1H, H3), 6.26 (br.s, 1H, H10), 5.31 (t, J = 4.58 Hz, 1H, H20), 5.20 (t, J = 5.20 Hz, 1H, H30), 4.56–4.51 (m, 1H, H40), 4.38 (br. s, 1H, H50 ABX system), 4.26 (d, J = 12.36 Hz, H50 ABX system), 2.13 (s, 3H, 13 Ac), 2.05 (s, 3H, Ac), 2.04 (s, 3H, Ac). C NMR (CDCl3), δ, ppm: 171.43 (C2), 169.60 (CO), 169.36 (CO), 165.73 (CO), 161.80 (CONH2), 141.66 (C6), 135.66 (C4), 121.59 (C3), 119.89 (C5), 87.96 (C10), 80.10 (C40), 74.13 (C20), 69.74 (C30), 62.74 (C50), 20.88 (Me), 20.48 (Me), 20.45 (Me). MS (ES): m/z 397.63 [M+ + H] and 419.56 [M+ + Na]. Synthesis of 6-pyridone-3-carboxamide-1-β-D-ribofuranoside (4) (Step-4): The triacetylated precursor (22) (0.360 g, 0.9 mmol) was dissolved in 2 mL ordinary methanol and stirred at rt for 15 min. After 15 min, 2 mL NH4OH (28–30%) was added, and the resulting mixture was stirred at rt for 4 h. The reaction progress was monitored by 1H NMR analysis of the crude reaction mixture. Upon completion of the reaction, the solvent was evaporated under reduced pressure, and the crude was kept at rt overnight before treating with acetone for 24 h. The solid was then filtered off and washed with fresh acetone to afford 6-PYR (4) 1 as a white solid. Yield 72%. H NMR (D2O), δ, ppm: 8.60 (s, 1H, H6), 7.84 (d, J = 9.2 Hz, 1H, H4), 6.54 (d, J = 9.44 Hz, 1H, H5), 6.01 (s, 1H, H10), 4.19–4.13 (m, H20, H30 & H40), 3.97 (d, J = 12.92 Hz, 1H, H50, ABX system), 3.80 (d, J = 12.88 Hz, 1H, H50, ABX system). 13C NMR (CDCl3), δ, ppm: 169.12 (CONH2), 163.80 (C2), 139.30 (C6), 139.02 (C4), 118.80 (C3), 114.30 (C5), 90.75 (C10), 83.56 (C40), 74.87 (C20), 68.24 (C30), 59.83 (C50), HRMS calcd for + C11H15N2O6 [M + H] 271.0930 found 271.0938.

Synthesis of O-methyl-4-pyridone-3-carboxylate-1-β-D-ribofuranoside (26) The synthesis of O-methyl-4-pyridone-3-carboxylate-1-β-D-ribofuranoside was conducted according to Scheme7. Int. J. Mol. Sci. 2021, 22, x Int. J. Mol. Sci. 2021, 22, 1145 19 of 2720 of 28

Scheme 7. SynthesisScheme of 7.theSynthesis methyl-ester of the methyl-ester and carboxylate and carboxylate ribosyl analogs. ribosyl analogs.

Silylated 4-pyridone-3-carboxylic methyl ester 23 (Step-1): O- Silylated 4-pyridone-3-carboxylic methyl ester (23) (Step-1):( O-)Methyl-4-pyridone-3-car-Methyl-4-pyridone-3- carboxylic ester (11) (3.0 g, 1.98 mmol) was dissolved in 50 mL of hexamethyl-disilazane boxylic ester (11(HMDS)) (3.0 g, in1.98 an RBFmmol) under was nitrogen, dissolved 5 mL in of 50 trimethylsilyl mL of hexamethyl-disilazane chloride (TMSCl) (0.494 mL, (HMDS) in an RBF0.423 under g, 3.9 mmol)nitrogen, was 5 then mL added. of trimethylsilyl The mixture waschloride refluxed (TMSCl) under nitrogen(0.494 mL, for 3 h at 0.423 g, 3.9 mmol)120–130 was ◦thenC. The added. excess reagentsThe mixture were evaporated was refluxed and co-evaporatedunder nitrogen with for toluene 3 h (10at mL). 1 120–130 °C. The excessThe product reagents was were a dark evapor yellowated oily residue.and co-evaporated Yield 90%. Hwith NMR toluene (400 M (10 Hz, mL). CDCl 3, δ, ppm): 8.92 (s, 1H, H2), 8.44 (d, J = 5.6 Hz, 1H, H6), 6.71 (d, J = 5.6 Hz, 1H, H5), 3.83 (s, 3H, The product was a dark yellow oily residue. Yield 90%. 1H NMR (400 MHz, CDCl3, δ, OMe), 0.27 (s, 9H, Si(CH3)3). ppm): 8.92 (s, 1H, H2), 8.44 (d, J = 05.60 Hz,0 1H, H6), 6.71 (d, J = 5.6 Hz, 1H, H5), 3.83 (s, 3H, Synthesis of 1-(2 ,3 ,5 -Tri-O-acetyl-β-D-ribofuranosyl)-4-pyridone-3-carboxylic methyl ester OMe), 0.27 (s, 9H,(24 Si(CH) (Step-2):3)3).Silylated 4-pyridone-3-carboxylic methyl ester (23) was dissolved in 25 mL of Synthesis of 1,2-dichloroethane1-(2′,3′,5′-Tri-O-acetyl- (DCE) underβ-D-ribofuranosyl)-4-pyridone-3-carboxylic nitrogen. 1,2,3,5-tetra-O-acetyl-δ-D-ribofuranoside methyl es- (6.30 g, ter (24) (Step-2): 1.98Silylated mmol) 4-pyridone-3-carboxylic was dissolved in DCE (25 methyl mL) and ester then ( added23) was to thedissolved ester solution. in 25 An mL of 1,2-dichloroethaneadditional (DCE 50 mL) ofunder DCE andnitrogen. 4.1 mL 1,2,3,5-tetra- of trimethylsilylO-acetyl- triflate (TMSOTf)δ-D-ribofuranoside (2.28 mmol) were ◦ (6.30 g, 1.98 mmol)added was to dissolved the mixture. in The DCE resulting (25 mL) mixture and then was stirred added at to 40 theC forester 5 h solution. under nitrogen. Upon completion of the reaction, the reaction mixture was cooled to 0 ◦C and poured into An additional 50iced mL water. of DCE The and reaction 4.1 mL flask of was trimethylsilyl rinsed with DCM triflate (50 mL)(TMSOTf) and added (2.28 to themmol) quenched were added to the mixture. The resulting mixture was stirred at 40 °C for 5 h under nitro- reaction solution. The mixture was neutralized with 35 mL of a saturated aq NaHCO3 gen. Upon completionsolution. of Thethe resultingreaction, phases the reaction separated, mixture and the was organic cooled layer to was 0 washed°C and withpoured saturated into iced water. NaClThe solutionreaction (50 flask mL). was Once rinsed separated, with the DCM organic (50 phase mL) wasand dried added over to anhydrous the quenched reactionsodium solution. sulfate, The filtered, mixture and evaporated.was neutralized The crude with product 35 mL was of purified a saturated by flash aq column chromatography using n-hexane and ethyl acetate as an eluent system to afford the desired NaHCO3 solution. The resulting phases separated, and the organic layer1 was washed with product (24) as a light brown crystalline solid (6.42 g, 75%). H NMR (400 M Hz, CDCl3, δ, saturated NaCl solutionppm): 8.40 (50 (d, mL). J = 2.5 Once Hz, 1H, separa H-2),ted, 7.45 the (dd, organic J = 7.9 and phase 2.5 Hz, was 1H, dried H-6), over 6.52 (d, anhy- J = 7.9 Hz, drous sodium sulfate,1H, H-5), filtered, 5.49 (d, and J = 5.9evaporated. Hz, 1H, H-1 The0), 5.30 crude (dd, product J = 5.6 and was 3.6 purified Hz, 1H, H-3by 0flash), 5.21 (dd, 0 0 0 0 column chromatographyJ = 5.9 and using 5.6 Hz, n-hexane 1H, H-2 ), 4.33–4.46 and ethyl (m, acetate 3H, H-5 A,as H-5an eluentB, H-4 ), system 3.88 (s, 3H,to afford O-CH3), 2.19 13 the desired product(s, 3H, (24 Ac),) as a 2.14 light (s, brown 3H, Ac), crystalline 2.10 (s, 3H, solid Ac); (6.42C NMR g, 75%). (100 M 1H Hz, NMR CDCl (4003, δ, MHz, ppm): 175.7 3 (COO), 170.1 (CO), 169.4 (CO), 169.3 (CO), 165.6 (C-4), 142.4 (C-2), 135.1 (C-6), 122.9 (C-5), CDCl , δ, ppm): 8.40 (d, J = 2.5 Hz, 1H,0 H-2), 7.450 (dd, J = 7.90 and 2.5 Hz,0 1H, H-6),0 6.52 (d,J 119.4 (C-3), 94.22 (C-1 ), 81.34 (C-4 ), 74.29 (C-2 ), 70.21 (C-3 ), 62.76 (C-5 ), 52.40 (–OCH3), = 7.9 Hz, 1H, H-5), 5.49 (d, J = 5.9 Hz, 1H, H-1′), 5.30 (dd, J = 5.6 and 3.6 Hz,+ 1H, H-3′), 5.21 20.47 (CH3), 20.31 (CH3); HRMS calcd for C18H22NO10 [M + H] 412.1243 found 412.1243. (dd, J = 5.9 and 5.6 Hz,Synthesis 1H, H-2 of′), O-methyl-4-pyridone-3-carboxylate 4.33–4.46 (m, 3H, H-5′A, H-5 ribonucleoside′B, H-4′), 3.88(25 )(s,(Step-3): 3H, O-CHA mixture3), of 0 0 0 13 2.19 (s, 3H, Ac), 2.141-(2 ,3(s,,5 3H,-tri- Ac),O-acetyl- 2.10β (s,-D -ribofuranosyl)-4-pyridone-3-carboxylic3H, Ac); C NMR (100 MHz, CDCl methyl3, δ, ppm): ester 175.7 (24) (3.20 g, (COO), 170.1 (CO),7.79 169.4 mmol), (CO), K2CO 169.33 (0.103 (CO), g, 0.78 165.6 mmol), (C-4), and 142.4 MeOH (C-2), (1.2 mL, 135.1 29.6 (C-6), mmol) 122.9 was ball-milled (C-5), for 119.4 (C-3), 94.2230 (C-1 min′), at 81.34 30 Hz. (C-4 Following′), 74.29 removal (C-2′ of), 70.21 methanol, (C-3 a′ brown), 62.76 solid (C-5 with′), 52.40 a taffy-like (–OCH consistency3), was obtained (1.81 g, 95%). 1H NMR (400 M Hz, MeOD, δ, ppm): 3.76 (dd, J = 12.1 and 20.47 (CH3), 20.31 (CH3); HRMS calcd for C18H22NO10[M + H]+ 412.1243 found 412.1243. 2.7 Hz, 1H, H-50A), 3.83 (s, 3H, -CH ), 3.85 (dd, J = 2.7 Hz and J = x, 1H, H-50B), 4.15 (dd, Synthesis of O-methyl-4-pyridone-3-carboxylate3 ribonucleoside (25) (Step-3): A mixture of J = 6.3 and 2.6 Hz, 1H, H-40), 4.17–4.22 (m, 2H, H-20, H-30), 5.49 (d, J = 5.1 Hz, 1H, H-10), 1-(2′, 3′, 5′-tri-O-acetyl-β-D-ribofuranosyl)-4-pyridone-3-carboxylic methyl ester (24) (3.20 g, 7.79 mmol), K2CO3 (0.103 g, 0.78 mmol), and MeOH (1.2 mL, 29.6 mmol) was ball-milled for 30 min at 30 Hz. Following removal of methanol, a brown solid with a taffy-like consistency was obtained (1.81 g, 95%). 1H NMR (400 MHz, MeOD, δ, ppm): 3.76 (dd, J = 12.1 and 2.7 Hz, 1H, H-5′A), 3.83 (s, 3H, -CH3), 3.85 (dd, J = 2.7 Hz and J = x, 1H, H-5′B), 4.15 (dd, J = 6.3 and 2.6 Hz, 1H, H-4′), 4.17–4.22 (m, 2H, H-2′, H-3′), 5.49 (d, J = 5.1 Hz, 1H, H- 1′), 6.53 (d, J = 7.7 Hz, 1H, H-5), 8.09 (dd, J = 7.7 and 2.3 Hz, 1H, H-6), 8.78 (d, J = 2.3 Hz, 1H, H-2);13C NMR (100 MHz, MeOD, δ, ppm): 50.89 (–OCH3), 61.19 (C-5′), 70.94 (C-3′),

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Int. J. Mol. Sci. 2021, 22, x 20 of 27

6.53 (d, J = 7.7 Hz, 1H, H-5), 8.09 (dd, J = 7.7 and 2.3 Hz, 1H, H-6), 8.78 (d, J = 2.3 Hz, 1H, 13 0 0 H-2); C NMR (100 M Hz, MeOD, δ, ppm): 50.89 (–OCH3), 61.19 (C-5 ), 70.94 (C-3 ), 76.33 (C-276.330), (C-2 87.11′), (C-487.110), (C-4 97.57′), (C-197.570), (C-1 117.7′), (C-3),117.7 120.6(C-3), (C-5),120.6 137.7(C-5), (C-6), 137.7 143.8(C-6), (C-2), 143.8 164.8(C-2), (C-4), 164.8 + + 177.4(C-4), (COO); 177.4 (COO); HRMS HRMS calcd for calcd C12 Hfor16 CNO12H7 16[MNO +7[M H] +286.0926 H] 286.0926 found found 286.0929. 286.0929. SynthesisSynthesis of 4-pyridone-3-carboxylic acid acid ribonucleoside ribonucleoside (26(26) (Step-4):) (Step-4): A mixtureA mixture of 4-pyrof 4- pyridone-3-carboxylicidone-3-carboxylic ester ester riboside riboside (25 (25) (2.43) (2.43 g, g, 8.85 8.85 mmol) mmol) was was dissolved dissolved in aa 1M1M NaOHNaOH aqueousaqueous solution solution (10 (10 mL) mL) and and stirred stirred for for 1 h.1 h. Upon Upon completion completion of of the the reaction, reaction, the the resulting result- mixtureing mixture was was neutralized neutralized with with 1 M HCl1 M HCl solution solution (5 mL) (5 andmL) evaporatedand evaporated on high on high vacuum vac- 1 1 (80%).uum (80%).H NMRH NMR (400 (400 M Hz, MHz, D2O, D2δO,, ppm): δ, ppm): 8.21 8.21 (d, (d, J = J 2.4= 2.4 Hz, Hz, 1H, 1H, H-2), H-2), 7.89 7.89 (dd, (dd, J J = = 7.6 7.6 andand 2.42.4 Hz,Hz, 1H,1H, H-6),H-6), 6.526.52 (d,(d, JJ == 7.6 Hz, 1H, H-5), 5.51 (d, J = 5.6 5.6 Hz, Hz, 1H, 1H, H-1 H-1′),0), 4.26 4.26 (dd, (dd, J J= = 5.6 5.6 and and 5.3 5.3 Hz, Hz, 1H, 1H, H-2 H-2′),0 ),4.20 4.20 (dd, (dd, J = J =5.3 5.3 and and 3.6 3.6 Hz, Hz, 1H, 1H, H-3 H-3′), 04.15), 4.15 (dd, (dd, J = J 8.3 = 8.3 and and 3.6 3.6Hz, Hz, 1H, 1H, H-4 H-4′), 3.820), 3.82 (dd, (dd, J = J12.8 = 12.8 and and 4.5 4.5Hz, Hz, 1H, 1H, H-5 H-5′B), 03.74B), 3.74 (dd, (dd, J = 12.8 J = 12.8 and and4.5 Hz, 4.5 Hz,1H, 013 13 0 0 0 1H,H-5 H-5′A); A);C NMRC NMR (100 (100MHz, M D Hz,2O, Dδ,2 ppm):O, δ, ppm): 60.97 60.97(C-5′), (C-5 70.03), (C-3 70.03′), (C-3 5.36), (C-2 5.36′), (C-2 85.81), (C-4 85.81′), (C-496.670), 96.67(C-1′), (C-1 118.90), 118.9(C-5), (C-5), 127.3 127.3 (C-6), (C-6), 138.4 138.4 (C-2), (C-2), 138.8 138.8 (C-3), (C-3), 172.0 172.0 (C-4), (C-4), 178.1 178.1 (COO).(COO). + HRMSHRMS calcdcalcd forfor CC1111HH1414NO77[M[M + +H] H]+ 272.0770272.0770 found found 272.0768. 272.0768.

SynthesisSynthesis ofof 2-pyridone-3-carboxamide2-Pyridone-3-Carboxamide Riboside Riboside (6 ()6) TheThe synthesissynthesis ofof 2-pyridone-3-carboxamide2-pyridone-3-carboxamide ribosideriboside waswas conductedconducted accordingaccording toto SchemeScheme8 .8.

Scheme 8. Synthesis of the 2-pyridone-3-carboxamide riboside. Scheme 8. Synthesis of the 2-pyridone-3-carboxamide riboside.

SynthesisSynthesis ofof 2-pyridone-3-carboxamide 2-pyridone-3-carboxamide(29 )((step-1)29) (step-1): 2-Hydroxypyridine-3-carbonyl: 2-Hydroxypyridine-3-carbonyl chlo- ridechloride (intermediate) (intermediate) was synthesizedwas synthesized via the via chlorination the chlorination of 2-hydroxy of 2-hydroxy nicotinic nicotinic acid (acid27) (1.0(27) g, (1.0 7.18 g, mmol)7.18 mmol) by using by using SOCl 2SOCl(10 mL).2 (10 mL). The reagentsThe reagents were were added added to a 50-mL to a 50-mL one-neck one- round-bottomneck round-bottom flask equippedflask equipped with a with condenser. a conden Theser. solution The solution was stirred was atstirred reflux at (90 reflux◦C) for(90 1–2 °C) h.for After 1–2 h. that After time, that the time, resulting the resultin mixtureg mixture with solid with residue solid residue was concentrated was concentrated on a rotaryon a rotary evaporator. evaporator. A yellowish-green A yellowish-green solid wassolid obtained, was obtained, which which was dissolved was dissolved immedi- im- atelymediately in 15 mLin 15 anhydrous mL anhydrous methanol methanol and stirred and stirred at rt for at 1 rt h forfor their1 h for conversion their conversion to methyl to 2-hydroxypyridine-3-carboxylatemethyl 2-hydroxypyridine-3-carboxylate (28). After (28 completion). After completion of the reaction, of the reaction, the solvent the was sol- removed,vent was removed, and the crude and the 2-hydroxypyridine-3-carboxylate crude 2-hydroxypyridine-3-carboxylate (28) was ( dissolved28) was dissolved in 10 mL in NH10 mL3 saturated NH3 saturated methanol, methanol, and then and the then round the bottom round flaskbottom was flask sealed was with sealed a septum. with a Thesep- resultingtum. The mixtureresulting was mixture stirred was at stirred rt for the at rt next for 60the h. next After 60 h. 60 After h, the 60 precipitated h, the precipitated white solidwhite was solid filtered was filtered off and off washed and washed with cold with methanol cold methanol (2–3 times) (2–3 times) and dried and dried to yield to yield 70% 1 of70% 2-oxo-1H-pyridine-3-carboxamide of 2-oxo-1H-pyridine-3-carboxamide (29). (H29 NMR). 1H NMR (DMSO- (DMSO-d6), δ,d ppm:6), δ, ppm: 12.3 (s, 12.3 1H, (s, NH), 1H, NH), 9.05 (s, 1H, NH), 8.29 (d, J = 5.28 Hz, 1H, H6), 7.66 (d, J = 4.32 Hz, 1H, H4), 7.51 (s, 1H, NH), 6.41 (t, J = 6.66 Hz, 1H, H5). 13C NMR (DMSO-d6), δ, ppm: 165.09 (CONH2), 162.71

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9.05 (s, 1H, NH), 8.29 (d, J = 5.28 Hz, 1H, H6), 7.66 (d, J = 4.32 Hz, 1H, H4), 7.51 (s, 1H, 13 NH), 6.41 (t, J = 6.66 Hz, 1H, H5). C NMR (DMSO-d6), δ, ppm: 165.09 (CONH2), 162.71 (CO), 144.69 (C4), 140.04 (C6), 121.27 (C3), 160.44 (C5); HRMS calcd for C6H6N2O2 [M + H]+ 139.0507 found 139.0493. Silylation of 2-pyridone-3-carboxamide (30) (Step-2): 2-pyridone-3-carboxamide (29) (426 mg, 3.08 mmol), hexamethyldisilazane (10 mL) and (NH4)2SO4 (cat. amount) were added sequentially to a 50-mL round bottom flask under nitrogen. The resulting mixture was stirred at 135 ◦C for 2 h. Upon completion of the reaction, the resulting solution was cooled to room temperature and then concentrated under vacuum. The residue was co-evaporated with anhydrous toluene (2 to 3 times) to afford the mixture of mono and bis-silylated 2-pyridone-3-carboxamide (30), which was used directly for the next step. Vorbrüggen glycosylation (Step-3): The crude mono and bis-silylated 2-pyridone-3- carboxamide (30) was dissolved in 10 mL of anhydrous CH3CN. Then, a solution of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranoside (1.017 g, 3.20 mmol) in 2 mL of anhydrous acetonitrile was added, followed by the addition of a solution trimethylsilyl triflate (1.67 mL, 9.24 mmol). The resulting mixture was stirred at rt for 24 h, and the reaction progress was monitored by NMR analysis of crude mixture. After completion of the reaction, the resulting solution was concentrated under vacuum, and the residue was purified by flash column chromatography using n-hexane: ethyl acetate (1:1.5) as an eluent to afford pure products (31) (81%). Deacetylation of compound 31 (Step 4): Compound 31 (1.01 g, 2.52 mmol) was dissolved in 10 mL methanol (saturated with ammonia) and stirred at rt for 48 h under sealed condition. The reaction progress was monitored by the NMR analysis of crude reaction mixture. Upon completion of the reaction, solvent was evaporated on high vacuum and the crude product was stirred with acetone and filtered off and washed with fresh acetone to afford 2-PYR (6) as white solid. The obtained product was enough pure. Yield 44%. 1H NMR (MeOD), δ, ppm: 8.46 (dd, J = 5.4 Hz & 7.04 Hz, 2H, H4 & H6), 6.55 (t, J = 6.96 Hz, 1H, 0 0 0 0 0 H5), 6.14 (s, 1H, H1 ), 4.12 (bs, 3H, H2 , H3 & H4 ), 3.97 (d, J = 12.9 Hz, 1H, Ha5 ), 3.80 (d, 0 13 J = 11.56 Hz, 1H, Hb5 ). C NMR (MeOD), δ, ppm: 166.62 (CONH2), 161.79 (CO), 144.02 (C6), 138.21 (C4), 119.69 (C3), 105.90 (C5), 91.11 (C10), 84.38 (C40), 75.71 (C20), 68.58 (C30), 0 + 59.88 (C5 ), HRMS calcd for C11H15N2O6 [M + H] 271.0930 found 271.0909 [32].

Synthesis of 2PYR and 6PYR via Fenton Chemistry Nicotinamide riboside chloride (0.20 g, 0.78 mmol) was dissolved in aqueous NaOH ◦ (1 M) at 0 C and rapidly added to a solution of K3[Fe(CN)6] (0.773 g, 2.35 mmol) in 10 mL ◦ H2O. The resulting mixture was stirred at 0 C for 1 h and then warmed to rt. After 1 h stirring at rt, 50 mL of MeOH was added. The mixture was stirred at rt for 2 h. A thick yellowish precipitate formed during stirring and was ﬁltered off and washed with MeOH (2–3 times). NMR of the crude reaction mixture showed the formation of 2PYR and 6PYR but not that of 4PYR.

4.2. Enzymatic Conversions 4.2.1. Synthesis of 4PYR via NQO2 Catalyzed Hyper-Oxidation of the Reduced Form of Nicotinamide Riboside (NR), Abbreviated NRH To prepare the sample, 1 mM NR in 450 µL buffer (50 mM potassium phosphate buffer, 125 mM NaCl, 5 µM FAD, 1.0 mg/mL BSA) was reacted with 20 µL of the NQO2 enzyme (Sigma-Aldrich, Q0380) and allowed to incubate overnight at room temperature. NRH was also incubated overnight with the same buffer composition for comparison. An Agilent 1200 series HPLC was used for the purification and isolation of compounds. Five microliters of each sample were injected onto an Agilent C18 reversed-phase column (2.1 × 150 mm) equipped with a C18 guard column using an autosampler. Solvent A consisted of H2O with 2.0% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA), and solvent B consisted of ACN with 2.0% H2O and 0.1% TFA. A flow rate of 100 µL per minute was used for the entirety of the run. The solvent was held at 5.0% B for the first 5 min, from Int. J. Mol. Sci. 2021, 22, 1145 23 of 28

5 to 20 min a gradient from 5.0 to 80.0% B was used, then solvents were held at 80% B for 5 min and returned to 5.0% B for the remainder of the run. Two injections were performed for each sample, one scanning at 260 nm (for the UV detection of NR and 4PYR) and the other injection scanning at 340 nm (for the UV detection of NRH). Two fractions were collected based on the retention time of previously analyzed standards. The ﬁrst from 5 to 8 min for the collection of NRH and 4PYR, and the second fraction from 19 to 26 min for the collection of NR. The collected fractions were dried using a speedvac concentrator (without heat applied). Samples were dissolved into 50 µL of 10 mM ammonium bicarbonate (ABC) for analysis by mass spectrometry.

4.2.2. LC-MS/MS Analyses Liquid chromatography-mass spectrometry analyses were performed on the individ- ual HPLC fractions that were previously prepared. Each fraction was analyzed separately. An Agilent 1200 Series HPLC coupled to a Thermo LTQ Orbitrap XL mass spectrometer was used for sample analysis. An autosampler was used to inject the samples onto an Agilent Zorbax 300SB-C18 reversed-phase column (2.1 × 150 mm, 5-micron) using a 4.0 µL injection volume for each. Solvent A consisted of 10 mM ABC in H2O, and Solvent B consisted of neat ACN. A flow rate of 50 µL per minute was used for the first 5 min of the run at 5.0% B solvent, followed by a gradient from 5.0–80.0% B from 5 to 15 min along with an increased flow rate of 100 µL per minute. The solvent was held at 80.0% B from 15 to 25 min and returned to 5.0% B for the remainder of the run. A HESI (heated electrospray ionization) source was used with positive polarity, a capillary temperature of 200 ◦C, the source voltage of 3.0 kV, tube lens voltage of 110, and a sheath gas flow rate of 8.0. One full scan from 80–800 m/z was performed at 15,000 resolution, followed by targeted MS2 scans of NR (255.1 m/z), NRH (257.1 m/z), and 4PYR (271.1 m/z) at 7500 resolution with an isolation width of 1.0 m/z. Normalized CID collision energy was set to 35. All scans were performed in the FTMS. The total run time was 30 min. A blank was run in-between each sample to minimize and monitor for carryover.

4.3. Cell-Based Assays 4.3.1. Cell Culture Human embryonic kidney containing the SV40 T-antigen (HEK293T) and the human hepatoma cell line (HepG3) were purchased from ATCC (Manassas, VA, USA). The cells ◦ were grown at 37 C in a 5% CO2 incubator in Dulbecco’s modiﬁed Eagle’s medium (DMEM, Hyclone, Logan, UT, USA; 4.5 g/L glucose and L-glutamine) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA), and 1% sodium pyruvate (Gibco, Carlsbad, CA, USA). Cells were routinely tested for mycoplasma contamination using the Lonza MycoAlert kit (Walkersville, MD, USA) and found to be free of mycoplasma contamination.

4.3.2. Cytotoxicity Studies Cell viability was determined by CellTiter-FluorTM Cell Viability assays (Promega Corporation, Madison, WI, USA). HEK293T and HepG3 cells were seeded at a density of 5000 cells/well and 10,000 cells/well respectively in a 96-well clear bottom black plate ◦ and incubated overnight (ON) at 37 C in a 5% CO2 incubator. For HEK293T cells, a poly D-lysine-coated black plate was used to prevent washing off the cells during medium replacement. For 24 h exposures, the cells were exposed to 100 µM concentrations alone or increasing doses (25, 50, 75, 100, and 150 µM) of pyridone derivatives (i.e., 2-PYR, 4-PYR, and 6-PYR) for 24 h, then medium containing NRH was replaced with fresh medium, and ◦ cells were further incubated for another 48 h (total 72 h) at 37 C in a 5% CO2 incubator. For continuous exposures, the cells were exposed to 100 µM concentrations alone or increasing doses (25, 50, 75, 100, and 150 µM) of pyridone derivatives (i.e., 2-PYR, 4-PYR, and 6-PYR) for 72 h. The 2-PYR, 4-PYR, and 6-PYR were dissolved in Dulbecco’s phosphate-buffered saline (PBS, Hyclone, Logan, UT, USA) at 10 mM and then diluted to the ﬁnal working Int. J. Mol. Sci. 2021, 22, 1145 24 of 28

concentrations in the cell growth medium. At the end of the 72-h incubation period, 100 µL of 2X CellTiter-FluorTM Viability reagent was added in each well, mixed briefly, and then ◦ the plates were incubated for another 30 min at 37 C in a 5% CO2 incubator. Fluorescence intensity was measured in a microplate reader (Infinite® M1000 PRO, TECAN, Männedorf, Switzerland) with a fluorometer at excitation/emission (Ex/Em) of 380/505 nm. Cells were plated in triplicate for each chemical concentration and exposure durations with at least three biological repeats performed. Fluorescent values are expressed as the number of cells in drug-treated wells relative to cells in vehicle-treated, control wells (%viability) ± standard error of the mean (SEM).

4.3.3. Clonogenic Assays A clonogenic assay was used to determine the cell growth and colony formation after exposure. HepG3 cells were plated at a density of 10,000 cells/well in each well of a 12-well ◦ plate and incubated ON at 37 C in a 5% CO2 incubator. The following day, cells were exposed to 25, 50, and 100 µM 4-PYR continuously for six days. At the end of the incubation period, plates were placed on ice, washed twice with ice-cold 1 X PBS before fixing the cells with ice-cold methanol for 10 min. Following the fixation step, cells were incubated with 0.5% crystal violet solution (made in 25% methanol) for 10 min at room temperature (RT, ~23 ◦C). The plates were rinsed with double-distilled water to remove the excess crystal violet stain and allowed to dry ON at RT. Stained plates were imaged using ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA). For analysis, the area fraction of the crystal violet-stained cells was generated from each image using NIS-Elements software. The percentage of the average area fraction of crystal violet staining per well was calculated. Each treatment was performed in technical triplicates and averaged over three biological replicates with final cell density percentage reported relative to control ± SEM.

4.3.4. Immunoblotting The cell death mechanism of 4-PYR induced cytotoxicity was assessed by immunoblotting. Brieﬂy, HepG3 cells were plated in 65-mm dishes at a density of 2 × 106 cells per dish ◦ and incubated ON at 37 C in a 5% CO2 incubator. The cells were treated with 100 µM 4-PYR for 48, 72, and 96 h. Cell pellets were collected at the end of the desired exposure period and stored at −80 ◦C. The cell pellets were thawed on ice and resuspended in a lysis buffer of 25 mM β-glycerolphosphate, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Triton X-100, and 0.3% NP-40 supplemented with 1X Halt protease and phosphatase inhibitor (Pierce, Waltham, MA, USA). Resuspended cells were incubated for 30 min on ice. Lysates were centrifuged at 12,000 rpm for 15 min at 4 ◦C, and the supernatant fraction containing protein was retained. Protein concentrations were quantiﬁed using Bradford Quick Start protein assay (Bio-Rad, Hercules, CA, USA). About 30 µg of each protein sample was separated on a 4–15% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio- Rad). Membranes were blocked in 5% skim milk in Tris-buffered saline (TBS, VWR Life Sciences) containing 0.1% Tween 20 (TBS-T) and incubated with the following antibodies: LC3B (1:1000, PA1-46286) from ThermoFisher (Rockford, IL, USA); Beclin-1 (1:1000) and phosphor-mTOR (1:1000, Ser2448, D92C) from Cell Signaling Technology, Inc. (Danvers, MA, USA); and α-tubulin (1:5000, T9026) from Millipore Sigma (St. Louis, MO, USA).

5. Conclusions Here, we have synthesized and systematically characterized all pyridones-derived from nicotinamide known to have some physiological relevance and sought the biochemical origin of the PYR series that had remained unknown. Indeed, we report that 4PYR can be generated from NRH by the FAD-dependent NQO2 enzyme and provide evidence that 2PYR and 6PYR can be generated from NR via Fenton chemistry, thus further supporting the hypothesis that reductive stress, as well as oxidative stress, promotes the formation of ribosylated pyridones. Int. J. Mol. Sci. 2021, 22, 1145 25 of 28

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-0 067/22/3/1145/s1. Figure S1: 1H NMR, 13C NMR and HRMS Spectra. Figure S2: NQO2 catalyzed 4PYR formation. Table S1: Isotopic pattern with relative abundance of pyridones and their intermediates. Author Contributions: Conceptualization, M.E.M.; methodology, N.R.G. and M.E.M.; validation, F.H., M.S. and M.V.M.; formal analysis, F.H. and M.S.; investigation, F.H., M.S., M.V.M. and P.M.; data curation, F.H. and M.S.; writing—original draft preparation, F.H., M.S., S.A.J.T.; writing—review and editing, N.R.G. and M.E.M.; supervision, N.R.G. and M.E.M.; project administration, M.E.M.; funding acquisition, M.E.M. All authors have read and agreed to the published version of the manuscript. Funding: We thank Elysium Health and the Mitchell Cancer Institute for the financial support of this work. Institutional Review Board Statement: The preliminary study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the University of Alabama (protocol code 19-465 and 12 August 2020). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Raw NMR and MS data files can be obtained upon request made directly to M.M. Acknowledgments: We thank the Mass Spectrometry Core facilities at the MCI and Lindsay Scham- beau for performing the mass spec. analyses. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations minute (min), hour (h), room temperature (rt), methanol (MeOH), dimethylsufoxide (DMSO), acetonitrile (CH3CN, ACN), Hydroxybenzotriazole (HOBt), dimethylformamide (DMF), dicyclohexyl- carbodiimide (DCC), dichloromethane (DCM), nicotinamide riboside (NR), nicotinamide riboside reduced form (NRH), nicotinamide adenine dinucleotide, (NAD), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide reduced form, (NADH), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), pyridone (PY), methyl-pyridones (N-Me-PY), pyridone ribosides (PYR), ﬂavin adenine dinucleotide (FAD), Electrospray ionization (ESI), mass spectrometry (MS), liquid chromatography coupled with mass spectrometry (LS-MS), proton nuclear magnetic resonance spectroscopy (1H NMR), calculated (calcd).

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