α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase

Article Cite This: J. Med. Chem. 2018, 61, 745−759

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α‑Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD) Inhibitors as Novel Modulators of De Novo Nicotinamide Adenine Dinucleotide (NAD+) Biosynthesis Roberto Pellicciari,*,† Paride Liscio,† Nicola Giacchè,† Francesca De Franco,† Andrea Carotti,‡ Janet Robertson,† Lucia Cialabrini,§ Elena Katsyuba,∥ Nadia Raffaelli,§ and Johan Auwerx*,∥ †

TES Pharma S.r.l., IT-06073 Corciano, Perugia, Italy Department of Pharmaceutical Sciences, University of Perugia, IT-06123 Perugia, Italy § Department of Agricultural, Food and Environmental Sciences, Polytechnic University of Marche, IT-60131 Ancona, Italy ∥ Laboratory of Integrative and Systems Physiology, Interfaculty Institute of Bioengineering, École Polytechnique Fédérale de Lausanne CH-1015 Lausanne, Switzerland ‡

S Supporting Information *

ABSTRACT: NAD+ has a central function in linking cellular metabolism to major cell-signaling and gene-regulation pathways. Defects in NAD+ homeostasis underpin a wide range of diseases, including cancer, metabolic disorders, and aging. Although the beneficial effects of boosting NAD+ on mitochondrial fitness, metabolism, and lifespan are well established, to date, no therapeutic enhancers of de novo NAD+ biosynthesis have been reported. Herein we report the discovery of 3-[[[5-cyano-1,6dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]phenylacetic acid (TES-1025, 22), the first potent and selective inhibitor of human ACMSD (IC50 = 0.013 μM) that increases NAD+ levels in cellular systems. The results of physicochemical-property, ADME, and safety profiling, coupled with in vivo target-engagement studies, support the hypothesis that ACMSD inhibition increases de novo NAD+ biosynthesis and position 22 as a first-class molecule for the evaluation of the therapeutic potential of ACMSD inhibition in treating disorders with perturbed NAD+ supply or homeostasis.

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INTRODUCTION

More than 95% of dietary tryptophan undergoes metabolism by the KP through the production of a large array of metabolites with various bioactivities.15−17 To date, indoleamine 2,3-dioxygenase-1 and -2 (IDO-1 and -2), kynurenine 3monooxygenase (KMO) and kynurenine aminotransferases I and II (KATI and KATII) are the most widely studied enzymes in this pathway that are potential druggable targets18−21 for cancer,22 immune dysfunctions,23,24 and central-nervous-system (CNS) disorders.16,25−27 Our early contributions in the discovery of potent and selective KMO28,29 and KATII30−32 inhibitors (Figure 1A) provided excellent chemical tools, which were instrumental in the development of more advanced preclinical candidate compounds (Figure 1B).33−35 In this context and as a continuation of our interest in the field, we have recently moved our attention toward unexplored

Originally described as an enzyme cofactor, nicotinamide adenine dinucleotide (NAD+) is a key cellular factor linking cellular metabolism to major cell-signaling and gene-regulation pathways.1−4 The medical importance of NAD+ was initially established in 1937 when Conrad Elvehjem and his team discovered that the devastating disease pellagra could be cured by nicotinic acid and nicotinamide, both NAD+ precursors.5 Today, deficits or disturbances in NAD+ homeostasis are associated with a wide range of diseases,6 including kidney dysfunction,7 metabolic disorders,8,9 muscle diseases,10 neurodegeneration,11 and aging.2,12−14 In humans, NAD+ is synthesized from four dietary sources: nicotinic acid, nicotinamide, and nicotinamide riboside (collectively known as vitamin B3), which are processed through metabolic pathways that salvage the preformed pyridine ring, and tryptophan (Trp), which is processed through the de novo NAD+ biosynthesis pathway (kynurenine pathway, KP).2,11 © 2018 American Chemical Society

Received: August 23, 2017 Published: January 18, 2018 745

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Figure 1. Early (a) and recently (b) disclosed KMO and KATII inhibitors.

Figure 2. Role of ACMSD as the branching point in the kynurenine pathway leading to de novo NAD+ biosynthesis.

ACMSD is a Zn2+-dependent amidohydrolase conserved in eukaryotes and several bacterial species.38 In mammals, the expression of the enzyme is restricted to the kidney, liver, and brain, with an expression ratio of 1300:30:1.39 The murine enzyme has been shown to be under nutritional and hormonal control, being upregulated by high protein diets40,41 and downregulated by dietary polyunsaturated fatty acids42 and cholesterol. 43 Notably, the enzyme is upregulated in streptozotocin-induced diabetic rats, and insulin treatment suppresses this elevation.41 As expected, changes in ACMSD activity are readily reflected in the serum levels of QUIN and PIC42,44 and in the rate of tryptophan-to-NAD+ conversion,45 confirming the role of the enzyme in regulating the production of metabolites whose levels are known to impact neuronal and metabolic functions. Indeed, increased QUIN levels have been associated with several neurological diseases.27 On the other hand, it is widely documented that enhanced NAD+ biosynthesis has both protective and therapeutic effects on a large spectrum of disorders, including metabolic,8,9 neurodegenerative,11 muscular,10 cardiac, and renal7 dysfunctions. Thus, the inhibition of ACMSD activity might be of therapeutic potential, because it should raise cellular NAD+ contents.

branches of the KP, focusing on the discovery of novel inhibitors of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD, EC 4.1.1.45), a critical enzyme in de novo NAD+ biosynthesis.36 (Figure 2) The enzyme catalyzes the decarboxylation of 2-amino 3carboxymuconate 6-semialdehyde (ACMS), an intermediate in the KP, to 2-aminomuconate-6-semialdehyde (AMS). AMS can either undergo spontaneous closure of the pyridine ring to form picolinic acid (PIC) or be oxidized to 2-aminomuconate, which is further metabolized so that it can enter the tricarboxylic acid (TCA) cycle (Figure 2). When the enzymatic capacity of ACMSD is exceeded, ACMS can also cyclize spontaneously to quinolinic acid (QUIN), which is further converted to the coenzyme NAD+ (Figure 2). Because the cyclization of ACMS into QUIN is a spontaneous reaction, the amount of ACMS undergoing this conversion and therefore leading to the production of NAD+ is primarily determined by the activity of ACMSD.37 Thus, the inhibition of ACMSD would channel ACMS toward de novo NAD+ biosynthesis, providing a novel way to boost NAD+ levels and to re-establish NAD+ homeostasis. 746

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Work carried out by Fukuwatari et al.46 identified phthalate monoesters, such as mono(2-ethylhexyl)phthalate (MEHP, 7, Figure 3), as the first examples of mammalian ACMSD

encouraged us to further investigate this structural class of druglike small-molecule ACMSD inhibitors. The preliminary observation of the induced fit of the docking pose of 10 in the hACMSD catalytic site from the 4IH3 X-ray (Figure 5) suggested that the majority of the binding affinity

Figure 3. Early ACMSD inhibitors.

inhibitors. Previous efforts in understanding the mechanism of recognition and binding of ligands to the active site of the human enzyme resulted in the release of the first cocrystal complex of the enzyme (PDB code 2WM1) with 1,3dihydroxyacetonephosphate (DHAP, 8, Figure 3).47 More recently, a second complex of human recombinant ACMSD (hACMSD) with a competitive inhibitor (PDB code 4IH3), namely pyridine-2,6-dicarboxylic acid (PDC, 9, Figure 3) was disclosed, refining the previous findings and highlighting the homodimeric nature of the enzyme.48 With only limited evidence of ACMSD inhibitors available to date in the literature, the quest for potent and selective inhibitors is of great interest in order to clarify the role of this unexplored enzyme in physiopathological conditions and to define its therapeutic potential. In this work, we describe our efforts spanning from hit identification to early chemical optimization, concluding with in vivo proof-of-concept studies that demonstrate that our compounds enhanced de novo NAD+ biosynthesis via ACMSD target engagement.

Figure 5. Docking model of 10 (pink) in the hACMSD active site (light green). The zinc ion is displayed as a gray ball. The hydrogen bonds engaging with the Lys41, Lys44, Arg47, and Trp191 residues are shown with yellow dashes.

was due to ionic interactions of the m-carboxylate group with the Zn2+ atom and Trp191 and Arg47 residues. Additionally, the 1,6-dihydropyrimidine nucleus engages in hydrogen-bond interactions with Lys41 and Lys44 (Figure 5). On the basis of these observations, medicinal chemistry efforts were focused on hit expansion with the primary goal of exploring structure−activity relationships (SAR) and corroborating the proposed binding hypothesis. To this end, we first investigated the role of the carboxylate functionality on the right-hand phenyl ring. Hydroxamic acid and 3-methyl-5-trifluoromethyl-[1,2,4]oxadiazole (TFMO)50 were selected as the most representative functional groups to probe the Zn2+-binding hypothesis of the carboxylate moiety of the starting compound, 10. The 2H-tetrazole nucleus was also investigated as a classical acid surrogate, concluding with the homologation of the meta-carboxylate group of 10. We then moved our attention to a short exploration of the 2thiopyrimidone-5-carbonitrile nucleus, producing SAR through systematic replacements at positions C-4, C-5, and C-6 (Table 1). Chemistry. The final compounds 10, 12, 13, 16−18, and 20−22 were easily prepared by regioselective S-alkylation reactions between a series of 5,6-disubstituted-2-thiopyrimidin4-ones (23 and 26−29) and commercially available 3(chloromethyl)benzoic acid (24) and benzyl bromide (25) or the appropriate chloro- or bromo-methyl phenyl intermediate (30−33) (Schemes 1−3). Chlorination in position C-6 of the 2-thiopyrimidone nucleus of compound 10 was accomplished by using POCl3, thus affording derivative 11 (Scheme 1). Halogen replacements in the C-5 positions, as in compounds 14 and 15, were obtained by reacting a suspension of compound 13 in AcOH with bromine or N-chlorosuccinimide, respectively, in the presence of PbO2 (Scheme 2). The synthesis of the hydroxamic acid derivative, 19, was described in Scheme 3. S-Alkylation of 4-oxo-6-(thiophen-2-yl)2-thioxo-pyrimidine-5-carbonitrile (29) with 3-chloromethyl-N(tetrahydro-pyran-2-yloxy)-benzamide (33) gave intermediate

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RESULTS AND DISCUSSION Hit Identification. Hit identification efforts started with the construction of an in silico model of the enzyme binding site, guided by the available structural information from the twocrystal complexes of hACMSD with 8 (DHAP) and 9 (PDC, Figure 3). The molecular-modeling workflow was first tested with a limited set of known binders (7−9) and then applied to TES Pharma’s internal database of compounds. The selected compounds were tested for their inhibitory effects on the catalytic activity of hACMSD by using an enzyme-coupled spectrophotometric assay.49 The virtual-screening campaign enabled the identification of 3-(5-cyano-6-oxo-4-phenyl-1,6dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic acid (10) as the first low-nanomolar hACMSD inhibitor (IC50 = 0.045 μM, Figure 4). Despite having a widely used 2-thiopyrimidone-5carbonitrile core, the meta-carboxyphenyl moiety of compound 10 conferred structural novelty to this class of derivatives and

Figure 4. hACMSD 2-thiopyrimidone screening hit, compound 10. 747

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Table 1. Biochemical Activities of the hACMSD Inhibitorsa

a The ACMSD enzymatic assay is described in the Experimental Section. The IC50 values are calculated with concentration−response curves, and the shown values are the means ± SD from three independent experiments.

Scheme 1. Synthetic Route to the 4-Phenyl-5-cyano-2-thiopyrimidone Derivatives 10−12a

a

Reagents and conditions: (a) K2CO3, CH3CN, 3-(chloromethyl)benzoic acid (24) or benzyl bromide (25), reflux; (b) POCl3, 70 °C.

34, which readily provided the desired hydroxamate compound, 19, after the acidic removal of the tetrahydropyran acetal protecting group. Furthermore, the 2-thiopyrimidin-4-one intermediates (23, 28, and 29) were prepared as described in Scheme 4. In

particular, the 5-cyano-6-aryl-2-thiopyrimidinones, 23, 28, and 29, were prepared following literature procedures by one-pot multicomponent condensations between the corresponding aldehyde, 35a or c; ethyl cyanoacetate, 36; and thiourea, 37, in the presence of potassium carbonate as the base.51 In a similar 748

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Scheme 2. Synthetic Route to the 4-Aryl-5-modified-2-thiopyrimidone Derivatives 13−15a

a

Reagents and conditions: (a) K2CO3, CH3CN, 24, reflux; (b) AcOH, PbO2, Br2 or NCS.

Scheme 3. Synthetic Route to the 5-Substituted-cyano-2-thiopyrimidone Derivatives 16−22a

a

Reagents and conditions: (a) K2CO3, CH3CN, reflux or DIPEA, DMSO, r.t.; (b) TFA, DCM, r.t..

carbodiimide (EDCI), or hydroxybenzotriazole (HOBT) were used (data not shown). Alternatively, NH2OH was protected by the tetrahydropyran acetal (NH2OTHP) following the literature procedure,53 and the amide-forming reaction with 24 proceeded smoothly via acyl chloride formation to give desired intermediate 33. Biochemical and SAR Evaluations. The newly synthesized compounds were evaluated for their abilities to inhibit the catalytic activity of hACMSD. IC50 values for tested compounds were calculated by assaying the enzyme activity at 10 μM concentrations of the substrate, which is very close to the Km value (6.5 μM, as reported in ref 39) As summarized in Table 1, we investigated variations around the functional groups presented by compound 10, starting with an investigation of variations at the C-6 position of the pyrimidone nucleus and at the meta carboxylic acid group, the two key pharmacophoric elements of our starting hit, 10. Chlorination of the carbonyl group at position C-6 resulted in detrimental effects on compound potency (derivative 11). The same trend was observed with the removal of the carboxylate at the meta-position. Indeed, compound 12, which lacked the acid moiety, was approximately 100-fold less potent than hit compound 10, thus supporting our initial binding hypothesis

manner, the reaction between commercially available ethylbenzoyl acetate (38) or 2-cyano-3-ethoxy-but-2-enoic acid ethyl ester (39) and 37 in basic conditions furnished 6-phenyl-4-oxo2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (26) and 6-methyl-4oxo-2-thioxo-pyrimidine-5-carbonitrile (27),52respectively, in acceptable yields (Scheme 4). Additionally, the bromo- or chloro-methyl phenyl intermediates, 30−33, were prepared using the chemistry described in Scheme 5. Treatment of meta-tolunitrile (40) with NH2OH· HCl quantitatively yielded the corresponding hydroxyl amidine, 41. Intramolecular cyclization to give the trifluoromethyl oxadiazole (TFMO) intermediate (42) was accomplished by treating 41 with trifluoroacetic anhydride using pyridine as the solvent. Finally, the benzylic bromination of 42 using standard conditions, such as NBS/DPO, provided target compound 30. 5-m-Tolyl-2H-tetrazole (43) was easily prepared by a 1,3dipolar cycloaddition reaction of nitrile 40 and sodium azide and further derivatized in the benzylic position to provide intermediate 31 in a satisfactory yield. In the same context, the bromination of commercially available phenylacetic acid, 44, cleanly gave intermediate 32. Attempts for the direct coupling of 24 with NH2OH failed, even when various promoting agents such as COCl 2 , 1-ethyl-3-(3-(dimethylamino)propyl)749

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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cycloalkyl, and bioisosteric replacements of the phenyl ring. Although the cyclohexyl substituent maintained the starting inhibitory potency with a slight decrease of the IC50 value (compound 17), a methyl replacement of the phenyl drastically reduced the starting potency, as shown in compound 16. Interestingly, a phenyl to 2-thienyl bioisosteric group manipulation at position C-4 of the 2-thiopyrimidone-5carbonitrile nucleus provided compound 18, which exhibited a 2-fold improvement in the ACMSD inhibitory potency (Table 1) with respect to the starting hit, 10. Interesting results were obtained by the two zinc-binding groups selected to work as carboxylate equivalents. Surprisingly, both the hydroxamic acid and the TFMO derivatives (compounds 19 and 20, respectively Table 1) recorded a dramatic drop in the inhibitory potency, thus pointing out the importance of the ionic characteristics of the interaction established between the carboxylate function with the zinc atom and key amino acid residues. Instead, the canonical replacement of the carboxylate group of 10 with an acid surrogate, such as the 2H-tetrazole motif, provided the 1,6-dihydro-6-oxo-2-[[[3-(2H-tetrazol-5-yl)phenyl]methyl]thio]-4-(2-thienyl)-5-pyrimidinecarbonitrile derivative, 21 (TES-991), which stands out as the most potent analogue of the series, suggesting an optimal fit for the tetrazole moiety within the catalytic binding site of the ACMSD enzyme (Figure 6a). These findings were confirmed by the homologation of the m-carboxylate group of compound 10, which enabled the discovery of 3-[[(5-cyano-1,6-dihydro-6-oxo-4-(2thienyl)-2-pyrimidinyl)thio] methyl] phenylacetic acid, 22 (TES-1025),54 (Table 1) a low nanomolar inhibitor of hACMSD suitable for future development. Furthermore, analysis of the docking poses of both 21 and 22 (Figure 6a,b) in our hACMSD model suggested that the excellent potency shown by the inhibitors may arise in part from the 2-thiophene ring better fitting the hydrophobic cleft defined by the Trp191 and Met180 residues, combined with

Scheme 4. Synthetic Route to the 5,6-Disubstituted-2thiopyrimidone Derivatives 23 and 26−29a

a

Reagents and conditions: (a) thiourea (37), K2CO3 or piperidine, EtOH, reflux; (b) 37, EtONa, EtOH, reflux.

and design strategy. Having established an SAR at the mposition of the right-hand phenyl ring, we turned our attention to the nitrile group at position C-5 of the 2-thio pyrimidinone scaffold. Interestingly, whereas bromine and chlorine in position C-5, 14 and 15, respectively, worked as nitrile bioisosteres resulting in low IC50 values, complete removal of the nitrile group drastically reduced the inhibitory potency, as shown in derivative 13. Continuing to probe the minimal requirements for the ACMSD-inhibitory activity of our starting chemotype, 10, we next set out to survey SAR at the C-4 position of the 2-thiopyrimidinone nucleus through alkyl, Scheme 5. Synthesis of Intermediates 30−33a

Reagents and conditions: (a) NH2OH·HCl, NaHCO3, EtOH; (b) (CF3CO)2O, pyridine, reflux; (c) NBS, DPO, CH3CN; (d) NaN3, Et3NH4+Cl−, toluene, reflux; (e) NBS, AIBN, CH3CN, reflux; (f) NBS, AIBN, CCl4, reflux; (g) CO2Cl2, DMF, DCM, Et3N.

a

750

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Figure 6. Docking models of 21 (gray, 6a) and 22 (green, 6b) in the hACMSD active site. The zinc ion is displayed as a gray ball. The hydrogen bonds engaged by the two compounds with Lys41, Lys44, Arg47, and Trp191 are shown with yellow dashes.

As our initial purpose was to develop a lead compound suitable for in vivo studies, the solubilities and passive cellular permeabilities of compounds 21 and 22 were evaluated as reported in Table 2. Furthermore, they were profiled over a panel of cytochrome P450s (CYP 3A4, 2D6, 2C19, and 3A1). Compound safety was evaluated in an in vitro human ERG potassium channel assay (hERG) for cardiotoxicity and in selected cellular lines (murine AML-12 and human HepG2) for hepatic toxicity. Selectivity to the target was initially assessed in an in-house nuclear receptor panel and then extended to a larger panel of about 126 off-targets including kinases, phosphatases, ion channels, and GPCRs (see the SI for details). As shown in Table 2, both compounds 21 and 22 exhibited good solubilities with low permeabilities and safe profiles in the cellular setting, being devoid of any additional off-target activities (see selectivity in Table 2). Whereas the free-acid compound, 22, was completely clean over all cytochrome P450s, compound 21 showed an inhibition of cytochrome P450 2C19, suggesting a possible involvement of the 2H-tetrazole motif in this interaction. In Vivo Studies. Compounds 21 and 22 were then subjected to in vivo pharmacokinetic studies, following intravenous (IV) and oral (PO) dosings of male CD-1 mice (Table 3). Because ACMSD is reported to be mostly expressed in the kidneys and liver, the penetration of both compounds in these two organs was evaluated. As shown in Table 3, comparable results were obtained for both the tetrazole (21) and acid (22) compounds. After the intravenous administration of 0.5 mg/kg, 21 and 22 showed low blood clearance, with low volumes of distribution and halflives (t1/2) of about 4.0 and 5.0 h, respectively, although after oral administration at 5 mg/kg, the blood concentrations of both compounds were quantifiable for up to 8 h (see Table 2S of the SI for the full PK profile). A moderate systemic exposure was observed for the 2H-tetrazole analogue, 21 (Cmax of 255 ng/mL, Table 3), and a good systemic exposure was recorded

orientation of the 2H-tetrazole or carboxymethylene motif, respectively, which optimize the interaction geometry. In Vitro Studies. To further validate the newly synthesized derivatives, we investigated whether the most potent compounds (21 and 22) were able to affect NAD+ levels in primary human hepatocytes. Inhibition of the ACMSD enzyme is expected to increase the rate of de novo NAD+ biosynthesis and should be reflected by an increase in intracellular NAD+ levels. The expression of ACMSD in these cells was confirmed by RT-PCR (not shown). Notably, compounds 21 and 22 were both able to significantly increase intracellular NAD+ levels, providing further proof of their mechanism of action (Figure 7).

Figure 7. NAD+ levels in primary cultured human hepatocytes incubated in the absence (NT) or presence of 21 or 22. One-way analysis of variance (ANOVA): Dunnett’s test. *p < 0.01.

These results establish for the first time that ACMSD inhibitor compounds are able to increase NAD+ contents in a cellular settings. Table 2. Measured Properties for Compounds 21 and 22 # 21 22

MW 393.4 383.4

Sa 1.0 0.8

Pappb

CYPsc

5.84 4.56

0.1 μM, 2C19 >10 μM

hERGd k

NA NAk

cytotoxicitye j

NT NTj

NRsf k

NA NAk

selectivityg NA < 10 μM NA < 10 μM

a

Solubility: mg/mL, PBS, pH 7.4. bPassive membrane permeability in MDCK-MDR1 cells: nm/sec (A > B). cCytochrome P-450 (1A2, 3A4, 2D6, and 2C19). dDetermined by a human ERG assay kit. eDetermined in AML-12 and Hep-G2 cell lines. fDetermined by an in-house nuclear-receptorselectivity panel (PXR, PPARs, LXR, VDR, GR, RXR, TR, and FXR). gDetermined by a Eurofin Safety Panel including 126 off-targets (for details, see Figures 1S−3S of the SI). kNA: not active. jNT: not toxic. 751

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Table 3. PK Profiles of Compounds 21 and 22 Following a Single IV or Oral Dose IVa

POb AUClast(h·ng/mL)

#

AUClast (h·ng/mL)

CL (mL/min/kg)

Vdss (L/kg)

t1/2 (h)

Cmax (ng/mL)

heart blood

liver

kidney

Tmax (h)

%F

21 22

6900 10 700

1.20 0.78

0.306 0.273

4.02 5.33

255 2570

1620 12 700

4290 19 200

2830 36 600

2.0 2.0

2.3 12

a

Mean IV: 0.5 mg/kg. Vehicle: saline water containing 5% (v/v) DMSO and 20% (v/v) PEG400. bMean PO: 5 mg/kg. Vehicle: HPMC 0.5% (w/v) in water containing 5% (v/v) DMSO.

for the free acid, 22, with a Cmax of 2570 ng/mL reached at 2 h after dosing (Table 3). The greater oral exposure of compound 22 was further confirmed in the liver and kidneys with AUC0−8h of 19 200 h·ng/mL and 36 600 h·ng/mL, respectively (Table 3). The liver-to-blood AUC ratio for compound 22 was 1.5, and the kidney-to-blood AUC ratio was 2.9, suggesting a peculiar tissue distribution for this derivative. The CNS penetration for both compounds was negligible and did not change in the presence of Elacridar (a known inhibitor of P-gp efflux transporters), suggesting no P-gp interactions for our compounds (see Table 2S of the SI), as demonstrated by the in vitro assay, and evidencing a restricted peripheral distribution of 21 and 22. According to their cellular-permeability profiles, the fraction absorbed for the 2H-tetrazole analogue, 21, was low (estimated %F of 2.3%, Table 3), and that for the free-acid compound, 22, was moderate, resulting in an estimated oral bioavailability of 12% (Table 3). On the basis of the better oral exposure of 22, this compound was chosen as the chemical tool in a target engagement study in order to validate in vivo its mechanism of action. To this end, ACMSD activity was measured in kidney and liver extracts prepared from mice at different times after PO dosings of 5 mg/kg. As shown in Figure 8, the enzyme activity was significantly reduced in kidneys from 0.5 to 2 h after the dosings of compound 22, thus proving the target engagement of ACMSD by 22 in vivo in the kidney.

In the livers of the control and treated mice, ACMSD activity was under the detection limit of the enzymatic assay; therefore, a pharmacodynamic validation of the enhancement of de novo NAD+ biosynthesis with compound 22 was performed by evaluating the NAD+ levels in the liver. Thus, male C57BL/6 mice were fed compound 22 as a diet supplement at 15 and 30 mg/kg/day for 10 days. At the end of the treatment, changes in NAD+ levels were measured in liver extracts and compared to those of the control mice (Figure 9).

Figure 9. NAD+ levels in liver extracts of mice fed compound 22 at 15 and 30 mg/kg/day for 10 days. Vehicle: 5% DMSO, 20% PEG. Oneway ANOVA: Dunnett’s test. *p < 0.05, **p < 0.01.

As illustrated in Figure 9, the liver NAD+ levels were significantly increased over those of the control animals at both doses of compound 22, giving us proof of the in vivo efficacy of our compound in the enhancement of de novo NAD+ biosynthesis via ACMSD inhibition.

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CONCLUSIONS In summary, we report herein the discovery, synthesis, and biological evaluation of a series of 2-thiopyrimidone-5-carbonitriles as the first class of small-molecule drug-like ACMSD inhibitors. SAR investigation around the screening hit, 10, led to the discovery of compound 22, the first low-nanomolar inhibitor54 of human ACMSD with a suitable overall balance of good physicochemical properties and in vitro safety profile. Proof-of-concept studies demonstrated for the first time that the inhibition of ACMSD by this class of compounds led to the modulation of intracellular NAD+ levels with consequent in vivo enhancement of de novo NAD+ biosynthesis via ACMSD target engagement. On the basis of the discovery of compound 22 and related analogues, we have established valuable tools for better understanding the therapeutic applications of ACMSD

Figure 8. ACMSD enzyme activity in kidneys of mice at different time points after PO dosings of 5 mg/kg of compound 22. Statistical oneway ANOVA: Dunnett’s test. **p < 0.01. 752

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

Journal of Medicinal Chemistry

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EtOAc (3 × 50 mL). The organic layers were combined and dried over sodium sulfate. The pure product was obtained by flash-column chromatography on silica gel and eluted with DCM/MeOH (from 5% to 10% for the product), and after the washing, it was eluted a mixture of EtO2/acetone if that was required. 3-[[(5-Cyano-1,6-dihydro-6-oxo-4-phenyl-2-pyrimidinyl)thio]methyl]benzoic Acid (10). Following general procedure A and starting from 23 (1.6 g, 6.98 mmol), K2CO3 (2.88 g, 20.9 mmol), and 3(chloromethyl)benzoic acid (24, 1.19 g, 6.98 mmol) in CH3CN (80 mL), the title compound (10) was obtained in a 40% yield (936 mg, 2.78 mmol) as a yellowish solid with a mp of 270−272 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.58 (s, 2H), 7.44 (t, J = 7.5 Hz, 1H), 7.54− 7.61 (m, 3H), 7.67 (d, J = 7.1 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.91 (d, J = 7.2 Hz, 2H), 8.04 (s, 1H), 13 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 33.5, 93.2, 115.6, 128.2, 128.4, 128.4, 128.5, 128.5, 128.6, 129.7, 130.8, 131.5, 133.3, 135.1, 137.4, 165.4, 166.8, 167.3. HPLC: 96.3%. HRMS (m/z): [MH]+ calcd for C19H13N3O3S: 364.0686, found: 364.075 75. 3-(6-Chloro-5-cyano-4-phenyl-pyrimidin-2-ylsulfanylmethyl)benzoic Acid (11). A stirred suspension of compound 10 (160 mg, 0.44 mmol) and POCl3 (3 mL) was heated at 70 °C for 6 h. The reaction crude was poured on crushed ice. The mixture was extracted with EtOAc (3 × 20 mL). The collected organic phase was washed with brine, dried over Na2SO4, and evaporated. After flashchromatography purification with CH2Cl2/MeOH as the eluent, the title compound (11) was obtained in a 36% yield (60 mg, 0.16 mmol) as a white solid with a mp of 170−174 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.58 (s, 2H), 7.44 (t, J = 7.7 Hz, 1H), 7.58−7.62 (m, 2H), 7.66 (d, J = 7.1 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 7.1 Hz, 2H), 8.08 (s, 1H), 12.98 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 34.9, 102.5, 115.2, 128.7, 129.2, 129.2, 129.6, 129.6, 130.4, 131.4, 132.7, 133.9, 134.7, 138.0, 162.9, 167.5, 169.0, 174.3. HPLC: 98.8%. HRMS (m/z): [MH]+ calcd for C19H12ClN3O2S: 382.035 24, found: 382.042 495. 1,6-Dihydro-6-oxo-4-phenyl-2-[(phenylmethyl)thio]-5-pyrimidinecarbonitrile (12). Following general procedure A and starting from 23 (300 mg, 1.3 mmol), K2CO3 (542 mg, 3.93 mmol), and benzyl bromide (25, 0.15 mL, 1.3 mmol) in CH3CN (15 mL), the title compound (12) was obtained in a 41% yield (172 mg, 0.53 mmol) as a yellowish solid with a mp of 194−196 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.53 (s, 2H), 7.25−7.32 (m, 3H), 7.40 (d, J = 6.9 Hz, 2H), 7.56−7.61 (m, 3H), 7.92 (d, J = 6.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 36.1, 95.1, 117.6, 129.3, 130.3, 130.3, 130.4, 130.4, 130.5, 130.5, 130.8, 130.8, 133.6, 137.1, 138.2, 162.9, 167.5, 169.1. HPLC: 98.3%. HRMS (m/z): [MH]+ calcd for C18H13N3OS: 320.078 46, found: 320.085 48. 3-(6-Oxo-4-phenyl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)benzoic Acid (13). Following general procedure A and starting from intermediate 26 (100 mg, 0.43 mmol), K2CO3 (178 mg, 1.2 mmol), and 24 (74 mg, 0.43 mmol) in CH3CN (15 mL), the title compound (13) was obtained in a 21% yield (30 mg, 0.08 mmol) as a white solid with a mp of 270−275 °C after being washed with a mixture of Et2O/ acetone. 1H NMR (400 MHz, DMSO-d6): δ 4.59 (s, 2H), 6.69 (s, 1H), 7.41 (m, 1H), 7.46 (m, 3H), 7.71 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 8.06 (m, 3H), 12.85 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 33.8, 127.3, 127.3, 128.5, 129.1, 129.1, 129.2, 130.1, 131.0, 131.3, 131.5, 133.6, 136.3, 138.8, 167.5. HPLC: 99.6%. HRMS (m/z): [MH]+ calcd for C18H14N2O3S: 339.073 37, found: 339.080 44. 3-(5-Bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic Acid (14). To a stirred solution of compound 13 (100 mg, 0.29 mmol) in acetic acid (5 mL), lead dioxide (77.2 mg, 0.32 mmol) and bromine (0.02 mL, 0.32 mmol) were added. Stirring was continued for 6 h at room temperature. The mixture was poured in a solution of Na2S2O5 and was extracted with EtOAc (3 × 20 mL). The collected organic phases were washed with water and brine and then were dried over Na2SO4. Washing with a mixture of Et2O/ acetone afforded the title compound (14) in a 33% yield (40 mg, 0.09 mmol) as a white solid with a mp of 246−250 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.44 (s, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.48 (m, 3H), 7.61−7.65 (m, 3H), 7.82 (d, J = 7.5 Hz, 1H), 8.0 (s, 1H), 13.1

inhibitors for disorders such as mitochondrial dysfunctions and metabolic and renal diseases, associated with the dysregulation of ACMSD activity or reduced NAD+ levels. The efficacy of compound 22 and compound 21 in restoring depleted NAD+ content in the context of animal models of acute kidney injuries (AKI) and non-alcoholic fatty liver disease (NAFLD) is now being tested and will be reported separately.

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EXPERIMENTAL SECTION

Chemistry. All commercial chemicals and solvents were reagent grade and were used without further treatment unless specified. 1H NMR spectra were recorded at 200 and 400 MHz, and 13C NMR spectra were recorded at 100.6 and 50.3 MHz using the solvents indicated below. Chemical shifts were reported in parts per million (ppm) calibrated against the solvent peak δ 2.50 (DMSO-d6) or δ 7.24 (CDCl3). All the reactions were performed under a nitrogen atmosphere using distilled solvents. All the tested compounds were found to have >95% purity determined either by HPLC or HRMS (HPLC/Q-TOF) analysis. The HPLC analytical-scale measurements were carried out on a Shimadzu LC Workstation Class LC-10 equipped with an SCL-10 A VP system controller, an LC-10AT VP high-pressure binary-gradient delivery system, an SPD-10A VP variable-wavelength UV−vis detector, and a Rheodyne 7725i injector with a 20 μL stainless-steel loop. The chromatographic profiles were obtained with the EZ Start software. All analytical runs were performed using a H2O/CH3CN/TFA (60/40/0.1, v/v/v) solution as the mobile phase. HPLC-grade water was obtained from a tandem Milli-Ro/Milli-Q apparatus. A Grace Smart C18 250 × 4.6 mm i.d., 5 μm, 100 Å analytical column was used after a previous conditioning of having the selected mobile phase pass through the column for at least 30 min. The UV detection wavelengths were set at 254 and 230 nm. Samples for the analytical-scale analyses were prepared in approximate concentrations between 0.1 and 0.5 mg/mL in filtered mobile-phase components with auxiliary DMSO, if necessary, and sonicated until completely dissolved. The HRMS (HPLC/Q-TOF) analyses were assessed by the method detailed below. The LC system was an Agilent 1290 Infinity module equipped with an autosampler, a binary pump, a thermostated column compartment, and a diode-array detector. The analytical column was a Zorbax Eclipse Plus (2.1 × 50 mm, 1.8 μm). The column temperature was maintained at 40 °C. The mobile phase consisted of eluent A (water containing 0.1% formic acid) and eluent B (acetonitrile plus 0.1% formic acid). At 0 min (B = 20%), a linear gradient at 80% B started within 4 min, and this mobile phase was maintained for 1 min; at the end of a run (5 min), it returned back to 20% B. The flow rate was 0.25 mL/min. The LC system was connected to a detector, an Agilent 6540 UHD Accurate-Mass QTOF/MS system equipped with a source dual-jet stream. The mass spectrometer operated with positive acquisition, a gas temp of 300 °C, a gas flow of 6.6 L/min, a nebulizer pressure of 16 psi, a sheath-gas temp of 290 °C, a fragmentor at 200 V, a skimmer at 65 V, an Octapole RFPeaks 750, a capillary voltage of 4000 V, a nozzle at 0 V, and reference masses of 121.050 87 and 922.009 798. The analyses were performed on a Mass Hunter workstation. General Procedure A. To a stirred suspension of the appropriate intermediate, 2-thiopyrimidone 23 or 26−29 (1 equiv), and K2CO3 (3 equiv) in CH3CN, 1 equiv of the corresponding chloro- or bromomethyl derivative, 24, 25, or 30, was added. Stirring was continued overnight at reflux. The volatiles were removed under vacuum. The crude was taken up with water, acidified to pH 5 with diluted hydrochloride acid (3 M), and washed with EtOAc. The pH was then adjusted to pH 3, and the mixture was extracted with EtOAc (3 × 50 mL). The organic layers were combined and dried over sodium sulfate. The final compounds were obtained with high purities after being triturated with hot acetone. General Procedure B. To a stirred suspension of intermediate 29 (1 equiv) and DIPEA (1 equiv) in DMSO, the corresponding chloroor bromo-methyl derivative, 31−33 (1 equiv), was added. Stirring was continued overnight at room temperature. The crude was poured in water, washed with EtOAc, then acidified to pH 3, and extracted with 753

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

Journal of Medicinal Chemistry

Article

(m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 33.9, 128.4, 128.4, 128.6, 129.14, 129.3, 129.3, 130.1, 130.2, 131.3, 133.9, 138.1, 138.4, 167.5. HPLC: 96.8%. HRMS (m/z): [MH] + calcd for C18H13BrN2O3S: 418.983 57, found: 418.988 86. 3-(5-Chloro-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic Acid (15). To a stirred solution of compound 13 (100 mg, 0.29 mmol) in acetic acid (5 mL), lead dioxide (55.8 mg, 0.35 mmol) and N-chlorosuccinimide (47 mg, 0.35 mmol) were added. Stirring was continued for 6 h at room temperature. The mixture was poured into water and was extracted with EtOAc (3 × 20 mL). The collected organic phases were washed with water and brine and dried over Na2SO4. Washing with a mixture of Et2O/acetone afforded the title compound (15) in a 37% yield (40 mg, 0.1 mmol) as a white solid with a mp of 278−280 °C. 1H NMR (400 MHz, DMSOd6): δ 4.47 (s, 2H), 7.43 (t, J = 7.7 Hz, 1H), 7.49 (m, 3H), 7.64 (d, J = 7.2 Hz, 1H), 7.71 (m, 2H), 7.83 (d, J = 7.5 Hz, 1H), 8.02 (s, 1H), 13.1 (s, 1H), 13.25 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 33.9, 128.4, 128.4, 128.4, 128.6, 129.1, 129.4, 129.4, 129.4, 130.2, 131.3, 133.8, 136.5, 138.3, 156.1, 167.5. HPLC: 95.3%. HPLC: 95.2%. HRMS (m/z): [MH]+ calcd for C18H13ClN2O3S: 373.034 54, found: 373.041 57. 3-(5-Cyano-4-methyl-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic Acid (16). Following general procedure A and starting from intermediate 27 (145 mg, 0.86 mmol), K2CO3 (599 mg, 4.33 mmol), and 24 (148 mg, 0.86 mmol) in CH3CN (15 mL), the title compound (16) was obtained in a 23% yield (60 mg, 0.2 mmol) as a white solid with a mp of 245−250 °C after flash-chromatography purification (elution with CH2Cl2/ MeOH and 3% AcOH). 1H NMR (400 MHz, DMSO-d6): δ 2.44 (s, 3H), 4.49 (s, 2H), 7.45 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 8.05 (m, 1H), 13.1 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 23.3, 33.9, 95.3, 115.6, 128.7, 129.1, 130.6, 131.2, 134.1, 138.0, 161.1, 165.7, 167.4, 170.9. HPLC:96.5%. HRMS (m/z): [MH]+ calcd for C14H11N3O3S: 302.052 82, found: 302.059 98. 3-(5-Cyano-4-cyclohexyl-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic Acid (17). Following general procedure A and starting from intermediate 28 (250 mg, 1.18 mmol), K2CO3 (495 mg, 3.56 mmol), and 24 (202 mg, 1.18 mmol), the title compound (17) was obtained in a 21% yield (90 mg, 0.24 mmol) as a white solid with a mp of 255−260 °C after being washed with hot Et2O. 1H NMR (400 MHz, DMSO-d6): δ 1.24 (m, 3H), 1.60 (m, 7H), 2.74 (m, 1H), 4.52 (s, 2H), 7.45 (t, J = 7.1 Hz, 1H), 7.67 (d, J = 6.8 Hz, 1H), 7.82 (d, J = 7.1 Hz, 1H), 8.04 (s, 1H), 13.0 (s, 1H). 13C NMR (100 MHz, DMSOd6): δ 25.4, 25.7, 25.7, 30.3, 30.3, 33.8, 44.9, 94.1, 115.3, 128.6, 129.2, 130.1, 131.3, 133.6, 138.5, 161.1, 166.2, 167.4, 177.9. HPLC: 98.1%. HRMS (m/z): [MH]+ calcd for C19H19N3O3S: 370.115 64, found: 370.122 71. 3-[[[5-Cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]benzoic Acid (18). Following general procedure A and starting from 29 (250 mg, 1.06 mmol), K2CO3 (440 mg, 3.18 mmol), and 24 (180 mg, 1.06 mmol) in CH3CN (15 mL), the title compound (18) was obtained in a 12% yield (45 mg, 0.12 mmol) as a yellowish solid with a mp of 267−270 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.62 (s, 2H), 7.33 (t, J = 4.3 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 8.05 (m, 2H), 8.26 (d, J = 3.8 Hz, 1H), 12.99 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 33.9, 88.7, 116.5, 128.8, 129.3, 129.9, 130.2, 131.5, 132.1, 133.7, 135.4, 137.9, 139.7, 159.0, 161.2, 165.3, 167.4. HPLC: 97.2%. HRMS (m/z): [MH]+ calcd for C17H11N3O3S2: 370.024 72, found: 370.031 88. 3-(5-Cyano-6-oxo-4-thiophen-2-yl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-N-hydroxy-benzamide (19). To a stirred suspension of intermediate 34 (103 mg, 0.21 mmol) in DCM (5 mL), TFA (0.14 mL, 2.1 mmol) was added. Stirring was continued at room temperature overnight. The volatiles were removed under vacuum. The crude was purified by flash chromatography (eluted with DCM/ MeOH, 10% for product). The title compound (19) was obtained in a 40% yield (30 mg, 0.07 mmol) as a yellowish solid with a mp of 240− 245 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.50 (s, 2H), 7.28 (t, J = 3.7 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.60 (d, J = 7.3 Hz, 2H), 7.84 (s, 1H), 7.93 (d, J = 4.3 Hz, 1H), 8.19 (d, J = 2.9 Hz, 1H), 9.02 (brs, 1H),

11.2 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 34.1, 87.4, 118.4, 126, 128.1, 128.9, 129.4, 130.1, 130.7, 132.0, 133.5, 138.6, 140.9, 159.1, 164.5, 168. HPLC: 95.1%. HRMS (m/z): [MH]+ calcd for C17H12N4O3S2: 385.036 08, found: 385.043 34. 6-Oxo-4-thiophen-2-yl-2-[3-(5-trifluoromethyl-[1,2,4]oxadiazol3-yl)-benzylsulfanyl]-1,6-dihydro-pyrimidine-5-carbonitrile (20). Following general procedure A and starting from intermediate 29 (146 mg, 0.62 mmol), K2CO3 (128 mg, 0.93 mmol), and intermediate 30 (200 mg, 0.65 mmol) in CH3CN (20 mL), the title compound (20) was obtained in a 39% yield (110 mg, 0.24 mmol) as a white solid with a mp of 244−248 °C after flash-chromatography purification with DCM/MeOH as the eluent (5% for product). 1H NMR (400 MHz, DMSO-d6): δ 4.68 (s, 2H), 7.36 (t, J = 4.2 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1 H), 7.8 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 4.9 Hz, 1H), 8.24 (s, 1H), 8.29 (d, J = 3.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 33.7, 88.8, 114.8, 116.5, 117.5, 125.1, 126.8, 128.2, 129.9, 130.3, 132.1, 133.4, 135.4, 139.2, 139.6, 159, 161.2, 165.2, 168.6. HPLC: >97.9%. HRMS (m/z): [MH]+ calcd for C19H10F3N5O2S2: 462.023 93, found: 462.031 09. 1,6-Dihydro-6-oxo-2-[[[3-(2H-tetrazol-5-yl)phenyl]methyl]thio]4-(2-thienyl)-5-pyrimidinecarbonitrile (21). Following general procedure B and starting from 29 (160 mg, 0.66 mmol), DIPEA (0.09 mL, 0.55 mmol), and intermediate 31 (171 mg, 0.55 mmol) in DMSO (3 mL), the title compound (21) was obtained in a 23% yield (90 mg, 0.22 mmol) as a white solid with a mp of 251−254 °C. 1H NMR (400 MHz, DMSO-d6): δ 4.67 (s, 2H), 7.33 (t, J = 6.3 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.7 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H), 8.04 (d, 5 Hz, 1H), 8.18 (s, 1H), 8.26 (d, J = 3.9 Hz, 1H), 13.9 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): δ 33.9, 88.7, 116.5, 124.9, 126.4, 127.8, 129.9, 130.1, 132, 132.1, 135.4, 138.7, 139.7, 157, 159.1, 161.2, 165.3. HPLC: 99.2%. HRMS (m/z): [MH]+ calcd for C17H11N7OS2: 394.047 36, found: 393.074 36. 3-[[[5-Cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]-phenylacetic Acid (22). Following general procedure B and starting from 29 (500 mg, 2.12 mmol), DIPEA (0.4 mL, 2.12 mmol), and intermediate 32 (487 mg, 2.12 mmol) in DMSO (5 mL), the title compound (22) was obtained in a 25% yield (200 mg, 0.52 mmol) as a pure yellowish solid with a mp of 237−239 °C. 1H NMR (400 MHz, DMSO-d6): δ 3.54 (s, 2H), 4.53 (s, 2H), 7.17 (d, J = 7.1 Hz, 1H), 7.36−7.37 (m, 3H), 8.06 (d, J = 4.4 Hz, 1H), 8.27 (d, J = 3 Hz, 1H), 12.5 (brs, 1H).13C NMR (100 MHz, DMSO-d6): δ 34.2, 40.9, 88.4, 116.9, 127.6, 128.9, 129.1, 129.8, 130.4, 131.8, 135.2, 135.7, 137.1, 140, 159, 162, 165.8, 172.9. HPLC: 97.1%. HRMS (m/z): [MH]+ calcd for C18H13N3O3S2: 384.039 53, found: 384.046 68 General Procedure C for the Synthesis of 4-Oxo-6substituted-2-thioxo-1,2,3,4-tetrahydro-pyrimidines-5-carbonitrile. To a stirred solution of the corresponding aldehyde, 35a−c (1 equiv); ethylcyanoacetate, 36 (1 equiv); and thiourea, 37 (1 equiv), in ethanol, K2CO3 (1.2 equiv) or piperidine (2 equiv) was added. Stirring was continued at reflux overnight. The pale yellow solid was collected after it cooled, taken up with boiling water, and filtered again. The aqueous phase was acidified to pH 5 with AcOH, and the precipitate was filtered and dried under vacuum to afford the desired compound in acceptable yields. 4-Oxo-6-phenyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile (23). Following general procedure C and starting from benzaldehyde (35a, 0.5 mL, 0.83 mL, 4.9 mmol), 36 (0.52 mL, 4.9 mmol), 37 (372 mg, 4.9 mmol), and K2CO3 (812 mg, 5.88 mmol) in ethanol (25 mL), the title compound (23) was obtained in a 44% yield (500 mg, 2.18 mmol) as a yellow solid with a mp of 286−288 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.48−7.61 (m, 3H), 7.70 (d, J = 7.6 Hz, 1H), 8.0 (d, J = 7.3 Hz, 1H), 8.27 (s, 1H), 12.1 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): δ 88.1, 117.3, 128.6, 128.8, 129.4, 130.7, 131.4, 133.1, 153.6, 161.1, 163.6. 6-Phenyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (26). To a solution of ethylbenzoylacetate (38, 2 g, 10.41 mmol) in EtOH (15 mL), EtONa (7 mL, 18.7 mmol) and 37 (1.18 g, 15.61 mmol) were added. Stirring was continued at reflux overnight. The solvent was removed under reduced pressure. The reaction was taken up with water, acidified to pH 3, extracted with EtOAc (3 × 20 mL), washed 754

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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with brine, and dried over Na2SO4. The crude was purified by flash chromatography with CH2Cl2/MeOH as the eluent (2.5% for product), affording the title compound (26) in a 24% yield (500 mg, 2.44 mmol) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 6.06 (s, 1H), 7.47 (m, 2H), 7.53 (d, J = 7.2 Hz, 1H), 7.68 (d, J = 7.6 Hz, 2H), 12.45 (s, 1H), 12.5 (s, 1H). 13C NMR (100 MHz, DMSOd6): δ 105.9, 130.3, 130.3, 131.4, 131.4, 133.6, 133.9, 155.7, 163.7, 179.3. 6-Methyl-4-oxo-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile (27). To a stirred solution of NaOEt (1.02 mL, 2.73 mmol) in EtOH abs (20 mL), compounds 2-cyano-3-ethoxy-but-2-enoic acid ethyl ester (39, 500 mg, 2.73 mmol) and 37 (207 mg, 2.73 mmol) were added. Stirring was continued at reflux for 4 h. The volatiles were removed under vacuum. The crude of the reaction was taken up with water and acidified with AcOH. The precipitate was collected, dissolved in water, and washed with a mixture of CHCl3 and MeOH. The aqueous phase was extracted with EtOAc (3 × 20 mL). The collected organic phase was washed with brine and dried over Na2SO4. The title compound (27) was obtained in a 55% yield (250 mg, 1.49 mmol) as a white solid with a mp >290 °C. 1H NMR (400 MHz, DMSO-d6): δ 2.32 (s, 3H), 13.03 (s, 1H), 13.08 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 128.7, 91.3, 114.7, 158.5, 162.7, 176.2. 6-Cyclohexyl-4-oxo-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5carbonitrile (28). Following general procedure C and starting from cyclohexanecarboxaldehyde (35b, 1.62 mL, 13.3 mL), 36 (1.42 mL, 13.37 mmol), 37 (1.01 g, 13.3 mmol), and piperidine (2.64 mL, 26.7 mmol) in ethanol (50 mL), the title compound (28) was obtained in a 30% yield (930 mg, 3.95 mmol) as a white solid with a mp >290 °C after flash-chromatography purification (CHCl3/MeOH as a gradient, from 0 to 2% for product). 1H NMR (400 MHz, DMSO-d6): δ 1.24 (m, 4H), 1.7 (m, 4H), 1.8 (t, J = 11.6 Hz, 1H), 12.9 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): δ 25.1, 25.9, 25.9, 28.6, 42.8, 89.7, 114.9, 159.1, 168.8, 177.1. 4-Oxo-6-thiophen-2-yl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5carbonitrile (29). Following general procedure C and starting from 2thiophenecarboxaldehyde (35c, 1 g, 0.83 mL), 36 (0.96 g, 8.8 mmol), 37 (672 mg, 8.8 mmol), and K2CO3 (1.57 g, 11.44 mmol) in ethanol (55 mL), the title compound (29) was obtained in a 49% yield (1 g, 4.25 mmol) as a yellowish solid with a mp of 286−288 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.3 (t, J = 4 Hz, 1H), 7.96 (d, J = 3.8 Hz, 1H), 8.8 (d, J = 4.9 Hz, 1H), 13.1 (brs, 2H). 13C NMR (100 MHz, DMSO-d6): δ 84.5, 115.9, 128.6, 129.9, 134, 134.3, 150.1, 154.3, 161.9. 3-(3-Bromomethyl-phenyl)-5-trifluoromethyl-[1,2,4]oxadiazole (30). To a stirred solution of intermediate 42 (390 mg, 1.71 mmol) and DPO (5 mg, 0.085 mmol) in CH3CN (15 mL), NBS (323 mg, 1.88 mmol) was added. Stirring was continued at 90 °C overnight. The solvent was removed under reduced pressure. The crude product was partitioned between water and NaHCO3(aq). The organic layer was washed with brine and dried over Na2SO4. The title compound (30) was obtained in a 93% yield (490 mg, 1.59 mmol) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 4.56 (s, 2H), 7.53 (t, J = 7.7 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 8.0 (d, J = 7.7 Hz, 1H), 8.17 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 32.1, 125.5 (JCF = 44 Hz), 127.6, 128.2, 128.9, 129.7, 130.4, 132.8, 139.1, 168.7. 5-(3-Bromomethyl-phenyl)-1H-tetrazole (31). To a suspension of compound 43 (300 mg, 1.87 mmol) in CH3CN (15 mL), AIBN (31 mg, 0.18 mmol) and NBS (333 mg, 1.87 mmol) were added. Stirring was continued at reflux overnight. The solvent was removed under reduced pressure. The reaction was taken up with water, extracted with EtOAc (3 × 20 mL), washed with brine, and dried over Na2SO4. The crude was purified by flash chromatography and eluted with CH2Cl2/ MeOH (7% for product), affording the title compound (31) in a 34% yield (150 mg, 0.62 mmol) as a yellow solid with a mp of 139−142 °C. 1 H NMR (400 MHz, DMSO-d6): δ 7.57−7.6 (m, 1H), 7.65 (d, J = 7.3 Hz, 1H), 7.96 (d, J = 7.4 Hz, 1H), 8.14 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 33.8, 124.5, 127.1, 128.1, 129.7, 130.2, 132.3, 139.8. (3-Bromomethyl-phenyl)-acetic Acid (32). To a suspension of compound 22 (750 mg, 5 mmol) in CCl4 (15 mL), AIBN (41 mg, 0.25 mmol) and NBS (933.7 mg, 5.24 mmol) were added. Stirring was continued at reflux overnight. The solvent was removed under reduced

pressure. The reaction was taken up with water, extracted with EtOAc (3 × 20 mL), washed with brine, and dried over Na2SO4. The crude was purified by flash chromatography and eluted with CH2Cl2/MeOH (3% for product), affording the title compound (32) in a 70% yield (800 mg, 3.49 mmol) as a white solid with a mp of 75−78 °C. 1H NMR (400 MHz, CDCl3): δ 3.67 (s, 2H), 4.25 (s, 2H), 7.23−7.25 (m, 1H), 7.33−7.34 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 33.1, 40.7, 128.1, 129.1, 129.4, 130, 133.7, 138.1, 177.5. GC/MS (m/z) 227.9 (M+). 3-Chloromethyl-N-(tetrahydro-pyran-2-yloxy)-benzamide (33). To a stirred solution of 24 (200 mg, 1.17 mmol) in DCM (20 mL), CO2Cl2 (0.13 mL, 1.52 mmol) was added. The mixture was cooled to 0 °C, and two drops of DMF were added. Stirring was continued at room temperature for 4 h. After the reaction completion, the solvent was removed under reduced pressure, and the crude acyl chloride was used for the next step. O-(tetrahydro-pyran-2-yl)-hydroxylamine (45, 123 mg, 1.05 mmol) was added dropwise to a solution of the above chloride (200 mg, 1.05 mmol) and Et3N (0.15 mL, 1.05 mmol) in DCM (15 mL). Stirring was continued at room temperature overnight. The solvent was removed under reduced pressure. The reaction was taken up with water, extracted with EtOAc (3 × 20 mL), washed with brine, and dried over Na2SO4.The crude was purified by flash chromatography and eluted with CH2Cl2/MeOH (1.5% for product), affording the title compound (33) in a 41% yield (130 mg, 0.48 mmol) as a white solid. 1H NMR (400 MHz, CDCl3): δ 1.52 (m, 3H), 1.68 (m, 3H), 3.51 (m, 1H), 4.03 (m, 1H), 4.80 (s, 2H), 4.98 (m, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.7 (t, J = 7.5 Hz, 1H), 7.82 (s, 1H), 11.71 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 13.9, 25, 28, 45, 62.5, 102.8, 127.1, 127.4, 129.2, 132.6, 138.2, 165.6. 3-(5-Cyano-6-oxo-4-thiophen-2-yl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-N-(tetrahydro-pyran-2-yloxy)-benzamide (34). Following general procedure B and starting from 29 (103 mg, 0.43 mmol), DIPEA (0.075 mL, 0.48 mmol), and intermediate 33 (130 mg, 0.48 mmol) in DMSO (5 mL), the title compound (34) was obtained in a 51% yield (103 mg, 0.22 mmol) as a yellowish solid with a mp of 250−255 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.52 (m, 3H), 1.69 (m, 3H), 3.5 (m, 1H), 4.02 (m, 1H), 4.59 (s, 2H), 4.94 (m, 1H), 7.33 (t, J = 4.7 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.64 (t, J = 6.4 Hz, 2H), 7.87 (s, 1H), 8.05 (d, J = 2.5 Hz, 1H), 8.25 (d, J = 3.9 Hz, 1H), 11.64 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 18.7, 25.1, 28.3, 34.1, 61.8, 88.5, 101.4, 116.9, 126.7, 128.4, 129.1, 129.9, 131.9, 132.4, 133.1, 135.2, 137.8, 140, 159.1, 161.9, 164.4, 165.8. N-Hydroxy-3-methyl-benzamidine (41). To a stirred solution of NH2OH·HCl (1.42 g, 20.5 mmol) and NaHCO3 (1.72 g, 20.5 mmol) in water (7 mL), a solution of m-tolunitrile (40, 2 mL, 17.0 mmol) in EtOH (13.3 mL) was gradually added. Stirring was continued at 80 °C for 4 h. The volatiles were removed under reduced pressure. The crude of the reaction was taken up with water and extracted with EtOAc (3 × 25 mL). The organic phase was collected, washed with brine, and dried over Na2SO4, affording the title compound (41) in a 95% yield (2.5 g, 9 mmol) as a white solid with a mp of 86−89 °C. 1H NMR (400 MHz, CDCl3): δ 2.39 (s, 3H), 4.85 (brs, 2H), 7.25 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.47 (s, 1H), 9.1 (brs, 1H). 13C NMR (100 MHz, CDCl3): δ 21.4, 123, 126.5, 128.5, 130.7, 132.3, 138.3, 152.7. 3-m-Tolyl-5-trifluoromethyl-[1,2,4]oxadiazole (42). To a solution of intermediate 41 (500 mg, 3.3 mmol) in pyridine (7.5 mL), (CF3CO2)O (1.4 mL, 9.9 mmol) was added dropwise at 0 °C. The reaction was slowly warmed to room temperature and further heated to 50 °C for 3 h. The reaction mixture was poured into ice water and adjusted to pH 4 by the addition of 1.5 N HCl. The product was purified by flash chromatography with PET/EtOAc. The title intermediate (42) was obtained in a 53% yield (400 mg, 1.75 mmol) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 2.45 (s, 3H), 7.38−7.44 (m, 2H), 7.93 (d, J = 6.9 Hz, 1H), 7.95 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 21.3, 116.1, 124.8, 128.2, 129, 133, 139, 165.7, 169.3. GC/MS (m/z) 228.1 (M+). 5-m-Tolyl-1H-tetrazole (43). A mixture of compound 40 (1,02 mL, 8.54 mmol), NaN3 (832 mg, 12.8 mmol), and Et3N*HCl (1.76 g, 12.8 mmol) in toluene (20 mL) was heated at reflux for 4 h. The solvent 755

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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Dunnett’s test. Differences were considered significant at p < 0.05 and p < 0.01. For the determination of NAD+ levels in liver extracts, C57BL/6 mice were fed 15 or 30 mg of 11 per kilogram per day as a dietary supplement for 10 days. After a 3 h fast, the animals were sacrificed humanely, and the livers were rapidly collected and snap-frozen in liquid nitrogen. Samples were ground in liquid nitrogen and NAD+ was extracted from liver N2-powders by sonication in HClO4, as described.61 NAD+ quantification was performed by ion-pair C18HPLC chromatography as previously reported.61 Values of NAD+ levels were referenced to total protein contents (BCA, Pierce) for both analyses. The in vivo study was performed following the Ethical Guidelines for animal-model studies authorized by Swiss Committee “Commission cantonale pour l’expérimentation animale” (license study number 2882). In Vitro ADME Profiling. Solubility Assay. The solubilities of compounds were estimated by mixing, incubating for 24 h, and filtering a solution in PBS buffer (pH 7.4, 1% DMSO) in a solubility filter plate (Multiscreen Filter plate, Millipore). The solutions were filtered into a 96-well collection plate (V-bottom collection plate, Geiner Bio) using vacuum filtration and then analyzed by UV/vis spectroscopy (UV Star analysis plate and EnSpire Multimode Plate Reader, PerkinElmer). The relative solubilities were then calculated using the sum of the recorded values as compared to a standard. Passive Membrane Permeability in the MDCKII-MDR1 Cell Line. This assay, performed by Aptuit (Verona, Italy, http://www.aptuit. com/), was designed to evaluate passive membrane permeability and whether test items are a P-gp substrates by using MDCKII-MDRI cells. A bidirectional assay (apical-to-basolateral [A > B] and basolateral-to-apical [B > A]) was run, in the absence and in the presence of a P-gp inhibitor (e.g., GFl209l8) in the MCDKII-MDRI cells, using Hank’s balanced salt solution (HBSS) as the transport medium at pH 7.4 (n = 3). Test items were investigated at 3 μM for 90 min. As reference compounds, amprenavir (a P-gp substrate), atenolol (a low-moderately permeable compound), and propranolol (a highly permeable compound) were included at a single concentration (3 μM) and at single time point (90 min). Amprenavir transport was evaluated in two directions (apical-to-basolateral [A > B] and basolateral-toapical [B > A]) (n = 3), whereas atenolol and propranolol were tested only in A > B direction (n = 3). Samples were analyzed by an LC-MS/ MS system to measure test items and reference-compound concentration levels, with compound concentrations expressed as area ratios determined by dividing the analyte-peak area with that of the internal-standard-peak area. At the end of permeability experiment, the integrity of the cell monolayer was evaluated using the paracellular permeability marker Lucifer yellow (LY) in the apical to basolateral direction in each well. Cytochrome P-450 Inhibition Analysis. Recombinant CYP450 proteins (baculosomes, Invitrogen) and fluorogenic substrates (Vivid, Invitrogen) were used in a fluorescent homogeneous assay. The concentrations of substrates used were below their Michaelis− Menten-constant values (Km) in a reaction with P450 isozymes, ensuring the detection of even weak CYP450 inhibitors. The assay was performed in triplicate in a black, low-volume 384-well plate, in 25 μL of the reaction mixtures according to the Thermo Fisher Scientific manual. The Z′ factor has been validated in the range of 0.66 to 0.82. The compounds were classified as follows: potent inhibitors, IC50 < 1 μM; moderate inhibitors, 1 μM < IC50 < 10 μM; and no or weak inhibitors, IC50 > 10 μM. Human ERG Assay (hERG). To determine the hERG channel binding affinities of the test compounds, a Predictor hERG assay kit (Thermo Fisher Scientific), which includes membrane preparations from Chinese hamster ovary cells stably transfected with the hERG potassium channel and a high-affinity red fluorescent hERG channel ligand (tracer), was used. Compounds binding to the hERG channel protein (competitors) were identified by their ability to displace the tracer, resulting in lower fluorescence polarization. The assays were performed according to the manufacturer’s protocol (Invitrogen). The final concentration of DMSO was 1%. Polarized fluorescence was read

was removed under vacuum. The crude was poured into water, acidified to pH 1 with 3 N HCl, and extracted with EtOAc (3 × 20 mL). The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The title compound (43) was obtained in an 89% yield (1.22 g, 7.6 mmol) as a white solid with a mp of 143−145 °C. 1H NMR (400 MHz, DMSO-d6): δ 2.40 (s, 3H), 7.39 (d, J = 7.5 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.8 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 21.3, 124.4, 127.7, 129.7, 132.2, 139.2, 155.7. Molecular Modeling Studies. The Maestro55 interface was used to build all the small molecules submitted to the in silico procedure. Protein complexes were retrieved by the Protein Databank and prepared using the default settings in the Protein Preparation Wizard tool of Schrodinger Suite 2017−1.56 All the protomers and tautomers of the screened compounds were assigned using the default settings of the Ligprep tool present in the same software package. To discard all the possible interfering compounds at an early stage of the hit identification process, all the small molecules were funneled through the PAINS filter57 present in the Canvas58 software package. The docking procedure consisted of generating a grid centered on the cocrystallized ACMSD−ligand and using the Glide59 tool in singleprecision (SP) mode to generate the putative binding pose. The bestranked molecules were then selected and biologically tested. The confirmed hits were further investigated in silico by applying an induced-fit procedure to obtain a more reliable binding pose through the relaxation of the binding site. In this step, all the amino acid side chains in within 6 Å of the ligand were left free to move. All the images produced in this paper were generated using Pymol software.60 hACMSD Preparation and Activity Assay. Recombinant hACMSD was expressed in Pichia pastoris and purified as previously described.39 Its enzyme activity was assayed by a coupled spectrophotometric assay.39 Briefly, in a pre-assay mixture, the ACMS substrate was generated from 10 μM 3-hydroxyanthranilic acid by recombinant 3-hydroxyanthranilate 3,4-dioxigenase from Ralstonia metallidurans. ACMS formation was monitored at 360 nm, and after the reaction was complete, an appropriate amount of ACMSD was added. Activity was calculated from the initial rate of the absorbance decrease subtracted from that of a control reaction mixture in the absence of ACMSD. The effects of the various compounds on the enzyme activity were tested by adding the compounds to the assay mixture along with ACMSD. For the IC50 evaluations for each compound, a serial dilution from a stock solution prepared in DMSO was tested, maintaining a DMSO concentration in all the reaction mixtures of 1.0%. One unit is defined as the amount of enzyme that consume 1 μmol of ACMS per minute at 37 °C. Kidney and liver extracts for the determination of ACMSD activity were prepared from CD1 mice dosed with 22 at 5 mg/kg using HPMC 0.5% (w/v) in water containing 5% (v/v) DMSO as the vehicle (for the tissues samples collected during the PK study, see the SI). After different times post the dosings, the animals were sacrificed humanely, and the organs were rapidly collected and snap-frozen in liquid nitrogen. Samples were ground in liquid nitrogen, and 100 mg of N2-powders were added with 0.3 mL of 50 mM tris buffer, pH 7.5, containing 0.15 M NaCl, 1 mM phenylmethylsulfonyl fluoride, and 0.02 mg/mL each of leupeptine, antipain, aprotinin, chymostatin, and pepstatin. Suspensions were homogenized and centrifuged at 38 000g for 20 min at 4 °C. Ten microliters of the supernatants were immediately assayed for ACMSD activity, as described above, in the presence of 30 μM 3-hydroxyanthranilic acid in a final assay-mixture volume of 500 μL. The activities were referenced to the protein concentrations measured in the supernatants (BCA, Pierce). NAD+ Determination in Human Hepatocytes and Mouse Liver Extracts. Human primary hepatocytes were cultured according to the manufacturer’s protocol (Biopredic International). Cells (2 ×104) were seeded in 96-well collagen-coated plates, and after an overnight incubation, they were stimulated with compounds 21 and 22. After 48 h of treatment, NAD+ levels were measured using the NAD/NADH-Glo Assay kit (Promega), according to manufacturer’s manual. The experiment was performed in quadruplicate. The statistical analysis was performed by a one-way ANOVA with 756

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with the use of an EnVision microplate analyzer (PerkinElmer Life and AnalyticalSciences). The Z′ value for E-4031 was 0.68, indicating a robust and good assay. Cytotoxicity. Cell viability was evaluated by measuring ATP levels using CellTiter-Glo (Promega), according to the manufacturer’s instructions. Tamoxifen (Sigma-Aldrich) was used as a control. Cell necrosis was evaluated by measuring the release of lactate dehydrogenase (LDH) from the necrotic cells using CytoTox-ONE, a homogeneous membrane-integrity assay (Promega), according to the manufacturer’s instructions. 5 × 103 HepG2 and AML12 cells (ATCC) were stimulated with the indicated concentrations of the test compounds in a white 384-well microplate for 4 h at 37 °C. Luminescence and fluorescence were read with a Victor V Multiplate Reader (PerkinElmer Life and Analytical Sciences). Selectivity. Serial dilutions (eight points) of compounds 21 and 22 and the reference standard were performed to obtain a nonlinear regression curve to assess the activity on a panel of nuclear receptors: PXR, PPARs, LXR, VDR, GR, RXR, FXR, TR. The assay was based on Alpha Technology (PerkinElmer). Off-Target Safety Panel. The GPCR, ion channel, kinase, and phosphatase assays were performed as described in the Eurofins GPCRProfiler, IonChannelProfiler, KinaseProfiler, and PhosphataseProfiler Services, (http://www.eurofins.com). The full data are included in Tables 1S−3S of the SI Pharmacokinetical Analysis. The pharmacokinetic (PK) analyses on compounds 21 and 22, via oral administration (PO) and intravenous injections (IV) were conducted at Aptuit (Verona, Italy, http://www.aptuit.com/) following relevant national and international guidelines according to Public Health Service Policy on Human Care and the Use of Laboratory Animals under Italian Legislative Decree no. 26/2014 and European Directive no. 2010/63/UE. The IV formulation was made in saline water containing 5% (v/v) DMSO and 20% (v/v) PEG400. The PO formulation was made in HPMC 0.5% (w/v) in water containing 5% (v/v) DMSO. PK experimental details and data tables have been included in the Supporting Information.

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dioxygenase-1; IDO2,indoleamine 2,3-dioxygenase-2; KMO,kynurenine 3-monooxygenase; KATI,kynurenine aminotransferase I; KATII,kynurenine aminotransferase II; CNS,central nervous system; ACMS,2-amino 3-carboxymuconate 6-semialdehyde; AMS,2-aminomuconate 6-semialdehyde; PIC,picolinic acid; TCA,tricarboxylic acid; QUIN,quinolinic acid; MEHP,mono(2-ethylhexyl)phthalate; DHAP,1,3-dihydroxyacetonephosphate; PDC,pyridine-2,6-dicarboxylic acid.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01254. Inhibition assays, experimental details of the pharmacokinetic analysis, mean pharmacokinetic parameters (PDF) Molecular formula string (CSV)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +39 075 6978111. E-mail: [email protected] (R.P.). *Tel.: +41 21 69 30951. E-mail: johan.auwerx@epfl.ch (J.A.). ORCID

Nicola Giacchè: 0000-0002-4810-3960 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is partially supported by grants from the École Polytechnique Fédérale de Lausanne and the Swiss National Science Foundation (31003A-140780).

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ABBREVIATIONS USED ACMSD,α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase; NAD+,nicotinamide adenine dinucleotide; Trp,tryptophan; KP,kynurenine pathway; IDO1,indoleamine 2,3757

DOI: 10.1021/acs.jmedchem.7b01254 J. Med. Chem. 2018, 61, 745−759

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