Identification of potent triazolylpyridine nicotinamide phosphoribosyltransferase (NAMPT) inhibitors bearing a 1,2,3-triazole tail group

European Journal of Medicinal Chemistry 181 (2019) 111576

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Identification of potent triazolylpyridine nicotinamide phosphoribosyltransferase (NAMPT) inhibitors bearing a 1,2,3-triazole tail group Cristina Travelli a, b, 1, Silvio Aprile a, 1, Daiana Mattoteia a, Giorgia Colombo a, Nausicaa Clemente c, Eugenio Scanziani d, e, Salvatore Terrazzino a, Maria Alessandra Alisi f, Lorenzo Polenzani f, Giorgio Grosa a, Armando A. Genazzani a, Gian Cesare Tron a, Ubaldina Galli a, *
del Piemonte Orientale, Largo Donegani 2, 28100, Novara, Italy Dipartimento di Scienze del Farmaco, Universita
degli Studi di Pavia, Viale Taramelli 12, 27100, Pavia, Italy Dipartimento di Scienze del Farmaco, Universita c
degli Studi del Piemonte Orientale, Via Solaroli 17, 28100, Novara, Italy Dipartimento di Scienze della Salute and IRCAD, Universita d
degli Studi di Milano, Via Celoria 10, 20133, Milano, Italy Dipartimento di Medicina Veterinaria, Universita e
degli Studi di Milano, Viale Ortles 22/4, 20139, Milano, Italy Mouse and Animal Pathology Lab (MAPLab), Fondazione Universita f Angelini RR&D (Research, Regulatory & Development), Angelini S.p.A, Piazzale della Stazione Snc, 00071, S. Palomba, Roma, Italy a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 June 2019 Received in revised form 29 July 2019 Accepted 30 July 2019 Available online 1 August 2019

The enzyme nicotinamide phosphoribosyltransferase is both a key intracellular enzyme for NAD biosynthesis (iNAMPT) and an extracellular cytokine (eNAMPT). The relationship between this latter role and the catalytic activity of the enzyme is at present unknown. With the intent of discovering inhibitors specifically able to target eNAMPT, we increased the polarity of MV78 (EC50 ¼ 5.8 nM; IC50 ¼ 3.1 nM), a NAMPT inhibitor previously discovered by us. The replacement of a phenyl ring with a 1,2,3-triazole bearing a protonable N,N-dialkyl methanamine group gave a series of molecules which maintained the inhibition of the enzymatic activity but were unable to cross the plasma membrane and affect cell viability in vitro. Compounds 30b and 30f can therefore be considered as the first experimental/pharmacological tools for scientists that wish to understand the role of the catalytic activity of eNAMPT. Serendipitously, we also discovered a compound (25) which, notwithstanding its high polarity, was able to cross the plasma membrane being cytotoxic, a potent NAMPT inhibitor and effective in reducing growth of triple negative mammary carcinoma in mice. In our hands, 25 lacked retinal and cardiac toxicity, although we observed a lesser toxicity of NAMPT inhibitors in general compared to other reports. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: NAD Nicotinamide phosphoribosyltransferase Inhibitors Cancer Click chemistry

1. Introduction

Abbreviations: DMF, N,N-dimethylformamide; t-BuOH, tert-butanol; EDCI, N-(3dimethylaminopropyl)-N0 -ethylcarbodimide hydrochloride; TMSN3, trimethylsilylazide; TMSA, trimethylsilylacetylene; HOBt, Hydroxybenzotriazole; EtOH, ethanol; MeOH, methanol; EtOAc, ethyl acetate; PE, petroleum ether; THF, tetrahydrofuran; Et2O, diethyl ether; DMSO, dimethylsulfoxide; DMAP, 4-dimethylaminopyridine; TEA, triethylamine; mCPBA, m-chloroperbenzoic acid; RLM, rat liver microsomes; HLM, human liver microsomes; PAK4, Serine/threonine-protein kinase 4; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. * Corresponding author. E-mail address: [email protected] (U. Galli). 1 C.T. and S.A. These authors contributed equally to this manuscript. https://doi.org/10.1016/j.ejmech.2019.111576 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that catalyses in cells the reaction between nicotinamide (1) and phosphoribosyl pyrophosphate (2) to form nicotinamide mononucleotide (NMN) (3), which is then further converted into NAD by nicotinamide mononucleotide adenylyl transferase (NMNAT) (Fig. 1) [1]. Over the last decades, NAMPT has been object of a number of medicinal chemistry programs, both from academia and industry, and a number of compounds have also entered clinical trials (FK866, teglarinad, KPT-9274) [2]. The rationale of targeting NAMPT in oncology is mainly given by the observation that cancer cells

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Fig. 1. The principal reaction catalysed by NAMPT.

over-express NAMPT [3]. Indeed, despite the presence of three pathways able to maintain NAD homeostasis in cells, namely i) the de novo pathway from tryptophan, ii) the salvage pathway from nicotinic acid, and iii) the salvage pathway from nicotinamide,1 the latter, in which NAMPT is the bottleneck enzyme, is by far the most important to sustain the tumultuous use and degradation of NAD in rapidly proliferating cells. An effect on myeloid-associated tumoral cells has also been postulated thereby also conferring immunotherapeutic potential to these compounds [4,5]. The same enzyme can also be released by a number of cell types [3]. This extracellular form (eNAMPT) has been shown to display pro-tumoral and pro-inflammatory activities [3e6], thereby making it an interesting target to evaluate. The mechanism of action by which eNAMPT exerts its effects is at present unclear. The demonstration that eNAMPT exists as a dimer [7], a necessary condition to display enzymatic activity, suggests that the extracellular protein can catalyse the formation of NMN. Although the availability of substrates is limited in the extracellular space [8], evidences also exist on a putative role of the catalytic activity in mediating the effects of eNAMPT [7e9]. For example, it has been shown that eNAMPT produced by differentiated adipocytes exhibits robust NMN production, higher than the intracellular form. Moreover, high concentrations of NMN circulate systemically in mouse plasma, revealing a possible functional mode of eNAMPT as an extracellular NAD biosynthetic enzyme [7]. On the other hand, eNAMPT activates several intracellular signaling pathways and elicits a number of effects that do not seem to correlate with the enzymatic activity, but may be reconducted to a more traditional cytokine-like role [3]. For example, a number of actions can be mimicked by the catalytically-dead mutated enzyme (H247E) [4]. At least two receptors have been postulated to mediate these effects, Toll-like receptor 4 (TLR4) and the CeC chemokine

receptor type 5 (CCR5), although conclusive evidence is still lacking [10,11]. eNAMPT is a putative druggable target both in cancer and in inflammation. Indeed, its levels are increased in serum of patients and, more importantly, they negatively correlate with disease outcome [2a,12]. To note, for example, that in several cancers with poor prognosis, levels of both intra- and extracellular NAMPT are deeply increased. On the other hand, rising levels of eNAMPT favor the inflammatory environment linked with carcinogenesis and appear to have an active role, favouring invasiveness and metastasis of tumours [13]. Although not all the effects mediated by eNAMPT can derive from its enzymatic nature, it is evident that molecules able to inhibit NAMPT should possess anti-proliferative and antiinflammatory effects, becoming important novel anticancer candidates thanks to their dual role in inhibiting intra and extracellular NAMPT, reducing the levels of intracellular NAD, and downregulating the inflammatory milieu [5,14]. Starting from FK866, the first NAMPT inhibitor discovered [15], several other NAMPT inhibitors have been brought to the attention of the scientific community [2]. To date, two NAMPT inhibitors have discontinued clinical trials, FK866 [15] (4) and teglarinad, the water soluble pro-drug of CHS828 [16] (5), while a dual PAK4/NAMPT inhibitor KPT-9274 [17] (6) is currently being evaluated (Fig. 2). Drawbacks associated with NAMPT inhibitors, highlighted mainly in recent pre-clinical investigations, have somehow reduced the hype on this class of compounds. Namely, hematological effects [2,18], cardiotoxicity [19], and retinopathy [20], have been suggested to be on-target side effects of all NAMPT inhibitors, although not all of them have been evaluated for these parameters. Furthermore, most of the reported inhibitors suffer from other drawbacks, including CYP inhibition [21] and poor

Fig. 2. NAMPT inhibitors advanced in clinical trials.

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pharmacokinetic properties (e.g. solubility) [22]. For this reason, the hunt for nanomolar NAMPT inhibitors with less side effects and a novel profile of eNAMPT vs iNAMPT selectivity is still open and welcomed. Our group, about eight years ago, reported the discovery of triazolylpyridines as potent NAMPT inhibitors exemplified by GPP78 (7) and MV78 (8) (Fig. 3). This class of inhibitors is characterized by a strong inhibitory activity on the enzyme (IC50 between 3 and 30 nM), the ability to deplete intracellular NAD at similar concentrations, and an effect on animal models of cancer and inflammation (e.g. inflammatory bowel disease) [23e25]. The modularity of our chemical approach along with the use of the archetypical click chemistry reaction, the Sharpless-Fokin 1,2,3-triazole synthesis [26], allowed us to prepare and validate more than three hundred compounds giving us a good knowledge of the structure activity relationship of triazolylpyridines and providing us with a platform to improve on our hit compounds. All NAMPT inhibitors characterized to date, including ours, are likely to inhibit both extracellular and intracellular NAMPT. Yet, the literature would benefit from inhibitors that solely target eNAMPT, as this would help understand the role of the catalytic activity in this latter form. Given the evidence, it could be envisaged that these molecules might have a therapeutic role, but this can only be confirmed when the appropriate experimental/pharmacological tools become available. For this reason, starting from the triazolylpyridines discovered by us, we attempted to design a series of novel inhibitors unable to cross the plasma membrane, being therefore able to act only against eNAMPT. At the beginning, we reasoned that the pyridine N-oxide of 8, the most druggable inhibitor in our hands, might be a good candidate due to its polar nature. The compound was easily prepared reacting MV78 (8) in the presence of mCPBA in dichloromethane affording the resulting N-oxide (9) in 91% yield [27] (Scheme 1). As expected the

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compound was devoid of any cytotoxic action but unfortunately was also inactive in the enzymatic test (see Biological Evaluation). We next tried i) to improve polarity of our molecules adding polar functional groups to the ring A of MV78 (8) and ii) to experiment a novel type of isosterism, replacing the key phenyl ring (A) of the biphenyl structure with a 1,2,3-triazole as in theory it might act as a non-classical isostere of the phenyl ring maintaining the planarity, the aromaticity, the ability to participate in p-p interactions, but imparting an increased polarity (Fig. 4) due to its high dipole moment (ca 5.0 Debye) [28]. Alongside, given that preliminary data (reported in the next sections) showed that a quinone metabolite was formed during the metabolism of MV78 (8) and this was potentially genotoxic, this replacement would have partially bypassed this problem. 2. Results and discussion 2.1. Chemistry A carboxylic acid at the ortho, meta and para-position (13, 19, 20), and an amide (14) were added on the phenyl ring A or a pyridine (21) replaced the benzene moiety according to the synthetic Scheme 2. Starting from intermediate 10, already prepared in a previous work [25], the amine was coupled with 11 in the presence of condensing agent EDCI to afford the desired amide 12 in 91% yield. Hydrolysis of the ester group afforded the carboxylic acid derivative 13 in 84%. This functional group was converted into an amide by reacting 13 in the presence of ammonium chloride, EDCI, Nmethylmorpholine and HOBt to afford compound 14 in 44% yield. Intermediate 10 was then coupled with 2-iodobenzoic acid to give amide 15, which was reacted with three different boronic acids (16, 17, 18) under standard Suzuki-Miyaura conditions to afford in discrete yields the final three compounds (19, 20, 21) of this series.

Fig. 3. Potent NAMPT inhibitors containing a triazolylpyridine moiety.

Scheme 1. Synthesis of the pyridine N-oxide (9) of MV78 (8).

Fig. 4. A novel attempted form of isosterism.

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Scheme 2. Synthetic route for the preparation of compounds 13, 14, 19, 20 and 21.

The two triazole isomers 25 and 28 were therefore synthesized as reported in Schemes 3 and 4 respectively. Starting from intermediate 10, the amine was coupled with 2-azido benzoic acid (22) [29] in the presence of condensing agent EDCI to afford the desired amide 23 in 68% yield. The azide was then reacted with trimethylsilylacetylene (TMSA) in the presence of copper (I) iodide by heating a solution of DMF/MeOH 1:1 at 100  C. Under these conditions we obtained the desired 1,2,3-triazole ring (24) in 68% yield. Finally, removal of the silyl group was accomplished in the presence of TBAF to afford compound 25 in 81% yield (Scheme 3). The synthesis of the other regioisomer commenced with a

Sonogashira reaction between compound 15 and trimethylsilylacetylene to afford the protected alkyne 26 in 70% yield. Removal of the silyl group afforded the terminal alkyne 27 in 61% yield, which was converted in 1,2,3-triazole 28 by reaction with trimethylsilylazide (TMSN3) in the presence of copper (I) iodide. To note that this reaction was rather capricious and after several attempts we were able to obtain 28 in only 9% yield (Scheme 4). Since 25 was around 3 times more potent than 28 (see Biological Evaluation), to further increase the polarity of the molecules, we opted to synthesize an extra series of derivatives having a 4substituted 1,2,3-triazole containing an aliphatic tertiary amine

Scheme 3. Synthetic route for the preparation of compound 25.

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Scheme 4. Synthetic route for the preparation of compound 28.

according to Scheme 5. Fig. 5 shows the N,N-dialkylpropynamines used. Apart compound 29c, which was commercially available, the others were easily prepared from reaction with propargyl bromide with the corresponding secondary amine in the presence of anhydrous K2CO3 according to the literature methods [30]. The final compounds 30a-i were prepared using classical Sharpless-Fokin reaction conditions (CuSO4ˑ5H2O, sodium ascorbate in t-BuOH-water) affording the required compounds in a range of yield of 54e84%. 2.2. Biological evaluation To evaluate the activity of our compounds, we estimated i) the cytotoxicity of our compounds on a cell line that we have previously shown to be highly sensitive to NAMPT inhibitors (SH-SY5Y, neuroblastoma) [25] and ii) the enzymatic inhibition of recombinant NAMPT in an enzymatic cell-free assay. A NAMPT inhibitor freely diffusible across membranes should be able to display EC50s for cytotoxicity in the same range as the IC50 for enzymatic inhibition. Indeed, our reference compounds for these assays, FK866 (4) and MV78 (8), display these characteristics. Conversely, an inhibitor that does not cross the plasma membranes should not be cytotoxic. Most of the compounds displayed a pattern compatible with a hampered membrane crossing (Table 1). The assays also provided new clues on the SAR. First, the hypothesis of substituting the phenyl ring A with a 1,2,3-triazole was successful; indeed, both the two regioisomers 25 and 28 were

active. Furthermore, the triazole can be substituted at the 4 position, with a N,N-dialkyl-methanamine group. Furthermore, activity on enzyme inhibition on phenyl ring A is retained only when it is substituted on the ortho-position (13 vs 19, 20). As postulated, the insertion of N,N-dialkyl-methanamine group tail group reduced the ability of compounds to cross the plasma membrane (30a, 30b, 30c, 30d, 30e, 30f, 30g, 30i). We can postulate that 30h, that displays similar EC50s and IC50s, undergoes an easy ester hydrolysis in the medium of cultured cells generating a zwitterion species again lipophilic enough to cross the cell membrane [31]. Compounds 30b and 30f showed a selectivity of at least 200-fold between enzymatic activity and cytotoxicity (Fig. 6A) that well fit with the inability of the compounds to cross membranes and, at the same time, to inhibit the enzymatic activity of NAMPT. In order to prove unequivocally the inability to cross the cell membrane for compounds 30b and 30f, we first investigated the ability of 30b and 30f to reduce NAD levels in cells. As shown in Fig. 6B, compound 30b has a moderate effect in reducing NAD levels at 16 h at 100 nM (30%), on the contrary compound 30f is devoid of any effect. Importantly, the IC50 of compound 4 in reducing NAD levels is in the low nanomolar range and therefore there is a two order of magnitude difference compared to similar IC50 values in the enzymatic test. To further prove the inability of compounds 30b and 30f to cross the cell membrane, we carried out a Caco-2 permeability assay (see supporting information for details). As reported in Fig. 6C, compound 4 and compound 25 were able to cross the Caco-2 monolayer, albeit at a lesser extent compared to the positive control (terbutaline). On the contrary neither 30b nor 30f

Scheme 5. General route for the synthesis of triazolylpyridines 30a-i containing an N,N-dialkyl-(1H-1,2,3-triazol-4-yl)methanamine tail group.

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Fig. 5. N,N-dialkylpropyn amines used to click compound 23.

Table 1 EC50 Values for Cytotoxicity in SH-SY5Y Cells treated for 48 h and IC50 Values for in vitro NAMPT Enzymatic Assay. Left column, Values of cytotoxicity (% of control) in SHSY5Y treated with 1 mM for 48 h; Middle column, EC50 were calculated with concentrationeresponse curves and using Kaleidagraph software. Shown are mean percentages ± SD of three independent experiments (n ¼ 12), Right column, IC50 were calculated with concentrationeresponse curves and using Kaleidagraph software. Shown are mean percentages ± SD of three independent experiments (n ¼ 6). nd ¼ not determined. * data of EC50 and IC50 already published25; # EC50 could not be calculated as compounds display a biphasic cytotoxicity pattern;  EC50 greater the 10 mM (cytotoxicity at 10 mM: 64.1 ± 6.2 for 30g; 61.9 ± 5.8 for 30e). Compound

SH-SY5Y cells viability (%) at 1 mM

SH-SY5Y cells viability EC50 [nM]

NAMPT inhibition IC50 [nM]

4 8* 9 13 14 19 20 21 25 28 30a# 30b 30c 30d# 30e 30f 30g 30h 30i#

9.7 ± 0.4 23.5 ± 2.8 103.2 ± 3.8 80.9 ± 5.4 65.7 ± 2.8 78.4 ± 1.9 22.5 ± 3.8 15.1 ± 3.2 10.5 ± 2.1 23.5 ± 4.3 37.8 ± 4.1 91.7 ± 3.6 86.6 ± 0.4 15.9 ± 2.1 88.9 ± 0.5 87.7 ± 5.1 80.2 ± 3.2 14.1 ± 2.8 30.0 ± 4.2

2.5 ± 0.4 5.8 ± 0.2 n.d. >500 289.4 ± 23.4 >500 224.0 ± 81.1 36.5 ± 5.6 35.4 ± 10.4 86.5 ± 7.5 >500 nM n.d. 285.2 ± 16.9 >500 nM n.d. n.d. n.d. 61.5 ± 10.6 >500 nM

2.7 ± 0.4 3.1 ± 4.1 >500 38.5 ± 6.4 87.9 ± 10.6 >500 >500 28.5 ± 12.7 3.4 ± 1.2 42.5 ± 8.4 73.8 ± 25.7 41.8 ± 4.1 42.4 ± 31.3 17.1 ± 5.0 25.4 ± 4.4 13.6 ± 2.9 24.7 ± 2.9 20.6 ± 4.1 49.6 ± 9.7

were not recovered on the basal side in the same experimental conditions. After these experiments, we feel that these two compounds could represent experimental tools to unravel the role of the enzymatic activity in mediating the functions of eNAMPT. To some surprise, notwithstanding its high polarity, compound 25 displayed properties (i.e. high potency, high membrane crossing) that were in line with traditional anti-tumoral NAMPT inhibitors, and indeed this was confirmed by its ability to cross the cell membrane in Caco-2 cells. Yet this compound caught our interest as its increased polarity should improve its solubility, one of the drawbacks of previously reported inhibitors. Given that iNAMPT inhibitors display other drawbacks, including cardiac and retinal toxicity, we therefore decided to fully characterize this compound. Previously unpublished genotoxicity experiments of 8 gave us another reason to explore compound 25. Indeed, when genotoxicity was evaluated in a mammalian cell-based assay 8 proved not be genotoxic, but its S9 metabolic activation mixture unravelled a genotoxicity with a lowest effective concentration of 125 mM, suggesting that a poorly-forming metabolite was genotoxic. When evaluating genotoxicity of 25, we were unable to unravel genotoxicity of the compound itself (up to 500 mM) or of the S9 metabolic activation mixture (up to 1 mM). In light of the revealed S9 bioactivation of 8, the putative reason of the favourable genotoxicity profile of 25 could be attributable to its improved metabolic stability (residual substrate 70% RLM, 94% HLM) compared to that

of 8 (45% RLM, 88% HLM) as the metabolic transformations remain unchanged. Indeed, the oxidative metabolic pathways were aliphatic hydroxylation of the paraffin linker, pyridine N-oxidation, and oxidative N-dealkylation (see Supporting Information). Moreover, given the presence of the biphenyl moiety, 8 could be prone to microsomal oxidation forming reactive quinone metabolites [32]. Indeed, albeit at low levels compared to pyridine N-oxidation [25], we were able to demonstrate in vitro this behaviour by detecting and characterizing by mass spectrometry the corresponding GSH hydroquinone adducts 8-GSH (see Supporting Information). Although genotoxicity is not necessarily a concern in anti-tumoural drugs, it should be noticed that NAMPT inhibitors have also been postulated as anti-inflammatory agents [10,25]. We next validated our hypothesis on solubility comparing compounds 4, 8, and 25 using the Millipore MultiScreen® kinetic solubility test (see Supporting Information for full details). For both 4 and 8 it was only possible to perform the test at 200 mM thus indicating their limited aqueous solubility while 25 gave concentration values similar to those of the calibrators (200 and 500 mM), demonstrating its aqueous solubility in this range. Thermodynamic solubility was also determined for 8 and 25 using the saturation shake flask method (see Supporting Information for full method). 25 resulted more than one order of magnitude more soluble in water compared to 8, being the concentrations of the saturated solutions 1.0 and 0.06 mM respectively. We also performed kinetic

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Fig. 6. Characterization of 30b and 30f. (A) Concentrationeresponse curves in SH-SY5Y cells and in vitro inhibition of NAMPT enzymatic activity. Shown are mean percentages ± SD of two independent experiments (n ¼ 8 and 6, respectively). (B) % of NAD level reduction after 16 h of treatment with compound 4, compound 30b and 30f (100 nM) in SH-SY5Y cells. Data are mean percentages ± SD of two independent experiments (n ¼ 6). (C) % of apical-to-basal permeability and Papp of compounds 4, 25, 30b, 30f and terbutaline, n ¼ 3.

solubility tests for 30a-i, and all the compounds gave concentration values similar to those of the calibrators (200 and 500 mM), confirming their aqueous solubility in this range (see Supporting Information). Next, we proceeded evaluating the pharmacokinetic parameters of 25 in mice. Briefly, mice were injected with compound 25 (i.v. 10 mg/kg, once) and serial blood sampling was performed. 25 showed a half-life of 3.5 h, with a clearance of 103 mL/ min/Kg and a volume of distribution of 31.3 L/Kg (Table 2). Given that retinotoxicity and cardiotoxicity have been reported as important on-target effects, we evaluated the effect of 25 compared to 4 and 5 in the relevant histological sections of treated animals. 4 and 25 were given IP at a concentration of 100 mg/kg while 5 was given by oral gavage (200 mg/kg). As shown in Fig. 7, we were unable to disclose any microscopic evidence of toxicity with any of the three compounds on the retina. It should be noticed that the dose and length of treatment used for 4 was similar to that used in the manuscript that initially reported this on-target toxicity [19,20]. We cannot exclude that the plasma exposure of the compounds is different in our experimental setting compared to the previous report given that the retinal toxicity has been well documented in preclinical models and has led ERG and visual activity monitoring to be included in phase I trial with NAMPT inhibitors [2c,33]. Unfortunately we were unable to test this hypothesis.

Table 2 Pharmacokinetic parameters of compounds 4 and 25. PK parameters

Compound 4

Compound 25

T1/2 (h) AUC (mg  h/L) CL (ml/min/kg) Vd (L/kg)

0.37 6466 22.8 0.8

3.51 1616 103.1 31.3

A recent paper [21] has shown CYP2C9 inhibition for NAMPT inhibitors which contain an exposed pyridine or a related heterocycle with an exposed and nucleophilic nitrogen atom. Unfortunately, these exposed and nucleophilic groups are a necessary pharmacophore for potent NAMPT cell-based inhibition and, while we have not explored this aspect, it is likely that our compounds might have similar less-then-ideal characteristics. In the heart, 4 elicited a modest histological disruption characterized by hyper-eosinophilia and an increased vacuolation of the cytoplasm of cardiomyocytes. No effect was observed in interstitial expansion. These data of compound 4 are in agreement with previous data [34]. Neither 5 nor 25 elicited any disruption in cardiac tissue. In our hands, therefore, NAMPT inhibitors appears less cardiotoxic and retinotoxic compared to the previous report, as we have been able only to unravel cardiotoxicity for FK866 (4), the pioneering molecule of this class of compounds, and not with 25 or with CHS828 (5). Given that i) the half-life of this compound appears reasonable for clinical applications, that ii) it shows a better solubility compared to other compounds and that iii) it is not genotoxic, we decided to investigate the effect of 25 in an allograft model of triple-negative breast cancer (4T1 cells). Given that we have shown that the half-life of 4 is significantly lower (half-life of 0.37 h [25], and that we flagged this compound as potentially cardiotoxic (as evidenced also by others) [19], we decided to use compound 5 (that in humans shows a half-life of approximately 2.1 h, no data is publicly available in rodents). Furthermore, it should be noted that when 4 was used in clinical trials, administration was by continuous infusion, hardly ideal for a drug. The two compounds were used at the same dose (10 mg/kg/twice daily). Briefly, compounds were dosed 14 days after implantation of cancer cells in the mammary gland, when the tumours were palpable. This provides a true translational approach, as we evaluate the ability of

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Fig. 7. Compound 25 does not present retinal or cardiac toxicity (A). Representative microscopic images from rat retina and heart after administration of compounds 4, 5 or 25 (H&E; 20  ). (B) Heart toxicity was characterized by the % of area with hyper-eosinophilia (left panel) and with vacuolation (right panel, H&E; 20x).

the drugs to act on an already formed cancer. Both 5 and 25 were able to significantly reduce tumour growth, both when following tumour volume throughout the experiment (Fig. 8A) and when weighting the tumour mass at the end of the treatment (24 days; Fig. 8B). Yet, 25 proved to be more efficacious. Similarly, the use of either of the two NAMPT inhibitors significantly reduced metastases formation (Fig. 8C). 3. Conclusions In the present manuscript, we describe a strategy to synthesize NAMPT inhibitors that can discriminate between the extracellular and intracellular form that might represent experimental/pharmacological tools for scientists that wish to understand the role of

the catalytic activity of eNAMPT in cell biology. Two compounds, 30b and 30f, have at least a 200-fold selectivity for the extracellular form based on their inability to cross freely the plasma membrane. It should be noticed that literature on NAMPT inhibitors has expanded in recent years and the initial screening for these compounds was usually on cell-based assays. Therefore, it is possible that other eNAMPT-selective inhibitors have been synthesized previously but have not been described as such. In only one case Authors have correlated the lack of cytotoxicity associated with a strong enzymatic inhibitory activity for permeable inhibitors with the lack of intracellular phosphoribosylation which prevented the reverse crossing of the plasma membrane and gives a tighter binding to the enzyme [35]. Last, a very recent paper has shown that NAMPT inhibitors can be coupled to antibodies to provide a

Fig. 8. Inhibition of mammary carcinoma growth in compound 25-treated mice. When tumours were palpable (at day 14), mice were randomized into vehicle and treated groups (4 mice/group). Compound 5 and 25 were administered intraperitoneally (10 mg/kg) twice a day for 12 consecutive days. Data shown are mean percentage ± of two independent experiments.

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payload for anticancer therapy [36]. It would be interesting to speculate that the use of cell-impermeant inhibitors might be even a safer manner to deliver NAMPT inhibition to cancer cells after internalization as their leakage from the antibody in the circulation should not have any cytotoxic effect. While in the process of developing the above inhibitors, we also synthesized a traditional NAMPT inhibitor that displays a significantly higher solubility, at least compared to others we had previously described. Compound 25 is not genotoxic, in our hands it does not present retinal or cardiac toxicity (although our model showed a lesser toxicity of the reference products compared to what reported previously) and is effective in reducing growth of triple negative mammary carcinoma in mice. Conflicts of interest The authors declare no competing financial interest. M.A.A. and L.P. are employees of Angelini Farmaceutici. Author contributions G.C.T., U.G., M.A.A. designed the compounds. U.G., D.M. synthesized the compounds. S.A., G.G., S.T., performed pharmacokinetic and metabolism studies. C.T., G.C., N$C., E.S. performed the biological assays. A.A.G., C.T., E.S., L.P. interpreted the biological assays. G.C.T. and A.A.G. wrote the manuscript. Acknowledgments We acknowledge funding support from the following sources: project code 14832 (Leonino Fontana and Maria Lionello FIRC fellowship to CT), AIRC grant (IG21842 to AAG), Fondazione Umberto Veronesi (Fellowship Grant 2018 to CT). This work was partly supported by Angelini S.p.A.

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[4]

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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111576.

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