Development of peptidomimetic boronates as proteasome inhibitors

European Journal of Medicinal Chemistry 64 (2013) 23e34

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

Original article

Development of peptidomimetic boronates as proteasome inhibitors Nicola Micale a, *, Roberta Ettari b, Antonio Lavecchia c, Carmen Di Giovanni c, Kety Scarbaci a, Valeria Troiano a, Silvana Grasso a, Ettore Novellino c, Tanja Schirmeister d, Maria Zappalà a a

Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy c Dipartimento di Farmacia, “Drug Discovery” Laboratory, Università degli Studi di Napoli Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy d Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, D-55099 Mainz, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2012 Received in revised form 1 March 2013 Accepted 20 March 2013 Available online 27 March 2013

Proteasome inhibition has emerged over the past decade as an effective therapeutic approach for the treatment of hematologic malignancies. It is a multicatalytic complex, whose proteolytic activity relies in three types of subunits: chymotrypsin-like (b5), trypsin-like (b2) and caspase-like (b1). Most important for the development of effective antitumor agents is the inhibition of the b5 subunits. In this context, the dipeptide boronate bortezomib (VelcadeÒ) represents the first proteasome inhibitor approved by the FDA and the lead compound in drug discovery. This paper describes the synthesis and biological evaluation of a series of conformationally constrained pseudopeptide boronates (1e3) structurally related to bortezomib. The synthesized compounds showed a promising inhibitory profile by blocking primarily the chymotrypsin-like activity of the proteasome with Ki values in submicromolar/micromolar range. These compounds also resulted quite selective since no significant inhibition was recorded in the test against bovine pancreatic a-chymotrypsin. The obtained results were rationalized by means of docking experiments based on a model of the crystal structure of bortezomib bound to the yeast 20S proteasome providing essential insights for further optimization of this class of inhibitors. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Peptidomimetic boronates Proteasome inhibitors Docking studies

1. Introduction The 26S proteasome is a multisubunit, multicatalytic threonine protease complex involved in different phases of cellular life-cycle. It is composed of a 20S catalytic core capped by two 19S regulatory complexes. Its proteolytic activity relies in three types of subunit (b1, b2 and b5) positioned into the barrel-like 20S core. These subunits possess chymotrypsin-like (ChT-L) (b5), trypsin-like (T-L) (b2), and post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (b1) activities, so named for the preferred substrate they hydrolyze. Targeting of eukaryotic proteins to this proteolytic system requires their prior marking by polyubiquitin chains. Proteasome is primarily responsible for the turnover of intracellular proteins that

Abbreviations: ChT-L, chymotrypsin-like; T-L, trypsin-like; PGPH, post-glutamyl peptide hydrolyzing; DMSO, dimethyl sulfoxide; DMF, dimethylformamide; Pd2(dba)3, tris(benzylideneacetone)dipalladium; EDC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; HOBt, N-hydroxybenzotriazole; DIPEA, N,Ndiisopropylethylamine. * Corresponding author. Tel.: þ39 090 6766419; fax: þ39 090 6766402. E-mail address: [email protected] (N. Micale). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.03.032

regulate cell proliferation and survival pathways. Thus, defects in the proteasome activity can lead to anarchic cell proliferation and tumor development. For these reasons, proteasome inhibition, in particular of the ChT-L activity, has become a significant strategy for drug development in cancer treatment [1e3]. In this context, we designed and synthesized conformationally constrained pseudopeptide boronates (1e3, Fig. 1) using bortezomib as lead compound, the first proteasome inhibitor approved by FDA in 2003 for the treatment of relapsed and refractory multiple myeloma [4] and mantle cell lymphoma [5]. These constrained surrogates should have in principle several advantages comparing to peptide inhibitors such as stability toward protease degradation, good cell permeability and target-selectivity by stabilizing a biologically active conformation [6]. This strategy has been accomplished by: i) maintaining the Leu-boronic electrophilic warhead at P1 due to its essentiality in binding interaction with the g-OH group of the N-terminal catalytic Thr; ii) substituting the Phe residue at P2 with less hindered residues such as Gly or Ala due to the fact that there is no much specificity assessed for this site; iii) enclosing the amide moiety at P3 in a 1H-pyridin-2-one ring to reduce the peptidic character of the compound as well as to reduce its

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Fig. 1. Structure of bortezomib and compounds 1e3.

conformational freedom; iv) maintaining a basic moiety corresponding to N4 of the pyrazine ring of bortezomib taking into account that the complex bortezomib-b5 subunit is stabilized through an interaction involving this nitrogen and the D114 residue of the adjacent b6 subunit [7]. Two series of compounds came out from our design. In the first series (i.e. 1aeg, Scheme 2), the constrained motif is represented by the sole 1H-pyridin-2-one scaffold bearing secondary or tertiary amines at C-4 (1beg) with the unsubstituted derivative 1a as reference compound to validate our hypothesis of the interaction with b6 subunit. In the second series [i.e. 2aec (Scheme 3) and 3 (Scheme 4)], the constrained motif is represented by a bicyclic 1,6naphthyridin-5(6H)-one scaffold.

2. Results and discussion 2.1. Chemistry The pyridone scaffolds 9aeg (Scheme 1) were synthesized by means of an initial nucleophilic aromatic substitution reaction of 2-chloropyridines (4e5) with benzyl alcohol in the presence of potassium hydride followed by debenzylation of the resulting Opyridyl benzyl ethers (6e7) under transfer hydrogenation conditions. The 1H-pyridin-2-one 9a was obtained directly by catalytic debenzylation of 2-benzyloxypyridine 6, whereas, for compounds 9beg, the debenzylation reaction was preceded by a microwavepromoted BuchwaldeHartwig amination at position 4 of 4chloro-2-benzyloxypyridine 7 in presence of Pd2(dba)3 as a catalyst, the DavePhos ligand and a suitable amine to give intermediates 8beg (Scheme 1), according to a procedure reported in literature [8]. The obtained 1H-pyridin-2-ones 9aeg were Nalkylated with ethyl bromoacetate in the presence of NaH to give esters 10aeg (Scheme 2), which were in turn smoothly converted into the corresponding acids 11aeg by alkaline hydrolysis with LiOH. Coupling reactions between acids 11aeg and pinanediol leucine boronate 12 [9] in the presence of EDC$HCl, HOBt and DIPEA gave the pinanediol esters 13aeg (Scheme 2), which were directly used for the successive trans-esterification reaction with isobutylboronic acid under acid conditions to provide the title boronic acids 1aeg. Synthesis of the boronic acids 2aec and 3 has been conducted with a similar approach starting from 1,6-naphthyridin-5(6H)-ones 14e15 and 1-methyl-1,2,3,4-tetrahydro-1,6-naphthyridin-5(6H)one 18 [10], as depicted in Schemes 3 and 4, respectively.

Scheme 1. Reagents and conditions: (a) BnOH, KH, THF, rt, 18 h, N2, 87e94%; (b) Pd2(dba)3, DavePhos, amine, t-BuONa, toluene, MW, 120e150  C, 15 min, N2, 65e88%; (c) H2, 10% Pd/C, MeOH/EtOAc (2:1), rt, 2 h, 95e99% (9bef) or BBr3, CH2Cl2, 78  C, 4 h, 99% (9g).

2.2. Biological activity The newly synthesized peptidomimetic boronates 1e3 have been tested for their inhibitory properties by using purified 20S proteasome isolated from human erythrocytes and the appropriate fluorogenic substrate for each of the proteolytic activities (i.e. SucLeu-Leu-Val-Tyr-AMC for ChT-L; Boc-Leu-Arg-Arg-AMC for T-L; ZLeu-Leu-Glu-AMC for PGPH). First, compounds 1e3 underwent a preliminary screening for ChT-L activity at 20 mM using an equivalent volume of dimethyl sulfoxide (DMSO) as a negative control. The screening showed that all compounds, with the exception of 1f, inhibited more than 40% of the enzyme activity. Continuous assays were then performed (progress curve method, at seven different concentrations, ranging from those that minimally inhibited to those that fully inhibited the enzyme, Fig. 2) to determine the Ki values (Fig. 3), reported in Table 1. As can be noticed, all compounds showed promising inhibitory properties toward the ChT-L activity of the proteasome, with Ki values in the submicromolar/micromolar range. The inhibitory activity toward the other two proteasome activities, as well as the selectivity of these compounds toward the target enzyme, by using bovine pancreatic a-chymotrypsin, was determined with the same method. None of these pseudopeptide boronates exhibited any inhibition in the T-L activity assay at 20 mM. Conversely, all of them but 1g inhibited the PGPH activity of the proteasome, although to a much lesser extent compared with the ChT-L activity inhibition (generally one or two order of magnitude), with the exception of 1c which is more potent in PGPH assay (Table 1). This outcome, per se, is extremely important since literature data suggest that the inhibition of only b5 subunits of the proteasome has a moderate effect on inhibition of protein degradation pathways [12], whereas the co-inhibition of b5 subunits with either b1 or b2 subunits exerts the maximal effect that is required to produce an antitumor response [13]. On the other hand, it is also known that the inhibition of all proteasome subunits is

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34

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Scheme 2. Reagents and conditions: (a) NaH, DMF, 0  C, 1 h; then ethyl bromoacetate, rt, 2 h, N2, 44e67%; (b) 1 N LiOH, EtOH, 0  C / rt, 4 h, 75e90%; (c) HOBt, CH2Cl2, 5  C, 20 min, then EDC$HCl, DIPEA, 12, 15  C / rt, 3 h, 83e99%; (d) iBuB(OH)2, 1 N HCl, MeOH/n-hexane (1:1), rt, 18 h, 30e50%.

generally cytotoxic [14]. The preliminary screening against achymotrypsin showed no significant inhibition for all compounds with the exception of 1e and 2b, which gave Ki values 12.7  7.1 and 9.16  7.53 mM, respectively. A survey of the results obtained for compounds 1aeg put in evidence that the 1H-pyridin-2-one nucleus as such is fundamental in determining the 20S proteasome inhibition. Indeed, 1a is one of the most active compounds of this series with a Ki ¼ 0.26 mM for the ChT-L activity and a Ki ¼ 1.38 mM for PGPH activity. The introduction of a tertiary amine at C-4 reduces the ChT-L and PGPH inhibition, in particular, the presence of a piperazine ring is decisively detrimental: no PGPH inhibition was detected with 1f and 1g, and only a fair ChT-L inhibition was recorded for 1g. Out of this trend lies the 4-(1-piperidinyl)-substituted derivative 1d which has an inhibition profile similar to that of 1a. The most relevant result for this series of compounds was obtained by introducing a secondary amine at C-4. As a matter of fact, the aniline derivative 1e maintained a micromolar inhibitory profile toward the PGPH activity of the 20S proteasome and reached nanomolar values of inhibition toward the ChT-L activity (Ki ¼ 0.098 mM). Compound 1e also showed a slight inhibition toward bovine pancreatic a-chymotrypsin (Ki ¼ 12.7 mM).

The condensation of a pyridine ring on the 1H-pyridin-2-one nucleus of 1a does not alter the inhibitory activity. In fact, the 1,6-naphthyridin-5(6H)-one 2a showed Ki values submicromolar and micromolar toward ChT-L and PGPH activities, respectively. By replacing the Gly residue at P2 site of 2a with an Ala residue (2b), both Ki values decreased of one order of magnitude and a comparable activity was also detected toward bovine pancreatic achymotrypsin (Ki ¼ 9.16 mM). An enhancement of the ChT-L inhibitory activity of 2a was achieved by introducing a bromine atom at C-8 of the 1,6-naphthyridin-5(6H)-one scaffold (i.e. 2c, Ki ¼ 0.17 mM). The presence of the bromine atom makes it possible to discriminate between b5 and b1 subunits of the proteasome with a difference of two orders of magnitude in Ki values. Differently, the N-methyl-tetrahydro derivative 3 showed a similar degree of inhibition of ChT-L and PGPH activities, both in a micromolar range. 2.3. Docking studies In order to help interpretation of biological activity data and to gain a better understanding of how the described peptidomimetic boronates might bind to the 20S proteasome, we carried out docking experiments of inhibitors 1aee and 2c into the binding

Scheme 3. Reagents and conditions: (a) NaH, DMF, 0  C, 1 h, then ethyl bromoacetate or methyl 2(R)-bromopropionate, rt, 2 h, N2, 34e65%; (b) 1 N LiOH, EtOH or MeOH, 0  C / rt, 6 h, 88e98%; (c) HOBt, CH2Cl2, 5  C, 20 min, then EDC$HCl, DIPEA, 12, 15  C / rt, 3 h, 70e100%; (d) iBuB(OH)2, 1 N HCl, MeOH/n-hexane (1:1), rt, 18 h, 32e34%.

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Scheme 4. Reagents and conditions: (a) NaH, DMF, 0  C, 1 h, then ethyl bromoacetate, rt, 2 h, N2, 78%; (b) 1 N LiOH, EtOH, 0  C / rt, 6 h, 95%. (c) HOBt, CH2Cl2, 5  C, 20 min, then EDC$HCl, DIPEA, 12, 15  C / rt, 3 h, 50%; (d) iBuB(OH)2, 1 N HCl, MeOH/n-hexane (1:1), rt, 18 h, 28%.

pocket of the b5 subunit of the previously reported crystal structure of bortezomib bound to the yeast 20S proteasome (PDB ID: 2F16) [7]. Because of the covalent binding of peptide boronic acids with proteasome, all the covalent docking simulations were performed by employing GOLD 5.1 [15,16] which has a unique function for handling covalent docking. The crystal structure of 20S/bortezomib complex revealed one well-defined water molecule in proximity to b6-D114, which coordinates a tight H-bonding network, interacting with b6-D114 Og, b5-A49 N and b5-A50 N of the protein and with the C]O oxygen of bortezomib [7]. Moreover, one of the pyrazine nitrogens of bortezomib interacted via a direct H-bond with the protonated b6-D114. It is to note that the pKa of a protonated pyrazine is <1 and thus presumably b6-D114 is protonated, since in the X-ray structure the OeN distance is 2.9  A, indicative of a strong H-bond. Accordingly, the intervening water molecule and the proper protonation state of D114 were included in the docking experiments. The Goldscore-CS docking protocol [17], was adopted in this study. In this protocol, the poses obtained with the original Goldscore function are rescored and reranked with the GOLD implementation of the Chemscore function [17e21]. To test the validity of this protocol for the 20S proteasome system, the crystal structure of bortezomib was first docked back into the binding pocket of the b5 subunit. In this docking run, the 200 poses produced by GOLD resulted in only one prevailing cluster on the basis of their conformations. The top-ranked pose obtained after rescoring with Chemscore function closely resembled the cocrystallized conformation with a heavy atom root-mean-square deviation (rmsd) of 1.5  A. Orientation of the P1eP3 substituents of bortezomib in the docked conformation were similar to that

observed in the crystal structure. H-bonds with b5-T21, b5-G47, b5A49, and b6-D114 of proteasome were also reproduced in the docked structure. Thus, this docking protocol was considered to be suitable enough to be used in the prediction of the binding modes of 1aee and 2c into the 20S proteasome. When 1aee and 2c were docked within the b5 subunit binding pocket, a convergent binding mode was largely adopted. All these proteasome inhibitors were found to be in the same location as bortezomib in the crystal structure. In particular, compounds 1ae d and 2c assumed a folded conformation stabilized by an intramolecular H-bond involving the pyridone C]O oxygen and one of the OH groups of the boronic acid fragment. On the contrary, the most active compound 1e adopted an extended conformation similar to that observed crystallographically for bortezomib. Fig. 4 depicts the binding mode of the representative compounds 1a, 1e and 2c into the proteasome binding site and their overlay with bortezomib. As expected, in all docked compounds, the boron atom formed a covalent interaction with the nucleophilic oxygen of b5 Og-T1, whereas the two acidic boronate OH groups engaged H-bonds with the T1 NH2 group and the backbone C]O of b5-Y168. Backbone of compounds 1aed and 2c formed two H-bonds with residues G47 (3.1  A) and T21 (2.9  A) of b5 subunit (Fig. 4a,e), whereas compound 1e established H-bonds with residue A49 (2.9  A) and the protonated D114 (3.0  A) in the b6 subunit (Fig. 4c). This latter interaction is crucial to retain the ChT-L inhibitory activity of 1e and further validates our docking results. In addition, the isopropyl substituent of all inhibitors was found to project into the S1 pocket adopting the same spatial arrangement of the P1-leucine side chain of bortezomib.

1400

F

1,8

1200

1,6

1000

1,4 1,2

800

b

1

600

0,8

400

0,6 0,4

200 0

0,2 0

200

400

600

Time (Seconds) Fig. 2. Progress curves of substrate hydrolysis in the presence of the inhibitor 2c. F ¼ fluorescence units. Inhibitor concentrations (from top to bottom): 0, 0.1, 0.5, 1.0, 5.0, 10.0, 15.0, 20.0 mM.

0

0

2

4

6

8

10

12

14

16

18

20

I Fig. 3. The slopes of the progress curve (b) from Fig. 1 were plotted against the inhibitor concentrations and fitted to the 4 parameter IC50 equation. Ki was obtained from the equation Ki ¼ IC50/(1 þ [S] K1 m ).

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34 Table 1 Inhibition of ChT-L and PGPH proteasome activities by boron tripeptides 1aeg, 2aec and 3. Comp

ChT-L Ki (mM)a

PGPH Ki (mM)a

1a 1b 1c 1d 1e 1f 1g 2a 2b 2c 3 Bortezomib

0.26  0.06 0.80  0.11 1.45  0.04 0.30  0.05 0.098  0.005 30% inhibition at 20 mM 5.90  1.47 0.44  0.21 1.34  0.12 0.17  0.01 1.62  0.44 0.0006b

1.38 4.42 0.52 1.42 5.14 n.d. n.i. 1.55 13.6 17.6 3.90

n.d. ¼ not determined; n.i. ¼ no inhibition. a Values represent the mean of three independent determinations. b Data reported in Ref. [11].

    

0.39 2.09 0.36 0.46 1.10

   

0.41 2.9 0.8 0.06

27

As depicted in Fig. 4d, the 4-phenylamino substituent of the most potent inhibitor 1e projected toward the deep specificity S3 binding pocket and was in close contact with residues of the adjacent b6 subunit. This result is in agreement with biochemical investigations showing that the size and length of the P3 side chain is crucial for inhibitor potency [22,23]. The pyridinone ring of 1a (Fig. 4b) and the naphthyridinone moiety of 2c (Fig. 4f) pointed toward the S2 pocket, a large cavity able to accept space-demanding substituents, occupying the spatial position of the P2 phenylalanine side chain of bortezomib. These moieties established hydrophobic and pep stacking interactions with T21, S129 and Y168 side chains. Notably, the 8-Br atom of 2c was engaged in a halogen bond with the backbone C]O oxygen of G23. The distance between the bromine and oxygen atoms is 3.1  A, which is significantly shorter than the sum of the van der Waals’ radii of the two atoms, consistent with quantum chemical calculations on the nature of halogen bonds [24]. The halogen-bond angle (CeBr/O) is 132 . Although halogen bonds are considered

Fig. 4. Binding modes of compounds 1a (a, green), 1e (c, magenta) and 2c (e, yellow) into the b5 (cyan)/b6 (white) active site of 20S proteasome represented as a ribbon model (PDB ID: 2F16) [7]. Only amino acids located within 4  A of the bound ligand are displayed (white) and labeled. Key H-bonds between the inhibitors and the protein are shown as dashed black lines. An overlay of 1a (b, docked pose), 1e (d, docked pose) and 2c (f, docked pose) with bortezomib (purple, X-ray crystal pose) is shown in the b5/b6 active site of 20S proteasome displayed as Connolly surface. The defined water molecule forming tight H-bonds to the protein is shown as a red sphere. Specificity pockets S1eS4 are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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weak interactions in comparison to classical H-bonds, they benefit from a lower desolvation penalty and may very well contribute to inhibitor specificity [25]. The slight decrease in activity in going from 2c to 2a is consistent with loss of the halogen bond upon replacement of bromine with hydrogen. Similar binding modes were also found for 1bed, where the substituent at position 4 of the pyridinone ring fitted into the S2 pocket and formed van der Waals and hydrophobic interactions with T21, S129 and Y168 residues of proteasome. It was proposed that these three residues and the P2 moiety of the inhibitors are exposed to the water in the central cavity of the b ring and act as a “gateway” of the pocket. It is worth noting that the more polar 4-morpholino moiety in 1c does not improve the inhibition of 20S proteasome activity in comparison with the 4-diethylamine and 4-piperidine moieties in 1b and 1d, respectively (see Table 1). Besides the hydrophilic differences, another explanation could be depicted: the electron-withdrawing effect of the oxygen on the morpholine system tends to decrease the electron density at the nitrogen making it less basic. If we compare the activity and pKa of 4-amino substituents of 1b (diethylamine, pKa ¼ 10.8), 1c (morpholine pKa ¼ 8.50), and 1d (piperidine pKa ¼ 11.1), it is observed that piperidine and diethylamine remain protonated up to physiologic pH. This could result in the formation of a cationep interaction with the phenyl ring of Y168, thus rationalizing the difference in potency of 1b and 1d compared to 1c. The obtained biological data and the consequent docking studies clearly indicate that the flexibility of these pseudopeptides at P2 constitutes the main issue on which future research efforts must be focused. As we noticed, in most cases our compounds adopted a folded conformation that flip the constrained scaffold to the S2 pocket instead of the originally assumed S3 pocket. One possibility to prevent the folding is that of introducing bulky hydrophobic residues at P2 that might fit with S2 pocket and obstacle the intramolecular H-bond formation (C]O/HOeB) at the same time. In this situation the constrained scaffold should be projected toward the intended S3 pocket as a consequence. Alternatively, the Gly residue at P2 of these compounds might be replaced with rigid synthons that likewise should impede the folding. 3. Conclusions In conclusion, we have identified two series of conformationally constrained analogs of bortezomib that efficiently inhibit two out of three of the proteolytic activities of the proteasome (mainly the ChT-L activity) and showed a good target-selectivity. In regard to the first series having a 1H-pyridin-2-one ring as a constrained motif, the 4-aniline-derivative 1e turned out to be the most active with a Ki value for ChT-L activity of proteasome in nanomolar range. With the support of docking studies we found out that 1e adopted an extended conformation within the binding pocket of b5 subunit (as for bortezomib) with the amine moiety interacting with the D114 residue of the adjacent b6 subunit, according to our initial design. This fact may explain its specificity toward the ChT-L activity of proteasome, that is two orders of magnitude higher with respect the PGPH activity. The other compounds of this series (1be g) as well as compounds of the 1,6-naphthyridin-5(6H)-one series (2aec and 3), although maintain a good inhibitory profile and target-selectivity, do not show a high subunit specificity due to the fact that they adopt a folded conformation within the binding pocket of the b5 subunit stabilized by an intramolecular H-bond involving the carbonyl oxygen at C2 of the pyridone ring and one of the OH groups of the boronic acid warhead. Only the C8eBr derivative 2c showed considerable subunit specificity (two orders of magnitude) due to additional interactions provided by the bromine atom. Therefore, 1e and 2c will be taken into account as new lead structures for further design of proteasome inhibitors.

4. Experimental protocols 4.1. Chemistry All reagents and solvents were obtained from commercial suppliers and were used without further purification. Reactions under microwave irradiation were performed on a CEM Discover apparatus. Elemental analyses were carried out on a C. Erba Model 1106 (Elemental Analyzer for C, H and N) and the obtained results are within 0.4% of the theoretical values. Merck Silica Gel 60 F254 plates were used as analytical TLC; flash column chromatography was performed on Merck Silica Gel (200e400 mesh). 1H and 13C NMR spectra were recorded on a Varian Gemini 300 MHz spectrometer using the residual signal of the deuterated solvent as internal standard. Splitting patterns are described as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multiplet (m) and broad singlet (bs). 1H and 13C chemical shifts are expressed in d (ppm) and coupling constants (J) in hertz (Hz). MS analyses were performed on a Varian 320-MS triple quadrupole mass spectrometer equipped with an electron spray ionization (ESI) source. 4.1.1. Synthesis of 1H-pyridin-2-ones 9aeg Compounds 9a [26], 9c, 9d, and 9f [8], were synthesized as reported in the literature. 4.1.2. 2-Benzyloxy-4-chloro-pyridine (7) A suspension of KH (1.08 g, 27 mmol) in dry THF (80 mL) at 0  C under N2, was treated with benzyl alcohol (1.7 mL, 16.2 mmol) and stirred for 1 h. Then, a solution of 2,4-dichloropyridine 5 (1.5 mL, 13.5 mmol) in THF (5 mL) was added and the mixture was stirred at room temperature for 18 h. The reaction was quenched with saturated NH4Cl (20 mL) and the organic solvent was removed in vacuo. The product was extracted from the residual aqueous layer with CH2Cl2 (3  50 mL), and the combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (light petroleum/ Et2O 8:2) to yield compound 7 as a pale yellow oil (2.58 g, 87%); Rf ¼ 0.56 (light petroleum/Et2O 8:2). 1H NMR (300 MHz, CDCl3) d 5.46 (s, 2H), 6.87e6.89 (m, 2H), 7.33e7.52 (m, 5H), 8.09 (dd, 1H, J ¼ 4.7, 1.8 Hz). 4.1.3. 2-Benzyloxy-4-diethylamino-pyridine (8b) To compound 7 (400 mg, 1.82 mmol) were added in sequence Pd2(dba)3 (1 mol%), DavePhos ligand (1.5 mol%), diethylamine (225 mL, 2.18 mmol) and t-BuONa (245 mg, 2.55 mmol). The mixture was suspended with toluene (4 mL) and degassed with N2 over 5 min. The resulting mixture was heated under microwave irradiation at 120e150  C for 15 min, then the suspension was taken up into EtOAc (100 mL), filtered through a short layer of celite and concentrated in vacuo. The crude product was purified by column chromatography (light petroleum/EtOAc 7:3) to give compound 8b (349 mg, 75%) as a yellow oil; Rf ¼ 0.64 (light petroleum/EtOAc 7:3). 1 H NMR (300 MHz, CDCl3) d 1.16 (t, 6H, J ¼ 7.0 Hz), 3.32 (q, 4H, J ¼ 7.0 Hz), 5.33 (s, 2H), 5.94 (d, 1H, J ¼ 2.2 Hz), 6.23 (dd, 1H, J ¼ 6.2, 2.0 Hz), 7.28e7.47 (m, 5H), 7.83 (d, 1H, J ¼ 6.2 Hz). 4.1.4. 2-Benzyloxy-4-phenylamino-pyridine (8e) Compound 7 (400 mg, 1.82 mmol) was reacted with aniline (197 mL, 2.18 mmol), according to the same procedure described for compound 8b. Compound 8e was obtained as a yellow oil (356 mg, 71%), after purification by column chromatography (CHCl3); Rf ¼ 0.16 (CHCl3). 1H NMR (300 MHz, CDCl3) d 5.35 (s, 2H), 6.07 (bs, 1H), 6.36 (d, 1H, J ¼ 1.3 Hz), 6.47 (dd, 1H, J ¼ 5.8, 1.8 Hz), 7.09e7.46 (m, 10H), 7.92 (d, 1H, J ¼ 5.8 Hz).

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4.1.5. 2-Benzyloxy-4-(4-allylpiperazin-1-yl)pyridine (8g) Compound 7 (400 mg, 1.82 mmol) was reacted with 1allylpiperazine (305 mL, 2.18 mmol), according to the same procedure described for compound 8b. Compound 8g was obtained as a yellow oil (421 mg, 75%), after purification by column chromatography (CHCl3/MeOH 95:5); Rf ¼ 0.27 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 2.52 (t, 4H, J ¼ 4.8 Hz), 3.02 (d, 2H, J ¼ 6.7 Hz), 3.30e3.90 (m, 4H, J ¼ 4.8 Hz), 5.16e5.24 (m, 2H), 5.34 (s, 2H), 5.80e 5.93 (m, 1H), 6.13 (d, 1H, J ¼ 1.9 Hz), 6.40 (dd, 1H, J ¼ 6.2, 1.9 Hz), 7.26e7.46 (m, 5H), 7.89 (d, 1H, J ¼ 6.2 Hz). 4.1.6. 4-Diethylamino-1H-pyridin-2-one (9b) To a solution of compound 8b (349 mg, 1.36 mmol) in MeOH/ EtOAc (2:1, 30 mL) at room temperature, 10% Pd/C (10 mol%) was added and the reaction mixture was fluxed with H2 under stirring for 2 h. The resulting mixture was filtered off through a short layer of celite and washed with EtOAc (150 mL). The solvent was removed in vacuo and the resulting residue was taken up into Et2O (15 mL) and filtered to provide compound 9b as a white powder (224 mg, 99%); Rf ¼ 0.48 (CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 1.05 (t, 6H, J ¼ 7.3 Hz), 3.26 (q, 4H, J ¼ 7.3 Hz), 5.13 (d, 1H, J ¼ 2.5 Hz), 5.80 (dd, 1H, J ¼ 7.8, 2.5 Hz), 7.05 (d, 1H, J ¼ 7.8 Hz), 10.52 (bs, 1H). 4.1.7. 4-Phenylamino-1H-pyridin-2-one (9e) Compound 9e was obtained as a white powder (236 mg, 99%) from 8e (356 mg, 1.29 mmol), by using the same procedure described for 9b; Rf ¼ 0.33 (CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 5.62 (d, 1H, J ¼ 1.2 Hz), 5.86 (dd, 1H, J ¼ 7.0, 1.8 Hz), 7.04 (t, 1H, J ¼ 7.0 Hz), 7.12e7.35 (m, 5H), 8.64 (bs, 1H), 10.66 (bs, 1H). 4.1.8. 4-(4-Allylpiperazin-1-yl)-1H-pyridin-2-one (9g) To a solution of compound 8g (421 mg, 1.36 mmol) in dry CH2Cl2 (70 mL), at 78  C was added dropwise a 1 M solution of BBr3 in CH2Cl2 (2.7 mL). After 3 h of stirring at 78  C the reaction was quenched with MeOH (20 mL) and the solvent was evaporated in vacuo to give compound 9g (296 mg, 99%) which was used directly for the next step without any purification; Rf ¼ 0.11 (CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6), 2.50 (t, 4H, J ¼ 5.3 Hz), 3.05 (d, 2H, J ¼ 6.5 Hz), 3.28 (t, 4H, J ¼ 5.2 Hz), 5.16e5.26 (m, 2H), 5.70 (d, 1H, J ¼ 2.6 Hz), 5.80e5.91 (m, 1H), 5.96 (dd, 1H, J ¼ 7.2, 2.6 Hz), 7.04 (d, 1H, J ¼ 7.2 Hz). 4.1.9. (2-Oxo-2H-pyridin-1-yl)-acetic acid ethyl ester (10a) A suspension of NaH (117 mg, 4.87 mmol) in dry DMF (10 mL) at 0  C under N2, was treated with a solution of pyridone 9a (386 mg, 4.06 mmol) in dry DMF (20 mL). After 1 h, ethyl bromoacetate (623 mL, 5.68 mmol) was added to the mixture and the reaction was stirred for a further 2 h at room temperature. The reaction was quenched with saturated NH4Cl (5 mL) and the product was extracted with CH2Cl2 (3  70 mL). The combined organic phases were washed with water (3  100 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by column chromatography (CHCl3/MeOH 95:5) to give compound 10a as a yellow oil (457 mg, 62%); Rf ¼ 0.70 (CHCl3/MeOH 9:1). 1H NMR (300 MHz, CDCl3) d 1.21 (t, 3H, J ¼ 7.3 Hz), 4.16e4.20 (m, 2H), 4.60 (s, 2H), 6.15e6.19 (m, 1H), 6.53e6.56 (m, 1H), 7.19e7.33 (m, 2H). 4.1.10. (4-Diethylamino-2-oxo-2H-pyridin-1-yl)-acetic acid ethyl ester (10b) Pyridone 9b (224 mg, 1.35 mmol) was reacted with ethyl bromoacetate (210 mL, 1.89 mmol), by employing the same procedure described for compound 10a, to provide compound 10b as a pale yellow oil (204 mg, 60%), after purification by column chromatography (CHCl3/MeOH 9:1); Rf ¼ 0.72 (CHCl3/MeOH 9:1). 1H NMR

29

(300 MHz, CDCl3) d 1.18 (t, 6H, J ¼ 7.3 Hz), 1.29 (t, 3H, J ¼ 7.3 Hz), 3.31 (q, 4H, J ¼ 7.3 Hz), 4.22 (q, 2H, J ¼ 7.3 Hz), 4.57 (s, 2H), 5.54 (d, 1H, J ¼ 2.6 Hz), 5.83 (dd, 1H, J ¼ 7.7, 2.9 Hz), 7.01 (d, 1H, J ¼ 7.7 Hz). 4.1.11. [4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid ethyl ester (10c) Pyridone 9c (223 mg, 1.24 mmol) was reacted with ethyl bromoacetate (193 mL, 1.74 mmol), by employing the same procedure described for compound 10a, to provide compound 10c as a yellow oil (144 mg, 44%); Rf ¼ 0.42 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 1.29 (t, 3H, J ¼ 7.0 Hz), 3.26 (t, 4H, J ¼ 5.3 Hz), 3.79 (t, 4H, J ¼ 5.3 Hz), 4.23 (q, 2H, J ¼ 7.0 Hz), 4.57 (s, 2H), 5.73 (d, 1H, J ¼ 3.1 Hz), 5.95 (dd, 1H, J ¼ 7.5, 3.1 Hz), 7.05 (d, 1H, J ¼ 7.5 Hz). 4.1.12. [4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid ethyl ester (10d) Pyridone 9d (270 mg, 1.51 mmol) was reacted with ethyl bromoacetate (234 mL, 2.11 mmol), by employing the same procedure described for compound 10a, to provide compound 10d as a yellow oil (265 mg, 67%); Rf ¼ 0.56 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 1.28 (t, 3H, J ¼ 7.0 Hz), 1.60e1.63 (m, 6H), 3.27e3.30 (m, 4H), 4.21 (q, 2H, J ¼ 7.1 Hz), 4.54 (s, 2H), 5.70 (d, 1H, J ¼ 2.8 Hz), 5.94 (dd, 1H, J ¼ 7.8, 2.8 Hz), 7.00 (d, 1H, J ¼ 7.8 Hz). 4.1.13. (4-Phenylamino-2-oxo-2H-pyridin-1-yl)-acetic acid ethyl ester (10e) Pyridone 9e (236 mg, 1.27 mmol) was reacted with ethyl bromoacetate (141 mL, 1.27 mmol), by employing the same procedure described for compound 10a, to provide compound 10e as a yellow oil (231 mg, 67%); Rf ¼ 0.42 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 1.27 (t, 3H, J ¼ 7.0 Hz), 4.21 (q, 2H, J ¼ 7.0 Hz), 4.56 (s, 2H), 5.91 (dd, 1H, J ¼ 7.0, 2.3 Hz), 5.99 (d, 1H, J ¼ 2.3 Hz), 6.52 (bs, 1H), 7.03 (d, 1H, J ¼ 7.0 Hz), 7.09e7.34 (m, 5H), 8.00 (bs, 1H). 4.1.14. [4-(4-Methyl-piperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid ethyl ester (10f) Pyridone 9f (230 mg, 1.19 mmol) was reacted with ethyl bromoacetate (185 mL, 1.67 mmol), by employing the same procedure described for compound 10a, to provide compound 10f as a white powder (167 mg, 50%); Rf ¼ 0.26 (CHCl3/MeOH 9:1). 1H NMR (300 MHz, CDCl3) d 1.25 (m, 3H), 2.27e2.47 (m, 7H), 3.26e3.36 (m, 4H), 4.17e4.21 (m, 2H), 4.54 (s, 2H), 5.71 (d, 1H, J ¼ 2.3 Hz), 5.95 (s, 1H), 7.02 (s, 1H). 4.1.15. [4-(4-Allyl-piperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid ethyl ester (10g) Pyridone 9g (296 mg, 1.35 mmol) was reacted with ethyl bromoacetate (269 mL, 2.43 mmol), by employing the same procedure described for compound 10a, to provide compound 10g as a white powder (235 mg, 57%); Rf ¼ 0.35 (CHCl3/MeOH 9:1). 1H NMR (300 MHz CDCl3) d 1.27 (t, 3H, J ¼ 7.0 Hz), 2.53 (t, 4H, J ¼ 5.3 Hz), 3.03 (d, 2H, J ¼ 6.5 Hz), 3.32 (t, 4H, J ¼ 5.3 Hz), 4.22 (q, 2H, J ¼ 7.0 Hz), 4.55 (s, 2H), 5.17e5.24 (m, 2H), 5.72 (d, 1H, J ¼ 2.9 Hz), 5.79e5.90 (m, 1H), 5.96 (dd, 1H, J ¼ 7.6, 2.9 Hz), 7.02 (d, 1H, J ¼ 7.6 Hz). 4.1.16. (2-Oxo-2H-pyridin-1-yl)-acetic acid (11a) A solution of ester 10a (457 mg, 2.52 mmol) in EtOH/H2O (1:1, 30 mL) at 0  C was treated with 1 N LiOH (5 mL) and stirred for 4 h at room temperature. The ethanol fraction was evaporated at reduced pressure and the residual aqueous solution was treated with 6 N HCl (pH w 6.8). The solution was concentrated in vacuo and the residue was purified by column chromatography (3% HCOOH in CHCl3/MeOH 9:1) to give compound 11a as a white powder (306 mg, 78%); Rf ¼ 0.27 (3% HCOOH in CHCl3/MeOH 9:1). 1 H NMR (300 MHz, acetone-d6) d 4.60 (s, 2H), 6.16 (t, 1H, J ¼ 5.9 Hz),

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N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34

6.55 (dd, 1H, J ¼ 7.4, 2.5 Hz), 7.20 (d, 1H, J ¼ 5.9 Hz), 7.33 (t, 1H, J ¼ 7.4 Hz). 4.1.17. (4-Diethylamino-2-oxo-2H-pyridin-1-yl)-acetic acid (11b) Ester 10b (204 mg, 0.81 mmol) was treated with 1 N LiOH (1.6 mL), according with the same procedure described for 11a. Title compound 11b was obtained as a white powder (137 mg, 75%); Rf ¼ 0.30 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 1.05 (t, 6H, J ¼ 7.0 Hz), 3.25 (q, 4H, J ¼ 7.0 Hz), 4.09 (s, 2H), 5.21 (d, 1H, J ¼ 2.6 Hz), 5.81 (dd, 1H, J ¼ 7.9, 2.9 Hz), 7.19 (d, 1H, J ¼ 7.7 Hz). 4.1.18. [4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid (11c) Ester 10c (144 mg, 0.54 mmol) was treated with 1 N LiOH (1.1 mL), according with the same procedure described for 11a. Title compound 11c was obtained as a white powder (104 mg, 87%); Rf ¼ 0.20 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 3.16 (t, 4H, J ¼ 4.8 Hz), 3.65 (t, 4H, J ¼ 4.4 Hz), 4.08 (s, 2H), 5.45 (d, 1H, J ¼ 3.1 Hz), 6.00 (dd, 1H, J ¼ 7.9, 2.7 Hz), 7.23 (d, 1H, J ¼ 7.9 Hz). 4.1.19. [4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid (11d) Ester 10d (265 mg, 1.0 mmol) was treated with 1 N LiOH (2 mL), according with the same procedure described for 11a. Title compound 11d was obtained as a white powder (180 mg, 76%); Rf ¼ 0.25 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 1.48e1.69 (m, 6H), 3.22e3.24 (m, 4H), 4.12 (s, 2H), 5.43 (d, 1H, J ¼ 2.7 Hz), 6.2 (dd, 1H, J ¼ 7.5, 2.7 Hz), 7.21 (d, 1H, J ¼ 7.5 Hz). 4.1.20. (4-Phenylamino-2-oxo-2H-pyridin-1-yl)-acetic acid (11e) Ester 10e (231 mg, 0.85 mmol) was treated with 1 N LiOH (1.7 mL), according with the same procedure described for 11a. Title compound 11e was obtained as a white powder (181 mg, 87%); Rf ¼ 0.31 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 4.13 (s, 2H), 5.79 (d, 1H, J ¼ 1.8 Hz), 6.08 (dd, 1H, J ¼ 7.0, 1.8 Hz), 6.98 (t, 1H, J ¼ 7.0 Hz), 7.16e7.31 (m, 5H). 4.1.21. [4-(4-Methylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid (11f) Ester 10f (167 mg, 0.60 mmol) was treated with 1 N LiOH (1.2 mL), according with the same procedure described for 11a, adjusting the residual aqueous solution to pH w6. Title compound 11f was obtained as a white solid (135 mg, 90%) after purification by column chromatography (CHCl3/MeOH 9:1 till all impurities came off, then 100% MeOH to elute the product); Rf ¼ 0.32 (MeOH). 1H NMR (300 MHz, CD3OD) d 2.34 (s, 3H), 2.54 (t, 4H, J ¼ 5.4 Hz), 3.41 (t, 4H, J ¼ 5.4 Hz), 4.41 (s, 2H), 5.71 (d, 1H, J ¼ 2.9 Hz), 6.27 (dd, 1H, J ¼ 7.6, 2.9 Hz), 7.37 (d, 1H, J ¼ 7.6 Hz). 4.1.22. [4-(4-Allyl-piperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid (11g) Ester 10g (235 mg, 0.77 mmol) was treated with 1 N LiOH (1.5 mL), according with the same procedure described for 11a, adjusting the residual aqueous solution to pH w7.2. Title compound 11f was obtained as a white solid (189 mg, 88%) after purification by column chromatography (CHCl3/MeOH 9:1 till all impurities came off, then 100% MeOH to elute the product); Rf ¼ 0.30 (MeOH). 1H NMR (300 MHz, CD3OD) d 2.57 (t, J ¼ 4.7 Hz 4H), 3.07 (d, 2H, J ¼ 7.0 Hz), 3.38 (t, 4H, J ¼ 4.7 Hz), 4.41 (s, 2H), 5.21e5.28 (m, 2H), 5.72 (d, 1H, J ¼ 2.3 Hz), 5.83e5.96 (m, 1H), 6.26 (dd, 1H, J ¼ 7.6, 2.3 Hz), 7.32 (d, 1H, J ¼ 7.6 Hz). 4.1.23. R-1-[2-(2-Oxo-2H-pyridin-1-yl)acetamido]-3methylbutylboronic acid pinanediol ester (13a) An ice-cold suspension (5  C) of acid 11a (100 mg, 0.65 mmol) in dry CH2Cl2 (20 mL) was treated with HOBt (211 mg, 1.56 mmol).

After 20 min, the reaction mixture was further cooled to 15  C and treated in sequence with EDC$HCl (242 mg, 1.56 mmol), a precooled (0  C) solution of commercially available pinanediol L-leucine boronate trifluoroacetate salt 12 (246 mg, 0.65 mmol) and DIPEA (136 mL, 0.78 mmol) in dry CH2Cl2 (5 mL). After stirring at 15  C for 1 h and additional 2 h at room temperature, the solution was washed with 0.1 M KHSO4, 5% NaHCO3 and brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was taken up into Et2O (20 mL), filtered and evaporated to give the crude product 13a (240 mg, 91%) as a pale yellow oil which was directly used in the next step without purification. Rf ¼ 0.82 (CHCl3/MeOH 95:5). 4.1.24. R-1-[2-(4-Diethylamino-2-oxo-2H-pyridin-1-yl) acetamido]-3-methylbutylboronic acid pinanediol ester (13b) Compound 11b (100 mg, 0.45 mmol) was reacted with HOBt (146 mg, 1.08 mmol), EDC$HCl (168 mg, 1.08 mmol), DIPEA (94 mL, 0.54 mmol) and amino boronate 12 (171 mg, 0.45 mmol) according with the same procedure described for 13a, to yield the crude product 13b (212 mg, 99%) which was directly used for the next step without purification. Rf ¼ 0.24 (EtOAc). 4.1.25. R-1-{2-[4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid pinanediol ester (13c) Compound 11c (100 mg, 0.42 mmol) was reacted with HOBt (136 mg, 1.01 mmol), EDC$HCl (157 mg, 1.01 mmol), DIPEA (88 mL, 0.50 mmol) and amino boronate 12 (159 mg, 0.42 mmol) according with the same procedure described for 13a, to yield the crude product 13c (204 mg, 99%) which was directly used for the next step without purification. Rf ¼ 0.50 (CHCl3/MeOH 9:1). 4.1.26. R-1-{2-[4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid pinanediol ester (13d) Compound 11d (100 mg, 0.42 mmol) was reacted with HOBt (136 mg, 1.01 mmol), EDC$HCl (157 mg, 1.01 mmol), DIPEA (88 mL, 0.50 mmol) and amino boronate 12 (159 mg, 0.42 mmol) according with the same procedure described for 13a, to yield the crude product 13d (203 mg, 99%)which was directly used for the next step without purification. Rf ¼ 0.52 (CHCl3/MeOH 9:1). 4.1.27. R-1-[2-(4-Phenylamino-2-oxo-2H-pyridin-1-yl)acetamido]3-methylbutylboronic acid pinanediol ester (13e) Compound 11e (100 mg, 0.41 mmol) was reacted with HOBt (132 mg, 0.98 mmol), EDC$HCl (152 mg, 0.98 mmol), DIPEA (85 mL, 0.49 mmol) and amino boronate 12 (155 mg, 0.41 mmol) according with the same procedure described for 13a, to yield the crude product 13e (197 mg, 97%) which was directly used for the next step without purification. Rf ¼ 0.49 (CHCl3/MeOH 95:5). 4.1.28. R-1-{2-[4-(4-Methylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid pinanediol ester (13f) Compound 11f (100 mg, 0.40 mmol) was reacted with HOBt (130 mg, 0.96 mmol), EDC$HCl (149 mg, 0.96 mmol), DIPEA (83 mL, 0.48 mmol) and amino boronate 12 (152 mg, 0.40 mmol) according with the same procedure described for 13a, to yield the crude product 13f (199 mg, 99%) which was directly used for the next step without purification. Rf ¼ 0.44 (CHCl3/MeOH 9:1). 4.1.29. R-1-{2-[4-(4-Allylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid pinanediol ester (13g) Compound 11g (100 mg, 0.36 mmol) was reacted with HOBt (116 mg, 0.86 mmol), EDC$HCl (133 mg, 0.86 mmol), DIPEA (75 mL, 0.43 mmol) and amino boronate 12 (136 mg, 0.36 mmol) according with the same procedure described for 13a, to yield the crude product 13g (157 mg, 83%) which was directly used for the next step without purification. Rf ¼ 0.46 (CHCl3/MeOH 9:1).

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4.1.30. R-1-[2-(2-Oxo-2H-pyridin-1-yl)acetamido]-3methylbutylboronic acid (1a) To a solution of 13a (240 mg, 0.6 mmol) in MeOH/n-hexane (1:1, 28 mL), isobutylboronic acid (306 mg, 3.0 mmol) and 1 N HCl (1.7 mL) were added. The reaction was stirred for 18 h at room temperature. The methanolic phase was washed with n-hexane (3  10 mL) and the n-hexane layer with MeOH (3  10 mL). The combined methanolic phases were evaporated in vacuo and the residue was taken up into CH2Cl2 (30 mL) and washed with 5% NaHCO3 (30 mL). The product was extracted with CHCl3 (3  30 mL) from the water layer and the combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The crude product was taken up in Et2O (20 mL), filtered off and concentrated in vacuo to give the title compound 1a as a white sticky solid (53 mg, 33%); Rf ¼ 0.29 (CHCl3/MeOH 9:1). MS (ESI) m/z 265.1 [M  H] (100%); 1 ½a27 D ¼ 4.0 (c 0.1, i-PrOH); H NMR (300 MHz, acetone-d6) d 0.87 (t, 6H, J ¼ 6.6 Hz), 1.29e1.72 (m, 3H), 3.39e3.44 (m, 1H), 4.06e4.92 (m, 2H), 6.25 (s, 1H), 6.39e6.47 (m, 1H), 7.45 (s, 1H), 7.60e7.64 (m, 1H);13C NMR (75 MHz, acetone-d6) d 23.4, 26.5, 32.0, 44.3, 51.8, 111.5, 118.8, 134.3, 138.7, 162.4, 170.4; Anal. Calcd. for C12H19BN2O4: C 54.16; H 7.20; N 10.53. Found: C 54.07; H 7.17; N 10.66. 4.1.31. R-1-[2-(4-Diethylamino-2-oxo-2H-pyridin-1-yl)acetamido]3-methylbutylboronic acid (1b) Ester 13b (212 mg, 0.45 mmol) was treated with isobutylboronic acid (229 mg, 2.25 mmol) and 1 N HCl (1 mL), by employing the same procedure described for 1a, to give the title compound 1b as a white sticky solid (61 mg, 40%); Rf ¼ 0.17 (CHCl3/MeOH 9:1). MS 1 (ESI) m/z 335.8 [M  H] (100%); ½a24 D ¼ 5.8 (c 0.4, MeOH); H NMR (300 MHz, acetone-d6) d 0.84 (t, 6H, J ¼ 5.6 Hz), 1.16 (t, 6H, J ¼ 6.2 Hz), 1.35e1.69 (m, 3H), 3.37e3.39 (m, 5H), 4.28e4.78 (m, 2H), 5.38 (d, 1H, J ¼ 2.6 Hz), 5.97 (dd, 1H, J ¼ 7.9, 2.9 Hz), 7.38 (d, 1H, J ¼ 7.7 Hz), 8.71 (bs, 1H); 13C NMR (75 MHz, acetone-d6) d 14.0, 23.4, 26.5, 32.0, 44.3, 44.6, 51.9, 86.2, 111.2, 134.3, 162.4, 166.1, 169.8; Anal. Calcd. for C16H28BN3O4: C 56.99; H 8.37; N 12.46. Found: C 56.91; H 8.47; N 12.44. 4.1.32. R-1-{2-[4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid (1c) To a solution of 13c (204 mg, 0.42 mmol) was treated with isobutylboronic acid (214 mg, 2.10 mmol) and HCl 1 N (1 mL), by employing the same procedure described for 1a, to give the title compound 1c as a white sticky solid (60 mg, 40%); Rf ¼ 0.28 (3% HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 350.2 [M  H] (100%); 1 ½a25 D ¼ 6.8 (c 0.4, MeOH); H NMR (300 MHz, acetone-d6) d 0.84 (t, 6H, J ¼ 5.6 Hz), 3.24e3.30 (m, 4H), 3.59e3.78 (m, 5H); 4.26e4.78 (m, 2H), 5.60 (s, 1H), 6.14 (s, 1H), 7.42 (s, 1H); 13C NMR (75 MHz CDCl3) d 14.0, 23.4, 26.5, 32.0, 44.3, 49.4, 60.0, 67.9, 86.2, 111.0, 134.3, 162.4, 166.2, 170.7; Anal. Calcd. for C16H26BN3O5: C 54.72; H 7.46; N 11.96. Found: C 54.71; H 7.53; N 11.84. 4.1.33. R-1-{2-[4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid (1d) Ester 13d (203 mg, 0.42 mmol) was treated with isobutylboronic acid (214 mg, 2.1 mmol) and 1 N HCl (1 mL), by employing the same procedure described for 1a, to give the title compound 1d as a white sticky solid (73 mg, 50%), Rf ¼ 0.32 (3% HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 348.1 [M  H] (100%); 1 ½a26 D ¼ 4.5 (c 0.5, MeOH); H NMR (300 MHz, acetone-d6) d 0.84 (t, 6H, J ¼ 7.0 Hz), 1.29e1.62 (m, 9H), 3.34e3.44 (m, 5H), 4.30e4.77 (m, 2H), 5.55e5.59 (m, 1H), 6.12 (t, 1H, J ¼ 7.5 Hz), 7.39 (d, 1H, J ¼ 7.5 Hz); 13C NMR (75 MHz, acetone-d6) d 23.3, 26.0, 26.2, 26.5, 32.0, 44.3, 48.4, 51.8, 86.3, 111.0, 134.3, 162.4, 166.2, 170.7; Anal. Calcd. for C17H28BN3O4: C 58.47; H 8.08; N 12.03. Found: C 58.52; H 8.06; N 12.09.

31

4.1.34. R-1-[2-(4-Phenylamino-2-oxo-2H-pyridin-1-yl)acetamido]3-methylbutylboronic acid (1e) Ester 13e (197 mg, 0.40 mmol) was treated with isobutylboronic acid (204 mg, 2 mmol) and 1 N HCl (1.2 mL), by employing the same procedure described for 1a, to give the title compound 1e as a white sticky solid (64 mg, 45%); Rf ¼ 0.20 (3% HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 356.2 [M  H] (100%); ½a27 D ¼ 11.9 (c 0.3, i-PrOH); 1 H NMR (300 MHz, acetone-d6) d 0.87 (m, 6H), 1.29e1.47 (m, 3H), 3.34 (m, 1 H), 4.05e4.50 (m, 2H), 5.88e6.10 (m, 2H), 7.10e7.43 (m, 6H); 13C NMR (75 MHz, acetone-d6) d 23.3, 26.4, 32.2, 44.3, 51.9, 88.4, 111.1, 117.1, 118.7, 128.8, 134.6, 144.9, 160.1, 162.3, 170.1; Anal. Calcd. for C18H24BN3O4: C 60.52; H 6.77; N 11.76. Found: C 60.50; H 6.89; N 11.69. 4.1.35. R-1-{2-[4-(4-Methylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid (1f) Ester 13f (199 mg, 0.4 mmol) was treated with isobutylboronic acid (204 mg, 2 mmol) and 1 N HCl (1.7 mL), by employing the same procedure described for 1a, to give the title compound 1f as a white sticky solid (41 mg, 30%); Rf ¼ 0.36 (3% HCOOH in CHCl3/MeOH 9:1). 1 MS (ESI) m/z 363.2 [M  H] (100%); ½a26 D ¼ 10.7 (c 0.4, MeOH); H NMR: (300 MHz) d 1.16 (t, 6H, J ¼ 5.4 Hz), 1.30e1.57 (m, 3H), 2.37 (s, 3H), 2.56 (t, 4H, J ¼ 5.3 Hz), 3.34e3.44 (m, 5H), 4.51 (s, 2H), 6.16 (d, 1H, J ¼ 2.9 Hz), 6.44 (dd, 1H, J ¼ 6.7, 2.9 Hz), 7.36 (d, 1H, J ¼ 6.7 Hz); 13 C NMR (75 MHz, CD3OD) d 23.3, 26.4, 32.2, 43.6, 44.3, 50.5, 51.9, 54.0, 86.2, 111.1, 134.6, 162.3, 166.3, 170.7; Anal. Calcd. for C17H29BN4O4: C 50.06; H 8.02; N 15.38. Found: C 50.01; H 8.09; N 15.42. 4.1.36. R-1-{2-[4-(4-Allylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl] acetamido}-3-methylbutylboronic acid (1g) Ester 13g (157 mg, 0.3 mmol) was treated with isobutylboronic acid (153 mg, 1.5 mmol) and 1 N HCl (1 mL), by employing the same procedure described for 1a, to give the title compound 1g as a white sticky solid (35 mg, 30%); Rf ¼ 0.47 (3% HCOOH in CHCl3/MeOH 9:1). 1 MS (ESI) m/z 389.2 [M  H] (100%); ½a26 D ¼ 12.3 (c 0.3, MeOH); H NMR (300 MHz, CD3OD) d 1.18 (t, 6H, J ¼ 6.2 Hz), 1.25e1.57 (m, 3H), 2.44 (t, 4H, J ¼ 4.7 Hz), 3.11 (d, 2H, J ¼ 7.0 Hz), 3.23e3.44 (m, 5H), 4.54 (s, 2H), 5.20e5.26 (m, 2H), 6.22 (d, 1H, J ¼ 2.3 Hz), 6.64e6.73 (m, 1H), 6.89 (dd, 1H, J ¼ 7.2, 2.3 Hz), 7.22 (d, 1H, J ¼ 7.2 Hz); 13C NMR (75 MHz, CD3OD) d 23.3, 26.4, 32.2, 43.6, 44.3, 50.5, 54.0, 51.2, 52.2, 53.8, 57.4, 86.2, 111.1, 116.1, 133.2, 134.6, 162.5, 166.4, 170.9; Anal. Calcd. for C19H31BN4O4: C 58.47; H 8.01; N 14.36. Found: C 58.34; H 8.05; N 14.28. 4.1.37. (5-Oxo-5H-1,6-naphthyridin-6-yl)acetic acid (16a) Step 1. Commercially available 1,6-naphthyridin-5(6H)-one 14 (200 mg, 1.37 mmol) and NaH (39 mg, 1.64 mmol) were suspended in dry DMF (10 mL) at 0  C and the reaction mixture was stirred for 1 h. After this time, a solution of ethyl 2-bromoacetate (212 mL, 1.92 mmol) in dry DMF (3 mL) was added and the reaction mixture was stirred for 2 h, then was quenched with saturated NH4Cl solution (5 mL) and extracted with CHCl3 (3  15 mL) and the organic phase was washed with saturated NaHCO3 solution (2  20 mL) and distilled water (2  20 mL). The organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The crude was purified by column chromatography (CHCl3/MeOH 95:5) to give (5-oxo-5H1,6-naphthyridin-6-yl)acetic acid ethyl ester as yellow crystals (190 mg, 60%); Rf ¼ 0.69 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 1.30 (t, 3H, J ¼ 7.3 Hz), 4.26 (q, 2H, J ¼ 7.3 Hz), 4.72 (s, 2H), 6.81 (d, 1H, J ¼ 7.7 Hz), 7.25 (d, 1H, J ¼ 7.7 Hz), 7.42 (dd, 1H, J ¼ 8.1, 4.4 Hz), 8.67 (dd, 1H, J ¼ 8.1, 1.8 Hz), 8.92 (dd, 1H, J ¼ 4.4, 1.8 Hz). Step 2. To a cooled solution (0  C) of ester intermediate (190 mg, 0.82 mmol) in EtOH (10 mL), 1 N LiOH (1.6 mL) was added, and the mixture was stirred at 25  C for 6 h, until TLC showed the complete

32

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hydrolysis of the starting material. Upon concentration in vacuo and pH regulation to 6.8, the aqueous layer was completely evaporated in vacuo and the resulting crude was purified by column chromatography (3% HCOOH in CHCl3/MeOH 9:1) to obtain the title compound 16a as a pale yellow solid (151 mg, 90%); Rf ¼ 0.27 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 4.57 (s, 2H), 6.67 (d, 1H, J ¼ 7.3 Hz), 7.50 (dd, 1H, J ¼ 7.9, 4.4 Hz), 7.68 (d, 1H, J ¼ 7.3 Hz), 8.24 (bs, 1H), 8.51 (dd, 1H, J ¼ 7.9, 1.8 Hz), 8.92 (dd, 1H, J ¼ 4.4, 1.8 Hz). 4.1.38. (S)-2-(5-Oxo-5H-1,6-naphthyridin-6-yl)propionic acid (16b) Step 1. Compound 16b was obtained in the same way as for 16a using 14 (200 mg, 1.37 mmol), methyl 2(R)-bromopropionate [27] (321 mg, 1.92 mmol) and NaH (39 mg, 1.64 mmol). Purification by column chromatography (EtOAc) provided the ester intermediate (110 mg, 34%); Rf ¼ 0.51 (EtOAc). 1H NMR (300 MHz, CDCl3) d 1.70 (d, 3H, J ¼ 7.5 Hz), 3.76 (s, 3H), 5.61 (q, 1H, J ¼ 7.5 Hz), 6.83 (d, 1H, J ¼ 7.5 Hz), 7.34 (d, 1H, J ¼ 7.5 Hz), 7.40 (dd, 1H, J ¼ 8.0, 4.4 Hz), 8.66 (dd, 1H, J ¼ 8.0, 1.8 Hz), 8.90 (dd, 1H, J ¼ 4.4, 1.8 Hz). Step 2. The hydrolysis was realized with the same procedure of 16a using the ester intermediate (110 mg, 0.47 mmol), 1 N LiOH (0.9 mL) and MeOH (10 mL) as solvent, to give the title compound 16b as a pale yellow solid (89 mg, 88%); Rf ¼ 0.24 (3% HCOOH in CHCl3/MeOH 9:1); 1H NMR (300 MHz, DMSO-d6) d 1.59 (d, 3H, J ¼ 6.9 Hz), 5.31 (q, 1H, J ¼ 6.9 Hz), 6.72 (d, 1H, J ¼ 7.4 Hz), 7.51 (dd, 1H, J ¼ 7.7, 4.4 Hz), 7.75 (d, 1H, J ¼ 7.4 Hz), 8.52 (dd, 1H, J ¼ 7.7, 1.8 Hz), 8.93 (dd, 1H, J ¼ 4.4, 1.8 Hz). 4.1.39. (8-Bromo-5-oxo-5H-1,6-naphthyridin-6-yl)acetic acid (16c) Step 1. Compound 16c was obtained in the same way as for 16a using 15 [10] (187 mg, 0.83 mmol), ethyl bromoacetate (129 mL, 1.16 mmol) and NaH (24 mg, 1.00 mmol). Purification by column chromatography (CHCl3/MeOH 95:5) provided the ester intermediate (168 mg, 65%); Rf ¼ 0.67 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 1.32 (t, 3H, J ¼ 7.0 Hz), 4.28 (q, 2H, J ¼ 7.0 Hz), 4.72 (s, 2H), 7.47e7.55 (m, 1H), 7.62 (s, 1H), 8.70 (d, 1H, J ¼ 7.5 Hz), 9.06 (s, 1H). Step 2. The successive hydrolysis was realized with the same procedure employed for 16a using the ester intermediate (168 mg, 0.54 mmol), 1 N LiOH (1.1 mL) and ethanol as solvent (10 mL), to give 16c as a pale pink solid (150 mg, 98%); Rf ¼ 0.14 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 4.64 (s, 2H), 7.63 (dd, 1H, J ¼ 7.9, 4.7 Hz), 8.22 (s, 1H), 8.57 (dd, 1H, J ¼ 7.9, 1.0 Hz), 9.04 (dd, 1H, J ¼ 4.7, 1.0 Hz). 4.1.40. R-1-[(5-Oxo-5H-1,6-naphthyridin-6-yl)acetamido]-3methylbutylboronic acid pinanediol ester (17a) To an ice-cold suspension (5  C) of [(6H)-5-oxo-1,6naphthyridinyl]acetic acid 16a (151 mg, 0.74 mmol) in dry CH2Cl2 (15 mL), was added HOBt (240 mg, 1.78 mmol). After 20 min, the reaction system was further cooled (15  C) and was treated with EDC$HCl (341 mg, 1.78 mmol). After that, a cold solution of pinanediol L-leucine boronate 12 (281 mg, 0.74 mmol) and DIPEA (155 mL, 0.89 mmol) in dry CH2Cl2 (5 mL) was added and the reaction mixture was stirred at 15  C for 1 h and then at room temperature for 2 h. Finally, the organic phase was washed with 0.1 M KHSO4, 5% NaHCO3 and brine, dried over Na2SO4 and filtered. The solution was evaporated to give a crude (334 mg, 100%) which was used directly in the next reaction without purification; Rf ¼ 0.42 (EtOAc/acetone/MeOH 80:15:5). 4.1.41. R-1-[(S)-2-(5-Oxo-5H-1,6-naphthyridin-6-yl) propanamido]-3-methylbutylboronic acid pinanediol ester (17b) Synthesis of compound 17b was performed following the same procedure employed for compound 17a using carboxylic acid 16b

(89 mg, 0.41 mmol), HOBt (132 mg, 0.98 mmol), EDC$HCl (188 mg, 0.98 mmol), DIPEA (85 mL, 0.49 mmol), pinanediol L-leucine boronate 12 (155 mg, 0.41 mmol). After work-up, the crude product (191 mg, 100%) was directly used in the next reaction without further purification; Rf ¼ 0.60 (CHCl3/MeOH 9:1). 4.1.42. R-1-[(8-Bromo-5-oxo-5H-1,6-naphthyridin-6-yl) acetamido]-3-methylbutylboronic acid pinanediol ester (17c) The synthesis of compound 17c was performed following the same procedure employed for compound 17a using carboxylic acid 16c (150 mg, 0.53 mmol), HOBt (243 mg, 1.27 mmol), EDC$HCl (1.27 mmol, 171 mg), DIPEA (112 mL, 0.64 mmol), pinanediol Lleucine boronate 12 (201 mg, 0.53 mmol). After work-up, the crude product (196 mg, 70%) was directly used in the next reaction without further purification; Rf ¼ 0.59 (CHCl3/MeOH 9:1). 4.1.43. R-1-[(5-Oxo-5H-1,6-naphthyridin-6-yl)acetamido]-3methylbutylboronic acid (2a) A solution of 17a (334 mg, 0.74 mmol) and isobutylboronic acid (377 mg, 3.70 mmol) in MeOH/n-hexane (1:1, 12 mL) was treated with 1 N HCl (1.8 mL), and the mixture was stirred at room temperature for 18 h. The methanolic phase was washed with n-hexane (3  10 mL) and the combined hexanic phases were re-extracted with MeOH (3  15 mL). The resulting methanolic layer was concentrated in vacuo and the residue was dissolved in CHCl3 (15 mL) and washed with 5% NaHCO3. The aqueous phase was extracted with CHCl3 (3  15 mL) and the organic layer was evaporated in vacuo to give a solid which was purified by trituration with Et2O to give title product 2a (75 mg, 32%); Rf ¼ 0.21 (3% HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 318.3 [M þ H]þ (100%); 1 ½a26 D ¼ 20.8 (c 0.2, i-PrOH); H NMR (300 MHz, acetone-d6) d 0.87 (d, 6H, J ¼ 6.7 Hz), 1.37 (q, 2H, J ¼ 7.5 Hz), 1.50e1.64 (m, 1H), 3.22e 3.29 (m, 1H), 4.63 (s, 2H), 6.57 (d, 1H, J ¼ 5.9 Hz), 6.82 (d, 1H, J ¼ 7.6 Hz), 7.40 (d, 1H, J ¼ 7.6 Hz), 7.42 (dd, 1H, J ¼ 8.2, 4.7 Hz), 8.66 (dd, 1H, J ¼ 8.2, 1.8 Hz), 8.92 (dd, 1H, J ¼ 4.7, 1.8 Hz); 13C NMR (75 MHz, acetone-d6) d 23.3, 26.1, 32.0, 44.5, 52.0, 105.4, 124.0, 124.1, 129.4, 138.0, 147.4, 156.4, 157.6, 170.8; Anal. Calcd. for C15H20BN3O4: C 56.81, H 6.36, N 13.25. Found: C 56.61, H 6.51, N 13.15. 4.1.44. R-1-[(S)-2-(5-Oxo-5H-1,6-naphthyridin-6-yl) propanamido]-3-methylbutylboronic acid (2b) Compound 2b was obtained following the same procedure employed for compound 2a using 17b (191 mg, 0.41 mmol), 2methylpropylboronic acid (209 mg, 2.05 mmol), 1 N HCl (1 mL) and a mixture of MeOH/n-hexane (1:1, 7 mL) as solvent. After purification, the reaction gave the title compound 2b (30 mg, 30%); Rf ¼ 0.40 (CHCl3/MeOH 9:1). MS (ESI) m/z 330.2 [M  H] (100%); 1 ½a27 D ¼ 41.6 (c 0.1, i-PrOH); H NMR (300 MHz, acetone-d6) d 0.76 (d, 6H, J ¼ 7.6 Hz), 1.24e1.33 (m, 2H), 1.37e1.45 (m, 1H), 1.66 (d, 3H, J ¼ 7.0 Hz), 3.34e3.46 (m, 1H), 5.77 (q, 1H, J ¼ 7.0 Hz), 6.75 (d, 1H, J ¼ 7.0 Hz), 7.46 (d, 1H, J ¼ 7.0 Hz), 7.71 (m, 1H), 8.56 (m, 1H), 8.89 (m, 1H); 13C NMR (75 MHz, (CD3)2CO) d 15.1, 23.3, 25.2, 30.2, 54.0, 72.1, 105.4, 124.0, 124.1, 129.4, 138.0, 147.4, 156.1, 157.6, 171.6; Anal. Calcd. for C16H22BN3O4: C 58.03, H 6.70, N 12.69. Found: C 58.28, H 6.89, N 12.47. 4.1.45. R-1-[(8-Bromo-5-oxo-5H-1,6-naphthyridin-6-yl) acetamido]-3-methylbutylboronic acid (2c) Compound 2c was obtained following the same procedure employed for compound 2a using 17c (196 mg, 0.37 mmol), isobutylboronic acid (188 mg, 1.85 mmol), 1 N HCl (0.9 mL) and a mixture of MeOH/n-hexane (1:1, 6 mL) as solvent. After purification, the reaction gave of the title compound 2c (50 mg, 34%); Rf ¼ 0.31 (CHCl3/MeOH 9:1). MS (ESI) m/z 395.05 [M  H] (100%); 1 ½a27 D ¼ 8.9 (c 0.3, i-PrOH); H NMR (300 MHz, acetone-d6) d 0.89

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34

(d, 6H, J ¼ 5.9 Hz), 1.37e1.44 (m, 2H), 1.55e1.66 (m, 1H), 3.22e3.29 (m, 1H), 4.59 (s, 2H), 7.46e7.59 (m, 1H), 7.78 (s, 1H), 8.69 (d, 1H, J ¼ 7.6 Hz), 9.02e9.14 (m, 1H); 13C NMR (75 MHz, acetone-d6) d 23.3, 25.2, 30.2, 46.8, 71.8, 103.9, 124.0, 124.1, 125.1, 138.0, 147.4, 156.4, 158.4, 169.6; Anal. Calcd. for C15H19BBrN3O4: C 45.49, H 4.84, N 10.61. Found: C 45.23, H 4.99, N 10.50. 4.1.46. (1-Methyl-1,2,3,4-tetrahydro-5-oxo-5H-1,6-naphthyridin6-yl)acetic acid (19) Step 1. Compound 19 was obtained in the same way as for 16a using 18 [10] (159 mg, 0.97 mmol), ethyl bromoacetate (151 mL, 1.36 mmol) and NaH (28 mg, 1.16 mmol). Purification by column chromatography (CHCl3/MeOH 95:5) provided the ester intermediate (190 mg, 78%); Rf ¼ 0.54 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3) d 1.28 (t, 3H, J ¼ 7.3 Hz), 1.85e1.96 (m, 2H), 2.58 (t, 2H, J ¼ 6.0 Hz), 2.95 (s, 3H), 3.22 (t, 2H, J ¼ 5.6 Hz), 4.22 (q, 2H, J ¼ 7.3 Hz), 4.56 (s, 2H), 5.85 (d, 1H, J ¼ 7.7 Hz), 6.95 (d, 1H, J ¼ 7.7 Hz). Step 2. The successive hydrolysis was performed in the same way as for 16a using the ester intermediate (190 mg, 0.76 mmol), 1 N LiOH (1.5 mL), and ethanol (10 mL) as solvent to give the title compound 19 as white solid (160 mg, 95%); Rf ¼ 0.30 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6) d 1.71e1.83 (m, 2H), 2.31 (t, 2H, J ¼ 5.9 Hz), 2.88 (s, 3H), 3.15 (t, 2H, J ¼ 4.7 Hz), 4.41 (s, 2H), 5.91 (d, 1H, J ¼ 7.6 Hz), 7.27 (d, 1H, J ¼ 7.6 Hz). 4.1.47. R-1-[(1-Methyl-1,2,3,4-tetrahydro-5-oxo-5H-1,6naphthyridin-6-yl)-acetamido]-3-methylbutylboronic acid (3) Step 1. Synthesis of compound 3 was performed following the same procedure employed for compound 17a using acid 19 (160 mg, 0.72 mmol), HOBt (234 mg, 1.73 mmol), EDC$HCl (332 mg, 1.73 mmol), DIPEA (150 mL, 0.86 mmol), pinanediol L-leucine boronate 12 (273 mg, 0.72 mmol). After work-up, the crude product was directly used in the next reaction without further purification (169 mg, 50%); Rf ¼ 0.57 (CHCl3/MeOH 9:1). Step 2. Compound 3 was obtained following the same procedure of compound 2a but using the crude pinanediol ester intermediate (169 mg, 0.36 mmol), isobutylboronic acid (183 mg, 1.80 mmol), 1 N HCl (0.9 mL) and a mixture of MeOH/n-hexane (1:1, 6 mL) as solvent. After purification, the reaction gave the title compound 3 as white solid (35 mg, 28%); Rf ¼ 0.25 (CHCl3/MeOH 9:1). MS (ESI) m/z 334.1 [M  H] 1 (100%); ½a27 D ¼ 27.7 (c 0.1, i-PrOH); H NMR (300 MHz, acetone-d6) d 0.86 (t, 6H, J ¼ 6.2 Hz), 1.36 (q, 2H, J ¼ 7.1 Hz), 1.49e1.62 (m, 1H), 1.87e1.97 (m, 2H), 2.58 (t, 2H, J ¼ 6.2 Hz), 2.95 (s, 3H), 3.14e3.28 (m, 3H), 4.28 (d, 1H, J ¼ 14.3 Hz), 4.46 (d, 1H, J ¼ 14.3 Hz), 5.98 (d, 1H, J ¼ 7.6 Hz), 7.30 (d, 1H, J ¼ 7.6 Hz); 13C NMR (75 MHz, acetone-d6) d 20.0, 23.3, 23.5, 26.1, 32.0, 38.5, 44.5, 47.8, 52.2, 106.5, 111.1, 134.5, 157.7, 157.9, 170.8; Anal. Calcd. for C16H26BN3O4: C 57.33, H 7.82, N 12.54. Found: C 57.15, H 7.72, N 12.77. 4.2. Computational chemistry Molecular modeling and graphics manipulations were performed using Maestro 9.2 (Schrödinger) [28] and UCSF-CHIMERA software packages [29], running on a E4 Computer Engineering E1080 workstation provided of an Intel Core i7-930 Quad-Core processor. GOLD 5.1 [15,16] was used for all docking calculations. Figures were generated using Pymol 1.0 [30]. 4.2.1. Protein and ligands preparation Coordinates for the chymotrypsin-like b5 subunit derived from the X-ray crystal structure of the yeast 20S proteasome determined at 2.8  A resolution (PDB ID: 2F16) were employed for the automated docking studies [7]. The protein setup was carried out by Protein Preparation Wizard in Maestro. Hydrogen atoms were added to the

33

protein consistent with the neutral physiologic pH. Arginine and lysine side chains were considered as cationic at the guanidine and ammonium groups, and the aspartic and glutamic residues were considered as anionic at the carboxylate groups. Since the X-ray structure of the proteasome/bortezomib complex is consistent with a protonated b6-D114, it was treated as protonated during docking simulations. The protonation and flip states of the imidazole rings of the histidine residues were adjusted together with the side chain amides of glutamine and asparagine residues in a H-bonding network optimization process. Successively, the protein hydrogens only were minimized using the Impref module of Impact with the OPLS_2005 force field. The initial structure of bortezomib was derived from the crystal complex coordinates, and the original structures of 1aee and 2c were constructed on the basis of the bortezomib crystal structure conformation followed by energy minimization with the PolakeRibiere conjugated gradient (PRCG) method with a convergence gradient of 0.001 kJ/mol  A. 4.2.2. Docking studies Compounds 1aee and 2c were covalently docked to the binding pocket of b5 subunit using GOLD, version 5.1 [15,16]. A radius of 20  A from the b5 catalytic N-terminal threonine was used to direct site location. For each of the genetic algorithm runs, a maximum of 100,000 operations were performed on a population of 100 individuals with a selection pressure of 1.1. Operator weights for crossover, mutation, and migration were set to 95, 95, and 10, respectively, as recommended by the authors of the software. The distance for H-bonding was set to 2.5  A, and the cutoff value for van der Waals calculation was set to 4  A. Covalent docking was applied, and the terminal boron atoms of all the ligands were bonded to the hydroxyl oxygen of b5-T1. The water molecule near b6-D114 (crystallographically determined for the proteasome/bortezomib complex) was specified in GOLD by switching state settings to ‘toggle’ and orientation mode to ‘spin’. The Goldscore-CS docking protocol [17], was adopted in this study. In this protocol, the poses obtained with the original GoldScore function are rescored and reranked with the GOLD implementation of the ChemScore function [17e21]. To perform thorough and unbiased search of the conformation space, each docking run was allowed to produce 200 poses without the option of early termination, using standard default settings. The top solution obtained after re-ranking of the poses with ChemScore was selected to generate the proteasome/ligand complexes. 4.3. Pharmacology 4.3.1. In vitro 20S proteasome inhibition assays Human 20S proteasome was obtained from Biomol GmbH, Hamburg, Germany. The three distinct proteolytic activities of the 20S proteasome were measured by monitoring the hydrolysis of the peptidyl 7-amino-4-methyl coumarin substrates (all obtained from Bachem) Suc-Leu-Leu-Val-Tyr-AMC, Boc-Leu-Arg-Arg-AMC, and Cbz-Leu-Leu-Glu-AMC for ChT-L, T-L and PGPH activity of the enzyme, respectively. Fluorescence of the product AMC of the substrates’ hydrolyzes was measured using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland) at 30  C with a 380 nm excitation filter and a 460 nm emission filter. The preliminary screening for the inhibition of the three proteolytic activities of the 20S proteasome was performed at 20 mM inhibitor concentrations using an equivalent amount of DMSO as a negative control. Compounds showing at least 40% inhibition at 20 mM were subjected to detailed assays. The dissociation constants Ki of the non covalent complex E$I were obtained from progress curves (10 min) at various concentrations of inhibitor by fitting the progress curves to a 4 parameter IC50 equation, and correction to zero substrate concentration from Ki ¼ IC50/(1 þ [S] K1 m ). The Km values

34

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were determined in separate experiments: ChT-L activity with SucLeu-Leu-Val-Tyr-AMC 13 mM, and PGPH activity with Cbz-Leu-LeuGlu-AMC 53 mM. 4.3.2. Assaying the chymotryptic activity of the 20S proteasome Human 20S proteasome was incubated at 30  C at a final concentration of 0.004 mg mL1 with test compound present at variable concentrations. The reaction buffer consisted of 50 mM Tris pH 7.5, 10 mM NaCl, 25 mM KCl, 1 mM MgCl2, 0.03% SDS, and 5% DMSO. Product release from substrate hydrolysis (75 mM) was monitored continuously over a period of 10 min. 4.3.3. Assaying the tryptic activity of the 20S proteasome Human 20S proteasome was incubated at 30  C at a final concentration of 0.0025 mg mL1 with test compound present at 20 mM. The reaction buffer consisted of 50 mM Tris buffer pH 7.4, 50 mM NaCl, 0.5 mM EDTA, 0.03% SDS, and 7.5% DMSO. Product release from substrate hydrolysis (85 mM) was monitored continuously over a period of 10 min.

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

4.3.4. Assaying the post-glutamyl peptide hydrolyzing activity of the 20S proteasome Human 20S proteasome was incubated at 30  C at a final concentration of 0.004 mg mL1 with the test compound present at variable concentrations. The reaction buffer consisted of 50 mM Tris buffer pH 7.5 containing 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.03% SDS, 5% DMSO. Product release from substrate hydrolysis (80 mM) was monitored continuously over a period of 10 min.

[15]

[16] [17]

[18]

4.3.5. Assays for bovine pancreatic a-chymotrypsin inhibition The enzyme (250 mg mL1) was incubated at 20  C with test compound. The reaction buffer consisted of 50 mM Tris buffer pH 8.0 containing 100 mM NaCl and 5 mM EDTA and 7.5% DMSO. Product release from substrate hydrolysis (75 mM final concentration, Suc-Leu-Leu-Val-Tyr-AMC from Bachem) was determined over a period of 10 min.

[19]

[20]

Acknowledgments This work was financially supported by the Ministero dell’Istruzione, dell’Università e della Ricerca Scientifica e Tecnologica (MIUR-PRIN2010-2011). We would like to thank the Deutscher Akademischer Austausch Dienst (DAAD) (Vigoni project 2011/12) for partial support of this work. TS thanks the Deutsche Forschungsgemeinschaft (DFG) for financial support. R.E. acknowledges the support of her postdoctoral fellowship from “Dote Ricerca”: FSE, Regione Lombardia.

[21]

[22]

[23]

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