lable at ScienceDirect

European Journal of Medicinal Chemistry 122 (2016) 702e722

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Research paper

Multifunctional diamine AGE/ALE inhibitors with potentialtherapeutical properties against Alzheimer's disease

Elodie Lohou a, N. Andr�e Sasaki a, *, Agn�es Boullier b, c, d, Pascal Sonnet a

a Universit�e de Picardie Jules Verne, Laboratoire de Glycochimie des Antimicrobiens et des Agroressouces, LG2A, UMR CNRS 7378, UFR de Pharmacie, 1 Ruedes Louvels, F-80037, Amiens Cedex 01, Franceb Universit�e de Picardie Jules Verne, UFR de M�edecine, 1 Rue des Louvels, F-80037, Amiens Cedex 01, Francec INSERM U1088, Centre Universitaire de Recherche en Sant�e (CURS), Avenue Ren�e La€ennec e Salouel, F-80054, Amiens Cedex 01, Franced CHU Amiens Picardie, Avenue Ren�e La€ennec e Salouel, F-80054, Amiens Cedex 01, France

a r t i c l e i n f o

Article history:Received 22 March 2016Received in revised form26 April 2016Accepted 28 April 2016Available online 30 April 2016Dedicated to the late Professor Pierre Potieron the occasion of the 10th anniversary ofhis decease.

Keywords:Alzheimer's diseaseAGE (Advanced Glycation Endproducts)ALE (Advanced Lipid peroxidationEndproducts)Carbonyl stressOxidative stressBiometal dyshomeostasisDiamine building blocksPhenolic acidsHydroxypyridinone (HOPO) ligands

* Corresponding author.E-mail address: andre.sasaki@u-picardie.fr (N.A. S

http://dx.doi.org/10.1016/j.ejmech.2016.04.0690223-5234/© 2016 Elsevier Masson SAS. All rights re

a b s t r a c t

An important part of pathogenesis of Alzheimer's disease (AD) is attributed to the contribution of AGE(Advanced Glycation Endproducts) and ALE (Advanced Lipid peroxidation Endproducts). In order toattenuate the progression of AD, we designed a new type of molecules that consist of two trapping partsfor reactive carbonyl species (RCS) and reactive oxygen species (ROS), precursors of AGE and ALE,respectively. These molecules also chelate transition metals, the promoters of ROS formation. In thispaper, synthesis of the new AGE/ALE inhibitors and evaluation of their physicochemical and biologicalproperties (carbonyl trapping capacity, antioxidant activity, Cu2þ-chelating capacity, cytotoxicity andprotective effect against in vitro MGO-induced apoptosis in the model AD cell-line PC12) are described. Itis found that compounds 40b and 51e possess promising therapeutic potentials for treating AD.

© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction

Alzheimer's disease (AD) is the most common neurodegenera-tive disease that leads to memory loss and progressive cognitiveimpairment. Two neuropathological hallmarks resulting fromabnormal protein deposits are identified in AD brains as extracel-lular amyloid b (Ab) plaques and intracellular tau-associatedneurofibrillary tangles (NFT) [1]. A critical imbalance between ce-rebral reactive oxygen species (ROS) production and endogenousantioxidant capacities associated with biometal dyshomeostasishas been suggested to be a driving force for AD onset and pro-gression [2e4]. Indeed, Ab-oligomers induce oxidative stresswhereas transition metals (Zn2þ, Cu2þ and Fe3þ) stimulate Ab

asaki).

served.

aggregation and APP (amyloid precursor protein) processing [2e4].Besides these various triggering factors at the early stages of AD,advanced glycation endproducts (AGE) induced by oxidative stressexacerbation are now considered to play an important role at thelate stages of pathogenesis [2]. Non-enzymatic condensation ofreducing carbohydrates (mainly glucose) with free nucleophilicfunctional groups of proteins such as amino and guanidino groupsof lysine and arginine, respectively, provides labile Schiff bases thatin turn rearrange to more stable a-ketoamines called Amadoriproducts [5]. Oxidation of Amadori products results in the forma-tion of very reactive a-oxoaldehydes, called as reactive carbonylspecies (RCS), such as glyoxal (GO), methylglyoxal (MGO) and 3-deoxyglucosone (3-DG). This process is known as “Maillard reac-tion”. Condensation of resulting a-oxoaldehydes with nucleophilicgroups of proteins or of nucleic acids further leads to structuralrearrangements to provide various products bearing nitrogen- andoxygen-containing heterocycles and representing the

Linker

Linker Linker

RCS trappingcapacity

ROS and biometalscavenging capacities

Linker

Fig. 1. New multifunctional diamine AGE/ALE inhibitors.

Fig. 2. Dap derivatives of previous AGE/ALE inhibitor series.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 703

heterogenous variety of AGE [6]. The slow oxidative degradation ofmonosaccharides like triose-phosphate intermediates in glycolyticpathway also forms the corresponding a-oxoaldehydes [7]. ALE(Advanced Lipid peroxidation Endproducts) correspond to theformation of similar irreversible covalent adducts. The lipid per-oxidation of polyunsaturated fatty acids leads to the production ofa,b-unsaturated aldehydes such as acrolein, malondialdehyde(MDA) and 4-hydroxy-2-nonenal (4-HNE) [5,7,8]. Michael additionof nucleophilic groups of biomolecules to thus formed RCS inducesthe formation of ALE. AGE/ALE formation plays an important role inage-related tissue and cell dysfunction As toxic mediator andoxidative stress promoter [6], carbonyl stress is also implicated inthe pathogenesis of diabetic microvascular complications (ne-phropathy, retinopathy and neuropathy) [9], atherosclerosis [10],cardiovascular and neurodegenerative diseases [8]. In order toprevent and treat these pathologies, several AGE inhibitors havealready been reported including aminoguanidine, pyridoxamine,carnosine, 2,3-diaminophenazine, tenilsetam and OPB-9195.However, clinical trials of these AGE inhibitors have been sus-pended up to date [8,11]. In AD, extensive AGE/ALE accumulationlinked to enhanced RCS level has been reported in senile plaquesand NFT [2,12]. The overproduction of MGO is due to the progres-sive decrease in efficacy of carbonyl-detoxification systems(glyoxalases), but also to a dysfunction of glycolytic pathway[12,13]. Furthermore, Ab-mediated membrane lipid peroxidationintensifies 4-HNE, acrolein andMDA synthesis [2,14]. Consequently,RCS accumulation takes part in the vicious downward redox amy-loid spiral leading to neurodegeneration. AGE/ALE contribute to ADpathogenesis through three main mechanisms. First, glycated Abcross-linking promotion accelerates its deposition and its proteaseresistance [12]. Secondly, AGE/ALE formation not only acceleratestau hyperphosphorylation, disturbs the neuronal membrane de-polarization process and the glucose transport but also exacerbatesglutamate-mediated excitotoxicity [12,13]. Thirdly, AGE promote

via their receptors RAGE oxidative stress and inflammation as wellas cell apoptosis [5,12,13].

New treatments are urgently needed since current AD therapiesoffer only short-term benefits to patients by transiently improvingthe cognitive symptoms [15]. Considering the multifactorial path-ogenesis of AD, an attractive strategy has recently emerged favoringthe design of multifunctional drugs [1]. With this in mind and as acontinuation of our effort in developing efficient AGE/ALE in-hibitors [16,17], we have designed a new type of molecules that aresimultaneously able to trap RCS as well as ROS and transitionmetals (Fig. 1).

Previously, we have successfully demonstrated the efficacy of2,3-diaminopropionic acid (Dap) derivatives to trap MGO and MDA(Fig. 2). In this paper, we report the synthesis of new AGE/ALE in-hibitors and the evaluation of these molecules in terms of theircapacities to trap RCS, ROS, and Cu2þ as well as their cytotoxicityand their protective effect against in vitro MGO-induced apoptosisin the model AD cell-line PC12.

2. Results and discussion

2.1. Chemistry

As shown in Scheme 1, our synthetic strategy involves: i) thetransformation of a-carbonyl group of easily available amino acids

n

Y and Z = CH2 or CO

N

R' =

O

OBn

OMe

OH

OBn

OBn

OBn,

X = NH or

NOBn

O

or

N N

BocHN

BocHN

ZX

Y

n = 1 to 3

R'

nBocHN

BocHNO

OH

R'-CH2NH2+

,

nBocHN

BocHNO

N

R'-COOH+

nBocHN

BocHN

NH2

R'-COOH+

NH

Diamine building blocks (A) Phenolic acid or HOPO ligands (B)

Scheme 1. Key coupling step between diamine building blocks (A) and phenolic acid or HOPO ligands (B).

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722704

(D or L) such as aspartic acid, glutamic acid, ornithine and lysineinto amine group in order to create vicinal diamines, ii) the use ofthe carboxylic or amino group of the side chain of these amino acidsfor linking the second functional moieties possessing antioxidantand siderophoric properties like phenolic acids or hydroxypyr-idinones (HOPO) [18e22]. The two functions are linked by differentspacers including amide, piperazine or simple alkyl chain. Thisstrategy also allows varying the size of such linkers.

2.1.1. Synthesis of diamine building blocks (A)2.1.1.1. Aspartic and glutamic acid derivatives. Different scaffoldsbearing diamine groupwere first synthesized starting from asparticacid, glutamic acid, ornithine or lysine (L or D). New compounds inwhich Z is a carbonyl group were obtained starting from asparticacid or glutamic acid (Scheme 1). The side-chain acid function wasselectively esterified and amine function was then protected by t-butoxycarbonyl (Boc) group to give compounds 3 and 4 (Scheme 2)[23,24]. At this stage, two methods were investigated to optimizethe synthetic pathway (Scheme 2). The route A provided com-pounds 5 and 6 in 77e81% after the NaBH4 reduction of theirsuccinimide active ester [25]. Following a procedure described byMarkidis et al., azide derivatives 9 and 10 were successfully pre-pared in two steps after the conversion of hydroxy group tomesylate [26,27]. Catalytic hydrogenation of the azide group in thepresence of (Boc)2O and subsequent hydrolysis of methyl esterafforded compounds 11 and 12 in 58e63% yield [28]. According tomethod B, treatment of acyl chlorides obtained from compounds 3and 4 with 25% ammonia solution gave the amide derivatives 13and 14 in 68e77% yield [29,30]. Corresponding cyano in-termediates 15 and 16were subsequently prepared by dehydrationusing trifluoroacetic anhydride and triethylamine (instead of pyri-dine) [29,30]. The nitriles 15 and 16 were transformed into N-Bocprotected diamines following the protocol of Caddick et al. [31]. Itwas found that method A gave compound 11 in 32% overall yield (vs20% by method B), whereas compound 12was obtained by methodB in better overall yield of 38% (vs 17% by method A). However,method B that required one step less was preferred.

The resulting diamine derivatives 11 and 12 were converted tothe succinimides and then coupled with 1-benzyloxycarbonyl-piperazine (1-Cbz-piperazine) in situ in the presence of triethyl-amine (introduction of which considerably improved the yield: 97%yield for compound 18 vs 53% without triethylamine) (Scheme 2).EDC/HOBt coupling gave less satisfactory result (77% yield forcompound 17 vs 24% with EDC/HOBt coupling) [16,17]. Catalytichydrogenation of compounds 17 and 18 afforded compounds 19and 20 in 100 and 96% yield, respectively.

2.1.1.2. Ornithine and lysine derivatives. Other new AGE/ALE in-hibitors in which Z is a methylene group were synthesized fromcommercially available Boc-Orn(Z)-OH and Boc-Lys(Z)-OH (Scheme1). In the samemanner as described for the synthesis of compounds11 and 12, two routes were performed to obtain diamine buildingblocks 29 and 30 (Scheme 3). In the synthetic pathway A, NaBH4reduction of acid function provided primary alcohols 21 and 22 in72e100% yield. Mesylate formation followed by treatment withNaN3 afforded azide intermediates 25 and 26 [32e34]. These werereduced to the corresponding amine derivatives by two mild pro-cedures involving a sodium borohydride/copper(II) sulfate systemor triphenylphosphine [32,33,35,36]. Following the syntheticpathway B, amide intermediates 31 and 32 were synthesizedstarting from Boc-Orn(Cbz)-OH and Boc-Lys(Cbz)-OH, respectively,under previously described conditions in 80e91% yield [29,37,38].Corresponding cyano intermediates 33 and 34 were similarly ob-tained and subsequently reduced to give compounds 27 and 28 inexcellent yields [29e31]. Catalytic hydrogenation of protected side-chain amine function thus afforded the starting building blocks 29and 30 in quantitative yield [33]. Finally, a comparative study ofboth methods A and B proved that the second one is preferable dueto its higher overall yields. (39e46% overall yield with route A vs68e83% with route B).

These diamine building blocks (A) were coupled with differentphenolic acid or HOPO ligands (B) to develop new multifunctionalAGE/ALE inhibitors.

OMeBocHN

HO

OMeBocHN

N3

OMeBocHN

MsO v

OHBocHN

BocHN

ivO

n n n

n

O O

O

5 7 9

11

13 15

BocHN

H2N O

n OMe

O

BocHN

CN

n OMe

Oviii

3 or 4

OHH2N

HO O

OMeH2N

HO Oi

OMeBocHN

HO Oii

iii

.HCl

O

n

Asp-OH

O O

n n

1 3n = 1

vii

Method B

Method A

ix

Method B

9 or 10

vi

Method A

15 or 16

n = 2 Glu-OH 2 4

6 8 10

12

14 16

n = 1n = 2

n = 1n = 2

n = 1n = 2

x

NBocHN

BocHN

n

O

1718

xi

NCbz

NBocHN

BocHN

n

O

1920

NH

Scheme 2. Reagents and conditions: (i) methanolic HCl (1.5 equiv), MeOH, 0 �C, 3 h then rt, 1e1.5 h, 100%; (ii) Boc2O (1.5 equiv), NaHCO3 (2.5 equiv), 1,4-dioxane/H2O 2:1, rt,20e24 h, 75e77%; (iii) 1) NHS (1.5 equiv), DCC (1.2 equiv), CH2Cl2, rt, overnight, 2) NaBH4 (2 equiv), THF/EtOH 3:2, 0 �C, 5 h, 77e81%; (iv) MsCl (1.2 equiv), Et3N (1.5 equiv), CH2Cl2,0 �C then rt, 1e18 h, 53e79%; (v) NaN3 (4 equiv), DMF, 60 �C, under Ar, 5e7 h, 70e80%; (vi) 1) H2, Pd/C (10% w/w), Boc2O (1.2 equiv), MeOH, rt, 6 h, 2) 4 N aqueous LiOH (4 equiv),THF/H2O 1:1, rt, 0.75e1 h, 58e63%; (vii) 1) ClCOOEt (1.4 equiv), Et3N (1.1 equiv), THF, �15 �C, 30 min, 2) 25% aqueous NH3 (2.5e2.7 equiv), �15 �C then rt, 18 h, 68e77%; (viii) TFAA(1.5 equiv), Et3N (3 equiv), THF, �10 �C, 2e4 h, 60e74%; (ix) 1) NaBH4 (8 equiv), NiCl2.6H2O (0.1 equiv), Boc2O (2 equiv), MeOH, 0 �C then rt, 3 h, 2) 4 N aqueous LiOH (4 equiv), THF/H2O 1:1, rt, 1e1.5 h, 50e67%; (x) 1) NHS (1.2e1.5 equiv), DCC (1.1e1.2 equiv), CH2Cl2, rt, overnight, 2) 1-Cbz-piperazine hydrochloride (1.2 equiv), Et3N (3 equiv), CH2Cl2, rt, 18 h,77e97%; (xi) H2, Pd/C (10% w/w), MeOH, rt, 6 h; 96e100%.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 705

2.1.2. Coupling of phenolic acid or HOPO ligands (B)2.1.2.1. Phenolic acid family. After DCC/NHS activation, antioxidantphenolic acids were coupled with different previously synthesizeddiamine scaffolds (A) in dichloromethane at room temperature (rt)to give amide intermediates 35 and 38 in good yields (Scheme 4).Whereas gallic acid had to be protected beforehand in its threephenolic functions according to a literature procedure [39],commercially available ferulic acid could be used directly.Furthermore, addition of triethylamine significantly improved theyield of condensation between gallic acid tribenzyl ether 37 andpiperazine derivative 20 (82% vs 21% without triethylamine).Ferulic acid compounds 36a-b were finally obtained as dihydro-chloride salts in 66e90% yield after the removal of N,N0-di-Boc bytreatment with 4N HCl in 1,4-dioxane [16,17]. Likewise, the syn-thesis of final products 40a-b was achieved by starting with gallicacid derivatives 38a-b that required the cleavage of benzyl groupsby catalytic hydrogenation followed by N,N0-di-Boc removal.

2.1.2.2. Hydroxypyridinone (HOPO) family. Hydroxypyridinone(3,2-HOPO and 2-methyl-3,4-HOPO) derivatives are reported to beexcellent iron chelators as well as radical scavengers with potentialprofile for therapeutic applications. We have anticipated that hy-bridization of such a moiety with vicinal alkyl diamines couldprovide a beneficial means to eradicate many deleterious RCS and

ROS. Accordingly, 3,2-HOPO and 2-methyl-3,4-HOPO derivativesthat possess carboxylic or amine group allowed the linkage for-mation with previously described diamine moiety. Taking this intoaccount, nitrile derivative 42 was prepared from 2,3-dihydroxypyridine and acrylonitrile using the protocole of Aru-mugam et al. [40]. After subsequent O-benzylation, compound 42was converted to carboxylic derivative 43 and amine derivative 44by alkaline hydrolysis and NaBH4 reduction followed by N-Bocremoval, respectively (Scheme 5) [31].

By the same token, analogous N-carboxyethyl and N-amino-propyl 2-methyl-3,4-HOPO derivatives 47 and 48were prepared bythe treatment of benzyl maltol with b-alanine or 1,3-diaminopropane under alkaline conditions, respectively (Scheme6) [41e44]. Compound 48 was further converted to hydrochloridesalt.

A number of new final products bearing hydropyridinonechelator group were synthesized by a coupling, O-debenzylationand diamine function deprotection sequence (Scheme 7). Twodifferent methods involving a preliminary NHS/DCC acid activationor a treatment with EDC/HOBt at rt were successfully carried out forthe condensation of carboxy ligands 43, 47 or 12 with variousamino compounds to give amide derivatives 49a-e and 52a-b[16,17,41]. Introduction of triethylamine in reaction mixture wasrequired when using N-succinimide intermediates to optimize

n = 3

NHCbzBocHN

HO

NHCbzBocHN

MsO

NHCbzBocHN

N3

NHCbzBocHN

BocHN

NH2BocHN

BocHN

21 23

27 29

25

ii

v

n n

n n

n

Orn(Cbz)-OH

Boc-

i

vi

Method B

Method A

ii

vii

NHCbzBocHN

H2N

n NHCbzBocHN

CN

n

31 33

O

33 or 34

25 or 26iv

viii

Method A

Method B

or

Boc-

Lys(Cbz)-OH

n = 2

4222 26

4323

0382

n = 3n = 2

n = 3n = 2

Scheme 3. Reagents and conditions: (i) 1) ClCOOEt (1 equiv), NMM (1 equiv), THF, �10 �C, 10 min, 2) NaBH4 (2.8e3 equiv), THF/MeOH 2:3, 0 �C, 10e15 min, 72e100%; (ii) MsCl(1e1.2 equiv), Et3N (1e1.1 equiv), CH2Cl2, 0 �C, 1.5e2 h, 84e92%; (iii) NaN3 (3 equiv), DMF, 60 �C, under Ar, 1.5e3 h, 62e78%; (iv) 1) NaBH4 (1 equiv), CuSO4.5H2O (0.01 equiv), MeOH,0 �C, 1 h, or PPh3 (2.5 equiv), Toluene/H2O 16:1, reflux, 18 h, 2) Boc2O (1e1.7 equiv), rt, 2 h, 76e95%; (v) H2, Pd/C (10% w/w), MeOH, rt, 6 h; 92e100%; (vi) 1) ClCOOEt (1.1 equiv), NMM(1.1 equiv), THF, �10 �C, 20 min, 2) 25% aqueous NH3 (2.4e2.6 equiv), �10 �C then rt, 4 h, 80e91%; (vii) TFAA (1.5 equiv), pyridine (3 equiv), THF, �10 �C, 2e4 h, 95e99%; (viii) 1)NaBH4 (7 equiv), NiCl2.6H2O (0.1 equiv), Boc2O (2 equiv), MeOH, 0 �C then rt, 1 h, 89e92%.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722706

yields (68% yield for compound 49e vs 12% without triethylamine).The synthesis of piperazine derivatives 49a-b revealed rather lowyields (39% and 25%, respectively). EDC/HOBt coupling method didnot improve the yield (11% yield with EDC/HOBt treatment forcompound 49b). Finally, benzyl and Boc-protecting group removalswere performed in excellent yields to provide compounds 51a-eand 54a-b as hydrochloride salts [16,17]. Another derivative 55used for structure-activity relationship studies was also obtainedafter quantitative N-Boc removal of intermediate 49d in 100% yield.

New hybrid diamine derivatives have thus been synthesized in10e32% overall yield for phenolic acid family (6e10 steps) vs 4e63%overall yield for HOPO one (7e10 steps). Their physicochemical andbiological properties as multifunctional AGE/ALE inhibitors havethen been investigated.

2.2. Physicochemical and biological evaluations

2.2.1. MGO and MDA trapping assayIn order to evaluate their carbonyl trapping capacity, the syn-

thesized compounds were incubated withMGO orMDA at 37 �C for24 h. Samples collected at regular time intervals were analyzed byLCMS. The identification of major adducts with MGO and MDA onmass spectra allowed to perform a kinetic study of adduct forma-tion (Figs. 3 and 4). Indeed, the primary vicinal diamine function-ality reacts with dicarbonyl compounds under physiologicalconditions to form stable 2,3-dihydropyrazine and 1,4-diazepineadducts, respectively. In the former case, adducts further reactwith a secondmolecule of MGO to end upwith pyrazine derivatives[16,17]. Area under the curve (AUC) of total peak of adducts wascompared with remaining free scavenger peak on UV chromato-gram at 190 nm [45]. Newly designed diamine compounds werefound to be very potent RCS scavengers, even more efficient thancarnosine used as reference and previously described Dap de-rivatives (i.e., Dap-Pip and Dap-(nBu)Pip) [17,46]. Indeed, more than

81% of MGO adduct formationwas observed in 15min of incubationwith all new compounds vs only up to 59% with Dap derivatives(Table 1). As expected, the removal of carbonyl group adjacent todiamine function in the new series greatly improved nitrogen atombasicity and reactivity. Results obtained for MDA trapping appearedmore contrasting. In fact, several interesting scavengers weredetected in the phenolic acid family as well as in HOPO one (Fig. 4).Galloyl compound 40b showed a good MDA trapping abilitycompared to feruloyl analog 36b and carnosine (84% vs 17% and 40%MDA adduct formation, respectively, in 1 h of incubation). Although3,2-HOPO derivatives 51a-b and 51e as well as 2-methyl-3,4-HOPOones 54a-b showed a potent MDA scavenging capacity (Table 1:more than 81% MDA adduct formation in 1 h of incubation), a lessinteresting result was obtainedwith 51c (7%MDA adduct formationin 1 h of incubation). There seems to be not significant differencebetween 3,2-HOPO and 2-methyl-3,4-HOPO derivatives in terms ofthe capacity of MDA adduct formation (Fig. 4: 54a vs 51d and 54b vs51e). Furthermore, in 3,2-HOPO series, the introduction of apiperazine cycle inside the linker did not decrease the activitywhereas the carbonyl position with respect to diamine functioncould affect it (Fig. 4: 51e vs 51b and 51c).

It is found that newly synthesized diamine derivatives trapMGOslightly faster than MDA (e.g., 95% vs 76% adduct formation,respectively, for 54b in 15min of incubation). Compounds 40b, 51a-b, 51e and 54a-b appeared as the most potent RCS scavengers. Now,a further screening by ORAC and Cu2þ-chelating assays is to becarried out to investigate their potential interest.

2.2.2. Oxygen radical absorbance capacity (ORAC) assayThe antioxidant activity of new hybrid diamine derivatives was

assessed by the oxygen radical absorbance capacity assay usingfluorescein (ORACFL) [47,48]. Peroxyl radicals generated from AAPHat 37 �C reacted with this fluorescent probe to form a nonfluores-cent product. The protective effect of the tested compounds were

Scheme 4. Reagents and conditions: (i) 1) NHS (1.1e1.2 equiv), DCC (1e1.1 equiv), 1,4-dioxane, rt, overnight, 2) 20 or 30 (1e1.2 equiv), Et3N (0e3 equiv), CH2Cl2, rt, 18 h, 55e82%; (ii)1) BnBr (4 equiv), K2CO3 (4 equiv), DMF, rt then 40 �C, 20 h, 2) 5 N aqueous NaOH (25 equiv), EtOH/H2O 1:1, reflux, 3 h, 3) conc. HCl (pH 2), H2O, rt, 30 min, 80%; (iii) H2, Pd/C (10% w/w), MeOH, rt, 6 h; 99e100%; (iv) 4 N HCl in 1,4-dioxane (20 equiv), 1,4-dioxane, rt, 0.75e2 h, 57e100%.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 707

determined following FL fluorescence decay in time and measuringAUC of the sample in comparisonwith the control corresponding toan absence of antioxidant (Fig. 5).

Trolox, a water-soluble vitamin E analog was used as standardfor the calculation of ORACFL values at 10 mM expressed as mmoltrolox equivalent (TE)/mmol of tested compound with respect to thelinear equation of its calibration curve (Net AUC vs concentration)

[49]. As shown in Fig. 6, the majority of newly designed compoundshad important radical scavenging capacity (Table 1: ORACFLvalues � 1 mmol TE/mmol) compared to a very weak activity ofcarnosine, cited as a good antioxidant [50]. Since Dap-Pip had noantioxidant activity as assessed by ORAC, the introduction ofphenolic or HOPOmoieties revealed to be crucial for the acquisitionof antioxidant properties. It is particularly interesting to note that

41

N

ii

OH

OH

N O

OH

CN42

N O

OBn

CN

43

N O

OBn

44

N O

OBn

BocHN

iii iv

v

45

N O

OBn

H2N

.HCl

i

HO O

2,3-dihydroxypyridine

Scheme 5. Reagents and conditions: (i) Acrylonitrile (3 equiv), CsF (0.1 equiv), MeCN, reflux, 16 h, 93%; (ii) BnBr (1.1 equiv), K2CO3 (1 equiv), MeCN, reflux, 18 h, 90%; (iii) NaOH(12.5 equiv), H2O, reflux, 1 h, 65%; (iv) NaBH4 (7 equiv), NiCl2.6H2O (0.1 equiv), Boc2O (2 equiv), MeOH, 0 �C then rt, 1 h, 84%; (v) 4 N HCl in 1,4-dioxane (20 equiv), 1,4-dioxane, rt, 2 h,100%.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722708

the galloyl derivative 40b was a more potent antioxidant than itsferuloyl analog 36b with (ORACFL values of 2.59 and 1.50 mmol TE/mmol, respectively). Judging from the good result obtainedwith 36a(Table 1), it is noted that both phenolic groups conserve theirantioxidant properties in the hybrid structure [18,19]. Furthermore,HOPO moiety, well-known as siderophore, also showed goodantioxidant activity [51]. In the present case, 3,2-HOPO derivative51e exhibited more interesting protective effects than its 2-methyl-3,4-HOPO analog 54b (ORACFL values of 1.96 and 1.01 mmol TE/mmol, respectively) contrary to results observed for RCS trapping.Finally, in the 3,2-HOPO series, the simple modification of carbonylposition inside the linker or the introduction of a piperazine cyclecould affect radical scavenging efficiency (Table 1: 51e vs 51c or51b).

So far, compounds 40b and 51e, each representing phenolic acidand HOPO family, respectively, appeared to be the most efficientROS scavengers (Fig. 6). Despite its good antioxidant activity, de-rivative 36a was ruled out for further investigations because of itsweak Cu2þ-chelating capacities.

46

O

OH

O

OBn

N

OBn

48

N

OBn

H2N

.HCl

i

O O

O

OHO

Maltoliii

ii

O

47

Scheme 6. Reagents and conditions: (i) BnCl (1.2 equiv), 10.5 N aqueous NaOH(1.1 equiv), MeOH, reflux, 18 h, 88%; (ii) b-alanine (2 equiv), NaOH (3 equiv), EtOH/H2O1:1, reflux, 18 h, 45%; (iii) 1) 1,3-diaminopropane (1.1 equiv), NaOH (0.5 equiv), EtOH/H2O 1:1, reflux, 3 h, 2) 4 N HCl in 1,4-dioxane (5 equiv), 1,4-dioxane, rt, 2 h, 48%.

2.2.3. Cu2þ-chelating assayCu2þ-chelating capacity of new hybrid diamine compounds was

assessed using murexide as complexometric indicator [52]. Testedcompounds at different concentrations were first incubated withCuSO4.5H2O before introduction of murexide that reacted withremaining free Cu2þ. The absorbance ratio A485/A520 (lmax of Cu2þ/murexide complex: 485 nm and lmax of free murexide: 520 nm)provided the remaining free Cu2þ concentration with respect tocalibration curves (A485/A520 vs Cu2þ concentration). Knowing thetotal quantity of metal ions introduced into the reaction mixture(control conditions without tested product), percentage of Cu2þ

chelation by tested compounds was estimated by difference [53].Except for 36b, all new hybrid diamine compounds showed Cu2þ-chelating capacity much superior to that of carnosine and Dapderivatives. This confirms the interest of newly designed pharma-comodulations (Fig. 7). In the phenolic acid family, galloyl com-pound 40b turned out to be almost as active as the positivestandard EDA (ethylenediamine) (Table 1: 87.39% and 100% Cu2þ

chelation at 200 mM, respectively). This result provides novel evi-dence that demonstrates the Cu2þ-chelating capacity of phenolicacids [54]. In HOPO family, 2-methyl-3,4-HOPO derivatives showedslightly better efficacy than 3,2-HOPO derivatives in complexingCu2þ (Table 1: 54a vs 51d and 54b vs 51ewith 74.70% vs 62.77% and66.70% vs 55.83% Cu2þ chelation at 200 mM, respectively). The na-ture and the length of the linker seem to have no particular role inCu2þ chelation (Fig. 7: 51e vs 51c or 51b). However, a questionarises as to the proportion of the Cu2þ chelation between thediamine function and HOPO moiety. Indeed, in case of Dap de-rivatives, the diamine function showed about 28% Cu2þ chelation at200 mM (Table 1). Similar chelating capacity (34%) was found withcompound 55 whose hydroxy group on HOPO is benzylated. Whenthe diamine function is protected by Boc group as seen in com-pound 50d, HOPO exhibited its chelating capacity in 55%. Nowwithcompound 51d in which both diamine and HOPO moiety bear noprotecting group, the chelating capacity of the entire moleculeslightly increased up to 63%. From this comparison, it is apparentthat with the present new compounds the role of HOPO moiety ismore important than that of diamine group as far as the Cu2þ-chelating capacity is concerned [20e22].

Finally, in view of the results obtained from the three differentphysicochemical assays, multifunctional scavengers 40b and 51e

Scheme 7. Reagents and conditions: (i) 1) NHS (1.2e1.5 equiv), DCC (1.1e1.2 equiv), 1,4-dioxane or CH2Cl2, rt, overnight, 2) 19, 20, 45 or 48 (1.2e1.7 equiv), Et3N (3e4 equiv), CH2Cl2,rt, 18 h, 25e68%; (ii) 29 or 30 (1 equiv), EDC (1.2 equiv), HOBt (1.1e1.2 equiv), Et3N (1.2 equiv), THF or DMF, 0 �C then rt, 4e18 h, 69e86%; (iii) H2, Pd/C (10% w/w), MeOH, rt, 6 h;60e100%; (iv) 4 N HCl in 1,4-dioxane (20 equiv), 1,4-dioxane, rt, 2 h, 88e100%.

0

20

40

60

80

100

0 5 10 15 20

% M

GO

Add

ucts

Time (h)

CarnosineDap-PipDap-(nBu)Pip36a (ND)36b40a40b51a51b51c51d51e54a54b

Fig. 3. Kinetic curves representing the formation of MGO adducts in the presence of different scavengers. Tested compounds (10 mM) dissolved in D-PBS (pH 7.4) wereincubated with MGO (20 mM) at 37 �C for 24 h. Samples collected at regular time intervals were subsequently analyzed by LCMS and AUC of MGO adduct peak and remaining freescavenger peak were measured on UV chromatogram at 190 nm. Data are expressed as % MGO adducts compared with remaining free scavenger for a representative single sample.ND: Not determined.

0

20

40

60

80

100

0 5 10 15 20

% M

DA

Add

ucts

Time (h)

CarnosineDap-PipDap-(nBu)Pip36a (ND)36b40a40b51a51b51c51d51e54a54b

Fig. 4. Kinetic curves representing the formation of MDA adducts in the presence of different scavengers. Tested compounds (10 mM) dissolved in D-PBS (pH 7.4) wereincubated with MDA (20 mM) at 37 �C for 24 h. Samples collected at regular time intervals were subsequently analyzed by LCMS and AUC of MDA adduct peak and remaining freescavenger peak were measured on UV chromatogram at 190 nm. Data are expressed as % MDA adducts compared with remaining free scavenger for a representative single sample.ND: Not determined.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722710

can be chosen as lead compounds representing phenolic and HOPOfamily, respectively.

2.2.4. Cell viability assayCytotoxicity of the newmultifunctional diamine derivatives was

evaluated in neuronal-like cell-line PC12 derived from primary ratpheochromocytoma, an in vitro model widely used to studyneurodegenerative diseases [55,56]. Cell viability was determinedusing the sensitive colorimetric CCK-8 (cell counting kit-8) assay[57]. In this test, the WST-8 reagent was reduced by mitochondrialdehydrogenases in cells to give awater-soluble formazan dye in thepresence of 1-methoxy PMS that reacts as an electron carrier. Aftercell treatment with various concentrations of tested compounds for

24 h, medium absorbance measurement provided an estimation ofthe number of living cells. A positive cytotoxicity standard wasassessed by adding 10% DMSO and cell viability was expressed as %of control conditions (non-treated cells). Except for less interestingcompound 51a, no cytotoxicity of new hybrid diamine derivativeswas observed in PC12 cells after 24 h of treatment at 10 mM as wellas at 100 mM (Fig. 8).

2.2.5. In vitro MGO-induced apoptosis inhibition assayAs AGE/ALE inhibitors, our original designed compounds might

be able to limit MGO-induced apoptosis in AD. In contrast to thecontradictory pro- or antioxidant behavior of phenolic acidsrecently reported in the literature [58,59], hydroxypyridinones are

Table 1RCS trapping, oxygen radical absorbance and Cu2þ-chelating capacities of new hybrid diamine derivatives and references.

Compound % MGO adduct formationa % MDA adduct formationb ORACFLc (mmol TE/mmol) % Cu2þ chelationd

Trolox ND ND 1 NDEDA ND ND ND 100Carnosine 46 40 0.08 ± 0.05 1.53 ± 1.18Dap-Pip ND 100 0 23.60 ± 0.59Dap-(nBu)Pip 59 ND ND 27.68 ± 0.2836a ND ND 2.05 ± 0.23 45.61 ± 0.8536b 100 17 1.50 ± 0.10 ND40a 81 68 0.89 ± 0.10 52.75 ± 0.9440b 90 84 2.59 ± 0.35 87.39 ± 1.3851a 99 87 0.58 ± 0.02 50.25 ± 1.2751b 97 81 0.99 ± 0.09 53.23 ± 0.4051c 97 7 1.03 ± 0.03 59.96 ± 0.3051d 98 69 1.21 ± 0.07 62.77 ± 0.1651e 97 81 1.96 ± 0.24 55.83 ± 0.1754a 99 88 1.24 ± 0.02 74.70 ± 0.2754b 95 92 1.01 ± 0.06 66.70 ± 0.1650d ND ND ND 54.61 ± 0.9855 ND ND ND 33.91 ± 0.33

TE: Trolox equivalent.ND: Not determined.Most interesting compounds are represented in bold.

a Data obtained in 15 min of incubation for a representative single sample.b Data obtained in 1 h of incubation for a representative single sample.c ORACFL values are expressed as means ± standard error of the mean (SEM) of three independent experiments performed in triplicate and calculated at 10 mM.d % Cu2þ chelation are expressed as means ± SEM of triplicates and measured at 200 mM.

Fig. 5. FL fluorescence decay curve induced by AAPH. Tested compounds (10 mM) or trolox standard (1e50 mM) were pre-incubated with FL (12 nM) in D-PBS (pH 7.4) at 37 �C forat least 30 min. AAPH reagent (30 mM), used as a peroxyl radical generator was added. This led to FL disappearance and the remaining FL fluorescence (lEx: 485 nm; lEm: 520 nm)was measured every 90 s for 60 cycles. Represented data show results obtained in the absence (control) or the presence of compound (A) 40b or (B) 51e at different concentrationsand are expressed as means ± SEM of triplicates that form a representative experiment among the at least performed three independent ones.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 711

0

20

40

60

80

100

0 0,5 1 1,5 2

% C

u2+

chel

atio

n

Concentration (mM)

EDA CarnosineDap-Pip Dap-(nBu)Pip36a 36b40a 40b51a 51b51c 51d51e 54a54b 50d55

Fig. 7. Evaluation of Cu2þ-chelating capacity of new hybrid diamine derivatives and references. Tested compounds (0e2 mM) were incubated with CuSO4.5H2O (120 mM) in a10 mM hexamine/HCl buffer containing 10 mM KCl (pH 5.0) or in a mixture buffer/MeOH 75:25 for 10 min at rt. Murexide (50 mM), used as a complexometric indicator reacting withremaining free Cu2þ, was added and the mixture was incubated for additional 1 min at rt. The absorbance ratio A485/A520 (lmax of Cu2þ/murexide complex: 485 nm and lmax of freemurexide: 520 nm) provided remaining free Cu2þ concentration with respect to calibration curves (A485/A520 vs Cu2þ concentration). Knowing the total quantity of metal ionsintroduced in the reaction mixture (control conditions without tested product), % Cu2þ chelation by tested compounds was calculated by difference. Data are presented asmeans ± SEM of triplicates.

Fig. 6. ORACFL values of new hybrid diamine derivatives and references. Bars represent the means ± SEM calculated at 10 mM of at least three independent experimentsperformed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001 vs Trolox (Student's t test: p values < 0.05 were considered significant).

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722712

considered as privileged chelating structures for designing medic-inal drugs [21,22]. Compound 51e was preferred to compound 40bon that account for biological studies. PC12 cells were thus pre-treated with the lead compound 51e at 37 �C before incubation inthe presence of MGO. In vitro MGO-induced apoptosis was thenmeasured using an ELISA detection of DNA fragmentation. Theoptical density (OD) reflected apoptosis level [59,60]. Cells incu-bated without MGO represented control conditions and a positiveapoptosis standard was also assessed in the presence of MGO, butin the absence of tested product. Finally, as shown in Fig. 9, MGO-induced apoptosis is attenuated in the presence of lead com-pound 51e at 100 mM on the model neuronal-like cell-linePC12 cells.

Together with the good physicochemical results, this new dataprovides the first promising biological evidence of the excellentcapacity of new diamine compounds to limit the vicious downwardcarbonyl redox amyloid spiral that lead to neurodegeneration inAD. Further investigations are required to evaluate their druglike-ness. However, a prediction of their ADME properties was

performed using QikProp, a Schr€odinger software. Thus, logPo/w(octanol/water partition coefficient) and logBB (brain/blood parti-tion coefficient) values of lead compounds 40b and 51e werecalculated (clogPo/w of �1801 and �2,110, respectively and clogBBof �2419 and �1,766, respectively). Despite their hydrophilicity,their clogBB appear in the range of QikProp-recommended values(�3<logBB<1,2). Nevertheless, we are aware that extended phar-macomodulations on the linker can improve their ADME propertiesand especially their capacity to cross the blood brain barrier. We arecurrently working on this point and the results will be disclosed indue course.

3. Conclusion

Taking into account the multifactorial pathogenesis of AD, thedevelopment of new multifunctional diamine derivatives is apromising therapy. In this work, we have designed new diaminestructures that act as AGE/ALE inhibitors (primary vicinal diaminefunction) with additional antioxidant and biometal chelating

Fig. 9. In vitro evaluation of MGO-induced apoptosis inhibition. PC12 cells wereincubated for 24 h in media (control), and with MGO (1 mM) in the absence or thepresence of either 10 mM or 100 mM of compound 51e. At the end of the incubation, thecells were lysed and analyzed for DNA fragmentation. *p < 0.05; **p < 0.01 vs control(Student's t test: p values < 0.05 were considered significant).

Fig. 8. Evaluation of cell viability. PC12 cells were incubated with 10 mM or 100 mM of the different compounds for 24 h. DMSO was used as a positive cytotoxicity control. Resultsare expressed as % of control (non-treated cells). Bars represent the mean ± SEM of at least three independent experiments performed in triplicate. *p < 0.05; **p < 0.01;***p < 0.001 vs control (Student's t test: p values < 0.05 were considered significant).

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 713

properties (phenolic acid or HOPO moiety). Diamine buildingblocks were first synthesized from various natural a-amino acidsand easily coupled with phenolic acids or HOPO ligands. Two leadcompounds 40b and 51e bearing galloyl or 3,2-HOPO group,respectively, demonstrated particularly interesting RCS, ROS andCu2þ-scavenging capacities. No cytotoxicity of this original hybridseries was observed in the model AD cell-line PC12. Furthermore,privileged compound 51e offered promising protective biologicalactivity after in vitro MGO-induced apoptosis. Although moreextended in vitro and in vivo biological investigations are required,the multifunctional diamine 51e seem to be able to prevent theoxidative stress extension and AGE/ALE accumulation that are bothimplicated in the pathogenesis of AD.

4. Experimental

All commercial reagents were purchased from SigmaeAldrich(Lyon, France) and were used without further purification unlessotherwise specified. Merck Silica gel 60 (40e63 mm) or a GraceReveleris® system in normal phase was used for column chroma-tography. Melting points (mp) were determined on a Stuart SMP3

apparatus and IR measurements were performed on a Jasco FT/IR-4200 system fitted with an ATR-golden gate. 1H and Q-DEPT NMRspectra were recorded on a Bruker AC600, 400, or 300 spectrometer.Chemical shifts (d) are expressed in parts per million (ppm)downfield from tetramethylsilane as an internal standard and thesignals are quoted as s (singlet), br s (broad singlet), d (doublet), t(triplet), br t (broad triplet), q (quartet) or m (multiplet) and np(negative peak). Coupling constant values are given in Hertz. Massspectra (MS) and high-resolution mass spectra (HRMS) (electro-spray in positive mode, ESI þ) were recorded on a Shimadzu LCMS-2020 system and a Micromass Q-TOF Ultima apparatus, respectively.For LCMS, UV chromatograms and mass spectra were obtained at190 nm and by positive ESI-MS interface (detection mode: scan,interface voltage: tuning file, DL voltage: 100 V, Q-array DC: 40 V,Q-array RF: 40 V) after gradient elution on aWaters Acquity columnusing an injection volume of 1 mL and a mobile phase composed ofwater/acetonitrile (solvent A/solvent B) with 0.1% formic acid (98:2during 2 min, 55:45 during 2 min and 45:55 during 3 min with aflow of 0.3 mL/min at 40 �C).

4.1. Chemistry

For compounds 1e7, 9e10, 21e26, 31e32, 34, 37, 41, 42 and46e48, NMR spectra were in full accordance with those reported inthe literature.

4.1.1. Synthesis of diamine building blocks (A)4.1.1.1. Method A

4.1.1.1.1. Synthesis of methyl 4-((t -butoxycarbonyl)amino)-5-((methylsulfonyl)oxy)pentanoate (8). Compound 6 (40.4 mmol) wasdissolved in CH2Cl2 (100 mL) and the solution was cooled at 0 �C.Triethylamine (1.5 equiv) and mesyl chloride (1.2 equiv) were thenadded. The mixture was stirred at rt for 18 h and the resultingproduct was extracted with CH2Cl2 (2 � 80 mL). The organic phasewas washed with 0.5 N HCl (2 � 80 mL), 5% NaHCO3 (2 � 80 mL)and brine (3 � 80 mL), dried over Na2SO4 and concentrated invacuo. The crude material was purified by column chromatographyon silica gel (CH2Cl2/MeOH 98:2) to give compound 8 as a yellowpasty solid in 53% yield. IR n (cm�1): 3417, 3231, 2918, 1714, 1415,1349, 1157, 1040, 963, 777. 1H NMR (CDCl3, 300 MHz) d (ppm):4.86e4.84 (br s, 1H), 4.20e4.13 (m, 2H), 3.87 (m, 1H), 3.63 (s, 3H),3.00 (s, 3H), 2.39 (t, J ¼ 7.2 Hz, 2H), 1.90e1.82 (m, 2H), 1.39 (s, 9H).Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 172.8 (np), 154.8 (np), 79.3(np), 70.5 (np), 51.2, 48.8, 36.8, 29.8 (np), 27.7 (3C), 25.7 (np). MS-

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722714

ESI m/z: [M� Boc þ 2H]þ 226.0, [MþH]þ 326.2, [MþNa]þ 348.2,[M þ MeCN þ Na]þ 389.2. HRMS-ESI m/z calcd for C7H16NO5S [M�Boc þ 2H]þ: 226.0749, found: 226.0738.

4.1.1.1.2. General procedure for synthesis of compounds 11 and 12.To a solution of compound 9 or 10 (8.5e16.9mmol) in drymethanol(40e60 mL) were added Boc2O (1.2 equiv) and Pd/C (10% w/w). Themixture was placed under H2 and stirred for 6 h at rt. After filteringthe catalyst and evaporatingmethanol, the residuewas dissolved inTHF/H2O 1:1 (30e50 mL) and 4 N aqueous LiOH (4 equiv) solutionwas added. The mixture was stirred for 0.75e1 h and THF wasevaporated in vacuo. The aqueous phase was adjusted to pH 12e13with 10% Na2CO3 and washed with Et2O (3 � 20e40 mL) to elim-inate remaining Boc2O and methyl ester derivative. After acidifi-cation of the aqueous phasewith 6 N HCl, the resulting product wasextracted with Et2O (2� 20e40 mL). The organic layer was washedwith brine (3 � 20e40 mL), dried over Na2SO4 and evaporated invacuo to give compound 11 or 12 in 58e63% yield.

4.1.1.1.2.1. 3,4-bis((t-Butoxycarbonyl)amino)butanoic acid (11)White solid was obtained from compound 9 (8.5 mmol)

(method A) or 15 (15.3 mmol) (method B) in 63 and 50% yield,respectively: mp 114.2 �C. IR n (cm�1): 3362, 2984, 1685, 1516, 1249,1160, 1051, 620. 1H NMR (CDCl3, 300 MHz) d (ppm): 8.70e8.61 (br s,1H), 5.54e5.41 (br s, 1H), 5.15 (br s, 1H), 4.00e3.98 (m, 1H),3.37e3.25 (m, 2H), 2.65e2.59 (m, 2H), 1.43 (s, 18H). Q-DEPT NMR(CDCl3, 75 MHz) d (ppm): 174.7 (np), 174.6 (np), 146.2 (np), 79.9(np), 79.8 (np), 48.0, 43.4 (np), 36.4 (np), 28.1 (6C). MS-ESI m/z:[MþH]þ 319.2, [MþNa]þ 341.2, [MþMeCNþNa]þ 382.2. HRMS-ESIm/z calcd for C14H26N2O6Na [MþNa]þ: 341.1689, found: 341.1676.

4.1.1.1.2.2. 4,5-bis((t-Butoxycarbonyl)amino)pentanoic acid (12)White solid was obtained from compound 10 (16.9 mmol)

(method A) or 16 (19.8 mmol) (method B) in 58 and 67% yield,respectively: mp 113.8 �C [61]. IR n (cm�1): 3360, 2982, 1677, 1521,1324, 1247, 1160, 1057, 628. 1H NMR (CDCl3, 300 MHz) d (ppm):8.35e8.28 (br s, 1H), 5.06 (br s, 1H), 4.99e4.96 (br s, 1H), 3.72e3.66(m, 1H), 3.18e3.16 (m, 2H), 2.43e2.37 (m, 2H), 1.87e1.76 (m, 2H),1.43 (s, 18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 177.4 (np),156.8 (np), 156.5 (np), 79.6 (np, 2C), 51.0, 44.6 (np), 30.5 (np), 28.3(6C), 27.7 (np). MS-ESI m/z: [MþNa]þ 355.2, [M þ MeCN þ Na]þ

396.2. HRMS-ESI m/z calcd for C15H28N2O6Na [MþNa]þ: 355.1845,found: 355.1855.

4.1.1.1.3. Synthesis of benzyl 4,5-bis((t-butoxycarbonyl)amino)-pentylcarbamate (27). To a stirred solution of CuSO4.5H2O(0.01 equiv) in dry methanol (20 mL) cooled at 0 �C was addedcompound 25 (5 mmol) dissolved in dry methanol (10 mL). NaBH4(1 equiv) was then added in several portions over a period of 1 h.After filtering through celite, Boc2O (1.7 equiv) was added to thereaction mixture that was stirred for 2 h at rt. Methanol wasevaporated under reduced pressure and the residue was dissolvedin EtOAc (2 � 25 mL). The organic phase was washed with brine(3 � 25 mL), dried over Na2SO4 and concentrated in vacuo to givecompound 27 as a white pasty solid in 76% yield. 1H NMR (CDCl3,300 MHz) d (ppm): 7.34e7.31 (m, 5H), 5.06 (s, 2H), 5.06 (br s, 1H)4.94 (br s, 1H), 4.80e4.77 (br s, 1H), 3.58 (m,1H), 3.21e3.12 (m, 4H),1.56e1.52 (m, 2H), 1.45e1.44 (m, 2H), 1.43 (s, 18H). Q-DEPT NMR(CDCl3, 75MHz) d (ppm): 172.0 (np),156.9 (np, 2C),136.9 (np),128.8(2C), 127.4 (3C), 79.7 (np, 2C), 67.0 (np), 51.5, 44.8 (np), 41.1 (np),30.4 (np), 28.7 (6C), 26.5 (np). MS-ESIm/z: [MþH]þ 452.1, [MþNa]þ

474.0. HRMS-ESI m/z calcd for C23H37N3O6Na [MþNa]þ: 474.2580,found: 474.2560.

4.1.1.1.4. Synthesis of benzyl 5,6-bis((t-butoxycarbonyl)amino)-hexylcarbamate (28). A solution of compound 26 (2.5 mmol) andtriphenylphosphine (2.5 equiv) in toluene (16 mL) was heatedunder reflux for 15 min and cooled to rt to add H2O (1 mL). Themixture was then heated under reflux for 18 h before adding Boc2O(1 equiv) at rt and stirred for further 2 h. Toluene was evaporated

under reduced pressure and the resulting product was extractedwith EtOAc (2 � 15 mL). The organic phase was washed with brine(3 � 15 mL), dried over Na2SO4 and concentrated in vacuo to givecompound 28 as a white solid in 95% yield after purification bycolumn chromatography on silica gel (EtOAc/Cyclohexane 40:60).mp 101.7 �C [33]. IR n (cm�1): 3358, 2929, 1680, 1247, 1161, 634. 1HNMR (CDCl3, 300 MHz) d (ppm): 7.34e7.29 (m, 5H), 5.07 (s, 2H),4.98e4.91 (br s, 2H), 4.72 (br s, 1H), 3.56 (m, 1H), 3.17e3.13 (m, 4H),1.46e1.41 (m, 6H), 1.41 (s, 18H). Q-DEPT NMR (CDCl3, 75 MHz)d (ppm): 176.3 (np), 155.8 (np, 2C), 136.0 (np), 127.8 (2C), 127.4 (3C),78.7 (np, 2C), 65.9 (np), 50.5, 43.9 (np), 39.8 (np), 31.6 (np), 29.1(np), 27.7 (6C), 22.1 (np). MS-ESI m/z: [MþH]þ 466.3, [MþNa]þ

488.3. HRMS-ESI m/z calcd for C24H39N3O6Na [MþNa]þ: 488.2737,found: 488.2730.

4.1.1.1.5. General procedure for synthesis of compounds 29 and 30.To a solution of compound 27 or 28 (1.1e5.7 mmol) in drymethanol(10e40 mL) was added Pd/C (10% w/w). The mixture was placedunder H2 and stirred for 6 h at rt. After filtering the catalyst andevaporating methanol, compound 29 or 30 was obtained in92e100% yield.

4.1.1.1.5.1. t-Butyl 5-aminopentane-1,2-diyldicarbamate (29)White pasty solid was obtained from compound 27 (5.7 mmol)

in 92% yield. 1H NMR (CDCl3, 400MHz) d (ppm): 4.98 (br s,1H), 4.96(br s, 1H), 3.56 (m, 1H), 3.18e3.12 (m, 2H), 2.74 (br s, 2H), 2.74 (m,2H), 1.48e1.43 (m, 4H), 1.41 (s, 18H). Q-DEPT NMR (CDCl3, 100MHz)d (ppm): 156.7 (np), 156.3 (np), 79.3 (2C), 51.2, 44.7 (np), 41.6 (np),30.1 (np), 29.3 (np), 28.4 (6C). MS-ESIm/z: [MþH]þ 318.2, [MþNa]þ

340.2. HRMS-ESI m/z calcd for C15H32N3O4 [MþH]þ: 318.2393,found: 318.2379.

4.1.1.1.5.2. t-Butyl 6-aminohexane-1,2-diyldicarbamate (30)White solid was obtained from compound 28 (1.1mmol) in 100%

yield: mp 81.4 �C [33]. IR n (cm�1): 3359, 2925, 1680, 1523, 1364,1246, 1158, 620. 1H NMR (CDCl3, 300 MHz) d (ppm): 4.99 (br s, 2H),4.76e4.73 (br s, 2H), 3.55 (m, 1H), 3.10 (m, 2H), 2.61e2.59 (m, 2H),1.57 (m, 2H), 1.37 (m, 22H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm):170.8 (np), 167.9 (np), 78.9 (2C), 51.0, 44.4 (np), 41.5 (np), 33.1 (np),32.4 (np), 28.0 (6C), 22.7 (np). MS-ESIm/z: [MþH]þ 332.2, [MþNa]þ

354.2. HRMS-ESI m/z calcd for C16H34N3O4 [MþH]þ: 332.2549,found: 332.2564.

4.1.1.2. Method B4.1.1.2.1. General procedure for synthesis of compounds 13 and 14.

A stirred solution of compound 3 or 4 (38.3e40.4 mmol) in dry THF(150 mL) was treated with triethylamine (1.1 equiv). Ethyl chlor-oformate (1.4 equiv) dissolved in THF (50 mL) was then addeddropwise at �15 �C. After pre-stirring for 30 min at �15 �C, 25% (v/v) ammonia (2.5e2.7 equiv) was added and the mixture was stirredat rt for 18 h. THF was evaporated under reduced pressure and afterneutralization of the aqueous phase with 1 N KHSO4, the resultingproduct was extracted with EtOAc (2 � 120 mL). The organic phasewas washedwith 10% NaHCO3 (2� 120mL) and brine (3� 120mL),dried over Na2SO4 and concentrated in vacuo to give compound 13or 14 in 68e77% yield.

4.1.1.2.1.1. Methyl 4-amino-3-(t-butoxycarbonylamino)-4-oxobutanoate (13)

White solid was obtained from compound 3 (40.4 mmol) in 68%yield: mp 108.6 �C. IR n (cm�1): 3395, 3337, 3205, 2921, 1732, 1639,1518, 1294, 1165, 993, 782, 620. 1H NMR (CDCl3, 300 MHz) d (ppm):6.60 (br s,1H), 6.21 (br s,1H), 5.80e5.77 (br s,1H), 4.50 (m.1H), 3.67(s, 3H), 2.92e2.67 (m, 2H), 1.38 (s, 9H). Q-DEPT NMR (CDCl3,75 MHz) d (ppm): 173.2 (np), 173.7 (np), 155.6 (np), 80.5 (np), 52.1,50.4, 35.9 (np), 28.3 (3C). MS-ESI m/z: [MþH]þ 247.1, [MþNa]þ

269.1, [M þ MeCN þ Na]þ 310.1. HRMS-ESI m/z calcd forC10H18N2O5Na [MþNa]þ ¼ 269.1113, found: 269.1103.

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 715

4.1.1.2.1.2. Methyl 5-amino-4-(t-butoxycarbonylamino)-5-oxopentanoate (14)

White pasty solid was obtained from compound 4 (38.3 mmol)in 77% yield [62]. IR n (cm�1): 3402, 3352, 3175, 1731, 1656, 1281,1159, 608. 1H NMR (d6-DMSO, 600 MHz) d (ppm): 7.26 (br s, 1H),7.01 (br s, 1H), 6.79e6.78 (br s, 1H), 3.89e3.85 (m. 1H), 3.58 (s, 3H),2.33e2.30 (m, 2H),1.91e1.72 (m, 2H), 1.38 (s, 9H). Q-DEPT NMR (d6-DMSO, 150 MHz) d (ppm): 174.1 (np), 174.0 (np), 155.8 (np), 78.5(np), 53.8, 51.8, 30.4 (np), 28.6 (3C), 27.6 (np). MS-ESI m/z: [MþH]þ

261.1, [MþNa]þ 283.1, [M þ MeCN þ Na]þ 324.1, [2M þ Na]þ 543.2.HRMS-ESIm/z calcd for C11H20N2O5Na [MþNa]þ ¼ 283.1270, found:283.1279.

4.1.1.2.2. General procedure for synthesis of compounds 15, 16 and33. To a solution of compound 13, 14 or 31 (8.1e28.8 mmol) in THF(70e100 mL) cooled at �10 �C were added trifluoroacetic anhy-dride (1.5 equiv) and triethylamine or pyridine (3 equiv). Themixture was stirred at �10 �C for 2e4 h. After evaporating THFunder reduced pressure, the residue was dissolved in EtOAc(2 � 50e80 mL). The organic phase was washed with 1 N KHSO4(2 � 50e80 mL), 10% NaHCO3 (2 � 50e80 mL) and brine(3 � 50e80 mL), dried over Na2SO4 and concentrated in vacuo togive compound 15, 16 or 33 in 60e99% yield after purification bycolumn chromatography on silica gel (CH2Cl2/MeOH 98:2), flashcolumn chromatography using Grace Reveleris® system in normalphase (CH2Cl2/MeOH 100:0 to CH2Cl2/MeOH 90:10) or recrystalli-zation in EtOAc/Cyclohexane 20:80.

4 .1.1. 2 . 2 .1. Me t hy l 3 - ( t - b u t o x y c a r b ony l am i n o ) - 3 -cyanopropanoate (15)

Yellow solid was obtained from compound 13 (26.4 mmol) in60% yield: mp 56.4 �C. IR n (cm�1): 3333, 2958, 1736, 1693, 1523,1372, 1252, 1154, 1055, 991, 849, 636. 1H NMR (CDCl3, 300 MHz)d (ppm): 5.61 (br s, 1H), 4.90 (m, 1H), 3.75 (s, 3H), 2.86e2.82 (m,2H), 1.39 (s, 9H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 159.0 (np),117.9 (np), 80.3 (np), 52.6, 37.4 (np), 28.4 (3C). MS-ESIm/z: [MþH]þ

229.2, [M þ MeCN þ Na]þ 292.1, [2M þ Na þ H]þ 480.2. HRMS-ESIm/z calcd for C10H16N2O4Na [MþNa]þ ¼ 251.1008, found: 251.1000.

4.1.1.2.2.2. Methyl 4-(t-butoxycarbonylamino)-4-cyanobutanoate(16)

Yellow solid was obtained from compound 14 (28.8 mmol) in74% yield: mp 83.7 �C. IR n (cm�1): 3375, 2982, 1727, 1687, 1507,1448, 1295, 1202, 1155, 1098, 978, 868. 1H NMR (CDCl3, 600 MHz)d (ppm): 5.41 (br s, 1H), 4.62 (m, 1H), 3.67 (s, 3H), 2.53e2.46 (m,2H), 2.13e2.10 (m, 2H), 1.41 (s, 9H). Q-DEPT NMR (CDCl3, 150 MHz)d (ppm): 172.6 (np), 154.5 (np), 118.8 (np), 81.2 (np), 52.0, 41.6, 29.6(np), 28.2 (3C), 28.2 (np). MS-ESI m/z: [MþH]þ 243.1, [MþNa]þ

265.1, [M þ MeCN þ Na]þ 306.1. HRMS-ESI m/z calcd forC11H18N2O4Na [MþNa]þ ¼ 265.1164, found: 265.1159.

4 .1.1. 2 . 2 . 3 . B e n z y l 4 - ( t - b u t o x y c a r b ony l am i n o ) - 4 -cyanobutylcarbamate (33)

White pasty solid was obtained from compound 31 (8.1 mmol)in 99% yield. 1H NMR (CDCl3, 600 MHz) d (ppm): 7.36e7.29 (m, 5H),5.11 (br s, 1H), 5.08 (s, 2H), 4.96 (br s, 1H), 4.55 (m, 1H), 3.24e3.23(m, 2H), 1.81e1.79 (m, 2H), 1.68e1.65 (m, 2H), 1.44 (s, 9H). Q-DEPTNMR (CDCl3, 150 MHz) d (ppm): 156.6 (np, 2C), 136.4 (np), 128.5(2C), 128.2, 128.1 (2C), 118.7, 79.1 (np), 66.8 (np), 42.0, 40.0 (np),29.6 (np), 28.2 (3C), 26.1 (np). MS-ESI m/z: [MþNa]þ 370.2,[2M þ Na]þ 717.3. HRMS-ESIm/z calcd for C18H25N3O4Na [MþNa]þ:370.1743, found: 370.1749.

4.1.1.2.3. General procedure for synthesis of compounds 11, 12, 27and 28. To a solution of compound 15, 16, 33 or 34 (8e19.8 mmol)in dry methanol (60e150 mL) cooled at 0 �C were added Boc2O(2 equiv) and NiCl2.6H2O (0.1 equiv). NaBH4 (7e8 equiv) was thenadded in several portions over a period of 1 h. The mixture wasstirred at rt for 1e3 h and diethylenetriamine (1e2 equiv) wasadded to quench NiCl2.6H2O. After stirring further for 0.5e1 h,

methanol was evaporated and the resulting product was extractedwith EtOAc (2 � 50e120 mL). The organic phase was washed withsaturated NaHCO3 solution (2 � 50e120 mL) and brine(3 � 50e120 mL), dried over Na2SO4 and concentrated in vacuo togive compound 27 or 28 in 89e92% yield. The intermediate ob-tained from 15 or 16was dissolved in THF/H2O 1:1 (40 mL) and 4 Naqueous LiOH (4 equiv) solution was added. The mixture was stir-red for 1e1.5 h and THF was evaporated in vacuo. The aqueousphasewas adjusted to pH 12e13with 10% Na2CO3 and washedwithEtOAc (3 � 30 mL) to eliminate remaining Boc2O and methyl esterderivative. After acidification of the aqueous phase with 6 N HCl,the resulting product was extracted with EtOAc (2 � 30 mL). Theorganic phase was washed with brine (3 � 30 mL), dried overNa2SO4 and evaporated in vacuo to give compound 11 or 12 in50e67% yield.

4.1.1.3. Synthesis of piperazine derivatives4.1.1.3.1. General procedure for synthesis of compounds 17 and 18.

To a solution of compound 11 or 12 (3.1e4.5 mmol) in CH2Cl2(25e30 mL) were added N-hydroxysuccinimide (NHS,1.2e1.5 equiv) and dicyclohexylcarbodiimide (DCC, 1.1e1.2 equiv).The mixture was stirred overnight at rt. After filtering off DCC ureaand evaporating CH2Cl2, the activated intermediate was addedwitha pre-stirred (30 min) solution of 1-Cbz-piperazine hydrochloride(1.2 equiv) and triethylamine (3 equiv) in CH2Cl2 (40 mL). Themixture was stirred at rt for 18 h and dilutedwith additional CH2Cl2(2 � 30 mL). The organic phase was washed with 1 N HCl(2 � 30 mL), saturated NaHCO3 solution (2 � 30 mL) and brine(3 � 30 mL), dried over Na2SO4 and concentrated in vacuo to givecompound 17 or 18 in 77e97% yield after purification by columnchromatography on silica gel (CH2Cl2/MeOH 98:2).

4.1.1.3.1.1. Benzyl 4-(3,4-bis(t-butoxycarbonylamino)butanoyl)piperazine-1-carboxylate (17)

White solid was obtained from compound 11 (3.1 mmol) 77%yield: mp 60.3 �C. IR n (cm�1): 3328, 2921, 1693, 1628, 1515, 1428,1363, 1230, 1161, 638. 1H NMR (CDCl3, 300 MHz) d (ppm): 7.33 (m,5H), 5.69 (br s, 1H), 5.12 (s, 2H), 5.03 (br s, 1H), 3.89 (m, 1H),3.62e3.45 (m, 10H), 2.65e2.41 (m, 2H), 1.40 (s, 18H). Q-DEPT NMR(CDCl3, 75 MHz) d (ppm): 173.0 (np), 163.7 (np), 161.1 (np), 147.7(np), 132.4 (np), 128.9 (2C), 128.5, 128.3 (2C), 81.2 (2C), 67.8 (np),49.7, 45.9 (np), 44.1 (np, 2C), 41.8 (np, 2C), 35.3 (np), 28.7 (6C). MS-ESI m/z: [MþH]þ 521.3, [MþNa]þ 543.2. HRMS-ESI m/z calcd forC26H40N4O7Na [MþNa]þ: 543.2795, found: 543.2794.

4.1.1.3.1.2. Benzyl 5-(4,5-bis(t-butoxycarbonylamino)pentanoyl)piperazine-1-carboxylate (18)

White solid was obtained from compound 12 (4.5 mmol) in 97%yield: mp 49.6 �C. IR n (cm�1): 3343, 2922, 1692, 1635, 1515, 1425,1363, 1229, 1163, 1026, 698. 1H NMR (CDCl3, 300 MHz) d (ppm):7.36e7.32 (m, 5H), 5.13 (s, 2H), 4.92 (br s, 2H), 3.60e3.44 (m, 9H),3.20e3.16 (m, 2H), 2.41e2.38 (m, 2H), 1.83e1.81 (m, 2H), 1.40 (s,18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 170.8 (np), 164.1 (np),163.8 (np), 154.7 (np), 136.0 (np), 128.2 (2C), 127.8, 127.6 (2C), 79.0(np, 2C), 67.1 (np), 51.1, 44.8 (np), 44.3 (np), 43.4 (np), 41.1 (np, 2C),29.2 (np), 28.0 (6C), 27.5 (np). MS-ESIm/z: [MþH]þ 535.3, [MþNa]þ

557.3. HRMS-ESI m/z calcd for C27H42N4O7Na [MþNa]þ: 557.2951,found: 557.2945.

4.1.1.3.2. General procedure for synthesis of compounds 19 and 20.To a solution of compound 17 or 18 (0.7e2.7mmol) in drymethanol(8e30 mL) was added Pd/C (10% w/w). The mixture was placedunder H2 and stirred for 6 h at rt. After filtering the catalyst andevaporating methanol, compound 19 or 20 were obtained in96e100% yield.

4.1.1.3.2.1. t-Butyl 4-oxo-4-(piperazin-1-yl)butane-1,2-diyldicarbamate (19)

White solid was obtained from compound 17 (2.7 mmol) 100%

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722716

yield: mp 74.5 �C. IR n (cm�1): 3320, 2934, 1691, 1626, 1514, 1439,1364,1246,1026, 645. 1H NMR (CDCl3, 300MHz) d (ppm): 7.33 (br s,1H), 5.69e5.66 (br s, 1H), 5.12 (br s, 1H), 3.89e3.88 (m, 1H),3.66e3.23 (m, 8H), 2.93e2.83 (m, 2H), 2.67e2.43 (m, 2H), 1.41 (s,18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 167.1 (np), 157.5 (np),155.4 (np), 79.1 (np, 2C), 48.6, 45.0 (np), 43.1 (np), 44.6 (np), 40.9(np, 2C), 34.8 (np), 27.9 (6C). MS-ESI m/z: [MþH]þ 387.3, [MþNa]þ

409.3. HRMS-ESI m/z calcd for C18H34N4O5Na [MþNa]þ: 409.2427,found: 409.2418.

4.1.1.3.2.2. t-Butyl 5-oxo-5-(piperazin-1-yl)pentane-1,2-diyldicarbamate (20)

White solid was obtained from compound 18 (0.7 mmol) in 96%yield: mp 65.3 �C. IR n (cm�1): 3326, 2932, 1691, 1626, 1517, 1441,1364,1246,1162,1009, 727, 626. 1H NMR (CDCl3, 300MHz) d (ppm):5.13e5.09 (br s, 2H), 3.56e3.52 (m, 3H), 3.39e3.36 (m, 2H),3.15e3.12 (m, 2H), 2.80e2.76 (m, 4H), 2.37e2.31 (m, 2H), 1.81e1.63(m, 2H), 1.37 (s, 18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 170.8(np), 156.5 (np), 156.2 (np), 80.3 (np), 79.1 (np), 51.3, 46.0 (np), 45.3(np), 44.5 (np), 42.6 (np), 41.5 (np), 29.4 (np), 28.2 (6C), 27.7(np).MS-ESIm/z: [MþH]þ 401.4, [MþNa]þ 423.3. HRMS-ESIm/z calcd forC19H36N4O5Na [MþNa]þ: 423.2583, found: 423.2573.

4.1.2. Coupling of phenolic acid or HOPO ligands (B)4.1.2.1. Synthesis of HOPO ligands

4.1.2.1.1. 3-(3-(Benzyloxy)-2-oxopyridin-1(2H)-yl)propanoic acid(43). To a solution of compound 42 (2 mmol) in water (40 mL) wasadded sodium hydroxide (12.5 equiv). The mixture was heatedunder reflux for 1 h and then extracted with EtOAc (3 � 30 mL).After acidification of the aqueous phase with 6 N HCl, the resultingproduct was extracted with EtOAc (2 � 30 mL). The organic phasewas washed with brine (3 � 30 mL), dried over Na2SO4 andconcentrated in vacuo to give compound 43 as a yellow solid in 65%yield [63]. 1H NMR (d6-DMSO, 300 MHz) d (ppm): 7.43e7.32 (m,5H), 7.25 (dd, J ¼ 6.9 Hz et J0 ¼ 1.5 Hz, 1H), 6.87 (dd, J ¼ 7.3 Hz etJ0 ¼ 1.8 Hz,1H), 6.09 (t, J¼ 7.2 Hz,1H), 4.99 (s, 2H), 4.07 (t, J¼ 6.9 Hz,2H), 2.65 (t, J ¼ 6.9 Hz, 2H). Q-DEPT NMR (d6-DMSO, 75 MHz)d (ppm): 171.9 (np), 156.5 (np), 147.5 (np), 136.2 (np), 130.1, 128.0(2C), 127.5, 127.4 (2C), 115.2, 103.5, 69.4 (np), 45.0 (np), 32.5 (np).MS-ESI m/z: [MþH]þ 273.8. HRMS-ESI m/z calcd for C15H15NO4Na[MþNa]þ: 296.0899, found: 296.0893.

4.1.2.1.2. t-Butyl (3-(3-(benzyloxy)-2-oxopyridin-1(2H)-yl)pro-pyl)carbamate (44). To a solution of compound 42 (7.9mmol) in drymethanol (80 mL) cooled at 0 �C were added Boc2O (2 equiv) andNiCl2.6H2O (0.1 equiv). NaBH4 (7 equiv) was then added in severalportions over a period of 30 min. The mixture was stirred at rt for1 h and diethylenetriamine (1 equiv) was added to quenchNiCl2.6H2O. After stirring for another 1 h, methanol was evaporatedand the resulting product was extracted with EtOAc (2 � 60 mL).The organic phase was washed with saturated NaHCO3 solution(2 � 60 mL) and brine (3 � 60 mL), dried over Na2SO4 andconcentrated in vacuo to give compound 44 as a white solid in 84%yield. 1H NMR (CDCl3, 400 MHz) d (ppm): 7.33 (d, J ¼ 7.6 Hz, 2H),7.25 (t, J¼ 6.8 Hz, 2H), 7.19 (t, J¼ 6.0 Hz, 1H), 6.80 (d, J¼ 6.3 Hz, 1H),6.55 (d, J¼ 7.4 Hz,1H), 5.96 (t, J¼ 7.3 Hz, 1H), 5.49 (br s, 1H), 5.01 (s,2H), 3.95 (t, J ¼ 6.3 Hz, 2H), 3.01e2.96 (m, 2H), 1.81e1.75 (m, 2H),1.33 (s, 9H). 13C NMR (CDCl3, 100 MHz) d (ppm): 158.3, 155.9, 148.5,135.8, 128.4, 128.3 (2C), 127.8, 127.1 (2C), 115.1, 105.2, 78.6, 70.5,46.0, 36.3, 29.7, 28.2 (3C). MS-ESI m/z: [MþH]þ 358.9, [MþNa]þ:380.9. HRMS-ESI m/z calcd for C20H26N2O4Na [MþNa]þ: 381.1790,found: 381.1782.

4.1.2.1.3. 1-(3-Aminopropyl)-3-(benzyloxy)pyridin-2(1H)-one,hydrochloride salt (45). To a solution of compound 44 (2.8 mmol)dissolved in 1,4-dioxane (10 mL) was added 4 N HCl solution in 1,4-dioxane (20 equiv). The mixture was stirred at rt for 2 h andconcentrated under reduced pressure. The residue was washed

with Et2O (2 � 8 mL) and after vacuum filtering, compound 45 wasobtained as a yellow precipitate in 100% yield: mp 162.3 �C [40]. 1HNMR (d6-DMSO, 300MHz) d (ppm): 8.06e8.05 (br s, 2H), 7.45e7.33(m, 6H), 6.93 (d, J ¼ 7.5 Hz, 1H), 6.18 (t, J ¼ 7.2 Hz, 1H), 5.01 (s, 2H),4.00 (t, J¼ 6.9 Hz, 2H), 2.75 (t, J¼ 7.2 Hz, 2H), 1.98e1.93 (m, 2H). 13CNMR (d6-DMSO, 75 MHz) d (ppm): 175.1, 147.6, 136.1, 129.5, 128.0(2C), 127.5 (3C), 115.3, 104.1, 69.4, 45.4, 35.9, 26.5. MS-ESI m/z:[MþH]þ 259.1, [M þ H þ MeCN]þ: 300.2. HRMS-ESI m/z calcd forC15H19N2O2 [MþH]þ: 259.1436, found: 259.1447.

4.1.2.2. General procedure for coupling of phenolic acid or HOPO li-gands. Method I: To a solution of ferulic, gallic acid, compound 12,43 or 47 (0.4e3.0 mmol) in CH2Cl2 or 1,4-dioxane (10e20 mL) wereadded NHS (1.1e1.5 equiv) and DCC (1e1.2 equiv). The mixture wasstirred overnight at rt. After filtering off DCC urea and evaporatingCH2Cl2, the activated intermediate was added with a pre-stirredsolution of compound 19, 20 or 30, 45 or 48 (1e1.7 equiv) andtriethylamine (0e4 equiv) in CH2Cl2 (8e30 mL). The mixture wasstirred at rt for 18 h and diluted with additional CH2Cl2(2 � 8e25 mL). The organic phase was washed with 1 N HCl(2� 8e25mL), saturated NaHCO3 solution (2� 8e25mL) and brine(3� 8e25mL), dried over Na2SO4 and concentrated in vacuo to givecompound 35a-b, 38a-b, 49a-b, 49e or 52b in 25e82% yield afterpurification by column chromatography on silica gel (CH2Cl2/MeOH98:2 or 95:5).

Method II: To a solution of compound 43 or 47 (1e6.5 mmol) inTHF or DMF (30e100 mL) cooled at 0 �C were added 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC,1.2 equiv) and 1-hydroxybenzotriazole monohydrate (HOBt,1.1e1.2 equiv). After pre-stirring at 0 �C for 30min, compound 29 or30 (1 equiv) and triethylamine (1.2 equiv) were added in themixture that was further stirred at rt for 4e18 h. The solvent wasevaporated under reduced pressure and the residue was diluted inEtOAc (2 � 20e80 mL). The organic phase was washed with brine(3 � 20e80 mL), dried over Na2SO4 and concentrated in vacuo togive compound 49c-d or 52a in 69e86% yield after purification bycolumn chromatography on silica gel (EtOAc/Cyclohexane 60:40 orEtOAc/MeOH 80:20).

4.1.2.2.1. di-t-Butyl (5-(4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)piperazin-1-yl)-5-oxopentane-1,2-diyl)-dicarbamate (35a).White solid was obtained by coupling ferulic acid and compound20 (0.4 mmol) according to method I in 55% yield: mp 76.5 �C. IR n

(cm�1): 3303, 2977, 1690, 1636, 1513, 1429, 1246, 1159, 1027, 626. 1HNMR (CDCl3, 300 MHz) d (ppm): 7.63 (d, J ¼ 15.0 Hz, 1H), 7.09 (dd,J¼ 8.4 Hz, J0 ¼ 1.8 Hz, 1H), 6.99 (d, J¼ 1.8 Hz, 1H), 6.91 (d, J¼ 8.1 Hz,1H), 6.67 (d, J ¼ 15.3 Hz, 1H), 6.08 (br s, 1H), 4.93 (br s, 2H), 3.92 (s,3H), 3.73e3.50 (m, 9H), 3.20 (t, J ¼ 5.4 Hz, 2H), 2.45e2.39 (m, 2H),1.86e1.80 (m, 2H), 1.42 (s, 18H). Q-DEPT NMR (CDCl3, 75 MHz)d (ppm): 170.8 (np), 170.5 (np), 165.5 (np, 2C), 147.2 (np), 146.4 (np),143.4, 127.1 (np), 121.7, 114.4, 113.3, 109.5, 77.5 (2C), 55.6, 51.0, 44.8(np), 44.2 (np, 2C), 41.3 (np, 2C), 29.1 (np), 27.9 (6C), 27.5 (np). MS-ESI m/z: [MþH]þ 577.3, [MþNa]þ 599.3. HRMS-ESI m/z calcd forC29H44N4O8Na [MþNa]þ: 599.3057, found: 599.3043.

4.1.2.2.2. di-t-Butyl (6-(3-(4-hydroxy-3-methoxyphenyl)acryl-amido)hexane-1,2-diyl) (E)-dicarbamate (35b). White solid wasobtained by coupling ferulic acid and compound 30 (0.5 mmol)according to method I in 71% yield: mp 52.1 �C. IR n (cm�1): 3319,2931, 1685, 1513, 1249, 1159, 1031, 661. 1H NMR (CDCl3, 300 MHz)d (ppm): 7.52 (d, J¼ 15.6 Hz,1H), 7.02 (dd, J¼ 8.1 Hz, J0 ¼ 1.8 Hz,1H),6.96 (d, J ¼ 1.8 Hz, 1H), 6.87 (d, J ¼ 8.1 Hz, 1H), 6.31 (d, J ¼ 15.6 Hz,1H), 6.28 (br s, 1H), 5.01e4.99 (br s, 1H), 4.84e4.82 (br s, 1H), 3.86(s, 3H), 3.59e3.58 (m, 1H), 3.34e3.33 (m, 2H), 3.16 (m, 2H),1.57e1.55 (m, 2H), 1.42 (m, 22H). Q-DEPT NMR (CDCl3, 75 MHz)d (ppm): 167.4 (np, 2C),157.3 (np), 148.3 (np), 147.8 (np), 141.6, 128.4(np), 122.9, 119.5, 115.8, 110.8, 80.4 (np, 2C), 56.8, 52.2, 45.3 (np),

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 717

39.9 (np), 33.1 (np), 30.6 (np), 29.3 (6C), 23.7 (np). MS-ESI m/z:[MþH]þ 508.3, [MþNa]þ 530.3. HRMS-ESI m/z calcd forC26H41N3O7Na [MþNa]þ: 530.2842, found: 530.2856.

4.1.2.2.3. di-t-Butyl (5-oxo-5-(4-(3,4,5-tris(benzyloxy)benzoyl)piperazin-1-yl)pentane-1,2-diyl)dicarbamate (38a). White solid wasobtained by coupling gallic acid and compound 20 (1.1 mmol) ac-cording to method I in 82% yield: mp 100.5 �C. IR n (cm�1): 3320,2918, 2849, 1699, 1625, 1428, 1244, 1167, 1108, 985, 735, 695. 1HNMR (CDCl3, 300 MHz) d (ppm): 7.40e7.33 (m, 12H), 7.28e7.27 (m,3H), 6.65 (s, 2H), 5.12 (s, 4H), 5.10 (s, 2H), 3.65e3.19 (m, 11H),2.42e2.39 (m, 2H), 1.90e1.84 (m, 2H), 1.70e1.64 (m, 2H), 1.43 (s,18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 171.0 (np), 169.9 (np),156.5 (np), 156.2 (np), 152.5 (np, 2C), 139.7 (np), 137.3 (np), 136.5(np, 2C), 129.9 (np), 128.4 (6C), 128.0, 127.8 (2C), 127.1 (6C), 107.1(2C), 79.2 (np, 2C), 75.0 (np), 71.0 (np, 2C), 51.2, 45.0 (np, 2C), 44.4(np), 41.5 (np, 2C), 33.7 (np), 29.3 (np), 28.2 (6C). MS-ESI m/z:[MþH]þ 823.3, [MþNa]þ 845.3. HRMS-ESI m/z calcd forC47H58N4O9Na [MþNa]þ: 845.4101, found: 845.4138.

4.1.2.2.4. di-t-Butyl (6-(3,4,5-tris(benzyloxy)benzamido)hexane-1,2-diyl)dicarbamate (38b). Yellow solid was obtained by couplinggallic acid and compound 30 (1.1 mmol) according to method I in58% yield: mp 156.0 �C. IR n (cm�1): 3345, 2929, 2849, 1679, 1526,1328, 1243, 1163, 1119, 850, 733, 695. 1H NMR (CDCl3, 300 MHz)d (ppm): 7.45e7.31 (m,12H), 7.28e7.26 (m, 3H), 7.17 (s, 2H), 6.62 (brs, 1H), 5.13 (s, 4H), 5.09 (s, 2H), 4.97 (br s, 1H), 4.78e4.75 (br s, 1H),3.63e3.62 (m, 1H), 3.40 (m, 2H), 3.20e3.18 (m, 2H), 1.62 (m, 2H),1.46e1.44 (m, 22H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 167.2(np), 156.2 (np, 2C), 152.8 (np, 2C), 141.1 (np), 137.6 (np), 136.9 (np,2C), 130.3 (np), 128.6 (6C), 128.3, 128.1 (2C), 127.7 (6C), 107.1 (2C),79.6 (np, 2C), 75.3 (np, 2C), 71.5 (np), 51.4, 44.2 (np), 39.5 (np), 32.1(np), 29.2 (np), 28.5 (6C), 22.8 (np). MS-ESI m/z: [M þ -3Bn þ 3H þ Na]þ 506.3, [M þ H þ Na]þ 777.2. HRMS-ESI m/z calcdfor C44H55N3O8Na [MþNa]þ: 776.3887, found: 776.3905.

4.1.2.2.5. di-t-Butyl (4-(4-(3-(3-(benzyloxy)-2-oxopyridin-1(2H)-yl)propanoyl)piperazin-1-yl)-4-oxobutane-1,2-diyl)dicarbamate(49a). Brown solid was obtained by coupling compounds 43 and 19(1 mmol) according to method I in 39% yield: mp 85.3 �C. IR n

(cm�1): 3321, 2931, 1698, 1624, 1435, 1364, 1244, 1162, 1020, 637. 1HNMR (CDCl3, 300 MHz) d (ppm): 7.32e7.18 (m, 5H), 7.01 (d,J ¼ 6.1 Hz, 1H), 6.56 (d, J ¼ 5.4 Hz, 1H), 5.92 (t, J ¼ 6.9 Hz, 1H),5.62e5.60 (br s, 1H), 5.03e4.97 (br s, 1H), 4.97 (s, 2H), 4.14 (t,J¼ 6.3 Hz, 2H), 3.81e3.79 (m,1H), 3.54e3.32 (m, 8H), 3.23e3.13 (m,2H), 2.79 (t, J¼ 6.0 Hz, 2H), 2.57e2.38 (m, 2H), 1.30 (s, 18H). Q-DEPTNMR (CDCl3, 75 MHz) d (ppm): 168.7 (np), 168.6 (np), 157.8 (np),156.2 (np), 155.2 (np), 142.0 (np), 135.6 (np), 129.8, 128.1 (2C), 127.5,126.8 (2C), 115.1, 104.1, 79.1 (np, 2C), 70.2 (np), 48.6, 46.7 (np), 44.8(np, 2C), 43.1 (np), 41.0 (np, 2C), 34.7 (np), 31.2 (np), 27.9 (6C). MS-ESI m/z: [MþH]þ 642.3, [MþNa]þ 664.3. HRMS-ESI m/z calcd forC33H47N5O8Na [MþNa]þ: 664.3322, found: 664.3322.

4.1.2.2.6. di-t-Butyl (5-(4-(3-(3-(benzyloxy)-2-oxopyridin-1(2H)-yl)propanoyl)piperazin-1-yl)-5-oxopentane-1,2-diyl)dicarbamate(49b). White solid was obtained by coupling compounds 43 and 20(1 mmol) according to method I in 25% yield: mp 63.0 �C. IR n

(cm�1): 3325, 2978, 1697, 1645, 1600, 1433, 1245, 1163, 1013, 731. 1HNMR (CDCl3, 300 MHz) d (ppm): 7.42 (d, J ¼ 6.6 Hz, 1H), 7.37e7.28(m, 3H), 7.11 (d, J ¼ 5.9 Hz, 1H), 6.65 (d, J ¼ 6.2 Hz, 1H), 6.02 (t,J ¼ 7.2 Hz, 1H), 5.08 (s, 2H), 4.94e4.93 (br s, 2H), 4.24 (t, J ¼ 6.3 Hz,2H), 3.54e3.32 (m, 9H), 3.17 (t, J ¼ 5.4 Hz, 2H), 2.89 (t, J ¼ 6.3 Hz,2H), 2.41e2.34 (m, 2H), 1.94e1.82 (m, 2H), 1.40 (s, 18H). Q-DEPTNMR (CDCl3, 75 MHz) d (ppm): 171.4 (np, 2C), 158.6 (np), 156.7 (np),149.1 (np), 140.1 (np), 138.2 (np), 130.7, 128.9 (2C), 128.4, 127.7 (2C),115.9, 104.9, 87.1 (np), 87.0 (np), 71.1 (np), 51.8, 47.6 (np), 45.9 (np,2C), 45.0 (np), 41.9 (np, 2C), 32.0 (np), 30.1 (np), 28.7 (6C), 28.3 (np).MS-ESIm/z: [MþH]þ 656.3, [MþNa]þ 678.3. HRMS-ESIm/z calcd forC34H49N5O8Na [MþNa]þ: 678.3479, found: 678.3474.

4.1.2.2.7. di-t-Butyl (5-(3-(3-(benzyloxy)-2-oxopyridin-1(2H)-yl)propanamido)pentane-1,2-diyl)dicarbamate (49c). White solid wasobtained by coupling compounds 43 and 29 (4 mmol) according tomethod II in 84% yield: mp 92.1 �C. 1H NMR (CDCl3, 300 MHz)d (ppm): 7.38e7.28 (m, 5H), 7.22e7.19 (br s, 1H), 7.08 (dd, J¼ 6.9 Hz,J0 ¼ 1.5 Hz,1H), 6.70 (dd, J¼ 7.5 Hz, J0 ¼ 1.2 Hz,1H), 6.04 (t, J¼ 7.2 Hz,1H), 5.07e5.01 (br s, 1H), 5.01 (s, 2H), 4.93e4.90 (br s, 1H),4.28e4.18 (m, 2H), 3.51e3.47 (m, 1H), 3.05e3.03 (m, 4H), 2.63 (t,J ¼ 6.0 Hz, 2H), 2.27e1.40 (s, 9H) 1.38 (s, 9H), 1.32e1.17 (m, 4H). Q-DEPT NMR (CDCl3, 75MHz) d (ppm): 169.7 (np, 2C),157.7 (np),156.1(np), 148.1 (np), 135.4 (np), 130.0, 128.1 (2C), 127.7, 127.2 (2C), 115.1,104.4, 78.7 (np, 2C), 70.3 (np), 50.5, 46.5 (np), 44.6 (np), 38.6 (np),34.5 (np), 29.4 (np), 27.9 (6C), 25.0 (np). MS-ESIm/z: [MþH]þ 573.2,[MþNa]þ 595.1. HRMS-ESI m/z calcd for C26H41N3O7Na [MþNa]þ:595.3108, found: 595.3098.

4.1.2.2.8. di-t-Butyl (6-(3-(3-(benzyloxy)-2-oxopyridin-1(2H)-yl)propanamido)hexane-1,2-diyl)dicarbamate (49d). Brown solid wasobtained by coupling compounds 43 and 30 (6.5 mmol) accordingtomethod II in 86% yield: mp 113.1 �C. IR n (cm�1): 3340, 2927,1680,1643, 1593, 1528, 1239, 1162, 1056, 741. 1H NMR (CDCl3, 300 MHz)d (ppm): 7.36e7.25 (m, 5H), 7.06 (dd, J¼ 6.9 Hz, J0 ¼ 1.5 Hz,1H), 6.99(br s, 1H), 6.66 (dd, J¼ 7.5 Hz, J0 ¼ 1.5 Hz, 1H), 6.00 (t, J¼ 7.2 Hz, 1H),5.00 (br s, 1H), 5.00 (s, 2H), 4.83e4.80 (br s, 1H), 4.21 (t, J ¼ 6.3 Hz,2H), 3.50e3.48 (m, 1H), 3.07e3.00 (m, 4H), 2.65 (t, J ¼ 6.0 Hz, 2H),1.38 (s, 18H), 1.33e1.23 (m, 6H). Q-DEPT NMR (CDCl3, 75 MHz)d (ppm): 170.1 (np, 2C), 158.1 (np, 2C), 148.5 (np), 135.9 (np), 130.3,128.5 (2C), 128.1, 127.4 (2C), 115.6, 104.7, 79.2 (np, 2C), 70.6 (np),51.1, 47.0 (np), 44.6 (np), 38.8 (np), 35.0 (np), 32.0 (np), 28.9 (np),28.3 (6C), 22.7 (np). MS-ESI m/z: [MþH]þ 587.5, [MþNa]þ 609.3.HRMS-ESI m/z calcd for C31H46N4O7Na [MþNa]þ: 609.3264, found:609.3254.

4.1.2.2.9. di-t-Butyl (5-((3-(3-(benzyloxy)-2-oxopyridin-1(2H)-yl)propyl)amino)-5-oxopentane-1,2-diyl)dicarbamate (49e). Whitesolid was obtained by coupling compounds 12 and 45 (2.4 mmol)according to method I in 68% yield: mp 143.3 �C. IR n (cm�1): 3336,2932, 1681, 1644, 1598, 1531, 1247, 1163, 1042, 751, 651. 1H NMR(CDCl3, 300 MHz) d (ppm): 7.40 (br s, 1H), 7.38e7.25 (m, 5H), 6.95(d, J ¼ 6.0 Hz, 1H), 6.67 (d, J ¼ 6.8 Hz, 1H), 6.10 (t, J ¼ 7.2 Hz, 1H),5.40e5.23 (br s, 2H), 5.03 (s, 2H), 4.00 (t, J ¼ 5.1 Hz, 2H), 3.61e3.59(m, 1H), 3.11 (m, 4H), 2.33e2.26 (m, 2H), 1.87e1.61 (m, 4H), 1.35 (s,18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 172.3 (np, 2C), 156.2(np), 156.4 (np), 148.2 (np), 135.6 (np), 128.5, 128.2 (2C), 128.0, 127.8(2C), 115.5, 105.8, 79.0 (np, 2C), 70.4 (np), 50.9, 46.8 (np), 44.2 (np),35.7 (np), 32.6 (np), 30.3 (np), 28.8 (np), 28.0 (6C). MS-ESI m/z:[MþH]þ 573.4, [MþNa]þ 595.4. HRMS-ESI m/z calcd forC31H46N4O7Na [MþNa]þ: 595.3108, found: 595.3078.

4.1.2.2.10. di-t-Butyl (6-(3-(3-(benzyloxy)-2-methyl-4-oxopyridin-1(4H)-yl)propanamido)hexane-1,2-diyl)dicarbamate(52a). White pasty solid was obtained by coupling compounds 47and 30 (1 mmol) according to method II in 69% yield. IR n (cm�1):3341, 2932, 1693, 1624, 1515, 1364, 1246, 1165, 731, 699. 1H NMR(CDCl3, 300 MHz) d (ppm): 7.70 (br s, 1H), 7.37e7.30 (m, 6H), 6.28(d, J ¼ 5.6 Hz, 1H), 5.40 (br s, 1H), 5.18 (br s, 1H), 5.06 (s, 2H),4.10e4.06 (m, 2H), 3.51e3.49 (m, 1H), 3.24e3.04 (m, 4H),2.65e2.50 (m, 4H), 2.12 (s, 3H), 1.44e1.37 (m, 4H), 1.37 (s, 18H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 173.6 (np), 169.1 (np), 157.0(np), 156.4 (np), 145.8 (np), 141.6 (np), 139.2, 137.2 (np), 128.9, 128.4(2C), 128.3 (2C), 116.9, 79.2 (np, 2C), 72.9 (np), 52.3, 50.1 (np), 44.4(np), 39.1 (np), 36.2 (np), 35.0 (np), 31.9 (np), 28.4 (6C), 22.7 (np),12.4. MS-ESI m/z: [MþH]þ 601.3, [MþNa]þ 623.3. HRMS-ESI m/zcalcd for C32H49N4O7 [MþH]þ: 601.3601, found: 601.3621.

4.1.2.2.11. di-t-Butyl (5-((3-(3-(benzyloxy)-2-methyl-4-oxopyridin-1(4H)-yl)propyl)amino)-5-oxopentane-1,2-diyl)dicarba-mate (52b). Yellow solid was obtained by coupling compounds 12and 48 (3 mmol) according to method I in 60% yield: mp 54.5 �C. IR

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722718

n (cm�1): 3279, 2976, 1693, 1625, 1510, 1246,1162, 743, 701. 1H NMR(CDCl3, 300 MHz) d (ppm): 7.58e7.55 (br s, 1H), 7.37e7.24 (m, 6H),6.32 (d, J ¼ 5.6 Hz, 1H), 5.47e5.45 (br s, 2H), 5.08 (s, 2H), 3.80e3.77(m, 2H), 3.54e3.53 (m, 1H), 3.24e3.10 (m, 4H), 2.22 (t, J ¼ 4.9 Hz,2H), 2.03 (s, 3H), 1.93e1.70 (m, 4H), 1.36 (s, 18H). Q-DEPT NMR(CDCl3, 75MHz) d (ppm): 173.8 (np),173.3 (np),156.9 (np, 2C),146.3(np), 141.4 (np), 139.0, 137.4 (np), 129.0 (2C), 128.4 (2C), 128.2, 117.1,79.6 (np, 2C), 73.1 (np), 51.9 (np), 51.3, 44.4 (np), 36.2 (np), 32.9 (np),30.7 (np), 29.2 (np), 28.4 (6C), 12.4. MS-ESI m/z: [MþH]þ 587.3,[MþNa]þ 609.3. HRMS-ESI m/z calcd for C31H47N4O7 [MþH]þ:609.3264, found: 609.3265.

4.1.2.3. General procedure for debenzylation of hydroxy group.To a solution of compound 38a-b, 49a-e or 52a-b (0.2e1.6 mmol) indry methanol (10e40 mL) was added Pd/C (10% w/w). The mixturewas placed under H2 and stirred for 6 h at rt. After filtering thecatalyst and evaporatingmethanol, compound 39a-b, 50a-e or 53a-b was obtained in 60e100% yield.

4.1.2.3.1. di-t-Butyl (5-oxo-5-(4-(3,4,5-trihydroxybenzoyl)piper-azin-1-yl)pentane-1,2-diyl)dicarbamate (39a). White solid was ob-tained from compound 38a (0.5 mmol) in 99% yield: mp 83.4 �C. IRn (cm�1): 3323, 2930, 1684, 1612, 1519, 1438, 1364, 1247, 1162, 1029,628. 1H NMR (CD3OD, 400 MHz) d (ppm): 6.45 (s, 2H), 3.59e3.43(m, 8H), 3.32e3.31 (m, 1H), 3.06e3.01 (m, 2H), 2.47e2.45 (m, 2H),2.01e1.70 (m, 2H), 1.43 (s, 18H). Q-DEPT NMR (CD3OD, 100 MHz)d (ppm): 172.4 (np), 171.9 (np), 157.3 (np), 157.0 (np), 145.8 (np, 2C),135.3 (np), 125.0 (np), 106.3 (2C), 78.7 (np, 2C), 50.5, 45.1 (np, 2C),43.8 (np), 42.3 (np, 2C), 33.5 (np), 29.1 (np), 27.4 (6C). MS-ESI m/z:[M�2Boc þ 3H]þ 352.9, [MþH]þ 553.1, [MþNa]þ 575.1. HRMS-ESIm/z calcd for C26H40N4O9Na [MþNa]þ: 575.2693, found: 575.2719.

4.1.2.3.2. di-t-Butyl (6-(3,4,5-trihydroxybenzamido)hexane-1,2-diyl)dicarbamate (39b). Orange solid was obtained from com-pound 38b (0.5 mmol) in 100% yield: mp 81.7 �C. IR n (cm�1): 3335,2931, 1681, 1515, 1364, 1248, 1160, 1035, 617. 1H NMR (CDCl3,300 MHz) d (ppm): 7.25 (br s, 1H), 6.84 (s, 2H), 5.43 (br s, 1H),5.24e5.21 (br s, 1H), 3.15e3.04 (m, 5H), 1.41e1.35 (m, 22H),1.20e1.16 (m, 2H). Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 168.4(np), 156.7 (np), 156.4 (np), 144.3 (np, 2C), 135.7 (np), 124.6 (np),106.9 (2C), 79.1(np, 2C), 50.7, 44.2 (np), 39.5 (np), 31.9 (np), 29.2(np), 27.9 (6C), 22.6 (np). MS-ESI m/z: [MþH]þ 484.2, [MþNa]þ

506.3. HRMS-ESI m/z calcd for C23H37N3O8Na [MþNa]þ: 506.2478,found: 506.2501.

4.1.2.3.3. di-t-Butyl (4-(4-(3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propanoyl)piperazin-1-yl)-4-oxobutane-1,2-diyl)dicarbamate (50a).Brown solid was obtained from compound 49a (0.2 mmol) in 100%yield: mp 83.2 �C. IR n (cm�1): 3326, 2978, 2933, 1692, 1627, 1514,1434, 1364, 1246, 1162, 1021, 661. 1H NMR (CDCl3, 600 MHz)d (ppm): 7.03e7.01 (m,1H), 6.80 (d, J¼ 7.3 Hz,1H), 6.10 (t, J¼ 7.1 Hz,1H), 5.66 (br s, 1H), 4.98 (br s, 1H), 4.27 (t, J ¼ 7.1 Hz, 2H), 3.89 (m,1H), 3.66e3.31 (m, 8H), 3.23e3.21 (m, 2H), 2.87e2.85 (m, 2H),2.67e2.46 (m, 2H), 1.39 (s, 18H). Q-DEPT NMR (CDCl3, 100 MHz)d (ppm): 169.5 (np), 169.0 (np), 157.0 (np), 156.1 (np), 146.5 (np),128.9, 115.1, 107.0, 79.8 (np), 79.7 (np), 49.5, 47.3 (np), 45.8 (np, 2C),43.9 (np), 41.8 (np, 2C), 35.4 (np), 32.1 (np), 28.6 (6C). MS-ESI m/z:[M� 2Boc þ 3H]þ 352.1, [M�Boc þ 2H]þ 452.2, [MþH]þ 552.2,[MþNa]þ 574.2. HRMS-ESI m/z calcd for C26H41N5O8Na [MþNa]þ:574.2853, found: 574.2849.

4.1.2.3.4. di-t-Butyl (5-(4-(3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propanoyl)piperazin-1-yl)-5-oxopentane-1,2-diyl)dicarbamate (50b).Brown solid was obtained from compound 49b (0.5 mmol) in 82%yield: mp 136.1 �C. IR n (cm�1): 3318, 2979, 1686, 1631, 1599, 1516,1428, 1244, 1161, 799. 1H NMR (CDCl3, 400 MHz) d (ppm): 7.01e7.00(m,1H), 7.76 (d, J¼ 7.1 Hz,1H), 6.09 (t, J¼ 6.9 Hz,1H), 5.04 (br s, 2H),4.23 (t, J ¼ 5.9 Hz, 2H), 3.67e3.36 (m, 9H), 3.12 (m, 2H), 2.83 (m,2H), 2.36e2.30 (m, 2H), 1.80e1.62 (m, 2H), 1.35 (s, 18H). Q-DEPT

NMR (CDCl3, 100 MHz) d (ppm): 171.3 (np), 169.0 (np), 158.6 (np),156.7 (np), 156.4 (np), 146.5 (np), 128.6, 114.8, 106.7, 79.3 (np, 2C),51.4, 47.1 (np), 45.2 (np), 44.4 (np, 2C), 41.6 (np, 2C), 31.7 (np), 29.4(np), 28.4 (6C), 27.7 (np). MS-ESI m/z: [MþH]þ 566.3, [MþNa]þ

588.3. HRMS-ESI m/z calcd for C27H43N5O8Na [MþNa]þ: 588.3009,found: 588.2980.

4.1.2.3.5. di-t-Butyl (5-(3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propanamido)pentane-1,2-diyl)dicarbamate (50c). White solid wasobtained from compound 49c (0.7 mmol) in 100% yield: mp 66.1 �C.IR n (cm�1): 3334, 2966, 1679, 1527, 1363, 1247, 1165, 1065, 742, 640.1H NMR (CDCl3, 600 MHz) d (ppm): 6.95 (d, J ¼ 6.7 Hz, 1H), 6.92 (brs, 1H), 6.82 (d, J ¼ 7.2 Hz, 1H), 6.14 (t, J ¼ 7.1 Hz, 1H), 5.18 (br s, 1H),5.04e5.03 (br s, 1H), 4.25 (m, 2H), 3.56e3.54 (m, 1H), 3.17e3.16 (m,2H), 3.09 (m, 2H), 2.68 (t, J¼ 6.4 Hz, 2H), 1.50e1.40 (s, 22H). Q-DEPTNMR (CDCl3, 150 MHz) d (ppm): 170.3 (np), 158.7 (np), 157.0 (np),156.7 (np), 146.7 (np), 128.6, 115.2, 107.2, 79.7 (np, 2C), 50.8, 47.3(np), 44.8 (np), 39.5 (np), 35.5 (np), 29.9 (np), 28.6 (6C), 25.7 (np).[MþNa] 505.3. MS-ESI m/z: [MþH]þ 483.3, [MþNa]þ 505.3. HRMS-ESI m/z calcd for C23H38N4O7Na [MþNa]þ: 505.2638, found:505.2644.

4.1.2.3.6. di-t-Butyl (6-(3-(3-hydroxy -2-oxopyridin-1(2H)-yl)propanamido)hexane-1,2-diyl)dicarbamate (50d). Yellow solid wasobtained from compound 49d (0.7 mmol) in 87% yield: mp133.2 �C. IR n (cm�1): 3343, 2931, 1681, 1603, 1365, 1246, 1160, 648.1H NMR (CDCl3, 300 MHz) d (ppm): 6.96 (dd, J ¼ 6.9 Hz andJ0 ¼ 1.5 Hz, 1H), 6.80 (dd, J ¼ 7.4 Hz and J0 ¼ 1.5 Hz, 1H), 6.52e6.51(br s, 1H), 6.12 (t, J¼ 7.2 Hz, 1H), 5.04 (br s, 1H), 4.88e4.86 (br s, 1H),4.26 (t, J ¼ 6.3 Hz, 2H), 3.55e3.53 (m, 1H), 3.13e3.08 (m, 4H), 2.68(t, J¼ 6.3 Hz, 2H),1.79e1.50 (m, 2H),1.40 (s,18H),1.35e1.23 (m, 4H).Q-DEPT NMR (CDCl3, 75 MHz) d (ppm): 169.7 (np, 2C), 158.3 (np,2C), 146.2 (np), 128.2, 114.6, 106.7, 79.5 (2C), 51.1, 46.9 (np), 44.3(np), 38.8 (np), 35.1 (np), 31.9 (np), 28.8 (np), 28.2 (6C), 22.6 (np).MS-ESI m/z: [M� 2Boc þ 3H]þ 296.9, [M� Boc þ 2H]þ 397.0,[MþH]þ 497.1, [MþNa]þ 519.1. HRMS-ESI m/z calcd forC24H40N4O7Na [MþNa]þ: 519.2795, found: 519.2801.

4.1.2.3.7. di-t-Butyl (5-((3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propyl)amino)-5-oxopentane-1,2-diyl)dicarbamate (50e).Brown oil was obtained from compound 49e (1.6 mmol) in 100%yield. IR n (cm�1): 3346, 2923, 1680, 1520, 1247, 1160, 637. 1H NMR(CDCl3, 300 MHz) d (ppm): 7.34 (br s, 1H), 6.89 (dd, J ¼ 6.9 Hz andJ0 ¼ 1.2 Hz, 1H), 6.83 (d, J ¼ 7.2 Hz, 1H), 6.19 (t, J ¼ 6.9 Hz, 1H),5.33e5.23 (br s, 2H), 4.06 (t, J ¼ 6.2 Hz, 2H), 3.67e3.64 (m, 1H),3.28e3.17 (m, 4H), 2.30 (t, J ¼ 7.2 Hz, 2H), 1.93 (t, J ¼ 6.3 Hz, 2H),1.95e1.60 (m, 2H), 1.40 (s, 18H). Q-DEPT NMR (CDCl3, 75 MHz)d (ppm): 173.3 (np, 172.4 (np), 158.6 (np), 156.8 (np), 146.5 (np),127.0, 114.6, 107.6, 79.4 (2C), 51.2, 47.1 (np), 44.5 (np), 35.9 (np), 33.0(np), 29.3 (np), 28.2 (6C), 25.4 (np). MS-ESI m/z: [MþH]þ 483.4,[MþNa]þ 505.3. HRMS-ESI m/z calcd for C23H38N4O7Na [MþNa]þ:505.2652, found: 505.2638.

4.1.2.3.8. di-t-Butyl (6-(3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propanamido)hexane-1,2-diyl)dicarbamate (53a).Orange oil was obtained from compound 52a (1.3 mmol) in 100%yield. IR n (cm�1): 3300, 2933, 1688, 1506, 1363, 1243, 1162, 1068,605. 1H NMR (CD3OD, 400 MHz) d (ppm): 7.56 (d, J ¼ 7.2 Hz, 1H),6.37 (d, J ¼ 7.2 Hz, 1H), 4.37e4.33 (m, 2H), 3.53 (m, 1H), 3.13e3.07(m, 2H), 3.00e2.95 (m, 2H), 2.64 (t, J¼ 6.5 Hz, 2H), 2.46 (s, 3H), 1.44(s, 18H), 1.44e1.40 (m, 4H), 1.40e1.23 (m, 2H). Q-DEPT NMR(CD3OD, 100 MHz) d (ppm): 171.5 (np), 170.8 (np), 158.5 (np), 158.2(np), 147.1 (np), 139.0, 132.5 (np), 112.5, 80.0 (np, 2C), 51.4, 50.3 (np),45.4 (np), 40.2 (np), 37.6 (np), 32.9 (np), 30.0 (np), 28.8 (6C), 24.3(np), 11.8. MS-ESIm/z: [MþH]þ 511.3, [MþNa]þ 533.2. HRMS-ESIm/z calcd for C25H42N4O7 [MþH]þ: 533.2951, found: 533.2935.

4.1.2.3.9. di-t-Butyl (5-((3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propyl)amino)-5-oxopentane-1,2-diyl)dicarbamate (53b).White solid was obtained from compound 52b (1.5 mmol) in 60%

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 719

yield: mp 134.6 �C. IR n (cm�1): 3322, 2977, 1679, 1638, 1508, 1227,1162, 1033, 834, 728. 1H NMR (CDCl3, 400 MHz) d (ppm): 7.39 (m,2H), 6.31 (m, 1H), 5.40e5.33 (br s, 2H), 3.95 (m, 2H), 3.55 (m, 1H),3.40e3.13 (m, 4H), 2.35 (s, 3H), 2.24 (m, 2H), 1.96e1.60 (m, 4H), 1.40(s, 18H). Q-DEPT NMR (CDCl3, 100 MHz) d (ppm): 174.2 (np), 169.8(np), 157.7 (np, 2C), 146.8 (np), 137.5, 129.3 (np), 111.6, 80.2 (np, 2C),52.2 (np), 51.6, 44.9 (np), 36.8 (np), 33.5 (np), 31.4 (np), 30.2 (np),28.7 (6C), 12.1. MS-ESI m/z: [MþH]þ 497.2, [MþNa]þ 519.3. HRMS-ESI m/z calcd for C24H40N4O7 [MþH]þ: 519.2795, found: 519.2795.

4.1.2.4. General procedure for N,N0-di-Boc removal. To a solution ofcompound 35a-b, 39a-b, 50a-e or 53a-b (0.2e1.7 mmol) dissolvedin 1,4-dioxane (5e10mL)was added 4 NHCl solution in 1,4-dioxane(20 equiv). The mixture was stirred at rt for 2 h and concentratedunder reduced pressure. The residue was washed with Et2O(2� 5e10mL) that was then evaporated in vacuo to give compound36a-b, 40a-b, 51a-e, 54a-b or 55 as precipitate in 57e100% yield.

4.1.2.4.1. 4,5-Diamino-1-(4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)piperazin-1-yl)pentan-1-one, dihydrochloride salt (36a).Yellow pasty solid was obtained from compound 35a (0.3 mmol) in90% yield. IR n (cm�1): 3330, 2927, 1625, 1513, 1244, 1125, 1025, 605.1H NMR (d6-DMSO, 300 MHz) d (ppm): 9.52 (br s, 1H), 8.57 (br s,2H), 8.46 (br s, 2H), 7.44 (d, J ¼ 15.0 Hz, 1H), 7.33 (s, 1H), 7.10 (m,2H), 6.79 (d, J¼ 8.4 Hz, 1H), 3.83 (s, 3H), 3.71e3.45 (m, 9H), 3.12 (m,2H), 2.60e2.58 (m, 2H), 1.89 (m, 2H). Q-DEPT NMR (d6-DMSO,75 MHz) d (ppm): 169.7 (np), 165.0 (np), 162.9 (np), 148.5 (np),142.4, 126.4 (np), 122.5, 115.3, 114.2, 111.2, 55.7, 48.7, 39.7 (np, 5C),28.1 (np), 25.3 (np). MS-ESIm/z: [MþH]þ 377.2. HRMS-ESIm/z calcdfor C19H29N4O4 [MþH]þ: 377.2189, found: 377.2198. LCMS: tr 6 min(49% A/51% B), 190 nm: > 93%.

4.1.2.4.2. N-(5,6-Diaminohexyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide, dihydrochloride salt (36b). Yellow solid was obtainedfrom compound 35b (0.3 mmol) in 66% yield: mp 133.5 �C. IR n

(cm�1): 2922, 1592, 1513, 1451, 1250, 1123, 1026, 611. 1H NMR (d6-DMSO, 300 MHz) d (ppm): 8.49 (br s. 4H), 8.11e8.09 (br s, 1H), 7.30(d, J ¼ 15.9 Hz, 1H), 7.11 (d, J ¼ 1.8 Hz, 1H), 6.97 (dd, J ¼ 8.4 Hz andJ0 ¼ 1.8 Hz, 1H), 6.79 (d, J¼ 8.1 Hz, 1H), 6.49 (d, J¼ 15.9 Hz, 1H), 3.78(s, 3H), 3.42e3.39 (m, 1H), 3.17e3.05 (m, 4H), 1.64e1.62 (m, 2H),1.46e1.40 (m, 4H). Q-DEPT NMR (d6-DMSO, 75MHz) d (ppm): 165.3(np),148.2 (np),147.7 (np),138.7,126.3 (np),121.4,119.0,115.6,110.7,55.4, 48.9, 38.1 (np, 2C), 29.5 (np), 28.6 (np), 21.7 (np). MS-ESI m/z:[MþH]þ 308.1. HRMS-ESI m/z calcd for C16H26N3O3 [MþH]þ:308.1974, found: 308.1986. LCMS: tr 4.6 min (52% A/48% B), 190 nm:> 99%.

4.1.2.4.3. 4,5-Diamino-1-(4-(3,4,5-trihydroxybenzoyl)piperazin-1-yl)pentan-1-one, dihydrochloride salt (40a). Yellow solid was ob-tained from compound 39a (0.4 mmol) in 100% yield: mp 128.2 �C.IR n (cm�1): 2927, 2851, 1576, 1439, 1315, 1200, 1028, 614. 1H NMR(d6-DMSO, 300MHz) d (ppm): 8.65 (br s, 2H), 8.55 (br s, 2H), 6.37 (s,2H), 3.49e3.48 (m, 9H), 3.13 (m, 2H), 2.59 (t, J ¼ 4.8 Hz, 2H),1.90e1.88 (m, 2H). Q-DEPT NMR (d6-DMSO, 75MHz) d (ppm): 169.5(np), 169.3 (np), 145.3 (np, 2C), 134.5 (np), 124.9 (np), 106.3 (2C),48.5, 40.0 (np, 2C), 39.7 (np, 2C), 38.3 (np), 27.8 (np), 25.0 (np). MS-ESI m/z: [MþH]þ 352.9. HRMS-ESI m/z calcd for C16H25N4O5

[MþH]þ: 353.1825, found: 353.1813. LCMS: tr 0.8 min (98% A/2% B),190 nm: > 95%.

4.1.2.4.4. N-(5,6-Diaminohexyl)-3,4,5-trihydroxybenzamide,dihydrochloride salt (40b). Yellow solid was obtained from com-pound 39b (0.4 mmol) in 57% yield: mp 81.3 �C. IR n (cm�1): 3024,2933, 1572, 1446, 1328, 976, 669. 1H NMR (d6-DMSO, 300 MHz)d (ppm): 8.47 (br s, 4H), 8.11 (br s, 1H), 6.82 (s, 2H), 3.24e3.07 (m,5H), 1.65e1.35 (m, 6H). Q-DEPT NMR (d6-DMSO, 75 MHz) d (ppm):166.5 (np), 145.4 (np, 2C), 136.1 (np), 125.0 (np), 106.8 (2C), 49.0,40.4 (np, 2C), 29.6 (np), 28.8 (np), 21.8 (np). MS-ESI m/z: [MþH]þ

284.2. HRMS-ESI m/z calcd for C13H22N3O4 [MþH]þ: 284.1610,

found: 284.1612. LCMS: tr 1.1 min (98% A/2% B), 190 nm: > 99%.4.1.2.4.5. 1-(3-(4-(3,4-Diaminobutanoyl)piperazin-1-yl)-3-

oxopropyl)-3-hydroxypyridin-2(1H)-one, dihydrochloride salt (51a).White pasty solid was obtained from compound 50a (0.2 mmol) in88% yield. IR n (cm�1): 3362, 2925, 1606, 1447, 1246, 1017, 623. 1HNMR (D2O, 400 MHz) d (ppm): 7.20 (d, J ¼ 6.4 Hz, 1H), 7.03 (d,J¼ 6.6 Hz,1H), 6.39 (t, J¼ 6.9 Hz,1H), 4.32e4.28 (m,1H), 4.06e4.02(m, 2H), 3.69e3.45 (m, 8H), 3.17e3.10 (m, 2H), 3.04e3.01 (m, 2H),2.99e2.95 (m, 2H). Q-DEPT NMR (D2O, 100 MHz) d (ppm): 172.8(np), 171.5 (np), 168.8 (np), 158.5 (np), 129.5, 118.9, 108.3, 47.1 (np),45.5, 45.3 (np), 44.7 (np), 44.3 (np), 41.5 (np), 40.7 (np), 33.2 (np),31.5 (np). MS-ESI m/z: [MþH]þ 352.2. HRMS-ESI m/z calcd forC16H26N5O4 [MþH]þ: 352.1985, found: 352.1977. LCMS: tr 2.4 min(90% A/10% B), 190 nm: > 92%.

4.1.2.4.6. 1-(3-(4-(4,5-Diaminopentanoyl)piperazin-1-yl)-3-oxopropyl)-3-hydroxypyridin-2(1H)-one, dihydrochloride salt (51b).Yellow pasty solid was obtained from compound 50b (0.3 mmol) in100% yield. IR n (cm�1): 3372, 2930, 1585, 1440, 1220, 1015, 750,606. 1H NMR (D2O, 400 MHz) d (ppm): 7.20 (d, J ¼ 6.3 Hz, 1H), 7.03(d, J ¼ 6.4 Hz, 1H), 6.39 (t, J ¼ 6.8 Hz, 2H), 4.30 (t, J ¼ 6.3 Hz, 2H),3.75e3.60 (m, 4H), 3.55e3.52 (m, 5H), 3.37e3.35 (m, 2H),2.98e2.95 (m, 2H), 2.74e2.68 (m, 2H), 2.11e2.03 (m, 2H). Q-DEPTNMR (D2O, 100 MHz) d (ppm): 172.7 (np), 161.7 (np), 158.8 (np),145.3 (np), 129.5, 118.6, 108.3, 49.0, 47.0 (np), 45.1 (np), 44.4 (np),41.5 (np), 41.2 (np), 40.6 (np), 31.4 (np), 28.3 (np), 25.2 (np). MS-ESIm/z: [MþH]þ 366.2, [MþNa]þ 388.2. HRMS-ESI m/z calcd forC17H28N5O4 [MþH]þ: 366.2141, found: 366.2134. LCMS: tr 3.8 min(58% A/42% B), 190 nm: > 98%.

4.1.2.4.7. N-(4,5-Diaminopentyl)-3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propanamide, dihydrochloride salt (51c). Yellow pasty solidwas obtained from compound 50c (0.5 mmol) in 100% yield. IR n

(cm�1): 3360, 2925, 1644, 1549, 1260, 1207, 1073, 749, 623. 1H NMR(D2O, 400 MHz) d (ppm): 7.16 (dd, J ¼ 6.8 Hz and J0 ¼ 1.4 Hz, 1H),7.05 (dd, J¼ 7.5 Hz and J0 ¼ 1.4 Hz,1H), 6.41 (t, J¼ 7.1 Hz,1H), 4.30 (t,J ¼ 6.4 Hz, 2H), 3.67e3.64 (m, 1H), 3.35e3.34 (m, 2H), 3.18 (t,J ¼ 6.7 Hz, 2H), 2.72 (t, J ¼ 6.4 Hz, 2H), 1.76e1.57 (m, 4H). Q-DEPTNMR (D2O, 100 MHz) d (ppm): 173.3 (np), 158.5 (np), 145.5 (np),129.4, 118.8, 108.5, 49.0, 47.4 (np), 40.9 (np), 38.7 (np), 35.3 (np),27.5 (np), 24.1 (np). MS-ESIm/z: [MþH]þ 283.1. HRMS-ESIm/z calcdfor C13H23N4O3 [MþH]þ: 283.1770, found: 283.1765. LCMS: tr1.3 min (98% A/2% B), 190 nm: > 96%.

4.1.2.4.8. N-(5,6-Diaminohexyl)-3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propanamide, dihydrochloride salt (51d). Yellow pastysolid was obtained from compound 50d (0.5 mmol) in 100% yield.IR n (cm�1): 3350, 2934, 1644, 1549, 1206, 1077, 747. 1H NMR (d6-DMSO, 300 MHz) d (ppm): 8.59 (br s, 4H), 8.10e8.07 (br s, 1H), 7.08(dd, J ¼ 6.9 Hz and J0 ¼ 1.5 Hz, 1H), 6.70 (dd, J ¼ 7.2 Hz andJ0 ¼ 1.5 Hz, 1H), 6.07 (t, J ¼ 7.2 Hz, 1H), 4.10 (t, J ¼ 6.9 Hz, 2H),3.40e3.38 (m,1H), 3.09e3.00 (m, 4H), 2.53e2.49 (m, 2H),1.63e1.58(m, 2H), 1.19 (m, 4H). Q-DEPT NMR (d6-DMSO, 75 MHz) d (ppm):169.6 (np), 157.8 (np), 146.8 (np), 128.6, 115.0, 105.4, 49.1, 45.9 (np),40.6 (np, 38.2 (np), 34.8 (np), 29.7 (np), 28.6 (np), 21.9 (np). MS-ESIm/z: [MþH]þ 296.9. HRMS-ESI m/z calcd for C14H25N4O3 [MþH]þ:297.1927. found: 297.1914. LCMS: tr 1.8 min (98% A/2% B), 190 nm: >93%.

4.1.2.4.9. 4,5-Diamino-N-(3-(3-hydroxy-2-oxopyridin-1(2H)-yl)propyl)pentanamide, dihydrochloride salt (51e). White pasty solidwas obtained from compound 50e (1.7 mmol) in 93% yield. IR n

(cm�1): 3346, 2923, 1680, 1520, 1247, 1160, 637. 1H NMR (d6-DMSO,300 MHz) d (ppm): 8.63 (br s, 2H), 8.54 (br s, 2H), 8.30 (br s, 1H),7.20 (dd, J ¼ 6.8 Hz and J0 ¼ 1.5 Hz, 1H), 6.72 (dd, J ¼ 7.2 Hz andJ0 ¼ 1.8 Hz,1H), 6.11 (t, J¼ 6.9 Hz,1H), 3.96e3.87 (m, 2H), 3.49e3.46(m,1H), 3.11e3.04 (m, 4H), 2.32e2.31 (m, 2H),1.90e1.75 (m, 4H). Q-DEPT NMR (d6-DMSO, 75MHz) d (ppm): 171.2 (np),157.6 (np),146.6(np), 128.2, 114.8, 105.5, 48.7, 46.6 (np), 40.3 (np), 35.8 (np), 30.8

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722720

(np), 28.7 (np), 26.1 (np). MS-ESIm/z: [MþH]þ 283.0. HRMS-ESIm/zcalcd for C18H23N2O [MþH]þ: 283.1810, found: 283.1800. LCMS: tr1.8 min (98% A/2% B), 190 nm: > 91%.

4.1.2.4.10. N-(5,6-Diaminohexyl)-3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propanamide, dihydrochloride salt (54a).White pasty solid was obtained from compound 53a (0.7 mmol) in100% yield. IR n (cm�1): 3341, 2938, 1630, 1505, 1336, 1249. 1H NMR(D2O, 400 MHz) d (ppm): 7.97 (d, J ¼ 6.6 Hz, 1H), 7.18 (d, J ¼ 7.0 Hz,1H), 4.68 (t, J ¼ 6.4 Hz, 2H), 3.71e3.65 (m, 1H), 3.38e3.37 (m, 2H),3.16 (t, J ¼ 6.7 Hz, 2H), 3.08e3.04 (br s, 1H), 2.88 (t, J ¼ 6.5 Hz, 2H),2.56 (s, 3H), 1.85e1.74 (m, 2H), 1.52e1.38 (m, 4H), 1.40e1.23 (m,2H). Q-DEPT NMR (D2O, 100 MHz) d (ppm): 171.3 (np, 2C), 158.2(np), 142.4 (np), 138.7, 110.9, 52.7 (np), 49.2, 40.8 (np), 38.9 (np),35.8 (np), 29.6 (np), 27.9 (np), 21.6 (np), 12.3. MS-ESI m/z: [MþH]þ

311.2. HRMS-ESI m/z calcd for C15H27N4O3 [MþH]þ: 311.2083,found: 311.2075. LCMS: tr 1.2 min (98% A/2% B), 190 nm: > 91%.

4.1.2.4.11. 4,5-Diamino-N-(3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propyl)pentanamide, dihydrochloride salt (54b).White solid was obtained from compound 53b (0.6 mmol) in 100%yield: mp 154.5 �C. IR n (cm�1): 2914, 2855, 1631, 1504, 1335, 1253,1118, 1030, 870, 832, 618. 1H NMR (D2O, 400 MHz) d (ppm): 8.28 (d,J ¼ 6.5 Hz, 1H), 7.13 (d, J ¼ 6.5 Hz, 1H), 4.44 (m, 2H), 3.33e3.29 (m,5H), 2.64 (s, 3H), 2.57 (m, 2H), 2.08e2.07 (m, 4H). Q-DEPT NMR(D2O, 100 MHz) d (ppm): 174.9 (np), 159.8 (np), 145.2 (np), 143.4(np), 139.4, 111.7, 55.7 (np), 50.6, 41.9 (np), 37.3 (np), 32.2 (np), 31.0(np), 27.1 (np), 12.8. MS-ESIm/z: [MþH]þ 297.2. HRMS-ESIm/z calcdfor C14H25N4O3 [MþH]þ: 297.1927, found: 297.1931. LCMS: tr1.2 min (98% A/2% B), 190 nm: > 99%.

4.1.2.4.12. 3-(3-(Benzyloxy)-2-oxopyridin-1(2H)-yl)-N-(5,6-diaminohexyl)propanamide, dihydrochloride salt (55). Orange pastysolid was obtained from compound 49d (0.3 mmol) in 100% yield,IR n (cm�1): 3391, 2931, 1644, 1550, 1455, 1226, 1053, 748, 697. 1HNMR (CD3OD, 400 MHz) d (ppm): 7.46 (br s, 2H), 7.37e7.27 (m, 3H),6.58 (m,1H), 5.21 (m, 2H), 4.40 (m, 2H), 3.62 (m,1H), 3.32e3.31 (m,2H), 3.19 (m, 2H), 2.77 (m, 2H), 1.80 (m, 2H), 1.53 (m, 4H). Q-DEPTNMR (CD3OD, 100 MHz) d (ppm): 171.4 (np), 157.1 (np), 147.2 (np),135.7 (np), 130.3, 128.6 (2C), 128.2, 127.8 (2C), 119.3, 109.5, 71.0 (np),49.6, 40.8 (np), 38.5 (np), 34.7 (np), 32.4 (np), 29.7 (np), 28.3 (np),21.8 (np). MS-ESI m/z: [MþH]þ 387.2. HRMS-ESI m/z calcd forC21H31N4O3 [MþH]þ: 387.2396, found: 387.2382.

4.2. Physicochemical and biological evaluations

4.2.1. MGO and MDA trapping assayAqueous 40% MGO solution was diluted in water (200 mM,

1.25mL) andMDA solution (200mM,1.25mL) was freshly preparedby hydrolyzing 1,1,3,3-tetraethoxypropane (250 mmol) with 1 N HCl(2 equiv) at rt for 1 h under vigorous stirring. A stock solution oftested compounds (30 mmol) was also prepared in D-PBS (20 mM,1.5 mL). Tested compounds (10 mM) were incubated with MGO orMDA (20 mM) at 37 �C for 24 h. The pH of the solutionwas adjustedto 7.4 (NaOH, 20 mM and final mixture volume, 1.25 mL). Samples(100 mL) were collected at regular time intervals (0.25, 0.5,1, 5, 24 h)and stored at �20 �C to stop the reaction. Finally, after leaving to rtand diluting with MeCN/H2O 2:98, they were submitted to LCMSanalysis (generally, trFree scavengers: 0.8e4.6 min, trAdducts:4.1e6.8 min). A blank solution without scavenger and a standardone with only scavenger were used as well. After identification ofunreacted free scavenger and adduct on mass spectrum, AUC oftotal peak of adducts and remaining free scavenger peak weremeasured on UV chromatogram to give % of adduct formation.

4.2.2. ORAC assayA fluorescein (FL) solution (12 nM, 150 mL) was introduced in a

black 96-well plate (Dutscher, Brumath, France). Then, tested

compounds (1e20 mM, 25 mL) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) standard(1e50 mM, 25 mL) dissolved in D-PBS were added to each well. Theplate was allowed to equilibrate by incubating for a minimum of30min at 37 �C. Reactions were initiated by the addition of a freshlyprepared 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH)solution (30 mM, 25 mL). The fluorescence (lEx: 485 nm; lEm:520 nm) was measured using a thermostated Tecan Infinite® 200PROmicroplate reader every 90 s for 60 cycles. All reaction mixtureswere prepared in triplicate, and at least three independent assayswere performed for each sample. FL fluorescence decay curve intime allowedmeasuring AUC of tested products in comparisonwiththe control corresponding to an absence of antioxidant. ORACFLvalues at 10 mMwere calculated with respect to the linear equationof trolox calibration curve (Net AUC vs concentration) andexpressed as mmol trolox equivalent (TE)/mmol of tested compound.

4.2.3. Cu2þ-chelating assayStock solutions of tested compounds (4.2 mM, 6.2 mL) and

CuSO4.5H2O (0.25 mM, 100 mL for the assay and 0.5 mM, 20 mL forthe calibration curve) were prepared in 10 mM hexamine/HClbuffer containing 10 mM KCl (pH 5.0) or in a mixture buffer/MeOH75:25 (medium used with less water-soluble compounds 36a, 50dand 55) as well as aqueous solution of murexide (1 mM, 10 mL).Tested compounds (0.02, 0.05, 0.08, 0.1, 0.2, 0.5, 1 and 2 mM) werediluted in buffer or buffer/MeOH 75:25 (Q.S. to 1mL) and incubatedwith CuSO4.5H2O (120 mM,1 mL) at rt for 10 min. Murexide (48 mM,0.1 mL), used as complexometric indicator was then added and themixture was incubated for another 1 min at rt. Absorbance of thesolution was recorded at 485 nm (lmax of Cu2þ/murexide complex)and 520 nm (lmax of free murexide) using a Jasco V-650 UV/Visspectrophotometer. A blank solution composed of buffer or buffer/MeOH 75:25 (2 mL) and water (0.1 mL) was required. Calibrationcurves (A485/A520 vs Cu2þ concentration) were previously plottedusing CuSO4.5H2O (0, 25, 50, 75, 100, 125 mM) in buffer or buffer/MeOH 75:25 (Q.S. to 2 mL) and murexide solution (0.1 mL).Knowing the total quantity of metal ions introduced into the re-action mixture (control conditions without tested product), % Cu2þ

chelation by tested compounds was estimated by difference.

4.2.4. Cell viability assayThe immortalized rat neuronal-like cell line PC12 Adh (CRL-

1721.1, LGC, Molsheim, France) is derived from primary rat pheo-chromocytoma. PC12 cells were cultured in F-12K medium sup-plemented with 2.5% heat-inactivated fetal bovine serum (FBS, PANbiotech., Dutscher, Brumath, France), 15% heat-inactivated horseserum (HS, Life technologies SAS, Saint Aubin, France), 100 IU/mLpenicillin and 100 mg/mL streptomycin at 37 �C in humidified at-mosphere containing 5% CO2.

Cell viability was assessed by measuring mitochondrial activityin the presence of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt (WST-8), that produces a water-soluble formazan dye uponbioreduction in the presence of an electron carrier, 1-methoxy-5-methylphenazinium methyl sulfate (1-Methoxy PMS). Briefly,cells (5.103 cells/well) were seeded in 96-well plates (Corning, VWR,Fontenay-sous-bois, France) in 100 mL of appropriate complete me-dium. Once cells have reached a 80% confluency, the medium waschanged and cells were treated with various concentrations oftested compounds (10 mM or 100 mM) for 24 h. A positive control ofcytotoxicity was used by adding 10% DMSO. The CCK8 solution(10 mL) was directly added to each well and incubated for another1 h at 37 �C. Absorbance was recorded at 450 nm using a PerkinElmer 2103 Envision® multilabel microplate reader and provided anestimation of formazan dye bioproduction that was directly

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722 721

proportional to the number of living cells. Cell viability wasexpressed as % of control (non-treated cells) and at least three in-dependent experiments were performed in triplicate.

4.2.5. In vitro MGO-induced apoptosis inhibitionMGO-induced apoptosis was measured by the detection of DNA

fragmentation. Briefly, cells were pre-incubated with tested com-pound 51e (10 mM or 100 mM) dissolved in D-PBS for 1 h at 37 �Cbefore adding MGO (2 mM) for another 24 h incubation. Apoptosiswas evaluated by an ELISA assay for cytoplasmic histone-associatedDNA fragments (mono- and oligonucleosomes), using a cell deathdetection kit from Roche Diagnostics (Meylan, France). Cell lysateswere applied to a plate coated with an anti-histone antibody. Aperoxidase labeled anti-DNA antibody was then added and detec-ted with diammonium 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) as a substrate. Reacting with hydrogen peroxide,it turned into a green soluble end-product whose optical density(OD) reflecting apoptosis level was read at 405 nm using a PerkinElmer 2103 Envision® multilabel microplate reader. Cells incubatedwithout MGO represented control conditions and a positiveapoptosis standardwas also assessed in the presence of MGO alone.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

We thank “Soci�et�e d'Acc�el�eration de Transfert de Technologies(SATT) Nord” for financial support of this study.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2016.04.069.

References

[1] M. Rosini, E. Simoni, A. Milelli, A. Minarini, C. Melchiorre, Oxidative stress inAlzheimer's disease: are we connecting the dots? J. Med. Chem. 57 (2014)2821e2831.

[2] D.A. Butterfield, J. Drake, C. Pocernich, A. Castegna, Evidence of oxidativedamage in Alzheimer's disease brain: central role for amyloid b-peptide,Trends Mol. Med. 7 (2001) 548e554.

[3] A. Tiiman, P. Palumaa, V. Tougu, The missing link in the amyloid cascade ofAlzheimer's disease e metal ions, Neurochem. Int. 62 (2013) 367e378.

[4] P. Faller, C. Hureau, A bioinorganic view of Alzheimer's disease: when mis-placed metal ions (re)direct the electrons to the wrong target, Chem. Eur. J. 18(2012) 15910e15920.

[5] C. Ott, K. Jacobs, E. Haucke, A. Navarrete Santos, T. Grune, A. Simm, Role ofadvanced glycation endproducts in cellular signaling, Redox Bio 2 (2014)411e429.

[6] A. Simm, B. Müller, N. Nass, B. Hofmann, H. Bushnaq, R.-E. Silber, B. Bartling,Protein glycation d Between tissue aging and protection, Exp. Gerontol. 68(2015) 71e75.

[7] M.P. Kalapos, Where does plasma methylglyoxal originate from? Diabetes Res.Clin. Pract. 99 (2013) 261e271.

[8] A. Negre-Salvayre, C. Coatrieux, C. Ingueneau, R. Salvayre, Advanced lipidperoxidation end products in oxidative damage to proteins. Potential role indiseases and therapeutic prospects for the inhibitors, Br. J. Pharm. 153 (2008)6e20.

[9] Q. Lv, C. Gu, C. Chen, Venlafaxine protects methylglyoxal-induced apoptosis inthe cultured human brain microvascular endothelial cells, Neurosci. Lett. 569(2014) 99e103.

[10] S. Del Turco, G. Basta, An update on advanced glycation endproducts andatherosclerosis, Biofactors 38 (2012) 266e274.

[11] R. Nagai, D.B. Murray, T.O. Metz, J.W. Baynes, Chelation: a fundamentalmechanism of action of AGE inhibitors, AGE breakers, and other inhibitors ofdiabetes complications, Diabetes 61 (2012) 549e559.

[12] M. Krautwald, G. Münch, Advanced glycation end products as biomarkers andgerontotoxins e a basis to explore methylglyoxal-lowering agents for Alz-heimer's disease? Exp. Gerontol. 45 (2010) 744e751.

[13] M. Tajes, A. Eraso-Pichot, F. Rubio-Moscardo, B. Guivernau, M. Bosch-Morato,

V. Valls-Comamala, F.J. Munoz, Methylglyoxal reduces mitochondrial poten-tial and activates Bax and caspase-3 in neurons: implications for Alzheimer'sdisease, Neurosci. Lett. 580 (2014) 78e82.

[14] E. Niedzielska, I. Smaga, M. Gawlik, A. Moniczewski, P. Stankowicz, J. Pera,M. Filip, Oxidative stress in neurodegenerative diseases, Mol. Neurobiol. Jul 22(2015) 1e32.

[15] H.-W. Klafki, M. Staufenbiel, J. Kornhuber, J. Wiltfang, Therapeutic approachesto Alzheimer's disease, Brain 129 (2006) 2840e2855.

[16] N.A. Sasaki, M.C. Garcia-Alvarez, Q. Wanga, L. Ermolenko, G. Franck, N. Nhiri,M.-T. Martin, N. Audic, P. Potier, N-Terminal 2,3-diaminopropionic acid (Dap)peptides as efficient methylglyoxal scavengers to inhibit advanced glycationendproduct (AGE) formation, Bioorg. Med. Chem. 17 (2009) 2310e2320.

[17] N. Audic, G. Potier, N.A. Sasaki, New 2,3-diaminopropionic acid inhibitors ofAGE and ALE formation, Org. Biomol. Chem. 11 (2013) 773e780.

[18] A. Sgarbossa, D. Giacomazza, M. di Carlo, Ferulic acid: a hope for Alzheimer'sdisease therapy from plants, Nutrients 7 (2015) 5764e5782.

[19] J. Teixeira, T. Silva, S. Benfeito, A. Gaspar, E.M. Garrido, J. Garrido, F. Borges,Exploring nature profits: development of novel and potent lipophilic anti-oxidants based on galloyl-cinnamic hybrids, Eur. J. Med. Chem. 62 (2013)289e296.

[20] A. Robert, Y. Liu, M. Nguyen, B. Meunier, Regulation of copper and iron ho-meostasis by metal chelators: a possible chemotherapy for Alzheimer's dis-ease, Acc. Chem. Res. 48 (2015) 1332e1339.

[21] M.A. Santos, S.M. Marques, S. Chaves, Hydroxypyridinones as “privileged”chelating structures for the design of medicinal drugs, Coord. Chem. Rev. 256(2012) 240e259.

[22] A. Gaeta, F. Molina-Holgado, X.L. Kong, S. Salvage, S. Fakih, P.T. Francis,R.J. Williams, R.C. Hider, Synthesis, physical-chemical characterisation andbiological evaluation of novel 2-amido-3-hydroxypyridin-4(1H)-ones: ironchelators with the potential for treating Alzheimer's disease, Bioorg. Med.Chem. 19 (2011) 1285e1297.

[23] S.S. More, R. Vince, Inhibition of glyoxalase I: the first low-nanomolar tight-binding inhibitors, J. Med. Chem. 52 (2009) 4650e4656.

[24] S. Jug�e, J. Bayardon, E. R�emond, M.-J. Ondel-Eymin, Versatile and Stereospe-cific Synthesis of Gamma,Delta -Unsaturated Amino Acids by Wittig Reaction,PCT (2013). WO 2013/030193 A1.

[25] C.S. Dexter, R.F.W. Jackson, Synthesis of enantiomerically pure b- and g-aminoacid derivatives using functionalized organozinc reagents, J. Org. Chem. 64(1999) 7579e7585.

[26] T. Markidis, G. Kokotos, A novel approach to the synthesis of chiral terminal1,2-diamines, J. Org. Chem. 66 (2001) 1919e1923.

[27] J.V. Duncia, J.B. Santella, D.A. Wacker, W. Yao, C. Zheng, Piperidine Amides asModulators of Chemokine Receptor Activity, PCT (2001). WO 01/98268 A2.

[28] E. Bellis, T. Markidis, G. Kokotos, Synthesis of chiral n,nþ1-diaminoacids andtheir application to the construction of dendrimers, Synthesis 10 (2002)1359e1364.

[29] A. Boeijen, R.M.J. Liskamp, J.A.J. Den Hartog, Monomers and Oligourea Pepti-domimetics and Process for the Preparation Thereof, PCT (2000). WO 00/64865 A1.

[30] V.V. Sureshbabu, S.A. Naik, G. Nagendra, Synthesis of Boc-amino tetrazolesderived from a-amino acids, Synth. Commun. 39 (2009) 395e406.

[31] S. Caddick, D.B. Judd, A.K. de K. Lewis, M.T. Reich, M.R.V. Williams, A genericapproach for the catalytic reduction of nitriles, Tetrahedron 59 (2003)5417e5423.

[32] D.W.P.M. Lowik, R.M.J. Liskamp, Synthesis of a- and b-substituted amino-ethane sulfonamide arginineeglycine mimics, Eur. J. Org. Chem. (2000)1219e1228.

[33] S. Schiesser, B. Hackner, M. Vrabel, W. Beck, T. Carell, Synthesis and DNA-damaging properties of cisplatin-N-mustard conjugates, Eur. J. Org. Chem.(2015) 2654e2660.

[34] P. Kumar, D.R. Jain, Cg-aminopropylene peptide nucleic acid (amp-PNA):chiral cationic PNAs with superior PNA: DNA/RNA duplex stability and cellularuptake, Tetrahedron 71 (2015) 3378e3384.

[35] H.S.P. Rao, P. Siva, Facile reduction of azides with sodium borohydride/cop-per(II) sulphate system, Synth. Comm. 24 (1994) 549e555.

[36] R.J. Cherney, J.B. Brogan, R. Mo, Y.C. Lo, G. Yang, P.B. Miller, P.A. Scherle,B.F. Molino, P.H. Carter, C.P. Decicco, Discovery of trisubstituted cyclohexanesas potent CC chemokine receptor 2 (CCR2) antagonists, Bioorg. Med. Chem.Lett. 19 (2009) 597e601.

[37] B. Peschke, M. Zundel, S. Bak, T.R. Clausen, N. Blume, A. Pedersen, F. Zaragozaa,K. Madsen, C-terminally PEGylated hGH-derivatives, Bioorg. Med. Chem. 15(2007) 4382e4395.

[38] A.K. Mallams, B. Dasmahapatra, B.R. Neustadt, M. Demma, H.A. Vaccaro,Quinazoline Derivatives Useful in Cancer Treatment, PCT (2007). WO 07/011623 A1.

[39] Y. Xie, Y. Zhao, Synthesis of 7-O-galloyl-D-sedoheptulose, Carbohydr. Res. 342(2007) 1510e1513.

[40] J. Arumugam, H.A. Brown, H.K. Jacobs, A.S. Gopalan, New synthetic approachfor the incorporation of 3,2-hydroxypyridinone (HOPO) ligands: synthesis ofstructurally diverse poly HOPO chelators, Synthesis 1 (2011) 57e64.

[41] B.L. Rai, L.S. Dekhordi, H. Khodr, Y. Jin, Z. Liu, R.C. Hider, Synthesis, physico-chemical properties, and evaluation of N-substituted-2-alkyl-3-hydroxy-4(1H)-pyridinones, J. Med. Chem. 41 (1998) 3347e3359.

[42] M.A. Santos, R. Grazina, A.Q. Neto, G. Cantinho, L. Gano, L. Patricio, Synthesis,chelating properties towards gallium and biological evaluation of two N-

E. Lohou et al. / European Journal of Medicinal Chemistry 122 (2016) 702e722722

substituted 3-hydroxy-4-pyridinones, J. Inorg. Biochem. 78 (2000) 303e311.[43] M.A. Santos, S. Gama, L. Gano, G. Cantinhoc, E. Farkas, A new bis(3-hydroxy-4-

pyridinone)-IDA derivative as a potential therapeutic chelating agent. Syn-thesis, metal-complexation and biological assays, Dalton Trans. (2004)3772e3781.

[44] J. Sebestik, M. Safarik, P. Bour, Ferric complexes of 3-Hydroxy-4-pyridinonescharacterized by density functional theory and Raman and UV-vis Spectros-copies, Inorg. Chem. 51 (2012) 4473e4481.

[45] L. Lv, X. Shao, L. Wang, D. Huang, C.-T. Ho, S. Sang, Stilbene glucoside frompolygonum multiflorum thunb.: a novel natural inhibitor of advanced glyca-tion end product formation by trapping of methylglyoxal, J. Agric. Food Chem.58 (2010) 2239e2245.

[46] A.R. Hipkiss, Energy metabolism, proteotoxic stress and age-relateddysfunction e protection by carnosine, Mol. Asp. Med. 32 (2011) 267e278.

[47] A. Davalos, C. Gomez-Cordoves, B. Bartolome, Extending applicability of theoxygen radical absorbance capacity (ORAC-Fluorescein) assay, J. Agric. FoodChem. 52 (2004) 48e54.

[48] R.L. Prior, X. Wu, K. Schaich, Standardized methods for the determination ofantioxidant capacity and phenolics in foods and dietary supplements, J. Agric.Food Chem. 53 (2005) 4290e4302.

[49] P. Meena, V. Nemaysh, M. Khatri, A. Manral, P.M. Luthra, M. Tiwari, Synthesis,biological evaluation and molecular docking study of novel piperidine andpiperazine derivatives as multi-targeted agents to treat Alzheimer's disease,Bioorg. Med. Chem. 23 (2015) 1135e1148.

[50] P. Larroque-Cardoso, C. Camar�e, F. Nadal-Wollbold, M.-H. Grazide, M. Pucelle,S. Garoby-Salom, P. Bogdanowicz, G. Josse, A.-M. Schmitt, K. Uchida,K. Zarkovic, R. Salvayre, A. N�egre-Salvayre, Elastin modification by 4-hydroxynonenal in hairless mice exposed to UV-A. Role in photoaging andactinic elastosis, J. Investig. Derm. 135 (2015) 1873e1881.

[51] R. Kayyali, A.S. Pannala, H. Khodr, R.C. Hider, Comparative radical scavengingability of bidentate iron(III) chelators, Biochem. Pharmacol. 55 (1998)1327e1332.

[52] X. Li, J. Lin, W. Han, W. Mai, L. Wang, Q. Li, M. Lin, M. Bai, L. Zhang, D. Chen,Antioxidant ability and mechanism of rhizoma Atractylodes macrocephala,

Molecules 17 (2012) 13457e13472.[53] M. Karamac, Chelation of Cu(II), Zn(II), and Fe(II) by tannin constituents of

selected edible nuts, Int. J. Mol. Sci. 10 (2009) 5485e5497.[54] P. Tamilmani, M.C. Pandey, Iron binding efficiency of polyphenols: compari-

son of effect of ascorbic acid and ethylenediaminetetraacetic acid on catecholand galloyl groups, Food Chem. 197 (2016) 1275e1279.

[55] K. Ohnuma, Y. Hayashi, M. Furue, K. Kaneko, M. Asashima, Serum-free cultureconditions for serial subculture of undifferentiated PC12 cells, J. Neurosci.Methods 151 (2006) 250e261.

[56] B. Cheng, H. Lu, B. Bai, J. Chen, D-b-Hydroxybutyrate inhibited the apoptosis ofPC12 cells induced by H2O2 via inhibiting oxidative stress, Neurochem. Int. 62(2013) 620e625.

[57] H. Chen, X. Liang, F. Xu, B. Xu, X. He, B. Huang, M. Yuan, Synthesis andcytotoxic activity evaluation of novel arylpiperazine derivatives on humanprostate cancer cell lines, Molecules 19 (2014) 12048e12064.

[58] B. Yang, F. Chen, Y. Hua, S.-S. Huang, S. Lin, L. Wen, Y. Jiang, Prooxidant ac-tivities of quercetin, p-courmaric acid and their derivatives analysed byquantitative structure-activity relationship, Food Chem. 131 (2012) 508e512.

[59] Q. Lv, C. Gu, C. Chen, Venlafaxine protects methylglyoxal-induced apoptosis inthe cultured human brain microvascular endothelial cells, Neurosci. Lett. 569(2014) 99e103.

[60] Apoptosis, Cytotoxicity and Cell Proliferation, Roche Diagnostics Brochure,fourth ed., 2008.

[61] A. Warshawsky, J. Altman, R. Arad-Yellin, H.E. Gottlieb, A. Deshe, N. Kahana,N. Shoef, M. Wilchek, Bifunctional chelating agents, Part 3: 4,5-bis[di(car-boxymethyl)amino]-N-(aminoalkyl)valeramide, an EDTA with an aminefunctionality and amide-type linker arm, J. Chem. Soc. Perkin Trans. I (1989)1781e1786.

[62] Y. Matsumura, Y. Satoh, K. Shirai, O. Onomura, T. Maki, New reaction condi-tions using trifluoroethanol for the E-I Hofmann rearrangement, J. Chem. Soc.Perkin Trans. I (1999) 2057e2060.

[63] Y. Gai, Z. Hu, Z. Rong, X. Ma, G. Xiang, A practical route for the preparation of1,4,7-triazacyclononanyl diacetates with a hydroxypyridinonate pendant arm,Molecules 20 (2015) 19393e19405.

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具