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European Journal of Medicinal Chemistry 46 (2011) 4676e4681

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European Journal of Medicinal Chemistry

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

Short communication

Synthesis and pharmacological assessment of diversely substitutedpyrazolo[3,4-b]quinoline, and benzo[b]pyrazolo[4,3-g][1,8]naphthyridinederivatives

Daniel Silva a,b,1, Mourad Chioua b,1, Abdelouahid Samadi b, M. Carmo Carreiras a,*, María-Luisa Jimeno c,Eduarda Mendes a, Cristóbal de los Ríos d,e, Alejandro Romero f,h, Mercedes Villarroya d,f,Manuela G. López f,g, José Marco-Contelles b,**

aResearch Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugalb Laboratorio de Radicales Libres y Química Computacional (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, SpaincCentro de Química Orgánica “Lora Tamayo” (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spaind Instituto de Investigación Sanitaria, Fundación de Investigación Biomédica, Hospital Universitario de la Princesa, C/Diego de León 62, 28006 Madrid, SpaineDepartamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcalá, Ctra. Barcelona, Km. 33.5, 28817 Alcalá de Henares, Spainf Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, SpaingHospital La Paz Health Research Institute-IdiPAZ, Madrid, SpainhDepartamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:Received 15 March 2011Received in revised form20 May 2011Accepted 26 May 2011Available online 7 June 2011

Keywords:PyrazolotacrinesAChE/BuChE inhibitorsOxidative stressNeuroprotectionAlzheimer’s disease

* Corresponding author. Tel.: þ351 21 7946475; fax** Corresponding author. Fax: þ34 91 5644853.

E-mail addresses: iqoc21@iqog.csic.es (M. Carmo C(J. Marco-Contelles).

1 Those authors have contributed equally to this w

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.05.068

a b s t r a c t

The synthesis and pharmacological analyses of a number of pyrazolo[3,4-b]quinoline and benzo[b]py-razolo[4,3-g][1,8]naphthyridine derivatives are reported. We have synthesized the diversely substitutedtacrine analogues 1e6, by Friedländer-type reaction of readily available o-amino-1-methyl-pyrazole-dicarbonitriles with cyclohexanone. The biological evaluation showed that pyrazolotacrines 1e6 areinhibitors of Electrophorus electricus acetylcholinesterase (EeAChE), in the micromolar range, and quiteselective in respect to serum horse butyrylcholinesterase (eqBuChE) inhibition; the most interestinginhibitor is N-(5-amino-1-methyl-6,7,8,9-tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naphthyridin-3-yl)acetamide (5) [IC50 (EeAChE) ¼ 0.069 � 0.006 mM; IC50 (eqBuChE) ¼ 6.3 � 0.6 mM]. Kinetic studiesshowed that compound 5 is a mixed-type inhibitor of EeAChE (Ki ¼ 155 nM). Inhibitor 5 showed a 45%neuroprotection value against rotenone/oligomycin A-induced neuronal death.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Alzheimer’s disease (AD) is the most prevalent neurodegener-ative disorder, and one of the most common of all diseases in theindustrialized world [1]. AD is characterized by the gradual devel-opment of forgetfulness, progressing to disturbances in language,dyscalculia/acalculia, visuospatial disorientation, ideational andideomotor apraxia, akinesia, and mutism. The symptomatic courseof disease is generally 5 or more years, although a 7-year preclinicalperiod of stepwise decline in memory and attention span has been

: þ351 21 7946470.

arreiras), mcdamaso@ff.ul.pt

ork.

son SAS. All rights reserved.

described [2]. Classical pathological hallmarks are senile plaques,consisting principally of amyloid-b (Ab), and neurofibrillarytangles, which are composed of phosphorylated tau. Furthermore,the basal nucleus of Meynert undergoes profound neuron loss, andthe neocortex exhibits a loss of cholinergic fibres and receptors anda decrease of both choline acetyltransferase and acetylcholines-terase (AChE) enzyme activity [2,3]. Since the symptoms of ADwereassociated with an altered cholinergic function, research hasfocused on the basal forebrain cholinergic system [3]. As a result,the so-called “cholinergic hypothesis” was developed, whichpostulated that a loss of cholinergic function in the central nervoussystem contributed significantly to cognitive decline associatedwith advanced age and AD [4]. Thus, drugs capable of inhibitingAChE might potentiate central cholinergic function, thereforeimproving cognition and perhaps even some of the behaviouralproblems experienced by AD patients [5]. AChE inhibitors (AChEI)

Table 1Inhibition of AChE from Electrophorus electricus (EeAChE) and horseserum butyrylcholinesterase (eqBuChE) by the pyrazolo-THA analogues 1e6 and THA.

Compound Structure IC50 (mM) EeAChE IC50 (mM) eqBuChE Selectivity BuChE/AChE

1

NNN

NH2

Me

NC

0.57 � 0.04 100 >175

2

NNN

NH2NC

Me

1.70 � 0.02 6.3 � 0.6 3.7

3

NNN

NH2C

Me

OH2N

0.6 � 0.1 5.4 � 0.6 9

4

NNN

HNCO

Me

OH2N

9.3 � 1.5 >100 > 10.7

5

N NNN

AcHN

Me

NH2

0.069 � 0.006 6.3 � 0.6 91.3

6

NNNN

NH2AcHN

Me

Ph

0.23 � 0.05 9.0 � 0.9 39

Tacrine

N

NH2

0.027 � 0.002 0.0052 � 0.0002 0.193

aData are expressed as the mean � SEM of at least three different experiments in quadruplicate.

Scheme 1. Synthesis of 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]quinoline-3-carbonitrile (1).

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681 4677

may inhibit AChE via a competitive mechanism, by interacting withthe catalytic centre (CC) of the enzyme, via a non-competitivemechanism, by binding with the peripheral anionic site (PAS), orvia both mechanisms, by exerting a dual binding AChE inhibition[5]. For a while the treatment with AChEI was reported to produceonly symptomatic improvement, having no effect in the course ofthe disease [6]. However, other studies indicated that AChE inter-acts with Ab by a hydrophobic environment close to the PAS, thuspromoting Ab fibril formation [7,8]. Besides, AChEeAb complexesincrease Ab-dependent neurotoxicity [9]. These reports arose a newenthusiasm in AChEI. Interestingly, early reports demonstrated theability of cholinesterase inhibitors (ChEI) to enhance the release ofnon-amyloidogenic soluble derivatives of amyloid precursorprotein (APP) in vitro and in vivo, and possibly to slow downformation of amyloidogenic compounds in the brain [10]. Theincrease of soluble APP (APPs) was also consistent with AChEinhibition [11]. Numerous clinical trials have demonstrated thesafety and efficacy of ChEI in the treatment of AD. Besides, there isgrowing evidence from preclinical studies indicating that these

agents can attenuate neuronal damage and death from cytotoxicinsults, and therefore might affect AD pathogenesis [12].

Considering the non-cholinergic aspects of AChE related to itsPAS [7e9] in associating with Ab, an attractive target for the designof new anti-dementia drugs emerged. Peripheral or dual siteinhibitors of AChE may simultaneously alleviate the cognitivedeficit in AD patients and prevent the assembly of Ab, which will

Scheme 2. Synthesis of 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]quinoline-3-carbonitrile (2), and 1-methyl-4-((6-phenoxyhexyl)amino)-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]quinoline-3-carboxamide (4).

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e46814678

delay the neurodegenerative process [13]. This strategy waspursued for several medicinal chemists who have been developingnew compounds with dual AChE inhibitory activity [14e17].

The mechanism of Ab toxicity also involves destabilization ofcellular calcium homeostasis resulting in elevated rest intracell-ular free calcium concentration ([Ca2þ)i] and increased [Ca2þ]iresponses to excitatory aminoacids and membrane depolarization.Thus, Ab renders neuronsmore vulnerable to environmental insults[18e20].

Due to the multiple events that take place in the cascade of theneurodegenerative disease new therapeutic strategies weredevised and the new drug candidates were designed to act onmultiple neural and biochemical targets for the treatment ofcognition impairment, motor dysfunction, depression and neuro-degeneration [21]. Examples include the development of singlemolecular entities that combine two or more of the followingproperties: cholinesterase inhibition, activation or inhibition ofspecific subtypes of acetylcholine receptors or a-adrenoceptors,anti-inflammatory activity, monoamine oxidase inhibition, neuro-protection, and anti-apoptotic activity among others [21]. There-fore, in order to address the different targets that contribute to ADpathogenesis a new generation of multipotent compounds for ADemerged [22e27]. The strategy of our group has been devoted tothe development of AChE inhibitors endowed with a neuronalCa2þchannel antagonist effect. Ca2þ modulates many neuralprocesses, including synaptic plasticity and apoptosis. Dysregula-tion of intracellular Ca2þ signalling has been implicated in thepathogenesis of AD. Increased intracellular Ca2þ elicits the char-acteristic lesions of this disorder, including the accumulation of Ab,the hyperphosphorylation of tau and neuronal death. Conversely,neurodegeneration that is induced by Ab or tau is probably medi-ated by changes in Ca2þ homeostasis. Disruption of Ca2þ regulationin the endoplasmic reticulum mediates the most significant signal-transduction cascades that are associated with AD. Moreover,mutations that cause familial AD have been linked to intracellularCa2þ signalling pathways. Destabilization of Ca2þ signalling seemsto be central to the pathogenesis of AD, and targeting this processmight be therapeutically beneficial [18,28,29]. In addition, Ca2þ

entry through L-type voltage-dependent Ca2þ channels (VDCC-L,Cav 1.1e1.4) causes both Ca2þ overload and mitochondrial disrup-tion, which leads to the activation of the apoptotic cascade and celldeath [30]. These data justify the research for a possible therapeuticeffect in brain ischemia of drugs that block the entry of Ca2þ intothe cell. The dihydropyridines (DHP) are primary modulators of the

Scheme 3. Synthesis of 4-amino-2-methyl-5,6,7,8-tetrahy

VDCC-L; hence they may prevent the Ca2þ overload of cells from ischemically compromised tissue, as well as the alteration of ener-getic metabolism of these cells by the Ca2þ overload of its mito-chondria [31]. Nimodipine, also a neuronal VDCC-L blocker protectsneurons from death evoked by focal cerebral ischemia [32].

In this context, some years ago we undertook a research projectdirected to the synthesis of multipotent compounds designed totarget both AChE and neuronal Ca2þ modulation, capable ofshowing neuroprotection and antioxidant properties. Hence, wehave synthesized and evaluated several families of hybridcompounds such as IeVIII [33e44], where the benzene ring intacrine (THA) was substituted by a 4H-pyran ring (I) [33e38],a pyridine ring (II) [34,36,37,39], a DHP ring (III) [40], a furan ring(IV) [35,41], a thiophene ring (V) [41], an oxazole ring (VI) [36,42],a 1H-chromeno[2,3-b]quinolin-1-one ring (VII) [43], and3,4,5,7,8,9,10,12-octahydrodibenzo[b,g][1,8]naphthyridin-1(2H)-one ring (VIII) [44], respectively (Chart 1). Other THA analogueshave also been reported from other research groups [45e47].

With these models in mind, and attempting to get more equi-potent AChE versus butyrylcholinesterase inhibitors (BuChEI),endowed with an additional neuroprotective profile, we have nowdesigned a series of new THA analogues showing pyrazole as theprivileged heterocyclic ring system [48e50]. Therefore, we havecarried out the synthesis and biological evaluation of the diverselysubstituted pyrazolotacrines (pyrazolo-THA) 1e6 (Table 1) bearingeither the pyrazolo[3,4-b]quinoline (IX) or benzo[b]pyrazolo[4,3-g][1,8]naphthyridine (X) (Chart 1) heterocyclic ring systems.

Barreiro and co-workers [51] designed a few positionalpyrazolo-THA analogues that were planned to investigate thecontribution of different N-phenyl orientations around the azahe-terocyclic system in the AChE activity. By performing molecularmodelling (MM) studies they have observed interaction of theazaheterocyclic ring of some pyrazolo derivatives in the crystallo-graphic structure of the complex THAeAChE. With the exception ofthe tetracyclic and the linear extended pyrazolo-THA derivatives,the AChE inhibitory activity of the synthesized compounds was, ingeneral, modest [51]. According to MM results the poor activitymight be ascribed to the angular pattern adopted by the N-phenylgroup attached to the pyrazolopyridine system, which introducedsevere steric constraints into the molecular fit with Ile439/His440and Tyr334/Trp432 on the active site of AChE. Interestingly,a possible neuroprotective effect was observed in two pairs of thepositional pyrazolo-THA isomers investigated; the results showedthey were not significantly toxic to neurons and revealed

dro-2H-pyrazolo[3,4-b]quinoline-3-carboxamide (3).

30

40

50

60

70 Control

10nM

30nM

100nM

300nM

1uM

(m

in

/A

U-1

)

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681 4679

a tendency to reduce lactate dehydrogenase (LDH) release belowcontrol levels in a concentration-dependent manner [51].

2. Chemistry

2.1. Pyrazolo [3,4-b]quinolines

The precursors for the synthesis of 4-amino-1-pyrazolo[3,4-b]quinoline derivatives (1e4) (Schemes 1e3)were the readily available3-amino-1-methyl-pyrazole-4,5-dicarbonitrile (7) and 5-amino-1-methyl-pyrazole-3,4-dicarbonitrile (8) [52]. The Friedländer reac-tion (FR) [53] of 3-amino-1-methyl-pyrazole-4,5-dicarbonitrile (7)with cyclohexanone, under the standard experimental conditions[33], gave a complex reactionmixture, fromwhichwewere unable toisolate the desired compound. However, the use of trimethylsilyltriflate (TMSOTf)-EtOAc [54] system to promote the reaction gave theexpected 4-amino-2-methyl-5,6,7,8-tetrahydro-2H-pyrazolo[3,4-b]quinoline-3-carbonitrile (1), albeit in poor yield (Scheme 1).

Conversely, the same reaction between the isomer 5-amino-1-methyl-pyrazole-3,4-dicarbonitrile (8) and cyclohexanone affordedthe required pyrazolo-THA 4-amino-1-methyl-5,6,7,8-tetrahydro-1H-pyrazolo[3,4-b]quinoline-3-carbonitrile (2) in 60% yield (Scheme 2).

The reaction of compound 7 with N-benzylpiperidin-4-one(9), using the TMSOTf/EtOAc reaction conditions [54], did notyield the expected product, but amide 10 (Scheme 3), as theresult of the regioselective hydrolysis of the nitrile at C5 incompound 7. The position of the carboxamido function was alsosupported by selective NOE experiments between the two singletamido protons and the NeMe group, although the unequivocalfinal confirmation was obtained from the FR between 10 andcyclohexanone using AlCl3 as promotor to give pyrazolo-THA 3(Scheme 3) in 28% yield (7% when using TMSOTf/EtOAc [54])(Supplementary material).

In order to prepare dual AChEI [55], pyrazolo-THA 2was reactedwith (6-bromohexyloxy)benzene (11) (Supplementary material), inthe presence of t-BuOH/t-BuOK. Under these conditions, the aminoat C4 was alkylated, and the nitrile group was transformed intoa carboxamide, to provide pyrazolo-THA 4 (Scheme 2) [56].

2.2. Benzo[b]pyrazolo[4,3-g][1,8]naphthyridines

Next, the N-(6-amino-5-cyano-1-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamides 17 and 18 [57] were used for the synthesisof the pyrazolo-THA 5 and 6 under the standard FR conditions(Scheme 4) (Supplementary material).

3. Results and discussion

3.1. Pharmacology

3.1.1. AChE/BuChE inhibitory activityPyrazolo-THA 1e6 have been evaluated as inhibitors of

AChE from Electrophorus electricus (EeAChE), and BuChE from

Scheme 4. Friedländer reaction of pyrazolo[3,4-b]pyridines 17 and 18.

horseserum (eqBuChE), according to Ellman’s protocol [58]. Thecorresponding IC50 values are shown in Table 1. Pyrazolo[3,4-b]quinolines 1e4 were less potent AChEI than benzo[b]pyrazolo[4,3-g][1,8]naphthyridines 5 and 6. Nevertheless, compounds 5/6 and2/3 revealed similar BuChE inhibitory activity. Among the pyrazolo[3,4-b]quinolines 1e4, the most potent AChEI 1 and 3 bear a methylgroup at N2. The presence of a methyl at N1 in compounds 2 and 4,as well as theN-alkylation in compound 4 are structural factors thatdecrease the AChE inhibitory potency. Interestingly, unlike theresults found with AChE, moving the methyl group from N2 to N1,BuChE inhibition was improved from more than 100 to 6 mM.Transformation of the nitrile into an amide group at C3 alsoincreased the BuChE inhibitory activity. Focussing on benzo[b]pyrazolo[4,3-g][1,8]naphthyridines 5 and 6, the presence ofa phenyl ring in compound 6 impaired AChE inhibition, ascompared to compound 5, which lacks this phenyl ring, whileBuChE inhibitory activity was quite similar. Overall, themost potentinhibitor for both enzymes was N-(5-amino-1-methyl-6,7,8,9-tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naphthyridin-3-yl)acetamide (5) [IC50 (EeAChE) ¼ 69 � 6 nM, and IC50(eqBuChE) ¼ 6.3 � 0.6 mM], 2.5-fold less active than THA, but muchless potent BuChEI.

THA is usually associated with hepatotoxicity. Though this workreports the in vitro investigation of a number of THA analogues asnew multi-target-directed-ligands, hepatotoxicity studies have notbeen included in this paper. In fact, THA leads to elevated amino-transferase levels in 50% of patients. In spite of these high enzymelevels, severe hepatoxicity is rarely seen, and enzyme values returnto normal following withdrawal of the drug [59]. THA undergoesextensive oxidative metabolism to a variety of mono- and dihy-droxylated metabolites in animals and humans [60e63]. THA bio-activation by cytochrome (CYP) P450 enzymes, mainly CYP1A2isoform is responsible for the protein-reactive metabolites (PRM)observed in microsomes of all animal species studied. Theproduction of PRM is supported by a two-step mechanism in thebioactivation of THA involving an initial 7-hydroxylation followedby a postulated 2-electron oxidation to yield a reactive quinonemethide [62,63]. However, due to the nature of the pyrazolederivatives structure a quinone methide is not feasible to beformed. Nevertheless drug metabolism studies are meant to becarried out after activity in vivo is confirmed. Besides, the fact thatTHA was shown to alter intracellular glutathione concentration incultured hepatocytes might suggest the involvement of generation

-4 -2 0 2 4 6 8 10

0

10

20

V-1

[ATCh]-1

(mM-1

)

Fig. 1. Steady-state inhibition of AChE hydrolysis of acetylthiocholine (ATCh) bycompound 5. LineweavereBurk reciprocal plots of initial velocity and substrateconcentrations (0.1e1 mM) are presented. Lines were derived from a weighted least-squares analysis of data.

N N

EtO2CAr NH2

X N

EtO2CAr NH2

N

NH2

Tacrine

III X= O

N

NH2

IV X= O

X

Ar

ArN

NH2

VI

N

OAr

X N

Ar NH2

VII X= O

O

( )n ( )n( )n ( )n

NH

O

NO2

OO

O

O

Nimodipine

NNN

NH2R1

R2(IX)

NNN

R1

R2(X)

N

NH2

III X= NH V X= S

VIII X= NH

( )n

Chart 1. General structure and heterocyclic ring systems for the tacrine analogues previously described by our group (IeVIII) (Refs. [13e24]), and reported in the work (IX, X).

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e46814680

of reactive oxygen species (ROS) and lipid peroxidation, becauseintracellular glutathione represents an important protective factoragainst oxidative damage. Regarding this hypothesis, it has beenpreviously reported that THA, at high concentrations induced lipidperoxidation in isolated rat hepatocytes, which was prevented bypreincubating the cells with vitamin E, a naturally occurring anti-oxidant [64e66]. Thus, some considerations can be done; if ROSgeneration was involved in pyrazolo-THAs toxicity, it could bespeculated that pyrazolo-THA 5 might not trigger this adverseeffect since this compound was shown to have antioxidant prop-erties on SH-SY5Y neuroblastoma, showing a protection of 48%.

3.1.2. Kinetic study of the AChE inhibition by compound 5The AChE inhibition mechanism accomplished by pyrazolo-THA

1e6was investigated using pyrazolo-THA 5, the most potent AChEI.The type of inhibition was elucidated from the analysis of Line-weavereBurk reciprocal plots (Fig. 1) showing both increasingslopes (lower Vmax) and intercepts (higher KM) with higher inhib-itory concentration [67]. This suggests a mixed-type inhibition. A Ki

value of 155 nMwas estimated from the slopes of double reciprocalplots versus compound 5 concentrations (Supplementary material).

3.1.3. Neuroprotective effect of compounds 1e6 against rotenone/oligomycin A-induced cell death in SH-SY5Y neuroblastoma cells

The exposure of SH-SY5Y cells to a mixture of rotenone plusoligomycin A (Rot/Olig) constitutes a goodmodel of oxidative stresshaving its origin inmitochondria [68]. Rotenone blocks complex I ofmitochondrial electron transport chain and oligomycin A inhibitscomplex V, thus ATP synthesis was completely disrupted; theyelicit neurotoxicity and allow the evaluation of potential neuro-protective AChEI drugs used to treat AD patients, such as galant-amine, donepezil, and rivastigmine. Cells exposed to Rot/Olig in theabsence of the compound showed 35% cell death with respect tocontrol cells (13%), measured as % of LDH release [69]. Measured at1 mM, only pyrazolo-THA 5was able to reduce the Rot/Olig-inducedneuronal death. In terms of protection, pyrazolo-THA 5 afforded48% protection. Melatonin, used as reference, protected a 48% at theconcentration of 10 nM (Supplementary material).

Encouraged by the neuroprotective activity of pyrazolo-THA 5against Rot/Olig neurotoxicity, its effect against a toxicitymodel wellrelated to AD, e.g. the exposure to okadaic acid (OA), was evaluated.

OA, a potent and non-selective inhibitor of serine/threonine phos-phatases, induces tau hyperphosphorylation and cell death in bothprimary cortical neurons and neuroblastoma cell lines, as well asin vivomodels [70e72]. In this model, measured with themethod ofMTT reduction, pyrazolo-THA 5 was not able to improve neuronalviability of cells damaged with OA (Supplementary material).

4. Conclusions

We have first reported the synthesis and pharmacologicalevaluation of a series of novel fused pyrazolo-THA possessing thepyrazolo[3,4-b]quinoline (IX), or benzo[b]pyrazolo[4,3-g][1,8]naphthyridine (X) heterocyclic ring systems. Pyrazolo-THA 1e6,designed as multipotent agents, since they combine ChE inhibitionwith a neuroprotective profile, were prepared by FR-type reactionof readily available precursors with cyclohexanone. The biologicalevaluation showed that some of these molecules were potent andselective AChEI; the most interesting inhibitor was N-(5-amino-1-methyl-6,7,8,9-tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naph-thyridin-3-yl)acetamide (5), a mixed-type inhibitor for EeAChE,showing a Ki of 155 nM. Interestingly, pyrazolo-THA 5 indicateda 45% neuroprotection value against rotenone/oligomycinA-induced cell death, but was not able to improve cell viability ofSH-SY5Y cells damaged with OA. To sum up, among all thesecompounds, pyrazolo-THA 5 can be considered as a promising hitfor further development of a new family of neuroprotective AChEI.

Acknowledgements

We thank V. Gómez and L. González-Lafuente for technicalsupport. D. Silva thanks Fundação para a Ciência e Tecnologia doMinistério da Ciência, Tecnologia e Ensino Superior of Portugalfor his grant belonging to project PTDC/SAU-NEU/64151/2006.M. Chioua thanks Instituto de Salud Carlos III (MICINN) fora “Sara Borrell” post-doctoral contract. JMC thanks MICINN(SAF2006-08764-C02-01; SAF2009-07271) financial support. MGLthanks MICINN Ref. SAF2009-12150. DS thanks COST Action D34.This work was partly supported by grants RD06/0026/1002(RENEVAS), ISCIII, MICINN, S/SAL-0275-2006, Comunidad deMadrid, and Fundación CIEN, project 1004040042. JMC thanks Prof.A. García for his continuous interest and support.

D. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4676e4681 4681

Appendix. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ejmech.2011.05.068.

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