Cannabinoid agonists showing BuChE inhibition as potential therapeutic agents for Alzheimer's disease

ACCEPTED MANUSCRIPT

1

Cannabinoid Agonists Showing BuChE Inhibition as Potential Therapeutic Agents for Alzheimer’s

Disease.

a

Pedro GonzálezꢀNaranjo , Natalia PérezꢀMacias , Nuria E. Campillo , Concepción Pérez , Vicente J. Arán ,

b

cꢀf

Rocio Girón , Eva SánchezꢀRobles , María Isabel Martín , María GómezꢀCañas , Moisés GarcíaꢀArencibia ,

cꢀf

a*

Javier FernándezꢀRuiz , and Juan A. Páez

a

b

Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006-Madrid, Spain. Departamento de Farmacología y

Nutrición-Unidad Asociada al IQM (CSIC), Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos. Avda.

c

Atenas s/n, 28922-Alcorcón, Spain. Instituto Universitario de Investigación en Neuroquímica, Departamento de

d

Bioquímica y Biología Molecular, Facultad de Medicina, Centro de Investigación Biomédica en Red sobre

e

Enfermedades Neurodegenerativas (CIBERNED), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), and

^fCEI-Moncloa UCM-UPM, Universidad Complutense, 28040-Madrid, Spain.

Abstract

Designing drugs with a specific multiꢀtarget profile is a promising approach against multifactorial illnesses as

Alzheimer’s disease. In this work, new indazole ethers that possess dual activity as both cannabinoid agonists

CB2 and inhibitors of BuChE have been designed by computational methods. On the basis of this knowledge,

the synthesis, pharmacological evaluation and docking studies of a new class of indazoles has been

3

performed. Pharmacological evaluation includes radioligand binding assays with [ H]ꢀCP55940 for CB1R and

CB2R and functional activity for cannabinoid receptors on isolated tissue. Additionally, in vitro inhibitory assays

of AChE/ BuChE and the corresponding competition studies have been carried out. The results of

pharmacological tests have revealed that three of these derivatives behave as CB2 cannabinoid agonists and

simultaneously show BuChE inhibition. In particular, compounds 3 and 24 have emerged as promising

candidates as novel cannabinoids that inhibit BuChE by a nonꢀcompetitive or mixed mechanism, respectively.

On the other hand, both molecules show antioxidant properties.

Keywords: Alzheimer's disease, antioxidant; BuChE inhibitor, CB2R agonist, drug design, indazole

ether, multitarget drug.

*

Instituto de Q. Médica (CSIC). Juan de la Cierva, 3, 28006-Madrid, Spain. jpaez@iqm.csic.es. Fax: +34

915644853; Tel.: +34 915622900

ACCEPTED MANUSCRIPT

2

1

. Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and is currently the leading cause of

dementia in the elderly. Despite of the devastating effects of this disease, therapeutic options for treating AD

remain limited. This disease is characterized by the formation of cortical amyloid plaques and neuroﬁbrillary

tangles in the brain. A biological function affected in AD is the cholinergic neurotransmission, which seems to

be closely related to pathological formation of betaꢀamyloid. This hypothesis asserts that most of the cognitive

impairments suffered by AD patients are the consequence of a deficit in acetylcholine (ACh). In this sense,

cholinergic drugs like acetylcholinesterase (AChE) inhibitors, including donepezil, rivastigmine, and

galantamine, are currently the most prescribed pharmaceuticals for AD [1ꢀ4].

Moreover, Alzheimer’s disease is characterized by a significant reduction in AChE activity and an increase in

butyrylcholinesterase (BuChE) activity (about 120%). This increase of BuChE activity, especially found in the

hippocampus, has been suggested to be related to the loss of episodic memory [5]. Likewise, it has been

reported that BuChE might facilitate the transformation of an initial benign form of senile plaque to a malignant

form associated with AD [6]. Therefore, the BuChE plays an important role in AD patients, what turns it into an

interesting target for the research in the field [5ꢀ8].

AChE is characterized by having two sites: a peripheral anionic site (PAS) located at the mouth of the active

site gorge, which is regarded as the recognition binding site; and the anionic subsite of the active site (CAS),

where acetylcholine (ACh)ꢀfixation is occurring before being hydrolysed in the active site. The interaction with

PAS is assumed to be determinant in both the association of AChE with ßꢀamyloid and the formation of

amyloid [9ꢀ11]. BuChE, also possessing PAS [12, 13] and CAS, is present in the neuritic plaques together with

AChE. By other hand, BuChEꢀselective inhibitors have been reported to reduce ßꢀamyloid (AB) and ßꢀamyloid

precursor (APP) secretion in vitro and in vivo [14, 15]

New pharmacological alternatives are focused on the development of new drugs capable of acting at different

targets [16] (i.e., multitarget drugs) as compounds with anticholinesterase and NMDA receptor antagonistic

activity [17]; dual inhibitors of histamine H3 receptors and AChE [18]; dual inhibitors of AChE and BACE1 [19];

dual acting hybrids BACE1 inhibitor and metal chelator [20]; dual acting hybrids gammaꢀsecretase modulators

with PPARγ activity[21] or acetylcholinesterase inhibitors and CB1R antagonist [22]. In this context,

cannabinoid compounds are very attractive candidates fit to fit into this paradigm since they could act on

different pharmacological targets within the soꢀcalled endocannabinoid signalling system, but also on other

targets not related to this system. It has been reported that the cannabinoid system is dramatically altered in

brains of AD patients [23]. Concerning CB1 receptor, the alteration of this receptor is not so clear. In fact, there

is some controversy regarding this issue [24]. Some studies suggest that CB1 receptors are intact in AD and

may play a role in preserving the cognitive function [25, 26], while other studies postulate that the expression

and distribution of CB1 are clearly different in AD [27ꢀ29] In any case, it is important to remark the fact that

ACCEPTED MANUSCRIPT

3

CB1 receptor is located preferentially in damaged neurons in cortical and subcortical areas, which is in

accordance to the expected decrease in the levels of this receptor type due to the progressive neuronal loss.

Regarding CB2 receptors, different studies show that they are abundantly expressed in neuritic plaqueꢀ

associated astrocytes and selectively expressed in reactive microglia [30ꢀ32] in brains of AD patients, in

contrast to their absence or poor expression in these glial cells under healthy conditions. Three potential

interventions have been identified using CB2R as target [24]. First, stimulation of CB2 receptors suppresses

microglial activation [33, 34] second, a CB2 receptor agonist such as JWHꢀ015 is capable of inducing the

removal of native betaꢀamyloid [25, 31, 35] and third, CB2 receptor agonists suppress the neuroinflammatory

process [36]. Taken together, these results suggest that CB1 receptor agonists may interrupt the mechanism

of excitotoxicity and CB2 receptor agonists may suppress neuroinflamation and lead to plaque removal.

On the other hand, our group has demonstrated that some cannabinoid agonists such as the

aminoalkyilindoles (AAI), JWHꢀ015 or WIN55212ꢀ2 (Fig. 1) inhibit the AChE in vitro [37].

Figure 1

Thus, continuing with our effort to develop an efficient strategy to Fig.ht against AD and taking advantage of

the demonstrated potential of the cannabinoids that are active at both the cholinergic transmission and at the

endocannabinoid system, our research group first disclosed a new family of indazole ethers with cholinergic

properties and cannabinoid effects showing multitarget drug profile, which opened a novel and interesting

therapeutic strategy for AD [38]. Subsequently, a similar approach was published by Rizzo et al. [39]. Herein,

we describe the synthesis and biological evaluation of a new family of compounds based on the indazole ether

scaffold.

2

. Result and Discussion

One possible way to find a hit consists of performing systematic modifications of the structure by variation of

functional groups at particular sites. In a first approach, three different positions of the indazole system were

considered for modulating the biological activity and were subject to modification. Toward this goal, we

proposed a procedure that encompassed docking studies of CB2 receptor model [40] and BuChE (pdb code:

1

P0I), synthesis and pharmacological evaluation of the effects of structure variations of the indazole system.

2

.1. Rational Design

Taking into account all above concepts and applying the bioisosterism criterion, we proposed to replace the

indole ring by an indazole core, and also introduce an aromatic ether that is well suited for interacting in the

binding site of CB2 receptor in a similar way to AAI. Based on the structural superposition of an AAI as JWHꢀ

0

15 with indazole ethers, we designed a virtual library of different indazole derivatives to make a preliminary

docking study. For this theoretical study, aimed at evaluating the binding capacity of this new backbone to the

ACCEPTED MANUSCRIPT

4

receptor, our theoretical model of CB2R* was employed (see details in computational section). The virtual

library was designed taking account three criteria: i) groups in position 1 and 3 that allow interactions with CB2

receptor, as previously noted in AAI structures; ii) structural variability and iii) synthetic accessibility.

Thus, this initial virtual library was assembled considering as structural variations a hydrogen, or nitro at

position 5; benzyl, diisopropylaminoethyl or piperidinoethyl groups at position 1; and benzyl, methoxybenzyl or

naphthylmethoxy at position 3 (Fig. 2). Preliminary modelling studies led us to find that several of the virtual

compounds showed a favourable energy of interaction compared to the AAI compounds and a similar

orientation of the indazole skeleton and the indole ring of AAI.

On the basis of these modelling results, new substituents were included. Thus, pentyl, cyclohexylmethyl,

benzyl, 4ꢀmethoxybenzyl, diisopropylaminoethyl, pyrrolidinylethyl, morpholinoethyl or piperidinoethyl were

considered as substituents at the 1ꢀposition of the indazole core. O-substituted groups as cyclohexylmethyl,

benzyl, 4ꢀmethoxybenzyl, 1ꢀnaphthylmethyl or 2ꢀnaphthylmethyl were selected as substituents at position 3

while hydrogen, bromo, nitro or amino were chosen as substituents at position 5 of the indazole ring (Fig. 2).

Figure 2

A docking study was performed using our CB2R model (see details in computational section), as target

against the virtual library. The results of the modelling studies indicated that most of the virtual compounds

show favourable energy of interaction and similar orientation of the indazole moiety compared to the indole

ring in related AAI, being stabilized by means of an aromatic interaction with Phe197. In addition, the groups in

R

2

and R are able to interact with key residues as Leu201 and Tyr190 in TM5 respectively. These initial

3

studies showed that, in general, the N1ꢀsubstituted derivatives have additional interactions with Met115 and

Ser161.

2

.2. Chemistry

Based on docking results we performed the synthesis of a representative set of compounds (1-25) as potential

candidates as cannabinoid ligands and ChEs inhibitors (Table 1). As it has been commented in the

introduction, this new family of 1ꢀsubstituted indazole ethers (1-25) has been claimed by us in a PCT patent

[38]. The synthetic methodology for accessing the target compounds 1-25 was based on the introduction of the

different groups at Nꢀ1 position or at the hydroxyl group of the indazole core through wellꢀestablished Nꢀ

alkylation or Oꢀalkylation procedures. Three possible strategies or routes were considered depending on the

nature of the desired substituents. The overall synthetic methodology showing the three possible routes is

presented in Scheme 1.

The direct baseꢀpromoted reaction of the starting indazole core with alkyl halides under refluxing ketone

solvent (route A) was found to be only useful to obtain disubstituted derivatives with identical groups at both

ACCEPTED MANUSCRIPT

5

Nꢀ and Oꢀpositions. For derivatives having different groups at Nꢀ1 and OH at C3, routes B and C were applied.

The synthetic route B consists of a twoꢀstep sequence involving initial alkylation with the a benzylic halide at

the more nucleophilic Nꢀ1 position of the indazole ethers, and then introduction of the substituent at position 3ꢀ

OH using different arylmethyl halides. The main limitation of this route is that it proved to be effective only for

selective Nꢀalkylation with highly reactive methyl or arylmethyl halides.

A more structurally versatile and general synthetic route for the formation of substituted indazoles, the route

C, comprises three steps involving protection of position Nꢀ1 as a carbamate, Oꢀalkylation to furnish the

corresponding ether and subsequent deprotection and introduction of the second substituent at the Nꢀ1

position.

Scheme 1

Following the route A, a wide variety of Nꢀ1 substituted indazole ethers were prepared by reaction of indazolol

2

6 or 5ꢀnitroindazolol 27 [41] with the corresponding halides in acetone or butanone and cesium or potassium

carbonate as base (Table 1, Scheme 2). Thus, the synthesis of indazole ethers 1, 2, 4, 8 were carried out

starting from 26 and cyclohexylmethyl, benzyl or 4ꢀmethoxybenzyl bromides or 1ꢀnaphthylmethyl chloride,

respectively. Similarly, the 5ꢀnitroindazole ethers 10, 12 [42] 16, 21 were prepared by reaction of 5ꢀnitroꢀ3ꢀ

indazolole 27 with the corresponding halides of pentyl, benzyl and 1 or 2ꢀnaphthylmethyl, respectively.

The first attempts to synthesize 1,3ꢀdisubstituted indazoles with different groups at Nꢀ1 position and ether

function were carried out following the synthetic route B starting from 5ꢀnitroindazolole 27. Thus, the reaction

of 27 with benzyl bromide in an aqueous solution of sodium hydroxide afforded the corresponding 1ꢀbenzylꢀ3ꢀ

indazolol 28 [43]. Then, subsequent Oꢀalkylation of 28 with cyclohexylmethyl or 2ꢀnaphthylmethyl bromide

afforded the indazole ethers 11 and 20.

As mentioned before, the route C was found to be a more versatile and suitable procedure for the sequential

twoꢀfold alkylation (at Nꢀ and Oꢀcenters) with different groups. This strategy resulted especially useful for the

preparation of indazole ethers bearing aminoethyl groups at Nꢀ1 position. Thus, standard N1ꢀprotection of

indazoles 26 and 27 with ethyl chloroformiate furnished the corresponding 1ꢀethoxycarbonyl derivatives 29

[43] and 30 [42], respectively. Next, the reaction of 29 or 30 with the corresponding halides in the presence of

K

2

CO as base afforded the 5ꢀunsubtituted derivatives of 4ꢀmethoxybenzyloxy 31, 1ꢀnaphthylmethoxy 32, 2ꢀ

3

naphthylmethoxy 33, or the 5ꢀnitro indazole ethers 34-36, respectively. All reactions were carried out under

reflux of butanone, except for derivatives 31 and 35, where 1,4ꢀdioxane and DMF were, respectively, the

solvents of choice. In some cases, migration of the arylmethyl substituent from the Oꢀ at C3 to N2 was

observed, leading to mixtures of 3ꢀarylmethyloxyꢀ1Hꢀindazole and N2ꢀalkylated 1,2ꢀdihydroꢀ3Hꢀindazolꢀ3ꢀone

derivatives (Scheme 2). For example, the reaction of derivatives 29 and 30 with the corresponding halides

yielded a mixture of the corresponding indazole ethers 31-36 and their corresponding Nꢀ2 substituted

ACCEPTED MANUSCRIPT

6

derivatives 37, 38ꢀ39 [44], 40, 41, and 42 [45], respectively. The relative ratio of the two isomers depended on

the conditions employed in each case. To the best of our knowledge, there is no reported procedure allowing

the selective Oꢀalkylation to afford the ether isomer in good yield. Nevertheless, by tuning the reaction

conditions we found conditions to obtain the desired indazole ethers 31-36 as the major component of the

mixture in all cases, which could be efficiently separated from the mixture by flash chromatography (see

experimental section for detailed conditions).

Scheme 2

For the majority of the derivatives, the best results were obtained using 2ꢀbutanone as solvent. However, for

the preparation of derivative 31 the use of 1,4ꢀdioxane was found to be essential for increasing the ratio of the

desired indazole ether over that of the N2ꢀalkylated product. As another exception to this general trend, the

synthesis of derivative 35 required harsher reaction conditions and a higher boiling point solvent (DMF). The

best O3ꢀ versus N2ꢀalkylation ratio was achieved for compounds 32 and 35 for which a relative ratio of 10:1

1

and 5:1, respectively, was measured by H NMR from the crude mixture.

With the 1Hꢀindazole 31ꢀ36 in hand, the last step to obtain the target compounds was the installation of the

aminoethyl group at N1 position. The synthesis of 4ꢀmethoxybenzyl derivative 3 and 4ꢀmethoxybenzylꢀ5ꢀ

nitroindazoles 13, 14 were carried out with diisoproylaminoethyl chloride and pentyl iodide, from 31 and 34,

respectively.

The reaction of the 1ꢀnaphthylmethoxy indazole 32 with the corresponding aminoethyl chlorides afforded the

diisopropylaminoethyl, pyrrolidinylethyl and piperidinoethyl derivatives 5-7, respectively. In a similar manner,

Nꢀalkylation of the 5ꢀnitroꢀ1ꢀnaphthylmethoxy indazole 35 with 2ꢀpirrolydinnoethyl chloride afforded the

corresponding ether 15. Finally, the preparation of the 3ꢀ(2ꢀnaphthylmethoxy)indazole derivative was carried

out from 33 by Nꢀalkylation with 2ꢀpiperidinoethyl chloride to afford the desired derivative 9.

Similarly, the reaction of the 5ꢀnitroꢀ2ꢀnaphthylmethoxyindazole 36 with the corresponding aminoethyl

chlorides and alkyl iodides afforded the pentyl, piperidinoethyl and morpholinoehtyl ethers 17-19, respectively.

Besides, we have performed novel modifications in the indazole ether system by bromination and reduction

reactions (Scheme 3). In this sense, the conversion of the nitro group at Cꢀ5 into the corresponding 5ꢀamino

derivative had not been reported before. Thus, to the preparation of the 5ꢀamino derivatives 22ꢀ24 a

modification of the standard ferric chlorideꢀpromoted procedure was necessary. Following the method

describes by Lauwiner et al. [46] for the reduction of monosubstituted nitrobenzenes, the ferric oxyhydroxide

FeO(OH) was prepared and used as catalyst by the treatment of FeCl with a NaOH solution.

3

Scheme 3

ACCEPTED MANUSCRIPT

7

Following this procedure the reaction of the 5ꢀnitro derivatives 12, 15 and 18 with hydrazine in MeOH in

presence of the ferric oxyhydroxide FeO(OH) as catalyst afforded the corresponding 5ꢀamino derivatives 22-

2

4, respectively. The preparation of 5ꢀbromo indazole 25 was achieved by bromination with Nꢀ

bromosuccinimide and ferric chloride from the 2ꢀnaphtylmethoxyꢀ1ꢀpiperidinoethylindazole 9.

The structures of synthesized compounds (1-25) were established as N1 and O3 disubstituted indazoles on

the basis of NMR data (Tables S1 and S2). The position of the 1 and 3 substituents can be distinguished by

¹³CꢀNMR spectra examining the chemical shifts (Table S2). Thus, the signals corresponding to aromatic

methylene group attach at Oꢀ3 (66ꢀ75 ppm) appears at lower field in relation to methylene Nꢀ1 (50ꢀ58 ppm). In

compounds bearing the methylene groups at Nꢀ2 the signals appearing at highest field (45 ꢀ47.5 ppm).

2

.3. Biological assays

2

.3.1. In vitro binding studies in cannabinoids receptors.

Radioligand displacement assays were used to evaluate the affinity of the new compounds 1-25 using

3

membranes from cells (HEK293EBNA) transfected with the CB1 or the CB2 receptors and [ H]ꢀCP55940 as

radioligand. Several indazole ethers showed low solubility at concentration greater than 10 µM under test

conditions (see material and methods section), therefore precluding the determination of their quantitative

value of IC50 for these compounds.

All synthesized compounds together with their binding affinity for the cannabinoid receptor CB1 and CB2 are

gathered in Table 1. Examination of these data indicates that the evaluated derivatives bind to the receptor

CB2 in µM range, except the disubstituted naphthyl derivatives 8 and 21. The comparison of the IC50 values

for inhibition of CB2R rather flat structureꢀactivity relationships, with values ranging from 0.24 to >10 ꢁM. The

situation is less pronounced for CB1 receptor, therefore some of the derivatives such as the

diisopropylaminoethyl derivatives 3, 5, and 14 do not exhibit affinity to 40 ꢁM. Regarding CB2 selectivity, it is

interesting to mark several compounds as the nitro compounds 11, 16, 20, and especially the 5Hꢀindazole 7

and the amino derivative 24 due to interesting pharmacological profiles that they will be discussed later.

Table 1

2

.3.2. AChE/BuChE Inhibitory Activity.

The derivatives 1-25 were subjected to Ellman’s test [47] in order to evaluate their potency to inhibit the AChE

from human and BuChE from horse serum and human using donepezil as reference standard. The displayed

IC50 values in Table 2 showing that several compounds exhibit inhibitory activity of AChE or BuChE at potency

levels in the submicromolar to micromolar range. Similarly to the case of cannabinoid binding assays, the

ACCEPTED MANUSCRIPT

8

quantitative value of IC50 for several derivatives was not possible to be determined for solubitlity reasons

Albeit, significant inhibition of AChE, with IC values in the range of 10 ꢁM, was observed for 5 and 7.

5

0

Regarding AChE inhibition, the results indicate that several indazole derivatives showed significant activity as

inhibitors of this enzyme being derivatives 1 and 2 which have lower IC50 values among the 5Hꢀindazoles.

In relation to BuChE inhibition, several indazole derivatives showing a significative IC50 value. The analysis of

the IC50 values reveals it is necessary but not sufficient, the presence at N1 position of a dialkylaminoethyl

group like demonstrated with the 5Hꢀindazole derivatives 3, 5, 6, 7, 9 and with the 5ꢀamino 23, 24 and 5ꢀ

bromo 25 compounds. It is interesting to mention that all the other 5Hꢀunsubstituted indazole derivatives

bearing the aminoethyl group behaved as BuChE inhibitors showing a marked selectivity towards BuChE. In

the case of 5ꢀnitroindazole derivatives only the piperidinoethyl 18 and pyrrolidinylethyl 15 showed inhibition of

BuChE. Noteworthy, these achieved values of inhibitory activity for BuChE are comparable to those published

from rivastigmine, a cholinesterase inhibitor used in Alzheimer’s disease. The IC50 of rivastigmine as AChE or

BuChE inhibitor is 1.5ꢀ48 ꢁM or 0.3ꢀ54 ꢁM respectively, depending on the assay conditions [48ꢀ50].

2

.3.3. Kinetic Study of BuChE/AChE Inhibition.

To gain further insight into the mechanism of action of this family of compounds, a kinetic study was carried

out with the most promising inhibitors of BuChE. Lineweaver–Burk plots were done for the multitarget

cannabinoids and donepezil as reference compound and are shown in material supplementary (Fig. S1 and

Fig. S2) and the data summarised in Table 2. The inhibition constants, K, for BuChE inhibitors (Table 2) were

i

determined by fitting the kinetic data to a competitive, noncompetitive, or mixed inhibition model by nonlinear

regression analysis using GraphPad Prism 5 [51]. Thus, the competition studies were carried out in all the

indazole derivatives with activity as BuChE inhibitors. Albeit deserve special attention those derivatives that

behave as cannabinoid agonists and BuChE inhibitors like 3, 6, 7, 9, 23 and 24 (Table 2).

Reciprocal plot for donepezil inhibition showed both increasing slopes (decreased at increasing inhibitor

concentrations) and increasing intercepts with higher inhibitor concentration (Fig. S1). These results indicate a

mixedꢀtype inhibition and are in agreement with patterns obtained with the same enzyme [52]. Therefore,

kinetic studies suggested two potential different site of interaction: the active site and the peripheral binding

site.

Regarding the inhibition type of BuChE, compounds 18 and 24 (Fig. S2) showed similar plots to donepezil

pointing to a mixed type competitive BuChE inhibition mode of action. Lineweaver–Burk plots obtained for 5, 6,

7

, 15, 23 and 25 (Fig. S2) increase in the slope at increasing inhibitor concentrations, typical for a pure

competitive BuChE inhibitor. Reciprocal plot for 3 and 9 (Fig. S2) showed unvaried yꢀintercepts and increasing

slopes at increasing inhibitor concentrations compatible with nonꢀcompetitive BuChE inhibitors.

ACCEPTED MANUSCRIPT

9

Moreover, complementary competition studies in AChE were performed with the indazole derivatives inhibitors

of BuChE that showed an IC50 about 10ꢁM as AChE inhibitors like 5 and 7. In a similar way to BuChE, the

analysis of the graphic profiles of 5 and 7 (Fig. S3) can be established that 5 and 7 showed a competitive

mode of action as AChE inhibitors.

2

.3.4. Cannabinoid Activity. Isolated Tissue Assays.

According to the objectives, all derivatives that showed simultaneously activity as cannabinoid ligands and as

inhibitors of BuChE have been studied in detail in isolated tissues. The functional activity of the new

compounds 3, 5-7, 9, 15, 18 and 23-25 has been tested on mouse vas deferens (MVD) a tissue commonly

used to study and characterize cannabinoid effects [53, 54]. In MVD cannabinoid agonists, acting at

prejunctional cannabinoid receptors, reduce ATP and noradrenaline release and inhibit the electrically evoked

smooth muscle contractions. In this tissue, cannabinoid receptor antagonists oppose the inhibitory effect of

agonists in a competitive and surmountable manner. CB1 and CB2ꢀlike cannabinoid receptors seem to be

involved in this effect [55, 56].

ꢀ7

ꢀ5

From the tested compounds, at the used concentrations (10 – 2×10 M), some derivatives were able to

significantly modify the electrically induced contractile response. As Fig. 3 shows, compounds 3, 6, 7, 9, 15

and 23-24 induced a concentrationꢀdependent inhibition of the electrically evoked contractions; from these

data, it could be established that these compounds behave as agonists. Furthermore, the effectiveness

(

maximum effect) of 6, 7, 9 and 23-24 was similar to that of the reference cannabinoid agonist WIN55,212ꢀ2.

Figure 3

Considering that compounds 3, 6, 7, 9, 15, 23 and 24 showed an interesting profile as potential cannabinoid

agonists, the blockade of their effect by the CB1 and CB2 receptors antagonists were tested, alone (AM251)

or in combination (AM251+AM630). These drugs were added to the organ bath before each concentration of

ꢀ7

ꢀ5

the new compounds (10 – 2×10 M).

Results showed a partial blockade of the effect of the new derivatives 3, 9 and 24 that was maximum at the

ꢀ6

concentration of 2,7 x 10 M (Fig. 4a), indicating that their mechanism of action includes the activation of the

cannabinoid receptors CB1 CB2 confirming our hypothesis. The lack of antagonist efficacy on the inhibition

/

ꢀ5

induced by the highest concentration of the new compounds (2 x 10 M) suggests a surmountable

antagonism, perhaps competitive (Fig. 4b).

Compounds 3, 9 and 24 had the better profile as cannabinoid agonists because they inhibited in a

concentrationꢀdependent manner the electrically evoked contractions on mouse vas deferens and this effect

was significantly blocked by CB1 and CB2 receptor antagonists.

ACCEPTED MANUSCRIPT

1

0

Figure 4

The inhibitory effects of the MVD contraction of compounds 6, 7 and 23 are not modified by the presence in

the organ bath of the cannabinoid antagonists, so they can be due to other mechanisms that not involve

cannabinoid receptors and that are not the object of study in this experimental work.

Taking in consideration our present aim, compounds 5, 15, 18 and 25 did not show an interesting profile

because their inhibitory effect is too slight.

2

.3.5. Antioxidant Activity.

The antioxidant activities of indazole ether derivatives 3, 6, 7, 9, 23 and 24 were evaluated by following the

wellꢀestablished ORACꢀFL method (oxygen radical absorbance capacity by fluorescence) [57, 58]. The results

shown in Table 2 indicate that indazole ether derivatives exhibit antioxidant properties with values higher than

4

5% in relation to trolox. The 5ꢀaminoindazole derivatives 23 and 24 protect much more efficiently against free

radicals (about to thirty percent) than the corresponding 5Hꢀindazoles 6 and 9, respectively.

In summary, the obtained results about indazole ethers derivatives that possess simultaneously activity as

cannabinoid agonists and as inhibitors of BuChE showed certain antioxidant capacity, in particular for the non

competitive 3 and competitive inhibitor of BuChE 23. This last compound showed the most antioxidant

capacity about 29% higher than Trolox.

2

.4. Computational studies

To rationalize the achieved biological properties for all derivatives that showed simultaneously activity as

cannabinoid agonists and as inhibitors of BuChE docking studies have been carried out employing both

targets, CB2R and BuChE. These simulations were attained employing FlexiDock module of the SYBYL 7.2

suite of programs [59].

2

.4.1. CB2R-ligands Complexes.

In our group we have been working on modelling the cannabinoid receptors for several years [40, 60]. The first

models in the inactive form were built with the pattern of bovine rhodopsin (pdb code: 1f88 [61]). Later, we

have modelled CB2 receptor in the active form (results not published). The docking and scoring runs were

performed using Flexidock program [59]. Fig. S4 allow us to verify that our indazole derivatives occupy a

similar area that the known agonist JWH015, showing similar interactions with the residues of the binding site

[

62] (see supporting information, Table S3). The results of the docking experiments for the compounds 3, 5-7,

, 15, 18 and 23-25 in CB2R* model in terms of key interactions are gathered in Table S3. The most important

difference is the displacement of the naphthyl group due to the replacement of carbonyl group by the ether

9

ACCEPTED MANUSCRIPT

1

function. Probably, this displacement is translated in a weak aromatic interaction with Phe197 and Trp258.

Observing the Fig. S4, this displacement is a bit more noticeable in the 2ꢀnaphthyl derivatives. Further

discussion of the docking studies with CB2R model is found in supplementary material.

2

.4.2. BuChE-ligands Complexes.

Regarding the recognition partners between the ChEs and the cannabinoid ligands, docking simulations were

done in the 3D structure of human AChE and BuChE (PDB codes: 1B41 [63] and 1P0I [64], respectively). The

interaction modes of active ChE inhibitors in AChE or BuChE were studied employing FlexiDock program [59]

(

see Experimental Section for details). The binding gorge of AChE, which is composed of the central catalytic

pocket (CAS) and the peripheral site (PAS), was taken as the binding site for docking studies. For each

inhibitor, conformation with the lowest interaction energy was taken out for further analysis. In a previous work

we studied an example of each known binding mode: donepezil (mixed), propidium (noꢀcompetitive) and

edrophonium (competitive) in order to test our docking protocol with good results [37]. Using the same

methodology, BuChE inhibitors 3, 5-7, 9, 15, 18 and 23-25 were studied and the k

with STC program [65] (Table 2). Highlight that the predicted k values are in agreement to the experimental

inhibition constants k as shown in the Fig. S5 (see supplementary material).

d

values were calculated

d

i

The best docking solutions of the most interesting compounds 3, 6, 7, 9, 15, 23 and 24 clearly show that these

derivatives are located along the tunnel between the sites of the PAS and CAS BuChE (Fig. 5, Table S4).

Visually, it is observed that indazole derivatives orientate the group indazole of the position in N1 towards the

interior of the channel, so that the indazole group is closed to Trp82. Another interesting observation is

regarding to the existence of two clusters of inhibitors due to the different group in R

orientation of the group, driven the kinetic of the inhibition. Thus, one of the cluster, it is formed by compounds

, 6, and 23 (1ꢀnaphthyl substituent on R ) behaving as competitive inhibitors and the second cluster, it is

3

and therefore different

7

3

formed by compound 3 (1ꢀmethoxyphenyl on R

3

) and 9 and 24 (2ꢀnaphthyl substituent on R ) behaving as

3

nonꢀcompetitive.

Figure 5

The group at R could be driving the type of inhibition since the 1ꢀnaphthyl derivatives behave as competitive

3

inhibitors meanwhile the 2ꢀnaphthyl derivatives as nonꢀcompetitiveꢀmixed inhibitors (Fig. 6). This fact could be

due to a relocation of the 2ꢀnaphthyl group to avoid a steric hindrance of the 2ꢀnapthyl ring with Thr284 and

Pro285. Thanks to this movement of the ring, two other interactions appear, one of them with Thr120 (by

hydrogen bond with the indazole nitrogen) and the other one with Ser287 (πꢀHB with 2ꢀnaphthyl) (Fig. S6) and

unlocking the entrance of the triad catalytic. Further discussion of the interactions of the most representative

compounds is displayed in the supporting information.

ACCEPTED MANUSCRIPT

1

2

Figure 6

3

. Conclusions

On the basis of this investigation, a novel and plausible approach based on the discovery of CB2 cannabinoid

agonists that behave as BuChE inhibitors is proposed. Docking techniques have been effectively used to

design new cannabinoids derivatives with an innovative indazole ether structure. Remarkably, more than 90%

of evaluated compounds have shown capacity to bind CB2 cannabinoid receptor. Among them, according to

tissue studies, compounds 3, 6, 7, 9, 23 and 24 behave as agonists showing a maximum effect similar to the

reference cannabinoid agonist WIN 55,212ꢀ2. The AChE and BuChE inhibition studies show that these

cannabinoids (3, 6, 7, 9, 23 and 24) are inhibitors of BuChE with comparable values to rivastigmine.

The kinetic and modelling studies have provided insights into the mode of action and the binding site of these

compounds. The full agonists 3 and 9 showed a nonꢀcompetitive inhibition mode, while 24 showed a mixed

type inhibition mode of action. Moreover, the BuChE inhibitors 6, 7 and 23 showed a competitive inhibition

mode of action. It is worth mentioning that the indazole derivative 7 inhibits AChE with a mixed action mode.

However, in despite of this different behaviour, the docking studies of indazole derivatives in BuChE have

allowed the identification of the amino acids involved in the binding site, suggesting that the six indazole

derivatives 3, 6, 7, 9, 23 and 24 are located along the tunnel from PAS to CAS interacting with different

residues of peripheral site. Derivatives, 3, 9, 24 behave as full agonist cannabinoids according to their capacity

to block the agonist effect caused by cannabinoids antagonists as AM251 and AM630. On the other hand,

compounds 3, 24, and 23 showed the most interesting antioxidant properties.

In summary, the cannabinoid effect, the inhibition of the BuChE of compounds 3 (non competitive inhibition

type) and 24 (mixed inhibition type) along with its antioxidant capacity suggest that these compounds can be

regarded as a potential agent useful for Alzheimer’s disease.

ACCEPTED MANUSCRIPT

1

3

4

. Experimental Section

.1. Chemistry

.1.1. General

Melting points were determined with a ReichertꢀJung Thermovar micro melting point apparatus and are

1

13

uncorrected. HꢀNMR spectra (300 MHz, DMSOꢀd ) and CꢀNMR spectra (75 MHz) were recorded on a

6

Gemini or Varian XLꢀ300 (Varian Unity 300 and 400 Varian Gemini and Bruker Avance 300 spectrometers)

and are reported in ppm. The signal of the solvent was used as reference. Mass spectra (electrospray

ionization) were determined on a MSDꢀSerie 1100 Hewlett Packard instrument. Elemental analyses were

performed on a Heraeus CHNꢀO Rapid Analysis in our Analytical Services at Centro de Química Orgánica

“

Manuel Lora Tamayo” (CSIC). Flash column chromatography was carried out using Merck silica gel 60 (230 –

00 mesh). HPLCꢀEMS was performed on an Sunfire C18 (4.6 x 50 mm, 3.5 mM) column at 30 ºC, with a flow

rate of 0.25 mL / min. with different gradients of CH CN with 0.08% of formic acid (solvent A) in 0.1% of formic

acid in H O (solvent B) were used as mobile phases . The initial conditions, time of gradient (gt) and time of

4

3

2

retention (rt) are specified in each case. The HPLC system was a Waters 2695 Separations Module coupled to

a Waters 2996 Photodiode Array detector and to a Waters micromass ZQ. The purity of all compounds was

>

95% prior to biological testing (Supporting Information).

All starting materials were commercially available in Aldrich or Alfa Aesar and used without further purification.

ꢀnitroꢀ3ꢀindazolꢀol (27) was prepared from the procedure reported by Pfannstiel [41], 1ꢀbenzylꢀ3ꢀbencyloxyꢀ5ꢀ

nitroindazole (12) and 1ꢀethoxycarbonylꢀ3ꢀindazolone (30) were prepared from the procedure reported by Arán

42], 1ꢀbenzylꢀ5ꢀnitroꢀ3ꢀindazolone (28) and 1ꢀethoxycarbonylꢀ3ꢀindazolone (29) were prepared from the

procedure reported by Palazzo [43].

.1.2. General procedure for the synthesis of indazole ethers 1-substituted 1, 2, 4, 8, 10, 16, 21 (R

Method A.

The corresponding halide was added to a suspension of the 1Hꢀ3ꢀindazolol derivative in acetone and Cs

5

[

4

2

= R ).

3

2

CO3.

The reaction mixture was refluxed, filtered and evaporated to dryness. The residue was purified by silica gel

column chromatography using a mixture of dichloromethane: hexane (1:1, 5:1) as eluent.

4

.1.3. General procedure for the synthesis of indazole ethers 1-substituted 3, 5-7, 9, 13-15, 17-19 from 1H-

indazole ethers. Method B.

A suspension of the 1Hꢀ3ꢀindazolol derivative in butanone and K

2

CO was treated with the corresponding

3

halide. The reaction mixture was refluxed, filtered and evaporated to dryness. The residue was recrystallized

or purified by silica gel column chromatography using the appropriate eluent.

4

.1.4. General procedure for the synthesis of indazole ethers 11 and 20 from N1-substituted indazolol

derivative 28. Method C.

ACCEPTED MANUSCRIPT

1

4

A suspension of 28 in butanone and K

2

CO

3

or acetone and Cs

2

CO was treated with the corresponding halide.

3

The reaction mixture was refluxed, filtered and evaporated to dryness. The residue was purified by silica gel

column chromatography for 11 and recrystallized for 20.

4

.1.5. General procedure for the synthesis of 5-aminoindazoles 22-24 from 5-nitroindazoles. Method D.

To a suspension of the corresponding 5ꢀnitroindazole 1Hꢀ3ꢀindazolol derivative in methanol and the modified

catalyst FeO(OH) under argon atmosphere, monohydrated hydrazine in excess was added. The reaction

mixture was achieved at 50 – 70 ºC for 24ꢀ30 h. Then, the suspension was filtered with zelite and the solvent

evaporated to dryness. The residue was treated with water and chloroform and the organic layers evaporated

to dryness yielding the corresponding indazole as red oil.

4

.1.6. General procedure for the synthesis of indazoles 31-42 from 1-ethoxycarbonyl derivatives. Method E.

The corresponding halide was added to a suspension of the 1ꢀethoxycarbonylꢀ3ꢀindazolol derivative in

acetone, K CO and catalytic amount of KI. The reaction mixture was refluxed until the end of reaction. The

2

3

reaction mixture was evaporated to dryness and the residue was treated with water and chloroform. The

organic layers were evaporated to dryness and a solution of 0.4 M KOH and ethanol were added. The solution

was stirred at room temperature for 1 hour and them evaporated to dryness and the residue was purified by

silica gel column chromatography using as eluent a mixture chloroform/methanol (100:0, 80:1, 50:1).

4

.1.7. 3-(cyclohexylmethoxy)-1-(cyclohexylmethyl)indazole (1).

Following the procedure A, from 26 (0.41 g, 3.01 mmol), cyclohexylmethyl bromide (1.20 mL, 8.67 mmol) and

1

K

2

CO

MHz, DMSOꢀd

CH ); 3.98 (d, 2H, N1ꢀCH

a); 127.0 (Cꢀ6); 119.2(Cꢀ5); 118.7 (Cꢀ4) 111.5 (Cꢀ3a) 109.4 (Cꢀ7); 73.9 (OꢀCH

3

(0.86 g, 6.20 mmol) in acetone (60 mL). Reaction time: 48 h. Yield: (0.10 g, 10 %). Oil. HꢀNMR (300

6

) δ: 7.52 (d, 1H, 4ꢀH); 7.44 (d, 1H, 7ꢀH); 7.30 (t, 1H, 5ꢀH); 6.95 (t,1H, 6ꢀH); 4.06 (d, 2H, Oꢀ

13

2

); 1.8ꢀ0.9 (m, 22H, 2Cy). CꢀNMR (300 MHz, DMSOꢀd

); 53.9 (N1ꢀCH

); 25.3 (CH ); 25.2 (CH

6

) δ: 154.8 (Cꢀ3); 141.4 (Cꢀ

); 38.3 (CH);

). HPLCꢀMS

7

3

2

7.0 (CH); 30.1 (2C, CH

2

); 29.2 (2C, CH

2

); 26.0 (2C, CH

2

); 25.9 (2C, CH

2

+

(

ES ): CH

3

CN/H

2

O 10:90, gt: 6 min; rt: 6,15, M+H] = 327,4.

4

.1.8. 1-benzyl-3-benzyloxyindazole (2).

Following the procedure A, from 26 (0.05 g, 0.38 mmol) (AlfaꢀAesar), benzyl bromide (0.215 mL, 0.90 mmol)

1

and Cs

2

CO

3

(0.51 g, 1.56 mmol) in acetone (60 mL). Reaction time: 48 h. Yield: (0.74 g, 62%). Oil. HꢀNMR

(

3

300 MHz, CDCl ) δ: 7.76 (d, 1H, 4ꢀH); 7.61 – 7.23 (m, 10H, 2Bn); 7.43 (d, 1H, 7ꢀH); 7.39 (t, 1H, 5ꢀH); 7.10 (t,

1

3

1

H, 6ꢀH); 5.52 (d, 2H, OꢀCH

2

); 5.48 (d, 2H, N1ꢀCH

2

); CꢀNMR (300 MHz, CDCl

3

) δ: 156.3 (Cꢀ3); 142.1 (Cꢀ7a);

28.4 (Cꢀ6); 120.7 (Cꢀ5); 119.7 (Cꢀ4); 113.6 (Cꢀ3a); 109.3 (Cꢀ7); 71.2 (OꢀC); 52.8 (N1ꢀC); 137.9 (Ar); 137.5 (C,

Ar); 129.0 (2C, Ar); 128.9 (2C, Ar); 128.6 (2C, Ar); 127.9 (2C, Ar); 127.5 (2C, Ar). HPLCꢀMS (ES⁺):

+

CH

3

CN/H

2

O 50:95, gt: 6 min; rt: 5.41, [M+H] =315.1.

ACCEPTED MANUSCRIPT

1

5

4

.1.9. 1-(2-diisopropylaminoethyl)-3-(4-methoxybenzyloxy)indazole (3).

Following the procedure B, from 31 (0.19 g, 0.80 mmol), 2ꢀdiisopropylaminoethyl chloride hydrochloride (0.16

g, 0.80 mmol) and K CO (0.62 g, 4.49 mmol) in 2ꢀbutanone (60 mL). The final product was purified by

2

3

chromatography column using as eluent dichloromethane : methanol (1:0, 30:1). Reaction time: 60 h. Yield:

1

(

0.10 g, 33%). Oil. HꢀNMR (300 MHz, CDCl

3

) δ: 7.63 (d, 1H, 4ꢀH); 7.47 (d, 2H, Ar); 7.45 (d, 1H, 7ꢀH); 7.32 (t,

); 4.16 (t, 2H, N1ꢀCH ); 3.01 (m, 2H, CH); 2.81

) δ: 159.5 (Ar); 155.6 (Cꢀ3); 141.7 (Cꢀ7a); 130.0

2C, Ar); 129,8 (2C, Ar); 129.4 (C, Ar); 126.9 (Cꢀ6); 120.0 (Cꢀ5); 118.6 (Cꢀ4); 113.8 (2C, Ar); 112.4 (Cꢀ3a);

1

H, 5ꢀH); 6.99 (t, 1H, 6ꢀH); 6.92 (d, 2H, Ar); 5.36 (s, 2H, OꢀCH

2

1

3

(

t, 2H, CH

2

); 0.98 (d, 12H, CH

3

). CꢀNMR (300 MHz, CDCl

3

1

08.8 (Cꢀ7); 70.4 (OꢀCH

2

); 55,3 (OꢀCH

3

2

); 50,5 (2C, CH); 48,9 (N1ꢀCH ); 44,9 (CH

2

); 20,8 (4C, CH ). HPLCꢀMS

3

+

(

ES ): CH

3

CN/H

2

O 10:90, gt: 6 min; rt: 2.94, [M+H] = 179.7.

4

.1.10. 1-(4-methoxybenzyl)-3-(4-methoxybenzyloxy)indazole (4).

Following the procedure A, from 26 (0.40 g, 2.94 mmol), 4ꢀmethoxybenzyl chloride (0.95 mL, 7.00 mmol) and

1

Cs

2

CO

3

(1.95 g, 6.00 mmol) in acetone (40 mL). Reaction time: 48 h. Yield: (0.68 g, 60%). Oil. HꢀNMR (300

MHz, DMSOꢀd

6

) δ: 7.65 (dd, 1H, 4ꢀH); 7.45 (d, 2H, Ar); 7.30 (t, 1H, 5ꢀH); 7.17 (dd, 1H, 7ꢀH); 7.13 (d, 2H, Ar);

); 5.34 (s, 2H, N1ꢀCH ); 3.82 (s, 3H, Oꢀ

) δ: 159.9 (CꢀOCH ); 159.4 (CꢀOCH ); 156.2 (Cꢀ3);

7

.01 (t, 1H, 6ꢀH); 6.91 (d, 2H, Ar); 6.81 (d, 2H, Ar); 5.36 (s, 2H, OꢀCH

2

1

3

CH

3

); 3.77 (s, 3H, OꢀCH

3

). CꢀNMR (300 MHz, DMSOꢀd

6

3

1

41.9 (Cꢀ7a); 129.9 (2C, Ar); 129.6 (Ar); 129.2 (Ar); 128.4 (2C, Ar); 127.2 (Cꢀ6); 120.1 (Cꢀ5); 119.0 (Cꢀ4);

13.9 (2C, Ar); 113.8 (2C, Ar); 113.2 (Cꢀ3a); 108.8 (Cꢀ7); 70.4 (OꢀCH

2

); 55.3 (OꢀCH

3

); 55.2 (OꢀCH ); 51.9 (N1ꢀ

3

+

CH

2

). HPLCꢀMS (ES ): CH

3

CN/H

2

O 20:100, gt: 20 min; rt: 15.18, [M+H] = 375.14.

4

.1.11. 1-(2-diisopropylaminoethyl)-3-(1-naphthylmethoxy)-indazole (5).

Following the procedure B, from 32 (0.15 g, 0.54 mmol), 2ꢀdiisopropylaminoethyl chloride hydrochloride (0.33

g, 2.42 mmol) and K CO (0.39 g, 2.82 mmol) in 2ꢀbutanone (60 mL). The final product was purified by

2

3

chromatography column using as eluent dichloromethane : methanol (1:0, 30:1). Reaction time: 24 h. Yield:

1

(

0.069 g, 32%). Oil. HꢀNMR (300 MHz, CDCl

3

) δ: 7.72 (dd, 1H, 4ꢀH); 7.60 (dd, 1H, 7ꢀH); 7.33 (t, 1H, 5ꢀH);

); 4.20 (t, 2H, N1ꢀCH ); 3.02 (t, 2H, CH ); 2.85 (t,

) δ: 155.6 (Cꢀ3); 141.7 (Cꢀ7a); 127.0 (Cꢀ6); 120.0 (Cꢀ

8

2

5

.21 (t, 1H, 6ꢀH); 7.91ꢀ7.40 (m, 7H, Ar); 5.88 (s, 2H, OꢀCH

2

1

3

H, CH); 0.99 (d, 12H, CH

3

). CꢀNMR (300 MHz, CDCl

3

); 118.7 (Cꢀ4); 112.4 (Cꢀ3a); 108.8 (Cꢀ7); 69.1 (OꢀCH

2

); 50.2 (2C, CH); 49.2 (N1ꢀCH

2

); 44.9 (CH

2

); 20.8 (4C,

CH ); 133.8 (C, Ar); 132.8 (C, Ar); 131.9 (C, Ar); 129.0 (C, Ar); 128.6 (C, Ar); 127.2 (C, Ar); 126.3 (C, Ar);

3

+

1

4

25.8 (C, Ar); 125.3 (C, Ar); 124.1 (C, Ar). HPLCꢀMS (ES ): CH

02,2.

3

CN/H

2

O 10:100, gt: 6 min; rt: 3,66, [M+H] =

.1.12. 3-(1-naphthylmethoxy)-1-(2-(pyrrolidin-1-yl)ethyl)indazole (6).

Following the procedure B, from 32 (0.20 g, 0.74 mmol), 2ꢀpirrolydinoethyl chloride hydrochloride (0.14 g, 0.79

mmol) and K CO (3.90 g, 2,82 mmol) in 2ꢀbutanone (60 mL). The final product was purified by

chromatography column using as eluent dichloromethane : methanol (1:0, 30:1). Reaction time: 60 h. Yield:

2

3

ACCEPTED MANUSCRIPT

1

6

1

(

0.21 g, 75%). Oil. HꢀNMR (300 MHz, CDCl

3

) δ: 7.71 (dd, 1H, 4ꢀH); 7.47 (dd, 1H, 7ꢀH); 7.36 (t, 1H, 5ꢀH); 7.00

); 4.44 (t, 2H, N1ꢀCH ); 3.05 (t, 2H, CH ); 2.63 (d, 4H,

) δ: 155.8 (Cꢀ3); 141.6 (Cꢀ7a); 127.4 (Cꢀ6); 120.2 (Cꢀ5);

); 54.9 (N1ꢀCH ); 54.3 (2C, CH ); 47.6 (CH ); 23.5 (2C,

); 133.7 (C, Ar); 132.7 (C, Ar); 131.9 (C, Ar); 129.0 (C, Ar); 128.6 (C, Ar); 127.1 (C, Ar); 127.2 (C, Ar);

(

t, 1H, 6ꢀH); 7.91ꢀ7.40 (m, 7H, Ar); 5.87 (s, 2H, OꢀCH

2

1

3

CH

2

); 1.80 (d, 4H, CH

2

). CꢀNMR (300 MHz, CDCl

3

1

19.1 (Cꢀ4); 112.8 (Cꢀ3a); 108.7 (Cꢀ7); 69.1 (OꢀCH

2

CH

2

+

1

3

4

26.4 (C, Ar); 125.9 (C, Ar); 125.3 (C, Ar); 124.1 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:100, gt: 6 min; rt:

2

+

.53, [M+H] = 372.3.

.1.13. 3-(1-naphthylmethoxy)-1-(2-piperidinoethyl)indazole (7).

Following the procedure B, from 32 (0.30 g, 1.11 mmol), 2ꢀpiperidinoethyl chloride hydrochloride (0.23 g, 1.20

mmol) and K CO (0.69g, 5,00 mmol) in 2ꢀbutanone (60 mL). The final product was purified by

2

3

chromatography column using as eluent dichloromethane: methanol (1:0, 30:1). Reaction time: 24 h. Yield:

1

(

0.25 g, 59%). Oil. HꢀNMR (300 MHz, CDCl

3

) δ: 7.89 (d, 1H, 4ꢀH); 7.62 (d, 1H, 7ꢀH); 7.49 (t, 1H, 5ꢀH); 7.00 (t,

); 4.40 (t, 2H, N1ꢀCH ); 2.89 (t, 2H, CH ); 2.54 (m, 4H,

). CꢀNMR (300 MHz, CDCl ) δ: 155.8 (Cꢀ3); 141.6 (Cꢀ7a); 127.1

); 57.5 (N1ꢀCH ); 54.7 (2C, CH ); 46.1

1

H, 6ꢀH); 8.20ꢀ7.34 (m, 7H, Ar); 5.86 (s, 2H, OꢀCH

2

1

3

CH

2

); 1.62 (m, 4H, CH

2

); 1.25 (m, 2H, CH

2

3

(

Cꢀ6); 120.1 (Cꢀ5); 119.1 (Cꢀ4); 112.7 (Cꢀ3a); 108.8 (Cꢀ7); 69.1 (OꢀCH

2

CH

2

); 25.8 (2C, CH

2

); 24.1 (CH); 133.7 (C, Ar); 132.7 (C, Ar); 131.9 (C, Ar); 129.0 (C, Ar); 128.6 (C, Ar); 127.1

+

C, Ar); 127.2 (C, Ar); 126.4 (C, Ar); 125.9 (C, Ar); 125.3 (C, Ar); 124.1 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O

2

+

2

0:100, gt: 20 min; rt: 9.57, [M+H] = 386.0.

4

.1.14. 3-(1-naphtylmethoxy)-1-(1-naphtylmethyl)indazole (8).

Following the procedure A, from 26 (0.050 g, 0.38 mmol), 1ꢀnaphtylmethyl chloride (0.150 g, 0.85 mmol) and

1

Cs

2

CO

3

(0.325 g, 1.00 mmol) in acetone (40 mL). Reaction time: 48 h. Yield: (0.09 g, 60%). Oil. HꢀNMR (300

MHz, DMSOꢀd

6

) δ: 7.71 (dd, 1H, 4ꢀH); 7.66 (d, 1H, 7ꢀH); 7.37 (t, 1H, 5ꢀH); 7.03 (t, 1H, 6ꢀH); 8.27 – 7.15 (m,

1

3

1

7

4H, Ar); 5.94 (s, 2H, OꢀCH

2

); 5.92 (s, 2H, N1ꢀCH

2

). CꢀNMR (300 MHz, DMSOꢀd

6

) δ: 155.9 (Cꢀ3); 142.0 (Cꢀ

); 133.8 (C,

a); 127.3 (Cꢀ6); 120.3 (Cꢀ5); 119.2 (Cꢀ4); 113.2 (Cꢀ3a); 109.0 (Cꢀ7); 69.2 (OꢀCH

2

); 50.9 (N1ꢀCH

2

Ar); 132.8 (C, Ar); 132.6 (C, Ar); 131.9 (C, Ar); 131.1 (C, Ar); 129.0 (C, Ar); 128.9 (C, Ar); 128.6 (C, Ar); 127.4

(C, Ar); 126.4 (2C, Ar); 125.9 (2C, Ar); 125.4 (2C, Ar); 125.3 (2C, Ar); 125.2 (2C, Ar); 124.1 (2C, Ar); 123.2

+

(

2C, Ar). HPLCꢀMS (ES ): CH

.1.15. 3-(2-naphthylmethoxy)-1-(2-piperidinoethyl)indazole (9).

Following the procedure B, from 33 (0.30 g, 1,10 mmol), 2ꢀpiperidinoethyl chloride hydrochloride (0.20 g, 1.10

mmol) and K CO (0.62g, 4.49 mmol) in 2ꢀbutanone (60 mL) The final product was purified by chromatography

column using as eluent dichloromethane : methanol (1:0, 30:1). Reaction time: 24 h. Yield: (0.27 g, 64%). Oil.

¹HꢀNMR (300 MHz, CDCl

) δ: 7.68 (dd, 1H, 4ꢀH); 7.35 (t, 1H, 5ꢀH); 7.27 (dd, 1H, 7ꢀH); 7.02 (t, 1H, 6ꢀH); 7.98ꢀ

.25 (m, 7H, Ar); 5.59 (s, 2H, OꢀCH ); 4.35 (t, 2H, N1ꢀCH ); 2.79 (t, 2H, CH ); 2.46 (m, 4H, CH ); 1.55 (m, 4H,

). CꢀNMR (300 MHz, CDCl ) δ: 155.6 (Cꢀ3); 141.6 (Cꢀ7a); 127.0 (Cꢀ6); 120.1 (Cꢀ5);

3

CN/H

2

O 10:90, gt: 6 min; rt: 6.66, [M+H] = 415.5.

4

2

3

7

2

1

3

CH

2

); 1.41 (m, 2H, CH

2

3

ACCEPTED MANUSCRIPT

1

7

1

19.0 (Cꢀ4); 112.6 (Cꢀ3a); 108.7 (Cꢀ7); 70.7 (OꢀCH

2

); 57.7 (N1ꢀCH ); 54.7 (2C, CH

2

); 46.3 (CH ); 25.8 (2C,

2

CH

2

); 24.1 (CH

2

); 134.6 (C, Ar); 133.3 (C, Ar); 133.1 (C, Ar); 128.1 (C, Ar); 128.0 (C, Ar); 127.7 (C, Ar); 127.2

+

(

C, Ar); 126.1 (C, Ar); 126.0 (C, Ar); 125.9 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:90, gt: 8 min; rt: 3.54,

2

+

[

M+H] = 386.3.

4

.1.16. 5–nitro-1-(pentyl)-3-(pentyloxy)indazole (10).

Following the procedure A, from 27 (0.37 g, 2.07 mmol), pentyl iodide (0.57 mL, 4.35 mmol) and K

2

CO (0.70

3

1

g, 5,07 mmol) in acetone (60 mL). Reaction time: 120 h. Yield: (0.42 g, 65%). Oil. HꢀNMR (300 MHz, DMSOꢀ

d

1

2

6

) δ: 8.47 (d, 1H, 4ꢀH); 8.18 (dd, 1H, 6ꢀH); 7.49 (d, 1H, 7ꢀH); 4.35 (t, 2H, OꢀCH

2

); 4.26 (t, 2H, N1ꢀCH

). CꢀNMR (300 MHz, DMSOꢀd ) δ: 156.9 (Cꢀ3);

); 48.1 (N1ꢀCH );

); 13.8 (CH ). HPLCꢀMS

2

); 1.79ꢀ

1

3

.11 (m, 12H, 2(CH

2

)

3

); 0.86 (t, 3H, CH

3

); 0.77 (t, 3H, CH

3

6

42.2 (Cꢀ7a); 140.1 (Cꢀ5); 122.0 (Cꢀ6); 117.5 (Cꢀ4); 110.7 (Cꢀ3a); 110.1 (Cꢀ7); 69.1 (OꢀCH

2

8.8 (CH

2

); 28.3 (CH

2

); 28.2 (CH

2

); 27.6 (CH ); 21.9 (CH

2

); 21.7 (CH

2

); 13.9 (CH

3

+

(

ES ): CH

3

CN/H

2

O 20:80, gt: 6 min; rt: 4.51, [M+H] = 320.3.

4

.1.17. 1-benzyl-3-cyclohexylmethoxy-5-nitroindazole (11).

Following the procedure C 28 (0.10 g, 0.38 mmol), cyclohexylmethyl bromide (0.10 mL, 0.70 mmol) and K

0.14 g, 0.98 mmol) in 2ꢀbutanone (60 mL). The product was purified by silica gel column chromatography

2

CO

3

(

using as eluent a mixture dichloromethane: hexane (1:1, 5:1). Reaction time: 24 h. Yield: (0.078 g, 60%). mp.

1

7

2 ꢀ 75 °C (2ꢀpropanol). HꢀNMR (300 MHz, CDCl

3

) δ: 8.67 (d, 1H, 4ꢀH); 8.17 (dd, 1H, 6ꢀH); 7.30 (d, 1H, 7ꢀH);

1

3

.29ꢀ7.15 (m, 5H, Ar); 5.41 (s, 2H, N1ꢀCH

2

); 4.20 (d, 2H, OꢀCH

2

); 1.93 – 1.09 (m, 11H, Cy). CꢀNMR (300

3

MHz, CDCl ) δ: 158.7 (Cꢀ3); 143.1 (Cꢀ5); 141.3 (Cꢀ7a); 137.6 (C, Ar); 129.3 (2C, Ar); 128.4 (C, Ar); 127.5 (2C,

Ar); 123.0 (Cꢀ6); 119.2 (Cꢀ4); 113.2 (Cꢀ3a); 109.2 (Cꢀ7); 71.5 (OꢀCH

2

); 53.3 (N1ꢀCH

2

); 38.0 (CH ); 30.1 (2C,

2

+

CH

2

); 26.9 (CH

3

); 26.2 (2C, CH

2

). HPLCꢀMS (ES ): CH

3

CN/H

2

O 10:90, gt: 8 min; rt: 7.07, [M+H] = 366.4.

4

.1.18. 3-(4-methoxybenzyloxy)-5-nitro-1-pentylindazole (13).

Following the procedure B, from 34 (0.20 g, 0.67 mmol), 1ꢀpentyl iodide (0.1 mL, 0.77 mmol) and K

2

CO (0.35

3

g, 2.50 mmol) in 2ꢀbutanone (60 mL). The final product was purified by chromatography column using as

1

eluent dichloromethane : hexane (1:1, 5:1). Reaction time: 72 h. Yield: (0.07 g, 30%). mp. 78 ꢀ 80 °C. HꢀNMR

(

300 MHz, CDCl

3

) δ: 8.66 (d, 1H, 4ꢀH); 8.20 (dd, 1H, 6ꢀH); 7.45 (2H, Ar); 7.23 (d, 1H, 7ꢀH); 6.93 (2H, Ar); 5.36

); 3.83 (s, 3H, OꢀCH ); 1.89 (q, 2H, CH ); 1.36 – 1.22 (m, 4H, 2CH ); 0.88

). CꢀNMR (300 MHz, CDCl ) δ: 159.8 (CꢀOCH ); 157.5 (Cꢀ3); 142.5 (Cꢀ7a); 140.7 (Cꢀ5); 130.1

s, 2H, OꢀCH ); 4.20 (t, 2H, N1ꢀCH

2

3

2

13

s, 3H, CH

3

2C, Ar); 128.4 (Ar); 122.4 (Cꢀ6); 118.8 (Cꢀ4); 113.9 (2C, Ar); 112.0 (Cꢀ3a); 108.4 (Cꢀ7); 70.9 (OꢀCH

2

); 55.31

+

OꢀCH

3

); 48.9 (N1ꢀCH

2

); 29.3 (CH

2

); 28.9 (CH

2

); 22.3 (CH

2

); 13.9 (CH

3

). HPLCꢀMS (ES ): CH

3

CN/H

2

O 20:100,

+

gt: 20 min; rt: 16,89, [M+H] = 370.3.

4

.1.19. 1-(2-diisopropylaminoethyl)-3-(4-methoxybenzyloxy)-5-nitroindazole (14).

Following the procedure B, from 34 (0.100 g, 0.33 mmol), 2ꢀdiisopropylaminoethyl chloride hydrochloride (0.07

g, 0.33 mmol) and K CO (0.12 g, 0.90 mmol) in 2ꢀbutanone (60 mL). The solvent was evaporated under

2

3

ACCEPTED MANUSCRIPT

1

8

reduced pressure. Then, the solid is suspended in water and extracted with diethilic ether. After evaporate the

ether fraction, the solid obtained is recristallized from 2ꢀpropanol. Reaction time: 6 h. Yield: (0.12 g, 82%). mp.

1

8

3 ꢀ 85 °C. HꢀNMR(300 MHz, CDCl

3

) δ: 8.63 (d, 1H, 4ꢀH); 8.18 (dd, 1H, 6ꢀH); 7.47 (d, 2H, Ar); 7.27 (d, 1H, 7ꢀ

); 4.17 (t, 2H, N1ꢀCH ); 3.83 (s, 3H, OꢀCH ); 2.95 (m, 2H, CH); 2.85 (t,

). CꢀNMR (300 MHz, CDCl ) δ: 160.2 (CꢀOCH ); 158.0 (Cꢀ3); 142.5 (Cꢀ7a);

40.7 (Cꢀ5); 130.1 (2C, Ar); 129.4 (Ar); 122.6 (Cꢀ6); 119.1 (Cꢀ4); 114.4 (2C, Ar); 112.2 (Cꢀ3a); 109.5 (Cꢀ7);

1.3 (OꢀCH ); 55.3 (OꢀCH ); 50.2 (N1ꢀCH ); 49.3 (2C, CH); 45.2 (CH ); 21.1 (4C, CH

). HPLCꢀMS (ES⁺):

CN/H O 10:90, gt: 6 min; rt: 3.36, [M+H] = 427.3.

.1.20. 3-(1-naphthylmethoxy)-5-nitro-1-(2-(pyrrolidin-1-yl)ethyl)indazole (15).

Following the procedure B, from 35 (0.20 g, 0.62 mmol), 2ꢀpirrolydinnoethyl chloride hydrochloride (0.17 g,

.00 mmol) and K CO (0,28 g, 2.00 mmol) in butanone (60 mL). The final product was purified by

H); 6.93 (d, 2H, Ar); 5.37 (s, 2H, OꢀCH

2

3

13

2

1

7

H, CH

2

); 0.91 (d, 12H, CH

3

2

3

2

3

+

CH

3

2

4

1

2

3

chromatography column using as eluent dichloromethane : methanol (1:0, 30:1). Reaction Time: 60 h. Yield:

1

(

0.20 g, 76%). Oil. HꢀNMR(300 MHz, CDCl

3

) δ: 8.64 (d, 1H, 4ꢀH); 8.29 (dd, 1H, 6ꢀH); 8.18 (dd, 1H, Ar); 7.51

); 4.84 (t, 2H, N1ꢀCH ); 3.49 (s, 2H, CH ); 2.84 (m,

) δ: 158.4 (Cꢀ3); 143.85 (Cꢀ7a); 142.1 (Cꢀ5); 123.9 (Cꢀ

); 54.3 (2C, CH); 53.9 (N1ꢀCH ); 45.8 (CH ); 23.7 (2C,

); 133.7 (C, Ar); 132.7 (C, Ar); 131.9 (C, Ar); 129.0 (C, Ar); 128.6 (C, Ar); 127.1 (C, Ar); 127.2 (C, Ar);

(

d, 1H, 7ꢀH); 7.93 – 7.58 (m, 6H, Ar); 5.91 (s, 2H, OꢀCH

2

1

3

4

6

H, CH

2

); 1.91 (m, 4H, CH

2

). CꢀNMR (300 MHz, CDCl

3

); 118.8 (Cꢀ4); 112.2 (Cꢀ7); 110.2 (C3a); 69.9 (OꢀCH

2

CH

2

+

1

3

4

26.4 (C, Ar); 125.9 (C, Ar); 125.3 (C, Ar); 124.1 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 15:95, gt: 6 min; rt:

2

+

.28, [M+H] = 417.4.

.1.21. 3-(1-naphtylmethoxy)-1-(1-naphthylmethyl)-5-nitroindazole (16).

Following the procedure A from 27 (0.35 g, 1.95 mmol), 1ꢀnaphtylmethyl chloride (0.83 g, 4.72 mmol) and

Cs

2

CO

3

(1.80 g, 5.52 mmol) in acetone (60 mL). Reaction time: 72 h. Yield: (0.717 g, 68%). mp 143 – 145 ºC*

1

(

decomp) (2ꢀpropanol). HꢀNMR (300 MHz, DMSOꢀd

6

) δ: 8.75 (d, 1H, 4ꢀH); 8.23 (dd, 1H, 6ꢀH); 7.25 (d, 1H, 7ꢀ

1

3

H); 8.30 – 7.37 (m, 14H, Ar); 6.06 (s, 2H, OꢀCH

) δ: 157.9 (Cꢀ3); 143.5 (Cꢀ5); 141.5 (Cꢀ7a); 122.6 (Cꢀ6); 118.7 (Cꢀ4); 112.7 (Cꢀ7); 109.0 (Cꢀ3a); 70.0 (Oꢀ

CH ); 52.1 (N1ꢀCH ); 133.8 (C, Ar); 133.7 (C, Ar); 131.7 (C, Ar); 131.6 (C, Ar); 131.4 (C, Ar); 131.0 (C, Ar);

29.4 (C, Ar); 129.0 (C, Ar); 128.9 (C, Ar); 128.7 (C, Ar); 127.6 (C, Ar); 126.7 (C, Ar); 126.6 (C, Ar); 126.1 (C,

2

); 6.05 (s, 2H, N1ꢀCH

2

). CꢀNMR (300 MHz, DMSOꢀ

d

6

2

1

Ar); 126.0 (C, Ar); 125.6 (C, Ar); 125.3 (C, Ar); 125.2 (C, Ar); 123.7 (C, Ar); 123.0 (C, Ar). HPLCꢀMS (ES⁺):

+

CH

3

CN/H

2

O, 10:90 gt: 6 min; rt: 6.65, [M+H] = 460.2.

4

.1.22. 3-(2-naphtylmethoxy)-5-nitro-1-pentylindazole (17).

Following the procedure B, from 36 (0.21 g, 0.67 mmol), 1ꢀpentyl iodide (0.1 mL, 0.76 mmol) and K

2

CO (0.39

3

g, 2,82 mmol) in acetone (60 mL). The final product was purified by chromatography column using as eluent

dichloromethane : hexane (1:1, 5:1). Reaction time: 120 h. Yield: (0.10 g, 58%). mp. 104 ꢀ 106 °C (2ꢀ

1

propanol). HꢀNMR (300 MHz, CDCl

3

) δ: 8.72 (d, 1H, 4ꢀH); 8.24 (dd, 1H, 6ꢀH); 7.52 (d, 1H, 7ꢀH); 7.98 – 7.23

ACCEPTED MANUSCRIPT

1

9

(

m, 7H, Ar); 5.61 (s, 2H, OꢀCH

2

); 4.21 (t, 2H, N1ꢀCH

). CꢀNMR (300 MHz, CDCl ) δ: 157.4 (Cꢀ3); 142.5 (Cꢀ7a); 140.8 (Cꢀ5); 122.4 (Cꢀ6); 118.8 (Cꢀ4); 111.9

); 49.0 (N1ꢀCH ); 29.3 (CH ); 28.9 (CH ); 22.5 (CH ); 13.9 (CH ) 133.7 (C,

Ar); 133.2 (C, Ar); 128.3 (C, Ar); 128.0 (C, Ar); 127.7 (C, Ar); 127.2 (C, Ar); 126.3 (C, Ar); 126.2 (C, Ar); 125.8

2

); 1.90 (q, 2H, CH

2

); 1.29 (m, 4H, 2CH

2

); 0.87 (t, 3H,

13

CH

3

(Cꢀ3a); 108.5 (Cꢀ7); 71.16 (OꢀCH

2

3

+

(

C, Ar). HPLCꢀMS (ES ): CH

3

CN/H

2

O 15:95, gt: 8 min; rt: 7.44, [M+H] = 390.4.

4

.1.23. 3-(2-naphthylmethoxy)–5–nitro-1-(2-piperidinoethyl)indazole (18).

Following the procedure B, from 36 (0.21 g, 0.67 mmol), 2ꢀpiperidinoethyl chloride hydrochloride (0.12 g, 0.67

mmol) and K CO (0.65g, 4.71 mmol) in 2ꢀbutanone (60 mL). The solvent was evaporated under reduced

2

3

pressure. Then, the solid is suspended in water and extracted with chloroform. After evaporate the organic

phase, the solid obtained is recristallized from 2ꢀpropanol. Reaction time: 72 h. Yield: (0.17 g, 60%). mp. 83 ꢀ

1

8

6 °C (2ꢀpropanol). HꢀNMR (300 MHz, CDCl

3

) δ: 8.70 (d, 1H, 4ꢀH); 8.22 (dd, 1H, 6ꢀH); 7.28 (d, 1H, 7ꢀH); 7.98

); 4.32 (t, 2H, N1ꢀCH ); 2.77 (t, 2H, CH ); 2.39 (d, 4H, CH ); 1.48 (d, 4H,

). CꢀNMR (300 MHz, CDCl ) δ: 157.5 (Cꢀ3); 143.1 (Cꢀ7a); 140.8 (Cꢀ5); 122.3 (Cꢀ6);

); 57.8 (N1ꢀCH ); 54.7 (2C, CH ); 46.3 (CH ); 25.8 (2C,

); 133.7 (C, Ar); 133.2 (C, Ar); 128.3 (C, Ar); 128.0 (C, Ar); 127.7 (C, Ar); 127.2 (C, Ar); 126.3

–

7.49 (m, 7H, Ar); 5.60 (s, 2H, OꢀCH

2

1

3

CH

2

); 1.39 (d, 2H, CH

2

3

1

18.6 (Cꢀ4); 112.0 (Cꢀ3a); 109.0 (Cꢀ7); 71.2 (OꢀCH

CH ); 24.1 (CH

C, Ar); 126.2 (C, Ar); 125.8 (C, Ar). HPLCꢀMS (ES ): CH

2

+

(

3

CN/H

2

O 5:95, gt: 20 min; rt: 12.54, [M+H] = 431.2.

4

.1.24. 1-(2-morpholinoethyl)-3-(2-naphthylmethoxy)-5-nitroindazole (19).

Following the procedure B, from 36 (0.09 g, 0.28 mmol), 2ꢀmorpholinoethyl chloride hydrochloride (0.07 g,

.40 mmol) and K CO (0.15g, 0.81 mmol) in 2ꢀbutanone (60 mL) The solvent was evaporated under reduced

0

2

3

pressure. Then, the solid is suspended in water and extracted with chloroform. After evaporate the organic

phase, the solid obtained is recristallized from 2ꢀpropanol. Reaction time: 98 h. Yield: (0.11 g, 94%). mp. 104 ꢀ

1

7

06 °C (2ꢀpropanol). HꢀNMR (300 MHz, CDCl

3

) δ: 8.71 (d, 1H, 4ꢀH); 8.23 (dd, 1H, 6ꢀH); 7.51 (d, 1H, 7ꢀH);

); 4.34 (t, 2H, N1ꢀCH ); 3.58 (d, 4H, CH ); 2.83 (t, 2H, CH ); 2.43

) δ: 157.5 (Cꢀ3); 143.1 (Cꢀ7a); 140.9 (Cꢀ5); 122.3 (Cꢀ6); 118.7 (Cꢀ4);

); 66.8 (OꢀCH ); 57.3 (N1ꢀCH ); 53.7 (2C, CH ); 46.7 (CH ); 133.6 (C,

.98 – 7.49 (m, 7H, Ar); 5.60 (s, 2H, OꢀCH

2

1

3

(

d, 4H, CH

2

). CꢀNMR (300 MHz, CDCl

3

1

12.1 (Cꢀ3a); 108.8 (Cꢀ7); 71.2 (2C, CH

2

Ar); 133.1 (C, Ar); 128.4 (C, Ar); 128.0 (C, Ar); 127.7 (C, Ar); 127.2 (C, Ar); 126.4 (C, Ar); 126.3 (C, Ar); 125.7

+

(

C, Ar). HPLCꢀMS (ES ): CH

3

CN/H

2

O 20:100, gt: 20 min; rt: 11.60, [M+H] = 433.45.

4

.1.25. 1-benzyl-3-(2-naphthylmethoxy)-5-nitroindazole (20).

Following the procedure C, from 28 (0.29 g, 1.07 mmol), 2ꢀnaphtylmethyl bromide (0.25 g, 1.12 mmol) and

Cs

2

CO

3

(1.20 g, 3.7 mmol) in acetone (60 mL). the product was purified by recristallization from 2ꢀpropanol.

1

Reaction time: 72 h. Yield: (0.201 g, 45%). mp. 100 ꢀ 103 °C (2ꢀpropanol). HꢀNMR (300 MHz, DMSOꢀd

6

) δ:

); 5.56 (s,

) δ: 156.8 (Cꢀ3); 142.6 (Cꢀ7a); 140.6 (Cꢀ5); 122.4 (Cꢀ6); 117.7 (Cꢀ

); 51.8 (N1ꢀCH ); 136.8 (C, Ar); 133.8 (C, Ar); 132.7 (C, Ar); 132.6

8

2

4

.60 (d, 1H, 4ꢀH); 8.22 (dd, 1H, 6ꢀH); 7.83 (d, 1H, 7ꢀH); 8.09 – 7.21 (m, 12H, Ar); 5.62 (s, 2H, OꢀCH

2

1

3

H, N1ꢀCH

2

). CꢀNMR (300 MHz, DMSOꢀd

6

); 112.8 (Cꢀ3a); 108.6 (Cꢀ7); 70.7 (OꢀCH

2

ACCEPTED MANUSCRIPT

2

0

(C, Ar); 128.5 (2C, Ar); 128.0 (C, Ar); 127.9 (C, Ar); 127.6 (C, Ar); 127.5 (C, Ar); 127.3 (2C, Ar); 127.0 (C, Ar);

+

1

4

26.3 (C, Ar); 126.0 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:90, gt: 6 min; rt: 6.40, [M+H] = 410.3.

2

.1.26. 3-(2-naphtylmethoxy)-1-(2-naphtylmethyl)-5-nitroindazole (21).

Following the procedure A, from 27 (0.31 g, 1.73 mmol), 2ꢀnaphtylmethyl bromide (1.21 mL, 5.48 mmol) and

K

2

CO

3

(0.73 g, 5.28 mmol) in acetone (60 mL). Reaction time: 72 h. Yield: (0.31 g, 30%). mp 101 ꢀ 103 °C (2ꢀ

1

propanol). HꢀNMR (300 MHz, DMSOꢀd

6

) δ: 8.73 (d, 1H, 4ꢀH); 8.18 (dd, 1H, 6ꢀH); 7.48 (d, 1H, 7ꢀH); 7.98 –

13

7

1

.23 (m, 14H, Ar); 5.64 (s, 2H, OꢀCH

42.8 (Cꢀ7a); 141.1(Cꢀ5); 122.7 (Cꢀ6); 118.7 (Cꢀ4); 112.7 (Cꢀ7); 108.9 (Cꢀ3a); 71.3 (OꢀCH

33.6 (C, Ar); 133.5 (C, Ar); 133.2 (C, Ar); 132.9 (C, Ar); 128.8 (C, Ar); 128.4 (C, Ar); 128.0 (C, Ar); 127.8 (C,

2

); 5.59 (s, 2H, N1ꢀCH

2

). CꢀNMR (300 MHz, DMSOꢀd

6

) δ: 157.6 (Cꢀ3);

2

); 53.2 (N1ꢀCH

2

);

Ar); 127.7 (C, Ar); 127.3 (C, Ar); 126.5 (C, Ar); 126.3 (C, Ar); 126.1 (C, Ar); 125.8 (C, Ar); 124.8 (C, Ar). HPLCꢀ

+

MS (ES ): CH

3

CN/H O 70:100, gt: 15 min; rt: 9.65, [M+H] = 460.2

2

4

.1.27. 5-amino-1-benzyl-3-benzyloxyindazole (22).

Following the procedure D, from 12 (0.22 g, 0.60 mmol), hydrazine monohydrated (0.60 mL, 1.6 mmol) and

FeO(OH) (0.05 g, 0.5 mmol) in methanol (30 mL). The product was purified by extraction water / chloroform

1

and evaporation of this one. Reaction time: 24 h. Yield: (0.191 g, 97%). Oil. HꢀNMR (300 MHz, DMSOꢀd

6

) δ:

); 5.30 (s,

) δ: 153.7 (Cꢀ3); 141.9 (Cꢀ5); 136.3 (Cꢀ7a);

7

2

1

.48 – 7.11 (m, 10H, Ar); 7.24 (d, 1H, 7ꢀH); 6.65 (d, 1H, 4ꢀH); 6.77 (dd, 1H, 6ꢀH); 5.32 (s, 2H, OꢀCH

2

1

3

H, N1ꢀCH

2

); 4.80 (br s, 2H, NH

2

). CꢀNMR (300 MHz, DMSOꢀd

6

19.1 (Cꢀ6); 112.9 (Cꢀ3a); 110.1 (Cꢀ7); 99.6 (Cꢀ4); 69.8 (OꢀCH

2

); 51.4 (N1ꢀCH

2

); 138.2 (C, Ar); 137.2 (C, Ar);

+

28.3 (2C, Ar); 127.9 (2C, Ar); 127.8 (C, Ar); 127.1 (2C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:100, gt: 6 min;

2

+

rt: 3.71, [M+H] =330.2.

4

.1.28. 5-amino-3-(1-naphthylmethoxy)-1-(2-(pyrrolidin-1-yl)ethyl)indazole (23).

Following the procedure D, from 15 (0.08 g, 0.20 mmol), hydrazine monohydrated (3.50 mL, 9.23 mmol) and

FeO(OH) (0.04 g, 0.4 mmol) in methanol (30 mL). The product was purified by cromatography column using

as eluent the mixture dichloromethane : methanol (1:0, 20:1). Reaction time: 30 h. Yield: (0.06 g, 85%). mp.

1

5

4

1

36 ꢀ 138 ºC. HꢀNMR (300 MHz, CDCl

3

) δ: 8.19– 7.45 (m, 7H, Ar); 7.16 (d, 1H, 7ꢀH); 6.84 (m, 2H, 4ꢀH, 6ꢀH);

); 4.37 (t, 2H, N1ꢀCH ); 3.57 (br s, 2H, NH ); 3.01 (t, 2H, CH ); 2.61 (s, 4H, CH ); 1.78 (d,

). CꢀNMR (300 MHz, CDCl ) δ: 155.3 (Cꢀ3); 139.6 (Cꢀ5); 137.9 (Cꢀ7a); 119.9 (Cꢀ6); 113.7 (Cꢀ3a);

10.0 (Cꢀ7); 103.4 (Cꢀ4); 69.5 (OꢀCH ); 55.3 (N1ꢀCH ); 54.7 (2C, CH ); 48.0 (CH ); 23.9 (2C, CH ); 133.7 (C,

.83 (s, 2H, OꢀCH

2

1

3

H, CH

2

3

2

Ar); 132.7 (C, Ar); 131.9 (C, Ar); 129.0 (C, Ar); 128.6 (C, Ar); 127.1 (C, Ar); 127.2 (C, Ar); 126.4 (C, Ar); 125.9

+

(

C, Ar); 125.3 (C, Ar); 124.1 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H

2

O 15:95, gt: 6 min; rt: 2,05, [M+H] = 387.4.

4

.1.29. 5-amino-3-(2-naphthylmethoxy)-1-(2-piperidinoethyl)indazole (24).

Following the procedure D, from 18 (0.11 g, 0.25 mmol), hydrazine monohydrated (1.20 mL, 3.16 mmol) and

FeO(OH) (0.02 g, 0.2 mmol) in methanol (30 mL). The product was purified by cromatography colmn using as

1

eluent the mixture dichloromethane : methanol (1 : 0, 20 : 1). Reaction time: 24 h. Yield: (0.07 g, 66%). Oil. Hꢀ

ACCEPTED MANUSCRIPT

2

1

NMR (300 MHz, CDCl

3

) δ: 7.96 – 7.47 (m, 7H, Ar); 7.11 (d, 1H, 7ꢀH); 6.91 (s, 1H, 4ꢀH); 6.83 (d, 1H, 6ꢀH); 5.54

); 4.27 (t, 2H, N1ꢀCH ); 3.26 (br s, 2H, NH ); 2.73 (t, 2H, CH ); 2.42 (d, 4H, CH ); 1.56 (d, 4H,

). CꢀNMR (300 MHz, CDCl ) δ: 154.9 (Cꢀ3); 139.2 (Cꢀ5); 137.7 (Cꢀ7a); 119.5 (Cꢀ6);

); 58.1 (N1ꢀCH ); 55.0 (2C, CH ); 46.7 (CH ); 26.1 (2C,

); 133.6 (C, Ar); 133.1 (C, Ar); 128.4 (C, Ar); 128.0 (C, Ar); 127.7 (C, Ar); 127.2 (C, Ar); 126.4

(

s, 2H, OꢀCH

2

13

CH

2

); 1.41 (d, 2H, CH

2

3

1

13.3 (Cꢀ3a); 109.9 (Cꢀ7); 103.2 (Cꢀ4); 71.0 (OꢀCH

CH ); 24.4 (CH

C, Ar); 126.3 (C, Ar); 125.7 (C, Ar). HPLCꢀMS (ES ): CH

2

+

(

3

CN/H

2

O 10:100, gt: 6 min; rt: 2.33, [M+H] =401.2.

4

.1.30. 5-bromo-3-(2-naphthylmethoxy)-1-(2-piperidinoethyl)indazole (25).

From 9 (0.09 g, 0.24 mmol) in acetonitrile (20 mL), iron trichloride hexahydrate (0.05 g, 0.17 mmol) was

added. The reaction mixture was heated to 80ºC and then, Nꢀbromosuccinimide (0.04 g, 0.20 mmol) was

added. The reaction was heated for 40 h. The solvent is evaporated under reduced pressure and the residue

is extracted with water / chloroform. After evaporation of the chloroform, The product was purified by

cromatography column using as eluent the mixture dichloromethane : methanol (1: 0, 10: 1). Reaction Time:

1

4

1

2

0 min. Yield: (0.03 g, 27%). Oil. HꢀNMR (300 MHz, CDCl

3

) δ: 7.83 (d, 1H, 4ꢀH); 7.41 (dd, 1H, 6ꢀH); 7.20 (dd,

); 4.35 (t, 2H, N1ꢀCH ); 2.81 (t, 2H, CH ); 2.43 (d, 4H, CH );

). CꢀNMR (300 MHz, CDCl ) δ: 154.8 (Cꢀ3); 140.3 (Cꢀ7a); 130.3 (Cꢀ6);

22.6 (Cꢀ4); 114.1 (Cꢀ5); 111.8 (Cꢀ3a); 110.4 (Cꢀ7); 70.8 (OꢀCH ); 57.5 (N1ꢀCH ); 54.7 (2C, CH ); 46.4 (CH );

H, 5ꢀH); 7.96ꢀ7.47 (m, 7H, Ar); 5.56 (s, 2H, OꢀCH

2

1

3

.55 (d, 4H, CH

2

); 1.39 (d, 2H, CH

2

3

2

5.5 (2C, CH

2

); 23.9 (CH

2

); 134.3 (C, Ar); 133.2 (C, Ar); 133.1 (C, Ar); 128.2 (C, Ar); 128.0 (C, Ar); 127.7 (C,

+

Ar); 127.0 (C, Ar); 126.2 (C, Ar); 126.1 (C, Ar); 125.8 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 5:80, gt: 20 min; rt:

2

+

1

5.28, [M+H] = 467.1.

4

.1.31. 3-(4-methoxybenzyloxy)-1H-indazole (31) and 2-(4-methoxybenziloxy)-1H-3-indazolone (37).

Following the procedure E, from 29 (1.50 g, 7.27 mmol), 4ꢀmethoxybenzyl bromide (1.3 mL, 9.0 mmol) and

K

2

CO

3

(2.20g, 16.16 mmol) in 1,4ꢀdioxane (100 mL). Reaction time: 24 h. Total yield: (1.42 g, 77%). Yield 31:

1

0

.61 g (33%). mp 73ꢀ76 ºC. HꢀNMR (300 MHz, CDCl

3

) δ: 8.86 (s, 1H, NH); 7.70 (d, 1H, 4ꢀH); 7.46 (d, 2H, Ar);

); 3.83 (s, 3H, Oꢀ

) δ: 159.6 (C, Ar) 157.6 (Cꢀ3); 142.6 (Cꢀ7a); 129.8 (2C, Ar); 129.1 (C, Ar);

); 55.3 (OꢀCH

O 5;95, gt: 20 min., rt: 13.31, [M+H] = 179.7. Yield 37: 0.81 g (44%). mp 165 ꢀ 167

7

.37 (t, 1H, 5ꢀH); 7.30 (d, 1H, 7ꢀH); 7.08 (t, 1H, 6ꢀH); 6.93 (d, 2H, Ar); 5.38 (s, 2H, OꢀCH

2

1

3

CH

3

). CꢀNMR (300 MHz, CDCl

3

1

27.8 (Cꢀ6); 120.0 (Cꢀ5); 119.8 (Cꢀ4); 113.9 (2C, Ar); 112.9 (Cꢀ3a); 109.6 (Cꢀ7); 70.4 (OꢀCH

2

3

)

+

HPLCꢀMS (ES ): CH

3

CN/H

2

1

ºC. HꢀNMR (300 MHz, DMSO) δ: 10,26 (br s, 1H, NH); 7.67 (d, 1H, 4ꢀH); 7.49 (t, 1H, 5ꢀH); 7.21 (d, 3H, 7ꢀH,

1

3

Ar); 7.09 (t, 1H, 6ꢀH); 6.90 (d, 2H, Ar); 4,91 (s, 2H, N2ꢀCH

2

); 3,72 (s, 3H, OꢀCH ). CꢀNMR (300 MHz, DMSO)

3

δ: 160.5 (C, Ar) 158.6 (Cꢀ3); 145.9 (Cꢀ7a); 131.3 (C, Ar); 129.1 (2C, Ar); 128.8 (Cꢀ6); 122.9 (Cꢀ5); 120.7 (Cꢀ4);

+

1

17.0 (Cꢀ3a); 113.8 (2C, Ar); 112.1 (Cꢀ7); 55.0 (N2ꢀCH

2

); 46.2 (OꢀCH

3

). HPLCꢀMS (ES ): CH

3

CN/H O 10:90,

2

+

gt: 6 min., rt: 3,70, [M+H] = 255.0.

ACCEPTED MANUSCRIPT

2

4

.1.32. 3-(1-naphthylmethoxy)-1H-indazole (32) and 2-(1-naphthylmethoxy)-1H-3-indazolone (38).

Following the procedure E, from 29 (0.89 g, 4.33 mmol), 1ꢀnaphtylmethyl chloride (0.76 g, 4.33 mmol) and

K

2

CO

3

(1.63g, 11.7 mmol) in butanone (80 mL). Reaction time: 48 h. (0.65 g, 54%). Total yield: (0,72 g, 54%).

1

Yield 32: (0.59 g, 49%). mp 100ꢀ103 ºC. HꢀNMR (300 MHz, DMSOꢀd

6

) δ: 12.00 (s, 1H, NH); 7.72 (d, 1H, 4ꢀH);

1

3

7

.52 (d, 1H, 7ꢀH); 7.35 (t, 1H, 5ꢀH); 6.98 (t, 1H, 6ꢀH); 8.15ꢀ7.30 (m, 7H, Ar); 5.85 (s, 2H, OꢀCH

2

). CꢀNMR

(

300 MHz, DMSOꢀd ) δ: 155.6 (Cꢀ3); 141.8 (Cꢀ7a); 127.1 (Cꢀ6); 119.0 (2C, Cꢀ4, Cꢀ5); 111.1 (Cꢀ7); 110.1 (Cꢀ

6

3

a); 68.3 (OꢀCH

2

); 133.3 (C, Ar); 132.5 (C, Ar); 131.3 (C, Ar); 128.8 (C, Ar); 128.5 (C, Ar); 127.0 (C, Ar); 126.5

+

(

C, Ar); 126.0 (C, Ar); 125.4 (C, Ar); 123.7 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:90, gt: 6 min; rt: 5.43,

2

+

1

[

M+H] = 275.2. Yield 38: 0.06 g (5%). mp 195 ꢀ 199 ºC. HꢀNMR (300 MHz, DMSO) δ: 8.35 (d, 1H, 4ꢀH); 7.32

1

3

(

t, 1H, 5ꢀH); 7.08 (d, 1H, 7ꢀH); 6.91 (t, 1H, 6ꢀH); 7.96 – 7.41 (m, 7H, Ar); 5.44 (s, 2H, N2ꢀCH ). CꢀNMR (300

2

MHz, DMSOꢀd6) δ: 159.6 (Cꢀ3); 146.2 (Cꢀ7a); 126.3 (Cꢀ6); 122.9 (Cꢀ5); 118.7 (Cꢀ4); 115.3 (Cꢀ3a); 112.4 (Cꢀ

7

); 44.9 (N2ꢀCH

2

); 133.2 (C, Ar); 132.8 (C, Ar); 130.8 (C, Ar); 130.0 (C, Ar); 128.4 (C, Ar); 128.1 (C, Ar); 127.0

+

(

C, Ar); 125.8 (C, Ar); 125,3 (C, Ar); 123.7 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:90, gt: 6 min; rt: 4.24,

2

+

[

M+H] = 275.0.

4

.1.33. 3-(2-naphthylmethoxy)-1H-indazole (33) and 2-(2-naphthylmethoxy)-1H-3-indazolone (39).

Following the procedure E, from 29 (0.84 g, 4.07 mmol), 2ꢀnaphthylmethyl bromide (0.95 g, 4.33 mmol) and

K

2

CO

3

(0.82 g, 5.92 mmol) in butanone (100 mL). Reaction time: 4 h. (0.55 g, 49%). Total yield: (0,72 g,

1

4

1

9%).Yield 33: (0.38 g, 34%). mp 114 ꢀ118 ºC. HꢀNMR (300 MHz, DMSOꢀd6) δ: 11.98 (s, 1H, NH); 7.65 (dd,

1

3

H, 4ꢀH); 7.34 (m, 2H, 5ꢀH, 7ꢀH); 7.02 (t, 1H, 6ꢀH); 8.04 – 7.50 (7H, Ar); 5.56 (s, 2H, OꢀCH

2

). CꢀNMR (300

3

MHz, CDCl ) δ: 155.5 (Cꢀ3); 141.7 (Cꢀ7a); 126.6 (Cꢀ6); 119.1 (Cꢀ5); 119.0 (Cꢀ4); 111.1 (Cꢀ3a); 110.0 (Cꢀ7);

6

9.9 (OꢀCH

2

); 134.7 (C, Ar); 132.7 (C, Ar); 132.5 (C, Ar); 127.9 (C, Ar); 127.7 (C, Ar); 127.5 (C, Ar); 127.0 (C,

+

CN/H

O 10:90, gt: 6 min; rt: 5.51, [M+H]⁺

Ar); 126.2 (C, Ar); 126.0 (C, Ar); 125.9 (C, Ar). HPLCꢀMS (ES ): CH

3

2

1

=

1

275.3. Yield 39: 0.17 g (15%). mp 206 – 210 ºC, 205ꢀ212 ºC). HꢀNMR (300 MHz, DMSOꢀd6) δ: 10.29 (s,

H, N1ꢀH); 7.71(d, 1H, 4ꢀH); 7.49 (t, 1H, 5ꢀH); 7.21 (d, 1H, 7ꢀH); 7.12 (t, 1H, 6ꢀH); 7.91 – 7.41 (m, 7H, Ar); 5.16

1

3

(

s, 2H, N2ꢀCH

2

). CꢀNMR (300 MHz, DMSOꢀd6) δ: 161.2 (Cꢀ3); 146.6 (Cꢀ7a); 126.7 (Cꢀ6); 123.5 (Cꢀ5); 121.3

(Cꢀ4); 117.5 (Cꢀ3a); 112.6 (Cꢀ7); 47.4 (N2ꢀCH

2

); 134.8 (C, Ar); 133.2 (C, Ar); 132.8 (C, Ar); 131.9 (C, Ar);

+

1

28.7 (C, Ar); 127.0 (C, Ar); 127.9 (C, Ar); 126.5 (C, Ar); 126.2 (C, Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:90,

2

+

gt: 6 min; rt: 4.30, [M+H] = 275.3.

.1.34. 3-(4-methoxybenzyloxy)-5-nitro-1H-indazole (34) and 2-(4-methoxybenzyloxy)-5-nitro-3-indazolone

40).

Following the procedure E, from 30 (0.35 g, 1.38 mmol), 4ꢀmethoxybenzyl bromide (0.35 mL, 2.50 mmol) and

4

(

K

2

CO

3

(0.26 g, 1.85 mmol) in butanone (30 mL). Reaction time: 24 h. Total yield: (0,72 g, 63%). Yield 34: (0.12

1

g, 30%). mp 129 – 132 ºC. HꢀNMR(300 MHz, DMSOꢀd

6

) δ: 12.72 (s, 1H, NH); 8.49 (d, 1H, 4ꢀH); 8.15 (dd, 1H,

13

6

ꢀH); 7.53 (d, 1H, 7ꢀH); 7.47 (d, 2H, Ar); 6.95 (d, 2H, Ar); 5.35 (s, 2H, OꢀCH

2

); 3.75 (s, 3H, OꢀCH ). CꢀNMR:

3

ACCEPTED MANUSCRIPT

2

3

(

300 MHz, DMSOꢀd

6

) δ: 159.3 (Ar); 157.6 (Cꢀ3); 143.1 (Cꢀ7a); 140.3 (Cꢀ5); 130.0 (2C, Ar); 128.4 (C, Ar );

1

22.0 (Cꢀ6); 117.4 (Cꢀ4); 113.8 (2C, C, Ar); 110.9 (Cꢀ7); 110.6 (Cꢀ3a); 70.3 (OꢀCH

2

); 55.1 (OꢀCH

3

). HPLCꢀMS

+

1

(

ES ): CH

3

CN/H

2

O 10:100, gt: 6 min., rt: 5.32, [M+H] = 300.2. Yield 40: 0.12 g (50%). 138 – 140 ºC. HꢀNMR

(

300 MHz, DMSO) δ: 8.51 (s, 1H, 4ꢀH); 8.26 (d, 1H, 6ꢀH) 7.35; (d, 1H, 7ꢀH); 7.23 (d, 2H, Ar); 6.91 (d, 2H, Ar);

1

3

5

.00 (s, 2H, N2ꢀCH

2

); 3,72 (s, 3H, OꢀCH ). CꢀNMR (300 MHz, DMSO) δ: 158.8 (C, Ar); 158.4 (Cꢀ3); 145.6

3

(Cꢀ7a); 140.3 (Cꢀ5); 129.1 (2C, Ar); 128.0 (C, Ar); 126.0 (Cꢀ6); 120.5 (Cꢀ4); 114.0 (2C, C, Ar); 111.9 (Cꢀ7);

+

1

3

4

11.6 (Cꢀ3a); 55.0 (N2ꢀCH

00.2.

2

); 46.4 (OꢀCH

3

). HPLCꢀMS (ES ): CH

3

CN/H O 10:90, gt: 6 min; rt: 3,91, [M+H] =

2

.1.35. 3-(1-naphthylmethoxy)-5-nitro-1H-indazole (35) and 2-(1-naphthylmethyl)-5-nitro-3-indazolone (41).

Following the procedure E, from 30 (0.90 g, 3.58 mmol), 1ꢀnaphthylmethyl chloride (0.91 g, 5.17 mmol) and

CO (0.72 g, 5.23 mmol) in DMF (60 mL). Reaction time: 24 h. Total yield: (0,72 g, 63%). Yield 35: (0.59g,

2%). mp.204 ꢀ 206 ºC. HꢀNMR (300 MHz, DMSOꢀd ) δ: 8.48 (d, 1H, 4ꢀH); 7.77 (d, 1H, 7ꢀH); 7.62 (dd, 1H, 6ꢀ

K

2

3

1

6

5

1

3

6

H); 8.17– 7.51 (m, 7H, Ar); 5.92 (s, 2H, OꢀCH

2

). CꢀNMR (300 MHz, DMSOꢀd ) δ: 157.6 (Cꢀ3); 143.4 (Cꢀ5);

); 133.3 (C, Ar); 132.0 (C, Ar);

31.2 (C, Ar); 129.0 (C, Ar); 128.5 (C, Ar); 127.3 (C, Ar); 126.7 (C, Ar); 126.0 (C, Ar); 125.4 (C, Ar); 123.7 (C,

1

40.2 (Cꢀ7a); 121.8 (Cꢀ6); 117.4 (Cꢀ4); 111.2 (Cꢀ7); 110.4 (Cꢀ3a); 68.9 (OꢀCH

2

+

Ar). HPLCꢀMS (ES ): CH

3

CN/H O 10:90, gt: 6 min; rt: 5.74.[M+H] = 320.4. Yield 41: (0.12 g, 11%) mp 212 ꢀ

2

1

2

7

1

13 ºC (decomp.). HꢀNMR (300 MHz, DMSO) δ: 11,9 (br s, 1H, NH); 8,55 (s, 1H, 4ꢀH); 8,26 (d, 1H, 6ꢀH);

1

3

,99–7,43 (m, 7H, Ar); 7,33 (d, 1H, 7ꢀH); 5,54 (s, 2H, N2ꢀCH

46,5 (Cꢀ5); 141,0 (Cꢀ7a); 126,5 (Cꢀ6); 121,0 (Cꢀ4); 112,5 (Cꢀ7); 112,3 (Cꢀ3a); 45,5 (N2ꢀCH

31,7 (C, Ar); 131,1 (C, Ar); 129,2 (C, Ar); 129,0 (C, Ar); 127,7 (C, Ar); 127,1 (C, Ar); 125,8 (C, Ar); 123,7 (C,

2

). CꢀNMR (300 MHz,DMSO) δ: 155,2 (Cꢀ3);

2

); 133,8 (C, Ar);

+

Ar). HPLCꢀMS (ES ): CH

.1.36. 3-(2-naphthylmethoxy)-5-nitro-1H-indazole (36) and 2-naphthylmetyl-5-nitro-3-indazolone (42).

Following the procedure E, from 30 (0.96 g, 3.82 mmol), 2ꢀnaphthylmethyl bromide (0.87 g, 3.94 mmol) and

3

CN/H

2

O 15:95, gt: 6 min; rt: 4,53,[M+H] = 320,4.

4

K

2

CO

3

(0.63 g, 4.52 mmol) in butanone (100 mL). Reaction time: 30 h. Total yield: (0.90 g, 74%). Yield 36:

1

0

1

3

1

.54 g, (45%) mp 161ꢀ163 ºC. HꢀNMR (300 MHz, CDCl

3

) δ: 9.27 (s, 1H, N1ꢀH); 8.75 (d, 1H, 4ꢀH); 8.27 (dd,

13

H, 6ꢀH); 7.50 (d, 1H, 7ꢀH); 7.98 – 7.33 (7H, Ar); 5.63 (s, 2H, OꢀCH

); 143.7 (Cꢀ5); 141.7 (Cꢀ7a); 123.1 (Cꢀ6); 118.4 (Cꢀ4); 112.3 (Cꢀ3a); 109.8 (Cꢀ7); 71.2 (OꢀCH

33.2 (C, Ar); 128.4 (C, Ar); 128.0 (C, Ar); 127.8 (C, Ar); 127.1 (C, Ar); 126.3 (C, Ar); 125.6 (C, Ar). HPLCꢀMS

2

). CꢀNMR (300 MHz, CDCl

3

) δ: 158.8 (Cꢀ

2

); 133.5 (C, Ar);

+

(

ES ): CH

3

CN/H O 10:90, gt: 8 min., rt: 5.53, [M+H] = 320.1. Yield 42: 0.36 g (29%). mp 230ꢀ232 ºC

2

1

(

decomp.). HꢀNMR (300 MHz, DMSO) δ: 12.88 (s, 1H, N1ꢀH); 8.55 (s, 1H, 4ꢀH); 8.26 (d, 1H, 6ꢀH); 7.44 (d,

1

3

1

H, 7ꢀH); 7.92 – 7.34 (7H, Ar); 5.28 (s, 2H, N2ꢀCH

40.8 (Cꢀ7a); 126.1 (Cꢀ6); 121.1 (Cꢀ4); 117.9 (Cꢀ3a); 112.4 (Cꢀ7); 47.5 (N2ꢀCH

32.8 (C, Ar); 128.8 (C, Ar); 128.0 (C, Ar); 127.9 (C, Ar); 126.9 (C, Ar); 126.8 (C, Ar); 126.6 (C, Ar). HPLCꢀMS

2

). CꢀNMR (300 MHz, DMSO) δ: 157.9 (Cꢀ3); 146.3 (Cꢀ5);

2

); 134.1 (C, Ar); 133.1 (C, Ar);

+

(

ES ): CH

3

CN/H O 10:90, gt: 8 min., rt: 4.42, [M+H] = 320.2.

2

ACCEPTED MANUSCRIPT

2

4

.2. Biological Assays

.2.1. Radioligand Binding Assays for CB

/CB2 receptor binding studies were performed using membrane fractions of human CB

transfected cells purchased from PerkinꢀElmer Life and Analytical Sciences (Boston, MA). HEK293EBNA

membranes were resuspended in Tris buffer (50 mM TrisꢀHCl, 2.5 mM EDTA, 5 mM MgCl , 0.5 mg/mL BSA

4

1

and CB2 Receptors

CB

1

/CB receptor

2

fatty acid free, pH 7.4). Fractions of the final membrane suspension (about 0.415 mg/mL of protein for CB1

3

and about 0.18 mg/mL of protein for CB2) were incubated at 30°C for 90 min with 0.54 nM [ H]ꢀCP55940

3

(

139.6 Ci/mmol) for CB1 and 0.33 nM [ H]ꢀCP55940 (139.6 Ci/mmol) for CB2 , in the presence or absence of

several concentrations of the competing drug, in a final volume of 0.2 mL for CB1 and 0.6 mL for CB2 of assay

buffer (50 mM TrisꢀHCl, 2.5 mM EDTA, 5 mM MgCl , 0.5 mg/mL BSA fatty acid free, pH 7.4). Nonspecific

2

binding was determined in the presence of 10 µM WIN 55,212ꢀ2. Silanized tubes were used throughout the

experiment to minimize receptor binding loss due to tube adsorption. The reaction was terminated by rapid

vacuum filtration with a filter mate Harvester apparatus (PerkinꢀElmer) through Filtermat A GF/C filters

presoaked in 0.05% polyethylenimine (PEI).

The filters were washed nine times with iceꢀcold buffer for CB1 (50 mM TrisꢀHCl, 2.5 mM EDTA, 5 mM MgCl

2

,

0

.5 mg/mL BSA fatty acid free, pH 7.4) for CB1 and CB2 (50 mM TrisꢀHCl, 2.5 mM EGTA, 5 mM MgCl , 1

2

mg/mL BSA fatty acid free, pH 7.5), and bound radioactivity was measured with a 1450 LSC & Luminiscence

counter Wallac MicroBeta TriLux (PerkinꢀElmer). The binding assay showed the appropriate sensitivity to CB1

and CB2 ligands. Thus, WIN55,212ꢀ2 inhibited the binding with a Ki value of 36.2 nM (CB1R) and WIN55,212ꢀ

2

and HUꢀ308 inhibited the binding with K

experiments, competition binding curves were analyzed by using an iterative curveꢀfitting procedure GraphPad

51], which provided IC50 values for test compounds. K values were determined by the method of Cheng and

Prusoff [66, 67].

.2.2. In vitro Cholinesterase Inhibition Assays.

i

values of 3.7 and 11.2 nM (CB2R), respectively. For all binding

[

i

4

AChE from human erythrocytes (min. 500 units/mg protein in buffered aqueous solution), BuChE from equine

serum (10 units/mg protein, lyophilized powder), and BuChE from human serum (3 units/mg protein,

lyophilized powder) were purchased from Sigma. Compounds were evaluated in 100 mM phosphate buffer pH

8

.0 at 30 ºC, using acetylthiocholine and butyrylthiocholine (0.4 mM) as substrates, respectively. In both

cases, 5,5ꢀdithioꢀbis(2ꢀnitrobenzoic)acid (DTNB, Ellmans reagent, 0.2 mM) was used and the values of IC50

were calculated by UV spectroscopy from the absorbance changes at 412 nm. Experiments were performed in

triplicate.

4

.2.3. Kinetic Study of BuChE/AChE Inhibition.

To investigate the mechanism of action of the compounds on AChE or BuChE, a kinetic analysis was

performed. The experiments were carried out using combinations of four substrate concentrations, and three

ACCEPTED MANUSCRIPT

2

5

inhibitor concentrations. Doubleꢀreciprocal Lineweaver–Burk plotting of the data obtained, in which each point

is mean of three different experiments, were analysed.

Competitive inhibitors have the same yꢀintercept as uninhibited enzyme (since Vmax is unaffected by

competitive inhibitors the inverse of Vmax also doesn't change) but there are different slopes and xꢀintercepts.

Nonꢀcompetitive inhibition produces plots with the same xꢀintercept as uninhibited enzyme (K is unaffected)

m

but different slopes and yꢀintercepts. Uncompetitive inhibition causes different intercepts on both the yꢀ and xꢀ

axes but the same slope. Mixed inhibitors causes intersect above or below the xꢀaxes.

The K

model by nonlinear regression analysis using GraphPad Prism [51].

.2.4. Functional activity for cannabinoid receptors on isolated tissue.

i

, values were determined by fitting the kinetic data to a competitive, noncompetitive, or mixed inhibition

4

The functional activity of the new compounds for CB1 receptors was evaluated on the mouse vas deferens

preparation. This is a nerveꢀsmooth muscle preparation that serves as a highly sensitive and quantitative

functional in vitro bioassay for cannabinoid receptor agonists. These ligands induce concentrationꢀrelated

decreases in the amplitude of electrically evoked contractions of the vas deferens by acting on naturally

expressed prejunctional neuronal cannabinoid receptors to inhibit release of the contractile neurotransmitters,

noradrenaline and ATP, that is provoked by the electrical stimulation [56].

For this study, male ICR mice weighing 25–30 g were used. Mouse vas deferens were isolated as described

by Hughes [68]. Tissues were suspended in a 10 mL organ bath containing 5 mL of Krebs solution (NaCl 118;

KCl 4.75; CaCl

2

2.54; KH

2

PO

4

1.19; MgSO

4

1.2; NaHCO 25; glucose 11 mM) that was continuously gassed

3

with 95% O and 5% CO

2

. Tissues were kept under 0.5 g of resting tension at 37 °C and were electrically

stimulated through two platinum ring electrodes. They were subjected to alternate periods of stimulation (trains

of five rectangular pulses of 70 V, 15 Hz and 2 ms duration each were applied every minute) and rest (10 min).

The isometric force was monitored by computer using a MacLab data recording and analysis system.

The effect of the synthetic cannabinoid agonist WIN 55,212ꢀ2 and that of the new compounds 3, 5-7, 9, 12, 15,

ꢀ7

ꢀ5

1

8, 23, and 24, (10 – 2×10 M) was tested by constructing concentration–response curves in a step by step

manner. Curves were carried out by the following protocol: WIN 55,212ꢀ2 or the new compounds were added

at a concentration to the organ bath 50 min after the beginning of electrical stimulation and their effect on the

electrically induced contractions was evaluated 10 min after their addition. Then, the electrical stimulation was

stopped, Krebs solution was replaced and the following concentration of the compound was added. This

protocol was repeated for every concentration of the curve.

To test the involvement of the CB1 and CB2 receptors in the effect of the most interesting compounds, they

ꢀ6

were tested in tissues incubated with the cannabinoid CB1 receptor antagonist AM251 (10 M) or the

ꢀ6

combination of both CB1 and CB2 receptor antagonists, AM251 and AM630 (10 M) respectively.

Concentration–response curves for the new compounds were constructed in a step by step manner as follows:

ACCEPTED MANUSCRIPT

2

6

AM251 or the combination of both antagonists was added to the organ bath 50 min after the beginning of

electrical stimulation and 10 minutes later, a dose of WIN 55, 212ꢀ2 or the new compounds was added and its

effect on the electrically induced contractions was tested 10 min later. Then, the electrical stimulation was

stopped, Krebs solution was replaced and this protocol was repeated for every dose of concentrationꢀ

response curve.

Results have been expressed as % of inhibition, taking the mean amplitude of the last five contractions before

the first addition of the agonist as 100%. Each tissue was employed to construct only one concentration–

response curve.

Drugs. WIN55,212ꢀ2, AM251 and AM630 were obtained from TOCRIS (Biogen Científica S.L., Madrid, Spain);

they were dissolved in ethanol 1mg:1 mL and subsequently in ethanol and Tween 80 (1:2) after which the

ethanol was evaporated [55] and saline solution (0.9%) was added.

ꢀ2

The new compounds were dissolved in ethanol, getting a first concentration of 10 M that was further diluted

in distillate water.

4

.2.5. Oxygen Radical Absorbance Capacity Assay.

The ORACꢀFL method of Ou et al. [57] partially modified by Dávalos et al. [58] was followed, using a Polarstar

Galaxy plate reader (BMG Labtechnologies GmbH, Offenburg, Germany) with 485ꢀP excitation and 520ꢀP

emission filters. The equipment was controlled by Fluorostar Galaxy software (version 4.11ꢀ0) for fluorescence

measurement.

2,2′ꢀAzobis(amidinopropane)

dihydrochloride

(AAPH),

(±)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀ

tetramethylchromaneꢀ2ꢀcarboxylic acid (Trolox), and fluorescein (FL) were purchased from SigmaꢀAldrich. The

reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 ꢀL.

Antioxidant (20 ꢀL) and FL (120 ꢀL, 70 mM, final concentration) solutions were placed in a black 96ꢀwell

microplate (96F untreat, Nunc). The mixture was preincubated for 15 min at 37 °C, and then AAPH solution

(

60 ꢀL, 12 mM, final concentration) was added rapidly using a multichannel pipet. The microplate was

immediately placed in the reader and the fluorescence recorded every minute for 80 min. The microplate was

automatically shaken prior each reading. Samples were measured at eight different concentrations (0.1−1

ꢀM). A blank (FL + AAPH in phosphate buffer) instead of the sample solution and eight calibration solutions

using Trolox (1−8 ꢀM) were also used in each assay. All the reaction mixtures were prepared in duplicate, and

at least three independent assays were performed for each sample. Raw data were exported from the Fluostar

Galaxy software to an Excel sheet for further calculations. Antioxidant curves (fluorescence vs time) were first

normalized to the curve of the blank corresponding to the same assay, and the area under the fluorescence

decay curve (AUC) was calculated. The net AUC corresponding to a sample was calculated by subtracting the

AUC corresponding to the blank. Regression equations between net AUC and antioxidant concentration were

calculated for all samples. ORACꢀFL values were expressed as Trolox equivalents by using the standard

curve calculated for each assay, where the ORACꢀFL value of Trolox was taken as 1.

ACCEPTED MANUSCRIPT

2

7

4

.3. Docking studies.

All calculations were performed using the SYBYL 8.1 program suite [Tripos Inc., St. Louis. Mo 1]. The docking

studies were achieved using the FlexiDock module of the SYBYL 8.1 suite of programs.

The structures of the ligands were built with standard bond lengths and angles using the molecular modelling

package SYBYL 8.1 and their energies were minimized using the conjugate gradient algorithm with a

conjugated gradient of <0.001 kcal/mol convergent criteria provided by the MMFF94 force field [69, 70] and

MMFF94 electrostatic charges.

The structure of AChE and BuChE (pdb code: 1B14 and 1P0I) were edited, protein hydrogen atoms were

added and partial charges were calculated using AMBER procedure. Positions of the hydrogen atoms were

refined with the use of AMBER force field.

The ligands were placed manually in the gorge limited by PAS and CAS in AChE and BuChE. The ligand–

receptor complex was subjected to energy minimization using the MMFF94 force field and MMFF94

electrostatic charges and their energies were minimized using the protocol previously indicated with a

conjugate gradient of <0.1 kcal/mol convergent criteria. A distanceꢀdependent dielectric constant was used in

all the calculations. These complexes were the input structure for docking using FlexiDock command. During

the flexible docking analysis, the ligands and a sphere of 6Å around the corresponding ligand were considered

flexibles. For each complex three flexible docking analysis were run. The default FlexiDock parameters were

utilized in all cases, with maximum and minimum iterations (MI) set for each complex according to MI= [Nº of

rotable bonds in the protein+ Nº of rotable bonds in the ligand+6 *1000ꢀ500, obtaining a series of model

complexes.

The conformations were analyzed in terms of interactions between ligand and the known key residues. We

choose the conformation with highest FlexiDock score (better interactions) and refine the minimization energy

step using a conjugate gradient of <0.01 kcal/mol convergent criteria. Analysis of the refined receptorꢀligand

complex models was based on hydrogen bond, aromatic and hydrophobic interactions predicted with the LPC

program [71] and the values of ∆G binding and dissociation constants obtained from the difference accessible

surface area method using the STC program [65].

Acknowledgments

The authors gratefully acknowledge the financial support of Spanish Ministry of Science and Innovation

(

project CTQ2009ꢀ07664) and from “Comunidad de Madrid, Programa de Biociencias (SꢀSALꢀ0261/2006) and

would like to thank to Guadalupe Pablo for her excellent technical assistance.

ACCEPTED MANUSCRIPT

2

8

References

1] J.A. Claassen, R.W. Jansen, Cholinergically mediated augmentation of cerebral perfusion in Alzheimer's

[

disease and related cognitive disorders: the cholinergicꢀvascular hypothesis, J Gerontol A Biol Sci Med

Sci, 61 (2006) 267ꢀ271.

[2] E. Coulthard, V. SinghꢀCurry, M. Husain, Treatment of attention deficits in neurological disorders, Curr

Opin Neurol, 19 (2006) 613ꢀ618.

[

3] M. Villarroya, A.G. Garcia, J. MarcoꢀContelles, M.G. Lopez, An update on the pharmacology of

galantamine, Expert Opin Investig Drugs, 16 (2007) 1987ꢀ1998.

[4] H.W. Klafki, M. Staufenbiel, J. Kornhuber, J. Wiltfang, Therapeutic approaches to Alzheimer's disease,

Brain, 129 (2006) 2840ꢀ2855.

[

5] M.A. Kamal, X. Qu, Q.S. Yu, D. Tweedie, H.W. Holloway, Y. Li, Y. Tan, N.H. Greig,

Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer

drug candidate, enzyme kinetic analysis, J Neural Transm, 115 (2008) 889ꢀ898.

[6] R.M. Lane, S.G. Potkin, A. Enz, Targeting acetylcholinesterase and butyrylcholinesterase in dementia,

Int J Neuropsychopharmacol, 9 (2006) 101ꢀ124.

7] R. Annicchiarico, A. Federici, C. Pettenati, C. Caltagirone, Rivastigmine in Alzheimer's disease: Cognitive

[

function and quality of life, Ther Clin Risk Manag, 3 (2007) 1113ꢀ1123.

[8] R. Bullock, R. Lane, Executive dyscontrol in dementia, with emphasis on subcortical pathology and the

role of butyrylcholinesterase, Curr Alzheimer Res, 4 (2007) 277ꢀ293.

9] A. Castro, A. Martinez, Peripheral and dual binding site acetylcholinesterase inhibitors: implications in

[

treatment of Alzheimer's disease, Mini Rev Med Chem, 1 (2001) 267ꢀ272.

[10] G.V. De Ferrari, M.A. Canales, I. Shin, L.M. Weiner, I. Silman, N.C. Inestrosa, A structural motif of

acetylcholinesterase that promotes amyloid betaꢀpeptide fibril formation, Biochemistry, 40 (2001) 10447ꢀ

1

0457.

11] N.C. Inestrosa, A. Alvarez, M.C. Dinamarca, T. PerezꢀAcle, M. Colombres, Acetylcholinesteraseꢀamyloidꢀ

betaꢀpeptide interaction: effect of Congo Red and the role of the Wnt pathway, Curr Alzheimer Res, 2

2005) 301ꢀ306.

12] P. Masson, M.T. Froment, C.F. Bartels, O. Lockridge, Asp7O in the peripheral anionic site of human

butyrylcholinesterase, Eur J Biochem, 235 (1996) 36ꢀ48.

[

(

[

13] P. Masson, W. Xie, M.T. Froment, V. Levitsky, P.L. Fortier, C. Albaret, O. Lockridge, Interaction between

the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of

substrates, Biochim Biophys Acta, 1433 (1999) 281ꢀ293.

[14] S. Diamant, E. Podoly, A. Friedler, H. Ligumsky, O. Livnah, H. Soreq, Butyrylcholinesterase attenuates

amyloid fibril formation in vitro, Proc Natl Acad Sci U S A, 103 (2006) 8628ꢀ8633.

[

15] N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu, J. Mamczarz, H.W. Holloway,

T. Giordano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi, D.K. Lahiri, Selective

butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer

betaꢀamyloid peptide in rodent, Proc Natl Acad Sci U S A, 102 (2005) 17213ꢀ17218.

[

16] M. Bajda, N. Guzior, M. Ignasik, B. Malawska, Multiꢀtargetꢀdirected ligands in Alzheimer's disease

treatment, Curr Med Chem, 18 (2011) 4949ꢀ4975.

[17] Y. Rook, K.U. Schmidtke, F. Gaube, D. Schepmann, B. Wunsch, J. Heilmann, J. Lehmann, T. Winckler,

Bivalent betaꢀcarbolines as potential multitarget antiꢀAlzheimer agents, J Med Chem, 53 3611ꢀ3617.

[

18] M. Incerti, L. Flammini, F. Saccani, G. Morini, M. Comini, M. Coruzzi, E. Barocelli, V. Ballabeni, S. Bertoni,

Dualꢀacting drugs: an in vitro study of nonimidazole histamine H3 receptor antagonists combining

anticholinesterase activity, ChemMedChem, 5 1143ꢀ1149.

[

19] L. Piazzi, A. Cavalli, F. Colizzi, F. Belluti, M. Bartolini, F. Mancini, M. Recanatini, V. Andrisano, A. Rampa,

Multiꢀtargetꢀdirected coumarin derivatives: hAChE and BACE1 inhibitors as potential antiꢀAlzheimer

compounds, Bioorg Med Chem Lett, 18 (2008) 423ꢀ426.

[

20] W. Huang, D. Lv, H. Yu, R. Sheng, S.C. Kim, P. Wu, K. Luo, J. Li, Y. Hu, Dualꢀtargetꢀdirected 1,3ꢀ

diphenylurea derivatives: BACE 1 inhibitor and metal chelator against Alzheimer's disease, Bioorg Med

Chem, 18 5610ꢀ5615.

Article Doi

Cannabinoid agonists showing BuChE inhibition as potential therapeutic agents for Alzheimer's disease

DOI: 10.1016/j.ejmech.2013.11.026

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2013.11.026

Full text of DOI:10.1016/j.ejmech.2013.11.026

Products guided by the article

R&D Labs maybe for 1286737-69-6

Relevant to this article

Hot Product