2,3,9- and 2,3,11-Trisubstituted tetrahydroprotoberberines as D2 dopaminergic ligands

European Journal of Medicinal Chemistry 68 (2013) 150e166

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European Journal of Medicinal Chemistry

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Original article

2,3,9- and 2,3,11-Trisubstituted tetrahydroprotoberberines as D₂

dopaminergic ligands

Javier Párraga ^a, Nuria Cabedo ^b, Sebastián Andujar ^c, Laura Piqueras ^d, Laura Moreno ^a,

Abraham Galán ^a, Emilio Angelina ^c, Ricardo D. Enriz ^c, María Dolores Ivorra ^a,

,

*

María Jesús Sanz ^{d e}, Diego Cortes ^a

^aDepartamento de Farmacología, Laboratorio de Farmacoquímica, Facultad de Farmacia, Universidad de Valencia, Burjassot, 46100 Valencia, Spain

^bCentro de Ecología Química Agrícola e Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Campus de Vera, Ediﬁcio 6C, 46022

Valencia, Spain

^cDepartamento de Química, Universidad Nacional de San Luís, Argentina

^dDepartamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain

^eInstitute of Health Research e INCLIVA, University Clinic Hospital of Valencia, Valencia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

Dopamine-mediated neurotransmission plays an important role in relevant psychiatric and neurological

disorders. Nowadays, there is an enormous interest in the development of new dopamine receptors (DR)

acting drugs as potential new targets for the treatment of schizophrenia or Parkinson’s disease. Previous

studies have revealed that isoquinoline compounds such as tetrahydroisoquinolines (THIQs) and tetra-

hydroprotoberberines (THPBs) can behave as selective D₂dopaminergic alkaloids since they share

structural similarities with dopamine. In the present study we have synthesized eleven 2,3,9- and 2,3,11-

trisubstituted THPB compounds (six of them are described for the ﬁrst time) and evaluated their po-

tential dopaminergic activity. Binding studies on rat striatal membranes were used to evaluate their

afﬁnity and selectivity towards D₁and D₂DR and establish the structureeactivity relationship (SAR) as

dopaminergic agents. In general, all the tested THPBs with protected phenolic hydroxyls showed a lower

afﬁnity for D₁and D₂DR than their corresponding homologues with free hydroxyl groups. In previous

studies in which dopaminergic afﬁnity of 1-benzyl-THIQs (BTHIQs) was evaluated, the presence of a Cl

into the A-ring resulted in increased afﬁnity and selectivity towards D₂DR. This is in contrast with the

current study since the existence of a chlorine atom into the A-ring of the THPBs caused increased af-

ﬁnity for D₁DR but dramatically reduced the selectivity for D₂DR. An OH group in position 9 of the THPB

(9f) resulted in a higher afﬁnity for DR than its homologue with an OH group in position 11 (9e) (250 fold

for D₂DR). None of the compounds showed any cytotoxicity in freshly isolated human neutrophils. A

molecular modelling study of three representative THPBs was carried out. The combination of MD

simulations with DFT calculations provided a clear picture of the ligand binding interactions from a

structural and energetic point of view. Therefore, it is likely that compound 9d (2,3,9-trihydroxy-THPB)

behave as D₂DR agonist since serine residues cluster are crucial for agonist binding and receptor

activation.

Received 13 May 2013

Received in revised form

23 July 2013

Accepted 25 July 2013

Available online 11 August 2013

Keywords:

Tetrahydroprotoberberines

Dopamine receptors

Structureeactivity relationships cytotoxicity

MTT and cytoﬂuorometric analysis

Theoretical calculations

1. Introduction

dopamine receptors (DR) as potential targets for treating schizo-

phrenia or Parkinson’s disease. Therefore, there is an increased

Dopamine-mediated neurotransmission plays an important role

in several psychiatric and neurological disorders affecting several

million people worldwide. Researchers have focused their efforts in

investigating the modulation of dopaminergic activity via the

interest in the discovery of novel dopaminergic ligands as potential

drug candidates in the therapy of these neurological disorders [1].

DR can be classiﬁed into two pharmacologic families (D₁and D₂-

like) that are encoded by at least ﬁve genes. From a therapeutical

point of view, drugs acting at D₂-like DR are more relevant than

those interacting with D₁-like DR [2]. In this context, whereas the

D₂-like DR antagonists are used in the treatment of schizophrenia

(antipsychotics), the agonists are used in the treatment of

* Corresponding author. Tel.: þ34 963 54 49 75; fax: þ34 963 54 49 43.

E-mail addresses: dcortes@uv.es, Diego.M.Cortes@uv.es (D. Cortes).

URL: http://www.farmacoquimicavalencia.es//

http://dx.doi.org/10.1016/j.ejmech.2013.07.036

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

151

Parkinson’s disease [1,3]. In addition, other studies have revealed

the potential role of D₂agonist in the treatment of depression. The

pathophysiology of depression has been classically assigned to the

noradrenaline and serotonine systems however, nowadays, pub-

lished reports also support a role of the dopaminergic system in

this disorder [2]. In regard to this, different selective D₂-type DR

agonists were found to display antidepressant-like actions in

several rodent models, suggesting a speciﬁc role of this receptor

subtype in their antidepressant efﬁcacy [4e8].

Previous studies from our group have revealed that isoquinoline

compounds such as tetrahydroisoquinolines (THIQs) have afﬁnity

for DR in striate membranes of rat brain tissue which was likely due

to their structural similarities to dopamine [8e18]. Likewise, tet-

rahydroprotoberberines (THPBs) are another group of natural and

synthetic isoquinoline alkaloids with a variety of powerful biolog-

ical activities, including dopaminergic activity [19e21]. Indeed,

coreximine, a natural tetrasubstituted THPB, and other analogues

were reported as selective D₂dopaminergic alkaloids [22].

different substitutions into the A-ring in natural and synthetic

isoquinoline alkaloids. We observed that the presence of hydroxyls

groups in this ring caused increased afﬁnity for the D₁-like and D₂-

like DR families, while blockade of these hydroxyls groups resulted

in decreased afﬁnity [8,12e15]. Moreover, the presence of a halogen

in the A-ring led to a selective binding at least to one of the two DR

subtypes investigated [8,16,17]. Therefore, we have prepared three

series of THPBs: 2,11-dihydroxy-3-chloro-THPB (series 1), 2,3-

dihydroxy-11-methoxy-THPB (series 2) and 2,3,11-trihydroxy-

THPB and 2,3,9-trihydroxy-THPB (series 3) and differently

substituted analogues (Fig. 1).

Inasmuch, at the concentrations tested none of the synthesized

THPBs affected human neutrophil apoptosis or survival, indicating

the absence of cytotoxicity for human cells in these in vitro ap-

proaches. Molecular modelling of the possible stereo-electronic

requirements for dopamine D₂receptor ligands of the THPBs syn-

thesized has been discussed based on the different afﬁnities

displayed.

Recently, L-stepholidine analogues have been synthetized display-

ing a dual dopaminergic activity, partial agonism at the D₁DR and

antagonism at D₂DR [23].

2.1. Chemistry

Since THPBs, THIQs and dopamine share structural similarities,

in the present study we have synthesized eleven 2,3,9- and 2,3,11-

trisubstituted THPB compounds (six of them are here described for

the ﬁrst time) and evaluated their potential dopaminergic activity.

Their structures were determined on the basis of their NMR spec-

tral data and mass spectrometry analysis. The structural features

that deﬁne the afﬁnity and selectivity of the three series of THPBs

synthesized for D₁/D₂receptors was determined analyzing the in-

ﬂuence of the substitution at the 2,3- and 9 or 11 positions, in order

to obtain dopaminergic ligands with increased speciﬁcity. There-

fore, all the synthesized compounds were tested for their ability to

displace the selective radioligands of D₁and D₂-like DR from their

speciﬁc binding sites in striatal membranes in order to establish the

structureeactivity relationship (SAR) as dopaminergic agents. In

addition, cytotoxicity studies in human cells were carried out. For

this purpose, we used the MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay and a cytoﬂuorometric anal-

ysis to determine their impact on human neutrophil apoptosis and

survival [24e26]. All of them were devoid of any toxic effect in

these in vitro assays.

The synthesis of the THPBs has been performed as outlined in

the Schemes 1e3. The THPBs of the series 1 (Scheme 1) have been

synthesized from 3-chloro-4-methoxybenzaldehyde as starting

material. 3-Chloro-4-methoxy-b-nitroestyrene was obtained from

3-chloro-4-methoxybenzaldehyde by a Henry’s reaction using

nitromethane, ammonium acetate and acetic acid as solvent [27],

and then, 3-chloro-4-methoxyphenylethylamine (1) was obtained

by reduction with lithium aluminium hydride [28]. This phenyl-

ethylamine (1), was treated with 3-methoxyphenylacetyl chloride

under SchotteneBaumann conditions to generate the N-(3-cloro-4-

methoxyphenylethyl)-b

-(3⁰-methoxyphenyl)acetamide (2) [29,30].

Next, the phenylacetamide (2) was converted into the corre-

sponding 1-benzyltetrahydroisoquinoline (BTHIQ) (3) using the

BischlereNapieralski cyclodehydration reaction. The use of a POCl₃

and P₂O₅mixture in dry toluene followed by NaBH₄reduction was

required since the presence of a halogen in position 3 of the phe-

nylacetamide can inactivate the BischlereNapieralski cyclo-

dehydration reaction due to the electro-attractive properties of the

halogen [8,31e33]. Once obtained, the BTHIQ 6-chloro-7-methoxy-

1-(3⁰-methoxybenzyl)-1,2,3,4-THIQ hydrochloride (3) was sub-

jected to Mannich cyclization conditions using 37% aqueous form-

aldehyde [34] to generate the corresponding 2,11-dimethoxy-3-

chloro-THPB (3a) with high yield (86%). Finally, the O-demethyla-

tion was performed by adding of 4 equivalents of BBr₃reagent for

2 h at room temperature [8] to obtain the 2,11-dihydroxy-3-chloro-

THPB (3b) (Scheme 1).

In order to better understand the molecular interactions stabi-

lizing and destabilizing the different THPBs/D₂DR complexes, a

molecular modelling study using molecular dynamic simulations

and quantum mechanic calculations was carried out for the most

representative compounds of this series. Thus, the possible stereo-

electronic requirements of the THPBs/D₂DR interactions have been

discussed based on their different afﬁnities.

The THPBs of the series 2 (Scheme 2) have been synthesized

using similar approaches to those previously described. A protec-

tive reaction of the hydroxyl groups was performed in 3,4-

dihydroxybenzaldehyde through dibenzylation [28]. Once pre-

2. Results and discussion

In the present study, the impact of different substituents into the

A- and D-ring of synthesized THPBs on dopaminergic afﬁnity was

evaluated. In previous reports, we determined the relevance of

pared the protected 3,4-dibenzyloxybenzaldehyde, a similar

sequence of synthesis steps were carried out to obtain the phen-

ylethylamine (4), the phenylacetamide (5), the BTHIQ (6) and

Fig. 1. THPBs series.

152

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

Scheme 1. Synthesis of THPBs 3a and 3b (Series 1). Reagents and conditions: (a) Nitromethane, NH₄OAc, AcOH, reﬂux, 4 h; (b) LiAlH₄, THF/Et₂O, N₂, reﬂux, 2 h; (c) CH₂Cl_2,3-

methoxyphenylacetyl chloride, 5% NaOH, rt, 3 h; (d) P₂O₅, POCl₃, Toluene, N₂, reﬂux, 8 h; (e) NaBH₄; MeOH, rt, 2 h; (f) HCHO, EtOH, H₂O, reﬂux, 5 h; (g) CH₂Cl₂, BBr₃, rt, 2 h.

ﬁnally, the corresponding 2,3-dibenzyloxy-11-methoxy-THPB (6a)

with good yields (63e93%). In this series, the BischlereNapieralski

cyclodehydration was performed using only POCl₃in dry acetoni-

trile since the presence of an alkoxy group in position 3 of the

phenylacetamide can activate the BischlereNapieralski cyclo-

dehydration reaction due to the electro-donor ability of the

oxygenated group. Then, this THPB was subjected to acidic condi-

tions in absolute ethanol [12e35] in order to deprotect the phenolic

hydroxyls groups yielding 2,3-dihydroxy-11-methoxy-THPB (6b). A

methylendioxy group was generated by CsF and dichloromethane

[13] from 6b to obtain the 2,3-methylendioxy-11-methoxy-THPB

(6c).

The THPBs of the series 3 (Scheme 3) have been synthesized

employing similar procedures to those followed in series 1 and 2.

The corresponding trisubstituted THPBs: 2,3,11-trimethoxy-THPB

(9a) and 2,3,9-trimethoxy-THPB (9b) were obtained from 3,4-

Scheme 2. Synthesis of THPBs 6ae6c (Series 2). Reagents and conditions: (a) Benzyl chloride, K₂CO₃, EtOH, reﬂux, 6 h; (b) Nitromethane, NH₄OAc, AcOH, reﬂux, 4 h; (c) LiAlH₄, THF/

Et₂O, N₂, reﬂux, 2 h; (d) CH₂Cl₂, 3-methoxyphenylacetyl chloride, 5% NaOH, rt, 3 h; (e) POCl₃, CH₃CN, N₂, reﬂux, 5 h; (f) NaBH₄; MeOH, rt, 2 h; (g) 37% HCHO, EtOH, H₂O, reﬂux, 5 h;

(h) EtOHeHCl, reﬂux, 3 h; (i) DMF, CH₂Cl₂, CsF, reﬂux, 3 h.

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

153

trimethoxy-THPB (9b) was produced (8% yield). The phenolic hy-

droxyl groups were deprotected through O-demethylation condi-

tions to obtain the corresponding 2,3,11-trihydroxy-THPB (9c)

and 2,3,9-trihydroxy-THPB (9d) respectively. Finally, the THPBs

with methylendioxy group were prepared as above described

to generate 2,3-methylendioxy-11-hydroxy-THPB (9e) and 2,3-

methylendioxy-9-hydroxy-THPB (9f) respectively.

2.2. Binding afﬁnities for dopamine receptors: structureeactivity

relationship

All the synthesized THPB compounds were assayed in vitro for

their ability to displace the selective ligands of D₁and D₂DR from

their respective speciﬁc binding sites in the striatal membranes. All

the compounds except 6a, were able to displace [³H]-SCH 23390

and [³H]-raclopride from their speciﬁc binding sites at micromolar

(mM) or nanomolar (nM) concentrations. This was not surprising

given the bulky substituents (Bn) at C3 and C4 in compound 6a.

The binding afﬁnities for D₁and D₂DR are summarized in

Table 1 illustrating some general trends of the structureeactivity

relationship.

2.2.1. Effect of the substituents into the A-ring of THPB

a) In general, all the tested THPBs with protected phenolic hy-

droxyls showed a lower afﬁnity for D₁and D₂DR than their

corresponding homologues with free hydroxyl groups (see 3a

vs 3b, 6a vs 6b, 9a vs 9c, 9b vs 9d in Table 1). The higher afﬁnity

for D₁and D₂DR of compounds with free phenolic group than

those without was previously described albeit in several iso-

quinolines [8,12,16,21].

b) In previous studies in which dopaminergic afﬁnity of BTHIQs

was evaluated, the presence of a chlorine atom into the A-ring

(ortho of oxygenated group) resulted in increased afﬁnity and

selectivity towards D₂DR [8,16e18]. This is in contrast with the

current study since the existence of a chlorine atom into the A-

Scheme 3. Synthesis of THPBs 9ae9f (Series 3). Reagents and conditions: (a) CH₂Cl_2,3-

methoxyphenylacetyl chloride, 5% NaOH, rt, 3 h; (b) POCl₃, CH₃CN, N₂, reﬂux, 5 h; and,

NaBH₄, MeOH, rt, 2 h; (c) HCHO, EtOH, H₂O, reﬂux, 5 h; (d) CH₂Cl₂, BBr₃, rt, 2 h; (e)

DMF, CH₂Cl₂, CsF, reﬂux, 3 h.

dimethoxyphenylethylamine (7). In this series, two different com-

pounds were obtained based on the two possible positions of the

Mannich cyclization. When cyclization was in para to the benzylic

methoxy group, the 2,3,11-trimethoxy-THPB (9a) was the major

product generated (77% yield). In contrast, when cyclization

occurred in ortho to the benzylic methoxy group then 2,3,9-

Table 1

Values of afﬁnity (K_i, pK_i) and selectivity (ratio K_iD₁/K_iD₂) determined in binding experiments to D₁and D₂DR of series 1e3.

THPB

Speciﬁc ligand D₁[³H]-SCH 23390

Speciﬁc ligand D₂[³H]-raclopride

K_iD₁/D₂

K_i(m

M)^m

pK_i^m

K_i(m

M)^m

pK_i^m

3a

3b

6a

6b

6c

9a

9b

9c

9d

9e

9f

3-Cl-2,11-diMeO

3-Cl-2,11-diOH

2,3-diBnO-11-MeO

2,3-diOH-11-MeO

2,3-OCH₂O-11-MeO

2,3,11-triMeO

2,3,9-triMeO

2,3,11-triOH

2,3,9-triOH

2,3-OCH₂O-11-OH

2,3-OCH₂O-9-OH

1.813 ꢀ 0.146

0.107 0.004

40.607 ꢀ 4.215

1.108 ꢀ 0.411

2.373 ꢀ 1.208

4.314 ꢀ 0.703

3.927 ꢀ 0.908

2.962 ꢀ 1.042

3.775 ꢀ 0.992

32.999 ꢀ 5.009

0.344 ꢀ 0.027

5.76 ꢀ 0.09

6.97 0.02^d,i

4.39 ꢀ 0.04^j

6.02 ꢀ 0.17^e,h

5.77 ꢀ 0.27^f

5. 38 ꢀ 0.07

5.44 ꢀ 0.11

5.58 ꢀ 0.15

5.46 ꢀ 0.14

4.49 ꢀ 0.07^d

6.47 ꢀ 0.03^f,g

2.046 ꢀ 0.347

0.188 ꢀ 0.021

40.866 ꢀ 1.461

0.078 0.017

1.331 ꢀ 0.536

8.543 ꢀ 4.709

0.393 ꢀ 0.115

0.396 ꢀ 0.028

0.093 0.024

7.506ꢀ1.591

5.70 ꢀ 0.07

0.9

0.6

1.0

14.2

1.8

0.5

10

7.5

40.6

4.4

10.0

6.73 ꢀ 0.05^b,i

4.38 ꢀ 0.02^j

7.13 0.09^b,d,h

5.94 ꢀ 0.17^l

5.29 ꢀ 0.36

6.44 ꢀ 0.12^b,c,k

6.41 ꢀ 0.03^a

7.07 0.14^b,d

5.16 ꢀ 0.09^d

7.55 0.16^a,f

0.035 0.012

ANOVA, post Newmann-Keuls multiple comparison tests.

a

p < 0.05.

p < 0.01, vs D₁-like DR.

p < 0.001 vs 9a.

p < 0.001 vs 9c.

p < 0.05 vs 9c.

p < 0.001 vs 9e.

p < 0.001 vs 9d.

p < 0.001 vs 6a, 6c, 9a.

p < 0.001 vs 3a.

p < 0.001 vs 6b.

p < 0.05 vs 9d.

p < 0.01 vs 9e.

b

c

d

e

f

g

h

i

j

k

l

m

Data were displayed as mean ꢀ SEM for 3e5 experiments.

154

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

ring of the THPBs caused increased afﬁnity for D₁DR but

dramatically reduced the selectivity for D₂DR (see 3b vs 9c in

Table 1).

To conﬁrm the absence of toxicity, we next investigated the

effect on neutrophil apoptosis and survival of the most active

THPBs on D₂DRs (6b, 9d and 9f). For this purpose a cytoﬂuorimetric

method was employed. As depicted in Fig. 6 none of the THPBs at

the concentrations assayed affected neutrophil apoptosis or sur-

vival indicating the lack of human cell toxicity of these compounds

in this in vitro approach.

c) Our results also showed that the presence of a methyl-

enedioxy group in 2,3 position decreased the selectivity for

D₂DR (see 6c vs 6b and 9e vs 9c in Table 1). This effect was

also observed when the substituent into the D-ring was in

position 9. In fact, when the dopaminergic afﬁnities of THPB

9d (2,3,9-triOH) and 9f (2,3-OCH₂O-, 9-OH) were compared,

9d exerted higher selectivity for D₂DR (K_iD₁/D₂¼ 40.6) than

9f (K_iD₁/D₂¼ 10). Moreover, the latter compound (9f) is the

THPB displaying the highest afﬁnity for both DR types

(K_i¼ 344 nM for D₁and K_i¼ 35 nM for D₂) (see Table 1 and

Fig. 2).

2.4. Molecular modelling

A molecular modelling study of three representative com-

pounds of these series was performed to add further support to the

results described in Section 2.2. Special attention was paid to the

effects exerted by the substituents into the D-ring of THPBs as well

as to the increased afﬁnity for the D₂DR detected by the 11-O-

protected derivatives. Therefore, compounds 9d, 9c and 6b were

selected for this comparative analysis due to their structural

differences.

First, MD calculations were carried out simulating the molecular

interactions between compounds 9d, 9c and 6b with the human

D₂DR (Fig. 7). In general, the three compounds displayed their

pharmacophoric portions in a closely related spatial form to that

reported for dopamine [17,18]. Consistent with previous experi-

mental [36] and theoretical [37] data, the simulation indicated the

relevance of the negatively charged aspartate 114 (D114) for ligand

binding. In this context, the highly conserved D114 in trans-mem-

brane helix 3 (TM3) is important for both D₂DR agonists and an-

tagonists binding [36,38], and its terminal carboxyl group may

function as an anchoring point for ligands with protonated amino

groups [39e41]. In the current study, all the compounds simulated

were docked into the receptor with the protonated amino group

close to D114. Although after 5 ns of MD simulations the ligands

slightly moved in a way different from the initial position, the

strong interaction with D114 was maintained for all the complexes

(see Fig. 7), reinforcing the role of the amino acid as an anchoring

point for ligands with protonated amino groups.

2.2.2. Effect of the 9 or 11 substituent into the D-ring of THPB

The present study demonstrated that the afﬁnity and the

selectivity for DR depended on the position of the oxygenated

substituents in positions 9 or 11 and on their protection or depro-

tection. In regard to this:

a) A hydroxyl group in position 9 of the THPB (9f) resulted in a

higher afﬁnity for D₁and D₂DR than its homologue with a

hydroxyl group in position 11 (9e). In this context, the afﬁnity

for D₂DR displayed by 9f was 214 fold higher than 9e (Table 1

and Fig. 3).

b) Surprisingly, either when this 2,3-methylenedioxy-11-OH-THPB

(9e) was compared with the protected 2,3-methylenedioxy-11-

OMe-THPB (6c), or when the 2,3,11-triOH-THPB (9c) was

compared with the protected 2,3-diOH-11-OMe-THPB (6b), an

increased afﬁnity for D₁and D₂DR was observed with the 11-O-

protected derivatives (6c and 6b, see Table 1 and Fig. 4).

2.3. Cytotoxicity studies

After determining the afﬁnity for DRs of the synthesized THPBs,

the potential cytotoxicity of these compounds was determined by

the use of the MTT assay. The concentrations tested were selected

based on their respective Ki value for D₂DR. None of the THPBs

evaluated displayed any cytotoxicity on freshly isolated human

neutrophils (Fig. 5).

Next, the relative energies (DDG values) obtained for the

different complexes were analysed. A 0.00, 12.14 and 2.58 kcal/mol

for complexes 9d/D₂DR, 9c/D₂DR and 6b/D₂DR, respectively were

obtained. These binding energies (BE) were in agreement with the

experimental data (see K_ivalues in Table 1). However, although BE

allowed to differentiate between very good and weak binders

3

HO

O

N

O

HO

2

OH

9

9f

9d

[³H]-SCH 23390

binding

[³H]-SCH 23390

binding

100

50

[³H]-raclopride

binding

0

-10

-9

-8

-7

-6

-5

-4

-10

-9

-8

-7

-6

-5

-4

log [THPB 9d] (M)

log [THPB 9f] (M)

Fig. 2. Displacement curves of [³H]-SCH 23390 (D₁) and [³H] raclopride (D₂) speciﬁc binding by compounds 9d and 9f. Data were displayed as mean ꢀ SEM for 3e5 experiments.

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

155

3

O

N

O

2

R₁

9f: (R₁= OH; R₂= H)

9

9e: (R₁= H; R₂= OH)

11

R₂

9f

9e

9f

9e

D₂

D₁

100

50

0

100

50

0

-10

-9

-8

-7

-6

-5

-4

-10

-9

-8

-7

-6

-5

-4

log [THPB] (M)

Fig. 3. Displacement curves of the speciﬁc binding of D₁and D₂DR ligands by the compounds 9e and 9f. Data were displayed as mean ꢀ SEM for 3e5 experiments.

(0.00 kcal/mol for compound 9d vs 12.14 kcal/mol for compound

9c), those with similar binding afﬁnities did not (0.0 kcal/mol for

compound 9d vs 2.58 kcal/mol for compound 6b). This was not

surprising since MD simulations poorly approximate features that

might be playing determinant roles such as lone pair directionality

afﬁnity relationship was further analysed. The THPB-residue

interaction spectra calculated by the free energy decomposition

suggested that the interaction spectra of compounds 9d, 9c and 6b

with D₂DR was closely related. Nevertheless, some subtle but

signiﬁcant differences were detected reﬂecting differential binding

features (Fig. 8).

in hydrogen bonds, explicit

p/p stacking polarization effects,

hydrogen bonding networks, induced ﬁt, and conformational en-

tropy. Collectively, the information obtained from MD simulations

might explain in part the differential D₂DR afﬁnity displayed by

compounds 9d and 9c.

To acquire more detailed insights into the mechanisms driving

the binding of the THPBs to the D₂DR active site, the structuree

In this regard, it is interesting to note that the only structural

difference between compounds 9d and 9c is the position of the OH

groups in the D ring (see Scheme 3). Despite of it, a signiﬁcant

difference was obtained for their respective calculated BE, indi-

cating that 9d/D₂DR complex was markedly more stable than 9c/

D₂DR. The comparison of both spectra (Fig. 8a and b) revealed that

3

HO

N

HO

2

9c: (R= H)

6b: (R= CH₃)

11

OR

9c

6b

9c

6b

D₁

D₂

100

50

0

50

0

-10

-9

-8

-7

-6

-5

-4

-10

-9

-8

-7

-6

-5

-4

log [THPB] (M)

Fig. 4. Displacement curves of the speciﬁc binding of D₁and D₂DR ligands by the compounds 6b and 9c. Data were displayed as mean ꢀ SEM for 3e5 experiments.

156

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

the OH group of compound 9d at position 9 (Fig. 8a) was forming a

150

100

50

strong hydrogen bond interaction with Y416. This is a particularly

strong stabilizing interaction which is due to the acidic character of

the oxygen atom of the Tyr residue. In contrast, the OH group of 9c/

D₂DR complex (Fig. 8b), was located within a hydrophobic pocket

where only weak hydrogen bonds (O/HeC with the side chains of

the amino acids) can take place (see the weak stabilizing interac-

tion with Y416, Fig. 8b). In addition, although the complex of

compound 9c displayed a moderated interaction with S193, there

was not a stabilizing interaction with S197. This is in sharp contrast

with 9d spectra since two strong interactions with S193 and Ser197

were encountered (Fig. 8a).

The remainder of the amino acids stabilizing these complexes

(D114, V111 V115, C118, C182, I183, I184 W382, H393, F389, F390

and T412) exerted closely related interactions. In light of these

observations, it is tempting to speculate that the different stabi-

lizing interactions of complexes 9d/D₂DR and 9c/D₂DR could

0

l

M

-

M

µM

µM µ

3µ

-

0µM

0.1µ

3µ

3µ 3µ

ntro

Co

10

0.3µ

0.1µ

-

DMSO

3a

3b

10

-

6c

9c

9b

9a

9e

9f - 0.1

6b

9d

6a

MeOH/H2O

Fig. 5. Effect of the synthesized THPBs on viability of human neutrophils. Data are

presented as mean ꢀ SEM of n ¼ 3 independent experiments.

Fig. 6. Percentage of apoptotic (A) and survival cells (B) after incubation with THPBs 6b, 9d and 9f. Early apoptotic cells were quantiﬁed as the percentage of total population of

annexin V^þ, PI^ꢁcells, late apoptotic, and/or necrotic cells as annexin V^þand PI^þ, and viable nonapoptotic cells as annexin V^ꢁand PI^ꢁat 24 h of culture of human neutrophils. The

columns are the means ꢀ SEM of n ¼ 3 independent experiments. Representative ﬂow cytometry panels showing the effects of compounds 6b, 9d and 9f on human neutrophil

apoptosis have been included.

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

157

Fig. 7. Spatial view of compound 9d (green)/D₂DR interaction. Magniﬁcation of the receptor active site at the right. (For interpretation of the references to colour in this ﬁgure

legend, the reader is referred to the web version of this article.)

explain both the different DDG values and the experimental afﬁn-

ities (Table 1).

catecholic ring are illustrated in Fig. 11a. The OH-2 establishes a

hydrogen bond with the carbonyl oxygen of the backbone of S193

(0.0246 au) but not with the side chain oxygen as found for 9d/

D₂DR. It also acts as a proton acceptor with H393 (0.0147 au) and

with a CeH bond of the side chain of S193 (0.0067 au). The OH-3

behaves as a proton acceptor for CeH interactions with F390 and

V115 without proton donor activity with any residue except a very

weak HeH contact with S197 (0.0016 au). In addition, this OH can

establish two OeO interactions with the carbonyl oxygen at S193

and the hydroxyl at S197. Such interactions suggest that this hy-

droxyl group is weakly bond at the binding site compared with 9d/

On the other hand, when the activities of the 2,3,11-triOH-THPB

(9c) and the protected 2,3-diOH-11-OMe-THPB (6b) were

compared, an increased afﬁnity for D₂DR was exerted by the 11-O-

protected derivative (Table 1). The analysis of the two spectra

(Fig. 8b and c) showed that compound 6b can establish two strong

molecular interactions with two serine residues (S193 and S197).

The average structures obtained for the complexes 9d/D₂DR, 9c/

D₂DR_,and 6b/D₂DR from the last ns of simulation are shown in

Fig. 9a, b and c, respectively. The salt bridge between the proton-

ated NeH group of BTHPs and the carboxyl group of D114 as well as

the rest of the interactions previously discussed can be appreciated

in this ﬁgure.

At this stage, the trend predicted for the MD simulations can be

considered as certainly signiﬁcant. However, it should be noted that

we were dealing with relatively weak interactions and therefore

MD simulations might underestimate such interactions. Therefore,

reduced models were constructed to perform more accurate DFT

calculations (B3LYP/6-1G(d)). These calculations allowed to

perform a QTAIM analysis for a further characterization of the most

critical interactions for these ligands.

D₂DR complex. In fact, while the sum of electronic density of all

P

OH-3 interactions in the binding site (

r (rb)) is 0.0247 for com-

pound 9c, the value for compound 9d was 0.0391 au. The OH-11 is

forming CeHeO type interactions with C182 (0.0031 au), I183

(0.0037 au) and F110 (0.0022 au), OeeO type interactions with the

carbonyl oxygen of backbone C182 (0.0044 au) and a weak HeH

interaction with V91 (0.0019 au) (Fig. 11b).

Fig. 12 shows the main interactions of compound 6b at the

binding pocket. The interaction pattern of this compound shares

similarities with those observed for compound 9d. In fact, OH-2

and OH-3 are acting as proton donors with S193 (0.0340 au)

and S197 (0.0308 au) and as proton acceptors with H393

(0.0064 au), I184 (0.0048 au), S193 (0.0114 au) and V115

(0.0065 au) (Fig. 12a). OMe-11 is only forming two very weak

hydrogen bonds with the CeH bonds of the side chain

The main interactions of compound 9d at the binding pocket

and the interactions net obtained are illustrated in Fig. 10. The OH-2

in the catecholic ring is acting as a proton donor with S193

(r(rb) ¼ 0.0207 au) and as a proton acceptor with two amino acids,

H393 (0.0187 au) and S194 (0.0012 au). The OH-3 behaves as a

proton donor with S197 (0.0238 au) and is also involved in three

weak CeH hydrogen bonds with S193, S194 and V115. The OH-9

possesses only one strong hydrogen bond with Y416 (0.0370 au).

It is noteworthy that the hydrogen bonds between the catecholic

hydroxyls (OH-2 and OH-3) with the serine residues (S193 and

S194) are facilitated by the adequate spatial orientation of the

molecule in which the atoms of rings A, B, C and D are making

further interactions with other residues (Fig. 10). These stabilizing

interactions include the strong salt bridge between the ammonium

group of 9d with the carboxyl group of D114, the weaker CeHeS

(

r

(rb) ¼ 0.0015) and the backbone (0.0011au) of I183 (Fig. 12b).

The CH₃group is interacting with C182 (CeHeS interaction

(0.0039 au)), V91 (CeH interaction (0.0085 au)) as well as with F110

(HeH interaction (r(rb) ¼ 0.0184 au). Taken together all these ob-

servations and the sum of the electronic density of all the in-

teractions (0.0150 au), it can be concluded that the binding strength

of OMe-11 to D₂DR is not relevant. In contrast, compound 6b in-

teractions seemed to be stronger when they are established be-

tween the catecholic hydroxyls and the serine residues. As oppose

to the OH-11 of 9c who remained almost ﬁxed with the same

orientation (Fig. 11b), the OMe-11 group in 6b is rotating almost

freely during the simulation for MD calculations. This is a striking

observation which may account for 9c and 6b differential D₂DR

afﬁnities. Finally, the QTAIM analysis seem to indicate that the

increased afﬁnities of compounds 9d and 6b compared with those

of 9c may rely on the stronger interactions established between the

catecholic hydroxyls and the serine residues.

interaction with S118 and the CeH–p interactions with F389 and

V115. These interactions are present with varying intensity in the

three compounds here analyzed.

The main interactions of compound 9c at the binding pocket are

shown in Fig. 11. To facilitate the description of the molecule in-

teractions, this ﬁgure was divided in two and the interactions of the

158

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

Fig. 9. Spatial view of the active D₂DR site for compounds 9d (a), 9c (b) and 6b (c). The

names of the residues involved in the main interactions are written in the ﬁgure.

Fig. 8. Histograms of interaction energies partitioned for D₂DR amino acids when

complexed with compound 9d (a), compound 9c (b) and compound 6b (c). The x-axis

denotes the residue number of D₂DR, and the y-axis shows the interaction energy

between the compound and the speciﬁc residue. Negative and positive values repre-

sent favourable or unfavourable binding, respectively.

In addition, none of the compounds evaluated showed any

cytotoxicity in freshly isolated human neutrophils. Finally, a mo-

lecular modelling study of three representative THPBs was carried

out. The QTAIM analysis allowed a further understanding of the

different experimental afﬁnities obtained for compounds 9d, 9c

and 6b. Since serine residues cluster seemed to be crucial for

agonist binding and receptor activation, it is likely that compounds

9d and 6b displayed such capability.

3. Conclusions

In summary, we have synthesized several THPBs with different

substituents into their A- and D- rings and evaluated the SAR

for their potential dopaminergic afﬁnity. Three series of THPBs

were obtained: 2,11-dihydroxy-3-chloro-THPBs (series 1), 2,3-

dihydroxy-11-methoxy-THPBs (series 2) and 2,3,11-trihydroxy-

THPBs and 2,3,9-trihydroxy-THPBs (series 3). Concerning to their

afﬁnity for DR: ﬁrst, the presence of hydroxyls groups into the A-

ring resulted in increased the DR afﬁnity and, blockade of these

groups blunted these responses; second, a halogen in the A-ring

shifted the selectivity of the compound towards one of the DR

investigated; third, while the presence of an OH at position 9

resulted in a positive effect on DR afﬁnities, its localization at po-

sition 11 was surprisingly detrimental, and the blockade of this

hydroxyl group at position 11 (MeO) reversed their DR afﬁnity.

4. Experimental section

4.1. General instrumentation

EIMS and FAB mass were recorded on a VG Auto Spec Fisons

spectrometer instruments (Fisons, Manchester, United Kingdom).

¹H NMR and ¹³C NMR spectra were recorded with CDCl₃or C₅D₅N

as a solvent on a Bruker AC-300, AC-400 or AC-500. Multiplicities of

¹³C NMR resonances were assigned by DEPT experiments. COSY,

HSQC and HMBC correlations were recorded at 400 MHz and

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

159

Fig. 10. Molecular graph of compound 9d interaction with the binding site. Large spheres represent attractors or nuclear critical points (3, ꢁ3) attributed to the atomic nuclei. The

connecting nuclei lines are bond paths and small spheres on them are bond critical points (3, ꢁ1).

500 MHz (Bruker AC-400^ꢂAC-500). The assignments of all com-

pounds were made by COSY, DEPT, HSQC and HMBC. All the re-

actions were monitored by analytical TLC by silica gel 60 F₂₅₄

used in the following step with no further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃): ), 7.59 (d,

¼ 7.91 (d, J ¼ 13.7 Hz, 1H, H-

J ¼ 2.2 Hz, 1H, H-2), 7.52 (d, J ¼ 13.7 Hz, 1H, H- ), 7.44 (dd, J ¼ 8.6,

2.2 Hz, 1H, H-6), 6.97 (d, J ¼ 8.6 Hz, 1H, H-5), 3.90 (s, 3H, OCH₃-4);

¹³C NMR (100 MHz, CDCl₃)

158.4 (C-4), 138.4 (CH- ), 136.4 (CH- ),

d

b

a

(Merck 5554). Residues were puriﬁed by sílica gel 60 (40e63 mm,

Merck 9385) column chromatography. Solvents and reagents used

were purchased from commercial sources. Quoted yields are of

puriﬁed material. The HCl salts of the synthesized compounds were

prepared from the corresponding base with 5% HCl in MeOH.

d

b

a

130.9 (CH-2), 130.1 (CH-6), 124.2 (C-1), 123.7 (C-3), 112.7 (CH-5),

56.8 (OCH₃-4); MS (EI) m/z (%): 213 (55) [M]^þ, 185 (100).

4.2.1.3. 3,4-Dibenzyloxy-

dehyde (1 g, 3.15 mmol) was subjected to similar conditions to those

above described to obtain the 3,4-dibenzyloxy- -nitrostyrene

(1.70 g, 99%) as yellow needles, which was used in the following step

with no further puriﬁcation. ¹H NMR (500 MHz, CDCl₃):

¼ 7.81 (d,

), 7.38e7.23 (m,10H,

b-nitrostyrene. 3,4-dibenzyloxybenzal-

4.2. Chemistry

b

4.2.1. General procedure for the synthesis of phenyethylamines (1

and 4)

d

4.2.1.1. 3,4-Dibenzyloxybenzaldehyde. A mixtureof 3,4-dihydroxyben-

zaldehyde (1 g, 7.24 mmol), benzyl chloride (3 mL, 24.51 mmol) and

anhydrous K₂CO₃(2.1 g, 14.3 mmol) in absolute EtOH (15 mL) was

reﬂuxed for 6 h. After being stirred, the reaction mixture was

concentrated to dryness, redissolved in 10 mL of CH₂Cl_2,and then 5%

aqueous NaOH (3 ꢃ 10 mL) was added. The combined organic layers

were dried over with anhydrous Na₂SO₄and evaporated to dryness.

The residue obtained was puriﬁed by silica gel column chromatog-

raphy (Hexane/EtOAc, 8:2) to obtain the 3,4-dibenzyloxybenzaldehyde

J ¼ 13.6 Hz,1H, H-

b

), 7.36 (d, J ¼ 13.6 Hz,1H, H-

a

2 ꢃ Ph), 7.01 (dd, J ¼ 8.3, 2.1 Hz,1H, H-6), 6.99 (d, J ¼ 2.1 Hz,1H, H-2),

6.87 (d, J ¼ 8.3 Hz, 1H, H-5), 5.15 (s, 2H, PhCH₂O-3), 5.10 (s, 2H,

PhCH₂O-4); ¹³C NMR (125 MHz, CDCl₃)

d

¼ 152.7 (C-4), 149.1 (C-3),

139.1 (CH-

b

),136.4 (C-1⁰),136.2 (C-1⁰⁰),135.3 (CH-

a

),129ꢁ127 (6C, C-

2⁰-4⁰y C-2⁰⁰-4⁰⁰), 124.7 (CH-6), 123.1 (C-1), 114.3 (CH-2), 114.2 (CH-5),

71.4 (PhCH₂O-4), 70.8 (PhCH₂O-3); ESMS m/z (%): 362 (100)

[M þ H]^þ.

(1.5 g, 65%) as a white solid. ¹H NMR (500 MHz, CDCl₃):

d

¼ 9.83 (s,1H,

4.2.1.4.

3-chloro-4-methoxy-

b

-(3-Chloro-4-methoxyphenyl)ethylamine (1). A solution of

-nitrostyrene (1 g, 4.7 mmol) in anhydrous

CHO), 7.56 (m, 2H, H-2, H-6), 7.48e7.27 (m, 10H, 2 ꢃ Ph), 7.02 (d,

b

J ¼ 13.7 Hz,1H, H-5), 5.22 (s, 2H, PhCH₂O-3), 5.17 (s, 2H, PhCH₂O-4); ¹³

C

THF (14 mL) was added dropwise to a well-stirred suspension of

LiAlH₄(0.7 g, 18.5 mmol) in anhydrous Et₂O (20 mL) under nitrogen

atmosphere, and reﬂuxed for 2 h. Then the reaction mixture was

cooled and the excess reagent was destroyed by a dropwise addi-

tion of H₂O (2 mL) and 15% aqueous NaOH (5 mL). After a partial

evaporation of the ﬁltered portion, the aqueous solution was

extracted with CH₂Cl₂(3 ꢃ 10 mL) and the organic layer was

extracted with 5% aqueous HCl (3 ꢃ 10 mL). The resulting aqueous

acid layer was made basic (5% aqueous NH₄OH to achieve pH z 9)

and extracted with CH₂Cl₂(3 ꢃ 10 mL). The organic layers were

washed with brine (2 ꢃ 10 mL) and H₂O (2 ꢃ 10 mL), dried over

with anhydrous Na₂SO₄and concentrated to dryness to obtain the

NMR (125 MHz, CDCl₃)

d

¼ 191.2 (CHO), 155.7 (C-4), 150.6 (C-3), 136.5

(C-1⁰), 136.3 (C-1⁰⁰), 130.5 (C-1), 129e127 (6C, C-2⁰-4⁰y C-2⁰⁰-4⁰⁰), 124.6

(CH-6), 116.9 (CH-5), 115.7 (CH-2), 71.3 (PhCH₂O-3), 71.1 (PhCH₂O-4);

ESMS m/z (%): 341 [M þ Na]^þ, 313 (100).

4.2.1.2. 3-Chloro-4-methoxy-b-nitrostyrene. A mixture of 3-chloro-

4-methoxy-benzaldehyde (1 g, 5.87 mmol), nitromethane (1 mL,

18.41 mmol) and NH₄OAc (1.2 g, 15.57 mmol) in AcOH (15 mL) was

reﬂuxed for 4 h. After this time, the reaction mixture was cooled to

room temperature, diluted with H₂O (10 mL), and extracted with

CH₂Cl₂(3 ꢃ 10 mL). The combined organic layer were washed with

brine (2 ꢃ 10 mL) and H₂O (2 ꢃ 10 mL), dried over with anhydrous

Na₂SO₄, and evaporated to dryness to obtain the 3-chloro-4-

b

-(3-chloro-4-methoxyphenyl)ethylamine (1) (630 mg, 72%) as a

yellow oil. ¹H NMR (500 MHz, CDCl₃):

¼ 7.33 (d, J ¼ 2.2 Hz, 1H, H-

2), 7.21 (dd, J ¼ 8.5, 2.2 Hz, 1H, H-6), 6.88 (d, J ¼ 8.5 Hz, 1H, H-5),

d

methoxy-b-nitrostyrene (1.1 g, 88%) as yellow needles, which was

160

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

Fig. 11. Molecular graph of compound 9c interaction with the binding site. The in-

teractions for the catecholic hydroxyls are shown in (a) and the interactions of OH-11

are shown in (b).

Fig. 12. Molecular graph for compound 6b interaction at the binding site. The in-

teractions for the catecholic hydroxyls are shown in (a) and the interactions of OMe-11

are shown in (b).

3.89 (s, 3H, OCH₃-4), 2.96 (m, 2H, H-

(125 MHz, CDCl₃):

¼ 153.8 (C-4), 133.3 (C-1), 130.8 (CH-2), 128.4

(CH-6), 122.6 (C-3), 112.5 (CH-5), 56.5 (OCH₃-4), 43.8 (CH₂- ), 39.1

(CH₂-

); MS (EI) m/z (%): 185 (45) [M]^þ.

b), 2.67 (m, 2H, H-a

); ¹³C NMR

d

b

a

4.2.2. General procedure for the synthesis of acetamides (2, 5 and

8) under SchotteneBaumann conditions

Formation of acetamides was carried out under Schottene

Baumann conditions using the appropriate phenylethylamine and

the corresponding acid chloride.

4.2.1.5.

-nitrostyrene (1.70 g, 4.7 mmol) was subjected to similar

conditions to those above described to obtain the -(3,4-

b-(3,4-Dibenzyloxy-phenyl)ethylamine (4). 3,4-dibenzyloxy-

b

dibenzyloxyphenyl)ethylamine (4) (1.44 g, 93%) as a yellow oil. ¹H

NMR (500 MHz, CDCl₃):

d

¼ 7.47 (m, 4H, H-2⁰, H-6⁰y H-2⁰⁰, H-6⁰⁰),

7.38 (m, 6H, H-3⁰, H-5⁰y H-3⁰⁰, H-5⁰⁰), 6.83e6.76 (m, 3H, H-2, H-5, H-

4.2.2.1. N-(3-chloro-4-methoxyphenylethyl)-

acetamide (2). An amount of 0.3 mL of 2-(3-methoxyphenyl)acetyl

chloride (2.55 mmol) was added dropwise at 0 ^ꢂC to a solution of

b-

b

-(3⁰-methoxyphenyl)

6), 5.16 (s, 2H, PhCH₂O-3), 5.15 (s, 2H, PhCH₂O-4), 2.98 (m, 2H, CH₂-

b

), 2.83 (m, 2H, CH₂-

a

); ¹³C NMR (125 MHz, CDCl₃):

d

¼ 149.7 (C-3),

147.0 (C-4), 136.7 (C-1⁰), 136.6 (C-1⁰⁰), 130.8 (C-1), 128.9e127.1 (6C,

C-2⁰, C-4⁰y C-2⁰⁰,C-4⁰⁰), 122.1 (CH-6), 112.8 (CH-2), 112.3 (CH-5),

121.9 (CH-6), 116.0 (CH-2), 112.8 (CH-4), 71.3 (PhCH₂O-4), 71.2

(3-chloro-4-methoxyphenyl)ethylamine (1) (500 mg, 2.69 mmol)

in CH₂Cl₂(20 mL) and 5% aqueous NaOH (4.4 mL) with stirring at

room temperature for 3 h. After the mixture was stirred, 2.5%

aqueous HCl was added and the organic solution was washed with

brine (2 ꢃ 10 mL) and H₂O (2 ꢃ 10 mL), dried over with anhydrous

(PhCH₂O-3), 41.9 (CH₂-b), 39.3 (CH₂-a); ESMS m/z (%): 333 (100)

[M þ H]^þ.

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

161

Na₂SO₄and evaporated to dryness. The residue obtained was pu-

riﬁed by sílica gel column chromatography (Hexane/CH₂Cl₂/EtOAc,

20:70:10) to afford the acetamide (2) (378 mg, 45%) as a white oil.

poured into a mixture of crushed ice. The solid residue was tritu-

rated with 20% aqueous NaOH to obtain a suspension (pH z 8e9)

and then extracted with CH₂Cl₂(3 ꢃ 15 mL). The combined CH₂Cl₂

extracts were dried over Na₂SO₄and the solvent was evaporated in

vacuo obtaining a reddish oil. The residue was dissolved in MeOH

(10 mL), cooled to ꢁ78 ^ꢂC and then treated with NaBH₄(76 mg,

2 mmol). The reaction mixture was stirred for 2 h. Thereafter,

H₂O (15 mL) was added and volatiles were evaporated under

reduced pressure. The aqueous phase was extracted with CH₂Cl₂

(3 ꢃ 15 mL), and the combined organic layers were dried over with

anhydrous Na₂SO₄and evaporated to dryness. The crude product

was puriﬁed by silica gel column chromatography (CH₂Cl₂/MeOH,

95:5) to furnish 6-chloro-7-methoxy-1-(3⁰-methoxybenzyl)-

1,2,3,4-THIQ (3) (155 mg, 54%) as a yellow oil. Then, the 3 hydro-

chloride salt was prepared for use in the next reaction. ¹H NMR

¹H NMR (500 MHz, CDCl₃):

d

¼ 7.15 (t, J ¼ 9.5 Hz, 1H, H-5⁰), 6.98 (d,

J ¼ 2.1 Hz, 1H, H-2), 6.74 (dd, J ¼ 8.4, 2.1 Hz, 1H, H-6), 6.70 (dd,

J ¼ 9.5, 3.3 Hz, 1H, H-4⁰), 6.68 (m, 1H, H-5), 6.65 (m, 1H, H-6⁰), 6.62

(t, J ¼ 3.3 Hz, 1H, H-2⁰), 5.50 (m, 1H, NH), 3.77 (s, 3H, OCH₃-4), 3.68

(s, 3H, OCH₃-3⁰), 3.33 (s, 2H, CH₂eCO), 3.31 (dd, J ¼ 12.9, 6.8 Hz, 2H,

H-

a

), 2.55 (t, J ¼ 6.8 Hz, 2H, H-

b

); ¹³C NMR (125 MHz, CDCl₃):

d

¼ 170.7 (CO), 159.9 (C-3⁰), 153.5 (C-4), 136.1 (C-1⁰), 131.7 (C-1),

130.4 (CH-2), 130.0 (CH-5⁰), 127.9 (CH-6), 122.2 (C-3), 121.6 (CH-6⁰),

114.9 (CH-2⁰), 112.8 (CH-4⁰), 112.1 (CH-5), 56.1 (OCH₃-4), 55.1

(OCH₃-3⁰), 43.8 (CH₂eCO), 40.6 (CH₂-

a), 34.3 (CH₂-b); MS (FAB) m/z

(%): 356 [M þ Na]^þ, 186 (85), 169 (100).

4.2.2.2. N-(3,4-Dibenzyloxyphenylethyl)-

b

-(3⁰-methoxyphenyl)

(500 MHz, CDCl₃) for the free base form:

d

¼ 7.25 (t, J ¼ 7.5 Hz, 1H,

acetamide (5).

1.5 mmol) was subjected to similar conditions to those above

described to obtain the N-(3,4-dibenzyloxyphenylethyl)-

-(3⁰-

b

-(3,4-dibenzyloxyphenyl)ethylamine (500 mg,

H-5⁰), 7.10 (s, 1H, H-5), 6.83 (d, J ¼ 7.5 Hz, 1H, H-6⁰), 6.80 (m, 1H, H-

4⁰), 6.79 (m, 1H, H-2⁰), 6.64 (s, 1H, H-8), 4.18 (dd, J ¼ 9.2, 4.7 Hz, 1H,

H-1), 3.80 (s, 3H, OCH₃-7), 3.77 (s, 3H, OCH₃-3⁰), 3.17e2.92 (m, 4H,

b

methoxyphenyl)acetamide (5). The residue was puriﬁed by silica

gel column chromatography (Hexane/CH₂Cl₂/EtOAc, 20:70:10), to

obtain the acetamide 5 (450 mg, 63%) as a white oil. ¹H NMR

H-3 y CH₂-a

), 2.71 (m, 2H, H-4); ¹³C NMR (125 MHz, CDCl₃):

d

¼ 159.8 (C-3⁰), 152.7 (C-7), 140.2 (C-1⁰), 137.8 (C-8a), 130.5 (CH-5),

129.6 (CH-5⁰), 128.2 (C-4a), 121.7 (CH-6⁰), 120.3 (C-6), 115.1 (CH-2⁰),

(500 MHz, CDCl₃):

d

¼ 7.45e7.30 (m, 10H, Ph-3 y Ph-4), 7.20 (t,

111.9 (CH-4⁰), 110.1 (CH-8), 56.9 (CH-1), 56.1 (OCH₃-7), 55.1 (OCH₃-

J ¼ 8.0 Hz, 1H, H-5⁰), 6.80 (m, 1H, H-5), 6.80 (m, 1H, H-6⁰), 6.78 (m,

1H, H-2⁰), 6.72 (m, 1H, H-2), 6.72 (m, 1H, H-4⁰), 6.52 (dd, J ¼ 8.1,

1.9 Hz, 1H, H-6), 5.43 (m, 1H, NH), 5.13 (s, 2H, PhCH₂O-4), 5.10 (s,

2H, PhCH₂O-3), 3.75 (s, 3H, OCH₃-3⁰), 3.47 (s, 2H, NHCOCH₂), 3.39

3⁰), 42.6 (CH₂-

a), 40.2 (CH₂-3), 28.7 (CH₂-4); MS (FAB) m/z (%): 318

[M þ H]^þ, 196 (100).

4.2.4. General procedure for the synthesis of 1,2,3,4-

tetrahydroisoquinoleines (6 and 9) by BischlereNapieralski

cyclisation

(q, J ¼ 6.7 Hz, 2H, H-

a

), 2.63 (t, J ¼ 6.7 Hz, 2H, H-

b

); ¹³C NMR

(125 MHz, CDCl₃):

d

¼ 170.6 (CO), 159.9 (C-3⁰), 149.0 (C-3), 147.6

(C-4), 137.4 (C-1⁰⁰⁰), 137.2 (C-1⁰⁰), 136.2 (C-1⁰), 132.0 (C-1), 129.9

(CH-5⁰), 128.5e127.3 (10CH, Ph-3 y Ph-4), 121.6 (CH-6), 121.5 (CH-

2⁰), 114.9 (CH-2), 114.9 (CH-4⁰), 112.8 (CH-5), 112.8 (CH-6⁰), 71.4

(PhCH₂O-4), 71.3 (PhCH₂O-3), 55.1 (OCH₃-3⁰), 43.8 (NHCOCH₂),

4.2.4.1. 6,7-Dibenzyloxy-1-(3⁰-methoxybenzyl)-1,2,3,4-THIQ

(6).

The corresponding acetamide 5 (300 mg, 0.645 mmol) was added

in dry acetonitrile (20 mL) to a 100 mL three-neck round-bottom

ﬂask at 0 ^ꢂC under N₂, and treated with POCl₃(0.45 mL, 4.5 mmol).

The mixture was stirred and reﬂuxed under N₂for 5 h and then

cooled to room temperature. Acetonitrile was concentrated under

reduced pressure and the reaction mixture was slowly poured into

a mixture of crushed ice. The solid residue was triturated with 10%

aqueous NaOH to obtain a suspension (pH z 8e9) which was then

extracted with CH₂Cl₂(3 ꢃ 15 mL). The combined CH₂Cl₂extracts

were dried over Na₂SO₄and the solvent was evaporated in vacuo

obtaining a reddish oil. The residue was dissolved in MeOH (10 mL),

cooled to ꢁ78 ^ꢂC and then treated with NaBH₄(76 mg, 2 mmol). The

reaction mixture was stirred for 2 h. Next, H₂O (15 mL) was added

and volatiles were evaporated under reduced pressure. The

aqueous phase was extracted with CH₂Cl₂(3 ꢃ 15 mL), and the

combined organic layers were dried over Na₂SO₄and evaporated to

dryness. The crude product was puriﬁed by silica gel column

chromatography (CH₂Cl₂/MeOH, 95:5) to furnish THIQ (6) (180 mg,

63%) as a yellow oil. Then, the 6 hydrochloride salt was prepared for

use in the next reaction. ¹H NMR (500 MHz, CDCl₃) for the free base

40.6 (CH₂-

a

), 34.8 (CH₂-

b

); MS (FAB) m/z (%): 504 [M þ Na]^þ, 413

(55), 322 (100).

4.2.2.3. N-(3,4-Dimethoxyphenylethyl)-b

-(3⁰-methoxyphenyl)acet-

amide (8). 3,4-dimethoxy-phenylethylamine (500 mg, 2.79 mmol)

was subjected to similar conditions to those above described to

obtain the N-(3,4-dimethoxyphenylethyl)-b

-(3⁰-methoxyphenyl)

acetamide (8). The residue was puriﬁed by silica gel column chro-

matography (Hexane/CH₂Cl₂/EtOAc, 20:70:10), to obtain the acet-

amide 8 (2 g, 91%) as a white oil. ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.21

(dd, J ¼ 8.3, 7.9 Hz, 1H, H-5⁰), 6.80 (dd, J ¼ 8.3, 1.9 Hz, 1H, H-4⁰), 6.73

(d, J ¼ 7.9 Hz, 1H, H-6⁰), 6.72 (m, 1H, H-2⁰), 6.70 (d, J ¼ 8.1 Hz, 1H, H-

2), 6.60 (d, J ¼ 1.9 Hz,1H, H-5), 6.53 (dd, J ¼ 8.1,1.9 Hz,1H, H-6), 5.44

(m, 1H, NH), 3.85 (s, 3H, OCH₃-4), 3.81 (s, 3H, OCH₃-3), 3.76 (s, 3H,

OCH₃-3⁰), 3.49 (s, 2H, NHCOCH₂), 3.43 (dd, J ¼ 12.8, 6.8 Hz, 2H, H-

a),

2.67 (t, J ¼ 6.8 Hz, 2H, H-

b

); ¹³C NMR (125 MHz, CDCl₃):

d

¼ 170.7

(CO), 159.9 (C-3⁰), 148.9 (C-3), 147.6 (C-4), 136.2 (C-1⁰), 131.1 (C-1),

129.9 (CH-5⁰), 121.6 (CH-6⁰), 120.5 (CH-6), 114.9 (CH-2), 112.8 (CH-

2⁰), 111.7 (C-4⁰), 111.2 (CH-5), 55.8 (OCH₃-4), 55.7 (OCH₃-3), 55.1

form:

d

¼ 7.45e7.30 (m, 10H, Ph-4 y Ph-3), 7.26 (m, 1H, H-5⁰), 6.81

(m, 1H, H-6⁰), 6.83 (m, 2H, H-2⁰, H-4⁰), 6.77 (s, 1H, H-5), 6.71 (s, 1H,

(OCH₃-3⁰), 43.9 (COCH₂), 40.7 (CH₂-

a

), 34.9 (CH₂-

b); MS (FAB) m/z

H-8), 5.15 (s, 2H, PhCH₂O-4), 5.11 (s, 2H, PhCH₂O-3), 4.11 (dd, J ¼ 9.5,

(%): 330 [M þ H]^þ, 182 (45), 165 (100).

4.2 Hz, 1H, H-1), 3.81 (s, 3H, OCH₃-3⁰), 3.18 (m, 1H, H-3

a), 3.10 (dd,

J ¼ 13.6, 4.2 Hz, 1H, H-

9.5 Hz, 1H, H-

⁰⁰), 2.73 (m, 1H, H-4

(125 MHz, CDCl₃):

a

⁰), 2.91 (m, 1H, H-3

b

), 2.85 (dd, J ¼ 13.6,

4.2.3. Synthesis of 1,2,3,4-tetrahydroisoquinoleines (3, 6 and 9) by

BischlereNapieralski cyclisation

a

a), 2.69 (m, 1H, H-4b

); ¹³C NMR

d

¼ 159.7 (C-3⁰), 147.6 (C-7), 146.8 (C-6), 140.6 (C-

4.2.3.1. 6-Chloro-7-methoxy-1-(3⁰-methoxybenzyl)-1,2,3,4-THIQ (3).

The acetamide 2 (300 mg, 0.93 mmol) was added in dry toluene

(10 mL) to a 100 mL three-neck round-bottom ﬂask at 0 ^ꢂC under N₂

and treated with P₂O₅(4.2 g, 15.02 mmol), which was added in

portions and followed by the dropwise addition of POCl₃(1.37 mL,

15.02 mmol). The mixture was stirred and reﬂuxed under N₂for 6 h,

and then cooled to room temperature. Toluene was concentrated

under reduced pressure and the reaction mixture was slowly

1⁰), 137.5 (C-1⁰⁰), 137.4 (C-1⁰⁰⁰), 131.4 (C-8a), 129.6 (CH-5⁰), 128.4e

127.3 (10CH, Ph-6 y Ph-7), 127.9 (C-4a), 121.7 (CH-6⁰), 115.5 (CH-4⁰),

115.0 (CH-5), 113.9 (CH-2⁰), 111.8 (CH-8), 71.8 (PhCH₂O-7), 71.3

(PhCH₂O-6), 56.7 (CH-1), 55.1 (OCH₃-3⁰), 42.6 (CH₂-

a), 40.7 (CH₂-3),

34.8 (CH₂-4); MS (FAB) m/z (%): 466 [M þ H]^þ, 344 (100).

4.2.4.2. 6,7-Dimethoxy-1-(3⁰-methoxybenzyl)-1,2,3,4-THIQ

(9).

N-(3,4-dimethoxy-phenyl-ethyl)-b

-(3⁰-methoxyphenyl)acetamide

162

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

(300 mg, 0.91 mmol) was subjected to similar conditions to those

above described to obtain the 6,7-dimethoxy-1-(3⁰-methox-

ybenzyl)-1,2,3,4-THIQ (9). The residue was puriﬁed by silica gel

column chromatography (CH₂Cl₂/MeOH, 95:5), to afford the THIQ

(9) (264 mg, 93%), as a yellow oil. Then, the 9 hydrochloride salt was

prepared for use in the next reaction. ¹H NMR (500 MHz, CDCl₃) for

(PhCH₂O-3), 71.1 (PhCH₂O-2), 59.4 (CH-14), 58.1 (CH₂-8), 55.2

(OCH₃-11), 51.4 (CH₂-6), 36.9 (CH₂-13), 28.9 (CH₂-5); MS (FAB) m/z

(%): 478 [M þ H]^þ, 344 (100). HRMS-FAB [M þ H]^þcalcd for

C₃₂H₃₂NO₃: 478.2377, found: 478.2389.

4.2.5.3. 2,3,11-Trimethoxytetrahydroprotoberberine (9a) and 2,3,9-

trimethoxy-tetrahydroprotoberberine (9b) by Mannich cyclisation.

6,7-dimethoxy-1-(3⁰-methoxybenzyl)-1,2,3,4-THIQ (9) (100 mg,

0.32 mmol) hydrochloride was subjected to similar conditions to

those above described to obtain the 2,3,11-trimethoxy-THPB (9a)

and 2,3,9-trimethoxy-THPB (9b). In this case, both compounds

were obtained as a consequence the two possible cyclization po-

sitions: in para position to the methoxyl group (THPB 9a, major

product) or in ortho position (THPB 9b, minor product).

the free base form:

d

¼ 7.23 (dd, J ¼ 7.6 Hz, 1H, H-5⁰), 6.83 (d,

J ¼ 7.6 Hz 1H, H-6⁰), 6.79 (m, 1H, H-2⁰), 6.77 (m, 1H, H-4⁰), 6.58 (s,

1H, H-5), 6.53 (s, 1H, H-8), 4.22 (dd, J ¼ 8.5, 5.2 Hz, 1H, H-1), 3.84 (s,

3H, OCH₃-6), 3.77 (s, 3H, OCH₃-3⁰), 3.76 (s, 3H, OCH₃-7), 3.18 (dt,

J ¼ 12.0, 5.9 Hz 1H, H-3

(dd, J ¼ 12.7, 8.5 Hz 1H, H-3

¹³C NMR (125 MHz, CDCl₃):

a

), 3.16 (dd, J ¼ 12.7, 5.2 Hz, 1H, H-

a

⁰), 2.99

b

), 2.96 (m, 1H, H-a

⁰⁰), 2.78 (m, 2H, H-4);

d

¼ 159.7 (C-3⁰), 147.6 (C-6), 147.0 (C-7),

140.1 (C-1⁰), 129.6 (CH-5⁰), 129.2 (C-8a), 126.6 (C-4a), 121.7 (CH-6⁰),

115.1 (CH-2⁰), 111.9 (CH-5), 111.7 (CH-4⁰), 109.5 (CH-8), 56.5 (CH-1),

55.8 (OCH₃-3⁰), 55.8 (OCH₃-7), 55.1 (OCH₃-6), 42.5 (CH₂-

a

), 40.3

4.2.5.4. 2,3,11-Trimethoxytetrahydroprotoberberine (9a). was puri-

ﬁed by silica gel column chromatography (Hexane/EtOAc, 50:50, to

obtain 9a as a white oil (76 mg, 77%). ¹H NMR (500 MHz, CDCl₃):

(CH₂-3), 28.7 (CH₂-4); MS (FAB) m/z (%): 314 [M þ H]^þ, 192 (100).

4.2.5. General procedure for the synthesis of

tetrahydroprotoberberines (3a, 6a, 9a and 9b) by Mannich

cyclisation

d

¼ 7.00 (d, J ¼ 8.3 Hz, 1H, H-9), 6.74 (s, 1H, H-1), 6.72 (m, 1H, H-10),

6.70 (m, 1H, H-12), 6.62 (s, 1H, H-4), 3.97 (d, J ¼ 14.5 Hz, 1H, H-8 ),

3.89 (s, 3H, OCH₃-2), 3.86 (s, 3H, OCH₃-3), 3.78 (s, 3H, OCH₃-11),

3.66 (d, J ¼ 14.5 Hz, 1H, H-8 ), 3.58 (dd, J ¼ 11.3, 3.5 Hz, 1H, H-14),

3.29 (dd, J ¼ 16.2, 3.4 Hz, 1H, H-13 ), 3.15 (m, 1H, H-6 ), 3.15 (m, 1H,

H-5 ), 2.65 (m,1H, H-5 ), 2.60

), 2.88 (dd, J ¼ 16.2,11.3 Hz,1H, H-13

(m,1H, H-6

); ¹³C NMR (125 MHz, CDCl₃):

¼ 157.9 (C-11),147.4 (C-

a

4.2.5.1. 2,11-Dimethoxy-3-chlorotetrahydroprotoberberine

(3a).

b

The corresponding THIQ 3 (100 mg, 0.313 mmol) hydrochloride was

added to a MeOH/HCl 37% (20:1) (pH 1e4) solution placed in a

100 mL round-bottom ﬂask and then evaporated to dryness. The

compound was then dissolved in absolute ethanol (2 mL), H₂O

(3 mL) and 37% aqueous formaldehyde (3 mL) and stirred and

reﬂuxed for 5 h. Thereafter, the reaction mixture was concentrated

to dryness. The residue was basiﬁed with a 5% aqueous NaOH and

the aqueous layer was extracted with CH₂Cl₂(3 ꢃ 15 mL).

The combined organic layers were dried over with anhydrous

Na₂SO₄and concentrated to dryness. The crude product was puri-

ﬁed by silica gel column chromatography (Hexane/CH₂Cl₂/EtOAc,

20:70:10) to obtain THPB (3a) (85.4 mg, 0.26 mmol, 86%). ¹H NMR

a

b

d

2), 147.4 (C-3), 135.5 (C-12a), 129.6 (C-14a), 127.1 (CH-9), 126.7 (C-

4a), 126.6 (C-8a), 113.2 (CH-12), 112.3 (CH-10), 111.3 (CH-4), 108.6

(CH-1), 59.5 (CH-14), 58.1 (CH₂-8), 56.0 (OCH₃-2), 55.8 (OCH₃-3),

55.2 (OCH₃-11), 51.4 (CH₂-6), 37.1 (CH₂-13), 29.0 (CH₂-5); MS (FAB)

m/z (%): 326 [M þ H]^þ, 192 (100). HRMS-FAB [M þ H]^þcalcd for

C

₂₀H₂₄NO₃: 326.1751, found: 326.1750.

4.2.5.5. 2,3,9-Trimethoxytetrahydroprotoberberine (9b). was puri-

ﬁed by silica gel column chromatography (Hexane/EtOAc, 50:50), to

obtain 9b as a yellow oil (8 mg, 0.0246 mmol, 8%). ¹H NMR

(500 MHz, CDCl₃):

6.75 (s, 1H, H-1), 6.73 (dd, J ¼ 8.4, 2.5 Hz, 1H, H-10), 6.70 (d,

J ¼ 2.5 Hz, 1H, H-12), 3.98 (d, J ¼ 14.5 Hz, 1H, H-8 ), 3.91 (s, 3H,

OCH₃-2), 3.79 (s, 3H, OCH₃-11), 3.70 (d, J ¼ 14.5 Hz, 1H, H-8 ), 3.64

),

d

¼ 7.14 (s, 1H, H-4), 7.00 (d, J ¼ 8.4 Hz, 1H, H-9),

(500 MHz, CDCl₃):

J ¼ 7.9 Hz, 1H, H-12), 6.75 (s, 1H, H-1), 6.70 (d, J ¼ 7.9 Hz, 1H, H-10),

6.62 (s,1H, H-4), 4.20 (d, J ¼ 15.8 Hz,1H, H-8 ), 3.89 (s, 3H, OCH₃-2),

3.87 (s, 3H, OCH₃-3), 3.83 (s, 3H, OCH₃-9), 3.60 (dd, J ¼ 11.1, 3.1 Hz,

1H, H-14), 3.46 (d, J ¼ 15.8 Hz, 1H, H-8 ), 3.30 (dd, J ¼ 16.1, 3.1 Hz,

1H, H-13 ), 3.20 (m, 1H, H-6 ), 3.15 (m, 1H, H-5

), 2.90 (dd, J ¼ 16.1,

11.1 Hz, 1H, H-13 ), 2.65 (m, 1H, H-5 ), 2.65 (m, 1H, H-6

); ¹³C NMR

(125 MHz, CDCl₃):

¼ 155.8 (C-9), 147.4 (C-2), 147.4 (C-3), 135.8 (C-

d

¼ 7.15 (t, J ¼ 7.9 Hz, 1H, H-11), 6.79 (d,

a

b

a

(dd, J ¼ 11.4, 3.6 Hz, 1H, H-14), 3.29 (dd, J ¼ 16.2, 3.6 Hz, 1H, H-13

a

3.15 (m, 1H, H-6

H-13 ), 2.67 (m, 1H, H-5

CDCl₃):

130.1 (CH-4), 127.7 (C-4a), 127.1 (CH-9), 126.3 (C-8a), 120.5 (C-3),

113.2 (CH-12), 112.4 (CH-10), 109.2 (CH-1), 59.5 (CH-14), 57.8 (CH₂-

8), 56.3 (OCH₃-2), 55.2 (OCH₃-11), 50.9 (CH₂-6), 36.7 (CH₂-13), 28.3

(CH₂-5); MS (FAB) m/z (%): 330 [M þ H]^þ, 196 (100); HRMS-FAB

[M þ H] ^þcalcd for C₁₉H₂₁NO₂Cl: 330.1255, found: 330.1262.

a

), 3.10 (m, 1H, H-5

a

), 2.93 (dd, J ¼ 16.2, 11.4 Hz, 1H,

b

), 2.60 (m, 1H, H-6

b

); ¹³C NMR (125 MHz,

a

d

¼ 158.1 (C-11), 153.2 (C-2), 137.2 (C-14a), 135.1 (C-12a),

b

d

12a), 129.7 (C-14a), 126.8 (C-4a), 126.7 (CH-11), 123.4 (C-8a), 120.8

(CH-12), 111.4 (CH-4), 108.6 (CH-1), 107.1 (CH-10), 58.9 (CH-14),

56.0 (OCH₃-2), 55.8 (OCH₃-3), 55.2 (OCH₃-9), 53.7 (CH₂-8), 51.4

(CH₂-6), 36.9 (CH₂-13), 29.1 (CH₂-5); MS (FAB) m/z (%): 326

[M þ H]^þ, 192 (100); HRMS-FAB [M þ H]^þcalcd for C₂₀H₂₄NO₃:

326.1751, found: 326.1754.

4.2.5.2. 2,3-Dibenzyloxy-11-methoxytetrahydroprotoberberine (6a).

6,7-dibenzyloxy-1-(3⁰-methoxybenzyl)-1,2,3,4-THIQ (6) (100 mg,

0.215 mmol) hydrochloride was subjected to similar conditions to

those above described to obtain the 2,3-dibenzyloxy-11-methoxy-

THPB (6a). The residue was puriﬁed by silica gel column chroma-

tography (CH₂Cl₂/MeOH, 99:1), to obtain THPB 6a (93 mg, 93%) as a

4.2.6. General procedure for the synthesis of THP 3b, 9c and 9d by

O-demethylation

4.2.6.1. 2,11-Dihydroxy-3-chloro-tetrahydroprotoberberine

(3b).

A solution of the appropriate THPB (3a) (50 mg, 0.15 mmol) in dry

CH₂Cl₂(5 mL) was cooled to ꢁ78 ^ꢂC. Then, BBr₃(0.4 mL, 1.02 mmol)

was dropwise added to the stirring solution. After 15 min, the re-

action mixture was left to warm up to room temperature and

stirred for 2 h. The reaction was terminated by the dropwise

addition of MeOH (1 mL) and the mixture was further stirred for

another 30 min. The solvent was concentrated to dryness. The

crude product was puriﬁed by silica gel column chromatography

(CH₂Cl₂:MeOH, 90:10), to obtain THPB (3b) (37 mg, 75%) as a yellow

yellow oil. ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.45e7.30 (m, 10H, Ph-2 y

Ph-3), 6.99 (d, J ¼ 8.4 Hz, 1H, H-9), 6.83 (s, 1H, H-1), 6.75 (m, 1H, H-

10), 6.72 (s,1H, H-4), 6.68 (m,1H, H-12), 5.16 (s, 2H, PhCH₂O-2), 5.12

(s, 2H, PhCH₂O-3), 3.95 (d, J ¼ 14.4 Hz, 1H, H-8

11), 3.63 (d, J ¼ 14.4 Hz, 1H, H-8 ), 3.53 (dd, J ¼ 11.2, 3.5 Hz, 1H, H-

14), 3.17 (dd, J ¼ 16.3, 3.5 Hz,1H, H-13 ), 3.13 (m, 1H, H-6 ), 3.10 (m,

1H, H-5 ), 2.60 (m, 1H, H-5 ),

), 2.82 (dd, J ¼ 16.3, 11.2 Hz, 1H, H-13

2.58 (m, 1H, H-6

); ¹³C NMR (125 MHz, CDCl₃):

¼ 157.9 (C-11),

a), 3.79 (s, 3H, OCH₃-

b

a

b

d

147.7 (C-2), 147.2 (C-3), 137.4 (C-1⁰⁰⁰), 137.2 (C-1⁰⁰), 135.5 (C-12a),

130.6 (C-14a), 128.4e127.3 (8 ꢃ CH-Ph, CH-9), 127.0 (C-4a), 126.7

(C-8a), 114.9 (CH-4), 113.4 (CH-10), 113.2 (CH-1), 112.1 (CH-12), 72.2

oil. ¹H NMR (500 MHz, C₅D₅N)

7.05 (m, 1H, H-10), 7.03 (m, 1H, H-9), 6.96 (m, 1H, H-12), 3.99 (d,

J ¼ 14.3 Hz, 1H, H-8 ), 3.61 (m, 1H, H-8 ), 3.56 (m, 1H, H-14), 3.26

d

¼ 7.22 (s, 1H, H-1), 7.18 (s, 1H, H-4),

a

b

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

163

(dd, J ¼ 16.1, 3.5 Hz, 1H, H-13

), 2.98 (dd, J ¼ 16.1, 11.6 Hz, 1H, H-13

(m, 1H, H-6

); ¹³C NMR (125 MHz, C₅D₅N):

(C-2), 138.1 (CH-14a), 135.9 (C-12a 8a), 130.0 (CH-4), 127.3 (CH-9),

126.7 (C-4a), 125.1 (C-12a), 119.6 (C-3), 115.7 (CH-12), 114.8 (CH-1),

114.4 (CH-10), 59.5 (CH-14), 57.9 (CH₂-8), 51.4 (CH₂-6), 36.9 (CH₂-

13), 28.3 (CH₂-5); MS (FAB) m/z (%): 302 [M þ H]^þ, 182 (100);

HRMS-FAB [M þ H]^þcalcd for C₁₇H₁₇NO₂Cl: 302.0942, found:

302.0946.

a

), 3.12 (m, 1H, H-5

), 2.60 (m, 1H, H-5

¼ 157.1 (C-11), 152.6

a

), 3.06 (m, 1H, H-

1H, H-6

b

), 2.40 (m, 1H, H-5

b

); ¹³C NMR (125 MHz, CDCl₃):

d

¼ 158.3

6

a

b

b), 2.52

(C-11), 143.6 (C-3), 143.5 (C-2), 134.9 (C-12a), 127.9 (C-14a), 127.1

(CH-9), 125.1 (C-4a), 124.9 (C-8a), 114.9 (CH-4), 113.5 (CH-12), 112.5

(CH-10), 111.9 (CH-1), 59.0 (CH-14), 57.4 (CH₂-8), 55.2 (OCH₃-11),

51.1 (CH₂-6), 35.4 (CH₂-13), 27.5 (CH₂-5); MS (FAB) m/z (%): 298

b

d

[M þ H]^þ, 164 (100); HRMS-FAB [M þ H] calcd for C₁₈H₂₀NO₃:

þ

298.1438, found: 298.1439.

4.2.7. General procedure for the synthesis of THP 6c, 9e and 9f by

formation of methylenedioxy group

4.2.6.2. 2,3,11-Trihydroxy-tetrahydroprotoberberine

(9c).

4.2.7.1. 2,3-Methylenedioxy-11-methoxytetrahydroprotoberberine

(6c). A solution of 2,3-dihydroxy-11-methoxy-THPB (6b) (100 mg,

0.34 mmol) in anhydrous DMF (3 mL) was treated with dichloro-

methane (10 mL, 155.6 mmol) and CsF (250 mg, 1.64 mmol). The

mixture was reﬂuxed for 3 h with stirring. After cooling, the reac-

tion mixture was extracted with CH₂Cl₂and the organic layer was

washed with 5% aqueous NaHCO₃and H₂O, dried over with anhy-

drous Na₂SO₄and concentrated in vacuo to dryness. The residue

was puriﬁed by silica gel column chromatography (CH₂Cl₂/MeOH

95:5) to obtain the 2,3-methylendioxy-11-methoxy-THPB (6c)

2,3,11-trimethoxy-THPB (9a) (50 mg, 0.15 mmol) was subjected

to similar conditions to those above described to obtain the

2,3,11-trihydroxy-THPB (9c). The same equivalents of BBr₃were

employed despite the presence of increased methoxyl group

numbers. The residue was puriﬁed by silica gel column chroma-

tography (CH₂Cl₂/MeOH, 90:10), to obtain the THPB 9c (43 mg, 99%)

as a white oil. ¹H NMR (500 MHz, C₅D₅N):

(m, 1H, H-9), 7.00 (s, 3H, OH), 6.99 (m, 1H, H-12), 6.96 (s, 1H, H-4),

6.92 (m, 1H, H-10), 4.26 (d, J ¼ 14.5 Hz, 1H, H-8 ), 3.99 (d,

J ¼ 10.8 Hz, 1H, H-14), 3.91 (d, J ¼ 14.5 Hz, 1H, H-8 ), 3.37 (m, 1H, H-

13 ), 3.33 (m, 1H, H-5 ), 3.32 (m, 1H, H-6 ), 3.14 (m, 1H, H-13 ),

2.89 (m, 1H, H-6 ), 2.71 (m, 1H, H-5

); ¹³C NMR (125 MHz, C₅D₅N):

d

¼ 7.15 (s, 1H, H-1), 7.01

a

b

(90 mg, 86%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃):

d

¼ 6.99 (d,

a

b

J ¼ 8.4 Hz, 1H, H-9), 6.74 (s, 1H, H-1), 6.72 (dd, J ¼ 8.4, 2.5 Hz, 1H, H-

10), 6.68 (d, J ¼ 2.5 Hz, 1H, H-12), 6.59 (s, 1H, H-4), 5.92 (s, 2H,

b

d

¼ 157.6 (C-11), 146.2 (C-2), 145.9 (C-3), 135.1 (C-12a), 127.5 (C-

OCH₂O), 3.95 (d, J ¼ 14.4 Hz, 1H, H-8

a), 3.79 (s, 3H, OCH₃-11), 3.65

14a), 127.5 (CH-9), 124.5 (C-8a), 122.9 (C-4a), 116.1 (CH-4), 115.7

(CH-10), 114.7 (CH-12), 113.5 (CH-1), 59.7 (CH-14), 57.2 (CH-8), 51.4

(CH₂-6), 36.1 (CH₂-13), 27.8 (CH₂-5); MS (FAB) m/z (%): 284

[M þ H]^þ, 164 (100); HRMS-FAB [M þ H]^þcalcd for C₁₇H₁₈NO₃:

284.1281, found: 284.1284.

(d, J ¼ 14.4 Hz, 1H, H-8

(dd, J ¼ 16.3, 3.5 Hz, 1H, H-13

), 2.87 (dd, J ¼ 16.3, 11.2 Hz, 1H, H-13

(m, 1H, H-6

); ¹³C NMR (125 MHz, CDCl₃):

b

), 3.57 (dd, J ¼ 11.2, 3.5 Hz, 1H, H-14), 3.25

), 3.12 (m, 1H, H-6 ), 3.09 (m, 1H, H-

), 2.63 (m, 1H, H-5 ), 2.60

¼ 158.0 (C-11), 146.1

a

5a

b

d

(C-2), 145.9 (C-3), 135.5 (C-12a), 130.8 (C-14a), 127.8 (C-4a), 127.1

(CH-9), 126.6 (C-8a), 113.2 (CH-12), 112.3 (CH-10), 108.4 (CH-4),

105.5 (CH-1), 100.7 (OCH₂O), 59.8 (CH-14), 58.0 (CH₂-8), 55.2

(OCH₃-11), 51.3 (CH₂-6), 37.2 (CH₂-13), 29.5 (CH₂-5); MS (FAB) m/z

(%): 310 [M þ H]^þ, 176 (100); HRMS-FAB [M þ H]^þcalcd for

4.2.6.3. 2,3,9-Trihydroxytetrahydroprotoberberine

(9d).

2,3,9-trimethoxy-THPB (9b) (50 mg, 0.15 mmol) was subjected to

similar conditions to those above described to obtain the 2,3,9-

trihydroxy-THPB (9d). The residue was puriﬁed by silica gel col-

umn chromatography (CH₂Cl₂/MeOH, 90:10), to obtain the THPB 9d

C₁₉H₂₀NO₃: 310.1438, found: 310.1440.

(42 mg, 97%) as a grey oil. ¹H NMR (500 MHz, C₅D₅N):

d

¼ 7.21 (s,

4.2.7.2. 2,3-Methylenedioxy-11-hydroxytetrahydroprotoberberine

(9e). 2,3,11-trihydroxy-THPB (9c) (100 mg, 0.353 mmol) was sub-

jected to similar conditions to those above described to obtain the

2,3-methylenedioxy-11-hydroxy-THPB (9e). The residue was puri-

ﬁed by silica gel column chromatography (CH₂Cl₂/MeOH, 98:2), to

obtain the THPB 9e (23 mg, 23%) as a white oil. ¹H NMR (500 MHz,

1H, H-1), 7.11 (t, J ¼ 7.8 Hz, 1H, H-11), 6.97 (s, 1H, H-4), 6.94 (d,

J ¼ 7.8 Hz, 1H, H-10), 6.71 (d, J ¼ 7.8 Hz, 1H, H-12), 6.00 (s, 3H, OH),

4.71 (d, J ¼ 15.7 Hz, 1H, H-8 ), 3.97 (dd, J ¼ 11.3, 3.4 Hz, 1H, H-14),

a

3.92 (d, J ¼ 15.7 Hz, 1H, H-8

3.38 (m, 1H, H-5 ), 3.36 (m, 1H, H-6

H-13 ), 2.87 (m, 1H, H-6

C₅D₅N):

(CH-11), 124.9 (C-14a), 123.7 (C-8a), 122.9 (C-4a), 119.6 (CH-12),

116.1 (CH-4), 113.6 (CH-1), 112.6 (CH-10), 59.6 (CH-14), 54.0 (CH-8),

51.8 (CH₂-6), 36.4 (CH₂-13), 28.2 (CH₂-5); MS (FAB) m/z (%): 284

[M þ H]^þ, 164 (100); HRMS-FAB [M þ H]^þcalcd for C₁₇H₁₈NO₃:

284.1281, found: 284.1282.

b

), 3.42 (dd, J ¼ 16.4, 3.4 Hz, 1H, H-13

a),

a

), 3.28 (dd, J ¼ 16.4, 11.3 Hz,1H,

b

), 2.68 (m, 1H, H-5

¼ 154.9 (C-9), 146.1 (C-2), 145.9 (C-3), 135.9 (C-12a), 127.5

b

); ¹³C NMR (125 MHz,

C₅D₅N):

1H, H-4), 5.96 (dd, J ¼ 4.1, 1.2 Hz, 2H, OCH₂O), 4.00 (d, J ¼ 14.3 Hz,

1H, H-8 ), 3.57 (d, J ¼ 11.4, 3.8 Hz,1H,

), 3.62 (d, J ¼ 14.3 Hz,1H, H-8

H-14), 3.33 (dd, J ¼ 16.1, 3.8 Hz, 1H, H-13 ), 3.12 (m, 1H, H-5 ), 3.04

(m, 1H, H-6 ), 2.58 (m, 1H, H-

), 2.99 (dd, J ¼ 16.1, 11.4 Hz, 1H, H-13

), 2.54 (m, 1H, H-6

); ¹³C NMR (125 MHz, C₅D₅N):

¼ 157.4 (C-

d

¼ 7.06 (m, 3H, H-9, H-10, H-12), 6.90 (s, 1H, H-1), 6.63 (s,

d

a

b

a

b

5b

b

d

11), 146.7 (C-2), 146.5 (C-3), 136.1 (C-12a), 131.3 (C-14a), 128.1 (C-

4a), 127.5 (CH-9), 125.3 (C-8a), 115.8 (CH-12), 114.6 (CH-10), 108.7

(CH-4),106.5 (CH-1),101.3 (OCH₂O), 60.2 (CH-14), 58.1 (CH₂-8), 51.5

(CH₂-6), 37.3 (CH₂-13), 29.6 (CH₂-5); MS (FAB) m/z (%): 296

[M þ H]^þ, 176 (100); HRMS-FAB [M þ H]^þcalcd for C₁₈H₁₈NO₃:

296.1281, found: 296.1282.

4.2.6.4. Synthesis of THP 6b by O-debenzylation. A solution of 2,3-

dibenzyloxy-11-methoxy-THPB (6a) (200 mg, 0.419 mmol) was

reﬂuxed for 3 h in absolute EtOH (25 mL) and concentrated HCl

(25 mL). The reaction mixture was evaporated to dryness, redis-

solved in CH₂Cl₂(10 mL) and made basic (15% aqueous NH₄OH). The

organic solution was washed with brine (2 ꢃ 5 mL) and H₂O

(2 ꢃ 5 mL), dried with anhydrous Na₂SO₄, and evaporated to dry-

ness. The crude product was puriﬁed by silica gel column chro-

matography (CH₂Cl₂/MeOH 95:5) to obtain the 2,3-dihydroxy-11-

methoxy-THPB (6b) (67 mg, 33%) as a yellow oil. ¹H NMR

4.2.7.3. 2,3-Methylenedioxy-9-hydroxytetrahydroprotoberberine

(9f). 2,3,9-trihydroxy-THPB (9d) (100 mg, 0.353 mmol) was sub-

mitted to the same conditions depicted above to obtain the 2,3-

methylenedioxy-9-hydroxy-THPB (9f). The residue was puriﬁed by

silica gel column chromatography (CH₂Cl₂/MeOH, 98:2), to obtain

the THPB 9f (58 mg, 56%) as a white oil. ¹H NMR (500 MHz, C₅D₅N):

(500 MHz, CDCl₃):

2.4 Hz,1H, H-10), 6.61 (d, J ¼ 2.4 Hz,1H, H-12), 6.51 (s,1H, H-1), 6.20

(s, 3H, H-4, 2 ꢃ OH), 3.93 (d, J ¼ 14.8 Hz, 1H, H-8 ), 3.73 (s, 3H,

OCH₃-11), 3.67 (d, J ¼ 14.8 Hz, 1H, H-8 ), 3.53 (dd, J ¼ 11.2, 3.4 Hz,

1H, H-14), 3.08 (dd, J ¼ 16.7, 3.4 Hz, 1H, H-13 ), 3.03 (m, 1H, H-6 ),

2.90 (m, 1H, H-5 ), 2.57 (m,

), 2.76 (dd, J ¼ 16.7, 11.2 Hz, 1H, H-13

d

¼ 6.95 (d, J ¼ 8.5 Hz, 1H, H-9), 6.70 (dd, J ¼ 8.5,

d

¼ 7.16 (t, J ¼ 7.8 Hz, 1H, H-11), 6.99 (d, J ¼ 7.8 Hz, 1H, H-10), 6.95 (s,

1H, H-1), 6.84 (d, J ¼ 7.8 Hz, 1H, H-12), 6.64 (s, 1H, H-4), 5.97 (dd,

J ¼ 4.9, 1.2 Hz, 1H, OCH₂O), 4.55 (d, J ¼ 15.6 Hz, 1H, H-8 ), 3.70 (d,

J ¼ 15.6 Hz, 1H, H-8 ), 3.57 (dd, J ¼ 11.1, 3.0 Hz, 1H, H-14), 3.38 (dd,

J ¼ 15.9, 3.0 Hz, 1H, H-13 ), 3.12 (m, 1H, H-6 ), 3.06 (m, 1H, H-5 ),

a

b

a

b

a

b

a

164

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

3.01 (dd, J ¼ 15.9,11.1 Hz, 1H, H-13

b

), 2.54 (m, 1H, H-5

¼ 155.0 (C-9), 146.7 (C-2),146.4

b

), 2.48 (m, 1H,

24 h. Then, 100

and incubated at 37 ^ꢂC for another 60 min. The supernatants were

discarded and 100 l of DMSO was added to each well to dissolve

the precipitated formazan. The optical densities at dual wave-

lengths (570 and 630 nm) were determined in a spectrophotometer

(Inﬁnite M200, Tecan, Mannedorf, Switzerland).

ml aliquot of MTT solution was added to each well

H-6b d

); ¹³C NMR (125 MHz, C₅D₅N):

(C-3), 136.8 (C-12a), 131.8 (C-14a), 128.4 (C-4a), 127.1 (CH-11), 123.1

(C-8a), 119.8 (CH-12), 112.5 (CH-10), 108.7 (CH-4), 106.3 (CH-1), 101.2

(OCH₂O), 59.9 (CH-14), 54.8 (CH₂-8), 51.8 (CH₂-6), 37.7 (CH₂-13), 29.9

(CH₂-5); MS (FAB) m/z (%): 296 [M þ H]^þ, 176 (100); HRMS-FAB

[M þ H]^þcalcd for C₁₈H₁₈NO₃: 296.1281, found: 296.1280.

m

4.3.2.2. Cytoﬂuorometric analysis of neutrophil apoptosis and sur-

vival. Freshly isolated neutrophils were resuspended in supple-

4.3. Bioassays

mented RPMI medium at 2 ꢃ 10⁶cells/mL. 25

ml were cultured in a

24-well plate containing 200 ml of supplemented RPMI medium for

4.3.1. Binding experiments

These experiments were performed on striatal membranes. Each

striatum was homogenized in 2 mL ice-cold TriseHCl buffer (50 mM,

pH ¼ 7.4 at 22 ^ꢂC) with a Polytron (4s, maximal scale) and immedi-

ately diluted with Tris buffer. The homogenate was centrifuged either

twice ([³H] SCH 23390 binding experiments) on four times ([³H]

raclopride binding experiments) at 20,000 g for 10 min at 4 ^ꢂC with

resuspension in the same volume of Tris buffer between centrifuga-

tions. For [³H] SCH 23390 binding experiments, the ﬁnal pellet was

resuspended in Tris buffer containing 5 mM MgSO₄, 0.5 mM EDTA

and 0.02% ascorbic acid (Tris-Mg buffer), and the suspension was

brieﬂy sonicated and diluted to a protein concentration of 1 mg/mL. A

24 h in the absence or presence of the most active THPBs on D₂DR

(6b, 9d and 9f). Assessment of apoptosis was performed by ﬂow

cytometry using annexin V- FITC and propidium iodide (PI). The

protocol indicated by the manufacturer (Annexin-Fluos; Roche

Applied Science) was used as outlined previously [45]. Cells

(1 ꢃ 10⁴) were analyzed in a Beckman Coulter Epics XL (Fullerton,

CA) and differentiated as early or viable apoptotic (annexin V^þand

PI^e), late apoptotic and/or necrotic (annexin V^þand PI^þ), and viable

nonapoptotic (annexin V^ꢁand PI^ꢁ) cells.

4.4. Molecular modelling

100

striatal protein) was incubated for 1 h at 25 ^ꢂC with 100

containing [³H] SCH 23390 (0.25 nM ﬁnal concentration) and 800

of Tris-Mg buffer containing the required drugs. Non-speciﬁc binding

m

L aliquot of freshly prepared membrane suspension (100

mg of

mL Tris buffer

4.4.1. Molecular dynamics simulations of complexes

A 3D model of the human D₂DR was used for the MD sim-

ulations. This model is based on the homology model from the

mL

was determined in the presence of 30

m

M SK&F 38393 and repre-

crystallized D₃DR, b2 adrenoceptor and A2a adenosine receptor

as templates [46]. (PMDB: http://mi.caspur.it/PMDB/codes:

PM0077430).

sented around 2e3% of total binding. For [³H] raclopride binding

experiments, the ﬁnal pellet was resuspended in Tris buffer con-

taining 120 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂and 0.1%

ascorbic acid (Tris-ions buffer), and the suspension was treated as

The complex geometries from docking were soaked in boxes of

explicit water using the TIP3P model [47] and subjected to MD

simulation. All MD simulations were performed with the Amber

software package using periodic boundary conditions and cubic

simulation cells. The particle mesh Ewald method (PME) [48] was

described above. A 200

suspension (200

g of striatal protein) was incubated for 1 h at 25 ^ꢂC

with 200

L of Tris buffer containing [³H] raclopride (0.5 nM, ﬁnal

concentration) and 400 L of Tris-ions buffer containing the drug

under investigation. Non-speciﬁc binding was determined in the

presence of 50 M apomorphine and represented around 5e7% of the

mL aliquot of freshly prepared membrane

m

ꢀ

m

applied using a grid spacing of 1.2 A, a spline interpolation order of

ꢀ

4 and a real space direct sum cutoff of 10 A. The SHAKE algorithm

m

was applied allowing for an integration time step of 2 fs. MD sim-

ulations were carried out at 300 K target temperature and extended

to 10 ns overall simulation time. The NPT ensemble was employed

using Berendsen coupling to a baro/thermostat (target pressure

1 atm, relaxation time 0.1 ps). Post MD analysis was carried out

with program PTRAJ.

total binding. In both cases, incubations were stopped by addition of

3 mL of ice-cold buffer (Tris-Mg buffer or Tris-ions buffer, as appro-

priate) followed by rapid ﬁltration through Whatman GF/B ﬁlters.

Tubes were rinsed with 3 mL of ice-cold buffer, and ﬁlters were

washed with 3 ꢃ 3 mL ice-cold buffer. After the ﬁlters had been dried,

radioactivity was counted in 4 mL BCS scintillation liquid at an efﬁ-

ciency of 45%. Filter blanks corresponded to approximately 0.5% of

total binding and were not modiﬁed by drugs.

4.4.2. MM-GBSA free energy decomposition

In order to determine the residues of the D₂DR active site

involved in the interactions, the residues proposed by Andujar et al.

[17] and Soriano-Ursua et al. were ﬁrst identiﬁed. Then MM-GBSA

free energy decomposition using the mm_pbsa program in

AMBER12 was employed to corroborate the amino acids interacting

with the ligands. This calculation can decompose the interaction

energies of each residue considering molecular mechanics and

solvation energies [49,50]. Each liganderesidue pair includes four

energy terms: van der Waals contribution (E_vdw), electrostatic

contribution (E_ele), polar desolvation term (G_GB) and nonpolar

desolvation term (G_SA), which are summarized in the following

4.3.2. In vitro cytotoxicity studies

Cytotoxicity of the THPBs was studied by the use of two different

in vitro approaches (MTT colorimetric assay and ﬂow cytometry

analysis) [42]. Cytotoxicity was evaluated on freshly isolated hu-

man peripheral blood neutrophils. Human neutrophils were ob-

tained from buffy coats of healthy donors by Ficoll-Hypaque

density gradient centrifugation, as previously described [43]. Cul-

tures were maintained at 37 ^ꢂC under 5% CO₂and 95% air atmo-

sphere in RPMI-1640 medium (SigmaeAldrich Chemical, USA).

equation:

D

G_{inhibitorꢁresidue}

¼

D

E_vdW

þ

D

E_ele

þ

D

G_GB

þ

DG_SA

4.3.2.1. MTT assay. The viability of neutrophils was determined

using the previously described MTT (3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide) colorimetric assay [44]. In this

assay, the yellow MTT is reduced to a blue formazan product by the

mitochondria of viable cells. MTT (SigmaeAldrich) was prepared at

For MM-GBSA methodology, snapshots were taken at 10 ps time

intervals from the corresponding last 1000 ps MD trajectories and

the explicit water molecules were removed from the snapshots.

4.4.3. Quantum mechanics calculations and topological study of the

electron charge density distribution

19 amino acids were included in this reduced model based on

the generated data. Three molecular complexes, 9d/D₂DR, 9c/D₂DR

and 6b/D₂DR, obtained for our “reduced model system”, were

1 mg/mL in RPMI and stored at 4 ^ꢂC in the dark. 100

ml of neutro-

phils suspension (2 ꢃ 10⁵cells/mL) was added to each well of a 96-

well microtiter plate followed by 20 ml of the appropriate concen-

tration of the THPBs tested. The mixture was incubated at 37 ^ꢂC for

J. Párraga et al. / European Journal of Medicinal Chemistry 68 (2013) 150e166

165

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selected due to their representative chemical features for the

calculation of the charge density. Single point calculations were

performed with Gaussian 03 and employing a hybrid B3LYP func-

tional and 6-31G(d) as basis set. This type of calculation has been

recently used in studies on the topology of (r) because it ensures a

reasonable compromise between the wave function quality

required to obtain reliable values of the derivatives of (r) and the

computer power available, due to the extension of the system in

study [17,18]. The topological properties of a scalar ﬁeld such as (r)

are summarized in terms of their critical points, i.e., the points r_c

where (r) ¼ 0. Critical points are classiﬁed according to their type (,)

by stating their rank, and signature. The rank is equal to the number

of nonzero eigenvalues of the Hessian matrix of (r) at r_c, while the

signature is the algebraic sum of the signs of the eigenvalues of this

matrix. Critical points of (3, ꢁ1) and (3, þ1) type describe saddle

points, where the (3, ꢁ3) is the maximum and (3, þ3) is the min-

imum in the ﬁeld. Among these critical points, the (3, ꢁ1) or bond

critical points are the most relevant ones since they are found be-

tween any two atoms linked by a chemical bond. The determina-

tion of all the bond critical points and the corresponding bond

paths connecting these point with bonded nuclei, were performed

with the AIMAll software. The molecular graphs were drafted using

the same program. Spatial views shown in Figs. 10 and 11 were

constructed using the UCSF Chimera program as graphic interface.

It should be noted that the THPBs here reported were enantio-

meric, they possessed one chiral centre and they could raise two

isomers (S and R). However, although an enantiomeric resolution

for the biological assays was not carried out, only one isomer of

each compound was evaluated in the calculations. The isomeric

forms selected were ﬁrst chosen on the basis of previously reported

results [8e14] and on the exploratory simulations in which the

spatially preferred form for these compounds was determined

(results not shown). In this context, Sun et al. [23] showed that

when a substitution was performed at the D ring, the S-isomers of

THPBs displayed stronger afﬁnities for the D₂DR than their corre-

sponding R-isomer. In addition, previous experimental ﬁndings

with BTHIQs also supported this hypothesis since S forms were the

preferred conformation of these compounds [13]. Finally, the pre-

liminary and exploratory MD simulations performed for the THPBs

here synthesized revealed that the DDG values obtained for the R-

isomers are at least 10 kcal/mol higher than their respective S-

isomers. Therefore, the spatial conformation adopted by the S

forms seemed to provide the adequate orientation of the molecules

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Acknowledgements

This study was supported by grants SAF2011-23777, Spanish

Ministry of Economy and Competitiveness, RIER RD08/0075/0016,

Carlos III Health Institute, Spanish Ministry of Health and the Eu-

ropean Regional Development Fund (FEDER). R.D.E. and S.A.A. are

staff members of the National Research Council of Argentina

(CONICET-Argentina).

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K. Chen, X. Zhen, H. Liu, Asymmetric total synthesis and identi ﬁ cation of

tetrahydroprotoberberine derivatives as new antipsychotic agents possessing

a dopamine D₁, D₂and serotonin 5-HT_1Amulti-action proﬁ le, Bioorg. Med.

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Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2013.07.036.

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2,3,9- and 2,3,11-Trisubstituted tetrahydroprotoberberines as D2 dopaminergic ligands

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