Synthesis of hexahydrocyclopenta[ ij ]isoquinolines as a new class of dopaminergic agents

Article abstract of DOI:10.1016/j.ejmech.2014.11.009

In this study, we have described the synthesis of the tricyclic 1,2,3,7,8,8a-hexahydrocyclopenta [ij]isoquinoline (HCPIQ). Herein, six differently substituted 5,6-dioxygenated-7-phenyl-HCPIQs have been synthesized using a new methodology via (E)-1-styryl-

Full text of DOI:10.1016/j.ejmech.2014.11.009

European Journal of Medicinal Chemistry 90 (2015) 101e106
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of hexahydrocyclopenta[ij]isoquinolines as a new class of
dopaminergic agents
a
a
b
,
, d, *
Javier Parraga , Abraham Galan , Maria Jesús Sanz c, Nuria Cabedo a
,
ꢀ
ꢀ
,
**
Diego Cortes a
a
ꢀ
Departamento de Farmacología, Facultad de Farmacia, Laboratorio de Farmacoquímica, Universidad de Valencia, 46100, Vicent Andres Estelles s/n,
Burjassot, Valencia, Spain
b Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46013, Valencia, Spain
c Institute of Health Research-INCLIVA, University Clinic Hospital of Valencia, Valencia, Spain
d
ꢀ
Centro de Ecología Química Agrícola-Instituto Agroforestal Mediterraneo, Universidad Politecnica de Valencia (UPV), Campus de Vera s/n, Edificio 6C,
46022, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we have described the synthesis of the tricyclic 1,2,3,7,8,8a-hexahydrocyclopenta [ij]iso-
quinoline (HCPIQ). Herein, six differently substituted 5,6-dioxygenated-7-phenyl-HCPIQs have been
synthesized using a new methodology via (E)-1-styryl-THIQ by FriedeleCrafts cyclization with Eaton's
reagent. Results showed that HCPIQs (3, 3aee) displayed a moderate affinity for D1 dopamine receptors
(DR) in the micromolar range, furthermore the catecholic HCPIQs 3a (NH), 3c (NCH3) and 3e (NCH2CH]
CH2) exhibited outstanding affinity and high selectivity towards D2 DR. Indeed, 3a, 3c and 3e showed Ki
values of 29 nM, 13 nM and 18 nM, respectively, and HCPIQs 3a (NH) and 3c (NCH3) displayed a
remarkable selectivity (Ki D1/D2 ratio ~ 1000e2500). In addition, none of the catecholic compounds
showed any cytotoxicity in freshly isolated human neutrophils. Although further studies are needed,
these compounds and particularly catecholic HCPIQs, show high potential in the treatment of Parkinson's
disease, psychosis or depression.
Received 18 September 2014
Received in revised form
29 October 2014
Accepted 5 November 2014
Available online 5 November 2014
Keywords:
Tetrahydroisoquinolines
Hexahydrocyclopenta[ij]isoquinolines
BischlereNapieralski cyclodehydration
FriedeleCrafts cyclization
Dopamine receptors
© 2014 Elsevier Masson SAS. All rights reserved.
Binding assays
1. Introduction
and synthetic THIQs, including 1-benzy-IQ, bisbenzyl-
isoquinolines, aporphines, tetrahydroprotoberberines and phen-
Tetrahydroisoquinoline (THIQ) alkaloids are biosynthetically
dopamine-derived metabolites which are present in many vegetal
species belonging to different families such as Papaveraceae, Ber-
beridaceae, Ranunculaceae and Aristolochiaceae [1e3]. This class of
alkaloids is relevant due to their diverse pharmacological activities
anthrenes [7e20]. We previously reported the synthesis of 1-butyl-
7-chloro-6-hydroxy-THIQ. At that time, it displayed the highest
affinity towards D2-like DR (Ki value of 66 nM) and the highest
selectivity (49-fold D2 vs D1) by in vitro binding experiments [9].
Afterwards, its evaluation in behavioural assays (spontaneous ac-
tivity and forced swimming test) in mice, showed an increased
that include
b
-adrenergic [4], inhibitor of
a-glucosidase enzyme [5],
as well as NMDA (N-methyl
D-aspartate) [6], serotoninergic [7] and
locomotor activity in a large dose range (0.04e25 mg/kg).
D1 and D2 dopaminergic receptor (DR) activities [7e20]. From a
therapeutical point of view, drugs acting at D2-like DR are more
important than those interacting with D1-like DR. In this context,
the D2-like DR antagonists are included in the therapy of schizo-
phrenia (antipsychotics), and the agonists are used in the treatment
of Parkinson's disease [21e25]. In the last two decades, our
research group has been studying dopaminergic activity of natural
Furthermore, this lead compound produced reduction in immo-
bility time in the forced swimming test at a dose of 0.01 mg/kg that
did not modify locomotor activity. Haloperidol (0.03 mg/kg), a D2
DR preferred antagonist, blocked the antidepressant-like effect of
this compound [9].
In the present study, we have considered the synthesis of a se-
ries of tricyclic alkaloids with 1,2,3,7,8,8a-hexahydrocyclopenta [ij]
isoquinoline (HCPIQ) skeleton. The HCPIQ system constitutes the
nucleus of the naturally occurring proaporphines (pronuciferin
type) [26,27], and besides, this system is structurally related to the
azafluoranthene alkaloids (Fig. 1) [28]. Although HCPIQs constitute
a small group into the IQ alkaloids, several synthesis have been
* Corresponding author. Centro de Ecología Química Agrícola-Instituto Agro-
ꢀ
forestal Mediterraneo, Universidad Politecnica de Valencia (UPV), Campus de Vera
s/n, Edificio 6C, Valencia 46022, Spain.
** Corresponding author.
E-mail addresses: ncabedo@ceqa.upv.es (N. Cabedo), dcortes@uv.es (D. Cortes).
http://dx.doi.org/10.1016/j.ejmech.2014.11.009
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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J. Parraga et al. / European Journal of Medicinal Chemistry 90 (2015) 101e106
102
is in pseudoaxial disposition (J8a,8 ¼ 11.8 Hz). In addition, the H-7
a
at 4.59 ppm must be pseudoaxial since its coupling constant value
is J7,8 ¼ 8.3 Hz, which was corroborated by NOE experiment be-
a
tween H-8a and H-7. Therefore, the phenyl group (pseudoequato-
rial) is cis to H-8a (pseudoaxial) (Fig. 2).
2.2. Dopaminergic receptors activity
In general, the HCPIQs with protected phenolic groups displayed
a lower affinity for D1 and principally D2 DR than their corresponding
homologues with free hydroxyl groups (see in Table 1, 3b vs 3c, and
3d vs 3e). Indeed, the higher affinity for D1 and D2 DR of catecholic
THIQs in comparison with those with blocked hydroxyl groups was
previously described in several isoquinolines [7,9,12,16,19]. All syn-
thesized HCPIQs (3, 3aee) were able to displace [3H]SCH 23390
(selective D1 DR ligand) and [3H] raclopride (selective D2 DR ligand)
from its binding sites in micromolar and nanomolar ranges,
respectively. Furthermore and surprisingly the catecholic HCPIQs 3a
(NH), 3c (NCH3) and 3e (NCH2CH]CH2) exhibited outstanding af-
finity and high selectivity towards D2 DR. Therefore, 3a, 3c and 3e
showed Ki values of 29, 13 and 18 nM, respectively. In addition,
HCPIQs 3a (NH) and 3c (NCH3) displayed a remarkable selectivity
with a Ki D1/D2 ratio of 2465 and 1010, respectively. For instance, the
huge selectivity of 3a and 3c is graphically represented in Fig. 3.
Regarding the N-substituent, we observed that in O-methylated
HCPIQs the affinity for DR did not change when a substituent like
methyl or allyl was located on the N atom (see in Table 1, 3 vs 3b and
3d), however in catecholic HCPIQs an increase of the affinity towards
D1 DR was observed (see in Table 1, 3a vs 3c and 3e). Moreover, the
potential cytotoxicity of these catecholic HCPIQs was determined by
the use of the MTT assay on freshly isolated human neutrophils [19].
The concentrations tested were selected based on their respective Ki
value for D2 DR. Results showed that none of the evaluated HCPIQs
displayed any cytotoxicity.
Fig. 1. THIQs skeletons: hexahydrocyclopenta [ij]isoquinoline (HCPIQ), proaporphine
and azafluoranthene.
reported [29e34] given their potential and useful biological prop-
erties, including the inhibition of phenylethanolamine N-methyl-
transferase (PNMT) [29] and poly(ADP-ribose) polymerases (PARPs)
[30] enzymes, as well as their antiprotozoal [35] activities.
Herein, we have synthesized six differently substituted 5,6-
dioxygenated-7-phenyl-HCPIQs in order to evaluate the ability of
this HCPIQ skeleton to bind to D1 and D2-like DR from rat striatal
membranes (Scheme 1). Substituents have been chosen on the
basis of previous reports with natural and synthetic IQ alkaloids,
which included computational analysis [11e20]. Therefore, we
have taken into account the relevance of the di-oxygenated IQ
nucleus, the nature of the N-substituents and the presence of a
hydrophobic part in the molecule to reach the dopaminergic ac-
tivity. These 7-phenyl-HCPIQs have been obtained using a new
methodology via (E)-1-styryl-THIQ intermediate by acid-catalysed
cyclization with Eaton's reagent [36]. Their structures were deter-
mined by NMR spectroscopy and HRESIMS spectrometry.
2. Results and discussion
2.1. Chemistry
The synthesis starts from the available 2-(3,4-dimethoxyphenyl)
ethylamine that reacts with cinnamoyl chloride under Schot-
3. Conclusions
teneBaumann
conditions
to
generate
the
N-(3,4-
We have synthesized a new molecular skeleton HCPIQ via (E)-1-
styryl-THIQ by intramolecular FriedeleCrafts cyclization using
Eaton's reagent. The six differently substituted 5,6-dioxygenated-7-
phenyl-HCPIQ displayed affinity towards both D1 and D2 DR re-
ceptors. Moreover, the three catecholic HCPIQs 3a (NH), 3c (NCH3)
and 3e (NCH2CH]CH2) showed D2 DR affinity in the nanomolar
range. Indeed, they exhibited higher affinity (2.3e5-fold) and
selectivity (Ki D1/D2 ratios ~1000e2500) for D2 DR than the 1-
butyl-7-chloro-6-hydroxy-THIQ, which is one of the most active
antidepressant THIQ synthesized by our group [9]. The major goal
of this study is the discovery of a new skeleton HCPIQs with both
high affinity and selectivity towards D2 DR in in vitro assays.
Although further studies are needed, these compounds and
particularly catecholic HCPIQs, which did not show cytotoxicity,
present high potential application in the treatment of Parkinson's
disease, psychosis or depression.
dimethoxyphenethyl)cinnamamide (1) in 91% yield [9,16]. Next,
the cinnamamide 1 was treated with POCl3 in dry acetonitrile to
perform the BischlereNapieralski cyclodehydration [11e13]. The
corresponding 1-styryl-dihydroisoquinoline was obtained and the
imine function reduced by using NaBH4 reagent to give the (E)-6,7-
dimethoxy-1-styryl-1,2,3,4-THIQ (2) in 81% yield. Compound 2 was
subjected to an intramolecular cyclization under mild FriedeleCrafts
conditions using Eaton's reagent [36] (1:10 w/w P2O5 in CH3SO3H) to
generate
the
5,6-dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahy-
drocyclopenta[ij] isoquinoline (3), containing the cyclopentane ring,
in 68% yield. Once synthesized the HCPIQ 3, we have introduced
different substituents on the nitrogen atom like methyl or allyl
groups. Therefore, we have obtained the corresponding N-methyl-
HCPIQ 3b (82%) using formaldehyde and formic acid in methanol
followed by reduction with NaBH4, besides the corresponding N-
allyl- HCPIQ 3d (89%) using allyl chloride and K2CO3 in acetonitrile.
Finally, these HCPIQs 3 (NH), 3b (NCH3) and 3d (NCH2CH]CH2) were
subjected to O-demethylation by addition of 4 equivalents of BBr3 for
2 h at room temperature [9] to obtain the catecholic HCPIQs 3a (88%),
3c (94%), and 3e (90%), respectively (Scheme 1).
4. Experimental section
4.1. General instrumentation
A single isomer has been produced during cyclization step given
that the 1H and 13C NMR spectrum of HCPIQ 3 showed only one set
of proton or carbon resonances. The relative stereochemistry of H-
8a and Ph was assigned by coupling constants values of H-8a, H-8
and H-7. The vicinal coupling constant values of H-8a at 4.24 ppm
High resolution (HRESIMS) data were recorded on a Waters
Xevo quadrupole time-of-flight (Q-TOF) spectrometer (Waters
Corp., Milford, MA, USA) coupled to an Acquity UPLC system (Wa-
ters Corp., Milford, MA, USA) via an electrospray ionization (ESI)
interface operating in positive mode and using a Waters Acquity
(dd, J ¼ 11.8 and 6.4 Hz) imply that H-8
b
at 2.50 ppm is located in
at 2.35 ppm
BEH C18 column (50 ꢀ 2.1 mm i.d., 1.7
m
m); 1H NMR spectra were
pseudoequatorial disposition (J8a,8 ¼ 6.4 Hz) and H-8
a
recorded with CDCl3 or Methanol-d4 as a solvent on a Bruker AC-
b

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J. Parraga et al. / European Journal of Medicinal Chemistry 90 (2015) 101e106
103
Scheme 1. Synthesis of HCPIQs (3, 3aee). Reagents and Conditions: (a) cinnamoyl chloride, NaOH 5%, CH2Cl2, rt, 3 h; (b) POCl3, CH3CN, N2, reflux, 5 h; (c) NaBH4; MeOH, rt, 2 h; (d)
Eaton's reagent (P2O5eCH3SO3H, 1:10 w/w), 45 ꢁC, 15 h; (e) BBr3, CH2Cl2, rt, 2 h; (f) HCHO, HCO2H, MeOH, reflux, 1 h, followed by NaBH4, reflux, 1 h; (g) K2CO3, allyl chloride, CH3CN,
reflux, 10 h.
500. The assignments were made by COSY, DEPT, HSQC and HMBC.
All the reactions were monitored by analytical TLC by silica gel 60
F254 (Merck 5554). Residues were purified by silica gel 60
CH2-2
), 2.72 (m,1H, CH2-3
(dd, J ¼ 11.8, 8.3 Hz, 1H, CH2-8
(296.1651, calc for C19H22NO2).
a
), 3.14 (dd, J ¼ 12.6, 10.5 Hz, 1H, CH2-2
b), 2.81 (m, 1H, CH2-
3a
b
), 2.50 (dd, J ¼ 11.8, 6.4 Hz,1H, CH2-8
a), 2.35
b
); HRESIMS m/z 296.1637 [M þ H]þ
(40e63 mm, Merck 9385) column chromatography. Solvents and
reagents used were purchased from commercial sources. Quoted
yields are of purified material.
4.3. 7-Phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]isoquinoline-
5,6-diol (3a)
4.2. FriedeleCrafts cyclization of (E)-6,7-dimethoxy-1-styryl-
1,2,3,4-tetrahydroisoquinoline (2) to obtain 5,6-dimethoxy-7-
phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]isoquinoline (3)
Brown oil (88% yield). 1H NMR (500 MHz, CD3OD):
d
¼ 7.25 (t,
J ¼ 7.7 Hz, 2H, CH-30 and CH-50), 7.19 (d, J ¼ 7.7 Hz, 2H, CH-20 and
An amount of 500 mg of (E)-6,7-dimethoxy-1-styryl-1,2,3,4-
tetrahydroisoquinoline (2) (1.69 mmol) was dissolved in 5 ml of
Eaton's reagent (P2O5eCH3SO3H, 1:10 w/w) at room temperature
and the solution was stirred for 15 h at 45 ꢁC. After, 5% aqueous
NaOH was added and the mixture was then extracted with CH2Cl2
(3 ꢀ 15 mL). The combined CH2Cl2 extracts were dried over Na2SO4
and the solvent evaporated in a vacuum to obtain a reddish oil. The
residue obtained was purified by silica gel column chromatography
(CH2Cl2/MeOH, 95:5) to obtain HCPIQ 3 as a brown oil (68% yield).
1H NMR (500 MHz, CDCl3):
d
¼ 7.27 (m, 2H, CH-30 and CH-50), 7.19
(m, 2H, CH-20 and CH-60), 7.18 (m,1H, CH-40), 6.64 (s,1H, CH-4), 4.59
(d, J ¼ 8.3 Hz, 1H, CH-7), 4.24 (dd, J ¼ 11.8, 6.4 Hz, 1H, CH-8a), 3.84
(s, 3H, OCH3-5), 3.59 (s, 3H, OCH3-6), 3.49 (dd, J ¼ 12.6, 6.7 Hz, 1H,
Fig. 2. Relative stereochemistry of HCPIQ 3.

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J. Parraga et al. / European Journal of Medicinal Chemistry 90 (2015) 101e106
104
Table 1
Values of affinity (Ki, pKi) and selectivity (ratio Ki D1/Ki D2) determined in binding experiments to D1 and D2 DR.
Cmpd
[3H]-SCH 23390a
Ki (
M)b
[3H]-raclopridea
Ki (
M)b
Ki ratio
m
pKib
m
pKib
D1/D2
3
33.373 6.891
71.493 16.281
23.780 4.549
13.136 2.452
18.620 4.822
6.873 1.391
4.499 0.104
4.174 0.117
4.640 0.084
4.895 0.076h
4.758 0.109
5.179 0.085h
10.977 1.933
0.029 0.009
4.188 1.348
0.013 0.002
3.514 0.654
0.018 0.007
4.972 0.071
7.593 0.184f
5.422 0.137g
7.890 0.083i
5.472 0.092g
7.800 0.160j
3.0c
2465.3e
5.7d
3a
3b
3c
3d
3e
1010.5e
5.29d
381.8e
a
Specific radioligands are [3H]-SCH 23390 and [3H]-raclopride for D1 and D2 dopamine receptors, respectively.
b
c
d
e
f
Data were displayed as mean SEM of n ¼ 3 independent experiments. ANOVA, post NewmanneKeuls multiple comparison tests.
p < 0.05 vs D1-like dopaminergic receptor.
p < 0.01 vs D1-like dopaminergic receptor.
p < 0.001 vs D1-like dopaminergic receptor.
p < 0.001 vs 3.
g
h
i
p < 0.05 vs 3.
p < 0.001 vs 3a.
p < 0.001 vs 3b.
p < 0.001 vs 3d.
j
3a
3c
D1
D2
D1
D2
100
50
0
100
50
0
-10
-8
-6
-4
-10
-8
-6
-4
log [HCPIQ] (M)
log [HCPIQ] (M)
Fig. 3. Displacement curves of [3H]-SCH 23390 (D1) and [3H] raclopride (D2) specific binding by compounds 3a vs 3c. Data were displayed as mean SEM for 3 experiments.
CH-60), 7.17 (m, 1H, CH-40), 6.70 (s, 1H, CH-4), 4.66 (dd, J ¼ 9.0,
6.6 Hz, 1H, CH-8a), 4.63 (d, J ¼ 8.1 Hz, 1H, CH-7), 3.78 (dd, J ¼ 13.1,
2.64 (dd, J ¼ 11.8, 6.4 Hz, 1H, CH2-3 ), 2.41 (dd, J ¼ 11.8, 5.9 Hz, 1H,
b
CH2-2
b
), 2.22 (m, 1H, CH2-8
b
); HRESIMS m/z 308.1641 [M þ H]þ
6.8 Hz, 1H, CH2-2
a
), 3.51 (ddd, J ¼ 13.1, 11.4, 6.8 Hz, 1H, CH2-2
b),
(308.1651, calc for C20H22NO2).
3.05 (dd, J ¼ 11.4, 6.8 Hz, 1H, CH2-3 ), 2.97 (dd, J ¼ 13.1, 6.8 Hz, 1H,
a
CH2-3
6.6 Hz, 1H, CH2-8
calc for C17H18NO2).
b
), 2.69 (dd, J ¼ 12.1, 8.1 Hz, 1H, CH2-8 ), 2.62 (d, J ¼ 12.1,
a
4.6. 5,6-Dimethoxy-1-methyl-7-phenyl-1,2,3,7,8,8a-
hexahydrocyclopenta-[ij]-isoquinoline (3b)
b
); HRESIMS m/z 268.1331 [M þ H]þ (268.1338,
White oil. (82% yield). HRESIMS m/z 310.1797 [M þ H]þ
4.4. 1-Methyl-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]
(310.1807, calc for C20H24NO2).
isoquinoline-5,6-diol (3c)
4.7. 1-Allyl-5,6-dimethoxy-7-phenyl-1,2,3,7,8,8a-
hexahydrocyclopenta[ij]-isoquinoline (3d)
Green oil (94% yield). 1H NMR (500 MHz, CD3OD):
d
¼ 7.26 (t,
J ¼ 7.5 Hz, 2H, CH-30 and CH-50), 7.16 (m, 2H, CH-20 and CH-60), 7.14
(m, 1H, CH-40), 6.48 (s, 1H, CH-4), 4.42 (d, J ¼ 8.4 Hz, 1H, CH-7), 3.38
Brown oil (89% yield). HRESIMS m/z 336.1956 [M þ H]þ
(m, 1H, CH-8a), 3.07 (m, 1H, CH2-2
a
), 2.72 (m, 1H, CH2-3
a
), 2.63 (m,
(336.1964, calc for C22H26NO2).
1H, CH2-3
NCH3), 2.14 (m, 1H, CH2-8
(282.1494, calc for C18H20NO2).
b), 2.49 (m, 1H, CH2-2b), 2.36 (m, 1H, CH2-8a), 2.20 (s, 3H,
b
); HRESIMS m/z 282.1495 [M þ H]þ
4.8. 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 ꢁC) with a Polytron (4s, maximal scale) and
immediately diluted with Tris buffer. The homogenate was centri-
fuged either twice ([3H] SCH 23390 binding experiments) on four
times ([3H] raclopride binding experiments) at 20000 g for 10 min
at 4 ꢁC with resuspension in the same volume of Tris buffer be-
tween centrifugations. 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
4.5. 1-Allyl-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]
isoquinoline-5,6-diol (3e)
Brown oil (90% yield). 1H NMR (500 MHz, CD3OD):
d
¼ 7.24 (t,
J ¼ 7.2 Hz, 2H, CH-30 and CH-50), 7.18 (d, J ¼ 7.2 Hz, 2H, CH-20 and
CH-60), 7.14 (m, 1H, CH-40), 6.52 (s, 1H, CH-4), 5.87 (dddd, J ¼ 17.3,
10.2, 8.0, 4.8 Hz, 1H, CH-200), 5.18 (d, J ¼ 17.3 Hz, 1H, CH2-300
a), 5.09
(d, J ¼ 10.2 Hz, 1H, CH2-300
), 4.49 (d, J ¼ 8.5 Hz, 1H, CH-7), 3.45 (dd,
b
J ¼ 10.4, 6.0 Hz, 1H, CH-8a), 3.39 (dd, J ¼ 13.8, 4.8 Hz, 1H, CH2-100
a),
3.16 (dd, J ¼ 11.8, 6.4 Hz,1H, CH2-2
a
), 2.72 (m,1H, CH2-3
a
), 2.68 (dd,
),
J ¼ 13.8, 8.2 Hz, 1H, CH2-100
b
), 2.65 (dd, J ¼ 16.4, 6.0 Hz, 1H, CH2-8
a
concentration of 1 mg/mL. A 100 mL aliquot of freshly prepared

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J. Parraga et al. / European Journal of Medicinal Chemistry 90 (2015) 101e106
105
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buffer containing [3H] raclopride (0.5 nM, final concentration) and
400 L of Tris-ions buffer containing the drug under investigation.
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apomorphine and represented around 5e7% of the total binding. In
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4.9. MTT assay
The viability of neutrophils was determined using the previ-
ously
described
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) colorimetric assay. In this assay,
the yellow MTT is reduced to a blue formazan product by the
mitochondria of viable cells. MTT (SigmaeAldrich) was prepared at
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1 mg/mL in RPMI and stored at 4 ꢁC in the dark. 100
ml of neutro-
phils suspension (2 ꢀ 105 cells/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 ꢁC for
24 h. Then, 100 l aliquot of MTT solution was added to each well
and incubated at 37 ꢁC for another 60 min. The supernatants were
discarded and 100 l of DMSO was added to each well to dissolve
m
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m
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the precipitated formazan. The optical densities at dual wave-
lengths (570 and 630 nm) were determined in a spectrophotometer
(Infinite M200, Tecan, Mannedorf, Switzerland).
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This work was supported by the Spanish Ministry of Education,
Culture and Sport, SAF2011-23777, Spanish Ministry of Economy
and Competitiveness, the European Regional Development Fund
(FEDER) and research grants from Generalitat Valenciana (GVA-
COMP2014-006). A.G. (FPU12/00744) is thankful to the Spanish
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program.
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