New serotonin 5-HT1A receptor agonists endowed with antinociceptive activity in vivo

Article abstract of DOI:10.1021/jm400766k

We report the synthesis of new compounds 4-35 based on two different openings (A and B) of the chromane ring present in the previously identified 5-HT_1A receptor (5-HT_1AR) ligand 3. The synthesized compounds were assessed for binding affinity, selectivity, and functional activity at the 5-HT_1AR. Selected candidates resulting from B opening were also evaluated for their potential antinociceptive effect in vivo and pharmacokinetic properties in vitro. Analogue 19 [2-(4-{[2-(2-ethoxyphenoxy) ethyl]amino}butyl)tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione] has been characterized as a high-affinity and potent 5-HT_1AR agonist (K _i = 2.3 nM; EC₅₀ = 19 nM). Pharmacokinetic studies indicated that compound 19 displays a good metabolic stability in human liver microsomes (t_1/2 ～ 3 h and CL_int = 3.5 mL/min/kg, at 5 μM), and a low level of protein binding (25%, at 5 μM). Interestingly, 19 (3 mg/kg, ip, and 30 mg/kg, po) caused significant attenuation of formalin-induced behavior in early and late phases of the mouse intradermal formalin test of pain, and this in vivo effect was reversed by the selective 5-HT_1AR antagonist WAY-100635. Thus, the new 5-HT_1AR agonist identified in this work, 19, exhibits oral analgesic activity, and the results herein represent a step toward identifying new therapeutics for the control of pain.

Full text of DOI:10.1021/jm400766k

Article
pubs.acs.org/jmc
New Serotonin 5‑HT1A Receptor Agonists Endowed with
Antinociceptive Activity in Vivo
Margarita Valhondo,†,∥ Isabel Marco,†,⊥ Mar Martín-Fontecha,† Henar Vazquez-Villa,† Jose A. Ramos,‡
́
́
Reinhard Berkels,§ Thomas Lauterbach,§ Bellinda Benhamu,*,† and María L. Lopez-Rodríguez*,†
́
́
†Departamento de Química Organ
́
ica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
‡Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense de Madrid, E-28040 Madrid,
Spain
§UCB Pharma GmbH, Alfred-Nobel-Strasse 10, 40789 Monheim, Germany
S
* Supporting Information
ABSTRACT: We report the synthesis of new compounds 4−35
based on two different openings (A and B) of the chromane ring
present in the previously identified 5-HT1A receptor (5-HT1AR)
ligand 3. The synthesized compounds were assessed for binding
affinity, selectivity, and functional activity at the 5-HT1AR. Selected
candidates resulting from B opening were also evaluated for their
potential antinociceptive effect in vivo and pharmacokinetic properties in vitro. Analogue 19 [2-(4-{[2-(2-ethoxyphenoxy)-
ethyl]amino}butyl)tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione] has been characterized as a high-affinity and potent
5-HT1AR agonist (Ki = 2.3 nM; EC50 = 19 nM). Pharmacokinetic studies indicated that compound 19 displays a good metabolic
stability in human liver microsomes (t1/2 ∼ 3 h and CLint = 3.5 mL/min/kg, at 5 μM), and a low level of protein binding (25%, at
5 μM). Interestingly, 19 (3 mg/kg, ip, and 30 mg/kg, po) caused significant attenuation of formalin-induced behavior in early
and late phases of the mouse intradermal formalin test of pain, and this in vivo effect was reversed by the selective 5-HT1AR
antagonist WAY-100635. Thus, the new 5-HT1AR agonist identified in this work, 19, exhibits oral analgesic activity, and the
results herein represent a step toward identifying new therapeutics for the control of pain.
produces analgesia as a “first-order” effect and hyperalgesia as a
“second-order” effect. The chronic treatment with opiods
attenuates the first-order analgesia and strengthens the second-
order hyperalgesia. According to this concept, it has been
proposed that 5-HT1AR activation has two effects that are
opposite from those produced by opioids: pain as a first-order
effect but also hypoalgesia as a second-order effect; with
chronicity, this second-order hypoalgesia counteracts the first-
order pain and remarkably develops into increasingly powerful
analgesia.26 This suggests that whereas repeated morphine
administration leads to a loss of analgesic activity (tolerance) and
even to hyperalgesia, repeated administration of a 5-HT1AR
agonist could produce the opposite effect, i.e., a progressive loss
of acute hyperalgesia and an increase in analgesia.27 These
considerations stimulated pharmaceutical drug discovery that
aimed to identify 5-HT1AR agonists with high selectivity. In
particular, the potent and selective 5-HT1AR agonist F-13640
[befiradol, 1 (Chart 1)] demonstrated analgesic activity in animal
models of acute and chronic pain comparable to those of large
doses of opioid painkillers, but with fewer and less prominent
side effects, as well as little or no development of tolerance with
repeated use.27,28 These studies represented a proof of concept
that the 5-HT1AR is involved in the regulation of nociceptive
INTRODUCTION
■
Serotonin (5-hydroxytryptamine, 5-HT) mediates a plethora of
physiological effects through at least 14 receptor subtypes, all but
one belonging to the G-protein-coupled receptor (GPCR) or
seven-transmembrane-spanning (7TM) receptor family. Defined
on the basis of molecular, pharmacological, and functional criteria,
5-HT receptors have been classified into seven discrete subfamilies
(5-HT1 to 5-HT7),1 and their study for almost three decades has
provided researchers with many targets for drug action. The
5-HT1A receptor (5-HT1AR) was the first 5-HT subtype to be fully
sequenced,2 and it has also been the most extensively studied
among serotonin receptors. Indeed, a large number of selective
or multitarget 5-HT1AR ligands have been developed so far with
the aim of identifying new drugs, and this receptor still represents
an attractive target for drug discovery.3−7 In particular, 5-HT1AR
agonists and partial agonists have long shown to be clinically
effective in the treatment of anxiety, depression, and psychosis.8−15
More recently, the neuroprotective properties observed for some
5-HT1AR agonists have suggested their utility for the treatment of
ischemic stroke,5,16−22 and these agents have also been proposed
as levodopa adjuvants in the alleviation of dyskinesia in
Parkinson’s disease.23,24 Moreover, one of the most attractive
therapeutic potentials that has recently emerged for 5-HT1AR
agonists is that they may rival the opioids in pain relief therapy.25
It is known that in nociceptive systems any input causes two
effects that are opposite in sign. Activation of opioid receptors
Received: May 23, 2013
© XXXX American Chemical Society
A
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pharmacokinetic properties in vitro. In particular, compound 19
was characterized as a high-affinity and potent 5-HT1AR agonist
that exhibited oral analgesic activity in the mouse intradermal
formalin test and a good pharmacokinetic profile.39
Chart 1. Structures of 5-HT1AR Agonists Endowed with
Antinociceptive Activity
RESULTS AND DISCUSSION
■
Synthesis. Target compounds 4−7 based on A opening
of the chromane ring in ligand 3 (Chart 2) were obtained by
nucleophilic substitution of 2-(4-bromobutyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (36) with the appropri-
ate commercial ω-(o-methoxyphenyl)alkylamines or amine
37 in acetonitrile, which was prepared starting from 3-(2-
methoxyphenyl)propanoic acid (Scheme 1). Final compounds
8−35 resulting from B opening of the chromane ring in ligand
3 (Chart 2) were synthesized starting from halogenated
intermediate 36, by reaction with appropriate 2-aryloxyethyl-
amines 38, or by alkylation of 2-(4-aminobutyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (39) with correspond-
ing 2-aryloxyethyl bromides 40, as described in Scheme 2.
Starting compound 39 was obtained by N-alkylation of
hydantoin 41 with 4-chlorobutanonitrile in the presence of
sodium hydride, followed by catalytic hydrogenation of inter-
mediate 42. Williamson reaction of appropriate phenols with
1,2-dibromoethane or 2-chloroacetamide in the presence of
potassium carbonate and catalytic potassium iodide in refluxing
2-butanone afforded 2-aryloxyethyl bromides 40a−t or inter-
mediate 2-aryloxyacetamides 43a−g. Subsequent reduction of
43a−g with the borane dimethyl sulfide complex in diglyme
yielded 2-aryloxyethylamines 38a−g. Intermediate amine
38h was prepared by Mitsunobu reaction starting from
8-hydroxyquinoline and N-(2-hydroxyethyl)phthalimide, followed
by phthalimide cleavage.
transmission. Therefore, 5-HT1AR activation was proposed as a
new molecular approach to the treatment of acute and chronic,
nociceptive and neuropathic pain states. Indeed, the concept has
been extended to other 5-HT1AR agonists such as Lu AA21004
[vortioxetine, 2 (Chart 1)] that has shown analgesic activity in
the formalin test.29,30 Given the complexity of the mechanisms
involved in pain modulation, it is not surprising that the
application of single analgesic agents is not always effective in
diverse painful conditions such as chronic pain syndromes. Thus,
it is becoming clear that the combination of different mecha-
nisms, which improves efficacy with reduced toxicity, is necessary
for the reliable pharmacotherapy of pain. At present, there is
evidence that the 5-HT1AR may be used as a target in the search
for new pharmacological approaches in the augmentation of
analgesia. The ongoing clinical trials with compound 131 have
certainly reinforced the interest of 5-HT1AR activation for the
treatment of pain conditions. In this context, our goal is the
development of a 5-HT1AR agonist as an antinociceptive agent.
Over the past several years, our efforts in the search for new
and selective 5-HT1AR ligands have led to the discovery of several
arylpiperazine-based 5-HT1AR agonists endowed with anxiolytic
properties.3,32−37 More recently, we have developed new non-
piperazine derivatives as second-generation 5-HT1AR agonists
exhibiting a neuroprotective effect against ischemic cell
damage.22,38 In these series, the modification of the different
structural moieties of the molecule led to highest-affinity ligand 3
(Chart 2), which was characterized as a potent 5-HT1AR agonist
Binding Affinities. New synthesized compounds 4−35 were
converted to hydrochloride salts, and their affinities were
evaluated at the 5-HT1AR by radioligand binding assays (see the
Supporting Information for details). High-affinity 5-HT1AR ligands
(Ki < 50 nM) were also assayed for selectivity over serotonin
5-HT2A, 5-HT3, 5-HT4, and 5-HT7 receptors.
Tables 1 and 2 show the calculated inhibition constants (Ki)40
for target compounds 4−35 obtained from A and B openings of
the chromane ring in ligand 3 (Chart 2). Clearly, A opening of
chromane ring leads to a marked decrease or loss of affinity for
the 5-HT1AR, affording inactive or poorly active derivatives
[Ki(4−7) ≥ 74 nM vs Ki(3) = 1.23 nM] (Table 1). On the other
hand, compound 9 that results from B opening of chromane ring
in compound 3 maintained a high affinity for the receptor [Ki(9) =
16.8 nM] (Table 1). Therefore, we decided to study the influence
of several substituents at different positions of the aromatic ring
in compounds 8 and 10−35. From data in Table 1, in general
ortho and meta positions are more favorable for 5-HT1AR affinity
than the para position, regardless of the nature of the substituent.
Thus, all para-substituted derivatives are inactive, including those
with substituents that provide high 5-HT1AR affinity [Ki(9) =
16.8 nM and Ki(10) = 30 nM vs Ki(11) = 742 nM; Ki(16) =
4.8 nM and Ki(17) = 34 nM vs Ki(18) > 1000 nM]. Also, the
presence of a substituent in the ortho position is in general more
favorable than that in the meta position (see, for example,
compounds 9 vs 10, 13 vs 14, and 16 vs 17). Electron-with-
drawing substituents seem to be detrimental for affinity even in
the preferred ortho position [Ki(24) > 1000 nM and Ki(27) =
510 nM]. Accordingly, compounds bearing ortho-alkoxy groups
displayed the highest 5-HT1AR affinities [Ki(16) = 4.8 nM, and
Ki(19) = 2.3 nM]. With regard to selectivity, in general, the new
Chart 2. Designed Compounds Based on A (4−7) or B
(8−35) Opening of the Chromane Ring Present in the
Previously Identified High-Affinity 5-HT1AR Ligand 3
(Ki = 1.23 nM; EC50 = 16.3 nM). In this work, we aimed to
generate structural variants of ligand 3, focusing our attention on
mimicking the chroman-2-ylmethyl group. In the new
compounds, two different openings (A and B) of the chromane
ring were considered (Chart 2). The synthesized compounds
have been assessed for binding affinity, selectivity, and functional
activity at the 5-HT1AR. Selected candidates have also been
evaluated for their potential antinociceptive effect in vivo and
B
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a
Scheme 1. Synthesis of Target Compounds 4−7
a
Reagents and conditions: (a) LiAlH4, Et2O, rt, 20 h, 48%; (b) SOCl2, toluene, pyridine, rt, 24 h, 77%; (c) KCN, KI, DMSO, rt, 3 h, 70%; (d)
LiAlH4, THF, rt, 3.5 h, 56%; (e) CH3CN, 60 °C, 15 h, 30−52%.
a
Scheme 2. Synthesis of Target Compounds 8−35
a
Reagents and conditions: (a) CH3CN, 60 °C, 15 h, 21−69%; (b) NaH, DMF, 110 °C, 4 h, 85%; (c) H2, PtO2, EtOH, rt, 15 h, 35%; (d)
Br(CH2)2Br, K2CO3, KI, 2-butanone, Δ, 48 h, 30−67%; (e) ClCH2CONH2, K2CO3, KI, 2-butanone, Δ, 48 h, 53−98%; (f) BH3·S(CH3)2, diglyme,
90 °C, 16 h, 61−88%; (g) N-(2-hydroxyethyl)phthalimide, Ph3P, DEAD, THF, Δ, 15 h, 50%; (h) N2H4·H2O, CH3COOH, EtOH, Δ, 3 h, 33%.
synthesized compounds were inactive or poorly active at other
5-HT receptor subtypes (Table 2). In particular, unsubstituted
compound 8 showed the best selectivity profile for the 5-HT1AR.
The new identified high-affinity 5-HT1AR ligands [Ki ≤ 25 nM
(Table 2)] were considered for functional characterization at the
5-HT1AR (Table 3), in parallel with in vitro pharmacokinetic
studies (Table 4).
Functional Activity at the 5-HT1AR. The functional role of
the new ligands at the 5-HT1AR was characterized both in vitro
and in vivo. The in vitro agonist action was studied by measuring
the inhibition of forskolin-stimulated cyclic adenosine mono-
phosphate (cAMP) formation in HeLa cells transfected with the
human 5-HT1AR (h5-HT1AR). The in vivo test of 5-HT1AR
stimulation consisted of the hypothermic response in mice
(sensitive to the selective 5-HT1AR antagonist WAY-100635).
Assayed compounds 8, 16, 19, and 32 behaved as agonists in the
cell line transfected with the h5-HT1AR, with EC50 values for
adenylyl cyclase inhibition in the range of 19−76 nM (Table 3).
Notably, the most potent alkoxyphenyl derivatives, 16 and 19
(EC50 values of 23 and 19 nM, respectively), also exhibited the
lowest minimal effective doses in the hypothermia assay (0.625
and 0.875 mg/kg, respectively).
equilibrated at 37 °C with NADPH and MgCl2 at concentrations
of 1 and 5 μM; RLM and HLM preparations were added to
initiate the reaction, and aliquots were withdrawn and quenched
at different times (see the Experimental Section for details). The
percent of parent compound remaining in the incubation mixture
was determined by high-performance liquid chromatography
and mass spectrometry (HPLC−MS) and plotted as a function
of time. The half-life time (t1/2) was determined from the slopes
of the peak areas over time, which was then used to calculate the
intrinsic clearance (CLint).41 From the results listed in Table 4,
microsomal metabolism was found to be more favorable for
HLMs than for RLMs. The data indicated that all assayed
compounds exhibited high rates of metabolic degradation in rats,
with t1/2 values of 8−13 min and CLint values of >100 mL/min/kg
(Table 4). However, good rates were observed in human
microsomes with t1/2 values of 2−4 h and CLint values of
<5 mL/min/kg. Interestingly, the most potent compounds, 16
and 19 (Ki = 4.8 and 2.3 nM and EC50 = 23 and 19 nM,
respectively), were also the most metabolically stable com-
pounds, with good half-life and clearance values (t1/2 = 3−4 h;
CLint = 2.6 and 3.5 mL/min/kg, respectively). In addition, the
stability rate of compound 19 in mouse liver microsomes was
2-fold higher than that determined in rats [t1/2 values of 29.5 and
13.1 min in mice and rats, respectively (Table 4)].
In Vitro Pharmacokinetics. High-affinity 5-HT1AR ligands
were also assessed for metabolic stability in vitro using rat and
human liver microsomes (RLMs and HLMs, respectively), as a
measure of first-pass metabolism. Test compounds were
An in vitro fluorescence-based inhibition assay was conducted
with cytochrome P450 2D6 (CYP2D6), one of the most impor-
C
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Table 1. 5-HT1AR Affinities of New Synthesized Compounds 4−35
a
b
Values are means of two to four experiments performed in triplicate. 8-OH-DPAT was used as a reference compound (Ki = 1.02 0.08 nM).
tant enzymes involved in drug metabolism, using human
recombinant microsomal CYP2D6 enzyme, AMMC {3-[2-
(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcou-
marin} as a substrate, and quinidine as a control inhibitor. Table 4
shows the remaining activities of CYP2D6 after a 30 min
incubation with assayed compounds at a concentration of
10 μM.
Selected compounds were also tested for interaction with
human serum albumina (HSA), at a concentration of 5 μM, and
the binding data are listed in Table 4. A correlation between
D
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Table 2. Selectivity of High-Affinity 5-HT1AR Ligands
a
b
Values are means of two to four experiments performed in triplicate. 8-OH-DPAT was used as a reference compound (Ki = 1.02 0.08 nM).
c
d
e
Ketanserin was used as a reference compound (Ki = 0.47 0.04 nM). Ondansetron was used as a reference compound (Ki = 0.77 0.01 nM). RS
f
39604 was used as a reference compound (Ki = 3.9 0.2 nM). 5-CT was used as a reference compound (Ki = 1.8 0.6 nM).
lipophilicity and protein binding can be observed, leading
compounds 9 and 32 to exhibit the highest values of both
clogP and HSA binding (clogP values of 2.33 and 2.48 and
HSA binding of 55 and 60%, respectively). It is noteworthy
that compounds 16 and 19, showing the lowest HSA bind-
ing (25%), are the most potent agonists in vivo [minimal
effective doses of 0.625 and 0.875 mg/kg, respectively
(see Table 3)].
Altogether, these pharmacokinetic studies indicate that the
new 5-HT1AR agonists identified in this work display a promising
pharmacokinetic profile in vitro.
Antinociceptive Activity in an in Vivo Model. According
to the in vitro results (Tables 3 and 4), compounds 8, 16, 19, and
32 endowed with high affinity, agonist potency for the 5-HT1AR,
and/or good pharmacokinetic properties were assessed for their
potential analgesic activity in vivo. The new compounds were
E
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Table 3. Functional Activities of High-Affinity 5-HT1AR
Ligands
hypothermia (minimal
effective dose, mg/kg)
a
b
compd
Ki SEM (nM)
EC50 (nM)
8
25.4 0.8
16.8 0.7
4.8 0.1
2.3 0.1
2.4 0.6
76
2.5
10
c
9
1195 (PA)
23
16
19
32
0.625
0.875
10
19
45
a
b
c
Values are means of two to four experiments performed in triplicate.
8-OH-DPAT was used as a reference compound (EC50 = 8 nM).
PA, partial agonist.
evaluated in the mouse formalin model of pain, which is an
effective test for screening compounds for nociception.42
Formalin injection induces a biphasic stereotypical nocifensive
behavior. Nociceptive responses are divided into an early, short-
lasting first phase (0−5 min), caused by formalin stimulus (phase
I or early acute phase), followed by a quiescent period and then a
second, prolonged phase (15−30 min) of persistent inflamma-
tory pain (phase II or late chronic phase). Test substances and
vehicle [0.2% hydroxypropyl methylcellulose (HPMC), 0.9%
NaCl] were each administered by intraperitoneal injection (ip)
or oral gavage (po) 15 or 30 min before subplantar injection of
formalin (0.02 mL, 2% solution, ip), respectively. Hind paw
licking time was recorded at 5 min intervals for 35 min following
formalin injection. Statistical analysis using one-way analysis of
variance (ANOVA) followed by Dunnett’s test was applied for
comparison between the test compound-treated and vehicle
control groups (significance was considered at the p < 0.05 level).
In these in vivo experiments, morphine, a substance widely used
as a drug with pain-attenuating effect in humans, and gabapentin,
a drug commonly used to relieve neuropathic pain, were also
assayed for comparative purposes. As shown in Figure 1, at a dose
of 3 mg/kg, the new 5-HT1AR agonists reduced licking time in
the second phase (15−30 min) of the nocifensive response to
formalin in mice. The most efficacious compound, 19, exhibited
an antinociceptive effect also against the first phase (0−5 min) of
formalin-induced nocifensive behavior (Figure 2A), when
administered ip at a 30-fold smaller dose than gabapentin.
Importantly, the selective 5-HT1AR antagonist WAY-100635
completely blocked the analgesic activity of 19, because no
significant decrease in the duration of licks was observed after
pretreatment with WAY-100635 (3 mg/kg, ip) 45 min before
injection of formalin (Figure 2B). These results support the idea
that the in vivo effect exhibited by compound 19 is mediated by
the serotonin 5-HT1AR. The specificity of agonist 19 was further
confirmed by the assessment of binding affinity toward other
nociception-related GPCRs.43 Indeed, compound 19 was found
to be inactive in α2-adrenergic, cannabinoid CB1 and CB2, GABAB,
muscarinic M2 and M4, δ2- and μ-opioid, and somatostatin
(SST) receptors (see Table S1 of the Supporting Information).
Figure 1. Effects of new 5-HT1AR agonists on formalin-induced licking
response in the late phase (15−30 min) of nociception. Vehicle (0.2%
HPMC, 0.9% NaCl), test compounds (3 mg/kg), morphine (8 mg/kg),
or gabapentin (100 mg/kg) was administered ip 15 min before
subplantar injection of formalin (0.02 mL, 2% solution, ip). Licking time
of the control group was 100%.
Moreover, orally administered 19 at a dose of 30 mg/kg was
associated with a significant antinociceptive effect in both early
and late phases (Figure 2C), and this analgesic activity was
comparable to that of gabapentin (100 mg/kg, ip). No significant
symptoms of toxicity were observed after the administration of
tested compounds.
CONCLUSIONS
■
Herein we report the synthesis of new compounds 4−35, based
on two different openings (A and B) of the chromane ring
present in the previously identified ligand 3 (Chart 2). The new
analogues were assessed for binding affinity at the 5-HT1AR and
selectivity over other serotonin receptors (Tables 1 and 2). The
newly characterized 5-HT1AR agonists were also evaluated for
their potential analgesic activity in vivo and pharmacokinetic
properties in vitro. Compounds 8, 16, 19, and 32 were identified
as high-affinity and potent 5-HT1AR agonists (Table 3) and
afforded an ip antinociceptive effect against the second phase of
the mouse intradermal formalin test (Figure 1). The antino-
ciceptive effect of the most efficacious compound 19 (3 mg/kg, ip)
was reversed by the selective 5-HT1AR antagonist WAY-100635
(Figure 2), and its oral activity (30 mg/kg, po) was comparable
to that of gabapentin, a drug commonly used to relieve neuro-
pathic pain. Pharmacokinetic studies indicated that compound
19 displays a good metabolic stability in HLMs (t1/2 ∼ 3 h and
CLint = 3.5 mL/min/kg at 5 μM) and a low level of protein
binding (25% at 5 μM). Thus, the new 5-HT1AR agonist
identified in this work, 19 (n = 2, X = O, and Ar = 2-ethoxyphenyl),
has been characterized as a high-affinity and potent 5-HT1AR
agonist (Ki = 2.3 nM; EC50 = 19 nM) endowed with oral
Table 4. In Vitro Pharmacokinetic Properties of New 5-HT1AR Agonists
RLM t1/2
(min)
CLint (mL/min/mg of
protein)
HLM t1/2
(min)
CLint (mL/min/mg of
protein)
CYP2D6 activity
remaining (%)
human serum albumin (HSA)
binding (%)
a
compd
clogP
8
10.0
10.7
9.7
140.3
131.2
144.7
107.1
171.1
151
137
237
179
123
4.1
4.6
2.6
3.5
5.1
24
13
48
18
6
38
55
25
25
60
1.31
2.33
1.04
1.57
2.48
9
16
19
32
b
13.1
8.1
a
b
Values were calculated using ChemBioDraw Ultra 11.0. Stability was also determined in mouse liver microsomes: t1/2 = 29.5 min.
F
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Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker
Avance 500 (1H, 500 MHz; 13C, 125 MHz), Bruker Avance 300-AM
(1H, 300 MHz; 13C, 75 MHz), or Bruker 200-AC (1H, 200 MHz; 13C,
50 MHz) spectrometer at the Universidad Complutense de Madrid’s
NMR facilities. Chemical shifts (δ) are expressed in parts per million
relative to internal tetramethylsilane; coupling constants (J) are in hertz.
The following abbreviations are used to describe peak patterns when
appropriate: s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet),
m (multiplet), and br (broad). Two-dimensional NMR experiments
(HMQC and HMBC) of representative compounds were conducted to
assign protons and carbons of the new structures. Elemental analyses
(C, H, N) were obtained on a LECO CHNS-932 apparatus at the
Universidad Complutense de Madrid’s analysis services and were within
0.5% of the theoretical values, confirming a purity of at least 95% for all
tested compounds. Analytical thin-layer chromatography (TLC) was
conducted on Merck silica gel plates (Kieselgel 60 F-254) with detection
by UV light (254 nm), a ninhydrin solution, or a 10% phosphomolybdic
acid solution in ethanol. Flash chromatography was performed on a
Varian 971-FP flash purification system using silica gel cartridges
(Varian, particle size of 50 μm). Unless stated otherwise, starting
materials, reagents, and solvents were purchased as high-grade
commercial products from Sigma-Aldrich, Acros, Lancaster, Scharlab,
or Panreac and were used without further purification. Anhydrous
tetrahydrofuran (THF) and diethyl ether were distilled from sodium
benzophenone ketyl and used immediately.
2-(4-Bromobutyl)tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-
dione (36)32 and tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-
dione (41)44 were synthesized according to described procedures, and
their spectroscopic data correspond with those previously reported.
Collected data for compounds 4−35 refer to free bases, and then
hydrochloride salts were prepared prior to mp determination, elemental
analyses, and biological assays. Spectroscopic data of all described
compounds were consistent with the proposed structures. For final
compounds 4−35, we include the data of 8, 9, 16, 19, and 32.
2-{4-[(2-Methoxybenzyl)amino]butyl}tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (4). Obtained from 36 and
2-methoxybenzylamine in 42% yield as an oil: chromatography in 1:9
hexane/ethyl acetate.
2-(4-{[2-(2-Methoxyphenyl)ethyl]amino}butyl)-tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (5). Obtained from 36
and 2-(2-methoxyphenyl)ethylamine in 30% yield as an oil: chromato-
graphy in 1:9 hexane/ethyl acetate.
2-(4-{[3-(2-Methoxyphenyl)propyl]amino}butyl)-tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (6). Obtained from 36
and 3-(2-methoxyphenyl)propylamine in 52% yield as an oil:
chromatography in 95:5 toluene/methanol.
2-(4-{[4-(2-Methoxyphenyl)butyl]amino}butyl)-tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (7). Obtained from 36
and 37 in 30% yield as an oil: chromatography in 95:5 chloroform/
methanol.
Figure 2. (A) Analgesic effect of compound 19 in the formalin test.
Vehicle (0.2% HPMC, 0.9% NaCl), 19, or gabapentin was administered
ip 15 min before subplantar injection of formalin (0.02 mL, 2% solution,
ip). (B) Effect of compound 19 (3 mg/kg, ip) during the late phase after
pretreatment with WAY-100635 (3 mg/kg, ip) 45 min before formalin
injection. (C) Analgesic effect of compound 19 in the formalin test when
administered po 30 min before formalin injection. In all cases, hind paw
licking time was recorded at 5 min intervals for 35 min following
formalin injection. A one-way ANOVA followed by Dunnett’s test was
applied; one, two, or three asterisks indicate significant differences
(p < 0.05, 0.01, or 0.001, respectively) vs vehicle control (n = 10).
2-{4-[(2-Phenoxyethyl)amino]butyl}tetrahydro-1H-pyrrolo-
[1,2-c]imidazole-1,3(2H)-dione (8). Obtained from 36 and 38a in
54% yield as a solid: mp 154−155 °C (diethyl ether); chromatography
in 9:1 dichloromethane/ethanol; IR (CHCl3) ν 3315 (NH), 1770, 1709
(CO), 1599, 1587, 1497 (Ar); 1H NMR (CDCl3) δ 1.47−1.77 (m, 5H,
(CH2)2, 1/2CH2cyc), 1.98−2.29 (m, 3H, CH2cyc, 1/2CH2cyc), 2.70 (t, J =
6.8, 2H, CH2NH), 2.99 (t, J = 4.9, 2H, CH2NH), 3.23 (ddd, J = 11.2, 7.6,
5.2, 1H, 1/2CH2cyc), 3.49 (t, J = 7.3, 2H, NCH2), 3.67 (dt, J = 11.2, 7.6,
1
1H, /2CH2cyc), 4.02−4.10 (m, 3H, CH, OCH2), 6.87−6.98 (m, 3H,
3CHAr), 7.23−7.32 (m, 2H, 2CHAr); 13C NMR (CDCl3,) δ 26.0, 27.1,
27.3, 27.7, 38.9, 45.7, 48.9, 49.4 (8CH2), 63.4 (CH), 67.3 (CH2), 114.7
(2CH), 121.0 (CH), 129.6 (2CH), 158.3 (C), 160.7, 174.0 (2CO).
Anal. (C18H25N3O3·HCl) C, H, N.
analgesic activity in vivo. The results reported herein suggest the
interest of compound 19 and represent a step toward identifying
new therapeutics for the control of pain.
2-(4-{[2-(2-Ethylphenoxy)ethyl]amino}butyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (9). Obtained from 36 and
38b in 69% yield as a solid: mp 183−185 °C (hexane); chromatography
in 9:1 dichloromethane/ethanol; IR (CHCl3) ν 3398 (NH), 1772, 1708
(CO), 1597, 1554, 1506 (Ar); 1H NMR (CDCl3) δ 1.19 (t, J = 7.4, 3H,
CH3), 1.59−1.74 (m, 5H, (CH2)2, 1/2CH2cyc), 1.97−2.09 (m, 2H,
CH2cyc), 2.17−2.28 (m, 1H, 1/2CH2cyc), 2.63 (q, J = 7.6, 2H, CH2CH3),
EXPERIMENTAL SECTION
■
Chemistry. Melting points (mp, uncorrected) were determined on a
Stuart Scientific electrothermal apparatus. Infrared (IR) spectra were
measured on a Shimadzu-8300 or Bruker Tensor 27 instrument
equipped with a Specac ATR accessory with a transmission range
of of 5200−650 cm−1; frequencies (ν) are expressed in cm−1
.
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2.80 (t, J = 7.1, 2H, CH2NH), 3.08 (t, J = 5.1, 2H, CH2NH), 3.17−3.29
2-(4-{[2-(3-Bromophenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (22). Obtained from 39
and 40i in 42% yield as a solid: mp 140−143 °C (hexane); chroma-
tography in ethyl acetate.
2-(4-{[2-(4-Bromophenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (23). Obtained from 39
and 40j in 46% yield as a solid: mp 166−167 °C (hexane); chroma-
tography in ethyl acetate.
2-(2-{[4-(1,3-Dioxotetrahydro-1H-pyrrolo[1,2-c]imidazol-
2(3H)-yl)butyl]amino}ethoxy)benzonitrile (24). Obtained from 39
and 40k in 42% yield as an oil: chromatography in ethyl acetate.
3-(2-{[4-(1,3-Dioxotetrahydro-1H-pyrrolo[1,2-c]imidazol-
2(3H)-yl)butyl]amino}ethoxy)benzonitrile (25). Obtained from 39
and 40l in 38% yield as an oil: chromatography in ethyl acetate.
4-(2-{[4-(1,3-Dioxotetrahydro-1H-pyrrolo[1,2-c]imidazol-
2(3H)-yl)butyl]amino}ethoxy)benzonitrile (26). Obtained from 39
and 40m in 43% yield as an oil: chromatography in ethyl acetate.
2-[4-({2-[2-(Trifluoromethyl)phenoxy]ethyl}amino)butyl]-
tetrahydro-1H-pyrrolo[1,2-c]-imidazole-1,3(2H)-dione (27). Ob-
tained from 39 and 40n in 30% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
1
(m, 1H, /2CH2cyc), 3.50 (t, J = 6.8, 2H, NCH2), 3.61−3.75 (m, 1H,
1/2CH2cyc), 4.02−4.15 (m, 3H, OCH2, CH), 6.82−6.94 (m, 2H,
2CHAr), 7.11−7.17 (m, 2H, 2CHAr); 13C NMR (CDCl3) δ 12.2 (CH3),
23.2, 25.7, 26.6, 27.0, 27.5, 38.6, 45.5, 48.5, 48.9 (9CH2), 63.3 (CH2,
CH), 111.3, 120.8, 126.8, 129.0 (4CH), 132.7, 156.3 (2C), 160.8, 167.4
(2CO). Anal. (C20H29N3O3·HCl) C, H, N.
2-(4-{[2-(3-Ethylphenoxy)ethyl]amino}butyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (10). Obtained from 39 and
40a in 43% yield as an oil: chromatography in ethyl acetate.
2-(4-{[2-(4-Ethylphenoxy)ethyl]amino}butyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (11). Obtained from 39 and
40b in 35% yield as an oil: chromatography in ethyl acetate.
2-(4-{[2-(2-Isopropylphenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (12). Obtained from 39
and 40c in 23% yield as a solid: mp 134−136 °C (diethyl ether); chro-
matography in ethyl acetate.
2-(4-{[2-(1,1′-Biphenyl-2-yloxy)ethyl]amino}butyl)-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (13). Ob-
tained from 39 and 40d in 35% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
2-[4-({2-[3-(Trifluoromethyl)phenoxy]ethyl}amino)butyl]-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (28). Ob-
tained from 39 and 40o in 30% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
2-(4-{[2-(1,1′-Biphenyl-3-yloxy)ethyl]amino}butyl)-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (14). Ob-
tained from 39 and 40e in 32% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
2-[4-({2-[4-(Trifluoromethyl)phenoxy]ethyl}amino)butyl]-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (29). Ob-
tained from 39 and 40p in 30% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
2-(4-{[2-(1,1′-Biphenyl-4-yloxy)ethyl]amino}butyl)-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (15). Ob-
tained from 39 and 40f in 30% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
2-(4-{[2-(2,3-Dimethylphenoxy)ethyl]amino}butyl)-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (30). Ob-
tained from 39 and 40q in 30% yield as an oil: chromatography in 9:1
ethyl acetate/ethanol.
2-(4-{[2-(5,6,7,8-Tetrahydronaphthalen-1-yloxy)ethyl]-
amino}butyl)tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-
dione (31). Obtained from 39 and 40r in 32% yield as an oil:
chromatography in 9:1 ethyl acetate/ethanol.
2-(4-{[2-(1-Naphthyloxy)ethyl]amino}butyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (32). Obtained from 36 and
38g in 48% yield as a solid: mp 163−164 °C (ethyl acetate); chro-
matography in 9:1 dichloromethane/ethanol; IR (CHCl3) ν 3354
(NH), 1771, 1707 (CO), 1582, 1508 (Ar); 1H NMR (CDCl3) δ 1.58−
1.77 (m, 5H, (CH2)2, 1/2CH2cyc), 1.93−2.30 (m, 3H, CH2cyc, 1/2CH2cyc),
2.86 (t, J = 7.1, 2H, CH2NH), 3.15−3.27 (m, 3H, CH2NH, 1/2CH2cyc),
3.49 (t, J = 6.8, 2H, NCH2), 3.60−3.73 (m, 1H, 1/2CH2cyc), 4.05 (dd, J =
9.0, 7.3, 1H, CH), 4.30 (t, J = 4.9, 2H, OCH2), 6.80 (dd, J = 8.5, 1.2, 1H,
CHAr), 7.31−7.53 (m, 4H, 4CHAr), 7.75−7.83 (m, 1H, CHAr), 8.22−
8.28 (m, 1H, CHAr); 13C NMR (CDCl3) δ 25.7, 26.3, 27.0, 27.5, 38.5,
45.5, 48.3, 48.8 (8CH2), 63.3 (CH), 66.7 (CH2), 104.9, 120.6, 121.9,
125.3 (4CH), 125.5 (C), 125.8, 126.4, 127.5 (3CH), 134.5, 154.3 (2C),
160.8, 174.0 (2CO). Anal. (C22H27N3O3·HCl) C, H, N.
2-(4-{[2-(2-Naphthyloxy)ethyl]amino}butyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (33). Obtained from 39 and
40s in 26% yield as an oil: chromatography in ethyl acetate to 9:1 ethyl
acetate/ethanol.
2-[4-({2-[(4-Methoxy-1-naphthyl)oxy]ethyl}amino)butyl]-
tetrahydro-1H-pyrrolo[1,2-c]-imidazole-1,3(2H)-dione (34). Ob-
tained from 39 and 40t in 21% yield as an oil: chromatography in ethyl
acetate to 9:1 ethyl acetate/ethanol.
2-(4-{[2-(8-Quinolinyloxy)ethyl]amino}butyl)tetrahydro-1H-
pyrrolo[1,2-c]imidazole-1,3(2H)-dione (35). Obtained from 36 and
38h in 38% yield as a solid: mp 189−191 °C; chromatography in 95:5 to
8:2 dichloromethane/methanol.
2-(4-{[2-(2-Methoxyphenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (16). Obtained from 36
and 38c in 56% yield as a solid: mp 126−127 °C (diethyl ether);
chromatography in 9:1 dichloromethane/ethanol; IR (CHCl3) ν 3385
(NH), 1771, 1709 (CO), 1493, 1445 (Ar); 1H NMR (CDCl3) δ 1.63−
1.71 (m, 5H, (CH2)2, 1/2CH2cyc), 1.99−2.29 (m, 3H, CH2cyc, 1/2CH2cyc),
2.78 (t, J = 6.8, 2H, CH2NH), 3.01−3.10 (m, 2H, CH2NH), 3.21 (ddd,
J = 11.2, 6.1, 5.6, 1H, 1/2CH2cyc), 3.57−3.80 (m, 3H, NCH2, 1/2CH2cyc),
3.83 (s, 3H, OCH3), 4.00−4.18 (m, 3H, OCH2, CH), 6.87−6.90 (m,
4H, 4CHAr); 13C NMR (CDCl3) δ 25.5, 25.6, 26.5, 27.4, 38.4, 45.4, 48.1,
48.6 (8CH2), 63.2 (CH), 67.7 (CH3), 71.0 (CH2), 111.8, 120.9, 125.9,
129.7 (4CH), 130.5, 147.8 (2C), 160.6, 173.9 (2CO). Anal.
(C19H27N3O4·HCl·4H2O) C, H, N.
2-(4-{[2-(3-Methoxyphenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (17). Obtained from 36
and 38d in 35% yield as a solid: mp 124−126 °C (diethyl ether); chro-
matography in 9:1 dichloromethane/ethanol.
2-(4-{[2-(4-Methoxyphenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (18). Obtained from 39
and 40g in 32% yield as an oil: chromatography in 9:1 dichloromethane/
ethanol.
2-(4-{[2-(2-Ethoxyphenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (19). Obtained from 36
and 38e in 30% yield as an oil: chromatography in ethyl acetate; IR
(CHCl3) ν 3422 (NH), 1773, 1707 (CO), 1593, 1541, 1505, 1445 (Ar);
1H NMR (CDCl3) δ 1.43 (t, J = 6.8, 3H, CH3), 1.62−1.72 (m, 5H,
(CH2)2, 1/2CH2cyc), 1.94−2.27 (m, 3H, CH2cyc, 1/2CH2cyc), 2.78 (t, J =
6.6, 2H, CH2NH), 3.06 (t, J = 5.1, 2H, CH2NH), 3.22 (ddd, J = 11.2, 7.3,
5.1, 1H, 1/2CH2cyc), 3.44−3.72 (m, 3H, NCH2, 1/2CH2cyc), 4.01−4.17
(m, 5H, OCH2, CH, CH2CH3), 6.87−6.92 (m, 4H, 4CHAr); 13C NMR
(CDCl3) δ 14.8 (CH3), 25.6, 26.4, 26.8, 27.4, 38.5, 45.4, 48.3, 48.7
(8CH2), 63.2 (CH), 64.3, 68.3 (2CH2), 113.6, 115.1, 120.9, 121.8
(4CH), 148.3, 149.1 (2C), 160.7, 173.8 (2CO). Anal. (C20H29N3O4·
HCl·4H2O) C, H, N.
Stability Assays in Rat and Human Liver Microsomes. Com-
pounds were incubated at 37 °C and a final concentration of 1 or 5 μM in
PBS together with a solution of NADPH in PBS (final concentration of
2 mM) and a solution of MgCl2 in PBS (final concentration of 5 mM).
Metabolic reactions were initiated by the addition of a suspension of
RLMs (male Sprague-Dawley rats pooled, Sigma-Aldrich) or HLMs
(male human pooled, Sigma-Aldrich), respectively, at a final protein
concentration of 1 mg/mL. The solutions were shaken in a vortex and
2-(4-{[2-(4-Fluoro-2-methoxyphenoxy)ethyl]amino}butyl)-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (20). Ob-
tained from 36 and 38f in 24% yield as a solid: mp 114−116 °C (diethyl
ether); chromatography in 95:5 dichloromethane/ethanol.
2-(4-{[2-(2-Bromophenoxy)ethyl]amino}butyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (21). Obtained from 39
and 40h in 48% yield as a solid: mp 98−99 °C (hexane); chroma-
tography in ethyl acetate.
H
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kept in a water bath open to the air at 37 °C. Aliquots of 100 μL were
quenched at time zero and at seven points ranging to 1 h (RLM) or 2 h
(HLM) when mixtures were poured into 100 μL of ice-cold acetonitrile.
Quenched samples were centrifuged at 10000g for 5 min, and the
supernatants were filtered through a polytetrafluoroethylene (PTFE)
membrane syringe filter (pore size of 0.2 μm, Albet Labscience).
The relative loss of parent compound over the course of the incuba-
tion was monitored by HPLC−MS using an Agilent 1200LC-MSD VL
instrument. HPLC separation was achieved with an Agilent Eclipse
XDB-C18 column (5 μm, 4.6 mm × 150 mm) together with a guard
column (5 μm, 4.6 mm × 12.5 mm). The gradient elution mobile phases
consisted of A (95:5 water/methanol) and B (95:5 methanol/water)
with 0.1% formic acid and 0.1% NH4OH as solvent modifiers. MS
analysis was performed with an electrospray ionization (ESI) source.
The capillary voltage was set to 3.0 kV, and the fragmentor voltage was
set at 70 eV. The drying gas temperature was 350 °C; the drying gas flow
rate was 10 L/min, and the nebulizer pressure was 20 psi. HPLC−MS
measurements were made by selected ion monitoring (SIM).
Concentrations were quantified by measuring the area under the peak
(M + H+) and converted to the percentage of compound remaining,
using the time zero peak area value as 100%. The natural logarithm of the
percentage remaining versus time data for each compound was fit to
linear regression, and the slope was used to calculate the degradation
half-life. The latter was then used to calculate the intrinsic plasma
clearance (CLint) according to the methods of Obach.41
HSA Binding Assay. The assessment of compounds binding to
HSA was performed by incubating a fixed concentration of the
compound with different concentrations of immobilized HSA, using the
TRANSILXL HSA Binding Kit (TMP-0210-2096, Sovicell). An eight-
well unit of the TRANSIL assay plate was used for each compound; six
wells contain increasing concentrations of HSA immobilized on silica
beads suspended in PBS at pH 7.4, and two wells contain buffer only and
serve as references to account for nonspecific binding.
injection (TOP Corp.), needle for oral administration (Natsume), and
stop watch (World Leader).
Test substances and vehicle were each administered to groups of
10 mice by ip injection (5 mL/kg) or po gavage (10 mL/kg) for 15 or
30 min, respectively, before subplantar injection of formalin (0.02 mL,
2% solution). In the vehicle group, half of the animals were treated with
0.2% HPMC and 0.9% NaCl via ip and the other half via po. Reduction
of the formalin-induced hind paw licking time was recorded at 5 min
intervals during the following 0−35 min after formalin injection. To
study the receptor specificity of compound 19, WAY-100635 was
administered ip 45 min before the injection of formalin. Statistical
analysis was performed by using a one-way ANOVA followed by
Dunnett’s test to compare the test compound-treated and vehicle
control groups. Significance is considered at the p < 0.05 level.
ASSOCIATED CONTENT
* Supporting Information
■
S
Binding affinity data of 19 toward other nociception-related
GPCRs, synthesis of compounds 4−35, 37, 38a−h, 39, 40a−t,
42, and 43a−g, spectral characterization data of compounds 4−
7, 10−15, 17, 18, 20−31, and 33−35, combustion analysis data
of final compounds 4−35, binding assays at 5-HT1A, 5-HT2A, 5-
HT3, 5-HT4, and 5-HT7 receptors, rectal temperatures, and
cAMP formation in HeLa cells transfected with the h5-HT1AR.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Phone: 34-91-3944239. Fax: 34-91-3944103. E-mail: mluzlr@
quim.ucm.es.
■
The TRANSIL assay plate was thawed for 3 h at room temperature
and centrifuged at 750g for 5 s. Then, 15 μL of an 80 μM stock solution
of the compound in PBS (for a final concentration of 5 μM) was added
to each well of the eight-well unit, and the plate was incubated on a plate
shaker at 1000 rpm for 12 min at room temperature. After this time, the
plate was centrifuged at 750g for 10 min, and 50 μL of the supernatants
was transferred for analytical quantification by HPLC−MS.
The binding percentage was calculated from the remaining free
compound concentration in the supernatant of each well, using the
spreadsheet and algorithms supplied with the kit.
*Phone: 34-91-3945156. Fax: 34-91-3944103. E-mail: bellinda.
benhamu@quim.ucm.es.
Present Addresses
∥M.V.: Department of Chemistry, Yale University, West Haven,
CT 06516.
⊥I.M.: Clarke, Modet & C°, 28001 Madrid, Spain.
Author Contributions
M.V. and I.M. contributed equally to this work.
Fluorescence-Based CYP2D6 Inhibition Assay. Effects on
CYP2D6 activity were determined at Suven Life Sciences (Banjara
Hills, Hyderabad, India). Briefly, after incubation for 30 min of the test
compound (10 μM) in microsomes containing human recombinant
CYP2D6 (BD Biosciences, 0.015 μM), the conversion of AMMC
(3 μM) to 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4 methylcoumar-
in (AHMC) was measured, using a Spectra Max Gemini EM spectro-
fluorometer with a 390 nm excitation filter and a 460 nm emission filter.
CYP2D6 activity is expressed as the percentage of the maximal effect
obtained with substrate and no inhibitor. Quinidine was used as a
control, and the IC50 value was found to be 0.002 μM, which is
consistent with the value reported in the kit.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from MINECO (SAF2010-
22198-C02-01), CAM (S2011/BMD2353), and UCB Pharma
GmbH. M.V. is grateful to UCB Pharma GmbH for a predoctoral
fellowship. We thank the NMR Core Facilities from Universidad
Complutense de Madrid.
ABBREVIATIONS
Formalin Test.45 Male CD-1 (Crl.)-derived mice weighing 24 2 g
were provided by BioLasco Taiwan (Charles River Laboratories
Technology Licensee). Space allocation for 10 animals was 47 cm ×
18 cm × 13 cm. All animals were maintained in a controlled temperature
(23−24 °C) and humidity (60−70%) environment with 12 h light/dark
cycles for at least 1 week in MDS Pharma Services Taiwan Laboratory
prior to use. Free access to standard lab chow for mice [MF-18 (Oriental
Yeast Co., Ltd.)] and RO water was granted. All aspects of the assay,
including housing, experimentation, and disposal of animals, were
performed in general accordance with the Guide for the Care and Use of
Laboratory Animals. Gabapentin was from Pfizer, and HMPC was from
Sigma. The following equipment was used for the assay: animal cage
(Allentown), beaker (1000 mL, Kimax), brown flask (50 mL),
glass syringe (1 mL, Top Corp.), hypodermic needle (25 gauge × 1 in.)
(TOP Corp.), mouse scale (Z-40, Taconic), needle for formalin
■
AHMC, 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methyl-
coumarin; AMMC, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-
7-methoxy-4-methylcoumarin; CLint, intrinsic clearance;
DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf
serum; h5-HT1AR, human 5-HT1A receptor; HLMs, human liver
microsomes; HPMC, hydroxypropyl methylcellulose; PTFE,
polytetrafluoroethylene; RLMs, rat liver microsomes
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