Development of a high-affinity ligand that binds irreversibly to Mel1b melatonin receptors.

Full text of DOI:10.1021/jm970437q

J . Med. Chem. 1997, 40, 4195-4198
4195
Communications to the Editor
Recently, it was reported that N-[2-(2,7-dimethoxy-
naphthyl)ethyl]propionamide (2) is a ligand that has
high affinity (Ki ) 0.07 ( 0.004 nM) for melatonin
receptors.14 It was postulated that the 2-methoxy group
binds to the accessory binding pocket at the receptor
site. Thus, we designed and synthesized N-[2-[2-(chlo-
roacetoxy)-7-methoxynaphthyl]ethyl]propionamide (3a )
and N-[2-[2-(bromoacetoxy)-7-methoxynaphthyl]ethyl]-
propionamide (3b, BMNEP) to alkylate the receptor at
the pocket.
Develop m en t of a High -Affin ity Liga n d
Th a t Bin d s Ir r ever sibly to Mel1b
Mela ton in Recep tor s
Paula A. Witt-Enderby,*,† Guo-Hua Chu,‡
Megan L. Gillen,† and Pui-Kai Li‡
Departments of Pharmacology and Toxicology and
of Medicinal Chemistry and Pharmaceutics,
School of Pharmacy, Duquesne University,
Pittsburgh, Pennsylvania 15282
Received J uly 2, 1997
Melatonin is a hormone that is synthesized and
secreted into the general circulation by the pineal
gland.1 The circadian production of melatonin is regu-
lated by the suprachiasmatic nucleus of the hypothala-
mus and is synchronized by environmental light.1,2
Melatonin entrains and synchronizes circadian and
seasonal rhythms2-4 and is involved in thermoregula-
tion,5,6 cardiovascular regulation,7 and oncogenesis.8
Melatonin may mediate its effects in vivo through
melatonin receptors. Presently, two melatonin receptor
subtypes have been defined as either the ML-1 or ML-2
type, based on pharmacological profiles.2 However, only
two human melatonin receptors have been cloned that
belong to the G-protein-coupled receptor class of the
The synthesis of target compounds 3a ,b is outlined
in Scheme 1. 7-Methoxy-2-naphthol (4) was used as a
starting material. Selective formylation of the 1-posi-
tion of compound 4 was accomplished by reaction with
chloroform in the presence of NaOH (Reimer-Tiemann
reaction),15 yielding the desired phenolic aldehyde 5.
The phenolic group in 5 was converted to benzyl ether
by reacting 5 with benzyl bromide using K2CO3 as a
base to yield compound 6 (100%). Condensation of
aldehyde 6 with nitromethane in the presence of NH4-
OAc afforded nitroalkene 7 (96.5%). Reduction of 7 with
LiAlH4 followed by acylation with propionyl chloride
furnished amide 8 (54.2% based on 7). Cleavage of the
benzyl ether in compound 8 by hydrogenation gave the
phenol 9 (100%). Reaction of 9 with chloroacetyl
chloride or bromoacetyl bromide using N,N-diisopropyl-
ethylamine as base yielded the target compounds 3a ,b,
respectively.
9
10
ML-1 type, defined as either the Mel1a or Mel1b
subtype. Little is known about the role that the Mel1b
melatonin receptor plays in vivo as “tools” (i.e., selective
Mel1b antibodies or affinity labels selective for the Mel1b
receptor) have not yet been developed. Melatonin
receptors of the Mel1b melatonin receptor subtype may
be involved in retinal physiology. This is supported by
competitive receptor binding assays using the radioli-
gand 2-[125I]iodomelatonin and competitive melatonin
receptor antagonists. It is shown that the pharmaco-
logical profile of these antagonists at the human Mel1b
melatonin receptors expressed in COS-7 cells is similar
to that of the functional presynaptic melatonin heter-
oreceptor of the rabbit retina.11 Additionally, the
expression of the Mel1b melatonin receptor mRNA is
predominantly in the retina.10
In the past, attempts have been made to develop an
affinity ligand (i.e., N-(bromoacetyl)-2-iodo-5-methox-
ytryptamine) for melatonin receptors.12,13 Because me-
latonin binds to its receptors in the picomolar to low-
nanomolar range, use of N-(bromoacetyl)-2-iodo-5-
methoxytryptamine12 has been minimal because of its
low (∼50 nM) affinity for melatonin receptors.3 In
addition, whether this ligand displays any selectivity
for either melatonin receptor subtype is unknown.
Here, we report the development of an affinity ligand
that binds specifically to and selectively alkylates Mel1b
melatonin receptors expressed in Chinese hamster
ovary (CHO) cells.
Saturation analysis, which was previously performed
on CHO cells expressing the human Mel1a receptor
(CHO-Mel1a ),16 was performed on human Mel1b mela-
tonin receptors expressed in the same cells (CHO-Mel1b)
using 2-[125I]iodomelatonin as already described.16
Briefly, CHO-Mel1b cells were resuspended in Tris (50
mM), pH 7.4, and added to tubes containing increasing
concentrations of 2-[125I]iodomelatonin (0-500 pM) in
the absence (total binding) or presence of melatonin
(nonspecific binding). All reactions were incubated for
1 h at room temperature, rapidly filtered, and counted
in a gamma counter as already described.16
To determine whether BMNEP alkylated melatonin
receptors, CHO-Mel1a or CHO-Mel1b cells were grown
to confluence on 6-well dishes and were preincubated
either with various concentrations of BMNEP (100 pM
to 1 µM) in the absence or presence of melatonin (1 µM)
* Corresponding author. Tel.: (412) 396-4346. Fax: (412) 396-5130.
E-mail: wittp@duq3.cc.duq.edu.
† Department of Pharmacology and Toxicology.
‡ Department of Medicinal Chemistry and Pharmaceutics.
S0022-2623(97)00437-8 CCC: $14.00 © 1997 American Chemical Society

4196 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Sch em e 1. Synthesis of Compounds 3a ,ba
Communications to the Editor
a
Reagents and conditions: (a) i. NaOH, CHCl3, H2O, ii. H3O+ (31.5%); (b) PhCH2Br, K2CO3, acetone, reflux, 5 h, 100%; (c) CH3NO2,
NH4OAc, reflux, 2 h, 96.5%; (d) i. LiAlH4, THF, 40 °C, 18 h, ii. C2H5COCl, Et3N, CH2Cl2, rt, 1 h, 54.2% from 7; (e) 5% Pd-C, MeOH, H2,
rt, 18 h, 100%; (f) XCH2COX (X ) Cl, Br), iPr2NEt, CH2Cl2, 45 min, 72% for 3a , 61% for 3b.
for 1 h at 37 °C or with 1 nM BMNEP for various periods
of time (0-120 min) at 37 °C. Following the incubation,
cells were washed with phosphate-buffered saline (137
mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 0.62 mM KH2-
PO4), lifted in buffer (10 mM K3PO4, 1 mM EDTA, pH
7.4), and added to tubes containing 2-[125I]iodomelatonin
(500 pM) and Tris (50 mM, pH 7.4) buffer in the absence
(total) or presence (nonspecific) of 1 µM melatonin.
Competition assays were performed as described
previously.16 Briefly, CHO-Mel1a cells were grown to
confluence in 10-cm diameter dishes and resuspended
in Tris-HCl (50 mM), pH 7.4. Cell lysates were added
to tubes containing Tris-HCl (50 mM) and 2-[125I]-
iodomelatonin (100 pM) in the absence (total) or pres-
ence of drugs (0.1 pM to 1 µM). All reactions were
incubated for 1 h at room temperature, rapidly filtered,
and counted in a gamma counter as already described.16
Bromoacetoxy and chloroacetoxy derivatives have
been widely used as alkylating moieties.17,18 This
F igu r e 1. Time-dependent alkylation of Mel1b melatonin
approach was used in the following study to develop
receptors by BMNEP. Following a 2-h exposure to 1 nM
BMNEP, no Mel1a receptors and 83 ( 4% of Mel1b melatonin
alkylating agents against melatonin receptors. Because
bromoacetoxy derivatives are more reactive than chlo-
roacetoxy derivatives, the focus of this study was on the
bromoacetoxy derivative BMNEP and its alkylating
effects on melatonin receptors.
receptors were alkylated by 5 min (t1/2 ) 3 min). Data were
normalized against control (untreated) cells and are expressed
as the mean ( SEM of 3-6 experiments. All data were
analyzed by one-way ANOVA and Student’s unpaired t-test
where significance (*) is defined as p < 0.05; (*) comparing
BMNEP-treated cells to control untreated cells.
The expression of human Mel1a melatonin receptors
in CHO cells did not affect the pharmacology of this
receptor as already described.16 The expression of
human Mel1b melatonin receptor cDNA10 in CHO cells
(developed in collaboration with Dr. Margarita Dub-
ocovich, Northwestern University Medical School) also
did not affect the pharmacology of this receptor. Bind-
ing of 2-[125I]iodomelatonin to CHO-Mel1b cells was
saturable, reversible, and of high affinity (KD ) 158 (
1 pM; Bmax ) 424 ( 115 fmol/mg of protein, n ) 3) which
is similar to that reported for the Mel1b receptors
expressed in COS-1 cells.10
where maximal inhibition (83 ( 4% Mel1b receptors
alkylated) occurred by 5 min (Figure 1). In addition,
increasing concentrations of BMNEP significantly de-
creased 2-[125I]iodomelatonin binding to CHO-Mel1b cells
compared to untreated control cells (p ) 0.0044, n )
3-7 experiments). Melatonin (1 µM), added in combi-
nation with BMNEP, prevented Mel1b receptors from
alkylation at the 1 nM (p ) 0.011, n ) 4), 30 nM (p )
0.025, n ) 3), and 1 µM (p ) 0.001, n ) 3) concentrations
of BMNEP (Figure 2B). The decrease in 2-[125I]iodome-
latonin binding to CHO-Mel1b cells following BMNEP/
melatonin exposure was not due to a downregulation
of Mel1b melatonin receptors as melatonin-pretreated
controls were run in parallel with untreated controls
Preincubation of CHO-Mel1b cells with BMNEP re-
sulted in a significant time-dependent decrease in total
2-[125I]iodomelatonin binding to Mel1b melatonin recep-
tors compared to untreated cells (n ) 3-6 experiments)

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4197
F igu r e 2. Concentration-dependent alkylation of Mel1b melatonin receptors expressed in CHO cells. (A) Following a 1-h exposure
to various concentrations of BMNEP (100 pM to 1 µM), no Mel1a receptors were alkylated by BMNEP. (B) In contrast, 50% of the
total Mel1b melatonin receptors were alkylated by 1 nM BMNEP where maximal alkylation (88 ( 5% Mel1b receptors alkylated)
occurred at 1 µM BMNEP. Melatonin (1 µM), added in combination with BMNEP, prevented the effect of BMNEP (1 nM, 30 nM,
and 1 µM)-mediated alkylation only at Mel1b receptors. Data were normalized against control (untreated) cells and are expressed
as the mean ( SEM of 3-7 experiments. All data were analyzed by one-way ANOVA and Student’s unpaired t-test where
significance (*) is defined as p < 0.05; (*) comparing BMNEP-treated cells to BMNEP/melatonin-treated cells.
Ta ble 1. Competition of Melatonin and Related Analogues for
our understanding of Mel1b melatonin receptors in vivo.
BMNEP could be used to characterize the Mel1b mela-
tonin receptor as affinity probes in the past have played
2-[125I]Iodomelatonin Binding to Me11a or Me11b Melatonin
Receptors Expressed in CHO Cellsa
important roles in molecular characterization of other
receptors. Such receptors include the adenosine recep-
tors,19 steroid receptors,20 and muscarinic receptors.21-24
In addition, because BMNEP selectively alkylated the
Mel1b and not the Mel1a receptors, this ligand may be
Ki (nM) (range of SEM)
used to determine the distribution of Mel1b receptors
compd
R
Me11a
Me11b
throughout the body even in tissues that coexpress Mel1a
melatonin receptors. In conclusion, use of this affinity
ligand will aid in a greater understanding of the
molecular characteristics of Mel1b melatonin receptors
both in vitro and in vivo.
2
CH3
0.099 (0.09-0.1) 0.38 (0.35-0.41)
3a
3b
COCH2Cl
COCH2Br
11 (2.3-51)
1.6 (0.86-3.0)
1.3 (0.84-2.0)
melatonin
0.08 (0.06-0.11) 0.12 (0.07-0.19)
a
All competition binding experiments were performed on CHO
whole cell lysates using 80-100 pM 2-[125I]iodomelatonin (NEN/
DuPont, Boston, MA; 2200 Ci/mmol) at room temperature. The
affinity of 2-[125I]iodomelatonin for Mel1a and Mel1b melatonin
receptors was 80 and 150 pM, respectively.
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binding occurred (data not shown). Fifty percent of the
total Mel1b melatonin receptors alkylated by BMNEP
occurred at ∼1 nM BMNEP (Figure 2B). In contrast,
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(Figure 1). In addition, increasing concentrations of
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