Substituted 2-amidomethyl[1,4]benzodioxins: Synthesis and binding affinity for the melatonin receptors

Article abstract of DOI:10.1211/146080899128734604

A novel series of melatonin analogues based on the benzodioxin nucleus is described. The compounds were prepared in several steps from 2-carbethoxy-1,4-benzodioxin. Some of these derivatives competitively inhibited 2-[¹²⁵I]-iodomelatonin bindin

Full text of DOI:10.1211/146080899128734604

Pharm. Pharmacol. Commun. 1999, 5: 199±206
# 1999 Pharm. Pharmacol. Commun.
Substituted 2-Amidomethyl[1,4]benzodioxins: Synthesis and
Binding Af®nity for the Melatonin Receptors
A. MAMAI, C. BENNEJEAN*, P. RENARD*, P. DELAGRANGE{, B. GUARDIOLA-LEMAITRE{,
H. E. HOWELL{, M. C. VIAUD AND G. GUILLAUMET
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Institut de Chimie Organique et Analytique, associe au CNRS, Universite d'Orleans, B.P. 6759, 45067
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Orleans Cedex 02, *ADIR, 1 rue Carle Hebert, 92415 Courbevoie Cedex, {I.R.I. Servier, 6, place des
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Pleiades, 92415 Courbevoie Cedex, France and {Rowett Research, Aberdeen, Scotland, UK
Abstract
A novel series of melatonin analogues based on the benzodioxin nucleus is described.
The compounds were prepared in several steps from 2-carbethoxy-1,4-benzodioxin.
Some of these derivatives competitively inhibited 2-[125I]-iodomelatonin binding to
chicken brain and ovine pars tuberalis membranes, albeit with very reduced af®nity
compared with melatonin.
The pineal hormone melatonin (Lerner et al 1958)
has a central role in the regulation of daily and
seasonal rhythms in vertebrates. The pineal gland
produces and releases melatonin during the hours
of darkness under the control of the circadian clock
in the suprachiasmatic nuclei of the hypothalamus.
As day length changes through the seasons, the
duration of the nightly elevation in circulating
melatonin alters, regulating many types of seasonal
behaviour, including reproduction, coat colour and
body weight in photoperiodic mammals (Bartness
et al 1993). Melatonin has found practical use in
inducing seasonally-breeding animals, such as
sheep, to breed out of season (Lincoln 1983). In
man, melatonin might have a variety of clinical
applications, for example in jet-lag and shift work
disturbances (Arendt 1991), and for circadian
rhythm control in the blind. Melatonin also has
hypnotic properties in animals and in man, and it
has been reported to have an oncostatic action and
to modulate the immune response (Holmes &
Sugden 1982; Waldhauser et al 1990; Maestroni &
Conti 1993).
important tools for the elucidation of the physio-
logical role of melatonin. Several indolic analogues
(Spadoni et al 1993; Garrat et al 1995; Henin et al
1997; Tarzia et al 1997; Davies et al 1998) of
melatonin have been found to act as ligands. Non-
indolic analogues of melatonin were designed and
synthesized as the naphthalenic bioisosteres (Yous
et al 1992; Depreux et al 1994; Langlois et al 1995)
and the conformationally-restricted analogues of
melatonin, such as carbazole (Garrat et al 1994;
Spadoni et al 1997), tetralin (Copinga et al 1993;
Leclerc et al 1996; Fourmaintraux et al 1998) and
amidoindan (Jansen et al 1996) derivatives.
With regard to the successful development of the
2H-1-benzopyran and 2,3-dihydro-2H-1-benzo-
pyran system as a structural base for the synthesis
of melatonin ligands (Sugden 1994; Viaud et al
1998) we have investigated the synthesis of the
benzodioxinic compounds as a template for non-
indolic melatonin-like agents. In this paper, we
describe the synthesis and pharmacological eva-
luation of a series of substituted 2-amidomethyl-
1,4-benzodioxin derivatives (Figure 1).
Despite renewed interest in melatonin in recent
years, much remains to be discovered, including its
mode of action and interaction with its receptor.
There is considerable interest in the search for new
molecules capable of mimicking or antagonizing
responses to melatonin. Such compounds are
Materials and Methods
Chemistry
Melting points were determined on a Ko¯er hot-
È
stage apparatus and are uncorrected. Proton NMR
were recorded on a Bruker 300 spectrometer.
Coupling constants are in Hz and chemical shifts
are d ppm down®eld from tetramethylsilane, which
Correspondence: M. C. Viaud, Institut de Chemie Organique
Â
et Analytique, associe au CNRS, Universite d'Orleans, B.P.
6759, 45067 Orleans Cedex 02, France.
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200
A. MAMAI ET AL
(10 mL). After cooling at 0ꢀC, 8% sodium hydro-
xide solution was added and then the reaction
mixture was stirred for 30 min at the same tem-
perature. The organic solvents were removed by
evaporation under reduced pressure and the residue
washed with ethyl acetate. The mixture was then
poured into excess ice-cold hydrochloric acid
(10%) and the resulting precipitate collected by
®ltration under reduced pressure, washed with
water and dried to afford quantitatively 3a as a
white solid, mp 197±198ꢀC. 1H NMR (CDCl3
‡ D2O) d 3Á73 (s, 3H, OCH3), 6Á36 (dd, 1H,
J ˆ 8Á7, 2Á8 Hz, H6), 6Á45 (d, 1H, J ˆ 2Á8 Hz, H2),
6Á67 (d, 1H, J ˆ 8Á7 Hz, H5), 7Á10 (s, 1H, H3). IR
Figure 1. Structure of 1,4-benzodioxins substituted at posi-
tion 2 by an amidomethyl group.
was used as an internal standard. IR spectra were
obtained using a Perkin-Elmer spectrophotometer
297. Mass spectra were recorded on an R 10-10 C
Nermag (70 eV) apparatus. Organic solvents were
puri®ed as necessary as described by Perrin et al
(1986) or purchased from Aldrich Chimie. All
solutions were dried over anhydrous magnesium
sulphate and evaporated on a BuÈchi rotatory eva-
porator. Analytical thin-layer chromatography was
carried out on precoated plates (silica gel, 60 F-
254), and spots were visualized with UV light or an
alcohol solution of ammonium cerium nitrate.
Column chromatography was performed with
Kieselgel 60 (70±230 mesh) silica gel (Merck) for
gravity columns and Kieselgel 60 (230±400 mesh)
silica gel (Merck) for ¯ash columns. All anhydrous
reactions were performed in oven-dried glassware
under an atmosphere of argon. The column chro-
matography solvents used were distilled and sol-
vent mixtures were reported as v=v ratios.
1
(KBr) n 3665±2465, 1710, 1260 cm . Calculated
for C10H8O5: C, 59Á46; H, 4Á54. Found: C, 59Á29;
H, 4Á45.
2-Carboxy-1,4-benzodioxin (3b)
Compound 3b was prepared as previously descri-
bed (Coudert et al 1978).
2-Carbomethoxy-7-methoxy-3-phenyl-1,4
benzodioxin (2c)
To a solution of lithium diisopropylamide (32Á5 mL,
2 M in THF=n-heptane, 65 mmol) in dry tetra-
hydrofuran (40 mL), 3Á85 g (18Á5 mmol) of the acid
3a in 20 mL tetrahydrofuran was added at 78ꢀC
under inert atmosphere. After stirring for 5 h at the
same temperature, 19Á6 mL (65 mL) tributyltin
chloride was added and the mixture was stirred for
a further 1 h. Saturated aqueous ammonium choride
solution was added and the mixture was extracted
with ethyl acetate. The combined organic extracts
were washed with water, dried and evaporated to
give crude stannylated acid, used without puri®ca-
tion in the next step. To this product (3Á67 g,
7Á40 mmol) dissolved in N,N-dimethylacetamide
(25 mL) were added 6Á21 g (74 mmol) sodium
hydrogenocarbonate and 2Á3 mL (37 mmol) iodo-
methane under inert atmosphere. The reaction
mixture was stirred for 4 days at room temperature
in darkness. The solvent was removed under
reduced pressure and the residue diluted with
water. The aqueous layer was extracted with
dichloromethane. The combined organic extracts
were dried over magnesium sulphate, ®ltered and
concentrated. The crude product was chromato-
graphed (eluent: ethyl acetate±dichloromethane±
petroleum ether, 2Á5:2Á5:95), to give 4a as pale oil.
To tetrakis(triphenylphosphine)palladium (0Á252 g,
0Á218 mmol) and copper iodide (0Á083 g,
0Á436 mmol) was added a solution of ester 4a
(2Á15 g, 4Á36 mmol) and iodobezene (1Á33 g,
2-Carbethoxy-7-methoxy-1,4-benzodioxin (2a)
To a solution of 2-carbethoxy-7-hydroxy-1,4-ben-
zodioxin 1 (2Á8 g, 12Á75 mmol) in N, N-dimethyl-
formamide (30 mL) were slowly added 0Á64 g
(16 mmol) sodium hydride, 60% in mineral oil, at
0ꢀC under inert atmosphere. After stirring for
30 min at the same temperature, 2Á28 g (16 mmol)
iodomethane was added. The reaction mixture was
stirred for 4 h at room temperature, and the solvent
evaporated under reduced pressure. The residue
was partitioned between ethyl acetate and water,
and the organic layer was washed with water. After
drying over magnesium sulphate and removal of
the solvent, the crude was submitted to column
chromatography (eluent: ethyl acetate±petroleum
ether, 3:7) to afford compound 2a in 97% yield as a
white solid, mp 64±65ꢀC. 1H NMR (CDCl3) d 1Á32
(t, 3H, J ˆ 6Á9 Hz, CH3), 4Á28 (q, 2H, J ˆ 6Á9 Hz,
CH2), 3Á70 (s, 3H, OCH3), 6Á37 (dd, 1H, J ˆ 9Á5,
2Á9 Hz, H6), 6Á44 (d, 1H, J ˆ 2Á9 Hz, H8), 6Á63 (d,
1H, J ˆ 9Á5 Hz, H5), 6Á96 (s, 1H, H3). IR (KBr) n
1
1725, 1260 cm . Calculatedfor C12H12O5: C, 61Á02;
H, 5Á12. Found: C, 61Á27; H, 5Á01.
2-Carboxy-7-methoxy-1,4-benzodioxin (3a)
Ester 2a (3 g, 12Á7 mmol) was dissolved in a mix-
ture of ethanol (30 mL) and tetrahydrofuran

SUBSTITUTED 2-AMIDOMETHYL[1,4]BENZODIOXINS
1
201
6Á54 mmol) in 1,4-dioxane (22 mL). After stirring
for 2 h at re¯ux, the salts were ®ltered and the
solvents removed under reduced pressure. The
residue was diluted with ethyl acetate and the
organic layer washed with saturated potassium
¯uoride solution. After drying over magnesium
sulphate and removal of the solvent, the resulting
oil was submitted to column chromatography
(eluent: ethyl acetate±petroleum ether, 15:85) to
afford compound 2c as a white solid in 49% overall
yield based from 3a, mp 82±83ꢀC. 1H NMR
(CDCl3) d 3Á65 (s, 3H, OCH3), 3Á76 (s, 3H, OCH3),
6Á41 (dd, 1H, J ˆ 8Á7, 2Á7 Hz, H6), 6Á51 (d, 1H,
J ˆ 2Á7 Hz, H8), 6Á71 (d, 1H, J ˆ 8Á7 Hz, H5),
7Á39±7Á49 (m, 5H, Harom). IR (KBr) n 1730,
mp 192±193ꢀC. H NMR (d6-DMSO) d 3Á67 (s,
3H, CH3), 6Á48 (dd, 1H, J ˆ 8Á7, 2Á7 Hz, H8), 6Á58
(d, 1H, J ˆ 2Á7 Hz, H6), 6Á82 (d, 1H, J ˆ 8Á7 Hz,
H5), 7Á34±7Á45 (m, 5H, Harom), 7Á57 (br s, 2H,
1
NH2). IR (KBr) n 3940±3420, 1645, 1260 cm .
Calculated for C16H13NO4: C, 63Á67; H, 4Á52; N,
5Á71. Found: C, 63Á81; H, 4Á42; N, 5Á87.
2-Carboxamide-3-phenyl-1,4-benzodioxin (5d)
This product was prepared from compound 2d
(2Á96 g, 11 mmol) and 28% aqueous ammonia
solution (70 mL) in ethanol (70 mL). After stirring
for 6 days at room temperature, the solvents were
removed under reduced pressure and the crude
product was chromatographed (eluent: methanol±
dichloromethane, 5:95) to give the desired product
5d as a white solid in 86% yield, mp 134±135ꢀC.
1H NMR (CDCl3) d 5Á50 (br s, 2H, NH2), 6Á86±
7Á01 (m, 4H, Harom), 7Á46±7Á62 (m, 5H, Harom). IR
1
1260 cm . Calculated for C17H14O5: C, 66Á95; H,
4Á45. Found: C, 67Á11; H, 4Á56.
2-Carbomethoxy-3-phenyl-1,4-benzodioxin (2d)
This compound was prepared from 3b according to
the procedure described for 2c (67% overall yield
based from 3b), mp 72±73ꢀC. 1H NMR (CDCl3) d
3Á71 (s, 3H, OCH3), 6Á81±6Á97 (m, 4H, Harom),
7Á46±7Á54 (m, 5H, Harom). IR (KBr) n 1740,
1
(KBr) n 3370±3175, 1640, 1250 cm . Calculated
for C15H11NO3: C, 71Á14; H, 4Á38; N, 5Á53. Found:
C, 70Á91; H, 4Á51; N, 5Á71.
2-Cyano-7-methoxy-1,4-benzodioxin (6a)
1
Amide 5a (1Á76 g, 8Á52 mmol) and phosphorus
oxychloride were dissolved in anhydrous pyridine
(40 mL). The reaction mixture was stirred at re¯ux
for 1 h under inert atmosphere. After removal of the
solvent under reduced pressure, the residue was
hydrolysed slowly with a saturated sodium hydro-
genocarbonate solution. The aqueous layer was
extracted with dichloromethane. The combined
organic layers were washed with water, dried over
magnesium sulphate and evaporated. Puri®cation
by column chromatography (eluent: ethyl acetate±
petroleum ether, 40:60) provided the nitrile 6a in
79% yield as a white solid, mp 91±92ꢀC. 1H NMR
(CDCl3) d 3Á72 (s, 3H, OCH3), 6Á80 (d, 1H,
J ˆ 3Á2 Hz, H8), 6Á39 (dd, 1H, J ˆ 9Á6, 3Á2 Hz,
H6), 6Á52 (s, 1H, H3), 6Á63 (d, 1H, J ˆ 9Á6 Hz, H5).
1250 cm . Calculated for C16H12O4: C, 66Á26; H,
4Á32. Found: C, 66Á09; H, 4Á41.
2-Carboxamide-7-methoxy-1,4-benzodioxin (5a)
To a solution of ester 2a (2Á90 g, 12Á35 mmol) in
ethanol (50mL) was added a 28% aqueous ammonia
solution. The reaction mixture was stirred for 40h at
room temperature. The solvents were removed
under reduced pressure and the residue was chro-
matographed (eluent: methanol±dichloromethane,
10:90) to give 5a as a white solid in 88% yield, mp
1
175±176ꢀC. H3 NMR (CDCl3) d 3Á60 (s, 3H,
CH3), 6Á35 (d, 1H, J ˆ 3Á0 Hz, H8), 6Á41 (dd, 1H,
J ˆ 9Á5, 3Á0 Hz, H6), 6Á69 (d, 1H, J ˆ 9Á5 Hz, H5),
6Á87 (s, 1H, H3), 7Á43 (br s, 2H, NH2), IR (KBr) n
1
1
IR (KBr) n 2220, 1260 cm . Calculated for
3490±3350, 1650, 1260 cm . Calculated for
C10H7NO3: C, 63Á08; H, 4Á24; N, 6Á54. Found: C,
C10H9NO4: C, 57Á97; H, 4Á38; N, 6Á76. Found: C,
63Á27; H, 4Á11; N, 6Á67.
57Á79; H, 4Á51; N, 6Á53.
2-Cyano-7-methoxy-3-phenyl-1,4-benzodioxin (6c)
This product was prepared according to the method
used for 6a from compound 5c (0Á24 g, 1Á5 mmol)
and phosphorus oxychloride (0Á9 mL, 7Á5 mmol).
The crude product was chromatographed (eluent:
ethyl acetate±petroleum ether, 25:75) to give the
desired nitrile 6c in 82% yield as a white solid, mp
94±95ꢀC. 1H NMR (CDCl3) d 3Á77 (s, 3H, OCH3),
6Á42 (d, 1H, J ˆ 2Á8 Hz, H8), 6Á48 (dd, 1H, J ˆ
8Á8, 2Á8 Hz, H6), 6Á82 (d, 1H, J ˆ 8Á8 Hz, H5),
2-Carboxamide-7-methoxy-3-phenyl-1,4-benzo-
dioxin (5c)
This compound was obtained from compound 2c
(3Á15 mmol) and 28% aqueous ammonia solution
(120 mL) in ethanol (35 mL). After stirring for 4
days at room temperature, the solvents was
removed under reduced pressure and the crude
product was puri®ed by column chromatography
(eluent: methanol±dichloromethane, 5:95) to
afford compound 5c as a white solid in 80% yield,

202
A. MAMAI ET AL
7Á45±7Á78 (m, 5H, Harom). IR (KBr) n 2220,
amine 7d was obtained in 68% yield as a yellow
1
1
1245 cm . Calculated for C6H11NO3: C, 65Á30; H,
oil. H NMR (CDCl3) d 1Á75±1Á92 (m, 2H, NH2),
4Á22; N, 6Á24. Found: C, 65Á09; H, 4Á13; N, 6Á39.
3Á42 (s, 2H, CH2N), 6Á61±7Á92 (m, 4H, Harom),
7Á23±7Á52 (m, 5H, Harom). IR (®lm) n 3510±3340,
1
1265 cm .
2-Cyano-3-phenyl-1,4-benzodioxin (6d)
The reaction was carried out in a similar manner to
that used for the reaction of 6c. Puri®cation of the
crude product by column chromatography (eluent:
ethyl acetate±petroleum ether, 40:60) gave 6d as a
N-((7-Methoxy-1,4-benzodioxin-2-yl)methyl)
acetamide (8a)
To a solution of amine 7a (0Á55 g, 3Á33 mmol) in
pyridine (10 mL) was added acetic anhydride
(0Á41 g, 4Á0 mmol) at 0ꢀC. The mixture was stirred
for 30 min at the same temperature under inert
atmosphere. The solvent was removed under
reduced pressure and the residue was diluted with
dichloromethane. The organic layer was acidi®ed
with 1 M HCl. The aqueous layer was extracted
with dichloromethane. The combined extracts were
washed and dried over magnesium sulphate. After
evaporation of solvents in-vacuo, the crude extract
was puri®ed by column chromatography (eluent:
methanol±dichloromethane, 10:90) to afford 8a in
1
white solid in 81% yield, mp 85±86ꢀC. H NMR
(CDCl3) d 6Á82±7Á01 (m, 4H, Harom), 7Á45±7Á82
1
(m, 5H, Harom). IR (KBr) n 2220, 1265 cm .
Calculated for C15H9NO2: C, 73Á76; H, 4Á13; N,
5Á73. Found: C, 73Á49; H, 3Á99; N, 5Á50.
2-Aminomethyl-7-methoxy-1,4-benzodioxin (7a)
The compound 6a (1 g, 6Á29 mmol) was dissolved
in anhydrous ether (50 mL). Lithium aluminium
hydride (0Á31 g, 8Á17 mmol) was added portionwise
with stirring under inert atmosphere. The mixture
was stirred at re¯ux for 3 h. After cooling, succes-
sively water (0Á31 mL), 15% sodium hydoxide
solution (0Á31 mL), and water (0Á93 mL) were
added very slowly. After stirring for 15 min at room
temperature, the salts were ®ltered and washed with
ether. The organic layer was dried over magnesium
sulphate and the solvent was removed to give the
expected amine 7a in 68% yield as a yellow oil. 1H
NMR (CDCl3) d 1Á58 (br s, 2H, NH2), 3Á16 (s, 2H,
CH2N), 3Á72 (s, 3H, OCH3), 5Á86 (s, 1H, H3), 6Á31
(d, 1H, J ˆ 2Á7 Hz, H8), 6Á39 (dd, 1H, J ˆ 8Á8,
2Á7 Hz, H6), 6Á57 (d, 1H, J ˆ 8Á8 Hz, H5). IR (®lm)
1
88% yield as a white solid, mp 105±106ꢀC. H
NMR (CDCl3) d 1Á95 (s, 3H, COCH3), 3Á64 (s, 3H,
OCH3), 3Á71 (d, 2H, J ˆ 4Á8 Hz, CH2N), 5Á71±5Á75
(m, 1H, NH), 5Á88 (s, 1H, H3), 6Á20 (d, 1H,
J ˆ 2Á9 Hz, H8); 6Á28 (dd, 1H J ˆ 8Á8, 2Á9 Hz, H6),
6Á49 (d, 1H, J ˆ 8Á8 Hz, H5). IR (KBr) n 3250±
1
3110, 1655, 1265 cm . MS (IC=NH3) m=z 236
(M ‡ 1). Calculated for C12H13NO4: C, 65Á50; H,
5Á50; N, 5Á59. Found: C, 65Á69; H, 5Á27; N, 5Á37.
N-((1,4-benzodioxin-2-yl)methyl)acetamide (8b)
This compound was prepared from 7b according to
the procedure described for 8a. The expected amide
8b was obtained after column chromatography
(eluent: ethyl acetate) in 85% yield as a white solid,
1
n 3530±3260, 1245 cm .
2-Aminomethyl-1,4-benzodioxin (7b)
The title compound was obtained by the previously
published procedure (Guillaumet et al 1984).
1
mp 111±112ꢀC. H NMR (CDCl3) d 2Á05 (s, 3H,
CH3), 3Á79 (d, 2H, J ˆ 5Á5 Hz, CH2N), 5Á72±5Á83
(m, 1H, NH), 5Á96 (s, 1H, H3), 6Á82±6Á86 (m, 4H,
1
H
arom). IR (KBr) n 3315±3230, 1645, 1260 cm .
MS (IC=NH3) m=z 206 (M ‡ 1). Calculated for
C11H11NO3: C, 62Á50; H, 5Á86; N, 7Á15. Found: C,
62Á71; H, 5Á71; N, 7Á01.
2-Aminomethyl-7-methoxy-3-phenyl-1,4-
benziodioxin (7c)
Reduction of 6c with lithium aluminium hydride as
described above gave 7c in 68% yield as a yellow
1
oil. H NMR (CDCl3) d 1Á88 (br s, 2H, NH2), 3Á99
(s, 2H, CH2N), 3Á75 (s, 3H, OCH3), 6Á38±6Á42 (m,
2H, H8 and H6), 6Á69 (d, 1H, J ˆ 8Á9 Hz, H5),
7Á36±7Á45 (m, 5H, Harom). IR (®lm) n 3490±3315,
N -((7-Methoxy-3-phenyl-1,4-benzodioxin-2-
yl)methyl)acetamide (8c)
The title compound was prepared from 7c by a
procedure similar to that described for 8b. The
expected product 8c was obtained after column
chromatography (eluent: ethyl acetate±petroleum
ether, 60:40) in 84% yield as a white solid, mp
172±173ꢀC. 1H NMR (CDCl3) d 2Á04 (s, 3H,
CH3CO), 3Á76 (s, 3H, OCH3), 4Á06 (d, 2H,
1
1260 cm .
2-Aminomethyl-3-phenyl-1,4-benzodioxin (7d)
The title compound was prepared from 6d accord-
ing to the procedure described for 7a. The expected

SUBSTITUTED 2-AMIDOMETHYL[1,4]BENZODIOXINS
203
J ˆ 5Á2 Hz, CH2N), 5Á69±5Á75 (m, 1H, NH), 6Á39
(d, 1H, J ˆ 2Á8 Hz, H8), 6Á42 (dd, 1H, J ˆ 8Á5,
2Á8 Hz, H6), 6Á70 (d, 1H, J ˆ 8Á5 Hz, H5), 7Á38±
7Á46 (m, 5H, Harom). IR (KBr) n 3390±3240, 1645,
until neutralization of the excess of isocyanate, then
the solvent was removed by evaporation. The crude
was puri®ed by column chromatography (eluent:
methanol±dichloromethane, 10:90) to give the
expected product 10 in 85% yield as a white solid,
1
1260 cm . MS (IC=NH3) m=z 312 (M ‡ 1). Cal-
1
culated for C18H17NO4: C, 69Á44; H, 5Á50; N, 4Á50.
Found: C, 69Á61; H, 5Á63; N, 4Á69.
mp 141±142ꢀC. H NMR (CDCl3) d 0Á92 (t, 3H,
J ˆ 7Á3 Hz, CH3), 1Á47±1Á56 (m, 2H, CH2), 3Á14 (q,
2H, J ˆ 6Á6 Hz, CH2), 3Á70 (s, 3H, OCH3), 3Á76 (d,
2H, J ˆ 5Á1 Hz, CH2N), 4Á47±4Á52 (m, 1H, NH2),
4Á61±4Á67 (m, 1H, NH), 5Á93 (s, 1H, H3), 6Á25 (d,
1H, J ˆ 2Á9 Hz, H8), 6Á33 (dd, 1H, J ˆ 8Á8, 2Á9 Hz,
H6), 6Á55 (d, 1H, J ˆ 8Á8 Hz, H5). IR (KBr) n 3450,
N-((3-Phenyl-1,4-benzodioxin-2-yl)methyl)acet-
amide (8d)
This compound was prepared from 7d according to
the procedure described for 8c (white solid, yield
83%), mp 181±182ꢀC. 1H NMR (CDCl3) d 2Á04 (s,
3H, CH3), 4Á06 (d, 2H, J ˆ 5Á2 Hz, CH2N), 5Á76 (br
s, 1H, NH), 6Á74±6Á90 (m, 4H, Harom), 7Á39±7Á51
(m, 5H, Harom). IR (KBr) n 3300±3160, 1640,
1
3055, 1615, 1265 cm . MS (IC=NH3) m=z 279
(M ‡ 1). Calculated for C14H18N2O4: C, 62Á50; H,
5Á86; N, 7Á15. Found: C, 62Á71; H, 5Á97; N, 7Á01.
1
1265 cm . MS (IC=NH3) m=z 282 (M ‡ 1). Cal-
Pharmacology
culated for C17H15NO3: C, 73Á33; H, 4Á58; N, 5Á46.
Found: C, 73Á57; H, 4Á41; N, 5Á57.
Melatonin receptor-binding assays. The af®nity of
the compounds was assessed in chicken brain or
ovine pars tuberalis membranes. Chickens (Gallus
domesticus), 12-days-old, were killed and the
brains were quickly removed, prepared at 4ꢀC
according to the method described by Yuan &
Pang (1991) and stored at 80ꢀC. Membrane
samples were incubated at 25ꢀC for 60 min with
50 pM 2-[125I]-iodomelatonin and two concentra-
N-((7-Methoxy-1,4-benzodioxin-2-yl)methyl)cyclo-
propylamide (9)
To a solution of amino compound 7a (0Á7 g,
3Á6 mmol) in anhydrous dichloromethane (15 mL)
were added slowly at 0ꢀC triethylamine (2Á5 mL,
18 mmol) and cyclopropanecarbonyl chloride
(0Á467 g, 4Á48 mmol). After stirring for 20 min at
the same temperature under inert atmosphere and
after addition of 1 M HCl to pH 1, the phases were
separated and the aqueous layer was extracted with
dichloromethane. Subsequently, the organic layers
were combined, dried over magnesium sulphate,
and evaporated in-vacuo to dryness. The residue
was chromatographed on silica gel (eluent:
methanol±dichloromethane, 2:98) to provide 9 in
8
6
tions (10 and 10 M) of competing ligand in
triplicate. The most potent compounds were eval-
uated at ten concentrations, in triplicate. Melatonin
was tested as described by Yuan & Pang (1991).
Non-speci®c binding was de®ned with 1 mM mela-
tonin. Results were expressed either as percentage
inhibition of speci®c binding or as IC50. Fresh
ovine pars tuberalis tissue was obtained from a
local abattoir from animals of mixed age and sex.
Preparation of membranes and primary culture of
pars tuberalis cells has been described (Morgan et
al 1989a, b). For competitive binding experiments
membranes were incubated with 50 pM 2-[125I]-
iodomelatonin and 11 concentrations of competing
ligand for 2 h at 37ꢀC. Melatonin was tested as
reference. Individual compounds were assayed in
triplicate and the values expressed as IC50.
1
82% yield as a white solid, mp 136±137ꢀC. H
NMR (CDCl3) d 0Á72±0Á78 (m, 2H, CH2), 0Á97±
1Á02 (m, 2H, CH2), 1Á32±1Á40 (m, 1H, CH), 3Á71 (s,
3H, OCH3), 3Á82 (d, 2H, J ˆ 5Á1 Hz, CH2N), 5Á80±
6Á01 (m, 1H, NH), 5Á94 (s, 1H, H3), 6Á28 (d, 1H,
J ˆ 2Á9 Hz, H8), 6Á35 (dd, 1H, J ˆ 8Á8, 2Á9 Hz,
H6), 6Á57 (d, 1H, J ˆ 8Á8 Hz, H5). IR (KBr) n
1
3345±3250, 1630, 1265 CM . MS (IC=NH3) m=z
262 (M ‡ 1). Calculated for C14H15NO4: C, 63Á33;
H, 5Á60; N, 5Á99. Found: C, 63Á07; H, 5Á67; N, 6Á12.
Cyclic AMP studies
The compounds (10 M) were tested seperately
5
9
and were compared with melatonin (10 M) for
N-((7-Methoxy-1,4-benzodioxin-2-yl)methyl)-N
-propylurea (10)
6
inhibitory potency against forskolin (10 M)-sti-
To a solution of compound 7a (1Á2 g, 1Á1 mmol) in
anhydrous toluene (15 mL) was added n-propyl
isocyanate (0Á78 g, 9Á17 mmol). The reaction mix-
ture was stirred for 4 h at room temperature under
inert atmosphere. A few drops of water were added
mulated cAMP production. The experiments were
carried out in triplicate. A cAMP index (F=D) was
determined for each compound. The melatonin
inhibition response is de®ned as an activity index of
1. Under these conditions, a compound which

204
A. MAMAI ET AL
mimics the effect of melatonin will produce an
activity index equal or close to 1. A weak agonist is
characterized by an activity index between 0Á4 and
0Á8.
sodium hydrogenocarbonate in dimethylacetamide.
The vinylstannanes were then engaged in a cou-
pling reaction with iodobenzene. The optimum
conditions for the reaction were obtained using
a freshly prepared tetrakis (triphenylphosphine)-
palladium catalyst in re¯uxing dioxane in the pre-
sence of copper iodide (Khatib et al 1997). Com-
pounds 3c and 3d were obtained in acceptable
overall yield from 3a and 3b.
Results and Discussion
Preparation of amino derivatives 7a, 7c and 7d is
reported in Figure 3. Ammonolysis in alcoholic
ammonia of the compounds 2a, 2c and 2d gave the
corresponding carboxamides in very good yields.
The treatment of 5a, 5c and 5d with phosphorus
oxychloride in pyridine at re¯ux provided com-
pounds 6a, 6c and 6d in 79±82% yields. Reduction
of these nitriles using lithium aluminium hydride in
ether gave amines 7a, 7c and 7d in 68% yield.
Amine 7b was synthesized by a procedure descri-
bed previously (Guillaumet et al 1984).
Figure 4 illustrates the synthesis of amides 8, 9
and 10. The acetylation of the amines 7 with acetic
anhydride in pyridine at 0ꢀC gave the correspond-
ing acetamides 8 in very good yields. The con-
densation of cyclopropanecarbonyl chloride with
amine 7a in a mixture of dichloromethane and
triethylamine gave compound 9 in 82% yield. The
urea 10 was prepared in satisfactory yield by
reaction of the amine 7a with n-propyl isocyanate
at room temperature in toluene.
Chemistry
The 3-phenyl-2-carbethoxy[1,4]benzodioxin deri-
vatives 2c and 2d were synthesized as shown in
Figure 2. O-Methylation of hydroxy ester 1 (Guil-
laumet et al 1994) was carried out in N,N-dime-
thylformamide in the presence of sodium hydride
and iodomethane to give 2a in 97% yield. Alkaline
hydrolysis of esters 2a and 2b (Coudert et al 1978)
at 0ꢀC, followed by acid work-up, provided the
corresponding acids 3a and 3b in excellent yields.
We adopted the palladium-catalysed cross-coupling
of organotin reagents with organic halides which
offers a method of C C bond formation remark-
able for its ef®ciency and selectivity (Stille 1986).
We had previously reported that 1,4-benzodioxin-
2-carboxylic acids were easily substituted at posi-
tion 3 via the corresponding lithio derivatives
(Guillaumet et al 1979). We expected that the
required vinylstannanes could be obtained in the
same way. When reacted with lithium diisopropy-
lamide at 78ꢀC, compounds 3a and 3b led to the
corresponding metalled heterocycles which were
reacted with tributyltin chloride providing vinyl-
stannanes. After hydrolysis, these were esterifed by
treatment with iodomethane in the presence of
Pharmacology
Compounds 8a±d, 9 and 10 were evaluated in
binding experiments on ovine pars tuberalis and
Figure 2. Synthesis of 3-amidomethyl-1,4-benzodioxin derivatives 2c and 2d.

SUBSTITUTED 2-AMIDOMETHYL[1,4]BENZODIOXINS
205
Figure 3. Preparation of amino derivatives 7a, 7c and 7d.
Figure 4. Synthesis of amides 8, 9 and 10.
Bartness, T. J., Powers, J. B., Hastings, M. H., Bittman, E. L.,
Goldman, B. D. (1993) The timed infusion paradigm for
melatonin delivery: what has it taught us about the melato-
nin signal, its reception, and the photoperiodic control of
seasonal responses? J. Pineal. Res. 15: 161±190
Copinga, S., Tepper, P. G., Grol, C. J., Horn, A. S., Dubocov-
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non-indolic melatonin-like agents. J. Med. Chem. 36: 2891±
2898
chicken brain membranes. Compared with mela-
tonin (IC50 ˆ 0Á15 and 0Á2 nM on ovine pars
tuberalis and chicken brain, respectively), all the
compounds presented a much lower af®nity for
melatonin receptors. Amides 8a±d were poorly
active (IC50 > 1 mM) but 9 and 10 showed greater
af®nity (IC50 ˆ 0Á33 and 0Á66 mM, respectively).
Of the derivatives studied, 10 presented a partial
agonist pro®le.
Coudert, G., Guillaumet, G., Loubinoux, B. (1978) Synthesis
of 1,4-benzodioxins. Tetrahedron Lett. 1059
In conclusion, this study represents a convenient
and effective synthetic pathway to substituted
benzodioxin derivatives bearing an amidomethyl
group at the 3 position. Unfortunately, only com-
pounds 9 and 10 showed af®nity vs melatonin in
the micromolar range.
Davies, D. J., Garrat, P. J., Tocker, D. A., Vonhoff, S., Davies,
J., Teh, M. T., Sugden, D. (1998) Mapping the melatonin
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