5-HT(1D) receptor agonist properties of novel 2-[5- [[(trifluoromethyl)sulfonyl]oxy]indolyl]ethylamines and their use as synthetic intermediates

Article abstract of DOI:10.1021/jm9604890

2-[5-[[(Trifluoromethyl)sulfonyl]oxy]-1H-indol-3-yl]ethylamine (18), its N,N'-di-n-propyl (12), N,N-diethyl (13), and N,N-dimethyl (14) derivatives, and 4-[3-[2-(N,N-dimethylamino)ethyl]-1H-indol-3-yl]-N-(p- methoxybenzyl)acrylamide (GR46611, 19) were syn

Full text of DOI:10.1021/jm9604890

J . Med. Chem. 1996, 39, 4717-4726
4717
5-HT1D Recep tor Agon ist P r op er ties of Novel
2-[5-[[(Tr iflu or om eth yl)su lfon yl]oxy]in d olyl]eth yla m in es a n d
Th eir Use a s Syn th etic In ter m ed ia tes
Tjeerd A. Barf,* Peter de Boer, and Ha˚kan Wikstro¨m
Department of Medicinal Chemistry, University of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, The
Netherlands
Stephen J . Peroutka
Spectra Biomedical Inc., 4040 Campbell Avenue, Menlo Park, California 94025
Kjell Svensson, Michael D. Ennis, Nabil B. Ghazal, J ames C. McGuire, and Martin W. Smith
CNS Diseases Research, Structural Analytical, and Medicinal Chemistry Cell Biology and Inflammation Research,
Chemical and Biological Screening, Pharmacia & Upjohn Inc., Kalamazoo, Michigan 49001
Received J uly 5, 1996X
2-[5-[[(Trifluoromethyl)sulfonyl]oxy]-1H-indol-3-yl]ethylamine (18), its N,N-di-n-propyl (12),
N,N-diethyl (13), and N,N-dimethyl (14) derivatives, and 4-[3-[2-(N,N-dimethylamino)ethyl]-
1H-indol-3-yl]-N-(p-methoxybenzyl)acrylamide (GR46611, 19) were synthesized and tested for
binding affinities to cloned 5-HT1A, 5-HT1DR, 5-HT1Dâ, and D2 receptors. In addition, the intrinsic
efficacy was measured as the reduction of forskolin-stimulated cAMP in cells transfected with
5-HT1DR and 5-HT1Dâ receptors in vitro. The 5-substituted indolylethylamines investigated
displayed agonist activity at the 5-HT1D receptors with varying degrees of preference for the
5-HT1DR vs the 5-HT1Dâ receptors. The primary amine and N,N-dimethyl substitution seemed
to be optimal for 5-HT1DR affinity. Furthermore, the N,N-diethyl (13) and N,N-dimethyl (14)
derivatives showed a 10-25 times preference for the 5-HT1DR vs the 5-HT1Dâ receptor. In
addition, all of the novel compounds showed affinity for the 5-HT1A receptor in vitro (Ki values
ranging from 18 to 40 nM). The most promising derivative 14 was virtually devoid of central
5-HT1A agonist activity in rats, as determined by in vivo biochemical assays. Paradoxically,
14, like 19, induced a hypothermic response and a decrease in 5-HIAA levels in the prefrontal
cortex and hypothalamus in guinea pigs after systemic administration. Sumatriptan failed to
produce either of these effects due to a poor brain penetration.
In tr od u ction
clinical effectiveness of these agents in the treatment
of migraine. In addition, the antimigraine action of
these agents has been attributed to other, both periph-
eral and central, mechanisms mediated by 5-HT1D
receptors.13 Moreover, the functional distinction be-
tween the 5-HT1DR and 5-HT1Dâ receptor subtypes is
unclear. Obviously, selective agonists and antagonists
are needed for unraveling the role of 5-HT1D receptors
in the CNS and in the periphery. 5-HT1D receptor
antagonists, such as (o-methoxyphenyl)piperazine de-
rivative 4 (GR127935)14 and (o-hydroxyphenyl)propyl-
amine derivative 5 (GR55562)15 are cautiously sug-
gested to serve as centrally acting antidepressants,
alone or in combination with SSRIs (selective serotonin
reuptake inhibitors).16
In almost all 5-HT1D receptor agonists reported to
date, a 5-substituted tryptamine serves as a structural
template. It seems that selectivity for either one of the
5-HT1 receptor subtypes can be achieved by modifying
substituents at the 5-position of the indole portion and
at the basic nitrogen atom of the ethylamino side
chain.17 Certain, mostly hydrogen bond accepting (aro-
matic), heterocycles have proven to be viable replace-
ments for the C-5 hydroxy substituent of serotonin
itself.18 Other groups embarked on the synthesis of
conformationally restricted tryptamine analogues.19
However, few data have been published on the affinity
5-HT1D receptors were first defined in bovine caudate
and subsequently in the brains of other species, includ-
ing human.1,2 The 5-HT1D receptor is the most abun-
dant 5-HT1 receptor subtype in the mammalian central
nervous system (CNS), existing as a presynaptic het-
eroreceptor or a terminal autoreceptor, activation of
which inhibits serotonin release.3,4 Human genes en-
coding for the 5-HT1DR and 5-HT1Dâ receptor have
recently been cloned,5,6 raising questions about which
of the two receptors is relevant to reported pharmaco-
logical effects. The mRNAs for the 5-HT1DR and 5-HT1Dâ
receptors appear to codistribute in the brain of non-
rodent species; however, the density of the 5-HT1DR
receptor mRNA is much lower.7 It seems that the
5-HT1Dâ receptors constitute the human counterpart of
rodent 5-HT1B receptors, and have also been identified
in vascular smooth muscle mediating contraction.8,9
Stimulation of the former receptors by selective 5-HT1Dâ
receptor agonists such as sumatriptan (1, Figure 1)10
and newer 5-C-substituted tryptamine derivatives such
as 2 (MK-462)11 and 3 (311C90)12 may account for the
* Address correspondence to T. A. Barf, Department of Medicinal
Chemistry, University of Groningen, Antonius Deusinglaan 1, 9713
AV Groningen, The Netherlands.
X Abstract published in Advance ACS Abstracts, October 15, 1996.
S0022-2623(96)00489-X CCC: $12.00 © 1996 American Chemical Society

4718 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Barf et al.
Ta ble 1. Physical Data of Compounds 6-14, 18, and 19
compd
R′
OBz
OBz
OBz
OH
R′′
salt
mp (°C)
formulaa
6
7
8
9
n-propyl oxalate 128-130 C23H30N2O‚C2H2O4
ethyl oxalate 154-156 C21H26N2O‚C2H2O4
methyl oxalate 167-170 C19H22N2O‚C2H2O4
n-propyl
ethyl
135-136 C16H24N2O
10 OH
11 OH
12 OTf
13 OTf
14 OTf
18 OTf
oxalate 226-228 C14H20N2O‚0.6C2H2O4
methyl oxalate 90-93 C12H16N2O‚C2H2O4‚H2O
n-propyl oxalate 148-150 C17H23N2O3SF3‚C2H2O4
ethyl
oxalate 142-145 C15H19N2O3SF3‚C2H2O4
methyl oxalate 168-171 C13H15N2O3SF3‚C2H2O4
H
oxalate 166-167 C11H11N2O3SF3‚C2H2O4‚
H2O
19 p-MBAAb methyl
C23H27N3O2‚0.5H2O
a
Empirical formula; errors for elemental analysis of all com-
pounds analyzed were within 0.4% of theory for C, H, N. b p-MBAA
means (p-methoxybenzyl)acrylamide.
Sch em e 1.a Synthetic Outline of Compounds 6-14
a
F igu r e 1.
Reagents: (a) Ammonium formate, 10% Pd/C, 96% EtOH; (b)
H2 (4 atm), Pd/C, MeOH; (c) PhN(SO2CF3)2, Et3N, CH2Cl2.
of ligands for 5-HT1DR and 5-HT1Dâ receptors individu-
ally. Sumatriptan, and other reported 5-HT1D receptor
ligands possess little or no selectivity for 5-HT1DR or
5-HT1Dâ receptor binding sites.
tamines, as is exemplified by the one-step synthesis of
the potent 5-HT1A/1D receptor agonist 19 from compound
14. The 5-HT1DR and 5-HT1Dâ receptor-mediated inhibi-
tion of forskolin-stimulated cAMP formation was mea-
sured for a series of prepared compounds. In case of
compounds 14 and 19, these data are substantiated by
neurochemical data and hypothermic effects.
Recently, the [(trifluoromethyl)sulfonyl]oxy (triflate)
group has been successfully applied as a bioisostere of
a hydroxy or a methoxy functionality in a number of
2-aminotetralins.20 These dopaminergic and seroton-
ergic ligands generally displayed improved pharmaco-
kinetic properties compared to their hydroxy/methoxy
analogues while exhibiting the same pharmacological
profile. The enhanced 5-HT1D receptor affinities of the
8-triflate-substituted 2-aminotetralins relative to the
hydroxy derivatives20 prompted us to replace the [(N-
methylamino)sulfonyl]methylene group of sumatriptan
by a triflate group, enabling us to investigate the affinity
Ch em istr y
The triflates 12-14 were synthesized starting from
the N,N-dialkylated 2-[5-(benzyloxy)indolyl]ethylamines,
which were prepared according to literature proce-
dures.23 Catalytic debenzylation in the presence of
ammonium formate or H2 atmosphere resulted in the
corresponding phenols. The triflates were synthesized
by treatment with N-phenyltrifluoromethanesulfo-
nimide and a base (Table 1).24 The steps of the process
are shown in Scheme 1. Since serotonin itself could not
be directly triflated, the amine functionality was pro-
tected first by a phthalimido group.25 After triflation,
the amine 18 was obtained by deprotection with hydra-
zine (Scheme 2).26 The synthesis of 19 was effected via
coupling of the triflate analogue 14 and (p-methoxyben-
zyl)acrylamide in the presence of Pd(OAc)2 and bis(1,3-
diphenylphoshino)propane (dppp).21
of these new tryptamine analogues to 5-HT1A, 5-HT1DR
,
and 5-HT1Dâ receptor subtypes. N,N-Dialkyl substitu-
ents were introduced in order to gain more insight into
the structure-affinity relationships (SAFIR) and struc-
ture-activity relationships (SAR) of tryptamine deriva-
tives. Furthermore, aromatic triflates may serve as key
intermediates in the synthesis of phenyl ring substi-
tuted compounds, as was shown in a number of triflated
2-aminotetralins21 and phenylpiperidines.22 Correspond-
ingly, this chemistry is applicable on triflated tryp-

5-HT1D Receptor Agonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4719
Sch em e 2.a Synthetic Outline of Compounds 14, 18, and 19
a
Reagents: (a) N-EtCO2-phth, 10% NaHCO3 (pH 8), THF/H2O; (b) PhN(SO2CF3)2, Et3N, CH2Cl2; (c) H2NNH2‚H2O, EtOH; (d) 37%
formaldehyde, NaCNBH3, pH 5, CH2Cl2; (e) (p-methoxybenzyl)acrylamide, Pd(OAc)2, dppp, DMF, 85 °C.
Ta ble 2. Affinities at Cloned 5-HT1A, 5-HT1DR/â, D2, 5-HT1DR, and 5-HT1Dâ Receptors
A. Cloned 5-HT1A, 5-HT1DR/â, and D2 Receptors
Ki (nM)a
select. 5-HT1DR
vs 5-HT1Dâ
b
b
b
c
compd
5-HT1A
5-HT1DR
5-HT1Dâ
D2
12
13
14
18
19
1
23 (19-27)
27 (20-35)
190 (161-224)
12 (11-14)
3.2 (2.8-3.6)
2.8 (2.5-3.1)
0.3 (0.2-0.5)
5.7 (2.9-9.5)
246 (198-306)
171 (140-210)
32 (28-36)
502 (354-712)
637 (479-994)
538 (379-764)
658 (472-918)
1.3
14.3
10.0
5.0
0.7
3.8
40 (32-50)
18 (15-22)
1.3 (0.8-2.1)
341 (283-522)
14 (11-16)
0.2 (0.1-0.3)
22 (19-27)
>1000
>218
B. Cloned 5-HT1DR and 5-HT1Dâ Receptors
IC50 (nM)d
e
e
f
compd
5-HT1DR
5-HT1Dâ
log D
6
7
8
470 ( 100
110 ( 7
25 ( 4
550 ( 200
260 ( 60
76 ( 10
2600 ( 500
690 ( 100
660 ( 50
530 ( 30
98 ( 6
3.08
2.18
2.28
1.24
0.28
2.21
1.31
1.41
-0.24
1.71
9
820 ( 100
77 ( 20
170 ( 10
21 ( 2
10
12
13
14
18
19
8.4 ( 1
NTg
NT
NT
NT
a
Ki values for displacement of the 5-HT1A receptor agonist [3H]-8-OH-DPAT, the 5-HT1DR/â agonist [3H]-5-HT, and the dopamine D2
receptor agonist [3H]U86170. b Method A; data from cloned human receptors expressed in CHO-K1 cells. Mean values (n ) 3). Parentheses
contain 95% confidence intervals. c Data from cloned rat receptors expressed in CHO-K1 cells. IC50 values for displacement of the 5-HT1DR
d
and 5-HT1Dâ receptor agonist [3H]-5-HT. e Method B; data from human 5-HT1DR and 5-HT1Dâ receptor clones, expressed in a human
embryonic kidney (HEK 293) cell line. Mean value ( SEM (n ) 3). f Calculated with Pallas 1.2 (CompuDrug Chemistry Ltd., 1994). NT
g
means not tested.
Ta ble 3. Intrinsic Efficacy in Cells Transfected with Human
5-HT1DR or 5-HT1Dâ Receptors
P h a r m a cology
cAMP (pmol)a
In Vitr o Bin d in g. The abilities of the new com-
pounds to displace the radioligands [3H]-8-OH-DPAT (5-
HT1A), [3H]-5-HT (5-HT1DR and 5-HT1Dâ), and [3H]U-
86170 (D2) were assessed in mammalian receptor clones
expressed in CHO cells (Table 2A, method A). In
addition, the compounds were evaluated for their in
vitro binding affinity at 5-HT1DR and 5-HT1Dâ human
receptor clones expressed in a human embryonic kidney
(HEK 293) cell line (Table 2B, method B).
compound
5-HT1DR (% 5-HT)
5-HT1Dâ (% 5-HT)
forskolin control
5-HT (1 µM)
1 (1 µM)
12 (10 µM)
13 (10 µM)
64 ( 3 (0)
166 ( 6 (0)
13 ( 1 (100)
24 ( 1 (93)
88 ( 11 (51)
37 ( 3 (84)
27 ( 1 (100)
30 ( 1 (92)
36 ( 4 (75)
29 ( 3 (94)
forskolin control
5-HT (1 µM)
1 (1 µM)
14 (10 µM)
18 (10 µM)
19 (1 µM)
61 ( 4 (0)
232 ( 8 (0)
17 ( 1 (100)
32 ( 2 (93)
28 ( 1 (95)
18 ( 2 (100)
NTc
20 ( 1 (100)
31 ( 4 (75)
30 ( 1 (75)
19 ( 2 (102)
cAMP Assa y. The functional cAMP assay using the
cloned human 5-HT1DR and 5-HT1Dâ receptors was
employed as previously described.27,28 Compounds 12,
13, 14, 18, and 19 were tested at 10 µM, and the agonist
inhibition was calculated as a percent of the 5-HT
control (Table 3).
(104)b
a
Values are expressed as means ( SEM (n ) 3). Data within
b
parentheses denote percent of 5-HT’s response. Tested in a
separate assay. c NT means not tested.
In Vivo Bioch em istr y. The synthesis rate of 5-HT
in terminal brain areas is inhibited by 5-HT1A receptor
agonists due to stimulation of the somatodendritic
5-HT1A receptors in the raphe nuclei.29 The effect of

4720 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Barf et al.
Ta ble 4. Effect of 14 on Rat Brain 5-HT Synthesis (5-HTP Accumulation) in Vivo in Reserpinized and Nonpretreated Rats
reserpinized (5-HTP accumulation)a
normal (5-HT levels)b
compound
striatum
accumbens
frontal cortex
hippocampus
10 µmol/kg
50 µmol/kg
saline
14
0.37 ( 0.03
0.28 ( 0.01
0.49 ( 0.07
0.40 ( 0.02
0.47 ( 0.09
0.37 ( 0.01
0.28 ( 0.02
0.28 ( 0.03
%
75.7
81.6
78.7
100.0
108 ( 10.5
75 ( 4.0
a
The animals received reserpine 24 h before decapitation and 45 and 30 min before the test drug (10 µmol/kg) and NSD 1015 (100mg/
kg) were administered, respectively. Shown are the [5-HTP] in µg/g wet tissue (means ( SEM) in striatum (n ) 8), accumbens (n ) 4),
b
frontal cortex (n ) 4), and hippocampus (n ) 4), after sc administration of saline or compound 14 (10 µmol/kg). Determined by in vivo
microdialysis. The values are percent of control 5-HT levels, means ( SEM (n ) 3), 1 h after sc administration of compound 14.
Ta ble 5. Effects on Guinea Pig Rectal Temperature and Brain 5-HT Turnover
neurochemistry (% of vehicle controls)b
hypothalamus
prefrontal cortex
dose,
hypothermia,a
compound
µmol/kg, sc
0-60 min, ∆ °C
5-HT
5-HIAA
5-HT
5-HIAA
saline
14
+0.18 ( 0.15
100 ( 6
97 ( 13
123 ( 16
NTc
121 ( 11
122 ( 5
134 ( 17
100 ( 5
66 ( 16
54 ( 16*
NT
49 ( 7**
133 ( 13
37 ( 7**
100 ( 5
104 ( 8
129 ( 1
NT
94 ( 6
119 ( 6
126 ( 2
100 ( 9
59 ( 9***
46 ( 6***
NT
37 ( 7***
129 ( 14
34 ( 8**
12.5
50
12.5
50
25
6.25
-1.70 ( 0.43*
-1.72 ( 0.36**
-1.62 ( 0.35**
-2.62 ( 0.05***
-0.14 ( 0.13
19
1
8-OH-DPAT
+0.22 ( 0.15
a
The hypothermia is presented as the difference in °C from control. The data represent a 60 min reading and are expressed as mean
b
( SEM (n ) 4-5). The animals were decapitated, 60 min after test drug treatment. Shown are the 5-HT and 5-HIAA levels expressed
as mean ( SEM (n ) 4-5), after sc administration of the test drugs. c NT means not tested. *p < 0.05, **p < 0.01, and ***p < 0.001 vs
vehicle-treated controls.
compound 14 on 5-HT synthesis was measured in four
brain areas in reserpinized rats. 5-Hydroxytryptophan
(5-HTP) accumulation, following decarboxylase inhibi-
tion by (3-hydroxybenzyl)hydrazine (NSD 1015), was
used as an indicator of the 5-HT synthesis rate (Table
4).30 The extracellular 5-HT levels in the hippocampus
were measured by in vivo microdialysis after subcuta-
neous (sc) administration of 14 in normosensitive rats
(Table 4). In addition, the 5-HT and 5-HIAA levels after
administration of 14 and 19 were determined in the
prefrontal cortex and the hypothalamus of guinea pigs
(Table 5).
5-oxygen is not of crucial importance for the 5-HT1D
receptor interaction. Obviously, the triflate derivatives
benefit from their two additional sulfonyl oxygens which
are capable of accepting a hydrogen bond. In addition,
the electron-withdrawing effect of the triflate substitu-
ent may contribute to a putative interaction of the indole
portion with the active-site surrounding amino acid
residues.
Since the novel triflate derivatives 12-14 and 18
displayed the most interesting binding profiles, these
compounds were subjected to the in vitro 5-HT1D recep-
tor agonist assays. In the in vitro binding assays, the
N,N-di-n-propyl-substituted analog 12 showed a com-
paratively weak affinity (Ki values around 200 nM) for
both 5-HT1D receptor subtypes, while the affinity for the
5-HT1A site was higher (23 nM, Table 2A). The IC50
values (Table 2B) were more distinct showing a 4-fold
preference for the the 5-HT1DR vs 5-HT1Dâ binding site
(170 and 660 nM, respectively). Despite these moderate
affinities, compound 12 reduced the formation of cAMP,
in both the 5-HT1DR and 5-HT1Dâ receptor assays;
however, the intrinsic activity appeared to be higher in
the former (Table 3).
Hyp oth er m ia . The ability of compounds 14 and 19
to induce hypothermia in guinea pigs after sc admin-
istration was tested in a 60-min reading (Table 5).
Resu lts a n d Discu ssion
Str u ctu r e-Affin ity a n d Str u ctu r e-Activity Re-
la tion sh ip s. From the data presented in Table 2, two
clear trends can be observed. First, the size of the N,N-
dialkyl group dramatically influences the affinity for
both 5-HT1D receptor subtypes, and second, the nature
of the 5-O-substituent is of major importance for both
the affinity and the selectivity for these subtypes. N,N-
Dimethyl substitution (IC50 values 8.4-25 nM) improves
the affinity for 5-HT1DR sites by approximately 20-fold
as compared to the N,N-di-n-propyl derivatives (Ki
values 170-820 nM), but only 7-fold in case of 5-HT1Dâ
receptors (Table 2B). Notably, bulk at the protonated
amine site of the tryptamine is poorly tolerated by the
latter receptor, resulting in moderate binding affinities
even for the N,N-dimethyl derivatives. Within the N,N-
di-n-propyl derivative series the triflate group enhances
the affinity for 5-HT1DR sites by 3- and 5-fold relative
to the benzyloxy and hydroxy substituents, respectively.
Similar trends are found in the N,N-diethyl- and N,N-
dimethyl-substituted tryptamines. The comparatively
low affinities of the 5-benzyloxy and 5-hydroxy deriva-
tives for the 5-HT1D receptor subtypes suggest that the
As already indicated, the 5-HT1DR receptor affinity
was greatly improved in the N,N-diethyl-substituted
analogue 13. This compound showed approximately a
15-25-fold preference for the 5-HT1DR vs 5-HT1Dâ recep-
tor, but only a 2-fold preference vs the 5-HT1A receptor
subtype. In the 5-HT1DR and 5-HT1Dâ receptor agonist
assay, a similar and slightly lower intrinsic activity was
observed, respectively, relative to sumatriptan when
tested at 10 µM. Increased affinity at the expense of
selectivity was observed in moving from N,N-diethyl to
N,N-dimethyl substituents. Compound 14 exhibited a
10-12-fold preference for the 5-HT1DR vs 5-HT1Dâ recep-
tor subypes (Ki values (IC50 values) of 3.2 nM (8.4 nM)
and 32 nM (98 nM), respectively). Interestingly, 14
showed both higher affinity for the 5-HT1DR receptor
subtype and a higher 5-HT1DR vs 5-HT1Dâ preference

5-HT1D Receptor Agonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4721
than sumatriptan (1). As expected, both 1 and 14 were
found to be agonists with similar intrinsic efficacy in
both cAMP assays. In addition, the primary amine 18
showed higher affinity for the 5-HT1DR site (Ki ) 2.8
nM). However, the affinities for the 5-HT1A and 5-HT1Dâ
sites were also increased (Ki values of 18 and 14 nM,
respectively). Compound 18 displayed a maximal in-
trinsic efficacy, similar to that of 5-HT in the cAMP
assay. It is noteworthy that even compounds with
moderate affinity for the 5-HT1D receptor subtypes can
fully inhibit the forskolin-stimulated cAMP formation.
F igu r e 2.
In conclusion, the triflate substituted tryptamines
investigated all display agonist activity at the 5-HT1D
receptors with varying degrees of preference for the
5-HT1DR vs the 5-HT1Dâ receptors in vitro. The primary
amine and N,N-dimethyl substitution seemed to be
optimal for 5-HT1DR affinity. Furthermore, the N,N-
dimethyl and N,N-diethyl analogs showed a 10-25-fold
selectivity for the 5-HT1DR vs 5-HT1Dâ receptor. In
addition, all of the compounds showed substantial
affinity for the 5-HT1A receptor in vitro (Ki values
ranging from 18 to 40 nM). Compound 14 seems to have
a weak inhibitory effect on the 5-HT turnover at doses
up to 50 µmol/kg (sc) in rats, but induces pronounced
decreases of 5-HT turnover in guinea pigs. This con-
trasting observation may be explained by the fact that
in these experiments the 5-HT turnover in rats is
predominantly mediated by 5-HT1A cell body autore-
ceptors, whereas the inhibitory presynaptic 5-HT1D
receptors have a major contribution in the observed
effect in guinea pigs. The potential antimigraine action
of compound 14 has been evaluated by means of a
porcine carotid blood flow model and appears to be
equipotent with sumatriptan.33 The question is whether
brain penetration is a desireable property of antimi-
graine agents or not. Animal studies have provided
evidence that if putative antimigraine drugs, such as
3, get access to the CNS, they display central neuronal
actions.13 Whether this provides increased clinical
efficacy in the treatment of migraine is still in debate.
Introduction of a hydrophobic tail on the 5-position
of the indole nucleus seems to favor 5-HT1Dâ affinity as
exemplified by 5-(nonyloxy)tryptamine.31 This 5-HT1D
receptor ligand binds with higher affinity at human
5-HT1Dâ receptors than 5-HT1DR receptors (Ki ) 1.2 vs
16 nM, respectively). Similarly, the (p-methoxybenzyl)-
acrylamido group of 19, allowing for hydrogen bond
formation, is well-tolerated by the 5-HT1A/1D receptor
subtypes. Table 2A shows that 19 potently displaced
the radioligands at these receptor subtypes, exhibiting
Ki values of 1.3 (5-HT1A), 0.3 (5-HT1DR), and 0.2 nM (5-
HT1Dâ), and was found to behave as a full 5-HT1DR
receptor agonist in the cAMP assay. Obviously, both
5-HT1D receptor binding sites contain a (lipophilic)
pocket which can accommodate bulky, hydrogen-bond-
accepting groups. None of the compounds tested showed
appreciable affinity for the dopamine D2 receptor (Table
2A).
P h a r m a cology. The 5-HT1D receptor agonist 14 of
this series was additionally screened for its (unwanted)
central 5-HT1A receptor activity by means of in vivo
biochemical models. In rats, 14 failed to significantly
inhibit the 5-HTP accumulation at a dose of 10 µmol/
kg in the investigated brain areas, although slight
decreases were observed in striatum, accumbens, and
frontal cortex. The inactivity at the 5-HT1A receptor is
substantiated by the microdialysis data, which showed
no differences compared to the control 5-HT levels in
the hippocampus after sc administration of 14 at a dose
of 10 and 50 µmol/kg (Table 4). However, like 19,
compound 14 has a pronounced effect on the 5-HT
turnover in the prefrontal cortex and the hypothalamus
of the guinea pig brain using similar doses. A lower
dose of 14 tested (3.1 µmol/kg, sc) did not produce a
statistical significant effect. As previously reported,
sumatriptan failed to affect guinea pig brain 5-HT
turnover after systemic administration. This is likely
explained by the fact that sumatriptan has a poor
penetration across the blood-brain barrier, as indicated
by its low calculated log D value (-0.53). This hypoth-
esis is further strengthened by the fact that sumatriptan
failed to produce hypothermia, while 19 and 14 signifi-
cantly produced a hypothermic response at the 12.5 and
50 µmol/kg dose, sc.32 The ability of 19 and 14 to lower
the central 5-HT turnover is likely the result from
activation of inhibitory presynaptic 5-HT1D or somato-
dendritic 5-HT1A and/or 5-HT1D receptors. Interestingly,
in contrast to rats and mice, the selective 5-HT1A
receptor agonist 8-OH-DPAT (20, Figure 2) failed to
induce hypothermia in the guinea pig, indicating that
5-HT1D receptors are of higher importance for the
temperature regulation in this species. The levels of
5-HIAA were reduced probably as a result of a stimula-
tion of 5-HT1A cell body autoreceptors.
Exp er im en ta l Section
Gen er a l. 1H and 13C NMR spectra were recorded at 200
and 50.3 MHz, respectively, on a Varian Gemini 200 spec-
trometer. CDCl3 was employed as the solvent unless otherwise
stated. Chemical shifts are given in δ units (ppm) and relative
to TMS or deuterated solvent. Coupling constants are given
in hertz. IR spectra were obtained on a ATI-Mattson spec-
trometer. Elemental analyses were performed in the Mi-
croanalytical department of the University of Groningen or
at Parke-Davis, Warner-Lambert Co. (Ann Arbor, MI) and
were within 0.4%. The chemical ionization (CI) mass spectra
were obtained on a Finnegan 3300 system. Melting points
were determined on a Electrothermal digital melting point
apparatus and are uncorrected. log D values were calculated
with Pallas version 1.2.34
Ma ter ia ls. All 2-[5-(benzyloxy)indolyl]ethylamines were
prepared according literature procedures starting from 5-(ben-
zyloxy)indole.23 Chemicals used were commercially available
(Aldrich) and were used without further purifation.
N ,N -D i-n -p r o p y l-2-[5-(b e n zy lo x y )-1H -in d o l-3-y l]-
eth yla m in e Oxa la te (6). N,N-Di-n-propyl-2-[5-(benzyloxy)-
1H-indol-3-yl]glyoxalylamide (2.20 g, 5.82 mmol) was dissolved
in dry Et2O (80 mL) and dry THF (20 mL) at room tempera-
ture. LiAlH4 (2.40 g, 11 equiv) was added portionwise, and
the reaction mixture was refluxed for 6 h under N2(g). After
the reaction mixture was cooled to room temperature, the
reaction was quenched with the addition of water (2.4 mL),
10% NaOH (2.4 mL), and water (7.2 mL) under N2(g). This
mixture was stirred until the Li salts had turned white. These
salts were filtered and washed with Et2O. The solvent of the
filtrate was evaporated under reduced pressure resulting in a

4722 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Barf et al.
yellow oil (1.83 g, 90%). Conversion into the oxalate and
recrystallization from acetonitrile gave pale brown crystals
(1.68 g, 66%): mp 128-130 °C; IR (KBr, cm-1) 1196 (C-O);
1H NMR (CD3OD) δ 0.94 (t, J ) 7.27, 6H), 1.57-1.76 (m, 4H),
3.03-3.14 (m, 6H), 3.27-3.34 (m, 2H), 5.09 (s, 2H), 6.87 (dd,
J 1 ) 8.98, J 2 ) 2.14, 1H), 7.12 (d, J ) 2.14, 1H), 7.15 (s, 1H),
7.26-7.47 (m, 6H); 13C NMR (CD3OD) δ 10.9, 17.8, 20.8, 53.7,
55.1, 71.7, 102.5, 109.7, 113.1, 113.5, 124.8, 128.1, 128.3, 128.4,
129.1, 133.3, 154.0, 166.5; MS (CI with NH3) m/e 351 (M + 1).
Anal. Calcd for C23H30N2O‚C2H2O4: C, H, N.
N ,N -D ie t h y l-2-[5-(b e n zy lo x y )-1H -in d o l-3-y l]e t h y l-
a m in e Oxa la te (7). Reduction of N,N-diethyl-2-[5-(benzyl-
oxy)-1H-indol-3-yl]glyoxalylamide (1.93 g, 5.51 mmol) was
performed according to the procedure given for the synthesis
of 6, resulting in a brown oil after evaporation of the solvents
(1.70 g, 96%). Conversion to the oxalate and recrystallization
from acetone gave pale green crystals (1.75 g, 77%): mp 154-
156 °C (lit.22 mp 161-162 °C); IR (KBr, cm-1) 1186 (C-O); 1H
NMR δ 1.14 (t, J ) 7.26, 6H), 2.72 (q, J ) 7.27, 4H), 2.79-
2.99 (m, 4H), 5.14 (s, 2H), 6.98 (m, 2H), 7.16-7.55 (m, 7H),
8.24 (br s, NH); 13C NMR δ 11.7, 22.8, 46.6, 53.3, 70.9, 103.4,
111.9, 112.8, 114.0, 122.4, 127.6, 127.7, 127.9, 128.5, 131.5,
137.6, 152.8; MS (CI with NH3) m/e 323 (M + 1). Anal. Calcd
for C21H26N2O‚C2H2O4: C, H, N.
N ,N -Dim e t h yl-2-[5-(b e n zyloxy)-1H -in d ol-3-yl]e t h yl-
a m in e Oxa la te (8). N,N-Dimethyl-2-[5-(benzyloxy)-1H-indol-
3-yl]glyoxalylamide (1.78 g, 5.53 mmol) was converted to 8
according to the procedure given for the synthesis of 6,
resulting in a colorless oil (1.53 g, 95%). This product was
converted to the oxalate salt, and recrystallization from MeOH/
Et2O gave a white solid (1.79 g, 85%): mp 167-170 °C (lit.22
mp 178-179 °C); IR (KBr, cm-1) 1186 (C-O); 1H NMR δ 2.38
(s, 6H), 2.66 (t, J ) 7.32, 2H), 2.95 (t, J ) 7.32, 2H), 5.11 (s,
2H), 6.90 (s, 1H) 6.96 (d, J ) 8.79, 1H), 7.17 (d, J ) 5.12, 2H),
7.41 (m, 5H), 9.03 (br s, NH); 13C NMR δ 23.3, 45.0, 59.9, 70.7,
102.1, 111.7, 112.2, 113.0, 122.5, 127.3, 127.4, 128.2, 131.6,
137.5, 152.5; MS (EIPI) m/e 294 (M+). Anal. Calcd for
C19H22N2O‚C2H2O4: C, H, N.
synthesis of 10, resulting in a purple oil (1.01 g) which
solidified upon standing. Pink crystals (70 mg) were obtained
while being stirred in MeOH (5 mL) and were collected by
filtration on a glass-sintered funnel. The filtrate was taken
up in 10% aqueous Na2CO3 (50 mL), and then the product
amine was extracted into EtOAc (3 × 30 mL). The organic
phases were separated, pooled, dried (MgSO4), and filtered.
The solvent of the filtrate was evaporated under reduced
pressure, leaving a brown oil (0.47 g, 65%): mp 90-93 °C (lit.22
mp 93-94 °C); IR (KBr, cm-1); 1H NMR (CD3OD) δ 2.23 (s,
6H), 2.54-2.82 (m, 4H), 6.71 (d, J ) 8.64, 1H), 6.94 (m, 2H),
7.15 (d, J ) 8.64, 1H); 13C NMR (CD3OD) δ 23.9, 45.0, 60.9,
103.3, 112.2, 112.5, 123.7, 129.0, 132.8, 150.8; MS (EIPI) m/e
204 (M+). Anal. Calcd for C12H16N2O‚C2H2O4‚H2O: C, H, N.
N,N-Di-n -p r op yl-2-[5-[[(tr iflu or om eth yl)su lfon yl]oxy]-
1H-in d ol-3-yl]eth yla m in e Oxa la te (12). N,N-Di-n-propyl-
5-hydroxytryptamine (9, 268 mg, 1.03 mmol), Et3N (290 µL,
2.52 mmol), and PhN(SO2CF3)2 (550 mg, 1.55 mmol) were
dissolved in CH2Cl2 (10 mL), and the mixture was stirred at
room temperature. After 3 h the mixture was diluted with
CH2Cl2 (20 mL) and washed with 10% aqueous Na2CO3 (2 ×
25 mL). The aqueous layers were once more extracted with
CH2Cl2 (30 mL), after which the organic layers were pooled,
washed with brine, and dried over MgSO4. The solvent was
removed in vacuo, leaving a yellow oil which was chromato-
graphed (SiO2, eluting with CH2Cl2/MeOH (5:1)). Pure frac-
tions were pooled and evaporated to dryness, yielding a pale
yellow oil (483 mg). The residual oil was converted to the
oxalate and recrystallized from MeOH/ether, giving white
crystals (251 mg, 51%): mp 148-150 °C; IR (KBr, cm-1) 1225,
1396 (O-SO2); 1H NMR (base) δ 0.92 (t, J ) 7.36, 6H), 1.46-
1.65 (m, 4H), 2.46-2.61 (m, 4H), 2.76-2.97 (m, 4H), 7.04 (dd,
J 1 ) 8.83, J 2 ) 2.37, 1H), 7.09 (s, 1H), 7.34 (d, J ) 8.83, 1H),
7.46 (d, J ) 2.37, 1H), 8.93 (br s, NH); 13C NMR (base) δ 11.8,
19.6, 22.2, 54.2, 55.9, 111.1, 112.2, 114.6, 114.9, 118.8 (q, J )
321, CF3), 124.4, 127.7, 135.0, 143.2; MS (CI with NH3) m/e
393 (M + 1). Anal. Calcd for C17H23N2O3SF3‚C2H2O4: C, H,
N.
N,N-Dieth yl-2-[5-[[(tr iflu or om eth yl)su lfon yl]oxy]-1H-
in d ol-3-yl]eth yla m in e Oxa la te (13). Triflation of the free
base of 10 (200 mg, 0.86 mmol) was performed according to
the procedure given for the synthesis of 12, yielding an oil (317
mg, 100%) after chromatography (SiO2, eluting with CH2Cl2/
MeOH (5:1)). This oil was converted to the oxalic acid salt
and recrystallized from MeOH/Et2O, giving white crystals (247
mg, 63%): mp 142-145 °C; IR (KBr, cm-1) 1221, 1415 (O-
SO2); 1H NMR (base) δ 1.16 (t, J ) 7.27, 6H), 2.84 (q, J ) 7.26,
4H), 2.93 (s, 4H), 6.96 (dd, J 1 ) 8.97, J 2 ) 2.14, 1H), 7.05 (s,
1H), 7.31 (d, J ) 8.97, 1H), 7.43 (d, J ) 2.13, 1H), 9.80 (br s,
NH); 13C NMR (base) δ 10.2, 21.5, 46.8, 52.6, 110.7, 112.5,
112.7, 114.7, 118.8 (q, J ) 321, CF3), 125.0, 127.3, 135.2, 143.2;
MS (CI with NH3) m/e 365 (M + 1). Anal. Calcd for
C15H19N2O3SF3‚C2H2O4: C, H, N.
N ,N -Di-n -p r op yl-2-(5-h yd r oxy-1H -in d ol-3-yl)e t h yl-
a m in e (9). The crystals of 6 (1.51 g, 3.43 mmol) were
dissolved in 95% EtOH (50 mL), after which ammonium
formate (2.16 g, 10 equiv) and Pd/C (10%, 100 mg) were added.
The reaction mixture was stirred at room temperature for 2
days. The solids were filtered over Celite, and the filtrate was
evaporated in vacuo leaving a brown oil. Next, 10% aqueous
Na2CO3 (50 mL) was added, and the product amine was
extracted with EtOAc (3 × 30 mL). The organic phases were
separated, pooled, dried (MgSO4), and filtered. The solvent
was removed under reduced pressure, yielding a pale brown
solid (0.79 g, 89%). Part of this solid (0.28 g) was recrystallized
from acetonitrile to give 0.24 g of brownish crystals: mp 135-
1
136 °C; IR (KBr, cm-1) 3236 (OH); H NMR (CD3OD) δ 0.92
(t, J ) 7.31, 6H), 1.49-1.61 (m, 4H), 2.49-2.57 (m, 4H), 2.71-
2.88 (m, 4H), 6.66 (dd, J 1 ) 8.55, J 2 ) 2.42, 1H), 6.91 (d, J )
2.56, 1H), 6.97 (s, 1H), 7.14 (d, J ) 8.45, 1H); 13C NMR (CD3-
OD) δ 12.0, 20.3, 22.8, 55.4, 56.8, 103.1, 112.0, 112.4, 112.9,
123.6, 129.0, 132.8, 150.7; MS (EIPI) m/e 260 (M+). Anal.
Calcd for C16H24N2O: C, H, N.
N,N-Dim et h yl-2-[5-[[(t r iflu or om et h yl)su lfon yl]oxy]-
1H-in d ol-3-yl]eth yla m in e Oxa la te (14 fr om 11). Triflation
of the free base of 11 (257 mg, 1.26 mmol) was performed as
above, yielding a colorless oil (394 mg, 93%) after column
chromatography (SiO2, eluting with CH2Cl2/MeOH (5:1)). This
oil (369 mg) was converted to its oxalate salt with oxalic acid
and recrystallized from MeOH/Et2O, yielding white crystals
(189 mg, 38%): mp 176-177 °C; IR (KBr, cm-1) 1221, 1415
N,N-Dieth yl-2-(5-h ydr oxy-1H-in dol-3-yl)eth ylam in e Ox-
a la te (10). The crystals of compound 7 (5.00 g, 12.14 mmol)
were dissolved in dry MeOH (200 mL) and hydrogenated over
10% Pd/C in a Parr apparatus under a H2 pressure of 4 atm.
After 4 h the reaction mixture was filtered over Celite by
suction, and after the solvent was evaporated under reduced
pressure a tarry pinkish oil (3.77 g) was obtained. Recrystal-
lization from MeOH/ether gave pale brown crystals (2.57 g;
1
(O-SO2); H NMR (CD3OD) δ 2.64 (s, 6H), 3.05 (s, 4H), 7.07
(d, J ) 8.78, 1H), 7.33 (s, 1H), 7.47 (d, J ) 8.79, 1H), 7.58 (s,
1H); 13C NMR (CD3OD) δ 22.4, 44.1, 59.6, 111.4, 112.3, 113.4,
115.2, 120.0 (q, J ) 321, CF3), 126.6, 128.2, 136.6, 144.3; MS
(CI with NH3) m/e 337 (M + 1). Anal. Calcd for C13H15
N2O3SF3‚C2H2O4: C, H, N.
-
1
66%): mp 226-228 °C; IR (KBr, cm-1) 3300 (OH); H NMR
(CD3OD) δ 1.09 (t, J ) 7.17, 6H), 2.64 (q, J ) 7.27, 4H), 2.72-
2.86 (m, 4H), 6.67 (dd, J 1 ) 8.65, J 2 ) 2.38, 1H), 6.92 (d, J )
2.23, 1H), 6.96 (s, 1H), 7.15 (d, J ) 8.68, 1H); 13C NMR (CD3-
OD) δ 11.0, 22.6, 47.4, 54.1, 103.1, 112.0, 112.4, 112.8, 123.6,
129.0, 132.8, 150.8; MS (CI with NH3) m/e 233 (M + 1). Anal.
Calcd for C14H20N2O‚0.6C2H2O4: C, H, N.
N ,N -D i m e t h y l-2-(5-h y d r o x y -1H -i n d o l-3-y l)e t h y l-
a m in e Oxa la te (11). Compound 8 (1.51 g, 3.93 mmol) was
converted to 11 according to the procedure given for the
N ,N -P h t h a lim id o-2-(5-h yd r oxy-1H -in d ol-3-yl)e t h yl-
a m in e (16). A stirred solution of serotonin creatine sulfate
monohydrate (15, 5.00 g, 12.35 mmol) in H2O (20 mL) and THF
(20 mL) was basified to pH 8 with 10% NaHCO3, after which
N-carbethoxyphthalimide (2.75 g, 12.35 mmol) was added. The
reaction mixture was stirred for 8 h, during which time a
bright yellow precipitate had formed. The organic solvent was
evaporated in vacuo, and the yellow solid (3.52 g, 94%) was
collected on
a
sintered-glass funnel (P3) and rinsed

5-HT1D Receptor Agonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4723
with Et2O. Recrystallization from EtOH (absolute) gave 3.01
g (80%) of yellow crystals: mp 213-216 °C (lit.25 mp 210 °C);
IR (KBr, cm-1) 1690 (CdO), 3370 (OH); 1H NMR (acetone-d6)
δ 3.06 (t, J ) 8.06, 2H), 3.92 (t, J ) 8.06, 2H), 6.71 (d, J )
8.42, 1H), 7.18 (d, J ) 8.79, 1H), 7.10 (d, J ) 8.42, 2H), 7.53
(br s, 1H), 7.81 (m, 4H), 9.63 (br s, NH); 13C NMR (acetone-d6)
δ 25.2, 39.3, 103.4, 103.5, 111.7, 112.6, 123.7, 124.2, 129.3,
133.3, 134.9, 151.7, 168.7, 206.0; MS (CI with NH3) m/e 393
(M + 1).
N,N-P h th alim ido-2-[5-[[(tr iflu or om eth yl)su lfon yl]oxy]-
1H-in d ol-3-yl]eth yla m in e (17). Triflation of 16 (1.33 g, 4.35
mmol) was performed according to the procedure given for the
synthesis of 12, affording a white solid after chromatography
(SiO2, eluting with CH2Cl2). The solid was recrystallized from
EtOH, yielding colorless needles (1.48 g, 78%): mp 165-166
°C; IR (KBr, cm-1) 1206, 1398 (O-SO2), 1719 (CdO); 1H NMR
δ 3.11 (t, J ) 7.69, 2H), 3.97 (t, J ) 7.69, 2H), 7.04 (d, J )
8.79, 1H), 7.13 (s, 1H), 7.30, (d, J ) 8.78, 1H), 7.55 (s, 1H),
7.67-7.81 (m, 4H), 8.38 (br s, NH); 13C NMR δ 21.6, 35.7,
108.7, 109.6, 110.7, 112.8, 116.3 (q, J ) 321, CF3), 120.7, 122.0,
125.2, 129.5, 131.4, 132.4, 141.0, 165.6; MS (EIPI) m/e 438
(M+). Anal. Calcd for C19H13N2O5SF3: C, H, N.
HRMS calcd (obsd) for C11H13NO2 191.0946 (191.0955). Anal.
Calcd for C11H13NO2: C, H, N.
4-[3-[2-(N,N-Dim eth yla m in o)eth yl]-1H-in d ol-5-yl]-N-(p-
m eth oxyben zyl)a cr yla m id e (19). A 25-mL, oven-dried two-
neck round bottom flask equipped with a water-cooled reflux
condenser was charged with 14 (500 mg, 1.5 mmol), (p-
methoxybenzyl)acrylamide (575 mg, 3.0 mmol), Pd(OAc)2 (13.3
mg, 4 mol %), and 1,3-bis(diphenylphosphino)propane (27 mg,
5 mol %). An argon atmosphere was established, DMF (5.0
mL) and Et3N (0.34 mL, 2.45 mmol) were added via a syringe,
and the reaction mixture was degassed with argon. The
mixture was heated to 85 °C for 18 h, cooled to ambient
temperature, and partitioned between aqueous NaOH (1 N)
and ethyl acetate. The aqueous layer was extracted with ethyl
acetate (2 × 50 mL), and the combined organic layers were
dried over Na2SO4, filtered, and concentrated to give a crude
oil (1.1 g). This oil was purified by flash chromatography on
200 g of silica gel, eluting with CH2Cl2/MeOH/NH4OH (92:7:
1; Rf 0.16) to give 252 mg (44%) of 19 as a tan foam: IR (mull,
cm-1) 1610, 1652; 1H NMR δ 2.34 (s, 6H), 2.63 (t, 2H), 2.93 (t,
J ) 7.3, 2H), 3.79 (s, 3H), 4.51 (d, J ) 5.6, 2H), 5.97 (br t, 1H),
6.37 (d, J ) 15.6, 1H), 6.87 (d, J ) 8.6, 2H), 7.01 (s, 1H), 7.26
(m, 3H), 7.35 (d, J ) 8.5, 1H), 7.72 (s, 1H), 7.80 (d, J ) 15.5,
1H), 8.46 (br s, 1H); 13C NMR δ 23.6, 43.3, 45.4, 55.3, 60.2,
111.6, 114.0, 115.0, 117.3, 119.8, 121.3, 122.5, 126.2, 127.7,
129.3, 130.5, 137.2, 142.9, 159.0, 166.4; HRMS calcd (obsd) for
C23H27N3O2 377.2103 (377.2104). Anal. Calcd for C23H27N3O2‚
0.5H2O: C, H, N.
P h a r m a cology. An im a ls. Adult male albino rats of a
Wistar-derived strain (Harlan, Zeist, The Netherlands) weigh-
ing 275-325 g were used. Until experiments, the rats were
housed in groups of six animals in plastic cages under
conditions of constant temperature (20 °C) and humidity with
lights on 06:30 and lights off 17:00. Food and water were
available ad libitum. Animal procedures were conducted in
accordance with guidelines published in the NIH Guide for
the Care and Use of Laboratory Animals, and all protocols were
approved by the Groningen University Institutional Animal
Care and Use Committee.
2-[5-[[(Tr iflu or om eth yl)su lfon yl]oxy]-1H-in dol-3-yl]eth -
yla m in e Oxa la te (18). The N,N-phthalimide 17 (1.07 g, 2.44
mmol) was dissolved in absolute EtOH (50 mL), after which
hydrazine hydrate (2.0 mL) was added. The reaction mixture
was stirred for 0.5 h at room temperature, after which time
the volatiles were removed in vacuo. The residue was refluxed
in CHCl3 for 0.5 h, cooled to ambient temperature, and filtered
in order to remove the solid phthalimidohydrazine. The
filtrate was evaporated in vacuo, leaving a colorless oil which
was converted to the oxalate. The oxalate salt was recrystal-
lized from EtOH/ether, giving 0.86 g (89%) of white crystals:
mp 166-167 °C; IR (KBr, cm-1) 1210, 1412 (O-SO2); 1H NMR
(base) δ 3.10-3.26 (m, 4H), 7.08 (dd, J 1 ) 8.79, J 2 ) 2.19, 1H),
7.36 (s, 1H), 7.48 (d, J ) 8.79, 1H), 7.57 (d, J ) 2.19, 1H); 13
C
NMR (base) δ 24.9, 41.1, 111.6, 111.7, 113.8, 115.6, 120.3 (q,
J ) 320, CF3), 127.4, 128.4, 137.1, 144.7, 166.7, 194.4; MS
(EIPI) m/e 308 (M+). Anal. Calcd for C11H11N2O3SF3‚C2H2-
O4‚H2O: C, H, N.
Dunkin-Hartley guinea pigs were ordered in from Kuipers
Rabbit Ranch (Gary, IN) with a weight range of 225-275 g
approximately 1 week before testing. On arrival the animals
were placed in standard guinea pig cages, six to seven animals
per cage. The ambient temperature of the housing room and
the testing room was 22.2 ( 2.0 °C. The humidity was kept
at 45-55%, and a 12 h light-dark regimen was employed
(lights on between 06:00 and 18:00).
N,N-Dim et h yl-2-[5-[[(t r iflu or om et h yl)su lfon yl]oxy]-
1H-in d ol-3-yl]eth yla m in e Oxa la te (14 fr om 18). To a
magnetically stirred solution of 18 (0.46 g, 1.5 mmol) and 37%
formaldehyde (aqueous, 1.2 mL) in acetonitrile (6 mL) was
added NaCNBH3 (0.29 g, 4.5 mmol). The mixture was acidified
to pH 5 with acetic acid and stirring continued for 3 h. NaOH
(10%, 30 mL) was added, after which the aqueous layer was
extracted with CH2Cl2 (3 × 20 mL). The organic layers were
combined and dried over MgSO4. Filtration and removal of
the solvent in vacuo gave an oil, which was subjected to column
chromatography (SiO2, eluting with CH2Cl2/MeOH (5:1)),
affording 193 mg (38%) of a colorless oil. Recrystallization of
the oxalate salt from MeOH/ether afforded 164 mg (26%) of
white crystals: mp 176-177 °C; all further spectroscopic data
were analoguous to that of 14 prepared from 11.
(p-Meth oxyben zyl)a cr yla m id e. A 500 mL, oven-dried
round bottom flask equipped with a pressure-equalizing drop-
ping funnel was charged with a solution of acryloyl chloride
(2.2 mL, 27 mmol) in CH2Cl2 (100 mL). The flask was cooled
to 15 °C (ethylene glycol/dry ice bath), and the dropping funnel
was charged with a solution of 4-methoxybenzylamine (3.27
mL, 25 mmol) and Et3N (3.5 mL) in CH2Cl2 (100 mL). This
solution was added over a period of 25 min to the acid chloride
solution, stirred for an additional 30 min at -15 °C, and then
slowly warmed to ambient temperature over 1.5 h. The
reaction mixture was poured into an aqueous NaOH solution
and extracted with CH2Cl2 (3 × 300 mL). The combined
organic phases were dried over Na2SO4, filtered, and concen-
trated, affording a pale yellow solid (5.1 g). This solid was
triturated with cyclohexane (4 × 450 mL) to give the title
compound (4.5 g, 97%) as an ivory solid: mp 100-101 °C; IR
(mull, cm-1) 1614, 1654, 1666 (amide); 1H NMR δ 3.79 (s, 3H),
4.42 (d, J ) 5.7, 2H), 5.64 (dd, J 1 ) 10.2, J 2 ) 1.6, 1H), 6.09
Ma ter ia ls. Sumatriptan was obtained from Glaxo (U.K.)
and serotonin, forskolin, and reserpine were purchased from
RBI (Natick, MA). The RIA kit for the cAMP assay was
purchased from Biomedical Technologies (Stroughton, MA).
The radioligand and other compounds were obtained from the
following sources: [
3H]-5-HT (28.2 Ci/mmol) from New En-
gland Nuclear (Boston, MA); 5-HT, pargyline, and Tris-HCl
from Sigma Chemical Co. (St. Louis, MO); STV, DME H-21,
and gentamicin from UCSF (San Francisco, CA); geneticin (G-
418 sulfate), fetal bovine serum, penicillin, streptomycin, and
HAMS #l2 DMEM (50:50) from GIBCO Laboratories (Grand
Island, NY); ascorbic acid from Mallinckrodt Inc. (Paris, KY).
All substances to be tested in rats were dissolved in saline
(0.9% NaCl in distilled water) and administered sc in a volume
of 1.0 mL/kg.
In guinea pig experiments, the compounds were made up
in a 0.25% methyl cellulose in water solution with the addition
of equimolar amounts of citric acid in case the test compound
was a free base. The volume of injection is 5 mL/kg for all
injections. The guinea pigs that were dosed subcutaneous
were injected with a Becton Dickinson 3 mL syringe with a
25 gage five-eighths in. Precision Glide needle both being
disposable.
In Vitr o Bin d in g Assa y. Meth od A. Competition radio-
ligand binding experiments employed 11 drug concentrations
run in duplicate. Radioligands used were [3H]-8-OH-DPAT
(br s, 1H), 6.10 (dd, J 1 ) 16.9, J 2 ) 10.2, 1H), 6.29 (dd, J 1
)
(5-HT1A, 85 Ci/mmol, 1.2 nM), [3H]-5-HT (5-HT1DR and 5-HT1Dâ
,
16.9, J 2 ) 1.5, 1H), 6.85 (d, J ) 8.6, 2H), 7.20 (d, J ) 8.6, 2H);
85 Ci/mmol, 2.6 nM), and [3H]U-86170 (D2-dopamine, 62 Ci/
13C NMR δ 42.9, 55.1, 126.4, 129.0, 129.9, 130.5, 158.8, 165.1;
mmol, 2 nM). Nonspecific binding (75-95% of total) was

4724 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Barf et al.
defined with the following cold compounds added in excess:
lisuride (5-HT1A), serotonin (5-HT1D), and haloperidol (D2).
Total binding was determined with buffer. Buffers (pH 7.4)
used were 50 mM Tris, 5 mM MgCl2 (5-HT1A), the same with
0.1% ascorbic acid (5-HT1D) and 20 mM HEPES, 10 mM MgSO4
(D2). Cloned human receptors permanently expressed in CHO
cells were the source of the 5-HT binding sites, except for the
dopamine D2 receptor which was cloned from the rat.27,35,36
Binding mixtures were made in 96-deep-well titer dishes by
the addition of 50 µL of drug, 50 µL of radioligand, and 800
µL of membranes (20-60 µg of protein) in binding buffer.
After room temperature incubation for 1 h (5-HT1D reactions
were protected from light), reactions were stopped by vacuum
filtration with a TomTec harvester. Counting was with a 1205
Betaplate using Meltilex as scintillant. IC50 values were
estimated by fitting the data to a one-site competition model:
for 5-HTP and L-DOPA (not shown). 5-HTP accumulation was
expressed as [5-HTP] in µg/g wet tissue.
Su r ger y a n d Micr od ia lysis Exp er im en ts. The microdi-
alysis probes that were used were of a ventrical, concentric
design.42 The exposed tip of the dialysis membrane was 4 mm.
The dialysis tube (i.d., 0.22 mm; o.d., 0.31 mm) was prepared
from polyacrylonitrile/sodium methyl sulfonate copolymer (AN
69, Hospal, Bologna, Italy). The microdialysis probes were
implanted under chloral hydrate anesthesia (400 mg/kg, ip)
at the following coordinates: AP -5.2, ML (4.8 relative to
bregma, and V -8.0 below dura (hippocampus). During
surgery, lidocaine HCl salt (6% in saline, brought to pH 6.0
with 1 N NaOH) was used as an adjuvant local anesthesic.
Probes were secured to the skull with two set screws and fast-
securing dental cement. Microdialysis experiments were
carried out 24-48 h after implantation of the probe. Samples
were collected on-line every 15 min in a 20 µL sample loop of
an HPLC system. In brief, the inlet of the microdialysis probe
was connected to a piece of polyethylene tubing (450 × 0.28
mm), whereas the outlet of the microdialysis probe was
connected to a piece of peek tubing (450 × 0.12 mm). The inlet
tube was connected to the perfusion pump and the peek tube
directly into the injection valve of the HPLC apparatus. The
connection with the HPLC equipment introduced a lag time
of about 8 min, for which the presented data are corrected.
With the help of an electronic timer, the injection valve was
held in the load position for 15 min, which was the time needed
to record a complete chromatogram. The perfusion was carried
out with an aCSF solution at a flow rate of 1.5 µL/min using
Carnegie CMA (Stockholm, Sweden) perfusion pump. The
composition of the aCSF solution was (mM): NaCl, 147.0; KCl,
4.0; CaCl2, 1.2; and MgCl2, 1.0. After finishing the experiment,
the rat was terminated with an overdose pentothal and the
brain was fixed with 4% paraformaldehyde via intracardiac
perfusion. Coronal sections (40 µm thick) were cut, and
dialysis probe placement was verified with the help of the atlas
of Paxinos and Watson.43
An a lysis of th e Dia lysa tes. 5-HT was quantified by
HPLC with electrochemical detection. A Shimadzu LC10-AD
pump was used in conjunction with an electrochemical detector
(Antec, Leiden, The Netherlands) set at 650 mV vs an Ag/AgCl
reference electrode. A reversed-phase C18 Supelco LC18DB
column (150 × 4.6 mm; 5 µm) was used. The mobile phase
consisted of an aquous solution of 2.0 g/L of citric acid, 5.0 g/L
of sodium acetate, 100 mg/L of EDTA, 300 mg/L of TMA, 300
mg/L of MSA, 10% methanol (v/v), and 5% acetronitile (v/v)
and was delivered at a flow rate of 1 mL/min. 5-HT eluted
after 8 min.
Gu in ea P ig Br a in Neu r och em istr y. Male Dunkin-Hart-
ley guinea pigs were decapitated 60 min after test drug
administration by means of guillotine. Their brains were
rapidly removed and put on an ice-chilled Petri dish. The
prefrontal cortex and the hypothalamus were dissected, and
the tissue parts were stored at -80 °C until further analysis.
The levels of 5-HT and 5-HIAA were measured by means of
HPLC with electrochemical detection according to methods
described in the literature with minor modifications.30
Hyp oth er m ia . At least 1 h before animals were to be
tested they were removed from the gang cages and placed in
individual plastic cages (20 × 30 × 15 cm) and then taken to
the testing room. The guinea pigs were tested in groups of
five animals per group. The zero rectal temperature was taken
using a Digital Thermometer VWR Scientific Inc. with a range
of -40 to a 300 °F, or -40 to 150 °C. The probe used in this
study is a Yellow Springs Instruments 423-series probe. The
probe was lubricated with a drop of silicon and then inserted
3-4 cm in the rectum and left until the reading on the Digital
Thermometer is stable, usually around 10 s. The temperature
is recorded to the nearest 0.1 °C. This is the control measure-
ment for the other time intervals used which are 30, 60, and
120 min after dosing. In the present study we present data
from the 60 min readings.
Y ) T/(1 + 10logX-logIC50
)
where Y is the specific CPM bound at the concentration X,
and T is the specific bound CPM in the absence of the
competitor. Inhibition constants (Ki) were calculated with the
Cheng-Prushoff equation.37
Meth od B. Human 5-HT1DR and 5-HTlDâ receptor clones
were expressed in a human embryonic kidney 293 (HEK 293)
cell line.38,39 The HEK 293 cells were grown as a monolayer
in 10 mL of HAMS #12 Dulbecco’s Modified Eagle Medium
(50:50) supplemented with 10% fetal bovine serum, penicillin
G (100 units/mL) and streptomycin (10 mg/mL). Confluent
monolayers of the cell lines were harvested (PBS containing
5 mM EDTA) and centrifuged at 480g for 10 min at 40 °C.
The cells were lysed in ice-cold buffer (50 mM Tris-HCl, pH
7.4 containing 5 mM EDTA), homogenized, and sonicated for
10 s. Nuclei and intact cells were removed by centrifugation
at 1000g for 10 min. The supernatant was spun at 35000g
for 30 min, and the pellet, containing the microsomal mem-
brane fraction, was resuspended binding buffer containing 50
mM Tris-HCl, 4 mM CaCl2, 0.1% ascorbic acid, 10 mM
pargyline, and 1 mM leupeptine. The microsomal membrane
suspension was stored at -70 °C.
Radioligand binding assays consisted of 0.1 mL of radioli-
gand (final concentrations: [3H]-5-HT, 0.01-150 nM, 0.8 mL
of tissue suspension (50 mg protein), and 0.1 mL of assay
buffer or displacing drug. All drugs were diluted in assay
buffer. After an incubation of 30 min at 25 °C, assay mixtures
were rapidly filtered through 132 glass fiber filters (Schleicher
and Schuell; Keene, NH) and washed two times with 5 mL of
50 mM Tris-HCl buffer (pH 7.8). The filters were transferred
to plastic counting vials, and radioactivity was measured by
liquid scintillation spectroscopy in 2.5 mL of Bio-Safe II
Scintillation Cocktail (Research Products International Corp.;
Mount Prospect, IL). Specific binding was defined as the
excess over blanks taken in the presence of 10-5 M 5-HT.
Radioligand binding data were analyzed by the EBDA40 and
LIGAND41 programs that utilize the nonlinear least-squares
curve-fitting technique with the Marquardt-Levenberg modi-
fication of the Gauss-Newton method.
F or sk olin -Stim u la ted cAMP In h ibition . The funcional
cAMP assay using the cloned human 5-HT1DR or 5-HT1Dâ
receptors was employed as previously described.26,27 Briefly,
confluent cells were preincubated with R-MEM 10 mM/
HEPES/1 mM IBMX for 10 min and then stimulated with 25
mM forskolin with or without test drug (either 1 µM 5-HT or
10 µM test compound) for 20 min. The reaction was quenched
with TCA and an aliquot assayed for cAMP using a RIA kit.
The results were expressed as pmol cAMP/well (n ) 3) and
agonist inhibition calculated as a percent of 5-HT response.
5-HTP Mea su r em en ts.30 Rats were reserpinized (5 mg/
kg, ip) 24 h prior to administration of the test compound. The
test compound was administered 15 min prior to the admin-
istration of 100 mg/kg NSD. After 30 min the rats were
decapitated and the brain quickly dissected on ice. Striatum,
accumbens, frontal cortex, and hippocampus were stored at
-70 °C until analysis. Before analysis the samples were
homogenized in TCA and centrifuged. The supernatant was
analyzed by means of HPLC with electrochemical detection
Exp r ession of Resu lts a n d Sta tistics. Differences be-
tween 5-HTP concentration of the control and drug treatment
were analyzed with one-way ANOVA followed by a posthoc
t-test. In microdialysis, the average of the last four stable

5-HT1D Receptor Agonists
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