Potent, selective tetrahydro-β-carboline antagonists of the serotonin 2B (5HT(2B)) contractile receptor in the rat stomach fundus

Article abstract of DOI:10.1021/jm960062t

A series of potent, selective 5HT(2B) receptor antagonists has been identified based upon yohimbine, with SAR studies resulting in a 1000-fold increase in 5HT(2B) receptor affinity relative to the starting structure (- log K(B)s > 10.0 have been obtained). These high-affinity tetrahydro-β- carboline antagonists are able to discriminate among the 5HT₂ family of serotonin receptors, with members of the series showing selectivities of more than 100-fold versus both the 5HT(2A) and 5HT(2C) receptors based upon radioligand binding and functional assays. As the first compounds reported with such selectivity and enhanced receptor affinity, these tetrahydro-β- carboline antagonists are useful tools for elucidating the role of serotonin acting at the 5HT(2B) receptor in normal and disease physiology.

Full text of DOI:10.1021/jm960062t

J . Med. Chem. 1996, 39, 2773-2780
2773
P oten t, Selective Tetr a h yd r o-â-ca r bolin e An ta gon ists of th e Ser oton in 2B
(5HT2B) Con tr a ctile Recep tor in th e Ra t Stom a ch F u n d u s
J ames E. Audia,* Deborah A. Evrard, Gwyn R. Murdoch, J ames J . Droste, J effrey S. Nissen, Kathy W. Schenck,
Pawel Fludzinski, Virginia L. Lucaites, David L. Nelson, and Marlene L. Cohen
Lilly Research Laboratories, A Division of Eli Lilly & Company, Indianapolis, Indiana 46285
Received J anuary 22, 1996X
A series of potent, selective 5HT2B receptor antagonists has been identified based upon
yohimbine, with SAR studies resulting in a 1000-fold increase in 5HT2B receptor affinity relative
to the starting structure (-log KBs >10.0 have been obtained). These high-affinity tetrahydro-
â-carboline antagonists are able to discriminate among the 5HT2 family of serotonin receptors,
with members of the series showing selectivities of more than 100-fold versus both the 5HT2A
and 5HT2C receptors based upon radioligand binding and functional assays. As the first
compounds reported with such selectivity and enhanced receptor affinity, these tetrahydro-â-
carboline antagonists are useful tools for elucidating the role of serotonin acting at the 5HT2B
receptor in normal and disease physiology.
Sch em e 1
In tr od u ction
Serotonin is a potent contractile agonist in isolated
smooth muscle from most tissues. The rat stomach
fundus is one of the tissues most sensitive to 5HT-
induced contraction with nanomolar concentrations
producing a pronounced effect.1-8 Considerable effort
from these laboratories and others has allowed for the
characterization of the stomach fundal receptor, cur-
rently designated the 5HT2B receptor.9 The rat 5HT2B
receptor and its human homolog have been recently
cloned, allowing for examination of the binding affinity
of receptor agonists and antagonists as well as studies
of receptor localization and effector coupling.10-14 Such
studies have established the correlation for a series of
ligands between the cloned 5HT2B receptor and the
receptor mediating the contraction in response to 5HT
in the rat stomach fundus.15 Both structurally and
pharmacologically, the 5HT2B receptor is similar to the
other 5HT2 receptors.9,16
Recently receptor agonists and antagonists have been
identified which begin to differentiate among these
closely related receptors (Scheme 1).17-20 To date, the
compounds reported have achieved useful (>100-fold)
selectivities versus the 5HT2A receptor, while discrimi-
nation versus the 5HT2C receptor has proven more
challenging and is only attained with agents with lower
receptor affinity.
Increasingly selective, high-affinity agents should
prove important as tools for the further study of the rat
receptor and its human homolog, for which the message
has been identified in human brain, liver, heart, kidney,
retina, and GI tract.21 To that end, we initiated
structure-activity relationship (SAR) studies targeted
at the development of potent and selective 5HT2B
receptor antagonists based upon the tetrahydro-â-
carboline alkaloid yohimbine (1). Among its multiple
pharmacologic effects, yohimbine has been shown to
antagonize 5HT-induced contractions in the rat stomach
fundus,22 an observation confirmed in our laboratory
(-log KB ) 6.92), although it had 8-fold higher affinity
utilizing the serotonin-induced contraction via the
5HT2B receptor in rat stomach fundus smooth muscle
strips in order to assess directly both affinity and
antagonist activity of the newly synthesized ligands
(Tables 1-5). We initiated such studies with a survey
of several partial structural analogs of yohimbine (2-
7), successively simplifying the molecule, to identify the
key structural elements necessary for potent antago-
nism of 5HT2B receptor-mediated contraction in the rat
stomach fundus.
Removal of the ring E ester and hydroxyl functionality
from yohimbine to afford the parent pentacycle (2,
Scheme 2) greatly simplified the structure and provided
the initial impetus for this exercise by virtue of a modest
increase in affinity relative to yohimbine (1, Table 1).
Further sequential removal of ring E (3) and ring D (4)
resulted in a loss of affinity in each case, suggesting a
possible role for ring E in the ligand-receptor interac-
tions. The related aryl E-ring yohimbine analog 5
possessed modest affinity. However, excising the meth-
ylene linker comprising ring D and exposing a secondary
amine (6) resulted in a 100-fold increase in affinity
relative to yohimbine. In contrast, the alternative ring
as an R2 adrenergic receptor antagonist (-log KB
)
7.77).23,24 Our SAR studies employed functional data,
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
S0022-2623(96)00062-3 CCC: $12.00 © 1996 American Chemical Society

2774 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Audia et al.
Sch em e 2
Sch em e 3
D methylene excision resulted in an inactive tertiary
amine analog (7). Our interest in 6 as a template for
SAR studies was further heightened by the increased
selectivity demonstrated for 5HT2B receptor antagonism
versus R2 receptor antagonism. Whereas yohimbine
possessed higher affinity for R2 receptors as determined
by blockade of the UK14301-induced inhibition of the
twitch response in the guinea pig ileum, the “partial
yohimbine” 6 showed more than 1000-fold selectivity for
the 5HT2B receptor blockade in the rat stomach fundus
condensing tryptamines directly with aza lactones under
our modified hydrolytic Pictet-Spengler protocol (Scheme
3),36 which was developed to increase the breadth of
these SAR studies. All tryptamines employed in this
study are known compounds and were prepared by
literature methods, most frequently Fisher-type indole
syntheses utilizing the requisite hydrazine and a suit-
able 4-aminobutyraldehyde equivalent (Scheme 4).48
Aza lactones were prepared from corresponding ben-
zaldehydes by condensation with N-acetylglycine in
acetic anhydride.37 The aza lactones were converted to
the corresponding phenylpyruvic acids for use in method
A by a two-step hydrolysis with aqueous sodium hy-
droxide followed by aqueous hydrochloric acid, or were
employed directly in method B. Analytical and physical
data for final compounds are reported in Tables 1-3.
(-log KB,
) 9.17 vs -log KB,R2 ) 6.13). Thus,
5HT2B
excision of ring D, as in 6, resulted in improved affinity
for the 5HT2B receptor with 1000-fold selectivity vis a
vis the R2 receptor. On the basis of upon these findings,
we pursued a more detailed examination of this “ABCE
partial yohimbine” tetracyclic system.
Ch em istr y
Resu lts a n d Discu ssion
The “partial yohimbines” 2,25 3,26 4,27 5,28 6,29 7,30 17,31
21,32 22,33 26,34 and 3235 are known compounds whose
syntheses have been reported previously. Novel tet-
rahydro-â-carbolines (18-20, 23-25, 27-31, and 33-
84) were prepared from their parent tryptamines and
suitable carbonyl precursors by two methods. Method
A involved a traditional Pictet-Spengler condensation
of aldehydes and reactive ketones with the requisite
tryptamines.29 Alternatively, method B was employed,
After a brief examination of the carbon tether between
the tetrahydro-â-carboline and the aromatic ring dem-
onstrated a significant advantage for a single methylene
unit (6, 17, 18), we turned our attention to the prelimi-
nary evaluation of substituent effects on the aromatic
ring E (Table 1). While the parent tetracycle 19 had
approximately 5-fold higher affinity than yohimbine,
addition of both electron-withdrawing and electron-
donating substituents to ring E resulted in further

Tetrahydro-â-carboline Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2775
Sch em e 4
Ta ble 1. Physical and Biological Data of Tetrahydro-â-carbolines
rat stomach
compd
n
Y
mol formula
mp (°C)
meth prep
fundus -log KB
1 (yohimbine)
2
3
4
5
6
7
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C21H26N2O3‚HCl
6.92 ( 0.12(3)
7.26 ( 0.06(3)
6.66 ( 0.24(3)
<5.52(3)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
19H24N2
15H18N2‚HCl
11H12N2‚HCl
21H22N2O2‚HCl
20H22N2O2‚HCl
20H22N2O2‚HCl
19H21N2O2‚HCl
21H23N2O2‚HCl
18H18N2‚HCl
<6.52(3)
1
3,4-OMe
9.17 ( 0.11(7)
<5.52(3)
<6.53(3)
0
2
1
1
1
1
1
1
1
1
1
1
1
1
3,4-OMe
3,4-OMe
H
4-Me
4-OMe
7.07 ( 0.18(6)
7.61 ( 0.09(3)
7.84 ( 0.06(3)
8.09 ( 0.07(3)
8.04 ( 0.20(3)
8.22 ( 0.1(3)
7.75 ( 0.07(4)
8.1 ( 0.23(5)
8.4 ( 0.2(3)
8.57 ( 0.11(9)
9.27 ( 0.12(9)
9.35 ( 0.12(9)
7.84 ( 0.12(3)
273-4
A
A
A
A
A
A
A
A
A
A
A
A
19H20N2‚HCl
250-1
19H20N2O‚HCl
19H20N2O‚HCl
18H16Cl2N2‚HCl
20H22N2O2‚HCl
21H24N2O3‚HCl‚H2O
250-1 dec
256-7
3-OMe
3,4-Cl
255-7
2,5-OMe
3,4,5-OMe
3,4-OCH2
3,4-OEt
2-Cl,3,4-OMe
2,3-benzo
3,4-benzo
222-5 dec
175-8
C19H18N2O2‚HCl
272-4 dec
210-2
C
C
C
C
22H26N2O2‚HCl
20H21ClN2O2‚HCl
22H20N2‚HCl
240-1
259-60
175 dec
22H20N2‚HCl
increases in affinity. The most significant positive
substituent effects correlated best with steric param-
eters and indicated that occupancy of the 2-, 3-, and
4-positions was favorable, while substitution at the
5-position was less advantageous. The 1-naphthyl
derivative 29 possessed the highest affinity of the
tetrahydro-â-carboline antagonists identified thus far.
This substituent, along with the 3,4-dimethoxyphenyl
(as in 6), was chosen for the ensuing phase of SAR
studies.
of oxygen substituents at this position (MeO, 31 and
BnO, 32) resulted in considerable loss of affinity relative
to 6 (Table 2).47 Further examination revealed a trend
with halogen substitution at C-6 showing F ≈ Cl < Br
< H < I, suggesting a negative effect of electron-
withdrawing substituents gradually overcome by a
positive steric effect. Also noteworthy were the effects
of the “soft” or polarizable substituents MeS (38) and I
(37), which resulted in a 5-fold increase in affinity
relative to the unsubstituted analogs. Introduction of
simple alkyl groups also had a remarkable positive
influence upon the 5HT2B receptor antagonist affinity.
The beneficial effect of alkyl substituents was limited,
however, to those of small size with the greatest effect
observed with the 6-Me substituent (39 and 40) result-
ing in a 8-fold increase in affinity. The 6-Et analogs
(42 and 43) showed improved affinity relative to the
We next examined substituent effects on the indole
portion of the tetrahydro-â-carboline template. Upon
even casual inspection, the superposition of serotonin
upon the tetracyclic tetrahydro-â-carboline framework
suggests a relationship between C-5 of the natural
ligand with C-6 of our antagonist structure. Thus, we
were somewhat surprised to discover that introduction

2776 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Ta ble 2. Physical and Biological Data of Tetrahydro-â-carbolines
Audia et al.
rat stomach
compd
series
X
mol formula
mp (°C)
meth prep
fundus -log KB
6
29
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
DMP
NP
H
H
C20H22N2O2‚HCl
9.17 ( 0.11(7)
9.35 ( 0.12(13)
8.48 ( 0.30(5)
6.73 ( 0.40(3)
8.49 ( 0.10(3)
8.36 ( 0.14(3)
9.16 ( 0.13(3)
8.80 ( 0.17(3)
9.52 ( 0.17(3)
9.56 ( 0.15(3)
9.86 ( 0.14(15)
9.75 ( 0.11(8)
<7.5(4)
9.46 ( 0.11(6)
9.65 ( 0.14(4)
8.64 ( 0.27(3)
<7.5(4)
8.50 ( 0.13(4)
<7.5
<7.0
7.31 ( 0.03(3)
9.72 ( 0.30(3)
9.10 ( 0.28(3)
9.0 ( 0.07(3)
9.3 ( 0.12(7)
9.69 ( 0.21(3)
9.34 ( 0.12(3)
9.71 ( 0.14(6)
9.61 ( 0.22(5)
10.12 ( 0.18(3)
9.29 ( 0.18(4)
9.22 ( 0.05(3)
9.60 ( 0.13(7)
8.92 ( 0.29(4)
8.82 ( 0.11(3)
9.02 ( 0.35(3)
8.56 ( 0.43(3)
C
C
C
C
C
C
C
C
C
C
C
22H20N2‚HCl
259-60
A
A
A
A
B
B
B
B
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
DMP
DMP
DMP
DMP
NP
DMP
DMP
DMP
DMP
NP
NP
DMP
NP
DMP
DMP
DMP
DMP
DMP
DMP
NP
6-OMe
6-OBn
6-F
6-Cl
6-Cl
6-Br
6-I
6-SMe
6-Me
6-Me
6-CO2H
6-Et
6-Et
6-n-Pr
6-n-Bu
6-i-Pr
6-c-Hex
6-Ph
21H24N2O3‚HCl
232-3
27H28N2O3‚HCl
232-3
20H21FN2O2‚HCl
20H21ClN2O2‚C4H4O4‚H2O
22H19ClN2‚HCl
238-40
220-2
280-5 dec
184-8
20H21BrN2O2‚C4H4O4
20H21IN2O2‚HCl
21H24N2O2S‚HCl‚1/2H2O
21H24N2O2‚HCl
270-3 dec
224-7
245-6 dec
276-80 dec
284-7 dec
185 dec
23H22N2‚HCl
C23H20N2O2‚HCl
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
22H26N2O2‚C4H4O4
24H24N2‚C4H4O4
23H28N2O2‚C4H4O4
24H30N2O2‚C4H4O4
23H28N2O2‚C4H4O4
26H32N2O2‚C4H4O4
26H26N2O2‚HCl
212-4 dec
195-7 dec
191-3
196-200
208-10 dec
253-5
7-OMe
8-OMe
8-Br
21H24N2O3‚HCl
223-4
23H22N2O‚C4H4O4
22H19BrN2‚HCl
21H24N2O2‚C4H4O4
21H23FN2O2‚C4H4O4
23H21FN2‚C4H4O4
22H26N2O2‚HCl
22H26N2O2‚C4H4O4
22H26N2O2‚HCl
22H26N2O2‚HCl
22H26N2O2‚HCl
24H24N2O2‚C4H4O4
20H20F2N2O2‚C4H4O4
21H23BrN2O2‚HCl
23H21BrN2‚C4H4O4
21H23BrN2O2‚HCl
23H21BrN2‚HCl
240-2 dec
229-35 dec
168 dec
NP
DMP
DMP
NP
8-Me
5-F,6-Me
5-F,6-Me
5,7-Me
6,7-Me
6,8-Me
7,8-Me
4,8-Me
7,8-benzo
6,8-F
6-Me,8-Br
6-Me,8-Br
7-Me,8-Br
7-Me,8-Br
191-4
253-5 dec
259-61
DMP
DMP
DMP
DMP
DMP
DMP
DMP
DMP
NP
197-200
257-9
278-80
275-7
187-9 dec
185-8 dec
253-5
208-11 dec
266-7
276-9 dec
DMP
NP
parent structures, but affinity rapidly diminished with
increased steric bulk, resulting in a loss of affinity with
the 6-nBu (45) and 6-cHex (47) analogs.
agreement with those obtained in our initial studies on
the unsubstituted indole series, with the 3-10-fold
increases in affinity attributed to the 6-methyl substitu-
ent maintained across a variety of E-ring substitution
patterns. Also interesting was the lack of tolerance of
alkylation on either nitrogen atom of the tetrahydro-â-
carboline, resulting in a 10- to >100-fold loss of affinity
(79-82). Likewise, introduction of C-1 methyl substitu-
tion resulted in similar dramatic loss of affinity (83).
With the publication of other 5HT2B(2C) receptor
antagonists, we sought to identify parallels that might
exist among the different series. Unfortunately, only
limited comparisons of SAR trends from our series to
those observed in the indolylurea or indolonaphthyri-
dine can be made at this time due to a shortage of
published SAR data on the latter series. However, SAR
data has been published for the 2-methyl-3-ethyl-5-
(dimethylamino)indole (“medmain”) series of 5HT2B
(“fundal”) receptor antagonists, from which the indolyl-
ureas were derived.20 The loss of 5HT2B receptor affinity
upon indole N-alkylation suggests that direct indole
superposition of the indolylureas (such as 9 or 10) and
the tetrahydro-â-carbolines is unlikely to reflect their
relative receptor-binding orientations, since N-alkyla-
Our examination of the effects of substitution at
alternate locations of the indole platform was less
detailed, but resulted in some interesting observations.
In similar fashion to the 6-OMe analog (31), the 7-OMe
derivative (49) showed diminished affinity relative to
the parent. In contrast, however, the 8-OMe analog (50)
was among the highest affinity compounds. Further
divergence of the C-8 SAR from the C-6 pattern of
substituent effects showed that substitution by halogen
(8-Br, 51) or alkyl (8-Me, 52) was without marked effect
on affinity. The effects of disubstitution on the indole
ring were also briefly investigated, and the results were
consistent with our earlier observations. Most striking
were the effects resulting from introduction of a 7-meth-
yl substituent, which increased affinity 10-fold in con-
junction with the 8-methyl group (58 versus 52), whereas
it had little effect in conjunction with the 6-methyl (56)
or the 8-bromo (64 and 65) analogs.
Our final efforts were directed to the examination of
the ring E substituent effects in the 6-methyltetrahydro-
â-carboline series (Table 3). The results were in good

Tetrahydro-â-carboline Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2777
Ta ble 3. Physical and Biological Data of Tetrahydro-â-carbolines
rat stomach
compd
X
Y
mol formula
mp (°C)
meth prep
fundus -log KB
39
40
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
6-Me
3′,4′-OMe
2′,3′-benzo
C21H24N2O2‚HCl
245-6 dec
276-80 dec
272-4
B
B
B
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
A
9.86 ( 0.14(15)
9.75 ( 0.11(8)
9.23 ( 0.24(3)
9.80 ( 0.15(4)
9.70 ( 0.22(4)
9.16 ( 0.21(3)
9.02 ( 0.08(4)
8.12 ( 0.16(7)
8.55 ( 0.10(4)
8.89 ( 0.12(4)
9.05 ( 0.03(4)
8.47 ( 0.24(5)
9.42 ( 0.18(5)
8.91 ( 0.17(7)
9.06 ( 0.27(3)
8.40 ( 0.40(3)
<7.52(4)
C
C
C
C
23H22N2‚HCl
3′-OH,4′-OMe
2′-Cl,3′,4′-OMe
2′-Br,3′,4′-OMe
2′-NO2-3′,4′-OMe
2′-NH2-3′,4′-OMe
5′-I,3′,4′-OMe
5′-NO2,3′,4′-OMe
5′-NH2,3′,4′-OMe
3′,5′-F
20H22N2O2‚HCl
21H23ClN2O2‚HCl
21H23BrN2O2‚HCl
244 dec
272-4 dec
233 dec
214 dec
163-6 dec
239-43
230-4
205 dec
216 dec
C21H23N3O4‚HCl
C21H25N3O2 ‚2HCl
C
C21H23N3O4‚HCl
C21H25N3O2 ‚2HCl
21H23IN2O2‚C4H4O4
C
C
C
19H18F2N2‚C4H4O4
19H18F2N2‚C4H4O4
20H21FN2O‚HCl
3′,4′-F
3′-F,4′-OMe
3′-CF3
277-9
252-3
C20H19F3N2‚HCl
3′,4′-Me
C
C
C
C
C
C
C
21H24N2‚HCl
285-7 dec
172-6 dec
227-9 dec
218-21
146-50
183-5
2,6-Me
2,6,9-Me
2,6-Me
6,9-Me
1,6-Me
6-Me
2′-Cl,3′,4′-OMe
2′-Cl,3′,4′-OMe
2′,3′-benzo
3′,4′-OMe
3′,4′-OMe
22H25ClN2O2‚C4H4O4
23H27ClN2O2‚HCl
24H24N2‚HCl
∼7.52(4)
22H27N2O2‚HCl
22H26N2O2‚C4H4O4
19H26N2‚HCl
<7.52(4)
<7.52(4)
8.32 ( 0.17(3)
“perhydro”
230 dec
Ta ble 4. 5HT2A and 5HT2C Receptor Binding Affinities and Rat 5HT2B (Fundus) Affinity of Tetrahydro-â-carbolines
-log Ki
compd
5HT2A
5HT2C
-log KB 5HT2B
ratio 2B/2A
ratio 2B/2C
1 (yohimbine)
6 (LY23728)
39
40
54
56
58 (LY287375)
64 (LY288345)
67 (LY266097)
68
5.79 ( 0.26(3)
7.18 ( 0.08(3)
7.65 ( 0.09(3)
8.45 ( 0.15(3)
8.57 ( 0.20(3)
8.33 ( 0.07(3)
8.06 ( 0.12(3)
8.53 ( 0.20(3)
7.71 ( 0.12(3)
7.78 ( 0.16(3)
<5.0
6.92 ( 0.12(3)
9.17 ( 0.11(7)
9.86 ( 0.14(15)
9.75 ( 0.11(8)
9.69 ( 0.21(3)
9.71 ( 0.14(6)
10.12 ( 0.18(3)
9.02 ( 0.35(3)
9.80 ( 0.15(4)
9.70 ( 0.22(4)
13.5
97.7
162.2
20.2
13.2
24.0
114.8
3.09
123.0
83.2
83.0
186.2
87.1
11.8
4.68
15.8
102.3
2.14
154.9
93.3
6.90 ( 0.07(3)
7.92 ( 0.05(3)
8.68 ( 0.10(3)
9.02 ( 0.20(3)
8.51 ( 0.03(3)
8.11 ( 0.08(3)
8.69 ( 0.14(3)
7.61 ( 0.04(3)
7.73 ( 0.05(3)
8, SDZ SER-082
9, SB 200646A
10, SB 206553
11, SB 204741
6.2 ( 0.1-0.2a
7.8 ( 0.1-0.2b
6.86 ( 0.07e
8.00 ( 0.02h
5.82 ( 0.02e
7.34 ( 0.1-0.2c
7.40 ( 0.06f
8.48 ( 0.05i
7.95 ( 0.11f
13.8
>158.5
489.8
0.35
3.50
3.02
<5.2d
5.79 ( 0.01g
<5.2d
>562
135
a
b
Binding affinity, rat cortex, [3H]ketanserin, ref 18. Binding affinity, pig choroid plexus, [3H]mesulergine, ref 18. c Rat stomach fundus,
ref 18. Binding affinity, human clone, [3H]ketanserin, ref 19. e Binding affinity, human clone, [3H]mesulergine, ref 19. f Rat stomach
d
fundus, ref 19. Binding affinity, human clone, [3H]ketanserin, ref 17. Binding affinity, human clone, [3H]mesulergine, ref 17. Rat
g
h
i
stomach fundus, ref 17.
tion of the indolylureas had little effect on affinity.
Likewise, the strong preference for the secondary amine
over the tertiary amine analogs in the tetrahydro-â-
carboline series would suggest a rather different envi-
ronment around the basic amine for the receptor-bound
ligand than that experienced by the indolonaphthyridine
(8). Further comparison among the series awaits
publication of more detailed SAR data for the indolyl-
ureas and indolonaphthyridines.
Having identified 5HT2B receptor antagonists with
high affinity, we examined the 5HT2A and 5HT2C recep-
tor binding affinity of selected compounds from the
series as a measure of selectivity within the 5HT2
receptor family. Table 4 illustrates the comparison of
radioligand binding data obtained at antagonist-labeled
rat 5HT2A and mouse 5HT2C receptors with functional
rat 5HT2B receptor antagonism. Interestingly, com-
pound 64 (LY288345) emerged as a high-affinity, non-
selective 5HT2 receptor antagonist. For many of the
remaining potent members of the series, however, there
was 80- to >180-fold selectivity for the 5HT2B receptor
relative to both the 5HT2A and 5HT2C receptors. Thus,
these compounds represent the first high affinity an-
tagonists to demonstrate substantial selectivity for
5HT2B receptor vis a vis both closely related 5HT2
receptor subtypes. Because of our awareness of the
issues surrounding the use of mixed data types (radio-
ligand binding versus functional studies, species differ-
ences, etc.) and to support further the observed selec-
tivity profile, we examined a set of compounds for their
functional 5HT2A receptor antagonism. To avoid species
differences, we utilized blockade of the serotonin-
induced contraction of the rat jugular vein (Table 5) for
in vitro assessment of the 5HT2A receptor-mediated
effects. The data obtained therein are also consistent
with 50-500-fold selectivity for the 5HT2B receptor-
mediated response and are in good agreement with our
binding data.

2778 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Audia et al.
Ta ble 5. Rat 5HT2B and Rat 5HT2A Affinity of
Tetrahydro-â-carboline Antagonists
sodium carbonate solution. The layers were separated, and
the aqueous layer was back extracted with 2 volumes of 5%
2-propanol/chloroform. The combined organic phases were
dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by flash chromatography
on silica, eluting with methanol/ or 2-propanol/chloroform
solutions (in the presence of 0.1% ammonium hydroxide
solution). Fractions containing product were pooled and
concentrated under reduced pressure. The free base was
dissolved in diethyl ether/methanol and treated either with
excess dry HCl gas to form the hydrochloride salt or with 1
equiv of maleic acid to prepare the maleate salt. The resulting
salt was isolated by filtration, washed with 2-propanol, and
dried under reduced pressure.
Gen er a l P r oced u r e for th e Hyd r olytic Con d en sa tion
of Aza La cton es w ith Tr yp ta m in es: Meth od B, w ith F r ee
Ba se Isola tion , P r ep a r a tion of Ma lea te Sa lt. To 20 mL
of a 1.0 N solution of hydrochloric acid was introduced 1.14 g
(5.41 mmol) of 5-methyltryptamine hydrochloride followed by
2.00 g (7.21 mmol) of aza lactone. Stirring was initiated, and
the mixture was heated to reflux for 12-72 h under an
atmosphere of nitrogen. The reaction was allowed to cool to
room temperature, diluted with 5% 2-propanol/chloroform, and
made basic by addition of saturated sodium bicarbonate
solution. The layers were separated, and the aqueous layer
was back extracted with 2 volumes of 5% 2-propanol/chloro-
form. The combined organic phases were dried over sodium
sulfate and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica, eluting with
methanol/ or 2-propanol/chloroform solutions (in the presence
of 0.1% ammonium hydroxide solution). Fractions containing
product were pooled and concentrated under reduced pressure.
The free base was dissolved in diethyl ether/methanol and
treated with 1 equiv of maleic acid with stirring. The resulting
salt was isolated by filtration, washed with 2-propanol, and
dried under reduced pressure.
Meth od B, w ith Dir ect P r ep a r a tion of Hyd r och lor id e
Sa lt. To 20 mL of a 1.0 N solution of hydrochloric acid was
introduced 1.0 g (4.75 mmol) of 5-methyltryptamine hydro-
chloride followed by 1.1-1.5 equiv of the appropriate aza
lactone. Stirring was initiated, and the mixture was heated
to reflux for 12-72 h under an atmosphere of nitrogen. The
reaction mixture was allowed to cool to room temperature, and
the resulting precipitate was collected by filtration. The solid
was washed with cold water, triturated with 2-propanol,
washed with diethyl ether, and dried in a vacuum oven. The
tetrahydro-â-carboline was isolated as its hydrochloride salt
with acceptable purity for biological evaluation. Physical data
are summarized in Tables 1, 2, and 3. Table 6, containing
MS and elemental analysis can be found in the Supporting
Information.
Gen er a l P r oced u r e for P r ep a r a tion of Aza La cton es
14. A solution of 0.16 mol of the aromatic carboxaldehyde
(freshly distilled or recrystallized before use) was dissolved in
147 mL of acetic anhydride. N-Acetylglycine (19.0 g, 0.16 mol)
was added followed by sodium acetate (13.1 g, 0.16 mol), and
the mixture was heated to 100 °C in an oil bath for 4 h. The
flask containing the reaction mixture was allowed to remain
in the oil bath under continued stirring during a slow
overnight cooling. During this time, the aza lactone precipi-
tated, typically as an orange/red granular solid. The solid was
isolated by filtration. Washing with 100 mL of cold diethyl
ether and drying under reduced pressure afforded aza lactone
of suitable purity for use in subsequent synthetic operations.
Gen er a l P r oced u r e for P r ep a r a tion of P h en ylp yr u vic
-log KB
5HT2B
5HT2A
compd
(rat stomach fundus)
(rat jugular vein)
1 (yohimbine)
6
39
40
67
6.92 ( 0.12(3)
9.17 ( 0.11(7)
9.86 ( 0.14(15)
9.75 ( 0.11(8)
9.80 ( 0.15(4)
6.37 ( 0.11(3)
7.43 ( 0.09(6)
7.21 ( 0.20(4)
8.07 ( 0.10(8)
8.14 ( 0.10(6)
For comparative purposes, it is apparent that certain
compounds from the present series have considerably
higher affinity for the 5HT2B receptor in the rat stomach
fundus than previously reported 5HT2B receptor an-
tagonists. Additionally, compounds 6 (LY23728), 58
(LY287375), and 67 (LY266097) are more than 100-fold
selective for the rat 5HT2B receptor versus both the 5HT2A
and 5HT2C receptors as demonstrated both by radioli-
gand binding and by functional measures. These com-
pounds should prove useful in elucidating the role of
serotonin acting at the 5HT2B receptors vis a vis 5HT2A
or 5HT2c receptors in normal and disease physiology.
Ultimately, such agents could have therapeutic poten-
tial in the treatment of anxiety,38 migraine,14,39-41 and
other disorders.
Con clu sion s
A series of potent tetrahydro-â-carboline 5HT2B recep-
tor antagonists has been identified on the basis of the
yohimbine platform, with SAR studies resulting in 1000-
fold increase in 5HT2B receptor affinity for several
compounds relative to yohimbine. High-affinity com-
pounds from this series have been identified which are
able to discriminate among the 5HT2 family of serotonin
receptors with >100-fold selectivity for 5HT2B receptors
over both 5HT2A and 5HT2C receptors as demonstrated
by radioligand binding and functional studies. More
detailed pharmacological investigation of members of
this series will be reported in due course.
Exp er im en ta l Section
Gen er a l. Solvents and reagents were obtained from com-
mercial suppliers and were used without further purification
except as noted. Benzaldehydes were purified by recrystalli-
zation from suitable solvents (hexanes, diethyl ether) or by
vacuum distillation prior to use. The tryptamines employed
were obtained from commercial suppliers or were prepared by
literature methods. Melting points were determined in open
capillary tubes on a Gallencamp melting point apparatus and
are uncorrected. 1H NMR were determined at 300 MHz in
CDCl3 or DMSO-d6 and chemical shifts reported downfield of
TMS. Elemental analyses were performed by the Physical
Chemistry Department of Eli Lilly and Company. TLC
analysis was conducted on silica gel plates visualized with UV
at 254 nM and/or staining with p-anisaldehyde solution and
heating. Preparative chromatography was carried out on
silica gel using modified flash chromatography conditions of
Still.42
Gen er a l P r oced u r e for th e Con d en sa tion of Ca r bon yl
Com p ou n d s w ith Tr yp ta m in es (Meth od A). To a solution
of tryptamine hydrochloride (1.0 g, 5.0 mmol) in 50 mL of
absolute ethanol was added phenylacetaldehyde (702 µL, 6.0
mmol) [alternatively, phenylpyruvic acids were employed in
equivalent fashion], and the solution was heated to reflux for
24-72 h under an atmosphere of nitrogen. The reaction was
allowed to cool to room temperature and was concentrated
under reduced pressure. The residue was dissolved in 5%
2-propanol/chloroform and made basic by addition of saturated
H). A suspension of aza lactone (0.125
Acid s (13, R ) CO2
mol) in 200 mL of 1.0 N sodium hydroxide solution was heated
on steam bath until homogeneous. The solution was
a
acidified with 5 N HCl solution until acidic by Congo Red
paper. Concentrated HCl (30 mL) was added followed by
water to a final volume of 500 mL, and the mixture was heated
to reflux for 4 h. The mixture was cooled to 4 °C, and the
product acid was isolated by filtration. Washing with water
and drying under reduced pressure afforded phenylpyruvic
acid of suitable purity for use in subsequent operations
(method A).

Tetrahydro-â-carboline Antagonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2779
Isola tion of Tissu e for Recep tor An ta gon ist Stu d ies.
Male Wistar rats (150-375 g; Harlan Sprague-Dawley, Inc.)
were sacrificed by cervical dislocation, and longitudinal sec-
tions of the stomach fundus were prepared for in vitro
examination. Four preparations were obtained from one rat
stomach fundus. In some experiments, external jugular veins
from the rats were dissected free of connective tissue, cannu-
lated in situ with polyethylene tubing (PE-50, o.d. ) 0.97 mm),
and placed in Petri dishes containing Krebs’ bicarbonate buffer
(see below). The tips of two 30-gauge stainless steel hypoder-
mic needles bent into an L-shape were slipped into the
polyethylene tubing. Vessels were gently pushed from the
cannula onto the needles. The needles were then separated
so that the lower one was tied with thread to a stationary rod
and the upper thread was tied to a transducer. Stomach strips
and vascular rings were mounted in organ baths containing
10 mL of modified Krebs’ solution of the following composition
(millimolar concentrations): NaCl, 118.2; KCl, 4.6; CaCl2,1.6;
KH2PO4, 1.2; MgSO4, 1.2; dextrose, 10.0; NaHCO3, 24.8.
Tissue bath solutions were maintained at 37 °C and equili-
brated with 95% O2-5% CO2. J ugular veins and stomach
strips were placed under optimum resting force (1g and 4g,
respectively) and were allowed to equilibrate for approximately
1 h before exposure to drugs. Isometric contractions were
recorded as changes in grams of force on a Beckman Dyno-
graph with Statham UC-3 transducers and a microscale
accessory attachment.
Deter m in a tion of Ap p a r en t 5HT2A a n d 5HT2B Recep tor
An ta gon ist Dissocia tion Con sta n ts. After control cumula-
tive contractile responses to serotonin were obtained in the
stomach fundus (5HT2B) and jugular vein (5HT2A), the tissues
were incubated with an appropriate concentration of antago-
nist for 1 h. Contractile responses to serotonin were then
repeated in the presence of the antagonist. Only one antago-
nist concentration was examined in each tissue. Apparent
antagonist dissociation constants (KB) were determined for
each concentration of antagonist according to the following
equation:
previously described.15 The level of nonspecific binding for
both assays was determined in the presence of 3 µM mianserin.
Ki values for the compounds were calculated using the Cheng-
Prusoff equation48 as previously described.15
Su p p or tin g In for m a tion Ava ila ble: Table 6, containing
1
MS and elemental analysis for compounds 19-84 and H NMR
(300 MHz) spectra for tetrahydro-â-carbolines 19-84 (69
pages). Ordering information is given on any current mast-
head page.
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