Binding of amine-substituted N1-benzenesulfonylindoles at human 5-HT6 serotonin receptors

Article abstract of DOI:10.1016/j.bmcl.2005.08.059

An examination of several amine-substituted analogs of N ₁-benzenesulfonylindoles reveals that although they bind at human 5-HT₆ serotonin receptors with high affinity, they are likely to bind in a dissimilar manner.

Full text of DOI:10.1016/j.bmcl.2005.08.059

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5298–5302
Binding of amine-substituted N1-benzenesulfonylindoles at
human 5-HT6 serotonin receptors
Manik Pullagurla,a Uma Siripurapu,a Renata Kolanos,a Mikhail L. Bondarev,a
Małgorzata Dukat,a V. Setola,b B. L. Rothb,c and Richard A. Glennona,*
aDepartment of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298-0540, USA
bDepartment of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA
cDepartment of Psychiatry and Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA
Received 11 July 2005; revised 9 August 2005; accepted 10 August 2005
Available online 23 September 2005
Abstract—An examination of several amine-substituted analogs of N1-benzenesulfonylindoles reveals that although they bind at
human 5-HT6 serotonin receptors with high affinity, they are likely to bind in a dissimilar manner.
Ó 2005 Elsevier Ltd. All rights reserved.
5-HT6 receptors represent one of seven (5-HT1–5-HT7)
major families of serotonin receptors, and are of interest
because of their possible involvement in certain neuro-
logical and neuropsychiatric disorders.1–3 The first
5-HT6 antagonists to be reported included Ro 04-6790
(1a Ki ca. 50 nM),4 Ro 63-0563 (1b Ki ca. 12 nM),4
MS-245 (2a Ki ca. 2 nM),5,6 and SB-271046 (3 Ki ca.
1 nM).7 Although these agents were independently iden-
tified, there are some conspicuous structural similarities
amongst them in that all of them possess an amine-
bearing bis-arylsulfonamide moiety. Despite attempts
to describe how such agents might bind relative to one
another, this issue has yet to be resolved. 5-HT6 recep-
tors are transmembrane-embedded G-protein coupled
receptors containing an aspartate moiety in TM helix
3 that, presumably, serves as a ligand–amine binding
site.1 Compounds 1 possess multiple basic amine func-
tions and it has been difficult identifying which is (are)
most important for binding.8 Compound 3 possesses
only two basic amines and some investigators view one
of the methylamino groups of 1 as mimicking the aryl
amine moiety of 3.9,10 QSAR studies suggest the aryl
amine might be the more important of the two, and
one model indicates that the nonaryl amine of 3-type
compounds even contributes negatively to binding.10
Yet, compounds 2 lack such an amine group. At this
time, it is not known how the various amine functions
influence affinity.
Evidently, one of the pyrimidine nitrogen atoms of 1a
(i.e., 1b) can be removed without detriment to affinity.4
One of the methylamino groups a to the pyridine nitro-
gen atom of 1b can also be eliminated.11 However, be-
cause this modification results in a symmetrically
substituted pyridine, it is not known which of the two
secondary amines is the more important. Is the remain-
ing ring nitrogen atom required? It might be argued, be-
cause 4 (Ki ca. 600 nM) and 5 (Ki ca. 200 nM)11 bind
with several-fold reduced affinity relative to 1, that this
could be the case. In contrast, neither 2 nor 3 possesses
a corresponding 6-membered heteroaryl ring. Further-
more, the high affinity of 6 and 7 (Ki ca. 30–40 nM)11
indicates that a ring nitrogen atom is not required for
binding.
N(CH3)2
NH
N
NHCH3
6
4
H3CO
H3CO
N
2
6
N
S
NH
S
NH
S
H3CHN
Z
O
O
O
S
O
O
O
CH3
R
H2N
Keywords: 5-HT6 serotonin receptors; Aminoindoles.
*
Cl
2a R = H
2b R = NH2
Corresponding author. Tel.: +1 804 828 8487; fax: +1 804 828
7404; e-mail: glennon@hsc.vcu.edu
3
1a Z = N
1b Z = CH
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.059

M. Pullagurla et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5298–5302
5299
Hirst et al.8 have suggested that the 2-methylamino
NO2
NO2
NO2
group of 1a interacts with the TM3 aspartate. Interest-
ingly, although the primary amine of 1 has not been
investigated, it has been mentioned (though no data
were provided) that its elimination results in loss of
affinity,11 suggesting it could be involved in binding.
However, we found that 8 (Ki = 230 nM), which lacks
the primary amine, and 9 (Ki = 200 nM), which bears
only the primary amine, bind with nearly identical affin-
ity and with an affinity comparable to that of 5.12 But,
the similar affinity of 2b (Ki = 0.8 nM)13 and 2a argues
that the primary amine is not necessary. Yet, it has been
demonstrated that 10 (Ki = 12 nM) (i.e., 2b minus the
tryptamine side chain) binds at 5-HT6 receptors and
behaves as an antagonist.13
OH
b
a
N
N
O2N
NH2
O2N
O2N
H
Ts
13
14
15
h
c
NH2
NO2
NO2
d
N
N
N
H2N
O2N
O
O2N
O
O
O
O
S
S
S
O
H3COCHN
e
H3COCHN
21
17
16
R1
d
f
4 R1=NHCH3, R2 = H, R3 = NH2
O
5 R1 = R2 = NHCH3, R3 = NH2
6 R1 = NHCH3, R2 = OCH, R3 = NH2
HN
OEt
N
NH2
NO2
R2
NH
7 R1 = NHCH3, R2 = CF3, R3 = NH2
O
S
OEt
8 R1 = NHCH3, R2 = R3 = H
O
9 R1 = R2 = H, R3 = NH2
N
O
N
H2N
N
O
O
O2N
O
O
H
S
S
R3
S
O
O
We have suggested that compounds such as 10 might
represent conformationally constrained analogs of 1.14
Thus, given this structural relationship, it should be pos-
sible to probe the importance to binding of the various
amine functions. The purpose of this investigation, then,
was to examine variously substituted amine analogs 11
to determine which amine moiety is most contributory
to binding. In addition, we examined the structurally
related aryl amine-bearing analogs 12b and c to deter-
mine if the presence of the aliphatic amine moiety de-
tracts from receptor affinity as earlier suggested.
H3COCHN
f
22
H2N
20
18
e
O
O
HN
OEt
N
HN
OEt
g
OEt
11a
11d
OEt
g
N
O
N
H
O
N
H
O
O
O
S
S
O
R1
H2N
CH3
19
23
11a R1 =R2 =NHCH3, R3 = NH2
11b R1 = NHCH3, R2 = H, R3 = NH2
Scheme 1. Reagents and conditions: (a) TsCl, 0 °C/30 min, 50 °C/1 h;
(b) KOH, 2-PrOH, D; (c) i—NaH, DMF, 80 °C; ii—N-acetylsulfanilyl
chloride, rt; (d) SnCl2Æ2H2O, EtOH, D; (e) ClCOOEt, DMF, Py; (f)
10% HCl, EtOH, D; (g) LiAlH4, THF, D; (h) i—NaH, DMF, 80 °C;
ii—PhSO2Cl, rt.
11c R1 = H, R2 = NHCH3, R3 = NH2
11d R1 = R2 = NHCH3, R3 = H
11e R1 = R2 = H, R3 = NH2
N
S
R2
N
S
O
O
O
O
R3
H2N
10
NH
NH
tion with benzenesulfonyl chloride (i.e., 21) and then
by conversion of the nitro groups to their corresponding
methylamino groups as described for 11a (Table 1).
N
N
12b R = H
12c R-= NH2
N
O
S
O
N
H
Compounds 11b (Scheme 2) and 11c (Scheme 3) were
prepared by a sequence of reactions somewhat similar
to those shown for the synthesis of 11a. Although 4-
methylaminoindole (25) has been previously described,16
in the present study it was prepared from commercially
available 4-aminoindole (24) by acylation with ethyl
chloroformate followed by reduction of the carbamate
with LiAlH4. Introduction of the arylsulfonyl group fol-
lowed by deprotection afforded 11b. The synthesis of 11c
was performed in nearly the reverse manner; that is,
6-nitroindole (28)17 was sulfonylated to arylsulfonyl
analog 29, the nitro group was reduced, and the
product converted to the target (Scheme 3).
12a
R
4,6-Dinitroindole (15)15 was prepared and sulfonylated
to arylsulfonyl analog 16 (Scheme 1); reduction of the
nitro groups, followed by treatment with ethyl chloro-
formate, afforded the protected indole derivative 18.
Deprotection of 18 followed by reduction of the carba-
mate groups afforded 11a. Compound 20 was obtained
from 16 by hydrolysis of the amide. Compound 11d also
was obtained from 4,6-dinitroindole (15) by sulfonyla-

5300
Table 1. Physicochemical and 5-HT6 receptor binding properties of target benzenesulfonylindoles and piperazinoindole 12a
NH
M. Pullagurla et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5298–5302
NH
R1
N
N
N
H
R2
N
S
N
S
O
O
O
O
R3
R
12b, 12c
12a
11, 20
Compound R1
R2
R3
R
Recrystallization Melting point (°C) Empirical formulaa
solvent
Ki (nM)
SEM
11a
11b
11c
11d
11e
12ab
12b
12c
20
–NHMe –NHMe –NH2
–NHMe –H –NH2
–H –NHMe –NH2
–NHMe –NHMe –H
—
—
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
—
145–146
181–182
103–105
149–151
88–90
C16H18N4O2S 2.75HCl
C15H15N3O2S 1.25HCl 0.25H2O
C15H15N3O2S 1.25C2H2O4
C16H17N3O2S 1.75C2H2O4
C14H12N2O2S
1.9
21
0.4
2
7.0
0.1
2
—
26
10
–H
—
–H
—
–NH2
—
—
3
500
—
–H
MeOH
MeOH/Et2O
—
—
2700
1.0
0.4
—
—
—
—
291–293
>250 (d)
230–231
C18H19N3O2S HCl
C18H20N4O2S HCl 0.5H2O
C14H10N4O6S
0.2
0.1
—
–NH2
–NH2 MeOH/Et2O
MeOH/CH2Cl2
–NO2
–NO2
—
980
20
1
a All compounds were homogeneous as determined using thin-layer chromatography; assigned structures are consistent with H NMR spectra, and
compounds analyzed within 0.4 for C, H, and N. C2H2O4 = oxalate salt.
b See Ref. 18.
O
a
b
CH3
NH2
H3C
N
NHCH3
N
N
N
O2N
H2N
O2N
O
O
O
O
H
S
S
a
b
28
N
N
N
H
H
H
26
24
25
H3COCHN
H3COCHN
30
29
c
c
O
CH3
H3C
N
e
d
OEt
OEt
11c
d
N
O
N
O
N
H
11b
O
N
O
H
S
S
N
O
O
O
O
S
H3COCHN
H2N
32
31
H3COCHN
27
Scheme 3. Reagents and conditions: (a) i—NaH, DMF, 80 °C; ii—N-
acetylsulfanilyl chloride, rt; (b) SnCl2Æ2H2O, EtOH, D; (c) ClCOOEt,
DMF, Py; (d) 10% HCl, EtOH, D; (e) LiAlH4, THF, D.
Scheme 2. Reagents and conditions: (a) i—ClCOOEt, DMF, Py, 0 °C;
ii—LiAlH4, THF, D; (b) AcCl, toluene, À10 °C; (c) i—NaH, DMF,
80 °C; ii—N-acetylsulfanilyl chloride, rt; (d) 10% HCl, EtOH, D.
11d; Ki = 26 nM) indicates that its presence is not essen-
tial for binding. For comparison, an analog of 11a was
examined where the methylamino groups were replaced
with nitro groups; dinitro compound 20 (Ki = 980 nM)
binds with >500-fold reduced affinity relative to 11a.
Compounds 12b and c (Table 1) were prepared from
12a18–20 as previously described in the patent literature.
Compound 11e (Ki = 10 nM; Table 1),21 the desmethyl
analog of 10,13 binds with an affinity comparable to
those of 10 (Ki = 12 nM) and 1b. Introduction of the
two methylamino functions found in 1 results in 5-fold
enhanced affinity (11a; Ki = 1.9 nM), suggesting that
the presence of one (or both) secondary amines might
make a minor (direct or indirect) contribution to bind-
ing. However, elimination of the primary amine (i.e.,
Which of the two secondary amines of 11a is the more
important? Introduction of the 6-methylamino group
(11c; Ki = 7.0 nM) has little effect on the affinity of 11e,
whereas introduction of a 4-methylamino group (i.e.,
11b; Ki = 21 nM) only halved affinity. The difference in
affinity of the mono-, di-, and tri-amino analogs 11 is quite

M. Pullagurla et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5298–5302
5301
small (range ꢀ 10-fold), making it nearly impossible to as-
cribe a major binding role to any single amine function
over the others. Comparing 11d and e, however, it would
seem unlikely that these compounds are binding at the
receptors in a similar fashion (i.e., with superimposable
indolic nuclei) if it is assumed they are interacting with a
common amine binding site. These findings are not unlike
what was found previously with 8 and 9.
Finally, the assumption has been made throughout that
each of these ligands binds to a common amine site.
However, there may be two accessible amine sites in
the binding pocket of 5-HT6 receptors: one in TM3
and another in TM7.27 The possibility exists, then, that
some ligands might utilize one or the other (or both) of
these sites. This remains to be further investigated.
Piperazinoindole 12a (Ki = 2700 nM; Table 1) lacks sig-
nificant affinity for 5-HT6 receptors. Incorporation of
the N1-benzenesulfonyl group results in >2500-fold en-
hanced affinity and 12b (Ki = 1 nM) binds with an affin-
ity comparable to that of 3. Introduction of the primary
amine (i.e., 12c; Ki = 0.4 nM) is tolerated, but the amine
is obviously not required for binding. If compound 12c
is viewed as an elaboration of 11b (Ki = 21 nM), it is also
evident that the presence of an intact piperazine ring
leads to a >50-fold enhancement in 5-HT6 receptor
affinity. That is, the presence of the alkyl amine group
does not detract from receptor affinity when compared
with 11b. Compounds 12b and c were also recently
reported by others to bind at human 5-HT6 receptors
with high affinity (Ki = 1 nM).19
Acknowledgment
This work was supported in part by MH 60599.
References and notes
1. Kroeze, W. K.; Kristiansen, K.; Roth, B. L. Curr. Top.
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shyn, L.; Savage, J. E.; Roth, B. L.; Hufesein, S.; Lee,
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7. Bromidge, S. M.; Brown, A. M.; Clarke, S. E.; Dodgson,
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9. Russell, M. G.; Baker, R. J.; Barden, L.; Beer, M. S.;
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Fairly apparent from this investigation is that all indole-
containing analogs likely do not interact with the recep-
tor with superimposed indolic nuclei, and that multiple
amine groups are not a requirement for binding. When
multiple amines are present, it remains to be determined
which is most critical for interaction with the TM3
aspartate; furthermore, the low affinity of 20 compared
with those of 11a and e suggests that the amine groups
might additionally have some effect on the electronic
character of the indole nucleus. Comparing the com-
pounds that were investigated, the only reasonable con-
clusion that can be drawn is that multiple modes of
binding are possible. As if to underscore this concept,
it has recently been shown that tryptamine analogs bear-
ing a arylsulfonamido group at the indole 5-position
also bind with nanomolar affinity at 5-HT6
receptors.22,23
11. Bo¨s, M.; Sleight, A. J.; Godel, T.; Martin, J. R.; Riemer,
C.; Stadler, H. Eur. J. Med. Chem. 2001, 36, 165.
12. Pullagurla, M. R. PhD Thesis, Virginia Commonwealth
University, December 2003.
13. Pullagurla, M.; Setola, V.; Roth, B. L.; Glennon, R. A.
Bioorg. Med. Chem. Lett. 2003, 13, 3355.
Initially, these findings are disturbing because they im-
ply that the sulfonamido moiety common to the various
agents will not be aligned. However, the present conclu-
sion is not inconsistent with observations that ꢀreverse
sulfonamidesꢁ24 and even sulfones25,26 bind at 5-HT6
receptors, and that the benzenesulfonyl group can be
effectively replaced with a benzyl group.3 In other
words, although the sulfonamido moiety might contrib-
ute to the affinity of some of these ligands, its presence
(or specific orientation) may not be essential for binding.
The possibility of multiple modes of binding is also dis-
appointing because it makes it rather difficult to utilize
or reliably extrapolate the structure–affinity findings
from one series to another for purposes of drug design.
This might be true even within a given series of agents
(e.g., compare 11d with e). Consequently, it will be nec-
essary to exercise caution when conducting QSAR inves-
tigations that require alignment of specific structural
features until it is known how such agents bind relative
to one another.
14. Pullagurla, M.; Dukat, M.; Roth, B. L.; Setola, V.;
Glennon, R. A. Med. Chem. Res., in press.
15. Samet, A. V.; Zakharov, E. P.; Semenov, V. V.; Buchanan,
A. C.; Gakh, A. A. Synth. Commun. 2001, 31, 1441.
16. Quick, J.; Saha, B. Tetrahedron Lett. 1994, 35, 8553.
17. Caixach, J.; Capell, R.; Galvez, C.; Gonzalez, A.; Roca,
N. J. Heterocycl. Chem. 1979, 16, 1631.
18. Compound 12a (commercially available from Bridge
Organics Co., Vicksburg, MI, as its dihydrochloride salt)
was purified by recrystallization from aqueous MeOH.
Compound 12b has been variously described in the patent
literature as
a low-melting hydrochloride salt (mp
60 °C),19 and as a dihydrochloride hydrate20 for which
no mp or other physicochemical data were provided.
Compound 12c, although also cited in the patent litera-
ture,19 was not characterized. In the present investigation,

5302
M. Pullagurla et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5298–5302
compounds 12b and c were prepared as described20 but
of total binding. Ki values are the result of triplicate
determinations.
were obtained as high-melting salts. Both compounds were
characterized by spectroscopic and elemental analysis;
compound 12b was obtained as its monohydrochloride
salt, and c as its monohydrochloride hemihydrate.
22. Holenz, J.; Merce, R.; Diaz, J. L.; Guitart, X.; Codony,
X.; Dordal, A.; Romero, G.; Torrens, A.; Mas, J.;
Andaluz, B.; Hernandez, S.; Monroy, X.; Sanchez, E.;
Hernandez, E.; Perez, R.; Cubi, R.; Sanfeliu, O.; Busch-
mann, H. J. Med. Chem. 2005, 48, 1781.
19. Kelly, M. G.; Cole, D. C. U.S. Patent US 2004/0192749,
2004.
20. Caldirola, P.; Johansson, G.; Nilsson, B. World Patent
WO0232864, 2002.
23. Cole, D. C.; Lennox, W. J.; Lombardi, S.; Ellingboe, J.
W.; Bernotas, R. C.; Tawa, G. J.; Mazandarani, H.;
Smith, D. L.; Zhang, G.; Coupet, J.; Schechter, L. E.
J. Med. Chem. 2005, 48, 353.
24. Bromidge, S. M.; Clarke, S. E.; Gager, T.; Griffith, K.;
Jeffrey, P.; Jennings, A. J.; Joiner, G. F.; King, F. D.;
Lovell, P. J.; Moss, S. F.; Newman, H.; Riley, G.; Rogers,
D.; Routledge, C.; Serafinowska, H.; Smith, D. R. Bioorg.
Med. Chem. Lett. 2001, 11, 55.
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Poli, S.; Porter, R. H. P.; Bo¨s, M. J. Med. Chem. 2003, 46,
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Setola, V.; Roth, B. L.; Glennon, R. A. Bioorg. Med.
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21. The h5-HT6 radioligand binding assay was performed as
previously described.28 In brief, h5-HT6 cDNA was
transiently expressed in HEK-293 cells using Fugene6
according to the manufacturerꢁs recommendations. Twen-
ty-four hours after transfection, the medium was replaced;
24 h later, medium containing dialyzed serum (to remove
5-HT) was added. At 75 h after transfection, cells were
harvested by scraping and centrifugation. Cells were then
washed by centrifugation and resuspension once in phos-
phate-buffered saline (pH = 7.40; PBS) and then frozen as
tight pellets at À80 °C until use. Binding assays were
performed at room temperature for 90 min in binding
buffer (50 mM Tris–Cl, 10 mM MgCl2, and 0.1 mM
EDTA, pH = 7.40) with [3H]LSD (1 nM final concentra-
tion) using 10 lM clozapine for non-specific binding.
Various concentrations of unlabeled test agent were used
for Ki determinations, with Ki values calculated using the
program LIGAND. Specific binding represented 80–90%
27. Pullagurla, M. R.; Westkaemper, R. B.; Glennon, R. A.
Bioorg. Med. Chem. Lett. 2004, 14, 4569.
28. Kohen, R.; Metcalf, M. A.; Khan, N.; Druck, T.; Huebner,
K.; Lachowicz, J. E.; Meltzer, H. Y.; Sibley, D. R.; Roth, B.
L.; Hamblin, M. W. J. Neurochem. 1996, 66, 47.