5-Chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2- benzothiophenesulfonamide (SB-271046): A potent, selective, and orally bioavailable 5-HT6 receptor antagonist [1]

Full text of DOI:10.1021/jm980532e

202
J . Med. Chem. 1999, 42, 202-205
Communications to the Editor
cholinergic neurotransmission and hence 5-HT6 receptor
antagonists may be useful for the treatment of memory
dysfunction.
5-Ch lor o-N-(4-m eth oxy-3-p ip er a zin -1-yl-
p h en yl)-3-m eth yl-2-ben zoth iop h en esu lfon -
a m id e (SB-271046): A P oten t, Selective,
a n d Or a lly Bioa va ila ble 5-HT6 Recep tor
An ta gon ist
Unfortunately, further pharmacological evaluation of
the function of the 5-HT6 receptor has been hampered
by the lack of selective ligands. Recently, the first
selective 5-HT6 antagonists, Ro 04-6790 and Ro 63-0563,
were reported.7 These compounds were found to have
moderate affinity for the rat 5-HT6 receptor but were
poorly brain penetrant (<1%). However, when Ro 04-
6790 was administered intraperitoneally to rats, suf-
ficient brain levels were achieved to evoke a statistically
significant effect on stretching similar to that seen
following treatment with antisense oligonucleotides.
Steven M. Bromidge,*,† Anthony M. Brown,‡
Stephen E. Clarke,§ Kathy Dodgson,‡ Tracey Gager,‡
Helen L. Grassam,† Phil M. J effrey,§
Graham F. J oiner,† Frank D. King,†
Derek N. Middlemiss,‡ Stephen F. Moss,†
Helen Newman,‡ Graham Riley,‡
Carol Routledge,‡ and Paul Wyman†
Departments of Medicinal Chemistry,
Neuroscience Research, and Drug Metabolism and
Pharmacokinetics, SmithKline Beecham Pharmaceuticals,
Discovery Research, New Frontiers Science Park,
Third Avenue, Harlow, Essex CM19 5AW, England
Received September 21, 1998
The 5-hydroxytryptamine (5-HT, serotonin) super-
family of receptors currently consists of 7 classes (5-
HT1-5-HT7) that embrace 14 human subclasses.1 The
most recent addition is the 5-HT6 receptor which was
first cloned from rat striatal mRNA in 1993 by two
independent groups.2 The human 5-HT6 receptor, cloned
in 1994 by Kohen et al.,3 is a 440-amino acid polypeptide
with seven transmembrane spanning domains typical
of G-protein-coupled receptors. Within the transmem-
brane region, the human 5-HT6 receptor shows 96%
identity to its rat homologue, but only 30-40% homol-
ogy to other human 5-HT receptors. The 5-HT6 receptor
is positively coupled to adenylyl cyclase.2
In the rat, the 5-HT6 receptor mRNA has its highest
abundance in the nucleus accumbens, striatum, cerebral
cortex, olfactory tubercle, and hippocampus.2,4 Although
the biological functions of the 5-HT6 receptor are poorly
understood, the distribution, together with its high
affinity for several therapeutically important antipsy-
chotic and antidepressant agents, suggests a possible
role for this receptor in the treatment of schizophrenia
and depression.2a,5 Most atypical antipsychotic drugs,
which lack extrapyrimidal side effects, bind with very
high affinity to the 5-HT6 receptor. In fact, the proto-
typic atypical antipsychotic agent, clozapine, exhibits
greater affinity for the 5-HT6 receptor than for any other
receptor subtype. Recent in vivo experiments demon-
strated that administration of antisense oligonucle-
otides, directed at 5-HT6 receptor mRNA, elicited a
behavioral syndrome in rats consisting of yawning,
stretching, and chewing which could be dose-depen-
dently blocked by the muscarinic antagonist atropine.6
This study implies that 5-HT6 receptors may modulate
High-throughput screening of the SmithKline Bee-
cham Compound Bank against the cloned human 5-HT6
receptor in HeLa cell membranes, using [3H]lysergic
acid diethylamide as radioligand,2a identified the bisaryl
sulfonamide 1. This compound (4-bromo-N-[4-methoxy-
3-(4-m et h ylpiper a zin -1-yl)ph en yl]ben zen esu lfon a -
mide) showed excellent affinity for the 5-HT6 receptor
(pKi 8.3) and greater than 50-fold selectivity over a
number of other key receptors including 10 other 5-HT
receptor subtypes (Table 1). The sulfonamide 1 was
further tested in a commercial screening package (Cerep)
and has been found to have no appreciable affinity for
a total of over 50 receptors, enzymes, or ion channels
so far tested.
1
Compound 1 was evaluated in a functional model of
5-HT6 receptor activation in which 5-HT-stimulated
adenylyl cyclase activity was determined by measuring
the conversion of [R-33P]ATP to [33P]cAMP in HeLa cells
expressing the cloned human 5-HT6 receptor.9 In this
system, 5-HT elicited a dose-dependent 3-5-fold in-
crease over basal cAMP levels which was surmountably
antagonized by clozapine, methiothepin, amitriptyline,
and 1. In the presence of compound 1, the 5-HT
concentration-response curve had the same maximal
response but was shifted rightward in a parallel manner
(Figure 1) with an apparent pKb of 7.8 ( 0.2 (n ) 3),
which is in agreement with the binding affinity. In
* To whom correspondence should be addressed.
† Department of Medicinal Chemistry.
‡ Department of Neuroscience Research.
§ Department of Drug Metabolism and Pharmacokinetics.
10.1021/jm980532e CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/05/1999

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 203
Ta ble 1. Receptor Binding Profile of Compounds 1, 2, and 15a
affinity (pKi)
Ta ble 2. Human Cytochrome P450 Inhibitory Potential of 1
and 15
IC50 (µM)
1
2
15
compd
1A2
2C9
2C19
2D6
3A4
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT1F
5-HT2A
5-HT2B
5-HT2C
5-HT4
6.6
6.4
6.6
5.8
6.5
5.9
6.2
6.0
6.3
6.1
6.7
5.6
6.6
6.0
6.0
6.3
6.4
6.1
6.6
<5.0
<6.0
<5.6
<5.4
5.7
1
15
10
66
>100
>100
46
26
11
32
6
40
a
The cytochrome P450 inhibitory potential was determined
using isoform-selective assays and heterologously expressed hu-
man CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. IC50's
were determined at the substrate Km as has been previously
described12 [CYP1A2, caffeine N3-demethylation (500 µM); CYP2C9,
tobutamide methylhydroxylation (100 µM); CYP2C19, S-mepheny-
toin 4-hydroxylation (100 µM); CYP2D6, bufuralol 1′-hydroxylation
(10 µM); CYP3A4, total cyclosporin oxidation (1 µM)]. These values
are the mean of duplicate determinations which did not vary by
more than 10%.
5.6
5.5
5.4
5-HT6
8.3 ( 0.2
(n > 10)
5.6
5.6
5.4
9.2 ( 0.1
(n ) 3)
5.5
5.7
6.1
8.9 ( 0.2
(n ) 3)
5.4
5.7
5.6
6.3
5-HT7
adrenergic R1B
dopaminergic D2
dopaminergic D3
6.1
6.7
Sch em e 1. Synthesis of Bisaryl Sulfonamides 1-13a
a
All values represent the mean of at least two determinations,
with each determination lying within 0.2 log unit of the mean.
Receptors and radioligands used in binding assay: 5-HT1A (human
cloned receptors in HEK 293 cells, [3H]-8-OH-DPAT); 5-HT1B
(human cloned receptors in CHO cells, [3H]-5-HT); 5-HT1D (human
cloned receptors in CHO cells, [3H]-5-HT); 5-HT1E (human cloned
receptors in CHO cells, [3H]-5-HT); 5-HT1F (human cloned recep-
tors in CHO cells, [3H]-5-HT); 5-HT2A (human cloned receptors in
HEK 293 cells, [3H]ketanserin); 5-HT2B (human cloned receptors
in HEK 293 cells, [3H]-5-HT); 5-HT2C (human cloned receptors in
HEK 293 cells, [3H]mesulergine); 5-HT6 (human cloned receptors
in HeLa cells, [3H]LSD); 5-HT7 (human cloned receptors in HEK
293 cells, [3H]-5-CT); D2 (human cloned receptors in CHO cells,
a
Reagents: (i) ArSO2Cl, acetone, rt, 18 h (65-92%).
(Fpo ) 12%, iv/po crossover study). The structure of 1
was deemed to be readily amenable to exploration of
structure-activity relationships (SAR) by rapid parallel
synthesis, and it was therefore selected as the starting
point for a chemical program.
[
[
125I]iodosulpride); D3 (human cloned receptors in CHO cells,
125I]iodosulpride).
The SAR around the lead structure 1 was investi-
gated by coupling 4-methoxy-3-(4-methylpiperazin-1-yl)-
aniline10 (14) in a parallel manner with commercially
available sulfonyl chlorides containing a wide variety
of aromatic nuclei (Scheme 1).11 The binding results on
a representative selection of compounds (2-13) are
shown in Table 3. A range of affinities for the 5-HT6
receptor were obtained with a number of analogues
including monocyclic and bicyclic aromatics demonstrat-
ing improved binding profiles relative to 1. The unsub-
stituted phenyl 10 with a pKi of 8.0 at the 5-HT6
receptor provides a baseline activity for comparison.
Lipophilic substituents, in particular halogen, were
beneficial to 5-HT6 activity (e.g., 4, 5, 7-9), whereas
polar groups were detrimental, e.g., the 3-cyano (11) and
4-nitro (12) analogues. The polar imidazole 13 also
showed very poor 5-HT6 receptor affinity. The 5-chloro-
3-methylbenzothiophene 2 was optimal in this study
demonstrating sub-nanomolar 5-HT6 receptor affinity
and greater than 300-fold selectivity against a range of
other receptors (totalling 13 subtypes). In the functional
adenylyl cyclase assay, 2 was found to be a competitive
antagonist with an apparent pKb of 8.5 ( 0.2 (n ) 3)
(Figure 1). Several iodophenyl analogues (e.g., 4, 5, 8)
were also identified with excellent 5-HT6 receptor af-
finity and selectivity. The use of [125I]-8 as a specific
radioligand will be reported elsewhere.
F igu r e 1. Effect of compounds 1 and 2 on 5-HT-stimulated
adenylyl cyclase activity in membranes from HeLa cells
transfected with the human 5-HT6 receptor. Data points
represent the mean of duplicate determinations from a typical
experiment which was repeated at least twice.
addition 1 showed no evidence of intrinsic activity in
this system as demonstrated by the lack of effect on
basal formation of cAMP with compound alone. Thus,
1 possesses the profile of a competitive antagonist.
The cytochrome P450 inhibitory potential of 1 was
determined using isoform-selective assays and heter-
ologously expressed human CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4 (Table 2) in order to assess the
potential likelihood of drug interactions.8 Low to moder-
ate levels of inhibition were seen at several of the major
human P450 enzymes with the highest level of inhibi-
tion seen against CYP3A4 (IC50 6 µM). Pharmacokinetic
studies at steady state in rats (n ) 3, following 8 h iv
infusion) demonstrated that 1 was moderately brain
penetrant (25%) but was subject to rapid blood clearance
(∼60 mL/min/kg) resulting in low oral bioavailability
R = Me
R = H
2
15 (SB-271046)

204 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Communications to the Editor
Ta ble 3. 5-HT6 Receptor Binding Affinity and Selectivitya of
Bisaryl Sulfonamides 2-13
Sch em e 2. Synthesis of 5-Chloro-N-(4-methoxy-3-
piperazin-1-ylphenyl)-3-methyl-2-benzothiophenesul-
fonamide 15a
selectivity vs 13
compd
Ar
pKi 5-HT6
receptor subtypes
2
9.2
>300
3
9.1
>250
4
5
9.1
9.0
>200
>125
a
Reagents: (i) (BOC)2O/K2CO3, THF/H2O, rt, 18 h (89%); (ii)
H2/10% Pd/C, EtOH, rt, 18 h (99%); (iii) 5-chloro-3-methylben-
zo[b]thiophene-2-sulfonyl chloride/pyridine, DCM, rt, 18 h (97%);
(iv) THF/cHCl (5:1), reflux, 2 h, recrystallization (EtOH/H2O)
(83%).
6
7
8.9
8.7
>300
>100
8
9
8.6
8.5
>160
>300
10
11
8.0
7.2
80
-
F igu r e 2. Effect of compound 15 (SB-271046) on 5-HT-
stimulated adenylyl cyclase activity in membranes from HeLa
cells transfected with the human 5-HT6 receptor. Data points
represent the mean of duplicate determinations from a typical
experiment which was repeated at least twice.
12
7.1
6.1
-
-
is in good agreement with its binding affinity (Figure
2). Linear regression analysis of Schild plot data re-
vealed a correlation coefficient of unity and a slope of
1.04. In addition to an excellent binding profile, com-
pound 15 demonstrated no significant inhibitory activity
at the major human P450 enzymes (Table 2). Pharma-
cokinetic studies demonstrated that 15 was moderately
brain penetrant (10%), subject to low blood clearance
(7.7 mL/min/kg) with a good half-life in rats (4.8 ( 0.1
h), and had excellent oral bioavailability (>80%).
In conclusion, a series of potent and selective N-
methylpiperazines has been developed from a high-
throughput screening lead 1. The benzothiophene 2
which was the most potent compound from this series
was metabolically demethylated in vivo. Consequently,
the NH-piperazine benzothiophene 15 was prepared and
found to be a high-affinity, selective, and orally bio-
available 5-HT6 receptor antagonist. Compound 15 is
currently being further evaluated for its therapeutic
potential.
13
a
Selectivity was determined against the 13 receptor subtypes
detailed in Table 1.
Pharmacokinetic studies at steady state in rats fol-
lowing a 16-h infusion (n ) 4) demonstrated that 2 was
moderately brain penetrant (18%) and, in contrast to
1, was subject to low blood clearance (12.5 mL/min/kg).
However, in rats 2 was metabolically N-dealkylated to
the corresponding NH-piperazine 15. As significant
levels of 15 were found in blood, it was synthesized and
its biological profile assessed. Thus, 5-chloro-N-(4-
methoxy-3-piperazin-1-ylphenyl)-3-methyl-2-benzothio-
phenesulfonamide (15) (SB-271046) was prepared via
the BOC-protected piperazine according to Scheme 2.
The receptor binding profile of 15 is shown in Table 1.
The 5-HT6 receptor affinity, although slightly reduced
relative to that of the N-methylpiperazine 2, remains
excellent (pKi 8.9), and furthermore 15 also has good
selectivity (>200-fold) against a total of over 50 recep-
tors, enzymes, or ion channels.
Su p p or tin g In for m a tion Ava ila ble: Experimental Sec-
tion containing the synthesis of compounds 2-5. This material
is available free of charge via the Internet at http://pubs.
acs.org.
In the functional adenylyl cyclase assay, 15 was found
to be a competitive antagonist with a pA2 of 8.7 which

Communications to the Editor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 205
of control or 5,7-dihydroxytryptamine-treated rats. Synapse
1996, 23, 164-173.
(5) Roth, B. L.; Craigo, S. C.; Choudhary, M. S.; Uluer, A.; Monsma,
F. J .; Shen, Y.; Meltzer, H. Y.; Sibley, D. R. Binding of typical
and atypical antipsychotic agents to 5-hydroxytryptamine-6 and
5-hydroxytryptamine-7 receptor. J . Pharmacol. Exp. Ther. 1994,
268, 1403-1410.
(6) Bourson, A.; Borroni, E.; Austin, R. H.; Monsma, F. J .; Sleight,
A. J . Determination of the role of the 5-HT6 receptor in the rat
brain: A study using antisense oligonucleotides. J . Pharmacol.
Exp. Ther. 1995, 274, 173-180. Sleight, A. J .; Monsma, F. J .;
Borroni, E.; Austin, R. H.; Bourson, A. Effects of altered 5-HT6
expression in the rat: Functional studies using antisense
oligonucleotides. Behav. Brain Res. 1996, 73, 245-248.
(7) Sleight, A. J .; Boess, F. G.; Bo¨s, M.; Levet-Trafit, B.; Riemer,
C.; Bourson, A. Characterization of Ro 04-6790 and Ro 63-
0563: potent and selective antagonists at human and rat 5-HT6
receptors. Br. J . Pharmacol. 1998, 124, 556-562.
Refer en ces
(1) Hoyer, D.; Martin, G. 5-HT receptor classification and nomen-
clature: towards a harmonisation with the human genome.
Neuropharmacology 1997, 36, 419-428. Hoyer, D.; Clarke, D.
E.; Fozard, J . R.; Hartig, P. R.; Martin, G. R.; Mylecharane, E.
J .; Saxena, P. R.; Humphrey, P. P. A. International union of
pharmacology classification of receptors for 5-hydroxytryptamine
(serotonin). Pharmacol. Rev. 1994, 46, 157-204.
(2) (a) Monsma, F. J .; Shen, Y.; Ward, R. P.; Hamblin, M. W.; Sibley,
D. R. Cloning and expression of a novel serotonin receptor with
high affinity for tricyclic psychotropic drugs. Mol. Pharmacol.
1993, 43, 320-327. (b) Ruat, M.; Traiffort, E.; Arrang, J .-M.;
Tardivel-Lacombe, J .; Diaz, J .; Leurs, R.; Schwartz, J .-C. A novel
serotonin (5-HT6) receptor: Molecular cloning, localisation and
stimulation of cAMP accumulation. Biochem. Biophys. Res.
Commun. 1993, 193, 269-276.
(8) Lewis, D. F. V. Cytochromes P450: Structure, Function and
Mechanism; Taylor and Francis Publishing: London, 1996.
(9) Salomon, Y. Adenylate Cyclase Assay. Adv. Cyclic Nucleotide
Res. 1979, 10, 35-55.
(10) Oxford, A. W.; Mitchell, W. L.; Bradshaw, J .; Clitherow, J . W.
EP Patent 533267, March 24, 1993.
(3) Kohen, R.; Metcalf, M. A.; Druck, T.; Huebner, K.; Sibley, D. R.;
Hamblin, M. W. Cloning and chromosomal localization of a
human 5-HT6 serotonin receptor. Soc. Neurosci. Abstr. 1994, 20,
476.8.
(4) Ward, R. P.; Hamblin, M. W.; Lachowicz, J . E.; Hoffman, B. J .;
Sibley, D. R.; Dorsa, D. M. Localization of serotonin subtype 6
receptor messenger RNA in the rat brain by in situ hybridization
histochemistry. Neuroscience 1995, 64, 1105-1111. Gerard, C.;
El Mestikawy, S.; Lebrand, C.; Adrien, J .; Ruat, M.; Traiffort,
E.; Hamon, M.; Martres, M.-P. Quantitative RT-PCR distribution
of serotonin 5-HT6 receptor mRNA in the central nervous system
(11) Bromidge, S. M.; King, F. D.; Wyman, P. A. WO Patent 98/27081,
J une 25, 1998.
(12) Baldwin, S. J .; Bloomer, J . C.; Smith, G. J .; Ayrton, A. D.; Clarke,
S. E.; Chenery, R. J . Ketoconazole and Sulfaphenazole as the
respective selective inhibitors of P453A and 2C9. Xenobiotica
1995, 25, 261-270.
J M980532E