N-Tetrahydrothiochromenoisoxazole-1-carboxamides as selective antagonists of cloned human 5-HT2B

Article abstract of DOI:10.1016/j.bmcl.2010.07.074

The serendipitous discovery of N-cyclohexyl-8-fluoro-3,3a,4,9b-tetrahydro- 1H-thiochromeno[4,3-c]isoxazole-1-carboxamide as a selective human serotonin 5-HT_2B antagonist with K_i of 42 ± 5 nM is reported herein. A subsequent functional assay indicated little agonist activity compared to 5-HT itself.

Full text of DOI:10.1016/j.bmcl.2010.07.074

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5488–5490
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Tetrahydrothiochromenoisoxazole-1-carboxamides as selective
antagonists of cloned human 5-HT2B
a
b
b
b
Yoon Jin Kwon a, Simon Saubern a, , James M. Macdonald , Xi-Ping Huang , Vincent Setola , Bryan L. Roth
*
a CSIRO Molecular and Health Technologies, Bag 10, Clayton MDC, Clayton, VIC 3169, Australia
b Department of Pharmacology School of Medicine, University of North Carolina Chapel Hill, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The serendipitous discovery of N-cyclohexyl-8-fluoro-3,3a,4,9b-tetrahydro-1H-thiochromeno[4,3-c]isox-
azole-1-carboxamide as a selective human serotonin 5-HT2B antagonist with Ki of 42 5 nM is reported
herein. A subsequent functional assay indicated little agonist activity compared to 5-HT itself.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 29 June 2010
Revised 15 July 2010
Accepted 16 July 2010
Available online 21 July 2010
The importance of selective serotonin 5-HT2B antagonists is evi-
dent from the on going research probing the significance of this
receptor in fields such as migraine, irritable bowel syndrome, pul-
monary hypertension, and hypertrophy.1–4 During the collection of
data in another project, we noted one tetrahydrothiochromenois-
oxazole-1-carboxamide that exhibited selective inhibition of sero-
tonin receptor 5-HT2B over 5-HT2A and 5-HT2C. While not the focus
of our project, we nevertheless recognized the importance of such
a selective inhibitor as a research probe and report our findings
here. By way of comparison with the tetrahydrothiochromenoisox-
azole-1-carboxamides disclosed here, Figure 1 lists the structures
of some commercially available, selective antagonists of 5-HT2B
used as assay standards.
for software tools suitable for this aim.5–8 This prompted us to
expand our existing software tools from an on-target/off-target
system to a more robust polypharmacology design system.9 In or-
der to further develop these software tools, we required assay
results against multiple targets for a common set of molecules.
A series of 8-fluorotetrahydrothiochromenoisoxazole-1-carbox-
amides 5–9 were prepared for this purpose in an analogous
manner to that previously reported for tetrahydrothiochromenois-
oxazoles as indicated in Scheme 1.10
2,5-Difluorobenzaldehyde 1 was treated with allyl mercaptan to
form the thioether 2. Treatment of 2 with 5-hydroxypentanal oxime
under Abiko conditions11 forms a nitrone that undergoes an intra-
molecular 1,3-dipolar cycloaddition reaction with the allyl moiety
to form the desired tetrahydrothiochromenoisoxazole ring system
3 with concomitant formation of an N-tetrahydropyranyl protecting
Recent growing interest in designing molecules that interact
with multiple receptors (polypharmacology) has identified a need
N
NH2
N
O
H
N
H
H
N
N
O
NH
O
N
S
N
F
RS 127445
LY 272015
SB 204741
Figure 1. Structures of some commonly available selective 5-HT2B antagonists. Source: Tocris Biosciences, UK.
* Corresponding author.
E-mail address: simon.saubern@csiro.au (S. Saubern).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.074

Y. J. Kwon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5488–5490
5489
Thp
O
N
O
S
O
F
(i)
(ii)
F
F
F
S
2
3
1
NHR
O
O
O
HN
S
N
(iii)
(iv)
F
F
S
5-9
4
Scheme 1. Reagents and conditions: (i) allyl mercaptan, K2CO3, DMF, 80 °C, 51%; (ii) 5-hydroxypentanal oxime, Bu2SnO, PhCH3, Dean–Stark; (iii) aq HCl, MeOH, rt, 81% (two
steps); (iv) RNCO, THF, rt, 77%.
Table 1
Selectivity of human 5-HT2 assay results for compounds 5–17
Compd
R
PDSPb
% Inhibition at 10
5HT2B
l
Ma
Kic (nM)
5HT2A
5HT2C
5HT2A
5HT2B
5HT2C
5
6
7
8
Ph
iPr
nPr
C6H11
nBu
Ph
iPr
nPr
C6H11
tBu
nBu
2-MePh
10,630
10,631
10,632
10,633
10,634
9397
9398
9399
9400
9401
33.6
2.2
95.3
89.3
93.0
102.5
92.0
71.6
14.8
53.5
75.5
43.0
35.4
52.2
61.2
55.4
37.4
30.0
34.9
56.7
28.9
14.7
28.1
25.9
23.4
40.2
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
223
569
384
42
5171
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
À23.2
6.2
9
À15.6
11.0
À0.2
2.4
5.5
12.0
0.8
489
11
12
13
14
15
16
17
1606
>10,000
6596
1773
>10,000
>10,000
3999
9402
9403
12.8
a
b
c
Average for n = 4. Assay details available at PDSP.
PDSP access code.
Binding values determined only when inhibition at 10 lM was over 50%.
group. The tetrahydropyranyl protecting group was removed with
aqueous acid to form 8-fluorotetrahydrothiochromenoisoxazole 4.
Treatment of 4 with an appropriate isocyanate in THF formed the
N-substituted isoxazolidine-1-carboxamides 5–9.12
Testing of the carboxamides 5–9 in a number of assays indi-
cated that the N-cyclohexyl isoxazolidine-1-carboxamide 8 com-
pletely inhibited 5-HT2B (102.5%) at the test concentration of
Table 2
Assay results for compound 8 showing selectivity for 5-HT2B
a
Receptor
% Inhibitionb
Ki (nM)
Receptor
% Inhibition
Ki (nM)
5ht1a
5ht1b
5ht1d
5ht1e
5ht2a
5ht2b
5ht2c
5ht3
5ht5a
5ht6
5ht7
Alpha1A
Beta1
Beta2
Beta3
D1
36.9
1.3
4.5
D5
À14.2
66.3
À3.2
À4.3
30.8
À10.0
À0.5
34.6
23.4
23.7
20.6
44.9
31.8
8.9
DAT
DOR
H1
H2
H3
H4
KOR
M1
M2
M3
>10,000
À7.8
10 lM, but not 5-HT2A (6.2%) or 5-HT2C (30.0%). A Ki value of
6.2
102.5
30.0
À8.7
46.6
À18.0
27.1
37.8
2.4
3.7
7.1
14.0
8.1
3.2
42
5
42 nM for carboxamide 8 was subsequently measured in a binding
experiment with 5-HT2B using [3H]LSD (1 nM) as the competitive
ligand. 5-HT itself exhibits Ki = 11 nM in this assay.
This selectivity prompted us to further explore the structure–
activity relationship of this group of molecules.
As can be seen from the results in Table 1, dramatic loss of
activity against 5-HT2B occurred when the N-cyclohexyl group
in 8 was replaced by short chain alkyl groups in compounds
6, 7 and 9. Replacement of the N-cyclohexyl group with a phenyl
group in 5 resulted in a fivefold reduction in binding affinity to
5-HT2B, and loss of selectivity with mild activity in the 5-HT2C
assay.
Compound 8 assay results for other receptors are shown in
Table 2, indicating a strong selectivity for 5-HT2B. Of note, the
cyclohexyl carboxamide 8 exhibited poor activity against other
M4
M5
MOR
NET
SERT
Sigma 1
Sigma 2
34.1
20.4
16.5
1.1
>10,000
>10,000
D2
D3
D4
12.0
a
Assay details available at PDSP. Data available as at July 2010.
% Inhibition at 10 lM. Average for n = 4.
b
serotonin receptors, even at the higher 10
lM concentration.
Compound 8 was then subjected to further testing in a
Dopamine, histamine, muscarinic, and opiate receptors show little
activity towards 8. Carboxamide 8 does not show activity in the
serotonin transporter assay.
functional assay to determine the nature of its activity against
5-HT2B. Compound 8 displays negligible activity in the 5-HT2B
agonist assay at 10 lM (0.5% efficacy compared to 5-HT), but high

5490
Y. J. Kwon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5488–5490
Y.J.K. is the thankful recipient of an IAESTE traineeship.
NHR
N
O
O
O
HN
O
Supplementary data
(i)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.074.
O
11-17
10
References and notes
Scheme 2. Reagents and conditions: (i) RNCO, THF, rt.
1. Berger, M.; Gray, J. A.; Roth, B. L. Annu. Rev. Med. 2009, 60, 355.
2. Giorgioni, G.; Accorroni, B.; Di Stefano, A.; Marucci, G.; Siniscalchi, A.; Claudi, F.
Med. Chem. Res. 2005, 14, 57.
3. Moss, N.; Choi, Y.; Cogan, D.; Flegg, A.; Kahrs, A.; Loke, P.; Meyn, O.; Nagaraja,
R.; Napier, S.; Parker, A.; Peterson, J. T.; Ramsden, P.; Sarko, C.; Skow, D.;
Tomlinson, J.; Tye, H.; Whitaker, M. Bioorg. Med. Chem. Lett. 2009, 19, 2206.
4. Huang, X.; Setola, V.; Yadav, P. N.; Allen, J. A.; Rogan, S. C.; Hanson, B. J.;
Revankar, C.; Robers, M.; Doucette, C.; Roth, B. L. Mol. Pharmacol. 2009, 76, 710.
5. Roth, B. L.; Sheffler, D. J.; Kroeze, W. K. Nat. Rev. Drug Disc. 2004, 3, 353.
6. Morphy, R.; Rankovic, Z. J. Med. Chem. 2005, 48, 6523.
7. Hopkins, A. L.; Mason, J. S.; Overington, J. P. Curr. Opin. Struct. Biol. 2006, 16, 127.
8. Keiser, M. J.; Setola, V.; Irwin, J. J.; Laggner, C.; Abbas, A. I.; Hufeisen, S. J.;
Jensen, N. H.; Kuijer, M. B.; Matos, R. C.; Tran, T. B.; Whaley, R.; Glennon, R. A.;
Hert, J.; Thomas, K. L. H.; Edwards, D. D.; Shoichet, B. K.; Roth, B. L. Nature 2009,
462, 175.
9. Polley, M.; Winkler, D.; Burden, F. J. Med. Chem. 2004, 47, 6230.
10. Saubern, S.; Macdonald, J. M.; Ryan, J. H.; Woodgate, R. C. J.; Louie, T. S.;
Fuchter, M. J.; White, J. M.; Holmes, A. B. Tetrahedron 2010, 66, 2761.
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activity in the antagonist assay (pIC50 = 7.37 0.07) (see Supple-
mentary data).
We had also prepared N-carboxamide derivatives of tetrahydro-
chromenoisoxazole 10 as part of our polypharmacology project,
and report their activities here by way of comparison. The parent
ring system 10 was prepared according to Abiko,11 then treated
with an appropriate isocyanate in THF to form the N-carboxamides
11–17 (Scheme 2).
Although a similar trend can be seen for the activity of this ser-
ies of compounds 11–17 against the 5-HT2 receptors (Table 1), the
activities are significantly lower, with compound 14, the series
analogue of 8, recording more than a 40-fold reduction in binding
activity against 5-HT2B
.
12. General procedure for carboxamide formation. N-cyclohexyl-8-fluoro-3,3a,4,9b-
tetrahydro-1H-thiochromeno[4,3-c]isoxazole-1-carboxamide 8: To a solution of 4
In summary, compound 8 was found to be an antagonist inhib-
itor of 5-HT2B with high selectivity over 5-HT2A and 5-HT2C
(53 mg, 0.25 mmol) in THF (2 ml) was added cyclohexyl isocyanate (34 ll,
.
0.26 mmol, 1.05 equiv) and the solution stirred overnight. Methanol (one drop)
was added to the reaction and stirring continued for 15 min. The reaction
solution was then filtered through a small plug of silica to remove polar
impurities using ethyl acetate as eluent. Concentration of the filtrate afforded 8
as a yellow oil (65 mg, 77%). MS (ESI+) m/z 337.2 (100%, M+H), 359.2 (70%,
M+Na), 673.3 (2M+H), 695.3 (2M+Na); 1H NMR (200 MHz, CDCl3) d 7.46 (ddd, J
9.9, 2.8, 0.8 Hz, 1H), 7.21 (dd, J 8.6, 5.5 Hz, 1H), 6.87 (dt, J 8.3, 2.9 Hz, 1H), 5.92
(d, J 8.3 Hz, 1H, NH), 5.31 (d, J 8.71 Hz, 1H), 4.00 (m, 2H); 3.70 (m, 1H), 3.30 (m,
1H), 3.03 (dd, J 13.0, 5.1 Hz, 1H), 2.63 (dd, J 13.0, 10.5 Hz, 1H), 2.10-1.00 (m,
10H); 13C-NMR (50 MHz, CDCl3) d 161.22 (d, JCF 245.9 Hz), 160.54, 136.89 (d, JCF
7.3 Hz), 129.40 (d, JCF 7.7), 128.96 (d, JCF 3.0), 117.47 (d, JCF 23.3), 114.60 (d, JCF
22.3), 74.07, 58.46, 49.17, 45.01, 33.67, 33.24, 30.26, 25.45, 24.88, 24.82; 19F
NMR {1H} (188 MHz, CDCl3) d À116.13.
Acknowledgements
Ki determinations, receptor binding profiles, agonist and antag-
onist functional data was generously provided by the National
Institute of Mental Health’s Psychoactive Drug Screening Program
(PDSP), Contract # NO1MH32004 (NIMH PDSP) and R01MH61887
to BLR. For experimental details please refer to the PDSP web site
http://pdsp.med.unc.edu/ and click on ‘Binding Assay’ or ‘Func-
tional Assay’ on the menu bar.