HTS and rational drug design to generate a class of 5-HT 2C-selective ligands for possible use in schizophrenia

Article abstract of DOI:10.1002/cmdc.201000186

(Chemical Equation Presented) Treating neurological conditions: Optimization of a previously identified lead 5-HT_2C agonist (left) led to the discovery of a highly selective 5-HT_2C agonist (right). Importantly, this compound is a 5-H

Full text of DOI:10.1002/cmdc.201000186

DOI: 10.1002/cmdc.201000186
HTS and Rational Drug Design to Generate a Class of 5-HT2C-Selective
Ligands for Possible Use in Schizophrenia.
Alan P. Kozikowski,*[a] Sung Jin Cho,[a] Niels H. Jensen,[b] John A. Allen,[b] Andreas M. Svennebring,[a] and
Bryan L. Roth[b]
The 5-hydroxytryptamine (1, 5-HT) 2C receptor (5-HT2C), a
prominent central serotonin receptor subtype, is widely distrib-
uted throughout the central nervous system (CNS) and is
thought to play a role in regulating a wide variety of behavio-
ral processes, such as mood, appetite, and sexual behavior.[1–4]
The 5-HT2A receptor mediates the hallucinogenic activity of
drugs, such as lysergic acid diethylamide (LSD), and is a major
target for treating schizophrenia, insomnia and other disor-
ders.[5–8] The 5-HT2B receptor mediates the potentially lethal val-
vulopathic side effects of several compounds that were used
as prescription drugs.[9,10]
5-HT2C agonists have demonstrated efficacy in preclinical
models of depression, obesity, addiction, and psychosis.[11–13]
Thus, targeting the 5-HT2C receptor appears to offer a promis-
ing means for developing novel therapeutics for the treatment
of CNS-related disorders. However, as this receptor is homolo-
gous to the two other family members, 5-HT2A and 5-HT2B,[14] it
is essential that 5-HT2C agonists being developed for clinical
use show little, if any, activity at these subtypes.[15] To date,
several 5-HT2C agonists have shown efficacy in preclinical
animal models (2–8),[16–18] and are currently undergoing human
trials.[16] In particular, one of the most advanced 5-HT2C ligands
is lorcaserin (2), which is being developed by Arena Pharma-
ceuticals and which has been demonstrated in two phase III
trials to be an orally active, antiobesity medication.[19]
quired to identify a potential clinical candidate. Currently, the
degree of selectivity that is actually needed to avoid side ef-
fects is unknown; however, this is a question that can ultimate-
ly be addressed only by studies in humans. Thus, we explored
additional, selected modifications of these trans-2-phenylcyclo-
propylmethylamine analogues in order to improve the subtype
selectivity. Our efforts to modify the 5-HT2C agonist 8 led to
the discovery of several new drug candidates with increased
subtype selectivity, including dual 5-HT2B antagonism/5-HT2C
agonism in the functional assays, and which were thus found
suitable for in vivo testing as detailed herein.
Based upon the identification of tranylcypromine as the ini-
tial hit from an HTS campaign employing a library of FDA-ap-
proved drugs, we undertook a structural optimization cam-
paign that led to a potent, but moderately selective, agonist
(8) with 120- and 14-fold selectivity over 5-HT2A and 5-HT2B, re-
spectively (EC50 =585, 65, and 4.8 nm at the 2A, 2B, and 2C
subtypes, respectively). Compound 8 (10–-60 mgkgꢀ1) was also
demonstrated to exhibit moderate antidepressant-like effects
in a commonly used behavioral assay.[18]
By stepwise structural modifications of the trans-(2-arylcyclo-
propyl)methylamine aromatic moiety, we found that potent, 5-
HT2C-selective compounds could be produced using 5-hydroxy-
or 5-fluoro-substituted systems with a medium sized 2-alkyloxy
group. The 2-cyclopropylmethyloxy-5-fluoro-substituted deriva-
tive 12 was synthesized according to the steps shown in
Scheme 1. Thus, the starting compound 9 was prepared by
employing a standard sequence of reactions as previously re-
ported.[18] The amino group of the phenolic derivative 9 was
protected using di-tert-butyl dicarbonate (Boc2O). The N-Boc-
protected derivative 10 was then alkylated with cyclopropyl-
methyl bromide followed by subsequent deprotection to pro-
vide the racemic compound 12.
However, because compound 8 fails to exhibit sufficient se-
lectivity over the 5-HT2B receptor, further optimization was re-
[a] Prof. Dr. A. P. Kozikowski, Dr. S. J. Cho, Dr. A. M. Svennebring
Drug Discovery Program, Department of Medicinal Chemistry and Pharma-
cognosy, College of Pharmacy, University of Illinois at Chicago
Illinois 60612-7230 (USA)
Fax: (+1)312-996-7107
E-mail: kozikowa@uic.edu
[b] Dr. N. H. Jensen, Dr. J. A. Allen, Prof. Dr. B. L. Roth
To synthesize the 2-cyclopropylmethyloxy-5-hydroxy substi-
tuted derivative 18, the 2-cyclopropylmethyloxy intermediate
16 was prepared in a straightforward manner through a se-
quence of selective protection and alkylation steps.[20] Next,
the pivaloyl and Boc protecting groups were removed sequen-
tially to afford the final product 18 (Scheme 2).
Departments of Pharmacology and Psychiatry, Comprehensive Cancer
Center, Center for Neurobiology, Division of Medicinal Chemistry and Natu-
ral Products and NIMH Psychoactive Drug Screening Program
University of North Carolina Medical School
Chapel Hill CB 7365, North Carolina 27599 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000186.
ChemMedChem 2010, 5, 1221 – 1225
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1221

MED
Scheme 1. Reagents and conditions: a) Boc2O, Et3N, CH2Cl2, 08C!RT, 5 h;
b) (bromomethyl)cyclopropane, K2CO3, DMF, 608C, 20 h; c) 2m HCl, RT, 48 h.
Scheme 3. Reagents and conditions: a) 2m HCl, RT, 48 h. Chiral separation
was carried out on a Chiralpak AD column (30ꢁ250 mm, DAICEL); mobile
phase: hexane/iPrOH, 4% isocratic. Optical purity: (+)-11: D=+47.28; Chiral
HPLC purity: 13.2 min, >99%; (ꢀ)-11: D=ꢀ45.98; Chiral HPLC purity:
16.8 min, >99%.
at the 5-HT2C receptor. The more active enantiomer (+)-12 also
showed the expected selectivity profile in these functional
assays. In contrast, the less active isomer (ꢀ)-12 had an EC50
value of 918 nm in the 5-HT2C assay. For comparison, we also
tested the 5-HT2C ligand 2, which is in phase III clinical trials for
obesity. In our hands, this compound has a low nanomolar po-
tency at the 5-HT2C receptor, however, it is also fairly active at
the 5-HT2B subtype, with an EC50 value of 85 nm and an Emax
value of 93%. As such, there may be some risk for possible val-
vulopathic side effects induced by this drug. Compound (+)-
12 has an even lower selectivity margin towards the 5HT2B-sub-
type, but due to its low maximal activity (Emax =21%), we be-
lieve this will be a very minor concern, especially in view of
the 2B isoform antagonist activity described below. Three
other known 5-HT2C agonists, vabicaserin (3) and the Way com-
pounds 4 and 5, along with 5-HT (1) are also included in
Table 1 for reference purposes.
Scheme 2. Reagents and conditions: a) 1. BBr3, CH2Cl2, ꢀ788C!RT, 6 h;
2. Boc2O, Et3N, CH2Cl2, 08C!RT, 1 h; b) Piv-Cl, CH2Cl2, Et3N, 08C!RT, 6 h;
c) (bromomethyl)cyclopropane, K2CO3, DMF, 608C, 20 h; d) NaOtBu, MeOH,
RT, 1 h; e) 2m HCl, RT, 48 h.
To prepare the optically pure enantiomers of 12 (Scheme 3),
we first carried out a chiral separation of the N-Boc-protected
fluoro derivative 11 using a Chiralpack AD column. Under iso-
cratic conditions (4% isopropanol in hexane), the individual
enantiomers could be conveniently separated in pure form
(>99%). Due to the high resolution, stacked injections could
be employed in order to increase throughput. The choice of in-
termediate 11 for the chiral resolution was based on the ease
of the separation and the ready cleavage of the resulting enan-
tiomers under acidic conditions.
In order to further characterize the pharmacology of the
lead compounds, both (+)-12 and (+)-18, which show minimal
5-HT2B activation, were tested for their ability to function as
5-HT2B antagonists. Compounds (+)-12 and (+)-18 were found
to shift the concentration curve of 5-HT in calcium flux assays
to the right, without depressing the maximal 5-HT response,
indicating fast binding kinetics (Figure 1). Schild analyses yield-
ed pA2 (ꢁSEM) values of 5.50ꢁ0.06, 5.79ꢁ0.07, and 5.92ꢁ
0.02, respectively (n=3). These results show that the com-
pounds tested act as moderately potent, full antagonists at the
5-HT2B receptor.
The resulting enantiomers (+)- and (ꢀ)-11 were then con-
verted individually to (ꢀ)- and (+)-trans-[2-(2-cyclopropylme-
thyloxy-5-fluorophenyl)cyclopropyl]methylamine
hydrochlor-
ides [(ꢀ)-12 and (+)-12], respectively, using the same method
as described above for the racemate. For preparation of the
pure enantiomers of compound 18, intermediate 17 was used
for the chiral separation.
The functional activity of these two sets of compounds was
determined by measuring Gaq-mediated intracellular calcium
mobilization in HEK-293 cells stably expressing the human
5-HT2A, human 5-HT2B, and human 5-HT2C (INI) receptors.[21] The
results are summarized in Table 1. In the first round of func-
tional assays, racemic 12 was found not to activate either the
5-HT2A or 5-HT2B receptors and to have an EC50 value of 19 nm
On the basis of their promising in vitro pharmacology, the
active enantiomers (+)-12 and (+)-18 were selected for further
in vivo studies. Specifically, we examined the ability of these
compounds to normalize disrupted prepulse inhibition (PPI) in
animals treated with phencyclidine (PCP; an NMDA receptor
antagonist).[22] The ability to normalize the effects of PCP-
1222
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1221 – 1225

Table 1. Functional activity and selectivity of racemic 12 and 18, their enantiomers, and comparison compounds at the Human 5-HT2A, 5-HT2B, and 5-HT2C
receptors in calcium flux assays using stably transfected HEK-293 cells.
5-HT2A
Emax [%]
5-HT2B
5-HT2C
Emax [%]
Selectivity
[b]
[b]
[b]
Compound[a]
EC50 [nm]
n[c]
EC50 [nm]
Emax [%]
n[c]
EC50 [nm]
n[c]
2A/2C
2B/2C
First round
1 (5-HT)
(+/-)-12
6.9ꢁ0.42
NA
761ꢁ160
264ꢁ31
NA
100
15
3
3
6
6
0.7ꢁ0.02
100
16
3
3
6
6
0.1ꢁ0.01
19ꢁ3.5
100
16
3
3
7
7
63
–
77
123
–
6.7
–
–
40
–
14
–
7ꢁ1.8
11ꢁ1.9
24ꢁ0.9
2ꢁ0.1
80ꢁ1.0
1ꢁ0.2
NA
6ꢁ0.6
2ꢁ0.9
93ꢁ1.2
5ꢁ1.0
92ꢁ1.3
6ꢁ1.3
68ꢁ5.2
68ꢁ4.5
99ꢁ1.0
95ꢁ0.8
97ꢁ1.6
80ꢁ1.1
(+/-)-18
NA
85ꢁ7.0
NA
15ꢁ1.6
NA
9.9ꢁ1.7
2.1ꢁ0.29
6.0ꢁ1.0
1.1ꢁ0.16
286ꢁ40
2 (lorcaserin)
3 (vabicaserin)
4 (WAY-161503)
5 (WAY 629)
Second round
1 (5-HT)
76ꢁ9.1
NA
3
6
3
6
4
7
70
–
6.1ꢁ0.38
894ꢁ91
NA
372ꢁ131
NA
136ꢁ24
NA
61ꢁ8.3
NA
100
9
3
3
3
3
3
3
3
3
0.8ꢁ0.08
289ꢁ34
NA
NA
NA
100
9
3
3
3
3
3
3
3
3
0.1ꢁ0.0
21ꢁ2.2
100
9
3
3
3
3
3
3
3
3
62
42
–
40
–
79
–
40
–
7.7
14
–
–
–
29
5.8
11
–
(+)-12
29ꢁ1.6
0ꢁ0.0
18ꢁ3.0
3ꢁ0.6
31ꢁ0.5
7ꢁ0.1
79ꢁ4.5
7ꢁ2.0
21ꢁ4.9
4ꢁ2.7
6ꢁ2.8
0ꢁ0.5
86ꢁ2.3
15ꢁ3.3
86ꢁ4.7
17ꢁ2.7
71ꢁ4.4
35ꢁ2.4
70ꢁ5.4
51ꢁ2.4
94ꢁ0.8
88ꢁ1.8
94ꢁ3.6
82ꢁ3.5
(ꢀ)-12
918ꢁ83
9.3ꢁ0.05
361ꢁ21
1.7ꢁ0.09
8.0ꢁ1.6
1.5ꢁ0.18
451ꢁ52
(+)-18
(ꢀ)-18
2 (lorcaserin)
3 (vabicaserin)
4 (WAY-161503)
5 (WAY 629)
50ꢁ10
47ꢁ6.4
16ꢁ3.2
> 5 mm
[a] Tested in two independent screening campaigns using different cell lines/passages; direct comparisons of the potencies and efficacies are only valid
within the bounds of each particular table section. [b] Percent of maximal activation by 5-HT (Emax); activation at 10 mm for compounds without EC50 value;
values are given ꢁSEM (standard error of the mean). [c] n: number of concentration curves from ꢂ2 (typically ꢂ3) independent experiments; values are
given ꢁSEM. NA: not applicable (Emax ꢃ12%). In contrast to binding affinities, the potencies in functional assays can vary strongly depending on cell type
and receptor-expression level.
induced disruption of PPI in mice is a well accepted model for
measuring atypical antipsychotic activity,[23,24] and it has been
used previously to characterize the antipsychotic activity of 5-
HT2C agonists.[25] Previously, we successfully used an identical
PPI testing paradigm to demonstrate clozapine, a classic atypi-
cal antipsychotic, robustly normalizes PCP-disrupted PPI.[26] We
tested the ability of compounds (+)-12 and (+)-18, as well as
the reference compound 3, to normalize PCP effects on PPI
(Figure 2). As is apparent from the accompanying figures, both
(+)-12 and (+)-18 are able to normalize PCP-disrupted PPI at
doses of 10 mgkgꢀ1 (and are comparable in activity to 3),
while at the 5 mgkgꢀ1 dose level, (+)-12 and 3 are still effec-
tive.
Because of its robust activity at normalizing PCP-disrupted
PPI, we carried out cytochrome P450 screening, metabolic sta-
bility studies, and hERG assays on (+)-12 in order to qualify it
as a possible candidate for further development. In the re-
combinant CYP inhibition test, (+)-12 showed relatively low in-
hibition against CYP2C9 (19.6%), CYP2D6 (27.0%), and CYP3A4
(21.7% and 25.1%) using midazolam and testosterone as the
substrates, respectively, at 10 mm (Table 2). Furthermore, com-
pound (+)-12 had an acceptable microsomal stability in a 1 h
assay (90.5% left in human microsomes and 66% in rat, re-
spectively) compared with the control drug, verapamil (39.6%
using human microsomes). Additionally, no hERG inhibition
was detected for (+)-12, (ꢀ)-12, or 18, nor for the comparison
Figure 1. 5-HT2B antagonism (Schild plots) of compounds a) (+)-18 (*, no
compounds 2, 3, and 5. However, compound 4 caused very
test compound; ^, 316 nm; *, 3.16 mm; &, 31.6 mm) and b) (+)-12 (*, no
modest inhibition (3.6 mm, 97%), and the standard cisapride
gave full inhibition (35 nm, 100%).
test compound; *, 3.16 mm; &, 31.6 mm). pA2 (ꢁSEM): 5.79ꢁ0.07 and
5.92ꢁ0.02; n=3.
ChemMedChem 2010, 5, 1221 – 1225
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1223

MED
Table 2. Cytochrome P450 inhibition profile of compound (+)-12.
CYP[a]
2C9
Substrate
Test inhibitors
Inhibition[b] [%]
tolbutamide
sulfaphenazole
(+)-12
quinidine
(+)-12
ketokonazole
(+)-12
ketokonazole
(+)-12
94.7
19.6
98.7
27.0
99.8
21.7
99.7
25.1
2D6
3A4
dextrometorphan
midazolam
testosterone
[a] Cytochrome P450 enzyme. [b] Percent inhibition was measured at
10 mm.
In conclusion, our optimization efforts led to the discovery
of the highly selective 5-HT2C agonists (ꢁ)-12 and (ꢁ)-18, and
to the identification of (+)-12 and (+)-18 as the more active
enantiomers. Compounds (+)-12 and (+)-18 were as effective
as the known reference compound 3 in the PCP-PPI animal
model of antipsychotic drug activity. Compound 3 was recently
in phase II clinical trials for the treatment of acute exacerba-
tions of schizophrenia. Compound (+)-12 has an acceptable
DMPK profile, while showing no adverse side effects in animals.
Compound (+)-12 represents a structurally simple 5-HT2C par-
tial agonist of excellent potency and high selectivity. The struc-
tural relationship of 12 with the marketed monamine oxidase
inhibitor tranylcypromine is also noteworthy, and when com-
bined with the information now in hand, bodes well for the
further development of this class of molecules. The study of
(+)-12 in other animal disease models, including cocaine
addiction, is underway.
Experimental Section
Full experimental protocols can be found in the Supporting Infor-
mation.
All experiments were conducted in accordance with National Insti-
tutes of Health (USA) guidelines and under an approved protocol
from the Institutional Animal Care and Use Committee at the Uni-
versity of North Carolina (USA).
Acknowledgements
This work was supported by NIH grants R01 DA022317 (A.P.K.)
and R01 MH61887, N01 MH80032, and U19 MH82441 (B.L.R.).
S.J.C. was supported by the Korea Research Foundation Grant
funded by the Korean Government (KRF-2007-357-C00071) and
the NIDA fellowship (2006–2007). J.A.A. was supported by fellow-
ship NICHD T32HD040127 and the UNC Neurodevelopmental Dis-
orders Research Center. We thank Drs. Arsen Gaysin and Rong He
for technical assistance. We thank Barbara Caldarone for review-
ing the article and providing comments.
Figure 2. Compounds (+)-12 and (+)-18 normalize PCP-disrupted PPI in
mice. Mice were administered (ip) with saline or 6 mgkgꢀ1 PCP and immedi-
ately tested for PPI of the acoustic startle response. The pretreatments of
mice (30 min) with a) (+)-12 or b) (+)-18 effectively normalize PCP-disrup-
tion of PPI. c) Compound 3, a positive reference control compound, also
normalizes PCP-disrupted PPI. , p<0.05 versus saline treated animals. Data
*
Keywords: 5-HT2C
chemistry · schizophrenia
· CNS · drug discovery · medicinal
are the mean ꢁSEM. n=9, 10 or 7 animals in each testing group for (+)-12,
(+)-18 or 3, respectively.
1224
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1221 – 1225

[1] J. M. Chou-Green, T. D. Holscher, M. F. Dallman, S. F. Akana, Physiol.
Behav. 2003, 78, 641.
[17] R. G. Booth, L. Fang, Y. Huang, A. Wilczynski, S. Sivendran, Eur. J. Phar-
macol. 2009, 615, 1.
[2] B. A. Rocha, E. H. Goulding, L. E. O’Dell, A. N. Mead, N. G. Coufal, L. H.
Parsons, L. H. Tecott, J. Neurosci. 2002, 22, 10039.
[3] L. H. Tecott, L. M. Sun, S. F. Akana, A. M. Strack, D. H. Lowenstein, M. F.
Dallman, D. Julius, Nature 1995, 374, 542.
[4] M. J. Millan, J. L. Peglion, G. S. L. Perrin-Monneyron, Eur. J. Pharmacol.
1997, 325, 9.
[5] D. E. Nichols, Pharmacol. Ther. 2004, 101, 131.
[18] J. S. Cho, H. N. Jensen, T. Kurome, S. Kadari, L. M. Manzano, E. J. Malberg,
B. Caldarone, L. B. Roth, P. A. Kozikowski, J. Med. Chem. 2009, 52, 1885.
[19] W. Wolfson, Chem. Biol. 2008, 15, 1139.
[20] Q. Yao, Angew. Chem. 2000, 112, 4060; Angew. Chem. Int. Ed. 2000, 39,
3896.
[21] N. H. Jensen, R. M. Rodriguiz, M. G. Caron, W. C. Wetsel, R. B. Rothman,
B. L. Roth, Neuropsychopharmacology 2008, 33, 2303.
[22] N. R. Swerdlow, D. L. Braff, N. Taaid, M. A. Geyer, Arch. Gen. Psychiatry
1994, 51, 139.
[23] M. A. Geyer, B. Ellenbroek, Prog. Neuro-Psychopharmacol. Biol. Psychiatry
2003, 27, 1071.
[6] B. M. Nilsson, J. Med. Chem. 2006, 49, 4023.
[7] E. Lacivita, M. Leopoldo, Curr. Top. Med. Chem. 2006, 6, 1927.
[8] S. Ahmad, K. Ngu, K. J. Miller, G. Wu, C. P. Hung, S. Malmstrom, G.
Zhang, E. O’Tanyi, W. J. Keim, M. J. Cullen, K. W. Rohrbach, M. Thomas, T.
Ung, Q. Qu, J. Gan, R. Narayanan, M. A. Pelleymounter, J. A. Robl, Bioorg.
Med. Chem. Lett. 2010, 20, 1128.
[24] M. A. Geyer, K. Krebs-Thomson, D. L. Braff, N. R. Swerdlow, Psychophar-
macology 2001, 156, 117.
[9] R. B. Rothman, M. H. Baumann, J. E. Savage, L. Rauser, A. McBride, S. J.
Hufeisen, B. L. Roth, Circulation 2000, 102, 2836.
[10] B. L. Roth, N. Engl. J. Med. 2007, 356, 6.
[11] J. Dunlop, K. L. Marquis, H. K. Lim, L. Leung, J. Kao, C. Cheesman, S.
Rosenzweig-Lipson, CNS Drug Rev. 2006, 12, 167.
[12] S. Burbassi, L. Cervo, Psychopharmacology 2008, 196, 15.
[13] K. J. Miller, Mol. Interventions 2005, 5, 282.
[25] L. K. Marquis, L. A. Sabb, F. S. Logue, A. J. Brennan, J. M. Piesla, A. T.
Comery, M. S. Grauer, R. C. Ashby, Q. H. Nguyen, A. L. Dawson, E. J. Bar-
rett, G. Stack, Y. H. Meltzer, L. B. Harrison, S. Rosenzweig-Lipson, J. Phar-
macol. Exp. Ther. 2006, 320, 486.
[26] I. A. Abbas, N. P. Yadav, W.-D. Yao, I. M. Arbuckle, G. N. S. Grant, G. M.
Caron, L. B. Roth, J. Neurosci. 2009, 29, 7124.
[14] W. K. Kroeze, K. Kristiansen, B. L. Roth, Curr. Top. Med. Chem. 2002, 2,
507.
Received: April 30, 2010
[15] M. Berger, J. A. Gray, B. L. Roth, Annu. Rev. Med. 2009, 60, 355.
[16] D. A. Wacker, K. J. Miller, Curr. Opin. Drug Discovery Dev. 2008, 11, 438.
Published online on June 8, 2010
ChemMedChem 2010, 5, 1221 – 1225
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1225