Synthesis and biological evaluation of novel hexahydro-pyrido[3′, 2′:4,5]pyrrolo[1,2-a]pyrazines as potent and selective 5-HT2C receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2005.11.083

Further lead optimization efforts on previously described 1,2,3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indoles led to the new class of 5,5a,6,7,8,9-hexahydro-pyrido[3′,2′:4,5]pyrrolo[1,2-a]pyrazines culminating in the discovery of (5aR,9R)-2-[(cyclopropylmethoxy)methyl]-5,5a,6, 7,8,9-hexahydro-9-methyl-pyrido[3′, 2′:4,5]pyrrolo[1,2-a]pyrazine 18 as a potent, full 5-HT_2C receptor agonist with an outstanding selectivity profile and excellent hERG and phospholipidosis properties.

Full text of DOI:10.1016/j.bmcl.2005.11.083

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1207–1211
Synthesis and biological evaluation of novel
hexahydro-pyrido[30,20:4,5]pyrrolo[1,2-a]pyrazines as potent
and selective 5-HT2C receptor agonists
Hans G. F. Richter,a,* D. R. Adams,b A. Benardeau,a M. J. Bickerdike,b J. M. Bentley,b,
T. J. Blench,b I. A. Cliffe,b C. Dourish,b P. Hebeisen,a G. A. Kennett,b A. R. Knight,b
C. S. Malcolm,b P. Mattei,a A. Misra,b J. Mizrahi,a N. J. T. Monck,b J.-M. Plancher,a
S. Roever,a J. R. A. Roffey,b S. Taylora and S. P. Vickersb
aF. Hoffmann-La Roche Ltd, Discovery Research, 4070 Basel, Switzerland
bVernalis Research Ltd, Oakdene Court, 613 Reading Road, Wokingham, RG41 5UA, UK
Received 4 October 2005; revised 10 November 2005; accepted 23 November 2005
Available online 19 December 2005
Abstract—Further lead optimization efforts on previously described 1,2,3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indoles led to the
new class of 5,5a,6,7,8,9-hexahydro-pyrido[30,20:4,5]pyrrolo[1,2-a]pyrazines culminating in the discovery of (5aR,9R)-2-[(cyclopro-
pylmethoxy)methyl]-5,5a,6,7,8,9-hexahydro-9-methyl-pyrido[30, 20:4,5]pyrrolo[1,2-a]pyrazine 18 as a potent, full 5-HT2C receptor
agonist with an outstanding selectivity profile and excellent hERG and phospholipidosis properties.
Ó 2005 Elsevier Ltd. All rights reserved.
Obesity is steadily increasing all over the world 1 and is a
major risk factor in the development of hyperglycemia,
hypertension, dyslipidemia, coronary artery disease,
and certain cancers.2
has been established through the use of 5-HT2C receptor
agonists, antagonists, and transgenic mouse models.3
The non-selective 5-HT2C receptor agonist m-chlorophe-
nylpiperazine (mCPP) reduces food intake in rats and
accelerates the appearance of the behavioral satiety se-
quence in rats. The anorectic action of mCPP is absent
in 5-HT2C receptor knockout mutant mice4 and is antag-
onized by the selective 5-HT2C receptor antagonist SB-
242084.5 In clinical studies, mCPP decreases food intake
and body weight of obese subjects.6 Phase IIa clinical
trials have demonstrated significant body weight loss
with the 5-HT2C receptor agonists BVT-933 (Biovitrum;
development stopped) and APD356 (Arena; Phase IIb
clinical trials initiated June 2005). The structures for
both compounds have not been published yet. In the
search for novel 5-HT2C receptor agonists, we recently
reported the discovery and synthesis of 1,2,3,4,10,10a-
hexahydro-1H-pyrazino[1,2-a]indoles as potent and
selective 5-HT2C receptor agonists such as 1.7 Although
compound 1 was a potent full agonist at the 5-HT2C
receptor and showed >10-fold selectivity against both
5-HT2A and 5-HT2B receptors, it was also found to
inhibit the hERG potassium channel in vitro with an
IC50 of 2.5 lM. Phospholipidosis was also induced by
The currently approved drugs for the long-term treat-
ment of obesity are the appetite suppressant Sibutr-
amineÒ,
a centrally acting mixed noradrenaline/
serotonin-reuptake inhibitor, and XenicalÒ, a non-sys-
temic acting lipase inhibitor which increases the fecal
loss of undigested triglycerides. Recent evidence of the
utility of 5-HT2C receptor agonists as appetite suppres-
sants in the management of obesity has led to a resur-
gence of interest in selective and safe 5-HT2C receptor
agonists.
In preclinical studies, the involvement of the 5-HT2C
receptor subtype in the control of feeding in animals
Keywords: 5-HT2C receptor agonist; Pyrido[30,20:4,5]pyrrolo[1,2-a]pyr-
azines; Obesity; Phospholipidosis; hERG.
*
Corresponding author. Tel.: +41 61 688 1330; fax: +41 61 688 6459;
e-mail: hans.richter@roche.com
Present address: GlaxoSmithKline S.p.A., Centre of Excellence for
Drug Discovery, Via Fleming 4, 37135 Verona, Italy.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.083

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H. G. F. Richter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1207–1211
1 in cultured fibroblasts8 at the lowest tested concentra-
tion of 2.5 lM (test range 2.5–20 lM). Striving for im-
proved properties with respect to both the hERG and
phospholipidosis flag we hypothesized that replacement
of the indoline core by an azaindoline system would
reduce lipophilicity and amphiphilicity and lead to
molecules with improved safety profile with respect to
these properties. This led us to the identification
of 5,5a,6,7,8,9-hexahydro-pyrido[30,20:4,5]pyrrolo[1,2-
a]pyrazines as a new class of potent human 5-HT2C
receptor agonists with improved binding selectivity pro-
files and good in vivo potency in rats after oral admin-
istration. In addition, several analogues in fact showed
significantly reduced hERG inhibition and phospholipi-
dosis induction in vitro.
tetrahydro-9-methyl-pyrido[30,20:4,5]pyrrolo[1,2-a]pyra-
zines, which after Boc-protection were further reduced
with NaCNBH3 in acetic acid to provide the (5aR,9R)-
diastereomer11 in an 8:1 excess over its (5aS,9R)-diaste-
reomer. The stereoisomers were separated by conventional
column chromatography on silica gel, which after
deprotection gave the final compounds 7 and 8. Similar-
ly the (5aS,9S)- and (5aR,9S)-diastereomers as well as
the 9-unsubstituted analogues could be synthesized from
either the (R)-5-methyl- or the 5-unsubstituted 2,2-
dioxo[1,2,3]oxathiazolidine-3-carboxylic acid tert-butyl
esters (not shown).
The Boc-protection of 7 yielded intermediate 9, which
turned out to be a versatile building block applicable
to various types of organometallic and transition met-
al-catalyzed reactions. Subsequent deprotection provid-
ed target molecules with more elaborate substitution
patterns (Scheme 2). For example, carbonylation of 9
with carbon monoxide under Pd catalysis in methanol
gave the corresponding methyl ester 15 which could be
reduced with DIBAL-H to give the 2-hydroxymethyl
compound which in turn could be further alkylated
deprotected to yield compounds 16–18. Alternatively
the ester 15 could be transformed into the Weinreb
amide using N,O-dimethylhydroxylamine and AlMe3,
which upon reaction with methyl magnesium bromide
gave the acetyl compound. Borane mediated reduction
of the acetyl derivative using chiral (R)-2-methyl-CBS-
oxazaborolidine catalyst led to the (S)-1-hydroxyethyl
intermediate in high diastereomeric excess (de > 99%).
Alkylation and deprotection led to the final derivative
20. Accordingly the (R)-1-hydroxyethyl diastereomer
could be obtained by using the (S)-2-methyl-CBS-oxaz-
aborolidine catalyst.
Synthesis of chiral 9-methyl-substituted hexahydro-pyr-
ido[30,20:4,5]pyrrolo[1,2-a]pyrazines was accomplished
using 1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid ethyl
ester 29 and recently developed 5-methyl-2,2-
dioxo[1,2,3]oxathiazolidine-3-carboxylic acid tert-butyl
ester 5 as a new type of crystalline and stable chiral
alkylating agent (Scheme 1).10 Chlorination or bromin-
ation of 2, ortho- to the pyridine nitrogen, was achieved
after Boc-protection, oxidation with m-CPBA, and sub-
sequent rearrangement of the N-oxide 3 with ethyl chlo-
roformate and benzoylbromide, respectively, in THF
with HMDS as a base. The 7-aza-indoles 4 were alkyl-
ated in excellent yields using the previously described
sulfamidate 5. In line with an SN2 reaction this proceed-
ed with inversion of configuration. Boc-deprotection of
the 1-alkylated 7-azaindole-2-carboxylates with trifluo-
roacetic acid in dichloromethane was followed by
cyclization to the dihydro-pyrido[30,20:4,5]pyrrolo[1,2-
a]pyrazinones 6 using potassium carbonate in methanol
as base. Reduction with LiAlH4 gave the corresponding
The compounds were screened at 1 lM for functional
activity at human recombinant 5-HT2C receptors ex-
pressed in CHO cells using a fluorimetric imaging plate
reader (FLIPR). The maximum fluorescent signal was
measured and compared with the response produced
by 10 lM 5-HT (defined as 100%).12 The active com-
pounds from this functional assay were then compared
Scheme 1. Reagents and conditions: (a) (Boc)2O, DMAP, CH3CN, rt, 99%; (b) m-CPBA, CH2Cl2, rt, 60%; (c) ClCO2Et, HMDS, THF, rt, 76% or (d)
BzBr, HMDS, THF, rt, 51%; (e) TFA, CH2Cl2, 0 °C to rt, 96–99%; (f) 5, NaH, DMF, 0 °C to rt, 93–98%; (g) TFA, CH2Cl2, 0 °C to rt, then MeOH,
K2CO3, rt, 74–87%; (h) LiAlH4, t-BuOMe, reflux, 15 min, 78–96%; (i) (Boc)2O, CH2Cl2, rt, 85–98%; (j) NaCNBH3, AcOH, rt, 66–88% for major
diastereomer (5aR,9R); (k) TFA, CH2Cl2, rt, 96%.

H. G. F. Richter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1207–1211
1209
Scheme 2. Reagents and conditions: (a) CuI, Pd2(dba)3, dppf, Et4NCN, dioxane, reflux; TFA, CH2Cl2, rt, 61% (10); (b) Pd2(dba)3, ( )-BINAP,
NaOt-Bu, toluene, benzophenone imine, reflux; Pd/C, NH4-formate, MeOH; AcCl, NEt3, CH2Cl2, rt; TFA, CH2Cl2, rt, 48% (11); (c) Pd2(dba)3,
BnOH, ( )-BINAP, NaOH, toluene, 70 °C; H2, Pd/C, MeOH/EtOAc, rt; MeI, NaH, DMF, rt; TFA, CH2Cl2, rt, 39% (12); (d) Pd[PPh3]4, Na2CO3,
(MeBO)3, DME, reflux; TFA, CH2Cl2, rt, 41% (13); (e) CF3CO2Na, CuI, NMP, 180 °C; TFA, CH2Cl2, rt, 20% (14); (f) CO (40 bar), dppf, Pd(OAc)2,
NEt3, MeOH, 80 °C, 74% (15); (g) DIBAL-H, THF, 0 °C to rt; TFA, CH2Cl2, rt, 17% (16); (h) DIBAL-H, THF, 0 °C to rt; MeI, NaH, DMF, rt;
TFA, CH2Cl2, rt, 77% (17); (i) DIBAL-H, THF; c-PrCH2Br, NaH, DMF, rt; TFA, CH2Cl2, rt, 41% (18); (j) HNMe(OMe), AlMe3, THF, 0 °C to rt,
94%; (k) MeMgBr, THF, ꢀ78 °C to rt, 92%; (l) (R)-Me-CBS-oxazaborolidine, BMS, THF, 0 °C, 98%; (m) TFA, CH2Cl2, 0 °C to rt, 74% (19) or
EtBr, NaH, DMF, 50 °C; TFA, CH2Cl2, 0 °C to rt, 52% (20).
in radioligand binding assays at recombinant human 5-
HT2A, 5-HT2B, and 5-HT2C receptors expressed in CHO
cells. For the 5-HT2A receptor [125I]-DOI was used as
the radioligand, whereas [3H]-5-HT was used for the 5-
HT2B and 5-HT2C receptor subtypes.13 The results are
shown in Tables 1 and 2. As described in Table 1 for
the 2-chloro substituted analogues, introduction of a
9R-methyl substituent increased the 5-HT2C receptor
binding affinity. As exemplified for the 2-chloro and 2-
bromo analogues (11 and 12, respectively), both the
(5aR,9R) and (5aS,9R) stereochemistries provided full
agonists. In contrast to the carba series7 however, the
(5aR,9R)-diastereomer provided both higher binding
affinity for the 5-HT2C receptor and significantly higher
binding selectivity over the other 5-HT2 receptor sub-
types compared to the (5aS,9R)-diastereomer. The
(5aR,9R)-7-bromo-9-methyl compound 7b was an
exceptional sub-nanomolar agonist with approximately
66-fold and 100-fold binding selectivity over the 5-
HT2A and 5-HT2B receptor, respectively.
rolo[1,2-a]pyrazine skeleton on receptor affinity and
selectivity is exemplified in Table 2. Small lipophilic sub-
stituents in the 2-position (CN, CF3, and Me), whether
electron withdrawing or donating, all led to high affinity
for the 5-HT2C receptor, while polar substituents led to a
comparative loss in affinity. Hydroxy (i.e., 16, 19) and
especially alkoxy substituents (i.e., 17, 18, and 20) were
also tolerated, however, only if the oxygen atom was not
directly attached to the core, as it is for compound 12.
From the branched ether analogues compound 20
had high binding affinity for the target receptor
(Ki 2.3 nM), 28-fold binding selectivity over the
5-HT2A receptor, and exceptionally high binding
selectivity of 128-fold over the 5-HT2B receptor. The
corresponding (R)-1-ethoxyethyl diastereomer 21 was
much less potent and selective, especially over the
5-HT2B receptor.
Further pharmacological characterization of 18 and 20
was performed using measurements of the stimulation
of intracellular calcium mobilization in stable CHO cell
lines expressing each of the human 5-HT2 receptor sub-
types14 and the results are summarized in Table 3. Both
The influence of several other substituents in the 2-position
of the 5,5a,6,7,8,9-hexahydro-9-methyl-pyrido[30,20:4,5]pyr-
Table 1. 5-HT2 receptor efficacy and binding of 5,5a,6,7,8,9-hexahydro-pyrido[30,20:4,5]pyrrolo[1,2-a]pyrazines—initial SAR
Compound Substituents Stereochemistry Relative efficacya h5-HT2C (%) Kib h5-HT2A (nM) Kic h5-HT2B (nM) Kic h5-HT2C (nM)
25
7a
2-Cl, 9-H
rac
NDd
98
217
42
54
53
58
19
221
48
131
1.6
52
0.8
21
1.9
0.3
2-Cl, 9-Me
2-Cl, 9-Me
2-Br, 9-Me
2-Br, 9-Me
5aR,9R
5aS,9R
5aR,9R
5aS,9R
4R,10aS
4R,10aR
8a
7b
8b
1
81
97
217
81
87
97
166
40
1
98
2.6
3.2
a Efficacy at 1 lM ligand concentration relative to 10 lM of 5-HT (100%).
b Displacement of [125I]-DOI.
c Displacement of [3H]-5-HT.
d ND, not determined.

1210
H. G. F. Richter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1207–1211
Table 2. 5-HT2 receptor efficacy and binding of 2-substituted (5aR,9R)-9-methyl-5,5a,6,7,8,9-hexahydro-pyrido[30,20:4, 5]pyrrolo[1,2-a]pyrazines
Compound
2-Substituent
Relative efficacya h5-HT2C (%)
Kib h5-HT2A (nM)
Kic h5-HT2B (nM)
Kic h5-HT2C (nM)
10
11
12
13
14
16
17
18
19
20
21
CN
NHAc
85
0
2547
NDd
511
136
43
1820
NDd
440
77
60
NDd
104
2.7
14
MeO
Me
77
101
96
88
97
87
101
83
86
CF3
CH2OH
68
942
565
74
1139
379
174
835
295
1306
54
16
CH2OMe
CH2OCH2c-Pr
(S)-CH3(OH)CH
(S)-CH3(OEt)CH
(R)-CH3(OEt)CH
3.6
39
392
65
2.3
93
2385
a Efficacy at 1 lM ligand concentration relative to 10 lM of 5-HT (100%).
b Displacement of [125I]-DOI.
c Displacement of [3H]-5-HT.
d ND, not determined.
Table 3. Functional activitiesa in stable CHO cell lines expressing
human 5-HT2 receptor subtypes
Several of the newly synthesized aza-analogues were
profiled under the same assay conditions and the results
are summarized in Table 5. Compared to 1, several of
the tested aza-analogues in fact showed substantially
attenuated hERG activity and exhibited a markedly re-
duced phospholipidosis potential that we attribute to
their reduced lipophilicity and amphiphilicity. Com-
pound 18 did not induce phospholipidosis up to the
highest test concentration of 20 lM and had a clean
hERG profile with an IC50 > 30 lM.
Compound
h5-HT2A
h5-HT2B
h5-HT2C
EC50
(nM)
Emax
EC50
(nM)
Emax
EC50
(nM)
Emax
18
23
N/Ab
N/Ab
9
6
23
26
31
56
15
90
93
6.2
a Efficacy was determined by receptor-induced mobilization of intra-
cellular Ca2+ using fluorometric imaging plate reader (FLIPRTM).
b N/A: insufficient efficacy for generating an EC50 value.
Several representatives showed good in vivo efficacy
after per os (po) application in a rodent food-deprived
model. For example, compound 18 was tested for its
ability to reduce food intake in 23 h food-deprived Lis-
ter Hooded rats after an acute administration.15 The
compound significantly reduced food intake in a dose-
dependent manner with a minimum effective dose
(MED) of 10 mg/kg po. This effect was antagonized by
the 5-HT2C receptor antagonist, SB-242084 (0.3, 1,
3 mg/kg sc), suggesting that the effects of 18 on food in-
take are mediated solely through activation of 5-HT2C
receptors.
compounds were found to be potent and full 5-HT2C
receptor agonists, weak partial agonists at the 5-HT2B
receptor, and failed to activate 5-HT2A receptors.
The lack of 5HT2A receptor agonism prompted us to
evaluate 18 for its ability to antagonize 5-HT-stimulated
calcium mobilization in a 5-HT2A receptor-expressing
cell line. Indeed, 18 also possessed 5-HT2A receptor
antagonist activity and exhibited appreciable affinity
with a pKB of 6.7. Ancillary binding studies addressing
selectivity of 18 toward a number of biogenic amine
binding sites and a broad spectrum of receptor, trans-
porter, and ion channel targets failed to reveal potent
binding affinity for any of the targets examined, indicat-
ing the compound to be highly selective for the 5-HT2C
receptor subtype (Table 4).
In conclusion, we have developed a novel series of chiral
5,5a,6,7,8,9-hexahydro-9-methyl-pyrido[30,20:4,5]pyrrolo-
[1,2-a]pyrazines as potent, selective, and in vivo active
Table 5. In vitro hERG and phospholipidosis results for selected
(5aR,9R)-9-methyl-5,5a,6,7,8,9-hexahydro-pyrido[30,20:4,5]pyrrolo[1,2-a]-
pyrazines
Although the original lead compound 1 was a potent 5-
HT2C receptor agonist with favorable 5-HT2 receptor
subtype selectivity, it also had some undesired effects,
as it induced phospholipidosis in vitro and inhibited
the hERG potassium channel with an IC50 of 2.5 lM.
Compound 2-Substituent
hERGa IC50
Phospholipidosisb
(lM)
1(4R,10aS)
2.5
21
+++ (7.5 lM)
Negative (20 lM)
+ (20 lM)
13
7a
18
20
Me
Cl
40
>30
Table 4. Ancillary receptor binding affinities determined for 18
CH2OCH2c-Pr
Negative (20 lM)
Target
Ki (nM)
(S)-CH3(OEt)CH 20% inhibition + (20 lM)
at 10 lMc
5-HT1D
5-HT1A/1B/5A/6; D4
5-HT3/7
>360
>720
a Inhibition of hERG K channel determined by whole-cell patch-clamp
experiments on a transfected CHO cell line.
>1000
>1000
>1000
>1000
a1, b1, l
5-HT-, NA-, DA-transporter
Na channel
b Induction of phospholipidosis in cultured fibroblasts: strong (+++),
moderate (++), and weak (+).
c Insufficient inhibition for generating an IC50 value.

H. G. F. Richter et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1207–1211
1211
Kennett, G. A.; Lightowler, S.; Mattei, P.; Mizrahi, J.;
Morley, T. J.; Plancher, J.-M.; Richter, H.; Roever, S.;
Taylor, S.; Vickers, S. P. WO2002051844A1; Chem. Abstr.
2002, 137, 78856.
5-HT2C receptor agonists. From this series, compound 18
emerged as a potent, full agonist with an outstanding
selectivity profile. Moreover, 18 did not induce phospho-
lipidosis in vitro and had a clean hERG profile with an
IC50 > 30 lM.
11. Enantiomeric excess in the SN2 reaction was determined
by means of chiral HPLC using 2% ethanol in heptane as
mobile and Chiralpak-AD as stationary phase. Confirma-
tion of the absolute configuration of the C5a- and C9
carbon was carried out by crystallization and X-ray of
(5aR,9R)-2-chloro-9-methyl-5a,6,8,9-tetrahydro-(5H)-pyr-
ido[30,20:4,5]pyrrolo[1,2-a]pyrazine-7-carboxylic acid 1,1-
dimethylethyl ester, which was synthesized according to
Scheme 2. CCDC 288646 contains the supplementary
crystallographic data for this paper. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/data_re-
quest/cif, by emailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223
336033.
Acknowledgments
Our warmest thanks and gratitude go to all members of
the 5-HT2C project teams of Vernalis and F. Hoffmann-
La Roche, respectively.
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