Indanyl piperazines as melatonergic MT2 selective agents

Article abstract of DOI:10.1016/S0960-894X(03)00090-8

Optimization of a benzyl piperazine pharmacophore produced N-acyl-4-indanyl-piperazines that bind with high affinity to melatonergic MT₂ receptors. (R)-4-(2,3-dihydro-6-methoxy-1H-inden-1-yl)-N-ethyl-1-piperazine-carboxamide fumarate (13) is a water soluble, selective MT₂ agonist, which produces advances in circadian phase in rats at doses of 1-56 mg/kg that are no different from those of melatonin at 1 mg/kg. Unlike melatonin, 13 produced only weak contractile effects in rat tail artery.

Full text of DOI:10.1016/S0960-894X(03)00090-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1199–1202
Indanyl Piperazines as Melatonergic MT2 Selective Agents
Ronald J. Mattson,a,* John D. Catt,a Daniel Keavy,a Charles P. Sloan,a
James Epperson,a Qi Gao,a Donald B. Hodges,a Lawrence Iben,a
Cathy D. Mahle,b Elaine Ryana and Frank D. Yoccaa
aBristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492-7660, USA
bBayer Pharmaceutical Research Center, West Haven, CT, USA
Received 11 September 2002; accepted 18 November 2002
Abstract—Optimization of a benzyl piperazine pharmacophore produced N-acyl-4-indanyl-piperazines that bind with high affinity
to melatonergic MT2 receptors. (R)-4-(2,3-dihydro-6-methoxy-1H-inden-1-yl)-N-ethyl-1-piperazine-carboxamide fumarate (13) is a
water soluble, selective MT2 agonist, which produces advances in circadian phase in rats at doses of 1–56 mg/kg that are no dif-
ferent from those of melatonin at 1 mg/kg. Unlike melatonin, 13 produced only weak contractile effects in rat tail artery.
# 2003 Elsevier Science Ltd. All rights reserved.
The secretion of melatonin in the pineal gland plays a
major role in the regulation of circadian and seasonal
behavior in mammals.1 The MT1 (formerly Mel1A) and
the MT2 (formerly Mel1B) melatonin receptors were
cloned2 from human hypothalamus in 1994–1995. While
the MT1 melatonin receptor was previously thought to
mediate the entrainment of circadian rhythms, melato-
nin shows phase shiftingactivity in knock-out mice
gives N-acyl-4-indanylpiperazines8 that bind with high
affinity to MT2 melatonergic receptors. Two other MT2
selective agonists have been described in the literature:
N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-
yl)ethyl amine,9 and N-[2-(7-methoxy-1-naphthyl)-
ethyl]cyclobutylcarboxamide.10
lackingthe MT
receptor,3 and the MT2 selective
1
antagonist, 4-P-PDOT,4 blocks the phase shiftingactiv-
ity of melatonin.5 This evidence supports the involve-
ment of MT2 melatonin receptors in the entrainment of
circadian rhythms.
In previous work, the 4-benzyl piperazine moiety was
identified as a new 5-HT1A pharmacophore.6 Further
optimization of this pharmacophore identified the
2-fluoro-5-methoxy substituent pattern as potentiating
bindingand igvinga compound with sub-nanomolar
affinity for the 5-HT1A receptor.7 Since melatonin is
structurally related to serotonin, we chose to investigate
whether this serotonergic pharmacophore could be
modified into a melatonergic pharmacophore by simple
acylation. We now report that while the N-acyl-4-ben-
zylpiperazines possess modest melatonergic affinity,
addingan element of structural rigidity by ringfusion
4-(2-Fluoro-5-methoxybenzyl)piperazines, 1-3, were
prepared by the method shown in Scheme 1. 4-Fluoro-
anisole was lithiated11 and reacted with DMF to give
the benzaldehyde. Reductive amination with piper-
azine12 and subsequent acylation gave the benzyl
piperazines 1–3. Compounds 1–3 were weakly active at
MT2 receptors, and we sought to improve their affinities
by conformational constraint. While (2,3-dihydro-6-
methoxy-1H-inden-1-yl) piperazine was previously pre-
pared by alkylation of piperazine with 1-chloro-6-
methoxyindane,13 this method could not be reproduced
in our hands and gave only dehydrochlorination. A tita-
nium(IV) isopropoxide reductive amination procedure14
*Correspondingauthor. Tel.: +1-203-677-6355; fax: +1-203-677-
7702; e-mail: mattsonr@bms.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00090-8

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R. J. Mattson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1199–1202
Table 1. Melatonin MT1 and MT2 receptor binding22 of Melatonin
(Mel) and Compounds 1–20
Compd
R
n
Chirality MT1 IC50 (nM) MT2 IC50 (nM)
Mel
1
2
3
4
5
6
7
8
—
Me
cPr
iPr
Me
Et
nPr
nBu
cPr
iPr
NH-Et
cPr
iPr
NH-Et
cPr
iPr
—
—
—
—
1
1
1
1
1
1
1
1
1
1
1
1
1
0.6
>1000
0.3
>1000
>1000
>1000
>1000
>1000
>1000
>1000
156
270
224
44
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
R
R
R
S
32
2.3
103
3.0
1.5
3.1
1.6
1.5
1.7
9
160
>1000
10
11
12
13
14
15
16
17
18
19
20
44.5
116
200
Figure 1. Agonist effects of melatonin and 13 on forskolin-induced
cAMP production in NIH3T3 cells expressingthe nhuman MT2 receptor.
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
S
S
>1000
>1000
75
94
90
>1000
NH-Et
cPr
NH-Et
cPr
NH-Et
2
2
—
—
ꢁ
ꢁ
azines (4–10), had good to excellent affinity for the MT2
receptor with little affinity for the MT1 receptor. MT2
bindingwas optimal with butyramide 6, cyclopropane
carboxamide 8, isobutyramide 9, and ethyl urea 10. MT2
affinity was attenuated with smaller amides 4 and 5, or
with larger amides, e.g., 7. MT2 and MT1 affinities were
found only in the R-enantiomers (11–13), while the
S-enantiomers (14–16), are inactive at both receptors.
The larger tetralin homologues (17–18), showed a
reduced level of MT2 affinity, while in the naphthyl ana-
logues (19–20), MT2 affinity was even further reduced.
Scheme 1. (a) Piperazine, NaBH3CN; (b) acylation; (c) NaBH4; (d)
SOCl2; (e) piperazine; (f) piperazine, Ti(OiPr)4, NaBH4, EtOH; (g)
resolution; (h) acylation with either an acid chloride, anhydride, or
isocyanate.
was modified15 as shown in Scheme 1 to give the desired
6-methoxyindanyl piperazine (80% yield), which was
resolved16 usingcamphorsulfonic acid and absolute ste-
reochemistry was determined by X-ray crystallography
of (ꢀ)-(10R)-(2,3-dihydro-6-methoxy-1H-inden-1-yl)pi-
perazine, (1S)-10-camphor-sulfonic acid salt.17 The tet-
ralin homologue was prepared in a similar manner from
7-methoxy-1-tetralone, and 7-methoxy-naphthyl-piper-
azine was prepared by literature methods.18 The sub-
stituted piperazines were acylated usingacid chlorides
or isocyanates to give the desired amide and urea pro-
ducts (1–20).19
A structurally similar series of amides, for example, 21,
has been described in the literature20 which have the
amide side chain in the same orientation as the indanyl
piperazines (11–13). Unlike the piperazine amides, 11–13,
which are MT2 selective, compound 21 binds with high
affinity to both MT1 and MT2 receptors.21
These bindingstudies identified 13 as a potent and
selective ligand for the MT2 receptor. Both MT1 and
MT2 are G-protein-coupled receptors and are negatively
linked to adenylyl cyclase by pertussis toxin sensitive
G-proteins.23 Compound 13 was further tested for
functional activity in a MT2 adenylyl cyclase assay24
(Fig. 1) and found to be a full agonist (EC50: 2.4ꢁ1.6
nM; intrinsic activity: 0.98ꢁ0.12).
Compounds 1–20 were evaluated for MT1 and MT2
bindingusingpublished assay methods ( Table 1).2 The
initial group of benzyl piperazine amides failed to
demonstrate potent affinity for melatonergic receptors.
Acetamide, 1, lacked affinity for either melatonergic
receptor, while the cyclopropylcarboxamide (2), and
isobutyramide, (3), had only modest affinity for the
MT2 receptor and no affinity for the MT1 receptor. In
contrast, the conformationally restricted indanyl piper-
In vascular tissue, melatonin potentiates the contractile
responses to serotonin.25 It has been proposed that MT2
receptors mediate relaxation and that the contractile
effects of melatonin may be mediated via MT1 recep-
tors.26 Supportingthis hypothesis, the MT agonist, 13,
2

R. J. Mattson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1199–1202
1201
Barrett, P.; Drew, J. E.; Delagrange, P.; Lesieur, D.; Morgan,
P. J. Eur. J. Pharm 2000, 390, 15.
11. Katsoulos, G.; Takagishi, S.; Schlosser, M. Synlett 1991,
10, 731.
12. A solution of 2-fluoro-5-methoxybenzaldehyde (5.0 g, 33
mmol), piperazine (26 g, 0.3 mol), and NaBH3CN (3.1 g, 50
mmol) in ethanol (400 mL) was heated to reflux for 18 h. The
reaction was concentrated in vacuo. The residue dissolved in
water and extracted with CH2Cl2. The extracts were con-
centrated and the residue was dissolved in 1 N HCl. The acidic
solution was washed with CH2Cl2 and then made basic with
NaOH. The product was extracted from the basic aqueous
solution with CH2Cl2. Concentratingthe extracts agve the
product as a light yellow oil (3.63 g, 50%).
13. (a) Manoury, P.; Binet, J.; Obitz, D.; Defosse, G.;
Dewitte, E.; Veronique, C. US Patent 4,963,680, 1990. Chem.
Abstr. 1990, 113, 59219. (b) El Ahmad, Y.; Laurent, E.; Mail-
let, P.; Talab, A.; Teste, J. F.; Dokan, R.; Tran, G.; Ollivier,
R. J. Med. Chem. 1997, 40, 952.
Figure 2. Acute effects of vehicle (5% DMSO+60% PEG-400+35%
saline), no injection, melatonin (Mel, 1 mg/kg), and 13 (1, 10, 20, and
56 mg/kg) on circadian phase advance.
produced only weak contractile effects in rat tail artery
(0.24 relative to melatonin).27
14. Mattson, R. J.; Pham, K. M.; Leuck, D. J.; Cowen, K. A.
J. Org. Chem. 1990, 55, 2552.
The effects of the selective MT2 agonist, 13, on circadian
phase advance28,29 were investigated (Fig. 2). Both
vehicle injection and no injection gave no significant
phase advance, while melatonin (1 mg/kg) gave a sig-
nificant phase advance of 28 min. Compound 13 at
doses from 1 to 56 mg/kg produced phase advances that
were not significantly different from that produced by
melatonin. This data further supports the involvement
of the MT2 receptor in the entrainment of circadian
rhythms.
15. (2,3-Dihydro-6-methoxy-1H-inden-1-yl) piperazine. An
intimate mixture of 6-methoxy-1-indanone (10 g, 62 mmol),
piperazine (53 g, 0.62 mol) and titanium(IV) isopropoxide
(17.6 g, 12.4 mmol) was heated on a steam bath for 10 min.
The IR spectrum of the mixture showed no carbonyl absorp-
tion. The material was dissolved in ethanol and sodium bor-
ohydride (2.5 g, 62 mmol) added. After stirring for 1 h, the
solution was heated to reflux and when a solution was
achieved, 15% sodium hydroxide (50 mL) was added. The
mixture was filtered and the filtrate was concentrated in vacuo.
The residue was dissolved in ether and washed with water and
1N HCl. The acid washes were made basic and the mixture
was extracted with CH2Cl2 to give the product [80%, mp: 150–
152 ꢂC (HCl salt)].
The present studies have identified the water-soluble30
compound, 13, as a potent MT2 agonist that produces
advances in circadian phase similar to those produced
by melatonin. Unlike melatonin, 13 produces only weak
contractile effects in vitro in rat tail artery. These studies
provide further support for the hypothesis that MT2
receptors mediate the entrainment of circadian rhythms
and not vasoconstriction.
16. Resolution of (2,3-dihydro-6-methoxy-1H-inden-1-yl)-
piperazine. (ꢀ)-(10R)-(2,3-dihydro-6-methoxy-1H-inden-1-
yl)piperazine (1S)-10-camphorsulfonic acid salt was recrys-
tallized from ethanol-water to a constant meltingpoint (mp:
234–235 ꢂC; [a]2D5 ꢀ36.3ꢂ,
c 2.51, MeOH). calcd for
ꢃ
C14H20N2O2 C10H10O4S: C, 62.04; H, 7.81; N, 6.03. Found:
C, 62.01; H, 7.96; N, 5.97.
A sample of the above salt was converted to the fumarate
salt and recrystallized from ethanol-water (mp: 170–172 ꢂC;
ꢂ
[a]2D5 ꢀ72.3 , c 2.66, MeOH). calcd for C14H20N2O2 C4H4O4:
ꢃ
References and Notes
C, 62.05; H, 6.94; N, 8.04. Found: C, 62.08; H, 6.61; N, 8.26.
(10S)-(2,3-dihydro-6-methoxy-1H-inden-1-yl)-piperazine (1R)-
(ꢀ)-10-camphor-sulfonic acid salt salt was recrystallized from
ethanol–water to a constant meltingpoint (mp: 234–235 ꢂC;
1. Morgan, P. J.; Barret, P.; Howell, H. E.; Helliwel, R. Neu-
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5. Dubocovich, M. L.; Yun, K.; Al-Ghoul, W. M.; Benlowcif,
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[a]2D5 +35.8ꢂ,
c 2.51, MeOH). calcd for C14H20N2O2
ꢃ C10H10O4S: C, 62.04; H, 7.81; N, 6.03. Found: C, 62.06; H,
7.73; N, 6.01.
A sample of the above salt was converted to the fumarate
salt and recrystallized from ethanol-water (mp: 165–166 ꢂC;
ꢂ
[a]2D5 +71.3 , c 2.66, MeOH). calcd for C14H20N2O2 C4H4O4:
ꢃ
C, 62.05; H, 6.94; N, 8.04. Found: C, 62.08; H, 6.61; N, 8.26.
17. Crystallographic data (excluding structure factors) has
been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication number CCDC 197571.
Copies of the data can be obtained, free of charge, on appli-
cation to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
[fax: +44 (0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].
18. Poindexter, G. S.; Bruce, M. A.; LeBoulluec, K. L.;
Monkovic, I. Tetrahedron Lett. 1994, 35, 7331.
19. N-Ethyl-4-(2,3-dihydro-6-methoxy-1H-inden-1-yl)-1-piper-
azinecarboxamide, 10. Ethyl isocyanate (0.27 mL, 3.4 mmol)
was added to a solution of (6-methoxy-1-indanyl)-1-piperazine
(0.8 g, 3.4 mmol) in CH2Cl2. After stirringfor 2 h, the solution
was washed with 1N HCl. The acid washes were made basic
6. Mattson, R. J.; Catt, J. D. US Patent 5,387,593, 1995;
Chem. Abstr. 1995, 123, 83213.
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R. B.; Gentile, A.; Mahle, C. D.; Matos, F. F.; McGovern, R.;
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10. Conway, S.; Canning, S. J.; Howell, H. E.; Mowat, E. S.;

1202
R. J. Mattson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1199–1202
with sodium hydroxide solution and the mixture was extracted
with CH2Cl2. The extracts were dried and concentrated. The
fumarate salt was prepared in methanol. (0.88 g, 85%, mp:
167–168 C). calcd for C4H4O4 0.1H2O: C, 59.87; H, 6.99; N,
9.97. Found: C, 59.65; H, 6.91; N, 9.81.
20. (a) Fukatsu, K.; Uchikawa, O.; Kawada, M.; Yamano, T.;
Yamashita, M.; Kato, K.; Hirai, K.; Hinuma, S.; Miyamoto,
M.; Ohkawa, S. J. Med. Chem. 2002, 45, 4212. (b) Takaki,
K.S.; Watson, B.T.; Poindexter, G.S.; Epperson, J.R. U.S.
Patent 5,661,186, 1997; Chem. Abstr. 1997, 127, 599243.
21. (R)-21, MT1 IC50=100 nM, MT2 IC50=16 nM; (S)-21,
MT1 IC50=0.3 nM, MT2 IC50=0.2 nM.
O2/ 5% CO2, pH 7.4) at 37 ꢂC. The rings were equilibrated for
1 h under restingtension of 0.7 g. The tension was measured
with a Grass FT.03 force development transducer and recor-
ded on a Sensormedic Dynograph R612 recorder. The rings
were precontracted with 2 mM PGF2A for 5 min, then melato-
nin or 13 (100 mM) was added after the contractile response
had stabilized. Data were expressed as grams of agonist
induced contraction (N=6): melatonin, 0.76 gꢁ0.14 g; 13,
0.18ꢁ0.13 g, 0.24 relative to melatonin.
ꢂ
ꢃ
28. Warren, W. S.; Hodges, D. B.; Cassone, V. M. J. Biol.
Rhythms 1993, 8, 233.
29. Circadian Running Wheel Method. Male hooded Long-
Evans rats were kept under a 12:12 h light-dark cycle sche-
dule until a reliable entrainment was established between the
lighting conditions and the onset of running-wheel activity
(ꢄtwo weeks). After entrainment, the rats were maintained
in constant darkness until the end of the experiment. Under
constant darkness, rats show an endogenous rhythm of run-
ning-wheel activity characterized by a predictable daily onset
of activity. This endogenously controlled rhythm under con-
stant environmental conditions is called a ‘free-running’
rhythm. After 2 wk in constant darkness, each rat was injected
approximately 2 h before the expected onset of running-wheel
activity for that individual animal. Seven groups of rats (9–11
rats each) were used: Group 1: no injection; Group 2: vehicle
(5% DMSO, 60% PEG 400, 35% saline); Group 3: melatonin
(1.0 mg/kg); Groups 4–7: 13 (1, 10, 20, & 56 mg/kg). All
injections were subcutaneous at CT 10. The onset of activity
was predicted usingthe wheel-runningactivity record (acto-
gram) and the rhythm period. Data Measurement and Ana-
lyses. Phase shift was calculated for each animal from the
difference between the expected onset of running-wheel activ-
ity and the real value obtained from the actogram for post
treatment recording. The data was analyzed for significance
usingANOVA followed by Bonferroni/Dunn post hoc analy-
sis when appropriate.
22. IC50 values are the mean of at least 3 determinations run
at five different concentrations with the radioligand at the Kd
concentration. Standard errors were typically ꢁ20% of the
mean value.
23. Li, P.-K.; Witt-Enderby, P. A. Drugs Future 2000, 25, 945.
24. MT2 Adenylyl Cyclase Functional Assay. Cyclic AMP
assays were performed usingNIH-3T3 cells stably expressing
human MT2 receptors. Cells were incubated in culture media
containing1 mM IBMX and 10 mM forskolin for 10 min at
37 ꢂC in the presence of increasingconcentrations of melato-
nin or 13. Assays were terminated by the addition of 0.1 N
HCl. Followingcentrifuagtion, supernatants were collected
and stored at ꢀ20 ꢂC until assayed. Cyclic AMP levels were
measured by radioimmunoassay (Amersham). Radioactivity
was quantitated by gamma emission spectrometry. Data were
analyzed by a 4-parameter logistic, non-linear least squares
regression to yield EC50 and Emax values.
25. Yang, Q.; Schalbert, E.; Delagrange, P.; Vanhoutte, P. M.;
O’Rourke, S. T. Am. J. Physiol. Heart. Circ. Physiol. 2001,
280, H76.
26. Doolen, S.; Krause, D. N.; Dubocovich, M. L.; Duckles,
S. P. Eur. J. Pharmacol. 1998, 345, 67.
27. Rat caudal artery. Rings of rat caudal artery (4 mm long)
were maintained in 30 mL organ baths containing PSS (conc.
in mM: NaCl, 115; KCl, 4.7; NaHCO3, 22; CaCl2, 1.6;
KH2PO4, 1.2; MgCl2, 1.2; and glucose, 11; aerated with 95%
30. The fumarate salt of 13 (10 mg) freely dissolved in water
(1 mL).