C-(4,5,6-trimethoxyindan-1-yl)methanamine: A mescaline analogue designed using a homology model of the 5-HT2A receptor

Article abstract of DOI:10.1021/jm060272y

A conformationally restricted analogue of mescaline, C-(4,5,6- trimethoxyindan-1-yl)-methanamine, was designed using a 5-HT_2A receptor homology model. The compound possessed 3-fold higher affinity and potency than and efficacy equal to that of

Full text of DOI:10.1021/jm060272y

J. Med. Chem. 2006, 49, 4269-4274
4269
C-(4,5,6-Trimethoxyindan-1-yl)methanamine: A Mescaline Analogue Designed Using a
Homology Model of the 5-HT2A Receptor
Thomas H. McLean, James J. Chambers, Jason C. Parrish, Michael R. Braden, Danuta Marona-Lewicka,
Deborah Kurrasch-Orbaugh, and David E. Nichols*
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue UniVersity,
West Lafayette, Indiana 47907-1333
ReceiVed March 10, 2006
A conformationally restricted analogue of mescaline, C-(4,5,6-trimethoxyindan-1-yl)-methanamine, was
designed using a 5-HT2A receptor homology model. The compound possessed 3-fold higher affinity and
potency than and efficacy equal to that of mescaline at the 5-HT2A receptor. The new analogue substituted
fully for LSD in drug discrimination studies and was 5-fold more potent than mescaline. Resolution of this
analogue into its enantiomers corroborated the docking experiments, showing the R-(+) isomer to have
higher affinity and potency and to have efficacy similar to that of mescaline at the 5-HT2A receptor.
Introduction
analogue 2, which maintains the embedded mescaline structure
in a low-energy conformation. Subsequent synthesis and testing
of (()-2 revealed that it possessed mescaline-like properties in
vitro and in vivo and had greater potency than 1 in several
assays.
The mechanism of action of mescaline (1), the hallucinogenic
component of psychoactive cactus Lophophora williamsii and
of most other drugs that possess hallucinogenic properties, is
believed to involve the activation of brain serotonin-2A (5-HT2A)
receptors.1 Recent SAR investigations in our laboratory into the
chemical and topological properties of potent hallucinogenic
phenethylamines have led to the design and synthesis of a
number of methoxylated phenethylamine analogues where the
methoxy groups have been constrained into furan or dihydro-
furan rings.2-6 Although this strategy has proven successful
when applied to 2,5-dimethoxy-substituted compounds, it failed
in the design of mescaline analogues.7 We therefore hypoth-
esized that when mescaline binds to the 5-HT2A receptor, the
distal 3,5-methoxy groups must be conformationally mobile or
adopt out-of-plane orientations.
Recently we developed a homology model of the 5-HT2A
receptor based on an in silico activated form of bovine
rhodopsin.8 A variety of agonist ligands, including mescaline,
LSD, and psilocin, were docked using an unbiased docking rou-
tine to give reasonable bound orientations of the ligands within
the receptor. When mescaline was docked into this model, we
observed that the 3- and 5-methoxy groups adopted out-of-plane
conformations (Figure 1A) allowing hydrogen bonding with
serine residues, yet maintaining the aromatic edge-face interac-
tion with F6.52(340), shown by mutagenesis studies to be essential
for agonist activity.9 Also, the side chain was in an approx-
imately gauche conformation, whereas in the 2,5-dimethoxy
compound series the side chain was in a more nearly anti orien-
tation. Single-point energy calculations performed on mescaline
in the docked conformation (isolated from the protein) indicate
that this conformation is higher in energy than the in vacuo
global conformational energy minimized form of mescaline in
which the side chain is extended into an anti conformation.
Results and Discussion
Computational Modeling. From docking studies of the
enantiomers of 2, we predicted that the R enantiomer would be
more potent than mescaline because of a predominance of highly
similar and plausible binding orientations obtained from unbi-
ased genetic algorithm searches. The docked conformer of (R)-2
(Figure 1B) most resembled that obtained for 1, indicating that
the amine-bearing side chain adopts a gauche orientation upon
binding to the receptor, apparently to optimize hydrogen bonding
of all three aromatic methoxy groups with the hydroxyl groups
of S3.36(159), T3.37(160), and S5.43(239), residues thought to be
potentially important for agonist binding to the 5-HT2A
receptor.6,9-11 In all docked conformations obtained for (R)-2,
the protonated amine of the ligand forms the essential ionic
interaction with the carboxylate of D3.32(155) 12
.
By contrast, unbiased docking of (S)-2 into the homology
model gave numerous orientations, none of which included the
salt bridge between the protonated amine of the ligand and the
carboxylate of D3.32(155). As this interaction is essential for
agonist activity, the docking routine was repeated with a 2-3
Å distance constraint between the amine nitrogen and carboxyl
carbon atoms, almost exclusively returning orientations with the
ligand tilted up to place the amine in the same position in space
as seen in the R isomer, at the expense of the hydrogen bonds
between the 6-methoxy and the hydroxy of S3.36(159) and
between the 4-methoxy and the hydroxy of S5.43(239). To test
this prediction, 2 was resolved into its enantiomers and each
isomer was assayed for 5-HT2A binding affinity and potency at
stimulating phosphoinositide turnover.
Synthesis. Racemic (()-2 was readily synthesized beginning
with indanone 3 (Scheme 1). Ketone 3 was functionalized utiliz-
ing reaction with trimethylsilyl cyanide and directly dehydrated
to indene-1-carbonitrile 4 by treatment with Amberlyst-15 acidic
resin. Subsequent two-step catalytic hydrogenation of 4, first
at low pressure over 5% Pd/C in ethanol to reduce the olefin,
followed by nitrile reduction over activated Raney nickel in
ethanol, yielded the final racemic compound (()-2.
Inspection of the putative bound conformation of 1 im-
mediately suggested the synthesis of conformationally restricted
Fractional crystallization of various diastereomeric salts of
(()-2 failed to resolve the enantiomers, as did attempted
* To whom correspondence should be addressed. Phone: 765-494-1461.
Fax: 765-494-1414. E-mail: drdave@pharmacy.purdue.edu.
10.1021/jm060272y CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/21/2006

4270 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
McLean et al.
Figure 1. (A) Top view of mescaline (1) virtually docked into the 5-HT2A receptor homology model with key residues known to be important for
binding. (B) Similar view of the docked R-enantiomer of 2. The view is from the extracellular face of the receptor, with ASP155 in TM3, PHE243
in TM5, and PHE339 and PHE340 in TM6.
Table 1. Results of Radioligand Competition Binding Studies at Cloned
Scheme 1. Synthesis of (()-2
Receptors
Ki (SEM), nM
compd
(()-2
(R)-(+)-2
(S)-(-)-2
mescaline (1)
psilocinb
LSDb
r5-HT2A (()-[125I]DOI
r5-HT2C (()-[125I]DOI
130(20)
69(6)
1120(180)
360(70)
25(5)
60(10)
nda
nda
380(60)
nda
3.5(0.6)
5.5(0.3)
a nd ) not determined. b Data from Chambers et al.6
Table 2. Results of the IP3 Accumulation Studies at Cloned Rat 5-HT2A
Receptors
Scheme 2. Synthesis of the Enantiomers of 2
EC50 at 5-HT2A
(SEM), nM
max 5-HT
stimulation (SEM), %
compd
(()-2
(R)-(+)-2
(S)-(-)-2
mescaline (1)
psilocina
LSDa
6100(740)
3200(400)
>50000
11300(1600)
2300(290)
9.8(3.7)
83(4)
88(1)
59(4)
86(10)
50(2)
20(3)
a Data from Chambers et al.6
Determination of the absolute configuration was accomplished
by X-ray crystallography of a single crystal of diastereomeric
intermediate (S,S)-(-)-7. This compound was obtained in
diastereopure form by the resolution of acid (()-6 by fractional
crystallization of its quinine salt from ethanol and condensation
of each enantiomer of 6 with (S)-1-phenethylamine. HPLC
analysis showed the diastereomeric excess of 7 to be greater
than 99%. X-ray crystallography of one diastereomer established
the configuration at the indan-1-position to be S, and by
inference, the other diastereomer must have the R configuration.
Reduction of each diastereomer of 7 by borane-dimethyl
sulfide in THF afforded secondary amines (S,R)- and (S,S)-8
without racemization. Once it was discovered that (()-8 could
be resolved by radial chromatography, the tedious fractional
crystallization step was omitted and (()-8 was synthesized in
racemic form and then resolved as shown in Scheme 2.
Pharmacological investigation of conformationally restricted
analogue (()-2 and its enantiomers was conducted, and the
results are compared directly to those obtained for mescaline
(Tables 1 and 2).
enantioselective acylation of the amine using Candida antarctica
B lipase (Novozyme 435).13 After several unsuccessful attempts
to induce chirality by stereoselective reduction of various
prochiral intermediates, we attempted the separation of diaster-
eomeric covalent derivatives of acid (()-6 as shown in Scheme
2.14 Unsaturated nitrile 4 was catalytically hydrogenated at low
pressure over Pd/C in ethanol, followed by alkaline hydrolysis
of the intermediate saturated nitrile to afford acid (()-6. The
mixture of diastereomeric amides (()-7, derived from coupling
of (()-6 with (S)-(+)-1-phenethylamine, was reduced using
borane, and the resulting secondary amines (S,R)- and (S,S)-8
were separated by radial chromatography on a silica rotor.
Catalytic N-debenzylation of the free base of each diastereomer
over Pd(OH)2 on carbon in wet methanol afforded the enanti-
omers of 2 in optically pure form.
The competition radioligand binding experiments confirmed
the model-derived qualitative prediction that 2 would possess
reasonable affinity for the 5-HT2A receptor. When compared to
1, racemic 2 possessed nearly 3-fold higher affinity for the
5-HT2A receptor, was 2 times more potent at stimulating IP3
accumulation, and had intrinsic activity comparable to that of
mescaline in this pathway.

C-(4,5,6-Trimethoxyindan-1-yl)methanamine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4271
redissolved in 40 mL of benzene, 0.5 g of Amberlyst-15 cation
exchange resin was added, and the flask was fitted with a Dean-
Stark trap. The solution was heated at reflux for 3 h until no more
water was collected. After filtration through diatomaceous earth to
remove the resin, the benzene was evaporated, and the resulting
residue was chromatographed on silica (EtOAc/hexanes, 1:1) to
give 4 (0.71 g, 68%) as a yellow oil that solidified on standing:
Pharmacological results also confirmed the prediction that
(R)-2 would be a better ligand at the 5-HT2A receptor than (S)-
2, displaying a 16-fold higher affinity and greater than 16-fold
higher potency than its distomer.
In vivo drug discrimination tests indicate that 2 substitutes
fully for LSD in an animal model of hallucinogenesis.15,16 In
particular, (()-2 had an ED50 (95% confidence interval) of 6.6
(3.8-11.5) µmol/kg, whereas mescaline had an ED50 of 34 (21-
54) µmol/kg. For comparison purposes, the potent hallucinogen
LSD had an ED50 of 0.037 (0.023-0.058) µmol/kg in this assay.
1
mp 95-97 °C; H NMR (300 MHz, CDCl3) δ 7.16 (t, J ) 2 Hz,
1 H, ArCCH), 6.78 (s, 1 H, ArH), 3.91 (s, 3 H, OCH3), 3.84 (s, 3
H, OCH3), 3.79 (s, 3 H, OCH3), 3.50 (d, J ) 2 Hz, 2 H, ArCH2);
low-resolution EIMS m/z (rel intensity) 231 (M+, 100). Anal.
(C13H13NO3) C, H, N.
Conclusion
(()-(2,3-Dihydro-4,5,6-trimethoxy-1H-inden-1-yl)amino-
methane Hydrochloride, (()-2. Unsaturated nitrile (()-4 (1.40
g, 6.05 mmol) dissolved in absolute EtOH (250 mL) was placed in
a 500 mL glass Parr hydrogenation flask containing 250 mg of 5%
Pd/C and shaken for 20 min under 20 psi of H2. The solution was
then filtered through diatomaceous earth to remove the catalyst and
placed back into the hydrogenation flask along with 1 g of fresh
activated Raney nickel 2800. Ammonia gas was bubbled through
the solution for 1 min, and the flask was then shaken under 45 psi
of H2 for 6 h. The catalyst was removed by filtration through
diatomaceous earth, and the solvent was evaporated to yield a clear
oil that was acidified with 1 N methanolic HCl and evaporated to
yield (()-2 hydrochloride (1.58 g, 96%) as a white solid. An
analytical sample was recrystallized from 95% EtOH (4.8 g,
Development of a human 5-HT2A receptor homology model
has provided an additional tool for the design of new chemical
probes for this receptor. Although 2 is a relatively simple
structure, it was designed on the basis of clues provided by the
model, and the side chain orientation was counterintuitive on
the basis of our previous SAR studies. The new ligand was
shown to possess higher affinity and potency than mescaline.
Furthermore, the model correctly predicted that (R)-2 would
be the more potent enantiomer, based on unbiased docking re-
sults and their correlation with key ligand-receptor interactions
previously identified by mutagenesis. Although this computer-
based model appears useful in explaining much of the empirical
data concerning the SAR of 5-HT2A receptor agonists, there is
little doubt that it does not accurately represent the exact
structure of the “activated” binding site. Nevertheless, it can
serve as a work in progress, subject to study and refinement
through an iterative procedure involving molecular modeling,
conformationally restricted analogue synthesis, and mutagenesis
studies. With this approach, the model will be incrementally
improved to expand our knowledge of the agonist binding site.
Until directly measured and detailed structural information for
this receptor becomes available, this method appears promising.
1
87%): mp 245 °C; H NMR (500 MHz, DMSO-d6) δ 1.82 (m, 1
H, ArCH2CH2, J ) 3.3 Hz), 2.34 (m, 1 H, ArCH2CH2, J ) 3.2
Hz), 2.85 (m, 1 H, ArCH2, J ) 3.1 Hz), 2.97 (dt, 2 H, CH2N, J )
5.0, 3.0 Hz), 3.20 (m, 1 H, ArCH2), 3.36 (m, 1 H, ArCH, J ) 6.3
Hz), 3.72 (s, 3 H, ArOCH3), 3.80 (s, 3 H, ArOCH3), 3.84 (s, 3 H,
ArOCH3), 6.65 (s, 1 H, ArH); MS (CI) m/z 238 (M + H), 221 (M
+ H - NH3). Anal. (C13H20ClNO2) C, H, N.
4,5,6-Trimethoxyindan-1-carboxylic Acid, (()-6. Indenecar-
bonitrile (()-4 (14.0 g, 60.5 mmol) was dissolved in absolute EtOH
(300 mL) and added to a glass hydrogenation bottle containing 1
g of 10% Pd/C. The mixture was shaken under 20 psi of H2 for 1
h at room temperature to reduce the olefin selectively. The catalyst
was removed by filtration through diatomaceous earth, and the
EtOH was removed by rotary evaporation to afford the intermediate
indan-1-carbonitrile as an oil. This residue was redissolved in 100
mL of MeOH in a flask with magnetic stirring, and 100 mL of 3
N aqueous KOH was added. The mixture was held at reflux under
argon for 12 h. The basic solution was washed once with 50 mL
of Et2O, then acidified carefully to pH 1 with concentrated HCl
and then extracted with 3 × 100 mL of Et2O. The ether extracts
were washed with brine, then dried over anhydrous Na2SO4 and
evaporated to give the acid as an oil that solidified upon standing
(14.3 g, 94%): mp 80-83 °C; 1H NMR (300 MHz, CDCl3) δ 6.73
(s, 1 H, ArH), 4.00 (dd, J ) 6 , 3 Hz, 1 H, ArCH), 3.87 (s, 3 H,
OCH3), 3.82 (s, 6 H, 2 × OCH3), 3.05 (m, 1 H, ArCH2), 2.87 (m,
1 H, ArCH2), 2.46-2.28 (m, 2 H, ArCH2CH2); low-resolution
ESIMS m/z (rel intensity) 253 (M + H, 100). Anal. (C13H16O5) C,
H.
Experimental Section
General. All reagents were commercially available and were
used without further purification unless otherwise indicated. In-
danone 3 was obtained in excellent yield by intramolecular
cyclization of 3-(2,3,4-trimethoxyphenyl)propionic acid by a modi-
fication of the method of Koo.17 Dry THF and diethyl ether were
obtained by distillation from benzophenone-sodium under nitrogen
immediately before use. Melting points were determined using a
1
Thomas-Hoover apparatus and are uncorrected. H NMR spectra
were recorded using either a 500 MHz Bruker DRX-500 or a 300
MHz Bruker ARX-300 NMR spectrometer. Chemical shifts are
reported in δ values (ppm) relative to an internal reference (0.03%
v/v) of tetramethylsilane (TMS) in CDCl3, except where noted.
Abbreviations used to report NMR peaks are as follows: bs ) broad
singlet, d ) doublet, dd ) doublet of doublets, dq ) doublet of
quartets, dt ) doublet of triplets, m ) multiplet, q ) quartet, s )
singlet, t ) triplet, td ) triplet of doublets. Chemical ionization
mass spectra (CIMS), using isobutane as the carrier gas, were
obtained with a Finnigan 4000 spectrometer. Elemental analyses
were performed by the Purdue University Microanalysis Laboratory,
and results are within (0.4% of the calculated values. Thin-layer
chromatography was performed using J. T. Baker flex silica gel
IB2-F, plastic-backed sheets with fluorescent indicator, visualizing
with UV light at 254 nm and eluting with 4:1 hexanes/ethyl acetate
unless otherwise noted. Column chromatography was carried out
using silica gel 60, 230-400 mesh (J. T. Baker). All reactions were
carried out under an inert atmosphere of argon unless otherwise
indicated.
Resolution of the Enantiomers of 4,5,6-Trimethoxyindan-1-
carboxylic Acid, (()-6. The racemic acid (11.6 g, 46.0 mmol) was
dissolved in 100 mL of THF and added to a solution of quinine
(14.9 g, 46.0 mmol) in 100 mL of THF. The precipitate that formed
was collected by suction filtration and dried to give the 1:1 mixture
of diastereomeric salts: [R]20D -113.1° (c 1.00, MeOH). The solid
was recrystallized five times from absolute EtOH to constant
specific rotation: [R]20 -108.9° (c 1.00, MeOH). The mother
liquors from all the EtOH recrystallizations were combined and
evaporated to near dryness. Addition of 200 mL of Et2O led to
formation of a precipitate, which was collected by suction filtration
and dried. The optical rotation for this fraction of the salt derived
4,5,6-Trimethoxy-3H-indene-1-carbonitrile, 4. To a solution
of 4,5,6-trimethoxyindanone18 3 (1.0 g, 4.5 mmol) in CH2Cl2 (60
mL) under argon were added ZnI2 (40 mg) and trimethylsilyl
cyanide (0.66 mL, 4.95 mmol). The mixture was heated at reflux
for 5 h, and then the solvent was evaporated. The resulting oil was
from the mother liquor was measured: [R]20 -116.8° (c 1.00,
D
MeOH). The salt obtained from the EtOH recrystallization and the
precipitate derived from the mother liquor fraction had melting
points of 256-260 °C, with decomposition. The free carboxylic
acid from each fraction was obtained by acidifying an aqueous

4272 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
McLean et al.
1
solution of the corresponding quinine salt to pH 1 with concentrated
HCl, extracting with CH2Cl2, and removing solvent. The rotation
of the acid derived from recrystallized salt (4.7 g) was [R]20D +4.97°
(c 1.00, benzene), and that of the acid derived from the mother
isomer. HPLC tR ) 9.85 min; mp 114-115 °C; H NMR (300
MHz, CDCl3) δ 7.27-7.17 (m, 5 H, ArH), 6.42 (s, 1 H, ArH),
5.75 (d, J ) 8 Hz, 1 H, NH), 5.12 (m, J ) 7 Hz, 1H, PhCHNH),
3.81 (s, 3 H, OCH3), 3.76 (s, 3 H, OCH3), 3.66 (s, 3 H, OCH3),
3.80-3.73 (t, 1 H, ArCH), 2.99-2.85 (m, 1 H, ArCH2), 2.82-
2.75 (m, 1 H, ArCH2), 2.38-2.18 (m, 2 H, ArCHCH2), 1.41 (d, 3
H, J ) 7 Hz, CH3); low-resolution ESIMS m/z (rel intensity) 356
(M + H, 100); [R]20D -8.96° (c 1.00, CHCl3). Anal. (C21H25NO4)
C, H, N.
liquor (6.8 g) was [R]20 -3.18° (c 1.00, benzene). The enantio-
D
meric purity of the acids was determined in the following step by
HPLC analysis of the corresponding R-methylbenzylamides 7.
(R)-(-)-4,5,6-Trimethoxy-N-((S)-1-phenethyl)-2,3-dihydroin-
dene-1-carboxamide, (R,S)-7. Carboxylic acid (R)-(+)-6 (2.0 g,
7.9 mmol) was dissolved in EtOAc (10 mL) with a drop of DMF
and stirred under argon. Oxalyl chloride (1.4 g, 11.1 mmol) was
added dropwise, and the solution was stirred vigorously for 15 min.
Approximately half of the solvent volume was removed by rotary
evaporation to azeotrope excess oxalyl chloride. Another 10 mL
of EtOAc was added to dissolve the intermediate acid chloride.
(S)-R-Methylbenzylamine (1.35 g, 11.1 mmol) was dissolved in
another 10 mL of EtOAc and added to the flask containing the
acid chloride, followed immediately by 20 mL of 2 N aqueous
NaOH. The flask was shaken vigorously on a Vortex mixer for 1
min, then poured into a separatory funnel. The solvent layers were
allowed to separate. The organic fraction was washed with 1 N
aqueous HCl to remove excess amine and then dried (Na2SO4),
filtered, and evaporated to give the crude amide. Column chroma-
tography (silica; hexanes/EtOAc, 3:2) provided the amide (R,S)-7
(2.0 g, 71%) as a white crystalline solid. A 1 mM solution in
acetonitrile was analyzed for diastereomeric purity by HPLC using
an analytical reversed-phase C8 silica column (3.0 mm × 150 mm)
with a 25 µL injection loop, UV (254 nm), and ESIMS detection.
Gradient elution over 25 min (35:65 to 50:50 acetonitrile/H2O
containing 0.1% trifluoroacetic acid) with a flow rate of 1 mL/min
provided adequate resolution to determine any contamination by
the other diastereomer. By comparison with a sample of the 1:1
mixture of diastereomeric amides, the absence of any shoulder peaks
indicated that the sample was >99% diastereomerically pure:
HPLC tR ) 9.73 min; mp 116-117 °C; 1H NMR (300 MHz,
CDCl3) δ 7.28-7.16 (m, 5 H, ArH), 6.45 (s, 1 H, ArH), 5.70 (d,
J ) 8 Hz, 1 H, NH), 5.12 (m, J ) 7 Hz, 1H, PhCHNH), 3.85 (s,
3 H, OCH3), 3.78 (s, 3 H, OCH3), 3.67 (s, 3 H, OCH3), 3.82-3.76
(t, 1 H, ArCH), 3.00-2.90 (m, 1 H, ArCH2), 2.83-2.75 (m, 1 H,
ArCH2), 2.45-2.34 (m, 1 H, ArCHCH2), 2.25-2.15 (m, 1 H,
ArCHCH2), 1.39 (d, 3 H, J ) 7 Hz, CH3); low-resolution ESIMS
m/z (rel intensity) 355 (M+, 100); [R]20D -35.73° (c 1.00, CHCl3).
Anal. (C21H25NO4) C, H, N.
(S)-(-)-4,5,6-Trimethoxy-N-((S)-1-phenethyl)-2,3-dihydroin-
dene-1-carboxamide, (S,S)-7. Carboxylic acid (S)-(-)-6 (2.44 g,
9.52 mmol) was dissolved with stirring under argon in EtOAc (15
mL) with a drop of DMF. Oxalyl chloride (1.69 g, 13.3 mmol)
was added dropwise, and the solution was stirred vigorously for
15 min. Approximately half of the solvent volume was removed
by rotary evaporation to azeotrope excess oxalyl chloride. Another
15 mL of EtOAc was added to dissolve the intermediate acid
chloride. (S)-R-Methylbenzylamine (1.62 g, 13.3 mmol) was
dissolved in another 15 mL of EtOAc and added to the flask
containing the acid chloride, followed immediately by 40 mL of 2
N aqueous NaOH. The flask was shaken vigorously on a Vortex
mixer for 1 min and then poured into a separatory funnel. The
solvent layers were allowed to separate. The organic fraction was
washed with 1 N aqueous HCl to remove excess amine and was
then dried (Na2SO4) and evaporated to afford the crude amide.
Column chromatography (silica; hexanes/EtOAc, 3:2) provided the
amide (S,S)-7 (2.84 g, 84%) as a white crystalline solid. Diaster-
eomeric purity determination using the same RP-HPLC method
described for the R,S amide above showed an ∼80:20 mixture of
amide diastereomers. A single recrystallization from absolute EtOH
was sufficient to bring the diastereomeric purity to >99% by HPLC
analysis, yielding 2.27 g (67%) of pure (S,S)-7.
(S)-(-)-N-(((R)-4,5,6-Trimethoxyindan-1-yl)methyl)-1-phen-
ethylamine, (S,R)-8. Amide (R,S)-7 (0.5 g, 1.4 mmol) was dissolved
in dry THF with magnetic stirring under argon at room temperature.
A 10 M BH3‚SMe2 complex solution (1 mL, 10 mmol) was added
in one portion, and the mixture was stirred for 2 h. Upon complete
consumption of starting material, indicated by TLC, 15 mL of 1 N
methanolic HCl was carefully added to quench excess borane
reagent. Concentrated HCl (3 mL) was added, and the solution was
brought to reflux for 20 min to hydrolyze the intermediate boramine
complex. The solvent was evaporated to near dryness, 10 mL of
MeOH was added, and the solvent was again evaporated to remove
residual trimethylborate by azeotropic distillation. The residue was
redissolved in 1 N aqueous HCl and extracted with Et2O to remove
nonpolar contaminants, and the solution was basified to pH 11 with
6 N aqueous NaOH. The free amine was extracted into 3 × 20 mL
of CH2Cl2. The combined extract was dried over anhydrous Na2-
SO4, filtered, and then evaporated to give (R,S)-8 as an oil (470
mg, 98%). An analytical sample was prepared by radial chroma-
tography on a 4 mm silica/gypsum rotor, eluting with EtOAc/
hexanes, 20:30. The hydrochloride proved too hygroscopic for
elemental analysis. The subsequent N-debenzylation step proceeded
much more smoothly with the free amine, but care had to be taken
to exclude air for the long-term storage of the free base: mp 220-
221 °C (hydrochloride); 1H NMR (hydrochloride, 300 MHz, CD3-
OD) δ 7.44-7.32 (m, 5 H, ArH), 6.51 (s, 1 H, ArH), 4.39 (q, 1 H,
CHNH, J ) 7 Hz), 3.72 (s, 3 H, OCH3), 3.69 (s, 3 H, OCH3), 3.65
(s, 3 H, OCH3), 3.41 (m, 1 H, ArCHCH2), 3.26-3.23 (m, 2 H,
CH2NH), 2.73 (m, 2 H, ArCH2), 2.57 (dd, 6 Hz, 1 H, ArCHCH2,
J ) 2 Hz), 2.28 (m, 1 H, ArCHCH2), 1.66 (d, 3 H, CH3, J ) 7
Hz); low-resolution ESIMS m/z (rel intensity) 342 (M + H, 100);
[R]20 -58.09° (c 1.00, MeOH, HCl salt). Anal. (C21H27NO3) C,
D
H, N.
(S)-(-)-N-(((S)-4,5,6-Trimethoxyindan-1-yl)methyl)-1-phen-
ethylamine, (S,S)-8. The same procedure was followed as for the
reduction of (S,S)-7 (0.5 g, 1.4 mmol). (S,S)-8 was obtained as an
oil (475 mg, 99%). An analytical sample was prepared by radial
chromatography on a 4 mm silica/gypsum rotor, eluting with
EtOAc/hexanes, 20:30: mp 225-227 °C (hydrochloride); 1H NMR
as hydrochloride (300 MHz, CD3OD) δ 7.52-7.38 (m, 5 H, ArH),
6.46 (s, 1 H, ArH), 4.41 (q, 1 H, CHNH, J ) 7 Hz), 3.74 (s, 3 H,
OCH3), 3.69 (s, 3 H, OCH3), 3.67 (s, 3 H, OCH3), 3.32 (m, 1 H,
ArCHCH2), 3.14-3.09 (dd, 4 Hz, 1 H, CH2NH, J ) 9 Hz), 2.97-
2.68 (m, 3 H, CH2NH, ArCH2), 2.26 (m, 1 H, ArCHCH2), 1.86
(m, 1 H, ArCHCH2), 1.66 (d, 3 H, CH3, J ) 7 Hz); low-resolution
ESIMS m/z (rel intensity) 342 (M + H, 100); [R]20 -15.77° (c
1.00, MeOH, as HCl salt). Anal. (C21H27NO3) C, H, N.
D
Alternative Procedure for Obtaining (S,R)-8 and (S,S)-8 by
Chromatographic Separation. The diastereomeric mixture of
amides (()-7, obtained by condensation of the racemic carboxylic
acid (()-6 with (S)-(+)-1-phenethylamine, as described for the
enantiomers above, was dissolved in THF with stirring under argon,
and 10 equiv of 1 M BH3-THF complex was added in one portion
at room temperature. Once consumption of starting material was
complete, as indicated by TLC, excess 1 N methanolic HCl was
carefully added to quench excess borane, and the solution was
brought to reflux for 30 min to hydrolyze the boramine complex.
The solvent was evaporated under reduced pressure, and the residue
was dissolved in aqueous 1 M NaOH and extracted with CH2Cl2.
The organic extracts were combined and evaporated to afford a
quantitative yield of the diastereomeric mixture as an oil. This
material was separated 500 mg at a time by radial chromatography
(3:2 hexanes/EtOAc; oven-dried 4 mm silica gel rotor). The S,S
A crystal of sufficient quality for X-ray structural determination
was produced by vapor diffusion of Et2O into a concentrated
solution of the amide in EtOAc. The absolute configuration at the
stereocenter adjacent to the amide carbonyl carbon was found to
be S, and by inference, the other amide must therefore be the R,S

C-(4,5,6-Trimethoxyindan-1-yl)methanamine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4273
diastereomer was the first to elute, followed by the S,R. In the case
of incomplete resolution, the fractions containing a mixture of
diastereomers were combined, evaporated, and rechromatographed.
Both diastereomers were analytically identical to the material
prepared by the previous method.
Radioligand Competition Binding Assays. Competition binding
experiments were carried out in a total volume of 500 µL with
0.20 nM [125I]DOI. Previously harvested cells were resuspended
in assay buffer and added to each well containing assay buffer (50
mM Tris, 0.5 mM EDTA, 10 mM MgCl2; pH 7.4), radioligand,
and test compound. Incubation was carried out at 25 °C for 60
min and terminated by rapid filtration using a prechilled Packard
96-well harvester with GF/B Uni-filters that had been preincubated
for 30 min in 0.3% polyethylenimine. The filters were rinsed using
chilled wash buffer (10 mM Tris, 154 mM NaCl) and left to dry
overnight. The following day, Microscint-O was added and
radioactivity was determined using a TopCount (Packard) scintil-
lation counter. GraphPad Prism (GraphPad Software, San Diego,
CA) was used to analyze the saturation and competition binding
curves.
Inositol Triphosphate Accumulation Studies in Cells Express-
ing the 5-HT2A Receptor. Accumulation of inositol phosphates
was determined using a modified version of a previously published
protocol.20 Briefly, cells expressing the rat 5-HT2A receptor were
labeled for 18-20 h in CRML medium containing 1.0 µCi/mL [3H]-
myoinositol. After the cells were pretreated with 10 µM pargyline/
10 mM LiCl for 15 min, the cells were exposed to a test drug for
30 min at 37 °C under an atmosphere of 5% CO2. The assay was
terminated by aspirating the medium and adding 10 mM formic
acid. After incubation for 16 h at 4 °C, the [3H]inositol phosphates
were separated from the cellular debris on Dowex-1 ion exchange
columns and eluted with 1.0 M ammonium formate and 0.10 M
formic acid. The vials were counted for tritium using a Beckman
LS6500 scintillation counter.
Drug Discrimination Studies. As a measure of hallucinogenic
activity, (()-2 was examined for its effects in rats that were trained
to discriminate a training drug from saline. A two-lever drug
discrimination paradigm was employed as a screen for potential
hallucinogenic activity. Briefly, rats were trained to discriminate
saline injection from the effects of ip injection of LSD tartrate (186
nmol/kg) for study of 5-HT2A receptor-mediated (LSD-like) effects.
The response of the animal to the test drug was quantified by
counting the number of presses on the appropriate drug lever.
Potencies of test drugs were measured using ED50 values for those
drugs that substituted completely for the training drug, LSD. Drugs
that do not substitute completely for the training drug are scored
as producing “partial substitution” (PS) or “no substitution” (NS).
At least 59% of the tested rats must have selected the drug lever
for a score of PS to be assigned, and 80% of those tested must
have selected the drug lever for full substitution. The precise
methodology has been described in detail elsewhere.15,16,21,22 The
target compounds that were tested in the drug discrimination assay
are presented along with their ED50 values and 95% confidence
intervals.
(R)-(-)-(4,5,6-Trimethoxyindan-1-yl)methanamine Hydro-
chloride, (R)-(-)-2‚HCl. (R,S)-8 free base (100 mg, 0.29 mmol)
was dissolved in 7 mL of MeOH with a drop of H2O and placed
into a 25 mL glass Ace hydrogenation flask containing a suspension
of 100 mg of wet 20% Pd(OH)2/C in 5 mL of MeOH and a
magnetic stirbar. The flask was pressurized to 40 psi of H2 and
stirred for 6 h. Once all starting material had been consumed, as
indicated by TLC, the reaction was filtered through diatomaceous
earth to remove the catalyst (Caution! Pd(OH)2 and MeOH
spontaneously ignite in air) and the solvent was evaporated to give
a clear oil. The hydrochloride salt was formed by dissolving the
free amine in MeOH and neutralizing to pH 4 with 1 N methanolic
HCl, then evaporating the solvent. The hydrochloride salt was
obtained in solid form by dissolving the residue in a minimum
amount of i-PrOH, followed by addition of Et2O and collection of
the resulting solid by suction filtration. The (R)-2‚HCl obtained
(77 mg, 96%), pure by TLC and NMR, was recrystallized from
absolute EtOH for elemental analysis to yield a white crystalline
solid (69 mg, 86%): mp 170-171 °C; 1H NMR (300 MHz, CD3-
OD) δ 6.64 (s, 1 H, ArH), 3.78 (s, 3 H, OCH3), 3.76 (s, 3 H, OCH3),
3.70 (s, 3 H, OCH3), 3.36-3.25 (m, 2 H, CH2NH3), 2.94-2.72
(m, 3 H, ArCH, ArCH2), 2.34-2.22 (m, 1 H, ArCH2CH2), 1.86-
1.75 (m, 1 H, ArCH2CH2); low-resolution ESIMS m/z (rel intensity)
238 (M + H, 30), 221 (M + H) - NH3, 100); [R]20 -14.40° (c
D
1.00, MeOH, as HCl salt). Anal. (C13H20ClNO3) C, H, N.
(S)-(+)-(4,5,6-Trimethoxyindan-1-yl)methanamine Hydro-
chloride, (S)-(+)-2‚HCl. Following a procedure similar to that used
for (R,S)-8 above, (S,S)-8 free base (150 mg, 0.44 mmol) was
dissolved in 10 mL of MeOH with a drop of H2O and placed into
a 25 mL glass Ace hydrogenation flask containing a suspension of
150 mg of wet 20% Pd(OH)2/C in 5 mL of MeOH and a magnetic
stir bar. The flask was pressurized to 40 psi of H2 and stirred for
6 h. Once all starting material had been consumed, as indicated by
TLC, the mixture was filtered through diatomaceous earth to remove
the catalyst and the solvent was evaporated to give a clear oil. The
hydrochloride salt was formed by dissolving the free amine in
MeOH and neutralizing to pH 4 with 1 N methanolic HCl, then
evaporating the solvent. The hydrochloride salt was recrystallized
from i-PrOH/Et2O. After filtration and drying under vacuum, 114
mg (95%) of (S)-(+)-2‚HCl was obtained as a white powder,
sufficiently pure for elemental analysis: mp 173-175 °C; 1H NMR
(300 MHz, CD3OD) δ 6.64 (s, 1 H, ArH), 3.78 (s, 3 H, OCH3),
3.76 (s, 3 H, OCH3), 3.70 (s, 3 H, OCH3), 3.36-3.25 (m, 2 H,
CH2NH3), 2.94-2.72 (m, 3 H, ArCH, ArCH2), 2.34-2.22 (m, 1
H, ArCH2CH2), 1.86-1.75 (m, 1 H, ArCH2CH2); low-resolution
ESIMS m/z (rel intensity) 238 (M + H, 30), 221 (M + H) - NH3,
100); [R]20D +14.10° (c 1.00, MeOH, as HCl salt). Anal. (C13H20-
ClNO3) C, H, N.
Pharmacology. Cells stably transfected to express the rat 5-HT2A
or rat 5-HT2C receptor19 were maintained in Dulbecco’s modified
Eagle medium, containing 10% dialyzed fetal bovine serum (Gibco
BRL) and supplemented with 2 mM L-glutamine, 50 units/L
penicillin, 50 µg/L streptomycin, and 300 mg/L Geneticin. The cells
were cultured at 37 °C in a H2O-saturated atmosphere of 95% air
and 5% CO2. For radioligand binding assays, cells were split into
100 mm2 culture dishes when they reached 90% confluency. Upon
reaching 100% confluency, the cells were washed with sterile
filtered phosphate-buffered solution and incubated in serum-free
Opti-MEM for 5 h. After incubation, the cells were scraped and
pelleted by centrifugation (2000g, 10 min), resuspended in assay
bugger, and pelleted again (15000g, 20 min). The supernatant was
aspirated, and the pellet was placed immediately in a freezer at
-80 °C until the assay was performed. For IP3 accumulation
experiments the cells were seeded into 48-well plates and assays
were performed when 70% confluency was achieved.
Molecular Modeling and Docking. Ligand structures were
drawn and energy-minimized (Powell method, 0.01 kcal mol-1 Å-1
gradient termination, MMFF94s force field, MMFF94 charges, 1000
maximum iterations) using the Sybyl 7.0 modeling program.23 The
in silico activated homology model of the h5-HT2A receptor was
prepared as previously described.8 Virtual dockings of energy-
minimized ligands to the h5-HT2A receptor were performed using
the program GOLD 2.224 and scored using GOLDScore with default
settings except for constraints. The GOLDScore fitness algorithm
was constrained to orientations containing ligand-protein interac-
tions implicated by site-directed mutagenesis and previous model-
ing8 to be essential for binding. Distance constraints of 2-3 Å were
set between the side chain carbonyl carbon of D155(3.32) and the
amine nitrogen of the ligand.11,25
The highest ranked docking output
structures were merged with the h5-HT2A receptor model and
analyzed with Sybyl.
Merged ligand-receptor structures were energy-minimized as a
subset based on the ligand molecule (aggregates set to monomers
of >6 Å radius from the ligand, monomers of >12 Å radius ignored,
Powell method, 0.1 kcal mol-1 Å-1 gradient termination, MMFF94s
force field, MMFF94 charges, distance dielectric of 4, 1000
maximum iterations). Constraints for subsequent molecular dynam-

4274 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
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meets function. Pharmacol. Ther. 1998, 79, 231-257.
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(11) Wang, C. D.; Gallaher, T. K.; Shih, J. C. Site-directed mutagenesis
of the serotonin 5-hydroxytrypamine2 receptor: identification of
amino acids necessary for ligand binding and receptor activation.
Mol. Pharmacol. 1993, 43, 931-940.
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C. Mapping the binding site pocket of the serotonin 5-hydroxy-
tryptamine2A receptor. Ser3.36(159) provides a second interaction
site for the protonated amine of serotonin but not of lysergic acid
diethylamide or bufotenin. J. Biol. Chem. 1996, 271, 14672-14675.
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catalyzed by lipase. Gaodeng Xuexiao Huaxue Xuebao 2001, 22,
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genic Phenethylamines as Functionally-selective 5-HT2A Agonists.
Ph.D. Thesis, Purdue University, West Lafayette, IN, 2006.
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(18) Safir, S. R. Sedative Indanones. U.S. Patent 3,454,565, October 5,
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5HT2 receptor defines a family of structurally distinct but functionally
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Signal transduction differences between 5-hydroxytryptamine type
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and antagonist effects of purported 5-hydroxytryptamine antagonists
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(23) Sybyl 7.0; Tripos Inc. (1699 South Hanley Road, St. Louis, MO
63144).
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J. E.; Glennon, R. A.; Roth, B. L. A highly conserved aspartic acid
(Asp-155) anchors the terminal amine moiety of tryptamines and is
involved in membrane targeting of the 5-HT(2A) serotonin receptor
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ics simulations and minimizations in Sybyl were defined as above
for GOLD; however, hydrogen bond constraints were defined as a
distance range constraint of 1-2 Å between each polar residue’s
hydrogen and the appropriate oxygen atom on the ligand. Con-
strained molecular dynamics simulations were then run on the
energy-minimized ligand-receptor structures (constraints, force
field, charges, and dielectric as outlined above, aggregates as above
plus backbone atoms, NTV ensemble at 300 K, Boltzmann
distribution of initial velocities, 5000 steps of 1 fs, and 5 fs
snapshots). Structures with lowest potential energy after the first
1000 fs equilibration period were then energy-minimized as outlined
above with defined constraints.
Acknowledgment. The authors are indebted to Stewart
Frescas for synthesis of the indanone starting material, for
funding by NIH Grant DA02189 from NIDA, a grant from the
Heffter Research Institute, a Purdue Presidential Distinguished
Fellowship to T.H.M., and a Purdue Research Foundation
Fellowship to J.J.C. The work was conducted in a facility
constructed with support from Research Facilities Improvement
Program Grant C06-14499 from the National Center for
Research Resources of the National Institutes of Health.
Supporting Information Available: X-ray crystallographic
coordinates for determination of absolute configuration; elemental
analysis results. This material is available free of charge via the
Internet at http://pubs.acs.org.
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