β-oxygenated analogues of the 5-HT2A serotonin receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane

Article abstract of DOI:10.1021/jm040082s

Activation of 5-HT_2A serotonin receptors represents a novel approach to lowering intraocular pressure. Because 5-HT_2A serotonin receptor agonists might also produce undesirable central effects should sufficient quantities enter the b

Full text of DOI:10.1021/jm040082s

6034
J. Med. Chem. 2004, 47, 6034-6041
â-Oxygenated Analogues of the 5-HT2A Serotonin Receptor Agonist
1-(4-Bromo-2,5-dimethoxyphenyl)-2-aminopropane
Richard A. Glennon,* Mikhail L. Bondarev, Nantaka Khorana, and Richard Young
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23298
Jesse A. May, Mark R. Hellberg, Marsha A. McLaughlin, and Najam A. Sharif
Ophthalmic Products Research, Alcon Research Ltd., Ft. Worth, Texas
Received April 13, 2004
Activation of 5-HT2A serotonin receptors represents a novel approach to lowering intraocular
pressure. Because 5-HT2A serotonin receptor agonists might also produce undesirable central
effects should sufficient quantities enter the brain, attempts were made to identify 5-HT2
serotonin receptor agonists with reduced propensity to penetrate the blood-brain barrier. 1-(4-
Bromo-2,5-dimethoxyphenyl)-2-aminopropan-1-ol (6), an analogue of the 5-HT2 serotonin
receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) bearing a benzylic
hydroxyl group, was identified as a candidate structure. Of the four optical isomers of 6, the
1R,2R-isomer (6d; Ki ) 0.5 nM) was found to bind at 5-HT2A receptors with an affinity similar
to that of R(-)DOB (Ki ) 0.2 nM). Like R(-)DOB, 6d behaved as a partial agonist (efficacy ca.
50%) in a 5-HT2-mediated calcium mobilization assay. However, in an in vivo test of central
action (i.e., stimulus generalization with rats as subjects), 6d was >15 times less potent than
R(-)DOB. O-Methylation of 6d (i.e., 7d; 5-HT2A Ki ) 0.3 nM) resulted in an agent that behaved
as a full (93% efficacy) agonist. Intraocular administration of 300 µg of 6d and 7d to ocular
hypertensive monkeys was shown to reduce intraocular pressure by 20-27%. Given the route
of administration (i.e., topical), and concentrations necessary to reduce intraocular pressure,
compounds such as 6d should demonstrate minimal central effects at potentially useful
therapeutic doses and offer useful leads for further development.
Compounds that function as efficient agonists at
5-HT2A serotonin receptors have been proposed as novel
agents with the potential to control intraocular pressure
in the treatment of ocular hypertension and glaucoma.1
Agents with agonist action at brain 5-HT2A receptors
have also been demonstrated to be psychoactive in
humans.2 However, a recent study has found that a local
ocular site of action seems to be sufficient for achieving
decreased intraocular pressure in a primate model of
ocular hypertension.1 Hence, a 5-HT2A serotonin recep-
tor agonist that does not readily penetrate the blood-
brain barrier should be effective following local ocular
application, and central side effects should be mini-
mized.
The goal of the present investigation was to develop
a novel 5-HT2A serotonin receptor agonist with reduced
ability to penetrate the blood-brain barrier. The general
approach to achieving this goal was to incorporate a
polar moiety into an agent that already possesses
5-HT2A serotonin receptor agonist actions in order to
decrease its lipophilicity. Pharmacophore-based design
might provide an effective means to accomplish this
task. Currently, the two largest categories of 5-HT2A
serotonin receptor agonists are the indolealkylamines
(e.g. tryptamine analogues) and the phenylalkylamines.2
Because the former are notoriously nonselective,2 we
focused on the latter, which includes agents such as 1-(4-
bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB, 1a)
and its iodo and methyl counterparts (1b and 1c,
respectively). We introduced [3H]DOB and [125I]DOI
years ago as radioligands for labeling 5-HT2 receptors,
and DOM and R(-)DOI are commonly employed as
5-HT2 serotonin receptor agonists in behavioral studies
(reviewed in ref 2). DOB (1a) is perhaps one of the best
studied 5-HT2A serotonin receptor agonists. In various
functional assays, DOB (1a) has been shown to behave
either as a full agonist or partial agonist,3,4 and this
property is associated principally with the R-(-)-isomer;
S(+)DOB is of lower efficacy than its enantiomer.3
Previous pharmacophoric studies have identified the
dimethoxy substitution pattern of DOB as contributing
to its actions.2 In fact, structure-affinity relationships
have been examined in detail, and nearly every position
of DOB (1a) has been investigated. The question at hand
was, where in the molecule might a polar substituent
* Correspondence author. Address: Department of Medicinal Chem-
istry, Box 980540, Virginia Commonwealth University, Richmond, VA
23298.Ph: 804-828-8487.Fax: 804-828-7404.E-mail: glennon@hsc.vcu.edu.
10.1021/jm040082s CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/16/2004

â-Oxygenated Analogues of DOB
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6035
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) Br2/CHCl3, 5 °C to room tem-
perature, 2 h; (b) (CH2)6N4/CHCl3, 50 °C, 1 h, then HCl/EtOH, 50
°C, 3 h; (c) NaBH4/MeOH, 0 °C; (d) ClCH2COCl/NaOH/H2O/
CH2Cl2, 0 °C to room temperature, 2 h; (e) KOH/EtOH, 12 h, room
temperature; (f) BH3-THF, reflux, 12 h.
be introduced without significant loss of agonist action?
For example, introduction of polar substituents, de-
pending upon the ring-position and the specific sub-
stituent, can result in phenylalkylamines that lack
affinity for 5-HT2A receptors;3 in other instances, action
has been converted from agonist to low-efficacy partial
agonist or even antagonist activity.5 Quaternization of
the terminal amine, a strategy often employed to reduce
blood-brain barrier permeability, was not an option
here, because the quaternary amine analogue of DOB
does not bind at 5-HT2A receptors.3 The one position of
DOB-type compounds that has not been extensively
investigated is the benzylic (i.e., C1 or â-) position.
Accordingly, we began this investigation by examining
the influence on 5-HT2A affinity and efficacy of polar
(i.e., oxygen-bearing) substituents at the benzylic posi-
tion of DOB-related compounds.
a Reagents and conditions: (a) (COCl)2/CH2Cl2, 1-bromo-2,5-
dimethoxybenzene/TiCl4, -50 °C to room temperature, 60 h; (b)
SiH(CH3)2Ph/TFA, -5 °C, 2 h then K2CO3, reflux, 2 h; (c) Ac2O,
room temperature to 110 °C, 1 h, then 60% H2SO4, 110 °C, 1 h;
(d) NaH/THF, 0 °C to room temperature 0.5 h, then MeI, reflux,
1 h.
rides. The acid chlorides were not purified but, following
evaporation of solvent, were subjected to a Friedel-
Crafts reaction with 1-bromo-2,5-dimethoxybenzene
under mild conditions with complete preservation of
configurational identity of the products S(-)13 and
R(+)13. The chiral purity of S(-)13 and R(+)13 was
established by examination of their proton NMR spectra
in the presence of the chiral shift reagent Eu(hfbc).12
Chiral shift NMR analysis revealed none of the opposite
enantiomer, indicating a chiral purity of >98% for each
isomer. Erythro isomers 6a and 6b were prepared by
the highly erythro-selective reduction13 of the corre-
sponding ketones R(+)13 and S(-) 13, respectively, with
dimethylphenylsilane in TFA. These erythro isomers
were also successfully converted into their correspond-
ing threo isomers 6c and 6d by utilizing a modification
of a procedure that was previously described for the
preparation of threo-norpseudoephedrine isomers.14 The
structures of all erythro and threo isomers were sup-
ported by 1H NMR spectrometry; the erythro isomer 6a
and 6b showed a signal at δ 5.06 (CH-OH), whereas
threo isomers 6c and 6d displayed a signal at δ 4.84
(CH-OH). Such a spectroscopic trend is consistent with
literature observations.15 Alkylation of compounds 6a-d
with CH3I afforded methoxy derivatives 7a-d. Al-
though the enantiomeric purity of the trifluoroacety-
lated intermediate leading to 5b was not examined, the
erythro isomer 5b was synthesized in a similar manner.
Chemistry
The synthesis of morpholine 2 (Scheme 1) began with
8; acetylation of commercially available 1-bromo-2,5-
dimethoxybenzene under Friedel-Crafts conditions ac-
cording to a literature procedure,6 followed by bromi-
nation, gave a bromoacetyl intermediate that was
transformed through a Sommelet reaction into amino
ketone 9 (isolated as its HCl salt). Crude compound 9
was reduced with NaBH4 to provide amino alcohol 3b.
Treatment of 3b with ClCH2COCl afforded the corre-
sponding N-chloroacetyl derivative 10, which was con-
verted into morpholinone 11 by base-catalyzed cycliza-
tion.7 Reduction of 11 with BH3-THF complex gave the
desired morpholine 2. Compound 3b was prepared as
an intermediate in the synthesis of 2, and compound
3c was prepared from 2,5-dimethoxy-4-bromobenzalde-
hyde as described in the literature.8
Compounds 12a and 12b (Scheme 2), as well as 12c
(where R ) S-(-)-Et), were prepared from commercially
available optically active amino acids by trifluroacety-
lation according to literature procedures.9-11 The result-
ing N-trifluroacetyl R-amino acids were then treated
with oxalyl chloride to form the appropriate acid chlo-
Results and Discussion
Radioligand binding data and the results of a func-
tional assay are provided in Table 1. One of the first
compounds prepared and examined in this investigation
was morpholine analogue 2. Compound 2 bears an ether

6036 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Glennon et al.
Table 1. Radioligand Binding and Functional Data for
displayed an efficacy (63%) greater than that of racemic
DOB (Table 1). These results suggested that not only
are polar substituents at the â-position of the phenyl-
alkylamines tolerated by 5-HT2A receptors, certain sub-
stituents might even enhance 5-HT2A serotonin receptor
agonist efficacy.
Lacking an amine substituent or an R-alkyl group, 3
might be prone to rapid metabolism in vivo by oxidative
deamination. Furthermore, although an R-methyl group
might not be required for the binding of DOB-type
agents at 5-HT2A receptors, we have previously shown
that its absence detracts somewhat from 5-HT2A selec-
tivity.18 Accordingly, we introduced small alkyl groups
at the R-position, namely an R-ethyl group and an
R-methyl group.
Compounds Examined
stereochemistry
Ki,
EC50
,
%
C1 (â) C2 (R)
nM SEM µMa SEM efficacy SEM
DOB (1a)
(()
0.10 0.04 0.06 0.03
0.20 0.04 0.02 0.01
20.6 1.8
38
51
4
1
2
1
1
1
3
6
2
R
2
(()
(()
(()
R
3b
3c
4
2.1 0.5
8
2.0 0.5
9.2 4.6
7.6 5.1
20.3 2.1
0.12 0.02
0.74 0.03
63
34
15
5
5a
5b
(()
S
R
Hydroxy Series
9.2 3.1
6a
6b
6c
6d
S
R
S
R
R
S
S
R
20
22
16
50
6
5
3
4
1.1 0.1
0.84 0.42
0.10 0.01
10.0 0.1
0.5 0.1
First, however, it was necessary to determine the role
of the 4-bromo group on efficacy and whether it should
be retained in the analogues to be investigated. Com-
pound 4 (Ki ) 9.2 nM), as previously shown,3 binds with
Methoxy Series
17.4 0.8
0.8 0.1
7a
7b
7c
7d
S
R
S
R
R
S
S
R
5.04 3.74
1.33 0.13
1.41 0.23
0.13 0.01
49
54
31
93
5
7
2
5
6.0 0.2
0.3 0.1
a Calcium-mobilization assay. EC50 value not determined where
efficacy e20%.
oxygen atom at the benzylic position that is tethered
to the terminal amine. However, its 5-HT2A affinity
(Ki ) 20.6 nM) is 100-fold lower than that of R(-)DOB
(Ki ) 0.2 nM), and its agonist efficacy in the 5-HT2-
mediated calcium mobilization assay is minimal (Table
1). There are several possible explanations for these
unfavorable findings. Apart from the molecule possess-
ing a chiral center, the ether oxygen atom might not be
tolerated by the receptor and/or the added ethylene
“bridge” might not be readily accommodated by the
receptor and its presence detracts from affinity. A more
plausible explanation, based on prior structure-affinity
investigations, is that the secondary amine of 2 con-
tributes to decreased affinity. That is, we have previ-
ously shown that addition of small N-alkyl substituents
can reduce the 5-HT2A receptor affinity of DOB and
DOB-related compounds.16,17
reduced affinity at 5-HT2A receptors relative to
R(-)DOB. Interestingly, the 4-bromo group does not
appear to contribute significantly to efficacy. Compound
4 (EC50 ) 0.74 µM), although 10-fold less potent than
R(-)DOB, approximated the efficacy of racemic DOB.
That is, the presence of the bromo group contributes
both to affinity and agonist potency, but somewhat less
to efficacy. Consequently, the bromo function was
retained and R-alkyl substituents were introduced.
Addition of an R-alkyl substituent introduces a second
chiral center, and four optical isomers are possible. For
purpose of comparison of the effect of an R-ethyl versus
an R-methyl group, we arbitrarily selected as targets
the 1R,2S isomers of a 4-brominated compound. Al-
though there was no a priori reason to suspect that this
stereochemistry would be optimal, our immediate goal
was simply to compare the influence on affinity/activity
of an R-ethyl versus an R-methyl substituent. Stan-
dridge et al.19 have previously synthesized a series of
R-ethyl phenylalkylamines including compound 5a. We
found compound 5a (Ki ) 7.6 nM) to bind with high
affinity, but with about 70-fold reduced affinity relative
to DOB. Furthermore, its 5-HT2A efficacy was only about
half that of DOB. Incorporation of the â-hydroxy group
reduced affinity (5b, 5-HT2A Ki ) 20.3 nM) and efficacy
(5%) by about 3-fold. In contrast, R-methyl compound
6b (Ki ) 1.1 nM) displayed 20 times the affinity of 5b
while retaining its efficacy.
Consequently, we turned our attention to simpler
phenylethylamine derivatives that lacked an N-alkyl
substituent (e.g. 3). Compound 3a, the R-(or C2-)-
desmethyl counterpart of DOB, binds at 5-HT2A sero-
tonin receptors with an affinity comparable to that of
DOB (1a);18 that is, the presence of the R-methyl group
of DOB is not required for binding. The â-(or C1-)-
hydroxy and -methoxy analogues 3b and 3c were
prepared for examination as their racemates; compound
3c had been previously synthesized by Lemaire et al.8
Hydroxy compound 3b (Ki ) 2.1 nM) displayed 10-fold
enhanced affinity relative to 2 but was also a low-
efficacy (8%) agonist. In contrast, methoxy compound
3c (Ki ) 2.0 nM) retained the affinity of 3b and
With information in hand that an R-methyl group is
favored over an R-ethyl group, that the 4-bromo sub-
stituent should be retained, and that â-hydroxy and
â-methoxy substituents are tolerated, the four optical
isomers of both â-hydroxy (i.e., 6) and â-methoxy (i.e.,
7) DOB were prepared for evaluation. As mentioned
above, earlier pharmacophoric investigations had al-
ready demonstrated that the R-isomers of DOB-related
agents bind with severalfold higher affinity than their
S-enantiomers. It was expected, then, that the 2R-

â-Oxygenated Analogues of DOB
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6037
isomers would bind with somewhat higher affinity than
their 2S counterparts. That the 1S-hydroxy-2R-methyl
compound 6a (Ki ) 9.2 nM) binds with nearly 10-fold
Figure 1. Results [% DOM-appropriate responding (SEM)]
of drug discrimination studies employing rats trained to
discriminate DOM (1c) (1.0 mg/kg) from saline vehicle. Stimu-
lus generalization was considered to have occurred when the
animals made g80% of their responses on the DOM-appropri-
ate lever.
lower affinity than its 1R-hydroxy-2S-methyl counter-
part 6b (Ki ) 1.1 nM) indicates that 5-HT2A receptors
do not readily accommodate the hydroxy group in the
S-configuration. Similar results were obtained with 1S-
hydroxy compound 6c (Ki ) 10 nM). Of the four isomers
of the hydroxy analogues, the highest affinity member
was 1R-hydroxy-2R-methyl compound 6d (Ki ) 0.5 nM).
Parallel results were obtained with the methoxy ana-
logues, and the highest affinity member of the methoxy
series was 1R-methoxy-2R-methyl compound 7d (Ki )
0.3 nM), and the affinity of 7d was comparable to that
of R(-) DOB (Ki ) 0.2 nM). It can be concluded that
introduction of a â-hydroxy group decreases the affinity
of R(-)DOB by 50-fold when in the S-configuration but
has little effect when in the R-configuration; likewise,
introduction of a â-methoxy group decreases affinity by
nearly 100-fold when in the S-configuration and is
tolerated when in the R-configuration.
All the hydroxy and methoxy isomers displayed
agonist action. But there is a broad span of potencies.
In general, the hydroxy compounds are not as potent
as the methoxy isomers; nevertheless, hydroxy com-
pound 6d (EC50 ) 0.10 µM), although 5-fold less potent
than R(-)DOB (EC50 ) 0.02 µM), was equiefficacious
(ca. 50%) in the calcium-mobilization assay. Methoxy
compound 7d possessed similar potency (EC50 ) 0.13
µM) but was more efficacious than R(-)DOB and
essentially behaved as a full (93%) agonist.
The intent of this investigation was to identify a
5-HT2A ligand with agonist character and reduced
propensity to penetrate the blood-brain barrier. Both
6d and 7d bear polar substituents at the benzylic
position of a phenylisopropylamine nucleus; although
both are similar in 5-HT2A serotonin receptor affinity
and agonist potency, 7d is a full agonist, whereas 6d
displays lower efficacy but an efficacy similar to that of
DOB. Because of the presence of the more polar hy-
droxyl group, 6d was selected for further evaluation,
despite its lower efficacy. Agents with 5-HT2A agonist
character have been shown to substitute for DOM (1c)
in rats trained to discriminate DOM (1c) from saline
vehicle in a two-lever drug discrimination task (re-
viewed in ref 20). The effect has been demonstrated to
be centrally mediated and is antagonized by 5-HT2A
serotonin receptor antagonists.20 Accordingly, we com-
pared the actions of 6d with that of the R-(-)-isomer of
DOB (1a). Using rats trained to discriminate 1.0
mg/kg of DOM (1c) from saline, administration of
R(-)DOB resulted in substitution (i.e., stimulus gen-
eralization; ED50 ) 0.09 mg/kg, 95% CL ) 0.06-0.16
Table 2. Intraocular Pressure (IOP, mmHg) Response after
Topical Ocular Administration to the Normal Eye of Conscious
Cynomolgus Monkeys
% IOP reduction (SEM)
baseline
compda
IOP
1 h
3 h
6 h
6d
37.3
38.0
35.5
10.7(4.1)
19.0b(4.7)
15.2(4.0)
19.0b(3.3)
27.5b(4.3)
31.0b(4.5)
22.1b(5.2)
25.5b(5.3)
34.4b(6.7)
7d
R(-)DOIc
a 300 µg in phosphate-buffered saline, pH 7.4. b p<0.05. c Data
from May et al.1 as assayed under comparable conditions.
mg/kg or 0.25 µmol/kg). Administration of 6d also
resulted in substitution (ED50 ) 1.4 mg/kg; 95% CL )
0.8-2.6 mg/kg, or 4.3 µmol/kg). Evidently, introduction
of the benzylic hydroxyl group resulted in a >15-fold
rightward shift of the dose-response curve (Figure 1).
Another way of viewing the results is that at doses
higher than that required for R(-)DOB to substitute
for the DOM stimulus, 6d still produced saline-ap-
propriate responding; higher doses of 6d were required
to produce >80% drug-appropriate responding. Given
the 5-HT2A affinities and efficacies of these two agents,
the results suggest that 6d might not penetrate the
blood-brain barrier as well as R(-)DOB.
Encouraged by the drug discrimination results, 6d
and 7d were assessed for their ability to lower pressure
in conscious cynomologus monkeys with laser-induced
ocular hypertension. Both compounds effectively de-
creased intraocular pressure (IOP) following topical
ocular application of a 300 µg dose (Table 2). Although
further dose-response studies are required to fully
characterize the peak IOP reduction, the efficacy of 6d
in the ocular hypertensive monkey coupled with the
drug discrimination results suggest that the IOP effect
of 5-HT2 serotonin receptor agonists is mediated by a
local rather than centrally mediated mechanism. The
maximum reduction in IOP observed for 7d of 27.5% is
similar to that achieved by R(-)DOI, an agent routinely
used as positive control in such studies.
In conclusion, introduction of a 1R-hydroxy (i.e.,
â-hydroxy) group to R(-)DOB is tolerated by 5-HT2A
receptors and has relatively little influence on agonist
potency or efficacy. In contrast, introduction of a 1R-
methoxy group, although tolerated by 5-HT2A receptors
and having relatively little influence on agonist potency,
tends to double efficacy. Additional studies are now
planned to further investigate the pharmacology of
compounds 6d (AL-34659) and 7d (AL-37662). Given the

6038 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Glennon et al.
in vacuo over CaCl2 to afford 10.25 g (66%) of crude product
as its HCl salt, which was used without further purification.
Sodium borohydride (12.48 g, 330 mmol) was added in small
portions to a stirred solution of the aminoacetophenone 9
(10.25 g, 33 mmol) in MeOH (250 mL) at 0 °C over 1.5 h. After
the addition was complete, the reaction mixture was allowed
to stir at 0 °C for an additional 2 h. Solvent was evaporated
under reduced pressure, H2O (150 mL) was added, and the
resulting mixture was extracted with CH2Cl2 (3 × 75 mL). The
combined CH2Cl2 portions were washed with H2O (3 × 50 mL)
and dried (MgSO4). Solvent was evaporated under reduced
pressure to give the crude free base of 3b as a yellow-white
solid which was purified by recrystallization from MeOH/Et2O
to give the free base of 3b as white crystals: mp 130-131 °C.
The free base in MeOH (50 mL) was treated with ethereal HCl
and solvent was evaporated under reduced pressure to give
8.56 g (83%) of 3b as off-white crystals following recrystalli-
affinity/potency of these agents and their increased
polarity, it is unlikely that they would produce centrally
mediated effects at the doses that would be required to
reduce intraocular pressure following topical applica-
tion.
Experimental Section
Chemistry. Melting points were taken in glass capillary
tubes on a Thomas-Hoover melting point apparatus and are
uncorrected. 1H NMR spectra were recorded with a Varian
EM-390 spectrometer, and peak position are given in parts
per million (δ) downfield from tetramethylsilane as the
internal standard. Microanalyses were performed by Atlantic
Microlab (GA) for the indicated elements, and the results are
within 0.4% of the calculated values. Chromatographic separa-
tions were performed on silica gel columns (Kieselgel 40,
0.040-0.063 mm, Merck) by flash chromatography. Reactions
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel precoated F254 Merck
plates.
1
zation from 2-PrOH: mp 204-207 °C; H NMR (DMSO-d6) δ
2.69-2.99 (m, 2H, CH2), 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
5.02 (m, 1H, CH-OH), 6.10 (br.s, 1H, OH, exchangeable), 7.20
(s, 1H, ArH), 7.22 (s, 1H, ArH), 8.02 (br.s, 3H, NH3+, exchange-
able). Anal. (C10H14BrNO3‚HCl‚0.5H2O) C, H, N.
Compounds 3a and 4, as their HCl salts, were on-hand from
previous investigations, and compound 5a‚HCl was a gift from
the Research Division of Bristol Laboratories.
(-)-erythro-(1R,2S)-1-Hydroxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminobutane Oxalate (5b). S(-)-2-[N-(Tri-
fluoroacetyl)amino]-1-(2,5-dimethoxy-4-bromophenyl)-1-bu-
tanone was prepared in 29% yield from S(+)-2-trifluoroacetyl-
aminobutyric acid,11 via trifluoroacetamide 12c, exactly as
described for the synthesis of S(-)13. The product was isolated
as a yellow-white powder: mp 92-94 °C; [R]D ) -5.7° (c 1,
MeOH); 1H NMR (CDCl3) δ 0.87 (t, J ) 7.6 Hz, 3H, CH3), 1.61
(m, 2H, CH2), 3.93 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 5.58 (m,
1H, CH), 7.28 (s, 1H, ArH), 7.41 (s, 1H, ArH), 7.44 (bs, 1H,
NHCO, exchangeable). The butanone was converted to 5b as
described for the synthesis of 6b, except that ethereal oxalic
acid was used in order to isolate the product as the oxalate
salt. The salt was recrystallized from MeOH/Et2O to afford
(()2-(4-Bromo-2,5-dimethoxyphenyl)morpholine Ox-
alate (2). A solution of 1 M BH3-THF complex (60 mL, 60.0
mmol) was added in a dropwise manner to a solution of 11
(3.16 g, 10.0 mmol) in THF (20 mL) at 0 °C under an N2
atmosphere. The reaction mixture was heated at reflux for 12
h under an N2 atmosphere, and concentrated HCl (15 mL) was
added in a dropwise manner at -5 °C. The mixture was heated
at reflux for an additional 1 h and then the THF was
evaporated under reduced pressure. The residue was made
alkaline with 1 N NaOH and extracted with CH2Cl2 (3 × 50
mL). The combined CH2Cl2 portions were washed with H2O
(3 × 50 mL) and dried (MgSO4), and the CH2Cl2 was evapo-
rated under reduced pressure to give a colorless oil. The oil in
Et2O/MeOH (4:1) was treated with ethereal oxalic acid; the
mixture was concentrated under reduced pressure and the
precipitated oxalate salt was collected by filtration, washed
with anhydrous Et2O (3 × 15 mL), and recrystallized twice
from 2-PrOH to afford 2.18 g (56%) of 2 as white crystals: mp
5b as white crystals in 76% yield: mp 203-205 °C; [R]D
)
-28.5° (c 1, MeOH); 1H NMR (DMSO-d6) δ 0.78 (t, J ) 7.3
Hz, 3H, CH3), 1.33 (m, 2H, CH2), 3.19 (m, 1H, CH-NH3+), 3.77
(s, 3H, OCH3), 3.80 (s, 3H, OCH3), 5.10 (m, 1H, CH-OH), 7.17
(s, 1H, ArH), 7.23 (s, 1H, ArH). Anal. (C12H18BrNO3‚C2H2O4)
C, H, N.
1
190-192 °C; H NMR (DMSO-d6) δ 2.85-3.31 (m, 4H, CH2),
(+)-erythro-(1S,2R)-1-Hydroxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane hydrochloride (6a) was pre-
pared from R(+)-2-[N-(trifluoroacetyl)amino]-1-(2,5-dimethoxy-
4-bromophenyl)-1-propanone (R(+)13), as described for 6b, as
white crystals in 68% yield: mp 194-196 °C; [R]D ) +42.9° (c
1, MeOH). Anal. (C11H16BrNO3‚HCl‚0.5H2O) C, H, N.
3.78 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.89-4.19 (m, 2H, CH2),
4.96 (d, J ) 9.0 Hz, 1H, OCH), 7.08 (s, 1H, ArH), 7.27 (s, 1H,
ArH). Anal. (C12H16BrNO3‚C2H2O4) C, H, N.
1-Hydroxy-1-(4-bromo-2,5-dimethoxyphenyl)-2-amino-
ethane Hydrochloride (3b). Bromine (7.99 g, 50 mmol) in
CHCl3 (20 mL) was added in a dropwise manner to a stirred
solution of 2,5-dimethoxy-4-bromoacetophenone6 (12.95 g, 50
mmol) in CHCl3 (100 mL) at 5 °C. After the addition was
complete, the reaction mixture was allowed to warm to room
temperature and stirred for an additional 2 h. The mixture
was poured onto crushed ice; the organic portion was separated
and washed with H2O (2 × 100 mL), saturated NaHCO3
solution (2 × 100 mL), and again with H2O (2 × 100 mL). The
solution was dried (MgSO4) and evaporated to dryness under
reduced pressure to give a crude brown/white product. The
product was recrystallized from MeOH to yield 14.70 g (87%)
of the desired bromoacetophenone product as a white solid:
(-)-erythro-(1R,2S)-1-Hydroxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane Hydrochloride (6b). Dimeth-
ylphenylsilane (1.70 g, 12.5 mmol) was added in a dropwise
manner to a solution of (S)-(-)-2-[N-(trifluoroacetyl)amino]-
1-(2,5-dimethoxy-4-bromophenyl)-1-propanone (S(-)13) (3.84
g, 10.0 mmol) in TFA (5 mL) at -5 °C under an N2 atmosphere.
The reaction mixture was allowed to warm to 0 °C, stirred for
an additional 2 h, poured onto crushed ice, and neutralized
with a saturated NaHCO3 solution. The solution was extracted
with CH2Cl2 (3 × 50 mL). The combined CH2Cl2 portions were
washed with saturated NaHCO3 solution (3 × 25 mL) and
brine (3 × 25 mL) and dried (MgSO4), and the solvent was
evaporated under reduced pressure. The resulting residue was
purified by flash chromatography with silica gel using, se-
quentially, CH2Cl2 and MeOH/CH2Cl2 (1:20) as eluants. The
crude product in MeOH (30 mL) was added to a stirred mixture
of K2CO3 (6.91 g, 50 mmol) in H2O (5 mL) and then heated at
reflux for 2 h. The MeOH was removed under reduced pressure
and the residue was extracted with CH2Cl2 (3 × 25 mL). The
combined organic portions were dried (MgSO4), and the solvent
was evaporated under reduced pressure to give the crude free
base of 6b as a yellow-white solid. The free base in anhydrous
Et2O (50 mL) was treated with ethereal HCl; the precipitated
HCl salt was collected by filtration, washed with anhydrous
Et2O (2 × 10 mL), and recrystallized from EtOAc to afford 2.28
g (70%) of 6b as white crystals: mp 197-199 °C; [R]D ) -37.1°
1
mp 122-123 °C; H NMR (CDCl3) δ 3.90 (s, 3H, OCH3), 3.93
(s, 3H, OCH3), 4.59 (s, 2H, CH2Br), 7.24 (s, 1H, ArH), 7.41 (s,
1H, ArH).
A solution of the above 2,5-dimethoxy-4-bromo-R-bromo-
acetophenone (16.90 g, 50 mmol) and hexamethylenetetramine
(7.00 g, 50 mmol) in CHCl3 (200 mL) was allowed to stir at 50
°C for 1 h. The reaction mixture was allowed to cool to the
room temperature, and the white precipitate was collected by
filtration and washed with CHCl3 (3 × 25 mL). The resulting
quaternary salt was suspended in a mixture of 95% EtOH (50
mL) and concentrated HCl (25 mL) and heated at 50 °C for 3
h. After 15 min, the reaction mixture was homogeneous and
the aminophenone hydrochloride began to crystallize. The
mixture was cooled to 0 °C and the white solid was collected
by filtration. The solid was recrystallized from H2O and dried

â-Oxygenated Analogues of DOB
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6039
1
(c 1, MeOH); H NMR (DMSO-d6) δ 0.92 (d, J ) 6.7 Hz, 3H,
in 52% yield: mp 115-118 °C; [R]D ) +51.7° (c 1, MeOH); 1H
NMR (DMSO-d6) δ 0.96 (d, J ) 6.7 Hz, 3H, CH3), 3.14 (s, 3H,
CH-OCH3) 3.40 (m, 1H, CH-NH3+), 3.78 (s, 3H, OCH3), 3.81
(s, 3H, OCH3), 4.55 (d, J ) 8.7 Hz, 1H, CH-OCH3), 6.96 (s,
1H, ArH), 7.32 (s, 1H, ArH). Anal. (C11H16BrNO3‚C2H2O4) C,
H, N.
CH3), 3.38 (m, 1H, CH-NH3+), 3.76 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 5.06 (m, 1H, CH-OH), 6.06 (d, J ) 3.3 Hz, 1H, OH,
exchangeable), 7.14 (s, 1H, ArH), 7.23 (s, 1H, ArH), 8.04 (br.s,
3H, NH3+, exchangeable). Anal. (C11H16BrNO3‚HCl) C, H, N.
(+)-threo-(1S,2S)-1-Hydroxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane Hydrochloride (6c). Acetic
anhydride (3.57 g, 35.0 mmol) was added to the free base of
(-)-erythro-(1R,2S)-1-hydroxy-1-(4-bromo-2,5-dimethoxyphe-
nyl)-2-aminopropane (6b) (2.90 g, 10.0 mmol) at room tem-
perature under an N2 atmosphere. The reaction mixture was
heated at 110 °C for 1 h and then cooled to 60-80 °C. Aqueous
H2SO4 (60%, 8 mL) was added and the reaction mixture was
heated at 110 °C for an additional 1 h. The mixture was
allowed to cool to room temperature, poured onto crushed ice,
and basified with 15% aqueous NaOH solution to pH ) 8. The
solution was extracted with CH2Cl2 (3 × 50 mL). The combined
CH2Cl2 portions were washed with brine (3 × 50 mL) and dried
(MgSO4), and solvent was evaporated under reduced pressure.
The resulting residue was purified by flash chromatography
[silica gel; CH2Cl2/MeOH (4:1)] to give an oil. The oil in
anhydrous Et2O (50 mL) was treated with ethereal HCl. The
precipitated HCl salt was collected by filtration, washed with
anhydrous Et2O (2 × 10 mL), and recrystallized from
Et2O/MeOH to afford 2.67 g (82%) of 6c as white crystals: mp
(-)-threo-(1R,2R)-1-Methoxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane oxalate (7d) was prepared,
as described for 7b, from (-)-threo-(1R,2R)-1-hydroxy-1-(4-
bromo-2,5-dimethoxyphenyl)-2-aminopropane (6d) as white
crystals in 73% yield: mp 115-118 °C; [R]D ) -52.2° (c 1,
MeOH). Anal. (C11H16BrNO3‚C2H2O4) C, H, N.
(()-2-(4-Bromo-2,5-dimethoxyphenyl)morpholin-5-
one (11). Chloroacetyl chloride (3.39 g, 30 mmol) was added
in a dropwise manner to a vigorously stirred mixture of NaOH
(0.94 g, 24 mmol) in H2O (100 mL) and the free base of
1-hydroxy-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminoethane (3b)
(6.25 g, 20 mmol) in CH2Cl2 (100 mL) at 0 °C. After the
addition was complete, the reaction mixture was allowed to
warm to room temperature and was stirred for an additional
6 h. The layers were separated, and the organic portion was
washed with 3% HCl (2 × 25 mL) and saturated NaHCO3
solution (2 × 25 mL) and dried (MgSO4), and solvent was
evaporated to dryness under reduced pressure to give 5.00 g
(71%) of the crude 1-hydroxy-1-(4-bromo-2,5-dimethoxyphe-
nyl)-2-(chloroacetylamino)ethane (10) as a yellow-white foamy
semisolid. The product was dissolved in 95% EtOH (50 mL)
and added in a dropwise manner to a stirred solution of KOH
(1.68 g, 30 mmol) in 95% EtOH (25 mL) at room temperature.
The reaction mixture was allowed to stir for an additional 12
h, concentrated under reduced pressure, and diluted with H2O
(80 mL). The mixture was extracted with CH2Cl2 (3 × 50 mL);
the combined CH2Cl2 portions were washed with H2O (3 × 50
mL) and dried (MgSO4), and CH2Cl2 was evaporated under
reduced pressure to give 3.58 g (80%) of 11 as white crystals:
mp 172-173 °C, after recrystallization from Et2O/hexanes: 1H
NMR (CDCl3) δ 3.24-3.65 (m, 2H, CH2), 3.80 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 4.31-4.51 (m, 2H, CH2), 5.00 (m, 1H,
O-CH), 6.47 (br s, 1H, NHCO, exchangeable), 7.07 (s, 1H, ArH),
7.09 (s, 1H, ArH).
1
213-214 °C; [R]D ) +30.9° (c 1, MeOH); H NMR (DMSO-d6)
δ 1.03 (d, J ) 6.7 Hz, 3H, CH3), 3.27 (m, 1H, CH-NH3+), 3.76
(s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.84 (m, 1H, CH-OH), 6.16
(d, J ) 3.3 Hz, 1H, OH, exchangeable), 7.14 (s, 1H, ArH), 7.25
(s, 1H, ArH), 7.98 (br.s, 3H, NH3+, exchangeable). Anal.
(C11H16BrNO3‚HCl) C, H, N.
(-)-threo-(1R,2R)-1-Hydroxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane hydrochloride (6d) was pre-
pared, as described for 6c, from (+)-erythro-(1S,2R)-1-hydroxy-
1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (6a) as white
crystals in 80% yield: mp 214-215 °C; [R]D ) -31.3° (c 1,
MeOH). Anal. (C11H16BrNO3 × HCl) C, H, N.
(+)-erythro-(1S,2R)-1-Methoxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane oxalate (7a) was prepared,
as described for 7b, from (+)-erythro-(1S,2R)-1-hydroxy-1-(4-
bromo-2,5-dimethoxyphenyl)-2-aminopropane (6a) as a white
crystals in 67% yield: mp 189-192 °C; [R]D ) +58.2° (c 1,
MeOH). Anal. (C11H16BrNO3‚C2H2O4) C, H, N.
S-(-)-2-[N-(Trifluoroacetyl)amino]-1-(2,5-dimethoxy-4-
bromophenyl)-1-propanone (S(-)13). Oxalyl chloride (11.64
g, 91.8 mmol) was added in one portion to a stirred mixture of
N-(trifluoroacetyl)-L-alanine9 (12a) (8.00 g, 43.2 mmol) and dry
pyridine (0.5 mL) in dry CH2Cl2 (300 mL) at 0 °C under an N2
atmosphere. The reaction mixture was allowed to warm to
room temperature and stirred for an additional 2 h. The
mixture was concentrated under reduced pressure at a tem-
perature below 30 °C to give an oil. The oil was mixed with
1-bromo-2,5-dimethoxybenzene (9.38 g, 43.2 mmol); the result-
ing mixture was dissolved in dry CH2Cl2 (25 mL) and added
in a dropwise manner to a stirred solution of 1 M TiCl4 in
CH2Cl2 (64.8 mL) at -50 °C under an N2 atmosphere. The
reaction mixture was allowed to warm to room temperature,
stirred for an additional 60 h, and poured onto crushed ice.
The organic portion was separated and washed successively
with 1 M HCl (2 × 50 mL), H2O (2 × 50 mL), and saturated
NaHCO3 solution (2 × 50 mL) and dried (MgSO4), and solvent
was removed by evaporation under reduced pressure to give
a crude, brown product. The product was purified by flash
chromatography (silica gel; CH2Cl2) and recrystallized from
Et2O/hexanes to yield 5.97 g (36%) of the title compound as a
(-)-erythro-(1R,2S)-1-Methoxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane Oxalate (7b). A solution of
free base of (-)-erythro-(1R,2S)-1-hydroxy-1-(4-bromo-2,5-
dimethoxyphenyl)-2-aminopropane (6b) (2.90 g, 10.0 mmol) in
THF (10 mL) was added in a dropwise manner to a suspension
of 95% NaH (0.38 g, 15.0 mmol) in THF (5 mL) at 0 °C under
an N2 atmosphere. After stirring at room temperature for 0.5
h, the reaction mixture was treated in a dropwise manner with
CH3I (1.42 g, 10.0 mmol) at 0 °C and then heated at reflux for
1 h. The mixture was cooled to room temperature and MeOH
(3 mL) was added to destroy excess NaH. The solution was
concentrated under reduced pressure and diluted with H2O
(10 mL). The resulting mixture was extracted with CH2Cl2 (3
× 25 mL); the combined CH2Cl2 portions were washed with
brine (3 × 25 mL) and dried (MgSO4), and solvent was
evaporated under reduced pressure to give a crude oil. The oil
was purified by flash chromatography (silica gel; CH2Cl2/
MeOH, 9:1), dissolved in anhydrous Et2O (50 mL), and treated
with an ethereal solution of oxalic acid. The precipitated
oxalate salt was collected by filtration, washed with anhydrous
Et2O (2 × 10 mL), and recrystallized from Et2O/MeOH to
afford 2.88 g (73%) of 7b as white crystals: mp 186-188 °C;
[R]D ) -59.8° (c 1, MeOH); 1H NMR (DMSO-d6) δ 0.95 (d, J )
6.8 Hz, 3H, CH3), 3.27 (s, 3H, CH-OCH3) 3.40 (m, 1H,
CH-NH3+), 3.78 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.75 (d, J
) 2.8 Hz, 1H, CH-OCH3), 6.91 (s, 1H, ArH), 7.30 (s, 1H, ArH).
Anal. (C12H18BrNO3‚C2H2O4) C, H, N.
1
white solid: mp 144-145 °C; [R]D ) -28.9° (c 1, MeOH); H
NMR (CDCl3) δ 1.43 (d, J ) 6.2 Hz, 3H, CH3), 3.90 (s, 3H,
OCH3), 3.95 (s, 3H, OCH3), 5.59 (m, 1H, CH), 7.26 (s, 1H, ArH),
7.41 (s, 1H, ArH), 7.61. (br s, 1H, NHCO, exchangeable).
R-(+)-2-[N-(Trifluoroacetyl)amino]-1-(2,5-dimethoxy-
4-bromophenyl)-1-propanone (R(+)13). An exact replica-
tion of the above procedure using N-(trifluoroacetyl)-D-
alanine10 (12b) in place of 12a gave 6.30 g (38%) of R(+)13 as
a white crystals: mp 144-145 °C; [R]D ) +28.4° (c 1, MeOH).
Determination of Binding to 5-HT2A Receptor. To
determine the relative affinities of serotonergic compounds at
the 5-HT2 receptors, their ability to compete for the binding
(+)-threo-(1S,2S)-1-Methoxy-1-(4-bromo-2,5-dimeth-
oxyphenyl)-2-aminopropane oxalate (7c) was prepared, as
described for 7b, from (+)-threo-(1S,2S)-1-hydroxy-1-(4-bromo-
2,5-dimethoxyphenyl)-2-aminopropane (6c) as white crystals

6040 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Glennon et al.
of the agonist radioligand [125I](()DOI to brain 5-HT2A recep-
tors was determined as described here with minor modification
of a literature procedure.21 Aliquots of post mortem rat cerebral
cortex homogenates (400 µL) dispersed in 50 mM Tris-HCl
buffer (pH 7.4) were incubated with [125I](()DOI (80 pM final)
in the absence or presence of methiothepin (10 µM final) to
define total and nonspecific binding, respectively, in a total
volume of 0.5 mL. The assay mixture was incubated for 1 h at
23 °C in polypropylene tubes, and the assays were terminated
by rapid vacuum filtration over Whatman GF/B glass fiber
filters previously soaked in 0.3% polyethyleneimine using ice-
cold buffer. Nonspecific binding was defined with 1-10 µM
methiothepin. Filter-bound radioactivity was determined by
liquid scintillation spectrometry on a â-counter. The data were
analyzed using a nonlinear, iterative curve-fitting computer
program22 to determine the compound’s affinity param-
eter. The concentration of the compound needed to inhibit the
sweetened condensed milk) using standard two-lever Coul-
bourn Instruments operant equipment as previously de-
scribed.23 Animal studies were conducted under an approved
Institutional Animal Care and Use Committee protocol.
In brief, animals were food-restricted to maintain their body
weights at approximately 80% of their free-feeding weight, but
were allowed access to water ad lib in their individual home
cages. Daily training sessions were conducted with the training
dose of the training drugs or saline. For approximately half
the animals, the right lever was designated as the drug-
appropriate lever, whereas the situation was reversed for the
remainder of the animals. Learning was assessed every fifth
day during an initial 2.5-min nonreinforced (extinction) session
followed by a 12.5-min training session. Data collected during
the extinction session included response rate (i.e., responses
per minute) and number of responses on the drug-appropriate
lever (expressed as a percent of total responses). Animals were
not used in the subsequent stimulus generalization studies
until they consistently made g80% of their responses on the
drug-appropriate lever after administration of training drug
and e20% of their responses on the same drug-appropriate
lever after administration of saline for several weeks. During
the stimulus generalization (i.e., substitution) phase of the
study, maintenance of the training drug/saline discrimination
was ensured by continuation of the training sessions on a daily
basis (except on a generalization test day). On one of the 2
days before a generalization test, approximately half the
animals would receive the training dose of training drug and
the remainder would receive saline; after a 2.5-min extinction
session, training was continued for 12.5 min. Animals not
meeting the original training criteria during the extinction
session were excluded from the subsequent generalization test
session. During the investigations of stimulus generalization,
test sessions were interposed among the training sessions.
Once per week, the animals were allowed 2.5 min to respond
under nonreinforcement conditions following administration
of a dose of DOM (1c), R(-)DOB‚HBr, or 6d; animals were
immediately returned to their individual home cages following
the 2.5-min test session An odd number of training sessions
(usually five) separated any two generalization test sessions.
Doses of test drugs were administered in a random order, using
a 15-min presession injection interval, to the group of rats.
Stimulus generalization was considered to have occurred when
the animals, after a given dose of drug, made g80% of their
responses (group mean) on the training drug-appropriate lever.
Animals making fewer than five total responses during the
2.5-min extinction session were considered as being disrupted.
Percent drug-appropriate responding refer only to animals
making five or more responses during the extinction session.
Where stimulus generalization occurred, an ED50 dose was
calculated by the method of Finney.24 The ED50 dose represents
the drug dose at which animals would be expected to make
50% of their responses on the drug-appropriate lever. All
solutions, in sterile 0.9% saline, were freshly prepared daily.
[
125I](()DOI binding by 50% of the maximum (IC50 value) was
determined for each compound.
Determination of 5-HT2 Activity: [Ca2+]i Mobilization
Assay. The receptor-mediated mobilization of intracellular
calcium ([Ca2+]i) was studied using a fluorescence imaging
plate reader (FLIPR). Rat vascular smooth muscle cells, A7r5,
were grown in a normal media of DMEM/10% FBS and 10
µg/mL gentamycin. Confluent cell monolayers were trypsinized,
pelleted, and resuspended in normal media. Cells were seeded
in a 50 µL volume at a density of 20 000 cells per well in a
black-walled, 96-well tissue culture plate and grown for 2 days.
On the day of the experiment, one vial of FLIPR Calcium
Assay Kit dye was resuspended in 50 mL of a FLIPR buffer
consisting of Hank’s balanced salt solution (HBSS), 20 mM
HEPES, and 2.5 mM probenecid, pH 7.4. Cells were loaded
with the calcium-sensitive dye by addition of an equal volume
(50 µL) to each well of the 96-well plate and incubated with
dye for 1 h at 23 °C. Typically, test compounds were stored at
25 µM in 50% DMSO/50% ethanol solvent. Compounds were
diluted 1:50 in 20% DMSO/20% ethanol. For dose-response
experiments, compounds were diluted 1:50 in FLIPR buffer
and serially diluted 1:10 to give a five- or eight-point dose-
response curve.
At the beginning of an experimental run, a signal test was
performed to check the basal fluorescence signal from the dye-
loaded cells and the uniformity of the signal across the plate.
The basal fluorescence was adjusted between 8000 and 12 000
counts by modifying the exposure time, the camera F-stop, or
the laser power. The instrument settings for a typical assay
were as follows: laser power, 0.3-0.6 W; camera F-stop, F/2;
and exposure time, 0.4 s. An aliquot (25 µL) of the test
compound was added to the existing 100 µL dye-loaded cells
at a dispensing speed of 50 µL/s. Fluorescence data were
collected in real-time at 1.0 s intervals for the first 60 s and
at 6.0 s intervals for an additional 120 s. Responses were
measured as peak fluorescence intensity minus basal and
where appropriate were expressed as a percentage of a
maximum 5-HT-induced response.
Acute IOP Response in Conscious Cynomolgus Mon-
keys. Intraocular pressure was determined with an Alcon
pneumatonometer after light corneal anesthesia with 0.1%
proparacaine. Eyes were rinsed with saline after each mea-
surement. After a baseline IOP measurement, test compound
was instilled in one 30 µL aliquot to the ocular hypertensive
eye of eight or nine cynomolgus monkeys. Vehicle was instilled
in the test eyes of five or six additional animals. Subsequent
IOP measurements were taken at 1, 3, and 6 h. A compound
is considered efficacious in hypertensive eyes if there is a
decrease from baseline IOP of at least 20% following topical
administration.
Drug Discrimination Studies. Seven male Sprague-
Dawley rats (Charles River Laboratories), weighing 250-300
g at the beginning of the study, were trained to discriminate
(15-min presession injection interval) 1.0 mg/kg of racemic
1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane HCl (DOM),
obtained as a gift from NIDA, from saline vehicle (sterile 0.9%
saline) under a variable-interval 15-s schedule of reward (i.e.,
Supporting Information Available: Analysis data for
2, 3, 5b, 6a-d, and 7a-d. This material is available free of
charge via the Internet at http://pubs.acs.org.
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