Structure-activity correlations for β-phenethylamines at human trace amine receptor 1

Article abstract of DOI:10.1016/j.bmc.2008.06.009

A cell line in which RD-HGA16 cells were stably transfected with the hTAAR 1 receptor was created and utilized to carry out a systematic evaluation of a series of β-phenethylamines. Fair agreement was observed with data obtained for aryl and ethylene chain substituted analogs in an AV12-664 cell line in which hemagglutinin-tagged hTAAR 1 was stably co-expressed with rat Gα_s. Analogs with multiple substituents as well as analogs with bulky groups were found to be partial agonists. Analogs in which the primary amino group was converted to a secondary or a tertiary amino group by N-methylation were also partial agonists. Comparative Molecular Field Analysis (CoMFA) using the potency data yielded a regression coefficient r² of 0.824. The steric field contribution to the model was 61% with the balance (39%) contributed by the electrostatic field. The collective results suggest that increasing steric bulk both at the amino nitrogen, particularly by N-dimethylation, and at the 4-position of the aromatic ring leads to low efficacy ligands.

Full text of DOI:10.1016/j.bmc.2008.06.009

Bioorganic & Medicinal Chemistry 16 (2008) 7415–7423
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity correlations for b-phenethylamines at human trace
amine receptor 1
*
Anita H. Lewin , Hernán A. Navarro, S. Wayne Mascarella
Center for Organic and Medicinal Chemistry, RTI International, 3040 Cornwallis Road, PO Box 12194, Research Triangle Park, NC 27709-2194, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A cell line in which RD-HGA16 cells were stably transfected with the hTAAR 1 receptor was created and
utilized to carry out a systematic evaluation of a series of b-phenethylamines. Fair agreement was
observed with data obtained for aryl and ethylene chain substituted analogs in an AV12-664 cell line
Received 9 May 2008
Revised 5 June 2008
Accepted 6 June 2008
Available online 13 June 2008
in which hemagglutinin-tagged hTAAR 1 was stably co-expressed with rat Gas. Analogs with multiple
substituents as well as analogs with bulky groups were found to be partial agonists. Analogs in which
the primary amino group was converted to a secondary or a tertiary amino group by N-methylation were
also partial agonists. Comparative Molecular Field Analysis (CoMFA) using the potency data yielded a
regression coefficient r2 of 0.824. The steric field contribution to the model was 61% with the balance
(39%) contributed by the electrostatic field. The collective results suggest that increasing steric bulk both
at the amino nitrogen, particularly by N-dimethylation, and at the 4-position of the aromatic ring leads to
low efficacy ligands.
Keywords:
b-PEA
Trace amine
hTAAR 1
CoMFA
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
of TAs in brain. Thus, it has been suggested that, in addition to
the above-mentioned conditions, TAs may be implicated in psy-
Whether amino acid metabolites such as b-phenethylamine,
p- and m- tyramine, octopamine, and tryptamine, which are pres-
ent at potentially physiologically relevant concentration in the
CNS, have a specific role in brain function has long been a matter
of speculation. These so called ‘trace amines (TAs)’ are known to
have important functions in invertebrates, particularly in insects.
The invertebrate receptors for trace amines, also referred to as
octopamine receptors, are known to belong to the family of G-pro-
tein coupled receptors (GPCRs).1 Four of them have been distin-
guished using pharmacological tools and they have been found to
show different couplings to second messenger systems, including
activation and inhibition of adenylyl cyclase, activation of phos-
pholipase C, and coupling to a chloride channel. Recently, octopa-
mine receptors from mollusks and insects have been cloned. In
humans and other mammals, the existence of receptors for TAs
had, until recently, only been hypothesized, although it has been
suggested that TAs may be involved in diverse conditions including
migraine, depression, Parkinson’s disease, and schizophrenia, as re-
viewed recently.2
chosis, ADHD, substance abuse, eating disorders, and epilepsy.4
Complete identification of all members of this novel GPCR family5
indicated the existence of three distinct subfamilies, with the first
subfamily containing four genes (or pseudogenes), the second con-
taining one gene, and the third containing 11 and 14 genes in
mouse and rat, respectively, but only five genes in chimpanzee
and human. Because not all of these receptors were activated by
TAs, the term TAAR (Trace Amine Associated Receptor) was pro-
posed for this family of receptors.5
The low TAAR densities in native tissue required the develop-
ment of expression systems for screening purposes. Rat (r), mouse
(m), rhesus monkey (rh) and human (h) TAAR 1 have been
cloned3,5–7; rTAAR 1 was stably expressed in HEK 293 cells,5,6 as
were mTAAR 15 and rhTAAR 1.8 Transient transfection has been re-
ported for hTAAR 13 but attempted stable in vitro expression was
frustrated by the lack of pharmacologic response to trace amines.6
This was attributed to intracellular sequestration of TAAR 1 in HEK
293 cells.9 A stable cell line with pharmacological properties
resembling those of mTAAR 1 was achieved by transfection of
HEK 293 cells with a human–rat chimera in which the coding
sequence was modified by the addition of an influenza hemagglu-
tinin viral leader sequence and the N-terminus, C-terminus, and
third intracellular loop were replaced with the corresponding
rTAAR 1 sequences.5 A stable cell line expressing hTAAR 1 in
The identification of a new family of mammalian GPCRs, two of
which are activated by TAs3 and their expression in amygdala, cer-
ebellum, dorsal root ganglia, hippocampus, hypothalamus, medul-
la, and pituitary has rekindled speculation regarding the function
rG
expressed unmodified hTAAR 1 in both CHO-K1 and RD-HGA16
a
sAV-12-664 cells has also been reported.10 We had stably
* Corresponding author. Tel.: +1 919 541 6691; fax: +1 919 541 8868.
E-mail address: ahl@rti.org (A.H. Lewin).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.009

7416
A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
Table 2
cells. The latter cell line was used to successfully create and vali-
date a functional high throughput assay based on the coupling of
the unmodified hTAAR 1 to Gq and the mobilization of internal cal-
cium.11 This assay was used to evaluate a series of b-phenethyl-
amine (b-PEA) analogs at hTAAR 1. The resulting potency data
were utilized in CoMFA 3D-QSAR studies aimed at the develop-
ment of a pharmacophore model of hTAAR 1.
Effect of Multiple Substituents on the Aromatic Moiety of b-PEA on Potency and
Efficacy at h TAAR 1
Compound No.
Substituent/position
2
3
4
5
6
EC50 (nM)
129 13
51
312 68
Emax (%)
1
H
Cl
Cl
F
F
Cl
H
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
OCH3
OH
H
Cl
H
H
Cl
Br
CH3
Et
H
H
H
H
Cl
F
100
94
9
9
26
27
28
29
30
31
32
33
34
35
36
37
38
39
9
94 11
90 12
103 14
H
H
H
H
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
F
F
1,400 680
767 180
1,160 210
3,430a
2. Results
H
H
H
H
H
H
H
H
H
H
84
8
78a
84a
69
The effect of substitution on the aromatic moiety of b-PEA on
both potency and efficacy at hTAAR 1 are shown in Tables 1 and
2. The results are consistent with the data reported by others.5,10
Nearly all of the analogs were partial or full agonists. Substitu-
tion on the ethylene chain of b-PEA indicates that the addition of a
single small substituent such as a methyl group to the b-carbon
(40, 41) is well tolerated, but greater bulk, for example, two methyl
groups (42) or a phenyl substituent, (43) greatly reduces potency
(Table 3). Similar, but somewhat larger, detrimental effects on
377a
2,010 120
3,750 460
7,190 880
6,478a
6,410 1,970
Inactive
3
6
5
30
22
36a
32
9
1
OCH3
-OCH2O-
5,030 4,000
63
a
Single determination.
potency result from introducing an
amphetamine (47, 48); even greater bulk, for example, two
-methyl groups (to give phentermine, 49), sharply reduces
potency. The -substituted compounds have reduced efficacy at
a-methyl group to give
a
Table 3
Effect of substitution on the ethylene chain of b-PEA on potency and efficacy at hTAAR 1
a
hTAAR 1 compared to the parent compound b-PEA (1). Converting
the primary amino group in b-PEA and in some analogs to a sec-
ondary amino group by N-methylation leads to minor (ꢀ3-fold)
reduction in potency, but further N-methylation, to afford the
tertiary amines, reduces potency by a factor of ꢀ30 (Table 4). As
shown in the steric CoMFA field map (Fig. 1) decreased potency
is associated with increased steric bulk essentially all around the
molecular perimeter (yellow colored regions) with the exception
Compound
Substituent
Position
EC50 (nM)
Emax (%)
100
104 10
97
70 14
79 22
1
H
129 13
129 33
361 60
9
40
41
42
43
44
45
46
47
48
49
(S)-CH3
(R)-CH3
(CH3)2
c(CH2)2
i-Pr
Ph
CH2
(S)-CH3
(R)-CH3
(CH3)2
b
b
b
b
b
b
8
7880 3600
5050 100
9950 2040
3400 1000
1510 120
935 260
920 270
5470 1900
46
6
70 11
a
a
a
a
/b
72
74
2
7
of an ortho position,
a-substitution, and N-substitution (green
colored regions). The electrostatic CoMFA filed map (Fig. 2) indi-
cates that negative charge along the ethylene chain is associated
with increased potency (red regions) while positive charge in the
3- and 4-positions of the phenyl ring is associated with increased
potency (blue regions).
64 10
68
4
cells, serves to validate both datasets. As observed by evaluating
cAMP accumulation in rG sAV12-664 cells stably expressing
a
3. Discussion
hTAAR 110 our data also show the positional aryl substituent ef-
fects on potency, that is, 2 > 3 > 4 for each substituent with the
exception of hydroxyl (–OH), where the potencies are essentially
the same at all positions. In addition, any substituent at either
the 4- or the 3-position of b-PEA serves to reduce potency at hTAAR
1. Some substituents at the 2-position, in particular, 2-halo (F (2),
The good correspondence between our results, obtained by
determining calcium flux in CHO cells stably expressing Ga16(RD-
HGA16 cells; Molecular Devices Corporation, Sunnyvale, CA) and
hTAAR 1,11 and the literature data10 determined in rG
asAV12-664
Table 1
Effect of single substituents on the aromatic moiety of b-PEA on potency and efficacy at hTAAR 1
Substituent
Position
2
EC50 (nM)
129 13
3
4
No.
Emax (%)
No.
EC50 (nM)
Emax (%)
No.
EC50 (nM)
Emax (%)
H
F
Cl
Br
1
2
5
8
11
12
100
91
72
9
2
1
9
1
3
6
9
129 13
150 13
189 20
398 114
100
93
9
2
1
4
7
129 13
100
81
104
98
9
3
7
7
65
77
2
5
740 210
2900 1000
1540 400
105 17
75
57 15
89
5
10
I
103a
109a
97
CH3
Et
355 76
7
13
638 160
93 12
14
15
16
19
22
24
25
4380 300
9100a
96
86a
ND
1
t-Bu
OCH3
OH
CF3
NO2
>50,000b
5980 580
731 220
11,600a
17
20
144 13
614 150
95
1
18
21
23
1444 25
1740 50
3950a
73
78
4
9
106
2
90 10
91 20
78a
NDd
69a
>50,000c
a
Single determination.
b
c
5,112,000 nM was the experimentally determined value used in the CoMFA.
167,386 nM was the experimentally determined value used in the CoMFA.
ND, not determined.
d

A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
7417
Table 4
Effect of N-methyl substituents of b-PEA on potency and efficacy at hTAAR 1
Compound
EC50 (nM)
Emax (%)
R = H
R0 = H
R = H
R0 = CH3
R = CH3
R0 = CH3
R = H
R0 = H
R = H
R0 = CH3
R = CH3
R0 = CH3
Structure
R
N
1
50
51
1
100
50
51
R'
R'
129 13
387 106a,b
100,000 50,000c
9
69 4a,b
64 7c
R
N
2
65
52
117 23
53
2
91
52
75
53
75
2
1290 400
2
2
3
1
2
3
9
5
4
F
R
N
F
3
54
530 128
55
3
93
54
88 15
55
78
R'
R'
150 13
4320 1800
R
N
4
56
57
4
81
56
63
57
71
740 213
1040 180
19,100 3000
6
6
5
6
9
9
5
F
R
N
5
77
58
160 38
59
687 210
5
72
58
70
59
79
R'
5
Cl
R
Cl
N
6
60
501 70
61
6
60
77
61
62 20
R'
189 19
5980 1280
105 17
R
N
7
62
63
7
104
62
67
63
43
R'
2860 1000
7430 2600
630,000 300,000
7
7
7
Cl
R
N
8
64
205 66
65
8
89
64
93
65
78
R'
57 15
3460 1200
9
5
7
Br
R
N
Br
9
66
67
9
75
66
71
67
88 10
R'
R'
398 114
1070 300
8300 2000
R
N
10
68
69
10
98
68
60
69
40
1540 400
6540 2300
24,400 6000
8
Br
a
EC50 = 160 40 nM; Emax = 100%.5
b
c
EC50 = 250 45 nM; Emax = 93.4 7.4%.10
EC50 = 1490 120 nM; Emax = 97.1 5.2%.10
Cl (5), Br (8) and I (11)), afford analogs that more potently activate
hTAAR 1 than the endogenous ligand b-PEA, while analogs 2-
substituted with a methyl (12) group show lower potency than
b-PEA. Analogs of b-PEA with multiple aromatic substituents exhi-
bit potencies that essentially represent the sum total of the effects
of individual substituents on potency to activate hTAAR 1. Thus, for
example, the EC50 value for 2,5-dimethoxy-b-PEA (33)
(2010 120 nM, see Table 2) is close to the sum of the EC50 values
of 2-methoxy-b-PEA (17) (144 13 nM, Table 1) and 3-methoxy-b-
PEA (18) (1444 259 nM, Table 1) and addition of a 4-chloro-(7) or
4-methyl (14) substituent (EC50 = 2900 1000, 4380 300 nM,
respectively, see Table 1) increases the EC50 by 2158 and 4890,
respectively. Addition of a 4-methoxy substituent (19), known to
exert a large negative effect on potency (EC50 = 5980 580 nM,
Table 1), leads to an inactive compound (38) (see Table 2).
Conversely, the negative effect of a 4-methoxy (19) substituent
on potency to activate hTAAR 1 is modulated by the enhancing
effect of a 2-fluoro (2) substituent (EC50 = 65 2 nM, Table 1) to
afford an EC50 = 1400 680 nM for 2,6-difluoro-4-methoxy-b-PEA
(28) (Table 2).
The most striking outcome of the CoMFA evaluation of this set
of analogs is that the steric constraints associated with hTAAR 1
binding are very severe, suggesting that analogs with large N-sub-
stituents may align differently from analogs with large substitu-
ents in the meta and/or para position. This may account for the
reduced potencies and efficacies (partial agonists) observed for
these analogs at hTAAR 1. These observations are also consistent
with the literature data10 determined for cAMP accumulation in
rG
a AV12-664 cells.
r
The effect of b-substitution on potency to activate hTAAR 1
(Table 3) is somewhat deleterious, but, interestingly, appears to
be stereospecific. Thus, in both cell lines (CHO-G and
sAV12-664) the S-enantiomer 40 is essentially equally as
a16
rGa
potent as b-PEA (1) while the R-enantiomer 41 is some 3-fold less
potent; both enantiomers are full agonists. By contrast, Wainscott
et al.10 claim stereospecificity for the effect of
a-substitution by a

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A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
but may be confounded by the fact that even larger differences
in potency between (S)- and (R)-amphetamine are observed in
HEK-293 cells heterologously and stably expressing rTAAR 1. Tran-
ylcypromine (49) a hybrid of
a and b substitution that is some 10-
fold less potent than b-PEA, also appears to be a partial agonist in
our calcium flux assay, but not in the cAMP assay in rG
cells.10
asAV12-664
The effects of N-methylation (Table 4), taken together with ef-
fects of aromatic substitution (Tables 1 and 2) are consistent with
the idea that b-PEA analogs with bulky substituents are poorly
accommodated due to steric constraints, as shown in the CoMFA
maps (Figs. 1 and 2). This results in decreased potency and effi-
cacy. Thus, b-PEA analogs with large single substituents, particu-
larly in the 4-position of the aromatic ring, as in 4-t-butyl b-PEA
(16) and 4-nitro-b-PEA (25) (Table 1), and with multiple aromatic
substituents, as in the series of 2,5-dimethoxy-b-PEA analogs (33,
38) (Table 2), have reduced efficacy. Increasing bulk at the amino
end of the molecule by N-methylation and N-dimethylation (Ta-
ble 4) has a profound effect on the potency of b-PEA with mini-
mal effect on efficacy. Increasing steric bulk at both the amino
nitrogen, particularly by N-dimethylation, and at the para posi-
tion of the aromatic ring, profoundly affects both potency and
efficacy.
Figure 1. Contour plot of the CoMFA model steric fields (StdDev * Coeff). The region
of favorable steric field interaction is indicated by green contours, while the region
of unfavorable steric interaction is indicated by yellow contours. b-PEA is shown for
reference.
4. Experimental
4.1. Chemicals
Derivativesand analogsof b-PEA were obtained fromcommercial
sources, from theNIDA ResearchTechnology Branch(Rockville, MD),
or by synthesis. Specifically, compounds that were purchased from
Sigma–Aldrich (St. Louis, MO) were as follows: 2-phenylethylamine
(1), 2-(2-fluorophenyl)ethylamine (2), 2-(3-fluorophenyl)ethyla-
mine (3), 2-(4-fluorophenyl)ethylamine (4), 2-(2-chlorophenyl)eth-
ylamine (5), 2-(3-clorophenyl)ethylamine (6) hydrochloride, 2-(4-
clorophenyl)ethylamine (7), 2-(2-bromopheneyl)ethylamine (8),
2-(3-bromophenyl)ethylamine (9), 2-(4-bromophenyl)ethylamine
(10), 2-(2-methylphenyl)ethylamine (12), 2-(3-methylphenyl)eth-
ylamine (13), 2-(4-methylphenyl)ethylamine (14), 2-(4-ethylphe-
nyl)ethylamine
(15)
hydrochloride,
2-(2-methoxyphenyl)
ethylamine (17), 2-(3-methoxyphenyl)ethylamine (18), 2-(4-
methoxyphenyl)ethylamine (19) hydrochloride, 2-(3-hydroxy-
phenyl)ethylamine (21) hydrochloride, 2-(4-hydroxyphenyl)ethyl-
amine (22), 2-(3-trifluoromethylphenyl)ethylamine (23), 2-(4-
trifluoromethyphenyl)ethylamine (24), 2-(4-nitrophenyl)ethyla-
mine (25), 2-(2,6-dichlorophenyl)ethylamine (26), 2-(2,6-difluoro-
4-methoxyphenyl)ethylamine (28) hydrochloride, 2-(2,3,-dichloro-
phenyl)ethylamine (30) hydrochloride, 2-(3,4,-dichlorophenyl)eth-
ylamine (31) hydrochloride, (S)-(ꢁ)-2-phenylpropylamine (40), (R)-
(+)-2-phenylpropylamine (41), 2,2-diphenylethylamine (45), trans-
2-phenylcyclopropylamine (46), N-methyl-2-phenylelthylamine
(50), N,N-dimethyl-2-phenylethylamine (51). The following two
compounds were purchased from Acros Organics (Gee, Belgium):
2-(2-chloro-6-fluorophenyl)ethylamine (27) hydrochloride and
2-(2,5-dimethylphenyl)ethylamine (32) hydrochloride; 2-(4-t-Bu-
phenyl)ethylamine (16) hydrochloride was purchased from ABCR
(Karlsruhe, Germany). Compounds obtained from the NIDA
ResearchTechnologyBranch (Rockville, MD) are: 2-(2,5-dimethoxy-
Figure 2. Contour plot of the CoMFA model electrostatic fields (StdDev * Coeff). The
region of favorable electrostatic interaction with external positive charge is
indicated by a blue contour, while regions of favorable interaction with external
negative charge are indicated by red contours. b-PEA is shown for reference.
methyl group on potency to activate hTAAR 1 by evaluating cAMP
accumulation in rG
asAV12-664 cells, with (R)-amphetamine (48)
being the less potent isomer, whereas our data, obtained by deter-
mining calcium flux in CHO cells stably expressing G 16and hTAAR
a
1, do not show this effect. Considering the magnitude of the error
bars associated with the two sets of data, there may in fact not be a
significant difference between them. At the same time, 3-fold
greater potency was reported for (S)-amphetamine (47) relative
to (R)-amphetamine (48) in HEK-293 cells heterologously and sta-
bly expressing h-rChTAAR 1 (human–rat chimera).12 However, the
significantly larger difference in potency observed between b-PEA
and p-tyramine (22) in the h-rChTAAR 1 cell line12 suggests that,
as has been noted,10 these effects may be due to the distinct spe-
cies differences observed for TAAR 1.
phenyl)ethylamine
(33),
2-(4-chloro-2,5-dimethoxyphenyl)
ethylamine (34), 2-(4-bromo-2,5-dimethoxyphenyl)ethylamine
(35), 2-(2,5-dimethoxy-4-methylphenyl)ethylamine (36), 2-(2,5-
dimethoxy-4-ethylphenyl)ethylamine (37), 2-(2,5-dimethoxy-4-
methoxylphenyl)ethylamine (38) hydrochloride, (S)-(+)-amphet-
amine (47) hydrochloride, (R)-(ꢁ)-amphetamine (47) hydrochlo-
ride, phentermine (49) hydrochloride. The remaining compounds
were synthesized and characterized as described below:
While stereospecific effects of
a-substituents on potency are
questionable, the effects on efficacy to activate hTAAR 1 may be
stereospecific. Our data are not strong in that regard, but this con-
clusion is supported by the agreement between our data and the
literature data10 for amphetamine (47, 48). Data obtained in the
h-rChTAAR 1 cell line12 are also consistent with this conclusion,

A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
7419
4.2. Synthesis
filtered through Celite, rinsed with CH3OH, and then concentrated
to a white solid. The HCl salt was generated using ethereal HCl
(ꢀ10 mL) to give a white solid (51 mg, 12%). Mp 187 °C; MS
(ESI): m/z calcd for C9H11F3N: 189.18, found (M+1): 190.4. Anal.
calcd for C9H11ClF3N: C, 47.91; H, 4.91; N, 6.21. Found: C, 47.7;
H, 4.83; N, 6.15. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.01 (s,
4H, CH2CH2), 7.51 (d, J = 7.8 Hz, 2H, ArH), 7.70 (d, J = 7.5 Hz, 2H,
ArH). 13C NMR (DMSO-d6, 75.5 MHz) d (ppm): 30.81, 38.10, 122.3
(q, JCF = 271.8 Hz, CF3), 123.6 (q, JFCCC = 3.77 Hz, ArC-3), 125.7 (q,
JFCC = 31.98 Hz, ArC-4), 127.9, (ArC-2), 140.8 (ArC-1).
Melting points were determined on either a Thomas-Hoover
capillary tube apparatus or on a Fisher-Johns Model 12-144 melt-
ing point apparatus. Thin layer chromatography was carried out
using Merck silica gel 60F254 TLC plates or Baker-flex silica gel
1B-F flexible sheets; visualization was under UV or in an iodine
chamber, as appropriate. Flash chromatography was conducted
on silica gel 60 (40 lM). Nuclear magnetic resonance (NMR) spec-
tra were determined on a Bruker Avance 300 MHz NMR spectrom-
eter. Mass spectra were obtained using a Perkin-Elmer Sciex API
150EX mass spectrometer outfitted with an APCI source. Microa-
nalyses were carried out by Atlantic Microlab, Inc.
4.2.4. 2-(2,6-Difluoro-4-hydroxyphenyl)ethylamine (29)
hydrochloride
A suspension of 2-(2,6-difluoro-4-methoxyphenyl)ethylamine
(28) HCl (132 mg, 0.630 mmol) in 48% HBr (2 mL) was heated at
145 °C for 1.5 h and then concentrated in vacuo to a pale orange
residue that was recrystallized twice from EtOH/ether. Mp
254 °C; MS (ESI): m/z calcd for C8H9F2NO: 173.0, found (M+1):
174.0. Anal. calcd for C8H10ClF2NO: C, 37.82; H, 3.97; N, 5.51.
Found: C, 37.87; H, 3.88; N, 5.43. 1H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.89–2.9 (m, 2H, CH2), 2.82–2.84 (m, 2H, CH2), 6.48 (d,
JHF = 21 Hz, 2H, ArH); 13C NMR (DMSO-d6, 75.5 MHz) d (ppm):
19.2, 37.7, 98.6 (d, JCCF = 27.6 Hz, ArC-3,5,) 101.8 (t, JCF = 21.1 Hz,
ArC-1), 157.8 (t, JFCCC = 15.1 Hz, ArC-4) 160.8 (dd, JCF = 241.6 Hz,
JCCF = 12.5 Hz, ArC-2,6).
4.2.1. 2-(2-Iodophenyl)ethylamine (11) hydrochloride
To a solution of (2-iodophenyl)acetonitrile (2.0 g, 0.008 mol) in
anhydrous THF (10 mL), BH3 THF (1.0 M, 20 mL, 20 mmol) was
slowly added. After refluxing for 16 h, the reaction mixture was
chilled to 0 °C and CH3OH (10 mL) was added dropwise. The result-
ing mixture was concentrated, CH3OH (15 mL) was added, and the
volatiles were removed in vacuo. The residue was treated with HCl
(0.5 N, 30 mL) and the solution was refluxed for 1 h. After cooling
to room temperature the mixture was washed with diethyl ether.
The aqueous phase was basified to pH 14 with 30% NaOH then ex-
tracted with ether (3ꢂ 10 mL). The combined extract was dried
over Na2SO4 and concentrated. The HCl salt was prepared by add-
ing 6% ethanolic HCl until no further precipitation occurred. The
precipitated solid was collected by filtration and recrystallized
from CH3OH/ether to yield 1.68 g (72%). Mp 213 °C [lit.13 251–
255 °C]; MS (ESI): m/z calcd for C8H11IN: 246.99, found (M+1):
248.10. Anal. calcd C8H10ClIN: C, 33.89; H, 3.91; N, 4.94. Found:
C, 34.01; H, 3.93; N 4.86. 1H NMR (DMSO-d6,300 MHz) d (ppm):
2.99 (s, 4H, CH2CH2), 7.8–7.3 (4H, ArH). 13C NMR (DMSO-
4.2.5. 2-Methyl-2-phenylpropylamine (42) hydrochloride
To a chilled solution of 1,1-dimethylbenzonitrile (145.13 mg,
1 mmol) and cobalt (II) chloride (237.9 mg, 1 mmol) in EtOH
(10 mL) was added NaBH4 (75.66 mg, 2 mmol) in portions. A pre-
cipitate was formed. After stirring the suspension for 3 h under
N2, it was poured into chilled 3 N HCl (5 mL) and then washed with
diethyl ether (2ꢂ 20 mL). The aqueous phase was basified with 20%
NaOH (pH 14) and extracted with diethyl ether (3ꢂ 30 mL). The
combined extract was dried over Na2SO4, filtered, and concen-
trated to an oil. The HCl salt was made using 6% methanolic HCl
(10 mL). The recovered solid was recrystallized from CH3OH/ether
to yield 28.5 mg (15%) of a white solid. Mp 204 °C; MS (ESI): calcd
for C10H15N: 149.1, found (M+1): 150.5. Anal. calcd for C10H16ClN:
C, 64.68; H, 8.68; N, 7.54. Found: C, 63.91; H, 8.58; N, 7.61. 1H NMR
(CDCl3, 300 MHz) d (ppm): 1.3 (s, 3H, CH3), 2.7 (s, 2H, CH2), 7.3 (s,
5H, ArH). 13C NMR (CDCl3, 75.5 MHz) d (ppm): 26.3, 37.3, 51.1,
125.9, 127.1, 128.9, 144.1.
d6,75.5 MHz)
d (ppm): 37.36, 37.67, 100.87, 128.84, 139.03,
129.37, 129.57, 129.97, 139.35, 139.74, 140.15.
4.2.2. 2-(2-Hydroxyphenyl)ethylamine (20) hydrochloride
A solution of 2-(2-methoxyphenyl)ethylamine (17) (484 mg,
3.30 mmol) in HCl (12 N, 0.7 mL) was placed in a thick walled micro-
wave test tube, covered with the test tube lid, lowered into the
microwave apparatus (CEM Explorer Microwave) and microwaved
for 10 min (83 psi, 160 °C). Reaction progress was followed by TLC
(90/10/1); CHCl3:CH3OH:NH4OH. The reaction was complete after
a total of 1 mL HCl was added and the heat increased to 170 °C.
The resulting mixture was chilled to 0 °C, basified with NaOH (pH
ꢀ10–1), and extracted with EtOAc (3ꢂ 20 mL). The combined organ-
ic extract was washed with H2O (30 mL) and saturated NaCl (30 mL)
then dried over Na2SO4. The solid remaining after evaporation of the
volatiles was taken up in EtOAc (5 mL) and treated with 6% ethanolic
HCl (10 mL). After freezing overnight (0 °C), the precipitated pink
solid was collected by filtration (133 mg, 23%). Mp 154 °C [lit.14
152–154 °C]; MS (ESI): m/z calcd for C8H11NO: 137.08, found
(M+1): 138.4. Anal. calcd for C8H12ClNO: C, 55.34; H, 6.97; N, 8.07.
Found: C, 55.12; H, 6.90; N, 7.97. 1H NMR (DMSO-d6, 300 MHz) d
(ppm): 2.80–2.83 (m, 2H, CH2), 2.85–2.92 (m, 2H, CH2), 6.71–6.76
(m, 1H, ArH), 6.85–6.88 (m, 1H, ArH), 7.04–7.09 (m, 2H, ArH). 13C
NMR (DMSO-d6, 75.5 MHz) d (ppm): 28.53, 38.84, 115.49, 119.33,
123.78, 128.15, 130.45, 155.80.
4.2.6. 1-Phenylcyclopropanemethylamine (43) hydrochloride
A
solution of 1-phenyl-1-cyclopropylacetonitrile (1 g,
0.007 mol) in anhydrous THF (10 mL) was added to a chilled solu-
tion of LAH (1.0 M in THF, 6.98 mL, 6.98 mmol). A precipitate was
formed. After stirring overnight under N2 at room temperature
the reaction mixture was poured over ice and 3 N HCl and ex-
tracted with ether (2ꢂ 10 mL). The aqueous phase was basified
to pH 14 with 30% NaOH, then extracted with ether (3ꢂ 10 mL),
dried over Na2SO4 and concentrated. The residual oil was treated
with 6% methanolic HCl. Evaporation of the solvent afforded a solid
that was recrystallized twice from CH3OH/ether (336 mg, 26%). Mp
176–177 °C [lit.15 175 °C]; MS (ESI): m/z calcd for C10H13N: 147.2,
found (M+1): 148.4. Anal. calcd C10H14ClN: C, 65.39; H, 7.68; N,
7.63. Found: C, 65.39; H, 7.84; N, 7.58. 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.03–1.12 (m, 4H, CH2CH2) 3.07 (s, 2H, CH2N), 7.2–7.4
(s, 5H, ArH). 13C NMR (CDCl3, 75.5 MHz) d (ppm): 13.0, 24.6,
49.4, 128.1, 129.3, 129.9, 140.4.
4.2.3. 2-(4-Trifluoromethylphenyl)ethylamine (24)
hydrochloride
To a solution of 4-(trifluromethylphenyl)acetonitrile (423 mg,
2.28 mmol), in methanolic ammonia (2 N, 10 mL), in a 250 mL Parr
flask was added a scoop of Raney Nickel (in water). The resulting
mixture was shaken overnight under hydrogen (50 psi), then
4.2.7. 3-Methyl-2-phenylbutylamine (44) hydrochloride
A
solution of 3-methyl-2-phenylbutyronitrile (500 mg,
3.14 mmol) in anhydrous THF (6 mL) was added dropwise to a
chilled solution of LAH in THF (1.0 M, 3.46 mL, 3.46 mmol). After

7420
A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
heating at 50 °C for 16 h the reaction mixture was slowly added to
chilled 3 N HCl, then washed with diethyl ether (3ꢂ 30 mL). The
aqueous phase was basified with chilled NaOH (pH 14) and ex-
tracted with diethyl ether (3ꢂ 30 mL). The combined organic ex-
tract was dried over Na2SO4, filtered, and then concentrated. The
HCl salt was generated by stirring the residue in methanolic HCl,
then concentrating the solution to a white solid that was recrystal-
lized from CH3OH/ether (202 mg, 32%). Mp 190–191 °C; MS (ESI):
m/z calcd for C11H17N: 163.1, 165.5, found (M+1): 164.4, 166.5.
Anal. calcd for C11H18ClN (+.13 mol H2O): C, 65.39; H, 9.11; N
6.93, Found: C, 65.39; H, 9.11; N, 6.95. 1H NMR (300 mHz,
DMSO-d6) d (ppm): 0.6 (d, 3H CH3), 0.88 (d, 3H, CH3), 1.9 (m, 1H,
CHCH (CH3)2), 2.79 (d, 1H, CHCH CH2) 3.1–3.2 (m, 2H, CH2),
7.25–7.45 (m, 5H, ArH). 13C NMR (DMSO-d6) d (ppm): 17.6, 19.7,
21.0, 30.7, 41.5, 49.9, 126.8, 128.3, 129.1, 139.6.
115.1, 115.4, 123.9, 124.0, 127.1, 127.4, 127.7, 127.8, 130.8,
130.9, 159.6, 162.8.
4.2.10. N-Methyl-2-(3-fluorophenyl)ethylamine (54)
hydrochloride
The free base 54 was prepared from 2-(3-fluorophenyl)ethyla-
mine (3) by a slight modification of Method A. Specifically, workup
of the borohydride reaction was by treatment of the cold reaction
mixture with H2O (15 mL), stirring at 0 °C for 5 min, then mixing
with NaOH (10%, 3 mL), stirring at 0 °C for 30 min, and extracting
with EtOAc (3ꢂ 15 mL). The combined extract was washed with
brine (3ꢂ 10 mL), dried over Na2SO4, concentrated in vacuo, leav-
ing a colorless oil (1.037 g, 94%). The crude product (906 mg,
5.91 mmol) was purified using flash silica gel chromatography,
eluting with hexane/EtOAc, to give a colorless oil (256 mg, 28%).
The hydrochloride salt was prepared as described in Method A.
Recrystallization from CH3OH/EtOAc (1:16) gave pure 54ꢃHCl. Mp
158–159 °C; MS (ESI): m/z calcd for C9H12FN: 153.2, found:
154.3 (M+1). Anal. calcd for C9H13ClFN: C, 57.00; H, 6.91; Cl,
18.69; F, 10.02; N, 7.39. Found: C, 56.77; H, 6.87; Cl, 18.94; F,
9.78; N, 7.34. 1H NMR (CD3OD, 300 MHz) d (ppm): 2.70 (s, 3H,
CH3), 3.03–2.97 (m, 2H, CH2), 3.30–3.22 (m, 2H, CH2), 7.11–6.97
(m, 3H, ArH-4,5,6), 7.38–7.31 (m, 1H, ArH-2). 13C NMR (CD3OD,
75.5 MHz) d (ppm): 33.2, 33.3, 34.1, 51.4, 115.3, 115.6, 116.9,
117.1, 126.0, 126.1, 132.1, 132.3, 140.7, 140.8, 163.3, 166.5.
4.2.8. N-Methyl-2-(2-fluorophenyl)ethylamine (52)
hydrochloride (Method A)
A
mixture of 2-(2-fluorophenyl)ethylamine (2) (1.066 g,
0.0077 mol), ethyl formate (24 mL, large excess), and formic acid
(3 drops) was refluxed under N2 for 5 h. The reaction mixture was
concentrated in vacuo, leaving an oil (1.339 g, 0.008 mol, 104%).
The bulk of the formamide intermediate (1.323 g, 0.0079 mol) was
dissolved in THF (anhydrous, 5 mL), cooled to 0 °C, mixed with bor-
an–tetrahydrofuran complex (1 M, 30 mL, 30 mmol). The mixture
was refluxed under N2 for 2 h. The reaction solution was cooled to
0 °C, mixed slowly with 10% methanolic HCl (6 mL), and refluxed
under N2 for 2 h. Removal of the volatiles afforded a brown solid
(3.386 g) that was mixed with H2O (20 mL) and 3 N HCl (8 mL),
washed with EtOAc (3ꢂ 10 mL), basified to pH 14 with NaOH
(10 N, 3 mL) and extracted with EtOAc (3ꢂ 15 mL). The combined
extract was washed with brine (3ꢂ 10 mL), dried over Na2SO4, con-
centrated in vacuo, leaving a colorless oil (518 mg, 3.38 mmol, 43%).
The crude product (510 mg, 3.33 mmol) was dissolved in 10% meth-
anolic HCl (6 mL) and the solution was refluxed under N2 for 2 h,
then concentrated in vacuo to give a light yellow solid (682 mg).
Recrystallization from MeOH/EtOAc (30:1) gave light yellow crys-
tals (308 mg). A solution of these crystals (300 mg) in 10% methano-
lic HCl (5 mL) was filtered through Celite then concentrated in
vacuo to give 52ꢃHCl as a white solid (290 mg, 22%). Mp 152–
153 °C; MS (ESI): m/z calcd for C9H12FN: 153.2, found (M+1):
154.1. Anal. calcd for C9H12FNꢃHCl: C, 57.00; H, 6.91; Cl, 18.69; F,
10.02; N, 7.39. Found: C, 57.01; H, 6.89; Cl, 18.91; F, 10.01; N,
7.49. 1H NMR (CD3OD, 300 MHz) d (ppm): 2.76 (s, 3H, CH3), 3.07–
3.19 (m, 2H, CH2), 3.25–3.34 (m, 2H, CH2), 7.11–7.19 (m, 2H, ArH),
7.33–7.39 (m, 2H, ArH). 13C NMR (CD3OD, 75.5 MHz) d (ppm):
27.2, 27.3, 34.2, 50.5, 116.8, 117.1, 124.7, 124.9, 126.2, 126.3,
130.9, 131.0, 132.6, 132.7, 161.4, 164.6.
4.2.11. N,N-Dimethyl-2-(3-fluorophenyl)ethylamine (55)
hydrochloride
Prepared from 2-(3-fluorophenyl)ethylamine (3) following
Method B. The HCl salt was prepared by dissolving a portion of
the oil (379 mg, 2.27 mmol) in CH3OH (2 mL), followed by addition
of 1 M HCl in Et2O (3 mL). The solvents were evaporated and the
product was obtained as white solid (409 mg, 89%). Mp 162–
163 °C; MS (ESI): m/z calcd for C10H14FN: 167.2, found (M+1):
168.5. Anal. calcd for C10H15ClFN: C, 58.97; H, 7.42; Cl, 17.41; F,
9.33; N, 6.88. Found: C, 58.96; H, 7.35; Cl, 17.42; F. 9.23; N, 6.79.
1H NMR (CD3OD, 300 MHz) d (ppm): 2.97 (s, 6H, (CH3)2), 3.15–
3.01 (m, 2H, CH2), 3.44–3.31 (m, 2H, CH2), 7.06–7.00 (m, 1H, ArH-
6), 7.33 (m, 1H, ArH-2), 7.18–7.12 (m, 2H, ArH-4,5). 13C NMR
(CD3OD, 75.5 MHz) d (ppm) 31.8, 44.1, 59.8, 115.3, 115.6, 117.0,
117.3, 126.21, 126.24, 132.1, 132.2, 140.5, 140.6, 163.3, 166.5.
4.2.12. N-Methyl-2-(4-fluorophenyl)ethylamine (56)
hydrochloride
Prepared from 2-(4-fluorophenyl)ethylamine (4) using a slight
modification of Method A. Specifically, for the workup the reaction
mixture was cooled to 0 °C then mixed slowly with 10% hydrogen
chloride methanol solution (12 mL). The mixture was refluxed un-
der N2 for 2 h, and concentrated in vacuo. Pure HCl salt (662 mg,
white solid, 46%) was obtained by recrystallization from CH3OH/
EtOAc (15/1). Mp 163–164 °C; MS (ESI): m/z calcd for C9H12FN:
153.2, found (M+1): 153.7. Anal. calcd for C9H13ClFN: C, 57.00; H,
6.91; Cl, 18.69; F, 10.02; N, 7.39. Found: C, 57.02; H, 6.91; Cl,
18.59; F, 10.10; N, 7.37. 1H NMR (CD3OD, 300 MHz) d (ppm):
2.73 (s, 3H, CH3), 2.95–3.03 (m, 2H, CH2), 3.22–3.33 (m, 2H, CH2),
7.06–7.13 (m, 2H, ArH-2,6), 7.31–7.36 (m, 2H, rH-3,5), 13C NMR
(CD3OD, 75.5 MHz) d (ppm): 32.8, 34.1, 51.8, 116.9, 117.2, 131.9,
132.1, 133.9, 134.0, 162.3, 165.5.
4.2.9. N,N-dimethyl-2-(2-fluorophenyl)ethylamine (53)
(Method B)
To
a cooled solution of 2-(2-fluorophenyl)ethylamine (2)
(1.066 g, 0.0077 mol) in formic acid (1.4 mL, 96%, 35 mmol) was
added formaldehyde (1.6 mL, 37%, 21.3 mmol) and the solution
was refluxed under N2 for 16 h. After cooling to 0 °C, HCl (3 N,
15 mL) was added and the mixture was washed with EtOAc (3ꢂ
10 mL). The aqueous phase was basified to pH 14 with NaOH
(10 N, 5 mL) then extracted with EtOAc (3ꢂ 15 mL). The combined
extract was washed with brine (3ꢂ 15 mL), dried over Na2SO4, and
concentrated in vacuo, leaving a colorless oil (814 mg, 63%); MS
(ESI): m/z calcd for C10H14FN: 167.2, found (M+1): 168.3. Anal.
calcd for C10H14FN: C, 71.82; H, 8.44; F, 11.36; N, 8.38. Found: C,
71.59; H, 8.50; F, 11.09; N, 8.32. 1H NMR (CDCl3, 300 MHz) d
(ppm): 2.30 (s, 6H, (CH3)2), 2.50–2.61 (m, 2H, CH2), 2.79–2.84 (m,
2H, CH2), 7.00–7.08 (m, 2H, ArH), 7.16–7.21 (m, 2H, ArH), 13C
NMR (CDCl3, 75.5 MHz) d (ppm): 27.48, 27.50, 45.4, 59.8, 59.9,
4.2.13. N,N-Dimethyl-2-(4-fluorophenyl)ethylamine (58)
hydrochloride
Prepared from 2-(4-fluorophenyl)ethylamine (4) using Method
B. The pure HCl salt (331 mg, pale yellow solid, 46%) was obtained
by recrystallization from CH3OH/EtOAc/Et2O (1:10:30). Mp 146–
147 °C; MS (ESI): m/z calcd for C10H14FN: 167.2, found (M+1):
168.2. Anal. calcd for C10H14FNꢃHCl: C, 58.97; H, 7.42; Cl, 17.41;

A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
7421
F, 9.33; N, 6.88. Found: C, 58.76; H, 7.38; Cl, 17.62; F, 9.51; N, 6.83.
1H NMR (CD3OD, 300 MHz) d (ppm): 2.97 (s, 6H, (CH3)2), 3.11–3.12
(m, 2H, CH2), 3.32–3.42 (m, 2H, CH2), 7.06–7.12 (m, 2H, ArH-2,6),
zation from CH3OH/EtOAc (1:30). Mp 137–138 °C; MS (ESI): m/z
calcd for C9H12ClN: 169.7, found (M+1): 170.3. Anal. calcd for
C9H13Cl2N: C, 52.45; H, 6.36; Cl, 34.40; N, 6.80. Found: C, 53.22;
H, 6.48; Cl, 33.17; N, 6.85. 1H NMR (CD3OD, 300 MHz) d (ppm):
2.76 (s, 3H, CH3), 3.07–3.19 (m, 2H, CH2), 3.25–3.34 (m, 2H, CH2),
7.11–7.19 (m, 2H, ArH), 7.33–7.39 (m, 2H, ArH). 13C NMR (CD3OD,
75.5 MHz) d 27.2, 27.3, 34.2, 50.5, 116.8, 117.1, 124.7, 124.9, 126.2,
126.3, 130.9, 131.0, 132.6, 132.7, 161.4, 164.6.
7.35–7.40 (m, 2H, ArH-3,5), 13C NMR (CD3OD, 75.5 MHz)
d
(ppm): 31.4, 44.0, 60.1, 116.9, 117.2, 132.1, 132.2, 133.7, 133.8,
162.3, 165.5.
4.2.14. N-Methyl-2-(2-chlorophenyl)ethylamine (58)
hydrochloride
The free base 58 was prepared from 5 following a slight modifica-
tion of Method A. Specifically, the workup involved mixing the
cooled reaction mixture with H2O (15 mL) stirring at 0 °C for
5 min, then mixing with NaOH (10%, 3 mL), stirring at 0 °C for
30 min, and extracting with EtOAc (3ꢂ 15 mL). The combined ex-
tract was washed with brine (3ꢂ 10 mL), dried over Na2SO4, concen-
trated in vacuo, leavinga colorlessoil (1.265 g, 97%). The HCl salt was
prepared by refluxing a solution of 58 in 10% methanolic hydrogen
chloride (10 mL) under N2 for 3 h and then concentrated in vacuo.
The residual light yellow solid was mixed with CH3OH (3 mL) and fil-
tered through a Celite pad. Pure HCl salt (209 mg, white solid, 30%)
was obtained by recrystallization from CH3OH/EtOAc (1:20). Mp
137–138 °C; MS (ESI): m/z calcd for C9H12ClN: 169.7, found (M+1):
170.3. Anal. calcd for C9H13Cl2N: C, 52.45; H, 6.36; Cl, 34.40; N,
6.80. Found: C, 52.43; H, 6.41; Cl, 34.15; N, 6.86. 1H NMR (CD3OD,
300 MHz) d (ppm): 2.64 (s, 3H, CH3), 3.03–3.20 (m, 2H, CH2CH2),
7.17–7.23 (m, 2H, ArH), 7.29–7.34 (m, 2H, ArH). 13C NMR (CD3OD,
75.5 MHz) d (ppm): 27.2, 27.3, 34.2, 50.5, 116.8, 117.1, 124.7,
124.9, 126.2, 126.3, 130.9, 131.0, 132.6, 132.7, 161.4, 164.6.
4.2.17. N,N-Dimethyl-2-(3-chlorophenyl)ethylamine (61)
hydrochloride
The free base 61 (726 mg, 3.95 mmol, 55%) was prepared from 5
following Method B. Pure HCl salt (487 mg, pale yellow solid) was
obtained by recrystallization from CH3OH/EtOAc (1/20). Mp 147–
148 °C; MS (ESI): m/z: calcd for C10H14ClN: 183.4, found (M+1):
184.3. Anal. calcd for C10H15Cl2N: C, 54.56; H, 6.87; Cl, 32.21; N,
6.36. Found: C, 54.74; H, 6.96; Cl, 32.17; N, 6.45. 1H NMR (CD3OD,
300 MHz) d (ppm): 2.97 (s, 6H, (CH3)2), 3.11–3.12 (m, 2H, CH2),
3.32–3.42 (m, 2H, CH2), 7.06–7.12 (m, 2H, ArH), 7.35–7.40 (m,
2H, ArH), 13C NMR (CD3OD, 75.5 MHz) d (ppm): 31.4, 44.0, 60.1,
116.9, 117.2, 132.1, 132.2, 133.7, 133.8, 162.3, 165.5.
4.2.18. N-Methyl-2-(4-chlorophenyl)ethylamine (62)
hydrochloride
The free base 62 was prepared from 6 as described in Method A
using a modified workup. Specifically, the workup involved mixing
the cooled reaction mixture with H2O (15 mL) stirring at 0 °C for
5 min, then mixing with NaOH (10%, 3 mL), stirring at 0 °C for
30 min, and extracting with EtOAc (3ꢂ 15 mL). The combined ex-
tract was washed with brine (3ꢂ 10 mL), dried over Na2SO4, con-
centrated in vacuo, leaving a colorless oil (1.273 g, 99%). The oil
(1.260 g) was dissolved in 10% methanolic hydrogen chloride
(10 mL). The solution was refluxed under N2 for 3 h, then concen-
trated in vacuo to a light yellow solid (1.919 g). This oil was mixed
with CH3OH (3 mL) and filtered through a Celite pad. Pure HCl salt
(348 mg, white solid) was obtained by recrystallization from
CH3OH/EtOAc (1:20). Mp 181–182 °C; MS (ESI): m/z calcd for
C9H12ClN: 169.7, found (M+1): 170.3. Anal. calcd for C9H13Cl2N:
C, 52.45; H, 6.36; Cl, 34.40; N, 6.80. Found: C, 52.39; H, 6.41; Cl,
34.23; N, 6.83. 1H NMR (CD3OD, 300 MHz) d (ppm): 2.57 (s, 3H,
CH3), 2.83–2.89 (m, 2H, CH2), 3.12–3.19 (m, 2H, CH2), 7.08–7.20
(m, 4H, ArH), 13C NMR (CD3OD, 75.5 MHz) d (ppm): 33.2, 34.2,
51.5, 128.75, 128.85, 130.3, 131.9, 136.1, 140.4.
4.2.15. N,N-Dimethyl-2-(2-chlorophenyl)ethylamine (59)
hydrochloride
The free base 59 was prepared in 74% from 4 following Method
B. The mixture obtained by dissolving 59 (970 mg, 5.28 mmol) in
Et2O (5 mL), and adding ethereal HCl (1 M, 8 mL) was centrifuged,
and the precipitated solid was collected, washed with EtOAc (2ꢂ
10 mL) and dried in vacuo to give the HCl salt (1.142 g, white solid,
98%). Mp 165–167 °C; MS (ESI): m/z calcd for C10H14ClN: 183.4,
found (M+1): 184.3. Anal. calcd for C10H15Cl2N: C, 54.56; H, 6.87;
Cl, 32.21; N, 6.36. Found: C, 54.43; H, 6.82; Cl, 32.01; N, 6.35. 1H
NMR (CD3OD, 300 MHz) d (ppm): 3.01 (s, 6H, (CH3)2), 3.23–3.51
(m, 4H, CH2CH2), 7.29–7.36 (m, 2H, ArH), 7.43–7.46 (m, 2H, ArH),
13C NMR (CD3OD, 75.5 MHz) d (ppm): 30.1, 44.2, 58.5, 129.2,
130.7, 131.3, 132.8, 135.42, 135.44.
4.2.16. N-Methyl-2-(3-chlorophenyl)ethylamine (60)
hydrochloride
4.2.19. N,N-Dimethyl-2-(4-chlorophenyl)ethylamine (63)
hydrochloride
A
mixture of 2-(3-chlorophenyl)ethylamine (6) (1.119 g,
The free base 63 was prepared from 7 following Method B. The
mixture obtained by dissolving the oil (946 mg, 5.15 mmol) in Et2O
(5 mL) and adding ethereal HCl (1 M, 8 mL) was centrifuged, and
the precipitated solid was collected, washed with EtOAc (2ꢂ
10 mL) and dried in vacuo (1.047 g, white solid, 92%). Mp 162–
163 °C; MS (ESI): m/z calcd for C10H14ClN: 183.4, found (M+1):
184.3. Anal. calcd for C10H15Cl2N: C, 54.56; H, 6.87; Cl, 32.21; N,
6.36. Found: C, 54.49; H, 6.94; Cl, 32.36; N, 6.38. 1H NMR (CD3OD,
300 MHz) d (ppm): 2.97 (s, 6H, (CH3)2), 3.11–3.12 (m, 2H, CH2),
3.32–3.42 (m, 2H, CH2), 7.06–7.12 (m, 2H, ArH), 7.35–7.40 (m,
2H, ArH), 13C NMR (CD3OD, 75.5 MHz) d (ppm): 31.4, 44.0, 60.1,
116.9, 117.2, 132.1, 132.2, 133.7, 133.8, 162.3, 165.5.
0.007 mol), ethyl formate (24 mL), and formic acid (3 drops) was
refluxed under N2 for 5 h. The reaction mixture was concentrated
in vacuo to give crude N-3-chlorophenethylformamide intermedi-
ate (light orange oil, 2.17 g, 0.012 mol). A solution of this interme-
diate (856 mg, 4.66 mmol) in THF (25 mL, anhydrous) was added
dropwise to a suspension of LiAlH4 (756 mg, 19.9 mmol, pellet)
in THF (10 mL, anhydrous) cooled to 0 °C under N2. The mixture
was refluxed under N2 for 4 h, then cooled to 0 °C, mixed slowly
with H2O (0.8 mL), NaOH (15%, 0.8 mL), and H2O (0.8 mL). The mix-
ture was stirred at RT for 10 min, filtered through a Celite pad, and
the solvent in the filtrate was evaporated. The residual colorless oil
was dissolved with HCl (3 N, 10 mL), washed with EtOAc (3ꢂ
10 mL), basified to pH 14 with NaOH (10 N, 4 mL), and extracted
with EtOAc (3ꢂ 10 mL). The combined extract was washed with
brine (3ꢂ 10 mL), dried over Na2SO4, and concentrated in vacuo,
leaving a colorless oil (588 mg, 29%). The free amine was converted
to HCl salt by dissolving the oil (576 mg, 3.40 mmol) in Et2O
(15 mL), and mixing with HCl in Et2O (1 M, 6 mL). The pure HCl salt
(234 mg, 1.14 mmol, white solid, 33%) was obtained by recrystalli-
4.2.20. N-Methyl-2-(2-bromophenyl)ethylamine (64)
hydrochloride
The free base 64 was prepared from 2-(2-bromophenyl)ethyla-
mine (8) using a slight modification of Method A. Specifically, the
chilled borohydride reaction mixture was treated with H2O
(10 mL) followed by 10% NaOH (until basic), stirred for 30 min at
0° under N2 and then extracted with EtOAc (3ꢂ 30 mL). The com-

7422
A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
bined organic extract was washed with H2O (20 mL), satd NaCl
(20 mL), then dried over Na2SO4 and concentrated to a solid. The
solid was taken up in diethyl ether (5 mL) and 6% ethanolic HCl
(10 mL) was added. Concentration afforded a solid that was recrys-
tallized from CH3OH/ether (69 mg, 8% yield). Mp 109 °C; MS (ESI):
m/z calcd for C9H11BrN: 213.02, 215.01., found (M+1): 214.1, 21.1.
Anal. calcd for C9H12BrClN: C, 43.14; H, 5.23; N, 5.59. Found: C,
43.07; H, 5.10; N, 5.53. 1H NMR (DMSO-d6, 300 MHz) d (ppm):
2.58 (s, 4H, CH2CH2), 3.1 (s, 3H, CH3), 7.25–7.27 (m, 1H, ArH),
7.3–7.4 (m, 2H, ArH), 7.62–7.65 (d, 1H, ArH). 13C NMR (DMSO-d6,
75.5 MHz) d (ppm): 32.02, 32.63, 47.74, 124.08, 128.5, 129.45,
131.29, 133.07, 136.79.
(20 mL), then dried over Na2SO4 and concentrated to a solid. The
solid was taken up in diethyl ether (5 mL) and 6% ethanolic HCl
(10 mL) was added. Concentration afforded a solid that was recrys-
tallized from CH3OH/ether (660 mg, 65%). Mp 196 °C; MS (ESI): m/z
calcd for C9H11BrN: 213.02, 215.1, found (M+1): 214.0, 216.1. Anal.
calcd for C9H12BrClN: C, 43.14; H, 5.23; N, 5.59. Found: C, 43.13; H,
5.22; N, 5.56. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.5 (s, 3H,
CH3), 2.9 (m, 2H, CH2), 3.1 (m, 2H, CH2), 7.23 (d, 2H, ArH), 7.54
(d, 2H, ArH). 13C NMR (DMSO-d6, 75.5 MHz) d (ppm): 31.03,
32.55, 49.04, 120.18, 131.28, 131.78, 137.0.
4.2.25. N,N-Dimethyl–2-(4-bromophenyl)ethylamine (69)
hydrochloride
4.2.21. N,N-Dimethyl-2-(2-bromophenyl)ethylamine (65)
hydrochloride
The free base 69 was prepared from 10 following Method B and
the hydrochloride salt (428 mg, 65%) was obtained by treatment of
69 with 6% ethanolic HCl (10 mL). Mp 196 °C; MS (ESI): m/z calcd
for C10H14BrN: 227.03, 229.03, found: 228.4, 230.5. Anal. calcd
for C10H15BrClN: C, 45.93; H, 5.71; N, 5.21. Found: C, 44.91; H,
5.68; N, 5.21. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.77 (s, 6H,
(CH3)2), 3.10, (m, 2H, CH2), 3.25 (m, 2H, CH2) 7.20 (d, 2H, ArH),
7.54 (d, 2H, ArH). 13C NMR (DMSO-d6, 75.5 MHz) d (ppm): 29.41,
42.19, 57.05, 120.22, 131.32, 131.81, 136.92.
The free base 65 was prepared from 8 following Method B.
Treatment of the oil with 6% ethanolic HCl (10 mL) afforded the
hydrochloride salt (363 mg, 40%). Mp 205 °C; MS (ESI): m/z calcd
for C10H14BrN: 227.03, 229.03, found: 228.4, 230.4. Anal. Calcd
for C10H15BrClN: C, 45.39; H, 5.71; N, 5.29. Found: C, 45.43; H,
5.69; N, 5.37. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.81 (s, 6H,
(CH3)2), 3.2 (m, 4H, CH2CH2), 7.37–7.39 (m, 1H, ArH), 7.39–7.41
(m, 2H, ArH), 7.62–7.65 (d, 1H, ArH). 13C NMR (DMSO-d6,
75.5 MHz) d (ppm): 30.54, 42.63, 56.18, 124.0, 125.77, 128.8,
129.71, 131.55, 133.55, 135.92.
4.3. hTAAR 1 activation assay
Receptor activation was assayed in CHO cells stably expressing
4.2.22. N-Methyl-2-(3-bromophenyl)ethylamine (66)
hydrochloride
G
(RD-HGA16 cells; Molecular Devices Corporation, Sunnyvale,
a
16
CA) and hTAAR 1 as previously described.11 Briefly, these cells were
plated in HAM’s F-12 medium with 10% FBS, 400 g/mL hygromy-
cin and 400 g/mL geneticin at 30,000 cells/well in 96-well black
The free base 66 was prepared from 2-(3-bromophenyl)ethyla-
mine (9) using a slight modification of Method A. Specifically, the
chilled borohydride reaction mixture was treated with H2O
(10 mL) followed by 10% NaOH (until basic), stirred for 30 min. at
0 °C under N2 and then extracted with EtOAc (3ꢂ 30 mL). The com-
bined organic extract was washed with H2O (20 mL), satd NaCl
(20 mL), then dried over Na2SO4 and concentrated to a solid. The
solid was taken up in diethyl ether (5 mL) and 6% ethanolic HCl
(10 mL) was added. Concentration afforded a solid that was recrys-
tallized from CH3OH/ether (600 mg, 50%). Mp 124 °C; MS (ESI): m/z
calcd for C9H11BrN: 213.02, 215.2, found (M+1): 214.1, 216.2. Anal.
calcd for C9H13BrClN: C, 43.14; H, 5.23; N, 5.59. Found: C, 43.04; H,
5.21; N, 5.59. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.56 (s, 2H,
CH3), 2.9 (m, 2H, CH2), 3.12 (m, 2H, CH2), 7.2–7.3 (m, 3H, ArH),
7.3–7.45 (m, 1H, ArH). 13C NMR (DMSO-d6, 75.5 MHz) d (ppm):
31.18, 32.69, 47.74, 122.17, 128.19, 129.96, 131.06, 131.71, 140.68.
l
l
clear-bottom plates and incubated at 37 °C, 5% CO2, the night be-
fore the assay. Activation of hTAAR 1 via test compounds was as-
sessed the next day using the Calcium 3 Assay Kit (Molecular
Devices; Sunnyvale, CA) per manufacturer’s specifications except
that the Calcium 3 dye was used at 1/3 the suggested concentra-
tion. The cells were incubated with the dye at 37 °C for 1 h. Test
compounds were evaluated using 7–8 different concentrations
run in duplicate and a six-point b-PEA curve was run on each assay
plate to monitor assay performance. The effect of test compound
on internal calcium mobilization was determined with a FlexStation
set for bottom read with 485 nm excitation and 525 nm emission
wavelengths with a 515 nm emission cutoff. Basal or unstimulated
fluorescence intensity was recorded for 13 s before the addition of
test compound. The effect of test compound was determined by sub-
tracting the minimum from the maximum fluorescence intensity
during a 47-s recording period following the addition of test com-
pound. A three-parameter logistic equation was fit to the data to cal-
culate the EC50 and Emax values (Prism version 4.0; GraphPad, Inc.
San Diego CA). The data represent the means SE from at least three
different experiments except where noted.
4.2.23. N,N-Dimethyl-2-(3-bromophenyl)ethylamine (67)
hydrochloride
The free base 67 was prepared from 9 following Method B and
the hydrochloride salt (395 mg, 60%) was obtained by treatment of
67 with 6% ethanolic HCl (10 mL). Mp 163 °C; MS (ESI): m/z calcd
for C10H14BrN: 227.03, 229.03, found: 228.4, 230.5. Anal. calcd
for C10H15BrClN: C, 45.39; H, 5.71; N, 2.29. Found: C, 45.39; H,
5.73; N, 5.29. 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.77 (s, 6H,
(CH3)2), 3.05 (m, 2H, CH2), 3.2 (m, 2H, CH2), 7.32–7.34 (m, 2H,
ArH), 7.45–7.48 (m, 1H, ArH), 7.61 (s, 1H, ArH). 13C NMR (DMSO-
d6, 75.5 MHz) d (ppm): 29.55, 42.19, 54.04, 122.19, 128.24, 130.0,
131.09, 131.79, 140.31.
4.4. Molecular modeling
The potency data (EC50) for the 68 b-phenethylamines (Tables
1–4) were analyzed using Comparative Molecular Field Analysis
(CoMFA). The starting conformation of each structure was set to
the global energy minimum conformation of the parent structure
(b-PEA) and then energy-optimized using the MMFF94 force field.
In addition, MMFF94 charges were calculated for each atom. The
CoMFA region was defined as extending 4 A beyond every mole-
cule in all directions. Lower corner coordinates = (ꢁ10.393577,
ꢁ8.368728, ꢁ6.689531), Top corner coordinates = (9.141720,
8.391055, 9.467717). The default grid spacing of 2 Å was used.
The default sp3 carbon probe atom was used with the default +1
charge. The structures were aligned by the common phenethyl-
amine substructure ([N]CCC@CC@CC@C) using the Sybyl ‘Align
4.2.24. N-Methyl-2-(4-bromophenyl)ethylamine (68)
hydrochloride
The free base 68 was prepared from 2-(4-bromophenyl)ethyla-
mine (10) using a slight modification of Method A. Specifically, the
chilled borohydride reaction mixture was treated with H2O
(10 mL) followed by 10% NaOH (until basic), stirred for 30 min at
0° under N2 and then extracted with EtOAc (3ꢂ 30 mL). The com-
bined organic extract was washed with H2O (20 mL), satd NaCl

A. H. Lewin et al. / Bioorg. Med. Chem. 16 (2008) 7415–7423
7423
Database’ command. The hTAAR 1 EC50 values (Tables 1–4) were
References and notes
utilized to calculate ‘pEC50’ (defined as ꢁlog(EC50)). The default
CoMFA row weighting (‘SAME_WEIGHTS_FOR_ALL’) and scaling
(‘COMFA_STANDARD’) were used. A validated PLS analysis was
performed on the dependent variable ‘pEC50’ on all 68 compounds
using the leave-one-out (LOO) method starting with eight compo-
nents; no compounds were left out. The optimum number of com-
ponents (5) was based on the cross validated standard error of
prediction (0.66 for five components). Carrying out the final PLS
analysis with five components yielded a regression coefficient r2
of 0.824 (predicted as actual pEC50). The steric field contribution
to the model was 61% with the balance (39%) contributed by the
electrostatic field. Actual values of StdDev * Coeff of the final five-
component model were contoured at 0.01 and ꢁ0.01 for both steric
and electrostatic fields; these are shown in Figures 1(steric) and 2
(electrostatic).
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Acknowledgments
Support for this research from the NIDA (R01-DA-19327-01) is
gratefully acknowledged. The authors thank Dr. Bruce Blough for
compounds from his collection in support of NIDA Grant R01-
DA12970, and D. Zhong, L.D. Mayer, M. Porter, and K. Warner for
their technical assistance. The authors also express their apprecia-
tion to the manuscript reviewers for their constructive suggestions.
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