Synthesis and evaluation of 3-trifluoromethyl-7-substituted-1,2,3,4- tetrahydroisoquinolines as selective inhibitors of phenylethanolamine N- methyltransferase versus the α2-adrenoceptor

Article abstract of DOI:10.1021/jm980734a

A series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquinolines was synthesized and evaluated as inhibitors of phenylethanolamine N- methyltransferase (PNMT) and as inhibitors of the binding of clonidine at the α₂-adrenoceptor. These compounds we

Full text of DOI:10.1021/jm980734a

J . Med. Chem. 1999, 42, 3315-3323
3315
Syn th esis a n d Eva lu a tion of 3-Tr iflu or om eth yl-7-su bstitu ted -
1,2,3,4-tetr a h yd r oisoqu in olin es a s Selective In h ibitor s of P h en yleth a n ola m in e
N-Meth yltr a n sfer a se ver su s th e r2-Ad r en ocep tor 1
Gary L. Grunewald,* Timothy M. Caldwell, Qifang Li, and Kevin R. Criscione
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045
Received December 30, 1998
A series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquinolines was synthesized and evaluated
as inhibitors of phenylethanolamine N-methyltransferase (PNMT) and as inhibitors of the
binding of clonidine at the R2-adrenoceptor. These compounds were found to be selective
inhibitors of PNMT due to their decreased affinity for the R2-adrenoceptor, which was attributed
to steric bulk intolerance around the 3-position of 1,2,3,4-tetrahydroisoquinoline (THIQ) at
the R2-adrenoceptor and to the decreased pKa of the THIQ amine due to the 3-trifluoromethyl
moiety. Overall, these compounds displayed less affinity for PNMT compared to previously
studied THIQ-type inhibitors, except for 16 which was found to have good affinity for PNMT
(PNMT Ki ) 0.52 µM). Compounds 14 and 16 proved to be the most selective inhibitors in this
small series of compounds and are some of the most selective inhibitors of PNMT known (14,
selectivity R2 Ki/PNMT Ki ) 700; 16, selectivity R2 Ki/PNMT Ki > 1900). Compounds 14 and 16
are also quite lipophilic due to the 3-trifluoromethyl moiety and represent promising new leads
for the development of new highly selective inhibitors of PNMT, which should be sufficiently
lipophilic to penetrate the blood-brain barrier.
In tr od u ction
Epinephrine (Epi) is known to comprise 5-10% of the
catecholamines in the mammalian central nervous
system (CNS).2,3 The final step in the biosynthesis of
Epi is the transfer of a methyl group from S-adenosyl-
L-methionine (AdoMet) to the primary amine of nor-
epinephrine (NE). This reaction is catalyzed by the
enzyme phenylethanolamine N-methyltransferase
(PNMT; EC 2.1.1.28). PNMT and Epi are co-localized
in the CNS, being sequestered principally in two specific
regions (C1 and C2) of the medulla oblongata.4,5 On the
basis of this CNS localization, Epi neurons have been
postulated to be involved in the regulation of a number
of physiological processes.4-13
highly selective inhibitor of its biosynthesis is required
(i.e., R2 Ki/PNMT Ki > 500).20
Previous studies from our laboratory and others found
that there were two areas on the tetrahydroisoquinoline
(THIQ) nucleus where substitution can increase both
potency for PNMT and selectivity versus the R2-adreno-
ceptor. The first area is at the 7-position,21 where it has
been determined for monosubstituted chloro-THIQs that
substitution at the 7-position was needed for optimal
potency for PNMT.22 Our laboratory has further exam-
ined how substitution at the 7-position of THIQ affects
both potency and selectivity by developing comparative
molecular field analysis23 (CoMFA) models (a type of
three-dimensional QSAR analysis) on a set of 30 7-sub-
stituted-THIQs24 for both the PNMT active site and the
R2-adrenoceptor. These models indicate that, depending
on the lipophilicity of the 7-substituent of THIQ, these
compounds may bind in two different orientations at
both the PNMT active site and the R2-adrenoceptor
(Figure 1). This study concluded that THIQs possessing
hydrophilic electron-withdrawing 7-substituents would
be both potent and selective for PNMT due to their
Undoubtedly the most thoroughly studied physiologi-
cal process to which central Epi has been linked is the
regulation of peripheral blood pressure. In vivo admin-
istration of centrally active PNMT inhibitors [SK&F
64139 (1),14 LY 134046 (2),15 and CGS 19281A (3)16] to
hypertensive rats has demonstrated dramatic decreases
in peripheral blood pressure. However, these results
have been complicated by the observation that all of
these inhibitors show low selectivity for the PNMT
active site versus the R2-adrenoceptor (Table 1). Thus,
the decrease in peripheral blood pressure in hyperten-
sive rat models by PNMT inhibitors may not be the
result of their ability to decrease Epi concentrations
within the medulla oblongata (i.e., to inhibit central
PNMT) but rather their ability to interact with the R2-
adrenoceptor at concentrations required for PNMT
inhibition.17-19 To unambiguously define the functions
of Epi within the CNS, the development of a potent yet
* To whom correspondence should be addressed. Phone: (785) 864-
4497. Fax: (785) 864-5326. E-mail: ggrunewald@ukans.edu.
10.1021/jm980734a CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/03/1999

3316 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Grunewald et al.
CoMFA study.24 The final PNMT CoMFA model con-
tained 80 compounds with 7 components being optimal,
a cross-validated r2 of 0.656 with a “press s” of 0.670
and a nonvalidated r2 of 0.906 with a standard error of
0.326.28 The R2-adrenoceptor CoMFA model contained
80 compounds, with 4 components being optimal, a
cross-validated r2 of 0.633 with a “press s” of 0.592 and
a nonvalidated r2 of 0.845 with a standard error of
0.385.28 The structures of the compounds used in this
study were constructed using the SYBYL 6.4 software
package, and the minimum energy conformations were
calculated with electrostatics using the Tripos force field
and charges calculated by the AM1 method in MOPAC
(SYBYL 6.4 implementation). The conformations of
compounds containing side chains were calculated by
the “systematic search” option in SYBYL to locate the
global minimum energy conformation. Side chains were
aligned so that they occupied the same region of space,
even though this sometimes resulted in the use of a local
minimum energy conformation (within 2 kcal/mol of the
corresponding global minimum).
Ta ble 1. In Vitro Activities of Some Well-Studied PNMT
Inhibitors for PNMT and for Inhibition of [3H]Clonidine
Binding at the R2-Adrenoceptora
Ki (µM)
selectivity
compd
PNMT
R2-adrenoceptor
R2/PNMT
SK&F 64139b (1) 0.22 ( 0.05
0.021 ( 0.005
4.5 ( 0.3
0.095
17
4.4
LY 134046b (2)
CGS 19281A (3)
0.26 ( 0.03
2.7 ( 0.1
12 ( 1
SK&F 29661c (4) 0.56 ( 0.04 100 ( 20
180
a
PNMT and R2-adrenoceptor Ki values for literature compounds
were determined in our laboratory for consistent internal com-
parison. Reference 45. c Reference 46.
b
Using these updated CoMFA models, compounds 12-
16 were proposed (Table 3).28 It should be noted that
CoMFA does not possess the ability to predict the Ki
values for racemic mixtures. Therefore, the Ki predic-
tions are given for each enantiomer. We have previously
investigated the enantioselectivity of other 3-substituted-
THIQs (for examples, see ref 27). The 3-trifluoromethyl
moiety found in these compounds was chosen29 because
(1) it is less bulky than the 3-ethyl group, which showed
decreased PNMT activity (Table 2), but still large
enough to take advantage of the area of steric bulk
intolerance around the 3-position of THIQ at the R2-
adrenoceptor indicated by our CoMFA model, (2) it is
more lipophilic (CF3, π ) 0.88) than the 3-hydroxy-
methyl moiety (CH2OH, π ) -1.03) of compounds 10
and 11, thereby increasing the probability that these
compounds may penetrate the BBB, (3) fluorine can act
as a hydrogen bond acceptor, as shown by studies of
fluorinated carbohydrates,30 thereby mimicking the
potential hydrogen-bonding interactions of the 3-hy-
droxymethyl substituent, and (4) studies from our
group31 and others32 have indicated that benzylamines
and THIQs appear to bind to PNMT in a neutral form.
The 3-trifluoromethyl moiety would decrease the pKa
of the THIQ amine to ca. 5.33 The 7-substituents that
were chosen for this study of 3-trifluoromethyl-THIQs
are 7-SO2CH3 (13), 7-NO2 (14), and 7-CN (15). These
substituents are all hydrophilic and electron-withdraw-
ing, which was previously shown in our CoMFA study
to increase the selectivity of THIQs for PNMT versus
the R2-adrenoceptor.24 These compounds were predicted
by our CoMFA models to have moderate to good
selectivity for PNMT (Table 3) and are less polar than
THIQs 4, 10, and 11. The 7-bromo-THIQ 16 was also
included in this study to further test the ability of our
CoMFA models to predict activity of lipophilic 7-sub-
stituted-THIQs at both PNMT and the R2-adrenoceptor.
The CoMFA models predict that 16 should not be
selective for PNMT (Table 3).
F igu r e 1. Two possible orientations of THIQ-type inhibitors
at PNMT and the R2-adrenoceptor.24 Alignment A is proposed
for lipophilic (+π) 7-substituents, while hydrophilic (-π)
7-substituents are proposed to bind in alignment B. Between
these two structures is a SYBYL-generated view of SK&F
64139 (1) in alignment A superimposed on SK&F 29661 (4) in
alignment B. The asterisk indicates the area in space where
the lone pairs of 1 in alignment A and 4 in alignment B may
overlap. (Adapted from ref 28, copyright 1999, with permission
from Elsevier Science.)
binding in a hydrophilic orientation at both PNMT and
the R2-adrenoceptor. SK&F 29661 (4) is an example of
this type of compound and is one of the most potent and
selective PNMT inhibitors currently reported (Table 1).
However, 4 has been shown in autoradiographic studies
to be unable to penetrate the blood-brain barrier (BBB),
presumably due to the high polarity of the 7-aminosul-
fonyl substituent.25
The other area where substitution on the THIQ
nucleus was found to affect both the potency and
selectivity for PNMT is the 3-position.26 These studies
indicated that substitution of a methyl or hydroxy-
methyl substituent at the 3-position of THIQ (5-7)
increased the potency of THIQs for PNMT while slightly
decreasing their potency at the R2-adrenoceptor (Table
2). However, this area of steric bulk tolerance at PNMT
was limited, with either a 3-methyl- or a 3-hydroxy-
methyl moiety (6 and 7) being ideal for potency, whereas
3-ethyl or 3-methoxymethyl27 substitution (8 and 9)
resulted in reduced potency for PNMT (Table 2).
It was later discovered that a synergistic effect was
achieved by combining both 3- and 7-substituents.27
Compounds 10 and 11 are examples of this synergism
and are the two most selective inhibitors of PNMT yet
known. However, these compounds are more polar than
4, due to the 3-hydroxymethyl moiety, and thus even
less likely to penetrate the BBB. Nonetheless, these
compounds did represent promising leads for the de-
velopment of new, more lipophilic inhibitors that have
the potential to penetrate into the CNS.
Ch em istr y
These compounds and others were added to our
previous CoMFA models for 7-substituted-THIQs and
were aligned according to the rules used in the original
Compounds 12-16 were synthesized as shown in
Schemes 1-3. Trifluoromethyl ketone 17 was formed

PNMT Inhibitors: 3-Trifluoromethyl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3317
Ta ble 2. In Vitro Inhibitory Potency of Various PNMT Inhibitors at PNMT and Binding of [3H]Clonidine at the R2-Adrenoceptora
Ki (µM)
compd
R3
R7
PNMT
10 ( 1
2.1 ( 0.1
1.1 ( 0.1
24 ( 1
9.2 ( 0.4
0.64 ( 0.04
0.34 ( 0.06
R2-adrenoceptor
selectivity R2/PNMT
5
6
7
8
9
H
H
H
H
H
H
0.35 ( 0.1
0.76 ( 0.08
6.6 ( 0.3
0.67 ( 0.11
2.8 ( 0.1
0.035
0.36
6.0
0.028
0.30
CH3
CH2OH
CH2CH3
CH2OCH3
CH2OH
CH2OH
10
11
SO2CH3
SO2NH2
660 ( 10
1400 ( 30
1000
4100
a
Reference 27.
Ta ble 3. Predicted and Actual in Vitro Activities of 3-Trifluoromethyl-7-substituted-THIQs as Inhibitors of PNMT and Binding of
[3H]Clonidine at the R2-Adrenoceptor
Ki SEM (µM)
PNMT
R2-adrenoceptor
obsd
selectivity R2/PNMT
compd
R
pred
obsd
15 ( 1
pred
pred
obsd
12 (()
H
400 ( 10
3900 ( 100
1400 ( 100
2900 ( 100
>1000a
27
110
R
4.0
44
2.0
2.1
0.50
0.050
S
13 (()
SO2CH3
NO2
CN
36 ( 3
R
0.93
8.1
180
180
190
22
S
14 (()
2.0 ( 0.2
13 ( 1
700
R
S
0.11
1.3
26
32
240
25
15 (()
220
R
0.40
4.8
5.8
6.8
15
1.4
S
16 (()
R
Br
0.52 ( 0.05
>1900
0.46
0.18
0.38
0.46
0.83
2.6
S
a
Precipitate was noted at 1000 µM in the R2-adrenoceptor assay.
Sch em e 1
Sch em e 2
Reduction of the lactam with BH3‚THF formed 3-tri-
fluoromethyl-THIQ (12) (Scheme 1).
by the reaction of ethyl trifluoroacetate with benzyl
Grignard.34 Treatment of 17 with hydroxylamine and
NaOAc in EtOH/water at reflux formed oxime 18, which
was reduced with LiAlH4 in THF to form 19.35 Amine
19 was treated with methyl chloroformate to form 20,
which was cyclized with PPA to form lactam 21.36
Chlorosulfonylation of lactam 21 with chlorosulfonic
acid (neat) formed 22. Chlorosulfone 22 was treated
with hydrazine in THF to form the hydrazinosulfone,
which was not isolated but was converted directly to
methyl sulfone 23 with NaOAc and MeI in EtOH.37

3318 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Grunewald et al.
Sch em e 3
underpredicted for the R2-adrenoceptor (Table 3). Nev-
ertheless, our CoMFA models were able to predict that
significant decreases in affinity for the R2-adrenoceptor
could be obtained through the combination of a hydro-
philic electron-withdrawing 7-substituent and the ap-
propriate 3-substituent as found in 13-15.
Examination of the affinities of 12-16 for the R2-
adrenoceptor suggests that the receptor may not only
be sensitive to substitution at the 3-position of THIQ
but also to the pKa of the THIQ amine. Therefore it may
be possible to modulate the pKa of THIQ in order to
decrease its affinity for the R2-adrenoceptor. For ex-
ample, compare the R2-adrenoceptor affinities of 12 (Ki
) 400 µM) and 6 (Ki ) 0.76 µM). These compounds
display a 500-fold difference in affinity, which cannot
be attributed only to unfavorable steric interactions
around the 3-position of THIQ, as it has been shown
previously that 3-ethyl-THIQ (8) has moderate affinity
for the R2-adrenoceptor (Ki ) 0.67 µM). Therefore, the
decreased affinity of 12 is most likely due to the decrease
in pKa for the THIQ amine (pKa ) 4.86 ( 0.02)33 and
not to deleterious steric interactions with the R2-
adrenoceptor. This is a logical assumption considering
that the natural ligands for the R2-adrenoceptor, NE and
Epi, have a pKa ca. 10 and would be protonated at
physiological pH.
Reduction of lactam 23 with BH3‚THF formed 13
(Scheme 2).
Electrophilic nitration of 21 formed lactam 24, which
was reduced with BH3‚THF to form 14 (Scheme 3).
Hydrogenation of the nitro moiety of 14 to the amine
followed directly by diazotization and a modified Sand-
meyer reaction formed 7-cyano-THIQ 15.38 Similar
procedures utilizing a Sandmeyer bromination were
used for the synthesis of 16 from 14 (Scheme 3).39
In general, this series of 3-trifluoromethyl-THIQs
(12-15) displayed decreased affinity for PNMT com-
pared to similarly substituted 3-methyl- and 3-hy-
droxymethyl-THIQs. A comparison of the PNMT affinity
of 3-trifluoromethyl-THIQ (12, Ki ) 15 µM) and 3-meth-
yl-THIQ (6, Ki ) 2.1 µM) shows a 7-fold decrease in
affinity. Examination of the activities of 12 and 3-hy-
droxymethyl-THIQ (7, Ki ) 1.1 µM) shows a 14-fold
decrease in PNMT affinity. The decreased affinity of
these 3-trifluoromethyl-THIQs for PNMT may be at-
tributed to three factors. First, it has been shown that
the amount of steric bulk tolerance at the 3-position of
THIQ is limited (e.g., methyl or hydroxymethyl, but not
ethyl) at the PNMT active site (Table 2). The trifluoro-
methyl moiety is larger (C-H bond ) 1.09 Å; C-F bond
) 1.34 Å) than a methyl group and may not be tolerated
as well by the PNMT active site. Second, the 3-trifluoro-
methyl moiety is quite lipophilic (π ) 0.88). It has been
previously shown that 3-hydroxymethyl-THIQs 10 and
11 (Table 2)27 are potent inhibitors of PNMT. In
contrast, the 3-hydroxymethyl substituent is quite hy-
drophilic (π ) -1.03). It is possible that by placing a
trifluoromethyl substituent at the 3-position we have
introduced a very lipophilic group into an area that
prefers a hydrophilic moiety. Third, the 3-trifluoro-
methyl group can only act as a hydrogen bond acceptor,
whereas the 3-hydroxymethyl moiety can act as both a
hydrogen bond acceptor and a hydrogen bond donor.
This argument is consistent with the decreased affinity
of 3-trifluoromethyl-THIQs 12-15 for PNMT. Com-
pound 16 is an exception to this trend and displayed
good affinity for PNMT (Ki ) 0.52 µM). Previously, it
had been postulated that PNMT binds THIQs in two
different orientations based on the lipophilicity of the
7-substituent (Figure 1).24 Therefore, since the 7-bromo
substituent has a positive π value (π ) +0.86), 16 may
be bound differently than 12-15. If 16 is bound in the
lipophilic alignment at the PNMT active site, this would
Bioch em istr y
All compounds were evaluated as their hydrochloride
or hydrobromide salts for their activity as inhibitors of
PNMT and the binding of [3H]clonidine to the R2-
adrenoceptor. Bovine adrenal PNMT was prepared
using the method of Connett and Kirshner through the
isoelectric precipitation step.40 The in vitro activity of
these compounds was determined using a standard
radiochemical assay which has been described previ-
ously.41 Inhibition constants were determined by using
three different concentrations of the inhibitor utilizing
phenylethanolamine as the variable substrate.
R2-Adrenoceptor binding assays were performed using
a standard radiochemical assay developed by U’Prichard
et al.42 that uses [3H]clonidine as the radioligand to
define specific binding and phentolamine to determine
the nonspecific binding affinity in order to simplify the
comparison with previous results.
Resu lts a n d Discu ssion
This series of 3-trifluoromethyl-THIQs (12-16) was
found to contain some of the most selective inhibitors
of PNMT known and represents promising new leads
for the development of even more selective inhibitors.
The high degree of selectivity for these compounds is
due to their decreased affinity for the R2-adrenoceptor
and not to their potency for PNMT (Table 3). All of the
compounds (12-16) in this study were found to display
decreased affinity for the R2-adrenoceptor (Ki > 400 µM).
These results are generally consistent with our R2-
adrenoceptor CoMFA model, which had predicted an
area of steric bulk intolerance around the 3-position of
THIQs aligned in the hydrophilic orientation B (Figure
1). However, the affinities of 12-16 were dramatically

PNMT Inhibitors: 3-Trifluoromethyl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3319
Hoover melting point apparatus calibrated with known com-
pounds but are otherwise uncorrected. Proton (1H NMR) and
carbon (13C NMR) nuclear magnetic resonance spectra were
taken on a Varian XL-300, a GE QE-300, or a Bruker DRX-
400 spectrophotometer. Proton chemical shifts are reported in
parts per million (ppm) relative to tetramethylsilane (TMS,
0.00 ppm) and carbon chemical shifts are reported in ppm
relative to CDCl3 (77.0 ppm) unless otherwise noted. For the
hydrochloride salts of these THIQs, NMR spectra were re-
corded in deuterated dimethyl sulfoxide (DMSO-d6) and the
chemical shifts are reported relative to DMSO (2.49 ppm for
1H and 39.5 ppm for 13C) or in deuterated MeOH (CD3OD) and
the chemical shifts are reported relative to MeOH (3.31 ppm
for 1H and 49.15 ppm for 13C). Multiplicity abbreviations are
as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad; ex, exchangeable. Infrared spectra were
obtained on a Perkin-Elmer 1420 infrared spectrophotometer.
Electron-impact mass spectra (EIMS), chemical-ionization
mass spectra (CIMS), and high-resolution mass spectra (HRMS)
were obtained on a Varian Atlas CH-5 or a Ribermag R 10-10
mass spectrophotometer. The intensity of each peak in the
mass spectrum relative to the base peak is reported in
parentheses. Microanalyses were performed on a Hewlett-
Packard model 185B CHN analyzer at the University of
Kansas. Bulb-to-bulb distillations were performed on a Kugel-
rohr distillation apparatus (Aldrich Chemical Co., Milwaukee,
WI), and oven temperatures were recorded. Flash chromatog-
raphy was performed using silica gel 60 (230-400 mesh)
supplied by Universal Adsorbents, Atlanta, GA.
All methanol (MeOH) and ethanol (EtOH) used were
anhydrous unless stated otherwise and were prepared by
distillation over magnesium. Solvents were routinely distilled
prior to use. Anhydrous tetrahydrofuran (THF) and diethyl
ether (Et2O) were distilled from sodium-benzophenone ketyl.
Methylene chloride (CH2Cl2) and chloroform (CHCl3) were
obtained by distillation from phosphorus pentoxide (P2O5). In
some cases anhydrous solvents were used directly from Aldrich
Sure Seal bottles. Hexanes refers to the mixture of hexane
isomers (bp 40-70 °C), and brine refers to a saturated solution
of NaCl. All reactions that required anhydrous conditions were
performed under a positive nitrogen (N2) flow, and all glass-
ware was either oven-dried or flame-dried before use.
F igu r e 2. 3-Trifluoromethyl-THIQs 16 and 13 are shown in
their proposed lipophilic and hydrophilic binding orientations,
respectively. According to our hypothesis, the 3-trifluoromethyl
moiety will occupy different regions in space (depicted by the
black ellipses), depending upon the orientation of the molecule.
place the 3-trifluoromethyl moiety in a totally different
area of space where it may not come in contact with
any of the negative interactions found for 12-15 (Figure
2).
Although previous results have indicated that THIQs
and benzylamines bind to PNMT in a neutral form,31,32
it was still not clear if this hypothesis was valid. Further
evidence in support of this hypothesis is that compounds
12-16 displayed moderate to good affinity for PNMT
(Table 3). The estimated pKa of these 3-trifluoromethyl-
THIQs (pKa ca. 5)33 implies these compounds should be
unprotonated at physiological pH when they interact
with the PNMT active site.
Overall, our CoMFA models for PNMT and the R2-
adrenoceptor were able to predict that 3-trifluoromethyl-
THIQs 12-15 would be selective inhibitors for PNMT.
The rank order of potency for compounds 12-16 was
predicted quite well by our PNMT CoMFA model,
whereas the R2-adrenoceptor model significantly under-
predicted the inhibition constants. Compounds 15 and
16 had the poorest predictions by the R2-adrenoceptor
model. According to our CoMFA models, THIQs with
lipophilic 7-substituents should be “nonselective” inhibi-
tors of PNMT. Compound 16 was predicted to have very
good potency at both PNMT and the R2-adrenoceptor,
but it was found to have almost no affinity for the R2-
adrenoceptor. This is an indication that some other
factor, not measured by our CoMFA models (e.g., the
decreased pKa of these compounds or the lipophilicity
of the 3-trifluoromethyl moiety), is affecting the potency
of these compounds at both PNMT and the R2-adreno-
ceptor.
S-Adenosyl-L-methionine used in the radiochemical assays
was obtained from Sigma Chemical Co. [3H]-S-Adenosyl-L-
methionine was purchased from American Radiolabeled Chemi-
cals, St. Louis, MO. [3H]Clonidine used in the R2-adrenoceptor
assays was purchased from New England Nuclear Corp.,
Boston, MA. Bovine adrenal glands were obtained from Davis
Meat Processing (Overbrook, KS).
1,1,1-Tr iflu or o-3-p h en ylp r op a n -2-on e (17). Compound
17 has been prepared previously34,43 but was synthesized using
the following modified procedure: 2 M benzylmagnesium
chloride in ether (24.5 mL, 49.0 mmol) was added dropwise to
a solution of ethyl trifluoroacetate (5.83 mL, 49.0 mmol) in
ether (100 mL) at -78 °C. After the addition, the reaction was
warmed to room temperature and stirred for 30 min. A
saturated solution of NH4Cl(aq) (40 mL) was added, followed
by the addition of 3 N HCl (25 mL). The mixture was stirred
for 5 min. The phases were separated, and the aqueous phase
was extracted with ether (2 × 25 mL). The organic extracts
were combined, washed with brine (50 mL), and dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure. The residue was purified by flash column chroma-
tography (silica gel) eluting with hexanes/EtOAc (6:1) to yield
a yellowish oil, which was distilled bulb-to-bulb (35 °C, 0.7
mmHg; lit.35 bp 51-52 °C, 2 mmHg) to yield 17 as a colorless
oil (7.9 g, 86%). All spectral data were consistent with
literature values.43
Su m m a r y a n d Con clu sion
Compounds 12-16 are some of the most selective
PNMT inhibitors known due to their reduced affinity
for the R2-adrenoceptor. Of these compounds, 14 and 16
were found to display selectivities (R2 Ki/PNMT Ki)
greater than 500 for PNMT. Due to the 3-trifluoro-
methyl moiety, both 14 and 16 should be sufficiently
lipophilic to penetrate the BBB, thus representing
important new leads in the development of selective
PNMT inhibitors.
1,1,1-Tr iflu or o-3-ph en ylpr opan -2-on e Oxim e (18). Oxime
18 has been prepared previously35 but was synthesized using
the following modified procedure: ketone 17 (6.92 g, 36.8
mmol) was dissolved in a solution of hydroxylamine hydro-
chloride (19.0 g, 272 mmol), NaOAc (22.3 g, 272 mmol), water
(100 mL), and EtOH (25 mL) and heated at reflux for 1 h. The
Exp er im en ta l Section
All of the reagents and solvents used were reagent grade
or were purified by standard methods before use. Melting
points were determined in open capillary tubes on a Thomas-

3320 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Grunewald et al.
solution was cooled and extracted with CHCl3 (3 × 100 mL).
The combined organic extracts were washed with brine (100
mL) and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to yield a yellowish residue.
The residue was purified by flash column chromatography
(silica gel) eluting with hexanes/EtOAc (6:1) to yield an oily
solid, which was distilled bulb-to-bulb (75-80 °C, 1 mmHg;
lit.35 bp 91-92 °C, 3 mmHg) to yield 18 as a colorless solid
(6.80 g, 91.5%): mp 38-40 °C (lit.35 mp 40-42 °C); IR (neat)
3300, 3100, 2920, 1700, 1600, 1500, 1450, 1190, 1130, 970, 750,
white solid, which was recrystallized from hexane to yield 21
as white needles (814 mg, 53.6%): mp 114-115 °C; IR (KBr)
3200, 3100, 2950, 1680, 1600, 1585, 1455, 1375, 1320, 1265,
1245, 1210, 1180, 1135, 1110, 1020, 900, 770, 740, 695 cm-1
;
1H NMR (CDCl3) δ 8.11-8.08 (m, 1H, ArH), 7.53-7.50 (m, 1H,
ArH), 7.47-7.39 (m, 1H, ArH), 7.25-7.23 (m, 1H, ArH), 6.53
(br ex s, 1H, NH), 4.22-4.18 (m, 1H, H-2), 3.40-3.17 (m, 2H,
H-3); EIMS m/z (relative intensity) 216 (M+ + 1, 13), 215 (M+,
48), 146 (100), 128 (70), 118 (25), 90 (45), 63 (20), 51 (12). Anal.
(C10H8NF3O) C, H, N.
1
700 cm-1; H NMR (CDCl3) δ 9.5 (br ex s, 1H, NOH), 7.34-
(()-3-Tr iflu or om eth yl-1,2,3,4-tetr a h yd r oisoqu in olin e
Hyd r och lor id e (12‚HCl). Lactam 21 (60 mg, 0.30 mmol) in
THF (5 mL) was added dropwise to a solution of BH3‚THF (1
M, 2.2 mL) in THF (10 mL). The reaction mixture was heated
at reflux for 2 h and cooled to 0 °C. MeOH (10 mL) was added
dropwise, and the solvent was removed under reduced pres-
sure. The residue was dissolved in MeOH (10 mL) and 6 N
HCl (10 mL) and heated at reflux for 3 h. The solution was
made basic with the addition of 10% NaOH. The reaction
mixture was extracted with CH2Cl2 (3 × 20 mL); the combined
organic extracts were washed with brine and dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure, and the residue was purified by flash chromatogra-
phy (silica gel) with CHCl3 as the eluent. A white solid was
obtained and dissolved in dry ether, and anhydrous HCl(g)
bubbled through the solution to form the hydrochloride salt.
The salt was recrystallized from EtOH/hexanes to yield 12‚
HCl (35.8 mg, 50.3%): mp 195-196 °C; IR (KBr) 3500-3300
(broad), 2920-2400 (broad), 1575, 1400, 1270, 1260, 1205,
1130, 1095, 1000, 755; 1H NMR (DMSO-d6) δ 11.5-10.6 (br
ex m, 2H, NH2+), 7.31 (m, 4H, ArH), 4.73-4.70 (m, 1H, H-3),
4.46-4.36 (m, 2H, H-1), 3.30-3.12 (m, 2H, H-4); EIMS m/z
(relative intensity) 202 (M+, 100), 185 (10), 133 (50), 105 (20),
91 (15), 77 (10). Anal. (C10H10NF3‚HCl) C, H, N.
(()-7-Ch lor osu lfon yl-3-tr iflu or om eth yl-3,4-d ih yd r oiso-
qu in olin -1(2H)-on e (22). Lactam 21 (450 mg, 2.09 mmol) was
dissolved in chlorosulfonic acid (3 mL) and heated at 80 °C
for 16 h. The reaction mixture was cooled to room temperature,
CHCl3 (10 mL) was carefully added, and the mixture was
slowly poured onto ice (25 g). The mixture was extracted with
CHCl3 (3 × 25 mL) and EtOAc (50 mL). The organic extracts
were combined, washed with brine (100 mL), and dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure, and the residue was purified by flash column
chromatography (silica gel) eluting with EtOAc/hexanes (3:1)
to yield a white solid that was recrystallized from EtOAc/
hexanes to yield 22 (455 mg, 69.6%) as white needles: mp
190-191 °C; IR (KBr) 3200, 3100, 2960, 1680, 1600, 1440,
1360, 1275, 1205, 1180, 1130, 1060, 990, 650; 1H NMR δ 8.78-
8.76 (m, 1H, ArH-8), 8.18-8.15 (m, 1H, ArH-6), 7.56-7.53 (m,
1H, ArH-5), 6.91 (br ex s, 1H, CONH), 4.38-4.25 (m, 1H, H-3),
3.58-3.34 (m, 2H, H-4); EIMS m/z (relative intensity) 313 (M+,
20), 278 (83), 262 (15), 244 (100), 214 (45), 145 (60), 117 (20),
89 (50), 63 (40). Anal. (C10H7NClF3O3S) C, H, N.
7.30 (m, 5H, ArH), 3.90 (s, 2H, ArCH2CN); 13C NMR (CDCl3)
δ 148.7 (q, J ) 95 Hz, CO), 133.4, 128.5, 128.3, 126.9, 120.4
(q, J ) 818 Hz, CF3), 29.3; HRMS (FAB) m/z calcd for C9H9-
NF3O (M+ + H) 204.0636, obsd 204.0610.
(()-1,1,1-Tr iflu or o-3-p h en yl-2-a m in op r op a n e Hyd r o-
ch lor id e (19‚HCl). Phenylpropylamine (19) has been pre-
pared previously35 but was synthesized using the following
modified procedure: a solution of oxime 18 (1.09 g, 5.38 mmol)
in THF (50 mL) was added dropwise to a mixture of LiAlH4
(406 mg, 10.7 mmol) in THF (50 mL) at 0 °C. The reaction
mixture was warmed to room temperature, after which it was
heated at reflux for 14 h. The reaction mixture was cooled in
an ice bath, and water was added dropwise until no H2
evolution was observed. At this point, 10% NaOH (10 mL) was
added and the solution was stirred for 30 min. The mixture
was filtered through Celite, and the Celite pad was rinsed with
Et2O (2 × 100 mL). The rinses and filtrate were combined and
dried over anhydrous Na2SO4 and the solvents removed under
reduced pressure. The resulting residue was distilled bulb-to-
bulb (35 °C, 0.05 mmHg; lit.35 bp 65-66, 2 mmHg) to yield a
colorless oil. The oil was dissolved in hexane, and anhydrous
HCl(g) was bubbled through the solution to form the hydro-
chloride salt, which was collected by filtration to yield 19‚HCl
as a white solid (0.91 g, 75%): mp dec 120 °C [lit.35 mp 203-
206 °C (sealed tube)]; IR (KBr) 3000, 2990, 2600, 1975, 1580,
1520, 1450, 1380, 1250, 1200, 1150, 1080, 1040, 740, 690, 660;
1H NMR (CDCl3) (free base) δ 7.42-7.20 (m, 5H, ArH), 3.48-
3.39 (m, 1H, H-2), 3.16-3.09 (m, 1H, H-3), 2.65-2.59 (m, 1H,
H-3), 1.28 (br ex s, 2H, NH2); EIMS m/z (relative intensity)
190 (M+ + 1, 12), 189 (M+, 56), 118 (21), 109 (12), 98 (83), 91
(100), 78 (14), 65 (20). Anal. (C9H10F3N‚HCl) C, H, N.
(()-Met h yl-N-(1,1,1-Tr iflu or o-3-p h en ylp r op -2-yl)ca r -
ba m a te (20). Compound 19‚HCl (500 mg, 2.22 mmol) was
dissolved in CHCl3 (40 mL) and pyridine (0.80 mL, 9.0 mmol)
and cooled to 0 °C in an ice bath. Methyl chloroformate (0.18
mL, 2.22 mmol) was added dropwise to the reaction mixture;
the solution was warmed to room temperature and stirred for
14 h. Ice water (30 mL) was added slowly to the mixture, and
the solution was stirred for 15 min. The organic phase was
removed, and the aqueous phase was extracted with chloro-
form (2 × 30 mL). The organic phases and extracts were
combined, washed with 3 N HCl (2 × 30 mL) and brine (30
mL), and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to yield a white solid which
was recrystallized from hexane to yield 20 as white needles
(535 mg, 97.0%): mp 94-95 °C; IR (KBr) 3300, 3060, 3010,
2950, 1690, 1600, 1540, 1490, 1440, 1360, 1300, 1250, 1200,
1160, 1120, 1050, 940, 870, 750, 700, 680, 640 cm-1; 1H NMR
(CDCl3) δ 7.36-7.19 (m, 5H, ArH), 4.76 (br ex s, 1H, NH),
4.66-4.52 (m, 1H, H-2), 3.61 (s, 3H, OCH3), 3.21-3.15 (m, 1H,
H-3), 2.80-2.75 (m, 1H, H-3); 13C NMR (CDCl3) δ 156.8, 135.4,
129.7, 129.5, 129.1, 128.9, 127.6, 125.6 (q, J ) 846 Hz, CF3),
54.1 (q, J ) 89 Hz, C-3), 53.0, 34.8; EIMS m/z (relative
intensity) 248 (M+ + 1, 10), 247 (M+, 46), 172 (70), 91 (100),
65 (20), 59 (27). Anal. (C10H12NF3O2) C, H, N.
(()-7-Meth ylsu lfon yl-3-tr iflu or om eth yl-3,4-d ih yd r oiso-
qu in olin -1(2H)-on e (23). Chlorosulfone 22 (1.65 g, 5.27
mmol) was dissolved in THF (20 mL) and cooled to 0 °C.
Hydrazine (0.47 mL, 14.7 mmol) was added dropwise to the
solution, which was stirred for 16 h. The solvent was removed
under reduced pressure and the residue dried under vacuum
to yield a white solid. The white solid was dissolved in EtOH
(25 mL). NaOAc (2.01 g, 24.5 mmol) and iodomethane (1.36
mL, 24.3 mmol) were added to the solution, and the resulting
suspension was heated at reflux overnight. The mixture was
cooled to room temperature, and water (50 mL) was added.
The solution was extracted with EtOAc (3 × 50 mL). The
combined organic extracts were washed with brine and dried
over anhydrous Na2SO4. The solvent was removed under
reduced pressure and the residue recrystallized from EtOAc/
hexane to yield 23 as white crystals (1.09 g, 70.6%): mp 226-
227 °C; IR (KBr) 3200, 3100, 1680, 1600, 1440, 1375, 1275,
(()-3-Tr iflu or om et h yl-3,4-d ih yd r oisoq u in olin -1(2H )-
on e (21). Carbamate 20 (1.75 g, 7.08 mmol) was dissolved in
cold polyphosphoric acid (45 g) and heated to 140 °C for 30
min. The mixture was poured onto ice water (100 mL). The
aqueous phase was extracted with chloroform (3 × 75 mL).
The combined organic extracts were washed with water (100
mL) and brine (100 mL) and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure to yield a
1
1180, 1130 cm-1; H NMR (CDCl3) δ 8.67 (d, J ) 2.0 Hz, 1H,
ArH-8), 8.11 (dd, J ) 2.0, 8.1 Hz, 1H, ArH-6), 7.49 (d, J ) 8.1
Hz, 1H, ArH-5), 6.47 (br ex s, 1H, NH), 4.26 (m, 1H, H-3),

PNMT Inhibitors: 3-Trifluoromethyl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3321
3.54-3.47 (m, 1H, H-4), 3.36-3.30 (m, 1H, H-4), 3.10 (s, 3H,
CH3); EIMS m/z (relative intensity) 294 (M+ + 1, 50), 293 (M+,
80), 224 (100), 145 (90). Anal. (C11H10NF3O3S) C, H, N.
(325 mg, 4.99 mmol) in water (5 mL) was prepared. Benzene
(5 mL) was added to the basic mixture and the suspension
chilled in an ice bath. A solution of Ni2SO4‚6H2O (192 mg,
0.110 mmol) in water (1 mL) was added to this suspension,
and the color of the biphasic solution changed to yellow-brown.
The diazonium salt solution was added dropwise to the basic
KCN solution. Brisk evolution of N2 was observed, and the
reaction mixture was warmed to room temperature over a
period of 2 h. The mixture was heated to 50 °C in an oil bath
for 1 h, cooled to room temperature, made basic (pH > 10)
with 1 N NaOH, and filtered through Celite. The Celite bed
was rinsed with CH2Cl2 (3 × 20 mL). The filtrate was extracted
with CH2Cl2 (3 × 30 mL). The organic rinses and extracts were
combined, washed with brine, and dried over anhydrous Na2-
SO4. The solvent was removed under reduced pressure, and
the crude product was purified by flash chromatography (silica
gel) with hexanes/EtOAc (3:1) to give a brown oil, which was
purified by flash column chromatography (silica gel) with CH2-
Cl2/hexanes (3:1) to yield a pale-yellow solid. The yellow solid
was recrystallized from CH2Cl2/hexane to yield 15 as pale-
yellow needles (116 mg, 30.0%): mp 116-118 °C. The hydro-
chloride salt was formed with HCl(g) in MeOH and was
recrystallized from MeOH/EtOAc/hexanes to yield 15‚HCl as
white crystals: IR (KBr) (salt) 2920, 2700, 2600, 2460, 2200,
1590, 1570, 1450, 1390, 1280, 1260, 1200, 1170, 1140, 1000,
(()-7-Meth ylsu lfon yl-3-tr iflu or om eth yl-1,2,3,4-tetr a h y-
d r oisoqu in olin e Hyd r och lor id e (13‚HCl). Lactam 23 (1.07
g, 3.66 mmol) was dissolved in THF (40 mL). BH3‚THF (1M)
(9.15 mL) was added dropwise, and the solution was heated
at reflux for 2 h. The solution was cooled to room temperature,
and MeOH (15 mL) was added dropwise. The solvent was
removed under reduced pressure, and MeOH (15 mL) and 6
N HCl (15 mL) were added. The solution was heated to reflux
for 3 h. A solution of 10% NaOH was added to the mixture
until the solution was basic (pH > 10). The solution was
extracted with EtOAc (4 × 30 mL). The combined organic
extracts were washed with brine and dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure and
the residue dissolved in CHCl3. Dry HCl(g) was bubbled
through the solution to form the HCl salt, which was recrys-
tallized from EtOH/hexanes to yield 13‚HCl as white crystals
(1.13 g, 97.9%): mp 253-254 °C; IR (KBr) 2900, 2700, 2600,
1425, 1300, 1180, 1130, 1010 cm-1; 1H NMR (DMSO-d6) δ 10.5
(br ex s, 2H, NH2+), 7.90 (s, 1H, ArH-8), 7.84 (d, J ) 8.1 Hz,
1H, ArH-6), 7.58 (d, J ) 8.1 Hz, 1H, ArH-5), 4.60 (m, 1H, H-3),
4.45 (s, 2H, H-1) 3.35 (m, 1H, H-4), 3.22 (s, 3H, CH3), 3.20 (m,
1H, H-4); EIMS m/z (relative intensity) 278 (M+ - 1, 35), 210
(100), 130 (75). Anal. (C11H12NF3O2S‚HCl) C, H, N.
1
900 cm-1; H NMR (DMSO-d6) δ 7.85 (s, 1H, H-8), 7.76 (d, J
(()-3-Tr iflu or om eth yl-7-n itr o-3,4-d ih yd r oisoqu in olin -
1(2H)-on e (24). Lactam 21 (403 mg, 1.87 mmol) was dissolved
in concentrated H2SO4 (5 mL) and cooled to 0 °C. KNO3 (207
mg, 2.06 mmol) was added in small portions over the course
of 30 min. The reaction mixture was warmed to room temper-
ature and stirred overnight. The mixture was poured slowly
onto ice (50 g). A white precipitate was formed, filtered off,
washed with water, and dried under vacuum. The aqueous
filtrate was collected and extracted with CHCl3 (3 × 25 mL).
The combined organic extracts were washed with brine and
dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure and the remaining yellow residue combined
with the previously isolated precipitate. The crude reaction
mixture was recrystallized from EtOAc/hexanes to yield 24 as
light-yellow needles (410 mg, 84.4%): mp 219-221 °C; IR
(KBr) 3190, 3090, 3010, 1675, 1600, 1510, 1430, 1370, 1340,
1250, 1200, 1170, 1120, 1050, 1000, 700 cm-1; 1H NMR (CDCl3)
δ 8.91 (s, 1 H, H-8), 8.38 (d, J ) 8.1 Hz, 1 H, H-6), 7.43 (d, J
) 8.1 Hz, 1 H, H-5), 6.48 (s, 1 H, NH), 4.12 (m, 1 H, H-3),
3.52-3.22 (m, 2 H, H-4); CIMS m/z (relative intensity) 278
(M + NH4+, 4), 261 (MH+, 20), 191 (100), 145 (30). Anal.
(C10H7F3N2O3) C, H, N.
) 8.0 Hz, 1 H, H-6), 7.28 (d, J ) 8.0 Hz, 1H, H-5), 4.80-4.73
(m, 1H, H-3), 4.49-4.39 (m, 2H, H-1), 3.42-3.22 (m, 2H, H-4);
13C NMR (DMSO-d6) δ 136.5, 131.8, 131.4, 131.2, 130.9, 124.7
(q, J ) 838 Hz, CF3), 119.3, 110.6 (CN), 52.9 (q, J ) 95 Hz,
C-3), 45.0, 25.7; CIMS m/z (relative intensity) 227 (MH+, 30),
226 (M+, 10), 225 (M+ - 1, 100), 155 (25), 129 (15). Anal.
(C11H9N2F3‚HCl) C, H, N.
(()-3-Tr iflu or om et h yl-7-br om o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (16‚HCl). Tetrahydroisoquinoline
14‚HCl (390 mg, 1.38 mmol) was placed in a Parr shaker bottle
and dissolved in MeOH (50 mL), and concentrated HCl (3 mL)
and water (8 mL) were added. After the addition of PtO2 (90
mg), the mixture was hydrogenated at 50 psi (4 h). The
solution was filtered and concentrated. The concentrated
solution was made basic with 10% NaOH solution. The basic
solution was extracted with CH2Cl2 (3 × 50 mL); the organic
extracts were combined and dried over anhydrous Na2SO4. The
solvent was removed, and the residue was dissolved in ice-
cold 48% HBr(aq) (1.0 mL) in water (3.0 mL). Sodium nitrite
(103 mg, 1.50 mmol) in water (2 mL) was added dropwise.
After 30 min, excess HNO2 was destroyed by the addition of
urea (a negative starch-iodide test was obtained at this time).
The diazonium salt solution was added to a mixture of CuBr
(600 mg, 4.18 mmol), 48% HBr(aq) (2.5 mL), and water (5 mL)
at 35 °C. After the addition of the diazonium salt solution, the
reaction mixture was warmed to 75-80 °C and stirred for 1.5
h. The reaction mixture was allowed to stand overnight at
room temperature and was cautiously made basic with a 50%
NaOH solution. Ethyl acetate (50 mL) was added, and the blue
copper salts were removed by filtration. The filtrate was
extracted with EtOAc (3 × 50 mL). The combined organic
extracts were dried over anhydrous Na2SO4 and evaporated
to give a dark oil. The crude product was purified by flash
chromatography (silica gel) with hexanes/EtOAc (6:1) to yield
a white solid, which was recrystallized from hexane to yield
16 as white crystals (292 mg, 75.8%): mp 77-79 °C. The
hydrochloride salt was formed in the same manner as 12‚HCl
and recrystallized from MeOH/EtOAc/hexane to give 16‚HCl
as white crystals: IR (KBr) 2920, 2850-2400 (broad), 1590,
1570, 1450, 1400, 1390, 1270, 1250, 1205, 1180, 1140, 1300,
(()-3-Tr iflu or om et h yl-7-n it r o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (14‚HCl). This compound was
prepared in a similar manner as 13 using the procedures
outlined previously but using lactam 24 (1.08 g, 4.15 mmol)
as the starting material. The hydrochloride salt was prepared
in a similar manner as 13‚HCl and was recrystallized from
MeOH/EtOAc to yield 14‚HCl as pale-yellow needles (1.02 g,
87.2%): mp dec 256-258 °C; IR (KBr) 3060, 2920, 2800-2400
(broad), 1570, 1510, 1420, 1345, 1275, 1255, 1170, 1125, 900
1
cm-1; H NMR (DMSO-d6) δ 8.23 (s, 1H, ArH-8), 8.10 (d, J )
8.6 Hz, 1H, ArH-6), 7.53 (d, J ) 8.6 Hz, 1H, ArH-5), 4.65 (m,
1H, H-3), 4.46 (s, 2H, H-1), 3.42-3.12 (m, 2H, H-4); CIMS m/z
(relative intensity) 247 (MH+, 100), 177 (30). Anal.
(C10H9N2F3O2‚HCl) C, H, N.
3-Tr iflu or om eth yl-7-cya n o-1,2,3,4-tetr a h yd r oisoqu in o-
lin e Hyd r och lor id e (15‚HCl). THIQ 14‚HCl (1.23 g, 4.99
mmol) was dissolved in MeOH (50 mL) and hydrogenated with
10% Pd/C (25 mg) at 50 psi. The solution was filtered through
Celite, the Celite bed was washed with MeOH (50 mL), and
the filtrate was evaporated to dryness. The residue was
dissolved in concentrated HCl (1.0 mL) and water (1 mL) and
stirred in an ice bath. NaNO2 (52.0 mg, 0.756 mmol), dissolved
in water (1 mL), was added dropwise. After stirring for 15 min,
a positive starch-iodide test was obtained and urea (20 mg,
0.34 mmol) was added to destroy any excess HNO2. In a second
flask, a solution of NaOH (0.20 g in 1.5 mL water) and KCN
1000, 900 cm-1 1H NMR (DMSO-d6) δ 7.58 (s, 1H, ArH-8),
;
7.50-7.48 (m, 1H, ArH-6), 7.28-7.26 (m, 1H, ArH-5), 4.72-
4.70 (m, 1 H, H-3), 4.44-4.34 (m, 2H, H-1), 4.37 (br ex s, 1 H,
NH), 3.27-3.3.08 (m, 2 H, H-4); 13C NMR (DMSO-d6) δ 132.0,
131.8, 131.3, 130.0, 129.9, 124.7 (q, J ) 837 Hz, CF3), 120.7,
53.2 (q, J ) 96 Hz, C-3), 45.0, 25.1; CIMS m/z (relative

3322 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Grunewald et al.
intensity) 283, (MH+ + 2, 10), 282 (MH+ + 1, 65), 281 (MH+,
15), 280 (M+, 100), 212 (10), 210 (10). Anal. (C10H9NBrF3‚HCl)
C, H, N.
(7) Crowley, W. R.; Terry, L. C. Effects of an Epinephrine Synthesis
Inhibitor, SKF64139, on the Secretion of Luteinizing Hormone
in Ovariectomized Female Rats. Brain Res. 1981, 204, 231-235.
(8) Trudeau, F.; Brisson, G. R.; Pe´ronnet, F. PNMT Inhibition
Decreases Exercise Performance in the Rat. Physiol. Behav.
1992, 52, 389-392.
(9) Mefford, I. N.; Lister, R. G.; Ota, M.; Linnoila, M. Antagonism
of Ethanol Intoxication in Rats by Inhibitors of Phenylethanol-
amine N-Methyltransferase. Alcoholism 1990, 14, 53-57.
(10) Masaharu, K.; Atobe, M.; Nakagawara, M.; Kariya, T. Effect of
Ra d ioch em ica l Assa y for P NMT Activity. The assay
used for this study has been described previously.41 A normal
assay tube mixture consists of 50 µL of 0.5 M phosphate buffer
(pH 8.0), 25 µL of 10 mM AdoMet, 5 µL of [3H]AdoMet that
contains 3 × 105 dpm (specific activity approximately 15 mCi/
mmol), 25 µL of substrate solution (phenylethanolamine), 25
µL of inhibitor solution, 25 µL of the enzyme preparation, and
water to a achieve a total volume of 250 µL. The mixture is
incubated for 30 min at 37 °C and quenched by the addition
of 250 µL of 0.5 M borate buffer (pH 10), and the mixture is
extracted with 2 mL of toluene/isoamyl alcohol (7:3). A 1-mL
aliquot of the organic layer is extracted, transferred to a
scintillation vial, and diluted with cocktail for counting. The
mode of inhibition for all of the inhibitors assayed was
determined to be competitive by inspection of the 1/V vs 1/S
plots of the data. All assays were run in duplicate with three
inhibitor concentrations over a 5-fold range. Ki values were
determined by a hyperbolic fit of the data.
a
Phenylethanolamine N-Methyltransferase Inhibitor, 2,3-
Dichloro-R-methylbenzylamine, on the Alpha-2-adrenoceptor
Function in the Hypothalamus in Rats. Neuropsychobiology
1996, 33, 132-137.
(11) Burke, W. J .; Chung, H. D.; Marshall, G. L.; Gillespie, K. N.;
J oh, T. H. Evidence for Decreased Transport of PNMT Protein
in Advanced Alzheimer’s Disease. J . Am. Geriatr. Soc. 1990, 38,
1275-1282.
(12) Burke, W. J .; Chung, H. D.; Strong, R.; Mattammal, M. B.;
Marshall, G. L.; Nakra, R.; Grossberg, G. T.; Haring, J . H.; J oh,
T. H. Mechanism of Degeneration of Epinephrine Neurons in
Alzeheimer’s Disease. In Central Nervous System Disorders of
Aging: Clinical Intervention and Research; Strong, R., et al.,
Eds.; Raven Press: New York, 1988; pp 41-70.
(13) Burke, W. J .; Galvin, N. J .; Chung, H. D.; Stoff, S. A.; Gillespie,
K. N.; Cataldo, A. M.; Nixon, R. A. Degenerative Changes in
Epinephrine Tonic Vasomotor Neurons in Alzheimer’s Disease.
Brain Res. 1994, 661, 35-42.
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Lowering of Blood Pressure in Hypertensive Rats by SKF 64139
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r2-Ad r en ocep tor Ra d ioliga n d Bin d in g Assa y. The ra-
dioligand binding assay was performed using the methods
developed by U’Prichard et al.42 Male Sprague-Dawley rats
were decapitated and the cortexes removed and homogenized
with 20 volumes (w/v) of ice-cold 50 mM Tris/HCl buffer (pH
7.7 at 25 °C). Homogenates were centrifuged three times for
10 min at 50000g with resuspension of the pellet in fresh buffer
between spins. The final pellet was homogenized in 200
volumes (w/v) of ice-cold 50 mM Tris/HCl buffer (pH 7.7 at 25
°C). Incubation tubes containing [3H]clonidine (specific activity
ca. 19.2 mCi/mmol, final concentration 2.0 nM), various
concentrations of the inhibitors, and an aliquot of freshly
suspended tissue (800 µL) to a final volume of 1 mL were used.
Tubes were incubated at 25 °C for 30 min, and the incubation
was terminated by rapid filtration under vacuum through
GF/B glass fiber filters. The filters were rinsed with three 5-mL
washes of 50 mM Tris/HCl buffer (pH 7.7 at 25 °C). The filters
were counted in vials containing premixed scintillation cock-
tail. Nonspecific binding was determined as the concentration
of ligand bound in the presence of 2 µM phentolamine. All
assays were examined by a log-probit analysis of the data to
determine the IC50 values, and Ki values were determined by
the equation: Ki ) IC50/(1 + [clonidine]/KD), as all the Hill
coefficients were approximately equal to 1.
Molecu la r Mod elin g. All molecular modeling and CoMFA
studies were carried out on a Silicon Graphics Indigo2 work-
station running SYBYL 6.4.44 All compounds in this updated
CoMFA study were aligned according to the rules used in the
previous study of 7-substituted-THIQs for PNMT and the R2-
adrenoceptor,24 and the details of these studies may be found
in ref 28.
(22) Bondinell, W. E.; Chapin, R. W.; Girard, G. R.; Kaiser, C.; Krog,
A. J .; Pavloff, A. M.; Schwartz, M. S.; Silvestri, J . S.; Vaidya, P.
D.; Lam, B. L.; Wellman, G. R.; Pendleton, R. G. Inhibitors of
Phenylethanolamine N-Methyltransferase and Epinephrine Bio-
synthesis. 1. Chloro-substituted 1,2,3,4-Tetrahydroisoquinolines.
J . Med. Chem. 1980, 23, 506-511.
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(24) Grunewald, G. L.; Dahanukar, V. H.; J alluri, R. K.; Criscione,
K. R. Synthesis, Biochemical Evaluation, and Classical and
Three-Dimensional Quantitative Structure-Activity Relation-
ship Studies of 7-Substituted-1,2,3,4-tetrahydroisoquinolines and
Their Relative Affinities toward Phenylethanolamine N-Meth-
yltransferase and the R2-Adrenoceptor. J . Med. Chem. 1999, 42,
118-134.
(25) Pendleton, R. G.; Gessner, G.; Weiner, G.; J enkins, B.; Sawyer,
J .; Bondinell, W.; Intoccia, A. Studies on SK&F 29661, an Organ-
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Ack n ow led gm en t. This research was supported by
USPHS Grant HL 34193.
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