3-Hydroxymethyl-7-(N-substituted aminosulfonyl)-1,2,3,4- tetrahydroisoquinoline inhibitors of phenylethanolamine N-methyltransferase that display remarkable potency and selectivity

Article abstract of DOI:10.1021/jm049368n

Six 3-hydroxymethyl-7-(N-substituted aminosulfonyl)-1,2,3,4- tetrahydroisoquinolines (16-21) were synthesized and evaluated for their phenylethanolamine N-methyltransferase (PNMT) inhibitory potency and affinity for the α₂-adrenoceptor. The add

Full text of DOI:10.1021/jm049368n

134
J. Med. Chem. 2005, 48, 134-140
3-Hydroxymethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinoline
Inhibitors of Phenylethanolamine N-Methyltransferase that Display
Remarkable Potency and Selectivity1
Gary L. Grunewald,* F. Anthony Romero, and Kevin R. Criscione
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045
Received August 4, 2004
Six 3-hydroxymethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (16-21)
were synthesized and evaluated for their phenylethanolamine N-methyltransferase (PNMT)
inhibitory potency and affinity for the R2-adrenoceptor. The addition of nonpolar substituents
to the sulfonamide nitrogen of 9 (3-CH2OH-7-SO2NH2-THIQ) led to inhibitors (16-21) that
have high PNMT inhibitory potency and high selectivity, and most of these (16-21) are
predicted, on the basis of their calculated log P values, to be able to penetrate the blood-brain
barrier. Compounds N-trifluoroethyl sulfonamide 20 (PNMT Ki ) 23 nM) and N-trifluoropropyl
sulfonamide 21 (PNMT Ki ) 28 nM) are twice as potent at inhibiting PNMT compared to 9
and display excellent selectivity (R2 Ki/PNMT Ki g 15 000).
Introduction
Epinephrine (Epi, 2) was identified within the central
nervous system (CNS) 50 years ago.2 Since that time,
it has been implicated in some of the neurodegeneration
seen in Alzheimer’s disease,3,4 as well as in the regula-
tion of blood pressure,5 respiration,6,7 body tempera-
ture,6,7 the R1-adrenoceptor,8 and the R2-adrenocep-
Figure 1. The terminal step in Epi (2) biosynthesis is the
tor.9,10 However, its role in the CNS still remains
unclear. One approach to study the effects of CNS Epi
transfer of a methyl group from AdoMet (3) to NE (1) to form
Epi (2) and the cofactor product AdoHcy (4).
(2) would be to inhibit the enzyme phenylethanolamine
N-methyltransferase (PNMT; EC 2.1.1.28).11 This en-
zyme catalyzes the last step in the biosynthetic pathway
of Epi (2) in which an activated methyl group from
S-adenosyl-L-methionine (3) is transferred to the pri-
mary amine of NE (1) to produce Epi (2) and S-adenosyl-
L-homocysteine (AdoHcy, 4; Figure 1).
results. Previous studies have shown that 3,7-disubsti-
tution on the THIQ nucleus increases selectivity for
PNMT versus the R2-adrenoceptor.21 For example, the
selectivity of 8 is much greater than that of either
monosubstituted 5 or 7.18 As such, the objective of this
research is to develop a 3,7-disubstituted-THIQ-type
PNMT inhibitor that is potent, selective, and capable
of penetrating the BBB.
Some of the first inhibitors of PNMT discovered were
benzylamines.12 Constraining benzylamines to 1,2,3,4-
tetrahydroisoquinolines (THIQs) increases their PNMT
inhibitory potency.13 SK&F 2966114 (5) and SK&F
6413915 (6) were some of the first examples of potent
THIQ-type PNMT inhibitors (Table 1). However, use of
these compounds as physiological probes to elucidate the
role of CNS Epi (2) has been complicated for two
different reasons. First, SK&F 29661 (5) is quite polar
(calculated log P ) -0.29) and does not cross the blood-
brain barrier (BBB).14 A previous study from our lab
using a BBB model16,17 on a small set of THIQs18 gave
a good (r ) 0.79) correlation between the calculated log
P19,20 of the compound and its permeability coefficient.
This correlation indicated that THIQs with a calculated
log P value of approximately 0.5 or greater should be
able to penetrate into the CNS. The second concern is
that SK&F 64139 (6), as well as many other THIQ-type
PNMT inhibitors, shows affinity for the R2-adrenoceptor,
which complicates interpretation of the biochemical
(()-7-Aminosulfonyl-3-fluoromethyl-THIQ (8) is a
highly potent and selective PNMT inhibitor, but this
compound is predicted to be too polar (calculated log P
) 0.00) to cross the BBB.18 An approach to increase the
lipophilicity of 8 that proved to be highly successful was
the addition of nonpolar substituents to the sulfonamide
nitrogen of 8.22 Not only are these inhibitors predicted
to penetrate the BBB, but N-trifluoroethyl sulfonamide
14 and N-trifluoropropyl sulfonamide 15 retained PNMT
inhibitory potency and dramatically increased selectiv-
ity compared to parent 8. Since the addition of nonpolar
substituents to 8 was successful, it was of interest to
determine if this same approach could be applied to
other PNMT inhibitors that are highly potent and
selective but incapable of penetrating the BBB. In this
paper, this strategy was applied to (()-7-aminosulfonyl-
3-hydroxymethyl-THIQ (9; PNMT Ki ) 52 nM, R2 Ki/
PNMT Ki ) 27 000) to increase its lipophilicity with the
expectation that N-substituted analogues of 9 could be
highly potent and selective PNMT inhibitors. Of the 27
(()-3-fluoromethyl-7-(N-substituted aminosulfonyl)-
THIQs prepared in the earlier study, six (10-15)
* To whom correspondence should be addressed. Phone: (785) 864-
4497. Fax: (785) 864-5326. E-mail: ggrunewald@ku.edu.
10.1021/jm049368n CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/13/2004

Remarkably Potent and Selective PNMT Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 135
Table 1. In Vitro PNMT and R2-Adrenoceptor Affinities of Some PNMT Inhibitors
Ki (µM) ( SEM
selectivity
(R2/hPNMT)
a
compd
R3
R7
R8
hPNMT
R2
ClogPb
5c,d
6d,e
7d,f
H
H
SO2NH2
H
Cl
H
H
H
H
H
H
H
H
H
0.28 ( 0.02
0.0031 ( 0.0006
0.82 ( 0.04
0.15 ( 0.01
0.052 ( 0.004
1.4 ( 0.1
100 ( 10
0.021 ( 0.005
3.8 ( 0.1
680 ( 10
1400 ( 200
550 ( 60
610 ( 60
190 ( 20
140 ( 20
1200 ( 100
660 ( 80
360
7
-0.29
2.75
1.84
0.00
-0.93
1.15
1.67
1.71
3.18
1.41i
1.39i
Cl
CH2F
CH2F
CH2OH
CH2F
CH2F
CH2F
CH2F
CH2F
CH2F
H
4.6
8d,f
SO2NH2
4500
27000
390
9d,g
10h
11h
12h
13d,f
14h
15h
SO2NH2
SO2NHCH2CH3
SO2NH(CH2)2CH3
SO2N(CH2CH2)2S
SO2NH-Ph-4-Cl
SO2NHCH2CF3
SO2NH(CH2)2CF3
1.7 ( 0.2
360
1.2 ( 0.2
160
0.27 ( 0.02
0.13 ( 0.02
0.22 ( 0.02
520
9200
3000
a In vitro activities reported for the inhibition of binding of [3H]clonidine at the R2-adrenoceptor. b Calculated log P; refs 19 and 20.
c Reference 14. d Biochemical data has been reported earlier for bovine PNMT. Here we report data for recombinant human PNMT.
e Reference 15. f Reference 18. g Reference 21. h Reference 22. i The trend in ClogP values is consistent with positioning a trifluoromethyl
group next to a heteroatom; ref 33.
Scheme 1a
Table 2. In Vitro Activities of
(()-3-Hydroxymethyl-7-(N-substituted
aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines
Ki (µM) ( SEM
selectivity
compd
R
hPNMT
R2
(R2/hPNMT) ClogPb
a
16 NHCH2CH3
0.17 ( 0.01
210 ( 20
230 ( 20
320 ( 30
1200
1200
1400
840
15000
19000
0.22
0.75
0.79
2.26
0.48c
0.46c
17 NHCH2CH2CH3 0.19 ( 0.01
18 N(CH2CH2)2S
19 NH(4-Cl-Ph)
20 NHCH2CF3
0.23 ( 0.01
0.063 ( 0.002 53 ( 5
0.023 ( 0.002 340 ( 40
21 NHCH2CH2CF3 0.028 ( 0.001 520 ( 50
a In vitro activities reported for the inhibition of binding of
[3H]clonidine at the R2-adrenoceptor. b Calculated log P. c The
trend in ClogP values is consistent with positioning a trifluoro-
methyl group next to a heteroatom; ref 33.
displayed high PNMT inhibitory potency and selectivity
(Table 1). Here we report the synthesis and biochemical
evaluation of the N-substituted analogues of 9 (16-21)
with these same substituents (Table 2).
Chemistry
The route for the synthesis of 16-21 is shown in
Schemes 1 and 2. Amino acid 22 was protected as the
acetamide (23) by treatment with acetyl chloride in an
acetone/NaOH mixture.23 The acid of 23 was protected
as the piperidinamide (24). Treatment of 24 with
chlorosulfonic acid afforded two sulfonyl chloride regio-
somers, 25 (44%) and 26 (15%), that were easily
separated by flash chromatography. Two-dimensional
NMR studies (HMQC, HMBC) indicated that 25 and 26
were substituted at the 7- and 6-positions of the
aromatic ring, respectively. Compound 25 was carried
forward in the synthesis and the substitution pattern
of 25 was further confirmed from an X-ray crystal
structure of THIQ 21. Compound 25 was treated with
the requisite amines to form sulfonamides 27-32.
Simultaneous hydrolysis of the acetamide and piper-
idinamide of 27-32 was accomplished with 6 N HCl to
a Reagents: (a) acetyl chloride, 2 N NaOH; (b) BOP, HOBt,
piperidine; (c) chlorosulfonic acid; (d) 1° or 2° amine; (e) 6 N HCl;
(f) BH3‚THF.
produce 33-38. The crude amino acid hydrochloride
salts (33-36) were directly reduced with BH3‚THF to
form the corresponding THIQs (16-19). Compounds 37
and 38 were insoluble in the solvent (THF) used for the
reduction and were converted to their methyl esters (39
and 40) with thionyl chloride and methanol, followed
by reduction with LiBH4 to produce the corresponding
THIQs (20 and 21).

136 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Grunewald et al.
Scheme 2a
a Reagents: (a) SOCl2, MeOH; (b) LiBH4.
Figure 2. This figure shows the amino acid residues that
could interact with SK&F 29661 (5) within the hPNMT27 active
site. A Connolly (solvent accessible) surface of the active site
revealing SK&F 29661 (5) is also shown. Yellow lines indicate
possible hydrogen bonds. Carbon is white, nitrogen is blue,
oxygen is red, and sulfur is yellow. Hydrogens are not shown
for clarity.
Biochemistry
In the current study, human PNMT (hPNMT) with a
C-terminal hexahistidine tag was expressed in Escheri-
chia coli.22,24 The radiochemical assay conditions, previ-
ously reported for the bovine enzyme,25 were modified
to account for the high binding affinity of some inhibi-
tors. Inhibition constants were determined using four
concentrations of phenylethanolamine as the variable
substrate and three concentrations of inhibitor.
R2-Adrenergic receptor binding assays were performed
using cortex obtained from male Sprague-Dawley
rats.26 [3H]Clonidine was used as the radioligand to
define the specific binding and phentolamine was used
to define the nonspecific binding. Clonidine was used
as the ligand to define R-adrenergic binding affinity to
simplify the comparison with previous results.
Results and Discussion
We note three general trends from comparing the
biochemical data of 9 and 16-21 (Table 2) with those
of 8 and 10-15 (Table 1). First, 9 and 16-21 are more
potent than their corresponding fluoromethyl analogues
(8, 10-15). Second, while 9 and 16-21 show increased
affinity for the R2-adrenoceptor compared to their cor-
responding fluoromethyl analogues (8, 10-15), they are
still up to 6-fold more selective than 8 and 10-15. Third,
the PNMT inhibitory potencies of 16-19 and 10-13
followed a similar trend compared to their parent 9 or
8, respectively, whereby the N-substituted compounds
were less potent than their parent. The exceptions are
N-trifluoroethyl sulfonamide 20 and N-trifluoropropyl
sulfonamide 21, which are twice as potent as unsubsti-
tuted sulfonamide 9. This is a different trend than that
observed for the addition of a 2,2,2-trifluoroethyl or a
3,3,3-trifluoropropyl substituent to the sulfonamide
nitrogen of 8, whereby the addition of these substituents
did not increase PNMT inhibitory potency over the
parent compound (8).22
Molecular modeling studies were performed to inves-
tigate the differences in PNMT inhibitory potencies
between 3-hydroxymethyl-7-(N-substituted aminosul-
fonyl)-THIQs (9, 16-21) and their corresponding fluo-
romethyl analogues (8, 10-15). The X-ray crystal
structure of PNMT27 cocrystallized with AdoHcy (4) and
SK&F 29661 (5) has been reported. This crystal struc-
ture was used for the docking (AutoDock 3.0)28 studies
of these inhibitors. The docking of inhibitors into the
PNMT active site in this paper was performed on the
R-enantiomer, since previous studies on 3-fluoromethyl-
Figure 3. This figure shows 20 docked into the active site of
hPNMT27 and the amino acid residues that could interact with
20. A Connolly (solvent accessible) surface of the active site
revealing 20 is also shown. The docking indicates how the
3-hydroxymethyl group may interact with Glu219. Yellow lines
indicate possible hydrogen bonds. Carbon is white, nitrogen
is blue, fluorine is green, oxygen is red, and sulfur is yellow.
Hydrogens are not shown for clarity.
and 3-hydroxymethyl-7-substituted-THIQs indicated
that the R-enantiomer of these 3-substituted-THIQs is
preferred over the S-enantiomer in the hPNMT active
site.29,30 Docking of 3-hydroxymethyl-7-(N-substituted
aminosulfonyl)-THIQs (Figure 3 shows 20) into the
PNMT active site indicates that the hydroxy moiety
appears to form a hydrogen bond with Glu219. In
contrast, docking studies on 3-fluoromethyl-7-(N-sub-
stituted aminosulfonyl)-THIQs (Figure 4 shows 14)
indicated that the 3-fluoromethyl moiety appears to
make a hydrophobic contact with Tyr222.22 The docking
studies also show that the substituent on the sulfona-
mide nitrogen of the 3-hydroxymethyl compounds ap-
pears to bind in the auxiliary binding pocket in a
manner similar to that of their fluoromethyl analogue22
(Figures 3 and 4). Hydrophobic contacts appear to be
made between the sulfonamide substituent and Val53
and Arg44. These docking studies are consistent with
the biochemical data that suggest that 3-hydroxym-
ethyl-7-(N-substituted aminosulfonyl)-THIQs (9, 16-21;
Table 2) are more potent than their corresponding
3-fluoromethyl analogues (8, 10-15; Table 1), since a

Remarkably Potent and Selective PNMT Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 137
(Whitehouse, NJ). Flash chromatography was performed using
silica gel 60 (230-400 mesh) supplied by Universal Adsor-
bents, Atlanta, GA. Anhydrous tetrahydrofuran (THF) and
diethyl ether (Et2O) were distilled from sodium-benzophenone
ketyl. Hexanes refers to the mixture of hexane isomers (bp
40-70 °C). All reactions that required anhydrous conditions
were performed under argon, and all glassware was either
oven-dried or flame-dried before use. (()-1,2,3,4-Tetrahydro-
3-isoquinoline carboxylic acid hydrochloride (22) was obtained
from Sigma-Aldrich (Milwaukee, WI). AdoMet was obtained
from Sigma-Aldrich (St. Louis, MO). [methyl-3H]AdoMet and
[3H]clonidine were obtained from Perkin-Elmer (Boston, MA).
Radiochemical Assay of PNMT Inhibitors. The assay
used in this study has been modified from that described
previously.25 A typical assay mixture consisted of 25 µL of 0.5
M phosphate buffer (pH 8.0), 25 µL of 50 µM unlabeled
AdoMet, 5 µL of [methyl-3H]AdoMet, containing approximately
3 × 105 dpm (specific activity approximately 15 Ci/mmol), 25
µL of substrate solution (phenylethanolamine), 25 µL of
inhibitor solution, 25 µL of enzyme preparation (containing
30 ng hPNMT and 25 µg of bovine serum albumin), and
sufficient water to achieve a final volume of 250 µL. After
incubation for 30 min at 37 °C, the reaction mixture was
quenched by addition of 250 µL of 0.5 M borate buffer (pH
10.0) and was extracted with 2 mL of toluene/isoamyl alcohol
(7:3). A 1 mL portion of the organic layer was removed,
transferred to a scintillation vial, and diluted with cocktail for
counting. The mode of inhibition was ascertained to be
competitive in all cases reported in Tables 1 and 2 by
examination of the correlation coefficients (r2) for the fit
routines as calculated in the Enzyme Kinetics module (version
1.1) in SigmaPlot (version 7.0).31 While all Ki values reported
were calculated using competitive kinetics, it should be noted
that there was not always a great difference between the r2
values for the competitive model versus the noncompetitive
model. 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 using the Single Substrate-
Single Inhibitor routine in the Enzyme Kinetics module
(version 1.1) in SigmaPlot (version 7.0). For inhibitors with
apparent IC50 values less than 0.1 µM (as determined by a
preliminary screen of the compounds to be assayed), the
Enzyme Kinetics Tight Binding Inhibition routine was used
to calculate the Ki values.
r2-Adrenoceptor Radioligand Binding Assay. The ra-
dioligand receptor binding assay was performed according to
the method of U’Prichard et al.26 Male Sprague-Dawley rats
were decapitated, and the cortexes were dissected out and
homogenized in 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 50 000g 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 approximately 55 Ci/mmol, final concentration
2.0 nM), various concentrations of drugs, and an aliquot of
freshly resuspended tissue (800 µL) in 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 ice-cold 50 mM Tris buffer (pH 7.7 at
25 °C). The filters were counted in vials containing premixed
scintillation cocktail. Nonspecific binding was defined as the
concentration of bound ligand in the presence of 2 µM of
phentolamine. All assays were run in quadruplicate with five
inhibitor concentrations over a 16-fold range. IC50 values were
determined by a log-probit analysis of the data and Ki values
were determined by the equation Ki ) IC50 /(1 + [clonidine]/
KD), as all Hill coefficients were approximately equal to 1.
Figure 4. This figure shows 14 docked into the active site of
hPNMT and the amino acid residues that could interact with
14. A Connolly (solvent accessible) surface of the active site
revealing 14 is also shown. The docking indicates how the
3-fluoromethyl group may interact with Tyr222. Yellow lines
indicate possible hydrogen bonds. Carbon is white, nitrogen
is blue, fluorine is green, oxygen is red, and sulfur is yellow.
Hydrogens are not shown for clarity.
hydrogen bond results in a significant gain in energy
(ca. 3-5 kcal/mol) compared to a hydrophobic contact.
For example, N-trifluoroethyl sulfonamide 20 (3-CH2-
OH) is approximately six times more potent at inhibit-
ing PNMT than its 3-fluoromethyl analogue 14. Veri-
fication of these interactions will require crystal
structures of hPNMT cocrystallized with a 3-fluorom-
ethyl-7-substituted-THIQ and a similarly 7-substituted
3-hydroxymethyl-THIQ. It is not readily apparent from
the modeling why N-trifluoroethyl sulfonamide 20
(Figure 3) and N-trifluoropropyl sulfonamide 21 are
more potent than parent 9 (3-CH2OH-7-SO2NH2) nor
why N-trifluoroethyl sulfonamide 14 (Figure 4) and
N-trifluoropropyl sulfonamide 15 are equipotent to
parent 8 (3-CH2F-7-SO2NH2). However, the biochemical
data suggest that the 3-hydroxymethyl group and
7-trifluoroalkyl aminosulfonyl substituent of N-trifluo-
roethyl sulfonamide 20 and N-trifluoropropyl sulfona-
mide 21 are highly favored in the PNMT active site.
In conclusion, the addition of nonpolar substituents
to the sulfonamide nitrogen of 7-aminosulfonyl-3-hy-
droxymethyl-THIQ (9) has produced PNMT inhibitors
that are more lipophilic than parent 9 and that retained
excellent PNMT inhibitory potency and selectivity.
Compounds 20 (PNMT Ki ) 23 nM) and 21 (PNMT Ki
) 28 nM) are the most potent and selective (R2 Ki/PNMT
Ki g 15 000) PNMT inhibitors reported to date that, on
the basis of their calculated log P values, should have
significant penetration into the CNS. These compounds
may prove useful as pharmacological tools to elucidate
the role of CNS Epi.
Experimental 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-
Hoover melting point apparatus calibrated with known com-
pounds but are otherwise uncorrected. All proton (1H NMR)
and carbon (13C NMR) nuclear magnetic resonance spectra
were taken on a Bruker DRX-400 or a Bruker AM-500
spectrometer. Unless otherwise noted, all spectra were col-
lected on the Bruker DRX-400. High-resolution mass spectra
(HRMS) were obtained on a VG Analytical ZAB. Elemental
analyses were performed by Quantitative Technologies, Inc.
Molecular Modeling. Calculated log P values and Con-
nolly surfaces were generated in SYBYL on a Silicon Graphics
Octane workstation.20 Docking of the various inhibitors into
the PNMT active site was performed using AutoDock 3.0.28
The default settings for AutoDock were used. The compound

138 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Grunewald et al.
to be docked was initially overlayed with cocrystallized ligand
δ 7.74-7.67 (m, 2H), 7.43 (d, J ) 7.7 Hz, 1H), 5.49 (m, 1H),
4.94-4.72 (m, 2H), 3.70-3.58 (m, 4H), 3.27-3.22 (m, 2H),
3.12-3.08 (m, 2H), 3.01-2.98 (m, 1H), 2.93-2.88 (m, 1H), 2.27
(s, 3H), 1.73-1.46 (m, 4H), 1.08 (t, J ) 7.2 Hz, 3H); HRMS
(FAB+) m/z calcd for C19H28N3O4S (MH+) 394.1801 obsd
394.1802.
(()-2-Acetyl-3-(1-piperidinocarbonyl)-7-(N-propylami-
nosulfonyl)-1,2,3,4-tetrahydroisoquinoline (28). n-Propy-
lamine (0.32 mL, 3.90 mmol) was used to yield 28 as an oily
foamy solid (300 mg, 57%): 1H NMR (500 MHz, DMSO-d6) δ
7.63 (ex s, 1H), 7.58 (d, J ) 7.4 Hz, 1H), 7.53 (m, 1H), 7.40 (d,
J ) 8.0 Hz, 1H), 5.53 (m, 1H), 4.91-4.88 (m, 1H), 4.59-4.55
(m, 1H), 3.52-3.44 (m, 4H), 3.05-2.96 (m, 2H), 2.73-2.51 (m,
2H), 2.15 (s, 3H), 1.61-1.54 (m, 4H), 1.42-1.37 (m, 4H), 0.81
(t, J ) 7.3 Hz, 3H); HRMS (FAB+) m/z calcd for C20H30N3O4S
(MH+) 408.1957 obsd 408.1957.
(()-2-Acetyl-3-(1-piperidinocarbonyl)-7-(N-thiomor-
pholinosulfonyl)-1,2,3,4-tetrahydroisoquinoline (29).
Thiomorpholine (0.39 mL, 3.88 mmol) was used to yield 29 as
an oily foamy solid (322 mg, 55%): 1H NMR (500 MHz, DMSO-
d6) δ 7.70 (s, 1H), 7.62 (m, 1H), 7.46 (m, 1H), 5.53 (m, 1H),
4.95-4.92 (m, 1H), 4.60-4.57 (m, 1H), 3.52-3.44 (m, 4H), 3.17
(m, 4H), 3.08-3.03 (m, 2H), 2.69-2.66 (m, 4H), 2.15 (s, 3H),
1.61-1.39 (m, 6H); HRMS (FAB+) m/z calcd for C21H30N3O4S
(MH+) 452.1678 obsd 452.1665.
(()-2-Acetyl-7-(N-4-chlorophenylaminosulfonyl)-3-(1-
piperidinocarbonyl)-1,2,3,4-tetrahydroisoquinoline (30).
4-Chloroaniline (500 mg, 3.90 mmol) and pyridine (5 mL)
were used to yield 30 as an oily foamy solid (360 mg, 58%):
1H NMR (CDCl3) δ 7.66 (ex s, 1H), 7.62 (d, J ) 7.4 Hz, 1H),
7.35 (m, 2H), 7.23-7.20 (m, 2H), 7.09-7.05 (m, 2H), 5.46 (m,
1H), 4.91-4.65 (m, 2H), 3.65-3.52 (m, 4H), 3.22-3.03 (m, 2H),
2.23 (s, 3H), 1.72-1.52 (m, 6H); HRMS (FAB+) m/z calcd for
C23H27N3O4SCl (MH+) 476.1411 obsd 476.1384.
(()-2-Acetyl-3-(1-piperidinocarbonyl)-7-(N-2,2,2-triflu-
oroethylaminosulfonyl)-1,2,3,4-tetrahydroisoquinoline
(31). 2,2,2-Trifluoroethylamine (0.310 mL, 3.90 mmol) and
pyridine (2 mL) were used to yield 31 as an oily foamy solid
(357 mg, 61%): 1H NMR (500 MHz, CDCl3) δ 7.64-7.61 (m,
2H), 7.30 (m, 1H), 6.50 (ex s, 1H), 5.70 (m, 1H), 4.72 (m, 2H),
3.54-3.39 (m, 5H), 3.34 (m, 1H), 3.12 (m, 2H), 2.22 (s, 3H),
1.65-1.62 (m, 6H); HRMS (FAB+) m/z calcd for C19H25N3O4F3S
(MH+) 448.1518 obsd 448.1526.
(()-2-Acetyl-3-(1-piperidinocarbonyl)-7-(N-3,3,3-triflu-
oropropylaminosulfonyl)-1,2,3,4-tetrahydroisoquino-
line (32). 3,3,3-Trifluoropropylamine hydrochloride (291 mg,
1.95 mmol) and pyridine (2 mL) were used to yield 32 as an
oily foamy solid (386 mg, 64%): 1H NMR (400 MHz, MeOH-
d4) δ 7.76-7.71 (m, 2H), 7.44 (m, 1H), 5.52 (m, 1H), 4.90-
4.77 (m, 2H), 3.69-3.22 (m, 6H), 3.11 (m, 2H), 2.42-2.36 (m,
2H), 2.31 (s, 3H), 1.84-1.53 (m, 6H); HRMS (FAB+) m/z calcd
for C20H27N3O4F3S (MH+) 462.1674 obsd 462.1659.
General Procedure for 16-19 (selected procedure for 16).
Compound 27 (335 mg, 0.852 mmol) was dissolved in 6 N HCl
(3 mL) and heated to reflux for 12 h. The solution was
concentrated and evaporated to complete dryness to yield 33‚
HCl. Dry THF (15 mL) and 1 M BH3‚THF (3.40 mL, 2.66
mmol) were added, and the mixture was heated to reflux for
12 h. The solution was cooled with an ice bath and quenched
by the slow addition of MeOH until bubbling ceased. The
solution was concentrated in vacuo, and MeOH (20 mL) and 6
N HCl (5 mL) were added. The reaction mixture was heated
to reflux for 3 h. The MeOH was removed and the solution
was made basic with 10 N NaOH. The solution was saturated
with NaCl and extracted with EtOAc (3 × 50 mL). The organic
layers were combined, washed with brine, and dried over
sodium sulfate. The solution was concentrated in vacuo and
the crude oil was purified by flash chromatography. The
purified free amine was converted to its HCl salt by dissolving
it in MeOH and bubbling HCl(g) through the solution.
SK&F 29661 (5) and minimized with the Tripos force field.
(()-2-Acetyl-1,2,3,4-tetrahydro-3-isoquinolinecarbox-
ylic Acid (23). A similar procedure was followed as that
described by Shinkai et al.23 Amino acid 22 (1.00 g, 4.67 mmol)
was added to a solution of acetone (20 mL) and 2 N NaOH (15
mL). Acetyl chloride (0.801 mL, 9.35 mmol) and 2 N NaOH
were added simultaneously slowly at room temperature. The
solution was maintained at a pH greater than 10 and stirred
for 1 h. The acetone was evaporated in vacuo and the
remaining solution was made acidic with 3 N HCl. The
aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic layers were washed with brine and dried
over NaSO4. The solution was concentrated in vacuo to yield
a light yellow solid (23), which needed no further purification
(923 mg, 90%): mp 170-171 °C (lit.32 mp 171-173); 1H NMR
(400 MHz, DMSO-d6) δ 7.28-7.13 (m, 4H), 5.39-5.37 (m, 1H),
4.66 (s, 2H), 3.34-3.29 (m, 1H), 3.15-3.10 (m, 1H), 2.28 (s,
3H); HRMS (FAB+) m/z calcd for C12H14NO3 (MH+) 220.0974
obsd 220.1231.
(()-2-Acetyl-3-(1-piperidinocarbonyl)-1,2,3,4-tetrahy-
droisoquinoline (24). Compound 23 (228 mg, 1.04 mmol),
1-hydroxybenzotriazole (155 mg, 1.14 mmol), and N-methyl-
morpholine (0.23 mL, 2.08 mmol) were dissolved in dichlo-
romethane (5 mL) and cooled to 0 °C. Benzotriazol-1-yloxytris-
(dimethylamino)phosphonium hexafluorophosphate (506 mg,
1.14 mmol) was added to the solution and the mixture stirred
for 15 min. Piperidine (0.134 mL, 1.35 mmol) was added and
the solution was stirred at room temperature overnight. The
solution was washed with 3 N HCl and brine and dried over
Na2SO4. The solution was concentrated in vacuo to give a light
brown oil. The oil was subsequently purified by flash chroma-
tography eluting with EtOAC f 95:5 EtOAc:MeOH to yield
24 as a clear foamy oil (308 mg, 98%): 1H NMR (500 MHz,
CDCl3) δ 7.22-7.13 (m, 4H), 5.55 (m, 1H), 4.69 (m, 2H), 3.60-
3.37 (m, 4H), 3.06 (m, 2H), 2.24 (s, 3H), 1.67-1.27 (m, 6H);
HRMS (FAB+) m/z calcd for C17H23N2O2 (MH+) 287.1760 obsd
287.1764.
(()-2-Acetyl-3-(1-piperidinocarbonyl)-7-sulfonyl chlo-
ride-1,2,3,4-tetrahydroisoquinoline (25). Compound 24
(8.24 g, 28.8 mmol) was dissolved in chlorosulfonic acid (100
mL) and stirred at room temperature overnight. The reaction
mixture was carefully pipetted onto ice. The mixture was
extracted with ethyl acetate (3 × 75 mL), and the combined
organic layers were washed with brine and dried over sodium
sulfate. The solution was concentrated in vacuo to give a yellow
foamy solid. The two regioisomers 25 and 26 were separated
by flash chromatography using a gradient of 40:60 hexanes:
EtOAc f 30:70 hexanes:EtOAc f 20:80 hexanes:EtOAc f 10:
90 hexanes:EtOAc f 5:95 hexanes:EtOAc f EtOAc. The
desired 7-sulfonyl chloride (25) eluted off the column first as
a light yellow oily foam (4.87 g, 44%), followed by the 6-sulfonyl
regioisomer (26) also as a light oily foam (750 mg, 15%).
Compound 25 data: 1H NMR (500 MHz, CDCl3) δ 7.88 (m,
1H), 7.77 (s, 1H), 7.44 (d, J ) 8.2 Hz, 1H), 5.83 (m, 1H), 4.83-
4.71 (m, 2H), 3.68-3.59 (m, 2H), 3.45-3.33 (m, 2H), 3.22-
3.11 (m, 2H), 2.26 (s, 3H), 1.71-1.53 (m, 6H); HRMS (FAB+)
m/z calcd for C17H22N2O4SCl (MH+) 385.0989 obsd 385.0971.
General Procedure for 27-32. Sulfonyl chloride 25 (500
mg, 1.30 mmol) was dissolved in EtOAc (25 mL) and sat.
NaHCO3 (10 mL). The requisite amine was added and the
biphasic solution was stirred for 12 h. The organic phase was
separated and the aqueous phase was saturated with sodium
chloride. The aqueous phase was extracted with EtOAc. The
combined organic layers were washed with 3 N HCl that was
saturated with sodium chloride, washed with brine, and dried
over sodium sulfate. After concentration in vacuo, the crude
oily foam was purified by flash chromatography eluting with
a gradient of 10:1 EtOAc:hexanes f EtOAc.
(()-2-Acetyl-7-(N-ethylaminosulfonyl)-3-(1-piperidi-
nocarbonyl)-1,2,3,4-tetrahydroisoquinoline (27). Ethy-
lamine hydrochloride (213 mg, 3.90 mmol) was used to give
27 as a oily foamy solid (335 mg, 65%): 1H NMR (MeOH-d4)
(()-3-Hydroxymethyl-7-(N-ethylaminosulfonyl)-1,2,3,4-
tetrahydroisoquinoline Hydrochloride (16‚HCl). The crude
free amine was purified by flash chromatography eluting with

Remarkably Potent and Selective PNMT Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 139
10:1 EtOAc:MeOH. The hydrochloride salt was recrystallized
from EtOH/hexanes to yield 16‚HCl as white crystals (49 mg,
19%): mp 249-250 °C; 1H NMR (500 MHz, DMSO-d6) δ 9.55
(br ex s, 2H), 7.71-7.47 (m, 3H), 7.45 (d, J ) 8.3 Hz, 1H), 5.57
(ex s, 1H), 4.38 (s, 2H), 3.81-3.44 (m, 3H), 3.08-2.98 (m, 2H),
2.79-2.75 (m, 2H), 0.99 (t, J ) 7.2 MHz, 3H); 13C NMR
(400 MHz, DMSO-d6) δ 139.7, 137.4, 130.8, 130.7, 126.2, 125.8,
61.3, 54.7, 44.7, 38.4, 28.2, 15.6; HRMS (FAB+) m/z calcd
for C12H19N2O3S (MH+) 271.1116 obsd 271.1102. Anal.
(C12H18N2O3S‚HCl) C, H, N.
(()-3-Hydroxymethyl-7-(N-propylaminosulfonyl)-1,2,3,4-
tetrahydroisoquinoline Hydrochloride (17‚HCl). Com-
pound 28 (280 mg, 0.912 mmol) and 1 M BH3‚THF (2.74 mL,
2.74 mmol) were used. The crude free amine was purified by
flash chromatography eluting with 10:1 EtOAc:MeOH. The
HCl salt was recrystallized from EtOH/hexanes to yield 17‚
HCl as white crystals (73 mg, 33%): mp 209-210 °C; 1H NMR
(500 MHz, DMSO-d6) δ 9.48 (br ex m, 2H), 7.71-7.64 (m, 3H),
7.46 (d, J ) 8.0 Hz, 1H), 5.58 (ex s, 1H), 4.40 (s, 2H), 3.78-
3.55 (m, 3H), 3.03 (m, 2H), 2.69 (m, 2H), 1.39 (m, 2H), 0.80 (t,
J ) 7.2 MHz, 3H); 13C NMR (500 MHz, DMSO-d6) δ 139.3,
136.9, 130.4, 130.2, 125.6, 125.3, 60.8, 54.2, 44.7, 44.2, 27.7,
22.8, 11.5; HRMS (FAB+) m/z calcd for C13H21N2O3S (MH+)
285.1273 obsd 285.1271. Anal. (C13H20N2O3S‚HCl) C, H, N.
(()-7-(N-3,3,3-Trifluoropropylaminosulfonyl)-1,2,3,4-
tetrahydro-3-isoquinolinecarboxylic Acid Methyl Ester
(40). Compound 40 was prepared in a similar manner as
described for 39, except 38 (330 mg, 0.717 mmol) was used.
The crude solid was recrystallized from EtOAc/hexanes to yield
40 as light yellow crystals (128 mg, 49%): mp 124-125 °C;
1H NMR (400 MHz, MeOH-d4) δ 7.64 (d, J ) 7.9 Hz, 1H), 7.61
(s, 2H), 7.38 (d, J ) 8.1 Hz, 1H), 4.19-4.06 (m, 2H), 3.84-
3.80 (m, 4H), 3.24-3.19 (m, 1H), 3.09-3.01 (m, 3H), 2.44-
2.32 (m, 2H); HRMS (FAB+) m/ z calcd for C14H18N2O4F3S
(MH+) 367.0939 obsd 367.0949.
(()-3-Hydroxymethyl-7-(N-2,2,2-trifluoroethylamino-
sulfonyl)-1,2,3,4-tetrahydroisoquinoline (20). Compound
39 (114 mg, 0.324 mmol) was dissolved in THF (15 mL), and
2 M LiBH4 (0.491 mL, 0.972 mmol) was added. The solution
was stirred at room temperature for 12 h and quenched with
MeOH. The MeOH and THF were removed, and MeOH (20
mL) and 6 N HCl (5 mL) were added. The solution was gently
heated to reflux for 3 h. The reaction workup was similar to
that described for 16. The crude free amine was purified by
flash chromatography eluting with a gradient of 12:1 DCM:
MeOH f 10:1 DCM:MeOH. The free amine was recrystallized
from EtOH/hexanes to yield 20 as white crystals (48 mg,
46%): mp 172-173 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.45
(br ex s, 1H), 7.55-7.52 (m, 2H), 7.15 (d, J ) 8.0 Hz, 1H), 4.74
(ex s, 1H), 4.03-3.90 (m, 2H), 3.67-3.62 (m, 2H), 3.46-3.33
(m, 3H), 2.84-2.74 (m, 2H); 13C NMR (500 MHz, DMSO-d6) δ
140.7, 138.0, 137.8, 130.3, 124.7 (q, J ) 277 Hz, CF3), 124.4,
124.0, 65.2, 54.9, 47.8, 43.8 (q, J ) 34 Hz, CF3CH2), 31.7;
HRMS (FAB+) m/z calcd for C12H16N2O3F3S (MH+) 325.0834
obsd 325.0833. Anal. (C12H15N2O3F3S) C, H, N.
(()-3-Hydroxymethyl-7-(N-thiomorpholinosulfonyl)-
1,2,3,4-tetrahydroisoquinoline Hydrochloride (18‚HCl).
Compound 29 (264 mg, 0.585 mmol) and 1 M BH3‚THF (1.76
mL, 1.76 mmol) were used. The crude free amine was purified
by flash chromatography eluting with a gradient of 10:1
EtOAc:MeOH f 10:2 EtOAc:MeOH. The HCl salt was recrys-
tallized from EtOH/hexanes to yield 18‚HCl as white crystals
1
(104 mg, 49%): mp 242-243 °C; H NMR (500 MHz, DMSO-
(()-3-Hydroxymethyl-7-(N-3,3,3-trifluoropropylamino-
sulfonyl)-1,2,3,4-tetrahydroisoquinoline (21). Compound
21 was prepared in a similar manner as described for 20,
except that 40 (115 mg, 0.314 mmol) and 2 M LiBH4 (0.471
mL, 0.940 mmol) were used. The crude free amine was purified
by flash chromatography eluting with 10:1 DCM:MeOH. The
free amine was recrystallized from EtOH/hexanes to yield 21
as white crystals (64 mg, 60%): mp 145-146 °C; 1H NMR (500
MHz, DMSO-d6) δ 7.77 (br ex s, 1H), 7.53-7.49 (m, 2H), 7.31
(d, J ) 8.0 Hz, 1H), 4.79 (ex s, 1H), 4.04-3.91 (m, 2H), 3.46-
3.33 (m, 3H), 2.95-2.92 (m, 2H), 2.85-2.74 (m, 2H), 2.47-
2.42 (m, 2H); 13C NMR (400 MHz, DMSO-d6) δ 141.1, 138.3,
137.5, 130.9, 127.3 (q, J ) 275 Hz, CF3), 125.0, 124.6, 65.7,
55.4, 48.3, 36.9, 33.9 (q, J ) 26.8 Hz, CF3CH2), 32.2; HRMS
(FAB+) m/z calcd for C13H18N2O3F3S (MH+) 339.0990 obsd
339.0973. Anal. (C13H17N2O3F3S) C, H, N.
d6) δ 9.48 (br ex s, 2H), 7.70 (s, 1H), 7.61 (m, 1H), 7.50 (m,
1H), 5.55 (ex s, 1H), 4.40 (m, 2H), 3.78 (m, 1H), 3.65 (m, 1H),
3.53 (m, 1H), 3.21 (m, 4H), 3.10-3.03 (m, 2H), 2.68 (m, 4H);
13C NMR (400 MHz, DMSO-d6) δ 138.6, 135.0, 131.7, 130.9,
126.9, 126.5, 61.4, 54.7, 48.7, 44.9, 28.4, 27.3; HRMS (FAB+)
m/z calcd for C14H21N2O3S2 (MH+) 329.0994 obsd 329.0975.
Anal. (C14H20N2O3S2‚HCl) C, H, N.
(()-3-Hydroxymethyl-7-(N-4-chlorophenylaminosulfo-
nyl)-1,2,3,4-tetrahydroisoquinoline Hydrochloride (19‚
HCl). Compound 30 (360 mg, 0.758 mmol) and 1 M BH3‚THF
(2.27 mL, 2.27 mmol) were used. The crude free amine was
purified by flash chromatography eluting with a gradient of
10:1 EtOAc:MeOH f 10:2 EtOAc:MeOH. The HCl salt was
recrystallized from EtOH/hexanes to yield 19‚HCl as white
1
crystals (36 mg, 12%): mp 219-220 °C; H NMR (500 MHz,
DMSO-d6) δ 9.44 (br ex s, 2H), 7.72 (s, 1H), 7.63 (d, J ) 7.7
Hz, 1H), 7.42 (d, J ) 8.0 Hz, 1H), 7.31 (d, J ) 8.6 Hz, 2H),
7.13 (d, J ) 8.6 Hz, 2H), 5.51 (ex s, 1H), 4.33 (m, 2H), 3.75 (m,
1H), 3.59-3.50 (m, 2H), 3.03-2.90 (m, 2H); 13C NMR (400
MHz, DMSO-d6) δ 138.5, 138.3, 137.5, 131.4, 130.9, 130.1,
128.9, 126.2, 126.0, 122.2, 61.4, 54.6, 44.9, 28.4; HRMS (FAB+)
m/z calcd for C16H18N2O3SCl (MH+) 353.0727 obsd 353.0712.
Anal. (C16H17N2O3SCl‚HCl) C, H, N.
Acknowledgment. This research was supported by
NIH Grant HL 34193. The 500 MHz NMR spectrometer
was partially funded by the National Science Founda-
tion Grant CHE-9977422. We thank David VanderVelde
and Sarah Neuenswander for obtaining the 2-D NMR
spectra. We thank Douglas Powell of the X-ray crystal-
lography laboratory (National Science Foundation Grant
CHE-0079282) for the crystal structure of 21. We also
thank both Gerald Lushington of the molecular graphics
and modeling laboratory and Todd Williams of the mass
spectrometry laboratory for their assistance.
(()-7-(N-2,2,2-Trifluoroethylaminosulfonyl)-1,2,3,4-tet-
rahydro-3-isoquinolinecarboxylic Acid Methyl Ester
(39). Compound 37 (286 mg, 0.640 mmol) was dissolved in 6
N HCl (3 mL) and heated to reflux for 12 h. The solution was
concentrated and evaporated to dryness. The crude amino acid
(43‚HCl) was dissolved in MeOH (10 mL), and thionyl chloride
(0.122 mL, 0.960 mmol) was carefully added. The solution was
heated to reflux for 12 h and the MeOH was subsequently
removed in vacuo. Saturated sodium bicarbonate (10 mL) was
added and the mixture was extracted with EtOAc (3 × 30 mL).
The combined organic layers were washed with brine and dried
over sodium sulfate. The solution was concentrated in vacuo
to produce a crude solid that was recrystallized from EtOAC/
hexanes to yield 39 as light yellow crystals (136 mg, 60%): mp
Supporting Information Available: All elemental analy-
ses (C, H, N) for assayed compounds and X-ray crystal
structure data for 21. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
1
155-156 °C; H NMR (400 MHz, MeOH-d4) δ 7.66-7.61 (m,
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1H); HRMS (FAB+) m/z calcd for C13H16N2O4F3S (MH+)
353.0783 obsd 353.0755.

140 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
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