Inhibitors of phenylethanolamine N-methyltransferase that are predicted to penetrate the blood-brain barrier: Design, synthesis, and evaluation of 3-fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines that possess low affinity toward the α2-adrenoceptor

Article abstract of DOI:10.1021/jm0400653

(±)-7-Aminosulfonyl-3-fluoromethyl-1,2,3,4-tetrahydroisoquinoline (7) is one of the most potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT) reported to date, but a blood-brain barrier (BBB) model indicates that it cannot penetrate the BBB. To increase the lipophilicity of 7 by addition of a nonpolar substituent to the sulfonamide nitrogen, a small library of (±)-3-fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4- tetrahydroisoquinolines was synthesized and evaluated as inhibitors of PNMT and for affinity at the α₂-adrenoceptor. In addition, this library probed the PNMT active site surrounding the sulfonamide nitrogen of 7. Bulky substituents on the sulfonamide nitrogen are disfavored at the PNMT active site more so than at the α₂-adrenoceptor (thus reducing selectivity). On the other hand, alkyl chains on the sulfonamide nitrogen that contain an electron dense atom, such as a fluorine, are favored in the PNMT active site and possess little α₂-adrenoceptor affinity, thereby conferring good selectivity (>500). Several members of the library (8, 14, 17, and 18) have excellent PNMT inhibitory potency and selectivity and are predicted, on the basis of their ClogP value (>0.5), to penetrate the BBB to a significant extent. Compounds 17 and 18 are the most potent and selective PNMT inhibitors reported to date.

Full text of DOI:10.1021/jm0400653

J . Med. Chem. 2004, 47, 4483-4493
4483
In h ibitor s of P h en yleth a n ola m in e N-Meth yltr a n sfer a se Th a t Ar e P r ed icted To
P en etr a te th e Blood -Br a in Ba r r ier : Design , Syn th esis, a n d Eva lu a tion of
3-F lu or om eth yl-7-(N-su bstitu ted a m in osu lfon yl)-1,2,3,4-tetr a h yd r oisoqu in olin es
Th a t P ossess Low Affin ity tow a r d th e r2-Ad r en ocep tor 1
F. Anthony Romero,† Steven M. Vodonick,† Kevin R. Criscione,† Michael J . McLeish,‡ and Gary L. Grunewald*,†
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, and College of Pharmacy,
University of Michigan, Ann Arbor, Michigan 48109
Received March 12, 2004
(()-7-Aminosulfonyl-3-fluoromethyl-1,2,3,4-tetrahydroisoquinoline (7) is one of the most potent
and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT) reported to date,
but a blood-brain barrier (BBB) model indicates that it cannot penetrate the BBB. To increase
the lipophilicity of 7 by addition of a nonpolar substituent to the sulfonamide nitrogen, a small
library of (()-3-fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines
was synthesized and evaluated as inhibitors of PNMT and for affinity at the R2-adrenoceptor.
In addition, this library probed the PNMT active site surrounding the sulfonamide nitrogen of
7. Bulky substituents on the sulfonamide nitrogen are disfavored at the PNMT active site
more so than at the R2-adrenoceptor (thus reducing selectivity). On the other hand, alkyl chains
on the sulfonamide nitrogen that contain an electron dense atom, such as a fluorine, are favored
in the PNMT active site and possess little R2-adrenoceptor affinity, thereby conferring good
selectivity (>500). Several members of the library (8, 14, 17, and 18) have excellent PNMT
inhibitory potency and selectivity and are predicted, on the basis of their ClogP value (>0.5),
to penetrate the BBB to a significant extent. Compounds 17 and 18 are the most potent and
selective PNMT inhibitors reported to date.
In tr od u ction
Phenylethanolamine N-methyltransferase (PNMT:
EC 2.1.1.28) is the enzyme that catalyzes the terminal
step in epinephrine (Epi, 2) biosynthesis in which a
methyl group from S-adenosyl-L-methionine (AdoMet,
3) is transferred to the amino group of norepinephrine
(NE, 1) to produce Epi (2) and the cofactor product
S-adenosyl-L-homocysteine (AdoHcy, 4, Figure 1).2 In
the mid-1950s, Epi (2) was discovered in the central
nervous system (CNS) and was found to constitute ca.
F igu r e 1. The terminal step in Epi (2) biosynthesis is
catalyzed by the enzyme PNMT whereby an activated methyl
group from AdoMet (3) is transferred to NE (1) to produce Epi
5% of the total catecholamine content in the mammalian
brain.3,4 Soon thereafter it was shown that peripheral
Epi (2) did not cross the blood-brain barrier (BBB) but
(2) and AdoHcy (4).
that CNS Epi was being synthesized in the brain.5-8
CNS Epi (2) has been implicated in some of the
PNMT because it does not cross the BBB as shown by
neurodegeneration seen in Alzheimer’s disease,9 as well
autoradiographic15 and BBB model studies.16 A selectiv-
as in the regulation of blood pressure,10 respiration,11,12
ity of >500 is desired for a PNMT inhibitor in order to
body temperature,11,12 and the R2-adrenoceptor.13,14 To
properly interpret biological results. It was of interest
to see if these structures could be optimized to develop
an inhibitor that is both potent and selective for PNMT
and is capable of penetrating the BBB.
help elucidate the role of CNS Epi (2), both a potent
and selective inhibitor of PNMT is desired.
Compounds 5 and 6 are two of the most potent PNMT
inhibitors yet reported (Table 1). Unfortunately, these
Prior to the availability of the X-ray crystal structure
compounds are unsuitable for the study of the observed
of PNMT,17,18 we have used molecular modeling tech-
effects from inhibiting CNS PNMT for different reasons.
niques, such as quantitative structure-activity rela-
tionship studies and comparative molecular field analy-
Compound 5 shows high affinity for the R2-adrenoceptor
(selectivity R2 Ki/PNMT Ki ) 7), which complicates
sis, to identify the factors that influence the binding of
biological interpretation, and 6 only inhibits peripheral
ligands to PNMT versus the R2-adrenoceptor.19,20 It was
found that substitution on both the 3- and 7-positions
* To whom correspondence should be addressed. Phone: (785) 864-
of the 1,2,3,4-tetrahydroisoquinoline (THIQ) nucleus
4497. Fax: (785) 864-5326. E-mail: ggrunewald@ku.edu.
resulted in dramatic increases in selectivity compared
† University of Kansas.
‡ University of Michigan.
to the monosubstituted analogues (e.g., 6 versus 7).21
10.1021/jm0400653 CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/04/2004

4484 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Romero et al.
Ta ble 1. In Vitro PNMT and R2-Adrenoceptor Affinities of Some PNMT Inhibitorsa
hPNMT
Ki ( SEM (µM)
selectivity
(R2/hPNMT)
compd
R3
R7
R8
R2 Ki (µM)b
ClogPc
5
H
H
CH2F
CH2F
Cl
SO2NH2
SO2NH2
SO2NH-Ph-4-Cl
Cl
H
H
H
0.0031 ( 0.0006
0.28 ( 0.02
0.021
100
680
7.0
360
4500
520
2.75
-0.29
0.00
6
7d
8d
0.15 ( 0.01
0.27 ( 0.03
140
3.18
a
b
Biochemical data have been reported earlier for bovine PNMT. Here, we report data for recombinant human PNMT. In vitro activities
reported for the inhibition of binding of [3H]clonidine at the R2-adrenoceptor. c Calculated log P. Reference 16.
d
More specifically, the addition of an electron-withdraw-
ing group, such as a fluoromethyl substituent, to the
3-position of THIQ has led to more selective inhibitors.16
While 7 is still too polar (ClogP ) 0.00) to cross the BBB
(vide infra), it provides a potential lead for increasing
lipophilicity to impart CNS penetration.16
An in vitro model of BBB permeability using cultured
bovine brain microendothelial cells grown on polycar-
bonate membranes was developed at the University of
Kansas.22,23 A study from our lab using this model on a
small set of THIQs gave a good (r ) 0.79) correlation
between the ClogP (calculated log P)24 of the compound
and its permeability coefficient.16 This correlation in-
dicated that THIQs with a ClogP value of ca. 0.5 or
greater should cross the BBB.
Previous results from our laboratory showed that 8
retained good PNMT inhibitory potency and selectivity
and is predicted to penetrate the BBB (ClogP ) 3.18).16
The addition of a 4-Cl-phenyl substituent to the sul-
fonamide nitrogen of 7 suggested that there may be an
auxiliary binding pocket in the PNMT active site
surrounding the 7-position of THIQ. In this paper, we
report the synthesis (10-20 and 22-34) and biochemi-
cal evaluation (9-34) of a small library of (()-3-
fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-
THIQs. This series of 7-N-substituted sulfonamides was
designed to explore both the effects of steric bulk and
lipophilicity on PNMT inhibitory potency and R2-
adrenoceptor affinity. Compounds 9-19 will provide
information on increased lipophilicity and the interac-
tion of conformationally flexible substituents within the
area of the binding site surrounding the sulfonamide
binding region of 7. Compounds 20-29 will explore
steric bulk tolerance, and 27-29 will also investigate
conformational aspects of substituents in this binding
pocket. The final series, 30-34, will explore the effects
of substitution on a phenyl ring in the binding pocket
compared to 8, which possesses good PNMT inhibitory
potency and selectivity.
biphasic mixture of saturated sodium carbonate and
ethyl acetate and treated with the requisite primary or
secondary amine to produce sulfonamides 36-54
(Scheme 1). Use of these same reaction conditions to
form the substituted N-phenylsulfonamides resulted in
partial recovery of the starting material. It was found
that when 35 was dissolved in pyridine and treated with
the appropriate anilines, 55-59 were produced in good
yield. The resulting sulfonamides (36-59) were reduced
with BH3‚THF to yield the corresponding THIQs (10-
20 and 22-34). In most cases, the intermediate lactam
was isolated and characterized, but in some instances
it was carried forward to the reduction reaction. Flash
column chromatography was often required to purify the
free amine before conversion to the HBr or HCl salt.
For those salts that were hygroscopic and unable to be
recrystallized, the compounds were recrystallized as the
free base.
The discussion herein will use the recently published
crystal structure of human PNMT17 (hPNMT) cocrys-
tallized with SK&F 29661 (6) and AdoHcy (4) to
interpret the biochemical results obtained.
Ch em istr y
Compounds 9 and 21 were synthesized previously,16
but their biochemical data for hPNMT are reported
herein for the first time. Sulfonyl chloride 3516 was a
common starting material that could be readily con-
verted to the desired sulfonamides. By use of Schotten-
Baumann reaction conditions, 35 was dissolved in a
The majority of the amines were commercially avail-
able. 3,3,3-Trifluoropropylamine (64) and 4,4,4-trifluoro-
butylamine (65) have been reported in the literature,
but because their syntheses were not practical or

PNMT Inhibitors Predicted To Penetrate the BBB
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4485
Sch em e 1a
a
Reagents: (A) 1° or 2° amines, EtOAc/Na2CO3 or pyridine; (B)
BH3·THF.
Sch em e 2a
F igu r e 2. Part A (top) shows the amino acid residues that
could interact with SK&F 29661 (6) within the hPNMT active
site. Part B (bottom) is a Connolly (solvent accessible) surface
of the active site revealing SK&F 29661 (6). Carbon is white,
nitrogen is blue, oxygen is red, and sulfur is yellow. Hydrogens
are not shown for clarity.
a
Reagents: (A) PCl5, ether; (B) BH3·THF; (C) LiAlH4.
involved low molecular weight azides,25,26 an alternative
method was used (Scheme 2). The corresponding acid
(60 or 61) was converted to the amide by a literature
procedure.26 The optimum conditions required the acid
to be heated at reflux in ether in the presence of PCl5
to yield the acid chloride that was then transformed in
situ to the amide by saturation with ammonia gas. 4,4,4-
Trifluorobutyroamide (63) was reduced smoothly with
lithium aluminum hydride to yield 4,4,4-trifluorobutyl-
amine (65). However, reduction of 3,3,3-trifluoropropi-
onamide (62) with lithium aluminum hydride yielded a
mixture of 64 and n-propylamine. 3,3,3-Trifluoropropi-
onamide (62) was instead reduced with BH3‚THF to give
3,3,3-trifluoropropylamine (64) in good yield.
ligand to define R-adrenergic binding affinity to simplify
the comparison with previous results.
Resu lts a n d Discu ssion
Figure 2A shows the interaction between SK&F
29661 (6) and the amino acid residues surrounding the
PNMT active site.17 A Connolly24 (solvent accessible)
surface of the active site revealing SK&F 29661 (6) is
shown in Figure 2B. The addition of a fluoromethyl
group to 6, compound 7, resulted in increased PNMT
inhibitory potency and selectivity. Compound 7 was
docked into the active site using FlexX,30 and it appears
that the 3-fluoromethyl moiety is making a hydrophobic
contact to Tyr222 (picture not shown, but the same
interaction can be seen with 18 in Figure 3A). We have
docked the (R)-enantiomer into the PNMT active site
because an earlier study31 showed that (R)-8 was more
potent at bovine PNMT than (S)-8.32
The effect of straight-chain alkyl groups attached to
the sulfonamide nitrogen is shown in Table 2. Figure
3B shows a Connolly surface of the hPNMT active site.
The small pocket to the left of the sulfonamide moiety
is where we propose substituents on the sulfonamide
nitrogen are binding. Compound 11 was docked into the
PNMT active site using FlexX and reveals that the side
chain appears to make hydrophobic contacts with Arg44
and Val53 (picture not shown, but it is similar to 18
shown in Figure 3A). An increase in PNMT inhibitory
potency is not observed with 10 or 11, even though a
hydrophobic interaction could be gained. In order for
the substituent on the sulfonamide nitrogen to bind in
the pocket, it must displace the water adjacent to
Tyr126, resulting in the loss of the water-mediated
hydrogen bond between the sulfonamide oxygen, Tyr126,
Bioch em istr y
All compounds were evaluated as their hydrochloride
(9, 11-19, 21, and 26-30) or hydrobromide (20) salts
or as the free base (10, 22-25, and 31-34). Stock
inhibitor solutions (10 mM) were made by dissolving the
compound in H2O (for salts) or in 0.01 N HClO4 (for free
amines). In the current study, hPNMT was expressed
in E. coli27 and purified with a C-teminal hexahistidine
tag (see Experimental Section). The radiochemical assay
conditions, previously reported for the bovine enzyme,28
were modified to account for the high binding affinity
of some inhibitors. 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.29
[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

4486 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Romero et al.
Ta ble 2. In Vitro Activities of (()-3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines: Alkyl
Substituents
hPNMT
Ki ( SEM (µM)
R2
selectivity
(R2/hPNMT)
compd
R
Ki ( SEM (µM)
ClogP
9a
10
11
12
13
14
15
16
17
18
19
NHCH3
NHCH2CH3
3.7 ( 0.5
1.4 ( 0.1
1.7 ( 0.2
3.4 ( 0.5
5.1 ( 0.7
0.56 ( 0.05
2.6 ( 0.2
5.9 ( 0.6
0.13 ( 0.02
0.22 ( 0.02
9.2 ( 1.7
310 ( 10
550 ( 60
610 ( 60
260 ( 20
430 ( 50
440 ( 40
750 ( 70
350 ( 40
1200 ( 100
660 ( 80
350 ( 50
84
390
360
76
0.55
1.15
1.60
2.13
0.80
1.19
0.98
1.51
1.41
1.39
1.61
NH(CH2)2CH3
NH(CH2)3CH3
NH(CH2)2OCH3
NH(CH2)2OEt
NH(CH2)3OCH3
NH(CH2)3OEt
NHCH2CF3
84
790
290
59
9200
3000
38
NH(CH2)2CF3
NH(CH2)3CF3
a
Reference 16.
Manual docking of 14 shows that the sulfonamide
substituent extends outside the auxiliary pocket (picture
not shown). The longer side chain may be folding back
over on itself in order to fit within the auxiliary pocket
or the enzyme may be undergoing a conformational
change in order to accommodate the side chain. Cocrys-
tallization of 14 with hPNMT will be required to further
address this point. The electrostatic potential of the
PNMT active site surface indicates that atoms with high
electron density would be favored within the auxiliary
binding pocket (Figure 3B) and may lead to increased
PNMT inhibitory potency. The addition of a trifluoro-
methyl group to the alkyl chain (17 and 18) resulted in
a significant increase in potency and a decrease in R2-
adrenoceptor affinity, affording very selective inhibitors.
Compounds 17 and 18 not only gain a hydrophobic
interaction but are also highly favored because of the
electron dense fluorines (Figure 3A). Extending the
alkyl chain to a trifluorobutyl (19) results in a loss of
PNMT inhibitory potency, suggesting an unfavorable
contact. In this series several compounds (14, 17, and
18) have selectivities greater than 500 and, on the basis
of their ClogP values, are predicted to penetrate the
BBB.
F igu r e 3. Part A (top) shows 18 docked into the hPNMT
active site. Part B (bottom) is an electrostatic potential mapped
on the Connolly surface of the hPNMT active site revealing
18. The red-orange area indicates where electron density is
favored, whereas the blue area indicates where electron
density is disfavored. Green indicates neutrality. Carbon is
white, nitrogen is blue, fluorine is green, oxygen is red, and
sulfur is yellow. Hydrogens are not shown for clarity.
To probe the width and depth of the auxiliary binding
pocket, various bulky substituents (20-26) were added
to the sulfonamide nitrogen (Table 3). All compounds
in this series exhibited a significant loss of PNMT
inhibitory potency and selectivity. This indicates that
the auxiliary binding pocket of PNMT is of limited size
and cannot accommodate bulky substituents.
To probe the auxiliary pocket further, we constrained
some of the alkyl sulfonamide substituents to a ring
(Table 4). While the data reported in Table 3 indicated
that bulky substituents were not well tolerated, we
obtained moderate PNMT inhibitory potency and se-
lectivity with 29. In this series, 27 possesses the lowest
PNMT inhibitory potency. As the lipophilicity increases
by substituting the oxygen with a carbon (28), PNMT
inhibitory potency increases. Replacement of this carbon
with a sulfur (29) leads to improved PNMT inhibitory
potency; however, an increase in R2-adrenoceptor affin-
ity is observed resulting in only modest selectivity. It
is evident from this series that the combination of a
lipophilic, electron dense atom on the sulfonamide
and Arg44 (Figure 2). The methyl group of 9 is not able
to extend into this pocket and does not make a favorable
contact in the active site. The butyl side chain of 12 may
be too large to bind in this pocket, and therefore, a
decrease in PNMT inhibitory potency is observed.
Extending the side chain from an ethyl substituent (10)
to a propyl substituent (11) lowers the R2-adrenoceptor
affinity. Substituting an oxygen at various points along
the alkyl side chain (13-16) also results in decreasing
R2-adrenoceptor affinity. The ethoxyethylsulfonamide
(14) maintains moderate PNMT inhibitory potency and
selectivity. Interestingly, the sulfonamide substituent
of 14 extends further than the butyl side chain of 12,
yet moderate PNMT inhibitory potency is observed.

PNMT Inhibitors Predicted To Penetrate the BBB
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4487
Ta ble 3. In Vitro Activities of (()-3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines: Bulky
Substituents
hPNMT
Ki ( SEM (µM)
R2
selectivity
(R2/hPNMT)
compd
R
Ki ( SEM (µM)
ClogP
20
NHCH(CH3)2
NHCH2Ph
NH(CH2)2Ph
66 ( 8
26 ( 3
11 ( 2
93 ( 10
40 ( 6
33 ( 5
88 ( 9
580 ( 60
110 ( 10
58 ( 6
8.8
4.2
5.3
0.073
0.30
0.039
1.1
1.45
2.31
2.49
3.55
3.45
2.94
3.12
21a
22
23
NH(CH2)4Ph
6.8 ( 0.6
12 ( 1
24
NH(2-naphthyl)
NH(2-tetralyl)
NH(1-adamantyl)
25b
26
1.3 ( 0.1
100 ( 10
a
b
Reference 16. Assayed as a mixture of diastereomers.
Ta ble 4. In Vitro Activities of (()-3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines: Nonaromatic
Rings
hPNMT
Ki ( SEM (µM)
R2
selectivity
(R2/hPNMT)
compd
R
Ki ( SEM (µM)
ClogP
27
28
29
N(CH2CH2)2O
N(CH2CH2)2CH2
N(CH2CH2)2S
36 ( 4
4.6 ( 0.6
1.2 ( 0.2
2100 ( 200
820 ( 100
190 ( 20
58
180
160
0.86
2.15
1.56
Ta ble 5. In Vitro Activities of (()-3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines: Substituted
Phenyl Moieties
hPNMT
Ki ( SEM (µM)
R2
selectivity
(R2/hPNMT)
compd
R
Ki ( SEM (µM)
ClogP
30
31
32
8b
33
34
NH(4-F-Ph)
1.3 ( 0.1
18 ( 2
7.7 ( 1.1
0.27 ( 0.02
890 ( 130
250 ( 30
86 ( 10
280 ( 10
>1000a
66
16
>130
520
>1.1
>4.0
2.54
1.46
2.44
3.18
3.18
2.44
NH(4-OH-Ph)
NH(4-NO2-Ph)
NH(4-Cl-Ph)
NH(3-Cl-Ph)
NH(3-NO2-Ph)
140 ( 20
>1000a
>1000a
a
b
Could not be exactly determined because of precipitation at high assay concentrations. Reference 16.
nitrogen increases PNMT inhibitory potency. Compound
29 is predicted (ClogP ) 1.56) to penetrate into the CNS.
Previously we have reported that adding a p-chlo-
rophenyl substituent to 7 to give N-(p-chlorophenyl)-
sulfonamide 8 resulted in submicromolar PNMT inhibi-
tory potency and good selectivity, and this compound is
predicted to cross the BBB.16 To investigate this region
of the binding site, several substituted N-phenylsulfon-
amides were evaluated (Table 5). Meta-substituted
N-phenylsulfonamides (33 and 34) resulted in a signifi-
cant decrease in PNMT inhibitory potency and R2-
adrenoceptor affinity. The auxiliary pocket of PNMT
appears to be too narrow to accommodate the steric
requirements of the meta substituent. In this series,
good PNMT inhibitory potency is only observed with 8.
Again, having a lipophilic electron dense substituent in
the auxiliary binding pocket seems to provide excellent
PNMT inhibitory potency. Manual docking of 8 (picture
not shown) reveals that the 4-Cl-phenyl ring is likely
making hydrophobic contacts with Arg44 and Val53.
In conclusion, we have prepared a small library of (()-
3-fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-
THIQs and evaluated them for their inhibitory potency
and selectivity for PNMT versus the R2-adrenoceptor.
Substituents on the sulfonamide nitrogen that are
lipophilic and electron dense are highly favored within
an auxiliary binding pocket of the PNMT active site.
Bulky substituents are generally disfavored because of
the limited size of this pocket. These conclusions are
consistent with previous QSAR studies.19 The N-4-Cl-
phenyl (8), N-ethyl (10), N-propyl (11), N-ethoxyethyl
(14), N-trifluoroethyl (17), N-trifluoropropyl (18), and
N-thiomorpholino (29) sulfonamides represent com-
pounds that are predicted to cross the BBB and retain
moderate to excellent PNMT inhibitory potency. Four
of these compounds (8, 14, 17, and 18) exhibit selectivi-
ties greater than 500. With the addition of nonpolar
substituents to the sulfonamide nitrogen of 7, we have
succeeded in producing inhibitors that are more lipo-
philic than 7, that maintain PNMT inhibitory potency,
and that have decreased R2-adrenoceptor affinity. To
date, 17 and 18 represent the most potent and selective
inhibitors of hPNMT that, on the basis of their ClogP
values, are predicted to penetrate the BBB.

4488 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Romero et al.
30 ng of 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 1-5 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).33 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 concen-
trations 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.
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-
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. High-resolution mass spectra (HRMS) were
obtained on a VG Analytical ZAB. Elemental analyses were
performed by Quantitative Technologies, Inc. (Whitehouse,
NJ ). Flash chromatography was performed using silica gel 60
(230-400 mesh) supplied by Universal Adsorbents, Atlanta,
GA. All methanol (MeOH) and ethanol (EtOH) used were
anhydrous and were prepared by distillation over magnesium.
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. AdoMet was obtained from Sigma-Aldrich (St.
Louis, MO). [Methyl-3H]AdoMet and [3H]clonidine were ob-
tained from PerkinElmer (Boston, MA). Compounds 37, 40-
42, 51, 52, and 59 could not be obtained in pure form and were
carried forward to the corresponding THIQ and subsequently
purified.
P r ep a r a tion of 6X-His-Ta gged h P NMT. With pMM03927
as the template, Pfu DNA polymerase and the QuikChange
site-directed mutagenesis kit (Stratagene, La J olla, CA) were
employed to introduce a XhoI restriction site at the C-terminus
of the hPNMT gene. The forward primer used for the muta-
genesis, with the XhoI site italicized and the lower case letters
indicating a base change from wild type, is as follows:
5′-GAAGGTTGGGCTcgaGGATCCGGCTGCTAAC-3′. The re-
sultant plasmid was digested with NdeI/XhoI, and the 850 bp
fragment ligated into NdeI/XhoI digested pET24b (Novagen,
Madison, WI), resulting in a plasmid denoted pET24PNMT-
his. The fidelity of the mutagenesis was confirmed by sequenc-
ing at the University of Michigan DNA sequencing facility.
The vector used for protein expression, pET17PNMT-his, was
prepared by ligation of a NdeI/BlpI fragment of pET24PNMT-
his, containing MAH-his, into NdeI/BlpI digested pET17b
(Novagen).
Exp r ession a n d P u r ifica tion of 6X-His-Ta gged h P -
NMT. For expression of the his-tagged enzyme, pET17PNMT-
his was transformed into E. coli strain BL21(DE3)pLysS. After
inoculation and growth as described for wild-type hPNMT,27
protein expression was induced by the addition of 0.5 mM
IPTG. The cells were grown at 28 °C for 4 h prior to harvest,
then resuspended in a buffer comprising 20 mM sodium
phosphate and 300 mM NaCl, pH 7.0 (buffer A) containing
0.1 mM PMSF. After sonication and centrifugation, the cleared
lysate was loaded onto a Sepharose CL-6B Ni/nitriloacetic acid
(Ni-NTA) column (Qiagen, Valencia, CA) that had been
equilibrated in buffer A containing 20 mM imidazole. The
column was washed with the same buffer and again with
buffer A containing 50 mM imidazole. Finally, the hPNMT-
his was eluted with buffer A containing 250 mM imidazole.
The fractions containing hPNMT-his were pooled and dialyzed
against a storage buffer consisting of 20 mM Tris (pH 7.2)/
15% glycerol/1 mM EDTA/0.5 mM DTT, before being concen-
trated and stored at 4 °C. The enzyme purified in this manner
was better than 95% pure by SDS-PAGE analysis (data not
shown). The kinetic constants were essentially identical (data
not shown) to those obtained for the wild-type enzyme from
earlier studies.27
R2-Ad r en ocep tor Ra d ioliga n d Bin d in g Assa y. The ra-
dioligand receptor binding assay was performed according to
the method of U’Prichard et al.29 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 thrice
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. 55 Ci/mmol, final concentration of 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 concen-
trations over a 16-fold range. IC50 values were determined by
a log-probit analysis of the data, and Ki values were deter-
mined by the equation Ki ) IC50 /(1 + [clonidine]/KD) because
all Hill coefficients were approximately equal to 1.
Molecu la r Mod elin g. All molecular modeling was per-
formed using the SYBYL software package on a Silicon
Graphics Octane or O2 workstation.24 The log P (ClogP) values
were calculated with the ClogP function in SYBYL. Docking
of the various inhibitors into the PNMT active site was
performed using FlexX30 with the CScore34 scoring function.
In some cases manual docking was performed. The default
settings for FlexX were used. A high-scoring conformation was
chosen, and the protein/ligand complex was minimized within
a 6 Å radius of the inhibitor using the MMFF94 force field.
Manual docking of other inhibitors involved the overlaying of
the inhibitor with a FlexX docked inhibitor followed by a
similar minimization of the protein/ligand complex. Connolly
surfaces were generated using the MOLCAD surface function
in SYBYL.
3,3,3-Tr iflu or op r op yla m in e Hyd r och lor id e (64‚HCl).26
3,3,3-Trifluoropropionic acid (60, 1.77 g, 14.1 mmol) was
dissolved in ether (70 mL) and was cooled in an ice-water
bath. Phosphorus pentachloride (2.68 g, 21.2 mmol) was added
in one portion, and the solution was heated to reflux for 2.5 h.
Ether (25 mL) was added to the solution, that was cooled with
an ice-water bath. Ammonia(g) was bubbled into the cooled
solution for 30 min. The solution was concentrated, and
saturated NaHCO3 (25 mL) was added. The solution was
extracted with EtOAc (4 × 35 mL) and dried over Na2SO4. The
crude solid was recrystallized in EtOAc/hexanes to yield the
Ra d ioch em ica l Assa y of P NMT In h ibitor s. The assay
used in this study has been modified from that described
previously.28 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

PNMT Inhibitors Predicted To Penetrate the BBB
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4489
amide (62) as clear needles (1.15 g, 65%). Trifluoropropiona-
mide (62, 599 mg, 4.72 mmol) was dissolved in THF (20 mL),
and BH3‚THF (14.2 mL, 14.2 mmol) was added. The solution
was stirred at 55 °C for 30 h. To this solution, MeOH (20 mL)
and concentrated HCl (10 mL) were added, and the solution
was heated to 60 °C for 4 h. The solution was then concen-
trated in vacuo. Water (15 mL) was added to the solution,
which was cooled in an ice-water bath and made basic (pH
14) with KOH pellets, not allowing the solution to get to warm.
The basic solution was extracted with ether (4 × 50 mL), and
the combined organic extracts were washed with brine and
dried over NaSO4. The solvent was filtered, and dry HCl(g) was
bubbled through the solution. The solvent was removed in
vacuo, and the solid was recrystallized from EtOH/EtOAc to
yield 64‚HCl as white crystals (426 mg, 61%): mp 125-126
°C; 1H NMR (400 MHz, DMSO-d6) δ 8.70 (ex s, 3H, NH), 3.03-
2.99 (m, 2H), 2.78-2.73 (m, 2H); 13C NMR (400 MHz, DMSO-
d6) δ 126.8 (q, J ) 274 Hz), 33.2 (d, J ) 4 Hz), 31.6 (q, J ) 29
Hz); HRMS (FAB+) m/z calcd for C3H6NF3 (MH+) 114.0531,
obsd 114.0539.
4,4,4-Tr iflu or ob u t yla m in e Hyd r och lor id e (65‚HCl).25
4,4,4-Trifluorobutyric acid (61, 1.08 g, 7.61 mmol) was dis-
solved in ether (50 mL) and was cooled in an ice-water bath.
Phosphorus pentachloride (3.16 g, 9.13 mmol) was added in
one portion, and the solution was heated to reflux for 3 h. Ether
(25 mL) was added to the solution, which was cooled with an
ice-water bath. Ammonia(g) was bubbled into the cooled
solution for 30 min. The solution was concentrated, and
saturated NaHCO3 (25 mL) was added. The solution was
extracted with EtOAc (4 × 35 mL) and dried over Na2SO4. The
crude solid was recrystallized in EtOAc/hexanes to yield the
amide (63) as clear needles (748 mg, 70%). Trifluorobutyroa-
mide (63, 1.30 g, 9.22 mmol) was dissolved in ether (75 mL),
and LiAlH4 (723 mg, 19.0 mmol) was added. The mixture was
heated to reflux for 6 h, the reaction was quenched using the
Fieser and Fieser method,35 and the mixture was stirred for
30 min. The mixture was filtered through a Celite pad and
concentrated in vacuo to yield the crude amine. The amine
was dissolved in ether, dry HCl(g) was bubbled through the
solution, and the solvent was removed under reduced pressure.
The solid was recrystallized from EtOAc to yield 65‚HCl as
white crystals (1.11 g, 74%): mp 130-131 °C; 1H NMR
(DMSO-d6) δ 8.32 (ex s, 3H, NH), 2.89-2.82 (m, 2H), 2.47-
2.34 (m, 2H), 1.85-1.77 (m, 2H); 13C NMR (DMSO-d6) δ 128.2
(q, J ) 275 Hz), 38.4, 30.7 (q, J ) 28 Hz), 31.6 (d, J ) 3 Hz);
HRMS (FAB+) m/z calcd for C4H8NF3 (MH+) 128.0687, obsd
128.0698.
4.42-4.35 (m, 1H, CH2F), 3.96-3.90 (m, 1H, H-3), 3.24-3.18
(m, 1H, H-4), 3.06-3.00 (m, 1H, H-4), 2.72 (q, J ) 6.2 Hz, 2H),
1.34 (quintet, J ) 6.8 Hz, 2H), 1.22 (sextet, J ) 7.2 Hz, 2H),
0.79 (t, J ) 7.3 Hz, 3H); HRMS (FAB+) m/z calcd for
C
14H20N2O3FS (MH+) 315.1179, obsd 315.1160.
(()-3-F lu or om eth yl-7-(N-3-m eth oxyp r op yla m in osu lfo-
n yl)-3,4-d ih yd r oisoqu in olin -1-(2H)-on e (41). Recrystalli-
zation from EtOH/hexanes yielded 41 as white crystals (152
1
mg, 64%): mp 161-162 °C; H NMR (DMSO-d6) δ 8.39 (ex s,
1H, NH), 8.24 (m, 1H), 7.88-7.85 (m, 1H), 7.71-7.68 (ex m,
1H), 7.56 (d, J ) 8.0 Hz, 1H), 4.54-4.47 (m, 1H, CH2F), 4.42-
4.36 (m, 1H, CH2F), 3.96-3.91 (m, 1H, H-3), 3.28-3.18 (m,
3H), 3.15 (s, 3H), 3.05-3.00 (m, 1H, H-4), 2.78 (q, J ) 6.7 Hz,
2H), 1.59 (quintet, J ) 6.6 Hz, 2H); HRMS (FAB+) m/z calcd
for C14H20N2O4FS (MH+) 331.1128, obsd 331.1141.
(()-3-F lu or om eth yl-7-(N-2,2,2-tr iflu or oeth yla m in osu l-
fon yl)-3,4-d ih yd r oisoqu in olin -1-(2H)-on e (43). Recrystal-
lization from EtOH/hexanes yielded 43 as white crystals (107
1
mg, 42%): mp 230-231 °C; H NMR (400 MHz, DMSO-d6) δ
8.73 (ex s, 1H, NH), 8.40 (ex s, 1H, NH), 8.26 (s, 1H), 7.91 (m,
1H), 7.56 (d, J ) 8.1 Hz, 1H), 4.51-4.49 (m, 1H, CH2F), 4.39-
4.37 (m, 1H, CH2F), 3.95-3.86 (m, 1H, H-3), 3.66 (m, 2H), 3.21
(m, 1H, H-4), 3.03 (m, 1H, H-4); HRMS (FAB+) m/z calcd for
C
12H13N2O3F4S (MH+) 341.0583, obsd 341.0574.
(()-3-F lu or om et h yl-7-(N-3,3,3-t r iflu or op r op yla m in o-
su lfon yl)-3,4-d ih yd r oisoqu in olin -1-(2H)-on e (44). Recrys-
tallization from EtOH/hexanes yielded 44 as white crystals
1
(100 mg, 39%): mp 140-141 °C; H NMR (400 MHz, MeOH-
d4) δ 8.42 (d, J ) 9.6 Hz, 1H), 7.99 (m, 1H), 7.59 (m, 1H), 4.58-
4.54 (m, 1H, CH2F), 4.46-4.42 (m, 1H, CH2F), 4.19-4.13 (m,
1H, H-3), 3.33-3.24 (m, 2H), 3.18-3.10 (m, 2H), 2.44-2.38
(m, 2H); HRMS (FAB+) m/z calcd for C13H15N2O3F4S (MH+)
355.0740, obsd 355.0734.
(()-3-F lu or om eth yl-7-(N-4,4,4-tr iflu or obu tyla m in osu l-
fon yl)-3,4-d ih yd r oisoqu in olin -1-(2H)-on e (45). Recrystal-
lization from EtOH/hexanes yielded 45 as white crystals (189
1
mg, 71%): mp 197-198 °C; H NMR (400 MHz, MeOH-d4) δ
8.41 (s, 1H), 7.97 (d, J ) 7.7 Hz, 1H), 7.56 (d, J ) 7.8 Hz, 1H),
4.56-4.53 (m, 1H, CH2F), 4.45-4.42 (m, 1H, CH2F), 4.04-
4.00 (m, 1H, H-3), 3.33-3.24 (m, 1H, H-4), 3.15-3.09 (m, 1H,
H-4), 2.97-2.94 (m, 2H), 2.23-2.14 (m, 2H), 1.73-1.69 (m, 2H);
HRMS (FAB+) m/z calcd for C14H17N2O3F4S (MH+) 369.0896,
obsd 369.0901.
(()-3-Flu or om eth yl-7-(N-1-m eth yleth ylam in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (46). Recrystallization
from EtOH/hexanes yielded 46 as white crystals (116 mg,
74%): mp 186-187 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.38
(ex s, 1H, NH), 8.27 (ex s, 1H, NH), 7.89 (d, J ) 6.5 Hz, 1H),
7.69 (d, J ) 7.2 Hz, 1H), 7.55 (d, J ) 8.0 Hz, 1H), 4.51-4.49
(m, 1H, CH2F), 4.39-4.38 (m, 1H, CH2F), 3.96-3.90 (m, 1H,
H-3), 3.28-3.18 (m, 2H), 3.03 (m, 1H, H-4), 0.95 (d, J ) 6.5
Hz, 6H); HRMS (FAB+) m/z calcd for C13H18N2O3FS (MH+)
301.1022, obsd 301.1028.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
36-59. Sulfonyl chloride 3516 (200 mg, 0.721 mmol) was
dissolved in
a biphasic mixture of EtOAc (15 mL) and
saturated Na2CO3 (10 mL). The requisite amine (3 equiv) was
added to the reaction, and the mixture was stirred for 4 h.
For compounds 43-45, pyridine (2 equiv) was also added to
the reaction mixture. The organic phase was removed, washed
with 3 N HCl (3 × 50 mL) and brine (30 mL), and dried over
Na2SO4. The solvent was removed under reduced pressure to
yield an off-white solid, which was purified by recrystallization
or column chromatography.
(()-3-Flu or om eth yl-7-(N-2-ph en yleth yla m in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (47). Recrystallization
from EtOAc/hexanes yielded 47 as white crystals (209 mg,
1
80%): mp 162-163 °C; H NMR (400 MHz, MeOH-d4) δ 8.37
(s, 1H), 7.93-7.91 (m, 1H), 7.50 (d, J ) 8.0 Hz, 1H), 7.25-
7.11 (m, 5H), 4.55 (m, 1H, CH2F), 4.44 (m, 1H, CH2F), 4.06-
3.98 (m, 1H, H-3), 3.26-3.22 (m, 1H, H-4), 3.14-3.06 (m, 3H),
2.76-2.72 (m, 2H); HRMS (FAB+) m/z calcd for C18H20N2O3-
FS (MH+) 363.1179, obsd 363.1176.
(()-3-Flu or om eth yl-7-(N-eth yla m in osu lfon yl)-3,4-d ih y-
dr oisoqu in olin -1-(2H)-on e (36). Recrystallization from EtOAc/
hexanes yielded 36 as white crystals (156 mg, 76%): mp 188-
1
189 °C; H NMR (400 MHz, DMSO-d6) δ 8.38 (ex s, 1H, NH),
8.25 (s, 1H), 7.88-7.86 (m, 1H), 7.65 (ex s, 1H, NH), 7.56 (d,
J ) 8.0 Hz, 1H), 4.54-4.47 (m, 1H, CH2F), 4.41-4.36 (m, 1H,
CH2F), 3.96-3.91 (m, 1H, H-3), 3.23-3.18 (m, 1H, H-4), 3.06-
3.00 (m, 1H, H-4), 2.77 (q, J ) 7.2 Hz, 2H), 0.97 (t, J ) 7.2
Hz, 3H); HRMS (FAB+) m/z calcd for C12H16N2O3FS (MH+)
287.0866, obsd 287.0846.
(()-3-Flu or om eth yl-7-(N-4-ph en ylbu tylam in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (48). Recrystallization
from EtOAc/hexanes yielded 48 as white crystals (188 mg,
81%): mp 159-160 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.39
(ex s, 1H, NH), 8.25 (s, 1H), 7.86 (d, J ) 7.7 Hz, 1H), 7.70-
7.68 (ex m, 1H, NH), 7.55 (d, J ) 7.9 Hz, 1H), 7.26 (m, 2H),
7.17-7.12 (m, 3H), 4.54-4.49 (m, 1H, CH2F), 4.42-4.35 (m,
1H, CH2F), 3.95-3.89 (m, 1H, H-3), 3.20 (m, 1H, H-4), 3.02
(m, 1H, H-4), 2.76 (q, J ) 6.2 Hz, 2H), 2.50-2.47 (m, 2H), 1.51
(quintet, J ) 7.9 Hz, 2H), 1.38 (q, J ) 7.1 Hz, 2H); HRMS
(FAB+) m/z calcd for C20H24N2O3FS (MH+) 391.1492, obsd
391.1507.
(()-3-F lu or om et h yl-7-(N-b u t yla m in osu lfon yl)-3,4-d i-
h yd r oisoqu in olin -1-(2H)-on e (38). Recrystallization from
EtOAc/hexanes yielded 38 as white crystals (188 mg, 83%):
1
mp 201-202 °C; H NMR (400 MHz, DMSO-d6) δ 8.45 (ex s,
1H, NH), 8.24 (s, 1H), 7.87 (d, J ) 7.9 Hz, 1H), 7.68-7.65 (ex
m, 1H, NH), 7.56 (d, J ) 8.0 Hz, 1H), 4.54-4.47 (m, 1H, CH2F),

4490 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Romero et al.
1
(()-3-F lu or om et h yl-7-(N-2-n a p h t h yla m in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (49). The crude residue
was purified by flash chromatography eluting with CH2Cl2/
MeCN (2.5:1) to yield an off-white solid. Recrystallization from
EtOAc/hexanes yielded 49 as white crystals (164 mg, 59%):
mp 209-210 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.60 (ex s,
1H, NH), 8.34 (ex s, 1H, NH), 8.30 (s, 1H), 7.87 (d, J ) 8.0 Hz,
1H), 7.82-7.76 (m, 3H), 7.58 (s, 1H), 7.48-7.29 (m, 4H), 4.48-
4.41 (m, 1H, CH2F), 4.34-4.29 (m, 1H, CH2F), 3.89-3.76 (m,
1H, H-3), 3.12 (m, 1H, H-4), 2.94 (m, 1H, H-4); HRMS (FAB+)
m/z calcd for C20H18N2O3FS (MH+) 385.1022, obsd 385.1040.
(()-3-F lu or om eth yl-7-(N-2-tetr a lyla m in osu lfon yl)-3,4-
d ih yd r oisoqu in olin -1-(2H)-on e (50). Compound 50 is re-
ported as a mixture of diastereomers. Recrystallization from
EtOAc/hexanes yielded 50 as white crystals (251 mg, 85%):
mp 158-159 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H,
NH), 8.32 (s, 1H), 7.95-7.93 (m, 2H), 7.56 (d, J ) 8.0 Hz, 1H),
7.05-7.03 (m, 3H), 6.94 (d, J ) 6.5 Hz, 1H), 4.53-4.51 (m,
1H, CH2F), 4.41-4.39 (m, 1H, CH2F), 3.97-3.91 (m, 1H, H-3),
3.34-3.32 (m, 1H), 3.22 (m, 1H, H-4), 3.04 (m, 1H, H-4), 2.79-
2.61 (m, 4H), 1.83-1.80 (m, 1H), 1.65-1.57 (m, 1H); HRMS
(FAB+) m/z calcd for C20H22N2O3FS (MH+) 389.1335, obsd
389.1351.
(()-3-F lu or om eth yl-7-(N-p ip er id in osu lfon yl)-3,4-d ih y-
dr oisoqu in olin -1-(2H)-on e (53). Recrystallization from EtOAc/
hexanes yielded 53 as white crystals (202 mg, 86%): mp 189-
190 °C; 1H NMR (400 MHZ, DMSO-d6) δ 8.44 (ex s, 1H, NH),
8.11 (d, J ) 1.7 Hz, 1H), 7.83 (m, 1H), 7.61 (d, J ) 8.0 Hz,
1H), 4.53-4.50 (m, 1H, CH2F), 4.41-4.39 (m, 1H, CH2F), 3.98-
3.92 (m, 1H, H-3), 3.23 (m, 1H, H-4), 3.05 (m, 1H, H-4), 2.91-
2.85 (m, 4H), 1.55-1.54 (m, 4H), 1.37-1.36 (m, 2H); HRMS
(FAB+) m/z calcd for C12H20N2O3FS (MH+) 327.1179, obsd
327.1104.
33%): mp 235-236 °C; H NMR (400 MHz, MeOH-d4) δ 8.45
(s, 1H), 8.21 (d, J ) 9.1 Hz, 2H), 8.01 (m, 1H), 7.51 (d, J ) 8.0
Hz, 1H), 7.33 (d, J ) 9.2 Hz, 2H), 4.55-4.47 (m, 1H, CH2F),
4.43-4.35 (m, 1H, CH2F), 4.02-3.94 (m, 1H, H-3), 3.21 (m,
1H, H-4), 3.03 (m, 1H, H-4); HRMS (FAB+) m/z calcd for
C
16H15N3O5FS (MH+) 380.0716, obsd 380.0701.
(()-3-Flu or om eth yl-7-N-(3-ch lor oph en ylam in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (58). Recrystallization
from EtOAc/hexanes yielded 58 as white crystals (218 mg,
82%): mp 114-115 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.66
(ex s, 1H, NH), 8.39 (ex s, 1H, NH), 8.24 (s, 1H), 7.84 (d, J )
7.8 Hz, 1H), 7.53 (d, J ) 8.0 Hz, 1H), 7.27 (t, J ) 8.1 Hz, 1H),
7.11-7.06 (m, 3H), 4.50-4.43 (m, 1H, CH2F), 4.39-4.31 (m,
1H, CH2F), 3.92-3.87 (m, 1H, H-3), 3.16 (m, 1H, H-4), 2.98
(m, 1H, H-4); HRMS (FAB+) m/z calcd forC16H15N2O3ClFS
(MH+) 369.0476, obsd 369.0491.
Gen er a l P r oced u r e for th e P r ep a r a tion of 10-20 a n d
22-34. The appropriate dihydroisoquinolone (36-59, 100-
250 mg) was dissolved in THF (25 mL), and 1 M BH3‚THF (3
equiv) was added. The solution was heated at reflux for 12 h.
The solution was cooled in an ice-water bath, and MeOH (10
mL) was carefully added dropwise until bubbling ceased. The
solvent was removed under reduced pressure. A solution of 6
N HCl (5 mL) and MeOH (20 mL) was added slowly to the
remaining reaction residue. The mixture was heated to reflux
for 3 h. The solution was concentrated under reduced pressure,
made basic with 15% KOH, and extracted with EtOAc (4 ×
50 mL). The combined organic extracts were washed with brine
and dried over Na2SO4. The solvent was removed under
reduced pressure to yield a crude solid, which often required
chromatography. The purified solid was dissolved in EtOH,
and HBr(g) or HCl(g) was bubbled through the solution. The
solvent was removed under reduced pressure, and the HBr or
HCl salt was purified by recrystallization.
(()-3-F lu or om eth yl-7-(N-th iom or p h olin osu lfon yl)-3,4-
d ih yd r oisoqu in olin -1-(2H)-on e (54). Recrystallization from
EtOAc/hexanes yielded 54 as white crystals (181 mg, 73%):
(()-3-F lu or om et h yl-7-(N-et h yla m in osu lfon yl)-1,2,3,4-
tetr a h yd r oisoqu in olin e (10). Recrystallization of the free
amine from EtOAc/hexanes yielded 10 as white crystals (106
mg, 76%): mp 139-140 °C; IR (Teflon film) 3296, 3048, 1600,
1471, 1379, 1348, 1041 cm-1; 1H NMR (400 MHz, DMSO-d6) δ
10.17 (br ex s, 1H, NH), 7.73 (s, 1H), 7.68-7.66 (m, 2H), 7.48
(d, J ) 8.0 Hz, 1H), 4.99-4.68 (m, 2H, CH2F), 4.48 (s, 2H,
H-1), 3.87-3.22 (m, 1H, H-3), 3.15-3.07 (m, 2H, H-4), 2.79-
2.74 (m, 2H), 0.99 (t, J ) 6.8 Hz, 3H); 13C NMR (DMSO-d6) δ
139.8, 136.8, 130.7, 130.7, 126.2, 125.8, 83.0 (d, J ) 168 Hz,
CH2F), 52.7 (d, J ) 19 Hz, C-3), 44.6, 38.4, 27.0, 15.6; HRMS
(FAB+) m/z calcd for C12H18N2O2FS (MH+) 273.1073, obsd
273.1060. Anal. (C12H17N2O2FS) C, H, N.
1
mp 210-211 °C; H NMR (400 MHz, DMSO-d6) δ 8.46 (ex s,
1H, NH), 8.12 (d, J ) 1.7 Hz, 1H), 7.85 (m, 1H), 7.63 (d, J )
8.0 Hz, 1H), 4.55-4.48 (m, 1H, CH2F), 4.44-4.36 (m, 1H,
CH2F), 3.99-3.93 (m, 1H, H-3), 3.27-3.20 (m, 5H), 3.06 (m,
1H, H-4), 2.68 (t, J ) 5.1 Hz, 4H); HRMS (FAB+) m/z calcd
for C14H18N2O3FS2 (MH+) 345.0743, obsd 345.0740.
Gen er a l P r oced u r e for th e P r ep a r a tion of 55-59.
Sulfonyl chloride 35 (200 mg, 0.721 mmol) was dissolved in
pyridine (5 mL), treated with the requisite aniline (4 equiva-
lents), and stirred for 4 h. A majority of the pyridine was
removed under reduced pressure, and the resulting residue
was dissolved in EtOAc (25 mL). The organic phase was
washed with 6 N HCl (3 × 50 mL), washed with brine (30 mL),
and dried over Na2SO4. The solvent was removed under
reduced pressure to yield a crude solid, which was purified by
recrystallization or column chromatography.
(()-3-F lu or om eth yl-7-(N-p r op yla m in osu lfon yl)-1,2,3,4-
tetr a h yd r oisoqu in olin e Hyd r och lor id e (11‚HCl). Com-
pound 37 was synthesized as described in the general proce-
dure (150 mg of crude). After reduction, recrystallization from
EtOH/hexanes yielded 11‚HCl as white crystals (70 mg, 60%
(()-3-Flu or om eth yl-7-(N-4-flu or oph en ylam in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (55). Recrystallization
from EtOH/hexanes yielded 55 as white crystals (102 mg,
40%): mp 188-189 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.30
(ex br s, 1H, NH), 8.37 (ex s, 1H, NH), 8.21 (s, 1H), 7.76 (m,
1H), 7.49 (d, J ) 8.4 Hz, 1H), 7.12-7.08 (m, 4H), 4.49-4.46
(m, 1H, CH2F), 4.37-4.35 (m, 1H, CH2F), 3.92-3.87 (m, 1H,
H-3), 3.18 (m, 1H, H-4), 3.03 (m, 1H, H-4); HRMS (FAB+) m/z
calcd for C16H15N2O3FS (MH+) 353.0771, obsd 353.0763.
(()-3-F lu or om eth yl-7-(N-4-h yd r oxyp h en yla m in osu lfo-
n yl)-3,4-d ih yd r oisoqu in olin -1-(2H)-on e (56). Recrystalli-
zation from EtOH/hexanes yielded 56 as light-brown crystals
1
from 35): mp 204-205 °C; H NMR (400 MHz, DMSO-d6) δ
10.04 (br ex s, 2H, NH2+), 7.73 (s, 1H), 7.69-7.64 (m, 2H), 7.48
(d, J ) 8.1 Hz, 1H), 4.68-4.97 (m, 2H, CH2F), 4.45 (s, 2H,
H-1), 3.92-3.82 (m, 1H, H-3), 3.17-3.06 (m, 2H, H-4), 2.69
(q, J ) 6.5 Hz, 2H), 1.39 (sextet, J ) 7.2 Hz, 2H), 0.81 (t, J )
7.3 Hz, 3H); 13C NMR (DMSO-d6) δ 139.9, 136.7, 130.7, 130.6,
126.2, 125.8, 83.5 (d, J ) 169 Hz, CH2F), 52.8 (d, J ) 18 Hz,
C-3), 45.2, 44.7, 26.9, 23.3, 12.0; HRMS (FAB+) m/z calcd for
C
13H20N2O2FS (MH+) 287.1229, obsd 287.1227. Anal.
(C13H19N2O2FS‚HCl) C, H, N.
(()-3-F lu or om eth yl-7-(N-bu tyla m in osu lfon yl)-1,2,3,4-
tetr a h yd r oisoqu in olin e Hyd r och lor id e (12‚HCl). Recrys-
tallization from EtOH/hexanes yielded 12‚HCl as white crys-
tals (115 mg, 75%): mp 118-119 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.12 (br ex s, 2H, NH2+), 7.73 (s, 1H), 7.66 (s,
2H), 7.48 (d, J ) 6.8 Hz, 1H), 5.04-4.70 (m, 2H, CH2F), 4.45
(s, 2H, H-1), 3.92-3.88 (m, 1H, H-3), 3.16-3.02 (m, 2H, H-4),
2.72 (d, J ) 4.9 Hz, 2H), 1.36 (s, 2H), 1.25 (d, J ) 5.6 Hz, 2H),
0.81 (s, 3H); 13C NMR (500 MHz, DMSO-d6) 139.4, 136.2,
130.2, 130.1, 125.6, 125.3, 82.5 (d, J ) 168 Hz, CH2F), 52.2 (d,
J ) 19 Hz, C-3), 44.1, 42.6, 31.5, 26.5, 19.6, 13.9; HRMS
1
(132 mg, 52%): mp 205-206 °C; H NMR (400 MHz, DMSO-
d6) δ 9.80 (s, 1H, NH), 9.33 (ex s, 1H, OH), 8.35 (s, 1H, NH),
8.17 (s, 1H), 7.68 (m, 1H), 7.47 (d, J ) 8.0 Hz, 1H), 6.83 (d, J
) 8.7 Hz, 2H), 6.60 (d, J ) 8.7 Hz, 2H), 4.49-4.45 (m, 1H,
CH2F), 4.38-4.33 (m, 1H, CH2F), 3.93-3.87 (m, 1H, H-3), 3.16
(m, 1H, H-4), 2.97 (m, 1H, H-4); HRMS (FAB+) m/z calcd for
C
16H16N2O4FS (MH+) 351.0815, obsd 351.0793.
(()-3-Flu or om eth yl-7-(N-4-n itr op h en yla m in osu lfon yl)-
3,4-d ih yd r oisoqu in olin -1-(2H)-on e (57). Recrystallization
from MeOH/ether yielded 57 as light-yellow crystals (91 mg,

PNMT Inhibitors Predicted To Penetrate the BBB
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4491
(FAB+) m/z calcd for C14H22N2O2FS (MH+) 301.1386, obsd
301.1377. Anal. (C14H21N2O2FS‚HCl) C, H, N.
) 8.2 Hz, 1H), 4.99-4.68 (m, 2H, CH2F), 4.44 (s, 2H, H-1),
3.93-3.87 (m, 1H, H-3), 3.74-3.66 (m, 2H, CF3CH2), 3.17-
3.03 (m, 2H, H-4); 13C NMR (500 MHz, DMSO-d6) δ 139.1,
136.4, 129.93, 129.86, 125.2, 124.9, 124.3 (q, J ) 279 Hz, CF3),
82.1 (d, J ) 539 Hz, CH2F), 51.8 (d, J ) 61 Hz, C-3), 43.7,
43.4 (q, J ) 108 Hz, CF3CH2), 26.1; HRMS (FAB+) m/z calcd
for C12H15N2O2F4S (MH+) 327.0790, obsd 327.0787. Anal.
(C12H14N2O2F4S‚HCl) C, H, N.
(()-3-F lu or om et h yl-7-(N-2-m et h oxyet h yla m in osu lfo-
n yl)-1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (13‚
HCl). Compound 39 was synthesized as described in the
general procedure (116 mg crude). After reduction, the crude
free amine was purified by flash chromatography eluting with
CH2Cl2/MeCN (5:1) to yield a clear residue. Recrystallization
of the HCl salt from EtOH/hexanes yielded 13‚HCl as white
crystals (99 mg, 27% from 35): mp 174-175 °C; 1H NMR (400
MHz, DMSO-d6) δ 10.43-9.95 (br ex m, 2H, NH2+), 7.81 (s,
1H), 7.74 (s, 1H, NH), 7.68 (d, J ) 7.6 Hz, 1H), 7.47 (d, J ) 8
Hz, 1H), 4.98-4.69 (m, 2H, CH2F), 4.44 (s, 2H, H-1), 3.90 (br
m, 1H, H-3), 3.31 (m, 2H), 3.18-2.97 (m, 5H), 2.90 (m, 2H);
13C NMR (500 MHz, DMSO-d6) δ 139.5, 136.2, 130.2, 130.1,
125.7, 125.3, 82.5 (d, J ) 168 Hz, CH2F), 70.9, 58.3, 52.2 (d, J
) 19 Hz, C-3), 44.1, 42.5, 26.5; HRMS (FAB+) m/z calcd for
(()-3-F lu or om et h yl-7-(N-3,3,3-t r iflu or op r op yla m in o-
su lfon yl)-1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e
(18‚HCl). Recrystallization from EtOH/hexanes yielded 18‚
HCl as white crystals (80 mg, 76%): mp 111-117 °C (dec); 1H
NMR (400 MHz, DMSO-d6) δ 9.69 (br ex s, 2H, NH2+), 7.98
(ex m, 1H, NH), 7.75 (s, 1H), 7.69 (d, J ) 8.1 Hz, 1H), 7.49 (d,
J ) 8.1 Hz, 1H), 4.93-4.63 (m, 2H, CH2F), 4.44 (s, 2H, H-1),
3.92-3.85 (m, 1H, H-3), 3.16-2.93 (m, 4H), 2.49-2.40 (m, 2H,
CF3CH2); 13C NMR (400 MHz, DMSO-d6) δ 139.1, 137.3, 131.2,
130.9, 128.3 (q, J ) 276 Hz, CF3), 126.1, 125.9, 83.2 (d, J )
168 Hz, CH2F), 52.8 (d, J ) 19 Hz, C-3), 44.9, 36.9, 34.1 (q, J
) 27 Hz, CF3CH2), 27.2; HRMS (FAB+) m/z calcd for
C
13H20N2O3FS (MH+) 303.1179, obsd 303.1167. Anal.
(C13H19N2O3FS‚HCl) C, H, N.
(()-3-Flu or om eth yl-7-(N-2-eth oxyeth yla m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (14‚HCl).
Compound 40 was synthesized as described in the general
procedure (234 mg crude). After reduction, the crude free
amine was purified by flash chromatography, eluting with CH2-
Cl2/MeCN (1:1) to yield a clear residue. Recrystallization of
the HCl salt from EtOH/hexanes yielded 14‚HCl as white
crystals (88 mg, 23% from 35): mp 168-169 °C; 1H NMR (400
MHz, DMSO-d6) δ 10.14 (br ex s, 2H, NH2+), 7.93 (m, 1H),
7.74 (s, 1H, NH), 7.69 (d, J ) 7.9 Hz, 1H), 7.47 (d, J ) 8 Hz,
1H), 4.98-4.68 (m, 2H, CH2F), 4.43 (s, 2H, H-1), 3.89 (br m,
1H, H-3), 3.37 (m, 4H), 3.16-3.02 (m, 2H, H-4), 2.90 (m, 2H),
1.06 (t, J ) 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) δ
139.5, 136.3, 130.2, 130.1, 125.7, 125.3, 82.5 (d, J ) 168 Hz,
CH2F), 68.9, 65.8, 52.2 (d, J ) 19 Hz, C-3), 44.1, 42.7, 26.5,
C
13H17N2O2F4S (MH+) 340.0947, obsd 340.0937. Anal.
(C13H16N2O2F4S‚HCl) C, H, N.
(()-3-F lu or om eth yl-7-(N-4,4,4-tr iflu or obu tyla m in osu l-
fon yl)-1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (19‚
HCl). Recrystallization from EtOH/hexanes yielded 19‚HCl as
white crystals (126 mg, 63%): mp 212-222 °C (dec); 1H NMR
(400 MHz, DMSO-d6) δ 9.75 (br ex s, 2H, NH2+), 7.98 (ex m,
1H, NH), 7.74 (s, 1H), 7.69 (d, J ) 8.1 Hz, 1H), 7.49 (d, J )
8.1 Hz, 1H), 4.96-4.65 (m, 2H, CH2F), 4.45 (m, 2H, H-1), 3.92
(br m, 1H, H-3), 3.16-3.00 (m, 2H, H-4), 2.84-2.81 (m, 2H),
2.27-2.20 (m, 2H), 1.65-1.60 (m, 2H); 13C NMR (400 MHz,
DMSO-d6) δ 139.6, 137.0, 130.8, 130.7, 128.3 (q, J ) 275 Hz,
CF3), 126.1, 125.8, 83.4 (d, J ) 168 Hz, CH2F), 52.8 (d, J ) 19
Hz, C-3), 44.7, 42.1, 30.6 (q, J ) 27 Hz, CF3CH2), 26.9, 22.9;
HRMS (FAB+) m/z calcd for C14H19N2O2F4S (MH+) 355.1103,
obsd 355.1103. Anal. (C14H18N2O2F4S‚HCl) C, H, N.
15.4; HRMS (FAB+) m/z calcd for
C
14H22N2O3FS (MH+)
317.1335, obsd 317.1336. Anal. (C14H21N2O3FS‚HCl) C, H, N.
(()-3-F lu or om eth yl-7-(N-3-m eth oxyp r op yla m in osu lfo-
n yl)-1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (15‚
HCl). The crude free amine was chromatographed, eluting
with CH2Cl2/MeCN (2:1) to yield a clear residue that solidified
upon standing. Recrystallization of the HCl salt from EtOH/
hexanes yielded 15‚HCl as white crystals (150 mg, 60%): mp
(()-3-Flu or om eth yl-7-(N-1-m eth yleth ylam in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r obr om id e (20‚HBr ).
Recrystallization from EtOH/hexanes yielded 20‚HBr as white
crystals (116 mg, 73%): mp 205-206 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.61 (br ex s, 2H, NH2+), 7.75 (s, 1H), 7.70 (d, J
) 8.1 Hz, 1H), 7.64 (ex d, J ) 7.1 Hz, 1H, NH), 7.48 (d, J )
8.1 Hz, 1H), 4.94-4.65 (m, 2H, CH2F), 4.50 (s, 2H, H-1), 3.99-
3.93 (m, 1H, H-3), 3.26-3.00 (m, 3H), 0.96 (m, 6H); 13C NMR
(400 MHz, DMSO-d6) δ 141.3, 136.4, 130.7, 130.3, 126.2, 125.7,
83.1 (d, J ) 169 Hz, CH2F), 52.9 (d, J ) 19 Hz, C-3), 46.1,
44.9, 26.9, 24.1; HRMS (FAB+) m/z calcd for C13H20N2O2FS
(MH+) 287.1229, obsd 287.1249. Anal. (C13H19N2O2FS‚HBr) C,
H, N.
1
166-167 °C; H NMR (400 MHz, DMSO-d6) δ 10.12 (br ex s,
2H, NH2+), 7.73-7.67 (m, 3H), 7.48 (d, J ) 7.1 Hz, 1H), 4.98-
4.70 (m, 2H, CH2F), 4.45 (s, 2H, H-1), 4.00-3.88 (m, 1H, H-3),
3.28 (s, 2H), 3.17-3.02 (m, 5H), 2.77 (s, 2H), 1.60 (s, 2H); 13
C
NMR (500 MHz, DMSO-d6) δ 139.3, 136.3, 130.2, 130.1, 125.7,
125.3, 82.5 (d, J ) 169 Hz, CH2F), 69.3, 58.2, 52.2 (d, J ) 19
Hz, C-3), 44.1, 29.6, 26.52, 26.47; HRMS (FAB+) m/z calcd for
C
14H22N2O3FS (MH+) 317.1335, obsd 317.1328. Anal.
(()-3-Flu or om eth yl-7-(N-2-ph en yleth yla m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (22). Recrystallization from
EtOAc/hexanes yielded 22 as white crystals (200 mg, 60%):
(C14H21N2O3FS‚HCl) C, H, N.
(()-3-Flu or om eth yl-7-(N-2-eth oxypr opylam in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (16‚HCl).
Compound 42 was synthesized as described in the general
procedure (303 mg crude). After reduction, the crude free
amine was purified by flash chromatography, eluting with CH2-
Cl2/MeCN (1:1) to yield a clear residue. Recrystallization of
the HCl salt from EtOH/hexanes yielded 16‚HCl as white
crystals (147 mg, 37% from 35): mp 175-176 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.27 (br ex s, 2H, NH2+), 7.76-7.66
(m, 3H), 7.48 (d, J ) 8 Hz, 1H), 4.98-4.62 (m, 2H, CH2F), 4.44
(s, 2H, H-1), 3.89 (br m, 1H, H-3), 3.16-3.06 (m, 2H, H-4),
2.78 (m, 2H), 2.09 (br s, 4H), 1.60 (t, J ) 6.1 Hz, 2H), 1.06 (t,
J ) 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) δ 139.3, 136.3,
130.23, 130.18, 125.7, 125.3, 82.5 (d, J ) 168 Hz, CH2F), 67.1,
65.6, 52.2 (d, J ) 19 Hz, C-3), 44.2, 31.1, 29.8, 26.5, 15.5;
HRMS (FAB+) m/z calcd for C15H24N2O3FS (MH+) 331.1492,
obsd 331.1495. Anal. (C15H23N2O3FS‚HCl) C, H, N.
1
mp 105-106 °C; H NMR (400 MHz, DMSO-d6) δ 7.63 (br ex
s, 1H, NH), 7.52 (d, J ) 8.0 Hz, 1H), 7.47 (s, 1H), 7.31-7.25
(m, 3H), 7.21-7.14 (m, 3H), 4.52-4.48 (m, 1H, CH2F), 4.41-
4.36 (m, 1H, CH2F), 4.01-3.89 (m, 2H, H-1), 3.14-3.09 (m,
1H, H-3), 2.93 (s, 2H), 2.77 (m, 1H, H-4), 2.68 (t, J ) 7.5 Hz,
2H), 2.56 (m, 1H, H-4); 13C NMR (500 MHz, DMSO-d6) δ 139.1,
138.7, 137.6, 137.2, 129.9, 128.7, 128.3, 126.2, 124.1, 123.8,
86.1 (d, J ) 169 Hz, CH2F), 52.2 (d, J ) 19 Hz, C-3), 47.1,
44.1, 35.2, 29.7; HRMS (FAB+) m/z calcd for C18H22N2O2FS
(MH+) 349.1386, obsd 349.1411. Anal. (C18H21N2O2FS‚0.25H2O)
C, H. N.
(()-3-Flu or om eth yl-7-(N-4-ph en ylbu tylam in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (23). Recrystallization from
EtOAc/hexanes and yielded 23 as white crystals (188 mg,
89%): mp 122-123 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.53-
7.48 (m, 3H), 7.34-7.24 (m, 3H), 7.18-7.13 (m, 3H), 4.53-
4.48 (m, 1H, CH2F), 4.41-4.36 (m, 1H, CH2F), 4.02-3.89 (m,
2H, H-1), 3.13-3.08 (m, 1H, H-3), 2.79-2.71 (m, 3H), 2.59-
2.47 (m, 3H), 1.51 (quintet, J ) 7.0 Hz, 2H), 1.38 (quintet, J
) 7.0 Hz, 2H); 13C NMR (500 MHz, DMSO-d6) δ 142.0, 138.9,
137.9, 137.2, 129.9, 128.24, 128.20, 125.6, 124.0, 123.8, 86.1
(d, J ) 166 Hz, CH2F), 52.3 (d, J ) 19 Hz, C-3), 47.2, 42.3,
(()-3-F lu or om eth yl-7-(N-2,2,2-tr iflu or oeth yla m in osu l-
fon yl)-1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (17‚
HCl). Recrystallization from EtOH/hexanes yielded 17‚HCl as
1
white crystals (67 mg, 63%): mp 120-130 °C (dec); H NMR
(400 MHZ, DMSO-d6) δ 10.21 (br ex s, 2H, NH2+), 8.75 (t, J )
6.6 Hz, 1H), 7.79 (s, 1H), 7.72 (d, J ) 8.1 Hz, 1H), 7.49 (d, J

4492 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Romero et al.
34.6, 29.7, 28.7, 28.0; HRMS (FAB+) m/z calcd for C20H26N2O2-
FS (MH+) 377.1699, obsd 377.1676. Anal. (C20H25N2O2FS‚
0.25H2O) C, H, N.
C-3), 47.0, 44.0, 26.5, 25.0, 23.3; HRMS (FAB+) m/z calcd for
C15H22N2O2FS (MH+) 313.1386, obsd 313.1359. Anal.
(C15H21N2O2FS‚HCl) C, H, N.
(()-3-F lu or om et h yl-7-(N-2-n a p h t h yla m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (24). The crude free amine
was purified by flash chromatography, eluting with CH2Cl2/
MeCN (1:2). The white solid was recrystallized from EtOAc/
hexanes and yielded 24 as white crystals (105 mg, 75%): mp
(()-3-F lu or om e t h yl-7-(N -t h iom or p h olin osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (29‚HCl).
Recrystallization from EtOH/hexanes yielded 29‚HCl as white
1
crystals (120 mg, 65%): mp 220-224 °C (dec); H NMR (400
MHz, DMSO-d6) δ 10.15 (br ex s, 2H, NH2+), 7.73 (s, 1H), 7.64
(d, J ) 8.1 Hz, 1H), 7.53 (d, J ) 8.2 Hz, 1H), 4.99-4.71 (m,
2H, CH2F), 4.52-4.42 (m, 2H, H-1), 3.95-3.88 (m, 1H, H-3),
3.21-2.97 (m, 6H), 2.68 (t, J ) 4.8 Hz, 4H); 13C NMR (500
MHz, DMSO-d6) δ 137.4, 134.9, 130.8, 130.4, 126.4, 126.1, 82.5
(d, J ) 166 Hz, CH2F), 52.1 (d, J ) 18 Hz, C-3), 48.2, 44.1,
26.8, 26.6; HRMS (FAB+) m/z calcd for C14H20N2O2FS2 (MH+)
331.0950, obsd 331.0929. Anal. (C14H19N2O2FS2‚HCl) C, H, N.
(()-3-Flu or om eth yl-7-(N-4-flu or oph en ylam in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (30‚HCl).
The crude free amine was chromatographed, eluting with CH2-
Cl2/MeCN (1:1) to yield a white solid. Recrystallization of the
HCl salt from EtOH/hexanes yielded 30‚HCl as white crystals
(97 mg, 30%): mp 125-137 °C (dec); 1H NMR (400 MHz,
DMSO-d6) δ 10.4 (ex s, 1H, NH), 9.64 (br ex s, 2H, NH2+), 7.69
(s, 1H), 7.63 (d, J ) 8.6 Hz, 1H), 7.45 (d, J ) 8.1 Hz, 1H),
7.14-7.10 (m, 4H), 4.89-4.59 (m, 2H, CH2F), 3.91-3.85 (m,
1H, H-3), 3.46-3.43 (m, 2H, H-1), 3.11 (m, 1H, H-4), 2.96 (m,
1H, H-4); 13C NMR (500 MHz, DMSO-d6) δ 159.0 (d, J ) 242
Hz, F-Ph), 137.8, 136.6, 133.8, 129.9, 125.4, 125.2, 122.64,
122.58, 115.9 (d, J ) 23 Hz, F-Ph), 82.6 (d, J ) 166 Hz, CH2F),
51.7 (d, J ) 19 Hz, C-3), 43.7, 26.1; HRMS (FAB+) m/z calcd
for C16H17N2O2F2S (MH+) 339.0979, obsd 339.0975. Anal.
(C16H16N2O2F2S‚HCl) C, H, N.
(()-3-F lu or om eth yl-7-(N-4-h yd r oxyp h en yla m in osu lfo-
n yl)-1,2,3,4-tetr a h yd r oisoqu in olin e (31). The crude solid
was purified by flash chromatography, eluting with CH2Cl2/
MeCN (1:1). This solid was recrystallized from EtOH/hexanes
and yielded 31 as off-white crystals (38 mg, 32%): mp 115-
116 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.88 (br ex s, 1H,
NH), 9.31 (ex s, 1H, OH), 7.40-7.37 (m, 2H), 7.24 (d, J ) 7.9
Hz, 1H), 6.97 (d, J ) 9.9 Hz, 2H), 6.60 (d, J ) 8.7 Hz, 2H),
4.51-4.45 (m, 1H, CH2F), 4.40-4.31 (m, 1H, CH2F), 3.94-
3.83 (m, 2H, H-1), 3.14-3.06 (m, 1H, H-3), 2.73 (m, 1H, H-4),
2.52 (m, 1H, H-4); 13C NMR (500 MHz, DMSO-d6) 155.1, 139.7,
137.4, 137.3, 130.1, 129.0, 124.6, 124.4, 124.1, 115.9, 86.4 (d,
J ) 166 Hz, CH2F), 52.5 (d, J ) 18 Hz, C-3), 47.4, 29.9; HRMS
(FAB+)m/z calcd for C16H18N2O3FS (MH+) 337.1022, obsd
337.1020. Anal. (C16H17N2O3FS) C, H, N.
(()-3-Flu or om eth yl-7-(N-4-n itr op h en yla m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (32). The crude oil was
chromatographed, eluting with CH2Cl2/MeCN (1:2) to yield a
yellow solid. Recrystallization from EtOH/hexanes yielded 32
as yellow crystals (57 mg, 40%): mp 205-206 °C; 1H NMR
(400 MHz, MeOH-d4) δ 8.11 (d, J ) 9.2 Hz, 2H), 7.69 (s, 1H),
7.67 (s, 1H), 7.33-7.27 (m, 3H), 4.65-4.35 (m, 2H, CH2F),
4.13-4.03 (m, 2H, H-1), 3.27-3.19 (m, 1H, H-3), 2.86 (m, 1H,
H-4), 2.72 (m, 1H, H-4); 13C NMR (500 MHz, DMSO-d6) δ
147.6, 140.8, 138.8, 138.1, 135.6, 130.0, 125.3, 124.4, 124.3,
118.0, 85.2 (d, J ) 166 Hz, CH2F), 52.1 (d, J ) 18 Hz, C-3),
46.4, 28.8; HRMS (FAB+) m/z calcd for C16H17N3O4FS (MH+)
366.0924, obsd 366.0935. Anal. (C16H16N3O4FS) C, H, N.
1
169-170 °C; H NMR (400 MHz, DMSO-d6) δ 7.80-7.76 (m,
3H), 7.58-7.55 (m, 3H), 7.46-7.30 (m, 3H), 7.23 (d, J ) 8.6
Hz, 1H), 4.46-4.42 (m, 1H, CH2F), 4.34-4.29 (m, 1H, CH2F),
3.94-3.82 (m, 2H, H-1), 3.07-3.00 (m, 1H, H-3), 2.68 (m, 1H,
H-4), 2.47 (m, 1H, H-4); 13C NMR (500 MHz, DMSO-d6) δ
139.8, 137.3, 136.8, 135.5, 133.3, 130.0, 129.9, 129.0, 127.5,
127.1, 126.6, 124.9, 124.3, 124.0, 120.1, 115.5, 86.0 (d, J ) 166
Hz, CH2F), 52.1 (d, J ) 19 Hz, C-3), 47.0, 29.5; HRMS (FAB+)-
m/z calcd for C20H20N2O2FS (MH+) 371.1230, obsd 371.1219.
Anal. (C20H19N2O2FS‚0.25H2O) C, H, N.
(()-3-F lu or om e t h yl-7-(N -2-t e t r a lyla m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (25). Recrystallization from
EtOAc/hexanes yielded 25 as white crystals (173 mg, 75%):
mp 173-174 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J )
6.3 Hz, 1H, NH), 7.60 (d, J ) 8.0 Hz, 1H), 7.55 (s, 1H), 7.33
(d, J ) 8.1 Hz, 1H), 7.08-7.01 (m, 3H), 6.96 (d, J ) 6.5 Hz,
1H), 4.53-4.49 (m, 1H, CH2F), 4.41-4.37 (m, 1H, CH2F), 3.97
(m, 2H, H-1), 3.34-3.30 (m, 1H), 3.12-3.09 (m, 1H, H-3), 2.81-
2.51 (m, 6H), 1.82-1.79 (m, 1H), 1.64-1.57 (m, 1H); 13C NMR
(400 MHz, DMSO-d6) δ 139.8, 139.7, 138.0, 136.1, 135.0, 130.8,
129.8, 129.3, 126.7, 126.5, 125.2, 124.9, 87.0 (d, J ) 166 Hz,
CH2F), 53.1 (d, J ) 18 Hz, C-3), 50.0, 48.0, 36.6, 30.5, 30.2,
27.9; HRMS (FAB+) m/z calcd for
C
20H24N2O2FS (MH+)
375.1543, obsd 375.1526. Anal. (C20H23N2O2FS) C, H, N.
(()-3-F lu or om eth yl-7-(N-1-a d a m a n ta n a m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e Hyd r och lor id e (26‚HCl).
Compound 51 was synthesized as described in the general
procedure (205 mg of crude). After reduction, recrystallization
from EtOH/hexanes yielded 26‚HCl as white crystals (141 mg,
47%, from 35): mp 175-183 °C (dec); 1H NMR (400 MHz,
DMSO-d6) δ 10.09 (br ex s, 2H, NH2+), 7.75-7.72 (m, 2H), 7.59
(s, 1H), 7.44 (d, J ) 8.0 Hz, 1H), 5.00-4.68 (m, 2H, CH2F),
3.92-3.87 (m, 1H, H-3), 3.44 (m, 2H, H-1), 3.16-3.01 (m, 2H,
H-4), 1.93 (s, 3H), 1.71 (s, 6H), 1.51 (q, J ) 12.1 Hz, 6H); 13C
NMR (500 MHz, DMSO-d6) 143.8, 135.6, 129.97, 129.86, 125.5,
124.6, 82.5 (d, J ) 539 Hz, CH2F), 56.4, 54.2, 52.2 (d, J ) 60
Hz, C-3), 44.2, 42.8, 35.9, 29.3, 26.5, 18.9; HRMS (FAB+) m/z
calcd for C20H28N2O2FS (MH+) 379.1856, obsd 379.1841. Anal.
(C20H27N2O2FS‚HCl) C, H, N.
(()-3-F lu or om eth yl-7-(N-m or p h olin osu lfon yl)-1,2,3,4-
tetr a h yd r oisoqu in olin e Hyd r och lor id e (27‚HCl). Com-
pound 52 was synthesized as described in the general proce-
dure (100 mg of crude). After reduction, recrystallization from
EtOH/hexanes yielded 27‚HCl as white crystals (40 mg, 32%,
from compound 35): mp 230-233 °C (dec); 1H NMR (400 MHz,
DMSO-d6) δ 10.19 (br ex s, 2H, NH2+), 7.72 (s, 1H), 7.63 (d, J
) 8.0 Hz, 1H), 7.55 (d, J ) 7.8 Hz, 1H), 4.99-4.71 (m, 2H,
CH2F), 4.48 (s, 2H, H-1), 3.92-3.89 (m, 1H, H-3), 3.64 (s, 4H),
3.20-2.99 (m, 2H, H-4), 2.87 (s, 4H); 13C NMR (400 MHz,
DMSO-d6) δ 138.0, 133.6, 131.3, 130.9, 127.4, 127.0, 85.0 (d,
J ) 169 Hz, CH2F), 66.1, 52.7 (d, J ) 19 Hz, C-3), 46.7, 44.7,
27.1; HRMS (FAB+) m/z calcd for
C
14H20N2O3FS (MH+)
315.1179, obsd 315.1178. Anal. (C14H19N2O3FS‚HCl‚0.67H2O)
(()-3-Flu or om eth yl-7-(N-3-ch lor oph en ylam in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (33). The crude residue was
purified by flash chromatography, eluting with CH2Cl2/MeCN
(3:1) to yield a light-yellow solid. Recrystallization from EtOAc/
hexanes yielded 33 as light-yellow crystals (96 mg, 48%): mp
C, H, N.
(()-3-F lu or om et h yl-7-(N-p ip er id in osu lfon yl)-1,2,3,4-
tetr ah ydr oisoqu in olin e Hydr och lor ide (28‚HCl). The crude
free amine was purified by flash chromatography, eluting with
CH2Cl2/MeCN (1:1). Recrystallization of the HCl salt from
EtOH/hexanes yielded 28‚HCl as white crystals (150 mg,
72%): mp 220-221 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.86
(br ex s, 2H, NH2+), 7.71 (s, 1H), 7.63 (d, J ) 8.0 Hz, 1H), 7.53
(d, J ) 8.2 Hz, 1H), 4.94-4.64 (m, 2H, CH2F), 4.55-4.45 (m,
2H, H-1), 3.94-3.90 (m, 1H, H-3), 3.18 (m, 1H, H-4), 3.05 (m,
1H, H-4), 2.89 (t, J ) 5.2 Hz, 4H), 1.55 (s, 4H), 1.36 (s, 2H);
13C NMR (500 MHz, DMSO-d6) δ 137.1, 134.4, 130.5, 130.3,
126.6, 126.3, 82.5 (d, J ) 166 Hz, CH2F), 52.1 (d, J ) 18 Hz,
1
166-167 °C; H NMR (400 MHz, DMSO-d6) δ 7.53-7.51 (m,
2H), 7.29-7.23 (m, 2H), 7.12-7.04 (m, 3H), 4.52-4.43 (m, 1H,
CH2F), 4.39-4.35 (m, 1H, CH2F), 3.98-3.85 (m, 2H, H-1), 3.45
(br ex s, 1H, NH), 3.12-3.06 (m, 1H, H-3), 2.76-2.72 (m, 1H,
H-4), 2.56-2.49 (m, 1H, H-4); 13C NMR (500 MHz, DMSO-d6)
140.3, 140.2, 137.8, 137.1, 133.7, 131.3, 130.5, 124.6, 124.3,
123.6, 119.1, 118.1, 86.4 (d, J ) 166 Hz, CH2F), 52.5 (d, J )
18 Hz, C-3), 47.4, 29.9; HRMS (FAB+) m/z calcd for C16H17N2O2-
ClFS (MH+) 355.0683, obsd 355.0671. Anal. (C16H16N2O2ClFS)
C, H, N.

PNMT Inhibitors Predicted To Penetrate the BBB
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4493
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Recognition of Sub-Micromolar Inhibitors by the Epinephrine
Synthesizing Enzyme Phenylethanolamine N-Methyltrasferase.
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(()-3-Flu or om eth yl-7-(N-3-n itr op h en yla m in osu lfon yl)-
1,2,3,4-tetr a h yd r oisoqu in olin e (34). Compound 59 was
synthesized as described in the general procedure (140 mg of
crude). After reduction, the crude residue was purified by flash
chromatography, eluting with CH2Cl2/MeCN (1:1) to yield a
yellow solid. Recrystallization from acetone/hexanes yielded
34 as yellow crystals (76 mg, 42%): mp 215-220 °C (dec); 1H
NMR (400 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.84-7.83 (m, 1H),
7.56-7.51 (m, 4H), 7.30 (d, J ) 8.2, 1H), 4.53-4.44 (m, 1H,
CH2F), 4.41-4.32 (m, 1H, CH2F), 4.00-3.87 (m, 2H, H-1), 3.45
(br ex s, 2H, NH), 3.15-3.07 (m, 1H, H-3), 2.76-2.72 (m, 1H,
H-4), 2.57-2.51 (m, 1H, H-4); 13C NMR (400 MHz, DMSO-d6)
149.0, 140.9, 140.7, 138.1, 137.5, 131.6, 131.1, 126.0, 125.1,
124.8, 118.7, 113.9, 86.7 (d, J ) 166 Hz, CH2F), 52.9 (d, J )
18 Hz, C-3), 47.8, 30.3; HRMS (FAB+) m/z calcd for C16H17N3O4-
FS (MH+) 366.0924, obsd 366.0919. Anal. (C16H16N3O4FS) C,
H, N.
Ack n ow led gm en t. This research was supported by
NIH Grant HL 34193 and NIH Predoctoral Training
Grant GM 07775 (F.A.R.). The 500 MHz NMR spec-
trometer was partially funded by the National Science
Foundation through Grant CHE-9977422. F.A.R. is both
an American Heart Association and American Founda-
tion for Pharmaceutical Education predoctoral fellow.
S.M.V. is an undergraduate research assistant.
Su p p or tin g In for m a tion Ava ila ble: Results from el-
emental analysis (C, H, N) for assayed compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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S-enantiomers and
a conformational analysis of the (R)-3-
fluoromethyl-7-(N-substituted-aminosulfonyl)-1,2,3,4-THIQs will
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J M0400653