Enantiospecific synthesis of 3-fluoromethyl-, 3-hydroxymethyl-, and 3- chloromethyl-1,2,3,4-tetrahydroisoquinolines as selective inhibitors of phenylethanolamine N-methyltransferase versus the α2-adrenoceptor

Article abstract of DOI:10.1021/jm990086a

An enantiospecific method was developed for the synthesis of 3- fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-7-substituted-1,2,3,4- tetrahydroisoquinolines (THIQs) from phenylalanine. Biochemical evaluation of the enantiomers of these compounds at both PNMT and the α₂-adrenoceptor indicates that both sites display similar stereoselectivity. Overall the R- enantiomer was usually the more potent enantiomer at both PNMT and the α₂- adrenoceptor for these 3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl- THIQs. The one exception is 3-hydroxymethyl-7-nitro-THIQ (9), which was found to display the opposite stereoselectivity at the α₂-adrenoceptor. A comparison of the PNMT inhibitory potency of the enantiomers of these 3- fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-THIQs indicates that all of the 3-substituted-THIQs displayed similar inhibitory potency for PNMT. However, the nature of the 3-substituent was found to have a major effect on the α₂-adrenoceptor affinity of these compounds with the 3-hydroxymethyl- and 3-fluoromethyl-THIQs having the highest affinity and THIQs containing the 3-chloromethyl moiety the least. Compounds R-3-fluoromethyl-7-cyano-THIQ (R- 12) and R-3-fluoromethyl-7-N-(4-chlorophenyl)aminosulfonyl-THIQ (R-13) and both enantiomers of 3-chloromethyl-7-nitro-THIQ (R- and S-30) are the most selective inhibitors in this study and display selectivities (α₂- adrenoceptor K(i)/PNMT K(i)) greater than 200. These compounds give important insight into the steric and stereochemical preferences of both PNMT and the α₂-adrenoceptor, which should assist in the development of new PNMT inhibitors.

Full text of DOI:10.1021/jm990086a

J . Med. Chem. 1999, 42, 4351-4361
4351
En a n tiosp ecific Syn th esis of 3-F lu or om eth yl-, 3-Hyd r oxym eth yl-, a n d
3-Ch lor om eth yl-1,2,3,4-tetr a h yd r oisoqu in olin es a s Selective In h ibitor s of
P h en yleth a n ola m in e N-Meth yltr a n sfer a se ver su s th e r2-Ad r en ocep tor 1
Gary L. Grunewald,* Timothy M. Caldwell, Qifang Li, Vilas H. Dahanukar, Brynn McNeil, and
Kevin R. Criscione
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045
Received February 24, 1999
An enantiospecific method was developed for the synthesis of 3-fluoromethyl-, 3-hydroxymethyl-,
and 3-chloromethyl-7-substituted-1,2,3,4-tetrahydroisoquinolines (THIQs) from phenylalanine.
Biochemical evaluation of the enantiomers of these compounds at both PNMT and the R2-
adrenoceptor indicates that both sites display similar stereoselectivity. Overall the R-enantiomer
was usually the more potent enantiomer at both PNMT and the R2-adrenoceptor for these
3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-THIQs. The one exception is 3-hy-
droxymethyl-7-nitro-THIQ (9), which was found to display the opposite stereoselectivity at the
R2-adrenoceptor. A comparison of the PNMT inhibitory potency of the enantiomers of these
3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-THIQs indicates that all of the
3-substituted-THIQs displayed similar inhibitory potency for PNMT. However, the nature of
the 3-substituent was found to have a major effect on the R2-adrenoceptor affinity of these
compounds with the 3-hydroxymethyl- and 3-fluoromethyl-THIQs having the highest affinity
and THIQs containing the 3-chloromethyl moiety the least. Compounds R-3-fluoromethyl-7-
cyano-THIQ (R-12) and R-3-fluoromethyl-7-N-(4-chlorophenyl)aminosulfonyl-THIQ (R-13) and
both enantiomers of 3-chloromethyl-7-nitro-THIQ (R- and S-30) are the most selective inhibitors
in this study and display selectivities (R2-adrenoceptor Ki/PNMT Ki) greater than 200. These
compounds give important insight into the steric and stereochemical preferences of both PNMT
and the R2-adrenoceptor, which should assist in the development of new PNMT inhibitors.
In tr od u ction
Phenylethanolamine N-methyltransferase (PNMT;
EC 2.1.1.28) catalyzes the final step in the biosynthesis
of the catecholamine epinephrine (Epi).2 Epi has been
determined to make up 5-10% of the catecholamines
found in the central nervous system (CNS).3 Epi and
PNMT are co-localized in the CNS4 and found in very
specific areas of the brain (e.g., the C1 and C2 regions
of the medulla).5 Although the presence of Epi in the
CNS is well-documented, its function is not very well-
understood. Therefore, primarily on the basis of the
location of these Epi-containing neurons and from
inhibition studies of PNMT, Epi has been postulated to
be involved in the regulation of a number of physiologi-
cal processes in the CNSsregulation of blood pressure
and respiration,5,6 secretion of hormones from the
pituitary gland,4,7 regulation of the R2-adrenoceptor,8
and some of the neurodegeneration seen in Alzheimer’s
disease.9 However, the inhibitors used in these studies
have been shown to display considerable affinity for the
R2-adrenoceptor, which complicates the interpretation
of these data.10 Therefore, to ascertain the role of Epi
in these processes, our laboratory is attempting to
develop highly selective inhibitors of PNMT versus the
R2-adrenoceptor that will have the ability to penetrate
the blood-brain barrier (BBB).
1,2,3,4-Tetrahydroisoquinoline (THIQ, 1) is an inhibi-
tor of PNMT (Table 1).11 SK&F 64139 (2) is a THIQ-
type inhibitor and is one of the most potent inhibitors
of PNMT known (Table 1). However, 2 also displays
considerable affinity for the R2-adrenoceptor (Table 1).12
SK&F 29661 (3) is a highly potent THIQ-type inhibitor
of PNMT but displays very poor affinity for the R2-
adrenoceptor (Table 1). Unfortunately, autoradiographic
studies have shown that 3 is unable to penetrate the
BBB,13 presumably due to the high polarity of the
sulfonamide, making it a poor candidate for physiologi-
cal studies.
Later studies investigating the structure-activity
relationships of THIQs have shown that substitution at
the 7-position was found to be the most important factor
influencing PNMT potency.14,15 To gain some under-
standing of the differences between the PNMT active
site and R2-adrenoceptor, we performed a comparative
molecular field analysis (CoMFA),16 a type of three-
dimensional QSAR study, for a set of 30 7-substituted-
THIQs for the R2-adrenoceptor and the PNMT active
site.17 In this study we proposed that THIQs could bind
in one of two different orientations at the PNMT active
site and at the R2-adrenoceptor, depending on the
* To whom correspondence should be addressed. Phone: (785)
864-4497. Fax: (785) 864-5326. E-mail: ggrunewald@ukans.edu.
10.1021/jm990086a CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/05/1999

4352 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grunewald et al.
Ta ble 1. In Vitro Activity of Several PNMT Inhibitors at
PNMT and Inhibition of [3H]Clonidine Binding at the
R2-Adrenoceptora
lack of a group (e.g., OH) that can participate in
hydrogen-bonding interactions with the PNMT active
site.
Ki, µM
B/A
compd
PNMT (A)
R2 (B)
selectivity
1b
2b
10 ( 1
0.35 (0.10
0.021 ( 0.005
100 ( 20
0.76 ( 0.08
6.6 ( 6.6
3.8 ( 0.1
0.67 ( 0.3
1400 ( 30
38 ( 1
0.35
0.095
180
0.36
6.0
0.22 ( 0.05
0.56 ( 0.04
2.1 ( 0.1
3c
4d
5d
1.1 ( 0.1
6e
1.5 ( 0.1
2.5
7d
24 ( 1
0.028
4100
160
14
1000
140
420
220
10
8d
(()
R-(+)
S-(-)
(()
0.34 ( 0.06
0.24 ( 0.03
0.90 ( 0.05
0.66 ( 0.10
0.54 ( 0.06
1.1 ( 0.1
9d
Later studies from this laboratory discovered that the
combination of a 3-substituent (i.e., 3-methyl or 3-hy-
droxymethyl) and a hydrophilic electron-withdrawing
7-substituent (i.e., 7-SO2NH2, 7-NO2) resulted in syn-
ergistic increases in selectivity for PNMT versus the R2-
adrenoceptor. Compound 8 is an example of this type
of inhibitor and is the most selective inhibitor of PNMT
yet reported (Table 1). However, studies have indicated
that 8 was too polar to penetrate the BBB.20
To decrease the polarity of the 3-hydroxymethyl-
THIQs (e.g., 8 and 9) to increase the possibility that
these compounds may penetrate the BBB, a series of
3-fluoromethyl-THIQs (10-13) were synthesized and
evaluated.20 A similar trend of increased selectivity was
found for THIQs in this study that combined a 3-fluo-
romethyl moiety and hydrophilic electron-withdrawing
7-substituents (10-13; Table 1), and these compounds
are some of the most selective inhibitors of PNMT
known.
9d
13 ( 1
10e
11e
12e
13e
15e
680 ( 10
76 ( 6
460 ( 10
160 ( 10
6.4 ( 0.2
(()
(()
(()
0.74 ( 0.07
0.64 ( 0.1
(()
a
PNMT and R2-adrenoceptor Ki values for literature compounds
were determined in our laboratory for consistent internal com-
parison. b Reference 11. c Reference 32. Reference 19. e Reference
d
20.
To determine the BBB permeability of some of these
inhibitors, we performed an in vitro study20 using a
model developed by Borchardt and Audus.21,22 The
results of this study indicated that there was a good
correlation between lipophilicity and BBB penetration
for THIQ-type inhibitors and that THIQs possessing
calculated partition coefficients (Clog P) in the range of
0.13-0.57 or greater should be able to gain some
penetration into the brain. Compounds 8-10 were
included in this study, and on the basis of the results
of this model, it was determined that 9 (Clog P ) +0.57)
should be able to penetrate the BBB, whereas 8 (Clog
P ) -1.01) and 10 (Clog P ) -0.07) probably would
not. 3-Fluoromethyl-THIQs, 11 (Clog P ) +1.51), 12
(Clog P ) +1.20), and 13 (Clog P ) +2.79), possess
calculated partition coefficients greater than 0.57 and
according to this model should be able to penetrate the
BBB. These compounds are also very selective inhibitors
of PNMT (Table 1) and represent promising new leads
in the development of selective PNMT inhibitors with
the ability to penetrate into the CNS.
F igu r e 1. Two proposed orientations of 7-substituted-THIQs
at PNMT and the R2-adrenoceptor, showing orientation A for
lipophilic (+π) 7-substituents and orientation B for hydrophilic
(-π) 7-substituents. Between the structures of 2 and 3 is a
SYBYL-generated view of the superimposition of 2 in orienta-
tion A on 3 in orientation B. The asterisk marks the area in
space where the lone pairs of both compounds may overlap.
(This figure was adapted from ref 31, copyright 1999, with
permission from Elsevier Science.)
lipophilicity of the 7-substituent (Figure 1). Thus,
THIQs containing a hydrophilic (-π) substituent are
thought to bind differently than THIQs possessing a
lipophilic (+π) substituent. Also, THIQs containing a
hydrophilic (-π) electron-withdrawing 7-substituent
were usually found to be selective for PNMT, while
THIQs possessing a lipophilic (+π) electron-withdraw-
ing 7-substituent were usually found to be nonselective
inhibitors of PNMT.
Further studies examining substitution on the THIQ
nucleus found that substitution of a (()-3-methyl (4) or
a (()-3-hydroxymethyl moiety (5) increased both po-
tency and selectivity relative to THIQ (1) (Table 1).18,19
However, it was also found that the area around the
3-position of THIQ at PNMT was found to exhibit
limited steric bulk tolerance with only a (()-3-methyl
(4), (()-3-hydroxymethyl (5),19 or (()-3-fluoromethyl
(6)20 moiety displaying good inhibitory potency (Table
1). Substitution of larger groups [e.g., (()-3-ethyl (7)]18
caused a significant decrease in PNMT inhibitory
potency (Table 1), which may be a result of an area of
steric bulk intolerance at the PNMT active site or the
However, most of these THIQs (8, 10-13) were
evaluated as racemates. Therefore, the influence of the
chirality of the 3-substituent of these compounds at both
the PNMT active site and the R2-adrenoceptor was
unknown. Due to the small amount of information
known about the effects of chirality on the potency and
selectivity of these 3-hydroxymethyl- and 3-fluoro-
methyl-THIQs for PNMT, we deemed it necessary to
evaluate the enantiomers of 8 and 11-13.
The enantiomers of 14 and 15 were also proposed for
synthesis, as in our previous studies of 3,7-disubstitut-
ed-THIQs19,20 the effects of chirality of the 3-substituent
of THIQ in combination with a lipophilic electron-

PNMT Inhibitors: 3-CH2OH, 3-CH2F, 3-CH2Cl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4353
Sch em e 1
Sch em e 2
F igu r e 2. Retrosynthetic analysis of target compounds R- or
S-3-hydroxymethyl-THIQs (8, 9, 14) and R- or S-3-fluoro-
methyl-THIQs (11-13, 15) formed from either D- or L-phen-
ylalanine (17), respectively. The asterisk denotes the chiral
center that is fixed by the chiral starting material.
withdrawing group (e.g., Br) had not been investigated.
Compound 15 had been evaluated previously as a
racemate (Table 1) and was found to be a nonselective
inhibitor of PNMT.20 This lack of selectivity was be-
lieved to be due to its ability to bind in the proposed
lipophilic orientation at the R2-adrenoceptor (see Figure
1). However, the influence of chirality on the activity of
this compound has not been investigated. Therefore, if
14 and 15 are bound differently at either the PNMT
active site or the R2-adrenoceptor (due to the lipophi-
licity of the 7-substituent), these compounds may dis-
play a different rank order of potency based on their
chirality as compared to THIQs that contain hydrophilic
7-substituents.
lactam intermediate 16 can act as a directing group for
electrophilic aromatic substitution reactions. Hence, the
7-position of these THIQs can be readily functionalized
without the formation of other regioisomers. Finally, a
large number of chiral 3-hydroxymethyl- and 3-fluo-
romethyl-7-substituted-THIQs can be made using the
same synthesis.
The previous synthesis used for the preparation of
3-hydroxymethyl-THIQs (8 and the enantiomers of 9)
possessed several limitations (racemization of a key
intermediate, multiple fractional recrystallizations, dif-
ficult separations of regioisomers, and low yields),19
while the synthesis used for the preparation of 3-fluo-
romethyl-THIQs (10-13, 15) could only produce these
products as racemic mixtures.20
Ch em istr y
Beginning with either D- or L-phenylalanine (17),
reduction with LiAlH4 formed R- or S-18, respectively.23
Treatment of R- or S-18 with 2 equiv of methyl chloro-
formate yielded R- or S-19. Carbamate 19 was cyclized
by treatment with phosphorus oxychloride at reflux
24
followed by the addition of SnCl4 at 0 °C to produce a
mixture of lactams R- or S-16 and R- or S-20 (3:1)
(Scheme 1), which were easily separated by flash column
chromatography (silica gel). It was noted that if the
SnCl4 was added to the phosphorus oxychloride and
carbamate 19 mixture while the mixture was still hot,
lactam 20 was obtained as the sole product.
Nitration of lactam R- or S-16 with KNO3 in H2SO4
formed R- or S-21. Reduction of the lactam and depro-
tection of the alcohol was accomplished in one step by
treatment with BH3‚THF to yield R- or S-9 (Scheme 2).
Treatment of lactam 16 with chlorosulfonic acid to add
a 7-chlorosulfonyl moiety to form an intermediate in the
Therefore, a new enantiospecific synthesis for the
production of both 3-hydroxymethyl- and 3-fluorometh-
yl-THIQs was proposed. Retrosynthetic analysis of R-
or S-3-hydroxymethyl-THIQs (8, 9, 14) and 3-fluoro-
methyl-THIQs (11-13, 15) indicates that both series of
compounds could be derived from a common intermedi-
ate (16), which could be synthesized in enantiomerically
pure form from either D- or L-phenylalanine (17) (Figure
2). This synthesis has several advantages over those
previously used. Most importantly, the absolute config-
uration of the final product is set with a starting
material of known chirality. Second, the carbonyl of

4354 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grunewald et al.
Sch em e 3
Sch em e 5
Sch em e 6
Sch em e 4
synthesis of 8 was unsuccessful and led only to decom-
position products. On the basis of our in vitro BBB
model studies, which indicated that 8 would not be able
to penetrate the CNS, we decided not to pursue the
synthesis of the enantiomers of 8. R- or S-3-Hydroxy-
methyl-7-bromo-THIQ (14) was synthesized using the
conditions outlined in Scheme 2. Hydrogenation of the
7-nitro substituent of R- or S-21 to the amine followed
by a Sandmeyer bromination formed R- or S-22.25
Reduction of the lactam and deprotection of the alcohol
was accomplished by treatment with BH3‚THF to yield
R- or S-14.
Beginning with lactam R- or S-16, hydrolysis of the
carbonate ester to the primary alcohol afforded R- or
S-23, which was treated with diethylaminosulfur tri-
fluoride (DAST) to yield R- or S-24.26 Reduction of the
lactam with BH3‚THF produced R- or S-6. Nitration of
R- or S-24 with KNO3 and H2SO4 yielded lactam R- or
S-25, which was reduced with BH3‚THF to form R- or
S-11 (Scheme 3).
could be isolated from the cyclization reaction that
formed key intermediate 16, it was decided to synthesize
several 3-chloromethyl-THIQs in order to determine
how these compounds would compare to similarly
substituted 3-hydroxymethyl- and 3-fluoromethyl-THIQs.
Reduction of lactam R- or S-20 with BH3‚THF yielded
R- or S-28 (Scheme 6). Nitration of R- or S-20 with
KNO3 and H2SO4 formed R- or S-29, which was reduced
with BH3‚THF to yield R- or S-30 (Scheme 6). Attempts
to synthesize a 7-bromo-3-chloromethyl-THIQ were
unsuccessful using the standard Sandmeyer bromina-
tion conditions.
Enantiomeric excess (ee) was determined for all final
compounds and was found to be greater than 95% by
chiral HPLC.
Bioch em istr y
The enantiomers of 3-fluoromethyl-THIQs 12 and 15
were synthesized using the procedures outlined in
Scheme 4. The hydrogenation of the nitro group of R-
or S-11 to the amine followed by a modified Sandmeyer
reaction yielded R- or S-12.27 A similar procedure was
used for the production of R- or S-15. Hydrogenation of
R- or S-11 followed by a Sandmeyer bromination gave
R- or S-15.25
We decided not to pursue the synthesis of the enan-
tiomers of 10 due to our in vitro BBB model studies,20
which had indicated that 10 would not be able to
penetrate into the CNS. However, R- or S-13 was
synthesized using the conditions shown in Scheme 5.
Chlorosulfonylation of R- or S-24 with chlorosulfonic
acid (neat) yielded R- or S-26. Treatment of R- or S-26
with 4-chloroaniline in pyridine formed R- or S-27,
which was reduced with BH3‚THF to produce R- or S-13.
Considering that chloromethyl lactam R- or S-20
All compounds were evaluated as their hydrochloride
or hydrobromide salts for their activity as inhibitors of
PNMT and inhibitors of the binding of [3H]clonidine to
the R2-adrenoceptor. Bovine adrenal PNMT was pre-
pared using the method of Connett and Kirshner
through the isoelectric precipitation step.28 The in vitro
activity of these compounds was determined using a
standard radiochemical assay that has been described
previously.29 Inhibition constants were determined by
using three different concentrations of the inhibitor
using phenylethanolamine as the substrate.
R2-Adrenoceptor binding assays were performed using
a standard radiochemical assay developed by U’Prichard
et al.30 that uses [3H]clonidine as the radioligand to
define specific binding and phentolamine to determine
the nonspecific binding affinity in order to simplify the
comparison with previous results.

PNMT Inhibitors: 3-CH2OH, 3-CH2F, 3-CH2Cl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4355
Ta ble 2. Biochemical Evaluation of 3,7-Disubstituted-1,2,3,4-tetrahydroisoquinolines
Ki, µM
B/A
compd
R3
R7
PNMT (A)
R2 (B)
selectivity
5a
R-CH2OH
S-CH2OH
R-CH2OH
S-CH2OH
R-CH2OH
S-CH2OH
R-CH2F
S-CH2F
R-CH2F
S-CH2F
R-CH2F
S-CH2F
R-CH2F
S-CH2F
R-CH2F
S-CH2F
R-CH2Cl
S-CH2Cl
R-CH2Cl
S-CH2Cl
S-CH3
H
H
NO2
NO2
Br
Br
H
H
NO2
NO2
CN
CN
SO2NHPh-4-Cl
SO2NHPh-4-Cl
Br
Br
H
H
0.56 ( 0.05
15 ( 2
4.4 ( 0.3
17 ( 1
7.9
1.1
160
14
9
14
6
0.24 ( 0.03
0.90 ( 0.05
0.38 ( 0.05
0.48 ( 0.05
0.69 ( 0.06
9.9 ( 0.4
38 ( 1
13 ( 1
1.2 ( 0.1
0.97 ( 0.20
2.9 ( 0.4
37 ( 5
3.2
2.0
4.2
3.7
11
12
13
15
28
30
4a
0.52 ( 0.07
0.88 ( 0.06
0.97 ( 0.08
3.5 ( 0.2
0.28 ( 0.04
1.9 ( 0.1
47 ( 3
90
140 ( 10
360 ( 30
580 ( 50
97 ( 10
101 ( 10
3.1 ( 0.1
17 ( 1
160
370
170
350
53
0.51 ( 0.06
0.47 ( 0.05
3.5 ( 0.2
6.1
36
2.3 ( 0.2
6.6 ( 0.4
100 ( 10
270 ( 10
0.49 ( 0.05
5.7 ( 0.3
19 (. 1
0.66
0.55
210
410
0.49
0.15
76
12 ( 1
NO2
NO2
H
H
NO2
NO2
0.47 ( 0.03
0.66 ( 0.04
1.0 ( 0.1
R-CH3
S-CH3
R-CH3
38 ( 2.0
0.25 ( 0.02
1.3 ( 0.1
31a
53 ( 2
41
a
Reference 19.
Resu lts a n d Discu ssion
(9), where the S-enantiomer was found to be the more
potent. This may be an indication that the S-3-hy-
droxymethyl substituent of 9 may be participating in a
type of hydrogen bond interaction with the R2-adreno-
ceptor that the R-enantiomer of 3-methyl-, S-enantiomer
of 3-fluoromethyl-, or S-enantiomer of 3-chloromethyl-
THIQs cannot. Examination of the R2-adrenoceptor
affinities of 14 and 15 indicates that 14 shows little
stereoselectivity, whereas 15 displays a 6-fold difference
in affinity between the R- and S-enantiomers. This also
may be an indication that a hydrogen-bonding interac-
tion may be taking place with the S-enantiomer of 14
increasing its affinity in a similar manner as that found
for S-9.
The results of the biochemical evaluation of com-
pounds from this study are shown in Table 2. R- and
S-3-Hydroxymethyl-THIQ (5), R- and S-3-methyl-THIQ
(4), and R- and S-3-methyl-7-nitro-THIQ (32) have been
synthesized previously19 and were included in the table
for comparison purposes. It should be noted that al-
though the absolute configurations of S-31 and R-9 are
oppositesdue to the Cahn-Ingold-Prelog rules for
assigning prioritysthe area of space in which the
3-substituent of these compounds is aligned is identical.
Direct comparison of all the PNMT inhibitory poten-
cies of these compounds (3-fluoromethyl-, 3-hydroxy-
methyl-, and 3-chloromethyl-THIQs) indicates that
PNMT displays the same stereoselectivity for all 3-sub-
stituted-THIQs, with the R-enantiomer being the more
potent. However, the amount of differentiation between
enantiomers seems to be dependent on the 7-substitu-
ent. PNMT seems to display very little stereoselectivity
for THIQs containing lipophilic 7-substituents (14 and
15), whereas the degree of differentiation between the
enantiomers is much larger for THIQs that either are
unsubstituted at the 7-positition (e.g., 4-6 and 28) or
contain hydrophilic electron-withdrawing 7-substituents
(e.g., 9-13, 30, and 31). This supports the hypothesis
that THIQs containing lipophilic 7-substituents may be
bound differently than THIQs that are unsubstituted
or contain hydrophilic 7-substituents as proposed previ-
ously for PNMT (Figure 1).17,20
Examination of the PNMT inhibitory potencies of the
7-nitro-THIQs 9, 11, 30, and 31 indicates that both
enantiomers of these compounds display similar activi-
ties. However, examination of their R2-adrenoceptor
affinities reveals that the 3-substituent has a large effect
on the potency of these compounds with S-31 > R-9 >
R-11 > R-30 and S-9 > R-31 > S-11 > S-30. Except for
3-hydroxymethyl-THIQ 9 which displays a reversal in
potency of the enantiomers, the overall trend for these
compounds indicates that as the 3-substituent gets
larger (methyl < fluoromethyl < chloromethyl), the R2-
adrenoceptor affinity decreases for both enantiomers.
This result would seem to confirm previous data that
had suggested that an area of limited steric bulk
tolerance surrounds the 3-position of THIQs possessing
hydrophilic 7-substituents at the R2-adrenoceptor.19,31
A similar trend in stereoselectivity was found for the
R2-adrenoceptor where the R-enantiomer is the more
potent for the majority of these 3-fluoromethyl-, 3-hy-
droxymethyl-, and 3-chloromethyl-THIQs. This trend is
similar to that found previously for 3-methyl-THIQs 4
and 31.19 The only exception is 3-hydroxymethyl-THIQ
Con clu sion s
An enantiospecific synthesis was developed for the
production of 3-hydroxymethyl-, 3-fluoromethyl-, and
3-chloromethyl-THIQs. Biochemical evaluation of the

4356 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grunewald et al.
1H and 49.2 ppm for 13C). Coupling constants (J ) are reported
in hertz (Hz), and s, d, t, q, m, br, and ex refer to singlet,
doublet, triplet, quartet, multiplet, broad, and exchangeable,
respectively. Infrared spectra (IR) were recorded on a Perkin-
Elmer IR 727 spectrophotometer. Chemical-ionization mass
spectra (CIMS) were obtained on a Varian Atlas CH-5 or a
Ribermag R 10-10 mass spectrophotometer. The intensity of
each peak in the spectrum is reported relative to the base peak
in parentheses. Microanalyses were performed on a Hewlett-
Packard model 185B CHN analyzer at the University of
Kansas. Optical rotations were recorded on a Perkin-Elmer
enantiomers of these compounds at both PNMT and the
R2-adrenoceptor indicates that both of these sites display
similar stereoselectivity where the R-enantiomer was
consistently the more potent. The one exception is
3-hydroxymethyl-7-nitro-THIQ (9), which displayed the
opposite stereoselectivity at the R2-adrenoceptor. The
amount of stereoselectivity displayed by the R2-adreno-
ceptor for these compounds is relatively the same for
all compounds, whereas the amount of stereoselectivity
for PNMT seems to be dependent on the lipophilicity of
the 7-substituent. Compounds containing hydrophilic
7-substituents display a fairly large degree of stereose-
lectivity, whereas compounds containing lipophilic 7-sub-
stituents display very little stereoselectivity. This may
be an indication that these two types of compounds are
binding differently at the PNMT active site. A compari-
son of 3-methyl, 3-fluoromethyl, 3-hydroxymethyl, and
3-chloromethyl substituents indicates that all of the
3-substituted-THIQs displayed similar inhibitory po-
tency for PNMT, whereas for the R2-adrenoceptor the
order of affinity from the highest to the least was found
to be 3-methyl > 3-hydroxymethyl = 3-fluoromethyl >
3-chloromethyl, which suggests that the R2-adrenoceptor
has a limited amount of steric bulk tolerance in the area
surrounding the 3-position of THIQ. Furthermore,
compounds R-3-fluoromethyl-7-cyano-THIQ (R-12; Clog
P ) 1.20), R-3-fluoromethyl-7-N-(4-chlorophenyl)ami-
nosulfonyl-THIQ (R-13; Clog P ) 2.63), and both enan-
tiomers of 3-chloromethyl-7-nitro-THIQ (R- and S-30;
Clog P ) 1.85) display selectivity ratios (R2-adrenoceptor
Ki/PNMT Ki) greater than 200 and according to our
previous BBB model study20 may be able to penetrate
into the CNS. These compounds give important insight
into the steric and stereochemical factors that determine
potency and selectivity for PNMT and are important
leads in the development of new, more selective inhibi-
tors of PNMT that should have the ability to penetrate
into the brain.
241 polarimeter using the sodium
D line. Chiral high-
performance liquid chromatography (HPLC) was performed
on a Shimadzu LC 6A system. The Chiralcel OJ column
(0.46 × 25 cm) was purchased from Daicel Inc., New York,
NY.
S-Adenosyl-L-methionine used in the radiochemical assays
was obtained from Sigma Chemical Co. [3H]-S-Adenosyl-L-
methionine was purchased from American Radiolabeled Chemi-
cals (St. Louis, MO). [3H]Clonidine used in the R2-adrenoceptor
assays was purchased from New England Nuclear Corp.
(Boston, MA). Bovine adrenal glands were obtained from Davis
Meat Processing (Overbrook, KS).
R-(+)-2-Am in o-3-p h en ylp r op a n ol (R-18). A solution of
lithium aluminum hydride (3.0 g, 78.9 mmol) in dry THF (150
mL) was stirred and cooled in an ice bath. D-(R)-Phenylalanine
(17) (9.00 g, 55.0 mmol) was added over 30 min. The mixture
was stirred at 0 °C for 2 h, warmed to room temperature, and
stirred an additional 2 h. The reaction mixture was heated at
reflux for 16 h. The solution was cooled in an ice bath and
diluted slowly with ether (100 mL). Water (4.5 mL), 15%
NaOH (4.5 mL), and water (12 mL) were added dropwise to
the solution in succession. The mixture was filtered through
Celite to remove the aluminum salts. The Celite was washed
with ether (200 mL). The filtrate was collected and the solvent
was removed under reduced pressure to yield a yellow solid.
Bulb-to-bulb distillation (120 °C, 0.1 mmHg) gave R-18 as a
white solid (6.67 g, 82%): mp 91-93 °C (lit.23 mp 90-91 °C);
[R]D ) +23.2° (c 1.0, EtOH) [lit.23 [R]D ) +23.8° (c 1.0,
EtOH)]; 1H NMR (CDCl3) δ 7.34-7.18 (m, 5H, ArH), 3.66-
3.62 (m, 1H, CH2O), 3.41-3.35 (m, 1H, CH2O), 3.14-2.98 (m,
1H, CHN), 2.83-2.77 (m, 1H, ArCH), 2.56-2.48 (m, 1H,
ArCH), 2.30-2.00 (ex br m, 3H, OH and NH2); 13C NMR
(CDCl3) δ 138.7, 129.2, 128.6, 126.4, 66.2, 54.2, 40.8. Anal.
(C9H13NO) C, H, N.
22
22
Exp er im en ta l Section
S-(-)-2-Am in o-3-p h en ylp r op a n ol (S-18). Compound S-18
was prepared in the same manner as R-18 using L-phenyl-
alanine (S-17) as the starting material: mp 92-94 °C (lit.23
All reactions that required inert atmospheric conditions
were performed under positive pressure of nitrogen in glass-
ware that was either flame-dried or oven-dried. All chemicals
used were of reagent grade unless stated otherwise. Solvents
were routinely distilled prior to use according to standard
methods. In some cases solvents were used directly from
Aldrich Sure Seal bottles. Hexanes refers to a mixture of
isomeric hexanes (bp 68-70 °C), and brine refers to a
saturated solution of NaCl. Melting points were determined
in open capillaries on a Thomas-Hoover melting point ap-
paratus and are otherwise uncorrected. Flash column chro-
matography was carried out using silica gel 60 (230-400 mesh)
purchased from Universal Adsorbents, Atlanta, GA. Bulb-to-
bulb distillations were carried out using a Kugelrohr distilla-
tion apparatus (Aldrich Chemical Co., Milwaukee, WI), and
the boiling range noted indicates the internal oven tempera-
ture. Proton nuclear magnetic resonance spectra (1H NMR)
and carbon nuclear magnetic resonance spectra (13C NMR)
were recorded on a Varian XL-300, a GE QE-300, or a Bruker
DRX-400 spectrophotometer, with CDCl3 as the solvent unless
otherwise noted. Proton chemical shifts are reported in parts
per million (ppm) relative to tetramethylsilane (TMS, 0.00
ppm). Carbon chemical shifts are reported in ppm relative to
CDCl3 (77.0 ppm). For the hydrochloride and hydrobromide
salts of these THIQs, NMR spectra were recorded in either
deuterated dimethyl sulfoxide (DMSO-d6) and the chemical
shifts reported relative to DMSO (2.49 ppm for 1H and 39.5
ppm for 13C) or deteurated methanol (CD3OD) (3.31 ppm for
mp 90-91 °C); [R]D ) -23.1° (c 1.0, EtOH) [lit.23 [R]D
)
20
25
-25.4° (c 1.22, EtOH)]. Anal. (C9H13NO) C, H, N.
R-(+)-N,O-Bis(m et h oxyca r b on yl)-2-a m in o-3-p h en yl-
p r op a n ol (R-19). A solution of R-18 (5.60 g, 37.0 mmol) in
dry CHCl3 (75 mL) and pyridine (15 mL) was cooled to 0 °C.
Methyl chloroformate (5.80 mL, 74.0 mmol) was added drop-
wise via an addition funnel. After the addition, the solution
was stirred at room temperature for 14 h. Ice water (50 mL)
was added slowly to the reaction mixture and the mixture was
stirred for 15 min. The organic phase was extracted and the
aqueous phase was extracted with CHCl3 (2 × 50 mL). The
organic extracts were combined and washed with 3 N HCl
(2 × 50 mL), 5% NaHCO3 (50 mL), and brine (50 mL). The
organic phase was dried over anhydrous Na2SO4 and the
solvent removed under reduced pressure. Bulb-to-bulb distil-
lation (140-150 °C, 0.5 mmHg) yielded R-19 as a white solid
(14.3 g, 99%): mp 83-84 °C; [R]D20 ) +3.9° (c 0.38, EtOH); IR
(KBr) 3350, 3080-2950, 1740, 1680, 975, 950, 790, 755, 700
1
cm-1; H NMR δ 7.65-7.18 (m, 5H, ArH), 4.80 (br ex s, 1H,
NH), 4.20-4.05 (m, 3H, CH(N)CH2O), 3.81 (s, 3H, CH3), 3.63
(s, 3H, CH3), 2.93-2.81 (m, 2H, ArCH2C); 13C NMR (CDCl3) δ
156.3, 155.5, 136.8, 129.1, 128.6, 126.7, 68.0, 54.9, 52.1, 51.2,
37.4; CIMS m/z (relative intensity) 285 (M + NH4+, 18), 268
(MH+, 11), 236 (57), 195 (12), 193 (12), 192 (86), 178 (27), 176
(15), 117 (10), 116 (13), 100 (100), 92 (11), 91 (60), 65 (12).
Anal. (C13H17NO5) C, H, N.

PNMT Inhibitors: 3-CH2OH, 3-CH2F, 3-CH2Cl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4357
S-(-)-N,O-Bis(m eth oxycar bon yl)-2-am in o-3-ph en ylpr o-
p a n ol (S-19). Using the same procedures as for R-19, S-18
was treated with methyl chloroformate to form S-19: mp 83-
S-(+)-O-Met h oxyca r b on yl-3-h yd r oxym et h yl-7-n it r o-
3,4-d ih yd r oisoqu in olin -1-2H-on e (S-21). Compound S-21
was prepared using the identical procedure as for R-21 using
S-16 as the starting material: mp 209-210 °C; [R]D ) +17°
(c 0.63, acetonitrile). Anal. (C12H12N2O6) C, H, N.
20
20
84 °C; [R]D ) -3.4° (c 0.50, EtOH). Anal. (C13H17NO5) C, H,
N.
R-(-)-O-Meth oxyca r bon yl-3-h yd r oxym eth yl-3,4-d ih y-
d r oisoqu in olin -1-2H-on e (R-16) a n d R-(-)-3-Ch lor om eth -
yl-3,4-d ih yd r oisoqu iolin -1-2H-on e (R-20). Freshly distilled
POCl3 (60 mL) was added to R-19 (9.01 g, 38.4 mmol). The
solution was heated at reflux (120 °C) for 24 h. The solution
was cooled in an ice bath and SnCl4 (7.0 mL, 60 mmol) was
added dropwise. The solution was stirred for 3 h at 0 °C. The
solution was warmed to room temperature and stirred for
another 2 h. The reaction mixture was poured onto ice (200 g)
and the solution stirred for 30 min. The solution was extracted
with CH2Cl2 (4 × 100 mL). The organic extracts were com-
bined, washed with brine, and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure to yield a
brown oil. The product was purified by flash column chroma-
tography (silica gel) with EtOAc/hexanes (1:1) to yield R-16
(3.18 g, 43%) and R-20 (1.05 g, 16%).
R-(+)-3-Hyd r oxym eth yl-7-n itr o-1,2,3,4-tetr a h yd r oiso-
qu in olin e Hyd r och lor id e (R-9‚HCl). Lactam R-21 (207 mg,
0.661 mmol) was suspended in a solution of anhydrous THF
(35 mL). 1 M BH3‚THF (4.0 mL) was added to the suspension
dropwise and the solution was heated at reflux for 16 h. It
was cooled in an ice bath and MeOH (5 mL) was added
dropwise. The solvent was removed under reduced pressure
to yield a white residue. This residue was dissolved in MeOH
(10 mL) and 12 M HCl (5 mL) and heated at reflux for 3 h.
The solution was concentrated to ca. 5 mL and the pH of the
solution was adjusted to 10 with 15% KOH. The solution was
extracted with EtOAc (3 × 50 mL). The organic extracts were
combined and dried over K2CO3. The solvent was removed to
yield a white solid, which was dissolved in dry EtOH, and dry
HCl(g) was used to form the hydrochloride salt. The solvent
was removed under reduced pressure and the residue was
recrystallized from EtOH to yield R-9‚HCl as off-white needles
(80.2 mg, 50%). This compound has been prepared previously
using different methodology and all spectral data were identi-
cal to those reported previously:19 mp 268 °C dec (lit.19 mp
20
R-16: mp 125-126 °C; [R]D ) -27° (c 0.29, EtOH); IR
(KBr) 3400, 3080, 2950, 1750, 1668, 1600, 1455, 1445, 1270,
1260, 1040, 970, 940, 785, 750 cm-1; 1H NMR δ 8.09-8.06 (d,
J ) 7.4 Hz, 1H, ArH-8), 7.70-7.45 (m, 1H, ArH-6), 7.40-7.33
(m, 1H, ArH-7), 7.23-7.21 (d, J ) 7.3 Hz, 1H, ArH-5), 6.35-
6.20 (br ex s, 1H, CONH), 4.31-4.25 (m, 1H, CH2O), 4.19-
4.15 (m, 1H, CH2O), 4.09-4.00 (m, 1H, H-3), 3.81 (s, 3H,
OCH3), 3.13-3.03 (m, 1H, H-4), 2.98-2.89 (m, 1H, H-4); 13C
NMR δ 165.7, 155.3, 136.4, 132.4, 128.1, 127.9, 127.6, 127.2,
68.7, 55.0, 49.6, 29.9; CIMS m/z (relative intensity) 236 (MH+,
53), 221 (23), 204 (100), 146 (25), 118 (30). Anal. (C12H13NO4)
C, H, N.
270 °C dec); [R]D ) +68° (c 0.31, MeOH) [lit.19 [R]D ) +62°
(c 0.28, MeOH)]. Anal. (C10H12N2O3‚HCl) C, H, N.
20
20
S-(-)-3-Hyd r oxym et h yl-7-n it r o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (S-9‚HCl). Compound S-9‚HCl
was prepared in the same manner as R-9‚HCl using S-21 as
the starting material: mp 268 °C dec (lit.19 mp 270 °C dec);
[R]D ) -62° (c 0.31, MeOH) [lit.19 [R]D ) -69° (c 0.28,
MeOH)]. Anal. (C10H12N2O3‚HCl) C, H, N.
20
20
20
R-20: mp 86-87 °C; [R]D ) -21° (c 0.99, EtOH); IR (KBr)
R-(+)-O-Meth oxyca r bon yl-3-h yd r oxym eth yl-7-br om o-
3,4-d ih yd r oisoqu in olin -1-2H-on e (R-22). Compound R-21
(160 mg, 0.572 mmol) was dissolved in MeOH (50 mL) and
Pd/C (10 mg) was added. The mixture was hydrogenated for 3
h at 50 psi. The solution was filtered through Celite and the
Celite bed rinsed with MeOH (50 mL). The solvent was
removed and the residue was dissolved in H2O (2.5 mL) and
48% HBr(aq) and cooled to 0 °C. KNO2 (40.4 mg, 0.580 mmol)
was dissolved in H2O (1 mL), added dropwise to the reaction
mixture, and stirred for 30 min. Excess HNO2 was destroyed
by the addition of urea (20 mg). This reaction mixture was
added dropwise to a warm (35 °C) solution of CuBr (252 mg,
1.76 mmol), 48% HBr (1.5 mL), and H2O (2 mL). After
completion of the addition, the resulting solution was warmed
to 75-80 °C and stirred for 1.5 h. The solution was cooled to
room temperature and stirred overnight. The solution was
made basic (pH > 10) with 50% KOH. The blue copper salts
were removed by filtration and rinsed with CH2Cl2 (30 mL).
The filtrate was extracted with CH2Cl2 (3 × 30 mL). The
organic rinse and extracts were combined and dried over
anhydrous Na2SO4 and the solvent was removed. The residue
was purified by flash chromatography (silica gel) eluting with
hexanes/EtOAc (4:1) to yield a white solid, which was recrys-
tallized from EtOAc/hexanes to yield R-22 as white needles
(85.2 mg, 48%): mp 139-141 °C; [R]D20 ) -27° (c 0.23, EtOH);
1H NMR (CDCl3) δ 8.23 (d, J ) 2.1 Hz, 1H, ArH-8), 7.61 (dd,
J ) 2.1, 8.1 Hz, 1H, ArH-6), 7.20 (d, J ) 8.1 Hz, 1H, ArH-5),
6.55 (br ex s, 1H, NH), 4.31-4.15 (m, 2H, CH2O), 4.07-4.03
3180, 3050, 2940, 1650, 1600, 1570, 1450, 1385, 1325, 775, 740
cm-1; 1H NMR (CDCl3) δ 8.05 (d, J ) 8.0 Hz, 1H, ArH-8), 7.51-
7.47 (m, 1H, ArH-6), 7.41-7.34 (m, 1H, ArH-7), 7.24 (d, J )
7.5 Hz, 1H, ArH-5), 6.38 (br ex s, 1H, NH), 4.02-3.94 (m, 1H,
H-3), 3.66-3.58 (m, 2H, CH2Cl), 3.18-3.03 (m, 2H, H-4); 13C
NMR (CDCl3) δ 166.1, 136.7, 133.1, 128.6, 128.5, 128.2, 127.9,
52.6, 46.6, 31.6; CIMS m/z (relative intensity) 196 (MH+, 60),
146 (100). Anal. (C10H10ClNO) C, H, N.
S-(+)-O-Meth oxyca r bon yl-3-h yd r oxym eth yl-3,4-d ih y-
d r oisoqu in olin -1-2H-on e (S-16) a n d S-(+)-3-Ch lor om eth -
yl-3,4-dih ydr oisoqu in olin -1-2H-on e (S-20). Compounds S-16
and S-20 were prepared using the identical procedure as for
R-16 and R-20 using S-19 as the starting material.
20
S-16: mp 125-126 °C; [R]D ) +28° (c 0.18, EtOH). Anal.
(C12H13NO4) C, H, N.
S-20: mp 85-86 °C; [R]D ) +21° (c 0.98, EtOH). Anal.
(C10H10ClNO) C, H, N.
20
R-(-)-O-Met h oxyca r b on yl-3-h yd r oxym et h yl-7-n it r o-
3,4-d ih yd r oisoqu in olin -1-2H-on e (R-21). Lactam R-16 (614
mg, 2.61 mmol) was dissolved in H2SO4 (10 mL) and cooled to
0 °C. KNO3 (257 mg, 2.54 mmol) was added in three portions
over 30 min. The mixture was warmed to room temperature
and stirred overnight. The mixture was poured onto ice and
stirred for 15 min as a white precipitate formed. The precipi-
tate was collected by filtration and dried. The aqueous filtrate
was extracted with EtOAc (4 × 50 mL). The combined organic
extractions were washed with brine and dried over anhydrous
Na2SO4. The solvent was removed and the residue combined
with the dried precipitate. The off-white solid was recrystal-
lized from acetone/hexanes to yield R-21 as fluffy white
crystals (580 mg, 79%): mp 209-210 °C; [R]D20 ) -17° (c 0.56,
acetonitrile); IR (KBr) 3190, 3090, 2980, 1740, 1680, 1610,
1520, 1430, 1345, 1260, 920, 790 cm-1; 1H NMR (CDCl3) δ 8.93
(d, J ) 2.3 Hz, 1H, ArH-8), 8.34 (dd, J ) 2.3, 8.3 Hz, 1H, ArH-
6), 7.44 (d, J ) 8.3 Hz, 1H, ArH-5), 6.40 (br ex s, 1H, CONH),
4.40-4.15 (m, 2H, CH2O), 4.14-4.08 (m, 1H, H-3), 3.83 (s, 3H,
OCH3), 3.21-3.05 (m, 2H, H-4); 13C NMR (DMSO-d6) δ 162.7,
155.3, 147.1, 145.4, 130.4, 130.3, 126.7, 122.0, 68.9, 55.2, 48.8,
29.8; CIMS m/z (relative intensity) 281 (MH+, 100), 249 (75),
163 (50). Anal. (C12H12N2O6) C, H, N.
(m, 1H, H-3), 3.83 (s, 3H, OCH3), 3.07-2.87 (m, 2H, H-4); 13
C
NMR (CDCl3) δ 164.6, 155.5, 135.8, 135.6, 131.5, 130.2, 129.8,
121.7, 69.2, 55.6, 50.2, 30.0; CIMS m/z (relative intensity) 316
(MH+ + 2, 100), 314 (MH+, 100), 301 (50), 299 (50), 284 (75),
282 (75). Anal. (C12H12NO4Br) C, H, N.
S-(+)-O-Meth oxyca r bon yl-3-h yd r oxym eth yl-7-br om o-
3,4-d ih yd r oisoqu in olin -1-2H-on e (S-22). Compound S-22
was prepared in the same manner as R-22 using S-21 as the
20
starting material: mp 139-141 °C; [R]D ) +26° (c 0.22,
EtOH). Anal. (C12H12NO4Br) C, H, N.
R-(+)-3-Hyd r oxym eth yl-7-br om o-1,2,3,4-tetr a h yd r oiso-
qu in olin e Hyd r och lor id e (R-14‚HCl). Compound R-22 (49.2
mg, 0.157 mmol) was dissolved in THF (15 mL) and 1 M

4358 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grunewald et al.
BH3‚THF (1.5 mL, 1.5 mmol) was added dropwise. The solution
was heated at reflux for 6 h and cooled in an ice bath. MeOH
(5 mL) was added to the reaction mixture. The solvent was
removed under reduced pressure and the residue dissolved in
MeOH (10 mL) and 6 N HCl (10 mL). The reaction mixture
was heated at reflux for 3 h. The solution was concentrated
(ca. 10 mL) and made basic (pH > 10) with 10% NaOH. This
basic solution was extracted with CH2Cl2 (3 × 20 mL). The
organic extracts were combined and dried over anhydrous
Na2SO4. The solvent was removed and the residue purified
by flash chromatography (silica gel) eluting with EtOAC/
hexanes (3:1) to yield a white solid. The solid was dissolved in
CH2Cl2 and dry HCl(g) was used to form the hydrochloride
salt. The salt was recrystallized from EtOH/hexanes to yield
R-14‚HCl as white needles (25.9 mg, 57%): mp 250 °C dec;
[R]D ) +48° (c 0.15, MeOH); H NMR (CD3OD) δ 7.34-7.33
(m, 2H, ArH-6,8), 7.07 (d, J ) 8.7 Hz, 1H, ArH-5), 4.27-4.26
(m, 2H, CH2OH), 3.85-3.80 (m, 1H, H-4), 3.60-3.54 (m, 1H,
H-4), 3.52-3.45 (m, 1H, H-3), 2.91-2.88 (m, 2H, H-1); CIMS
m/z (relative intensity) 244 (MH+ + 2, 100), 242 (MH+, 100),
212 (45), 210 (45). Anal. (C10H12NOBr‚HCl) C, H, N.
synthesis of racemic 620 to yield R-6‚HCl as white needles (32.0
mg, 66%). All spectral data were identical to those reported
20
previously: mp 241 °C; [R]D ) +54° (c 0.20, MeOH). Anal.
(C10H12NF‚HCl) C, H, N.
S-(-)-3-F lu or om et h yl-1,2,3,4-t et r a h yd r oisoq u in olin e
Hyd r och lor id e (S-6‚HCl). Compound S-6 was prepared in
the same manner as racemic 620 using S-24 as the starting
20
material: mp 241 °C; [R]D ) -52° (c 0.28, MeOH). Anal.
(C10H12NF‚HCl) C, H, N.
R-(-)-3-F lu or om eth yl-7-n itr o-3,4-d ih yd r oisoqu in olin -
1-2H-on e (R-25). Lactam R-24 (200 mg, 1.12 mmol) was
nitrated using the same procedures used for the synthesis of
racemic 2520 to yield R-25 as light yellow needles (132 mg,
53%). All spectral data were identical to those reported
20
previously: mp 209 °C; [R]D ) -3.4° (c 0.53, acetone). Anal.
20
1
(C10H9NO3F) C, H, N.
S-(+)-3-F lu or om eth yl-7-n itr o-3,4-d ih yd r oisoqu in olin -
1-2H-on e (S-25). Compound S-25 was prepared in the same
manner as racemic 2520 using S-24 as the starting material:
20
mp 209 °C; [R]D ) +3.7° (c 0.43, acetone). Anal. (C10H9NO3-
F) C, H, N.
S-(-)-3-Hyd r oxym eth yl-7-br om o-1,2,3,4-tetr a h yd r oiso-
qu in olin e Hyd r och lor id e (S-14‚HCl). The same procedure
used for the synthesis of R-14 was used for S-14 with S-22 as
R-(+)-3-Flu or om eth yl-7-n itr o-1,2,3,4-tetr ah ydr oisoqu in -
olin e Hyd r och lor id e (R-11‚HCl). Lactam R-25 (141 mg,
0.629 mmol) was reduced in the same manner as used for the
synthesis of racemic 1120 to yield S-11‚HCl as off-white crystals
(75.0 mg, 48%). All spectral data were identical to those
20
the starting material: mp 250 °C dec; [R]D ) -49° (c 0.12,
MeOH). Anal. (C10H12NOBr‚HCl) C, H, N.
R-(-)-3-Hyd r oxym eth yl-3,4-d ih yd r oisoqu in olin -1-2H-
on e (R-23). A solution of R-16 (541 mg, 2.30 mmol) in MeOH
(20 mL) and 6 N HCl (20 mL) was heated at reflux overnight.
The MeOH was removed under reduced pressure. The solution
was neutralized with 6 N NaOH and extracted with EtOAc
(4 × 50 mL). The extracts were combined, washed with brine,
and dried over anhydrous Na2SO4. The solvent was removed
and the residue was purified by a flash column chromatogra-
phy (silica gel) with EtOAc as the eluent to yield R-23 as a
white solid (365 mg, 90%): mp 141-142 °C; [R]D20) +14°
20
reported previously: mp 260 °C dec; [R]D ) +72° (c 0.52,
EtOH). Anal. (C10H11N2O2F‚HCl) C, H, N.
S-(-)-3-Flu or om eth yl-7-n itr o-1,2,3,4-tetr ah ydr oisoqu in -
olin e Hyd r och lor id e (S-11‚HCl). Compound S-11 was pre-
pared in the same manner as racemic 1120 using S-25 as the
starting material: mp 260 °C dec; [R]D20 ) -66° (c 0.55, EtOH).
Anal. (C10H11N2O2F‚HCl) C, H, N.
R-(+)-3-F lu or om et h yl-7-cya n o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (R-12‚HCl). THIQ R-11‚HCl (1.23
g, 4.99 mmol) was converted to nitrile R-12 using the same
procedures reported for the synthesis of racemic 1220 to yield
R-12 (256 mg, 27%) as a pale brown solid. The hydrochloride
salt was formed in MeOH using dry HCl(g). The solvent was
removed under reduced pressure and the crude hydrochloride
salt was recrystallized from MeOH/EtOAc to yield R-12‚HCl
(267 mg, 100%). All spectral data were identical to those
reported previously: mp 240 °C dec; (free base) [R]D20 ) +110°
(c 0.14, CHCl3). Anal. (C11H11N2F‚HCl) C, H, N.
1
(c 1.1, EtOH); H NMR (DMSO-d6) δ 7.78 (d, J ) 6.9 Hz, 1H,
ArH-8), 7.67 (br ex s, 1H, NH), 7.43-7.38 (m, 1H, ArH-7), 7.25
(m, 2H, ArH-5,6), 4.9 (br ex s, 1H, OH), 3.51-3.50 (m, 1H,
H-3), 3.49-3.25 (m, 2H, CH2O), 2.96-2.76 (m, 2H, H-4); 13C
NMR (DMSO-d6) δ 164.8, 138.3, 132.3, 129.3, 128.4, 127.3,
127.0, 63.5, 52.4, 30.0; CIMS m/z (relative intensity) 178 (MH+,
60), 146 (100), 128 (20), 89 (15). Anal. (C10H11NO2) C, H, N.
S-(-)-3-Hyd r oxym eth yl-3,4-d ih yd r oisoqu in olin -1-2H-
on e (S-23). Compound S-23 was prepared in the same manner
as R-23 using S-16 as the starting material: mp 141-142 °C;
S-(-)-3-F lu or om et h yl-7-cya n o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (S-12‚HCl). Compound S-12 was
20
[R]D ) -14° (c 1.1, EtOH). Anal. (C10H11NO2) C, H, N.
prepared using the same methods as racemic 1220 using S-11
R-(-)-3-F lu or om e t h yl-3,4-d ih yd r oisoq u in olin -1-2H -
on e (R-24). Compound R-23 (600 mg, 3.38 mmol) was added
to dry CH2Cl2 (10 mL) and cooled to -78 °C. Diethylamino-
sulfur trifluoride (DAST; 0.90 mL, 6.8 mmol) was added
dropwise to the suspension. The mixture was slowly warmed
to room temperature and stirred for 16 h. Ice water (10 mL)
was added and the reaction mixture was stirred for 15 min.
The organic phase was separated and the aqueous phase was
extracted with CH2Cl2 (2 × 20 mL). The organic phase and
extracts were combined and washed with 3 N HCl (50 mL),
saturated NaHCO3 (50 mL), and brine (50 mL). The organic
phase was dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The residue was purified by
flash chromatography eluting with hexane/EtOAc (1:1) to yield
a white solid that was recrystallized from hexanes to yield
R-24 as white needles (281 mg, 46%). This compound has been
synthesized in racemic form20 and all spectral data were
identical to those reported previously: mp 120-121 °C;
20
as the starting material: mp 240 °C dec; (free base) [R]D
-110° (c 0.15, CHCl3). Anal. (C11H11N2F‚HCl) C, H, N.
)
R-(+)-3-F lu or om et h yl-7-b r om o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (R-15‚HCl). THIQ R-11‚HCl (804
mg, 3.26 mmol) was converted to R-15 using the same methods
reported for the synthesis of racemic 1520 to yield R-15 as a
white solid (527 mg, 67%): mp 91-92 °C. The hydrochloride
salt was formed in MeOH using dry HCl(g). The solvent was
removed and the crude hydrochloride salt recrystallized from
MeOH and ether to yield R-15‚HCl as white needles (604 mg,
100%). This compound was synthesized in racemic form20 and
all spectral data were identical to those reported previously:
20
mp 258 °C dec; [R]D ) +56° (c 0.50, MeOH). Anal. (C10H11
-
NFBr‚HCl) C, H, N.
S-(-)-3-F lu or om et h yl-7-b r om o-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (S-15‚HCl). Compound S-15 was
prepared using the same methods as for racemic 1520 using
20
20
[R]D ) -22° (c 0.36, EtOH). Anal. (C10H10NOF) C, H, N.
S-11‚HCl as the starting material: mp 258 °C dec; [R]D
-52° (c 0.62, MeOH). Anal. (C10H11NFBr‚HCl) C, H, N.
)
S -(+)-3-F lu or om e t h yl-3,4-d ih yd r oisoq u in olin -1-2H -
on e (S-24). Lactam S-24 was prepared in the same manner
as racemic 2420 using S-23 as the starting material: mp 119-
R-(-)-3-F lu or om eth yl-7-ch lor osu lfon yl-3,4-d ih yd r oiso-
qu in olin -1-2H-on e (R-26). Lactam R-24 (338 mg, 1.89 mmol)
was chlorosulfonylated using the procedures reported for the
synthesis of racemic 2620 to yield R-26 as white crystals (342
mg, 65%). All spectral data were identical to those reported
20
120 °C; [R]D ) +21° (c 0.30, EtOH). Anal. (C10H10NOF) C,
H, N.
R-(+)-3-F lu or om et h yl-1,2,3,4-t et r a h yd r oisoq u in olin e
Hyd r och lor id e (R-6‚HCl). Lactam R-24 (43.3 mg, 0.241
mmol) was reduced using the same procedures as for the
20
previously: mp 183 °C; [R]D ) -13° (c 0.20, acetonitrile).
Anal. (C10H9NO3FClS) C, H, N.

PNMT Inhibitors: 3-CH2OH, 3-CH2F, 3-CH2Cl-THIQs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4359
S-(+)-3-F lu or om eth yl-7-ch lor osu lfon yl-3,4-d ih yd r oiso-
qu in olin -1-2H-on e (S-26). Compound S-26 was prepared
using the same methods as for racemic 2620 using S-24 as the
starting material: mp 183 °C; [R]D ) +13° (c 0.16, acetoni-
trile). Anal. (C10H9NO3FClS) C, H, N.
mp 180-182 °C; [R]D20 ) +4.8° (c 1.0, acetone); IR (KBr) 3200,
3080, 2950, 1670, 1600, 1500, 1430, 1340, 740 cm-1; 1H NMR
(DMSO-d6) δ 8.54 (d, J ) 2.5 Hz, 1H, H-8), 8.50 (ex s, 1H,
NH), 8.33 (dd, J ) 2.5, 8.3 Hz, 1H, H-6), 7.41 (d, J ) 8.3 Hz,
1H, H-5), 4.08-3.99 (m, 1H, H-3), 3.75-3.68 (m, 2H, CH2Cl),
3.32-3.13 (m, 2H, H-4); 13C NMR (DMSO-d6) δ 163.1, 147.6,
145.7, 130.8, 130.6, 127.3, 122.4, 51.3, 47.6, 30.8; CIMS m/z
(relative intensity) 241 (MH+, 100), 191 (45), 145 (15). Anal.
(C10H9ClN2O3) C, H, N.
20
R-(+)-3-F lu or om eth yl-7-N-(4-ch lor op h en yl)a m in osu l-
fon yl-3,4-d ih yd r oisoqu in olin -1-2H-on e (R-27). Compound
R-26 (500 mg, 1.80 mmol) was transformed into sulfonamide
R-27 using the same procedures reported20 for the production
of racemic 27 to yield compound R-27 as light orange crystals
(639 mg, 1.74 mmol, 97%). All spectral data were identical to
S-(-)-3-Ch lor om eth yl-7-n itr o-3,4-d ih yd r oisoqu in olin -
1-2H-on e (S-29). This compound was prepared in the same
manner as R-29 using lactam S-20 as the starting material:
20
those reported previously: mp 197-198 °C; [R]D ) +13°
20
(c 0.55, acetone). Anal. (C16H14N2O3FClS) C, H, N.
mp 180-182 °C; [R]D ) -4.9° (c 1.0, acetone). Anal. (C10H9-
ClN2O3) C, H, N.
S-(-)-3-F lu or om eth yl-7-N-(4-ch lor op h en yl)a m in osu l-
fon yl-3,4-d ih yd r oisoqu in olin -1-2H-on e (S-27). Compound
R-(+)-3-Ch lor om eth yl-7-n itr o-1,2,3,4-tetr ah ydr oisoqu in -
olin e Hyd r och lor id e (R-30‚HCl). Lactam R-29 (616 mg, 2.56
mmol) was dissolved in dry THF (30 mL) and 1 M BH3‚THF
(7.7 mL, 7.7 mmol) was added dropwise to the solution. The
mixture was heated at reflux for 15 h. It was cooled in an ice
bath and MeOH (5 mL) was added dropwise. The solvent was
removed under reduced pressure to yield a white residue. This
residue was dissolved in MeOH (10 mL) and 6 N HCl (10 mL)
and heated to reflux for 3 h. The MeOH was removed under
vacuum and the solution was made basic (pH > 10) with 15%
NaOH. The aqueous solution was extracted with EtOAc (3 ×
50 mL). The combined organic extracts were washed with brine
(50 mL) and dried over anhydrous K2CO3. The solvent was
removed under reduced pressure to yield R-30 as a white solid,
from which the hydrochloride salt was formed in using dry
HCl(g). The solvent was removed and the residue recrystallized
from EtOH/hexanes to yield R-30‚HCl as white needles (524
S-27 was prepared using the same methods as for racemic 2720
20
using S-26 as the starting material: mp 197-198 °C; [R]D
-13° (c 0.55, acetone). Anal. (C16H14N2O3FClS) C, H, N.
)
R-(+)-3-F lu or om eth yl-7-N-(4-ch lor op h en yl)a m in osu l-
fon yl-1,2,3,4-tetr a h yd r oisoqu in olin e (R-13). Lactam R-27
(639 mg, 1.74 mmol) was reduced using the same procedures
reported previously for the synthesis of racemic 2720 to yield
R-13 as white crystals (248 mg, 36%). It should be noted that
R-13 was isolated and characterized as its free base. All
spectral data were identical to those reported previously: mp
20
178-179 °C; [R]D ) +50° (c 0.29, MeOH). Anal. (C16H16N2-
O2FClS) C, H, N.
S-(-)-3-F lu or om eth yl-7-N-(4-ch lor op h en yl)a m in osu l-
fon yl-1,2,3,4-t et r a h yd r oisoq u in olin e (S-13). Compound
S-13 was prepared using the same methods as for racemic 1320
20
using S-27 as the starting material: mp 178-179 °C; [R]D
-56° (c 0.23, MeOH). Anal. (C16H16N2O2FClS) C, H, N.
)
20
mg, 78%): mp 258-260 °C; [R]D ) +53° (c 1.0, MeOH); IR
(KBr) 3000-2600 (broad), 2480, 1575, 1510, 1495, 1435, 1345,
1330, 770, 735 cm-1; 1H NMR (DMSO-d6) δ 10.43 (br ex s, 2H,
NH2+), 8.23 (br s, 1H, ArH-6), 8.09 (br s, 1H, ArH-8), 7.55 (br
s, 1H, H-5), 4.49 (s, 2H, H-1), 4.14 (m, 2H, CH2Cl), 3.96 (m,
1H, H-3), 3.24 (m, 2H, H-4); 13C NMR (DMSO-d6) δ 146.9,
140.3, 131.5, 137.2, 122.9, 122.7, 53.3, 44.7, 44.7, 29.5; CIMS
R-(+)-3-Ch lor om et h yl-1,2,3,4-t et r a h yd r oisoqu in olin e
Hyd r och lor id e (R-28‚HCl). Lactam R-20 (310 mg, 1.58
mmol) was dissolved in dry THF (20 mL) and 1 M BH3‚THF
(5 mL) was added dropwise to the solution. The mixture was
heated at reflux for 15 h. It was cooled in an ice bath and
MeOH (5 mL) was added dropwise. The solvent was removed
under reduced pressure to yield a white residue. This residue
was dissolved in MeOH (10 mL) and concentrated HCl (5 mL)
and heated at reflux for 3 h. The MeOH was removed under
reduced pressure and 15% KOH was added until the solution
was at pH > 10. The aqueous phase was extracted with EtOAc
(3 × 50 mL). The combined organic extracts were washed with
brine (50 mL) and dried over anhydrous K2CO3. The solvent
was removed under reduced pressure to yield R-28 as a white
solid, from which the hydrochloride salt was formed in MeOH
using HCl(g). The solvent was removed and the residue
recrystallized from EtOH/hexanes to yield R-28‚HCl as white
m/z (relative intensity) 227 (MH+, 100), 177 (75). Anal. (C10
11ClN2O2‚HCl) C, H, N.
-
H
S-(-)-3-Ch lor om eth yl-7-n itr o-1,2,3,4-tetr ah ydr oisoqu in -
olin e Hyd r och lor id e (S-30‚HCl). Compound S-30‚HCl was
prepared using the same methods as for R-30‚HCl using S-29
20
as the starting material: mp 258-260 °C; [R]D ) -54°
(c 1.0, MeOH). Anal. (C10H11ClN2O2‚HCl) C, H, N.
Deter m in a tion of En a n tiom er ic Excess. Enantiomeric
excess (ee) was determined for every compound to be greater
than 95% and was assessed in the following manner. Using
Chiral HPLC analysis (Chiralcel OJ ) eluting with hexanes/
isopropyl alcohol/NH(Et)2, all compounds appeared as a single
distinct peak. However, for the racemic mixtures of these
compounds, baseline separation could not be obtained due to
tailing caused by the THIQ amine. Several solutions of each
isolated enantiomer (2.0 mg in 5 mL of isopropyl alcohol) were
prepared and mixed in the following proportions (95:5, 97.5:
2.5, and 98.75:1.25). These solutions were analyzed, and it was
found each enantiomer could easily be detected as a shoulder
in the 97.5:2.5 mixture but not in the 98.75:1.25 mixture,
which implied that the ee of each of these compounds was at
a minimum greater than 95%.
Ra d ioch em ica l Assa y for P NMT Activity. The assay
used for this study has been described previously.29 A normal
assay tube mixture consisted of 50 µL of 0.5 M phosphate
buffer (pH 8.0), 25 µL of 10 mM AdoMet, 5 µL of [3H]AdoMet
that contains 3 × 105 dpm (specific activity approximately 15
mCi/mmol), 25 µL of substrate solution (phenylethanolamine),
25 µL of inhibitor solution, 25 µL of the enzyme preparation,
and water to achieve a total volume of 250 µL. The mixture
was incubated for 30 min at 37 °C, quenched by the addition
of 250 µL of 0.5 M borate buffer (pH 10), and extracted with 2
mL of toluene/isoamyl alcohol (7:3). A 1-mL aliquot of the
organic layer was removed, transferred to a scintillation vial,
and diluted with cocktail for counting. The mode of inhibition
for all of the inhibitors assayed was determined to be competi-
20
crystals (286 mg, 83%): mp 226-228 °C; [R]D ) +38° (c 1.0,
MeOH); IR (KBr) 3380, 2880, 2580, 2480, 2400, 1575, 1495,
1430, 1395, 1000, 750, 690 cm-1; 1H NMR (DMSO-d6) δ 10.23
(br ex s, 2H, NH2+), 7.26-7.24 (m, 4H, Ar-H), 4.34 (m, 2H,
H-1), 4.11-4.01 (m, 2H, CH2Cl), 3.88 (m, 1H, H-3), 3.17-3.08
(m, 2H, H-4); 13C NMR (DMSO-d6) δ 131.9, 129.6, 129.3, 128.3,
127.5, 127.4, 53.9, 45.1, 45.0, 29.3; CIMS m/z (relative inten-
sity) 182 (MH+, 85), 132 (100), 104 (45), 78 (20). Anal. (C10
12ClN‚HCl) C, H, N.
-
H
S-(-)-3-Ch lor om et h yl-1,2,3,4-t et r a h yd r oisoq u in olin e
Hyd r och lor id e (S-28‚HCl). This compound was prepared in
the same manner as R-28‚HCl using lactam S-20 as the
20
starting material: mp 226-228 °C; [R]D ) -40° (c 1.0,
MeOH). Anal. (C10H12ClN‚HCl) C, H, N.
R-(+)-3-Ch lor om eth yl-7-n itr o-3,4-d ih yd r oisoqu in olin -
1-2H-on e (R-29). Lactam R-20 (620 mg, 3.17 mmol) was
dissolved in concentrated H2SO4 (12 mL) at 0 °C and KNO3
(385 mg, 8.80 mmol) was added to the solution in one portion.
The reaction mixture was stirred overnight at room temper-
ature. The reaction solution was poured onto ice carefully and
the resulting solution was extracted with EtOAc (4 × 50 mL).
The combined organic extractions were washed with brine and
dried over anhydrous Na2SO4. The solvent was removed to
yield a yellow solid, which was recrystallized with EtOAc/
hexanes to yield R-29 as pale yellow needles (641 mg, 84%);

4360 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grunewald et al.
Alpha-2-adrenoceptor Function in the Hypothalamus of Rats in
Comparison with SKF29661, SKF64139 and Yohimbine. Neu-
ropsychobiology 1996, 34, 82-89.
tive by inspection of the 1/V versus 1/S plots of the data. All
assays were run in duplicate with three inhibitor concentra-
tions over a 5-fold range. Ki values were determined by a
hyperbolic fit of the data.
(9) (a) Burke, W. J .; Chung, H. D.; Marshall, G. L.; Gillespie, K. N.;
J oh, T. H. Evidence for Decreased Transport of PNMT Protein
in Advanced Alzheimer’s Disease. J . Am. Geriatr. Soc. 1990, 38,
1275-1282. (b) Burke, W. J .; Chung H. D.; Strong, R.; Mattam-
mal, M. B.; Marshall, G. L.; Nakra, R.; Grossberg, G. T.; Haring,
J . H.; J oh, T. H. Mechanism of Degeneration of Epinephrine
Neurons in Alzheimer’s Disease. In Central Nervous System
Disorders of Aging: Clinical Intervention and Research; Strong,
R., Wood, W. G., Burke, W. J ., Eds.; Raven Press: New York,
1988; pp 41-70. (c) Burke, W. J .; Chung H. D.; Huang, J . S.;
Huang, S. S.; Haring J . H.; Strong, R.; Marshall, G. L.; J oh, T.
H. Evidence for Retrograde Degeneration of Epinephrine Neu-
rons in Alzheimer’s Disease. Ann. Neurol. 1988, 24, 532-536.
(d) Burke, W. J .; Galvin, N. J .; Chung, H. D.; Stoff, S. A.;
Gillespie, K. N.; Cataldo, A. M.; Nixon, R. A. Degenerative
Changes in Epinephrine Tonic Vasomotor Neurons in Alz-
heimer’s Disease. Brain Res. 1994, 661, 35-42.
(10) Drew, G. M. R2-Adrenoceptor-blocking Action of the Phenyl-
ethanolamine N-Methyltransferase Inhibitor SKF 64139. J .
Pharm. Pharmacol. 1981, 33, 187-188.
(11) Grunewald, G. L.; Dahanukar, V. H.; Ching, P.; Criscione, K.
R. Effect of Ring Size or an Additional Heteroatom on the
Potency and Selectivity of Bicyclic Benzylamine-type Inhibitors
of Phenylethanolamine N-Methyltransferase. J . Med. Chem.
1996, 39, 3539-3546.
(12) Pendleton, R. J .; Hieble, J . P. Studies on the Adrenergic Receptor
Specificity of Inhibitors of Phenylethanolamine N-Methyltrans-
ferase. Res. Commun. Chem. Pathol. Pharmacol. 1981, 34, 399-
408.
(13) Pendleton, R. G.; Gessner, G.; Weiner, G.; J enkins, B.; Sawyer,
J .; Bondinell, W.; Intoccia, A. Studies on SK&F 29661, an Organ-
Specific Inhibitor of Phenylethanolamine N-Methyltransferase.
J . Pharmacol. Exp. Ther. 1979, 208, 24-30.
(14) Bondinell, W. E.; Chapin, R. W.; Girard, G. R.; Kaiser, C.; Krog,
A. J .; Pavloff, A. M.; Schwartz, M. S.; Silvestri, J . S.; Vaidya, P.
D.; Lam, B. L.; Wellman, G. R.; Pendleton, R. G. Inhibitors of
Phenylethanolamine N-Methyltransferase and Epinephrine Bio-
synthesis. 1. Chloro-Substituted 1,2,3,4-Tetrahydroisoquinolines.
J . Med. Chem. 1980, 23, 506-511.
(15) Singh, P. Quantitative Structure-Activity Relationship Studies
of 1,2,3,4-Tetrahydroisoquinoline Derivatives as Inhibitors of
Phenylethanolamine N-Methyltransferase. Indian J . Biochem.
Biophys. 1983, 20, 397-399.
r2-Ad r en ocep tor Ra d ioliga n d Bin d in g Assa y. The ra-
dioligand binding assay was performed using the methods
developed by U′Prichard et al.30 Male Sprague-Dawley rats
were decapitated and the cortexes removed and homogenized
with 20 volumes (w/v) of ice-cold 50 mM Tris/HCl buffer (pH
7.7 at 25 °C). Homogenates were centrifuged three times for
10 min at 50000g with resuspension of the pellet in fresh buffer
between spins. The final pellet was homogenized in 200
volumes (w/v) of ice-cold 50 mM Tris/HCl buffer (pH 7.7 at 25
°C). Incubation tubes containing [3H]clonidine (specific activity
ca. 19.2 mCi/mmol, final concentration 2.0 nM), various
concentrations of the inhibitors, and an aliquot of freshly
suspended tissue (800 µL) to a final volume of 1 mL were used.
Tubes were incubated at 25 °C for 30 min, and the incubation
was terminated by rapid filtration under vacuum through
GF/B glass fiber filters. The filters were rinsed with three 5-mL
washes of 50 mM Tris/HCl buffer (pH 7.7 at 25°C). The filters
were counted in vials containing premixed scintillation cock-
tail. Nonspecific binding was determined as the concentration
of ligand bound in the presence of 2 µM phentolamine. All
assays were examined by a log-probit analysis of the data to
determine the IC50 values, and Ki values were determined by
the equation: Ki ) IC50/(1 + [clonidine]/KD), as all Hill coef-
ficients were approximately equal to 1.
Ack n ow led gm en t. This research was supported by
NIH Grant HL 34193, NIH Pre-Doctoral Training Grant
GM 07775 (T.M.C.), and an American Federation for
Pharmacy Education Pre-Doctoral Fellowship (T.M.C.).
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