3,7-Disubstituted-1,2,3,4-tetrahydroisoquinolines display remarkable potency and selectivity as inhibitors of phenylethanolamine N- methyltransferase versus the α2-adrenoceptor(1a)

Article abstract of DOI:10.1021/jm9807252

3-Hydroxymethyl-1,2,3,4-tetrahydroisoquinoline (4) is a more selective inhibitor (PNMT K(i) = 1.1 μM, α₂ K(i) = 6.6 μM, selectivity (α₂ K(i)/PNMT K(i)) = 6.0) of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28), with respec

Full text of DOI:10.1021/jm9807252

1982
J . Med. Chem. 1999, 42, 1982-1990
3,7-Disu bstitu ted -1,2,3,4-tetr a h yd r oisoqu in olin es Disp la y Rem a r k a ble P oten cy
a n d Selectivity a s 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 1a
Gary L. Grunewald,* Vilas H. Dahanukar, Bee Teoh,1b and Kevin R. Criscione
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045
Received December 23, 1998
3-Hydroxymethyl-1,2,3,4-tetrahydroisoquinoline (4) is a more selective inhibitor (PNMT Ki )
1.1 µM, R2 Ki ) 6.6 µM, selectivity (R2 Ki/PNMT Ki) ) 6.0) of phenylethanolamine N-
methyltransferase (PNMT, EC 2.1.1.28), with respect to its R2-adrenoceptor affinity, than is
3-methyl-1,2,3,4-tetrahydroisoquinoline (2; PNMT Ki ) 2.1 µM, R2 Ki ) 0.76 µM, selectivity )
0.36) or 1,2,3,4-tetrahydroisoquinoline (1, THIQ; PNMT Ki ) 9.7 µM, R2 Ki ) 0.35 µM, selectivity
) 0.036). Evaluation of the O-methyl ether derivative of 4 suggested that the 3-hydroxymethyl
substituent might be involved in a hydrogen-bond donor-type of interaction at a sterically
compact region in the PNMT active site. The directionality of the steric bulk tolerance at both
the PNMT active site and the R2-adrenoceptor appears to be the same. Since the presence of
a hydrophilic electron-withdrawing substituent (such as NO2, SO2CH3, or SO2NH2) at the
7-position of THIQ reduced the binding affinity toward the R2-adrenoceptor, we investigated
the combination of both a hydrophilic electron-withdrawing 7-substituent and a 3-alkyl
substituent on a THIQ nucleus. A synergistic effect in increasing the PNMT-inhibitory potency
of the THIQ nucleus and reducing the affinity toward the R2-adrenoceptor was observed with
this 3,7-disubstitution. Remarkably, 7-aminosulfonyl-3-hydroxymethyl-THIQ (12; PNMT Ki )
0.34 µM, R2 Ki ) 1400 µM, selectivity ) 4100) displayed a 23-680-fold enhanced selectivity
over the parent compounds 27 (SK&F 29661; PNMT Ki ) 0.55 µM, R2 Ki ) 100 µM, selectivity
) 180) and 4 (selectivity ) 6.0) and is thus the most selective PNMT inhibitor yet reported.
Ta ble 1. In Vitro Activitiesa of
In tr od u ction
3-Alkyl-1,2,3,4-tetrahydroisoquinolines as Inhibitors of PNMT
and of the Binding of [3H]Clonidine to the R2-Adrenoceptor
We have targeted the enzyme phenylethanolamine
N-methyltransferase (PNMT, EC 2.1.1.28), the enzyme
involved in the biosynthesis of epinephrine, to define
the role of epinephrine in the central nervous system
(CNS). Inhibition of PNMT offers a unique way of
studying the pharmacology of central epinephrine with-
out significantly affecting the levels of other brain
catecholamines.2,3 On the basis of the innervation of
various brain nuclei, epinephrine is speculated to be
involved in the control of oxytocin secretion, food and
water intake, gonadotrophin secretion, blood pressure,
and respiration, along with sleep and wakefulness.4
Also, more recent studies have implied that PNMT may
play a role in the degeneration of epinephrine-contain-
ing neurons in the progression of Alzheimer’s and
Parkinson’s diseases.5,6 Unfortunately, the high affinity
of most of the well-studied PNMT inhibitors toward the
R2-adrenoceptor has been the major barrier in unam-
biguously defining the role of epinephrine in the central
nervous system.7
compd
R3
PNMT Ki ( SEMb
R2 Ki ( SEMc
1
2
3
4
5
6
7
H
10 ( 1
3.0 ( 0.2
24 ( 1
0.35 ( 0.10d
0.76 ( 0.08
0.66 ( 0.11
6.6 ( 0.3
CH3
CH2CH3
CH2OH
CO2H
CO2CH3
CONH2
2.4 ( 0.2
>2000
70 ( 6
41 ( 2
11 ( 1
250 ( 20
a
All values are µM. b Reference 11. c Determined in our labora-
d
tory. Reference 10.
on the THIQ nucleus.9 Introduction of a methyl sub-
stituent at position 1 or 4 led to a 10-20-fold reduction
in PNMT-inhibitory activity as compared to 3-methyl-
THIQ (2). The increased potency of 2 (Ki ) 3.0 µM,
molar refractivity (MR) for CH3 ) 5.65) over 1 (Ki ) 10
µM) was attributed to a favorable steric interaction at
the PNMT active site.10 Previously, a variety of sub-
stituents at the 3-position on THIQ had been synthe-
sized11 to probe this site and the nature of the interac-
tion that enhanced PNMT-inhibitory potency (Table 1).
An ethyl substituent is not very sterically demanding
(MR ) 10.3) relative to a hydroxymethyl substituent
(MR ) 7.19), but 3 (3-ethyl-THIQ) was found to be 10
times less active (Ki ) 24 µM) at inhibiting PNMT than
4 (3-hydroxymethyl-THIQ; Ki ) 2.4 µM). This result
A number of potent inhibitors of PNMT contain the
1,2,3,4-tetrahydroisoquinoline (THIQ, 1) nucleus.8 It
was established by Kaiser et al.8 that a chlorine at the
7-position on the THIQ nucleus is optimal as compared
to the 5-, 6-, or 8-position in regard to PNMT inhibition.
Subsequently, we reported that the 3-position was best
for an alkyl substituent as compared to position 1 or 4
* To whom correspondence should be addressed. Phone: (785) 864-
4497. Fax: (785) 864-5326. E-mail: ggrunewald@ukans.edu.
10.1021/jm9807252 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/18/1999

3,7-Disub-THIQs as Selective PNMT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1983
Sch em e 1
argued for a hydrogen-bonding-type of interaction that
overcomes steric bulk intolerance and increases PNMT-
inhibitory activity. Evaluation of THIQs bearing car-
bonyl substituents, such as CO2CH3 and CONH2, indi-
cated that not only the amount of steric bulk but also
the spatial orientation of the steric bulk was important.
Linear chains of limited steric bulk were tolerated better
than the bent carbonyl chains.
Introduction of a 3-hydroxymethyl substituent onto
the potent PNMT inhibitor SK&F 64139 (8) to produce
3-hydroxymethyl-7,8-dichloro-THIQ (9) was expected to
further enhance the PNMT-inhibitory activity. However,
9 (Ki ) 0.38 µM)11 was found to exhibit similar potency
as a PNMT inhibitor compared to 8 (Ki ) 0.20 µM),10
while the R2-adrenoceptor affinity of 9 (R2 Ki ) 0.15
µM)12 was found to be about 7 times lower than that of
8 (R2 Ki ) 0.022 µM),10 resulting in an overall gain in
selectivity (8, selectivity (R2 Ki/PNMT Ki) ) 0.11 vs 9,
selectivity ) 0.39). In light of our previous study on
7-substituted-THIQs,13 it seemed of interest to probe the
effect of the 3-hydroxymethyl substituent on THIQs
bearing a hydrophilic electron-withdrawing substituent
at the 7-position that previously had been shown to
confer enhanced selectivity (e.g., NO2, SO2NH2, or
SO2CH3).
Sch em e 2
Most of the classical methods for the preparation of
THIQs (e.g., Pictet-Spengler, Bischler-Napieralski,
and Pomeranz-Fritsch reactions) involve the participa-
tion of the aromatic ring π electrons in the cyclization
step. Therefore, these methods cannot be used in the
synthesis of THIQs bearing strong electron-withdrawing
substituents on the aromatic ring. In the synthesis of
11, a direct approach based on electrophilic aromatic
nitration of 5 was undertaken (Scheme 2). Electrophilic
substitution reactions such as nitration,17,18 chlorosul-
fonation,19 and Friedel-Crafts acylation20 have been
quite successful in introducing a substituent at the
7-position on THIQ. To minimize racemization of 5, an
unconventional nitration was carried out with com-
mercially available nitronium tetrafluoroborate21 using
nonaqueous and nonacidic conditions. The mixture of
5- and 7-nitro amino alcohols 15a ,b obtained could not
be separated by column chromatography. Thus, the
mixture was converted to the N-benzyloxycarbonyl
derivatives 16a ,b, followed by esterification to the
methyl esters 17 and 18, which enabled the successful
separation of the regioisomers by medium-pressure
liquid chromatography (MPLC).22 (It was interesting to
note that the N-tert-butyloxycarbonyl methyl ester
derivative of 5 could not be cleanly separated by MPLC.)
The regiochemistry of the isolated products was unam-
biguously established only after their conversion to the
The 3-substituted-THIQs evaluated above were race-
mates, and thus stereoselectivity in binding remained
undetermined. On the basis of these considerations, the
enantiomers resulting from the combination of a 3-
methyl or a 3-hydroxymethyl substituent with a 7-sub-
stituent on THIQ were investigated. Also, the O-methyl
ether derivative of 4 (compound 10) was synthesized to
probe the nature of the hydrogen-bonding interaction.
Ch em istr y
Compound 5 was prepared from phenylalanine by
treatment with formaldehyde and concentrated hydro-
chloric acid (Scheme 1), according to literature proce-
dure.14 However, with enantiopure phenylalanine, par-
tial racemization of 5 was reported to occur under the
acidic reaction conditions.14b Repeated crystallization of
5 from aqueous ethanol (to constant optical rotation)
permitted the isolation of the enantiomers in enantio-
merically pure form, albeit in modest yields. The borane-
mediated reduction of amino acid 5 was facilitated by
the addition of BF3‚Et2O15 which formed a THF-soluble
complex with 5. Preferential O-methylation of 4 to yield
10 was accomplished quantitatively using the reaction
conditions described by Meyers et al.16

1984 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Grunewald et al.
Sch em e 3
Sch em e 4
As mentioned earlier, electrophilic substitution reac-
tions such as chlorosulfonation and Friedel-Crafts
acylation on N-acetyl-THIQ are known to produce the
desired 7-regioisomer. However, nitration of 2 or its
N-acetyl derivative gave an inseparable mixture of
regioisomers. Nitration of 3,4-dihydro-2H-isoquinolin-
1-one has been reported26 to produce only the 7-nitro
product in excellent yields. Racemic 3-methylisoqui-
nolone 24 and its enantiomers were readily prepared
by a literature method27 and nitrated cleanly to yield a
single regioisomer 25 and its enantiomers (Scheme 4).
The presence of the most downfield doublet at 8.55 ppm
(due to H-8 which is ortho to both the nitro and the
amido group) in the 1H NMR spectrum of 25 is in
accordance with the assigned regiochemistry. Borane
reduction of amide 25 gave 14, with no competing
reduction of the nitro group noted.
Bioch em istr y. All the compounds were evaluated as
their hydrochloride salts. In vitro PNMT activity was
assessed by use of a standard radiochemical assay that
has been described previously for both substrates28 and
inhibitors.29 Bovine adrenal PNMT used for the in vitro
assay was purified according to the procedure of Connett
and Kirshner30 through the isoelectric precipitation
step. Inhibition constants were determined by using at
least three different concentrations of inhibitor, as
previously described,29 with phenylethanolamine as the
variable substrate. R2-Adrenergic receptor binding as-
says were performed using cortex obtained from male
Sprague-Dawley rats.31 [3H]Clonidine was used as the
radioligand to define the specific binding, and phentol-
amine was used to define the nonspecific binding.
Clonidine was used as the ligand to define R-adrenergic
binding affinity to simplify the comparison with previ-
ous results.
corresponding amino alcohols. Reduction of the ester
group in 18 followed by cleavage of the benzyloxycar-
bonyl group under basic conditions23 gave the desired
compound 11. On the basis of the aromatic splitting
1
pattern in the H NMR spectrum and the results of a
one-dimensional difference nuclear Overhauser effect
(NOE) experiment on 11, the nitro group was assigned
to the 7-position (see Experimental Section).
Chlorosulfonation of the aromatic nucleus provided
a convenient way of introducing a sulfur substituent.
The reaction of 4 with carbonyldiimidazole24 gave ox-
azolidinone 19sthe key intermediate for the synthesis
of 7-sulfur-substituted 3-hydroxymethyl-THIQs (Scheme
3). The protection of 4 as an oxazolidinone avoided any
side reactions in the subsequent chlorosulfonation proc-
ess. Unlike the chlorosulfonation of N-acetyl-THIQ,
which gave only the 7-regioisomer,13 oxazolidinone 19
gave a mixture of regioisomers after treatment with
chlorosulfonic acid. Fortunately, the desired 7-chloro-
sulfonyl regioisomer 20 could be purified by fractional
crystallization from ethyl acetate in a low yield of 16%.
The regiochemistry of 20 was confirmed by a one-
dimensional difference NOE experiment (see Experi-
mental Section). Formation of sulfonamide 21 from 20,
followed by basic hydrolysis of the oxazolidinone, fur-
nished the desired aminosulfonyl alcohol 12. The trans-
formation of the sulfonyl hydrazide derivative 22 to the
methanesulfonyl oxazolidinone 23 was conveniently
achieved by heating an ethanolic suspension of 22 with
methyl iodide and sodium acetate. In this general
method,25 the initially formed N-methylsulfonyl hy-
drazide is thought to undergo an acetate anion-mediated
elimination to the sulfinate anion which is then alkyl-
ated by methyl iodide to afford the methyl sulfone.
Removal of the oxazolidinone protecting group from 23
gave sulfone 13.
Resu lts a n d Discu ssion
The results of the biochemical evaluations are re-
ported in Table 2. For comparison, the PNMT Ki values
for the enantiomers of 210 are included. For internal
consistency, the PNMT Ki values given in Table 2 for
racemic 2 and 4 were determined in assays done
simultaneously with their enantiomers, rather than
those reported previously.9,11 Also, the PNMT Ki value
reported for 1 was determined in an assay run concur-
rently with its R2 assay,10 rather than that reported
previously.11
In comparison with unsubstituted THIQ (1; PNMT
Ki ) 9.7 µM, R2 Ki ) 0.35 µM, selectivity (R2 Ki/PNMT
Ki) ) 0.036),10 racemic 2 and 4 were more potent at
PNMT and were also more selective (PNMT vs R2-
adrenoceptor affinity). Although the absolute configu-

3,7-Disub-THIQs as Selective PNMT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1985
Ta ble 2. In Vitro Activities of 3-Alkyl-7-substituted-1,2,3,4-tetrahydroisoquinolines as Inhibitors of PNMT and of the Binding of
[3H]Clonidine at the R2-Adrenoceptor
compd
R3
CH3
R7
stereo
PNMT Ki (A) ( SEM (µM)
R2 Ki (B) ( SEM (µM)
B/A selectivity
2
H
(()
2.1 ( 0.1
38 ( 2
1.0 ( 0.1
0.76 ( 0.08
5.7 ( 0.3
0.49 ( 0.05
31 ( 1
53 ( 2
19 ( 1
0.36
0.15
0.49
63
41
76
6.0
7.9
1.1
66
160
14
4100
1000
0.30
R-(-)
S-(+)
(()
14
4
CH3
NO2
H
0.49 ( 0.05
1.3 ( 0.1
R-(-)
S-(+)
(()
R-(+)
S-(-)
(()
R-(+)
S-(-)
(()
(()
(()
0.25 ( 0.02
1.1 ( 0.1
CH2OH
CH2OH
6.6 ( 0.3
4.4 ( 0.3
17 ( 1
0.56 ( 0.05
15 ( 2
11
NO2
0.29 ( 0.04
0.24 ( 0.03
0.90 ( 0.05
0.34 ( 0.06
0.64 ( 0.04
9.2 ( 0.4
19 ( 1
38 ( 1
13 ( 1
12
13
10
CH2OH
CH2OH
CH2OCH3
SO2NH2
SO2CH3
H
1400 ( 30
660 ( 10
2.8 ( 0.1
ration of the more active enantiomer was the opposite
in the case of 2 and 4 (due to the Cahn-Ingold-Prelog
priority rule), the side chains are oriented in the same
region of space. As such, the more active enantiomer in
each case at PNMT was the (+)-enantiomer. Unfortu-
nately, in the case of both 2 and 4, the enantiomer that
was more active at PNMT also had the higher R2-
adrenoceptor affinity (lower R2 Ki) and, therefore, lower
overall selectivity. It can be inferred that the direction-
ality of steric bulk tolerance of the 3-substituted-THIQs
at the PNMT active site and at the R2-adrenoceptor
appeared to be the same. Higher selectivity (PNMT vs
R2-adrenoceptor affinity) of (R)-(+)-4 over (S)-(+)-2
might be attributed to two factors: (1) higher potency
at PNMT due to a hydrogen-bonding-type of interaction
of the 3-hydroxymethyl substituent of 4 and (2) lower
affinity at the R2-adrenoceptor due to unfavorable steric
interactions of the 3-substituent (the hydroxymethyl
group is larger than the methyl group).
To probe the nature of hydrogen-bonding interactions
(i.e., a hydrogen bond acceptor-type or donor-type of
interaction), the O-methyl ether derivative 10 was
prepared in racemic form. In comparison with (()-4, (()-
10 (PNMT Ki ) 9.2 µM, R2 Ki ) 2.8, selectivity ) 0.30)
was less potent at PNMT and had a 2-fold higher
affinity toward the R2-adrenoceptor. On the basis of
these results, it appeared that, at the PNMT active site,
there is either a limited amount of steric bulk tolerance
at the 3-position, as in the case of the 3-ethyl compound
3, or a hydrogen bond donor-type of interaction that
might be responsible for the increase in potency, as in
the case of the 3-methoxymethyl compound 10. The
2-fold enhancement in affinity of 10 toward the R2-
adrenoceptor might result from the increased lipo-
philicity of the CH2OCH3 (π ) -0.78)32 substituent over
the CH2OH substituent (π ) -1.03).32 This hypothesis
was consistent with the observed higher R2-adrenoceptor
affinity of (()-2, wherein the 3-substituent was more
lipophilic (π for CH3 ) 0.56).32
synergistic effect was not observed for 9. We have
proposed that THIQs bind in two different orientationss
lipophilic (orientation A) and nonlipophilic (orientation
B), depending on the lipophilicity of the 7-substituent
on the aromatic ring.13 Thus, the lack of synergism is
not surprising, given that 9 (π ) 0.71 for 7-Cl) is
proposed to bind in orientation A, which would place
the 3-hydroxymethyl substituent in a different region
of space (Figure 1a).
Accordingly, the hydrophilicity of the 7-nitro group
will direct the binding of the THIQ in orientation B
(Figure 1b). The effect of adding a 7-nitro substituent
to 2 (compound 14) in reducing the R2-adrenoceptor
affinity was more pronounced than adding a 7-nitro
substituent to 4 (compound 11). As shown by the
stereoselectivity ratios (as measured by the ratio of the
Ki values of the two enantiomers) in Table 3, the general
trend seen in this series was that the enantiomer which
was more active in inhibiting PNMT also had the higher
affinity toward the R2-adrenoceptor, although this did
not hold true for (R)-(+)-11.
Sulfonyl substituents (such as the methylsulfonyl and
aminosulfonyl groups) markedly affect the potency and
selectivity of the THIQ nucleus, and a remarkable
synergism is displayed when a 3-hydroxymethyl sub-
stituent is combined with a 7-sulfonyl substituent. The
3-hydroxymethyl analogue (12; PNMT Ki ) 0.34 µM) of
SK&F 29661 (27) was about 1.6 times more potent than
27 (PNMT Ki ) 0.55 µM)13 in inhibiting PNMT. More
importantly, its affinity toward the R2-adrenoceptor was
significantly decreased (12, R2 Ki ) 1400 µM vs 27, R2
Ki ) 100 µM), resulting in a tremendous gain in
selectivity (12, R2/PNMT ) 4100 vs 27, R2/PNMT ) 180).
Compound 12 is thus the most selective inhibitor of
PNMT yet reported.
The synergistic effect was less dramatic in the case
of the 3-hydroxymethyl analogue (13) of 7-methylsul-
fonyl-THIQ (28). In comparison with 28 (PNMT Ki )
1.34 µM, R2 Ki ) 160 µM, selectivity ) 120),13 a 2-fold
enhancement in PNMT-inhibitory activity and a 4-fold
loss in affinity toward the R2-adrenoceptor were ob-
served for 13 (PNMT Ki ) 0.64 µM, R2 Ki ) 660 µM,
selectivity ) 1000). It previously had been shown that
the 7-methylsulfonyl group was more effective than the
The combination of a nitro group at the 7-position and
a 3-alkyl substituent on THIQ enhanced selectivity
(PNMT vs R2-adrenoceptor affinity) as compared to
3-alkyl-THIQs or 7-nitro-THIQ (26; PNMT Ki ) 0.41
µM, R2 Ki ) 4.3 µM, selectivity ) 10)13 alone. This

1986 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Grunewald et al.
yl-THIQs. At the PNMT active site, the hydroxymethyl
substituent may be involved in a hydrogen bond donor-
type of interaction. The combination of a 7-electron-
withdrawing substituent and a 3-alkyl substituent on
the THIQ ring not only enhanced PNMT-inhibitory
potency but also reduced the R2-adrenoceptor affinity.
In general, the trend in stereoselectivity in binding of
7-nitro-3-alkyl-THIQs at the PNMT active site remained
the same as observed for the 3-alkyl-THIQs. A tremen-
dous gain in selectivity was observed when a hydroxy-
methyl substituent was introduced on 27, resulting in
the most selective (PNMT vs R2-adrenoceptor affinity)
compound (12) yet reported. Unfortunately, previous
studies have shown that 27 is unable to penetrate the
blood-brain barrier (BBB),33 presumably due to its high
polarity. Therefore, it is also likely that 12 would be
unable to penetrate the BBB, which would make it an
unsuitable pharmacological tool to define the role of
epinephrine in the CNS. It is hoped that other combina-
tions of 3,7-disubstitution on THIQ will produce a
selective inhibitor with sufficient lipophilicity to pen-
etrate the BBB.
Exp er im en ta l Section
All reagents and solvents were reagent grade or were
purified by standard methods before use. Melting points were
determined in open capillaries on a Thomas-Hoover melting
point apparatus calibrated with known compounds but are
otherwise uncorrected. Proton nuclear magnetic resonance
spectra (1H NMR) were recorded on a Varian XL-300 or GE
QE-300 spectrometer with CDCl3 as the solvent, and chemical
shifts are reported in parts per million (ppm) relative to
tetramethylsilane (TMS, 0.00 ppm). Carbon nuclear magnetic
resonance spectra (13C NMR) were recorded on a Varian XL-
300 spectrometer with CDCl3 as the solvent, and the chemical
shifts are reported in ppm relative to CDCl3 (77.0 ppm). For
the hydrobromide salts of the phenolic amines, NMR spectra
were recorded in deuterated dimethyl sulfoxide (DMSO-d6),
and the chemical shifts are reported relative to DMSO (2.49
ppm for 1H and 39.50 ppm for 13C). Multiplicity abbrevia-
tions: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
bs, broad singlet; e, exchangeable. Infrared spectra were
obtained on a Perkin-Elmer 1420 infrared spectrophotometer.
Electron-impact mass spectra (EIMS) were obtained on a
Ribermag R10-10 mass spectrometer. The relative intensities
of the mass spectrum peaks are listed in parentheses. Medium-
pressure liquid chromatography (MPLC), using an adaptation
of the apparatus of Meyers and co-workers,22 was performed
using silica gel 60 (230-400 mesh) supplied by Universal
Adsorbents, Atlanta, GA. Preparative centrifugal thin-layer
chromatography (PCTLC) was performed on a Harrison model
7924 chromatotron (Harrison Research, Palo Alto, CA) using
Merck silica gel 60 PF254/CaSO4‚0.5H2O binder on 1-, 2-, or
4-mm thickness plates. Analytical TLC was performed by
using silica gel with a fluorescent indicator coated on 1- × 3-in.
glass plates in 0.2-mm thickness (Whatman MKGF silica gel
200 µm). Bulb-to-bulb distillations were carried out on a
Kugelrohr distillation apparatus (Aldrich Chemical Co., Mil-
waukee, WI), and oven temperatures were recorded. High-
F igu r e 1. (a) Putatively more active R enantiomer of 9, in
orientation A, wherein it cannot approach the hydrogen-
bonding site and was therefore unable to exhibit a synergistic
effect at the PNMT active site. (b) More active R enantiomer
of 11, in orientation B, in which the side chain can participate
in a hydrogen-bonding interaction and enhance the PNMT-
inhibitory potency in a synergistic manner. (c) SYBYL-gener-
ated stereoview of (R)-9 (orientation A: dark) superimposed
on (R)-11 (orientation B: light). The optimized geometries
(Tripos force field) of 9 and 11 were fitted using three points:
both ends of a normal (2 Å long) passing through the centroid
of the THIQ aromatic ring and the end of the axial lone pair
(2.4 Å long) on the THIQ nitrogen.
Ta ble 3. Stereoselectivity Ratiosa for PNMT and
R2-Adrenoceptor Activities
compd
PNMT (-)/(+)
R2 (-)/(+)
2
4
11
14
38
27
3.8
5.2
12
3.9
0.34
2.8
a
Ratios of the Ki values of the enantiomers.
7-aminosulfonyl group in reducing the R2-adrenoceptor
affinity of the THIQ nucleus (compare 27 and 28).13 In
contrast, the reverse was found to be true in the case of
the 3-hydroxymethyl-THIQs. The cause of further re-
duction in the binding affinity of 12 was not readily
understood. It might be possible that 12 is binding in a
different fashion or at some other allosteric site at the
R2-adrenoceptor.
performance liquid chromatography was performed on
a
Shimadzu LC 6A system. The Chiralcel OJ column (0.46 × 25
cm) was purchased from Diacel Inc., New York. Optical
rotations were recorded on a Perkin-Elmer 241 polarimeter
using the sodium D line as the light source. Combustion
analyses were performed on a Hewlett-Packard model 185B
CHN analyzer at the University of Kansas by Dr. Tho Ngoc
Nguyen.
Amine hydrochloride salts were prepared by adding a
solution of methanolic HCl to a methanolic solution of the
amine, followed by crystallization of the resulting hydrochlo-
ride from MeOH-ether.
Su m m a r y a n d Con clu sion
It appears that the R2-adrenoceptor displays more
stereoselectivity in the binding of ligands than does the
active site of PNMT. The directionality of steric bulk
tolerance at the PNMT active site and at the R2-
adrenoceptor appears to be the same. The 3-hydroxy-
methyl substituent was more effective in increasing the
PNMT-inhibitory activity of THIQ as well as reducing
the R2-adrenoceptor affinity, as compared to the 3-meth-

3,7-Disub-THIQs as Selective PNMT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1987
The compounds SK&F 64139 (8) and SK&F 29661 (27) were
kindly provided by Smith Kline and French Laboratories,
Smith Kline Corp., Philadelphia, PA. S-Adenosyl-L-methionine
(AdoMet) was obtained from Sigma Chemical Co. [methyl-3H]-
S-Adenosyl-L-methionine that was used in the radiochemical
assay was purchased from New England Nuclear Corp.
(Boston, MA). Bovine adrenal glands were obtained from Davis
Meat Processing (Overbrook, KS). [3H]Clonidine used in the
R2-adrenoceptor binding assay was purchased from Amersham
Corp. (Arlington Heights, IL).
onto ice, and 10% NH4OH was added dropwise until Congo
red paper no longer turned blue. The solid was collected by
suction filtration and was successively washed with water,
EtOH, and ether. After air-drying 15a ,b was obtained as a
pale-yellow solid (3.09 g, 98.6%): mp > 300 °C dec; IR (KBr)
2940, 2760, 2640, 2500, 1620 (CO), 1530 (NO2), 1450, 1390,
1350 (NO2), 1310, 1080, 860, 830, 780, 730 cm-1
.
To a rapidly stirred suspension of 15a ,b (0.50 g, 2.2 mmol)
and NaHCO3 (0.51 g, 6.1 mmol) in water (10 mL) was added
benzyl chloroformate (0.48 g, 2.8 mmol) in small portions. The
reaction mixture was stirred for 2 h at room temperature, and
then the reaction mixture was washed with ether (thrice). The
aqueous layer was adjusted to pH 2 with 1 N HCl, and the
aqueous layer was extracted with EtOAc (thrice). The com-
bined EtOAc layers were washed with brine and dried and
the solvent removed by rotary evaporation to yield 16a ,b (0.53
g, 69%) as a thick red oil: IR (film) 3300-2800, 1700 (CO),
1660 (NCO), 1520 (NO2), 1345 (NO2), 1225, 1105, 1000, 730
cm-1; MS (CI, NH3), m/z 374 (M + NH4+, 97), 357 (M+ + 1,
19), 313 (27), 221 (15), 177 (34), 145 (100), 106 (75), 91 (10),
52 (52).
A solution of 16a ,b (2.08 g, 5.83 mmol), K2CO3 (1.61 g, 11.7
mmol), and MeI (2.48 g, 1.09 mL, 17.5 mmol) in dry acetone
(100 mL) was heated at reflux for 30 min under N2. After
cooling to room temperature the solvent was removed by rotary
evaporation, water was added, and the mixture was extracted
with CH2Cl2 (thrice). The combined CH2Cl2 layers were dried
and evaporated to afford a mixture of 17 and 18 as an orange
oil (2.59 g). The separation of the regioisomers was ac-
complished by MPLC using hexane/EtOAc (3:1) as the eluent,
yielding 17 (0.810 g, 37.5%) and 18 (1.09 g, 50.6%).
(()-3-H yd r oxym 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 (4‚HCl). A suspension of 514a (1.10 g, 5.64
mmol) in dry THF (15 mL) was stirred under N2, and BF3‚
Et2O (1.0 mL, 8.1 mmol) was added to it via syringe. After
the mixture stirred for 30 min, BH3‚THF complex (1 M in THF,
11 mL, 11 mmol) was added and the mixture was stirred
overnight. The clear solution was cooled in an ice bath, and
MeOH (5 mL) was added dropwise. After the effervescence
ceased the solvent was removed on a rotary evaporator to
afford a pale-yellow viscous oil that was heated with 6 N NaOH
(10 mL) to reflux under N2 for 6 h. The reaction mixture was
cooled and extracted with ether (four times). The ether layers
were combined, washed with brine, and dried over anhydrous
MgSO4. Evaporation of the etheral extract afforded a viscous
yellow oil (1.10 g) that was distilled bulb-to-bulb (115-120 °C,
0.3 mmHg) to obtain a pale-yellow oil that solidified on cooling
(0.64 g, 69%): mp 84-86 °C; IR (KBr) 3240 (NH), 3150-3050
(OH), 2900, 2820, 1450, 1320, 1060, 1040, 740 cm-1; 1H NMR
(CDCl3) δ 7.13-6.98 (m, 4 H, ArH), 3.98 (s, 2 H, H-1), 3.68
(dd, 1 H, J ) 11.0, 3.7 Hz, OCH2), 3.48 (dd, 1 H, J ) 11.0, 7.5
Hz, OCH2), 3.02-2.95 (m, 1 H, H-3), 2.62-2.55 (m, 2 H, H-4);
the two exchangeable protons NH and OH were not observed
and might be rapidly exchanged; 13C NMR (CDCl3) δ 135.2,
133.9, 129.2, 126.1, 126.0, 125.7, 65.3 (CH2O), 55.0 (C-3), 47.8
(C-1), 30.8 (C-4). The free base was converted to the hydro-
chloride salt and recrystallized from MeOH-Et2O: mp 196-
198 °C, lit.11 mp 195-196 °C.
(()-17 was obtained as a thick yellow oil: IR (film) 2950,
1740 (CO2Me), 1700 (CON), 1520 (NO2), 1340 (NO2), 1300,
1200, 1100, 1010, 730, 695 cm-1 1H NMR (CDCl3) δ 8.08-
;
8.05 (m, 2 H, H-6 and H-8), 7.41-7.24 (m, 6 H, H-7 and C6H5),
5.33-5.20 (m, 2 H, OCH2Ph), 5.16-5.10 (m, 1 H, H-3), 4.91
and 4.71 (d, 2 H, J ) 18.0 Hz, H-1, conformers), 3.64 and 3.58
(s, 3 H, OCH3, conformers), 3.45-3.22 (m, 2 H, H-4); MS (CI,
NH3) m/z 388 (M + NH4+, 11), 371 (M+ + 1, 16), 341 (9), 279
(49), 205 (11), 190 (24), 164 (53), 157 (100), 129 (26), 126 (13),
110 (26), 102 (13), 85 (32), 79 (63), 49 (98). Anal. (C19H18N2O6)
C, H, N.
(R)-(+)-3-Hyd r oxym eth yl-1,2,3,4-tetr a h yd r oisoqu in o-
lin e Hyd r och lor id e (4‚HCl). Similarly (R)-(+)-514b (0.40 g,
2.2 mmol) gave (R)-(+)-4 (0.22 g, 60%): mp 113-114 °C
(recrystallized from PhH), lit.34 mp 116 °C. The free base was
converted to its HCl salt: mp 240-241 °C dec; [R]24 ) 56° (c
D
0.23, EtOH). Anal. (C10H13NO‚HCl) C, H, N.
(()-18 was crystallized from EtOAc-hexanes as a pale-
yellow solid: mp 135-136 °C; IR (KBr) 3060, 2960, 1730
(CO2Me), 1700 (CON), 1520 (NO2), 1410, 1340 (NO2), 1320,
(S)-(-)-3-H yd r oxym et h yl-1,2,3,4-t et r a h yd r oisoq u in o-
lin e Hyd r och lor id e (4‚HCl). Similarly (S)-(-)-514b (0.5 g, 2.8
mmol) gave (S)-(-)-4 (0.33 g, 72%): mp 115-116 °C (recrystal-
lized from PhH), lit.35 mp 118-119 °C (recrystallized from
PhH). The free base was converted to its HCl salt: mp 240-
1240, 1200, 1100, 1010, 900, 840 cm-1 1H NMR (CDCl3) δ
;
8.04-7.99 (m, 2 H, H-6 and H-8), 7.41-7.31 (m, 6 H, H-5 and
C6H5), 5.31-5.19 (m, 2 H, OCH2Ph), 5.10-5.07 (m, 1 H, H-3),
4.93 and 4.69 (d, 2 H, J ) 17 0.0 Hz, H-1, conformers), 3.64
and 3.57 (s, 3 H, OCH3, conformers), 3.44-3.20 (m, 2 H, H-4);
MS (CI, NH3) m/z 388 (M + NH4+, 13), 371 (M+ + 1, 6), 341
(10), 279 (11), 207 (24), 145 (11), 108 (13), 91 (13), 52 (100).
Anal. (C19H18N2O6) C, H, N.
241 °C dec; [R]24 ) -56° (c 0.23, MeOH). Anal. (C10H13NO‚
D
HCl) C, H, N.
(()-3-Met h oxym 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 (10‚HCl). The amino alcohol 4 (1.00 g, 6.16
mmol) was methylated according to the procedure described
by Meyers et al.16 for the O-methylation of amino alcohols with
methyl iodide. After bulb-to-bulb distillation (100-105 °C, 0.25
mmHg) of the crude product, a colorless oil was obtained (1.00
g, 91.7%): IR (film) 3320 (NH), 2910, 2880, 1450, 1320, 1100,
(R)-(-)-7-Nitr o-N-ben zyloxycar bon yl-3-m eth oxycar bon -
yl-1,2,3,4-tetr a h yd r oisoqu in olin e (18). Using an identical
procedure as for the racemic compound, (R)-(+)-514b yielded
(R)-(-)-18 as a thick yellow oil: [R]22D ) -46° (c 0.20, CHCl3).
Anal. (C19H18N2O6) C, H, N.
1
790, 740 cm-1; H NMR (CDCl3) δ 7.11-6.98 (m, 4 H, ArH),
4.06 and 4.01 (AB q, 2 H, J AB ) 15.6 Hz, H-1), 3.49 (dd, 1 H,
J ) 9.28, 3.8 Hz, CH2O), 3.38-3.33 (m, 4 H, OCH3 and CH2O),
3.14-3.08 (m, 1 H, H-3), 2.78-2.50 (m, 2 H, H-4), 2.05 (bs, e,
1 H, NH); 13C (CDCl3) δ 135.4, 133.8, 129.0, 125.9, 125.8, 125.5,
76.4 (CH2O), 58.8 (OCH3), 52.9 (C-3), 47.9 (C-1), 31.2 (C-4).
The hydrochloride salt was crystallized as colorless small
crystals: mp 214-215 °C dec; EIMS m/z 178 (M+ + 1, 1), 177
(M+, 1), 133 (11), 144 (4), 134 (100), 117 (11), 115 (12), 104
(14), 77 (11), 45 (14). Anal. (C11H15NO‚HCl) C, H, N.
(()-5- a n d (()-7-Nitr o-N-ben zyloxyca r bon yl-3-m eth -
oxyca r bon yl-1,2,3,4-tetr a h yd r oisoqu in olin e (17 a n d 18).
To an ice-cold suspension of (()-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5)14a (2.50 g, 14.1 mmol) in dry acetonitrile
(20 mL) was added nitronium tetrafluoroborate (2.43 g of 85%
NO2+BF4-, 15.5 mmol) in one portion under N2 with rapid
stirring. After stirring for 1 h the reaction mixture was poured
(S)-(+)-7-Nitr o-N-ben zyloxycar bon yl-3-m eth oxycar bon -
yl-1,2,3,4-tetr a h yd r oisoqu in olin e (18). Using an identical
procedure as for the racemic compound, (S)-(-)-514b yielded
22
(S)-(+)-18 as a thick yellow oil: [R]D ) 52° (c 0.20, CHCl3).
Anal. (C19H18N2O6) C, H, N.
(()-3-Hyd r oxym eth yl-7-n itr o-1,2,3,4-tetr a h yd r oisoqu i-
n olin e Hyd r och lor id e (11‚HCl). To a rapidly stirred ice-
cold solution of (()-18 (1.10 g, 2.97 mmol) in dry THF (30 mL)
was added LiBH4 solution (2.0 M solution in THF, 5.9 mL, 12
mmol) dropwise under a positive pressure of N2. The reaction
mixture was allowed to stir at room temperature for 3 h and
quenched with MeOH and 1 N HCl. Evaporation of the
reaction mixture gave a yellow oil which was treated with
water and extracted with CH2Cl2 (thrice). Drying followed by
evaporation of the combined CH2Cl2 extracts furnished a
yellow semisolid (0.84 g, 82%).

1988 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Grunewald et al.
In a thick-walled tube the yellow semisolid (0.70 g, 2.0
mmol) was dissolved in DME (7 mL), and to it KOH (0.34 g,
6.1 mmol) was added as a solution in water (3 mL). The tube
was flushed with N2, and the orange-colored reaction mixture
was heated at about 100 °C for 12 h. The resulting black-brown
solution was cooled, made acidic with 6 N HCl, and extracted
with CH2Cl2 (thrice) to remove color impurities. The aqueous
solution was cooled, made alkaline with KOH pellets, and
extracted with EtOAc (thrice). The combined EtOAc extracts
were washed with brine (once), dried, and evaporated to yield
a yellow-brown solid (0.25 g, 58%), which was crystallized from
EtOAc-hexanes: mp 172-174 °C; IR (KBr) 3230 (NH), 3100
(OH), 2880, 2820, 1540 (NO2), 1345 (NO2), 1080, 1060, 1040,
830, 740 cm-1; 1H NMR (DMSO-d6 and CDCl3) δ 7.94 (m, 2 H,
H-6 and H-8), 7.27 (d, 1 H, J ) 8.5 Hz, H-6), 4.17 and 4.12
(AB q, 2 H, J AB ) 16.2 Hz, H-1), 3.73 (dd, 1 H, J ) 10.7, 3.8
Hz, OCH2), 3.60-3.36 (m, 2 H, OCH2 and NH), 3.09-3.00 (m,
1 H, H-3), 2.85-2.62 (m, 2 H, H-4); 13C NMR (DMSO-d6 and
CDCl3) δ 145.0, 142.1, 136.4, 129.4, 120.4, 120.1, 64.4 (OCH2),
bath, and the excess BH3 was quenched carefully by dropwise
addition of MeOH. The reaction mixture was evaporated to
dryness, then methanolic HCl (15 mL) was added, and the
reaction mixture was heated to reflux for 6 h. The solvent was
removed on a rotary evaporator, and the solid obtained was
suspended in water, made alkaline with solid Na2CO3, and
extracted with CHCl3 (four times). The organic layers were
combined and dried (K2CO3), and the solvent was removed by
rotary evaporation to yield a yellow-red solid. Purification by
PCTLC (silica, 2 mm) using CH2Cl2/MeOH/NH4OH (250:17:
1) as the eluent gave a pale-yellow solid (0.34 g, 92%) which
was recrystallized from EtOAc-hexanes as pale-buff needles:
mp 96-98 °C; IR (KBr) 3200 (NH), 2950, 2910, 1520 (NO2),
1335 (NO2), 1090, 810, 735 cm-1 1H NMR (CDCl3) δ 7.99-
;
7.92 (m, 2 H, H-6 and H-8), 7.21 (d, 1 H, J ) 8.3 Hz, H-5),
4.15 (s, 2 H, H-1), 3.08-3.01 (m, 1 H, H-3), 2.88 (dd, 1 H, J )
17.1, 3.9 Hz, H-4), 2.57 (dd, 1 H, J ) 17.1, 10.7 Hz, H-4), 1.66
(bs, e, 1 H, NH), 1.28 (d, 3 H, J ) 6.3 Hz, CH3); 13C NMR
(CDCl3) δ 146.0, 142.9, 136.8, 129.9, 121.2, 121.0, 48.7 (C-1),
48.4 (C-3), 37.3 (C-4), 22.2 (CH3). The hydrochloride salt was
obtained as a colorless solid: mp 284-286 °C dec; EIMS m/z
193 (M+ + 1, 4), 192 (M+, 7), 178 (11), 177 (100), 149 (16), 131
(16), 130 (16), 103 (18), 91 (20), 77 (16). Anal. (C10H12N2O2‚
HCl) C, H, N.
(R)-(-)-3-Met h yl-7-n it r o-1,2,3,4-t et r a h yd r oisoq u in o-
lin e Hyd r och lor id e (14‚HCl). Using the same procedure as
above, (R)-(-)-25 was reduced to the desired amine (-)-14: mp
99-100 °C, mp (HCl) 283 °C dec; [R]D22 ) -91° (c 0.20, MeOH).
Anal. (C10H12N2O2‚HCl) C, H, N.
53.9 (C-3), 47.1 (C-1), 30.7 (C-4); MS (CI, NH3) m/z 209 (M+
+
1), 169 (43), 104 (15), 102 (100), 85 (98).
The regiochemistry of the product was confirmed by a one-
dimensional difference NOE experiment. When the protons at
approximately 4.00 ppm were irradiated (corresponding to the
1-position on the THIQ nucleus), a positive NOE response was
detected at 7.94 ppm (corresponding to the 8-position on the
THIQ nucleus). This singlet was the only NOE enhancement
observed. The hydrochloride salt was obtained as a pale-brown
solid: mp 253-268 °C dec. Anal. (C10H12N2O3‚HCl) C, H, N.
(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 (11‚HCl). Application of the above
procedure to (R)-(-)-18 gave (R)-(+)-11: mp 171-173 °C dec,
(S)-(+)-3-Met h yl-7-n it r o-1,2,3,4-t et r a h yd r oisoq u in o-
lin e Hyd r och lor id e (14‚HCl). Using the same procedure as
above, (S)-(+)-25 was reduced to the desired amine (+)-14: mp
mp (HCl) > 270 °C dec; [R]22 ) 62° (c 0.28, CHCl3). Anal.
22
D
98-99 °C, mp (HCl) 278-279 °C dec; [R]D ) 89° (c 0.20,
(C10H12N2O3‚HCl) C, H, N.
MeOH). Anal. (C10H12N2O2‚HCl) C, H, N.
(S)-(-)-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 (11‚HCl). Application of the above
procedure to (S)-(+)-18 gave (S)-(-)-11: mp 174-176 °C dec,
(()-1,4,9,9a-Tetr ah ydr o-2-oxa-3a-azacyclopen ta[b]n aph -
th a len -3-on e (19). To a solution of (()-4 (9.40 g, 57.6 mmol)
in dry THF (100 mL) was added 1,1′-carbonyldiimidazole (11.2
g, 69.1 mmol), and the mixture was heated to reflux under N2
overnight. The reaction mixture was cooled, and 1 N HCl (100
mL) was added. The acidic aqueous layer was extracted with
CH2Cl2 (thrice). The organic layers were combined, washed
with brine, and dried, and the solvent was removed by rotary
evaporation to yield a pale-yellow solid (9.87 g). Recrystalli-
zation of this solid from EtOAc gave 19 as a colorless
crystalline solid (8.36 g, 77.2%): mp 120-122 °C; IR (KBr)
mp (HCl) > 270 °C dec; [R]22 ) -69° (c 0.28, CHCl3). Anal.
D
(C10H12N2O3‚HCl) C, H, N.
(()-3,4-Dih yd r o-3-m et h yl-7-n it r oisoq u in olin -1-(2H )-
on e (25). The amide 3,4-dihydro-3-methylisoquinolin-1-(2H)-
one27b (24; 0.90 g, 5.6 mmol) was dissolved in concentrated
H2SO4 (4 mL) and cooled in an ice bath. Potassium nitrate
(0.63 g, 6.2 mmol) was added in three portions to the stirred
ice-cold reaction mixture. After stirring for 4 h at room
temperature, the reaction mixture was poured onto ice to yield
a yellow solid which was filtered, dried, and crystallized (with
charcoal treatment) from aqueous EtOH to yield a golden-
yellow crystalline solid (0.83 g, 72%): mp 242-243 °C; IR
(KBr) 3180 (NH), 3070, 2960, 1660 (CO), 1510 (NO2), 1335
1
2940, 1735 (CO), 1430, 1265, 1080, 1000, 950, 750 cm-1; H
NMR (CDCl3) δ 7.27-7.13 (m, 4 H, ArH), 4.81 (d, 1 H, J )
16.7 Hz, H-4), 4.57 (t, 1 H, J ) 8.3 Hz, CH2O), 4.35 (d, 1 H, J
) 16.5 Hz, H-4), 4.15-4.10 (m, 1 H, CH2O), 3.99-3.92 (m, 1
H, H-9a), 2.97-2.80 (m, 2 H, H-9); 13C NMR (CDCl3) δ 157.3
(CO), 131.6, 131.2, 129.3, 126.9, 126.7, 126.3, 68.3 (CH2O), 51.0
(C-3), 42.9 (C-1), 33.9 (C-4); EIMS m/z 190 (M+ + 1, 18), 189
(M+, 52), 188 (M+ - 1, 10), 144 (9), 128 (14), 116 (19), 105
(12), 104 (100), 78 (20), 51 (11). Anal. (C11H11NO2) C, H, N.
1
(NO2), 1060, 920, 850, 805, 740 cm-1; H NMR (DMS0-d6) δ
8.55 (d, 1 H, J ) 2.4 Hz, H-8), 8.35-8.30 (m, 2 H, H-7 and
NH), 7.63 (d, 1 H, J ) 8.4 Hz, H-5), 3.81-3.74 (m, 1 H, H-3),
3.14 (dd, 1 H, J ) 16.5, 4.3 Hz, H-4), 2.82 (dd, 1 H, J ) 16.4,
9.9 Hz, H-4), 1.22 (d, 3 H, J ) 6.4 Hz, CH3); 13C NMR (DMSO-
d6) δ 162.6 (CO), 146.6, 146.0, 130.1, 129.6. 126.2, 121.5, 45.8
(C-3), 35.1 (C-4), 20.8 (CH3); EIMS m/z 207 (M+ + 1, 3), 206
(M+, 8), 192 (13), 191 (100), 163 (52), 145 (26), 135 (12), 89
(37), 77 (15), 69 (11). Anal. (C10H10N2O3) C, H, N.
(()-6-Ch lor osu lfon yl-1,4,9,9a -tetr a h yd r o-2-oxa -3a -a za -
cyclop en ta [b]n a p h th a len -3-on e (20). Compound 19 (2.0 g,
11 mmol) was dissolved in CHCl3 (30 mL), and the solution
was chilled in a dry ice-acetone bath. Chlorosulfonic acid (8.5
g, 4.8 mL, 73 mmol) was added dropwise, and the reaction
mixture was allowed to warm and stir at room temperature
for 14 h. The reaction mixture was poured carefully onto ice
and extracted with CHCl3 (four times). The combined CHCl3
extracts were washed with brine, dried, and evaporated to
yield a yellow oil (2.82 g). Fractional crystallization from
EtOAc gave a single regioisomer 20 as a colorless crystalline
solid (0.50 g, 16%): mp 190-192 °C dec; IR (KBr) 3180, 3160,
1720 (CO), 1460, 1410, 1365 (SO2), 1160 (SO2), 1070, 1020,
950, 900, 820, 760, 710 cm-1; 1H NMR (CDCl3) 7.88-7.85 (m,
2 H, H-5 and H-7), 7.43 (d, 1 H, J ) 7.8 Hz, H-8), 4.98 (d, 1 H,
J ) 17.5 Hz, H-4), 4.64 (t, 1 H, J ) 8.3 Hz, CH2O), 4.47 (d, 1
H, J ) 17.6 Hz, H-4), 4.24-4.19 (m, 1 H, CH2O), 4.06-4.00
(m, 1 H, H-9a), 3.15-2.94 (m, 2 H, H-9); 13C NMR (CDCl3)
157.0 (CO), 143.1, 140.2, 133.7, 131.0, 125.2, 125.1, 68.1, 50.3,
43.0, 34.0; EIMS m/z 289 (M+ + 2, 15), 287 (M+, 39), 272 (10),
(R)-(-)-3,4-Dih ydr o-3-m eth yl-7-n itr oisoqu in olin -1-(2H)-
on e (25). Nitration as described above on (R)-(-)-24 {mp 145-
23
146 °C; [R]D ) -91° (c 0.20, MeOH)} gave a golden-yellow
23
crystalline solid: mp 232-233 °C dec; [R]D ) -106° (c 0.20,
MeOH). Anal. (C10H10N2O3) C, H, N.
(S)-(+)-3,4-Dih ydr o-3-m eth yl-7-n itr oisoqu in olin -1-(2H)-
on e (25). Nitration as described above on (S)-(+)-24 {mp 143-
144 °C, lit.27b mp 147 °C; [R]D ) 91° (c 0.22, MeOH)} gave a
23
23
golden-yellow crystalline solid: mp 232-233 °C dec; [R]D
100° (c 0.20, MeOH). Anal. (C10H10N2O3) C, H, N.
)
(()-3-Meth yl-7-n itr o-1,2,3,4-tetr ah ydr oisoqu in olin e Hy-
d r och lor id e (14‚HCl). The suspension of amide (()-25 (0.40
g, 1.9 mmol) in dry THF (5 mL) was heated to reflux with BH3‚
THF complex (1 M solution in THF, 6 mL, 6 mmol) under N2
for 14 h. The resulting yellow solution was cooled in an ice

3,7-Disub-THIQs as Selective PNMT Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1989
252 (M+ - Cl, 13), 204 (30), 202 (73), 188 (M+ - SO2Cl, 15),
solvent was removed on a rotary evaporator to yield a yellow
solid. Recrystallization of this solid from EtOH-water gave a
pale-yellow crystalline solid (0.20 g, 83%): mp 212-213 °C;
IR (KBr) 3000, 2920, 1750 (CO), 1440, 1290 (SO2), 1150 (SO2),
1070, 775 cm-1; 1H NMR (DMSO-d6) δ 7.77-7.72 (m, 2 H, H-5
and H-7), 7.41 (d, 1 H, J ) 7.8 Hz, H-8), 4.88 (d, 1 H, J ) 17.1
Hz, H-4), 4.62 (t, 1 H, J ) 8.3 Hz, CH2O), 4.46 (d, 1 H, J )
17.6 Hz, H-4), 4.24-4.19 (m, 1 H, CH2O), 4.08-4.02 (m, 1 H,
H-9a), 3.14-2.88 (m, 5 H, H-9 and SO2CH3); 13C NMR (DMSO-
d6) δ 156.8 (CO), 139.0, 138.9, 133.2, 130.5, 125.3, 124.8, 68.1
(CH2O), 50.0 (C-3), 43.6, 42.5, 33.1 (C-4); EIMS m/z 268 (M++1,
4), 267 (M+, 24), 252 (M+-CH3, 5), 223 (11), 208 (5), 194 (21),
182 (57), 143 (23), 115 (51), 103 (27), 102 (23), 91 (100), 77
(82), 65 (33), 63 (61), 51 (33). Anal. (C12H13NO4S) C, H, N.
167 (12), 143 (13), 127 (12), 115 (46), 103 (86), 102 (34), 91
(20), 86 (28), 77 (100), 63 (34), 51 (70), 49 (94). Anal. (C11H10
ClNO4S) C, H, N.
-
The regiochemistry of the product was confirmed by a one-
dimensional difference NOE experiment. When the protons at
approximately 4.47 and 4.98 ppm (corresponding to the two
protons at H-4) were irradiated, a positive NOE response was
detected at 7.84 ppm (singlet; corresponding to the proton at
H-5).
(()-6-Am in osu lfon yl-1,4,9,9a -tetr a h yd r o-2-oxa -3a -a za -
cyclop en ta [b]n a p h th a len -3-on e (21). Compound 20 (1.30
g, 4.52 mmol) was suspended in acetonitrile (20 mL) and
heated to reflux with concentrated NH4OH (20 mL) overnight.
The solvent was removed by rotary evaporation to yield a
colorless solid. The solid was filtered, washed thoroughly with
MeOH and water, and dried in vacuo (1.20 g, 86.3%): mp 228-
230 °C dec; IR (KBr) 3220 (NH2), 1710 (CO), 1320 (SO2), 1270,
(()-7-Meth ylsu lfon yl-3-h yd r oxym eth yl-1,2,3,4-tetr a h y-
d r oisoqu in olin e Hyd r och lor id e (13‚HCl). The oxazolidi-
none 23 (0.20 g, 0.74 mmol) was hydrolyzed with KOH (0.2 g)
by using the procedure as described for compound 12‚HCl. The
hydrochloride salt was isolated as colorless crystals (0.11 g,
52%): mp 225-226 °C dec; IR (KBr) 3360 (OH), 2980, 2920,
2760, 1320, 1305 (SO2), 1150 (SO2), 1130, 1080, 1050, 960, 770
1160 (SO2), 1080, 760 cm-1 1H NMR (DMSO-d6) 7.70-7.63
;
(m, 2 H, H-5 and H-7), 7.38 (d, 1 H, J ) 7.9 Hz, H-8), 7.34 (s,
2 H, SO2NH2), 4.69 (d, 1 H, J ) 17.5 Hz, H-4), 4.55 (t, 1 H, J
) 8.2 Hz, CH2O), 4.40 (d, 1 H, J ) 17.5 Hz, H-4), 4.18-4.13
(m, 1 H, CH2O), 4.02-3.99 (m, 1 H, H-9a), 3.04 (dd, 1 H, J )
16.1, 3.9 Hz, H-9), 2.87-2.75 (m, 1 H, H-9); 13C NMR (DMSO-
d6) 157.7 (CO), 151.3, 143.3, 137.6, 133.3, 130.9, 124.6, 68.9
(CH2O), 51.0 (C-3), 43.4 (C-1), 33.8 (C-4); EIMS m/z 269 (M+
+ 1, 14), 268 (59), 253 (11), 224 (15), 195 (23), 183 (100), 115
(14), 103 (10), 91 (10), 77 (10). Anal. (C11H12N2O4S) C, H, N.
1
cm-1; H NMR (DMSO-d6) δ 9.93 (bs, 2 H, NH2+), 7.86-7.76
(m, 2 H, H-6 and H-8), 7.48 (d, 1 H, J ) 7.8 Hz, H-6), 5.60 (bs,
1 H, OH), 4.35 (s, 2 H, H-1), 3.77-3.67 (m, 2 H, CH2O), 3.50-
3.32 (m, 1 H, H-3), 3.19 (s, 3 H, SO2CH3), 3.19-3.05 (m, 2 H,
H-4); 13C NMR (DMSO-d6) δ 139.0, 138.2, 130.4, 130.0, 125.6,
125.4, 60.4 (CH2O), 53.7 (C-1), 43.5, 27.6 (C-4); MS (CI, NH3)
m/z 242 (M+ + 1, 35), 211 (12), 210 (100), 131 (17), 130 (29),
129 (12). Anal. (C11H15NO3S‚HCl) C, H, N.
(()-7-Am in osu lfon yl-3-h yd r oxym eth yl-1,2,3,4-tetr a h y-
d r oisoqu in olin e Hyd r och lor id e (12‚HCl). A solution of
KOH (0.17 g, 3.0 mmol) in water (3 mL) was added to a
suspension of oxazolidinone 21 (0.33 g, 1.2 mmol) in EtOH (5
mL). The resulting pale-yellow solution was heated to reflux
under N2 for 10 h. The reaction mixture was cooled and made
acidic with concentrated HCl. Loss of CO2 occurred, and the
reaction mixture was concentrated to yield a solid which was
heated in absolute EtOH, cooled, and filtered to remove KCl.
The filtrate was evaporated to dryness and crystallized from
90% EtOH-Et2O to afford colorless needles (0.15 g, 44%): mp
218-220 °C dec; IR (KBr) 3480, 3200, 2930, 1335, 1150, 1025,
Ra d ioch em ica l Assa y for P NMT Activity. The assay
used in this study has been described elsewhere.29 Briefly, a
typical assay mixture consisted of 50 µL of 0.5 M phosphate
buffer (pH 8.0), 25 µL of 10 mM unlabeled AdoMet, 5 µL of
[methyl-3H]AdoMet, containing approximately 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 sufficient water
to achieve a final volume of 250 µL. After incubation for 30
min at 37 °C, the reaction mixture was quenched by addition
of 250 µL of 0.5 M borate buffer (pH 10.0) and was extracted
with 2 mL of toluene/isoamyl alcohol (7:3). A 1-mL portion of
the organic layer was removed, transferred to a scintillation
vial, and diluted with cocktail for counting. The mode of
inhibition was ascertained to be competitive in all cases
reported in Tables 1 and 2 by inspection of the 1/V vs 1/S plots
of the data. All assays were run in duplicate with three
inhibitor concentrations over a 5-fold range. Ki values were
determined by a hyperbolic fit of the data.
800, 720 cm-1 1H NMR (DMSO-d6) δ 9.95-9.60 (m, 2 H,
;
NH2+), 7.73-7.65 (m, 2 H, H-6 and H-8), 7.45-7.39 (m, 3 H,
H-5 and SO2NH2), 5.60 (bs, 1 H, OH), 4.34 (s, 2 H, H-1), 3.85-
3.66 (m, 2 H, CH2O), 3.52-3.42 (m, 1 H, H-3), 3.3 (d, 2 H, J )
7.2 Hz, H-4); 13C NMR (DMSO-d6) δ 142.4, 136.1, 129.7, 129.6,
124.60 123.9, 60.5 (CH2O), 53.9 (C-3), 43.7 (C-1), 27.5 (C-4);
MS (CI, NH3) m/z 243 (M+ + 1, 100), 211 (6), 52 (7). Anal.
(C10H14N2O3S‚HCl‚0.25H2O) C, H, N.
(()-6-Hyd r a zin osu lfon yl-1,4,9,9a -tetr a h yd r o-2-oxa -3a -
a za cyclop en ta [b]n a p h th a len -3-on e (22). To an ice-cold
solution of sulfonyl chloride 20 (0.50 g, 1.7 mmol) in THF (10
mL) was added hydrazine (0.15 mL, 4.8 mmol), and the
mixture was stirred at room temperature for 8 h. The solvent
was removed, and the resulting semisolid was crystallized from
aqueous MeOH as a colorless crystalline solid (0.26 g, 53%):
mp 178-179 °C; IR (KBr) 3420, 3380, 3300 (NH), 2900, 1750
(CO), 1620, 1430, 1330 (SO2), 1280, 1150 (SO2), 940, 730, 700,
r2-Ad r en ocep tor Ra d ioliga n d Bin d in g Assa y. The ra-
dioligand receptor binding was performed according to the
method of U’Prichard et al.31 Male Sprague-Dawley rats were
decapitated, and the cortexes were dissected out and homog-
enized 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. 19.2 mCi/mmol, final concentration 4.0 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), as all
Hill coefficients were approximately equal to 1.
670 cm-1 1H NMR (DMSO-d6) δ 8.39 (bs, 1 H, NH), 7.69-
;
7.60 (m, 2 H, H-5 and H-7), 7.41 (d, 1 H, J ) 7.9 Hz, H-8),
4.73 (d, 1 H, J ) 17.5 Hz, H-4), 4.55 (t, 1 H, J ) 8.2 Hz, CH2O),
4.42 (d, 1 H, J ) 17.2 Hz, H-4), 4.19-3.99 (m, 3 H, CH2O,
H-9a and NH), 3.36 (bs, 1 H, NH), 3.15-3.04 (m, 1 H, H-9),
2.88-2.79 (m, 1 H, H-9); 13C NMR (DMSO-d6) δ 156.8 (CO),
137.6, 136.4, 132.6, 130.1, 125.9, 125.5, 68.0 (CH2O), 50.1 (C-
3), 42.5 (C-1), 33.0 (C-4). Anal. (C11H13N3O4S) C, H, N.
(()-6-Meth ylsu lfon yl-1,4,9,9a -tetr a h yd r o-2-oxa -3a -a za -
cyclop en ta [b]n a p h th a len -3-on e (23). To a suspension of
sulfonylhydrazide 22 (0.25 g, 0.88 mmol) in EtOH (5 mL) were
added NaOAc (0.48 g, 5.85 mmol) and MeI (0.62 g, 0.27 mL,
4.8 mmol). The resulting suspension was heated to reflux for
14 h yielding a yellow solution which was cooled and concen-
trated to yield a semisolid. Water (15 mL) was added to the
semisolid, and the mixture was extracted with EtOAc (four
times). The organic layers were combined and dried, and the

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