Synthesis of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines and comparison with their isosteric 1,2,3,4-tetrahydroisoquinolines as inhibitors of phenylethanolamine N-methyltransferase

Article abstract of DOI:10.1016/j.bmc.2007.08.066

A series of substituted 4,5,6,7-tetrahydrothieno[3,2-c]pyridines (THTPs) was synthesized and evaluated for their human phenylethanolamine N-methyltransferase (hPNMT) inhibitory potency and affinity for the α₂-adrenoceptor. The THTP nucleus was

Full text of DOI:10.1016/j.bmc.2007.08.066

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 542–559
Synthesis of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines
and comparison with their isosteric 1,2,3,4-tetrahydroisoquinolines
as inhibitors of phenylethanolamine N-methyltransferaseq
Gary L. Grunewald,* Mitchell R. Seim, Seema R. Bhat,
Marc E. Wilson and Kevin R. Criscione
Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, USA
Received 6 June 2007; revised 23 August 2007; accepted 28 August 2007
Available online 11 October 2007
Abstract—A series of substituted 4,5,6,7-tetrahydrothieno[3,2-c]pyridines (THTPs) was synthesized and evaluated for their human
phenylethanolamine N-methyltransferase (hPNMT) inhibitory potency and affinity for the a2-adrenoceptor. The THTP nucleus was
suggested as an isosteric replacement for the 1,2,3,4-tetrahydroisoquinoline (THIQ) ring system on the basis that 3-thienylmethyl-
amine (18) was more potent as an inhibitor of hPNMT and more selective toward the a2-adrenoceptor than benzylamine (15).
Although the isosterism was confirmed, with similar influence of functional groups and chirality in both systems on hPNMT inhib-
itory potency and selectivity, the THTP compounds proved, in general, to be less potent as inhibitors of hPNMT than their THIQ
counterparts, with the drop in potency being primarily attributed to the electronic properties of the thiophene ring. A hypothesis for
the reduced hPNMT inhibitory potency of these compounds has been formed on the basis of molecular modeling and docking stud-
ies using the X-ray crystal structures of hPNMT co-crystallized with THIQ-type inhibitors and S-adenosyl-L-homocysteine as a
template.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
As one approach to elucidate the role(s) of epinephrine
in the CNS, our laboratory has targeted phenylethanol-
amine N-methyltransferase (PNMT; EC 2.1.1.28). This
enzyme catalyzes the terminal step in the biosynthesis
of epinephrine (Fig. 1).
Figure 1. The terminal step in epinephrine biosynthesis.
Compounds based on the 1,2,3,4-tetrahydroisoquinoline
(THIQ, 5, Table 1) nucleus have been found to be some
of the most potent inhibitors of human PNMT
(hPNMT) yet reported. The addition of hydrophilic or
lipophilic electron-withdrawing substituents to the 7-po-
sition of 5 leads to enhanced hPNMT inhibitory po-
tency,1 as illustrated by compounds 6–9 (Table 1).
THIQs having lipophilic 7-substituents, such as halides
(6 and 7), are generally more potent at hPNMT than
those having hydrophilic groups (8 and 9). However,
THIQs bearing lipophilic 7-substituents are less selective
than THIQs bearing hydrophilic 7-substituents due to
significant affinity for the a2-adrenoceptor.1 The addi-
tion of small substituents (e.g., methyl, 10) to the 3-posi-
tion of THIQ increases potency at hPNMT while
decreasing affinity for the a2-adrenoceptor.2,3 The
absolute stereochemistry at the 3-position of THIQs
Keywords: Phenylethanolamine N-methyltransferase; Enzyme inhibi-
tors; 4,5,6,7-Tetrahydrothieno[3,2-c]pyridine; Isosteric substitution;
Stacking interactions.
is important and is discussed below. A synergistic
q
The contents of this paper were taken in large part from the Ph.D.
effect is achieved by adding both 3- and 7-substituents
dissertation (University of Kansas, 2006) of Mitchell R. Seim.
to THIQ, resulting in multiplicative increases in
PNMT inhibitory potency and selectivity versus the
*
Corresponding author. Tel.: +1 785 864 4497; fax: +1 785 864
5326; e-mail: ggrunewald@ku.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.066

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
543
Table 1. In vitro human PNMT (hPNMT) inhibitory potency and a2-adrenoceptor affinity of previously studied inhibitors
Compound
hPNMT Ki (lM) SEMa
a2-Adrenoceptorb Ki (lM) SEMa
Selectivity (a2/PNMT)
THIQ (5)c,d
SK&F 64139 (6)c,f
7c,h
5.8 0.5e
0.0031 0.0006g
0.056 0.003i
0.28 0.02g
0.12 0.01i
0.35 0.11
0.021 0.005
0.23 0.13
100 10
0.060
7
77
SK&F 29661 (8)c,j
9c,h
360
36
4.3 0.3
0.76 0.08
1.1 0.1
( )-10c,k
1.4 0.01i
0.54
65
( )-11i
0.017 0.005
0.072 0.005i
0.067 0.006
( )-12c,l
31
0.20 0.02
3
430
3.0
SK&F 7698 [( )-13]c,m
a Standard error of the mean.
b In vitro activities for the inhibition of [3H]clonidine binding to the a2-adrenoceptor (rat cortex).
c The data for this compound were originally reported for the inhibition of bovine PNMT.
d Ref. 5.
e Ref. 6.
f Ref. 7.
g Ref. 8.
h Ref. 1.
i Ref. 9.
j Ref. 10.
k Ref. 11.
l Ref. 4.
m Ref. 12.
a2-adrenoceptor compared to the similarly substituted
3- or 7-monosubstituted THIQs.4 This is illustrated by
comparing the inhibitory potency and selectivity of
disubstituted-THIQs 11 or 12 with their respective
monosubstituted analogs 7 and 10 or 9 and 10.
to be 3-fold more potent at hPNMT than the isostere
with the methylamine group on the 2-position (2-thie-
nylmethylamine, 17). Compared to benzylamine (15),
we were pleased to observe that 18 has 50% more
hPNMT inhibitory potency and twice the selectivity
for hPNMT versus the a2-adrenoceptor. Previous stud-
ies have shown that constraining the methylamine side
chain of benzylamine (15) to form THIQ (5) increases
its hPNMT inhibitory potency 29-fold.15 Because 18
was found to be more potent and selective for hPNMT
than 15, we predicted that constraining the side chain
of 18 into a 4,5,6,7-tetrahydrothieno[3,2-c]pyridine
(THTP, 14) ring system would likely result in superior
hPNMT inhibitory potency and selectivity versus 5.
The X-ray crystal structures of hPNMT co-crystallized
with THIQs, such as 613 (Fig. 2) and 8,14 were consistent
with SAR studies at PNMT. 3-Substituents of THIQ are
able to occupy a lipophilic channel between the norepi-
nephrine (1) binding site and the S-adenosyl-L-methio-
nine (3) binding site and the 7-substituents are able to
occupy an auxiliary binding pocket (Fig. 2).
Because the THIQ nucleus can be viewed as either a
constrained benzylamine or a constrained 2-phenyleth-
ylamine and a common isosteric replacement for a ben-
zene ring is a thiophene ring, the commercially available
thiophene analogs of benzylamine (15) and 2-phenyleth-
ylamine (16) were evaluated at hPNMT and the a2-adre-
noceptor (Table 2). As expected, 2-(2-thienyl)ethylamine
(19) was found to have hPNMT inhibitory potency that
was similar to phenylethylamine 16. The thiophene iso-
stere of benzylamine, with the methylamine group on
the 3-position (3-thienylmethylamine, 18), was found
An additional reason for using a thiophene ring is that
replacement of the benzene ring of 10 with a benzothi-
ophene ring resulted in a 21-fold increase in hPNMT
inhibitory potency (SK&F 7698, 13). Prior to the avail-
ability of the crystal structure of hPNMT, it was

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G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
active site as the chlorines of 6. It thus appeared that the
isosteric substitution of the benzene ring of THIQ-type
inhibitors of hPNMT with a thiophene ring could lead
to potent inhibitors of hPNMT. Bioisosteric substitu-
tion of a THIQ ring system with a THTP ring system
has previously been applied toward the design of potent
j opioid selective analgesics.18
Crystallographic studies suggest that the positions of
both the aromatic ring and the 7-substituent of THIQ-
type hPNMT inhibitors is important in the binding of
these compounds to hPNMT.13,14,19 Also, the preferred
configuration is (S) for a 3-methyl substituent [e.g.,
(S)-12 is 100-fold more potent than (R)-12 (Section 4
and Table 3)]. The preferred binding conformer of
3-substituted-THIQs is a half-chair conformation in
which the 3-substituent is in an equatorial position
and the nitrogen lone pair is axial.20,21 A molecular
overlay of (S)-2-bromo-6-methyl-THTP and (S)-7-bro-
mo-3-methyl-THIQ in this energy minimized conforma-
tion (Fig. 3) shows that 2,6-disubstituted-THTP
superimposes well with 3,7-disubstituted-THIQ when
the molecules are fitted using the respective substituents
and the axial lone pair on the nitrogen. As the addition
of appropriate substituents to the 3- and 7-positions of
THIQ is known to confer both potency and selectivity
for PNMT, it was proposed that 2,6-disubstituted-
THTPs could be highly potent and selective inhibitors.
Figure 2. The active site of hPNMT co-crystallized with SK&F 64139
(6) and S-adenosyl-L-homocysteine (4) and the amino acid residues
that form key interactions with 6.13 A Connolly (solvent accessible)
surface exposing 6 and 4 is also shown and indicates the presence of a
lipophilic channel adjacent to the 3-position of 6 and an auxiliary
binding pocket adjacent to the 7-position of 6. A lipophilic potential is
mapped on the Connolly surface (areas shown in green are neutral,
blue are hydrophilic, and brown are lipophilic). Carbons are shown in
white, nitrogen in blue, oxygen in red, chlorine in green, and sulfur in
yellow. Hydrogens are not shown for clarity.
The 2- and 6-substituents for the proposed THTPs (20–
31, below) were selected on the basis of previous SAR data
for THIQs.1,4 Methyl and trifluoromethyl groups were
added to the 6-position of THTP and while these groups
are not likely to be the optimal substituents for the 6-posi-
tion of THTP, the methyl group is optimal for determin-
ing the effect of 6-substitution with minimal steric
interference, while the trifluoromethyl group is suitable
for determining the influence of the pKa of the THTP
amine on a2-adrenoceptor affinity. The decreased affinity
for the a2-adrenoceptor associated with reduction in the
pKa caused by the addition of fluorinated methyl groups
to the 3-position of THIQ is well established.23–25 In addi-
tion, 32 (Table 3), in which the methyl group of 13 is re-
placed with a trifluoromethyl group, was prepared in
order to decrease the a2-adrenoceptor affinity of this
highly potent, but nonselective, inhibitor.
Table 2. In vitro hPNMT inhibitory potency and a2-adrenoceptor
affinity of benzylamine, 2-phenylethylamine, and their analogous
thiophenes
Compound hPNMT Ki
(lM) SEMa (lM) SEMa
a2-Adrenoceptorb Ki Selectivity
a2/PNMT
15c,d
16c,e
17
170 10
220 10
450 20
110 10
300 20
20
4.0 0.4
1
0.12
0.018
0.12
0.29
27
32
3
3
18
19
1.2 0.1
0.0040
a Standard error of the mean.
b In vitro activities for the inhibition of [3H]clonidine binding to the a2-
adrenoceptor (rat cortex).
c Data for this compound were originally reported for the inhibition of
bovine PNMT.
d Ref. 16.
e Ref. 17.
proposed that the increased potency of 13 versus 10 and
that of 6 versus 5 could be due to similar favorable
hydrophobic interactions with the enzyme. A molecular
overlay of 13 and 6 (not shown) suggested that the thi-
ophene ring of 13 could be binding in the same area of
the active site as the benzene ring of 6 and that the ben-
zene ring of 13 could be binding in the same area of the
2. Chemistry
The 4,5,6,7-tetrahydrothieno[3,2-c]pyridine (THTP)
ring system can be synthesized in a variety of
ways.26–32 For the synthesis of THTPs having nitro,
cyano, methyl, acetyl, and carboxamide groups in the

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
545
Table 3. In vitro hPNMT inhibitory potency and a2-adrenoceptor affinity of 2,6-disubstituted-THTPs and their analogous 3,7-disubstituted-THIQs
b
R2 for THTPs R6 for THTPs THTP
hPNMT Ki
a2 Ki
Selectivity
THIQ
hPNMT
hPNMT Ki ratio
THTP/THIQ
R7 for
THIQs
R3 for
THIQs
Compound (lM) SEMa (lM) SEMa (a2/hPNMT) Compound Ki (lM)
SEMa
H
H
CH3
14c
37
5.8 0.3
40
1
1.5 0.1
29
0.041
5.0
38
5
5.8 0.5
1.4 0.01
23 2e
6.3
4.1
1.7
H
20c
21
3
10
H
Br
CF3
H
1
1500 100
0.38 0.04
60d
7
22f
23g
24
1.2 0.1
2.6 0.1
0.32
16
0.056 0.003c 21
NO2
CN
H
18
16
2
1
9
0.12 0.01
1.5 0.1
22
10
1.3
12
13
H
15
100 10
2
1.1
61h,i
62h,i
63h,i
64h,i
11
(C@O)NH2
(C@O)CH3
CH3
Br
H
25
26
4.1 0.4
9.8 1.0
0.49 0.05
2.3 0.2
>1000
0.041
0.16
0.020
3.2
72
6
H
61
25
3
1
5.0 0.3
2.0 0.1
H
CH3
27
28
0.73 0.04
21
0.017 0.005 43
Br
CF3
29
1
>47
1.4
65d
(R)-12
(S)-12
—
3.2 0.3e
1.6 0.1
6.6
6.0
NO2
NO2
CN
(R)-CH3
(S)-CH3
(R)-CH3
(S)-CH3
Compound 32
(R)-30
(S)-30
(R)-31
(S)-31
9.6 0.5
0.48 0.04
150 10
13
29
47
1
3
5
60
0.016 0.001 30
0.31
39
—
—
—
—
—
—
CN
3.3 0.3
17
130 10
>500
—
—
1
>29
a Standard error of the mean.
b In vitro activities for the inhibition of [3H]clonidine binding to the a2-adrenoceptor (rat cortex).
c Ref. 27.
d Ref. 24.
e Ref. 25.
f Ref. 50.
g Ref. 51.
h Ref. 1.
i The data for this compound were originally reported for the inhibition of bovine PNMT.
Figure 3. Overlay of the energy minimized conformations22 of 3,7-
disubstituted-THIQ (S)-11 (THIQ nucleus is shown in cyan) and the
corresponding 2,6-disubstituted-THTP (S)-28 (THTP nucleus is shown
in orange). Hydrogens are not shown for clarity. The overlays show
the structural alignment with the aromatic rings of (S)-11 and (S)-28 in
the plane of the page (left) and the endview (right). These compounds
were fitted using the (S)-3-methyl substituent of 11 and the (S)-6-
methyl substituent of 28, the 7-bromo substituent of 11 and the 2-
Scheme 1. Reagents and conditions: (a) BtsCl, dioxane, NaOH (aq);
(b) POCl3ÆDMF; then NaOAc (aq); (c) Na2SÆ9H2O; then BrCH2R;
then NaOH (aq) for the preparation of 40 and 41; (d) PhSH, K2CO3,
DMF.
˚
bromo substituent of 28, and the end of the axial lone pair (1 A long,
shown in magenta) on the nitrogen.
2-position (Schemes 1–3), we applied the 2-substituted
thiophene synthesis developed by Cagniant and Kirsch33
to the THTP preparation method developed by Mat-
sumura et al.34 This synthetic route was particularly
advantageous as it allowed for the enantioselective syn-
thesis of 2,6-disubstituted-THTPs. For THTPs having a
hydrogen or bromine in the 2-position and the trifluo-
romethyl analog of 13, the Pictet-Spengler27 reaction
was used to obtain the THTP ring system (Scheme 4).
The synthesis of THTPs 23–26 is shown in Scheme 1.
This reaction sequence required a protecting group for
the amine which would be stable in the acidic conditions

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G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
Scheme 2. Reagents and conditions: (a) Na2SÆ9H2O; then ethyl-2-
bromopropionate; (b) EtOH, H2O, NaOH; then HCl; (c) PhSH,
K2CO3, DMF.
Scheme 4. Reagents and conditions: (a) Na2CO3, thiophene or
benzothiophene; (b) trifluoroacetic acid, CH2Cl2; (c) LiAlH4; (d)
formaldehyde; then HCl; (e) Br2, AcOH.
the use of hydrogenation for the deprotection step was
avoided to reduce possible complications of reductive
ring opening. The benzothiazole-2-sulfonyl (Bts) group,
which is most commonly used in peptide synthesis, met
these criteria.35 4-Piperidone (35) was first protected
with BtsCl in a 3:1 solution of dioxane and aqueous
sodium hydroxide. For optimum yield, it was important
to maintain the pH between 10 and 11 with the addition
of aqueous 1 M NaOH to neutralize the HCl by-prod-
uct. The resulting Bts-amine (36) was subjected to Vils-
meyer-Haack formylation to yield chloroformyl
derivative 37.36 Compound 37 was reacted with sodium
sulfide to generate the thiol in situ. Addition of various
alkylbromides resulted in nucleophilic displacement of
the bromine by the intermediate thiol, followed by a
base catalyzed condensation reaction to form the cyc-
lized THTPs 38–41.33 Removal of the Bts group with
PhSH and K2CO3 yielded the desired THTPs 23–26.37
Compound 37 (Scheme 2) was reacted with sodium
sulfide to generate the thiol intermediate in situ, which
was subsequently reacted with ethyl-2-bromopropionate
to afford crude 42, which was cyclized and hydrolyzed
under basic conditions, followed by the decarboxylation
and condensation of the resulting acid under acidic con-
ditions to yield the protected 2-methyl-THTP (43).
Removal of the Bts group with PhSH and K2CO3
yielded THTP 27.37,38
Scheme 3. Reagents and conditions: (a) vinylMgBr, THF, then (S)-a-
methylbenzylamine, H2O; (b) H2, Pd(OH)2, THF; then NvocCl, THF,
NaOH (aq); (c) POCl3ÆDMF; then NaOAc (aq); (d) Na2SÆ9H2O,
DMF; then BrCH2NO2 or BrCH2CN; then NaOH (aq); (e) BF3ÆEt2O,
EtSH.
The enantiospecific synthesis of THTPs 30, 31, 33, and
34 is shown in Scheme 3. Attempts to carry out the reac-
tion sequence in Scheme 3 using Bts, allyloxycarbonyl,
benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, or eth-
oxycarbonyl protecting groups for the amine were
of step (b), the basic conditions of step (c), and could be
removed using conditions which would allow for the
preparation of a variety of 2-substituted-THTPs. Also,

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
547
unsuccessful due to an inability to separate the regioi-
somers formed in step (c). The mixture of regioisomers
obtained from step (c) was subjected to step (d), but
the products from this reaction were also inseparable.
Ultimately, the reaction sequence was successfully
completed using the 6-nitroveratryloxycarbonyl (Nvoc)
protecting group. Nvoc-amines are stable under acidic
or basic conditions and are most commonly employed
due to their photochemical lability.39 The highly crystal-
line nature of the Nvoc derivatives allowed for the
separation of the regioisomers formed in step (d) by
fractional recrystallization.
compounds is now reported for comparison purposes.
In the current study, human PNMT (hPNMT) with a
C-terminal hexahistidine tag was expressed in Esche-
richia coli.45,46 The radiochemical assay conditions,
previously reported for the bovine enzyme,47 were
modified to account for the high binding affinity of
some inhibitors.8,45 Inhibition constants were deter-
mined using four concentrations of phenylethanol-
amine as the variable substrate, and three concentra-
tions of inhibitor.
a2-Adrenergic receptor binding assays were performed
using cortex obtained from male Sprague–Dawley
rats.48 [3H]Clonidine was used as the radioligand to de-
fine the specific binding and phentolamine was used to
define the nonspecific binding. Clonidine was used as
the ligand to define a2-adrenergic binding affinity to sim-
plify the comparison with previous results.49
Weinreb amide 44 was reacted with vinylmagnesium
bromide in THF to form 1,4-hexadien-3-one in situ, to
which (S)-a-methylbenzylamine and water were added.
This yielded a mixture of (R)- and (S)-2-methyl-1-((S)-
1-phenylethyl)piperidin-4-one (45)40,41 that was sepa-
rated by flash chromatography. Compound (R,S)- or
(S,S)-45 was deprotected by hydrogenation with Pearl-
man’s catalyst at ambient temperature and pressure,
and the resulting crude amine was reacted with 6-nitro-
veratryl chloroformate (NvocCl) to yield (R)- or (S)-46.
Compound (R)- or (S)-46 was subjected to Vilsmeier
conditions to yield a mixture of regioisomers, (R)-47
and (R)-48, or (S)-47 and (S)-48,36 which were separated
by fractional crystallization. Compound (R)- or (S)-47
was reacted with sodium sulfide to generate the thiol
in situ. Addition of bromonitromethane or bromoaceto-
nitrile resulted in nucleophilic displacement of the
bromine by the intermediate thiol, followed by a base
catalyzed condensation reaction to afford the cyclized
6-methyl-2-substituted-THTP products (R)- or (S)-49
or (R)- or (S)-50, respectively.33 (R)- or (S)-48 was re-
acted under the same conditions used to convert 47 to
6-methyl-THTPs 49 and 50, to yield the cyclized
4-methyl-2-substituted-THTP products (R)- or (S)-51
or (R)- or (S)-52, respectively. Deprotection of THTPs
49–52 with BF3ÆEt2O in EtSH afforded THTPs 30, 31,
33, and 34.42 The stereochemistry and regiochemistry
of (S)-52 have been verified by X-ray crystallography
(see Section 7). This structure was then used to define
the stereochemistry and regiochemistry of the final com-
pounds in Scheme 3.
4. Results and discussion
The biochemical data for THTPs 14 and 20–32 are shown
in Table 3 along with the data for their corresponding
isosteric THIQs for comparison purposes. Unfortu-
nately, the prediction that constraining the methylamine
side chain of 18 to form the THTP ring system (14) would
result in a large increase in potency, as is observed for
benzylamine (15) versus THIQ (5), was incorrect. Only
a small increase in hPNMT inhibitory potency (3-fold,
as compared to the 29-fold increase in potency of 15 ver-
sus 5) is observed, resulting in the parent THTP (14) hav-
ing 6-fold less potency at hPNMT than the parent THIQ
(5). In fact, all of the THTPs examined were found to be
significantly less potent as inhibitors of hPNMT than
their corresponding THIQs, suggesting that the THIQ
nucleus has greater intrinsic affinity for hPNMT than
the THTP nucleus. Given the structural similarity of
THTPs and THIQs and their molecular overlay in Figure
3, we concluded that the most likely reason for the de-
creased hPNMT inhibitory potency of the THTPs was
fundamental differences between the thiophene ring of
THTPs and the benzene ring of THIQs.
4.1. Docking studies comparing the binding of THTPs
with THIQs
The synthesis of THTPs 20, 21, 28, 29, and 32 is shown in
Scheme 4. The reaction of 53 with Na2CO3 yields1,1,1-tri-
fluoro-2-nitroso-2-propene.43 This heterodiene reacted
with thiophene to afford the bicyclic 1,2-oxazine 54, which
was converted directly to oxime 55 with trifluoroacetic
acid. Reduction of 55 with LiAlH4 yielded amine 56,
which was cyclized by a Pictet-Spengler reaction to afford
THTP 21. Reaction of 21 or 2044 with bromine in acetic
acid yielded 2-bromo-THTPs 29 and 28, respectively.
Compound 32 was also prepared from 53 and benzothi-
ophene via the procedure used for the synthesis of 21.
Docking studies with the crystal structure of hPNMT
predict that substituents on the 2- and 6-positions of
the THTP nucleus are binding in the same area in the ac-
tive site as substituents on the 7- and 3-positions of the
THIQ nucleus (Fig. 4). The 2-bromo substituent of 28 is
predicted to form favorable hydrophobic interactions
with Val53 in the same way as the 7-bromo substituent
of 11 (Fig. 4A).9 Docking studies52 (not shown) with 2-
nitro-THTPs 23 and 30 predicted that (similarly to 7-ni-
tro-THIQs)9 the nitro group could form hydrogen
bonding interactions with Lys57. The 6-methyl group
of (S)-28 is predicted to form favorable hydrophobic
interaction with Tyr222 in a similar way as the 3-methyl
group of (S)-11 (Fig. 4A). The interaction between
Tyr222 and lipophilic 3-substituents of THIQ is well
established.9,23,45
3. Biochemistry
The hPNMT inhibitory potency for compounds 13,12
15,16 and 1617 was originally determined using bovine
PNMT. The hPNMT inhibitory potency for these

548
G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
Figure 4. (S)-7-Bromo-3-methyl-THIQ [(S)-11] and (S)-2-bromo-6-methyl-THTP [(S)-28] docked into the active site of hPNMT [from the X-ray
structure of hPNMT co-crystallized with SK&F 64139 (6) and S-adenosyl-L-homocysteine (4)]13 and the amino acid residues that form key
interactions with (S)-11 and (S)-28. Nitrogen is blue, oxygen is red, bromine is magenta, sulfur is yellow, carbons of amino acid side chains are gray,
carbons of (S)-11 are cyan, and carbons of (S)-28 are orange. Hydrogens are not shown for clarity. (A) Stereoview of an overlay of (S)-11 and (S)-28.
The 7-bromo and (S)-3-methyl substituents of (S)-11 are predicted to bind in the same area in the active site as the 2-bromo and (S)-6-methyl
substituents of (S)-28. (B) The interaction of (S)-11 with Phe182, the aromatic ring of which is shown in the plane of the paper. The benzene ring of
˚
(S)-11 forms a parallel displaced p-stacking interaction with Phe182 at a distance of 3.7 A. (C) The interaction of (S)-28 with Phe182, the aromatic
˚
ring of which is shown in the plane of the paper. The distance between the thiophene ring of (S)-28 and Phe182 is 3.7 A. The partially negatively
charged sulfur of (S)-28 is in the face of Phe182, which results in a less favorable p-stacking interaction as compared to that of (S)-11.
Crystallographic studies have shown that the benzene
ring of substrates53 or THIQ-type inhibitors6,13,14,19 is
positioned between the side chains of Phe182 and
Asn39. The distance between the aromatic ring of 613
(Fig. 2) or 814 and the aromatic ring of Phe182 is 3.8
electrostatic potentials predicted for THIQ and THTP
(Fig. 5). According to docking studies with (S)-28, the
sulfur atom is in the face of Phe182 (Fig. 4C). Although
p-stacking interactions between benzene and thiophene
rings have not been thoroughly investigated, it appears
that this p-stacking interaction with the THTP sulfur in
the face of Phe182 would be less favorable than the paral-
lel displaced p-stacking orientation observed between the
benzene ring of THIQs and Phe182. This is likely the
molecular basis for the THIQ nucleus having greater
intrinsic affinity for hPNMT than the THTP nucleus.
˚
or 3.7 A, respectively, which implies the presence of a
p-stacking interaction. Within the hydrophobic environ-
ment of a protein, the preferred geometry of a Phe–Phe
interaction is in either a T-shaped edge-to-face or a par-
allel displaced p-stacking orientation, with the latter
being more energetically favored.54,55 This is the ob-
served orientation between Phe182 and the benzene ring
of 6 (Fig. 2) and the predicted orientation between
Phe182 and (S)-11 (Fig. 4B). However, it is likely that
the benzene ring of (S)-11 is able to make a more favor-
able p-stacking interaction with Phe182 than the thio-
phene ring of (S)-28 (Fig. 4C).
4.2. Effects of substitution on THTP and THIQ
4.2.1. 2-Substituted-THTPs versus 7-substituted-THIQs.
The addition of bromo, nitro, and cyano groups (22–24)
to the 2-position of THTP (14) increased the hPNMT
inhibitory potency 31-, 14-, and 2.5-fold, respectively.
The addition of the same groups to the 7-position (7,
9, and 61) of THIQ (5) increased the hPNMT inhibitory
potency 100-, 48-, and 4-fold, respectively. Thus, the
addition of electron-withdrawing substituents to the
2-position of THTP improved the hPNMT inhibitory
potency of 14, but not to the same degree as the addition
of the same substituents to the 7-position of 5.
Unlike benzene, thiophene has a dipole moment.56,57
The charge distribution of the p cloud of benzene is in
the center of the ring, which is surrounded by elec-
tron-poor hydrogens. The charge distribution of the p
cloud of thiophene, however, is shifted away from the
center of the ring toward the sulfur atom, giving the sul-
fur a partial negative charge. This is consistent with the

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
549
ring of THIQs. In Figure 4, the centroid of (S)-28 is
˚
0.5 A from the centroid of (S)-11. Because these changes
in binding orientation are very subtle, co-crystallization
of a 2-substituted-THTP and its corresponding 7-substi-
tuted-THIQ with hPNMT would be required to verify
this conclusion.
4.2.2. 6-Substituted-THTPs versus 3-substituted-THIQs.
The addition of a methyl group to the 6-position of
THTPs (14, 22, and 23) results in a similar improvement
in the hPNMT inhibitory potency as does the addition
of a methyl group to the 3-position of THIQs (5, 7,
and 9), with the increase being virtually the same for
each corresponding 2- or 7-substituent [e.g., 27 (6-fold)
vs 10 (4-fold), 28 vs 11 (2-fold for each), and (S)-31 vs
(S)-12 (5-fold for each)]. These observations are sup-
ported by the docking results (Fig. 4A), which show that
the (S)-methyl groups of 11 and 28 occupy the same area
in the active site of hPNMT and are able to make the
same favorable interactions with Tyr222.
The replacement of the 6-methyl group of 28 with a tri-
fluoromethyl group results in a 29-fold reduction in
hPNMT inhibitory potency for 29 and the replacement
of the methyl group of SK&F 7698 (13) with a trifluo-
romethyl group results in a 250-fold reduction in po-
tency for 32. This was not unexpected because
previous studies have shown that the replacement of
the 3-methyl group of 11 with a trifluoromethyl group
results in a 190-fold reduction in hPNMT inhibitory po-
tency for 65 (Table 3).25 In these studies, we attributed
the reduction in potency of 3-trifluoromethyl-THIQs
versus 3-methyl-THIQs to the increased steric bulk of
the trifluoromethyl group, specifically from unfavorable
interactions with Tyr222.23,25 Similarly, docking studies
(not shown) indicate that the decrease in hPNMT inhib-
itory potency of the 6-trifluoromethyl-THTPs is most
likely due to these same factors.
Figure 5. Electrostatic potential diagram of unsubstituted THIQ (5,
top) and unsubstituted THTP (14, bottom). Electronegative areas are
shown in blue and electropositive areas are shown in red (see Section
6.4). Carbon is white, nitrogen is blue, sulfur is yellow, and hydrogen is
cyan. As discussed in the text, the charge distribution of the p cloud of
the benzene ring of 5 is in the center of the ring, and the charge
distribution of the p cloud of the thiophene ring of 14 is shifted away
from the center of the ring toward the sulfur atom.
The (S)-enantiomers of 30 and 31 are considerably more
potent (20- and 45-fold, respectively) at hPNMT than
the corresponding (R)-enantiomers. This is consistent
with hPNMT data for enantiomers of various 3-substi-
tuted-THIQs. For compounds (R)- and (S)-12, the
(S)-enantiomer is 100-fold more potent at hPNMT than
the (R)-enantiomer. Analysis of the PNMT inhibition
data for 3-trifluoromethyl-,25 3-fluoromethyl-,3,20 and
3-hydroxymethyl-THIQs3 shows that the (R)-enantio-
mers are more potent at hPNMT than the (S)-enantio-
mers. Although the absolute configuration of the more
active enantiomer was opposite for these compounds
(due to the Cahn-Ingold-Prelog priority rules), the 3-
substituents are oriented in the same region of space
as the methyl groups of (S)-30, (S)-31, and (S)-12. Sim-
ilarly to 3-substituted THIQs,20 docking studies (not
shown) on 6-methyl-THTP show that the 6-(S)-methyl
substituent and the ring nitrogen can favorably interact
with Tyr222 and Glu219, respectively, whereas those
groups on 6-(R)-methyl-THTP cannot.
Crystallographic studies with THIQ inhibitors show
that the position of both the benzene ring and the 7-sub-
stituent are important in the binding of these com-
pounds.13,14,19 Thus, the molecular basis for the less
substantial improvement in potency for 2-bromo-, ni-
tro-, and cyano-THTP in comparison to the correspond-
ing 7-substituted-THIQs is either (1) the position of the
thiophene ring of the THTPs is adversely affecting the
orientation of the 2-substituent in the active site such
that it cannot make optimal interactions with key resi-
dues, or (2) that the orientation of the 2-substituent is
forcing the THTP ring into a position in which the thi-
ophene ring makes less optimal interactions with Phe182
in comparison to the benzene ring of THIQs. Docking
studies suggest that the latter reason is more probable
because they predict that 2-substituents of THTP are
binding in the same area of the active site as 7-substitu-
ents of THIQ, which shifts the centroid of the thiophene
ring of THTPs relative to the centroid of the benzene
Collectively, the similar effects on hPNMT inhibitory
potency from the addition of methyl or trifluoromethyl
groups to the 6-position of THTP and the 3-position

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G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
of THIQ, and the observation that the S-enantiomer is
more potent for both 6-methyl-THTPs and 3-methyl-
THIQs, indicate that 6-substituents of THTP are bind-
ing in the same area of the hPNMT active site as the
3-substituents of THIQ as predicted by the docking
studies. Thus, the optimization of THTPs by the addi-
tion of appropriate substituents to the 6-position is
likely to proceed similarly to the optimization of
3-substituted-THIQs, but as discussed in the previous
section, the optimization of THTPs by the addition of
substituents to the 2-position is not as effective as the
optimization of THIQs at the analogous 7-position.
compounds. Proton (1H NMR) and carbon (13C NMR)
nuclear magnetic resonance spectra were taken on a
Bruker DRX-400, Bruker AM-500, or Bruker AV-800
spectrophotometer. High resolution mass spectra
(HRMS) were obtained on a Ribermag R 10-10 mass
spectrophotometer. Thin-layer chromatography (TLC)
˚
was performed on K6F silica gel 60 A (Whatman) glass
backed plates. Flash chromatography was performed
using silica gel 60 (230–400 mesh) supplied by Universal
Adsorbents, Atlanta, Georgia.
Anhydrous methanol and ethanol were used unless sta-
ted otherwise, and were prepared by distillation over
magnesium. Other solvents were routinely distilled prior
to use. Anhydrous tetrahydrofuran (THF) and diethyl
ether (Et2O) were distilled from sodium-benzophenone
ketyl. Hexanes refer to the mixture of hexane isomers
(bp 40–70 ꢁC) and brine refers to a saturated solution
of NaCl. All reactions that required anhydrous condi-
tions were performed under a positive nitrogen or argon
flow, and all glassware was either oven-dried or flame-
dried before use. [methyl-3H]AdoMet and [3H]clonidine
were obtained from Perkin-Elmer (Boston, MA).
At the a2-adrenoceptor, the addition of a trifluoro-
methyl group to the 6-position of THTP causes a dra-
matic reduction in affinity, most likely due to the
decrease in the pKa of the THTP amine. Similar results
have previously been reported for 3-difluoromethyl-
THIQs,23 3-trifluoromethyl-THIQs,24,25 and fluori-
nated benzazapines.58 Replacement of the methyl
group of 20, 28 or 13 with a trifluoromethyl group re-
sults in a 1000-fold (21), >430-fold (29) or >2500-fold
(32) decrease in a2-adrenoceptor affinity for these com-
pounds. Unfortunately, the reduction in hPNMT
inhibitory potency that results from the addition of a
trifluoromethyl group makes them poor candidates
for in vivo investigations.
6.2. Radiochemical assay of PNMT inhibitors
A typical assay mixture consisted of 25 lL of 0.5 M
phosphate buffer (pH 8.0), 25 lL of 50 lM unlabeled
AdoMet, 5 lL of [methyl-3H]AdoMet, containing
approximately 3 · 105 dpm (specific activity approxi-
mately 15 Ci/mmol), 25 lL of substrate solution (phen-
ylethanolamine), 25 lL of inhibitor solution, 25 lL of
enzyme preparation (containing 30 ng hPNMT and
25 lg of bovine serum albumin), and sufficient water
to achieve a final volume of 250 lL. After incubation
for 30 min at 37 ꢁC, the reaction mixture was quenched
by addition of 250 lL 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 cock-
tail for counting. The mode of inhibition was ascer-
tained to be competitive in all cases reported in Tables
1–3 by examination of the correlation coefficients (r2)
for the fit routines as calculated in the Enzyme Kinetics
module (version 1.1) in SigmaPlot (version 7.0).31 While
all Ki values reported were calculated using competitive
kinetics, it should be noted that there was not always a
great difference between the r1 values for the competitive
model versus the noncompetitive model. All assays were
run in duplicate with three inhibitor concentrations over
a 5-fold range. Ki values were determined by a hyper-
bolic fit of the data using the Single Substrate–Single
Inhibitor routine in the Enzyme Kinetics module (ver-
sion 1.1) in SigmaPlot (version 7.0). For inhibitors with
apparent IC50 values less than 0.1 lM (as determined by
a preliminary screen of the compounds), the Enzyme
Kinetics Tight Binding Inhibition routine was used to
calculate the Ki values.
5. Conclusion
A series of substituted THTPs were shown to have con-
siderably less hPNMT inhibitory potency than their cor-
responding isosteric THIQs. The observation that
unsubstituted THTP (14) is 6-fold less potent than
unsubstituted THIQ (5) and the overall data trend of
THTPs having diminished hPNMT inhibitory potency
versus their corresponding THIQs suggest that the
THIQ nucleus has greater intrinsic affinity for hPNMT
than does the THTP nucleus. Docking studies in con-
junction with analysis of the electrostatic potential of
THTPs and THIQs suggest that the benzene ring of
THIQs is able to make a more favorable p-stacking
interaction with Phe182 than the thiophene ring of
THTPs. The analysis of the effects of substitution on
THTP and THIQ strongly supports the binding orienta-
tion predicted by the docking studies, however, the co-
crystallization of one or more of these THTPs with
hPNMT would be helpful in verifying our conclusions.
While we were able to increase the potency of 14 by add-
ing appropriate substituents to the 2- and 6-positions,
the decreased potency of these compounds versus the
analogous THIQs did not merit further optimization.
6. Experimental
6.1. General methods
All reagents and solvents were of reagent grade or were
purified by standard methods before use. Melting points
were determined in open capillary tubes on a Thomas-
Hoover melting point apparatus calibrated with known
6.3. a2-Adrenoceptor radioligand binding assay
The radioligand receptor binding assay was performed
according to the method of U’Prichard et al.28 Male

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
551
Sprague–Dawley rats were decapitated, and the cortexes
were dissected out and homogenized in 20 volumes (w/v)
of ice-cold 50 mM Tris/HCl buffer (pH 7.7 at 25 ꢁC).
Homogenates were centrifuged thrice for 10 min at
50,000g with resuspension of the pellet in fresh buffer be-
tween spins. The final pellet was homogenized in 200
volumes (w/v) of ice-cold 50 mM Tris/HCl buffer (pH
7.7 at 25 ꢁC). Incubation tubes containing [3H]clonidine
(specific activity approximately 55 Ci/mmol, final con-
centration 2.0 nM), various concentrations of drugs,
and an aliquot of freshly resuspended tissue (800 lL)
in a final volume of 1 mL were used. Tubes were incu-
bated at 25 ꢁC for 30 min and the incubation was termi-
nated 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 pre-
mixed scintillation cocktail. Nonspecific binding was de-
fined as the concentration of bound ligand in the
presence of 2 lM of phentolamine. All assays were run
in quadruplicate with five inhibitor concentrations over
a 16-fold range. IC50 values were determined by a log-
probit analysis of the data and Ki values were deter-
mined by the equation Ki = IC50/(1 + [clonidine]/KD),
as all Hill coefficients were approximately equal to 1.
(500 MHz, CDCl3) d 205.0 164.1, 152.3, 136.1, 127.8,
127.5, 125.1, 122.1, 46.1, 40.6; HRMS (FAB+) m/z Calcd
for C12H13N2O3S2 (MH+) 297.0368 obsd 297.0357.
6.5.2. N-Bts-4-chloro-1,2,5,6-tetrahydropyridine-3-car-
boxaldehyde (37). DMF (2.70 mL, 35.4 mmol) was dis-
solved in trichloroethylene (30 mL) and cooled to 5 ꢁC.
POCl3 (2.42 mL, 26.0 mmol) was added dropwise and
the reaction mixture was warmed to ambient tempera-
ture and stirred for 10 min. Compound 36 (3.50 g,
11.8 mmol) was dissolved in trichloroethylene (20 mL)
and added dropwise to the POCl3-DMF solution. The
reaction was continued for 12 h and then poured into
an ice-cold solution of sodium acetate (9.0 g) in water
(200 mL) and stirred vigorously for 20 min. This mix-
ture was extracted with CH2Cl2 (4· 50 mL). The com-
bined organic extracts were washed with brine (50 mL)
and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure and the crude product
was purified by flash chromatography eluting with hex-
anes/EtOAc/CHCl3 (3:1:1) to yield 37 (3.25 g,
9.48 mmol, 80%) as a pale yellow solid: 1H NMR
(500 MHz, CDCl3) d 10.10 (s, 1H), 8.19–8.17 (m, 1H),
8.02–8.00 (m, 1H), 7.65–7.58 (m, 2H), 4.22 (t, 2H,
J = 2.2 Hz), 3.77 (t, 2H, J = 5.8 Hz), 2.90–2.87 (m,
2H); 13C NMR (500 MHz, CDCl3) d 188.1, 163.5,
152.3, 148.2, 136.1, 129.6, 127.8, 127.5, 125.1, 122.1,
44.3, 43.6, 35.0; HRMS (FAB+) m/z Calcd for
C13H12ClN2O3S2 (MH+) 342.9978 obsd 342.9962.
6.4. Molecular modeling
Connolly surfaces were generated in SYBYLꢂ on a Sil-
icon Graphics Octane workstation.22 Docking of the
various inhibitors into the hPNMT active site was per-
formed using AutoDock 3.0.59 The default settings for
AutoDock 3.0 were used. The compound to be docked
was initially overlayed with the co-crystallized ligand
and minimized with the Tripos force field. The docking
of inhibitors into the hPNMT active site was performed
on the S-enantiomer as the S-enantiomer is preferred
over the R-enantiomer in the hPNMT active site. Figure
4 was drawn with Molscript60 and Raster3D.61 For Fig-
ure 5, the electrostatic potential was mapped on a Con-
nolly surface generated in SYBYLꢂ.22 Atomic charges
were calculated using the Gasteiger–Marsili method.62
6.5.3. N-Bts-2-nitro-4,5,6,7-tetrahydrothieno[3,2-c]pyri-
dine (38). To a stirred solution of aldehyde 37 (350 mg,
1.02 mmol) in 10 mL DMF at 0 ꢁC was added a solution
of sodium sulfide nonahydrate (368 mg, 1.53 mmol) in
water (2 mL) and the reaction mixture was stirred for
15 min. Bromonitromethane (0.285 mL) was added
dropwise and the reaction mixture was warmed to ambi-
ent temperature and stirred for 1 h. The reaction mix-
ture was poured into ice water (75 mL) and a white
precipitate formed. The mixture was extracted with
CH2Cl2 (4· 50 mL) and the combined organic extracts
were washed with brine (50 mL) and dried over anhy-
drous Na2SO4. The solvent was removed under reduced
pressure and the crude product was purified by flash
chromatography eluting with hexanes/EtOAc (3:1) to
6.5. Synthesis
6.5.1. N-Bts-4-piperidone (36). To a stirred solution of 4-
piperidoneÆH2OÆHCl (35, 4.20 g, 27.9 mmol), dioxane
(15 mL), and 0.25 M NaOH (45 mL) at 5 ꢁC was added
8.50 g (36.3 mmol) of benzothiazole-2-sulfonylchloride.
The reaction mixture was stirred for 2 h at 5 ꢁC, and
for an additional 10 h at ambient temperature while
maintaining the pH between 10 and 11 with the addition
of aqueous 1 M NaOH. The reaction mixture was acid-
ified with 1 M HCl and then extracted with CHCl3 (3·
50 mL). The combined organic extracts were washed
with 1 M HCl (50 mL) and brine (50 mL) and dried over
anhydrous Na2SO4. The solvent was removed under re-
duced pressure and the crude product was purified by
flash chromatography eluting with hexanes/ether (1:1)
to yield 36 (7.25 g, 24.5 mmol, 88%) as a white solid: mp
yield a pale yellow solid. Recrystallization from
EtOAc/hexanes yielded 38 (175 mg, 0.46 mmol, 45%)
as white crystals: mp 170–172 ꢁC; H NMR (500 MHz,
CDCl3) d 8.32–8.30 (m, 1H), 8.20–8.18 (m, 1H), 8.01
(s, 1H), 7.72–7.65 (m, 2H), 4.57 (s, 2H), 3.77 (t, 2H,
J = 5.8 Hz), 3.03 (t, 2H, J = 5.8 Hz); 13C NMR
(500 MHz, CDCl3) d 164.1, 152.4, 149.0, 143.2, 136.2,
131.7, 128.5, 128.3, 128.3, 125.0, 123.7, 45.7, 43.8,
25.4; HRMS (FAB+) m/z Calcd for C14H12N3O4S3
(MH+) 381.9990 obsd 381.9969.
1
6.5.4. General procedure for Bts deprotection (selected
procedure for 23ÆHCl). To a stirred solution of 38
(70 mg, 0.18 mmol) in 2 mL dry DMF was added
37 lL (40 mg, 0.36 mmol) thiophenol and 75 mg
(0.54 mmol) K2CO3 at ambient temperature. The sus-
pension was stirred for 1 h and then diluted with water
(20 mL) and ether (20 mL). The ether layer was removed
1
119–121 ꢁC; H NMR (500 MHz, CDCl3) d 8.20–8.18
(m, 1H), 8.03–8.00 (m, 1H), 7.67–7.59 (m, 2H), 3.85 (t,
4H, J = 6.2 Hz), 2.63 (t, 4H, J = 6.2 Hz); 13C NMR

552
G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
and the aqueous layer was extracted with ether (3·
20 mL). The combined organic extracts were then ex-
tracted with 0.5 M HCl (2· 50 mL). The combined
aqueous extracts were made basic (pH ꢀ 11) with
1 M NaOH and extracted with CH2Cl2 (3· 50 mL).
The combined organic extracts were washed with brine
(50 mL) and dried over Na2SO4. Removal of the sol-
vent yielded 23 as the free amine, which was redis-
solved in CHCl3 (20 mL). Dry HCl(g) was bubbled
through this solution to form the hydrochloride salt,
which was recrystallized from EtOH/hexanes to yield
23ÆHCl51 (40 mg, 0.18 mmol, 96%) as light brown
crystals.
for C8H9ClN2SÆ1/3H2O: C, 46.49; H, 4.71; N, 13.55.
Found: C, 46.37; H, 4.37; N, 13.28.
6.5.8. N-Bts-2-carboxamide-4,5,6,7-tetrahydrothieno[3,2-
c]pyridine (40). To a stirred solution of aldehyde 37
(70.0 mg, 0.204 mmol) in 3 mL DMF at 0 ꢁC was added
a solution of sodium sulfide nonahydrate (73.6 mg,
0.306 mmol) in water (0.5 mL) and the reaction mixture
was stirred for 15 min. Bromoacetamide (112 mg,
0.817 mmol) was added dropwise and the reaction mix-
ture was warmed to ambient temperature and stirred for
1 h. To this reaction mixture was added 0.2 M NaOH
(15 mL). The reaction mixture became cloudy and after
1 min was poured into a saturated ammonium chloride
solution (50 mL). The aqueous mixture was extracted
with CH2Cl2 (3· 30 mL) and the combined organic ex-
tracts were washed with brine (25 mL) and dried over
anhydrous Na2SO4. The solvent was removed under re-
duced pressure and the crude product was purified by
flash chromatography eluting with EtOAc to yield a
white solid. Recrystallization from acetone yielded 40
(45.0 mg, 0.119 mmol, 58%) as white crystals: mp 214–
216 ꢁC; 1H NMR (500 MHz, DMSO-d6) d 8.31–8.29
(m, 1H), 8.21–8.19 (m, 1H), 7.86 (br, 1H), 7.71–7.65
(m, 2H), 7.46 (s, 1H), 7.34 (br, 1H), 4.52 (s, 2H), 3.73
(t, 2H, J = 5.8 Hz), 2.94 (t, 2H, J = 5.7 Hz); 13C NMR
(500 MHz, DMSO-d6) d 164.3, 163.0, 152.4, 138.2,
137.9, 136.1, 131.2, 128.3, 128.2, 126.8, 125.0, 123.6,
45.9, 44.3, 25.0; HRMS (FAB+) m/z Calcd for
C15H14N3O3S3 (MH+) 380.0197 obsd 380.0189.
6.5.5. 2-Nitro-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydro-
chloride (23ÆHCl). See general procedure for Bts depro-
1
tection: mp 199–201 ꢁC; H NMR (500 MHz, DMSO-
d6) d 9.64 (br, 2H), 8.04 (s, 1H), 4.20 (s, 2H) 3.44 (t,
2H, J = 6.0 Hz), 3.15 (t, 2H, J = 6.0 Hz); 13C NMR
(500 MHz, DMSO-d6) d 149.5, 142.3, 129.6, 128.6,
41.9, 40.5, 22.5; HRMS (FAB+) m/z Calcd for
C7H9N2O2 S (MH+) 185.0385 obsd 185.0371; Anal.
Calcd for C7H9ClN2O2S: C, 38.10; H, 4.11; N, 12.69.
Found: C, 38.44; H, 3.89; N, 12.52.
6.5.6. N-Bts-2-cyano-4,5,6,7-tetrahydrothieno[3,2-c]pyri-
dine (39). To a stirred solution of aldehyde 37 (310 mg,
0.904 mmol) in 10 mL DMF at 0 ꢁC, was added a solu-
tion of sodium sulfide nonahydrate (326 mg, 1.36 mmol)
in water (2 mL) and the reaction mixture was stirred for
15 min. Bromoacetonitrile (0.285 mL) was added drop-
wise and the reaction mixture was warmed to ambient
temperature and stirred for 1 h. The reaction mixture
was poured into ice water (75 mL) and a white precipi-
tate formed. The mixture was extracted with CH2Cl2
(4· 50 mL) and the combined organic extracts were
washed with brine (50 mL) and dried over anhydrous
Na2SO4. The solvent was removed under reduced pres-
sure and the crude product was purified by flash chro-
matography eluting with hexanes/EtOAc (3:1) to yield
a pale yellow solid. Recrystallization from EtOAc/hex-
anes yielded 39 (190 mg, 0.53 mmol, 58%) as white crys-
tals: mp 184–186 ꢁC; 1H NMR (500 MHz, CDCl3) d
8.31–8.30 (m, 1H), 8.19–8.17 (m, 1H), 7.76 (s, 1H),
7.72–7.65 (m, 2H), 4.57 (s, 2H), 3.76 (t, 2H,
J = 5.8 Hz), 3.02 (t, 2H, J = 5.8 Hz); 13C NMR
(500 MHz,) d 164.1, 152.4, 142.1, 137.3, 136.1, 132.1,
128.3, 128.2, 125.0, 123.7, 114.8, 106.2, 45.9, 44.0,
25.2; HRMS (FAB+) m/z Calcd for C15H12N3O2S3
(MH+) 362.0092 obsd 362.0077.
6.5.9. 2-Carboxamide-4,5,6,7-tetrahydrothieno[3,2-c]pyr-
idine hydrochloride (25ÆHCl). Compound 40 (75.0 mg,
0.198 mmol) was converted to THTP 25 according to
the general procedure for Bts deprotection. The hydro-
chloride salt was recrystallized from EtOH/hexanes to
yield 25ÆHCl (32.2 mg, 0.147 mmol, 74%) as white crys-
tals: mp dec 303–305 ꢁC; 1H NMR (500 MHz, CD3OD)
d 7.51 (s, 1H), 4.32 (s, 2H), 3.59 (t, 2H, J = 6.2 Hz), 3.21
(t, 2H, J = 6.2 Hz); 13C NMR (500 MHz, CD3OD) d
164.4, 138.0, 137.4, 128.2, 126.8, 42.5, 41.4, 21.6; HRMS
(FAB+) m/z Calcd for C8H11N2OS (MH+) 183.0592
obsd 183.0584; Anal. Calcd for C8H11ClN2OSÆ1/4H2O:
C, 43.05; H, 5.19; N, 12.55. Found: C, 43.42; H, 5.11;
N, 12.17.
6.5.10. N-Bts-2-aceto-4,5,6,7-tetrahydrothieno[3,2-c]pyri-
dine (41). To a stirred solution of aldehyde 37 (140 mg,
0.408 mmol) in 5 mL DMF at 0 ꢁC was added a solution
of sodium sulfide nonahydrate (147 mg, 0.612 mmol) in
water (0.5 mL) and the reaction mixture was stirred for
15 min. Chloroacetone (0.130 mL, 1.63 mmol) was
added dropwise and the reaction mixture was warmed
to ambient temperature and stirred for 1 h. To this reac-
tion mixture was added 0.2 M NaOH (25 mL). The reac-
tion mixture became cloudy and was then diluted to
100 mL with water. This aqueous mixture was extracted
with EtOAc (3· 50 mL) and the combined organic ex-
tracts were washed with brine (50 mL) and dried over
anhydrous Na2SO4. The solvent was removed under re-
duced pressure and the crude product was purified by
flash chromatography eluting with hexanes/EtOAc
(2:1) to yield a white solid. Recrystallization from
6.5.7. 2-Cyano-4,5,6,7-tetrahydrothieno[3,2-c]pyridine
hydrochloride (24ÆHCl). Compound 37 (65 mg,
0.18 mmol) was converted to THTP 24 according to
the general procedure for Bts deprotection. The hydro-
chloride salt was recrystallized from EtOH/hexanes to
yield 24ÆHCl (35 mg, 0.19 mmol, 97%) as white crystals:
1
mp dec 303–305 ꢁC; H NMR (500 MHz, DMSO-d6) d
9.76 (br, 2H), 7.81 (s, 1H), 4.20 (s, 2H), 3.41 (t, 2H,
J = 6.0 Hz), 3.15 (t, 2H, J = 5.9 Hz); 13C NMR
(500 MHz, DMSO-d6) d 141.4, 137.5, 130.1, 114.6,
106.8, 42.0, 40.6, 22.3; HRMS (FAB+) m/z Calcd for
C8H9N2S (MH+) 165.0486 obsd 165.0468; Anal. Calcd

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
553
EtOAc/hexanes yielded 41 (90 mg, 0.238 mmol, 58%) as
white crystals: mp 189–191 ꢁC; 1H NMR (500 MHz,
CDCl3) d 8.17–8.15 (m, 1H), 8.01–7.99 (m, 1H), 7.65–
7.57 (m, 2H), 7.37 (s, 1H), 4.65 (s, 2H), 3.84 (t, 2H,
J = 5.7 Hz), 3.07 (t, 2H, J = 5.7 Hz), 2.52 (s, 3H); 13C
NMR (500 MHz, CDCl3) d 190.2, 163.8, 152.4, 142.5,
141.9, 136.1, 131.4, 129.7, 127.7, 127.5, 125.1, 122.0,
45.8, 44.1, 26.5, 25.7; HRMS (FAB+) m/z Calcd for
C16H15N2O3S3 (MH+) 379.0245 obsd 379.0228.
6.5.13. 2-Methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine
hydrochloride (27ÆHCl). Compound 43 (80.0 mg,
0.228 mmol) was converted to THTP 27 according to
the general procedure for Bts deprotection. The hydro-
chloride salt was recrystallized from EtOH/hexanes to
yield 27ÆHCl (40.4 mg, 0.212 mmol, 93%) as white crys-
1
tals: mp 227–229 ꢁC; H NMR (500 MHz, CD3OD) d
6.59 (s, 1H), 4.21 (s, 2H), 3.53 (t, 2H, J = 6.2 Hz), 3.09
(t, 2H, J = 6.1 Hz), 2.45 (s, 3 H); 13C NMR (500 MHz,
CD3OD) d 139.5, 129.1, 127.0, 122.6, 42.8, 41.8, 21.3,
13.6; HRMS (FAB+) m/z Calcd for C8H12NS (MH+)
6.5.11. 2-Aceto-4,5,6,7-tetrahydrothieno[3,2-c]pyridine
hydrochloride (26ÆHCl). Compound 41 (75.0 mg,
0.198 mmol) was converted to THTP 26 according to
the general procedure for Bts deprotection. The hydro-
chloride salt was recrystallized from EtOH/hexanes to
yield 26ÆHCl (36.0 mg, 0.165 mmol, 84%) as white crys-
154.0690
obsd
154.0683;
Anal.
Calcd
for
C8H12ClNSÆ1/4H2O: C, 49.48; H, 6.49; N, 7.21. Found:
C, 49.51; H, 6.23; N, 7.15.
6.5.14. (R)- and (S)-2-Methyl-1-((S)-1-phenylethyl)pip-
eridin-4-one [(R,S)-45 and (S,S)-45]. To a stirred solution
of 44 (8.00 g, 61.9 mmol) in dry THF (150 mL) at 0 ꢁC
was added 1 M vinylmagnesium bromide in THF
(68.0 mL, 68.0 mmol) and the reaction mixture was
warmed to ambient temperature. The reaction mixture
was stirred for 1 h and the disappearance of starting
material was observed according to TLC. To the reac-
tion mixture were added [S]-a-methylbenzylamine
(16.0 mL, 124 mmol) and water (15 mL). The reaction
mixture was stirred for 1 h and the THF was removed
under reduced pressure. Water (150 mL) was added
and the aqueous mixture was extracted with CH2Cl2
(3· 100 mL). The combined organic extracts were
washed with brine (100 mL) and dried over anhydrous
Na2SO4. The solvent was removed under reduced pres-
sure to yield a brown oil, which was purified by flash
chromatography eluting with hexanes/EtOAc (4:1) to
yield the (R,S)-45 (3.72 g, 17.1 mmol, 28%, Rf = 0.25
1
tals: mp 257–259 ꢁC; H NMR (500 MHz, CD3OD) d
7.71 (s, 1H), 4.35 (s, 2H), 3.59 (t, 2H, J = 6.2 Hz), 3.23
(t, 2H, J = 6.2 Hz), 2.56 (s, 3H); 13C NMR (500 MHz,
CD3OD) d 191.2, 143.2, 141.0, 131.1, 128.9, 42.5, 41.3,
25.0, 22.0; HRMS (FAB+) m/z Calcd for C9H12NOS
(MH+) 182.0640 obsd 182.0621; Anal. Calcd for
C9H12ClNOSÆ1/8H2O: C, 49.14; H, 5.61; N, 6.37.
Found: C, 49.03; H, 5.53; N, 6.20.
6.5.12. N-Bts-2-methyl-4,5,6,7-tetrahydrothieno[3,2-c]
pyridine (43). To a stirred solution of aldehyde 37
(280 mg, 0.816 mmol) in 10 mL DMF at 0 ꢁC was
added a solution of sodium sulfide nonahydrate
(300 mg, 1.25 mmol) in water (1 mL) and the reaction
mixture was stirred for 15 min. Ethyl-2-bromopropio-
nate (0.425 mL, 3.26 mmol) was added dropwise and
the reaction mixture was warmed to ambient tempera-
ture and stirred for 2 h. The reaction mixture was
poured into ice water (50 mL) and a white precipitate
formed. The mixture was extracted with ether (3·
50 mL) and the combined organic extracts were washed
with water (4· 30 mL) and brine (30 mL), and dried
over anhydrous Na2SO4. The solvent was removed un-
der reduced pressure and the crude product was puri-
fied by flash chromatography eluting with hexanes/
EtOAc (3:1) to yield the sulfide intermediate (42) as a
clear oil. This oil was dissolved in ethanol (30 mL)
and cooled to 0 ꢁC. NaOH (1 M, 100 mL) was added
to this solution and the reaction was continued for
5 min. The reaction mixture was then made acidic
(pH ꢀ 1) with 3 M HCl and was stirred for 15 min at
ambient temperature. The resulting cloudy reaction
mixture was extracted with CH2Cl2 (3· 50 mL) and
the combined organic extracts were washed with brine
(25 mL) and dried over anhydrous Na2SO4. The sol-
vent was removed under reduced pressure and the
crude product was purified by flash chromatography
eluting with hexanes/EtOAc (3:1) to yield 43 (195 mg,
0.556 mmol, 68%) as a white solid: mp 138–140 ꢁC;
1H NMR (500 MHz, CDCl3) d 8.19–8.17 (m, 1H),
8.00–7.98 (m, 1H), 7.64–7.56 (m, 2H), 6.42 (s, 1H),
4.53 (s, 2H), 3.79 (t, 2H, J = 5.7 Hz), 2.94 (t, 2H,
J = 5.6 Hz), 2.41 (s, 3H); 13C NMR (500 MHz, CDCl3)
d 164.0, 152.5, 138.2, 136.1, 129.9, 129.7, 127.5, 127.3,
125.1, 122.5, 122.0, 46.1, 44.6, 25.0, 15.1; HRMS
(FAB+) m/z Calcd for C15H15N2O2S3 (MH+)
351.0296 obsd 351.0280.
in 4:1 hexanes/EtOAc) as a yellow solid: mp 70–72 ꢁC;
24
½aꢁ ꢂ11.8ꢁ (c 0.12, CH2Cl2); 1H NMR (500 MHz,
D
CDCl3) d 7.45–7.44 (m, 2H), 7.35–7.32 (m, 2H), 7.27–
7.24 (m, 1H), 4.03–3.99 (m, 1H), 3.41–3.34 (m, 1H),
2.78–2.72 (m, 1H), 2.69–2.62 (m, 3H), 2.35–2.30 (m,
1H), 2.26–2.17 (m, 2H), 1.34–1.33 (m, 3H), 1.15–1.13
(m, 3H); 13C NMR (500 MHz, CDCl3) d 210.5, 145.2,
128.3, 127.2, 126.9, 57.6, 52.4, 48.8, 43.9, 41.3, 16.3,
16.2; HRMS (ES+) m/z Calcd for C14H19NO (MH+)
219.1545 obsd 219.1537. and (S,S)-45 (3.90 g, 17.9
mmol, 29%, Rf = 0.35 in 4:1 hexanes/EtOAc) as a yellow
24
D
1
oil: ½aꢁ ꢂ4.0ꢁ (c 21.4, CH3OH); H NMR (500 MHz,
CDCl3) d 7.36–7.31 (m, 4H), 7.28–7.24 (m, 1H), 3.94–
3.90 (m, 1H), 3.18–3.13 (m, 1H), 2.99–2.91 (m, 2H),
2.59–2.50 (m, 2H), 2.34–2.29 (m, 1H), 2.12–2.08 (m,
1H), 1.43–1.42 (m, 3H), 1.04 (d, 3H, J = 6.5 Hz); 13C
NMR (500 MHz, CDCl3) d 210.5, 144.0, 128.4, 127.2,
127.1, 58.7, 52.3, 48.2, 43.1, 41.1, 21.8, 14.6; HRMS
(ES+) m/z Calcd for C14H19NO (MH+) 219.1545 obsd
219.1538.
6.5.15. (R)-N-Nvoc-2-methyl-4-piperidone [(R)-46]. A
solution of (R,S)-45 (1.38 g, 6.24 mmol) in dry THF
(10 mL) was hydrogenated over Pd(OH)2 (200 mg) for
4 h at ambient temperature and pressure. The reaction
mixture was filtered through Celite and washed with
THF (2· 20 mL). To the resulting THF solution
(50 mL), were added water (50 mL), NvocCl (2.07 g,
7.49 mmol), and 4 M NaOH (to maintain the pH at

554
G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
ꢀ11). After 1 h, the reaction mixture was partitioned be-
tween CH2Cl2 (250 mL) and brine (100 mL). The organ-
ic layer was removed and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure to
yield the crude product as a yellow solid, which was
purified by flash chromatography eluting with hexanes/
ether/CH2Cl2 (1:1:1) to yield (R)-46 (1.92 mg,
HRMS (ES+) m/z Calcd for C17H20ClN2O7 (MH+)
399.0959 obsd 399.0952. The solvent was removed from
the filtrate to yield a yellow solid, which was dissolved in
the minimum amount of EtOAc. Hexanes were added
until the solution became slightly cloudy. Yellow crys-
tals of (R)-48 (291 mg, 0.73 mmol, 26%) formed over
23
D
2–3 days: mp 132–134 ꢁC; ½aꢁ
ꢂ107.4ꢁ (c 0.50,
5.44 mmol, 87%) as light yellow crystals: mp 134–
CHCl3); 1H NMR (500 MHz, CDCl3) d 10.14 (s,
1H), 7.73 (s, 1H, major rot. 70%), 7.68 (s, 1H, minor
rot. 30%), 7.04 (s, 1H, major rot. 70%), 6.94 (s, 1H,
minor rot. 30%), 5.63–5.49 (m, 2H), 5.16–5.10 (m,
1H), 4.37–4.33 (s, 1H, major rot. 70%), 4.19–4.15 (s,
1H, minor rot. 30%), 4.00 (s, 3H), 3.97 (s, 3H), 3.30–
3.20 (m, 1H, major and minor rot.), 2.94–2.80 (m,
1H, major and minor rot.), 2.58–2.48 (m, 1H, major
and minor rot.), 1.34 (d, 3H, J = 7.0 Hz, major rot.
70%), 1.30 (d, 3H, J = 7.0 Hz, minor rot. 30%); HRMS
(ES+) m/z Calcd for C17H20ClN2O7 (MH+) 399.0959
obsd 399.0948.
24
D
136 ꢁC; ½aꢁ
ꢂ5.0ꢁ (c 4.7, CHCl3); 1H NMR
(500 MHz, CDCl3) d 7.68 (s, 1H), 6.97 (s, 1H), 5.57–
5.50 (m, 2H), 4.78–4.71 (m, 1H), 4.31–4.27 (m, 1H),
3.96 (s, 3H), 3.95 (s, 3H), 3.44–3.39 (m, 1H), 2.71–2.67
(m, 1H), 2.53–2.47 (m, 1H), 2.40–2.35 (m, 1H), 2.31–
2.27 (m, 1H), 1.23 (d, 3H, J = 6.9 Hz); 13C NMR
(500 MHz, CDCl3) d 207.0, 154.7, 153.4, 148.7, 140.8,
127.0, 11.6, 108.6, 64.7, 56.5, 56.4, 48.5, 46.5, 40.5,
38.8, 19.1; HRMS (ES+) m/z Calcd for C16H21N2O7
(MH+) 353.1349 obsd 353.1334.
6.5.16. (S)-N-Nvoc-2-methyl-4-piperidone [(S)-46]. Com-
pound (S)-46 was prepared in the same manner as (R)-
46 using (S,S)-45 as the starting material. mp 134–
6.5.18. (S)-N-Nvoc-4-chloro-6-methyl-1,2,5,6-tetrahydro-
pyridine-3-carboxaldehyde [(S)-47] and (S)-N-Nvoc-4-
chloro-2-methyl-1,2,5,6-tetrahydropyridine-3-carboxalde-
hyde [(S)-48]. Compounds (S)-47 and (S)-48 were pre-
pared in the same manner as (R)-47 and (R)-48 using
(S)-46 as the starting material. (S)-47: mp 153–155 ꢁC;
24
1
136 ꢁC; ½aꢁ ꢂ5.2ꢁ (c 1.6, CHCl3); H NMR and 13C
D
NMR data were identical; HRMS (ES+) m/z Calcd for
C16H21N2O7 (MH+) 353.1349 obsd 353.1339.
24
1
6.5.17. (R)-N-Nvoc-4-chloro-6-methyl-1,2,5,6-tetrahydro-
pyridine-3-carboxaldehyde [(R)-47] and (R)-N-Nvoc-4-
chloro-2-methyl-1,2,5,6-tetrahydropyridine-3-carboxalde-
hyde [(R)-48]. To a stirred solution of DMF (0.542 mL,
7.10 mmol) in CHCl3 (15 mL) at 5 ꢁC was added POCl3
(0.463 mL, 4.97 mmol) dropwise. The reaction mixture
was warmed to ambient temperature and stirred for
10 min. Compound (R)-46 (1.00 g, 2.84 mmol) was dis-
solved in CHCl3 (8 mL) and added dropwise to the
POCl3-DMF solution. The reaction was continued for
10 h and then the mixture was poured into an ice-cold
solution of sodium acetate (4.0 g) in water (100 mL)
and stirred vigorously for 20 min. This mixture was ex-
tracted with CH2Cl2 (4· 50 mL). The combined organic
extracts were washed with brine (50 mL) and dried over
anhydrous Na2SO4. The solvent was removed under re-
duced pressure and the crude product was partially puri-
fied by flash chromatography eluting with hexanes/
CHCl3/ether (1:1:2) to separate the unreacted starting
material (R)-46 (151 mg, 0.429 mmol, 15%) from a mix-
ture of (R)-47 and (R)-48 (770 mg, 1.93 mmol, 68%) as a
pale yellow solid. The mixture of regioisomers (R)-47
and (R)-48 was separated by fractional crystallization.
The mixture was first dissolved in the minimum amount
of ether. Hexanes (a volume equal to that of the ether)
were added and yellow needles of (R)-47 (460 mg,
½aꢁ ꢂ75.3ꢁ (c 0.50, CHCl3); H NMR and 13C NMR
D
data were identical; HRMS (ES+) m/z Calcd for
C17H20ClN2O7 (MH+) 399.0959 obsd 399.0959. (S)-48:
24
mp 133–135 ꢁC; ½aꢁ +105.0ꢁ (c 0.50, CHCl3); 1H
D
NMR and 13C NMR data were identical; HRMS
(ES+) m/z Calcd for C17H20ClN2O7 (MH+) 399.0959
obsd 399.0952.
6.5.19. (R)-N-Nvoc-6-methyl-2-nitro-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(R)-49]. To a stirred solution of
aldehyde (R)-47 (135 mg, 0.339 mmol) in 5 mL DMF
at 0 ꢁC was added a solution of sodium sulfide nonahy-
drate (98 mg, 0.406 mmol) in water (0.5 mL) and the
reaction mixture was stirred for 15 min. Bromonitrome-
thane (0.20 mL) was added dropwise and the reaction
mixture was warmed to ambient temperature and stirred
for 1 h. The reaction mixture was poured into ice water
(75 mL) and a white precipitate formed. The mixture
was extracted with CH2Cl2 (4· 50 mL) and the com-
bined organic extracts were washed with brine (50 mL)
and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure and the crude product
was purified by flash chromatography eluting with hex-
anes/EtOAc (1:1) to yield a pale yellow solid. Recrystal-
lization from EtOAc/hexanes yielded (R)-49 (102 mg,
0.233 mmol, 69%) as light yellow crystals: mp 161–
23
D
1.15 mmol, 41%) formed overnight: mp 153–155 ꢁC;
163 ꢁC; ½aꢁ
+51.1ꢁ (c 0.50, CH2Cl2); 1H NMR
24
½aꢁ +79.7ꢁ (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3)
(500 MHz, CDCl3) d 7.71 (s, 1H), 7.68 (s, 1H), 6.99 (s,
1H), 5.60–5.55 (m, 2H), 4.96 (br, 1H, minor rot. 40%),
4.93 (br, 1H, major rot. 60%), 4.19 (br, 1H, major rot.
60%), 4.16 (br, 1H, minor rot. 40%), 3.98 (s, 3H), 3.97
(s, 3H), 3.19–3.17 (m, 1H, minor rot. 40%), 3.16–3.14
(m, 1H, major rot. 60%), 2.72 (s, 1H, major rot. 60%),
2.68 (s, 1H, minor rot. 40%), 1.23 (d, 3H, J = 6.9 Hz);
13C NMR (500 MHz, CDCl3) d 154.6, 153.3, 150.0,
148.8, 140.8, 140.5, 130.8, 126.7, 126.0, 111.7, 108.6,
64.7, 56.4, 56.3, 45.2, 40.0, 30.8, 17.4; HRMS (ES+)
D
d 10.19 (s, 1H), 7.73 (s, 1H), 7.04 (s, 1H), 5.56 (s, 2H),
4.76–4.72 (m, 2H, major and minor rot.), 4.01 (s, 3H),
3.98 (s, 3H), 3.83 (br, 1H, major rot. 60%), 3.80 (br,
1H, minor rot. 40%), 3.13–3.11 (br, 1H, minor rot.
40%), 3.08–3.05 (br, 1H, major rot. 60%), 2.41 (s, 1H,
major rot. 60%), 2.37 (s, 1H, minor rot. 40%), 1.23 (d,
3H, J = 7.0 Hz); 13C NMR (500 MHz, CDCl3) d
188.4, 154.2, 153.6, 148.4, 147.2, 140.2, 129.5, 127.2,
110.6, 108.5, 64.6, 56.4, 56.4, 45.2, 40.6, 38.6, 17.6;

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
555
m/z Calcd for C18H20N3O8S (MH+) 438.0971 obsd
438.0965.
ture was warmed to ambient temperature and stirred for
1 h. The reaction mixture was poured into ice water
(75 mL) and a white precipitate formed. The mixture
was extracted with CH2Cl2 (4· 50 mL) and the com-
bined organic extracts were washed with brine (50 mL)
and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure and the crude product
was purified by flash chromatography eluting with hex-
anes/EtOAc (1:1) to yield a pale yellow solid. Recrystal-
lization from EtOAc/hexanes yielded (R)-50 (110 mg,
6.5.20. (S)-N-Nvoc-6-methyl-2-nitro-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(S)-49]. Compound (S)-49 was pre-
pared in the same manner as (R)-49 using S-47 as the
23
starting material. Mp 160–162 ꢁC; ½aꢁ ꢂ52.3ꢁ (c 0.17,
D
1
CH2Cl2); H NMR and 13C NMR data were identical;
HRMS (ES+) m/z Calcd for C18H20N3O8S (MH+)
438.0971 obsd 438.0963.
0.264 mmol, 78%) as white crystals: mp 152–154 ꢁC;
23
6.5.21. General procedure for Nvoc deprotection (selected
procedure for (R)-30ÆHCl). To a stirred solution of 49
(70 mg, 0.16 mmol) in CH2Cl2 (0.5 mL) was added eth-
anethiol (1.0 mL) at 0 ꢁC. BF3ÆEt2O (0.60 mL) was
added dropwise and the reaction mixture was warmed
to ambient temperature and continued for 10 h. The eth-
anethiol and CH2Cl2 were blown off with a stream of N2
and the reaction was slowly quenched with H2O (5 mL).
This mixture was then diluted with H2O (50 mL), made
basic (pH ꢀ 12) with 1 M NaOH, and extracted with
CH2Cl2 (3· 25 mL). The combined organic extracts
were washed with brine (25 mL) and dried over Na2SO4.
The solvent was removed under reduced pressure and
the crude product was purified by flash chromatography
eluting with CH2Cl2/MeOH (10:1) to yield the free base,
which was redissolved in CHCl3 (20 mL). Dry HCl(g)
was bubbled through this solution to form the hydro-
chloride salt, which was recrystallized from EtOH/hex-
anes to yield (R)-30ÆHCl (28.0 mg, 0.119 mmol, 75%)
as yellow crystals.
½aꢁ +60.2ꢁ (c 0.50, CH2Cl2); 1H NMR (500 MHz,
D
CDCl3) d 7.71 (s, 1H), 7.35 (s, 1H), 6.98 (s, 1H), 5.60–
5.54 (m, 2H), 4.98 (br, 1H, minor rot. 40%), 4.94 (br,
1H, major rot. 60%), 4.20 (br, 1H, major rot. 60%),
4.17 (br, 1H, minor rot. 40%), 3.98 (s, 3H), 3.96 (s,
3H), 3.17–3.15 (m, 1H, minor rot. 40%), 3.14–3.12 (m,
1H, major rot. 60%), 2.74 (s, 1H, major rot. 60%),
2.71 (s, 1H, minor rot. 40%), 1.21 (d, 3H, J = 6.9 Hz);
13C NMR (500 MHz, CDCl3) d 154.6, 153.3, 148.7,
140.7, 140.1, 134.8, 131.5, 126.8, 114.0, 111.6, 108.6,
107.9, 64.6, 56.4, 56.3, 45.3, 40.1, 30.7, 17.3; HRMS
(ES+) m/z Calcd for C19H20N3O6S (MH+) 418.1073
obsd 418.1069.
6.5.25. (S)-N-Nvoc-2-cyano-6-methyl-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(S)-50]. Compound (S)-50 was pre-
pared in the same manner as (R)-50 using (S)-47 as the
23
starting material. Mp 152–154 ꢁC; ½aꢁ ꢂ56.1ꢁ (c 0.15,
D
1
CH2Cl2); H NMR and 13C NMR data were identical;
HRMS (ES+) m/z Calcd for C19H20N3O6S (MH+)
418.1073 obsd 418.1069.
6.5.22.
(R)-6-Methyl-2-nitro-4,5,6,7-tetrahydrothieno
[3,2-c]pyridine hydrochloride [(R)-30ÆHCl]. See general
6.5.26.
(R)-2-Cyano-6-methyl-4,5,6,7-tetrahydrothieno
24
procedure for Nvoc deprotection: mp 210–112 ꢁC; ½aꢁ
[3,2-c]pyridine hydrochloride [(R)-31ÆHCl]. Compound
(R)-50 (100 mg, 0.240 mmol) was converted to (R)-31
according to the general procedure for Nvoc deprotec-
tion. The free amine was purified by flash chromatogra-
phy eluting with CH2Cl2/MeOH (8:1) and the
hydrochloride salt was recrystallized from EtOH/hex-
D
ꢂ72.6ꢁ (c 0.46, CH3OH); 1H NMR (500 MHz, CD3OD)
d 7.64 (s, 1H), 4.45–4.33 (m, 2H), 3.79–3.72 (m, 1H),
3.38–3.34 (m, 1H), 3.00–2.94 (m, 1H), 1.54 (d, 3H,
J = 6.5 Hz); 13C NMR (500 MHz, CD3OD) d 140.1,
135.5, 128.4, 113.1, 108.7, 49.9, 42.0, 29.0, 16.9; HRMS
(ES+) m/z Calcd for C8H11N2O2S (MH+) 199.0541 obsd
199.0545; Anal. Calcd for C8H11ClN2O2SÆ1/4H2O: C,
40.17; H, 4.85; N, 11.71. Found: C, 39.95; H, 4.67; N,
11.47.
anes to yield (R)-31ÆHCl (43.5 mg, 0.203 mmol, 84%)
24
D
as yellow crystals: mp 277–279 ꢁC; ½aꢁ ꢂ94.5ꢁ (c 0.36,
CH3OH); 1H NMR (500 MHz, CD3OD) d 7.64 (s,
1H), 4.45–4.33 (m, 2H), 3.79–3.72 (m, 1H), 3.38–3.34
(m, 1H), 3.00–2.94 (m, 1H), 1.54 (d, 3H, J = 6.5 Hz);
13C NMR (500 MHz, CD3OD) d 140.1, 135.5, 128.4,
113.1, 108.7, 49.9, 42.0, 29.0, 16.9; HRMS (ES+) m/z
Calcd for C9H11N2S (MH+) 179.0643 obsd 179.0644;
Anal. Calcd for C9H11ClN2S: C, 50.34; H, 5.16; N,
13.05. Found: C, 50.14; H, 5.03; N, 12.91.
6.5.23. (S)-6-Methyl-2-nitro-4,5,6,7-tetrahydrothieno[3,2-
c]pyridine hydrochloride [(S)-30ÆHCl]. Compound (S)-
30ÆHCl was prepared in the same manner as (R)-30 using
24
D
(S)-49 as the starting material. Mp 212–214 ꢁC; ½aꢁ
+73.2ꢁ (c 0.40, CH3OH); H NMR and 13C NMR data
were identical; HRMS (ES+) m/z Calcd for
C8H11N2O2S (MH+) 199.0541 obsd 199.0547; HRMS
(ES+) m/z Calcd for C8H11N2O2S (MH+) 199.0541 obsd
199.0545; Anal. Calcd for C8H11ClN2O2S: C, 40.94; H,
4.72; N, 11.94. Found: C, 40.68; H, 4.42; N, 11.71.
1
6.5.27.
(S)-2-Cyano-6-methyl-4,5,6,7-tetrahydrothieno
[3,2-c]pyridine hydrochloride [(S)-31ÆHCl]. Compound
(S)-31ÆHCl was prepared in the same manner as (R)-
31ÆHCl using (S)-50 as the starting material. Mp 276–
24
D
278 ꢁC; ½aꢁ +92.4ꢁ (c 0.25, CH3OH); 1H NMR and
6.5.24. (R)-N-Nvoc-2-cyano-6-methyl-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(R)-50]. To a stirred solution of
aldehyde (R)-47 (135 mg, 0.339 mmol) in 5 mL DMF
at 0 ꢁC was added a solution of sodium sulfide nonahy-
drate (98 mg, 0.406 mmol) in water (0.5 mL) and the
reaction mixture was stirred for 15 min. Bromoacetonit-
rile (0.20 mL) was added dropwise and the reaction mix-
13C NMR data were identical; HRMS (ES+) m/z Calcd
for C9H11N2S (MH+) 179.0643 obsd 179.0648; Anal.
Calcd for C9H11ClN2S: C, 50.34; H, 5.16; N, 13.05.
Found: C, 50.36; H, 5.04; N, 12.97.
6.5.28. (R)-N-Nvoc-4-methyl-2-nitro-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(R)-51]. To a stirred solution of

556
G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
aldehyde (R)-48 (135 mg, 0.339 mmol) in 5 mL DMF at
0 ꢁC was added a solution of sodium sulfide nonahy-
drate (98 mg, 0.406 mmol) in water (0.5 mL) and the
reaction mixture was stirred for 15 min. Bromonitrome-
thane (0.20 mL) was added dropwise and the reaction
mixture was warmed to ambient temperature and stirred
for 1 h. The reaction mixture was poured into ice water
(75 mL) and a white precipitate formed. The mixture
was extracted with CH2Cl2 (4· 50 mL) and the com-
bined organic extracts were washed with brine (50 mL)
and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure and the crude product
was purified by flash chromatography eluting with hex-
anes/EtOAc (1:1) to yield a pale yellow solid. Recrystal-
lization from CH2Cl2/hexanes yielded (R)-51 (96 mg,
6.5.32. (R)-N-Nvoc-2-cyano-4-methyl-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(R)-52]. To a stirred solution of
aldehyde (R)-48 (160 mg, 0.401 mmol) in 5 mL DMF
at 0 ꢁC was added a solution of sodium sulfide nonahy-
drate (116 mg, 0.481 mmol) in water (0.5 mL) and the
reaction mixture was stirred for 15 min. Bromoacetonit-
rile (0.168 mL) was added dropwise and the reaction
mixture was warmed to ambient temperature and stirred
for 1 h. The reaction mixture was poured into ice water
(50 mL) and a white precipitate formed. Brine (50 mL)
was added and the aqueous mixture was extracted with
CH2Cl2 (3· 50 mL). The combined organic extracts
were washed with brine (50 mL) and dried over anhy-
drous Na2SO4. The solvent was removed under reduced
pressure and the crude product was purified by flash
chromatography eluting with hexanes/EtOAc (1:1) to
0.219 mmol, 65%) as light yellow crystals: mp 168–
23
D
170 ꢁC; ½aꢁ ꢂ111.8ꢁ (c 0.50, CH2Cl2); 1H NMR
yield a pale yellow solid. Recrystallization from
EtOAc/hexanes yielded (R)-52 (138 mg, 0.331 mmol,
(500 MHz, CDCl3) d 7.71 (s, 1H), 7.66 (s, 1H), 6.99 (s,
1H), 5.61–5.52 (m, 2H), 5.24 (br, 1H, major and minor
rot.), 4.48 (br, 1H, major and minor rot.), 3.98 (s, 3H),
3.97 (s, 3H), 3.23–3.17 (m, 1H), 2.98–2.91 (m, 1H, major
and minor rot.), 2.84–2.80 (m, 1H, major and minor
rot.), 1.47 (d, 3H, J = 6.8 Hz); 13C NMR (CDCl3) d
154.2, 153.2, 149.8, 148.6, 141.7, 141.1, 137.3, 126.6,
126.3, 111.5, 108.4, 64.5, 56.3, 56.2, 48.9, 36.9, 25.5,
20.2; HRMS (ES+) m/z Calcd for C18H20N3O8S
(MH+) 438.0971 obsd 438.0971.
23
D
82%) as white crystals: mp 150–152 ꢁC; ½aꢁ ꢂ97.1ꢁ (c
1
0.50, CH2Cl2); H NMR (500 MHz, CDCl3) d 7.70 (s,
1H), 7.33 (s, 1H), 6.98 (s, 1H), 5.60–5.51 (m, 2H), 5.26
(br, 1H, major and minor rot.), 4.33 (br, 1H, major
and minor rot.), 3.98 (s, 3H), 3.96 (s, 3H), 3.22–3.16
(m, 1H), 2.95–2.89 (m, 1H, major and minor rot.),
2.85–2.79 (m, 1H, major and minor rot.), 1.45 (d, 3H,
J = 6.8 Hz); 13C NMR (CDCl3) d 154.4, 153.3, 148.7,
141.3, 140.7, 138.1, 135.3, 126.9, 114.0, 111.6, 108.6,
107.8, 64.6, 56.4, 56.3, 49.2, 37.2, 25.5, 20.5; HRMS
(ES+) m/z Calcd for C19H20N3O6S (MH+) 418.1073
obsd 418.1060.
6.5.29. (S)-N-Nvoc-4-methyl-2-nitro-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(S)-51]. Compound (S)-51 was pre-
pared in the same manner as (R)-51 using (S)-48 as the
23
starting material. Mp 168–170 ꢁC; ½aꢁ +108.1ꢁ (c 0.18,
6.5.33. (S)-N-Nvoc-2-cyano-4-methyl-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine [(S)-52]. Compound (S)-52 was pre-
pared in the same manner as (R)-52 using (S)-48 as the
D
1
CH2Cl2); H NMR and 13C NMR data were identical;
HRMS (ES+) m/z Calcd for C18H20N3O8S (MH+)
438.0971 obsd 438.0975.
23
starting material. Mp 152–154 ꢁC; ½aꢁ +103.4ꢁ (c 0.50,
D
1
CH2Cl2); H NMR and 13C NMR data were identical;
HRMS (ES+) m/z Calcd for C19H20N3O6S (MH+)
418.1073 obsd 418.1071. The structure of (S)-52 has
been confirmed by X-ray crystallography (Section 7).
6.5.30.
(R)-4-Methyl-2-nitro-4,5,6,7-tetrahydrothieno
[3,2-c]pyridine hydrochloride [(R)-33ÆHCl]. Compound
(R)-51 (85.0 mg, 0.194 mmol) was converted to (R)-33
according to the general procedure for Nvoc deprotec-
tion. The free amine was purified by flash chromatogra-
phy eluting with CH2Cl2/MeOH (10:1) and the
hydrochloride salt was recrystallized from EtOH/hex-
6.5.34.
(R)-2-Cyano-4-methyl-4,5,6,7-tetrahydrothieno
[3,2-c]pyridine hydrochloride [(R)-34ÆHCl]. Compound
(R)-52 (100 mg, 0.240 mmol) was converted to (R)-34
according to the general procedure for Nvoc deprotec-
tion. The free amine was purified by flash chromatogra-
phy eluting with CH2Cl2/MeOH (8:1) and the
hydrochloride salt was recrystallized from EtOH/hex-
anes to yield (R)-33ÆHCl (37.0 mg, 0.158 mmol, 81%)
24
as yellow crystals: mp 202–204 ꢁC; ½aꢁ +58.4ꢁ (c 0.45,
D
CH3OH); 1H NMR (500 MHz, CD3OD) d 8.00 (s,
1H), 4.63–4.59 (m, 1H), 3.78–3.73 (m, 1H), 3.57–3.51
(m, 1H), 3.26–3.23 (m, 2H), 1.70 (d, 3H, J = 6.9 Hz);
13C NMR (500 MHz, CD3OD) d 150.8, 140.3, 133.3,
126.2, 50.4, 39.8, 21.9, 17.1; HRMS (ES+) m/z Calcd
for C8H11N2O2S (MH+) 199.0541 obsd 199.0548; Anal.
Calcd for C8H11ClN2O2S: C, 40.94; H, 4.72; N, 11.94.
Found: C, 40.87; H, 4.58; N, 11.74.
anes to yield (R)-34ÆHCl (42.2 mg, 0.197 mmol, 82%)
24
as yellow crystals: mp 232–234 ꢁC; ½aꢁ +58.7ꢁ (c 0.68,
D
CH3OH); 1H NMR (500 MHz, CD3OD) d 7.74 (s,
1H), 4.64–4.60 (m, 1H), 3.77–3.72 (m, 1H), 3.55–3.49
(m, 1H), 3.27–3.23 (m, 2H), 1.69 (d, 3H, J = 6.8 Hz);
13C NMR (500 MHz, CD3OD) d 140.2, 135.5, 134.1,
113.1, 109.6, 50.7, 40.4, 21.8, 17.3; HRMS (ES+) m/z
Calcd for C9H11N2S (MH+) 179.0643 obsd 179.0646;
Anal. Calcd for C9H11ClN2SÆ1/8H2O: C, 49.82; H,
5.23; N, 12.91. Found: C, 49.77; H, 5.12; N, 12.71.
6.5.31. (S)-4-Methyl-2-nitro-4,5,6,7-tetrahydrothieno[3,2-
c]pyridine hydrochloride [(S)-33ÆHCl]. Compound (S)-
33ÆHCl was prepared in the same manner as (R)-33 using
24
D
(S)-51 as the starting material. Mp 202–204 ꢁC; ½aꢁ
ꢂ56.4ꢁ (c 0.30, CH3OH); H NMR and 13C NMR data
were identical; HRMS (ES+) m/z Calcd for
C8H11N2O2S (MH+) 199.0541 obsd 199.0548; Anal.
Calcd for C8H11ClN2O2SÆ1/2H2O: C, 39.43; H, 4.96;
N, 11.49. Found: C, 39.72; H, 4.80; N, 11.41.
6.5.35.
(S)-2-Cyano-4-methyl-4,5,6,7-tetrahydrothieno
1
[3,2-c]pyridine hydrochloride [(S)-34ÆHCl]. Compound
(S)-34ÆHCl was prepared in the same manner as (R)-34
using (S)-52 as the starting material. Mp 234–236 ꢁC;
24
½aꢁ ꢂ57.5ꢁ (c 0.30, CH3OH); 1H NMR and 13C
D

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
557
NMR data were identical; HRMS (ES+) m/z Calcd for
C9H11N2S (MH+) 179.0643 obsd 179.0645; Anal. Calcd
for C9H11ClN2SÆ1/4H2O: C, 49.31; H, 5.29; N, 12.78.
Found: C, 49.31; H, 5.24; N, 12.65.
product was purified by flash chromatography eluting
with hexanes/EtOAc (6:1) to yield 21 as white crystals.
This free base was dissolved in Et2O and dry HCl(g)
was bubbled through this solution to form the hydro-
chloride salt, which was recrystallized from EtOH/hex-
anes to yield 21ÆHCl (49.5 mg, 0.203 mmol, 22% from
55) as white crystals: mp 183–185 ꢁC; 1H NMR
(400 MHz, CD3OD) d 7.47 (d, J = 4.8 Hz, 1H), 6.97
(d, J = 4.8 Hz, 1H), 4.66–4.57 (m, 1H), 4.53–4.43 (m,
2H), 3.51–3.24 (m, 2H); HRMS (FAB+) m/z Calcd for
C8H9F3NS (MH+) 208.0408 obsd 208.0388; Anal. Calcd
for C8H9ClF3NS: C, 39.43; H, 3.72; N, 5.75. Found: C,
39.10; H, 3.17; N, 5.46.
6.5.36. 1,1,1-Trifluoro-3-(thiophen-2-yl)-propan-2-one
oxime (55). 3-Bromo-1,1,1-trifluoropropan-2-one oxime
(53, 764 mg, 3.71 mmol), thiophene (8.20 mL,
103 mmol), powdered Na2CO3 (5.89 g, 55.6 mmol),
and DCE (100 mL) were reacted in a thick-walled pyrex
bottle sealed with a Teflon screw cap at 100 ꢁC for 72 h.
The reaction mixture was cooled and filtered through
Celite, which was washed with CH2Cl2 (3· 50 mL).
The solvent was removed under reduced pressure to
yield oxazine 54. This crude product was dissolved in
CHCl3 and treated with trifluoroacetic acid (1 mL) at
ambient temperature for 1 h. The reaction mixture was
diluted with CHCl3 (50 mL) and then extracted with
water (50 mL) and brine (50 mL). The organic layer
was dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The crude product
was purified by flash chromatography eluting with hex-
anes/EtOAc (10:1) to yield 55 (389 mg, 1.86 mmol, 50%
for 2 steps) as colorless crystals: mp 43–45 ꢁC; 1H NMR
(400 MHz, CDCl3) d 8.11 (s, 1H), 7.22–7.20 (m, 1H),
6.97–6.95 (m, 2H), 4.04 (s, 2H).
6.5.38. ( )-2-Bromo-6-trifluoromethyl-4,5,6,7-tetrahydro-
thieno[3,2-c]pyridine hydrochloride (29ÆHCl). To a stirred
solution of 21 (36.4 mg, 0.176 mmol) in glacial acetic
acid (1.5 mL),
a solution of bromine (11.8 lL,
0.229 mmol) in glacial acetic acid (1.0 mL) was added
dropwise at 0 ꢁC. The reaction mixture was stirred at
ambient temperature for 3 h. The reaction mixture was
poured into saturated solution of NaHCO3 (50 mL)
and then extracted with EtOAc (3· 50 mL). The com-
bined organic extracts were washed with brine (50 mL)
and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure and the crude product
was purified by flash chromatography eluting with hex-
anes/EtOAc (1:1) to yield 29 as white crystals. This free
base was dissolved in Et2O and dry HCl(g) was bubbled
through this solution to form the hydrochloride salt,
which was recrystallized from EtOH/hexanes to yield
29ÆHCl (35.2 mg, 0.123 mmol, 70%) as white crystals:
mp, sublimes above 185 ꢁC; 1H NMR (400 MHz,
CD3OD) d 6.97 (s, 1H), 4.32–4.22 (m, 2H), 3.72–3.64
(m, 1H), 3.19–3.14 (m, 1H), 2.84–2.78 (m, 1H); HRMS
(FAB+) m/z Calcd for C8H8BrF3NS (MH+) 285.9513
obsd 285.9514; Anal. Calcd for C8H8BrClF3NS: C,
29.79; H, 2.50; N, 4.34. Found: C, 29.89; H, 2.23; N, 4.22.
6.5.37.
( )-6-Trifluoromethyl-4,5,6,7-tetrahydrothieno
[3,2-c]pyridine hydrochloride (21ÆHCl). To a slurry of
LiAlH4 (107 mg, 2.82 mmol) in Et2O (20 mL) was added
a solution of 55 (197 mg, 0.942 mmol) in Et2O (8 mL) at
0 ꢁC. The reaction mixture was allowed to warm to
ambient temperature and then refluxed at 40 ꢁC for
2.5 h. The reaction mixture was cooled to ambient tem-
perature and then quenched with the successive drop-
wise addition of water (0.1 mL), 15% NaOH (0.1 mL),
and water (0.3 mL). The reaction mixture was filtered
through Celite, which was washed with Et2O (3·
50 mL). The resulting organic solution was washed with
brine (50 mL), dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure. The crude
product was purified by flash chromatography eluting
with hexanes/EtOAc (9:1) to yield amine 56 (100 mg,
0.512 mmol) as a colorless oil: 1H NMR (400 MHz,
CDCl3) d 7.24 (d, J = 4.9 Hz, 1H), 7.00–6.95 (m, 2H),
3.64–3.60 (m, 1H), 3.49–3.45 (m, 1H), 2.94–2.88 (m,
1H). To this amine was added 20% formaldehyde
(0.428 mL) dropwise at 0 ꢁC. This mixture was allowed
to warm to ambient temperature and then refluxed at
100 ꢁC for 3 h. The reaction mixture was cooled and
then partitioned between Et2O (50 mL) and brine
(50 mL) to remove excess formaldehyde. The organic
layer was removed, dried over anhydrous Na2SO4, and
the solvent was removed under reduced pressure to yield
the imine intermediate (120 mg). To this crude product
was added 5 M HCl (1 mL) dropwise at 0 ꢁC. The reac-
tion mixture was permitted to warm to ambient temper-
ature and stirred for 3 h. The reaction mixture was
diluted with water (50 mL), made basic (pH ꢀ 10) with
1 M NaOH, and extracted with CH2Cl2 (3· 50 mL).
The combined organic extracts were washed with brine
(50 mL) and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure and the crude
6.5.39. ( )-2-Bromo-6-methyl-4,5,6,7-tetrahydrothieno
[3,2-c]pyridine hydrobromide (28ÆHBr). Compound 28
was prepared from 20 (115 mg, 0.750 mmol) using the
same procedure for the conversion of 21–29. The crude
product was purified by flash chromatography eluting
with EtOAc to yield 28 as white crystals. This free base
was dissolved in Et2O and dry HBr(g) was bubbled
through this solution to form the hydrobromide salt,
which was recrystallized from EtOH/hexanes to yield
28ÆHBr (60.2 mg, 0.224 mmol, 30%) as white crystals:
mp 199–201 ꢁC; 1H NMR (400 MHz, CD3OD) d 6.98 (s,
1H), 4.33–4.23 (m, 2H), 3.74–3.67 (m, 1H), 3.20–3.16 (m,
1H), 2.86–2.79 (m, 1H), 1.51 (d, J = 6.5 Hz, 3H); HRMS
(FAB+) m/z Calcd for C8H11BrNS (MH+) 231.9795 obsd
231.9766; Anal. Calcd for C8H11Br2NS: C, 30.69; H,
3.54; N, 4.47. Found: C, 31.09; H, 3.25; N, 4.38.
6.5.40. 3-(Benzo(b)thiophen-2-yl)-1,1,1-trifluoropropan-2-
one oxime (58). Compound 58 was prepared from 53
(678 mg, 3.29 mmol), benzo[b]thiophene (2.0 mL,
18 mmol), powdered Na2CO3 (3.49 g, 32.9 mmol), and
CH2Cl2 (25 mL) using the same procedure for the con-
version of 53–55. Compound 58 (76.3 mg, 29.4 mmol,
1
9% from 53) was obtained as a white solid: H NMR

558
G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
3. Grunewald, G. L.; Caldwell, T. M.; Li, Q. F.; Dahanukar,
V. H.; McNeil, B.; Criscione, K. R. J. Med. Chem. 1999,
42, 4351.
(400 MHz, CDCl3) d 8.44 (s, 1H), 7.90 (d, J = 7.7 Hz,
1H), 7.84 (d, J = 7.8 Hz, 1H), 7.25 (s, 1H), 4.10 (s, 2H).
4. Grunewald, G. L.; Dahanukar, V. H.; Teoh, B.; Criscione,
K. R. J. Med. Chem. 1999, 42, 1982.
5. Sall, D. J.; Grunewald, G. L. J. Med. Chem. 1987, 30,
2208.
6. Grunewald, G. L.; Seim, M. R.; Regier, R. C.; Martin, J.
L.; Gee, C. L.; Drinkwater, N.; Criscione, K. R. J. Med.
Chem. 2006, 49, 5424.
7. Pendleton, R. G.; Kaiser, C.; Gessner, G. J. Pharmacol.
Exp. Ther. 1976, 197, 623.
8. Wu, Q.; Criscione, K. R.; Grunewald, G. L.; McLeish, M.
J. Bioorg. Med. Chem. Lett. 2004, 14, 4217.
9. Grunewald, G. L.; Romero, F. A.; Chieu, A. D.; Fincham,
K. J.; Bhat, S. R.; Criscione, K. R. Bioorg. Med. Chem.
2005, 13, 1261.
10. Pendleton, R. G.; Gessner, G.; Weiner, G.; Jenkins, B.;
Sawyer, J.; Bondinell, W.; Intoccia, A. J. Pharmacol. Exp.
Ther. 1979, 208, 24.
6.5.41. ( )-3-Trifluoromethyl-1,2,3,4-tetrahydrobenzothi-
eno[3,2-c]pyridine hydrochloride (32ÆHCl). Compound 32
was prepared from 58 (75.0 mg, 0.289 mmol) using the
same procedure for the conversion of 55–21. The hydro-
chloride salt was recrystallized from EtOH/hexanes to
yield 32ÆHCl (26.0 mg, 0.0885 mmol, 31% from 58) as
1
white crystals: mp, sublimes above 195 ꢁC; H NMR
(500 MHz, CD3OD) d 7.94–7.92 (m, 1H), 7.83–7.81
(m, 1H), 7.52–7.44 (m, 2H), 4.83–4.79 (m, 1H), 4.77 (s,
2H), 3.60–3.56 (m, 1H), 3.30–3.24 (m, 1H); 13C NMR
(500 MHz, CD3OD) d 139.1, 137.3, 127.3, 125.4,
124.8, 124.6, 123.7 (q, J = 280 Hz), 122.3, 120.9, 53.9
(q, J = 32.6 Hz), 43.4, 20.8; HRMS (FAB+) m/z Calcd
for C12H11F3NS (MH+) 258.0564 obsd 258.0556. Anal.
Calcd for C12H11ClF3NSÆ1/4H2O: C, 48.33; H, 3.89;
N, 4.70. Found: C, 48.40; H, 3.60; N, 4.55.
11. Grunewald, G. L.; Sall, D. J.; Monn, J. A. J. Med. Chem.
1988, 31, 433.
12. Pendleton, R. G.; Kaiser, C.; Gessner, G.; Finlay, E.;
Green, H. J. Pharmacol. Exp. Ther. 1974, 190, 551.
13. Wu, Q.; Gee, C. L.; Lin, F.; Tyndall, J. D.; Martin, J. L.;
Grunewald, G. L.; McLeish, M. J. J. Med. Chem. 2005,
48, 7243.
14. Martin, J. L.; Begun, J.; McLeish, M. J.; Caine, J. M.;
Grunewald, G. L. Structure 2001, 9, 977.
15. Bondinell, W. E.; Chapin, F. W.; Girard, G. R.; Kaiser,
C.; Krog, A. J.; Pavloff, A. M.; Schwartz, M. S.; Silvestri,
J. S.; Vaidya, P. D. J. Med. Chem. 1980, 23, 506.
16. Fuller, R. W.; Molloy, B. B.; Day, W. A.; Roush, B. W.;
Marsh, M. M. J. Med. Chem. 1973, 16, 101.
17. Fuller, R. W.; Mills, J.; Marsh, M. M. J. Med. Chem.
1971, 14, 322.
7. X-ray crystallography
The structure of compound (S)-52 has been deposited in
the Cambridge Crystallographic Data Centre, reference
number CCDC 638032.
Acknowledgments
This research was supported by National Institutes of
Health (NIH) Grant HL 34193. The funding for M.R.S.
was also supported by NIH Predoctoral Training Grant
GM 07775 and the American Foundation for Pharmaceu-
tical Education. We thank David VanderVelde and Sarah
Neuenswander of the University of Kansas Nuclear Mag-
netic Resonance Laboratory for their assistance. The
500 MHz NMR spectrometer was partially funded by
National Science Foundation Grant CHE-9977422. We
thank Douglas Powell and Victor Day of the University
of Kansas X-ray Crystallography Laboratory, Gerald
Lushington of the University of Kansas Molecular
Graphics and Modeling Laboratory, and Todd Williams
of the University of Kansas Mass Spectrometry Labora-
tory for their assistance. We also thank Ernst Scho¨nbrunn
for assistance with molecular modeling and David Ben-
son for various helpful discussions.
18. Vecchietti, V.; Clarke, G. D.; Colle, R.; Giardina, G.;
Petrone, G.; Sbacchi, M. J. Med. Chem. 1991, 34, 2624.
19. McMillan, F. M.; Archbold, J.; McLeish, M. J.; Caine, J.
M.; Criscione, K. R.; Grunewald, G. L.; Martin, J. L.
J. Med. Chem. 2004, 47, 37.
20. Grunewald, G. L.; Romero, F. A.; Criscione, K. R.
J. Med. Chem. 2005, 48, 1806.
21. Gee, C. L. D. N.; Tyndall, J. D. A.; Grunewald, G. L.;
Wu, Q.; McLeish, M. J.; Martin, J. L. J. Med. Chem. 2007,
50, 4845.
22. SYBYLꢂ 7.0 Tripos, Inc., 1699 South Hanley Rd., St.
Louis, MO 63144, USA.
23. Grunewald, G. L.; Seim, M. R.; Lu, J.; Makboul, M.;
Criscione, K. R. J. Med. Chem. 2006, 49, 2939.
24. Grunewald, G. L.; Caldwell, T. M.; Li, Q. F.; Criscione,
K. R. J. Med. Chem. 1999, 42, 3315.
25. Grunewald, G. L.; Lu, J.; Criscione, K. R.; Okoro, C. O.
Bioorg. Med. Chem. Lett. 2005, 15, 5319.
26. Herz, W. J. Am. Chem. Soc. 1951, 73, 351.
27. Gronowitz, S.; Sandberg, E. Arkiv foer Kemi 1970, 32,
217.
28. Eloy, F.; Deryckere, A. Bull. Soc. Chim. Belg. 1970, 79,
415.
29. Maffrand, J. P.; Eloy, F. J. Heterocycl. Chem. 1976, 13,
1347.
Supplementary data
Supplementary Information Available: Biochemical
data for the enantiomers of 33 and 34. This material is
available free of charge via the Internet. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2007.08.066.
30. Warm, A. Heterocycles 1992, 34, 2263.
31. Maffrand, J. P.; Biogegrain, R. Heterocycles 1979, 12,
1479.
References and notes
32. Satake, K.; Imai, T.; Kimura, M.; Morosawa, S. Hetero-
cycles 1981, 16, 1271.
33. Cagniant, P.; Kirsch, G. Comptes Rendus des Seances de
l’Academie des Sciences, Serie C: Sciences Chimiques 1975,
281, 35.
1. Grunewald, G. L.; Dahanukar, V. H.; Jalluri, R. K.;
Criscione, K. R. J. Med. Chem. 1999, 42, 118.
2. Grunewald, G. L.; Sall, D. J.; Monn, J. A. J. Med. Chem.
1988, 31, 824.

G. L. Grunewald et al. / Bioorg. Med. Chem. 16 (2008) 542–559
559
34. Matsumura, T.; Hirai, Y.; Nakamoto, M.; Sasaki, T.;
Yokozuka, A.; Shinohara, T. Patent JP 86-146356
63002992, 1988, 5 pp.
35. Vedejs, E.; Lin, S.; Klapars, A.; Wang, J. J. Am. Chem.
Soc. 1996, 118, 9796.
47. Grunewald, G. L.; Borchardt, R. T.; Rafferty, M. F.;
Krass, P. Mol. Pharmacol. 1981, 20, 377.
48. U’Prichard, D. C.; Greenberg, D. A.; Snyder, S. H. Mol.
Pharmacol. 1977, 13, 454.
49. Grunewald, G. L.; Romero, F. A.; Seim, M. R.; Criscione,
K. R.; Deupree, J. D.; Spackman, C. C.; Bylund, D. B.
Bioorg. Med. Chem. Lett. 2005, 15, 1143.
36. Al-Awar, R. S.; Joseph, S. P.; Comins, D. L. Tetrahedron
Lett. 1992, 33, 7635.
37. Vedejs, E.; Kongkittingam, C. J. Org. Chem. 2000, 65,
2309.
50. Halczenko, W.; Stump, C.A. Application: Patent WO
9927929, 1999, 141 pp.
38. Kikuchi, C.; Ando, T.; Fuji, K.; Okuno, M.; Satoh, E.;
Shiiyama, M.; Ushiroda, O.; Koyama, M.; Hiranuma, T.
Patent WO 9933804, 1999, 139 pp.
39. Amit, B.; Zehavi, U.; Patchornik, A. J. Org. Chem. 1974,
39, 192.
51. Koike, H.; Asai, F.; Sugidachi, A.; Kimura, T.; Inoue, T.;
Nishino, S.; Tsuzaki, Y. Patent CA 2077695, 1993, 180 pp.
52. These docking studies were performed using the crystal
structure of hPNMT co-crystallized with SK&F 29661 (8).
See Ref. 6.
40. Grishina, G. V.; Potapov, V. M.; Abdulganeeva, S. A.;
Gudasheva, T. A.; Leshcheva, I. F.; Sergeev, N. M.;
Kudryavtseva, T. A.; Korchagina, E. Y. Khim. Geter-
otsikl. Soedin. 1983, 1693.
41. Grishina, G. V.; Potapov, V. M.; Gudasheva, T. A.;
Abdulganeeva, S. A. Khim. Geterotsikl. Soedin. 1985,
1378.
53. Gee, C. L.; Tyndall, J. D.; Grunewald, G. L.; Wu, Q.;
McLeish, M. J.; Martin, J. L. Biochemistry 2005, 44,
16875.
54. McGaughey, G. B.; Gagne, M.; Rappe, A. K. J. Biol.
Chem. 1998, 273, 15458.
55. Chelli, R.; Gervasio, F. L.; Procacci, P.; Schettino, V.
J. Am. Chem. Soc. 2002, 124, 6133.
42. Bose, D. S.; Thurston, D. E. Tetrahedron Lett. 1990, 31,
6903.
56. Barton, T. J.; Roth, R. W.; Verkade, J. G. J. Am. Chem.
Soc. 1972, 94, 8854.
43. Zimmer, R.; Reissig, H. U. J. Org. Chem. 1992, 57, 339.
44. Elmore, S.W.; Cooper, C.S.; Schultz, C.C.; Hutchinson,
D.K.; Donner, P.L.; Green, B.E.; Anderson, D.D.; Xie,
Q.; Dinges, J.; Lynch, L.M. Patent WO 2001032655, 2001,
294 pp.
45. Romero, F. A.; Vodonick, S. M.; Criscione, K. R.;
McLeish, M. J.; Grunewald, G. L. J. Med. Chem. 2004,
47, 4483.
57. Marino, G. J. Heterocycl. Chem. 1972, 9, 817.
58. Grunewald, G. L.; Caldwell, T. M.; Li, Q. F.; Criscione,
K. R. J. Med. Chem. 2001, 44, 2849.
59. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Hart, W.
E.; Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19,
1639.
60. Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 945.
61. Merrit, E. A.; Bacon, D. J. Methods Enzymol. 1997, 277,
505.
46. Caine, J. M.; Macreadie, I. G.; Grunewald, G. L.;
McLeish, M. J. Protein Expression Purif. 1996, 8, 160.
62. Gasteiger, J.; Marsili, M. Tetrahedron 1980, 36, 3219.