Lobelane analogues as novel ligands for the vesicular monoamine transporter-2

Article abstract of DOI:10.1016/j.bmc.2005.04.013

A series of lobelane analogues has been synthesized and their structure-activity relationships at the vesicular monoamine transporter-2 (VMAT2) have been evaluated. The most potent analogues in this series were the cis-2,6-piperidino analogues, 25b, 27b, 28b, and 30b, with K_i values ranging from 430 to 580 nM.

Full text of DOI:10.1016/j.bmc.2005.04.013

Bioorganic & Medicinal Chemistry 13 (2005) 3899–3909
Lobelane analogues as novel ligands for the
vesicular monoamine transporter-2
Guangrong Zheng, Linda P. Dwoskin, Agripina G. Deaciuc, Jun Zhu,
Marlon D. Jones and Peter A. Crooks*
College of Pharmacy, University of Kentucky, Rose Street, Lexington, KY 40536-0082, USA
Received 18 March 2005; revised 7 April 2005; accepted 7 April 2005
Available online 29 April 2005
Abstract—A series of lobelane analogues has been synthesized and their structure–activity relationships at the vesicular mono-
amine transporter-2 (VMAT2) have been evaluated. The most potent analogues in this series were the cis-2,6-piperidino analogues,
25b, 27b, 28b, and 30b, with Ki values ranging from 430 to 580 nM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the cytosolic pool, which undergoes reverse transport
via the DA transporter, leading to an increase in DA
concentrations in the extracellular space.5
Psychostimulant abuse is a serious and escalating world-
wide problem.1 Methamphetamine is an addictive
psychostimulant drug and chronic use may cause long-
term neural damage in humans, with concomitant dele-
terious effects on cognitive processes such as memory
and attention.2 Despite the serious consequences of
methamphetamine abuse, currently there are no ac-
cepted pharmacological treatments for methamphet-
amine addiction. As a result, there is increasing
interest in identifying underlying mechanisms of meth-
amphetamine action and relevant pharmacological tar-
gets to promote the development of novel therapeutic
agents to treat methamphetamine abuse.
(À)-Lobeline (the 2R,6S,10S-stereoisomer, 1; Fig. 1), the
major alkaloid in Lobelia inflata, decreases both the
stimulant and rewarding effects of methamphetamine,
and does not act as a substitute reinforcer.7–10 The
mechanism underlying the lobeline-induced inhibition
of these effects of methamphetamine has been suggested
to be due to a noncompetitive inhibition of VMAT2.11
The observation that lobeline is not self-administered
is consistent with findings that lobeline does not evoke
DA release.10–12 Furthermore, the observation that
lobeline inhibits methamphetamine-evoked DA release
from superfused rat striatal slices7 is in agreement with
its ability to decrease methamphetamine self-administra-
tion.9 These studies clearly suggest the significance of
The abuse liability of psychostimulants is thought to re-
sult from alterations in the brainꢀs dopaminergic system,
which is generally accepted as being responsible for the
rewarding effects of these abused drugs.3 Through an
interaction with the vesicular monoamine transporter-2
(VMAT2), amphetamine and methamphetamine pro-
mote dopamine (DA) release from the synaptic vesicles
into the cytosol of the dopaminergic presynaptic termi-
nals.4,5 These psychostimulants also inhibit monoamine
oxidase,6 resulting in an increase in DA concentration in
Keywords: Lobeline; Methamphetamine; Vesicular monoamine trans-
porter; Structure–activity relationships.
*
Figure 1. Structures of lobeline (1), MTD (2), lobelane (3), and N-
methyl-2,6-cis-di-(naphthylene-1-ethyl)piperidine (1-NAP-lobelane, 4).
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257
7585; e-mail: pcrooks@email.uky.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.013

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G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
VMAT2 as a potential target for the development of
agents to treat methamphetamine abuse. To date, there
are very few VMAT2 ligands reported in the litera-
ture.13–15 Thus, lobeline analogues with selectivity for
VMAT2 would provide a novel structural class of li-
gands as new tools to probe VMAT2 and as potential
leads for therapeutic development.
OH
O
N
N
CH3
CH3
5
6
N
N
CH3
CH3
Due to the high affinity of lobeline for several neuronal
nicotinic acetylcholine receptor (nAChR) subtypes,16–18
studies have been conducted in our laboratory, which
have focused on structural modification of the lobeline
molecule to increase affinity and selectivity for
VMAT2.17,19 Systematic structural modification of lobe-
line afforded meso-transdiene (MTD, 2) (Fig. 1), a more
selective ligand than lobeline at VMAT2; however,
MTD was slightly less potent than lobeline at
VMAT2.17,19 Interestingly, the defunctionalized, satu-
rated lobeline analogue, lobelane (3) (Fig. 1), had higher
affinity than lobeline and good selectivity for the
[3H]dihydrotetrabenazine ([3H]DTBZ) binding site on
VMAT2 compared with lobeline. To further explore
the structural features that might improve affinity and
selectivity for VMAT2, we prepared compound 4 (1-
NAP-lobelane, Fig. 1) where the 2,6-phenyl groups in
lobelane have been replaced by 1-naphthyl moieties to
determine if enhancing p–p interaction at the VMAT2
binding site would improve affinity.19 While retaining
affinity at VMAT2, compound 4 had increased selectiv-
ity at VMAT2 over a4b2* and a7* nAChRs, indicating
that the VMAT2 binding site can tolerate structural
modifications in the phenyl groups of lobelane.
8
7
Figure 2. Structural fragments of the lobeline (5 and 6), MTD (7), and
lobelane (8) molecules.
interaction, we synthesized analogues 5–8 (Fig. 2), which
are structural fragments of lobeline (5 and 6), MTD (7),
and lobelane (8). Compounds 523 and 824 were synthe-
sized as previous reported. Compounds 625 and 726 were
synthesized via initial Sonogashira cross-coupling of 2-
bromopyridine (9) and phenylacetylene to form com-
pound 10.27 Compound 10 was then hydrolyzed to the
keto compound 11 with 60% sulfuric acid under reflux.
Hydrogenation of 11 with Adamꢀs catalyst and then
N-methylation afforded compound 6. The keto group
in compound 6 was reduced to a hydroxyl group and
the resulting product dehydrated to compound 7
(Scheme 1). Compounds 5 and 6, which represent the
hydroxyl containing fragment and the keto containing
fragment of lobeline (1), showed diminished affinity at
both a4b2* and a7* nAChRs. Interestingly, compound
8, in which one of the piperidine ring side chains of lobe-
lane (3) has been removed, showed 3-fold higher affinity
at a4b2* compared to lobelane, and exhibited no affinity
at a7*. Compound 7, in which a double bond was intro-
duced, showed no affinity at either a4b2* or a7* nAC-
hRs. None of these fragmented compounds showed
any affinity at VMAT2 (Table 1). These results suggest
that both the C2 and C6 side chains of the piperidine
ring of lobeline, MTD, or lobelane are essential for
VMAT2 affinity and selectivity.
The objective of the present study was to prepare an ex-
panded series of lobelane analogues containing a variety
of substituents in the phenyl rings of lobelane, and to as-
sess their affinity and selectivity for VMAT2. In addi-
tion, to further develop structure–activity relationships
within the lobeline/lobelane series of analogues and to
define the structural requirement for VMAT2 binding,
we prepared additional analogues. These included (1)
structural fragments of the lobeline, MTD, and lobelane
molecules, (2) stereoisomeric analogues at the piperidino
C2 and C6 positions, (3) replacement of the piperidine
ring with a piperizino ring, and (4) replacement of the
N-methyl group of lobelane with either a hydrogen,
ethyl, or propyl group.
To further understand the importance of the juxtaposi-
tion and stereochemistry of the piperidino C2 and C6
substituents in the lobelane molecule, and the impor-
tance of the intramolecular distance between the nitro-
gen atom and the phenyl rings of lobelane, compounds
14a,b, 15a,b, 18a–c, 19a–c, and 20a–c were synthesized.
2. Results and discussion
Fragments of the lobeline structure, such as compounds
5 and 6, have been shown previously to have diminished
affinity at nAChRs.20–22 Additionally, MTD (2) and
lobelane (3), which are defunctionalized lobeline ana-
logues, had markedly diminished nAChR affinity.
Importantly, both MTD and lobelane had good affinity
at VMAT2. Furthermore, MTD and lobelane were
more selective ligands than lobeline at this transporter.
Lobelane exhibited a significant, but small increase in
affinity at VMAT2, compared to lobeline.17 In order
to determine if the structure of the whole lobeline/
MTD/lobelane molecule is required for potent VMAT2
Scheme 1. Reagents and conditions: (a) phenylacetylene,
Pd(PPh3)2Cl2, CuI, Et3N, THF, rt; (b) 60% H2SO4, reflux; (c) (i)
HCl; (ii) H2, PtO2, MeOH; (d) HCOOH, 37% HCHO, reflux; (e)
NaBH4, EtOH; (f) 85% H3PO4, 60 ꢁC.

G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
Table 1. Inhibition constants (Ki) for lobelane analogues at the
3901
[3H]NIC binding site (a4b2* nAChR) and the [3H]MLA binding site
(a7* nAChR) on rat brain membranes, and at the [3H]DTBZ binding
site (VMAT2) on rat synaptic vesicle membranes
Compound
Ki (lM) SEMa
[3H]NIC
binding
[3H]MLA
binding
[3H]DTBZ
binding
1
0.004 0.000
11.6 2.01
14.9 1.67
>100
6.26 1.30
>100
2.76 0.64
9.88 2.22
0.97 0.19
0.63 0.16
>100
2
3
26.0 6.57
>100
>100
4
5
>100
6
25.9 1.62
4.52 0.58
>100
>100
>100
>100
>100
>100
7
8
>100
>100
14a
15a
14b
15b
14c
15c
18a
19a
20a
18b
19b
20b
18c
19c
20c
>100
>100
6.11 0.92
11.7 0.65
1.36 0.11
2.62 0.51
36.7 20.3
6.09 0.19
3.32 0.74
3.11 0.51
3.35 0.80
3.95 0.61
10.5 1.58
4.04 1.76
1.87 0.25
3.28 1.65
2.47 0.70
>100
>100
>100
>100
>100
>100
>100
5.06 0.70
>100
>100
>100
66.9 27.4
>100
Scheme 2. Reagents and conditions: (a) 12a: N-benzylidene-4-chloro-
aniline, t-BuOK, DMF, 90 ꢁC; 12b and 12c: benzaldehyde, Ac2O,
reflux; (b) H2, PtO2, HOAc, 45 psi, rt; (c) (CH2O)n, NaCNBH3,
MeOH, rt.
>100
>100
9.02 1.54
>100
>100
>100
>100
>100
>100
7.62 1.47
>100
>100
>100
>100
a Each Ki value represents data from at least four independent experi-
ments, each performed in duplicate.
In these compounds, the C2, C6 phenethyl groups of
lobelane (3) were moved to the C3, C5; C2, C4; N1,
C2; N1, C3; or N1, C4 positions of the piperidine ring.
The synthesis of 14b and 15b was initiated from the con-
densation reaction of 2,4-lutidine with benzaldehyde.28
The resulting highly conjugated product, 13b, was re-
duced to compound 14b under Adamꢀs reduction condi-
tions.28 N-Methylation was then carried out utilizing
NaCNBH3/para-formaldehyde to form compound 15b
(Scheme 2). Similar procedures were employed for the
synthesis of compounds 14a and 15a. N-Benzylidene-4-
chloroaniline was used as an activated form of benzalde-
hyde in the condensation reaction, due to the low activ-
ity of the pyridinyl 3-methyl group.29 Compounds 17a–c,
which were synthesized from 2-, 3-, or 4-picoline utiliz-
ing a similar procedure as that utilized for the synthesis
of 14a and 14b (Scheme 3), were transformed into the N-
phenethyl compounds 18a, 18b, and 18c via reductive
amination, or transformed into 19a, 19b, and 19c by
alkylation with trans-cinnamyl bromide. Catalytic hydro-
genation of 19a, 19b, or 19c afforded compound 20a, 20b,
or 20c, respectively (Scheme 3). [3H]Nicotine ([3H]NIC),
[3H]methyllycaconitine ([3H]MLA), and [3H]dihydrotet-
rabenazine ([3H]DTBZ) binding data for compounds
14a–20c are summarized in Table 1. As expected, most
of these compounds showed no affinity at either a4b2*
or a7* nAChRs. All of these analogues retained affinity
at VMAT2 but were generally less potent than lobelane;
affinity for the [3H]DTBZ binding site on VMAT2 was
within one order of magnitude of that for lobelane (3)
(Table 1). Analogues bearing a side chain at the piper-
Scheme 3. Reagents and conditions: (a) H2, PtO2, HOAc, 45 psi, rt;
(b) phenylacetaldehyde, NaCNBH3, MeOH, rt; (c) trans-cinnamyl
bromide, K2CO3, acetone; (d) H2, 10% Pd/C, EtOH, 15 psi, rt.
idino C3 position, that is, 14a, 15a, 18b, 19b, and 20b,
exhibited slightly lower potency at VMAT2 compared
with lobelane (3). Thus, the position of the piperidine
N atom relative to the C2 and C6 side chains does not
appear to be critical for VMAT2 interaction. The best
compound in this series was 14b (Ki = 1.36 lM), in
which the two side chains are located on the piperidino
C2 and C4 positions. These results also indicate that the
VMAT2 binding site can tolerate apparent changes in

3902
G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
distance between the piperidine nitrogen and the two
phenyl rings in these molecules. However, the above
molecules are very flexible, and can exist in a number
of conformational forms. Thus, it is possible that the
VMAT2 pharmacophore may require a fixed phenyl
ring–piperidine nitrogen distance, and that the above
molecules may undergo conformational changes to
accommodate this pharmacophoric requirement.
might offer improved potency and selectivity at
VMAT2. Unfortunately, compound 4 has very low
water solubility, which precluded further evaluation of
the pharmacological properties of this compound. Nev-
ertheless, the pharmacological properties of compound
4 strongly suggest that the phenyl groups of lobelane
might be amenable to structural modification. Thus, a
series of phenyl ring substituted lobelane analogues were
synthesized and evaluated at the [3H]NIC binding site
(a4b2* nAChR) and the [3H]MLA binding site (a7*
nAChR) on rat brain membranes, and at the [3H]DTBZ
binding site (VMAT2) on rat synaptic vesicle mem-
branes. These data are summarized in Table 2. The syn-
thesis of these compounds was carried out using an
approach similar to that utilized for compounds 14b
and 15b (see Scheme 2). The general structures of these
compounds are given in Table 2. All the compounds
listed in Table 2 showed little or no affinity for both
a4b2* and a7* nAChRs, but retained VMAT2 affinity,
which was comparable to lobelane. All these phenyl
substituted lobelane analogues exhibited Ki values in
the VMAT2 assay within a range of about one order
of magnitude. The 2-methoxy substituted analogue 28b
had the highest affinity (Ki = 0.43 lM) at the [3H]DTBZ
binding site on VMAT2. Interestingly, in these ana-
logues, there were no marked differences in VMAT2
Compounds 14c and 15c, in which the piperidine ring of
lobelane has been replaced by a piperazine ring, were
synthesized by employment of a similar procedure as
was utilized for the preparation of 14b and 15b (Scheme
2). Compounds 14c and 15c were about 36- and 6-fold
less potent than lobelane at VMAT2, respectively. In
addition, compound 15c had 3-fold higher affinity than
lobelane at a4b2* (Table 1).
In a previous SAR study, we prepared compound 4 (Fig.
1, Table 1) in which the phenyl groups in lobelane were
replaced with 1-naphthyl moieties.19 We found that
compound 4 had increased affinity and selectivity at
VMAT2 compared with lobeline, and had slightly better
affinity than lobelane at VMAT2. This led us to con-
clude that modification of the phenyl rings in lobelane,
and specifically introduction of an extended p system
Table 2. Inhibition constants (Ki) for lobelane analogue at [3H]NIC (a4b2* nAChR) and [3H]MLA (a7* nAChR) binding sites on rat brain
membranes, and inhibition of the [3H]DTBZ binding site on VMAT2 on rat synaptic vesicle membranes
Compound
R
R1
R2
R3
Ki (lM) SEMa
[3H]NIC binding
[3H]MLA binding
[3H]DTBZ binding
22
23
CH3CH2
CH3CH2CH2
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.41 0.67
1.87 0.25
1.60 0.10
1.07 0.07
1.60 0.08
0.57 0.07
1.25 0.08
0.98 0.31
1.87 0.19
0.58 0.04
1.67 0.07
0.43 0.03
3.32 0.82
1.73 0.20
2.34 0.16
0.52 0.25
3.23 0.10
4.36 0.18
9.90 2.01
1.51 0.07
1.32 0.18
1.04 0.06
>100
H
H
H
18.6 2.87
>100
>100
24a
24b
25a
25b
26a
26b
27a
27b
28a
28b
29a
29b
30a
30b
31a
31b
32a
32b
33a
33b
34a
34b
35
H
F
H
H
CH3
H
F
H
H
H
F
H
49.2 21.3
16.6 3.47
>100
CH3
H
H
F
H
H
H
F
CH3
H
H
H
F
>100
>100
CH3O
CH3O
H
H
H
CH3
H
H
H
29.7 7.73
>100
CH3O
CH3O
H
H
CH3
H
H
H
25.1 2.23
>100
>100
H
CH3O
CH3O
CH3
H
H
H
H
–OCH2O–
–OCH2O–
CH3
>100
CH3
H
H
18.2 6.87
>100
>100
H
H
CH3
H
H
H
CH3
H
H
CF3
CF3
H
>100
>100
CH3
H
H
H
Cl
Cl
Cl
H
>100
>100
CH3
H
H
Cl
Ph
H
>100
>100
CH3
H
H
H
Ph
CH3COO
OH
10.7 6.60
5.96 1.76
5.26 0.47
H
H
>100
>100
36
H
H
H
a Each Ki value represents data from at least four independent experiments, each performed in duplicate.

G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
3903
affinity between analogues bearing electron donating or
withdrawing substituents. However, compounds 34a
and 34b, in which a phenyl group is attached to the para
position of the lobelane phenyl rings, showed reduced
affinity, and a lack of affinity at VMAT2, respectively.
This may indicate that the VMAT2 binding site can
not accommodate bulky substituents at the para posi-
tion of the phenyl rings.
viously reported, and their characterization data are in
agreement with reported values.
4.1. General procedure for synthesis of compounds 14a–c,
17a–c, nor-lobelane, 24a–34a, and 35
The synthetic procedure utilized was a modification of a
previously reported general procedure.28 2,4-Lutidine
(1 equiv for synthesis of 14b), or 2,6-dimethylpyrazine
(for synthesis of 14c), or 2-picoline (for synthesis of
17a), or 4-picoline (for synthesis of 17c), or 2,6-lutidine
(for synthesis of nor-lobelane, 24a–34a, and 35), was
mixed with the appropriate aryl aldehyde (1.05 equiv
for picolines and 2.1 equiv for 2,6-dimethylpyrazine
and lutidines) (for compound 35, 4-hydroxybenzalde-
hyde was used) and acetic anhydride. The mixture was
refluxed under N2 for 48–72 h and then cooled to room
temperature. The solidified mixture was transferred to a
suction filter and the filter cake was washed with 95%
ethanol. If the product did not solidify during cooling,
then excessive ethanol (95%) was added and the mixture
stirred until the product was precipitated. The crude
product was recrystallized from benzene or ethanol to
afford compounds 13b,c, 16a,c, and 21. For compound
13a or 16b, N-benzylidene-4-chloroaniline was used as
an activated form of benzaldehyde, t-BuOK was used
as base, as reported previously.29
N-Methyl lobelane analogues were often slightly more
corresponding
potent
than
the
nor-lobelane
(R1 = R2 = R3 = R = H, see structure in Table 2) ana-
logues. This encouraged us to synthesize N-ethyl lobe-
lane (22) and N-propyl lobelane (23) in order to
determine if the methyl group is critical for recognition
by the VMAT2 bind site. Compounds 22 and 23 were
equipotent, but slightly less potent than lobelane at
VMAT2, indicating that the N-methyl group plays a
role in VMAT2 binding, but is not critical for binding
site recognition.
3. Summary
Defunctionalization of the lobeline molecule markedly
decreases affinity for a4b2* and a7* nAChRs, while
increasing affinity for VMAT2. The most potent and
selective compounds were lobelane analogues bearing
methoxy, methylenedioxy, and fluoro substituents in
the aromatic rings. The complete lobelane structure ap-
pears to be critical for high affinity binding at VMAT2,
since fragments of lobelane or MTD exhibited signifi-
cantly decreased affinity for VMAT2. Modification of
the phenyl rings in lobelane by introduction of an ex-
tended p system also appears to afford an improvement
in potency and selectivity at VMAT2. Compound 28b
appears to be a promising lead compound for further
development of second generation of VMAT2 ligands.
Each of the above conjugated products (10 mmol) was
dissolved in glacial acetic acid (30 mL) and PtO2 (1–
2% w/w) was added. The resulting mixture was hydroge-
nated on a Parr hydrogenation apparatus (45 psi) for
12–48 h. The catalyst was removed by filtration through
a Celite pad. The filter cake was rinsed with methanol,
and the combined organic liquors were concentrated un-
der reduced pressure. The resulting residue was basified
with saturated aqueous K2CO3 and the aqueous solu-
tion was extracted with CHCl3 (3 · 50 mL). The com-
bined organic extracts were dried (Na2SO4), filtered,
and concentrated. The crude product was purified by
column chromatography to afford the title compounds.
4. Experimental
All reagents and chemicals were purchased from Aldrich
Chemical Co., Milwaukee, WI; Acros Organics, Somer-
ville, NJ; or Lancaster Synthesis, Windham, NH and
were used without further purification. Flash column
chromatography was carried out using ICN SILITECH
4.1.1. 3,5-cis-Diphenethylpiperidine (14a). Mp 98–99 ꢁC
(HCl salt); 1H NMR (300 MHz, CDCl3): d 0.73 (m,
1H), 1.40–1.60 (m, 6H), 2.00 (br d, J = 12.9 Hz, 1H),
2.19 (dd, J = 11.7, 10.5 Hz, 2H), 2.50–2.71 (m, 5H),
3.13 (br d, J = 9.0 Hz, 2H), 7.13–7.32 (m, 10H) ppm;
13C NMR (75 MHz, CDCl3): d 33.3, 36.8, 37.0, 38.7,
52.8, 125.8, 128.4, 128.5, 142.6 ppm; MS (EI) m/z
293 (M+). Anal. Calcd for C21H27NÆHClÆ1/3H2O: C,
75.09; H, 8.60; N, 4.17. Found: C, 74.86; H, 8.79; N,
4.15.
˚
32–63, 60 A silica gel. TLC analyses were carried out on
EMD Chemicals Inc. glass plates precoated with 250 lm
silica gel 60 F254. Melting points were determined on a
Fisher Scientific melting point apparatus and are uncor-
rected. NMR spectra were recorded in CDCl3 on a Var-
ian 300 MHz instrument and are reported in ppm
relative to TMS as internal standard. Mass spectra were
recorded on a JEOL JMS-700T MStation or on a Bru-
ker Autoflex MALDI-TOF MS. GC–mass spectra were
recorded on an Agilent 6890 GC incorporating an Agi-
lent 7683 autosampler and an Agilent 5973 MSD. All of
the final amine compounds were converted to their
hydrochloride salts with 2 N HCl in Et2O. Elemental
analyses were carried out on a COSTECH elemental
combustion system and are within 0.4% of theoretical
values. Compounds 5,23 6,25 7,24 and 826 have been pre-
4.1.2. 2,4-cis-Diphenethylpiperidine (14b). 1H NMR
(300 MHz, CDCl3): d 0.83 (dd, J = 24.0, 11.7 Hz, 1H),
1.10 (ddd, J = 24.3, 12.3, 4.2 Hz, 1H), 1.39 (m, 1H),
1.55 (m, 2H), 1.62–1.98 (m, 5H), 2.40–2.76 (m, 6H),
3.11 (ddd, J = 12.0, 4.2, 2.4 Hz, 1H), 7.10–7.36 (m,
10H) ppm; 13C NMR (75 MHz, CDCl3): d 32.6, 33.1,
33.4, 36.1, 39.3, 39.4, 39.9, 47.0, 56.4, 125.7, 125.9,
128.4, 128.5, 142.3, 142.8 ppm; MS (EI) m/z 293 (M+).
Anal. Calcd for C21H27NÆHClÆ0.5H2O: C, 74.42; H,
8.62; N, 4.13. Found: C, 74.44; H, 8.50; N, 4.08.

3904
G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
4.1.3. 2,6-cis-Diphenethylpiperizine (14c). Mp 251–
252 ꢁC (HCl salt); H NMR (300 MHz, DMSO-d6): d
4.1.8. 2,6-cis-Di-(m-methoxyphenethyl)piperidine (28a).
Mp 113–114 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.08 (ddd, J = 24.0, 12.9, 3.6 Hz, 2H), 1.32
(ddt, J = 25.8, 12.9, 3.6 Hz, 1H), 1.62–1.75 (m, 6H),
1.79 (ddd, J = 13.2, 6.3, 3.0 Hz, 1H), 2.50 (m, 2H),
2.61 (t, J = 8.1 Hz, 4H), 3.78 (s, 6H), 6.70–6.80 (m,
6H), 7.16–7.26 (m, 4H) ppm; 13C NMR (75 MHz,
CDCl3): d 25.0, 32.8, 32.9, 39.1, 55.3, 56.9, 111.2,
114.2, 120.9, 129.4, 144.0, 159.7 ppm; MS (EI) m/z 353
(M+). Anal. Calcd for C23H31NO2ÆHClÆ1/3H2O: C,
69.77; H, 8.32; N, 3.54. Found: C, 69.65; H, 8.54; N,
3.72.
1
1.95 (m, 2H), 2.13 (m, 2H), 2.62–2.86 (m, 4H), 3.14 (t,
J = 12.3 Hz, 2H), 3.34–3.68 (m, 4H), 7.17–7.38 (m,
10H) ppm; 13C NMR (75 MHz, DMSO-d6): d 30.2,
31.4, 43.0, 52.0, 126.0, 128.1, 128.3, 140.2 ppm; MS
(EI) m/z 294 (M+). Anal. Calcd for C20H26N2Æ2HClÆ
0.25H2O: C, 64.60; H, 7.73; N, 7.53. Found: C, 64.56;
H, 7.44; N, 7.61.
4.1.4. 2,6-cis-Di-(o-fluorophenethyl)piperidine (24a). Mp
1
228–229 ꢁC (HCl salt); H NMR (300 MHz, CDCl3): d
1.08 (ddd, J = 12.9, 4.0, 3.9 Hz, 2H), 1.33 (ddt,
J = 25.8, 12.9, 3.9 Hz, 1H), 1.59–1.75 (m, 6H), 1.79
(ddd, J = 12.9, 6.0, 3.0 Hz, 1H), 2.50 (m, 1H), 2.69 (t,
J = 7.8 Hz, 4H), 6.96–7.09 (m, 4H), 7.12–7.22 (m, 4H)
ppm; 13C NMR (75 MHz, CDCl3): d 25.0, 25.7, 32.8,
37.9, 56.8, d (115.150, 115.454), d (124.050, 125.095), d
(127.497, 127.603), d (129.031, 129.243), d (130.534,
130.595), d (159.525, 162.760) ppm; MS (EI) m/z 329
(M+). Anal. Calcd for C21H25F2NÆHCl: C, 68.94; H,
7.16; N, 3.83. Found: C, 69.46; H, 7.13; N, 4.05.
4.1.9. 2,6-cis-Di-(p-methoxyphenethyl)piperidine (29a).
1
Mp 205–206 ꢁC (lit.28 mp 209–211 ꢁC) (HCl salt); H
NMR (300 MHz, CDCl3): d 1.08 (ddd, J = 23.7, 13.2,
3.3 Hz, 2H), 1.31 (ddt, J = 25.8, 13.2, 3.3 Hz, 1H), 1.41
(br s, 1H), 1.58–1.73 (m, 6H), 1.78 (ddd, J = 12.9, 6.3,
3.3 Hz, 1H), 2.48 (m, 2H), 2.57 (t, J = 8.1 Hz, 4H),
3.77 (s, 6H), 6.82 (dd, J = 8.7, 3.0 Hz, 4H), 7.09 (dd,
J = 8.7, 3.3 Hz, 4H) ppm; 13C NMR (75 MHz, CDCl3):
d 25.0, 31.8, 32.9, 39.4, 55.4, 56.8, 113.9, 129.3, 134.4,
157.8 ppm; MS (EI) m/z 353 (M+). Anal. Calcd for
C23H31NO2ÆHClÆ1/3H2O: C, 69.77; H, 8.32; N, 3.54.
Found: C, 69.82; H, 8.77; N, 3.81.
4.1.5. 2,6-cis-Di-(m-fluorophenethyl)piperidine (25a). Mp
1
202–203 ꢁC (HCl salt); H NMR (300 MHz, CDCl3): d
1.07 (ddd, J = 24.0, 13.2, 3.6 Hz, 2H), 1.33 (ddt,
J = 25.8, 12.9, 3.6 Hz, 1H), 1.58–1.75 (m, 6H), 1.80
(ddd, J = 12.9, 6.3, 3.3 Hz, 1H), 2.50 (m, 2H), 2.64 (t,
J = 7.8 Hz, 4H), 6.83–7.00 (m, 6H), 7.23 (m, 2H) ppm;
13C NMR (75 MHz, CDCl3): d 24.9, 32.4, 32.8, 38.9,
56.7, d (112.645, 112.918), d (115.135, 115.409), d
(124.065, 124.095), d (129.805, 129.912), d (144.855,
144.961), d (164.317, 164.567) ppm; MS (EI) m/z
329 (M+). Anal. Calcd for C21H25F2NÆHCl: C, 68.94;
H, 7.16; N, 3.83. Found: C, 68.98; H, 7.42; N,
4.07.
4.1.10. 2,6-cis-Di-(3,4-methylenedioxyphenethyl)piperidine
1
(30a). Mp 207–208 ꢁC (lit.28 215–216 ꢁC) (HCl salt); H
NMR (300 MHz, CDCl3): d 1.06 (ddd, J = 24.0, 12.9,
3.6 Hz, 2H), 1.31 (ddt, J = 5.8, 12.9, 3.6 Hz, 1H), 1.52–
1.73 (m, 6H), 1.78 (ddd, J = 13.2, 6.3, 3.0 Hz, 1H),
2.47 (m, 2H), 2.55 (t, J = 8.1 Hz, 4H), 5.90 (s, 4H),
6.62 (dd, J = 7.8, 1.8 Hz, 2H), 6.67 (d, J = 1.8 Hz, 2H),
6.72 (d, J = 7.8 Hz, 2H) ppm; 13C NMR (75 MHz,
CDCl3): d 25.0, 32.4, 32.9, 39.4, 56.7, 100.9, 108.3,
108.9, 121.1, 136.1, 145.6, 147.6 ppm; MS (EI) m/z 381
(M+). Anal. Calcd for C23H27NO4ÆHClÆ1/3H2O: C,
65.16; H, 6.82; N, 3.30. Found: C, 65.35; H, 7.20; N,
3.52.
4.1.6. 2,6-cis-Di-(p-fluorophenethyl)piperidine (26a). Mp
1
194–195 ꢁC (HCl salt); H NMR (300 MHz, CDCl3): d
1.08 (ddd, J = 24.0, 12.9, 3.6 Hz, 2H), 1.33 (ddt,
J = 26.4, 12.9, 3.6 Hz, 1H), 1.58–1.74 (m, 6H), 1.80
(ddd, J = 13.2, 5.7, 3.6 Hz, 1H), 2.49 (m, 2H), 2.61 (t,
J = 7.8 Hz, 4H), 6.96 (tt, J = 9.0, 2.4 Hz, 4H), 7.12
(dd, J = 9.0, 5.4 Hz, 4H) ppm; 13C NMR (75 MHz,
CDCl3): d 25.0, 31.9, 32.9, 39.4, 56.8, 115.1, 115.4,
129.7, 129.8, 137.8, 137.9, 159.6, 162.9 ppm; MS (EI)
m/z 329 (M+). Anal. Calcd for C21H25F2NÆHClÆ0.2H2O:
C, 68.26; H, 7.20; N, 3.79. Found: C, 68.35; H, 7.42; N,
4.07.
4.1.11. 2,6-cis-Di-(p-methylphenethyl)piperidine (31a).
Mp 221–222 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.08 (ddd, J = 24.0, 12.9, 3.3 Hz, 2H), 1.32
(m, 1H), 1.58–1.73 (m, 6H), 1.78 (m, 1H), 2.31 (s, 6H),
2.49 (m, 2H), 2.59 (t, J = 8.1 Hz, 4H), 7.08 (s, 8H)
ppm; 13C NMR (75 MHz, CDCl3): d 21.3, 25.1, 32.3,
32.9, 39.3, 56.9, 128.3, 129.2, 135.3, 139.3 ppm; MS
(EI) m/z 321 (M+). Anal. Calcd for C23H31NÆHClÆ0.5-
H2O: C, 75.28; H, 9.06; N, 3.82. Found: C, 75.49; H,
9.09; N, 4.19.
4.1.7. 2,6-cis-Di-(o-methoxyphenethyl)piperidine (27a).
Mp 141–142 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.09 (ddd, J = 24.0, 12.6, 3.6 Hz, 2H), 1.29
(ddt, J = 25.5, 12.6, 3.6 Hz, 1H), 1.58–1.76 (m, 6H),
1.76 (ddd, J = 12.6, 6.3, 3.0 Hz, 1H), 2.45 (m, 2H),
2.65 (m, 4H), 3.79 (s, 6H), 6.82 (d, J = 8.1 Hz, 2H),
6.87 (dt, J = 7.5, 1.2 Hz, 2H), 7.10–7.20 (m, 4H) ppm;
13C NMR (75 MHz, CDCl3): d 25.0, 26.6, 32.8, 37.8,
55.3, 56.8, 110.2, 120.5, 127.0, 129.8, 130.7, 157.3 ppm;
MS m/z 353 (M+). Anal. Calcd for C23H31NO2ÆHClÆ
1/3H2O: C, 69.77; H, 8.32; N, 3.54. Found: C, 69.71;
H, 8.44; N, 3.88.
4.1.12. 2,6-cis-Di-(m-trifluoromethylphenethyl)piperidine
(32a). Mp 162–163 ꢁC (HCl salt); 1H NMR (300
MHz, CDCl3): d 1.08 (ddd, J = 24.3, 12.9, 3.9 Hz, 2H),
1.35 (ddt, J = 26.1, 12.9, 3.9 Hz, 1H), 1.61–1.78 (m,
6H), 1.82 (ddd, J = 13.2, 6.3, 3.0 Hz, 1H), 2.52 (m,
2H), 2.71 (t, J = 8.1 Hz, 4H), 7.34–7.58 (m, 8H) ppm;
13C NMR (75 MHz, CDCl3): d 24.9, 32.5, 32.9, 39.1,
56.7,
q
(118.961, 122.560, 126.174, 129.773),
q
(122.757, 122.803, 122.848, 122.909),
q
(125.081,
125.126, 125.187, 125.233), 128.9, q (130.138, 130.563,
130.988, 131.398), 131.8, 143.3 ppm; MS (EI) m/z 430
(MÀ1)+. Anal. Calcd for C23H25F6NÆHClÆ1/3H2O: C,

G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
3905
58.54; H, 5.70; N, 2.97. Found: C, 58.34; H, 5.97; N,
3.21.
C22H29NÆHCl: C, 76.83; H, 8.79; N, 4.07. Found: C,
76.55; H, 8.83; N, 4.12.
4.1.13. 2,6-cis-Di-(2,4-dichlorophenethyl)piperidine (33a).
Mp 183–184 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.09 (ddd, J = 24.0, 12.6, 3.6 Hz, 2H), 1.35
(ddt, J = 26.1, 12.9, 3.6 Hz, 1H), 1.56–1.77 (m, 6H),
1.83 (ddd, J = 13.2, 6.6, 3.3 Hz, 1H), 2.53 (m, 2H),
2.74 (dd, J = 10.7, 7.2 Hz, 4H), 7.15 (s, 2H), 7.16 (d,
J = 1.8 Hz, 2H), 7.35 (dd, J = 1.8, 0.6 Hz, 2H) ppm;
13C NMR (75 MHz, CDCl3): d 24.9, 29.8, 32.7, 37.5,
56.8, 127.2, 129.3, 129.4, 131.1, 132.2, 134.6,
138.5 ppm; MS (EI) m/z 428/430/432/434/436 (MÀ1)+.
Anal. Calcd for C21H23Cl4NÆHCl: C, 53.93; H, 5.17;
N, 2.99. Found: C, 54.33; H, 5.25; N, 3.14.
4.2.2. N-Methyl-2,4-cis-diphenethylpiperidine (15b). 1H
NMR (300 MHz, CDCl3):
d 1.14 (dd, J = 23.4,
11.7 Hz, 1H), 1.24–1.46 (m, 2H), 1.59 (dd, J = 15.3,
6.6 Hz, 2H), 1.66–1.98 (m, 5H), 2.11 (dt, J = 12.0,
2.4 Hz, 1H), 2.30 (s, 3H), 2.50–2.82 (m, 4H), 2.95 (m,
1H), 7.12–7.40 (m, 10H) ppm; 13C NMR (75 MHz,
CDCl3): d 31.4, 32.4, 33.3, 35.7, 36.0, 37.7, 38.9, 42.8,
57.7, 63.5, 125.77, 125.83, 128.38, 128.43, 128.5,
142.8 ppm; MS (EI) m/z 307 (M+). Anal. Calcd for
C22H29NÆHClÆ0.2H2O: C, 76.03; H, 8.82; N, 4.03.
Found: C, 76.38; H, 8.73; N, 4.19.
4.2.3. 1,4-Dimethyl-3,5-cis-diphenethylpiperizine (15c).
Mp 179–180 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.70 (m, 2H), 1.93 (m, 2H), 2.01 (t,
J = 11.4 Hz, 2H), 2.25 (s, 3H), 2.30 (s, 3H), 2.40 (m,
2H), 2.55–2.83 (m, 6H), 7.13–7.39 (m, 10H) ppm; 13C
NMR (75 MHz, CDCl3): d 32.2, 33.8, 35.7, 46.3, 58.9,
61.9, 125.9, 128.3, 128.4, 142.3 ppm. MS (EI) m/z 308
(M+). Anal. Calcd for C22H30N2Æ2HClÆH2O: C, 63.91;
H, 8.29; N, 6.78. Found: C, 64.05; H, 8.43; N, 6.76.
4.1.14. 2,6-cis-Di-(p-phenylphenethyl)piperidine (34a).
Mp 253–254 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.11 (ddd, J = 23.7, 12.6, 3.3 Hz, 2H), 1.34
(ddt, J = 26.1, 12.6, 3.3 Hz, 1H), 1.58–1.88 (m, 7H),
2.54 (m, 2H), 2.68 (t, J = 7.8 Hz, 4H), 7.22–7.59 (m,
18H) ppm; 13C NMR (75 MHz, CDCl3): d 25.0, 32.3,
32.9, 39.1, 56.9, 127.0, 127.1, 127.2, 128.8, 128.9,
138.8, 141.1, 141.4 ppm; MS (EI) m/z 445 (M+). Anal.
Calcd for C33H35NÆHCl: C, 82.21; H, 7.53; N, 2.91.
Found: C, 82.51; H, 7.46; N, 2.87.
4.2.4. 1,2-Diphenethylpiperidine (18a). Mp 157–158 ꢁC
1
(HCl salt); H NMR (300 MHz, CDCl3): d 1.27–1.98
4.1.15. 2,6-cis-Di-(p-acetoxyphenethyl)piperidine (35).
Mp 238–239 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.07 (ddd, J = 23.4, 12.6, 3.6 Hz, 2H), 1.33
(ddt, J = 25.8, 12.9, 3.6 Hz, 1H), 1.80 (ddd, J = 10.2,
5.7, 3.0 Hz, 1H), 2.28 (s, 6H), 2.51 (m, 2H), 2.64 (t,
J = 7.8 Hz, 4H), 6.99 (dt, J = 9.0, 2.4 Hz, 4H), 7.18
(dt, J = 9.0, 2.4 Hz, 4H) ppm; 13C NMR (75 MHz,
CDCl3): d 21.4, 24.9, 32.0, 32.8, 39.2, 56.8, 121.5,
129.3, 139.9, 148.8, 169.7 ppm; MS (EI) m/z 408
(MÀ1)+. Anal. Calcd for C25H31NO4ÆHCl: C, 67.33;
H, 7.23; N, 3.14. Found: C, 67.15; H, 7.54; N, 3.40.
(m, 8H), 2.35–3.00 (m, 9H), 7.10–7.39 (m, 10H) ppm;
13C NMR (75 MHz, CDCl3): d 23.8, 25.7, 30.3, 32.1,
32.4, 33.3, 52.1, 55.7, 59.4, 125.9, 126.0, 128.4, 128.5,
128.8, 140.8, 142.8 ppm; MS (EI) m/z 292 (MÀ1)+.
Anal. Calcd for C21H27NÆHCl: C, 76.45; H, 8.55; N,
4.25. Found: C, 76.34; H, 8.82; N, 4.21.
4.2.5. 1,3-Diphenethylpiperidine (18b). Mp 187–188 ꢁC
(HCl salt); 1H NMR (300 MHz, CDCl3): d 0.91 (m,
1H), 1.42–1.76 (m, 6H), 1.84 (dd, J = 12.9, 1.5 Hz,
1H), 1.94 (dt, J = 11.4, 2.4 Hz, 1H), 2.52–2.68 (m, 4H),
2.80 (m, 2H), 2.97 (m, 2H), 7.10–7.35 (m, 10H) ppm;
13C NMR (75 MHz, CDCl3): d 25.6, 31.2, 33.5, 33.8,
36.1, 36.8, 54.5, 60.5, 61.3, 125.8, 126.1, 128.4, 128.5,
128.8, 140.6, 142.7 ppm; MS (EI) m/z 292 (MÀ1)+.
Anal. Calcd for C21H27NÆHCl: C, 76.45; H, 8.55; N,
4.25. Found: C, 76.21; H, 8.63; N, 4.07.
4.2. General procedure for synthesis of compounds 15a–c,
18a–c, and 24b–34b
NaCNBH3 (188 mg, 3.00 mmol) was added to a mixture
of the appropriate nor-compound (1.00 mmol), para-
formaldehyde (150 mg, 5.00 mmol) (for 18a–c, phenyl-
acetaldehyde was used) and methanol (10 mL). The mix-
ture was stirred at room temperature for 2–12 h. The
solvent was then evaporated under reduced pressure.
The residue was dissolved in water (30 mL) and the
aqueous solution was extracted with CHCl3
(3 · 20 mL). The combined organic extracts were dried
(Na2SO4), filtered, and concentrated. The crude product
was purified by column chromatography to give the title
compounds.
4.2.6. 1,4-Diphenethylpiperidine (18c). Mp 178–179 ꢁC
(HCl salt); 1H NMR (300 MHz, CDCl3): d 1.48 (m,
1H), 1.69 (dd, J = 15.3, 6.9 Hz, 2H), 1.84–2.18 (m,
4H), 2.54–2.72 (m, 4H), 3.06–3.34 (m, 4H), 3.62 (br d,
J = 11.4 Hz, 2H), 7.08–7.40 (m, 10H) ppm; 13C NMR
(75 MHz, CDCl3): d 29.2, 30.5, 32.8, 33.8, 37.1, 53.4,
59.0, 126.1, 127.4, 128.3, 128.6, 128.8, 129.1, 136.3,
141.5 ppm; MS (EI) m/z 292 (MÀ1)+. Anal. Calcd for
C21H27NÆHCl: C, 76.45; H, 8.55; N, 4.25. Found: C,
76.68; H, 8.74; N, 4.21.
4.2.1. N-Methyl-3,5-cis-diphenethylpiperidine (15a). Mp
1
151–152 ꢁC (HCl salt); H NMR (300 MHz, CDCl3): d
4.2.7. N-Methyl-2,6-cis-di-(o-fluorophenethyl)piperidine
(24b). Mp 181–182 ꢁC (HCl salt); H NMR (300 MHz,
CDCl3): d 1.32–1.52 (m, 5H), 1.68 (m, 2H), 1.74–1.87
(m, 3H), 2.18 (s, 3H), 2.37 (m, 2H), 2.70 (m, 4H),
6.96–7.06 (m, 4H), 7.12–7.24 (m, 4H) ppm; 13C NMR
(75 MHz, CDCl3): d 25.2, 25.7, 27.0, 30.9, 35.0, 62.7, d
(115.214, 115.435), d (124.035, 124.073), d (127.443,
1
0.57 (dd, J = 24.0, 12.0 Hz, 1H), 1.44–1.61 (m, 6H),
1.68 (m, 2H), 1.93 (br d, J = 13.2 Hz, 1H), 2.30 (s,
3H), 2.56–2.72 (m, 4H), 2.93 (br d, J = 10.2 Hz, 2H),
7.12–7.35 (m, 10H) ppm; 13C NMR (75 MHz, CDCl3):
d 33.5, 36.0, 36.9, 37.7, 46.7, 62.4, 125.8, 128.4, 128.5,
142.6 ppm; MS (EI) m/z 307 (M+). Anal. Calcd for

3906
G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
127.527), d (129.849, 130.009), d (130.889, 130.943), d
(160.167, 162.604) ppm; MS (EI) m/z 343 (M+). Anal.
Calcd for C22H27F2NÆHCl: C, 69.55; H, 7.43; N, 3.69.
Found: C, 69.18; H, 7.21; N, 3.89.
4.2.13. N-Methyl-2,6-cis-di-(3,4-methylenedioxyphenethyl)-
piperidine (30b). Mp 195–196 ꢁC (HCl salt); H NMR
1
(300 MHz, CDCl3): d 1.34–1.48 (m, 6H), 1.44–1.68 (m,
2H), 1.74–1.91 (m, 2H), 2.18 (s, 3H), 2.37–2.49 (m, 2H),
2.54–2.64 (m, 4H), 5.91 (s, 4H), 6.64 (dd, J = 7.8,
1.8 Hz, 2H), 6.70 (d, J = 1.8 Hz, 2H), 6.72 (d,
J = 7.8 Hz, 2H) ppm; 13C NMR (75 MHz, CDCl3): d
25.1, 26.7, 30.4, 32.3, 36.6, 62.6, 100.9, 108.2, 109.1,
121.2, 136.6, 145.6, 147.6 ppm; MS (MALDI) m/z 396
(M+1)+. Anal. Calcd for C24H29NO4ÆHClÆ0.5H2O: C,
65.37; H, 7.09; N, 3.18. Found: C, 65.13; H, 7.38; N, 3.57.
4.2.8. N-Methyl-2,6-cis-di-(m-fluorophenethyl)piperidine
1
(25b). Mp 140–141 ꢁC (HCl salt); H NMR (300 MHz,
CDCl3) d 1.33–1.50 (m, 5H), 1.65 (m, 2H), 1.78 (m,
1H), 1.84 (m, 2H), 2.15 (s, 3H), 2.37 (m, 2H), 2.67 (m,
4H), 6.84–6.94 (m, 4H), 6.97 (d, J = 5.7 Hz, 2H), 7.22
(dt, J = 5.7, 4.5 Hz, 2H) ppm; 13C NMR (75 MHz,
CDCl3): d 25.2, 26.7, 30.3, 32.1, 36.1, 62.3, d (112.558,
112.770), d (115.351, 115.556), d (124.270, 124.293), d
(129.781, 129.865), d (145.707, 145.775), d (161.883,
164.319) ppm; MS (EI) m/z 343 (M+). Anal. Calcd for
C22H27F2NÆHClÆH2O: C, 66.40; H, 7.60; N, 3.52.
Found: C, 66.50; H, 7.68; N, 3.70.
4.2.14. N-Methyl-2,6-cis-di-(p-methylphenethyl)piperidine
1
(31b). Mp 203–204 ꢁC (HCl salt); H NMR (300 MHz,
CDCl3): d 1.26–1.48 (m, 5H), 1.63 (m, 2H), 1.75–1.92
(m, 3H), 2.16 (s, 3H), 2.31 (s, 6H), 2.36 (br t,
J = 6.0 Hz, 2H), 2.50–2.71 (m, 4H), 7.09 (s, 8H) ppm;
13C NMR (75 MHz, CDCl3): d 21.3, 25.3, 27.1, 31.0,
32.1, 36.8, 62.7, 128.4, 129.1, 135.1, 139.9 ppm; MS
(EI) m/z 335 (M+). Anal. Calcd for C24H33NÆHClÆ
2/3H2O: C, 75.07; H, 9.27; N, 3.65. Found: C, 74.73;
H, 9.27; N, 4.05.
4.2.9. N-Methyl-2,6-cis-di-(p-fluorophenethyl)piperidine
1
(26b). Mp 185–186 ꢁC (HCl salt); H NMR (300 MHz,
CDCl3): d 1.30–1.51 (m, 5H), 1.63 (m, 2H), 1.72–1.91
(m, 3H), 2.15 (s, 3H), 2.37 (m, 2H), 2.64 (m, 4H), 6.95
(tt, J = 9.0, 2.4 Hz, 4H), 7.15 (dt, J = 9.0, 3.0 Hz, 4H);
13C NMR (75 MHz, CDCl3): d 25.3, 26.8, 30.5, 31.7,
36.7, 62.4, d (114.9, 115.2), d (129.8, 129.9), 138.5, d
(159.6, 162.8); MS (EI) m/z 343 (M+). Anal. Calcd for
C22H27F2NÆHClÆ2/3H2O: C, 67.42; H, 7.59; N, 3.57.
Found: C, 67.50; H, 7.44; N, 3.94.
4.2.15. N-Methyl-2,6-cis-di-(m-trifluoromethylphenethyl)-
1
piperidine (32b). Mp 152–153 ꢁC (HCl salt); H NMR
(300 MHz, CDCl3): d 1.39–1.57 (m, 5H), 1.67 (m, 2H),
1.74–1.96 (m, 3H), 2.17 (s, 3H), 2.42 (m, 2H), 2.75 (t,
J = 8.4 Hz, 4H), 7.36–7.50 (m, 8H) ppm; 13C NMR
(75 MHz, CDCl3): d 25.4, 26.4, 29.7, 32.2, 36.2, 62.1, q
(122.575, 122.621, 122.666, 122.727),
q (125.233,
4.2.10. N-Methyl-2,6-cis-di-(o-methoxyphenethyl)piperi-
dine (27b). Mp 92–93 ꢁC (HCl salt); 1H NMR
(300 MHz, CDCl3): d 1.36–1.56 (m, 5H), 1.64 (m, 2H),
1.74–1.90 (m, 3H), 2.21 (s, 3H), 2.35 (br s, 2H), 2.66
(t, J = 8.4 Hz, 4H), 3.81 (s, 6H), 6.80–6.92 (m, 4H),
7.10–7.20 (m, 4H) ppm; 13C NMR (75 MHz, CDCl3):
d 25.4, 27.2, 27.7, 31.8 34.9, 55.5, 63.4, 110.3, 120.5,
126.9, 129.9, 131.5, 157.5 ppm; MS (EI) m/z 367 (M+).
Anal. Calcd for C24H33NO2ÆHClÆ1.25H2O: C, 67.59;
H, 8.63; N, 3.28. Found: C, 67.67; H, 8.77; N, 3.73.
125.293, 125.339, 125.384), 126.2, 128.8, q (130.001,
130.411, 130.836, 131.261), 132.0, 143.8 ppm; MS (EI)
m/z 444 (MÀ1)+. Anal. Calcd for C24H27F6NÆHClÆ1/
3H2O: C, 59.32; H, 5.95; N, 2.88. Found: C, 59.24; H,
6.32; N, 3.14.
4.2.16. N-Methyl-2,6-cis-di-(2,4-dichlorophenethyl)piper-
idine (33b). Mp 181–182 ꢁC (HCl salt); 1H NMR
(300 MHz, CDCl3): d 1.30–1.56 (m, 5H), 1.65 (m, 6H),
1.71–1.86 (m, 3H), 2.18 (s, 3H), 2.38 (m, 2H), 2.66–
2.84 (m, 4H), 7.14–7.22 (m, 4H), 7.35 (dd, J = 1.8,
0.6 Hz, 2H) ppm; 13C NMR (75 MHz, CDCl3): d 25.3,
26.9, 29.8, 30.6, 34.4, 62.5, 127.1, 129.3, 131.4, 132.1,
134.6, 139.1 ppm; MS (EI) m/z 442/444/446/448/450
(MÀ1)+. Anal. Calcd for C22H25Cl4NÆHCl: C, 54.85;
H, 5.44; N, 2.91. Found: C, 55.04; H, 5.61; N, 3.11.
4.2.11. N-Methyl-2,6-cis-di-(m-methoxyphenethyl)piperi-
dine (28b). Mp 157–158 ꢁC (HCl salt); 1H NMR
(300 MHz, CDCl3): d 1.40–1.62 (m, 3H), 1.73 (m, 2H),
1.82–1.95 (m, 3H), 2.17 (m, 2H), 2.50 (s, 3H), 2.67 (m,
4H), 3.81 (s, 6H), 6.73–6.82 (m, 6H), 7.21 (dd, J = 9.0,
7.5 Hz, 2H) ppm; 13C NMR (75 MHz, CDCl3): d 22.8,
24.1, 32.4, 33.6, 55.6, 64.7, 112.4, 113.8, 120.7, 129.8,
141.2, 159.9 ppm; MS (EI) m/z 367 (M+). Anal. Calcd
for C24H33NO2ÆHClÆH2O: C, 68.31; H, 8.60; N, 3.32.
Found: C, 68.25; H, 9.03; N, 3.43.
4.2.17. N-Methyl-2,6-cis-di-(p-phenylphenethyl)piperidine
(34b). Mp 215–216 ꢁC (HCl salt); 1H NMR (300
MHz, CDCl3): d 1.32–1.47 (m, 4H), 1.64–1.95 (m,
6H), 2.21 (s, 3H), 2.42 (m, 2H), 2.62–2.80 (m, 4H),
7.26–7.60 (m, 18H) ppm; 13C NMR (75 MHz, CDCl3):
d 25.3, 26.9, 30.7, 32.0, 36.4, 62.6, 126.9, 127.2, 127.3,
128.9, 129.0, 138.8, 141.3, 142.3 ppm; MS (MALDI)
m/z 460 (M+1)+. Anal. Calcd for C34H37NÆHClÆ1.5H2O:
C, 78.06; H, 7.90; N, 2.68. Found: C, 78.20; H, 8.42; N,
3.05.
4.2.12. N-Methyl-2,6-cis-di-(p-methoxyphenethyl)piperi-
dine (29b). Mp 175–176 ꢁC (lit.28 mp 176–178 ꢁC) (HCl
salt); 1H NMR (300 MHz, CDCl3): d 1.40 (m, 4H),
1.55–1.70 (m, 3H), 1.70–1.90 (m, 3H), 2.16 (s, 3H),
2.37 (m, 2H), 2.60 (m, 4H), 3.78 (s, 6H), 6.82
(d, J = 8.4 Hz, 4H), 7.12 (d, J = 8.4 Hz, 4H) ppm; 13C
NMR (75 MHz, CDCl3): d 25.4, 27.0, 30.9, 31.6, 36.9,
55.5, 62.6, 113.8, 129.4, 135.1, 157.6 ppm; MS (EI) m/z
367 (M+). Anal. Calcd for C24H33NO2ÆHClÆ0.5H2O: C,
69.80; H, 8.54; N, 3.39. Found: C, 69.60; H, 8.66; N,
3.53.
4.3. General procedure for synthesis of compounds 19a–c,
and 20a–c
trans-Cinnamyl bromide (394 mg, 2.00 mmol) was
added dropwise to a mixture of the appropriate

G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
3907
nor-compound (17a,b, or 17c) (378 mg, 2.00 mmol),
K2CO3 (552 mg, 4.00 mmol) and acetone (15 mL). The
mixture was stirred at room temperature for 4 h. The
solvent was then evaporated under reduced pressure.
The residue was dissolved in CHCl3 (40 mL) and washed
with water (20 mL) and saturated aqueous NaCl
(20 mL). The organic phase was dried (Na2SO4), fil-
tered, and concentrated. The crude product was purified
by column chromatography to give the title compounds
19a–c. Compounds 19a,b, or 19c (305 mg, 1 mmol) in
ethanol (20 mL) with 10% Pd/C (20 mg) was hydroge-
nated on a Parr hydrogenation apparatus (15 psi) for
4 h. The catalyst was removed by filtration through a
Celite pad. The filter cake was rinsed with ethanol,
and the combined organic liquors were concentrated un-
der reduced pressure. The resulting residue was purified
by column chromatography to give the title compounds
20a–c.
4.3.5. 1-trans-Cinnamyl-4-phenethylpiperidine (19c). Mp
160–161 ꢁC (HCl salt); H NMR (300 MHz, CDCl3): d
1
1.26–1.65 (m, 5H), 1.79 (br d, J = 13.2 Hz, 2H), 2.10
(m, 2H), 2.63 (t, J = 7.8 Hz, 2H), 3.10 (br d,
J = 12.0 Hz, 2H), 3.26 (dd, J = 6.9, 0.9 Hz, 2H), 6.35
(dt, J = 15.9, 6.9 Hz, 1H), 6.54 (d, J = 15.9 Hz, 1H),
7.10–7.43 (m, 10H) ppm; 13CNMR (75 MHz, CDCl3):
d 31.8, 33.3, 35.0, 38.3, 53.8, 61.3, 124.9, 125.8, 126.6,
127.9, 128.40, 128.43, 128.7, 134.4, 136.6, 142.5 ppm;
MS (EI) m/z 305 (M+). Anal. Calcd for C22H27NÆHCl:
C, 73.41; H, 8.40; N, 3.89. Found: C, 73.46; H, 8.73;
N, 3.68.
4.3.6. 1-(3-Phenylpropyl)-4-phenethylpiperidine (20c).
Mp 152–153 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 1.24–1.43 (m, 3H), 1.56 (m, 2H), 2.38 (m,
2H), 1.73 (br d, J = 10.8 Hz, 2H), 1.80–1.98 (m, 4H),
2.56–2.66 (m, 4H), 2.95 (br d, J = 12.0 Hz, 2H), 7.08–
7.32 (m, 10H) ppm; 13C NMR (75 MHz, CDCl3): d
28.7, 32.3, 33.3, 34.1, 35.5, 38.5, 54.1, 58.6, 125.7,
125.8, 128.38, 128.41, 142.1, 142.7 ppm; MS (EI) m/z
307 (M+). Anal. Calcd for C22H29NÆHCl: C, 76.83; H,
8.79; N, 4.07. Found: C, 76.79; H, 8.76; N, 3.98.
1
4.3.1. 1-trans-Cinnamyl-2-phenethylpiperidine (19a). H
NMR (300 MHz, CDCl3): d 1.35 (m, 1H), 1.52–2.10
(m, 7H), 2.40 (m, 1H), 2.48–2.63 (m, 2H), 2.76 (m,
1H), 2.99 (dt, J = 11.4, 4.5 Hz, 1H), 3.31 (ddd,
J = 13.8, 7.8, 1.2 Hz, 1H), 3.57 (ddd, J = 13.8, 6.9,
2.1 Hz, 1H), 6.27 (ddd, J = 15.9, 7.5, 6.3 Hz, 1H), 6.54
(d, J = 15.9 Hz, 1H), 7.14–7.39 (m, 10H) ppm; 13C
NMR (75 MHz, CDCl3): d 23.2, 24.9, 29.6, 31.9, 32.6,
52.0, 55.8, 60.0, 124.4, 126.0, 126.5, 127.8, 128.3,
128.5, 128.6, 134.3, 136.6, 142.0 ppm; MS (EI) m/z 305
(M+). Anal. Calcd for C22H27NÆHCl: C, 73.41; H,
8.40; N, 3.89. Found: C, 73.08; H, 8.54; N, 3.87.
4.4. N-Ethyl-2,6-cis-diphenethylpiperidine (22)
A mixture of nor-lobelane (440 mg, 1.50 mmol), iodo-
ethane (2.34 g, 15.0 mmol), and K2CO3 (414 mg,
3.0 mmol) in THF (10 mL) was stirred under reflux for
3 days. The reaction mixture was allowed to cool and
the solvent was removed under reduced pressure. The
residue was dissolved in water (40 mL) and the aqueous
solution was extracted with CHCl3 (3 · 20 mL). The
combined organic extracts were dried (Na2SO4), filtered,
and concentrated. The crude product was purified by
column chromatography (40:1 CHCl3–CH3OH) to give
22 (356 mg, 74%) as a white solid. Mp 186–187 ꢁC
(lit.28 187–188 ꢁC) (HCl salt); 1H NMR (300 MHz,
CDCl3): d 0.94 (t, J = 7.2 Hz, 3H), 1.28–1.48 (m, 3H),
1.56–1.82 (m, 5H), 1.86–2.02 (m, 2H), 2.46–2.80 (m,
8H), 7.13–7.31 (m, 10H) ppm; 13C NMR (75 MHz,
CDCl3): d 12.6, 24.5, 28.8, 32.7, 36.0, 39.6, 60.7, 125.8,
128.4, 142.7 ppm; MS (EI) m/z 321 (M+). Anal. Calcd
for C23H31NÆHClÆ0.5H2O: C, 75.28; H, 9.06; N, 3.82.
Found: C, 75.26; H, 9.16; N, 4.08.
1
4.3.2. 1-(3-Phenylpropyl)-2-phenethylpiperidine (20a). H
NMR (300 MHz, CDCl3): d 1.47 (m, 1H), 1.74–2.18 (m,
9H), 2.47–3.03 (m, 8H), 3.15 (m, 1H), 7.08–7.32 (m,
10H) ppm; 13C NMR (75 MHz, CDCl3): d 21.3, 22.6,
24.8, 26.9, 30.4, 31.8, 33.1, 51.6, 60.6, 126.5, 128.3,
128.71, 128.74, 139.8, 140.0 ppm; MS (EI) m/z 307
(M+). Anal. Calcd for C22H29NÆHClÆ0.5H2O: C, 74.87;
H, 8.85; N, 3.97. Found: C, 75.02; H, 8.96; N, 3.77.
4.3.3. 1-trans-Cinnamyl-3-phenethylpiperidine (19b). Mp
1
170–171 ꢁC (HCl salt); H NMR (300 MHz, CDCl3): d
0.91 (ddd, J = 22.8, 12.3, 4.5 Hz, 1H), 1.42–1.74 (m,
6H), 1.78–1.96 (m, 2H), 2.61 (m, 2H), 2.96 (m, 2H),
3.14 (d, J = 6.6 Hz, 2H), 6.30 (dt, J = 15.9, 6.6 Hz,
1H), 6.50 (d, J = 15.9 Hz, 1H), 7.10–7.40 (m, 10H)
ppm; 13C NMR (75 MHz, CDCl3): d 25.7, 31.2, 33.4,
36.1, 36.8, 54.5, 60.6, 61.8, 125.7, 126.4, 127.0, 127.5,
128.4, 128.6, 132.9, 137.1, 142.7 ppm; MS (EI) m/z 305
(M+). Anal. Calcd for C22H27NÆHClÆ1/3H2O: C, 75.95;
H, 8.30; N, 4.03. Found: C, 75.58; H, 8.62; N, 4.11.
4.5. N-(n-Propyl)-2,6-cis-diphenethylpiperidine (23)
Compound 23 was prepared as described for compound
22 from nor-lobelane and 1-iodopropane. Mp 176–
177 ꢁC (lit.28 174–176 ꢁC) (HCl salt); 1H NMR
(300 MHz, CDCl3): d 0.78 (t, J = 7.5 Hz, 3H), 1.28–
1.47 (m, 5H), 1.52–1.89 (m, 7H), 2.41–2.78 (m, 8H),
7.14–7.33 (m, 10H) ppm; 13C NMR (75 MHz, CDCl3):
d 12.1, 22.3, 24.6, 28.1, 33.1, 36.5, 48.3, 61.6, 125.8,
128.46, 128.51, 142.8 ppm; MS (EI) m/z 335 (M+). Anal.
Calcd for C24H33NÆHClÆ2/3H2O: C, 75.07; H, 9.27; N,
3.65. Found: C, 74.83; H, 9.65; N, 3.87.
4.3.4. 1-(3-Phenylpropyl)-3-phenethylpiperidine (20b).
Mp 158–159 ꢁC (HCl salt); 1H NMR (300 MHz,
CDCl3): d 0.89 (ddd, J = 22.8, 12.3, 4.8 Hz, 1H), 1.40–
1.72 (m, 6H), 1.74–1.92 (m, 4H), 2.36 (m, 2H), 2.52–
2.66 (m, 4H), 2.90 (m, 2H), 7.10–7.34 (m, 10H) ppm;
13C NMR (75 MHz, CDCl3): d 25.5, 28.7, 31.1, 33.4,
34.0, 35.9, 36.7, 54.4, 58.7, 60.4, 125.7, 125.8, 128.3,
128.4, 142.1, 142.6 ppm; MS (EI) m/z 307 (M+). Anal.
Calcd for C22H29NÆHClÆ0.5H2O: C, 74.87; H, 8.85; N,
3.97. Found: C, 74.68; H, 8.75; N, 3.74.
4.6. 2,6-cis-Di-(p-hydroxyphenethyl)piperidine (36)
A solution of compound 35 (710 mg, 1.73 mmol) in
methanol (30 mL) was treated with NaHCO3 (1.45 g,

3908
G. Zheng et al. / Bioorg. Med. Chem. 13 (2005) 3899–3909
17.30 mmol) for 4 h at ambient temperature. The sol-
vent was then evaporated under reduced pressure. The
residue was dissolved in water (30 mL) and the resulting
solution was acidified with 2 N HCl to pH 2.0. The
aqueous solution was extracted with CHCl3
(3 · 20 mL). The combined organic extracts were dried
(Na2SO4), filtered, and concentrated. The crude product
was purified by recrystallization from methanol/diethyl
ether to give 36 (480 mg, 77 %) as a white solid. Mp
263–264 ꢁC (HCl salt); 1H NMR (300 MHz, DMSO-
d6) (HCl salt): d 1.26–1.46 (m, 3H), 1.64–1.85 (m, 3H),
1.85–2.06 (m, 4H), 2.41–2.66 (m, 4H), 2.92 (br s, 2H),
6.69 (dt, J = 8.7, 2.5 Hz, 4H), 6.99 (dt, J = 8.7, 2.4 Hz,
4H), 8.64 (m, 2H), 9.23 (s, 2H) ppm; 13C NMR
(75 MHz, DMSO-d6): d 21.9, 27.4, 29.7, 34.8, 56.3,
115.0, 128.8, 130.6, 155.3 ppm; MS (MALDI) m/z 326
(M+1)+. Anal. Calcd for C21H27NO2ÆHClÆ1.2H2O: C,
65.77; H, 7.99; N, 3.65. Found: C, 65.91; H, 8.12; N,
4.03.
4.8. [3H]DTBZ binding assay
Synaptic vesicles were prepared as previously de-
scribed.12 Briefly, fresh whole rat brain (excluding cere-
bellum and brain stem) was homogenized in 20 vol of
ice-cold 0.32 M sucrose using a glass homogenizer (se-
ven strokes of a Teflon pestle, clearance = 0.003 in.).
Homogenates were centrifuged at 1000g for 12 min at
4 ꢁC. Resulting supernatants were centrifuged at
22,000g for 10 min. Resulting pellets, containing the
synaptosomes, were resuspended in 18 mL ice-cold
Milli-Q water for 5 min with seven strokes of the Teflon
pestle homogenizer. Osmolarity was restored by imme-
diate addition of 2 mL of 25 mM HEPES and 100 mM
K2-tartrate buffer (pH 7.5). Samples were centrifuged
at 20,000g for 20 min. MgSO4 (final concentration,
1 mM) was added to the resulting supernatants. Final
centrifugations were performed at 100,000g for 45 min.
Pellets were resuspended immediately in ice-cold buffer
(25 mM HEPES, 100 mM K2-tartrate, 5 mM MgSO4,
0.1 mM EDTA, and 0.05 mM EGTA, pH 7.5, 25 ꢁC)
providing ꢀ15 lg protein/100 lL. [3H]DTBZ binding
to synaptic vesicle membranes was performed according
to previously described procedures.31 Briefly, 100 lL of
vesicles suspension was incubated in assay buffer (see
above) in the presence of 5 nM [3H]DTBZ and 1 nM–
1 mM lobeline analogues (final concentrations) for
30 min at room temperature. Nonspecific binding was
determined in the presence of 20 lM tetrabenazine
(TBZ). Assays were performed in duplicate using Unifil-
ter 96-well GF/B filter plates (presoaked in 0.5%
polyethylenimine) and terminated by harvesting using
the FilterMate harvester. After washing 5 times with
350 lL of the ice-cold wash buffer (25 mM
HEPES, 100 mM K2-tartrate, 5 mM MgSO4, and
10 mM NaCl, pH 7.5), filter plates were dried,
bottoms-sealed and each well filled with 40 lL Micro-
Scint 20 cocktail (Packard). Bound [3H]DTBZ was
measured using a Packard TopCount NXT scintill-
ation counter and a Packard Windows NT-based
operating system. Data were analyzed as described
above.
4.7. [3H]NIC and [3H]MLA binding assay
For the [3H]NIC and [3H]MLA binding assays, rat
whole brain (excluding cerebellum) from two to four
rats was homogenized using a Tekmar polytron (Tek-
mar-Dohrmann, Mason, OH) in 10 vol of ice-cold mod-
ified Krebs-HEPES buffer (20 mM HEPES, 118 mM
NaCl, 4.8 mM KCl, 2.5 mM CaCl2, and 1.2 mM
MgSO4, pH 7.5). Homogenates were incubated in a
water bath for 5 min at 37 ꢁC and centrifuged (16,000g
for 17 min at 4 ꢁC). Resulting pellets were resuspended
in 20 vol of ice-cold MilliQ water (Millipore Corpora-
tion, Molsheim, France), incubated for 10 min at
37 ꢁC and centrifuged (16,000g for 17 min at 4 ꢁC). Fi-
nal pellets were stored at À20 ꢁC in Krebs-HEPES buf-
fer until use. Prior to assay, pellets were resuspended in
20 vol of Krebs-HEPES buffer. Samples (100 lL con-
taining 100–140 lg of membrane protein) were added
to assay tubes containing analogue (7–9 concentrations,
1 nM–1 mM) and 3 nM [3H]NIC or [3H]MLA for a final
assay vol of 200–250 lL, and incubated for 90 and
120 min, respectively. Nonspecific binding was deter-
mined in the presence of 10 lM NIC for the [3H]NIC
binding assay and in the presence of 10 lM MLA or
1 mM NIC for the [3H]MLA binding assays. Reactions
were terminated by addition of ice-cold buffer and rapid
filtration onto either Whatman GF/B glass fiber filters
presoaked in 0.5% polyethylenimine using a Brandel
Harvester (Biomedical Research and Development Lab-
oratory, Inc., Gaithersburg, MD or onto Unifilter-96
GF/B 96-well filter plates also presoaked in 0.5% poly-
ethylenimine using a Packard Filter Mate Harvester
(Packard BioScience Co., Meriden, CT). Bound radio-
activity was determined via liquid scintillation spectro-
metry. Specific [3H]NIC and [3H]MLA binding were
determined by subtracting nonspecific binding from to-
tal binding. Concentrations of inhibitor that produced
50% inhibition (IC50 values) were determined from the
concentration effect curves via an iterative curve-fitting
program (Prism 3.0; GraphPad Software Inc., San Die-
go, CA). Inhibition constants (Ki values) were deter-
mined using the Cheng–Prusoff equation.30
Acknowledgements
This research was supported by NIH grants DA
00399 and DA 13519. The authors also grate-
fully acknowledge the generous gift of [3H]DTBZ
from Dr. Michael R. Kilbourn (supported by NIH
Grant MH 47611). The authors also thank Mr. Robert
King of the Environmental Research and Training
Laboratory, University of Kentucky for performing
the elemental analyses. For purposes of full dis-
closure, the University of Kentucky holds patents on
lobeline and lobeline analogues, which have been
licensed by Yaupon Therapeutics Inc. (Lexington,
KY). A potential royalty stream to G.Z., L.P.D.,
M.D.J., and P.A.C. may occur consistent with Univer-
sity of Kentucky policy. Both L.P.D. and P.A.C. are
founders of, and have financial interest in Yaupon Thera-
peutics Inc.

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3909
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