N-hydroxyalkyl derivatives of 3β-phenyltropane and 1-methylspiro[1H- indoline-3,4'-piperidine]: Vesamicol analogues with affinity for monoamine transporters

Article abstract of DOI:10.1021/jm970326r

As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol [vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3β-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands.

Full text of DOI:10.1021/jm970326r

J . Med. Chem. 1997, 40, 3905-3914
3905
N-Hyd r oxya lk yl Der iva tives of 3â-P h en yltr op a n e a n d
1-Meth ylsp ir o[1H-in d olin e-3,4′-p ip er id in e]: Vesa m icol An a logu es w ith Affin ity
for Mon oa m in e Tr a n sp or ter s
Simon M. N. Efange,*,† Ashok P. Kamath,† Anil B. Khare,† Mei-Ping Kung,‡ Robert H. Mach,§ and
Stanley M. Parsons∇
Departments of Radiology, Medicinal Chemistry, and Neurosurgery and Graduate Program in Neuroscience, University of
Minnesota, Minneapolis, Minnesota 55455, Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania
19104, Departments of Radiology and Physiology & Pharmacology, Bowman Gray School of Medicine, Wake Forest University,
Winston-Salem, North Carolina 27157, and Department of Chemistry and Graduate Program in Biochemistry and Molecular
Biology, University of California, Santa Barbara, California 93106
Received May 15, 1997X
As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter
ligand 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives
of 3â-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4′-piperidine], 7, were synthesized and
tested for binding in vitro. Although a few compounds displayed moderately high affinity for
the vesicular acetylcholine transporter, no compound was more potent than the prototypical
vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed
higher affinity for the dopamine transporter than cocaine. We conclude that modification of
the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter
ligands.
Ch a r t 1
In tr od u ction
Newly synthesized acetylcholine is actively trans-
ported from its site of synthesis in the cytosol into
synaptic vesicles by
a
vesicular acetylcholine
transporter1-3 which is localized exclusively in cholin-
ergic neurons.4,5 As the synaptic vesicle is the primary
vehicle for impulse-mediated release of acetylcholine,
vesicular transport of acetylcholine constitutes an im-
portant component of neurotransmission at the cholin-
ergic synapse. Consequently, vesicular acetylcholine
transporter ligands will affect acetylcholine storage and
related aspects of cholinergic function.
The tertiary amino alcohol 2-(4-phenylpiperidinyl)-
cyclohexanol (vesamicol, AH5183, 1; Chart 1) binds to
the vesicular acetylcholine transporter with moderately
high affinity.6,7 In laboratory animals, 1 causes respira-
tory paralysis, convulsions, and death.8,9 The pharma-
cological actions of this molecule have been attributed
to inhibition of acetylcholine transport into synaptic
vesicles and the subsequent quantal release of acetyl-
choline.1-3 However, vesamicol also displays R-adreno-
ceptor activity10 and high affinity for σ1 and σ2 recep-
tors.11 Consequently, a number of efforts have been
launched to develop more selective ligands for the
vesicular acetylcholine transporter.
Previously,6,12,13 we and others have shown through
the synthesis of analogues such as 3-5 that the con-
formational restriction of selected fragments of 1 can
yield vesicular acetylcholine transporter ligands of
comparable or higher potency than vesamicol. In the
* Address inquiries to this author at Department of Radiology, Mayo
Box 292, UMHC, 420 Delaware St. S.E., Minneapolis, MN 55455. Tel:
(612) 626-6646. Fax: (612) 624-8495. E-mail: efang001@
maroon.tc.umn.edu.
present communication, we report our attempts to
extend the conformational restriction strategy by re-
placing the 4-phenylpiperidyl fragment of 1 with the 3â-
phenyltropanyl fragment 6. We also report our at-
tempts to develop a new series of vesicular acetylcholine
transporter ligands from the spirobase 7.
† University of Minnesota.
‡ University of Pennsylvania.
§ Wake Forest University.
∇ University of California.
X Abstract published in Advance ACS Abstracts, November 1, 1997.
S0022-2623(97)00326-9 CCC: $14.00 © 1997 American Chemical Society

3906 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Sch em e 1. Synthesis of N-Hydroxyalkyl/aryltropanesa
Efange et al.
a
(a) Ethyl bromoacetate, NaH, THF, rt; (b) Na, urea, EtOH, reflux; (c) LiAlH4, THF, reflux; (d) substituted epoxide, EtOH, reflux; (e)
K2CO3, EtOH, reflux; (f) HCl(g), EtOAc, 0 °C; (g) 4-fluoro- or 3-iodobenzyl bromide, K2CO3, DMF, rt.
Ch em istr y
Sch em e 2. Synthesis of Spiro[indoline-1,4′-piperidine]
Analoguesa
The target compounds were synthesized as outlined
in Schemes 1 and 2. For the first series of target
compounds, 3â-phenyltropane 6 was synthesized from
3-phenyltropidine as described.14 The reaction of 6 with
various epoxides provided the corresponding amino
alcohols 10a -k (Chart 2) in low to moderate yields. In
contrast to 4-phenylpiperidine, the reaction of 6 with
these epoxides was generally sluggish, requiring pro-
longed heating. For the piperidinols 10c,d , 1,2,3,6-
tetrahydropyridine was converted to the BOC-protected
derivative as previously described,16 and the latter was
reacted with mCPBA to generate the requisite epoxide.
As reported earlier,16 the presence of the tert-butoxy-
carbonyl protecting group favors nucleophilic attack at
the C4 position of the epoxide, resulting in a predomi-
nance of the desired regioisomer.
For the second series of compounds, 1-methylspiro-
[indoline-1,4′-piperidine] dihydrochloride 7 was pre-
pared as elaborated in Scheme 2. The reaction of
1-methyl-2-oxindole with a large excess of ethyl bro-
moacetate provided a 2:1 mixture of the di- and
monoalkylated products from which 15 was separated
by HPLC in 28% yield. Base-catalyzed reaction of the
latter with urea provided the imide 16 which was
subsequently reduced with LiAlH4 to yield 7 in an
overall yield of 7%. The target amino alcohols were
subsequently obtained by reaction of 7 with the corre-
a
(a) PhLi, ether; (b) 48% HBr, rt; (c) i. vinyl chloroformate, ii.
sponding epoxides. Compounds 20a and 21a (Chart 3)
2 N HCl, EtOH, reflux; (d) 10% Pd-C/H2, EtOH; (e) epoxide,
were obtained as described for 10c,d , respectively.
Yields were typically low, even with long reaction times,
suggesting that both the tropane 6 and the spirobase 7
EtOH, reflux; (f) NaHCO3, aq EtOH, reflux; (g) KI,CH3CN, Et3N,
reflux; (h) 3 N HCl, EtOH, reflux; (i) NaBH4, EtOH; (j) HCl(g),
EtOAc, 0 °C; (k) K2CO3, EtOH, reflux.

New Vesamicol Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3907
Ch a r t 2
are significantly less nucleophilic than 4-phenylpiperi-
dine under these conditions. Reaction of 7 or 2,3-
dihydrospiro[indene-1,4′-piperidine] with the relevant
epoxides provided compounds 22a ,b and 23a ,b in low
to moderate yield. These two spirobases were also used
to synthesize 24a ,b using the procedure outlined for 13
(Scheme 1).
of 1-methylspiro[1H-indoline-3,4′-piperidine] (7) as con-
formationally restricted vesamicol analogues. The lat-
ter study is an extension of our previous investigation13
of spirovesamicol analogues such 3-5. The target
compounds were tested for binding to the vesicular
acetylcholine transporter of Torpedo synaptic vesicles.
Binding to σ receptors was also evaluated because
vesamicol and some of its analogues display moderate
to high affinity for these sites.11 The compounds were
also tested for binding to monoamine transporters
because the 3â-phenyltropanyl moiety is found in many
inhibitors of monoamine reuptake.17-20 Finally, the
affinity of these compounds for dopamine D2 receptors
was evaluated because of behavioral effects in rodents
(data not shown). Since the reference ligand [125I]-
NCQ298 does not discriminate between dopamine D2
and D3 receptors, the data obtained with this radioli-
gand reflect binding to both dopamine receptor sub-
types.
Resu lts a n d Discu ssion
For the purpose of this discussion, the structure of
vesamicol has been divided into three major frag-
ments: A, B, and C (Chart 1). Although many vesa-
micol analogues have been synthesized in an effort to
expand our understanding of the structure-activity
relationships for binding to the vesicular acetylcholine
transporter, the vast majority contains an unmodified
B fragment, and the rest contains only single-point
modifications of this fragment. Consequently, little is
known about the interaction between this region of the
molecule and the vesicular acetylcholine transporter. In
previous studies,6,12,13 we and others utilized a strategy
of conformational restriction (in fragment A and/or C)
to develop selective high-affinity ligands for the vesicu-
lar acetylcholine transporter. To test the limits of this
strategy, we chose to extend our studies to fragment B.
In our first attempt, we replaced the piperidyl frag-
ment B with a tropanyl moiety. Formally, tropane may
be regarded as 2,6-ethanopiperidine. Since the ethylene
bridge prevents the interconversion of piperidine con-
formers, the tropanyl fragment may be regarded as a
conformationally restricted piperidyl residue. In a
parallel effort, we also synthesized several derivatives
Although many of the tropane analogues displayed
moderate affinity for the vesicular acetylcholine trans-
porter, none of the new compounds was more potent
than vesamicol. In fact, compared to the corresponding
piperidyl analogues, most of the new compounds fared
rather poorly. Thus, while compound 10a displayed
4-fold lower affinity than vesamicol, 10b-d were at
least 2 orders of magnitude less potent than benzo-
vesamicol (2), 25, and 26, respectively (Table 1). These
disparities persisted even when the cyclohexyl moiety
was replaced with a hydroxyethyl group (27 vs 10f-k )
but disappeared when the N-alkyl substituent became
more flexible (28 vs 10e). Taken together, the foregoing

3908 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Efange et al.
Ch a r t 3
suggests that the two-carbon bridge (which converts the
piperidyl into a tropanyl fragment) is profoundly det-
rimental to the ligand-receptor interaction. As the two-
carbon bridge increases the steric volume of fragment
B, one plausible explanation for the adverse effect of
this bridge may be that fragment B fits into a narrow
pocket within the binding site.
While some of the compounds caused a reduction in
locomotor activity in animals, none of the hydroxylalkyl
derivatives displayed even moderate affinity for dopam-
ine D2/D3 receptors. However, moderate to high affin-
ity was observed with the butyrophenones 12 and 24a ,b.
The latter observation is not particularly surprising,
given the striking similarity between these compounds
and the potent dopamine antagonists spiperone and
haloperidol.24 The latter also displays high affinity for
σ receptors.21
Among the phenyltropanes, all analogues containing
a cyclic B fragment (cyclohexyl or piperidyl) displayed
poor affinity for the dopamine transporter (DAT). In
contrast, all analogues lacking the cylohexyl group
displayed moderate to high affinity for the DAT and
serotonin transporter (SERT) and higher affinity for the
DAT than (-)-cocaine. With the respect to N-substitu-
tion, the moderate to high affinity of these compounds
for the DAT is consistent with the previously25 observed
bulk tolerance of the DAT around the nitrogen atom of
cocaine. However, the absence of a C2 substituent
requires further comment.
Although spirovesamicols such as 3-5 display com-
parable or higher affinity for the vesicular acetylcholine
transporter than vesamicol, all analogues of 7 were
significantly less potent than vesamicol. Moreover, the
new compounds generally displayed 1-2 orders of
magnitude lower potency than the corresponding carbon
analogues (compare 18a vs 4, 19a vs 19b, 20a vs 20b,
and 21a vs 21b). While the disparity between the two
series of compounds was obscured by the absence of a
cyclohexyl group (compare 22a vs 22b and 23a vs 23b),
it is clear that 7 is not a suitable replacement for the
A-B fragment in vesamicol.
All compounds tested showed moderate to weak
affinity for both σ1 and σ2 receptors. In addition,
several of the analogues (10e,g,h ,j,k , 22a , and 23b)
displayed 6-20-fold higher affinity for σ2 receptors than
σ1 sites. The moderate affinity of these compounds is
consistent with previous findings11 and the view that
the 4-phenylpiperidyl fragment constitutes an impor-
tant recognition element for the σ receptor.21-23
Previous studies of 3â-aryl-2â-carbomethoxytropanes
(the WIN series of cocaine analogues) clearly demon-
strate that replacement of the 2â-carbomethoxy group
with a halovinyl17 or propionyl18 moiety does not ad-
versely affect affinity for the DAT. While the present

New Vesamicol Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3909
Ta ble 1. Relative Affinities (Ki ( SEM, nM) of Vesamicol Analogues at Selected Receptors
compd
VR
σ1
σ2
D2/D3
>38000
3385 ( 429
8795 ( 2339
1967 ( 357
1490 ( 399
DAT
SERT
10a
10b
10c
10d
10e
10f
10 g
10h
10i
10j
10k
11
8.5 ( 2.7
8.5 ( 0.94
325 ( 49
14.5 ( 3.1
10.3 ( 4.4
474 ( 150
130 ( 30
298 ( 106
144 ( 43
411 ( 170
60 ( 8.3
380 ( 36
175 ( 21.4
118 ( 18
175 ( 12.4
260.1 ( 4.2
275.1 ( 21.6
4.95 ( 0.34
32.7 ( 9.3
7.71 ( 1.12
9.87 ( 0.03
11.28 ( 1.85
15.87 ( 3.74
18.8 ( 2.6
73.33 ( 8.52
15.36 ( 1.87
2813 ( 316
1066 ( 131
4183 ( 500
1069 ( 143
306 ( 20
41.55 ( 7.1
67.25 ( 24.2
23.6 ( 3.2
73.8 ( 11
3422 ( 286
1673 ( 221
9201 ( 2313
4434 ( 178
234 ( 20
240.3 ( 44.9
194.5 ( 32.2
30.89 ( 6.72
40.4 ( 11.3
95.69 ( 11.40
117 ( 24.2
2658 ( 1051
5092 ( 2062
27 ( 3
30.05 ( 0.75
17.49 ( 1.94
302.4 ( 51.7
249.3 ( 38.2
280.75 ( 15.29
34.10 ( 9.49
42.05 ( 2.0
23.65 ( 3.1
221.7 ( 34.2
129 ( 6.7
3400 ( 3000
594 ( 94
12
13
16.3 ( 6.5
88.55 ( 8.77
380 ( 115
74 ( 9.9
463.6 ( 77.2
18a
19a
20a
21a
22a
23a
24a
4
19b
20b
21b
22b
23b
24b
25
286.4 ( 35.1
357.3 ( 23.7
>1000
77 ( 9.5
>1000
>1000
>1000
>1000
>1000
33 ( 4.8
17 ( 2.2
121.8 ( 14.8
191.0 ( 9.2
517.7 ( 46.9
110.4 ( 29.6
21 ( 4
139.2
420 ( 39
22.9 ( 3.1
99.2 ( 14.2
60.8
182 ( 32
570 ( 61
56.5 ( 24.5
7.60 ( 0.99a
0.36 ( 0.15a
0.36 ( 0.10a
0.67 ( 0.19a
86 ( 14
30 ( 8
>1000
>1000
>1000
>1000
>1000
>1000
25 ( 1
283 ( 20
8.5 ( 0.9
106.8 ( 22.5
1.8 ( 0.1
67 ( 7
224 ( 49
8.8 ( 3.6
12.7 ( 1.6
3.3 ( 0.2
600 ( 49
85 ( 17
25.3 ( 5.4
0.44 ( 0.11b
0.13 ( 0.03b
30 ( 5c
26
27
28
170 ( 20c
2.0 ( 1.0d
0.055 ( 0.01d
(()-vesamicol
benzovesamicol
26 ( 8
34 ( 2
a
b
d
Reference 13. Reference 11. c Reference 15. Reference 6. KD values for (-)-cocaine, haloperidol, [125I]IPT (DAT), [125I]IPT (SERT),
and [125I]NCQ298 (D2/D3) are 373 ( 150, 1.21 ( 0.2, 0.25, 1.2, and 0.05 nM, respectively.
thesis (Windham, MA) and used as received. Tetrahydrofuran
(THF) was distilled from sodium hydride immediately prior
to use. All other reagents and solvents were purchased as
reagent grade and used without further purification.
study would appear to go even further by suggesting
that a C2 substituent is not required for binding to the
DAT, the situation appears to be significantly more
complex.
Removal of the 2â-carbomethoxy group of (-)-cocaine
results in a 50-fold reduction in affinity for the DAT.26
On the other hand, both 3R-(diphenylmethoxy)tro-
panes27,28 and 3R-(diphenylmethoxy)-2â-carbomethoxy-
tropanes29 display high affinity for the DAT. Taken
together, the foregoing suggests that the requirement
for a C2 substituent is partly governed by the C3
substituent. Previous investigators29 appear to have
arrived at a similar conclusion.
In summary, 3â-phenyltropanyl derivatives of vesa-
micol exhibit lower affinity for the vesicular acetylcho-
line transporter than the parent compound. Conse-
quently, the introduction of a two-carbon bridge across
the C2 and C6 positions of the piperidyl moiety of
vesamicol is not a suitable strategy for enhancing
affinity for the vesicular acetylcholine transporter or
increasing selectivity for the vesicular acetylcholine
transporter relative to σ receptors. The introduction of
an aminomethyl bridge into the C fragment of vesamicol
(to yield analogues of 7) is also unsuitable for similar
reasons. Since the results obtained with the tropanes
suggest that fragment B binds to a narrow pocket within
the binding site, we suggest that further modification
of this fragment should be limited to single-point
substitution.
All air-sensitive reactions were carried out under nitrogen.
Standard handling techniques for air-sensitive materials were
employed throughout this study. Yields were not optimized.
Melting points were determined on a Haake-Buchler or Mel-
Temp melting point apparatus and are uncorrected. 1H NMR
spectra were recorded on a 200-MHz IBM-Brucker spectrom-
eter or
a 300-MHz GE spectrometer. NMR spectra are
referenced to the deuterium lock frequency of the spectrometer.
With this condition, the chemical shifts (in ppm) of residual
solvents are observed at 7.26 (CHCl3) and 4.78 (CD3OH). The
following abbreviations are used to describe peak patterns
wherever appropriate: b ) broad, d ) doublet, t ) triplet, q
) quartet, m ) multiplet. Preparative chromatography was
performed on a Harrison Research Chromatotron using Merck
60 PF254 silica gel or a preparative HPLC system (Rainin
Instrument Co.) using a 41.1-mm i.d. Dynamax silica gel
column (delivering solvent at 80 mL/min). Analytical TLC was
carried out on Analtech GHLF silica gel glass plates, and
visualization was aided by UV and/or methanolic iodine.
P r oced u r e A. Rep r esen ta tive P r oced u r e for th e Syn -
th esis of Ep oxid es. 3-Chloroperbenzoic acid (3.71 g of 57%
mCPBA, 9.8 mmol) was added portionwise over a 15-min
period to a cold (ice bath) stirring solution of 4-bromostyrene
(1.5 g, 8.2 mmol) in CH2Cl2 (50 mL). The reaction mixture
was gradually warmed to room temperature and allowed to
stir for 15 h, at which time it was diluted with CCl4 (50 mL),
and the precipitated 3-chlorobenzoic acid was removed by
suction filtration. The filtrate was washed with a 1:1 mixture
of 5% aqueous NaHCO3 and 5% aqueous NaHSO3 (3 × 25 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure to yield the epoxide as a colorless liquid (1.48 g, 91%).
Exp er im en ta l Section
Gen er a l. Synthetic intermediates were purchased from
P r oced u r e B. Syn th esis of Vicin a l Am in o Alcoh ols.
Aldrich Chemical Co. (Milwaukee, WI) and Lancaster Syn-
tr a n s-2-(3â-P h en yltr opan -8-yl)cycloh exan ol (10a). A mix-

3910 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Efange et al.
ture of 3â-phenyltropane hydrochloride (0.57 g, 2.55 mmol) and
cyclohexene oxide (0.32 g, 3.31 mmol) was refluxed in EtOH
(15 mL) and Et3N (1 mL) for 5 days. (Subsequent reactions
were only refluxed for 20 h.) Solvent was evaporated under
reduced pressure, and the residue was purified by radial flow
chromatography [hexane(9)-acetone(1)] to yield 0.3 g (37%)
of free base. The corresponding hydrochloride was obtained
by bubbling dry HCl(g) through a cold solution of the free base
in MeOH followed by concentration of the resulting acidic
solution and subsequent recrystallization from i-PrOH-
Et2O: mp 234-236 °C; 1H NMR (CDCl3) δ 1.17-1.73 (m, 8H,
cyclohexyl), 2.12-3.78 (m, 13H, cyclohexyl methine Hs, tro-
panyl Hs), 4.67 (s, 1H, OH), 7.15-7.29 (m, 5H, phenyl). Anal.
(C19H27NO‚HCl‚H2O) C, H, N.
tr a n s-2-H yd r oxy-3-(3â-p h en ylt r op a n -8-yl)-1,2,3,4-t et -
r a h yd r on a p h th a len e h yd r och lor id e (10b): procedure B,
yield 8%; mp 228-231 °C (i-PrOH-Et2O); 1H NMR (CDCl3) δ
1.61-1.91 (m, 4H, tropanyl), 2.41-3.72 (m, 15H, tropanyl,
tetrahydronaphthyl Hs), 4.47 (m, 1H, N-CH-), 4.89 (bs, 1H,
OH), 6.10-7.31 (m, 9H, phenyl). Anal. (C23H27NO‚HCl‚0.5
H2O) C, H, N.
1-(4-Br om op h en yl)-3-(3â-p h en ylt r op a n -8-yl)p r op a n -
2-ol h yd r och lor id e (10e): procedure B, yield 26%; mp 221-
223 °C; 1H NMR (CHCl3) δ 1.52-2.46 (m, 12H), 2.72-3.29 (m,
3H), 3.51-3.78 (m, 1H, CH-OH), 4.38 (bs, 1H, OH), 7.16 (d,
2H, 4-Br Ar-H), 7.25-7.37 (m, 5H, Ar-H), 7.42 (d, 2H, 4-Br
Ar-H). Anal. (C22H26NOBr‚HCl‚1/4H2O) C, H, N.
1-P h en yl-2-(3â-p h en yltr op a n -8-yl)eth a n ol h yd r och lo-
r id e (10f): procedure B, yield 0.26 g (45%); mp 179-182 °C;
1H NMR (CDCl3) δ 1.54-2.65 (m, 9H), 3.11-3.60 (m, 4H), 4.66
(dd, 1H, CH-OH), 4.77 (bs, 1H, OH), 7.18-7.39 (m, 10H, Ar-
H). Anal. (C21H26NOCl‚0.25 H2O) C, H, N.
2H), 3.40 (m, 1H), 4.55 (dd, 1H, CH-OH), 4.58 (bs, 1H, OH),
7.18-7.49 (m, 8H, Ar-H). Anal. (C21H24NOCl3) C, H, N.
tr a n s-4-H yd r oxy-3-(3â-p h en ylt r op a n -8-yl)p ip er id in e
Dih yd r och lor id e (29). A mixture of 3â-phenyltropane hy-
drochloride (1.14 g, 6.1 mmol), 2.15 g (7.6 mmol) of the isomeric
trans-bromohydrins 1-(tert-butoxycarbonyl)-3(4)-bromo-4(3)-
hydroxypiperidine,16 and potassium carbonate (1.68 g, 14.0
mmol) was refluxed in ethanol (50 mL) for 4 days. After
cooling, the insoluble material was removed by filtration, and
the filtrate was concentrated to yield a tan residue. The latter
was passed through a short column of silica gel using 25%
acetone-hexane (plus trace Et3N). The eluent was concen-
trated, and the residue thus obtained was purified by HPLC
(5% i-PrOH-hexane, plus trace Et3N) to afford 0.24 g (10%)
of the title compound as a clear syrup: 1H NMR (CDCl3) δ
1.43 (s, 9H, BOC), 1.5-1.95 (m, 6H, piperidyl CH2), 2.13-3.15
(m, 8H, piperidyl CH2), 3.38-3.56 (m, 3H, piperidyl CH), 3.85-
4.2 (bs, 3H, OH, CH-N, CH-OH), 7.18-7.36 (m, 5H, Ar-H).
Similar yields were obtained from subsequent runs. For
removal of the butoxycarbonyl protecting group, a stirring
solution of this intermediate (1.0 g, 0.25 mmol) in EtOAc was
cooled in an ice bath, and dry HCl(g) was bubbled vigorously
through it for 30 min. Stirring and cooling were continued
for an additional 30 min, at which time the mixture was
concentrated under reduced pressure. Coevaporation of the
residual solvent with toluene (3 × 25 mL) and subsequent
drying yielded crude 29 as a white solid (0.91 g, 99%). The
latter was used without further purification.
3,4-tr a n s-1-(4-F lu or ob en zyl)-4-h yd r oxy-3-(3â-p h en yl-
tr op a n -8-yl)p ip er id in e Dih yd r och lor id e (10c). 4-Fluo-
robenzyl bromide (0.26 g, 1.37 mmol) was added to a mixture
of 29 (0.41 g, 1.14 mmol) and anhydrous potassium carbonate
(0.4 g, 2.85 mmol) in DMF (20 mL). The resulting mixture
was stirred at room temperature for 18 h, diluted with
methylene chloride (25 mL), and filtered. The filtrate was
concentrated under reduced pressure and the tan residue
purified by radial flow chromatography using a mobile phase
1-(2-Br om oph en yl)-2-(3â-ph en yltr opan -8-yl)eth an ol h y-
d r och lor id e (10 g): procedure B: yield 49%; mp 184-188 °C;
1H NMR (CDCl3) δ 1.51-2.53 (m, 8H), 2.81 (dd, 2H, bridge
CH), 3.03-3.19 (m, 2H), 3.58 (t, 1H), 4.72 (bs, 1H, OH), 5.01
(dd, 1H, CH-OH), 7.12-7.70 (m, 9H, Ar-H). Anal. (C21H24
-
NOBr‚HCl‚1/4H2O) C, H, N.
N) to afford 10c as a
of 15% acetone-hexane (plus trace Et3
1-(3-Br om oph en yl)-2-(3â-ph en yltr opan -8-yl)eth an ol h y-
d r och lor id e (10h ): procedure B, yield 27%; mp 171-173 °C;
1H NMR (CDCl3) δ 1.54-2.71 (m, 8H), 3.06-3.57 (m, 4H), 4.58
(dd, 2H, CH-OH), 4.81 (bs, 1H, OH), 7.18-7.56 (m, 9H, Ar-
H). Anal. (C21H25NOBrCl‚0.5H2O) C, H, N.
1-(4-Br om oph en yl)-2-(3â-ph en yltr opan -8-yl)eth an ol h y-
d r och lor id e (10i): procedure B, yield 35%; mp (MeOH-Et2O)
200-202 °C; 1H NMR (free base) (CDCl3) δ 1.49-2.63 (m, 8H),
3.05-3.55 (m, 4H), 4.58 (dd, 2H, CH-OH), 4.81 (bs, 1H, OH),
7.16-7.35 (m, 7H, Ar-H), 7.49 (d, 2H, 4-Br Ar-H). Anal.
(C21H25NOBrCl) C, H, N.
solid (0.27 g, 64%). The free base was converted to the
corresponding hydrochloride with cold methanolic HCl, and
recrystallization was performed in i-PrOH-ether: mp 183-
186 °C; 1H NMR (CDCl3) δ 1.53-2.10 (m, 10H, piperidinyl
CH2), 2.41-2.50 (m, 3H, piperidinyl CH2, CH), 2.70-3.1 (m,
3H, piperidinyl CH2, CH), 3.2-3.49 (m, 5H, benzyl CH2,
piperidinyl CH, OH), 4.22 (s, 1H), 6.94-7.31 (m, 9H, Ar-H).
Anal. (C25H33N2OFCl2‚0.5H2O) C, H, N.
3,4-tr a n s-1-(3-Iod oben zyl)-4-h yd r oxy-3-(3â-p h en yltr o-
p a n -8-yl)p ip er id in e Dih yd r och lor id e (10d ). Alkylation of
29 with 3-iodobenzyl bromide as described for 10c above
provided an 89% yield of 10d as a pale oil. Conversion to the
hydrochloride and subsequent recrystallization were per-
formed as described above: mp 180-184 °C; 1H NMR (CDCl3)
δ 1.55-2.10 (m, 10H, piperidinyl CH2), 2.39-3.15 (m, 6H,
piperidinyl CH2, CH), 3.2-3.49 (m, 5H, benzyl CH2, piperidinyl
CH, OH), 4.20 (s, 1H), 7.02-7.31 (m, 7H, Ar-H), 7.57-7.65
(m, 2H, Ar-H). Anal. (C25H33N2OICl2‚0.5H2O) C, H, N.
8-[(4-F lu or oben zoyl)m eth yl]-3â-p h en yltr op a n e Hyd r o-
ch lor id e (11). A mixture of 3â-phenyltropane (0.4 g, 2.14
mmol), 2-bromo-4′-fluoroacetophenone (0.56 g, 2.57 mmol), and
NaHCO3 (0.91 g, 8.6 mmol) was refluxed in 25% aqueous EtOH
(40 mL) for 17 h. The reaction mixture was cooled to room
temperature, diluted with water (75 mL), and extracted with
CH2Cl2 (2 × 50 mL). The combined organic extracts were dried
(Na2SO4) and concentrated under reduced pressure to a dark
yellow residue. Purification of the crude product by radial flow
chromatography [hexane(80)-acetone(20)-Et3N(1)] afforded
0.35 g (50%) of the title compound as a pale yellow syrup: 1H
NMR (CDCl3) δ 1.48-1.61 (m, 4H, tropane CH2-CH2), 2.02-
2.06 (m, 2H, tropane CH2-CH-CH2), 2.39-2.58 (m, 2H, tropane
CH2-CH-CH2), 3.07-3.12 (m, 1H, tropane CH-Ph), 3.35 (m, 2H,
bridge CH), 3.72 (s, 2H, N-CH2), 7.09-7.28 (m, 7H, Ar-H),
8.10-8.17 (m, 2H, fluorophenyl Ar-H). The hydrochloride was
prepared in methanolic HCl and recrystallized from MeOH-
Et2O: mp 121-124 °C. Anal. (C21H23NOClF‚3/4H2O) C, H, N.
3-(4-F lu o r o b e n zo y l)-1-(3â-p h e n y lt r o p a n -8-y l)p r o -
p a n e (12). A mixture of 4-chloro-1,1-ethylenedioxy-1-(4-
1-(2,6-Dich lor op h en yl)-2-(3â-p h en ylt r op a n -8-yl)et h a -
n ol h yd r och lor id e (10j): procedure B, yield 28%; mp 180-
1
182 °C; H NMR (CDCl3) δ 1.48-3.40 (m, 13H), 4.38 (bs, 1H,
OH), 5.47 (dd, 1H, CH-OH), 7.11-7.31 (m, 8H, Ar-H). Anal.
(C21H24NOCl3‚1.5H2O) C, H, N.
1-(3,4-Dich lor op h en yl)-2-(3â-p h en ylt r op a n -8-yl)et h a -
n ol h yd r och lor id e (10k ). Sodium borohydride (2.0 g, 53
mmol) was added portionwise to a solution of 3′,4′-dichloro-
acetophenone (2.5 g, 13 mmol) in MeOH (30 mL). After
completion of addition the mixture was stirred at room
temperature for 1 h, and the solvent was removed in vacuo.
The residue was partitioned between CH2Cl2 (50 mL) and
water (50 mL); the organic layer was separated, dried (Na2-
SO4), and concentrated under reduced pressure to afford the
desired alcohol (2.25 g, 89%) as a chromatographically homo-
geneous sample. The latter was combined with p-TsOH (0.23
g, 1.2 mmol) and benzene (50 mL), and the mixture was
refluxed for 4 h with azeotropic distillation of water. The
reaction mixture was subsequently cooled to room tempera-
ture, washed with 5% aqueous NaHCO3 (3 × 25 mL), dried
over anhydrous Na2SO4, and concentrated to yield 3,4-dichlo-
rostyrene as a colorless liquid (1.5 g, 68%): 1H NMR (CDCl3)
δ 5.4 (d, 1H, -CHdCH-H), 5.8 (d, 1H, -CHdCH-H), 6.6 (dd,
1H, -CHdCH2), 7.4-7.6 (m, 3H, phenyl).
The title compound was synthesized from 3,4-dichlorosty-
rene as described in procedure B: yield 0.13 g (17%); mp 204-
206 °C; 1H NMR (CDCl3) δ 1.53-2.60 (m, 10H), 3.05-3.16 (m,

New Vesamicol Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3911
fluorophenyl)butane30 (0.52 g, 2.14 mmol), 3â-phenyltropane
(0.4 g, 2.14 mmol), KI (0.36 g, 2.14 mmol), and Et3N (3 mL)
was refluxed in MeCN (15 mL) for 22 h. After the mixture
cooled to room temperature, the solvent was evaporated in
vacuo and the residue reconstituted in CH2Cl2 (25 mL) and
washed successively with 5% aqueous NaHCO3 and water. The
organic extract was dried (Na2SO4) and concentrated in vacuo
to afford a pale brown residue of the alkylated ketal which
was sufficently pure for the next reaction. The ketal was
refluxed in 3 N HCl (10 mL) for 20 min, and the solution was
concentrated under reduced pressure. The residue was treated
with saturated aqueous NaHCO3 solution and extracted with
CH2Cl2 (2 × 25 mL). The combined organics were dried over
anhydrous Na2SO4 and concentrated under reduced pressure
to give a yellow syrup which was purified by radial flow
chromatography (15% acetone-hexane) to provide 0.36 g (48%)
of the title compound: 1H NMR (CDCl3) δ 1.38 (m, 5H), 1.85-
1.99 (m, 4H), 2.27-2.42 (m, 4H), 2.87-3.10 (m, 2H), 3.26 (m,
2H), 7.09-7.31 (m, 7H, Ar-H), 8.01-8.08 (m, 2H, p-F-phenyl
Ar-H); mp (hydrochloride) 195-198 °C. Anal. (C23H26NOF‚
HCl‚0.25H2O) C, H, N.
1-(4-Flu or oph en yl)-3-(3â-ph en yltr opan -8-yl)bu tan ol Hy-
d r och lor id e (13). To a methanolic solution of 12 (0.33 g, 0.93
M) was added NaBH4 (0.15 g, 3.72 mmol) portionwise at room
temperature. The reaction mixture was then stirred at room
temperature for an additional 15 min. The solvent was
evaporated in vacuo, the white residue dissolved in CH2Cl2
(50 mL), and the resulting solution washed with water (2 ×
25 mL). Drying of the organic extract (Na2SO4) followed by
solvent removal under reduced pressure afforded the title
compound as a pale yellow syrup (0.31 g, 94%): 1H NMR
(CDCl3) δ 1.47-2.02 (m, 9H), 2.16-2.46 (m, 5H), 3.17-3.39
(m, 3H), 4.69-4.73 (dd, 1H, CH-OH), 6.96-7.39 (m, 9H, Ar-
H), 8.51 (bs, 1H, OH); mp (hydrochloride) 130-134 °C. Anal.
(C23H28NOF‚HCl‚0.75H2 O) C, H, N.
phenyl), 7.33-7.38 (m, 1H, phenyl). Anal. (C13H11N2O2) H,
N; C: calcd, 63.93; found, 62.94.
1-Meth ylsp ir o[in d olin e-1,4′-p ip er id in e] (7). A solution
of 16 (0.60 g, 2.46 mM) in 10 mL of dry THF was added
dropwise to a stirring suspension of lithium aluminum hydride
(0.93 g, 24.6 mM) in dry THF (20 mL) cooled in an ice bath.
At the end of the addition, the reaction mixture was warmed
to room temperature, refluxed for 6 h, and cooled to room
temperature. The reaction was then quenched by dropwise
addition of water (1 mL), 15% NaOH (1 mL), and water (3
mL), consecutively. The resulting precipitate was removed by
suction filtration, washed with EtOAc (25 mL), and discarded,
while the filtrate was dried over Na2SO4 and concentrated
under reduced pressure to yield a pale brown syrup (0.46 g,
93%): 1H NMR (CDCl3) δ 1.70-1.98 (m, 4H, piperidyl), 2.74
(s, 3H, N-CH3), 2.76-2.81 (m, 2H, piperidyl), 3.07-3.23 (m,
2H, piperidyl), 3.35 (s, 2H, indoline CH2), 4.09 (bm, 1H, N-H),
6.46 (d, 1H, phenyl), 6.67-6.74 (m, 1H, phenyl), 7.08-7.14 (m,
2H, phenyl).
P r oced u r e C. Syn th esis of Sp ir oin d olin e-Con ta in in g
Am in o Alcoh ols. 1′-(1-Hyd r oxycycloh ex-2-yl)-1-m eth yl-
sp ir o[in d olin e-3,4′-p ip er id in e] Dih yd r och lor id e (18a ). A
mixture of 7 (0.49 g, 2.4 mmol), cyclohexene oxide (0.35 g, 3.2
mmol), and triethylamine (5 mL) was refluxed in absolute
ethanol (50 mL) for 3 days. The reaction mixture was cooled
to room temperature and concentrated to a residue which was
passed through a short column of silica gel [eluting with
hexane(70)-acetone(30)-Et3N(1)]. Concentration of the elu-
ent yielded a residue which was further purified by radial flow
chromatography, using a mobile phase of hexane(85)-acetone-
(15)-Et3N(1), to provide 0.40 g (55%) of the title compound.
The latter was converted to the corresponding hydrochloride
as elaborated above and recrystallized from i-PrOH-ether: mp
233-235 °C; 1H NMR (CDCl3) δ (free base) 1.17-3.70 (m, 23H),
4.09 (bs, 1H, OH), 6.45 (d, 1H, indoline Ar-H), 6.70 (t, 1H,
indoline Ar-H), 7.04-7.13 (m, 2H, indoline Ar-H). Anal. Calcd
for C19H28N2O‚2HCl ‚H2O: C, 58.31; H, 8.24; N, 7.15. Found
C, 58.42; H, 7.02; N, 6.20.
1′-(2-Hyd r oxy-1,2,3,4-tetr a h yd r on a p h th -3-yl)-1-m eth yl-
sp ir o[in d olin e-3,4′-p ip er id in e] h yd r och lor id e (19a ): pro-
cedure C, purified by radial flow chromatography [acetone-
(20)-hexane(80)-Et3N(1)], yield: 20%; 1H NMR (CDCl3) δ
1.77-1.89 (m, 6H, piperidyl), 2.75 (s, 3H, CH3), 2.78-3.38 (m,
9H, piperidyl, tetrahydronaphthyl, OH, indoline CH2), 3.71 (m,
1H, CH-N), 3.93 (m, 1H, CH-OH), 6.46 (m, 1H, indoline
phenyl), 6.71 (m, 1H, indoline phenyl), 7.07-7.50 (m, 6H,
indoline, tetrahydronaphthyl ArH). The corresponding hy-
drochloride was obtained in methanolic HCl and recrystallized
from MeOH-Et2O as a dark yellow solid: mp 242-246 °C.
Anal. (C23H30N2OCl2‚3/4H2O) C, H, N.
1′-(4-H yd r oxyp ip er id in -3-yl)-1-m et h ylsp ir o[in d olin e-
3,4′-p ip er id in e] Tr ih yd r och lor id e (17). A mixture of 7 (1.5
g, 7.5 mmol), 1-(tert-butoxycarbonyl)-3,4-epoxy-1,2,3,6-tetrahy-
dropiperidine (1.8 g, 9.0 mmol), and K2CO3 (1.15 g, 8.0 mmol)
was refluxed in absolute EtOH (50 mL) for 2 days. At this
stage another batch of epoxide (1.8 g, 9.0 mmol) was added,
and heating was continued for 1.5 days. The reaction mixture
was then cooled to room temperature, concentrated under
reduced pressure, and passed through a short silica gel column
(eluting with 25% acetone-hexane plus trace Et3N). The
above desired regioisomer was isolated as a liquid by prepara-
tive HPLC [Et3N(1)-isopropyl alcohol(3)-hexane(97); reten-
tion time, 8.95 min]: yield 0.45 g (16%);1H NMR (CDCl3) δ
1.46 (s, 9H, tert-butyl H), 1.77-2.67 (m, 12H), 2.78 (s, 3H,
N-Me), 2.84-3.74 (m, 5H), 4.06 (bs, 2H, CH-OH, OH), 6.45
(d, 1H, Ar-H), 6.70 (t, 1H, Ar-H), 7.02-7.14 (m, 2H, Ar-H).
The product (0.45 g, 1.12 mmol) was dissolved in EtOAc (40
mL) and cooled in an ice bath. Hydrogen chloride gas was
passed through this solution for 0.5 h, after which the reaction
mixture was allowed to stir under ice bath conditions for an
additional 0.5 h. Concentration of the mixture yielded a pale
yellow solid which was dried to give 0.42 g (91%) of the
product: 1H NMR (CD3OD) δ 1.93-2.60 (m, 8H), 3.14-4.20
(m, 16H), 7.56 (bs, 4H, Ar-H).
3,3-Bis(ca r beth oxym eth yl)-1-m eth yloxin d ole (15).
A
suspension of sodium hydride (28.2 g of a 50% dispersion in
mineral oil, 1.172 mol) was washed with dry THF (20 mL) and
resuspended in the same solvent (40 mL). To this stirring
suspension was added a solution of N-methyloxindole31 (43.0
g, 0.293 mol) in 50 mL of dry THF dropwise under nitrogen
for 20 min. This was followed by the dropwise addition of a
solution of ethyl bromoacetate (147.0 g, 0.88 mol) in 30 mL of
THF, after which the reaction mixture was stirred at room
temperature. for 1.15 h. The reaction was then quenched with
100 mL of saturated aqueous NH4Cl, diluted with water, and
extracted with CH2Cl2 (5 × 50 mL). The combined organic
extracts were dried over anhydrous Na2SO4 and concentrated
under reduced pressure to a syrup. The latter was applied
onto a short silica gel column which was eluted with 25%
acetone-hexane to afford 76.6 g of a product containing both
the mono- and dialkylation reaction products (approximately
1:2) along with unreacted ethyl bromoacetate. The desired
compound was separated by preparative HPLC (10% i-PrOH-
hexane with trace Et3N) in 28% yield (17.8 g): 1H NMR
(CDCl3) δ 1.01 (t, 6H, COOCH2CH3), 2.88-3.11 (bq, 4H, CH2-
CO), 3.23 (s, 3H, N-CH3), 3.84 (q, 4H, COOCH2), 6.80 (d, 1H,
phenyl), 6.95-7.33 (m, 3H, phenyl).
1-Met h yl-2,2′,6′-t r ioxosp ir o[in d olin e-1,4′-p ip er id in e]
(16). The compound was synthesized according to a previously
published procedure.32 To a refluxing solution of sodium (0.5
g, 2.2 g-atom) in absolute EtOH (35 mL) was added a solution
of 15 (3.0 g, 9.4 mmol) in absolute EtOH (15 mL) and urea
(0.85 g, 14.1 mmol). The resulting mixture was refluxed under
a N2 atmosphere for 6.5 h. After the reaction cooled to room
temperature, the volatiles were evaporated in vacuo to provide
a tan solid. This was treated with 6 N HCl (25 mL), and the
resulting mixture was extracted consecutively with CH2Cl2 (3
× 25 mL) and EtOAc (50 mL). The combined organic extracts
were dried (Na2SO4) and evaporated under reduced pressure.
The residue thus obtained was passed through a short silica
gel column using 25% acetone-hexane to give 0.62 g (27%) of
a pale brown solid: mp 199-201 °C; yields of 41% were
subsequently obtained upon scaleup; 1H NMR (CDCl3) δ 2.53
(d, 2H, CH2), 2.94 (d, 2H, CH2), 3.24 (s, 3H, N-CH3), 6.90 (d,
1H, phenyl), 7.05-7.10 (m, 1H, phenyl), 7.20-7.24 (m, 1H,
1′-[1-(4-F lu or ob e n zyl)-4-h yd r oxyp ip e r id in -3-yl]-1-
m eth ylsp ir o[in d olin e-3,4′-p ip er id in e] Tr ih yd r och lor id e

3912 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Efange et al.
(20a ). A mixture of 17 (0.21 g, 0.51 mM) and 4-fluorobenzyl
bromide (0.12 g, 0.61 mmol) was stirred in DMF (5 mL)
containing anhydrous K2CO3 (0.36 g, 2.55 mmol) at room
temperature for 18 h. The reaction mixture was diluted with
CH2Cl2 (30 mL), and the inorganics were filtered. The filtrate
was evaporated under reduced pressure and the compound
purified from the crude residue by radial flow chromatography
[acetone(20)-hexane(80)-Et3N(1)]: yield 85 mg (21%); 1H
NMR (CDCl3) δ 1.23-2.24 (m, 11H, piperidine), (m, 13H,
piperidine, N-Me, CH-OH, OH, benzyl CH2), 6.43 (d, 1H,
indoline Ar-H), 6.64 (t, 1H, indoline Ar-H), 6.99-7.25 (m, 6H,
indoline, phenyl Ar-H). The corresponding hydrochloride was
prepared in methanolic HCl and recrystallized from i-PrOH-
Et2O: mp 190-194 °C. Anal. (C25H33N3OF‚3HCl‚1.5H2O) C,
H, N.
g, 2 mmol), 1-(4-bromophenyl)propane 1,2-dioxide (0.6 g, 2.6
mmol), and anhydrous potassium carbonate (0.7 g, 5 mmol)
was refluxed in anhydrous EtOH (50 mL) for 21 h and cooled
to room temperature. Removal of the insoluble material by
filtration followed by concentration of the filtrate and purifica-
tion of the residue by radial flow chromatography (10%
acetone-hexane with trace Et3N) afforded 0.36 g (41%) of the
desired compound as a pale brown liquid: 1H NMR (CDCl3) δ
(free base) 1.25 (t, 2H, piperidyl), 1.96-3.97 (m, 16H), 7.13-
7.35 (m, 8H, Ar-H). The hydrochloride was prepared with cold
methanolic HCl and recrystallized from MeOH-Et2O: mp
196-200 °C. Anal. (C22H26NOBr‚HCl) C, H, N.
2,3-Dih yd r o-1′-[2-(3-b r om op h en yl)-2-h yd r oxyet h yl]-
spir o[1H-in den e-1,4′piper idin e] h ydr och lor ide (23b): syn-
thesized following the procedure described for 22b, yield 0.22
1
1′-[1-(3-Iod oben zyl)-4-h yd r oxyp ip er id in -3-yl]-1-m eth -
ylsp ir o[in d olin e-3,4′-p ip er id in e] Tr ih yd r och lor id e (21a ).
A mixture of 17 (0.19 g, 0.47 mmol) and 3-iodobenzyl bromide
(0.16 g, 0.55 mmol) was stirred in DMF (5 mL) containing
anhydrous K2CO3 (0.33 g, 2.35 mmol) at room temperature
for 18 h. The reaction mixture was diluted with CH2Cl2 (30
mL), and the inorganics were removed by filtration. The
filtrate was concentrated under reduced pressure and the
compound purified from the crude residue by radial flow
chromatography (20% acetone-hexane with trace Et3N): yield
0.16 g (24%); mp 225-228 °C. 1H NMR (CDCl3) δ 1.23-2.24
(m, 11H, piperidine), (m, 13H, piperidine, N-Me, CH-OH, OH,
& benzyl CH2), 6.34 (d, 1H, indoline Ar-H), 6.58 (t, 1H, indoline
Ar-H), 6.91-7.15 (m, 2H, indoline Ar-H), 7.20-7.56 (m, 4H,
phenyl Ar-H). Anal. (C25H33N3OI.3HCl‚ 0.75H2O) C, H, N.
1′-[3-(1-(3-Br om op h en yl)-2-h yd r oxyp r op yl]]-1-m eth yl-
sp ir o[in d olin e-3,4′-p ip er id in e] (22a ): procedure C, purified
by radial flow chromatography [acetone(20)-hexane(80)-
Et3N(1)], yield 0.26 g (46%); mp (hydrochloride) 209-212 °C
g (26%); mp 218-221 °C; H NMR (CDCl3) δ (free base) 1.25
(t, 2H, piperidyl), 1.96-3.62 (m, 14H), 7.24-7.56 (m, 8H, Ar-
H). Anal. (C21H24NOBr‚HCl) C, H, N.
2,3-Dih yd r o-1′-[3-(4-flu or oben zoyl)p r op yl]sp ir o[1H-in -
d en e-1,4-′p ip er id in e] (24b). A mixture of 2,3-dihydrospiro-
[1H-indene-1,4′-piperidine] hydrochloride (0.25 g, 1.2 mmol)
and 4-chloro-1,1-ethylenedioxy-1-(4-fluorophenyl)butane (0.31
g, 1.2 mmol) was refluxed in MeCN (25 mL) containing KI (0.2
g, 1.2 mmol) and Et3N (2 mL) for 23 h. After the reaction
mixture cooled to room temperature, the volatiles were re-
moved under reduced presure. The residue was redissolved
in CH2Cl2 (50 mL), and the resulting solution was washed
successively with saturated aqueous NaHCO3 (2 × 25 mL) and
water (25 mL), dried over Na2SO4, and concentrated in vacuo
to a yellow syrup. The latter was refluxed in 3 N HCl (10 mL)
for 20 min, after which the volatiles were evaporated in vacuo.
The residue was then treated with saturated aqueous NaHCO3
and extracted with CH2Cl2 (3 × 25 mL). The combined
extracts were dried (Na2SO4) and concentrated in vacuo to
provide a tan residue. The title compound was isolated as a
hydrochloride (0.12 g, 27%) by recrystallization from
MeOH-Et2O: mp 218-220 °C; 1H NMR (CD3OD) δ 1.79-2.20
(m, 7H), 2.93-3.70 (m, 11H), 7.19-7.28 (m, 6H, Ar-H), 8.06-
8.13 (m, 2H, Ar-H). Anal. (C23H26NOF.HCl‚0.5‚H2O) C, H;
N: calcd, 3.53; found, 4.10.
1
(MeOH-ether); H NMR (CDCl3) δ 1.20-3.56 (m, 17H), 3.99
(bm, 2H, OH, CH-OH), 6.45 (d, 1H, indoline Ar-H), 7.01 (t,
1H, indoline Ar-H), 7.09-7.45 (m, 6H, indoline, bromophenyl
Ar-H). Anal. (C22H27BrN2O) H; C: calcd, 63.62; found, 62.59.
N: calcd, 6.74; found, 5.74.
1′-[2-(1-(3-Br om op h en yl)-1-h yd r oxyet h yl]-1-m et h yl-
sp ir o[in d olin e-3,4′-p ip er id in e] (23a ): procedure C (26 h),
purified by radial flow chromatography [acetone(15)-hexane-
(85)-Et3N(1)], yield 27%; hydrochloride recrystallized from
MeOH-Et2O; mp 215-219 °C dec; 1H NMR (CDCl3) δ 1.78
(m, 2H, piperidyl), 1.95 (m, 2H, piperidyl), 2.43-2.60 (m, 4H,
piperidyl), 2.80 (s, 3H, N-CH3), 3.22 (s, 2H, indoline CH2), 3.60
(m, 2H, N-CH2), 4.20 (bs, 1H, OH), 4.65 (dd, 1H, CH-OH), 6.45
(d, 1H, indoline Ar-H), 6.75 (m, 1H, indoline Ar-H), 7.10-7.60
(m, 6H, indoline Ar-H, styryl Ar-H). Anal. (C21H27N2-
OBrCl2‚0.25H2O) C, H, N.
1′-[3-(4-Flu or oben zoyl)p r op yl]-1-m eth ylsp ir o[in dolin e-
3,4′-p ip er id in e] (24a ). A mixture of 4-chloro-1,1-ethylene-
dioxy-1-(4-fluorophenyl)butane30 (0.43 g, 1.7 mmol), 7 (0.35 g,
1.7 mmol), KI (0.12 g, 1.7 mmol), and Et3N (3 mL) was refluxed
in MeCN (10 mL) for 22 h. After the reaction mixture cooled
to room temperature, the solution was taken to dryness in
vacuo and the residue was reconstituted in CH2Cl2 (25 mL).
The resulting solution was washed successively with 5%
aqueous NaHCO3 and water, dried (Na2SO4), and concentrated
in vacuo to yield the alkylated ketal as a pale brown residue.
The crude ketal was refluxed in 3 N HCl (10 mL) for 30 min,
and the reaction mixture was concentrated to a residue. The
latter was treated with saturated aqueous NaHCO3 and the
resulting mixture extracted with CH2Cl2 (2 × 25 mL). The
combined organic extracts were dried and concentrated in
vacuo to provide a tan syrup which was purified by radial flow
chromatography (10% acetone-hexane, trace Et3N) to give
0.16 g (25%) of the desired compound. The hydrochloride was
prepared in methanolic HCl and recrystallized from MeOH-
diethyl ether: mp 226-228 °C; 1H NMR (2HCl) (CD3OD) δ
0.64-3.45 (m, 19H), 6.17 (m, 1H, indoline Ar-H), 6.40 (m, 1H,
indoline Ar-H), 6.8 (m, 4H, indoline, phenyl Ar-H), 7.74 (m,
2H, phenyl Ar-H). Anal. (C23H27N2OF.2HCl‚3/4H2O) C, H, N.
2,3-Dih yd r o-1′-[3-(4-br om op h en yl)-2-h yd r oxyp r op a n -
1-yl]sp ir o[1H-in d en e-1,4′-p ip er id in e] (22b). A mixture of
2,3-dihydrospiro[1H-indene-1,4′piperidine] hydrochloride23 (0.5
Biologica l Testin g. All compounds were tested in the form
of the corresponding hydrochlorides.
Vesicu la r Acetylch olin e Tr a n sp or ter Bin d in g. Dis-
sociation constants of novel compounds were determined by
competition against the binding of [3H]vesamicol to electric
organ synaptic vesicles at 22 °C, after 24 h of incubation, by
the method of Rogers et al.6
σ Recep tor Bin d in g. σ1 binding sites were labeled with
the σ1- selective radioligand [3H]-(+)-pentazocine (DuPont-
NEN) in guinea pig brain membranes (Rockland Biological)
according to published procedures.33,34 σ-2 sites were assayed
in rat liver membranes, a rich source of these sites, with [3H]-
DTG (DuPont-NEN) in the presence of (+)-pentazocine (100
nM).
Mem br a n e P r ep a r a tion . The crude P2 membrane frac-
tion was prepared from frozen guinea pig brains minus
cerebellum. Brains were allowed to thaw slowly on ice before
homogenization. The crude P2 membrane fraction was also
prepared from the livers of male Sprague-Dawley rats (175-
225 g). Animals were sacrificed by decapitation and the livers
removed and minced before homogenization.
Tissue homogenization was carried out at 4 °C in 10 mL/g
of tissue weight 10 mM Tris-HCl/0.32 M sucrose, pH 7.4, using
a Porter-Elvehjem tissue grinder. The crude homogenate was
centrifuged for 10 min at 1000g and the supernatant saved
on ice. The pellet was resuspended in 2 mL/g of tissue weight
ice-cold 10 mM Tris-HCl/0.32 M sucrose, pH 7.4, by vortexing.
After centrifugation at 1000g for 10 min, the pellet was
discarded and the supernatants were combined and centri-
fuged at 31000g for 15 min. The pellet was resuspended in 3
mL/g 10 mM Tris-HCl, pH 7.4, by vortexing, and the suspen-
sion was allowed to incubate at 25 °C for 15 min. Following
centrifugation at 31000g for 15 min, the pellet was resus-
pended by gentle homogenization to 1.53 mL/g in 10 mM Tris-
HCl (pH 7.4), and aliquots were stored at -80 °C until used.
The protein concentration of the suspension was determined

New Vesamicol Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3913
by the method of Bradford and generally ranged from 6 to 11
mg of protein/mL.
ments were performed in the buffer containing 50 mM Tris-
HCl, pH 7.4, and 120 mM NaCl with 0.2 M [125I]IPT (for
striatal homogenates) or 0.5 M [125I]IPT (for cortical homoge-
nates) and 8-10 concentrations (10-10-10-5 M) of competing
drugs. Nonspecific binding was defined in the presence of 40
µM (-)-cocaine. Competition experiments were analyzed using
the iterative nonlinear least-squares curve-fitting program
LIGAND.
σ1 Bin d in g Assa y. Guinea pig membranes (100 µg of
protein) were incubated with 3 nM [3H]-(+)-pentazocine (31.6
Ci/mmol) in 50 mM Tris-HCl, pH 8.0, at 25 °C for either 120
or 240 min. Test compounds were dissolved in ethanol and
then diluted in buffer to give a total incubation volume of 0.5
mL. Assays were terminated by the addition of ice-cold 10
mM Tris-HCl, pH 8.0, followed by rapid filtration through
Whatman GF/B glass filters (presoaked in 0.5% poly(ethylen-
imine)) using a Brandel harvester (Gathersburg, MD). Filters
were washed twice with 5 mL of ice-cold buffer. Nonspecific
binding was determined in the presence of 10 µM (+)-
pentazocine. Liquid scintillation counting was carried out in
Ecolite(+) (ICN Radiochemicals, Costa Mesa, CA) using a
Beckman LS 6000IC spectrometer with a counting efficiency
of 50%. Typical counts were 70 dpm/µg of protein for total
binding, 6 dpm/µg for nonspecific binding, and 64 dpm/µg for
specific binding.
σ2 Bin d in g Assa y. Rat liver membranes (35 µg of protein)
or guinea pig brain membranes (360 µg) were incubated with
3 nM [3H]DTG (38.3 Ci/mmol) in the presence of 100 nM (+)-
pentazocine to mask σ1 sites. Incubations were carried out
in 50 mM Tris-HCl, pH 8.0, for 120 min at 25 °C in a total
incubation volume of 0.5 mL. Assays were terminated by the
addition of ice-cold 10 mM Tris-HCl, pH 8.0, followed by rapid
filtration through Whatman GF/B glass filters (presoaked in
0.5% poly(ethylenimine)) using a Brandel harvester (Gaith-
ersburg, MD). Filters were then washed twice with 0.5 mL of
ice-cold buffer. Nonspecific binding was determined in the
presence of 5 µM DTG. Liquid scintillation counting was
carried out in Ecolite(+) (ICN Radiochemicals, Costa Mesa,
CA) using a Beckman LS 6000IC spectrometer with a counting
efficiency of 50%. Typical counts for rat liver were 297 dpm/
µg of protein for total binding, 11 dpm/µg for nonspecific
binding, and 286 dpm/µg for specific binding. Typical counts
for guinea pig brain were 16 dpm/µg of protein for total
binding, 2 dpm/µg for nonspecfic binding, and 14 dpm/µg for
specific binding.
Da ta An a lysis. The IC50 values for σ sites were determined
in triplicate by nonlinear regression analysis using J MP (SAS
Institute, Cary, NC), with 5-10 concentrations of each ace-
tylcholine compound. Ki values were calculated using the
Cheng-Prusoff equation35 and represent mean values ( SEM.
All assays were done in triplicate unless otherwise noted. For
the radioligands, the following previously33,34 reported Kd
values were employed: [3H]DTG, 17.9 nM (rat liver); [3H]-(+)-
pentazocine, 4.8 nM (guinea pig brain). The Kd value for [3H]-
DTG in guinea pig brain was determined by a Scatchard
analysis to be 21.6 nM.
Dop a m in e D2/D3 Recep tor Bin d in g Assa y. The frozen
membrane preparations from Spodoptera frugiperda (Sf9)
insect cells expressing rat dopamine D2 receptors were kindly
provided by Dr. Perry Molinoff (Bristol-Myers Squibb Phar-
maceutical Research Inc. Wallingford, CT). Rat striatal ho-
mogenates were prepared as described previously.36 Compe-
tition experiments were carried out with 0.2 nM [125I]NCQ298
(for Sf9/D2 receptors) or 0.05 nM [125I]NCQ298 (for D2/D3
receptors in striatal homogenates) as the radioligand and 8-10
concentrations (10-10-10-5 M) of competing drugs (serially
diluted in Tris-HCl buffer containing 0.1% BSA) in these cell
membranes as described previously.37 Nonspecific binding was
defined with 1 µM spiperone. Incubation was carried out at
37 °C for 30 min. The reaction was terminated by separation
of bound from free radioligand by filtration through glass fiber
filters (no. 25, Schleicher & Schuell, Keene, NH) presoaked
with 1% poly(ethylenimine). The filters were then washed
three times with 3 mL of ice-cold 20 mM Tris buffer and
counted in a gamma counter (Packard 5000) with 70% ef-
ficiency. Competition experiments were analyzed using the
iterative nonlinear least-squares curve-fitting program
LIGAND.38
Ack n ow led gm en t. Financial support for this work
was provided by the NINDS (Grant NS28711 to
S.M.N.E.) and NIA (Grant AG13621 to S.M.N.E.).
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