Structural requirements for 2,4- and 3,6-disubstituted pyran biomimetics of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine compounds to interact with monoamine transporters

Article abstract of DOI:10.1016/j.bmc.2004.07.069

Structure-activity relationship study of cis-(6-benzhydryl-tetrahydropyran- 3-yl)-(4-fluorobenzyl)-amine derivatives and their bioisosteric analogs for the monoamine transporters in the central nervous system. In our effort to delineate novel pharmacophor

Full text of DOI:10.1016/j.bmc.2004.07.069

Bioorganic & Medicinal Chemistry 12 (2004) 6301–6315
Structural requirements for 2,4- and 3,6-disubstituted pyran
biomimetics of cis-(6-benzhydryl-piperidin-3-yl)-
benzylamine compounds to interact with monoamine transporters
Shijun Zhang,a Juan Zhen,b,c Maarten E. A. Reithb,c and Aloke K. Duttaa,*
aDepartment of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, USA
bIllinois State University, Department of Biological Sciences, Normal, IL 61790, USA
cNew York University School of Medicine, Millhauser Laboratories, Department of Psychiatry, New York, NY 10016, USA
Received 24 March 2004; accepted 16 July 2004
Available online 28 September 2004
Abstract—In our effort to delineate novel pharmacophoric configuration of bioisosteric pyran versions of cis-(6-benzhydryl-piper-
idin-3-yl)-benzylamine derivatives in interacting with the monoamine transporter, further structure–activity relationship study was
carried out. Both cis and trans 2,4- and 3,6-disubstituted derivatives were synthesized to determine the positional importance of N-
substitution on affinity for monoamine transporters, that is the dopamine transporter (DAT), the serotonin transporter (SERT), and
the norepinephrine transporter (NET) in rat brain. For that purpose, the potency of compounds was determined in competing for
the binding of [3H]WIN 35,428, [3H]citalopram, and [3H]nisoxetine, respectively. Selected compounds were also evaluated for their
activity in inhibiting the uptake of [3H]DA by DAT. Our binding results demonstrated potency in 3,6-disubstituted derivatives while
2,4-disubstituted derivatives failed to exhibit any appreciable binding affinity. Further structural exploration of the exocyclic N-
atom in 3,6-disubstituted derivatives produced compounds potent at both DAT and NET. Compounds 16h and 16o with hydroxyl
and amino groups in the phenyl moiety of the benzyl group produced the highest activity for the NET. In this regard, compound 16e
with a methoxy substituent produced weak affinity at NET, which upon conversion into a hydroxyl functionality as in 16h produced
potent affinity for the NET. Various indole derivatives displayed different interactions; the 5-substituted indole derivative 16n
exerted potent affinity for NET, confirming the bioisosteric equivalence between this indole moiety and the phenyl-4-hydroxy group
in 16h.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
mainly from binding to the dopamine transporter
(DAT).4–7 Such a role for DAT is strongly supported
by various experimental evidences.8–10 However, this
does not rule out the involvement of nondopaminergic
systems in cocaine reward, and for example, the seroton-
ergic system has been shown to modulate some of co-
caineꢀs effects.11,12
Cocaine, a naturally occurring alkaloid, is well known
for its powerful abuse and addiction potential. Cocaine
dependence is a major problem in our society today,
inflicting severe medical, social, judicial, and financial
costs.1,2 Currently, no effective medication is available
for the treatment of cocaine addiction and there is an
urgent need to develop a suitable medication to treat
this chronic disorder.3
Many efforts have been directed toward the develop-
ment of molecules targeting DAT and a great number
of structurally diverse compounds have already been
synthesized with an aim to develop effective pharmaco-
therapies for cocaine addiction. These compounds
include tropane, benztropine, mazindol, or methyl-
phenidate derivatives, and also piperazine or piperidine
derivatives of GBR 12935. Detailed description of SAR
studies on these compounds is provided in recent review
papers.13–15 The existence of this wide variety of molec-
ular structures might indicate the existence of flexible
binding pockets in the DAT, which can accommodate
Extensive studies have been conducted so far to under-
stand the mechanism of action of cocaine, which might
eventually lead to the development of a much needed
medication for cocaine dependence. Cocaine binds to
all three monoamine transporter systems in the brain
but its central reinforcing action is thought to be derived
*
Corresponding author. Tel.: +1 313 577 1064; fax: +1 313 577
2033; e-mail: adutta@wayne.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.069

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S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
different molecular templates. Our efforts to develop
molecules targeting DAT started with piperidine ana-
logs of GBR 12909. A large number of potent and selec-
tive piperidine analogs have been synthesized and
biologically characterized.16–19 Most of these molecules
possess a high degree of structural flexibility, and conse-
quently, it was difficult to elucidate their biologically
active conformation for interacting with DAT. Re-
cently, we converted one of our lead piperidine analogs
into structurally constrained 3,6-disubstituted piperidine
derivatives possessing cis- and trans-structures.20 The re-
sults demonstrated that preferential affinity for the DAT
lied with the cis-structure compared to the trans-struc-
ture (Fig. 1). Further SAR study on the cis-template
produced derivatives with higher affinity for the DAT
confirming the cis-structure as a novel template for the
DAT.21
compound. In previous studies with tropane and
benztropine analogs, transformation of certain DAT
selective 3-aryltropane and benztropine analogs into
oxy-3-aryltropane and oxy-benztropine analogs was car-
ried out, which resulted in divergent results.23,24 Thus
transformation of tropane to oxy-3-aryltropane had a
minimal influence on activity at DAT compared to its
parent bioisosteric N-analog.23 On the other hand, sim-
ilar transformation of benztropine to oxy-benztropine
analogs resulted in loss of potency for the DAT.24 Both
oxy-3-aryltropane and oxy-benztropine have a con-
strained tetrahydro-pyran moiety albeit oxy-3-aryltro-
pane analogs contain additional substitutions. These
results point to the N-atom in benztropine as a critical
requirement for binding to the DAT, whereas the N-
atom in 3-aryltropane analogs may not be so critical,
consonant with the existence of flexible binding pockets
in the DAT. These results also indicate that a structur-
ally constrained pyran moiety requires more molecular
specificity for exhibiting activity at DAT compared to
a structurally constrained piperidine motif. These differ-
ences in activity at the DAT might be due to the fact
that several changes can occur in the pharmacodynamic
properties upon the replacement of an N-atom by a less
basic O-atom. Consequently, different modes of interac-
tion with DAT could occur for pyran and their bioiso-
steric piperidine counterparts. These two types of
compounds may also produce different pharmacokinetic
properties.
In a recent preliminary study, we demonstrated that the
piperidine ring in our structurally constrained 3,6-disub-
stituted piperidine derivatives can be replaced by a pyr-
an moiety while preserving DAT activity in the same
stereochemical cis-structural preference (Fig. 2).22 How-
ever, the relative activity was some what greater in the
piperidine derivatives, for example the IC50 for, inhibit-
ing radioligand binding to DAT for 1a was 31.5nM ver-
sus 52.6nM for 16c, indicating the potential importance
of the more basic N-atom in interacting with DAT. Our
earlier study reported the synthesis and biological char-
acterization of a trans-3,6-disubstituted pyran derivative
and a limited number of cis-3,6-disubstituted pyran
derivatives. The result demonstrated that the cis deriva-
tive was approximately two times as potent as the trans
In our current study we wanted to explore further sub-
stitution on the exocyclic N-atom in 3,6-disubstituted
derivatives to gain more insight in the molecular deter-
F
F
H C
3
R
N
HN
COOMe
OCOPh
O
N
N
N
H
1a, R = CN
1b, R = F
1c, R = OMe
(-)-Cocaine
GBR 12909
Figure 1. Molecular structure of dopamine transporter blockers.
F
F
NH
HN
N
O
NH
H
N
III
F
II
I
Figure 2. Rational modification of flexible piperidine molecules into constrained structures.

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
6303
minants required for activity. In addition, we wanted to
map out the positional requirement of the amino moiety
on the pyran ring for interaction with the DAT by var-
ying its location. For this purpose, we have designed, in
addition to 3,6-disubstituted derivatives, 2,4-disubsti-
tuted pyran derivatives in their cis- and trans-isomeric
forms. The results from these studies should shed more
light on the dynamics of molecular interactions of these
novel pyran derivatives with monoamine transporters.
6a and 6b were synthesized from 3a and 3b, respectively,
in high yields by three steps that involved first mesyla-
tion with methanesulfonyl chloride in dry dichlorometh-
ane to produce 4a and 4b, which were then treated with
sodium azide in DMF to produce azides 5a and 5b with
inversion of configuration.27 This azido displacement
reaction resulted in production of the cis-isomer 5a from
trans-4a and the trans-isomer 5b from cis-4b. Finally,
catalytic hydrogenation of the azides 5a and 5b with
Pd/C produced the amine precursors 6a and 6b in good
yield. Reductive amination of 6a and 6b by following a
procedure described by us earlier furnished 7a and 7b,
respectively, in 72.6% and 54% yield.
2. Chemistry
Target compounds 7a,b and 16a–p were synthesized by
following the synthetic procedures shown in Schemes
1–5.
Scheme 2 delineates the preparation of the key pyran
3,6-disubstituted intermediate 11 with trans-stereochem-
istry. Briefly, aldehyde 1 was converted into 8 by react-
ing with the in situ prepared Grignard reagent
4-bromomagnesium-1-butene, prepared from 4-bromo-
1-butene and magnesium in dry ether in 91% yield.
O-vinyllation of 8 with ethyl vinyl ether in the presence
of Hg(OCOCF3)2 at room temperature produced 9 in
66% yield.28 Ring closing metathesis of 9 in presence
of a Grubbꢀs catalyst in refluxing benzene afforded olefin
10 in 92.6% yield.29 Hydroboration of 10 with 9-BBN
in THF, followed by oxidation gave exclusively trans-
isomer 11 in 93.5% yield.30 Compound 11 was used
next as a starting precursor for the synthesis of various
derivatives with different substitutions at the exocyclic
Synthesis of the target compounds 7a and 7b, shown in
Scheme 1, was accomplished in high yields by following
efficient synthetic routes. The basic pyranose ring struc-
ture in compound 2 was achieved by [4+2] hetero-Diels–
Alder cycloaddition between Danishefskyꢀs diene and
aldehyde 1 in the presence of BF3ÆEt2O, which produced
2 in 80% yield.25,26 Reduction of 2 with NaCNBH3 in
presence of BF3ÆEt2O in THF produced racemic cis-
and trans-mixture of 3a and 3b (2.5:1) in 96% yield.
The two isomers were separated by careful flash chro-
matography, and their structures were assigned by
NMR and NOE (see Supplementary data). Compounds
O
OMe
OH
a
b
OHC
+
+
HO
O
O
O
Me SiO
3b
2
3
3a
Danishefsky's
diene
1
OMs
f
d
e
c
H N
2
N
3
3a
O
O
O
4a
6a
5a
F
N
H
O
7a
NH
N
f
2
3
e
d
c
MsO
3b
O
O
O
6b
5b
4b
F
NH
O
7b
Scheme 1. Reagents and conditions: (a) Danishefskyꢀs diene (trans-1-methoxy-3-trimethylsilyloxy-1,3-butadiene), BF3/Et2O, dry ethyl ether, ꢀ78 to
0ꢁC, 4h, 80.2%; (b) BF3/Et2O, NaCNBH3, dry THF, ꢀ78ꢁC to room temperature, 2h, 96%; (c) CH3SO2Cl, Et3N, (d) NaN3, DMF, 10 0ꢁC, 4h,
82.7%; (e) H2/Pd–C, MeoH, 60psi, 4h, quantitative yields; (f) 4-fluorobenzaldehyde, AcOH, NaCNBH3, ClCH2CH2Cl, room temperature, 4h, 54–
72.6%.

6304
S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
b
a
OHC
HO
O
8
9
1
c
d
HO
O
11
O
10
Scheme 2. Reagents and conditions: (a) 4-bromo-1-butene, Mg, Et2O, ꢀ78ꢁC to room temperature, 4h, 91%; (b) ethyl vinyl ether, Hg(OCOCF3)2,
room temperature, overnight, 66%; (c) Grubbꢀs catalyst (benzylidene-bis(tricyclohexylphosphine)-dichlororuthenium), benzene, 80ꢁC, 20h, 92.6%;
(d) (i) 9-BBN, THF, room temperature, overnight; (ii) NaOH, H2O2, 55ꢁC, 1h, 93.5% overall yield.
H
N
F
O
16a
O
b
a
HO
O
O
12
11
O
NH
16b
F
Scheme 3. Reagents and conditions: (a) oxalyl chloride, DMSO, Et3N, CH2Cl2, ꢀ78ꢁC to room temperature, 30min, 91%; (b) 4-fluorobenzylamine,
AcOH, NaCNBH3, ClCH2CH2Cl, room temperature, 4h, 15–45%.
a
b
MsO
HO
O
O
14
O
N3
13
11
c
d
O
O
NH2
NH
R
15
16b-n
16c R=
16d R=
NO2 16e R=
OCH3
CN
16b R=
F
Cl
Cl
16i R=
16g R=
16h R=
OH
16f R=
N
H
N
H
F
16m R=
I
16l R=
Br
16k R=
16j R=
F
16n R=
N
H
Scheme 4. Reagents and conditions: (a) Ch3SO2Cl, Et3N, CH2Cl2, room temperature, 4h, 77.8%; (b) NaN3, DMF, 10 0ꢁC, 4h, 92%; (c) H2, Pd–C,
MeOH, 60psi, 4h, 78% (d) aldehyde, AcOH, ClCH2CH2Cl, NaCNBH3, MeOH, room temperature, 4h, 82%.

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
6305
a
O
O
NH
NH
16o
O N
2
H N
2
16d
c
O
O
b
O
NH
NH
F
F
NH
2
O
16p
15
17
Scheme 5. Reagents and conditions: (a) SnCl2Æ2H2O, EtOH/EtOAc (7:3), reflux, 1.5h, 60%; (b) 4-fluorophenylacetyl chloride, Et3N, CH2Cl2, room
temperature, 80%; (c) NaBH4, BF3ÆEt2O, THF, reflux, overnight, 81%.
N-atom as shown in Schemes 3 and 4. First, compound
11 was subjected to Swern oxidation reaction condition,
which produced ketone 12 in 91% yield. Reductive ami-
nation of 12 with 4-fluorobenzylamine produced 16a as
a major product in 45% yield (Scheme 3). As described
in the synthesis of compound 6a–b in Scheme 1, com-
pound 11 was next converted into a cis-amine intermedi-
ate 15 via three steps consisting of first mesylation with
methanesulfonyl chloride in dry dichloromethane fol-
lowed by displacement with sodium azide in DMF and
finally, catalytic hydrogenation with Pd–C in methanol
(Scheme 4). Reductive amination of 15 with various
aldehydes furnished target compounds 16b–n in good
yield (Scheme 4).
steric heterocyclic moieties and other substituted benzyl
derivatives. Results from these derivatives will allow us
to compare any similarity or dissimilarity in molecular
interaction between the piperidine and pyran series of
compounds, which in turn could provide a unique phar-
macophoric models for pyran derivatives.
Additionally, we wanted to investigate the positional
importance of the exocyclic N-substituted moiety on
the pyran ring. This exploration was thought to be nec-
essary since any loss in potency from transforming the
piperidine to a pyran moiety might lead to a less than
optimal interaction at the 3-amino substituent site on
the pyran ring. This could potentially arise as the O-
atom in the pyran ring, being less basic than the piper-
idine N-atom, may interact with (a) different residue
(s) of the DAT. A shift of the N-substituent to the adja-
cent position could be thought to compensate for this,
leading to enhanced interaction. Moreover, testing the
effect of a positional shift of the amino substituent will
answer the question of whether the 3,6-disubstituted
configuration is required as an optimal pharmacophore
configuration for binding interaction. In an attempt to
address these questions, 2,4-disubstituted derivatives in
their cis- and trans-forms were designed and synthesized.
The synthesis of compounds 16o and 16p is described in
Scheme 5. The compound 16o was synthesized by the
reduction of 16d with tin(II) chloride dihydrate in etha-
nol and ethyl acetate in 60% yield. Amide intermediate
17 was obtained from the reaction of amino-compound
15 with 4-fluorophenylacetyl chloride. Reduction of 17
with freshly generated borohydrate gave the target com-
pound 16p.
3. Results and discussions
Following synthesis of the 2,4-disubstituted cis and trans
compounds 7a and 7b, their potencies were determined
in binding assays for the three monoamine transporters
(Table 1). The results indicated that the positional
change from 3,6-disubstitution to 2,4-disubstitution ad-
versely affected the binding activity of these two mole-
cules. It is interesting to note that even though the
activity was low in the 2,4-disubstituted compounds,
the preferential affinity for DAT was still exhibited in
the cis version. These results confirmed that the cis-form
of the 3,6-disubstituted pyran template contributes to
the basic pharmacophore for interacting with DAT.
As an extension of our studies on structurally con-
strained piperidine derivatives, we have developed novel
3,6-disubstituted pyran molecules as potential blockers
of monoamine transporters. Preliminary binding results
of the compounds at monoamine transporters indicated
a positive correlation with the results from our structur-
ally constrained 3,6-disubstituted piperidine template
including the cis-isomeric preference for higher activity.
However, in comparison to their piperidine counter-
parts, these compounds were somewhat less potent at
DAT. This might indicate that even though the N-
and O-atoms in the piperidine and pyran rings are bio-
isosteres, the existence of different interaction modes
with the monoamine transporter systems can not be
ruled out as the physical properties such as basicity of
these two atoms are quite different. Consequently, in
our current SAR study we wanted to examine additional
derivatives. These derivatives were synthesized by func-
tionalizing the exocyclic N-atom with various bioiso-
In the 3,6-disubstituted version, as we reported in
our preliminary communication,22 replacement of the
fluoro substituent by electron withdrawing substituents
resulted in more potent compounds for the DAT as
illustrated in the cyano-substituted molecule 16c and
the nitro-substituted molecule 16d. Nitro-substitution
produced the most potent compound among these

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S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
Table 1. Affinity of drugs at dopamine, serotonin, and norepinephrine transporters in rat striatum
Compd
Inhibition of [3H]Win 35,
428 binding
to DAT, IC50 (nM)a
Inhibition of
[3H]citalopram binding
to SERT, IC50 (nM)a
Inhibition of
[3H]nisoxetine
binding to NET,
IC50 (nM)a
Inhibition of [3H]DAa
uptake by DAT, IC50 (nM)
Cocaine
GBR 12909
I
266 37
10.6 1.9
32.5 12.6
1302 68
1581 283
313 71
550 737 1603130
132
0
496 22
1020 72
2220 590
3313 170
4778 1808
8410 163
1860 22
45.7 5.1
7a
5101 1037
17,543 2153
12,700 3180
232 46
7b
16ab
16bb
16cb
16db
16e
163 29
156 36
58.6 13.2
52.6 5.9
38.3 3.9
84 6.5
863 52
1580 89
968 98
738 164
1180 269
2590 1410
1640 448
398 22
102
7
1550 682
1860 847
401 96
59.5 11.6
135.2 47.5
880 136
16f
16g
794 111
227 67
16h
16i
78.4
400 31
9
22.6 1.4
144 25
780 84
16j
368 85
303 14
202 13
319 21
587 66
151 13
129 58
777 41
3520 831
1577 97
2363 92
2477 145
325 2056
1690 169
3950 660
695 142
274 29
592 12
16k
16l
242 39
251 14
500 34
16m
16n
234 17
6
160
16p
123 10
5210 678
155 14
15
251 31
a For binding, the DAT was labeled with [3H]WIN 35,428, the SERT with [3H]citalopram and the NET with [3H]nisoxetine. For uptake by DAT,
[3H]DA accumulation was measured. Results are average SEM of three to eight independent experiments assayed in triplicate.
b See Ref. 22.
Table 2. Selectivity of various drugs for their activity at monoamine
transporters
synthesized analogs for the DAT (IC50 = 38.3nM). This
trend agrees with our previous data for the piperidine
counterparts. On the other hand, the electron donating
methoxy substituent in 16e produced comparable
potency at the DAT (IC50 = 84nM).22 Similar relative
differences in potency were observed for piperidine
derivatives.21 Introduction of 3,4-difluoro substituents
in 16j reduced potency at all three transporters com-
pared to the 4-fluoro compound 16b. For the dichloro-
substituted compound 16i, no improvement in potency
at DAT was observed compared to the unsubstituted
16k, suggesting a different mode of binding interaction
compared to tropane- and methylphenidate-type of
compounds.31,32 As far as other halogen derivatives
are concerned, the bromo compound 16l exhibited
somewhat higher activity at DAT compared to unsubsti-
tuted 16k whereas the iodo compound 16m displayed
comparable potency.
Compound SERT binding/ NET binding/ [3H]DA uptake/
DAT binding
DAT binding DAT binding
Cocaine 2.8
GBR 12909 12.5
11.8
46.8
I
7a
68.3
2.5
3
31.4
3.9
1.4
7b
11.1
40.6
1.4
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
15
26.9
11.4
16.4
19.3
14
0.96
301.1
25.3
18.5
2.3
2.7
0.71
3.3
7.2
5.1
1.9
9.6
1.8
0.60
0.29
0.36
1.9
5.20
0.90
2.93
0.73
0.09
0.81
40.4
0.79
1.24
1.56
11.69
7.76
0.55
Compared to the methoxy substituted compound 16e,
the hydroxy substituted compound 16h retained potency
at DAT (IC50 = 78.4nM for 16h and IC50 = 84nM for
16e), but its selectivity was shifted in favor of norepi-
nephrine transporter (NET) shown by the much higher
activity at NET (IC50 = 22.6nM for the NET, NET/
DAT = 0.29) (Table 2). The amino-substituted
compound 16o also exhibited high potency at NET.
Hydroxy or amino substituents can act as both hydro-
gen-bond donor or acceptor sites, although in different
capacity. The appreciable shift toward potency and
selectivity at NET caused by these two polar substitu-
ents might indicate a critical involvement of hydrogen
11.19
30.6
1.02
0.32
bonding in interacting with NET. However, similar re-
sults were not observed in the structurally constrained
piperidine analogs, reflecting the existence of different
interaction modes between these two templates.21 Since
a high degree of homogeneity has been demonstrated

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
6307
between the DAT and NET structural sequence, it is of
interest to observe that a subtle change in pyran struc-
ture can induce differential interactions in favor of the
NET.33,34
In order to gain further insight into the hydrophobic
nature of the interaction between the aromatic moiety
and monoamine transporters, we decided to replace
the phenyl aromatic moiety in the benzyl group by bio-
isosteric indole moieties. Thus, replacement with 2- and
3-indole moieties as illustrated in compounds 16g and
16f, led to moderate to diminished potency at DAT.
Interestingly, as was seen with our piperidine derivative
counterparts, the 2-indole substituted derivative 16g was
3.5-fold more active at DAT compared to the 3-substi-
tuted 16f (227 vs 794nM) and was also more active than
the unsubstituted derivative 16k. A similar increase in
affinity for the NET was also observed for the 2-substi-
tuted indole compared to the 3-substituted compound
(401 vs 1860nM). In our further attempt to test the
importance of the position of the indole N-atom along
with its hydrophobicity, the 5-substituted indole deriva-
tive 16n was designed and synthesized. In this regard, 5-
substitution was chosen as a bioisosteric configuration
of the p-hydroxy-phenyl moiety of 16h. The binding re-
sults for 16n indicated high affinity, similar to 16h, for
the NET, indicating the involvement of H-bonding with
the indole amino moiety. This result further demon-
strates the existence of a H-bond donor or acceptor site
in the NET, which when oriented correctly with respect
to ligandꢀs H-bond forming functionality, can provide
potent interaction.
Figure 3. Three-dimensional orientation of the lowest energy con-
formers and the overlapped ligands: (A) lowest energy conformer from
compound 7a; (B) lowest conformer from compound 16b; (C)
overlapped ligands based on two conformers A and B.
pound 16b were chosen for this study. Compounds
were minimized first with the SYBYL molecular modeling
program (version 6.9, 2002, Tripos Associates, Inc., St.
Louis, MO). Minimized molecules obtained from this
operation were next subjected to a grid search protocol
to search for the lowest energy conformer. Grid search
operation was carried out with the change of torsional
angle from 0ꢁ to 360ꢁ with an increment of 10ꢁ compris-
ing of atoms a–b–c–d as shown in Figures 3A and B for
both 7a and 16b. This operation resulted in the genera-
tion of 3.16kcal/mol lowest energy for 7a with a corre-
sponding torsional angle of 77.8ꢁ and 5.61kcal/mol for
16b with a torsional angle of 300ꢁ. In the final step,
the two minimized structures were overlapped with the
alignment program (see Fig. 3C). It was quite evident
that the exocyclic amino substituents in the two com-
pounds were oriented very differently in the two different
directions.
In compound 16p, the fluorobenzyl moiety was replaced
by a 4-fluorophenylethyl moiety which did not result,
surprisingly, in decreased activity at DAT compared to
16b. This result was in contrast to the results observed
in the constrained piperidine counterpart where a drop
in DAT activity resulted from such modification.21 This
result likely indicates that a different pharmacophoric
optimization is required, probably via a distance
geometry approach, to produce optimum activity in
the pyran template. As we expected, the exocyclic-N-
substitution with an aromatic moiety is necessary in
pyran derivatives for their activity at monoamine trans-
porter systems, as compound 15 exhibited little or no
activity.
Selected compounds with relatively higher affinity for
DAT were tested in the DA uptake assay. For the most
part no differential uptake and binding activity was ob-
served with the exception of compound 16d, which
showed a 3-fold higher potency in inhibiting binding
than uptake.
5. Conclusion
In this report, we have outlined the cis-3,6-disubstituted
tetrahydro-pyran template as a pharmacophore for
activity at the monoamine transporter systems. The
SAR exploration with this template with various substit-
uents on the exocyclic N-atom produced potencies at
both DAT and NET. Compound 16d with the electron
withdrawing nitro-substituent turned out to be the most
potent for the DAT. Interestingly, the compounds 16h
and the 16o with para-hydroxy and para-amino substit-
uents exhibited high potency for the NET, indicating
formation of H-bonding. This was further confirmed
by the bioisosteric version 16n, which exhibited strong
selective potency at NET. The SAR results for the
4. Molecular modeling
In order to test for a difference in spatial distribution in
the lowest energy conformers between 3,6-disubstituted
and 2,4-disubstituted pyran derivatives, we have carried
out a preliminary molecular modeling study. 2,4-Disub-
stituted compound 7a and the 3,6-disubstituted com-

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S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
current pyran molecules do not correspond with those
for the piperidine derivatives, indicating differential
interaction modes with monoamine transporters. Our
ongoing studies at different molecular centers on this
pyran ring to probe and identify optimum pharmaco-
phoric structure will shed more light on their nature of
interaction with monoamine transporters.
In the next step, using the grid search protocol, a con-
formational search on each minimized molecule was
performed by rotating the torsion angle of compounds
7a and 16b formed by atoms a–b–c–d (see Fig. 3) from
0ꢁ to 360ꢁ by 10ꢁ increments. This method was used to
perform a simple systematic search such that each spec-
ified torsion angle is varied over a grid of equally space
value. While searching for the lowest energy conformer,
a cutoff value of 8kcal/mol was specified relative to the
lowest conformer, and charges were computed using
the Gasteiger–Huckel method. Also, the six-membered
pyran ring was treated as an aggregate. For compound
7a, a conformer with torsional angle 77.8ꢁ was found
to have lowest energy 3.16kcal/mol, whereas compound
16b produced lowest energy 5.61kcal/mol with a torsion
angle 300ꢁ (see Supplementary data for detail energy dis-
tribution). These two lowest energy conformers were
used next for overlapping.
6. Experimental details
Reagents and solvents were obtained from commercial
suppliers and used as received unless otherwise indi-
cated. Dry solvent was obtained according to the stand-
ard procedure as described in Vogelꢀs book. All
reactions were performed under inert atmosphere (N2)
unless otherwise noted. Analytical silica gel-coated
TLC plates (Si 250F) were purchased from Baker,
Inc. and were visualized with UV light or by treatment
with phosphomolybdic acid (PMA). Flash chromato-
graphy was carried out on Baker Silica Gel 40mM.
1H NMR spectra were routinely obtained at Var-
ian 400MHz FT NMR. The NMR solvent used was
CDCl3 as indicated. TMS was used as an internal
standard. Elemental analyses were performed by Atlan-
tic Microlab, Inc. and were within 0.4% of the theore-
tical value.
During overlapping, the alignment program within
SYBYL 6.9 was employed, and the approach used was
the common structure method. The compound 16b
was used as a template molecule and the six-membered
pyran ring was used as a common substructure for
overlapping.
6.1.1. Synthesis of 2-benzhydryl-2,3-dihydro-4H-pyran-4-
one (2). A solution of boron trifluoride diethyl etherate
(7.80g, 55mmol) in dry ether (50mL) was added to a
stirred mixture of E-1-methoxy-3-trimethylsilyloxy-
buta-1,3-diene (Danishefskyꢀs Diene) (8.30g, 48mmol),
diphenylacetaldehyde 1 (11.40g, 58mmol), and dry
ether (300mL) cooled to ꢀ78ꢁC. After 1h, the mixture
was allowed to reach 0ꢁC for 3h. The deep red reaction
mixture was quenched with saturated aqueous NaH-
CO3, and the mixture was allowed to come to room tem-
perature. The organic phase was separated and the
aqueous phase was extracted with ether (3 · 70mL).
Combined organic phase was washed with brine, and
dried over anhydrous Na2SO4. Evaporation of the sol-
vent under reduced pressure and purification of the
crude product by chromatography (hexane/ethyl acetate
8:2) gave 2-benzhydryl-2,3-dihydro-4H-pyran-4-one 2
(10.20g, 80.2%, yield) as a yellow solid.
[3H]WIN 35,428 (86.0Ci/mmol), [3H]nisoxetine (80.0Ci/
mmol), and [3H]dopamine (48.2Ci/mmol) were ob-
tained from Dupont-New England Nuclear (Boston,
MA, USA). [3H]citalopram (85.0Ci/mmol) was from
Amersham Pharmacia Biotech Inc. (Piscataway, NJ,
USA). Cocaine hydrochloride was purchased from
Mallinckrodt Chemical Corp. (St. Louis, MO, USA.).
WIN 35,428 napthalene sulfonate was purchased from
Research Biochemicals, Inc. (Natick, MA, USA). (ꢀ)-
Cocaine HCl was obtained from the National Institute
on Drug Abuse. GBR 12909 Dihydrochloride (1-[2-
[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]-
piperazine) was purchased from SIGMA-ALDRICH
(#D-052; St. Louis, MO).
6.1. Molecular modeling
Molecular modeling investigation was performed by
using the SYBYL molecular modeling package (version
6.9, 2002, Tripos Associates, Inc., St. Louis, MO).
Modeling was carried out on Silicon Graphics Octane
IRIX 6.5 workstation. The compounds were sketched
in appropriate stereochemistry.
1H NMR (400MHz, CDCl3): 2.38 (dd, J = 3.20,
16.80Hz, 1H, H-3), 2.51 (m, 1H, H-3), 4.23 (d,
J = 9.20Hz, 1H, (Ph)2CH), 5.15 (dt, J = 3.20, 8.80Hz,
1H, H-2), 5.44 (d, J = 6.40Hz, 1H, H-5), 7.16–7.38 (m,
11H, H-6, aromatic-CH).
6.1.2. Synthesis of cis- and trans-2-benzhydryl-tetrahy-
dropyran-4-ol (3a) and (3b). NaCNBH3 (0.75g, 12mmol)
was added portionwise to a mixture of 2-diphenyl-
methyl-2,3-dihydro-4H-pyran-4-one 2 (1.05g, 4mmol)
and boron trifluoride etherate (1.99g, 14mmol) in dry
THF (50mL) cooled to ꢀ78ꢁC. The reaction mixture
was allowed to reach room temperature and the reaction
was quenched with saturated aqueous NaHCO3
(30mL). The organic phase was separated, and the aque-
ous phase was extracted with ethyl ether (3 · 20mL).
The organic phase was combined and dried over anhy-
drous Na2SO4. Removal of the solvent under reduced
First, each structure was fully minimized using standard
Tripos force field with a distance-dependent dielectric
˚
function, a 0.05kcal/molA energy gradient convergence
criterion was used and the six-membered pyran ring was
treated as an aggregate. The Powell method was used
during minimization, and charges were computed using
the Gasteiger–Huckel method within SYBYL 6.9. The
number of iteration was 1000. After minimization the
energy for 2,4-disubstituted molecule 7a was 5.85kcal/
mol and the energy for 3,6-disubstituted molecule 16b
was 5.63kcal/mol.

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
6309
pressure, and purification by flash chromatography
(hexane/ethyl acetate 7:3) first afforded trans-2-benzhy-
dryl-tetrahydropyran-4-ol 3a (0.73g, 68% yield).
brine, and then dried over anhydrous Na2SO4. Removal
of the solvent and purification by flash chromatography
(hexane/ethyl acetate 9:1) afforded compound 5a (0.23g,
82.7% yield) as a liquid.
1H NMR (400MHz, CDCl3): 1.50–1.58 (m, 4H, H-3, H-
5eq, OH), 1.84 (m, 1H, H-5ax), 3.79 (m, 1H, H-6eq),
3.88 (d, J = 8.80Hz, (Ph)2CH), 3.91 (dt, J = 3.20,
11.20Hz, 1H, H-6ax), 4.18 (m, 1H, H-4eq), 4.52 (dt,
J = 4.00, 8.80Hz, 1H, H-2), 7.16–7.38 (m, 10H, aro-
matic-CH).
1H NMR (500MHz, CDCl3): 1.32 (q, J = 11.00Hz, 1H,
H-3ax), 1.61 (dq, J = 5.50, 13.00Hz, 1H, H-5ax), 1.82
(m, 1H, H-3eq), 1.90(m, 1H, H-5eq), 3.44–3.50(m,
2H, H-4, H-6ax), 3.96 (d, J = 8.50Hz, 1H, (Ph)2CH),
4.03 (dt, J = 2.00, 9.00Hz, 1H, H-2), 4.08 (ddd,
J = 2.00, 5.50, 12.50Hz, 1H, H-6eq), 7.16–7.38
(m,10H, aromatic-CH).
Eluted second was cis-2-benzhydryl-tetrahydropyran-
4-ol, 3b (0.30g, 28.1% yield).
6.1.6. Synthesis of trans-4-azido-2-benzhydryl-tetrahyd-
ropyran (5b). Methanesulfonic acid cis-2-benzhydryl-
tetrahydro-pyran-4-yl ester 4b (0.38g, 1.10mmol) was
reacted with sodium azide (0.29g, 4.4mmol) in dry
DMF (Procedure B) to yield compound 5b (0.26g,
80%) as a liquid.
1H NMR (400MHz, CDCl3): 1.22 (q, J = 12.00Hz, 1H,
H-3ax), 1.46 (dq, J = 4.80, 12.00Hz, 1H, H-5ax), 1.74–
1.86 (m, 2H, H-3eq, H-5eq), 3.40(dt,
J = 2.00,
12.00Hz, 1H, H-6ax), 3.71 (m, 1H, H-4), 3.94–4.04
(m, 2H, H-6eq, (Ph)2CH), 7.15–7.4 (m, 10H, aromatic-
CH).
1H NMR (400MHz, CDCl3): 1.50–1.68 (m, 3H, H-3, H-
5eq), 1.86 (m, 1H, H-5ax), 3.74–3.86 (m, 2H, H-6), 3.87
(d, J = 9.20Hz, 1H, (Ph)2CH), 4.02 (m, 1H, H-4), 4.39
(dt, J = 3.20, 13.00Hz, 1H, H-2), 7.16–7.38 (m, 10H,
aromatic-CH).
6.1.3. Procedure A. Synthesis of methanesulfonic acid
trans-2-benzhydryl-tetrahydro-pyran-4-yl ester (4a).
Methanesulfonyl chloride (0.62g, 5.41mmol) in dry
methylene chloride (10mL) was added dropwise to a
mixture of trans-2-benzhydryl-tetrahydropyran-4-ol 3a
(0.73g, 2.70mmol), triethylamine (0.41g, 4.06mmol) in
methylene chloride (10mL) and was cooled to 0 ꢁC.
After 1h, the reaction was gradually allowed to reach
room temperature over a period of 4h. Additional meth-
ylene chloride (20mL) was added to the reaction mix-
ture, and the mixture was washed in turn with
saturated aqueous sodium bicarbonate, brine and water,
then dried over anhydrous sodium sulfate. The solvent
was removed under reduced pressure, and purification
by flash chromatography gave compound 4a (0.93g,
99.9% yield) as an oil.
6.1.7. Procedure C. Synthesis of cis-(2-benzhydryl-tetra-
hydropyran-4-yl)-amine (6a). cis-4-Azido-2-benzhydryl-
tetrahydropyran 5a (0.23g, 0.78mmol) was hydrogen-
ated (60psi) in the presence of 10% Pd–C (0.02g,
10%wt) for 4h. Reaction mixture was filtered through
a short bed of Celite and removal of the solvent afforded
0.21g (quantitative yield) product. This product was
pure enough for continuation to the next reaction step.
1H NMR (400MHz, CDCl3): 1.15–1.25 (m, 1H, H-3),
1.40–1.52 (m, 1H, H-3), 1.70–1.88 (m, 2H, H-5), 2.99
(m, 1H, H-4), 3.41 (dt, J = 2.00, 12.40Hz, 1H, H-6ax),
3.90–4.06 (m, 3H, H-2, H-6ax, (Ph)2CH), 4.70(br s,
2H, NH2), 7.16–7.38 (m, 10H, aromatic-CH).
1H NMR (300MHz, CDCl3): 1.61 (m, 1H, H-3ax), 1.80–
1.96 (m, 4H, –OH, H-3eq, H-5), 2.96 (s, 3H, CH3SO2),
3.80–3.94 (m, 3H, H-6, (Ph)2CH), 4.46 (dt, J = 2.00,
10.00Hz, 1H, H-2), 5.10 (m, 1H, H-4), 7.16–7.38 (m,
10H, aromatic-CH).
6.1.8. Synthesis of trans-(2-benzhydryl-tetrahydropyran-
4-yl)-amine (6b). trans-4-Azido-2-benzhydryl-tetrahy-
dropyran 5b (0.26g, 0.89mmol) was hydrogenated (Pro-
cedure C) to yield compound 6b (0.24g, quantitative).
6.1.4. Synthesis of methanesulfonic acid cis-2-benzhydryl-
tetrahydro-pyran-4-yl ester (4b). cis-2-Benzhydryltetra-
hydro-pyran-4-ol 3b (0.30g, 1.12mmol) was reacted
with methanesulfonyl chloride (0.26g, 2.24mmol) (Pro-
cedure A) to give compound 4b (0.38g, 98%) as an oil.
1H NMR (300MHz, CDCl3): 1.21–1.40(m, 4H, H-3,
NH2), 1.59 (m, 1H, H-5ax), 1.87 (m, 1H, H-5eq), 3.37
(m, 1H, H-4), 3.77 (m, 1H, H-6eq), 3.91 (dt, J = 2.40,
11.70Hz, 1H, H-6ax), 3.94 (d, J = 9.30Hz, 1H,
(Ph)2CH), 4.56 (dt, J = 2.40, 10.20Hz, 1H, H-2), 7.16–
7.38 (m, 10H, aromatic-CH).
1H NMR (300MHz, CDCl3): 1.54 (m, 1H, H-3ax), 1.82
(m, 1H, H-5ax), 1.95 (m, 1H, H-3eq), 2.10(m,1H, H-
5eq), 2.95 (s, 3H, CH3SO2), 3.46 (dt, 1H, H-6ax), 3.96
(d, 1H, (Ph)2CH), 4.10(m, 2H, H-2, H-6eq), 4.83 (m,
1H, H-4), 7.15–7.38 (m, 10H, aromatic-CH).
6.1.9. Procedure D. Synthesis of cis-(2-benzhydryl-tetra-
hydropyran-4-yl)-(4-fluorobenzyl)-amine (7a). Into
a
solution of cis-(2-benzhydryl-tetrahydro-pyran-4-yl)-
amine 6a (0.20g, 0.75mmol), 4-fluorobenzaldehyde
(0.83g, 0.67mmol), and glacial acetic acid (0.45g,
0.75mmol) in 1,2-dichloroethane (20mL) was added
portion wise NaCNBH3 (0.57g, 0.90mmol) dissolved
in methanol (5mL). After 4h, water was added to
quench the reaction and the mixture was stirred for
30min at 0 ꢁC. Then the mixture was basified with
6.1.5. Procedure B. Synthesis of cis-4-azido-2-benzhydryl-
tetrahydropyran (5a). Into a solution of methanesulfonic
acid trans-2-diphenylmethylpyran-4-yl ester 4a (0.33g,
0.95mmol) in dry DMF (40mL) was added sodium
azide (0.18g, 2.85mmol). The mixture was heated to
100ꢁC and stirred for 4h. The mixture was diluted with
ethyl ether, washed with 2M aqueous NaHCO3 and

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S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
saturated aqueous NaHCO3 and extracted thrice with
methylene chloride (3 · 30mL). The combined organic
phase was washed with brine, water and dried over
anhydrous Na2SO4. Solvent was removed in vacuo to
collect the crude residue. The residue was purified by
flash chromatography (hexane/ethyl acetate/triethylam-
ine 3:2:0.2) to give cis-(2-benzhydryl-tetrahydro-pyran-
4-yl)-(4-fluorobenzylamino)-tetrahydropyran 7a (0.20g,
72.6%) as a liquid.
sure, and flash chromatography of the crude residue
(SiO2, hexane/ethyl acetate 9:1) gave 1,1-diphenyl-hex-
5-en-2-ol 8 (0.68g, 91%) as a liquid.
1H NMR (400MHz, CDCl3): 1.45–1.70(m, 2H, H-3),
1.69 (br d, –OH), 2.10–2.40 (m, 2H, H-4), 3.91 (d,
J = 8.40Hz, 1H, H-1), 4.39 (m, 1H, H-2), 4.95–5.10
(m, 2H, H-6), 5.81 (m, 1H, H-5), 7.16–7.38 (m, 10H,
aromatic-CH).
1H NMR (500MHz, CDCl3): 1.13 (q, J = 10.50Hz, 1H,
H-3ax), 1.32 (broad, NH), 1.38 (dq, J = 5.00, 12.50Hz,
1H, H-5ax), 1.74 (m, 1H, H-3eq), 1.87 (m, 1H, H-5eq),
2.72 (tt, J = 4.00, 11.50Hz, 1H, H-4), 3.44 (dt,
J = 2.00, 12.00Hz, 1H, H-6ax), 3.68 (d, J = 13.50Hz,
1H, (F)Ph–CH), 3.75 (d, J = 13.00Hz, 1H, (F)Ph–
CH), 3.94 (d, J = 9.00Hz, 1H, (Ph)2CH), 4.00–4.08 (m,
2H, H-2, H-6eq), 6.90–7.38 (m, 14H, aromatic-CH).
6.1.12. Synthesis of 1,1-diphenyl-2-(1-ethenoxy)-hex-5-
ene (9). Into a mixture of 1,1-diphenyl-hex-5-en-2-ol 8
(7.00g, 27.78mmol) in ethyl vinyl ether (250mL) was
added Hg(OCOCF3)2 (2.37g, 5.56mmol) and was stir-
red overnight at room temperature. The reaction mix-
ture was neutralized by addition of saturated aqueous
NaHCO3. Organic phase was separated and the aqueous
layer was extracted with ethyl ether, dried over anhy-
drous Na2SO4. Removal of the solvent and purification
by flash chromatography (hexane/ethyl acetate 20:1)
gave 1,1-diphenyl-2-(1-ethenoxy)-hex-5-ene 9 (5.10g,
66%) as a liquid.
Free base was converted into its oxalate salt: mp 177–
181ꢁC, Anal. [C25H26NOFÆ(COOH)2] C, H, N.
6.1.10. Synthesis of trans-(2-benzhydryl-tetrahydropyran-
4-yl)-(4-fluorobenzyl)-amine (7b). trans-(2-Benzhydryl-
tetrahydro-pyran-4-yl)-amine 6b (0.24g, 0.90mmol)
was reacted with 4-fluorobenzaldehyde (0.11g,
0.90mmol) in presence of acetic acid (0.05g, 0.90mmol),
and then reduced with NaCNBH3 (0.07g, 1.08mmol) to
yield compound 7b (0.18g, 54%) (Procedure D).
1H NMR (400MHz, CDCl3): 1.58–1.78 (m, 2H, H-3),
2.08–2.30 (m, 2H, H-4), 3.86 (dd, J = 1.60, 8.40Hz,
1H, H-20), 4.15 (d, J = 8.00Hz, 1H, H-1), 4.25 (dd,
J = 1.60, 14.00Hz, 1H, H-20), 4.50(m, 1H, H-2), 5.0
(m, 2H, H-6), 5.77 (m, 1H, H-5), 6.15 (dd, J = 6.80,
14.80Hz, 1H, H-1 0), 7.16–7.38 (m, 10H, aromatic-CH).
1H NMR (400MHz, CDCl3): 1.24 (br s, 1H, –NH), 1.28
(m, 1H, H-3), 1.45–1.58 (m, 2H, H-3, H-5eq), 1.83 (tt,
J = 4.00, 13.00Hz, 1H, H-5ax), 3.07 (m, 1H, H-4),
3.65 (s, 2H, (F)Ph–CH2), 3.75 (m, 1H, H-6eq), 3.91 (d,
J = 9.60Hz, 1H, (Ph)2CH), 3.94 (dt, J = 2.40, 12.00Hz,
1H, H-6ax), 4.59 (dt, J = 3.20, 9.60Hz, 1H, H-2),
6.90–7.40 (m, 14H, aromatic-CH).
6.1.13. Synthesis of 2-benzhydryl-3,4-dihydro-2H-pyran
(10). A solution of 1,1-diphenyl-2-(1-ethenoxy)-hex-5-
ene 9 (5.10g, 18.30mmol) and Grubb ꢀs catalyst, benzyl-
idene-bis(tricyclohexylphosphine)dichloro
ruthenium
(1.50g, 1.83mmol) in benzene (200mL) was heated
under reflux for 20h. The solvent was removed under
vacuo and the residue was chromatographed over silica
gel (hexane/ethyl acetate 20:1) to give 2-benzhydryl-3,4-
dihydro-2H-pyran 10 (4.25g, 92.6%) as a liquid.
Free base was converted into its oxalate salt: mp 185–
187ꢁC, Anal. [C25H26NOFÆ(COOH)2] C, H, N.
1H NMR (400MHz, CDCl3): 1.52–1.66 (m, 1H, H-3),
1.76–1.84 (m, 1H, H-3), 1.92–2.14 (m, 2H, H-4), 4.08
(d, J = 9.20Hz, 1H, Ph2CH), 4.59 (dt, J = 2.40,
8.80Hz, 1H, H-2), 4.72 (m, 1H, H-5), 6.38 (d,
J = 6.04Hz, 1H, H-6), 7.16–7.50 (m, 10H, aromatic-
CH).
6.1.11. Synthesis of 1,1-diphenyl-hex-5-en-2-ol (8). A dry
three-neck, round-bottom flask fitted with a reflux con-
denser, air-balance drop funnel and nitrogen inlet was
charged with Mg (0.11g, 4.44mmol) and a crystal of
I2. The flask was warmed (heat gun) to volatilize the I2
under vacuum, and was allowed to cool. Dry ethyl ether
(10mL) was added next followed by introduction of cat-
alytic neat 4-bromo-1-butene (0.02g). The reaction was
initiated by brief warming and then the rest of total
amount of bromide (0.40g, 2.96mmol) in dry ethyl ether
(5mL) was added dropwise over 5min. The mixture was
refluxed for 30min and then was allowed to reach 0 ꢁC.
Into the stirred Grignard reagent solution was added
dropwise a solution of diphenylacetaldehyde 1 (0.64g,
3.26mmol) in dry ethyl ether (5mL), and the reaction
mixture was stirred for an additional 3.5h at room tem-
perature. Saturated aqueous NaHCO3 was added to the
reaction mixture at 0ꢁC, organic phase was separated
and the aqueous phase was extracted thrice with ethyl
ether (3 · 20mL). Combined organic phase was washed
with brine and water, then dried over anhydrous
Na2SO4. The solvent was removed under reduced pres-
6.1.14. Synthesis of trans-6-benzhydryl-tetrahydropyran-
3-ol (11). Into a solution of 0.5M 9-BBN–THF complex
(24mL, 12mmol) in dry THF (20mL) was added in a
drop wise manner 2-diphenyl-3,4-dihydro-2H-pyran 10
(1.00g, 4mmol) dissolved in dry THF (10mL). The mix-
ture was kept under stirring at room temperature. After
the completion of initial addition reaction, the inter-
mediate reaction mixture was oxidized with 5.3mL 3N
sodium hydroxide and 3mL of 30% hydrogen peroxide.
The reaction was continued at 55ꢁC for 1h to insure the
completion of oxidation. After the mixture was diluted
with satd aqueous NaHCO3, the organic layer was
separated, and the aqueous layer was extracted with
ethyl acetate (3 · 40mL). The combined extract was
dried over anhydrous Na2SO4. The solvent was removed
in vacuo and the crude product was purified by flash

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
6311
chromatography (hexane/ethyl acetate 7:3) to furnish
trans-6-benzhydryl-tetrahydropyran-3-ol 11 (1.00g,
93.5%) as a liquid.
1H, Ph2CH), 3.99 (dt, J = 3.00, 8.70Hz, 1H, H-6), 4.08
(m, 1H, H-2eq) 6.90–7.38 (m, 14H, aromatic-CH).
Free base was converted into oxalate: mp 141–143ꢁC,
Anal. [C25H26NOFÆ(COOH)20.65H2O] C, H, N.
1H NMR (300MHz, CDCl3): 1.32–1.44 (m, 2H, H-5),
1.54–1.64 (m, 1H, H-4), 1.75 (br s, 1H, OH), 2.02–2.14
(m, 1H, H-4), 3.14 (t, J = 10.20Hz, 1H, H-2ax), 3.67
(m, 1H, H-3), 3.90(d, J = 9.30Hz, 1H, Ph2CH), 3.95–
4.04 (m, 2H, H-2eq, H-6), 7.16–7.38 (m, 10H, aro-
matic-CH).
6.1.17. Synthesis of methanesulfonic acid trans-6-
benzhydryl-tetra-hydropyran-3-yl ester (13). Methane-
sulfonyl chloride (0.33g, 2.87mmol) was reacted with
trans-6-benzhydryl-tetrahydropyran-3-ol 11 (0.38g,
1.43 mmol) in the presence of triethylamine (0.22g,
2.15mmol) in methylene chloride (10mL) to give trans-
6.1.15. Synthesis of 6-benzhydryl-dihydro-pyran-3-one
(12). Into a solution of DMSO (0.13g, 1.64mmol) in
methylene chloride (5mL) at ꢀ78ꢁC was added a solu-
tion of oxalyl chloride (0.11g, 0.82mmol) in methylene
chloride (1mL) in a drop wise manner. A solution of
trans-2-diphenylmethyl-tetrahydropyran-5-ol 11 (0.20g,
0.75mmol) in methylene chloride (2mL) was added
next. The reaction was continued for 15min, triethylam-
ine (0.38g, 3.73mmol) was next added portion wise and
the reaction mixture was allowed to come to room tem-
perature for over a period of 30min. Additional methyl-
ene chloride (10mL) was added, and washed with satd
aqueous NaHCO3, brine, and then dried over anhy-
drous Na2SO4. Removal of the solvent and purification
by flash chromatography (SiO2, hexane/ethyl acetate
8.5:1.5) gave 6-benzhydryl-dihydro-pyran-3-one 12
(0.18g, 91%) as a liquid.
6-benzhydryl-tetrahydropyran-3-yl methanesulfonate
13 (0.39g, 77.8%) as an oil (Procedure A).
1H NMR (400MHz, CDCl3): 1.47 (m, 1H, H-5), 1.62–
1.78 (m, 2H, H-5, H-4), 2.25 (m, 1H, H-4), 2.96 (s,
3H, CH3SO2), 3.36 (t, J = 10.40Hz, 1H, H-2ax), 3.89
(d, J = 8.80Hz, 1H, Ph2CH), 4.00 (dt, J = 2.00,
9.60Hz, 1H, H-6), 4.14 (m, 1H, H-2eq), 4.61 (m, 1H,
H-3), 7.16–7.38 (m, 10H, aromatic-CH).
6.1.18. Synthesis of cis-3-azido-6-benzhydryl-tetrahydro-
pyran (14). trans-6-Diphenylmethyl-tetrahydropyran-
3-yl methanesulfonate 13 (0.39g, 1.12mmol) in dry
DMF (50mL) was reacted with sodium azide (0.22g,
3.35mmol) to yield cis-3-azido-6-diphenylmethyl-tetra-
hydropyran 14 (0.30g, 92%) as an oil (Procedure B).
1H NMR (300MHz, CDCl3): 1.90–1.98 (m, 2H, H-5),
2.38–2.62 (m, 2H, H-4), 4.00 (d, J = 17.10Hz, 1H, H-
2), 4.05 (d, J = 9.00Hz, 1H, Ph2CH), 4.17 (dd,
J = 1.80, 16.20Hz, 1H, H-2), 4.44 (dt, J = 5.20,
8.40Hz, 1H, H-6), 7.16–7.38 (m, 10H, aromatic-CH).
1H NMR (400MHz, CDCl3): 1.34 (m, 1H, H-5), 1.63
(m, 1H, H-5), 1.76 (m, 1H, H-4), 1.96 (m, 1H, H-4),
3.53 (m, 1H, H-3), 3.61 (dd, J = 2.00, 12.60Hz, 1H, H-
2), 3.95–4.10(m, 3H, H-2, H-6, Ph CH), 7.16–7.38 (m,
2
10H, aromatic-CH).
6.1.16. Synthesis of trans-(6-benzhydryl-tetrahydropyran-
3-yl)-(4-fluorobenzy)-amine (16a). 6-Benzhydryl-dihy-
dro-pyran-3-one 12 (0.18g, 0.68mmol) was reacted with
4-fluorobenzylamine (0.08g, 0.68mmol) in the presence
of glacial acetic acid (0.04g, 0.68mmol) in 1,2-dichloro-
ethane (10mL) at room temperature, and then reduced
by NaCNBH3 (0.05g, 0.81mmol) (Procedure D) to yield
a mixture of 16a and 16b. cis-(6-Benzhydryl-tetrahydro-
pyran-3-yl)-(4-fluorobenzyl)-amine 16b was eluted first
(0.04g, 15%).
6.1.19. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-amine (15). cis-5-Azido-2-diphenylmethyl-tetrahy-
dropyran 14 (0.30g, 1.02mmol) in methanol (25mL)
was hydrogenated in presence of 10% Pd–C (0.03g,
10%wt) for 4h (Procedure C) to give cis-(6-benzhy-
dryl-tetrahydropyran-3-yl)-amine 15 (0.21g, 78%) as
an oil.
1H NMR (400MHz, CD3OD): 1.31 (m, 1H, H-5eq),
1.54 (m, 1H, H-5ax), 1.70–1.86 (m, 2H, H-4), 2.90 (br
s, 1H, H-3), 3.66–3.84 (m, 2H, H-2), 3.98 (d,
J = 9.20Hz, 1H, Ph2CH), 4.18 (dt, J = 2.00, 9.60Hz,
1H, H-6), 7.10–7.40 (m, 10H, aromatic-CH).
1H NMR (300MHz, CDCl3): 1.33 (m, 1H, H-5), 1.46–
1.72 (m, 2H, H-5, H-4), 1.94 (m, 1H, H-4), 2.03 (br m,
1H, NH), 2.64 (m, 1H, H-3), 3.57 (dd, J = 1.80,
11.40Hz, 1H, H-2ax), 3.75 (m, 2H, (F)Ph–CH2), 3.95–
4.14 (m, 3H, H-6, H-2eq, Ph2CH), 6.90–7.38 (m, 14H,
aromatic-CH).
Free base was converted to HCl salt: mp 260–261ꢁC,
Anal. [C18H21NOÆHClÆ0.2H2O] C, H, N.
6.1.20. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-fluorobenzyl)-amine (16b). cis-(6-Benzhydryl-tetra-
hydropyran-3-yl)-amine 15 (0.21g, 0.79mmol) was
reacted with 4-fluorobenzaldehyde (0.10g, 0.79mmol)
in the presence of glacial acetic acid (0.05g, 0.79mmol)
in 1,2-dichloroethane (20mL), and then reduced by
NaCNBH3 (0.06g, 0.95mmol) in methanol (5mL)
(Procedure D) to give compound 16b (0.24g, 82%).
Free base was converted into oxalate: mp 229–230ꢁC,
Anal. [C25H26NOFÆ(COOH)2] C, H, N.
Eluted second was trans-(6-benzhydryl-tetrahydro-pyr-
an-3-yl)-(4-fluorobenzyl)-amine 16a (0.11g, 45%).
1H NMR (300MHz, CDCl3): 1.24–1.44 (m, 2H, H-5),
1.55 (m, 1H, H-4), 1.75 (br m, NH), 2.02 (m, 1H, H-
4), 2.68 (m, 1H, H-3), 3.11 (t, J = 10.80Hz, 1H, H-
2ax), 3.76 (s, 2H, (F)Ph–CH2), 3.89 (d, J = 9.00Hz,
1H NMR (300MHz, CDCl3): 1.33 (m, 1H, H-5), 1.46–
1.72 (m, 2H, H-5, H-4), 1.94 (m, 1H, H-4), 2.03 (br m,

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S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
1H, NH), 2.64 (m, 1H, H-3), 3.57 (dd, J = 1.80,
11.40Hz, 1H, H-2ax), 3.75 (m, 2H, (F)Ph–CH2), 3.95–
4.14 (m, 3H, H-6, H-2eq, Ph2CH), 6.90–7.38 (m, 14H,
aromatic-CH).
Free base was converted into oxalate: mp 215–217ꢁC,
Anal. [C26H29NO2Æ(COOH)2] C, H, N.
6.1.24. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(3-indole-methyl)-amine (16f). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.12g, 0.45mmol) was
reacted with 3-indole-carboxaldehyde (0.07g, 0.45
mmol) in the presence of glacial acetic acid (0.03g,
0.45mmol) in 1,2-dichloroethane (20mL), and NaCN-
BH3 (0.03g, 0.54mmol) in methanol (5mL) (Procedure
D) to give compound 16f (0.15g, 82%) as an oil.
Free base was converted into oxalate: mp 229–230ꢁC,
Anal. [C25H26NOFÆ(COOH)2] C, H, N.
6.1.21. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-cyano-benzyl)-amine (16c). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.15g, 0.56mmol) was
reacted with 4-cyanobenzaldehyde (0.07g, 0.56mmol)
in the presence of glacial acetic acid (0.03g, 0.56mmol)
in 1,2-dichloroethane (20mL), and NaCNBH3 (0.04g,
0.67mmol) in methanol (5mL) (Procedure D) to give
compound 16c (0.17g, 80%) as an oil.
1H NMR (300MHz, CDCl3): 1.33 (m, 1H, H-5), 1.48–
1.76 (m, 2H, H-5, H-4), 1.99 (m, 1H, H-4), 2.27 (br s,
1H, NH), 2.79 (m, 1H, H-3), 3.60(dd, J = 1.80,
12.30Hz, 1H, H-2ax), 4.00 (s, 2H, indole-3-CH ),
2
4.02–4.20 (m, 3H, H-6, H-2eq, Ph2CH), 7.00–7.80 (m,
14H, aromatic-CH), 8.42 (s, 1H, indole-NH).
1H NMR (300MHz, CDCl3): 1.36 (m, 1H, H-5), 1.46–
1.58 (m, 1H, H-5), 1.58–1.74 (m, 1H, H-4), 1.93 (m,
1H, H-4), 2.62 (br m, 1H, H-3), 3.59 (dd, J = 1.80,
11.70Hz, H-2ax), 3.83 (m, 2H, (CN)Ph–CH2), 3.95–
4.16 (m, 3H, H-6, H-2eq, Ph2CH), 7.16–7.62 (m, 14H,
aromatic-CH).
Free base was converted into oxalate: mp 177–179ꢁC,
Anal. [C27H28N2OÆ(COOH)20.5H2O] C, H, N.
6.1.25. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(2-indole-methyl)-amine (16g). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.07g, 0.25mmol) was
Free base was converted into oxalate: mp 241–242ꢁC,
Anal. [C26H26N2OÆ(COOH)2] C, H, N.
reacted
with
2-indole-carboxaldehyde
(0.04g,
0.25mmol) in the presence of glacial acetic acid (0.02g,
0.25mmol) in 1,2-dichloroethane (20mL), and then re-
duced by NaCNBH3 (0.02g, 0.3mmol) in methanol
(5mL) (Procedure D) to give compound 16g (0.08g,
82%) as an oil.
6.1.22. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-nitro-benzyl)-amine (16d). cis-(6-Benzhydryl-tetra-
hydropyran-3-yl)-amine 15 (0.10g, 0.38mmol) was
reacted with 4-nitrobenzaldehyde (0.06g, 0.38mmol)
in the presence of glacial acetic acid (0.02g, 0.38mmol)
in 1,2-dichloroethane (20mL), and then reduced by
NaCNBH3 (0.03g, 0.45mmol) in methanol (5mL)
(Procedure D) to give compound 16d (0.12g, 80%) as
an oil.
1H NMR (300MHz, CDCl3): 1.34 (m, 1H, H-5), 1.56
(m, 1H, H-5), 1.69 (tt, J = 3.60, 13.50Hz, 1H, H-4),
1.99 (m, 1H, H-4), 2.27 (br m, 1H, NH), 2.79 (br s,
1H, H-3), 3.60(dd, J = 10.70, 1.60Hz, 1H, H-2ax),
3.96 (s, 2H, 2-indole-CH2), 3.92–4.14 (m, 3H, H-6, H-
2eq, Ph2CH), 6.35 (s, 1H, indole-3-H), 7.05–7.60 (m,
14H, aromatic-CH), 9.1 (s, 1H, indole-NH).
1H NMR (300MHz, CDCl3): 1.35 (m, 1H, H-5), 1.53
(m, 1H, H-5), 1.67 (tt, J = 3.60, 13.50Hz, 1H, H-4),
1.91 (m, 2H, H-4, NH), 2.62 (m, 1H, H-3), 3.58 (dd,
J = 1.80, 9.60Hz, 1H, H-2ax), 3.87 (m, 2H, (NO2)Ph–
CH2), 3.92–4.14 (m, 3H, H-6, H-2eq, Ph2CH), 7.14–
7.54, 8.12–8.20(m, 14H, aromatic-CH).
Free base was converted into oxalate: mp 215–216ꢁC,
Anal. [C27H28N2OÆ(COOH)20.5H2O] C, H, N.
6.1.26. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-hydroxy-benzyl)-amine (16h). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.15g, 0.56mmol) was
reacted with 4-hydroxybenzaldehyde (0.07g, 0.56mmol)
in the presence of glacial acetic acid (0.03g, 0.56mmol)
in 1,2-dichloroethane (20mL), and NaCNBH3 (0.04g,
0.67mmol) in methanol (5mL) (Procedure D) to give
compound 16h (0.17g, 80%) as an oil.
Free base was converted into oxalate: mp 236–238ꢁC,
Anal. [C25H26N2O3Æ(COOH)2] C, H, N.
6.1.23. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-methoxy-benzyl)-amine (16e). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.15g, 0.56mmol) was
reacted with 4-methoxybenzaldehyde (0.08g, 0.56mmol)
in the presence of glacial acetic acid (0.03g, 0.56mmol)
in 1,2-dichloroethane (20mL), and NaCNBH3 (0.04g,
0.67mmol) in methanol (5mL) (Procedure D) to give
compound 16e (0.17g, 78%) as an oil.
1H NMR (400MHz, CDCl3): 1.34 (m, 1H, H-5), 1.50
(m, 1H, H-5), 1.67 (tt, J = 4.00, 13.60Hz, 1H, H-4),
2.02 (m, 1H, H-4), 2.71 (m, 1H, H-3), 3.56 (dd,
J = 1.60, 11.60Hz, 1H, H-2ax), 3.64 (m, 2H, (HO)Ph–
CH2), 3.95 (d, J = 8.00Hz, 1H, Ph2CH), 4.02–4.14 (m,
2H, H-6, H-2eq), 6.52 (m, 2H, aromatic-CH), 6.90–
7.38 (m, 12H, aromatic-CH).
1H NMR (300MHz, CDCl3): 1.35 (m, 1H, H-5), 1.48–
1.76 (m, 2H, H-5, H-4), 1.88–2.02 (m, 1H, H-4), 2.68
(br s, 1H, H-3), 3.59 (dd, J = 12.30, 2.40Hz,1H,
H-2ax), 3.76 (d, J = 7.20Hz, 2H, (CH3O)Ph–CH2),
3.83 (s, 3H, CH3O), 3.98–4.16 (m, 3H, H-6,
H-2eq, Ph2CH), 6.88–6.94, 7.18–7.44 (m, 14H, aro-
matic-CH).
Free base was converted into oxalate: mp 136–138ꢁC,
Anal. [C25H27NO2Æ(COOH)2] C, H, N.

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
6313
6.1.27. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(3,4-dichloro-benzyl)-amine (16i). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.10g, 0.38mmol) was
in 1,2-dichloroethane (20mL), and NaCNBH3 (0.01g,
0.18mmol) in methanol (5mL) (Procedure D) to give
compound 16l (0.05g, 80%) as an oil.
reacted
with
3,4-dichlorobenzaldehyde
(0.07g,
0.38mmol) in the presence of glacial acetic acid (0.02g,
0.38mmol) in 1,2-dichloroethane (20mL), and NaC-
NBH3 (0.03g, 0.45mmol) in methanol (5mL) (Proce-
dure D) to give compound 16i (0.12g, 75%) as an oil.
1H NMR (400MHz, CDCl3): 1.31 (m, 1H, H-5), 1.50
(m, 1H, H-5), 1.64 (m, 1H, H-4), 1.80(br s, 1H, NH),
1.90(m, 1H, H-4), 2.61 (m, 1H, H-3), 3.56 (dd,
J = 1.60, 11.60Hz, 1H, H-2ax), 3.72 (m, 2H, (Br)Ph–
CH2), 3.94–4.30(m, 2H, Ph 2CH, H-2eq), 4.07 (dt,
J = 1.60, J = 9.60Hz, 1H, H-6), 7.00–7.42 (m, 14H, aro-
matic-CH).
1H NMR (500MHz, CDCl3): 1.34 (m, 1H, H-5), 1.52
(m, 1H, H-5), 1.66 (m, 1H, H-4), 1.79 (br s, 1H, NH),
1.91 (m, 1H, H-4), 2.61 (m, 1H, H-3), 3.57 (dd,
J = 1.50, 11.50Hz, 1H, H-2ax), 3.72 (m, 2H,
(Cl,Cl)Ph–CH2), 3.94–4.05 (m, 2H, H-2eq, Ph2CH),
4.08 (dt, J = 2.00, 8.50Hz, 1H, H-6), 7.10–7.50 (m,
14H, aromatic-CH).
Free base was converted into oxalate: mp 250–252ꢁC,
Anal. [C25H26BrNOÆ(COOH)2] C, H, N.
6.1.31. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-iodo-benzyl)-amine (16m). cis-(6-Benzhydryl-tetra-
hydropyran-3-yl)-amine 15 (0.04g, 0.15mmol) was
reacted with 4-iodobenzaldehyde (0.05g, 0.15mmol) in
the presence of glacial acetic acid (0.01g, 0.15mmol) in
1,2-dichloroethane (20mL), and NaCNBH3 (0.01g,
0.18mmol) in methanol (5mL) (Procedure D) to give
compound 16m (0.06g, 81%) as an oil.
Free base was converted into oxalate: mp 251–252ꢁC,
Anal. [C25H25NOCl2Æ(COOH)2] C, H, N.
6.1.28. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(3,4-difluorobenzyl)-amine (16j). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.10g, 0.38mmol) was
reacted
with
3,4-difluorobenzaldehyde
(0.06g,
0.38mol) in the presence of glacial acetic acid (0.02g,
0.38mmol) in 1,2-dichloroethane (20mL), and NaC-
NBH3 (0.03g, 0.45mmol) in methanol (5mL) (Proce-
dure D) to give compound 16j (0.12g, 80%).
1H NMR (400MHz, CDCl3): 1.28 (m, 1H, H-5), 1.50
(m, 1H, H-5), 1.64 (m, 1H, H-4), 1.72 (br s, 1H, NH),
1.90(m, 1H, H-4), 2.60(m, 1H, H-3), 3.56 (dd,
J = 1.60, 12.40Hz, 1H, H-2ax), 3.71 (m, 2H, (I)Ph–
CH2), 3.92–4.02 (m, 2H, Ph2CH, H-2eq), 4.06 (dt,
J = 1.60, 9.20Hz, 1H, H-6), 7.00–7.70 (m, 14H, aro-
matic-CH).
1H NMR (300MHz, CDCl3): 1.34 (m, 1H, H-5), 1.52
(m, 1H, H-5), 1.66 (tt, J = 3.60, 13.50Hz, 1H, H-4),
1.76 (br s, 1H, NH), 1.92 (m, 1H, H-4), 2.61 (m, 1H,
H-3), 3.57 (dd, J = 1.80, 11.40Hz, 1H, H-2ax), 3.72
(m, 2H, (F,F)Ph–CH2), 3.94–4.14 (m, 3H, H-6, H-2eq,
Ph2CH), 6.90–7.38 (m, 14H, aromatic-CH).
Free base was converted into oxalate: mp 243–244ꢁC,
Anal. [C25H26INOÆ(COOH)2] C, H, N.
6.1.32. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(1H-iodo-5-ylmethyl)-amine (16n). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.05g, 0.19mmol) was
Free base was converted into oxalate: mp 234–235ꢁC,
Anal. [C25H25NOF2Æ(COOH)2] C, H, N.
reacted
with
5-indole-carboxaldehyde
(0.03g,
6.1.29. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-benzyl-amine (16k). cis-(6-Benzhydryl-tetrahydropy-
ran-3-yl)-amine 15 (0.03g, 0.11mmol) was reacted with
benzaldehyde (0.01g, 0.11mmol) in the presence of gla-
cial acetic acid (0.01g, 0.11mmol) in 1,2-dichloroethane
(20mL), and NaCNBH3 (0.01g, 0.14mmol) in methanol
(5mL) (Procedure D) to give compound 16k (0.03g,
85%).
0.19mmol) in the presence of glacial acetic acid
(0.01g, 0.19mmol) in 1,2-dichloroethane (20mL), and
NaCNBH3 (0.02g, 0.37mmol) in methanol (5mL)
(Procedure D) to give compound 16n (0.06g, 82%) as
an oil.
1H NMR (400MHz, CDCl3): 1.32 (m, 1H, H-5), 1.50–
1.70(m, 2H, H-5, H-4), 1.95 (m, 2H, H-4, NH), 2.71
(br s, 1H, H-3), 3.57 (dd, J = 2.00, 12.00Hz, 1H, H-
2ax), 3.88 (m, 2H, indole-CH2), 3.96–4.12 (m, 3H,
Ph2CH, H-2eq, H-6), 6.51, 7.10–7.40, 7.57 (m, 15H, aro-
matic-CH), 8.36 (br s, 1H, NH).
1H NMR (300MHz, CDCl3): 1.30(m, 1H, H-5), 1.44–
1.70(m, 2H, H-5, H-4), 1.80(br s, 1H, NH), 1.92 (m,
1H, H-4), 2.64 (m, 1H, H-3), 3.55 (dd, J = 1.80,
11.70Hz, 1H, H-2ax), 3.77 (m, 2H, Ph–CH2), 3.92–
4.10(m, 3H, Ph CH, H-6, H-2eq), 7.00–7.38 (m, 15H,
2
aromatic-CH).
Free base was converted into oxalate: mp 128–130ꢁC,
Anal. [C27H28N2OÆ(COOH)20.5H2O] C, H, N.
Free base was converted into oxalate: mp 208–210ꢁC,
Anal. [C25H27NOÆ(COOH)2] C, H, N.
6.1.33. Synthesis of cis-(6-benzhydryl-tetrahydropyran-
3-yl)-(4-amino-benzyl)-amine (16o). A mixture of cis-(6-
benzhydryl-tetrahydropyran-3-yl)-(4-nitro-benzyl)-amine
(16f) (0.16g, 0.39mmol) and SnCl2/2H2O (0.35g,
1.55mmol) in EtOH/EtOAc (20mL, 7:3) was heated to
reflux for 1.5h (monitored by TLC, Hex/EtOAc/Et3N
5:5:0.4). After removal of the solvent, the residue was
diluted with 10% NaHCO3 and EtOAc and stirred
6.1.30. Synthesis of cis-(6-benzhydryl-tetrahydropyran-3-
yl)-(4-bromo-benzyl)-amine (16l). cis-(6-Benzhydryl-
tetrahydropyran-3-yl)-amine 15 (0.04g, 0.15mmol) was
reacted with 4-bromobenzaldehyde (0.03g, 0.15mmol)
in the presence of glacial acetic acid (0.01g, 0.15mmol)

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S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
vigorously for 30min. After filtration the organic phase
was separated and the aqueous phase was extracted
with EtOAc (20mL · 2). The combined organic phase
was dried over Na2SO4. After removal of the solvent,
flash chromatography gave 16o, cis-(6-benzhydryl-
tetrahydropyran-3-yl)-(4-amino-benzyl)-amine (0.09g,
60%).
tracted with dichloromethane (3 · 20mL). The organic
phase was dried over anhydrous Na2SO4, and the sol-
vent was removed in vacuo. Flash chromatography gave
16p cis-(6-benzhydryl-tetrahydropyran-3yl)-[2-(4-fluoro-
phenyl)-ethyl]-amine (0.13g, yield 81%).
1H NMR (300MHz, CDCl3): 1.20–1.42 (m, 2H, H-5,
NH), 1.61 (m, 1H, H-5), 1.88 (m, 2H, H-4), 2.64 (m,
1H, H-3), 2.72–2.82 (m, 4H, Ph–CH2CH2), 3.55 (dd,
J = 1.80, 11.70Hz, 1H, H-2ax), 3.86–3.98 (m, 2H,
Ph2CH, H-2eq), 4.03 (dt, J = 3.00, 10.00Hz, 1H, H-6),
6.90–7.40 (m, 14H, aromatic-CH).
1H NMR (400MHz, CDCl3): 1.30(m, 1H, H-5), 1.47
(m, 1H, H-5), 1.64 (tt, J = 4.00, 12.80Hz, 1H, H-4),
1.90(m, 1H, H-4), 2.53–2.70(m, 3H, H-3, (NH 2)–
PhCH2), 3.54 (dd, J = 1.60, 11.20Hz, 1H, H-2ax),
3.92–4.00 (m, 2H, Ph2CH, H-2eq), 4.06 (dt, J =
2.40, 9.60Hz, 1H, H-6), 7.06–7.38 (m, 14H, aromatic-
CH).
Free base was converted into oxalate: mp 240–242ꢁC,
Anal. [C26H28NOFÆ(COOH)2] C, H, N.
Free base was converted into oxalate: mp 151–153ꢁC,
Anal. [C25H28N2OÆ2(COOH)20.3H2O] C, H, N.
6.2. Biology
The affinity of test compounds in binding to rat DAT,
SERT, and NET was assessed by measuring inhibition
of binding of 5.0nM [3H]WIN 35,428, 3.5nM [3H]citalo-
pram, and 1.1nM [3H]nisoxetine, respectively, exactly as
described by us previously. Briefly, rat striatum was the
source for DAT, and cerebral cortex for SERT and
NET. Final [Na+] was 30mM for DAT and SERT as-
says, and 152mM for NET assays. All binding assays
were conducted at 0–4ꢁC, for a period of 2h for
[3H]WIN 35,428 and [3H]citalopram binding, and 3h
for [3H]nisoxetine binding. Nonspecific binding of
[3H]WIN 35,428 and [3H]citalopram binding was de-
fined with 100lM cocaine, and that of [3H]nisoxetine
binding with 1lM desipramine. Radioligand Kd values
were 2.1, 3.2, and 2.2nM, respectively. Test compounds
were dissolved in dimethyl sulfoxide (DMSO) and di-
luted out in 10% (v/v) DMSO. Additions from the latter
stocks resulted in a final concentration of DMSO of
0.5%, which by itself did not interfere with radioligand
binding. At lease five triplicate concentrations of each
test compound were studied, spaced evenly around the
IC50 value. For DAT uptake assays, uptake of 50nM
[3H]DA into rat striatal synaptosomes was measured ex-
actly as described by us previously. Briefly, rat striatal
P2 membrane fractions were incubated with test com-
pounds for 8min followed by the additional presence
of [3H]DA for 4min at 25ꢁC. Nonspecific uptake was
defined with 100lM cocaine. Construction of inhibition
curves and dissolvement of test compounds were as de-
scribed above.
6.1.34. Synthesis of cis-N-(6-benzhydryl-tetrahydro-
pyran-3-yl)-2-(4-fluorophenyl)-acetamide (17). Into
a
solution of 4-fluorophenylacetic acid (0.23g, 1.46mmol)
in dichloromethane (25mL) was added oxalyl chloride
(0.22g, 1.76mmol) dissolved in dichloromethane
(5mL) at 0ꢁC, which was followed by addition of one
drop of DMF. The reaction mixture was allowed to
reach at room temperature over a period of 2h. The sol-
vent was removed in vacuo, and the residue was dis-
solved in dichloromethane (5mL) and was added into
a solution of cis-(6-benzhydryl-tetrahydropyran-3-yl)-
amine (0.26g, 0.96mmol) and triethylamine (0.31g,
1.46mmol) in dichloromethane (25mL) at 0ꢁC. After
20min the reaction mixture was allowed to come to
room temperature. After 3h, more dichloromethane
was added and the mixture was washed in turn with
1M NaHCO3, H2O, and brine, then dried over anhy-
drous Na2SO4. The solvent was removed under vacuo,
and the residue was purified by flash chromatography
(hexane/ethyl acetate 7:3) to give cis-N-(6-benzhydryl-
tetrahydropyran-3-yl)-2-(4-fluorophenyl)-acetamide 17
(0.31g, yield 80%) as an oil.
1H NMR (300MHz, CDCl3): 1.10–1.40 (m, 2H, H-5),
1.60–1.93 (m, 2H, H-4), 3.49 (s, 2H, Ph–CH2CO), 3.63
(dd, J = 1.80, 11.70Hz, 1H, H-2ax), 3.70–3.85 (m, 2H,
Ph2CH, H-3), 3.90–4.08 (m, 2H, H-6, H-2eq), 6.90–
7.40(m, 14H, aromatic-CH).
6.1.35. Synthesis of cis-(6-benzhydryl-tetrahydropyran-
3yl)-[2-(4-fluorophenyl)-ethyl]-amine (16p). Into a sus-
pension of NaBH4 (0.21g, 3.33mmol) in dry THF
(20mL) was added BF3ÆEt2O drop wise at 0ꢁC. The mix-
ture was stirred for 1.5h at room temperature and
cooled to 0ꢁC. A solution of cis-N-(6-benzhydryl-tetra-
hydropyran-3-yl)-2-(4-fluorophenyl)-acetamide (0.17g,
0.42mmol) in dry THF (10mL) was added drop wise
into the solution. The mixture was refluxed overnight
and cooled to room temperature. Methanol was added
to quench the reaction followed by removal of solvent
in vacuo. Into the residue was added 20mL 10% HCl/
MeOH and refluxed for 1h. The reaction mixture was
cooled down to room temperature and solid NaHCO3
was added at 0ꢁC to pH9. The aqueous phase was ex-
Acknowledgements
This work was supported by the National Institute of
Drug Abuse, Grant No DA 12449 (A.K.D.). We thank
Janet L. Berfield and Lijuan C. Wang for conducting
many of the binding and uptake assays.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2004.07.069.

S. Zhanget al. / Bioorg. Med. Chem. 12 (2004) 6301–6315
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69.57 6.07 3.01 69.66 6.06 3.01
69.68 6.17 3.04 69.66 6.06 3.01
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70.36 6.68 3.03 69.96 6.31 3.02
62.52 5.23 2.66 62.805.27 2.71
67.09 5.70 2.88 67.07 5.63 2.90
71.86 6.65 3.11 71.88 6.57 3.10
61.57 5.36 2.65 61.605.36 2.66
56.43 4.94 2.45 56.55 4.92 2.45
70.05 6.29 5.40 70.29 6.30 5.65
16o 0.3H2O 62.11 5.73 4.92 62.42 5.89 5.02
16p
15 0.2H2O
69.76 6.34 2.907.103 6.31 2.92
70.41 7.57 4.17 70.32 7.34 4.55
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