The design and synthesis of novel derivatives of the dopamine uptake inhibitors GBR 12909 and GBR 12935. High-affinity dopaminergic ligands for conjugation with highly fluorescent cadmium selenide/zinc sulfide core/shell nanocrystals

Article abstract of DOI:10.1016/S0040-4020(03)01179-7

There is a growing demand for compounds with very high affinities for the dopamine transporter protein (DAT) that can be conjugated to fluorescent markers such as cadmium selenide/zinc sulfide core/shell nanocrystals. This paper describes the design and synthesis of two derivatives of the DAT antagonists GBR 12935 and GBR 12909. These compounds have a high biological affinity for DAT and may be conjugated to nanocrystals via a thiol linkage without a significant reduction in their biological activity. Such conjugates may be used in fluorescent imaging studies.

Full text of DOI:10.1016/S0040-4020(03)01179-7

Tetrahedron 59 (2003) 8035–8047
The design and synthesis of novel derivatives of the dopamine
uptake inhibitors GBR 12909 and GBR 12935. High-affinity
dopaminergic ligands for conjugation with highly fluorescent
cadmium selenide/zinc sulfide core/shell nanocrystals
Ian D. Tomlinson,a John Mason,b Jon N. Burton,a Randy Blakelyb and Sandra J. Rosenthala
,
*
aDepartment of Chemistry, Vanderbilt University, Station B, 351822, Nashville, TN 37235-1822, USA
bDepartment of Pharmacology, Vanderbilt University School of Medicine, Vanderbilt University, Nashville, TN, USA
Received 17 August 2002; revised 28 July 2003; accepted 28 July 2003
Abstract—There is a growing demand for compounds with very high affinities for the dopamine transporter protein (DAT) that can be
conjugated to fluorescent markers such as cadmium selenide/zinc sulfide core/shell nanocrystals. This paper describes the design and
synthesis of two derivatives of the DAT antagonists GBR 12935 and GBR 12909. These compounds have a high biological affinity for DAT
and may be conjugated to nanocrystals via a thiol linkage without a significant reduction in their biological activity. Such conjugates may be
used in fluorescent imaging studies.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
source to be used for simultaneous excitation of all sizes of
nanocrystals.
Semiconductor nanocrystals, or ‘quantum dots’, are crystals
with diameters on the 1–10 nm scale and consist of between
a few hundred to several thousand atoms. These materials
have the same crystal packing as the bulk material;
however, their small size gives them unique physical
properties.1 For instance, when a cadmium selenide
nanocrystal core is passivated with a shell of a wider band
gap material such as zinc sulfide, highly fluorescent
cadmium selenide/zinc sulfide core/shell nanocrystals are
obtained. These core/shell nanocrystals have several unique
optical properties.2–4 They have fluorescent properties that
are superior to organic dyes such as increased photo stability
and brightness. This enables fluorescence experiments
with core/shell nanocrystals to be performed over longer
time-periods and with lower concentrations than can be
achieved with conventional dyes. Additionally the fluor-
escence emission band is size tunable and narrow.1 Small
cores give rise to fluorescence at blue wavelengths while
larger cores give rise to emission at red wavelengths. Their
narrow emission bands enable several different sizes of core/
shells to be used at once in multiplexing experiments.5,6
Finally, the absorption of core/shell nanocrystals is a
continuum above the band gap, enabling a single light
We are interested in using cadmium selenide/zinc sulfide
core/shell nanocrystals as probes for integral membrane
proteins involved in neural signaling. In order to make
nanocrystals biologically active, a biologically active
molecule must be attached to the surface of the nanocrystal
without loosing its activity. Several groups have reported
attaching proteins and antibodies to fluorescent nanocrystals
and have demonstrated that these are biologically active.5–8
Our approach is to attach small molecules such as drugs and
neurotransmitters to nanocrystals. We have recently demon-
strated that derivatives of serotonin may be attached to
nanocrystals while maintaining activity.9 Using these
conjugates we have visualized serotonin transporter proteins
on the surface of living HEK cells.10 In addition to enabling
dynamic imaging experiments in living tissue, drug-
conjugated fluorescent nanocrystals could potentially be
utilized in high throughput screening assays for drug
discovery, cell sorting applications via flow cytometry,
and in vivo imaging.
Changes in dopamine receptor distribution and in the
dopamine transporter system have been shown to be
important factors in a number of different disease states
including Parkinson’s disease, Huntington’s chorea and
schizophrenia.11 Further, it has been shown that the
dopamine transporter system may be responsible for
the locomotor and reinforcing effects of cocaine.12 The
Keywords: cadmium selenide/zinc sulfide core/shell nanocrystals; thiol;
dopamine transporter; ligand; inhibition.
*
Corresponding author. Tel.: þ1-615-322-2633; fax: þ1-615-343-1234;
e-mail: sjr@femto.cas.vanderbilt.edu
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)01179-7

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Figure 1. Two potent inhibitors of dopamine reuptake GBR 12909 and GBR 12935.
dopamine transporter complex (DAT) is a protein located on
the presynaptic membranes of mesotelencephalic dopamin-
ergic neurons. These neurons are located in the substantia
nigra and ventral tegmentum in the midbrain and converge
in the basal ganglia of the forebrain.11 DAT is a member of
the family of Naþ Cl2 dependent transporters and transports
dopamine via a coupled sodium chloride transport mecha-
nism. We are developing high-affinity ligands for DAT that
may be conjugated to cadmium selenide/zinc sulfide core/
shell nanocrystals in order to image the localization and
distribution of the dopamine transporter protein within
neuronal cells. By imaging neurons containing DAT we
hope to understand more about these disease states and the
dopaminergic role in cocaine addiction.
Many different classes of compounds have been demon-
strated to bind to DAT.13–16 The DAT antagonists GBR
12909 and GBR 12935 (Fig. 1) have been extensively
studied.17–19 These compounds have been shown to have a
high affinity for DAT and slowly dissociate from the
transporter, resulting in a long duration of action.20 The
biological properties of these compounds make the GBR
compounds ideal candidates for ligands that can be attached
to fluorescent core/shell nanocrystals. They bind tightly to
Figure 2. The DAT antagonists prepared in this study.

I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
8037
Table 1. The IC50 values obtained for the compounds used in this study
(NA, not applicable)
activity.24 Consequently, it was decided to attach a linker
arm to this position. The other end of the linker arm is
designed so that it can be attached to the surface of the
nanocrystal. A thiol is used for the point of attachment to
the nanocrystal, as thiols are known to bind tightly to the
surfaces of cadmium selenide/zinc sulfide core/shell
nanocrystals. It is hoped that the linker arm will reduce
steric interactions between the drug and nanocrystal and the
biological activity of the bound ligand will be comparable to
that of the free ligand. Our synthetic sequence is designed so
that the linker arm is attached to the derivative of GBR
12909 or GBR 12935 at the end of the synthetic scheme.
Figure 2 shows the different ligands designed during the
course of this study and their biological activities are
summarized in Table 1.
Compound no.
IC50 of free ligand (nM)
IC50 of bound ligand (nM)
4
7
12
18
30
6000
18
NA
NA
32
10
140
DAT and their IC50 values are in the nanomolar range. This
should enable the development of fluorescence assays with
high sensitivity and selectivity, with little or no binding to
other transporter proteins, such as the serotonin transporter
(SERT). In this paper we report the synthesis of analogues
of GBR 12909 and GBR 12935 designed such that they can
be attached to the surface of cadmium selenide/zinc sulfide
core/shell nanocrystals.
2. Results and discussion
We require ligands that have a high affinity for DAT when
bound to the nanocrystal and are inexpensive to synthesize
from commercially available reagents. Structure activity
relations for the GBR compounds21–23 suggest bulky
substituents at the para position on the phenyl propyl
moiety are tolerated without a large reduction in biological
We considered many different methodologies for attaching
the linker arm to the drug and concluded that an amide
linkage would be the simplest way of attaching a linker arm.
In order to ascertain whether or not an amide linkage would
reduce biological activity we synthesized compound 4. The
synthesis of this compound is outlined in Scheme 1. In this
Scheme 1. (i) (a) Oxalyl chloride, DMF, (b) CH2Cl2, triethylamine, reflux 18 h, yield¼99%; (ii) AlH3, yield¼68%; (iii) SnCl2, yield¼78.6%; (iv) acetyl
chloride, triethylamine, CH2Cl2, yield¼25%.

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I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
Scheme 2. (i) Chromium (IV) oxide, H2SO4, yield¼93%; (ii) 4-methoxy-a-toluenethiol, sodium ethoxide, yield¼94%; (iii) (a) oxalyl chloride, DMF,
(b) triethylamine, (6), CH2Cl2, reflux 4 days, yield¼28.9%.
synthetic scheme, 1-[2-[bisphenylmethoxy]ethyl]piperazine
was synthesized using the method described by Van Der Zee
et al.21 This was reacted with the acid chloride derivative of
4-nitrophenyl propionic acid, giving 1-[2-[bisphenyl-
methoxy]ethyl]-4-(3-(4-nitrophenyl)-1-oxopropyl)pipera-
zine (1) in a 99% yield. The amide was reduced using
alane–N,N-dimethylamine complex in THF25 and 1-[2-[bis-
phenylmethoxy]ethyl]-4-(3-(4-nitrophenyl)propyl)pipera-
zine (2) was obtained in a 68% yield. The para-nitro group
was reduced using tin(II)chloride giving 1-[2-[bisphenyl-
methoxy]ethyl]-4-(3-(4-aminophenyl)propyl)piperazine (3)
as a red oil in a 78.6% yield. Finally, the amine was acylated
using acetyl chloride, giving N-(4-{3-[4-(2-benzhydryloxy-
ethyl)-piperazin-1-yl]-propyl}-phenyl)-acetamide (4) in a
25% yield. Compound 4 was assayed against the transporter
protein by using a competition assay with tritiated dopamine
and was shown to have an IC50 of 30 nM. This compares
favorably with the reported IC50 of 3.7 nM26 for GBR
12935. Thus we concluded that an amide linkage attached to
the GBR compounds would not be detrimental to biological
activity.
Our first ligand, N-(4-(3-[4-(2-benzhydryloxyethyl)pipera-
zine-1-yl]propyl)phenyl-2-[2-(2-mercaptoethoxy)ethoxy]-
acetamide (2), was designed so that it is comprised of a
polyethylene glycol chain that was attached to a
derivative of GBR 12935. The other end of the polyethylene
chain terminated in a thiol and was to be the point of
attachment to the nanocrystal. A polyethylene glycol linker
was selected as it was hoped that this would enhance
the water solubility of the nanocrystal conjugate by
interacting with solvated cations in a manner similar to
crown ethers.
We developed two synthetic routes that can be used to
attach generic polyethylene glycol linker arms to the GBR
derivative, these are shown in Schemes 2 and 3. Both routes
use 2-(2-(2-chloroethoxy)ethoxy)ethanoic acid (5), which
was obtained in a 93% yield via a Jones oxidation27 of 2-(2-
(2-chloroethoxy)ethoxy)ethanol. In our first route (Route 1),
the chlorine atom in 2-(2-(2-chloroethoxy)ethoxy)ethanoic
acid was displaced with the sodium salt of 4-methoxybenzyl
mercaptan by refluxing for 18 h in absolute ethanol. This
Scheme 3. (i) CDI, 2-(2-(2-chloroethoxy)ethoxy)ethanoic acid, THF, 18 h, yield¼42%; (ii) potassium thioacetate, DMF, 18 h, yield¼73%; (iii) ammonia,
yield¼94%.

I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
8039
Scheme 4. (i) Method A: (a) SOCl2, DMF, (b) Et3N, CH2Cl2, 4 days 258C, yield¼23%. Method B: 11-bromoundecanoic acid, CDI, DMF, yield¼28%;
(ii) potassium thioacetate, DMF, 18 h, yield¼31%; (iii) ammonia, yield¼51%.
gave {2-[2-(4-methoxy-benzylsulfanyl)-ethoxy]-ethoxy}-
acetic acid (6) in a 94% yield as a colorless oil. We decided
to use a thiol protected with the 4-methoxybenzyl group, as
it can be removed with several reagents, including mercury
or silver salts, as well as by acid hydrolysis.28 We
considered using other protecting groups for the thiol,
such as disulfides,29,30 we rejected these as they required
strong acids and high temperatures. We thought that the
bisphenyl methyl ether in the GBR derivative might be
cleaved under such conditions.
propyl}-phenyl)-2-[2-(2-chloro-ethoxy)-ethoxy]-acetamide
(8) in a 42% yield as a clear oil. The chlorine atom in 8
was displaced using thioacetate to give thioacetic acid
S-(2-{2-[(4-{3-[4-(2-benzhydryloxy-ethyl)-piperazin-1-yl]-
propyl}phenylcarbomyl)-methoxy]-ethoxy}ethyl) ester (9)
as a brown oil in a 73% yield. Compound 9 was hydrolyzed
using methanolic ammonia to give the desired product (7) in
a 94% yield.
The IC50 value for compound 7 was 6 mM. We hypothe-
sized that this low biological activity may be due to the
polyethylene glycol chain interacting with the transporter
protein, thereby reducing the affinity of this ligand for DAT.
Alternatively the conformation of the PEG linker may result
in unfavorable steric interactions with the DAT protein.
Such steric interactions could reduce the affinity of the
compound for DAT. The ability of PEG to interact with
cations may also reduce the affinity of this compound for the
DAT protein by interacting with cations. Such complexes
may have lower affinity for DAT if the compound is binding
to region that carries a positive charge. As this ligand had
too low an affinity for DAT to be used in a fluorescence
assay system, it was decided to replace the polyethylene
glycol chain with a simple alkyl chain. It has been shown
that alkyl substituents in this region do not significantly
reduce biological activity and we thought an analogue of
We discovered that when the acid chloride of compound 6
was added to 1-[2-[bisphenylmethoxy]ethyl]-4-(3-(4-amino-
phenyl)propyl)piperazine (3) and stirred at reflux in
methylene chloride in the presence of triethylamine under
argon for 4 days, 7 was produced in a 28.9% yield. Thus, we
were able to couple the linker arm and deprotect the thiol in
one pot. Whilst this route worked the percentage yield of
final product was low. When we resynthesized the final
product 7, we observed that the reaction gave the product in
a low to moderate yield intermittently. In order to overcome
this variability we developed our second route (Route 2).
In this route 2-(2-(2-chloroethoxy)ethoxy)ethanoic acid
(5) was coupled to 1-[2-[bisphenylmethoxy]ethyl]-4-(3-(4-
aminophenyl)propyl)piperazine (3) using CDI in THF. This
gave N-(4-{3-[4-(2-benzhydryloxy-ethyl)piperazin-1-yl]-

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I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
Scheme 5. (i) 11-Bromoundecanoic acid, CDI, DMF, yield¼30%; (ii) potassium thioacetate, DMF, 18 h, yield¼54%; (iii) ammonia, yield¼51%.
GBR 12935 with a simple alkyl chain at this position might
retain activity.
sulfide core/shell nanocrystals an IC50 of 32 nM was
obtained. These results appear to confirm our original
hypothesis that the biological activity of compound 7 is
reduced due to unfavorable interactions with the DAT
transporter and the polyethylene glycol chain.
We decided to synthesize 11-mercaptoundecanoic acid
(4-{3-[4-(2-benzhydryloxyethyl)-piperazin-1-yl]-propyl}-
phenyl)-amide (12) as 11-bromoundecanoic acid is com-
mercially available and inexpensive. The route we used to
synthesize this ligand is shown in Scheme 4. Initially 1-[2-
[bisphenylmethoxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (3) was coupled to the acid chloride of
11-bromoundecanoic acid in methylene chloride at room
temperature to give 11-bromoundecanoic acid (4-{3-[4-(2-
benzhydryloxy-ethyl)-piperazine-1-yl]-propyl}-phenyl)-
amide (10) in a 23% yield. We tried to increase this yield by
coupling the 11-bromoundecanoic acid to 3 in dry DMF
using CDI (Method B).31 This increased the yield of 10
slightly to 28%. In the next step, we displaced the bromine
atom in 10 using potassium thioacetate in dry DMF. This
nucleophilic displacement gave thioacetic acid S-[10-(4-{3-
[4-(2-benzhydryloxy-ethyl)-piperazin-1-yl]-propyl}-phenyl-
carbamoyl)-decyl] ester (11) in a 31% yield. The thioacetate
was hydrolyzed using ammonia dissolved in methanol under
argon at room temperature to give compound 12 in a 51%
yield. The biological activity of compound 12 was measured
and it was found to have an IC50 of 18 nM. After
conjugating compound (12) to cadmium selenide/zinc
The fluorinated derivative of GBR 12935, GBR 12909, has
been shown to have a greater selectivity for DAT over the
serotonin transporter (SERT) than GBR 12935.32 Conse-
quently we decided to synthesize a derivative of this ligand,
as it may be of interest in future studies to compare the
nanocrystal conjugates of the two different ligands in
fluorescent biological assays. Our synthetic route is outlined
in Scheme 5. 1-[2-[4,40-Fluorobenzhydryloxy]ethyl]pipera-
zine was synthesized using the method described by
Buzas.33 This was coupled to 4-nitrophenyl propionic acid
via the acid chloride to give 1-[2-[4,40-fluorobenzhydryloxy]-
ethyl]-4-(3-(4-nitrophenyl)-1-oxopropyl)piperazine (13) in
a 97% yield. Alane was used to reduce the resulting amide
giving an 80% yield of 1-[2-[4,40-fluorobenzhydryl-
oxy]ethyl]-4-(3-(4-nitrophenyl)propyl)piperazine (14).
Reduction of the nitro functionality in 14 was accomplishe0d
by refluxing 14 in ethanol with tin(II)chloride. 1-[2-[4,4 -
Fluorobenzhydryloxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (15) was obtained in a 92% yield. We coupled
11-bromoundecanoic acid to compound 15 using CDI,

I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
8041
giving 11-bromoundecanoic acid (4-{3-[4-(4,40-fluorobenz-
hydryloxy-ethyl)-piperazine-1-yl]-propyl}-phenyl)-amide
(16) in a 28% yield. The thioacetate was synthesized by
reacting 16 with potassium thioacetate giving thioacetic
acid S-[10-(4-{3-[4-(2-(4,40-fluorobenzhydryloxy)-ethyl)-
piperazin-1-yl}-propyl}-phenylcarbamoyl)-decyl] ester
(17) in a 54% yield. Subsequent hydrolysis of 17 with
ammonia resulted in a 51% yield of 11-mercapto-undeca-
noic acid (4-{3-[4-(2-(4,40-fluorobenzhydryloxy)-ethyl)-
piperazin-1-yl]-propyl}-phenyl)-amide (18). When 18
was assayed it was found to have an IC50 of 10 nM
which compares favorably with GBR 12909 which has an
IC50 3.7 nM.32 Once again this was attached to quantum
dots and the IC50 for the conjugated dots was found to be
140 nM.
process generated the sodiated molecular ion (usually as the
singly-charged species), denoted as (MþNa)þ. However, in
some cases, acetic acid or trifluoroacetic acid was used to
generate the protonated molecular ion (MþH)þ instead.
Electron impact (EI) ionization was performed with a
Kratos MS-25, using 70 eV ionization conditions. Samples
for elemental analysis were routinely dried at ca. 10 mm Hg.
Elemental analysis was performed by Atlantic Microlabs,
Georgia and the analysis is corrected for the presence of
sodium and water when necessary. IR was performed using
silver chloride plates with neat samples on a ATI Mattson
Genesis Series FT IR machine.
4.1. Data for compounds
4.1.1. 1-[2-[Bisphenylmethoxy]ethyl]-4-(3-(4-nitrophenyl)-
1-oxopropyl)piperazine (1). para-Nitrohydrocinnamic
acid (2.8 g, 9.5 mmol) is added to dry toluene (100 mL),
in a 250 mL round bottomed flask equipped with a stirrer
and a reflux condenser. Oxalyl chloride (1 mL) was added,
after which a catalytic quantity of dry DMF (2 drops) was
also added. Then the mixture was stirred at room
temperature for 2 h. The solvent was removed by evapo-
ration and the crude acid chloride was dissolved in dry
dichloromethane (100 mL). Dry triethylamine (10 mL) and
1-[2-[bisphenylmethoxy]ethyl]piperazine (1.84 g, 9.5 mmol)
were dissolved in dry dichloromethane (50 mL) and added
to the solution of p-nitrohydrocinnamyl chloride. The
mixture was heated at reflux for 18 h under argon in a
250 mL round bottomed flask equipped with a stirrer and
reflux condenser. Then the solvent was removed under
reduced pressure and the product was purified using silica
gel chromatography eluted with dichloromethane 96%–
methanolic ammonia. The resulting yellow oil was
converted to the yellow maleate salt by dissolving the
base in diethyl ether (50 mL) and adding an ethereal
solution of maleic acid. The resulting solid was collected by
filtration and washed with diethyl ether (2£150 mL). The
maleate was dried under reduced pressure, to yield 4.3 g
3. Conclusions
We have developed a synthetic methodology for two ligands
with high affinity for the DAT transporter protein. We have
shown that when these ligands are conjugated to cadmium
selenide/zinc sulfide core/shell nanocrystals the biological
activity of the ligand remains high. Such conjugates will be
used for fluorescent imaging of neuronal cells in future
studies.
4. Experimental
2-(2-(2-Chloroethoxy)ethoxy)ethanol, alane, 11-bromo-
undecanoic acid, potassium thioacetate and tin(II)chloride
were purchased from the Aldrich Chemical Company.
4-Nitrophenyl propionic acid was obtained by nitrating
hydrocynamic acid and the melting point of the resulting
product was consistent with commercially available
4-nitrophenyl propionic acid. Reagents were used as they
were received. Cadmium selenide/zinc sulfide core/shell
nanocrystals were provided by Quantum Dot Corporation.
Thin-layer chromatography was carried out on precoated
plates and the products were visualized using UV light.
Column chromatography on silica refers to silica gel
obtained from Scientific Adsorbents Inc. catalogue number
02826-25. All NMR data were obtained using CDCl3 as
a solvent unless otherwise stated. All NMR measurements
were performed using a Brucker 300 MHz machine.
Chemical shifts were measured relative to TMS and
coupling constants are measured in Hz. Low resolution
mass spectra (MS) were obtained from a Finnegan Thermo
quest TSQ 7000 triple quadrapole LC-MS equipped with an
API-1 electrospray ionization source (ES). High resolution
MS were obtained either at the University of Notre Dame
mass spectrometry facility using a JOEL GCmate employ-
ing FAB as the ionization method, or at Ohio State
University using electrospray ionization (ESI) mass spec-
trometry. ESI analyses were performed with a 3-Tesla
Finnigan FTMS-2000 Fourier Transform mass spectro-
meter. Samples were sprayed from a commercial Analytica
electrospray ionization source, and then focused into the
FTMS cell using a home-built set of ion optics. For ESI
analysis, most compounds were sprayed from a micromolar
concentration of the analyte in various solvent mixtures,
such as tetrahydrofuran/CH3OH, with added NaCl. This
1
(99%) of the product, mp 122–1238C. H NMR (CDCl3)
d 2.45 (t, J¼4.9 Hz, 4H), 2.66–2.68 (m, 4H), 3.06 (t,
J¼7.6 Hz, 2H), 3.95 (t, J¼4.9 Hz, 2H), 3.60–3.65 (m, 4H),
5.36 (s, 1H), 7.19–7.41 (m, 12H), 8.08–8.12 (d, 2H); 13C
NMR (CDCl3) d 30.7, 33.7, 41.5, 45.1, 41.5, 45.1, 52.9,
53.3, 57.5, 66.6, 83.6, 123.3, 126.6, 127.2, 128.1, 129.1,
141.9, 146.1, 149.2, 169.3; calculated for C28H31N3O4 m/z
FAB (MþH) 474.2393; found 474.2398; IR (neat) CH2
2821–2938 cm21 (m), CvO 1641 cm21 (s).
4.1.2. 1-[2-[Bisphenylmethoxy]ethyl]-4-(3-(4-nitrophenyl)-
propyl)piperazine (2). 1-[2-[Bisphenylmethoxy]ethyl]-4-
(3-(4-nitrophenyl)-1-oxopropyl)piperazine (5 g, 14.8 mmol)
was dissolved in dry THF (100 mL) in a 250 mL round
bottomed flask equipped with a stirrer and a reflux
condenser. Alane in toluene (0.5 M, 59 mL) was added
and stirred at room temperature for 30 min. The reaction
was quenched with sodium hydroxide solution (10%,
200 mL) and the aqueous solution was extracted with
diethyl ether (3£150 mL). It was dried over magnesium
sulfate filtered and evaporated and the product was purified
using silica gel chromatography eluted with a gradient
system eluted with ethyl acetate 90%–methanol to ethyl
acetate 87%–methanol 10%–triethylamine. This gave

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I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
1
3.35 g (68%) of the product as a pale yellow oil. H NMR
(CDCl3) d 1.78 (t, J¼6.5 Hz, 4H), 2.30 (t, J¼7.1 Hz, 2H),
2.43 (br s, 4H), 2.53 (br s, 4H), 2.67 (t, J¼2.9 Hz, 2H), 3.59
(t, J¼3.0 Hz, 2H), 5.37 (s, 1H), 7.15–7.35 (m, 12H), 8.04–
8.06 (d, 2H); 13C NMR (CDCl3) d 27.7, 33.0, 52.8, 53.3,
57.0, 57.6, 66.6, 83.5, 123.1, 126.6, 127.0, 127.9, 128.8,
141.0, 145.8, 149.8; calculated for C28H33N3O3 m/z ESI
(MþNa) 482.2420; found 482.2392; IR (neat) CH2 2939–
2809 cm21 (m).
solution of sulfuric acid (1.5 M, 60 mL) containing
chromium VI oxide (5.79 g, 38 mmol) at 08C. Upon
complete addition of the alcohol the solution was allowed
to warm to room temperature and stirred at room
temperature for 18 h. Inorganic chromium salts were
removed by filtration and the solution was concentrated
under reduced pressure.9 The crude product was extracted
from the solution using dichloromethane (3£100 mL) and
the combined extracts were dried over magnesium sulfate.
After filtration and evaporation under reduced pressure the
crude product was obtained as a colorless oil 1.7 g (93%).
This was used without further purification. 1H NMR
(CDCl3) d 3.90–3.54 (m, 8H), 3.95 (s, 2H), 8.56 (s, 1H);
13C NMR (CDCl3) d 44.2, 69.9, 72.0, 72.5, 72.9, 175.8;
calculated for C6H11ClO4 m/z ESI (MþNa) 205.0238; found
205.0230; IR (neat) OH 3150 cm21 (br s), CH2 2920 (m),
CvO 1737 cm21 (s).
4.1.3. 1-[2-[Bisphenylmethoxy]ethyl]-4-(3-(4-aminophe-
nyl)propyl)piperazine (3). 1-[2-[Bisphenylmethoxy]-
ethyl]-4-(3-(4-nitrophenyl)-1-oxopropyl)piperazine (0.9 g,
2.8 mmol) was dissolved in absolute ethanol (10 mL) in a
100 mL round bottomed flask equipped with a stirrer and
reflux condenser. Tin(II)chloride dihydrate (2.6 g) was
added and the mixture was heated at reflux for 90 min.
The solution was poured into crushed ice and a solution of
sodium carbonate (5%) in water was added until a pH of 8 is
obtained. The aqueous solution was extracted with ethyl
acetate (3£200 mL) and this was dried over magnesium
sulfate. The product was purified using column chromato-
graphy on silica gel eluted with ethylacetate 92%–methanol
5%–triethylamine, to give 0.66 g (78.6%) of the product as
a pale yellow oil. The base was converted to the oxalate salt
by adding oxalic acid (4 g) dissolved in methanol (50 mL)
to a solution of the base dissolved in methanol (50 mL). The
resulting salt was collected by filtration and washed with
methanol (2£50 mL). After drying under reduced pressure
the oxalate was obtained as a brown solid, mp 156–1588C.
1H NMR (CDCl3) d 1.73 (t, J¼6.2 Hz, 4H), 2.32 (t, J¼
6.2 Hz, 2H), 2.38–2.51 (m, 8H), 2.65 (t, J¼5.9 Hz, 2H),
3.57 (t, J¼5.9 Hz, 2H), 3.64 (br, NH2), 5.35 (s, 1H), 6.51–
6.54 (d, 2ArH), 6.91–6.94 (d, 2ArH), 7.18–7.34 (m,
10ArH); 13C NMR (CDCl3) d 28.5, 32.5, 52.9, 53.3, 57.6,
60.0, 66.6, 83.5, 114.8, 126.7, 127.1, 127.7, 128.0, 131.5,
142.0, 144.1; calculated for C28H35N3O m/z FAB (MþH)
430.2858; found 430.2862; IR (neat) NH2 3349 cm21 (br s),
CH2 2809–2938 (m).
4.1.6. {2-[2-(4-Methoxybenzylsulfanyl)-ethoxy]-ethoxy}-
acetic acid (6). Sodium (0.253 g, 1.1 mmol) was added
to absolute ethanol (50 mL) at 08C and stirred for 30 min.
4-Methoxy-a-toluenethiol (0.78 mL, 6 mmol) was added
and this mixture was stirred at room temperature for 30 min.
2-(2-(2-Chloroethoxy)ethoxy)ethanoic acid (1 g, 5.5 mmol)
was added and the mixture was heated at reflux for 18 h. The
mixture was cooled to room temperature poured into
distilled water (100 mL) and acidified with hydrochloric
acid (2 M, 1£50 mL). The product was extracted with
dichloromethane (2£100 mL) and the organic solution was
dried over magnesium sulfate. After filtering the organic
solution, it was evaporated under reduced pressure. The
product was purified using column chromatography on
silica eluted with a gradient system running from pure
dichloromethane to dichloromethane 90%–methanol 10%.
This gave 1.24 g (94%) of the product as a colorless oil. 1H
NMR (CDCl3) d 2.58 (t, J¼6.8 Hz, 2H), 3.54–3.59 (m, 4H),
3.67 (br s, 4H), 4.14 (s, 2H), 6.81 (d, 2ArH), 7.2 (d, 2ArH);
13C NMR (CDCl3) d 29.6, 35.2, 54.7, 67.6, 69.4, 70.0, 70.2,
113.3, 129.6, 129.7, 157.9, 173.3; CHS (0.5H2O) calculated
for C14H20O5S, C¼54.35, H¼6.84, S¼10.36; found
C¼54.46, H¼6.63, S¼10.90; IR (neat) OH 3300–
3000 cm21 (br s), CH2 2916–2837 (m), CvO 1739 cm21
(s).
4.1.4. N-(4-{3-[4-(2-Benzhydryloxy-ethyl)-piperazin-1-
yl]-propyl}-phenyl)-acetamide (4). 1-[2-[Bisphenyl-
methoxy]ethyl]-4-(3-(4-aminophenyl)propyl)piperazine
(0.5 g, 1.2 mmol) was dissolved in dry dichloromethane.
Triethylamine (1 mL) was added followed by acetyl
chloride (1 mL). The mixture was stirred at room tempera-
ture overnight then evaporated under reduced pressure. The
product was purified by column chromatography on silica
gel eluted with methanol 4%–methylene chloride. This
4.1.7. N-(4-(3-[4-(2-Benzhydryloxyethyl)piperazin-1-yl]-
propyl)phenyl-2-[2-(2-mercaptoethoxy)ethoxy]aceta-
mide (7). Route 1. 8-(4-Methoxybenzylthio)-3,6-dioxa-
octanoic acid (0.6 g, 2.2 mmol) was dissolved in dry
toluene (50 mL) under nitrogen in a 100 mL round
bottomed flask equipped with a stirrer and a reflux
condenser. Oxalyl chloride (0.5 mL) and a catalytic quantity
of dimethyl formamide (1 drop) were added. The solution
was stirred at room temperature for 2 h, then evaporated
under reduced pressure. The resulting crude 8-(4-methoxy-
benzylthio)-3,6-dioxaoctonyl chloride was dissolved in dry
dichloromethane (100 mL) in a 250 mL round bottomed
flask equipped with a reflux condenser and a stirrer. 1-[2-
[Bisphenylmethoxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (0.66 g, 2.2 mmol) in dry tetrahydrofuran and
triethylamine (5 mL) are added and the mixture was allowed
to reflux under nitrogen for 4 days. Then the solvent was
evaporated and the product was columned on silica eluted
with ethyl acetate 93%–methanol 5%–triethylamine 2%.
1
gave 0.14 g (25%) of the product as a pale brown oil. H
NMR (CDCl3) d 1.75–1.85 (m, 2H), 2.20 (s, 3H), 2.38 (t,
J¼7.9 Hz, 2H), 2.40–2.67 (m, 14H), 3.55 (t, J¼5.7 Hz),
5.29 (s, 1H), 6.92 (d, 2ArH), 7.17–7.28 (m, 12ArH); 13C
NMR (CDCl3) d 26.9, 27.8, 33.1, 52.7, 53.0, 57.6, 57.6,
66.7, 83.9, 126.9, 127.4, 128.3, 128.4, 129.7, 137.1, 142.0,
142.6, 173.0; calculated for C30H37N3O2 m/z ESI (MþH)
472.2964; found 472.2973; IR (neat) C(CvO)NH
3249 cm21 (br s), CH2 2939–2816 (m), CvO 1676 cm21
(s).
4.1.5. 2-(2-(2-Chloroethoxy)ethoxy)ethanoic acid (5).
2-(2-(2-Chloroethoxy)ethoxy)ethanol (1.69 g, 10 mmol)
was dissolved in acetone (50 mL) and added dropwise to a

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8043
˚
The crude product was converted to the oxylate salt by
dissolving it in methanol (50 mL) and adding oxalic acid
(1 g) dissolved in methanol (20 mL). The resulting solid was
left standing at room temperature for 18 h and removed by
filtration. The oxalate salt was converted back to the base
and the product was purified by column chromatography on
silica eluted with a gradient system running from dichloro-
methane 90%–methanol 10% to dichloromethane 87%–
methanol 10%–triethylamine 3%. This gave the product as
a yellow oil; it was converted back to the oxylate salt, as
described above and this was filtered and air dried to yield
5.2 mmol) and 4 A molecular sieves (8 pellets) were added.
The mixture was stirred at room temperature over night and
filtered. Diethyl ether (100 mL) was added to the filtrate and
it was washed with sodium bicarbonate solution (2 M,
1£50 mL). After drying the organic solution over mag-
nesium sulfate the organic solvent was evaporated under
reduced pressure and the product was purified by column
chromatography on a silica column eluted with dichloro-
methane–methanol (5%). This gave 0.60 g (73%) of the
product as a pale brown oil. 1H NMR (CDCl3) d 1.72–1.82
(m, 2H), 2.25 (s, 3H), 2.30–2.60 (m, 15H), 2.66 (t, J¼
5.9 Hz, 2H), 3.10 (t, J¼6.4 Hz, 2H), 3.56–3.62 (m, 8H),
4.06 (s, 2H), 5.36 (s, 1H), 7.10 (d, 2ArH), 7.12–7.34 (m,
10ArH), 7.44 (d, 2ArH), 8.70 (br s, NH);); 13C NMR
(CDCl3) d 28.2, 30.1, 32.7, 52.8, 53.2, 53.3, 57.4, 57.5, 66.6,
69.2, 69.3, 70.1, 70.6, 83.4, 119.6, 126.6, 127.0, 127.9,
128.4, 134.7, 137.9, 141.9, 167.4, 194.8; IR (neat)
C(vO)N–H 3347 cm21 (br s), CH2 2940–2816 cm21
(m), CvO 1690 cm21 (s); calculated for C36H47N3O5S
m/z ESI (MþNa) 656.3129; found 656.3100.
1
0.28 g (28.9%) of the product as a yellow solid. H NMR
(CDCl3) d 1.80 (t, J¼9.0 Hz, 4H), 2.20 (s, 3H), 2.38 (t,
J¼8.0 Hz, 2H), 2.50–2.60 (m, 8H), 2.65 (t, J¼5.7 Hz, 2H),
3.55 (t, J¼5.8 Hz, 2H), 5.29 (s, 1H), 6.89–6.90 (d, 2ArH),
7.13–7.27 (m, 12ArH); 13C NMR (CDCl3) d 26.9, 27.8,
33.1, 52.7, 53.0, 57.6, 57.6, 66.7, 83.9, 126.9, 127.4, 128.3,
128.4, 129.7, 137.1, 142.0, 142.6, 173.0; m/z ES (Mþ)
591.66 (low res); calculated for C34H45N3O4S m/z ESI
(MþNa) 614.3023; found 614.3070; IR (neat) C(vO)N–H
3300 cm21 (br s), CH2 2900–2750 cm21 (m), CvO
1680 cm21 (s).
4.1.10. 11-Bromoundecanoic acid (4-{3-[4-(2-benzhydryl-
oxyethyl)-piperazin-1-yl]-propyl}-phenyl)-amide (10).
Method A. 11-Bromoundecanoic acid (0.42 g, 1.6 mmol)
was dissolved in dry dichloromethane (50 mL) and thionyl
chloride (1 mL) was added. A catalytic quantity of dry
dimethyl formamide (1 drop) was added and the mixture
was heated at reflux for 30 min. The solvent was removed
under reduced pressure and the acid chloride was dissolved
in dry dichloromethane (20 mL). This solution was added
dropwise to a methylene chloride solution containing 1-[2-
[bisphenylmethoxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (0.64 g, 1.5 mmol) and triethylamine (1 mL).
The solution was stirred at room temperature for 4 days.
Then the solvent was removed under reduced pressure and
the product was purified on a silica column eluted with
a gradient system running from dichloromethane to
dichloromethane–methanol (5%). This yielded 0.19 g
(23%) of the product as a pale yellow oil. 1H NMR
(CDCl3) 1.15–1.4 (m, 12H), 1.50–1.62 (m, 6H), 2.19–2.55
(m, 14H), 2.59 (t, J¼5.8 Hz, 2H), 3.29 (t, J¼6.7 Hz, 2H),
3.52 (t, J¼5.8 Hz, 2H), 5.28 (s, 1H), 6.99 (d, 2ArH), 7.11–
7.26 (m, 10ArH), 7.34 (d, 2ArH), 7.63 (br s, NH); 13C NMR
(CDCl3) d 25.5, 28.0, 28.3, 28.5, 29.1, 29.2, 32.6, 32.9,
33.9, 37.4, 52.9, 53.3, 57.7, 66.7, 83.7, 119.8, 126.8, 127.2,
128.2, 128.6, 135.8, 137.6, 142.0, 171.4; calculated for
C39H54BrN3O2 m/z FAB (MþH) 676.3478; found
676.3454; IR (neat) C(vO)N–H 3290 cm21 (br s), CH2
2920–2812 cm21 (m), CvO 1676 cm21 (s).
Route 2. Thioacetic acid S-(2-{2-[(4-{3-[4-(2-benzhydryl-
oxy-ethyl)-piperazin-1-yl]-propyl}phenylcarbomyl)-meth-
oxy]-ethoxy}ethyl) ester (0.6 g, 0.9 mmol) was dissolved in
methanolic ammonia (50 mL) and stirred under nitrogen at
room temperature for 4 h. Then the solvent was removed by
evaporation under reduced pressure and purified by column
chromatography on silica eluted with methylene chloride
94%–methanol 5%–triethylamine 1%. This gave 0.5 g
(94%) of the product as a pale brown oil.
4.1.8. N-(4-{3-[4-(2-Benzhydryloxy-ethyl)piperazin-1-
yl]-propyl}-phenyl)-2-[2-(2-chloro-ethoxy)-ethoxy]-aceta-
mide (8). 2-(2-(2-Chloroethoxy)ethoxy)ethanoic acid
(0.8 g, 4.3 mmol) was dissolved in dry THF (10 mL) and
carbonyl diimidazole (0.7 g, 4.3 mmol) was added. This
mixture was stirred at room temperature for 5 min then 1-[2-
[bisphenylmethoxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (1.8 g, 4.3 mmol) dissolved in dry THF (10 mL)
was added and the mixture was stirred at room temperature
overnight. The THF was removed under reduced pressure
and the product was purified via column chromatography on
silica gel eluted with dichloromethane–methanol (5%).
This yielded 1 g (42%) of the product as a colorless oil. 1H
NMR (CDCl3) d 1.75–1.85 (m, 2H), 2.35 (t, J¼8.2 Hz, 2H),
2.47–2.63(m, 12H), 2.69 (t, J¼6.1 Hz, 2H), 3.61–3.80 (m,
8H), 4.10 (s, 2H), 5.4 (s, 1H), 7.15 (d, 2ArH), 7.23–7.37 (m,
10ArH), 7.54 (d, 2ArH), 8.67 (br s, NH); 13C NMR (CDCl3)
28.4, 32.9, 42.5, 52.9, 53.4, 57.6, 57.7, 66.7, 69.9, 70.3,
70.7, 71.1, 83.6, 119.8, 126.8, 127.2, 128.1, 128.6, 134.8,
138.2, 142.0, 167.6; IR (neat) C(vO)N–H 3360 cm21 (br
s), CH2 2940–2813 cm21 (m), CvO 1686 cm21 (s);
calculated for C34H44ClN3O4 m/z ESI (MþNa) 616.2913;
found 161.2939.
Method B. 11-Bromoundecanoic acid (1.06 g, 4 mmol) was
dissolved in dry tetrahydrofuran (10 mL) and 1,10-carbo-
nyldiimidazole (0.65 g, 4 mmol) was added. The resulting
solution was stirred at room temperature for 1 h, then 1-[2-
[bisphenylmethoxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (1.7 g, 4 mmol) was dissolved in dry tetra-
hydrofuran (10 mL). The solution was stirred at room
temperature for 4 days, after which the solvent was removed
under reduced pressure and the product was purified on a
silica column eluted with a gradient system running
from dichloromethane to dichloromethane–methanol
(5%). This yielded 0.75 g (28%) of the product as a pale
yellow oil.
4.1.9. Thioacetic acid S-(2-{2-[(4-{3-[4-(2-benzhydryl-
oxy-ethyl)-piperazin-1-yl]-propyl}phenylcarbomyl)-
methoxy]-ethoxy}ethyl) ester (9). N-(4-{3-[4-(2-Benz-
hydryloxy-ethyl)piperazin-1-yl]-propyl}-phenyl)-2-[2-(2-
chloro-ethoxy)-ethoxy]-acetamide (1 g, 1.8 mmol) was dis-
solved in dry DMF (10 mL), potassium thioacetate (0.6 g,

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I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
4.1.11. Thioacetic acid S-[10-(4-{3-[4-(2-benzhydryloxy-
ethyl)-piperazin-1-yl}-propyl}-phenylcarbamoyl)-decyl]
ester (11). 11-Bromoundecanoic acid (4-{3-[4-(2-benz-
hydryloxy-ethyl)-piperazine-1-yl]-propyl}-phenyl)-amide
(0.19 g, 0.35 mmol) was dissolved in dry dimethyl forma-
mide (4 mL) and potassium thioacetate (0.08 g, 0.7 mmol)
was added. The mixture was stirred under nitrogen for 48 h
and then it was diluted with diethyl ether (100 mL). This
was filtered and evaporated under reduced pressure. The
product was purified by column chromatography on silica
gel eluted with a gradient system running from dichloro-
methane to dichloromethane–methanol 5%. This gave
0.058 g (31%) of the product as a pale yellow oil. 1H
NMR (CDCl3) d 1.20–1.45 (m, 12H), 1.50–52 (m, 2H),
1.55–1.71 (m, 4H), 2.27–2.35 (m, 5H), 2.44–2.61 (m,
15H), 2.66 (t, J¼6.0 Hz, 2H), 2.85 (t, J¼7.3 Hz, 2H), 3.59
(t, J¼5.9 Hz, 2H), 5.36 (s, 1H), 7.09 (d, 2ArH), 7.18–7.35
(m, 8ArH), 7.41 (d, 2ArH), 7.73 (s, NH); 13C NMR (CDCl3)
d 25.5, 28.4, 28.6, 28.8, 29.0, 29.1, 29.1, 29.2, 29.3, 30.5,
32.9, 37.4, 53.0, 53.5, 57.8, 66.8, 83.7, 119.8, 126.9, 127.3,
128.1, 128.6, 135.8, 137.7, 142.1, 171.3, 195.9; calculated
for C41H57N3O3S m/z FAB (MþH) 672.4199; found
672.4178; IR (neat) C(vO)N–H 3300 cm21 (br s), CH2
2927–2812 cm21 (m), CvO 1690 cm21 (s).
was stirred at room temperature for 2 h. The solvent was
removed by evaporation, and the crude acid chloride was
dissolved in dry dichloromethane (100 mL). Dry triethyl-
amine (10 mL) and 1-[2-[4,40-fluorobenzhydryloxy]ethyl]-
piperazine (4.68 g, 15.4 mmol) was dissolved in dry
dichloromethane (50 mL) and added to the solution of
p-nitrohydrocinnamyl chloride. The mixture was heated at
reflux for 18 h under argon in a 250 mL round bottomed
flask equipped with a stirrer and reflux condenser. Then
the solvent was removed under reduced pressure and the
product is purified using silica gel chromatography eluted
with dichloromethane 96%–methanol. 8.0 g (97%) of the
1
product was obtained as a yellow oil. H NMR (CDCl3) d
2.37 (br s, 4H), 2.55–2.60 (m, 4H), 2.99 (t, J¼7.5 Hz, 2H),
3.3 (t, J¼4.6 Hz, 2H), 3.45 (t, J¼5.6 Hz, 2H), 3.51 (t, J¼
4.9 Hz, 2H), 5.24 (s, 1H), 6.88–6.94 (m, 4ArH), 7.16–7.20
(m, 4ArH), 7.30 (d, 2ArH), 8.04 (d, 2ArH); 13C NMR
(CDCl3) d 30.8, 33.9, 41.6, 45.3, 53.1, 53.5, 57.6, 66.8,
82.5, 115.1, 115.4, 123.6, 128.4, 128.5, 129.3, 137.6,
137.7, 146.4, 149.3, 160.4, 163.7, 169.5; calculated for
C28H29F2N3O4 m/z ESI (MþNa) 532.2024; found 532.2057,
(MþH) calculated 510.2204; found 510.2192; CHN
(0.1H2O) calculated C¼65.71, H¼5.69, N¼8.22; found
C¼65.77, H¼5.69, N¼8.18; IR (neat) CH2 2821–
2939 cm21 (m), CvO 1640 cm21 (s).
4.1.12. 11-Mercaptoundecanoic acid (4-{3-[4-(2-benz-
hydryloxyethyl)-piperazin-1-yl]-propyl}-phenyl)-amide
(12). Thioacetic acid S-[10-(4-{3-[4-(2-benzhydryloxy-
ethyl)-piperazin-1-yl}-propyl}-phenylcarbamoyl)-decyl]
ester (0.058 g, 0.11 mmol) was dissolved in degassed
methanol (10 mL) and methanolic ammonia (10 mL) was
added. The mixture was stirred at room temperature under
nitrogen for 2 h and evaporated. The product was purified by
column chromatography on silica gel eluted with a gradient
system running from dichloromethane to dichloromethane–
methanol 7%–triethylamine 3%. The base was obtained as a
yellow oil and this was converted to the oxalate salt, by
dissolving the base in methanol (50 mL) and adding a
solution of oxalic acid (1 g) dissolved in methanol. The
resulting oxalate salt precipitated from methanol. This was
removed by filtration and dried under reduced pressure. This
gave 30 mg (51%) of the product as a white solid. 1H NMR
(CDCl3) d 1.27–1.31 (m, 15H), 1.30–1.69 (m, 6H), 2.50–
2.59 (m, 16H), 2.69 (t, J¼6.0 Hz, 2H), 3.59 (t, J¼6.0 Hz,
2H), 5.36 (s, 1H), 5.47 (br s, NH), 7.10 (d, 2ArH), 7.22–
7.40 (m, 12ArH); 13C NMR (CDCl3) d 24.5, 25.5, 28.1,
28.2, 28.9, 29.1, 29.2, 29.3, 29.3, 32.9, 33.9, 37.6, 52.7,
53.1, 57.5, 57.6, 55.7, 83.8, 119.9, 126.9, 127.3, 128.2,
128.7, 135.8, 137.7, 142.1, 171.3; calculated for
C39H55N3O2S m/z FAB (MþH) 630.4093; found
630.4085; CHN (0.75 H2O) calculated C¼72.80%,
H¼8.85%, N¼6.53%; found C¼72.96%, H¼8.67%,
N¼6.41%; IR (neat) C(vO)N–H 3299 cm21 (br s), CH2
2926 – 2810 cm21 (m), SH 2360 cm21 (s), CvO
1659 cm21 (s).
4.1.14. 1-[2-[4,40-Fluorobenzhydryloxy]ethyl]-4-(3-(4-
nitrophenyl)propyl)piperazine (14). 1-[2-[4,40-Fluoro-
benzhydryloxy]ethyl]-4-(3-(4-nitrophenyl)-1-oxopropyl)-
piperazine (8 g, 15 mmol) was dissolved in dry THF
(100 mL) in a 250 mL round bottomed flask equipped
with a stirrer and a reflux condenser. Alane in toluene
(0.5 M, 59 mL) was added and stirred at room temperature
for 30 min. The reaction was quenched with sodium
hydroxide solution (10%, 200 mL). The aqueous solution
was extracted with diethyl ether (3£150 mL), dried over
magnesium sulfate filtered and evaporated. The product was
purified using silica gel chromatography eluted with
methylene chloride 96%–methanol. This yielded 6.14 g
(80%) of the product as a pale yellow oil. 1H NMR (CDCl3)
d 1.79–1.88 (m, 2H), 2.35 (t, J¼7.6 Hz, 2H), 2.46–22.54
(m, 8H), 2.67 (t, J¼6.0 Hz, 2H), 2.74 (t, J¼7.7 Hz, 2H),
3.57 (t, J¼5.0 Hz, 2H), 5.34 (s, 1H), 6.97–7.02 (m, 4ArH),
7.26–7.35 (m, 6ArH), 8.13 (d, 2ArH); 13C NMR (CDCl3) d
28.0, 33.4, 53.1, 53.5, 57.3, 57.7, 66.8, 82.4, 115.0, 115.3,
123.5, 128.4, 128.5, 129.1, 137.7, 137.8, 146.2, 150.0,
160.4, 163.6; calculated for C28H31F2N3O3 m/z ESI
(MþNa) 518.2231; found 518.2252, (MþH) calculated
496.2412; found 496.2442; CHN calculated C¼67.86,
H¼6.31, N¼8.48; found C¼67.75, H¼6.39, N¼8.26; IR
(neat) CH2 2775–2941 cm21 (m).
4.1.15. 1-[2-[4,40-Fluorobenzhydryloxy]ethyl]-4-(3-(4-
aminophenyl)propyl)piperazine (15). 1-[2-[4,40-Fluoro-
benzhydryloxy]ethyl]-4-(3-(4-nitrophenyl)-1-oxopropyl)-
piperazine (6.14 g, 12 mmol) was dissolved in absolute
ethanol (10 mL) in a 100 mL round bottomed flask equipped
with a stirrer and reflux condenser. Tin(II)chloride dihydrate
(11.14 g) was added and the mixture was heated at reflux for
90 min. The solution was poured into crushed ice and a
solution of sodium carbonate (5%) in water was added until
a pH of 8 is obtained. The aqueous solution was extracted
with ethyl acetate (3£200 mL) and this was dried over
4.1.13. 1-[2-[4,40-Fluorobenzhydryloxy]ethyl]-4-(3-(4-
nitrophenyl)-1-oxopropyl)piperazine (13). para-Nitro-
hydrocinnamic acid (3 g, 15.4 mmol) was added to dry
toluene (100 mL), in a 250 mL round bottomed flask
equipped with a stirrer and a reflux condenser. Oxalyl
chloride (2 mL) was added, after which a catalytic quantity
of dry DMF (2 drops) was also added, and the mixture

I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
8045
magnesium sulfate. The product was purified using column
chromatography on silica gel eluted with methylene
chloride 96%–methanol. This gave 5.11 g (92%) of the
product as a red oil. 1H NMR (CDCl3) d 1.59–1.70 (m, 2H),
2.23 (t, J¼8.0 Hz, 2H), 2.30–2.49 (m, 10H), 2.54 (t, J¼
5.9 Hz, 2H), 3.45 (t, J¼5.6 Hz, 4H), 5.21 (s, 1H), 6.47 (d,
2ArH), 6.83–6.90 (m, 6ArH), 7.13–7.18 (m, 4ArH); 13C
NMR (CDCl3) d 28.6, 32.6, 53.0, 53.4, 57.6, 57.8, 66.6,
82.2, 114.8, 114.9, 115.1, 128.4, 128.5, 128.9, 131.7, 137.6,
137.7, 144.1, 160.2, 163.5; calculated for C28H33F2N3O m/z
ESI (MþNa) 488.2489; found 488.2494, (MþH) calculated
466.2670; found 466.2677; CHN (0.25 H2O) calculated
C¼71.53, H¼7.08, N¼8.94; found C¼71.53, H¼7.17,
N¼8.78; IR (neat) NH2 3200–3400 cm21 (m), CH2
2775–2941 cm21 (m).
C41H55F2N3O3S m/z ESI (MþNa) 730.3830; found
730.3795, (MþH) calculated 708.4010; found 708.3994;
IR (neat) C(vO)N–H 3303 cm21 (br s), CH2 2929–
2812 cm21 (m), CvO 1689 cm21 (s).
4.1.18. 11-Mercaptoundecanoic acid (4-{3-[4-(2-(4,40-
fluorobenzhydryloxy)-ethyl)-piperazin-1-yl]-propyl}-
phenyl)-amide (18). Thioacetic acid S-[10-(4-{3-[4-(2-
(4,40fluorobenzhydryloxy)-ethyl)-piperazin-1-yl}-propyl}-
phenylcarbamoyl)-decyl] ester (50 mg, 0.076 mmol) was
dissolved in degassed methanol (10 mL) and methanolic
ammonia (10 mL) was added. The mixture was stirred at
room temperature under nitrogen for 2 h and evaporated.
The product was purified by column chromatography on
silica gel eluted with a gradient system of methylene
chloride then methylene chloride (96%)–methanol, giving
24 mg (51%) as a red oil. 1H NMR (CDCl3) d 1.21–1.35 (m,
15H), 1.54–1.63 (m, 4H), 1.83–1.86 (m, 2H), 2.28 (t,
J¼7.6 Hz, 2H), 2.44 (t, J¼7.0 Hz, 2H), 2.52 (t, J¼7.2 Hz,
2H), 2.58–2.66 (m, 10H), 3.51 (t, J¼5.5 Hz), 5.25 (s, 1H),
6.89–6.93 (m, 4ArH), 7.02 (d, 2ArH), 7.16–7.28 (m,
4ArH), 7.39 (d, 2ArH), 7.61 (s, NH); 13C NMR (CDCl3) d
25.64, 27.3, 28.4, 29.1, 29.2, 29.3, 29.4, 32.7, 37.7, 39.2,
52.3, 52.5, 57.4, 66.6, 82.6, 115.2, 115.5, 120.0, 128.5,
128.6, 128.7, 136.2, 137.6, 137.6, 160.5, 163.8, 171.6;
calculated for C39H54F2N3O2S m/z ESI (MþH) 666.3905;
found 666.3901; IR (neat) IR C(vO)N–H 3305 cm21 (br
s), CH2 2928–2818 cm21 (m), CvO 1687 cm21 (s).
4.1.16. 11-Bromoundecanoic acid (4-{3-[4-(4,40-fluoro-
benzhydryloxy-ethyl)-piperazine-1-yl]-propyl}-phenyl)-
amide (16). 11-Bromoundecanoic acid (0.65 g, 2.4 mmol)
was dissolved in dry tetrahydrofuran (10 mL) and carbonyl
diimidazole (0.4 g, 2.4 mmol) was added. The mixture was
stirred at room temperature for 30 min. Then 1-[2-[4,40-
fluorobenzhydryloxy]ethyl]-4-(3-(4-aminophenyl)propyl)-
piperazine (0.57 g, 1.2 mmol) in dry tetrahydrofuran
(10 mL) was added. The solution was stirred at room
temperature for 4 days, and then the solvent was removed
under reduced pressure. The product was purified on a silica
column eluted with dichloromethane–methanol (5%). This
yielded 280 mg (30%) of the product as a pale yellow oil. 1H
NMR (CDCl3) d 1.1–1.25 (m, 12H), 1.54–1.67 (m, 6H),
2.16–2.47 (m, 14H), 2.56 (t, J¼5.8 Hz, 2H), 3.46 (t,
J¼6.0 Hz), 3.80 (t, J¼6.9 Hz, 2H), 5.23 (s, 1H), 6.86–7.00
(m, 6ArH), 7.15–7.20 (m, 4ArH), 7.38 (d, 2ArH), 8.62 (br s,
NH); 13C NMR (CDCl3) d 25.4, 26.1, 28.2, 28.6, 30.0, 29.4,
30.7, 32.8, 37.2, 52.8, 53.2, 57.6, 57.7, 66.5, 82.3, 114.9,
115.2, 119.0, 119.8, 128.3, 128.4, 128.5, 136.0, 137.1,
137.5, 137.6, 160.4, 163.6, 171.5; calculated for C39H52-
BrF2N3O2 m/z ESI (MþH) 712.3289; found 712.3261; IR
(neat) N–H 3300–3400 cm21 (br s), CH2 2815–2928 cm21
(m), CvO 1650 cm21 (s).
4.2. Nanocrystal ligand exchange methodology
The ligand exchanges were performed using a 1.9 mM
solution of trioctylphosphine oxide (TOPO) coated cad-
mium selenide/zinc sulfide nanocrystals dissolved in
hexanes. These dots had a fluorescence emission wave-
length of 625.8 nm and an absorption maximum at 610 nm.
0.5 mL of this solution was added to 20 mL of methanol at
which point the TOPO coated dots precipitated out of
solution and were collected by centrifugation. The methanol
was decanted and the dots were washed with another 20 mL
of methanol. After centrifugation and decanting the
methanol, the dots were dissolved in pyridine (0.5 mL).
This solution was stirred at 608C for 18 h. The solution was
allowed to cool to room temperature and the dots were
precipitated by adding hexanes (20 mL). This process was
repeated three times and then the precipitated dots were
dissolved in pyridine (0.5 mL). The concentration was
determined using UV–vis spectroscopy based upon an
extinction coefficient of 700000.34 After measuring the
concentration we were able to calculate the number of moles
of dots present in the pyridine solution. Then we added
100 equiv. of ligand dissolved in 0.1 mL of methylene
chloride and this mixture was stirred at 608C for 2 h. After
cooling to room temperature hexanes (20 mL) were added
to the dots and the resultant precipitate was collected by
centrifugation. The core/shells were washed with ethyl
acetate (3£20 mL) to remove unbound ligand. Then they
were dissolved in dimethyl formamide (0.5 mL) and
thioacetic acid (0.5 mL) was added. The thioacetic acid
was added as a co-solubility ligand in order to increase the
water solubility of the nanocrystal ligand conjugate. This
mixture was stirred at ambient temperature for 24 h and then
potassium tertiary butoxide (1.8 g) dissolved in methanol
4.1.17. Thioacetic acid S-[10-(4-{3-[4-(2-(4,40-fluorobenz-
hydryloxy)-ethyl)-piperazin-1-yl}-propyl}-phenylcarba-
moyl)-decyl] ester (17). 11-Bromoundecanoic acid (4-{3-
[4-(2-(4,40-fluorobenzhydryloxy)-ethyl)-piperazine-1-yl]-
propyl}-phenyl)-amide (0.20 g, 0.28 mmol) was dissolved
in dry dimethyl formamide (4 mL) and potassium thioace-
tate (0.065 g, 0.56 mmol) was added. The mixture was
stirred under nitrogen for 48 h, and then it was diluted
with diethyl ether (100 mL). This was filtered and evapo-
rated under reduced pressure. The product was purified
by column chromatography on silica gel eluted with
dichloromethane–methanol 5%. This gave 100 mg (54%)
of the product as a pale yellow oil. 1H NMR (CDCl3) d 1.2–
1.35 (m, 12H), 1.44–1.50 (m, 2H), 1.60–1.78 (m, 4H),
221–2.28 (m, 5H), 2.32 (t, J¼6.0 Hz, 2H), 2.40–2.53 (m,
6H), 2.32 (t, J¼6.0 Hz, 2H), 2.78 (t, J¼ 5.5 Hz, 2H), 3.49 (t,
J¼4.4 Hz, 2H), 5.25 (s, 1H), 6.90–6.95 (m, 4ArH), 7.03 (d,
2ArH), 7.18–7.24 (m, 4ArH), 7.27 (s, NH), 7.35 (d, 2ArH);
13C NMR (CDCl3) d 25.6, 28.4, 28.6, 28.9, 29.0, 29.1, 29.2,
29.2, 29.4, 33.0, 37.6, 53.0, 53.4, 57.6, 57.7, 66.7, 82.4,
115.0, 115.3, 119.8, 128.4, 128.5, 128.7, 135.8, 137.7,
137.8, 160.4, 163.7, 171.4, 196.1; calculated for

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I. D. Tomlinson et al. / Tetrahedron 59 (2003) 8035–8047
7. Chunyang, Z.; Hui, M.; Nie, S.; Yao, D.; Leland, J.; Dieyan, C.
The Analyst 2000, 125, 1029.
(20 mL) was added at 08C. The solid was removed by
centrifugation and excess potassium tertiary butoxide was
removed by washing with methanol (2£20 mL). Then the
solid was dried under reduced pressure and dissolved in
DI water. It was kept at 48C and used when required in
biological assays.
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scattering Spectroscopy (RBS) as previously described.10,35
for the GBR 12909 derivative (compound 18) conjugated to
CdSe/ZnS core/shell nanocrystals. The fluorine atoms in 18
serve as a marker to quantify the coverage of the ligand, as
N and O are always present in the RBS spectrum due to
exposure of the substrate to air. In this experiment, samples
were prepared by dropping 0.2 mL of a concentrated
nanocrystal solution onto a 1 cm2 graphite substrate and
wicking off excess solution. RBS experiments were
performed in a high vacuum chamber (,1027 Torr) with
a 1.8 MeV He ion beam at normal incidence. Scattered ions
were collected on axis at 1808 with a solid-state detector. To
obtain the F and Zn ratios in order to determine ligand
coverage, the individual peaks in the RBS spectrum are
integrated and normalized by the square of their atomic
numbers (the intensity of the peaks in the spectrum is
proportional to the square of the atomic number of the
element and its relative abundance). The core/shell
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4840–4849.
˚
˚
nanocrystals have a 48 A CdSe core and a 15 A ZnS shell.
To obtain the percentage ligand coverage from the Zn/F
ratio, the core/shells are assumed to be spherical. In this way
we find ,20 ligands on the surface of the nanocrystal.
Addition of mercaptoacetic acid during the ligand exchange
procedure does not displace the ligand from the surface of
the nanocrystals, as verified by using RBS to analyze for F
in the methanol washings.
18. Lewis, D. B.; Matecka, D.; Zhang, Y.; Hsin, L.; Dersch, C. M.;
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Acknowledgements
22. Benedetti, P.; Mannhold, R.; Cruciani, G.; Pastor, M. J. Med.
Chem. 2002, 45, 1577–1584.
We would like to thank Quantum Dot Corporation for
supplying the core shell nanocrystals used in this study. We
would also like to thank Dr Marcel Bruchez of Quantum Dot
Corporation for helpful advice during the course of this
study and, Qiao Han for assistance measuring the IC50
values used in this study. Jon Burton would like to thank
Vanderbilt University for a summer research stipend. This
work was supported by grants from the National Institutes of
Health and Quantum Dot Corporation.
23. Dutta, A. K.; Davis, M. C.; Fei, X.; Beardsley, P. M.; Cook,
C. D.; Reith, M. E. A. J. Med. Chem. 2002, 45, 654–662.
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