Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmc.2008.10.045

A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR.

Full text of DOI:10.1016/j.bmc.2008.10.045

Bioorganic & Medicinal Chemistry 16 (2008) 10295–10300
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Polyethylene glycol-based homologated ligands for nicotinic
acetylcholine receptors q
Bradley A. Scates, Bethany L. Lashbrook, Benjamin C. Chastain , Kaoru Tominaga , Brandon T. Elliott ,
*
Nicholas J. Theising , Thomas A. Baker , Richard W. Fitch
Department of Chemistry, Indiana State University, 600 Chestnut Street, Science S35E, Terre Haute, IN 47809, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand
series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-
4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain sig-
nificant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such com-
pounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and
multivalent ligands for nAChR.
Received 16 August 2008
Revised 16 October 2008
Accepted 18 October 2008
Available online 1 November 2008
Keywords:
Pyridyl ethers
A-84543
SAR
Ó 2008 Elsevier Ltd. All rights reserved.
ADMP
DMPP
1. Introduction
fects, while central neuronal receptors, especially
ered to mediate the cognitive enhancing and addictive properties
of nicotine.4 The
7 homopentamer has been implicated in inflam-
mation and schizophrenia and it has been observed that a high per-
centage (>80%) of schizophrenics smoke heavily, possibly as a form
of self-medication.5 Some of the major foci for the development of
drugs for nAChR include analgesia, cognitive enhancement, and
smoking cessation.2,3
a4b2 are consid-
Nicotinic acetylcholine receptors (nAChR) are a subclass of
receptors activated by the neurotransmitter acetylcholine (ACh).
These ligand-gated ion channels are activated by the tobacco alka-
loid nicotine, as opposed to muscarinic acetylcholine receptors
(mAChR), which are G-protein coupled receptors (GPCR) and are
activated by the mushroom muscarine.1
a
Nicotinic receptors are important targets in the development of
therapeutics for a variety of neurological disorders.1b,2,3 They are
involved in cellular signaling in both the peripheral (PNS) and cen-
tral (CNS) nervous systems. Nicotinic receptors exist as a number
of different subtypes based on subunit constitution of this penta-
meric protein. At present there are approximately 20 different
characterized subtypes and differential activation/inhibition of
these are considered to be responsible for nicotine’s myriad phar-
While the analgesic activity of nicotine has been known for
many decades, its clinical use is precluded by its toxicity and
addictive liability.3 However, since discovery of epibatidine, a
highly potent nicotinic receptor agonist from a poison frog,6 the
field of nAChR-mediated analgesia has become quite active. Epi-
batidine and several derived analogs are sensitive nAChR probes
many with significant subtype selectivity (Fig. 1). Notably, the pyr-
idyl ethers developed by Abbott have been shown to have useful
analgesic and cognitive enhancing effects,6 though groups at sev-
eral companies have entered the market with nAChR based there-
apeutics, notably Pfizer, with their antismoking drug varenicline
(ChantixÒ).8
We are interested in the development of nicotinic ligands with
external tethers, with potential applications to fluorescent and/or
affinity tags,9 as well as multivalent ligands.10 As a prerequisite
to such development, it is necessary to demonstrate the ability to
homologate a particular ligand of interest such that it retains affin-
ity for the receptor of interest. Such a ligand serves as the recogni-
tion element for receptor binding, but appropriate point(s) of
attachment must first be selected such that the tether is placed
macological effects. In the neuromuscular a1b1c(e)d nAChR medi-
ate muscle contraction by the triggering of action potentials (in
concert with voltage-gated sodium channels) at the muscle end-
plate.1b In the CNS, nAChR mediate much of the fast synaptic trans-
mission in several areas of the brain. Nicotine’s effects on
ganglionic receptors are thought to mediate some of its toxic ef-
q
Presented at the 233rd National Meeting of the American Chemical Society,
Chicago, IL, March 24–29, 2007 (Poster MEDI 0291).
* Corresponding author. Tel.: +1 812 237 2244; fax: +1 812 237 2232.
E-mail address: rfitch@indstate.edu (R.W. Fitch).
Undergraduate student.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.045

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B. A. Scates et al. / Bioorg. Med. Chem. 16 (2008) 10295–10300
O
R
R
X
CH3
CH3
N
N
CH3
N
CH3
CH3
Acetylcholine (ACh) R = CH3, X = O
Carbamylcholine (CaCh), R = NH2, X = O
Acetylaminocholine (AaCh), R = CH3, X = NH
Succinyldicholine (SuCh),
Acetyldimethylpiperazinium (ADMP) R = Ac
Dimethylphenylpiperazinium (DMPP), R = Ph
R = (CH3)3N+CH2CH2OC(=O)CH2CH2, X = O
R
Cl
n
H
O
R2
N
R
N
N
N
3
5
R1
CH3
N
N
Nicotine (R = H)
Epibatidine (R = H)
A-85380 (n = 0, R1 = R2 = H)
A-84543 (n = 1, R1 = Me, R2 = H)
≡
SIB-1508Y (R = C CH)
Figure 1.
in a non-congested location when the ligand binds to the receptor,
such that affinity is retained. Further the tether composition must
be compatible with receptor, biological system, and solvent for
both reactivity and physical property considerations (solubility,
lipophilicity).11 We thus examined potential sites of homologation
for several nicotinic ligands. Our first interest was in quaternary
ammonium compounds analogous to ACh or its bioisosteric coun-
terparts carbamylcholine (CaCh) or acetylaminocholine (AaCh,
Fig. 2). We initially considered that these ligands would be amena-
ble to homologation based on the observation that succinyldicho-
line (SuCh), a paralytic agent employed during surgery, and other
bivalent ACh homologs demonstrate the ability to substitute ACh
that hydrocarbon backbones would be undesirable due to increas-
ing hydrophobicity and the anticipated potential for undesired
interactions with membranes and solubility problems. Polyethyl-
ene glycols (PEGs), on the other hand, have been shown to be a
hydrophilic, biocompatible polymer scaffold with a number of
applications.11b We describe herein a series of highly water soluble
homologated ligands (Fig. 2) based on AaCh (1a–g), ADMP (2a–g),
and A-84543 (3a–g), and their activity at nicotinic receptors. We
anticipate these ligands to have significant utility as models for
application to other receptor systems.
2. Results and discussion
2.1. Chemistry
at the
a
-carbon of the acetyl group.2 It has also been shown that
the ester linkage may be replaced by amide.12 Along this line, rigid
bioisosteric quaternary analogs of acetyldimethypiperazinium
(ADMP), a well-known nAChR-selective quaternary agonist were
of interest to us as they showed increased potency over the acyclic
amide or acetylcholine itself.13 Given the structural similarities to
We initially approached the synthesis of the quaternary AaCh
and ADMP homologs. For our purposes, we felt that PEG deriva-
tives of this type should be accessible in straightforward fashion
by reaction of N,N-dimethylethylenediamine, or 1-methylpipera-
zine with an appropriate PEG-based acylating agent. For examina-
tion of the effect of tether length, we chose to use polyethylene
glycol monomethyl ethers (mPEG)nOH where n = 0–6 (4a–g) as
the homologous series. Monomethyl ethers 4b–d are available15
commercially while the higher oligomers 4e–g with 4–6 PEG units
were synthesized by alkylation of tetraethylene glycol with methyl
ACh, we anticipated these would tolerate substitution at the a-car-
bon of the acetyl group as well. Finally, a series of homologated
versions of both epibatidine and A-84543, a pyridyl ether from Ab-
bott, were recently described.14 These derivatives were appended
with
x-alcohol and hydrocarbon moieties up to ten carbons. In-
deed, Abbott had described 5-substituted derivatives of their pyr-
idyl ether ligands previously.7b However, we anticipated the need
for very long tethers (>30 atoms) for our purposes and postulated
O
O
≡
C CCH2R
O
CH3
CH3
CH3
R
N
R
X
N
CH3
N
N
R1
N
CH3
1
2
3
R
X
Aminocholines Piperazines Pyridines
CH3O
NH
NH
NH
NH
NH
NH
NH
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
CH3(OCH2CH2)O
CH3(OCH2CH2)2O
CH3(OCH2CH2)3O
CH3(OCH2CH2)4O
CH3(OCH2CH2)5O
CH3(OCH2CH2)6O
1g
2g
3g
Figure 2.

B. A. Scates et al. / Bioorg. Med. Chem. 16 (2008) 10295–10300
10297
iodide (for 4e) or triethylene glycol with mPEG mesylates 5c,d (for
4f,g).15–18
With the alcohols in hand, we set about synthesizing the
amides corresponding to the two quaternary series (Scheme
1).12,13,19 There are two two possible ways to approach this. The lit-
erature describes formation of 4-methylpiperazine-1-carbonyl
chloride from methylpiperazine using phosgene or the more
user-friendly di- or triphosgene compounds,20 which we envi-
sioned using in situ to acylate the mPEG alcohols 4 to produce
the intermediate urethanes 6 and 7. However, in our hands this
was complicated significantly by formation of the symmetrical ur-
eas, leading to reduced yields. Fortunately, formation of the mPEG
chloroformates by reaction of mPEG alcohols 4 with phosgene in
toluene proceeded smoothly overnight and in situ acylation of
N,N-dimethylethylenediamine afforded a convenient one-pot syn-
thesis of the mPEG-acyldimethylethylenediamines 6b–g in good
yield.12 Acylation of N,N-dimethylethylenediamine proceeded like-
wise, affording the acylmethylpiperazines 7b–g.13 Predictably,
aqueous workups of these highly water soluble compounds gave
poor yields, affording only 7–15% recoveries even after saturating
with NaCl, while direct flash chromatography of the reaction mix-
tures gave 60–70% yields. The amines were subsequently quatern-
ized using methyl iodide in acetone to produce 1b–g and 2b–g
smoothly, though in several cases oils were obtained rather than
the expected crystalline compounds.21 The parent compounds
AaCh, ADMP, and the corresponding methoxycarbonyl analogs 1a
and 2a were prepared in analogous fashion by acylation of N,N-
dimethylethylenediamine or 1-methylpiperazine respectively with
acetyl chloride or methyl chloroformate, to produce compounds 8,
9, 6a, and 7a, which were quaternized as above to provide crystal-
line methiodides.12,13
Figure 3.
containing subtypes, especially a4b2 receptors, which are the ma-
jor subtype present in cortex and thought to be responsible for
antinociception and cognitive enhancement.4 Two of these have
gone into clinical trials (ABT-089 and ABT-594). It has also been
shown that in this series, the 5-position of the pyridine ring is ame-
nable to substitution.7 Indeed, another compound, SIB-1508Y
shows substitution tolerance in nicotine at the 5- position.22 Re-
cently, it was demonstrated that similar substitution of both A-
84543 and epibatidine can be achieved and enhances selectivity
for
a
4b2 receptors.14
We prepared a series of homologated ligands analogous to this
series with oligomeric mPEG side chains (3a–g) based on similar
chemistry (Scheme 2). We chose the A-84543 homologs as the
chemistry is simplified relative to the azetidine clinical candidates
noted above. We prepared 5-bromo-A-84543 (10) using an SNAr
reaction essentially as described from commercially available
3,5-dibromopyridine and (S)-N-methylprolinol using NaH in
DMF.7a,14 Initial attempts to conjugate mPEG alcohols 4a–f with
10 in the same manner were unsuccessful due to the increasing
deactivation of the pyridine ring to a second SNAr substitution by
the presence of the electron-donating ether installed from the first
displacement. We observed no reaction at room temperature in
DMF for extended reaction times and elevated temperature led
only to decomposition products. We then examined the Sonogash-
ira reaction as described for the installation of the hydrocarbon
side chains.7,14 Alkylation of mPEG alcohols with propargyl bro-
mide afforded the alkyne coupling partners (11b–g).23 Sonogashira
coupling proceeded in moderate yield with an excess of alkyne to
A variety of pyridyl ethers were developed by Abbott laborato-
ries in the mid 1990s and several have advanced to the clinic with
useful cognitive enhancing and/or analgesic properties.2a,3a These
compounds can be looked at as homologs of nicotine and/or bio-
isosteric variants of epibatidine (Fig. 3). Several of these com-
pounds have been shown to have significant selectivity for b2-
H
HO
O
H3CO
H3CO
MsCl
Et3N
Ms
H
H3CO
3 , NaH
H
O
O
O
n
n
n
THF
4f n = 5
4g n = 6
4b n = 1
4c n = 2
5b,c
O
O
H3CO
H
O
n
H3CO
Cl
toluene
r.t.
Cl
O
Cl
n
4b-g
not isolated
CH3
I-
CH3
N
CH3
CH3
CH3
O
O
N
CH3
H2N
N
CH3I
R
N
H
CH3
R
N
H
acetone
CH2Cl2
6a-g R = MeO(CH2CH2O)n
1a-g R = MeO(CH2CH2O)n
AaCh R = Me
O
8
R = Me
Cl
R
O
O
I-
R
N
HN
N CH3
R
N
CH3
CH3I
N
N
CH3
acetone
CH2Cl2
CH3
7a-g R = MeO(CH2CH2O)n
R = Me
2a-g R = MeO(CH2CH2O)n
ADMP R = Me
9
Scheme 1. Synthesis of quaternary ammonium compounds.

10298
B. A. Scates et al. / Bioorg. Med. Chem. 16 (2008) 10295–10300
and may not accurately represent the full receptor, in spite of the
homology of the native protein to the extracellular portion of the
7 receptor.22 These models are typically produced by virtual
NaH/DMF
Br
Br
O
HO
N
CH3
N
CH3
Br
a
N
mutation of residues with the homologous counterparts from the
receptor subtype of interest. We had originally designed the length
of the tethers to reach the channel lumen through the channels
proposed by Unwin based on electron diffraction data obtained
on the Torpedo receptor.26a However, the models based on AChBP
and newer data from the Unwin group now show the binding site
to be accessed from the opposite side of the protein extending di-
rectly into the extracellular space to the side of the receptor.26b
Nonetheless, we find that the tethers are easily accommodated
and the uniform decrease in affinity suggests a nonselective effect
and no sharp transition in affinity to indicate emergence from a
tunnel into the extracellular space, and is thus consistent with
the current model. However, this issue highlights the need for
high-resolution X-ray data for this receptor and it is hoped that
newer techniques in the crystallization of membrane proteins
may resolve this problem.
10
N
1. NaH/THF
MeO
H
MeO
O
O
n
n
≡
2. BrCH2C CH
11b-g
4b-g
12, Pd/C, CuI, K2CO3
RO
O
RO
N
CH3
DME/H2O
N
R = H, Me,
Me(OCH2CH2)n
3a-g R =Me(OCH2CH2)n
3h R = H
Scheme 2. Synthesis A-84543 homologs.
afford the corresponding ethynylpyridines (3b–g).24 As reference
compounds, we prepared propargylic alcohol (3h) and correspond-
ing methyl ether (3a) in analogous fashion using commercially
available propargyl alcohol and methoxypropyne, respectively, as
described.14
3. Conclusions
We have successfully prepared and evaluated homologated nic-
otinic receptor ligands based on three binding motifs. Of these, the
pyridyl ether series showed the strongest binding in the 1–10 nM
range. Functionalized variants of these ligands should have signif-
icant utility as models for the design of multivalent ligands and/or
affinity probes for nicotinic receptors.
2.2. Pharmacology
With the compounds in hand, we examined their affinity for
nicotinic receptors in rat cerebral cortex (Table 1).25 The acylami-
nocholine analogs (1a–g) were found to be poor ligands, having
4. Experimental
4.1. General
affinities 15–200
affinities 1 to 40
l
l
M. The piperazinium series fared better, with
M. In each series, exchange of the acetyl for
Commercially available reagents were used as received unless
otherwise noted. All moisture sensitive reactions were carried
out under nitrogen or argon in oven dried glassware. Tetrahyrofu-
ran (THF) was freshly distilled from sodium/benzophenone under
an argon atmosphere. Dichloromethane and acetonitrile were dis-
tilled from CaH2 under an argon atmosphere. Dimethoxyethane
(DME) was dried and freed of peroxides by filtration through
Al2O3. CeliteÒ filtrations utlilized Johns-Manville 545 grade. Flash
column chromatography utilized 230–400 mesh silica gel EM Sci-
ence 9385, while silica filtrations utilized EM Science 7734 (70–
230 mesh) material. Thin layer chromatography was performed
using silica gel plates (EM Science 5715), visualized with UV light,
and stained with KMnO4, iodine, or ninhydrin as appropriate. NMR
spectra were obtained on Bruker AC-250 or Bruker Avance II 400
spectrometers. Chemical shifts are reported in ppm relative to
TMS as calibrated by internal TMS for CDCl3 and by the residual
proton signals for other solvents. 13C spectra in D2O are referenced
to the deuterium signal and are uncorrected. Coupling constants
methoxycarbonyl (1a, 2a) led to an improvement in potency. The
A-84543 analogs (3a–g) were very potent, with Ki values in the
2–15 nM range. In all three series, increasing chain length pro-
duced a regular but modest decrease in potency (a factor of 1.7–
2.2 per PEG unit), losing just over an order of magnitude over the
series. However, the retention of affinity, especially the high affin-
ity for the A-84543 homologs suggests that very long tethers may
be appended to these molecules thus making them useful for tag-
ging nAChR. We are currently evaluating this behavior and results
will be forthcoming.
The retention of affinity for these series is in keeping with the
idea that these tethers extend out of the binding pocket of the
receptor into the extracellular space. The currently used models
for nicotinic receptors account for this as the binding site extends
directly into the extracellular space without little protein interac-
tion, save for the recognition motif.14,26b Of course, one must al-
ways be cautious as to such interpretation, as the majority of
these models are based on the acetylcholine binding protein27
Table 1
Pharmacologic data.
Compound
Ki (nM)
Compound
Ki (nM)
Compound
Ki (nM)
AaCh
1a
1b
1c
1d
1e
1f
63,000 16,000
14,000 5000
54,000 12,000
32,000 8000
70,000 15,000
15,0000 37,000
190,000 50,000
30,000 2000
ADMP
2a
2b
2c
2d
2e
2f
2g
3600 1500
670 130
3h
3a
3b
3c
3d
3e
3f
3.1 1.2
4.7 1.5
3000 1700
8200 3900
48,000 23,000
26,000 10,000
38,000 5000
19,000 5000
15
7
7.5 4.7
15
30
40
63
1
1
4
3
1g
3g
Data represent mean SEM for three experiments conducted in triplicate. Ki values were determined in rat cerebral cortex against 200 pm [3H]( )-epibatidine hydrochloride
with Kd taken as 40 pM (observed IC50 for cold ( )-epibatidine hydrochloride was determined to be 230–310 pM).

B. A. Scates et al. / Bioorg. Med. Chem. 16 (2008) 10295–10300
10299
are reported in Hz. Carbon signals marked with an asterisk repre-
4.5. General procedure for the synthesis of mPEG quaternary
salts
sent methyl and methine carbons, and quaternary carbons are des-
ignated with a (q) as determined on the basis of DEPT-edited
experiments. FTIR spectra were obtained with a Midac M200
instrument and are expressed in cmꢀ1. Optical rotations were ob-
tained with a Perkin-Elmer 241 Polarimeter operating at 589 nm
at ambient room temperature. Boiling points were obtained by
fractional or kugelrohr distillation as noted and are uncorrected.
Melting points were obtained with an Electrothermal Mel-Temp
apparatus and are uncorrected. Unit mass resolution electron im-
pact mass spectra (EIMS) were taken with a Thermo-Finnigan
GCQ instrument consisting of a programmable capillary GC inter-
faced to an ion trap autotuned against standard perfluorotributyl-
amine. Where applicable, unit resolution chemical ionization mass
spectra (CIMS) were obtained using ammonia as the reagent gas.
High resolution mass spectrometry (HRMS) was performed by
the Washington University Center for Biomedical and Bioorganic
Mass Spectrometry (NIH Research Resource, Grant No.
P41RR0954). Mass spectral formulas are calculated according to
the most abundant isotopic formula and m/z determined relative
to exact 12C.
To a dry culture tube was added 30–50 mg of amine (3b–g, 5b–
g). The compounds were suspended in 1 mL of acetone (dried over
0
4 AÅ molecular sieves). Iodomethane (3–5 equiv) was added to the
solutions and the reactions were allowed to stand in the dark over-
night. Anhydrous diethyl ether was added dropwise as needed to
promote precipitation, and the reactions were then returned to
the dark for 48 h. The supernatant was removed and the salts were
washed with ether and dried under high vacuum. While the lighter
amines gave crystalline salts, many of the higher quaternary salts
failed to crystallize, thus precluding a precise yield determination.
Melting points are noted where crystalline materials were
obtained.
4.6. Pharmacology
Binding assays in rat cerebral cortex were performed essentially
as described using [3H]epibatidine as the radioligand.25 Functional
fluorescence assays for the measurement of intracellular calcium
dynamics were performed essentially as described28 in HEK cells
4.2. General procedure for the preparation of mPEG propargyl
ethers.23
expressing rat a3 and b4 subunits for neuronal nicotinic acetylcho-
line receptors.29 Detailed procedures are provided in the Supple-
mentary material.
To suspension of sodium hydride (1.2 equiv, 0.7 M) in THF
(cooled in an ice bath) was added a solution of a oligoethylene gly-
col monomethyl ether (1.0 equiv, 0.5 M) in THF. The reaction was
stirred for fifteen minutes and propargyl bromide (80% in toluene,
1.1 equiv) was added. The ice bath was removed and the reaction
allowed to warm to room temperature. When the reaction was
judged to be complete by TLC, the reaction was carefully quenched
with water (2 equiv) and the product isolated. For the lower oligo-
mers (n = 1–3), the reaction was performed on 40 mmol scale and
the product was isolated by aqueous-organic extraction and distil-
lation. For the higher oligomers (n = 4–6) the reaction was per-
formed on 1–2 mmol scale and the workup consisted of filtration
concentration and the product was purified by flash column chro-
matography (EtOAc/MeOH).
Acknowledgments
Mr. Ying Chau (Allen) Liu, Ms. Kiersten Yake, Mr. Fernand M.
Bedi, and Ms. Amanda Clay are acknowledged for performing
exploratory experiments related to this work. A generous gift of
HEK cells expressing various subunit combinations for nicotinic
receptors by Drs. Kenneth J. Kellar and Yingxian Xiao of George-
town University Medical School is gratefully acknowledged.29 Dr.
John W. Daly is acknowledged for many helpful discussions, and
his passing is deeply mourned. This work was performed with
startup funds from the Department of Chemistry and College of
Arts and Sciences at Indiana State University, as well as the Na-
tional Institute for General Medical Sciences (1R21GM072780),
and the National Science Foundation (CHE0521075 for
400 MHz NMR spectrometer at Indiana State University).
a
4.3. General procedure for Sonogashira coupling reactions
The reactions were done in parallel analogous to the literature
procedure for related compounds.14 To a dry reaction vial was
added in the following sequence, 6 (70 mg, 0.25 mmol), K2CO3
(90 mg, 0.65 mmol), triphenylphosphine (10 mg, 0.04 mmol), al-
kyne (1.5–4 mmol),23 CuI (10 mg, 0.05 mmol), and 10% Pd/C
(10 mg, 0.009 mmol). The compounds were suspended in a 1:1
mixture of dimethoxyethane/water the suspensions were sparged
with Ar, then sealed and heated to 80 °C with stirring on a heating
block for 6 days. The reactions were then cooled, filtered through
CeliteÒ, and the products isolated by flash column chromatography
with an EtOAc/MeOH gradient.
Supplementary data
Detailed experimental procedures and NMR spectra for com-
pounds and intermediates are provided. This material accompanies
the online version of this article. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2008.10.045.
References and notes
1. (a) Nicotinic see: Brown, J. H.; Taylor, P. In Goodman and Gilman’s
Pharmacological Basis of Therapeutics, 10th ed.; Hardman, J. G.; Limbird, L. E.,
Eds., New York: McGraw-Hill, Chapter 9, pp. 193–213.; (b) Muscarinic, see:
Brown, J. H.; Taylor, P. In Goodman and Gilman’s Pharmacological Basis of
Therapeutics, 10th ed.; Hardman, J. G.; Limbird, L. E., Eds., New York: McGraw-
Hill, Chapter, Chapter 7 pp. 154–173.
4.4. General procedure for the preparation of mPEG urethanes
To neat 1a–f (1.0 mmol) at 0 °C under N2 was added a solution
of phosgene (0.50 mL, 1.9 M in toluene, 0.95 mmol). The reaction
was allowed to reach room temperature and stirred overnight.
The reaction was sparged with dry N2 briefly to expel unreacted
phosgene, cooled in an ice bath, dry CH2Cl2 (1 mL) was added
and the corresponding amine (2.5 mmol) was added dropwise.
The reaction was warmed to room temperature and the reaction
stirred at room temperature for 3–12 h. The product was isolated
from the solution by flash column chromatography with a CHCl3/
MeOH gradient to yield 3b–g, 5b–g as colorless to pale yellow oils.
2. (a) Arneric, S. P.; Holladay, M.; Williams, M. Biochem. Pharmacol. 2007, 74,
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