Guanidine-acylguanidine bioisosteric approach in the design of radioligands: Synthesis of a tritium-labeled NG-propionylargininamide ([3H]-UR-MK114) as a highly potent and selective neuropeptide Y Y1 receptor antagonist

Article abstract of DOI:10.1021/jm801018u

Synthesis and characterization of (R)-N^α-(2,2- diphenylacetyl)-N-(4-hydroxybenzyl)-N^ω-([2,3- ³H]-propanoyl)-argininamide ([³H]-UR-MK114), an easily accessible tritium-labeled NPY Y₁ receptor (Y₁R) antagonist (K_B: 0.8 nM, calcium assay, HEL cells) derived from the (R)-argininamide BIBP 3226, is reported. The radioligand binds with high affinity (K_D, saturation: 1.2 nM, kinetic experiments: 1.1 nM, SK-N-MC cells) and selectivity for Y₁R over Y₂, Y ₄, and Y₅ receptors. The title compound is a useful pharmacological tool for the determination of Y₁R ligand affinities, quantification of Y₁R binding sites, and autoradiography.

Full text of DOI:10.1021/jm801018u

8168
J. Med. Chem. 2008, 51, 8168–8172
Guanidine-Acylguanidine Bioisosteric Approach in the Design of Radioligands: Synthesis of a
Tritium-Labeled NG-Propionylargininamide ([3H]-UR-MK114) as a Highly Potent and Selective
Neuropeptide Y Y1 Receptor Antagonist
Max Keller,† Nathalie Pop,† Christoph Hutzler,† Annette G. Beck-Sickinger,‡ Gu¨nther Bernhardt,† and Armin Buschauer*,†
Department of Pharmaceutical/Medicinal Chemistry II, Faculty of Chemistry and Pharmacy, UniVersity of Regensburg, UniVersita¨tsstrasse 31,
D-93053 Regensburg, Germany, Institute of Biochemistry, UniVersity of Leipzig, Bru¨derstrasse 34, D-04103 Leipzig, Germany
ReceiVed August 13, 2008
Synthesis and characterization of (R)-NR-(2,2-diphenylacetyl)-N-(4-hydroxybenzyl)-Nω-([2,3-3H]-propanoyl)-
argininamide ([3H]-UR-MK114), an easily accessible tritium-labeled NPY Y1 receptor (Y1R) antagonist
(KB: 0.8 nM, calcium assay, HEL cells) derived from the (R)-argininamide BIBP 3226, is reported. The
radioligand binds with high affinity (KD, saturation: 1.2 nM, kinetic experiments: 1.1 nM, SK-N-MC cells)
and selectivity for Y1R over Y2, Y4, and Y5 receptors. The title compound is a useful pharmacological tool
for the determination of Y1R ligand affinities, quantification of Y1R binding sites, and autoradiography.
Chart 1. Structures and Y1R Affinities of BIBP 3226 (14) and
Selected NG-Substituted Derivatives, Arranged According to
Increasing Size of the Substituent “R”
Introduction
Neuropeptide Y (NPYa), a 36-amino acid peptide, is one of
the most abundant neuropeptides in the central and peripheral
nervous system. NPY is involved in the regulation of numerous
(patho)physiological functions such as food intake, blood
pressure, stress, pain, and hormone secretion. In humans, the
biological effects of NPY are mediated by four receptor
subtypes, termed Y1, Y2, Y4, and Y5. Recently, the Y1 receptor
(Y1R) was reported to be expressed in different tumors such as
breast cancer,1 prostate cancer,2 and adrenal tumors3 and
therefore proposed as a potential tumor marker.4 The (R)-
configured argininamide BIBP 3226 (14, Chart 1), the first
highly potent and selective nonpeptide Y1R antagonist,5 has been
commonly used as a pharmacological tool for studying the
physiological role of the Y1R. The compound is considered a
mimic of the C-terminus, i.e., Arg35 and Tyr36, in NPY.6 On
one hand, the guanidino group in 14 has been considered
important for the biological activity due to interaction with
Asp287 of the human Y1R; on the other hand, the strongly basic
group is a major drawback with respect to oral availability and
brain penetration. Recently, we prepared a series of NG-
substituted derivatives of 14, which revealed a preference for
electron-withdrawing substituents in terms of retaining or even
increasing the Y1R affinity regardless of the by 4-5 orders of
magnitude reduced basicity.7 Especially the introduction of
carbamoyl residues into the NG position yielded Y1R antagonists
with considerably increased affinity (Chart 1),7,8 These findings
a Determined on SK-N-MC cells with [3H]-propionyl-pNPY as radio-
ligand (1 nM).7 The subnanomolar affinities of the carbamoylated argini-
namides 19 and 20 were confirmed using 8b (1.5 nM) as radioligand.
support the concept that the acylguanidines are bioisosteres of
guanidines. The high affinities and selectivities achieved with
this class of compounds prompted us to develop a Y1R selective
antagonistic radioligand.
* To whom correspondence should be addressed. Phone: +49-941
9434827. Fax: +49-941 9434820. E-mail: armin.buschauer@
chemie.uni-regensburg.de.
†
Department of Pharmaceutical/Medicinal Chemistry II, Faculty of
Chemistry and Pharmacy, University of Regensburg.
Results and Discussion
‡
Institute of Biochemistry, University of Leipzig.
NG-acetyl (15), NG-methoxycarbonyl (16), and NG-propyl (17)
substituted argininamides (Chart 1) exhibit lower Y1R affinities
(two-digit nM range) than the parent compound BIBP 3226 (14),
whereas the NG-propionyl derivative 8a turned out to be a highly
potent Y1 antagonist with an affinity of about 1 nM. Obviously,
the extension of the acyl substituent by one methylene group
enables an additional hydrophobic interaction, which compen-
sates for the moderate acylation-induced decrease in affinity
observed for the lower homologue 15. This was also true for
a Abbreviations: Bmax, maximum number of binding sites; CDI, carbo-
nyldiimidazole; CRC, concentration-response curve; DCC, dicyclohexyl-
carbodiimide; EDAC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride; DMAP, 4-(dimethylamino)pyridine; FAB, fast atom bom-
bardement; FmocOSu, 9-fluorenylmethyl succinimidyl carbonate; H&E,
hematoxylin and eosin; hPP, human pancreatic polypeptide; HR-MS, high
resolution mass spectrometry; kob, observed association rate constant; koff
,
dissociation rate constant; kon, association rate constant; NPY, neuropeptide
Y; PET, positron emission tomography; pNPY, porcine NPY; rt, room
temperature; tR, retention time; Y1R, Y2R, Y4R, and Y5R, NPY receptor
subtypes Y1, Y2, Y4, and Y5.
10.1021/jm801018u CCC: $40.75
2008 American Chemical Society
Published on Web 11/21/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8169
Scheme 1. Synthetic Route for the Preparation of the
NG-Propanoyl Argininamides 8a,ba
Figure 1. HPLC purity control of the freshly synthesized (I, dashed
line) and long-term (18 months) stability control (II, solid line) of the
tritiated Y1R antagonist 8b with radiometric detection, shown for
different chromatographic conditions to avoid coincidence of the peaks.
Conditions: I, purity control: eluent: mixtures of acetonitrile + 0.05%
TFA (A) and 0.05% aq TFA (B), gradient: 0-30 min: A/B 20/80 to
90/10, 30-38 min: 90/10, tR ) 16.7 min; II, long term stability control:
eluent: mixtures of acetonitrile (A) and 0.05% aq TFA (B), gradient:
0-30 min: A/B 20/80 to 50/50, 30-35 min: 50/50 to 90/10, 35-45
min: 90/10, tR ) 26.5 min. The identity of 8b was confirmed by spiking
with larger amounts of the “cold” analogue 8a, which allowed UV
detection.
Table 1. NPY Receptor Selectivity Data of 8a/8b
Y1 KD [nM]a
Y2 Ki [nM]b
Y4 Ki [nM]c
Y5 Ki [nM]b
>2000
1.2
>10000
>10000
a Binding of 8b to SK-N-MC neuroblastoma cells. b Flow cytometric
binding assay on CHO-Y2 and HEC-1B-Y5 cells using Dy-635-pNPY or
Cy5-pNPY as labeled ligands (10 nM). c Flow cytometric binding assay
on CHO-Y4 cells and Cy5-[K4]-hPP as fluorescent ligand (5 nM).
guanidine derivatives can be N-alkylated with alcohols under
Mitsunobu conditions.13 Acyl-substituted alkylguanidines are
accessible if one of the protecting groups of the guanidinylation
reagent is replaced with the pertinent acyl residue.8 However,
such a synthetic route is not well suited for the preparation of
a tritiated propionylguanidine because the radiolabel is prefer-
ably introduced in a final one-pot reaction. Therefore, direct
attachment of the acyl substituent to the guanidine group in 14
(Chart 1) was performed using the NG-Boc-O-tert-butyl pro-
tected analogue 7 as a precursor (Scheme 1). This method
afforded the target compounds in good (up to 80%) yields and
has been frequently used in our laboratory to synthesize NG-
acylated arginine derivatives. Carboxylic acids can be attached
to the NG-Boc-alkylguanidine scaffold with the aid of coupling
reagents from peptide chemistry (EDAC, CDI) or using
anhydrides, chlorides, and active esters. However, the reaction
rate is noticeably slower compared to the coupling with amines.
a Reagents and conditions: (i) Cbz-Cl (45% in toluene), NaOH 0.5 N,
K2CO3, CuSO4, 20 h, 4 °C to rt; (ii) titriplex III, H2O, 60 min, 100 °C,
83%; (iii) FmocOSu, H2O/dioxane, 20 h, rt, 96%; (iv) CDI, THF, 20 h, rt,
62%; (v) diethylamine, DCM, 4 h, rt, 76%; (vi) NEt3, 1,2-dimethoxyethane,
20 h, 35 °C, 84%; (vii) Pd/C, ammonium formate, MeOH, 20 h, rt, 85%;
(viii) NEt3, CH2Cl2, 20 h, rt; (ix) Pd/C, H2, MeOH, 3.5 h, rt, 84%; (x)
NEt3, MeCN, 20 h, rt; (xi) TFA, MeCN, 3 h, 50 °C, 70% (8a), 34% (8b).
the methoxycarbonyl derivative 16 (Ki ) 33 nM) compared to
the ethoxycarbonyl homologue 18 (Ki ) 4.5 nM). Acylation
was superior to alkylation at the NG-position (8a vs 17, Chart
1), but an exchange of the R-CH2 group by an oxygen atom,
resulting in an ester group, was again less favorable (16, 8a,
Chart 1). Surprisingly, the corresponding NG-carbamoyl sub-
stituted analogues (19, 20), containing a R-NH group, showed
significantly higher affinities. Presumably, the presence of this
H-donor group enables additional affinity-enhancing interactions,
resulting in a different favorable orientation of the flexible amide
substituent in 19, 20 compared to the acyl residues in 8a,b.
Considering Y1R affinity and selectivity as well as synthetic
feasibility, the NG-[2,3-3H]-propionyl-substituted argininamide
8b ([3H]-UR-MK114), the “hot” form of 8a, was considered
most attractive, particularly because tritiated propionic acid
succinimidyl ester is commercially available as a radiolabeling
reagent. In principle, this labeling strategy can be transferred
to other classes of compounds containing guanidine as a
pharmacophoric group.
The precursor 7 can be obtained from amine 6 by reaction
with a guanidinylation reagent containing a Boc and a Cbz
protecting group, followed by hydrogenolytic Cbz deprotection.
For the preparation of amine 6, starting from D-ornithine, the
R-amino group was protected with Fmoc, orthogonally to Cbz
and the tert-butyl phenyl ether.8 A conversion of the alcohol
corresponding to amine 6 (obtained via reduction of D-
glutamate) into acylated guanidines under Mitsunobu condition
was reported not to give the desired argininamides14 and was
therefore not explored any longer as an alternative synthetic
route. The guanidinylation reagent 13 was prepared from
aminoguanidine carbonate following reported protocols.10 All
other required compounds (9, 10, 11) were prepared according
to standard procedures.
Chemistry. Alkylguanidines are conventionally prepared
from amines and bis-urethane (Boc, Cbz) protected guanidiny-
lation reagents, for example, S-methyl isothioureas9 or pyra-
zol-1-carboxamidines10,11 or triflylguanidines.12 Alternatively,

8170 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Brief Articles
Figure 3. Association and dissociation kinetics for the specific Y1R
binding of 8b on SK-N-MC cells (65th passage) at 24 °C. (A)
Radioligand (c ) 3 nM) association as a function of time, Inset: ln[Beq/
(Beq - B)] versus time, slope ) kob ) 0.79 min-1, kon ) (kob - koff)/
[L] ) 0.19 min-1 ·nM-1. (B) Radioligand (pre-incubation: 6 nM)
dissociation as a function of time, monophasic exponential decay, t1/2
Figure 2. (A) Concentration-response curves (CRCs) of pNPY from
a fura-2 assay on HEL cells. The presence of 8a led to a parallel
rightward shift of the curves. (B) Schild regression: log(r - 1) plotted
against log[antagonist]; the concentration ratios r (r ) 10∆pEC50) were
calculated from the rightward shifts (∆pEC50) of the CRCs in the
presence of 8a as shown in Figure 2A. The slope of the line equals
unity, therefore the pA2 value corresponds to pKB: pA2 ≈ pKB ) 9.1
(slope forced to unity), KB ) 0.8 nM (mean values ( SEM or
propagated error, n ) 2).
) 2.2 min, Inset: ln(B/B0) versus time, slope(-1) ) koff ) 0.22 min-1
Mean values ( SEM, n ) 3.
.
Depending on the source of succinimidyl [2,3-3H]-propionate,
GE Healthcare (Amersham) and ARC (American Radiolabeled
Chemicals, Inc.), the tritiated compound 8b was obtained with
specific activities of 97 Ci/mmol and 51 Ci/mmol, respectively.
The radioligand 8b was isolated by HPLC with a radiochemical
purity of 99% (Figure 1, dashed line) and showed a high long-
term stability when stored in ethanol at -20 °C over a period
of 1.5 years (Figure 1, solid line).
Pharmacology. The selectivity of the new Y1R antagonist
8a for human NPY Y1 over Y2, Y4, and Y5 receptors was proven
by flow cytometric binding assays based on fluorescence-labeled
pNPY (Y2R, Y5R) or [K4]-hPP (Y4R) according to previously
described methods.15-17 As summarized in Table 1, comparable
to the parent compound BIBP 3226, the NG-propionylated
derivative (8a) shows a clear binding preference for the Y1R.
The Y1R antagonism of 8a was investigated in a fura-2-based
Ca2+-assay on HEL cells.18 Concentration-response curves of
pNPY were constructed in the absence and presence of 8a at
different concentrations (Figure 2A), and the data were subjected
to Schild analysis19 (Figure 2B). Because the slope in this linear
plot nearly equals unity, a competitive antagonism of 8a is very
likely. The pKB (in this case identical to pA2) reflects the affinity
of the antagonist and the resulting KB value (0.8 nM) is in very
good agreement with the KD value (1.2 nM) from saturation
binding experiments (Figure 3). A plausible reason for the
depressed maxima of the concentration effect curves (Figure
2A) is hemi-equilibrium.19 Because of the rapid onset of the
calcium response in the functional assay, the time window is
too narrow to enable an equilibrium among the receptors, the
Figure 4. (A) Saturation curves for binding of 8b to MCF-7 (176th
passage) and SK-N-MC (67th passage) cells revealing KD values of
2.2 nM and 0.85 nM, respectively. Estimated Bmax (sites/cell): 120000
(MCF-7) and 52000 (SK-N-MC). The cell number was determined in
at least 4-6 wells. (B) Scatchard plot for the binding of 8b to MCF-7
cells, KD ) -1/slope ) 1.7 nM. (C) Scatchard plot for the binding to
SK-N-MC cells, KD ) -1/slope ) 1.1 nM (mean values ( SEM or
propagated error, n ) 3).
agonist pNPY (slow binding kinetics20) and the antagonist 8a
(high on- and off-rate, Figure 3).
The results of kinetic studies of 8b at 24 °C are presented in
Figure 3. Association and dissociation at the Y1R were in the

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8171
Table 2. Y1R Binding and Functional Characteristics of 8a/8b
cancer cells express about 100000-150000 sites/cell (fluctua-
tions are probably caused by a slightly varying estrogen stimulus
provided by the fetal calf serum supplement of the culture
medium). Estrogen stimulation of MCF-7 cells leads to Y1R
up-regulation to 300000 sites/cell. Y1R up-regulation has been
already reported on the mRNA level22 and could be confirmed
on intact cells at the protein level using the radioligand 8b as
an NPY Y1R probe. On the basis of these results, an assay has
been developed for the functional characterization of estrogens
and antiestrogens (to be reported elsewhere). Binding and
functional Y1R data of 8a/8b are summarized in Table 2.
Competition binding experiments of 8b with the antagonist
BIBP 3226 and the agonist pNPY yielded Ki values (1.5 ( 0.2
nM and 0.5 ( 0.02 nM, respectively) consistent with reported
data (Figure 5).21,23
KD
KD
KB
KD
kon
koff
[nM]a
[nM]b
[nM]c
[nM]d [min-1 ·nM-1 e
]
[min-1 f
]
2.9 ( 0.4 1.2 ( 0.1 0.8 ( 0.1 1.1 ( 0.1
0.19 ( 0.01
0.22 ( 0.01
a Equilibrium dissociation constant determined on MCF-7 cells, mean
value ( SEM from 5 independent experiments, each in triplicate.
b Equilibrium dissociation constant determined on SK-N-MC cells, mean
value ( SEM from 6 independent experiments, each in triplicate. c Schild
analysis derived dissociation constant of 8a, mean value ( SEM from 2
independent experiments (calcium assay using HEL cells). d Kinetically
derived dissociation constant ( propagated error. e Association rate constant
( standard error from linear regression. f Dissociation rate constant (
standard error from linear regression.
To explore the applicability of 8b to autoradiographic binding
studies cryosections of a human MCF-7 tumor, subcutaneously
grown in a nude mouse, and of a rat brain were incubated with
the new radioligand. In rat brain, Y1R binding sites were clearly
detected in the cerebral cortex in layers I-III (I: molecular layer;
II: external granular layer; III: layer of medium-sized pyramidal
cells), in the thalamus, and in the hippocampus (Figure 6A,B)
as reported from binding studies with [125I][Leu31,Pro34]PYY
and [3H]-BIBP3226.24,25 The results indicate a very high Y1R
expression in the tumor, as there is a strong difference between
total and non-specific binding (Figure 6C).
Figure 5. Displacement of the tritiated Y1R antagonist 8b (c ) 1.5
nM) by the agonist pNPY, the antagonist BIBP 3226 and the nonlabeled
analogue 8a. The competition assay was performed on SK-N-MC cells
at 24 °C with incubation periods of 20 min (BIBP 3226, 8a) and 120
Conclusion
NG-propionylation of BIBP 3226 leads to a highly potent and
selective Y1R antagonist (8a). Direct NG-acylation of NG-Boc,
O-tert-butyl protected BIBP 3226 (7) allows a convenient
preparation of a tritium-labeled radioligand from succinimidyl
[2,3-3H]-propionate with specific activities of up to 100 Ci/
mmol. Because of the practicability and low costs of this labeling
strategy, 8b ([3H]-UR-MK114) is a highly attractive alternative
to (the formerly commercially available) [3H]-BIBP 3226. 8b
has also advantages over radiolabeled peptides addressing the
Y1R due to stability, selectivity, mode of action (antagonist),
and costs. [3H]-UR-MK114 can be used for the quantification
of Y1R binding sites as well as for autoradiography experiments
on tissues. Because of the attenuated basicity (pKa ) 7-8) of
the acyl-guanidine moiety, 8a is supposed to exhibit an improved
pharmacokinetic profile over the strongly basic BIBP 3226 (pKa
≈ 13). Generally, radioligands capable of penetrating into the
brain would provide interesting tools for the study of Y1R
binding by ex vivo (autoradiography) and in vivo (PET)
experiments. Recent findings in the NPY Y2R14 as well as in
the histamine H2R and H4R field26 support the hypothesis that
the concept of guanidine-acylguanidine bioisosterism may be
of general interest to design receptor ligands including radiotrac-
ers with high affinity, reduced basicity and improved pharma-
cokinetic properties.
min (pNPY). BIBP 3226: pIC50 ) 8.44, Ki ) 1.5 nM; pNPY: pIC50
)
9.01, Ki ) 0.5 nM. 8a: pIC50 ) 8.65, Ki ) 1.0 nM (mean values (
SEM, n ) 3).
Figure 6. (A) Total and nonspecific binding of the tritiated Y1R
antagonist 8b to adjacent coronal sections of rat brain (male Wistar
rat, 10 month old). (B) H & E stained adjacent section, (a) cerebral
cortex, (b) third ventricle, (c) hippocampus, (d) thalamus. (C) Total
and nonspecific binding of 8b to adjacent tumor sections (sc human
MCF-7 mammary carcinoma from a NMRI (nu/nu) mouse). (D)
Masson-Goldner (Jerusalem’s modification) stained adjacent tumor
section, (e) connective and adipose tissue, (f) necrotic regions.
Experimental Section
range of minutes (kon ) 0.19 min-1 ·nM-1, koff ) 0.22 min-1
)
(R)-Nr-(2,2-Diphenylacetyl)-N-(4-hydroxybenzyl)-Nω-([2,3-
3H]-propanoyl)argininamide (8b). Here, only a brief experimental
protocol is given. A more detailed procedure is provided as
Supporting Information. 8b was prepared from two different batches
of succinimidyl-[2,3-3H]-propionate (11b), one from GE Healthcare
(Amersham) provided in toluene (185 MBq/5 mL, as ) 3.48 TBq/
mmol), and a second one from American Radiolabeled Chemicals
provided in ethyl acetate (185 MBq/1 mL, as ) 2.22 TBq/mmol).
The labeling reaction was performed in a 1.5 mL Eppendorf reaction
vessel (screw top) in acetonitrile using 40 equiv of the labeling
precursor 7. NEt3 was added to ensure deprotonation of 7. A portion
as typical for antagonists. The kinetically derived KD (koff/kon
) 1.1 nM) is in excellent agreement with the KD from saturation
binding experiments (1.2 nM).
Saturation analysis of radioligands provides dual information,
the equilibrium dissociation constant KD and the maximum
number of binding sites (Bmax). Multiple saturation experiments
with 8b on SK-N-MC neuroblastoma cells and MCF-7 breast
cancer cells afforded KD values of 1.2 and 2.9 nM, respectively
(Figure 4). The Bmax value for SK-N-MC cells of about 50000
sites per cell fit well with data from literature.21 The breast

8172 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Brief Articles
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of this mixture (precursor 7 and NEt3) was added to the solution
of 11b in either toluene or ethyl acetate, and the solvents were
removed by a slight stream of nitrogen and in a vacuum concentra-
tor, respectively. The residue was dissolved in acetonitrile (≈ 80
µL) and remaining fractions of 7 and NEt3 were added. The reaction
mixture was stirred at rt for 20 h, then TFA was added and stirring
was continued under moderate heating (≈ 50 °C) for 2-3 h. 8b
was purified by reversed-phase HPLC (column: Agilent Scalar C18,
250 mm × 4.6 mm, 5µm). The quantity was calculated from an
HPLC standard curve (8a) and the specific activity was determined
to be 3.6 and 1.9 TBq/mmol, respectively. 8b is stored in ethanol
with a TFA additive (100 µM) at -20 °C. Yield of purified product:
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equation.27 For Schild analysis log10(r - 1) was plotted against
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experiments were processed with the aid of the WinMDI 2.7
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- t0)/t0.
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Acknowledgment. We are grateful to Dr. Thilo Spruss for
the preparation of the cryosections, to Dr. Chiara Cabrele for
the synthesis of pNPY, to Matthias Freund for synthetic help,
to Elvira Schreiber, Susanne Bollwein, Brigitte Wenzl, and Franz
Wiesenmeyer for expert technical assistance, and to Laura
Emmerich and Dorothea Wedler for their assistance in the Schild
analysis. This work was supported by the Graduate Training
Program (Graduiertenkolleg) GRK 760, “Medicinal Chemistry:
Molecular Recognition-Ligand-Receptor Interactions”, of the
Deutsche Forschungsgemeinschaft.
Supporting Information Available: General experimental
conditions, experimental details and analytical data for 8a, inter-
mediates and building blocks 1-7 and 9-13, chromatogram of 8a
(purity control), detailed experimental procedure for the synthesis
of 8b, and pharmacological methods. This material is available free
of charge via the Internet at http://pubs.acs.org.
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