A study of the structure-activity relationship of GABAA-benzodiazepine receptor bivalent ligands by conformational analysis with low temperature NMR and X-ray analysis

Article abstract of DOI:10.1016/j.bmc.2008.08.072

The stable conformations of GABA_A-benzodiazepine receptor bivalent ligands were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations in solution correlated well with those in th

Full text of DOI:10.1016/j.bmc.2008.08.072

Bioorganic & Medicinal Chemistry 16 (2008) 8853–8862
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A study of the structure–activity relationship of GABAA–benzodiazepine
receptor bivalent ligands by conformational analysis with low temperature
NMR and X-ray analysis
Dongmei Han a, , F. Holger Försterling a, , Xiaoyan Li a, Jeffrey R. Deschamps b, Damon Parrish b,
Hui Cao a, Sundari Rallapalli a, Terry Clayton a, Yun Teng a, Samarpan Majumder c,
Subramaniam Sankar d, Bryan L. Roth c, Werner Sieghart e, Roman Furtmuller e,
James K. Rowlett f, Michael R. Weed g, James M. Cook a,
*
a Department of Chemistry, University of Wisconsin-Milwaukee, PO Box 413, Milwaukee, WI 53211, USA
b Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375, USA
c Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
d Department of Biochemistry, CWRU Medical School, Cleveland, OH 44106, USA
e Division of Biochemistry & Molecular Biology, Center for Brain Research, Medical University Vienna, A-1090 Vienna, Austria
f Harvard Medical School, New England Primate Research Center, Southborough, MA 01772, USA
g Johns Hopkins Bayview Medical Center, Baltimore, MD 21224, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The stable conformations of GABAA–benzodiazepine receptor bivalent ligands were determined by low
temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations
in solution correlated well with those in the solid state. The linear conformation was important for these
Received 16 May 2008
Revised 21 August 2008
Accepted 28 August 2008
Available online 2 September 2008
dimers to access the binding site and exhibit potent in vitro affinity and was illustrated for a5 subtype
selective ligands. Bivalent ligands with an oxygen-containing linker folded back upon themselves both
in solution and the solid state. Dimers which are folded do not bind to Bz receptors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Conformation
Benzodiazepines
NMR
GABA
1. Introduction
and new anticonvulsant compounds with decreased side ef-
fects.13–17
The GABAA/BzR complex contains a chloride ion channel which
comprises part of the major inhibitory neurotransmitter system in
the CNS.1 This system regulates numerous neurological functions
including convulsions, anxiety, and sleep activity, as well as mem-
ory and learning processes.2–5 This membrane-bound heteropenta-
Currently, transforming monomers into a bivalent ligand is one
of the successful strategies for developing potent ligands with en-
hanced selectivity.18–20 Bivalent ligands are defined as compounds
which contain two pharmacophores joined through a connecting
unit or linker. The general structure for bivalent ligands is de-
scribed as P-X-P (P, pharmacophore; X, linker) (see Table 1). The
proper selection of a suitable linker X is crucial for potent receptor
binding.
meric protein polymer is composed principally of
a, b, and c
subunits. Recombinant receptors containing these subunits closely
mimic the biological, electrophysiological and pharmacological
properties of native GABAA receptors.5–7 Agents selective for spe-
cific BzR subtypes may permit one to separate out the pharmaco-
logical activities of these different isoforms.8–12 This is a goal of
paramount importance in the search for new anxiolytic agents
Recent studies on the binding selectivity of the inverse agonist
RY-80 (1) indicated preferential binding to
a5 BzR/GABAA sub-
types.21,22 Therefore, the bivalent ligand Xli093 (2) was developed
by incorporating the pharmacophore of 1 with a three-carbon lin-
ker.22 This bivalent ligand exhibited selective affinity for the
a5
subtype and behaved as a selective antagonist of the effects of
diazepam in oocytes at this 5 subtype. Effects at the other three
diazepam-sensitive sites were minimal. Encouraged by this, a
* Corresponding author. Tel.: +1 414 229 5856; fax: +1 414 229 5530.
E-mail address: capncook@uwm.edu (J.M. Cook).
These authors contributed equally to this work.
a
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.072

8854
D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
Table 1
linker
mono-
unit 1
mono-
unit 2
O
N
O
N
O
N
N
O
N
O
N
O
N
N
N
H
H
O
O
A
B
C
Compound
Monounit 1
Monounit 2
Spanner
Stable Conformation
Ki (anb3c2) = nM
X-ray
NMR
a1
a
2
a3
a4
a
5
a6
1a
2
3
A
A
A
C
A
B
B
B
—
A
A
C
A
—
B
C2H5
(CH2)3
(CH2)5
(CH2)3
CH2OCH2
C2H5
(CH2)2O(CH2)2
(CH2)3
—
Linear
—
—
—
—
Folded
Linear
—
28.4
>1000
231
1852
3795
287
21.4
>1000
661
4703
2694
45
25.8
858
2666
8545
1864
96
5.3
1550
ND
ND
ND
1504
ND
>5000
0.49
2 8.8
>2000
54.22
5000
ND
1000
5000
>5000
Linear
Linear
Linear
Folded
—
15
5.4
4
100.5
76.14
13.8
5000
194.2
5
6a
7
Folded
Linear
460
236
5000
7.4
ND
272
8
B
a
Monomer.
series of new bivalent ligands were designed and synthesized
(Table 1).
design. The influence of the molecular structure of the linker on the
conformation is also discussed.
It was hoped that these new dimers might exhibit enhanced
selectivity and potency at the a5 BzR/GABAA subtypes. It was also
2. Results
expected that insertion of an oxygen atom into the linker might in-
crease water solubility and hence enhance molecular hydrophilic-
ity which should play an important role in the pharmacokinetic
properties of the ligand.
The nature of the functional groups in a ligand plays an impor-
tant role in receptor binding, of course, as well as the conformation
in solution. The more information about the stable conformation(s)
of molecules the better the understanding of the structure–activity
relationships. It was essential from the beginning of the present
study to determine the conformation of these bivalent ligands,
which contained 3–5 atom linkers, since the steric requirements
for affinity to the Bz receptor must be satisfied.23
In traditional medicinal chemistry, computer assisted molecular
modeling programs and X-ray analysis contribute greatly in the
search for stable conformations. However, some problems with
these methods should not be neglected. Using computer modeling
to determine the stable conformation of molecules containing
many freely rotating bonds, such as those contained in bivalent li-
gands, is difficult. Although X-ray crystallography is the ultimate
arbiter of chemical structure, it has many limitations beyond the
obvious need for crystals: it often does not reflect accurately the
conformation in solution, nor is it informative regarding conforma-
tional equilibria. This information is crucial in drug design. How-
Recently it was shown the active dimer 2 existed in a linear
conformation in the solid state while dimer 7 with an oxygen-
containing linker folded back upon itself, as illustrated in
Figure 1.25
Since the bioactive conformation in solution may or may not
parallel that in the crystal structure, the lowest energy solution
structure must be established in order to correlate conformation
with biological activity. Thus, NMR experiments at variable tem-
peratures were performed and data were collected in different sol-
vents. In methylene chloride or chloroform at room temperature,
only a single set of signals was detected for both bivalent ligands
2 and 7. At low temperature, it was found that the linear dimer 2
exhibited only a small splitting of about 3 Hz for some of the aro-
matic protons in the 1H NMR spectra,25 while two clearly separated
sets of signals were observed for the folded dimer 7 (Fig. 2). For
example, as seen in Figure 2 for 7, the signal of H1 (7.92 ppm at
298 K) was split into two peaks at d 7.91 and 7.88 ppm, respec-
tively, at 193 K. Similar results were observed in the 13C spectrum
where C1 (134.9 ppm at 298 K) split into two signals at 135.3 and
135.4 ppm at 198 K. The doubling of the signals is consistent with
disruption of the symmetry between the two domains of the mol-
ecule as expected if 7 adopted a static folded structure similar to
the crystalline state.
ever, NMR spectroscopy is
a powerful technique in drug
discovery and its role in conformational analysis cannot be sur-
However, the possibility could not be ruled out that the split
in the signals was caused by slowing a dynamic process within
each domain, such as conformational interconversion of the se-
ven-membered ring. In order to investigate this possibility, the
NMR spectra of the monomer 6 were run at low temperature
as well. At temperatures as low as 173 K only one set of signals
was observed in both the 1H and 13C NMR spectra of 6 (Fig. 3).25
At lower temperatures, however, this was quite different from
what was observed for dimer 7 at room temperature; the spectra
of 6 and 7 were indistinguishable at 25 °C. Moreover, some addi-
passed by other spectroscopic methods.24
Herein is described a method utilizing low temperature NMR
for the determination of the solution stable conformation of a ser-
ies of GABAA–benzodiazepine bivalent ligands with different
monomeric units and linkers. The conformations in solution were
determined by NMR spectroscopy and compared with those in
the crystal structure. The combination of low temperature NMR
and X-ray analysis provided accurate structural information re-
quired for understanding structure–activity relationships and drug

D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
8855
2 (XLi-093)
7 (dm-III-96)
8 (DMH-D-053)
Figure 1. Crystal structure of 2 (left top), 7 (right) and 8 (left bottom).
298 K
263 K
193 K
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
ppm
Figure 2. Aromatic region of 1H NMR spectra of 7 in CD2Cl at variable temperatures.
2
tional line broadening of some of the aromatic signals was
observed at the lowest temperature in both the monomer 6
and in its dimer 7.
The study was then expanded by varying the nature of the lin-
ker and monomer. Dimers 3 and 5 contain the same monomeric
unit as 2, whereas 8 contains an all carbon linker. It was found that
2, 3, 4, and 8 exhibited only one set of NMR signals at low temper-
ature, whereas the NMR signals of ligands 5 and 7 split into two
sets at low temperature. Low temperature NMR studies were per-
formed in CD2Cl2. It was concluded that 5 and 7 preferred a folded
conformation, while 2, 3, 4, and 8 assumed a linear conformation.
These conclusions are supported by a crystal structure obtained for
bivalent ligand 8 which indicated 8 was present in a linear confor-
mation in the solid state. These results are illustrated in Table 1.
Since the goal was to design and synthesize bivalent ligands for
biological applications in aqueous solution, the question arose: Do
the conformations in CDCl3 or CD2Cl2 resemble those in aqueous
solution? Attempts to run the NMR experiments in water failed
since the ligands were not sufficiently soluble in D2O. However,
The analysis of these data indicated that the line broadening at
the lowest temperature was due to one of the conformational pro-
cesses mentioned above, whereas the doubling of the peaks in 7
was caused by the presence of two domains. Certainly an interdo-
main interaction existed between the two heterocyclic units of 7,
but not in monomer 6. It was therefore concluded the internal
mobility of the molecule decreased when the temperature was
lowered which permitted observation of the two sets of signals
of 7 on the NMR time scale. It was thus suggested that only when
the molecule preferred the folded conformation in solution were
two sets of signals observed. The preferred conformations of the
molecules in CDCl3 and CD2Cl2 correlated quite well with those ob-
served in the crystal structures (Table 1).

8856
D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
298 K
278 K
173 K
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
ppm
Figure 3. Aromatic region of 1H NMR spectra of 6 in CD2Cl2 at different temperatures.
the spectra of both the linear and folded dimers 2 and 7 could be
carried out in MeOH-d4. The solvating properties of methanol, of
course, more closely resemble those of water, and this more closely
mimics aqueous physiological condition as well. The conforma-
tions of dimers in MeOH-d4 were consistent with conformations
in hydrophobic solvents.
ferred a linear structure in the solid state and in lipophilic solvents,
only one set of signals was observed at room temperature in
MeOH-d4 (Fig. 7).
It is interesting to note that in the preparation of the samples,
dimer 2 was less soluble in MeOH-d4 than was 7. It has been estab-
lished that ligands are easier to dissolve in a solvent when the li-
gands surface energy can be minimized. Bivalent ligand 7 was
more polar than dimer 2 and has more tendencies to fold back.
Consequently, 7 was presumably, easier to dissolve in methanol
because its surface energy was minimized. On the other hand,
when ligand 2 was dissolved in a more polar solvent such as meth-
anol, the surface energy may be forced to be minimized.
Since the conformation of molecules 2 and 7 in methanol agree
with those in CD2Cl2 or CDCl3, the behavior of these ligands in
CD2Cl2 or CDCl3 should reflect those in aqueous solution. The stable
conformation of the compounds determined in CD2Cl2 or CDCl3
were correlated with the newly generated receptor binding data
3. Discussion
From the results described above, it is clear that dimers which
contain an oxygen atom in the linker tend to adopt a folded confor-
mation. Analysis of these data indicated in the hydrophilic solvent,
7 also had a higher tendency to fold back upon itself than 2, as it
did in the hydrophobic media (Figs. 4 and 5). In fact, the tendency
of 7 to assume a folded structure appeared to be higher in metha-
nol than in CD2Cl2 or CDCl3, as the free rotation of the molecule
was limited (Fig. 6). On the other hand, for dimer 2, which pre-
298 K
278 K
258 K
8.6
8.5
8.4
8.3
8.2
8.1
8.0
7.9
7.8
7.7
7.6
7.5
ppm
Figure 4. Aromatic region of 1H NMR spectra of 2 in MeOH-d4 at different temperatures.

D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
8857
298 K
278 K
253 K
8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 ppm
Figure 5. Aromatic region of proton spectra of 7 in MeOH-d4 at different temperatures.
H9
H9
H10
H10
ppm
116
118
120
122
124
126
128
130
132
134
136
138
140
142
144
8.3
8.2
8.1
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
ppm
Figure 6. Partial HSQC spectrum of 7 in MeOH-d4 at 298 K.
(Table 1). In the pharmacophore/receptor model, the bivalent li-
gands in the linear conformation align well (Fig. 7).
in a linear arrangement of atoms. From examination of the linkers
(Fig. 8), it was easily seen that linkers C and D can be regarded as
oligomers of oxymethylene (OCH2)2 and oxyethylene (OCH2CH2)2.
It was well documented that the preference for the gauche–
gauche conformation28–30 of a simple open-chain acetal such as
dimethoxymethane (CH3OCH2OCH3, 9) could be predicted on the
basis of the anomeric effect and related stereoelectronic effects.
In this conformation the polar CO bonds are favorably oriented
such that a lone pair orbital of the oxygen atom was almost anti-
periplanar to the CO bond (Fig. 9, lone pair orbital and CO bond
in same color). This permits maximum overlap of the n orbital of
the oxygen atom with the r* orbital of the CO bond. This was not
possible in the anti–anti or gauche–trans conformations and the
two rabbit-ear interactions28–32 (Fig. 9) engendered by each pair
of adjacent oxygen atoms in the anti-anti conformer are avoided.
Importantly, bivalent ligands 2, 3, 4, and 8 with carbon only
linkers preferred the linear conformation as the stable conforma-
tion, independent of the number of linker atoms. In contrast,
replacing the middle carbon of either linker (CH2)3 or (CH2)5 with
an oxygen atom altered the stable conformation of the molecules
5 and 7 from linear to folded. The only difference between bivalent
pairs 2 and 5 with linear and folded conformations as the stable
ones, respectively, was the center atom. In compound 2, the middle
atom was carbon, while in 5, oxygen was present. Consequently, it
was decided to focus attention on the conformational difference
between carbon and oxygen-containing linkers.
It was well known26,27 that the carbon chain in both small mol-
ecules and polymers favored the anti conformation which results

8858
D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
1,5-CHO interaction38–40 within the molecule were responsible
for the gauche-rich conformations.
Based on this pioneering work, the correlation was made that
the conformation of the linkers in the bivalents 25 and 78 adopt
anti (B), gauche–gauche (C), trans–gauche–trans (D) conformations,
respectively, regardless of the monomeric units which comprise
them. The arrangement in space (disregarding the direction) of
every unit in the linkers is depicted in Figure 10. The end-to-end
distance of each unit (C and D) which adopted the gauche confor-
mation was shorter than the one in the anti conformer. The more
units in the linker, the shorter the gauche linker. Moreover, it
was recognized that the CO bond length (1.43 Å) is often apprecia-
bly shorter than the CC bond (1.54 Å).41 Therefore, it was believed
that the linkers with the oxygen atom in the middle favored the
helical conformation and rendered the two monomeric units in
each dimer sufficiently close to each other with suitable dihedral
angles to facilitate the intramolecular lipophilic–lipophilic
(aromatic–aromatic) interaction. This was regarded as one of the
most important factors to stabilize the folded structure as the
preferred conformation.42–46 For these same reasons, bivalent
ligands with the linker B adopted the linear conformation.
For the higher analog of POM and POE, namely, poly(trimethyl-
ene oxide) [ (CH2)3O]x (POM3), trans-gauche-gauche-trans was
sightly preferred over all-trans and trans–trans–gauche–trans con-
formers in the crystalline state.27 The preference for the gauche state
in this case was only 0.2 kcal/mol where as in the POM and POE
examples was 1.5 and 0.4 kcal/mol, respectively.33 Since the energy
difference between gauche and trans was low, the linker A had more
flexibility than the other linkers (C and D) to rotate freely and less
tendency to occur as gauche. This could lead to improper end-to-
end distances or dihedral angles for the interaction between the
aromatic monounits. Hence, dimers connected with linker A could
not be stabilized in the folded conformation even though the same
aromatic monounits were contained in the molecules.
Figure 7. Bivalent ligand
pharmacophore/receptor model for the
2 (Xli093) aligned in the included volume of the
a5b3
c
2 subtype.
O
O
O
O
O
O
O
b
b
d
d
b
a
d
f
a
g
O
c
e
c
e
e
O
O
O
O
d
O
b
f
g
O
a
O
c
O
e
a
c
O
O
Bivalent ligands were evaluated in competition binding assays
for specific GABAA membrane proteins using [3H]flunitrazepam
as the radiolabel. This assay measures the ability of the ligand to
displace flunitrazepam. Data are reported as Ki according to the
Cheng–Prusoff equation.47 Biological data are presented in Table
1 for dimers synthesized using different spanners (or linkers).
The linkers in 5 and 7 contained an oxygen atom. Compounds 2,
3, 4, and 8 contained all carbon linkers. Binding data indicate de-
creased affinity when bivalents contain an oxygen atom in the lin-
Figure 8. The linkers of the bivalent ligands.
H3C
O
ker. Compounds 2 and 3 showed increased selectivity for the
a5
subtype as compared to parent monomer 1. Compound 5 which
was analogous to 2 with the exception of the oxygen atom present
CH3
H3C
H
O
H
in the linker, bound with less affinity at the a5 subtype. Likewise
CH3
bivalent 8 showed increased selectivity versus monomer 6. How-
ever, the bivalent 7 containing an oxygen atom in the linker did
not bind. The data suggest that dimers which contain a single oxy-
gen atom in the linker bind with less decreased affinity to the Bz
receptor. This is due to their propensity to adopt a folded confor-
mation. A strategy to increase hydrophilicity and avoid a folded
anti-anti
gauche-gauche
Figure 9. The conformations for dimethoxymethane.
Furthermore, in the related polymer of 9 with the two-bond
repeating sequence, poly(oxymethylene) (POM), the gauche con-
formation was, in fact, markedly preferred over trans and the poly-
mer existed in a helical (all gauche) conformation27,33–35 rather
than in the all-anti one.
Similarly, much effort has been spent on the investigation of the
conformational characteristics of 1,2-dimethoxyethane (glyme,
CH3OCH2CH2OCH3, 10) as a model molecule for understanding
the conformations of poly(oxyethylene) (POE). It had long been
established that POE chains have a large fraction of bonds in gauche
conformations and assumed a helical conformation overall.26,27,33–35
It has been proposed that the oxygen gauche effect27,36,37 and
gauche
gauche
anti
Oa
Oa
Oa
H2
d
C
H
H
c
H
O
c
c
b
d
b
b
H
H
H
H
H
H
d
H2C
H
B
C
D
Figure 10. Newman projection for linkers B, C, and D.

D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
8859
conformation is to extend the linker length and insert two oppos-
ing oxygen atoms. This research is currently underway.
DPX300 NMR spectrometer equipped with a z-gradient broadband
(BBO) probe or on a Bruker DRX500 NMR spectrometer with either
a triple axis gradient inverse (BBI) probe or a broadband observe
(BBO) probe. Infrared spectra were recorded on a Nicolet DX FTIR
BX V5.07 spectrometer or a Mattson Polaris IR-10400 instrument.
Low-resolution mass spectral data (EI/CI) were obtained on a Hew-
lett–Packard 5985B GC–mass spectrometer, while high resolution
mass spectral data were taken on a VG autospectrometer (Double
Focusing High Resolution GC/Mass Spectrometer, UK). Microanaly-
ses were performed on a CE Elantech EA1110 elemental analyzer.
4. Conclusion
In summary, comparison of the results of low temperature NMR
studies to crystal structures has provided enough information to
demonstrate that low temperature NMR can be used as a quick
method to identify dimeric ligands with a tendency to fold back
upon themselves, as compared with those preferring a linear con-
formation. A correlation with binding data shows that the suitabil-
5.3. Competition binding assays
ity of a ligand in the
a5 BzR/Gabaergic subtype is heavily
influenced by its conformation in solution. Variable temperature
NMR thus can be used as a tool for screening bivalent ligands for
their in vivo suitability. It is also clear the presence of one oxygen
atom in the linker was the principle cause for the dimer to fold
back onto itself. Ligands which contain two offsetting oxygen
atoms in the linker are now under study in our laboratory.
Competition binding assays were performed in a total volume
of 0.5 mL at 4 °C for 1 h using [3H]flunitrazepam as the radioligand.
For these binding assays, 20–50 mg of membrane protein har-
vested with hypotonic buffer (50 mM Tris–acetate, pH 7.4, at
4 °C) was incubated with the radiolabel as previously described.53
Non-specific binding was defined as radioactivity bound in the
presence of 100 lM diazepam and represented less than 20% of
5. Experimental
5.1. Synthesis
total binding. Membranes were harvested with a Brandel cell
harvester followed by three ice-cold washes onto polyethyleneim-
ine-pretreated (0.3%) Whatman GF/C filters. Filters were dried
overnight and then soaked in Ecoscint A liquid scintillation cocktail
(National Diagnostics; Atlanta, GA). Bound radioactivity was quan-
tified by liquid scintillation counting. Membrane protein concen-
trations were determined using an assay kit from Bio-Rad
(Hercules, CA) with bovine serum albumin as the standard.
Inverse agonist 1 (RY080) was synthesized via the reported pro-
cedure.3,48 Hydrolysis of the ester function of 1 provided the acid 9
in excellent yield and this material was subjected to a standard
CDI-mediated coupling reaction to furnish bivalent ligands 2–5 in
60% yield (Scheme 1).22,49
The acid 11, obtained from the ester 10, which was available
from the literature,3,50 was stirred with CDI in DMF, followed by
stirring with the required diol and DBU to provide bromide dimers
12 or 13, respectively. They were converted into the trimethylsilyl-
acetylenyl 14 or 15, respectively, under standard conditions
(Pd-mediated, Heck-type coupling).51,52 The bisacetylene 7 or 8
(individually) was easily obtained by treatment of the trimethyl-
silyl ligand 14 or 15 with fluoride anion, as shown in Scheme 2.
5.4. Radioligand binding assays (Drs. McKernan and Atack)49
In brief, the affinity of compounds for human recombinant GA-
BA(A) receptors was measured by competition binding using
0.5 nM [3H]flunitrazepam. Transfected HEK Cells (b2
c
2 and de-
sired
a subtype) were harvested into phosphate-buffered saline,
centrifuged at 3000g and stored at À70 °C until required. On the
day of the assay, pellets were thawed and re-suspended in suffi-
cient volume of 50 mM Tris/acetate (pH 7.4 at 4 °C) to give a total
binding of approximately 1500–2000 dpm. Non-specific binding
was defined in the presence of 100 mM (final concentration) diaz-
epam. Test compounds were dissolved in DMSO at a concentration
of 10 mM and diluted in assay buffer to give an appropriate con-
centration range in the assay, such that the final DMSO concentra-
tion in the assay was always less than 1%. Total assay volume was
0.5 mL and assays were carried out in 96-well plates and incuba-
tion time started by the addition of 0.1 mL of re-suspended cell
membranes. Following incubation for 1 h at 4 °C, assays were ter-
minated by filtration through GF/B filters, washed with 10 mL
5.2. Materials and general instrumentation
Chemicals were purchased from Aldrich Chemical Co. or Tokyo
Chemical Industries and were used without further purification ex-
cept where otherwise noted. Anhydrous THF was distilled from so-
dium/benzophenone ketyl. TLC analyses were carried out on Merck
Kieselgel 60 F254, and flash column chromatography was performed
on silica gel 60b purchased from E.M. Laboratories. Melting points
were taken on a Thomas–Hoover melting point apparatus or an
Electrothermal Model IA8100 digital melting point apparatus and
are reported uncorrected. NMR spectra were acquired on a Bruker
O
N
O
N
ºC;
2 N NaOH / EtOH, 70
10% aq HCl
N
CH2CH3
N
O
OH
N
N
CH3
CH3
O
O
1
9
O
O
N
N
O
N
O
X
N
CDI, DMF;
diol, DBU
n
N
N
CH3
H3C
O
O
2 X = CH2, n = 1
3 X = CH2, n = 3
5 X = O, n = 1
Scheme 1. Synthesis of bivalent analogs of Xli093.

8860
D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
N
N
O
COOC2H5
COOH
O
N
N
N
N
N
2N NaOH / EtOH, reflux ;
O
O
X
CDI / DMF;
diol,DBU
N
N
N
Br
N
Br
10% aq. HCl
N
Br
Br
10
11
12 X = CH2OCH2
13 X = CH2
O
O
O
O
N
N
N
N
N
H
(H3C)3Si
X
O
O
X
O
O
N
N
N
N
Pd(OAc)2(PPh3)2
.
xH2O,
TBAF
Et3N, CH3CN
N
N
-78 ºC
THF,
N
reflux
TMS
TMS
14 X = CH2OCH2
15 X = CH2
7 X = CH2OCH2,
8 X = CH2
Scheme 2. Synthesis of bivalent analogs of DMH-D-053.
ice-cold buffer, dried and then counted using a liquid scintillation
counter. The percentage inhibition of [3H]flunitrazepam binding,
the IC50 and the Ki values were calculated using the Activity Base
Software Package (ID Business Solutions, Guildford, UK) according
to the Cheng–Prusoff equation.47
vide 3 as a white solid in 89.2% yield. 3: mp 132–138 °C; IR
(KBr) 3422, 3280, 2931, 1714, 1635, 1487, 1249, 1064 cmÀ1
;
1H NMR (500 MHz, CDCl3) d 1.90 (m, 4H), 3.24 (s,6H), 3.52 (s,
2H), 4.39 (s, 8H), 5.29 (s, 2H), 7.36 (dd, 2H, J = 8.1, 16 Hz), 7.70
(m, 2H) 7.70 (m, 2H), 7.86 (s, 2H), 8.18 (s, 2H); MS (FAB, NBA)
m/e (relative intensity) 631 (M++1, 13). Anal. Calcd for
5.4.1. 1,3-Bis(8-acetyleno-5,6-dihydro-5-methyl-6-oxo-4H-
imidazo[1,5-a][1,4]benzodiazepine-3-carboxy) propyl diester
(2) (XLi093) (Procedure A)
C
35H30N6O6Á5/3 H2O: C, 63.61; H, 4.83; N, 12.72. Found: C,
63.16; H, 4.72; N, 13.06.
To a solution of carbonyl diimidazole (230.3 mg, 0.57 mmol) in
anhydrous DMF (5 mL) was added 8-ethynyl-5,6-dihydro-5-
methyl-6-oxo-4H-imidazo[1,5-a][1,4]-benzodiazepine-3-carbox-
ylic acid 9 (200 mg, 0.71 mmol). The solution which resulted was
stirred for 2 h at rt. Analysis by TLC (silica gel) indicated the absence
of starting material. To the solution which resulted was then added
1,3-propanediol (27.1 mg, 0.36 mmol) in dry DMF (0.5 mL) and also
DBU (114.2 mg, 0.75 mmol) in dry DMF (0.10 mL) at rt. The mixture
was stirred at rt for 4.5 h until analysis by TLC (silica gel) indicated
the reaction was complete. The reaction mixture was then poured
into ice water (30 mL) and extracted with CH2Cl2 (3Â 50 mL). The
combined organic layer was washed with H2O (5Â 50 mL), brine
and dried (Na2SO4). The solvent was removed under reduced pres-
sure and the residue was purified by flash chromatography (silica
gel, EtOAc/CH3OH, 4:1) to provide 2 (157 mg) as a white solid in
73.4% yield. 2: mp >230 °C (dec.); IR (NaCl) 3247, 1725, 1641,
1359, 1253, 1061 cmÀ1; H NMR (300 MHz, CDCl3) d 2.37 (m, 2H),
3.24 (s, 2H), 3.26 (s, 6H), 4.40 (s, 2H), 4.57 (t, 4H, J = 6.2 Hz), 5.31
(br, 2H), 7.41 (d, 2H, J = 8.3 Hz) 7.72 (dd, 2H, J = 6.43, 1.86 Hz),
7.89 (s, 2H), 8.19 (d, 2H, J = 1.76 Hz); 13C NMR (75.5 MHz, CDCl3)
d 26.2, 34.4, 40.7, 60.2, 78.7, 79.7, 120.4, 121.6, 127.1, 127.7,
130.1, 133.4, 134.1, 134.9, 161.3, 164.1; MS (FAB, NBA) m/e (relative
intensity) 603 (M++1, 100). This material was employed for the X-
ray crystal structure. It was homogenous in two independent TLC
systems [Rf = 0.31 in EtOAc/CH3OH, 4:1; Rf = 0.32 in CH2Cl2/CH3OH,
9:1]. Anal. Calcd for C33H26N6O6Á2/3 CH3OH: C, 64.81; H, 4.63; N,
13.47. Found: C, 64.56; H, 4.72; N, 13.76.
5.4.3. 1,3-Bis(8-ethyl-5,6-dihydro-5-methyl-6-oxo-4H-imi-
dazo[1,5-a][1,4]benzodiazepine-3-carboxy) propyl diester
(4) (XLi356)
1,3-Bis(8-acetyleno-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-
a][1,4]-benzodiaze-pine -3-carboxy) propyl diester
2 (500 mg,
0.83 mmol) was dissolved in EtOH (150 mL) after which Pd/C
(176 mg) was added in solution at rt. The slurry was stirred for 5 h un-
der one atmosphere of H2 (bench top, balloon of H2). The catalyst was
removed by filtration and washed with EtOH. The EtOH was removed
under reduced pressure to furnish a residue. This material was puri-
fied by flash chromatography (silica gel, EtOAc/EtOH 8:2) to provide
4 (504 mg, 99%) as white crystals: mp 125–133 °C; IR (NaCl) 3407,
1
2964, 2358, 1725, 1640, 1499 cmÀ1; H NMR (CDCl3) d 1.29 (m, 6H),
2.39(m, 2H), 2.78 (dd, 4H, J = 7.5, 15.1 Hz), 3.26 (s, 6H), 4.48 (br,
2H), 4.56 (t, 4H, J = 6.1 Hz, 12.2 Hz), 5.16 (br, 2H), 7.33 (d, 2H,
J = 8.2 Hz), 7.48 (d, 2H, J = 1.8 Hz), 7.89 (t, 4H, J = 3.2 Hz, 5.3 Hz),
8.15; MS(EI) m/e (relative intensity) 611 (M++1, 100). Anal. Calcd for
C
33H34N6O6Á2H2O: C, 61.33; H, 5.92; N, 13.00. Found: C, 61.74; H,
5.91; N, 12.63.
5.4.4. Bis(8-acetyleno-5,6-dihydro-5-methyl-6-oxo-4H-
imidazo[1,5-a][1,4]benzodiazepine-3-carboxy) dimethyl
glycol diester (5) (XLi374)
Ligand 9 (100 mg, 0.356 mmol) was dissolved in DMF (10 mL),
and to this solution dibromodimethyl ether (36.3 mg, 0.178 mmol)
was added, followed by TEA(2.0 mL). After 26 h of stirring at ambi-
ent temperature, the solvent was evaporated under reduced pres-
sure and the residue was distributed between CHCl3 and water.
The organic phase was washed multiple times with water and
dried over Na2SO4, and dried. The dried crude ether was applied
on a column of silica gel to afford dimer 5 (86 mg) in 80% yield:
mp >220 °C (dec.); IR (KBr) 3419, 3237, 2910, 1714, 1635, 1561,
5.4.2. 1,5-Bis(8-acetyleno-5,6-dihydro-5-methyl-6-oxo-4H-
imidazo[1,5-a][1,4]benzodiazepine-3-carboxy) pentyl diester
(3) (XLi210)
Ligand 3 was prepared by following the procedure A and acid
9 by replacing the 1,3-propanediol with 1,5-pentanediol to pro-

D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
8861
1498 cmÀ1; 1H NMR (500 MHz, CD2Cl) d d 3.18 (s, 6H), 3.31 (s, 2H),
4.37 (br, 2H), 5.29 (br, 2H), 5.74 (s, 4H), 7.40 (d, 2H, J = 8.1 Hz), 7.74
(dd, 2H, J = 1.6, 8.2 Hz), 7.90 (s, 2H), 8.15 (s, 2H); MS(FAB,NBA) m/e
(relative intensity) 605 (M++1, 100). Anal. Calcd for C32H24N6O7 Á3/
2 CH3COOC2H5: C, 61.99; H, 4.93; N, 11.41. Found: C, 61.36; H,
4.52; N, 11.96.
2H), 4.05 (d, 2H, J = 12.6 Hz), 4.55 (m, 4H), 6.02 (d, 2H,
J = 12.6 Hz), 7.37–7.55 (m, 14H), 7.75 (dd, 2H, J = 1.8, 8.4 Hz),
7.94 (s, 2H); 13C NMR (75.5 MHz, CDCl3) d 0.3, 28.3, 44.9, 61.4,
97.4, 102.3, 122.4, 122.6, 128.0, 128.3, 129.0, 129.4, 130.5, 134.1,
134.9, 135.1, 139.0, 139.2, 139.2, 162.6, 168.5; MS (FAB, NBA) m/
e (relative intensity) 839 (M++1, 100). Anal. Calcd for C49H46N6O4-
Si2: C, 70.14; H, 5.53; N, 10.02. Found: C, 69.97; H, 5.35; N,
9.77.
5.4.5. 8-Bromo-6-phenyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-
3-carboxylic acid (11)
The ester 10 (2 g) was dissolved in EtOH (50 mL) and aq sodium
hydroxide (10 mL, 2 N) was added to the solution. The mixture was
heated to reflux for 0.5 h. After the EtOH was removed under re-
duced pressure, the solution was allowed to cool. The pH value
was adjusted to 4 by adding 10% aq HCl dropwise. The mixture
was filtered and the solid was washed with water and ethyl ether.
The solid was dried to provide 11 (1.8 g, 96.6%): mp >250 °C; IR
5.4.8. 1,3-Bis(8-acetylenyl-6-phenyl-4H-benzo[f]imidazo[1,5-
a][1,4]diazepine-3-carboxy)propyl diester (8) (DMH-D-053)
(Procedure D)
A solution of bistrimethylsilyl dimer 15 (330 mg, 0.4 mmol) in
THF (70 mL) was stirred with tetrabutylammonium fluoride hy-
drate (250 mg, 0.96 mmol) at À78 °C for 5 min. After this, H2O
(35 mL) was added to the solution to quench the reaction and stir-
ring continued at low temperature for one half hour. The solution
was extracted with EtOAc (3Â 100 mL), and the organic layer was
washed with water. After removal of the solvent under reduced
pressure, ethyl ether was added to the residue to precipitate a so-
lid. The mixture was filtered and the solid was washed with
(KBr) 3450 (b), 2844, 1707, 1615, 1493, 1166, 700 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6) d 4.14 (d, 1H, J = 12.6 Hz), 5.79 (d, 1H,
12.6 Hz), 7.41–7.54 (m, 6H), 7.88 (d, 1H, J = 8.7 Hz), 8.03 (dd, 1H,
J = 8.7, 2.1 Hz), 8.47 (s, 1H); MS (EI) m/e (relative intensity) 381
(M+, 20), 383 (19).
CH2Cl2/Et2O (ca. 1:15) to provide the bisacetylenyl dimer
8
(DMH-D-053, 220 mg, 80%) as a yellow solid which crystallizes in
5.4.6. 1,3-Bis(8-bromo-6-phenyl-4H-benzo[f]imidazo[1,5-a]
[1,4]diazepine-3-carboxy) propyl diester (13) (DMH-D-070)
(Procedure B)
CH2Cl2: mp 172–175 °C; IR (KBr) 3450, 3280, 2950, 1720, 1715,
1495, 1250, 1120, 1050 cmÀ1 1H NMR (300 MHz, CDCl3) d 2.35
;
(m, 2H), 3.18 (s, 2H), 4.08 (d, 2H, J = 12.3 Hz), 4.56 (m, 4H), 6.04
(d, 2H, J = 12.6 Hz), 7.36–7.59 (m, 14H), 7.78 (dd, 2H, J = 8.4,
1.7 Hz), 7.95 (s, 2H); 13C NMR (75.5 MHz, CDCl3) d 28.8, 45.4,
61.9, 80.2, 81.3, 121.4, 122.7, 128.1, 128.3, 129.0, 129.3, 130.5,
134.2, 135.2, 135.3, 135.6, 138.9, 139.2, 162.6, 168.5; MS (FAB,
The carboxylic acid 11 (2 g, 5.2 mmol) was dissolved in DMF
(20 mL), after which CDI (1.02 g, 6.3 mmol) was added at rt and
the mixture was stirred for 2 h. Then 1,3-propanediol (0.19 mL,
2.6 mmol) and DBU (0.78 mL, 5.2 mmol) were added to the mix-
ture and stirring continued overnight. The reaction solution was
then cooled with an ice-water bath, after which water was added
to precipitate a solid. This material was purified further by flash
chromatography on silica gel (gradient elution, EtOAc/EtOH 20:1,
15:1, 10:1) to provide the bisbromide 13 (DMH-D-070) as a white
solid (1.3 g, 61.9%): mp 187.5–189 °C; IR (KBr) 3112, 2968, 1708,
NBA) m/e (relative intensity) 695 (M++1, 100). Anal. Calcd for
1
C
43H30N6O4Á 4CH2Cl2: C, 72.63; H, 4.30; N, 11.75. Found: C,
72.36; H, 4.27; N, 11.36.
5.4.9. Bis(8-bromo-6-phenyl-4H-benzo[f]imidazo[1,5-a][1,4]-
diazepine-3-carboxy) diethylene glycol diester (12) (DM-III-93)
Ligand 12 was prepared from acid 11, under the same condi-
tions employed in procedure B, by replacing 1,3-propanediol with
diethylene glycol to yield a yellow solid (93.7%) 12: mp 165–
168 °C; IR (KBr) 3060, 2956, 1725, 1610, 1558, 1491, 1267, 1161,
1610, 1559, 1491, 1269, 1160, 1123, 1073 cmÀ1
;
1H NMR
(300 MHz, CDCl3) d 2.35 (m, 2H), 4.08 (d, 2H, J = 12.6 Hz), 4.55
(m, 4H), 6.05 (d, 2H, J = 12.6 Hz), 7.37–7.53 (m, 12H), 7.6 (d, 2H,
J = 2.1 Hz), 7.81 (dd, 2H, J = 2.1, 8.6 Hz), 7.93 (s, 2H); 13C NMR
(75.5 MHz, CDCl3) d 28.2, 44.9, 61.4, 120.7, 124.2, 128.3, 129.0,
129.3, 129.6, 130.6, 134.1, 134.4, 134.7, 135.0, 138.9, 138.9,
162.6, 167.9; MS (FAB, NBA) m/e (relative intensity) 803 (M++1,
15), Anal. Calcd for C39H28N6O4Br2: C, 58.23; H, 3.51; N, 10.45.
Found: C, 57.92; H, 3.43; N, 10.29.
; d 3.93 (t, 4H,
1123, 1074 cmÀ1 1H NMR (300 MHz, CDCl3)
J = 4.8 Hz), 4.06 (d, 2H, J = 12.6 Hz), 4.54 (m, 4H), 6.05 (d, 2H,
J = 12.6 Hz), 7.39–7.50 (m, 12H), 7.57 (d, 2H, J = 2.7 Hz), 7.80 (dd,
2H, J = 2.1, 8.4 Hz), 7.90 (s, 2H); 13C NMR (75.5 MHz, CDCl3) d
44.9, 63.6, 69.0, 120.7, 124.2, 128.3, 129.0, 129.3, 129.6, 130.6,
134.1, 134.4, 134.6, 135.0, 138.9, 139.0, 162.5, 167.9; MS (FAB,
NBA) m/e (relative intensity) 833 (M++1, 5). Anal. Calcd for
5.4.7. 1,3-Bis(8-trimethylsilylacetylenyl-6-phenyl-4H-benzo-
[f]imidazo[1,5-a][1,4]-diazepine-3-carboxy) propyl diester
(15) (DMH-D-048) (Procedure C)
C
40H30Br2N6O5Á0.15CHCl3: C, 56.72; H, 3.57; N, 9.88. Found: C,
56.61; H, 3.55; N, 9.92.
To a suspension of bisbromide 13 (1.005 g, 1.25 mmol) in ace-
tonitrile (50 mL) and triethylamine (65 mL), was added bis(tri-
phenylphosphine)-palladium (II) acetate (0.15 g, 0.2 mmol). The
solution which resulted was degassed and trimethylsilylacetylene
(0.7 mL, 5 mmol) was added after which it was degassed again (ar-
gon followed by vacuum). The mixture was heated to reflux and
stirring maintained overnight. After removal of the solvent under
reduced pressure, the residue was dissolved in CH2Cl2 and washed
with water. 3-Mercaptopropyl functionalized silica gel (0.6 g) was
added into the organic layer and stirring continued for 1 h. The sil-
ica gel/Pd complex was removed by filtration and the filtrate was
concentrated under reduced pressure. The residue was purified
by flash column chromatography on silica gel (gradient elution,
EtOAc/EtOH 20:1, 15:1, 10:1) to furnish the bistrimethylsilyl dimer
15 (DMH-D-048, 680 mg, 60.8%) as a white solid: mp 169–172 °C;
IR (KBr) 3449, 2950, 1725, 1720, 1715, 1496, 1250, 1160, 1080,
5.4.10. Bis(8-trimethylsilylacetylenyl-6-phenyl-4H-benzo-
[f]imidazo[1,5-a][1,4]diazepine-3-carboxy) diethylene glycol
diester (14) (DM-III-94)
Ligand 14 was prepared from dibromide 12, under the same
conditions employed in Procedure C by replacing 1,3-propanediol
with diethylene glycol to produce a yellow solid (49.5%) 14: mp
205–208 °C; IR (KBr) 3433, 2960, 1730, 1700, 1612, 1493, 1255,
1169, 1120, 1071, 847 cmÀ1 1H NMR (300 MHz, CDCl3) d 0.25 (s,
;
18H), 3.93 (t, 4H, J = 5.4 Hz), 4.04 (d, 2H, J = 12.6 Hz), 4.55 (m,
4H), 6.04 (d, 2H, J = 12.6 Hz), 7.37–7.53 (m, 14H), 7.74 (dd, 2H,
J = 1.2, 8.4 Hz), 7.91 (s, 2H); 13C NMR (75.5 MHz, CDCl3) d 0.3,
45.0, 63.6, 69.0, 97.5, 102.4, 122.5, 122.7, 128.1, 128.3, 129.0,
129.4, 130.5, 134.2, 135.0, 135.1, 135.2, 139.1, 139.3, 162.7,
168.6; MS (FAB, NBA) m/e (relative intensity) 869 (M++1, 100).
1
Anal. Calcd for C50H48N6O5Si2Á 4H2O: C, 68.81; H, 5.60; N, 9.62.
847 cmÀ1 1H NMR (300 MHz, CDCl3) d 0.25 (s, 18H), 2.35 (m,
;
Found: C, 68.88; H, 5.66; N, 9.51.

8862
D. Han et al. / Bioorg. Med. Chem. 16 (2008) 8853–8862
Macaulay, A.; Brown, N.; Howell, O.; Moore, K. W.; Carling, R. W.; Street, L. J.;
Castro, J. L.; Ragan, C. I.; Dawson, G. R.; Whiting, P. J. Nat. Neurosci. 2000, 3, 587.
12. Möhler, H.; Fritschy, J. M.; Rudolph, U. J. Pharmacol. Exp. Ther. 2002, 300, 2.
13. Liu, R. Y.; Zhang, P. W.; McKernan, R. M.; Wafford, K.; Cook, J. M. Med. Chem.
Res. 1995, 5, 700.
14. Liu, R. Y.; Hu, R. J.; Zhang, P. W.; Skolnick, P.; Cook, J. M. J. Med. Chem. 1996, 39,
1928.
15. Yu, S.; Ma, C. R.; He, X. H.; McKernan, R.; Cook, J. M. Med. Chem. Res. 1999, 9,
71.
5.4.11. Bis(8-acetylenyl-6-phenyl-4H-benzo[f]imidazo[1,5-
a][1,4]diazepine-3-carboxy) diethylene glycol diester (7) (dm-
III-96)
Ligand 7 was prepared from diester 14, under the same condi-
tions employed in procedure B, by replacing 1,3-propanediol with
diethylene glycol to provide a yellow solid (81.6%) 7: mp 173–
177 °C; IR (KBr) 3432, 3280, 1720, 1715, 1496, 1254, 1175, 1120,
1074 cmÀ1 1H NMR (300 MHz, CDCl3) d 3.12 (s, 2H), 3.93 (t, 4H,
;
16. Bailey, D. J.; Tetzlaff, J. E.; Cook, J. M.; He, X. H.; Helmstetter, F. J. Neurobiol.
Learn. Mem. 2002, 78, 1.
J = 4.5 Hz), 4.06 (d, 2H, J = 12.6 Hz), 4.55 (m, 4H), 6.05 (d, 2H,
J = 12.6 Hz), 7.38–7.56 (m, 14H), 7.75 (dd, 2H, J = 8.4, 1.8 Hz),
7.91 (s, 2H); 13C NMR (75.5 MHz, CDCl3) d 45.0, 63.6, 69.0, 79.8,
81.3, 121.3, 122.7, 128.1, 128.3, 129.0, 129.3, 130.5, 134.2, 135.2,
135.3, 135.6, 139.0, 139.1, 162.6, 168.4; MS (FAB, NBA) m/e (rela-
tive intensity) 725 (M++1, 63). Anal. Calcd for C44H32N6O5Á
17. Platt, D. M.; Rowlett, J. K.; Spealman, R. D.; Cook, J.; Ma, C. R.
Psychopharmacology 2002, 164, 151.
18. Erez, M.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem. 1982, 25, 847.
19. Portoghese, P. S.; Larson, D. L.; Yim, C. B.; Sayre, L. M.; Ronsisvalle, G.;
Lipkowski, A. W.; Takemori, A. E.; Rice, K. C.; Tam, S. W. J. Med. Chem. 1985, 28,
1140.
20. Portoghese, P. S.; Larson, D. L.; Sayre, L. M.; Yim, C. B.; Ronsisvalle, G.; Tam, S.
W.; Takemori, A. E. J. Med. Chem. 1986, 29, 1855.
21. Roth, B. 2006, unpublished results.
22. Li, X. Y.; Cao, H.; Zhang, C. C.; Furtmueller, R.; Fuchs, K.; Huck, S.; Sieghart, W.;
Deschamps, J.; Cook, J. M. J. Med. Chem. 2003, 46, 5567.
23. Huang, Q.; He, X. H.; Ma, C. R.; Liu, R. Y.; Yu, S.; Dayer, C. A.; Wenger, G. R.;
McKernan, R.; Cook, J. M. J. Med. Chem. 2000, 43, 71.
1
4EtOAcÁ3/2 H2O: C, 69.89; H, 4.82; N, 10.87. Found: C, 70.12; H,
4.45; N, 10.58.
6. X-ray crystallographic data
24. Burger’s Medicinal Chemistry & Drug Discovery, 6th ed.; John Wiley & Sons Inc.,
2003..
25. Han, D. M.; Forsterling, F. H.; Li, X. Y.; Deschamps, J. R.; Cao, H.; Cook, J. M.
Bioorg. Med. Chem. Lett. 2004, 14, 1465.
26. Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; Wiley
Interscience: NY, 1994.
27. Flory, P. J. Statistical Mechanics of Chain Molecules; Interscience Publishers: NY,
1968.
28. Lemieux, R. U. Molecular Rearrangement; Interscience Publishers: NY, 1964.
29. Eliel, E. L. J. Acc. Chem. Res 1970, 3, 1.
30. Graczyk, P. P.; Mikolajczyk, M. Topics in Stereochemistry; John Wiley & Sons,
1994.
31. Hutchins, R. O.; Kopp, L. D.; Eliel, E. L. J. Am. Chem. Soc. 1968, 90, 7174.
32. Eliel, E. L.; Giza, C. A. J. Org. Chem. 1968, 33, 3754.
33. Uchida, T.; Kurita, Y.; Kubo, M. J. Polym. Sci. 1956, 19, 365.
34. Abe, A.; Mark, J. E. J. Am. Chem. Soc. 1976, 98, 6468.
35. Ohsaku, M. Macromolecules 1978, 11, 970.
Crystallographic data (excluding structure factors) for the struc-
tures in this paper have been deposited with the Cambridge Crys-
tallographic Data Centre as supplementary publication Nos.
687205(DMH-D-053), 222395(Xli093), and 222396(DM-III-96).
Copies of the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44 (0)
1223 336033 or email: deposit@ccdc.cam.ac.uk).
Acknowledgments
The authors thank NIMH (MH-46851), the Research Growth
Initiative of the University of Wisconsin-Milwaukee, NSF (instru-
mentation grant NSF-9512622), NIDA (DA-11792) and NCRR
(RR-00168) for support of this work. Moreover, the authors acknowl-
edge NIDA and ONR for support of the X-ray crystallography.
36. Wolfe, S. Acc. Chem. Res. 1972, 5, 102.
37. Jaffe, R. J.; Smith, G. D.; Yoon, D. Y. J. Phys. Chem. 1993, 97, 12745.
38. Bultinck, P.; Van Alsenoy, C.; Goeminne, A. J. Phys. Chem. 2001, 105, 9203.
39. Bultinck, P.; Goeminne, A.; Van de Vondel, D. J. Mol. Struct. 1999, 467, 211.
40. Glendening, E. D.; Feller, D.; Thompson, M. A. J. Am. Chem. Soc. 1994, 116,
10657.
41. Glasstone, S.; Laidler, K. J.; Eyring, H. The Theory of Rate Processes; McGraw-Hill:
NY, 1941.
42. Azumaya, I.; Uchida, I.; Kato, T.; Yokoyama, A.; Tanatani, A.; Takayanagi, H.;
Yokozawa, T. Angew. Chem. Int. Ed. 2004, 43, 1360.
43. Rashkin, M. J.; Waters, M. L. J. Am. Chem. Soc. 2002, 124, 1860.
44. Tsuzuki, S.; Honda, K.; Uchimaru, T.; Mikami, M.; Tanabe, K. J. Am. Chem. Soc.
2002, 124, 104.
45. Hobza, P.; Selzle, H. L.; Schlag, E. W. J. Am. Chem. Soc. 1994, 116, 3500.
46. Burley, S. K.; Petsko, G. A. Science 1985, 229, 23.
47. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
48. Fryer, R. I.; Schmidt, R. A.; Sternbach, L. H. J. Pharm. Sci. 1964, 53, 264.
49. Li, X. Synthesis of Selective Ligands for GABAA/Benzodiazepine Receptors;
University of Wisconsin-Milwaukee: Milwaukee, 2004.
50. Sternbach, L. H.; Fryer, R. I.; Metlesics, W.; Reeder, E.; Sach, G.; Saucy, G.;
Stempel, A. J. Org. Chem. 1962, 27, 3788.
References and notes
1. Squires, R. F.; Braestrup, C. Nature 1977, 266, 732.
2. Ninan, P. T.; Insel, T. M.; Cohen, R. M.; Cook, J. M.; Skolnick, P.; Paul, S. M. Science
1982, 218, 1332.
3. Wong, G.; Skolnick, P. Eur. J. Pharmacol.—Mol. Pharmacol. Sect. 1992, 225, 63.
4. Mendelson, W. B.; Cain, M.; Cook, J. M.; Paul, S.; Skolnick, P. Science 1983, 219,
414.
5. Venault, P.; Chapouthier, G.; de Carvalho, L. P.; Simiand, J.; Morre, M.; Dodd, R.
H.; Rossier, J. Nature 1986, 321, 864.
6. Sieghart, W. Pharmacol. Rev. 1995, 47, 181.
7. Nayeem, N.; Green, T. P.; Martin, I. L.; Barnard, E. A. J. Neurochem. 1994, 62, 815.
8. Griebel, G.; Perrault, G.; Letang, V.; Granger, P.; Avenet, P.; Schoemaker, H.;
Sanger, D. J. Psychopharmacology 1999, 146, 205.
9. Benson, J. A.; Low, K.; Keist, R.; Mohler, H.; Rudolph, U. FEBS Lett. 1998, 431, 400.
10. Rudolph, U.; Crestani, F.; Benke, D.; Brunig, I.; Benson, J. A.; Fritschy, J. M.;
Martin, J. R.; Bluethmann, H.; Möhler, H. Nature 1999, 401, 796.
11. McKernan, R. M.; Rosahl, T. W.; Reynolds, D. S.; Sur, C.; Wafford, K. A.; Atack, J.
R.; Farrar, S.; Myers, J.; Cook, G.; Ferris, P.; Garrett, L.; Bristow, L.; Marshall, G.;
51. Austin, W. B.; Bilow, N.; Kelleghan, W. J.; Lau, K. S. Y. J. Org. Chem. 1981, 46,
2280.
52. Heck, R. F. Palladium Reagents in Organic Synthesis; Academic Press, 1985.
53. Choudhary, M. S.; Craigo, S.; Roth, B. L. Mol. Pharmacol. 1992, 42, 627.