Novel benzodiazepine photoaffinity probe stereoselectively labels a site deep within the membrane-spanning domain of the cholecystokinin receptor

Article abstract of DOI:10.1021/jm049072h

An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active

Full text of DOI:10.1021/jm049072h

850
J. Med. Chem. 2006, 49, 850-863
Articles
Novel Benzodiazepine Photoaffinity Probe Stereoselectively Labels a Site Deep within the
Membrane-Spanning Domain of the Cholecystokinin Receptor
Elizabeth M. Hadac,† Eric S. Dawson,‡ James W. Darrow,§ Elizabeth E. Sugg,| Terry P. Lybrand,‡ and Laurence J. Miller*,†
Department of Molecular Pharmacology and Experimental Therapeutics and Cancer Center, Mayo Clinic, Scottsdale, Arizona 85259,
Department of Chemistry and Center for Structural Biology, Vanderbilt UniVersity, NashVille, Tennessee 37235-1822, Glaxo-SmithKline
Research Laboratories, Research Triangle Park, North Carolina, and CGI Pharmaceuticals, Inc., Branford, Connecticut 06405
ReceiVed NoVember 17, 2004
An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties
and for development of more potent and selective molecules that act at the same biological target. In this
work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone
derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A
cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands
were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported
in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of
attachment for the photolabile moiety of the antagonist probe deep within the receptor’s membrane-spanning
region at Leu88, a residue within transmembrane segment two. In contrast, the agonist probe labeled a region
including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent
attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular
models for the antagonist-receptor complex. These models provided a means for visualization of physically
plausible ligand-receptor interactions in the context of all currently available biological data that address
small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile
compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces
powerful tools and a strategy for direct and selective investigation of nonpeptidyl ligand binding to peptide
receptors.
Introduction
have also been successful in radiolabeling them with 14C,
without changing their agonist or antagonist properties and with
maintenance of adequate binding affinity.1 Despite the extremely
low specific radioactivity of these probes (0.05 Ci/mmol),
representing less than one thirty-thousandth of the activity of
the radioiodinated peptide probes we have previously used in
affinity labeling this receptor,4-7 we have been successful in
using these unique ligands to photoaffinity label the CCK
receptor.1
In this work, through the use of resolved enantiomeric CCK-A
ligands, we have been able to extend the insights possible with
the racemic compounds that we initially described in a prior
publication.1 The previously reported racemic photolabile benzo-
phenone derivatives of the 1,5-benzodiazepine agonist (mixture
of compounds 5 and 6, Figure 1) and antagonist (mixture of
compounds 7 and 8, Figure 1) were separated into their enantio-
meric components, and each chiral compound was then assayed
for biological activity, binding affinity and CCK receptor
labeling efficiency. For the subsequent photoaffinity labeling
work, the racemic intermediate amines (shown in Scheme 1, 1,
2 and 3, 4) were resolved into their respective enantiomers and
converted into 14C analogues of each of the four separated
enantiomers (shown in Scheme 2, 5, 6, 7, and 8).
Understanding of the molecular basis of drug action is ex-
tremely important, contributing to refinement of the specificity,
affinity, and potency of lead compounds, and even to the rational
design of new drugs. Insights into this can come from the com-
plementary approaches of receptor mutagenesis and photoaf-
finity labeling. Loss of function in a receptor mutant can be
explained either by direct or allosteric effects, while clean photo-
affinity labeling of specific residues is direct and unambiguous,
but can be quite difficult to achieve. Typical challenges include
identification of a small molecule ligand that can accommodate
both a photolabile functional group and a radiolabel, while
retaining receptor binding affinity and biological activity.
We have previously reported the development of structurally
related 1,5-benzodiazepine ligands that act as agonists and
antagonists at the type A cholecystokinin receptor.1 This is a
guanine nucleotide-binding protein (G protein)-coupled receptor
in the rhodopsin-â-adrenergic receptor family.2 It normally binds
and responds to a peptide hormone that is important in the
regulation of gallbladder contraction, exocrine pancreatic secre-
tion, gastric emptying, enteric transit, and satiety.3 We have been
able to incorporate photolabile benzophenone and diazirine
moieties into these nonpeptidyl CCKa receptor ligands.1 We
When the racemic mixture demonstrating agonist activity was
separated into its components, only the putative (S)-enantiomer
(6) displayed biological agonist activity, and it was also the
* To whom correspondence should be addressed. Tel: (480) 301-6650.
Fax: (480) 301-6969. E-mail: miller@mayo.edu.
† Mayo Clinic.
‡ Vanderbilt University.
a Abbreviations used: CCK, cholecystokinin; Alexa488-CCK, Alexa488
-
| Glaxo-SmithKline Research Laboratories.
§ CGI Pharmaceuticals, Inc.
Gly-[(Nle28,31)CCK-26-33]; CHO, Chinese hamster ovary; KRH, Krebs-
Ringers-HEPES medium.
10.1021/jm049072h CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/07/2006

SelectiVe Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 851
Figure 1. Chemical structures of nonpeptidyl CCK receptor ligands used in the current series of studies. The racemic mixture of compounds 5 and
6 having an agonist profile was separated into its components (5, (R)-form with no agonist activity and 6, (S)-form with agonist activity), and the
racemic mixture of compounds 7 and 8 having an antagonist profile was similarly separated into its components (7, (R)-form and 8, (S)-form). Also
illustrated is the structure of a related 1,4-benzodiazepine CCK receptor antagonist that was developed by Merck, initially called L-364,718 and
subsequently referred to as devazepide (9). This was used in pharmacological competition assays and molecular model-building studies.
isomer that bound more tightly to the receptor. Therefore, the
corresponding (R)-enantiomer in this series (5) actually repre-
sents a functional antagonist. Of interest, this (S)-enantiomer
(6) was substantially less efficient in covalent labeling of the
CCK receptor than was the (R)-enantiomer (5). In contrast, both
antagonist enantiomers (7 and 8) had similar binding affinities
for the CCK receptor, but the proposed (S)-enantiomer (8) was
substantially more efficient in covalent labeling of the receptor
than the corresponding (R)-enantiomer (7). Covalent attachment
sites for these structurally similar agonist and antagonist probes
were characterized, and the results indicate that agonist and
antagonist ligands covalently labeled distinct regions within the
CCK receptor. The site labeled by the antagonist was determined
definitively with proteolytic peptide mapping, followed by
Edman degradation sequencing of the labeled fragment. The
antagonist was covalently attached to Leu88 within the second
transmembrane helical segment, clearly establishing a nonpep-
tidyl antagonist binding site that is distinct from the extramem-
branous receptor regions involved in binding the natural peptide
agonist ligand.8,9 The nonpeptidyl agonist ligand covalently
labeled a different portion of the receptor, believed to represent
the cyanogen bromide fragment that includes the first extracel-
lular loop and the upper portion of the third transmembrane
segment. The receptor fragments covalently labeled by agonist
and antagonist probes were distinct, but coincubation of the
receptor preparation with a competing concentration (1 µM) of
the endogenous peptide ligand, CCK, along with the benzodi-
azepine probes, did not block photoaffinity labeling by either
probe. However, competitive coincubation with the nonpeptidyl
antagonist, devazepide (also referred to in the literature as
L-364,718 and MK329) (9), blocked photoaffinity labeling by
both agonist and antagonist probes in a concentration-dependent
manner. On the basis of these results, we propose that both
agonist and antagonist probes probably bind to receptor sites
that are composed of partially overlapping regions within the
CCK receptor membrane-spanning domains.
The data obtained from photoaffinity labeling studies with
these compounds were used to construct three-dimensional
molecular models for ligand-receptor complexes. Small mol-
ecule probes containing a fused benzodiazepine ring system have
far less conformational flexibility than peptide ligands. There-
fore, they proved amenable to conformational search strategies
that are impractical for peptide ligands. For example, methods
were applied that evaluated various conformations of the ligands
within the context of the putative receptor binding site. The
predominantly alpha-helical transmembrane domain where the
nonpeptidyl ligands bind also possesses far fewer degrees of
conformational freedom than the flexible extracellular loops
shown to be important for peptide binding.8,9 Therefore, we were
able to generate plausible three-dimensional models for these
ligand-receptor complexes using only the photoaffinity labeling
results, along with mutagenesis data from earlier studies for the
closely related antagonist, devazepide.
Results
Scheme 1 illustrates the strategy utilized for the preparation
of the key racemic amine intermediates (1, 2 and 3, 4) to be
used in the synthesis of the photolabile chiral probes (5, 6, 7,
and 8). The known bromoacetamides (12 and 13) were prepared
in high yield from bromoacetyl bromide and the corresponding

852 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Hadac et al.
Scheme 1a
a (a) C6H5NHNH2, EtOH, H2O, HCl; (b) PCl5, CCl4; (c) bromoacetyl bromide, DIPEA, DCM; (d) N-phenyl phenylenediamine, NaH, DMF; (e) THF, e4
°C; (f) Zn dust, AcOH.
Scheme 2a
a (a) Phosgene, 3-aminobenzophenone, base, <4 °C; (b) chiral reverse-phase HPLC purification.
N-methyl or N-isopropyl secondary amine using the general
procedure as described in Aquino et al.10 Subsequent alkylation
of commercially available N-phenylphenylenediamine with the
appropriate bromoacetamide yielded the substituted phenylene-
diamine intermediates (14 and 15). The known 2-(phenylhy-
drazono-propanedioyldichloride (17) was prepared in two steps
from the commercially available ketomalonic acid using the
procedure outlined in Aquino et al.10 and condensed with the
phenylenediamine intermediates (14 and 15) to afford the
corresponding hydrazones (18 and 19). Zinc reduction of the
hydrazones yielded the racemic amines (1, 2 and 3, 4).
With the amine racemates in hand, we approached the synthe-
sis of the final resolved chiral photoaffinity labeling probes (5,
6, 7, and 8) using two complementary routes, as shown in
Scheme 2. Both methods provide pure, separated photoaffinity
ligands with similar synthetic efficiency and enantiopurity.
As illustrated by the first method shown in Scheme 2, initially
for the bioactivity and binding experiments, the racemic amine
intermediates were cleanly converted to the racemic benzophe-
none ureas (5, 6 and 7, 8) using a phosgene equivalent to couple
the amines with commercially available 3-aminobenzophenone.
Subsequently, chiral chromatography resolved each of these two
racemic benzophenone-1,5-benzodiazepine urea pairs into the
final four enantiomers (5, 6, 7, and 8) in high enantiomeric
excess and complete baseline peak separation under chiral HPLC
conditions.
For further exploration with modeling analysis, and in the
absence of being able to obtain adequate crystallographic data
on the resolved enantiomers, we provisionally assigned the
absolute configurations of our compounds based on comparison
to reported X-ray structures for highly analogous CCK ligands.
Within the racemate originally displaying agonist activity
(racemate 5, 6), after enantiomer separation, the antagonist
compound 5 was putatively assigned the (R)-configuration based
on its strong structural homology with devazepide (9), as well
as other benzodiazepine CCK antagonists having X-ray struc-
tures reported in the literature.11-13 The only agonist ligand in
the set, compound 6, was correspondingly assigned the (S)-
configuration. Comparing, under the same stationary and mobile
phase conditions (see Experimental Section), the relative chiral

SelectiVe Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 853
Figure 2. Binding and biological activity of CCK receptor ligands. Shown are curves representing the abilities of different concentrations of CCK
receptor ligands (the racemic mixture having agonist action and its enantiomeric components in the top left panel, and the racemic mixture having
antagonist action and its enantiomeric components in the bottom left panel) to compete for the saturable binding of 125I-D-Tyr-Gly-[(Nle28,31)CCK-
26-33]. 100% represents total saturable binding of this CCK-like radioligand in the absence of competitor and 0% represents the binding in the
presence of 1 µM CCK, with this value always less than 10% of total bound radioactivity. Shown in the right panels are biological activity curves,
representing the abilities of these compounds to stimulate amylase secretion from dispersed rat pancreatic acini. 100% represents the maximal
response of these cells to CCK, occurring at 0.1 nM CCK. Shown are means ( SEM of the values from three independent experiments.
HPLC retention times of these two compounds (41.3 for
compound 5 and 48.3 min for compound 6) with those observed
for the two antagonist enantiomers (49.9 min for 7 and 82.2
min for 8) allowed for the provisional assignment of compounds
7 and 8 to be (R) and (S), respectively.
enantiomeric final products previously obtained using method
1, and each was determined to be >96.5% enantiomeric excess
(ee). Using this methodology, the enantiopurity of the final ureas
synthesized from the resolved amine intermediates closely
matched the enantiopurities of the respective intermediates
themselves, indicating no significant reduction in enantiomeric
excess during the final urea formation and purification. Further
experiments, in particular the treatment of the separated enan-
tiomers under basic conditions (with TEA or DIPEA), caused
rapid racemization and the appearance of both parent peaks in
the chiral HPLC traces, yet no degradation of compound to other
species by HPLC, NMR, or MS analysis. Method 2 was then
applied directly to the synthesis of the 14C incorporated
photoaffinity probes (5, 6, 7, and 8), and the previously
synthesized and purified nonradioactive, “cold” analogues were
then used as comparative standards during the synthesis and
purification of the radiolabeled versions.
As can be seen in Figure 2, after separating the racemic
mixture that demonstrated agonist activity, only the putative
(S)-enantiomer (6) displayed intrinsic agonist activity (EC50 150
( 36 nM). The corresponding (R)-enantiomer (5) showed no
ability to stimulate an increase in pancreatic acinar cell amylase
secretion in concentrations as high as 1 µM. These two enan-
tiomers also displayed distinct binding affinities, with compound
6 having a higher affinity than compound 5 (IC50s of 61 ( 17
nM for compound 6 and 710 ( 420 nM for compound 5). The
data for compound 5 are consistent with the pharmacological
profile of a type A CCK receptor antagonist. Figure 2 also shows
binding data for the pair of compounds separated from the
antagonist racemic mixture. Both compounds 7 and 8 had similar
apparent binding affinities for the CCK receptor, with IC50s for
inhibition of the binding of the CCK-like peptide radioligand
of 490(55 nM for the proposed (S)-enantiomer (8) and 470 (
66 nM for the corresponding (R)-enantiomer (7).
To minimize the use of radioactivity during the synthesis and
purification of the chiral photoaffinity probes, we employed the
second method shown in Scheme 2 whereby the racemic amine
intermediates were separated into their respective enantiomers
prior to phosgene coupling with the 3-aminobenzophenone. This
approach ultimately allowed facile incorporation of 14C into the
molecules during the last possible step, thus providing more
efficient handling and purification of radioactive compounds.
However, to validate the ultimate enantiopurity of this
synthetic methodology, each chiral amine was first cleanly
converted into its respective chiral urea (5, 6, 7, and 8) using
nonradioactive phosgene and 3-aminobenzophenone. The puri-
fied compounds synthesized using this approach were compared
via chiral HPLC analysis to the corresponding separated
Each of the four 14C-tagged probes labeled the CCK receptor,
as demonstrated by the covalent labeling of a band migrating
at Mr ) 85000-95000 (Figure 3). This band had a core protein
migrating at Mr ) 42000 (data not shown). These match the
established electrophoretic migrations of this receptor glyco-
protein and its deglycosylated core protein, as well as the broad
nature of those bands resolved under these conditions, as
previously reported.14 Each probe labeled the receptor with
distinct efficiencies, as reflected by the relative intensities of
the bands on autoradiographs, as seen and quantified in Figure
3. Compound 8 (the proposed (S)-enantiomer antagonist)
reproducibly generated the strongest receptor signal in experi-
ments utilizing the same amounts of receptor-bearing membrane
and similar amounts of probes having similar specific radioac-
tivity.
Inhibition of affinity labeling of the CCK receptor by each
of the four probes was attempted with various concentrations
of CCK and devazepide (9). Of note, no inhibition was achieved
for any probe after competitive coincubation with concentrations

854 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Hadac et al.
compounds (Figure 4), suggesting some shared commonality
of binding site between the nonpeptide ligands.
The CCK receptor bands labeled by the agonist (6) and the
corresponding (S)-enantiomer antagonist (8) were cleaved using
cyanogen bromide (Figure 5). The resultant labeled receptor
fragments were both small, migrating on the gel below the 6
kDa protein standard. On the basis of differential electrophoretic
migration, these fragments appeared to be distinct peptide
segments.
Only the antagonist-labeled receptor fragment was recovered
in sufficient quantities for detailed sequence characterization.
After purification by reversed-phase capillary HPLC, this
fragment was sequenced by Edman degradation (Figure 6). The
sequencing results show definitively that the antagonist co-
valently labels a peptide fragment contained within transmem-
brane segment two (residues Arg73 through Met89 of the mature
receptor). This sequence was verified in two independent
preparations. Of particular interest, this sequence was clean and
absolute, until it ended abruptly after residue Asp87, suggesting
that the labeled residue was either Leu88 or Met89. However,
the Met89 residue cannot be the site of labeling, since its modi-
fication would interfere with cyanogen bromide cleavage at this
site. On the other hand, covalent attachment of the photoaffinity
label to Leu88 would result in the release of a residue in that
cycle of Edman degradation that would not be recognized as a
standard amino acid, which supports the cleavage results
observed experimentally. The presumptive labeling of Leu88 was
not directly confirmed by radiochemical sequencing, due to the
extremely low specific radioactivity of the probe.
Figure 3. Efficiency of photoaffinity labeling of the CCK receptor.
Shown in the upper panel is a typical autoradiograph of an SDS-
polyacrylamide gel used to separate the products of affinity labeling
of the CCK receptor expressed on CHO-CCKR cell membranes. The
arrowhead marks the typical migration of the labeled CCK receptor.
Shown in the lower panel is the relative densitometric quantitation of
the covalent labeling of the CCK receptor by these probes, with the
labeling by compound 8 always found to be the darkest, and considered
to represent 100 percent. Values reflect means ( SEM of results from
four independent experiments.
The agonist (6) probe labeled a much larger receptor
fragment, and analysis of cyanogen bromide cleavage sites
within the receptor suggest this fragment is most likely either
a peptide segment extending from Pro96 to Met121 (loop one
and top of transmembrane segment 3) or a segment comprised
of the endogenous peptide ligand, CCK, as high as 1 µM (data
not shown). In contrast, coincubation with the 1,4-benzodiaz-
epine, devazepide, effectively inhibited the covalent labeling
of this receptor by each of the 1,5-benzodiazepine probe
Figure 4. Inhibition of affinity labeling of the CCK receptor using devazepide (9). Shown are representative autoradiographs of SDS-polyacrylamide
gels used to separate products of photoaffinity labeling experiments performed with each of the 14C-tagged CCK receptor probes, using as competitor
in the incubation the noted molar concentrations of devazepide.

SelectiVe Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 855
that implicate CCK-A receptor residues Cys94, Met121, Ser124
,
Val125, Ile330, Asn334, Leu357, and Ser364 in ligand binding16-19
and the successful identification of the covalent attachment site
(Leu88) for compound 8 reported in this study. Initial automated
docking attempts for the 1,5-benzodiazepines with our unmodi-
fied starting receptor structure were unsuccessful, since the
cavities within the membrane-spanning domain were not quite
large enough to accommodate these ligands. Therefore, initial
refinements of the membrane-spanning domain required the use
of interactive molecular graphics techniques to manually dock
the closely related antagonist, devazepide, using the aforemen-
tioned mutagenesis data available from binding studies with this
molecule to guide the docking process (Figure 7). The receptor
backbone atoms were initially held fixed in our starting model,
and amino acid side chain conformations were adjusted using
a well-documented rotamer library16 to create a binding pocket
for devazepide, as suggested by the mutagenesis data (see
Methods). The location of this putative antagonist-binding site
correlates well with our photoaffinity labeling data, as the Leu88
side chain projects directly into this pocket. After manual model
building, the complexes were refined with energy minimization
and molecular dynamics. All residues implicated in previously
reported mutagenesis studies featuring the devazepide antagonist
were colocalized in a pocket within the membrane-spanning
domain. These amino acids experienced subtle conformational
changes that were necessary in the initial refinement of this
binding site to accommodate small molecule ligands. The
addition of a nondisruptive hydrogen bonding interaction
between the hydroxyl of Ser124 and the backbone carbonyl of
Phe120 was the only observed change in intramolecular hydrogen
bonding as a result of these initial refinements. The resulting
Figure 5. Identification of the regions of the CCK receptor that were
labeled with (S)-1,5-benzodiazepine compounds (6 and 8). Shown is a
representative autoradiograph of an SDS-polyacrylamide gel used to
resolve the cyanogen bromide fragments of the CCK receptor that were
covalently labeled using two photolabile (S)-enantiomeric CCK receptor
probes representing agonist (6) and antagonist (8). These bands were
clearly distinct, based on their electrophoretic migration.
of Gln206 to Met225 (transmembrane segment 5). Partial sequenc-
ing of this fragment, furthermore, clearly established the first
10 residues as Pro96 through Lys105, although it was not possible
to sequence the entire fragment since the signal blended into
background after that point. However, since the covalent label
attachment site would have to occur after Lys105, these results
indicate that the agonist probe (6) labels the receptor at a residue
near the top of transmembrane segment 3.
Molecular models were constructed for receptor complexes
with both the agonist and antagonist photoaffinity labeling
probes, using previous models for the receptor-peptide complex
as a starting point.15 There are more extensive experimental
constraint data for the antagonist complex, due to earlier
mutagenesis studies for the closely related antagonist, devazepide,
Figure 6. Localization of the site of labeling with the (S)-1,5-benzodiazpine antagonist (compound 8). Shown is a diagram of the rat type A CCK
receptor, with the separations of the peptide chain representing sites of cyanogen bromide cleavage. The cyanogen bromide fragment of the receptor
that was covalently labeled by compound 8 (residues 73 through 89) is highlighted in black circles with white lettering. After this was resolved by
SDS-polyacrylamide gel (shown in Figure 5), it was eluted and further resolved by microbore C-18 reversed-phase HPLC. The entire eluent from
this separation was collected directly onto a moving PVDF membrane that was exposed to autoradiography. The single dominant radioactive peak
was sequenced by Edman degradation to yield the expected residues from Arg73 through Asp87, where the sequence stopped abruptly. This is
consistent with Leu88 having its migration and identification altered by the covalent attachment of the probe to this residue. The receptor fragment
(residues 96 through 121) that was covalently labeled by compound 6 is also illustrated in this diagram.

856 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Hadac et al.
Figure 7. Devazepide (9)-refined type A CCK receptor model. Amino acid residues implicated in mutagenesis studies (referenced above) with
contributions to the small molecule binding site are labeled near their alpha carbon positions in the membrane-spanning helical coils (translucent
green). Nonpolar residues are shown in orange; polar residues appear in red. The left panel is a view of the binding site from the extracellular loops
that are clipped away for a clear view of the small molecule binding site. The right panel is a view of the same ligand-receptor complex from the
space between membrane-spanning helices two (left of ligand) and three (right of ligand).
Figure 8. (S)-1,5-Benzodiazpine antagonist (8)-refined type A CCK receptor model. For comparison with Figure 7, the left and right panel views
correspond to those of the compound 9 ligand-receptor complex (shown above). In our refined model, the antagonist benzophenone moiety is
positioned only 3.5 Å away from the Cγ-H bond of Leu88 (the photoaffinity label attachment site), as shown by the white dashed line. Intermolecular
hydrogen bonds are formed during structural refinement between the side chain amide group of Asn334 (in helix six) and the methoxy group of the
(S)-antagonist photoprobe, as well as between the hydroxyl group of Ser364 (in helix seven) and a carbonyl group of the diazepine ring moiety of
the (S)-antagonist photoprobe. Hydrogen bonds are highlighted with pink, dashed lines. Key binding site residues are labeled and colored as described
for Figure 7.
receptor complex structure (Figure 7) differed from our previ-
ously reported model15 by only 0.97 Å RMSD in the membrane-
spanning region.
(isoleucine, leucine, and valine) or next to heteroatoms, for
example, peptide backbone CR-H bonds, glycine CR-H bonds,
and the Cγ-H bonds of methionine.20 The 25 best-scoring
complexes from both shape-based and energy-based DOCK 4.0
scoring functions were examined and those that were consistent
with the antagonist photoaffinity labeling data were retained as
starting points for further consideration. None of the structures
generated by automated docking positioned the 1,5-benzodiaz-
epine in perfect position to form a covalent bond with the target
receptor residue, Leu88. However, one of the top-scoring docked
complexes did orient the ligand such that the benzophenone
substituent was properly aligned for covalent insertion in the
Cγ-H bond of Leu88, although the interatomic separation was
too long (10 Å) to be consistent with the benzophenone reaction
mechanism. This complex was further refined with manual
adjustments and restrained molecular dynamics simulations.
The refined structure displays a 3.5 Å separation between
the benzophenone carbonyl carbon and the reactive Cγ-H bond
of Leu88 (Figure 8), quite consistent with the labeling results
Automated docking calculations for the agonist compound
produced many equally plausible docked conformers with high
scores that were all consistent with labeling of a receptor
fragment composed of portions of the third transmembrane helix
and the first extracellular loop regions. The agonist models were
not refined further due to the lack of an identified site of covalent
attachment for the agonist photoaffinity ligand. On the basis of
the limited nature of the current data, we cannot rule out a
possible attachment site for the agonist photoaffinity probe in
the first extracellular loop of the receptor, although the failure
of coincubation with the peptide agonist to block photolabeling
by the 1,5-benzodiazepine agonist probe would appear to
preclude a direct interaction in the first extracellular loop.
The benzophenone photophore reacts with the receptor
through a bond-insertion mechanism that targets electron-rich
C-H bonds such as those found in residues with tertiary centers

SelectiVe Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 857
and the photochemistry involved. In addition, two intermolecular
hydrogen bonds are formed in this docked complex with residues
Asn334 and Ser364 (Figure 8) that were both previously implicated
in a mutagenesis study by Smeets et al.21 Furthermore, favorable
van der Waals interactions are maintained with all above
referenced residues from mutagenesis studies that were used in
the initial refinement of the membrane-spanning region of our
model using devazepide. The refined receptor complex structure
for the putative (S)-1,5-benzodiazepine antagonist probe (8)
(Figure 8) differed from the starting structure (Figure 7) by 2.57
Å RMSD in the membrane-spanning regions. This difference
reflects some minor repacking of the model in this region. The
only significant changes observed were a bending of the second
transmembrane helix at Pro96 to form favorable packing
interactions with the (S)-1,5-benzodiazepine antagonist probe
(8) and a disruption of one turn of the sixth transmembrane
helix by the formation of a hydrogen bond between the hydroxyl
of Ser360 and the backbone carbonyl of Ile356. The docked
complex was subsequently refined at two different temperatures,
20 K (not shown) and 300 K (Figure 8) and both of the features
detailed above were present in both of the resulting models,
which differed by only 1.2 Å RMSD in their membrane-
spanning regions. Therefore, it is likely that these features of
our refined model are mostly due to the presence of the larger
(S)-antagonist photoprobe ligand (8) that is now docked into a
previously poorly refined region of the small molecule binding
site. Of note, docking of the (R)-antagonist photoprobe ligand
(7) using similar protocols (data not shown) did not result in
the formation of significant intermolecular hydrogen bonds with
polar residues or produce favorable van der Waals contacts with
a majority of the binding site residues implicated by previous
mutagenesis studies, as did the docking of the experimentally
more efficient (S)-antagonist photoprobe (8).
Many previous studies of small molecule complexes with type
A CCK receptor relied primarily on site-directed mutagenesis
and other indirect structural probes to investigate ligand-
receptor complexes.19 These data are quite useful, but interpreta-
tion of results can be difficult, and it is generally not possible
to build unique structural models based on indirect data alone.
Nonetheless, these data have provided important clues about
the location and nature of the small molecule binding site in
the type A CCK receptor. For example, residues His381 and
Val354 in the rat type B CCK receptor align with type A receptor
residues Leu357 and Ile330. In type B CCK receptor mutant
proteins, changes at these positions to the corresponding type
A residues shifted the pharmacological profile of devazepide
substantially toward that of the type A CCK receptor.18,19
Previous alanine-scanning mutagenesis experiments impli-
cated the polar residues Ser124, Asn334, and Ser364 in binding
to devazepide.21 Mutation of residues Cys94, Met121, and Val125
in the type A CCK receptor abolished binding for the non-
peptidyl agonist, SR-146,131.17 All these residues impli-
cated in previous mutagenesis studies are colocalized in a cavity
within the membrane-spanning domain of our previous type A
CCK receptor model. The key Leu88 residue covalently labeled
by our antagonist probe is also present in this cavity, and our
initial manual docking studies were guided and constrained by
these observations. Both the devazepide antagonist ligand 9
(Figure 7) and the 1,5-benzodiazepine antagonist photoaffinity
probe (Figure 8) form plausible interactions with these binding
pocket residues in our refined receptor complex models. These
current models are also fully compatible with all previously
reported structure-activity and photoaffinity labeling data for
the type A CCK receptor. However, this current study provides
the first direct experimental evidence for a specific ligand
contact with a CCK receptor residue deep inside the transmem-
brane domain.
Discussion
The methodological difficulties and uniqueness of this effort
should be noted. Photoaffinity probes require the incorporation
of both photolabile and indicator moieties. In peptide and protein
ligands, it is often feasible to incorporate a photolabile amino
acid derivative and a site for radioiodination that can be utilized
in autoradiography. Indeed, a single radioiodine per probe
molecule provides a specific radioactivity of 2000 Ci/mmol.
Small drug candidates are often much less able to accommodate
extraneous groups. We were fortunate that structure-activity
considerations allowed the incorporation of a benzophenone
moiety to confer photolability, without having substantial
negative impact on either biological activity or binding affinity.
However, for practical reasons, we were able to incorporate only
a single 14C into this compound, yielding a specific radioactivity
of 0.05 Ci/mmol. This resulted in a specific radioactivity that
was 30000-fold lower than that of any of the peptide photoaf-
finity labeling probes previously utilized to label this receptor
and its subdomains and residues.4-7,9,23 Nonetheless, we were
still able to demonstrate specific photoaffinity labeling of this
receptor with the benzodiazepine probes, and we were also able
to localize the site of covalent attachment for the antagonist
probe that labeled this receptor.
G protein-coupled receptors are remarkable for the structural
diversity of natural ligands that bind and activate these
molecules, ranging from photons, odorants, and small biogenic
amines to larger peptides, proteins, and even viral particles.22
The CCK receptor is a member of the rhodopsin/â-adrenergic
receptor family of G protein-coupled receptors.2 Its natural
ligand is a small linear peptide hormone. A series of receptor
mutagenesis and photoaffinity labeling studies have localized
the peptide-binding domain of this receptor to the extracellular
surface of the membrane, where the peptide ligand interacts with
regions of the receptor amino terminus and loops.6,7,9,23 We have
previously generated and refined three-dimensional CCK pep-
tide-receptor complex models that are consistent with all
existing structure-activity data and direct biophysical measure-
ments, including photoaffinity labeling and fluorescence reso-
nance energy transfer experiments.9,15
We have reported previously that the bovine rhodopsin crystal
structure24 is probably not an ideal template for homology
modeling of peptide receptors such as the type A CCK receptor,
since the extracellular loops that comprise the peptide hormone
binding site probably adopt quite different conformations in
rhodopsin and CCK.9 Therefore, we have used de novo model-
ing techniques, together with constraint data from mutagenesis
and biophysical experiments, to generate three-dimensional
models for peptide complexes with the CCK receptor.9 The new
photoaffinity labeling data reported here for nonpeptidyl CCK
ligands is especially useful, as these data provide specific
constraint information for regions of the type A CCK receptor
that were not well defined in previous models.
While it is well recognized that different G protein-coupled
receptors bind their natural ligands in different ways and in
different domains, correlating with the distinct chemical char-
acteristics of their ligands,22 our data present a clear, direct
demonstration of ligand binding to two distinct domains within
the same receptor. Our current study has directly established
an intramembranous binding site with moderately high affinity
for the putative (S)-1,5-benzodiazpine antagonist (compound 8)

858 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Hadac et al.
that is clearly distinct from the site of action of the natural
peptide ligand. Unfortunately, the same robust results could not
be definitively confirmed for the structurally related agonist
compound (6). At this stage, we cannot define a precise binding
position or mode for the agonist, since the current constraint
data for probe attachment spans a 15 amino acid residue
fragment (Asp106-Met121). This may be due in large measure to
the low efficiency of photoaffinity labeling by the agonist probe
molecule and could easily reflect unfavorable interatomic
distances or directionality between the binding site residues and
the photoreactive ketone of the benzophenone moiety. This result
underscores the importance of localization of the site of probe
molecule covalent attachment to a single receptor amino acid.
Recent reports on agonist-induced conformational changes in
the â2-adrenergic receptor indicated that even structurally distinct
agonists of the same receptor could stabilize different receptor
conformational states, and that a full agonist may stabilize
multiple receptor states that have various kinetic lifetimes.25,26
Additionally, fluorescence lifetime data from the adrenergic
receptor studies suggest that high affinity agonist-receptor
complexes would likely be the shortest lived of two observed
states.20 These reports may explain the difficulty encountered
in the current work to obtain high efficiency receptor labeling
with the biologically active, (S)-agonist probe (compound
6).
probes, will likely serve as an important tool for gaining further
insights into the structures of membrane-spanning domains of
G protein-coupled receptors that bind peptide hormones.
Experimental Section
Reagents. All chemicals and solvents are reagent grade unless
otherwise specified. Synthetic sulfated cholecystokinin octapeptide
(CCK) was purchased from Peninsula Chemical Co. The radioio-
dinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33], was
synthesized in our laboratory as we have described.4,28 The CCK
antagonist, devazepide, was provided by Merck Research Labora-
tories (West Pointe, PA). Cyanogen bromide (CNBr) and the solid-
phase oxidant, N-chlorobenzenesulfonamide (Iodobeads), were from
Pierce Chemical Co. (Rockville, IL), wheat germ agglutinin-agarose
was from E-Y Laboratories (San Mateo, CA), purified type II
collagenase and soybean trypsin inhibitor (STI) were from Wor-
thington Biochemicals (Lakewood, NJ), and formic acid was from
Fluka Chemical Co. (Milwaukee, WI). Endoglycosidase F was
prepared in our laboratory, as we have described.29 The following
solvents and reagents have been abbreviated: acetic acid (AcOH),
dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dichlo-
romethane (DCM), diisopropylethylamine (DIPEA), ethanol (EtOH),
ethyl ether (Et2O), ethyl acetate (EtOAc), 2-propanol (IPA),
methanol (MeOH), tetrahydrofuran (THF), trifluroacetic acid (TFA),
triethylamine (TEA). Nonradioactive phosgene was purchased
commercially as a 20% solution in toluene from Fluka Chemical
Co, while 14C-labeled phosgene was available from American
Radiolabeled Chemicals, Inc. (St. Louis, MO). Reactions were
monitored by thin-layer chromatography (TLC) on 0.25 mm silica
gel plates (60F-254, E. Merck) and visualized with UV light.
Analytical Purity was assessed by RP-HPLC using a Waters 600E
system equipped with a Waters 990 diode-array spectrophotometer.
The stationary phase was a Vydac C-18 column (4.6 mm × 200
mm), the mobile phase employed was 0.1% aqueous TFA with
acetonitrile, and the flow rate was maintained at 1.0 or 1.5 mL/
min (t0 ) 3 min).
Evidence exists in the literature to support distinct binding
sites in the type A CCK receptor for nonpeptidyl agonists and
antagonists;27 however, our results do not distinguish between
the possibility for the small molecule agonist to bind at a site
that includes residues from the first extracellular loop versus a
binding site contained entirely within the transmembrane
domain. Our results suggest that the small molecule agonist
binding site may not overlap with the peptide agonist binding
site, since micromolar concentrations of the endogenous peptide
failed to competitively block the affinity labeling observed with
the (S)-agonist photoaffinity probe (compound 6), a compound
that retained full biological activity at reasonably low concentra-
tions (EC50 ∼ 150 nM). Therefore, some questions remain
regarding whether the same peptide receptor may be similarly
activated by binding of different types of ligands to distinct
receptor regions. Certainly, the small molecule, antagonist-
binding site is clearly within the membrane-spanning helical
bundle domain, while the natural peptide ligand binds to the
ectodomain of this receptor. Our photoaffinity labeling data
confirm this for the (S)-1,5-benzodiazepine antagonist (com-
pound 8).
1H NMR spectra were recorded on a Varian Unity-300 instru-
ment. Chemical shifts are reported in parts per million (ppm, δ
units). Coupling constants are reported in units of Hertz (Hz).
Splitting patterns are designated as s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; b, broad. Low resolution mass spectra (MS)
were recorded on a JEOL JMS-AX505HA, a JEOL SX-102, or a
SCIEX-APIiii spectrometer. Mass spectra were acquired in either
positive ion mode or negative ion mode under electrospray
ionization (ESI) or atmospheric pressure chemical ionization
conditions (APCI). Combustion analyses were performed by
Atlantic Microlabs, Inc., Norcross, GA, and the results reported in
the form “Anal. (C39H33N5O6) C, H, N” where the observed results
were within (0.4% of calculated values.
Synthesis and Separation of Individual Nonpeptidyl Enan-
tiomers for Use as Photolabile Radioligand Probes. Representa-
tive Procedure for Formation of Bromoacetamides (12 and 13).
2-Bromo-N-isopropyl-N-(4-methoxyphenyl)acetamide (12). Pro-
cedure was followed as described in Aquino et al.,10 wherein
N-isopropyl-N-(4-methoxyphenyl)amine (10) was prepared by
reductive animation of 4-methoxyphenylamine with acetone, em-
ploying the method of Abdel-Magid et al.30,31 Subsequent reaction
of compound 10 with bromoacetyl bromide at e4 °C cleanly
provided the previously described compound 2-bromo-N-isopropyl-
N-(4-methoxyphenyl)acetamide (12).10
2-Bromo- N-(4-methoxyphenyl)-N-methylacetamide (13). Us-
ing the same procedure for the preceding example, reaction of
bromoacetyl bromide with commercially available N-(4-methox-
yphenyl)-N-methylamine (Sigma-Aldrich) provided 2-bromo-N-(4-
methoxyphenyl)-N-methylacetamide (13) in 85% overall yield after
chromatography: 1H NMR (300 MHz, CDCl3) δ 3.22 (s, 3H), 3.61
(s, 2H), 3.79 (s, 3H), 6.89 (d, 2H, J ) 8.8 Hz), 7.15 (d, 2H, J )
8.8 Hz); 13C NMR (100.58 MHz, CDCl3) δ 26.89, 38.20, 55.52,
115.01, 128.11, 135.68, 159.33, 166.83; LRMS (APCI) m/z 279.9,
281.8 (Br isotopes) [M + Na]+; TLC Rf ) 0.68 (EtOAc:Hex, 1:2)
In conclusion, we have added to our understanding of the
multiple ways that ligands can bind to the type A CCK receptor
by reporting direct evidence for a small molecule antagonist
binding site deep within the transmembrane domain. Addition-
ally, the photoaffinity labeling data reported here for nonpeptidyl
CCK ligands provide specific constraint information for regions
of the type A CCK receptor that were not well defined in
previous receptor models.
However, much remains to be explained regarding the
molecular basis for differences between small molecule agonist
and antagonist ligand binding. We have taken an important
initial step in this direction with refinement of the transmem-
brane domain of our previous CCK receptor model structures.
Here, we have employed a conformationally constrained small
molecule antagonist with a well-defined point of interaction in
the receptor membrane-spanning domain as a tool in the
refinement of our evolving model of the CCK receptor. This
type of direct biophysical data, collected using novel molecular

SelectiVe Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 859
2-(Phenylhydrazono)malonic Acid (16) and 2-(Phenylhydra-
zono)propanedioyl Dichloride (17). To a vigorously stirred
suspension of ketomalonic acid disodium salt (5.0 g, 30.9 mmol)
cooled to e4 °C in an ice bath was added dropwise a solution of
hydrochloric acid (1.0 N, 62.0 mL, 62.0 mmol). After the suspension
had dissolved, ethanol (20 mL) was added, and the reaction
proceeded as outlined in Aquino et al.10
4) product (95.3% yield) was obtained. 1H NMR (300 MHz, CD3-
OD) δ 3.20 (s, 3H), 3.87 (s, 3H), 4.45 (d, 1H, J ) 21.0 Hz), 4.72
(d, 1H, J ) 21.0 Hz), 4.75 (b, 1H), 6.70-7.50 (m, 13H); LRMS
(APCI) m/z 445.2 [M + H]+; TLC Rf ) 0.47 (EtOAc:Hex, 1:2);
Anal. (C25H24N4O4) C, H, N.
Method 1: General Procedure for Racemic Benzophenone
Urea Formation and Subsequent Chiral Resolution. 2-{3-[3-
(4-Benzoylphenyl)ureido]-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-
benzo[b][1,4]diazepin-1-yl}-N-isopropyl-N-(4-methoxyphenyl)acet-
amide (Racemate 5, 6). Under anhydrous conditions, triphosgene
(0.5 mmol) was dissolved in anhydrous acetonitrile (1 mL) and
the stirring solution cooled to e4 °C under dry N2. A solution
containing commercially available 3-aminobenzophenone (0.10 g,
0.5 mmol) and diisopropylethylamine (DIPEA, 0.1 mL) in anhy-
drous acetonitrile (1 mL) was added dropwise to the stirring
reaction. The reaction was allowed to stir under dry N2 at e4 °C
for 30 min, at which time a second solution containing the 1,5-
benzodiazepine amine racemate (1, 2) (0.5 mmol) and DIPEA (0.1
mL) in dry acetonitrile (2 mL) was added dropwise to the cooled,
stirring reaction. Stirring continued at e4 °C for 3-6 h until the
reaction was complete as monitored by TLC. The product urea
precipitated out of acetonitrile during the course of the reaction
and was purified by filtration. Subsequent further precipitation of
product from filtrate occurred upon standing for several hours at
room temperature. The white solid was washed thoroughly with
acetonitrile and dried under vacuum to provide 350 mg white solid
racemate (5, 6). No further purification was required as the solids
isolated were observed to be pure by HPLC, NMR and combustion
analysis. 1H NMR (300 MHz, CDCl3) δ 0.91 (d, 3H, J ) 6.8 Hz),
0.94 (d, 3H, J ) 6.8 Hz), 3.78 (s, 3H), 4.16 (d, 1H, J ) 16.8 Hz),
4.58 (d, 1H, J ) 16.8 Hz), 4.74 (septet, 1H, J ) 6.8 Hz), 4.99 (d,
1H, J ) 8.0 Hz), 6.91 (t, 2H, J ) 8.8 Hz), 7.43 (d, 2H, J ) 8.8
Hz), 7.19-7.58 (m, 15H), 7.65 (t, 1H, J ) 7.2 Hz), 7.68 (d, 2H, J
) 7.2 Hz), 7.79 (s, 1H), 9.44 (s, 1H); LRMS (APCI) m/z 717.9 [M
+ Na]+, 694.3 [M]-; Anal. (C41H37N5O6) C, H, N.
2-[2,4-Dioxo-5-phenyl-3-(phenylhydrazono)-2,3,4,5-tetrahydro-
benzo[b][1,4]diazepin-1-yl]-N-isopropyl-N-(4-methoxyphenyl)acet-
amide (18). As per the modified procedure of Hirst and co-
workers,32 commercially available N-phenyl-l,2-phenylenediamine
(5.0 g, 27.1 mmol) was dissolved in DMF (25 mL) and stirred at
room temperature. 1.4 molar equiv of bromoacetamide (12) were
added to this solution. Solid K2CO3 (8.0 g, 57.9 mmol) was then
added and the resulting suspension stirred vigorously overnight at
room temperature. Upon completion as monitored by TLC (1:2,
EtOAc:Hex), the DMF was evaporated and the resulting oil
partitioned into ice water/EtOAc. The organic layer was washed
twice with ice-water then once with brine, and the organic layer
was then dried over MgSO4 and evaporated to a reddish oil. The
crude intermediate thus obtained was pure enough to use directly
for the formation of the 1,5-benzodiazepine ring scaffold. Solutions
of compound 14 (6.0 g, 16.6 mmol) in THF (40 mL) and compound
17 (5.1 g, 20.8 mmol) in THF (40 mL) were added concomitantly
dropwise with cooling in an ice/methanol bath over 30 min. The
solution was allowed to warm to room temperature and stirred 16
h. Upon completion of reaction, as monitored by TLC, the solution
was evaporated down to a yellow solid that was then resuspended
in EtOAc and washed twice with 0.02 N sodium hydroxide solution
and once with brine. The organic layer was dried over MgSO4 and
the solvent removed. The resulting solid was purified by flushing
through a pad of silica gel with (1:1) EtOAc:Hex as eluent After
drying under vacuum overnight, the corresponding compounds 18
were obtained as yellow solids in 90% yield (8.4 g) and high purity
1
(as approximately 1:1 mixtures of syn- and anti-hydrazone). H
NMR (300 MHz, CDCl3) δ 1.09 (m, 6H), 3.88 (s, 3H), 3.90 (m,
1H), 4.20-4.60 (m, 2H), 6.90-7.65 (m. 18H), 10.80 (s, 0.5H),
11.54 (s, 0.5H]; LRMS (APCI) m/z 584.0 [M + Na]+; TLC Rf )
0.27 (EtOAc:Hex, 1:2). 1:1 Mixture of cis- and trans-hydrazones.
2-[2,4-Dioxo-5-phenyl-3-(phenylhydrazono)-2,3,4,5-tetrahydro-
benzo[b][1,4]diazepin-1-yl]-N-(4-methoxyphenyl)-N-methylacet-
amide (19). Prepared using the procedure described for 18 to
Chiral Resolution Procedure and Results for Resolved Com-
pound 5 (and 6). The isopropyl racemic mixture of 5 and 6 was
separated into the resolved enantiomers using semipreparative chiral
RP-HPLC and monitored by UV absorbance (Dynamax C-8
column, 25 cm × 4.1 mm, 8 mL/min flow rate, ramping from [75%
CH3CN/25% (H2O with 0.1% TFA)] up to 100% CH3CN over 30
min). An amount of 100 mg of the parent N-isopropyl racemate
(5, 6) was separated into its purified component enantiomers, which
were isolated as white solids (30 mg of compound 5, and 25 mg of
compound 6). Enantiomer purity and enantiomeric excess were
confirmed by chiral RP-HPLC analysis using an alternate isocratic
analytical method (Analytical Chiralpak AD C-18 column, with
mobile phase (65:35, Hex:IPA) eluting at 1 mL/min flow rate).
Compound 5 eluted at 41.28 min, while compound 6 eluted at 48.33
min (showing good baseline peak separation); coinjections with
each other and with the parent racemate confirmed chiral purity.
Compound 5 (and 6): 1H NMR (300 MHz, CDCl3) δ 0.92 (d, 3H,
J ) 6.8 Hz), 0.95 (d, 3H, J ) 6.8 Hz), 3.78 (s, 3H), 4.17 (d, 1H,
J ) 16.8 Hz), 4.58 (d, 1H, J ) 16.8 Hz), 4.76 (septet, 1H, J ) 6.8
Hz), 5.00 (d, 1H, J ) 8.0 Hz), 6.92 (t, 2H, J ) 8.8 Hz), 7.43
(d, 2H, J ) 8.8 Hz), 7.19-7.58 (m, 15H), 7.65 (t, 1H, J ) 7.2
Hz), 7.68 (d, 2H, J ) 7.2 Hz), 7.80 (s, 1H), 9.44 (s, 1H); LRMS
(APCI) m/z 718.1 [M + Na]+, 694.3 [M]-; Anal. (C41H37N5O6) C,
H, N.
2-{3-[3-(4-Benzoylphenyl)ureido]-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydrobenzo[b][1,4]diazepin-1-yl}-N-(4-methoxyphenyl)-N-
methylacetamide (Racemate 7, 8). Reaction with commercially
available 3-aminobenzophenone and the intermediate amine race-
mate (3, 4), using the procedure outlined for racemate (5, 6),
provided 300 mg of the racemic N-methyl analogues (7, 8) as a
white solid that was pure by NMR, LCMS, and combustion
analysis. 1H NMR (300 MHz, CDCl3) δ 3.10 (s, 3H), 3.77 (s, 3H),
4.35 (d, 1H, J ) 16.8 Hz), 4.64 (d, 1H,J ) 16.8 Hz), 5.01 (d, 1H,
J ) 8.0 Hz), 6.88 (d, 1H, J ) 8.0 Hz), 6.92 (d, 1H, J ) 7.6 Hz),
7.03, (d, 1H, J ) 8.0 Hz), 7.21 (d. 1H, J ) 7.6 Hz), 7.24 (d, 1H,
J ) 7.6 Hz), 7.30-7.48 (m, 13H), 7.51 (t, 1H, J ) 7.6 Hz); 7.63
1
provide 90% yield (8.0 g) of a light yellow solid (19). H NMR
(300 MHz, CDCl3) δ 3.39 (m, 3H), 3.89 (s, 3H), 4.30-4.65 (m,
2H), 6.90-7.65 (m, 18H)0.10.80 (s, 0.5H). 11.54 (s, 0.5H); LRMS
(APCI) m/z 556.3 [M + Na]+; TLC Rf ) 0.12 (EtOAc:Hex, 1:2).
1:1 Mixture of cis- and trans-hydrazones.
Procedure for Cleavage of Hydrazone To Provide Racemic
Benzodiazepine Amines. 2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydrobenzo[b][1,4]diazepin-1-yl)-N-isopropyl-N-(4-meth-
oxyphenyl)acetamide (Racemate 1, 2). To a vigorously stirred
solution of hydrazone 18 (4.27 g, 8.0 mmol) in glacial acetic acid
(50 mL) at room temperature was added anhydrous zinc dust (4.20
g, 64.3 mmol) portionwise. After addition, the solution was stirred
3-5 h until the color of the slurry changed from green to yellow.
The zinc was removed by filtration through Celite and the cake
washed with EtOAc (2 × 75 mL). The filtrate was concentrated,
adsorbed onto silica gel and washed with EtOAc:Hex (1:2) to
remove more polar impurities. The product was then eluted with
10% methanol in DCM and concentrated in vacuo to give a
yellowish-orange oil which was dried under high vacuum to provide
the corresponding racemic amine intermediate (1, 2) in good yield
(3.38 g product as a white amorphous solid, 93.7% yield). 1H NMR
(300 MHz, CD3OD) δ 1.09 (m, 6H), 3.87 (s, 3H), 4.37 (d, 1H, J
) 21.0 Hz), 4.60 (d, 1H, J ) 21.0 Hz), 4.35 (m, 1H), 4.85 (m,
1H), 6.80-7.50 (m, 13H); LCMS (APCI) m/z 473.3 [M + H]+;
TLC Rf ) 0.45 (EtOAc:Hex, 1:2); Anal. (C27H28N4O4) C, H, N.
2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydrobenzo[b]-
[1,4]diazepin-1-yl)-N-(4-methoxyphenyl)-N-methylacetamide
(Racemate 3, 4). Using the procedure for the racemate (1, 2) above
and starting with 8 mmol of hydrazone 19, 3.22 g of racemate (3,

860 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Hadac et al.
Table 1
compound retention time, min purity, % enantiomeric excess, %
(t, 1H, J ) 7.2 Hz), 7.68 (d, 2H, J ) 7.2 Hz), 7.79 (s, 1H), 9.43
(s, 1H); LRMS (APCI) m/z 689.9 [M + Na]+, 666.7 [M]-; Anal.
(C39H33N5O6) C, H, N.
5
6
7
8
41.3
48.3
49.9
82.2
>99
>99
>99
>99
>99
>99
97.0
96.5
Chiral Resolution Procedure and Results for Resolved
Compound 7 (and 8). Following the procedure for the resolved
enantiomers 5 and 6, 100 mg of the parent N-methyl racemate (7,
8) was separated into its purified component enantiomers, which
were isolated as white solids (35 mg of compound 7, and 25 mg of
compound 8). Enantiomer purity and enantiomeric excess were
confirmed by chiral RP-HPLC analysis using an alternate isocratic
analytical method (Analytical Chiralpak AD C-18 column, with
mobile phase (65:35, Hex:IPA) eluting at 1 mL/min flow rate).
Compound 7 eluted at 49.94 min, while compound 8 eluted at 82.17
min (showing good baseline peak separation); coinjections with
each other and with the parent racemate confirmed chiral purity.
Compound 7 (and 8): 1H NMR (300 MHz, CDCl3) δ 3.09 (s, 3H),
3.76 (s, 3H), 4.37 (d, 1H, J ) 16.8 Hz), 4.67 (d, 1H, J ) 16.8 Hz),
5.02 (d, 1H, J ) 8.0 Hz), 6.88 (d, 1H, J ) 8.0 Hz), 6.92 (d, 1H,
J ) 7.6 Hz), 7.04, (d, 1H, J ) 8.0 Hz), 7.21 (d. 1H, J ) 7.6 Hz),
7.24 (d, 1H, J ) 7.6 Hz), 7.30-7.48 (m, 13H), 7.50 (t, 1H, J )
7.6 Hz); 7.63 (t, 1H, J ) 7.2 Hz), 7.69 (d, 2H, J ) 7.2 Hz), 7.79
(s, 1H), 9.43 (s, 1H); LRMS (APCI) m/z 689.9 [M + Na]+, 666.7
[M]-; Anal. (C39H33N5O6) C, H, N.
4.41 (d, 1H, J ) 21.0 Hz), 4.69 (d, 1H, J ) 21.0 Hz), 4.75 (b,
1H), 6.70-7.50 (m, 13H). LRMS (APCI) m/z 445.2 [M + H]+;
TLC Rf ) 0.47 (EtOAc:Hex, 1:2); Anal. (C25H24N4O4) C, H, N.
Representative Procedure for Chiral Urea Formation. 2-{3-
[3-(4-Benzoylphenyl)ureido]-2,4-dioxo-5-phenyl-2,3,4,5-tetra-
hydrobenzo[b][1,4]diazepin-1-yl}-N-isopropyl-N-(4-methoxy-
phenyl)acetamide (5). 3-Aminobenzophenone (0.016 g, 0.08
mmol) was dissolved in anhydrous CH3CN (1 mL) and stirred under
N2. DIPEA (0.010 g, 0.08 mmol, 14 µL) was then added directly
into the reaction solution, and the reaction solution was cooled to
e4 °C in an ice-water bath. Phosgene (20% in toluene, 0.08 mmol,
42.1 µL) was quickly syringed directly into the reaction solution
and the reaction stirred at e4 °C under N2 for 1 h. Dropwise the
chiral amine hydrochloride salt of compound 1 (0.038 g, 0.08 mmol)
in 2 mL of dry CH3CN was added to the stirring reaction solution.
After 2-3 min, 1 equiv of DIPEA (0.010 g, 0.08 mmol, 14 µL)
was added to the e4 °C solution while stirring under N2. After an
additional 2 min, the final 1 equiv of DIPEA (0.010 g, 0.08 mmol,
14 µL) was added and the reaction allowed to stir at e4 °C under
N2 for 2 h.
For each of the isopropyl compounds (5 and 6), the product
precipitated out of solution and was filtered and washed with dry
acetonitrile and then dried to constant weight under vacuum.
Analytical data for the isolated products matched the data obtained
for the same materials synthesized via method 1.
For the methyl compounds (7 and 8), the solvent was evaporated
using a stream of dry N2, then the resulting oil was chromatographed
on silica gel using (55% EtOAc/45% Hex) as eluent. The purified
material was evaporated to a white solid and then dried to constant
weight under vacuum. As with the isopropyl compounds, the
analytical data for the isolated products matched the data obtained
for the same materials synthesized via method 1 previously shown.
In all four examples (5, 6, 7, and 8), pure white solids were typically
obtained in 80% yields. This procedure was used for subsequent
14C synthesis of the enantiomers 5, 6, 7, and 8, merely substituting
14C-labeled phosgene solution for the nonradioactive phosgene used
above.
Method 2: General Procedure for Resolving the Enantio-
meric Amine Intermediates Prior to Forming Chiral Ureas.
Resolved (R)- and (S)-2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydrobenzo[b][1,4]diazepin-1-yl)-N-isopropyl-N-(4-meth-
oxyphenyl)acetamide (1 and 2). For use in the synthesis of 14C-
labeled (R)- and (S)-photoaffinity probes, the isopropyl racemic
mixture of amines 1 and 2 were then separated into the resolved
enantiomers using semipreparative chiral RP-HPLC and monitored
by UV absorbance, showing good baseline peak separation (Dy-
namax C-8 column, 25 cm × 4.1 mm, 8 mL/min flow rate, ramping
from [40% CH3CN/60% (H2O with 0.1% TFA)] up to 100% CH3-
CN over 30 min). Each enantiomer was collected in a flask
containing at least 1 equiv of acid (HCl) in order to prevent
epimerization of the purified enantiomers upon sample concentra-
tion. Chiral HPLC purification of the original 500 mg of parent
racemate yielded 202 mg of compound 1 and 165 mg of compound
2. Under standard achiral NMR conditions and LCMS conditions,
both enantiomers of each pair gave identical spectra to each other
as well as to that of the parent racemate (1, 2) provided above.
Enantiomer purity and enantiomeric excess were confirmed by
chiral RP-HPLC analysis, showing good baseline peak separation
using an alternate isocratic analytical method (Analytical Chiralpak
AD C-18 column, with mobile phase (3:1, Hex:IPA) eluting at 1
mL/min flow rate). Compound 1 eluted at 20.60 min, while
compound 2 eluted at 31.76 min; coinjections with the parent
The enantiopurity of the final compounds was verified using
reverse phase HPLC (Analytical Chiralpak AD C-18 column,
isocratic method with 1 mL/min flow rate, eluent ) 65% hexane/
35% IPA). Retention times for the isolated enantiomers obtained
by using method 2 are given in Table 1.
1
racemate confirmed chiral purity. Compound 1 (and 2) H NMR
(300 MHz, CD3OD) δ 1.10 (m, 6H), 3.87 (s, 3H), 4.35 (d, 1H,
J ) 21.0 Hz), 4.61 (d, 1H, J ) 21.0 Hz), 4.35 (m, 1H), 4.87
(m, 1H), 6.80-7.50 (m, 13H); LCMS (APCI) m/z 473.3 [M + H]+;
TLC Rf ) 0.45 (EtOAc:Hex, 1:2); Anal. (C27H28N4O4) C, H,
N.
Enantiomer Racemazation Conditions. The purified enanti-
omer (1, 2, 3, 4, 5, 6, 7, or 8) (0.1 g) was dissolved in DCM and
1.5 equiv of TEA or DIPEA added at room temperature. The
reaction was allowed to stir at room temperature in open to air for
several hours, and the conversion back to the parent racemate was
followed by chiral HPLC, using the conditions described previously
for the individual enantiomers. In all cases, conversion was allowed
to proceed until approximately 1:1 ratio of stereoisomers was
achieved. The residual solvent and base were then removed in
vacuo, and the newly formed racemate analyzed for degradation
products. In all cases, each newly formed racemate was clean of
impurities and provided the identical analytical results to those
obtained for the original parent racemate given above.
Receptor-Bearing Cell Line. The Chinese hamster ovary cell
line stably expressing the type A rat CCK receptor (CHO-CCKR)
that has previously been fully characterized was utilized as source
of receptor for the biochemical and receptor domain-mapping
studies.14 This cell line was cultured in Ham’s F-12 medium
supplemented with 5% fetal clone-2 (Hyclone Laboratories, Logan,
UT) at 37 °C in an environment of 5% CO2 in Costar tissue culture
plasticware. Cells were harvested mechanically and passaged twice
weekly.
Resolved (R)- and (S)-2-(3-Amino-2,4-dioxo-5-phenyl-2,3,4,5-
tetrahydrobenzo[b][1,4]diazepin-1-yl)-N-(4-methoxyphenyl)-N-
methylacetamide (3 and 4). The methyl racemate (3, 4) was
separated similarly into its respective enantiomeric amines 3 and 4
using the procedure outlined for racemate (1, 2). Chiral HPLC
purification of the original 500 mg parent racemate yielded 185
mg of (3) and 242 mg of (4). As with the isopropyl analogues 1
and 2, under standard achiral NMR conditions and LCMS condi-
tions, both enantiomers of each pair gave identical spectra to each
other as well as to that of the parent racemate (1, 2) provided above.
Enantiomer purity and enantiomeric excess were confirmed by
chiral RP-HPLC analysis using an alternate isocratic analytical
method (Analytical Chiralpak AD C-18 column, with mobile phase
(3:1, Hex:IPA) eluting at 1 mL/min flow rate). Compound 3 eluted
at 36.44 min, while compound 4 eluted at 42.03 min; coinjections
with the parent racemate confirmed chiral purity. Compound 3 (and
1
4): δ H NMR (300 MHz, CD3OD) δ 3.15 (s, 3H), 3.82 (s, 3H),

SelectiVe Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 861
Radioligand Binding Studies. Membranes were prepared from
the CHO-CCKR cells, as we have previously described.14 For
binding characterization, membranes (3-5 µg) were incubated with
a constant amount of the CCK-like radioligand, 125I-D-Tyr-Gly-
[(Nle28,31)CCK-26-33] (2-5 pM), in the absence or presence of
variable concentrations of unlabeled competing ligand. Incubations
were performed to attain steady state, achieved after incubation
for 3 h at 30 °C in Krebs-Ringers-HEPES (KRH) medium (25
mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1.2 mM MgSO4,
2 mM CaCl2, 1 mM KH2PO4, 0.2% bovine serum albumin, 0.01%
soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride).
Bound and free radioligand were separated using a Skatron cell
harvester (Molecular Devices, Sunnyvale, CA) with receptor-
binding filtermats that had been presoaked with 0.2% bovine serum
albumin. Bound radioactivity was quantified in a gamma-
spectrometer. Nonspecific binding was determined in the presence
of 1 µM CCK and represented less than 10% of total bound
radioactivity.
Affinity Labeling Studies. For covalent labeling studies,
substantially greater amounts of receptor-bearing membranes (100-
200 µg) were incubated with 1-10 µM radioligand, representing
the 14C-labeled compounds described above, in the absence or
presence of competing unlabeled CCK receptor ligands in KRH
medium for 3 h at 30 °C. Tubes were exposed to photolysis for 30
min at 4 °C in a Rayonet photochemical reactor equipped with 3,-
500 Å lamps (Southern New England Ultraviolet, Hamden, CT).
Membranes were then isolated by centrifugation, washed twice in
KRH medium, and solubilized with 1% NP-40 in KRH for 18 h at
4 °C with constant mixing.
Solubilized affinity-labeled receptor was diluted with an equal
volume of KRH medium and one-tenth volume of a 50% slurry of
WGA-agarose, with the incubation allowed to proceed for 24 h at
4 °C with constant mixing. The WGA-agarose was pelleted and
washed with a solution containing 0.5 M NaCl and 0.1% NP-40,
followed by a water wash. In preparation for electrophoresis,
reducing sample buffer was added to the pelleted WGA-agarose
beads, and this was allowed to sit for at least 30 min at room
temperature. Samples were applied to a 10% SDS-polyacrylamide
gel for electrophoresis using the conditions described by Laemmli.33
At the completion of electrophoresis the receptor band was excised
for further study or the gel was dried in preparation for autorad-
iography. Dried gels were placed into film cassettes containing
BioMax MS X-ray film and a BioMax TranScreen-LE (Eastman
Kodak Company, Rochester, NY). Cassettes were kept at -80 °C
for 1-6 weeks before the film was developed.
Gel-purified affinity-labeled receptor was deglycosylated with
endoglycosidase F and/or cleaved with cyanogen bromide using
conditions we have previously reported, with minor modifications.34
Gel regions containing receptor bands were excised, diluted with
water, and treated with dounce homogenization. Gel pellets were
removed by centrifugation, and the supernatants containing the
labeled receptor or its fragments were lyophilized to dryness.
Detergent was removed from the samples by precipitation with 85%
ethanol at -20 °C for 18 h. Precipitates were pelleted, dried, and
then resuspended in 70% formic acid containing 2.5 mg of cyanogen
bromide. Cleavage was allowed to proceed for 18 h under nitrogen
at room temperature. Acid was then removed from the samples by
three cycles of water washes and vacuum centrifugation. Sample
buffer was added to tubes in preparation for electrophoresis on a
10% NuPAGE gel (Invitrogen, Carlsbad, CA) with MES running
buffer. A small portion of the sample was run in a separate lane so
it could be dried and used for autoradiography to identify the
position of the cleavage fragment. The remaining portion of the
gel was frozen, and, once the position of the labeled fragment of
interest was identified, that region of the gel could be excised and
the sample eluted for further purification by HPLC.
a Perkin-Elmer/Brownlee reversed-phase C18 column, measuring
0.5 × 150 mm with 5 µm beads and a pore size of 300 Å. The
buffer composition was 0.1% TFA as buffer A and 0.085% TFA/
80% acetonitrile as buffer B. The sample was injected at 10% B,
and the system was allowed to run for 20 min before beginning a
230 min gradient to 90% B. The flow rate was 5 µL/min, and
samples were collected directly on poly(vinylidene difluoride)
(PVDF) membranes. The UV detector monitored absorbance at 210
nm. The PVDF sample membrane was exposed to BioMax MS
X-ray film and a BioMax TranScreen-LE. The major radioactive
spot was localized and excised from the PVDF membrane and was
then subjected to Edman degradation sequencing using an Applied
Biosystems automated instrument.
Biological Activity Studies. Rat pancreatic acini were freshly
prepared from 80 to 100 g male Sprague Dawley rats. All studies
were reviewed and approved by the Mayo Clinic Animal Care and
Use Committee. Pancreatic tissue was harvested and placed in iced,
oxygenated KRH medium. The pancreas was trimmed of fat and
connective tissue and injected with 600 units of purified collagenase
diluted to 6 mL with KRH medium. The tissue was minced,
oxygenated, and incubated at 37 °C for 5 min in a shaking water
bath. Additional digestion took place with 10 min of shaking by
hand before the acini were washed and filtered through 200 µm
nytex mesh. The health of the acini was assessed morphologically,
and the cells were suspended in KRH medium for use.
One milliliter aliquots of cells were added to tubes containing
various concentrations of secretagogues. The samples were incu-
bated for 30 min at 37 °C in a shaking water bath. The secretion
assay was terminated by centrifugation of an aliquot of the cell
suspension over Nyosil oil (William F. Nye, Inc., New Bedford,
MA). Amylase measurement utilized the Phadebas amylase reagent
(Pharmacia Diagnostics, Uppsala, Sweden). Tablets were dissolved
in assay buffer (0.02 M NaH2PO4, 0.05 M NaCl, 0.02% NaN3, pH
7.0). Two milliliters of Phadebas suspension was added to aliquots
of secretion samples in lysis buffer (0.01 M NaH2PO4, pH 7.8,
containing 0.1% SDS and 0.1% bovine serum albumin). Samples
were incubated at 37 °C for 15 min before the incubation was
terminated by addition of 0.5 M NaOH. Samples were measured
with a spectrophotometer at 620 nm, and values were expressed as
percentages of amylase release in response to maximal stimulation
with CCK (0.1 nM CCK).
Molecular Modeling. Our current model for the high affinity
agonist-bound type A CCK receptor served as a starting point for
the construction of molecular models that incorporated the photo-
affinity labeling data reported in this study.15 The receptor’s small
molecule binding site was manually adjusted via interactive
computer graphics techniques to accommodate small molecule
ligands within the membrane-spanning domain. These adjustments
were performed using the most probable side chain rotamer
conformations from the backbone-dependent amino acid rotamer
library of Dunbrack and Karplus,16 without alteration of the
positions of protein backbone atoms. We targeted stable, low energy
structure modifications by choosing only combinations of rotamers
with the three highest probabilities based on a statistical analysis
of experimentally observed conformations in the above library of
side chain dihedral angles for each of the following amino acid
side chains (Thr118, Ser124, Asn334, Arg337, Tyr339, Thr341, Ile356).
We next performed manual docking studies for the nonpeptidyl
antagonist, devazepide,35 a 1,4-benzodiazepine that competitively
blocks 1,5-benzodiazepine agonist and antagonist photoaffinity
labeling. Manual docking of devazepide (compound 9) was guided
by mutagenesis data from previous ligand-binding studies described
above.
Limited energy minimization and constrained molecular dynam-
ics simulations (5 ps at 20 K) of the devazepide ligand-receptor
complex were performed in vacuo with a distance dependent
dielectric constant to relieve some minor steric and conformational
strain introduced during initial ligand docking. In this initial
refinement of the membrane-spanning region, previously published
constraints were used to maintain the overall conformation of the
receptor starting structure.15 Snapshots were taken from the
HPLC Purification and Sequence Analysis. Prior to having
components resolved by HLPC, the samples were applied to a
Dispo-Biodialyzer (The Nest Group, Inc., Southborough, MA) with
a molecular weight cutoff of 1 kDa, and samples were dialyzed
for 24 h against 2 L of water. Capillary HPLC was performed using

862 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Hadac et al.
molecular dynamics trajectories; each molecular dynamics snapshot
was energy minimized, and then the devazepide antagonist ligand
(9) was removed to generate target receptor structures for automated
ligand docking calculations for the 1,5-benzodiazepines (5, 6, 7,
and 8).
( SEM. Binding and biological activity curves were analyzed and
plotted using the nonlinear regression analysis routine in the Prism
software package (GraphPad Software, San Diego, CA). Binding
kinetics were determined by analysis with the LIGAND program.44
The setup for DOCK 4.036 calculations involved the generation
of a negative image of potential ligand binding sites in the receptor
by filling the area within the confluence of membrane-spanning
helices with spheres. Boundaries for agonist and antagonist binding
sites were defined using photoaffinity-labeling data for the respec-
tive ligands, and sphere generation was confined to bounding boxes
centered at the receptor positions for agonist and antagonist covalent
labeling. Although the bounding boxes for the agonist and
antagonist sites have significant overlap, the antagonist photoaf-
finity-labeling data clearly precludes any receptor contacts for
compound 8 in the first extracellular loop. Therefore, the bounding
box for the antagonist photoaffinity label was limited to the
membrane-spanning receptor domain, while the bounding box (and
corresponding sphere set) for the agonist photoaffinity ligand
contained portions of the receptor membrane-spanning domain and
the extracellular loop regions.
Docking calculations were performed using low energy confor-
mations for each compound. Geometry optimizations were per-
formed using ab initio Hartree-Fock calculations with a 3-21G*
basis set. Quantum mechanical electrostatic potentials were calcu-
lated for each molecule using a 6-31G* basis set and then used to
fit atom-centered point charges with the RESP module of AMBER
7.37 Conformational searches in torsion angle space for all rotatable
bonds of each ligand were conducted using the torsion-drive feature
in DOCK 4.0.36 Most sp2-sp3 bonds were scanned at 60°
increments, and all sp3-sp3 bonds were probed at -60°, +60°,
and 180°. Additionally, energy minimization of the ligand conform-
ers generated from this search were performed ‘on the fly’ in DOCK
to facilitate the identification of low energy complexes for each
ligand with the receptor model. Docked complexes were evaluated
according to the DOCK 4.0 shape-based and energy-based scoring
functions.36 Representative receptor-ligand complex structures
generated with DOCK were then refined in vacuo with limited (500
steps) energy minimization. For the receptor-(S)-antagonist (8)
docked complex, a limited number of additional dihedral angle
adjustments were performed (following the above outlined strategy)
for the following protein side chains prior to energy refinements
to relieve unfavorable van der Waals contacts around the photo-
affinity-labeling site (Leu88, Cys91, Cys94, Met121, Leu357, Ser364).
Molecular dynamics simulations for the (S)-antagonist (8) complex
were performed in vacuo with a distance-dependent dielectric
constant at temperatures of 20 K and 300 K for 100 ps. Since our
model does not include membrane lipids or solvent, weak (1-10
kcal/mol) harmonic constraints were gradually imposed for the 300
K simulations over a 50 ps time frame to stabilize the conformation
of the membrane-spanning helix bundle at the higher temperatures.
An NMR-style energy restraint (5 kcal/mol) was also imposed on
the distance between the ketone carbonyl of the photolabile
benzophenone moiety and the Cγ-H bond of Leu88 to maintain a
physically realistic photoincorporation distance consistent with the
experimental data in this report.
Acknowledgment. We acknowledge the expert contribution
of Manon Villeneuve and Itzela Correa of Glaxo-SmithKline
Research Laboratories, who performed the chiral separations
and the analysis and radiolabeling of the compounds used in
this study. We thank Dr. Maoqing Dong for his advice and help
with preparing this manuscript. This work was supported by
grants from the National Institutes of Health (DK32878 to L.J.M.
and NS33290 to T.P.L.), Glaxo-SmithKline, and the Fiterman
Foundation.
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