Probing the steric space at the floor of the D1 dopamine receptor orthosteric binding domain: 7α-, 7β-, 8α-, and 8β-methyl substituted dihydrexidine analogues

Article abstract of DOI:10.1021/jm200334c

To probe the space at the floor of the orthosteric ligand binding site in the dopamine D₁ receptor, four methylated analogues of dihydrexidine (DHX) were synthesized with substitutions at the 7 and 8 positions. The 8α-axial, 8β-equatorial, and

Full text of DOI:10.1021/jm200334c

ARTICLE
pubs.acs.org/jmc
Probing the Steric Space at the Floor of the D1 Dopamine Receptor
Orthosteric Binding Domain: 7r-, 7β-, 8r-, and 8β-Methyl Substituted
Dihydrexidine Analogues
Juan Pablo Cueva,‡ Alejandra Gallardo-Godoy,§ Jose I. Juncosa, Jr.,† Pierre A. Vidi,† Markus A. Lill,†
Val J. Watts,† and David E. Nichols*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University,
RHPH Pharmacy Building, Room 506C, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States
‡Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, United Kingdom
§Small Molecule Discovery Center (SMDC), School of Pharmacy, University of California, San Francisco, San Francisco,
California 94143, United States
ABSTRACT:
To probe the space at the floor of the orthosteric ligand binding site in the dopamine D1 receptor, four methylated analogues of
dihydrexidine (DHX) were synthesized with substitutions at the 7 and 8 positions. The 8R-axial, 8β-equatorial, and 7R-equatorial
were synthesized by photochemical cyclization of appropriately substituted N-benzoyl enamines, and the 7β-axial analogue was
prepared by an intramolecular Henry reaction. All of the methylated analogues displayed losses in affinity when compared to DHX
(20 nM): 8β-Meax-DHX (270 nM), 8R-Meeq-DHX (920 nM), 7β-Meeq-DHX (6540 nM), and 7R-Meax-DHX (>10000 nM).
Molecular modeling studies suggest that although the disruption of an aromatic interaction between Phe2035.47 and Phe2886.51 is
the cause for the 14-fold loss in affinity associated with 8β-axial substitution, unfavorable steric interactions with Ser1073.36 result in
the more dramatic decreases in binding affinity suffered by the rest of the analogues.
’ INTRODUCTION
with improved pharmacological properties. Modifications of the
dihydrexidine template have already led to compounds with
The development of the selective dopamine D1 receptor full
agonist dihydrexidine, 1, has enabled numerous studies aimed at
investigating the physiological role of the D1 dopamine receptor
and the potential of this receptor subtype as a therapeutic target.
Dihydrexidine has been shown to possess profound antiparkin-
sonian effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine (MPTP) lesioned monkey model of Parkinson’s disease,1
underscoring the importance of this receptor in the volitional
control of motor function (Figure 1). D1 receptor activation in
the prefrontal cortex also has been shown to be essential for the
proper performance of cognitive functions such as working
memory and attention.2,3 Recently, a small clinical trial of dihydrex-
idine demonstrated that a single dose significantly increased
prefrontal cortical blood flow in schizophrenia patients.4 Reward
phenomena also are thought to be dependent upon D1 receptor
activation.5
increased D1 selectivity and distinct metabolic profiles.6
A
pharmacophoric moiety has been proposed that provides for
binding interactions between polar residues of the binding site
and catechol functions, electrostatic interactions between a
putative anionic function to engage the protonated ligand amine,
and an accessory binding region that accommodates the pendant
phenyl ring. In essence, we have successfully employed this
“β-phenyldopamine” pharmacophore for the design of several
D1 dopamine-selective full agonists.7,8 The original conceptual
model of the receptor has been applied to understand the
molecular structure of the G protein-coupled D1 receptor itself,
with the purpose of identifying the nature of the binding site.
Prior mutagenesis studies have identified key polar residues that
line the orthosteric binding domain. That is, aspartate 103 in
Increasing awareness of the physiological roles of the dopa-
mine D1 receptor has made evident the need to develop ligands
Received: May 3, 2011
Published: June 29, 2011
r
2011 American Chemical Society
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Figure 1. Dihydrexidine and the synthesized 7- and 8-methyl analogues.
Scheme 1. Synthesis of Diastereomeric Amides 11a and 11b
transmembrane helix 3 forms a salt bridge with the protonated
amine of the ligand, and serines 198, 199, and 202 in transmem-
brane helix 5 interact with the catechol moiety. Mutant human
dopamine D1 receptors S198A, S199A, and S202A previously
have been examined using the 10- and 11-monohydroxy analo-
gues of 1 to establish that S198 and S199 interact with the 11-OH
group, whereas S202 engages the 10-hydroxy group.9 These
results parallel early mutagenesis studies of the β-adrenergic
receptors, where it was shown that analogous serine residues in
transmembrane domain 5 (TM5) interacted with the catechol
moiety of β-adrenergic receptor agonists.10 These studies clearly
identify the area within the receptor where a catechol agonist
ligand must bind. Additionally, it has been suggested that
phenylalanines 203, 288, and 289 in transmembrane domains
5 and 6 form aromatic interactions with the catechol ring, and
one or more hydrophobic and/or aromatic residues in helix 7
(Phe313) and extracellular loop 2 (Leu190) form the binding
site for the pendant accessory (e.g., phenyl) ring.11ꢀ15
Thus, to continue developing the structureꢀactivity relation-
ships of these compounds and to obtain a better understanding of
the topography of the D1 dopamine receptor binding site, we
now report on the synthesis and receptor affinities of four
analogues of dihydrexidine bearing a methyl substituent on the
unsaturated B-ring, namely compounds 2a, 2b, 3a, and 3b. We
also report on their affinity at D1- and D2-like receptors. Finally,
using an in silico activated homology model of the D1 dopamine
receptor based on the first X-ray crystal structure of the
β2-adrenergic receptor,11 we provide likely explanations for the
observed relative affinities of the target compounds. The present
study reveals that modifications at the 7- or 8-positions lead to a
significant loss of receptor affinity, and molecular modeling
suggests that the regional space constraints are sufficient only
to accommodate the catechol ring.
’ CHEMISTRY
In early studies, Grol and Rollema16 had proposed a region of
steric intolerance below the catechol ring, and this feature was
incorporated into a working model by McDermed,17 who
demonstrated an area of steric intolerance within “dopamine
receptors,” specifically located adjacent to the dopamine 5-posi-
tion, which corresponds to position 9 in 1. We have assumed (but
not tested the hypothesis) that this spatial restriction applies to
the D1 dopamine receptor (e.g., see Brewster et al.18), but in fact
very little is known of the floor of the orthosteric binding site in
thedopamineD1 receptor. Therefore, we were interested in probing
this area of the receptor, with the goal of possibly exploiting this
region to develop additional selective agonists.
Synthesis of 2a and 2b. The synthesis of the 8-Me-DHX
analogues 2a and 2b is shown in Schemes 1 and 2. Commercially
available 3,4-dimethoxyphenylacetic acid was iodinated using
iodine monochloride to provide the iodophenylacetic acid 5,
which was esterified prior to Heck olefination with methyl
crotonate19 to afford methyl cinnamate 7. This compound
could be reduced with H2 over 10% PdꢀC, but reduction over
Raney nickel in EtOH gave a slightly better yield. Intramolecular
Dieckmann condensation gave an intermediate ketoester that
was decarboxylated by treatment with lithium chloride and HCl
in boiling N-methylpyrrolidinone. Treatment of the resulting
β-tetralone 9 with benzylamine, followed by benzoyl chloride,
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Scheme 2. Final Synthesis of the 8r,β-Methyl Analogues of DHX
Scheme 3. Synthesis of the 7-Axial Methyl Analogue of DHX
gave access to enamide 10, which was irradiated using a 450 W
medium-pressure quartz mercury-vapor lamp to induce
cyclization,18ꢀ20 giving a 50/50 mixture of the R- and β-methyl
trans-lactams 11 in moderate yield. Chromatographic separation
of this mixture allowed isolation of small amounts of pure axial
β-methyl lactam 11a (Scheme 2). The separation process did
not, however, allow recovery of a pure sample of the equatorial
R-methyl-substituted diastereomer 11b. Compound 11a gave
crystals suitable for X-ray structure determination, which un-
ambiguously allowed a determination of its stereochemistry. This
pure axial-methyl lactam 11a was then reduced with borane in
THF, and N-debenzylated by catalytic hydrogenation to yield
amine 12a, which was demethylated with boron tribromide to
afford the catecholamine hydrobromide final compound 2a.
To obtain the desired 8R-Meeq-DHX, the mixture 11a,b
obtained by photocyclization was reduced with borane in
THF, and the intermediate tertiary amine 13a,b was N-deben-
zylated and Boc-protected in one step to yield carbamates 14a
and 14b. This mixture was separable by flash column chroma-
tography, which allowed isolation of both the 8R-axial (14a) and
8R-equatorial (14b) carbamates. Once purified and separated,
the equatorial carbamate 14b was simultaneously demethylated
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Scheme 4. Proposed Mechanism for the Isomerization and the 7 Position under Photochemical Conditions
and N-deprotected using boron tribromide to give 2b as the
hydrobromide salt in excellent yield. It also was possible to obtain
2a using this route.
Synthesis of Analogues 3a and 3b. Common intermediate 6
provided the starting point for the synthesis of 7R-Meax-DHX
(Scheme 3), which was synthesized using methodology similar to
that used to prepare 2a and 2b. Heck coupling of 6 with methyl
methacrylate gave the methyl cinnamate derivative 15, which was
reduced by catalytic hydrogenation to provide the saturated
diester 16. Dieckmann condensation and decarboxylation al-
lowed access to β-tetralone 17, which was converted to the
enamide 18.
Unexpectedly, irradiation of 18 as performed previously gave
exclusively the 7R-methyl lactam 19a in 60% yield. The diaster-
eomeric purity of this product suggests that epimerization of the
7β-methyl into the 7R-methyl must have occurred at some point
Figure 2. Crystal structure of 20a.
during this transformation. As this photocyclization likely pro-
ceeds by an electrocyclic mechanism (Scheme 4) that involves
the imidate resonance structure of 18,21 it is conceivable that the
7-methine hydrogen, adjacent to the methyl group, is abstracted,
giving a resonance stabilized anion B that would be free to
epimerize. Molecular modeling of this intermediate anion sug-
gests that the methyl group of this species will adopt an R-axial
conformation to avoid a steric clash with the N-benzyl substi-
tuent. Selective protonation from the β side of the moiety,
followed by suprafacial [1,5] hydride shift,22 would give diaste-
reomer 19a exclusively.
Borane reduction of 19a, followed by debenzylation yielded
the amine 20a, which gave crystals suitable for X-ray crystal
structure determination of its stereochemistry, confirmed the
axial orientation of its methyl substituent (Figure 2). Treatment
of this amine with boron tribromide furnished the 7R-Meax-
DHX, 3a, as the hydrobromide salt.
Given the unexpected diastereomeric selectivity of the photo-
cyclization reaction leading to the formation of 3a, we were
unable to produce the 7β-methyl diastereomer 3b using this
approach. To prepare 3b, we modified our recently reported23
synthesis of dihydrexidine based on the intramolecular Henry
reaction of key nitrobenzophenone 28 (Scheme 5).
To prepare key synthon 28, 3,4-dimethoxybenzaldehyde was
converted to methylcinnamic acid 22 by Knoevenagel condensa-
tion with methylmalonic acid.24 Attempts to reduce this decep-
tively ordinary olefin using usual catalytic hydrogenation
conditions (Pd/C, H2, EtOH), similar to those employed for
other cinnamic acids, were unsuccessful. Instead, the conditions
reported by Schrecker25 were employed, which involved vigor-
ous stirring of a highly basic aqueous NaOH solution of the
compound with NiꢀAl alloy to effect reduction. This procedure
provided the saturated acid 23 in quantitative yield. Further
reduction with borane in THF gave the alcohol 24, which was
converted to the corresponding bromide using triphenylpho-
sphine and carbon tetrabromide. Alkyl bromide 25 was dissolved
in DMF along with NaNO2 to effect nitration,26 leading only to a
41% yield of the expected nitro compound 26 but with recovery
of significant amounts of starting material.
FriedelꢀCrafts acylation of 26 with phthaloyl dichloride
yielded benzophenone 27. This intermediate was esterified (28)
to facilitate the subsequent intramolecular Henry cyclization,
which was promoted by a catalytic amount of DBU in a protic
solvent. The reduction of the resulting nitroalkene 29 (Scheme 6)
was considerably slower than previously reported for the des-
methyl analogue,23 taking about four days at reflux in 2-propanol
and a very large excess of sodium borohydride for complete
reduction. Quenching of the resulting mixture using a concen-
trated solution of urea in dilute aqueous acetic acid27 allowed
recovery of the inseparable 1,2-trans diastereomeric mixture 30a,
b but in only a disappointing 21% yield. This mixture was
esterified and treated with borane in THF to produce two
diastereomeric lactams, 32a and 32b, which were separable by
chromatography. Inspection of the key methyl signal in the NMR
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Scheme 5. Synthesis of Nitrodihydrotetralin Intermediate 29
Scheme 6. Final Synthesis of the 7-Equatorial Methyl Analogue of DHX
spectrum of the mixture revealed that these compounds had been
carried through the synthesis from the point of diastereomer
formation as a 5:1 mixture favoring the axial R-methyl compound
32a over the desired equatorial-β-methyl 32b. These lactams
were separated by flash column chromatography, obtaining
amounts that confirmed the 5:1 diastereomeric ratio and allow-
ing a positive determination of the product’s stereochemistry by
examination of the NMR signal corresponding to the methine
hydrogen at the R-position relative to the amide nitrogen. In the
case of the 7β-equatorial-methyl 32b, this signal appeared as a
triplet with a single coupling constant, in accordance with its
double-trans relationship with vicinal hydrogens.
The 5:1 diastereomeric ratio was likely set during reduction of
nitroalkene 29. From the structure of the axial-methyl isomer
30a, which was the major product of the reduction, it is evident
that hydride transfer had occurred preferentially from the same
face as the methyl substituent. Molecular modeling of 3a and 3b
also suggests that the predominant diastereomer is not the
thermodynamically favored product, supporting speculation that
the actual species that undergoes reduction is probably a high
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Table 1. D1- and D2-Like Affinity of Test Compounds
and had a greater than 30-fold loss of D2 affinity. The presence of
the 7β-equatorial methyl in the DHX template (3b) caused a
greater than 300-fold decrease in D1 affinity and more than 35-
fold decrease in D2 affinity.
Substitution at the 8-position with axial and equatorially
oriented methyl groups also gave marked decreases in D1 and
D2 affinity compared to DHX. The 8β-axial-methyl analogue 2a
had the highest affinity of the methylated series but still suffered a
more than 13-fold decrease in D1 affinity compared to DHX. The
loss of affinity of the 8R-equatorial-methyl analogue 2b was more
substantial, being about 40-fold lower than the unsubstituted
parent ligand.
To gain a better understanding of the potential mode of
binding of the parent ligand DHX to the D1 receptor and to
rationalize the relative decrease in affinity observed for the test
compounds 2a,b and 3a,b, we constructed a homology model of
the D1 receptor based on an in silico activated model of the first
published X-ray crystal structure of the β2-adrenergic receptor28
andusedanunbiasedcomputer-baseddockingroutinetodetermine
probable binding orientations of DHX to the D1 binding site.
After molecular dynamics (MD) and energy minimization, it was
observed that the D1 receptor binding site accommodates the
active (6aR,12bS)-(+) isomer of DHX within a cavity located
between transmembrane helices 3, 5, and 6 that is lined by several
residues that have been shown by site-directed mutagenesis
experiments to be involved in ligand binding (Figure 3). Aspar-
tate 103, a residue located in the third transmembrane domain
that interacts with the protonated amine of the ligand, is highly
conserved among monoamine receptors and has been shown
by mutagenesis studies to be instrumental in the binding of
catecholamines.11ꢀ15
binding in porcine striatal homogenatesa
D1-like
D2-like
fold selectivity
(D2/D1)
ligand
Ki (nM)
Ki (nM)
DHX
20 ( 2
240 ( 40
5530 ( 3180
3630 ( 550
7380 ( 2440
8670 ( 2290
ND
12 ( 2
20 ( 12
3.9 ( 0.6
<0.74
8β-Meax-DHX, 2a
8R-Meeq-DHX, 2b
7R-Meax-DHX, 3a
7β-Meeq-DHX, 3b
SCH-23390
270 ( 34
920 ( 46
>10000
6540 ( 1120
0.5 ( 0.1
ND
1.3 ( 0.4
ND
chlorpromazine
8 ( 2
ND
a All results shown are the mean ( SEM for four independent
experiments.
Mutagenesis studies of the D1 receptor have identified three
serine residues in the fifth transmembrane domain that are of
importance in ligand binding: Ser 198, Ser 199, and Ser 202,
which are thought to hydrogen-bond to the catechol moiety of
DHX.14 The resulting energy-minimized docked conformation
involves a complex hydrogen bonding network between polar
residues in TMs 5 (serines 198, 199, and 202) and 6 (asparagine
292) and the catechol hydroxyl groups.
Figure 3. Structure of DHX docked into the activated homology model
of the human dopamine D1 receptor. Key polar residues involved in
binding are identified.
molecular weight acyloxyborohydride. If that is the case, perhaps
this species undergoes a kinetically favored intramolecular hy-
dride addition that would account for the observed diastereo-
meric product ratio.
To complete the synthesis, the 7β-equatorial-methyl lactam
32b was reduced with borane in THF to the amine 20b, which
was then demethylated using boron tribromide to afford the
desired 3b, as the hydrobromide salt, thus completing the series
of 7- and 8-methyl substituted dihydrexidine analogues.
To begin with, the previously known17 steric restriction in
the region of the catechol ring (position 9 in DHX) can be
rationalized as an interaction between the ligand aromatic ring
and residue Phe203. Our experimental data clearly show that
there are strict steric limits that extend outward from the bottom
of the catechol ring into the region below the 7,8-ethyl bridge in
DHX. Figure 4 illustrates structures of 2a,b and 3a,b in their
minimized binding poses, superimposed on the docked structure
of DHX (1).
’ RESULTS AND DISCUSSION
Competition binding assays using porcine striatal homogenate
preparations revealed that all four methyl-substituted dihydrex-
idine analogues had lower affinity at D1- and D2-like receptors
than the parent unsubstituted compound DHX (Table 1).
Comparison of the minimum energy models of DHX, 2a, 2b,
3a, and 3b in vacuum (not shown), and in a model receptorꢀ
membrane complex (Figure 3), revealed that all compounds
share a near-identical conformation of the tetracyclic ring
system. Thus, the dramatic loss of D1- and D2-like affinity
observed for the methyl substituted analogues can only be
attributed to steric effects caused by the presence of the
methyl substituents.
According to our model, although all ligands form the critical
salt bridge with Asp103, there are differences in the binding
orientation and receptorꢀligand interactions of the synthesized
compounds that might help to explain their differences in
binding affinity relative to DHX. In the case of the original
template, DHX, the interaction pattern is as follows. Aside from
the conserved salt bridge between the amine group and Asp103, a
complex hydrogen bonding network is observed around the
catechol moiety.11 The para-hydroxyl group serves as a donor in
a hydrogen bond with Ser202, which interacts with Thr108 in the
same fashion. The meta-hydroxyl group, on the other hand, has a
more intricate interaction pattern: besides a hydrogen bond
accepted by Ser198, it is involved in a water-mediated interaction
with Asn2926.55 (stable during the time course of the MD
The loss of affinity was most dramatic for the 7-methyl
substituted compounds 3a and 3b. The 7R-axial-methyl ligand
3a had no appreciable affinity at D1-like receptors (>10 μM Ki)
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Table 2. D1-Like Affinity of Test Compounds and Root-
Mean-Square Deviation (rmsd) of the Modeling Poses Re-
lative to DHX
ligand
Ki (nM)
rmsd
DHX
20 ( 2
0.000
0.732
1.146
1.798
1.923
8β-Meax-DHX, 2a
8R-Meeq-DHX, 2b
7R-Meax-DHX, 3a
7β-Meeq-DHX, 3b
270 ( 34
920 ( 46
>10000
6540 ( 1120
adjacent TM3, forcing a significant shift in the position of the
ligand. To avoid a steric clash between the methyl group and
Ser107, the molecule moves slightly toward the extracellular
space while keeping the salt bridge intact. This movement
happens in such a way that the interaction between the para-
hydroxyl group and Ser202 is replaced with hydrogen bonds
involving Ser198 and Ser199. Consequently, the catechol ring
is no longer in its optimal location within the binding pocket
(as exemplified by DHX), disrupting the aromatic interactions
in this region and resulting in a ligand with poor binding
properties.
One notable aspect of the biological activity of these analogues
is the fact that the D2 receptor was found to be much more
sensitive to these substitutions than the D1 receptor. Even
though the compounds displayed a range of affinities from
midnanomolar to micromolar at the latter, affinities at the former
were all in the micromolar range. It is likely that the larger residue
at position 3.36 (a cysteine instead of a serine) expands the
region of steric restriction at the bottom of the binding site,
therefore causing a more dramatic loss in affinity upon substitu-
tion at the 7 and 8 positions.
The differences in affinity for the synthesized compounds
also can be rationalized by analyzing the root-mean-square
deviation for the heavy carbon atoms of the poses of the
analogues with respect to DHX. That is, if one assumes that
DHX has a near optimal complementarity to the orthosteric
binding site, then the greater the deviation of its congeners
from this binding orientation, the greater the decrease in
binding energy one ought to expect. Qualitatively, the trends
are similar to what is observed with the binding affinities. In
the case of the 8-methyl substituted analogues, the axial
compound 2a deviates the least from the position of DHX in
the binding pocket, followed by the equatorial compound 2b
(Table 2). Both of these show a smaller rmsd than the 7-sub-
stituted compounds. The latter, however, show a slightly differ-
ent profile in rmsd than they do for affinity, with the lower affinity
axial compound 3a having a smaller deviation. This occurrence
might result from the fact that displacement of the 7-axial
compound from the position of DHX is more vertical, rather
than lateral, which more markedly pushes the catechol moiety
out of its binding region. In other words, the receptor may
have greater tolerance for pulling the catechol away from TM5
without changing the binding pattern, whereas a vertical
movement (toward the extracellular space) of the catechol
will more readily force the molecule into a different, less optimal
binding pose.
Figure 4. Binding poses for compounds DHX 1 (green), 2a (yellow),
2b (blue), 3b (red), and 3a (orange), in order of decreasing D1-like
binding affinity. One can gain the general impression from visual
inspection that the lower the affinity of the molecule, the more it
diverges from the docked pose of DHX.
simulations). We note that a recent report cites the residue at this
location in the D2 L receptor (His6.55) as a major determinant of
ligand-based signaling.15
The binding profile of the 8β-axially substituted compound
2a, in which the methyl group points away from TM3, is similar
to DHX with respect to the salt bridge and the catechol hydrogen
bonding network. The alkyl substituent appears only mildly
obtrusive, moderately disrupting an edgeꢀface aromatic inter-
action between Phe288 and Phe203 by acting as a wedge
between the phenyl rings. Considering that these residues are
part of a larger cluster of aromatic residues involved in the
binding of the catechol ring, it is possible that this disruption
could be the cause of the observed nearly 14-fold loss in affinity.
This value translates into about 1.6 kcal/mol of free energy of
binding, which is in the range of what could be expected from the
loss of an aromatic interaction.29
In the case of the equatorially substituted compounds, 2b and
3b, the methyl groups generally lie in the plane of the catechol
ring. The extra bulk of the substituent clashes with TM3 in the
vicinity of Ser107. Because of its location on the framework of the
ligand, a methyl at the 8-position displays slightly less overlap
with the aforementioned residue than when it is at the 7-position,
which might explain the observed differences in affinity. In both
cases, the steric clash causes a twist of the molecule around the
amineꢀcatechol axis so that the methyl group moves away from
TM3 and the pendant phenyl ring moves in the opposite
direction. This rotation increases the distance between the
meta-hydroxyl group and Asn292, making it too long for the
water-mediated hydrogen bond to occur. In the case of the 7β-
equatorially substituted compound, 3b, another distinct shift
can be observed, in this case a translational movement away
from TM5, further impairing its binding affinity due to an
increase in the distance between the catechol moiety and
serines 198, 199, and 202. Notably, for this compound, Ser202
does not form a direct interaction with the ligand and displays
a different rotameric state than in the prototypical DHX-
bound structure.11
From a quantitative point of view, the rmsd values correlated
extremely well to the free energies of binding of the correspond-
ing compounds (R2 = 0.9851). The previously discussed incon-
gruencies between the rmsd and the biological data for the
Finally, in the case of compound 3a, with a 7R-axially located
methyl group, the alkyl substituent projects directly toward the
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7-methylated compounds resulted in only slight deviation from
linearity.
stirred for 1 h, and the solvents removed by rotary evaporation. The
residue was redissolved in 15 mL of MeOH, and the solvents were
removed by rotary evaporation. This procedure was repeated twice, and
the remaining residue was put under high vacuum for 8 h to afford 110
1
mg (100%) of the desired hydrobromide salt; mp 199ꢀ201 °C. H
NMR (D2O): δ 7.53 (m, 4H, Ar2H), 6.96 (s, 1H, ArH), 6.93 (s, 1H,
ArH), 4.48 (bs, 2H, Ar2CH2N), 4.35 (d, 1H, Ar2CHCHN, J = 11.40
Hz), 3.30ꢀ3.10 (m, 2H, Ar2CHCHN and ArCH(CH3)CH2CHN),
2.20 (m, 1H, ArCH(CH3)CH2CHN), 2.00 (m, 1H, ArCH(CH3)-
CH2CHN), 1.31 (d, 3H, ArCH(CH3)CH2CHN, J = 6.88 Hz). ESIMS:
282 (M + H+, 100). Anal. (C18H20BrNO2 + 2H2O) (C, H, N).
(()-trans-10,11-Dihydroxy-8R-equatorial-methyl-5,6,6a,7,8,12b-
hexahydrobenzo[a] phenanthridine (2b). Compound 14b (75 mg,
0.18 mmol) was dissolved in 20 mL of CH2Cl2, and the solution was
cooled to ꢀ78 °C. Through a syringe, a 1.0 M solution of BBr3 in
CH2Cl2 (3.0 mL, 3.0 mmol) was added slowly. The solution was left to
warm to RT, and it was stirred overnight. The reaction was quenched by
addition of methanol (5.0 mL). The solvent was removed under vacuum,
and the product was recrystallized from 2-propanol. The crystals were
collected by filtration and dried under high vacuum to obtain 63 mg
(95%) of the desired hydrobromide salt 2b; mp 200ꢀ201 °C. 1H NMR
(300 MHz, CD3OD): δ 7.51 (d, 1H, Ar2H, J = 7.24 Hz), 7.39 (m, 3H,
Ar2H), 6.85 (s, 1H, ArH), 6.81 (s, 1H, ArH), 4.45 (bs, 2H, Ar2CH2N),
4.25 (d, 1H, Ar2CHCHN, J = 11.14 Hz), 3.13 (m, 1H, Ar2CHCHN),
3.00 (m, 1H, ArCH(CH3)CH2CHN), 2.40 (m, 1H, ArCH(CH3)CH2-
CHN), 1.75 (m, 1H, ArCH(CH3)CH2CHN), 1.38 (d, 3H, ArCH-
(CH3)CH2CHN, J = 6.96 Hz). ESIMS: 282 (M + H+, 100). Anal.
(C18H20BrNO2 + 2H2O) C, H, N.
’ CONCLUSION
In conclusion, we have shown that there is little steric
tolerance at the floor of the orthosteric binding site in the
dopamine D1 (and D2) receptor, beyond the ethyl bridge that
exists in DHX. There is clearly a region of restricted tolerance at
the bottom of the orthosteric binding site that is comprised of
Phe2035.47, Phe2886.51, and Ser1073.36, with the latter apparently
being responsible for most of the observed losses in affinity in the
present series. In the case of 8β-axially substituted compound 2a,
the absence of an unfavorable steric interaction between Ser107
and the ligand allowed the affinity to be only modestly impacted
compared to the other analogues. In general, as the overlap of the
substituent with the proposed location of TM3 increased, the
affinity of the ligand for the receptor decreased, with the effect
most evident for 7R-axially methylated compound 3a, whose
affinity was beyond the range of detection.
10,11-Dihydroxy-7R-axial-methyl-5,6,6a,7,8,12b-hexahydrobenzo-
[a]phenanthridine Hydrobromide (3a). Compound 20a (0.30 g, 0.867
mmol) was dissolved in 20 mL of CH2Cl2, and the solution was cooled
to ꢀ78 °C. A 1.0 M solution of BBr3 in CH2Cl2 (6.0 mL, 6.0 mmol) was
added slowly to the solution over 30 min. The solution was allowed to
warm up to room temperature and was stirred overnight. The reaction
was quenched by addition of dry methanol (10 mL). The solvent was
removed under vacuum, and the crude product was recrystallized from
2-propanol. Drying under high vacuum left 260 mg (95%) of the desired
1
hydrobromide salt as a yellowish solid; mp 272ꢀ274 °C. H NMR
(DMSO-d6): δ 9.13 (bs, 1H, NH), 8.70 (bs, 2H, OH), 7.40 (m, 4H,
Ar2H), 6.68 (s, 1H, ArH), 6.50 (s, 1H, ArH), 4.40 (bs, 2H, Ar2CH2N),
4.14 (d, 1H, Ar2CHCHN, J = 11.4 Hz), 3.48ꢀ3.35 (m, 1H, Ar2CH-
CHN), 3.10 (m, 1H, ArCH2CH(CH3)CHN), 2.92 (dd, 1H, ArCH2-
CH(CH3)CHN, J = 7.20 Hz, J0 = 16.8 Hz), 2.52 (m, 1H,
ArCH2CH(CH3)CHN), 1.09 (d, 3H, ArCH2CH(CH3)CHN, J =
’ EXPERIMENTAL SECTION
Chemistry. All reagents were commercially available and were used
without further purification unless otherwise indicated. Anhydrous THF
was obtained by distillation from benzophenone-sodium under nitrogen
immediately before use. Melting points were determined using a
ThomasꢀHoover apparatus and are uncorrected. 1H NMR spectra
were obtained with a Bruker ARX300 (300 MHz) instrument. Chemical
shifts are reported in δ values (ppm) relative to an internal reference of
TMS. Chemical ionization mass spectra (CIMS), using isobutene as the
carrier gas, were obtained with a Finnigan 4000 spectrometer. Elemental
analyses were performed by the Purdue University Microanalysis
Laboratory and are within (0.4% of the calculated values. Thin-layer
chromatography was performed using J.T. Baker flex silica gel IB2-F,
plastic-blacked sheets with fluorescent indicator, visualizing with UV
light at 254 nm. Column chromatography was carried out using Silica
Gel 60, 230ꢀ400 mesh (J.T. Baker). All reactions were carried out under
an inert atmosphere of argon unless otherwise indicated.
(()-trans-10,11-Dihydroxy-8β-axial-methyl-5,6,6a,7,8,12b-hexa-
hydrobenzo[a]phenanthridine (2a). The free base 12a (100 mg,
0.32 mmol) was dissolved in 20 mL of CH2Cl2, and the solution was
cooled to ꢀ78 °C. Through a syringe, a 1.0 M solution of BBr3 in
CH2Cl2 (3.0 mL, 3.0 mmol) was added slowly to the solution. The
reaction was left to warm to room temperature and was stirred overnight.
The reaction was then quenched by addition of methanol (15 mL),
6.30 Hz). ESIMS: 282 (M + H+, 100). Anal. (C18H20BrNO2
H2O) C, H, N.
+
(()-trans-10,11-Dihydroxy-7β-methyl-5,6,6a,7,8,12b-hexahydrobenzo-
[a]phenanthridine Hydrobromide (3b). The HCl salt 20b (50 mg,
0.144 mmol) was dissolved in water, and 5 mL of conc NaHCO3 were
added. This suspension was extracted into 10 mL of CH2Cl2, dried over
MgSO4, and filtered. This solution was cooled to ꢀ78 °C, and 0.6 mL
(0.6 mmol) of a 1 M solution of BBr3 in CH2Cl2 was added. This
solution was stirred overnight at RT. It was then quenched by addition of
dry methanol and the solvents removed by rotary evaporation. Another
5 mL of dry methanol were added to the residue, and the solution was
reduced under reduced pressure to leave a residue containing trace
methanol. Addition of EtOAc to this residue induced crystallization of
the product, which was collected by filtration to yield 38 mg (72.5%
yield) of 3b as a yellow powder. 1H NMR (DMSO-d6): δ 9.25 (br, 2H,
+NH2), 8.88 (s, 2H, 2ArOH), 7.46ꢀ7.36 (m, 4H, 4ArH), 6.69 (s, 1H,
ArH), 6.60 (s, 1H, ArH), 4.40 (s, 2H, CH2N), 4.18ꢀ4.14 (d, 1H,
ArCHAr, Jtrans = 11.1 Hz), 2.80 (dd, 1H, ArCHequat, Jgem = 15.9 Hz, Jvic
5.4 Hz), 2.59 (t, 1H, CHN, Jtrans = 11.1 Hz), 2.40 (dd, 1H, ArCHaxial
Jgem = 15.9 Hz, Jvic = 7.8 Hz), 2.15 (m, 1H, CCHC), 1.07 (d, 3H, CH3).
=
,
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Journal of Medicinal Chemistry
ARTICLE
ESIMS: 282 (M+, 100). High resolution ESIMS for C18H21NO2 (M+):
calcd 282.1494, found 282.1496.
3.50 mL, 25.0 mmol) was added. The mixture was cooled in an ice
bath. Benzoyl chloride (3.50 g, 3.0 mL, 25.0 mmol) in CH2Cl2 (3.0 mL)
was added dropwise to the cold solution. After complete addition, the
mixture was allowed to warm to RT and was left to stir overnight. The
mixture was the diluted in CH2Cl2, and the organic layer was washed
with 2 M HCl (2 ꢁ 15 mL), 1 M NaOH (2 ꢁ 10 mL), and brine (2 ꢁ
10 mL). The mixture was dried with MgSO4 and filtered, and the solvent
was evaporated. The product was then purified by column chromatog-
raphy on silica, using EtOAc:hexanes (10:90) as the eluent to yield a
yellowish oil, 6.29 g (67%). 1H NMR (CDCl3): δ 7.60 (d, 2H, Ar2H, J =
7.41 Hz), 7.43ꢀ7.22 (m, 8H, Ar2H and Ar3H), 6.58 (s, 1H, ArH), 6.38
(s, 1H, ArH), 6.02 (s, 1H, ArCHCN), 4.98 (d, 2H, Ar3CH2N, J = 4.30
Hz), 3.84 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 2.69 (m, 1H, ArCH-
(CH3)CH2CN), 2.26 (dd, 1H, ArCH(CH3)CH2CN, J = 6.80, J0 = 16.50
Hz), 1.92 (dd, 1H, ArCH(CH3)CH2CN, J = 6.80, J0 = 16.50 Hz), 0.89
(d, 3H, ArCH(CH3)CH2CN, J = 6.89 Hz). CIMS: 414 (M + H+, 100).
Anal. (C27H27NO3) C, H, N.
N-Benzyl-10,11-dimethoxy-8-methyl-6a,7,8,12b-tetrahydro-6H-
benzo[a]-phenanthridin-5-one (11a,b). A solution of 10 (1.0 g, 2.40
mmol) in dry THF (250 mL) was introduced into a 250 mL photo-
chemical reactor. The solution was stirred under argon and irradiated for
1 h with a 450 W Hanovia medium-pressure quartz mercury-vapor lamp
seated in a water-cooled quartz immersion well. The solution was
concentrated in vacuo, and the product mixture (50/50% cis/trans by
1H NMR) was purified by column chromatography on silica, using
EtOAc:hexanes (20:80) to yield the mixture of stereoisomers that
crystallized as white needles 0.50 g (50%).
N-Benzyl-10,11-dimethoxy-8β-axial-methyl-6a,7,8,12b-tetrahydro-
6H-benzo[a]-phenanthridin-5-one (11a). The mixture 11a,b (3 g) was
subjected to rotary chromatography eluting with EtOAc:hexanes (1:99)
as obtaining a trace amount of crystalline 11a, which was used to seed its
selective crystallization from the mixture 11a,b, obtaining 1.1 g of pure
11a as white crystals. 1H NMR (CDCl3): δ 8.21 (dd, 1H, Ar2H, J = 1.70
Hz, J0 = 7.20 Hz), 7.59 (d, 1H, Ar2H,J = 7.30 Hz), 7.52ꢀ7.40 (m, 2H,
Ar2H), 7.34ꢀ7.18 (m, 5H, Ar3H), 6.93 (s, 1H, ArH), 6.65 (s, 1H, ArH),
5.36 (d, 1H, Ar3CH2N, J = 16.0 Hz), 4.75 (d, 1H, Ar3CH2N, J = 16 Hz),
4.30 (d, 1H, Ar2CHCHN, J = 11.30 Hz), 4.00 (m, 1H, Ar2CHCHN),
3.89 (s, 6H, 2OCH3), 3.00 (m, 1H, ArCH(CH3)CH2CHN), 2.09 (m,
1H, ArCH(CH3)CH2CHN), 1.95 (m, 1H, ArCH(CH3)CH2CHN),
1.10 (d, 3H, ArCH(CH3)CH2CHN, J = 7.27 Hz); mp 215ꢀ217 °C.
CIMS: 414 (M + H+, 100). Anal. (C27H27NO3) C, H, N.
(E)-3-(4,5-Dimethoxy-2-methoxycarbonylmethyl-phenyl)-but-2-
enoic Acid Methyl Ester (7). 2-Iodo-4,5-dimethoxyphenylacetic acid
methyl ester 619 (10.0 g, 30.0 mmol) was dissolved in 50 mL of CH3CN
under an inert atmosphere. Into this solution was added Et3N (24.0 mL,
0.16 mol), methyl crotonate (26 mL, 0.230 mol), and Pd(PPh3)2Cl2
(0.60 g, 0.85 mmol). The reaction mixture was heated at reflux for 4 days
and then cooled to RT. The solvent was evaporated, and the residue was
dissolved in ether. The undissolved solids were removed by filtration,
and the filtrate was washed with 2 M HCl (2 ꢁ 100 mL) and H2O (2 ꢁ
100 mL). The organic layer was dried (MgSO4) and filtered, and the
solvent was evaporated. The crude mixture was purified by column
chromatography on silica, using ethyl acetate:hexanes (10:90) as eluent,
to yield the product as a colorless oil 4.95 g (54%). 1H NMR (CDCl3):
δ 6.79 (s, 1H, ArH), 6.63 (s, 1H, ArH), 5.78 (d, 1H, ArC(CH3)-
CHCO2CH3, J = 1.40 Hz), 3.89 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.58 (s, 2H, ArCH2CO2CH3),
2.45 (d, 3H, ArC(CH3)CHCO2CH3, J = 1.35 Hz). ESIMS: 331 (M +
Na+, 100). Anal. (C16H20O6) C, H, N.
3,(4,5-Dimethoxy-2-methoxycarbonylmethyl-phenyl)-butyric Acid
Methyl Ester (8). The olefin 7 (9.0 g, 29.2 mmol) was dissolved in
EtOH (200 mL) and shaken in a Parr hydrogenator at 60 psi over 1.0 g of
Ra-Ni until H2 uptake ceased. The suspension was filtered over a pad of
Celite, and the filtrate was evaporated. Bulb to bulb distillation
(140ꢀ145 °C, 0.2 Torr) gave the pure product as a colorless oil (8.9
g, quant). 1H NMR (CDCl3): δ 6.72 (s, 1H, ArH), 6.68 (s, 1H, ArH),
3.87 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.78 (s, 1H, ArCH2CO2CH3),
3.70 (s, 3H, OCH3), 3.63 (s, 1H, ArCH2CO2CH3), 3.61 (s, 3H, OCH3),
3.51ꢀ3.40 (m, 1H, ArCH(CH3)CH2CO2CH3), 2.57 (m, 2H, ArCH-
(CH3)CH2CO2CH3), 1.25 (d, 3H, ArCH(CH3) CH2CO2CH3, J = 6.83
Hz). ESIMS: 333 (M + Na+, 100). Anal. (C16H22O6) C, H, N.
6,7-Dimethoxy-4-methyl-3,4-dihydro-1H-naphthalen-2-one (9).
To a mixture of potassium tert-butoxide (3.90 g, 35.0 mmol) in dry Et2O
(150 mL), under inert atmosphere and protected from light, a solution
of 8 (10.5 g, 33.83 mmol) in Et2O (150 mL) was added dropwise
through a dropping funnel. The reaction was left to stir for 45 min at RT.
The solids were separated by filtration, washed on the filter with Et2O,
and dried under high vacuum and protected from light to afford a
quantitative yield of the potassium enolate as an off-white powder. This
potassium salt (10.5 g, 33.19 mmol) and LiCl (1.70 g, 40.0 mmol) were
dissolved in NMP (100 mL). Concentrated HCl (4.0 mL, 40.0 mmol)
was added, turning the color of the solution from dark-red to yellow. The
flask was then placed in an oil bath preheated to 125 °C and heated at
reflux overnight under and inert atmosphere. The mixture was allowed
to cool to RT (protected from light). Then, the mixture was diluted with
EtOAc (50 mL) and the organic layer was washed with H2O (3 ꢁ
15 mL) and brine (3 ꢁ 10 mL). The organic layer was dried (MgSO4)
and simultaneously decolorized with charcoal. After filtration over
Celite, the solvent was evaporated. Bulb to bulb distillation
(125ꢀ130 °C, 0.1 Torr) gave a yellowish oil (6.0 g, 82.1%). 1H NMR
(CDCl3): δ 6.78 (s, 1H, ArH), 6.62 (s, 1H, ArH), 3.91 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.55 (d, 2H, ArCH2CO, J = 3.52 Hz), 3.22 (m, 1H,
ArCH(CH3)CH2CO), 2.71 (dd, 1H, ArCH(CH3)CH2CO, J = 5.33 Hz,
J0 = 16.0 Hz), 2.37 (dd, 1H, ArCH(CH3)CH2CO, J = 6.50, J0 = 16.0 Hz),
1.30 (d, 3H, ArCH(CH3CH2)CO, J = 7.0 Hz). CIMS: 221 (M+ + H,
100). Anal. (C13H1603) C, H, N.
(()-trans-10,11-Dimethoxy-8β-axial-methyl-5,6,6a,7,8,12b-hexahy-
drobenzo[a]phenanthridine (12a). A solution of 11a (1.1 g, 2.66
mmol) in dry THF (100 mL) was cooled in an iceꢀsalt bath, and
6.0 mL of a 1 M solution of BH3 in THF was added through a syringe and
the reaction was heated at reflux overnight. Carefully, 10 mL of a 1 M
solution of HCl in MeOH was added to the solution and the reaction
was stirred at reflux for 1 h. The solvents were removed by rotary
evaporation, and the product was dissolved in 15 mL of dry EtOH and
rotary evaporated. The residue was dissolved in 15 mL of dry EtOH and
rotary evaporated three more times. The remaining material was then
crystallized from EtOH/Et2O to afford 0.9 g (78%) of yellowish crystals
of 6-benzyl-10,11-dimethoxy-8β-axial-methyl-5,6,6a,7,8,12b-hexahydro
benzo[a]phenanthridine hydrochloride; mp (free amine) 190ꢀ192 °C.
1H NMR (CDCl3, free amine): δ 7.48 (d, 1H, Ar2H, J = 7.50 Hz),
7.42ꢀ7.20 (m, 7H, Ar2H and Ar3H), 7.08 (d, 1H, Ar2H, J = 7.32 Hz),
6.92 (s, 1H, ArH), 6.87 (s, 1H, ArH), 4.14 (d, 1H, Ar2CH2N, J = 10.73
Hz), 4.00 (d, 1H, Ar2CH2N, J = 13.21 Hz), 3.95 (s, 3H, OCH3), 3.85 (m,
1H, Ar2CHCHN), 3.79 (s, 3H, OCH3), 3.50 (d, 1H, Ar3CH2N, J =
15.21 Hz), 3.30 (d, 1H, Ar3CH2N, J = 13.23 Hz), 3.10 (m, 1H,
Ar2CHCHN), 2.50 (m, 1H, ArCH(CH3)CH2CHN), 2.22 (m, 1H,
ArCH(CH3)CH2CHN), 1.82 (m, 1H, ArCH(CH3)CH2CHN), 1.45
(d, 3H, ArCH(CH3)CH2CHN, J = 6.84 Hz). EIMS: 400 (M + H+).
N-Benzyl-N-(6,7-dimethoxy-4-methyl-3,4-dihydro-naphthalen-2-yl)-
benzamide (10). In a flask equipped with a condenser and a Deanꢀ
Stark trap, a solution of 9 (5.0 g, 22.7 mmol) in toluene (80 mL) was
added benzylamine (2.60 g, 2.70 mL, 24.0 mmol). The solution was
heated at reflux overnight with continuous azeotropic removal of water.
The reaction was allowed to cool to RT, and the solvent was evaporated.
The residue was dissolved in CH2Cl2, and triethylamine (2.50 g,
Anal. (C27H29NO2
+
1/3H2O) C, H, N. To a solution of this
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hydrochloride salt (0.8 g, 1.83 mmol) in 95% ethanol (250 mL) was
added 100 mg of 10% PdꢀC catalyst and shaken at room temperature
under 60 psi of H2 for 6 h. After removal of the catalyst by filtration, the
solution was concentrated to dryness under vacuum. The remaining
residue was crystallized from EtOH/Et2O to afford 0.6 g (95.2%) of the
title compound product; mp (free amine) 131ꢀ133 °C. 1H NMR (300
MHz, CDCl3, free amine): δ 7.41 (d, 1H, Ar2H, J = 7.37 Hz), 7.02 (m,
2H, Ar2H), 7.10 (d, 1H, Ar2H, J = 7.12 Hz), 6.86 (s, 1H, ArH), 6.79 (s,
1H, ArH), 4.00 (m, 2H, Ar2CH2N), 3.84 (s, 3H, OCH3), 3.80 (d, 1H,
Ar2CHCHN, J = 11.0 Hz), 3.69 (s, 3H, OCH3), 3.00 (m, 1H,
Ar2CHCHN), 2.60 (m, 1H, ArCH(CH3)CH2CHN), 1.85ꢀ1.65 (m,
2H, ArCH(CH3)CH2CHN), 1.35 (d, 3H, ArCH(CH3)CH2CHN, J =
6.82 Hz). CIMS: 310 (M + H+).
(()-trans-10,11-Dimethoxy-8R-equatorial-methyl-6a,7,8,12b-tet-
rahydro-5H-benzo[a]phenanthridine-6-carboxylic Acid tert-Butyl Es-
ter (14b). A solution of 11a,b (1.1 g, 2.66 mmol) in dry THF (100 mL)
was cooled in an iceꢀsalt bath and 6.0 mL (6.0 mmol) of a 1 M solution
of BH3 in THF was added through a syringe. The reaction was heated at
reflux overnight. Slowly, 10 mL of a 1 M solution of HCl in MeOH was
added to the solution and the reaction was stirred at reflux for 1 h. The
solvents were removed by rotary evaporation, and the crude product was
dissolved in 15 mL of dry EtOH and rotary evaporated. The residue was
dissolved in 15 mL of dry EtOH and rotary evaporated three more times.
The diastereomeric product mixture 13a,b was crystallized from EtOH/
Et2O to afford 0.9 g (78%) that was used without further purification for
the next step.
A solution of the free base of 11a,b (3.0 g, 7.50 mmol), t-Boc
anhydride (3.0 g, 14.0 mmol), and Pd (10% on carbon) in dry THF
(10.0 mL) was hydrogenated under 1 atm H2 for 2 days. The suspension
was filtered through a pad of Celite and the filtrate concentrated to afford
2.70 g (88%) of product as a mixture of diastereomers. Silica-gel flash
column chromatography eluting with EtOAc:hexanes 20:80 allowed
separation of this mixture to afford 1.2 g (39%) of 14b and 1.5 g of 14a
(49%). For 14b: mp 133ꢀ134 °C. 1H NMR (CDCl3): δ 7.50 (d, 1H,
Ar2H, J = 7.60 Hz), 7.25 (m, 3H, Ar2H), 6.97 (s, 1H, ArH), 6.66 (s, 1H,
ArH), 5.05 (d, 1H, Ar2CHCHN, J = 14.64 Hz), 4.15 (d, 1H, Ar2CH2N,
J = 14.60 Hz), 4.02 (d, 1H, Ar2CH2N, J = 11.70 Hz), 3.85 (s, 3H,
OCH3), 3.78 (s, 3H, OCH3), 3.44 (m, 1H, Ar2CHCHN), 2.99 (m, 1H,
ArCH(CH3)CH2CHN), 2.50 (d, 1H, ArCH(CH3)CH2CHNJ), 1.84
(m, 1H, ArCH(CH3)CH2CHN), 1.39 (s, 9H, NBoc), 1.30 (d, 3H,
ArCH(CH3)CH2CHN, J = 10.84 Hz). CIMS: 410 (M + H+). Anal.
(C25H31NO4) C, H, N.
3,(4,5-Dimethoxy-2-methoxycarbonylmethyl-phenyl)-2-methyl-
acrylic Acid Methyl Ester (15). Under an inert, dry atmosphere, 2-iodo-
4,5-dimethoxyphenylacetic acid methyl ester 619 (10.0 g, 30.0 mmol)
was dissolved in 150 mL of CH3CN. To the solution was added Et3N
(12.0 mL, 89.0 mmol), methyl methacrylate (13.0 mL, 0.120 mol), and
Pd(PPh3)2Cl2 (0.42 g, 0.60 mmol). The reaction mixture was heated at
reflux overnight. The mixture was left to cool to RT, and the solvent was
removed under reduced pressure. The remaining material was dissolved
in 50 mL of CH2Cl2, and the solution was washed with H2O (2 ꢁ
50 mL), 2 M HCl (2 ꢁ 50 mL), and H2O (2 ꢁ 30 mL). The organic
layer was dried (MgSO4) and filtered, and the solvent was evaporated.
The crude product was purified by silica gel flash column chromatog-
raphy eluting with EtOAc:hexanes 90:10 to afford 7.0 g (76%) of a white
solid; mp 31ꢀ32 °C. 1H NMR (CDCl3): δ 7.70 (d, 1H, ArCH, J = 1.2
Hz), 6.82 (s, 1H, ArH), 6.75 (s, 1H, ArH), 3.87 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 3.80 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.55 (s, 2H,
ArCH2CO2CH3), 1.96 (d, 3H, ArCHC(CH3)CO2CH3, J = 1.2 Hz).
ESIMS: 331 (M + Na+, 100). Anal. (C16H20O6) C, H, N.
hydrogenator with H2 at 60 psi until H2 uptake had ceased. The suspension
was filtered over Celite, and the solvent was evaporated. Bulb to bulb
distillation (145ꢀ150 °C/0.2 Torr) gave, quantitatively, a colorless oil
that solidified; mp 33ꢀ35 °C. 1H NMR (CDCl3): δ 6.67 (s, 1H, ArH),
6.63 (s, 1H, ArH), 3.86 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.69 (s, 3H,
OCH3), 3.63 (s, 3H, OCH3), 3.59 (s, 2H, ArCH2CO2CH3), 3.00 (m,
1H, ArCH2CH(CH3)CO2CH3), 2.68 (m, 2H, ArCH2CH(CH3)CO2-
CH3 and ArCH2CH(CH3)CO2CH3), 1.19 (d, 3H, ArCH2CH(CH3)-
CO2CH3, J = 6.6 Hz. ESI-MS (m/z) 333 (M+ + Na+, 100). Anal.
(C16H22O6) C, H, N.
6,7-Dimethoxy-3-methyl-3,4-dihydro-1H-naphthalen-2-one (17).
To a suspension of potassium tert-butoxide (2.3 g, 20.0 mmol) in dry
Et2O (30 mL) under an inert atmosphere and shielded from light, a
solution of 16 (6.0 g, 19.33 mmol) in Et2O (50 mL) was added dropwise
through a dropping funnel. The reaction was stirred for 45 min at RT.
The solids were collected by filtration, washed on the filter with Et2O,
and dried under high vacuum, shielded from light, affording quantitative
yield of the potassium enolate as an off-white powder. This potassium
salt (2.0 g, 6.32 mmol) and LiCl (0.42 g, 10.0 mmol) were dissolved
under an inert atmosphere in NMP (20 mL). Concentrated HCl
(1.0 mL, 10.0 mmol) was added, turning the color of the solution from
dark-red to yellow. The flask was then placed in an oil bath preheated to
125 °C and heated at reflux overnight under argon. While shielded from
light, the mixture was allowed to cool to RT and it was diluted with ethyl
acetate (50 mL). This solution was washed with H2O (3 ꢁ 30 mL) and
brine (3 ꢁ 10 mL). It was then dried over MgSO4 and simultaneously
decolorized with charcoal. After filtration over Celite, the solvent was
evaporated to obtain 0.90 g of an off-white oil that rapidly crystallized
(64%); mp 85ꢀ87 °C. 1H NMR (CDCl3): δ 6.68 (s, 1H, ArH), 6.58 (s,
1H, ArH), 3.86 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.51 (d, 2H,
ArCH2CO, J = 2.40 Hz), 3.00 (dd, 1H, ArCH2CH(CH3)CO, J = 5.70
Hz, J0 = 15.3 Hz), 2.75 (dd, 1H, ArCH2CH(CH3)CO, J = 10.8 Hz, J0 =
15.3 Hz), 2.61ꢀ2.51 (m, 1H, ArCH2CH(CH3)CO), 1.17 (d, 3H,
ArCH2CH(CH3)CO, J = 6.6 Hz). CI-MS 221 (M+ + H+, 100). Anal.
(C13H16O3) C, H, N.
N-Benzyl-N-(6,7-dimethoxy-3-methyl-3,4-dihydro-naphthalen-2-yl)-
benzamide (18). In a flask equipped with a condenser and a Deanꢀ
Stark trap containing a solution of 17 (5.0 g, 22.7 mmol) in toluene
(80.0 mL), benzylamine (2.60 g, 2.70 mL, 24.0 mmol) was added. The
solution was heated at reflux overnight with continuous azeotropic
removal of water. The reaction was allowed to cool to RT, and the
solvent was evaporated. The remaining brown oil was dissolved in
CH2Cl2 (20 mL) and triethylamine (2.50 g, 3.50 mL, 25.0 mmol) and
cooled in an ice bath. Benzoyl chloride (3.50 g, 3.0 mL, 25.0 mmol) in
CH2Cl2 (3.0 mL) was added dropwise to the cold solution. After
complete addition, the mixture was allowed to warm to room tempera-
ture and was left to stir overnight. The mixture was the diluted in
CH2Cl2, and the organic layer was washed with 2 M HCl (2 ꢁ 15 mL), 1
M NaOH (2 ꢁ 10 mL), and brine (2 ꢁ 10 mL). The mixture was dried
with MgSO4 and filtered, and the solvent was evaporated. The residue
was dissolved in Et2O, and the solution was filtered over a Celite. After
evaporating the solvent, the product (6.2 g, 66%) was crystallized from
Et2O; mp: 118ꢀ120 °C. 1H NMR (CDCl3): δ 7.70 (d, 2H, Ar2H, J = 7.8
Hz), 7.45ꢀ7.27 (m, 8H, Ar2H and Ar3H), 6.51 (s, 1H, ArH), 6.41 (s,
1H, ArH), 6.13 (s, 1H, ArCHCN), 5.31 (d, 1H, Ar3CH2N, J = 15.0 Hz),
4.71 (d, 1H, Ar3CH2N, J = 15.0 Hz), 3.81 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 2.52 (dd, 1H, ArCH2CH(CH3)CHN, J = 6.4, J0 = 15.1 Hz),
2.25 ꢀ 2.11 (m, 2H, ArCH2CH(CH3)CN and ArCH2CH(CH3)CN),
0.75 (d, 3H, ArCH2CH(CH3)CN, J = 6.6 Hz). ESIMS: 414 (M + H+,
100). Anal. (C27H27NO3) C, H, N.
3,(4,5-Dimethoxy-2-methoxycarbonylmethyl-phenyl)-2-methyl-
propionic Acid Methyl Ester (16). The olefin 15 (9.0 g, 29.2 mmol) was
dissolved in hot EtOH (200 mL), and to this solution was added 10%
palladium on carbon (1.0 g). The reaction mixture was shaken in a Parr
(()-trans-N-Benzyl-10,11-dimethoxy-7R-methyl-6a,7,8,12b-tetra-
hydro-6H-benzo[a]phenanthridin-5-one (19a). A solution of 18 (1.0 g,
2.40 mmol) in dry THF (250 mL) was placed into a 250 mL photo-
chemical reactor. The solution was stirred under argon and irradiated for
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Journal of Medicinal Chemistry
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1 h with a 450 W Hanovia medium-pressure quartz mercury-vapor lamp
seated in a water-cooled quartz immersion well. The solution was
concentrated in vacuo and crystallized from Et2O to provide yellowish
crystals (600 mg, 60%); mp 214ꢀ216 °C. 1H NMR (CDCl3): δ 8.20 (dd,
1H, Ar2H, J = 1.60, J0 = 7.36 Hz), 7.51 (d, 1H, Ar2H, J = 7.46 Hz), 7.45 (m,
2H, Ar2H), 7.30ꢀ7.10 (m, 5H, Ar3H), 6.95 (s, 1H, ArH), 6.60 (s, 1H,
ArH), 5.59 (d, 1H, Ar3CH2N, J = 16.0 Hz), 4.30 (d, 2H, Ar3CH2N and
Ar2CHCHN, J = 16.0 Hz and J = 12.8 Hz), 3.90 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 3.76 (dd, 1H, ArCH2CH(CH3)CHN, J = 3.1, J0 = 15.1 Hz),
2.81 (dd, 1H, ArCH2CH(CH3)CHN, J = 3.70, J0 = 16 Hz), 2.55ꢀ2.43
(m, 2H, ArCH2CH(CH3)CHN and ArCH2CH(CH3)CHN), 1.00 (d,
3H, ArCH2CH(CH3)CHN, J = 6.6 Hz). ESIMS: 436 (M + Na+, 100).
Anal. (C27H27NO3) C, H, N.
ArCHequat, Jgem = 16.2 Hz, Jvic = 5.1 Hz), 2.54 (t, 1H, CHN, Jtrans = 11.1
Hz), 2.33 (dd, 1H, ArCHaxial, Jgem = 16.2 Hz, Jvic = 9.6 Hz), 2.10 (m, 1H,
CCHC), 1.07 (d, 3H, CH3). ESIMS: 310 (M+, 100). High resolution
ESIMS for C20H24ClNO2 (M+): calcd 310.4101; found 309.4103.
3-(3,4-Dimethoxyphenyl)-2-methylacrylic Acid (22). The procedure
of Gensler and Berman24 was used to obtain a 57% yield; mp 141 °C. 1H
NMR (CDCl3): δ 7.78 (s, 1H, ArCH), 7.12ꢀ7.08 (dd, 1H, ArH, Jvic
=
8.4 Hz, Jw = 2.1 Hz), 7.00 (d, 1H, ArH, Jw = 2.1 Hz), 6.94ꢀ6.91 (d, 1H,
ArH, Jvic = 8.4 Hz), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 2.19 (s,
3H, CH3). Anal. (C12H14O4) C, H.
3-(3,4-Dimethoxyphenyl)-2-methylpropanoic Acid (23). The reduc-
tion procedure of Schrecker25 was employed. In a three-neck flask, 4 g
(18.02 mmol) of methyl cinnamic acid 22 were dissolved in a solution of
13.3 g of NaOH in 120 mL of water. This solution was heated to 90 °C.
With vigorous mechanical stirring and providing a safe escape route for
the evolving hydrogen by means of a plastic hose directly attached to the
flask neck and to the fume hood output, a total of 12 g of Ni/Al alloy was
added portionwise over 20 min. This suspension was stirred for 1.5 h and
then cooled to RT. The crude aqueous suspension was filtered, adding
water and never allowing the filtered solids to dry. The filtrate was cooled
in an ice bath and acidified to pH 1 with conc HCl. The mixture was then
extracted several times with Et2O. The pooled ethereal extracts were washed
once with water and then dried over MgSO4. The mixture was then filtered
and freed of solvent by rotary evaporation and then placed under high
vacuum overnight. The remaining oil crystallized on standing to give 4.00 g
of a white solid (99.1% yield). EIMS: 224 (M+, 100). Anal. (C12H16O4) C, H.
3-(3,4-Dimethoxyphenyl)-2-methylpropan-1-ol (24). The methyl-
propanoic acid 23 (3.95 g, 17.64 mmol) was dissolved in 30 mL of dry
THF, and 30 mL (30 mmol) of a 1 M solution of BH3 in THF was added.
The mixture was stirred at RT overnight and was then quenched by
addition of water. The mixture was then reduced to one-third of its
original volume by rotary evaporation and was extracted three times with
EtOAc. The extracts were washed once with water and then once with
brine and dried over MgSO4. This mixture was filtered and freed of
solvent to yield 3.52 g (95%) of a clear oil. 1H NMR (CDCl3): δ 6.76 (m,
1H, ArH), 6.68 (m, 2H, 2ArH), 3.85 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 3.52ꢀ3.45 (m, 2H, HOCH2), 2.67 (dd, 1H, ArCH), 2.35 (dd,
1H, ArCH), 1.90 (m, 1H, CCHC), 0.89 (d, 3H, CH3). CIMS: 193 (M +
H+ ꢀ H2O, 100); 211 (M + H+, 51). Anal. (C12H18O3) C, H.
(()-trans-10,11-Dimethoxy-7R-methyl-5,6,6a,7,8,12b-hexahydrobenzo-
[a]phenanthridine (20a). A solution of 19a (2.0 g, 4.80 mmol) in dry
THF (100 mL) was cooled in an iceꢀsalt bath, and 10.0 mL of a 1 M
solution of BH3 in THF was added via a syringe. The reaction was heated
at reflux overnight. Carefully, 15 mL of a 1 M solution of HCl in MeOH
was added to the solution and the reaction was stirred at reflux for 1 h.
The solvents were removed under reduced pressure, and the remaining
solids were again dissolved in 20 mL of dry EtOH. The solvents were
removed under reduced pressure. The remaining material was crystallized
from EtOH/Et2O to afford 1.72 g (85%) of (()-trans-N-benzyl-10,11-
dimethoxy-7R-methyl-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine
as colorless crystals; mp: 208ꢀ211 °C. 1H NMR (300 MHz, CDCl3): δ
7.68 (dd, 2H, Ar2H, J = 1.80, J = 6.90 Hz), 7.50 (m, 2H, Ar2H),
7.43ꢀ7.35 (m, 5H, Ar3H), 6.88 (s, 1H, ArH), 6.55 (s, 1H, ArH), 4.83
(dd, 1H, Ar2CH2N, J = 4.51, J0 = 14.7 Hz), 4.70 (dd, 1H, Ar2CH2N, J =
2.75, J0 = 12.30 Hz), 4.36 (d, 1H, Ar3CH2N, J = 14.87 Hz), 4.28 (d, 1H,
Ar3CH2N, J = 11.76 Hz),), 3.88 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
3.50 (m, 1H, Ar2CHCHN), 2.87 (m, 1H, Ar2CHCHN), 2.55 (dd, 1H,
ArCH2CH(CH3)CHN, J = 4.30, J0 = 16.20 Hz), 2.50 (dd, 1H, ArCH2-
CH(CH3)CHN, J = 2.0 Hz, J0 = 17.0 Hz), 1.72 (m, 1H, ArCH2CH-
(CH3)CHN), 1.30 (d, 3H, ArCH2CH(CH3)CHN, J = 6.79 Hz. CIMS:
400.20 (M + H+, 100). To a solution of this hydrochloride salt (1.0 g,
2.30 mmol) in 95% ethanol (250 mL) was added 0.1 g of 10% Pd/C, and
the solution was shaken at room temperature under 60 psi of H2 for 8 h.
After removal of the catalyst by filtration over Celite, the solution was
concentrated to dryness under vacuum and the residue crystallized from
EtOH/Et2O to afford 0.86 mg (89.5%) of crystalline salt; mp 149ꢀ
152 °C. 1H NMR (DMSO-d6): δ 7.46ꢀ7.31 (m, 4H, Ar2H), 6.86 (s, 1H,
ArH), 6.84 (s, 1H, ArH), 4.41 (s, 2H, Ar2CH2N), 4.24 (d, 1H,
Ar2CHCHN), 3.77 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.36 (bs,
1H, Ar2CHCHN), 3.15 (dd, 1H, ArCH2CH(CH3)CHN, J = 4.57 Hz,
J0 = 11.35 Hz), 3.03 (dd, 1H, ArCH2CH(CH3)CHN, J = 4.90 Hz, J0 =
15.48 Hz), 2.56 (m, 1H, ArCH2CH(CH3)CHN), 1.10 (d, 3H,
ArCH2CH(CH3)CHN, J = 6.62 Hz). CIMS: 310 (M + H+, 100). Anal.
(C20H23NO2) C, H, N).
4-(3-Bromo-2-methylpropyl)-1,2-dimethoxybenzene (25). The al-
cohol 24 (13.44 g, 63.92 mmol) was dissolved in 150 mL of CH2Cl2,
along with 23.31 g (70.29 mmol) of CBr4. This solution was cooled to
0 °C, and 18.44 g (70.30 mmol) of PPh3 were added slowly. The
solution was stirred for 2 h, and the solvents were removed under
reduced pressure. A quick elution through a short silica gel column using
1:1 EtOAc:hexanes gave a solution that was PPh3O-free and which was
concentrated by rotary evaporation. Distillation of the remaining crude
oil at 0.1 Torr and 50 °C accomplished removal of CBr4 and CHBr3 to
leave a residue of 15.05 g (86%) of pure 25 as a pale-yellow oil. 1H NMR
(CDCl3): δ 6.78 (m, 1H, ArH), 6.70 (m, 2H, 2ArH), 3.86 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 3.39ꢀ3.26 (m, 2H, CH2Br), 2.70ꢀ2.45
(2dd, 2H, ArCH2), 2.03 (m, 1H, CCHC), 1.02 (d, 3H, CH3). EIMS: 272
(M+, 100). Anal. (C12H17BrO2) C, H.
(()-trans-10,11-Dimethoxy-7β-methyl-5,6,6a,7,8,12b-hexahydrobenzo-
[a]phenanthridine Hydrochloride (20b). The 7β-methyl lactam 32b
(70 mg, 0.216 mmol) was dissolved in 10 mL of dry THF, and 1 mL
(1 mmol) of a 1 M solution of BH3 in THF was added. This solution was
stirred at reflux for 14 h, and water was added to quench the reaction.
The mixture was extracted three times with 5 mL of CH2Cl2, and the
organic solvents were pooled and washed once with water. The solution
was dried over MgSO4, filtered, and the solvent removed to leave a clear
residue. This residue was treated with 5 mL of a 2 M solution of HCl in
EtOH and heated at reflux for 10 min. EtOAc was added to this solution
and, after cooling to RT overnight, produced a precipitate, which was
collected by filtration. A second crystallization gave a total of 60 mg
(80% yield) of the HCl salt 20b as a white powder; mp 246ꢀ252 °C dec.
1H NMR (D2O): δ 7.31ꢀ7.18 (m, 4H, 4ArH), 6.86 (s, 1H, ArH), 6.76
1,2-Dimethoxy-4-(2-methyl-3-nitropropyl)-benzene (26). The bro-
mide 25 (1 g, 3.66 mmol) was dissolved in 25 mL of DMF and cooled to
0 °C. Into this flask, 328 mg (4.76 mmol) of NaNO2 were introduced
and the solution was stirred at RT for 6.5 h and then poured onto ice.
Water (100 mL) was added to the reaction mixture, which was extracted
three times with CH2Cl2 (15 mL). The organic layer was then washed
four times with 15 mL of water, dried over MgSO4, filtered, and the
solvents were removed by rotary evaporation. Flash column chroma-
tography using silica gel eluting with 2:1 hexanes:EtOAc provided 354
mg (40.4%) of the nitroalkane 26 as a yellowish oil and 482 mg of
(s, 1H, ArH), 4.31 (s, 2H, CH2N), 4.16ꢀ4.12 (d, 1H, ArCHAr, Jtrans
=
1
11.1 Hz), 3.67 (s, 3H, OCH3), 3.63 (s, 3H, OCH3), 2.80ꢀ2.73 (dd, 1H,
unreacted starting material. H NMR (CDCl3): δ 6.78 (d, 1H, ArH),
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6.68 (m, 2H, 2ArH), 4.32ꢀ4.16 (m, 2H, CH2NO2), 3.86 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 2.61ꢀ2.53 (m, 3H, ArH, CCHC), 1.01
(d, 3H, CH3). EIMS: 239 (M+, 100). Anal. (C12H17NO4) C, H, N.
2-(4,5-Dimethoxy-2-(2-methyl-3-nitropropyl)benzoyl)benzoic Acid
(27). Nitroalkane 26 (7.93 g, 33.16 mmol) was dissolved in 150 mL of
CH2Cl2 and cooled to 0 °C. Into this solution, 7.6 mL (66.31 mmol) of
SnCl4 were added, and then 7.2 mL (49.73 mmol) of phthaloyl
dichloride were added dropwise. The reaction was stirred at RT for 10
h and was poured onto ice. The layers were separated, and the organic
later was washed twice with 30 mL of a 2 M HCl solution. The solvent
was then extracted four times with 75 mL of conc NaHCO3. The
combined basic aqueous extracts were washed once with 30 mL of Et2O,
cooled in an ice bath, and made strongly acidic with conc HCl. This
acidic mixture was extracted three times with CH2Cl2. The combined
extracts were dried over MgSO4, filtered, and the solvents removed to
yield 6.75 g (52.5%) of a yellow residue. An analytical sample could be
obtained by crystallization from methanol; mp 160ꢀ161 °C. 1H NMR
(CDCl3): δ 8.03 (dd, 1H, ArH), 7.65 (dt, 1H, ArH), 7.55 (dt, 1H, ArH),
7.38 (dd, 1H, ArH), 6.78 (s, 1H, ArH), 6.69 (s, 1H, ArH), 4.38ꢀ4.32 (m,
2H, CH2NO2), 3.93 (s, 3H, OCH3), 3.58 (s, 3H, OCH3), 3.07 (m, 2H,
ArCH2), 2.78 (m, 1H, CCHC), 1.11 (d, 3H, CH3). EIMS: 272 (M+,
100). Anal. (C20H21NO7) C, H, N.
trans-1-(2-Carboxyphenyl)-2-nitro-6,7-dimethoxy-3R-methyl-1,2,3,4-
tetrahydronaphthalene (32a). The diastereomeric mixture 30a,b (636
mg, 1.72 mmol) was dissolved in 20 mL of CH2Cl2. Methanol (0.08 mL,
1.88 mmol), DCC (388 mg, 1.88 mmol), and DMAP (48 mg, 0.17
mmol) were then added. A precipitate began to form almost immedi-
ately, and the mixture was stirred for 3 h. The solvents were then
removed under reduced pressure, the residue was dissolved in 10 mL of
Et2O, and the suspension was filtered to remove DCU. This solution was
concentrated to dryness to afford the crude diastereomeric ester mixture,
which was dissolved in 40 mL of acetic acid, 2 g of zinc powder was
added, and the reaction was stirred for 4 h. This suspension was filtered,
the zinc and zinc salts washed on the filter with THF, and the filtrate
concentrated under reduced pressure. The residue was made strongly
basic with 20 mL of a 2 M solution of NaOH, and this mixture was
extracted twice with 15 mL of CH2Cl2. The organic extracts were pooled
and washed with water, dried over MgSO4, filtered, and the solvents
removed under reduced pressure. The residue was dissolved in 20 mL of
MeOH and heated at reflux overnight, producing a white precipitate.
The flask was then cooled, and the white solid collected by filtration.
This solid was revealed by TLC to be a diastereomeric mixture. Retrieval
of the remaining product from the mother liquor gave a total yield of 458
mg (82.7%). Isolation of the major product by chromatography gave 381
mg of 32a as a white powder; mp 239 °C. 1H NMR (CDCl3): δ 8.10 (d,
1H, ArH), 7.68 (d, 1H, ArH), 7.53 (t, 1H, ArH), 7.40 (t, 1H, ArH), 6.97
(s, 1H, ArH), 6.73 (br, 1H, CONH), 6.67 (s, 1H, ArH), 4.19 (d, 1H,
ArCHAr, Jtrans = 12.3 Hz), 3.91 (s, 3H, OCH3), 3.90 (s, 3H, OCH3),
3.82 (dd, 1H, NCH, Jtrans = 12.3 Hz, Jvic < 3 Hz), 3.10 (dd, 1H, ArCHeq,
1-(2-Carboxyphenyl)-2-nitro-6,7-dimethoxy-3-methyl-3,4-dihydro-
naphthalene (29). To a dry flask containing 4.12 g (10.63 mmol) of
benzoic acid 27 was added 30 mL of SOCl2, and the reaction was stirred
overnight. Benzene was added to the solution, and the acid chloride was
freed of excess SOCl2 and benzene by distillation. The residue was
dissolved in dry MeOH, whereupon the pure product immediately
crystallized. The crystals were collected by filtration and air-dried to
yield 3.612 g (84.7%) of the ester 28 as a white powder; mp 102 °C.
ESIMS: 424 (M + Na+, 100). Anal. (C21H23NO7) C, H, N. This ester
(3.60 g, 9.75 mmol) was dissolved in a warm mixture of 150 mL of THF
and 150 mL of MeOH. DBU (2.4 mL, 19.491 mmol) was added, and the
reaction was stirred overnight at RT. The mixture was then reduced to
one-fourth of its volume under reduced pressure, diluted with 200 mL of
water, and washed twice with 20 mL of Et2O. The aqueous solution was
acidified to pH 6 with conc HCl and extracted with CH2Cl2 (3 ꢁ
20 mL). The organic extracts were washed once with 2 M HCl, then
once with brine, dried over Na2SO4, filtered, and the solvents were
removed to leave 3.21 g (96.9%) of a bright-yellow solid; mp
J
gem = 15.9 Hz, Jvic < 3 Hz), 2.58 (d, 1H, ArCHaxial, Jgem = 15.9 Hz), 2.34
(m, 1H, CCHC), 1.04 (d, 3H, CH3). Anal. (C20H21NO3) C, H, N.
(()-trans-10,11-Dimethoxy-7β-methyl-6,6a,7,8,12b-tetrahydro-6H-
benzo[a]phenanthridin-5-one (32b). Chromatographic separation of
the crude product described above also gave 76 mg of 32b as a white
powder; mp 255ꢀ257 °C. 1H NMR (CDCl3): δ 8.10 (dd, 1H, ArH),
7.64 (d, 1H, ArH), 7.50 (dt, 1H, ArH), 7.40 (t, 1H, ArH), 6.96 (s, 1H,
ArH), 6.72 (br, 1H, CONH), 6.67 (s, 1H, ArH), 4.28 (d, 1H, ArCHAr,
Jtrans = 11.4 Hz), 3.90 (s, 6H, 2OCH3), 3.33 (t, 1H, NCH, Jtrans = 11.4 Hz),
2.75 (dd, 1H, ArCHequat, Jgem = 16.2 Hz, Jvic = 4.2 Hz), 2.58 (dd, ArCHaxial
,
Jgem = 16.2 Hz, Jvic = 11.7 Hz), 2.07 (m, 1H, CCHC), 1.19 (d, 3H, CH3, J =
6.3 Hz). CIMS: 324 (MH+, 100). Anal. (C20H21NO3) C, H, N.
Pharmacology. Materials. Porcine striatal tissue was obtained
from the Purdue Butcher Block and prepared as previously described.7
[3H]Spiperone (79 Ci/mmol) and [3H]SCH-23390 (65 Ci/mmol)
were purchased from Amersham Biosciences (Piscataway, NJ). Unless
otherwise stated, other reagents were purchased from Sigma-Aldrich (St.
Louis, MO).
1
187ꢀ192 °C. H NMR (CDCl3): δ 8.18 (d, 1H, ArH), 7.63 (dt, 1H,
ArH), 7.50 (dt, 1H, ArH), 7.13 (d, 1H, ArH), 6.74 (s, 1H, ArH), 5.94 (s,
1H, ArH), 3.90 (s, 3H, CH3O), 3.47 (s, 3H, CH3O), 3.36ꢀ3.29 (m, 2H,
ArCH2), 2.66 (d, 1H, CCHC), 1.26 (d, 3H, CH3). ESIMS: 392 (M +
Na+, 100). Anal. (C20H19N06) C, H, N.
Competition Binding Experiments. Radioligand binding assays were
performed as described.7 Briefly, porcine striatal membrane pellets were
resuspended in receptor binding buffer (50 mM Hepes, 4 mM MgCl2,
pH 7.4). As determined with receptor isotherms, Kd for D1-like and D2-
like receptor sites were 0.44 nM ([3H]SCH-23390) and 0.075 nM
([3H]spiperone), respectively. Drug dilutions were made in receptor
binding buffer and added to assay tubes containing 75 μg of protein and
either 1 nM [3H]SCH-23390 or 0.15 nM [3H]spiperone in a total
volume of 500 μL. Binding experiments were incubated for 30 min at
37 °C and were terminated by rapid filtration onto FB glass fiber plates
with ice-cold wash buffer (10 mM TRIS, 0.9% NaCl) using a cell
harvester (FilterMate, Packard). Radioactivity was determined using a
Packard TopCount scintillation counter. Nonspecific binding was
defined with 5 μM butaclamol. D2-like receptor binding assays were
performed in the presence of 50 nM ketanserin to mask 5-HT2A sites.
Data Analysis. The Prism software (GraphPad Software, San Diego,
CA) was used to generate competition binding curves. Nonspecific
binding values were used to set the bottom of the curves. Apparent Ki
values were calculated using the ChengꢀPrusoff equation.
(()-trans-1-(2-Carboxyphenyl)-2-nitro-6,7-dimethoxy-3-methyl-
3,4-dihydronaphthalene (30a,b). The nitroalkene 29 (3.4 g, 9.21
mmol) was dissolved in 450 mL of boiling iPrOH and stirred at reflux.
Every 8 h, over a period of 48 h, 600 mg of NaBH4 were added (for a total
of 3.6 g, 95.24 mmol). The milky solution was then reduced to one-third
of its volume and quenched by addition of a concentrated solution of
urea in aqueous 1% CH3COOH. This mixture was extracted with
CH2Cl2 (15 mL ꢁ 3) and then washed with water (5 mL ꢁ 3). The
organic layer was dried over MgSO4, filtered, and reduced to dryness to
yield a residue that upon dissolution in EtOAc formed a white crystalline
solid. NMR spectroscopy revealed this solid to be a mixture of the trans
diastereomers 30a and 30b. Retrieval of the remaining unresolvable
diastereomers from the crude mother liquor by chromatography yielded
a total of 738 mg (21%), which was used as a mixture in the next step; mp
236ꢀ240 °C. 1H NMR (CDCl3): δ 7.96 (bd, 1H, ArH), 7.29ꢀ7.21 (m,
2H, ArH), 6.69 (m, 1H, ArH), 6.56 (s, 1H, ArH), 6.24 (bs, 1H, ArH),
5.63 (br, 1H, CH2NO2), 4.82 (br, 1H, ArCHAr), 3.80 (s, 3H, OCH3),
3.59 (s, 3H, OCH3), 2.82 (m, 1H, ArCH), 2.43 (m, 1H, ArCH), 2.33 (m,
1H, CCHC), 1.01 (d, 3H, CH3). Anal. (C20H21NO6) C, H, N.
5519
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Computational Chemistry. General Methods. The complete
details of the in silico activation of the β2-adrenergic receptor are
provided in Bonner et al. 2011.11 All renderings were performed in
PyMol,30 and trajectories were viewed using Visual Molecular Dynamics
(VMD).37 Simulations were performed based on the crystal structure of
the β2-adrenergic receptor (RCSB pdb ID 2rh1).28
’ REFERENCES
(1) Taylor, J. R.; Lawrence, M. S.; Redmond, D. E., Jr.; Elsworth,
J. D.; Roth, R. H.; Nichols, D. E.; Mailman, R. B. Dihydrexidine, a full
dopamine D1 agonist, reduces MPTP-induced parkinsonism in mon-
keys. Eur. J. Pharmacol. 1991, 199, 389–391.
(2) Goldman-Rakic, P. S.; Castner, S. A.; Svensson, T. H.; Siever,
L. J.; Williams, G. V. Targeting the dopamine D(1) receptor in
schizophrenia: insights for cognitive dysfunction. Psychopharmacology
(Berlin, Ger.) 2004, 3–16.
(3) Williams, G. V.; Goldman-Rakic, P. S. Modulation of memory
fields by dopamine D1 receptors in prefrontal cortex. Nature 1995,
376, 572–575.
(4) Mu, Q.; Johnson, K.; Morgan, P. S.; Grenesko, E. L.; Molnar,
C. E.; Anderson, B.; Nahas, Z.; Kozel, F. A.; Kose, S.; Knable, M.;
Fernandes, P.; Nichols, D. E.; Mailman, R. B.; George, M. S. A single 20
mg dose of the full D1 dopamine agonist dihydrexidine (DAR-0100)
increases prefrontal perfusion in schizophrenia. Schizophrenia Res. 2007,
94, 332–341.
(5) Schultz, W. Getting formal with dopamine and reward. Neuron
2002, 36, 241–263.
(6) Knoerzer, T. A.; Watts, V. J.; Nichols, D. E.; Mailman, R. B.
Synthesis and biological evaluation of a series of substituted benzo-
[a]phenanthridines as agonists at D1 and D2 dopamine receptors.
J. Med. Chem. 1995, 38, 3062–3070.
(7) Cueva, J. P.; Giorgioni, G.; Grubbs, R. A.; Chemel, B.; Watts,
V. J.; Nichols, D. E. trans-2,3-Dihydroxy-6a,7,8,12b-tetrahydro-6H-
chromeno[3,4-c]isoquinoline: Synthesis, Resolution, and Preliminary
Pharmacological Characterization of a New Dopamine D1 Receptor Full
Agonist. J. Med. Chem. 2006, 49, 6848–6857.
(8) Grubbs, R. A.; Lewis, M. M.; Owens-Vance, C.; Gay, E. A.;
Jassen, A. K.; Mailman, R. B.; Nichols, D. E. 8,9-Dihydroxy-1,2,3,11b-
tetrahydrochromeno[4,3,2,-de]isoquinoline (dinoxyline), a high affinity
and potent agonist at all dopamine receptor isoforms. Bioorg. Med. Chem.
2004, 12, 1403–1412.
(9) Jassen, A. K., Lewis, M. M., Miller, D. W., Leonard, S., Nichols,
R. A., Nichols, D. E., Tropsha, A., Suzuki, K., Mailman, R. B. Molecular
and pharmacological characterization of D1-like dopamine receptors
reveals structural requirements for critical amino acid residues. Soc.
Neurosci. Abstr. 2000, Abstract 532.9.
(10) Strader, C. D.; Candelore, M. R.; Hill, W. S.; Sigal, I. S.; Dixon,
R. A. Identification of two serine residues involved in agonist activation
of the beta-adrenergic receptor. J. Biol. Chem. 1989, 264, 13572–13578.
(11) Bonner, L. A.; Laban, U.; Chemel, B. R.; Juncosa, J. I.; Lill,
M. A.; Watts, V. J.; Nichols, D. E. Mapping the catechol binding site in
dopamine D1 receptors: synthesis and evaluation of two parallel series of
bicyclic dopamine analogues. ChemMedChem 2011, 6, 1024–1040.
(12) Kong, M. M. C.; Fan, T.; Varghese, G.; O’Dowd, B. F.; George,
S. R. Agonist-induced cell surface trafficking of an intracellularly
sequestered D1 dopamine receptor homo-oligomer. Mol. Pharmacol.
2006, 70, 78–89.
Membrane Simulations. Membrane simulations were performed
using GROMACS 4.0 (GROningen MAchine for Chemical Simulations),32
using the AMBER03 force field port33 (from http://ffamber.cnsm.csulb.
edu/) with optimized parameters for united-atom POPC (palmitoyl
oleoyl phosphatidylcholine) lipids (from http://www.bioinf.uni-sb.de/
RB/).34 Ligand parameters were created with the AmberTools 1.4
package35,36 based on an ab initio HF/6-31G* optimization37 performed
on Gaussian0338 and subsequent RESP (restrained electrostatic potential)
fitting.39,40 Details of the in silico activation process, using Restrained
Molecular Dynamics for 368 ns, have been previously described.11
Homology Models. Homology models of the D1 receptor were
created with Modeler 9 version 2.41,42 Sequence alignments were made
manually, utilizing key conserved residues as references. Protein se-
quences were obtained from the Protein Information Resource.43,44 The
model with the lowest internal score out of 1000 was inspected, and
extracellular loop 3 (EL3) was refined with Modeler, taking the best out
of 1000 structures ranked by internal score. Necessary torsional mod-
ifications were carried out in order to preserve relevant motifs. The
molecule was embedded in the same membrane system as the original
template. The receptor was equilibrated for 40 ns with the agonist
doxanthrine (DOX) in the binding site, as previously detailed.11
Docking. Flexible Docking of the (6aR,12bS) enantiomers of DHX
and its methylated analogues (constructed and energy minimized in
vacuum using SYBYL 8.1 with the MMFF94s force field)45 in the
receptor binding site was performed using the GOLD program (Genetic
Optimization for Ligand Docking) version 3.2.46,47 A distance constraint
was used to preserve the known salt bridge between D103 and the ligand
ammonium moiety, and a water molecule present in the vicinity of
Ser199 and Asn292 was included. The best five out 100 docking poses
were inspected and found to be very similar, so the docking pose with the
best GOLD score was used for further study. The protein side chain
torsions were modified according to the GOLD output as appropriate
using SYBYL, and then the previous ligand was replaced by the docked
structure in the system coordinate file using the text editor. The system
was then energy minimized and MD simulations were performed without
any proteinꢀligand restraints until convergence was achieved, monitored
by plateauing of the rmsd over time, typically 6ꢀ8 ns. Energy minimization
was performed again and the output structures were used for evaluation.
Root-mean-square deviation calculations of the ligands relative to
DHX were performed based on all heavy atoms but excluding the variable
methyl group. Data were processed using Microsoft Excel. The correlation
between ΔGbind and rmsd was obtained using linear regression.
(13) Nichols, D. E. Dopamine Receptor Subtype-Selective Drugs:
D1-Like Receptors. In The Dopamine Receptors; Neve, K. A., Ed.;
Humana Press: Totowa, NJ, 2010; pp 75ꢀ99.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (765) 494-1461. Fax: (765) 494-1414 E-mail: drdave@
pharmacy.purdue.edu.
(14) Pollock, N. J.; Manelli, A. M.; Hutchins, C. W.; Steffey, M. E.;
Mackenzie, R. G.; Frail, D. E. Serine mutations in transmembrane-V of
the dopamine D1-receptor affect ligand interactions and receptor
activation. J. Biol. Chem. 1992, 267, 17780–17786.
(15) Tschammer, N.; Bollinger, S.; Kenakin, T.; Gmeiner, P. Histi-
dine 6.55 Is a Major Determinant of Ligand-Biased Signaling in
Dopamine D-2L Receptor. Mol. Pharmacol. 2011, 79, 575–585.
(16) Grol, C. J.; Rollema, H. Conformational analysis of dopamine by
the INDO molecular orbital method. J. Pharm. Pharmacol. 1977,29,153–156.
(17) McDermed, J. D.; Freeman, H. S.; Ferris, R. M. Enantioselec-
tivity in the binding of (+) and (ꢀ)-2-amino-6,7-dihydroxy-1,2,3,4-
tetrahydronapthalene and related agonists to dopamine receptors. In
Catecholamines: Basic and Clinical Frontiers; Usdin, E., Kopin, I. J.;
Barchas, J., Eds.; Pergamon Press: New York, 1978; pp 568ꢀ570.
’ ACKNOWLEDGMENT
This work was funded by NIH grants MH42705 (D.E.N.),
GM085604 (M.A.L.), and MH60397 (V.J.W.).
’ ABBREVIATIONS USED
DHX, dihydrexidine; DOX, doxanthrine; MD, molecular dy-
namics; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;
rmsd, root-mean-square deviation; TM5, transmembrane domain 5
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ARTICLE
(18) Brewster, W. K.; Nichols, D. E.; Riggs, R. M.; Mottola, D. M.;
Lovenberg, T. W.; Lewis, M. H.; Mailman, R. B. trans-10,11-Dihydroxy-
5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine: a highly potent se-
lective dopamine D1 full agonist. J. Med. Chem. 1990, 33, 1756–1764.
(19) Qandil, A. M.; Miller, D. W.; Nichols, D. E. A Practical and
Cost-Effective Synthesis of 6,7-Dimethoxy-2-tetralone. Synthesis 1999,
12, 2033–2035.
(20) Ninomiya, I.; Naito, T.; Mori, T. Photochemical reactions of
N-acylenamines. Tetrahedron Lett. 1969, 2259–2262.
(21) Ninomiya, I.; Naito, T.; Mori, T. Photocyclization of enam-
ides.1. Photochemical reactions of N-acyl enamines. J. Chem. Soc., Perkin
Trans. 1 1973, 505–509.
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich,
S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.;
Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.;
Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian
03, Revision E.01; Gaussian, Inc.: Wallingford, CT, 2007.
(39) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A well-
behaved electrostatic potential based method using charge restraints for
deriving atomic charges—the Resp Model. J. Phys. Chem. 1993, 97,
10269–10280.
(22) Woodward, R. B.; Hoffmann, R. The Conservation of Orbital
Symmetry; Academic Press: New York, 1970.
(23) Cueva, J. P.; Nichols, D. E. A novel and efficient synthesis of
dihydrexidine. Synthesis 2009, 715–720.
(24) Gensler, W. J.; Berman, E. Decarboxylative condensation—
alpha-alkylcinnamic acids from aromatic aldehydes and alkylmalonic
acids. J. Am. Chem. Soc. 1958, 80, 4949–4954.
(25) Schrecker, A. W. Resolution and rearrangement of alpha-
methylhydrocinnamic acid and of Its 3,4-dimethoxy derivative. J. Org.
Chem. 1957, 22, 33–35.
(40) Wang, J. M.; Cieplak, P.; Kollman, P. A. How well does a
restrained electrostatic potential (RESP) model perform in calculating
conformational energies of organic and biological molecules? J. Comput.
Chem. 2000, 21, 1049–1074.
(41) Fiser, A.; Do, R. K.; Sali, A. Modeling of loops in protein
structures. Protein Sci. 2000, 9, 1753–1773.
(42) Sali, A.; Blundell, T. L. Comparative protein modelling by
satisfaction of spatial restraints. J. Mol. Biol. 1993, 234, 779–815.
(43) Wu, C. H.; Huang, H. Z.; Arminski, L.; Castro-Alvear, J.; Chen,
Y. X.; Hu, Z. Z.; Ledley, R. S.; Lewis, K. C.; Mewes, H. W.; Orcutt, B. C.;
Suzek, B. E.; Tsugita, A.; Vinayaka, C. R.; Yeh, L. S. L.; Zhang, J.; Barker,
W. C. The Protein Information Resource: an integrated public resource
of functional annotation of proteins. Nucleic Acids Res. 2002, 30, 35–37.
(44) Wu, C. H.; Yeh, L. S. L.; Huang, H. Z.; Arminski, L.; Castro-
Alvear, J.; Chen, Y. X.; Hu, Z. Z.; Kourtesis, P.; Ledley, R. S.; Suzek, B. E.;
Vinayaka, C. R.; Zhang, J.; Barker, W. C. The Protein Information
Resource. Nucleic Acids Res. 2003, 31, 345–347.
(45) SYBYL 8.1; Tripos International: St. Louis, MO, 2009.
(46) Jones, G.; Willett, P.; Glen, R. C. Molecular recognition of
receptor sites using a genetic algorithm with a description of desolvation.
J. Mol. Biol. 1995, 245, 43–53.
(47) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible docking.
J. Mol. Biol. 1997, 267, 727–748.
(26) Kornblum, N. The synthesis of aliphatic and alicyclic nitro
compounds. Organic Reactions; Cope, A. C., Ed.; Wiley & Sons: New
York, 1962, Vol. 12, pp 101ꢀ156.
(27) Kornblum, N.; Graham, G. E. The regeneration of nitropar-
affins from their salts. J. Am. Chem. Soc. 1951, 73, 4041–4043.
(28) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen,
S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.;
Kobilka, B. K.; Stevens, R. C. High-resolution crystal structure of an
engineered human beta2-adrenergic G protein-coupled receptor. Science
2007, 318, 1258–1265.
(29) McGaughey, G. B.; Gagne, M.; Rappe, A. K. Pi-stacking interactions—
alive and well in proteins. J. Biol. Chem. 1998, 273, 15458–15463.
(30) The PyMOL Molecular Graphics System [0.99rc6]; Schr€odinger,
LLC, New York, 2006.
(31) Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular
dynamics. J. Mol. Graphics 1996, 14, 33–38.
(32) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GRO-
MACS 4: Algorithms for highly efficient, load-balanced, and scalable
molecular simulation. J. Chem. Theory Comput. 2008, 4, 435–447.
(33) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang,
W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.;
Kollman, P. A point-charge force field for molecular mechanics simula-
tions of proteins based on condensed-phase quantum mechanical
calculations. J. Comput. Chem. 2003, 24, 1999–2012.
(34) Berger, O.; Edholm, O.; Jahnig, F. Molecular dynamics simula-
tions of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydra-
tion, constant pressure, and constant temperature. Biophys. J. 1997, 72,
2002–2013.
(35) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case,
D. A. Development and testing of a general Amber force field. J. Comput.
Chem. 2004, 25, 1157–1174.
(36) Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. Automatic
atom type and bond type perception in molecular mechanical calcula-
tions. J. Mol. Graphics Modell. 2006, 25, 247–260.
(37) Kuyper, L. F.; Hunter, R. N.; Ashton, D.; Merz, K. M.; Kollman,
P. A. Free-Energy Calculations on the Relative Solvation Free-Energies
of Benzene, Anisole, and 1,2,3-Trimethoxybenzene—Theoretical and
Experimental-Analysis of Aromatic Methoxy Solvation. J. Phys. Chem.
1991, 95, 6661–6666.
(38) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A. Jr.; Vreven, T.; Kudin,
K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.;
Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.;
5521
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