Scaffold ranking and positional scanning utilized in the discovery of nAChR-selective compounds suitable for optimization studies

Article abstract of DOI:10.1021/jm401543h

Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The α4β2 nAChR is the most prevalent subtype in the brain and possesses the most evidence linking it to nicotine seeking behavior. Herein we report the use of mixture based combinatorial libraries for the rapid discovery of a series of α4β2 nAChR selective compounds. Further chemistry optimization provided compound 301, which was characterized as a selective α4β2 nAChR antagonist. This compound displayed no agonist activity but blocked nicotine-induced depolarization of HEK cells with an IC₅₀ of approximately 430 nM. 301 demonstrated nearly 500-fold selectivity for binding and 40-fold functional selectivity for α4β2 over α3β4 nAChR. In total over 5 million compounds were assessed through the use of just 170 samples in order to identify a series of structural analogues suitable for future optimization toward the goal of developing clinically relevant smoking cessation medications.

Full text of DOI:10.1021/jm401543h

Article
pubs.acs.org/jmc
Scaffold Ranking and Positional Scanning Utilized in the Discovery
of nAChR-Selective Compounds Suitable for Optimization Studies
Jinhua Wu,† Yaohong Zhang,†,‡ Laura E. Maida,† Radleigh G. Santos,† Gregory S. Welmaker,†
Travis M. LaVoi,† Adel Nefzi,† Yongping Yu,†,‡ Richard A. Houghten,† Lawrence Toll,*,†
and Marc A. Giulianotti*,†
†Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, Florida 34987, United States
‡Institute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P.R. China
S
* Supporting Information
ABSTRACT: Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The
α4β2 nAChR is the most prevalent subtype in the brain and possesses the most evidence linking it to nicotine seeking behavior.
Herein we report the use of mixture based combinatorial libraries for the rapid discovery of a series of α4β2 nAChR selective
compounds. Further chemistry optimization provided compound 301, which was characterized as a selective α4β2 nAChR
antagonist. This compound displayed no agonist activity but blocked nicotine-induced depolarization of HEK cells with an IC50
of approximately 430 nM. 301 demonstrated nearly 500-fold selectivity for binding and 40-fold functional selectivity for α4β2
over α3β4 nAChR. In total over 5 million compounds were assessed through the use of just 170 samples in order to identify a
series of structural analogues suitable for future optimization toward the goal of developing clinically relevant smoking cessation
medications.
or maintaining addiction to nicotine or other abused drugs.
Pharmacological and genetic evidence has suggested that the
α3β4* nAChR, found primarily in the medial habenula (MHb)
and intepedunclear nucleus, is involved in addiction to
cigarettes and other abused drugs. The relatively nonselective
α3β4 antagonist 18-methoxycoronaridine (18-MC) blocks the
self-administration and conditioned place preference of many
abused drugs.9 The more selective and higher affinity α3β4
nAChR ligand AT-1001 potently blocks nicotine self-
administration and reinstatement.10 These data are consistent
with Genome Wide Association Studies (GWAS) that have
implicated variants in the α3-α5-β4 gene cluster on
chromosome 15 to be associated with an increased risk of
whether a smoker becomes nicotine dependent and to smoking
a greater number of cigarettes per day.11,12 The GWAS,
therefore, also suggest a possible involvement of the α5 subunit
in tobacco dependence. This is consistent with behavioral
experiments. In α5 null mice, there is an increase in nicotine
conditioned place preference and nicotine self-administration at
INTRODUCTION
■
Neuronal nicotinic acetylcholine receptors (nAChRs) are
ligand-gated ion channels that mediate cation flux and are
activated by nicotine, the major constituent of tobacco leading
to addiction.1,2 In mammalian neural tissue, the nAChR is a
pentamer usually made up of a combination of α and β
subunits,3 although there are also fully functional homomeric
receptor proteins, particularly the prominent α7 nAChR. The
α4β2* nAChR (where * denotes the possible presence of an
additional subunit) is by far the most prevalent in the CNS,4,5
being expressed in large numbers throughout the brain.
Addiction to cigarettes is primarily mediated by nicotine-
induced activation of nAChR and, as with other abused drugs,
nicotine addiction is thought to be primarily due to the
activation of the dopaminergic mesocorticolimbic pathway.6,7
The α4β2* nAChR is the most prevalent nicotinic receptor
subtype in this region.4,8
Despite the prevalence of the α4β2* nAChR in the drug
reward pathway, a considerable amount of research has been
conducted on many different nAChR receptor types and
individual subunits, with many different receptor types and
subunits having been demonstrated to be involved in acquiring
Received: October 3, 2013
Published: November 25, 2013
© 2013 American Chemical Society
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higher nicotine levels.13,14 Furthermore, viral vector-mediated
reintroduction of α5 subunits into the MHb recovers wild-type
nicotine self-administration behavior.14 The studies indicate
that nAChRs containing the α5 subunit may be involved in
limiting the reward mediated by higher nicotine doses. There is
also considerable evidence that the α6 subunit, which is also
found in high concentrations in the mesolimbic dopamine
pathway, is involved in nicotine reward. α6 knockout (KO)
mice do not self-administer nicotine, and inhibition of
α6-containing nAChR using conotoxin also blocks nicotine
self-administration.15
Nevertheless, the greatest body of evidence accumulated for
the involvement of a particular nAChR in smoking pertains to
the α4β2* nAChR. Neither β2- nor α4-knockout mice self-
administer nicotine.16,17 In addition, transgenic mice with a
sensitized α4 subunit self-administer nicotine at lower doses,18
indicating that changes in the α4 alone is sufficient to modify
nicotine self-administration. Pharmacological evidence for the
involvement of the α4β2* nAChR also exists. The selective
α4β2 antagonist dihydro-β-erythroidine hydrobromide
(DHβE) blocks nicotine self-administration in rats,19 and
most important of all, the α4β2 partial agonist varenicline is
clinically used as a smoking cessation medication.
Unfortunately, although varenicline exhibits binding selectiv-
ity, this selectivity is not observed in functional assays. In
functional assays, it is a full agonist at both α3β4 and α7
nAChR with comparable potency to that observed at α4β220,21
Varenicline users haves also experienced significant adverse
psychiatric effects, including depression and suicidal thoughts,
limiting its clinical usefulness.22 Although other high affinity
α4β2 agonists and antagonists have been reported, generally,
the selectivity compared to α3β4 nAChR and functional activity
at α3β4 nAChR were generally not characterized.23−25 Here we
report novel selective α4β2 nAChR antagonists, derived from
compounds originally identified from combinatorial libraries
containing mixtures of small molecules.
Starting from a small molecule mixture-based combinatorial
scaffold ranking library with more than 5 million individual
compounds arranged into 34 samples, we selected a mixture-
based library comprised of 45864 compounds systematically
arranged into 110 mixtures for further screening. Upon
screening of this positional scanning library, a number of
mixtures showed affinity and selectivity for α4β2 nAChR. From
this single library, we identified 18 individual compounds with
modest affinity but some with excellent selectivity for α4β2
nAChR over α3β4 nAChR. These lead compounds were
modified to produce antagonists with high affinity and
impressive selectivity in both receptor binding studies and in
functional activity in vitro. The success of this approach shown
herein demonstrates the ability of mixture-based combinatorial
libraries to identify individual novel structures in the context of
any low- to medium-throughput assay (Figure 1).
Figure 1. Approach used to identify potent and selective (over α3β4)
α4β2 ligands. Starting with just 34 samples comprised of >5 million
compounds in the Scaffold Ranking Library, a specific Positional
Scanning Library (1169) was identified for screening. This library
contained 110 samples, and the results from screening the library were
used in the selection of 18 compounds that were synthesized and
tested (Deconvolution). On the basis of these results, eight new
optimized compounds were synthesized and tested and found to be
potent and selective (over α3β4) α4β2 ligands. In total, only 170
samples were tested; this shows the broad utility of this approach for
many assay formats, especially those limited for whatever reason by
throughput.
the advantages and limitations of the approach, elsewhere.27
The scaffold ranking library utilized in this work contained 34
mixture samples that in total contained more than 5 million
individual compounds (Supporting Information Table S1). As
previously described,26 each mixture sample contains only
compounds with the same specific core scaffold, so only
close structural analogues are contained in each mixture sample.
The mixture samples tested in this work contained anywhere
from as few as 10800 individual compounds (Library 1409
Supporting Information Table S1) to as many as 738192
(Libraries 1343−1347 Supporting Information Table S1). The
median complexity was 45864 compounds per sample. Each of
these individual compounds is represented at an approximately
equal molar concentration within a given sample. Because the
samples are organized to contain only structural analogues, if a
particular individual compound is active then the mixture
sample containing that compound may possesses many more
structural analogues that are also active. Therefore the effective
concentration of active compounds in a given mixture is, in general,
significantly higher than it would be if only one compound in the
mixture was active.32 In this manner, we are able to utilize these 34
mixture samples to prioritize which chemical scaffolds are likely to
contain active individual compounds.
These 34 mixtures were screened at the four different
concentrations 10, 5, 1, and 0.5 μg/mL for their ability to block
[3H]epibatidine binding to α4β2 and α3β4 nAChR, as
described in Materials and Methods. Shown in Figure 2 are
the results from the 10 μg/mL screening (data from all doses is
shown in Supporting Information Table S2).
1169 Library Screening. We identified positional scanning
library 1169 for screening based on the results of the scaffold
ranking library. Library 1169 contains 45864 individual bis-
cyclic guanidines (see Scheme 1) systematically formatted into
a positional scanning library containing 110 mixture samples
(Table 1). For example, sample 1 contains in approximately
equal molar amounts the 1092 individual compounds that have
(1-phenyl-cyclopropyl)-methyl fixed at the R1 position (Table 1).
In this manner, the first 42 samples of library 1169 contain all
45864 compounds in the library sorted by R1 functionality.
Samples 43−68 contain the same 45864 compounds sorted by R2
functionality, and samples 69−110 are sorted by R3 functionality.
The 110 samples were screened for binding affinity exactly as
conducted on the original scaffold ranking library. To use
RESULTS AND DISCUSSION
■
Scaffold Ranking. To reduce the number of samples to
test, we began by using our scaffold ranking library.26,27 As
reported elsewhere, we have successfully utilized the scaffold
ranking approach in efforts directed toward the identification
of metalloprotease ADAM 17 ligands,28 antimicrobials that
inhibit tyrosine recombinases and Holliday junction-resolving
enzymes,29,30 and novel antinoceptives through the use of a
direct in vivo assay.31 We have described in some detail the
method for constructing a scaffold ranking library,26 as well as
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Figure 2. Scaffold Ranking Library data for the 10 μg/mL dose (approximately 20 μM assuming an average molecular weight of 500 Da for each
compound in a given mixture). Values are shown as percent inhibition of [3H]epibatidine binding to α4β2 (red) and α3β4 (blue) nAChR. The
numbers along the x-axis represent the specific Scaffold Ranking Library sample, sc = solvent control (0.1%DMF), and the EPI (no.) are different
concentrations of epibatidine (EPI(1) = 1 nM, (10) = 10 nM, (100) = 100 nM, (1000) = 1000 nM).
Scheme 1. Synthesis of Bis-cyclic Guanidines
Harmonic Meaning to confirm the integrity of the screening,32
percent inhibition values were converted to xIC50 values,
assuming a Hill coefficient of one (see Materials and Methods).
The Harmonic Mean was then taken in each position for each
receptor. The calculated μM harmonic mean values at the R1,
R2, and R3 positions were (respectively) 26.6, 28.0, and 34.1 for
the α4β2 receptor, and 78.6, 82.4, and 73.2 for the α3β4
receptor; the deviation of at most 27% at each receptor
represents excellent evidence of the integrity of the synthesis
and screening. The xIC50 values for each sample are shown in
Table 1 as well as the fold selectivity. In general, the samples
produced a stronger response against α4β2 than against α3β4.
The data shows significant differentiation in activity levels
among the samples tested at each position, a key feature for
deconvoluting a positional scanning library.27 Moreover, some
preliminary SAR can be seen from the library screening. For
example when an aromatic group with a halogen substitution is
fixed in the R1 position (Table 1: 5, 6, 9, 11, and 15), the
sample produced strong inhibition for α4β2. However, while
these same fixed functionalities provided activity at the R3
position (Table 1: 73, 74, 77, 79, and 83) against α4β2, a
number of other samples with different functionalities fixed at
this position also produced strong inhibition. At the R2
position, the profile indicates that for activity an R configuration
could be preferred over S for α4β2 (Table 1: 53 over 44, 59
over 51, 62 over 61, 66 over 65, and 68 over 67). The
screening data also identifies several active samples that are
highly selective for α4β2 over α3β4 (>4-fold selectivity, such as
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Table 1. The 110 Samples That Make up Library 1169, Their Associated xIC50 Values for α4β2 and α3β4 nAChR, and Fold
a
Selectivity Are Reported
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Table 1. continued
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Table 1. continued
a
Library 1169 is made up of 45864 bis-cyclic guanidines all containing the same central scaffold shown at the top of Table 1. The compounds are
systematically synthesized into a positional scanning library containing the 110 samples shown in the table. By way of example, sample 1 is an
approximate equal molar mixture of 1092 compounds. The 1092 compounds contain (1-phenyl-cyclopropyl)-methyl fixed in the R1 position, and all
1092 combinations of the 26 R2 (shown in Table 1 43−68) and 42 functionalities at R3 (Table 1 69−110). Similarly, sample 83 contains the 1092
compounds generated from fixing R3 with 3,4-dichlorophenethyl and utilizing all 1092 combinations of the 42 R1 functionalities and 26 R2
functionalities. The xIC50 and fold selectivity values were calculated as described in the Materials and Methods section. The xIC50 values are further
color coded using a three-color scale: red (most active), white, blue (least active). The fold selectivity values are also color coded using a three-color
scale: green (more selective for α4β2), yellow, red (less selective for α4β2). A value of 0 for fold selectivity means a sample produced the same
activity in both receptors.
Table 2. The Functionalities, Ki Values, and Selectivity (Both Experimentally Determined and Projected) for the 18 Individual
a
Compounds Synthesized Based on the Data Generated from the Screening of 1169 are Shown
a
The functionalities correspond to the core scaffold shown in Table 1. The activity data is presented as a Ki (μM) for each individual compound
against each of the two targets. The experimental selectivity is the value obtained from (Ki α3β4 − Ki α4β2)/(Ki α4β2). The projected relative
selectivity value is generated from using only library 1169 data as described in the Materials and Methods section. The Ki values are further color
coded using a three-color scale: red (most active), white, blue (least active). The fold selectivity values are also color coded using a three-color scale:
green (more selective for α4β2), yellow, red (less selective for α4β2). A value of 0 for fold selectivity means a sample produced the same activity in
both receptors. Note the strong correlation between the experimental and projected selectivity values (R2 = 0.831), also shown in Figure 3.
Table 1: 5, 21, 26, 53, 68, and 106). There were some samples
that showed activity against both targets providing low levels of
selectivity (<1.5-fold selectivity, Table 1: 15, 65, 66, 74, 79, and 83).
Selection of Individual Compounds for Synthesis. On
the basis of the 1169 library screening data, 18 individual
compounds were selected for synthesis (Scheme 1). These 18
compounds were selected based on three different criteria. The
first set was chosen by selecting functionalities that were active
at both α4β2 and α3β4 nAChR, showing little selectivity for
one receptor over the other. For the R1 position, 3,4-
dichlorophenethyl (15) was selected. While two other samples
that also have aromatic functionalities with halogen groups 2-
(3-fluoro-phenyl)-ethyl (5) and 2-(3-bromo-phenyl)-ethyl (6)
were actually more active at α4β2 than 15, they were also more
selective for α4β2 in the library screening (Table 1). From the
R2 position, R-2-naphthylmethyl (66) and S-2-naphthylmethyl
(65) were selected due to the fact they showed activity at both
receptors. Additionally, by using these chiral isomers, the
preference for the R configuration for activity at α4β2 could be
confirmed with individual compounds. From the R3 position,
three different halogen substituted aromatic functionalities that
showed activity and low selectivity in the library screening were
selected: 2-(3-bromo-phenyl)-ethyl (74), 2-(4-bromo-phenyl)-
ethyl (79), and 3,4-dichlorophenethyl (83). There are therefore
only six compounds in this set (1 × 2 × 3), identified as
compounds 201 through 206 in Table 2. While this set of
individual compounds was chosen with the purpose of reducing
receptor selectivity, it should also be noted that this is the set of
compounds that would have been made if the sole purpose was
to choose the compounds most likely to be active at α3β4. In
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contrast to the first set of compounds, the second set was
chosen in order to maximize affinity at α4β2 as well as
selectivity for α4β2 over α3β4. Additionally, consideration was
made to explore chemical diversity where appropriate. For the
R1 position in this set, three different functionalities were
chosen: 2-(3-fluoro-phenyl)-ethyl (5), heptyl (21), and
4-methylpentyl (26). While sample 5 (Table 1) was not the
only sample in the library with a substituted aromatic
functionality at R1 that showed activity and selectivity at α4β2,
it was the most active/selective sample and therefore was
selected. The two aliphatic functionalities, heptyl and
4-methylpentyl, were chosen because they showed strong
selectivity for α4β2 (7.20- and 12.32-fold, respectively) and
provided a different chemical characteristic. Likewise for the R2
position in this set, two chemically diverse functionalities were
selected: R-benzyl and R-cyclohexyl. Both mixture samples with
these defined functionalities, 53 and 68, respectively, showed
affinity for α4β2 and strong selectivity. For the R3 position, in
general, mixture samples with aliphatic groups defined showed
slightly less potency but more selectivity for α4β2 than samples
defined with aromatic groups. Therefore, for this set, 4-tert-butyl-
cyclohexyl-methyl was utilized, as it was among the most active
mixtures with an aliphatic defined R3 group and the most
selective R3 sample (R3 samples are samples 69−110 in
Table 1). This second set of individuals is shown in Table 2 as
compounds 207−212. A final set of six compounds was selected
for synthesis from the library screening, compounds 213−218
shown in Table 2. This set contains the same functionalities at
R1 and R2 as the previous set but replaces the R3 group with
2-(3-bromo-phenyl)-ethyl (Table 1: 74). This substitution
changes the functionality used in the R3 group from the
most selective R3 functionality in the library at this position to
the most active at α4β2 regardless of selectivity. In so doing,
this set contains among it, the compound that combines the
most active functionalities at each position (Table 2: 213)
against α4β2.
Screening of Individual Compounds. The 18 individual
compounds were synthesized and screened against α4β2 and
α3β4 nAChR (Table 2). The results further confirmed the SAR
identified from the library screening. The first set of six
compounds proved to be the least selective of the three sets,
the second set the most selective, and the third set of six
showed moderate selectivity. The predicted selectivity versus
experimental results is further shown in Figure 3. The
experimental selectivity exhibited a high correlation of R =
0.91 to predicted values (R2 = 0.83), demonstrating the validity
of choosing compounds to synthesize based upon the affinities
of the individual functionalities in positions 1, 2, and 3 derived
from mixture screening as well as the validity of our
methodology of predicting binding affinities. Of the 18
individual compounds tested in these sets, the highest affinity
compounds against α3β4 came from the first set (201, 202, and
205); this was to be expected as this set contained the
compounds that matched the most active functionalities against
α3β4 in the library screening. From the first set of six
compounds, 201−206, the apparent preference for R over S
absolute configuration at the R2 position that was seen at the
library level for α4β2 is also observed. When comparing the
affinities of the second set of six compounds (207−212)
against the last set of six (213−218), we note that the
respective affinities against α4β2 remain similar but the
selectivity in general decreases significantly. The only difference
in these two sets is the functionality used in the R3 position. For
Figure 3. The values for “Projected Selectivity” and “Experimental
Selectivity” shown in Table 2 for the 18 compounds are graphed in the
scatter plot. Each dot corresponds to one of the 18 compounds. The
Experimental Selectivity value is calculated using the following equation
(Ki α3β4 − Ki α4β2)/(Ki α4β2). The Projected Selectivity value is a value
“projected” from using the 1169 library data. The methodology used to
determine the Projected Selectivity is detailed in Materials and Methods.
In brief, first a projected activity value for each compound is determined
for both receptors. This is done by taking the product of the activity value
of the 1169 positional scanning sample that corresponds with the
functionality used in the Table 2 individual compounds. Once this value is
obtained for each of the compounds and for both receptors, the Projected
Selectivity value can be determined using the same selectivity formula
used for the Experimental Selectivity values.
compounds 207−212, the aliphatic functionality 4-tert-butyl-
cyclohexyl-methyl was utilized whereas the halogen substituted
aromatic 2-(3-bromo-phenyl)-ethyl group was used for
compounds 213−218. The results indicate that the R3 group
change has little effect on the affinity of the compounds against
α4β2 but has a dramatic effect on their affinity for α3β4. We
sought to further exploit these observations in order to produce
a set of compounds with increased affinity for α4β2 as well as
selectivity for α4β2 over α3β4.
Optimization of Individuals. Utilizing the acquired data,
compounds 207 and 208 were selected for further
optimization. Compound 207 was selected because it exhibits
the highest affinity for α4β2, is among the most selective for
α4β2, and has an aromatic functionality at the R1 position that
we identified for modification. Compound 208 was selected, as
it is the α4β2 selective analogue of 207 containing the same
aromatic group in the R1 position. The focus of the
optimization was the modification of the R1 functionality.
While the 1169 library contains a diverse set of aromatic
functionalities in the R1 position, it does not contain any
pyridyl groups. A number of reported nAChR ligands contain
pyridyl groups, and some of the most potent, including nicotine,
contain a 3-pyridyl group or substituted 3-pyridyl group such as
epibatidine.33 Using the same two-carbon spacer off the core
cyclic guanidine scaffold, the 3-fluoro-phenyl group of 207 was
replaced with a 3-pyridyl group in 301 (see Figure 4, Table 3).
This substitution drastically increased its affinity at α4β2, from
1587 to 118 nM while remaining relatively inactive at α3β4,
61750 nM for 207 and 56480 nM for 301, producing a high
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Figure 4. Hit optimization of compounds 207 and 208. The colored stars and arrows correspond to site specific analogue modifications.
affinity and highly selective compound. The same R1
modification to compound 208 also dramatically increased its
α4β2 affinity and selectivity, 306 (see Figure 4, Table 3).
Modifying the R2 chirality of 301 and 306 from R to S,
compounds 302 and 307, respectively, decreases the affinity at
α4β2 in line with what is observed in both the 1169 library
samples (Table 1) as well as the individual compounds (Table 2)
with regards to this change in absolute configuration at the R2
position. Eliminating the R2 position entirely from 301 and 306
provides the same analogue, 303, which has a lower affinity to
α4β2 and selectivity than either parent compound. A change
in absolute configuration in the core scaffold of 301 from S
to R, compound 304, also decreased its affinity at α4β2,
however, the same change to 306 provided a slightly more
potent α4β2 ligand, 308. Combining the change in absolute
configuration in the core scaffold from S to R with the
elimination of the R2 functionality provides the same
analogue, 305, which has the lowest affinity at α4β2 of all
the analogues tested in this optimization phase. Therefore,
from this set of optimized hits, compound 301 was selected
for further evaluation.
Table 3. Binding Affinity, Selectivity, and Antagonist Activity
of Selected Individual Compounds
a
[3H]epibatidine binding
selectivity
(fold)
(α3β4
α4β2 Ki
α4β2 antagonist
compd
α3β4 Ki (nM)
(nM)
Ki/α4β2 Ki) potency IC50 (nM)
207
208
301
302
303
304
305
306
307
308
DHβE
61750 36030
88895 18005
56480 8920
23765 5345
27550 7150
63320 3560
51893 27353
59678 15253
78085 8355
33353 4373
>10000
1587 301
3731 237
39
24
26320 980
5319 346
514 84
118
2
478
74
323 152
433 79
1465 43
2820 82
3024 125
2953 594
2830 75
3405 125
1686 373
624 24
64
307 78
206
23
2217 1194
340 148
611 220
257 113
485 33
176
128
130
>20
a
Experiments were conducted a minimum of two times by methods
described in Materials and Methods. Values shown are mean SEM
for experiments conducted in triplicate at least twice.
Biological Profiling of 301. Compound 301 was further
evaluated for binding affinity against α3β4α5 nAChR and found
to be weakly active with a Kd/Ki of 9062 nM. Compound 301
was tested for functional activity by measuring the ligand-
induced change in membrane potential in intact HEK cells
transfected with α3β4 and α4β2 nAChR. Membrane potential
changes were determined using a FlexStation, as described
previously10 and in Materials and Methods. Compound 301
displayed no agonist activity in either cell line. As seen in
Figure 5 and Table 3, 301 blocks nicotine stimulation at α4β2
nAChR with an IC50 of approximately 500 nM, which is slightly
more potent than the selective α4β2 nAChR antagonist DHβE.
It is also very selective for α4β2 nCAhR, as it did not inhibit
α3β4 nAChR activation by 50% up to 10 μM, the highest
concentration tested (data not shown). 301 has no apparent
agonist activity, so there is no reason to believe that this
compound induces desensitization rather than direct inhibition
of nicotine activity. To determine if antagonist activity is
competitive, nicotine dose response curves were constructed in
the presence of various concentrations of 301. As seen in
Figure 6, 301 causes a small rightward shift in the nicotine dose
response curve but to a greater extent a decrease in maximal
activity. These results appear to suggest a noncompetitive
inhibition of nicotine’s activity.
DISCUSSION AND CONCLUSION
■
Screening to identify novel structures with high affinity to
nAChR started with a “scaffold ranking library,” which is a
collection of 34 samples. Each of these 34 samples contains a
mixture of individual compounds arranged so that compounds
with the same structural core “scaffold” are contained in the
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functional selectivity is encouraging because some very selective
compounds, such as varenicline, have excellent binding
selectivity but very poor functional selectivity.21
Interestingly, inhibition of nicotine stimulation appears to be
noncompetitive, as increasing concentrations of 301 induce a
progressive decrease in the maximal activity rather than a
parallel shift to the right of a nicotine dose response curve. It
seems unlikely that this would be due to desensitization of
the receptor because 301 does not activate the receptor in these
experiments, although it is possible that desensitization of the
α4β2 nAChR might occur. The α4β2 partial agonist sazetidine
A was originally described as a nonactivating desensitizer, but it
was later determined that it was in fact a partial or full agonist
in other cellular assays.38,39 Additional studies of 301 will be
required to determine if it has agonist activity under some
circumstances.
Figure 5. Antagonist activity of compound 301. 301 has no agonist
activity but potently blocks nicotine-induced increase in membrane
potential in α4β2-containing cells. 301 has potential inverse agonist
activity at very high concentrations.
In conclusion, through the use of mixture-based libraries a
total over 5 million compounds were assessed through the use
of only 170 samples in order to provide a series of nAChR-
selective compounds. One compound, 301, has moderately
high affinity and binding and functional selectivity for α4β2
over α3β4 nAChR; additional studies are underway on
congeners and fragments of 301. The success of this study
demonstrates conclusively that mixture-based combinatorial
libraries utilizing the scaffold ranking and positional scanning
approaches can successfully be used in assays of low to medium
throughput to develop novel lead compounds for further
research.
Figure 6. Compound 301 inhibits nicotine-induced α4β2 nAChR
stimulation in a dose-dependent manner. Increasing concentrations of
301 induce a progressive decrease in maximal activity with a slight
change in the EC50. EC50 and maximum stimulation were: 779 nM and
100%, 999 nM and 83%, 1809 nM and 38%, EC50 not measurable,
maximum 0, for 0, 0.3, 1.0, and 3.0 μM 301, respectively.
MATERIALS AND METHODS
■
Chemistry General. The 1H and 13C NMR spectra were obtained
utilizing the Bruker 400’54 Ascend (400 and 100 MHz, respectively).
NMR chemical shifts were reported in δ (ppm) using the δ 7.26 signal
of CHCl3 (1H NMR), the δ 4.79 signal of D2O (1H NMR), and the δ
77.16 signal of CDCl3 (13C NMR) as internal standards. 13C NMR
spectra in D2O were not adjusted (representative NMR is available in
the Supporting Information). Mass spectra data was acquired using
Shimatzu LCMS-2010. The reported final purity of the compounds
were verified by Shimadzu HPLC and mass spectra under the following
conditions: column, Phenomenex Luna 150 mm × 21.20 mm, 5 μm,
C18; mobile phase, (A) H2O (+0.1% formic acid)/(B) MeCN (+0.1%
formic acid). Three gradient methods were used based on hydro-
phobicity (2% B to 20% B, 11 min) (25% B to 45% B, 31 min) (45% B
to 65% B, 21 min); flow rate, 12 mL/min; detection, UV 214 nm. The
purity of all final compounds were >95%. All chirality was generated
from the corresponding amino acids. Under the reaction conditions
described, no epimerization was observed and, for those compounds
with multiple chiral centers, a single diastereomer was obtained.
same sample, overall more than 5 million individual compounds
are contained in the 34 mixture samples. From the initial 34
mixtures, we identified a sample that demonstrated moderate
affinity for α4β2 nAChR. Therefore the complete positional
scanning library associated with this sample was chosen for
further screening. This positional scanning library, 1169,
contains 45864 individual bis-cyclic guanidines systematically
formatted into 110 samples allowing for the identification of
individual compounds of high affinity, by methods that were
discussed above and described previously.26,34−36 The series of
individual compounds identified by the library provided useful
SAR that was utilized to produce a series of further optimized
compounds with high affinity and selectivity for nAChR
subtypes. The highest affinity and most selective compounds
appear to be pure antagonists when measuring membrane
potential changes in HEK cells transfected with α4β2 nAChR.
These compounds have low affinity (IC50 > 10 μM) for α3β4
nAChR.
As we have demonstrated previously, mixture-based libraries
provide a method for the efficient and rapid identification of
novel structures that can be used directly from the libraries or
for further chemical modification.26,37 Of the compounds
described here, 301 is a novel compound, never previously
demonstrated to bind to nAChR. In FlexStation assays, 301
appears to have no agonist activity at α4β2 nAChR up to
10 μM. When cells are pretreated with 301, the increase in
membrane potential induced by nicotine is blocked at
concentrations of 301 similar to binding affinity. 301 has an
IC50 for inhibition of nicotine activity of approximately 430 nM.
This is at least 20-fold selective over its ability to block nicotine
activity in cells transfected with α3β4 nAChR. This 20-fold
Synthesis of Library 1169 and Individual Compounds and
Construction of Scaffold Ranking Plate. General Synthesis of Bis-
cyclic Guanidines F. Library 1169 and all individual compounds
reported (201−218 and 301−308) were synthesized following the
same general scheme (Scheme 1).40,41 The solid phase synthesis was
performed using the “tea-bag” methodology.42 Initially, 100 mg of
p-methylbenzdrylamine (MBHA) resin (1.1 mmol/g, 100−200 mesh)
was sealed in a mesh “tea-bag,” neutralized with 5% diisopropylethyl-
amine (DIEA) in dichloromethane (DCM) and subsequently swelled
with additional DCM washes. Fmoc-Lys(Boc)-OH (Fmoc-L-Lys(Boc)
for all compounds reported within except 304, 305, and 308 where
Fmoc-D-Lys(Boc) was used) (6 equiv) was coupled in dimethylfor-
mamide (0.1 M DMF) for 120 min in the presence of
diisopropylcarbodiimide (DIC, 6 equiv) and 1-hydroxybenzotriazole
hydrate (HOBt, 6 equiv) (A, Scheme 1). The Boc protecting group
was removed with 55% TFA/DCM for 30 min and subsequently
neutralized with 5% DIEA/DCM (3×). Boc-amino acids were coupled
utilizing standard coupling procedures (6 equiv) with DIC (6 equiv)
and HOBt (6 equiv) in DMF (0.1M) for 120 min (B, Scheme 1). The
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Boc protecting group was removed with 55% trifluoroacetic acid
(TFA) in DCM for 30 min and subsequently neutralized with 5%
DIEA/DCM (3×). Carboxylic acids are coupled using (6 equiv) in the
presence of DIC (10 equiv) and HOBt (10 equiv) in DMF (0.1M) for
120 min (C, Scheme 1). The Fmoc protecting group was removed
with 20% piperidine in DMF for 20 min, and the R1 carboxylic acids
were coupled in 10 equiv under the same solvent and activating
reagent conditions. All coupling reactions were monitored for
completion by the ninhydrin test. The reduction was performed in a
4000 mL Wilmad LabGlass vessel under nitrogen. A borane in 1.0 M
tetrahydrofuran complex solution was used in 40-fold excess for each
amide bond. The vessel was heated to 65 °C and maintained at
temperature for 72 h. The solution was then discarded, and the bags
were washed with THF and methanol. Once completely dry, the bags
were treated overnight with piperidine at 65 °C and washed several
times with methanol, DMF, and DCM (D, Scheme 1). Before
proceeding, completion of reduction was monitored by a control
cleavage and analyzed by LCMS. Urea cyclization (E, Scheme 1) was
performed with a 5-fold excess of cyanogen bromide (CNBr) in a
0.1 M anhydrous DCM solution for each of the cyclization sites
overnight. Following the cyclization, the bags were rinsed with DMF
and DCM. The resin was cleaved with HF in the presence of anisole in
an ice bath at 0 °C for 90 min (F, Scheme 1).
7.27−7.25 (m, 2H), 7.21 (d, J = 5.6 Hz, 2H), 7.18−7.13(m, 1H), 7.03
(d, J = 6.4 Hz, 1H), 4.15−4.02 (m, 1H), 3.94−3.85 (m, 2H), 3. 56−3.47
(m, 3H), 3.35−3.25 (m, 5H), 3.20−3.13 (m, 2H), 2.97−2.92 (m, 1H),
2.88−2.70 (m, 4H), 1.58−1.50 (m, 1H), 1.42−1.25 (m, 3H), 1.17−1.05
(m, 2H). 13C NMR (100 MHz, CDCl3) δ 159.17, 157.03, 138.83, 138.57,
134.06, 132.42, 132.21, 131.60, 131.31, 131.05, 130.91, 130.82, 130.64,
130.50, 129.35, 129.02, 128.77, 128.42, 127.77, 126.90, 126.24, 125.72,
122.65, 58.79, 57.20, 50.99, 46.26, 45.11, 44.48, 43.01, 35.66, 32.75, 31.29,
26.52, 24.80, 20.72. MS (ESI) m/z [M + H]+: 709.0.
(S)-3-(3-Bromophenethyl)-1-(4-((S)-3-(3,4-dichlorophenethyl)-2-
iminoimidazolidin-4-yl)butyl)-4-(naphthalen-1-ylmethyl)-
imidazolidin-2-imine (204). Using general scheme for the synthesis
of F, compound 204 was synthesized using the following reagents:
3,4-dichlophenylacetic acid (R1), Boc-L-2-naphthylalanine (R2),
3-bromophenylacetic acid (R3). Final crude product was purified by
HPLC as described above. 1H NMR (400 MHz, CDCl3): δ 8.64
(s, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.65 (d, J =
8.0 Hz, 1H), 7.55−7.47 (m, 2H), 7.39 (s, 1H), 7.38 (d, J = 8.0 Hz,
1H), 7.38 (d, J = 3.2 Hz, 1H), 7.30−7.26 (m, 2H), 7.19 (d, J = 7.2 Hz,
1H), 7.16 (d, J = 8.4 Hz, 1H), 7.12−7.08 (m, 2H), 4.13−4.10 (m,
1H), 3.92−3.84 (m, 1H), 3.82−3.75 (m, 1H), 3.65−3.53 (m, 2H),
3.46 (dd, J = 13.6, 4.0 Hz, 1H), 3.38−3.25 (m, 4H), 3.19−3.12 (m,
3H), 2.91 (dd, J = 13.6, 9.2 Hz, 1H), 2.84−2.78 (m, 4H), 1.57−1.48
(m, 1H), 1.46−1.35 (m, 3H), 1.20−1.10 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 159.25, 157.01, 140.73, 138.88, 134.06, 132.28,
131.96, 131.65, 131.35, 131.08, 130.68, 130.61, 130.55, 130.07, 129.33,
129.00, 128.40, 127.86, 127.81, 126.90, 126.21, 125.73, 122.71, 58.78,
57.35, 50.86, 46.21, 45.07, 44.71, 42.98, 35.58, 33.32, 32.73, 31.30,
26.54, 20.66. MS (ESI) m/z [M + H]+: 719.0.
(R)-3-(3-Bromophenethyl)-1-(4-((S)-3-(3,4-dichlorophenethyl)-2-
iminoimidazolidin-4-yl)butyl)-4-(naphthalen-1-ylmethyl)-
imidazolidin-2-imine (201). Using general scheme for the synthesis
of F, compound 201 was synthesized using the following reagents:
3,4-dichlophenylacetic acid (R1), Boc-D-2-naphthylalanine (R2),
3-bromophenylacetic acid (R3). Final crude product was purified by
1
(S)-3-(4-Bromophenethyl)-1-(4-((S)-3-(3,4-dichlorophenethyl)-2-
iminoimidazolidin-4-yl)butyl)-4-(naphthalen-1-ylmethyl)-
imidazolidin-2-imine (205). Using general scheme for the synthesis
of F, compound 205 was synthesized using the following reagents:
3,4-dichlophenylacetic acid (R1), Boc-L-2-naphthylalanine (R2),
4-bromophenylacetic acid (R3). Final crude product was purified by
HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.51 (s,
2H), 7.86 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.0
Hz, 1H), 7.55 (t, J = 7.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.38 (t, J =
8.0 Hz, 2H), 7.32−7.29 (m, 3H), 7.20 (d, J = 6.8 Hz, 2H), 7.15−7.10
(m, 2H), 4.15−4.08 (m, 1H), 3.85−3.80 (m, 2H), 3.65−3.58 (m, 2H),
3.49 (dd, J = 13.6, 4.4 Hz, 1H), 3.40−3.28 (m, 4H), 3.24−3.14 (m,
3H), 2.94−2.88 (m, 3H), 2.82−2.72 (m, 2H), 1.65−1.54 (m, 1H),
1.50−1.35 (m, 3H), 1.25−1.10 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 158.97, 156.94, 140.78, 138.74, 134.10, 132.31, 132.01,
131.67, 131.27, 131.09, 130.71, 130.63, 130.61, 130.10, 129.36, 129.04,
128.44, 127.87, 127.81, 126.92, 126.23, 125.74, 122.74, 58.99, 57.37,
50.83, 46.14, 44.97, 44.65, 43.00, 35.58, 33.39, 32.72, 31.23, 26.52,
20.64. MS (ESI) m/z [M + H]+: 719.0.
(R)-3-(4-Bromophenethyl)-1-(4-((S)-3-(3,4-dichlorophenethyl)-2-
iminoimidazolidin-4-yl)butyl)-4-(naphthalen-1-ylmethyl)-
imidazolidin-2-imine (202). Using general scheme for the synthesis of
F, compound 202 was synthesized using the following reagents:
3,4-dichlophenylacetic acid (R1), Boc-D-2-naphthylalanine (R2),
4-bromophenylacetic acid (R3). Final crude product was purified by
HPLC as described above. 1H NMR (400 MHz, CDCl3): δ 8.52
(s, 1H), 8.34 (s, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.76 (d, J = 8.4 Hz,
1H), 7.65 (d, J = 8.0 Hz, 1H), 7.55−7.49 (m, 2H), 7.41 (s, 1H), 7.38
(d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6 Hz, 2H), 7.30 (d, J = 8.0 Hz, 1H),
7.20 (d, J = 7.2 Hz, 2H), 7.08 (d, J = 7.6 Hz, 2H), 4.15−4.12 (m, 1H),
3.92−3.84 (m, 2H), 3.65−3.57 (m, 2H), 3.51 (dd, J = 13.6, 4.0 Hz,
1H), 3.42−3.25 (m, 5H), 3.22−3.15 (m, 2H), 2.94−2.90 (m, 1H),
2.88−2.75 (m, 4H), 1.65−1.56 (m, 1H), 1.50−1.37 (m, 3H), 1.27−
1.13 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 158.89, 157.01, 138.81,
137.38, 134.12, 132.28, 131.95, 131.67, 131.33, 131.11, 130.91, 130.71,
130.60, 129.35, 129.10, 128.42, 127.78, 126.90, 126.24, 125.75, 122.73,
120.83, 59.05, 57.14, 50.87, 46.09, 44.92, 44.45, 43.01, 35.55, 33.25,
32.72, 31.15, 26.51, 20.57. MS (ESI) m/z [M + H]+: 718.9.
1
HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.62 (s,
2H), 7.88−7.85 (m, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz,
1H), 7.56−7.50 (m, 2H), 7.41−7.39 (m, 2H), 7.36 (d, J = 8.4 Hz,
2H), 7.31 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 6.8 Hz, 1H), 7.18 (d, J = 8.4
Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 4.20−4.16 (m, 1H), 3.91−3.89 (m,
2H), 3.58−3.55 (m, 2H), 3.51 (dd, J = 14.0, 4.8 Hz, 1H), 3.38−3.31
(m, 4H), 3.21−3.18 (m, 3H), 2.95 (dd, J = 13.6, 8.8 Hz, 1H), 2.85−
2.80 (m, 4H), 1.61−1.50 (m, 1H), 1.50−1.38 (m, 3H), 1.25−1.13 (m,
2H). 13C NMR (100 MHz, CDCl3): δ 159.28, 157.01, 138.76, 137.18,
134.13, 132.38, 132.00, 131.66, 131.26, 131.10, 130.86, 130.72, 130.69,
129.38, 128.98, 128.49, 127.81, 126.94, 126.29, 125.76, 122.69, 120.92,
58.76, 57.20, 50.96, 46.19, 45.17, 44.66, 43.07, 35.64, 33.22, 32.81,
31.05, 26.53, 20.56. MS (ESI) m/z [M + H]+: 718.9.
(S)-3-(3,4-Dichlorophenethyl)-1-(4-((S)-3-(3,4-dichloropheneth-
yl)-2-iminoimidazolidin-4-yl)butyl)-4-(naphthalen-1-ylmethyl)-
imidazolidin-2-imine (206). Using general scheme for the synthesis
of F, compound 206 was synthesized using the following reagents:
3,4-dichlophenylacetic acid (R1), Boc-L-2-naphthylalanine (R2),
3,4-dichlorophenylacetic acid (R3). Final crude product was purified
1
by HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.62
(s, 2H), 7.83 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.68 (d, J =
8.0 Hz, 1H), 7.55−7.47 (m, 2H), 7.38−7.35 (m, 2H), 7.28−7.25 (m,
2H), 7.22 (d, J = 7.2 Hz, 1H), 7.20 (s, 1H), 7.15 (d, J = 7.6 Hz, 1H),
7.01 (d, J = 8.0 Hz, 1H), 4.13−4.02 (m, 1H), 4.00−3.90 (m, 1H),
3.87−3.77 (m, 1H), 3. 56−3.47 (m, 3H), 3.29−3.26 (m, 5H), 3.20−
3.17 (m, 2H), 2.97 (q, J = 8.8 Hz, 1H), 2.85−2.70 (m, 4H), 1.58−1.50
(m, 1H), 1.47−1.28 (m, 3H), 1.17−1.05 (m, 2H). 13C NMR (100
MHz, CDCl3): δ 159.19, 157.03, 138.84, 138.54, 134.09, 132.48,
132.26, 131.63, 131.36, 131.07, 130.91, 130.88, 130.82, 130.67, 130.55,
129.38, 129.00, 128.76, 128.45, 127.80, 126.93, 126.27, 125.75, 122.66,
58.79, 57.21, 51.03, 46.20, 45.11, 44.50, 43.00, 35.73, 32.74, 31.31,
26.52, 20.68. MS (ESI) m/z [M + H]+: 709.0.
(R)-3-(3,4-Dichlorophenethyl)-1-(4-((S)-3-(3,4-dichloropheneth-
yl)-2-iminoimidazolidin-4-yl)butyl)-4-(naphthalen-1-ylmethyl)-
imidazolidin-2-imine (203). Using general scheme for the synthesis
of F, compound 203 was synthesized using the following reagents:
3,4-dichlophenylacetic acid (R1), Boc-D-2-naphthylalanine (R2),
3,4-dichlorophenylacetic acid (R3). Final crude product was purified
(R)-4-Benzyl-3-((4-tert-butylcyclohexyl)methyl)-1-(4-((S)-3-(3-flu-
orophenethyl)-2-iminoimidazolidin-4-yl)butyl)imidazolidin-2-imine
(207). Using general scheme for the synthesis of F, compound 207
was synthesized using the following reagents: 3-fluorophenylacetic acid
1
by HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.61
(s, 2H), 7.83 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.0
Hz, 1H), 7.54−7.46 (m, 2H), 7.40−7.38 (m, 1H), 7.35 (d, J = 7.6 Hz, 1H),
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(R1), Boc-D-phenylalanine (R2), 4-tert-butyl-cyclohexanecarboxylic
acid, predominantly trans, 98+% (R3). Final crude product was
purified by HPLC as described above. 1H NMR (400 MHz, CDCl3): δ
8.53 (s, 1H), 8.25 (s, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.24 (d, J = 7.6 Hz,
1H), 7.20 (t, J = 6.8 Hz, 1H), 7.12 (d, J = 7.2 Hz, 2H), 7.07 (d, J = 7.6
Hz, 1H), 7.03 (d, J = 10.0 Hz, 1H), 6.84 (t, J = 8.4 Hz, 1H), 4.04−3.99
(m, 1H), 3.92−3.84 (m, 1H), 3.81−3.61 (m, 1H), 3. 53−3.42 (m,
3H), 3.37−3.26 (m, 2H), 3.24−3.17 (m, 3H), 3.11−2.95 (m, 2H),
2.90−2.80 (m, 2H), 2.73−2.65 (m, 1H), 1.83−1.75 (m, 1H), 1.73−
1.68 (m, 2H), 1.65−1.45 (m, 4H), 1.43−1.30 (m, 3H), 1.20−1.11 (m,
2H), 1.08 (d, J = 12.8 Hz, 1H), 1.03−0.98 (m, 1H), 0.95−0.90 (m,
2H), 0.81 (s, 9H). MS (ESI) m/z [M + H]+: 589.2.
(m, 1H), 1.00−0.97 (m, 3H), 0.86 (d, J = 6.8 Hz, 6H), 0.83 (s, 9H).
MS (ESI) m/z [M + H]+: 551.2.
(R)-3-((4-tert-Butylcyclohexyl)methyl)-4-(cyclohexylmethyl)-1-(4-
((S)-2-imino-3-(4-methylpentyl)imidazolidin-4-yl)butyl)-
imidazolidin-2-imine (212). Using general scheme for the synthesis of
F, compound 212 was synthesized using the following reagents:
4-methylvaleric acid (R1), Boc-D-cyclohexylalanine (R2), 4-tert-butyl-
cyclohexanecarboxylic acid, predominantly trans, 98+% R3). Final
1
crude product was purified by HPLC as described above. H NMR
(400 MHz, CDCl3): δ 8.54 (s, 1H), 8.28 (s, 2H), 3.84−3.78 (m, 2H),
3.66 (t, J = 9.2 Hz, 2H), 3.48−3.42 (m, 4H), 3.34−3.30 (m, 1H),
3.19−3.13 (m, 1H), 3.05 (dd, J = 14.4, 7.6 Hz, 1H), 2.95 (dd, J = 14.8,
5.6 Hz, 1H), 1.80−1.77 (m, 1H), 1.71 (d, J = 10.4 Hz, 6H), 1.64−1.63
(m, 2H), 1.60 (d, J = 8.0 Hz, 4H), 1.53 (q, J = 6.0 Hz, 4H), 1.49−1.44
(m, 2H), 1.33−1.29 (m, 3H), 1.27−1.24 (m, 1H), 1.23−1.21 (m, 3H),
1.20−1.18 (m, 1H), 1.17−1.15 (m, 2H), 1.13−1.10 (m, 1H), 1.07−
0.1.05 (m, 1H), 1.02−0.99 (m, 2H), 0.96−0.92 (m, 3H), 0.86 (d, J =
6.8 Hz, 6H), 0.82 (s, 9H). MS (ESI) m/z [M + H]+: 557.2.
(R)-3-((4-tert-Butylcyclohexyl)methyl)-4-(cyclohexylmethyl)-1-(4-
((S)-3-(3-fluorophenethyl)-2-iminoimidazolidin-4-yl)butyl)-
imidazolidin-2-imine (208). Using general scheme for the synthesis
of F, compound 208 was synthesized using the following reagents:
3-fluorophenylacetic acid (R1), Boc-D-cyclohexylalanine (R2), 4-tert-
butyl-cyclohexanecarboxylic acid, predominantly trans, 98+% (R3).
1
(R)-4-Benzyl-3-(3-bromophenethyl)-1-(4-((S)-3-(3-fluoropheneth-
yl)-2-iminoimidazolidin-4-yl)butyl)imidazolidin-2-imine (213).
Using general scheme for the synthesis of F, compound 213 was
synthesized using the following reagents: 3-fluorophenylacetic acid
(R1), Boc-D-phenylalanine (R2), 3-bromophenylacetic acid (R3). Final
Final crude product was purified by HPLC as described above. H
NMR (400 MHz, D2O): δ 7.44 (q, J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz,
1H), 7.12 (t, J = 8.0 Hz, 2H), 4.06−4.00 (m, 1H), 3.80 (q, J = 9.6 Hz,
2H), 3.72−3.66 (m, 2H), 3.62−3.55 (m, 1H), 3.48−3.38 (m, 1H),
3.36−3.31 (m, 2H), 3.29−3.21 (m, 1H), 3.16 (d, J = 7.6 Hz, 1H), 2.99
(t, J = 6.4 Hz, 2H), 2.75−2.73 (m, 1H), 1.86−1.71 (m, 8H), 1.66−
1.54 (m, 8H), 1.43−1.37(m, 2H), 1.31−1.25 (m, 5H), 1.10−1.08 (m,
2H), 1.04−1.03 (m, 2H), 0.99−0.93 (m, 2H), 0.89 (s, 9H). MS (ESI)
m/z [M + H]+: 595.2.
(R)-4-Benzyl-3-((4-tert-butylcyclohexyl)methyl)-1-(4-((S)-3-heptyl-
2-iminoimidazolidin-4-yl)butyl)imidazolidin-2-imine (209). Using
general scheme for the synthesis of F, compound 209 was synthesized
using the following reagents: heptanoic acid (R1), Boc-D-phenylalanine
(R2), 4-tert-butyl-cyclohexanecarboxylic acid, predominantly trans, 98+
% (R3). Final crude product was purified by HPLC as described above.
1H NMR (400 MHz, CDCl3): δ 8.54 (s, 1H), 8.00 (s, 2H), 7.32 (t, J =
7.2 Hz, 2H), 7.27−7.25 (m, 1H), 7.14 (d, J = 7.2 Hz, 2H), 4.06−4.02
(m, 1H), 3.85−3.80 (m, 1H), 3.73−3.65 (m, 2H), 3.55−3.47 (m, 2H),
3.40−3.32 (m, 2H), 3.29−3.22 (m, 2H), 3.12−3.05 (m, 2H), 3.03−
2.97 (m, 1H), 2.76−2.68 (m, 1H), 1.83−1.75 (m, 1H),1.71−1.65 (m,
3H),1.57−1.41 (m, 7H), 1.27−1.25 (m, 11H), 1.13−1.04 (m, 2H),
1.00−0.95 (m, 2H), 0.86 (d, J = 6.4 Hz, 3H), 0.81 (s, 9H). MS (ESI)
m/z [M + H]+: 565.2.
1
crude product was purified by HPLC as described above. H NMR
(400 MHz, CDCl3): δ 8.54 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.31
(s, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.24 (d, J = 7.2 Hz, 1H), 7.22−7.19
(m, 2H), 7.15 (t, J = 7.6 Hz, 2H), 7.03 (d, J = 6.8 Hz, 3H), 6.85 (t, J =
8.0 Hz, 1H), 4.03−3.98 (m, 1H), 3.87−3.80 (m, 1H), 3.71−3.64 (m,
1H), 3.62−3.50 (m, 2H), 3.36 (t, J = 9.6 Hz, 2H), 3.32−3.20 (m, 4H),
3.12 (dd, J = 9.6, 6.4 Hz, 1H), 2.91 (d, J = 4.0 Hz, 1H), 2.87−2.80 (m,
4H), 2.60 (d, J = 13.2, 7.6 Hz, 1H), 1.62−1.50 (m, 1H), 1.45−1.30
(m, 3H), 1.21−1.14 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 162.98
(J = 244.2 Hz), 159.00, 156.85, 140.99 (J = 7.6 Hz), 140.77, 135.27,
132.02, 130.61, 130.35 (J = 7.1 Hz), 130.07, 129.19 (J = 4.0 Hz),
128.02, 127.50, 125.04, 122.67, 121.63, 115.95 (J = 20.9 Hz), 113.68
(J = 21.7 Hz), 58.94, 58.22, 50.64, 46.10, 45.02, 44.43, 43.12, 38.30,
33.20, 31.05, 26.42, 20.55. MS (ESI) m/z [M + H]+: 619.0.
(R)-3-(3-Bromophenethyl)-4-(cyclohexylmethyl)-1-(4-((S)-3-(3-flu-
orophenethyl)-2-iminoimidazolidin-4-yl)butyl)imidazolidin-2-imine
(214). Using general scheme for the synthesis of F, compound 214
was synthesized using the following reagents: 3-fluorophenylacetic acid
(R1), Boc-D-cyclohexylalanine (R2), 3-bromophenylacetic acid (R3).
1
(R)-3-((4-tert-Butylcyclohexyl)methyl)-4-(cyclohexylmethyl)-1-(4-
((S)-3-heptyl-2-iminoimidazolidin-4-yl)butyl)imidazolidin-2-imine
(210). Using general scheme for the synthesis of F, compound 210
was synthesized using the following reagents: heptanoic acid (R1),
Boc-D-cyclohexylalanine (R2), 4-tert-butyl-cyclohexanecarboxylic acid,
predominantly trans, 98+% (R3). Final crude product was purified by
Final crude product was purified by HPLC as described above. H
NMR (400 MHz, CDCl3): δ 8.56 (s, 2H), 7.35 (s, 1H), 7.32 (d, J =
8.0 Hz, 1H), 7.22−7.19 (m, 2H), 7.14 (t, J = 7.6 Hz, 1H), 7.02 (t, J =
8.0 Hz, 2H), 6.84 (t, J = 8.0 Hz, 1H), 3.95−3.85 (m, 2H), 3.58−3.50
(m, 2H), 3.50 (d, J = 8.8 Hz, 1H), 3.46−3.42 (m, 2H), 3.37−3.30 (m,
3H), 3.25−3.17 (m, 1H), 3.04−3.00 (m, 1H), 2.87−2.78 (m, 4H),
1.67−1.62 (m, 4H), 1.50−1.45 (m, 6H), 1.21 (d, J = 6.4 Hz, 2H),
1.16−1.11 (m, 5H), 0.91 (t, J = 11.6 Hz, 1H), 0.76 (t, J = 6.8 Hz, 1H).
13C NMR (100 MHz, CDCl3) δ 162.96 (J = 244.2 Hz), 159.10,
156.94, 141.01 (J = 7.3 Hz), 140.80, 131.98, 130.53, 130.30 (J = 8.1
Hz), 130.01, 127.93, 124.98 (d, J = 2.0 Hz), 122.69, 115.90 (J = 20.7
Hz), 113.64 (J = 20.7 Hz), 58.83, 55.56, 51.89, 46.13, 45.14, 43.94,
43.07, 39.87, 34.28, 34.11, 33.20, 32.32, 31.30, 26.54, 26.27, 26.16,
20.66. MS (ESI) m/z [M + H]+: 625.0.
1
HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.58 (s,
1H), 3.83−3.79 (m, 2H), 3.66 (t, J = 8.8 Hz, 2H), 3.50−3.44 (m, 4H),
3.37−3.30 (m, 1H), 3.19−3.13 (m, 1H), 3.09−3.03 (m, 1H), 2.96 (dd,
J = 15.2, 6.0 Hz, 1H), 1.83−1.77 (m, 2H), 1.75−1.72 (m, 2H), 1.71−
1.67 (m, 4H), 1.66−1.64 (m, 2H), 1.62−1.59 (m, 3H), 1.57−1.48 (m,
4H), 1.37−1.32 (m, 2H), 1.30−1.22 (m, 11H), 1.20−1.16 (m, 2H),
1.14−1.11 (m, 1H), 1.06 (d, J = 8.8 Hz, 1H), 1.03−1.00 (m, 2H),
0.97−0.93 (m, 3H), 0.88−0.86 (m, 3H), 0.82 (s, 9H). MS (ESI) m/z
[M + H]+: 571.3.
(R)-4-Benzyl-3-((4-tert-butylcyclohexyl)methyl)-1-(4-((S)-2-imino-
3-(4-methylpentyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine
(211). Using general scheme for the synthesis of F, compound 211
was synthesized using the following reagents: 4-methylvaleric acid
(R1), Boc-D-phenylalanine (R2), 4-tert-butyl-cyclohexanecarboxylic
acid, predominantly trans, 98+% (R3). Final crude product was
purified by HPLC as described above. 1H NMR (400 MHz, CDCl3): δ
8.55 (s, 1H), 7.33 (t, J = 7.2 Hz, 2H), 7.28−7.25 (m, 1H), 7.13 (d, J =
7.2 Hz, 2H), 4.07−4.02 (m, 1H), 3.83−3.80 (m, 1H), 3.71−3.64 (m,
1H), 3.51−3.45 (m, 2H), 3.30−3.21 (m, 4H), 3.10−3.06 (m, 2H),
3.04−2.98 (m, 2H), 2.76−2.68 (m, 1H), 1.83−1.78 (m, 1H), 1.71 (d,
J = 10.4 Hz, 1H), 1.66−1.58 (m, 3H), 1.56−1.50 (m, 5H), 1.44−1.38
(m, 2H), 1.27−1.23 (m, 2H), 1.16 (q, J = 7.2 Hz, 3H), 1.07−1.04
(R)-4-Benzyl-3-(3-bromophenethyl)-1-(4-((S)-3-heptyl-2-imino-
imidazolidin-4-yl)butyl)imidazolidin-2-imine (215). Using general
scheme for the synthesis of F, compound 215 was synthesized using
the following reagents: heptanoic acid (R1), Boc-D-phenylalanine (R2),
3-bromophenylacetic acid (R3). Final crude product was purified by
1
HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.56 (s,
1H), 8.06 (s, 2H), 7.33 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H),
7.23 (t, J = 7.2 Hz, 2H), 7.15 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 6.8 Hz,
2H), 4.17−4.05 (m, 1H), 3.85−3.83 (m, 1H), 3.65−3.63 (m, 2H),
3.52−3.48 (m, 1H), 3.37−3.30 (m, 5H), 3.11 (dd, J = 9.6, 6.4 Hz,
1H), 3.07−3.03 (m, 1H), 2.90 (d, J = 4.4 Hz, 1H), 2.87 (d, J = 4.0 Hz,
2H), 2.59 (dd, J = 13.6, 8.0 Hz, 1H), 1.67−1.65 (m, 1H), 1.49−1.41
(m, 5H), 1.26−1.23 (m, 10H), 0.83 (t, J = 6.8 Hz, 3H). 13C NMR
(100 MHz, CDCl3): δ 158.93, 156.87, 140.78, 135.19, 132.00, 130.56,
10113
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Journal of Medicinal Chemistry
Article
130.02, 129.18, 129.13, 127.99, 127.48, 122.61, 58.22, 58.15, 50.57,
46.03, 45.01, 44.32, 41.97, 38.26, 33.17, 31.81, 31.22, 29.16, 27.19,
26.61, 22.68, 20.60, 14.17. MS (ESI) m/z [M + H]+: 595.0.
δ 158.23, 156.14, 148.37, 146.78, 138.85, 135.69, 134.87, 129.71, 128.84,
127.23, 124.73, 58.87, 56.84, 49.54, 47.88, 45.68, 44.10, 43.60, 42.76,
36.63, 31.72, 30.37, 29.92, 29.90, 27.56, 26.84, 25.71, 20.34. MS (ESI)
m/z C35H54N7 [M + H]+: 572.2.
(R)-3-(3-Bromophenethyl)-4-(cyclohexylmethyl)-1-(4-((S)-3-hep-
tyl-2-iminoimidazolidin-4-yl)butyl)imidazolidin-2-imine (216).
Using general scheme for the synthesis of F, compound 216 was
synthesized using the following reagents: heptanoic acid (R1), Boc-D-
cyclohexylalanine (R2), 3-bromophenylacetic acid (R3). Final crude
(S)-4-Benzyl-3-((4-tert-butylcyclohexyl)methyl)-1-(4-((S)-2-imino-
3-(2-(pyridin-3-yl)ethyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine
(302). Using general scheme for the synthesis of F, compound 302
was synthesized using the following reagents: 3-pyridinylacetic acid
(R1), Boc-L-phenylalanine (R2), 4-tert-butyl-cyclohexanecarboxylic
acid, predominantly trans, 98+% (R3). Final crude product was
purified by HPLC as described above. 1H NMR (400 MHz, CDCl3): δ
8.62 (s, 1H), 8.53 (s, 1H), 8.40 (d, J = 3.6 Hz, 1H), 7.78 (d, J = 8.0
Hz, 1H), 7.33 (d, J = 7.2 Hz, 2H), 7.28−7.21 (m, 2H), 7.14 (d, J = 6.8
Hz, 2H), 4.10−4.03 (m, 1H), 4.01−3.96 (m, 1H), 3.76 (dd, J = 15.2,
8.4 Hz, 1H), 3.65−3.55 (m, 2H), 3.48 (t, J = 9.6 Hz, 2H), 3.43−3.39
(m, 1H), 3.32−3.19 (m, 3H), 3.09−3.06 (m, 1H), 3.05−3.03 (m, 1H),
2.92−2.86 (m, 2H), 2.72 (dd, J = 13.6, 8.4 Hz, 1H), 1.79−1.77 (m,
2H), 1.72 (d, J = 11.6 Hz, 2H), 1.60 (t, J = 7.6 Hz, 2H), 1.51−1.47 (m,
1H), 1.42 (t, J = 7.2 Hz, 2H), 1.21−1.18 (m, 2H), 1.10−1.02 (m, 2H),
0.97−0.96 (m, 3H), 0.82 (s, 9H). 13C NMR (100 MHz, D2O): δ
158.22, 156.49, 148.49, 146.86, 138.60, 135.65, 134.72, 129.60, 128.80,
127.19, 124.60, 58.93, 57.44, 49.69, 48.59, 47.58, 45.77, 44.17, 42.80,
36.54, 35.80, 31.67, 30.59, 30.52, 29.97, 27.05, 26.47, 25.74, 20.48. MS
(ESI) m/z calcd for [M + H]+: 572.2.
(S)-1-((4-tert-Butylcyclohexyl)methyl)-3-(4-(2-imino-3-(2-(pyridin-
3-yl)ethyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine (303). Using
general scheme for the synthesis of F, compound 303 was synthesized
using the following reagents: 3-pyridinylacetic acid (R1), Boc-glycine
(R2), 4-tert-butyl-cyclohexanecarboxylic acid, predominantly trans,
98+% (R3). Final crude product was purified by HPLC as described
above. 1H NMR (400 MHz, D2O): δ 8.54−8.50 (m, 2H), 7.93 (d, J =
7.6 Hz, 1H), 7.55 (dd, J = 7.6, 5.2 Hz, 1H), 3.96−3.89 (m, 1H), 3.73
(d, J = 9.2 Hz, 1H), 3.71−3.67 (m, 4H), 3.64−3.62 (m, 1H), 3.37−
3.31 (m, 4H), 3.13 (d, J = 7.2 Hz, 2H), 3.07−3.03 (m, 2H), 1.85−1.82
(m, 2H), 1.76 (d, J = 10.0 Hz, 2H), 1.69−1.51 (m, 6H), 1.31−1.25
(m, 2H), 1.01 (d, J = 4.4 Hz, 3H), 0.97−0.94 (m, 1H), 0.86 (s, 9H).
13C NMR (100 MHz, D2O): δ 158.24, 157.19, 148.17, 146.57, 138.99,
1
product was purified by HPLC as described above. H NMR (400
MHz, CDCl3): δ 8.56 (s, 1H), 8.31 (s, 1H), 7.35 (s, 1H), 7.33 (d, J =
8.0 Hz, 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 3.96−
3.94 (m, 1H), 3.85−3.83 (m, 1H), 3.71−3.64 (m, 1H), 3.53 (t, J = 8.8
Hz, 2H), 3.44−3.42 (m, 2H), 3.35−3.31 (m, 3H), 3.04 (t, J = 8.0 Hz,
2H), 2.87−2.82 (m, 2H), 1.70−1.65 (m, 4H), 1.55−1.44 (m, 8H),
1.26−1.23 (m, 11H), 1.18−1.10 (m, 5H), 0.95−0.89 (m, 1H), 0.84
(t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 159.19, 156.98,
140.80, 131.98, 130.53, 129.99, 127.94, 122.67, 58.09, 55.56, 51.90,
46.05, 45.21, 43.93, 41.91, 39.89, 34.28, 34.11, 33.19, 31.82, 31.32,
29.15, 27.21, 26.65, 26.26, 26.16, 25.96, 22.69, 20.66, 14.18. MS (ESI)
m/z [M + H]+: 601.1.
(R)-4-Benzyl-3-(3-bromophenethyl)-1-(4-((S)-2-imino-3-(4-
methylpentyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine (217).
Using general scheme for the synthesis of F, compound 217 was
synthesized using the following reagents: 4-methylvaleric acid (R1),
Boc-D-phenylalanine (R2), 3-bromophenylacetic acid (R3). Final crude
product was purified by HPLC as described above. 1H NMR (400 MHz,
CDCl3): δ 8.57 (s, 1H), 8.26 (s, 1H), 7.34 (s, 1H), 7.32−7.31 (m, 1H),
7.28 (d, J = 7.2 Hz, 2H), 7.24 (d, J = 7.2 Hz, 1H), 7.22−7.20 (m, 1H),
7.16 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 7.2 Hz, 2H), 4.13−4.07 (m, 1H),
3.85−3.82 (m, 1H), 3.65−3.60 (m, 2H), 3.51−3.48 (m, 1H), 3.37−3.33
(m, 5H), 3.12 (dd, J = 9.6, 6.4 Hz, 1H), 3.06−3.03 (m, 1H), 2.90 (d, J =
4.4 Hz, 1H), 2.87 (d, J = 4.0 Hz, 2H), 2.59 (dd, J = 13.6, 8.0 Hz, 1H),
1.67−1.60 (m, 1H), 1.52−1.46 (m, 4H), 1.42−1.36 (m, 2H), 1.26−1.23
(m, 2H), 1.18−1.12 (m, 2H), 0.84 (d, J = 6.8 Hz, 6H). 13C NMR (100
MHz, CDCl3): δ 159.11, 156.90, 140.76, 135.21, 132.01, 130.58, 130.03,
129.19, 129.14, 127.99, 127.49, 122.62, 58.16, 50.61, 46.04, 45.05, 44.35,
42.11, 38.28, 35.58, 33.17, 31.25, 27.93, 26.60, 25.02, 22.67, 22.59, 20.58.
MS (ESI) m/z [M + H]+: 581.0.
(R)-3-(3-Bromophenethyl)-4-(cyclohexylmethyl)-1-(4-((S)-2-
imino-3-(4-methylpentyl)imidazolidin-4-yl)butyl)imidazolidin-2-
imine (218). Using general scheme for the synthesis of F, compound
218 was synthesized using the following reagents: 4-methylvaleric acid
(R1), Boc-D-cyclohexylalanine (R2), 3-bromophenylacetic acid (R3).
Final crude product was purified by HPLC as described above.
1H NMR (400 MHz, CDCl3): δ 8.57 (s, 1H), 8.25 (s, 1H), 7.35 (d, J =
8.0 1H), 7.32 (s, 1H), 7.22 (d, J = 7.2 Hz, 1H), 7.15 (t, J = 8.0 Hz,
1H), 3.96−3.92 (m, 1H), 3.85−3.83 (m, 1H), 3.75−3.65 (m, 1H),
3.53 (t, J = 8.8 Hz, 2H), 3.45−3.41 (m, 2H), 3.38−3.31 (m, 3H), 3.03
(t, J = 8.0 Hz, 2H), 2.86−2.84 (m, 2H), 1.70−1.63 (m, 4H), 1.54−
1.43 (m, 10H), 1.38−1.25 (m, 2H), 1.20−1.09 (m, 8H), 0.84 (d, J =
6.4 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 159.13, 156.95, 140.86,
131.99, 130.54, 129.98, 127.97, 122.66, 58.09, 55.57, 51.89, 46.05,
45.23, 43.96, 42.11, 39.88, 35.59, 34.28, 33.21, 32.31, 31.30, 27.93,
26.71, 26.16, 25.96, 25.02, 22.67, 20.63. MS (ESI) m/z [M + H]+:
587.1.
134.90, 124.73, 58.89, 50.95, 47.93, 47.62, 46.14, 45.73, 45.38, 42.65,
35.52, 31.68, 31.53, 30.43, 30.40, 26.89, 26.80, 26.31, 20.45. MS (ESI)
m/z [M + H]+: 482.1.
(R)-4-Benzyl-3-((4-tert-butylcyclohexyl)methyl)-1-(4-((R)-2-imino-
3-(2-(pyridin-3-yl)ethyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine
(304). Using general scheme for the synthesis of F, compound 304 was
synthesized using the following reagents: 3-pyridinylacetic acid (R1),
Boc-D-phenylalanine (R2), 4-tert-butyl-cyclohexanecarboxylic acid, pre-
dominantly trans, 98+% (R3). The only variation of the general
compound synthesis for compound 304 was utilizing Fmoc-D-Lys(Boc)-
OH instead of Boc-L-Lys(Fmoc)-OH. Final crude product was purified
1
by HPLC as described above. H NMR (400 MHz, CDCl3): δ 8.93
(s, 2H), 8.61 (s, 1H), 8.52 (s, 1H), 8.41 (s, 1H), 7.76 (d, J = 6.8 Hz,
1H), 7.33 (t, J = 7.2 Hz, 2H), 7.27 (d, J = 6.4 Hz, 1H), 7.24−7.20 (m,
1H), 7.14 (d, J = 7.2 Hz, 2H), 4.10−4.04 (m, 1H), 4.01−3.96 (m, 1H),
3.76 (dd, J = 14.4, 8.4 Hz, 1H), 3.65−3.57 (m, 2H), 3.47 (t, J = 9.6 Hz,
2H), 3.43−3.39 (m, 1H), 3.29−3.23 (m, 3H), 3.09−3.06 (m, 1H),
3.05−3.02 (m, 1H), 2.94−2.84 (m, 2H), 2.72 (dd, J = 13.2, 8.4 Hz, 1H),
1.79−1.77 (m, 2H), 1.72 (d, J = 11.2 Hz, 2H), 1.65−1.55 (m, 2H),
1.51−1.37 (m, 3H), 1.24−1.16 (m, 2H), 1.08−1.04 (m, 2H), 0.99−0.94
(m, 3H), 0.82 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 159.35, 157.28,
150.20, 147.99, 137.26, 135.16, 133.97, 129.30, 129.21, 127.58, 123.90,
58.63, 57.78, 50.34, 48.82, 47.87, 46.27, 45.19, 43.03, 37.76, 36.18, 32.54,
31.20, 30.71, 27.66, 26.85, 26.50, 20.61. MS (ESI) m/z [M + H]+: 572.2.
(R)-1-((4-tert-Butylcyclohexyl)methyl)-3-(4-(2-imino-3-(2-(pyridin-
3-yl)ethyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine (305). Using
general scheme for the synthesis of F, compound 305 was synthesized
using the following reagents: 3-pyridinylacetic acid (R1), Boc-glycine
(R2), 4-tert-butyl-cyclohexanecarboxylic acid, predominantly trans,
98+% (R3). The only variation of the general compound synthesis
for compound 305 was utilizing Fmoc-D-Lys(Boc)-OH instead of Boc-
L-Lys(Fmoc)-OH. Final crude product was purified by HPLC as
(R)-4-Benzyl-3-((4-tert-butylcyclohexyl)methyl)-1-(4-((S)-2-imino-
3-(2-(pyridin-3-yl)ethyl)imidazolidin-4-yl)butyl)imidazolidin-2-imine
(301). Using general scheme for the synthesis of F, compound 301
was synthesized using the following reagents: 3-pyridinylacetic acid
(R1), Boc-D-phenylalanine (R2), 4-tert-butyl-cyclohexanecarboxylic
acid, predominantly trans, 98+% (R3). Final crude product was
1
purified by HPLC as described above. H NMR (400 MHz, D2O): δ
8.55−8.45 (m, 2H), 7.87 (d, J = 8.0 Hz, 1H), 7.51 (dd, J = 7.2, 5.2 Hz,
1H), 7.42 (t, J = 7.2 Hz, 2H), 7.36 (t, J = 6.8 Hz, 1H), 7.29 (d, J = 6.8
Hz, 2H), 4.36−4.30 (m, 1H), 3.85−3.79 (m, 1H), 3.73−3.65 (m, 3H),
3.61−3.54 (m, 1H), 3.45 (dd, J = 10.4, 4.8 Hz, 1H), 3.30−3.25 (m,
3H), 3.06−3.02 (m, 5H), 2.94 (dd, J = 14.4, 4.0 Hz, 1H), 1.86 (d, J =
2.4 Hz, 1H), 1.80 (d, J = 7.6 Hz, 2H), 1.75 (d, J = 12.8 Hz, 1H), 1.69−
1.65 (m, 1H), 1.60−1.57 (m, 1H), 1.47−1.39 (m, 1H), 1.37−1.23
(m, 2H), 1.10−0.96 (m, 7H), 0.87 (s, 9H). 13C NMR (100 MHz, D2O):
1
described above. H NMR (400 MHz, D2O): δ 8.54−8.46 (m, 2H),
10114
dx.doi.org/10.1021/jm401543h | J. Med. Chem. 2013, 56, 10103−10117

Journal of Medicinal Chemistry
Article
7.98 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 6.4 Hz, 1H), 3.95−3.91 (m, 1H),
3.74 (d, J = 9.2 Hz, 1H), 3.73−3.67 (m, 5H), 3.65−3.59 (m, 1H),
3.37−3.31 (m, 3H), 3.13 (d, J = 7.2 Hz, 2H), 3.08−3.05 (m, 2H),
1.87−1.80 (m, 2H), 1.76 (d, J = 10.8 Hz, 3H), 1.69−1.62 (m, 4H),
1.59−1.51 (m, 1H), 1.34−1.25 (m, 2H), 1.01 (d, J = 4.4 Hz, 3H), 0.96
(d, J = 12.4 Hz, 1H), 0.87 (s, 9H). 13C NMR (100 MHz, CCl3D): δ
159.41, 158.06, 150.16, 147.98, 137.22, 134.05, 123.91, 58.64, 51.72,
47.91, 46.51, 46.20, 45.70, 45.41, 42.91, 36.26, 32.54, 31.36, 30.89,
30.78, 27.67, 26.78, 26.70, 20.73. MS (ESI) m/z [M + H]+: 482.1.
(R)-3-((4-tert-Butylcyclohexyl)methyl)-4-(cyclohexylmethyl)-1-(4-
((S)-2-imino-3-(2-(pyridin-3-yl)ethyl)imidazolidin-4-yl)butyl)-
imidazolidin-2-imine (306). Using general scheme for the synthesis
of F, compound 306 was synthesized using the following reagents:
3-pyridinylacetic acid (R1), Boc-D-cyclohexylalanine (R2), 4-tert-butyl-
cyclohexanecarboxylic acid (R3). Final crude product was purified by
compounds from the screening of the library.26,35,36 Equimolar
isokinetic ratios have been previously determined and calculated for
each of the amino and carboxylic acids utilized for the respective
mixtures.43,44 The bis-cyclic guanidine library 1169 has a total diversity
of 45864 compounds (42 × 26 × 42 = 45864). The R1 and R3
positions as shown in Scheme 1 (F) each consist of 42 carboxylic acids
and the R2 contains 26 amino acids.
Scaffold Ranking Library. The scaffold ranking library contains one
sample representing each of the positional scanning libraries. This sample
contains an approximate equal molar amount of each compound in that
library. So, for example, the sample 1169 in the scaffold ranking library
contains 45864 compounds in approximately equal molar amounts. These
samples can be prepared by mixing the cleaved products of the complete
positional scanning library, as was the case for sample 1169, or they can be
synthesized directly as a single mixture.26,27
Calculation of xIC50s. It has been shown32 that the harmonic
mean of the IC50s of constituents within a mixture accurately models
the resulting IC50 of the mixture. Because percent responses are
generally used as the initial screening metric, it is often necessary (as it
was in this case) to extrapolate IC50s for samples using the equation
1
HPLC as described above. H NMR (400 MHz, D2O): δ 8.55−8.45
(m, 2H), 7.88 (d, J = 7.6 Hz, 1H), 7.51−7.49 (m, 1H), 4.00−3.90 (m,
2H), 3.82 (t, J = 9.2 Hz, 1H), 3.77−3.72 (m, 1H), 3.68 (t, J = 7.2 Hz,
1H), 3.62−3.55 (m, 1H), 3.37−3.33 (m, 4H), 3.24−3.11 (m, 2H),
3.06−2.97 (m, 2H), 1.92−1.75 (m, 5H), 1.74−1.60 (m, 8H), 1.58−
1.50 (m, 2H), 1.43−1.36 (m, 2H), 1.32−1.25 (m, 4H), 1.17 (d, J =
10.8 Hz, 1H), 1.02−0.92 (m, 7H), 0.87 (s, 9H). 13C NMR (100 MHz,
D2O): δ 158.27, 156.96, 148.71, 146.99, 138.36, 124.56, 59.01, 55.68,
51.42, 48.32, 47.54, 45.81, 44.50, 42.87, 39.08, 36.01, 34.02, 33.63,
32.17, 31.89, 30.71, 30.12, 27.28, 26.68, 26.09, 25.85, 20.59. MS (ESI)
m/z [M + H]+: 578.2.
⎛
⎞
⎛
⎝
⎞
⎠
100
⎜
xIC50 = min [x]
⎜
⎟
⎟
⎠
⎜
⎟
− 1 × 1000
⎜
⎟
%
Resp
⎝
Here [x] is the concentration tested and %resp is the percent inhibition
value obtained at that concentration. Obviously, these values are
extrapolations and not a substitute for actual experimentally determined
IC50s, but they are sufficient for the order-of-magnitude estimation used in
the Harmonic Mean Analysis of position scanning libraries.
Prediction of Selectivity. For a sample with IC50 x1 against α3β4
and x2 against α4β2, we define the selectivity over α3β4 as
(S)-3-((4-tert-Butylcyclohexyl)methyl)-4-(cyclohexylmethyl)-1-(4-
((S)-2-imino-3-(2-(pyridin-3-yl)ethyl)imidazolidin-4-yl)butyl)-
imidazolidin-2-imine (307). Using general scheme for the synthesis
of F, compound 306 was synthesized using the following reagents:
3-pyridinylacetic acid (R1), Boc-L-cyclohexylalanine (R2), 4-tert-butyl-
cyclohexanecarboxylic acid, predominantly trans, 98+% (R3). Final
1
x1 − x2
x2
crude product was purified by HPLC as described above. H NMR
s =
(400 MHz, CCl3D): δ 8.97 (s, 1H), 8.61 (s, 1H), 8.52 (m, 1H), 8.42
(s, 1H), 7.75 (d, J = 6.8 Hz, 1H), 7.25−7.22 (m, 1H), 4.01−3.93
(m, 1H), 3.82 (d, J = 7.2 Hz, 2H), 3.65 (t, J = 9.2 Hz, 2H), 3.58−3.52
(m, 3H), 3.44−3.37 (m, 2H), 3.25−3.21 (m, 1H), 3.16 (t, J = 8.0 Hz,
1H), 2.97 (dd, J = 14.8, 5.6 Hz, 1H), 2.90−2.86 (m, 1H), 1.81−1.78
(m, 2H), 1.72 (d, J = 10.8 Hz, 5H),1.65−1.63 (m, 4H), 1.56−1.48 (m,
3H), 1.33−1.14 (m, 8H), 1.03−0.93 (m, 7H), 0.81 (s, 9H). 13C NMR
(100 MHz, CDCl3): δ 159.41, 157.33, 150.19, 147.99, 137.24, 134.03,
58.63, 55.21, 51.81, 48.40, 47.87, 46.27, 45.31, 43.00, 39.55, 36.09,
34.43, 34.38, 32.53, 31.38, 31.21, 30.72, 27.66, 26.87, 26.59, 26.26,
26.06, 20.69. MS (ESI) m/z [M + H]+: 578.2.
To predict the selectivity of an individual compound based on the
values of the positional scanning mixture, we used the standard
biometrical analysis approach45−47 of using the product of the mixture
activity values. In particular, if a compound has functionality A1 at
position R1, A2 at position R2, and A3 at position R3, then its
Biometrical Analysis Score would be
1/3
⎛
⎞
⎟
⎟
(Activity of A1 Mixture at the R1 Position)×
⎜
⎜
BA = (Activity of A2 Mixture at the R2 Position)×
⎜
⎜
⎟
⎟
(R)-3-((4-tert-Butylcyclohexyl)methyl)-4-(cyclohexylmethyl)-1-(4-
((R)-2-imino-3-(2-(pyridin-3-yl)ethyl)imidazolidin-4-yl)butyl)-
imidazolidin-2-imine (308). Using general scheme for the synthesis
of F, compound 308 was synthesized using the following reagents:
3-pyridinylacetic acid (R1), Boc-D-cyclohexylalanine (R2), 4-tert-butyl-
cyclohexanecarboxylic acid, predominantly trans, 98+% (R3). The only
variation of the general compound synthesis for compound 308 was
utilizing Fmoc-D-Lys(Boc)-OH instead of Boc-L-Lys(Fmoc)-OH. Final
(Activity of A3 Mixture at the R3 Position)
⎝
⎠
Given BA values for α3β4 and α4β2, the predicted selectivity is then
found using the same formula as above:
BA1 − BA2
sp =
BA2
1
crude product was purified by HPLC as described above. H NMR
Cell Culture. KXα3β4R2 and KXα4β2R2 cells, containing rat α3β4
and α4β2 nAChR, respectively (obtained from Drs. Kenneth Kellar
and Yingxian Xiao, Georgetown University), were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with
10% fetal bovine serum (FBS), 0.5% penicillin/streptomycin, and 0.4
mg/mL of geneticin and were maintained in an atmosphere of 7.5%
CO2 in a humidified incubator at 37 °C. For binding assays, cells were
passaged on 150 mm dishes and harvested when confluent.
Binding Assays. Cells were harvested by scraping the plates with a
rubber policeman, suspended in 50 mM Tris buffer pH 7.4,
homogenized using a Polytron homogenizer, and the centrifugation
was repeated twice at 20000g (13500 rpm) for 20 min. For binding,
the cell membranes were incubated with the test compounds or
mixtures in the presence of 0.3 nM of [3H]epibatidine. After 2 h of
incubation, at room temperature, samples were filtered, using a
Tomtec cell harvester, through glass fiber filters that had been
presoaked in 0.05% polyethyleneimine. Filters were counted on a betaplate
reader (Wallac). Nonspecific binding was determined by using 1 μM of
(400 MHz, CDCl3): δ 8.59 (s, 1H), 8.54−8.48 (m, 1H), 8.40 (s, 1H),
7.78 (d, J = 7.2 Hz, 1H), 7.23−7.21 (m, 1H), 4.03−3.96 (m, 1H), 3.82
(d, J = 8.4 Hz, 1H), 3.66 (t, J = 9.2 Hz, 3H), 3.61−3.58 (m, 2H),
3.54−3.52 (m, 1H), 3.46−3.40 (m, 2H), 3.26−3.23 (m, 1H), 3.17
(t, J = 8.0 Hz, 1H), 2.97 (dd, J = 15.2, 5.6 Hz, 1H), 2.91−2.85 (m.
1H), 1.78−1.61 (m, 12H), 1.56−1.51 (m, 4H), 1.29−1.24 (m, 4H),
1.23−1.13 (m, 4H), 1.05−0.99 (m, 3H), 0.96−0.93 (m, 2H), 0.80
(s, 9H). 13C NMR (100 MHz, CDCl3): δ 159.34, 157.26, 150.23,
147.98, 137.33, 133.96, 123.87, 58.66, 55.21, 51.84, 48.43, 47.84,
46.33, 45.43, 43.10, 39.55, 36.09, 34.37, 32.53, 31.39, 31.18, 30.83,
30.69, 27.66, 26.87, 26.73, 26.25, 26.05, 20.73. MS (ESI) m/z calcd for
C35H60N7 [M + H]+, 578.49; found, 578.15.
Positional Scanning Library 1169. Utilizing Scheme 1, positional
scanning library 1169 was synthesized. The positional scanning library
incorporates both individual and mixtures of amino acids (R2) and
carboxylic acid (R1 and R3). The synthetic technique facilitates the
generation of information regarding the likely activity of individual
10115
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Journal of Medicinal Chemistry
Article
AT-1001 or 0.1 μM of unlabeled epibatidine. IC50 values were determined
by using the program Graphpad/PRISM. Ki values were calculated using
the Cheng−Prusoff transformation: Ki = IC50/(1 + L/ Kd)48 where, L is
radioligand concentration and Kd is the binding affinity of the radioligand,
as determined previously by saturation analysis.
BA, biometric analysis; DMEM, Dulbecco’s Modified Eagle’s
Medium; FBS, fetal bovine serum; HBSS, Hank’s Balanced Salt
Solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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change, which can be directly read by Molecular Devices Membrane
Potential Assay Kit (Blue Dye) (Molecular Devices), using the
FlexStation 3 microplate reader (Molecular Devices). The HEK cells
transfected with α3β4 or α4β2 nAChR were seeded in a 96-well plate
(4000 cells per well) one day prior to the experiments. For agonist
assays, after brief washing, the cells were loaded with 225 μL of HBSS
assay buffer (Hank’s Balanced Salt Solution with 20 mM of HEPES,
pH 7.4), containing the blue dye, and incubated at 370C. After 30 min,
25 μL of the appropriate compounds were dispensed into the wells by
the FlexStation and nAChR stimulation-mediated membrane potential
change was recorded every 3 s for 120 s by reading 550−580 nM
fluorescence excited at 530 nM wavelength. For the antagonist assay,
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and incubated at 37 °C. After 20 min, 25 μL of test compounds were
added, and after another 10 min, 25 μL of epibatidine (or nicotine) was
added by the FlexStation to a final concentration of 100 nM, with
fluorescence measured as described above. The change in fluorescence
represents the maximum response minus the minimum response for each
well. Graphpad PRISM was used to determine the EC50 and IC50 values.
■
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ASSOCIATED CONTENT
* Supporting Information
Detailed description of the scaffold ranking library samples and
their corresponding dose response data against α4β2 and α3β4
nAChR as well as 1H and 13C NMR spectra for key compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
*For M.A.G.: phone, 772-345-4729; E-mail, mgiulianotti@
tpims.org, marcello@tpims.org.
*For L.T. (pharmacology questions): phone, 772-345-4714;
E-mail, ltoll@tpims.org.
Author Contributions
All authors have given approval to the final version of the
manuscript.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a NIH-NIDA grant R01-
DA031370 and the State of Florida, Executive Office of the
Governor’s Department of Economic Opportunity. We also
thank Clemencia Pinilla and Jon Appel for their critical reading
of the manuscript and insightful suggestions.
ABBREVIATIONS USED
■
nAChR, nicotinic acetylcholine receptors; DHβE, dihydro-β-
erythroidine hydrobromide; 18-MC, 18-methoxycoronaridine;
GWAS, Genome Wide Association Studies; MHb, medial
habenula; KO, knockout; NAc, nucleus accumben; ADAM 17,
ADAM metallopeptidase domain 17; MBHA, p-methylbenzdryl-
amine resin; DIEA, disopropylethylamide; DCM, dichloromethane;
Fmoc, g-fluoromethyloxycarbonyl; Boc, tert-butyloxycarbonyl;
DMF, dimethylformamide; DIC, diisopropylcarbodiimide; HOBt,
hydroxybenzotriazole hydrate; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; LCMS, liquid chromatograph/mass spectrometer;
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