Design, synthesis, and biological evaluation of aminoalkylindole derivatives as cannabinoid receptor ligands with potential for treatment of alcohol abuse

Article abstract of DOI:10.1021/jm400268b

Attenuation of increased endocannabinoid signaling with a CB1R neutral antagonist might offer a new therapeutic direction for treatment of alcohol abuse. We have recently reported that a monohydroxylated metabolite of the synthetic aminoalkylindole cannabinoid JHW-073 (3) exhibits neutral antagonist activity at CB1Rs and thus may serve as a promising lead for the development of novel alcohol abuse therapies. In the current study, we show that systematic modification of an aminoalkylindole scaffold identified two new compounds with dual CB1R antagonist/CB2R agonist activity. Similar to the CB1R antagonist/inverse agonist rimonabant, analogues 27 and 30 decrease oral alcohol self-administration without affecting total fluid intake and block the development of alcohol-conditioned place preference. Collectively, these initial findings suggest that design and systematic modification of aminoalkylindoles such as 3 may lead to development of novel cannabinoid ligands with dual CB1R antagonist/CB2R agonist activity with potential for use as treatments of alcohol abuse.

Full text of DOI:10.1021/jm400268b

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Article
Design, Synthesis and Biological Evaluation of
Aminoalkylindole Derivatives as Cannabinoid Receptor
Ligands with Potential for Treatment of Alcohol Abuse
Tamara Vasiljevik, Lirit N. Franks, Benjamin M. Ford, Justin T. Douglas,
Paul L. Prather, William E. Fantegrossi, and Thomas E. Prisinzano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400268b • Publication Date (Web): 30 Apr 2013
Downloaded from http://pubs.acs.org on May 17, 2013
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Design, Synthesis and Biological Evaluation of
Aminoalkylindole Derivatives as Cannabinoid
Receptor Ligands with Potential for Treatment of
Alcohol Abuse
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a
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Tamara Vasiljevik , Lirit N. Franks , Benjamin M. Ford , Justin T. Douglas , Paul L. Prather ,
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William E. Fantegrossi , and Thomas E. Prisinzano
a
Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence,
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Kansas, Department of Pharmacology and Toxicology, College of Medicine, University of
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Arkansas for Medical Sciences, Little Rock, Arkansas, and The University of Kansas, NMR
Core Laboratory, Lawrence, Kansas
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Abstract. Attenuation of increased endocannabinoid signaling with a CB1R neutral antagonist
might offer a new therapeutic direction for treatment of alcohol abuse. We have recently
reported that a mono-hydroxylated metabolite of the synthetic aminoalkylindole cannabinoid
JHW-073 (3) exhibits neutral antagonist activity at CB1Rs and thus may serve as a promising
lead for the development of novel alcohol abuse therapies. In the current study, we show that
systematic modification of an aminoalkylindole scaffold identified two new compounds with
dual CB1R antagonist/CB2R agonist activity. Similar to the CB1R antagonist/inverse agonist
rimonabant, analogues 27 and 30 decrease oral alcohol self-administration, without affecting
total fluid intake and block the development of alcohol-conditioned place preference.
Collectively, these initial findings suggest that design and systematic modification of
aminoalkylindoles such as 3 may lead to development of novel cannabinoid ligands with dual
CB1R antagonist/CB2R agonist activity with potential for use as treatments of alcohol abuse.
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Introduction
The abuse of drugs and alcohol is often associated with substantial psychiatric comorbidity and
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exacerbates the spread of HIV/AIDS and drug resistant tuberculosis. Estimates of the total
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overall cost of substance abuse in the United States, including productivity, health- and crime-
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related costs, exceeds $600 billion annually. This includes approximately $193 billion for use
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of illicit drugs, $193 billion for tobacco, and $235 billion for alcohol.
The World Health Organization estimates that approximately 2.5 million people die from
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alcohol use every year. While several therapeutic options are available for the treatment of
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alcohol abuse, all existing therapies have only modest efficacy. A growing body of evidence
suggests that the endocannabinoid system is involved in some of the abuse related effects of drug
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and alcohol dependence. For example, studies have shown that chronic ethanol (EtOH)
exposure down-regulates cannabinoid 1 receptors (CB1Rs) and increases the brain concentration
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of endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG).
It has also
been demonstrated that CB1R knock-out mice exhibit reduced voluntary alcohol consumption as
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compared to wild type mice. Furthermore, the CB1R antagonist/inverse agonist rimonabant
(SR141716A, Figure 1) reduces EtOH intake in C57B1/6J mice to levels comparable with that of
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CB1 mice. Cannabinoid 2 receptors (CB2Rs) have also been implicated in substance abuse.
Systematic administration of the CB2R agonist 1 (JWH-133) dose-dependently inhibits
intravenous cocaine self-administration in wild-type and CB1R-deficient mice, but not in CB2R-
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deficient mice and this effect was blocked by the CB2R antagonist 2 (AM630).
To
determine if this effect was mediated by antagonism of central or peripheral CB2Rs, the authors
microinjected 2 into the nucleus accumbens and again observed antagonism of the reduction in
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cocaine self-administration induced by systemic administration of the CB2R agonist 1.
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Collectively, these studies suggest that targeting the central cannabinoid signaling system has
potential in treating drug and alcohol dependence.
One approach to developing cannabinoid-based drugs for treatment of alcohol abuse is to
target a molecule that possesses both CB1R neutral antagonist and CB2R agonist activity. This
combined pharmacological profile offers two advantages over presently available CB1R
antagonists/inverse agonists. Firstly, it provides an opportunity to avoid the side effect profile
seen with rimonabant. During the first year of a RIO-Europe clinical trial several moderate side
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effects of rimonabant treatment were observed, including nausea, dizziness, and diarrhea.
However, upon completion of a meta-analysis study in 2007, it was concluded that a therapeutic
dose of rimonabant produced very serious psychiatric side effects such as depression, anxiety
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and suicide.
However, subsequent studies in animals indicate that a neutral antagonist might
be expected to retain the therapeutic properties of rimonabant, but be associated with fewer
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adverse effects resulting from inverse agonism at CB1Rs.
Secondly, incorporation of CB2
agonism into the same molecule provides an opportunity to harness a potential new direction in
developing anti-addiction therapies. Our approach to identifying such a molecule has been to
investigate synthetic cannabinoids.
Synthetic cannabinoids derived from the aminoalkylindole structural class have received
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increased attention due to recent widespread abuse of the incense blends known as K2/Spice.
Despite the recreational popularity of these commercial preparations, very little is known
regarding K2/Spice metabolism, pharmacology, and toxicity. Among the most common
components of K2/Spice are naphthalen-1-yl(1-pentyl-1H-indol-3-yl)methanone (JWH-018), (1-
butyl-1H-indol-3-yl)(naphthalen-1-yl)methanone (JWH-073), (1-(2-morpholinoethyl)-1H-indol-
3
-yl)(naphthalen-1-yl)methanone (JWH-200), and 2-((1S,3R)-3-hydroxycyclohexyl)-5-(2-
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methyloctan-2-yl)phenol (CP-47,497, Figure 2). These agents exhibit differential selectivity
towards CB1Rs and CB2Rs and have been shown to be both more potent than the classical
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cannabinoid ∆ -THC.
Recently, our group reported that a mono-hydroxylated metabolite of
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aminoalkylidole 3 (JWH-073), compound 4 (JWH-073-M4), retains relatively high affinity for
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CB1Rs, but lacks intrinsic activity when examined in the [ S]GTPγS functional assay. These
results indicate that this metabolite may act as a neutral antagonist at CB1Rs.
Given our results that 4 acts as a neutral antagonist at CB1Rs and the known promiscuity of
the aminolakylindoles for both CB1 and CB2 receptors, we hypothesize that further structure-
activity relationships (SAR) would identify analogues with dual CB1R neutral antagonist/CB2R
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agonist activity.
Results from our initial results are described below.
Chemistry
Synthesis of analogues based on scaffold 4 began with the use of commercially available 7-
methoxyindole (5), which was subjected to mild alkylating conditions to afford compound (6),
an important intermediate in 74-93% yield for the different substituents, respectively (Scheme 1).
In addition, commercially available 7-ethylindole was also subjected to the aforementioned
conditions to yield compound (6) with an ethyl substitution at the 7-position of the indole ring in
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3% yield. Intermediate 6 was then subjected to Friedel-Crafts like acylation conditions, using
dimethyaluminum chloride and the appropriate acid chloride at 0 °C to afford many of the
analogues prepared, structurally represented as intermediate (7), in yields ranging from 9-68%.
Compound 7 was shown to be a versatile intermediate that can undergo several reactions to yield
various scaffolds. Specifically, it undergoes O-demethylation in the presence of BBr to afford
3
compound (8) in yields ranging from 38%-90% as well as LAH reduction conditions to afford
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compound (9) in 20% yield. 1-butyl-5-methoxy-1H-indole, 11 synthesized from N-alkylation of
commercially available 5-methoxyindole 10, was subjected to Friedel-Crafts like acylation
conditions to produce compound (12) in 58% yield, which was then O-demethylated using BBr3
to afford compound (13) in 90% yield (Scheme 2). Compound 5 was additionally used in the
synthesis of intermediate (14) utilizing a one-pot N-alkylation and 3-indole iodination in 69%
yield. Intermediate 14 was then subjected to Suzuki coupling conditions utilizing different
boronic acids to afford analogues (15) and (16) in 37% and 50% yield, respectively. Twenty-one
analogues were synthesized and the structures of the analogues are schematically represented in
Table 1.
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Results and Discussion
In our quest for novel dual activity compounds with CB1R neutral antagonist/CB2R agonist
properties, twenty-one analogues were prepared using step-wise molecular investigation of the
elements present in the scaffold of compound 4. The analogues prepared were designed to
investigate the SAR at several positions on the scaffold of interest. Firstly, we wanted to
examine the necessity of the carbonyl moiety as well as the optimal length of the linker from the
indole core to the naphthalene ring. We also sought out to explore the possibility for addition of
rotational bonds at this position of the molecule. Secondly, we set to explore the requirement of
the naphthalene ring through selective introduction of electron withdrawing and electron
donating groups. Our initial design strategy was to limit the use of charged species and keep
steric demands to a minimum. Lastly, we wanted to investigate the positioning of the hydroxyl
moiety around the indole core and its necessity for activity at CBRs. The prepared analogues
were subjected to in vitro pharmacological characterization, which included receptor binding and
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functional assays for both cannabinoid receptors. Initial receptor binding screens for both
CB1Rs and CB2Rs were conducted for all twenty-one analogues prepared. These initial binding
screens were conducted by examining the ability of a single analogue concentration of 1 µM to
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compete for receptor binding with the high affinity CB1/CB2R agonist [ H]CP-55,940.
This
approach allowed us to quickly attain an approximate affinity of all compounds tested at the
cannabinoid receptors. Compounds were selected for further assays to determine intrinsic
activity at CB1 and CB2Rs if a 1 µM concentration produced over 50% displacement of a 0.2
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nM concentration of [ H]CP-55,940, indicating a relatively high sub-micromolar affinity. For
example, employing the conditions used for this screen, it would be predicted by the Cheng-
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Prusoff equation that the concentration of a compound producing 50% displacement of [ H]CP-
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5,940 from a receptor will estimate the compounds affinity for that receptor. Data presented
in Table 1 shows that a 1µM concentration of most analogues examined produced greater than
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50% displacement of [ H]CP-55,940 from both CB1R and CB2Rs. As a result, out of the
twenty-one analogues that were screened, sixteen bind to CB1R and eighteen bind to CB2R with
sub-micromolar affinity. Several analogues exhibited very high affinity for either of the two
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receptors tested, which can be predicted from their near 100% displacement of [ H]CP-55,940
from the receptors. Based on the above criteria, thirteen compounds were chosen for further
evaluation.
A functional assay to screen for the inhibition of adenylyl cyclase (AC) activity by CBRs was
selected for determination of intrinsic activity of the analogues that demonstrated sub-
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micromolar affinity for CB1R and CB2Rs. Since all analogues examined are predicted to bind
to CBRs with sub-micromolar affinity, a 10 µM concentration of all compounds was used to
achieve full-receptor occupancy. It is assumed that full-receptor occupancy will produce
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maximal efficacy. The non-selective CB1R/CB2R full agonist CP-55,940 inhibited AC-activity
via CB1Rs endogenously expressed in Neuro2A cells by 45% and 37% in CHO cells transfected
with CB2 receptors (Table 1). Most compounds examined exhibited levels of AC-inhibition
similar to that produced by the full agonist CP-55,940. Unlike our prior report demonstrating
that metabolite 4 exhibited neutral antagonist properties in assays examining G-protein
activation, in this study 4 interestingly acts as a full CB1R agonist to inhibit AC-inhibition in
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Neuro2A cells. However, compounds 17, 27, and 30, produced significantly less AC-inhibition
via CB1Rs when compared to the full agonist CP-55,940 of -4, 18, and 16% inhibition,
respectively. Despite little or no AC-inhibition when examined at CB1Rs, these compounds
exhibited significantly different results when tested for activity at CB2Rs. Specifically, the three
compounds 17, 27, and 30 produced levels of AC-inhibition that were similar to inhibition
resulting from CP-55,940 administration of 22.1, 33.2, and 20.8%, respectively. Collectively,
these data indicate that compounds 17, 27, and 30 display only neutral antagonist to weak partial
agonist activity at CB1Rs, while exhibiting partial to full agonist efficacy at CB2Rs.
Based on efficacy predicted from functional experiments examining AC-inhibition, together
with the initial screen for sub-micromolar receptor affinity, several compounds were selected to
determine actual K values by conducting full concentration effect curves for inhibition of
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[
H]CP-55,940 binding to CBRs. Even though compound 17 produced minimal AC-inhibition
via CB1Rs, it was shown to bind to both CB1 and CB2Rs with a relatively low affinity of 387
and 281 nM, respectively (Table 2). However, analogue 19 demonstrated high affinity for CB1
and CB2Rs in the low nanomolar range of 1.7 nM and 0.81 nM, respectively. Compounds 27
and 30 displayed similar affinity for both receptors, however binding with slightly higher affinity
to the CB2R. Specifically, 27 bound to CB1Rs with a K value of 15.4 nM, while binding to
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CB2Rs with a K value of 10.9 nM. Compound 30 demonstrated an affinity of 37.2 nM and 26.5
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nM for CB1R and CB2Rs, respectively.
The four lead compounds exhibiting relatively high affinity as indicated by K values were
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examined together with metabolite 4 to determine their potency to inhibit AC-activity via CB1
and CB2Rs. Similar to data reported in the initial screen for AC-inhibition, 4 and 19 produced
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0-60% inhibition of AC-activity via CB1Rs with IC values of 225 and 45 nM, respectively.
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Conversely, 27 and 30 produced very little inhibition of AC-activity, which was only observed at
very high concentrations of both analogues (Figure 3A). In support of the observed functional
activity indicating neutral antagonism, the two lead compounds and the CB1R antagonist O-2050
(1 µM concentration) significantly antagonized AC-inhibition produced by the CB1R full agonist
3
(Figure 3B). In contrast to neutral antagonism at CB1Rs, all of the compounds examined
produced 40-50% inhibition of AC-activity at CB2Rs in CHO-hCB2 cells with potency similar
to their rank order of K values reported in Table 2 (Figure 4A). To examine if the observed
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agonist activity in CHO-hCB2 cells is due to activation of CB2Rs, the AC-inhibition assay was
conducted in CHO-WT cells not expressing CB2Rs. As observed in Figure 4B, neither 27 nor
30 significantly modified AC-activity in CHO-WT cells indicating that the agonist activity
observed for both compounds in CHO-hCB2 cells is indeed due to their activation of CB2Rs.
The dual CB1R neutral antagonist/CB2 agonist activity displayed by compounds 27 and 30 in
the in vitro assays prompted us to select these analogues for further evaluation in vivo using
murine subjects in two complementary models relevant to alcohol abuse. Our hypothesis is that
an analogue with dual activity as CB1R neutral antagonist/CB2R agonist will reduce both the
reinforcing effects of EtOH and the conditioned rewarding effects of EtOH in mice. The
compounds selected were initially tested for potential cannabinoid receptor antagonist activity
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using a thermoregulation assay. The hypothermic effects of cannabinoid agonists are well
established, and are blunted by prior treatment with CB1R antagonists, allowing us to rapidly
determine whether or not our selected compounds display in vivo effects consistent with CB1R
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antagonism.
The assay was conducted using glass-encapsulated radiotelemetry probes
surgically implanted into the peritoneal cavity of each mouse, which monitored core body
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temperature in response to drug administration. Intraperitoneal administration of 10 mg/kg of
the full CB1R agonist 3, elicited a profound hypothermic effect as seen in Figure 5 (black
circles). Importantly, thirty minute pretreatment with either 27 (Figure 5, gray triangle) or 30
(
Figure 5, white circles), significantly decreased the hypothermic effects elicited by subsequent
administration of compound 3, confirming the in vivo cannabinoid activity of 27 and 30, and
illustrating apparent antagonist effects against CB1R agonist-induced hypothermia.
Having established apparent in vivo antagonism, 27 and 30 were further tested in two
complementary models of alcohol abuse: oral self-administration (SA) and alcohol conditioned
place preference (CPP). These assays are important models for the study of alcoholism because
they capture several aspects of this condition including voluntary EtOH drinking (SA) and
conditioned EtOH reward (CPP). The effects of compounds 27 and 30 on voluntary 10% EtOH
drinking were studied using a two-bottle choice procedure (ethanol vs. water) similar to those
described by Keane and coworkers, as well as by Cunningham and coworkers, using 10 mg/kg
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rimonabant as a positive control.
Across five observations conducted under baseline “no
injection” conditions it can be observed in Figures 6A, and 6D (black circles) that EtOH
preference and total fluid consumption (Figure 6B, Figure 6E; black circles) were steadily
maintained during the entire treatment period. Consistent with previous reports, daily treatment
with 10 mg/kg rimonabant decreased EtOH preference (Figure 6A, Figure 6D, triangles) without
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altering total fluid intake (Figure 6B, Figure 6E, triangles). However, consisted with previously
published reports, rimonabant decreased body weight across the treatment period (Figure 6C,
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Figure 6F, triangles).
After ten day treatment with rimonabant, mice were returned to
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baseline “no injection” conditions where their weights increased and voluntary EtOH drinking
resumed. After five such observations, representing a two week drug “washout" period, daily
treatments with 10 mg/kg of 27 or 30 were initiated. As previously observed with rimonabant,
both compounds 27 and 30 reduced EtOH preference (Figure 6A, inverted triangles; Figure 6D,
gray triangles, respectively) without affecting total fluid consumption (Figure 6B, inverted
triangles; Figure 6E, gray triangles). However, unlike rimonabant, compounds 27 and 30 did not
decrease body weight of mice tested (Figure 6C, inverted triangles; Figure 6E, gray triangles).
The last treatment was a daily injection of the rimonabant/27/30 vehicle, which was 8%
Tween/92% sterile water. With the exception of the first observation period for compound 27
(perhaps representing a persistent effect of 27), EtOH preference (Figure 6A, gray circles-
compound 27; Figure 6D, gray circles-compound 30), total fluid consumption (Figure 6B, gray
circles-compound 27; Figure 6E, gray circles-compound 30), and mean body weight (Figure 6C,
gray circles-compound 27; Figure 6F, gray circles-compound 30) were not affected by vehicle
injections. This study demonstrates that a JWH-derived compound devoid of inverse agonist
activity can replicate the effects of rimonabant on alcohol self-administration in mice.
To further explore the in vivo effects of these compounds, an alcohol conditioned place
preference assay was performed with compounds 27, 30, and rimonabant. This assay tested the
effects of these compounds on the conditioned rewarding effects of EtOH. Training of mice
involved daily pairings of 2 mg/kg EtOH with novel contextual cues within a spatial
conditioning chamber, and daily pairings of saline with distinct contextual cues within the same
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chamber. Following four such pairings, an increase in time spent in EtOH-paired chamber was
observed, indicating the development of conditioned place preference (Figure 7, “No inj” bar).
Separate groups of mice were conditioned as previously described, with the exception that 10
mg/kg 27, 10 mg/kg 30 or 10 mg/kg rimonabant were administered one hour before each EtOH
pairing. In contrast to the ~400 sec preference elicited by EtOH in the absence of cannabinoid
administration, mice treated with 10 mg/kg rimonabant prior to each EtOH pairing did not
exhibit a significant preference for the EtOH-paired chamber, which is in agreement with
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previously reported data (Figure 7, “10 RIM” bar).
Similar to the effects of rimonabant
produced in the present study, the conditioned rewarding effects of EtOH were also blocked by
the administration of either 10 mg/kg 27 or 10 mg/kg 30 prior to each EtOH pairing. In total,
these studies demonstrate that a novel indole-derived compound with cannabinoid activity
lacking inverse agonist activity can replicate the effects of rimonabant on alcohol induced CPP in
mice.
Conclusions
CBR ligands that attenuate endocannabinoid signaling have been shown to produce
16, 39
effects in several disorders, among which are drug abuse and alcohol dependence.
Our goal
is to design and synthesize a dual activity CB1R neutral antagonist/CB2R agonist that would
reduce increased endocannabinoid signaling without affecting intrinsic CB1R activity, and also
be able to modulate the rewarding effects of alcohol abuse. As an initial step towards achieving
this goal, we conducted experiments to synthesize compounds that exhibit dual CB1R neutral
antagonist/CB2R agonist activity based on the scaffold of compound 4. Initial studies explored
the scaffold of 4 by utilizing a molecular dissection approach to better understand the elements
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involved in producing cannabinoid activity. The necessity of the naphthalene ring and the
electronic potential at this position, the necessity of the carbonyl moiety, the length of the linker
between the indole core and the naphthalene substituent, as well as the necessity of the hydroxyl
moiety on the 7-position of the indole ring were all explored. Similar to the parent compound 3,
the majority of the analogues examined exhibited relatively high affinity for both CB1R and
CB2Rs. Of all of the compounds tested, two analogues, 27 and 30, appeared to have the most
promise. For example, compounds 27 and 30 displayed similar high affinity for both receptors,
with K values of 27 of 15.4 nM at CB1R and 10.9 nM for CB2R and K values of 30 of 37.2 and
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6.5 nM for CB1R and CB2R, respectively. In AC-inhibition assays, both compounds produced
very little inhibition of AC-activity via CB1Rs, consistent with neutral antagonism. However, 27
and 30 both produced 40-50% inhibition of AC-activity via CB2Rs, similar to that of the full
agonist CP-55,940 and thus consistent with partial to full agonism. Based on promising results
from these in vitro studies, compounds 27 and 30 were selected for testing in two in vivo models
of alcohol abuse: oral self-administration and alcohol conditioned place preference. Similar to
the actions produced by the CB1R antagonist/inverse agonist rimonabant, both 27 and 30 were
shown to decrease alcohol self-administration, without affecting total fluid intake. Unlike
rimonabant, 27 and 30 did not alter body weight during the treatment period. Interestingly, both
2
7 and 30 decreased alcohol conditioned place preference in a similar manner to rimonabant,
despite not possessing any apparent inverse agonist activity in vitro. As has been previously
reported for rimonabant, results from the present study collectively demonstrate that both the
reinforcing and conditioning rewarding effects of alcohol can also be significantly blunted by
40
treatment with novel JWH-derived cannabinoids, devoid of inverse agonist activity at CB1Rs.
Such compounds with dual CB1R neutral antagonist/CB2R agonist activity may indeed represent
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potential leads in an ongoing search for new and improved alcohol abuse therapies. Additional
data will be reported in due course.
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Experimental section
General Methods. Unless otherwise indicated, all reagents were purchased from
commercial sources and were used without further purification. Melting points were determined
on a Thomas-Hoover capillary melting apparatus. NMR spectra were recorded on a Bruker
DRX-400 with qnp probe or a Bruker AV-500 with cryoprobe using δ values in ppm (TMS as
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internal standard) and J (Hz) assignments of H resonance coupling. High resolution mass
spectrometry data were collected on either a LCT Premier (Waters Corp., Milford, MA) time of
flight mass spectrometer or an Agilent 6890 N gas chromatograph in conjunction with a quarto
Micro GC mass spectrometer (Micromass Ltd, Manchester UK). Thin-layer chromatography
(
TLC) was performed on 0.25 mm plates Analtech GHLF silica gel plates using ethyl acetate/n-
hexanes, in 20%:80% ratio as the solvent unless otherwise noted. Spots on TLC were visualized
by UV (254 or 365 nm), if applicable, and phosphomolybdic acid in ethanol. Column
chromatography was performed with Silica Gel (40-63 µ particle size) from Sorbent
Technologies (Atlanta, GA). Analytical HPLC was carried out on an Agilent 1100 Series
Capillary HPLC system with diode array detection at 254 nm on an Agilent Eclipse XDB-C18
column (250 x 10 mm, 5 µm) with isocratic elution in 80% CH CN/20% H O (0.1% Formic
3
2
acid) unless otherwise specified.
General Procedure A: Indole N-alkylation. To a suspension of KOH (5 equiv) in DMF (13
mL) was added 5-methoxyindole, 7-methoxyindole or 7-ethylindole (1 equiv). After stirring at
2
1 °C for an hour, 1-bromobutane (1.375 equiv) was added and the reaction mixture was heated
to 50 °C and stirred overnight. Upon completion, the resulting mixture was poured into H O (15
2
mL) and extracted with DCM (3 x 15 mL). Combined organic extracts were washed with water,
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dried over anhydrous Na SO , concentrated under reduced pressure and the resulting residue was
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4
purified by flash column chromatography on silica gel using EtOAc/n-hexanes.
-Butyl-7-methoxy-1H-indole (6ac). Compound 6ac was synthesized from commercially
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available 7-methoxyindole using general procedure A and 1-bromobutane to afford 0.51 g (74%
yield) as a clear oil. TLC system: 10% EtOAc/90% n-hexanes. Spectral data matched
4
1
previously reported data.
1
-Butyl-7-ethyl-1H-indole (6bc). Compound 6bc was synthesized from commercially
available 7-ethylindole using general procedure A and 1-bromobutane to afford 0.64 g (93%
1
yield) as a clear oil. TLC system: 10% EtOAc/90% n-hexanes. H NMR (500 MHz, CDCl ) δ
3
7
3
3
.47 (dd, J = 7.6, 1.4 Hz, 1H), 7.05 – 6.95 (m, 3H), 6.47 (d, J = 3.1 Hz, 1H), 4.31 – 4.21 (m, 2H),
.03 (q, J = 7.5 Hz, 2H), 1.83 – 1.69 (m, 2H), 1.35 (td, J = 7.6, 2.7 Hz, 5H), 0.94 (t, J = 7.4 Hz,
13
H). C NMR (126 MHz, CDCl ) δ 133.57, 130.09, 129.60, 127.30, 122.42, 119.49, 119.00,
3
101.40, 48.75, 34.47, 25.67, 20.04, 16.08, 13.77. [M+H] calcd for C H N, 202.1590; found
1
4
19
2
02.1596.
-(2-(7-Methoxy-1H-indol-1-yl)ethyl)morpholine (6ad). Compound 6ad was synthesized
4
from commercially available 7-methoxyindole using general procedure A and 4-(2-
42
bromoethyl)morpholine that was made utilizing conditions reported by Cai J., et al. to afford
0
.51 g (74% yield) as an oil with a slightly orange tint. TLC system: 40% EtOAc/60% n-
1
hexanes. H NMR (500 MHz, CDCl ) δ 7.20 (dd, J = 7.9, 0.9 Hz, 1H), 7.01 – 6.99 (m, 1H), 6.97
3
(d, J = 7.8 Hz, 1H), 6.61 (dd, J = 7.8, 0.9 Hz, 1H), 6.42 (d, J = 3.1 Hz, 1H), 4.53 – 4.46 (m, 2H),
1
3
3.93 (s, 3H), 3.74 – 3.68 (m, 4H), 2.78 – 2.71 (m, 2H), 2.53 – 2.47 (m, 4H). C NMR (126
MHz, CDCl ) δ 147.33, 130.92, 129.19, 125.48, 119.75, 113.77, 102.14, 101.33, 67.02, 60.15,
3
5
5.15, 53.93, 46.65. [M+H] calcd for C H N O , 261.1598; found 261.1603.
15 21 2 2
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1
-Butyl-5-methoxy-1H-indole (11). Compound 11 was synthesized from commercially
available 5-methoxyindole using general procedure A and 1-bromobutane to afford 1.28 g (92%
1
yield) as a clear oil. TLC system: 10% EtOAc/90% n-hexanes. H NMR (500 MHz, CDCl ) δ
3
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.18 (d, J = 8.9, 1H), 7.02 (dd, J = 2.7, 13.3, 2H), 6.81 (dd, J = 2.4, 8.9, 1H), 6.34 (dd, J = 0.7,
3.0, 1H), 4.02 (t, J = 7.1, 2H), 3.79 (s, 3H), 1.75 (ddd, J = 7.3, 11.2, 14.8, 2H), 1.32 – 1.23 (m,
13
2
1
H), 0.87 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 154.09, 131.56, 129.06, 128.55,
3
11.93, 110.35, 102.71, 100.53, 56.12, 46.54, 32.63, 20.43, 13.95. [M+H] calcd for C H NO,
13 18
204.1383; found 204.1386.
Preparation of 1-butyl-3-iodo-7-methoxy-1H-indole (14). A round bottom flask containing
7
1
3
-methoxyindole (0.22 mL, 1.70 mmol, 1 equiv) in DMF at 21 °C was stirred with KOH (0.10 g,
.78 mmol, 1.05 equiv) for 15 min and then treated with I (0.44g, 1.73 mmol, 1.02 equiv). After
2
0 min, NaH (60% dispersion in mineral oil; 0.082 g, 2.04 mmol, 1.2 equiv) was added portion-
wise. After an additional 15 min, 1-bromobutane (0.2 mL, 1.87 mmol, 1.1 equiv) was added and
the reaction mixture was stirred until completion. Upon completion (TLC monitoring), water
was added and allowed to stir for 15 min, upon which the mixture was extracted with DCM and
the layers were separated. Aqueous layer was washed with DCM (3 x 15 mL) and the combined
organic layers were washed with H O (2 x 15 mL), dried over anhydrous Na SO and
2
2
4
concentrated under reduced pressure. The resulting residue was purified by silica gel column
chromatography using EtOAc/n-hexanes to afford 0.45g (80% yield) as an clear oil with a
1
yellow tint that was used immediately. TLC system: 10% EtOAc/90% n-hexanes. H NMR
(500 MHz, CDCl ) δ 7.08 (t, J = 3.8, 2H), 7.04 (dd, J = 1.1, 8.0, 1H), 6.68 (d, J = 7.5, 1H), 4.38
3
(t, J = 7.2, 2H), 3.95 (s, 3H), 1.78 (dt, J = 7.5, 12.7, 2H), 1.33 (td, J = 7.5, 15.0, 2H), 0.94 (t, J =
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.4, 3H). C NMR (126 MHz, CDCl3) δ 146.67, 132.31, 131.99, 125.18, 119.88, 113.27,
02.43, 54.80, 48.90, 33.67, 19.20, 13.13.
General Procedure B: O-Demethylation procedure. A solution of BBr (1 M in DCM, 6
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equiv) in DCM was added dropwise to a solution of methyl ether (1 equiv) in DCM at -78 °C.
The mixture was then allowed to warm to 21 °C overnight, and upon completion, NaHCO (6
3
equiv) was added. The resulting mixture was then cooled in an ice-bath and MeOH (20 mL) was
added dropwise and then stirred for 30 min. The reaction mixture was then warmed up to 21 °C
and stirred for an additional hour. Upon that, the reaction was quenched with H O and the
2
separated aqueous phase was extracted with DCM (3 x 15 mL). Combined organic extracts were
dried over anhydrous Na SO , and concentrated under reduced pressure. The resulting residue
2
4
was purified by flash chromatography on silica gel using mixtures of EtOAc/n-hexanes.
General Procedure C: Acid chloride formation. The appropriate acid (1 equiv) was placed
in a round bottom flask and flushed twice with Argon (Ar). Anhydrous DCM (7 mL) was then
added followed by the dropwise addition of oxalyl chloride (2 M in DCM, 3.1 equiv). After 5
min, 3 drops of anhydrous DMF were added to the reaction mixture and once the fizzing
stopped, the reaction was allowed to stir overnight at 21 °C. Solvent was then evaporated using
reduced pressure and the crude residue was used immediately without any further purification.
General Procedure D: 3’-indole acylation. To a solution of the appropriate indole (1 equiv)
in DCM at 0 °C under an Ar atmosphere was added Me AlCl (1.5 equiv) dropwise and the
2
solution was allowed to stir at that temperature for 30 min, after which a solution of the
appropriate acid chloride (1.2 equiv) in DCM was added dropwise. Reaction was monitored by
TLC and upon completion, was carefully poured into an ice-cold 1N HCl solution and then
extracted with DCM (3 x 15 mL). Combined organic layers were then washed with NaHCO (2
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x 15 mL), brine, and dried over anhydrous Na SO The solvent was evaporated under reduced
2
4.
pressure and the resulting residue was purified by flash column chromatography on silica gel
using mixtures of EtOAc/n-hexanes to afford the desired product.
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-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(4-fluorophenyl)ethanone (17). Compound 17 was
synthesized from compound 6 using general procedure D and 4-fluorophenylacetyl chloride to
1
afford 0.08g (9% yield) isolated as an brown oil. H NMR (500 MHz, CDCl ) δ 7.98 (dd, J =
3
0
.8, 8.1, 1H), 7.64 (s, 1H), 7.29 – 7.25 (m, 2H), 7.16 (t, J = 8.0, 1H), 7.02 – 6.96 (m, 2H), 6.71
(d, J = 7.5, 1H), 4.39 (t, J = 7.2, 2H), 4.09 (s, 2H), 3.93 (s, 3H), 1.85 – 1.77 (m, 2H), 1.36 – 1.27
13
(
m, 2H), 0.94 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 192.32, 162.72, 160.77, 147.31,
3
1
35.78, 131.58, 131.55, 130.88, 130.82, 129.07, 126.33, 123.30, 115.76, 115.38, 115.21, 115.12,
1
04.31, 55.36, 50.28, 45.87, 33.81, 19.78, 13.68. HRMS (m/z): [M+K] calcd for C H FKNO ,
2
1
22
2
3
78.1272; found 378.1315. HPLC t = 15.031 min; purity = 95.4% using 70% CH CN/30% H O
R 3 2
(0.1% Formic acid).
1
-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(4-methoxyphenyl)ethanone (18). Compound 18
was synthesized from compound 6 using general procedure D and 4-methoxyphenylacetyl
1
chloride to afford 0.63 g (59.2% yield) isolated as a reddish solid, mp = 72-74 °C. H NMR (500
MHz, CDCl ) δ 8.00 (dd, J = 0.8, 8.1, 1H), 7.63 (s, 1H), 7.25 – 7.21 (m, 2H), 7.15 (t, J = 8.0,
3
1H), 6.87 – 6.81 (m, 2H), 6.70 (d, J = 7.8, 1H), 4.37 (t, J = 7.2, 2H), 4.06 (s, 2H), 3.92 (s, 3H),
1
3
3
.77 (s, 3H), 1.84 – 1.76 (m, 2H), 1.35 – 1.26 (m, 2H), 0.93 (t, J = 7.4, 3H). C NMR (126
MHz, CDCl ) δ 193.44, 158.73, 147.69, 136.29, 130.73, 129.59, 128.50, 126.69, 123.58, 116.22,
3
115.62, 114.40, 104.62, 55.76, 55.69, 50.63, 46.48, 34.22, 20.19, 14.11. HRMS (m/z): [M+Na]
calcd for C H NNaO , 374.1732; found 374.1783. HPLC t = 7.490 min; purity = 99.9%.
2
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-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(2,3-difluorophenyl)ethanone (19). Compound 19
was synthesized from compound 6 using general procedure D and 1-naphthoyl chloride to afford
1
2
.00 g (53% yield) isolated as pale brown solid, mp = 108-110 °C. H NMR (500 MHz, CDCl )
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0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
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7
8
9
0
δ 8.19 (d, J = 8.4, 1H), 8.11 (d, J = 7.4, 1H), 7.96 (d, J = 8.2, 1H), 7.91 (d, J = 7.8, 1H), 7.65 (dd,
J = 1.1, 7.0, 1H), 7.56 – 7.45 (m, 3H), 7.28 – 7.24 (m, 2H), 7.22 (s, 1H), 6.79 (d, J = 7.7, 1H),
4
.30 (t, J = 7.3, 2H), 3.96 (s, 3H), 1.80 – 1.71 (m, 2H), 1.32 – 1.23 (m, 2H), 0.89 (t, J = 7.4, 3H).
1
3
C NMR (126 MHz, CDCl ) δ 192.08, 147.45, 139.32, 139.07, 133.81, 130.89, 129.96, 129.46,
3
128.22, 126.79, 126.61, 126.34, 126.14, 125.89, 124.65, 123.60, 117.42, 115.35, 104.66, 55.50,
0.34, 33.86, 19.83, 13.73. HRMS (m/z): [M+Na+CH CN] calcd for C H N NaO 421.1892;
5
3
26 26
2
2
found 421.1843. HPLC t = 14.576; purity = 95.8%.
R
1
-(1-Butyl-7-hydroxy-1H-indol-3-yl)-2-(4-fluorophenyl)ethanone (20). Compound 20 was
synthesized from 7 using general procedure B to afford 0.10g (38% yield) isolated as an off-
1
brown solid, mp = decomposition at 204-206 °C. H NMR (500 MHz, DMSO) δ 9.91 (s, 1H),
8
6
.44 (s, 1H), 7.62 (dd, J = 0.9, 8.0, 1H), 7.39 – 7.33 (m, 2H), 7.12 (ddd, J = 2.6, 5.9, 8.9, 2H),
.94 (t, J = 7.8, 1H), 6.61 (dd, J = 0.9, 7.7, 1H), 4.44 (t, J = 7.0, 2H), 4.09 (s, 2H), 1.85 – 1.75
1
3
(
m, 2H), 1.31 – 1.21 (m, 2H), 0.90 (t, J = 7.4, 3H). C NMR (126 MHz, DMSO) δ 191.61,
61.72, 159.80, 144.49, 137.94, 132.50, 132.48, 131.05, 130.99, 128.73, 125.46, 122.80, 114.76,
114.59, 114.50, 112.34, 108.26, 48.70, 44.52, 33.29, 18.97, 13.39. HRMS (m/z):
1
[
M+Na+CH CN] calcd for C H FN2NaO , 389.1641; found 389.1711. HPLC t = 6.491 min;
3
22 23
2
R
purity = 99.9% using 70% CH CN/30% H O (0.1% Formic acid).
3
2
1-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-phenylethanone (21).
Compound 21 was
synthesized from compound 6 using general procedure D and phenyacetyl chloride to afford 0.13
1
g (17% yield) isolated as an off-white solid with a pinkish tint, mp = 65-68 °C. H NMR (500
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MHz, CDCl ) δ 8.03 (dd, J = 0.8, 8.1, 1H), 7.66 (s, 1H), 7.36 – 7.30 (m, 4H), 7.26 – 7.22 (m,
3
1
1
H), 7.18 (t, J = 8.0, 1H), 6.72 (d, J = 7.8, 1H), 4.39 (t, J = 7.2, 2H), 4.14 (s, 2H), 3.94 (s, 3H),
1
3
.87 – 1.77 (m, 2H), 1.32 (dq, J = 7.4, 14.8, 2H), 0.95 (t, J = 7.4, 3H). C NMR (126 MHz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
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41
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43
44
45
46
47
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49
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59
60
CDCl ) δ 192.72, 147.37, 136.13, 136.06, 129.43, 129.25, 128.62, 126.64, 126.38, 123.30,
3
115.96, 115.30, 104.34, 55.44, 50.32, 47.09, 33.88, 19.86, 13.78. HRMS (m/z): [M+Na] calcd
for C H NNaO , 344.1626; found 344.1656. HPLC t = 8.114 min; purity = 99.9%.
2
1
23
2
R
1
-(1-Butyl-7-hydroxy-1H-indol-3-yl)-2-phenylethanone (22).
Compound 22 was
synthesized from compound 7 using general procedure B to afford 0.17 g (68% yield) isolated as
1
a fluffy brown solid, mp = decomposition at 197-200 °C. H NMR (500 MHz, DMSO) δ 9.91 (s,
1H), 8.44 (s, 1H), 7.62 (dd, J = 0.9, 8.0, 1H), 7.36 – 7.31 (m, 2H), 7.28 (dd, J = 4.9, 10.3, 2H),
7
4
.20 (dd, J = 4.3, 11.6, 1H), 6.93 (t, J = 7.8, 1H), 6.61 (dd, J = 0.9, 7.7, 1H), 4.44 (t, J = 7.0, 2H),
1
3
.08 (s, 2H), 1.84 – 1.75 (m, 2H), 1.30 – 1.20 (m, 2H), 0.90 (t, J = 7.4, 3H). C NMR (126
MHz, DMSO) δ 191.75, 144.49, 137.99, 136.39, 129.20, 128.76, 128.00, 126.01, 125.46, 122.77,
14.60, 112.35, 108.24, 48.67, 45.62, 33.28, 18.95, 13.38. HRMS (m/z): [M+Na] calcd for
C H NNaO , 330.1470; found 330.1514. HPLC t = 6.458 min; purity = 99.8% using 70%
1
20 21
2
R
CH CN/30% H O (0.1% Formic acid).
3
2
1
-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(naphthalen-1-yl)ethanone (23). Compound 23 was
synthesized from compound 6 using general procedure D and 1-naphthoylacetyl chloride to
1
afford 0.09 g (12% yield) isolated as an amorphous yellow solid, mp = 110-113 °C. H NMR
(
500 MHz, CDCl ) δ 8.07 – 8.00 (m, 2H), 7.89 – 7.85 (m, 1H), 7.79 (dd, J = 2.4, 7.0, 1H), 7.72
3
(s, 1H), 7.51 – 7.46 (m, 2H), 7.46 – 7.43 (m, 2H), 7.18 (t, J = 8.0, 1H), 6.73 (d, J = 7.6, 1H), 4.61
(s, 2H), 4.38 (t, J = 7.1, 2H), 3.95 (s, 3H), 1.85 – 1.77 (m, 2H), 1.34 – 1.24 (m, 2H), 0.94 (t, J =
1
3
7
.4, 3H).
C NMR (126 MHz, CDCl ) δ 192.76, 147.41, 135.82, 133.97, 132.76, 132.55,
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29.27, 128.76, 127.93, 127.63, 126.32, 126.30, 125.73, 125.57, 124.28, 123.31, 115.99, 115.33,
04.33, 55.45, 50.32, 44.80, 33.84, 19.84, 13.77. HRMS (m/z): [M+K] calcd for C H KNO ,
25 25
2
10.1522; found 410.1527. HPLC t = 11.439 min; purity = 99.8%
R
0
1
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8
9
0
1
2
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8
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0
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0
1
-(1-Butyl-7-hydroxy-1H-indol-3-yl)-2-(naphthalen-1-yl)ethanone (24). Compound 24 was
synthesized from compound 7 using general procedure B to afford 0.07 g (55% yield) isolated as
an off brown solid, mp = decomposition at 213-216 °C. TLC system: 30% EtOAc/70% n-
1
hexanes. H NMR (500 MHz, DMSO) δ 9.32 (s, 1H), 8.85 (s, 1H), 8.65 – 8.58 (m, 1H), 8.39 –
8.33 (m, 1H), 8.28 (dt, J = 4.3, 8.6, 2H), 8.00 – 7.88 (m, 4H), 7.42 (t, J = 7.8, 1H), 7.15 (d, J =
7
1
1
1
.6, 1H), 5.13 (s, 2H), 5.02 (t, J = 7.1, 2H), 2.41 – 2.33 (m, 2H), 1.81 (dq, J = 7.4, 14.9, 2H),
1
3
.40 (t, J = 7.4, 3H). C NMR (126 MHz, DMSO) δ 191.47, 144.32, 136.79, 133.93, 133.52,
32.76, 129.74, 128.34, 128.04, 127.00, 125.69, 125.40, 125.36, 124.74, 122.71, 115.59, 113.81,
08.35, 49.30, 43.73, 33.85, 19.44, 13.07. HRMS (m/z): [M+Na+CH CN] calcd for
3
C H N NaO , 421.1892; found 421.1924. HPLC t = 6.458 min; purity = 99.8% using 70%
2
6
26
2
2
R
CH CN/30% H O (0.1% Formic acid).
3
2
(
1-Butyl-5-methoxy-1H-indol-3-yl)(naphthalen-1-yl)methanone (25). Compound 25 was
synthesized from compound 11 using general procedure D and 1-naphthoylacetyl chloride to
1
afford 0.36 g (16% yield) isolated as an amorphous yellow oil. H NMR (500 MHz, CDCl ) δ
3
8.18 (d, J = 8.5, 1H), 8.01 (d, J = 2.5, 1H), 7.96 (d, J = 8.2, 1H), 7.90 (d, J = 7.6, 1H), 7.64 (dd, J
=
1.2, 7.0, 1H), 7.54 – 7.44 (m, 3H), 7.28 (d, J = 2.2, 1H), 7.25 (s, 1H), 6.98 (dd, J = 2.6, 8.9,
1
H), 4.02 (t, J = 7.2, 2H), 3.92 (s, 3H), 1.81 – 1.71 (m, 2H), 1.32 – 1.22 (m, 2H), 0.88 (t, J = 7.4,
1
3
3H). C NMR (126 MHz, CDCl ) δ 192.12, 156.75, 139.25, 138.00, 133.81, 131.99, 130.88,
3
1
5
29.96, 128.24, 127.90, 126.80, 126.37, 126.11, 125.83, 124.65, 117.25, 114.28, 110.95, 103.99,
5.93, 47.23, 31.95, 20.11, 13.65. HRMS (m/z): [M+Na] calcd for C H N NaO , 421.1892;
26 26
2
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found 421.1930. HPLC t = 16.771 min; purity = 99.9% using 70% CH CN/30% H O (0.1%
R
3
2
Formic acid).
1-Butyl-5-hydroxy-1H-indol-3-yl)(naphthalen-1-yl)methanone (26). Compound 26 was
(
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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41
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44
45
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48
49
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52
53
54
55
56
57
58
59
60
synthesized from compound 12 using general procedure B to afford 0.59 g (90% yield) isolated
1
as a lightly yellow solid, mp = 209-212 °C. TLC system: 30% EtOAc/70% n-hexanes. H NMR
(500 MHz, DMSO) δ 9.20 (s, 1H), 8.07 (d, J = 7.8, 1H), 8.02 (d, J = 7.9, 1H), 7.98 (d, J = 8.5,
1
H), 7.74 (d, J = 2.4, 1H), 7.65 – 7.59 (m, 3H), 7.58 – 7.54 (m, 1H), 7.50 (ddd, J = 1.4, 6.8, 8.2,
1H), 7.41 (d, J = 8.8, 1H), 6.80 (dd, J = 2.4, 8.8, 1H), 4.11 (t, J = 7.2, 2H), 1.69 – 1.61 (m, 2H),
13
1
1
1
.23 – 1.14 (m, 2H), 0.81 (t, J = 7.4, 3H). C NMR (126 MHz, DMSO) δ 190.38, 153.63,
38.93, 138.61, 133.14, 130.83, 129.92, 129.30, 128.09, 127.39, 126.46, 126.06, 125.31, 125.20,
24.82, 115.24, 112.81, 111.40, 106.01, 45.86, 31.33, 19.16, 13.27.
HRMS (m/z):
[
M+Na+CH CN] calcd for C H N NaO , 407.1735; found 407.1777. HPLC t = 13.460 min;
3 25 24 2 2 R
purity = 100.0% using 60% CH CN/40% H O (0.1% Formic acid).
3
2
1
-Butyl-7-methoxy-3-(naphthalen-1-ylmethyl)-1H-indole (27). LiAlH (4.9 mL, 4.85 mmol,
4
4
equiv) was dissolved in THF (1M) and a solution of AlCl (1.94 g, 14.5 mmol, 12 equiv) in
3
THF (8 mL) was added dropwise at 0 °C. After 30 min, indole 3 (0.433 g, 1.21 mmol, 1 equiv)
in THF (9 mL) was added to the reaction mixture and allowed to stir at 21 °C for 48 hrs. Upon
completion, reaction mixture was cooled in an ice-bath and carefully quenched with H O and
2
acidified with 1 N HCl to pH = 3. The organic phase was then separated and washed with
NaHCO , brine, and dried over anhydrous Na SO . The solvent was evaporated under reduced
3
2
4
pressure and the resulting residue was purified by flash column chromatography on silica gel
using mixtures of EtOAc/n-hexanes to afford 0.07 g (18% yield) isolated as an pinkish oil. TLC
1
system: 10% EtOAc/90% n-hexanes. H NMR (500 MHz, CDCl ) δ 8.11 (d, J = 8.0, 1H), 7.91 –
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0
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.85 (m, 1H), 7.76 (d, J = 8.0, 1H), 7.46 (tt, J = 3.5, 8.3, 2H), 7.42 – 7.34 (m, 2H), 7.21 (dd, J =
.8, 8.0, 1H), 7.00 (t, J = 7.8, 1H), 6.65 (d, J = 7.5, 1H), 6.50 (s, 1H), 4.51 (s, 2H), 4.24 (t, J =
.2, 2H), 3.94 (s, 3H), 1.70 (dt, J = 7.4, 14.8, 2H), 1.24 (dq, J = 7.4, 14.7, 2H), 0.87 (t, J = 7.4,
0
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8
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7
8
9
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4
5
6
7
8
9
0
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5
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7
8
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3
H). C NMR (126 MHz, CDCl ) δ 147.93, 137.47, 134.22, 132.61, 130.65, 128.93, 128.32,
3
127.11, 126.92, 126.31, 126.09, 126.05, 125.83, 124.87, 119.55, 113.82, 112.31, 102.72, 55.68,
+
4
9.26, 34.70, 29.37, 20.24, 14.16. HRMS (m/z): [M ] calcd for C H NO, 343.1936; found
2
4
25
3
43.1887. HPLC t = 12.038 min; purity = 99.9% using 90% CH CN/10% H O (0.1% Formic
R 3 2
acid).
(
7-Methoxy-1-(2-morpholinoethyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone
(28).
Compound 28 was synthesized from compound 6 using general procedure D and 1-
naphthoylacetyl chloride to afford 0.16 g (16% yield) isolated as an yellow solid, mp = 149-152
1
°
C. TLC system: 50% EtOAc/50% n-hexanes. H NMR (500 MHz, CDCl ) δ 8.18 – 8.10 (m,
3
2H), 7.95 (d, J = 8.2, 1H), 7.89 (d, J = 7.5, 1H), 7.63 (dd, J = 1.2, 7.0, 1H), 7.54 – 7.41 (m, 3H),
7
2
1
1
.27 – 7.23 (m, 3H), 6.77 (d, J = 7.4, 1H), 4.40 (t, J = 6.5, 2H), 3.94 (s, 3H), 3.60 – 3.49 (m, 4H),
1
3
.67 (t, J = 6.6, 2H), 2.42 – 2.33 (m, 4H). C NMR (126 MHz, CDCl ) δ 192.18, 147.29,
3
40.00, 139.28, 133.80, 130.86, 130.00, 129.34, 128.26, 126.86, 126.45, 126.38, 126.06, 125.82,
24.55, 123.67, 117.60, 115.47, 104.70, 66.99, 59.29, 55.48, 53.77, 47.37. HRMS (m/z): [M+H]
+
calcd for C H N O , 415.2016; found 415.1985. HPLC t = 7.049 min; purity = 95.0% using
2
6
27
2
3
R
3
0% CH CN/70% H O (0.1% Formic acid).
3 2
1
-(1-Butyl-7-hydroxy-1H-indol-3-yl)-2-(4-hydroxyphenyl)ethanone (29). Compound 29
was synthesized from compound 7 using general procedure B to afford 0.05 g (17% yield)
isolated as an off-white solid, mp = decomposition at 197-200 °C. TLC system: 30%
1
EtOAc/70% n-hexanes. H NMR (500 MHz, Acetone) δ 8.86 (s, 1H), 8.23 (s, 1H), 8.16 (s, 1H),
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.86 (dd, J = 0.9, 8.0, 1H), 7.21 (d, J = 8.6, 2H), 6.98 (t, J = 7.8, 1H), 6.80 – 6.73 (m, 2H), 6.68
(dd, J = 0.9, 7.6, 1H), 4.54 (t, J = 7.1, 2H), 4.01 (s, 2H), 1.94 – 1.85 (m, 2H), 1.39 – 1.29 (m,
1
3
2
1
H), 0.94 (t, J = 7.4, 3H). C NMR (126 MHz, Acetone) δ 193.14, 156.81, 145.21, 137.93,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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31.20, 130.75, 128.46, 126.87, 123.57, 116.43, 115.91, 115.82, 114.76, 109.25, 50.17, 46.31,
34.79, 20.37, 13.99. HRMS (m/z): [M+Na] calcd for C H N NaO , 387.1685; found 387.1658.
2
2
24
2
3
HPLC t = 4.998 min; purity = 99.4% using 60% CH CN/40% H O (0.1% Formic acid).
R
3
2
1
-Butyl-7-methoxy-3-(naphthalen-2-yl)-1H-indole (30). Pd(OAc) (2 mg, 0.01 mmol, 0.01
2
equiv), SPhos (7.4 mg, 0.02 mmol, 0.02 equiv), K PO (379 mg, 1.79 mmol, 2 equiv), and 2-
3
4
naphthaleneboronic acid (230 mg, 1.34 mmol, 1.5 equiv) were placed into a round bottom
pressure flask and flushed with Ar. Intermediate 14 (294 mg, 0.89 mmol, 1 equiv) in toluene (1
mL) was then added and the reaction mixture was allowed to stir at 100 °C overnight. Reaction
was monitored via TLC and upon completion, it was allowed to cool to 21 °C and diluted with
Et O (10mL). Mixture was then filtered through a pad of Celite and concentrated under reduced
2
pressure. The resulting residue was purified by flash column chromatography on silica gel using
mixtures of DCM/pentane, to afford 0.15 g (50% yield) isolated as an clear amorphous oil, TLC
1
system: 15% DCM/85% n-hexanes. H NMR (500 MHz, CDCl ) δ 8.10 (s, 1H), 7.93 – 7.84 (m,
3
4
H), 7.80 (dd, J = 1.8, 8.4, 1H), 7.65 (dt, J = 3.2, 6.3, 1H), 7.47 (dddd, J = 1.3, 6.9, 8.0, 16.2,
2H), 7.29 (s, 1H), 7.12 (t, J = 7.9, 1H), 6.72 (d, J = 7.7, 1H), 4.46 (t, J = 7.2, 2H), 3.99 (s, 3H),
1
3
1
1
.92 – 1.83 (m, 2H), 1.45 – 1.36 (m, 2H), 0.98 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ
3
47.84, 134.10, 133.46, 131.93, 128.72, 128.20, 127.80, 127.75, 127.51, 126.71, 126.56, 126.12,
125.11, 125.07, 120.43, 116.57, 112.74, 102.83, 55.45, 49.49, 34.43, 20.06, 13.91. HRMS (m/z):
+
[
M ] calcd for C H NO, 329.1780; found 329.1747. HPLC t = 12.570 min; purity = 96.7%
2
3
23
R
using 90% CH CN/10% H O (0.1% Formic acid).
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(
1-Butyl-7-methoxy-1H-indol-3-yl)(4-fluorophenyl)methanone (31). Compound 31 was
synthesized from compound 6 using general procedure D and 4-fluorobenzoyl chloride to afford
1
0
8
.28 g (49% yield) isolated as an pinkish solid, mp = 91-94 °C. H NMR (500 MHz, CDCl ) δ
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0
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3
4
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6
7
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.02 (dd, J = 0.8, 8.0, 1H), 7.89 – 7.84 (m, 2H), 7.45 (s, 1H), 7.26 (t, J = 8.0, 1H), 7.22 – 7.17
(m, 2H), 6.80 (d, J = 7.4, 1H), 4.44 (t, J = 7.2, 2H), 4.00 (s, 3H), 1.91 – 1.81 (m, 2H), 1.43 – 1.32
13
(
m, 2H), 0.98 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 189.45, 165.64, 163.64, 147.43,
3
1
37.73, 137.34, 137.32, 131.17, 131.10, 129.80, 126.50, 123.39, 115.45, 115.31, 115.28, 115.13,
104.57, 55.49, 50.37, 33.97, 19.90, 13.79. HRMS (m/z): [M+Na+CH CN] calcd for
3
C H FN NaO , 389.1641; found 389.1608. HPLC t = 8.765 min; purity = 99.8%.
2
2
23
2
2
R
1
-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(2-fluorophenyl)ethanone (32). Compound 32 was
synthesized from compound 6 using general procedure D and an acid chloride made in situ from
4
3
2
-(2-fluorophenyl)acetic acid to afford 0.30 g (35% yield) isolated as a darker yellow solid, mp
1
= 86-88 °C. H NMR (500 MHz, CDCl ) δ 8.04 (dd, J = 0.8, 8.1, 1H), 7.78 (s, 1H), 7.41 (td, J =
3
1
1
0
1
.7, 7.6, 1H), 7.29 – 7.23 (m, 1H), 7.21 (t, J = 8.0, 1H), 7.16 – 7.07 (m, 2H), 6.75 (d, J = 7.4,
H), 4.44 (t, J = 7.2, 2H), 4.20 (s, 2H), 3.97 (s, 3H), 1.91 – 1.82 (m, 2H), 1.42 – 1.32 (m, 2H),
1
3
.99 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 190.37, 160.78, 158.83, 146.34, 135.04,
3
35.03, 130.71, 130.67, 128.14, 127.54, 127.48, 125.39, 123.22, 123.19, 122.30, 122.13, 122.01,
114.61, 114.37, 114.19, 114.17, 103.31, 54.40, 49.30, 38.49, 38.48, 32.82, 18.80, 12.73.
[
M+Na] calcd for C H FNNaO , 362.1532; found 362.1513. HPLC t = 8.732 min; purity =
21 22 2 R
9
9.6%.
1-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(3-fluorophenyl)ethanone (33). Compound 33 was
synthesized from compound 6 using general procedure D and an acid chloride made in situ from
4
3
1
2
-(3-fluorophenyl)acetic acid to afford 0.21 g (27% yield) isolated as an yellow oil. H NMR
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(
500 MHz, CDCl ) δ 7.98 (dd, J = 0.8, 8.1, 1H), 7.63 (s, 1H), 7.28 – 7.21 (m, 2H), 7.16 (t, J =
3
8
1
0
.0, 1H), 7.08 (d, J = 8.1, 1H), 7.03 (d, J = 9.8, 1H), 6.91 (td, J = 1.8, 8.3, 1H), 6.71 (d, J = 7.5,
H), 4.38 (t, J = 7.2, 2H), 4.10 (s, 2H), 3.92 (s, 3H), 1.85 – 1.75 (m, 2H), 1.36 – 1.25 (m, 2H),
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32
33
34
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36
37
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
.93 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 191.85, 163.92, 161.97, 147.41, 138.42,
3
138.36, 135.97, 130.00, 129.94, 129.16, 126.44, 125.18, 125.16, 123.45, 116.54, 116.37, 115.86,
15.21, 113.69, 113.52, 104.46, 55.46, 50.40, 46.55, 46.54, 33.88, 19.87, 13.77. HRMS (m/z):
M+Na] calcd for C H FNNaO , 362.1532; found 362.1503. HPLC t = 13.931 min; purity =
1
[
21 22
2
R
99.9% using 50% CH CN/50% H O (0.1% Formic acid).
3
2
1
-(1-Butyl-7-ethyl-1H-indol-3-yl)-2-(4-fluorophenyl)ethanone (34). Compound 34 was
synthesized from compound 6 using general procedure D and 4-fluorophenylacetyl chloride to
1
afford 0.34 g (39% yield) isolated as an white powder, mp = 67-70 °C. H NMR (500 MHz,
CDCl ) δ 8.34 (dd, J = 1.2, 8.0, 1H), 7.72 (s, 1H), 7.32 – 7.27 (m, 2H), 7.25 – 7.20 (m, 1H), 7.10
3
(d, J = 6.5, 1H), 7.04 – 6.98 (m, 2H), 4.35 – 4.28 (m, 2H), 4.12 (s, 2H), 3.02 (q, J = 7.5, 2H),
13
1
1
1
.87 – 1.78 (m, 2H), 1.42 – 1.32 (m, 5H), 0.98 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ
3
92.35, 162.81, 160.87, 136.80, 134.58, 131.73, 131.70, 130.95, 130.88, 128.22, 127.79, 124.78,
23.06, 120.82, 115.82, 115.50, 115.33, 49.80, 46.00, 34.07, 25.52, 20.01, 16.28, 13.75. HRMS
(
m/z): [M+Na+CH CN] calcd for C H FN NaO, 401.2005; found 401.1976. HPLC t = 9.645
3 24 27 2 R
min; purity = 99.9%.
-Butyl-7-methoxy-3-(naphthalen-1-yl)-1H-indole (35). Intermediate 14 (512 mg, 1.56
1
mmol, 1 equiv) and Pd(PPh ) (54 mg, 0.05 mmol, 0.03 equiv) were placed into a round bottom
3 4
flask and flushed with Ar. Solvent (DME, 6 mL) was then added and allowed to stir for 10 min
upon which the solution was degassed by bubbling with Ar for 15 min. Sodium carbonate (2M
[
1.06 g in 5 mL H O, 1.6 ml], 3.11mmol, 2 equiv) and 1-naphthaleboronic acid (401 mg, 2.33
2
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mmol, 1.5 equiv) in EtOH (1 mL) were added and the reaction mixture was refluxed. Reaction
was monitored via TLC and upon completion, it was allowed to cool to 21 °C and EtOAc was
added. Mixture was then filtered through a pad of Celite and concentrated under reduced
pressure. The resulting residue was purified by flash column chromatography on silica gel using
0
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mixtures of EtOAc/n-hexanes, to afford 0.17 g (37% yield) isolated as an yellow solid, mp = 74-
1
7
1
6 °C. TLC system: 10% DCM/90% n-hexanes. H NMR (500 MHz, CDCl ) δ 8.10 (d, J = 8.2,
3
H), 7.92 (d, J = 8.2, 1H), 7.85 (d, J = 8.0, 1H), 7.53 (dddd, J = 1.3, 6.9, 8.1, 23.6, 3H), 7.41
(ddd, J = 1.3, 6.8, 8.2, 1H), 7.19 (s, 1H), 7.09 (dd, J = 0.9, 8.0, 1H), 7.00 (t, J = 7.8, 1H), 6.70 (d,
J = 7.3, 1H), 4.50 (t, J = 7.1, 2H), 4.01 (s, 3H), 1.95 – 1.86 (m, 2H), 1.47 – 1.37 (m, 2H), 0.99 (t,
1
3
J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 147.75, 134.08, 133.36, 132.64, 130.54, 128.76,
3
1
5
28.28, 127.75, 126.92, 126.80, 125.90, 125.70, 125.67, 125.65, 119.90, 114.86, 113.25, 102.56,
+
5.47, 49.36, 34.47, 20.08, 13.93. HRMS (m/z): [M ] calcd for C H NO, 329.1780; found
2
3
23
329.1747. HPLC t = 11.727 min; purity = 95.4% using 90% CH CN/10% H O (0.1% Formic
R
3
2
acid).
-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(3,4-difluorophenyl)ethanone (36). Compound 36
1
was synthesized from compound 6 using general procedure D and an acid chloride made in situ
43
from 2-(3,4-difluorophenyl)acetic acid to afford 0.07 g (8% yield) isolated as an off-white
1
solid, mp = 88-90 °C. H NMR (500 MHz, CDCl ) δ 8.01 (dd, J = 0.8, 8.1, 1H), 7.68 (s, 1H),
3
7
7
.21 (t, J = 8.0, 1H), 7.19 – 7.14 (m, 1H), 7.14 – 7.09 (m, 1H), 7.06 (d, J = 2.2, 1H), 6.76 (d, J =
.8, 1H), 4.44 (t, J = 7.2, 2H), 4.11 (s, 2H), 3.97 (s, 3H), 1.92 – 1.79 (m, 2H), 1.37 (dq, J = 7.4,
1
3
14.8, 2H), 0.99 (t, J = 7.4, 3H). C NMR (126 MHz, CDCl ) δ 191.44 , 150.14 (dd, J = 247.8,
3
1
1
2.8 Hz), 149.30 (dd, J = 246.7, 12.6 Hz), 147.32 , 135.70 , 132.64 (dd, J = 6.0, 4.0 Hz), 128.98 ,
26.38 , 125.38 (dd, J = 6.1, 3.6 Hz), 123.43 , 118.36 (d, J = 17.3 Hz), 117.08 (d, J = 17.1 Hz),
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15.66 , 115.02 , 104.41 , 55.37 , 50.34 , 46.07 – 44.76 (m), 33.81 , 19.80 , 13.68 . HRMS (m/z):
[M+Na+CH CN] calcd for C H F N NaO , 421.1704; found 421.1678. HPLC t = 8.678 min;
3 23 24 2 2 2 R
purity = 100.0%.
-(1-Butyl-7-methoxy-1H-indol-3-yl)-2-(2,3-difluorophenyl)ethanone (37). Compound 37
was synthesized from compound 6 using general procedure D and an acid chloride made in situ
10
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18
19
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58
59
60
1
4
3
from 2-(2,3-difluorophenyl)acetic acid to afford 0.23 g (24% yield) isolated as an off-brown
1
solid. H NMR (500 MHz, CDCl ) δ 8.02 (dd, J = 0.8, 8.1, 1H), 7.78 (s, 1H), 7.21 (t, J = 8.0,
3
1H), 7.16 (t, J = 6.7, 1H), 7.13 – 7.03 (m, 2H), 6.76 (d, J = 7.4, 1H), 4.45 (t, J = 7.2, 2H), 4.23
13
(d, J = 1.3, 2H), 3.98 (s, 3H), 1.92 – 1.83 (m, 2H), 1.43 – 1.33 (m, 2H), 0.99 (t, J = 7.4, 3H). C
NMR (126 MHz, CDCl ) δ 190.40 , 150.68 (dd, J = 222.0, 13.0 Hz), 148.72 (dd, J = 220.5, 13.1
3
Hz), 147.32 , 135.89 (d, J = 1.6 Hz), 129.00 , 126.37 , 126.29 (t, J = 3.2 Hz), 125.38 (d, J = 12.6
Hz), 123.91 (dd, J = 6.9, 4.6 Hz), 123.37 , 115.65 (d, J = 17.1 Hz), 115.44 , 115.03 , 104.36 ,
55.36 , 50.32 , 39.10 (t, J = 1.8 Hz), 33.78 , 19.76 , 13.67 . HRMS (m/z): [M+Na+CH CN] calcd
3
for C H F N NaO , 421.1704; found 421.1687. HPLC t = 8.827 min; purity = 100.0%.
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3
24
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2
2
R
2
7
Membrane preparation. Mouse brain homogenates for in vitro assays were prepared as
2
7
previously described. Briefly, whole brains were harvested from B6SJL mice, snap-frozen in
liquid nitrogen and stored at -80 °C. On the day membrane homogenates were to be prepared,
brains were thawed on ice, then pooled in a 40 mL Dounce glass homogenizer and suspended in
5
volumes of ice-cold homogenization buffer (50mM HEPES, pH 7.4, 3 mM MgCl , and 1 mM
2
EGTA). Brains were then subjected to 10 complete strokes with an A pestle, followed by
centrifugation at 40,000 x g for 10 min at +4 °C. Resulting supernatants were discarded, and the
pellet was resuspended, homogenized and centrifuged similarly twice more, with supernatants
being discarded. For the final resuspension and homogenization with a B pestle, ice-cold 50 mM
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