Identification of Psychoplastogenic N, N-Dimethylaminoisotryptamine (isoDMT) Analogues through Structure-Activity Relationship Studies

Article abstract of DOI:10.1021/acs.jmedchem.9b01404

Ketamine, N,N-dimethyltryptamine (DMT), and other psychoplastogens possess enormous potential as neurotherapeutics due to their ability to potently promote neuronal growth. Here, we report the first-ever structure-Activity relationship study with the explicit goal of identifying novel psychoplastogens. We have discovered several key features of the psychoplastogenic pharmacophore and used this information to develop N,N-dimethylaminoisotryptamine (isoDMT) psychoplastogens that are easier to synthesize, have improved physicochemical properties, and possess reduced hallucinogenic potential as compared to their DMT counterparts.

Full text of DOI:10.1021/acs.jmedchem.9b01404

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Article
Identification of Psychoplastogenic
N,N‑Dimethylaminoisotryptamine (isoDMT) Analogues through
Structure−Activity Relationship Studies
Lee E. Dunlap, Arya Azinfar, Calvin Ly, Lindsay P. Cameron, Jayashri Viswanathan, Robert J. Tombari,
Douglas Myers-Turnbull, Jack C. Taylor, Ana Cristina Grodzki, Pamela J. Lein, David Kokel,
and David E. Olson*
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ABSTRACT: Ketamine, N,N-dimethyltryptamine (DMT), and other
psychoplastogens possess enormous potential as neurotherapeutics
due to their ability to potently promote neuronal growth. Here, we
report the first-ever structure−activity relationship study with the
explicit goal of identifying novel psychoplastogens. We have
discovered several key features of the psychoplastogenic pharmaco-
phore and used this information to develop N,N-dimethylaminoiso-
tryptamine (isoDMT) psychoplastogens that are easier to synthesize,
have improved physicochemical properties, and possess reduced
hallucinogenic potential as compared to their DMT counterparts.
scopolamine8 and GLYX-13 (i.e., rapastinel)9 have demon-
strated psychoplastogenic properties, and this class of
compounds has enormous potential for treating a variety of
neuropsychiatric diseases. Our group has demonstrated that
classic serotonergic psychedelics are among the most potent
psychoplastogens, producing effects on neuronal structure
comparable to ketamine in both cellular assays and in vivo.10
As pyramidal neurons in the PFC are known to exhibit top-
down control over areas of the brain controlling motivation,
fear, and reward, these results provide a potential explanation
for the known antidepressant, anxiolytic, and antiaddictive
effects of psychedelics in the clinic.11
Careful inspection of the molecular structures of psychedelic
compounds reveals that N, N-dimethyltryptamine (DMT, 1) is
a core feature of many of these molecules (Figure 1).12 As
DMT produces antidepressant and anxiolytic behavioral effects
in rodents13,14 and a DMT-containing tisane has demonstrated
clinical efficacy for treatment-resistant depression,15−17 we
reasoned that DMT was an excellent starting point for
medicinal chemistry efforts aimed at identifying novel
psychoplastogenic therapeutics. However, the synthesis of
DMT derivatives from simple indoles is typically accomplished
using the method of Speeter and Anthony,18 which requires
INTRODUCTION
■
Major depressive disorder and related neuropsychiatric
diseases are among the leading causes of disability worldwide.1
Despite the prevalence of these illnesses, we still lack broadly
efficacious treatments capable of producing both fast-acting
and sustained effects. Recently, the Food and Drug
Administration (FDA) approved the dissociative anesthetic
esketamine for treatment-resistant depression, making it the
first mechanistically distinct medicine to be introduced to
psychiatry in nearly 30 years. Accumulating evidence suggests
that ketamine is capable of rectifying the deleterious changes in
neuronal structure that are associated with depression.2,3 Such
structural alterations include the loss of dendritic spines and
synapses in the prefrontal cortex (PFC), as well as reductions
in dendritic arbor complexity.4,5 While the advent of ketamine
represents an incredibly important milestone in the history of
neuropsychiatric disease drug discovery, ketamine is an
imperfect drug with potential for abuse,6 and moreover, its
dissociative effects necessitate the hospitalization of patients
during treatment. Therefore, the identification of safer
alternatives to ketamine is an incredibly important goal.
Until recently, relatively few compounds were known to
possess neural plasticity-promoting properties comparable to
ketamine. Known as psychoplastogens,7 these molecules
promote neuronal growth through a mechanism involving
the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepro-
pionic acid (AMPA) receptors, the tropomyosin receptor
kinase B (TrkB), and the mammalian target of rapamycin
(mTOR). In addition to ketamine, the tropane alkaloid
Received: September 6, 2019
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Table 1. Optimization of Indole N-Alkylation
equiv
of 3
temp
(°C)
yield
d
a
b
c
base
solvent
additive
(%)
1.3
1.1
1.1
1.1
1.1
1.1
1.1
3
NaH (2.6)
NaH (2.6)
KOH (5)
KOH (5)
KOH (5)
KOH (5)
KOH (5)
KOH (5)
THF (0.5)
66
23
23
23
23
23
23
23
66
67
31
51
69
24
38
69
DMF (0.4)
DMF (0.4)
DMSO (0.4)
DMSO (0.4)
DMSO (0.1)
DMSO (1.0)
DMSO (0.4)
Kl (1.1)
Kl (1.1)
Kl (1.1)
Kl (3)
Figure 1. Structures of compounds possessing the DMT pharmaco-
phore. (A) The DMT structure (highlighted in black) is the core
scaffold of several known psychoplastogenic compounds. (B) The
only difference between the chemical structures of DMT (1) and
N,N-dimethylaminoisotryptamine (isoDMT) (2) is that the C1 and
C3 substituents of the indole are transposed. Predicted chemical
properties and calculated MPO scores are shown. clog D = calculated
log D; TPSA = total polar surface area; HBD = hydrogen bond donor;
and MPO = multiparameter optimization score.
a
b
c
Number of equivalents of the base are shown in parentheses.
Reaction molarities based on indole are shown in parentheses.
Number of equivalents of the additive are shown in parentheses.
Yields are based on H nuclear magnetic resonance (NMR) spectra
obtained after aqueous workup with 6-flouroindole serving as an
d
1
internal standard.
multiple steps, harsh reaction conditions, and electron-rich
indoles, thus limiting the number of derivatives we would be
able to access rapidly for structure−activity relationship (SAR)
studies. Moreover, many DMT derivatives are well known to
be potent hallucinogens.
critical, and ultimately, we were able to obtain 2 in good yield
using KOH as the base and KI to enhance reactivity via an in
situ Finkelstein reaction. Maintaining the reaction at 0.4 M
proved optimal with both higher and lower concentrations
resulting in a reduction of yield.
When considering how we could address these issues, we
were inspired by the elegant work of Glennon and co-
workers.19 By transposing the N1 and C3 substituents of
DMT, they produced a small series of N,N-dimethylaminoiso-
tryptamine (isoDMT, 2) analogues with reduced hallucino-
genic potential as measured by their abilities to substitute for
known hallucinogens in rodent drug discrimination assays. In
principle, related analogues could be accessed in a single step
through N-alkylation of the corresponding indoles or related
heterocycles. Additionally, several isoDMTs have been shown
to possess a comparable affinity for serotonin receptors as
compared to their DMT counterparts.19,20 Our group has
demonstrated that the 5-HT2A receptor is necessary for the
psychoplastogenic effects of DMT.10 As isoDMTs are known
to bind to 5-HT2A receptors,20 we hypothesized that isoDMT
analogues would still be capable of promoting neuronal growth
despite lacking indole N−H bonds. Furthermore, isoDMT
analogues are likely to exhibit improved physicochemical
properties as the loss of a hydrogen bond donor decreases total
polar surface area and improves central nervous system
multiparameter optimization (MPO) scores (Figure 1).21
Here, we describe our efforts to develop an efficient method
to access a variety of isoDMT derivatives as well as their
subsequent evaluation in cellular neural plasticity assays.
Previously published methods for the synthesis of isoDMT
have either required purification via column chromatography
or vacuum distillation followed by oxalate salt formation.19,22,23
As indole was cleanly converted to 2 using our reaction
conditions, we reasoned that crystallization following a simple
aqueous workup might obviate the need for further
purification. Using this operationally simple method, we
synthesized 20 isoDMT analogues in modest to good yields
without the need for chromatography (Figure 2). Low yielding
reactions could often be attributed to difficulties with
crystallization, as the NMR yields of those reactions were
often substantially higher than the isolated yields. An
additional four compounds proved recalcitrant toward
crystallization and hence were isolated following column
chromatography on silica gel. The method is quite general,
with electron-rich (4−11, 16) and electron-poor (12−15, 17−
21) indoles performing equally well. Additionally, substitution
on the indole did not have an obvious impact on reaction
performance with substitution at all positions being tolerated.
Finally, related heterocycles including benzimidazole, pyrrole,
and carbazole are efficiently alkylated using these same
conditions (22−24).
We hypothesized that the efficiency of the alkylation was due
to the formation of a reactive aziridinium intermediate (Figure
3A). However, the use of 1-chloro-3-methylbutane as the
alkylating agent results in a comparable yield (Figure 3B).
Moreover, increasing the distance between the electrophilic
carbon bearing the halide and the nucleophilic nitrogen does
not drastically reduce reaction performance (Figure 3B). These
results suggest that the reaction is proceeding through a
traditional second-order nucleophilic substitution (SN2)
reaction with negligible to no enhancement via neighboring
group participation.
RESULTS AND DISCUSSION
■
Chemistry. Surprisingly, there have been relatively few
reports concerning the synthesis of isoDMT or related
analogues, and they all require multiple steps or employ
harsh reaction conditions.19,22,23 Therefore, we sought to
develop an operationally simple and robust method for
synthesizing a variety of isoDMTs under mild reaction
conditions. We screened several conditions for performing
the desired N-alkylation of indole, including the previously
reported methods (Table 1), and found that alkylation could
be achieved without using NaH or refluxing the reaction. The
use of dimethyl sulfoxide (DMSO) as the solvent proved to be
Dendritogenesis Assays. Phenotypic screening has
historically proven more successful than target-based ap-
proaches for identifying drugs with novel mechanisms of
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Figure 2. Substrate scope for the N-alkylation of various indoles with 3. Percent yields following crystallization are indicated. Values in parentheses
denote yields based on 1H NMR spectra obtained after aqueous workup with indole serving as an internal standard. Note: 6-fluoroindole was used
as the internal standard when determining the yield of 2. An asterisk indicates that a compound was purified via chromatography.
potential hydrogen bonding interaction must not be critical for
a compound to induce plasticity, as both 27 and 2 increased
dendritic arbor complexity to a comparable extent as 1, despite
lacking an indole N−H bond (Figure 4).
Next, we were interested in performing head-to-head
comparisons between DMT analogues and their isoDMT
counterparts, as Glennon and co-workers previously used
serotonin receptor binding affinities and drug discrimination
assays to demonstrate that these pairs of compounds can
exhibit bioisosterism.19 We chose 5-MeO-DMT (28) and 6-F-
DMT (29) as electron-rich and electron-poor DMT analogues,
Figure 3. Neighboring group participation has a minimal impact on
respectively. Compound 28 has been reported to promote
reaction performance. (A) Hypothesized reactive aziridinium
neuritogenesis in the dentate gyrus28 and alleviate symptoms of
intermediate. (B) Reaction efficiency remains high when using
depression and anxiety in humans.29,30 Compound 29 is
alkylating agents that cannot form a reactive aziridinium intermediate.
predicted to be nonhallucinogenic, as fluorination of DMT
analogues is known to attenuate their hallucinogenic
potential.31 When compared directly, isoDMT analogues 6
and 13 performed identically to 28 and 29 (Figure 5),
suggesting that SAR data related to neuronal growth obtained
using derivatives of the isoDMT scaffold could be applied to
derivatives of the DMT scaffold through analogy due to the
isosteric nature of the two structures.
Confident that the DMT and analogous isoDMT derivatives
would behave similarly, we next attempted to use various
isoDMT analogues to establish the key features of the
psychoplastogen pharmacophore (Figure 6). Removing the
basic amine of isoDMT to produce 25 yielded a molecule that
did not promote dendritogenesis. Furthermore, compound
31the N,N-dimethylamide analogue of isoDMTdid not
promote neuronal growth, confirming our hypothesis that a
basic nitrogen is necessary to promote plasticity (Figure 6A,B).
Extending the distance between the aromatic ring and the
amine by one carbon (26) resulted in only a slight decrease in
the Nmax value (Figure 6C).
Percent yields following crystallization are indicated. Values in
parentheses denote yields based on H NMR spectra obtained after
aqueous workup with 6-fluoroindole serving as an internal standard.
An asterisk indicates that a compound was purified via chromatog-
raphy.
1
action.24,25 Having established a simple and robust method for
accessing isoDMT analogues, we next tested their ability to
increase dendritic arbor complexity in cultures of cortical
neurons using a phenotypic assay. Following treatment,
neurons were fixed and visualized using an antibody against
MAP2, a cytoskeletal protein localized to the somatodendritic
compartment of neurons.26 Sholl analysis27 was then
performed, and the maximum number of crossings (Nmax
)
was used as a quantitative metric of dendritic arbor complexity.
For statistical comparisons between specific compounds, we
compared the raw Nmax values; however, we have also
calculated a percent efficacy as well as an MPO score for
every compound tested in this study (Figure S1). Percent
efficacies were determined by setting the Nmax values for the
vehicle (DMSO) and positive (ketamine) controls equal to 0
and 100%, respectively.
We began our SAR studies by comparing the effect of DMT
(1) to that of 1-Me-DMT (27) and isoDMT (2). While DMT
has the potential to serve as a hydrogen bond donor when
bound to its target receptors, 27 and 2 do not. Therefore, this
Modification of the aromatic ring was generally well
tolerated (Figure 6C). Converting the indole into a
benzimidazole (22), pyrrole (23), or carbazole (24) had a
minimal effect on the ability of these molecules to promote
neuronal growth. Moreover, substitution at the 2- and 3-
positions of the indole (16 and 21, respectively) was well
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Figure 4. Indole N−H of tryptamine derivatives is not necessary to promote dendritogenesis. (A) Representative images of cortical neurons
(DIV6) treated with compounds. See Figure S2 for the entire field of view from which these neurons were selected. (B) Sholl analysis demonstrates
that 1-Me-DMT (27) and isoDMT (2) increase dendritic arbor complexity to a comparable extent as DMT (1) (n = 46−79 neurons). (C) The
maximum number of crossings (Nmax) of the Sholl plots in B. Data are represented as mean standard error of the mean (SEM). *p < 0.05, **p <
0.01, ***p < 0.001, ****p < 0.0001, as compared to the vehicle control following a one-way analysis of variance (ANOVA) with Dunnett’s post
hoc test (F = 9.702; DFn = 4; DFd = 304; p-value < 0.0001). VEH = vehicle, KET = ketamine. Scale bar = 20 μm.
tolerated. Taken together, the minimal psychoplastogen
pharmacophore appears to involve a modifiable aromatic ring
separated from a basic nitrogen by a short linker.
Substitution on the benzene ring of both DMTs and
isoDMTs is known to impact hallucinogenic potential.19,32 For
example, 5-MeO-DMT (28) substitutes for the hallucinogen
2,5-dimethoxy-4-methylamphetamine (DOM) in rats trained
to discriminate DOM from saline, whereas 6-MeO-DMT does
not.32 Similarly, 6-MeO-isoDMT (5) substitutes for a
hallucinogenic training drug, while 5-MeO-isoDMT (6) does
not.19 Therefore, we desired to test the effects of steric and
Figure 5. DMT and isoDMT analogues produce comparable effects
on dendritic arbor complexity. (A) Chemical structures of DMT
derivatives and analogous isoDMTs. (B) The maximum number of
electronic perturbations at positions 4−7 of the indole. We
synthesized and tested three series of analogues substituted
crossings (Nmax) of the Sholl analysis for cortical neurons treated with
with either methoxy (electron-donating; 4−7), benzyloxy
compounds (n = 82−95 neurons). Data are represented as mean
(electron-donating, but sterically demanding; 8−11), or fluoro
SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as
(electron-withdrawing; 12−15) groups (Figure 7). We found
that substitutions of the 5-, 6-, and 7-positions were well
tolerated regardless of the substituent. However, substitution at
the 4-position resulted in compounds incapable of increasing
dendritic arbor complexity. This result was quite striking as the
specific electronic or steric properties of the substituents were
compared to the vehicle control following a one-way ANOVA with
Dunnett’s post hoc test (F = 11.17; DFn = 5; DFd = 524; p-value <
0.0001). VEH = vehicle, KET = ketamine.
Figure 6. Establishment of the essential psychoplastogen pharmacophore. (A) Chemical structures of nonbasic analogues of isoDMT 2. (B, C) The
maximum number of crossings (Nmax) of the Sholl plots for cortical neurons treated with compounds (n = 46−85 neurons). The effects of nitrogen
basicity and modifications to the aromatic ring were assessed in B and C, respectively. Data are represented as mean SEM. *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001, as compared to the vehicle control following a one-way ANOVA with Dunnett’s post hoc test. [For B: F = 19.03;
DFn = 4; DFd = 273; p-value < 0.0001. For C: F = 6.933; DFn = 8; DFd = 599; p-value < 0.0001.] VEH = vehicle, KET = ketamine.
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Figure 7. Impact of indole substitution on the ability of isoDMTs to promote neuronal growth. The maximum number of crossings (Nmax) of the
Sholl plots for cortical neurons treated with compounds (n = 39−93 neurons). Data are represented as mean SEM. *p < 0.05, **p < 0.01, ***p <
0.001, ****p < 0.0001, as compared to the vehicle control following a one-way ANOVA with Dunnett’s post hoc test. (For R = OMe: F = 13.85;
DFn = 5; DFd = 493; p-value < 0.0001. For R = OBn: F = 15.44; DFn = 5; DFd = 372; p-value < 0.0001. For R = F: F = 13.24; DFn = 5; DFd =
506; p-value < 0.0001.) VEH = vehicle, KET = ketamine.
Figure 8. Concentration−response experiments demonstrate that DMTs and isoDMTs have similar psychoplastogenic potencies. The maximum
number of crossings (Nmax) of the Sholl plots for cortical neurons treated with compounds at concentrations ranging from 10 μM to 10 pM (n =
66−123 neurons). Data are represented as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to the vehicle control
following a one-way ANOVA with Dunnett’s post hoc test (F = 15.40; DFn = 24; DFd = 2,276; p-value < 0.0001). V = vehicle, K = ketamine.
inconsequential. Even a fluorine substituent with a very small
van der Waals radius (1.2 and 1.47 for H and F, respectively)33
was not tolerated. Careful inspection of the 5-HT2B crystal
structure bound to lysergic acid diethylamide (LSD) suggests
that the 7-position of the LSD indole abuts helix V and thus is
not likely to tolerate any substituent at that position.34
Assuming that DMTs and isoDMTs bind to 5-HT2 receptors
in a similar conformation as LSD, we would predict that
substitution at the 4-position of an isoDMT (corresponding to
the 7-position of the LSD indole) would not be tolerated.
However, this hypothesis is purely speculative at the moment,
as there are no published crystal structures of DMTs or
isoDMTs bound to 5-HT2 receptors.
to its low hallucinogenic potential in both drug-discrim-
ination19 and head-twitch response (HTR) assays (Figure 11).
Previously, we demonstrated that DMT and other
psychedelic compounds promote increased dendritic arbor
complexity, dendritic spine density, and synaptogenesis
through a 5-HT2A-dependent process.10 As expected, we
found that pretreating cortical cultures with a 5-HT2A
antagonist blocked the ability of 5-MeO-DMT (28) to
increase dendritic growth (Figure 9). Importantly, the
psychoplastogenic effects of isoDMTs were also blocked
under these conditions, suggesting that 5-HT2A receptors
may be involved in their mechanism of action (Figure 9).
However, the involvement of other receptors cannot be ruled
out at this time.
To determine if DMT and isoDMT derivatives exhibited
differences in psychoplastogenic potency, we performed
concentration−response experiments (Figure 8). We found
that isoDMTs (2 and 6) produced comparable maximum
efficacies and had similar potencies as isosteric DMTs (1 and
28). Moreover, they were capable of increasing dendritic arbor
complexity at concentrations as low as 1 nM. We also observed
that these compounds exhibited comparable efficacies and
potencies to ketamine, further emphasizing their potential as
antidepressants. Finally, compound 5 proved to be an
exceptional psychoplastogen, which is highly significant due
Zebrafish Behavioral Assays. While our cellular den-
dritogenesis assays indicated that isosteric molecules from the
DMT and isoDMT classes performed comparably, we wanted
to demonstrate bioisosterism in a different context. To this
end, we decided to employ an in vivo zebrafish behavioral
assay that has been previously validated for assessing
similarities between compounds.35 Due to the short timescale
of the zebrafish behavioral effects (hours) relative to changes in
neuronal structure (days), it is unlikely that psychoplastoge-
nicity plays a role in modulating acute zebrafish behavior.
Larval zebrafish were treated with 5-MeO-DMT (28), 6-MeO-
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concentration-dependent manner (Figure 10B) with the
largest effects (ECmax) being observed at 200 μM for each
compound.
To test our hypothesis that pairs of isosteric compounds
would produce behavioral phenotypes similar to each other but
dissimilar from nonisosteric compounds, we trained a multi-
classification model to choose between animals treated with 5-
MeO-DMT (28), 6-MeO-isoDMT (6), 6-MeO-DMT (30),
and 5-MeO-isoDMT (5) at 200 μM. We found that
misclassification of isosteric pairs [e.g., 5-MeO-DMT (28)
and 6-MeO-isoDMT (6); or 6-MeO-DMT (30) and 5-MeO-
isoDMT (5)] was more likely (Figure 10C; 50.4% higher, p =
0.154 by the permutation test), indicating that isosteric
compounds produce more similar behavioral phenotypes.
Though the acute behavioral changes in zebrafish are unlikely
the result of compound-induced neural plasticity, these results
suggest that the biological effects of isoDMT compounds are
likely to be similar to their DMT isosteres.
Figure 9. Psychoplastogenic effects of isoDMTs are blocked by a 5-
HT2A antagonist. The maximum number of crossings (Nmax) of the
Sholl plots for cortical neurons treated with compounds (n = 45−63
neurons) in the presence (+) or absence (−) of the 5-HT2A
antagonist ketanserin. Data are represented as mean SEM. ****p <
0.0001, as compared to the vehicle control following a one-way
ANOVA with Dunnett’s post hoc test (F = 13.92; DFn = 8; DFd =
461; p-value < 0.0001). V = vehicle, K = ketamine, KTSN =
ketanserin.
Hallucinogenic Potential. While Glennon and co-workers
have shown that isoDMTs such as 5 and 6 exhibit reduced
hallucinogenic potential than their DMT counterparts in drug-
discrimination assays,19 we were interested in testing their
abilities to elicit a mouse head-twitch response (HTR)a
well-validated behavioral proxy for hallucinations.36 The
isoDMT (6), 6-MeO-DMT (30), or 5-MeO-isoDMT (5) and
video-recorded during a 17 min battery of acoustic and visual
stimuli (Figures 10A and S3). Aggregate locomotion over time
was determined per well. All compounds tested induced
significant behavioral effects relative to the vehicle control in a
Figure 10. Zebrafish behavioral assays demonstrate the similarity between isosteres. (A) An example motion trace with associated stimuli. Top:
locomotion in wells treated with the vehicle (gray) or 5-MeO-DMT (28, 200 μM, red). Individual traces for 5, 6, and 30 are shown in Figure S3.
Bottom: stimuli applied over time. Colors indicate bright light-emitting diode (LED) light of respective colors. Black traces represent the waveform
of acoustic stimuli, and gray vertical lines indicate physical tapping as secondary acoustic stimuli. (B) Concentration−response curves for DMT and
isoDMT analogues (n = 21 wells per condition). The y axis is the mean accuracy of classification against vehicle controls. Gray lines are the mean
standard deviation (STD) under bootstrap. (C) Confusion matrix showing that isosteric pairs produce similar behavioral phenotypes (n = 18 wells
per condition). Darkness indicates the percentage of wells classified; the maximum is 50.7% (5-MeO-DMT as itself).
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Figure 11. Mouse HTR assays demonstrate that psychoplastogenic isoDMTs exhibit reduced hallucinogenic potential. Male and female mice were
administered drugs via intraperitoneal injection, and the number of head-twitches was recorded over the next 20 min (n = 3−8 mice per condition).
Data are represented as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as compared to the vehicle control following a one-
way ANOVA with Dunnett’s post hoc test. V = vehicle.
Nuclear magnetic resonance (NMR) spectra were acquired on
either a Bruker 400 operating at 400 and 100 MHz, a Varian 600
operating at 600 and 150 MHz, or a Bruker 800 operating at 800 and
known hallucinogenic compound 5-MeO-DMT (28) produces
a robust, dose-dependent HTR that was greater in female mice.
However, the isosteric compound 6-MeO-isoDMT (6) is
significantly less potent (Figure 11). As expected based on
drug-discrimination data,32 6-MeO-DMT (30) did not
produce a HTR. Finally, potent plasticity-promoting com-
pound 5-MeO-isoDMT (5) did not produce any HTR (Figure
11), demonstrating that hallucinogenic potential and psycho-
plastogenicity can be decoupled.
1
200 MHz for H and 13C, respectively, and are referenced internally
1
according to residual solvent signals. Data for H NMR are recorded
as follows: chemical shift (δ, ppm), multiplicity (s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet), integration, coupling constant
(Hz). Data for 13C NMR are reported in terms of chemical shift (δ,
ppm). Infrared spectra were recorded using a Thermo Nicolet iS10
Fourier transform infrared (FT-IR) spectrometer with a Smart iTX
Accessory [diamond attenuated total reflection (ATR)] and are
reported in the frequency of absorption (ν, cm−1). Liquid
chromatography−mass spectrometry (LC−MS) was performed
using a Waters LC−MS with an ACQUITY Arc QDa detector.
Ketamine was purchased from Fagron. DMT (1) and 6-F-DMT (29)
were synthesized using previously published methods [purity >99% as
determined by ultra-high-performance liquid chromatography
(UHPLC)].13,37 For cellular plasticity assays (i.e., dendritogenesis),
all compounds were dissolved in DMSO and stored as 10 mM stock
solutions in the dark at −20 °C.
All compounds tested in cellular assays were confirmed to be of
>95% purity based on UHPLC analysis (Waters ACQUITY Arc)
measuring absorbance at 254 and 280 nm. Mobile phase A consisted
of 0.01% formic acid in water, and mobile phase B consisted of 0.01%
formic acid in acetonitrile. All samples were injected at a volume of 5
μL, and the column temperature was maintained at 40 °C. One of the
three methods was used depending on the specific compound.
Method A utilized a CORTECS C18, 2.7 μm, 4.6 × 50 mm2 column,
a flow rate of 0.6 mL/min, and a gradient from 10 to 90% mobile
phase B over 3 min, which was maintained for an additional 2 min.
Method B utilized an XBridge BEH C18 2.5 μm, 2.1 × 100 mm2
column, a flow rate of 0.6 mL/min, and a gradient from 10 to 90%
mobile phase B over 0.5 min, which was maintained for an additional
4.5 min. Method C utilized a CORTECS C18, 2.7 μm, 4.6 × 50 mm2
column, a flow rate of 0.2 mL/min, and a gradient from 10 to 90%
mobile phase B over 4 min, which was maintained for an additional 2
min. As most compounds reported in this study were isolated as the
fumarate salts, peaks in UHPLC traces corresponding to fumaric acid
were not included in the calculation of purity.
CONCLUSIONS
■
While the potent psychoplastogenic properties of psychedelics
have been hypothesized to play a key role in their therapeutic
mechanism of action, the hallucinogenic effects of these
compounds have limited their potential to serve as widespread
medicines. Moreover, very little is known about how the
structures of these compounds impact their abilities to
promote dendritic growth. Here, we address both of these
issues by demonstrating that isoDMT derivatives with low
hallucinogenic potential are capable of promoting dendrito-
genesis to a comparable extent as the psychedelic DMT and
the state-of-the-art fast-acting antidepressant ketamine. Our
SAR studies have defined the minimal psychoplastogen
pharmacophore as an aromatic ring separated from a basic
nitrogen by a short linker. Additionally, we have discovered
that substitution at the 4-position of isoDMT derivatives
renders them devoid of psychoplastogenic properties.
Importantly, this work is the first to demonstrate that a
psychedelic compound (i.e., DMT) can be engineered to lack
hallucinogenic potential while retaining the ability to promote
neural plasticity (e.g., 5). All told, our studies will inform future
medicinal chemistry efforts to identify novel psychoplastogens
with improved safety profiles for treating a variety of
neuropsychiatric disorders.
Synthesis of isoDMTs. To a solution of respective indole or
related heterocycle in DMSO (0.4 M) were added 2-chloro-N,N-
dimethylethylamine hydrochloride (1.1 equiv), potassium iodide (1.1
equiv), and potassium hydroxide pellets (5.0 equiv). The reaction was
stirred at room temperature for 24 h before being diluted with 1.0 M
NaOH(aq). The aqueous phase was extracted three times with
dichloromethane (DCM). The organic extracts were combined, dried
over Na2SO4, filtered, and concentrated under reduced pressure to
yield an oil. The unpurified oil was dissolved in a minimal amount of
acetone and added dropwise to a boiling solution of fumaric acid (1.0
equiv) in acetone. In most cases, a precipitate formed immediately,
which was stored at −20 °C overnight. The resulting crystals were
filtered and washed with several portions of ice-cold acetone to yield
the desired product. In cases where the desired product did not
EXPERIMENTAL SECTION
■
Chemistry (General). All reagents were obtained from
commercial sources and were used without purification unless
otherwise noted. DMSO was purified by passage under 12 psi N2
through activated alumina columns. Reactions were performed using
glassware that was flame-dried under reduced pressure (∼1 Torr).
Chromatography was performed using Fisher Chemical Silica Gel
Sorbent (230−400 mesh, grade 60). Compounds purified by
chromatography were dissolved in a minimal amount of chloroform
for loading. Thin-layer chromatography (TLC) was performed on
Millipore silica gel 60 F254 plates. Visualization of the developed
chromatogram was accomplished by fluorescence quenching or by
staining with ninhydrin.
G
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Article
readily crystalize as the fumarate salt, the oil was subjected to column
chromatography (9:1 CH2Cl2/MeOH/1% NH4OH(aq)) unless noted
otherwise.
1% NH4OH(aq)); 1H NMR (600 MHz, DMSO-d6) δ 7.26 (d, 1H, J =
3.1 Hz), 7.09−7.03 (m, 2H), 6.60 (s, 2H), 6.52 (dd, 1H, J = 6.1, 1.3
Hz), 6.41 (dd, 1H, J = 3.0, 0.7 Hz), 4.29 (t, 2H, J = 6.8 Hz), 3.86 (s,
3H), 2.75 (t, 2H, J = 6.8 Hz), 2.29 (s, 6H) ppm: 13C NMR (150
MHz, DMSO-d6) δ 166.46, 152.80, 137.02, 134.24, 127.14, 121.98,
118.38, 103.09, 99.16, 97.88, 57.83, 54.89, 44.60, 43.15 ppm; IR
(diamond, ATR) ν 3435, 3034, 2653, 1705 cm−1. LRMS (ES+) calcd
for C13H18N2O+ 219.15, found 220.40 (MH+); mp 120−123 °C.
Benzyloxy Indoles. The 4-, 5-, 6-, and 7-OBn indoles were
synthesized using methods published previously.38
2-(1H-Indol-1-yl)-N,N-dimethylethan-1-amine Fumarate
Salt (1:1) (2). Reaction performed using indole (100 mg, 0.85
mmol) and purified via crystallization. Yield = 175 mg, 67%. Purity
>99%. TLC Rf (free base) = 0.50 (9:1 CH2Cl2/MeOH/1%
1
NH4OH(aq)); H NMR (600 MHz, DMSO-d6) δ 7.54 (d, 1H, J =
7.6 Hz), 7.48 (d, 1H, J = 7.6 Hz), 7.38 (d, 1H, J = 3.1 Hz), 7.13 (ddd,
1H, J = 7.6, 1.0 Hz), 7.01 (dd, 1H, J = 7.6, 1.0 Hz), 6.61 (s, 2H), 6.42
(d, 1H J = 3.1 Hz), 4.29 (t, 2H, J = 6.7 Hz), 2.70 (t, 2H, J = 6.7 Hz),
2.26 (s, 6H) ppm; 13C NMR (150 MHz, DMSO-d6) δ 166.32, 135.66,
134.20, 128.82, 128.06, 120.96, 120.38, 118.88, 109.68, 100.54, 58.09,
44.83, 43.11 ppm; IR (diamond, ATR) ν 3100, 2923, 2393, 1705
cm−1; low-resolution mass spectrometry (LRMS) (ES+) calcd for
2-(4-(Benzyloxy)-1H-indol-1-yl)-N,N-dimethylethan-1-
amine Fumarate Salt (1:1) (8). Reaction performed using 4-
benzyloxyindole (200 mg, 0.89 mmol) and purified via crystallization.
Yield = 120 mg, 46%. Purity >99%. TLC Rf (free base) = 0.42 (9:1
1
CH2Cl2/MeOH/1% NH4OH(aq)); H NMR (400 MHz, CD3OD) δ
+
C12H16N2 188.13, found 189.38 (MH+); mp = 147−149 °C.
7.49 (d, 2H, J = 7.5 Hz), 7.37 (t, 2H, J = 7.5 Hz), 7.31 (t, 1H, J = 7.5
Hz), 7.20 (d, 1H, J = 3.26 Hz), 7.12 (m, 2H), 6.72 (s, 1H), 6.66 (m,
1H), 5.22 (s, 2H), 4.57 (t, 2H, J = 6.7 Hz), 3.50 (t, 2H, J = 6.7 Hz),
2.81 (s, 6H) ppm; 13C NMR (100 MHz, CD3OD) δ 169.90, 153.99,
139.05, 138.85, 135.75, 129.47, 128.78, 128.47, 127.21, 124.18,
121.16, 103.87, 102.67, 101.07, 70.95, 57.32, 43.90, 42.46 ppm; IR
(diamond, ATR) ν 2918, 2493, 1701, 1639 cm−1. LRMS (ES+) calcd
for C19H22N2O+ 294.17, found 295.24 (MH+); mp = 145−150 °C.
2-(5-(Benzyloxy)-1H-indol-1-yl)-N,N-dimethylethan-1-
amine Fumarate Salt (1:1) (9). Reaction performed using 5-
benzyloxyindole (287 mg, 1.3 mmol) and purified via crystallization.
Yield = 133 mg, 25%. Purity >99%. TLC Rf (free base) = 0.47 (9:1
2-(4-Methoxy-1H-indol-1-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (1:1) (4). Reaction performed using 4-methoxyindole
(100 mg, 0.68 mmol) and purified via crystallization. Yield = 95 mg,
42%. Purity = 96%. TLC Rf (free base) = 0.35 (9:1 CH2Cl2/MeOH/
1% NH4OH(aq)); 1H NMR (600 MHz, DMSO-d6) δ 7.26 (d, 1H, J =
3.1 Hz), 7.10 (d, 1H, J = 7.8 Hz), 6.90 (t, 1H, J = 7.8 Hz), 6.66 (d,
1H, J = 7.8 Hz), 6.60 (s, 2H), 6.35 (d, 1H, J = 3.1 Hz), 4.49 (t, 2H, J
= 7.0 Hz), 3.89 (s, 3H), 2.76 (t, 2H, J = 7.0 Hz), 2.32 (s, 6H) ppm;
13C NMR (150 MHz, DMSO-d6) δ 166.30, 146.96, 134.17, 130.46,
129.79, 124.94, 119.64, 113.33, 102.42, 100.93, 59.61, 55.32, 45.63,
44.63 ppm; IR (diamond, ATR) ν 2929, 2455, 1712, 1644 cm−1.
LRMS (ES+) calcd for C13H18N2O+ 219.15, found 220.33 (MH+);
mp = 140−145 °C.
2-(5-Methoxy-1H-indol-1-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (1:1) (5). Reaction performed using 5-methoxyindole
(100 mg, 0.68 mmol) and purified via crystallization. Yield = 111 mg,
49%. Purity = 98%. TLC Rf (free base) = 0.66 (9:1 CH2Cl2/MeOH/
1% NH4OH(aq)); 1H NMR (600 MHz, DMSO-d6) δ 7.38 (d, 1H, J =
8.9 Hz), 7.33 (d, 1H, J = 3.0 Hz), 7.04 (d, 1H, J = 2.4 Hz), 6.77 (dd,
1H, J = 8.9, 2.4 Hz), 6.6 (s, 2H), 6.33 (d, 1H, J = 3.0 Hz), 4.29 (t, 2H,
J = 6.8 Hz), 3.74 (s, 3H), 2.79 (t, 2H, J = 6.8 Hz), 2.30 (s, 6H) ppm;
13C NMR (150 MHz, DMSO-d6) δ 166.58, 153.42, 134.32, 130.90,
1
CH2Cl2/MeOH/1% NH4OH(aq)); H NMR (400 MHz, CD3OD) δ
7.44 (d, 2H, J = 7.5 Hz), 7.36 (m, 3H), 7.29 (d, 2H, J = 7.5 Hz), 7.24
(d, 1H, J = 3.2 Hz), 7.15 (d, 1H, J = 2.4 Hz), 6.95 (dd, 1H, J = 2.4,
8.9 Hz), 6.72 (s, 2H), 6.43 (d, 1H, J = 3.2 Hz), 5.07 (s, 2H), 4.54 (t,
2H, J = 6.8 Hz), 3.45 (t, 2H, J = 6.8 Hz), 2.78 (s, 6H) ppm; 13C NMR
(200 MHz, CD3OD) δ 171.06, 154.78, 139.26, 136.11, 132.77,
130.89, 130.81, 129.44, 128.73, 128.61, 113.97, 110.95, 105.71,
103.24, 71.86, 57.66, 44.16, 42.77 ppm; IR (diamond, ATR) ν 2916,
2516, 1698, 1639 cm−1. LRMS (ES+) calcd for C19H22N2O+ 294.17,
found 295.17 (MH+); mp = 133−135 °C.
2-(6-(Benzyloxy)-1H-indol-1-yl)-N,N-dimethylethan-1-
amine (10). Reaction performed using 6-benzyloxyindole (370 mg,
1.7 mmol) and purified via chromatogrpahy. Yield = 184 mg, 38%.
Purity >97%. TLC Rf (free base) = 0.45 (9:1 CH2Cl2/MeOH/1%
129.20, 128.48, 111.12, 110.39, 102.13, 100.34, 57.68, 55.30, 44.44,
42.91 ppm; IR (diamond, ATR) ν 3035, 2923, 2446, 1715 cm−1.
LRMS (ES+) calcd for C13H18N2O+ 219.15, found 220.19 (MH+);
mp = 140−142 °C.
1
NH4OH(aq)); H NMR (600 MHz, CD3OD) δ 7.47 (d, 2H, J = 7.4
Hz), 7.40 (d, 2H, J = 8.6 Hz), 7.37 (t, 1H J = 7.4 Hz), 7.30 (t, 1H, J =
7.4 Hz), 7.08 (d, 1H, J = 3.8 Hz), 6.96 (s, 1H), 6.78 (d, 1H, J = 8.6
Hz), 6.35 (d, 1H, J = 3.8 Hz), 5.15 (s, 2H), 4.22 (t, 2H, J = 7.3 Hz),
2.66 (t, 2H, J = 7.3 Hz), 2.26 (s, 6H) ppm; 13C NMR (150 MHz,
CDCl3) δ 156.39, 137.60, 136.66, 128.69, 127.97, 127.67, 127.22,
123.24, 121.68, 109.95, 101.31, 94.71, 70.87, 58.92, 45.91, 44.90 ppm;
IR (diamond, ATR) ν 3030, 2952, 2768, 1621 cm−1. LRMS (ES+)
calcd for C19H22N2O+ 294.17, found 295.10 (MH+).
2-(6-Methoxy-1H-indol-1-yl)-N,N-dimethylethan-1-amine
(6). Reaction performed using 6-methoxyindole (147 mg, 1.0 mmol)
and purified via chromatography. Yield = 148 mg, 68%. Purity >99%.
TLC Rf (free base) = 0.32 (9:1 CH2Cl2/MeOH/1% NH4OH(aq)); 1H
NMR (600 MHz, CDCl3) δ 7.49 (d, 1H, J = 7.8 Hz), 7.02 (d, 1H, J =
8.2 Hz), 6.82 (s, 2H), 6.78 (dd, 1H, J = 7.5 Hz), 6.42 (d, 1H, J = 7.8
Hz), 4.17 (t, 2H, J = 7.0 Hz), 2.31 (s, 3H), 2.69 (t, 2H, J = 7.0 Hz),
2.31 (s, 6H) ppm; 13C NMR (150 MHz, CDCl3) δ 156.30, 136.77,
127.04, 123.00, 121.67, 109.22, 101.31, 93.14, 59.00, 55.91, 45.94,
44.91 ppm; IR (diamond, ATR) ν 2940, 2859, 2769, 1602 cm−1.
LRMS (ES+) calcd for C13H18N2O+ 219.15, found 220.33 (MH+).
The free base was used for dendritogenesis assays.
2-(7-(Benzyloxy)-1H-indol-1-yl)-N,N-dimethylethan-1-
amine (11). Reaction performed using 7-benzyloxyindole (119 mg,
0.53 mmol) and purified via chromatography. Yield = 51 mg, 23%.
Purity >99%. TLC Rf (free base) = 0.48 (9:1 CH2Cl2/MeOH/1%
1
NH4OH(aq)); H NMR (600 MHz, CDCl3) δ 7.49 (d, 2H, J = 7.4
2-(6-Methoxy-1H-indol-1-yl)-N,N-dimethylethan-1-amine
Oxalate (6). Reaction performed using 6-methoxyindole (250 mg,
1.7 mmol) and purified via crystallization. Yield = 221 mg, 42%.
Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.34 (t, 1H, J = 7.4 Hz), 7.22 (d, 1H, J =
7.9 Hz), 7.01 (d, 1H, J = 3.0 Hz), 6.97 (t, 1H, J = 7.8 Hz), 6.70 (d,
1H, J = 7.8 Hz), 5.19 (s, 2H), 4.45 (t, 2H, J = 7.4 Hz), 2.62 (t, 2H, J =
7.4 Hz), 2.09 (s, 6H) ppm; 13C NMR (150 MHz, CDCl3) δ 150.59,
146.71, 137.11, 131.25, 129.45, 128.75, 128.24, 128.15, 119.82,
114.16, 103.35, 101.60, 70.55, 61.02, 47.54, 45.64 ppm; IR (diamond,
ATR) ν 2940, 2821, 1575, 1439 cm−1. LRMS (ES+) calcd for
C19H22N2O+ 294.17, found 295.24 (MH+).
2-(4-Fluoro-1H-indol-1-yl)-N,N-dimethylethan-1-amine Fu-
marate Salt (1:1) (12). Reaction performed using 4-fluoroindole
(135 mg, 1.0 mmol) and purified via crystallization. Yield = 164 mg,
51%. Purity >99%. TLC Rf (free base) = 0.39 (9:1 CH2Cl2/MeOH/
1% NH4OH(aq)); 1H NMR (400 MHz, DMSO-d6) δ 7.44 (d, 1H, J =
2.3 Hz), 7.35 (d, 2H, J = 8.3 Hz), 7.10 (dd, 2H, J = 7.4, 7.25 Hz),
6.79 (t, 2H, J = 9.5 Hz), 6.60 (s, 2H), 6.49 (d, 2H, J = 2.3 Hz), 4.32
1
Purity = 98%. H NMR (600 MHz, CD3OD) δ 7.42 (d, 1H, J = 8.5
Hz), 7.16 (d, 1H, J = 2.9 Hz), 7.02 (s, 2H), 6.74 (d, 1H, J = 8.5 Hz),
6.44 (d, 1H, J = 2.9 Hz), 4.58 (t, 2H, J = 6.8 Hz), 3.87 (s, 3H), 3.56
(t, 2H, J = 7.0 Hz), 2.86 (s, 6H) ppm; 13C NMR (150 MHz,
CD3OD) δ 166.72, 158.20, 138.02, 127.52, 124.45, 122.61, 111.06,
103.58, 93.83, 57.22, 56.19, 44.04, 42.17 ppm; IR (diamond, ATR) ν
3129, 3014, 2641, 1727 cm−1. LRMS (ES+) calcd for C13H18N2O+
219.15, found 220.05 (MH+); mp = 165−167 °C. The oxalate salt
was used for HTR assays.
2-(7-Methoxy-1H-indol-1-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (1:1) (7). Reaction performed using 7-methoxyindole
(100 mg, 0.68 mmol) and purified via crystallization. Yield = 162 mg,
72%. Purity >99%. TLC Rf (free base) = 0.44 (9:1 CH2Cl2/MeOH/
H
https://dx.doi.org/10.1021/acs.jmedchem.9b01404
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(t, 2H, J = 6.7 Hz), 2.74 (t, 2H, J = 6.8 Hz), 2.28 (s, 6H) ppm; 13C
NMR (150 MHz, DMSO-d6) δ 166.37, 156.67, 154.34, 138.56,
138.44, 134.21, 129.35, 121.63, 121.55, 116.74, 116.51, 106.48,
106.45, 103.70, 103.51, 96.24, 57.90, 44.73, 43.40 ppm; IR (diamond,
ATR) ν 3123, 2389, 1702, 1660 cm−1. LRMS (ES+) calcd for
2-(4,6-Difluoro-1H-indol-1-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (1:1) (18). Reaction performed using 4,6-
difluoroindole (153 mg, 1.0 mmol) and purified via crystallization.
Yield = 265 mg, 78%. Purity >99%. TLC Rf (free base) = 0.35 (9:1
CH2Cl2/MeOH/1% NH4OH(aq)); 1H NMR (400 MHz, DMSO-d6) δ
7.44 (d, 1H, J = 2.8 Hz), 7.32 (d, 1H, J = 10.1 Hz), 6.83 (t, 1H, J =
10.1 Hz), 6.60 (s, 2H), 6.49 (d, 1H, J = 2.8 Hz), 4.31 (t, 2H, J = 6.6
Hz), 2.78 (t, 2H, J = 6.6 Hz), 2.31 (s, 6H) ppm; 13C NMR (150
MHz, DMSO-d6) δ 166.55, 159.33, 159.21, 156.99, 156.87, 156.03,
155.88, 153.58, 153.43, 137.50, 137.36, 137.22, 134.29, 129.82,
129.79, 113.42, 113.20, 96.61, 94.53, 94.30, 94.24, 94.00, 93.32, 93.28,
93.06, 93.02, 57.49, 44.49, 43.23 ppm; IR (diamond, ATR) ν 3026,
+
C12H16FN2 207.13, found 208.32 (MH+); mp = 145−149 °C.
2-(5-Fluoro-1H-indol-1-yl)-N,N-dimethylethan-1-amine Fu-
marate Salt (1:1) (13). Reaction performed using 5-fluoroindole
(135 mg, 1.0 mmol) and purified via crystallization. Yield = 145 mg,
45%. Purity >99%. TLC Rf (free base) = 0.35 (9:1 CH2Cl2/MeOH/
1% NH4OH(aq)); 1H NMR (400 MHz, DMSO-d6) δ 7.50 (dd, 1H, J =
4.5, 4.3 Hz), 7.46 (d, 1H, J = 2.1 Hz), 7.29 (d, 1H, J = 9.5 Hz), 6.97
(t, 1H, J = 9.5 Hz), 6.60 (s, 2H), 6.41 (d, 1H, J = 2.1 Hz), 4.32 (t, 2H,
J = 6.7 Hz), 2.79 (t, 2H, J = 6.7 Hz), 2.31 (s, 6H) ppm; 13C NMR
(150 MHz, DMSO-d6) δ 166.59, 159.61, 158.06, 135.77, 135.69,
134.33, 129.54, 129.51, 124.72, 121.39, 121.32, 107.48, 107.32,
100.97, 96.32, 96.15, 57.66, 44.58, 43.00 ppm; IR (diamond, ATR) ν
+
2398, 1706, 1640 cm−1. LRMS (ES+) calcd for C12H16F2N2 224.11,
found 225.28 (MH+); mp = 141−145 °C.
N,N-Dimethyl-2-(6-nitro-1H-indol-1-yl)ethan-1-amine Fu-
marate Salt (1:1) (19). Reaction performed using 6-nitroindole
(43.6 mg, 0.269 mmol) and purified via crystallization. Yield = 52 mg,
55%. Purity >96%. TLC Rf (free base) = 0.48 (9:1 CH2Cl2/MeOH/
1% NH4OH(aq)); 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, 1H, J =
2.0 Hz), 7.90 (dd, 1H, J = 8.8, 2.0 Hz), 7.82 (d, 1H, J = 3.0 Hz), 7.72
(d, 1H, J = 8.8 Hz), 7.74 (s, 1H), 6.65 (d, 1H, J = 3.0 Hz), 6.60 (s,
2H), 4.44 (t, 2H, J = 6.3 Hz), 2.68 (t, 2H, J = 6.3 Hz), 2.23 (s, 6H)
ppm; 13C NMR (600 MHz, DMSO-d6) δ 166.66, 136.18, 134.76,
134.59, 133.41, 121.04, 114.52, 107.53, 102.25, 58.98, 45.50, 44.18
ppm; IR (diamond, ATR) ν 3048, 2922, 1704, 1607 cm−1. LRMS (ES
+) calcd for C12H16N3O2+ 233.12, found 234.25 (MH+); mp = 159−
164 °C.
+
3036, 2049, 1723, 1663 cm−1. LRMS (ES+) calcd for C12H16FN2
207.13, found 207.40 (MH+); mp = 145−148 °C.
2-(6-Fluoro-1H-indol-1-yl)-N,N-dimethylethan-1-amine Fu-
marate Salt (1:1) (14). Reaction performed using 6-fluoroindole
(100 mg, 0.739 mmol) and purified via crystallization. Yield = 145
mg, 61%. Purity = 97%. TLC Rf (free base) = 0.45 (9:1 CH2Cl2/
1
MeOH/1% NH4OH(aq)); H NMR (600 MHz, DMSO-d6) δ 7.52
(dd, 1H, J = 7.0, 3.0 Hz), 7.39−7.37 (m, 2H), 6.88−6.85 (m, 1H),
6.59 (s, 2H), 6.44 (d, 1H, J = 3.1 Hz), 4.29 (t, 2H, J = 6.8 Hz), 2.77
(t, 2H, J = 6.8 Hz), 2.30 (s, 6H) ppm; 13C NMR (150 MHz, DMSO-
d6) δ 166.59, 159.61, 158.06, 136.16, 134.75, 129.95, 125.14, 121.78,
107.91, 107.74, 101.39, 96.74, 96.57, 57.66, 44.58, 43.00 ppm; IR
(diamond, ATR) ν 3058, 2385, 1698, 1634 cm−1. LRMS (ES+) calcd
2-(5-Bromo-1H-indol-1-yl)-N,N-dimethylethan-1-amine Fu-
marate Salt (1:1) (20). Reaction performed using 5-bromoindole
(56.1 mg, 0.281 mmol) and purified via crystallization. Yield = 60 mg,
55%. Purity >99%. TLC Rf (free base) = 0.49 (9:1 CH2Cl2/MeOH/
+
for C12H16FN2 207.13, found 208.39 (MH+); mp = 141−147 °C.
1
1% NH4OH(aq)); H NMR (600 MHz, DMSO-d6) δ 7.72 (d, J = 1.9
2-(7-Fluoro-1H-indol-1-yl)-N,N-dimethylethan-1-amine Fu-
marate Salt (1:1) (15). Reaction performed using 7-fluoroindole
(135 mg, 1.0 mmol) and purified via crystallization. Yield = 172 mg,
53%. Purity = 98%. TLC Rf (free base) = 0.45 (9:1 CH2Cl2/MeOH/
Hz, 1H), 7.48 (d, 1H, J = 8.7 Hz), 7.44 (d, 1H, J = 3.1 Hz), 7.23 (dd,
1H, J = 8.7, 1.9 Hz), 6.60 (s, 3H), 6.41 (d, 1H, J = 3.1 Hz), 4.27 (t,
2H, J = 6.6 Hz), 2.67 (t, 2H, J = 6.6 Hz), 2.22 (s, 6H) ppm; 13C NMR
(600 MHz, DMSO-d6) δ 166.24, 134.47, 134.15, 130.46, 129.89,
123.81, 122.54, 111.87, 111.56, 100.28, 58.09, 44.86, 43.32 ppm; IR
(diamond, ATR) ν 2959, 2443, 1705, 1661 cm−1. LRMS (ES+) calcd
1
1% NH4OH(aq)); H NMR (400 MHz, DMSO-d6) δ 7.41 (s, 1H),
7.35 (d, 1H, J = 7.6 Hz), 6.98−6.88 (m, 2H), 6.61 (s, 2H), 6.48 (s,
1H,), 4.37 (t, 2H, J = 6.7 Hz), 2.69 (t, 2H, J = 6.7 Hz), 2.23 (s, 6H)
ppm; 13C NMR (100 MHz, CD3OD) δ 169.37, 134.60, 133.26,
129.51, 119.90, 119.46, 116.82, 116.79, 107.06, 106.88, 102.78, 57.61,
57.61, 43.71, 43.67, 42.90 ppm; IR (diamond, ATR) ν 3040, 2429,
+
for C12H16BrN2 266.04, found 267.26 (MH+); mp = 140−142 °C.
2-(1-(2-(Dimethylamino)ethyl)-1H-indol-3-yl)-N,N-dimethyl-
2-oxoacetamide (21). Reaction performed using 2-(1H-indol-3-yl)-
N,N-dimethyl-2-oxoacetamide (synthesized using the method devel-
oped by Speeter et. al.)18 (200 mg, 0.92 mmol) and purified via
chromatography. Yield = 92 mg, 35%. Purity = 98%. TLC Rf (free
+
1718, 1661 cm−1. LRMS (ES+) calcd for C12H16FN2 207.13, found
207.33 (MH+); mp = 168−170 °C.
N,N-Dimethyl-2-(2-methyl-1H-indol-1-yl)ethan-1-amine Fu-
marate Salt (1:1) (16). Reaction performed using 2-methylindole
(100 mg, 0.76 mmol) and purified via crystallization. Yield (172 mg,
71%). Purity >99%. TLC Rf (free base) = 0.47 (9:1 CH2Cl2/MeOH/
1% NH4OH(aq)); 1H NMR (400 MHz, DMSO-d6) δ 7.41 (d, 1H, J =
7.8 Hz), 7.37 (d, 1H, J = 7.8 Hz), 7.06 (t, 1H, J = 7.8 Hz), 6.96 (t,
1H, J = 7.8 Hz), 6.61 (s, 2H), 6.20 (d, J = 1.3 Hz, 1H), 4.24 (t, 2H, J
= 7.0 Hz), 2.65 (t, 2H, J = 7.0 Hz), 2.42 (s, 3H), 2.31 (s, 6H) ppm;
13C NMR (150 MHz, DMSO-d6) 167.03, 137.08, 136.72, 134.75,
1
base) = 0.38 (9:1 CH2Cl2/MeOH/1% NH4OH(aq)); H NMR (600
MHz, CDCl3) δ 8.32 (s, 1H), 7.92 (s, 1H), 7.37−7.29 (m, 3H), 4.21
(t, 2H, J = 7.0 Hz), 3.08 (s, 3H), 3.04 (s, 3H), 2.71 (t, 2H, J = 7.0
Hz), 2.27 (s, 6H) ppm; 13C NMR (150 MHz, CDCl3) δ 185.64,
167.70, 138.82, 137.01, 126.36, 124.00, 123.28, 122.48, 113.45,
110.06, 58.40, 45.68 45.42, 37.59, 34.51 ppm; IR (diamond, ATR) ν
2981, 1734 1631, 1525 cm−1. LRMS (ES+) calcd for C16H21N3O2+
287.16, found 288.25 (MH+).
2-(1H-Benzo[d]imidazol-1-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (1:1) (22). Reaction performed using benzimidazole
(200 mg, 1.6 mmol) and purified via crystallization. Yield = 218 mg,
45%. Purity = 98%. TLC Rf (free base) = 0.42 (9:1 CH2Cl2/MeOH/
128.09, 120.58, 119.65, 119.62, 119.40, 109.61, 100.14, 57.82, 45.18,
12.76 ppm; IR (diamond, ATR) ν 3040, 2489, 1700, 1606 cm−1.
+
LRMS (ES+) calcd for C13H18N2 203.15, found 204.43 (MH+); mp
1
= 131−133 °C.
1% NH4OH(aq)); H NMR (400 MHz, DMSO-d6) δ 8.23 (s, 1H),
2-(5,6-Difluoro-1H-indol-1-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (1:1) (17). Reaction performed using 5,6-
difluoroindole (153 mg, 1.0 mmol) and purified via crystallization.
Yield = 147 mg, 43%. Purity = 98%. TLC Rf (free base) = 0.35 (9:1
CH2Cl2/MeOH/1% NH4OH(aq)); 1H NMR (400 MHz, DMSO-d6) δ
7.64 (dd, 1H, J = 7.0, 4.7 Hz), 7.51 (dd, 1H, J = 8.5, 2.1 Hz), 7.45 (d,
1H, J = 2.1 Hz), 6.60 (s, 2H), 6.43 (d, 1H, J = 2.1 Hz), 4.28 (t, 2H, J
= 6.5 Hz), 2.73 (t, 2H, J = 6.5 Hz), 2.27 (s, 6H) ppm; 13C NMR (150
MHz, DMSO-d6) δ 166.37, 134.21, 131.09, 130.99, 134.70, 130.67,
123.21, 123.12, 106.86, 106.67, 100.98, 100.94, 98.30, 98.08, 57.86,
44.71, 43.36 ppm; IR (diamond, ATR) ν 3051, 2392, 1712, 1658
cm−1. LRMS (ES+) calcd for C12H16F2N2+ 224.11, found 225.28
(MH+); mp = 162−165 °C.
7.71−7.56 (m, 2H), 7.34−7.10 (m, 2H), 6.61 (s, 2H), 4.41 (t, 2H, J =
6.5 Hz), 2.83 (dd, 2H, J = 7.0, 6.0 Hz), 2.32 (s, 6H) ppm; 13C NMR
(100 MHz, CD3OD) δ 170.14, 144.78, 143.90, 135.81, 134.62,
124.80, 124.04, 120.36, 111.29, 57.46, 44.39, 41.80 ppm; IR
(diamond, ATR) ν 3054, 2384, 1707, 1654 cm−1. LRMS (ES+)
+
calcd for C11H15N3 189.13, found 190.23 (MH+); mp = 171−178
°C.
N,N-Dimethyl-2-(1H-pyrrol-1-yl)ethan-1-amine Fumarate
Salt (1:1) (23). Reaction performed using pyrrole (0.103 mL, 1.5
mmol) and purified via crystallization. Yield = 126 mg, 33%. Purity
>99%. TLC Rf (free base) = 0.45 (9:1 CH2Cl2/MeOH/1%
1
NH4OH(aq)); H NMR (600 MHz, CD3OD) δ 6.80 (t, 2H, J = 2.2,
Hz), 6.72 (s, 2H), 6.14 (t, 2H. J = 2.2, Hz), 4.34 (t, 2H, J = 6.4 Hz),
I
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3.50 (dd, 2H, J = 6.4 Hz), 2.78 (s, 6H) ppm; 13C NMR (100 MHz,
CD3OD) δ 170.26, 136.21, 122.18, 110.89, 59.35, 45.83, 44.45 ppm;
IR (diamond, ATR) ν 2998, 2532, 1662, 1421 cm−1. LRMS (ES+)
calcd for C8H14N2+ 138.12, found 139.29 (MH+); mp = 174−180 °C.
2-(9H-Carbazol-9-yl)-N,N-dimethylethan-1-amine Fumarate
Salt (1:1) (24). Reaction performed using carbazole (100 mg, 0.57
mmol) and purified via crystallization. Yield = 102 mg, 51%. Purity
>99%. TLC Rf (free base) = 0.42 (9:1 CH2Cl2/MeOH/1%
equiv) followed by 37% formaldehyde(aq) (0.16 mL, 1.9 mmol, 2.6
equiv). The reaction was stirred at room temperature for 5 h before
being concentrated under reduced pressure. The unpurified material
was then diluted with CH2Cl2 (50 mL) and 1 M NaOH(aq) (100 mL).
The phases were separated, and the aqueous phase was extracted with
CH2Cl2 (2 × 50 mL). The organic extracts were combined, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
unpurified material was dissolved in acetone (5 mL) and was added to
a boiling solution of fumaric acid (0.088 g, 1 mmol, 1 equiv) in
acetone (20 mL). A precipitate formed immediately, and the solution
was cooled to room temperature prior to being filtered. The resulting
white solid was dried under reduced pressure to yield the pure
compound as the fumarate salt (1:1). Yield = 0.108 g, 65%. Purity
>99%. TLC Rf (free base) = 0.19 (9:1 CH2Cl2/MeOH/1%
1
NH4OH(aq)); H NMR (600 MHz, DMSO-d6) δ 8.15 (d, 2H, J =
7.8 Hz), 7.61 (d, 2H, J = 8.2 Hz), 7.48−7.44 (m, 2H), 7.23−7.19 (m,
2H), 6.61 (s, 2H), 4.52 (t, 2H, J = 7.0 Hz), 2.73 (t, 2H, J = 7.0 Hz),
2.31 (s, 6H) ppm; 13C NMR (100 MHz, DMSO-d6) δ 169.78, 139.86,
134.76, 125.83, 123.17, 119.99, 119.39, 108.34, 54.39, 42.61, 37.85
ppm; IR (diamond, ATR) ν 3053, 2405, 1720, 1660 cm−1. LRMS (ES
+) calcd for C16H18N2+ 238.15, found 239.34 (MH+); mp = 182−184
°C.
1-Isopentyl-1H-indole (25). Reaction performed using indole
(100 mg, 0.85 mmol) and 1-chloro-3-methylbutane (0.11 mL mg,
0.94 mmol, 1.1 equiv) and purified via chromatography (4:1 hexanes/
EtOAc). Yield = 85 mg, 53%. Purity = 97%. TLC Rf = 0.70 (7:3
hexanes/EtOAc); 1H NMR (600 MHz, CDCl3) δ 7.65 (d, 1H, J = 8.0
Hz), 7.36 (d, 1H, J = 8.0 Hz), 7.22 (t, 1H, J = 6.9 Hz), 7.11 (m, 2H),
6.49 (d, 1H, J = 3.1 Hz), 4.15 (t, 2H, J = 7.5 Hz), 1.74 (dd, 2H, J =
6.7 Hz), 1.62 (quint, 2H, J = 6.7 Hz), 0.98 (d, 1H, J = 6.7 Hz) ppm;
13C NMR (150 MHz, CDCl3) δ 135.78, 128.46, 127.58, 121.20,
1
NH4OH(aq)); H NMR (600 MHz, CD3OD) δ 7.60 (d, 1H, J = 8.0
Hz), 7.37 (d, 1H, J = 8.0 Hz), 7.20 (t, 1H, J = 8.0 Hz), 7.14 (s, 1H),
7.09 (t, 1H, J = 8.0 Hz), 6.69 (s, 2H), 3.78 (s, 3H), 3.42 (t, 2H, J =
7.8 Hz), 3.20 (t, 2H, J = 7.8 Hz), 2.91 (s, 6H) ppm; 13C NMR (150
MHz, CD3OD) δ 171.44, 138.80, 136.23, 128.67, 128.59, 122.95,
120.20, 119.27, 110.53, 109.11, 59.12, 43.41, 32.77, 21.72 ppm; IR
(diamond, ATR) ν 3435, 3034, 2653, 1705 cm−1. LRMS (ES+) m/z
+
calcd for C13H18N2 202.15, found 203.37 (MH+); mp = 167−170
°C.
2-(5-Methoxy-1H-indol-3-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (2:1) (5-MeO-DMT, 28). To an ice-cold solution of
5-methoxytryptamine (0.50 g, 2.2 mmol) and glacial acetic acid (0.60
mL, 11 mmol, 5.0 equiv) in MeOH (44 mL) was added sodium
cyanoborohydride (0.305 g, 4.8 mmol, 2.2 equiv) followed by 37%
formaldehyde(aq) (0.46 mL, 5.7 mmol, 2.6 equiv). The reaction was
stirred at room temperature for 5 h before being concentrated under
reduced pressure. The residue was diluted with CH2Cl2 (50 mL) and
1 M NaOH(aq) (100 mL). The phases were separated, and the
aqueous phase was extracted with CH2Cl2 (2 × 50 mL). The organic
extracts were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The unpurified material was dissolved in
acetone (5 mL) and was added to a boiling solution of fumaric acid
(0.26 g, 2.2 mmol, 0.7 equiv) in acetone (35 mL). A precipitate
formed immediately, and the solution was cooled to room
temperature prior to being filtered. The resulting white solid was
dried under reduced pressure to yield the pure compound as the
fumarate salt (2:1). Yield = 0.49 g, 80%. Purity = 98%. TLC Rf (free
120.83, 119.05, 109.28, 100.78, 44.44, 40.72, 38.92, 25.61, 22.37 ppm;
IR (diamond, ATR) ν 3054, 2955, 2927, 2869 cm−1. LRMS (ES+)
calcd for C13H17N+ 187.14, found 188.39 (MH+).
3-(1H-Indol-1-yl)-N,N-dimethylpropan-1-amine Fumarate
Salt (1:1) (26). Reaction performed using indole (100 mg, 0.85
mmol) and 3-chloro-N,N-dimethylpropan-1-amine (160 mg, 0.98
mmol, 1.1 equiv) and purified via crystallization Yield = 107 mg, 48%.
Purity = 98%. TLC Rf (free base) = 0.38 (9:1 CH2Cl2/MeOH/1%
NH4OH(aq)); 1H NMR (600 MHz, DMSO-d6) δ 7.54 (d, 1H, J = 7.8
Hz), 7.48 (d, 1H, J = 7.8 Hz), 7.36 (d, 1H, J = 3.1 Hz), 7.13 (td, 1H, J
= 7.4, 1.0 Hz), 7.01 (td, 1H, J = 7.4, 1.0 Hz), 6.55 (s, 2H), 6.43 (dd,
1H, J = 3.1, 1.0 Hz), 4.21 (t, 2H, J = 6.8 Hz), 2.56 (t, 2H, J = 7.4 Hz),
2.40 (s, 6H), 2.00 (tt, 2H, J = 7.4, 6.8 Hz) ppm; 13C NMR (150 MHz,
DMSO-d6) δ 167.19, 135.60, 134.62, 128.49, 128.07, 120.99, 120.41,
118.90, 109.67, 100.61, 54.88, 43.41, 43.07, 26.26 ppm; IR (diamond,
ATR) ν 3435, 3034, 2653, 1705 cm−1. LRMS (ES+) calcd for
+
1
C13H18N2 203.15, found 204.36 (MH+); mp = 129−131 °C.
base) = 0.20 (9:1 CH2Cl2/MeOH/1% NH4OH(aq)); H NMR (600
tert-Butyl (2-(1H-Indol-3-yl)ethyl)carbamate. To an ice-cold
solution of tryptamine (0.50 g, 3.1 mmol) and triethylamine (0.68
mL, 9.4 mmol, 3 equiv) in CH2Cl2 (44 mL) was added Boc2O (0.77
g, 3.7 mmol, 1.2 equiv). The reaction was warmed to room
temperature, stirred overnight, and then quenched with H2O (200
mL). The organic phase was separated, and the aqueous phase was
extracted with CH2Cl2 (2 × 50 mL). The organic extracts were
combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure to afford an oil that was purified by chromatography
on silica gel (8:2 hexanes/EtOAc); white solid (0.69 g, 85%).
N-Methyl-2-(1-methyl-1H-indol-3-yl)ethan-1-amine. To an
ice-cold solution of sodium hydride (0.23 g, 5.8 mmol, 2.2 equiv)
in dimethylformamide (DMF) (3 mL) was added tert-butyl (2-(1H-
indol-3-yl)ethyl)carbamate (0.69 g, 2.6 mmol) in DMF (3 mL). The
reaction mixture was allowed to stir at room temperature before being
cooled to 0 °C. Methyl iodide (0.4 mL, 5.8 mmol, 2.2 equiv) was
added dropwise. The reaction was stirred at room temperature for 20
h. Next, the reaction was cooled to 0 °C, quenched with
trifluoroacetic acid (TFA) (2 mL), and stirred for 30 min. The
mixture was diluted with 1.0 M NaOH(aq) (600 mL) and extracted
with CH2Cl2 (3 × 75 mL). The organic phases were combined, dried
over Na2SO4, filtered, and concentrated under reduced pressure to
afford an oil that was used without further purification (0.45 g, 90%).
N,N-Dimethyl-2-(1-methyl-1H-indol-3-yl)ethan-1-amine Fu-
marate Salt (1:1) (1-Me-DMT, 27). To an ice-cold solution of N-
methyl-2-(1-methyl-1H-indol-3-yl)ethan-1-amine (0.14 g, 0.70 mmol)
and glacial acetic acid (0.22 mL, 11 mmol, 5.0 equiv) in MeOH (12
mL) was added sodium cyanoborohydride (0.10 g, 1.6 mmol, 2.1
MHz, CD3OD) δ 7.24 (d, 1H, J = 8.8 Hz), 7.14 (s, 1H), 7.07 (s, 1H),
6.78 (d, 1H, J = 8.8 Hz), 6.70 (s, 1H), 3.83 (s, 3H), 3.29 (m, 2H),
3.13 (t, 2H, J = 7.9 Hz), 2.83 (s, 6H) ppm; 13C NMR (150 MHz,
CD3OD) δ 174.36, 155.27, 137.09, 133.44, 128.49, 124.79, 113.20,
112.96, 110.05, 101.05, 59.25, 56.37, 43.56, 22.12 ppm; IR (diamond,
ATR) ν 3436, 3034 2654, 1705 cm−1. LRMS (ES+) m/z calcd for
C13H18N2O+ 218.14, found 219.34 (MH+); mp = 175−177 °C.
2-(6-Methoxy-1H-indol-3-yl)-N,N-dimethylethan-1-amine
Fumarate Salt (2:1) (6-MeO-DMT, 30). To an ice-cold solution of
6-methoxytryptamine (0.40 g, 2.1 mmol) and glacial acetic acid (0.60
mL, 10 mmol, 5.0 equiv) in MeOH (42 mL) was added sodium
cyanoborohydride (0.29 g, 4.6 mmol, 2.2 equiv) followed by 37%
formaldehyde(aq) (0.44 mL, 5.5 mmol, 2.6 equiv). The reaction was
stirred at room temperature for 5 h before being concentrated under
reduced pressure. The residue was diluted with CH2Cl2 (45 mL) and
1 M NaOH(aq) (100 mL). The phases were separated, and the
aqueous phase was extracted with CH2Cl2 (2 × 45 mL). The organic
extracts were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The unpurified material was dissolved in
acetone (5 mL) and was added to a boiling solution of fumaric acid
(0.26 g, 2.2 mmol, 0.7 equiv) in acetone (35 mL). A precipitate
formed immediately, and the solution was cooled to room
temperature prior to being filtered. The resulting white solid was
dried under reduced pressure to yield the pure compound as the
fumarate salt (2:1). Yield = 0.320 g, 55%. Purity = 95%. TLC Rf (free
1
base) = 0.31 (9:1 CH2Cl2/MeOH/1% NH4OH(aq)); H NMR (600
MHz, CD3OD) δ 7.44 (d, 1H, J = 8.6 Hz), 7.04 (s, 1H), 6.88 (s, 1H),
6.70 (m, 2H), 3.78 (s, 3H), 3.32 (t, 2H, J = 7.5 Hz), 3.12 (t, 2H, J =
J
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7.5 Hz), 2.84 (s, 6H) ppm; 13C NMR (150 MHz, CD3OD) δ 173.79,
157.87, 138.97, 136.93, 122.89, 122.53, 119.64, 110.40, 109.99, 95.62,
59.12, 55.94, 43.36, 21.96 ppm; IR (diamond, ATR) ν 2915, 2836,
1691, 1559 cm−1. LRMS (ES+) m/z calcd for C13H18N2O+ 218.14,
found 219.29 (MH+); mp = 173−176 °C.
(1:10 000; EnCor, CPCA-MAP2). The next day, plates were washed
three times with DPBS and once with 2% ADB in DPBS. Plates were
incubated for 1 h at room temperature in ADB containing an anti-
chicken IgG secondary antibody conjugated to Alexa Fluor 488 (Life
Technologies, 1:500) and washed five times with DPBS. After the
final wash, 100 μL of DPBS was added per well and imaged on an
ImageXpress Micro XL High-Content Screening System (Molecular
Devices, Sunnyvale, CA) with a 20× objective. Images were analyzed
using ImageJ Fiji (version 1.51W). First, images corresponding to
each treatment were sorted into individual folders that were then
blinded for data analysis. Plate controls (both positive and negative)
were used to ensure that the assay was working properly as well as to
visually determine appropriate numerical values for brightness/
contrast and thresholding to be applied universally to the remainder
of the randomized images. Next, the brightness/contrast settings were
applied, and approximately 1−2 individual pyramidal-like neurons per
image (i.e., no bipolar neurons) were selected using the rectangular
selection tool and saved as separate files. Neurons were selected that
did not overlap extensively with other cells or extend far beyond the
field of view. The threshold settings were then applied to the
individual images. The paintbrush tool was used to eliminate artifacts
and dendritic processes originating from adjacent neurons (cleanup
phase). See Figure S2 for a visual explanation of how neurons were
selected and processed for data analysis. Next, the point tool was used
to select the center of the neuron, and the images were saved and
processed using the following Sholl analysis batch macro:
2-(1H-Indol-1-yl)-N,N-dimethylacetamide (31). To a solution
of indole (117 mg, 1.0 mmol) in DMSO (2.5 mL, 0.4 M) were added
2-chloro-N,N-dimethylacetamide (0.11 mL, 1.1 mmol, 1.1 equiv),
potassium iodide (182 mg, 1.1 mmol, 1.1 equiv), and potassium
hydroxide pellets (280 mg, 5 mmol, 5.0 equiv). The reaction was
stirred at room temperature for 24 h before being diluted with 1.0 M
NaOH(aq). The aqueous phase was extracted three times with DCM.
The organic extracts were combined, dried over Na2SO4, filtered, and
concentrated under reduced pressure to yield an oil. The oil was
purified via chromatography (3:2 hexanes/EtOAc). Yield = 175 mg,
1
57%. Purity = 99%. TLC Rf = 0.15 (3:2 hexanes/EtOAc); H NMR
(600 MHz, CD3OD) δ 7.53 (d, 1H, J = 7.9 Hz), 7.27 (d, 1H, J = 7.9
Hz), 7.12 (m, 2H), 7.01 (t, 1H, J = 7.5, Hz), 6.46 (d, 1H, J = 2.9 Hz),
5.01 (s, 2H), 3.15 (s, 3H), 2.96 (s, 3H) ppm; 13C NMR (150 MHz,
CDCl3) δ 167.4, 136.7, 128.7, 128.5, 122.0, 121.2, 119.8, 109.1, 102.4,
48.2, 36.7, 36.1 ppm; IR (diamond, ATR) ν 3021, 2922, 2877, 1648
cm−1. LRMS (ES+) calcd for C12H14N2O+ 202.11, found 203.17
(MH+); mp = 58−61 °C.
Animals. For the dendritogenesis experiments, timed pregnant
Sprague Dawley rats were obtained from Charles River Laboratories
(Wilmington, MA). For the head-twitch response assay, male and
female C57BL/6J mice were obtained from Jackson Laboratory
(Sacramento, CA). Mice were housed in a temperature- and
humidity-controlled room maintained on a 12 h light/dark cycle in
groups of four to five (same sex). Animals weighed between 17 and 30
g at the time of the experiments. All experimental procedures
involving rodents were approved by the UC Davis Institutional
Animal Care and Use Committee (IACUC) and adhered to principles
described in the National Institutes of Health Guide for the Care and
Use of Laboratory Animals. Zebrafish experiments were performed in
accordance with established protocols approved by the UCSF IACUC
and adhered to principles described in the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. The
University of California, Davis and the University of California, San
Francisco are accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care International (AAALAC).
Dendritogenesis Experiments. Dendritogenesis experiments
were performed following a previously published method with slight
modifications.10 Neurons were plated in a 96-well format (200 μL of
media per well) at a density of approximately 15 000 cells/well in
Neurobasal (Life Technologies) containing 1% penicillin−streptomy-
cin, 10% heat-inactivated fetal bovine serum, and 0.5 mM glutamine.
After 24 h, the medium was replaced with Neurobasal containing 1×
B27 supplement (Life Technologies), 1% penicillin−streptomycin, 0.5
mM glutamine, and 12.5 μM glutamate. After 3 days in vitro (DIV3),
the cells were treated with compounds. All compounds tested in the
dendritogenesis assays were treated at 10 μM unless noted otherwise.
Stock solutions of the compounds in DMSO were first diluted 100-
fold in Neurobasal before an additional 10-fold dilution into each well
(total dilution = 1:1000; 0.1% DMSO concentration). Treatments
were randomized. After 1 h, the media was removed and was replaced
with new Neurobasal media containing 1× B27 supplement, 1%
penicillin−streptomycin, 0.5 mM glutamine, and 12.5 μM glutamate.
The cells were allowed to grow for an additional 71 h. At that time,
neurons were fixed by removing 80% of the media and replacing it
with a volume of 4% aqueous paraformaldehyde (Alfa Aesar) equal to
50% of the working volume of the well. Then, the cells were incubated
at room temperature for 20 min before the fixative was aspirated and
each well was washed twice with Dulbecco’s phosphate-buffered saline
(DPBS). Cells were permeabilized using 0.2% Triton X-100
(ThermoFisher) in DPBS for 20 min at room temperature without
shaking. Plates were blocked with antibody diluting buffer (ADB)
containing 2% bovine serum albumin (BSA) in DPBS for 1 h at room
temperature. Then, plates were incubated overnight at 4 °C with
gentle shaking in ADB containing a chicken anti-MAP2 antibody
run(″Sholl Analysis...″, ″starting=0 ending=NaN radius_step=2
#_samples=1 integration=Mean enclosing=1 #_primary=4 infer fit
linear polynomial=[Best fitting degree] most semi-log normal-
izer=Area create background=228 save do″);
Sholl analysis circle radii = 2 pixel increments = 0.67 μm. All
images were taken and analyzed by an experimenter blinded to
treatment conditions. The number of crossings for each neuron at
each distinct radius was averaged to produce an average Sholl plot for
each treatment. The Nmax values were simply determined by
identifying the maximum of each plot. For each treatment, neurons
were selected from at least six wells spread across two plates (9 sites/
well × 3 wells/plate × 2 plates). Each plate was prepared using
neurons obtained from independent pregnant dams.
Ketanserin Blocking Experiments. For the ketanserin blocking
experiments (Figure 9), a slightly modified method was employed. On
DIV3, neurons were first treated with ketanserin (10 μM) for 1 h
followed by a 1 h incubation with the drug (1 μM) and ketanserin (10
μM) (final concentration of DMSO = 0.2%). After 1 h, the media was
removed and replaced with new Neurobasal media containing 1× B27
supplement, 1% penicillin−streptomycin, 0.5 mM glutamine, and 12.5
μM glutamate. The cells were allowed to grow for an additional 71 h
before being fixed, stained, and imaged.
Zebrafish Behavioral Experiments. At 7 days post-fertilization,
healthy zebrafish larvae (wild-type Singapore strain) were sorted from
unhealthy fish, and their mobility was reduced by transferring
approximately 500 fish to a plate containing a 2:1 ratio of 4 °C to
room temperature egg water.39 Using a pipette, eight larvae were
carefully distributed into each well of a 96-well plate (GE Healthcare
Life Sciences) in 300 μL aliquots. Plates were incubated at room
temperature for 1 h, at which time the animals were mobile. Stock
solutions of the compounds in DMSO (1.2 μL) were mixed into the
well (0.4% DMSO concentration), and plates were incubated at room
temperature for 1 h prior to behavioral experiments. DMSO (Sigma-
Aldrich 472301) and eugenol (100 μM; Tokyo Chemical Industries
A0232) were used as vehicle and lethal controls, respectively.
Treatments were spread across seven plates (3 replicate wells per
treatment condition, 3 lethal control wells, and 21 DMSO wells on
each plate) and were randomized on the plates using a Biomek FXP
liquid handler. Plates were recorded at approximately 30 min intervals
between the hours of 16:00 and 20:00 on a single day.
After 1 h of incubation, treated plates were positioned in an
automated behavioral instrument and acclimated in darkness for 5
min. The plates were illuminated from below with 760 nm light
through an acrylic diffuser and recorded with an overhead PointGrey
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Grasshopper GS3-U3-41C6M-C camera (FLIR Integrated Imaging
Solutions Inc., Richmond, BC, Canada) mounted to a telecentric lens
(Opto Engineering) through an infrared filter (LEE Filters LE8744
polyester #87). The camera captured 1600 × 1068 8-bit-deep images
at 100 Hz. Stimulus lights included 623 nm (DigiKey 1537-1041-
ND), 525 nm (DigiKey 1537-1039-ND), 460 nm (DigiKey 1537-
1037-ND), 400 nm (Mouser LZ4-40UB00-00U7), 355 nm (Mouser
416-LST101G01UV01), and 4000 K white (Mouser 416-
0D0BN240E-SB01) LEDs, which were positioned overhead. Audio
stimuli were presented through two 5 W transducers (Generic) with
an APA150 150 W power amplifier. Two 36 V SparkFun Electronics
push−pull solenoids were used to deliver tapping stimuli. All stimuli
were verified with sensors. A pixel intensity threshold was set such
that values below the threshold represented noise. Animal locomotion
was calculated as the number of pixels that changed with intensity
greater than or equal to that threshold. Vectors were interpolated
using nanosecond-resolved timestamps from the image sensor and
aligned to the stimuli.
Distances from vehicle controls were estimated from the out-of-bag
accuracy of random forest models trained to distinguish between the
motion vectors of compound-treated and vehicle-treated wells.
Models (12 total, 2000 trees each) were trained and averaged per
problem (compound−concentration pair), sampling wells without
replacement to avoid class imbalance, but maintaining the same
number per plate. One plate was removed from further analysis when
an independent visualization showed that all its vehicle-treated,
compound-treated, and eugenol-treated wells clustered together. For
multiclassification, a random forest model with 10 000 trees was
trained on all 18 replicates per compound at 200 μM. For all models,
scikit-learn 0.21.3 defaults were used for all remaining hyper-
parameters.
Head-Twitch Response Experiments. Mice (9−10 weeks old)
were injected intraperitoneally with compound (injection volume 5
mL/kg), placed in an empty cage, and filmed for 20 min. Cages were
cleaned with 70% ethanol between experiments. Each video was
scored for the number of head-twitches by two trained observers
blinded to treatment condition (Pearson correlation coefficients =
0.91 and 0.99 for males and females, respectively), and these results
were averaged.
Statistical Analysis. Treatments were randomized, and data were
analyzed by experimenters blinded to treatment conditions. Statistical
analyses were performed using GraphPad Prism (version 8.1.2). The
specific tests used, F-statistics, degrees of freedom, and main effect p-
values are indicated in the figure legends where appropriate. All
comparisons were planned prior to performing each experiment. For
dendritogenesis experiments, a one-way ANOVA with Dunnett’s post
hoc test was deemed most appropriate, as our research question
focused on determining whether or not a particular treatment
promoted neuronal growth to a greater extent than the vehicle
control. Ketamine was included as a positive control to ensure that the
assay was working properly.
Center for Neuroscience, University of California, Davis, Davis,
California 95616, United States; orcid.org/0000-0002-
4517-0543; Email: deolson@ucdavis.edu
Authors
Lee E. Dunlap − Department of Chemistry, University of
California, Davis, Davis, California 95616, United States
Arya Azinfar − Department of Chemistry, University of
California, Davis, Davis, California 95616, United States
Calvin Ly − Department of Chemistry, University of California,
Davis, Davis, California 95616, United States
Lindsay P. Cameron − Neuroscience Graduate Program,
University of California, Davis, Davis, California 95618, United
States
Jayashri Viswanathan − Department of Chemistry, University of
California, Davis, Davis, California 95616, United States
Robert J. Tombari − Department of Chemistry, University of
California, Davis, Davis, California 95616, United States;
orcid.org/0000-0002-6561-8181
Douglas Myers-Turnbull − Quantitative Biosciences
Consortium and Institute for Neurodegenerative Diseases,
University of California, San Francisco, San Francisco,
California 94143, United States
Jack C. Taylor − Institute for Neurodegenerative Diseases,
University of California, San Francisco, San Francisco,
California 94143, United States
Ana Cristina Grodzki − Department of Molecular Biosciences,
School of Veterinary Medicine, University of California, Davis,
Davis, California 95616, United States
Pamela J. Lein − Department of Molecular Biosciences, School of
Veterinary Medicine, University of California, Davis, Davis,
California 95616, United States; orcid.org/0000-0001-
7665-7584
David Kokel − Institute for Neurodegenerative Diseases and
Department of Physiology, University of California, San
Francisco, San Francisco, California 94143, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.9b01404
Author Contributions
DEO and LED were responsible for the overall experimental
design. LED performed the majority of the chemistry with help
from AA. LED performed the majority of the dendritogenesis
experiments with help from CL. ACG and PJL supervised the
high-content imaging experiments. DM-T, JCT, and RJT
performed the zebrafish behavioral experiments and were
supervised by DK. LPC and JV performed the HTR
experiments. DEO conceived the project and supervised the
experiments. DEO and LED wrote the manuscript with input
from all authors.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01404.
■
sı
Funding
CNS MPO scores and maximum psychoplastogenicity
(Figure S1); process for converting images into files
suitable for data analysis (Figure S2); zebrafish motion
This work was supported by funds from the National Institutes
of Health (R01GM128997 to DEO and R01AA022583 to
DK), a UC Davis Miller Symposium Graduate Fellowship
(LED), a UC Davis Provost Undergraduate Fellowship (AA),
an NIH Chemical Biology T32 Training Grant
(T32GM113770 to CL and RJT), a UC Davis Science
Translation and Innovative Research (STAIR) Grant (DEO), a
Hellman Fellowship (DEO), The Paul G. Allen Family
Foundation (DK), the Genentech Foundation (DK), and the
National Institute of Child Health and Human Development
(U54 HD079125).
1
traces with associated stimuli (Figure S3); H and 13C
NMR spectra (PDF)
Molecular formula strings for all compounds (CSV)
AUTHOR INFORMATION
Corresponding Author
David E. Olson − Department of Chemistry, Department of
Biochemistry & Molecular Medicine, School of Medicine, and
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(9) Liu, R. J.; Duman, C.; Kato, T.; Hare, B.; Lopresto, D.; Bang, E.;
Burgdorf, J.; Moskal, J.; Taylor, J.; Aghajanian, G.; Duman, R. S.
GLYX-13 Produces Rapid Antidepressant Responses with Key
Synaptic and Behavioral Effects Distinct from Ketamine. Neuro-
psychopharmacology 2017, 42, 1231−1242.
Notes
The authors declare the following competing financial
interest(s): DEO is the president and chief scientific officer
of Delix Therapeutics, Inc.
(10) Ly, C.; Greb, A. C.; Cameron, L. P.; Wong, J. M.; Barragan, E.
V.; Wilson, P. C.; Burbach, K. F.; Soltanzadeh Zarandi, S.; Sood, A.;
Paddy, M. R.; Duim, W. C.; Dennis, M. Y.; McAllister, A. K.; Ori-
McKenney, K. M.; Gray, J. A.; Olson, D. E. Psychedelics Promote
Structural and Functional Neural Plasticity. Cell Rep. 2018, 23, 3170−
3182.
ACKNOWLEDGMENTS
■
We thank members of the Olson Laboratory for helpful
discussions and Steven Chen and Michelle Arkin for access to
the Biomek liquid handler.
́
(11) dos Santos, R. G.; Osorio, F. L.; Crippa, J. A.; Riba, J.; Zuardi,
A. W.; Hallak, J. E. Antidepressive, Anxiolytic, and Antiaddictive
Effects of Ayahuasca, Psilocybin and Lysergic Acid Diethylamide
(LSD): A Systematic Review of Clinical Trials Published in the Last
25 Years. Ther. Adv. Psychopharmacol. 2016, 6, 193−213.
(12) Cameron, L. P.; Olson, D. E. Dark Classics in Chemical
Neuroscience: N,N-Dimethyltryptamine (DMT). ACS Chem. Neuro-
sci. 2018, 9, 2344−2357.
ABBREVIATIONS
■
DMT, N,N-dimethyltryptamine; PFC, prefrontal cortex; 5-
HT2A, serotonin 2A; MPO, multiparameter optimization;
LSD, lysergic acid diethylamide; TPSA, total polar surface
area; MAP2, microtubule-associated protein 2; Nmax, maximum
number of crossings; 5-HT2B, serotonin 2B; DIV, days in
vitro; VEH, vehicle; KET, ketamine; SEM, standard error of
the mean; ANOVA, analysis of variance; DOM, 2,5-
dimethoxy-4-methylamphetamine; OMe, methoxy; OBn,
benzyloxy; F, fluoro; μM, micromolar; nM, nanomolar; pM,
picomolar; V, vehicle; K, ketamine; ATR, attenuated total
reflectance; FT-IR, Fourier transform infrared spectroscopy;
UHPLC, ultra-high-performance liquid chromatography;
LRMS, low-resolution mass spectrometry; IACUC, institu-
tional animal care and use committee; AAALAC, Association
for Assessment and Accreditation of Laboratory Animal Care;
BSA, bovine serum albumin; DPBS, Dulbecco’s phosphate-
buffered saline; mTOR, mammalian target of rapamycin;
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid; TrkB, tropomyosin receptor kinase B; HTR, head-twitch
response
(13) Cameron, L. P.; Benson, C. J.; Dunlap, L. E.; Olson, D. E.
Effects of N,N-Dimethyltryptamine (DMT) on Rodent Behaviors
Relevant to Anxiety and Depression. ACS Chem. Neurosci. 2018, 97,
1582−1590.
(14) Cameron, L. P.; Benson, C. J.; DeFelice, B. C.; Fiehn, O.;
Olson, D. E. Chronic, Intermittent Microdoses of the Psychedelic
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and Anxiety in Rodents. ACS Chem. Neurosci. 2019, 10, 3261−3270.
(15) Palhano-Fontes, F.; Barreto, D.; Onias, H.; Andrade, K. C.;
Novaes, M. M.; Pessoa, J. A.; Mota-Rolim, S. A.; Osor′io, F. L.;
Sanches, R.; Dos Santos, R. G.; Tof′oli, L. F.; de Oliveira Silveira, G.;
Yonamine, M.; Riba, J.; Santos, F. R.; Silva-Junior, A. A.; Alchieri, J.
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