Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines

Article abstract of DOI:10.1016/j.bmcl.2015.12.053

N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20 mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT_1Aand 5-HT_2Breceptors, while the affinity of DALT itself at 5-HT_1Areceptors was slightly lower at 100 nM. Among the 5-HT₂subtypes, the weakest affinity was at 5-HT_2Areceptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT_1D, 5-HT₆, and 5-HT₇receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT_1B, 5-HT_1E, and 5-HT_5Asubtypes and little or no affinity for the 5-HT₃subtype. These compounds also had generally nanomolar affinities for adrenergic α_2A, α_2B, and α_2Creceptors, sigma receptors σ₁and σ₂, histamine H₁receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated correlations among physicochemical properties of the congeneric 5-substituted-DALT compounds. The descriptors included electronic (σ_p), hydrophobic (π), and steric (CMR) parameters. The binding affinity at 5-HT_1A, 5-HT_1D, 5-HT₇, and κ opioid receptors was positively correlated with the steric volume parameter CMR. At α_2A, α_2B, and α_2Creceptors, and at the histamine H₁receptor, binding affinity was correlated with the Hammett substituent parameter σ_p; higher affinity was associated with larger σ_pvalues. At the σ₂receptor, higher affinity was correlated with increasing π. These correlations should aid in the development of more potent and selective drugs within this family of compounds.

Full text of DOI:10.1016/j.bmcl.2015.12.053

Accepted Manuscript
Receptor binding profiles and quantitative structure-affinity relationships of
some 5–substituted-n,n-diallyltryptamines
Nicholas V. Cozzi, Paul F. Daley
PII:
S0960-894X(15)30367-X
http://dx.doi.org/10.1016/j.bmcl.2015.12.053
BMCL 23414
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 November 2015
14 December 2015
16 December 2015
Please cite this article as: Cozzi, N.V., Daley, P.F., Receptor binding profiles and quantitative structure-affinity
relationships of some 5–substituted-n,n-diallyltryptamines, Bioorganic & Medicinal Chemistry Letters (2015), doi:
http://dx.doi.org/10.1016/j.bmcl.2015.12.053
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RECEPTOR BINDING PROFILES AND QUANTITATIVE STRUCTURE-AFFINITY RELATIONSHIPS OF
SOME 5-SUBSTITUTED-N,N-DIALLYLTRYPTAMINES
Nicholas V. Cozzi,a,b,* Paul F. Daleya
aThe Alexander Shulgin Research Institute
1483 Shulgin Road
Lafayette, CA 94549
bNeuropharmacology Laboratory
2695 Medical Sciences Center
Department of Cell and Regenerative Biology
University of Wisconsin School of Medicine and Public Health
1300 University Avenue
Madison, WI 53706
*Corresponding author
Keywords: DALT; psychedelic; QSAR; tryptamine; N,N-diallyltryptamine; 5-methoxy-N,N-
diallyltryptamine; hallucinogen; receptor binding; psychopharmacology

ABSTRACT
N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two
tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT
was reported to be psychoactive in the 12-20 mg range, while the unsubstituted compound
DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used
in nonmedical settings for its psychoactive effects, but these effects have been poorly
characterized and little is known of its pharmacological properties. We extended the work of
Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and
5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based
on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-
DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin
5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly
lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors,
spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nanomolar range
for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had
the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-
HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also
had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1
and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They
also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding
results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are
important for the psychoactive effects of DALT drugs. To learn whether any quantitative
structure-affinity relationships existed, we evaluated correlations among physicochemical
properties of the congeneric 5-substituted-DALT compounds. The descriptors included
electronic (σp), hydrophobic (π), and steric (CMR) parameters. The binding affinity at 5-HT1A, 5-
HT1D, 5-HT7, and κ opioid receptors was positively correlated with the steric volume parameter
CMR. At α2A, α2B, and α2C receptors, and at the histamine H1 receptor, binding affinity was
correlated with the Hammett substituent parameter σp; higher affinity was associated with
larger σp values. At the σ2 receptor, higher affinity was correlated with increasing π. These
correlations should aid in the development of more potent and selective drugs within this
family of compounds.

The pharmacology of naturally occurring and synthetic psychoactive N,N-dialkyltryptamines has
been a matter of investigation since the 1950s.1-3 The discovery of serotonin in the brain4 and
the subsequent discovery that hallucinogenic snuffs prepared by people in South America,
Haiti, and Puerto Rico contained a close congener of serotonin, namely N,N-
dimethyltryptamine (DMT),5 led Szára in Hungary to perform self-experiments with DMT. After
his discovery that parenteral DMT produced psychedelic effects similar to those produced by
mescaline and LSD,6 the synthesis and pharmacological characterization of numerous
N,N-disubstituted tryptamines, including N,N-diallyltryptamine (DALT), followed. However,
with the exception of some early reports on DALT,7-9 information about the chemistry and
pharmacology of DALT and ring-substituted DALT compounds remains sparse.
As part of his systematic structure-activity studies of hallucinogenic tryptamines, Alexander
Shulgin synthesized and self-tested two N,N-diallyltryptamines, namely DALT itself and the ring-
substituted 5-MeO-DALT (Fig. 1). While Szára reported that DALT was "psychotropic" at oral or
intramuscular doses of 60 mg,9 Shulgin concluded that DALT had few discernible effects when
taken orally in the 42-80 mg range and that 5-MeO-DALT had short-lived psychoactive effects in
a range of 12-20 mg orally (A.T. Shulgin, personal communication). The psychoactive effects of
5-MeO-DALT reported by Shulgin were not well-characterized but they seemed to consist of an
intoxication that was devoid of the usual visual imagery and cognitive effects associated with
psychedelic agents. Nevertheless, 5-MeO-DALT has recently been identified as a compound
used outside of medical settings for its psychoactive effects.10,11 The European Monitoring
Centre for Drugs and Drug Addiction (EMCDDA) received the first notification of 5-MeO-DALT
detection in 2007,12 followed by 4-acetoxy-N,N-diallyltryptamine (4-AcO-DALT) in 2012,13 and
DALT itself in 2014.14 The availability of 5-MeO-DALT in adult shops in Tokyo was reported in
200715 and analytical data was published in 2008.16 Since then, the detection and analysis of 5-
MeO-DALT, either on its own or present in other products, has been reported relatively
frequently.17-21
The pharmacological properties of DALT compounds are largely unexplored. A recent metabolic
study of DALT and 5-MeO-DALT in rats identified numerous metabolites and proposed the
involvement of several metabolic pathways in the biotransformation of DALT compounds.22
Several pharmacodynamic studies of 5-MeO-DALT have been reported to date. In one study, 5-
MeO-DALT did not exhibit appreciable monoamine uptake inhibition or releasing activity in
synaptosomes,15 while in another study, 5-MeO-DALT was observed to stimulate [35S]GTPγS
binding in rat brain membranes, possibly including agonist activity at the 5-HT1A receptor.23 A
third study evaluating the binding affinity of 5-MeO-DALT at various cloned receptors and
transport proteins, as well as its ability to mobilize calcium via cloned serotonin 5-HT2A, 5-HT2B,
and 5-HT2C receptors, has been reported.18 In this report, 5-MeO-DALT had the highest affinity

at 5-HT2B, 5-HT6, and 5-HT7 receptor subtypes and displayed the weakest affinity at 5-HT2A and
5-HT2C receptors. In a calcium mobilization assay, 5-MeO-DALT was a full 5-HT2 agonist at low
nanomolar concentrations but was less potent that serotonin.18
The relative lack of pharmacological data for DALT compounds, especially given their growing
occurrence and recreational use, motivated the present study. To identify other biological
targets for these tryptamines, we synthesized DALT compounds with various indole ring
substitutions and presented these substances to the National Institute of Mental Health's
Psychoactive Drug Screening Program (NIMH PDSP) for binding analysis at 45 cloned human
receptors and transport proteins. Here, we report the binding profiles and quantitative
structure-affinity analysis (QSAR) of a congeneric set of six DALT compounds, namely DALT, 5-F-
DALT, 5-Cl-DALT, 5-Br-DALT, 5-Me-DALT, and 5-MeO-DALT (Fig. 1).
1
4
2
5
3
6
Fig. 1. Chemical structures of 5-substituted-N,N-diallyltryptamines.
After binding targets were identified, we examined the data set for correlations among
physicochemical descriptors and binding affinity. The physicochemical properties analyzed were
electronic (σp), hydrophobic (π), and steric (calculated molar refractivity; CMR) parameters. The
electronic value σp, also called the Hammett substituent constant, is a measure of the change in
free-energy that accompanies formation of complexes between ligands and their target
proteins.24-26 Positive σ values are associated with electron-withdrawing substituents, negative
σ values indicate electron-donating substituents, and the absolute value of σ indicates the
strength of the electronic effect. In our analysis, we treat the sigma value of the 5-substituent
as para (σp) to the indole nitrogen (N1). There are several reports that in indole-containing
compounds, N1 is involved in hydrogen bond formation with binding targets, and that the
electronic influences of 5-substituents are best explained if they are transmitted to N1 via both

inductive and resonance effects, i.e. the 5-substituent is para to N1.27-29 The hydrophobic
parameter π quantifies the degree to which the addition of a given substituent changes the
partitioning of a compound between water and n-octanol and thus reflects the relative
tendency of a compound to partition into nonpolar environments compared to the
unsubstituted parent compound.25,26,30 Finally, the calculated molar refractivity (CMR)
estimates the spatial volume occupied by an atom or substituent group, with higher CMR
values representing greater steric bulk. Correlations of binding with substituent CMR reveals
potential steric interactions between a ligand and its binding site.26,31
The DALT compounds were synthesized by adapting our published methods (Scheme 1).32
Briefly, using the method of Speeter and Anthony,33 the 5-substituted-glyoxylamides (1b-6b)
were obtained by acylation of the 5-substituted-indoles (1a-6a) with oxalyl chloride, followed
by reaction with N,N-diallylamine to give the 5-substituted-N,N-diallylglyoxylamides (1c-6c). The
N,N-diallylglyoxylamides were rapidly reduced to the N,N-diallyltryptamines (1-6) using lithium
aluminum hydride in sealed glass tubes under microwave-accelerated conditions as
described.32 Compounds were isolated as hydrochloride salts and were analyzed by gas
chromatography-ion trap-mass spectrometry, liquid chromatography-high-resolution
electrospray quadrupole time-of-flight mass spectrometry (LC-HR-MS/MS) with diode array
detection, 1H nuclear magnetic resonance spectroscopy, and infrared spectroscopy. Analytical
results agreed with the predicted structures and will be published elsewhere.
CH2
O
O
R
R
R
Cl
N
a
b
CH2
O
O
N
N
H
N
H
H
1a-6a
1b-6b
1c-6c
CH2
c
R
N
CH2
N
H
1-6
Scheme 1. Synthesis of 5-substituted-N,N-diallyltryptamines. Reagents and conditions: (a)
oxalyl chloride, diethyl ether, 0 °C, 4 h, 90-92%; (b) N,N-diallylamine, THF, 0 °C, 4 h, 55-64%; (c)
LAH, THF, 150 °C, microwave power 250 W, maximum pressure 280 psi, ramp time 5 min, hold
time 5 min, 68-70%. R = H, F, Cl, Br, CH3, CH3O.

Binding assays were performed by the National Institute of Mental Health's Psychoactive Drug
Screening Program (NIMH PDSP, Contract # HHSN-271-2013-00017-C). Details of individual
binding assay conditions and protocols are available at the NIMH PDSP website:
http://pdsp.med.unc.edu/pdspw/binding.php. Briefly, DALT compounds were screened at 10
μM for their abilities to compete with various target-selective radioactive probe compounds at
45 cloned receptors and transporter proteins. In this primary assay target screening, an IC50
value of 10 μM was established as the threshold for further analysis. If a DALT compound
displaced more than 50% of the radioligand probe at 10 μM, secondary assays were performed
at the identified receptor or transporter using 12 concentrations of the DALT compound to
generate competition binding isotherm curves. Ki values were then obtained from nonlinear
regression of these binding isotherms. Ki values are calculated by the NIMH PDSP from best-fit
IC50 values using the Cheng-Prusoff equation.34 Ki values were converted to pKi values for data
analysis.
Correlations were evaluated between in vitro pKi values and physicochemical parameters to
assess quantitative structure-affinity relationships (QSAR). Specifically, electronic (σp),
hydrophobic (π), and molar volume (CMR) values of the substituents at the 5-position of the
tryptamine ring were examined by linear regression analysis for correlations to binding affinity
at target proteins using commercial computer software (GraphPad Prism, San Diego, CA, USA).
The regression analysis tests the null hypothesis that the slope of the least-squares regression
line is different from zero, with P < 0.05 considered significant. Hammett σp and π values were
obtained from Hansch et al.26, and CMR values were calculated using commercial computer
software (ChemBioDraw Ultra 13, PerkinElmer Informatics, Waltham Massachusetts 02451
USA).

Although N,N-diallyltryptamine compounds were briefly explored over 50 years ago, it is only
recently that one of them, 5-MeO-DALT, has been reportedly used for its psychoactive
effects.11,12 Very little is known of its pharmacology.15,18,23 To more completely characterize the
binding targets for this substance and related DALT compounds, we synthesized and
characterized 5-substituted-DALTs and engaged the NIMH PDSP to identify protein binding
sites. We then employed QSAR analysis to elucidate structural determinants for affinity at the
identified binding sites. There were four main findings. First, all six DALT compounds exhibited
less than 10 μM binding affinity at most sites tested, with some of the highest affinities
observed at 5-HT1A, 5-HT2B, 5-HT7, H1, and σ1 receptors, and the serotonin uptake transporter
(SERT) (Table 1). Second, QSAR analysis identified a significant correlation between the steric
parameter (CMR) of the 5-substituents and binding affinity at 5-HT1A, 5-HT1D, 5-HT7, and κ
opioid receptors (Table 1, Fig. 2). Third, pKi values at α2A, α2B, α2C, and H1 receptors were
positively correlated with the Hammett constant, σp (Table 1, Fig. 3). Finally, binding at σ2
receptors was correlated with the hydrophobic constant, π (Table 1, Fig. 4). These results show
that steric bulk, electron-withdrawing power, and hydrophobicity of the 5-substituent of the
DALT scaffold are key determinants for in vitro selectivity among these binding sites.

Table 1. In vitro binding constants (pKi ± SEM) for DALT compounds with Ki values of 10 μM or lower at various receptors
and transporters.a
5-substituted DALT compounds
3
1
2
4
5
6
Physicochemical
descriptorb
π
σp
0
0
0.14
0.06
0.71
0.23
0.86
0.23
0.56
-0.17
-0.02
-0.27
CMR
Binding site
7.8344
7.8499
8.3258
8.6114
8.2982
8.4513
pKi ± SEM at binding sitea
c
5-HT1A
7.00 ± 0.06
> 5.00
7.10 ± 0.07
5.75 ± 0.04
6.12 ± 0.09
6.32 ± 0.04
6.61 ± 0.05
7.78 ± 0.06
6.99 ± 0.05
5.37 ± 0.07
7.13 ± 0.06
6.40 ± 0.06
5.90 ± 0.06
> 5.00
7.23 ± 0.05
6.05 ± 0.04
6.67 ± 0.09
6.45 ± 0.05
6.39 ± 0.05
7.52 ± 0.04
6.69 ± 0.05
5.68 ± 0.06
7.16 ± 0.07
7.13 ± 0.06
6.28 ± 0.05
6.10 ± 0.20
5.88 ± 0.09
7.09 ± 0.05
6.77 ± 0.04
6.20 ± 0.10
5.73 ± 0.04
6.85 ± 0.04
7.00 ± 0.10
5.80 ± 0.04
6.09 ± 0.07
5.55 ± 0.04
7.01 ± 0.06
6.98 ± 0.08
6.10 ± 0.20
6.20 ± 0.20
7.39 ± 0.06
8.00 ± 0.10
6.02 ± 0.04
6.90 ± 0.10
6.29 ± 0.04
6.32 ± 0.05
7.28 ± 0.05
6.45 ± 0.05
5.62 ± 0.06
6.88 ± 0.08
7.31 ± 0.06
6.20 ± 0.06
5.70 ± 0.20
5.95 ± 0.09
7.08 ± 0.06
6.64 ± 0.04
6.40 ± 0.10
5.36 ± 0.05
6.62 ± 0.04
7.00 ± 0.20
5.83 ± 0.04
6.05 ± 0.07
5.76 ± 0.03
6.99 ± 0.06
7.01 ± 0.06
5.60 ± 0.10
6.00 ± 0.20
6.90 ± 0.05
7.50 ± 0.10
5.87 ± 0.04
6.40 ± 0.10
6.31 ± 0.03
6.14 ± 0.05
7.22 ± 0.05
6.50 ± 0.08
5.49 ± 0.04
6.13 ± 0.03
6.81 ± 0.07
5.90 ± 0.06
5.75 ± 0.09
> 5.00
7.70 ± 0.10
6.13 ± 0.04
7.00 ± 0.10
6.30 ± 0.05
6.66 ± 0.08
7.23 ± 0.05
6.34 ± 0.08
5.48 ± 0.04
6.81 ± 0.03
7.05 ± 0.07
> 5.00
5-HT1B
c
5-HT1D
6.16 ± 0.09
6.42 ± 0.04
6.15 ± 0.05
7.21 ± 0.05
6.41 ± 0.05
> 5.00
5-HT1E
5-HT2A
5-HT2B
5-HT2C
5-HT5A
5-HT6
5.76 ± 0.04
> 5.00f
c
5-HT7
α1A
α1B
α1D
5.78 ± 0.05
5.90 ± 0.20
> 5.00
> 5.00
> 5.00
> 5.00
d
α2A
α2B
6.91 ± 0.06
6.52 ± 0.06
6.05 ± 0.06
> 5.00
6.93 ± 0.05
6.66 ± 0.04
6.10 ± 0.10
5.61 ± 0.04
6.92 ± 0.06
7.10 ± 0.20
5.68 ± 0.04
5.66 ± 0.07
> 5.00
6.71 ± 0.06
6.36 ± 0.04
5.96 ± 0.07
5.29 ± 0.09
6.16 ± 0.07
6.17 ± 0.05
> 5.00
6.67 ± 0.07
6.14 ± 0.04
5.83 ± 0.06
> 5.00
d
d
α2C
D2
D3
6.17 ± 0.06
6.90 ± 0.10
> 5.00
> 5.00
d
H1
6.30 ± 0.06
5.77 ± 0.04
5.95 ± 0.07
> 5.00
H3
κORc
μOR
σ1
5.61 ± 0.08
> 5.00
5.91 ± 0.07
> 5.00
7.00 ± 0.06
6.45 ± 0.09
5.90 ± 0.10
6.00 ± 0.20
6.82 ± 0.05
7.07 ± 0.06
6.52 ± 0.05
5.70 ± 0.10
5.70 ± 0.20
7.44 ± 0.06
7.13 ± 0.08
6.66 ± 0.06
5.50 ± 0.10
> 5.00
6.52 ± 0.06
6.60 ± 0.05
5.50 ± 0.20
> 5.00
e
σ2
DAT
NET
SERTd
6.44 ± 0.06
6.30 ± 0.05
apKi values determined by NIMH PDSP. bValues of π and σp from Hansch et al., 1973; values of CMR from ChemBioDraw
Ultra 13. cpKi correlates with CMR. dpKi correlates with Hammett substituent constant σp. epKi correlates with π. fValue
excluded from QSAR analysis; see text.

5-HT1D
5-HT1A
9.0
8.5
8.0
7.5
9.0
8.5
8.0
7.5
Br
CH3O
CH3
Br
Cl
CH3
CH3O
Cl
CMR = 0.7903pK i + 3.059
CMR = 0.7584pKi + 2.6
F
R2 = 0.8566
P = 0.0081
R2 = 0.8313
P = 0.0113
F
H
H
6.00
6.25
6.50
6.75
7.00
6.75 7.00 7.25 7.50 7.75 8.00 8.25
7.25
pKi
pKi
5-HT7
κOR
9.0
8.5
8.0
7.5
9.0
Br
Br
CH3O
CH3
CH3O
8.5
8.0
7.5
CH3
Cl
Cl
CMR = 1.464pK i - 0.3771
CMR = 0.7700pKi + 2.964
R2 = 0.8421
P = 0.0099
R2 = 0.9064
P = 0.0125
F
F
H
6.25
6.50
6.75
7.00
7.25
7.50
5.50
5.75
6.00
6.25
pKi
pKi
Fig. 2. Correlational analysis between CMR and pKi values from Table 1. The figure shows the
correlation between spatial volume (CMR, ordinate) of the 5-substituent and binding affinity
(pKi, abscissa) at 5-HT1A, 5-HT1D, 5-HT7, and κ opioid receptors.

α2B
α2A
0.4
0.3
0.4
0.3
Br
Br
0.2
0.2
Cl
Cl
F
0.1
0.1
F
0.0
0.0
H
H
-0.1
-0.2
-0.3
-0.4
-0.1
-0.2
-0.3
-0.4
CH3
σp = 0.8399pK i - 5.458
σp = 1.145pK i - 7.884
CH3
R2 = 0.8941
P = 0.0044
R2 = 0.9885
P < 0.0001
CH3O
CH3O
6.50
6.75
7.00
7.25
6.00
6.25
6.50
pKi
6.75
7.00
pKi
H1
α2C
0.4
0.3
0.4
0.3
Cl
Br Cl
0.2
0.2
Br
F
F
0.1
0.1
H
0.0
0.0
H
-0.1
-0.2
-0.3
-0.4
-0.1
-0.2
-0.3
-0.4
CH3
σp = 0.4408pK i - 2.960
σp = 0.9748pK i - 5.923
CH3
R2 = 0.7478
P = 0.0262
R2 = 0.8791
P = 0.0057
CH3O
6.50
CH3O
5.75
6.00
6.25
6.50
6.00
6.25
6.75
7.00
7.25
pKi
pKi
Fig. 3. Correlational analysis between the Hammett parameter σp and pKi values from Table 1.
The figure shows the correlation between electron-withdrawing strength (σp, ordinate) of the
5-substituent and binding affinity (pKi, abscissa) at α2A, α2B, and α2C adrenergic receptors, and at
histamine H1 receptors.

σ2
1.00
0.75
0.50
0.25
0.00
-0.25
-0.50
Br
CH3
Cl
F
H
CH3O
π = 1.468pK i - 9.463
R2 = 0.8249
P = 0.0122
6.25
6.50
6.75
pKi
7.00
7.25
Fig. 4. Correlational analysis between π and pKi values from Table 1. The figure shows the
correlation between hydrophobicity (π, ordinate) of the 5-substituent and binding affinity (pKi,
abscissa) at σ2 receptors.

The DALT compounds were initially screened at 45 different receptors and transporters. From
this set, 27 sites met the 10 μM threshold criterion for secondary analysis and Ki determination.
Notably, none of the following sites met the 10 μM threshold: beta adrenergic (β1-β3);
dopaminergic D1, D4, D5; GABAergic (flunitrazepam GABAA, muscimol GABAA, and peripheral
benzodiazepine sites); muscarinic (M1-M5); 5-HT3; δ-opioid; histaminergic H2 and H4 receptors.
All of the test compounds exhibited Ki values of less than 10 μM at the following targets:
serotonergic receptors 5-HT1A, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT6; adrenergic
receptors α2A, α2B, and α2C; histamine receptor H1; κ opioid receptor (κOR); sigma receptors σ1
and σ2; DAT and SERT monoamine uptake transporters. The highest measured affinities, which
were in the 10-100 nM range, were at the 5-HT1A and 5-HT2B subtypes. In addition, several
compounds had sub-100 nM affinity at 5-HT6, 5-HT7, α2A, and σ1 receptors and the SERT. The 5-
substituted-DALT compounds are thus relatively nonselective over a broad range of biogenic
amine receptors and transporters, as well as opioid and sigma receptors.
Evidence from both human and animal studies indicates that, at a minimum, binding and partial
agonist activity at serotonergic 5-HT1A and 5-HT2A receptors are required for the manifestation
of psychoactive effects from psychedelic tryptamines such as DMT, 5-MeO-DMT, and psilocin.35-
38 These two receptors, as well as several other 5-HT receptors, σ1 receptors, and the SERT were
previously identified as high affinity targets for 5-MeO-DALT.18,23 The present study is in good
agreement with these earlier findings and identifies additional binding sites that are likely to
play a role in the psychoactive effects of DALT compounds. In particular, our data indicate that
5-MeO-DALT binds with the highest affinity at 5-HT1A sites (19 nM) and has 150 nM or better
affinity at 5-HT1D, 5-HT2B, 5-HT6, and 5-HT7 receptors; its affinity for 5-HT2A receptors is
somewhat weaker at 218 nM (Table 1). Nonserotonergic targets identified in the present study
are also likely to contribute to the psychoactive effects of DALT drugs. Notably, binding potency
at some of these other sites is as high or higher than potency at serotonergic sites. Thus, α2A,
H1, σ1, and σ2 receptors are all comparatively high-affinity targets for the DALT compounds
(Table 1). Although the present work did not examine the functional consequences of 5-
substituted-DALT binding, the receptor and transporter targets identified herein are all
expressed in brain tissue and drugs that bind to these receptors are well-known to affect
neuronal function, producing effects on mood, alertness, perception, memory, and cognition.39-
43 Our results therefore suggest that multiple serotonin receptors, as well as several
nonserotonergic sites, are involved in the psychoactive effects of DALT drugs. While it is not
possible to predict from our data whether the DALT binding profiles correlate with specific
cognitive changes, it seems likely that perhaps nine 5-HT receptors, as well as α2A, H1, σ1, and σ2
receptors and the SERT all contribute to the psychoactive effects of these drugs at
physiologically meaningful concentrations. Additional studies, including behavioral and

pharmacokinetic studies, will be needed to clarify what roles these binding sites play in the
psychopharmacology of DALT compounds.
To identify factors that influence binding affinity, a quantitative structure-affinity analysis was
conducted for the six DALT compounds with respect to three physicochemical parameters.
These physicochemical parameters are steric bulk (CMR), electronic (σp), and hydrophobic (π)
constants. Of the parameters analyzed, CMR and the Hammett constant σp correlated with
binding affinity at multiple receptors, and one correlation was detected between π and binding
affinity at the σ2 receptor. Note that NIMH PDSP reported that DALT (1) had no appreciable
affinity for 5-HT7 receptors, although all of the other compounds had pKi values in the 6.4-7.3
(50-400 nM) range. Compound 1 was therefore excluded from the QSAR analysis at this
particular receptor. Significant correlations were found between CMR of the 5-substituent and
pKi values at 5-HT1A (R2 = 0.8313, P = 0.0113), 5-HT1D (R2 = 0.8566, P = 0.0081), 5-HT7 (R2 =
0.9064, P = 0.0125), and κOR (R2 = 0.8421, P = 0.0099) receptors (Fig. 2, Table 1). At these
receptors, increased steric bulk is associated with higher affinity. The spatial volume of the
5-substituent thus plays a key role in binding at these sites, suggesting a topological
correspondence between the DALT 5-position and the binding pocket within these four
receptors. As long as the spatial limits of the binding pocket are not exceeded, we would
anticipate that DALT compounds with even larger CMR values (e.g. 5-I-DALT, 5-EtO-DALT, 5-Pr-
DALT) would have very high, possibly subnanomolar, affinity at 5-HT1A receptors and increased
affinity at the other three receptors as well. The electron-withdrawing capacity of the 5-
substituent, described by the Hammett constant σp, was positively correlated with pKi values at
α2A (R2 = 0.9885, P < 0.0001), α2B (R2 = 0.8941, P = 0.0044), and α2C (R2 = 0.8791, P = 0.0057)
adrenergic receptors, and the histamine H1 (R2 = 0.7478, P = 0.0262) receptor (Fig. 3, Table 2).
At SERT, there was a trend to increased affinity with σp, but this correlation did not quite reach
significance (R2 = 0.6427, P = 0.0551). We would expect that DALT compounds having 5-
substituents with larger σp values, (e.g. 5-CN-DALT, 5-CF3-DALT, 5-NO2-DALT), would exhibit
even higher affinity at these sites. There are several plausible mechanisms whereby larger σp
values are associated with higher binding affinity. First, one or more positively charged amino
acid residues within the target binding pockets in the vicinity of the DALT 5-substituent could
account for the correlation between σp and binding affinity at the receptors. Based upon
multiple sequence alignments among these four receptors, six positively-charged conserved
arginine and lysine residues were identified as potential candidates at positions 3.50, 4.41, 5.60,
6.29, 6.31, and 6.32.44-46 Second, favorable π-π stacking interactions between the indole ring of
the DALT compounds and conserved binding site aromatic amino acid residues could also
contribute to the observed higher affinity with electron-withdrawing 5-substituents, either
through an electrostatic mechanism47,48 or through a direct interaction of the 5-substituent
with hydrogen atoms of the amino acid aromatic nucleus.49 To this point, two conserved
aromatic residues are located at positions 5.47 and 5.48 and six additional conserved aromatic

residues are located nearby within a stretch of eight amino acids in the 6.48-6.55 ligand-binding
regions of these receptors.44-46 Finally, hydrogen bond strength between the tryptamine N1 and
a fully conserved threonine residue (3.37)29,44-46 within the orthosteric binding site of these four
receptors could be increased by higher electron-withdrawing power of the 5-substitutent,
transmitted to N1 via inductive and resonance effects.
In summary, we have identified a number of receptor and monoamine transporter binding sites
for a series of 5-substituted-DALT compounds, and we found associations between binding
affinity and physicochemical properties at some of these sites. The correlations between CMR,
σp, and π and receptor binding affinity should be of value in the design of more potent and
selective drugs within the DALT family of compounds.
ACKNOWLEDMENTS
We thank Simon D. Brandt, Ph.D. for excellent synthetic and analytical chemistry assistance and
we thank the National Institute of Mental Health for supporting the receptor binding assay
program at PDSP.

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