Synthesis and Structure-Activity Relationships of Tool Compounds Based on WAY163909, a 5-HT2C Receptor Agonist

Article abstract of DOI:10.1021/acschemneuro.6b00439

The development of probe molecules that can be used to investigate G protein-coupled receptor (GPCR) pharmacology, trafficking, and relationship with other GPCRs is an important and growing area of research. Here, we report the synthesis of analogues of t

Full text of DOI:10.1021/acschemneuro.6b00439

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Letter
Synthesis and structure-activity relationships of tools
based on WAY163909, a 5-HT2c receptor agonist
Ying-Chu Chen, Rachel M. Hartley, Noelle C. Anastasio, Kathryn A. Cunningham, and Scott R. Gilbertson
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00439 • Publication Date (Web): 17 Apr 2017
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Synthesis and structure activity relationships of tools based on WAY163909, a 5ꢀHT2C
receptor agonist
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YingꢀChu Chen1, Rachel M. Hartley2, Noelle C. Anastasio2,3, Kathryn A. Cunningham2,3, and
Scott R. Gilbertson1,2
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1Department of Chemistry, University of Houston, Houston TX Center for Addiction Research,
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3Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston,
TX
Abstract
The development of probe molecules that
can be used to investigate G proteinꢀ
coupled receptor (GPCR) pharmacology,
trafficking and relationship with other
GPCRs is an important and growing area of
research. Here, we report the synthesis of
analogs of the known selective serotonin
(5ꢀHT) 5ꢀHT2C receptor (5ꢀHT2CR) agonist WAY163909 which were designed to allow for the
attachment of a second ligand, signaling or reporter molecules as well as immobilization agents
to the parent molecule with the maintenance of agonist activity. This goal was accomplished by
the synthesis of novel molecules in which sites aꢀd were modified and resulting compounds
were analyzed pharmacologically in vitro.
Keywords
Serotonin, 5ꢀHT2C receptor agonist, WAY163909 derivatives
Introduction
Serotonin (5ꢀhydroxytryptamine, 5ꢀHT) receptors are implicated in a wide variety of
physiological functions in both the central and peripheral nervous systems. We are focused on
the development of tool compounds to study two 5ꢀHT GPCRs, the 5ꢀHT2A receptor (5ꢀHT2AR)
and 5ꢀHT2CR. These receptors exhibit an oppositional regulatory role over multiple behaviors
such that selective 5ꢀHT2AR antagonists and 5ꢀHT2CR agonists exert similar, and synergistic
effects, upon behavioral outcomes in preclinical studies.1ꢀ2 Furthermore, the 5ꢀHT2AR and 5ꢀ
HT2CR are proposed to form and/or function within both homoꢀ and/or heteromeric protein
complexes.3ꢀ12 The functional and/or physiological impact of these types of multimeric receptors
are not fully understood and the development of tool molecules to biochemically and/or
pharmacologically distinguish between the types of oligomeric receptor complexes is an
important goal. Thus, we have initiated a program to develop ligands capable of being linked to
other molecules, including a second ligand, a reporter molecule (e.g., fluorescent dyes) or
immobilization agents (e.g., biotin). To accomplish this goal, it is critical to identify locations on
known 5ꢀHT2R ligands that will allow for linking such groups without alteration of ligand binding
and/or functional activity. Our initial paper reported the synthesis and in vitro pharmacology of a
homobivalent 5ꢀHT2AR antagonist.13 Here, we report the synthesis and structureꢀactivity
relationships (SAR) for derivatives of the selective 5ꢀHT2CR agonist WAY163909 (1)1, 14ꢀ27 which
can be used to attach tethers [e.g., polyethylene glycol (PEG)] to link these molecules to various
other partners. Versions of the basic structure were synthesized in which locations around the
molecule, sites aꢀd (2), were modified to determine the viability of these locations as points for
the attachment of a necessary linker.
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Results and Discussion
The initial step was to identify a site on WAY163909 capable of modification without significant
loss of activity. This work can present challenges as the derivatives not only must bind to the 5ꢀ
HT2CR orthosteric site but, for some studies, the ligands should also retain their agonist activity.
The SAR data from the original work on these structures indicated that ring D (Figure 1 cpd 2)
was rather insensitive to different sizes, so a synthetic plan was selected to allow for the
synthesis of derivatives of ring D at a late stage in the route.27 The synthetic approach taken
was a modified version of the published route (Scheme 1)28 selected because ring D is formed
late in the synthesis by a Fischer indole reaction, using a ketone as its source. Given the variety
of ketones available, this provides access to a number of different substituted D rings. For the
first example, 1ꢀ(benzyloxycarbonyl)ꢀ4ꢀpiperidinone (7) was reacted with hydrazine 6 to provide
8 in a 64% yield. The hydrazine 6 was obtained from isatoic anhydride (3) and glycine followed
by reduction of the diamide, selective acetylation of the diamine, reaction with sodium nitrite,
and reduction with titanium tetrachloride and magnesium. After formation of the indole, the
hydroxyl group was converted to a methyl ether with sodium hydride and methyl iodide. This
was followed by reduction of the double bond in 8 with sodium borohydride and trifluoroacetic
acid and then hydrogenolysis of the Cbz group to give the desired product, 9. The piperidinone
was selected so the amine could be used as a handle for the attachment of linkers. Additionally,
4ꢀ(methoxy) cyclohexanone was used in this sequence to provide 11 with an ether to serve as a
protected alcohol.
Reagents and conditions: (i) a. 6N NaOH, 100 ˚C, 12 h, b. glycine, 6N NaOH, H2O, reflux, 12 h, then Lꢀtartaic acid,
100 ˚C, 2 h, 86%; (ii) a. LAH, THF, 24 h, 93%; b. CbzCl, Et3N, CH2Cl2, rt, 18 h, 88%; (iii) a. NaNO2, 1,4ꢀdioxane, b.
1N HCl, rt, 24 h, 98%; c. TiCl4, Mg, CH2Cl2, Et2O, rt, 0.75 h, 97% crude product; (iv) ketone 8, acetic acid, reflux, 18 h,
64%; (v) a. NaH, CH3I, DMF, 0 ˚Cꢀrt 16h; b. NaBH4, TFA, 18 h, rt, 78%; c. 10% Pd/C, H2, EtOH, rt, 48 h, 88%; (vi) 4ꢀ
(methoxy)cyclohexanone, acetic acid, reflux, 16 h, 45%; (vii) a. NaBH4, TFA, 18 h, rt, 70%; b. 10% Pd/C, H2, EtOH, rt,
48 h 42%.
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The ability of the molecules to act as agonists to induce 5ꢀHT2CRꢀmediated intracellular calcium
(Cai++) release13, 29ꢀ31 was conducted in a U2OS cell line stably expressing the human 5ꢀHT2CR.
While the potency for the novel molecules was lower relative to 5ꢀHT or the parent WAY163909,
the derivatives maintained full efficacy (Table 1 and Supporting Information Figure 1).
However, since the potency of the first set of synthesized derivatives (9, 11, 24, 25 and 26) was
in the range of 1ꢀ10 ꢁM, significantly reduced relative to 5ꢀHT (23) and WAY163909 (1) (Table
1), the decision was made to examine other attachment points.
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An alternative location investigated for the attachment of a tether to the WAY163909 scaffold
was the secondary amine (site b, cpd 2). The necessary molecules were synthesized by the
route outlined in Scheme 1. The two versions examined (Figure 2; 12 and 13) both
demonstrated reduced potency (EC50 >10 µM) in the Cai++ assay (data not shown).
Reagents and conditions: (i) a. triphosgene, THF, rt, 18 h, 97%; b. glycine, 6N NaOH, H2O, reflux, 12 h, then Lꢀ
tartaric acid, reflux, 2 h, 77%; (ii) a. LAH, THF, reflux, 48 h, 98%; b. benzyl chloroformate, Et3N, CH2Cl2, rt, 18 h, 77%;
(iii) NaNO2, 1N HCl, H2O, 1,4ꢀdioxane, rt, 15 min, 99%; (iv) TiCl4, Mg, CH2Cl2, diethyl ether, rt, 15 min, 99% crude
product; (v) cyclopentanone, pꢀTSA, toluene, reflux, 3 h, 48% over steps iv and v; (vi) a. NaBH4, TFA, rt, 10 min,
99%; b. 10% Pd/C, H2, MeOH, rt, 3 h, 99%. Steps for 14 are the same as 15. Yields and details are reported in the
supporting information.
We reasoned that a methoxy group would be a good representation of the polyether type
connection ultimately desired to link this molecule to others. Thus, versions with a methoxy
group at locations c and d were synthesized (Scheme 2). The two derivatives were synthesized
using two different starting materials, 2ꢀaminoꢀ4ꢀmethoxy benzoic acid (16) or 2ꢀaminoꢀ5ꢀ
methoxyꢀbenzoic acid (17). Reaction of 16 with triphosgene provided the benzoxazanone, which
was reacted with glycine to form the bisamide (18). Exhaustive reduction of both amides then
provided the diamine, which was selectively protected as the Cbz carbamate (19). Formation of
the nitroso compound (20) followed by reduction with titanium tetrachloride and magnesium
metal provided the hydrazine (21) necessary for the Fischer indole synthesis step. Reduction of
the double bond (22) with sodium borohydride in the presence of trifluoroacetic acid and
hydrogenolysis of Cbz carbamate afforded 15. Compound 14 was obtained by the same route
with comparable yields.
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Table 1. Effects of WAY163909 derivatives on intracellular calcium release in h5ꢀHT2CRꢀU2OS
cells
b,c
b,c
Cpd #
Structure
EC50 (nM)a
(95% C.I.)
Emax
Cpd #
Structure
EC50 (nM)a
(95% C.I.)
Emax
7
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9
23
0.44
(0.1ꢀ0.8)
100
26 (±)
>1 ꢁM
ND
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122.6
(92ꢀ153)
95.3 ± 2.3
101 ± 7.3
1 (±)
9 (±)
18.4
(16ꢀ21)
100
ND
31 (±)
32
1.3
(0.5ꢀ2.0)
>10 ꢁM
>1 ꢁM
72.9
(34ꢀ112)
94.9 ± 6.4
11 (±)
ND
33
1487
(810ꢀ2163)
97.6 ± 2.7
53.4
(26ꢀ80)
96.5 ± 2.2
96.8 ± 5.2
94 ± 2.5
14 (±)
15 (±)
40 (±)
41 (±)
42 (±)
2.2
(1.3ꢀ3.1)
103 ± 3.8
236.7
(37ꢀ437)
647.3
(271ꢀ1024)
24 (±)
25 (±)
>1 ꢁM
ND
ND
>10 ꢁM
a EC50 is presented as the mean; 95% confidence interval (CI) is presented as the range
Emax is presented as mean ± SEM; % Cai++ release of compounds (except 5ꢀHT) normalized to 1 ꢁM of
b
WAY163909 (1)
c ND = not determined
The product from 2ꢀaminoꢀ5ꢀmethoxyꢀbenzoic acid, (14) proved to be less potent (EC50 ~1.5
ꢁM) relative to WAY163909 (EC50~18.4 nM), but exhibited efficacy (Emax ~97%) similar to that
observed at 1 ꢁM of WAY163909 (Supporting Information Figure 2). The product with the
methoxy group in the 4ꢀposition (15) demonstrated an increase in potency (EC50 ~2.2 nM)
relative to WAY163909 with no change in efficacy (Emax ~103%; Supporting Information,
Figure 2). Thus, this small change in moving the methoxy group over one carbon (14 versus
15) resulted in a significant change in the potency, but not the efficacy, of the derivative.
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Table 2. Deprotection of the methyl ether
Equivalents
Equivalents of EtSH Time (hr) 29:30 Isolated Yield
of AlCl3
4 added 2X
24 X 2
48
24
18
18
18
1:1.3
NA
1:25
1:120
1:16
36%
No reaction
95%
1.5 added 2X
20 X 2
4*
4
6
4
2
6
97%
87%
Starting material and AlCl3 where premixed for 1 hour at 0 ˚C
The next step in the process was the deprotection of the methoxy group to provide a phenol,
which could then be connected to a PEG linker. Initial attempts to remove the methyl group
resulted in loss of the Cbz group. A wide variety of reactions were tested but either no reaction,
or decomposition with no significant identifiable products were observed. Details of the various
reaction conditions are presented in the Supporting Information. Consequently, the Cbz group
was exchanged for an acetate. Through careful control of the reaction conditions, the methoxy
group could be cleaved with aluminum chloride and ethanethiol in methylene chloride. Initially,
the free phenol product (30) was obtained as a mixture with the ethylthioether (29). After
optimization of the reaction conditions, the desired product (30) was obtained in up to 97%
isolated yield (Table 2).
Given the previous demonstration that WAY163909 exhibits differential pharmacological
properties, the two enantiomers of compound 15 were resolved.25 The originally reported
resolution of WAY163909 was performed by synthesizing the diastereomeric salt with
dibenzoylꢀ
desired separation. A variety of chiral acids were tested with a number of different solvents.
Success was ultimately achieved with diꢀpꢀtoluoylꢀ ꢀtartaric acid in isopropanol. The absolute
Lꢀtartaric acid. However, in the case of molecule 15, this method failed to provide the
L
stereochemistry was determined by Xꢀray diffraction of both enantiomers after separation. The
potencies of the two enantiomers of compound 15 (32 and 33) are considerably different
(EC50~1.3 nM and ~72.9 nM, respectively), but both retained full efficacy (Emax~100% and 95%,
respectively) (Table 1; Figure 3). The two enantiomers (32 and 33) were submitted to CEREP
(www.crep.fr) to determine receptor selectivity to displace binding to the 5ꢀHT2AR, 5ꢀHT2BR and
5ꢀHT2CR. Compounds were tested at a single concentration of 10 ꢁM to determine their ability to
displace [125I]ꢀ(±)ꢀ2,5ꢀdimethoxyꢀ4ꢀiodoamphetamine ([125I]ꢀ(±)ꢀDOI) binding according to their
standard assay protocols. The (R,R)ꢀenantiomer (32) displaced ~85ꢀ100% of specific binding at
5ꢀHT2AR, 5ꢀHT2BR and 5ꢀHT2CR, indicating a lack of selectivity across these three receptors
(Table 3). However, the (S,S)ꢀenantiomer (33) was selective for displacing ~91% of [125I]ꢀ(±)ꢀ
DOI binding to the 5ꢀHT2CR, with ~5ꢀ20% displacement of binding to the 5ꢀHT2AR or 5ꢀHT2BR
(Table 3).
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Table 3. Selectivity profile of WAY163909 derivatives at 5ꢀHT2R
Cpd #
Structure
5ꢀHT2A
R
5ꢀHT2B
R
5ꢀHT2C
R
32
86.5
98.6
100.2
33
22.8
67.1
4.8
91.5
99.6
±
41 ( )
67.7
Compounds were submitted to CEREP to determine receptor selectivity at the 5ꢀHT2
Compounds were tested at a single concentration of 10 ꢁM in duplicate to determine their
ability to displace [125I] (+/ꢀ) DOI. Data are presented as mean % inhibition of control specific
binding for compound tested at each receptor subtype. Significant inhibition is considered
>50%; 25ꢀ50%inhibition is indicative of weak to moderate effects.
The next step in the process was the attachment of a molecule that can be used to link either
two WAY163909 derivatives together or attach another molecule. This was accomplished by
reaction of the free phenol (30) with the alkyl tosylates 34ꢀ36 in DMF with sodium hydride as
base (Scheme 4). In all three cases investigated, the alkylation of the phenol proceeded in
excellent to good yield. The length of the alkyne tether proportionately impacted the potency of
the WAY163909 derivatives with little to no effect on efficacy (40ꢀ42, Table 1; Figure 4). In
CEREP assays, 41(±) (10 ꢁM) displaced 100% of [125I]ꢀ(±)ꢀDOI binding to the 5ꢀHT2CR, and
~70% of binding to the 5ꢀHT2AR and 5ꢀHT2BR (Table 3). It should be noted that 41(±) is a
racemic mixture of both enantiomers. Given the observation that in the case of 32 and 33 one
enantiomer (33) is much more selective for the 5ꢀHT2CR versus the other (32), it is completely
reasonable that the racemic mixtures of any of these compounds will be less selective than the
pure enantiomer. In future work only the most selective enantiomer will be used.
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Through the synthesis of a number of analogs of WAY163909, we have identified a site on the
scaffold of WAY163909 for the attachment of a tether to create tool compounds to bind to other
molecules. Derivatives where site d (2, Figure 1) has a methoxyl group or PEG chain attached
retain nanomolar potency with little effect on efficacy. The identification that site d can be
modified without significant loss of activity will allow for the synthesis of molecules that bind to
the 5ꢀHT2CR and retain activity as full agonists. Versions where the PEG chain is terminated
with an alkyne will be valuable as tools to link WAY163909 derivatives to other biologically
active molecules, fluorescent tags, or affinity probes through the alkyne/azide click reaction. The
pharmacological characterization of these molecules are an active research focus of our group
and future biological studies will be reported as they are completed.
Materials and Methods
Cell lines and cell culture. The PathHunter® U2OS HTR2C βꢀArrestin cell line (5ꢀHT2CRꢀU2OS;
DiscoveRx) stably express the nonꢀedited (INI) human 5ꢀHT2CR isoform (h5ꢀHT2CR). The 5ꢀ
HT2CꢀINIRꢀU2OS cells were grown in Assay Complete™ U2OS Medium 31 (DiscoveRx) at 37°C,
5% CO2 and 85% relative humidity per manufacturer’s recommendations utilizing
AssayComplete™ Cell Detachment Reagent (DiscoveRx). Cells were passaged at 70ꢀ80%
confluence and all experiments were conducted using cells in log phase growth.
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Intracellular calcium assay. Intracellular calcium (Cai++) release was monitored using the FLIPR
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4
5
6
Calcium 4 Assay Kit (Molecular Devices) according to previously published protocols with minor
29ꢀ31
modifications.13,
Cells were plated at 5,000ꢀ7,000 cells/well in Assay Complete™ Cell
Plating Reagent 16 (DiscoveRx) in blackꢀsided, clear bottomed 96ꢀwell tissue culture plates and
allowed to adhere overnight. Medium was removed and replaced with 40 ꢁl Hank’s balanced
salt solution without calcium, magnesium and phenol red (HBSS; Corning) plus 40 ꢁl Calcium 4
dye solution in Buffer B supplemented with 2.5 mM probenecid (SigmaꢀAldrich) to inhibit
extracellular dye transport. Plates were incubated for 60 min at 37 °C followed by 30 min at
room temperature in the dark. Fluorescence (λex = 485 nm, λem = 525 nm) was measured using
a FlexStation3 (Molecular Devices). Baseline was established for 17 sec before addition of 20 ꢁl
vehicle (HBSS without calcium or magnesium) or 5x concentrated compound. Addition of 5ꢀHT,
WAY163909, or compound occurred at the 17ꢀsec timepoint and fluorescence was recorded
every 1.7 sec for 120 sec. Maximum peak height was determined using FlexStation software
(SoftMax Pro 5.4). After the final readings, cells were fixed in 2% paraformaldehyde overnight.
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Data analysis. Peak responses from each well were normalized to total cell mass as determined
with crystal violet staining.30 The Emax is defined as the maximum possible Cai++ response and
data are expressed as a percent of the Cai++ release (mean ± SEM) obtained with 1 ꢁM of
WAY163909. Potency of the compounds was determined using the EC50 (concentration of
compound required to achieve halfꢀmaximal response). The EC50 values were determined using
4ꢀparameter nonlinear regression analysis (GraphPad Prism Version 7.02) and calculated from
at least three independent experiments, each conducted in triplicate, and are presented as the
mean and the 95% confidence interval.32ꢀ33 An EC50 or Emax value was not calculated for ligands
that failed to reach a plateau [reported as not determined (ND) in Table 1].
CEREP binding assays. Compounds 32, 33 and 41 were submitted to CEREP to determine
receptor selectivity to displace binding to the 5ꢀHT2AR, 5ꢀHT2BR and 5ꢀHT2CR per their standard
assay
protocols.
(http://www.cerep.fr/cerep/users/pages/Downloads/Documents/Marketing/Pharmacology%20&
%20ADME/Assay%20lists/Binding%20assays_2013.pdf). Compounds were tested at a single
concentration of 10 ꢁM in duplicate to determine their ability to displace [125I] (+/ꢀ) DOI. Data
are presented as mean % inhibition of control specific binding for compound tested at each
receptor subtype. Significant inhibition is considered > 50%; 25ꢀ50% inhibition is indicative of
weak to moderate effects.
Chemistry. Details for the synthesis of the individual compounds are presented in the
Supplementary Information. The characterization data for the five most active compounds are
presented below.
7ꢀmethoxyꢀ2,3,4,7b,8,9,10,10aꢀoctahydroꢀ1Hꢀcyclopenta[b][1,4]diazepino[6,7,1ꢀhi]indole
(racemate of 32/33) 1H NMR (500 MHz, CDCl3) δ 6.80 (d, J = 8.1 Hz, 1H), 6.22 (d, J = 8.1 Hz,
1H), 3.98ꢀ3.90 (m, 2H), 3.84ꢀ3.76 (m, 4H), 3.68 (d, J = 15.2 Hz, 1H), 3.28ꢀ3.22 (m, 1H), 3.19ꢀ
3.15 (m, 1H), 2.85 (p, J = 11.3 Hz, 2H), 1.95ꢀ1.80 (m, 2H), 1.76ꢀ1.53 (m, 4H). 13C NMR (126
MHz, CDCl3) δ 154.7, 153.9, 127.7, 121.5, 119.4, 101.0, 73.4, 56.9, 55.1, 54.3, 51.2, 43.7, 34.2,
33.4, 24.6. HRMS (ESIꢀTOF) Calcd. for C15H20N2O [M+H]+: 245.1648; found: 245.1650.
7ꢀ(2ꢀ(propꢀ2ꢀynyloxy)ethoxy)ꢀ2,3,4,7b,8,9,10,10aꢀoctahydroꢀ1Hꢀ
1
cyclopenta[b][1,4]diazepino[6,7,1ꢀhi]indole (40): H NMR (500 MHz, CDCl3) δ 6.76 (d, J =
8.2 Hz, 1H), 6.19 (d, J = 8.2 Hz, 1H), 4.19 (d, J = 2.3 Hz, 2H), 4.11 (td, J = 4.8, 2.6 Hz, 2H),
3.94ꢀ3.89 (m, 2H), 3.83 (t, J = 5.0 Hz, 2H), 3.80 (dd, J = 9.0, 3.1 Hz, 1H), 3.74 (dd, J = 5.8, 3.6
Hz, 2H), 3.71ꢀ3.65 (m, 7H), 3.30ꢀ3.23 (m, 1H), 3.20ꢀ3.14 (m, 1H), 2.95 (br, 1H), 2.90ꢀ2.82 (m,
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2H), 2.42 (t, J = 2.3 Hz, 1H), 1.93ꢀ1.79 (m, 2H), 1.76ꢀ1.50 (m, 4H). 13C NMR (126 MHz, CDCl3)
δ 154.4, 153.9, 128.6, 122.1, 114.7, 102.7, 79.5, 74.5, 73.4, 70.8, 70.6, 70.4, 69.7, 69.0, 67.4,
58.3, 53.9, 52.4, 49.9, 43.8, 34.4, 33.3, 24.5. HRMS (ESIꢀTOF) Calcd. for C23H32N2O4 [M+H]+:
401.2435; found: 401.2440.
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7ꢀ(2ꢀ(2ꢀ(propꢀ2ꢀynyloxy)ethoxy)ethoxy)ꢀ2,3,4,7b,8,9,10,10aꢀoctahydroꢀ1Hꢀ
cyclopenta[b][1,4]diazepino[6,7,1ꢀhi]indole (41): H NMR (500 MHz, CDCl3) δ 6.80 (d, J =
1
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8.3 Hz, 1H), 6.22 (d, J = 8.2 Hz, 1H), 4.96 (br, NH), 4.21 (d, J = 2.3 Hz, 2H), 4.16ꢀ4.09 (m, 2H),
4.04 (d, J = 15.1 Hz, 1H), 3.92 (dd, J = 8.8, 5.1 Hz, 1H), 3.84 (t, J = 4.9 Hz, 2H), 3.81 (dd, J =
8.8, 3.2 Hz, 1H), 3.77ꢀ3.74 (m, 2H), 3.74ꢀ3.69 (m, 3H), 3.39 (dd, J = 13.2, 3.1 Hz, 1H), 3.20 (dd,
J = 12.6, 2.5 Hz, 1H), 3.03ꢀ2.88 (m, 2H), 2.43 (t, J = 2.3 Hz, 1H), 1.93ꢀ1.80 (m, 2H), 1.77ꢀ1.60
(m, 3H), 1.60ꢀ1.49 (m, 1H). 13C NMR (126 MHz, CDCl3) δ 154.3, 153.9, 128.4, 122.1, 115.7,
102.6, 79.6, 74.5, 73.5, 70.6, 69.8, 69.1, 67.5, 58.4, 54.5, 52.7, 50.0, 43.8, 34.4, 33.3, 24.5.
HRMS (ESIꢀTOF) Calcd. for C21H28N2O3 [M+H]+: 357.2173; found: 357.2178.
7ꢀ(2ꢀ(2ꢀ(2ꢀ(propꢀ2ꢀynyloxy)ethoxy)ethoxy)ethoxy)ꢀ2,3,4,7b,8,9,10,10aꢀoctahydroꢀ1Hꢀ
cyclopenta[b][1,4]diazepino[6,7,1ꢀhi]indole (42):1H NMR (500 MHz, CDCl3) δ 6.76 (d, J = 8.2
Hz, 1H), 6.19 (d, J = 8.2 Hz, 1H), 4.19 (d, J = 2.3 Hz, 2H), 4.11 (td, J = 4.8, 2.6 Hz, 2H), 3.94ꢀ
3.89 (m, 2H), 3.83 (t, J = 5.0 Hz, 2H), 3.80 (dd, J = 9.0, 3.1 Hz, 1H), 3.74 (dd, J = 5.8, 3.6 Hz,
2H), 3.71ꢀ3.65 (m, 7H), 3.30ꢀ3.23 (m, 1H), 3.20ꢀ3.14 (m, 1H), 2.95 (br, 1H), 2.90ꢀ2.82 (m, 2H),
2.42 (t, J = 2.3 Hz, 1H), 1.93ꢀ1.79 (m, 2H), 1.76ꢀ1.50 (m, 4H). 13C NMR (126 MHz, CDCl3) δ
154.4, 153.9, 128.6, 122.1, 114.7, 102.7, 79.5, 74.5, 73.4, 70.8, 70.6, 70.4, 69.7, 69.0, 67.4,
58.3, 53.9, 52.4, 49.9, 43.8, 34.4, 33.3, 24.5. HRMS (ESIꢀTOF) Calcd. for C23H32N2O4 [M+H]+:
401.2435; found: 401.2440.
Author Contributions
YꢀC.C. performed the chemical syntheses and analyses and drafted the manuscript; R.M.H.
performed the in vitro biological assays and analyses and drafted the manuscript; N.C.A.
conducted pharmacological analyses; N.C.A., K.A.C. and S.R.G. conceptualized the project,
oversaw experimental design/interpretation/analyses, and wrote/edited the manuscript.
Corresponding Author: Eꢀmail srgilbe2@central.uh.edu. Voice 832ꢀ842ꢀ8821
Funding Sources
This work was supported by NIDA grant P50 DA033935.
Conflicts of Interest
The remaining authors declare no competing financial interests.
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