Synthesis and activity of functionalizable derivatives of the serotonin (5-HT) 5-HT2A receptor (5-HT2AR) antagonist M100907

Article abstract of DOI:10.1016/j.bmcl.2018.02.058

The approach of tethering together two known receptor ligands, to be used as molecular probes for the study of G protein-coupled receptor (GPCR) systems, has proven to be a valuable approach. Selective ligands that possess functionality that can be used to link to other ligands, are useful in the development of novel antagonists and agonists. Such molecules can also be attached to reporter molecules, such as fluorophores, for the study of GPCR dimerization and its role in signaling. The highly selective serotonin (5-HT) 5-HT_2A receptor (5-HT_2AR) antagonist M100907 (volinanserin) is of clinical interest in the treatment of neurological and mental health disorders. Here, we synthesized the most active (+)-M100907 enantiomer as well as a series of derivatives that possessed either an alkyne or an azide. The triazole resulting from the dipolar cycloaddition of these groups did not interfere with the ability of the bivalent ligand to act as an antagonist. Thus, we have synthesized a number of compounds which will prove useful in elucidating the role of the 5-HT_2AR in the central nervous system.

Full text of DOI:10.1016/j.bmcl.2018.02.058

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activity of functionalizable derivatives of the serotonin
(5-HT) 5-HT2A receptor (5-HT2AR) antagonist M100907
a
b
a
d
Scott R. Gilbertson a,b, , Ying-Chu Chen , Claudia A. Soto , Yaxing Yang , Kenner C. Rice ,
⇑
Kathryn A. Cunningham b,c, Noelle C. Anastasio b,c
a Department of Chemistry, University of Houston, Houston, TX, United States
b Center for Addiction Research, United States
c Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, United States
d Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, Bethesda, MD, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The approach of tethering together two known receptor ligands, to be used as molecular probes for the
study of G protein-coupled receptor (GPCR) systems, has proven to be a valuable approach. Selective
ligands that possess functionality that can be used to link to other ligands, are useful in the development
of novel antagonists and agonists. Such molecules can also be attached to reporter molecules, such as flu-
orophores, for the study of GPCR dimerization and its role in signaling. The highly selective serotonin (5-
HT) 5-HT2A receptor (5-HT2AR) antagonist M100907 (volinanserin) is of clinical interest in the treatment
of neurological and mental health disorders. Here, we synthesized the most active (+)-M100907 enan-
tiomer as well as a series of derivatives that possessed either an alkyne or an azide. The triazole resulting
from the dipolar cycloaddition of these groups did not interfere with the ability of the bivalent ligand to
act as an antagonist. Thus, we have synthesized a number of compounds which will prove useful in elu-
cidating the role of the 5-HT2AR in the central nervous system.
Received 3 February 2018
Accepted 28 February 2018
Available online xxxx
Keywords:
Serotonin
M100907
Volinanserin
Bivalent
Ó 2018 Published by Elsevier Ltd.
M100907 (1), also known as volinanserin, is a highly selective
5-HT2AR receptor antagonist developed initially by Sanofi-Aventis
for the treatment of schizophrenia1 and sleep disorders.2 Our group
reported that M100907 derivatives substituted at the methoxy
group of the catechol ring retain the 5-HT2AR antagonist activity
with either the ketone (2) or racemic hydroxyl group (3) at the
benzylic position.3,4 Reported here is the installation of a polyethy-
lene glycol (PEG) linker substituted at the methoxy group of
M100907 and the chiral resolution of the molecule to provide a
version of M100907 possessing an ether tether that is terminated
with an azide (4) or an alkyne (5). The azide on (4) will be used
for future connection to other molecules such as fluorophores,
affinity tags and other receptor ligands (Fig. 1).5,6
with TBDPSCl, imidazole and catalytic amount of DMAP at room
temperature for 24 h to generate compound 6 in 98% yield. The
silyl protected 6 was regioselectively ortho-lithiated by n-butyl
lithium with TMEDA for 2 h at room temperature. Then Weinreb
amide 7 was added at -70 °C and the mixture was stirred at room
temperature for 21 h to produce the ketone 8. Following reaction
with the lithiated 6, the Boc group of 8 was removed with TFA to
give 9. Sodium borohydride reduction then provided the racemic
alcohol 10 (Scheme 1).
Weinreb amide 7 was synthesized from isonipecotic acid
(Scheme 2). BOC protection of isonipecotic acid by reaction of di-
tertbutyldicarbonate, in a mixed solvent of 1,4-dioxane, acetoni-
trile and water in the presence of 1 N NaOH gave compound 12
which was then reacted with N,O-dimethylhydroxylamine
hydrochloride, and the coupling reagent HBTU, in the presence of
DIPEA to give the desired Weinreb amide 7 in ꢀ80% yield over
the two steps.
For the chiral resolution of a later synthetic intermediate by the
Rice group,7 methanol was used as the solvent to obtain the
resolved salt with (R)-mandelic acid. However, the solubility of
the diastereomeric salt formed from compound 10 and (R)-man-
delic acid was sufficiently high in methanol that the yield of recov-
ered material was low. Using a 1:1 ratio of acetonitrile:methanol
for the first recrystallization and 1:2 ratio for the second
The synthetic route to M100907 developed by Rice7 was uti-
lized, however, the chiral resolution was carried out at an earlier
stage to provide the possibility of introducing different sub-
stituents onto the piperidinyl group. The conditions for this resolu-
tion were different from previously reported.7
The route to M100907-azide 4, began with the protection of
commercially available guaiacol (Scheme 1). Guaiacol was reacted
⇑
Corresponding author at: Department of Chemistry, University of Houston,
Houston, TX, United States.
E-mail address: srgilbe2@central.uh.edu (S.R. Gilbertson).
https://doi.org/10.1016/j.bmcl.2018.02.058
0960-894X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Gilbertson S.R., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.058

2
S.R. Gilbertson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Scheme 2. Synthesis of compound 7.
Scheme 3. Chiral resolution of compound 10.
Fig. 1. Structures of M100907 (1) and derivatives (2–5) which retained 5-HT2A
R
antagonist properties.
recrystallization, provided one diastereomeric salt in 36% overall
yield as white crystals (Scheme 3). Aqueous workup of the
diastereomeric salt with ammonium hydroxide afforded the enan-
tiomer in a purity of >95% ee (Fig. 2).
The optical purity was evaluated with (R)-(ꢁ)-1,10- binaphthyl-
2,20-diyl hydrogen phosphate [(ꢁ)-BNP] as a 1H NMR shift reagent
(Fig. 2).8 The benzylic proton adjacent to the hydroxyl group gave a
doublet near 4.6 ppm that was resolved from all other aliphatic
signals. This peak was followed on adding (ꢁ)-BNP. When one
equivalent of (ꢁ)-BNP was added to the racemic 10 in CDCl3, the
benzylic signal separated into 2 doublets, with R-enantiomer at
4.4 ppm and the S-enantiomer at 4.5 ppm. Chemical shift changes
of this signal were linear relative to the concentration of amine and
(ꢁ)-BNP. Higher concentrations of (ꢁ)-BNP resulted in more signif-
icant proton shifting but with broadening of the proton signal.8
The resolved diastereomeric salt was partitioned between
ammonium hydroxide and dichloromethane to obtain a single
enantiomer amine (R)-10, which underwent N-alkylation with
(2-tosylethyl)-4-fluorobenzene 11 to generate compound 12.
Removal of the TBDPS group and reaction with linker 13 provided
azide-terminated M100907 (4) (Scheme 4).
The alkyne needed to form the homobivalent 15 was synthe-
sized from intermediate 12 through cleavage of the silyl group
and alkylation with the tosylated PEG-alkyne 14 (Scheme 5). The
bivalent was then synthesized by formation a 1,2,3-triazole ring
generated from the dipolar cycloaddition between the azide and
the alkyne.5 This reaction was carried out by adding copper sulfate
with sodium ascorbate in a mixture of 4 and 5 DMF and water at
room temperature to form the triazole homodimer 15.
Fig. 2. Determination of the optical purity of 10. The proton NMR shift of benzylic
proton of compound 10 after mixing with same weight of (ꢁ)-BNP in CDCl3.
Inhibition of 5-HT2AR-mediated signaling was determined by
measuring the reduction of 5-HT (1 mM) stimulated intracellular
calcium (Ca2+) release in CHO-K1 cells stably expressing the 5-
HT2AR.3,4 Serotonin induces a concentration-dependent increase
in Cai2+ release with an EC50 of 4.2 nM (pEC50 = 8.38 0.10)
and 1 mM of 5-HT exhibited maximal intracellular Cai2+ release
Scheme 1. Synthesis of the precursor 10 of M100907 derivative 4.
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S.R. Gilbertson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Scheme 4. Synthesis of the target molecule, M100907-azide 4 from the chiral resolved 10.
Scheme 5. Synthesis of M100908-alkyne 5 and M100907 homodimer 15 with the heterocyclic 1,2,3-triazole ring.
Fig. 3. Representative Cai2+ release in 5-HT2AR-CHO cells. [A] 5-HT evokes a concentration-dependent elevation of Cai2+ release (pEC50 = 8.38 0.10; EC50 = 4.2 nM) and 1 mM
of 5-HT induces maximal Cai2+ release. [B] M100907 derivatives induce a concentration-dependent inhibition of 1 mM of 5-HT. pIC50 and IC50 values are listed in Table 1.
(Fig. 3A). As expected, the active isomer (+)-M1009077 displayed
low nanomolar potency in inhibiting 5-HT (1 mM)-evoked Cai2+
release (IC50 = 4.8 nM; pIC50 = 8.32 0.40; Fig. 3B). None of the
compounds displayed activity in the absence of 5-HT (data not
shown) and all compounds retained sub-micromolar antagonist
activity as shown in Table 1. Compound 4 retained antagonist
activity suggesting that tethering diethylene-azido linker on the
catechol ring of M100907 does not disrupt antagonist properties.
Although compounds 5 and 15 displayed slightly lower potency
when compared to parent (+)-M100907, these compounds
retained comparable potency to a previously published series of
5-HT2AR bivalent ligands which contain 14 and 17 ethylene glycol
linkers.3,4 These results indicate that the 1,2,3-triazole ring formed
in the click reaction does not significantly affect 5-HT2AR antago-
nist activity.
Derivatives of (+)-M100907 that possess either an alkyne or an
azide have been synthesized. The most active enantiomer was
obtained by resolution of a relatively early intermediate in the syn-
thesis. The ability of these molecules to maintain 5-HT2AR antago-
nist properties as (+)-M100907 was demonstrated together with
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S.R. Gilbertson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 1
Effect of M100907 derivatives on Intracellular Calcium Release in 5-HT2AR-expressing cells.
Compound Number
Structure
pIC50 SEMa (IC50 nM)
1 (+)-M100907
8.32 0.33
(4.8 nM)
4
7.33 0.18
(46.7 nM)
5
7.25 0.13
(56.6 nM)
15
7.44 0.24
(36.5 nM)
16c
17c
7.53 0.18b
(29.5 nM)
7.40 0.28
(39.8 nM)
a
b
c
pIC50 is presented as mean SEM (n = 3–5 independent experiments), except for 16; IC50 values were calculated from averaged pIC50 values.
Compound 16 was run in two independent experiments; pIC50 reported SD.
Data previously published.3,4
the ability to use dipolar cycloaddition between the alkyne and
azide to link these molecules to form bivalent antagonists. The
1,2,3-triazole ring generated in the dipolar cycloaddition used to
link to the M100907 derivatives does not interfere with the ability
of the bivalent ligand to act as an antagonist. Thus, functional ver-
sions of M100907 have been synthesized with tethers possessing
azides or alkynes, allowing the application of these molecules in
the synthesis of dimeric ligands as well as conjugation of the 5-
HT2AR antagonist to reporter molecules, such as fluorophores, for
the study of GPCR dimerization and their role in signaling.
cals, Atlanta GA), 100 mg/ml hygromycin (Mediatech, Manassas
VA) and were passaged when they reached 80% confluence.
Intracellular calcium assay
Intracellular calcium (Cai2+) release was monitored using FLIPR
Calcium 4 Assay Kit (Molecular Devices) according to previously
published protocols with minor modifications.3,4,10–13 Cells were
plated in serum-replete medium at 16–20 K cells in 150 ml in
black-sided, clear bottom 96-well tissue culture plates. Cells were
fed 24 h later with serum-free medium and, following 3 h incuba-
tion, medium was removed and replaced with 40 ml of fresh serum-
free medium plus 40 ml of Calcium 4 dye solution supplemented
with 2.5 mM of water-soluble probenicid (Invitrogen, Carlsbad
CA) to inhibit extracellular transport of the dye. Plates were incu-
bated for 50 min at 37 °C followed by 60 min at room temperature
in the dark. Fluorescence (kex = 485 nm, kem = 525 nm) was mea-
sured with a FlexStation 3 (Molecular Devices). Baseline was estab-
lished for 17 s before addition of 20 ml of vehicle (HBSS without
calcium of magnesium) or 5ꢂ concentrated test compound and flu-
orescence was recorded every 1.7 s for a total of 60 s to detect any
intrinsic activity of the test compounds. Fifteen minutes after test
compound addition, a second 17 s baseline was recorded again
immediately before addition of 25 ml of 5 mM 5-HT (final concen-
tration = 1 mM). Fluorescence was subsequently measured for an
additional 60 s. Maximum peak height was determined by the
FlexStation software (SoftMax Pro 5.2) for each well. Concentra-
tion-response curves (10ꢁ10 to 10ꢁ5 M) were performed for each
Materials and methods
Cell lines and cell culture
A CHO-K1 cell line stably transfected with the 5-HT2AR (5-HT2A
-
R-CHO cells; FA4 line) was a generous gift of K. Berg and W. Clarke
(University of Texas Health Science Center at San Antonio). This
line expresses transfected human (h)5-HT2AR in the p198-DHFR-
Hygro vector containing a hygromycin resistance gene.9 Reverse
transcription of RNA followed by a quantitative real time PCR assay
confirmed that 5-HT2AR-CHO cells expressed 5-HT2AR mRNA (esti-
mated to be approximately 3–4% of the mRNA level of the house-
keeping gene cyclophilin), but not mRNA for other members of
the 5-HT2R family (i.e., 5-HT2BR or 5-HT2CR) (data not shown).
Levels of 5-HT2AR protein expression (200 fmol/mg protein) which
approximates physiological levels in brain, have been reported9
and immunoblot analysis in our hands confirmed moderate 5-
HT2AR protein expression (data not shown). Cells were grown at
compound. A full 5-HT concentration response curve (10ꢁ10
–
37 °C, 5% CO2 and 85% relative humidity in GlutaMax
a
-MEM
10ꢁ5 M) was performed in each experiment to establish assay
and cell performance.
(Invitrogen, Carlsbad CA), 5% fetal bovine serum (Atlanta Biologi-
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S.R. Gilbertson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Data analysis
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Funding sources
This work was supported by National Institute on Drug Abuse
Grants P50 DA0333935 (K.A.C., S.R.G., and N.C.A.), T32 DA07287
(C.A.S.), and K05 DA020087 (K.A.C.). A portion of this work was
supported by the NIH Intramural Research Programs of the
National Institute on Drug Abuse and the National Institute of Alco-
hol Abuse and Alcoholism (K.C.R.).
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and structure–activity relationships of tool compounds based on WAY163909,
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The authors declare no competing financial interests.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.02.058.
Please cite this article in press as: Gilbertson S.R., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.02.058