Discovery of a Highly Selective Sigma-2 Receptor Ligand, 1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-one (CM398), with Drug-Like Properties and Antinociceptive Effects In Vivo

Article abstract of DOI:10.1208/s12248-020-00472-x

The sigma-2 receptor has been cloned and identified as Tmem97, which is a transmembrane protein involved in intracellular Ca²⁺ regulation and cholesterol homeostasis. Since its discovery, the sigma-2 receptor has been an extremely controversial target, and many efforts have been made to elucidate the functional role of this receptor during physiological and pathological conditions. Recently, this receptor has been proposed as a potential target to treat neuropathic pain due to the ability of sigma-2 receptor agonists to relieve mechanical hyperalgesia in mice model of chronic pain. In the present work, we developed a highly selective sigma-2 receptor ligand (sigma-1/sigma-2 selectivity ratio > 1000), 1-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H- benzo[d]imidazol-2(3H)-one (CM398), with an encouraging in vitro and in vivo pharmacological profile in rodents. In particular, radioligand binding studies demonstrated that CM398 had preferential affinity for sigma-2 receptor compared with sigma-1 receptor and at least four other neurotransmitter receptors sites, including the norepinephrine transporter. Following oral administration, CM398 showed rapid absorption and peak plasma concentration (Cmax) occurred within 10?min of dosing. Moreover, the compound showed adequate, absolute oral bioavailability of 29.0%. Finally, CM398 showed promising anti-inflammatory analgesic effects in the formalin model of inflammatory pain in mice. The results collected in this study provide more evidence that selective sigma-2 receptor ligands can be useful tools in the development of novel pain therapeutics and altogether, these data suggest that CM398 is a suitable lead candidate for further evaluation.

Full text of DOI:10.1208/s12248-020-00472-x

The AAPS Journal
(2020) 22:94
DOI: 10.1208/s12248-020-00472-x
Research Article
Discovery of a Highly Selective Sigma-2 Receptor Ligand,
1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2
(1H)-yl)butyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-one (CM398),
with Drug-Like Properties and Antinociceptive Effects In Vivo
Sebastiano Intagliata,1 Abhisheak Sharma,2 Tamara I. King,2 Christophe Mesangeau,3 Michael Seminerio,4
Frederick T. Chin,5 Lisa L. Wilson,6 Rae R. Matsumoto,4,7 Jay P. McLaughlin,6
Bonnie A. Avery,2 and Christopher R. McCurdy1,3,8
Received 1 May 2020; accepted 16 June 2020
Abstract.
The sigma-2 receptor has been cloned and identified as Tmem97, which is a
transmembrane protein involved in intracellular Ca2+ regulation and cholesterol homeostasis.
Since its discovery, the sigma-2 receptor has been an extremely controversial target, and
many efforts have been made to elucidate the functional role of this receptor during
physiological and pathological conditions. Recently, this receptor has been proposed as a
potential target to treat neuropathic pain due to the ability of sigma-2 receptor agonists to
relieve mechanical hyperalgesia in mice model of chronic pain. In the present work, we
developed a highly selective sigma-2 receptor ligand (sigma-1/sigma-2 selectivity ratio >
1000), 1-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H-
benzo[d]imidazol-2(3H)-one (CM398), with an encouraging in vitro and in vivo pharmaco-
logical profile in rodents. In particular, radioligand binding studies demonstrated that CM398
had preferential affinity for sigma-2 receptor compared with sigma-1 receptor and at least
four other neurotransmitter receptors sites, including the norepinephrine transporter.
Following oral administration, CM398 showed rapid absorption and peak plasma concentra-
tion (Cmax) occurred within 10 min of dosing. Moreover, the compound showed adequate,
absolute oral bioavailability of 29.0%. Finally, CM398 showed promising anti-inflammatory
analgesic effects in the formalin model of inflammatory pain in mice. The results collected in
this study provide more evidence that selective sigma-2 receptor ligands can be useful tools in
the development of novel pain therapeutics and altogether, these data suggest that CM398 is
a suitable lead candidate for further evaluation.
KEY WORDS: sigma receptors; sigma-2 receptor; neuropathic pain; formalin assay; pharmacokinetic.
INTRODUCTION
Neuropathic pain is a major clinical problem that results
1 Department of Medicinal Chemistry, College of Pharmacy, Univer-
sity of Florida, Gainesville, Florida 32610, USA.
in a drastic reduction to the quality of life in patients who are
affected with this chronic condition (1,2). It represents one of
the most frequent causes of adult disability and a consider-
able health-care cost which consists of medical expenses and
lost workdays. It has been estimated that approximately 20
million individuals in the USA suffer from some form of
peripheral neuropathy (3). The most common reasons of
developing peripheral neuropathy include physical injury
(trauma), chronic diseases (diabetic neuropathy), and expo-
sure to toxins (3). Despite the fact that neuropathic pain is a
common medical problem, there are only a few effective
treatment options, each with their own limitations, thus the
management of chronic pain is quite complicated and
challenging. First-line treatments include antidepressant
2 Department of Pharmaceutics, College of Pharmacy, University of
Florida, Gainesville, Florida 32610, USA.
3 Department of BioMolecular Sciences, School of Pharmacy, The
University of Mississippi, University, Mississippi 38677, USA.
4 Department of Basic Pharmaceutical Sciences, West Virginia
University, Morgantown, West Virginia 26506, USA.
5 Department of Radiology, Stanford University School of Medicine,
Stanford, California 94305, USA.
6 Department of Pharmacodynamics, College of Pharmacy, University
of Florida, Gainesville, Florida 32610, USA.
7 Present Address: Dean’s Office, Touro University California College
of Pharmacy, Vallejo, CA 94592, USA.
8 To whom correspondence should be addressed. (e–mail:
CMccurdy@cop.ufl.edu)
#
1550-7416/20/0000-0001/0
2020 American Association of Pharmaceutical Scientists

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agents such as tricyclic antidepressants (TCAs) and liver, and kidney) (9). Two different receptor subtypes were
serotonin-norepinephrine reuptake inhibitors (SNRIs) or reported and identified as sigma-1 and sigma-2. The sigma-1
anticonvulsant drugs such as calcium channel alpha-2-delta receptor was cloned over two decades ago, and the human
ligands (gabapentinoids) (4). Nonsteroidal anti-inflammatory crystal structure has been reported (10,11), whereas purifica-
drugs (NSAIDs) may be used for mild pain relief, whereas tion and cloning of the sigma-2 receptor have only recently
narcotic agents are generally prescribed for pain that does not been published in 2017 (12). The sigma-2 receptor has been
respond to the first-line treatments (4). Although most of the confirmed as Tmem97 (transmembrane protein 97) (12).
prescribed drugs are effective for alleviating pain symptoms, Several literature reports suggest a role for the sigma-1
they cause several side effects and possess notable liabilities receptor in pain modulation, and either selective or non-
that include sedation, diplopia, and dizziness in case of selective sigma-1 receptor ligands have shown antinociceptive
antiepileptic drugs (5), or tolerance, respiratory depression, effects in animal models (13–16). In particular, the sigma-1
dependence, and addiction in case of opioids (6). Therefore, receptor antagonist S1RA (E-52862) (Fig. 1) has completed
there is a strong need for new therapeutics with fewer side successfully phase I clinical trials studies and is currently
effects and increased effectiveness for pain management. To under phase II investigation (17,18). Another example of
this end, several new potential targets to treat neuropathic sigma-1 receptor antagonist is [18F]FTC-146 (Fig. 1) which
pain have been proposed, and novel agents have shown has entered into clinical trials as a positron emission
efficacy in preclinical models, and some have currently tomography and magnetic resonance imaging (PET/MRI)
reached investigation in clinical trials (7). Among these diagnostic agent to pinpoint peripheral nerve injury (19–22).
approaches, sigma receptor ligands emerged as promising Despite many years of research efforts focused on the
tools for alleviating chronic pain (8). Sigma receptors are validation of the sigma-1 subtype as an effective target to
transmembrane proteins present throughout the central treat neuropathic pain (23), the evaluation of the sigma-2
nervous system as well as in peripheral tissues (e.g., spleen, receptor potential as a therapeutic target for pain target has
Fig. 1. Chemical structure and affinity values of selective sigma-1 antagonists S1RA (E-52862) and [18F]FTC-146 which
are current under clinical studies; selective sigma-2 agonist UKH-1114 and non- selective sigma-1/sigma-2 ligand AZ66
which have showed antineuropathic pain effects in mice; selective sigma-2 benzimidazolone-based analog CM397

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also started to gain momentum. Most recently, the phenyl 2 as an orange oil (2.00 g, 93%). 1H NMR (400 MHz,
methanobenzazocine derivative (UKH-1114, Fig. 1) as sigma- DMSO-d6) δ 8.14 (d, J 0 5.5 Hz, 1H), 8.01 (dd, J 0 8.6,
2/Tmem97 agonist was reported to be able to produce 1.5 Hz, 1H), 7.49 (ddd, J 0 8.3, 6.9, 1.5 Hz, 1H), 6.91 (dd, J 0
antinociceptive effects when administered IT to spared nerve 8.8, 1.3 Hz, 1H), 6.62 (ddd, J 0 8.3, 7.0, 1.2 Hz, 1H), 2.91 (d,
injury (SNI) mice, suggesting antineuropathic pain effects of J 0 5.0 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 146.59,
sigma-2 ligands (24). Moreover, the non-selective sigma-1/ 137.19, 131.48, 126.72, 115.49, 114.80, 30.24. MS (ESI+) m/z
sigma-2 ligand AZ66 (Fig. 1) demonstrated the ability to 153 [M + H]+.
alleviate multiple modalities of chronic pain with reduced
liabilities compared to opioids (16).
N1-Methylbenzene-1,2-diamine (3). A solution of 2
Our research group has been working for many years in
the development of selective sigma receptor ligands, including
benzimidazolone-based selective sigma-2 receptor ligands
(25,26). Several of our compounds have been used as
effective chemical probes in the elucidation of the putative
roles of the sigma receptor subtypes in many diseases such as
cancer, drug addiction, and pain (27–31). More recently, our
efforts have been focused on the translational research of
many of our lead molecules into effective diagnostic or
pharmacotherapies through a multi-disciplinary and inte-
grated approach. Herein, we report the synthesis, in vitro/in
vivo pharmacology evaluation, and preclinical pharmacoki-
netic studies of CM398, a highly selective sigma-2 receptor
ligand (sigma-1/sigma-2 selectivity ratio > 1,000), that demon-
strates anti-inflammatory analgesic effects in mice as an initial
proof-of-concept.
(2.00 g, 13.14 mmol) in methanol (20 mL) was added 10%
palladium on carbon (0.10 g) in a portion wise manner. After
addition and stirring for an additional 2 h under hydroge-
nated (H2) atmosphere at room temperature, the reaction
mixture was then filtered through a pad of celite, and the
filtrate was concentrated in vacuo to afford 3 as brown
residue, which was used in the next step without further
purification. 1H NMR (400 MHz, DMSO-d6) δ 6.55 (t, J 0 7.3
Hz, 2H), 6.48–6.35 (m, 2H), 4.56 (d, J 0 5.9 Hz, 1H), 4.42 (s,
2H), 2.70 (d, J 0 3.6 Hz, 3H).13C NMR (101 MHz, DMSO-d6)
δ 137.85, 135.75, 118.40, 117.37, 114.46, 109.78, 30.91. MS
(ESI+) m/z 123 [M + H]+.
1-Methyl-1H-benzo[d]imidazol-2(3H)-one (4). A mix-
ture of 3 (2.00 g, 16.40 mmol), 1,1′-carbonyldiimidazole
(3.18 g, 19.60 mmol) in anhydrous tetrahydrofuran (25 mL)
was stirred at 65°C for 5 h. The mixture was poured onto
water and extracted with ethyl acetate (3 × 50 mL). The
extract was washed with a saturated aqueous solution of
sodium chloride (35 mL) and dried over anhydrous sodium
sulfate. The solvent was removed in vacuo, and the obtained
residue was crystallized from hexane-ethyl acetate (1:1) to
MATERIALS AND METHODS
Chemistry
Reagents and starting materials were obtained from
commercial suppliers and were used without purification.
Precoated silica gel GF Uniplates from Analtech were used
for thin-layer chromatography (TLC). Column chromatog-
raphy was performed on silica gel 60 (Sorbent Technolo-
1
afford 4 as white crystals (1.90 g, 79%). H NMR (400 MHz,
DMSO-d6) δ 10.82 (s, 1H), 7.06–6.93 (m, 4H), 3.25 (s, 3H).13C
NMR (101 MHz, DMSO-d6) δ 155.07, 131.58, 128.85, 121.47,
121.13, 109.23, 108.21, 27.03. MS (ESI+) m/z 149 [M + H]+.
1
gies). H and 13C NMR spectra were obtained on a Bruker
1-(4-Bromobutyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-
one (5). K2CO3 (0.56 g, 4.05 mmol) and 1,4-dibromobutane
(1.12 mL, 9.45 mmol) were added, under mechanical stirring,
to a solution of 4 (0.20 g, 1.35 mmol) in anhydrous DMF
(8 mL). The reaction mixture was heated at 60°C for 3 h.
After cooling, the mixture was poured into 100 mL of water,
extracted with ethyl acetate (3–40 mL), washed with a
saturated aqueous solution of sodium chloride, and dried
over anhydrous sodium sulfate. The solvent was removed in
vacuo, and the residue was purified by flash column
chromatography (petroleum ether/ethyl acetate 7:3) to afford
5 as a colorless oil (0.27 g, 70%). 1H NMR (400 MHz, CDCl3)
d 7.06–7.03 (m, 2H), 6.96–6.91 (m, 2H), 3.88–3.86 (m, 2H),
3.41–3.38 (m, 2H), 3.63 (s, 3H), 1.89–1.85 (m, 4H). 13C NMR
(101 MHz, CDCl3) d 154.41, 130.10, 129.17, 121.26, 121.23,
107.53, 107.50, 40.08, 33.13, 29.66, 27.15, 26.98. MS (ESI) m/z
305 [M + Na]+ (79Br) and 307 [M + Na]+ (81Br).
APX400 (400 and 100 MHz, respectively) in CDCl3 and
DMSO-d6 solution. Chemical shift (δ) values are given in
parts per million (ppm) using tetramethylsilane (TMS) and
DMSO or CHCl3 as the internal standard; coupling
constants (J values) are given in hertz (Hz). For signal
multiplicities, the following abbreviations are used as
follows: s (singlet), d (doublet), dd (doublets of doublet),
ddd (doublet of doublet of doublets), t (triplet), br s (broad
singlet), and m (multiplet). The mass spectra (MS) were
recorded on a WATERS ACQUITY Ultra Performance LC
with ZQ detector in ESI or APCI mode. The high
resolution mass spectra (HRMS) were recorded on a Waters
Micromass Q-TOF Micro mass spectrometer with a lock
spray source. Chemical names were generated using
ChemDraw Ultra (CambridgeSoft, version 10.0).
N-Methyl-2-nitroaniline (2). A solution of 1-fluoro-2-
nitro-benzene (2.00 g, 14.17 mmol) in water (5 mL) was
added 40% aqueous methylamine (5 mL) at room temper-
ature, and the reaction mixture stirred at room temperature
under nitrogen. After 2 h, the reaction mixture was poured
into a saturated aqueous solution of sodium chloride
(50 mL) and extracted with ethyl acetate (3 × 50 mL). The
organic layer was dried over anhydrous sodium sulfate,
concentrated in vacuo, and the residue was purified by flash
column chromatography (hexane/ethyl acetate 9:1) to afford
1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)butyl)-3-methyl-1H- benzo[d]imidazol-2(3H)-one Hydro-
chloride (CM398). K2CO3 (0.08 g, 0.62 mmol) and 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride
(0.05 g, 0.21 mmol) were added, under mechanical stirring,
to a solution of 5 (0.05 g, 0.18 mmol) in anhydrous DMF
(3 mL). The reaction mixture was heated at 40°C for 1 h.

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After cooling, the mixture was poured into 20 mL of water, binding, and chemical instability of the compound in the
extracted with ethyl acetate (3–30 mL), washed with a reaction mixture. In a 96-well plate, 178 μL of buffer, 2 μL of
saturated aqueous solution of sodium chloride, and dried CM398/verapamil stock solution (100 μM), and 10 μL of liver
over anhydrous sodium sulfate. The solvent was removed in microsomes (protein 20 mg/mL) were added and equilibrated
vacuo, and the residue was purified by flash column for 5 min in a shaking water bath at 37°C. Total organic
chromatography (methylene chloride/methanol 95:5) to af- content in reaction mixture was 1%. The reaction was started
ford 6, which was converted into the hydrochloride salt by by adding 10 μL of NADPH (40 mM). For the negative
addition of HCl/dioxane and isolated as a white solid (0.026 g, control reaction, volume of NADPH was replaced by
34%). 1H NMR (400 MHz, DMSO-d6) d 11.26 (br s, 1H), phosphate buffer. An aliquot (20 μL) from the incubation
7.24–7.22 (m, 1H), 7.16–7.14 (m, 1H), 7.08–7.06 (m, 2H), 6.79 mixture was quenched at 0, 5, 10, 15, 30, and 45 min with
(s, 1H), 6.77 (s, 1H), 4.35–4.32 (m, 1H), 4.13–4.09 (m, 1H), 80 μL of ice cold acetonitrile containing internal standard (IS)
3.87 (t, J 0 7.2 Hz, 2H), 3.73–3.56 (m, 7H), 3.33–3.19 (m, 7H), to terminate the microsomal reaction. The samples were
2.89–2.85 (m, 1H), 1.83–1.71 (m, 4H). 13C NMR (101 MHz, centrifuged at 4°C for 15 min at 12,000 rpm, and supernatants
DMSO-d6) d 153.49, 148.16, 147.51, 129.57, 128.65, 123.23, were analyzed using UPLC/MS-MS for residual CM398
120.72, 119.77, 111.40, 109.65, 107.64, 107.60, 55.47, 55.41, content. The positive and negative controls were also
54.22, 50.99, 48.51, 39.65, 26.77, 25.11, 24.14, 20.49. HRMS processed similarly. The area under the curve (AUC) was
calcd for C23H30N3O3 [M + H]+ 396.2287, found 396.2268.
calculated for the analyte and IS in MassLynx, and the ratio
of the analyte to IS AUC was used to plot relative
concentration vs. time and calculate percent degradation of
CM398 in the reaction mixture. The elimination half-life
(T1/2) was calculated as Eq. 1.
Competition Binding Assays
T1=2 ¼ −0:693=k
ð1Þ
Radioligand binding studies at sigma and non-sigma
receptors were performed using competition binding assays
in homogenates of rat brain tissues and following previously
reported procedures (26,32). Specific information regarding
tissues, radioligands, and drugs used in the assays are
reported in Table I.
Where k is the slope of the line obtained by plotting natural
logarithmic of percentage of CM398 remaining in the reaction
mixture vs. incubation time. Intrinsic clearance (CLint) and
whole liver clearance (CLint,H) were calculated from the
following equations:
Metabolic Stability in Rat Liver Microsomes
CLint ¼ k  ðIncubation volumeÞ=ðMicrosomal proteinÞ ð2Þ
Phase-I metabolism stability of CM398 was studied in rat
liver microsomes. The incubation mixture consisted of liver
microsomal protein (1 mg/mL), CM398 (1 μM), phosphate
buffer (100 mM, pH 7.4), and reduced nicotinamide adenine
dinucleotide phosphate (NADPH, 2 mM). Verapamil (1 μM)
was used as a positive control to assess the metabolizing
capacity of rat liver microsomes. NADPH deficient micro-
somal reaction was performed as a negative control to reveal
the non-NADPH-dependent degradation, non-specific
CLint;H ¼ CLint  MPPGL  liver scaling factor
ð3Þ
where MPPGL represents the microsomal protein per gram of
Sprague Dawley rat liver (45 mg/g) (33), and liver scaling factor
(40 g/Kg) (34) represents the liver weight per body weight of
Sprague Dawley rats. In vivo hepatic clearance was extrapolated
through well-stirred model (Eq. 4) including microsomal protein
binding (fumic) (35). The fumic was derived using the equation
Table I. Binding affinities of CM398 for sigma and non-sigma receptors and specific conditions used for the competition binding assays
Target
Ki a(nM)
Tissue
Radioligand
Nonspecific binding
Sigma receptors
Sigma-1
Sigma-2
560 8.72
Rat brain
5 nM [3H](+)-pentazocine
10 μM
haloperidol
0.43 0.015 Rat brain
3 nM [3H]di-o-tolylguanidine 10 μM
haloperidol
50 μM cocaine
Monoamine transporters
Dopamine
Serotonin
Norepinephrine
32.90 1.9 Rat striatum
244.2 2.4 Rat brainstem
0.5 nM [3H]WIN 35,428
0.2 nM [3H]paroxetine
1.5 μM imipramine
4 μM desipramine
1 μM haloperidol
1 μM mianserin
10 μM cyclazocine
1 μM naloxone
> 1000
> 1000
Rat cerebral cortex 0.5 nM [3H]nisoxetine
Other neurotransmitter receptors Dopamine (D2)
Rat brain
Rat brain
Rat brain
Rat brain
5 nM [3H](−)sulpiride
2 nM [3H]ketanserin
5 nM [3H]TCP
Serotonin (5-HT2) > 1000
NMDA
Opioid
> 10,000
>1000
1 nM [3H]naloxone
a Affinities (Ki values in nanomolar) were determined in brain tissue homogenates
The values represent S.E.M. from replicate assays. Values of > 10,000 represent less than 30% displacement of the radioligand at that
concentration.

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(Eq. 5) of Hallifex and Houstan (36). Hepatic extraction ratio was metabolic cages with mesh floor and receptacles for urine
also determined using Eq. 6.
and feces. Rodent feed was removed from the metabolic
cages 12–18 h prior to oral (P.O.) dosing, and feed was again
provided 4 h post-dose. Animals in the (I.V.) administration
study were allowed constant access to standard feed. All
animals always had access to water ad libitum. Solution
formulations of CM398 were prepared in water (5 mg/mL)
and normal saline (1 mg/mL) for P.O. and I.V. PK studies,
respectively. The formulation for the I.V. study was filtered
through 0.2 μm syringe filter. Both formulations were
analyzed for CM398 content. The I.V. solutions were
administered via caudal vein. The P.O. solution was admin-
istered via oral gavage. Blood samples were collected using
the indwelling cannula. An initial blood volume of 0.05 mL
was withdrawn to clear the line of heparinized saline. Using a
fresh syringe, 0.15 mL of blood was withdrawn and placed in
heparin coated micro-centrifuge tube. The cannula was then
flushed with 0.20 ml of heparinized saline. For the P.O. study,
blood samples were taken pre-dose and at 0.17, 0.33, 0.5, 0.75,
1, 2, 4, 6, 8, 12, and 24 h post-dose. For the I.V. study, blood
samples were taken pre- dose and at 0.083, 0.17, 0.33, 0.5, 1, 2,
4, 6, 8, 12, and 24 h post-dose. Rat plasma was separated by
centrifugation of blood at 4000g for 10 min at 4°C and stored
at − 80°C refrigerator until analysis. Plasma samples were
processed and analyzed using an UPLC-MS/MS method as
described in supporting information. Plasma concentration-
time data and PK parameters are represented as mean
standard error of the mean (SEM). Peak plasma concentra-
tion (Cmax) and time to reach Cmax (Tmax) were obtained
directly from the concentration-time data plot. Concentration
vs. time data was subjected to noncompartmental analysis
using Phoenix WinNonlin (version 6.3; Certara Inc, Missouri,
USA). The area under plasma concentration-time curve
(AUC) of CM398 was calculated using the linear trapezoidal
method. Clearance (CL) was calculated as a ratio of dose and
AUC. Absolute oral bioavailability (%F) was calculated
using Eq. 9.
Hepatic Clearance ðCLHÞ
¼ ðQ  fu  CLint=fumicÞ=ðQ þ ðfu  CLint=fumicÞÞ
ð4Þ
^
0:072ÂðlogP=DÞ 2þ0:067ÂlogP=D−1:126
ð
Þ
fumic ¼ 1=1 þ C Â 10
ð5Þ
ð6Þ
Hepatic extraction ratio ¼ CLH=Q
where fu (6.2%) is the unbound fraction of CM398 in rat
plasma, logP/D (3.1) is the partition coefficient, C is the
microsomal protein concentration, and Q is the hepatic blood
flow in Sprague Dawley rats (4.8 L/h/kg) (33).
Plasma Protein Binding Studies
The protein binding of CM398 was evaluated in vitro
using an ultra-filtration method at concentrations of 1 and
10 μM. The desired concentrations (1 and 10 μM) were
obtained by spiking the required volume of stock solutions in
rat plasma. Spiked organic content was ≤ 0.5% v/v. The
spiked plasma samples were placed in Centrifree® devices
(YM-30, Millipore, Bedford, USA) and incubated at 37°C for
30 min. The samples were then centrifuged for 10 min at
1000 g, and ultrafiltrates were collected. Spiked plasma and
plasma filtrate samples were processed and analyzed using
the UPLC-MS/MS method as described in supporting infor-
mation. Nonspecific binding was also determined and incor-
porated in the calculation of plasma protein binding values.
The fraction unbound (fu) and plasma protein binding were
calculated as described in Eqs. 7 and 8, respectively (37).
Â
Ã
fu ¼ ½Cf=ð1−NSBÞ=CiŠ
ð7Þ
ð8Þ
%F ¼ Doseintravenous  AUCðlastÞoral=DoseðoralÞ Â AUC
 100
ð9Þ
ðlastÞintravenous
Protein binding ¼ ð1−fuÞ Â 100
Where Ci is the initial concentration, Cf is the concentration
in filtrate or free compound, NSB is the nonspecific binding
fraction, and fu is the percent unbound drug concentration.
In Vivo Characterization in a Model of Inflammatory Pain
and Statistical Analysis
Thirty-five 10 weeks old male CD-1 mice obtained from
the Charles River Laboratories, Wilmington, Massachusetts,
USA ,were used in the formalin assay. Mice were housed five
Pharmacokinetic Studies in Rats
Oral (20 mg/kg) and intravenous (1 mg/kg) pharmacoki- to a cage in a temperature and humidity-controlled room at
netic (PK) studies of CM398 were evaluated in male Sprague the University of Florida vivarium on a 12:12-h light/dark
Dawley rats (N05, each). Right jugular vein cannulated rats cycle (lights off at 19:00 h) with free access to food and water
(225 25 g) were purchased from Envigo (Indianapolis, except during experimental sessions. All procedures were
USA). Prior to the PK study, the animals were quarantined pre-approved and carried out in accordance with the UF
in the University of Mississippi vivarium for 48 h with a 12-h Institutional Animal Care and Use Committee as specified by
dark/light cycle and allowed access to standard feed and the 2011 National Institutes of Health Guide for the Care and
water ad libitum. All animal experiments were performed in Use of Laboratory Animals. Animal studies are reported in
accordance with the University of Mississippi Institutional compliance with the ARRIVE guidelines (38,39). Sample
Animal Care and Use Committee (IACUC) pre-approved sizes (i.e., number of animals) were predetermined by power
protocol. Animals were housed in individual Nalgene analysis, and animals were assigned to groups randomly. Drug

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treatment experiments were conducted in a blinded fashion. closure using 1,1′-carbonyldiimidazole (CDI) in anhydrous
No animals were excluded from statistical analysis. tetrahydrofuran (THF) and heated to 65°C to obtain 1-
After seven mice each received a 10-min pre-treatment methyl-2-benzimidazolinone (4). Treatment of compound 4
with vehicle (saline, 0.9% as a control group), morphine with 1,4-dibromobutane in the presence of anhydrous
(23.2 μmol/kg, i.p. as a positive control), or CM398 (0.23, potassium carbonate in anhydrous dimethylformamide
2.32, or 23.2 μmol/kg, i.p.), formalin (10 μL of a 10% (DMF) gave 5. The bromo derivative (5) was then coupled
solution) was injected into the hind paw as described with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline in the
previously (40,41). Paw-licking duration (in s) during 5 min presence of potassium carbonate in DMF to afford the
intervals was recorded for 70 min. The summated response target compound (CM398). Spectral analysis and high-
during the final 60 min was analyzed, consistent with resolution mass spectra of CM398 were consistent with its
nociception attributed to Phase II inflammation (41,42).
All paw-licking data for formalin testing are reported as
summed paw licking as area under the curve (AUC) by each
animal across the 60-min measured response, SEM. Mor-
phine data vs. saline was analyzed by Student’s t test. CM398
data was analyzed by a one-way ANOVA (factor:dose) with
significant results between groups further analyzed with
Tukey’s multiple comparisons post hoc test. The data and
statistical analysis comply with the recommendations on
experimental design and analysis in pharmacology (43). All
data are presented as mean SEM, with a significance set at
P<0.05, denoted by the asterisk (*).
assigned structure.
Competition Binding Assays
The binding affinity of CM398 for sigma-1 and sigma-2
receptor subtypes is summarized in Table I. CM398 possess
subnanomolar affinities for the sigma-2 receptor and over
1000-fold selectivity for the sigma-1 receptor. Moreover,
binding affinities versus other off-target proteins, including
monoamine transporter and other neurotransmitter recep-
tors, were also reported (Table I). The receptor binding
profile of CM398 is indicative of its highly preference for the
sigma-2 compared with the sigma-1 and to the other non-
sigma proteins (selectivity ratio > 1000). Furthermore, CM398
resulted highly selective also for the norepinephrine trans-
porter receptor (Ki > 1,000 nM) and for the serotonin
RESULT
Chemistry
The straightforward synthetic scheme for CM398 is transporter receptor (500-fold); on the other hand, CM398
outlined in Fig. 2. Treatment of 1-fluoro-2-nitro-benzene (1) displayed significant affinity for the dopamine transporter (Ki
with 40% aqueous methylamine at room temperature gave 0 32.90 nM). However, because dopamine transmission is not
N-methyl-2-nitroaniline (2), which was subjected to hydro- strictly related to nociception but rather it may reinforce the
genation (H2) in presence of 10% palladium on activated noradrenergic effects to inhibit certain type of pain (46),
charcoal to afford N1-methylbenzene-1,2-diamine (3) CM398 represented a suitable probe for the scope of this
(25,44,45). The later intermediate was subjected to ring study.
Fig. 2. Synthesis of CM398. Reagents and conditions: a CH3NH2, H2O, 2 h, r.t. b 10% Pd/C, H2 (1 atm), MeOH, 2 h. c CDI,
THF, 65°C, 18 h. d 1,4-dibromobutane, K2CO3, DMF, 60°C, 2 h. e 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, K2CO3,
DMF, 60°C, 1 h

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Metabolic Stability in Rat Liver Microsomes
Pharmacokinetic Studies in Rats
The PK properties of CM398 were studied following the
It is important to understand the fate of a discovery
compound undergoing hepatic metabolism; therefore, meta- P.O. (20 mg/kg) and I.V. (1 mg/kg) administration of the
bolic stability of CM398 was performed in rat liver micro- compound to male Sprague Dawley rats. After dosing of
somes. In vitro metabolic stability method is able to CM398, close and continuous visual monitoring of the
determine the metabolic depletion of a compound by animals revealed that there was no severe acute toxicity
cytochrome P450 enzymes in a closed system without response, as none of the animals showed any signs of
considering the physiological factors like blood flow or drug behavioral or neurological toxicity during the entire study
binding within the matrix (35). The in vitro half-life of period. The plasma concentration-time profiles are presented
CM398, when incubated in rat liver microsomes, was found in Fig. 3. PK parameters were estimated using non-
to be 0.10 0.01 h. The CLint,H derived from in vitro half-life compartmental analysis with Phoenix WinNonlin and are
and scaling factors is 12.8 0.3 L/h/kg. Calculated value of presented in Table II. CM398 shows a very rapid absorption
fumic was 0.63, which also suggests that only 37% of the as Cmax (2052.8 252.8 ng/mL) occurred at 0.17 0.00 h
compound in liver will be available for metabolism by the (Tmax) post-dose. Oral absorption is so fast, that the
liver enzymes. In vitro CLint,H was also successfully extrap- absorption phase cannot be captured. Absolute oral bioavail-
olated to hepatic clearance and extraction ratio. Compound ability was calculated to be 29.0%, indicating adequate total
CM398 exhibited both high hepatic clearance (CLH, 4.6
exposure after the oral dose. Total body CL (2.1 0.1 L/h/kg)
0.0 L/h/Kg) and extraction ratio (0.96 0.01). The high of CM398 was lower than the total hepatic blood flow (4.8 L/
extraction ratio suggests that only approximately 4% of h/kg) (33) in rats, indicating negligible extra-hepatic elimina-
CM398 can escape unchanged after a single pass through tion of the compound. The volume of distribution (Vd, 5.3
the liver following its phase I metabolism.
0.9 L/kg) of CM398 was larger than the total blood volume of
rats (0.085 L/kg) (34), showing extra-vascular distribution of
the compound. After P.O. and I.V. dosing, the mean half- life
(T1/2) was found to be 1.9 0.2 and 1.7 0.3 h, respectively.
Plasma Protein Binding Studies
The reversible binding of a drug to plasma proteins is
an important determinant of its PK and pharmacodynamics
characteristics. The protein binding of CM398 was evalu-
ated in vitro using ultra-filtration method. The
concentration-dependent Fu of CM398 was studied in rat
plasma. At concentrations of 1 and 10 μM, the Fu values
were 6.7 1.1 and 5.6 0.1%, respectively. Average plasma
protein binding of CM398 was found to be 93.8 0.9%.
Non-specific binding of CM398 to Centrifree® devices was
less than 5.0%.
In Vivo Characterization
The analgesic effects in vivo of CM398 were tested using
the formalin model of inflammatory pain in mice. Mice (n 0 7)
were pretreated with vehicle (saline, 0.9%), morphine
(23.2 μmol/kg), or CM398 (0.23, 2.32, or 23.2 μmol/kg)
through the intraperitoneal (i.p.) route and 10 min later,
administered formalin (10 μL in the hind paw). Mice
pretreated with saline spent an average of 205.1 32.6 s
Fig. 3. Mean plasma concentration-time profile of CM398 in male Sprague Dawley rats
(N 0 5, each) after a single oral (20 mg/kg) and intravenous (1 mg/kg) administration. Bar
represents SEM

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Page 8 of 11
The AAPS Journal
(2020) 22:94
nociception (8,47). In particular, the high expression of
sigma-1 receptors in specific areas of the brain such as
dorsal spinal cord, thalamus, periaqueductal gray,
basolateral amygdala, and rostroventral medulla (48), as
well as in peripheral tissues (especially dorsal root ganglia
neurons) (49), clearly suggests its involvement in pain
modulation.
From a pharmacological point of view, inhibition of
sigma-1 receptor produces a decrease of nociception stimuli
through attenuated expression of pain behaviors in several
animal models, including mechanical hypersensitivity (cap-
saicin-induced test), inflammatory pain (formalin assay),
and neuropathic pain models (SNI in mice) (50). Moreover,
these findings were supported by previous studies which
used sigma-1 receptor KO mice (51–53).
In a more recent study, Sahn et al. have tested the effect
of IT injection of different sigma-1 and sigma-2 receptor
ligands in the mouse SNI model (24). As a result, both the
sigma-1 and the sigma-2/Tmem97 preferring ligands pro-
duced antinociceptive effects, which were significantly
different from vehicle, whereas the moderate selective
sigma-2 receptor agonist, siramesine, showed only a modest
inhibitory effect on mechanical hypersensitivity (24). These
results led to further investigations into the putative role of
sigma-2 receptors in the modulation of pain. In fact, unlike
the results obtained for sigma-1 receptor antagonists, which
are consistent with previously reported literature, those
obtained by using slightly preferring sigma-2 receptor
ligands did not completely clarify if a mixed sigma-1/
sigma-2 or a selective sigma-2 compound might be benefi-
cial to treat pain. Unfortunately, as is often the case, the
lack of very selective ligands for the protein target adds
complexity for elucidating their function. From this per-
spective, the main goal of this work was the preliminary
characterization of a highly selective sigma-2 receptor
ligand with drug-like properties, serving as a lead candidate
for further preclinical and clinical development.
Table II. Pharmacokinetic parameters of CM398 after oral dose and
intravenous administration in male Sprague Dawley ratsa
Parameters
Oral
Intravenous
Cmax (ng/mL)
Tmax (h)
AUC0 → t (ng h/L)
Vd (L/kg)
2052.8 252.8
0.17 0.00
2733.6 293.4
6.2 0.6
–
–
471.3 23.6
5.3 0.9
1.7 0.3
2.1 0.1
–
T1/2 (h)
CL (L/h/kg)
Bioavailability (%)
1.9 0.2
2.2 0.2
29.0
a Each value represents the average of five rats dosed oral (20 mg/kg)
and intravenous (1 mg/kg); values are mean SEM.
AUC area under the plasma concentration-time curve, CL clearance,
Cmax plasma peak concentration, Tmax time to Cmax, T1/2 elimination
half-life, Vd volume of distribution
licking the injected hindpaw. Mice pretreated with morphine
spent significantly less time licking their formalin-treated
hindpaw (205.1 32.6 s; p 0 0.0002, Student’s t test). Pretreat-
ment with CM398 dose dependently reduced the time spent
licking the hind paw (Fig. 4), demonstrating a significant
antinociceptive effect doses of 2.32 and 23.2 μmol/kg, i.p.
(F(3, 24) 0 7.66, p 0 0.0009; one-way ANOVA with Tukey’s
post hoc test).
DISCUSSION
Previous research and preclinical studies have eluci-
dated the role of the sigma-1 receptor in modulating
The radioligand binding assays revealed that CM398
displayed subnanomolar affinities (Ki 0 0.43 nM) for the
sigma-2 receptor and very low affinities (Ki 0 560 nM) for
the sigma-1 receptor, thus, to the best of our knowledge,
CM398 represents the most selective sigma-2 receptor ligand
reported to date with regard to sigma-1/sigma-2 selectivity
ratio (1000-fold) (54). The binding properties of CM398 were
consistent with those of a recently reported set of structurally
related benzimidazolone-based analogs (25). Structure-
affinity relationships (SARs) studies suggested that the
presence of both the benzimidazolone as a scaffold and the
6,7-disubstituted tetrahydroisoquinoline as a cyclic amine
fragment was optimal in term of sigma binding profile.
Specifically, replacement of the 1-(4-fluorophenyl)piperazine
(CM397, Fig.1) with the 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline moiety significantly increased the
affinity for the sigma-2 receptor (Ki 0 2.5 and 0.43 nM) and
the selectivity over the sigma-1 receptor subtype (179-fold
and > 1000 fold, respectively).
Fig. 4. Antinociceptive effects of CM398 in the formalin test. Values
represent summed time licking across 60 min testing period following
5% formalin injection into the mouse hindpaw. n 0 7 (each); *p 0 0.02
vs. vehicle response; one-way ANOVA with Tukey’s post hoc test for
CM398, or p 0 0.0002 vs. vehicle response; Student’s t test for
morphine
CM398 also possessed negligible affinities for D2, 5-HT2,
NMDA, opioid receptors, and norepinephrine transporters
(1000–10,000 nM). CM398 did demonstrate notable affinity for
dopamine (Ki 0 32.90 nM) and serotonin transporters (Ki 0
244.2 nM), but these are still 76-fold and > 500-fold over the

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94
Page 9 of 11
affinity for sigma-2 receptors. As mentioned above, the in vitro
pharmacologic profile of CM398 was well suited to unveil the
utility of sigma-2 ligands as potential therapeutic for pain
treatment, especially due to the lack of affinity with those
target proteins mainly involved in pain relief mechanisms, for
instance, opioid receptors, noradrenaline transporters, D2, and
NMDA receptors (55–59).
In regard to the pharmacokinetic evaluation, CM398
demonstrated adequate oral exposure, extravascular distribu-
tion, and negligible extra hepatic elimination in rats indicating
satisfactory pharmacokinetic properties to support its further
development as a pharmacological tool and potential orally
active drug candidate.
Finally, we decided to test the analgesic properties of
CM398 in a tonic pain stimuli induced by formalin injection
rather than allodynia elicited by chronic nerve injuries. The
formalin test is a standard animal model of inflammation-
induced nociception and frequently included in the battery of
behavioral pharmacology tests of pain (60). The 2.32 and 23.2
μmol/kg i.p. doses of CM398 in mice produced a significant
reduction of the time spent licking as compared with saline,
through attenuation of the localized inflammatory pain
produced after injection of formalin in the paw of mice.
These results suggest that targeting sigma-2 receptor with a
highly selective ligand, like CM398, may be effective in
alleviating inflammatory pain. Notably, this initial testing was
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This study was supported, in part, by grants from the
National Institutes of Health NIDA R01 DA023205 (CRM,
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