Structural Optimization of the Diarylurea PSNCBAM-1, an Allosteric Modulator of Cannabinoid Receptor 1

Article abstract of DOI:10.1016/j.curtheres.2019.100574

Background: Structure–activity relationship studies improve the pharmacological and pharmacokinetic properties of a lead compound such as PSNCBAM-1, an allosteric modulator of the cannabinoid receptor 1. Objectives: Here, several derivatives of PSNCBAM-1 were synthesized with the aim of reducing the number of rings within its structure and enhancing the solubility of the compounds. The derivatives studied contain substituents previously shown to enhance binding of agonists (ie, a cyano group and a pyrimidine ring), with a reduced number of rings compared with the parent compound, PSNCBAM-1. Methods: The synthesized compounds were tested for the enhancement of the binding of orthosteric cannabinoid receptor 1 agonist CP55,940 in the presence of varying concentrations of each test compound. Select compounds were also tested for their effects on cannabinoid receptor 1 inverse agonist SR141716A binding. The compounds were also subjected to computational analysis of drug-like properties and solubility. Results: Consistent with a positive allosteric modulator for orthosteric ligand binding, compounds LDK1317 (12a), LDK1320 (12b), LDK1321 (6a), LDK1323 (8a), and LDK1324 (6b) all enhanced the binding of agonist CP55,940 to some degree. Reduction in the number of rings did not abolish the activity. The new lead compounds LDK1317 (12a) and LDK1321 (6a) showed improved drug-like properties and enhanced solubility in silico. Conclusions: In contrast to PSNCBAM-1, the synthesized compounds are analogs with fewer rings. The compounds LDK1317 (12a) and LDK1321 (6a) contained only 2 or 3 rings, respectively, and showed the binding parameters (K_B = 110 nM, α = 2.3, and K_B = 85 nM, α = 5.9). Further, the computationally predicted drug-like properties and solubility suggest these compounds are acceptable new lead compounds for further development of cannabinoid receptor 1 allosteric modulators. (Curr Ther Res Clin Exp. 2020; 81:XXX–XXX)

Full text of DOI:10.1016/j.curtheres.2019.100574

Journal Pre-proof
Structural Optimization of the Diarylurea PSNCBAM-1, an Allosteric
Modulator of Cannabinoid Receptor 1
Rachel Dopart , Sri Sujana Immadi , Dai Lu , Debra A. Kendall
PII:
S0011-393X(19)30023-2
DOI:
Reference:
https://doi.org/10.1016/j.curtheres.2019.100574
CUTHRE 100574
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Current Therapeutic Research
Received date:
Accepted date:
25 June 2019
15 December 2019
Please cite this article as: Rachel Dopart , Sri Sujana Immadi , Dai Lu , Debra A. Kendall , Struc-
tural Optimization of the Diarylurea PSNCBAM-1, an Allosteric Modulator of Cannabinoid Receptor 1,
Current Therapeutic Research (2019), doi: https://doi.org/10.1016/j.curtheres.2019.100574
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Highlights


PSNCBAM-1 is an allosteric modulator of the cannabinoid receptor 1
Derivatives of PSNCBAM-1 were made, to reduce the
total rings in the structure


Several derivatives maintained allosteric activity, as shown by
binding experiments
Some calculated physicochemical properties for these derivatives are provided

Structural Optimization of the Diarylurea PSNCBAM-1, an Allosteric Modulator of
Cannabinoid Receptor 1
Rachel Dopart1, Sri Sujana Immadi2, Dai Lu2, and Debra A. Kendall1*
1Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road
Unit 3092 Storrs, Connecticut, 06269, USA
2Rangel College of Pharmacy, Health Science Center, Texas A&M University, 1010 W. Ave. B
Kingsville, Texas, 78363, USA
Author email addresses:
Rachel Dopart: rachel.dopart@uconn.edu
Sri Sujana Immadi: immadi@tamhsc.edu
Dai Lu: dlu@tamhsc.edu
Debra A. Kendall: debra.kendall@uconn.edu
*Corresponding author:
Debra A. Kendall, Department of Pharmaceutical Sciences, 69 N. Eagleville Road, Storrs, CT
06269-3092. Phone: (860) 486-1891; Email: debra.kendall@uconn.edu

Abstract
Background
Structure-activity relationship studies improve the pharmacological and pharmacokinetic
properties of a lead compound such as PSNCBAM-1, an allosteric modulator of the cannabinoid
receptor 1 (CB1).
Objectives
Here, several derivatives of PSNCBAM-1 were synthesized with the aim of reducing the number
of rings within its structure and enhancing the solubility of the compounds. The derivatives
studied contain substituents previously shown to enhance binding of agonists (i.e. a cyano group,
and a pyrimidine ring), with a reduced number of rings compared to the parent compound,
PSNCBAM-1.
Methods
The synthesized compounds were tested for the enhancement of the binding of orthosteric CB1
agonist CP55,940 in the presence of varying concentrations of each test compound. Select
compounds were also tested for their effects on CB1 inverse agonist SR141716A binding. The
compounds are also subjected to computational analysis of drug-like properties and solubility.
Results
Consistent with a positive allosteric modulator for orthosteric ligand binding, compounds
LDK1317 (12a), LDK1320 (12b), LDK1321 (6a), LDK1323 (8a), and LDK1324 (6b) all
enhanced the binding of agonist CP55,940 to some degree. Reduction in the number of rings did

not abolish the activity. The new lead compounds LDK1317 (12a) and LDK1321 (6a) showed
improved drug-like properties and enhanced solubility in silico.
Conclusions
In contrast to PSNCBAM-1, the synthesized compounds are analogs with fewer rings. The
compounds LDK1317 (12a) and LDK1321 (6a) contained only 2 or 3 rings, respectively, and
showed the binding parameters (KB = 110 nM,  = 2.3, and KB = 85 nM, = 5.9). Further, the
computationally predicted drug-like properties and solubility suggest these compounds are
acceptable new lead compounds for further development of CB1 allosteric modulators.
Keywords
allosteric modulator, cannabinoid, CB1 receptor, diarylurea

Introduction
The cannabinoid receptor 1 (CB1) is a rhodopsin-like G protein coupled receptor
(GPCR)1, and is the most abundant GPCR in the brain.2,3 Orthosteric ligands for CB1 include the
endogenous cannabinoids 2-arachidonyl glycerol and anandamide, as well as the
phytocannabinoid, 9-tetrahydrocannabinol, and synthetic compounds such as the agonist
CP55,940 and the inverse agonist SR141716A.4-10 CB1 is implicated in pathways involving pain,
hunger, emotional state, and neurodegenerative disease, thus making it an attractive therapeutic
target for maladies impacting these pathways.11-13
In addition to orthosteric compounds, which bind the site where the endogenous ligands
bind, several allosteric modulators for CB1 have been identified such as ORG2756914 and
PSNCBAM-1.15 An allosteric modulator binds to a site which is topographically distinct from
the orthosteric site.14,16 There are several advantages to targeting the allosteric site, such as
subtype selectivity, spatiotemporal control, pathway selectivity, and a ceiling effect which may
minimize overdose risk.17-20
PSNCBAM-1 is an allosteric modulator of CB1, first characterized by Horswill and
colleagues in 2007.15 It displayed properties characteristic of a compound which promotes an
active conformation of CB1 in that it enhanced binding of the CB1 agonist CP55,940, while
reducing the binding of the inverse agonist SR141716A.15 However, PSNCBAM-1 had non-
competitive, inhibitory effects in GTPγS and cAMP assays, and caused reduced food intake and
body weight in rats.15
Several SAR studies have been performed on derivatives of PSNCBAM-1.21-23 A finding
from these studies is that a non-cyclic substitution in the 2-pyrrolidinylpyridine position, such as
a dimethylamino, is favored.21 In addition, it was suggested that the electron-withdrawing cyano

group may play additional roles such as replacing the water molecule in the receptor-ligand
complex, which in turn improved the potency of the modulator compared to the original chloro
group of PSNCBAM-1.21, 22 In binding experiments of CB1 using agonist CP55,940 as the tracer,
the EC50 of the derivative of PSNCBAM-1 which was cyano-substituted was 55 nM, compared
to the EC50 of PSNCBAM-1, which was 167 nM,21 suggesting it is important for the cyano group
to maintain its position. By shifting the substituent to the meta position, the affinity for CB1
decreases.22 The NH group of the urea also appears to be essential for the compound to influence
CP55,940 binding.23 Khurana and colleagues22 replaced the pyridine ring of PSNCBAM-1 with
a pyrimidine ring. Two scaffolds were made where the nitrogens of the pyrimidine ring were in
different positions. For both of these newly synthesized PSNCBAM-1 derivatives, they
maintained the ability to positively modulate the binding of the orthosteric agonist CP55,940.
Although the compounds had a lowered binding affinity, they showed a greater degree of
positive cooperativity than the parent compound, indicated by a higher α value.22In the lead
selection and optimization for central nervous system (CNS) drug discovery, the preferred
number of rings within a structure is up to three.24 The lead compound PSNCBAM-1 possesses
four rings. In this study, several derivatives of PSNCBAM-1 with reduced numbers of rings (two
or three) were synthesized and tested for their ability to potentiate orthosteric agonist CP55,940
binding. All compounds in this series have a cyano group which replaces the chloro group of the
lead compound PSNCBAM-1, while some compounds in this series feature a pyrimidine ring as
opposed to the pyridine ring of the lead compound (see Figure 1). Our work in reducing the rings
of PSNCBAM-1 to optimize the scaffold met the endpoints of this study that aim at enhancing
the drug-like properties25 and improving aqueous solubility.

Materials and Methods
Compound Synthesis
The target compounds were synthesized according to the methods illustrated in Scheme 1-3.
Generally, the target compounds 6, 10 and 12 were prepared from coupling a commercially
available (i.e., 9 -) 1or synthesized arylamine (i.e. 4) with 4-Cyano isocyanate (5). To synthesize
the target compound 8, the diaryl urea 6 was further reacted with ethanolamine in heated
anhydrous N,N-dimethyl formaldehyde (DMF) in the presence of potassium carbonate.
Scheme 1: Synthesis Route for Pyridinyl and Pyrimidinyl Biphenyl Ureas 6 and 8a

Scheme 2: Synthesis Route for Diaryl Urea 10
Scheme 3: Synthesis Route for Diaryl Urea 12
General Procedure A for the synthesis of diarylurea compounds (6a-6b, 10a-10b, 12a-12b).
To the solution of 1.5 mmol of amine in anhydrous DCM (5 − 8 mL) was added the selected
isocyanate 5 (1.8 mmol, 1.2 equiv.) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min
and then at room temperature for 2 h-4 h. The reaction was monitored by TLC (30% acetone in
hexane or 50 − 70% ethyl acetate in hexane). After completion of the reaction, the suspension
was filtered. The filtered solid was further washed with dichloromethane (2 mL) and diethyl
ether (5 mL) successively and then dried in a vacuum oven to provide the desired compounds.

2-Chloro-4-(3-nitrophenyl)pyrimidine (3a). In a 3 neck round bottomed flask, argon gas was bubbled
through a mixture of 3-nitrophenylboronic acid (1, 2.68 g, 16.10 mmol), 2,4-dichloropyrimidine (2a, 2 g,
13.42 mmol), Na2CO3 (4.26 g, 40.26 mmol), dimethoxyethane (80 mL), and H2O (7 mL) for 20-25 min.
Then the palladium catalyst Pd(PPh3)4 (1.54 g, 1.34 mmol) was added, and the reaction mixture was
refluxed for 12 hours and monitored by TLC. Upon completion of the reaction, the mixture was allowed
to cool to room temperature and was filtered through a small Celite pad. The filtrate was washed with
water (2 X 20 mL), and the organic compound was extracted with ethyl acetate (3 × 30 mL). The
combined organic phase was washed with water, brine and dried over Na2SO4. Filtration and removal of
solvent in vacuo provided the crude product, which was purified by silica gel Combiflash
chromatography (0-70% dichloromethane in hexane) to afford the compound 3a (1.88 g, 59.8%) as a
white solid; mp 147−149 °C. 1H NMR (300 MHz, CDCl3): δ 8.95 (t, J= 1.8 Hz, 1H), 8.78 (d, J= 5.4 Hz,
1H), 8.52 (dt, J= 7.8, 0.9 Hz, 1H), 8.42 (ddd, J= 8.1, 2.1, 0.9 Hz, 1H), 7.74-7.79 (m, 2H). MS (ESI): m/z
= 236.015[M+H]+.
2-Chloro-6-(3-nitrophenyl)pyridine (3b). The compound 3b was synthesized from 3-
nitrophenylboronic acid (952 mg, 5.75 mmol), 2-bromo-6-chloropyridine 2b (850 mg, 4.42 mmol),
Na2CO3 (1.39 g, 13.26 mmol), Pd(PPh3)4 (508 mg, 0.44 mmol), dimethoxyethane (25 mL), and H2O (2.3
mL) according to the procedure described for compound 3a. The crude compound was purified by
Combiflash chromatography (0- 30 % ethyl acetate in hexane) to afford the compound 3b (540 mg,
52.2%) as white solid; mp 128- 129 °C. 1H NMR (300 MHz, CDCl3): δ 8.86 (t, J = 1.9 Hz, 1H, CH), 8.40
(dt, J = 7.8, 1.1 Hz, 1H, CH), 8.31 (ddd, J = 8.2, 2.1, 0.8 Hz, 1H, CH), 7.66-7.84 (m, 3H, CH), 7.38 (dd,
J = 7.5, 1.0 Hz, 1H, CH). MS (ESI): m/z = 235.020[M+H]+
3-(2-Chloropyrimidin-4-yl)aniline (4a). The 3-nitrophenyl)pyrimidine 3a (815mg, 3.46 mmol) in the
mixture of DCM and methanol (1:1) was added SnCl22H2O (4.68g, 20.76 mmol) and stirred at 0 oC for 1
h and then at room temperature. The reaction was monitored by TLC (30% ethyl acetate in hexane).
Upon completion of the reaction, it was cooled to room temperature and condensed in vacuo. It was then

treated with saturated NaHCO3 solution (60 mL) and the solid precipitated out. The solid was
filtered under vacuum and the filtrate was extracted with ethyl acetate (2 X 40 mL). The organic
layer was washed with water, brine and dried over anhydrous Na2SO4. Filtration and removal of
solvent provided the crude solid, which was purified using silica gel Combiflash chromatography (0-
1
30 % ethyl acetate in hexane) to provide 4a (348 mg, 48 %) as a light yellow solid; mp 101-105 °C; H
NMR (300 MHz, CDCl3): δ 8.63 (d, J = 6.0 Hz, 1H, CH), 7.63 (d, J = 5.3 Hz, 1H, CH), 7.50 (t, J = 3.0
Hz, 1H, CH), 7.40 (dt, J = 9.0, 3.0 Hz, 1H, CH), 7.31 (d, J = 9.0 Hz, 1H, CH), 6.86 (ddd, J = 7.8, 2.4, 0.9
Hz, 1H, CH), 3.9 (s, 2H, NH). MS (ESI): m/z = 206.041[M+H]+.
3-(6-Chloropyridin-2-yl)aniline (4b). The compound 3-nitrophenyl)pyridine 3b (100 mg, 0.43 mmol) in
the mixture of EtOAc and EtOH (1:1) was added with SnCl2.2H2O (298.3, 2.98 mmol) at room
temperature. The reaction mixture was then refluxed for 6-8 h and monitored by TLC (30% ethyl acetate
in hexane). Upon completion of the reaction, the reaction mixture was cooled to room temperature and
condensed in vacuo and treated with saturated NaHCO3 solution (60 mL) and filtered. The filtrate was
then extracted with ethyl acetate (2 x 40 mL). The combined organic layer was washed with water, brine
and dried over anhydrous Na2SO4. Filtration and removal of solvent provided the crude solid, which was
purified using silica gel Combiflash chromatography (0-30% ethyl acetate in hexane) to provide 4b (68
mg, 77 %) as a light yellow solid; mp 76–80 °C; 1H NMR (300 MHz, CDCl3): δ 7.62-7.73 (m, 2H, CH),
7.42 (t, J = 1.8 Hz, 1H, CH), 7.23-7.34 (m, 3H, CH), 6.77(dd, J = 7.8, 1.2 Hz, 1H, CH), 3.84 (brs, 2H,
NH). MS (ESI): m/z = 205.045[M+H]+.
1-(3-(2-Chloropyrimidin-4-yl)phenyl)-3-(4-cyanophenyl)urea (6a, LDK1321). The compound 6a was
synthesized from amine 4a (308.46 mg, 1.5 mmol), and the 4-cyanophenyl isocyanate 5 (259.43 mg, 1.8
mmol) in anhydrous DCM according to general procedure A. The crude compound was purified using
Combiflash chromatography (0-50% ethyl acetate in hexane) to provide product 6a (302 mg, 57%) as a
1
white solid; mp 241-243 °C. H NMR (300 MHz, DMSO-d6): δ 8.79 (d, J= 5.4 Hz, 1H), 8.73 (s, 1H),

8.63 (s, 1H), 8.41 (t, J = 1.9 Hz, 1H), 8.01(d, J = 5.4 Hz, 1H), 7.68-7.88 (m, 6H), 7.52 (t, J = 7.9 Hz,
1H). MS (ESI): m/z = 350.073[M+H]+.
1-(3-(6-Chloropyridin-2-yl)phenyl)-3-(4-cyanophenyl)urea (6b, LDK1324). The compound 6b was
synthesized from amine 4b (145 mg, 0.71 mmol), and 4-cyanophenyl isocyanate 5 (122.5 mg, 0.85 mmol)
in anhydrous DCM according to general procedure A. The crude compound was purified using flash
chromatography (0-50% ethyl acetate in hexane) to provide product 6b (180 mg, 72%) as a white solid;
1
mp 180-184 °C. H NMR (300 MHz, DMSO-d6): δ 9.20 (s, 1H, NH), 9.13 (s, 1H, NH), 8.19 (t, J = 1.8
Hz, 1H, CH), 7.96 (d, J = 3.3 Hz, 1H, CH), 7.95 (s, 1H, CH), 7.57 - 7.76 (m, 6H, CH), 7.45 (dd, J = 5.9,
2.4 Hz, 1H, CH), 7.42-7.47 (m, 1H, CH). MS (ESI): m/z = 349.078[M+H]+.
1-(4-Cyanophenyl)-3-(3-(2-((2-hydroxyethyl)amino)pyrimidin-4-yl)phenyl)urea (8a, LDK1323). The
solution of 6a (50mg, 0.14 mmol) in anhydrous DMF (2.5 mL) was added to ethanolamine 7 (17.71 mg,
0.29 mmol) and K2CO3 (40 mg, 0.29 mmol). The reaction mixture was stirred and heated at 110 °C for 8
hours. The reaction was monitored by TLC (10% methanol in dichloromethane). Upon completion
of the reaction, it was cooled to room temperature and quenched by adding 30 mL of water and
extracted with ethyl acetate (3 x 12mL). The organic layer was washed with water, brine and
dried over anhydrous Na2SO4. Filtration and removal of the solvent in vacuo provided the crude
compound. The crude compound was purified using Combiflash chromatography (0-10% MeOH in
1
dichloromethane) to provide the product 8a (37 mg, 70%) as a light yellow solid; mp 189-193 °C. H
NMR (300 MHz, DMSO-d6): δ 9.27 (s, 1H), 9.04 (s, 1H), 8.35 (d, J = 5.1 Hz, 1H), 8.18 (s, 1H), 7.76 -
7.64 (m, 6H), 7.43 (t, J = 6.0 Hz, 1H), 7.07 (d, J = 4.5 Hz, 1H), 4.71 (t, J= 5.4 Hz, 1H), 3.59 (brs, 2H),
3.42 (brs, 2H). MS (ESI): m/z = 375.149 [M+H]+ .
1-(4-Cyanophenyl)-3-(3-(6-((2-hydroxyethyl)amino)pyridin-2-yl)phenyl)urea (8b, LDK1325). The
compound 8b was synthesized from 6b (100 mg, 0.29 mmol) and ethanolamine 7 (35 mg, 0.58 mmol) in
anhydrous DMF (5 mL) in the presence of K2CO3 (80 mg, 0.58 mmol) according to the procedure for 8a.

The crude compound was purified by Combiflash chromatography (0-10% MeOH in DCM) to provide
product 8b (25 mg, 23%) as a light yellow solid; mp 225-230 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.46
(s, 1H, NH), 9.14 (s, 1H, NH), 8.04 (s, 1H, CH), 7.32-7.75 (m, 8H), 6.97 (d, J = 6.0 Hz, 1H), 6.56 (t, J =
5.1 Hz, 1H), 6.46 (d, J = 8.3 Hz, 1H), 4.76 (t, J = 5.2 Hz, 1H, OH), 3.59 (brs, 2H), 3.42 (brs, 2H). MS
(ESI): m/z = 374.154 [M+H]+.
1-(4-Cyanophenyl)-3-(1H-indol-6-yl)urea (10a, LDK1319). The compound 10a was synthesized from
amine 9a (50 mg, 0.38 mmol), and 4-cyanophenyl isocyanate 5 (65 mg, 0.45 mmol) in anhydrous
dichloromethane according to the general procedure A. The pure product was obtained as a white solid
1
(97 mg, 92%); mp 235-238 °C. H NMR (300 MHz, DMSO-d6): δ 10.97 (s, 1H), 9.73 (s, 1H), 8.74 (s,
1H), 7.62-7.78 (m, 5H), 7.42 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 6.86 (dd, J = 8.5, 1.7 Hz, 1H), 6.35 (s,
1H). MS (ESI): m/z = 277.101[M+H]+.
1-(4-Cyanophenyl)-3-(1H-indol-5-yl)urea (10b, LDK1318). The compound 10b was synthesized from
amine 9b (50 mg, 0.38 mmol), and 4-cyanophenyl isocyanate 5 (66 mg, 0.46 mmol) in anhydrous
dichloromethane according to the general procedure A. The pure product was obtained as a white solid
(96 mg, 91.4%); mp 238-240 °C. 1H NMR (300 MHz, DMSO-d6): δ10.99 (s, 1H, NH), 9.11 (s, 1H, NH),
8.60 (s, 1H, NH), 7.62-7.73 (m, 5H, CH), 7.30-7.33 (m, 2H, CH), 7.08 (dd, J = 8.7, 1.6 Hz, 1H, CH),
6.36 (s, 1H, CH). MS (ESI): m/z = 277.101[M+H]+.
N-(3-(3-(4-Cyanophenyl)ureido)phenyl)acetamide (12a, LDK1317). The biphenyl urea 12a was
synthesized from N-(3-aminophenyl)acetamide 11a (0.050 g, 0.33 mmol) and 4-isocyanatobenzonitrile 5
(0.056 g, 0.39 mmol) in 8 mL of – anhydrous dichloromethane according to the general procedure A. The
crude product was purified by trituration with diethyl ether to provide 12a (85 mg, 87.5%) as a yellow
solid; mp 240-243 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.94 (s, 1H), 9.10 (s, 1H), 8.90 (s, 1H), 7.77 (s,
1H), 7.72 (d, J = 8.55 Hz, 2H), 7.62(d, J = 8.55 Hz, 2H), 7.20-7.16 (m, 3H), 2.02 (s, 3H). MS (APCI):
m/z = 295.11 (M+H+).

1-(4-Cyanophenyl)-3-(4-(dimethylamino)phenyl)urea (12b, LDK1320). The biphenyl urea 12b was
synthesized from dimethyl aniline 11b (0.1 g, 0.73 mmol) and 4-isocyanatobenzonitrile 5 (0.115 g, 0.80
mmol) in 10 mL of dichloromethane according to the general procedure A. The crude product was
purified by multiple titrations with diethyl ether to provide 12b (85 mg, 87.5%) as a white solid; mp 219-
221 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.06 (s, 1H), 8.49 (s, 1H), 7.59-7.77 (m, 4H), 7.25 (d, J = 8.94
Hz, 2H), 6.70 (d, J = 9.03 Hz, 2H), 2.83 (S, 6H). MS (APCI): m/z = 281.13 (M+H+).
Receptor Expression and Membrane Preparation
Human embryonic kidney 293T (HEK293T) cells were seeded at 1,000,000 cells/ 100-mm plate,
and grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, and 3.5
mg/mL glucose. They were incubated at 37 ˚C with 5% carbon dioxide. The next day, cells were
transfected with 20 µg of human CB1 receptor cloned into pcDNA3.1, using the calcium
phosphate method.26 Transfected cells were harvested, and the membranes prepared as
previously described,27 21 hours after transfection.
Equilibrium Binding Assays
CB1-expressing membrane preparations (5 µg) were incubated with nine concentrations of the
allosteric modulator (1 nM-10 µM). In each reaction, [3H]CP55,940 (150.2 Ci/mmol; Perkin
Elmer), a radiolabeled tracer which is an orthosteric agonist of CB1, was also added at 0.5 nM.
Or, for select allosteric compounds (LDK1321, LDK1323, or LDK 1324), [3H]SR141716A (56
Ci/mmol; Perkin Elmer), a radiolabeled tracer which is an orthosteric inverse agonist of CB1,
was added at a concentration of 1 nM instead of the orthosteric agonist. Nonspecific binding was
determined by treatment of the membranes with 10 µM of either untritiated CP55,940 (Tocris) or
untritiated SR141716A (Tocris). Membranes were incubated for 60 minutes, at 30oC. The
reaction was terminated by the addition of 300 µL of TME (Tris-Mg2+-EDTA) buffer with 5%
bovine serum albumin (BSA). Harvesting of the mixture was performed with a Brandel cell

harvester with Whatman GF/C filter paper. Measurement of bound radioactivity was performed
using liquid scintillation counting.
Data Analysis
The binding data collected were subjected to nonlinear regression, fitted to a log (dose) versus
response curve to determine the EC50 using Prism 7.02 (Graphpad Software, LaJolla, CA).
Binding analysis was performed in the graphs as the mean ± S.E. (error bars) and summarized in
Table 1 as the corresponding 95% confidence limits. The physicochemical properties including
solubility were obtained from computational prediction using the ChemAxon program
(Chemicalize, SanDiego, CA).

Results and Discussion
The compounds reported in this study (Figure 1) were generated to explore whether
reducing the number of rings within the scaffold of PSNCBAM-1 was possible and to identify
lead compounds with improved drug-like properties. Eight analogs of PSNCBAM-1 and the
parent compound were tested using equilibrium binding for their ability to enhance binding of
the CB1 agonist CP55,940. For comparison, PSNCBAM-1 was tested and consistent with the
literature value,22 KB= 55 nM. The cooperativity factor,  = 3.6, indicates positive cooperativity
with agonist CP55,940 (Figure 2A, Table 1).
All eight analogs also featured a cyano group which was previously shown to enhance the
allosteric properties of PSNCBAM-1-derived compounds.22 Of these derivatives, three
compounds displayed no binding: LDK1318 (10b), LDK1319 (10a), and LDK1325 (8b). The
compounds LDK1318 (10b) and LDK1319 (10a) were structurally similar in that both featured
an indole ring (Figure 1). LDK 1323 (8a) and LDK1325 (8b) featured an ethanolamine attached
to the pyrimidine or pyridine ring, respectively (Figure 1). All of these compounds were
optimized from PSNCBAM-1 with reduced number of rings (two or three rings), which is
preferred for therapeutic agents used in the CNS.24
Compounds LDK1321 (6a) and LDK1324 (6b) were designed to retain the three aromatic
rings of PSNCBAM-1 while its pyrrolidinyl ring was removed. LDK1321 (6a) showed a KB = 85
nM and  = 5.9 suggesting that this compound retained key features of receptor modulation of
PSNCBAM-1 (Table 1). LDK1324 (6b) maintained the pyridine ring PSNCBAM-1 and also
featured a chlorine substituent (Figure 1), and enhanced binding of CP55,940 (Figure 2B). In the
CP55,940 binding experiments, the KB = 300 nM and the cooperativity factor, α = 5.6, indicative
of positive cooperativity (Table 1). While the KB is higher than that of the parent compound,

PSNCBAM-1 (55 nM), the cooperativity factor for LDK1324 (6b) is greater than one, indicating
that this compound maintains the ability to positively enhance the binding of CP55,940 (Table
1).
LDK1320 (12b) featured a dimethylamino group which replaced the two rings (i.e. the
pyridine ring and the pyrrolidine ring) (Figure 1). LDK1320 (12b) enhanced binding of agonist
CP55,940 (Figure 2C), and showed positive cooperativity, as indicated by its α = 5.4 (Table 1).
However, the KB = 830 nM, thus the binding affinity is weaker than PSNCBAM-1 (Table 1).
Replacement of the pyridine ring (Figure 1) with an acetamide group maintains some
enhancement of binding of CP55,940 as with LDK1317 (12a), which exhibited with a KB = 110
nM and an α = 2.3 (Figure 2D, Table 1). Results from testing these two compounds indicate that
diarylureas which possess only two aromatic rings may still be capable of binding the allosteric
site and cause positive binding cooperativity.
Compound LDK1323 (8a) featured a pyrimidine ring in place of the pyridine ring of the
parent compound, and also had an ethanolamine group attached to the pyrimidine ring (Figure 1).
This compound has a modest, but positive impact on the binding of agonist CP55,940 (Figure
2E). The compound in this series with both the lowest KB (85 nM) and the highest cooperativity
factor (5.9) was LDK1321 (6a) (Table 1). This compound, like LDK1323 (8a), has a pyrimidine
ring instead of the pyridine ring of PSNCBAM-1, but with a chlorine replacing the pyrrolidine
group (Figure 1), and was successful in enhancing the binding of agonist CP55,940 (Figure 2F).
Select compounds were also tested for their ability to decrease the binding of the inverse
agonist SR141716A. LDK1323 (8a) displayed a modest decrease in SR141716A binding, while
LDK1321 (6a) and LDK1324 (6b) demonstrated a robust, dose-dependent decrease of
SR141716A binding (Figure 3A-C). All compounds with this orthosteric inverse agonist had KB

values in the micromolar range (1.4, 6.8. and 1.8 µM for LDK1321 (6a), LDK1323 (8a), and
LDK1324 (6b), respectively), and cooperativity factors of less than 1, which is indicative of
negative binding cooperativity (Figure 3). This negative cooperativity of binding with an inverse
agonist is a characteristic of other positive allosteric modulators of CB1, including PSNCBAM-
1.14,15 Since the positive allosteric modulator stabilizes CB1 in an activated form that enhances
CP55,940 binding, one would expect the modulator to have a lower affinity for an inverse
agonist (e.g. SR141716A) in its presence relative to its absence and a negative cooperativity
factor. As one would expect, SR141716A binding is decreased as one employs a higher
concentration of allosteric modulator (Figure 3A-C).
For those where there is binding, it is established by the binding assay to CP55,940 with
allosteric modulator that there is positive cooperativity. That is expected for an activated
receptor. That the CP55,940 (agonist) binding has an alpha factor greater than 1.0 argues that
the allosteric modulator activates the receptor and therefore is positive allosteric modulator
(PAM)-like. That SR141716A (inverse agonist) binds an activated receptor less well and the
alpha factor is less than one agrees with that.28
In order to investigate whether the structural optimization improves the drug-like
properties, the compounds shown in Table 1 were assessed with a computational program for
their drug-like properties and solubility. The results are shown in Table 2. Compound LDK1317
(12a) (KB= 110 nM, α= 2.3) and LDK 1321 (6a) (KB= 85 nM, α= 5.9) can serve as lead
compounds for further development of allosteric modulators from the diarylurea scaffold. Based
on the computationally calculated drug-like properties of these molecules in Table 2, reducing
the number of rings within the structures of the diarylurea analogs makes these satisfy the
Lipinski rule of five and leads to improvement of the distribution constant (LogD) and intrinsic

solubility of the compounds (Table 2). The calculated LogD values obtained in two different pH
conditions (pH 7.4 and pH 1.7) indicates that the synthesized compounds could be ionized in
acidic media (e.g. the stomach) except compounds LDK1319 (10a) and LDK1318 (10b). The
calculated LogD and LogP values obtained at pH=7.4 are identical. This indicated that the
compounds are in neutral unionized forms in aqueous media at pH 7.4 (e.g. the blood).
Introducing ethanolamine into compounds LDK1321 (6a) and LDK1324 (6b) enhanced the calculated
intrinsic solubility (i.e. LDK1323 (8a) and LDK1325 (8b)) by approximately 20-fold with the cost that
LDK1325 (8b) lost its binding affinity for the allosteric site. It is noteworthy that reducing the number
of rings of diarylurea analogs to 2 significantly enhanced intrinsic solubility by about 400-fold
(ie. PSNCBAM-1, 0.000203 mg/mL vs LDK1320 (12b), 0.0873 mg/mL, Table 2).
Conclusions
In this preliminary study, it was found that that reducing the number of rings within the scaffold of
PSNCBAM-1 is a viable approach to generate novel lead compounds for developing allosteric modulators
of the CB1 receptor. A fewer number of rings likely holds the key for improving the drug-like properties
and solubility. By continuing structure-activity relationship studies based on the allosteric
modulator scaffold of PSNCBAM-1, we can develop new lead compounds with improved drug-
like properties that target the pharmacologically important CB1 receptor.
Acknowledgements
The authors acknowledge that this research was supported in part by the National Institutes of
Health (DA039942).
Conflict of Interest
The authors have no conflicts of interest regarding the content of this article.

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Figure Legends
Figure 1
Structures of PSNCBAM-1 and the analogs tested for allosteric modulator binding.
Figure 2
The influence of allosteric modulators on orthosteric agonist [3H]CP55940 specific binding to
CB1. Shown are binding assays with [3H]CP55940 as a tracer with varying concentrations of (A)
PSNCBAM-1, (B) LDK1324 (6b), (C) LDK1320 (12b), (D) LDK1317 (12a), (E) LDK1323 (8a),
and (F) LDK1321 (6a). Results determined from at least three experiments performed in
duplicate, and data are presented as the mean ± S.E. (error bars).
Figure 3
The influence of allosteric modulators on orthosteric inverse agonist [3H]SR141716A specific
binding to CB1. Shown are equilibrium binding assays with [3H]SR141716A as a tracer with
varying concentrations of (A) LDK1321 (6a), (B) LDK1323 (8a), and (C) LDK1324 (6b).
Results are determined from at least three experiments performed in duplicate, and data are
presented as the mean ± S.E. (error bars).

Table 1. Binding parameters of PSNCBAM-1 analogs.
Compound code
KB (nM)a
αb
55
(26-120)
3.6
(2.6-6.1)
PSNCBAM-1
85
(41-180)
5.9
(2.8-12)
6a, LDK1321
6b, LDK1324
5.6
(3.4-9.4)
300
(150-630)
200
(19-1,900)
2.4
(1.4-4.2)
8a, LDK1323
8b, LDK1325
NBc
NBc
10a, LDK1319
10b, LDK1318
12a, LDK1317
NBc
NBc
NBc
NBc
2.3
(1.6-3.3)
110
(24-490)
830
(240-3,000)
5.4
(2.7-11)
12b, LDK1320
aKB: equilibrium dissociation.
bα: cooperativity factor for the allosteric modulator tested.
The allosteric parameters, KB and α, were determined using [3H]CP55,940 as the orthosteric
ligand. KB and  values are given with 95% confidence intervals in parentheses.
cNB: no detectable binding of the orthosteric agonist [3H]CP55,940 in the presence of the
test compound up to 10 µM.

Table 2. Calculated physicochemical properties and solubility of the synthesized analogs of
PSNCBAM-1.a
Lipinski
Rule
of Five
satisfaction
Intrinsic
Solubilityb
(pH = 7.4)
Compound
Code
Log D
(pH = 7.4)
Log D
(pH = 1.7)
Log P
(pH = 7.4)
Rings
PSNCBAM-
1
0.000203
mg/mL
4
3
3
3
3
3
3
2
2
No
5.64
3.99
4.61
2.55
3.15
0.94
0.94
0.08
0.95
3.69
3.97
4.59
1.14
1.21
0.94
0.94
0.08
0.99
5.64
3.99
4.61
2.55
3.15
0.94
0.94
0.08
0.95
6a,
LDK1321
0.000207
mg/mL
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
6b,
LDK1324
0.000219
mg/mL
8a,
LDK1323
0.00381
mg/mL
8b,
LDK1325
0.00402
mg/mL
10a,
LDK1319
0.00758
mg/mL
10b,
LDK1318
0.00758
mg/mL
12a,
LDK1317
0.0493
mg/mL
12b,
LDK1320
0.0873
mg/mL
aThe parameters were obtained from computational prediction using the ChemAxon program.
bThe intrinsic solubility is the equilibrium solubility of the compound at the pH where it is fully
unionized.