Synthesis and biological evaluation of a ring analogs of the selective CB2 inverse agonist SMM-189

Bioorg. Med. Chem. 33 (2021) 116035

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Synthesis and biological evaluation of a ring analogs of the selective CB2

inverse agonist SMM-189

Sahar S. Alghamdi, Suni M. Mustafa, Bob M. Moore II^*

Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA

A R T I C L E I N F O

A B S T R A C T

Keywords:

Microglia are the principle cell type driving sustained neuroinﬂammation in neurodegenerative diseases such as

Alzheimer’s, Parkinson’s, and Multiple Sclerosis. Interestingly, microglia locked into a chronic M1 pro-

inﬂammatory phenotype signiﬁcantly up-regulate the cannabinoid receptor 2 (CB2) expression. Our approach

to exploiting CB2 as a therapeutic target in neuroinﬂammatory diseases focuses on the development of selective

CB2 inverse agonists to shift microglia bias to a M2 pro-wound healing phenotype. Herein we report work

designed to reﬁne the structure activity relationship of the 2,6-dihydroxy-biphenyl-aryl-methanone CB2 inverse

agonist scaffold. A series of analogs of our lead compound SMM-189 were synthesized and measured for afﬁnity/

selectivity, potency, and efﬁcacy in regulating cAMP production and β-arrestin recruitment. In this series com-

pound 40 demonstrated a signiﬁcant increase in potency and efﬁcacy for cAMP stimulation compared to SMM-

189. Akin to our lead SMM-189, this compound was highly efﬁcacious in biasing microglia to an M2 pro-wound

healing phenotype in LPS stimulated cell lines. These results advance our understanding of the structure–activity

relationship of the 2,6-dihydroxy-biphenyl-aryl-methanone scaffold and provide further support for regulating

microglia activation using CB2 inverse agonists.

Cannabinoids

Inverse agonist

CB2 receptor

cAMP

β-Arrestin

Microglia

Polarization

1. Introduction

of the BBB declines in many neurodegenerative disorders, repurposing

existing anti-inﬂammatory and/or immune regulatory drugs has proven

Inﬂammation and dysregulated immune responses are an underlying

pathology in a broad range of diseases such as inﬂammatory bowel

disease,¹cancer,²and neurodegenerative disorders.³Non-steroidal anti-

inﬂammatory drugs and glucocorticoids are the mainstays for acute

inﬂammation; however, side effects from long-term NSAIDs therapy, e.g.

gastrointestinal tract side-effects,⁴or glucocorticoid-induced osteopo-

rosis⁵can complicate their use in chronic inﬂammatory diseases.

Disease-modifying drugs, such as methotrexate and leﬂunomide, are

effective in treating autoimmune diseases but can exhibit reduced efﬁ-

cacy over time.⁶More recently biologicals, e.g. Inﬂiximab and Humira,

have had a signiﬁcant impact in regulating several peripherally medi-

ated inﬂammatory/immune responses in diseases including rheumatoid

arthritis, psoriasis, and, Crohn’s disease.⁷Most of these approaches

however have limited efﬁcacy in the treatment of inﬂammation and

dysregulated immune responses in the central nervous system (CNS).

The limited efﬁcacy is in large part caused by the blood–brain barrier

(BBB) that excludes approximately 98 percent of small molecule drugs

and all of the high molecular weight biologicals.⁸Although the integrity

challenging.⁹Unfortunately, neuroinﬂammation is an underlying pa-

thology in CNS diseases, such as traumatic brain injury,¹⁰Alzheimer’s¹¹

and Parkinson’s diseases¹², which contributes to disease progression. As

such, there remains a critical unmet need for new therapeutic ap-

proaches to regulate neuroinﬂammation.

Acute neuroinﬂammation plays a key role in resolving and healing

damage to the brain; however, the progression into chronic inﬂamma-

tion ultimately exacerbates the initial injury. The principal meditator of

neuroinﬂammation are microglia,¹³the CNS equivalent of the macro-

phage, which is the central target of many anti-inﬂammatory strategies.

For example, in neurodegenerative disorders, several existing drugs,

such as thalidomide,¹⁴minocycline,¹⁵psychiatric drugs,¹⁶and thiazo-

lidinediones¹⁷have been investigated as potential regulators of micro-

glial activation. In vitro, these compounds are effective in reducing the

pro-inﬂammatory M1 microglia activation state and, in some cases,

promoting the pro-wound healing M2 state. However, the efﬁcacy in

halting neuroinﬂammation of Alzheimer’s, Parkinson’s, and traumatic

brain injury remains controversial. More recently the cannabinoid

* Corresponding author at: 881 Madison Ave., Room 563, Memphis, TN 38163, USA.

E-mail address: bmoore@uthsc.edu (B.M. Moore II).

https://doi.org/10.1016/j.bmc.2021.116035

Received 17 December 2020; Received in revised form 14 January 2021; Accepted 16 January 2021

Available online 25 January 2021

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

receptor 2 (CB2) has emerged as an important target for the develop-

ment of therapeutics to treat microglia-mediated neuroinﬂammation.¹⁸

Interestingly, in the resting state, microglia express low levels of CB2 but

the expression is signiﬁcantly up-regulated in response to CNS insult.

Evidence indicates that CB2 upregulation is a conserved microglia

response to activation. Speciﬁcally, CB2 up-regulation on microglia has

been found in post-mortem brains of Parkinson’s,¹⁹Alzheimer’s,²⁰and

human immunodeﬁciency virus encephalitis,²¹and in the spinal cords of

multiple sclerosis and amyotrophic lateral sclerosis.²²Presumably, CB2

is also up-regulated in traumatic brain injury because post-mortem

brains of patients also exhibit microglia activation.²³In sum, the

expression of CB2 on activated microglia present a unique target for

regulating the progressive CNS damage associated with chronic

neuroinﬂammation.

reﬁne the structure–activity relationship of the scaffold, a series of A

ring-substituted analogs of SMM-189 were synthesized. Analog biolog-

ical activity was measured in CB1 and CB2 binding assays, functional

activity in the ACTOne cAMP assay, and inhibition of β-arrestin

recruitment in the Tango-BLA. Finally, LPS stimulated murine and

human microglial cell lines were assayed for M1 versus M2 bias

following treatment with compound 40, the most potent compound in

the series.

2. Results and discussion

2.1. Chemistry

Our ﬁrst series of 2,6-dihydroxy-biphenyl-aryl-methanone CB2 in-

verse agonists were designed to test the effects of C ring substituents on

biological activity.⁴²Out of the 21 compounds in the series, the 4-ethyl-

phenyl and 4-propylphenyl C ring analogs were measured to have

increased afﬁnity, potency, and efﬁcacy at CB2 relative to SMM-189.

Additionally, the resorcinol hydroxyl groups were discovered to be

essential since methylation of one or both hydroxyl groups abolished

activity. Within this series the 3,5-dichlorophenyl A ring of SMM-189

was preserved, which left open the question of the effects of A ring

substitution on CB1/CB2 receptor activity. To reﬁne the SAR of this

structural class of CB2 inverse agonists, the second series of A ring-

substituted analogs were synthesized. The C ring substitution in this

series was phenyl or thiophenyl, the latter of which demonstrated efﬁ-

cacy in our selective CB2 agonist.⁴³

The CB2 receptor is a constitutively active G-protein coupled re-

ceptor that couples to G_α.^24–26The receptor is widely distributed in the

peripheral immune cellsⁱmaking it an important target for regulating

inﬂammation and immune activity.^27,28Selective agonists of CB2 are by

far the most structurally diverse and mature class of ligands representing

at least 12 candidates that have entered into clinical trials (reviewed by

Dhopeshwarkar and Mackie²⁹and Tabrizi and colleagues³⁰). However,

the constitutive activity of CB2 also allows for the design of inverse

agonists providing an alternate mechanism to regulate CB2 func-

tion.^31,32This class of CB2 selective ligands, which includes SR144528,

Sch.336, and JTE-907 (Fig. 1), have been reported to regulate immune

cell function and/or inﬂammation.^33–35We have also developed a se-

lective CB2 inverse agonist based on the 2,6-dihydroxy-biphenyl-aryl-

methanone scaffold. Our lead compound, SMM-189 (Fig. 1) is a potent

in vitro inhibitor of microglia polarization to the M1 pro-inﬂammatory

phenotype while promoting an M2 pro-healing phenotype.³⁶The com-

pound also inhibits the expression of pro-inﬂammatory cytokines and

chemokines in primary human and murine microglia.^36,37In vivo, SMM-

189 alleviated or reduced the adverse physiological and psychological

effect of mild traumatic brain injury in mice.^37–40Recently, we reported

the administration of SMM-189 in the kainate-induced mouse status

epileptics model protected against neuronal damage and reduced in-

ﬂammatory markers.⁴¹The promising in vivo activity of SMM-189

demonstrates the potential of the 2,6-dihydroxy-biphenyl-aryl-metha-

none scaffold for developing novel CB2 inverse agonists. Therefore, to

The 2,6-dihydroxy-biphenyl-aryl-methanone compounds were pre-

viously synthesized starting from syringaldehyde; however, removal of

the methyl protecting groups required heating to 220 ^◦C in pyridine

hydrochloride.⁴²While successful, to obtain products of 96 percent or

greater purity for biological testing, some of the ﬁnal products required

re-crystallization and/or preparative HPLC puriﬁcation. To circumvent

the challenging deprotection, the methoxymethyl (MOM) group was

selected due to milder deprotection conditions. Syringaldehyde was

unsuitable as a starting material since 4 steps would be required to ex-

change the methyl groups for MOM. Additionally, the Suzuki–Miyaura

cross-coupling was carried out using the pseudohalide triﬂate ester. The

formation of the ester required triﬂic anhydride which we sought to

Fig. 1. Selective CB2 inverse agonists.

2

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

eliminate to reduce synthetic cost. Based on the functional group re-

quirements to conduct the transformation necessary to prepare the

target compounds intermediate 3 was identiﬁed.

Synthesis of intermediate 3 was accomplished in 4 steps beginning

with the regiospeciﬁc 4-bromination 3,5-dihydroxybenzoic acid (1)

according to the method of Fillion and Fishlock.⁴⁴Introduction of the

MOM groups was carried out using in situ generated MOMCl as reported

by Berliner and Belecki.⁴⁵This approach limited exposure to the

MOMCl, which is toxic and carcinogenic, and provided a highly reactive

reagent compared to some commercial sources. Reduction of the MOM

ester with DIBAL and subsequent oxidation using PCC provided inter-

mediate 3 in 61% overall yield (Scheme 1). The C ring was introduced in

2 steps via a reaction of 3 with either phenyl magnesium bromide or 2-

thiophenyl magnesium bromide followed by PCC oxidation to yield in-

termediate ketones 4 and 5 (Scheme 2). Introduction of the A ring was

accomplished via a Suzuki–Miyaura cross-coupling using Buchwald

precatalysts Pd-G3 SPhos in a THF-0.5 N potassium triphosphate solvent

system (Scheme 3). Deprotection using 3 N hydrochloric acid in ethanol

yielded the target resorcinol products.

Scheme 2. Reagents and conditions: (a) phenyl-or thienyl-magnesium bro-

mide, THF, 0 ^◦C → r.t. ; (b) PCC, DCM, r.t.

cyano (28), 3,4-methylenedioxy (25), and 2,3-dihydro-benzo[1,4]

dioxin (26) analogs for hCB2. These results suggest that in the ligand-

binding pocket the A ring occupies a sterically limited site. Replace-

ment of the phenyl A ring with a pyridyl ring (29) was equally inef-

fective. The addition of electronegative groups to the pyridyl ring, i.e.

ﬂuorine (30) and chlorine (31), in the 3-position relative to compounds

20 and 22, abolished binding to both hCB1 and hCB2. It is presumed

that the pyridyl group would be protonated under the assay conditions

suggesting unfavorable charge-charge interactions in the ligand-binding

pocket of both receptors. Interestingly replacement of the A ring with

the neutral, ring contracted heterocycles furanyl (33) and thiophenyl

(32) shifted receptor activity. In the case of 33 (K_i= 1.33 µM) the

functional activity in the cAMP assay switched from an inverse agonist

to a partial agonist. The thiophenyl substitution (33) abolished binding

to hCB1 and hCB2. The results for the 5 membered heterocycles suggest

that compound 33 binds in a different orientation to hCB2 relative to the

phenyl ring analogs.

2.2. Receptor binding

The binding afﬁnities of compounds 20–33 and 40–45 for the hCB1

and hCB2 receptors were measured in competition radioligand

displacement assays using membrane preparations from the ACTOne

cell lines and [³H] CP 55,940.⁴²Our lead compound SMM-189 con-

tained a 3,5-dichlorophenyl A ring suggesting electronegative groups

are favored. However, the substitution of this group with 3,5-diﬂuoro-

phenyl (21) surprisingly reduced hCB2 afﬁnity by an order of magni-

tude (121.3 nM versus 1,634 nM) and increased hCB1 afﬁnity from 4.78

µM to 1.08 µM with a hCB1/hCB2 selectivity ratio (SR) of 0.66 compared

to 39.4 for SMM-189 (Table 1). The decrease in the afﬁnity of 21 may

reﬂect a reduction in the size of the electronegative group which is

supported by compound 23 which contains the 3,5-bis-(triﬂuoromethyl)

phenyl group. Speciﬁcally, this A ring substitution restored hCB2 af-

ﬁnity (Ki = 215 nM) but negatively affected selectivity (SR = 8.86) due

to increased binding to hCB1 (K_i= 1.90 µM). Removal of the tri-

ﬂuoromethyl group in the 5 positions (20) eliminated binding to CB1

while retaining afﬁnity for hCB2 (K_i= 204 nM) increasing the SR to 49.

Substitution of the 3-triﬂuoromethyl with 3-methyl (24) modestly

decrease hCB2 afﬁnity (Ki = 320 nM) with a concomitant increase for

hCB1 (K_i= 4.01 µM). Interestingly, the 2,5-dichlorophenyl (22) caused a

slight decrease in hCB2 binding (K_i= 233 nM) but increased SR to 42.9

by inhibiting hCB1 binding. The A ring in 22 can be alternatively

numbered 3,6-dichlorophenyl suggesting that electronegative groups in

the 3 position of the A ring are important for receptor selectivity.

We previously reported that SMM-189 exhibits orthosteric binding to

hCB2 in competition functional assays against CP-55,940.⁴²This

determination, in combination with the presence of a methylenehydroxy

group in HU-210, suggested that an A ring containing a 3-hydroxy-

methylene (27) group would enhance afﬁnity for hCB2. Surprisingly,

compound 27 had a poor afﬁnity for hCB2 (Ki = 2.49 µM) and did not

displace CP 55,940 at CB1. Larger functional groups on the A ring and

substitution of the 4 positions were equally detrimental to hCB2 binding

as demonstrated by the poor afﬁnity (1.84 to 4.74 µM) of the 4-ﬂuoro-3-

The results from our earlier work on C1^′substituted classical can-

nabinoids⁴⁶and biaryl-aryl hCB2 agonist⁴³revealed that a thiophene

ring increased hCB2 afﬁnity. To determine if this transformation could

improve inverse agonist activity compounds 40–45 were synthesized

wherein the phenyl C ring was replaced by thiophenyl. In the case of the

3-triﬂuoromethyl (40) and 3,5-triﬂuoromehtyl (42) analogs, the thio-

phenyl group increased binding to 154 and 187 nM, respectively, with

SRs of 64.9 and 20 (Table 2). However, this transformation resulted in a

3-fold decrease in hCB2 afﬁnity in the 3-methylphenyl (41) analog (K_i=

975 nM) compared to the phenyl C analog (24) afﬁnity of 320 nM and a

2 fold decrease in hCB1 binding (4.02 µM versus 9.53 µM). In a similar

trend, the thiophenyl C ring decreased the afﬁnity of the 2,5-dichloro-

phenyl (43) 2-fold, the 3-hydroxymethylene phenyl (44) 3-fold, and

abolished binding in the 3-pyridyl analog (45). Overall, replacement of

the 3,5-dichlorophenyl A ring in SMM-189 with different functional

groups did not improve hCB2 afﬁnity; however, the 3-triﬂuoromethyl

group did abolish hCB1 afﬁnity providing SRs of 49 and 65 for com-

pounds 20 and 40, respectively, compared to 39.4 for SMM-189.

2.3. Functional assays

The ACTOne functional assay was used to measure the potency and

efﬁcacy of compounds 20–33 and 40–45 in regulating cAMP production

in cells expressing either hCB1 or hCB2 or the parental line containing

Scheme 1. Reagents and conditions: (a) 20% HCl, Br₂, Δ; (b) i. dimethoxymethane, acetylchloride, zinc bromide, 0 ^◦C, ii. DCM, 4-bromo-3,5-dihydroxybenzoic acid,

iii. DIEA, 0 ^◦C → r.t.; (c) DIBAL, THF, 0 ^◦C → r.t.; (d) PCC, DCM, r.t.

3

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

Scheme 3. Reagents and conditions: (a) aryl/heteroaryl boronic acid, Pd-G3 SPhos, THF, 0.5 N K₃PO₄, r.t.; (b) 3 N HCl in ethanol.

only the cAMP gated ion channel. The reference compound CP-55,940

SR-144528, activated β-arrestin recruitment in either hCB1 or hCB2

assays. The absence of activity at hCB2 was consistent with inverse

agonist activity wherein the ligands bind to inactive receptor confor-

mations. Based on these results we conducted competition assays in the

presence of CP-55,940 at an EC₈₀concentration in the hCB2 cell line.

The pEC₅₀of CP-55,940 was signiﬁcantly reduced in the presence of

SMM-189, analogs 20–24, 40–43, and SR-144528 with values tightly

clustered from 5 to 5.9. Compound 28 failed to inhibit β-arrestin

recruitment presumably due to low afﬁnity and efﬁcacy, as measured in

the ACTOne assay. A linear regression of log(1/K_i) versus log(1/Efﬁ-

cacy) exhibits good correlation with an R²= 0.72 which is consistent

with higher afﬁnity compounds displacing a greater fraction of the CP-

55,940. In sum, the results from the binding, cAMP, and β-arrestin

functional assays establish the SMM-189 analogs as selective inverse

agonists at hCB2.

was a potent agonist at hCB1 (pEC₅₀= 8.70) and hCB2 (pEC₅₀

=

8.20) with cAMP suppression efﬁcacies of ꢀ 94.6 and ꢀ 77.8, respec-

tively (Table 4). In the hCB1 assay, none of the compounds were active

(Tables 3 and 4) nor was there response in the parental cell line indi-

cating no off-target effects. A linear regression of log(1/K_i) versus log(1/

EC₅₀) for compounds active in the hCB2 cAMP assay yielded an R²

=

0.56 wherein the compounds outside the 95% conﬁdence interval were

the thiophenyl analogs 40–43 and compound 24 (Fig. 2). There are

insufﬁcient compounds in the thiophenyl series to determine if separate

correlations can be established between the series. Notwithstanding, the

thiophenyl C ring (compounds 40, 41, and 43) improves potency

compared to the phenyl C ring analogs (compounds 20, 22, and 24) for

the 3-triﬂuoromethyl- (pEC₅₀7.41 versus 6.62), 3-methyl- (pEC₅₀6.70

versus 5.78), and the 2,5-dichloro- (pEC₅₀6.85 versus 6.44) functional

groups. Within this group of compounds, only compound 40 had a sta-

tistically signiﬁcant increase in potency with p values ranging from 0.05

to 0.0001. In terms of efﬁcacy (% max), none of the compounds

signiﬁcantly increased cAMP levels above the lead compound SMM-189

and a signiﬁcant decrease was measured for compound 24 (p = 0.05 to

0.001). The conversion of the C ring phenyl to thiophenyl in the 3,5-tri-

ﬂuoromethyl analogs, 23 and 42 respectively, did not signiﬁcantly

affect potency but did signiﬁcantly increase efﬁcacy (p = 0.0001) in the

thiophenyl analog. Although the replacement of the 3,5-dichlorophenyl

ring in SMM-189 with 3,5-diﬂuorophenyl (21) negatively affected af-

ﬁnity and potency, the efﬁcacy was not signiﬁcantly different from

potent and efﬁcacious compounds such as 40. Surprisingly, compound

33, wherein the A ring is 3-furanyl, was measured to have weak agonist

activity with a pEC₅₀of 5.56 and efﬁcacy of ꢀ 18.5.

2.4. Effects of compound 40 on LPS activated microglia polarization

The impetus behind the continued reﬁnement of the structur-

e–activity relationship of the 2,6-dihydroxy-biphenyl-aryl-methanone

scaffold is the remarkable activity of CB2 inverse agonists, e.g. SMM-

189 and SR-144528, to bias activated microglia to a pro-healing M2

phenotype.³⁶To continue validating therapeutic potential CB2 inverse

agonists, compound 40 was selected to measure the effects on LPS

activated murine and human microglia. Compound 40 was ﬁrst tested

for M1/M2 phenotype bias using the C8B4 microglia cell line, which is

reported to express the closest features to the native mouse microglia.⁴⁸

To assess M1/2 surface markers, C8B4 cells were stimulated with LPS (1

μ

g/ml) for 1 h. The cells were then treated with 20 μM compound 40,

Based on receptor theory, inverse agonists do not bind to an active

conformation of a constitutively active G-protein coupled receptor, nor

do they cause a conformational change leading to an active conforma-

tion. In turn, recruitment of β-arrestin would not be predicted since the

C-terminus would not be accessible for phosphorylation by G-protein

receptor kinases. To measure β-arrestin recruitment we selected the

Tango-BLA β-arrestin functional assay which has been reported to pro-

vide reliable EC₅₀s for cannabinoid receptor inverse agonists.⁴⁷Treat-

ment of cells expressing either hCB1 or hCB2 with the non-selective

agonist CP-55,940 resulted in cleavage of the CCF4/AM ﬂuorogenic

reporter substrate. The pEC₅₀in the hCB1 assay was 8.24 and 7.43 in the

hCB2 assay with efﬁcacies of 4.51 and 3.70, respectively (Table 5). To

measure the efﬁcacy of our inverse agonist in this assay, we selected

compounds SMM-189, 20–24, 28, and 40–43 based on afﬁnity and

potency in ACTOne functional assay. Dose-response studies revealed

that none of the nine compounds, or the selective CB2 inverse agonist

based on the EC₅₀in cytotoxicity assays, and incubated for 24 h. The

effects on the M1 pro-inﬂammatory (CD 16/32 and CD 68) and M2 anti-

inﬂammatory (CD 204 and CD 206) markers were evaluated using the

on-cell polarization assay previously developed in our lab.³⁶Consistent

with our previous ﬁndings with SMM-189, compound 40 signiﬁcantly

suppressed the M1 associated markers CD 16/32 (p < 0.01) and CD 68

(p < 0.001) relative to LPS (Fig. 3). Moreover, treatment signiﬁcantly

enhanced the expression of M2 markers CD 204 and CD 206 in com-

parison to LPS (P < 0.0001). To determine if inter-species differences in

microglial responses to CB2 inverse agonist could change the M1/M2

phenotype bias, the polarization studies were repeated in SV40

immortalized human microglia. In this study, cells were activated with

LPS (100 ng/ml) for 1 h followed by treatment with 40 (35 μM) at the

previously determined cytotoxic EC₅₀. Rewardingly, the expression of

M1 markers CD 16 (p < 0.001) and CD 68 (p < 0.0001) were decreased

relative to LPS control (Fig. 4). Additionally, the expression of M2

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S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

Table 1

Table 2

Binding afﬁnities of compounds 40–45 for the hCB1 and hCB2 receptors.

Binding afﬁnities of compounds 20–33 for the hCB1 and hCB2 receptors.

Compound

R₂

CB1 K_i(nM)

>10,000

CB2 K_i(nM)

204 ± 40.6

CB1:CB2

ratio

Compound

R₂

CB1 K_i(nM)

>10,000

CB2 K_i(nM)

154 ± 1.09

CB1:CB2 ratio

64.9

40

20

49.0

41

42

9,526 ± 19.2

3,735 ± 2.39

975 ± 1.18

187 ± 1.11

9.77

20.0

21

1,076 ± 91.4

1,634 ± 45.8

0.66

22

23

>10,000

233 ± 45.5

215 ± 54.4

42.9

8.86

1,904 ± 77.7

43

44

>10,000

447 ± 1.18

22.4

1.19

8,383 ± 50.1

24

25

4,015 ± 142.9

>10,000

320 ± 59.2

>10,000

12.6

1

45

>10,000

1

measure motility and histochemical analysis of cell morphology. In the

scratch assay, SV40 immortalized human microglia cells were untreated,

treated with LPS (100 ng/ml), or a combination of LPS and 40 (100 ng/

26

27

>10,000

4,740 ± 74.9

2,494 ± 117.6

2.11

4

ml and 10 μM, respectively). Following a 24-hour incubation period, cell

counting revealed signiﬁcant migration (p = 0.001) of microglia treated

with LPS relative to controls (Fig. 5). In contrast, treatment with 40

signiﬁcantly reduced migration compared to LPS alone (p = 0.001) with

no signiﬁcant difference relative to controls. To conﬁrm that the

migration of microglia in the LPS treatment group correlated with an M1

phenotype, and not an M2 activation state, histochemical analysis of

control, LPS treated, and LPS plus 40 was conducted. Analysis of the LPS

treated group revealed enlarged, ﬂat, rounded cell bodies with fewer

projections, i.e. amoeboid morphology (Fig. 6), which is consistent with

an LPS induced M1 phenotype.⁴⁹Conversely, treatment with compound

40 following activation induced a distinct transformation of microglia

into a ramiﬁed cell morphology with a small cell body and several

elongated projections, indicating microglia adopted an M2 wound-

healing phenotype.⁵⁰It is worth noting that all the three treatment

groups showed similar nucleus distribution conﬁrming that morpho-

logical changes are due to alteration in cell size and shape rather than a

change in cell number.

28

>10,000

1,839 ± 61.2

5.44

29

30

>10,000

1

31

>10,000

1

32

>10,000

1

33

>10,000

1,328 ± 56.8

121.3 ± 20.6

7.53

39.4

The migration and morphology of activated microglia are but one set

of markers used in phenotype characterization. Mediators generated or

secreted by M1 activated microglia also exhibit phenotype-speciﬁc

proﬁles such as increased pro-inﬂammatory cytokines, nitric oxide,

and reactive oxygen species (ROS). Of note, ROS has been implicated as

a major contributor to neuronal death in many neurological diseases.⁵¹

Therefore, to measure the efﬁcacy of 40 to modulate ROS production,

and provide corroborating support of the M2 activation state, we tested

40 in a ROS assay using the immortalize human microglia. At 50 min

SMM-189

4,778 ± 246

markers CD 204 and CD 206 were signiﬁcantly enhanced in comparison

to LPS (P < 0.01). The polarization of murine and human microglia from

a pro-inﬂammatory M1 phenotype to a pro-wound healing M2 pheno-

type was further validated in microscopy studies using the human

microglial cell line.

post LPS (100 ng/ml) plus 40 (10 μM) there was a signiﬁcant decrease (p

< 0.01) in ROS generation relative to the LPS alone group (Fig. 7). After

a 24-hour incubation period, the addition of 40 following LPS activation

signiﬁcantly (p < 0.0001) suppressed ROS expression. Our ﬁndings are

consistent with the results reported by Ribeiro et al. wherein SR-144528,

a CB2 inverse agonist, signiﬁcantly inhibits ROS production in LPS

Migration and changes in morphology are additional measures of the

polarization state of microglia. Therefore, to obtain secondary mea-

surements to support M2 polarization, we conducted scratch assays, to

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Bioorganic & Medicinal Chemistry 33 (2021) 116035

Table 3

Table 4

Functional activities of compounds 20–33 in the hCB1 and hCB2 ACTOne cAMP

Functional activities of compounds 40–45 in the hCB1 and hCB2 ACTOne cAMP

assay.

Compound

R₂

Potency

Efﬁcacy

Potency

Efﬁcacy

Compound

R₂

Potency

Efﬁcacy

Potency

Efﬁcacy

(pEC₅₀

CB1

)

(% max.)

(pEC₅₀

CB2

)

(% max.)

(pEC₅₀

CB1R

n.a.

)

(% max.)

(pEC₅₀

CB2R

)

(% max.)

40

41

42

n.a.

7.41 ±

57.2 ±

20

21

n.a.

6.62 ±

66.0 ±

5.3 ^****

0.05

3.5 ^****

0.06

n.a.

6.70 ±

51.7 ±

n.a.

5.77 ±

63.0 ±

2.8 ^****

0.13

1.7 ^****

0.14

6.17 ±

49.7 ±

22

23

n.a.

6.44 ±

66.5 ±

0.37

3.7 ^****

0.08

2.9 ^****

6.43 ±

22.9 ±

0.07

2.4^****

43

44

n.a.

6.85 ±

61.0 ±

0.15

3.4 ^****

n.a.

24

25

n.a.

5.78 ±

40.3 ±

1.09

3.4 ^****

45

n.a.

CP-

8.70 ±

ꢀ 94.6 ±

8.20 ±

ꢀ 77.8 ±

55,940

0.11

0.11 ^***

0.06

6.5 ^***

26

27

n.a.

5.31 ±

49.0 ±

2.0 ^****

***

0.39

p < 0.001 in comparison to parental HEK-CNG.

p < 0.0001 in comparison to parental HEK-CNG.

****

n.a.

28

n.a.

5.55 ±

52.3 ±

0.27

2.4 ^****

29

30

n.a.

31

n.a.

32

n.a.

33

5.56 ±

ꢀ 18.5 ±

0.46

0.3 ^**

Fig. 2. Linear regression of afﬁnity versus potency showing compounds outside

SMM-189

6.81 ±

54.8 ±

0.09

3.2^**

the 95% conﬁdence interval (dotted lines).

damaging effects of chronically M1 activated microglia.

**

****

p < 0.01 in comparison to parental HEK-CNG.

p < 0.0001 in comparison to parental HEK-CNG.

3. Conclusions

stimulated murine BV-2 and primary rat microglia.⁵²The inhibition of

ROS production is consistent with the results of the cell surface marker,

motility, and morphology studies indicating that 40 biases activated

microglia to an M2 pro-wound healing phenotype. In sum, the study

ﬁndings, combined with our work on SMM-189 and SR144528, reveal

the therapeutic potential of CB2 inverse agonists to slow or halt the

Cannabinoid receptor 2 inverse agonists are a promising new class of

therapeutic agents for the treatment of chronic neuroinﬂammation in

debilitating diseases such as Alzheimer’s and Parkinson’s diseases and

traumatic brain injury. We previously identiﬁed a new class of CB2 in-

verse agonists based on the 2,6-dihydroxy-biphenyl-aryl-methanone

scaffold, represented by SMM-189, which was highly efﬁcacious in

6

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

Table 5

Compound

R₂

Potency (pEC₅₀

CB1

)

Efﬁcacy (Emission Ratio)

Potency (pEC₅₀

CB2

)

Efﬁcacy (Emission Ratio)

CP-55,940

20

8.24 ± 0.27

4.51 ± 0.09

7.43 ± 0.03

5.46 ± 0.21

3.70 ± 0.06

n.a.

1.41 ± 0.52 ^****

21

n.a.

5.05 ± 4.27

2.22 ± 0.44 ^****

22

23

n.a.

5.22 ± 0.55

5.85 ± 0.07

1.03 ± 0.20 ^****

0.91 ± 0.19 ^****

24

n.a.

<5

2.30 ± 0.12 ^****

28

n.a.

SMM-189

6.81 ± 0.09

0.66 ± 0.08 ^****

40

41

42

n.a.

5.07 ± 0.67

5.21 ± 0.81

5.89 ± 0.06

1.19 ± 0.23 ^****

2.68 ± 0.09 ^****

0.94 ± 0.09 ^****

43

n.a.

<5

1.46 ± 0.22 ^****

0.65 ± 0.18 ^****

SR-144528

5.93 ± 0.18

****

p < 0.0001 in comparison to CP-55,940.

biasing microglial activation to an M2 phenotype in vitro and in vivo. To

reﬁne the structure–activity relationship of this structural class, a series

of twenty SMM-189 analogs were synthesized. The analogs focused on

the effects of various A ring functional groups, and C ring thiophenyl

substitution, on hCB1/hCB2 afﬁnity/selectivity and potency and efﬁ-

cacy in regulating cAMP production and β-arrestin recruitment. Analysis

of the actives of the twenty analogs reveals that substitution of the A ring

in the 3-position with electronegative groups approximating the size of

chlorine, e.g. triﬂuoromethyl group in 20 and 40 is optimum for activity.

Further improvement in activity was achieved by replacing the C ring

phenyl (20) with the 2-thiophenyl functional group (40). In general,

larger substituents, e.g. 27, or substitution of the 4-position (25) nega-

tively impacted receptor afﬁnity as did replace chlorine with ﬂuorine

(21). Substitution of the A ring with heterocycles abolished receptor

afﬁnity and functional activity. The notable exception was the 2-furanyl

in compound 33 that resulted in CB2 partial agonist. Of the compounds

synthesized in this series compound, 40 had a signiﬁcant increase in

potency and efﬁcacy for cAMP stimulation relative to SMM-189.

In contrast to the cAMP up-regulation, selected compounds did no

stimulate the recruitment of β-arrestin, consistent with binding of an

7

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

Fig. 3. On cell polarizations assays. Treatment of LPS stimulated murine C8B4

microglia with compound 40 caused a signiﬁcant decrease in M1 markers

CD16/32 and CD68 (black bars) 24 h post treatment. In contrast, a signiﬁcant

increase in M2 markers CD204 and CD206 (checkered bars) was measured in

the same time interval. Asterisks above columns are comparisons to LPS control

with signiﬁcance designated as ** P < 0.01, *** P < 0.001, and **** P <

0.0001. Data represents 6–9 biological replicates and error is SEM.

Fig. 5. Measurement of immortalized human microglia migration in the

scratch assay. Control cells did not exhibit extensive migration 24 h after

application of the scratch (Panel A). Stimulation with LPS triggered a signiﬁcant

migration of microglia after 24 h incubation (Panels B and D). Treatment with

compounds 40 signiﬁcantly inhibited LPS stimulated migration to a level which

was not signiﬁcantly different than controls (Panels C and D). Cell migration

from 7 biological replicates were measured using ImageJ. Error is reported as

SEM, signiﬁcance is **** P < 0.0001, the white bar at the bottom of the images

Fig. 4. On cell polarizations assays. Treatment of LPS stimulated immortalized

human microglia with compound 40 caused a signiﬁcant decrease in M1

markers CD16 and CD68 (black bars) 24 h post treatment. In contrast, a sig-

niﬁcant increase in M2 markers CD204 and CD206 (checkered bars) was

measured in the same time interval. Asterisks above columns are comparisons

to LPS control with signiﬁcance designated as ** P < 0.01 and **** P < 0.0001.

Data represents 6–9 biological replicates and error is SEM.

represents 1,000 μm.

expect that validation of the biological activity in combination with

functional activity will guide our development of the structure–activity

relationship of the 2,6-dihydroxy-biphenyl-aryl-methanone scaffold

leading to a newer more potent CB2 inverse agonist.

inverse agonist to an inactive receptor conformation. The SMM-189

analogs did inhibit CP-55,940 stimulated recruitment of β-arrestin,

albeit with low EC₅₀s. This is presumably due to an almost order of

magnitude difference in the CB2 afﬁnities of the analogs compared to

CP-55,940. To further support our hypothesis that CB2 inverse agonists

provide a novel approach to regulating microglia function, the effects of

40 on LPS stimulated microglia were measured. All the phenotypical

markers evaluated in this work demonstrate the efﬁcacy of 40 in biasing

microglia to a pro-wound healing M2 phenotype. This is evidenced by

signiﬁcant decreases in CD 16/32 and CD 68 (M1 markers), cell

migration, and ROS production. Concomitantly, expression of the M2

markers CD 204 and CD 206 signiﬁcantly increased and a shift from an

amoeboid to a ramiﬁed (M2 phenotype) morphology was observed. We

4. Experimental

4.1. Synthesis reagents and supplies

All chemicals and reagents were purchased from Sigma–Aldrich or

Fisher Scientiﬁc Inc. Anhydrous solvents were prepared by distillation

over sodium metal or calcium hydride prior to use. Moisture or oxygen-

sensitive reactions were carried out using oven-dried glassware under

dry conditions under an argon atmosphere. Analytical thin-layer

8

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

Fig. 6. Representative micrographs of immortalized human microglia visualized using DAPI (nuclear stain) and phalloidin F-actin stain. Control cells did not exhibit

any morphological changes over the 24 h time course (Panel A). Stimulation with LPS triggered a signiﬁcant increase in cell size with rounded shape morphology

(yellow arrows) after 24 h incubation (Panel B). Treatment with compounds 40 signiﬁcantly increased the ramiﬁed and rod-shaped microglia morphology (magenta

arrows) with multiple projections (Panel C). White bars in each photomicrograph represent 100

μm with ten ﬁelds collected per group. (For interpretation of the

references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

chromatography was performed on Silica G plates Sorbent Technologies

(Atlanta, GA) and was visualized by ﬂuorescence quenching under UV

light. Compound puriﬁcation was performed on a Biotage SP1 Flash

Chromatography Puriﬁcation System (Charlotte, NC) using Biotage

SNAP Ultra columns. NMR spectra were acquired on a Bruker Ascend

400 (Billerica, MA) spectrometer (400 MHz for ¹H and 100 MHz for ¹³C).

Coupling constants (J) are expressed in hertz (Hz) and chemical shifts

are reported in ppm on the δ scale and referenced to the appropriate

solvent peak. Routine mass spectra were collected on a Bruker anaZon

SL electrospray/ion trap instrument (Billerica, MA). High-resolution

mass spectrometer (HRMS) data and purity were acquired on a Waters

Xevo G2 + S QTOF (Milford, MA) system equipped with a Waters

Acquity UPLC system. A reversed-phase BEH C18 analytical column

(2.1 mm × 50 mm, 1.7 mm particle size) (Waters, Milford, MA) was used

for the LC separation. Acetonitrile and 95% water with 5% acetonitrile

were used as the organic (solution B) and aqueous (solution A) mobile

phase solutions, respectively. A 5-min gradient elution program was as

following: from start – 1.5 min, 20% B; 1.5 min – 3.0 min, 20% to 100%

B; 3.0 min-4 min, 100%B; 4–4.5 min 100% to 20% B back to initial

condition and ﬁnish at 5 min. The total ﬂow rate was at 0.3 mL/min. All

target compounds were found to be ≥ 95% pure by UPLC.

Fig. 7. Measurement of immortalized human microglia reactive oxygen species

production. Treatment with LPS caused a signiﬁcant increase in ROS compared

to control cells at 50 min and 24 h (black bars). Compound 40 treatment

following activation with LPS signiﬁcantly suppressed ROS generation at both

time points compared to LPS alone (checkered bars). Asterisks above columns

are comparisons to LPS control with signiﬁcance designated as ** P < 0.01 and

**** P < 0.0001. Data represents 6 biological replicates and error is SEM.

4.2. 4.2 Synthesis

4.2.1. 4-Bromo-3,5-dihydroxybenzoic acid

Into a three-necked 2 L bottom tap ﬂask ﬁtted with a mechanical

stirrer and reﬂux condenser was charged 1 L of 20% HCl, 3,5-dihydroxy

benzoic acid (1, 100 g, 0.60 mol) and Br₂(105 g, 0.65 mol). With

9

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

vigorous stirring the suspension was heated to reﬂux, after 1.5 h the

solids dissolved and at 3 h a white solid began to form. The reaction was

cooled to room temperature and stirred vigorously overnight. The ﬂask

was tapped, and the efﬂuent was directed into a Buchner funnel and the

collected solid as air-dried. The light-yellow solid was recrystallized

from methanol to yield 127.9 g (91%) of the desired product as a white

solid. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 9.16 (br. s, 2H, ArOH), 7.20 (s,

2H, ArH), MS: m/z (ESI, neg.) = 232.8 [Mꢀ 1]^ꢀ.

was taped off, organic phases combined, then the aqueous phase was

removed and allowed to stand to collect waste chromium (III) as a green

solid. The combined organic fraction was returned to the ﬂask and 2 L of

saturated sodium bicarbonate was added and stirred vigorous for 15

min. The organic phase was removed via the tap and passed over a

column of celite/silica then the solvent was removed from the colorless

ﬁltrate to yield 90.9 g (93%) of 3 was a white solid. ¹H NMR, (400 MHz,

CDCl₃), δ 9.91 (s, 1H, CHO), 7.34 (s, 2H, ArH), 5.34 (s, 4H, OCH₂O),

3.54 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 328.9 [M + 23]⁺.

4.2.2. (Methoxy)methyl 3,5-bis(methoxymethoxy)-4-bromobenzoate (2)

Into a 3 L jacketed three neck bottom tap ﬂask equipped with a

mechanical stirrer, condenser, and dropping funnel was charged dime-

thoxymethane (130 g, 151 mL 1.71 mol) and anhydrous zinc bromide

(72 mg, 0.32 mmol). The ﬂask was cooled via a circulating ice-cold brine

solution. Acetyl chloride (134 g, 121 mL, 1.88 mol) was added to the

dropping funnel and an initial aliquot of 6 mL was added followed by

drop wise addition of the remaining reagent over 30 min. Two 215 mL

aliquots of anhydrous dichloromethane were passed through the funnel

and added all at once into the reaction mixture. To the mixture was

added 4-bromo-3,5-dihydroxybenzoic acid (107 g, 0.46 mol) and the

resulting slurry stirred vigorously. The dropping funnel was charged

with N,N-diisopropylethylamine (237 g, 317 mL, 1.24 mol) then added

dropwise to the mixture over a 1 h period after which cooling was dis-

continued and the reaction was stirred at room temperature for 12 h.

The reaction was quenched with saturated ammonium chloride (200

mL) to which 200 mL of water was then added. The organic phase was

collected via the ﬂask tap and the aqueous phase was extracted in he

ﬂask with dichloromethane (3 × 200 mL). The organic fractions were

pooled, dried over sodium sulfate, treated with decolorizing carbon,

then ﬁltered through a silica/celite column. The volume was reduced to

approximately 200 mL under reduced pressure then cooled in an ice

bath. The solution was triturated with hexane then the dichloromethane

was slowly removed by passing a stream of air over the ice-cold solution.

The white crystals were collected by ﬁltration and dried to yield 143.8 g

(86%) of 2. ¹H NMR, (400 MHz, CDCl₃), δ 7.52 (s, 2H, ArH), 5.48(s, 2H,

CO₂CH₂), 5.27 (s, 4H, OCH₂O), 3.54 (s, 9H, OCH₃), MS: m/z (ESI, pos.)

= 388.9 [M + 23]⁺.

4.2.5. (4-Bromo-3,5-bis-methoxymethoxy-phenyl)-phenyl-methanol

Glassware was dried overnight in a 110 ^◦C oven, assembled hot, and

allowed to come to ambient temperature under dry positive nitrogen

pressure. A solution of 3 (3.18 g, 10.4 mmol) in 60 mL of anhydrous THF

was cooled in an ice bath to which phenyl magnesium bromide (15.6

mL, 15.6 mmol, 1 M solution in THF) was added dropwise. The reaction

was stirred at 0 ^◦C for 1 h the allowed to warm to ambient temperature

then stirred for an additional 3 h. The reaction was quenched with

saturated ammonium chloride (60 mL), layers separated, and the

aqueous phase extracted with THF (2 × 50 mL), and the organic phase

extracted with brine (50 mL) then dried over sodium sulfate. Solvent

was removed and the residue was loaded onto a SNAP Ultra 50 g silica

column and eluted with a hexane:ethyl acetate gradient (5% to 5% 1

column volume, 5% to 40% 10 column volumes, 40% to 40% 2 column

volumes). Product fractions were pooled and dried yielding 3.42 g

(86%) of the product as a white solid. ¹H NMR, (400 MHz, CDCl₃), δ 7.35

(m, 5H, ArH), 6.88 (s, 2H, ArH), 5.77 (d, J = 4 Hz, 1H, CHOH), 5.21 (s,

4H, OCH₂O), 3.5 (s, 6H, OCH₃), 2.28 (d, J = 3.6 Hz,1H, CHOH), MS: m/z

(ESI, pos.) = 406.9 [M + 23]⁺.

Utilizing 2-thienyl magnesium (1 M solution in THF) following

alcohol was similarly prepared.

4.2.6. (4-Bromo-3,5-bis-methoxymethoxy-phenyl)-thiophen-2-yl-methanol

Pale yellow solid (1.57 g, 70%) ¹H NMR, (400 MHz, CDCl₃), δ 7.26

(dd, J = 3.2 Hz , 1.2 Hz, 1H, ThiophenylH), 6.93 (s, 2H, ArH), 6.89 (dt, J

= 3.3, 0.8 2H, ThiophenylH), 5.97 (d, J = 3.6 Hz, 1H, CHOH), 5.25 (s,

4H, OCH₂O), 3.49 (s, 6H, OCH₃), 2.74 (d, J = 4.0 Hz, 1H, CHOH), MS:

m/z (ESI, pos.) = 412.9 [M + 23]⁺.

4.2.3. (3,5-bis(methoxymethoxy)-4-bromophenyl) methanol

Into a 5 L bottom tap three necked ﬂask ﬁtted with a mechanical

stirrer, dropping funnel, and gas in let was charged 2 (143 g, 0.39 mol)

and 1.65 L anhydrous THF. The reaction apparatus was ﬂushed with N₂

then DIBAL (890 mL, 1.1 M solution in cyclohexane, 0.98 mol) was

cannula transferred to the funnel. The solution was cooled in a brine/ice

bath and DIBAL was added dropwise over 1 h. The ice bath was

removed, and the reaction allowed to warm to room temperature. After

stirring 2 h at room temperature the reaction was quenched, with

cooling, using 50 mL methanol followed by a saturated solution of so-

dium potassium tartrate (550 g in 800 mL water). The mixture was

stirred vigorously for 18 h, phases separated, the organic phase dried

over sodium sulfate, and the solvent removed under reduced pressure to

yield 98.8 g (83%) of the product as a white solid. ¹H NMR, (400 MHz,

CDCl₃), δ 6.85 (s, 2H, ArH), 5.26 (s, 4H, OCH₂O), 4.65 (d, J = 5.2 Hz, 2H,

CH₂OH), 3.52 (s, 6H, OCH₃), 1.96 (t, 1H, J = 5.6 Hz, CH₂OH), MS: m/z

(ESI, pos.) = 330.9 [M + 23]⁺.

4.2.7. (4-Bromo-3,5-bis-methoxymethoxy-phenyl)-phenyl-methanone (4)

Into a 500 mL ﬂask was charged PCC (3.58 g, 16.7 mmol) and 120

mL of dichloromethane. The mixture was brieﬂy heated to reﬂux then

(4-bromo-3,5-bis-methoxymethoxy-phenyl)-phenyl-methanol (3.04 g,

7.93 mmol) dissolved in 60 mL of dichloromethane was added and

stirred for 1 h at ambient temperature. To the mixture was added 150 mL

of 1 N sodium hydroxide and the mixture stirred for 45 min. The phases

were separated, and the green aqueous phase extracted with dichloro-

methane (2 × 100 mL) and combined organic phase dried with sodium

sulfate. The solvent was removed and the remaining material was loaded

onto a 50 g SNAP Ultra column and eluted with a hexane:ethyl acetate

gradient (5% to 5% 1 column volume, 5% to 40% 10 column volumes,

40% to 40% 2 column volumes). Product fractions were pooled and

dried yielding 2.72 g (90%) of the product as a white solid. ¹H NMR,

(400 MHz, CDCl₃), δ 7.81 (d, J = 8.4 Hz, 2H, ArH), 7.60 (t, J = 7.4 Hz,

1H, ArH), 7.50 (t, J = 7.6 Hz, 2H, ArH), 7.24 (s, 2H, ArH), 5.27 (s, 4H,

OCH₂O), 3.51 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 404.9 [M + 23]⁺.

The following ketone was similarly prepared.

4.2.4. 3,5-bis(methoxymethoxy)-4-bromobenzaldehyde (3)

Into a 5 L three necked bottom tapped ﬂask equipped with a me-

chanical stirrer was charged PCC (153 g, 0.71 mol) and 2 L of

dichloromethane. A solution of (3,5-bis(methoxymethoxy)-4-bromo-

phenyl) methanol in 500 mL of dichloromethane was added to the

vigorously stirred mixture and allowed to stir for 20 h at room tem-

perature. The tap port was cleared, and the mixture was removed. The

tap was closed and 1.5 L of dichloromethane and 2 L of 1 N sodium

hydroxide were added and the mixture vigorously stirred until all the

solids dissolved and the aqueous phase turned green. The organic phase

4.2.8. (4-Bromo-3,5-bis-methoxymethoxy-phenyl)- thiophen-2-yl-

methanone (5)

Pale yellow solid (1.07 g, 73%) ¹H NMR, (400 MHz, CDCl₃), δ 7.74

(dd, J = 4.9, 1.0 Hz, 1H, ThiophenylH), 7.67 (dd, J = 3.7, 1.0 Hz, 1H,

ThiophenylH), 7.33 (s, 2H, ArH), 7.18 (dd, J = 5.0, 3.8 Hz, 1H, Thio-

phenylH), 5.31 (s, 4H, OCH₂O), 3.54 (s, 6H, OCH₃), MS: m/z (ESI, pos.)

= 410.7 [M + 23]⁺.

10

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

4.2.9. (2,6-Bis-methoxymethoxy-3^′-trifuoromethyl-biphenyl-4-yl)-phenyl-

methanone (6)

= 7.6 Hz, 2H, ArH), 7.59 (t, J = 7.6 Hz, 1H, ArH), 7.49 (t, J = 7.6 Hz, 2H,

ArH), 7.31 (s, 2H, ArH), 6.93 (d, J = 8.8 Hz, 1H, ArH), 6.91 (s, 1H, ArH),

6.87 (d, J = 8.8 Hz, 1H, ArH) 5.08 (s, 4H, OCH₂O), 4.31 (s, 4H,

ArOCH₂CH₂O), 3.34 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 437.5 [M +

1]⁺.

Tetrahydrofuran and 0.5 N K₃PO₄were degassed via 3 cycles of

evacuation and nitrogen ﬂush with sonication. Into an oven dried re-

action tube and stirring bar was charged 4 (42.3 mg, 0.11 mmol), 3-tri-

ﬂuoromethyl phenyl boronic acid (32.6 mg, 0.17 mmol), and Pd-G3

SPhos (1.6 mg, 2

μmol) and the tube was sealed with a septum. The

4.2.16. (3^′-Hydroxymethyl-2,6-bis-methoxymethoxy-biphenyl-4-yl)-

phenyl-methanone (13)

tube was evacuated and ﬂushed with nitrogen 3 times then degassed

THF (0.5 mL) followed by 0.5 N tribasic potassium phosphate (2 mL)

were added via syringe. The mixture was stirred at ambient temperature

for 8 h after which ethyl acetate (5 mL) was added. Phases were sepa-

rated and the aqueous phase extracted with ethyl acetate (2 × 5 mL),

combine organic phase dried with sodium sulfate, and the solvent was

removed. The remaining material was loaded onto a 10 g SNAP Ultra

column and eluted with a hexane:ethyl acetate gradient (2% to 2% 1

column volume, 2% to 20% 10 column volumes, 20% to 20% 2 column

volumes). Product fractions were pooled and dried yielding 38.7 mg

(78%) of the product as a colorless viscous oil. ¹H NMR, (400 MHz,

CDCl₃), δ 7.88 (d, J = 7.6 Hz, 2H, ArH), 7.81 (d, J = 7.6 Hz, 1H, ArH),

7.68 (s, 1H, ArH), 7.59 (m, 3H, ArH), 7.53 (t, J = 7.6 Hz, 2H, ArH), 7.31

(s, 2H, ArH), 5.08 (s, ,4H, OCH₂O), 3.07 (s, 6H, OCH₃), MS: m/z (ESI,

pos.) = 447.3 [M + 1]⁺.

White solid (20.8 mg, 42%) ¹H NMR, (400 MHz, CDCl₃), δ 7.87 (d, J

= 7.6 Hz, 2H, ArH), 7.62 (t, J = 7.2 Hz, 1H, ArH), 7.50 (t, J = 7.6 Hz, 2H,

ArH), 7.41 (t, J = 6.0 Hz, 1H, ArH), 7.39 (m, 2H, ArH), 7.33 (t, J = 1.6

Hz, 1H, ArH) 7.31 (s, 2H, ArH) 5.05 (s, 4H, OCH₂O), 4.75 (s, 2H,

CH₂OH), 3.38 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 431.1 [M + 23]⁺.

4.2.17. 4^′-Benzoyl-4-fuoro-2^′,6^′-bis-methoxymethoxy-biphenyl-3-

carbonitrile (14)

White solid (29.3 mg, 21%) ¹H NMR, (400 MHz, CDCl3), δ 7.87 (d, J

= 8.0 Hz, 2H, ArH), 7.63 (m, 3H, ArH), 7.52 (t, J = 7.6 Hz, 2H, ArH),

7.30 (s, 2H, ArH), 7.23 (s, 1H, ArH) 5.06 (s, 4H, OCH₂O), 3.38 (s, 6H,

OCH₃), MS: m/z (ESI, pos.) = 444.3 [M + 23]⁺.

4.2.18. (3,5-Bis-methoxymethoxy-4-pyridin-3-yl-phenyl)-phenyl-

methanone (15)

Utilizing the appropriate aryl/heteroaryl boronic acids the following

methanones were prepared.

White solid (18.3 mg, 45%) ¹H NMR, (400 MHz, CDCl₃), δ 8.65 (d, J

= 2.0 Hz, 1H, PyridylH), 8.59 (dd, J = 4.8, 1.6 Hz, 1H, PyridylH), 7.88

(d, J = 8.0 Hz , 2H, ArH), 7.74 (dt, J = 8.0, 1.6 Hz, 1H, PyridylH), 7.61 (t,

J = 7.2 Hz, 1H, ArH), 7.52 (t, J = 6.4, 2H, ArH), 7.39 (dd, J = 7.6, 2.8 Hz,

1H, PyridylH), 7.33(s, 2H, ArH), 5.10 (s, 4H, OCH₂O), 3.38 (s, 6H,

OCH₃), MS: m/z (ESI, pos.) = 380.1 [M + 1]⁺.

4.2.10. (3^′,5^′-Difuoro-2,6-bis-methoxymethoxy-biphenyl-4-yl)-phenyl-

methanone (7)

Colorless resin (31.8 mg, 72%). ¹H NMR, (400 MHz, CDCl₃), δ 7.87

(d, J = 8.0 Hz , 2H, ArH), 7.63 (t, J = 7.2 Hz, 1H, ArH), 7.52 (t, J = 8.0

Hz, 2H, ArH), 7.29 (s, 2H, ArH), 6.90 (d, J = 7.2 Hz, 2H, ArH), 6.79 (t, J

= 8.8 Hz, 1H, ArH), 5.1 (s, 4H, OCH₂O), 3.33 (s, 6H, OCH₃), MS: m/z

(ESI, pos.) = 437.3 [M + 23]⁺.

4.2.19. [4-(2-Fluoro-pyridin-4-yl)-3,5-bis-methoxymethoxy-phenyl]-

phenyl-methanone (16)

White solid (13.8 mg, 27%) ¹H NMR, (400 MHz, CDCl₃), δ 8.27 (d, J

= 5.2 Hz, 1H, PyridylH), 7.86 (d, J = 8.0 Hz , 2H, ArH), 7.60 (t, J = 7.2

Hz, 1H, ArH), 7.51 (t, J = 7.2 Hz, 2H, ArH), 7.30 (s, 2H, ArH), 7.22 (dt, J

= 5.2, 1.2 Hz, 1H, PyridylH), 6.99 (s, 1H, PyridylH), 5.11 (s, 4H,

OCH₂O), 3.34 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 420.2 [M + 23]⁺.

4.2.11. 4.2.11 (2^′,5^′-Dichloro-2,6-bis-methoxymethoxy-biphenyl-4-yl)-

phenyl-methanone (8)

Colorless resin (17.6 mg, 41%) ¹H NMR, (400 MHz, CDCl₃), δ 7.88 (d,

J = 8.0 Hz, 2H, ArH), 7.61 (t, J = 7.6 Hz ,1H, ArH), 7.50 (t, J = 7.6 Hz,

2H, ArH), 7.42 (d, J = 8.4 Hz, 1H, ArH), 7.30 (s, 2H, ArH), 7.28–7.24 (m,

2H, ArH), 5.1 (s, 4H, OCH₂O), 3.48 (s, 6H, OCH₃), MS: m/z (ESI, pos.) =

447.4 [M + 1]⁺.

4.2.20. [4-(2-Chloro-pyridin-4-yl)-3,5-bis-methoxymethoxy-phenyl]-

phenyl-methanone (17)

White solid (7.1 mg, 17%) ¹H NMR, (400 MHz, CDCl₃), δ 8.45 (d, J =

4.4 Hz, 1H, PyridylH), 7.86 (d, J = 8.0 Hz , 2H, ArH), 7.62 (t, J = 7.6 Hz,

1H, ArH), 7.52 (t, J = 8.0 Hz, 2H, ArH), 7.38 (s, 1H, PyridylH), 7.29 (s,

2H, ArH), 7.26 (m, 1H, PyridylH), 5.11 (s, 4H, OCH₂O), 3.34 (s, 6H,

OCH₃), MS: m/z (ESI, pos.) = 414.2 [M + 1]⁺.

4.2.12. (2,6-Bis-methoxymethoxy-3^′,5^′-bis-trifuoromethyl-biphenyl-4-yl)-

phenyl-methanone (9)

Off white solid (73.9 mg, 60%) ¹H NMR, (400 MHz, CDCl₃), δ 7.87

(m, ,5H, ArH), 7.62 (t, J = 7.6 Hz,1H, ArH), 7.51 (t, J = 7.6 Hz , 2H,

ArH), 7.30 (s, 2H, ArH), 5.11 (s ,4H, OCH₂O), 3.37 (s, 6H, OCH₃), MS:

m/z (ESI, pos.) = 515.3 [M + 1]⁺.

4.2.21. (3,5-Bis-methoxymethoxy-4-thiophen-3-yl-phenyl)-phenyl-

methanone (18)

4.2.13. (2,6-Bis-methoxymethoxy-3^′-methyl-biphenyl-4-yl)-phenyl-

methanone (10)

Colorless viscous oil (12.1 mg, 33%) ¹H NMR, (400 MHz, CDCl₃), δ

7.86 (d, J = 8.4 Hz, 2H, ArH), 7.62 (t, J = 7.6 Hz, 1H, ArH), 7.52 (m,3H,

ArH/thiophenylH), 7.35 (m, 2H, thiophenylH), 7.31 (s, 2H, ArH), 5.15

(s ,4H, OCH₂O), 3.37 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 385.3 [M +

1]⁺.

Colorless resin (17.2 mg, 44%) ¹H NMR, (400 MHz, CDCl₃), δ 7.88 (d,

J = 8.0 Hz, 2H, ArH), 7.62 (t, J = 7.6 Hz, 1H, ArH), 7.50 (m, 5H, ArH),

7.33 (s, 2H, ArH) 7.24 (s, 1H, ArH), 5.06 (s, 4H, OCH₂O), 3.50 (s, 6H,

OCH₃), 2.41 (s, 3H, Me) , MS: m/z (ESI, pos.) = 415.3 [M + 23]⁺.

4.2.22. (4-Furan-3-yl-3,5-bis-methoxymethoxy-phenyl)-phenyl-methanone

(19)

4.2.14. (4-Benzo[1,3]dioxol-5-yl-3,5-bis-methoxymethoxy-phenyl)-

phenyl-methanone (11)

Colorless viscous oil (29.5 mg, 71%) ¹H NMR, (400 MHz, CDCl₃), δ

7.97 (s, 1H, FuranylH),7.84 (d, J = 8.0 Hz ,2H, ArH), 7.61 (t, J = 7.2 Hz ,

1H, ArH), 7.48 (m, 3H, ArH/furanylH), 7.31 (s, 2H, ArH), 6.97 (s,1H,

FuranylH), 5.19 (s, 4H, OCH₂O), 3.43 (s, 6H, OCH₃), MS: m/z (ESI, pos.)

= 369.3 [M + 1]⁺.

Colorless resin (36.4 mg, 81%) ¹H NMR, (400 MHz, CDCl₃), δ 7.85 (d,

J = 8.1 Hz, 2H, ArH), 7.58 (t, J = 6.4 Hz ,1H, ArH), 7.49 (t, J = 8.0 Hz,

2H, ArH), 7.31 (s, 2H, ArH), 6.87 (m, 3H, ArH), 6.00 (s, 2H, ArOCH₂O),

5.07 (s ,4H, OCH₂O), 3.38 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 445.2

[M + 23]⁺.

4.2.23. (2,6-Bis-methoxymethoxy-3^′-triﬂuoromethyl-biphenyl-4-yl)-

thiophen-2-yl-methanone (34)

4.2.15. [4-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)-3,5-bis-methoxymethoxy-

phenyl]-phenyl-methanone (12)

Pale yellow resin (34.4 mg, 71%) 1H NMR, (400 MHz, CDCl₃), δ

7.76–7.73 (m, 3H, ArH/ThiophenylH), 7.67 (s, 1H, ArH), 7.61 (m, 2H,

White solid (21.7 mg, 43%) ¹H NMR, (400 MHz, CDCl₃), δ 7.86 (d, J

11

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

ArH), 7.33 (s, 2H, ArH), 7.19 (m, 1H, ThiophenylH), 5.11 (s, 4H,

4.2.30. (3^′,5^′-Diﬂuoro-2,6-dihydroxy-biphenyl-4-yl)-phenyl-methanone

(21)

OCH₂O), 3.38 (s, 6H, OCH₃), MS: m/z (ESI, pos.) = 475.3 [M + 23]⁺.

Off white solid (14.9 mg, 60%) Analytical UPLC retention time 2.68

min; purity 99.8%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.81(d, J = 8.4 Hz,

2H, ArH), 7.67 (t, J = 7.4, 1H, ArH), 7.57 (t, J = 7.7 Hz, 2H, ArH), 7.13

(m, 2H, Ar’H), 6.97, (t, J = 9.1 Hz, 1H, Ar’H), 6.96, (s, 2H, ArH). ¹³C

NMR (100 MHz, (CD₃)₂CO), δ 195.8, 164.6, 164.5, 162.2, 161.1, 156.1,

139.3, 138.6, 138.5, 133.1, 130.4, 129.1, 118.9, 114.8, 114.7, 114.6,

109.6, 103.1, 102.8, 102.6. HRMS: m/z calculated for C₁₉H₁₃O₃F₂,

[M+H]⁺327.0833, found: 327.0834.

4.2.24. (2,6-Bis-methoxymethoxy-3^′-methyl-biphenyl-4-yl)-thiophen-2-yl-

methanone (35)

Pale yellow solid (34.1 mg, 79%) ¹H NMR, (400 MHz, CDCl₃), δ 7.75

(dd, J = 3.6, 1.2 Hz, 1H, ThiophenylH), 7.72 (dd, J = 5.2, 1.2 Hz, 1H,

ThiophenylH), 7.40 (s ,2H, ArH), 7.33 (t, J = 6.6 Hz, 1H, ArH), 7.18 (m,

4H, ArH/thiophenylH), 5.08 (s, 4H, OCH₂O), 3.39 (s, 6H, OCH₃), 2.48

(s, 3H, Me),MS: m/z (ESI, pos.) = 399.3 [M + 1]⁺.

4.2.25. (2,6-Bis-methoxymethoxy-3^′,5^′-bis-trifuoromethyl-biphenyl-4-yl)-

thiophen-2-yl-methanone (36)

4.2.31. (2^′,5^′-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-phenyl-methanone

(22)

Pale yellow solid (14.7 mg, 25%) ¹H NMR, (400 MHz, CDCl₃), δ 7.88

(s, 3H, ArH), 7.76 (dd, J = 5.0, 1.2 Hz, 1H,ThiophenylH), 7.74 (dd, J =

3.8 Hz, 1.2 Hz, 1H,ThiophenylH) 7.39 (s, 2H, ArH), 7.20 (dd , J = 5.0,

3.6 Hz, 1H, ThiophenylH), 5.14 (s, 4H, OCH₂O), 3.36 (s, 6H, OCH₃), MS:

m/z (ESI, pos.) = 543.1 [M + 23]⁺.

Off white solid (10.6 mg, 70%) Analytical UPLC retention time 3.31

min; purity 100%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.81(d, J = 8.4 Hz,

2H, ArH), 7.67 (t, J = 7.5, 1H, ArH), 7.55 (m, 3H, ArH), 7.41 (m, 2H,

ArH), 6.94, (s, 2H, ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 195.9, 156.5,

139.8, 138.7, 136.5, 134.2, 133.13, 133.1, 132.5, 131.5, 130.4, 129.7,

129.2, 118.1, 109.6. HRMS: m/z calculated for C₁₉H₁₃O₃Cl₂, [M+H]⁺

359.0242, found: 359.0247.

4.2.26. (2^′,5^′-Dichloro-2,6-bis-methoxymethoxy-biphenyl-4-yl)-thiophen-

2-yl-methanone (37)

Pale yellow resin (29.6 mg, 59%) ¹H NMR, (400 MHz, CDCl₃), δ 7.75

(overlapping dd, J = 4.8, 1.2 Hz , 2H, ThiophenylH), 7.42 (d, J = 8.4 Hz,

1H, ArH), 7.39 (s, 2H, ArH), 7.33–7.26 (m, 2H,ArH), 7.19 (dd, J = 5.2,

3.8 Hz, 1H, ThiophenylH), 5.12 (s, 4H, OCH₂O), 3.36 (s, 6H, OCH₃), MS:

m/z (ESI, pos.) = 475.1 [M + 23]⁺.

4.2.32. (2,6-Dihydroxy-3^′,5^′-bis-trifuoromethyl-biphenyl-4-yl)-phenyl-

methanone (23)

White solid (16.0 mg, 59%) Analytical UPLC retention time 3.91

min; purity 100%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.15 (s, 2H, Ar’H),

7.99 (s, 1H, Ar’H), 7.83 (d, J = 7.0, 1H, ArH), 7.68 (t, J = 7.4 Hz, 1H,

ArH), 7.57 (t, J = 7.3 Hz, 2H, ArH), 7.00, (s, 2H, ArH). ¹³C NMR (100

MHz, (CD₃)₂CO), δ 195.8, 156.2, 139.9, 138.5, 137.6, 133.3, 132.5,

131.5, 131.2, 130.5, 129.2, 126.1, 121.33, 121.3, 117.9, 109.7. HRMS:

m/z calculated for C₂₁H₁₃O₃F₆, [M+H]⁺427.0769, found: 427.0777.

4.2.27. (3^′-Hydroxymethyl-2,6-bis-methoxymethoxy-biphenyl-4-yl)-

thiophen-2-yl-methanone (38)

Pale yellow resin (12.5 mg, 29%) ¹H NMR, (400 MHz, CDCl₃), δ 7.75

(dd, J = 4.0, 1.2 Hz , 1H, ThiophenylH), 7.37 (dd, J = 4.8, 1.2 Hz, 1H,

ThiophenylH), 7.44 (t, J = 8.4 Hz, 1H, ArH), 7.40 (s, 2H, ArH), 7.38 (m,

2H, ArH), 7.31 (d, J = 7.6 Hz, 1H, ArH), 7.19 (dd, J = 5.0, 3.8 Hz, 1H,

ThiophenylH), 5.09 (s, 4H, OCH₂O), 4.76 (s, 2H, CH₂OH), 3.36 (s, 6H,

OCH₃), MS: m/z (ESI, pos.) = 437.1 [M + 23]⁺.

4.2.33. (2,6-Dihydroxy-3^′-methyl-biphenyl-4-yl)-phenyl-methanone (24)

White solid (11.6 mg, 87%) Analytical UPLC retention time 3.20

min; purity 100%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.81 (d, J = 7.6 Hz,

2H, ArH), 7.65 (t, J = 7.3, 1H, ArH), 7.56 (t, J = 7.3 Hz, 2H, ArH), 7.27

(m, 3H, Ar’H), 7.14 (d, J = 7.4 Hz, 1H, Ar’H) 6.95, (s, 2H, ArH), 2.37 (s,

3H, CH₃Ar). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 196.0, 156.3, 138.9,

138.4, 138.0, 134.4, 133.0, 132.3, 130.4, 129.1, 128.8, 128.7, 121.7,

109.7, 21.5. HRMS: m/z calculated for C₂₀H₁₇O₃, [M+H]⁺305.1178,

found: 305.1185.

4.2.28. (3,5-Bis-methoxymethoxy-4-pyridin-3-yl-phenyl)-thiophen-2-yl-

methanone (39)

Pale yellow resin (18.6 mg, 47%) ¹H NMR, (400 MHz, CDCl₃), δ 8.65

(dd, J = 2.0, 0.4 Hz, 1H, PyridylH), 8.59 (dd, J = 4.8, 2.0 Hz, 1H,

PyridylH), 7.75 (m, 3H, PyridylH/ThiophenylH), 7.41(s, 2H, ArH), 7.39

(dd, J = 7.9, 0.8 Hz, 1H, PyridylH), 7.20 (dd, J = 5.0, 4.0, Hz, 1H,

ThiophenylH), 5.12 (s, 4H, OCH₂O), 3.35 (s, 6H, OCH₃), MS: m/z (ESI,

pos.) = 408.0 [M + 23]⁺.

4.2.34. (4-Benzo[1,3]dioxol-5-yl-3,5-dihydroxy-phenyl)-phenyl-

methanone (25)

White solid (16.8 mg, 58%) Analytical UPLC retention time 2.49

min; purity 99.4%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.81 (d, J = 7.8 Hz,

2H, ArH), 7.66 (t, J = 7.4, 1H, ArH), 7.56 (t, J = 7.5 Hz, 2H, ArH),

4.2.29. (2,6-Dihydroxy-3^′-trifuoromethyl-biphenyl-4-yl)-phenyl-

methanone (20)

6.96–6.89 (m, 3H, Ar’H), 6.94, (s, 2H, ArH), 6.03 (s, 2H, Ar’OCH₂O). ¹³

C

To a suspension of 6 (38.7 mg, 86.7

μmol) in methanol (3 mL) was

NMR (100 MHz, (CD₃)₂CO), δ 195.9, 156.4, 148.3, 147.7, 138.9, 138.3,

133.0, 130.4, 129.1, 127.8, 125.2, 112.1, 109.7, 108.7, 101.9. HRMS:

m/z calculated for C₂₀H₁₅O₅, [M+H]⁺335.0919, found: 335.0916.

added 3 N hydrochloric acid in methanol (0.5 mL) and the mixture

heated to reﬂux. The solids dissolved in 1 h and after 2 h water (5 mL)

and ethyl acetate (5 mL) were added, phases separated, and the aqueous

phase extracted with ethyl acetate (2 × 5 mL) and organic phase dried

with sodium sulfate. The solvent was removed and the residue applied to

a 10 g SNAP Ultra column and eluted with a hexane:ethyl acetate

gradient (5% to 5% 1 column volume, 5% to 40% 10 column volumes,

40% to 40% 2 column volumes). Product fractions were pooled and

dried yielding 28.2 mg (91%) of the product as a colorless glass.

Analytical UPLC retention time 2.90 min; purity 98.2%. ¹H NMR, (400

MHz, (CD₃)₂CO), δ 7.84–7.78 (m, 4H, ArH), 7.67 (m, 3H, ArH), 7.57 (t,

J = 6.8 Hz, 2H, ArH), 6.98 (s, 2H, ArH). ¹³C NMR, (100 MHz, (CD₃)₂CO),

δ 195.8, 156.2, 139.1, 138.7, 135.9, 135.7, 133.1, 130.4, 130.1, 129.3,

129.1, 128.4, 128.4, 124.4, 124.4, 109.7. HRMS: m/z calculated for

4.2.35. [4-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)-3,5-dihydroxy-phenyl]-

phenyl-methanone (26)

White solid (16.7 mg, 96%) Analytical UPLC retention time 2.41

min; purity 98.2%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.81 (d, J = 7.2 Hz,

2H, ArH), 7.66 (t, J = 7.4, 1H, ArH), 7.56 (t, J = 7.6 Hz, 2H, ArH), 6.94

(t, J = 1.8, 1H, Ar’H), 6.93, (s, 2H, ArH), 6.92 (t, J = 2 Hz, 1H, Ar’H),

6.86 (d, J = 8.3, 1H, Ar’H), 2.85 (m, 4H, Ar’OCH₂CH₂O). ¹³C NMR (100

MHz, (CD₃)₂CO), δ 195.9, 170.9, 156.3, 144.2, 143.9, 138.9, 138.3,

132.9, 130.4, 129.1, 127.1, 124.8, 120.5, 117.4, 109.6, 65.3, 65.1.

HRMS: m/z calculated for

C

₂₁H₁₇O₅, [M+H]⁺349.1076, found:

349.1074.

C

₂₀H₁₄F₃O₃, [M+H]⁺359.0895, found: 359.0892.

The following resorcinols were similarly prepared.

4.2.36. (2,6-Dihydroxy-3^′-hydroxymethyl-biphenyl-4-yl)-phenyl-

methanone (27)

White solid (8.2 mg, 54%) Analytical UPLC retention time 2.09 min;

12

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

purity 95.4%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.82 (d, J = 7.8 Hz, 2H,

ArH), 7.66 (t, J = 7.5, 1H, ArH), 7.56 (t, J = 7.7 Hz, 2H, ArH), 7.45 (s,

1H, Ar’H), 7.40–7.32 (m, 3H, Ar’H), 6.94, (s, 2H, ArH), 4.68 (s, 2H,

CH₂OH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 196.0, 156.3, 142.9, 138.9,

138.5, 134.4, 133.0, 130.4, 130.2, 129.9, 129.1, 128.5, 126.3, 121.7,

109.7, 64.8. HRMS: m/z calculated for C₂₀H₁₇O₄, [M+H]⁺321.1127,

found: 321.1114.

141.9, 139.0, 137.0, 132.9, 130.4, 129.1, 118.6, 109.9. HRMS: m/z

calculated for C₁₇H₁₃O₄, [M+H]⁺281.0814, found: 281.0819.

4.2.43. (2,6-Dihydroxy-3^′-triﬂuoromethyl-biphenyl-4-yl)-thiophen-2-yl-

methanone (40)

Pale yellow solid (18.3 mg, 66%). Analytical UPLC retention time

2.81 min; purity 98.8%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.00 (dd, J =

4.8, 1.0 Hz, 1H, ThiophenylH), 7.83 (dd, J = 3.7, 1.0 Hz, 1H, Thio-

phenylH), 7.80 (m, 2H, Ar’H), 7.66 (m, 2H, Ar’H), 7.29 (dd, J = 4.8, 3.8

Hz, 1H, ThiophenylH), 7.09 (s, 2H, ArH). ¹³C NMR (100 MHz,

(CD₃)₂CO), δ 187.4, 170.9, 156.4, 156.3, 144.3, 139.6, 135.9, 135.8,

135.7, 135.5, 135.4, 130.5, 130.1, 129.4, 129.1, 128.5, 128.4, 126.9,

124.5, 1244, 124.3, 124.2119.6, 108.5. HRMS: m/z calculated for

4.2.37. 4^′-Benzoyl-4-ﬂuoro-2^′,6^′-dihydroxy-biphenyl-3-carbonitrile (28)

White solid (3.2 mg, 26%) Analytical UPLC retention time 2.52 min;

purity 98%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.91 (m, 2H, Ar’H), 7.81

(d, J = 7.6, 2H, ArH), 7.67 (t, J = 7.2 Hz, 1H, ArH), 7.57 (t, J = 7.6, 2H,

ArH), 7.48 (t, J = 9.2 Hz, 1H, Ar’H), 6.97 (s, 2H, ArH). ¹³C NMR (100

MHz, (CD₃)₂CO), δ 195.7, 156.2, 139.4, 139.38, 139.3, 138.6, 136.9,

133.2, 130.5, 129.2, 116.7, 116.5, 109.7, 79.3, 79.0. HRMS: m/z

calculated for C₂₀H₁₃FNO₃, [M+H]⁺334.0879, found: 334.0871.

C

₁₈H₁₂O₃F₃S, [M+H]⁺365.0459, found: 365.0468.

4.2.44. (2,6-Dihydroxy-3^′-methyl-biphenyl-4-yl)-thiophen-2-yl-methanone

(41)

4.2.38. (3,5-Dihydroxy-4-pyridin-3-yl-phenyl)-phenyl-methanone (29)

White solid (11.5 mg, 85%) Analytical UPLC retention time 2.32

min; purity 96.2%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.70 (d, J = 2.2 Hz,

1H, PyridylH), 8.50 (dd, J = 4.9 Hz, 1.7 Hz, 1H, PyridylH), 7.88 (dt, J =

7.7 Hz, 2.1 Hz, 1H, PyridylH), 7.82 (d, J = 7.8, 2H, ArH), 7.66 (t, J = 7.4

Hz, 1H, ArH), 7.56 (t, J = 7.7, 2H, ArH), 7.41 (dd, J = 8.0 Hz, 2.8, 1H,

PyrH), 6.98 (s, 2H, ArH).

Pale yellow solid (17.3 mg, 65%). Analytical UPLC retention time

2.64 min; purity 98.1%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.98 (dd, J =

5.0, 1.0 Hz, 1H, ThiophenylH), 7.82 (dd, J = 4.0, 1.0 Hz, 1H, Thio-

phenylH), 7.33–7.23 (m, 4H, Ar’H/ThiophenylH), 7.14 (d, J = 7.4 Hz,

1H, Ar’H), 7.05 (s, 2H, ArH), 2.37 (s, 3H, Ar’CH₃). ¹³C NMR (100 MHz,

(CD₃)₂CO), δ 187.5, 156.4, 144.4, 138.9, 138.0, 135.4, 135.1, 134.3,

132.3, 129.8, 128.5, 128.8, 128.7, 128.6, 121.6, 108.9, 21.5. HRMS: m/

z calculated for C₁₈H₁₅O₃S, [M+H]⁺311.0742, found: 311.0707.

¹³C NMR (100 MHz, (CD₃)₂CO), δ 195.9, 157.9, 156.5, 152.3, 148.6,

139.2, 139.0, 138.7, 133.1, 130.8, 130.5, 129.2, 123.6, 118.0, 109.6.

HRMS: m/z calculated for C₁₈H₁₄NO₃, [M+H]⁺292.0974, found:

292.0966.

4.2.45. (2,6-Dihydroxy-3^′,5^′-bis-triﬂuoromethyl-biphenyl-4-yl)-thiophen-

2-yl-methanone (42)

Pale yellow solid (13.1 mg, 96%). Analytical UPLC retention time

3.11 min; purity 99.3%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.16 (s, 2H,

Ar’H), 8.02 (dd, J = 5.0, 1.1 Hz, 1H, ThiophenylH), 7.99 (s, 1H, Ar’H),

7.83 (dd, J = 3.8, 1.0 Hz, 1H, ThiophenylH), 7.30 (dd, J = 5.0, 3.8 Hz,

1H, ThiophenylH), 7.10 (s, 2H, ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ

187.3, 170.9, 156.3, 144.2, 140.4, 137.6, 135.7, 135.6, 132.5, 131.8,

131.5, 131.2, 130.9, 129.2, 126.1, 123.4, 121.4, 121.3, 121.2, 117.8,

108.9. HRMS: m/z calculated for C₁₉H₁₁O₃F₆S, [M+H]⁺433.0333,

found: 433.0334.

4.2.39. [4-(2-Fluoro-pyridin-4-yl)-3,5-dihydroxy-phenyl]-phenyl-

methanone (30)

White solid (3.6 mg, 20%) Analytical UPLC retention time 2.67 min;

purity 95.6%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.43 (d, J = 5.2 Hz, 1H,

PyridylH), 7.82 (d, J = 7.8, 2H, ArH), 7.68 (t, J = 7.2 Hz, 1H, ArH), 7.57

(m, 3H, ArH/PyridylH), 7.52 (dd, J = 5.0 Hz, 1.5, 1H, PyridylH), 6.96 (s,

2H, ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 195.7, 156.2, 151.4, 149.8,

146.6, 138.5, 133.3, 130.5, 129.2, 126.9, 126.0, 109.6. HRMS: m/z

calculated for C₁₈H₁₃NO₃F, [M+H]⁺310.0879, found: 310.0890.

4.2.46. (2^′,5^′-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-thiophen-2-yl-

methanone (43)

4.2.40. [4-(2-Chloro-pyridin-4-yl)-3,5-dihydroxy-phenyl]-phenyl-

methanone (31)

Pale yellow solid (24.3 mg, 98%). Analytical UPLC retention time

2.72 min; purity 97.9%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.99 (dd, J =

4.9, 1.0 Hz, 1H, ThiophenylH), 7.82 (dd, J = 3.8, 1.1 Hz, 1H, Thio-

phenylH), 7.54 (dd, J = 6.5, 2.4 Hz, 1H, Ar’H), 7.42 (dd, J = 7.2, 2.4 Hz,

2H, Ar’H), 7.29 (dd, J = 5.0, 3.8, Hz, 1H, ThiophenylH), 7.05 (s, 2H,

ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 187.4, 170.9, 156.5, 144.3,

140.3, 136.5, 135.6, 135.4, 134.2, 133.1, 132.5, 131.5, 129.7, 129.1,

117.9, 108.5. HRMS: m/z calculated for C₁₇H₁₁O₃SCl₂, [M+H]⁺

364.9806, found: 364.9796.

White solid (3.6 mg, 20%) Analytical UPLC retention time 2.51 min;

purity 97.0%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.25 (d, J = 5.6 Hz, 1H,

PyridylH), 7.82 (d, J = 7.2, 2H, ArH), 7.67 (t, J = 7.6 Hz, 1H, ArH), 7.57

(t, J = 7.2 Hz, 2H, ArH), 7.46 (d, J = 5.0 Hz, 1H, PyridylH), 7.19 (s, 1H,

PyridylH) 6.97 (s, 2H, ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 195.7,

156.2, 147.6, 146.2, 138.5, 133.3, 130.5, 129.2, 125.0, 109.6. HRMS:

m/z calculated for C₁₈H₁₃NO₃Cl, [M+H]⁺326.0584, found: 326.0578.

4.2.41. (3,5-Dihydroxy-4-thiophen-3-yl-phenyl)-phenyl-methanone (32)

Off white solid (5.9 mg, 64%) Analytical UPLC retention time 2.99

min; purity 100%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.80 (d, J = 7.4,

2H, ArH), 7.72 (dd, J = 2.9, 1.3 Hz, 1H, ThiophenylH), 7.65 (t, J = 7.3

Hz, 1H, ArH), 7.56 (t, J = 7.6 Hz, 2H, ArH), 7.51 (dd, J = 5.2, 1.2 Hz, 1H,

ThiophenylH), 7.45 (dd, J = 5.0, 3.0 Hz, 1H, ThiophenylH), 6.96 (s, 2H,

ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 195.8, 156.4, 138.9, 138.0,

133.7, 132.9, 131.2, 130.4, 129.1, 126.4, 124.1, 109.9. HRMS: m/z

calculated for C₁₇H₁₃O₃S, [M+H]⁺297.0585, found: 297.0584.

4.2.47. (2,6-Dihydroxy-3^′-hydroxymethyl-biphenyl-4-yl)-thiophen-2-yl-

methanone (44)

Pale yellow solid (7.2 mg, 73%). Analytical UPLC retention time

2.16 min; purity 99.8%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 7.98 (dd, J =

5.0, 1.0 Hz, 1H, ThiophenylH), 7.83 (dd, J = 3.8, 1.1 Hz, 1H, Thio-

phenylH), 7.45 (s, 1H, Ar’H), 7.39–7.32 (m, 2H, Ar’H) 7.29 (dd, J = 5.2,

3.8, 1H, Ar’H), 7.29 (dd, J = 5.0, 3.7, Hz, 1H, ThiophenylH), 7.06 (s, 2H,

ArH), 4.68 (s, 2H, CH₂OH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 187.5,

173.3, 156.4, 144.4, 142.9, 138.9, 135.4, 135.2, 134.3, 130.2, 129.9,

129.1, 128.6, 126.2, 121.6, 108.9. HRMS: m/z calculated for C₁₈H₁₅O₄S,

[M+H]⁺327.0691, found: 327.0677.

4.2.42. 4.2.42 (4-Furan-3-yl-3,5-dihydroxy-phenyl)-phenyl-methanone

(33)

Off white solid (13.4 mg, 60%). Analytical UPLC retention time 2.96

min; purity 96.1%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.21 (s, 1H, Fur-

anylH), 7.79 (d, J = 7.2, 2H, ArH), 7.65 (t, J = 7.3 Hz, 1H, ArH),

7.59–7.53 (m, 3H, ArH/FuranylH), 7.26 (d, J = 2 Hz, 1H, FuranylH),

6.97 (s, 2H, ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 195.8, 156.4, 144.5,

4.2.48. (3,5-Dihydroxy-4-pyridin-3-yl-phenyl)-thiophen-2-yl-methanone

(45)

Pale yellow solid (9.4 mg, 66%). Analytical UPLC retention time

2.09 min; purity 99.5%. ¹H NMR, (400 MHz, (CD₃)₂CO), δ 8.70 (dd, J =

13

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

2.0 Hz, 0.4 Hz, 1H, PyridylH), 8.50 (dd, J = 4.7 Hz, 2.0 Hz, 1H, PyrH),

7.99 (dd, J = 5.1, 1.0 Hz, 1H, ThiophenylH), 7.88 (dt, J = 7.9, 1.7 Hz,

1H, PyridylH), 7.83 (dd, J = 3.8, 1.1 Hz, 1H, ThiophenylH), 7.42 (dd, J

= 7.9, 0.8 Hz, 1H, PyridylH), 7.29 (dd, J = 5.0, 3.7, Hz, 1H, Thio-

phenylH), 7.09 (s, 2H, ArH). ¹³C NMR (100 MHz, (CD₃)₂CO), δ 187.4,

170.9, 156.6, 152.4, 148.6, 144.3, 139.7, 138.9, 135.5, 135.4, 130.7,

129.1, 123.6, 117.9 108.9. HRMS: m/z calculated for C₁₆H₁₂NO₃S,

[M+H]⁺298.0538, found: 298.0535.

2–3 days or when conﬂuence reached 80%. Cell culture ﬂasks were

changed every ﬁve passages or after two weeks of use.

Unless otherwise stated, all cell culture experiments wherein incu-

bation is stated used humidiﬁed incubation chambers maintained at

37 ^◦C and 5% CO₂.

4.3.2. Membrane preparations

The membrane preparations were prepared from the functional cell

lines HEK-CNG + CB1 or HEK-CNG + CB2 cell lines. Brieﬂy, HEK-CNG

+ CB1 or HEK-CNG + CB2 cells were plated in 500 cm²culture dishes

and incubated for two days or when it reached 80 to 90% conﬂuence.

Media was removed and cells were washed with 10 mL of ice-cold PBS.

Cells were scrapped after the addition of 10 mL of ice-cold PBS with 10

mmol/L EDTA and the cell suspension was centrifuged at 4 ^◦C, 500g for

5 min. The supernatant was discarded, and the pellet homogenized in

10 mL of solution A (50 mM Tris HCL, 320 mM sucrose, 2 mM EDTA, and

5 mM MgCl2). The cell suspension was centrifuged at 4 ^◦C, 1600g for 10

min. The supernatant was saved, and the cell pellet washed three times

in 8 mL of solution A. All supernatants were combined and centrifuged

at 4 ^◦C, 50,228g for 3 h. The supernatant was discarded, and the pellet

was suspended in 3 mL of solution B (50 mM Tris HCL, 10% sucrose, 2.5

mM EDTA, and 5 mM MgCl2). Protein concentration was determined

using a Pierce BCA protein assay kit and membrane aliquots were stored

at ꢀ 80 ^◦C. Scatchard analysis was carried out on CB1 and CB2 mem-

brane preparations yielding a K_d= 1.4 nmol/L and B_max= 1.7 pmol/mg

for CB1 and for CB2 a K_d= 2.3 ± 0.8 nmol/L with B_max1.25 ± 0.35

pmol/mg.

4.3. Biological reagent and supplies

The HEK-CNG parental, HEK-CNG + CB1 (human), and HEK-CNG +

CB2 (human) cell lines, and ACTOne Membrane Potential Dye were

purchased from Codex BioSolutions, Inc (Gaithersburg, MD). Tango™

CNR2-bla and CNR1-bla U2OS human cell lines were purchased from

Invitrogen (Carlsbad, CA). C8-B4 (ATCC® CRL-2540™) was purchased

from ATCC (Manassas, Virginia). Immortalized Human Microglia SV40

was purchased from ABM (Richmond, BC). Puromycin, DMEM, peni-

cillin/streptomycin, gentamicin, PBS, 0.25% Trypsin/EDTA, Hank’s

Buffer, HEPES, EDTA, Tris base, sucrose, MgCl₂, Millipore ﬁlter plates

and punch kits, EcoLite scintillation cocktail, and Poly-d-lysine coated

96 well plates catalog # 356,461 were purchased from Fisher Scientiﬁc

(Waltham, MA). G418 was purchased from KSE Scientiﬁc (Durham, NC).

FBS was purchased from Atlanta Biologicals (Flowery Branch, GA). Ro

20-1724, acetonitrile, DMSO, polyethylene amine, and fatty acid-free

BSA were purchased from Sigma Aldrich (St. Louis, MO). Forskolin

and CP 55,940 were purchased from Tocris (Bristol, UK). LiveBLAzer™-

FRET B/G Loading Kit, Solution D, Recovery™ Cell Culture Freezing

Medium, FreeStyle™ Expression Medium, McCoy’s 5A Medium (modi-

ﬁed) (1X), fetal bovine serum (FBS) dialyzed, Non-essential amino acids

(NEAA), sodium pyruvate, HEPES (1 M, pH 7.3), 0.05% Trypsin/EDTA,

Zeocin ™, Hygromycin, Geneticin were purchased from Invitrogen

(Carlsbad, CA). Black-wall, clear-bottom, 96-well assay plates #3603

were purchased from Corning (Corning, NY). Tritiated [³H]-CP 55,940

was purchased from PerkinElmer (Waltham, MA). The 96-well ﬁlter

plates and punch tips were purchased from Millipore (Billerica, MA).

Pierce BCA protein assay kit and Fisherbrand™ 7 mL Borosilicate Glass

Scintillation Vial were purchased from Thermoﬁsher (Waltham, MA).

Ecolite(+) LC Fluid, MP Biomedicals was purchased from Tocris (Min-

neapolis, MN). Dojindo Molecular Technologies IncSupplier Diversity

Partner Cell Counting Kit-8 (NC9864731) was purchased from Ther-

moﬁsher (Waltham, MA). Lipopolysaccharide (LPS) from Escherichia

coli O111: B4 (L2630) was purchased from Sigma Aldrich (St. Louis,

MO). Antibodies against murine CD 16/32 (ab25235), CD 68

(ab213363), CD 204 (ab123946) and CD 206 (ab64693) were purchased

from Abcam (Cambridge, UK). The human CD16 antibody was pur-

chased from R&D systems. Meso-Scale Discovery (MSD) high bind plates

(L15XB), anti-rat (R32AH-5), and anti-rabbit (R32AB-5) SULFO-TAG

antibodies were purchased from Meso-Scale Discovery (Gaithersburg,

MD). Prigrow III Medium (TM003) was purchased from ABM (Rich-

mond, BC).

4.3.3. Receptor binding assays

Millipore ﬁlter plates were ﬁlled with 210 μL/well of 0.05% (w/v)

polyethylene amine in deionized water and incubated for 1 h at room

temperature. Plates were washed ﬁve times with 250 L/well of

μ

deionized water then ﬁltered using a vacuum manifold before starting

the experiment. After plate coating, 125

each well, followed by the addition of 5

μL binding buffer was added to

μ

L of [³H]-CP 55,940, 10

μg of

membrane protein in binding buffer, and 50 μL of a test compound in

binding buffer (ﬁnal concentration ranging from 1 nmol/L to 10 μmol/

L). Plates were incubated at 30 ^◦C for 1.5 h, then solutions were removed

via vacuum and washed nine times with binding buffer. The plates were

vacuumed dry and punch tips were used to eject the ﬁlters into 5 mL of

Eco-lite scintillation cocktail in 7 mL scintillation vials. Counts per

minute (CPM) were measured the next day using PerkinElmer Liquid

Scintillation Analyzer Tri-Carb 2810TR, 3-min dwell time. At least six

biological replicates were used for data analysis.

4.3.4. Actone cAMP functional assay

The HEK-CNG (parental cell line), HEK-CNG + CB1, and HEK-CNG +

CB2 cells were plated in clear poly-^D-lysine coated 96-well plates in

plating media (DMEM, 10% FBS, 1% P/S) at 50,000 cells/100

μ

L me-

dium, and plates were incubated for 16–18 h. The next day, 100

μ

L of

ACTOne membrane potential dye was added to each well and the plates

were maintained at room temperature for 1 h in the dark. 5X of tested

compounds were prepared from 1 × 10^{ꢀ 6}to 1 × 10^{ꢀ 9}containing 25

4.3.1. Cell culture

μmol/L Ro 20–1724, and 800 nmol/L forskolin in PBS with 2.5% (v/v)

CNR2-bla and CNR1-bla U2OS cell lines were cultured in McCoy’s 5A

medium with 10% dialyzed FBS, 1%P/S, 0.1 mM NEAA, 25 mM HEPES,

DMSO. Following the addition of the test compounds, plates were read

every 5 min using a BioTek (Winooski, VT) plate reader (Ex 540 nm, Em

590 nm) for 1 h. The data at 50 mins were combined and analyzed to

calculate the EC₅₀. Six to nine biological replicates were used for data

analysis. The non-selective agonist CP 55,940 was used as a positive

control in each experimental run. An assay was considered acceptable if

the EC₅₀s for CP 55,940 ranged from 1 to 10 nM and % cAMP sup-

pression was 70 to 95 for both cell lines.

1 mM sodium pyruvate, 200

100

μg/ml Zeocin ™, 50 μg/ml Hygromycin,

μ

g/ml Geneticin. CNR2-bla and CNR1-bla U2OS cells were passaged

every 2 to 3 days or when conﬂuence reach 80%. HEK-CNG + CB1 and

HEK-CNG + CB2 cells were cultured in DMEM with 10% FBS, 1% P/S,

250 µg/ml G418, and 5 μg/mL puromycin. HEK-CNG cells were cultured

in DMEM with 10% FBS, 1% P/S, and 250 µg/ml G418. HEK-CNG + CB1

and HEK CNG + CB2 cells were passaged every 2–3 days or when

conﬂuence reached 80%. C8B4 cells were cultured in DMEM with 10%

FBS, 1% P/S, and passaged every one or two weeks or when conﬂuence

reaches 80%. Immortalized SV40 human microglia were maintained in

Prigrow III media with 1% P/S and 10% FBS and were passaged every

4.3.5. Tango™ -BLA β-Arrestins functional assay

CNR2-bla and CNR1-bla U2OS cells were plated in black-wall, clear-

bottom, 96 well assay plates in FreeStyle™ Expression Medium at

14

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

50,000 cells/100 µL well and incubated for 16–18 h. A 10X stock of test

compounds was prepared in FreeStyle™ Expression Medium with 0.5%

DMSO. The ﬁnal concentrations of test compounds ranged from 1 ×

10^{ꢀ 6}to 10^{ꢀ 9}M and the internal control CP-55,940 at 5 µM. Additional

controls contained assay medium with 0.5% DMSO was added in the

cell-free wells and to unstimulated cell wells. The plates were incubated

for 5 h then 20 µL of a 6X loading solution, prepared according to the

manufacture’s protocol, was added to each well. The plates were incu-

bated at room temperature for an additional 2 h in the dark. Plates were

read using Synergy 2 BioTek (Winooski, VT) plate reader (Ex 409/20

nm, Em 460/40 nm, Em 530/30 nm) with ﬂuorescence reading set to a

bottom-read mode with optimal gain and 5 reads. CP 55,940 served as a

positive control for all runs. An acceptable run was deﬁned by CP 55,940

EC₅₀between 30 and 40 nM for CB2 and 1–10 nM for CB1, with %

activation range between 80 and 100% for both cell lines. Three to nine

biological replicates were used for data analysis.

Prigrow III media containing LPS, was added to the treatment group.

Plates were incubated for an additional 24 h. Images from 7 biological

replicates were taken at 0 h and 24 h and the number of cells migrated

was analyzed using ImageJ.

4.3.9. Microglia morphology – Cytoskeleton staining

SV40 cells were plated on 12 well collagen I coated plates and

incubated for 18–24 h. The following day, Prigrow III 10% FBS media

was replaced with 1% FBS media, and the plates were incubated for an

additional 24 h. The cells were then stimulated with LPS (1

μ

g/ml) or a

combination of compound 40 (at 10 μM) + LPS (1 μg/ml) or left un-

treated. Plates were incubated for an additional 24 h. Cells were stained

using actin cytoskeleton staining kit (FAK100) according to manufac-

turer protocol. TRITC-conjugated Phalloidin was used to detect F-actin

and DAPI was used to stain the nucleus. Ten ﬁelds were taken for each

group using a BZ-X ﬂuorescence microscope.

For the competition (antagonist) assays cells were treated with

compound concentrations ranging from 1 × 10^{ꢀ 6}to 10^{ꢀ 9}M and incu-

bated for 30 min. To the cells was then added 10 µL of CP 55,940 so-

lution (10X the EC₈₀) prepared in an expression assay medium with

0.5% DMSO and allowed to incubate for 5 h. After the incubation in-

terval, 6X loading solution was added to each well and the plates were

incubated at room temperature for an additional 2 h protected from

light. Plates were read using as before from at least four biological

replicates.

4.3.10. Measurement of ROS

SV40 cells were plated at 30,000 cells/ml on 96 well black wall clear

bottom collagen I coated plate and incubated for 24 h. The following day

the Prigrow III 10% FBS media was replaced with serum-free media and

the plates were incubated for 18–24 h. The media was then removed,

and dye was added to the cells at 10 μM concentration and incubated for

30 min. Plates were washed twice with serum-free media (phenol red-

free) and then treated with serum-free media (negative control), LPS

(100 ng/ml), or compound 40 (at 10 μM) + LPS (100 ng/ml). Reading

4.3.6. Cytotoxicity assays

was taken at 50 min and 24 h. The ﬂuorescence measurement was made

using BioTek Synergy 2 (BioTek Instruments, Inc. Winooski, VT) with

485/20 excitation and 528/20 emission ﬁlters set to bottom read.

C8B4 or SV40 cells were plated at 5000–10,000 cells/well (100 μL/

well) on a 96 well plate and incubated for 24 h. On the second day, the

10% FBS media was replaced with 1% FBS media, and plates were

incubated overnight. Compound 40 was added at ﬁnal concentrations of

10^{ꢀ 4}to 10^{ꢀ 6.5}and plates were incubated for an additional 24 h. To the

plates was added 10 µL of CCK-8 Reagent to each well and the plate was

incubated for at least 2 h in the incubator followed by absorbance

measurement at 450 nm wavelength using BioTek plate reader.

4.3.11. Data analysis

Binding assay K_ivalues were determined using Cheng and Prusoff

equation.⁵³For the ACTOne data, the analysis was done using non-linear

regression analysis and ﬂuorescent measurements at 50 mins. The data

were normalized using a scaling equation:

(X ꢀ Xmin)(B ꢀ A)

X^′= A +

4.3.7. M1 and M2 murine and human microglia cell surface markers

C8B4 or SV40 cells were plated on 96-well Meso Scale Discovery high

bind plate at a density of 20,000 cells in 50 µL (10,000 cells in 100 µL)

per well and incubated overnight. The 10% FBS medium was removed

Xmax ꢀ Xmin

where A = Blank well-containing dye and cells only, B = Forskolin

stimulation, X^′= Normalized value, X max = 100%, X = min 0%. The

Tango-BLA assay analysis on the ﬂuorescent measurements was carried

out by subtracting the background (cell-free wells) from both emission

channels (460 nm and 530 nm). Blue/Green Emission Ratio was calcu-

lated for each well by dividing the background-subtracted blue emission

values by the background-subtracted green emission values.

and replaced with 100

μL of 1% FBS medium and the plates were

incubated for 18–24 h. The following day LPS (ﬁnal concentration 100

ng/mL) was added to each well and incubated for 1 h (except control

wells received 1% FBS media). After 1 h of LPS stimulation, compound

40 was added at 20 μM for C8B4 and 35 μM for SV40 cells, and plates

were incubated for an additional 24 h. The expression of M1 (CD 16/32,

CD 16, CD 68) and M2 markers (CD 204, and CD 206) was measured

using 30 µL per well of antibody prepared in PBS at a concentration of 2

µg/mL. After a 2 h incubation on a shaker plate at 130 rpm, wells were

washed using 150 µL PBS, and 30 µL per well of secondary antibody

(anti-rat or anti-rabbit) containing Sulfo-tag in PBS was added at a

concentration of 2 µg/mL. Plates were then incubated for an additional

2 h, washed three times with 150 µL per well of PBS, and read in 2X

surfactant-free read-buffer using a SECTOR Imager 2400 (Meso Scale

Discovery).

Background-Subtracted Fluorescence (Fl) = Fl _Sample– Fl _{Cell-free control}

Coumarin Emission (460 nm)

Em ission Ratio (ER) =

Fluroescin Emission (530 nm)

Funding

This research did not receive any speciﬁc grant from funding

agencies in the public, commercial, or not-for-proﬁt sectors.

Declaration of Competing Interest

4.3.8. Scratch assay

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

SV40 cells were plated at 50,000 cells/ml on 12 wells collagen I

coated plates and incubated overnight. The next day Prigrow III 10%

FBS media was replaced with 1% FBS media and the plates were incu-

bated for 18–24 h. Following the equilibration period, a scratch was

made using 100 μL pipette tip and wells were gently washed twice with

Acknowledgments

1% FBS Prigrow III media to remove detached and ﬂoating cells. Cells

were then stimulated with LPS (100 ng/ml) for LPS control and treated

wells for 30 mins (control wells received 1% FBS media only). After 30

This research was supported by Dr. Kennard Brown and the Plough

Center for Sterile Drug Delivery Systems, University of Tennessee Health

Science Center. The fellowship for S.S.A. was provided by King Saud Bin

min LPS stimulation, compound 40 (10

μM), prepared in 1% FBS

15

S.S. Alghamdi et al.

Bioorganic & Medicinal Chemistry 33 (2021) 116035

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