Synthesis and biological evaluation of (3′,5′-dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone selective CB2 inverse agonist

Article abstract of DOI:10.1016/j.bmc.2015.07.057

Cannabinoid receptor 2 (CB2) selective agonists and inverse agonists possess significant potential as therapeutic agents for regulating inflammation and immune function. Although CB2 agonists have received the greatest attention, it is emerging that inverse agonists also manifest anti-inflammatory activity. In process of designing new cannabinoid ligands we discovered that the 2,6-dihydroxy-biphenyl-aryl methanone scaffold imparts inverse agonist activity at CB2 receptor without functional activity at CB1. To further explore the scaffold we synthesized a series of (3′,5′-dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone analogs and evaluated the CB1 and CB2 affinity, potency, and efficacy. The studies reveal that an aromatic C ring is required for inverse agonist activity and that substitution at the 4 position is optimum. The resorcinol moiety is required for optimum CB2 inverse agonist activity and selectivity. Antagonist studies against CP 55,940 demonstrate that the compounds 41 and 45 are noncompetitive antagonists at CB2.

Full text of DOI:10.1016/j.bmc.2015.07.057

Accepted Manuscript
Synthesis and Biological Evaluation of 3’,5’-Dichloro-2,6-dihydroxy-biphen-
yl-4-yl)-aryl/alkyl-methanone Selective CB2 Inverse Agonist
Chaela S. Presley, Suni M. Mustafa, Ammaar H. Abidi, Bob M. Moore II
PII:
S0968-0896(15)00641-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.057
BMC 12486
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
9 June 2015
16 July 2015
25 July 2015
Please cite this article as: Presley, C.S., Mustafa, S.M., Abidi, A.H., Moore, B.M. II, Synthesis and Biological
Evaluation of 3’,5’-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone Selective CB2 Inverse Agonist,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.057
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Synthesis and Biological Evaluation of 3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone Selective CB2 Inverse Agonist
Chaela S. Presley,a
Suni M. Mustafa,a
Ammaar H. Abidi,a
Bob M.Moore, II,a*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, 38163, USA
*Corresponding author: 847 Monroe Ave. Room 327D, Memphis, TN 38163, USA
Tel.: 1-901-448-6085; fax: 1-901-448-6828; e-mail: bmoore@uthsc.edu

Abstract: Cannabinoid receptor 2 (CB2) selective agonists and inverse agonists possess significant potential as therapeutic agents for regulating
inflammation and immune function. Although CB2 agonists have received the greatest attention, it is emerging that inverse agonists also
manifest anti-inflammatory activity. In process of designing new cannabinoid ligands we discovered that the 2,6-dihydroxy-biphenyl-aryl
methanone scaffold imparts inverse agonist activity at CB2 receptor without functional activity at CB1. To further explore the scaffold we
synthesized a series of 3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone analogs and evaluated the CB1 and CB2 affinity,
potency, and efficacy. The studies reveal that an aromatic C ring is required for inverse agonist activity and that substitution at the 4 position is
optimum. The resorcinol moiety is required for optimum CB2 inverse agonist activity and selectivity. Antagonist studies against CP 55,940
demonstrate that the compounds 41 and 45 are noncompetitive antagonists at CB2.
Keywords: Cannabinoid; Inverse agonist; CB2 receptor, cAMP stimulation, ACTOne Assay

1. Introduction
The scope of therapeutic potential of cannabinoid based drugs has evolved rapidly following the identification of the cannabinoid receptors 1
and 2 (CB1 and CB2).1-3 Both receptors, either individually or in combination, have been identified as potential targets for intervention of human
diseases including neurodegenerative4-6 and cardiovascular disease,7-9 diabetes,10-12 and cancer.13-16 Within this spectrum of diseases,
intervention in neurodegenerative disorders using CB2 agonists and inverse agonists is arguably one of the most exciting developments in the
cannabinoid field. Specifically, microglia, the resident immune and inflammatory mediators in the central nervous system (CNS), express CB2 in
Alzheimer’s17, 18 and Parkinson’s disease19 and amyotrophic lateral sclerosis.6, 20 The CB2 is also up-regulated during microglial migration,
bacterial insult, and in response to CNS trauma.21-23 The apparent selective up-regulation of CB2 on microglia in response to insult indicates that
CB2 ligands would provide selective effects in only the damaged CNS tissue. In fact, both agonists and inverse agonists of CB2 have been
evaluated for efficacy in ameliorating disease progression in in vivo models of traumatic brain injury,24, 25 Alzheimer's 17, 18, 26-30 and Parkinson’s
disease, 19, 31, 32 amyotrophic lateral sclerosis, 6, 20 and multiple sclerosis.5, 33-39 The comparable efficacy of agonists and inverse agonists highlights
one of the major unanswered questions in this field, what is the optimum functional activity of CB2 ligands?
Agonists of CB2 have received the greatest attention in terms ligand design and evaluation as therapeutic agents, a recent review by Jones and
colleagues highlights the progress in this area.40 A review by Lunn however highlights the emerging interest in the development of CB2 inverse
agonists.41 The prototypical examples of a CB2 inverse agonist are the biaryl pyrazoles, and closely related triazoles, exemplified by SR144528
(Figure 1). Compounds based on the indole, benzimidazole, and isatin scaffold have also been described such as compound 1.42 CB2 inverse
agonist activity has been achieved using a 2- (JTE-907) and 4-oxoquinoline, and related 2-oxopyrine based derivatives.43 The triaryl bis-sulfones
(Sch 414319), and our 2,6-dihydroxy-biphenyl-aryl-methanone analog (SMM-189), represent additional scaffolds that are being investigated as
CB2 inverse agonists.41, 44 Raloxifen, himbacine and cannabidiol have also been reported to function as inverse agonist of CB2.45-47 Undoubtedly
the diversity of CB2 inverse agonist scaffolds will continue to expand owing to the as yet untapped potential in treating inflammation and
immune system disorders.

Figure 1. Structures of Selected CB2 Inverse Agonists
We previously reported the synthesis, and CB1 and CB2 receptor binding, of a small group of triaryl based cannabinoid ligands.48 In the
evaluation of the functional activity of the compounds it was discovered that SMM-189 exhibited selective inverse agonist activity at CB2.44
Based on the potential of CB2 inverse agonist in treating CNS diseases, we demonstrated that SMM-189 beneficially down-regulates cytokine
and chemokine production in LPS stimulated primary human microglia.25, 44 Subsequent testing of SMM-189 in the murine model of mild
traumatic brain injury (mTBI), and pre-clinical evaluation of biopharmaceutical properties, demonstrated the protective effects of SMM-189 in
mTBI and indicated that the 2,6-dihydroxy-biphenyl-aryl-methanone scaffold has acceptable drug-like properties to warrant further
investigation.25 The potential of the scaffold for developing selective CB2 inverse agonist prompted us to investigate the functional group
requirements required for activity. A set of compounds were synthesized and evaluated for CB1 and CB2 receptor binding in ACTOne membrane
preparations and functional activity using the ACTOne cAMP assay. The determination of affinity, potency, and efficacy revealed that the
resorcinol group and 4 substitutions are required for optimum CB2 inverse agonist activity. Furthermore, compounds 41 and 45 function as
noncompetitive antagonists when assayed against CP 55,940 at CB2.

2. Results and Discussion
2.1 Chemistry
The design of the 2,6-dihydroxy-biphenyl-aryl-methanone scaffold arose from a combination of our work on the C1’-aryl substituted classical
cannabinoids 49 and the biaryl ligands reported by Makriyannis.50 The hypothesis was that the hexyl group in the gem-dimethylheptyl side chain
could be replaced by a phenyl ring thus increasing CB2 selectivity. Unexpectedly, the deprotected intermediate (SMM-189) wherein the gem-
dimethyl group was replaced by a ketone, yielded a CB2 inverse agonist with no functional activity at CB1. The original design hypothesized an
overlapping binding pocket with classical and non-classical cannabinoids which was supported by the fact that SMM-189 is a orthosteric
antagonist of CP 55,940.44 Therefore, aliphatic substitution of the phenyl ring in SMM-189 was predicted increase affinities for CB2, as was
observed in the C1’ 3- and 4-alkylphenyl substituted analogs of Δ8-THC.49 To test this hypothesis a series of alkylphenyl analogs of SMM-189
were synthesized. The effect of hydrogen bonding and/or electron density on affinity and potency was evaluated by introducing hydroxyl,
chloro, and trifluoromethyl groups. Furthermore, it is well established that gem-dimethylheptyl and gem-dimethylcycloalkyl side chains afford
high affinity and potency in classical and non-classical CB1 and CB2 ligands.51, 52 Thus, the heptan-1-one and cyclohexylmethanone functional
groups were introduced to determine if the aromatic ring was required for affinity and potency. The requirement of the resorcinol functional
group was also examined by testing hydroxymethoxy and dimethoxy analogs. We took advantage of the by-products isolated from efforts to
optimize the boron tribromide deprotection of 21, 30, and 33 to test the resorcinol functional group requirement.
The synthetic methodology for the 3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl/alkyl-methanone analogs was revised from the previous
synthesis due to low yields in the Suzuki–Miyaura cross-coupling, final deprotection step, and to reduce cost. Formation of the trifluoro-
methanesulfonic acid ester of syringe aldehyde (2) was conducted using dichloromethane/pyridine as described by McLure and Young (Scheme
1).53 The modification was aimed at reducing cost by substituting pyridine for collidine. The microwave cross-coupling did not scale nor did the
addition of LiCl improve the reaction.54 An increase in yields to over 80 percent was achieved using a modification of the Suzuki–Miyaura cross-
coupling described by Leblond et al.55 The order of addition of the reagents proved to be important wherein the sequential addition of triflate,
boronic acid, water, and finally sodium carbonate provided optimum yields. However, the reaction did not scale beyond 1 gram thus reactions
were conducted in parallel as opposed to investigating alternative Pd(0) catalysts. The 2,6-dimethoxy protected intermediates were prepared
by reacting 3 with the appropriately substituted Grignard reagent followed by PCC oxidation. The oxidation reaction was modified from our
previous method to reduce reaction time and simplify workup. Specifically, the reaction was accelerated by first heating the
PCC/dichloromethane to reflux and cooling before addition of the alcohol. Addition of 1 N sodium hydroxide for workup yielded a clear almost
colorless organic phase, green aqueous, without confounding tarry residues previously obtained. Deprotection of the dimethoxy intermediates
with 2.5 equivalents boron tribromide yielded starting material, mono- and di-deprotected products. Extending the reaction time and increasing
the equivalents of boron tribromide, to compensate for coordination to the additional Lewis bases, decreased the yield of the desired product.

The mono-deprotected products (49-51) from optimization reactions on the p-chloro, p-methyl, and p-butyl were isolated (Scheme 2), albeit in
low yields, and subsequently used to examine the effect of methoxy group(s) on receptor affinity and potency. Deprotection was achieved using
molten pyridine hydrochloride 56 providing the desired resorcinol in a relatively short reaction time (2 hours) in good to high yields.

Scheme 1.
Scheme 2.
2.2 Receptor Binding and Functional Assays
The CB1 and CB2 receptor affinities, potency, and efficacy of compounds 34-51 and SMM-189, using the ACTOne functional assay cell lines and
membranes preparations derived from these cells. We have previously reported this method as part of our pre-clinical evaluation of SMM-
189.44 The method provided good correlation between the Ki and EC50 values; however, the Ki deviated significantly from the previous

determination 48 in part due to differences in Kd and Bmax of the preparations. A second consideration stemmed from the identification that
SMM-189 is a non-competitive antagonist against CP 55,940, thus indicating an overlapping binding site. This prompted us to consider the
possibility that the initial binding is to a ground state receptor and inverse agonist activity is induced by ligation. Thus binding of different
ligands, and the subsequent conformational change, could lead to one or more inactive conformations with differing efficacy in G-protein
coupling.57, 58 The identification of ligand functional groups that affect G-protein coupling, and ultimately efficacy, is an important component in
the drug design process. However, elucidating these effects in vitro can be challenging depending on the assay format and sensitivity. Our
approach to this challenge was to minimize potential variables, e.g. affinities determined using CHO cell preparation and functional activity
determined in HEK cells, by employing a single background to determine our compound affinity, potency, and efficacy at CB1 and CB2.
Using membrane preparation for the CB1 and CB2 lines, binding affinities were determined by measuring the displacement of [3H]CP 55,940 by
increasing concentrations of the of the compounds. The first question to be addressed was the effect hexyl and cyclohexyl substitution affinities
for CB1 and CB2. The hexyl analog (34) exhibited no affinity (>10 µM, Table 1) for the receptors compared to the gem-dimethylheptyl analog (Ki
= 2.6 nM and 0.6 nM, CB1 and CB2, respectively) reported by Makriyannis.50 Considering our initial hypothesis of an overlapping ligand binding
pocket (LBP) with classical cannabinoids this is not unexpected considering a keto group at C1’ is disfavored in the classical scaffold.59-61 It was
therefore surprising that the cyclohexyl group in 35 improved CB1 and CB2 affinities (Kis = 298.6 and 1589 nM, respectively). However,
compound 35 potency at CB1 and CB2 was >10 µM and 2.54 µM, respectively, without a significant difference in efficacy relative to controls (see
below). This may suggest an off-target effect at high concentrations of 35. Notwithstanding, this is intriguing in that cyclohexyl group is
sterically comparable to the C ring phenyl yet the cyclohexyl ring introduction does not result in inverse agonist activity at CB2 and yields an
antagonist at CB1.

Table 1.
Affinity - Ki (nM)
Potency EC50 (nM)
Efficacy (% max)
Potency EC50 (nM)
Efficacy (% max)
Compound
number
CB1:CB2
ratio
R1
R2
R
CB1
CB2
CB1
CB2
34
35
H
H
H
H
n-hexyl
>10,000
>10,000
1
-
NS
NS
-
NS
NS
cyclohexyl
298.6 ± 53.75
1589 ± 438.4
0.19
>5,000
2540 ± 42.4
36
37
38
39
40
41
H
H
H
H
H
H
H
H
H
H
H
H
4-methylphenyl
3-methylphenyl
9429 ± 364.8
>10,000
257.6 ± 70.75
2618 ± 368.8
>10,000
36.6
3.82
1
-
-
-
NS
NS
NS
118± 5.05
230 ± 5.23†††
-
-
NS
NS
3,5-dimethylphenyl
4-trifluoromethylphenyl
3-trifluoromethylphenyl
4-ethylphenyl
>10,000
>10,000
>10,000
1
>5,000
-
37.0 ± 1.55†††
NS
>5,000
-
49.7 ± 2.93†††
NS
4134 ± 767.5
1138 ± 216.2
>10,000
0.41
30.3
37.56 ± 4.91
-
NS
29.5 ± 4.83
>5,000
28.8 ± 3.84
182 ± 9.38
254 ± 3.79
3970 ± 158
>5,000
89.7 ± 9.09
>5,000
>5,000
>5,000
-
52.2 ± 2.13†††
184 ± 7.81†††
50.0 ± 3.49††
56.0 ± 3.66††
176 ± 5.19†††
275 ± 23.3†††
75.6 ± 7.96†††
119 ± 5.49†††
129 ± 7.15†††
108 ± 6.19†††
97.5 ± 5.46†††
NS
42
43
44
45
46
47
48
49
50
51
21
30
33
H
H
H
H
H
H
H
H
H
H
H
H
3-ethylphenyl
4-propylphenyl
4-isopropylphenyl
4-n-butylphenyl
4-tert-butylphenyl
4-hydroxyphenyl
4-chlorophenyl
4-methylphenyl
4-n-butylphenyl
4-chlorophenyl
4-methylphenyl
4-n-butylphenyl
4-chlorophenyl
>10,000
>10,000
1717 ± 376.5
59.91 ± 5.43
160.7 ± 39.1
464.4 ± 75.9
3166 ± 418.9
>10,000
5.82
166.9
20.7
21.5
0.8
1
-
NS
-
NS
H
3326 ± 1088
>10,000
-
NS
H
-
NS
H
2529 ± 1185
>10,000
-
NS
H
-
NS
3280 ± 82
169 ± 14.6†††
H
4645 ± 2540
>10,000
120.7 ± 19
>10,000
38.5
1
CH3
CH3
CH3
-
NS
>10,000
>10,000
1
-
NS
>10,000
>10,000
1
2880 ± 30.4
71.3 ± 3.79†††
CH3 CH3
CH3 CH3
CH3 CH3
>10,000
>10,000
1
-
-
-
NS
NS
NS
>10,000
>10,000
1
-
NS
>10,000
>10,000
1
-
NS

-
-
NS
NS
153 ± 22.3
11.5 ± 3.29
54.8 ± 3.23†
80.8 ± 3.18††
SMM-189
4778 ± 246
>10,000
121.3 ± 20.6
18.65 ± 1.43
39.4
SR 144528
536.2
†p < 0.05, †† p < 0.01, ††† p < 0.001 in comparison to parental HEK-CN

The introduction of substituents in the 3, 4, and 3,5 positions of the C ring phenyl group significantly
altered CB2 affinities (Kis = 37.6 nM to >10 µM). The substituent effects were not nearly as pronounced
for CB1, although substitution at the 3 and 4 positions did yield 8 compounds with micromolar affinities.
In terms of alkyl groups, the 4-alkylphenyl analogs exhibited superior affinity for CB1 and CB2 compared
to the to the 3-alkylphenyl while the 3,5-dimehtylphenyl (38) abolished affinity. Increased CB2 affinity,
relative to SMM-189 (Ki = 121.3 nM), was observed in the 4-methyl (36), 4-ethyl (41), 4-propyl (43), and
isopropyl (44) analogs demonstrating Ki values of 257, 37.6, 59.9, and 160 nM, respectively. Selectivity
for the CB2 receptor peaked with compound 43 (ratio = 167) but was 3 fold lower than our reference
inverse agonist SR 144528 (ratio = 536). Compound 41 had comparable CB2 affinity to SR 144528 (Ki =
18.7 nM) but increased CB1 affinity imparted by the ethyl group decreased the ratio to 30. The affinity
of these compounds for CB1, while not optimal, revealed a modest preference for bulky versus linear
aliphatic groups, e.g. 4-n-butyl (45, Ki > 10 µM) versus 4-tert-butyl (46, Ki = 2.53 µM). Introduction of a
methyl (37) or ethyl (42) group into the 3 position blocked binding to CB1. CB2 receptor affinity was
impacted similarly wherein a 10 and 46 fold reduction in the Ki occurred relative to the 4 position
isomers (36 and 41). Modification of the resorcinol group was not tolerated as demonstrated by
absence of affinity for CB1 and CB2 in the hydroxymethoxy (49, 50, 51) or dimethoxy (21, 30, 33)
analogs.
The 4-hydroxyl, 3- and 4-tifluoromethyl, and 4-chloro groups were introduced to examine the
contribution of electron density and hydrogen bonding to receptor affinity. Surprisingly, the 4-chloro
analog (48) had a 2 fold higher CB2 affinity than the 4-methyl analog (36, Kis = 121 and 257 nM,
respectively) while the 4-hydroxy (47) and 4-trifluoromethyl (39) demonstrated no affinity for either
receptor. The functional groups have comparable van der Waal radii, i.e. 1.4 to 2.0 Å, thus steric
interactions are unlikely to be responsible for reduced affinity. The charge density rank order of CF3 > Cl
> OH disfavors an effect due to repulsive interactions. The formation of a classical hydrogen bond would
be predicted to increase affinity however this is not observed with 4-hydroxyl or 4-trifluoromethyl,
although the existence of an intermolecular F to H bond in a drug-protein complex is rare based on the
analysis reported by Dunitz and Taylor.62 This raises the intriguing possibility that a multipolar
interaction of a carbonyl oxygen with the σ hole in the aromatic chloro enhances receptor affinity.63 It is
possible for trifluoromethyl to interact analogous to chloro; however, the preferred parallel C-Cl to O=C
interaction may be favorable over the orthogonal O=C to C-CF3 interaction.64 The CB1 affinities for the 4-
hydroxy (47) and 4-trifluoromethyl (39) analogs were equally low; however, the Kis for the 4-chloro (48)
and 3-trifluoromethyl (40) derivatives were improved to 4.13 and 4.64 µM.
The potency and efficacy of the compounds were determined using the CB1 and CB2 ACTOne assay
system (for selected compounds 41, 45, SMM-189, and SR 144528, see Figure 2 A, B and C). The assay
employs a cyclic nucleotide gated (CNG) ion channel which opens in response to intracellular cAMP
levels. The assay permits real time monitoring of intracellular cyclic nucleotides using a fluorescent
membrane potential dye to detect polarization or depolarization associated with closing or opening of
the channel. CB1 and CB2 agonists trigger a decrease in cAMP driven fluorescent signal via activation of
Gi/o, antagonists yield no response, and inverse agonists cause an increase in fluorescence. At CB1 all

but three compounds, the 4-trifluoromethyl (39), 4-chloro (48), and mono methoxy 4-chloro (51)
analogs, failed to significantly alter the fluorescence signal above HEK-CNG controls (Table 1). The
potency of the three active compounds was low (EC50 2.8 to > 5 µM) yet a significant increase in cAMP
production was detected with efficacies from 37 to 169 percent maximal. Interestingly, compound 48
had comparable Ki and EC50 values and good efficacy compared to the indeterminate affinity and
potency of 39 and 51. The indeterminate Ki for these compounds likely reflects poor binding
interactions that are not sufficient to displace CP 55,940 at CB1 and CB2. However, in the absence of a
high affinity ligand the weak ligand-receptor interactions are sufficient to produce a significant
functional response, albeit with low potency.

Figure 2. Representative Functional Activity Graphs for A) CB2, B) CB1, and C) CNG. White squares are
41, black triangles are 45, black circles are SMM-189, and Xs are SR 144528. N = 6-17, error is SEM.
It was noted in the discussion of affinities, the cyclohexyl analog (35) demonstrated no functional
activity. While this may be related to the low affinity (Ki = 1.59 µM), it is not consistent with the
comparable Ki (3.17 µM) and EC50 (3.97 µM) of the tert-butyl analog (46) – which possess the highest
efficacy – thus indicating 35 may be a neutral antagonist. The progression from cyclohexyl to

substituted phenyl yielded inverse agonists with potencies ranging from 28.8 to 3,968 nM. The 4-ethyl
(41) and 4-propyl (43), EC50 values 29.5 and 28.8 nM, manifest the highest potency and were
comparable to the 11.5 nM potency of the standard inverse agonist SR144528 (Table 1, Figure 2A -X);
however, efficacy of 41 (Figure 2A – white boxes) and 43 were significantly lower than SR 144528 (mean
50 versus 81 percent). In contrast the effect of compounds 36, 42, 45 (Figure 2A – black triangles), 46,
and 48 on cAMP stimulation was significantly higher (119 to 230 percent) than SR 144528 despite
significantly lower potencies. A linear analysis of the Ki versus EC50 of 36, 41, 44, 45, 48 and SMM-189
(compounds 39, 42, and 47 were excluded due to overweighting of the regression) revealed a modest
correlation (R2 = 0.69) while potency versus efficacy did not correlate (R2 = 0.18). The hydroxymethoxy
compounds (49, 50, 51), 4-trifluoromethyl (39), and 4-hydroxy (47) are interesting in that affinity and
potency is poor yet a significant increases in cAMP production occurs (49 to 130 percent). The basis for
this effect is not clear, but is very interesting, thus prompting us to begin to investigate the effects of
mono-alkylation of the resorcinol to further elucidate structure activity relationships.
Compounds 41 (EC50 = 29.5 nM, %max = 52.2) , 45 (EC50 = 254 nM, %max = 176), and SMM-189 (EC50
=
153 nM, %max = 54.8) were further evaluated in competition assays against CP 55,940 to determine the
mode of inhibition at CB2. The study was designed to examine the three clusters of functional activities,
i.e. high potency-modest efficacy, modest potency-high efficacy, and modest potency-modest efficacy to
determine if the activities correlated with mode of inhibition. All the compounds exhibited profiles
consistent with noncompetitive antagonism (Figure 3), although only 45 and SMM-189 were orthosteric
antagonists as well according to Schild analysis (Figure 3B and C insets). This raised an intriguing
question regarding the presence of overlapping yet unique ligand binding sites in CB2. Specifically,
agonists couple to G-proteins via an active receptor conformation while inverse agonists signal through
an inactive state. One possible interpretation of the result, and the absence of correlation between
potency and efficacy, may be derived from the studies of on G-protein coupling of CB1 and
conformational states of ligand binding.57, 58 It was suggested that ligands induce distinct
conformational states resulting in different efficacies in G-protein coupling. One possible interpretation
is that ligands bind to a ground state receptor which then induces a ligand dependent change in
conformation. If induction of confirmation is occurring in CB2 then this may suggest that initial binding
is to a ground state, i.e. correlation between Ki and EC50, but the efficacy in coupling to G-proteins is
dependent on substituent induced inactive conformations of CB2. While speculative, functional group
modification to induce unique conformations with varying efficacy in G-protein coupling may be
valuable in the future development of CB2 ligands.

Figure 3. Antagonist studies of selected triaryl analogs at CB2. A) 41 B) 45 C) SMM-189 versus CP 55,940.
Insets are Schild analyses of antagonist interactions (black circles) with a reference line with the slope of
1 denoting orthosteric interactions (white squares). N = 6, error is SEM.
3. Conclusions
A series of alkyl and substituted phenyl analogs of SMM-189 were synthesized and evaluated for CB1
and CB2 affinity, potency, and efficacy. The n-hexyl (34) analog was inactive at CB1 and CB2 while the
cyclohexyl (35) exhibited neutral antagonist activity. Substitution of the cyclohexyl with an aromatic ring
provided compounds with inverse agonist activity thus demonstrating the requirement for an aromatic
C ring in the biaryl methanone scaffold. Affinity of the aromatic analogs for CB1 was poor while affinity
for CB2 was highly dependent on phenyl ring substitution. The introduction of alkyl groups at the 4
position was optimal with the ethyl (41) and propyl (43) groups yielding the highest affinity and potency.
With the exception of the 4-chloro (48) analog, electron density on the phenyl ring was not tolerated
and significantly decreased activity. Interestingly the addition of larger alkyl groups, e.g. 45 and 46, at
the 4 position of the phenyl ring decreased potency but increased efficacy. We hypothesize this effect

may be related to induction of unique inactive state conformations resulting different efficacies in G-
protein coupling. The resorcinol ring is required for optimum inverse agonist activity at CB2. The
introduction of the monomethoxy (49, 50, 51) significantly decreased affinity and potency but
interestingly the compounds manifest good efficacy. The same was not true for the dimethoxy analogs
in that no activity was observed at the receptors. We believe that continued development of the 3',5'-
dichloro-2,6-dihydroxy-biphenyl-4-yl)-aryl –methanone scaffold will yield not only valuable information
on the structure activity relationship but also provide new probes for mechanistic studies on CB2
receptor conformation and G-protein coupling.
4. Experimental
4.1 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
sensitive reactions were carried out using oven dried glassware under dry conditions under an argon
atmosphere. Analytical thin layer 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 Grace Reveleris 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 Brucker ESQUIRE electrospray/ion trap instrument (Billerica,
MA). High resolution mass spectrometer (HRMS) data were acquired on a Waters Xevo G2+S QTOF
(Milford, MA) system. Preparative HPLC was performed on a Gilson system (Middleton, WI) equipped
with a 322 pump, 155 UV-Vis detector, and a 215 liquid handler, using a Phenomenex Luna PFP 100A
250 X 21.2 mm column and isocratic elution acetonitrile:water (9:1) flow rate 10 ml/min. Analytical
HPLC was performed on a Waters e2695 HPLC (Milford, MA) with a 2998 photodiode array detector and
and isocratic elution acetonitrile:water (9:1) flow rate 0.3
ml/min. All target compounds were found to be ≥ 95% pure by analytical HPLC.
4.2 Synthesis
Trifluoro-methanesulfonic acid 4-formyl-2,6-dimethoxy-phenyl ester
The starting material syringaldehyde (2, 23 g, 126 mmol) was dissolved in dry dichloromethane (200 mL)
to which 36 ml dry pyridine (0.46 mol) was added and the solution was cooled to 0 °C. To the cooled
solution was added triflic anhydride (40g, 140 mmol) dropwise, when addition was complete the
reaction was allowed to warm to ambient temperature then stirred for 2 hours. To the reaction mixture
was added 600 mL of ethyl acetate followed by extraction with 1 N HCl (2 X 200 mL), sat. NaHCO3 (200
mL), and the organic layer was dried over sodium sulfate and evaporated. The red solid was dissolved in
30 mL dichloromethane, insoluble material was removed by filtration, and loaded onto a Grace Reveleris
330g silica column, and eluted with a hexane:ethyl acetate gradient (10% to 10% 2 column volumes,
10% to 80% 10 column volumes, 80% to 80% 2 column volume, flow rate 100 mL per min.). Product

1
fractions were pooled and dried yielding 23.7 g (60%) of as white crystals. H NMR (400 MHz, CDCl3): 
9.94 (s, 1H, CHO); 7.17 (s, 2H, ArH), 3.84 (s, 6H, -OCH3).
3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-carbaldehyde (3)
Reactions were conducted in an 8 well aluminum block in 25 X 150 test tubes and 8 reaction were
pooled for workup. To degassed dimethylformamide (14 mL) was added
tetrakis(triphenylphosphine)palladium(0) (184 mg, 0.16 mmol) and the mixture was heated to 40 °C for
30 minutes to dissolve the catalyst then cooled to room temperature. To the solution was added 2 (1 g,
3.18 mmol) with stirring to dissolve then 3,5-dichlorophenylboronic acid (1.21 g, 6.36 mmol), degassed
water (3 mL), and sodium carbonate (675 mg, 6.36 mmol) were added sequentially. Tubes were blanked
with nitrogen, sealed with a septum, and heated to 40 °C for 16 hours. Reactions were pooled and
filtered through a 0.5 X 4.5 cm Celite pad and the pad washed with 40 mL ethyl acetate. To the filtrate
was added 100 mL water and the mixture was extracted with ethyl acetate (3 X 40 mL). The organic
phase was extracted with water (3 X 30 mL) and brine (30 mL) and dried with sodium sulfate. The
residue after solvent removal was dissolved in dichloromethane (10 mL) and loaded on a Grace Reveleris
120g silica column and eluted with a hexane:ethyl acetate gradient (5% to 5% 2 column volumes, 5% to
40% 10 column volumes, 40% to 40% 2 column volume, flow rate 80 mL per min.). Product fractions
were pooled and dried yielding 6.52 g (82%) of 3 as white crystals. 1H NMR, (400 MHz, CDCl3), 9.98 (s,
1H, CHO), 7.34 (t, J = 2.0 Hz, 1H, 4’ArH), 7.21 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.15 (s, 2H, 2,6ArH), 3.83 (s, 6H,
OCH3), MS: m/z (ESI, pos.) = 333.6 [M+23]+
1-(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-heptan-1-ol (4)
Glassware and magnesium turnings were dries overnight in a 110°C oven, assembled hot, and allowed
to come to ambient temperature under dry positive nitrogen pressure. To the magnesium turning (290
mg, 12.1 mmol) was added dry THF (12 mL), n-bromohexane (2 g, 12.1 mmol), and 1 drop of 1,2-
dibromoethane and the mixture was heated to reflux, the reaction was determined to be complete
when the magnesium turnings were consumed. The solution was allowed to cool to ambient
temperature then 6.5 mL (6.5 mmol) of the Grignard reagent was added dropwise to an ice cold solution
of 3 (1 g, 3.2 mmol) in dry THF (3.5 mL). The solution was allowed to warm to ambient temperature
then stirred for an additional 3 hours. The reaction was quenched with saturated ammonium chloride
(15 mL), layers separated, and the aqueous phase extracted with THF (2 X 15 mL), and the organic phase
extracted with brine (15 mL) then dried over sodium sulfate. Solvent was removed and the residue was
loaded onto a Grace Reveleris 12g silica column and eluted with a hexane:ethyl acetate gradient (5% to
5% 2 column volumes, 5% to 40% 10 column volumes, 40% to 40% 2 column volume, flow rate 36 mL
1
per min.). Product fractions were pooled and dried yielding 1.10 g (73%) of 4 as a white solid. H NMR,
(400 MHz, CDCl3), 7.30 (t, J = 2.0 Hz, 1H, 4’ArH), 7.21 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 6.62 (s, 2H, 2,6ArH),
5.30 (s, 1H, CHOH), 4.69 (t, 1H, CHOH), 3.83 (s, 6H, OCH3), 1.78 (m, 2H, CH2C5H11), 1.31 (m, 8H, C4H8),
0.89 (t, J = 7 Hz, 3H, CH3), MS: m/z (ESI, pos.) = 419.5 [M+23]+

Utilizing cyclohexylbromide or the appropriately substituted bromo-benzene derivatives the following
alcohols were similarly prepared.
Cyclohexyl-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanol (5)
1
White solid (0.49 g, 38%) H NMR, (400 MHz, CDCl3), 7.29 (t, J = 2.0 Hz, 1H, 4’ArH), 7.23 (d, J = 2.0 Hz,
2H, 2’,6’ArH), 6.51 (s, 2H, 2,6ArH), 4.31 (t, 1H, CHOH), 3.83 (s, 6H, OCH3), 1.73 (m, 1H, CHC5H10), 1.61 (m,
4H, CH2), 1.08 (m, 6H, CH2), MS: m/z (ESI, pos.) = 417.5 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-p-tolyl-methanol (6)
1
Off white solid (0.87 g, 69%) H NMR, (400 MHz, CDCl3), 7.32 (d, J = 8.0 Hz, 2H, 9,13ArH) 7.29 (t, J = 2.0
Hz, 1H, 4’ArH), 7.23 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.19 (d, J = 8.0 Hz, 2H, 10,12ArH), 6.68 (s, 2H, 2,6ArH),
5.83 (t, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.37 (s, 3H, ArCH3), MS: m/z (ESI, pos.) = 425.4 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-m-tolyl-methanol (7)
1
Off white solid (0.91 g, 70%) H NMR, (400 MHz, CDCl3), 7.29 (t, J = 2.1 Hz, 1H, 4’ArH) 7.27 (d, J = 7.5
Hz, 1H, 12ArH), 7.24 (m, 2H, 11,13ArH), 7.22 (d, J = 2.0 Hz, 2H, 2’6’ArH), 7.14 (d, J = 7.7 Hz, 1H, 9ArH),
6.68 (s, 2H, 2,6ArH), 5.82 (d, J = 3.3, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.38 (s, 3H, ArCH3), MS: m/z (ESI,
pos.) = 425.3 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(3,5-dimethyl-phenyl)-methanol (8)
1
Off white solid (1.25 g, 94%) H NMR, (400 MHz, CDCl3), 7.29 (t, J = 1.9 Hz, 1H, 4’ArH), 7.21 (d, J = 2.0
Hz, 2H, 2’,6’ArH), 7.03 (s, 2H, 9,13ArH), 6.96 (s, 1H, 11ArH), 6.69 (s, 2H, 2,6ArH), 5.78 (d, J = 3.3 Hz, 1H,
CHOH), 3.74 (s, 6H, OCH3), 2.06 (s, 6H, ArCH3), MS: m/z (ESI, pos.) = 439.6 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-trifluoromethyl-phenyl)-methanol (9)
1
White solid (1.03 g, 70%) H NMR, (400 MHz, CDCl3),  7.65 (d, J = 8.2 Hz, 2H, 10,12ArH), 7.57 (d, J = 8.3
Hz, 2H,
7.30 (t, J = 1.9 Hz, 1H, 4’ArH), 7.20 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 6.65 (s, 2H, 2,6ArH),
5.90 (d, J = 3.24 Hz, 1H, CHOH), 3.73 (s, 6H, OCH3), MS: m/z (ESI, pos.) = 481.3 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(3-trifluoromethyl-phenyl)-methanol (10)
1
White solid (1.21 g, 82%) H NMR, (400 MHz, CDCl3),  7.76 (s, 1H, 9ArH), 7.60 (t, J = 7.9 Hz, 2H,
11,13ArH), 7.51 (d, J = 7.9Hz, 1H, 12ArH), 7.30 (t, J = 1.8 Hz, 1H, 4’ArH), 7.21 (d, J = 1.8 Hz, 2H, 2’,6’ArH),
6.65 (s, 2H, 2,6ArH), 5.89 (d, J = 3.14 Hz, 1H, CHOH), 3.73 (s, 6H, OCH3), MS: m/z (ESI, pos.) = 481.4
[M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-ethyl-phenyl)-methanol (11)
1
White crystals (0.80 g, 60%) H NMR, (400 MHz, CDCl3),  7.34 (d, J = 8.0 Hz, 2H, 9,13ArH), 7.29 (t, J =
2.0 Hz, 1H, 4’ArH), 7.22 (d, J = 8.0 Hz, 2H, 10,12ArH), 7.21 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 6.69 (s, 2H,

2,6ArH), 5.83 (d, J = 3.4 Hz, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.66 (q, J = 7.7 Hz, 2H, CH2CH3), 1.25 (t, J = 7.7
Hz, 3H, CH2CH3), MS: m/z (ESI, pos.) = 437.5 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(3-ethyl-phenyl)-methanol (12)
1
White crystals (1.24 g, 92%) H NMR, (400 MHz, CDCl3),  7.36 (d, J = 2.0 Hz 1H, 12ArH), 7.29 (t, J = 2.0
Hz, 1H, 4’ArH), 7.24 (t, J = 1.6 Hz, 1H, 9ArH), 7.22 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.17 (d, J = 7.4 Hz, 1H,
13ArH), 7.11 (d, J = 7.0 Hz, 1H, 11ArH), 6.69 (s, 2H, 2,6ArH), 5.83 (s, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.68
(q, J = 7.7 Hz, 2H, CH2CH3), 1.27 (t, J = 7.6 Hz, 3H, CH2CH3), MS: m/z (ESI, pos.) = 439.3 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-propyl-phenyl)-methanol (13)
1
White solid (1.15 g, 82%) H NMR, (400 MHz, CDCl3),  7.33 (d, J = 8.0 Hz, 2H, 9,13ArH), 7.29 (t, J = 2.1
Hz, 1H, 4’ArH), 7.21 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.20 (d, J = 8.0 Hz, 2H, 10,12ArH), 6.69 (s, 2H, 2,6ArH),
5.83 (d, J = 3.4 Hz, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.60 (t, J = 8.0 Hz, 2H, CH2CH2CH3), 1.65 (sxt, J = 7.7 Hz,
2H, CH2CH2CH3), 0.95 (t, J = 7.7 Hz, 3H, CH2CH2CH3), MS: m/z (ESI, pos.) = 453.2 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-isopropyl-phenyl)-methanol (14)
1
White crystals (0.94 g, 68%) H NMR, (400 MHz, CDCl3),  7.35 (d, J = 8.2 Hz, 2H, 9,13ArH), 7.29 (t, J =
2.0 Hz, 1H, 4’ArH), 7.25 (d, J = 8.2 Hz, 2H, 10,12ArH), 7.21 (d, J = 2.0 Hz, 2H, 2’,6’ArH), ), 6.70 (s, 2H,
2,6ArH), 5.83 (d, J = 3.5 Hz, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.92 (spt, J = 7.1 Hz, 1H, CH(CH3)2), 1.26 (d, J =
7.0 Hz, 6H, CH(CH3)2), MS: m/z (ESI, pos.) = 453.8 [M+23]+
(4-Butyl-phenyl)-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanol (15)
1
Off white crystals (1.25 g, 87%) H NMR, (400 MHz, CDCl3),  7.41 (d, J = 8.2 Hz, 2H, 9,13ArH), 7.33 (d, J
= 8.2 Hz, 2H, 10,12ArH), 7.29 (t, J = 2.1 Hz, 1H, 4’ArH), 7.21 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 6.69 (s, 2H,
2,6ArH), 5.83 (s, 1H, CHOH), 3.73 (s, 6H, OCH3), 2.62 (t, J = 7.9 Hz, 2H, CH2CH2CH2CH3), 1.61 (p, J = 8.0 Hz,
2H, CH2CH2CH2CH3), 1.37 (sxt, J = 8.0 Hz, 2H, CH2CH2CH2CH3), 0.94 (t, J = 7.5 Hz, 3H, CH2CH2CH2CH3), MS:
m/z (ESI, pos.) = 467.1 [M+23]+
(4-tert-Butyl-phenyl)-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanol (16)
1
White crystals (1.34 g, 94%) H NMR, (400 MHz, CDCl3),  7.41 (d, J = 8.2 Hz, 2H, 9,13ArH), 7.36 (d, J =
8.2 Hz, 2H, 10,12ArH), 7.29 (t, J = 2.0 Hz, 1H, 4’ArH), 7.21 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 6.71 (s, 2H,
2,6ArH), 5.84 (d, J = 3.0, 1H, CHOH), 3.74 (s, 6H, OCH3), 1.33 (s, 9H, C(CH3)3), MS: m/z (ESI, pos.) = 467.3
[M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-methoxy-phenyl)-methanol (17)
1
White solid (0.81 g, 60%) H NMR, (400 MHz, CDCl3),  7.33 (d, J = 8.5 Hz, 2H, 9,13ArH), 7.29 (t, J = 2.0
Hz, 1H, 4’ArH), 7.21 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 6.90 (d, J = 8.6 Hz, 2H, 10,12ArH), 6.67 (s, 2H, 2,6ArH),
5.84 (d, J = 3.4, 1H, CHOH), 4.05 (s, 3H, 11ArOCH3), 3.74 (s, 6H, 3,6ArOCH3), MS: m/z (ESI, pos.) = 441.2
[M+23]+

(4-Chloro-phenyl)-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanol (18)
1
White solid (1.60g, 56%) H NMR, (400 MHz, CDCl3), 7.37 (s, 2H, 10,12ArH), 7.36 (s, 2H, 9,13ArH), 7.29
(t, J = 2.0 Hz, 1H, 4’ArH), 7.20 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 6.64 (s, 2H, 2,6ArH), 5.83 (d, J = 3.5 Hz, 1H,
CHOH), 3.73 (s, 6H, OCH3). MS: m/z (ESI, pos.) = 445.6 [M+23]+
1-(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-heptan-1-one (19)
A mixture of pyridinium chlorochromate (218 mg, 1 mmol) in dichloromethane (4 mL) in a 25 X 150 test
tube was heated to reflux then cooled to ambient temperature. A solution of 4 (202 mg, 0.51 mmol) in
dichloromethane (2 mL) was added dropwise and the mixture was stirred at ambient temperature for
1.5 hours. The reaction was quenched with 1N sodium hydroxide (10 mL), separated, the aqueous
phase extracted with dichloromethane (2 X 10 mL), the combined organic phase dried with sodium
sulfate, and the solvent removed. The residue was loaded onto a Grace Reveleris 12g silica column and
eluted with a hexane:ethyl acetate gradient (5% to 5% 2 column volumes, 5% to 40% 10 column
volumes, 40% to 40% 2 column volume, flow rate 36 mL per min.). Product fractions were pooled and
1
dried yielding 95.3 mg (47%) of 19 as a white solid. H NMR, (400 MHz, CDCl3), 7.34 (t, J = 2.0 Hz, 1H,
4’ArH), 7.23 (s, 2H, 2,6ArH), 7.22 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 3.82 (s, 6H, OCH3), 3.00 (t, J = 7.5 Hz, 2H,
C(O)CH2C5H11), 1.78 (p, J = 7.6 Hz, 2H, CH2CH2C4H9), 1.38 (m, 6H, CH2CH2(CH2)3CH3), 0.92 (t, J = 7.9, 3H,
C5H10CH3), MS: m/z (ESI, pos.) = 417.0 [M+23]+
Utilizing the appropriate alcohol the following ketones were prepared
Cyclohexyl-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanone (20)
1
Off white solid (123 mg, 61%) H NMR, (400 MHz, CDCl3), 7.34 (t, J = 2.0 Hz, 1H, 4’ArH), 7.22 (d, J = 2.0
Hz, 2H, 2’,6’ArH), 7.20 (s, 2H, 2,6ArH), 3.82 (s, 6H, OCH3), 3.25 (tt, J = 3.2, 11.5 Hz, 1H, C(O)CHC5H10), 1.92
(m, 2H, CH2), 1.78(m, 2H, CH2), 1.44 (m, 6H, C3H6), MS: m/z (ESI, pos.) = 415.0 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-p-tolyl-methanone (21)
1
White solid (138 mg, 69%). H NMR, (400 MHz, CDCl3), 7.79 (d, J = 8.2 Hz, 2H, 9,13ArH), 7.35 (t, J = 1.9
Hz, 1H, 4’ArH), 7.33 (d, J = 8.2 Hz, 2H, 10,12ArH), 7.25 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.03 (s, 2H, 2,6ArH),
3.77 (s, 6H, OCH3), 2.48 (s, 3H, ArCH3). 13C NMR, (100 MHz, CDCl3), 195.9, 157.1, 143.7, 139.6, 139.2,
135.4, 134.2, 130.4, 129.3, 129.1, 125.7, 115.5, 105.8, 56.1, 21.8. HRMS: m/z calculated for C22H19Cl2O3S,
[M+H]+401.0711, Found: 401.0710.
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-m-tolyl-methanone (22)
1
White solid (576 mg, 71%) H NMR, (400 MHz, CDCl3), 7.71 (m, 1H, 9ArH), 7.64 (d, J = 7.6 Hz, 1H,
13ArH), 7.44 (m, 1H, 12ArH), 7.41 (d, J = 7.6, 1H, 11ArH), 7.35 (t, J = 1.9 Hz, 1H, 4’ArH), 7.26 (d, J = 1.9 Hz,
2H, 2’,6’ArH), 7.06 (s, 2H, 2,6ArH), 3.77 (s, 6H, OCH3), 2.46 (s, 3H, ArCH3), MS: m/z (ESI, pos.) = 423.2
[M+23]+

(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(3,5-dimethyl-phenyl)-methanone (23)
1
Light yellow solid (151 mg, 76%) H NMR, (400 MHz, CDCl3), 7.47 (s, 2H, 9,13ArH), 7.35 (t, J = 1.9 Hz,
1H, 4’ArH), 7.27 (s, 1H, 11ArH), 7.26 (d, J = 1.8 Hz, 2H, 2’,6’ArH), ), 7.05 (s, 2H, 2,6ArH), 3.77 (s, 6H,
OCH3), 2.41 (s, 6H, ArCH3), MS: m/z (ESI, pos.) = 437.3 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-trifluoromethyl-phenyl)-methanone (24)
1
White crystals (151 mg, 76%) H NMR, (400 MHz, CDCl3), 7.97 (d, J = 8.2 Hz, 2H, 9,13ArH), 7.80 (d, J =
8.2 Hz, 2H, 10,12ArH), 7.36 (t, J = 1.8 Hz, 1H, 4’ArH), 7.25 (d, J = 1.8 Hz, 2H, 2’,6’ArH), 7.04 (s, 2H,
2,6ArH), 3.78 (s, 6H, OCH3), MS: m/z (ESI, pos.) = 477.0 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(3-trifluoromethyl-phenyl)-methanone (25)
1
White crystals (174 mg, 85%) H NMR, (400 MHz, CDCl3), 8.14 (s, 1H, 9ArH), 8.07 (d, J = 7.8 Hz, 1H,
11ArH), 7.90 (d, J = 7.8 Hz, 1H, 13ArH), 7.69 (t, J = 7.8 Hz, 1H, 12ArH), 7.36 (t, J = 1.9 Hz, 1H, 4’ArH), 7.26
(d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.04 (s, 2H, 2,6ArH), 3.78 (s, 6H, OCH3), MS: m/z (ESI, pos.) = 477.0 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-ethyl-phenyl)-methanone (26)
1
White crystals (155 mg, 78%) H NMR, (400 MHz, CDCl3), 7.82 (d, J = 8.3 Hz, 2H, 9,13ArH), 7.36 (t, J =
1.9 Hz, 1H, 4’ArH), 7.35 (m, 2H, 10,12ArH), 7.26 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.05 (s, 2H, 2,6ArH), 3.78 (s,
6H, OCH3), 2.77 (q, J = 7.7 Hz, 2H, CH2CH3), 1.32 (t, J = 7.6 Hz, 3H, CH2CH3), MS: m/z (ESI, pos.) = 437.5
[M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(3-ethyl-phenyl)-methanone (27)
1
Off white solid (116 mg, 56%) H NMR, (400 MHz, CDCl3), 7.73 (s, 1H, 9ArH), 7.68 (dt, J = 1.7, 7.3 Hz,
1H, 13ArH), 7.48 (m, 1H, 11ArH), 7.44 (t, J = 7.5, 1H, 12ArH), 7.35 (t, J = 2.1 Hz, 1H, 4’ArH), 7.27 (d, J = 2.0
Hz, 2H, 2’,6’ArH), 7.06 (s, 2H, 2,6ArH), 3.78 (s, 6H, OCH3), 2.76 (q, J = 7.6 Hz, 2H, CH2CH3), 1.30 (t, J = 7.7
Hz, 3H, CH2CH3), MS: m/z (ESI, pos.) = 437.1 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-propyl-phenyl)-methanone (28)
1
Off white solid (125 mg, 62%) H NMR, (400 MHz, CDCl3), 7.82 (d, J = 8.3 Hz, 2H, 9,13ArH), 7.35 (t, J =
2.0 Hz, 1H, 4’ArH), 7.33 (d, J = 8.3, 2H, 10,12ArH), 7.26 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.04 (s, 2H, 2,6ArH),
3.78 (s, 6H, OCH3), 2.70 (t, J = 7.5 Hz, 2H, CH2CH2CH3), 1.72 (sxt, J = 7.7 Hz, 2H, CH2CH2CH3), 1.00 (t, J =
7.4, 3H, CH2CH2CH3), MS: m/z (ESI, pos.) = 451.1 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-isopropyl-phenyl)-methanone (29)
1
White solid (123 mg, 62%) H NMR, (400 MHz, CDCl3), 7.83 (d, J = 8.3 Hz, 2H, 9,13ArH), 7.38 (d, J = 8.3
Hz, 2H, 10,12ArH), 7.35 (t, J = 2.0 Hz, 1H, 4’ArH), 7.26 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.05 (s, 2H, 2,6ArH),
3.78 (s, 6H, OCH3), 3.03 (spt. J = 6.8 Hz, 1H, CH(CH3)2), 1.32 (d, J = 6.8 Hz, 6H, CH(CH3)2), MS: m/z (ESI,
pos.) = 451.1 [M+23]+

(4-Butyl-phenyl)-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanone (30)
1
White solid (460 mg, 40%). H NMR, (400 MHz, CDCl3), 7.82 (d, J = 8.0 Hz, 2H, 9,13ArH), 7.34 (d, J = 8.0
Hz, 2H, 10,12ArH), 7.32 (t, J = 2.1 Hz, 1H, 4’ArH), 7.26 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 7.05 (s, 2H, 2,6ArH),
3.78 (s, 6H, OCH3), 2.73 (t, J = 8.8 Hz, 2H, CH2(CH2)2CH3), 1.67 (p, J = 8.0, 2H, CH2CH2CH2CH3), 1.41 (sxt. J =
8.4, 2H, CH2CH2CH2CH3), 0.97 (t, J = 7.6, 3H, (CH2)3CH3). 13C NMR, (100 MHz, CDCl3), 195.8, 157.1,
148.6, 139.2, 136.3, 135.4, 129.2, 128.5, 127.3, 112.7, 105.7, 56.2, 35.8, 33.3, 22.4, 13.95. HRMS: m/z
calculated for C25H25Cl2O3, [M+H]+ 443.1181, Found: 443.1182.
(4-tert-Butyl-phenyl)-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanone (31)
1
White solid (146 mg, 73%) H NMR, (400 MHz, CDCl3), 7.84 (d, J = 8.6 Hz, 2H, 9,13ArH), 7.54 (d, J = 8.6,
2H, 10,12ArH), 7.35 (t, J = 2.0 Hz, 1H, 4’ArH), 7.26 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.05 (s, 2H, 2,6ArH), 3.78
(s, 6H, OCH3), 1.39 (s, 9H, C(CH3)3), MS: m/z (ESI, pos.) = 465.1 [M+23]+
(3',5'-Dichloro-2,6-dimethoxy-biphenyl-4-yl)-(4-methoxy-phenyl)-methanone (32)
1
White solid (140 mg, 72%) H NMR, (400 MHz, CDCl3), 7.89 (d, J = 8.8 Hz, 2H, 9,13ArH), 7.35 (t, J = 2.1
Hz, 1H, 4’ArH), 7.26 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 7.00 (s, 2H, 2,6ArH), 6.99 (d, J = 8.8 Hz, 2H, 10,12ArH),
3.78 (s, 6H, OCH3), 4.15 (s, 3H, OCH3), MS: m/z (ESI, pos.) = 439.8 [M+23]+
(4-Chloro-phenyl)-(3',5'-dichloro-2,6-dimethoxy-biphenyl-4-yl)-methanone (33)
1
Off white solid (140 mg, 72%). H NMR, (400 MHz, CDCl3), 7.82 (d, J = 8.9 Hz, 2H, 9,13ArH), 7.50 (d, J =
8.9 Hz, 2H, 10,12ArH), 7.34 (t, J = 2.1 Hz, 1H, 4’ArH), 7.24 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 7.01 (s, 2H,
2,6ArH), 3.78 (s, 6H, OCH3). 13C NMR, (100 MHz, CDCl3), 194.8, 157.4, 139.2, 138.5, 136.2, 135.8,
134.2, 131.4, 129.2, 128.8, 127.4, 121.0, 105.8, 56.2. HRMS: m/z calculated for C21H16Cl3O3, [M+H]+
421.0165, Found: 421.0163.
1-(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-heptan-1-one (34)
Into a 8mm X 10.2cm pressure tube (Ace Glass) with stirring bar was loaded 19 (60 mg, 0.15 mmol) and
pyridine hydrochloride (520 mg, 4.6 mmol), flushed with nitrogen, sealed, and heated to 220° C in an oil
bath. Immediately following the melt the mixture was shaken to thoroughly mix the reactants and
returned to the bath for 2 hours. The reaction was allowed to cool to ambient temperature then water
(2 mL) and diethyl ether (2 mL) were added to dissolve the brown crystalline material. Ten milliliters of
water were added and the aqueous phase was extracted with diethyl ether (3 X 5 mL) and the combined
organic phases were dried over sodium sulfate and the solvent removed. The residue was loaded onto a
Grace Reveleris 12g silica column and eluted with a hexane:methyl t-butyl ether gradient (5% to 5% 4
column volumes, 5% to 80% 13 column volumes, 80% to 80% 2 column volume, flow rate 36 mL per
min.). Product fractions were pooled and dried yielding 47.0 mg (84%) of 34 as a pink solid. A sample of
34 was recrystallized from methanol:water to yield a white solid.
1
Analytical HPLC retention time 7.40 min; purity 99.0%, H NMR, (400 MHz, d6-acetone), 7.44 (d, J = 1.9
Hz, 2H, 2’,6’ArH), 7.42 (t, J = 1.9 Hz, 1H, 4’ArH), 7.15 (s, 2H, 2,6ArH), 2.93 (t, J = 7.3 Hz, 2H,

C(O)CH2(CH2)4CH3), 1.68 (p, J = 7.5 Hz, 2H, CH2CH2(CH2)3CH3), 1.34 (m, 6H, CH2CH2(CH2)3CH3), 0.89 (t, J =
7.2 Hz, 3H, (CH2)5CH3). 13C NMR, (100 MHz, d6-acetone), 206.2, 156.5, 139.4, 138.6, 134.8, 130.5,
127.6, 118.9, 107.9, 39.1, 32.5, 25.2, 23.3, 14.0. HRMS: m/z calculated for C19H21Cl2O3, [M+H]+ 367.0868,
Found: 367.0858
Using pyridine HCl the following compounds were prepared.
Cyclohexyl-(3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-methanone (35)
Pink solid (56.2 mg, 89%). The material was future purified by preparative reverse phase HPLC.
1
Analytical HPLC retention time 7.00 min; purity 97.5%, H NMR, (400 MHz, d6-acetone), 7.45 (d, J = 2.2
Hz, 2H, 2’,6’ArH), 7.43 (t, J = 2.2 Hz, 1H, 4’ArH), 7.16 (s, 2H, 2,6ArH), 3.22 (m, 1H, C(O)CHC5H10), 1.85 (m,
2H, CH2CH2(CH2)3), 1.71 (m, 2H, CH2CH2(CH2)3), 1.42 (m, 6H, CH2CH2(CH2)3). 13C NMR, (100 MHz, d6-
acetone), 203.8, 156.4, 139.4, 138.4, 134.5, 130.5, 127.4, 118.8, 108, 46.8, 30.5, 26.8, 26.5, HRMS: m/z
calculated for C19H19Cl2O3, [M+H]+ 365.0711, Found: 365.0707
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-p-tolyl-methanone (36)
Pale pink solid solid (49.0 mg, 81%). The material was future purified by preparative reverse phase
1
HPLC. Analytical HPLC retention time 6.64 min; purity 99.6% H NMR, (400 MHz, d6-acetone), 7.72 (d,
J = 8.0 Hz, 2H, 9,13ArH), 7.48 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.43 (t, J = 1.9 Hz, 1H, 4’ArH), 7.38 (d, J = 8.0
Hz, 2H, 10,12ArH), 6.93 (s, 2H, 2,6ArH), 2.44 (s, 3H, ArCH3)13C NMR, (100 MHz, CDCl3), 195.6, 156.1,
143.9, 140.0, 138.6, 135.9, 134.7, 130.7, 130.5, 129.8, 127.43, 109.6, 109.5, 21.5. HRMS: m/z calculated
for C20H15Cl2O3, [M+H]+ 373.0398, Found: 373.0392
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-m-tolyl-methanone (37)
Off white solid (49.6 mg, 52%). The material was recrystallized from methanol:water to yield a white
1
solid. Analytical HPLC retention time 6.61 min; purity 96.5% H NMR, (400 MHz, , d6-acetone), 7.63
(m, 1H, 9ArH), 7.59 (d, J = 7.4 Hz, 1H, 13ArH), 7.49 (m, 1H, 12ArH), 7.48 (t, J = 1.9 Hz, 1H, 4’ArH), 7.47 (m,
1H, 11ArH), 7.44 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 6.95 (s, 2H, 2,6ArH), 2.43 (s, 3H, ArCH3). 13C NMR, (100
MHz, CDCl3), 195.9, 156.2, 139.7, 139.0, 138.8, 138.6 , 134.8, 133.9, 130.9, 130.6, 129.1, 127.8, 127.5,
109.8, 21.4. HRMS: m/z calculated for C20H15Cl2O3, [M+H]+ 373.0398, Found: 373.0396
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(3,5-dimethyl-phenyl)-methanone (38)
Pale pink solid (62.9 mg, 71%). The material was recrystallized from methanol:water to yield a white
solid. Analytical HPLC retention time 6.98 min; purity 96.3% 1H NMR, (400 MHz, d6-acetone), 7.48 (d, J
= 2.0 Hz, 2H, 2’,6’ArH), 7.43 (t, J = 2.0 Hz, 1H, 4’ArH), 7.41 (m, 2H, 9,13ArH), 7.30 (m, 1H, 11ArH), 6.95 (s,
2H, 2,6ArH), 2.38 (s, 6H, ArCH3). 13C NMR, (100 MHz, d6-acetone), 196.0, 156.2, 139.8, 138.8, 138.5,
134.7, 134.6, 130.5, 128.2, 127.5, 118.4, 109.7, 21.2. HRMS: m/z calculated for C21H17Cl2O3, [M+H]+
387.0555, Found: 387.0555
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(4-trifluoromethyl-phenyl)-methanone (39)

Pink solid (63.5 mg, 85%). The material was recrystallized from methanol:water to yield a white solid.
1
Analytical HPLC retention time 6.80 min; purity 95.3%. H NMR, (400 MHz, d6-acetone), 8.01 (d, J = 8.0
Hz, 2H, 9,13ArH), 7.94 (d, J = 8.0 Hz, 2H, 10,12ArH), 7.49 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.45 (t, J = 1.9 Hz,
1H, 4’ArH), 7.00 (s, 2H, 2,6ArH). 13C NMR, (100 MHz, d6-acetone), 194.9, 156.3, 142.3, 138.6, 138.2,
134.8, 133.5, 130.9, 130.4, 127.6, 126.3, 123.6, 119.2, 109.3. HRMS: m/z calculated for C20H12Cl2F3O3,
[M+H]+ 427.0116, Found: 427.0111
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(3-trifluoromethyl-phenyl)-methanone (40)
Pink solid (66.8 mg, 86%). The material was future purified by preparative reverse phase HPLC.
1
Analytical HPLC retention time 6.78 min; purity 97.7%. H NMR, (400 MHz, d6-acetone), 8.11 (s, 1H,
9ArH), 8.08 (d, J = 7.8 Hz, 1H, 11ArH), 8.02 (d, J = 7.8 Hz, 1H, 13ArH), 7.84 (t, J = 8.1 Hz, 1H, 12ArH), 7.48
(d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.44 (t, J = 2.0 Hz, 1H, 4’ArH), 6.98 (s, 2H, 2,6ArH). 13C NMR, (100 MHz, d6-
acetone), 194.5, 156.4, 139.5, 138.6, 138.3, 134.7, 134.2, 131.3, 131.0, 130.4, 129.6, 127.6, 126.8,
123.6, 109.7. HRMS: m/z calculated for C20H12Cl2F3O3, [M+H]+ 427.0116, Found: 427.0119
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(4-ethyl-phenyl)-methanone (41)
Pink solid (66.1mg, 78%). The material was recrystallized from methanol:water to yield a white solid.
1
Analytical HPLC retention time 6.97 min; purity 96.5%. H NMR, (400 MHz, d6-acetone), 7.76 (d, J = 7.9
Hz, 2H, 9,13ArH), 7.48 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.43 (t, J = 2.0 Hz, 1H, 4’ArH), 7.41 (d, J = 7.9 Hz, 2H,
10,12ArH), 6.94 (s, 2H, 2,6ArH), 2.75 (q, J = 7.5 Hz, 2H, CH2CH3), 1.27 (t, J = 7.7 Hz, 3H, CH2CH3). 13C NMR,
(100 MHz, d6-acetone), 195.4, 156.1, 150.1, 139.9, 138.5, 136.2, 134.6, 130.8, 130.5, 128.7, 127.4,
109.6, 29.6, 15.7. HRMS: m/z calculated for C21H17Cl2O3, [M+H]+ 387.0555, Found: 387.0545
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(3-ethyl-phenyl)-methanone (42)
Pink solid (59.2 mg, 96%). The material was future purified by preparative reverse phase HPLC.
1
Analytical HPLC retention time 7.01 min; purity 99.3%. H NMR, (400 MHz, d6-acetone), 7.67 (s, 1H,
9ArH), 7.62 (dt, J = 1.6, 7.6 Hz, 1H, 13ArH), 7.53 (m, 1H, 11ArH), 7.49 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.48 (d,
J = 7.6, 1H, 12ArH), 7.44 (t, J = 1.9 Hz, 1H, 4’ArH), 6.97 (s, 2H, 2,6ArH), 2.75 (q, J = 7.6 Hz, 2H, CH2CH3),
1.27 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR, (100 MHz, d6-acetone), 195.9, 156.1, 139.7, 138.8, 138.5,
134.7, 132.7, 130.5, 129.7, 129.1, 128.0, 127.4, 126.9, 126.7, 109.7, 29.6, 16.0. HRMS: m/z calculated for
C21H17Cl2O3, [M+H]+ 387.0555, Found: 387.0545
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(4-propyl-phenyl)-methanone (43)
Pink solid (65.6 mg, 85%). The material was recrystallized from methanol:water to yield a white solid.
1
Analytical HPLC retention time 7.43 min; purity 99.1%. H NMR, (400 MHz, d6-acetone), 7.74 (d, J = 8.1
Hz, 2H, 9,13ArH), 7.48 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.43 (t, J = 1.9 Hz, 1H, 4’ArH), 7.39 (d, J = 8.1, 2H,
10,12ArH), 6.94 (s, 2H, 2,6ArH), 2.70 (t, J = 7.9 Hz, 2H, CH2CH2CH3), 1.70 (sxt, J = 7.7 Hz, 2H, CH2CH2CH3),
0.96 (t, J = 7.4, 3H, CH2CH2CH3). 13C NMR, (100 MHz, d6-acetone), 195.4, 156.2, 148.6, 139.9, 138.5,
136.1, 134.7, 130.8, 130.5, 129.3, 127.4, 118.3, 109.6, 38.5, 25.1, 14.0. HRMS: m/z calculated for
C22H19Cl2O3, [M+H]+ 401.0711, Found: 401.0707

(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(4-isopropyl-phenyl)-methanone (44)
Off white solid (39.1 mg, 70%). The material was recrystallized from methanol:water to yield a white
1
solid. Analytical HPLC retention time 7.31 min; purity 97.7%. H NMR, (400 MHz, d6-acetone), 7.76 (d,
J = 8.4 Hz, 2H, 9,13ArH), 7.48 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.45 (t, J = 2.0 Hz, 1H, 4’ArH), 7.43 (d, J = 8.4,
2H, 10,12ArH), 6.95 (s, 2H, 2,6ArH), 3.04 (spt. J = 6.8 Hz, 1H, CH(CH3)2), 1.30 (d, J = 6.8 Hz, 6H, CH(CH3)2).
13C NMR, (100 MHz, d6-acetone), 195.4, 156.2, 154.6, 139.9, 138.5, 136.3, 134.7, 130.9, 130.5, 127.4,
127.2, 118.3, 109.6, 34.9, 24.0. HRMS: m/z calculated for C22H19Cl2O3, [M+H]+ 401.0711, Found:
401.0700
(4-Butyl-phenyl)-(3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-methanone (45)
Off white solid (44.1 mg, 47%). The material was recrystallized from methanol:water to yield a white
solid. Analytical HPLC retention time 8.08 min; purity 99.2%. 1H NMR, (400 MHz, d6-acetone), 7.75 (d,
J = 8.4 Hz, 2H, 9,13ArH), 7.48 (d, J = 2.0 Hz, 2H, 2’,6’ArH), 7.43 (t, J = 2.0 Hz, 1H, 4’ArH), 7.40 (d, J = 8.4
Hz, 2H, 10,12ArH), 6.94 (s, 2H, 2,6ArH), 2.73 (t, J = 7.8 Hz, 2H, CH2(CH2)2CH3), 1.66 (p, J = 7.8, 2H,
CH2CH2CH2CH3), 1.39 (sxt. J = 7.4, 2H, CH2CH2CH2CH3), 0.94 (t, J = 7.4, 3H, (CH2)3CH3). 13C NMR, (100
MHz, d6-acetone), 195.4, 156.1, 148.9, 139.9, 138.5, 136.1, 134.7, 130.8, 130.5, 129.2, 127.4, 118.3,
109.6, 36.2, 34.2, 23.0, 14.2. HRMS: m/z calculated for C23H21Cl2O3, [M+H]+ 415.0868, Found: 415.0865
(4-tert-Butyl-phenyl)-(3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-methanone (46)
Pale pink solid (49.8 mg, 77%). The material was recrystallized from methanol:water to yield a white
1
solid. Analytical HPLC retention time 7.73 min; purity 98.9%. H NMR, (400 MHz, d6-acetone), 7.77 (d,
J = 8.8 Hz, 2H, 9,13ArH), 7.61 (d, J = 8.8 Hz, 2H, 10,12ArH), 7.49 (d, J = 2.1 Hz, 2H, 2’,6’ArH), 7.44 (t, J =
2.1 Hz, 2H, 4’ArH), 6.95 (s, 2H, 2,6ArH), 1.38 (s, 9H, C(CH3)3). 13C NMR, (100 MHz, d6-acetone), 195.4,
156.7, 156.1, 139.8, 138.5, 135.9, 134.7, 130.6, 130.5, 127.4, 126.1, 109.6, 35.6, 31.4. HRMS: m/z
calculated for C23H21Cl2O3, [M+H]+ 415.0868, Found: 415.0868
(3',5'-Dichloro-2,6-dihydroxy-biphenyl-4-yl)-(4-hydroxy-phenyl)-methanone (47)
Pink solid (61.0 mg, 85%). The material was future purified by preparative reverse phase HPLC.
1
Analytical HPLC retention time 5.94 min; purity 99.4%. H NMR, (400 MHz, d6-acetone), 7.79 (d, J = 8.7
Hz, 2H, 9,13ArH), 7.49 (d, J = 1.8 Hz, 2H, 2’,6’ArH), 7.44 (t, J = 1.8 Hz, 1H, 4’ArH), 6.99 (d, J = 8.6 Hz, 2H,
10,12ArH), 6.90 (s, 2H, 2,6ArH). 13C NMR, (100 MHz, d6-acetone), 194.4, 162.5, 156.1, 140.6, 138.6,
134.6, 133.3, 130.5, 130.0, 127.4, 117.8, 115.9, 109.4. HRMS: m/z calculated for C19H13Cl2O4, [M+H]+
375.0191, Found: 375.0178
(4-Chloro-phenyl)-(3',5'-dichloro-2,6-dihydroxy-biphenyl-4-yl)-methanone (48)
Pale pink solid (40.4mg, 89%). The material was recrystallized from methanol:water to yield a white
1
solid. Analytical HPLC retention time 6.83 min; purity 100%. H NMR, (400 MHz, d6-acetone), 7.83 (d, J
= 9.1 Hz, 2H, 9,13ArH), 7.61 (d, J = 9.1 Hz, 2H, 10,12ArH), 7.47 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.44 (t, J = 1.9
Hz, 1H, 4’ArH), 6.94 (s, 2H, 2,6ArH). 13C NMR, (100 MHz, d6-acetone), 194.8, 156.3, 138.7, 134.7,

134.2, 132.6, 132.2, 130.4, 129.4, 127.5, 118.8, 109.7. HRMS: m/z calculated for C19H12Cl3O3, [M+H]+
392.9852, Found: 392.9831
(3',5'-Dichloro-2-hydroxy-6-methoxy-biphenyl-4-yl)-p-tolyl-methanone (49)
A solution of 21 (21.7 mg, 54.0 mol) in dry dichloromethane (3 mL) was cooled to -78° C followed by
the dropwise addition of BBr3 (200 L, 1M solution, 85 mol, 3 eq). The solution was stirred at this
temperature for 1 hour then allowed to warm to ambient temperature and stirred 12 hours. The
solution was cooled to -78° C and 1 piece of ice (~1 mL) was added followed by warming to ambient
temperature. Five milliliters of water and 3 mL of dichloromethane were added, layers separated,
aqueous phase extracted 2 X 3 mL dichloromethane, organic phases combined and dried over sodium
sulfate and the solvent removed. The residue was loaded onto a Grace Reveleris 12g silica column and
eluted with a hexane:methyl t-butyl ether gradient (5% to 5% 4 column volumes, 5% to 80% 13 column
volumes, 80% to 80% 2 column volume, flow rate 36 mL per min.). Dark pink solid (4.7 mg, 24%).
The compound was future purified by preparative reverse phase HPLC. Analytical HPLC retention time
1
9.05 min; purity 100%. H NMR, (400 MHz, CDCl3), 7.78 (d, J = 8.2 Hz, 2H, 9,13ArH), 7.43 (t, J = 1.9 Hz,
1H, 4’ArH), 7.32 (d, J = 8.2 Hz, 2H, 10,12ArH), 7.31 (d, J = 1.9 Hz, 2H, 2’,6’ArH), 7.02 (d, J = 1.5 Hz, 1H,
2ArH), 6.99 (d, J = 1.5 Hz, 1H, 6ArH), 3.80 (s, 3H, OCH3), 2.45 (s, 3H, ArCH3). 13C NMR, (100 MHz, CDCl3),
195.6, 157.3, 153.1, 143.6, 139.5, 135.5, 135.2, 134.5, 130.3, 129.1, 128.4, 118.4, 111.1, 104.4, 56.1,
21.7. HRMS: m/z calculated for C21H17Cl2O3, [M+H]+ 387.0555, Found: 387.0556
(4-Butyl-phenyl)-(3',5'-dichloro-2-hydroxy-6-methoxy-biphenyl-4-yl)-methanone (50)
Dark pink solid (7.5 mg, 38%). The compound was future purified by preparative reverse phase HPLC.
1
Analytical HPLC retention time 10.72 min; purity 100%. H NMR, (400 MHz, CDCl3), 7.79 (d, J = 8.1 Hz,
2H, 9,13ArH), 7.42 (t, J = 2.0 Hz, 1H, 4’ArH), 7.32 (d, J = 8.1 Hz, 2H, 10,12ArH), 7.31 (d, J = 2.0 Hz, 2H,
2’,6’ArH), 7.02 (d, J = 1.3 Hz, 1H, 2ArH), 7.00 (d, J = 1.3 Hz, 1H, 6ArH), 3.79 (s, 3H, OCH3), 2.71 (t, J = 8.2
Hz, 2H, CH2(CH2)2CH3), 1.66 (p, J = 7.7 Hz, 2H, CH2CH2CH2CH3), 1.40 (sxt. J = 7.7, 2H, CH2CH2CH2CH3), 0.96
(t, J = 7.2 Hz, 3H, (CH2)3CH3). 13C NMR, (100 MHz, CDCl3), 195.6, 157.4, 153.1, 148.6, 139.5, 135.4,
134.7, 130.3, 129.1, 128.5, 128.4, 125.7, 118.5, 111.1, 104.4. HRMS: m/z calculated for C24H23Cl2O3,
[M+H]+ 429.1024, Found: 429.1025
(4-Chloro-phenyl)-(3',5'-dichloro-2-hydroxy-6-methoxy-biphenyl-4-yl)-methanone (51)
Dark pink solid (32.1 mg, 33%). The compound was future purified by preparative reverse phase HPLC.
1
Analytical HPLC retention time 9.48; purity 97.8%. H NMR, (400 MHz, CDCl3), 7.80 (d, J = 8.4 Hz, 2H,
9,13ArH), 7.48 (d, J = 8.4 Hz, 2H, 10,12ArH), 7.42 (t, J = 2.1 Hz, 1H, 4’ArH), 7.29 (d, J = 2.1 Hz, 2H,
2’,6’ArH), 6.99 (d, J = 1.3 Hz, 1H, 2ArH), 6.96 (d, J = 1.3 Hz, 1H, 6ArH), 3.78 (s, 3H, OCH3). 13C NMR, (100
MHz, CDCl3), 194.7, 157.6, 153.3, 139.3, 138.7, 135.5, 135.4, 135.0, 131.5, 129.0, 128.8, 128.5, 118.9,
111.1, 104.3, 56.2. HRMS: m/z calculated for C20H14Cl3O3, [M+H]+ 407.0009, Found: 407.0010
4.3 Receptor Binding
Filter plates were filled with 210 L/well of 0.05% (w/v) polyethyleneamine in deionized water and
incubated for 1 hour at room temperature. Plates were then filtered using a vacuum manifold and

washed 5 more times with 250 µL/well of deionized water prior to assay. Each well contained 125 µL
binding buffer, 5 µL of [3H]-CP 55,940 (final concentration 1 nM), 10 µg (in 20 µL) of membrane protein
homogenized in binding buffer [see 44 for membrane preparation methods], and 50 µL test ligand in
binding buffer (final concentrations: 1 nM to 10 µM). Plates were incubated at 30°C for 90 minutes.
Following incubation, solutions were removed via vacuum and wells were washed 9X with 250 µL/well
of binding buffer. Plate backing was then removed, vacuumed dry, and individual filters punched out
using punch tips into 5 mL of Eco-lite scintillation cocktail in 7 mL scintillation vials. Vials were left
overnight and read the next day on PerkinElmer Liquid Scintillation Analyzer Tri-Carb 2810TR using a 3
minute dwell time. All binding studies were carried out with a minimum of 6 biological replicates. CB1
Kd was 1.98 ± 0.6 nM, Bmax 8.47 ± 2.35 pmol/mg. CB2 Kd was 1.65 ± 0.5 nM, Bmax 3.18 ± 0.1 pmol/mg.
4.4 ACTOne functional assay
In order to determine functional pharmacology of the tested compound, HEK-CNG, HEK-CNG+CB1, and
HEK-CNG+CB2 cells were plated at 50,000 cells/100 µL medium in clear Poly-D-lysine coated 96 well
plates in DMEM 10% FBS, 1% P/S medium the day before experiments were performed. The day of the
experiment, ACTOne formulation Membrane Potential Dye was warmed to 37°C and 100 µL added to
each well, followed by 1 hour incubation in the dark at room temperature. Test ligands were tested at
final concentrations from 5x 10-6 to 5 x 10-10 with 25 µM Ro 20-1724, and 800 nM forskolin in DPBS with
2.5% (v/v) DMSO. Once test ligands, Ro, forskolin, and buffer were added, plates were read using a
BioTek (Winooski, VT) plate reader (Ex 540 nm, Em 590 nm) at 50 mins. At least six biological replicates
were used for subsequent data analysis. Data analysis was done in GraphPad Prism 6.0 with non-linear
analysis.
In order to determine antagonist pharmacology, 30 minutes after adding cation sensitive dye, 20 µL of
4-ethyl (41) or 4-n-butyl (45) in DPBS with 2.5% DMSO was added to each well and incubated for an
additional 30 minutes in the dark at room temperature prior to addition of CP 55,940. Concentrations of
41 or 45 were chosen to span concentrations roughly one log above and below the previously
determined EC50. To initiate antagonist studies, 30 µL of CP 55,940 made in DPBS with 2.5% DMSO
buffer, Ro 20-1724 (final concentration 25 µM), and forskolin (final concentration 800 nM) was added to
each well, and the plate was read at 50 minutes. Six independent experiments were used for
subsequent data analysis. Schild analysis was done with sigmoidal curve fit for Log (DR-1) values.
Significance comparisons were done using sum of squares in comparison to the logEC50 from agonist
alone curve.
5. Acknowledgements
This research was supported by the College of Pharmacy and Neuroscience Institute of the University of
Tennessee Health Science Center.

References
1. Devane, W.A., Dysarz, F.A., 3rd, Johnson, M.R., Melvin, L.S., Howlett, A.C., Mol Pharmacol, 1988, 34,
605.
2. Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., Bonner, T.I., Nature, 1990, 346, 561.
3. Munro, S., Thomas, K.L., Abu-Shaar, M., Nature, 1993, 365, 61.
4. Amenta, P.S., Jallo, J.I., Tuma, R.F., Elliott, M.B., J Neurosci Res, 2012, 90, 2293.
5. Arevalo-Martin, A., Vela, J.M., Molina-Holgado, E., Borrell, J., Guaza, C., J Neurosci, 2003, 23, 2511.
6. Kim, K., Moore, D.H., Makriyannis, A., Abood, M.E., Eur J Pharmacol, 2006, 542, 100.
7. Steffens, S., Veillard, N.R., Arnaud, C., Pelli, G., Burger, F., Staub, C., Karsak, M., Zimmer, A., Frossard,
J.L., Mach, F., Nature, 2005, 434, 782.
8. Lake, K.D., Compton, D.R., Varga, K., Martin, B.R., Kunos, G., J Pharmacol Exp Ther, 1997, 281, 1030.
9. Niederhoffer, N., Szabo, B., Br J Pharmacol, 1999, 126, 457.
10. Matias, I., Gonthier, M.P., Orlando, P., Martiadis, V., De Petrocellis, L., Cervino, C., Petrosino, S.,
Hoareau, L., Festy, F., Pasquali, R., Roche, R., Maj, M., Pagotto, U., Monteleone, P., Di Marzo, V., J Clin
Endocrinol Metab, 2006, 91, 3171.
11. Bermudez-Silva, F.J., Sanchez-Vera, I., Suarez, J., Serrano, A., Fuentes, E., Juan-Pico, P., Nadal, A.,
Rodriguez De Fonseca, F., Eur J Pharmacol, 2007, 565, 207.
12. Romero-Zerbo, S.Y., Garcia-Gutierrez, M.S., Suarez, J., Rivera, P., Ruz-Maldonado, I., Vida, M.,
Rodriguez De Fonseca, F., Manzanares, J., Bermudez-Silva, F.J., J Neuroendocrinol, 2012, 24, 1106.
13. Duntsch, C., Divi, M.K., Jones, T., Zhou, Q., Krishnamurthy, M., Boehm, P., Wood, G., Sills, A., Moore,
B.M., 2nd, J Neurooncol, 2006, 77, 143.
14. Gurley, S.N., Abidi, A.H., Allison, P., Guan, P., Duntsch, C., Robertson, J.H., Kosanke, S.D., Keir, S.T.,
Bigner, D.D., Elberger, A.J., Moore, B.M., 2nd, J Neurooncol, 2012, 110, 163.
15. Martinez-Martinez, E., Gomez, I., Martin, P., Sanchez, A., Roman, L., Tejerina, E., Bonilla, F., Merino,
A.G., De Herreros, A.G., Provencio, M., Garcia, J.M., Oncoscience, 2015, 2, 131.
16. Mcallister, S.D., Murase, R., Christian, R.T., Lau, D., Zielinski, A.J., Allison, J., Almanza, C., Pakdel, A.,
Lee, J., Limbad, C., Liu, Y., Debs, R.J., Moore, D.H., Desprez, P.Y., Breast Cancer Res Treat, 2011, 129, 37.
17. Aso, E., Juves, S., Maldonado, R., Ferrer, I., J Alzheimers Dis, 2013, 35, 847.
18. Cheng, Y., Dong, Z., Liu, S., Pharmacology, 2014, 94, 1.
19. Garcia-Arencibia, M., Gonzalez, S., De Lago, E., Ramos, J.A., Mechoulam, R., Fernandez-Ruiz, J., Brain
Res, 2007, 1134, 162.
20. Shoemaker, J.L., Seely, K.A., Reed, R.L., Crow, J.P., Prather, P.L., J Neurochem, 2007, 101, 87.
21. Maresz, K., Carrier, E.J., Ponomarev, E.D., Hillard, C.J., Dittel, B.N., J Neurochem, 2005, 95, 437.
22. Cabral, G.A., Raborn, E.S., Griffin, L., Dennis, J., Marciano-Cabral, F., Br J Pharmacol, 2008, 153, 240.
23. Ashton, J.C., Glass, M., Curr Neuropharmacol, 2007, 5, 73.
24. Amenta, P.S., Jallo, J.I., Tuma, R.F., Hooper, D.C., Elliott, M.B., J Neuroinflammation, 2014, 11, 191.
25. Reiner, A., Heldt, S.A., Presley, C.S., Guley, N.H., Elberger, A.J., Deng, Y., D'surney, L., Rogers, J.T.,
Ferrell, J., Bu, W., Del Mar, N., Honig, M.G., Gurley, S.N., Ii, B.M., Int J Mol Sci, 2015, 16, 758.
26. Cao, C., Li, Y., Liu, H., Bai, G., Mayl, J., Lin, X., Sutherland, K., Nabar, N., Cai, J., J Alzheimers Dis, 2014,
42, 973.
27. Esposito, G., Iuvone, T., Savani, C., Scuderi, C., De Filippis, D., Papa, M., Di Marzo, V., Steardo, L., J
Pharmacol Exp Ther, 2007, 322, 1144.
28. Fakhfouri, G., Ahmadiani, A., Rahimian, R., Grolla, A.A., Moradi, F., Haeri, A., Neuropharmacology,
2012, 63, 653.
29. Martin-Moreno, A.M., Brera, B., Spuch, C., Carro, E., Garcia-Garcia, L., Delgado, M., Pozo, M.A.,
Innamorato, N.G., Cuadrado, A., De Ceballos, M.L., J Neuroinflammation, 2012, 9, 8.

30. Wu, J., Bie, B., Yang, H., Xu, J.J., Brown, D.L., Naguib, M., Neurobiol Aging, 2013, 34, 791.
31. Garcia, C., Palomo-Garo, C., Garcia-Arencibia, M., Ramos, J., Pertwee, R., Fernandez-Ruiz, J., Br J
Pharmacol, 2011, 163, 1495.
32. Price, D.A., Martinez, A.A., Seillier, A., Koek, W., Acosta, Y., Fernandez, E., Strong, R., Lutz, B.,
Marsicano, G., Roberts, J.L., Giuffrida, A., Eur J Neurosci, 2009, 29, 2177.
33. Arevalo-Martin, A., Molina-Holgado, E., Guaza, C., Neuropharmacology, 2012, 63, 385.
34. Baker, D., Pryce, G., Croxford, J.L., Brown, P., Pertwee, R.G., Huffman, J.W., Layward, L., Nature,
2000, 404, 84.
35. Carrillo-Salinas, F.J., Navarrete, C., Mecha, M., Feliu, A., Collado, J.A., Cantarero, I., Bellido, M.L.,
Munoz, E., Guaza, C., PLoS One, 2014, 9, e94733.
36. Docagne, F., Muneton, V., Clemente, D., Ali, C., Loria, F., Correa, F., Hernangomez, M., Mestre, L.,
Vivien, D., Guaza, C., Mol Cell Neurosci, 2007, 34, 551.
37. Gijsen, H.J., De Cleyn, M.A., Surkyn, M., Van Lommen, G.R., Verbist, B.M., Nijsen, M.J., Meert, T.,
Wauwe, J.V., Aerssens, J., Bioorg Med Chem Lett, 2012, 22, 547.
38. Mestre, L., Docagne, F., Correa, F., Loria, F., Hernangomez, M., Borrell, J., Guaza, C., Mol Cell
Neurosci, 2009, 40, 258.
39. Ni, X., Geller, E.B., Eppihimer, M.J., Eisenstein, T.K., Adler, M.W., Tuma, R.F., Mult Scler, 2004, 10,
158.
40. Han, S., Thatte, J., Buzard, D.J., Jones, R.M., J Med Chem, 2013, 56, 8224.
41. Lunn, C.A., Reich, E.P., Fine, J.S., Lavey, B., Kozlowski, J.A., Hipkin, R.W., Lundell, D.J., Bober, L., Br J
Pharmacol, 2008, 153, 226.
42. Pagé, D., Brochu, M.-C., Yang, H., Brown, W., St-Onge, S., Martin, E., Salois, D., Lett. Drug Des. Discov,
2006, 3, 298.
43. Iwamura, H., Suzuki, H., Ueda, Y., Kaya, T., Inaba, T., J Pharmacol Exp Ther, 2001, 296, 420.
44. Presley, C.S., Abidi, A.H., Suryawanshi, S., Mustafa, S., Meibohm, B., Moore, B.M., 2nd, Pharmacol
Res Perspect, 2015.
45. Kumar, P., Song, Z.H., Biochem Biophys Res Commun, 2013, 435, 76.
46. Darroch, S.A., Taylor, W.C., Choo, L.K., Mitchelson, F., Eur J Pharmacol, 1990, 182, 131.
47. Thomas, A., Baillie, G.L., Phillips, A.M., Razdan, R.K., Ross, R.A., Pertwee, R.G., Br J Pharmacol, 2007,
150, 613.
48. Bhattacharjee, H., Gurley, S.N., Moore, B.M., 2nd, Bioorg Med Chem Lett, 2009, 19, 1691.
49. Krishnamurthy, M., Gurley, S., Moore, B.M., 2nd, Bioorg Med Chem, 2008, 16, 6489.
50. Makriyannis, A., Lai, X., Lu, C., 2004.
51. Nadipuram, A.K., Krishnamurthy, M., Ferreira, A.M., Li, W., Moore, B.M., Bioorg Med Chem, 2003,
11, 3121.
52. Pertwee, R.G., Handb Exp Pharmacol, 2005, 1.
53. Mclure, K., Young, P.R., 2013.
54. Fujinaga, M., Kumata, K., Yanamoto, K., Kawamura, K., Yamasaki, T., Yui, J., Hatori, A., Ogawa, M.,
Yoshida, Y., Nengaki, N., Maeda, J., Zhang, M.R., Bioorg Med Chem Lett, 2010, 20, 1565.
55. Leblond, B., Taverne, T., Beausoleil, B., Chauvignac, C., Casagrande, S., Desire, L., 2013.
56. Kulkarni, P.P., Kadam, A.J., Mane, R.B., Desai, U.V., Wadgaonkar, P.P., J. Chem Res Synop, 1999, 394.
57. Houston, D.B., Howlett, A.C., Cell Signal, 1998, 10, 667.
58. Turu, G., Hunyady, L., J Mol Endocrinol, 2010, 44, 75.
59. Krishnamurthy, M., Ferreira, A.M., Moore, B.M., 2nd, Bioorg Med Chem Lett, 2003, 13, 3487.
60. Ferreira, A.M., Moore, B.M., 2nd, Krishnamurthy, M., Journal of Molecular Structure (Theochem),
2004, 131.
61. Papahatjis, D.P., Kourouli, T., Abadji, V., Goutopoulos, A., Makriyannis, A., J Med Chem, 1998, 41,
1195.