Chain substituted cannabilactones with selectivity for the CB2 cannabinoid receptor

Article abstract of DOI:10.3390/molecules24193559

In earlier work, we reported a novel class of CB2 selective ligands namely cannabilactones. These compounds carry a dimethylheptyl substituent at C3, which is typical for synthetic cannabinoids. In the current study with the focus on the pharmacophoric side chain at C3 we explored the effect of replacing the C1′-gem-dimethyl group with the bulkier cyclopentyl ring, and, we also probed the chain's length and terminal carbon substitution with bromo or cyano groups. One of the analogs synthesized namely 6-[1-(1,9-dihydroxy-6-oxo-6H-benzo[c]chromen-3-yl) cyclopentyl] hexanenitrile (AM4346) has very high affinity (K_i = 4.9 nM) for the mouse CB2 receptor (mCB2) and 131-fold selectivity for that target over the rat CB1 (rCB1). The species difference in the affinities of AM4346 between the mouse (m) and the human (h) CB2 receptors is reduced when compared to our first-generation cannabilactones. In the cyclase assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC₅₀ = 3.7 ± 1.5 nM, E_(max) = 89%). We have also extended our structure-activity relationship (SAR) studies to include biphenyl synthetic intermediates that mimic the structure of the phytocannabinoid cannabinodiol.

Full text of DOI:10.3390/molecules24193559

molecules
Article
Chain Substituted Cannabilactones with Selectivity
for the CB2 Cannabinoid Receptor
Shakiru O. Alapafuja 1, Spyros P. Nikas 1,
*
, Thanh C. Ho 1 , Fei Tong 1, Othman Benchama 1
and Alexandros Makriyannis 1,2,
*
1
Center for Drug Discovery and Department of Pharmaceutical Sciences, Northeastern University,
Boston, MA 02115, USA; s.alapafuja@northeastern.edu (S.O.A.); t.ho@northeastern.edu (T.C.H.);
tong.f@husky.neu.edu (F.T.); othmanb@gmail.com (O.B.)
Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, USA
Correspondence: s.nikas@northeastern.edu (S.P.N.); a.makriyannis@northeastern.edu (A.M.);
Tel.: +1617-373-3125 (S.P.N.); +1617-373-4200 (A.M.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Spyros P. Nikas
Received: 23 July 2019; Accepted: 27 September 2019; Published: 1 October 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In earlier work, we reported a novel class of CB2 selective ligands namely cannabilactones.
These compounds carry a dimethylheptyl substituent at C3, which is typical for synthetic cannabinoids.
In the current study with the focus on the pharmacophoric side chain at C3 we explored the
effect of replacing the C10-gem-dimethyl group with the bulkier cyclopentyl ring, and, we also
probed the chain’s length and terminal carbon substitution with bromo or cyano groups. One of
the analogs synthesized namely 6-[1-(1,9-dihydroxy-6-oxo-6H-benzo[c]chromen-3-yl) cyclopentyl]
hexanenitrile (AM4346) has very high affinity (Ki = 4.9 nM) for the mouse CB2 receptor (mCB2) and
131-fold selectivity for that target over the rat CB1 (rCB1). The species difference in the affinities
of AM4346 between the mouse (m) and the human (h) CB2 receptors is reduced when compared
to our first-generation cannabilactones. In the cyclase assay, our lead compound was found to be a
highly potent and efficacious hCB2 receptor agonist (EC50 = 3.7
extended our structure-activity relationship (SAR) studies to include biphenyl synthetic intermediates
that mimic the structure of the phytocannabinoid cannabinodiol.
±
1.5 nM, E(max) = 89%). We have also
Keywords: cannabinoid receptors; CB2 selective ligands; synthesis; cannabilactones; structure-activity
relationship studies
1. Introduction
The two G-protein coupled receptors (GPCRs) termed cannabinoid receptor 1 (CB1) and cannabinoid
receptor 2 (CB2) are principal components of the endocannabinoid biosignaling system and molecular
targets for the psychoactive constituent of cannabis
CB2 also bind the FDA approved drug Nabilone and many other exogenous (plant-derived and synthetic)
cannabinoids. The CB1 receptor retains a high ( 97%) degree of amino acid sequence identity across
mouse, rat, and human [ ]. In contrast, the human CB2 receptor displays only 82% and 81% amino
acid identity with mouse [ ] and rat [ ] respectively. The CB1 receptor was found to be localized
primarily in the brain and it is one of the most abundant GPCRs in the central nervous system (CNS) [ ].
This receptor is also found in peripheral tissues and organs [ ]. Activation of the central CB1 receptors
∆ ∆ 1–3]. Both CB1 and
9-tetrahydrocannabinol ( 9-THC) [
≥
4
5
6
4
7
mediates most of the cannabinoid psychotropic and behavioral effects. Conversely, the CB2 receptors
are detectable at very low levels in brain and they are expressed predominantly in immune cells and
in the periphery [8]. Nevertheless, the CB2 receptors may be induced in the CNS under pathologic
conditions. Collaborative efforts, including our laboratory, have led to the first crystal structures of the
agonist and antagonist bound human CB1, while recently we published the first crystal structure of the
Molecules 2019, 24, 3559; doi:10.3390/molecules24193559
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3559
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human CB2 receptor in a complex with the antagonist AM10257. This breakthrough work provides a
molecular basis for predicting the binding modes of cannabinoids with their target proteins, and offers
invaluable information for target-based drug design [9–12].
Selective CB2 receptor activation is a very promising method of modulating the endocannabinoid
system because the pharmacologic effects are devoid of centrally mediated liabilities that are correlated
with CB1 receptor modulation either directly by exogenous cannabinoids or indirectly through
functional interactions with other receptor systems [13]. Selective CB2 activation would decrease
CB1 associated ataxia, hypothermia, mood and memory disturbances, and abuse potential [14
Currently, the CB2 receptor has emerged as an attractive therapeutic target for the treatment of
inflammatory and neuropathic pain, as well as neurodegenerative disorders [20 23], and cancer [24 26].
–19].
–
–
Our group has identified the cannabilactones (Figure 1), as a new class of CB2 agonists with
structural similarities to the 6,6-dimethyl counterpart and phytocannabinoid cannabinol (Figure 2).
Two first generation analogs within this class of compounds namely AM1714 and AM1710 (Figure 1)
have become important pharmacological tools in establishing proof of concept for the usefulness
of the CB2 receptor activation approach [27–33]. Both these CB2 selective agonists are found to
possess potent peripheral analgesic activity in several animal models of inflammatory and neuropathic
pain. Moreover, functional studies with a lead compound have highlighted the potential of the
cannabilactone based CB2 agonists to behave as neutral antagonists/low potency inverse agonists at
CB1, a unique property which imparts very high functional selectivity for CB2 over CB1 [30,31,33].
Thus, from a medicinal chemistry perspective, the class of cannabilactone compounds remains an
attractive structural motif for further development of CB2 selective and efficacious agonists.
Figure 1. Design of chain substituted cannabilactones and structures of the first-generation analogs
AM1710 and AM1714.
Figure 2. Chemical structures of the phytocannabinoids cannabinol (CBN), cannabidiol (CBD),
and cannabinodiol (CBND).
The present work is informed by our findings with analogs of (
(THC) and (
)-hexahydrocannabinol (HHC) with cyclic moieties at the C10 position of the side chain
pharmacophore [34 37] (Figure 1). These studies have shown that analogs with six- to eight-atoms-long
−
)-∆
8-tetrahydrocannabinol
−
–
side chains substituted at C10 with a cyclopentyl ring exhibit remarkably high affinities for CB1 and
CB2 receptors. Taken together, in this study our design replaces the 10,10-gem-dimethyl group in the
cannabilactone scaffold with the larger and sterically more confined cyclopentyl group (Figure 1).
Additionally, we have explored the pharmacophoric limits of side chain length, while the polar

Molecules 2019, 24, 3559
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characteristics have been enhanced by incorporating bromo- and cyano-substituents at the terminal
carbon atom [37 38]. All synthesized compounds were assessed for their binding affinities at rat CB1
,
(rCB1) and mouse CB2 (mCB2) receptors while the most promising analogs were also tested in human
CB2 (hCB2) to identify potential species differences between the mouse and human clone. One of the
analogs synthesized namely AM4346 has very high affinity (Ki = 4.9 nM) and 131-fold selectivity for
mCB2 over rCB1 and behaves as a potent CB2 agonist in the cyclase assay. The species difference in the
affinities of AM4346 between the mouse and the human CB2 receptors is reduced when compared to
our first-generation compound AM1714, while AM4346 is endowed with enhanced polarity due to the
presence of the cyano group. Additionally, we extended our SAR to include the biphenyl synthetic
intermediates 23a–23d as they encompasses the biaryl subunit which is a privileged structure (biaryls
found in 4.3% of known drugs), and also, they have structural similarities with the phytocannabinoid
cannabidiol and its oxidative metabolite cannabinodiol (Figure 2) [39–42].
2. Results and Discussion
2.1. Chemistry
We conjectured that cannabilactones would be assessible from biphenyl compounds
7 via
lactonization (Scheme 1). The bromo- or cyano-group on the C3-side chain of cannabilactones would
be introduced to either the biphenyl or the cannabilactone template via substitution. Disconnection of
biphenyl compounds 7 through a Suzuki coupling led to boronic acid 8 and aryl bromides 9.
OH
OMe
Suzuki cross-coupling
lactonization
OH
OMe
OMe
OMe
Br
B(OH)2
+
3
X
Y
Y
( )n
( )n
MeO
( )n
O
O
N
O
N
O
OMe
substitution
cannabilactones (3)
n = 2–4, X = Br, CN, Me
7
8
9
Y = OPh, Me
Scheme 1. Retrosynthetic analysis of cannabilactones.
The synthesis of aryl bromides 9a–9d is summarized in Scheme 2. Bromides 9a–9c were prepared
in a general approach starting from commercially available 4-bromo-3,5-dihydroxybenzoic acid (10).
Following the procedure of Luning et al. [43], etherification of the phenolic hydroxyl groups as well
as, esterification of the carboxylic acid was accomplished in a single step by refluxing a mixture of
10, potassium carbonate, and dimethyl sulfate in acetone. Reduction of the resulting ester 11 with
diisobutylaluminum hydride (DIBAL-H) gave benzylic alcohol 12. Although the conversion of 12
to the benzyl chloride has been reported [44], we adopted a more convenient and higher yielding
approach. This involved refluxing a mixture of 12 and triphenylphosphine in dry carbon tetrachloride
for two hours [35
sulfoxide afforded benzyl nitrile 14 in 87% yield [35
,
45
,
46] to give 13 in 93% yield. Treatment of 13 with sodium cyanide in dimethyl
44]. Sequential deprotonation of 14 using potassium
,
bis(trimethylsilyl)amide and cyclobisalkylation using 1,4-dibromobutane in tetrahydrofuran (THF) at
0 ◦C produced cyclopentyl nitrile 15 in 70% yield [35
yield with DIBAL-H at −78 ◦C [48].
,47]. This was transformed to aldehyde 16 in 85%

Molecules 2019, 24, 3559
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OH
OMe
OMe
OMe
OMe
Br
Br
Br
Br
Br
d
a
b
c
OH
HO
OMe
OH
Cl
CN
MeO
MeO
MeO
MeO
O
O
10
11
12
13
14
OMe
OMe
OMe
OMe
Br
Br
Br
Br
g
h
e
f
O
2'
3'
CN
OPh
( )n
MeO
MeO
H
MeO
MeO
( )
n OPh
15
16
17a
17b
9a
: n = 3
9b
: n = 4
: n = 2
: n = 3
OMe
OMe
OMe
OMe
Br
Br
MeO
Br
Br
k
O
H
i
O
j
O
2'
MeO
OEt
MeO
OR
MeO
OH
3'
20
16
18
19a: R = Et
19b
: R = Me
OMe
OMe
Br
Br
l
m
MeO
Br
MeO
OPh
21
9c
OMe
OMe
Br
MeO
n
MeO
22
9d
Scheme 2. Synthesis of aryl bromides a.
a Reagents and conditions: (a) Me2SO4, K2CO3, acetone, reflux, 92%; (b) DIBAL-H, THF, rt, 1 h,
85%; (c) Ph3P, CCl4, reflux, 93%; (d) NaCN, DMSO, rt, overnight, 87%; (e) KHMDS, 1,4-dibromobutane,
THF, 0 ◦C, 5 min, 70%; (f) DIBAL-H, CH2Cl2,
−
78 ◦C, 1 h, 85%; (g) (3-phenoxypropyl)- or
◦
(4-phenoxybutyl)triphenylphosphonium bromide, KHMDS, THF, 0 C, 10 min, 94% for 17a and
93% for 17b; (h) H2, Pd/C, EtOAc, rt, 12 h, 95% for 9a and 94% for 9b; (i) triethyl phosphonoacetate,
NaH, THF, 92%; (j) for 19a: H2, Pd/C, EtOAc, 30 psi, 95%; for 19b: Mg/MeOH, 0 ◦C, 2 h, then rt, 12 h,
80%; (k) DIBAL-H, THF, rt, 1 h, 86% from 19a, 85% from 19b; (l) Ph3P/CBr4, 0 ◦C
DMSO, rt, 20 h, 50%; (n) Br2, CCl4, 0 ◦C, 1 h, 78%.
→
rt, 85%; (m) NaOPh,
Wittig olefination of aldehyde 16 with (3-phenoxypropyl)- and (4-phenoxybutyl)
triphenylphosphonium bromides gave exclusively cis-alkenes 17a and 17b, respectively, in 93–94%
yields. The geometry of the newly formed double bond in 17a and 17b was assigned based on
1
the H NMR spectra of crude products (3JH2 −H3 ~ 11.3 Hz). Reduction of these alkenes by
catalytic hydrogenation afforded the corresponding aryl bromides 9a and 9b. Wittig olefination
of aldehyde 16 with (2-phenoxyethyl)triphenylphosphonium bromide was not successful because
vinyltriphenylphosphonium bromide was generated under the reaction conditions [49]. To prepare
aryl bromide 9c, a slightly modified route was followed. Horner-Wardsworth-Emmons olefination
0
0
of aldehyde 16 with commercially available triethyl phosphonoacetate [50–52] produced exclusively
trans-conjugated ester 18 in 92% yield (3JH2 −H3 ~ 15.8 Hz). Palladium-catalyzed hydrogenation of 18 in
ethyl acetate required high pressure (30 psi) to produce ethyl ester 19a in 95% yield, whereas exposure
of 18 to magnesium turnings in methanol gave methyl ester 19b in 80% yield [53]. Reduction of
either 19a or 19b with DIBAL-H at room temperature gave alcohol 20 (85–86% yield). This was
followed by treatment of 20 with triphenylphosphine and carbon tetrabromide to afford 21 in 85%
yield. Phenoxide displacement of 21 in dimethyl sulfoxide provided aryl bromide 9c in moderate yield
0
0
(50%). Additionally, aryl bromide 9d was prepared via bromination of resorcinol dimethyl ether 22 [35]
using bromine in carbon tetrachloride (78% yield) [54].

Molecules 2019, 24, 3559
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With aryl bromides 9a
–
9d in hand, we proceeded to complete the synthesis of cannabilactones
(Scheme 3). Suzuki cross-coupling of aryl bromides 9a
–
9d with boronic acid
8, which was prepared
from commercially available 4-methoxybenzoic acid [55
,
56], furnished the sterically hindered biphenyls
7a–7d in 48–50% yields.
OMe
OH
OH
OMe
OH
OH
OMe
Br
a
b
c
OPh
( )n
X
X
X
( )n
( )n
( )n
MeO
N
O
N
O
O
O
OMe
OH
9a: n = 3, X = OPh
9b
7a: n = 3, X = OPh
7b
23a: n = 3, X = Br
23b
3a: n = 3, X = Br
3b
: n = 4, X = OPh
9c: n = 2, X = OPh
9d
: n = 4, X = OPh
7c: n = 2, X = OPh
7d
: n = 4, X = Br
23c: n = 2, X = Br
23d
: n = 4, X = Br
3c: n = 2, X = Br
3d
: n = 4, X = Me
: n = 4, X = Me
: n = 4, X = Me
: n = 4, X = Me
OH
OH
OMe
d
CN
( )n
O
O
B(OH)2
3e: n = 3
3f
N
O
: n = 4
3g: n = 2
8
Scheme 3. Synthesis of cannabilactones a.
a Reagents and conditions: (a)
50% for 7b from 9b, 50% for 7c from 9c, and 50% for 7d from 9d; (b) BBr3, CH2Cl2,
92% for 23a from 7a, 90% for 23b from 7b, 91% for 23c from 7c, and 89% for 23d from 7d; (c) AcOH,
8
, Ba(OH)2, DME/H2O, Pd(Ph3P)4, 80 ◦C, 6 h, 48% for 7a from 9a
,
−
78 ◦C, rt, 12 h,
reflux, 24 h, 89% for 3a from 23a, 90% for 3b from 23b, 91% for 3c from 23c, and 91% for 3d from 23d
(d) NaCN, DMSO, rt, 24 h, 72% for 3e from 3a, 69% for 3f from 3b, and 70% for 3g from 3c.
;
Exposure of 7a
yields, in which cleavage of all methyl ether groups and of the phenolic ether with introduction of
the bromide in the case of 7a 7c took place in a single step. Acetic acid mediated lactonization of
polyphenols 23a 23d afforded cannabilactones 3a 3d in 89–91% yields. Subsequent exposure of 3a 3c
–7d to boron tribromide led to trihydroxybiphenyl intermediates 23a–23d in 89–92%
–
–
–
–
to sodium cyanide in dimethyl sulfoxide gave nitriles 3e–3g.
2.2. Affinity for Cannabinoid Receptors
The abilities of the cannabilactone analogs 3a
CP-55,940 from membranes prepared from rat brain (a source of CB1) and HEK293 cells expressing
mouse CB2 were determined as described earlier [38 57 58]. Inhibition constant values (Ki) from the
–3g to displace the radiolabeled CB1/CB2 agonist
,
,
respective competition binding curves are listed in Table 1 in which our first generation cannabilactone
analog AM1714 is included for comparison. The current data of AM1714 for mCB2 are slightly different
when compared to those we published earlier [27]. This is because the compound was first assayed in
a different mCB2 receptor preparation, e.g., mouse spleen membrane.
It should also be noted that the rat, mouse, and human CB1 receptors have 97–99% sequence
identity across species and, as shown earlier (see for example [12,37,38,57]), are not expected to
exhibit variations in their Ki values. However, the CB2 receptor shows less homology (~82%)
between species than does CB1 (97–99%), and that variability could cause species-related differences in
affinity. Indeed, in our original work on the cannabilactone class of compounds, we have identified
species-specific variation in CB2 affinity [27]. For this reason, the key compounds were also assayed
using membranes from HEK293 cells expressing human CB2 (hCB2). Data from the latter preparation
are listed in Table 2.
As shown in Table 1, replacement of the C10-gem-dimethyl group with the bulkier, more sterically
confined cyclopentyl ring produces cannabilactone analogs with enhanced affinity and selectivity for

Molecules 2019, 24, 3559
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the mCB2 relative to the rCB1 receptors. We also observe that this trend for mCB2 selectivity can be
optimized by varying the length of the side chain and the substituent at the terminal carbon atom.
Thus, analogs carrying five- to seven-atoms long side chains terminated with a bromine atom or a
cyano group (3c
, 3a, 3b, 3g, and 3e) exhibit 16- to 26-fold selectivity for mCB2 over rCB1. The more
lipophilic C10-gem-dimethyl-heptyl (1b) and C10-cyclopentyl-heptyl (3d) analogs have comparable
affinity (Ki = 4.7
±
1.8 nM and 8.4
±
2.5 nM) and selectivity (21- to 32-fold) for mCB2 over rCB1.
Within these series, however, analog AM4346 with its longer chain (eight atoms) and C60-cyano
substituent has optimal properties: maximal binding affinity for mCB2 and minimal affinity for rCB1.
In fact, AM4346 exhibits a remarkable 131-fold selectivity for mCB2 over rCB1.
The binding affinities for the human CB2 (hCB2) receptor of the three key analogs AM1714, AM4348,
and AM4346 are listed in Table 2. We observe that although all analogs exhibit somewhat reduced
affinity for hCB2 as compared to mCB2 (Table 1), the C10-cyclopentyl-analogs show a pronounced
reduction in the affinity differences between mCB2 and hCB2 as compared to C10-gem-dimethyl analog.
Thus, AM1714 has 18-fold greater affinity for mCB2 than for hCB2, but this preference is reduced to
3-fold for AM4348 and 7-fold for AM4346. With a roughly 19-fold preference, analog AM4346 has the
highest selectivity for hCB2 over rCB1.
Table 1. Affinities of side chain-modified cannabilactone analogs for rCB1 and mCB2 cannabinoid
receptors (± 95% confidence limits).
OH
OH
O
O
R
Ki (nM) 1
Compound
rCB1/mCB2
R
rCB1
mCB2
1b
AM1714
100 ± 24
4.7 ± 1.8
21.3
16.7
3c
Br
405 ± 122
24.3 ± 3.9
20.8 ± 5.9
16.3 ± 5.2
17.9 ± 5.8
11.8 ± 4.1
4.9 ± 1.2
8.4 ± 2.5
AM4354
Br
3a
AM4350
390 ± 132
370 ± 130
290 ± 64
304 ± 73
640 ± 152
266 ± 58
18.8
22.7
16.2
25.7
130.6
31.7
3b
AM4345
Br
3g
AM4355
CN
CN
3e
AM4351
3f
CN
AM4346
3d
AM4348
1
Affinities were determined using rat brain (CB1) or membranes from HEK293 cells expressing mouse CB2 and
[3H]CP-55,940 as the radioligand following previously described procedures [38
57 58]. Data were analyzed using
,
,
nonlinear regression analysis. Ki values were obtained from three independent experiments performed in triplicate
and are expressed as the mean of the three values.

Molecules 2019, 24, 3559
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Table 2. Affinities of key cannabilactone analogs for hCB2 cannabinoid receptors ( 95% confidence limits).
±
OH
OH
O
O
R
Ki (nM) 1
Compound
rCB1/hCB2
hCB2/mCB2
R
rCB1
hCB2
100 ± 24
640 ± 152
266 ± 58
83.6 ± 23.2
1.2
17.7
1b
AM1714
CN
33.8 ± 10.4
28.0 ± 9.7
18.9
9.5
6.9
3.3
3f
AM4346
3d
AM4348
1
Cannabinoid receptors were prepared as described for Table 1, except that hCB2 was from HEK293 expressing
the human and not the mouse protein. Binding affinities were determined, and data were analyzed as for Table 1.
Affinities for rCB1 (from Table 1) are included for comparison.
Synthesized as precursors to cannabilactones, binding affinities of the intermediate biphenyl
compounds 23a–23d for rCB1 and mCB2 were also determined (Table 3). To our surprise, these
biphenyl analogs bind mCB2 with high affinity and substantial selectivity for that receptor over rCB1.
In this series, the C10-cyclopentyl-heptyl analog AM4347 has both the greatest binding affinity (Ki = 5.7
± 1.5 nM) for mCB2 and the highest selectivity (60-fold) for that receptor over rCB1.
Table 3. Affinities of biphenyl analogs for rCB1 and mCB2 cannabinoid receptors (
±
95% confidence limits).
OH
OH
N
O
R
OH
Ki (nM) 1
Compound
rCB1/mCB2
R
rCB1
mCB2
Br
229 ± 70
702 ± 220
745 ± 215
341 ± 100
10.5 ± 3.5
21.8
31.3
42.8
59.8
23c
AM4353
Br
22.4 ± 4.5
17.4 ± 4.8
5.7 ± 1.5
23a
AM4349
Br
23b
AM4344
23d
AM4347
1
Cannabinoid receptors were prepared, binding affinities measured, and data analyzed as described for Table 1.

Molecules 2019, 24, 3559
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2.3. Functional Characterization
Functional characterization of three key cannabilactones (AM1710, AM1714, and AM4346) for the
hCB2 receptor was carried out by measuring the decrease in forskolin stimulated cAMP, as detailed
earlier [38,57]. Data are listed in Table 4 in which the standard cannabinoid agonist CP-55,940 is
included for comparison. Our testing results show that all three compounds potently decreased the
levels of cAMP, indicating that within this signaling mechanism these compounds behaved as potent
agonists at the hCB2 receptor with the C10-cyclopentyl-analog AM4346 being more potent (EC50 = 3.7
±
1.5 nM, E(max) = 89%) than the C10-gem-dimethyl-analogs AM1710 and AM1714 (EC50 = 10.5
(max) = 73% and EC50 = 36.9 ± 6.8 nM, E(max) = 77% respectively).
In the same assay, one of the biphenyl analogs, compound 23d (AM4347), failed to show any
±
2.5 nM,
E
responses in concentrations up to 2 µM.
Table 4. Functional potencies (EC50) of key cannabilactones and CP-55,940 for the hCB2 cannabinoid
receptor (± 95% confidence limits).
EC50 (nM)1 (classification)
E(max) (%) 2
Compound
hCB2
3.4 ± 1.2 (agonist)
10.5 ± 2.5 (agonist)
100
73
CP-55,940
1a
AM1710
1b
AM1714
3f
AM4346
36.9 ± 6.8 (agonist)
3.7 ± 1.5 (agonist)
77
89
1
Functional potencies at hCB2 receptor were determined by measuring the decrease in forskolin-stimulated
cAMP levels [38,57]. EC50 values were calculated using nonlinear regression analysis. Data are the average of
two independent experiments run in triplicate. 2Forskolin stimulated cAMP levels were normalized to 100%.
E(max) is the maximum inhibition of forskolin stimulated cAMP levels and is presented as the percentage of
CP-55,940 response at 500 nM.
3. Experimental Section
Materials: all reagents and solvents were purchased from Aldrich Chemical Company, unless
otherwise specified, and used without further purification. All anhydrous reactions were performed
under a static argon atmosphere in flame-dried glassware using scrupulously dry solvents. Flash column
chromatography employed silica gel 60 (230–400 mesh). All compounds were demonstrated to
be homogeneous by analytical TLC on pre-coated silica gel TLC plates (Merck, 60 F245 on glass,
layer thickness 250 µm), and chromatograms were visualized by phosphomolybdic acid staining.
Melting points were determined on a micro-melting point apparatus and are uncorrected. IR spectra
were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer. NMR spectra were recorded in
CDCl3, unless otherwise stated, on a Bruker Ultra Shield 400 WB plus (1H at 400 MHz, 13C at 100 MHz)
or on a Varian INOVA-500 (1H at 500 MHz) spectrometers and chemical shifts are reported in units
of
δ relative to internal TMS. Multiplicities are indicated as br (broadened), s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet) and coupling constants (J) are reported in hertz (Hz). Low and
high-resolution mass spectra were performed in School of Chemical Sciences, University of Illinois at
Urbana-Champaign. Mass spectral data are reported in the form of m/z (intensity relative to base = 100).
4-Bromo-3,5-dimethoxybenzyl
chloride
(13).
To
a
stirred
solution
of
4-bromo-3,5-dimethoxyphenylmethanol (12) (4.74 g, 19.2 mmol) in anhydrous carbon tetrachloride
(200 mL) at room temperature under an argon atmosphere was added triphenylphosphine (11.6 g,
42.2 mmol). The reaction mixture was refluxed for 45 min and then cooled to room temperature with
spontaneous precipitation of triphenylphosphine oxide. To this suspension was added anhydrous
hexane, the white precipitate was filtered off, washed with hexane, and the combined filtrate was
evaporated under reduced pressure. The residue purified by flash column chromatography on silica

Molecules 2019, 24, 3559
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gel (20% diethyl ether in petroleum ether) to give compound 13 (4.74 g, 93% yield) as a colorless oil
which crystallized on standing. mp 65–67 ◦C (lit [44]. mp 69 ◦C); 1H NMR (500 MHz, CDCl3)
6.60 (s,
2H, 2-H, 6-H), 4.55 (s, 2H, -CH2Cl), 3.92 (s, 6H, OMe). The 4-bromo-3,5-dimethoxyphenylmethanol
12) was synthesized in two steps from commercially available 4-bromo-3,5-dihydroxybenzoic acid
δ
(
(10), following previously reported procedures [43].
4-Bromo-3,5-dimethoxyphenylacetonitrile (14). To a stirred suspension of sodium cyanide (2.9 g,
40.8 mmol) in DMSO (80 mL) at room temperature was added a solution of 13 (4.3 g, 16.2 mmol) in
DMSO (80 mL) over a period of 10 min. The reaction mixture was stirred vigorously overnight and
then diluted by adding ice, saturated aqueous NaCl solution and diethyl ether. The organic layer
was separated, and the aqueous phase was extracted with diethyl ether. The combined organic layer
was washed with brine, dried over MgSO4 and the solvent was evaporated under reduced pressure.
The residue purified by flash column chromatography on silica gel (50 % diethyl ether in petroleum
ether) to give 14 in 87% yield (3.6 g). mp 123–125◦C (lit [44]. mp 125–126 ◦C); 1H NMR (500 MHz,
CDCl3) δ 6.52 (s, 2H, 2-H, 6-H), 3.92 (s, 6H, OMe), 3.74 (s, 2H, -CH2CN).
1-(4-Bromo-3,5-dimethoxyphenyl)cyclopentanecarbonitrile (15). To a solution of 14 (3.42 g,
13.36 mmol) in anhydrous THF (120 mL), at 0 ◦C under an argon atmosphere, was added potassium
bis(trimethylsilyl)amide (8 g, 40.2 mmol). The resulting slurry was stirred at the same temperature
for 10 min, and then a solution of 1,4-dibromobutane (3.46 g, 16 mmol) in anhydrous THF (20 mL)
was added dropwise. The reaction was stirred for an additional 10 min at 0 ◦C and then quenched by
adding saturated aqueous NH4Cl solution (80 mL). The mixture was warmed to room temperature
and diluted with diethyl ether (200 mL). The organic layer was separated and the aqueous phase
extracted with diethyl ether. The combined organic layer was washed with brine and dried over
MgSO4 and the solvent evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (25% diethyl ether in hexane) to give 15 as a white solid in 70% yield
(2.9 g). mp 135-138 ◦C; 1H NMR (500 MHz, CDCl3)
δ 6.65 (s, 2H, 2-H, 6-H), 3.92 (s, 6H, OMe), 2.54–2.45
(m, 2H of the cyclopentane ring), 2.14–2.04 (m, 4H of the cyclopentane ring), 2.02–1.94 (m, 2H of the
cyclopentane ring); mass spectrum m/z (relative intensity) 311 (M++2, 99), 309 (M+, 100), 270 (80), 268
(81), 257 (6), 255 (6), 245 (7), 243, (7), 218 (10), 216 (10), exact mass calculated for C14H16BrNO2 309.0364,
found 309.0362.
1-(4-Bromo-3,5-dimethoxyphenyl)cyclopentaneca◦rboxaldehyde (16). To a stirred solution of
15 (1.40 g, 4.52 mmol) in dry CH2Cl2 (50 mL), at 78 C, under an argon atmosphere, was added
−
diisobutylaluminum hydride (1 M solution in hexanes, 12 mL) dropwise. The reaction mixture was
stirred at the same temperature for 1 h, and then quenched by the dropwise addition of potassium
sodium tartrate (10% solution in water). The mixture was warmed to room temperature and stirred
vigorously for 1 h. The organic layer was separated, and the aqueous phase extracted with diethyl ether.
The combined organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo.
The crude product was purified by flash column chromatography on silica gel (25% diethyl ether in
hexane) to give 16 as a white solid in 85% yield (1.2 g). mp 88–91 ◦C; 1H NMR (400 MHz, CDCl3)
δ
9.39 (s, 1H, CHO), 6.44 (s, 2H, 2-H, 6-H), 3.90 (s, 6H, OMe), 2.56–2.47 (m, 2H of the cyclopentane ring),
1.95–1.86 (m, 2H of the cyclopentane ring), 1.84–1.73 (m, 2H of the cyclopentane ring), 1.72–1.62 (m, 2H
of the cyclopentane ring); mass spectrum m/z (relative intensity), 314 (M++2, 20), 312 (M+, 21), 285
(100), 283 (100), 270 (6), 268 (6); exact mass calculated for C14H17BrO3 312.0361, found 312.0361.
2-Bromo-1,3-dimethoxy-5-[1-(1,2-cis-4-phenoxy-buten-1-yl)cyclopentyl]benzene (17a). To a stirred
suspension of 3-(phenoxypropyl)triphenylphosphonium bromide (2.30 g 4.82 mmol) in dry THF
(50 mL) at 0 ◦C, under an argon atmosphere was added potassium bis(trimethylsilyl)amide (0.94 g,
4.72 mmol). To the resulting slurry was added a solution of 16 (0.30 g, 0.96 mmol) in dry THF (5 mL)
dropwise. Stirring was continued for an additional 10 min at the same temperature and the reaction was
quenched by adding saturated aqueous NH4Cl (20 mL). The mixture was warmed to room temperature
and diluted with diethyl ether (40 mL). The organic layer was separated and the aqueous phase
extracted with diethyl ether. The combined organic layer was washed with brine, dried over MgSO4

Molecules 2019, 24, 3559
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and the solvent evaporated under reduced pressure. The residue was chromatographed through a
column of silica gel (10% diethyl ether in petroleum ether) to give 17a as a colorless liquid in 94% yield
(390 mg); 1H NMR (400 MHz, CDCl3)
δ 7.23 (t, J = 8.1 Hz, 2H, 3-H, 5-H, OPh), 6.91 (t, J = 8.1 Hz, 1H,
4-H, OPh), 6.75 (d, J = 8.1 Hz, 2H, 2-H, 6-H, OPh), 6.61 (s, 2H, ArH), 5.90 (d, J = 11.3 Hz, 1H, 20-H), 5.46
(dt, J = 11.3 Hz, J = 7.3 Hz, 1H, 30-H), 3.86 (s, 6H, OMe), 3.74 (t, J = 6.6 Hz, 2H, 50-H)), 2.20 (q, J = 6.8 Hz,
2H, 40-H), 2.19-1.93 (m, 4H of the cyclopentane ring), 1.85–1.70 (m, 4H of the cyclopentane ring); mass
spectrum m/z (relative intensity) 432 (M++2, 68), 430 (M+, 67), 338 (62), 309 (35), 258 (65), 231 (90), 229
(90), 176 (52), 121 (100), 90 (78), 77 (72); exact mass calculated for C23H27BrO3 430.1144, found 430.1146.
2-Bromo-1,3-dimethoxy-5-[1-(1,2-cis-5-phenoxy-penten-1-yl)cyclopentyl]benzene
(17b).
The synthesis was carried our as described for 17a using (4-phenoxybutyl)triphenylphosphonium
bromide (3.18 g, 6.47 mmol) in anhydrous THF (40 mL), potassium bis(trimethylsilyl)amide (1.72 g,
6.36 mmol), and a solution of 16 (0.42 g, 1.35 mmol) in anhydrous THF (5 mL). The crude product
obtained after work up was chromatographed through a column of silica gel (10% diethyl ether in
1
petroleum ether) to give 17b as a colorless liquid in 93% yield (0.56 g). H NMR (400 MHz, CDCl3)
δ
7.26 (t, J = 8.0 Hz, 2H, 3-H, 5-H, OPh), 6.91 (t, J = 8.0 Hz, 1H, 4-H, OPh), 6.79 (d, J = 8.0 Hz, 2H, 2-H,
6-H, OPh), 6.60 (s, 2H, ArH), 5.79 (d, J = 11.3 Hz, 1H, 20-H), 5.37 (dt, J = 11.3 Hz, J = 7.3 Hz, 1H, 30-H),
3.86 (s, 6H, OMe), 3.70 (t, J = 6.5 Hz, 2H, 60-H), 2.05–1.87 (m, 6H, 4H of the cyclopentane ring and 40-H,
overlapping), 1.80-1.78 (m, 4H of the cyclopentane ring), 1.63 (qt, J = 6.9 Hz, 2H, 50-H); mass spectrum
m/z (relative intensity) 446 (M++2, 9), 444 (M+, 9), 348 (12), 314 (21), 312 (21), 285 (99), 283 (100), 252
(12), 231 (25), 229 (25), 204 (44), 176 (27), 133 (8), 94 (48), 67 (49); exact mass calculated for C24H29BrO3
444.1300, found 444.1301.
2-Bromo-1,3-dimethoxy-5-[1-(4-phenoxybutyl)cyclopentyl]benzene (9a). A mixture of 17a (360 mg,
0.84 mmol) and 10% Pd/C (54 mg) in ethyl acetate (20 mL) was stirred vigorously under hydrogen
atmosphere (room temperature overnight). The catalyst was removed by filtration through celite and
the filtrate was evaporated under reduced pressure to give the product 9a as a viscous liquid (346 mg,
1
95% yield) which was used in the next step without further purification. H NMR (400 MHz, CDCl3)
δ
7.26 (t, J = 8.1 Hz, 2H, 3-H, 5-H, OPh), 6.91 (t, J = 8.1 Hz, 1H, 4-H, OPh), 6.83 (d, J = 8.1 Hz, 2H, 2-H,
6-H, OPh), 6.49 (s, 2H, ArH), 3.87 (s, 6H, OMe), 3.85 (t, J = 6.4 Hz, 2H, 50-H), 1.95–1.88 (m, 2H of the
cyclopentane ring), 1.87–1.79 (m, 2H of the cyclopentane ring), 1.77–1.59 (m, 8H, 4H of the cyclopentane
ring and 4H of the 4-phenoxybutyl group), 1.22–1.14 (m, 2H of the 4-phenoxybutyl group); mass
spectrum m/z (relative intensity) 434 (M++2, 24), 432 (M+, 24), 342 (16), 340 (16), 285 (100), 283 (97), 231
(24), 229 (24), 204 (32), 176 (18), 149 (19), 91 (21), 67 (34); exact mass calculated for C23H29BrO3 432.1300,
found 432.1303.
2-Bromo-1,3-dimethoxy-5-[1-(5-phenoxypentlyl)cyclopentyl]benzene (9b). The title compound
was synthesized as described for 9a, using 17b (550 mg, 1.26 mmol) and 10% Pd/C (80 mg) in EtOAc
(20 mL) and gave 9b as a white solid in 94% yield (529 mg). mp 65–67 ◦C; 1H NMR (500 MHz, CDCl3)
δ
7.26 (t, J = 8.0 Hz, 2H, 3-H, 5-H, OPh), 6.92 (t, J = 8.0 Hz, 1H, 4-H, OPh), 6.84 (d, J = 8.0 Hz, 2H,
2-H, 6-H, OPh), 6.49 (s, 2H, ArH), 3.88 (s, 6H, OMe), 3.86 (t, J = 6.5 Hz, 2H, 60-H), 1.94–1.86 (m, 2H
of the cyclopentane ring), 1.85–1.77 (m, 2H of the cyclopentane ring), 1.75–1.63 (m, 6H, 4H of the
cyclopentane ring and 2H of the 5-phenoxypentyl group, overlapping), 1.62–1.56 (m, 2H, 20-H), 1.34
(qt, J = 7.6Hz, 2H of the 5-phenoxypentyl group), 1.10–1.00 (m, 2H of the 5-phenoxypentyl group);
mass spectrum m/z (relative intensity) 448 (M++2, 7), 446 (M+, 8), 381 (5), 331 (6), 279 (12), 231 (14), 149
(55), 119 (34), 69 (100); exact mass calculated for C24H31BrO3 446.1457, found 446.1457.
trans-3-[1-(4-Bromo-3,5-dimethoxyphenyl)cyclopentyl]acrylic acid ethyl ester (18). To a solution of
triethyl phosphonoacetate (2.78 g, 12.42 mmol) in dry THF (50 mL), at 0 ◦C, under an argon atmosphere,
was added sodium hydride (497 mg, 12.42 mmol, 60% dispersion in mineral oil). The mixture was
stirred for 15 min at the same temperature and a solution of 16 (1.11 g, 3.55 mmol) in dry THF (10 mL)
was added dropwise. Stirring was continued for an additional 10 min, and the reaction was quenched
by adding saturated aqueous NH4Cl (20 mL). The mixture was warmed to room temperature and
diluted with diethyl ether (100 mL). The organic phase was separated and the aqueous phase extracted

Molecules 2019, 24, 3559
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with diethyl ether. The combined organic layer was washed with brine, dried over MgSO4 and
evaporated under reduced pressure. The crude product was purified by flash column chromatography
on silica gel (35% diethyl ether in hexane) to give 18 as a white solid in 92% yield (1.25 g). mp
68–72 ◦C; 1H NMR (400 MHz, CDCl3)
δ 7.04 (d, J = 15.8 Hz, 1H, >CH=CH<), 6.48 (s, 2H, ArH), 5.62 (d,
J = 15.8 Hz, 1H, >CH=CH<), 4.16 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.88 (s, 6H, OMe), 2.18–2.08 (m, 2H of
the cyclopentane ring), 2.07–1.96 (m, 2H of the cyclopentane ring), 1.84–1.72 (m, 4H of the cyclopentane
ring), 1.27 (t, J = 7.1 Hz, 3H, OCH2CH3); mass spectrum m/z (relative intensity) 384 (M++2, 100), 382
(M+, 100), 353 (16), 339 (22), 337 (22); exact mass calculated for C18H23BrO4 382.0780, found 382.0778.
3-[1-(4-Bromo-3,5-dimethoxyphenyl)cyclopentyl]propionic acid ethyl ester (19a). A mixture of 18
(0.5 g, 1.3 mmol) and 10% Pd/C (100 mg) in ethyl acetate (20 mL) was placed in a Parr apparatus (Parr
Instrument Co, Moline, IL) and treated with hydrogen at 30 psi for 6 h. The catalyst was removed
by filtration through a pad of celite and the filtrate was evaporated under reduced pressure to give
19a as a white solid (472 mg, 95% yield) which was used in the next step without further purification.
mp 78–80 ◦C; 1H NMR (500 MHz, CDCl3)
δ 6.48 (s, 2H, ArH), 4.03 (q, J = 7.1 Hz, 2H, OCH2CH3),
3.89 (s, 6H, OMe), 2.04–1.98 (m, 2H, 30-H), 1.96–1.89 (m, 4H, 2H of the cyclopentane ring and 20-H,
overlapping), 1.86–1.65 (m, 6H of the cyclopentane ring), 1.20 (t, J = 7.1 Hz, 3H, OCH2CH3); mass
spectrum m/z (relative intensity), 386 (M++2, 16), 384 (M+, 15), 341 (8), 339 (8), 285 (37), 283 (36), 231
(8), 229 (8), 204 (15), 176 (9), 115 (7), 101 (9), 77 (20), 67 (100); exact mass calculated for C18H25BrO4
384.0936, found 384.0934.
3-[1-(4-Bromo-3,5-dimethoxyphenyl)cyclopentyl]propionic acid methyl ester (19b). A mixture
of 18 (0.7 g, 1.83 mmol) and magnesium turnings (132 mg, 5.5 mmol), in dry methanol (20 mL) was
stirred at 0 ◦C for 2 h and at room temperature for an additional 12 h. The solvent was evaporated
under reduced pressure and the residue diluted with water (20 mL) and diethyl ether (50 mL). To this
mixture was added 5% aqueous HCl (10 mL), the organic layer was separated, and the aqueous layer
extracted with diethyl ether. The combined ethereal layer was successively washed with NaHCO3 and
brine, dried over MgSO4 and evaporated under reduced pressure to give the product 19b as a white
solid (543 mg, 80% yield) which was used in the next step without further purification. mp 71–74 ◦C;
1H NMR (500 MHz, CDCl3)
δ 6.49 (s, 2H, ArH), 3.91 (s, 6H, OMe), 3.60 (s, 3H, COOCH3), 2.09–2.02
(m, 2H, 30-H), 1.99–1.90 (m, 4H, 2H of the cyclopentane ring and 20-H, overlapping), 1.88–1.65 (m, 6H,
of the cyclopentane ring); mass spectrum m/z (relative intensity) 372 (M++2, 98), 370 (M+, 100), 341
(19), 339 (20); exact mass calculated for C17H23BrO4 370.0780, found 370.0783.
3-[1-(4-Bromo-3,5-dimethoxyphenyl)cyclopentyl]propan-1-ol (20). To a stirred solution of 19b
(520 mg, 1.4 mmol) in dry THF (20 mL), at room temperature, under an argon atmosphere, was added
diisobutylaluminum hydride (3.7 mL, 1M solution in hexanes) over a period of 15 min. The mixture
was stirred at the same temperature for 1 h, and then cooled to 0 ◦C, and the reaction was quenched by
dropwise addition of aqueous potassium sodium tartrate (10% solution in water, 10 mL). The resulting
mixture was warmed to room temperature, diluted with ethyl acetate (10 mL) and stirred vigorously for
1h. The organic layer was separated, and the aqueous phase extracted with ethyl acetate. The combined
organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (40% ethyl acetate in hexane) to give 20 as a
1
colorless liquid in 85% yield (408 mg). H NMR (500 MHz, CDCl3)
δ 6.51 (s, 2H, ArH), 3.91 (s, 6H,
OMe), 3.53 (t, J = 6.4 Hz, 2H, 40-H), 1.98-1.91 (m, 2H of the cyclopentane ring), 1.89–1.83 (m, 2H of the
cyclopentane ring), 1.81–1.63 (m, 6H, 4H of the cyclopentane ring and 20-H, overlapping, especially
1.66, m, 20-H), 1.34–1.26 (m, 2H, 30-H); mass spectrum m/z (relative intensity) 342 (M+, 100), 297 (26),
285 (10), 283 (10); exact mass calculated for C16H23BrO3 342.0831, found 342.0829. This compound was
also synthesized by using 19a (462 mg, 1.2 mmol) in anhydrous THF (10 mL) and diisobutylaluminum
hydride (3 mL, 1M solution in hexanes) and gave 354 mg (86% yield) of product (20).
2-Bromo-1,3-dimethoxy-5-[1-(3-bromopropyl)cyclopentyl]benzene (21). To a stirred solution of 20
(400 mg, 1.17 mmol) and carbon tetrabromide (465 mg, 1.40 mmol) in anhydrous CH2Cl2 (10 mL) at
0 ◦C, under a nitrogen atmosphere, was added triphenylphosphine (368 mg, 1.40 mmol) portionwise.

Molecules 2019, 24, 3559
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After the addition was completed, the reaction mixture was stirred for an additional 20 min, whereupon
the solvent was removed in vacuo. The residue was purified by flash column chromatography on
1
silica gel (10% diethyl ether in hexane) to give 21 as a colorless liquid in 85% yield (403 mg). H NMR
(500 MHz, CDCl3)
δ
6.48 (s, 2H, ArH), 3.89 (s, 6H, OMe), 3.27 (t, J = 6.4 Hz, 2H, 40-H), 1.96–1.88 (m,
2H of the cyclopentane ring), 1.86–1.64 (m, 8H, 6H of the cyclopentane ring and 20-H, overlapping),
1.60–1.52 (m, 2H, 30-H); mass spectrum m/z (relative intensity) 408 (M++4, 13), 406 (M++2, 29), 404 (M+,
14), 327 (26), 325 (26), 285 (99), 283 (100), 233 (29), 231 (29), 204 (45), 289 (18), 176 (25), 109 (24), 67 (36);
exact mass calculated for C16H22Br2O2 403.9987, found 403.9987.
2-Bromo-1,3-dimethoxy-5-[1-(3-phenoxypropyl)cyclopentyl]benzene (9c). To a stirred solution of
21 (250 mg, 0.62 mmol) in DMSO (6 mL) at room temperature, under an argon atmosphere, was added
sodium phenoxide trihydrate (530 mg, 3.10 mmol). The reaction mixture was stirred vigorously for
24 h and then diluted by adding ice water (5 mL) and diethyl ether. The organic layer was separated,
and the aqueous phase was extracted with diethyl ether. The combined organic layer was washed
with brine, dried over MgSO4 and evaporated under reduced pressure. Purification by flash column
chromatography on silica gel (10% diethyl ether in hexane) gave 9c as a colorless liquid in 50% yield
(130 mg). 1H NMR (500 MHz, CDCl3)
δ 7.24 (t, J = 7.5 Hz, 2H, 3-H, 5-H, OPh), 6.91 (t, J = 7.5 Hz,
1H, 4-H, OPh), 6.81 (d, J = 7.5 Hz, 2H, 2-H, 6-H, OPh), 6.51 (s, 2H, ArH), 3.89 (s, 6H, OMe), 3.80 (t,
J = 6.5 Hz, 2H, 40-H), 1.97-1.91 (m, 2H of the cyclopentane ring), 1.89–1.83 (m, 2H of the cyclopentane
ring), 1.79–1.65 (m, 6H, 4H of the cyclopentane ring and 20-H, overlapping), 1.53–1.46 (m, 2H, 30-H);
mass spectrum m/z (relative intensity) 420 (M++2, 21), 418 (M+, 21), 327 (62), 325 (62), 285 (71), 283 (71),
231 (99), 229 (100), 204 (38), 151 (47), 109 (10), 67 (35); exact mass calculated for C22H27BrO3 418.1144,
found 418.1142.
2-Bromo-1,3-dimethoxy-5-(1-hexylcyclopentyl)benzene (9d). To a solution of 22 (1 g, 3.45 mmol)
in anhydrous carbon tetrachloride (40 mL) at 0 ◦C, under an argon atmosphere was added bromine
(7 mL, 1 M solution in CCl4). The reaction mixture was stirred at the same temperature for 20 min and
the solvent was removed in vacuo. The residue was purified by flash column chromatography on
silica gel (5% diethyl ether in hexane) to give 9d as a white solid in 78% yield (0.99 g). mp 38–40 ◦C; 1H
NMR (500 MHz, CDCl3) δ 6.49 (s, 2H, ArH), 3.89 (s, 6H, OMe), 1.91–1.85 (m, 2H of the cyclopentane
ring), 1.84–1.76 (m, 2H of the cyclopentane ring), 1.75–1.60 (m, 4H of the cyclopentane ring), 1.58–1.50
(m, 2H, 20-H), 1.25–1.09 (m, 6H, 40-H, 50-H, 60-H), 1.01-0.93 (m, 2H, 30-H), 0.83 (t, J = 7.2 Hz, 3H, 70-H).
mass spectrum m/z (relative intensity) 370 (M++2, 30), 368 (M+, 30), 285 (100), 283 (98), 233 (20), 231
(20), 204 (36), 176 (21), 67 (25); exact mass calculated for C19H29BrO2 368.1351, found 368.1351.
40-[1-(4-Phenoxybutyl)cyclopentyl]-20,5,60-trimethoxy-N,N-diisopropyl-[1,10-biphenyl]-2-carboxamide
(7a). A degassed mixture of 9a (135 mg, 0.31 mmol), boronic acid
8 (125 mg, 0.45 mmol), Ba(OH)2
(160 mg, 0.93 mmol), and Pd(PPh3)4 (70 mg, 0.06 mmol) in DME (5 mL)/water (1 mL) was flushed with
argon, and heated in a sealed tube at 80 ◦C for 6 h with vigorous stirring. The reaction mixture was
cooled to room temperature and diluted with EtOAc (30 mL) and the catalyst removed by filtration
through celite. The filtrate was diluted with brine, and the organic layer was separated, dried (MgSO4),
and concentrated in vacuo. Purification by flash column chromatography on silica gel (30% diethyl
ether in hexane) gave 7a as a viscous liquid in 48% yield (87 mg). IR (neat) 2927, 2857, 1625, 1607, 1576,
1337, 1241, 1128, 1032 cm−1; 1H NMR (400 MHz, CDCl3)
δ 7.29 (t, J = 7.3 Hz, 2H, 3-H, 5-H, -OPh group),
7.27 (d, J = 7.2 Hz, 1H, 3-H, Ph-Ph group), 6.95 (t, J = 7.3 Hz, 1H, 4-H, -OPh group), 6.92–6.86 (d and dd
overlapping, 3H, 2-H, 6-H of the -OPh group and 4-H of the Ph-Ph group), 6.82 (d, J = 2.5 Hz, 1H, 6-H,
Ph-Ph group), 6.52 (s, 1H, 30-H or 50-H, Ph-Ph group), 6.51 (s, 1H, 50-H or 30-H, Ph-Ph group), 3.89 (t,
J = 6.4 Hz, 2H, -CH2OPh), 3.84 (s, 3H, OMe), 3.74 (s, 3H, OMe), 3.73 (s, 3H, OMe), 3.71 (qt, J = 6.5 Hz,
1H, -CH(CH3)2), 3.16 (qt, J = 6.5 Hz, 1H, -CH(CH3)2), 2.08-1.97 (m, 2H of the cyclopentane ring),
1.91–1.60 (m, 10H, 6H of the cyclopentane ring and 4H of the 4-phenoxybutyl group, overlapping), 1.46
(d, J = 6.5 Hz, 3H, -CH(CH3)2), 1.38–1.25 (m, 2H of the 4-phenoxybutyl group), 1.10 (d, J = 6.5 Hz, 3H,
-CH(CH3)2), 0.92 (d, J = 6.5 Hz, 3H, -CH(CH3)2), 0.58 (d, J = 6.5 Hz, 3H, -CH(CH3)2); mass spectrum

Molecules 2019, 24, 3559
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m/z (relative intensity) 588 (M++1, 22), 587 (M+, 54), 586 (29), 556 (10), 487 (84), 395 (100), 323 (12), 283
(15), 262 (44), 183 (20), 91 (11), 67 (9); exact mass calculated for C37H49NO5 587.3611, found 587.3612.
40-[1-(5-Phenoxypentyl)cyclopentyl]-20,5,60-trimethoxy-N,N-diisopropyl-[1,10-biphenyl]-2-
carboxamide (7b). The synthesis was carried out as described for 7a, using 9b (260 mg, 0.58 mmol),
8
(242 mg, 0.87 mol), Ba(OH)2 (300 mg, 1.74 mmol), and tetrakis(triphenylphosphine)palladium(0)
(48 mg, 0.04 mmol) in DME (5 mL) and water (2 mL). The crude product obtained after workup was
chromatographed through a column of silica gel (40% ethyl acetate in petroleum ether) to give 7b as a
viscous liquid in 50% yield (175 mg). IR (neat) 2924, 2855, 1624, 1602, 1572, 1335, 1241, 1127, 1030 cm−1
;
1H NMR (400 MHz, CDCl3)
δ 7.19 (t, J = 7.3 Hz, 2H, 3-H, 5-H, -OPh group), 7.18 (d, J = 7.0 Hz, 1H, 3-H,
Ph-Ph group), 6.85 (t, J = 7.3 Hz, 1H, 4-H, -OPh group), 6.82–6.77 (d and dd overlapping, 3H, 2-H,
6-H of the -OPh group and 4-H of the Ph-Ph group), 6.72 (d, J = 2.6 Hz, 1H, 6-H, Ph-Ph group), 6.41
(s, 1H, 30-H or 50-H, Ph-Ph group), 6.40 (s, 1H, 50-H or 30-H, Ph-Ph group), 3.82 (t, J = 6.5 Hz, 2H,
-CH2OPh), 3.73 (s, 3H, OMe), 3.64 (s, 3H, OMe), 3.63 (s, 3H, OMe), 3.61 (qt, J = 6.6 Hz, 1H, -CH(CH3)2),
3.06 (qt, J = 6.6 Hz, 1H, -CH(CH3)2), 1.95–1.83 (m, 2H of the cyclopentane ring), 1.81–1.51 (m, 10H, 6H
of the cyclopentane ring and 4H of the 5-phenoxypentyl group, overlapping), 1.37 (d, J = 6.6 Hz, 3H,
-CH(CH3)2), 1.25 (qt, J = 7.7 Hz, 2H of the 5-phenoxypentyl group), 1.10–0.95 (m and d overlapping, 5H,
especially 1.03, d, J = 6.6 Hz, 3H, -CH(CH3)2), 0.82 (d, J = 6.6 Hz, 3H, -CH(CH3)2), 0.47 (d, J = 6.6 Hz,
3H, -CH(CH3)2); mass spectrum m/z (relative intensity) 602 (M++1, 56), 601 (M+, 84), 600 (49), 570 (20),
530 (9), 501 (100), 407 (12), 323 (16), 297 (22), 269 (53), 257 (32), 95 (10), 67 (10); exact mass calculated for
C38H51NO5 601.3767, found 601.3765.
40-[1-(3-Phenoxypropyl)cyclopentyl]-20,5,60-trimethoxy-N,N-diisopropyl-[1,10-biphenyl]-2-
carboxamide (7c). The synthesis was carried out as described for 7a, using 9c (90 mg, 0.21 mmol),
8
(108 mg, 0.39 mol), Ba(OH)2 (125 mg, 0.74 mmol) and tetrakis(triphenylphosphine)palladium(0)
(48mg, 0.04mmol) in DME (5 mL) and water (1 mL). The crude product obtained after workup was
chromatographed through a column of silica gel (40% ethyl acetate in petroleum ether) to give 7c as a
viscous liquid in 50% yield (60 mg). IR (neat) 2932, 2858, 1624, 1605, 1575, 1342, 1243, 1127, 1032 cm−1
;
1H NMR (500 MHz, CDCl3)
δ 7.26 (t, J = 7.7 Hz, 2H, 3-H, 5-H, -OPh group), 7.25 (d, J = 8.5 Hz, 1H,
3-H, Ph-Ph group), 6.92 (t, J = 7.7 Hz, 1H, 4-H, -OPh group), 6.87 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, 4-H
of the Ph-Ph group), 6.83 (d, J = 7.7 Hz, 2H, 2-H, 6-H of the -OPh group), 6.79 (d, J = 3.0 Hz, 1H,
6-H, Ph-Ph group), 6.52 (s, 1H, 30-H or 50-H, Ph-Ph group), 6.50 (s, 1H, 50-H or 30-H, Ph-Ph group),
3.81 (t, J = 6.7 Hz, 2H, -CH2OPh), 3.80 (s, 3H, OMe), 3.72 (s, 3H, OMe), 3.71 (s, 3H, OMe), 3.69 (qt,
J = 6.5 Hz, 1H, -CH(CH3)2), 3.14 (qt, J = 6.5 Hz, 1H, -CH(CH3)2), 2.04–1.94 (m, 2H of the cyclopentane
ring), 1.88–1.81 (m, 2H of the cyclopentane ring), 1.80–1.65 (m, 6H, 4H of the cyclopentane ring
and –CH2CH2CH2OPh, overlapping), 1.60–1.52 (m, 2H, –CH2CH2CH2OPh), 1.44 (d, J = 6.5 Hz, 3H,
-CH(CH3)2), 1.07 (d, J = 6.5 Hz, 3H, -CH(CH3)2), 0.90 (d, J = 6.5 Hz, 3H, -CH(CH3)2), 0.57 (d, J = 6.5 Hz,
3H, -CH(CH3)2); mass spectrum m/z (relative intensity) 574 (M++1, 23), 573 (M+, 65), 572 (36), 542 (13),
500 (11), 473 (82), 415 (19), 379 (22), 323 (13), 269 (100), 241 (23), 149 (32), 100 (27); exact mass calculated
for C36H47NO5 573.3454, found 573.3455.
40-(1-Hexyl-cyclopentyl)-20,5,60-trimethoxy-N,N-diisopropyl-[1,10-biphenyl]-2-carboxamide (7d).
The synthesis was carried out as described for 7a, using 9d (90 mg, 0.21 mmol), 8 (108 mg, 0.39 mol),
Ba(OH)2 (125 mg, 0.74 mmol), and tetrakis(triphenylphosphine)palladium(0) (48 mg, 0.04 mmol) in
DME (5 mL) and water (1 mL). The crude product obtained after workup was chromatographed
through a column of silica gel (40% ethyl acetate in petroleum ether) to give 7d as a viscous liquid
in 50% yield (60 mg). IR (neat) 2929, 2857, 1605, 1578, 1345 cm−1; 1H NMR (400 MHz, CDCl3)
δ 7.26
(d, J = 8.4 Hz, 1H, 3-H, Ph-Ph group), 6.87 (dd, J = 8.4 Hz, J = 2.6 Hz, 1H, 4-H, Ph-Ph group), 6.79
(d, J = 2.6 Hz, 1H, 6-H, Ph-Ph group), 6.48 (s, 1H, 30-H or 50-H, Ph-Ph group), 6.47 (s, 1H, 50-H or
30-H, Ph-Ph group), 3.81 (s, 3H, OMe), 3.72 (s, 3H, OMe), 3.71 (s, 3H, OMe), 3.69 (qt, J = 6.5 Hz, 1H,
-CH(CH3)2), 3.17 (qt, J = 6.5 Hz, 1H, -CH(CH3)2), 1.97–1.88 (m, 2H of the cyclopentane ring), 1.84–1.76
(m, 2H of the cyclopentane ring), 1.75-1.59 (m, 4H of the cyclopentane ring), 1.57–1.52 (m, 2H, 20-H,
hexyl group), 1.46 (d, J = 6.5 Hz, 3H, -CH(CH3)2), 1.30-1.13 (m, 6H, 40-H, 50-H, 60-H, hexyl group), 1.09

Molecules 2019, 24, 3559
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(d, J = 6.5 Hz, 3H, -CH(CH3)2), 1.10-1.01 (m, 2H, 30-H, hexyl group), 0.90 (d, J = 6.5 Hz, 3H, -CH(CH3)2),
0.87 (t, J = 7.1 Hz, 3H, 70-H, hexyl group), 0.56 (d, J = 6.5 Hz, 3H, -CH(CH3)2); mass spectrum m/z
(relative intensity) 524 (M++1, 8), 523 (M+, 24) 522 (18), 492 (6), 450 (5), 423 (100), 337 (5), 269 (5), 235
(5), 192 (8), 135 (35), 94 (27), 57 (19); exact mass calculated for C33H49NO4 523.3662, found 523.3664.
40-[1-(4-Bromobutyl)cyclopentyl]-20,5,60-trihydroxy-N,N-diisopropyl-[1,10-biphenyl]-2-carboxamide
(23a). To a stirring solution of 7a (80.0 mg, 0.14 mmol) in anhydrous CH2Cl2 (5 mL), at
78 ◦C, under
−
an argon atmosphere, was added boron tribromide (0.7 mL, 1 M solution in CH2Cl2). The mixture
was gradually warmed to room temperature and stirred for an additional 12 h. Unreacted boron
tribromide was destroyed by the addition of methanol at 0 ◦C. The resulting mixture was warmed to
room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate
and washed with saturated NaHCO3 and brine. The organic layer was dried over MgSO4 and the
solvent evaporated under reduced pressure. The crude product obtained after workup was purified by
flash column chromatography on silica gel (50% diethyl ether in petroleum ether) to give 23a as a
white solid in 92% yield (68 mg). mp 183–185 ◦C; IR (neat) 3305 (br, OH), 2928, 2852, 1631, 1616, 1570,
1340 cm−1; 1H NMR (400 MHz, CDCl3)
δ 7.92 (br s, 1H, OH), 7.12 (d, J = 8.1 Hz, 1H, 3-H, Ph-Ph group),
6.88 (dd, J = 8.1 Hz, J = 2.5 Hz, 1H, 4-H of the Ph-Ph group), 6.77 (d, J = 2.5 Hz, 1H, 6-H, Ph-Ph group),
6.54 (d, J = 1.2 Hz, 1H, 30-H or 50-H, Ph-Ph group), 6.46 (d, J = 1.2 Hz, 1H, 50-H or 30-H, Ph-Ph group),
6.19 (br s, 1H, OH), 4.54 (br s, 1H, OH), 3.79 (qt, J = 6.8 Hz, 1H, -CH(CH3)2), 3.30 (qt, J = 6.8 Hz, 1H,
-CH(CH3)2), 3.26 (t, J = 6.5 Hz, 2H, -CH2Br), 1.93–1.85 (m, 2H of the cyclopentane ring), 1.76–1.53 (m,
10H, 6H of the cyclopentane ring and 4H of the 4-bromobutyl group, overlapping), 1.44 (d, J = 6.8 Hz,
3H, -CH(CH3)2), 1.30–1.21 (m, 2H of the 4-bromobutyl group), 1.07 (d, J = 6.8 Hz, 3H, -CH(CH3)2),
1.06 (d, J = 6.8 Hz, 3H, -CH(CH3)2), 0.86 (d, J = 6.8 Hz, 3H, -CH(CH3)2); mass spectrum m/z (relative
intensity) 533 (M++2, 7), 531 (M+, 7), 451 (16), 433 (10), 431 (10), 351 (18), 295 (21), 268 (8), 241 (12), 102
(32), 86 (100); exact mass calculated for C28H38BrNO4 531.1984, found 531.1980.
40-[1-(5-Bromopentyl)cyclopentyl]-20,5,60-trihydroxy-N,N-diisopropyl-[1,10-biphenyl]-2-carboxamide
(23b). The synthesis was carried out as described for 23a, using 7b (165 mg, 0.27 mmol) and BBr3
(1.7 mL, 1M solution in CH2Cl2) in anhydrous CH2Cl2 (5 mL). The crude product obtained after
workup was purified by flash column chromatography on silica gel (50% diethyl ether in petroleum
ether) to give 23b as a white solid in 90% yield (130 mg). mp 190–192 ◦C; IR (neat) 3312 (br, OH), 2925,
2857, 1632, 1615, 1567, 1343 cm−1; 1H NMR (400 MHz, CDCl3)
δ 8.16 (br s, 1H, OH), 7.11 (d, J = 8.4 Hz,
1H, 3-H, Ph-Ph group), 6.84 (dd, J = 8.4 Hz, J = 2.7 Hz, 1H, 4-H of the Ph-Ph group), 6.68 (d, J = 2.7 Hz,
1H, 6-H, Ph-Ph group), 6.59 (d, J = 1.3 Hz, 1H, 30-H or 50-H, Ph-Ph group), 6.47 (d, J = 1.3 Hz, 1H, 50-H
or 30-H, Ph-Ph group), 6.37 (br s, 1H, OH), 4.74 (br s, 1H, OH), 3.70 (qt, J = 7.0 Hz, 1H, -CH(CH3)2),
3.34 (qt, J = 7.0 Hz and t J = 6.8 Hz overlapping, 3H, -CH(CH3)2, -CH2Br), 1.94–1.85 (m, 2H of the
cyclopentane ring), 1.79–1.52 (m, 10H, 6H of the cyclopentane ring and 4H of the 5-bromopentyl
group, overlapping), 1.46 (d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.28 (qt, J = 6.9 Hz, 2H of the 5-bromopentyl
group), 1.07 (d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.06 (d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.05–0.97 (m, 2H of the
5-bromopentyl group), 0.87 (d, J = 7.0 Hz, 3H, -CH(CH3)2); mass spectrum m/z (relative intensity) 547
(M++2, 8), 545 (M+, 8), 465 (8), 447 (17), 445 (16), 367 (8), 335 (16), 295 (23), 267 (8), 241 (15), 101 (24), 86
(100); exact mass calculated for C29H40BrNO4 545.2141, found 545.2145.
40-[1-(3-Bromopropyl)cyclopentyl]-20,5,60-trihydroxy-N,N-diisopropyl-[1,10-biphenyl]-2-carboxamide
(23c). The synthesis was carried out as described for 23a, using 7c (52 mg, 0.09 mmol) and BBr3 (0.6 mL,
1M solution in CH2Cl2) in anhydrous CH2Cl2 (5 mL). The crude product obtained after workup was
purified by flash column chromatography on silica gel (50% diethyl ether in petroleum ether) to give
23c as a white solid in 91% yield (42 mg). mp 178–180 ◦C; IR (neat) 3312 (br, OH), 2924, 2855, 1630,
1617, 1572, 1335 cm−1; 1H NMR (500 MHz, CDCl3)
δ 8.14 (br s, 1H, OH), 7.13 (d, J = 8.5 Hz, 1H, 3-H,
Ph-Ph group), 6.86 (dd, J = 8.5 Hz, J = 2.7 Hz, 1H, 4-H of the Ph-Ph group), 6.65 (d, J = 2.7 Hz, 1H, 6-H,
Ph-Ph group), 6.60 (d, J = 1.5 Hz, 1H, 30-H or 50-H, Ph-Ph group), 6.48 (d, J = 1.5 Hz, 1H, 50-H or 30-H,
Ph-Ph group), 6.30 (br s, 1H, OH), 4.72 (br s, 1H, OH), 3.71 (qt, J = 7.0 Hz, 1H, -CH(CH3)2), 3.33 (qt,
J = 7.0 Hz, 1H, -CH(CH3)2), 3.23 (t, J = 6.5 Hz, 2H, -CH2Br), 1.98–1.88 (m, 2H of the cyclopentane ring),

Molecules 2019, 24, 3559
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1.78–1.62 (m, 8H, 6H of the cyclopentane ring and -CH2CH2CH2Br, overlapping), 1.59–1.53 (m, 2H,
-CH2CH2CH2Br), 1.45 (d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.07 (d, J = 7.0 Hz, 6H, -CH(CH3)2), 0.87 (d,
J = 7.0 Hz, 3H, -CH(CH3)2); mass spectrum m/z (relative intensity) 519 (M++2, 4), 517 (M+, 4), 437 (28),
336 (56), 296 (22), 294 (24), 241 (9), 102 (43), 86 (100); exact mass calculated for C27H36BrNO4 517.1828,
found 517.1830.
40-(1-Hexyl-cyclopentyl)-20,5,60-trihydroxy-N,N-diisopropyl-[1,10-biphenyl]-2-carboxamide (23d).
The synthesis was carried out as described for 23a, using 7d (57 mg, 0.11 mmol) and BBr3 (0.7 mL,
1M solution in CH2Cl2) in anhydrous CH2Cl2 (5 mL). The crude product obtained after workup was
purified by flash column chromatography on silica gel (50% diethyl ether in petroleum ether) to give
23d as a white solid in 89% yield (47 mg). mp 185–187 ◦C; IR (neat) 3295 (br, OH), 2925, 2858, 1632, 1617,
1565, 1340 cm−1; 1H NMR (400 MHz, CDCl3)
δ 8.00 (br s, 1H, OH), 7.15 (d, J = 8.5 Hz, 1H, 3-H, Ph-Ph
group), 6.89 (dd, J = 8.5 Hz, J = 2.7 Hz, 1H, 4-H of the Ph-Ph group), 6.75 (d, J = 2.7 Hz, 1H, 6-H, Ph-Ph
group), 6.60 (d, J = 1.5 Hz, 1H, 30-H or 50-H, Ph-Ph group), 6.47 (d, J = 1.5 Hz, 1H, 50-H or 30-H, Ph-Ph
group), 5.63 (br s, 1H, OH), 4.64 (br s, 1H, OH), 3.71 (qt, J = 7.0 Hz, 1H, -CH(CH3)2), 3.31 (qt, J = 7.0 Hz,
1H, -CH(CH3)2), 1.95-1.85 (m, 2H of the cyclopentane ring), 1.78–1.59 (m, 6H of the cyclopentane ring),
1.57–1.49 (m, 2H, 20-H), 1.47 (d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.25–1.10 (m, 6H, 40-H, 50-H, 60-H), 1.08
(d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.07 (d, J = 7.0 Hz, 3H, -CH(CH3)2), 1.04-0.97 (m, 2H, 30-H), 0.87 (d,
J = 7.0 Hz, 3H, -CH(CH3)2), 0.82 (t, J = 7.2 Hz, 3H, 70-H). mass spectrum m/z (relative intensity) 482
(M++1, 15), 481 (M+, 37), 86 (100); exact mass calculated for C30H43NO4 481.3192, found 481.3191.
3-[1-(4-Bromobutyl)cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3a). A stirred
solution of 23a (50 mg, 0.094 mmol) in glacial acetic acid (4 mL) and water (1 mL) was refluxed
for 24 h. The reaction mixture was cooled to room temperature and diluted with aqueous NaHCO3
solution (5 mL) and ethyl acetate (20 mL). The organic phase was separated, and the aqueous phase
extracted with ethyl acetate. The combined organic layer was washed with brine, dried over MgSO4
and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel
◦
(60% diethyl ether in hexanes) to give 3a as a white solid in 89%yield (36 mg). mp 96–98 C; IR
(neat) 3272 (br, OH), 2941, 1670 (>C=O), 1611, 1272, 1110 cm−1; 1H NMR (400 MHz, CDCl3)
δ 8.45
(d, J = 2.4 Hz, 1H, 10-H), 8.33 (d, J = 8.7 Hz, 1H, 7-H), 7.01 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H, 8-H),
6.88 (d, J = 1.6 Hz, 1H, 4-H or 2-H), 6.64 (d, J = 1.6 Hz, 1H, 2-H or 4-H), 6.32–5.50 (br s, 2H, OH),
3.29 (t, J = 6.8 Hz, 2H, -CH2Br), 1.97–1.87 (m, 2H of the cyclopentane ring), 1.86–1.51 (m, 10H, 6H
of the cyclopentane ring and 4H of the 4-bromobutyl group, overlapping), 1.21–1.10 (m, 2H of the
4-bromobutyl group); 13C NMR (100 MHz, CD3OD)
δ 165.2, 163.9, 157.7, 154.0, 153.0, 138.9, 133.3,
117.0, 113.9, 113.1, 111.6, 107.9, 105.8, 52.4, 42.0, 38.7, 34.5, 34.2, 25.2, 24.2; mass spectrum m/z (relative
intensity) 432 (M++2, 8), 430 (M+, 8), 350 (11), 321 (33), 295 (100), 267 (15), 91(3). exact mass calculated
for C22H23BrO4 430.0780, found 430.0778.
3-[1-(5-Bromopentyl)cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3b). The synthesis
was carried out as described for 3a using 23b (55 mg, 0.10 mmol) in glacial acetic acid (4 mL) and
water (1 mL) and gave 3b as a white solid in 90% yield (40 mg). mp 101-103 ◦C; IR (neat) 3275 (br, OH),
2923, 1668 (>C=O), 1607, 1272, 1105 cm−1; 1H NMR (500 MHz, CDCl3)
δ 8.37 (d, J = 2.1 Hz, 1H, 10-H),
8.27 (d, J = 8.3 Hz, 1H, 7-H), 6.94 (dd, J = 8.3 Hz, J = 2.1 Hz, 1H, 8-H), 6.81 (d, J = 1.1 Hz, 1H, 4-H or
2-H), 6.56 (d, J = 1.1 Hz, 1H, 2-H or 4-H), 6.02 (br s, 1H, OH), 5.87 (br s, 1H, OH), 3.24 (t, J = 6.7 Hz, 2H,
-CH2Br), 1.89–1.80 (m, 2H of the cyclopentane ring), 1.76–1.48 (m, 10H, 6H of the cyclopentane ring
and 4H of the 5-bromopentyl group, overlapping), 1.22 (qt, J = 6.5 Hz, 2H of the 5-bromopentyl group),
1.01–0.89 (m, 2H of the 5-bromopentyl group); 13C NMR (100 MHz, CD3OD)
δ 165.2, 163.9, 157.7, 154.0,
153.2, 138.9, 133.3, 116.9, 113.9, 113.1, 111.6, 107.9, 105.8, 52.4, 42.8, 38.7, 34.3, 33.9, 29.8, 25.8, 24.2; mass
spectrum m/z (relative intensity) 446 (M++2, 10), 444 (M+, 10), 364 (11), 335 (28), 295 (100), 267 (16), 241
(42), 91(5), 67 (14); exact mass calculated for C23H25BrO4 444.0936, found 444.0930.
3-[1-(3-Bromopropyl)cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3c). The synthesis
was carried out as described for 3a using 23c (30 mg, 0.058 mmol) in glacial acetic acid (4 mL) and
water (1 mL) and gave 3c as a white solid in 91% yield (22 mg). mp 105–106 ◦C; IR (neat) 3285 (br,

Molecules 2019, 24, 3559
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1
OH), 2928, 1668 (>C=O), 1610, 1272, 1110 cm−1; H NMR (500 MHz, CDCl3)
δ
8.49 (d, J = 2.0 Hz,
1H, 10-H), 8.32 (d, J = 8.3 Hz, 1H, 7-H), 7.00 (dd, J = 8.3 Hz, J = 2.0 Hz, 1H, 8-H), 6.67 (d, J = 1.4 Hz,
1H, 4-H or 2-H), 6.52 (d, J = 1.4 Hz, 1H, 2-H or 4-H), 6.35–5.89 (br s, 2H, OH), 3.25 (t, J = 6.5 Hz, 2H,
-CH2Br), 1.96–1.86 (m, 2H of the cyclopentane ring), 1.77–1.60 (m, 8H, 6H of the cyclopentane ring
and -CH2CH2CH2Br, overlapping), 1.57–1.52 (m, 2H, -CH2CH2CH2Br), mass spectrum m/z (relative
intensity) 418 (M++2, 6), 416 (M+, 6), 336 (9), 307 (25), 295 (100), 267 (18), 91(3); exact mass calculated
for C21H21BrO4 416.0623, found 416.0627.
3-(1-Hexyl-cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3d). The synthesis was
carried out as described for 3a, using 23d (40 mg, 0.083 mmol) in glacial acetic acid (4 mL) and water
(1 mL), and gave 3d as a white solid in 91% yield (28 mg). mp 153–155 ◦C; IR (neat) 3278 (br, OH), 2925,
1667 (>C=O), 1614, 1384, 1275, 1108 cm−1; 1H NMR (500 MHz, CDCl3)
δ 8.41 (d, J = 2.4 Hz, 1H, 10-H),
8.33 (d, J = 8.7 Hz, 1H, 7-H), 7.00 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H, 8-H), 6.90 (d, J = 1.6 Hz, 1H, 4-H or
2-H), 6.61 (d, J = 1.6 Hz, 1H, 2-H or 4-H), 5.65 (br s, 1H, OH), 5.50 (br s, 1H, OH), 1.94–1.87 (m, 2H of
the cyclopentane ring), 1.82–1.62 (m, 6H of the cyclopentane ring), 1.60–1.52 (m, 2H, 20-H), 1.24–1.12
(m, 6H, 40-H, 50-H, 60-H), 1.03–0.94 (m, 2H, 30-H), 0.82 (t, J = 6.9 Hz, 3H, 70-H); 13C NMR (100 MHz,
CD3OD)
δ 165.2, 164.0, 157.7, 153.9, 153.3, 139.0, 133.3, 116.9, 113.9, 113.0, 111.7, 107.9, 105.7, 52.5, 43.0,
38.8, 32.9, 31.0, 26.5, 24.3, 23.7, 14.4; mass spectrum m/z (relative intensity) 381 (M++1, 6), 380 (M+,
18), 295 (4), 270 (23), 241 (11), 205 (71), 149 (62), 135 (80), 91 (63), 83 (54), 69 (37), 59 (100); exact mass
calculated for C24H28O4 380.1988, found 380.1986.
3-[1-(4-Cyanobutyl)cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3e). To a stirring
solution of 3a (30 mg, 0.07 mmol) in anhydrous DMSO (5 mL), at room temperature, under an argon
atmosphere, was added and NaCN (17 mg, 0.35 mmol). The reaction mixture was stirred vigorously
for 24 h and then diluted by adding ice water (2 mL) and diethyl ether (10 mL). The organic layer was
separated, and the aqueous phase was extracted with diethyl ether (2
×
5 mL). The combined organic
layer was washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude
material was purified by flash column chromatography on silica gel (40% ethyl acetate in petroleum
ether) gave 3e as a white solid in 72% yield (19 mg). mp 138–140 ◦C; IR (neat) 3275 (br, OH), 2925,
2853, 2259 (w, C δ 9.82 (br s,
N), 1684 (>C=O), 1602, 1270, 1107 cm−1; 1H NMR (400 MHz, CDCl3)
≡
1H, OH), 9.78 (br s, 1H, OH), 8.60 (d, J = 2.3 Hz, 1H, 10-H), 8.22 (d, J = 8.7 Hz, 1H, 7-H), 6.98 (dd,
J = 8.7 Hz, J = 2.3 Hz, 1H, 8-H), 6.78 (d, J = 1.7 Hz, 1H, 4-H or 2-H), 6.77 (d, J = 1.7 Hz, 1H, 2-H or
4-H), 2.24 (t, J = 7.1 Hz, 2H, -CH2CN), 1.99–1.89 (m, 2H of the cyclopentane ring), 1.80–1.57 (m, 8H, 6H
of the cyclopentane ring and 2H of the 4-cyanobutyl group, overlapping), 1.53 (qt, J = 7.6 Hz, 2H of
the 4-cyanobutyl group), 1.23–1.11 (m, 2H of the 4-cyanobutyl group); mass spectrum m/z (relative
intensity) 378 (M++1, 8), 377 (M+, 27), 368 (5), 309 (5), 295 (100), 279 (98), 241 (27), 224 (31), 205 (20), 91
(13), 67 (14); exact mass calculated for C23H23NO4 377.1627, found 377.1625.
3-[1-(5-Cyanopentyl)cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3f). The synthesis
was carried out as described for 3e, using 3b (31 mg, 0.07 mmol) and NaCN (17 mg, 0.35 mmol) in
DMSO (5 mL), and gave 3f as a white solid in 69% yield (19 mg). mp 176–178 ◦C; IR (neat) 3281 (br,
OH), 2923, 2854, 2259 (w, C
≡
N), 1683 (>C=O), 1618, 1598, 1271, 1216, 1103 cm−1; 1H NMR (500 MHz,
CDCl3) 8.45 (d, J = 2.0 Hz, 1H, 10-H), 8.33 (d, J = 8.2 Hz, 1H, 7-H), 7.02 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H,
δ
8-H), 6.88 (d, J = 1.2 Hz, 1H, 4-H or 2-H), 6.65 (d, J = 1.2 Hz, 1H, 2-H or 4-H), 6.20 (br s, 1H, OH), 5.87
(br s, 1H, OH), 2.26 (t, J = 6.8 Hz, 2H, -CH2CN), 1.97-1.90 (m, 2H of the cyclopentane ring), 1.81–1.52 (m,
10H, 6H of the cyclopentane ring and 4H of the 5-cyanopentyl group, overlapping), 1.35 (qt, J = 7.3 Hz,
2H of the 5-cyanopentyl group), 1.10–1.03 (m, 2H of the 5-cyanopentyl group); 13C NMR (100 MHz,
CD3OD)
δ 165.2, 163.9, 157.7, 154.0, 153.1, 138.9, 133.3, 121.2, 116.9, 113.9, 113.1, 111.6, 107.9, 105.8, 52.4,
42.7, 38.7, 30.4, 26.4, 25.9, 24.2, 17.3; mass spectrum m/z (relative intensity) 392 (M++1, 15), 391 (M+, 44),
349 (4), 306 (100), 295 (15), 266 (11), 252 (28), 240 (12), 190 (4), 149 (6), 67 (10); exact mass calculated for
C24H25NO4 391.1784, found 391.1781.
3-[1-(3-Cyanopropyl)cyclopentyl]-1,9-dihydroxy-6H-dibenzo[b,d]pyran-6-one (3g). The synthesis
was carried out as described for 3e, using 3c (18 mg, 0.043 mmol) and NaCN (11 mg, 0.22 mmol) in

Molecules 2019, 24, 3559
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DMSO (5 mL) and gave 3g as a white solid in 70% yield (13 mg). mp 118–120 ◦C; IR (neat) 3285 (br,
OH), 2928, 2853, 2260 (w, C
N), 1683 (>C=O), 1599, 1272, 1105 cm−1; 1H NMR (500 MHz, CDCl3)
≡
δ
8.47 (d, J = 2.1 Hz, 1H, 10-H), 8.31 (d, J = 8.3 Hz, 1H, 7-H), 6.98 (dd, J = 8.3 Hz, J = 2.1 Hz, 1H, 8-H),
6.64 (d, J = 1.3 Hz, 1H, 4-H or 2-H), 6.49 (d, J = 1.3 Hz, 1H, 2-H or 4-H), 6.22 (br s, 1H, OH), 5.78 (br s,
1H, OH), 2.23 (t, J = 6.7 Hz, 2H, -CH2CN), 1.94-1.83 (m, 2H of the cyclopentane ring), 1.74–1.56 (m, 8H,
6H of the cyclopentane ring and -CH2CH2CH2CN, overlapping), 1.55–1.49 (m, 2H, -CH2CH2CH2CN);
mass spectrum m/z (relative intensity) 364 (M++1, 7), 363 (M+, 22), 336 (67), 309 (24), 295 (100), 279 (48),
241 (47), 165 (10), 129 (24); exact mass calculated for C22H21NO4 363.1471, found 363.1470.
Radioligand binding assays: the affinities (Ki) of the new compounds for rat CB1 receptor as well
as for mouse and human CB2 receptors were obtained by using membrane preparations from rat brain
or HEK293 cells expressing either mCB2 or hCB2 receptors, respectively, and [3H]CP-55,940 as the
radioligand, as previously described [38,57]. Results from the competition assays were analyzed using
nonlinear regression to determine the IC50 values for the ligand [59]; Ki values were calculated from the
IC50 (Prism by GraphPad Software, Inc.). Each experiment was performed in triplicate and Ki values
determined from three independent experiments and are expressed as the mean of the three values.
cAMP assay: [57,60] HEK293 cells stably expressing hCB2 receptors were used for the studies.
The cAMP assay was carried out using PerkinElmer’s Lance ultra cAMP kit following the protocol of
the manufacturer. Briefly, the assays were carried out in 384-well plates using 1000–1500 cells/well.
The cells were harvested with non-enzymatic cell dissociation reagent Versene, washed once with HBSS
and resuspended in the stimulation buffer. The various concentrations of the test compound (5
forskolin (2 M final concentration) containing stimulation buffer were added to the plate followed by
the cell suspension (5 L). Cells were stimulated for 30 min at room temperature. Eu-cAMP tracer
working solution (5 L) and Ulight-anti-cAMP working solution (5 L) were then added to the plate
µL) in
µ
µ
µ
µ
and incubated at room temperature for 60 min. The data were collected on a Perkin-Elmer Envision
instrument. The EC50 values were determined by non-linear regression analysis using GraphPad
Prism software (GraphPad Software, Inc., San Diego, CA, USA).
4. Conclusions
This study expands on earlier work on the CB2-selective cannabilactone prototype. We explored
the effects of modifications to the pharmacophoric side chain. In particular, we replaced the
C10-gem-dimethyl group in AM1714 with the bulkier cyclopentyl ring, varied the chain’s length,
and substituted a bromo or cyano group for the terminal carbon. We identified the analog
6-[1-(1,9-dihydroxy-6-oxo-6H-benzo[c]chromen-3-yl)cyclopentyl]hexanenitrile (AM4346) as a high-affinity
ligand to mCB2 (Ki = 4.9 nM) with 131-fold selectivity for mCB2 over rCB1. Moreover, the species difference
in the affinities of AM4346 between the mouse and the human CB2 receptors is reduced when compared
to our first-generation compound AM1714. Importantly, in the cyclase assay AM4346 was found to be
a highly potent and efficacious hCB2 receptor agonist (EC50 = 3.7
±
1.5 nM, E(max) = 89%). Our lead
cannabilactone analog, AM4346, is also endowed with enhanced polarity due to the incorporation of the
cyano group. Extension of our SAR to include the biphenyl synthetic intermediates has revealed new
compounds that bind mCB2 with high affinity and substantial selectivity for that receptor over rCB1.
Author Contributions: Conceptualization, A.M. and S.P.N.; Methodology, S.O.A. and S.P.N.; Validation, A.M.,
S.P.N., S.O.A., T.C.H.; Formal Analysis, A.M., S.P.N. and S.O.A.; Biological Investigation, F.T., O.B. and S.P.N.;
Writing-Original Draft Preparation, S.O.A., S.P.N, and T.C.H.; Writing-Review and Editing, S.P.N.; Supervision,
A.M. and S.P.N.; Funding Acquisition, A.M.
Funding: This research was funded by the National Institute on Drug Abuse: grand numbers DA009158,
DA045020, and DA041307-01.
Conflicts of Interest: The authors declare no conflict of interest.

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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).