CB2-selective cannabinoid receptor ligands: Synthesis, pharmacological evaluation, and molecular modeling investigation of 1,8-naphthyridin-2(1 H)-one-3-carboxamides

Article abstract of DOI:10.1021/jm500807e

We have recently identified 1,8-naphthyridin-2(1H)-one-3-carboxamide as a new scaffold very suitable for the development of new CB2 receptor potent and selective ligands. In this paper we describe a number of additional derivatives in which the same central scaffold has been variously functionalized in position 1 or 6. All new compounds showed high selectivity and affinity in the nanomolar range for the CB2 receptor. Furthermore, we found that their functional activity is controlled by the presence of the substituents at position C-6 of the naphthyridine scaffold. In fact, the introduction of substituents in this position determined a functionality switch from agonist to antagonists/inverse agonists. Finally, docking studies showed that the difference between the pharmacology of these ligands may be in the ability/inability to block the Toggle Switch W6.48(258) (χ1 g+ → trans) transition.

Full text of DOI:10.1021/jm500807e

Article
pubs.acs.org/jmc
CB2-Selective Cannabinoid Receptor Ligands: Synthesis,
Pharmacological Evaluation, and Molecular Modeling Investigation
of 1,8-Naphthyridin-2(1H)‑one-3-carboxamides
Valentina Lucchesi,† Dow P. Hurst,‡ Derek M. Shore,‡ Simone Bertini,† Brandie M. Ehrmann,‡
Marco Allara,§ Lyle Lawrence,‡ Alessia Ligresti,§ Filippo Minutolo,† Giuseppe Saccomanni,† Haleli Sharir,∥
̀
Marco Macchia,† Vincenzo Di Marzo,§ Mary E. Abood,∥ Patricia H. Reggio,*,‡ and Clementina Manera*,†
†Dipartimento di Farmacia, Universita
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
‡Center for Drug Discovery, University of North Carolina Greensboro, Greensboro, North Carolina 27402, United States
§Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34,
80078 Pozzuoli, Napoli, Italy
∥Center for Substance Abuse Research, Temple University, Philadelphia, Pennsylvania 19140, United States
S
* Supporting Information
ABSTRACT: We have recently identified 1,8-naphthyridin-
2(1H)-one-3-carboxamide as a new scaffold very suitable for
the development of new CB2 receptor potent and selective
ligands. In this paper we describe a number of additional
derivatives in which the same central scaffold has been variously
functionalized in position 1 or 6. All new compounds showed
high selectivity and affinity in the nanomolar range for the CB2
receptor. Furthermore, we found that their functional activity is
controlled by the presence of the substituents at position C-6 of
the naphthyridine scaffold. In fact, the introduction of
substituents in this position determined a functionality switch from agonist to antagonists/inverse agonists. Finally, docking
studies showed that the difference between the pharmacology of these ligands may be in the ability/inability to block the Toggle
Switch W6.48(258) (χ1 g+ → trans) transition.
inflammatory pain, obesity, and neurodegenerative disease.12 At
the present time, CB2R has gained attention as a potential
target for immunoregulation. Recent advances suggest a role for
INTRODUCTION
■
Cannabinoids are a unique family of terpenophenolic active
constituents of Cannabis sativa; Δ9-tetrahydrocannabinol
CB2R within the nervous system, particularly in inflammatory
conditions such as neurodegenerative disease (Parkinson’s
disease, Alzheimer’s disease, Huntington’s disease, multiple
sclerosis, etc.), since CB2R is up-regulated in the brain under
these conditions and disease states. This finding is supported by
observing the pattern of expression of CB2R during microglia
differentiation using an in vitro model of multistep
activation.13−15 Additional data suggest that CB2R-selective
agonists show promise for suppressing inflammatory and
neuropathic pain states. Behavioral, electrophysiological, and
neurochemical studies all support a role for CB2R activation in
modulating inflammatory nociception. Moreover, recent re-
views have focused on the evidence for the functional neuronal
presence and the emerging role of CB2R in neuropsychiatric
disorders.16,17 Finally, CB2R is overexpressed in several tumor
cells, and various in vitro studies and animal models have shown
(THC) is the most relevant member, owing to its psychoactive
effects and a wide variety of pharmacological effects.1,2 The
isolation and characterization of THC3 allowed for the
identification of two distinct cannabinoid receptors (CBRs),
named CB1 receptor (CB1R) and CB2 receptor (CB2R), that
have been cloned and characterized from mammalian tissues.4,5
CB1R is abundantly expressed in the central nervous system
(CNS), with the highest densities in the hippocampus,
cerebellum, and striatum.6 Locations outside the brain have
also been indicated, including adipose tissue, liver, muscle, the
gastrointestinal tract, pancreas, urinary bladder, lung, heart,
adrenal gland, testis, uterus, and prostate.7−10 In contrast,
CB2R has been reported to be essentially limited to the cells
associated with the immune system, such as spleen, thymus,
and tonsils,5 but it also has been found in low concentrations in
the brain.11
Since the discovery of the CBRs and their endogenous
ligands, numerous studies implicate the endocannabinoid
system in several physiological and pathological processes,
including cancer, appetite, fertility, memory, neuropathic and
Received: March 31, 2014
© XXXX American Chemical Society
A
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that activation of the CB2R induces apoptosis, inhibits tumor
growth, and inhibits neo-angiogenesis.1,18
Scheme 1. Synthesis of 1,8-Naphthyridin-2(1H)-one-3-
carboxamide Derivatives 1−13
a
The effectiveness of selective CB2R agonists as neuro-
protective and anticancer agents prompted us to report our
efforts in this field. Our aim was to identify new selective CB2R
agonists as potential drugs devoid of the psychotropic side
effects associated with CB1R.
Recently we described the synthesis, binding affinities, and
pharmacological characterization of a novel series of 1,8-
naphthyridin-2(1H)-one-3-carboxamides of general structure A
(Figure 1), acting as potent and selective CB2R ligands.19
Figure 1. General structure of compounds A.
Furthermore, the concentration-dependent inhibitory action on
human basophils activation and the concentration-dependent
decrease of cell viability in Jurkat cells shown by one of these
derivatives strongly suggest that these compounds possess
agonist properties on CB2R.19
In an effort to develop improved naphthyridine-based CB2R
ligands and also to develop structure−activity relationships
(SARs) for both CB1R and CB2R, the present paper describes
the synthesis and the pharmacological properties of a number
of additional 1,8-naphthyridin-2(1H)-one-3-carboxamide deriv-
atives, 1−26 (summarized in Tables 1 and 2, below), in which
the central naphthyridine scaffold has been variously function-
alized with different substituents in position 1 or 6. The 4-
methylcyclohexyl carboxamide group in position 3 has been
selected on the basis of binding results obtained for derivatives
A. The new compounds were tested in competitive binding
assays toward both human recombinant CB1R and CB2R
expressed in HEK-293 cells and were found to be selective for
CB2R. Furthermore, the functional activity of the most
representative compounds was determined by a β-arrestin 2
recruitment assay using U2OS cells co-expressing CB2R and β-
arr2/GFP as well as with a forskolin-stimulated cAMP assay,
demonstrating that the functionality of tested compounds is
controlled by the presence of the substituents at position C-6 of
the naphthyridine scaffold.
a
Reagents and conditions: (i) Cs2CO3, R1Cl or R1Br, DMF, 50 °C, 12
h, 23%−85%; (ii) NaOH aq. 10%. 110 °C, 5h, 92%−95%.
methanesulfonyl chloride in anhydrous dichloromethane and
triethylamine at room temperature for 6 h generated mesylates
28−30. Exposure of 28−30 to tetrabutylammonium fluoride in
THF at reflux for 4 h provided derivatives 14−16.
The synthetic route to obtain 6-substituted 1,8-naphthyridin-
2(1H)-one-3-carboxamide derivatives 17−26 is outlined in
Scheme 3. 2-Aminonicotinaldehyde was treated with bromine
in glacial acetic acid at room temperature for 24 h to obtain the
corresponding 6-bromo derivative 31, which was refluxed with
diethyl malonate and in the presence of piperidine in EtOH for
24 h to afford ethyl 6-bromo-1,8-naphthyridin-2(1H)-one-3-
carboxylate 32. The reaction of ethyl ester 32 with a cis/trans
diastereoisomeric mixture of 4-methylcyclohexylamine in a
sealed tube for 24 h at 150 °C provided the desired
carboxamide 33. N-Alkylation of 33 in anhydrous DMF with
p-fluorobenzyl chloride or 4-(2-chloroethyl)morpholine in the
presence of cesium carbonate afforded the desired 1,8-
naphthyridin-2-one derivatives 17 and 18, respectively. To
obtain compounds 19−26, 6-bromo derivatives 17 or 18 were
subjected to a cross-coupling reaction with suitable boronic
acids in dioxane under Suzuki conditions by generating in situ
Pd(PPh3)4 as the catalyst and aqueous Na2CO3 (2 M) as the
base. These reactions were carried out in a microwave reactor
(CEM). Each crude mixture was purified by flash chromatog-
raphy. For compounds 17, 20, and 22, the separation of cis and
trans isomers was also obtained.
Finally, docking studies were performed in order to analyze
both the complex of the CB2R in its inactive state (R) with
antagonists/inverse agonists and the complex of the CB2R in
its activated state (R*) with agonists. These studies
demonstrated that the difference between the pharmacology
of these ligands may be in the ability/inability to block the
Toggle Switch W6.48(258) (χ1 g+ → trans) transition.
CHEMISTRY
■
The synthesis of compounds 1−26 is depicted in Schemes 1−3.
As reported in Scheme 1 the N1-alkylation of 1,8-
naphthyridine-3-carboxamide 2719 in anhydrous DMF with
the suitable halogenated reagent in the presence of cesium
carbonate at 50 °C for 12 h afforded the desired compounds
1−10. The carboxylic acid derivatives 11−13 were obtained
from the corresponding esters 8−10 by alkaline hydrolysis
followed by acidification.
RESULTS AND DISCUSSION
■
CB1R and CB2R Affinity. The binding affinities (Ki values)
of target compounds 1−26 were evaluated by competitive
radioligand displacement assays against the human CB1R and
CB2R using [3H]CP-55,940 as the radioligand for both
receptors.19 The results are summarized in Tables 1 and 2,
together with the Ki values of previously reported morpholi-
The synthesis of fluorine derivatives 14−16 is outlined in
Scheme 2. Treatment of alcohol derivatives 5−7 with
B
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a
Scheme 2. Synthesis of 1,8-Naphthyridin-2(1H)-one-3-carboxamide Derivatives 14−16
a
Reagents and conditions: (i) CH2Cl2, Et3N, MsCl, r.t., 6 h, 37%−86%; (ii) TBAF, CH2Cl2, reflux, 4 h, 47%−49%.
a
Scheme 3. Synthesis of 1,8-Naphthyridin-2(1H)-one-3-carboxamide Derivatives 17−26
a
Reagents and conditions: (i) Br2, AcOH, 24 h, r.t., 73%; (ii) diethyl malonate, piperidine, EtOH, reflux, 12 h, 90%; (iii) 4-methylcyclohexylamine,
150 °C, 24 h, 65%; (iv) Cs2CO3, p-fluorobenzyl chloride or 4-(2-chloroethyl)morpholine, DMF, 50 °C, 12 h, 71%, 92%; (v) Ph3P, Pd(OAc)2,
dioxane, Na2CO3, suitable boronic acid, 150 °C, microwave (200 W, 100 psi, 15 min, under stirring), 41−94%.
noethyl and p-fluorobenzyl derivatives A1 and A2, respec-
tively,19 and reference compounds SR14452820 and JWH133.21
First efforts to improve the CB2R affinity and selectivity were
focused on the introduction of a large variety of alkyl
substituents in position N-1 of the 1,8-naphthyridine nucleus.
Compounds bearing n-butyl (1) and n-pentyl chains (2) display
excellent affinities for CB2R and low affinities at CB1R, so
these compounds behave similarly to previously studied
compounds A1 and A2.
Interestingly, compounds 3−7, characterized by a hydrox-
yalkyl chain, show no affinity toward CB1R, while their CB2R
affinity increases with the elongation of the alkyl chain length.
In fact, the replacement of the hydroxyethyl group of 3 with a
three-carbon (hydroxypropyl in compound 4), a four-carbon
linker (hydroxybutyl in compound 5), or a five-carbon linker
(hydroxypentyl in compound 6) progressively increased CB2R
affinity (Ki values varying from 2096 nM to 3.60 nM). On the
contrary, compound 7 possessing a further elongated
hydroxyhexyl chain, displays a slight loss in CB2R affinity (Ki
= 18.0 nM).
The replacement of the hydroxy group by a fluorine atom
allows to increase affinity for CB2R, but significantly decreases
the selectivity toward this receptor (see compounds 15, 16),
with the exception of compound 14, which has the highest
selectivity obtained for fluoroalkyl compounds (Ki(CB1)/
Ki(CB2) = 743).
Finally, the esters 8−10 display a good CB2R affinity and no
affinity toward CB1R (Ki > 10 000 nM), thus showing an
C
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a
Table 1. Radioligand Binding Data of 1,8-Naphthyridin-2(1H)-one-3-carboxamide Derivatives 1−16
Ki (nM)
b
c
compd
R1
CB1
CB2
Ki(CB1)/Ki(CB2)
1
2
3
4
5
6
7
8
9
CH2(CH2)2CH3
CH2(CH2)3CH3
CH2CH2OH
2314 108
908 19.3
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
1011 46.5
22.59 1.02
35.67 1.09
1000
3.90 0.17
2.82 0.08
2096 98.8
129 5.55
53.3 1.22
3.60 0.13
18.0 0.26
176 7.3
62.6 0.79
27.1 0.77
1394 62.3
2466 95.5
1341 53.4
1.36 0.053
0.56 0.013
1.46 0.014
1.90
593
322
>5
CH2(CH2)2OH
CH2(CH2)3OH
CH2(CH2)4OH
CH2(CH2)5OH
CH2CH2COOEt
CH2(CH2)2COOEt
CH2(CH2)3COOMe
CH2CH2COOH
CH2(CH2)2COOH
CH2(CH2)3COOH
CH2(CH2)3F
>77
>187
>2778
>554
>57
>160
>369
>7
10
11
12
>4
13
>7
14
743
40.3
24.4
526
222
728
226
15
CH2(CH2)4F
16
CH2(CH2)5F
A1
morpholinoethyl
p-fluorobenzyl
A2
200
0.90
SR144528
JWH133
437
0.60
677
3.00
a
Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM) for CB1R and CB2R
b
binding assays. Affinity of compounds for CB1R was evaluated using membranes from HEK-293 cells transfected with CB1R and [3H]CP-55,940.
c
Affinity of compounds for CB2R was evaluated using membranes from HEK-293 cells transfected with CB2R and [3H]CP-55,940.
a
Table 2. Radioligand Binding Data of 6-Substituted 1,8-Naphthyridin-2(1H)-one-3-carboxamide Derivatives 17−26
Ki (nM)
b
c
compd
R1
R2
CB1
CB2
Ki(CB1)/Ki(CB2)
17
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
morpholinoethyl
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
p-fluorobenzyl
morpholinoethyl
morpholinoethyl
morpholinoethyl
morpholinoethyl
Br
Br
Br
Br
96.1 1.99
166 7.7
121 5.32
750 26.5
3262 101
3280 116
430 20.2
472 20.6
>10 000
0.18 0.002
1.12 0.01
0.12 0.002
1.26 0.04
3.83 0.05
1.85 0.06
65.6 2.41
0.96 0.035
2.17 0.09
0.67 0.009
94.2 2.47
0.27 0.004
1.47 0.05
0.17 0.002
0.68 0.009
0.35 0.01
534
148
17-trans
17-cis
18
1010
595
19
p-methoxyphenyl
2-thienyl
851
20
1773
6.5
20-trans
20-cis
21
2-thienyl
2-thienyl
491
p-fluorophenyl
2-furyl
>4608
5140
>106
15 022
>6802
192
22
3444 170.5
>10 000
22-trans
22-cis
23
2-furyl
2-furyl
4056 200
>10 000
p-methoxyphenyl
2-thienyl
24
32.7 1.48
66.7 2.33
52.9 1.90
25
p-fluorophenyl
2-furyl
98
26
151
a
Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM) for CB1R and CB2R
b
binding assays. Affinity of compounds for CB1R was evaluated using membranes from HEK-293 cells transfected with CB1R and [3H]CP-55,940.
c
Affinity of compounds for CB2R was evaluated using membranes from HEK-293 cells transfected with CB2R and [3H]CP-55,940.
D
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important degree of CB2R selectivity. The corresponding
carboxylic acids 11−13 show no affinity toward CB1R (Ki > 10
000 nM) but also very low CB2R affinity.
Next, with the aim to investigate the impact on the CB2R
affinity and selectivity within this series of 1,8-naphthyridin-
2(1H)-one-3-carboxamides, various lipophilic groups were
introduced at the position C-6 (Table 2). In details,
compounds 17−26 are characterized by the presence of the
common 4-methylcyclohexyl carboxamide moiety at position
C-3 together with a p-fluorobenzyl or a morpholinoethyl group
at position N-1 of the naphthyridine scaffold, since these groups
had been shown to be important for CB2R affinity in our
previous studies.19
All of these 6-substituted analogues (17−26) maintain high
CB2R affinities (Ki < 4 nM) relative to the corresponding
compounds lacking the substituent in position C-6 (A1 and A2,
Table 1), with the exception of compounds 20-trans and 22-
trans.
With regard to the N-p-fluorobenzyl derivatives, the
compounds substituted in position 6 with a bromine atom
(17) exhibits a Ki value at CB2R (0.18 nM) slightly lower than
that of its reference analogue A2 (0.90 nM), while the
selectivity for CB2R of this new compound is considerably
greater (A2, Ki(CB2)/Ki(CB1) = 222; 17, Ki(CB2)/Ki(CB1) =
534). The introduction of aromatic groups, such as p-
methoxyphenyl (19), thienyl (20), p-fluorophenyl (21), or
furyl (22), in position 6 of the naphthyridine nucleus generally
results in a significantly reduced affinity for the CB1R, when
compared to that of unsubstituted compound A2 or 6-bromine-
substituted 17. Moreover, for some of these compounds (17,
20, and 22) the cis and trans isomers were separated in order to
assess the effect of stereoselectivity on the CB2R affinity. Pure
isomers 17-cis, 20-cis, and 22-cis showed 9-fold, 68-fold, and
349-fold increases in their affinity for the CB2R when
compared with their corresponding diastereoisomers 17-trans,
20-trans, and 22-trans. These data confirm our previously
hypothesis that the cis conformation is the preferred one for the
interaction of 4-methylcyclohexyl carboxamide derivatives at
CB2R.19
Figure 2. Concentration−response curves depicting βarr2-GFP
recruitment following treatment with CB2R agonists. EC50 values
are reported in Table 3. Activity values were normalized to the
agonist’s response (30 nM WIN-55,212−2 was considered as 100%).
Data are the mean SE of three experiments carried out in duplicate.
as antagonists (Figure 3, Table 3). The functionality of the
cannabinoid agonist WIN-55,212-2 and the antagonist
SR144528 were determined as reference compounds.
Figure 3. WIN-55,212−2 mediated βarr2-GFP recruitments is blocked
by CB2R antagonists. IC50 values are reported in Table 3. Activity
values were normalized to the agonist’s response (30 nM WIN-
55,212−2 was considered as 100%). Data are the mean SE of three
experiments carried out in duplicate.
More surprisingly, substitution in position C-6 of morpho-
linoethyl derivatives (18, 23−26) did not significantly alter
CB2R binding affinity respect to the corresponding N-p-
fluorobenzyl-substituted derivatives, but determined a general
enhancement of the affinity on CB1R, with the exception of
compound 23, which has a Ki for CB1R higher than 10 000
nM.
CB2R Functional Activity. The potency and efficacy of the
new ligands 5, 14, 17, 18, and 23 and of previously studied
derivatives A1 and A2 were characterized with a β-arrestin 2
assay using U2OS cells co-expressing CB2R and β-arr2/GFP as
previously described.22 We chose these representative com-
pounds in order to assess the impact of the substitution in
positions 1 and 6 of the naphthyridine nucleus on the
functional activity. Furthermore, we have compared the
pharmacology observed using the β-arrestin 2 recruitment
assay for compounds A1, A2, 14, and 18 with the effects on
forskolin-stimulated cAMP levels (Perkin-Elmer LANCE) in
the same cell line (Table 3).
The ability of selected compounds A1, A2, 14, and 18 to
activate CB2R was assessed in a functional cAMP assay using
U2OS cells stably expressing human CB2R. Unfortunately,
compounds 5, 17, and 23 proved to be insoluble under the test
conditions. Similar to the agonist WIN-55,212-2 (EC50 = 17.3
nM), A1, A2, and 14 potently inhibited forskolin-mediated
cAMP production by human CB2R, with EC50 = 28.0, 29.6, and
20.6 nM, respectively (Figure 4, Table 3). The inhibition curve
of antagonist/inverse agonists 18 on human CB2R stimulated
with WIN-55,212-2 (30 nM) is reported in Figure 5, and IC50 =
59.6 nM.
Table 3 shows an overall good correlation between the EC50
and IC50 values obtained using all three methodologies. With
the cell-based activity data, we demonstrated an analogue
functional activity for compounds A1, A2, 14, and 18, which
indicates that both pathways, β-arrestin and GPCR, are
pharmacologically modulated by these ligands. Replacement
of p-fluorobenzyl (A2) and morpholinoethyl (A1) groups with
alkyl groups, such as 4-hydroxybutyl (5) and 4-fluorobutyl
(14), in position N-1 was well tolerated, maintaining agonist
activity and potency in the β-arrestin and cAMP assays
(compare A1 and A2 with 5 and 14). Interestingly the tested
Using the β-arrestin 2 assay, four agonists at CB2R were
determined, A1, A2, 5, and 14, with CB2R EC50 values ranging
from 17.6 to 29.6 nM (Figure 2, Table 3), while 17, 18, and 23
showed a reduction of basal β-arrestin recruitment, therefore
suggesting that these compounds act as inverse agonists as well
E
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rhodopsin mutant, the transition of the ligand from 11-cis-
retinal to all-trans-retinal releases W6.48(265). The β-ionone
ring shifts 4.3 Å toward the cleft between TMH5 and TMH6
and the W6.48(265) indole ring moves 3.6 Å away from its
ground-state position when rhodopsin is activated.27 Mutation
studies have suggested that F3.36(200) serves the same
function in the CB1R as the β-ionone ring serves in
rhodopsin.28,29 These residues form a “toggle switch” in
which the CB1R F3.36(200) χ1 must undergo a trans → g+
conformational change in order for the χ1 of W6.48(356) to
undergo its g+ → trans transition. In CB2R, F3.36(117) appears
to serve a similar function in holding W6.48(258) in a g+ χ1
conformation. Agonist binding promotes a conformational
Figure 4. CB2R Agonists inhibit cAMP formation. Higher TR-FRET
signals correlates with lower cAMP levels. EC50 Values are reported in
Table 3. Data are the mean SE of three experiments carried out in
triplicate.
change in these residues (F3.36(117) χ1 trans → g+
;
W6.48(258) χ1 g+→ trans). The W6.48 χ1 has not been
found in the trans conformation in recent X-ray crystal
structures of GPCR activated states. However, in their meta-
rhodopsin II crystal structure paper, Choe and co-workers note
that the W6.48(265) χ1 g+ → trans change may be transient
and therefore not captured in the crystalline state.30 In fact, in
molecular dynamics simulations of cannabinoid CB2R
activation by its endogenous ligand (2-AG), we observed
such a transient change in W6.48(258).31 Interestingly, while
the results of mutagenesis studies suggest that the toggle switch
found in the cannabinoid receptors is comprised of F3.36(117)
and W6.48(265), these residues do not necessarily form the
toggle switch in all GPCRs. For example, Kobilka and co-
workers have reported that in the β2 adrenergic receptor, the
residues F6.48(286) and F6.52(290) may form a rotamer
toggle switch that changes conformation upon receptor
activation.32 These results may suggest that while the identity
of the participating residues may vary, the functional role of the
toggle switch appears to be conserved among numerous
GPCRs.
Figure 5. Treatment with the CB2R antagonist/inverse agonist 18
inhibits the response to WIN-55,212−2 in the cAMP assay. IC50 value
is reported in Table 3. Data are the mean SE of three experiments
carried out in triplicate.
Glide Docking Studies Suggest the Difference
between Inverse Agonists and Agonists May Depend
on Interaction with Toggle Switch. Glide docking studies
in our previously published model of the CB2R inactive and
active states31 using the global minimum energy conformer
revealed that both the antagonists/inverse agonists 17, 18, and
23, and the agonists A2, A1, 5, and 14, bind in the TMH2-3-6-
7 region of CB2R. Modeling studies suggested that the
difference between the pharmacology of the CB2R ligands
synthesized here (antagonist/inverse agonist vs agonist) may be
in the ability/inability to block the Toggle Switch W6.48(258)
(χ1 g+ → trans) transition. Compounds 23 and A1 form an
antagonist/agonist pair of structurally related compounds.
Figure 6A,B illustrates the complex of the CB2R with
antagonist/inverse agonist 23. Compound 23 binds in the
TMH2-3-6-7 region of CB2R, establishing a hydrogen bond
with K3.28(109) via the 1,8-naphthyridine carbonyl at C2.
Compound 23 also forms a hydrogen bond with S7.39(285) via
the carboxamide oxygen. The 1,8-naphthyridine ring also has a
tilted-T aromatic stack with F2.57(87) and a tilted-T aromatic
stack with W5.43(194). The 6 position p-methoxyphenyl
substituent has a direct tilted-T aromatic stack with the
aromatic ring of W6.48(258) and an offset parallel aromatic
stack with W5.43(194).
compounds acted as agonists or antagonists/inverse agonists in
functional activity assays, depending on the presence of the
substituents at position C-6 of the naphthyridine scaffold. In
fact, the introduction of substituents (bromine or p-
methoxyphenyl) in this position determined a functionality
switch from agonist to antagonists/inverse agonists (see
compounds 17, 18, and 23).
Docking Study. The Key Molecular Features That
Discriminate 1,8-Naphthyridin-2(1H)-one-3-carboxa-
mide Derivative Agonists from Antagonists/Inverse
Agonists. As we reported previously,19,23 the cis structural
isomer of the 4-methylcyclohexyl substituent has higher affinity
for CB2R than the trans structural isomer by 7−13-fold.
Therefore, in the docking studies reported below, the lowest
energy cis positional isomer was used. The conformational
analysis of antagonists/inverse agonists 17, 18, 23, and the
agonists A2, A1, 5, 14 is reported in Supporting Information.
Molecular Toggle Switch. Agonist binding triggers the
changes in the intracellular region of a GPCR that leads to the
activated state. The CB2R TMH6 flexible hinge (CWXP)
residue, W6.48(258), in the R (inactive) state, has a g+ χ1
dihedral angle. (Please see Experimental Section for explanation
of Ballesteros−Weinstein residue nomenclature.) In the Class A
GPCR, rhodopsin, the β-ionone ring of the covalently bound
ligand, 11-cis-retinal, sterically keeps W6.48(265) in a g+
χ1.24−26 In the X-ray crystal structure of a constitutively active
Figure 6C,D illustrates the complex of the CB2R with the
agonist A1. In the TMH2-3-6-7 region of CB2R, A1 establishes
a hydrogen bond with K3.28(109) via the 1,8-naphthyridine
carbonyl oxygen at C2 and a hydrogen bond with S7.39(285)
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Table 3. Affinities and Potencies of CB2 Ligands Obtained from Binding, Second Messenger, and Receptor β-Arrestin
a
Interaction Assays
a
Data represent mean values for at least three separate experiments performed in duplicate.
via the carboxamide oxygen. A1 does not form aromatic
stacking interactions with W5.43(194) or W6.48(258).
Compounds 23 and A1 differ only in their C-6 substituent.
In 23, the C-6 substituent is a p-methoxyphenyl group, while at
A1, the substituent is hydrogen. Because the 6 position points
directly intracellular, it extends deep enough in the binding
pocket to block the movement of W6.48(258) (χ1 g+ → trans),
thus rendering 23 an antagonist/inverse agonist (see Figure
6B). In contrast, since A1 lacks the large 6-substituent, it does
not extend deep enough to influence W6.48(258), leaving this
residue free to change conformation and therefore for the
receptor to activate (see Figure 6D).
Similarly, the antagonists/inverse agonists 17 and 18 have
large bromo substituents at the 6 position that would also block
toggle switch transition (for compound 17 see Supporting
Information, Figure S6), whereas agonists A1, A2, 5, and 14 do
not (see Supporting Information, discussion on ligand
complexes and Figure S5).
Furthermore, the functional activity of more representative
compounds was determined by the β-arrestin 2 assay using
U2OS cells co-expressing CB2R and β-arr2/GFP and the
forskolin-stimulated cAMP assay. The potency values (IC50 or
EC50) of the novel compounds measured in functional assays
are in the nanomolar range and are closely correlated with the
high-affinity values (expressed as Ki). An interesting observa-
tion in our SAR is the significant difference in the functional
activity between the 6-substituted derivatives and the
compounds lacking this substituent. In fact, the functionality
of this new series of 1,8-naphthyridine was not affected by
several modifications in position N-1, showing agonist behavior,
while all the 6-substituted derivatives tested possess antagonist/
inverse agonist properties at CB2R.
To better understand the SAR results, we performed a
docking study for the tested compounds using a CB2R
homology model. In particular, the complex of CB2R in its
inactive state (R) with antagonist and the complex of CB2R in
its activated state (R*) with agonist were analyzed. The results
showed that both the antagonists/inverse agonist and the
agonist bind in the TMH2-3-6-7 regions of CB2R. The
difference between the pharmacology of these ligands
(antagonist/inverse agonist vs agonist) may be in the ability/
inability to block the Toggle Switch W6.48(258) (χ1 g+ →
trans) transition. We demonstrated that the substituent at the
C-6 position of the central nucleus is crucial for the
functionality, identifying it as the key molecular feature that
discriminates 1,8-naphthyridin-2(1H)-one-3-carboxamide ago-
nists from antagonists/inverse agonists.
CONCLUSION
■
In our study, in order to improve the CB2R affinity and
selectivity and to develop an exhaustive structure−activity
relationship (SAR) for the series of 1,8-naphthyridin-2(1H)-
one-3-carboxamides as CB2R ligands, we synthesized and
evaluated several new derivatives belonging to this chemical
class. These compounds are characterized by the same central
scaffold and are variously functionalized with different
substituents in position N-1 or C-6. Generally, the new
compounds exhibited good selectivities and remarkable
affinities for the CB2R. In particular, all the 6-substituted
analogues showed high CB2R affinity, with Ki < 4 nM.
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Figure 6. Binding sites and spatial relationship with W6.48(258) of the antagonist/inverse agonist 23 and the agonist A1, docked in CB2R (inactive)
model. (A,C) The view is from lipid looking toward TMH6 and TMH7 (transparent for clarity). 23 is shown in pink; A1 is shown in blue. Toggle
switch residues are shown in orange; aromatic cluster residues are shown in lavender; residues that each ligand forms a hydrogen bond with are
shown in yellow. Hydrogen bonds are shown as yellow dashed lines. (B,D) The view is from lipid looking toward TMH4 and TMH6 (TMH5 is
omitted for clarity). In (B), the p-methoxyphenyl C6 substituent of 23 penetrates deeply into the binding pocket, deep enough to block any possible
movement of W6.48(258). The ability of this substituent to block W6.48(258) likely renders 23 an antagonist/inverse agonist. In contrast, in (D) it
is clear that A1 does not penetrate the binding pocket deeply enough to sterically block W6.48(258), permitting this compound to act as an agonist.
General Procedure for the Synthesis of N-1-Substituted N-
EXPERIMENTAL SECTION
■
(4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-
Chemistry. Melting points were determined on a Kofler hot-stage
carboxamide (1−10). A solution of 1,8-naphthyridine-3-carbox-
1
apparatus and are uncorrected. H NMR and 13C NMR spectra were
amide 30 (1.42 g, 5.0 mmol) in anhydrous DMF (20 mL) was treated
recorded on a Varian Gemini 200 MHz spectrometer in δ units with
TMS as an internal standard. Mass spectra were obtained with a
Hewlett-Packard MS system 5988. TLC was performed on silica gel
sheets (silica gel 60 F254, Merck, Germany). Microwave-assisted
reactions were run in a CEM microwave synthesizer. The system for
isocratic flash chromatography includes a Buchi Pump Module C-601
(continuous flow of solvents up to 250 mL/min at a maximum of 10
bar) and Buchi prepacked cartridges (silica gel 60, particle size 40−63
μm).
The analytical HPLC system consisted of a Thermo Finnigan
Spectra System SN4000 system controller, coupled to a P2000 pump,
a SCM1000 degasser, and a UV2000 UV detector at operation
wavelengths of 220 and 280 nm (Thermo Finnigan, Waltham, MA).
Separation was performed on a 150 mm × 4.6 mm Luna column
packed with 5 μm C18 particles. The mobile phase, delivered at
isocratic flow, consisted of methanol (15−30%) and water (85−70%).
HPLC-grade methanol was acquired from Sigma-Aldrich (Sydney,
Australia), and the water used was of Milli-Q grade purified by a Milli-
Q UV purification system (Millipore Corp., MA). For all compounds,
1.0 mg was dissolved in 2.0 mL of MeOH, and an amount of 20 μL
was injected in analytical HPLC. Comparing the chromatograms, we
were able to estimate the purity of each compound that appeared
>96%. High-resolution mass spectra (HRMS) were recorded on a
Thermo Scientific Q Exactive Plus mass spectrometer (ThermoFisher,
Breman, Germany) equipped with an electrospray ionization source.
with cesium carbonate (0.43 g, 14.0 mmol) at room temperature for 1
h. The appropriate reagent (10.0 mmol) was added, and the mixture
was stirred for 12 h at 50 °C. After cooling, the reaction mixture was
evaporated in vacuo, yielding the crude products which were purified
by crystallization or flash chromatography.
1-Butyl-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyri-
dine-3-carboxamide (1). Purified by flash chromatography (hexane/
ethyl acetate 2:1). Yield 61%; MS m/z 341 (M+). 1H NMR (CDCl3):
δ 10.03 and 9.65 (2m, 1H, NH), 8.88 (s, 1H, Ar), 8.73 (dd, J = 4.6 and
1.8 Hz, 1H, Ar), 8.07 (dd, J = 7.4 and 2.0 Hz, 1H, Ar), 7.27 (m, 1H,
Ar), 4.61 (t, J = 7.6 Hz, 2H, CH2), 4.26 and 3.95 (2m, 1H, CH), 1.84−
0.89 (m, 19H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ
162.16, 162.02, 152.09, 149.66, 140.96, 138.71, 123.47, 119.23, 115.11,
49.84, 45.97, 38.54, 34.21, 33.41, 32.21, 31.38, 30.43, 30.55, 29.90,
29.71, 24.78, 22.31, 21.85, 14.20. HRMS-ESI: m/z calcd for
C20H27N3O2 [M+H]+, 342.2182; found 342.2171.
N-(4-Methylcyclohexyl)-2-oxo-1-pentyl-1,2-dihydro-1,8-naph-
thyridine-3-carboxamide (2). Yield 85%; crystallized from diisopropyl
1
ether; MS m/z 355 (M+). H NMR (CDCl3): δ 10.05 and 9.69 (2m,
1H, NH), 8.85 (s, 1H, Ar), 8.71 (m, 1H, Ar), 8.06 (m, 1H, Ar), 7.27
(m, 1H, Ar), 4.58 (t, J = 7.3 Hz, 2H, CH2), 4.25 and 3.96 (2m, 1H,
CH), 1.84−0.92 (m, 21H, cyclohexyl + CH2 + CH3). 13C NMR
(CDCl3): δ 162.34, 162.01, 152.15, 149.80, 141.79, 138.65, 123.36,
119.15, 115.21, 49.63, 45.49, 38.93, 34.22, 33.08, 32.15, 31.87, 30.55,
30.48, 29.91, 29.66, 24.23, 23.61, 22.14, 21.38, 14.35. HRMS-ESI: m/z
calcd for C21H29N3O2 [M+H]+, 356.2338; found 356.2326.
H
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1-(2-Hydroxyethyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-
Ethyl 4-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyri-
1,8-naphthyridine-3-carboxamide (3). Purified by flash chromatog-
din-1(2H)-yl)butanoate (9). Purified by flash chromatography
raphy (hexane/ethyl acetate 2:3). Yield 55%; MS m/z 329 (M+). H
(hexane/ethyl acetate 1:1). Yield 28%; MS m/z 399 (M+). H NMR
1
1
NMR (DMSO): δ 10.00 and 9.62 (2m, 1H, NH), 8.88 (s, 1H, Ar),
8.78 (m, 1H, Ar), 8.07 (m, 1H, Ar), 7.44 (m, 1H, Ar), 4.62 (m, 2H,
CH2), 4.26 and 3.95 (2m, 1H, CH), 4.17 (exchangeable proton, 1H,
(CDCl3): δ 10.04 and 9.67 (2m, 1H, NH); 8.89 (s, 1H, Ar); 8.70 (dd,
J = 4.6 and 1.8 Hz, 1H, Ar); 8.10 (dd, J = 7.4 and 2.0 Hz, 1H, Ar); 7.26
(m, 1H, Ar); 4.69 (m, 2H, CH2); 4.15 (m, 2H, CH2); 4.28 and 3.95
(2m, 1H, CH); 2.45 (m, 2H, CH2); 2.15 (m, 2H, CH2); 1.84−0.89
(m, 15H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 173.59, 162.34,
162.21, 152.34, 149.56, 142.09, 138.77, 123.41, 119.21, 115.31, 61.43,
49.88, 45.82, 40.89, 34.22, 33.31, 32.42, 31.37, 31.43, 30.44, 29.87,
23.35, 22.13, 21.46, 15.63. HRMS-ESI: m/z calcd for C22H29N3O4 [M
+H]+, 400.2236; found 400.2224.
OH), 3.67 (m, 2H, CH2), 1.84−0.89 (m, 12H, cyclohexyl + CH3). 13
C
NMR (CDCl3): δ 162.45, 162.07, 152.25, 149.34, 141.89, 138.64,
123.23, 119.21, 115.22, 61.71, 49.55, 45.41, 41.72, 34.45, 33.72, 32.42,
31.73, 30.42, 29.96, 22.15, 21.38. HRMS-ESI: m/z calcd for
C18H23N3O3 [M+H]+, 330.1818; found 330.1807.
1-(3-Hydroxypropyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (4). Purified by flash chromatog-
raphy (hexane/ethyl acetate 1:1.5). Yield 51%; MS m/z 343 (M+). 1H
NMR (DMSO): δ 10.00 and 9.62 (2m, 1H, NH), 8.88 (s, 1H, Ar),
8.78 (m, 1H, Ar), 8.07 (m, 1H, Ar), 7.44 (m, 1H, Ar), 4.55 (m, 2H,
CH2), 4.26 and 3.95 (2m, 1H, CH), 4.19 (exchangeable proton, 1H,
OH), 3.52 (m, 2H, CH2), 1.84−0.89 (m, 14H, cyclohexyl + CH2 +
CH3). 13C NMR (CDCl3): δ 162.71, 162.12, 152.11, 149.34, 141.65,
138.64, 123.23, 119.21, 115.12, 62.07, 49.00, 45.91, 41.46, 34.15,
33.28, 32.12, 31.30, 30.63, 30.54, 29.44, 22.14, 21.26. HRMS-ESI: m/z
calcd for C19H25N3O3 [M+H]+, 344.1974; found 344.1963.
Methyl 5-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyr-
idin-1(2H)-yl)pentanoate (10). Purified by flash chromatography
1
(hexane/ethyl acetate 1:2). Yield 23%; MS m/z 399 (M+). H NMR
(CDCl3): δ 10.03 and 9.66 (2m, 1H, NH); 8.88 (s, 1H, Ar); 8.71 (dd,
J = 4.6 and 1.8 Hz, 1H, Ar); 8.09 (dd, J = 7.4 and 2.0 Hz, 1H, Ar); 7.27
(m, 1H, Ar); 4.63 (m, 2H, CH2); 4.28 and 3.95 (2m, 1H, CH); 3.68
(1s, 3H, CH3); 2.45 (m, 2H, CH2); 1.82−0.83 (m, 16H, cyclohexyl +
CH2 + CH3). 13C NMR (CDCl3): δ 173.61, 162.42, 162.23, 152.55,
149.36, 141.39, 138.87, 123.24, 119.21, 115.34, 55.82, 49.97, 45.69,
40.39, 34.27, 33.28, 33.19, 32.12, 31.43, 30.95, 30.47, 29.86, 27.41,
23.35, 22.31, 21.46. HRMS-ESI: m/z calcd for C22H29N3O4 [M+H]+,
400.2236; found 400.2223.
General Procedure for the Synthesis of the Acids 11−13. A
mixture of 0.21 mmol of ester derivatives 8−10 in 15.0 mL of NaOH
10% was heated at 110 °C for 5 h. After cooling, the reaction mixture
was treated with water and then with concentrated HCl until pH 2−3.
The precipitate formed was filtered and treated with Et2O to give the
acid derivatives 11−13.
3-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyridin-
1(2H)-yl)propanoic Acid (11). Yield 96%; MS m/z 357 (M+). 1H
NMR (DMSO): δ 12.55 (s, 1H, OH); 9.83 and 9.68 (2m, 1H, NH);
8.86 (s, 1H, Ar); 8.76 (m, 1H, Ar); 8.48 (m, 1H, Ar); 7.42 (m, 1H,
Ar); 4.68 (m, 2H, CH2); 4.25 and 3.97 (2m, 1H, CH); 2.90 (m, 2H,
CH2); 1.86−0.90 (m, 12H, cyclohexyl + CH3). 13C NMR (DMSO): δ
177.83, 161.48, 161.22, 150.42, 148.45, 142.11, 138.20, 122.44, 117.23,
113.13, 47.68, 44.97, 41.74, 37.80, 34.12, 33.31, 32.42, 31.63, 29.76,
29.25, 22.31, 21.36. HRMS-ESI: m/z calcd for C19H23N3O4 [M+H]+,
358.1767; found 358.1756.
4-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyridin-
1(2H)-yl)butanoic Acid (12). Yield 92%; MS m/z 371 (M+). 1H NMR
(DMSO): δ 12.06 (s, 1H, OH); 9.96 and 9.68 (2m, 1H, NH); 8.90 (s,
1H, Ar); 8.78 (m, 1H, Ar); 8.48 (m, 1H, Ar); 7.44 (m, 1H, Ar); 4.54
(m, 2H, CH2); 4.24 and 3.95 (2m, 1H, CH); 2.49 (m, 2H, CH2);
1.94−0.92 (m, 14H, cyclohexyl + CH2 + CH3). 13C NMR (DMSO): δ
177.88, 161.53, 161.36, 150.46, 148.55, 142.21, 138.22, 122.24, 117.28,
113.16, 47.72, 44.95, 43.12, 36.92, 34.15, 33.12, 32.34, 31.33, 29.65,
29.19, 23.79, 22.32, 21.34. HRMS-ESI: m/z calcd for C20H25N3O4 [M
+H]+, 372.1923; found 372.1912.
5-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyridin-
1(2H)-yl)pentanoic Acid (13). Yield 95%; MS m/z 385 (M+). 1H
NMR (DMSO): δ 12.05 (s, 1H, OH); 9.94 and 9.58 (2m, 1H, NH);
8.90 (s, 1H, Ar); 8.80 (dd, J = 4.6 and 1.8 Hz, 1H, Ar); 8.49 (dd, J =
7.4 and 2.0 Hz, 1H, Ar); 7.45 (m, 1H, Ar); 4.49 (m, 2H, CH2); 4.12
and 3.85 (2m, 1H, CH); 2.29 (m, 2H, CH2); 1.97−0.88 (m, 16H,
cyclohexyl +2CH2 + CH3). 13C NMR (DMSO): δ 177.89, 161.42,
161.21, 150.40, 148.68, 142.00, 138.00, 122.43, 117.23, 113.19, 47.68,
44.97, 41.74, 37.82, 34.35, 33.21, 32.20, 31.33, 29.89, 29.43, 27.85,
24.09, 22.31, 21.46. HRMS-ESI: m/z calcd for C21H27N3O4 [M+H]+,
386.2080; found 386.2068.
General Procedure for the Synthesis of Mesylates 28−30.
Triethylamine (0.82 mL,0.526 g, 5.89 mmol) was added at 0 °C to a
solution of suitable alcohol 5−7 (0.98 mmol) and methanesulfonyl
chloride (0.23 mL, 0.34 g, 2.94 mmol) in anhydrous dichloromethane
(8.0 mL), and the mixture was stirred for 6 h at ambient temperature.
After addition of ethyl acetate (30 mL), the mixture was washed with
water (20.0 mL) and then with brine (20.0 mL). The organic layer was
dried over MgSO4 and evaporated to dryness to give a residue which
was purified by flash column chromatography.
1-(4-Hydroxybutyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (5). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:2). Yield 63%; MS m/z 357 (M+). H
NMR (DMSO): δ 10.01 and 9.61 (2m, 1H, NH), 8.81 (s, 1H, Ar),
8.66 (dd, J = 4.4 and 1.7 Hz, 1H, Ar), 8.04 (dd, J = 7.2 and 2.0 Hz, 1H,
Ar), 7.24 (m, 1H, Ar), 4.55 (m, 2H, CH2), 4.28 and 3.89 (2m, 1H,
CH), 4.18 (exchangeable proton, 1H, OH), 3.73 (m, 2H, CH2), 1.84−
0.89 (m, 16H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ
162.54, 162.12, 151.97, 149.64, 141.87, 138.81, 123.09, 119.27, 115.12,
62.56, 49.12, 45.79, 41.14, 34.33, 33.51, 32.12, 31.11, 30.44, 29.43,
29.86, 25.75, 22.37, 21.22. HRMS-ESI: m/z calcd for C20H27N3O3 [M
+H]+, 358.2131; found 358.2120.
1-(5-Hydroxypentyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (6). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:2). Yield 85%; MS m/z 371 (M+). H
NMR (CDCl3): δ 10.03 and 9.64 (2m, 1H, NH); 8.87 (s, 1H, Ar);
8.72 (m, 1H, Ar); 8.08 (m, 1H, Ar); 7.28 (m, 1H, Ar); 4.61 (m, 2H,
CH2); 4.27 and 3.89 (2m, 1H, CH); 4.15 (exchangeable proton, 1H,
OH) 3.69 (m, 2H, CH2); 1.82−0.92 (m, 18H, cyclohexyl + CH2 +
CH3). 13C NMR (CDCl3): δ 162.21, 162.04, 152.2, 149.58, 142.06,
138.34, 123.05, 119.37, 115.22, 62.68, 49.35, 45.92, 40.89, 34.21,
33.12, 32.33, 31.13, 30.46, 29.92, 29.78, 27.79, 25.20, 22.26, 21.35.
HRMS-ESI: m/z calcd for C21H29N3O3 [M+H]+, 372.2287; found
372.2275.
1-(6-Hydroxyhexyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (7). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:2). Yield 71%; MS m/z 385 (M+). H
NMR (CDCl3): δ 10.00 and 9.65 (2m, 1H, NH), 8.89 (s, 1H, Ar),
8.74 (m, 1H, Ar), 8.09 (m, 1H, Ar), 7.29 (m, 1H, Ar), 4.62 (m, 2H,
CH2), 4.26 and 3.89 (2m, 1H, CH), 4.16 (exchangeable proton, 1H,
OH), 3.69 (m, 2H, CH2), 1.83−0.96 (m, 20H, cyclohexyl + CH2) +
CH3). 13C NMR (CDCl3): δ 162.32, 162.25, 152.00, 149.65, 141.48,
138.50, 123.44, 119.21, 115.10, 62.72, 49.40, 45.92, 40.90, 34.19,
33.12, 32.33, 31.43, 30.47, 29.95, 29.79, 27.68, 27.61, 25.49, 22.35,
21.48. HRMS-ESI: m/z calcd for C22H31N3O3 [M+H]+, 386.2444;
found 386.2430.
Ethyl 3-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyri-
din-1(2H)-yl)propanoate (8). Purified by flash chromatography
(toluene/ethyl acetate 1:2). Yield 33%; MS m/z 385 (M+). 1H
NMR (CDCl3): δ 10.01 and 9.62 (2m, 1H, NH); 8.90 (s, 1H, Ar);
8.71 (dd, J = 4.6 and 1.8 Hz, 1H, Ar); 8.08 (dd, J = 7.4 and 2.0 Hz, 1H,
Ar); 7.27 (m, 1H, Ar); 4.90 (m, 2H, CH2); 4.14 (m, 2H, CH2); 4.25
and 3.97 (2m, 1H, CH); 2.80 (m, 2H, CH2); 1.84−0.89 (m, 15H,
cyclohexyl + CH3). 13C NMR (CDCl3): δ 173.69, 162.34, 162.18,
152.28, 149.54, 141.78, 138.58, 123.37, 119.29, 115.39, 61.47, 49.37,
45.79, 40.95, 34.26, 33.36, 33.19, 32.52, 31.43, 30.85, 30.45, 29.78,
22.23, 21.16, 15.67. HRMS-ESI: m/z calcd for C21H27N3O4 [M+H]+,
386.2080; found 386.2066.
I
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4-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyridin-
29.90, 28.06, 26.97, 25.26, 22.72, 21.76. HRMS-ESI: m/z calcd for
C22H30FN3O2 [M+H]+, 388.2400; found 388.2387.
1(2H)-yl)butyl Methanesulfonate (28). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:1). Yield 86%; MS m/z 435 (M+). H
2-Amino-5-bromonicotinaldehyde (31). To a stirred solution of 2-
aminopyridine-3-carboxaldehyde (0.40 g, 3.30 mmol) in 15 mL of
glacial acetic acid was added bromine (0.16 mL, 3.16 mmol), and the
reaction mixture was stirred at room temperature for 24 h. The
precipitate obtained was filtered off and washed with ether. The filter
cake was poured into water and treated with solid NaOH until pH 7−
8, and the mixture was extracted with dichloromethane. The organic
layer was dried with MgSO4 and evaporated to dryness under reduced
pressure. The crude solid was purified by crystallization in acetonitrile
NMR (CDCl3): δ 9.98 and 9.63 (2m, 1H, NH); 8.83 (s, 1H, Ar); 8.67
(m, 1H, Ar); 8.08 (m, 1H, Ar); 7.26 (m, 1H, Ar); 4.61 (m, 2H, CH2);
4.30 (m, 2H, CH2); 4.28 and 3.89 (2m, 1H, CH); 3.01(s, 3H, CH3);
1.88−0.87 (m, 16H, cyclohexyl + CH2+ CH3). 13C NMR (CDCl3): δ
162.73, 161.96, 152.22, 149.78, 142.00, 138.69, 123.34, 119.35, 115.11,
69.94, 49.03, 45.83, 41.21, 37.60, 34.14, 33.21, 32.25, 31.36, 30.45,
29.88, 26.84, 24.29, 22.50, 21.87.
5-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyridin-
1
to give 31 (0.48 g, 73%): mp 147−150 °C; MS m/z 199 (M+). H
1(2H)-yl)pentyl Methanesulfonate (29). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:1). Yield 43%; MS m/z 449 (M+). H
NMR (DMSO): δ 9.83 (s, 1H, CHO), 8.33 (s, 1H, Ar); 8.27 (s,1H,
Ar); 5.27 (br, 2H, NH2).
NMR (CDCl3): δ 9.99 and 9.61 (2m, 1H, NH); 8.86 (s, 1H, Ar); 8.70
(m, 1H, Ar); 8.07 (m, 1H, Ar); 7.26 (m, 1H, Ar); 4.57 (m, 2H, CH2);
4.29 (m, 2H, CH2); 4.18 and 3.89 (2m, 1H, CH); 3.00(s, 3H, CH3);
1.97−0.89 (m, 18H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ
162.73, 162.05, 152.20, 149.78, 142.00, 138.62, 123.41, 119.24, 115.13,
70.05, 49.05, 45.90, 41.71, 37.64, 34.18, 33.25, 32.27, 31.32, 30.48,
29.86, 29.04, 27.44, 23.29, 22.51, 21.79.
Ethyl 6-Bromo-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxy-
late (32). To a solution of 31 (1.50 g, 7.5 mmol) in ethanol (20
mL) were added diethyl malonate (1.80 g, 11.25 mmol) and 0.21 mL
of piperidine (0.182 g, 2.14 mmol), and the mixture was stirred under
reflux for 20 h. After cooling, the solid obtained was filtered, washed
with ethanol, and dried. The crude product was used without further
1
purifications (1.99 g, 90%): mp 200−203 °C; MS m/z 296 (M+). H
6-(3-(4-Methylcyclohexylcarbamoyl)-2-oxo-1,8-naphthyridin-
1(2H)-yl)hexyl Methanesulfonate (30). Purified by flash chromatog-
NMR (DMSO): δ 12.64 (br, 1H, NH); 8.69 (d, J = 2.2 Hz, 1H, Ar);
8.56 (d, J = 2.2 Hz, 1H, Ar); 8.45 (s, 1H, Ar); 4.28 (q, J = 7.3 Hz, 2H,
CH2); 1.31 (t, J = 7.1 Hz, 3H, CH3).
1
raphy (hexane/ethyl acetate 1:1). Yield 37%; MS m/z 463 (M+). H
NMR (CDCl3): δ 9.96 and 9.63 (2m, 1H, NH); 8.82 (s, 1H, Ar); 8.65
(m, 1H, Ar); 8.01 (m, 1H, Ar); 7.20 (m, 1H, Ar); 4.50 (m, 2H, CH2);
4.28 (m, 2H, CH2); 4.17 and 3.89 (2m, 1H, CH); 2.96 (s, 3H, CH3);
1.99−0.76 (m, 20H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ
162.65, 162.07, 152.13, 149.82, 141.80, 138.53, 123.41, 119.12, 115.09,
70.21, 49.02, 45.92, 41.88, 37.64, 34.18, 33.21, 32.49, 31.25, 30.45,
29.81, 29.29, 27.84, 26.68, 25.42, 22.49, 21.72.
6-Bromo-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naph-
thyridine-3-carboxamide (33). A mixture of 4 (0.40 g, 1.35 mmol)
and 4-methylcyclohexylamine (0.76 g, 6.75 mmol) was heated in a
sealed tube at 150 °C for 24 h. After cooling, the reaction mixture was
treated with diethyl ether to give a solid residue which was collected by
filtration. The product was crystallized from ethyl acetate (0.32 g,
65%): MS m/z 363 (M+). 1H NMR (DMSO): δ 12.09 (br, 1H, NH);
10.06 and 9.57 (2m, 1H, NH); 8.73−8.83 (m, 3H, Ar); 4.12 and 3.78
(2m, 1H, CH); 1.90−0.89 (m, 12H, cyclohexyl + CH3).
General Procedure for the Preparation of Fluorine Deriva-
tives 14−16. To a solution of suitable mesylate 28−30 (0.6 mmol) in
anhydrous THF (10 mL) was added tetrabutylammonium fluoride
(1.00 N) in THF (0.35 mL, 0.314 g, 1.2 mmol), and the mixture was
refluxed for 4 h. After cooling, the solvent was removed under vacuum,
and the residue was solubilized in chloroform (10 mL), washed with
water (20 mL), and then washed with brine (20 mL). The organic
layer was dried over MgSO4 and evaporated to dryness to give crude
product which was purified by flash column chromatography.
1-(4-Fluorobutyl)-1,2-dihydro-N-(4-methylcyclohexyl)-2-oxo-1,8-
naphthyridine-3-carboxamide (14). Purified by flash chromatog-
raphy (hexane/ethyl acetate 1.5:1). Yield 56%; MS m/z 359 (M+). 1H
NMR (CDCl3): δ 10.01 and 9.62 (2m, 1H, NH); 8.88 (s, 1H, Ar);
8.71 (m, 1H, Ar); 8.08 (m, 1H, Ar); 7.28 (m, 1H, Ar); 4.66 (m, 2H,
CH2); 4.42 (m, 2H, CH2); 4.26 and 3.93 (2m, 1H, CH); 1.92−0.91
(m, 16H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ 162.76,
162.06, 152.15, 149.91, 141.89, 138.59, 123.47, 119.19, 115.13, 85.61,
82.33, 49.07, 45.94, 41.59, 34.20, 33.27, 32.29, 31.34, 30.48, 29.88,
28.44, 24.23, 22.52, 21.78. HRMS-ESI: m/z calcd for C20H26FN3O2
[M+H]+, 360.2087; found 360.2076.
General Procedure for the Synthesis of 6-Bromo Derivatives
17 and 18. A solution of 6-bromo-N-(4-methylcyclo-hexyl)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide 33 (1.42 g, 5.0 mmol) in
anhydrous DMF (20 mL) was treated with cesium carbonate (0.43 g,
14.0 mmol) at room temperature for 1 h. The suitable chloride (10
mmol) was added, and the mixture was stirred for 12 h at 50 °C. After
cooling, the reaction mixture was evaporated in vacuo, yielding the
crude products which were purified by flash chromatography.
6-Bromo-1-(4-fluorobenzyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-di-
hydro-1,8-naphthyridine-3-carboxamide (17). Purified by flash
chromatography (toluene/ethyl acetate 14:1 and 1% of acetic acid).
1
Yield 71%. H NMR (CDCl3): δ 9.92 and 9.52 (2m, 1H, NH); 8.75
(m, 2H, Ar); 8.20 (s, 1H, Ar); 7.52 (m, 2H, Ar); 7.03 (m, 2H, Ar);
5.73 (s, 2H, CH2), 4.26 and 3.95 (2m, 1H, CH); 1.80−0.91(m, 12H,
cyclohexyl + CH3). 13C NMR (CDCl3): δ 164.39, 162.75, 161.82,
152.57, 151.49, 142.35, 140.92, 138.96, 129.12, 124.75, 116.33, 115.28,
114.16, 49.47, 46.16, 44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86,
22.56, 21.80. HRMS-ESI: m/z calcd for C23H23BrFN3O2 [M+H]+,
472.1036; found 472.1027.
1-(5-Fluoropentyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carboxamide (15). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:1). Yield 49%; MS m/z 373 (M+). H
trans-6-Bromo-1-(4-fluorobenzyl)-N-(4-methylcyclohexyl)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (17-trans) and cis-6-
Bromo-1-(4-fluorobenzyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihy-
dro-1,8-naphthyridine-3-carboxamide (17-cis). Compounds 17-
trans and 17-cis were obtained from derivative 17 by flash
chromatography on a silica gel (toluene/ethyl acetate 14:1 and 1%
of acetic acid).
NMR (CDCl3): δ 10.03 and 9.64 (2m, 1H, NH); 8.88 (s, 1H, Ar);
8.71 (m, 1H, Ar); 8.09 (m, 1H, Ar); 7.26 (m, 1H, Ar); 4.59 (m, 2H,
CH2); 4.37 (m, 2H, CH2); 4.28 and 3.95 (2m, 1H, CH); 1.82−0.92
(m, 18H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ 162.77,
162.11, 152.15, 149.95, 141.84, 138.59, 123.42, 119.17, 115.15, 85.82,
82.55, 49.73, 45.96, 41.92, 34.21, 33.27, 32.30, 31.24, 30.62, 30.44,
29.88, 27.76, 24.23, 22.72, 21.76. HRMS-ESI: m/z calcd for
C21H28FN3O2 [M+H]+, 374.2244; found 374.2232.
1
17-trans. Yield 17%; MS 471 m/z (M+); mp 163−165 °C. H
NMR (CDCl3): δ 9.52 (m, 1H, NH); 8.75 (m, 2H, Ar); 8.20 (s, 1H,
Ar); 7.45 (m, 2H, Ar); 7.02 (m, 2H, Ar); 5.72 (s, 2H, CH2), 3.95 (m,
1H, CH); 1.79−0.91 (m, 12H, cyclohexyl + CH3). 13C NMR
(CDCl3): δ 164.39, 162.75, 161.82, 152.57, 151.49, 142.35, 140.92,
138.96, 129.12, 124.75, 116.33, 115.28, 114.16, 46.16, 44.88, 33.25,
31.28, 29.86, 21.80.
1-(6-Fluorohexyl)-N-(4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide (16). Purified by flash chromatog-
1
raphy (hexane/ethyl acetate 1:1). Yield 47%; MS m/z 387 (M+). H
NMR (CDCl3): δ 10.03 and 9.65 (2m, 1H, NH); 8.87 (s, 1H, Ar);
8.71 (m, 1H, Ar); 8.08 (m, 1H, Ar); 7.28 (m, 1H, Ar); 4.59 (m, 2H,
CH2); 4.34 (m, 2H, CH2); 4.27 and 3.93 (2m, 1H, CH); 1.81−0.91
(m, 20H, cyclohexyl + CH2 + CH3). 13C NMR (CDCl3): δ 162.77,
162.15, 152.15, 149.95, 141.90, 138.55, 123.42, 119.10, 115.11, 85.93,
82.65, 49.05, 45.968 42.02, 34.21, 33.27, 32.30, 31.24, 30.78, 30.48,
1
17-cis. Yield 8%; MS 471 m/z (M+); mp 158−160 °C. H NMR
(CDCl3): δ 9.92 (m, 1H, NH); 8.75 (m, 2H, Ar); 8.20 (s, 1H, Ar);
7.45 (m, 2H, Ar); 7.02 (m, 2H, Ar); 5.72 (s, 2H, CH2), 4.26 (m, 1H,
CH); 1.79−0.93 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ
J
dx.doi.org/10.1021/jm500807e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
164.39, 162.75, 161.82, 152.57, 151.49, 142.35, 140.92, 138.96, 129.12,
124.75, 116.33, 115.28, 114.16, 49.47, 44.88, 34.21, 32.20, 30.54,
22.56.
1-(4-Fluorobenzyl)-6-(4-fluorophenyl)-N-(4-methyl-cyclohexyl)-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (21). Purified by
flash chromatography (toluene/ethyl acetate 9:1). Yield 94%; MS 487
1
m/z (M+). H NMR (CDCl3): δ 10.01 and 9.59 (2m, 1H, NH); 8.91
6-Bromo-N-(4-methylcyclohexyl)-1-(2-morpholinoethyl)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (18). Yield 92%
(crystallized by acetonitrile); MS 476 m/z (M+). 1H NMR
(CDCl3): δ 9.97 and 9.57 (2m, 1H, NH); 8.75 (m, 2H, Ar); 8.19
(s, 1H, Ar); 4.73 (t, J = 6.8 Hz, 2H, CH2), 4.24 and 3.96 (2m, 1H,
CH); 3.51 (m, 4H, morpholine), 2.57 (m, 6H, morpholine + CH2);
1.87−0.95 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 162.45,
161.52, 152.43, 148.38, 141.11, 139.93, 124.45, 116.31, 114.26, 67.24,
56.12, 54.12, 49.13, 45.89, 39.30, 34.16, 33.21, 32.25, 31.34, 30.41,
29.90, 22.52, 21.91. HRMS-ESI: m/z calcd for C22H29BrN4O3 [M
+H]+, 477.1501; found 477.1491.
General Procedure for the Synthesis of 6-Substituted
Derivatives 19−26. A mixture of 50 mg of Ph3P (0.20 mmol) and
10 mg of Pd(OAc)2 (0.04 mmol) in dioxane (1.0 mL) was stirred
under N2 for 10 min. Then the approriate 6-bromo derivative 17 or 18
(0.41 mmol) in 1 mL of MeOH, 0.85 mL of Na2CO3 (10%), and
suitable boronic acid (0.82 mmol) were added. The mixture was
heated by microwave radiation (CEM) at 150 °C for 10 min (power
200 W, pressure 100 psi, stirring on). The reaction mixture was then
cooled to room temperature, treated with water, and extracted with
dichloromethane. The combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, and evaporated to dryness
to obtain a residue which was purified by flash chromatography on
silica gel.
(m, 2H, Ar); 8.18 (s, 1H, Ar); 7.56 (m, 3H, Ar); 7.21 (m, 2H, Ar);
6.97 (m, 2H, Ar); 5.81 (s, 2H, CH2), 4.26 and 3.92 (2m, 1H, CH);
1.82−0.92 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 164.82,
162.72, 162.14, 161.87, 150.70, 149.24, 142.55, 140.65, 139.67, 136.26,
132.55, 130.42, 129.16, 124.27, 116.88, 115.69, 115.29, 49.47, 46.16,
44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56, 21.80. HRMS-
ESI: m/z calcd for C29H27F2N3O2 [M+H]+, 488.2150; found
488.2137.
1-(4-Fluorobenzyl)-6-(furan-2-yl)-N-(4-methylcyclohexyl)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (22). Purified by flash
chromatography (toluene/ethyl acetate 8:1). Yield 78%. 1H NMR
(CDCl3): δ 9.99 and 9.60 (2m, 1H, NH); 8.98 (m, 2H, Ar); 8.29 (s,
1H, Ar); 7.49 (m, 3H, Ar); 6.99 (m, 2H, Ar); 6.80 (d, J = 3.2 Hz, 1H,
Ar); 6.56 (m, 1H, Ar); 5.79 (s, 2H, CH2), 4.27 and 3.93 (2m, 1H,
CH); 1.83−0.91 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ
164.39, 162.75, 162.12, 154.33, 150.90, 142.47, 142.09, 140.56, 134.66,
133.26, 128.86, 124.55, 122.8, 116.33, 115.28, 109.35, 107.89, 49.40,
46.26, 44.88, 34.31, 33.28, 32.00, 31.78, 30.24, 29.67, 22.50, 21.76.
HRMS-ESI: m/z calcd for C27H26FN3O3 [M+H]+, 460.2036; found
460.2023.
trans-1-(4-Fluorobenzyl)-6-(furan-2-yl)-N-(4-methylcyclohexyl)-
2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (22-trans)
and cis-1-(4-Fluorobenzyl)-6-(furan-2-yl)-N-(4-methylcyclohexyl)-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (22-cis). Com-
pounds 22-trans and 22-cis were obtained from derivative 22 by flash
chromatography on a silica gel (toluene/ethyl acetate 8:1).
1-(4-Fluorobenzyl)-6-(4-methoxyphenyl)-N-(4-methylcyclohexyl)-
2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (19). Purified
by flash chromatography (hexane/ethyl acetate 2:1). Yield 41%; MS
1
m/z 499 (M+). H NMR (DMSO): δ 10.02 and 9.61 (2m, 1H, NH);
1
22-trans. Yield 33%; MS 459 m/z (M+); mp 175−178 °C. H
8.92 (m, 2H, Ar); 8.18 (s, 1H, Ar); 7.55 (m, 4H, Ar); 7.05 (m, 4H,
Ar); 5.82 (s, 2H, CH2), 3.95 and 4.26 (2m, 1H, CH); 3.88 (s, 3H,
CH3); 1.60−0.92 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ
164.39, 162.75, 162.12, 161.09, 150.99, 142.35, 140.87, 134.66, 133.80,
129.12, 128.89, 128.13, 125.76, 124.75, 116.33, 115.28, 115.07, 55.78,
49.47, 46.16, 44.88, 34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56,
21.80. HRMS-ESI: m/z calcd for C30H30FN3O3 [M+H]+, 500.2349;
found 500.2337.
NMR (CDCl3): δ 9.60 (m, 1H, NH); 8.98 (m, 2H, Ar); 8.29 (s, 1H,
Ar); 7.49 (m, 3H, Ar); 6.99 (m, 2H, Ar); 6.80 (d, J = 3.2 Hz, 1H, Ar);
6.56 (m, 1H, Ar); 5.79 (s, 2H, CH2), 3.93 (m, 1H, CH); 1.83−0.91
(m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 164.39, 162.75,
162.12, 154.33, 150.90, 142.47, 142.09, 140.56, 134.66, 133.26, 128.86,
124.55, 122.8, 116.33, 115.28, 109.35, 107.89, 46.26, 44.88, 33.28,
31.78, 29.67, 21.76.
1
22-cis. Yield 27%; MS 459 m/z (M+); mp 168−170 °C. H NMR
1-(4-Fluorobenzyl)-N-(4-methylcyclohexyl)-2-oxo-6-(thiophen-2-
yl)-1,2-dihydro-1,8-naphthyridine-3-carboxamide (20). Purified by
flash chromatography (toluene/ethyl acetate 8:1). Yield 68%. 1H
NMR (CDCl3): δ 9.98 and 9.47 (2m, 1H, NH); 8.95 (m, 2H, Ar);
8.21 (s, 1H, Ar); 7.51 (m, 3H, Ar); 7.19 (m, 2H, Ar); 6.99 (m, 2H,
Ar); 5.80 (s, 2H, CH2), 4.26 and 3.94 (2m, 1H, CH); 1.82−0.95 (m,
12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 164.39, 162.75,
162.12, 150.90, 142.47, 140.56, 138.85, 134.66, 133.26, 128.86, 127.92,
127.63, 125.56, 124.55, 122.8, 116.33, 115.28, 49.47, 46.16, 44.88,
34.21, 33.25, 32.20, 31.28, 30.54, 29.86, 22.56, 21.80. HRMS-ESI: m/z
calcd for C27H26FN3O2S [M+H]+, 476.1808; found 476.1797.
trans-1-(4-Fluorobenzyl)-N-(4-methylcyclohexyl)-2-oxo-6-(thio-
phen-2-yl)-1,2-dihydro-1,8-naphthyridine-3-carboxamide (20-
trans) and cis-1-(4-Fluorobenzyl)-N-(4-methylcyclohexyl)-2-oxo-6-
(thiophen-2-yl)- 1,2-dihydro-1,8-naphthyridine-3-carboxamide (20-
cis). Compounds 20-trans and 20-cis were obtained from derivative 20
by flash chromatography on a silica gel (toluene/ethyl acetate 8:1).
(CDCl3): δ 9.99 (m, 1H, NH); 8.98 (m, 2H, Ar); 8.29 (m, 1H, Ar);
7.49 (m, 3H, Ar); 6.99 (m, 2H, Ar); 6.80 (d, J = 3.2 Hz, 1H, Ar); 6.56
(m, 1H, Ar); 5.79 (s, 2H, CH2), 4.27 (m, 1H, CH); 1.83−0.91 (m,
12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 164.39, 162.75,
162.12, 154.33, 150.90, 142.47, 142.09, 140.56, 134.66, 133.26, 128.86,
124.55, 122.8, 116.33, 115.28, 109.35, 107.89, 49.40, 44.88, 34.31,
32.00, 30.24, 22.50.
6-(4-Methoxyphenyl)-N-(4-methylcyclohexyl)-1-(2-morpholi-
noethyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (23).
Purified by flash chromatography (hexane/ethyl acetate 2:1). Yield
1
48%; MS 504 m/z (M+). H NMR (CDCl3): δ 10.04 and 9.63 (2m,
1H, NH); 8.90 (m, 2H, Ar); 8.18 (s, 1H, Ar); 7.56 (m, 2H, Ar); 7.06
(m, 2H, Ar); 4.77 (t, J = 6.7 Hz, 2H, CH2), 4.22 and 3.96 (2m, 1H,
CH); 3.89 (s, 3H, CH3); 3.68 (m, 4H, morpholine); 2.73 (m, 6H,
morpholine + CH2); 1.95−0.87 (m, 12H, cyclohexyl + CH3). 13C
NMR (CDCl3): δ 162.45, 161.87, 161.06, 152.45, 148.89, 139.93,
128.72, 128.57, 125.98, 124.45, 116.31, 115.36, 114.81, 67.44, 56.34,
55. 91, 54.32, 49.63, 45.82, 39.60, 34.16, 33.12, 32.25, 31.34, 30.41,
29.90, 22.43, 21.78. HRMS-ESI: m/z calcd for C29H36N4O4 [M+H]+,
505.2815; found 505.2801.
1
20-trans. Yield 24%; MS 475 m/z (M+); mp 169−171 °C. H
NMR (CDCl3): δ 9.47 (m, 1H, NH); 8.97 (m, 2H, Ar); 8.22 (s, 1H,
Ar); 7.46 (m, 3H, Ar); 7.19 (m, 2H, Ar); 6.96 (m, 2H, Ar); 5.79 (s,
2H, CH2), 3.97 (m, 1H, CH); 1.80−0.92 (m, 12H, cyclohexyl + CH3).
13C NMR (CDCl3): δ 164.39, 162.75, 162.12, 150.90, 142.47, 140.56,
138.85, 134.66, 133.26, 128.86, 127.92, 127.63, 125.56, 124.55, 122.8,
116.33, 115.28, 55.78, 46.16, 44.88, 33.25, 31.28, 29.86, 21.80.
N-(4-Methylcyclohexyl)-1-(2-morpholinoethyl)-2-oxo-6-(thio-
phen-2-yl)-1,2-dihydro-1,8-naphthyridine-3-carboxamide (24). Pu-
rified by flash chromatography (hexane/ethyl acetate 2:1). Yield 78%;
1
MS 480 m/z (M+). H NMR (CDCl3): δ 10.02 and 9.61 (2m, 1H,
1
20-cis. Yield 21%; MS 475 m/z (M+); mp 163−165 °C. H NMR
NH); 8.94 (m, 2H, Ar); 8.20 (s, 1H, Ar); 7.41 (d, J = 4.4 Hz, 1H, Ar);
7.27−7.14 (m, 2H, Ar); 4.80 (t, J = 6.6 Hz, 2H, CH2), 4.28 and 3.97
(2m, 1H, CH); 3.69 (m, 4H, morpholine); 2.74 (m, 6H, morpholine +
CH2); 1.82−0.88 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ
162.45, 161.87, 152.45, 148.89, 139.93, 138.31, 127.92, 127.65, 125.53,
122.68, 124.45, 116.31, 115.36, 67.44, 56.34, 54.32, 49.63, 45.82,
(CDCl3): δ 9.47 (m, 1H, NH); 8.97 (m, 2H, Ar); 8.22 (s, 1H, Ar);
7.46 (m, 3H, Ar); 7.19 (m, 2H, Ar); 6.96 (m, 2H, Ar); 5.79 (s, 2H,
CH2), 3.97 (m, 1H, CH); 1.80−0.92 (m, 12H, cyclohexyl + CH3). 13
C
NMR (CDCl3): δ 164.39, 162.75, 162.12, 150.90, 142.47, 140.56,
138.85, 134.66, 133.26, 128.86, 127.92, 127.63, 125.56, 124.55, 122.8,
116.33, 115.28, 55.78, 49.47, 44.88, 34.21, 32.20, 30.54, 22.56.
K
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39.60, 34.16, 33.12, 32.25, 31.34, 30.41, 29.90, 22.63, 21.80. HRMS-
ESI: m/z calcd for C26H32N4O3S [M+H]+, 481.2273; found 481.2270.
6-(4-Fluorophenyl)-N-(4-methylcyclohexyl)-1-(2-morpholinoeth-
yl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (25). Puri-
fied by flash chromatography (hexane/ethyl acetate 1:1). Yield 53%;
dissociation constant of the radioligand−receptor complex. Data are
the mean SEM of at least n = 3 experiments.36
Cell Line. U2OS cells (osteosarcoma cell line) permanently
expressing h-CB2R and βarr2-GFP (green fluorescent protein) were
obtained from Drs. Larry Barak and Marc Caron (Duke University).
They were maintained at 37 °C in a humidified atmosphere containing
5% CO2 in Dulbecco’s modified Eagle medium nutrient mixture F-12
HAM, supplemented with 10% fetal bovine serum, 0.6% zeocin, and
400 mg/mL of G418. Cells were then harvested using trypsin-EDTA
(Gibco catalog number 25300-054), and viable cells were assessed
using trypan blue dye exclusion.
1
MS 492 m/z (M+). H NMR (CDCl3): δ 10.00 and 9.62 (2m, 1H,
NH); 8.87 (m, 2H, Ar); 8.17 (s, 1H, Ar); 7.57 (m, 2H, Ar); 7.20 (m,
2H, Ar); 4.79 (t, J = 6.6 Hz, 2H, CH2), 4.27 and 3.96 (2m, 1H, CH);
3.68 (m, 4H, morpholine); 2.68 (m, 6H, morpholine + CH2); 1.80−
0.89 (m, 12H, cyclohexyl + CH3). 13C NMR (CDCl3): δ 164.55,
162.24, 161.68, 152.35, 148.92, 139.88, 132.21, 129.32, 125.98, 124.65,
116.31, 116.13, 115.30, 67.48, 56.65, 54.42, 49.33, 45.75, 39.45, 34.09,
33.22, 32.64, 31.54, 30.22, 29.89, 22.64, 21.80. HRMS-ESI: m/z calcd
for C28H33FN4O3 [M+H]+, 493.2615; found 493.2601.
β-Arrestin Assay. The assays were performed using a procedure
described previously.22 U2OS cells permanently expressing h-CB2R
and βarr2-GFP were detached using trypsin-EDTA, seeded onto glass
coverslips at 80−85% confluence, and placed in 24-well plates (BD
Falcon). After incubation at 37 °C (5% CO2, 95% relative humidity)
overnight, cells were washed with Hanks’s balanced salt solution
(HBSS) before drug application. Test compounds and reference
cannabinoid compounds were dissolved in DMSO, and dilutions were
made in HBSS. In order to detect agonist-stimulated redistribution of
βarr2-GFP, the cells were stimulated with various concentrations drug
at room temperature for 40 min. Then the suspension was removed,
paraformaldehyde (4% in HBSS, p/v) was added, and the incubation
continued at room temperature for 25 min. Finally the cells were
washed with PBS three times and once with double-distilled water.
The antagonism protocol included 15 min of pre-incubation with the
antagonist, followed by a 40 min co-incubation of antagonist and
agonist (30 nM WIN-55,212-2). Glass coverslips were mounted onto
slides and imaged using a fluorescence microscope (Nikon E1000;
Tokyo, Japan) using a 40× oil objective and 488 nm excitation for
GFP. The redistribution of diffuse β-arrestin-GFP from the cytoplasm
to agonist- or antagonist-occupied receptor-containing pits or vesicles
was imaged using a fluorescence microscope (Nikon E1000; using a
40× oil objective and 488 nm excitation for GFP, Tokyo, Japan). The
RGB color images captured from the fluorescent microscope were
transformed into 8-bit gray-scale images using the Automate-Batch
function in Adobe Photoshop CS5. To quantify βarr2-GFP aggregates,
gray-scale images were processed through ImageJ software (http://
rsbweb.nih.gov/ij/), using a custom-written plug-in provided by
Pingwei Zhao (Temple University). Curves were fit by nonlinear
regression using the sigmoidal dose−response equation in GraphPad
Prism Version 5.0 (GraphPad, San Diego, CA). Activity values were
normalized to the agonist’s response (30 nM WIN-55,212-2 was
considered as 100%).
6-(Furan-2-yl)-N-(4-methylcyclohexyl)-1-(2-morpholinoethyl)-2-
oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide (26). Purified by
flash chromatography (hexane/ethyl acetate 1:1). Yield 79%; MS 464
1
m/z (M+). H NMR (CDCl3): δ 10.04 and 9.64 (2m, 1H, NH); 8.99
(m, 2H, Ar); 8.28 (s, 1H, Ar); 7.57 (d, J = 3.2 Hz, 1H, Ar); 6.80 (d, J =
3.2 Hz, 1H, Ar); 6.56 (m, 1H, Ar); 4.77 (t, J = 6.7 Hz, 2H, CH2), 4.22
and 3.96 (2m, 1H, CH); 3.69 (m, 4H, morpholine); 2.77 (m, 6H,
morpholine + CH2); 1.82−0.92 (m, 12H, cyclohexyl + CH3). 13C
NMR (CDCl3): δ 162.31, 162.00, 152.67, 148.920, 142.77, 139.91,
124.46, 122.87, 116.33, 115.31, 109.67, 107.54, 67.68, 56.33, 54.21,
49.21, 45.65, 39.59, 34.15, 33.18, 32.57, 31.32, 30.49, 29.99, 22.63,
21.82. HRMS-ESI: m/z calcd for C26H32N4O4 [M+H]+, 465.2502;
found 465.2490.
CB1 and CB2 Receptor Binding Assays. The new compounds
were evaluated in CB1R and CB2R binding assays using membranes
from HEK-293 cells transfected with cDNAs encoding the human
recombinant CB1R (Bmax = 2.5 pmol/mg protein) and human
recombinant CB2R (Bmax = 4.7 pmol/mg protein) (Perkin-Elmer,
Italy). These membranes were incubated with [3H]-(−)-cis-3-[2-
hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)-
cyclohexanol ([3H]CP-55,940) (0.14 nM/Kd = 0.18 nM and 0.084
nM/Kd = 0.31 nM for CB1R and CB2R, respectively) as high-affinity
ligand33 and displaced with 100 nM (R)-(+)-[2,3-dihydro-5-methyl-3-
(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naph-
thalenylmethanone (WIN-55,212-2)34 as heterologous competitor for
nonspecific binding (Ki = 9.2 and 2.1 nM, respectively, for CB1R and
CB2R). All compounds were tested following the procedure described
by the cell membrane manufacturer.35
CB1R binding protocol involves the use of the same solution buffer
used for both incubation and washing reaction (Tris-HCl, 50 mM;
EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM
for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10
μM), and finally 8 μg/sample membrane in a total volume of 200 μL.
CB2R binding assays were carried out with two different buffers:
incubation buffer (Tris-HCl, 50 mM; EGTA, 2.5 mM; MgCl2, 5 mM;
BSA, 1 mg/mL at pH 7.4) and washing buffer (Tris-HCl, 50 mM;
EGTA, 2.5 mM; MgCl2, 5 mM; BSA, 2% at pH 7.4). The assay
mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test
substances (concentrations from 0.001 to 10 μM), and 4 μg/sample
membrane in a total assay volume of 600 μL. Assay tubes were
prepared in duplicate and incubated for 90 min at 30 °C. The reaction
was terminated by addition of ice-cold buffer followed by rapid
filtration under vacuum through Whatman GF/C filters (pretreated
for 2 h with 0.05% aqueous polyethyleneimine) using a 12-well
harvester from Millipore. After washing, radioactivity associated with
the filters was counted on a liquid scintillation analyzer (Tri-Carb 2100
TR, Perkin-Elmer). Specific binding was determined by subtracting
nonspecific binding from total binding in the absence of competing
ligand. The percentage displacement of specific binding was calculated
for the amount of radiolabel bound in the presence of unlabeled
displacing ligand. Displacement IC50 values were determined by linear
regression analysis of log concentration−percent displacement data
using GraphPad Prism. Ki values were calculated by applying the
Cheng−Prusoff equation, Ki = IC50/(1 + L/KD), where L is the
concentration of the radioligand, IC50 is the concentration of drug
causing 50% inhibition of specific radioligand binding, and KD is the
cAMP Assay. These assays were performed using LANCE Ultra
cAMP kit (catalog number TRF0262; Perkin-Elmer Inc., Boston, MA)
according to the manufacturer’s protocol.
U2OS cells expressing the hCB2R were detached using trypsin-
EDTA, washed with HBSS, and counted, and cell viability was
determined using Trypan Blue stain. Cells were resuspended in
stimulation buffer (HBSS, 1X; BSA stabilizer, 0.1%; IBMX, 0.5 mM;
HEPES, 5 mM; pH 7.4) at a concentration of 600 cells/μL. In order to
detect the agonist-induced reduction in cAMP levels, 5 μL of the cell
suspension (3000 cells/well) was stimulated with forskolin (10 μM
final concentration) and with various concentrations of test ligands in
white Optiplate-384 wells at room temperature for 30 min. Functional
antagonism of the cannabinoid CB2R antagonist response was
measured by incubating the suspension cells with drug dilutions,
forskolin (10 μM final concentration), and the reference agonist WIN-
55,212-2 (30 nM final concentration) at room temperature for 30 min.
After the incubation, 5 μL of europium chelated labeled cAMP tracer
solution in detection buffer and then 5 μL of the cAMP-specific
monoclonal antibodies (labeled with ULight-dye) solution in detection
buffer were added to the wells. The reaction was allowed to incubate
for 1 h at room temperature in the dark. Time-resolved fluorescence
signals were detected on an EnVision multiplate reader (Perkin-Elmer,
CA,USA) at 615 and 665 nm emission. The amounts of cAMP
produced in the stimulated cells were determined according to the
cAMP standard curves. Antagonism in the cAMP assay has been
expressed as percent of inhibition of WIN-55,212-2. Inhibition curves
L
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were analyzed by nonlinear regression using GraphPad Prism Version
5.0 software (GraphPad, San Diego, CA), and data were fitted to
sigmoidal concentration−response curves to obtain IC50 values. Also
for agonist, the sigmoidal dose−response equation was used to
determine EC50 values. In this case, the logarithmic value of agonist
concentrations is plotted against the TR-FRET signal normalized to
the response of forskolin.
Ballesteros−Weinstein Nomenclature. Here, the Ballesteros−
Weinstein numbering system for GPCR amino acid rersidues is used.
In this numbering system, the label 0.50 is assigned to the most highly
conserved Class A residue in each transmembrane helix (TMH).37
This is preceded by the TMH number. In this system, for example, the
most highly conserved residue in TMH6 is P6.50. The residue
immediately before this would be labeled 6.49, and the residue
immediately after this would be labeled 6.51. When referring to a
specific CB2 residue, the Ballesteros−Weinstein name is followed by
the absolute sequence number given in parentheses (e.g.,
K3.28(109)); however, when referring to a highly conserved residue
among Class A GPCRs (and not a specific residue in CB2), only the
Ballesteros−Weinstein name is given.
Modeling Methods. Conformational Search. The structures of
ligands were built in Spartan’08 (Wave function, Inc., Irvine, CA).
Initial conformational analyses of these compounds were performed
using the semiempirical method AM1 encoded in Spartan’08.
Conformational searches were performed (using 3−8-fold rotations)
for each rotatable bond. All unique conformers identified were then
optimized with ab initio Hartree−Fock calculations at the 6-31G*
level, except for 17 and 18, which required 6-311G* to accommodate
a bromine atom. To calculate the difference in energy between the
global minimum energy conformer of each compound and its final
docked conformation, rotatable bonds in the global minimum energy
conformer were driven to their corresponding value in the final docked
conformation, and the single-point energy of the resultant structure
was calculated at the HF 6-31G* level, or for 17 and 18 at the 6-
311G* level.
change was the development of additional helical turns in the IC-3
(TMH5-TMH6) loop after the original end of TMH5.31
CB2R Activated State Model. The CB2R activated state model
(R*) used here for docking studies was produced from the inactive
state model described above via a multimicrosecond-long molecular
dynamics simulation of the interaction of the endogenous CB2R
ligand, 2-AG, with CB2R in a palmitoyl-oleoyl-phosphatidylcholine
(POPC) bilayer. In these simulations, 2-AG entered the binding
pocket via the lipid bilayer between TMH6 and TMH7 and activated
the CB2R. Ligand entry resulted in changes on the intracellular end of
the receptor. Here the R3.55(136)/D6.30(240) ionic lock was broken
as TMH6 straightened and moved its IC end away from the TMH
bundle. Ligand entry also resulted in changes in the binding pocket, as
toggle switch residue W6.48(258) underwent a χ1 torsion angle change
from g+ to trans. This change was transitory, with W6.48(258)
reverting to a g+ χ1. The R* model used for docking studies here was
taken from the section of the trajectory in which the W6.48(258) χ1
was trans.31
Ligand/CB2R Complexes. The inactive state model was used to
dock compounds 17, 18, and 23, while A1, A2, 5, and 14 were docked
in the activated state model described above. In addition, to probe the
origins of the antagonism vs agonism of 23 and A1, each compound
was docked in our CB2R inactive state model. The automatic docking
program Glide v5.8 (Schrodinger Inc., Portland, OR) was used to
explore possible binding conformations or receptor site interactions
with flexible docking.31,32 Because S7.39(285) has been shown to be a
ligand interaction site in CB2R,37 S7.39(285) was defined as a required
interaction during the initial flexible docking procedure. Glide was
used to generate a grid based on the centroid of the ligand in the
binding site. Any hydrophobic region defined in the grid generation
that contacted the ligand was selected as important to the flexible
docking procedure. The box for flexible docking was defined to be 26
Å in the x, y, and z dimensions. Extra precision (XP) was selected with
scaling of VdW radii and flexible docking invoked.34 These Glide
docking studies consistently identified an interaction with K3.28(109).
A second Glide docking study was initiated in which K3.28(109) and
S7.39(285) were defined as required interactions during the flexible
docking procedure. Extra precision (XP) was selected and flexible
docking invoked. This second run resulted in improved Glide scores,
particularly for ligands with higher CB2R binding affinities.
For each receptor−ligand complex, the complex with the best Glide
score was minimized using the OPLS2005 all-atom force field in
Macromodel 9.9 (Schrodinger Inc.). An 8.0 Å nonbonded cutoff
(updated every 10 steps), a 20.0 Å electrostatic cutoff, and a 4.0 Å
hydrogen bond cutoff were used in each stage of the calculation. The
first stage consisted of 3500 steps of Polak−Ribier conjugate gradient
minimization using a distance-dependent dielectric function with a
base constant of 2. No harmonic constraints were placed on the side
chains, but 100 kJ/mol torsional constraints were applied to hold all
the backbone ϕ/ψ torsion angles. During the second stage of 500
steps, all torsional constraints were released. To relax the loops, an
additional 1000-step Polak−Ribier conjugate gradient minimization of
the loop regions was performed. The loop and termini regions were
left free, while the transmembrane regions were not allowed to move
during this final minimization. An 8.0 Å extended nonbonded cutoff
(updated every 10 steps), 20.0 Å electrostatic cutoff, and 4.0 Å
hydrogen bond cutoff were used in this calculation, and the
generalized Born/surface area (GB/SA) continuum solvation model
for water available in Macromodel was employed.
Model Development. Complete details on the generation of the
inactive and activated state CB2R models used here are available in our
previous publication.31 We provide a synopsis below.
CB2R Inactive State Model. The crystal structure of the Class A
GPCR, rhodopsin in the dark state was used as the template for the
creation of our CB2R inactive state model.26 This template was chosen
because no mutations or modifications were made to its structure for
crystallization. In addition, the cannabinoid receptors and rhodopsin
share some unusual sequence motifs. These receptors share a TMH4
GWNC motif at their extracellular ends. Here a TRP forms an
aromatic stacking interaction with Y5.39, influencing the EC positions
of TMH3-4-5. The initial homology model was refined by calculating
the low free energy conformations for any TMH with an important
sequence divergence from rhodopsin and replacing the corresponding
helix from the initial model with one that more accurately reflects the
sequence dictated TMH geometries in CB2R. This includes TMH2
(GG helix distorting motif in Rho vs no PRO or GG in CB2R) and
TMH5 (PRO at 5.50 in Rho vs no PRO at 5.50 in CB2R). The
resultant CB2R model has been tested using results from substituted
cysteine accessibility studies to identify binding pocket facing
residues,38,39 from mutation studies of key ligand interactions
sites,38−41 and from covalent labeling studies of CB2R42 that support
a lipid entry pathway for CB2R ligands.
To permit adjustment to a lipid bilayer environment, the resultant
model was pre-equilibrated in a stearoyl-docosahexaenoylphosphati-
dylcholine (SDPC) bilayer for 300 ns.31 While the toggle switch
residue, W6.48(258), remained in its inactive state g+ χ1 dihedral angle
after the equilibration in SDPC, some notable changes did occur
during this equilibration. The R3.50(131) and D6.30(240) salt bridge
at the intracellular ends of TMHs3/6 (analogous to the R3.50(135)/
E6.30(247) salt bridge in the dark state of rhodopsin) rearranged
quickly to form a salt bridge between R3.55(136) and D6.30(240),
with Y3.51(132) supporting the salt bridge by hydrogen bonding to
the exposed backbone carbonyl of L6.29(239). A second notable
ASSOCIATED CONTENT
* Supporting Information
■
S
Table T1, dihedral definitions of the global minima energy
conformers of 17, 18, 23, A1, A2, 14, and 5; Table T2,
energetic cost of docked ligand conformations; Tables T3 and
T4, hydrogen bond parameters between each compound and
K3.28(109) and S7.39(285), respectively; Table T5, ligand−
receptor aromatic stacking interactions; Figure S1, drawings of
the 1,8-naphthyridine scaffolds; Figures S2 and S3, global
M
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Journal of Medicinal Chemistry
Article
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minima for antagonists 17, 18, and 23 and for agonists A1, A2,
5, and 14, respectively; discussion of ligand/receptor
complexes, including detailed description of the binding pocket
interactions; Figures S4 and S5, models of the antagonists/
inverse agonists 17, 18, and 23 docked in CB2R (inactive) and
of the agonists A1, A2, 14, and 5 docked in CB2R* (active),
respectively; Figure S6, spatial relationship between compound
17 and the toggle switch residue W6.48(258). This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*P.H.R.: phone, (336) 334-5333; e-mail, phreggio@uncg.edu.
*C.M.: phone, +39(0)502219548; e-mail, clementina.manera@
farm.unipi.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH grants RO1 DA003934 and
KO5 DA021359 (PHR), R01 DA023204 and P30 DA013429,
and by National Interest Research Projects (PRIN 2010-2011,
Grant 20105YY2HL_008).
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