Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as potent and selective CB2 receptor ligands: The structural elements controlling binding affinity, selectivity and functionality

Article abstract of DOI:10.1016/j.ejmech.2016.07.012

CB2-based therapeutics show strong potential in the treatment of diverse diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this reason, research activities are currently directed towards the development of CB2 selective ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported. A set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity. Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (K_i= 7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity (～70 and ～200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series presented the most potent and selective CB2 ligand in this series (K_i= 2.15 nM and CB2 subtype selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2 receptor activity modulators.

Full text of DOI:10.1016/j.ejmech.2016.07.012

Accepted Manuscript
Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as
potent and selective CB2 receptor ligands: The structural elements controlling binding
affinity, selectivity and functionality
Noha A. Osman, Alessia Ligresti, Christian D. Klein, Marco Allarà, Alessandro
Rabbito, Vincenzo Di Marzo, Khaled A. Abouzid, Ashraf H. Abadi
PII:
S0223-5234(16)30559-1
10.1016/j.ejmech.2016.07.012
EJMECH 8730
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 May 2016
Revised Date: 5 July 2016
Accepted Date: 7 July 2016
Please cite this article as: N.A. Osman, A. Ligresti, C.D. Klein, M. Allarà, A. Rabbito, V. Di Marzo, K.A.
Abouzid, A.H. Abadi, Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as
potent and selective CB2 receptor ligands: The structural elements controlling binding affinity, selectivity
and functionality, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.012.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
The molecular surface of 6b
CB2 selective agonist
hCB2 Ki = 2.15 nM,
The molecular surface of 19b
CB2 inverse agonist
hCB2 Ki = 6.15 nM,
hCB1 Ki /hCB2 Ki SI 195
hCB1 Ki /hCB2 Ki SI 469

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Discovery of Novel Tetrahydrobenzo[b]thiophene and Pyrrole
Based Scaffolds as Potent and Selective CB2 Receptor Ligands:
The Structural Elements Controlling Binding Affinity,
Selectivity and Functionality.
Noha A. Osmana, Alessia Ligrestic, Christian D. Kleinb, Marco Allaràc, Alessandro Rabbitoc,
Vincenzo Di Marzoc, Khaled A. Abouzid*d, Ashraf H. Abadia
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt.
bMedicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg
University , Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
cEndocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale
delle Ricerche, Pozzuoli, Italy.
dPharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo
11566, Egypt.
*To whom correspondences should be addressed: email: khaled.abouzid@pharma.asu.edu.eg;
Tel: +202 224051150; Fax: +202 2405110
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ABSTRACT: CB2-based therapeutics show strong potential in the treatment of diverse
diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis
and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this
reason, research activities are currently directed towards the development of CB2 selective
ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported.
A
set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-
sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-
tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed
compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity.
Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (Ki =
7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity ( 70 and
200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series
presented the most potent and selective CB2 ligand in this series (Ki = 2.15 nM and CB2 subtype
selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while
compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The
present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds
with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2
receptor activity modulators.
KEYWORDS: CB2 selective ligands; CB2 agonist; CB2 inverse agonist; cannabinoids
ABBREVIATIONS
CB1: Cannabinoid receptor 1; CB2: Cannabinoid receptor 2;; DIPEA: N,N-
Diisopropylethylamine; EDC: 1-Ethyl-3-(3’-dimethylamino)carbodiimide HCl salt; HATU: 1-
[Bis(dimethylamino)methylene]-1H-1,2,3
triazolo[4,5-b]pyridinium
3-oxid
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hexafluorophosphate;
HBTU:
O-(1H-benzotriazol-1-yl)-N,N,N’,N’
tetramethyluronium
hexafluorophosphate; HEK-293: Human embryonic kidney 293 cells; Hz: Hertz; IBD:
Inflammatory Bowel Disease; IC50: Half maximal (50%) inhibitory concentration; Ki: Inhibition
constant; m.p.: Melting point; M.wt: Molecular weight; nM: Nanomolar; SI: Selectivity index;
TLC: thin layer chromatography
INTRODUCTION
Cannabinoid receptors, endogenous cannabinoid receptor ligands “endocannabinoids”, and
enzymes catalyzing their formation and degradation collectively constitute the endocannabinoid
system. The potential modulation of this ubiquitous system for therapeutic gain has become a
central focus of research during the last decade. Two cannabinoid receptor subtypes have been
identified, to date: CB1 and CB2. Both are G protein-coupled receptors which have variable
tissue distribution. Although the CB1 receptor is present in various peripheral tissues, its highest
expression is in the CNS where it mediates the psychotropic effects of ∆9-THC. Such
psychotropic effects include euphoria, drowsiness, memory lapses, disruption of motor skills,
lack of concentration and disorientation1-5. Conversely, the CB2 subtype is predominantly, but
not exclusively, expressed in the periphery, primarily in cells of the immune system. It has also
been found to be expressed in osteoclasts, and osteoblasts, as well as in various tumors and the
tumor cell microenviroment6. Interestingly, in the case of colorectal and endometrial carcinoma,
the CB2 receptor is over-expressed and its levels correlate with tumor malignancy7, 8. This
cellular distribution has increased the popularity of CB2 receptors for their immunomodulatory9,
anti-inflammatory10, analgesic11, bone remodeling12 and anti-tumor effects13. In addition, studies
have demonstrated that CB2 receptors are overexpressed in chronically activated microglial
cells, which are thought to play an important role in neurodegenerative disorders14. Hence,
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targeting this receptor also holds promise for the treatment of neuro-inflammatory disorders such
as dementia, multiple sclerosis and Alzheimer’s15, 16
.
We are thus left with the conclusion that the CB2 receptor represents an attractive therapeutic
target for the treatment of many conditions with important unmet medical needs. More
importantly, because this receptor is significantly found outside the brain, compounds selective
for the CB2 receptor do not exhibit the same psychotropic side effects that have plagued CB1
receptor-based therapeutics.5 This has, consequently, prompted the development of several
chemical classes of CB2 receptor selective ligands.
Over the past few years, a diverse number of CB2 ligands have been developed, either as
agonists, partial agonists, or antagonists/inverse agonists (Figure 1). Among the well-known CB2
agonists are the classical cannabinoids (CC); (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-
tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (1, JWH-133) in this class has shown to
suppress colitis in several experimental models of IBD in rodents. {4-[4-(1,1-dimethylheptyl)-
2,6-dimethoxy-phenyl]-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl}-methanol (2, HU-308) is a
bicyclic CC analogue that exhibits a 400 fold higher selectivity for the CB2 receptor subtype
over CB1.17 The aminoalkylindoles (AAI) is also one of the most extensively studied classes
represented by R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4
benzoxazin-6-yl]-1-naphthalenylmethanonemesylate (3, R-(+)-WIN55212) which exhibits a
slightly higher affinity towards CB2 versus CB1.18 Second-generation CB2 agonists based on
AAI structure-activity relationship (SAR) studies is exemplified by novel compounds such as (1-
(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl) methanone (4, A-
796260). It has been found to display analgesic activity in inflammatory, osteoarthritic,
neuropathic and postoperative rodent pain models.19 With respect to selective CB2 antagonists/
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inverse agonists, fewer classes have been reported. Among these are the diarylpyrazole
carboxamides represented by the first to be discovered and one of the most potent members: 5-
(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,4R,6S)-1,5,5-trimethyl-6-
bicyclo[2.2.1]-heptanyl]pyrazole-3-carboxamide (5, SR144528).20 More recently, other series
have been disclosed which include the quinolone amide derivatives. Compound 6 is a
representative of this series endowed with a high affinity (Ki = 0.6 nM and selectivity (>16,666-
fold) for the CB2 receptor over CB1.21 Also, the 1,8-Naphthyridin-2(1H)-one-3-carboxamide
series has been recently identified to act as potent and selective CB2 ligands, where it has been
shown that their functional activity is controlled by the presence of the substituents at certain
positions of the naphthyridine scaffold. Thus, it is pretty clear that the functional activity can be
modulated by changing the nature of substituents around the heterocyclic scaffold.22
In the light of these findings and as an extension of our research project aimed at the
identification of novel CB2-selective chemotypes, we decided to design and synthesise a novel
series of pyrrole-3-carboxamide derivatives, introducing structural modifications, which have
been previously reported to increase CB2 receptor subtype affinity and selectivity (Figure 2).23
Moreover, it was noted that both 1,3-thiazole- and indole-based chemical scaffolds presented
potent and selective CB2 ligands. What is common among these scaffolds is the presence of
either a single or fused heterocycle with an amide substituent at the heterocyclic 2-position
(Figure 2)23. Such observations encouraged us to embark an isosterism approach and to
synthesize another novel series of amide derivatives in which a thiophene or a
tetrahydrobenzo[b]thiophene ring represents their heterocyclic cores. A thiophene ring replaced
the 1,3-thiazole ring while a tetrahydrobenzo[b]thiophene ring was chosen to replace the indole
ring. It is worth to note that, in the latter case, the disruption of planarity was due to the fusion of
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the heterocycle with a cyclohexane ring in the tetrahydrobenzo[b]thiophene scaffold, versus an
aromatic benzene ring in the original indole scaffold. Additionally, in order to obtain greater
chemical diversity and based on recent findings that indicated the presence of sulphonamide
functionalities in promising CB2 ligands24-26, we also synthesized a series of thiophene and
tetrahydrobenzo[b]thiophene-based scaffolds that displayed a substituted sulphonamide in place
of the classical amide functionality. The general structures of the novel chemical scaffolds are
shown in Figure 2.
RESULTS AND DISCUSSION
Chemistry
Synthesis
of
2-(Acylamino/Sulphonylamino)-thiophene
derivatives
and
2-
(Acylamino/Sulphonylamino)-tetrahydrobenzo[b]thiophene derivatives was afforded via a two-
step synthetic route, outlined in Scheme 1. The first step involved the synthesis of the 2-
aminothiophene intermediates 2a,b adapting the famous one –pot Gewald reaction. This multi-
component reaction involves 3 components: aldehydes or ketones, α-activated acetonitriles and
sulphur in the presence of a base such as morpholine or diethylamine. Reaction takes place in
solvents like methanol, ethanol or DMF, usually at 50-60 °C, in two subsequent steps –
Knoevenagel-Cope condensation and intramolecular ring closure of formed sulfanyl substituted
α,β-unsaturated nitrile. The reaction generally produces polysubstituted 2-aminothiophenes in
yields varying between 35-90%.27, 28
Thus, condensation of phenylacetaldehyde or cyclohexanone with ethyl cyanoacetate in the
presence of sulfur and a base such as morpholine or diethyl amine yielded the ethyl 2-
aminothiophene-3-carboxylate derivatives 2a and 2b, respectively. Several variations of the
reaction were tried in order to optimise reaction yield and shorten reaction time. In an attempt to
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prepare compound 2a, the respective reactants were all added in one pot using ethanol as a
solvent and left to stir overnight at room temperature. TLC monitoring showed that no new
product was formed, even after it was left to stir over another night. The same reaction was
repeated by overnight heating at 60-65°C in an oil bath. TLC monitoring revealed that reaction
completion occurred after 24 h. By pouring the reaction mixture onto ice-water, a significant
amount of precipitate was noticed, producing the target compound in 90% yield. To further
optimise reaction conditions, the microwave-assisted Gewald synthesis was attempted. Here, the
multi-reactants vial was submitted to microwave irradiation for 30 min at 77 °C (Pmax = 80 W),
while monitoring reaction by TLC. It was obvious that nearly all reactants disappeared after 20
minutes of microwave irradiation. Reaction workup was done in the same way but this time
giving the target products in 96% yield. Therefore, this shows that the microwave-accelerated
method provides a straightforward and a very efficient preparation of the target compounds in
high yields.
Secondly, the 2-aminothiophene derivatives (2a,b) were then reacted with different carboxylic
acids/acyl chlorides and sulphonyl chlorides yielding carboxamides (3a,b-6b) and
sulphonamides (7a,b-12a), respectively. Carboxamide derivatives were produced either by
coupling with carboxylic acids using a coupling agent (HATU, HBTU or EDCI/HOBt in the
presence of DIPEA) or by direct reaction with acyl chlorides in the presence of TEA. Generally,
reaction yields did not exceed 30% using the former method (irrespective of the coupling agent
type). In contrast to this, the latter method gave yields that reached up to 90%. The reason for
this is that the 2-amino group is of poor nucleophilicity due to resonance with the aromatic
thiophene system and thus needed the more reactive acyl chlorides for the reaction to happen in a
good yield. Sulphonamides (7a,b-11b) were produced by reaction with sulphonyl chlorides in
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the presence pyridine, while stirring at room temperature overnight. In a few cases, the N,N-
disulphonylamino product (12a) was isolated instead of the monosulphonylamino product. The
increased acidity of the monosulphonylamino derivative promoted further deprotonation of the
sulphonamide and subsequent formation of the N,N-disulphonylamino product.
Synthesis of the desired 2,5-Dialkyl-1-phenyl-1H-pyrrole-3-carboxamide derivatives was
carried using a four-step procedure that is depicted in Scheme 2. Ethylacetoacetate or
ethylpropionylacetate (β-keto-ester) was first alkylated with chloroacetone (haloketone) by
refluxing in dry acetone in the presence of potassium carbonate and potassium fluoride. Reaction
was monitored by TLC and required refluxing for 48-72 h to yield the corresponding 1,4-
diketoesters (14a,b). The intermediate compounds 14a,b were then allowed to undergo Paal-
Knorr reaction, by refluxing with aniline in acetic acid for ≈ 5 h, to afford the corresponding
ethyl 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxylates (15a,b) in an average yield of 55-65%.
Successful reaction was evident from 1H-NMR spectra of 15a and 15b. 1H-NMR of 15a showed
a singlet peak at 2.27 ppm that corresponds to the methyl protons at the pyrrole-2-position. On
1
the other hand, H-NMR of 15b showed a quartet signal at 2.70 ppm, J = 7.5 Hz and a triplet
signal at 0.97 ppm, J = 7.5 Hz, corresponding to the methylene and methyl protons of the ethyl
group at the pyrrole-2-position. Ester hydrolysis of 15a,b was accomplished, in a nearly 100%
yield, through refluxing with 10% aqueous NaOH, followed by neutralization with 10% HCl to
afford the corresponding carboxylic acids (16a,b). Coupling 16a,b with the appropriate amines
in the presence of HBTU and DIPEA, while stirring in DMF at room temperature, successfully
afforded the corresponding amide derivatives (17a,b-22a,b) in an average yield of 80%.
In Vitro CB1 and CB2 receptor binding affinity and structure–activity relationships. All
newly synthesized compounds were evaluated in radioligand binding assays for their ability to
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displace [3H]-CP-55,940 (a high affinity radioligand; 0.14 nM / Kd = 0.18 nM and 0.084nM / Kd
= 0.31 nM, respectively for CB1 and CB2 receptor) from human recombinant CB1 and CB2
receptors. Preliminary screening assays for hit discovery were run at 1 and 10 µM. Compounds
showing greater than 50% displacement at the screening doses were tested in a dose response
curve to determine their IC50 values. Ki values were calculated by applying the Cheng-Prusoff
equation to the IC50 values. Selectivity indices (Ki CB1/Ki CB2) were also calculated. The
binding affinities are reported in Table 1.
Regarding Scheme 1 compounds, binding data showed that most compounds with a 5-
phenyl thiophene scaffold (4a, 7a-10a) did not show any binding affinity to the CB2 receptor. In
fact, only compound 3a, with a 1-naphthylamide substituent at position 2, displayed a Ki on CB2
= 59.81 nM and ˃ 167-fold selectivity for CB2 over CB1. It was also evident from the binding
data (Table 1) that replacing the 5-phenyl-thiophene substituent with a tetramethylene chain
linking the 4- and 5- positions of the thiophene ring yielded the tetrahydrobenzo[b]thiophene
derivatives (3b-11b), which was accompanied in most cases (3b-6b, 8b and 10b) with a
noticeable increase in the CB2 receptor affinity, irrespective of the functional group at position 2.
To explore the effect of having variable functionalities at position 2 of the thiophene/
tetrahydrobenzo[b]thiophene scaffold, both amide and sulphonamide derivatives were
synthesized. It was clear that having a sulphonamide functionality at position 2, generally,
provided compounds with a lower (8b and 10b) or a nearly abolished affinity to the CB2
receptor (7b, 9b, 11b, 7a-10a and 12a). Comparing 8b and 10b, we can find that Ki values
dropped from 1993.94 nM to 800.00 nM when the 1-phenylsulphonamide substituent in 8b was
replaced by the bulkier 1-naphthylsulphonamide substituent in 10b.
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For compounds having an amide functionality at position 2 (3b-6b and 3a), a marked
enhancement in the CB2 receptor affinity and selectivity was noted. Interestingly, compound 6b
was the most potent and selective compound of the current series, showing a remarkably high
affinity at the CB2 receptor (Ki = 2.15 nM) and a CB2 receptor subtype selectivity that is almost
500-fold over CB1. Such an affinity is seemingly owed to the 1-admantyl substituent of the
amide functionality. Pharmacomodulations of the substituent on the carboxamide function was
also carried out. Replacing the 1-adamantyl group with a 1-naphthyl (aromatic) group yielded
compound, 3b, the second most potent and selective compound of the current class (Ki = 16.80
nM and 80-fold selectivity for CB2 over CB1. Substituting the 1-naphthyl group with its
positional isomer, 2-naphthyl (4b), led to a 100-fold lower CB2 affinity, which reflects that the
1-naphthyl moiety is more favourable compared to its 2-naphthyl isomer. Substitution with other
lipophilic groups such as 2-phenylethyl (5b) in the same position led to relatively lower CB2
binding affinity (Ki = 111.87 nM), however displaying a CB2 selectivity profile that is almost
identical to that of 3b. It can be, therefore, concluded that the type of the heteroayl nucleus
together with its substitution pattern, type of functionality at the 2-position and the substituents
attached to such functionalities are all crucial modulators of the CB2 receptor biniding affinity
and selectivity profiles.
Regarding the second set of compounds synthesized, Scheme 2 was carried out to afford the
2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamide derivatives. Two series of these derivatives
were synthesized that were characterized by either having a 2-methyl pyrrole substituent,
represented as “series a” or a 2-ethyl pyrrole substituent represented as “series b”. It is worth to
note that all compounds in both series showed quite impressive CB2 receptor binding affinities,
all being in the nanomolar range. Some CB2 subtype selectivities reached almost as high as 200
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fold over CB1. Biological results in Table 1 revealed that a bulky, lipophilic 1-adamantly amide
gives a very potent CB2 ligand when either an ethyl (19b) or a methyl group (19a) was used at
the 2-position of the pyrrole ring. In particular, compound 19b (Ki = 6.15 nM, CB2 selectivity of
almost 200-fold over CB1) showed a slightly higher CB2 receptor affinity and a much higher
CB2 receptor subtype selectivity, when compared to 19a (Ki = 7.59 nM, 68-fold selectivity for
CB2 over CB1). In an effort to test the effect of less bulkier aliphatic amides, cyclohexyl amide
analogues (22a and 22b) were prepared. As evident from Table 1, reduced CB2 receptor affinity
and selectivity were observed. In an attempt to explore the affinity of aromatic carboxamide
substituents, 2-naphthylamide derivatives were prepared; yielding compounds 18a and 18b with
a CB2 receptor affinity and selectivity that is higher when compared to their cyclohexyl
counterparts while lower than their 1-adamantyl counterparts. Replacing the 2-naphthyl
substituent of 18b (Ki = 57 nM) with its 1-naphthyl positional isomer (17b, Ki = 20.02 nM)
demonstrated an enhancement of the CB2 receptor affinity. However, an opposite effect was
observed in series a when 18a (Ki = 58.28 nM) was compared to 17a (Ki = 84.22 nM). In order
to further evaluate the importance of the hydrophobic character of the amide substituent, we also
synthesized 1-phenyl ethyl (21a and 21b) and 2-phenyl ethyl amide derivatives (20a and 20b). Ki
values revealed deterioration in the CB2 affinity of both derivatives when compared to their
naphthyl analogues. This effect was less marked with the 1-phenyl ethyl derivatives (21a, Ki =
546.44 nM and 21b, Ki = 540.10 nM) than their 2-phenylethyl counterparts (20a, Ki = 584.17 nM
and 20b, Ki = 718.20 nM). Therefore, we can say that increasing spacer length from 1- to 2-C
atoms detrimentally affects the CB2 receptor affinity.
In summary, it is evident that the combination of a 1-aryl substituent with small alkyl
substituents on positions 2 and 5 of the pyrrole-3-carboxamide nucleus led to very potent and
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selective CB2 ligands. Lipophilic, aliphatic and aromatic, carboxamide substituents both yielded
potent CB2 ligands. The best results in this series being demonstrated by compounds displaying
the 1-adamantyl susbtituent (19a and 19b) at the pyrrole-3-carboxamide position. In addition, the
abolished CB2 affinity exhibited by the pyrrole-3-carboxylic acid intermediates (16a and 16b)
(Ki ˃ 10000 nM) highlights the importance of the amide substitution at this position. It is also
worth noting that, in general, series b compounds displayed either a very similar or a higher CB2
binding affinity and selectivity compared to series a. However, exceptions to this were only
noted with the 2-phenyl ethyl amide (20a and 20b) and the cyclohexyl amide (22a and 22b)
derivatives. In the former, compound 20a gave a 1.2-fold higher CB2 receptor affinity than 20b
while in the latter, compound 22a showed more than double the CB2 receptor selectivity of 22b.
In vitro CB2 functional activity. Compounds 6b, 19a and 19b, taken as representatives of
relatively potent and selective CB2 ligands from Scheme 1 and 2 respectively, were subject to
further in vitro pharmacological evaluation. We performed the Hunter™ eXpress GPCR assay to
measure the modulation of intracellular cAMP levels induced by the water-soluble analog of
forskolin, NKH-477. Opposite to compound 6b, which showed a typical agonist behavior by
reducing the NKH-477 induced cAMP (IC50 of 474.9 nM); compound 19a and 19b showed a
similarity with the reference compound SR144528 (in terms of both potency and functionality),
as indicated by the stimulation of cAMP production over the NKH-477 stimulus (IC50 of 108.6
and 398.3 nM, respectively). With the aim of investigating whether these compounds were able
to antagonize CB2 receptor activation, we tested compounds also in the presence of an EC80
concentration of a selective CB2 agonist (JWH-133) as indicated in the Methods section. As
shown in Figure 3 (A-B) compounds 19a and 19b increased cAMP levels far beyond that
induced by NKH-477 both in absence and in presence of the EC80 ligand challenge suggesting
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that they act orthosterically as inverse agonists. Conversely, compound 6b reduced the cAMP
levels induced by the NKH-477 stimulus as expected for an orthosteric Gi agonist. Consistently,
this compound showed no activity in presence of the EC80 ligand challenge (Figure 3, C-D).
Thus, it is obvious that the functional activity of these ligands is dictated by the nature of their
heterocyclic cores as well as the substituents around these cores. Ligands with a pyrrole core that
has a pendant benzene ring at the 1-position were shown to be inverse agonists. It is interesting
to note that the latter ligands confer structural similarity to the well-known potent CB2 inverse
agonist/antagonist; SR14452820 (Figure 1). In the light of these findings, it can be deduced that
an N-containing 5-membered heteroaryl central scaffold with a 1-phenyl/benzyl substituent are
crucial elements that can be utilised to direct the functionality of the designed CB2 ligands
towards an inverse agonist/ antagonist activity. Conversely, replacing the pyrrole ring with its
isostere “thiophene”, fusing the latter with a cyclohexane ring while abolishing the pendant
phenyl substituent from the central heterocyclic scaffold has led to a full agonist functionality.
Taken together, the present study confirms that these novel chemotypes are attractive leads that
can be further optimised to produce selective CB2 ligands, acting either as full or inverse
agonists.
CONCLUSION
2-(acylamino)tetrahydrobenzo[b]thiophene and pyrrole-3-carboxamide series were designed
and synthesised as CB2 receptor ligands, tested in radioligand binding studies, and functionally
characterized in adenylate cyclase assays. Both heterocyclic-based scaffolds presented novel
chemical classes of potent and selective CB2 ligands, displaying Ki values in the nanomolar
range and CB2 selectivities reaching up to 500-fold over CB1. In both series, best results were
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demonstrated by compounds having the bulky 1-adamantyl substituent (6b, 19a and 19b)
attached to the heterocyclic-amide functionality. The latter two compounds behaved as potent
inverse agonists in functional assay, whereas the former one behaved as an agonist. Along with
these data, we also confirmed specific structural elements that lead to agonism/inverse agonism
activity. These findings pave the way to the design and/or optimisation of heterocyclic-based
scaffolds with lipophilic carboxamide and/or retroamide substituents that can be exploited as
potential CB2 receptor activity modulators and hence evaluating the therapeutic utility of such
modulators in various disease settings.
ACKNOWLEDGMENTS
The authors gratefully acknowledge DiscoveRx for its willingness to share their expertise and
particularly to Dr. Sumeer Dhar, Dr. Neil Charter and Dr. Daniel Bassoni for the helpful
discussions.
EXPERIMENTAL
Chemistry. All starting materials, reactants and solvents were obtained from Sigma–Aldrich and
were used without further purification. Melting points were determined on Buchi B-540 Melting
1
Point apparatus and are uncorrected. H NMR and 13C-NMR spectra were recorded on Varian
300 MHz spectrometer or 500 MHz spectrometer using CDCl3 as a solvent; chemical shifts (δ)
were reported in parts per million (ppm) downfield from TMS; multiplicities are abbreviated as:
s: singlet; bs: broad singlet; d: doublet; q: quartet; p: pintet; m: multiplet; dd: doublet of doublet;
ddd: doublet of doublet of doublet, dt: doublet of triplet and all coupling constants (J) are given
in Hz. HR-ESI mass spectra were recorded on Bruker micrOTOF-Q II instrument and were
acquired either in positive or in negative mode and data are reported as m/z. All masses were
reported either as (M+H)+, (M+Na)+ or (M+K)+ in case of positive mode or as (M-H)- in case of
14

ACCEPTED MANUSCRIPT
negative mode. Flash chromatography was performed using Biotage Isolera One purification
system using silica gel (0.06-0.2 mm) cartridges (KP-SIL) and UV monitoring at 254-280 nm.
Reaction progress was monitored by TLC performed on Merck silica gel plates 60 and detection
of the components was made by UV light (254 nm). Microwave irradiations were conducted
using a Monowave 300 synthesis reactor with IR temperature sensor from Anton Paar. All
reactions were carried out under nitrogen when inert atmosphere was needed.
General procedure for the synthesis of 2-Amino-4/5-substituted-thiophene-3-carboxylic
acid ethyl ester (2a,b)29. A mixture of the respective aldehyde or ketone (4 mmol),
ethylcyanoacetate (4 mmol), S8 (0.14 g, 4.4 mmol), and morpholine or diethylamine (5 mL) in
EtOH (7 mL) was put in a vial (G30 size) and submitted to microwave irradiation for 20 minutes
at 77 °C (Pmax = 80 W). After cooling, the solution was poured onto 50 mL ice water to yield a
precipitate which was filtered, washed with water and dried under vacuum. The solid was used
without further purification.
2-Amino-5-phenyl-thiophene-3-carboxylic acid ethyl ester (2a).30 Brown powder, 95%; MS
(HR-ESI) m/z calcd for C13H13NNaO2S (M+Na)+: 270.0559, found: 270.0562.
2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl ester (2b).30 Yellow
powder, 96%; MS (HR-ESI) m/z calcd for C11H15NNaO2S (M+Na)+: 248.0716, found: 248.0709.
General procedure for the synthesis of 2-(Acylamino)-4/5-substituted-thiophene-3-
carboxylic acid ethyl ester (3b, 6b). The appropriate acyl chloride (1.5 mmol) was slowly
added under a nitrogen atmosphere to a cooled (0 °C) solution of aminothiophene derivative 21a
or 21b (1 mmol) and TEA (1.5 mmol) in dry DCM (10 mL). After being stirred at room
temperature for 18 h, the solution was washed with 1 N HCl, saturated solution of NaHCO3 and
15

ACCEPTED MANUSCRIPT
brine, then dried over anhydrous MgSO4 and evaporated to dryness. The crude product was
purified by flash column chromatography using the reported eluent system.
2-[(Naphthalene-1-carbonyl)-amino]-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic
o
1
acid ethyl ester (3b). Yellow solid, 90%; m.p.: 126-128 C; H-NMR (300 MHz) δ 11.88 (s,
1H), 8.54 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.89 – 7.79 (m, 2H), 7.60 – 7.44 (m, 3H),
4.27 (q, J = 7.1 Hz, 2H), 2.79 – 2.76 (m, 2H), 2.69-2.66 (m, 2H), 1.86 – 1.71 (m, 4H), 1.31 (t, J
13
= 7.1 Hz, 3H); C-NMR (75 MHz) δ 166.61, 165.57, 147.58, 133.86, 131.99, 131.93, 131.09,
130.50, 128.38, 127.51, 127.09, 126.61, 126.02, 125.50, 124.77, 112.16, 60.52, 26.42, 24.43,
23.02, 22.88, 14.28; MS (HR-ESI) m/z calcd for C22H21NNaO3S (M+Na)+: 402.1134, found:
402.1132.
2-[(Adamantane-1-carbonyl)-amino]-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic
acid ethyl ester (6b). White solid, 91%; m.p.: 133-135 oC; 1H-NMR (300 MHz) δ 11.54 (s, 1H),
4.34 (q, J = 7.1 Hz, 2H), 2.78-2.75 (m, 2H), 2.65-2.67 (m, 2H), 2.10 (s, 3H), 1.98 (d, J = 2.7 Hz,
6H), 1.91 (dd, J = 15.8, 2.7 Hz, 1H), 1.76 (bs, 9H), 1.38 (t, J = 7.1 Hz, 3H); MS (HR-ESI) m/z
calcd for C22H29NNaO3S (M+Na)+: 410.1754, found: 410.1760.
General procedure for the synthesis of 2-(Acylamino)-4/5-substituted-thiophene-3-
carboxylic acid ethyl ester (3a, 4a-b, 5b). To a magnetically stirred solution of the respective
aminothiophene derivative (1.20 mmol) in anhydrous DMF (15 mL) were successively added
N,N-diisopropylethylamine (Hunig’s base) (3.60 mmol), HBTU (1.2 mmol) in case of 5b/ HATU
(1.2 mmol) in case of 4b/ EDCI and HOBt (1.2 mmol each) in case of 3a and 4a , and the acid
derivative (1 mmol). After stirring overnight, the resulting mixture was concentrated in vacuo.
The residue was dissolved in EtOAc, successively washed with aqueous NaHCO3 solution,
16

ACCEPTED MANUSCRIPT
water, and brine, dried over MgSO4, filtered, and concentrated to give a crude solid. This solid
was further purified by flash chromatography using the reported eluent system.
2-[(Naphthalene-1-carbonyl)-amino]-5-phenyl-thiophene-3-carboxylic acid ethyl ester (3a).
Orange solid, 30%; m.p.: 150-152 oC; 1H-NMR (300 MHz) δ 11.71 (s, 1H), 8.65 (d, J = 8.2 Hz,
1H), 8.05 (d, J = 8.2 Hz, 1H), 7.94 (dd, J = 7.3, 1.1 Hz, 2H), 7.70–7.75 (m, 5H), 7.52 (s, 1H),
7.46 – 7.38 (m, 2H), 7.35 – 7.29 (m, 1H), 4.38 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H); MS
(HR-ESI) m/z calcd for C24H19NNaO3S (M+Na)+: 424.0978, found: 424.0974.
2-[(Naphthalene-2-carbonyl)-amino]-5-phenyl-thiophene-3-carboxylic acid ethyl ester (4a).
o
1
Brown solid, 12%; m.p.: 218-220 C; H-NMR (500 MHz) δ 12.18 (s, 1H), 8.59 (s, 1H), 8.09 –
8.03 (m, 2H), 7.99 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.66 – 7.64 (m, 2H), 7.63 – 7.57
(m, 2H), 7.50 (s, 1H), 7.44 – 7.38 (m, 2H), 7.32 – 7.28 (m, 1H), 4.45 (q, J = 7.1 Hz, 2H), 1.47 (t,
J = 7.1 Hz, 3H); 13C-NMR (126 MHz) δ 165.99, 163.64, 148.58, 135.30, 134.03, 133.79, 132.67,
129.43, 129.19, 128.99, 128.97, 128.84, 128.38, 127.83, 127.47, 127.03, 125.51, 123.27, 119.34,
114.14, 61.00, 14.45; MS (HR-ESI) m/z calcd for C24H19NNaO3S (M+Na)+: 424.0978, found:
424.0962.
2-[(Naphthalene-2-carbonyl)-amino]-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic
o
1
acid ethyl ester (4b). Brown powder, 13%; m.p.: 174-176 C; H-NMR (300 MHz) δ 12.47 (s,
1H), 8.56 (s, 1H), 8.08 – 7.88 (m, 4H), 7.63-7.57 (m, 2H), 4.41 (q, J = 7.1 Hz, 2H), 2.89 – 2.79
(m, 2H), 2.74-2.69 (m, 2H), 1.87-1.79 (m, 4H), 1.43 (t, J = 7.1 Hz, 3H); MS (HR-ESI) m/z calcd
for C22H21NNaO3S (M+Na)+: 402.1134, found: 402.1127.
2-(3-Phenyl-propionylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl
1
ester (5b). Yellow resin, 15%; H-NMR (300 MHz) δ 11.27 (s, 1H), 7.33 – 7.16 (m, 5H), 4.30
17

ACCEPTED MANUSCRIPT
(q, J = 7.1 Hz, 2H), 3.11 – 3.02 (m, 2H), 2.82 – 2.70 (m, 4H), 2.69 – 2.59 (m, 2H), 1.82 – 1.74
(m, 4H), 1.36 (t, J = 7.1, 3H); MS (HR-ESI) m/z calcd for C20H23NNaO3S (M+Na)+: 380.1296,
found: 380.1291.
General procedure for the synthesis of 2-(Sulphonylamino)-4/5-substituted-thiophene-3-
carboxylic acid ethyl ester (7a,b-10a,b; 11b and 12a)31. The appropriate sulphonyl chloride
(1.5 mmol equiv) was slowly added to a 0 °C solution of thiophene-2-amine derivative (1 mmol)
in pyridine (10 mL). The reaction mixture was stirred at room temperature under inert
atmosphere overnight. After addition of EtOAc, the solution was washed with 1 N HCl and
brine. The organic phase was dried over MgSO4 and evaporated under vacuum. The residue was
purified by flash chromatography using the reported eluent system.
2-Methanesulphonylamino-5-phenyl-thiophene-3-carboxylic acid ethyl ester (7a). Brown
o
1
solid, 60%; m.p.: 155-157 C; H-NMR (300 MHz) δ 7.64 (s, 1H), 7.61 – 7.55 (m, 2H), 7.46 –
7.35 (m, 3H), 4.39 (q, J = 7.1 Hz, 2H), 3.52 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H); 13C-NMR (75MHz)
δ 161.50, 145.06, 133.81, 132.59, 129.31, 129.21, 126.15, 123.77, 61.57, 43.26, 14.43; MS (HR-
ESI) m/z calcd for C14H14NO4S2 (M-H)-: 324.0370, found: 324.0369.
2-Methanesulphonylamino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl
o
1
ester (7b). Yellow crystalline solid, 30%; m.p.: 74-76 C; H-NMR (300 MHz) δ 10.16 (s, 1H),
4.30 (q, J = 7.1 Hz, 2H), 3.05 (s, 3H), 2.77 – 2.68 (m, 2H), 2.64 – 2.55 (m, 2H), 1.83 – 1.69 (m,
4H), 1.35 (t, J = 7.1 Hz, 3H); MS (HR-ESI) m/z calcd for C12H17NNaO4S2 (M+Na)+: 326.0491,
found: 326.0493.
2-Benzenesulphonylamino-5-phenyl-thiophene-3-carboxylic acid ethyl ester (8a). Red sticky
powder, 22%; m.p.: 72-74 oC; 1H-NMR (500 MHz) δ 7.98 – 7.94 (m, 1H), 7.83 – 7.80 (m, 1H),
18

ACCEPTED MANUSCRIPT
7.62 – 7.46 (m, 4H), 7.42 – 7.27 (m, 4H), 7.24 (s, 1H), 4.29 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1
Hz, 3H); MS (ESI) m/z calcd for C19H16NO4S2 (M-H)-: 386.1, found: 386.0.
2-Benzenesulphonylamino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl
o
1
ester (8b). White solid, 48%; m.p.: 116-118 C; H-NMR (300 MHz) δ 10.48 (s, 1H), 7.96 –
7.89 (m, 2H), 7.62 – 7.53 (m, 1H), 7.53 – 7.45 (m, 2H), 4.25 (q, J = 7.1 Hz, 2H), 2.70 –2.56 (m,
4H), 1.83 – 1.66 (m, 4H), 1.32 (t, J = 7.1 Hz, 3H); MS (HR-ESI) m/z calcd for C17H19NNaO4S2
(M+Na)+: 388.0648, found: 388.0646.
2-(4-Chloro-benzenesulphonylamino)-5-phenyl-thiophene-3-carboxylic acid ethyl ester
(9a). Red powder, 22%; m.p.: 92-94 oC; 1H-NMR (500 MHz) δ 7.91 – 7.86 (m, 1H), 7.62 – 7.50
(m, 1H), 7.47 – 7.41 (m, 2H), 7.40 – 7.27 (m, 4H), 7.24 (s, 1H), 7.20 (t, J = 7.4 Hz, 1H) 4.30 (q,
J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H); MS (HR-ESI) m/z calcd for C19H15ClNO4S2 (M-H)-:
420.0137, found: 420.0143.
2-(4-Chloro-benzenesulphonylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic
o
acid ethyl ester (9b). Orange solid, 28%; m.p.: 95-97 C; 1H-NMR (500 MHz) δ 10.50 (s, 1H),
7.86 – 7.82 (m, 2H), 7.46 – 7.42 (m, 2H), 4.24 (q, J = 7.1 Hz, 2H), 2.68 – 2.63 (m, 2H), 2.61 –
2.56 (m, 2H), 1.78 – 1.70 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H); 13C-NMR (126 MHz) δ 166.04,
147.38, 140.00, 137.52, 132.30, 129.56, 128.90, 127.23, 114.06, 60.91, 26.53, 24.64, 22.94,
22.66, 14.31; MS (HR-ESI) m/z calcd for C17H17ClNO4S2 (M-H)-: 398.0293, found: 398.0291.
2-(Naphthalene-1-sulphonylamino)-5-phenyl-thiophene-3-carboxylic acid ethyl ester (10a).
Yellow solid, 30%; m.p.: 163-165 oC; 1H-NMR (300 MHz) δ 10.68 (s, 1H), 8.69 (d, J = 9.3 Hz,
1H), 8.40 (dd, J = 7.4, 1.2 Hz, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.96 – 7.89 (m, 1H), 7.79 – 7.70 (m,
19

ACCEPTED MANUSCRIPT
1H), 7.66 – 7.27 (m, 7H), 7.19 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H); MS
(HR-ESI) m/z calcd for C23H18NO4S2 (M-H)-: 436.0683, found: 436.0672.
2-(Naphthalene-1-sulphonylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid
o
1
ethyl ester (10b). Yellow solid, 31%; m.p.: 175-177 C; H-NMR (500 MHz) δ 10.95 (s, 1H),
8.68 – 8.64 (m, 1H), 8.35 (dd, J = 7.4, 1.2 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.2 Hz,
1H), 7.74 – 7.69 (m, 1H), 7.61 – 7.57 (m, 1H), 7.54 – 7.50 (m, 1H), 4.18 (q, J = 7.1 Hz, 2H),
2.61 – 2.48 (m, 4H), 1.72 – 1.63 (m, 4H), 1.25 (t, J = 7.1 Hz, 3H); 13C-NMR (126 MHz) δ
165.91, 147.82, 135.10, 134.34, 133.52, 132.04, 130.94, 129.19, 128.74, 128.09, 127.10, 126.34,
124.41, 124.11, 113.02, 60.74, 26.45, 24.55, 22.96, 22.66, 14.29; MS (HR-ESI) m/z calcd for
C21H21NNaO4S2 (M+Na)+: 438.0804, found: 438.0802.
2-(Naphthalene-2-sulphonylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid
o
1
ethyl ester (11b). White powder, 63%; m.p.: 173-175 C; H-NMR (300 MHz) δ 10.60 (s, 1H),
8.51 (s, 1H), 8.02 – 7.83 (m, 4H), 7.69 – 7.56 (m, 2H), 4.22 (q, J = 7.1 Hz, 2H), 2.69 – 2.48 (m,
4H), 1.80 – 1.61 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H); MS (HR-ESI) m/z calcd for C21H20NO4S2 (M-
H)-: 414.0839, found: 414.0815.
2-(Bis-naphthalene-1-sulphonylamino)-5-phenyl-thiophene-3-carboxylic acid ethyl ester
o
1
(12a). Brown solid, 57%; m.p.: 113-115 C; H-NMR (300 MHz) δ 8.56 (d, J = 11.5 Hz, 2H),
8.06 – 7.90 (m, 7H), 7.75 – 7.55 (m, 7H), 7.53 – 7.35 (m, 4H), 3.47 (q, J = 7.1 Hz, 2H), 0.87 (t, J
= 7.1 Hz, 3H); MS (HR-ESI) m/z calcd for C33H25NNaO6S3 (M+Na)+: 650.0736, found:
650.0722.
General procedure for the synthesis of the diketoesters (14a,b)32. A mixture of
Ethylacetoacetate (for 14a) or Ethylpropionylacetate (for 14b) (1.0 mmol), anhydrous potassium
20

ACCEPTED MANUSCRIPT
carbonate (2.0 mmol), and potassium fluoride (1.0 mmol) in dry acetone was refluxed under
nitrogen atmosphere and continuously stirred for half an hour, and then a solution of
chloroacetone (1.1 mmol) in dry acetone was added, refluxing for another 48-72 h and
monitoring the reaction by TLC. The reaction mixture was filtered, the residue was washed with
acetone, and the combined filtrate and washings were evaporated. The residue was dissolved in
ethylacetate, and the solution was washed with water, dried and evaporated under reduced
pressure.
2-Acetyl-4-oxo-pentanoic acid ethyl ester (14a)32. Brown resin, 78%; MS (ESI) m/z calcd for
C9H14NaO4 (M+Na)+: 209.0, found: 209.1.
3-Oxo-2-(2-oxo-propyl)-pentanoic acid ethyl ester (14b)33. Brown resin, 96%; MS (ESI) m/z
calcd for C10H16NaO4 (M+Na)+: 223.0, found: 223.1.
General procedure for the synthesis of 2,5-Dialkyl-1-phenyl-1H-pyrrole-3-carboxylic acid
ethyl ester (15a,b).34 To a solution of 14a,b (11 mmol) in acetic acid (40 mL), aniline (11
mmol) was added. The mixture was heated under reflux for 5-6 h until disappearance of reactants
(TLC monitorage). Water was then added to the resulting solution and the solution extracted
with ethylacetate (3 times). The combined organic layer was then dried using MgSO4,
evaporated under reduced pressure and the residue purified by flash chromatography according
to the reported eluent.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester (15a).35 Yellow liquid, 55%;
MS (ESI) m/z calcd for C15H18NO2 (M+H)+: 244.1, found: 244.1.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester (15b). Brown resin,
64%; 1H-NMR (500 MHz) δ 7.50 – 7.42 (m, 3H), 7.21 – 7.16 (m, 2H), 6.37 (d, J = 0.9 Hz, 1H),
21

ACCEPTED MANUSCRIPT
4.28 (q, J = 7.1 Hz, 2H), 2.70 (q, J = 7.5 Hz, 2H), 1.91 (d, J = 0.9 Hz, 3H), 1.35 (t, J = 7.1 Hz,
3H), 0.97 (t, J = 7.4 Hz, 3H); MS (HR-ESI) m/z calcd for C16H20NO2 (M+H)+: 258.1489, found:
258.1475.
General procedure for the synthesis of 2,5-Dialkyl-1-phenyl-1H-pyrrole-3-carboxylic acid
(16a,b). The appropriate ethyl ester (5.8 mmol) was refluxed for 3-4 h in a mixture of aqueous
10% sodium hydroxide (20 mL) and ethanol (20 mL). After cooling, the solution was adjusted to
pH 3 with aqueous 10% hydrochloric acid. The resulting precipitate was collected by filtration,
washed with H2O, dried under vacuum and used without further purification.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid (16a).36 White powder, 95%; m.p.: 216-
o
1
218 C; H-NMR (300 MHz) δ 7.56 – 7.44 (m, 3H), 7.23 – 7.18 (m, 2H), 6.44 (d, J = 0.9 Hz,
1H), 2.32 (s, 3H), 1.99 (d, J = 0.9 Hz, 3H); MS (HR-ESI) m/z calcd for C13H12NO2 (M-H)-:
214.0874, found: 214.0866.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid (16b). Brown powder, 100%; m.p.:
o
1
197-199 C; H-NMR (500 MHz) δ 7.52 – 7.44 (m, 3H), 7.23 – 7.18 (m, 2H), 6.39 (d, J = 0.9
Hz, 1H), 2.72 (q, J = 7.5 Hz, 2H), 1.93 (d, J = 0.9 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H); MS (HR-
ESI) m/z calcd for C14H15NNaO2 (M+Na)+: 252.0995, found: 252.0989.
General procedure for the synthesis of 2,5-Dialkyl-1-phenyl-1H-pyrrole-3-carboxamides
(17a,b; 18a,b; 19a,b; 20a,b; 21a,b and 22a, b).37 To a magnetically stirred suspension of the
carboxylic acid (1.6 mmol) in anhydrous DMF were successively added N,N-
diisopropylethylamine (Hunig’s base) (6 eq) and O-benzotriazol-1-yl-N,N,N¢,N¢-
tetramethyluronium hexafluorophosphate (HBTU) (1.2 eq) and the mixture was allowed to stir at
room temperature for 30 miuntes. The appropriate amine (1.2 equiv) was then added, and the
22

ACCEPTED MANUSCRIPT
solution was stirred at room temperature overnight. The mixture was diluted with EtOAc,
washed with aqueous NaHCO3 solution, 1N HCl and brine, dried over MgSO4 and concentrated
to give a crude resin. The crude product was further purified by flash chromatography according
to the reported eluent.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid naphthalen-1-ylamide (17a). Grey
o
1
solid, 81%; m.p.: 145-147 C; H-NMR (300 MHz) δ 8.08 (d, J = 8.4 Hz, 1H), 7.61 – 7.48 (m,
5H), 7.45 – 7.38 (m, 1H), 7.30 – 7.19 (m, 5H), 6.67 (s, 1H), 2.34 (s, 3H), 2.05 (s, 3H); MS (HR-
ESI) m/z calcd for C23H20N2NaO (M+Na)+: 363.1468, found: 363.1474.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid naphthalen-1-ylamide (17b).
Beige solid, 40%; m.p.: 106-108 oC; 1H-NMR (300 MHz) δ 8.08 (d, J = 8.4 Hz, 1H), 7.99 – 7.86
(m, 1H), 7.61 – 7.48 (m, 5H), 7.45 – 7.38 (m, 1H), 7.30 – 7.26 (m, 1H), 7.26 – 7.18 (m, 3H),
6.67 (d, J = 0.9 Hz, 1H), 2.73 (q, J = 7.4 Hz, 2H), 2.02 (d, J = 0.9 Hz, 3H), 1.02 (t, J = 7.4 Hz,
3H); MS (HR-ESI) m/z calcd for C24H22N2NaO (M+Na)+: 377.1624, found: 377.1614.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid naphthalen-2-ylamide (18a). Beige
o
1
solid, 83%; m.p.: 142-144 C; H-NMR (300 MHz) δ 8.08 (d, J = 8.4 Hz, 1H), 7.61 – 7.37 (m,
7H), 7.27 – 7.19 (m, 4H), 6.67 (d, J = 0.9 Hz, 1H), 2.34 (s, 3H), 2.05 (d, J = 0.9 Hz, 3H); MS
(HR-ESI) m/z calcd for C23H20N2NaO (M+Na)+: 363.1468, found: 363.1461.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid naphthalen-2-ylamide (18b).
Brown resin, 35%; 1H-NMR (300 MHz) δ 8.08 (d, J = 8.4, 1H), 7.87 – 7.19 (m, 11H), 6.67 (d, J
= 1.0 Hz, 1H), 2.74 (q, J = 7.4 Hz, 2H), 2.02 (d, J = 1.0 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H); MS
(HR-ESI) m/z calcd for C24H23N2O (M+H)+: 355.1805, found: 355.1795.
23

ACCEPTED MANUSCRIPT
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid adamantan-1-ylamide (19a). Yellow
resin, 81%; 1H-NMR (300 MHz) δ 7.52 – 7.41 (m, 3H), 7.20 – 7.12 (m, 2H), 6.02 (d, J = 0.9 Hz,
1H), 2.29 (s, 3H), 2.13 (bs, 9H), 1.96 (bs, 3H), 1.72 (bs, 6H); 13C NMR (75 MHz) δ 165.66,
133.79, 129.45, 128.52, 128.48, 128.40, 115.32, 104.58, 51.80, 42.23, 36.66, 29.73, 27.07, 12.83,
12.41; MS (HR-ESI) m/z calcd for C23H29N2O (M+H)+: 349.2274, found: 349.2272.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid adamantan-1-ylamide (19b).
White solid, 40%; m.p.: 143-144 oC; 1H-NMR (300 MHz) δ 7.66 – 7.55 (m, 3H), 7.35 – 7.29 (m,
2H), 6.13 (d, J = 0.8 Hz, 1H), 2.87 (q, J = 7.4 Hz, 2H), 2.25 (bs, 9H), 2.07 (d, J = 0.7 Hz, 3H),
1.82 (bs, 6H), 1.11 (t, J = 7.4 Hz, 3H); MS (HR-ESI) m/z calcd for C24H31N2O (M+H)+:
363.2431, found: 363.2430.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid phenethyl-amide (20a). Yellow oil,
1
100%; H NMR (300 MHz) δ 7.53 – 7.40 (m, 3H), 7.37 – 7.29 (m, 2H), 7.29 – 7.20 (m, 3H),
7.20 – 7.13 (m, 2H), 5.95 (s, 1H), 3.67 (t, J = 6.9 Hz, 2H), 2.92 (t, J = 6.9 Hz, 2H), 2.29 (s, 3H),
1.96 (s, 3H); MS (HR-ESI) m/z calcd for C21H22KN2O (M+K)+: 357.1364, found: 357.1363.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid phenethyl-amide (20b). Beige
o
1
solid, 42%; m.p.: 139-141 C; H NMR (300 MHz) δ 7.55 – 7.43 (m, 3H), 7.38 – 7.15 (m, 7H),
5.93 (s, 1H), 3.67 (t, J = 6.8 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H), 2.75 (q, J = 7.4 Hz, 2H), 1.92 (s,
3H), 0.97 (t, J = 7.4 Hz, 3H); 13C NMR (75 MHz) δ 165.78, 140.29, 139.57, 137.91, 129.38,
129.01, 128.73, 128.67, 128.58, 126.50, 113.53, 104.38, 40.51, 36.30, 19.16, 14.86, 12.78; MS
(HR-ESI) m/z calcd for C22H24N2NaO (M+Na)+: 355.1781, found: 355.1776.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid (1-phenyl-ethyl)-amide (21a). Beige
crystalline solid, 50%; m.p.: 149-151 oC; 1H NMR (300 MHz) δ 7.55 – 7.26 (m, 8H), 7.20 – 7.12
24

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(m, 2H), 6.07 (s, 1H), 5.31 (m, 1H), 2.31 (s, 3H), 1.97 (s, 3H), 1.58 (d, J = 6.8 Hz, 3H); MS
(HR-ESI) m/z calcd for C21H23N2O (M+H)+: 319.1805, found: 319.1795.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid (1-phenyl-ethyl)-amide (21b).
Beige crystalline solid, 32%; m.p.: 168-170 oC; 1H NMR (300 MHz) δ 7.54 – 7.44 (m, 3H), 7.43
– 7.30 (m, 4H), 7.29 – 7.16 (m, 3H), 6.07 (s, 1H), 5.38 – 5.26 (m, 1H), 2.85 – 2.68 (m, 2H), 1.94
(s, 3H), 1.57 (d, J = 6.9 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H); MS (HR-ESI) m/z calcd for
C22H24N2NaO (M+Na)+: 355.1781, found: 355.1785.
2,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid cyclohexylamide (22a). White solid,
87%; m.p.: 115-117 oC; 1H NMR (300 MHz) δ 7.51 – 7.37 (m, 3H), 7.19 – 7.10 (m, 2H), 6.06 (d,
J = 0.9 Hz, 1H), 4.01 – 3.86 (m, 1H), 2.30 (s, 3H), 2.07 – 1.96 (m, 2H), 1.96 (d, J = 0.9 Hz, 3H),
13
1.79 – 1.57 (m, 3H), 1.49 – 1.32 (m, 2H), 1.27 – 1.10 (m, 3H); C NMR (75 MHz) δ 165.17,
137.88, 133.78, 129.38, 128.56, 128.45, 128.28, 114.49, 104.35, 47.77, 33.62, 26.97, 25.77,
25.11, 12.76, 12.26; MS (HR-ESI) m/z calcd for C19H24N2NaO (M+Na)+: 319.1781, found:
319.1782.
2-Ethyl-5-methyl-1-phenyl-1H-pyrrole-3-carboxylic acid cyclohexylamide (22b). White
crystalline solid, 45%; m.p.: 129-131 oC; 1H NMR (300 MHz) δ 7.52 – 7.40 (m, 3H), 7.22 – 7.15
(m, 2H), 6.04 (d, J = 0.8 Hz, 1H), 5.63 (d, J = 6.9 Hz, 1H), 4.02 – 3.86 (m, 1H), 2.75 (q, J = 7.4
Hz, 2H), 2.07 – 1.96 (m, 2H), 1.94 (d, J = 0.8 Hz, 3H), 1.79 – 1.57 (m, 3H), 1.49 – 1.32 (m, 2H),
1.27 – 1.10 (m, 3H), 0.98 (t, J = 7.4 Hz, 3H); 13C NMR (75 MHz) δ 164.90, 140.17, 137.92,
129.35, 128.62, 128.57, 113.80, 104.32, 47.82, 33.69, 25.82, 25.17, 19.17, 14.82, 12.73; MS
(HR-ESI) m/z calcd for C20H26N2NaO (M+Na)+: 333.1937, found: 333.1934.
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Competition Binding Assay. Membranes from HEK-293 cells over-expressing the respective
human recombinant CB1 receptor (Bmax= 2.5pmol/mg protein) and human recombinant CB2
receptor (Bmax= 4.7pmol/mg protein) were incubated with [3H]-CP-55,940 (0.14nM/Kd=0.18nM
and 0.084nM/Kd=0.31nM, respectively for CB1 and CB2 receptor) as the high affinity ligand.
Competition curves were performed by displacing [3H]-CP-55,940 with increasing concentration
of the newly synthesized compounds (0.1nM–10ꢀM). Nonspecific binding was defined by 10µM
of WIN55,212-2 as the heterologous competitor (Ki values 9.2nM and 2.1nM respectively for
CB1 and CB2 receptor). All compounds were tested following the procedure described by the
manufacturer (Perkin Elmer, Italy). Displacement curves were generated by incubating drugs
with [3H]-CP-55,940 for 90 minutes at 30ºC. Ki values were calculated by applying the Cheng-
Prusoff equation to the IC50 values (obtained by GraphPad) for the displacement of the bound
radioligand by increasing concentrations of the test compound. Data represent mean values for at
least three separate experiments performed in duplicate and are expressed as Ki (nM), average
SEM < 10%.
Functional Activity at CB2 Receptors in Vitro. The cAMP Hunter™ assay enzyme fragment
complementation chemiluminescent detection kit was used to characterize the functional activity
in CB2 receptor-expressing cell lines. Gi-coupled cAMP modulation was measured following the
manufacturer’s protocol (DiscoveRx, Fremont, CA). Briefly, CHO-K1 cells overexpressing the
human CB2 receptor were plated into a 96 well plate (30,000 cells/well), and incubated
overnight at 37 °C, 5% CO2. Media was aspirated and replaced with 30ꢀl of assay buffer. Cells
were incubated 30 min at 37°C with 15µl of 3x dose-response solutions of samples prepared in
presence of cell assay buffer containing a 3x of 25µM NKH-477 solution (a water soluble
analogue of Forskolin) to stimulate adenylate cyclase and enhance basal cAMP levels. We
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ACCEPTED MANUSCRIPT
further investigated the effect upon receptor activation by testing compounds in the presence of
JWH-133 selective agonist. Cells were pre-incubated with samples (15 min at 37°C at 6x the
final desired concentration) followed by 30 min incubation with JWH-133 agonist challenge at
the EC80 concentration (EC80=4µM, previously determined in separate experiments) in
presence of NKH-477 to stimulate adenylate cyclase and enhance cAMP levels. For all
protocols, following stimulation, cell lysis and cAMP detection were performed as per the
manufacturer’s protocol. Luminescence measurements were measured using a GloMax Multi
Detection System (Promega, Italy). Data are reported as mean ± SEM of three independent
experiments conducted in triplicate and were normalized considering the NKH-477 stimulus
alone as 100% of the response. The percentage of response was calculated using the following
formula: % RESPONSE = 100% x (1- (RLU of test sample - RLU of NKH-477 positive control)
/ (RLU of vehicle - RLU of NKH-477 positive control). When tested in presence of JWH-133,
the percentage of response was calculated using the following formula: % RESPONSE = 100% x
(RLU test sample - RLU of EC80 control) / (mean RLU NKH-477 positive control - RLU EC80
control). The data were analyzed using PRISM software (GraphPad Software Inc, San Diego,
CA).
SUPPLEMENTARY DATA
Supplementary data associated with this article can be found in the online version.
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