Synthesis of novel 2-(1-adamantanylcarboxamido)thiophene derivatives. Selective cannabinoid type 2 (CB2) receptor agonists as potential agents for the treatment of skin inflammatory disease

Article abstract of DOI:10.1016/j.ejmech.2018.09.070

A set of CB2R ligands, based on the thiophene scaffold, was synthesized and evaluated in in vitro assays. Compounds 8c-i, k, l, bearing the 3-carboxylate and 2-(adamantan-1-yl)carboxamido groups together with apolar alkyl/aryl substituents at 5-position or at 4- and 5-positions of the thiophene ring possess high CB2R affinity at low nanomolar concentration, good receptor selectivity, and agonistic functional activity. The full agonist 8g, showing the best balance between receptor affinity and selectivity, was tested in vitro in an experimental model of allergic contact dermatitis and proved to be able to block the release of MCP-2 in HaCaT cells at 10 μM concentration.

Full text of DOI:10.1016/j.ejmech.2018.09.070

Accepted Manuscript
Synthesis of novel 2-(1-adamantanylcarboxamido)thiophene derivatives. Selective
cannabinoid type 2 (CB2) receptor agonists as potential agents for the treatment of
skin inflammatory disease
Claudia Mugnaini, Alessandro Rabbito, Antonella Brizzi, Nastasja Palombi, Stefania
Petrosino, Roberta Verde, Vincenzo Di Marzo, Alessia Ligresti, Federico Corelli
PII:
S0223-5234(18)30857-2
10.1016/j.ejmech.2018.09.070
EJMECH 10781
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2018
Revised Date: 24 September 2018
Accepted Date: 28 September 2018
Please cite this article as: C. Mugnaini, A. Rabbito, A. Brizzi, N. Palombi, S. Petrosino, R. Verde, V. Di
Marzo, A. Ligresti, F. Corelli, Synthesis of novel 2-(1-adamantanylcarboxamido)thiophene derivatives.
Selective cannabinoid type 2 (CB2) receptor agonists as potential agents for the treatment of skin
inflammatory disease, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.070.
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Graphical abstract
O
S
NH
CO2Me
8g

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Synthesis of Novel 2-(1-Adamantanylcarboxamido)thiophene Derivatives. Selective
Cannabinoid Type 2 (CB2) Receptor Agonists as Potential Agents for the Treatment of Skin
Inflammatory Disease
Claudia Mugnainia,*, Alessandro Rabbitob, Antonella Brizzia, Nastasja Palombia, Stefania
Petrosinob, Roberta Verdeb, Vincenzo Di Marzob, Alessia Ligrestib,**, Federico Corelli a
aDipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo
Moro 2, 53100 Siena, Italy
bEndocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle
Ricerche, Via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
Abbreviations: CB1R, cannabinoid type-1 receptor; CB2R, cannabinoid type-2 receptor; ClogP,
calculated logarithm of the partition coefficient (P); CNS, central nervous system; DCM,
dichloromethane; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s Modified Eagle’s Medium;
ECS, endocannabinoid system; DNFB, 2,4-dinitrofluorobenzene; FCS, fetal calf serum; MCP-2,
human monocyte chemotactic protein-2; PET, positron emission tomography; SAR, structure-
activity relationship; SI, selectivity index; TPSA, topological polar surface area.
*Corresponding author. Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi
di Siena, Via Aldo Moro 2, 53100 Siena, Italy.
**Corresponding author. Endocannabinoid Research Group, Istituto di Chimica Biomolecolare,
Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
E-mail addresses: claudia.mugnaini@unisi.it (C. Mugnaini), aligresti@icb.cnr.it (A. Ligresti).

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ABSTRACT
A set of CB2R ligands, based on the thiophene scaffold, was synthesized and evaluated in in vitro
assays. Compounds 8c-i, k, l, bearing the 3-carboxylate and 2-(adamantan-1-yl)carboxamido
groups together with apolar alkyl/aryl substituents at 5-position or at 4- and 5-positions of the
thiophene ring possess high CB2R affinity at low nanomolar concentration, good receptor
selectivity, and agonistic functional activity. The full agonist 8g, showing the best balance between
receptor affinity and selectivity, was tested in vitro in an experimental model of allergic contact
dermatitis and proved to be able to block the release of MCP-2 in HaCaT cells at 10 µM
concentration.
Keywords:
CBR ligands
CB2R agonists
Skin inflammation
Chemokine release

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1. Introduction
In 1988, twenty-four years after the isolation of ∆9-THC [1], the main active ingredient of
Cannabis, type-1 cannabinoid receptor (CB1R) was discovered in the brain where it regulates the
release of a number of neurotransmitters controlling emotions, pain, movement, memory, food
intake [2]. In 1993, a second receptor was identified, predominantly expressed at a peripheral level,
in immune cells, gastrointestinal tract, liver, heart, kidney, bones [3]. More recently, it was
demonstrated that type-2 cannabinoid receptor (CB2R) is also expressed in the CNS during
inflammatory processes [4]. Both CBRs share an overall homology of 44%, but the similarity in the
7-transmembrane region, where the ligand-binding domain has been identified, reaches 68%.
In the same years, the first endogenous ligands of CBRs, namely anandamide [5] and 2-
arachidonoylglycerol (2-AG) [6] were also discovered. Since then, new ligands have been identified
able to modulate CBRs together with the enzymes responsible for their synthesis and degradation,
making the complex picture of endocannabinoid system (ECS) increasingly exhaustive [7]. Due to
their wide distribution, CBRs are implicated in a number of physiological and pathological
conditions both centrally and peripherally [8], and hence they are considered interesting targets for
the design of drug candidates potentially useful for the treatment of disorders such as chronic
inflammation [9], pain [10], multiple sclerosis [11], cancer [12], and addiction [13].
Despite the initial controversy on the presence of functional cannabinoid CB2R in the CNS, it is
now well accepted that this receptor is present in limited amounts and distinct locations in the brain
of several animal species, including humans [14]. The inducible nature of these receptors under
neuroinflammatory conditions and the lack of the undesired psychoactive effects make them
attractive targets for the development of novel therapeutic approaches for the treatment of various
pathological conditions, such as neurodegenerative disorders, chronic and inflammatory pain,
ischemic stroke, diabetic neuropathy and nephropathy, liver cirrhosis, pruritus, osteoporosis, and
cancers [15]. Of current interest are also skin inflammatory diseases, such as allergic contact
dermatitis, that affect a significant percentage of population, and mainly women, in industrialized

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countries, where are among the main causes of occupational diseases. Notably, the CB2R agonist S-
777469 has been advanced into a phase II clinical trial to assess its efficacy in subjects with mild to
moderate atopic dermatitis [16].
Because of the high homology between the ligand-binding domains of the two receptors and the
overall higher tissue expression of CB1R, the development of selective CB2R ligands, devoid of the
centrally CB1R-mediated psychotropic effects, is a challenging task [17]. Despite the plethora of
structurally diverse, selective CB2R agonists and inverse agonists unveiled so far and the progress
of some of them into clinical trials [18], none of these bioactive compounds has been further
developed into a successful clinical drug. Although it is unclear why some of the candidates have
failed, generally the main reasons for failure can be found in low receptor selectivity, poor in vitro
DMPK and pharmacokinetics, off-target activity, deficiencies in the predicative utility of the
preclinical models as well as in the understanding of the molecular mode of action of CB2R
agonists [17].
Fig. 1. Representative examples of quinolones and thiophene derivatives previously developed

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Intensive research efforts have been devoted in our laboratories to the design and synthesis of new
compounds showing high CB2R affinity and selectivity over the CB1R subtype. In particular,
within a significantly large set of 4-quinolone-3-carboxamides [19a,b] and 4-hydroxy-2-quinolone-
3-carboxamides [19c] described in the last years, a number of compounds proved to possess a
remarkable in vitro profile, with binding constant (Ki) values at CB2R usually in the low nanomolar
range and selectivity indexes (SI = KiCB1/KiCB2) even >2,000, as exemplified by compounds 1
[19b] and 2 [19c] (Fig. 1). The 6-isopropyl analog 3 (usually referred to as COR167) [19a] was
further characterized in in vitro, ex vivo, and in vivo assays, where it elicited: a) potent
immunonomodulatory activity on immune cells from healthy subjects and patients with multiple
sclerosis [20]; b) protective effects on rat brain tissues toward ischemia and reperfusion-induced
injury [21]; c) anti-nociceptive properties [22], thereby validating the CB2R as a therapeutic target
and becoming a useful pharmacological tool for further investigations.
More recently, we engaged also in the evaluation of alternative scaffolds to develop new CB2R
selective ligands. Among the diverse building blocks that are of more prospective use in medicinal
chemistry, the 2-aminothiophene scaffolds, widely represented in several classes of compounds
possessing various biological activities [23a-c], including CB2R agonism [23d,e], have kindled our
attention [24-26]. In 2016, Oman et al. reported the synthesis of pyrrole- and
tetrahydrobenzo[b]thiophene-carboxamides as CB2R ligands [27]. While aroylamides and
sulfonamides showed modest or no CBRs affinity, the amides and retroamides bearing the
adamantan-1-yl moiety elicited high affinity and selectivity. In particular, compound 4 (Fig. 1)
exhibited the highest CB2R affinity (Ki = 2.15 nM), as well as the highest CB2R/CB1R subtype
selectivity (SI = 469). In addition, due to the current interest in CB2R as potential indicator of
neurodegeneration [28], recently two thiophene-based radioligands [11C]-5 and [11C]-6 were
developed as potential positron emission tomography (PET) tracers to visualize CB2R in the brain
[29].
Given the promising findings obtained with compound 4 and bearing in mind the remarkably

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positive effect that the adamantyl group can exert in terms of both CBRs affinity and selectivity
[19,23d,23e,27], we embarked on the synthesis and biological evaluation of a family of
amidothiophene derivatives characterized by the adamantan-1-carbonyl moiety. Furthermore, also
the methyl ester group was retained in all of the compounds as they showed lower lipophilicity and
comparable CB2R affinity than corresponding amides such as the n-propylamide [23d]. Although
esters of carboxylic acids, particularly methyl esters, are functional groups featuring “hot spots”
from the metabolic viewpoint, because they usually suffer from limited chemical and/or enzymatic
stability, the methyl ester group in these thiophene compounds proved to be stable toward plasma
esterases [26] as well as to hydrolysis in DMSO/water solution.
Major aim was to evaluate in vitro the biological profile of the new compounds and possibly to
establish their structure-activity relationship (SAR). In addition, we decided also to test the effects
on biological activity of the isosteric replacement of the thiophene ring with other heterocyclic
scaffolds as well as the modification of the amide functionality into a urea or carbamate group to
modulate lipophilicity and introduce further H-bond donor/acceptor groups.
2. Results and discussion
2.1. Chemistry
Among the aminothiophene derivatives 7 (Scheme 1) used as starting substrates, compound 7a was
commercially available, while the analogs 7b-k were prepared in-house according to the Gewald
procedure, as previously reported for the synthesis of 7g [26]. Condensation of methyl cyanoacetate
in the presence of sulfur and morpholine with the appropriate aldehydes or ketones yielded
intermediates 7b-d and 7e-k, respectively. Similarly, Gewald reaction between methyl cyanoacetate
and cyclohexanone or tetrahydro-4H-thiopyran-4-one afforded the bicyclic aminothiophene
intermediates 7l [26] and 7m, respectively. Subsequent acylation of 7a-m with adamantan-1-
carbonyl chloride was best performed in refluxing 1,4-dioxane as solvent in the absence of any
added base. Under these conditions, the final amidothiophene derivatives 8a-m were obtained in

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13-95% yield by avoiding a possible bis-acylation of the amino group of 7, as observed previously
when this reaction was carried out in the presence of a base at room temperature [25,26].
Compound 8m was converted into the corresponding sulfone derivative 8n by oxidation with
Oxone®. In addition, two more compounds 8o [26] and 8p were prepared by acylation of 7g with
4-chlorobenzoyl chloride or 4-fluorobenzoyl chloride, respectively, to compare their biological
activity to that of 8g.
Scheme 1. Synthesis of 2-(1-adamantanylcarboxamido)thiophene derivatives.a
aReagents and conditions: (i) methyl cyanoacetate, S8, morpholine, MeOH, reflux, overnight; (ii)
adamantane-1-carbonyl chloride, 1,4-dioxane, 100 °C, 1-2 h; (iii) 4-chlorobenzoyl chloride or 4-
fluorobenzoyl chloride, 1,4-dioxane, 100 °C, 1-2 h; (iv) Oxone , MeOH, H2O, rt, overnight.
According to the scaffold hopping approach [30], although not exhaustively applied, the thiophene
nucleus was replaced with some other heterocycles able to provide final compounds with different

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electronic properties but basically similar steric characteristics. As starting substrates for the
preparation of compounds 9-13 (Scheme 2), we used heterocyclic amino esters commercially
available, such as methyl 2-aminopyridine-3-carboxylate (14), or synthesized following published
procedures, as for methyl 3-amino-1H-pyrrole-2-carboxylate (15) [31], methyl 4-amino-1-methyl-
1H-pyrazole-5-carboxylate (16), and the isomeric methyl 4-amino-1-methyl-1H-pyrazole-3-
carboxylate (17) [32].
Scheme 2. Synthesis of isosteric compounds 9-13.a
aReagents and conditions: (i) adamantane-1-carbonyl chloride, TEA, DCM, 0 °C to rt, 18 h; (ii)

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adamantane-1-carbonyl chloride, 1,4-dioxane, 100 °C, 18 h; (iii) adamantane-1-carbonyl chloride,
NaH, dry THF, rt to reflux, 2 h.
For the synthesis of methyl 5-amino-3-methyl-4-isoxazolecarboxylate (18), we adopted a reaction
sequence (Scheme 3) slightly modified compared to those described in the only two literature
reports concerning its preparation [33,34], and entailing the reaction of methyl cyanoacetate with
triethyl orthoacetate to give methyl 2-cyano-3-ethoxy-2-butenoate (19) followed by
cyclocondensation with hydroxylamine. Precursors 14-18 were then converted into the
corresponding adamantanecarboxamides 9-13 under appropriate acylation conditions.
Finally, compounds 20-22 (Scheme 4), where the amide functionality is replaced by urea moieties,
were also synthesized in 50-87% yield by reaction of 1-aminoadamantane, morpholine, and
piperidine with phenyl carbamate 23, in turn obtained by acylation of 7l with phenyl chloroformate.
Scheme 3. Synthesis of methyl 5-amino-3-methylisoxazole-4-carboxylate.a
aReagents and conditions: (i) triethyl orthoacetate, Ac2O, reflux, 3 h; (ii) hydroxylamine
hydrochloride, dry pyridine, reflux, 4 h.

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Scheme 4. Synthesis of ureido derivatives 20-22.a
aReagents and conditions: (i) phenyl chloroformate, 1,4-dioxane, 100 °C, overnight; (ii) 1-
aminoadamantane, 1,4-dioxane, 100 °C, overnight; (iii) morpholine, 1,4-dioxane, 100 °C, 5 h; (iv)
piperidine, 1,4-dioxane, 100 °C, 1 h.
2.2. Binding affinity for cannabinoid receptors and SAR
The binding affinities (Ki values) of compounds 8a-p, 9-13, and 20-24 for human recombinant
CB1R and CB2R are reported in Table 1. The tested compounds were evaluated in parallel with 24
(SR144528) [35a] and 25 (AM630) [35b] as reference CB2R ligands.
Table 1.
CB1R and CB2R affinity values for compounds 8a-p, 9-13, and 20-24.a,b
CB1Rc
CB2Rd
Compd
Structure
SIe
Kif (nM)
Kif (nM)
O
8a
S
>10,000
1324.12 ± 283.34 >68
NH
OMe
O

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O
8b
8c
8d
8e
8f
S
NH
OMe
>10,000
23.09 ± 2.93
>433
186
O
O
S
NH
1339.29 ± 230.43 7.21 ± 0.77
OMe
O
O
S
NH
>10,000
6.48 ± 1.88
9.24 ± 1.45
5.92 ± 0.21
2.46 ± 2.08
4.28 ± 0.65
>1540
>1080
358
OMe
O
O
S
NH
OMe
>10,000
O
O
S
NH
OMe
2120 ± 110
185.14 ± 31.49
1100 ± 120
O
O
8g
8h
S
NH
OMe
75
O
O
S
257
NH
OMe
O

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O
8i
8j
S
NH
OMe
58.89 ± 6.03
1.72 ± 0.14
34
O
O
S
NH
OMe
>10,000
35.00 ± 1.95
>285
O
O
8k
S
NH
OMe
518.57 ± 165.43
8.03 ± 0.05
65
O
O
8l
S
>10,000
>10,000
5.08 ± 0.03
>1968
>197
NH
OMe
O
8m
50.70 ± 13.62
8n
8o
>10,000
>10,000
1673.74 ± 312.44 >6
621.00 ± 150.85
>16

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8p
>10,000
495.77 ± 208.59
>20
9
>10,000
1220.19 ± 281.21 >8
2141.79 ± 167.09 >4
1081.88 ± 232.16 >9
O
10
11
12
13
NH
OMe
>10,000
>10,000
>10,000
>10,000
HN
O
O
NH
OMe
N
N
O
O
NH
OMe
845.42 ± 34.25
64.72 ± 3.93
>11
N
N
O
O
O
>154
>477
NH
OMe
N
O
20
O
NH
>10,000
20.97 ± 1.81
S
NH
OMe
O

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21
22
>10,000
2494.08 ± 32.50
195.70 ± 43.70
>4
>10,000
>51
O
O
23
>10,000
244.06 ± 49.88
>41
S
NH
OMe
O
24
116.8
5152
1.8
64.8
165
(SR144528)g,h
25
31.2
(AM630)g,h
aData represent mean values for at least three separate experiments performed in duplicate and are
b
expressed as Ki (nM). For both receptor binding assays, the new compounds were tested using
membranes from HEK cells transfected with either the CB1R or CB2R and [3H]-(–)-cis-3-[2-
hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP-55,940).
d
e
cCB1R: human cannabinoid type 1 receptor. CB2R: human cannabinoid type 2 receptor. SI:
f
selectivity index for CB2R, calculated as Ki(CB1R)/Ki(CB2R) ratio. Ki: “Equilibrium dissociation
constant”, that is, the concentration of the competing ligand that will bind to half the binding sites at
g
h
equilibrium in the absence of radioligand or other competitors. CB2 reference compound. The

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binding affinities of reference compound were evaluated in parallel with test compounds under the
same conditions.
As to the thiophene derivatives 8a-p, they showed higher affinity for CB2R than CB1R, with
Ki(CB2) values spanning three orders of magnitude (from 1.72 to 1673 nM), while Ki(CB1) is
mostly >1,000 nM, excepted compounds 8g, 8i, and 8k which exhibited affinity binding to CBR1
of 185.14, 58.89, and 518.57 nM, respectively. In particular, nine compounds (8c-i and 8k,l) out of
sixteen are the most potent CB2R ligands [(Ki(CB2) <10 nM] showing comparable affinity to the
reference compound SR144528, and three compounds (8b, 8j, and 8m) show noticeable CB2R
affinity ranging between 23.09 and 50.70 nM. As a result of the capacity of compounds 8a-p to
bind preferentially to CB2R, SI values range from 34 to approximately 2,000, with SI >1,000 for
compounds 8d, 8e, and 8l, which therefore can be considered the most selective CB2R ligands.
Compound 8a, the prototypical element in the series 8a-p, was only marginally active, but its
conversion into the corresponding 5-methyl analog 8b resulted in a 50-fold boost of potency, an
outcome that traces back to the so-called “magic methyl” effect [36]. Although the important role
played by methyl groups in biologically active small molecules is well documented [37], a survey
of the medicinal chemistry literature performed by Jorgensen and co-workers has suggested that the
introduction of a methyl group in a lead molecule could give rise to a 10-fold increase in activity in
8% of the time, while 100-fold gain in potency is far less likely, occurring with a 0.4% frequency
[38]. Thus, the transition from 8a to 8b features an extreme example of boostering effect of the
methyl substituent, that cannot be ascribed merely to the slight increase in lipophilicity or to
favourable conformational changes, but might be sooner attributed to the ability of 8b to place its
methyl nicely into the receptor binding site, so as to establish profitable hydrophobic interactions.
This hypothesis seems to be corroborated by replacing the methyl group of 8b with the n-propyl
chain or the phenyl ring to produce compounds 8c and 8d, respectively, which gained a further 3-
fold increase in potency. The progression from 8a to 8b, to 8c, and to 8d maintained receptor

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selectivity, probably because the increasingly larger and more hydrophobic substituents at position
5 of the thiophene ring well sit within the CB2R pocket, but are unfavourable or uninfluential for
interaction with CB1R. The shift of the phenyl substituent from the 5 (compound 8d) to the 4
position (compound 8j) on thiophene did not invert receptor selectivity, despite CB2R affinity was
descreased by five times. 4,5-Disubstituted thiophene derivatives generally displayed better CB2R
affinity compared to the monosubstituted thiophene derivatives, although with some loss of receptor
selectivity for compounds 8g, 8i, and 8k. Although the CB1R affinity of these three compounds
cannot be definitely rationalized, we can envisage that the lengthening of the 5-chain to four/five
carbon atoms (8i and 8k) or the homologation of the methyl into an ethyl group at 4 position (8g)
have an influence on selectivity by conferring higher CB1R affinity. On the other hand, further
homologation of the 4-ethyl group of 8g into a 4-n-propyl chain (8h) led to a more selective CB2R
ligand, though with a modest decrease in potency. Compound 8l, featuring the annulation product
of 8a with cyclohexane, showed a boost in potency of 260 times with respect to the prototype.
Interestingly, the binding profile of 8l is much more similar to that of 8f than 8g, with the complete
loss of affinity for CB1R (Ki >10,000 nM). It is not easy to explain why the replacement of a
methylene group with a sulfur atom (from 8l to 8m), that resulted in a slight decrease of ClogP
(from 4.77 to 4.30) and no change in TPSA [39], was able to cause a 10-fold decrease in CB2R
affinity. The same result was obtained by Nelson et al. when in a compound similar to 8l a
methylene group was replaced by an oxygen atom [23d], which however cause a more pronounced
decrease of ClogP and a slight increase of TPSA compared to a sulfur atom. It is worth mentioning
that 8l showed an increase in selectivity, accompanied by reduced affinity, compared to compound
4, differing only in the ester functionality. As expected, the oxidation of 8m to the corresponding
sulfone 8n produced a basically inactive compound, probably because it possesses a polar group
that would project inside a very hydrophobic pocket of the receptor. Finally, the biological
evaluation of compounds 8o and 8p, bearing a 4-substituted phenyl ring instead of the adamantyl
group, once more confirmed the positive impact of the adamantylamide on CB2R binding [19].

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The isosteric analogs 9-12 elicited no affinity for CB1R, but also low binding ability to CB2R with
Ki approximately around 1,000 nM. This finding seems to be due to the polarity of these
compounds, that show TPSA values higher than that of thiophene derivatives. However, the
isoxazole derivative 13, though even more polar compared to the other isosteres 9-12, is a selective
CB2R ligand with significant affinity of 64.72 nM. At moment, we are not able to make any
plausible conjecture to explain the discrepant behaviour of 13 with respect to compounds 9-12.
Within the other subset of carbamate and ureido analogs 20-23, while compounds 21-23 only
elicited moderate affinity for CB2R, compound 20 still represents a potent and selective CB2R
ligand, with an SI value >477 and only 4-fold decreased CB2R affinity compared to 8l.
It is interesting to compare the biological profile of the new thiophene compounds with that of
quinolones 1-3 that we have previously investigated. Thus, while quinolones 1 and 2 appear to be
superior in terms of both affinity and selectivity, most of the new compounds compare well with
quinolone 3 and exhibit in some cases better affinity or selectivity. Finally, thiophene derivatives
have the advantage over quinolones of being attainable through a simple two-step synthesis rather
than a more demanding preparation.
In vitro CB2R functional activity
The most potent compounds 8e, 8f, 8g, 8i and 8l were subjected to further in vitro pharmacological
evaluation in order to assess their capability of activating CB2R. We performed the cAMP
Hunter™ assay enzyme fragment complementation chemiluminescent detection kit to measure
whether compounds modulate intracellular cAMP levels in NKH-477-stimulated CHO cells
overexpressing CB2R.

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Fig. 2. Concentration-response curves of compounds in cAMP Hunter™ assay enzyme fragment
complementation chemiluminescent detection kit.
As shown in Fig. 2, all compounds displayed a typical agonist behavior by reducing cAMP levels
induced by the NKH-477 (a water-soluble analog of forskolin) as expected for an orthosteric Gi
agonist. However, we noticed that the nature of substituent on the position 4 and 5 of the thiophene
ring influenced the compounds activity, being the compound 8g the most active full agonist (EC50 =
197 nM) followed by compounds 8e and 8f (EC50 318 and 371 nM, respectively). The elongation of
the acyl chain in position 5 or the insertion of the cyclohexyl moiety were instead detrimental for
functional activity, as compound 8i and 8l acted as partial agonists (EC50 1135 and 1531 nM,
respectively) since they were not able to decrease cAMP content under basal conditions not even at
the highest concentrations tested (Fig. 2). Consistently, all compounds showed no activity in
presence of an EC80 CB2-ligand challenge (3 µM of JWH-133, not shown).
The curves show the effect of increasing concentrations of compounds on NKH-477-induced cAMP
levels in stable CHO cells expressing the human CB2R. Data were normalized to the maximal and
minimal response observed Data are reported as mean ± SEM of three independent experiments

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conducted in triplicate and were normalized considering the NKH-477 stimulus alone as 100% of
the response. The percentage of response was calculated as indicated in the experimental section.
Effect on MCP-2 chemokine release in polyinosinic polycytidylic acid [poly-(i:c)]-stimulated
human keratinocyte (HaCaT) cells
Endocannabinoids and their receptors constitute part of an adaptive system that also regulates
cutaneous inflammation. Cannabinoids exert their anti-inflammatory effects in skin via their actions
on keratinocyte cytokine production or their capability of modulating immune cells [40].
Pharmacological inhibition of CBR augments 2,4-dinitrofluorobenzene (DNFB)-induced dermatitis
in mice [41], suggesting a potential therapeutical effect for CB2R agonists in skin inflammation
disease. For this reason we tested the effect of the most active full agonist compound of the series
on the Poly-(I:C)-induced release of human monocyte chemotactic protein-2 (MCP-2) in HaCaT
cells, a widely used in vitro model of allergic contact dermatitis that highly expresses CB2R. Upon
a 100 µg/mL stimulus of Poly-(I:C), we found a significative increase of MCP-2 release (p<0.001)
that was completely abolished (p<0.001) in presence of 10 µM of compound 8g (Fig. 3). Co-
incubation with the CB2R antagonist AM630 (0.1 µM) did not affect per se the release of pro-
inflammatory cytokine MCP-2, but was able to significantly revert (p<0.001) the anti-inflammatory
effect of the full agonist 8g.

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Fig. 3. Effect of the full agonist 8g (10 µM) on Poly-(I:C)-induced release of MCP-2 in HaCaT
cells in presence and absence of per se inactive concentration (0.1 µM) of the CBR2 antagonist
AM630.
Student’s t test: *** p< 0.001 versus Vehicle; °°° p<0.001 versus Poly-(I:C) and §§§ p< 0.001
versus compound 8g under Poly-(I:C) stimulus.
3. Conclusions
Thiophene-2-carboxamide derivatives were designed, synthesized, and evaluated in vitro for their
ability to bind selectively at CB2R over CB1R. In the whole set of compounds both 3-carboxylate
and 2-(adamantan-1-yl)carboxamide groups were kept unchanged, whereas different substituents
were introduced at positions 4 and 5 of the heterocyclic ring to get some insight into SAR. Thus,
compound 8a, having no substituents at positions 4 and 5, showed only week affinity for CB2R,
demonstrating that the 3-carboxylate and 2-carboxamido moieties are not decisive for receptor
binding. On the other hand, the insertion of substituents at position 5 seems to be much more
fruitful, as a progressive increase of CB2R affinity, often accompanied by higher selectivity, can be
noticed in compounds 8b, 8c, 8d bearing an increasingly larger substituent. A further gain in
affinity/selectivity was achieved by substituting also the position 4 of the ring, as in compounds 8e-i

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and 8k,l. However, polar groups, such as the sulfone group of 8n, or the isosteric replacement of
thiophene with more polar heterocycles led to far less active compounds. All the most potent CB2R
ligands showed a functional profile as agonists, being capable of activating this receptor. In
conclusion, a set of potent and selective CB2R agonists, based on a novel chemotype for
cannabimimetic agents, was obtained. In an experimental model of allergic contact dermatitis, the
potent full agonist 8g was able to completely abolish the MCP-2 release in HaCaT cells at 10 µM
concentration, demonstrating to have a potential for the treatment of skin inflammatory disease.
4. Experimental
4.1. Chemistry
Reagents were purchased from commercial suppliers and used without further purification.
Anhydrous reactions were run under a positive pressure of dry N2. Merck silica gel 60 was used for
1
13
flash chromatography (23-400 mesh). H NMR and C NMR were recorded at 400 and 100 MHz
on a Bruker Advance DPX400. Chemical shifts are reported relative to tetramethylsilane at 0.00
ppm. Mass spectral (MS) data were obtained using Agilent 1100 LC/MSD VL system (G1946C)
with a 0.4 mL/min flow rate using a binary solvent system of 95:5 methanol/water. UV detection
was monitored at 254 nm. Mass spectra were acquired either in positive or in negative mode
scanning over the mass range of 105-1500. High resolution molecular ion determinations (HRMS)
were performed using a Dionex Ultimate 3000 RS UHPLC system coupled to an Orbitrap™ Q-
Exactive high-resolution mass spectrometer (Thermo Scientific) operating in ESI positive full scan
(m/z range 100-800 at resolution 70,000 FWHM at 200 m/z). Melting points were determined on a
Gallenkamp apparatus and are uncorrected. Elemental analyses were performed on a Perkin-Elmer
PE 2004 elemental analyzer and the data for C, H, and N are within 0.4% of the theoretical values.
The chemical purity of the target compounds was determined using the following conditions: an
Agilent 1100 series LC/MSD with a Lichrocart 125-4 Lichrospher 100 RP-18 (4.6 mm x 100 mm, 5

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µm) reversed phase column; method: 86% (v/v) of MeOH in H2O, isocratic, flow rate of 1 mL/min,
UV detector, 254 nm. The purity of each compound was ≥95% in either analysis.
4.1.1. General procedure for the synthesis of aminothiophene precursors 7a-m
Most of these compounds were commercially available or were prepared according to known
procedures [42]. As an example, the preparation of the new compound 7h is reported below.
4.1.1.1. Methyl 2-Amino-5-methyl-4-(n-propyl)thiophene-3-carboxylate (7h). A mixture of 3-
hexanone (1.0 g, 10 mmol), methyl cyanoacetate (882 µL, 10 mmol), sulfur (321 mg, 10 mmol),
and morpholine (872 µL, 10 mmol) in MeOH (4 mL) was refluxed overnight. After cooling, the
reaction mixture was poured into ice (50 g) and the precipitate was filtered and dried under vacuum.
The crude product was purified by silica gel chromatography (PE/AcOEt 9:1 as eluent) to give the
title compound (230 mg, 10%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 5.87 (bs, 2H), 3.79
(s, 3H), 2.59 (t, J = 8.0 Hz, 2H), 2.14 (s, 3H), 1.45 (m, 2H), 0.91 (t, J = 8.0 Hz, 3H).
4.1.2. General procedure for the synthesis of methyl 2-(acylamino)thiophene-3-carboxylate 8a-m,
8o, 8p and related isosteres 10-12.
A solution of the appropriate acyl chloride (adamantane-2-carbonyl chloride or 4-substituted
benzoyl chloride, 1.3 mmol) in dry dioxane (3 mL) was added dropwise to a solution of 7a-m, 15,
16, 17 (1.0 mmol) in 10 mL of the same solvent, maintained at 70 °C. After the addition was
complete, the mixture was refluxed until the formation of hydrogen chloride stopped (1-18 h). The
solution was concentrated in vacuo and the residue was dissolved in DCM (30 mL), washed with
NaHCO3 solution (10 mL), dried over anhydrous Na2SO4, and evaporated to dryness. The crude
product was purified by recrystallization from MeOH or flash column chromatography on silica gel
using the reported eluent system.

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4.1.2.1. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]thiophene-3-carboxylate (8a). Yield: 50%.
Mp 207-208 °C. 1H NMR (400 MHz, CDCl3): δ 7.17 (d, J = 5.6 Hz, 1H), 6.70 (d, J = 5.6 Hz, 1H),
3.88 (s, 3H), 2.10-1.99 (m, 9H), 1.79-1.72 (m, 6H). MS (ESI): m/z 342 [M + Na]+.
4.1.2.2. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-5-methylthiophene-3-carboxylate (8b).
Eluent: DCM. Yield: 56%. Mp 133-134 °C. 1H NMR (400 MHz, CDCl3): δ 11.10 (br s, 1H), 6.76
(s, 1H), 3.80 (s, 3H), 2.30 (s, 3H), 2.20 (m, 7H), 2.04 (m, 2H), 1.93 (m, 6H). MS (ESI): m/z 334 [M
+ H]+, 356 [M + Na]+, 372 [M + K]+.
4.1.2.3. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-5-(n-propyl)-thiophene-3-carboxylate (8c).
Eluent: DCM. Yield: 56%. Mp 124-127 °C. 1H NMR (400 MHz, CDCl3): δ 11.12 (br s, 1H), 6.78
(s, 1H), 3.80 (s, 3H), 2.61-2.58 (t, J = 7.4 Hz, 2H), 2.03 (m, 2H), 1.93 (m, 7 H), 1.69 ( m, 6H), 1.62-
1.56 (q, J = 7.3 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). MS (ESI): m/z 362 [M + H]+, 384 [M + Na]+, 400
[M+K]+.
4.1.2.4. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-5-phenylthiophene-3-carboxylate (8d).
1
Yield: 69%. Mp 167-168 °C. H NMR (400 MHz, CDCl3): δ 11.23 (br s, 1H), 7.52-7.50 (m, 2H),
7.32-7.28 (m, 2H), 7.19 (m, 2H), 3.85 (s, 3H), 2.06 (m, 2H), 1.96 (m, 6H), 1.72 (m, 7H). MS (ESI):
m/z 418 [M + Na]+.
4.1.2.5. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-4,5-dimethylthiophene-3-carboxylate (8e).
1
Yield: 84%. Mp 155-156 °C. H NMR (400 MHz, CDCl3): δ 3.87 (s, 3H), 2.23 (s, 3H), 2.21 (s,
3H), 2.12-1.96 (m, 9H), 1.78-1.72 (m, 6H). MS (ESI): m/z 348 [M + H]+, 370 [M + Na]+.
4.1.2.6. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-5-ethyl-4-methylthiophene-3-carboxylate
1
(8f). Yield: 13%. Mp 99-100 °C. H NMR (400 MHz, CDCl3): δ 11.54 (s, 1H), 3.88 (s, 3H), 2.67

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(q, J = 7.6 Hz, 2H), 2.23 (s, 3H), 2.09 (br s, 3H), 1.97 (br s, 6H), 1.75 (br s, 6H), 1.19 (t, J = 7.6 Hz,
3H). MS (ESI): m/z 362 [M + H]+, 384 [M + Na]+, 400 [M + K]+.
4.1.2.7. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-4-ethyl-5-methylthiophene-3-carboxylate
(8g). Yield: 98%. Mp 112-116 °C. 1H-NMR (400 MHz, CDCl3) δ 11.49 (s, 1H), 3.83 (s, 3H), 2.65
(q, J = 8.0 Hz, 2H), 2.18 (s, 3H), 2.03 (br s, 3H), 1.91 (br s, 6H), 1.69 (br s, 6H), 0.98 (t, J = 8.0 Hz,
3H). MS (ESI): m/z 362 [M + H] +.
4.1.2.8.
Methyl
2-[((Adamantan-1-yl)carbonyl)amino]-5-methyl-4-(n-propyl)-thiophene-3-
carboxylate (8h). Yield: 95%. Mp 75-76 °C. 1H NMR (400 MHz, CDCl3): δ 3.88 (s, 3H), 2.67 (t, J
= 8.1 Hz, 2H), 2.24 (s, 3H), 2.09 (m, 3H), 1.98 (m, 6H), 1.75 (m, 6H), 1.46 (m, 2H), 0.90 (t, J = 8.0
Hz, 3H). MS (ESI): m/z 376 [M + H] +, 398 [M + Na] +, 414 [M + K]+.
4.1.2.9. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-5-(n-butyl)-4-methylthiophene-3-carboxylate
1
(8i). Eluent: DCM. Yield: 51%. Mp 80-82 °C. H NMR (400 MHz, CDCl3): δ 3.82 (s, 3H), 2.60-
2.56 (t, J= 7.5 Hz, 3H), 2.17 (s, 3H), 2.03 (m, 2H), 1.92 (m, 7H), 1.69 (m, 6H), 1.53-1.46 (m, 2H),
1.33-1.24 (m, 2H), 0.86-0.82 (t, J= 7.2 Hz , 2H). MS (ESI): m/z 391 [M + H]+, 413 [M + Na]+, 429
[M + K]+.
4.1.2.10. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-4-phenylthiophene-3-carboxylate (8j).
1
Yield: 58%. Mp 194-196 °C. H NMR (400 MHz, CDCl3): δ 11.23 (br s, 1H), 7.29-7.19 (m, 5H),
6.51 (s, 1H), 3.63 (s, 3H), 2.06 (m, 2H), 1.96 (m, 7H), 1.72 (m, 6H). MS (ESI): m/z 418 [M + Na]+.
4.1.2.11.
Methyl
2-[((Adamantan-1-yl)carbonyl)amino]-5-(n-pentyl)-4-phenylthiophene-3-
carboxylate (8k). Eluent: DCM. Yield: 69%. Mp 103-104 °C. 1H NMR (400 MHz, CDCl3): δ 7.29-
7.22 (m, 3H), 7.07-7.05 (d, J = 6.9 Hz, 2H), 3.40 (s, 3H), 2.41-2.38 (t, J = 7.8 Hz, 3H), 2.05 (m,

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2H), 1.95-1.94 (m, 7H), 1.71 (m, 6H), 1.50-1.42 (m, 2H), 1.13-1.11 (m, 4H), 0.75-0.72 (m, 2H).
MS (ESI): m/z 466 [M + H]+, 488 [M + Na]+, 504 [M + K]+.
4.1.2.12. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
1
carboxylate (8l). Yield: 56%. Mp 90-91 °C. H NMR (400 MHz, CDCl3): δ 11.51 (s, 1H), 3.86 (s,
3H), 2.74-2.72 (m, 2H), 2.63-2.60 (m, 2H), 2.09-1.97 (m, 9H), 1.77-1.72 (m, 10H). MS (ESI): m/z
396 [M + Na]+.
4.1.2.13. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-4,7-dihydro-5H-thieno[2,3-c]thiopyran-3-
carboxylate (8m). Yield: 75%. Mp 149-150 °C. 1H NMR (400 MHz, CDCl3): δ 3.80 (s, 3H), 3.00
(m, 2H), 2.83 (m, 2H), 2.14 (m, 3H), 1.92 (m, 6H), 1.69 (m, 6H). MS (ESI): m/z 414 [M + Na]+.
4.1.2.14. Methyl 2-[((4-Chlorophenyl)carbonyl)amino]-4-ethyl-5-methylthiophene-3-carboxylate
(8o). Its physicochemical properties have been described previously [26].
4.1.2.15. Methyl 4-Ethyl-2-[((4-fluorophenyl)carbonyl)amino]-5-methylthiophene-3-carboxylate
1
(8p). Yield: 90%. Mp 126-128 °C. H NMR (400 MHz, CDCl3): δ 8.02-7.98 (m, 2H), 7.19-7.15
(m, 2H), 3.92 (s, 3H), 2.29 (s, 3H), 2.74 (q, J = 7.4 Hz, 2H), 1.07 (t, J = 7.5 Hz, 3H). MS (ESI): m/z
322 [M + H]+, 344 [M + Na]+.
4.1.2.16. Methyl 3-[((Adamantan-1-yl)carbonyl)amino]-1H-pyrrole-2-carboxylate (10). Eluent:
PE/AcOEt (2:1). Yield: 46%. Mp 221-224 °C. 1H NMR (200 MHz, CDCl3): δ 9.52 (s, 1H), 8.89 (s,
1H), 7.06-7.03 (m, 1H), 6.81-6.78 (m, 1H), 3.84 (s, 3H), 2.05-1.96 (m, 9H), 1.71-1.65 (m, 6H). MS
(ESI): m/z 303 [M + H]+.

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4.1.2.17. Methyl 4-[((Adamantan-1-yl)carbonyl)amino]-1-methyl-1H-pyrazole-5-carboxylate (11).
1
Yield: 64%. Mp 205-206 °C. H NMR (400 MHz, CDCl3): δ 9.06 (s, 1H), 8.19 (s, 1H), 4.00 (s,
3H), 3.90 (s, 3H), 2.00 (s, 3H), 1.87 (m, 6H), 1.70-1.63 (m, 6H). MS (ESI): m/z 319 [M + H]+.
4.1.2.18. Methyl 4-[((Adamantan-1-yl)carbonyl)amino]-1-methyl-1H-pyrazole-3-carboxylate (12).
1
Mp 214-216 °C. H NMR (400 MHz, CDCl3): δ 9.26 (s, 1H), 8.13 (s, 1H), 3.90 (s, 3H), 3.85 (s,
3H), 2.02 (s, 3H), 1.90 (m, 6H), 1.69 (m, 6H). MS (ESI): m/z 319 [M + H]+.
4.1.3. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]-4,7-dihydro-5H-thieno[2,3-c]thiopyran-3-
carboxylate 6,6-dioxide (8n)
A solution of Oxone (466 mg, 0.75 mmol) in water (3 mL) was slowly added to a solution of 8m
(100 mg, 0.25 mmol) in MeOH (3 mL) and the mixture was maintained under stirring at room
temperature overnight. After evaporation of MeOH, the aqueous phase was extracted with DCM
(3x10 mL) and the organic layer was washed with water (10 mL) and dried over anhydrous sodium
sulfate. Evaporation of the solvent left a residue which was purified by flash chromatography on
silica gel with DCM/MeOH (95:5) as eluent. Fractions containing the higher spot were pooled and
solvent was removed under reduced pressure. The solid residue was further purified by trituration
with PE to give 76 mg (70% yield) of the sulfone 8n. Mp 263-264 °C. 1H NMR (400 MHz, CDCl3):
δ 7.20 (s, 1H), 4.14 (s, 2H), 3.85 (s, 3H), 3.42 (m, 2H), 3.16 (m, 2H), 2.07 (m, 3H), 1.92 (m, 6H),
1.71 (m, 6H). MS (ESI): m/z 446 [M + Na]+, 462 [M + K]+.
4.1.4. Methyl 2-[((Adamantan-1-yl)carbonyl)amino]pyridine-3-carboxylate (9)
A solution of 1-adamantanecarbonyl chloride (198 mg, 1 mmol) in dry DCM (1 mL) was slowly
added to a cooled (0-5 °C) solution of methyl 2-aminopyridine-3-carboxylate (14) (152 mg, 1
mmol) and TEA (210 µL, 1.5 mmol) in dry DCM (3 mL). After stirrring for 18 h, the solution was
diluted with DCM (5 mL), washed with saturated solution of NaHCO3 (5 mL) and brine (5 mL),

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then dried over anhydrous sodium sulfate, and evaporated to dryness. The crude residue was
purified by recrystallization from MeOH to afford the title compound in 40% yield. Mp 183-184
1
°C. H NMR (200 MHz, CDCl3): δ 10.88 (s, 1H), 8.64 (m, 1H), 8.30-8.27 (m, 1H), 7.04-7.01 (m,
1H), 3.94 (s, 3H), 2.10-2.02 (m, 9H), 1.76-1.74 (m, 6H). MS (ESI): m/z 337 [M+Na]+.
4.1.5. Methyl 5-[((Adamantan-1-yl)carbonyl)amino]-3-methylisoxazole-4-carboxylate (13)
Sodium hydride powder (31 mg, 1.29 mmol) was added to a solution of 18 (100 mg, 0.64 mmol) in
dry THF (4 mL) and the mixture was stirred at rt under nitrogen for 15 min. Then a solution of 1-
adamantanecarbonyl chloride (140 mg, 0.7 mmol) in dry THF (2 mL) was added dropwise and the
reaction mixture was stirred at rt for 30 min and then was refluxed for 2 h. After removal of most of
solvent, the residue was diluted with water (15 mL), neutralized by adding 1 N HCl, and extracted
with DCM (3x10 mL). The organic phase was washed with water (10 mL), dried over anhydrous
sodium sulfate, and evaporated to give a crude solid which was purified by flash column
chromatography on silica gel (PE/AcOEt 1:1 as eluent) to obtain pure 13 in 41% yield. Mp 168-169
1
°C. H NMR (400 MHz, CDCl3): δ 10.06 (s, 1H), 3.82 (s, 3H), 2.31 (s, 3H), 1.96 (br s, 3H), 1.90
(br s, 6H), 1.70-1.68 (m, 6H). MS (ESI): m/z 320 [M + H]+.
4.1.6. Methyl 5-Amino-3-methylisoxazole-4-carboxylate (18)
A solution of methyl cyanoacetate (2 mL, 20 mmol) and triethyl orthoacetate (4 mL, 22 mmol) in
acetic anhydride (12 mL) was refluxed for 3 h. After cooling, the solution was poured into ice (150
g) and the precipitated solid was filtered and washed with water (100 mL). The crude product was
purified by trituration with diethyl ether to give 616 mg (20% yield) of methyl 2-cyano-3-ethoxy-2-
butenoate (19). Mp 87-88 °C. A solution of 19 (600 mg, 3.6 mmol) and hydroxylamine
hydrochloride (380 mg, 5.4 mmol) in pyridine (6 mL) was refluxed for 4 h, then poored into ice (50
g) and extracted with AcOEt (3x20 mL). The organic layer was separated, washed with brine (20
mL), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was

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purified by flash column chromatography on silica gel eluting with PE/AcOEt 1:1 to afford 214 mg
(38% yield) of 18 as a colorless solid, mp 137-138 °C (lit. [33]: 140-141 °C).
4.1.7. Methyl 2-[(Phenoxycarbonyl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate
(23)
To a solution of 7l (300 mg, 1.42 mmol) in dry dioxane (6 mL) warmed at 70 °C were added
DIPEA (495 µL, 2.84 mmol) and then phenyl chloroformate (267 µL, 2.13 mmol). After heating at
100 °C overnight, volatiles were removed under vacuum and the residue was purified by flash
column chromatography on silica gel (PE/DCM 2:1 as eluent) to provide the title compound 23 in
1
99% yield (465 mg) as a white solid, mp 124-125 °C. H NMR (400 MHz, CDCl3): δ 10.75 (br s,
1H), 7.34-7.30 (m, 2H), 7.19-7.14 (m, 3H), 3.81 (s, 3H), 2.70- 2.68 (m, 2H), 2.58-2.55 (m, 2H),
1.73-1.72 (m, 4H). MS (ESI): m/z 354 [M+Na]+.
4.1.8. General Procedure for the preparation of ureido derivatives 20-22
1-Aminoadamantane, morpholine, or piperidine (1.3 mmol) was added to a solution of 23 (331 mg,
1 mmol) in dioxane (4 mL) and the reaction mixture was refluxed for the appropriate time (18 h, 5
h, and 1 h, respectively). After cooling to rt, the solution was diluted with DCM (10 mL) and
washed with 2 N NaOH (5 mL), 2 N HCl (5 mL), and brine (5 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated to dryness lo leave a residue which was purified as
described below.
4.1.8.1. Methyl 2-[((1-Adamantylamino)carbonyl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (20). Crystallized from MeOH. Yield: 87%. White solid, mp 125-129 °C. 1H NMR 400
MHz, CDCl3): δ 10.31 (br s, 1H), 3.63 (s, 3H), 2.64-2.62 (m, 2H), 2.52-2.49 (m, 2H), 2.02-1.94 (m,
9H), 1.72-1.61 (m, 11H). MS (ESI): m/z 389 [M+H]+, 411 [M+Na]+.