Design, Synthesis, and in vitro Evaluation of P2X7 Antagonists

Article abstract of DOI:10.1002/cmdc.202000303

The P2X7 receptor is a promising target for the treatment of various diseases due to its significant role in inflammation and immune cell signaling. This work describes the design, synthesis, and in vitro evaluation of a series of novel derivatives bearing diverse scaffolds as potent P2X7 antagonists. Our approach was based on structural modifications of reported (adamantan-1-yl)methylbenzamides able to inhibit the receptor activation. The adamantane moieties and the amide bond were replaced, and the replacements were evaluated by a ligand-based pharmacophore model. The antagonistic potency of the synthesized analogues was assessed by two-electrode voltage clamp experiments, using Xenopus laevis oocytes that express the human P2X7 receptor. SAR studies suggested that the replacement of the adamantane ring by an aryl-cyclohexyl moiety afforded the most potent antagonists against the activation of the P2X7 cation channel, with analogue 2-chloro-N-[1-(3-(nitrooxymethyl)phenyl)cyclohexyl)methyl]benzamide (56) exhibiting the best potency with an IC₅₀ value of 0.39 μΜ.

Full text of DOI:10.1002/cmdc.202000303

Accepted Article
Title: Design, synthesis, and in vitro evaluation of novel P2X7
antagonists
Authors: Maria Koufaki, Dimitra Pournara, Anna Durner, Eftichia Kritsi,
Alexios Papakostas, Panagiotis Zoumpoulakis, and Annette
Nicke
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000303
Link to VoR: https://doi.org/10.1002/cmdc.202000303
01/2020

10.1002/cmdc.202000303
ChemMedChem
FULL PAPER
Design, synthesis, and in vitro evaluation of novel P2X7
antagonists
Dimitra T. Pournara,[a] Anna Durner,[b] Eftichia Kritsi,[a] Alexios Papakostas,[a] Panagiotis
Zoumpoulakis,[a] Annette Nicke,[b]* and Maria Koufaki[a]*
[a]
Dr. D. T. Pournara, Dr. E. Kritsi, M.Sc. A. Papakostas, Dr. P. Zoumpoulakis, Dr. M. Koufaki*
Institute of Chemical Biology
National Hellenic Research Foundation,
48 Vassileos Constantinou Ave., 11635 Athens, Greece
M. Sc. A. Durner, Prof. Dr. nat. phil. A. Nicke*
[b]
Walther-Straub-Institut für Pharmakologie und Toxikologie
Ludwig-Maximilians-Universität München
Nußbaumstr. 26, 80336 München, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: The P2X7 receptor is a promising target for the
treatment of various diseases due to its significant role in
inflammation and immune cell signaling. This work describes the
design, synthesis, and in vitro evaluation of a series of novel
derivatives bearing diverse scaffolds as potent P2X7 antagonists.
Our approach was based on structural modifications of reported
(adamantan-1-yl)methyl-benzamides able to inhibit the receptor
activation. The adamantane moieties and the amide bond were
replaced, and the replacements were evaluated by a ligand-based
pharmacophore model. The antagonistic potency of the
synthesized analogues was assessed by two-electrode voltage
clamp experiments, using Xenopus laevis oocytes that express the
human P2X7 receptor. SAR studies suggested that the
replacement of the adamantane ring by an aryl-cyclohexyl moiety
afforded the most potent antagonists against the activation of the
P2X7 cation channel, with analogue 2-chloro-N-[1-(3-
to desensitization.[9] This non-selective macropore facilitates
the influx of large ions and hydrophilic solutes (up to 900 Da)
that may cause cell death due to apoptosis and necrosis.[13]
Specifically, it has been shown that the efflux of K+ ions through
the P2X7 macropore triggers the activation of the most widely
characterized inflammasome,[14] Nod-like receptor family pyrin
domain containing protein 3 (NLRP3), leading to the ultimate
release of pro-inflammatory factors such as active caspase-1
and interleukin-1β (IL-1β).[15]
During the last decade, numerous classes of P2X7
antagonists featuring drug-like properties have been
generated.[16] Particularly, three small antagonists have
entered clinical trials for the treatment of CNS disorders.[17]
However, AZD9056 and CE-224535 were proven inefficient in
Phase II clinical studies for rheumatoid arthritis,[18,19] while
GSK1482160 was proved to be unsafe for neuropathic pain
inhibition.[20]
(nitrooxymethyl)phenyl)cyclohexyl)methyl]benzamide
exhibiting the best potency with an IC50 value of 0.39 μΜ.
(56)
In this study, we report the design, synthesis, and in vitro
evaluation of novel P2X7 antagonists based on diverse
structural motifs and supported by structure–activity
relationship (SAR) data. The novel analogues bear
substituents that could release nitric oxide (NO) or hydrogen
sulfide (H2S),[21] aiming to enhance their anti-inflammatory
effect, as experimental data have shown that both NO and H2S
downregulate the IL-1β secretion and caspase-1 activation.[22–
Introduction
P2X receptors are trimeric, ligand-gated, non-selective
cation channel receptors with seven clearly established
subtypes (P2X1–7) that are involved in diverse physiological
functions, including the cardiovascular, neuronal, and immune
system.[1] The P2X7 subtype is the largest member of the P2X
receptor family, composed of a 595-amino acid polypeptide
chain.[2] Its long intracellular C terminus distinguishes the P2X7
receptor from the other P2X family members and has been
reported to be involved in pore formation,[3] protein-protein
interactions,[4] as well as activation of downstream signaling
pathways[5] such as cytokine release, modulation of cell
proliferation and phagocytosis,[6] and cell death. Moreover,
P2X7 is present in a wide variety of cells in the human body[7]
and has been associated with multiple diseases, including
inflammatory[8] and CNS disorders,[9,10] inflammatory pain,
rheumatoid arthritis,[11] and cardiovascular diseases.[12]
25]
A recent study also revealed that P2X7 can be blocked by
endogenous H2S, thus limiting the IL-1β secretion involved in
the pathogenesis of secondary brain injury.[26]
Results and Discussion
Experiment design
Our approach towards the development of potent P2X7
antagonists was based mainly on reported adamantane
analogues able to inhibit the receptor activation (Figure 1).
Specifically, analogue AZ1, developed by Furber et al. from
Astra Zeneca R&D U.K., has shown significant selectivity to
human P2X7 (hP2X7) compared to related P2X receptors.[27]
Moreover, an X-ray structure of AZ1 revealed that the amide
carbonyl is twisted by 44° out of the plane of the phenyl group
The activation of P2X7 requires high extracellular ATP
concentrations (1 mM), in contrast to concentrations <100 μΜ
needed to activate other P2X subtypes. Τhe activation of P2X7
leads to the formation of a large conductance pore rather than
1
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10.1002/cmdc.202000303
ChemMedChem
FULL PAPER
by the ortho-chloro substituent, supporting its P2X7 selectivity.
This structural feature has been observed in all the analogues
of these series and was also associated with high potency at
the P2X7 receptor.[27]
Adamantane-based putative P2X7 antagonists
Initially, three adamantyl amide analogues were
synthesized; AZ1, which was used as positive control
throughout the study, and two additional compounds bearing a
group that could release NO (4) or H2S (5) at the 5-position of
the phenyl ring, respectively. 1-Adamantylmethanamine (1)
was obtained in quantitative yield from 1-adamantaneacetic
acid via a Schmidt reaction. Coupling of 1 with 2-chloro-5-
methylbenzoic acid in the presence of 1-ethyl-3-(3-
(dimethylamino)propyl) carbodiimide hydrochloride (EDC
hydrochloride) and triethylamine (Et3N) gave amide 2. Radical
bromination of the methyl group with azobisisobutyronitrile
(AIBN) as initiator and N-bromosuccinimide (NBS) afforded
bromide 3, which then reacted with piperazine,[27]
methanesulfonothioate sodium salt, or AgNO3 to afford
analogues AZ1, 4, and 5, respectively (Scheme 1).
NH2
N
O
H
N
H
H
N
Cl
N
O
Cl
O
O
Cl
O
O
N
H
O
N
N
OH
H
N
H
N
H
H
Cl
N
O
NH
N
H
N
N
N
H
O
H
O
N
NH2
OH
a
b
O
Cl
AZ1
2
1
Figure 1. Structures of known adamantane-based P2X7 antagonists.
c
ONO2
Br
Additional structural features of the reported analogues
(Figure 1) were also exploited to design novel adamantane-
based antagonists. Particularly, it has been observed that the
methylene linker to adamantane is essential for high
antagonistic potency,[28,29] while the potency is not affected by
reversing the connectivity of the amide.[27] However, chain
extension between the amide and the adamantane from
methylene to ethylene resulted in a significant decrease in the
antagonistic potency.[29] Similarly, N-methyl substituted amides
were not tolerated, whereas an ortho-substitution of the aryl
moiety by a chloro or methyl group was proven to be essential
for efficient P2X7 inhibition.[30] It should also be noted that the
ortho-chloro substitution has been related to enhanced activity
of other benzamide classes.[31–33]
Therefore, in order to develop novel P2X7 antagonists, the
present SAR study involved structural modifications (Figure 2)
based on these earlier findings and the main scaffold of AZ1.
Furthermore, the replacement of the amide bond and the
adamantane moieties was evaluated using a ligand-based
pharmacophore model (Supporting Information), utilized as a
3D search query to screen the ZINC chemical database that
led to the identification of analogues with the most promising
pharmacophore-fit scores (Tables 1 and 2).
H
H
N
N
e
f
O
Cl
O
Cl
3
4
d
NH
N
O
S
S
O
H
N
H
N
O
Cl
O
Cl
AZ1
5
Scheme 1. Reagents and conditions: (a) NaN3, H2SO4, H2O, CHCl3, 50 °C–
r.t., 5.5 h, 86%; (b) 2-chloro-5-methylbenzoic acid, EDC hydrochloride, Et3N,
CH2Cl2, r.t., 22 h, 63%; (c) NBS, AIBN, CHCl3, reflux, 4 h, obtained as
mixture with 2; (d) piperazine, CH3OH, 0 °C–r.t., 3 h, 29%; (e) AgNO3,
CH3CN, 60 °C, 2 h, 50%; (f) sodium methanesulfonothioate; DMF, 60 °C,
5 h, 58%.
Table 1. Pharmacophore-fit scores of the synthesized adamantane-based
analogues.
Adamantane-
based
analogues
Adamantane-
based
analogues
Pharmacophore-
fit score
Pharmacophore-
fit score
Cl or CH3
Cl
NO or H2S
methylene
linker
H
donors
N
AZ1
4
36.28
44.57
46.12
37.34
36.03
35.96
19a
19b
23b
24a
24b
45.48
37.46
44.78
25.49
25.39
O
replacement of
the amide bond
5
replacement of
adamantane
11
Figure 2. Structural modifications of analogue AZ1. The structural features
required for high antagonistic potency are indicated in red, purple, and grey.
Green and blue colors indicate the structural characteristics that were
replaced during the design of the novel analogues.
18a
18b
2
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Replacement of the amide bond by the 1,2,3-triazole
ring
cross-coupling reaction to give the intermediates 13a and 13b,
respectively. After reducing the methyl esters with LiAlH4 in
THF, the TMS-protective group was cleaved under basic
conditions. The isolated alkynes (15a–b) afforded triazoles 16a
and 16b after a click reaction with azide 9. Bromination of the
benzyl alcohols was achieved using PBr3 in anhydrous CH2Cl2.
Treatment of analogues 17a and 17b with AgNO3 or sodium
methanesulfonothioate yielded compounds 18a/19a and
18b/19b, respectively (Scheme 3).
Although hundreds of benzamide analogues have been
reported as P2X7 antagonists in research papers and
patents,[34] and 1,2,4-triazoles have been reported as isosteres
of tetrazoles several years ago,[35] information on derivatives in
which the amide bond is replaced by the bioisosteric 1,2,3-
triazole is scarce.[36] Given also that the use of 1,2,3-triazoles
has attracted increasing research interest for the development
of new therapeutic agents,[37] we replaced the amide bond of
AZ1 by its bioisostere 1,2,3-triazole ring. The new analogues
preserved the necessary structural features, i.e., a relatively
bulky ortho-substitution (chloro or methyl) and a nitrate ester
or a (methylsulfonyl)thio moiety at the meta-position of the
phenyl ring.
OH
OH
O
O
O
OH
a,b
c,d
e
N
N
N
R
I
R
R
R
TMS
13a-b
15a-b
16a-b
f
ONO2
Br
As shown in Scheme 2, adamantyl azide 9 was synthesized
using the commercially available 1-adamantanecarboxylic
acid. The 1,2,3-triazoles 11, 18a–b, 19a–b, and 24a–b were
g
N
N
N
N
N
N
R
R
then obtained via
cycloaddition (click reaction) between azide
appropriate aryl alkyne under microwave conditions.
Compound 11, bearing only an ortho-chloro-substituted
benzene, was synthesized from 1-chloro-2-iodobenzene,
a
copper-catalyzed azide-alkyne
and the
18a-b
17a-b
h
9
12a-19a. R = Me
12b-19b.
O
S
S
R = Cl
O
N
N
which was coupled with ethynyltrimethylsilane via
a
N
R
Sonogashira reaction using Pd(II)/Cu(I) as the catalytic
system. Cleavage of the TMS group by tetrabutylammonium
fluoride (TBAF) in THF afforded an intermediate alkyne, which
was not isolated but used directly for the cycloaddition due to
its low boiling point (Scheme 2).
19a-b
Scheme 3. Reagents and conditions: (a) CH3OH, H2SO4, reflux, 24 h, 98%;
(b) ethynyltrimethylsilane, CuI, Pd(PPh3)2Cl2, Et3N/THF, 60 °C, 86–95%; (c)
LiAlH4, THF, 0 °C–r.t., 45 min; (d) KOH (aq.), CH3OH, r.t., 15 min, 99% over
two steps; (e) 9, sodium ascorbate, CuSO4·5H2O, tBuOH/H2O, mw, 80 Watt,
90 °C, 30 min, 79–85%; (f) PBr3, CH2Cl2, 0 °C–r.t., 2 h, 84–91%; (g) AgNO3,
CH3CN, 60 °C, dark, 2–17 h, 35–71%; (h) sodium methanethionosulfonate,
DMF, 70 °C, 2–5 h, 23–51%.
O
OH
OH
OMs
a,b
c
7
8
d
N3
To investigate the effect of bulky substituents at the meta-
position of the phenyl ring, 3-(hydroxymethyl)-4-phenyl-1,2,5-
oxadiazole 2-oxide ring (20), a well-known NO donor,[38] was
also employed. Compound 20 was synthesized from cinnamyl
alcohol after cyclization with NaNO2 and glacial CH3COOH.[39]
Despite the reported tautomerism,[39] only the indicated isomer
20 was obtained under these conditions,[40] which was used for
the synthesis of analogues 24a–b (Scheme 4). Then, the TMS-
protected methyl esters 13a–b yielded alkynes 21a–b upon
treatment with a saturated aqueous KOH solution. A click
reaction with 9 followed and the obtained esters (22a–b) were
hydrolyzed to the corresponding carboxylic acids with LiOH
(2 N). The subsequent Steglich esterification of 23a–b with 20
in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and 4-
dimethylaminopyridine (DMAP) afforded adamantyl analogues
24a and 24b.
Cl
9
Cl
I
N
e
f
Cl
Cl
N
N
g
10
TMS
H
11
Scheme 2. Reagents and conditions: (a) EtOH, H2SO4, 70 °C, 18 h, 95%;
(b) LiAlH4, THF, 0 °C–r.t., 1.5 h, 97%; (c) methanesulfonyl chloride, Et3N,
0 °C–r.t., 21 h, 95%; (d) NaN3, DMF, mw, 100 Watt, 130 °C, 1 h, 84%; (e)
ethynyltrimethylsilane, CuI, Pd(PPh3)2Cl2, Et3N/THF, 60 °C, 22 h, 64%; (f)
TBAF, THF, r.t., 1 h; (g) sodium ascorbate, CuSO4·5H2O, mw, 80 Watt,
90 °C, 30 min, 15%.
For the preparation of analogues 18a–b and 19a–b, the
methyl esters of 4-methyl-3-iodobenzoic acid or 4-chloro-3-
iodobenzoic acid were coupled with ethynyltrimethylsilane in
the presence of PdCl2(PPh3)2 and Cu(I) via a Sonogashira
3
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OH
N
O
N
a
O
OH
O
O
O
O
O
OH
20
O
O
O
O
N
R
N
e
R
R
O
O
b
c
d
N
N
N
N
N
N
N
N
13a-24a.
R = Me
13b-24b. R = Cl
N
TMS
R
13a-b
22a-b
21a-b
23a-b
24a-b
Scheme 4. Reagents and conditions: (a) glacial acetic acid, NaNO2 (aq.), 0 °C–r.t., 24 h, 50%; (b) KOH (aq.), CH3OH, r.t., 20–30 min, 70–98%; (c) 9, sodium
ascorbate, CuSO4·5H2O, tBuOH/H2O, mw, 80 Watt, 90 °C, 30 min, 91–93%; (d) LiOH (2 N), THF, r.t., 24 h, 87–95%; (e) DMAP, DCC, CH2Cl2, 0 °C–r.t., 2 h, 48–
71%.
Structurally modified putative P2X7 antagonists
Table 2. Pharmacophore-fit scores of the structurally modified analogues.
Structurally
modified
analogues
Structurally
modified
analogues
The adamantane ring has been earlier replaced by other
polycyclic scaffolds[41] or fluorine bioisosteres.[42] In our study,
the ZINC chemical database was virtually screened to identify
alternative scaffolds of adamantane, indicating the (i) 3-fluoro-
4-(trifluoromethyl)phenyl, (ii) aryl-substituted cyclohexyl, and
(iii) 1,2,5-oxadiazole-2-oxide (furoxan) moieties as potential
units (Figure 3). Especially the heteroaryl-substituted
cyclohexyl group has earlier been employed for research on
the P2X7 receptor.[43] Examination of their structural and
pharmacophore features revealed that all three moieties
shared the necessary hydrophobic features, while the fluorine
atoms along with the nitrogen and oxygen heteroatoms fulfilled
the required hydrogen bond acceptor (HBA) features.
Pharmacophore-
fit score
Pharmacophore-
fit score
AZ1
30
36.28
35.67
62.13
62.35
52.28
52.77
53.63
45.31
60
61
63
64
67
68
70
43.69
35.81
44.27
43.32
62.39
46.22
52.53
35
42a
42b
43
53
methylene
linker
methylene
linker
R
R
56
O
N
N O
F
F
R
aryl-substituted cyclohexyl
F
methylene
linker
F
Replacement of adamantane by the 3-fluoro-4-
(trifluoromethyl)phenyl moiety
3-fluoro-4-(trifluoromethyl)phenyl
1,2,5-oxadiazole-2-oxide
Figure 3. The 3-fluoro-4-(trifluoromethyl)phenyl, aryl-substituted cyclohexyl,
and furoxan scaffolds used for the replacement of the adamantane ring.
In line with the adamantane-based analogues, both amides
and 1,2,3-triazoles were synthesized, while the ortho- and
meta-substitutions on the phenyl ring were maintained. 2-(3-
Fluoro-4-(trifluoro-methyl)phenyl)acetic acid yielded amine 25
through an one-step Schmidt reaction. The methyl ester of 2-
chloro-5-methylbenzoic acid (26) was prepared upon
treatment with CH3OH and H2SO4. Bromination of the aryl
methyl group followed using AIBN and NBS in anhydrous
CHCl3, giving the intermediate 27. Hydrolysis of the carboxylic
acid group with LiOH (2 N) in THF resulted in the concurrent
replacement of the bromine atom by a hydroxyl group. The
isolated benzoic acid 28 was then successfully coupled with 25
using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b] pyridinium 3-oxide hexafluorophosphate (HATU) and N,N-
diisopropylethylamine (DIPEA) in DMF.[44] The in situ formation
of the bromide using PPh3 and NBS in an anhydrous
CH3CN/CH2Cl2 mixture and the subsequent addition of AgNO3
afforded the desired compound 30 (Scheme 5).[45]
Thus, a series of novel, structurally modified 1,2,3-triazoles
and amides that exhibited the highest pharmacophore-fit
scores (Table 2) were synthesized by replacing the
adamantane ring with the identified units (Figure S83). In
agreement with the above results, the nitrogen atoms of the
triazole analogues serve as HBAs, while the substituted
benzene rings display hydrophobic interactions. In addition,
the 3-fluoro-4-(trifluoromethyl)phenyl derivatives 30, 35, 42a–
b, and 43 display three hydrophobic regions and two HBAs. In
the aryl-substituted cyclohexyl derivatives 53, 56, 67, 68, and
70, the cyclohexyl ring does not develop interactions, whereas
the carbonyl oxygen atom of the amide serves as HBA and the
halogen benzene ring constitutes a hydrophobic region (Figure
S83) with the fluorine atoms in 67, 68, and 70 serving as HBAs.
Additionally, the pharmacophore model of the furoxan
analogues (60, 61, 63, and 64) suggests that the ring’s
heteroatoms, the carbonyl oxygen atom, and the triazole ring’s
nitrogen atom act as HBAs, while the halogen benzene rings
develop hydrophobic interactions (Figure S83).
4
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OH
O
O
OH
Br
N3
NH2
OH
a-c
d
e
a
N
OH
N
N
R
F
F
F
F
F
F
25
39
CF3
CF3
CF3
38
CF3
CF3
F3
C
40a-b
H
N
e
O
Cl
f
ONO2
Br
F
OH
g
CF3
HO
O
HO
O
b-d
29
N
N
N
N
N
R
N
R
F
F
41a-b
42a-b
Cl
Cl
28
F3
C
F3C
f
h
ONO2
O
S
S
O
40a-42a.
40b-42b.
R = Me
R = Cl
H
N
O
Cl
N
N
N
F
F
CF3
30
43
F3C
Scheme 5. Reagents and conditions: (a) H2SO4, NaN3, CHCl3, H2O, 50 °C,
5 h, 50%; (b) CH3OH, H2SO4, 70 °C, 18 h, 77%; (c) NBS, AIBN, CHCl3,
reflux, 2.5 h, 53%; (d) LiOH (2 N), THF, 40 °C, 20 h, 92%; (e) HATU, DIPEA,
DMF, r.t., 72 h, 41%; (f) PPh3, NBS, AgNO3, dark, CH2Cl2/CH3CN, 0–50 °C,
24 h, 39%.
Scheme 7. Reagents and conditions: (a) CH3OH, H2SO4, 60 °C, 24 h, 83%;
(b) LiAlH4, THF, 0 °C–r.t., 1.5 h, 82%; (c) PBr3, CH2Cl2, 0 °C–r.t., 2.5 h, 26%;
(d) NaN3, DMF, 40 °C, 3 h, 50%; (e) 15a–b, sodium ascorbate,
CuSO4·5H2O, tBuOH/H2O, mw, 80 Watt, 90 °C, 30 min, 52–55%; (f) PBr3,
CH2Cl2, 0 °C–r.t., 2.5 h, 31–50%; (g) AgNO3, CH3CN, 60 °C, dark, 2.5 h, 42–
82%; (h) sodium methanesulfonothioate, DMF, 60 °C, 2.5 h, 47%.
To study the influence of the structure of the amide bond on
the potency, a reverse amide was also synthesized, using 2-
(3-fluoro-4-(trifluoromethyl) phenyl)acetic acid and aniline 33.
The nitro-group of the methyl ester 31 was reduced under mild
conditions with Fe(0) and an aqueous solution of NH4Cl in
ethanol, resulting in ester 32. Reduction of the ester group with
LiAlH4 in anhydrous THF afforded aniline 33. 2-(3-Fluoro-4-
(trifluoro-methyl)phenyl) acetic acid was then heated overnight
with 33 and HATU/DIPEA in DMF to give 34. The in situ
formation of the bromide and subsequent addition of AgNO3 in
a mixture of anhydrous CH2Cl2/CH3CN afforded the nitrate
ester 35 (Scheme 6).
Replacement of adamantane by the aryl-substituted
cyclohexyl moiety
5-(4-Hydroxyphenyl)-3H-1,2-dithiol-3-thione (ADT-OH),[46]
a known H2S-releasing agent, [47] was used for the synthesis of
analogue 49. Elimination of the α-hydrogen atoms of
cyanomethyl propionate by Cs2CO3, followed by a double
attack from 1,5-dibromopentane, resulted in compound 44.
Reduction of the nitrile with LiAlH4 afforded amine 45, which
was further protected using Boc2O. A Mitsunobu reaction
between the Boc-protected derivative 46 and ADT-OH
afforded compound 47, which was deprotected by
trifluoroacetic acid (TFA). Amine 48 was then coupled with 2-
chloro-5-methylbenzoic acid using EDC hydrochloride and
Et3N in anhydrous CH2Cl2 to afford analogue 49 (Scheme 8).
HO
OH
OMe
O
O
a,b
c
NH2
O2N
H2N
33
32
EtOOC CN
HO
NH2
O
a
b
O
OH
O
ONO2
NC
COOEt
OH
HN
HN
d
e
44
45
F
F3
F
F
c
C
F3C
CF3
34
35
HO
NHBoc
ADTO
NHBoc
ADTO
NH2
d
e
Scheme 6. Reagents and conditions: (a) CH3OH, H2SO4, reflux, 18 h, 94%;
(b) Fe(0), NH4Cl (aq.), EtOH, reflux, 3 h, 86%; (c) LiAlH4, THF, 0 °C–r.t.,
1.5 h, 100%; (d) 33, HATU, DIPEA, DMF, 70 °C, 15 h, 67%; (e) PPh3, NBS,
AgNO3, CH2Cl2/CH3CN, 0–50 °C, dark, 5 h, 29%.
46
48
47
f
S
S
S
Cl
O
In addition, three triazoles were prepared as corresponding
analogues of 18a–b and 19a. The methyl ester (36) of 3-fluoro-
4-(trifluoromethyl)benzoic acid was reduced with LiAlH4 in
anhydrous THF to give benzyl alcohol 37. Bromination of 37
with PBr3 afforded analogue 38, which was further heated with
NaN3 in DMF to give azide 39. Microwave irradiation was then
applied to obtain the ortho-methyl and -chloro 1,2,3-triazoles
40a and 40b, respectively. Upon treatment with PBr3, bromides
41a–b were obtained and reacted with AgNO3 or sodium
methanesulfonothioate to afford analogues 42a–b and 43,
respectively (Scheme 7).
ADTO
NH
HO
ADT-OH
49
Scheme 8. Reagents and conditions: (a) 1,5-dibromopentane, Cs2CO3,
DMF, 0 °C–r.t., 24 h, 79%; (b) LiAlH4, THF, 0 °C–r.t., 29 h, 98%; (c) Boc2O,
THF, r.t., 23 h, 99%; (d) ADT-OH, PPh3, DIAD, THF, r.t., 72 h, 29%; (e) TFA,
CH2Cl2, r.t., 1 h, 95%; (f) 2-chloro-5-methylbenzoic acid, EDC hydrochloride,
Et3N, CH2Cl2, r.t., 20 h, 28%.
5
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10.1002/cmdc.202000303
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FULL PAPER
Analogues 53 and 56 were synthesized bearing the nitrate
ester group on opposite phenyl rings (Scheme 9) to examine
the effect of the substitution position on the antagonistic
potency. NaH was used for the α-deprotonation of (4-
methylphenyl)acetonitrile to yield analogue 50. Reduction of
the nitrile was achieved with BH3·S(CH3)2 in THF. Amine 51
was then coupled with 28 using HATU/DIPEA in anhydrous
DMF. Ιn situ bromination of the benzylic hydroxyl group with
PPh3 and NBS in an anhydrous CH2Cl2/CH3CN mixture,
followed by the formation of the nitrate ester, afforded amide
53. For the synthesis of compound 56, amine 51 was coupled
with 2-chlorobenzoic acid using EDC hydrochloride and
hydroxybenzotriazole (HOBt) as the coupling agent in
anhydrous DMF. Radical bromination of 54 with AIBN and NBS
afforded amide 55, which was subsequently refluxed with
AgNO3 to yield analogue 56 (Scheme 9).
N3
OH
Br
b
a
N
O
N
N
N
O
N
N
O
O
58
e
O
O
20
57
c
NH2
N
Cl
N
N
N
d
N
O
N
O
N
O
O
61
59
Cl
O
NH
O
N
N
O
60
Scheme 10. Reagents and conditions: (a) PBr3, CH2Cl2, 0 °C–r.t., 3.5 h,
55%; (b) NaN3, DMF, 40 °C, 4 h, 99%; (c) PPh3, H2O, THF, r.t., 22 h,
obtained as mixture with PPh3O; (d) 2-chloro-5-methylbenzoic acid, EDC
hydrochloride, Et3N, CH2Cl2, r.t, 24 h, 21% over two steps; (e) 1-chloro-2-
ethynylbenzene, sodium ascorbate, CuSO4·5H2O, tBuOH/H2O, mw,
80 Watt, 90 °C, 30 min, 45%.
Cl
O
CN
NH2
e
a
b
NH
CN
54
51
50
f
c
Br
Cl
The furoxan ring (20) was also used in place of the phenyl
ring of the 3-fluoro-4-(trifluoromethyl)phenyl and aryl-
substituted cyclohexyl derivatives to further investigate its role
on the potency. Thus, the furoxan carboxylic acid (62) was
quantitatively obtained by the oxidation of 20 with the Jone’s
reagent, which gave analogue 63 using HATU/DIPEA.[51] In the
case of 64, the coupling reaction was achieved using
EDC/Et3N in anhydrous CH2Cl2 (Scheme 11).
Cl
O
O
NH
NH
HO
55
52
d
g
Cl
ONO2
O
Cl
O
NH
NH
O
O
N
O2NO
53
H
N
N
56
O
O
O
F
O
N
O
N
N
N
Scheme 9. Reagents and conditions: (a) 1,5-dibromopentane, NaH (60%),
DMF, 0 °C–r.t., 4 h, 67%; (b) BH3·S(CH3)2, THF, 0 °C–r.t., 3 h, 49%; (c) 28,
HATU, DIPEA, DMF, r.t., 48 h; (d) PPh3, NBS, AgNO3, CH2Cl2/CH3CN, 0–
50 °C, dark, 24 h, 21%; (e) 2-chlorobenzoic acid, HOBt, EDC hydrochloride,
Et3N, DMF, r.t., 22 h, 45%; (f) NBS, AIBN, CHCl3, reflux, 90 min, 57%; (g)
AgNO3, CH3CN, 60 °C, dark, 1 h, 63%.
b
c
CF3
HO
63
a
HO
O
20
62
NH
N
O
N
O
O
64
Replacement of adamantane by the furoxan ring
Scheme 11. Reagents and conditions: (a) Jone’s reagent, acetone, 0 °C–
r.t., 3.5 h, 74%; (b) 25, HATU, DIPEA, DMF, r.t., 22 h, 73%; (c) 51, HOBt,
EDC hydrochloride, Et3N, DMF, r.t., 20 h, 26%.
Furoxan analogues are interesting pharmacophores,
endowed with various biological effects such as anticancer[48]
and antimicrobial.[49,50] However, P2X7 antagonists bearing
this group have not been reported so far. Thus, in this study,
adamantane was replaced by the furoxan ring to afford an
amide and a 1,2,3-triazole derivative. In particular, bromide 57
reacted with NaN3 in DMF to afford azide 58. Αmine 59 was
obtained via a Staudinger reaction of azide 58 with PPh3 and
H2O in THF and was subsequently used for the synthesis of
analogue 60 (Scheme 10). Microwave irradiation of 58 and the
chloro-substituted alkyne (isolated from 10, as in Scheme 2) in
the presence of Cu(II) and sodium ascorbate in a mixture of
tBuOH/H2O afforded analogue 61.
Combination of the 3-fluoro-4-(trifluoromethyl)phenyl
and aryl-substituted cyclohexyl moieties
Finally, three additional analogues that combined the 3-
fluoro-4-(trifluoromethyl)phenyl and aryl-substituted cyclohexyl
moieties were synthesized to elucidate the structural features
responsible for the interactions with the P2X7 receptor. Thus,
the cyclohexyl ring of 65 was prepared using the commercially
available 2-(3-fluoro-4-(trifluoromethyl)phenyl)acetonitrile and
1,5-dibromopentane in the presence of NaH in anhydrous
DMF. Reduction of the nitrile with BH3·S(CH3)2 in THF afforded
amine 66, which was then coupled with 2-chlorobenzoic acid
or 2-chloro-5-methylbenzoic acid to yield compounds 67 and
68, respectively. Analogue 68 was further radically brominated
with AIBN and NBS in anhydrous CHCl to afford compound 69
3
6
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10.1002/cmdc.202000303
ChemMedChem
FULL PAPER
as a non-separable mixture with the starting material (68).
Upon treatment with AgNO3 analogue 70 was obtained
(Scheme 12).
F3C
F3C
NH2
F3C
F
a
b
CN
F
F
CN
66
65
c
d
O
Cl
O
Cl
F3C
F
F3C
F
HN
HN
68
67
e
O
Cl
Figure 4. Antagonistic potencies of AZ1 and the indicated analogues by
TEVC. Xenopus leavis oocytes expressing the hP2X7 receptor were
clamped at −70 mV. 10 μM of the indicated compounds were preincubated
for 3 min before application of 300 μM ATP. Responses are normalized to
ATP-evoked current responses in the absence of antagonist. Error bars
represent S.D. AZ1 (n=4), 4 (n=8), 5 (n=3), 11 (n=6), 18a (n=3), 18b (n=3),
19a (n=7), 19b (n=4), 24a (n=4), 23b (n=6), and 24b (n=3).
O
Cl
F3
C
F
HN
F3C
F
HN
f
ONO2
69
70
Br
Scheme 12. Reagents and conditions: (a) 1,5-dibromopentane, NaH (60%),
DMF, 0 °C–r.t., 1 h, 81%; (b) BH3·S(CH3)2, THF, reflux, 1 h, 81%; (c) 2-
chlorobenzoic acid, HOBt, EDC hydrochloride, Et3N, DMF, r.t., 18 h, 55%;
(d) 2-chloro-5-methyl benzoic acid, HOBt, EDC hydrochloride, Et3N, DMF,
r.t., 22 h, 30%; (e) NBS, AIBN, CHCl3, reflux, 2 h, obtained as mixture with
68; (f) AgNO3, CH3CN, 50 °C, dark, 30 min, 10% over two steps.
Since seven of these analogues led to a reduction of ATP-
induced current responses of at least 50%, their dose–
response relationships were determined (Figure 5). The IC50
values (Table 3) confirmed that analogues 4 and 18a were the
most potent adamantane-based P2X7 antagonists of the
current study, with about five- and ten-fold reduced potency
compared to AZ1, respectively. However, except for analogue
18a, the replacement of the amide bond by the 1,2,3-triazole
ring significantly reduced the potency. Complete absence of
the meta-substitution (11) as well as a bulkier substituent, such
as the (methylsulfonyl)thio group (19a), negatively affected the
analogues’ antagonistic abilities. Moreover, the IC50 values of
amides 4 (0.34 μΜ) and 5 (2.72 μΜ), as well as those of
triazoles 18a (0.74 μΜ) and 18b (8.00 μΜ), were remarkably
different, suggesting that the nature of the ortho- and meta-
substitution might affect the interaction with the receptor and
thus the antagonistic potency.
In vitro evaluation of the antagonistic potency -
SAR study
The synthesized analogues were evaluated in vitro by two-
electrode voltage clamp (TEVC) experiments, using Xenopus
laevis oocytes that express the human P2X7 (hP2X7) receptor.
The already reported P2X7 antagonist AZ1 was used as a
positive control to allow the comparison of the isolated
compounds’ antagonistic potencies. In general, the
synthesized analogues featured drug-like properties based on
predictions of their absorption, distribution, metabolism, and
excretion (ADME) properties (Table S1).
The blocking potency was initially estimated by incubating
hP2X7 expressing oocytes for 3 min with 10 μM of each
compound. AZ1 completely blocked ATP-induced (300 μM)
current responses under these conditions, while analogues 4
and 5, bearing a chloro group at the ortho-position of the
phenyl ring, were equally potent, irrespective of the benzyl
meta-substitution (Figure 4). Replacement of the amide bond
by its bioisostere 1,2,3-triazole ring in analogues that bear the
same meta-substitution and an ortho-chloro or -methyl
substitution (11, 18a–b, and 19a–b) impaired the antagonistic
effect compared to the control (AZ1). According to the data
shown in Figure 4, the methyl group in analogues 18a and 19a
probably favored the interaction with the receptor compared to
the chloro group (18b and 19b). Furthermore, the presence of
the (methylsulfonyl)thio group reduced the antagonistic
potency, especially in the case of analogue 19b.
Additionally, the evaluation of analogues 24a–b, bearing
the furoxan ring, confirmed the results of the pharmacophore
model that bulky substituents do not improve the compound’s
activity (Figure 4). However, the congener of 24b, compound
23b, displayed potent inhibition of hP2X7, suggesting that
inactive esters may be hydrolyzed after their insertion into the
host and result in a more active form of the initial analogue.
Figure 5. Dose–response curves of compound AZ1 and the indicated
analogues. All compounds were preincubated for 3 min and the current
responses of the oocyte-expressed hP2X7 receptor to 300 μM ATP were
recorded by TEVC at −70 mV. Each point represents the average of at least
three measurements and error bars represent S.D.
7
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10.1002/cmdc.202000303
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FULL PAPER
increased potency and indicated again the aryl-cyclohexyl
Table 3. IC50 values with 95% confidence intervals (CI) of the most active
moiety as
a suitable scaffold to replace adamantane.
adamantane-based analogues.
Surprisingly, the aryl-cyclohexyl analogues bearing the fluoro-
substituted aromatic moiety (67, 68, and 70) exhibited
moderate potency, while the lack of the meta-substitution (67
and 70) seemingly favored the interaction with the receptor
compared to analogue 68 (Figure 6).
Dose-response analysis (Figure 7, Table 4) further
confirmed that the aryl-cyclohexyl moiety is an ideal substitute
of adamantane. Specifically, analogue 56 had the lowest IC50
value (0.39 μΜ), while analogue 53, bearing the nitrate ester
on the non-chloro phenyl ring, had an IC50 value of 1.43 μΜ.
Compound
IC50 (μM)
0.065
0.339
2.716
4.355
0.735
8.004
3.859
1.712
95% CI (μΜ)
0.054–0.077
0.253–0.452
1.901–3.799
3.761–5.039
0.611–0.894
5.990–10.69
3.027–4.920
1.442–2.030
AZ1
4
5
11
18a
18b
19a
23b
Additionally,
it was
shown that the (3-fluoro-4-
(trifluoromethyl)phenyl moiety did not eliminate the potency
when used as a substituent, giving compounds with IC50 values
from 0.69 μΜ (67) to 3.24 μΜ (70) and 4.80 μΜ (68). For all the
aryl-cyclohexyl analogues, it was revealed that the substitution
at the 5-position of the phenyl ring results in lower potency (53,
68, and 70). Furthermore, analogue 64, bearing the bulkier
furoxan moiety in place of the phenyl ring, was effective in
inhibiting P2X7 activation with an IC50 value of 0.88 μM, which
was similar to those of the aryl-cyclohexyl analogues 56 and
67.
Regarding the scaffold exchange strategy of adamantane,
its replacement by the (3-fluoro-4-(trifluoromethyl)phenyl
moiety in analogues 30, 35, 42a–b, and 43 resulted in very low
potencies for both amides and 1,2,3-triazoles, compared to the
control (AZ1) (Figure 6). Instead, the replacement of
adamantane by the aryl-cyclohexyl moiety improved the
analogues’ potency. Except for analogue 49, which could not
be tested due to its low solubility confirming the ADME
predictions (Table S1), the aryl-cyclohexyl amides bearing a
nitrate ester group (53 and 56) were potent hP2X7 antagonists
irrespective of the substituents’ position on the phenyl ring
(Figure 6).
100
AZ1
53
56
64
67
68
70
80
60
40
20
0
-10
-8
-6
-4
log [M]
Figure 7. Dose–response curves of compound AZ1 and the indicated
analogues. All compounds were preincubated for 3 min and the current
responses of the oocyte-expressed hP2X7 receptor to 300 μM ATP were
recorded by TEVC at −70 mV. Each point represents the average of at least
three measurements and error bars represent S.D.
Table 4. IC50 values with 95% confidence intervals (CI) of the most active
analogues in μΜ after the replacement of the adamantane ring.
Compound
IC50 (μM)
0.065
1.430
0.385
0.878
0.686
4.804
3.243
95% CI (μΜ)
0.054–0.077
1.240–1.648
0.332–0.443
0.784–0.983
0.399–1.176
3.514–7.012
2.515–4.164
Figure 6. Antagonistic potencies of AZ1 and the indicated analogues by
TEVC. Xenopus leavis oocytes expressing the hP2X7 receptor were
clamped at −70 mV. 10 μM of the indicated compounds were preincubated
for 3 min before application of 300 μM ATP. Responses are normalized to
ATP-evoked current responses in the absence of antagonist. Error bars
represent S.D. AZ1 (n=4), 30 (n=3), 35 (n=3), 42a (n=3), 42b (n=3), 43
(n=3), 53 (n=4), 56 (n=3), 60 (n=4), 61 (n=4), 63 (n=4), 64 (n=3), 67 (n=3),
68 (n=3), and 70 (n=3).
ΑΖ1
53
56
64
67
68
70
The replacement of adamantane by the furoxan ring led to
analogues with no inhibition at 10 μM (60 and 61) (Figure 6).
No blocking effect was also observed by replacing the phenyl
ring of the (3-fluoro-4-(trifluoromethyl)phenyl-based analogues
with the furoxan ring (63), suggesting that this moiety
deteriorates the interactions with the hP2X7 receptor. In
contrast, a similar replacement in the aryl-cyclohexyl-based
compounds afforded analogue 64, which exhibited significantly
8
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10.1002/cmdc.202000303
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FULL PAPER
Macherey-Nagel) under the following conditions: gradient elution 50/50
H2O_0.1%TFA/CH3CN_0.1%TFA to 0/100 H2O_0.1%TFA/CH3CN
_0.1%TFA over 20 min; flow rate: 1.2 mL/min; detection at 216 nm. All
the synthesized analogues exhibited a purity >97%, unless otherwise
reported. The microwave-assisted experiments were carried out with a
CEM Discover 300W monomode microwave instrument. The melting
points were measured on a Büchi 510 Apparatus and are not corrected.
Summarizing, the replacement of the amide bond by its
bioisostere 1,2,3-triazole resulted in adamantyl analogues with
decreased antagonistic effect (11, 18a–b, 19a–b, and 24a–b).
The use of the (3-fluoro-4-(trifluoromethyl)phenyl moiety in
place of adamantane afforded novel amides and 1,2,3-
triazoles with very low potency (30, 35, 42a–b, 43, and 63).
Furthermore, replacement of the adamantane ring by the
furoxan moiety did not favor the blocking effect of the 60 and
61. Nevertheless, satisfying results were obtained by replacing
adamantane by the aryl-cyclohexyl moiety (53, 56, 64, 67, 68,
and 70), leading to potent P2X7 antagonists with analogue 56
exhibiting the lowest IC50 value of 0.39 μΜ. Furthermore, the
IC50 value of analogue 56 was equivalent to that of analogue 4
(0.34 μΜ).
Synthesis of analogues AZ1, 4, 5, 11, 18a–b, 19a–b,
24a–b
N-[(Adamantan-1-yl)methyl]-2-chloro-5-(piperazin-1-
ylmethyl)benzamide (AZ1)
To
a
stirred solution of piperazine (11 mg, 0.13 mmol) in 2 mL
solution of N-[(adamantan-1-yl)methyl]-5-
anhydrous CH3OH,
a
(bromomethyl)-2-chlorobenzamide (3) (25 mg, 0.06 mmol) in
anhydrous CH3OH was added at 0 °C. The mixture was allowed to
warm to room temperature, while stirring for 3 h. The solvent was
removed in vacuo and AZ1 was obtained by flash column
chromatography (CH2Cl2/CH3OH, 90:10 to 20:80) as white sticky solid
(29%). HPLC: tR: 15.4 min, purity 86%; 1H NMR (600 MHz, CD3OD):
δ = 7.44–7.40 (m, 3H, ArH), 3.62 (s, 2H, CH2NCH2), 3.23–3.21 (m, 4H,
CH2-piperazine), 3.07 (s, 2H, CH2NHCO), 2.70 (bs, 4H, CH2-
piperazine), 1.99 (bs, 3H, CH-adamantane), 1.79–1.63 (m, 12H, CH2-
adamantane); 13C NMR (150 MHz, CD3OD): δ = 170.2 (C=O), 138.2,
137.9, 132.5, 131.0, 130.7, 130.4, 62.1 (CH2N-piperazine), 52.6, 50.7,
45.0 (CH2NH), 41.5, 38.1, 35.7, 29.8; HRMS (ESI): m/z calcd for
C23H32ClN3O+H+: 402.2307 [M+H]+, found: 402.2310. Data are in
accordance with the literature.[27]
Conclusions
Although P2X7 is an important drug target and various
classes of P2X7 antagonists have already been described,
successful clinical candidates are missing. Moreover, neither
papers nor patents have reported SAR studies on the class of
compounds described in this work. Herein, by replacing
several structural features of the Astra Zeneca derivative (AZ1)
with moieties that have not yet been examined to block the
activation of the P2X7 channel and based on an in silico refined
SAR study, a series of hit compounds were developed as novel
putative P2X7 antagonists bearing low micromolar to high
nanomolar potencies. Our data showed that analogue 4
bearing a nitrate ester group in place of piperazine could
effectively inhibit P2X7 activation (IC50 = 0.34 μΜ), while the
aryl-cyclohexyl group was proved to be the most promising
alternative for adamantane with analogue 56 exhibiting an
IC50 value of 0.39 μΜ. Most importantly, this work provides
different structural starting points for further hit-to-lead
optimization towards the development of derivatives with
improved structural features that can effectively block the
activation of the P2X7 ion channel with nanomolar potency.
N-[(Adamantan-1-yl)methyl]-2-chloro-5-
(nitrooxymethyl)benzamide (4)
N-[(Adamantan-1-yl)methyl]-5-(bromomethyl)-2-chlorobenzamide (3)
(41 mg) was dissolved in 4 mL of anhydrous CH3CN, followed by the
addition of AgNO3 (20 mg, 0.12 mmol). The suspension formed was
heated in the dark at 60 °C for 2 h. Upon completion of the reaction, the
solvent was removed under reduced pressure and the crude material
was purified by flash column chromatography (n-hexane/EtOAc 85:15
to 80:20) as white solid (50%). m.p. 118.0–120.0 °C; HPLC: tR:
10.4 min; 1H NMR (300 MHz, CDCl3): δ = 7.72 (s, 1H, ArH), 7.45–7.37
(m, 2H, ArH), 6.31 (s, 1H, CH2NH), 5.40 (s, 2H, CH2ONO2), 3.16 (d,
J = 6.2 Hz, 2H, CH2NH), 2.00 (bs, 3H, CH-adamantane), 1.75–1.58 (s,
12H, CH2-adamantane); 13C NMR (75 MHz, CDCl3): δ = 165.9 (C=O),
136.1, 131.8, 131.7, 131.5, 131.1, 130.9, 73.3 (CH2ONO2), 51.9
(CH2NH), 40.4, 37.0, 34.0, 28.3; HRMS (ESI): m/z calcd for
C19H23ClN2O4+H+: 379.1419 [M+H]+; found: 379.1419.
Experimental Section
General chemistry
N-[(Adamantan-1-yl)methyl]-2-chloro-5-
[(methylsulfonyl)thio]methyl)benzamide (5)
Commercial reagents and solvents were obtained from Acros
Organics, Merck, Sigma-Aldrich or Fluorochem in the qualities puriss,
p.a. or purum and used without further purification. All non-aqueous
reactions were set up under argon atmosphere, utilizing glassware that
was flame-dried and cooled under vacuum. Thin layer chromatography
(TLC) was performed using precoated SiO2 aluminum plates
(Macherey–Nagel Sil G-25 UV254), while the chromatographic
N-[(Adamantan-1-yl)methyl]-5-(bromomethyl)-2-chlorobenzamide (3)
(15 mg, 0.04 mmol) was dissolved in anhydrous DMF (2 mL) and
sodium methanesulfonothioate (8 mg, 0.06 mmol) was added. The
mixture was heated at 60 °C for 5 h. Afterwards, it was washed several
times with H2O and saturated aqueous NaCl solution, and the aqueous
layer was extracted with EtOAc. The combined organic layers were
dried over Na2SO4, filtered, and concentrated under reduced pressure.
Flash column chromatography (n-hexane/EtOAc 80:20) afforded 5 in
58% yield (white solid, 10 mg). m.p. 125.0–127.0 °C; HPLC: tR: 9.1 min;
1H NMR (600 MHz, CDCl3): δ = 7.73 (s, 1H, ArH), 7.39 (s, 2H, ArH),
6.27 (s, 1H, CH2NH), 4.35 (s, 2H, CH2S), 3.16 (d, J = 6.3 Hz, 2H,
CH2NH), 3.10 (s, 3H, S2O2CH3), 2.00 (bs, 3H, CH-adamantane), 1.73–
1.57 (m, 12H, CH2-adamantane); 13C NMR (150 MHz, CDCl3):
δ = 165.9 (C=O), 136.1, 134.9, 131.9, 131.3, 131.1, 130.5, 52.0
(SCH3), 51.4 (CH2NH), 40.5, 39.7, 37.0, 34.1, 28.4; HRMS (ESI): m/z
calcd for C20H26ClNO3S2+H+: 428.1115 [M+H]+; found: 428.1116.
purifications were performed with silica gel (200–400 mesh). 1H and 13
C
spectra were recorded on Varian spectrometers operating at 300 MHz
or 600 MHz/75 MHz or 150 MHz, respectively, at 25 °C using CDCl3,
DMSO-d6, CD3OD, acetone-d6, or CD2Cl2. The processing and
evaluation of the spectra were performed using the program
MestReNova 9.0. The resonance multiplicity is indicated as s (singlet),
d (doublet), t (triplet), and m (multiplet) or combinations of them, and
the coupling constants (J) are given in Hz. The mass spectra were
obtained on HPLC–MSn Fleet-Thermo in the ESI mode. The HRMS
spectra were recorded in the ESI mode, on a UPLC-MSn Orbitrap
Velos-Thermo instrument. The purity of the tested compounds was
determined by HPLC (Thermo Scientific HPLC Spectra System) using
a column EC 250/4.6 Nucleosil 100-5 C18 HD (particle size 5 μm,
1-(Adamantan-1-yl)methyl)-4-(2-chlorophenyl)-1H-1,2,3-
triazole (11)
9
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10.1002/cmdc.202000303
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To a stirred solution of [(2-chlorophenyl)ethynyl]trimethylsilane (10)
(50 mg, 0.24 mmol) in 1.5 mL anhydrous THF, TBAF (0.17 mL,
0.60 mmol) was added. After stirring at room temperature for 1 h, the
crude intermediate solution was transferred in a microwave tube. 1-
Adamantantylmethyl azide (9) (46 mg, 0.24 mmol), sodium ascorbate
(29 mg, 0.15 mmol), and CuSO4·5H2O (18 mg, 0.07 mmol) were added
and the mixture was irradiated at 90 °C (80 Watt) for 30 min.
Purification by flash column chromatography (n-hexane/EtOAc 95:5 to
85:15) yielded compound 11 as white sticky solid (12 mg, 15%). HPLC:
tR: 15.1 min; 1H NMR (300 MHz, CDCl3): δ = 8.28 (dd, J = 7.8, 1.7 Hz,
1H, ArH), 8.10 (s, 1H, CH-triazole), 7.45 (dd, 1H, ArH), 7.37 (m, 1H,
ArH), 7.26 (m, 1H, ArH), 4.09 (s, 2H, CH2NCH), 2.01 (bs, 3H, CH-
adamantane), 1.73–1.56 (m, 12H, CH2-adamantane); 13C NMR
(75 MHz, CDCl3): δ = 143.4 (C–N), 131.3, 130.3, 129.9, 129.6, 129.0,
127.3, 124.8, 62.5 (CH2N), 40.4, 36.7, 34.4, 28.2; HRMS (ESI): m/z
calcd for C19H22ClN3+H+: 328.1575 [M+H]+; found: 328.1573.
1-(Adamantan-1-yl)methyl)-4-[2-chlorophenyl-5-
(methylsulfonyl)thio)methyl)]-1H-1,2,3-triazole (19b)
Analogue 19b was prepared following the synthetic procedure for
analogue 19a and was isolated by flash column chromatography (n-
hexane/EtOAc 80:20) as white solid (18 mg, 23%). m.p. 139.0–
140.0 °C;1H NMR (300 MHz, CDCl3): δ = 8.34–8.33 (m, 1H, ArH), 8.12
(s, 1H, CH-triazole), 7.45 (d, J = 8.2 Hz, 1H, ArH), 7.31 (dd, J = 8.2,
2.2 Hz, 1H, ArH), 4.41 (s, 2H, CH2S), 4.09 (s, 2H, CH2NCH), 3.10 (s,
3H, S2O2CH3), 2.01 (bs, 3H, CH-adamantane), 1.74–1.56 (m, 12H,
CH2-adamantane); 13C NMR (75 MHz, CDCl3): δ = 142.7 (C–N), 134.5,
131.1, 131.0, 130.31, 130.27, 129.5, 125.0, 62.5 (CH2N), 51.4 (SCH3),
40.4, 40.1, 36.6, 34.4, 28.2; HRMS (ESI): m/z calcd for
C21H26ClN3O2S2+H+: 452.1228 [M+H]+; found: 452.1231.
[1-(Adamantan-1-yl)methyl]-3-[[5-[(4-methylbenzoyloxy)-4-phenyl-
1,2,5-oxadiazole-2-oxide]-2-methylphenyl]-1H-1,2,3-triazole (24a)
3-(Hydroxymethyl)-4-phenyl-1,2,5-oxadiazole 2-oxide (20) (31 mg,
0.16 mmol) and DMAP (2 mg, 0.008 mmol) were added to a stirred
1-(Adamantan-1-yl)methyl)-4-[2-methylphenyl-5-
(nitrooxymethyl)]-1H-1,2,3-triazole (18a)
solution of
3-[1-[(adamantan-1-yl)methyl]-1H-1,2,3-triazol-4-yl]-4-
methylbenzoic acid (23a) (28 mg, 0.08 mmol) in anhydrous CH2Cl2
(3 mL). DCC (18 mg, 0.09 mmol) was added at 0 °C and the reaction
mixture was stirred at this temperature for 5 min and then at room
temperature for 2 h. Upon completion of the reaction, the mixture was
filtered, and the filtrate was extracted with CH2Cl2. The combined
organic phases were dried over Na2SO4, filtered, and concentrated in
vacuo. The crude product was purified by flash column chromatography
(n-hexane/EtOAc 50:50) to afford 24a (30 mg, 71%) as colorless oil.
1H NMR (300 MHz, CDCl3): δ = 8.33 (s, 1H, ArH), 7.85 (d, J = 8.0 Hz,
1H, ArH), 7.77–7.74 (m, 2H, ArH-furoxan), 7.60 (s, 1H, CH-triazole),
7.53–7.50 (m, 3H, ArH-furoxan), 7.34 (d, 1H, ArH), 5.38 (s, 2H, OCΗ2),
4.09 (s, 2H, CH2NCH), 2.55 (s, 3H, ArCH3), 2.01 (bs, 3H, CH-
adamantane), 1.74–1.55 (m, 12H, CH2-adamantane); 13C NMR
(75 MHz, CDCl3): δ = 165.6 (C=O), 157.0, 145.6, 142.4, 131.5, 130.8,
130.5, 129.6, 129.3, 127.8, 126.7, 126.2, 123.6, 111.5, 62.4 (CH2O),
54.8 (CH2N), 40.4, 36.6, 34.4, 28.2; HRMS (ESI): m/z calcd for
C30H31N5O4+Na+: 548.2268 [M+Na]+; found: 548.2270.
To a stirred solution of 1-(adamantan-1-yl)methyl)-4-[5-(bromomethyl)-
2-methylphenyl]-1H-1,2,3-triazole (17a) (44 mg, 0.11 mmol) in
anhydrous CH3CN (5 mL), AgNO3 (20 mg, 0.13 mmol) was added and
the reaction mixture was heated at 60 °C in the dark for 17 h. The
resulting mixture was filtered through celite and washed with EtOAc,
and the obtained filtrate was extracted with EtOAc. The combined
organic layers were then washed with saturated aqueous NaCl
solution, dried over Na2SO4, filtered, and concentrated in vacuo to
afford the nitrate ester derivative 18a as yellow solid (31 mg, 71%).
m.p. 115.0–118.0 °C;
HPLC:
tR: 12.9 min,
purity
90%;
1H NMR (300 MHz, CDCl3): δ = 7.88 (s, 1H, ArH), 7.59 (s, 1H, CH-
triazole), 7.29 (s, 2H, ArH), 5.44 (s, 2H, CH2ONO2), 4.08 (s, 2H,
CH2NCH), 2.48 (s, 3H, ArCH3), 2.01 (bs, 3H, CH-adamantane), 1.74–
1.56 (m, 12H, CH2-adamantane); 13C NMR (75 MHz, CDCl3): δ = 145.6
(C–N), 137.0, 131.6, 130.8, 130.1, 129.8, 128.7, 123.5, 74.8
(CH2ONO2), 62.4 (CH2N), 40.4, 36.7, 34.4, 28.2, 21.5; HRMS (ESI):
m/z calcd for C21H26N4O3+H+: 383.2078 [M+H]+; found: 383.2080.
1-(Adamantan-1-yl)methyl)-3-[5-[(4-methylbenzoyloxy)-4-phenyl-
1,2,5-oxadiazole-2-oxide]-2-chlorophenyl]-1H-1,2,3-triazole (24b)
Analogue 24b was prepared following the procedure of analogue 24a.
Purification by flash column chromatography (CH2Cl2 to CH2Cl2/CH3OH
99:1) afforded 24b (50 mg, 48%) as white powder. 1H NMR (300 MHz,
CDCl3): δ = 8.86 (d, J = 2.2 Hz, 1H, ArH), 8.09 (s, 1H, CH-triazole),
7.85 (dd, J = 8.4, 2.2 Hz, 1H, ArH), 7.76–7.73 (m, 2H, ArH-furoxan),
7.53–7.50 (m, 4H, ArH, ArH-furoxan), 5.40 (s, 2H, OCΗ2), 4.09 (s, 2H,
CH2NCH), 2.01 (bs, 3H, CΗ-adamantane), 1.73–1.55 (m, 12H, CΗ2-
adamantane); 13C NMR (75 MHz, CDCl3): δ = 164.8 (C=O), 156.9,
142.3, 136.8, 131.5, 131.3, 130.7, 130.2, 129.9, 129.6, 127.9, 127.8,
126.1, 125.0, 111.3, 62.5 (CH2O), 55.1 (CH2N), 40.4, 36.6, 34.4, 28.2;
HRMS (ESI): m/z calcd for C29H28ClN5O4+H+: 546.1903 [M+H]+; found:
546.1928.
1-(Adamantan-1-yl)methyl)-4-[2-chlorophenyl-5-(nitrooxymethyl)]-
1H-1,2,3-triazole (18b)
Analogue 18b was prepared following the synthetic procedure for
analogue 18a and was obtained as pale yellow solid without any further
purification (24 mg, 35%). m.p. 142.0–144.0 °C; HPLC: tR: 15.3 min;
1H NMR (300 MHz, CDCl3): δ = 8.35–8.34 (m, 1H, ArH), 8.12 (s, 1H,
CH-triazole), 7.48 (d, J = 8.2 Hz, 1H, ArH), 7.30 (dd, J = 8.2, 2.0 Hz,
1H, ArH), 5.46 (s, 2H, CH2ONO2), 4.10 (s, 2H, CH2NCH), 2.01 (bs, 3H,
CH-adamantane), 1.74–1.56 (m, 12H, CH2-adamantane); 13C NMR
(75 MHz, CDCl3): δ = 142.7 (C–N), 132.2, 131.7, 130.8, 130.5, 130.0,
129.1, 124.9, 73.8 (CH2ONO2), 62.5 (CH2N), 40.3, 36.6, 34.4, 28.2;
HRMS (ESI): m/z calcd for C20H23ClN4O3+H+: 403.1531 [M+H]+; found:
403.1534.
1-(Adamantan-1-yl)methyl)-4-[5-(methylsulfonyl)thio)methyl)-2-
methylphenyl]-1H-1,2,3-triazole (19a)
Synthesis of analogues 30, 35, 42a–b, 43, 49, 53, 56,
60, 61, 63, 64, 67, 68, 70
To a stirred solution of 1-(adamantan-1-yl)methyl)-4-[5-(bromomethyl)-
2-methylphenyl]-1H-1,2,3-triazole (17a) (64 mg, 0.16 mmol) in 1.2 mL
anhydrous DMF, sodium methanesulfonothioate (32 mg, 0.24 mmol)
was added and the mixture was heated at 70 °C for 5 h. Extraction with
EtOAc followed and the combined organic layers were washed with
saturated aqueous NaCl solution, dried over Na2SO4, filtered, and
concentrated under reduced pressure. Flash column chromatography
(n-hexane/EtOAc 80:20 to 70:30) afforded 19a as white solid in 51%
yield (35 mg). m.p. 124.0–125.0 °C; HPLC: tR: 10.9 min, purity 96%;
1H NMR (300 MHz, CDCl3): δ = 7.86 (s, 1H, ArH), 7.58 (s, 1H, CH-
triazole), 7.26–7.25 (m, 2H, ArH), 4.38 (s, 2H, CH2S), 4.07 (s, 2H,
CH2NCH), 3.02 (s, 3H, S2O2CH3), 2.45 (s, 3H, ArCH3), 2.00 (bs, 3H,
CH-adamantane), 1.72–1.55 (m, 12H, CH2-adamantane); 13C NMR
(75 MHz, CDCl3): δ = 145.5 (C–N), 135.5, 132.7, 131.6, 130.9, 129.3,
128.5, 123.4, 62.3 (CH2N), 51.1 (SCH3), 40.5, 40.3, 36.5, 34.3, 28.2,
21.3; HRMS (ESI): m/z calcd for C22H29N3O2S2+H+: 432.1774 [M+H]+;
found: 432.1774.
2-Chloro-N-(3-fluoro-4-(trifluoromethyl)benzyl)-5-
(nitrooxymethyl)benzamide (30)
To a stirred solution of 2-chloro-N-(3-fluoro-4-(trifluoromethyl)benzyl)-
5-(hydroxymethyl) benzamide (29) (18 mg, 0.05 mmol) in a mixture of
anhydrous CH2Cl2/CH3CN (1:2.5), PPh3 (13 mg, 0.05 mmol) and NBS
(9 mg, 0.05 mmol) were added at 0 °C and the mixture was stirred at
room temperature for 2 h. AgNO3 (10 mg, 0.06 mmol) was then added
and the resulting deep red solution was heated at 50 °C in the dark for
24 h. Afterwards, the mixture was filtered through celite and was
purified by flash column chromatography (n-hexane/EtOAc 70:30) to
afford 8 mg (39%) of 30 as white solid. m.p. 114.0–116.0 °C; HPLC:
tR: 13.3 min; 1H NMR (600 MHz, CDCl3): δ = 7.79 (s, 1H, ArH), 7.60 (t,
J = 7.6 Hz, 1H, ArH), 7.49–7.43 (m, 2H, ArH), 7.27–7.23 (m, 2H, ArH),
6.72 (bs, 1H, CH2NHCO), 5.42 (s, 2H, CH2ONO2), 4.72 (d, J = 6.0 Hz,
10
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10.1002/cmdc.202000303
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2H, CH2NHCO); 13C NMR (75 MHz, CDCl3): δ = 165.9 (C=O), 144.9,
134.8, 132.2, 132.1, 131.9, 131.2 (d, J = 4.4 Hz), 127.8–127.7 (m),
123.2 (d, J = 3.5 Hz), 116.1 (d, J = 21.1 Hz), 73.1 (CH2ONO2), 43.5
(CH2NH); 19F NMR (282 MHz, CDCl3): δ = −61.3 (d, J = 12.4 Hz, CF3),
−113.5–−113.7 (m, C–F); HRMS (ESI): m/z calcd for
C16H11ClF4N2O4−H−: 405.0271 [M−H]−; found: 405.0263.
δ = −61.5 (t, J = 10.5 Hz, CF3), −112.3–−112.4 (m, C–F); HRMS (ESI):
m/z calcd for C19H17F4N3O2S2+H+: 460.0771 [M+H]+; found: 460.0770.
2-Chloro-5-methyl-N-[1-(4-(3-thioxo-3H-1,2-dithiol-5-
yl)phenoxy)methyl)cyclohexyl)methyl]benzamide (49)
A mixture of 5-[4-(1-(aminomethyl)cyclohexyl)methoxy)phenyl]-3H-1,2-
dithiol-3-thione (48) (43 mg, 0.12 mmol), 2-chloro-5-methylbenzoic
acid (42 mg, 0.24 mmol), EDC hydrochloride (70 mg, 0.37 mmol), and
Et3N (0.10 mL, 0.73 mmol) in 4 mL of anhydrous CH2Cl2 was stirred at
room temperature for 20 h. Afterwards, the reaction was washed with
H2O and the aqueous phase was extracted with CH2Cl2. The organic
layer was washed with saturated aqueous NaCl solution and the
combined organic phases were dried over Na2SO4, filtered, and
concentrated in vacuo. The crude material was purified by flash column
chromatography (n-hexane/EtOAc, 85:15 to 75:25) to afford 49 as
orange oil (17 mg, 28%). 1H NMR (600 MHz, CDCl3): δ = 7.59 (d,
J = 8.7 Hz, 2H, ArH), 7.48 (s, 1H, ArH), 7.37 (s, 1H, CH-dithiol-thione),
7.25 (d, J = 5.6 Hz, 1H, ArH), 7.15 (d, J = 6.8 Hz, 1H, ArH), 6.97 (d,
J = 8.7 Hz, 2H, ArH), 6.57 (s, 1H, CH2NHCO), 3.92 (s, 2H, CH2O), 3.65
(d, J = 6.1 Hz, 2H, CH2NHCO), 2.32 (s, 3H, ArCH3), 1.63–1.49 (m, 10H,
CH2-cyclohexyl); 13C NMR (150 MHz, CDCl3): δ = 215.3 (C=S), 173.0
(C–S), 166.8 (C=O), 162.4 (C–O), 137.4, 134.9, 134.85, 134.79, 132.3,
131.2, 130.1, 128.7, 127.3, 124.6, 115.7, 74.6 (CH2O), 45.5 (CH2N),
38.2, 31.2, 26.2, 21.5; HRMS (ESI): m/z calcd for C25H26ClNO2S3+H+:
504.0887 [M+H]+; found: 504.0890.
2-[3-Fluoro-4-(trifluoromethyl)phenyl]-N-[2-methylphenyl-5-
(nitrooxymethyl)] acetamide (35)
Analogue 35 was prepared following the synthetic procedure for
analogue 30 and was isolated by flash column chromatography (n-
hexane/EtOAc, 60:40) as white solid (10 mg, 29%). m.p. 162.0–
164.0 °C; 1H NMR (600 MHz, CDCl3): δ = 7.67 (s, 1H, ArH), 7.65 (t,
J = 7.8 Hz, 1H, ArH), 7.27–7.25 (m, 2H, ArH), 7.20 (d, J = 7.7 Hz, 1Η,
ArH), 7.12 (d, J = 8.1 Hz, 1H, ArH), 6.92 (bs, 1H, CH2CONH), 5.38 (s,
2H, CH2ONO2), 3.82 (s, 2H, CH2CONH), 2.12 (s, 3H, ArCH3); 13C NMR
(75 MHz, CD3OD): δ = 171.2 (C=O), 160.9 (d, 1J = 256.1 Hz, C–F)
144.6, 137.1, 135.6, 132.4, 132.1, 128.3, 127.7, 126.6 (d, J = 3.5 Hz),
118.8 (d, J = 21.2 Hz), 75.6 (CH2ONO2), 43.3 (CH2CO), 17.9; 19F NMR
(282 MHz, CDCl3): δ = −61.4 (t, J = 13.1 Hz, CF3), −113.0–−113.1 (m,
C–F); HRMS (ESI): m/z calcd for C17H14F4N2O4−H−: 385.0817 [M−H]−;
found: 385.0806.
1-(3-Fluoro-4-(trifluoromethyl)benzyl)-4-[5-(nitrooxymethyl)-2-
methylphenyl)]-1H-1,2,3-triazole (42a)
Analogue 42a was prepared following the synthetic procedure for
analogue 18a and was obtained by flash column chromatography (n-
hexane/EtOAc, 95:5 to 70:30) as colorless oil (10 mg, 42%). 1H NMR
(300 MHz, CDCl3): δ = 7.83 (s, 1H, ArH), 7.68–7.61 (m, 2H, CH-
triazole, ArH), 7.30 (s, 2H, ArH), 7.19–7.11 (m, 2H, ArH), 5.66 (s, 2H,
CH2NCH), 5.43 (s, 2H, CH2ONO2), 2.46 (s, 3H, ArCH3); 13C NMR
(75 MHz, CDCl3): δ = 160.2 (d, 1J = 260.6 Hz, C–F), 147.4, 141.6 (d,
J = 8.1 Hz), 137.2, 131.7, 130.3 (d, J = 15.5 Hz), 129.8 (d, J = 3.5 Hz),
129.1, 128.32–128.29 (m), 122.7 (dd, J = 180.6, 3.9 Hz), 116.4 (d,
J = 21.8 Hz), 74.6 (CH2ONO2), 53.1 (CH2N), 21.4; 19F NMR (282 MHz,
CDCl3): δ = −61.5 (d, J = 12.5 Hz, CF3), −112.31–−112.33 (m, C–F);
HRMS (ESI): m/z calcd for C18H14F4N4O3+H+: 411.1075 [M+H]+; found:
411.1072.
2-Chloro-5-(nitrooxymethyl)-N-[1-(4-
methylphenyl)cyclohexyl)methyl]benzamide (53)
Analogue 53 was prepared following the synthetic procedure for
analogue 30. Purification by flash column chromatography (n-
hexane/EtOAc, 70:30) afforded 8 mg of 53 as colorless oil (21%).
HPLC: tR: 11.7 min; 1H NMR (600 MHz, CDCl3): δ = 7.58 (s, 1H, ArH),
7.37–7.34 (m, 2H, ArH), 7.28 (d, J = 8.2 Hz, 2H, ArH), 7.17 (d,
J = 7.9 Hz, 2H, ArH), 5.77 (bs, 1H, CH2NHCO), 5.36 (s, 2H, CH2ONO2),
3.60 (d, J = 5.9 Hz, 2H, CH2NHCO), 2.33 (s, 3H, ArCH3), 2.13–1.43 (m,
10H, CH2-cyclohexyl); 13C NMR (75 MHz, CDCl3): δ = 165.7 (C=O),
141.2, 136.0, 135.9, 131.8, 131.7, 131.4, 130.9, 129.7, 126.9, 73.3
(CH2ONO2), 51.2 (CH2NH), 42.0, 34.2, 26.5, 22.2, 21.0. HRMS (ESI):
m/z calcd for C22H25ClN2O4+H+: 417.1576 [M+H]+; found: 417.1574.
1-(3-Fluoro-4-(trifluoromethyl)benzyl)-4-[2-chlorophenyl-5-
(nitrooxymethyl)]-1H-1,2,3-triazole (42b)
2-Chloro-N-[1-(3-
Analogue 42b was prepared following the synthetic procedure for
analogue 18a and was obtained by flash column chromatography (n-
hexane/EtOAc, 80:20) as yellowish solid (14 mg, 82%). m.p. 84.0–
85.0 °C. 1H NMR (300 MHz, CDCl3): δ = 8.34 (d, J = 2.2 Hz, 1H, ArH),
8.23 (s, 1H, CH-triazole), 7.64 (t, J = 7.6 Hz, 1H, ArH), 7.50–7.47 (m,
1H, ArH), 7.33 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 7.18–7.11 (m, 2H, ArH),
5.67 (s, 2H, CH2NCH), 5.46 (s, 2H, CH2ONO2); 13C NMR (75 MHz,
CDCl3): δ = 160.1 (d, 1J = 260.5 Hz, C–F), 144.1, 141.3, 132.0 (d,
J = 35.9 Hz), 130.8, 130.3, 129.4, 128.3–128.1 (m), 123.5, 123.1 (d,
J = 3.9 Hz), 116.2 (dd, J = 65.0, 22.4 Hz), 73.6 (CH2ONO2), 53.0
(CH2N); 19F NMR (282 MHz, CDCl3): δ = −61.5 (d, J = 12.5 Hz, CF3),
−112.3–−112.4 (m, C–F); HRMS (ESI): m/z calcd for
C17H11ClF4N4O3+H+: 431.0529 [M+H]+; found: 431.0529.
(nitrooxymethyl)phenyl)cyclohexyl)methyl]benzamide (56)
Analogue 56 was prepared following the synthetic procedure for
analogue 18a and was obtained by flash column chromatography (n-
hexane/EtOAc, 80:20) as white solid (23 mg, 63%). m.p. 76.0–77.0 °C;
HPLC: tR: 9.3 min; 1H NMR (600 MHz, CDCl3): δ = 7.53 (dd, J = 7.6,
1.7 Hz, 1H, ArH), 7.45 (d, J = 8.3 Hz, 2H, ArH), 7.39 (d, J = 8.3 Hz, 2H,
ArH), 7.33–7.25 (m, 3H, ArH), 5.77 (bs, 1H, CH2NHCO), 5.41 (s, 2H,
CH2ONO2), 3.63 (d, J = 6.3 Hz, 2H, CH2NHCO), 2.13–1.39 (m, 10H,
CH2-cyclohexyl); 13C NMR (150 MHz, CDCl3): δ = 166.4 (C=O), 146.1,
135.0, 131.2, 130.4, 130.2, 130.1, 130.0, 129.6, 127.5, 127.0, 74.5
(CH2ONO2), 42.5 (CH2NH), 33.9, 29.7, 26.2, 22.0. HRMS (ESI): m/z
calcd for C21H23ClN2O4+H+: 403.1419 [M+H]+; found: 403.1415.
3-[(2-Chloro-5-methyl)-(4-phenyl-1,2,5-oxadiazole-2-
1-(3-Fluoro-4-(trifluoromethyl)benzyl)-4-[5-
oxide)methyl]benzamide (60)
(methylsulfonyl)thio)methyl)-2-methylphenyl]-1H-1,2,3-
triazole (43)
Analogue 60 was prepared following the synthetic procedure for
analogue 49 and was isolated by flash column chromatography (n-
hexane/EtOAc, 80:20) as white solid (82 mg). m.p. 129.0–130.0 °C;
HPLC: tR: 6.97 min; 1H NMR (600 MHz, CDCl3): δ = 7.95–7.94 (m, 2H,
ArH-furoxan), 7.59–7.57 (m, 3H, ArH-furoxan), 7.45 (s, 1H, ArH), 7.27
(d, J = 8.2 Hz, 1H, ArH), 7.18 (d, J = 7.9 Hz, 1H, ArH), 7.07 (bs, 1H,
CH2NHCO), 4.76 (d, J = 6.1 Hz, 2H, CH2NHCO), 2.33 (s, 3H, ArCH3);
13C NMR (150 MHz, CDCl3): δ = 166.9 (C=O), 156.5, 137.4, 133.1,
132.8, 131.4, 130.9, 130.2, 129.5, 128.1, 127.7, 126.1, 113.3, 32.9
(CH2NH), 20.8; HRMS (ESI): m/z calcd for C17H14ClN3O3+H+: 344.0796
[M+H]+; found: 344.0794.
Analogue 43 was prepared following the synthetic procedure for
analogue 19a and was isolated by flash column chromatography
(CH3OH 0.2% in CH2Cl2) as colorless oil (13 mg, 47%). 1H NMR
(300 MHz, CDCl3): δ = 7.82 (s, 1H, ArH), 7.69 (s, 1H, CH-triazole), 7.64
(t, J = 7.5 Hz, 1H, ArH), 7.31–7.28 (m, 2H, ArH), 7.19–7.13 (m, 2H,
ArH), 5.66 (s, 2H, CH2NCH), 4.39 (s, 2H, CH2S), 3.02 (s, 3H, S2O2CH3),
2.45 (s, 3H, ArCH3); 13C NMR (150 MHz, CDCl3): δ = 160.2 (d,
1J = 260.3 Hz, C–F), 147.4, 141.6 (d, J = 8.0 Hz), 135.9, 133.0, 131.9,
130.4, 129.6 (d, J = 3.9 Hz), 129.1, 129.0 (d, J = 22.0 Hz), 128.32–
128.29 (m), 123.4 (d, J = 3.8 Hz), 122.1, 116.4 (d, J = 21.7 Hz), 53.1
(CH2N), 51.3 (SCH3), 40.5 (CH2S), 21.4; 19F NMR (282 MHz, CDCl3):
11
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10.1002/cmdc.202000303
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FULL PAPER
4-(2-Chlorophenyl)-1-[(4-phenyl-1,2,5-oxadiazole 2-oxide)methyl]-
1H-1,2,3-triazole (61)
2-Chloro-N-[1-(3-fluoro-4-
(trifluoromethyl)phenyl)cyclohexyl)methyl]-5-methylbenzamide
(68)
Analogue 61 was prepared following the synthetic procedure for
analogue 11 using a tBuOH/H2O (1:1) mixture as the reaction solvent.
Purification by flash column chromatography (n-hexane/EtOAc, 80:20)
yielded 61 (37 mg, 45%) as white solid. m.p. 102.0–104.0 °C; 1H NMR
(600 MHz, CDCl3): δ = 8.48 (s, 1H, CH-triazole), 8.18 (d, J = 7.8 Hz,
1H, ArH), 7.92–7.91 (m, 2H, ArH-furoxan), 7.60–7.59 (m, 3H, ArH-
furoxan), 7.45 (d, J = 8.1 Hz, 1H, ArH), 7.36 (t, J = 7.5 Hz, 1H, ArH),
7.29 (t, J = 7.6 Hz, 1H, ArH), 5.64 (s, 2H, CH2N); 13C NMR (150 MHz,
CDCl3): δ = 156.7, 145.0, 131.9, 131.6, 130.4, 130.0, 129.8, 129.6,
128.7, 128.3, 127.3, 125.5, 124.5, 111.5, 42.3 (CH2N); HRMS (ESI):
m/z calcd for C17H12ClN5O2+H+: 354.0752 [M+H]+; found: 354.0752.
Analogue 68 was prepared following the synthetic procedure for
analogue 64 and was obtained by flash column chromatography (n-
hexane/EtOAc, 90:10 to 80:20) as colorless oil (69 mg, 30%). 1H NMR
(600 MHz, CDCl3): δ = 7.57 (t, J = 7.9 Hz, 1H, ArH), 7.35 (s, 1H, ArH),
7.28 (d, J = 8.3 Hz, 1H, ArH), 7.24–7.19 (m, 2H, ArH), 7.12–7.10 (m,
1H, ArH), 5.84 (bs, 1H, CH2NHCO), 3.60 (d, J = 6.3 Hz, 2H,
CH2NHCO), 2.29 (s, 3H, ArCH3), 2.11–1.23 (m, 10H, CH2-cyclohexyl);
13C NMR (75 MHz, CDCl3): δ = 166.7 (C=O), 141.6, 137.4, 134.4,
132.3, 131.0, 130.1, 127.5–127.2 (m), 122.9, 116.0 (d, J = 20.9 Hz),
50.8 (CH2N), 43.4, 34.0, 26.2, 22.1, 20.8; HRMS (ESI): m/z calcd for
C22H22ClF4NO+H+: 428.1399 [M+H]+; found: 428.1400.
N-(3-Fluoro-4-(trifluoromethyl)-3-(4-phenyl-1,2,5-oxadiazole-2-
oxide)benzamide (63)
2-Chloro-N-[1-(3-fluoro-4-
To a stirred solution of 4-carboxy-3-phenyl-1,2,5-oxadiazole 2-oxide
(62) (10 mg, 0.05 mmol) in 1.5 mL anhydrous DMF, 3-fluoro-4-
(trifluoromethyl)phenyl)methanamine (25) (19 mg, 0.10 mmol), HATU
(37 mg, 0.10 mmol), and anhydrous DIPEA (0.02 mL, 0.10 mmol) were
added and the mixture was stirred at room temperature for 22 h. The
resulting mixture was washed with H2O and saturated aqueous NaCl
solution, and the aqueous layers were extracted with EtOAc. The
combined organic phases were dried over Na2SO4, filtered, and
concentrated in vacuo. The crude material was purified by column
chromatography (n-hexane/EtOAc, 8:2) to afford 63 (14 mg, 73%) as
yellowish solid. m.p. 108.0–110.0 °C; 1H NMR (600 MHz, CDCl3):
δ = 7.80 (d, 2H, ArH-furoxan), 7.57–7.52 (m, 2H, ArH, ArH-furoxan),
7.46–7.43 (m, 2H, ArH-furoxan), 7.21–7.17 (m, 2H, ArH), 6.70 (bs, 1H,
CH2NHCO), 4.67 (d, J = 5.4 Hz, 2H, CH2NHCO); 13C NMR (150 MHz,
CDCl3): δ = 167.8 (C=O), 160.1 (d, 1J = 260.1 Hz, C–F) 145.7–145.6
(m), 133.9, 132.1, 128.9, 127.7–127.6 (m), 127.1 (d, J = 6.3 Hz), 123.1,
120.2, 116.0 (d, J = 22.2 Hz), 43.2 (CH2NH); 19F NMR (282 MHz,
CDCl3): δ = −61.3 (t, J = 11.9 Hz, CF3), −113.7–−113.9 (m, C–F).
(trifluoromethyl)phenyl)cyclohexyl)methyl]-5-(nitrooxymethyl)-
benzamide (70)
Analogue 70 was prepared following the synthetic procedure for
analogue 18a and was obtained by flash column chromatography (n-
hexane/EtOAc, 90:10 to 80:20) in 10% yield. HPLC: tR: 12.1 min, purity
80%; 1H NMR (600 MHz, CDCl3): δ = 7.61–7.58 (m, 2H, ArH), 7.39–
7.35 (m, 2H, ArH), 7.29 (d, J = 8.3 Hz, 1H, ArH), 7.23 (s, 1H, ArH), 5.83
(bs, 1H, CH2NHCO), 5.36 (s, 2H, CH2ONO2), 3.63 (d, J = 6.3 Hz, 2H,
CH2NHCO), 2.12–1.25 (m, 10H, CH2-cyclohexyl); 13C NMR (75 MHz,
CDCl3): δ = 166.9 (C=O), 141.6, 137.4, 134.4, 132.3, 131.0, 130.5,
127.5–127.2 (m), 122.9, 116.0 (d, J = 20.9 Hz), 78.9 (CH2ONO2), 50.9
(CH2NH), 43.2, 33.8, 26.2, 22.9; HRMS (ESI): m/z calcd for
C22H21ClF4N2O4+H+: 489.1199 [M+H]+; found: 489.1200.
In silico studies
Ligand-based pharmacophore model generation and validation
The LigandScout 4.0 Advanced software, available from InteLigand,
GmbH, Vienna, Austria (http://www.inteligand.com/ligandscout) was
used for the generation and validation of the pharmacophore model.[52]
A detailed description of the experimental procedure is referred to the
section Pharmacophore Model generation and validation in the
Supporting Information.
4-Phenyl-(1,2,5-oxadiazole-2-oxide)-3-[N-(1-(4-
methylphenyl)cyclohexyl)methyl]benzamide (64)
4-Carboxy-3-phenyl-1,2,5-oxadiazole 2-oxide (62) (30 mg, 0.15 mmol)
was dissolved in 3 mL anhydrous DMF. HOBt (27 mg, 0.18 mmol) was
added and the mixture was stirred at room temperature for 10 min,
followed by the addition of EDC hydrochloride (31 mg, 0.16 mmol).
After 10 min, amine 51 (30 mg, 0.15 mmol) and Et3N (0.02 mL,
0.16 mmol) were added and the resulting mixture was stirred at room
temperature for 20 h. The mixture was washed several times with
saturated aqueous NaCl solution, and the aqueous phase was
extracted with EtOAc. The combined organic layers were dried over
Na2SO4, filtered, and concentrated in vacuo. Flash column
chromatography (n-hexane/EtOAc, 90:10) afforded 64 (15 mg, 26%) as
white solid. m.p. 103.0–105.0 °C; HPLC: tR: 10.3 min; 1H NMR
(300 MHz, CDCl3): δ = 7.58 (d, J = 7.4 Hz, 2H, ArH-furoxan), 7.44 (t,
J = 7.4 Hz, 1H, ArH-furoxan), 7.38–7.35 (m, 2H, ArH-furoxan), 7.30 (d,
J = 8.1 Hz, 2H, ArH), 7.22 (d, J = 8.0 Hz, 2H, ArH), 5.68 (bs, 1H,
CH2NHCO), 3.55 (d, J = 6.1 Hz, 2H, CH2NHCO), 2.36 (s, 3H, ArCH3),
2.10–1.38 (m, 10H, CH2-cyclohexyl); 13C NMR (75 MHz, CDCl3):
δ = 167.4 (C=O), 141.4, 136.0, 134.9, 131.4, 129.7, 128.6, 126.85,
126.82, 50.7 (CH2NH), 42.4, 34.0, 26.5, 22.2, 21.0.
In vitro studies
Two-electrode voltage clamp recordings
cDNA encoding hP2X7 was obtained from Life Technologies and
cloned into a modified pUC19 vector via Gibson Assembly (New
England Biolabs). cRNA was synthesized from linearized plasmids with
T7
RNA
polymerase
using
the
mMessageMachinekit
(Invitrogen/Thermo Fisher). Xenopus laevis (NASCO) oocytes were
provided by Prof. Dr. Luis Pardo (Max Planck Institute for Experimental
Medicine, Göttingen) and injected with 50 nL aliquots of cRNA
(0.5 μg/μL). Two-electrode voltage clamp recordings in oocytes were
performed 1–10 days after cRNA injection at a holding potential of
−70 mV using a Turbo Tec-05X Amplifier (npi electronic, Tamm,
Germany). The currents were filtered at 1 kHz and digitized at 200 Hz,
using CellWorks
E 5.5.1 software. The perfusion medium was
2-Chloro-N-[1-(3-fluoro-4-
automatically switched between ND96 [NaCl (96 mM), KCl (2 mM),
MgCl2 (1 mM), CaCl2 (1 mM), HEPES (5 mM)] and ATP (300 μM)
containing low divalent buffer in which MgCl2 was omitted and CaCl2
was reduced to 0.5 mM (in order to reduce inhibition of the P2X7
receptor by divalent cations). A fast and reproducible solution exchange
(<300 ms) was achieved using a 50 μL funnel-shaped oocyte chamber
combined with a fast solution flow (~150 μL/s) fed through a custom-
made manifold mounted immediately above the oocyte. ATP was
applied for 2 s in 4-min intervals. After each application, the cell was
superfused for 54 s with agonist-free ND96, and the flow was then
stopped for 3 min.[53] When the agonist responses were stabilized, the
potential antagonists were mixed from a 10-fold stock into the static
(trifluoromethyl)phenyl)cyclohexyl)methyl]benzamide (67)
Analogue 67 was prepared following the synthetic procedure for
analogue 64 and was obtained by flash column chromatography (n-
hexane/EtOAc 80:20) as colorless oil (64 mg, 55%). HPLC: tR:
11.1 min; 1H NMR (600 MHz, CDCl3): δ = 7.60–7.54 (m, 2H, ArH),
7.39–7.24 (m, 5H, ArH), 5.83 (bs, 1H, CH2NHCO), 3.63 (d, J = 6.3 Hz,
2H, CH2NHCO), 2.12–1.24 (m, 10H, CH2-cyclohexyl); 13C NMR
(75 MHz, CDCl3): δ = 168.0 (C=O), 141.6, 137.4, 136.5, 134.2, 132.3,
130.9, 130.1, 127.5–127.2 (m), 115.8 (d, J = 20.9 Hz), 50.8 (CH2NH),
34.0, 26.0, 21.9; HRMS (ESI): m/z calcd for C21H20ClF4NO+H+:
414.1242 [M+H]+; found: 414.1245.
12
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10.1002/cmdc.202000303
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FULL PAPER
bath and preincubated for 3 min. The dose–response curves were fit to
the data by the equation Y = Bottom + (Top-Bottom)/(1+10^((LogIC50-
X)*HillSlope)) using Prism software (GraphPad Software, Inc., Version
8.3.0, San Diego, CA).
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Acknowledgments
B. B. Mishra, V. A. K. Rathinam, G. W. Martens, A. J. Martinot, H.
Kornfeld, K. A. Fitzgerald, C. M. Sassetti, Nat. Immunol. 2013, 14,
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This work was partly supported by an Alexander S. Onassis
Public Benefit Foundation fellowship of D.P. and the Deutsche
Forschungsgemeinschaft
(DFG,
German
Research
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Foundation) - Project-ID: 335447717 - SFB 1328. This work
was related to the Ph.D. thesis of D.P. (National Hellenic
Research Foundation, Institute of Chemical Biology).
Conflict of interest statement
The authors declare no conflict of interest.
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Keywords: adamant-1-yl • aryl-cyclohexyl • P2X7
antagonists • structure–activity relationship • two-electrode
voltage clamp
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Entry for the Table of Contents
H
HBA
H
H
Structural
modifications
hP2X7
H
inhibition
HBA
A series of novel derivatives were synthesized through successive modifications of reported (adamantan-1-yl)methyl-benzamides to
study their potency as potential hP2X7 receptor antagonists. In vitro evaluation of the synthesized analogues by two-electrode voltage
clamp experiments revealed that the replacement of adamantane by an aryl-cyclohexyl moiety resulted in the most potent derivatives
with IC50 values up to the low micromolar range.
15
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