Synthesis and Pharmacological Evaluation of Novel Non-nucleotide Purine Derivatives as P2X7 Antagonists for the Treatment of Neuroinflammation

Article abstract of DOI:10.1021/acs.jmedchem.0c02145

The ATP-gated P2X7 purinergic receptor (P2X7) is involved in the pathogenesis of many neurodegenerative diseases (NDDs). Several P2X7 antagonists have been developed, though none of them reached clinical trials for this indication. In this work, we designed and synthesized novel blood-brain barrier (BBB)-permeable derivatives as potential P2X7 antagonists. They comprise purine or xanthine cores linked to an aryl group through different short spacers. Compounds were tested in YO-PRO-1 uptake assays and intracellular calcium dynamics in a human P2X7-expressing HEK293 cell line, two-electrode voltage-clamp recordings in Xenopus laevis oocytes, and in interleukin 1β release assays in mouse peritoneal macrophages. BBB permeability was assessed by parallel artificial membrane permeability assays and P-glycoprotein ATPase activity. Dichloroarylpurinylethanones featured a certain P2X7 blockade, being compound 6 (2-(6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one), named ITH15004, the most potent, selective, and BBB-permeable antagonist. Compound 6 can be considered as a first non-nucleotide purine hit for future drug optimizations.

Full text of DOI:10.1021/acs.jmedchem.0c02145

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Article
Synthesis and Pharmacological Evaluation of Novel Non-nucleotide
Purine Derivatives as P2X7 Antagonists for the Treatment of
Neuroinflammation
́
Francesco Calzaferri, Paloma Narros-Fernandez, Ricardo de Pascual, Antonio M.G. de Diego,
Annette Nicke, Javier Egea, Antonio G. García,* and Cristóbal de los Ríos*
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ABSTRACT: The ATP-gated P2X7 purinergic receptor (P2X7) is
involved in the pathogenesis of many neurodegenerative diseases
(NDDs). Several P2X7 antagonists have been developed, though
none of them reached clinical trials for this indication. In this work,
we designed and synthesized novel blood−brain barrier (BBB)-
permeable derivatives as potential P2X7 antagonists. They
comprise purine or xanthine cores linked to an aryl group through
different short spacers. Compounds were tested in YO-PRO-1
uptake assays and intracellular calcium dynamics in a human
P2X7-expressing HEK293 cell line, two-electrode voltage-clamp
recordings in Xenopus laevis oocytes, and in interleukin 1β release
assays in mouse peritoneal macrophages. BBB permeability was assessed by parallel artificial membrane permeability assays and P-
glycoprotein ATPase activity. Dichloroarylpurinylethanones featured a certain P2X7 blockade, being compound 6 (2-(6-chloro-9H-
purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one), named ITH15004, the most potent, selective, and BBB-permeable antagonist.
Compound 6 can be considered as a first non-nucleotide purine hit for future drug optimizations.
is structurally characterized by a dolphin-like shape and
assembles into homotrimers that present three extracellular
ATP-binding pockets at the subunit interfaces.9 Two main
allosteric binding sites have been described so far,10−12 which
are located in the extracellular domain along the longitudinal
axis of the receptor. Most of the antagonists developed so far
bind to the allosteric pocket placed in the upper vestibule of
the receptor, hindering the conformational changes that lead to
the pore opening.
Concerning AD, an in vivo study using the Tg2576 AD
mouse model reported that P2X7s are overexpressed in both
astrocytes and microglia around Aβ senile plaques13 as was also
observed in AD patients.14 Pharmacological inhibition of P2X7
using Brilliant Blue G (BBG) (Chart 1) in the transgenic J20
mice carrying the human APP protein induced a significant
decrease of hippocampal amyloid plaques.15 Moreover, the
P2X7 antagonist GSK1482160 (Chart 1) reduced ATP-
INTRODUCTION
■
Brain disorders affect 1 billion people around the world.1
Neurodegenerative diseases (NDDs) are the most common
form of brain disorder and, since they are age-dependent, this
results in a high socio-economic burden as the longevity of the
global population increases.2 Current available medicines only
mitigate some of the symptoms. For this reason, new
innovative, more potent, and selective drugs are urgently
needed to hinder disease progression. The NDDs, such as
Alzheimer’s disease (AD), Parkinson’s disease (PD), amyo-
trophic lateral sclerosis (ALS), and multiple sclerosis (MS),
feature diverse symptomatology and physiopathology. How-
ever, recent evidences focus on neuroinflammation as a
common central stage in their pathogenesis.
In this context, the ATP-gated P2X7 purinergic ion channels
(P2X7) are emerging as important gatekeepers of neuro-
inflammation3 and as novel therapeutic targets for the above-
mentioned diseases.4 P2X7 is a non-selective ligand-operated
ion channel, physiologically activated by ATP. They are
permeable to Na+, Ca2+, and K+ as well as to small molecules
up to 900 Da of molecular weight.5,6 P2X7s have a low affinity
for ATP. Thus, they are considered as important sensors of
tissue damage and inflammation, conditions where extracellular
ATP levels considerably rise. P2X7 activation triggers immune
responses that mediate maturation and secretion of inter-
leukins, such as interleukin-1β (IL-1β).7,8 Each P2X7 subunit
Received: December 11, 2020
Published: February 9, 2021
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KN-62 derivatives,30 the discovery of berberine alkaloids as
P2X7 blockers,31 and the development of adamantine-32 and o-
chlorobenzamide-based new ligands as well as their juxtapo-
sition products (1, Chart 1).25 Several other scaffolds have
been explored, such as 4,5-diarylimidazolines,33 pyrazolodia-
zepines,34 1-piperidinylimidazoles,35 quinolones and quinoli-
nones,36,37 arylboronic acids,38 thiadiazoles,39 and tetrazoles,
yielding the derivative A438079.40 They all have different
structural motifs, but we noticed that the o-chlorobenzamide
and its isosteres are recurrent patterns. For instance, it is
present in a series of triazole rings disclosed by Janssen41 as
well as in the pyroglutamic acid amide analogue GSK1482160
(Chart 1)42 as a superior homologue and an inverse isostere.
Also, the heteroaryl-cyanoguanidines A74000343 and A804598
(Chart 1)44 can be considered as o-chlorobenzamide isosteres
since both the cyano group and ortho-halogen induce the
dihedral torsion of the aromatic ring over the vicinal guanidine
or carbonyl group, respectively. Finally, the two Janssen
derivatives JNJ-5530894245 and JNJ-5417544646 (Chart 1)
also contain an o-halobenzamide moiety. The former passed
three phase I clinical trials,47 while the latter is currently in a
phase II trial for major depression.48 Apart from this, the other
few P2X7 antagonists that have entered clinical trials were
intended for the treatment of peripheral disorders.49,50 Hence,
there is still a great need for defining accurate drug-like
properties to target the CNS, such as sufficient lipophilicity
and brain tissue half-life. Our research group has recently
embarked on a project aiming at the development of new P2X7
antagonists with enhanced BBB penetration for the potential
treatment of brain diseases such as NDDs. We designed a
novel series of compounds that combined the essential
structural features for their inhibitory capacity on P2X7: the
presence of a heteroaromatic cycle bound to a halobenzene
through a non-complex spacer (Figure 1).
Chart 1. Chemical Structure of Some P2X7 Antagonists,
Including the Adamantane-o-chlorobenzamide Scaffold
Reported by Astra-Zeneca (1)25
induced microglia migration and restored their phagocytic
activity in mice.14
As for PD, P2X7s are overexpressed in PD patients,16 and
their activation is cytotoxic for dopaminergic SN4741
neurons.17 BBG succeeded in preventing dopaminergic neuron
loss in the 6-hydroxydopamine-injected rat model of PD.18,19
P2X7s have an important role in ALS neuroinflammation,20
and their expression is up-regulated in the post-mortem spinal
cord tissue of ALS patients.21 The treatment with BBG
improved motoneuron survival and motor performance deficit
of the treated SOD1G93A mice,22 a disease model for ALS,
though no effect was observed when JNJ-47965567 (Chart 1),
a more potent and selective antagonist, was used.23 Finally, a
high expression of P2X7 in activated microglia and astrocytes
of post-mortem MS patients has been reported.21,24
The earliest attempts to synthesize P2X7 antagonists based
on nucleotide analogues resulted in the irreversible inhibitor o-
ATP (Chart 1).26 Then, the discovery of the triphenylmethane
dye BBG, which is blood−brain barrier (BBB)-permeable27
but a non-selective P2X7 antagonist, constituted a real
breakthrough. Other first-generation P2X7 antagonists were
the non-selective pyridoxalphosphate derivative PPADS,28 the
naphthylsulfonate suramine, and its isoquinoline derivative
KN-6229 (Chart 1). During the last 2 decades, the search for
more potent, selective, and CNS-penetrant P2X7 antagonists
has been a continuous challenge, which has led to a series of
Figure 1. Scheme of the designed molecules. A purine scaffold (blue)
was linked to an o-halophenyl (green) through three different spacers
(red): carbonyl (A), ethanoyl (B), or sulfonyl (C) groups.
On this basis, we surprisingly noticed that the purine scaffold
had been only explored in the earliest nucleotide P2X
ligands,51 but it had been never linked to the o-
chlorobenzamide moiety. We then synthesized such non-
nucleotide purine derivatives, hypothesizing that the haloben-
zamide substructure would afford potency and selectivity,
whereas the purine-like heterocycles would contribute to favor
BBB penetration.
RESULTS
■
Chemistry. Following the chemical design (Figure 1),
several structures were proposed. At a first glance, the simplest
design was based on connecting a purine analogue with o-
chlorobenzamide through benzoylation. 6-Chloropurine was
selected as a substrate to achieve regioselective nucleophilic
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acyl substitution, assuming that chlorine at that position would
hinder the N7 nucleophilic center. We first selected o-
chlorobenzoic acid as the electrophile, assisted by cyanuric
chloride,52 unsuccessfully. With the use of o-chlorobenzoyl
chloride as the acylating reagent, several bases, for example,
pyridine or TEA, and solvents such as DMF, CH2Cl2, or
MeCN were probed, achieving the best yield (78%) for the
synthesis of 2 with the TEA/DMAP system as the base in THF
(Scheme 1). Analogously, 6-methoxypurine (3), prepared by
aromatic nucleophilic substitution with Na in MeOH,
furnished benzoyl purine 4 in moderate yields (53%, Scheme
1).
S1, Supporting Information), easily accessible through Suzuki
coupling of the THP-protected 6-chloropurine 7 with
phenylboronic acid that gives rise to intermediate 8 and the
subsequent deprotection to form 6-phenylpurine 9. Com-
pound 17 (R1 = Cl, R2 = F, R3, R4 = Cl, Scheme 2) was
synthesized from 6-chloro-2-fluoropurine 10, which was
obtained from 6-chloropurin-2-amine and an HF solution in
pyridine. Compound 18 (R1 = I, R2, R3, R4 = Cl, Scheme 2)
was synthesized from 2-chloro-6-iodopurine 11, which was
prepared by treating 2,6-dichloropurine with HI. Compounds
19 and 20, bearing a methylamine at C6 or an amine at C2,
respectively, were prepared through nucleophilic substitution
of commercial purines (2-chloro-N-methylpurin-6-amine and
6-chloropurin-2-amine, respectively) over the alkyl chloride 5,
assisted by tetrabutylammonium hydrogensulfate (TBAHS)
phase transfer catalysis (Scheme 2). As expected, the presence
of amines, susceptible to compete with N9 as nucleophile
centers, lowered the chemical yield of the reactions (Table 2).
X-ray analysis of 19 confirmed the expected regioselectivity of
the reaction toward the N9-alkylation (Figure 2). To
a
Scheme 1. Synthesis of o-Chlorobenzoylpurines 2 and 4
a
Reagents and conditions: (a) TEA, DMAP, THF, 4.5−6 h, 0 °C to
rt; (b) Na, MeOH, 2 h, 60 °C.
Subsequently, the homologous derivatives of N-acylpurines
were synthesized to assess the effect of the spacer lengthening
over the P2X7 antagonist capability and to improve the critical
chemical stability (acid-catalyzed hydrolysis of the amide) of
N-acylpurines. The nucleophilic substitution of 6-chloropurine
on 2-chloro-1-(2,4-dichlorophenyl)ethan-1-one (5) assisted by
NaH as the base in DMF afforded 6 (R1, R3, R4 = Cl, R2 = H,
Scheme 2).
Figure 2. X-ray diffraction of compounds 19 and 31. Purine and
theophylline scaffolds used in this study have the same influence on
N7 and N9 nucleophilicity for all the respective derivatives.
understand whether the chlorine at the ortho position was
relevant for blocking the P2X7 opening, we synthesized other
derivatives of compound 6 bearing only the halogen at the para
position (R3 = H, i.e., 12 and 13, Scheme 2).
Scheme 2. Synthesis of Purinyl-2′,4′-disubstituted
a
Phenylethanones (6 and 12−20)
We also explored if lengthening the spacer present in
compound 6 affected the pharmacological activity by inserting
a propanone instead of an ethanone as the spacer between the
purine core and the aryl group (Scheme 3). We prepared
Scheme 3. Synthesis of 3-(6-Chloro-9H-purin-9-yl)-1-(2′,4′-
a
dichlorophenyl)-propan-1-one 22
a
Reagents and conditions. For the synthesis of 6 and 12−18: (a)
NaH, DMF, 15−40 h, 0 °C to rt. For the synthesis of 19: (a) KOH,
Bu4NHSO4, DMF, 26 h, 0 °C to rt. For the synthesis of 20: (a)
K2CO3, Bu4NHSO4, DMF, 30 h, 0 to 80 °C.
a
When tested on the selected pharmacological assays
described below, purinylethanone 6 presented the most
promising P2X7 antagonist profile. For this reason, we
synthesized several new analogues of 6 to optimize its
pharmacological properties (12−20). Modifications of 6
mainly pursued to probe the effect of substitution at C2 (R2,
Scheme 2) and the replacement of chlorine at C6 (R1, Scheme
2). Compounds 12−20 were obtained in low to good yields
(Table 2) following the experimental procedures depicted in
Scheme 2. Compound 14 (R1 = Ph, R2 = H, R3, R4 = Cl,
Scheme 2) was synthesized from 6-phenylpurine (9, Scheme
Reagents and conditions: (a) AlCl3, 4 h, rt to 60 °C; (b) 6-
chloropurine, NaH, DMF, 18 h, 0 °C to rt.
compound 22 through a Friedel−Crafts reaction of 1,3-
dichlorobenzene with 3-chloropropanoyl chloride to form
intermediate 21 and the subsequent nucleophilic substitution
with 6-chloropurine (Scheme 3).
Another strategy to modify the spacer and to improve the
stability was the insertion of a sulfonamide moiety (Scheme 4),
which is considered a non-classical bioisostere of amides. For
this purpose, 6-chloropurine reacted with o-chlorobenzenesul-
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Scheme 4. Synthesis of 2-Chlorophenylsulfonyl Purines
(23−27)
Scheme 5. Synthesis of 2′,4′-Dichlorophenylethanones and
2′-Chlorobenzamides, Using Xanthine Derivatives as the
a
a
Starting Material
a
Reagents and conditions: (a) TEA, DMAP, CH2Cl2/THF, 0 °C to
rt, 2−4 h.
fonyl chloride to give 23 (R1 = Cl, R2 = H) in low yield when
NaH was used as a base. Yield was enhanced (77%) by using
the TEA/DMAP system (Scheme 4). Although compound 23
did not show a relevant P2X7 blockade (Table 1), we prepared
Table 1. P2X7 Antagonist Profile of the First Purine and
Xanthine Derivatives Assessed by the YO-PRO-1 Aye Assay
in hP2X7-HEK293 Cells and by TEVC in hP2X7-Expressing
X. laevis Oocytes
a
c
compd.
scaffold
spacer
YO-PRO-1 (%)
IATP (%)
a
Reagents and conditions: (a) 2-chlorobenzoyl chloride, TEA,
DMAP, THF, 48 h, 0 to 70 °C; (b) 2-chloro-1-(2,4-dichlorophenyl)-
ethan-1-one (5), K2CO3, Bu4NHSO4, DMF, 1−6 h, 150 °C.
JNJ
2
4
56 5***
49 4*
purine
purine
purine
purine
theobromine
theobromine
theophylline
theophylline
purinone
−CO−
−CO−
−CH2CO−
−SO2
−CO−
−CH2CO−
−CH2CO−
−SO2−
7
1
2
8
5
0
2
1
b
6
63 4***
47 1***
due to the low nucleophilic strength of N1. Analogously but
using theophylline as the starting material, we isolated 30 in
moderate yields (Scheme 5, 45%). Only one of the two
possible regioisomers was obtained. NOESY analysis did not
show any spatial coupling between methylene and the methyl
group at N3 (Figure S1, Supporting Information), confirming
the more probable N7 alkylation.
23
28
29
30
31
40
17
5
6
10
9
5
2
7
8
2
2
nb
0
8
10
b
52 4***
16
28 2**
−CH2−
6
9
2
a
The incorporation of the o-chlorophenylsulfonyl moiety to
theophylline, following the procedure shown in Scheme 6, gave
hP2X7-HEK293 cells stimulated with BzATP 30 μM. Compound
concentration was 10 μM, except for JNJ-47965567 (JNJ, 1 μM).
b
Data are mean S.E.M. of triplicates of three different cultures. IC50
data of 6 and 31 were calculated, being 9
3 and 10
4 μM,
Scheme 6. Synthesis of Arylsulfonyl Theophyllines (31−
c
a
respectively. Oocytes expressing hP2X7 stimulated with ATP 0.3
mM. Compound concentration was 100 μM, except for JNJ (1 μM).
Data are mean S.E.M. of triplicates of three different oocytes from
two different frogs. *P < 0.05, **p < 0.01, ***p < 0.001, nb = no
blockade.
38)
new purine derivatives bearing a sulfonyl spacer by considering
the good biological results obtained with sulfonamide 31.
Thus, we replaced the chlorine at C6 of 23 (R1, Scheme 4) by
polar groups and evaluated the influence of substitutions at C2
(R2, Scheme 4) over the pharmacological activity. Following
the synthetic procedure provided in Scheme 4, analogues of
compound 23 (24−27) were prepared in low to good yields
and mild conditions.
The xanthine scaffold was also considered to build ligands
under the chemical design outlined in Figure 1. Xanthine
derivatives (e.g., theobromine, theophylline, caffeine, and so
on) are substances with well-known CNS effects, thus eligible
as the starting point to develop drugs with extended brain
penetration. Hence, theobromine suffered benzoylation with o-
chlorobenzoyl chloride under the conditions outlined in
Scheme 5 to yield benzoyltheobromine 28 (Scheme 5).
Unlike 6-chloropurine, theobromine reacted better with 5
when using K2CO3 as the base in DMF at 150 °C under phase
transfer catalysis (TBAHS) to form 29 (Scheme 5) presumably
a
Reagents and conditions: (a) NaH, THF, 4−48 h, 0 °C to rt.
31 in a regioselective fashion, as proved by X ray diffraction
(Figure 2). We thus confirmed that N7 is the most
nucleophilic center in theophylline as proposed for 30. The
theophylline 31 was the most promising analogue among the
xanthine derivatives (Table 1). For this reason, we proposed
tiny chemical modifications to increase its potency over P2X7.
In this case, we focused on modifying substitutions at the
pending phenyl ring (Scheme 6). Compounds 32−38 were
obtained following the procedure shown in Scheme 6 with
good yields.
Finally, to further explore possible modifications on the
spacer and to evaluate whether a more stable amide than the
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one present in benzoylpurines 2 and 4 could be essential for
P2X7 antagonism, we designed a compound starting from 6-
purinone and that included a methylene group as purinylar-
ylethanones 6 and 12−20. The result was the synthesis of 40
by nucleophilic substitution of 6-purinone 3953 over 1-chloro-
2-(chloromethyl)benzene (Scheme 7).
YO-PRO-1 uptake and ATP-activated currents were not
affected by purines 2, 4, 23, and the N1-benzyl-6-purinone 40.
By contrast, the ethanoyl spacer successfully contributed to the
blockade of YO-PRO-1 uptake exerted by purine derivative 6
(63% at 10 μM, Table 1) but not in xanthine derivatives 29
and 30. The sulfonyl spacer was only effective when linked to
theophylline, as in 31, which halves the YO-PRO-1 uptake at
10 μM and reduces ATP currents in hP2X7-expressing oocytes
(Table 1). Those compounds possessing a %blockade of YO-
PRO-1 uptake higher than 40% at 10 μM were subjected to
concentration−response experiments. Thus, compounds 6 and
31 presented an IC50 defined as their concentration capable of
reducing the BzATP-induced YO-PRO-1 uptake by 50% of 9
Scheme 7. Synthesis of 2-Chloro-1-(2′-chlorobenzyl)-9-
a
methyl-1,9-dihydro-6H-purin-6-one (40)
3 and 10
4 μM, respectively. Interestingly, the two
screening methods confirmed each other’s outcomes despite
the experimental differences.
a
Reagents and conditions: (a) basic alumina, MeOH, 24 h, 70 °C; (b)
1-chloro-2-(chloromethyl)benzene, NaHCO3, DMF, overnight, 75
Considering the promising pharmacological profile of 6 as
P2X7 antagonist, its analogues 12−20 were prepared and
biologically assessed (Table 2). Most of the compounds 12−
20 did not reach the blocking properties of their head
compound 6 (Table 2), as measured by the YO-PRO-1 assay
and two-electrode voltage-clamp (TEVC). Compounds 12, 14,
and 16 presented an IC50 to block the BzATP-induced YO-
PRO-1 uptake of 10 2, 6 1, and 13 2 μM, respectively,
comparable data to that of compound 6. In any case, the use of
the ethanoyl spacer seemed appropriate since most of them
reduced the YO-PRO-1 uptake in a significant manner.
Generally, we noticed that an increase in polarity of the
substituents on the purine scaffold lowered the inhibitory
potential. Moreover, hydrophobic bulky substituents at the C6
position (R1) were well tolerated, like the phenyl present in 14.
By contrast, only the highly halogenated derivatives 16 and 17
showed a decent mitigation of the ATP-evoked currents,
°C.
Biological Evaluation. Compounds 2, 4, 6, 23, 28−31,
and 40 were initially assessed as P2X7 antagonists by two
experimental procedures, YO-PRO-1 dye uptake assay54,55 and
inhibition of ATP-activated currents in hP2X7-expressing
Xenopus laevis oocytes (Table 1). In the former, the potent
P2X7 agonist benzoyl-ATP (BzATP) activates the human
P2X7 receptors (hP2X7), stably transfected in HEK293 cells.
After channel opening, the dye enters the cell and binds to
nucleic acids, giving a fluorescent signal. In the latter, ionic
currents are evoked by ATP (300 μM) in the presence or
absence of the assessed compounds to test their ability to
inhibit channel opening. The percentages of blockade in both
assays are shown in Table 1. JNJ-47965567 (Chart 1) was used
as the reference P2X7 antagonist.
Table 2. P2X7 Antagonist Profile of Purinylarylethanones 12−20, Analogues of 6, Assessed by the YO-PRO-1 Dye Uptake
Assay in hP2X7-HEK293 Cells and by TEVC in hP2X7-Expressing X. laevis Oocytes
a
b
d
compd.
R1
R2
R3
R4
yield (%)
YO-PRO-1 (%)
IATP (%)
JNJ
6
56 5***
63 4***
55 4***
34 7**
62 3***
25 3***
41 3***
27 5**
49 4**
37 1***
c
Cl
Cl
Cl
Ph
MeO
Cl
Cl
I
NHMe
Cl
H
H
H
H
H
Cl
F
Cl
Cl
NH2
Cl
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
45
48
83
41
28
40
39
28
22
10
c
12
13
14
15
16
17
18
19
20
11
7
11
3
2
2
c
14 2*
24 2**
24 3*
c
14
2
4
9
6
5
6
2
7
4
4
16
a
b
Chemical yields of the reaction in Scheme 2. hP2X7-HEK293 cells stimulated with BzATP 30 μM. Compounds assayed at 10 μM, except for
c
JNJ-47965567 (JNJ, 1 μM). Data are mean S.E.M. of triplicates of three different cell cultures. IC50 data of 6, 12, 14, and 16 were calculated,
being 9 3, 10 2, 6 1, and 13 2 μM, respectively. Oocytes expressing the hP2X7 stimulated with ATP 0.3 mM. Compounds assayed at 100
d
μM, except for JNJ (1 μM). Data are mean S.E.M. of triplicates of three different oocytes from two different frogs. *P < 0.05, **p < 0.01, ***p <
0.001.
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slightly worse than that exerted by compound 6. Interestingly,
substitutions at C2 (R2) decreased ligands’ potency,
presumably due to steric effects since halogens are better
tolerated than an amine, but no substitution still affords the
best result.
Table 4. P2X7 Antagonist Profile of
Arylsulfonyltheophyllines 32−38, Analogues of 31, Assessed
by the YO-PRO-1 Dye Uptake Assay in hP2X7-HEK293
Cells and by TEVC Analysis in hP2X7-Expressing X. laevis
Oocytes
According to ATP current data of compounds 12 and 13,
the removal of chlorine at C2′ of the aryl residue leads to a
decrease in potency, meaning that the chlorine is necessary for
antagonism, and confirming our hypothesis that an o-haloaryl
moiety (or its structural isostere-like aryl-cyanoguanidine) is
essential for the P2X7 blocking activity, as represented in the
majority of P2X7 antagonists.
a
b
d
Compound 22, the superior homologue of compound 6,
scarcely affected both YO-PRO-1 uptake (19 9% blockade)
compd.
R1
R2
R3 yield (%) YO-PRO-1 (%) IATP (%)
JNJ
31
32
33
34
35
36
37
38
56 5***
52 4***
49 4*
28 2**
and ATP currents (6
1% blockade), indicating that
c
Cl
Cl
Cl
Cl
F
Me
Cl
H
H
Cl
F
CF3
F
F
H
H
H
H
H
H
Cl
H
69
74
54
75
72
78
73
81
lengthening the spacer is not beneficial to inhibit the P2X7.
Hence, we did not launch further similar analogues.
As for analogues of arylsulfonylpurine 23 (24−27), the
insertion of polar substituents hindered the P2X7 inhibition
(Table 3). Apparently, the addition of a chlorine atom at C2
7
20
5
7
23
4
3
4
3
4
7
5
1
9
1
nb
nb
1
nb
nb
nb
2
H
F
Table 3. P2X7 Antagonist Profile of 2-
10
Chlorophenylsulfonylpurines 24−27, Analogues of 23,
Assessed by the YO-PRO-1 Dye Uptake Assay in hP2X7-
HEK293 Cells and by TEVC Analysis in hP2X7-Expressing
X. laevis Oocytes
a
b
Chemical yields of the reaction in Scheme 6. hP2X7-HEK293 cells
stimulated with BzATP 30 μM. Compounds assayed at 10 μM, except
for JNJ-47965567 (JNJ, 1 μM). Data are mean S.E.M. of triplicates
c
of three different cell cultures. IC50 of 31 was calculated, being 10
4 μM. hP2X7-expressing oocytes stimulated with ATP 0.3 mM.
d
Compounds assayed at 100 μM, except for JNJ (1 μM). Data are
mean
S.E.M. of triplicates of three different oocytes from two
different frogs. *P < 0.05, **p < 0.01, ***p < 0.001.
subfamily dramatically impairs the favorable interactions of
ligands with the binding site of the receptor.
Cytosolic Ca2+ concentration ([Ca2+]c) changes were
assessed in the presence of most of the synthesized compounds
to corroborate the pharmacological results acquired (Table S1,
Supporting Information). The [Ca2+]c was monitored in
hP2X7-expressing HEK293 cells with the fluorescent dye
FURA-2 after P2X7 activation by BzATP. Compound 6
showed the best receptor inhibition (Figure 3), in agreement
with previous assays. It exerted a concentration-dependent
P2X7 blockade with an IC50 of 13.7 μM. The activity of
compound 14 in the Ca2+ dynamics assay is in line with the
YO-PRO-1 data (Table S1, Supporting Information) but not
with those of TEVC (Table 2). This incongruence could be
ascribed to the high concentration used in the TEVC
experiments, which would be reaching the solubility threshold
of 14.
The potency of a selection of the novel antagonists reported
in this paper was then evaluated in a well-known model of
P2X7-induced inflammation,56,57 which is the quantification of
IL-1β released by murine peritoneal macrophages (MPMs),
after LPS priming, and ATP stimulation (Figure 4). Only
compounds 6 and 31 reduced IL-1β release in a concentration-
dependent fashion (Figure 4A,B, respectively), in agreement
with their P2X7-blocking properties found in the previous
assays. In addition, these experiments demonstrated that the
two most promising hit compounds are also active on Mus
musculus P2X7.
a
b
d
compd.
R1
R2
yield (%) YO-PRO-1 (%) IATP (%)
56 5*** 49 4*
17
JNJ
23
24
25
26
27
Cl
Cl
H
Cl
H
NH2
Cl
77
47
37
50
48
6
9
7
1
8
c
46 4**
2
MeO
Cl
NHMe
nb
8
9
4
9
8
Nb
2
1
a
b
Chemical yields of the reaction in Scheme 4. hP2X7-HEK293 cells
stimulated with BzATP 30 μM. Compounds assayed at 10 μM, except
for JNJ-47965567 (JNJ, 1 μM). Data are mean S.E.M. of triplicates
of three different cell cultures. IC50 of 24 was calculated, being 10.2
0.6 μM. hP2X7-expressing oocytes stimulated with ATP 0.3 mM.
Compounds assayed at 100 μM, except for JNJ (1 μM). Data are
S.E.M. of triplicates of three different oocytes from two
different frogs. *P < 0.05, **p < 0.01, ***p < 0.001, nb = no
c
d
mean
blockade.
(R2 = Cl, Scheme 4), as in compound 24, conferred potency to
these derivatives according to the YO-PRO-1 assay. For this
reason, its IC50 was calculated, being 10.2 0.6 μM, a similar
value to those found for the best compounds of these series.
However, considering that these data were not confirmed by
TEVC (Table 3), we speculate that the potency of compound
24 is not strictly related to P2X7 pore opening.
Derivatives 32−38 were prepared and pharmacologically
assessed according to the apparent blocking properties of
arylsulfonyltheophylline 31 (Scheme 6). However, they
elicited a very weak blockade of P2X7 (Table 4). It seems
that the substitution at the phenyl ring in the theophylline
All the pharmacological data classified by chemical families
have been collected and reorganized in Table S1 (Supporting
Information). Since compound 6 stood out as the most potent
P2X7 antagonist, we evaluated its selectivity in three rat P2X
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Figure 3. (A) Raw data representation of a FURA-2 intracellular Ca2+
dynamics experiment in the presence of increasing concentrations of
compound 6 (ITH15004). The BzATP (100 μM) control response is
shown in black. (B) Dose−response curve of 6 (ITH15004) in
[Ca2+]c dynamics. Dots represent the mean
S.E.M. of three
triplicates of three different cell cultures.
receptor subtypes by TEVC in X. laevis oocytes (Figure 5).
Compound 6 showed high selectivity for the human P2X7
against rat P2X1, P2X2, and P2X4 receptors. The selectivity is
unlikely to be ascribable to species difference since compound
6 could still inhibit the rat P2X7, apparently with a higher
potency (Figure S2, Supporting Information).
To direct the synthesis of the novel compounds toward
BBB-permeable ligands, we had initially evaluated their
physico-chemical properties, such as lipophilicity, number of
hydrogen bonds donors and acceptors, and the molecular
weight.
Later, we took advantage of the parallel artificial membrane
permeability assay (PAMPA), which measures the molecular
ability of compounds to cross a lipophilic membrane by passive
diffusion. We selected the best inhibitory compounds among
all the families. The majority of the tested compounds showed
an elevated permeability that should guarantee their distribu-
tion within the CNS according to Di et al.58 (Figure 6).
Curiously, compound 16 has a lower permeability than the
other structural analogues, although it bears an additional
chlorine atom with respect to 6. Replacement of chlorine with
fluorine (17) leads to better permeability but not as much as
when a hydrogen occupies the C2 position of the purine core.
N-Alkylation and N-sulfonylation of theophylline induced a
huge increase of BBB permeability as initially hypothesized
(compounds 29−31). Indeed, compound 31 showed a huge
permeability rate. These derivatives, together with 6, showed
better permeability than the P2X7 antagonist JNJ-47965567
(JNJ).
Figure 4. IL-1β release (%) from LPS-primed, ATP-stimulated MPMs
in comparison to control (only ATP; black bar). (A) Cells were
primed with LPS for 4 h and the compounds were added for 15 min
at 1 μM. Cells were then stimulated with ATP for 30 min and the
levels of IL-1β in medium were determined by ELISA. (B) Dose−
response effect of compound 6. (C) Dose−response effect of
compound 31. JNJ-47965567 (JNJ, tested at 0.1 μM) was used as
the reference compound. Data are mean
S.E.M. of triplicates of
three different mice. *P < 0.05, **p < 0.01, ***p < 0.001 with respect
to control (black bar).
endure in the CNS, we measured in addition their ability to
stimulate the P-glycoprotein (Pgp) ATPase activity as potential
substrates of this efflux pump, highly expressed in the BBB and
responsible of expulsion of most of xenobiotics from the CNS.
We also measured the effect of the well-known Pgp substrate
verapamil and the P2X7 blocker JNJ-47965567 (JNJ), all of
them tested at 10 μM. The Pgp substrate verapamil59 up-
regulated the ATPase activity of Pgp, measured as the variation
in luminescence (R.L.U., Figure 7). Surprisingly, JNJ-
47965567 strongly activated Pgp ATPase, too, meaning that
it is likely to be an important substrate of the efflux pump. This
was not reported yet in the literature, although ex vivo
radioligand assays in rat brains demonstrated the capacity of
JNJ-47965567 to endure in the CNS for at least 2 h,60 with a
continuous decrease in concentration during the time.61 Unlike
JNJ-47965567, compounds 6 and 31 did not affect Pgp
ATPase activity in statistical significance (P = 0.09 and 0.16,
respectively, Figure 7). Considering that human and rat P-
Compounds 6 and 31 showed an interesting inhibitory
activity on P2X7 and high permeability through lipid
membranes. In order to predict whether they are able to
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Figure 7. Effect of compounds 6 and 31 on Pgp ATPase activity
compared with those of the Pgp substrate verapamil and the P2X7
blocker JNJ-47965567 (JNJ). Data are presented as the mean
S.E.M. of triplicates of three individual experiments. *P < 0.05.
core deep into the pocket, interacting in a presumably π−π
stacking with phenylalanines 95 (F95), 103 (F103), and 293
(F293), as previously described for Calmidazolium.11 Besides,
all the antagonists crystallized in the pdP2X7 show their most
lipophilic moiety in this vestibule.9 The purine core, instead, is
placed among tyrosine 295 (Y295) and 298 (Y298) as well as
isoleucine 310 (I310) and methionine 105 (M105). The top
area of the binding pocket is defined by the presence of
phenylalanines 88 (F88) and 108 (F108), which suggests a
halogen-aromatic interaction of the chlorine at C6 with these
residues (Figure 8). This would explain why the compounds
with polar groups at this position lose their activity. Moreover,
the replacement of the chloride with a bulkier iodine at C6
reduces the potency, considering that the distance between
halogen and F88/F108 is about 3.5 Å.65 Noteworthily, many
residues of this pocket have shown an essential contribution
for receptor blockade in mutagenesis experiments.9,11 Further
investigation is needed to validate this in silico prediction and
the potential involvement of these residues in ligand binding.
Figure 5. Evaluation of 6 by TEVC analysis. (A) Typical TEVC
recording of ATP (300 μM, 2 s)-activated hP2X7 currents in the
presence (red) and absence (black) of 6 at 100 μM. (B) Selective
inhibition of hP2X7 but not of rP2X1, rP2X2, and rP2X4 by 6 at 100
μM. Data represent the mean S.E.M. of three oocytes from two
different frogs, normalized to control current amplitude (marked as a
horizontal dashed red line). *P < 0.001 compared to control.
DISCUSSION
■
Despite efforts carried out by Big Pharma and Academia in the
last decade, no P2X7 antagonists have reached clinical trials for
the treatment of NDDs. Only JNJ-54175446 (Chart 1) drew
attention for the potential treatment of a CNS disease, that is,
major depression.47,48 Indeed, the few candidates blocking
P2X7 studied in clinical trials have focused on peripheral
disorders.49,50,66 This fact evidences the critical role that
pharmacokinetics properties play in the selection of a drug
candidate for NDDs besides high potency, selectivity, and an
interspecies blocking effect. Most of the studies reported in
literature to find a proper P2X7 antagonist for NDDs have
focused only on pharmacological potency. When a head
compound was selected, it commonly lacked an adequate
pharmacokinetic profile to penetrate the CNS, forcing
medicinal chemists to rethink all the design programs. Such
is the case of the adamantine series, which showed a good
P2X7 antagonist effect but a mediocre pharmacokinetic profile
(for instance, t1/2 = 0.22 h).32
In this work, we have paid attention to brain penetration,
starting from the molecular design. First, we selected well-
known scaffolds that are present in drugs with recognized CNS
activity. Then, we corroborated that the target compounds
fulfilled Lipinski’s rule of five. These preliminary considerations
guided us to synthesize and study the pharmacological scope of
novel non-nucleotide purine derivatives as potential antago-
nists of P2X7. The purine scaffold of these novel compounds
has been linked to a lipophilic group, that is, a substituted
Figure 6. Permeability values of selected new compounds (reddish
bars are purine analogues and bluish bars are xanthine analogues),
measured by PAMPA experiments. Theophylline (T), piroxicam (P),
and verapamil (V) were used as low-, middle-, and high-permeability
standards, respectively. Their permeability constants in 10−6·cm·s−1
are 0.12 (T), 2.5 (P), and 16 (V), similar to those described by Di et
al.58 The red line at 4·10−6·cm·s−1 marks the theoretical threshold for
guaranteeing CNS permeability. Data are presented as the mean
S.E.M. of triplicates of three individual experiments performed in
three different days.
glycoprotein activities are usually comparable,62−64 our data
suggest that our compounds could persist in the CNS similar
to or even better than JNJ-47965567 despite its enhancing
activity on the Pgp.
Computational Study. Finally, we wanted to predict the
binding pose of compound 6, being the most potent P2X7
ligand discovered in this work. Most of the antagonists
developed so far act in a highly lipophilic allosteric binding
pocket located between neighboring subunits in the extrac-
ellular domain (Figure 8). We ran docking experiments in this
binding site using the model described by Bin Dayel and co-
workers (Figure 8 and Video S1, Supporting Information).11
The majority of the poses obtained placed the dichloroaryl
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their inhibitory potency, and we consider that it might interact
differently with P2X7 than purinylethanones. Compound 6 (2-
(6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one),
which we have named ITH15004, has a chlorine at C6, which
can be replaced by other halogens such as iodine. However,
this results in a decrease in potency. Substitution of the
chlorine with a phenyl, like in 14, might be permitted, but
adding some polar substituent to increase compound solubility
was an issue that led to contradictory data in this work. Also,
the insertion of smaller halogens such as fluorine or non-
voluminous alkyl chains at this position has not been tested yet
as well as the effect of substituents at position 8 of the purine
core. Small substituents at C2 are accepted with a decrease in
potency, but a reduction in BBB-permeation is expected as
happened for compound 16 in PAMPA. The removal of the
chlorine at C2′ at the aryl group of the ethanoyl derivatives
seems detrimental to blockade. This might be due to lipophilic
interactions with V12 (Figure 8), one of the residues
responsible for P2X7 inhibition with other antagonists.9,11
Compound 6 (ITH15004) elicits a concentration-depend-
ent blockade of BzATP-evoked [Ca2+]c transients in hP2X7-
HEK293 cells, with an IC50 of 13.7 μM, and blocks by 63% at
10 μM the BzATP-induced YO-PRO-1 uptake in the same
cellular model. At 100 μM, it also decreases by 47% the ATP
(300 μM)-activated currents in Xenopus laevis oocytes
expressing hP2X7. It was also shown to be P2X7-selective as
it did not block rat P2X1, P2X2, or P2X4 but rat P2X7.
Compound 6 also blocked mouse P2X7, halving the ATP-
induced IL-1β release in LPS-primed MPMs, and exhibits a
high lipophilic membrane permeability in the PAMPA,
suggesting that it can permeate BBB and gain access to the
brain.
Although the potency of compound 6 is in the micromolar
range, its pharmacodynamic profile and BBB permeability
suggest that this non-nucleotide purine derivative could serve
as the starting point to design and synthesize new P2X7
blockers with moderate potency. This is relevant in the context
of drug side effects and safety, which can be compromised with
highly potent compounds that target P2X7 in the low
nanomolar range. Moreover, it was observed that the complete
removal of P2X7 in a mouse model of multiple sclerosis caused
an exacerbation of the disease, probably due to P2X7-
dependent changes in lymphocytic homeostasis,67 which
could also be affected by a chronic administration of a potent
antagonist. Hence, we consider that a modulation of P2X7 by a
chronic administration of a mild potent antagonist could
potentially lead to better results. This could presume a point in
favor for compounds 6 and 31 that confirmed the success of
our chemical design, aimed at developing a P2X7 antagonist
with improved pharmacokinetic profile and high BBB
permeability rate. These features represent a fundamental
issue that affects the development of agents targeting the CNS,
such as the well-known P2X7 antagonist JNJ-47965567, which
was never included in a clinical trial. Although it was
demonstrated that it reaches the CNS,60 we have observed
for the first time that it also increases the Pgp ATPase activity,
which could jeopardize its positive effects on CNS disorders.
Pursuing this hypothesis is out of the scope of this work, but it
underlines the necessity of a deeper focus on the
pharmacokinetic parameters of potential new CNS drugs, as
we have intended.
Figure 8. Lateral (A) and top (B) views of the ribbon-like
representation of P2X7. The predicted binding site of compound 6
is indicated by the black frame. The residues defining the binding
pocket are shown in C. The protein model was provided by Dayel et
al.11 with permission (crystal templates PDB IDs: 5U1U,5U1V,
5U1W, 5U1X, 5U1Y).
benzene, through a small spacer, namely, a carbonyl, a sulfonyl,
and an ethanoyl moiety. All the data obtained through the
pharmacological assays revealed that these features fit the
structural properties necessary to inhibit the P2X7 but that
more investigation is needed to fully define them. The most
potent compound of our series, 6, belongs to the family that
possesses an ethanoyl group as the spacer. Only compound 31,
which bears a sulfonyl spacer and a theophylline core in its
structure, showed a certain blockade. All its derivatives lose
In conclusion, in this work, we have designed and
synthesized a novel series of non-nucleotide purine derivatives
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(gradient of EtOAc in hexane, 18/82 to 100/0), giving compound
7 as a transparent oil that solidified upon standing (1.45 g, 62%). H
NMR (300 MHz, CDCl3): δ 8.75 (s, 1H, H2), 8.34 (s, 1H, H8), 5.80
(dd, J = 10.2 Hz, 2.6 Hz, 1H, CHO), 4.20 (dd, J = 12.8, 3.0 Hz, 1H,
CH2O), 3.79 (td, J = 11.4, 3.1 Hz, 1H, CH2O), 2.28−1.96 and 1.94−
1.63 (2m, 6H, (CH2)3CH2O).
that show a certain effect as the P2X7 antagonist when an
ethanoyl moiety is used as a spacer according to our designed
model (Figure 1). Most of them have high lipophilicity, being
adequate to target the CNS. ITH15004 was the best antagonist
of the series and could be used as a tool for further in vitro and
in vivo studies in neurodegenerative diseases where chronic
administration and P2X7 modulation might be preferred.
Future studies should be addressed to further explore the
structure−activity relationships of this scaffold and evaluate
other important features such as metabolism and CNS
stability, without affecting its gained selectivity and good
BBB permeability.
1
6-Phenyl-9-(tetrahydro-2H-pyran-2-yl)purine (8). Intermediate 7
(138 mg, 0.58 mmol) was dissolved in freshly distilled toluene (6 mL)
under dry and inert conditions. K2CO3 (116 mg, 0.84 mmol) and
phenylboronic acid (102 mg, 0.84 mmol) were added, and the
mixture was sonicated for 5 min under Ar bubbling. Tetrakis-
(triphenylphosphine)palladium(0) (33 mg, 29 μmol) was added, and
the reaction was refluxed overnight. After 15 h, the mixture was
filtered, and the filtrate was concentrated under reduced pressure. The
residue was purified by column chromatography (gradient of EtOAc
in hexane, 18/82 to 100/0), obtaining 8 as a transparent oil that
solidified upon standing (117 mg, 72%). 1H NMR (300 MHz,
acetone-d6): δ 9.01−8.92 (m, 3H, H2′ and H2), 8.65 (s, 1H, H8),
7.63−7.52 (m, 3H, H3′, H4′), 5.90 (dd, J = 10.9, 2.4 Hz, 1H, CHO),
4.17−4.07 (m, 1H, CH2O), 3.82 (td, J = 11.5, 3.1 Hz, 1H, CH2O),
2.45−2.23, 2.20−2.08, 1.99−1.81, 1.79−1.60 (4m, 6H,
(CH2)3CH2O).
EXPERIMENTAL SECTION
■
General Procedures and Materials. Solvents and starting
materials were used as supplied by Sigma-Aldrich, Merck KGaA
(Madrid, Spain), unless otherwise indicated. Anhydrous and inert
conditions for specific reactions were obtained using a Schlenk line
(vacuum purges and Ar atm), and solvents were dried and freshly
distilled over its corresponding adsorbent. Reactions were controlled
by TLC (silica gel plates from Sigma-Aldrich, Merck KGaA).
Detection was made with a UV light lamp at a wavelength of 254
nm. Flash normal-phase CC (CC) was carried out on an Isolera One
(Biotage) using silica gel pre-packaged cartridges unless otherwise
6-Phenylpurine (9). Intermediate 8 (117 mg, 0.42 mmol) was
suspended in MeOH (4 mL). Acetyl chloride (8.3 μL, 0.12 mmol)
was added, and the reaction was stirred at rt for 72 h. Then, the
solvent was evaporated under reduced pressure, and the residue was
washed with water (2 × 4 mL) and concentrated under vacuum,
1
indicated. H and 13C NMR spectra were obtained in a spectrometer
1
giving 9 as a white powder (69 mg, 84%). H NMR (300 MHz,
Bruker AVANCE 300 MHz and presented in ppm using the residual
signal of the proton of the corresponding deuterated solvent as the
internal standard. MS spectra were obtained in an ABSciex QSTAR
spectrometer under the high-resolution configuration with electro-
spray as the ionization source. Melting points were obtained in an
apparatus Stuart SMP-10 and are uncorrected. X-ray diffractions for
selected compounds were performed using a Bruker D8 Kappa series
apparatus, with radiation of graphite monochromated Mo Kα at a λ =
0.71073 Å. Full details of data collection and refinement have been
described in the Supporting Information. CIF files have been
deposited in the Cambridge Crystallographic Data Centre (CCDC)
with the codes CCDC2023914 for the compound 19 X ray structure
and CCDC2023913 for the compound 31 X ray structure. The tested
compounds did not present potential PAINS activity according to
screening in http://zinc15.docking.org/patterns/home.68 The purity
of the tested compounds was determined by elemental organic
analysis on a station LECO-CHNS-932 or by HPLC-UV with a
Varian Prostar analytical liquid chromatographer. Values of C, H, and
N from elemental analyses data were within 0.4% of the calculated
values for each final compound. The purity of all tested compounds is
>95%.
acetone-d6): δ 9.04−8.94 (m, 2H, H2′), 8.93 (s, 1H, H2), 8.56 (s, 1H,
H8), 7.65−7.52 (m, 3H, H3′, H4′).
6-Chloro-2-fluoropurine (10). Following the procedure described
by Kim et al.,69 2-amino-6-chloropurine (100 mg, 0.59 mmol) was
added to a solution of hydrofluoric acid in pyridine (70%, 1.5 mL) at
−50 °C. The reaction was allowed to warm up to −30 °C, and tert-
butyl nitrite (105 μL, 0.88 mmol) was added. The reaction was stirred
at −30 °C for 20 min; then, it was quenched by adding water and ice
(4 mL). The mixture was extracted with CHCl3 (5 × 3 mL), and the
organic fractions were washed with brine, dried over anhydrous
Na2SO4, and filtered. After solvent evaporation under vacuum, the
residue was purified by column chromatography (gradient of MeOH
in DCM, 1/99 to 8/92), giving 10 as a white solid (43 mg, 42%). 1H
NMR (300 MHz, acetone-d6): δ 8.60 (s, 1H, H8).
2-Chloro-6-iodopurine (11). Following the procedure described by
Tobrman et al.,70 2,6-dichloropurine (100 mg, 0.53 mmol) was
suspended in hydriodic acid (1 mL) at 0 °C. After 5 h of stirring, the
reaction was diluted with water (2 mL) and NH4OH was added until
pH = 9. The mixture was decanted overnight, then filtered, washed
with water (1 mL), and dried under vacuum, obtaining 11 as a
1
yellowish powder (123 mg, 83%). H NMR (300 MHz, acetone-d6):
Preparation of Synthetic Precursors. 6-Methoxypurine (3).
Metallic Na (200 mg, 9.1 mmol) was slowly added to MeOH (10
mL) in a flask under dry and inert conditions. After 30 min of stirring
at rt, 6-chloropurine (350 mg, 2.3 mmol) was added. The mixture was
heated to 60 °C and stirred for 2 h. The solvent was then evaporated
under reduced pressure, and the crude was purified by filtration
through a silica plug (gradient of methanol in CH2Cl2, 0/100 to 20/
80). The filtrate was evaporated under vacuum obtaining a white
δ 8.60 (s, 1H, H8).
3-Chloro-1-(2,4-dichlorophenyl)propan-1-one (21). AlCl3 (252
mg, 1.9 mmol) was suspended in 1,3-dichlorobenzene (920 μL, 8
mmol) under dry and inert conditions. 3-Chloropropanoyl chloride
(150 μL, 1.6 mmol) was added dropwise to the mixture, which was
subsequently heated up to 60 °C and stirred for 4 h. Then, the
mixture was cooled down to 0 °C and water (2 mL) was slowly added
to quench the reaction. The mixture was extracted with EtOAc (3 × 3
mL), and the combined organic fraction was dried over anhydrous
Na2SO4 and filtered, and the solvent was evaporated under reduced
pressure. The residue was purified by CC (gradient of EtOAc in
hexane, 2/98 to 20/80). After solvent evaporation under vacuum,
intermediate 21 was obtained as a clear amber oil (307 mg, 82%). 1H
NMR (300 MHz, CDCl3): δ 7.52 (d, J = 8.4 Hz, 1H, H6′), 7.46 (d, J
= 2.0 Hz, H3′), 7.34 (dd, J = 8.4, 2.0 Hz, 1H, H5′), 3.88 (t, J = 6.6
Hz, 2H, CH2CO), 3.76 (t, J = 6.6 Hz, 1H, CH2CO), 3.44 (t, J = 6.5
Hz, 1H, CH2Cl), 2.87 (t, J = 6.6 Hz, 1H, CH2Cl).
1
powder that corresponded to 3 in quantitative yield. H NMR (300
MHz, CD3OD): δ 8.49 (s, 1H, H2), 8.27 (s, 1H, H8), 4.18 (s, 3H,
CH3).
6-Chloro-9-(tetrahydro-2H-pyran-2-yl)purine (7). 6-chloropurine
(1.5 g, 9.7 mmol) and p-toluenesulfonic acid (PTSA, 33 mg, 0.19
mmol) were suspended in dry EtOAc (16 mL) in dry and inert
conditions. The mixture was heated up to 50 °C, and 3,4-
dihydropyran (DHP, 992 μL, 11 mmol) was added dropwise over 5
min. The reaction was stirred at 50 °C for 64 h. Then, aqueous
ammonia was added until pH = 8. The mixture was diluted with
EtOAc (10 mL) and water (10 mL). Then, the organic layer was
separated, washed again with water and brine, dried over anhydrous
Na2SO4, and filtered, and the solvent was evaporated under reduced
pressure. The crude was purified by column chromatography
2,6-Dichloropurin-9-ium Chloride. 2,6-Dichloropurine (100 mg,
0.53 mmol) was dissolved in a 3 M solution of HCl in
cyclopentylmethylether (1 mL) under dry and inert conditions. The
reaction was stirred at rt for 24 h and filtered. The resulting solid was
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washed with diethyl ether (2 × 1 mL) and dried under vacuum,
yielding 12 as a yellowish powder (84 mg, 70%). 1H NMR (300 MHz,
D2O): δ 8.64 (s, 1H, H8).
mixture was stirred at rt for 30 min.71 The corresponding 2-chloro-1-
arylethan-1-one (1.5 equiv), dissolved in dry DMF (1 mL), was added
dropwise at 0 °C over 45 min. The reaction was allowed to reach rt.
After completion (15−40 h, TLC), the solvent was evaporated under
vacuum and the residue was dissolved in EtOAc (30 mL) and water
(20 mL). The layers were decanted, and the aqueous phase was
extracted with EtOAc (2 × 10 mL). The combined organic fraction
was washed with brine, dried over anhydrous Na2SO4, and filtered,
and the solvent was evaporated under reduced pressure. The crude
was purified by CC or trituration. Method B. The purine or xanthine
derivative used as the starting material (1 equiv), 2-chloro-1-(2,4-
dichlorophenyl)ethan-1-one (1.2 equiv), Bu4NHSO4 (0.05 equiv),
and K2CO3 (1 equiv) were dissolved in dry DMF (0.2 M) under dry
and inert conditions. The mixture was then heated up to 150 °C in a
sealed tube. After completion (1−6 h, TLC), the solvent was
evaporated under vacuum, and the residue was retrieved with CH2Cl2
(10 mL) and washed with water (2 × 4 mL). The organic fraction was
dried over anhydrous Na2SO4 and filtered, and the solvent was
evaporated under reduced pressure. The crude was eventually purified
by CC.
2-Chloro-9-methyl-1,9-dihydro-6H-purin-6-one (39). Following
the procedure described by Tumma et al.,53 2,6-dichloropurin-9-ium
chloride (84 mg, 0.37 mmol) and basic alumina (38 mg) were
suspended in MeOH (2 mL). The reaction was heated up to 70 °C
and stirred for 24 h. The reaction was interrupted by solvent
evaporation under reduced pressure, and the residue was purified by
CC (gradient of MeOH in CH2Cl2, 2/98 to 16/84), yielding 39 as a
white powder (26 mg, 38%). 1H NMR (300 MHz, acetone-d6): δ 8.32
(s, 1H, H8), 4.15 (s, 3H, CH3).
General Procedure for the Synthesis of 2-Chlorobenza-
mides 2, 4, and 28. The 6-substituted purine or theobromine (1
equiv) was dissolved in freshly distilled THF (0.1 M) under dry and
inert conditions. DMAP (0.05 equiv) and TEA (2 equiv) were
injected, and the mixture was cooled down to 0 °C. 2-Chlorobenzoyl
chloride (1 equiv) was added dropwise for 30 min, and the mixture
was allowed to reach rt while stirring. After the reaction was
completed (4−6 h for purines, 24 h at rt plus 24 h at 70 °C when
theobromine was used as the substrate, TLC), the solvent was
evaporated under reduced pressure, EtOAc (20 mL) and water (20
mL) were added to the crude, and the two layers were separated. The
aqueous layer was extracted again with EtOAc (10 mL), and the
combined organic fraction was washed with brine, dried over
anhydrous Na2SO4, and filtered. The solvent was concentrated
under reduced pressure, and the residue was purified by trituration in
hexane several times to yield a pure product.
(6-Chloro-9H-purin-9-yl)(2-Chlorophenyl)methanone (2). 6-
Chloropurine (227 mg, 1.5 mmol), 2-chlorobenzenesulfonyl chloride
(186 μL, 1.5 mmol), DMAP (9 mg, 73.7 μmol), and TEA (410 μL, 3
mmol) in THF (15 mL) yielded 2 as a white powder (337 mg,
78%).1H NMR (300 MHz, acetone-d6): δ 9.02 (s, 1H, H2), 8.61 (s,
1H, H8), 7.88−7.52 (m, 4H, Ar). 13C NMR (75.4 MHz, acetone-d6):
δ 164.0, 154.1, 152.1, 151.9, 145.5, 134.4, 133.7, 133.6, 132.3, 131.3,
131.0, 128.4. HRMS (ESI+) mass calcd for C12H6Cl2N4O (m/z)
292.9991 [M + H]+; found, 292.9994. mp 133−135 °C. Anal. Calcd
for C12H6Cl2N4O: C, 49.17; H, 2.06; N, 19.12. Found: C, 48.78; H,
2.40; N, 19.50.
(2-Chlorophenyl)(6-methoxy-9H-purin-9-yl)methanone (4). 6-
Methoxypurine (3, 100 mg, 0.67 mmol), 2-chlorobenzenesulfonyl
chloride (84.5 μL, 0.67 mmol), DMAP (4 mg, 33.3 μmol), and TEA
(177 μL, 1.3 mmol) in THF (6.5 mL) yielded 4 as a white powder
(102 mg, 53%). 1H NMR (300 MHz, acetone-d6): δ 8.74 (s, 1H, H2),
8.34 (s, 1H, H8), 7.80−7.52 (m, 4H, Ar), 4.15 (s, 3H, CH3). 13C
NMR (75.4 MHz, acetone-d6): δ 164.6, 162.3, 154.3, 152.3, 145.5,
142.1, 134.4, 134.1, 132.2, 131.1, 130.9, 128.4, 123.4, 54.8. HRMS
(ESI+) mass calcd for C13H9ClN4O2 (m/z) 311.0306 [M + Na]+;
found, 311.0302 [M + Na]+. mp 169−171 °C. Anal. Calcd for
C13H9ClN4O2: C, 54.09; H, 3.14; N, 19.41. Found: C, 54.00; H, 3.06;
N, 18.95.
2-(6-Chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one
(6). Following method A, 6-chloropurine (100 mg, 0.65 mmol), 2-
chloro-1-(2,4-dichlorophenyl)ethan-1-one (224 mg, 0.97 mmol), and
NaH (60% dispersion in mineral oil, 31 mg, 776 μmol) in dry DMF
(11 mL) yielded 6 as a white powder (50 mg, 45%) after purification
1
by CC (gradient of EtOAc in hexane, 16/84 to 100/0). H NMR
(300 MHz, acetone-d6): δ 8.71 (s, 1H, H2), 8.55 (s, 1H, H8), 8.03 (d,
J = 8.4 Hz, 1H, H6′), 7.73 (d, J = 2.0 Hz, 1H, H3′), 7.63 (dd, J = 8.4,
2.0 Hz, 1H, H5′), 6.01 (s, 2H, CH2). 13C NMR (75.4 MHz, acetone-
d6): δ 193.4, 153.6, 152.8, 151.0, 148.3, 139.4, 134.9, 133.9, 132.7,
132.2, 131.8, 128.8, 53.2. HRMS (ESI+) mass calcd for C13H7Cl3N4O
(m/z) 340.9758 [M + H]+; found, 340.9759. mp 166−168 °C. Anal.
Calcd for C13H7Cl3N4O: C, 45.71; H, 2.07; N, 16.40. Found: C,
45.69; H, 1.89; N, 16.50.
2-(6-Chloro-9H-purin-9-yl)-1-(4-chlorophenyl)ethan-1-one (12).
Following method A, 6-chloropurine (100 mg, 0.65 mmol), 2-chloro-
1-(4-chlorophenyl)ethan-1-one (183 mg, 0.97 mmol), and NaH (60%
dispersion in mineral oil, 31 mg, 796 μmol) in dry DMF (10 mL)
yielded 12 as a white powder (95 mg, 48%) after purification by CC
(gradient of acetone in hexane, 10/90 to 80/20). 1H NMR (300
MHz, acetone-d6): δ 8.69 (s, 1H, H2), 8.53 (s, 1H, H8), 8.20 (d, J =
8.6 Hz, 2H, H2′), 7.68 (d, J = 8.6 Hz, 2H, H3′), 6.12 (s, 2H, CH2).
13C NMR (75.4 MHz, acetone-d6): δ 191.6, 153.8, 152.7, 150.9,
148.5, 141.0, 134.0, 132.2, 131.0, 130.3, 50.8. HRMS (ESI+) mass
calcd for C13H8Cl2N4O (m/z) 307.0147 [M + H]+; found, 307.0161.
mp 217 °C (dec.). Anal. Calcd for C13H8Cl2N4O: C, 50.84; H, 2.63;
N, 18.24. Found: C, 50.77; H, 2.78; N, 18.26.
2-(6-Chloro-9H-purin-9-yl)-1-(4-fluorophenyl)ethan-1-one (13).
Following method A, 6-chloropurine (100 mg, 0.65 mmol), 2-
chloro-1-(4-fluorophenyl)ethan-1-one (167 mg, 0.97 mmol), and
NaH (60% dispersion in mineral oil, 31 mg, 796 μmol) in dry DMF
(10 mL) yielded 13 as a white powder (156 mg, 83%) after
purification by CC (gradient of EtOAc in hexane, 12/88 to 100/0).
1H NMR (300 MHz, acetone-d6): δ 8.69 (s, 1H, H2), 8.54 (s, 1H,
H8), 8.27 (ddd, J = 8.9, 4.6, 1.7 Hz, 2H, H2′), 7.46−7.34 (m, 2H,
H3′), 6.12 (s, 2H, CH2). 13C NMR (75.4 MHz, acetone-d6): δ 191.0,
167.1 (d, J = 253.3 Hz), 153.6, 152.5, 150.8, 148.4, 132.1 (d, J = 9.8
Hz), 131.9, 131.9, 116.9 (d, J = 21.9 Hz), 50.6. HRMS (ESI+) mass
calcd for C13H8ClFN4O (m/z) 291.0443 [M + H]+; found, 291.0454.
mp 162−165 °C. Anal. Calcd for C13H8ClFN4O: C, 53.72; H, 2.77;
N, 19.27. Found: C, 53.86; H, 2.93; N, 19.37.
1-(2-Chlorobenzoyl)-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-
dione (28). Theobromine (3,7-dimethyl-3,7-dihydro-1H-purine-2,6-
dione, 200 mg, 1.1 mmol), 2-chlorobenzoyl chloride (141 μL, 1.1
mmol), DMAP (7 mg, 57.3 μmol), and TEA (309 μL, 2.2 mmol) in
THF (11 mL) yielded 28 as a white powder (196 mg, 55%). 1H NMR
(300 MHz, acetone-d6): δ 8.01 (d, J = 7.9 Hz, 1H, H6′), 7.95 (s, 1H,
H8), 7.70−7.60 (m, 2H, Ar), 7.48 (t, J = 7.4 Hz, 1H, Ar), 3.97 (s, 1H,
N7CH3), 3.47 (s, 1H, N3CH3). 13C NMR (75.4 MHz, acetone-d6): δ
167.8, 154.6, 151.2, 150.8, 144.5, 135.5, 134.9, 133.6, 132.6, 132.6,
128.4, 108.1, 33.8. HRMS (ESI+) mass calcd for C14H11ClN4O3 (m/
z) 341.0411 [M + Na]+; found, 341.0398. mp 182−183 °C. Anal.
Calcd for C14H11ClN4O3: C, 52.76; H, 3.48; N, 17.58. Found: C,
52.66; H, 3.38; N, 17.56.
General Procedure for the Preparation of Purinyl/Xanthin-
yl-2,4-dichlorophenylethanones/propanones 6, 12−18, 20,
22, 29, and 30. Method A. The purine derivative used as the
starting material (1 equiv) was dissolved in dry DMF (0.05 M) under
dry and inert conditions and cooled down to 0 °C. NaH (60%
dispersion in mineral oil, 1.2 equiv) was added, and the resulting
1-(2,4-Dichlorophenyl)-2-(6-phenyl-9H-purin-9-yl)ethan-1-one
(14). Following method A, 6-phenylpurine (9, 55 mg, 0.28 mmol), 2-
chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 97 mg, 0.42 mmol),
and NaH (60% dispersion in mineral oil, 13 mg, 336 μmol) in dry
DMF (7 mL) yielded 14 as a yellowish powder (55 mg, 41%) after
purification by CC (gradient of EtOAc in hexane, 15/85 to 100/0).
1H NMR (300 MHz, acetone-d6): δ 9.04−8.98 (m, 2H, C6Ar), 8.94
(s, 1H, H2), 8.56 (s, 1H, H8), 8.04 (d, J = 8.4 Hz, 1H, H6′), 7.73 (d,
J = 2.0 Hz, 1H, H3′), 7.68−7.51 (m, 4H, C6Ar, H5′), 5.99 (s, 2H,
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CH2). 13C NMR (75.4 MHz, acetone-d6): δ 194.0, 154.4, 154.1,
153.1, 147.3, 139.3, 137.0, 135.2, 133.8, 132.6, 131.9, 131.7, 131.5,
130.8, 129.4, 128.8, 52.7. HRMS (ESI+) mass calcd for
C19H12Cl2N4O (m/z) 383.0460 [M + H]+; found, 383.0465. mp
160−162 °C. Anal. Calcd for C19H12Cl2N4O: C, 59.55; H, 3.16; N,
14.62. Found: C, 59.58; H, 3.43; N, 14.48.
1-(2,4-Dichlorophenyl)-2-(6-methoxy-9H-purin-9-yl)ethan-1-one
(15). Following method A, 6-methoxypurine (3, 100 mg, 0.67 mmol),
2-chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 223 mg, 1.0 mmol),
and NaH (60% dispersion in mineral oil, 32 mg, 799 μmol) in dry
DMF (11 mL) yielded 15 as a yellowish powder (53 mg, 24%) after
purification by CC (gradient of acetone in hexane, 12/88 to 80/20).
1H NMR (300 MHz, acetone-d6): δ 8.47 (s, 1H, H2), 8.25 (s, 1H,
H8), 7.99 (d, J = 8.4 Hz, 1H, H6′), 7.71 (d, J = 2.1 Hz, 1H, H3′),
7.61 (dd, J = 8.3, 2.2 Hz, 1H, H5′), 5.89 (s, 2H, CH2), 4.15 (s, 3H,
CH3). 13C NMR (75.4 MHz, acetone-d6): δ 194.0, 161.9, 153.7,
152.7, 144.7, 139.1, 135.2, 133.6, 132.5, 131.6, 128.7, 121.8, 54.4,
52.8. HRMS (ESI+) mass calcd for C14H10Cl2N4O2 (m/z) 337.0253
[M + H]+; found, 337.0256. mp 158 °C (dec). Anal. Calcd for
C14H10Cl2N4O2: C, 49.87; H, 2.99; N, 16.62. Found: C, 50.01; H,
3.11; N, 16.38.
(d, J = 8.4 Hz, 1H, H6′), 7.70 (d, J = 2.0 Hz, 1H, H3′), 7.60 (dd, J =
8.4, 2.0 Hz, 1H, H5′), 6.17 (br, 2H, NH2), 5.69 (s, 2H, CH2). 13C
NMR (75.4 MHz, acetone-d6): δ 194.1, 161.1, 155.6, 151.3, 144.3,
139.2, 135.3, 133.7, 132.6, 131.6, 128.8, 125.1, 52.6. HRMS (ESI+)
mass calcd for C13H8Cl3N5O (m/z) 355.9867 [M + H]+; found,
355.9873. mp 208−209 °C (dec). HPLC-UV (λ = 306 nm), purity
99%.
3-(6-Chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)propan-1-one
(22). Following method A, 6-chloropurine (75 mg, 0.49 mmol),
intermediate 21 (174 mg, 0.73 mmol), and NaH (60% dispersion in
mineral oil, 23 mg, 586 μmol) in dry DMF (10 mL) yielded 22 as a
white powder (40 mg, 23%) after purification by CC (gradient of
EtOAc in hexane, 20/80 to 100/0). 1H NMR (300 MHz, acetone-d6):
δ 8.70 (s, 1H, H2), 8.55 (s, 1H, H8), 7.72 (d, J = 8.4 Hz, 1H, H6′),
7.58 (d, J = 2.1 Hz, 1H, H3′), 7.48 (dd, J = 8.4, 2.0 Hz, 1H, H5′),
4.80 (t, J = 6.3 Hz, 2H, NCH2), 3.81 (t, J = 6.3 Hz, 2H, CH2CO). 13
C
NMR (75.4 MHz, acetone-d6): δ 198.3, 152.3, 151.4, 149.7, 147.2,
137.2, 136.6, 131.9, 131.5, 130.9, 130.3, 127.5, 41.3, 39.1. HRMS
(ESI+) mass calcd for C14H9Cl3N4O (m/z) 354.9914 [M + H]+;
found, 354.9919. mp 140 °C (dec). HPLC-UV (λ = 256 nm), purity
99%.
1-[2-(2,4-Dichlorophenyl)-2-oxoethyl]-3,7-dimethyl-3,7-dihydro-
1H-purine-2,6-dione (29). Following method B, theobromine (3,7-
dimethyl-3,7-dihydro-1H-purine-2,6-dione, 70 mg, 0.39 mmol), 2-
chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 104 mg, 0.47 mmol),
Bu4NHSO4 (7 mg, 19 μmol), and K2CO3 (54 mg, 0.39 mmol) in dry
DMF (2 mL) yielded 29 as a brown powder (36 mg, 25%) after
purification by CC (gradient of MeOH in EtOAc, 0/100 to 4/96) and
2-(2,6-Dichloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-
one (16). Following method A, 2,6-dichloropurine (100 mg, 0.53
mmol), 2-chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 183 mg, 0.79
mmol), and NaH (60% dispersion in mineral oil, 25 mg, 635 μmol) in
dry DMF (18 mL) yielded 16 as a yellowish powder (65 mg, 40%)
after purification by CC (gradient of EtOAc in hexane, 12/88 to 100/
1
0) and trituration in diethyl ether. H NMR (300 MHz, acetone-d6):
1
trituration in diethyl ether. H NMR (300 MHz, acetone-d6): δ 7.89
δ 8.57 (s, 1H, H8), 8.05 (d, J = 8.4 Hz, 1H, H6′), 7.73 (d, J = 2.0 Hz,
1H, H3′), 7.63 (dd, J = 8.4, 2.0 Hz, 1H, H3′), 6.01 (s, 2H, CH2). 13
C
(s, 1H, H8), 7.86 (d, J = 8.3 Hz, 2H, H6′), 7.67 (d, J = 2.0 Hz, 1H,
H3′), 7.58 (dd, J = 8.4, 2.0 Hz, 1H, H5′), 5.28 (s, 2H, CH2), 3.99 (s,
3H, N7CH3), 3.51 (s, 3H, N3CH3). 13C NMR (75.4 MHz, acetone-
d6): δ 195.4, 155.3 152.0, 150.2, 143.9, 138.4, 136.6, 133.1, 132.1,
131.3, 128.6, 50.2, 33.8. HRMS (ESI+) mass calcd for C15H12Cl2N4O3
(m/z) 367.0359 [M + H]+; found, 367.0367. mp 196 °C (dec.). Anal.
Calcd for C15H12Cl2N4O3: C, 49.07; H, 3.29; N, 15.26. Found: C,
49.05; H, 3.39; N, 15.19.
9-[2-(2,4-Dichlorophenyl)-2-oxoethyl]-1,3-dimethyl-3,7-dihydro-
1H-purine-2,6-dione (30). Following method B, theophylline (1,3-
dimethyl-3,9-dihydro-1H-purine-2,6-dione, 70 mg, 0.39 mmol), 2-
chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 104 mg, 0.47 mmol),
Bu4NHSO4 (7 mg, 19 μmol), and K2CO3 (54 mg, 0.39 mmol) in dry
DMF (2 mL) yielded 30 as a white powder (65 mg, 46%) after
purification by CC (gradient of MeOH in EtOAc, 1/99 to 10/90) and
trituration in diethyl ether. 1H NMR (300 MHz, acetone-d6): δ 8.04−
7.94 (m, 2H, H8, H6′), 7.69 (d, J = 2.1 Hz, 1H, H3′), 7.62 (dd, J =
8.4, 2.1 Hz, 1H, H5′), 5.87 (s, 2H, CH2), 3.53 (s, 3H, N3CH3), 3.26
(s, 3H, N1CH3). 13C NMR (75.4 MHz, acetone-d6): δ 194.1, 156.2,
152.4, 149.7, 144.0, 139.1, 135.6, 133.5, 132.7, 131.5, 128.8, 107.7,
55.8, 28.0. HRMS (ESI+) mass calcd for C15H12Cl2N4O3 (m/z)
367.0359 [M + H]+; found, 367.0368. mp 198−199 °C. HPLC-UV (λ
= 260 nm), purity 97%.
2-[2-Chloro-6-(methylamino)-9H-purin-9-yl]-1-(2,4-
dichlorophenyl)ethan-1-one (19). 2-Chloro-N-methylpurin-6-amine
(60 mg, 0.33 mmol), Bu4NHSO4 (5.5 mg, 16 μmol), and KOH (18
mg, 0.33 mmol) were suspended in dry DMF (2 mL) under dry and
inert conditions. 2-Chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 80
mg, 0.36 mmol) was dissolved in dry DMF (0.5 mL) and added
dropwise to the reaction at 0 °C over 45 min. The reaction was stirred
at rt for 26 h and then it was diluted with cold water (6 mL) and
EtOAc (7 mL). The mixture was stirred for 30 min after which the
two layers were separated. The aqueous fraction was extracted with
EtOAc (3 × 3 mL), and the combined organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure. The crude was purified by CC (gradient of EtOAc
in hexane, 18/82 to 100/0). After solvent evaporation, the solid was
triturated with diethyl ether, furnishing 19 as a white solid (27 mg,
22%). The compound was recrystallized in EtOAc by slow
evaporation to obtain crystals for X-ray diffraction (experimental
details in the Supporting Information). 1H NMR (300 MHz, acetone-
NMR (75.4 MHz, acetone-d6): δ 192.9, 155.2, 153.1, 151.7, 149.3,
139.6, 134.6, 134.1, 132.9, 131.9, 131.6, 128.9, 53.4. HRMS (ESI+)
mass calcd for C13H6Cl4N4O (m/z) 374.9368 [M + H]+; found,
374.9374. Mp 136−138 °C. Anal. calcd for C13H6Cl4N4O: C, 41.53;
H, 1.61; N, 14.90. Found: C, 41.78; H, 1.93; N, 14.81.
2-(6-Chloro-2-fluoro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)-
ethan-1-one (17). Following method A, 6-chloro-2-fluoropurine (10,
42 mg, 0.24 mmol), 2-chloro-1-(2,4-dichlorophenyl)ethan-1-one (5,
82 mg, 0.36 mmol), and NaH (60% dispersion in mineral oil, 15 mg,
365 μmol) in dry DMF (4 mL) yielded 17 as a yellow oil (34 mg,
39%) after purification by CC (gradient of acetone in hexane, 6/94 to
1
50/50). H NMR (300 MHz, acetone-d6): δ 8.55 (s, 1H, H8), 8.04
(d, J = 8.4 Hz, 1H, H6′), 7.73 (d, J = 2.0 Hz, 1H, H3′), 7.63 (dd, J =
8.4, 2.0 Hz, 1H, H5′), 5.98 (s, 2H, CH2). 13C NMR (75.4 MHz,
acetone-d6): δ 191.9, 157.9 (d, J = 214.9 Hz), 154.7 (d, J = 17.3 Hz),
151.4 (d, J = 18.1 Hz), 148.2 (d, J = 3.0 Hz), 138.5, 133.6, 133.0,
131.8, 130.8, 130.1 (d, J = 6.8 Hz), 127.8, 52.3. HRMS (ESI+) mass
calcd for C13H6Cl3FN4O (m/z) 358.9663 [M + H]+; found,
358.9657. mp 134 °C. HPLC-UV (λ = 260 nm): purity 98%.
2-(2-Chloro-6-iodo-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-
1-one (18). Following method A, 2-chloro-6-iodopurine (11, 69 mg,
0.25 mmol), 2-chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 85 mg,
0.37 mmol), and NaH (60% dispersion in mineral oil, 15 mg, 369
μmol) in dry DMF (4 mL) yielded 18 as a yellowish powder (30 mg,
1
28%) after purification by trituration in diethyl ether. H NMR (300
MHz, acetone-d6): δ 8.55 (s, 1H, H8), 8.04 (d, J = 8.4 Hz, 1H, H6′),
7.73 (d, J = 2.1 Hz, 1H, H3′), 7.63 (dd, J = 8.4, 2.1 Hz, 1H, H5′),
5.97 (s, 2H, CH2). 13C NMR (75.4 MHz, acetone-d6): δ 191.9, 151.7,
150.1, 147.5, 138.5, 138.0, 133.5, 133.0, 131.8, 130.8, 127.8, 121.7,
52.2. HRMS (ESI+) mass calcd for C13H6Cl3IN4O (m/z) 466.8724
[M + H]+; found, 466.8721. mp 214−215 °C (dec). HPLC-UV (λ =
284 nm): purity 100%.
2-(2-Amino-6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)-
ethan-1-one (20). Following method B, 6-chloropurin-2-amine (100
mg, 0.53 mmol), 2-chloro-1-(2,4-dichlorophenyl)ethan-1-one (5, 145
mg, 0.65 mmol), Bu4NHSO4 (10 mg, 29 μmol), and K2CO3 (81 mg,
0.59 mmol) in dry DMF (3 mL) yielded 20 as a brown powder (15
mg, 10%) after 30 h of reaction time at 80 °C and purification by CC
(gradient of MeOH in CH2Cl2, 1/99 to 8/92) and trituration in cold
1
EtOAc. H NMR (300 MHz, acetone-d6): δ 8.03 (s, 1H, H8), 7.96
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d6): δ 8.02 (s, 1H, H8), 7.98 (d, J = 8.4 Hz, 1H, H6′), 7.70 (d, J = 2.1
Hz, 1H, H3′), 7.61 (dd, J = 8.4, 2.1 Hz, 1H, H5′), 7.24 (br s, 1H,
NH), 5.75 (s, 2H, CH2), 3.12 (s, 3H, CH3). 13C NMR (75.4 MHz,
acetone-d6): δ 193.9, 157.1, 154.3, 142.4, 139.1, 135.1, 133.6, 132.5,
131.5, 128.6, 119.2, 52.5, 27.7. HRMS (ESI+) mass calcd for
C14H10Cl3N5O (m/z) 370.0023 [M + H]+; found, 370.0041. mp 213
°C (dec). Anal. Calcd for C14H10Cl3N5O·H2O: C, 43.27; H, 3.11; N,
18.02. Found: C, 43.33; H, 2.80; N, 17.99.
7.85−7.77 (m, 2H, H4′, H5′), 7.66 (dd, J = 7.8, 1.5 Hz, 1H, H3′),
4.13 (s, 3H, CH3). 13C NMR (75.4 MHz, acetone-d6): δ 162.2, 154.5,
151.5, 142.3, 138.0, 134.9, 134.6, 133.2, 133.1, 129.0, 122.6, 54.9.
HRMS (ESI+) mass calcd for C12H9ClN4O3S (m/z) 346.9976 [M +
Na]+; found, 346.9980. mp 158 °C (dec). Anal. Calcd for
C12H9ClN4O3S: C, 44.38; H, 2.79; N, 17.25; S, 9.87. Found: C,
44.48; H, 2.99; N, 16.94; S, 9.93.
6-Chloro-9-[(2-chlorophenyl)sulfonyl]-9H-purin-2-amine (26).
Following method A, 6-chloropurin-2-amine (100 mg, 0.59 mmol),
2-chlorobenzenesulfonyl chloride (161 μL, 1.2 mmol), DMAP (14
mg, 118 μmol), and TEA (164 μL, 1.2 mmol) in CH2Cl2/THF (6
mL) yielded 26 as a white powder (102 mg, 50%) after purification by
General Procedure for the Synthesis of Sulfonamides 23−
27 and 31−38. Method A. The purine derivative (1 equiv) was
dissolved in freshly distilled CH2Cl2 and THF (1:1, 0.1 M) under dry
and inert conditions. DMAP (0.2 equiv) and TEA (2 equiv) were
added, and the mixture was cooled down to 0 °C. 2-
Chlorobenzenesulfonyl chloride (2 equiv) was added dropwise for
30 min. The mixture was stirred at rt, and after completion (2−4 h,
TLC), the solvent was removed under reduced pressure. The crude
was dissolved in EtOAc (30 mL) and water (20 mL), and the two
layers were separated. The aqueous layer was extracted again with
EtOAc (10 mL), and the combined organic fraction was washed with
brine, dried over anhydrous Na2SO4, and filtered. The solvent was
evaporated under reduced pressure and the residue purified by CC or
trituration to yield the pure product. Method B. Theophylline (1,3-
dimethyl-3,9-dihydro-1H-purine-2,6-dione, 1 equiv) was suspended in
freshly distilled THF (0.05 M) in dry and inert conditions. The
mixture was cooled down to 0 °C and NaH (60% dispersion in
mineral oil, 1.2 equiv) was added, allowing the reaction to stir at rt for
30 min. The corresponding arylsulfonyl chloride derivative (1.2
equiv) was dissolved in dry THF (1 mL) and added to the reaction
dropwise at 0 °C for 15 min. The reaction was stirred at rt, and after
completion (4−48 h, TLC), the solvent was evaporated under
reduced pressure. The residue was dissolved in EtOAc (30 mL) and
water (30 mL). The layers were separated, and the aqueous layer was
extracted once again with EtOAc (20 mL). The combined organic
fraction was washed with brine, dried over anhydrous Na2SO4, and
filtered. The solvent was evaporated under reduced pressure, and the
residue was purified by CC to yield the pure product.
6-Chloro-9-[(2-chlorophenyl)sulfonyl]-9H-Purine (23). Following
method A, 6-chloropurine (100 mg, 0.65 mmol), 2-chlorobenzene-
sulfonyl chloride (176 μL, 1.3 mmol), DMAP (16 mg, 129 μmol),
and TEA (180 μL, 1.3 mmol) in CH2Cl2/THF (6.5 mL) yielded 23
as a white powder (165 mg, 77%) after trituration with cold MeOH.
1H NMR (300 MHz, acetone-d6): δ 9.05 (s, 1H, H2), 8.70 (s, 1H,
H8), 8.60 (dd, J = 7.9, 1.9 Hz, 1H, H6′), 7.93−7.74 (m, 2H, H4′,
H5′), 7.67 (dd, J = 7.9, 1.5 Hz, 1H, H3′). 13C NMR (75.4 MHz,
acetone-d6): δ 154.2, 152.1, 151.4, 145.5, 138.3, 134.6, 134.5, 133.4,
133.1, 129.1. HRMS (ESI+) mass calcd for C11H6Cl2N4O2S (m/z)
328.9661 [M + H]+; found, 328.9663. mp 172 °C (dec). Anal. Calcd
for C11H6Cl2N4O2S: C, 40.14; H, 1.84; N, 17.02; S, 9.74. Found: C,
40.12; H, 2.06; N, 16.66; S, 9.75.
1
CC (gradient of EtOAc in hexane, 12/88 to 85/15). H NMR (300
MHz, acetone-d6): δ 8.51−8.42 (m, 2H, H8, H6′), 7.84 (td, J = 7.7,
1.7 Hz, 1H, H5′), 7.77−7.62 (m, 2H, H3′, H4′), 6.52 (br s, 2H,
NH2). 13C NMR (75.4 MHz, CDCl3): δ 161.8, 153.5, 152.5, 140.8,
137.9, 135.0, 134.7, 133.3, 133.2, 128.9, 125.1. HRMS (ESI+) mass
calcd for C11H7Cl2N5O2S (m/z) 343.9770 [M + H]+ and 365.9589
[M + Na]+; found, 343.9765 and 365.9573. mp 200 °C (dec). Anal.
Calcd for C11H7Cl2N5O2S: C, 38.39; H, 2.05; N, 20.35; S, 9.32.
Found: C, 38.66; H, 2.37; N, 19.91; S, 9.23.
2-Chloro-9-[(2-chlorophenyl)sulfonyl]-N-methyl-9H-purin-6-
amine (27). Following method A, 2-chloro-N-methylpurin-6-amine
(100 mg, 0.54 mmol), 2-chlorobenzenesulfonyl chloride (148 μL, 1.1
mmol), DMAP (13 mg, 118 μmol), and TEA (152 μL, 1.1 mmol) in
CH2Cl2/THF (6 mL) yielded 27 as a white powder (94 mg, 48%)
after purification by CC (gradient of EtOAc in hexane, 12/88 to 100/
0). 1H NMR (300 MHz, acetone-d6): δ 8.55−8.46 (m, 2H, H8, H6′),
7.91−7.71 (m, 2H, H5′, H4′), 7.67 (dd, J = 7.9, 1.4 Hz, 1H, H3′),
7.55 (br s, 1H, NH), 3.07 (d, J = 4.4 Hz, 3H, CH3). 13C NMR (75.4
MHz, DMSO-d6): δ 155.4, 154.9, 147.9, 139.6, 137.4, 133.2, 133.1,
132.4, 131.3, 126.2, 118.4, 27.2. HRMS (ESI+) mass calcd for
C12H9Cl2N5O2S (m/z) 357.9926 [M + H]+; found, 357.9932. mp 235
°C (dec). HPLC-UV (λ = 260 nm), purity 96%.
7-[(2-Chlorophenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-1H-pu-
rine-2,6-dione (31). Following method B, theophylline (60 mg, 0.33
mmol), 2-chlorobenzenesulfonyl chloride (55 μL, 0.40 mmol), and
NaH (60% dispersion in mineral oil, 16 mg, 0.4 mmol) in THF (5.5
mL) yielded 31 as a white powder (82 mg, 69%) after purification by
CC (gradient of EtOAc in hexane, 12/88 to 100/0). The compound
was recrystallized in CH2Cl2 and acetone by slow evaporation to
obtain crystals for X-ray diffraction (Supporting Information). 1H
NMR (300 MHz, acetone-d6): δ 8.70 (s, 1H, H8), 8.60 (dd, J = 8.1,
1.7 Hz, 1H, H6′), 7.88−7.84, 7.76−7.66 (2m, 3H, Ar), 3.52 (s, 3H,
N3CH3), 3.16 (s, 3H, N1CH3). 13C NMR (75.4 MHz, CDCl3): δ
153.6, 152.0, 151.8, 151.3, 145.4, 138.0, 136.1, 134.6, 133.3, 132.8,
128.6, 28.5. HRMS (ESI+) mass calcd for C13H11ClN4O4S (m/z)
377.0081 [M + Na]+; found, 377.0070. mp 212 °C (dec). HPLC-UV
(λ = 287 nm), purity 100%.
2,6-Dichloro-9-[(2-chlorophenyl)sulfonyl]-9H-purine (24). Fol-
lowing method A, 2,6-dichloropurine (100 mg, 0.53 mmol), 2-
chlorobenzenesulfonyl chloride (144 μL, 1.1 mmol), DMAP (13 mg,
106 μmol), and TEA (147 μL, 1.1 mmol) in CH2Cl2/THF (5.3 mL)
yielded 24 as a white powder (90 mg, 47%) after purification by CC
(gradient of EtOAc in hexane, 0/100 to 50/50). 1H NMR (300 MHz,
acetone-d6): δ 9.07 (s, 1H, H8), 8.58 (dd, J = 8.0, 1.9 Hz, 1H, H6′),
7.95−7.75 (m, 2H, H4′, H5′), 7.70 (dd, J = 7.9, 1.2 Hz, 1H, H3′).
13C NMR (75.4 MHz, acetone-d6): δ 154.7, 153.0, 152.8, 146.2,
138.6, 134.8, 134.4, 133.7, 133.6, 132.9, 129.3. HRMS (ESI+) mass
calcd for C11H5Cl3N4O2S (m/z) 362.9271 [M + H]+; found,
362.9285. Mp 156 °C (dec). Anal. Calcd for C11H5Cl3N4O2S: C,
36.34; H, 1.39; N, 15.41; S, 8.82. Found: C, 36.06; H, 1.58; N, 15.20;
S, 9.14.
7-[(2,4-Dichlorophenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (32). Following method B, theophylline (100 mg,
0.55 mmol), 2,4-dichlorobenzenesulfonyl chloride (163 mg, 0.67
mmol), and NaH (60% dispersion in mineral oil, 27 mg, 0.67 mmol)
in THF (11 mL) yielded 32 as a white powder (160 mg, 74%) after
purification by CC (gradient of EtOAc in hexane, 12/88 to 95/5). 1H
NMR (300 MHz, CDCl3): δ 8.59 (d, J = 9.2 Hz, 1H, H6′), 8.43 (s,
1H, H8), 7.61−7.47 (m, 2H, H3′, H5′), 3.60 (s, 3H, N3CH3), 3.30
(s, 3H, N1CH3). 13C NMR (75.4 MHz, CDCl3): δ 152.9, 151.2,
150.6, 144.0, 142.9, 136.2, 133.9, 132.0, 131.7, 127.9, 105.7, 30.3,
28.6. HRMS (ESI+) mass calcd for C13H10Cl2N4O4S (m/z) 410.9692
[M + Na]+; found, 410.9687. mp 206−208 °C. Anal. Calcd for
C13H10Cl2N4O4S: C, 40.12; H, 2.59; N, 14.40; S, 8.24. Found: C,
40.19; H, 2.71; N, 14.22; S, 8.25.
9-[(2-Chlorophenyl)sulfonyl]-6-methoxy-9H-purine (25). Follow-
ing method A, 6-methoxypurine (3, 90 mg, 0.60 mmol), 2-
chlorobenzenesulfonyl chloride (163 μL, 1.2 mmol), DMAP (15
mg, 120 μmol), and TEA (167 μL, 1.2 mmol) in CH2Cl2/THF (6
mL) yielded 25 as a white powder (72 mg, 37%) after purification by
trituration with cold MeOH. 1H NMR (300 MHz, acetone-d6): δ 8.76
(s, 1H, H2), 8.57 (dd, J = 7.9, 1.9 Hz, 1H, H6′), 8.43 (s, 1H, H8),
7-[(2-Chloro-4-fluorophenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-
1H-purine-2,6-dione (33). Following method B, theophylline (100
mg, 0.55 mmol), 2-chloro-4-fluorobenzenesulfonyl chloride (97 μL,
0.67 mmol), and NaH (60% dispersion in mineral oil, 27 mg, 0.67
mmol) in THF (11 mL) yielded 33 as a white powder (112 mg, 54%)
after purification by CC (gradient of EtOAc in hexane, 12/88 to 100/
1
0). H NMR (300 MHz, CDCl3): δ 8.69 (dd, J = 9.0, 5.7 Hz, 1H,
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H6′), 8.43 (s, 1H, H8), 7.34−7.20 (m, 2H, H3′, H5′), 3.60 (s, 3H,
N3CH3), 3.29 (s, 3H, N1CH3). 13C NMR (75.4 MHz, CDCl3): δ
166,4 (d, J = 263.9 Hz), 153.0, 151.3, 150.6, 144.0, 138.0 (d, J = 10.6
Hz), 135.2 (d, J = 11.3 Hz), 134.3, 129.8, 119.6 (d, J = 25.6 Hz),
115.1 (d, J = 21.9 Hz), 105.8, 30.3, 28.6. HRMS (ESI+) mass calcd for
C13H10ClFN4O4S (m/z) 394.9987 [M + Na]+; found, 394.9980. mp
220−222 °C. Anal. Calcd for C13H10ClFN4O4S: C, 41.89; H, 2.70; N,
15.03; S, 8.60. Found: C, 41.85; H, 2.82; N, 14.85; 8.72.
222 °C (dec). Anal. Calcd for C13H10Cl2N4O4S: C, 40.12; H, 2.59; N,
14.40; S, 8.24. Found: C, 40.34; H, 2.78; N, 14.23; S, 8.52.
7-[(4-Fluorophenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-1H-pu-
rine-2,6-dione (38). Following method B, theophylline (100 mg, 0.55
mmol), 4-fluorobenzenesulfonyl chloride (130 mg, 0.67 mmol), and
NaH (60% dispersion in mineral oil, 27 mg, 0.67 mmol) in THF (11
mL) yielded 38 as a white powder (152 mg, 81%) after purification by
1
CC (gradient of EtOAc in hexane, 10/90 to 100/0). H NMR (300
MHz, CDCl3): δ 8.42−8.21 (m, 3H, H2′, H8), 7.36−7.20 (m, 2H,
H3′), 3.57 (s, 3H, N3CH3), 3.36 (s, 3H, N1CH3). 13C NMR (75.4
MHz, CDCl3): δ 166.9 (d, J = 260.13), 153.1, 151.3, 150.8, 142.2,
133.0 (d, J = 10.6 Hz), 132.4 (d, J = 3.1 Hz), 117.1 (d, J = 22.6 Hz),
106.1, 30.3, 28.7. HRMS (ESI+) mass calcd for C13H11FN4O4S (m/z)
361.0377 [M + Na]+ and 699.0862 [2M + Na]+; found, 361.0366 and
699.0841. mp 223 °C (dec). Anal. Calcd for C13H11FN4O4S: C,
46.15; H, 3.28; N, 16.56; S, 9.48. Found: C, 46.05; H, 3.41; N, 16.45;
S, 9.21.
7-([2-Chloro-4-(trifluoromethyl)phenyl]sulfonyl)-1,3-dimethyl-
3,7-dihydro-1H-purine-2,6-dione (34). Following method B, theo-
phylline (100 mg, 0.55 mmol), 2-chloro-4-(trifluoromethyl)-
benzenesulfonyl chloride (186 mg, 0.67 mmol), and NaH (60%
dispersion in mineral oil, 27 mg, 0.67 mmol) in THF (11 mL) yielded
34 as a white powder (177 mg, 75%) after purification by CC
(gradient of EtOAc in hexane, 15/85 to 100/0). 1H NMR (300 MHz,
CDCl3): δ 8.80 (d, J = 8.4 Hz, 1H, H6′), 8.46 (s, 1H, H8), 7.84 (dd, J
= 8.4, 1.7 Hz, 1H, H5′), 7.75 (d, J = 1.7 Hz, 1H, H3′), 3.60 (s, 3H,
N3CH3), 3.28 (s, 3H, N1CH3). 13C NMR (75.4 MHz, CDCl3): δ
153.0, 151.2, 150.6, 144.0, 138.0 (q, J = 31.7 Hz), 136.92, 136.1,
133.9, 129.0 (q, J = 3.8 Hz), 124.4 (q, J = 3.8 Hz), 122.2 (d, J = 273.7
Hz), 105.8, 30.4, 28.6. HRMS (ESI+) mass calcd for
C14H10ClF3N4O4S (m/z) 444.9955 [M + Na]+; found, 444.9954.
mp 210 °C (dec). Anal. Calcd for C14H10ClF3N4O4S: C, 39.77; H,
2.38; N, 13.25; S, 7.58. Found: C, 39.93; H, 2.55; N, 12.99; S, 7.76.
7-[(2,4-Difluorophenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (35). Following method B, theophylline (100 mg,
0.55 mmol), 2,4-difluorobenzenesulfonyl chloride (141 mg, 0.67
mmol), and NaH (60% dispersion in mineral oil, 27 mg, 0.67 mmol)
in THF (11 mL) yielded 35 as a white powder (142 mg, 72%) after
purification by CC (gradient of EtOAc in hexane, 10/90 to 80/20).
1H NMR (300 MHz, CDCl3): δ 8.48 (ddd, J = 9.0, 8.1, 5.9 Hz, 1H,
H6′), 8.37 (d, J = 1.2 Hz, 1H, H8), 7.16 (dddd, J = 8.9, 7.6, 2.4, 1.1
Hz, 1H, H5′), 6.93 (ddd, J = 10.4, 8.2, 2.4 Hz, 1H, H3′), 3.60 (s, 3H,
N3CH3), 3.31 (s, 3H, N1CH3). 13C NMR (75.4 MHz, CDCl3): δ
167.8 (dd, J = 12.1, 262.4 Hz), 160.5 (dd, J = 12.8, 261.6 Hz), 153.0,
151.3, 150.6, 143.0, 135.8 (d, J = 11.31 Hz), 120.8 (dd, J = 3.8, 12.1
Hz), 112.7 (dd, J = 3.8, 22.6 Hz), 105.9 (dd, J = 24.9, 26.4 Hz), 105.8,
30.3, 28.6. HRMS (ESI+) mass calcd for C13H10F2N4O4S (m/z)
379.0283 [M + Na]+; found, 379.0269. Mp 194 °C (dec). Anal. Calcd
for C13H10F2N4O4S: C, 43.82; H, 2.83; N, 15.72; S, 9.00. Found: C,
43.83; H, 2.96; N, 15.63; S, 9.12.
Synthesis of 2-Chloro-1-(2-chlorobenzyl)-9-methyl-1,9-di-
hydro-6H-purin-6-one (40). Following the procedure described
by Szarek et al.,72 NaHCO3 (35 mg, 0.42 mmol) was added to a
solution of intermediate 39 (24 mg, 0.13 mmol) in DMF (1 mL). The
reaction was stirred at rt for 30 min. 1-Chloro-2-(chloromethyl)-
benzene (40 μL, 0.31 mmol) was added dropwise, and the mixture
was heated up to 75 °C and stirred overnight. The solvent was
evaporated under reduced pressure, and the residue was suspended in
water (3 mL) and extracted with EtOAc (3 × 2 mL). The combined
organic layer was washed with brine, dried over anhydrous Na2SO4,
and filtered, and the solvent was evaporated under reduced pressure.
Purification was performed by CC (gradient of EtOAc in hexane, 15/
1
85 to 80/20), giving 40 as a brownish powder (10 mg, 32%). H
NMR (300 MHz, acetone-d6): δ 8.44 (s, 1H, H8), 7.51 (dd, J = 7.8,
1.5 Hz, 1H, H3′), 7.35 (m, 2H, H4′, H5′), 7.08 (dd, J = 7.6, 1.9 Hz,
1H, H6′), 5.77 (s, 2H, CH2), 4.07 (s, 3H, CH3). 13C NMR (75.4
MHz, acetone-d6): δ 161.4, 153.8, 152.3, 143.8, 133.5, 132.8, 129.9,
129.8, 129.7, 127.6, 120.3, 54.4, 45.0. HRMS (ESI+) mass calcd for
C13H10Cl2N4O (m/z) 309.0304 [M + H]+; found, 309.0308. mp
146−149 °C. HPLC-UV (λ = 250 nm): purity 95%.
HEK293-hP2X7 Cell Culture. The human embryonic kidney cell
line that stably expresses the human P2X7 isoform A (hP2X7-
HEK293) was generously donated by Francesco di Virgilio’s group.
HEK293-hP2X7 cells were kept in 75 cm2 flasks under DMEM-F12
culture medium enriched with 50 units/mL of penicillin and 50 μg/
mL of streptomycin, 10% FBS bovine serum, and 0.2 mg/mL of
geneticin (G-418 sulfate, Roche Diagnostics GmbH, Mannheim,
Germany), which was used as a selection reagent. Cells were allowed
to grow up to 95% confluence in an incubator with a constant
atmosphere of 5% CO2, saturated humidity, and at a temperature of
37 °C. For culture plating, they were dissociated from the flask using
Trypsin−EDTA 0.05%, counted, and seeded in 96-well black, clear-
bottomed plates (Conning Inc. Kennebunk ME, USA) at a different
density depending on the experiment. Experiments were performed
24−48 h after plating.
YO-PRO-1 Uptake. hP2X7-HEK293 cells were seeded at a density
of 400,000 cells/well. After 48 h, the culture medium was harvested
and the cells were incubated with Mg2+-free Krebs-HEPES buffer
containing (in mM) 144 NaCl, 5.9 KCl, 11 glucose, 10 HEPES, and
0.2 CaCl2 at pH 7.4. Test groups were incubated with the compounds
for 20 min; then, the YOPRO-1 probe (2 μM) was applied, and
subsequently, the cells were stimulated with BzATP (30 μM).
Changes in the fluorescence (excitation at 485 nm and emission at
520 nm) were measured for 25 min using a fluorescence plate reader
(Fluostar, BMG Labtech, Offenburg, Germany). Responses of each
well were normalized with respect to the maximum (Fmax) and the
minimum (Fmin) fluorescence values obtained by adding 0.5% Triton
X-100 and then 2 M MnCl2. Data were calculated with the formula F
= (Fx − F0)/(Fmax − Fmin), where Fx is the maximum fluorescence
obtained and F0 is the averaged fluorescence before Bz-ATP injection.
Fura-2 Calcium Measurements. hP2X7-HEK293 cells were
seeded at a density of 50,000 cells/well and after 24−48 h were
loaded with 8 μM Fura-2-AM (Invitrogen or Biotium, USA) for 1 h at
7-[(4-Fluoro-2-methylphenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-
1H-purine-2,6-dione (36). Following method B, theophylline (100
mg, 0.55 mmol), 4-fluoro-2-methylbenzenesulfonyl chloride (97 μL,
0.67 mmol), and NaH (60% dispersion in mineral oil, 27 mg, 0.67
mmol) in THF (11 mL) yielded 36 as a white powder (152 mg, 78%)
after purification by CC (gradient of EtOAc in hexane, 10/90 to 80/
1
20). H NMR (300 MHz, CDCl3): δ 8.57 (dd, J = 9.0, 5.5 Hz, 1H,
H6′), 8.38 (s, 1H, H8), 7.17 (ddd, J = 9.5, 7.6, 2.6 Hz, 1H, H5′), 6.99
(dd, J = 9.0, 2.6, 1H, H3′), 3.59 (s, 3H, N3CH3), 3.30 (s, 3H,
N1CH3), 2.51 (s, 3H, C2′CH3). 13C NMR (75.4 MHz, CDCl3): δ
166.6 (d, J = 263.1 Hz), 153.0, 151.3, 150.6, 142.7, 142.2 (d, J = 9.8
Hz), 137.1 (d, J = 10.6 Hz), 130.3 (d, J = 3.0 Hz), 119.7 (d, J = 22.6
Hz), 114.1 (d, J = 21.9 Hz), 106.1, 30.3, 28.6, 20.4 (d, J = 1.1 Hz).
HRMS (ESI+) mass calcd for C14H13FN4O4S (m/z) 375.0533 [M +
Na]+; found, 375.0535. mp 216−218 °C (dec). Anal. Calcd for
C14H13FN4O4S: C, 47.72; H, 3.72; N, 15.90; S, 9.10. Found: C, 47.63;
H, 3.83; N, 15.64; S, 9.18.
7-[(2,5-Dichlorophenyl)sulfonyl]-1,3-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (37). Following method B, theophylline (100 mg,
0.55 mmol), 2,5-dichlorobenzenesulfonyl chloride (163 mg, 0.67
mmol), and NaH (60% dispersion in mineral oil, 27 mg, 0.67 mmol)
in THF (11 mL) yielded 37 as a white powder (157 mg, 73%) after
purification by CC (gradient of EtOAc in hexane, 15/85 to 100/0).
1H NMR (300 MHz, CDCl3): δ 8.62 (d, J = 2.5 Hz, 1H, H6′), 8.43
(s, 1H, H8), 7.60 (dd, J = 8.6, 2.5 Hz, 1H, H4′), 7.42 (d, J = 8.6 Hz,
1H, H3′), 3.59 (s, 3H, N3CH3), 3.30 (s, 3H, N1CH3). 13C NMR
(75.4 MHz, CDCl3): δ 152.8, 151.3, 150.5, 144.0, 136.4, 134.9, 134.8,
133.8, 132.8, 131.1, 105.8, 30.3, 28.6. HRMS (ESI+) mass calcd for
C13H10Cl2N4O4S (m/z) 410.9692 [M + Na]+; found, 410.9686. mp
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37 °C. After incubation, the loading medium was washed with Tyrode
solution without Mg2+ (in mM): 137 NaCl, 10 HEPES, 2 CaCl2, 4
KCl, and 10 glucose at pH 7.4, for 15−30 min. Test molecules were
incubated during this period. Fura-2 calcium measurements were
carried out with a fluorescence multi-well plate reader (Fluostar
Optima, BMG, Germany). Cells were alternatively illuminated by 340
and 380 nm wavelength lights, and the emitted fluorescence was
detected through a 520/10 nm filter for both excitation wavelengths.
After a few cycles of basal fluorescence recording, the P2X7 agonist
BzATP was injected to get a final concentration of 100 μM.
Fluorescence was transformed to [Ca2+]c with Grynkiewitz’s
equation.73 Each experimental condition was performed in triplicate
and averaged. Blockade is expressed as a percentage of the maximum
[Ca2+]c increase elicited by BzATP in control Tyrode-only wells.
Electrophysiological Measurements. cDNA of human His-
P2X7 was synthesized (GeneArt String DNA fragment, Life
Technologies/Thermo Fisher Scientific Inc.) and cloned together
with the poly-A tail from the pNKS2 vector74 into a modified pUC19
vector. Rat P2X7 cDNA was present in pSGem,75 and rat P2X1 and
P2X2 subunits were present in pNKS2.74 Capped cRNAs were
formed from linearized templates using the mMESSAGE mMA-
CHINE Kit (Ambion, Austin, Texas, USA). Oocytes were kindly
donated by Prof. Luis Pardo, MPI for Experimental Medicine,
IL-1β detection. IL-1β (pg/mL) was detected in the collected cell
supernatants using a specific ELISA kit. Supernatant samples were
obtained from the treated MPMs and subjected to the ELISA analysis
according to the supplier recommendations (R&D systems).
Parallel Artificial Membrane Permeability Assay. The
lipophilic membrane was prepared by dissolving porcine brain polar
lipid extract (Avanti, Merck) in dodecane (Sigma-Aldrich) at a
concentration of 20 mg/mL and injecting 4 μL of it on the membrane
of each well of a 96-well Multiscreen Filter Plate (Reference:
MAIPNTR10, Millipore, Merck). Compound solutions were prepared
at 100 μM in PBS at pH 7.4 (Sigma-Aldrich, Merck). The blank
solution contained 1% of DMSO. Each well was then filled with 180
μL of compounds or blank solution, whereas the corresponding wells
of the acceptor Multiscreen 96-well tray (MAMCS9610, Millipore,
Merck) were filled with an equal amount of PBS at pH 7.4. The filter
plate was inserted in the acceptor tray and incubated at rt for 4 h. A
volume of 150 μL of acceptor plate solutions was transferred to a UV-
transparent 96-well plate (MSCPNUV40, Millipore, Merck) together
with compound solutions at initial and equilibrium concentrations
(100 μM and 50 μM, respectively). Compound absorption spectra
were recorded by a SPECTROstar Nano Microplate Reader (BMG
Labtech, Orterberg, Germany) and corrected by blank absorption
spectra. Concentrations and permeability values were calculated with
the following equations
̈
Gottingen or from EcoCyte Bioscience, Dortmund, Germany),
P(10−6·cm·sec−1) = K·−ln(1 − Cac/Ceq)
injected with 50 nL of cRNA (0.5 mg/mL for rP2X1, rP2X4 and
hP2X7; 0.1 mg/mL for rP2X2; 25 μg/mL for rP2X7), and incubated
for 1−2 days at 16−18 °C before recordings were performed as
described76 in a low divalent-cation solution (90 mM NaCl, 1 mM
KCl, 0.5 mM CaCl2, 5 mM HEPES). Current responses to ATP (300
μM for hP2X7, 30 μM for rP2X4 and rP2X7, and 10 μM for rP2X2
and rP2X1) were measured by TEVC at −70 mV using a Turbo Tec
05X Amplifier (NPI Electronic, Tamm, Germany) and Cell Works
software. A rapid exchange of solution, about 300 ms, was reached
with a 50 μL oocyte chamber with a funnel shape, combined with a
fast solution flow (150 μL/s), fed through a manifold built
immediately above the oocyte. Compounds were directly diluted in
the recording chamber and incubated for 3 min without perfusion.
ATP pulses were applied for 2 s (3 s for rP2X7 subtype) followed by
58 s of perfusion at 4-min intervals. Data were shown as means
S.E.M. from at least three oocytes from two different frogs.
V
K =
Cac = Aac·C0/A0
2 · a · t
Ceq = Aeq·C0/A0
where V is the solution volume (180 μL); a is the membrane area in
cm2; t is the time of incubation in seconds; C0 is the starting
concentration of compound solutions; A0 is the measured absorbance
of compound solutions at starting concentration; Aeq is the measured
absorbance of the solution with concentration at equilibrium point,
that is, when the compound completely permeates the membrane;
and Aac is the measured absorbance of solutions in the acceptor plate.
For each compound, the wavelength at maximum absorbance was
considered for calculations (240 nm for JNJ-47965567; 264 nm for 6,
16, 23, 29, 30, and 40; 274 nm for theophylline, 17, and 24; 284 nm
for verapamil and 31; 365 nm for piroxicam). All data are presented as
Isolation of MPMs. Elicited MPMs were obtained from twelve (n
= 12) 6−8 week-old C57BL/6 male mice. Twenty-four hours before
macrophages extraction, mice were injected intraperitoneally with 1
mL of 3.8% Brewer’s thioglycolate medium in order to attract
circulating macrophages to the peritoneal cavity. The day after, the
mice were euthanized by cervical dislocation and the abdominal skin
was retracted to expose the peritoneal wall. The highest efforts were
made to mitigate animal suffering according to the EU Council
Directive guidelines. All experiments with animals were carried out in
accordance with the ARRIVE guidelines, the International Council for
Laboratory Animal Science and the European Union 2010/63/EU
Guidelines. The experimental protocol was approved by the
Institutional Ethical Committee for Animal Research at the
mean
S.E.M. of triplicates of three individual experiments
performed in three different days.
Pgp ATP-ase Activity Measurements. Human P-glycoprotein
ATPase activity was measured using the luminescence-based Pgp-glo
Assay System (Promega, Madison, WI, USA). The assay was
performed according to manufacturer’s protocol (freely available at
Promega webpage). Compounds were tested at 10 μM, and MgATP
was incubated for 1.5 h. Data were acquired using a microplate
luminometer (Orion II, Berthold Technologies GmbH& Co. KG,
Germany) and processed according to the manufacturer’s protocol.
Briefly, basal relative luminescence unit (R.L.U) variation was
calculated as the R.L.U. difference of Na3VO4-treated wells and
non-treated wells R.L.U. Compounds R.L.U. variation was calculated
as the R.L.U. difference of Na3VO4-treated wells and compound-
treated wells.
Data Analysis. The statistically significant differences were
analyzed by the one sample t-test or the one-way analysis of variance
(ANOVA), followed by the Dunnett post hoc test, using Prism 5.0
(Graph Pad) under a Mac OS X-operated computer. Groups of data
were considered statistically different when p < 0.05.
Molecular Docking. The ligand was docked with the consent of
Dr. Ralf Schmid in the homology model of the hP2X7 published by
Dayel et al.11 Molecules’ 3D conformations were generated using
Open Babel.77 The ligand was docked in two previously described
allosteric binding pockets of hP2X79−11 and in the orthosteric binding
pocket using a squared grid with a side of 22−26 Å. Docking was
performed using AutodockTools and Autodock Vina78 and
represented using UCSF Chimera.79
́
Universidad Autonoma de Madrid (UAM). Ten milliliters of cold
sterile PBS were injected in the peritoneal cavity with a 20-G needle,
and the peritoneal content was collected by aspirating the fluid with
the same syringe and needle. The peritoneal exudate cells were
centrifuged at 4 °C for 10 min at 1000 rpm, and the cell pellet was
resuspended in 1 mL of cold DMEM high glucose (Gibco)
supplemented with 10% FBS (Gibco), 100 U/mL penicillin/
streptomycin (Lonza). Cells were counted with a Neubauer chamber,
and the cell concentration was adjusted to 1−3 × 106 cells/mL.
Macrophages were seeded in 24-well plates at a density of 3 × 105
cells/well and primed with 1 μg/mL LPS (Escherichia coli 026:B6
serotype; Sigma-Aldrich) in DMEM high glucose +10% SBF for 4 h.
The LPS-containing medium was removed and fresh DMEM without
FBS containing the different compounds was added for 15 min. ATP
5 mM (Sigma-Aldrich) was then added for 30 min. Cell supernatants
were collected and frozen at −20 °C for IL-1β detection.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02145.
ACKNOWLEDGMENTS
■
■
sı
This work has been supported by the following grants: EU
Horizon 2020 Research and Innovation Program under Marie
Skłodowska-Curie, Grant Agreement N. 766124 to AGG and
AN, and Ministerio de Economía y Competitividad, Spain,
Grant Number SAF2016-78892R to AGG; Deutsche For-
schungsgemeinschaft (DFG, German Research Foundation)
Project-ID: 335447717, SFB 1328 (TP15) to A.N.; Instituto
de Salud Carlos III, Ministerio de Economía y Competitividad,
Spain, Grant Numbers PI16/01041 and PI19/01724 (Co-
funded by FEDER) to CdlR; Instituto de Salud Carlos III,
Ministerio de Economía y Competitividad, Spain, Grant
Numbers PI16/00735 and PI19/00082 (Co-funded by
FEDER) to J.E. We thank Fundación Teófilo Hernando for
continuous support. We thank Prof. Francesco de Virgilio and
his research group for the generous donation of hP2X7-
HEK293 cells. We thank Prof. Luis Pardo for the generous
donation of X. laevis oocytes. We thank Dr. Ralf Schmid for
permission to use the homology model of hP2X7. We thank
Dr. Eva M. Garcia-Frutos (ICMM, CSIC, Spain) for X-ray
diffraction crystal preparation. We thank Dr. Juan Felipe
Franco-Gonzalez (CIB, CSIC, Spain) for assistance with
computational docking analyses.
Scheme for the synthesis of 14, summary of pharmaco-
logical data of the tested compounds, NMR spectra,
current inhibition in rat P2X7 by 6, HPLC, and X-ray
(PDF)
Molecular formula strings (CSV)
PDB coordinates for the computational model used,
modified from the protein model by Dayel et al.11 with
permission (crystal templates PDB IDs 5U1U,5U1V,
5U1W, 5U1X, 5U1Y) (TXT)
Compound 6 pose at the P2X7 binding site (MP4)
AUTHOR INFORMATION
Corresponding Authors
■
Antonio G. García − Instituto-Fundación Teófilo Hernando
and Departamento de Farmacología, Facultad de Medicina,
Universidad Autónoma de Madrid, 28029 Madrid, Spain;
Instituto de Investigación Sanitaria, Hospital Universitario de
La Princesa, 28006 Madrid, Spain; Phone: (+34)
914975397; Email: agg@uam.es; Fax: (+34)-914973453
Cristóbal de los Ríos − Instituto-Fundación Teófilo Hernando
and Departamento de Farmacología, Facultad de Medicina,
Universidad Autónoma de Madrid, 28029 Madrid, Spain;
Instituto de Investigación Sanitaria, Hospital Universitario de
La Princesa, 28006 Madrid, Spain; orcid.org/0000-
0002-6456-7589; Phone: (+34)-914972765;
ABBREVIATIONS
■
Aβ, amyloid β peptide; AD, Alzheimer’s disease; ALS,
amyothrophic lateral sclerosis; BBB, blood−brain barrier;
BBG, brilliant blue G; CC, column chromatography; CNS,
central nervous system; DMEM, Dulbecco-modified Eagle’s
medium; FBS, fetal bovine serum; IL-1β, interleukin 1β;
MPMs, mouse peritoneal macrophages; NDDs, neurodegener-
ative diseases; PAMPA, parallel artificial membrane perme-
ability assay; PD, Parkinson’s disease; P2X, type 2 ionotropic
purinergic receptor
Email: cristobal.delosrios@inv.uam.es
Authors
Francesco Calzaferri − Instituto-Fundación Teófilo Hernando
and Departamento de Farmacología, Facultad de Medicina,
Universidad Autónoma de Madrid, 28029 Madrid, Spain;
orcid.org/0000-0002-4781-2925
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https://pubs.acs.org/10.1021/acs.jmedchem.0c02145
Notes
The authors declare no competing financial interest.
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