Development of novel oxazolo[5,4-d]pyrimidines as competitive CB2 neutral antagonists based on scaffold hopping

Article abstract of DOI:10.1016/j.ejmech.2018.01.034

A series of novel oxazolo[5,4-d]pyrimidines was designed via a scaffold hopping strategy and synthesized through a newly developed approach. All these compounds were evaluated for their biological activity toward CB₁/CB₂ cannabinoid receptors, their metabolic stability in mice liver microsomes and their cytotoxicity against several cell lines. Eight compounds have been identified as CB₂ ligands with K_i values less than 1 μM. It is noteworthy that 2-(2-chlorophenyl)-5-methyl-7-(4-methylpiperazin-1-yl) oxazolo[5,4-d]pyrimidine 47 and 2-(2-chlorophenyl)-7-(4-ethylpiperazin-1-yl)- 5-methyloxazolo[5,4-d]pyrimidine 48 showed CB₂ binding affinity in the nanomolar range and significant selectivity over CB₁ receptors. Interestingly, functionality studies imply that they behave as competitive neutral antagonists. Moreover, all tested compounds are devoid of cytotoxicity toward several cell lines, including Chinese hamster ovary cells (CHO) and human colorectal adenocarcinoma cells HT29.

Full text of DOI:10.1016/j.ejmech.2018.01.034

Accepted Manuscript
Development of novel oxazolo[5,4-d]pyrimidines as competitive CB neutral
2
antagonists based on scaffold hopping
Wei Tuo, Mélanie Bollier, Natascha Leleu-Chavain, Lucas Lemaire, Amélie Barczyk,
Xavier Dezitter, Frédérique Klupsch, Fabien Szczepanski, John Spencer, Philippe
Chavatte, Régis Millet
PII:
S0223-5234(18)30047-3
10.1016/j.ejmech.2018.01.034
EJMECH 10111
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2017
Revised Date: 10 January 2018
Accepted Date: 11 January 2018
Please cite this article as: W. Tuo, Mé. Bollier, N. Leleu-Chavain, L. Lemaire, Amé. Barczyk, X.
Dezitter, Fréé. Klupsch, F. Szczepanski, J. Spencer, P. Chavatte, Ré. Millet, Development of novel
oxazolo[5,4-d]pyrimidines as competitive CB neutral antagonists based on scaffold hopping, European
2
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.034.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Table of Content

ACCEPTED MANUSCRIPT
Development of novel oxazolo[5,4-d]pyrimidines as competitive CB2 neutral
antagonists [1] based on scaffold hopping
Wei Tuo,a Mélanie Bollier,a Natascha Leleu-Chavain,a Lucas Lemaire,a Amélie
Barczyk,a Xavier Dezitter,a Frédérique Klupsch,a Fabien Szczepanski,a John Spencer,b
Philippe Chavatte,a and Régis Milleta,*
a
ICPAL, Univ. Lille, Inserm, U995-LIRIC-Lille Inflammation Research International
Center, 3 Rue du Professeur Laguesse, BP83, F-59006 Lille, France
b
Department of Chemistry, School of Life Sciences, University of Sussex, Falmer,
Brighton, East Sussex BN1 9QJ, U.K.
Abstract: A series of novel oxazolo[5,4-d]pyrimidines was designed via a scaffold
hopping strategy and synthesized through a newly developed approach. All these
compounds were evaluated for their biological activity toward CB1/CB2 cannabinoid
receptors, their metabolic stability in mice liver microsomes and their cytotoxicity
against several cell lines. Eight compounds have been identified as CB2 ligands with
Ki
values
less
than
1
µM.
It
is
noteworthy
that
2-(2-chlorophenyl)-5-methyl-7-(4-methylpiperazin-1-yl) oxazolo[5,4-d]pyrimidine 47
Abbreviations: Boc, tert-butyloxycarbonyl; cAMP, cyclic adenosine monophosphate; CHO, Chinese
hamster ovary cells; CHO-CB2, Chinese hamster ovary cells expressing CB2 receptors; CHO-WT,
Chinese hamster ovary cells wild type; DIEA, N,N-diisopropylethylamine; ECS, endocannabinoid
system; KHMDS, potassium bis(trimethylsilyl)amide; SAR, structure-activity relationship; TFA,
trifluoroacetic acid.
*
Corresponding author.
E-mail address: regis.millet@univ-lille2.fr (R. Millet).

ACCEPTED MANUSCRIPT
and 2-(2-chlorophenyl)-7-(4-ethylpiperazin-1-yl)- 5-methyloxazolo[5,4-d]pyrimidine
48 showed CB2 binding affinity in the nanomolar range and significant selectivity
over CB1 receptors. Interestingly, functionality studies imply that they behave as
competitive neutral antagonists. Moreover, all tested compounds are devoid of
cytotoxicity toward several cell lines, including Chinese hamster ovary cells (CHO)
and human colorectal adenocarcinoma cells HT29.
Key words: Cannabinoid, CB2 receptor, neutral antagonist, oxazolo[5,4-d]pyrimidine
1. Introduction
The endocannabinoid system (ECS), present in mammals and comprising two
primary G protein-coupled receptors, is linked with endogenous lipid mediators and
relevant enzymes responsible for the synthesis and degradation of lipid mediators [2].
Due to its functions in the regulation of pain, inflammation, motor behavior, emotion,
appetite and addiction, the ECS has been regarded as an ideal target with broad
therapeutic potential [2-4]. The two G protein-coupled cannabinoid CB1 and CB2
receptors, are predominantly expressed in the central nervous system (e.g. brain) and
immune cells (e.g. macrophages), respectively [3, 5-7]. CB1 receptor antagonists have
shown potential in the treatment of addiction and obesity. It has been demonstrated
that the administration of a CB1 antagonist (e.g., rimonabant) brings about a reduction
of drug-seeking behaviors and weight loss [8-11]. By contrast, CB2 receptor agonists
lead to down regulation of inflammation and pain by restoring the equilibrium
between pro- and anti-inflammatory cytokines [3, 5, 7, 12]. Moreover, several studies

ACCEPTED MANUSCRIPT
have shown that CB2 inverse agonists can regulate bone proliferation and relieve
inflammation through modulating the migration of immune cells [13-15]. However,
research on CB2 neutral antagonists is rare, and their therapeutic potential remains
unknown. In some cases, CB2 neutral antagonists can be considered as ideal tools for
investigating the biological function of receptors due to their ability to combine with
receptors without altering corresponding biomolecular levels [16, 17]. In addition,
targeting CB1 receptors rather than CB2 receptors has been demonstrated to induce
tetrahydrocannabinol-like side effects (e.g., hypomotility, hypothermia, catalepsy)
[18]. Thus, the development of selective CB2 ligands has been considered as an
interesting therapeutic strategy.
Over the course of the last few decades, the development of CB2 ligands has
significantly progressed. Numerous selective CB2 ligands have shown therapeutic
potential in a wide range of in vivo models, including pain, inflammation, cancer,
immune disorders, neurodegenerative diseases and bone disorders [14, 19-26].
Specifically, more than ten CB2 selective agonists have been reported to be under
evaluation in clinical trials for the treatment of inflammation and pain [12]. For
instance, compound
1
(GRC10693, Figure 1), developed by Glenmark
Pharmaceuticals, successfully completed Phase I trials in 2009 [27]. It was intended to
be used for pain remission, including neuropathic pain and inflammatory pain.
Moreover, Eli Lilly and Co. disclosed a series of purines as selective CB2 agonists
[28-31]. Of note, compound 2 (LY2828360, Figure 1), derived from the lead
compound 3 (Figure 1), has been recognized as a potent and efficacious analgesic

ACCEPTED MANUSCRIPT
agent in a rat model of osteoarthritis related chronic pain at a 1 mg/kg oral dose [30].
Accordingly, clinical trials for the treatment of osteoarthritic knee pain were
performed [32]. Additionally, it was reported that compound 4 (AM630, Figure 2),
one of the most potent CB2 inverse agonist identified to date, can attenuate
osteoporosis [25, 33, 34]. Two other notable CB2 inverse agonists, 5 (Sch225336,
Figure 2) and 6 (Sch414319, Figure 2), have been shown to reduce inflammation
through modulating the recruitment of immune cells [13, 15].
Figure 1. Structures of representative CB2 agonists evaluated in clinical trials.
Figure 2. Representative CB2 inverse agonists.
Scaffold hopping is a useful and powerful strategy for drug discovery. It has been
widely used for the design of potential bioactive molecules [35-37]. In general,
scaffold hopping incorporate heterocycle replacement, ring opening and closure,
pseudopeptides and peptidomimetics, and topology/shape-based scaffold hopping [36].

ACCEPTED MANUSCRIPT
Herein, we applied the heterocycle replacement-based scaffold hopping strategy
toward compound 3, a CB2 agonist developed by Eli Lilly and Co [30].
Oxazolo[5,4-d]pyrimidine is a versatile scaffold used for the design of bioactive
ligands against enzymes or receptors [38-40]. Hence, we replaced the purine core by
this scaffold, with the intention of observing its influence on bioactivity toward CB2
receptors (Figure 3). Afterwards, we carried out pharmacomodulations around the
oxazolopyrimidine core (Figure 4). The newly synthesized compounds were tested for
their CB2 vs CB1 affinity in a binding assay, their efficacy toward CB2, and their
metabolic stability in mice liver microsomes.
Figure 3. Heterocycle replacement-based scaffold hopping toward compound 3.

ACCEPTED MANUSCRIPT
Figure 4. Structural modifications of substituents on the oxazolo[5,4-d]pyrimidine core.
2. Results and discussion
2.1. Chemistry
Compounds 11-14 were prepared by a cyclisation reaction between benzoyl
chloride or ortho-halogenated benzoyl chloride derivatives and aminomalononitrile
p-toluenesulfonate (75-90% yields). Starting from these compounds, target
compounds were obtained through three different synthetic pathways (Scheme 1): (I)
5-amino-2-(2-chlorophenyl)oxazole-4-carbonitrile (13) reacted with formamidine
acetate to generate 2-(2-chlorophenyl)oxazolo[5,4-d]pyrimidin-7-amine 15 (86%
yield), which was further involved in a nucleophilic substitution using 15 wt. %
potassium bis(trimethylsilyl)amide (KHMDS) solution in toluene and brominated
derivatives to either give target compounds 17-20 (37-52% yields) directly, or to

ACCEPTED MANUSCRIPT
afford a tert-butyloxycarbonyl (Boc)-protected precursor 16 (43% yield).
Deprotection of the Boc group of 16 using trifluoroacetic acid (TFA) followed by
neutralisation yielded compound 21 (99% yield). (II) Alternatively, compounds 11-14
were cyclized using formic acid to give 2-phenyloxazolo[5,4-d]pyrimidin-7-ones
22-25 (33-46% yields), respectively. Afterwards, POCl3-dependent chlorination
converted these 2-phenyloxazolo[5,4-d]pyrimidin-7-ones into compounds 26-29
(72-85% yields). The chloride group of 26-29 was substituted by a piperazine or
morpholine derivative to either give target compounds 32-42 (98-99% yields) directly,
or to afford Boc-protected compound 30 (98% yield). Compound 43 (98% yield) was
obtained after deprotection of the piperazine group of compound 30 using TFA
followed by a nucleophilic substitution on bromopropane. (III) Additionally,
compound 13 was acetylated to afford 44 (78% yield) in the presence of acetyl
chloride and N,N-diisopropylethylamine (DIPEA). A solution of 44 in 30% NaOH and
35% H2O2 was refluxed to give 45 (69% yield), which was further chlorinated by
POCl3 (77% yield) and substituted with piperazine derivatives to produce compounds
47-48 (98-99% yields).

ACCEPTED MANUSCRIPT
Scheme 1. Synthetic routes to prepare compounds 17-21, 32-43, and 47-48. (a)
aminomalononitrile p-toluenesulfonate, NMP, microwave, 80 °C, 25 min, 75-90%. (b)
formamidine acetate, NMP, 100 °C, 3 h, 86%. (c) KHMDS, 15 wt. % in toluene, brominated
derivative, DMF, room temperature (rt), 3 h, 37-52%. (d) TFA, CH2Cl2, rt, 3 h, 99%. (e) formic
acid, reflux, overnight, 31-37%. (f) POCl3, DIPEA, toluene, rt, 2 h, 72-85%. (g) piperazine
derivative or morpholine, Cs2CO3, DMF, 100 °C, 30 min, 98-99%. (h) bromopropane, Cs2CO3,
DMF, 100 °C, 30 min, 99%. (i) acetyl chloride, DIPEA, NMP, 45 °C, 6 h, 78%. (j) 30% NaOH
and 35% H2O2 in water, reflux, 2 h, 61%.
2.2. In vitro binding assays and cytotoxicity
The affinities of each synthesized compound for CB receptors were determined

ACCEPTED MANUSCRIPT
by a competitive radioligand displacement assay using the dual CB1/CB2 ligand
[3H]-CP55,940 [22, 41]. All compounds were first screened at the concentration of 1
µM. The binding affinities (Ki) were calculated using the Cheng-Prusoff equation
based on the experimental IC50 values for compounds exhibiting a competitive
binding superior to 50% for CB2. Kd values of CP-55,940 towards CB1 and CB2 are
0.62 and 0.85 nM, respectively.
As illustrated in Table 1, compounds 47 and 48 bearing a chlorine at the ortho
position of the phenyl ring, a C5-substituted methyl group, and a C7-substituted
piperazine moiety, possess the best affinities toward CB2 receptors, in the double-digit
nanomolar range, with good selectivity over CB1 receptors (SI > 12). The removal of
the C5-substituted methyl group of 47 and 48 (Ki = 27.5 nM and 23 nM, respectively)
leads to a sharp decrease in CB2 binding affinities (i.e. compounds 40 and 41, Ki =
350 and 329 nM, respectively). These results imply that the methyl group at the C5
position plays a crucial role in binding affinity for CB2, probably due to hydrophobic
interactions with the receptor. In general, C7-substituted piperazine derivatives (40-42,
Ki values range from 350 nM to 678 nM) are preferred over morpholine (39, Ki >
1000 nM) or secondary amine substituents (17-21, Ki > 1000 nM). Of note,
compounds bearing an N-ethylpiperazine at the C7 position (37, 41, and 48, Ki = 95.5
nM, 329 nM, and 23 nM, respectively) possess slightly superior CB2 binding affinities
than the corresponding compounds bearing a N-methylpiperazine (36, 40, and 47, Ki =
155 nM, 350 nM, and 27.5 nM, respectively) and are approximately 2-3 times more
potent CB2 ligands than compounds bearing a N-acetylpiperazine substituent (38 and

ACCEPTED MANUSCRIPT
42, Ki = 254 nM and 678 nM, respectively). Specifically, extending the carbon chain
on the nitrogen at the C7 position of 41 (Ki = 329 nM) from 2C to 3C (43, Ki > 1000
nM) leads to almost complete loss of CB2 binding affinity. Moreover, large ortho
substituents (such as CF3, 36-38, Ki values range from 95.5 nM to 254 nM, and Cl,
40-42, Ki values range from 329 nM to 678 nM) on the phenyl ring contribute to
better CB2 binding affinities in comparison with small groups (such as F, 34-35, Ki >
1000 nM, and H, 32-33, Ki > 1000 nM). This observation might be attributed to the
fact that larger groups make stronger hydrophobic interactions with the CB2 receptor.
Table 1. Affinities (Ki values) of compounds 17-21, 32-43, 47-48, and reference compounds 3, 4,
WIN 55,212-2 toward hCB2 and hCB1 cannabinoid receptors, selectivity ratios hCB2 versus hCB1,
and cytotoxicity on CHO-WT, CHO-CB2, and HT29 Cells.
Binding assays (nM)a
Cytotoxicity assays % inhibition at 10 µM
CB1/CB2
ratio
R1
R2
Compounds
Ki hCB2
> 1000
Ki hCB1
> 1000
> 1000
> 1000
CHO-WT
CHO-CB2
HT29
Cl
N.D.b
N.D.b
N.D.b
0
0
6
18
0
0
0
17
18
19
Cl
Cl
> 1000
> 1000
13
10
Cl
Cl
> 1000
> 1000
> 1000
> 1000
N.D.b
N.D.b
0
0
10
9
0
20
21
41
R1
R3
H
H
> 1000
> 1000
> 1000
> 1000
N.D.b
N.D.b
0
0
0
0
0
0
32
33

ACCEPTED MANUSCRIPT
F
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
N.D.b
N.D.b
> 6.4
> 10.4
> 3.9
N.D.b
> 2.8
> 3.0
> 1.4
N.D.b
8
10
3
7
8
0
24
0
34
35
36
37
38
39
40
41
42
43
F
> 1000
CF3
CF3
CF3
Cl
155 ± 8
95.5 ± 11.5
254 ± 26
> 1000
0
10
12
0
5
1
1
12
5
10
0
Cl
350 ± 33
329 ± 61
678 ± 84
> 1000
0
8
Cl
1
9
4
Cl
23
0
11
2
0
Cl
R1
Cl
0
R4
27.5 ± 0.5
23 ± 4
> 1000
293
> 36.3
12.7
0
0
4
24
16
47
48
Cl
16
3 (agonist)
51 ± 9
> 1000
> 1000
> 19.6
> 11.0
30
0
0
0
0
0
4 (inverse agonist)
WIN55212-2
(agonist)
91 ± 11
3.01 ± 0.99
a Data represent the mean ± SEM of at least three experiments performed in duplicate or triplicate.
b N.D. means not determined.
In summary, the structure-activity relationship (SAR) studies indicate that
piperazine moieties at the C7 position favor CB2 binding affinities of the molecules.
Notably, lipophilic piperazine moieties (i.e., methylpiperazine and ethylpiperazine)
are preferred for CB2 affinity rather than a hydrophilic piperazine moiety (i.e.,
acetylpiperazine). However, there seems to be steric limitations due to a sudden

ACCEPTED MANUSCRIPT
decrease in CB2 binding affinity caused by extending the N-substituted alkyl chain
from 2C to 3C. Moreover, large ortho substituents on the phenyl ring are beneficial
for CB2 binding affinities. The introduction of a methyl group at the C5 position
seems to significantly improve the CB2 binding affinities of the molecules (Figure 5).
It is noteworthy that the two most potent compounds 47 and 48 (Ki = 27.5 nM and 23
nM, respectively) manifest CB2 binding affinities twice as potent as the reference
compound 3 (Ki = 51 nM). This result implies that oxazolo[5,4-d]pyrimidine-based
compounds can act as effective bioisosteres of purines in terms of CB2 binding.
Additionally, compounds 47 (SI > 36) and 48 (SI = 12.7) are selective CB2 ligands
over CB1 receptors.
Figure 5. Structure-affinity relationships of oxazolo[5,4-d]pyrimidine-based CB2 ligands.
The cytotoxicity of these compounds was determined at 10 µM using a cell
proliferation assay on Chinese hamster ovary cells wild type (CHO-WT), Chinese
hamster ovary cells expressing CB2 receptors (CHO-CB2), and human colorectal
adenocarcinoma cells HT29. This test is based on a colorimetric method, which
measures the activity of cellular enzymes that reduce the tetrazolium dye (MTS,
yellow) to its insoluble formazan, giving a purple color. This assay measures cellular

ACCEPTED MANUSCRIPT
metabolic activity via NADPH-dependent cellular oxidoreductase enzymes and
reflects, under defined conditions, the number of viable cells [5, 42]. No significant
cytotoxicity over 72h was observed for these new compounds.
2.3. Cyclic adenosine monophosphate (cAMP) assays
Eight compounds displaying potent CB2 affinity were further studied for their
functionality by cAMP assays in CHO-CB2 cells. Cells were treated with forskolin (3
µM) in order to activate adenylyl cyclase and thereby producing cAMP [43-45]. CB2
ligands can be classified as agonists, neutral antagonists, or inverse agonists regarding
to their functional effects on cAMP formation. Agonists inhibit forskolin-induced
cAMP production, whereas inverse agonists promote cAMP production. On the
contrary, neutral antagonists do not significantly alter cAMP levels [46, 47]. As shown
in Table 2, the maximum efficacy (Emax) of a compound represents the maximum
response at 1 µM and is expressed as the percentage of forskolin-induced cAMP
production [5]. Despite their potent binding affinities toward CB2 receptors, these
compounds did not significantly affect CB2-mediated regulation of cAMP
accumulation (Emax values range from 88% to 131%), compared with forskolin
(100%). Data in Figure 6 further demonstrated that compounds 47 and 48 showed
competitive antagonist properties. These two compounds remarkably antagonized
CB2 agonist (0.1 µM WIN 55,212-2)-induced inhibition of cAMP formation at 1 µM
and almost completely restored forskolin-induced cAMP levels at 10 µM. IC50 values
of these two compounds are 0.93 µM (47) and 0.12 µM (48), respectively.

ACCEPTED MANUSCRIPT
Interestingly, although replacement of the purine core by oxazolo[5,4-d]pyrimidine
does not alter the binding affinity of the molecule toward CB2, it appears to alter the
biological response from a CB2 agonist to competitive neutral antagonist.
Table 2. Maximum efficacy (Emax) of compounds 36-38, 40-42, 47-48, and reference compounds
3, 4, WIN 55,212-2 at 1 µM.
Compounds
CB2 cAMP assays Emax (%)a
36
129 ± 3
117 ± 15
107 ± 16
131 ± 13
120 ± 8
117 ± 7
94 ± 2
37
38
40
41
42
47
48
88 ± 7
forskolin
100
3 (agonist)
4 (inverse agonist)
71 ± 3
863 ± 28
WIN55212-2 (agonist) 64 ± 12
a
Data represent the mean ± SEM of at least three independent experiments performed in duplicate or
triplicate. Emax values are expressed as the percentage of forskolin-induced cAMP production. Forskolin
(3 µM) effect was set to 100%.
Figure 6. Inhibition of forskolin-stimulated cAMP formation by WIN 55,212-2 (0.1 µM) and
antagonism by 47 and 48 (0.1-10 µM) in CB2-CHO cells. Forskolin (3 µM) effect was set to 100%.

ACCEPTED MANUSCRIPT
Data represent the mean ± SEM of at least three experiments performed in duplicate.
2.4. Metabolic stability in mice liver microsomes
These eight potent CB2 ligands have also been examined for their in vitro
metabolic stability in the presence of mice liver microsomes (Figure 7, Table 3). It
was found that almost all of compounds 36, 37, 40 and 41 were rapidly eliminated
under 5 min (t1/2 ≈ 1 min). Compounds 47 and 48 showed lower intrinsic clearance
(t1/2 = 4.1 and 8.1 min respectively) than compounds 40 and 41 (t1/2 = 1.3 and 1.2 min
respectively). This observation implies that a C5-substituted methyl group confers
higher metabolic stability to the molecules[48], although the clearance values are still
rather high. In addition, the replacement of an N-alkyl group by an N-acetyl group on
the piperazine moiety (38 and 42, t1/2 = 12.5 and 12.2 min respectively) has been
deemed to endow the molecules with significantly improved metabolic stability.
Although these eight newly synthesized compounds are less metabolically stable than
compounds 3 in mice liver microsomes, the study of their structure-stability
relationships may be helpful to further improve the metabolic stability of
oxazolo[5,4-d]pyrimidine derivatives.

ACCEPTED MANUSCRIPT
Figure 7. Metabolic stability of compounds 36-38, 40-42, 47, 48, 3, and propranolol.
Table 3. Clint and t1/2 of compounds 36-38, 40-42, 47, 48, and 3 in the presence of mice liver
microsomes.
Compounds Clint (µL/min/mg)a t1/2 (min)
36
37
38
40
41
42
47
48
3
1257
1236
174
0.9
1.0
12.5
1.3
1.2
12.2
4.1
8.1
44
1178
1101
194
502
263
54
a Clint = dose/AUC, expressed as µL/min/mg proteins.
3. Conclusion and perspective
A novel series of oxazolo[5,4-d]pyrimidines has been synthesized. Some of them
display potent affinity toward CB2 at nanomolar concentrations and significant
selectivity over CB1. Especially, compounds 47 and 48 have been identified as
competitive
neutral
antagonists.
Our
studies
indicate
that
oxazolo[5,4-d]pyrimidine-based compounds can act as effective bioisosteres of

ACCEPTED MANUSCRIPT
purines in CB2 binding. Interestingly, such a replacement changes the biological
response from a CB2 agonist to a competitive neutral antagonist.
There are many reports on CB2 agonists and inverse agonists but few in the case
of neutral antagonists. During the last few years, several clinical trials of CB2 agonists
have been launched. The therapeutic potential of CB2 inverse agonists has also
progressed. To the best of our knowledge, research on neutral antagonists is rare.
Indeed, the first reported antagonist SR144528 was then identified to behave as an
inverse agonist [16, 49]. Some purported “antagonists” are actually inverse agonists
[50-54]. The real first reported CB2 neutral antagonist may be WIN55212-3 (Figure 8),
an enantiomer of WIN55212-2, but it showed weak affinity toward CB2 (Ki > 1 µM).
Another reported CB2 neutral antagonist is XIE35-1001 (Figure 8) [55]. However,
data in a cAMP assay or [35S]-GTPγS assay are missing. Recently, Bertini and
coworkers described a non-competitive CB2 neutral antagonist (49, Figure 8) [56],
which significantly reversed CB2 agonist (1 nM HU-210) responses at 10 µM but did
not alter maximal responses of the agonist (0.1 µM HU-210) even at 20 µM in a
[35S]-GTPγS assay. On the contrary, our two neutral antagonists 47 and 48 remarkably
reversed CB2 agonist (0.1 µM WIN 55,212-2)-induced inhibition of cAMP formation
at 1 µM. This observation makes compounds 47 and 48 useful tools for studying the
function of CB2 receptors at the cellular level.
In this context, we identified that oxazolo[5,4-d]pyrimidine could be a useful
scaffold for the development of new selective neutral antagonists toward CB2.

ACCEPTED MANUSCRIPT
Moreover, although CB2 inverse antagonists have shown therapeutic potential in
bone-dependent disorders and inflammation through somehow modulating the
migration of immune cells, relevant mechanisms remain unclear. The discovery of
new CB2 neutral antagonists with high affinity might be helpful for elucidating how
CB2 inverse agonists regulate bone proliferation and the migration of immune cells.
Notably, compounds 47 and 48, which display nanomolar potency and behave as
competitive neutral antagonists toward CB2, may serve as lead compounds for further
studies. Furthermore, we will continue the structural optimization of these
oxazolepyrimidines to improve their metabolic stability so that they can be suitable
for systemic applications.
Figure 8. Structures of representative CB2 neutral antagonists.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
All reactions involving air or moisture-sensitive compounds were performed
under argon atmosphere. Solvents and reagents were purchased from commercial
suppliers and were used without further purification. The thin layer chromatography

ACCEPTED MANUSCRIPT
was performed using silica gel plates (Polygram Sil G/UV254, thickness: 0.2 mm, ref.
805023 Macherey Nagel). The revelation was made under UV (253 and 366 nm). The
purifications by column chromatography were performed using silica gel 60,
granulometry 40-63 µm (ref. 9385.5000 Merck). The microwave reactions were
performed using a CEM DiscoverSP. Melting points were recorded on a Büchi
melting point apparatus and are uncorrected. They are expressed in degrees Celsius.
NMR spectra were recorded at 300 (1H) and 75 (13C) MHz on a Bruker AC300P
Fourier transform spectrometer. Chemical shifts (δ) are reported relative to
tetramethylsilane (TMS) as the internal standards in the NMR laboratory of Physical
Chemistry of University Lille 2. Spectra were recorded at ambient temperature. Each
signal is identified by its chemical shift δ expressed in parts per million (ppm).
Coupling constant (J) is reported in Hertz (Hz). All target compounds were
characterized by LC-MS and HRMS. The high performance liquid chromatograph
(ODS column, mobile phase: H2O/CH3CN/HCOOH using gradient method) is
coupled to a UV detector and a type of APCI+ (atmospheric pressure chemical
ionization) mass detector. HRMS analyzes were performed under ESI (electrospray
ionization) using a TOF analyzer in V mode with a mass resolution of 9000. The
spectra were recorded at the Centre Universitaire des Mesures Analytique of
University Lille 2 using Thermo Electron Surveyor MSQ mass spectrometer. The
purity of all compounds was determined by HPLC using a Chromazing column (4.6 x
150 mm, 5 µm, 100 Å, mobile phase: CH3CN/H2O/HCOOH = 100/25/1) and a
WATERS 600 pump chromatograph equipped with a WATERS 2487 dual absorption

ACCEPTED MANUSCRIPT
wavelength UV detector (λ = 254 nm and 366 nm). Retention time was obtained with
flow rates of 1 mL/min. The acquisition time is 20 min.
4.1.2. Synthesis of 5-amino-2-(2-substituted phenyl)oxazole-4-carbonitriles (11-14)
A solution of benzoyl chloride or ortho halogenated benzoyl chloride (4.8 mmol,
1.2 equiv) and aminomalononitrile p-toluenesulfonate (4 mmol, 1 equiv) in 15 mL
NMP was stirred at room temperature for 10 min, and then heated by microwave
irradiation at 80 °C for 25 min. At the end of the reaction, after cooling, the solution
was diluted with 100 mL saturated brine and neutralized (pH ~8) to give a white
precipitate, which was further recrystallized in CH2Cl2 to afford compounds 11-14.
These compounds were used for the next reaction.
4.1.3. Synthesis of 2-(2-chlorophenyl)oxazolo[5,4-d]pyrimidin-7-amine (15)
A solution of compound 13 (2.5 mmol, 1 equiv) and formamidine acetate (3
mmol, 1.2 equiv) in 15 mL NMP was stirred and heated at 100 °C for 3 h. At the end
of the reaction, after cooling, the solution was diluted with 100 mL saturated brine to
give a white precipitate, which was recrystallized in CH2Cl2 to afford compound 15.
This compound was used for the next reaction.
4.1.4. Synthesis of 2-(2-chlorophenyl)-N-alkyloxazolo[5,4-d]pyrimidin-7-amines
(16-20)
To a solution of compound 15 (0.25 mmol, 0.1 equiv) and brominated derivative
(0.2 mmol, 0.8 equiv) in 15 mL DMF, cooled to 0 °C, 15 wt. % KHMDS (0.3 mmol,

ACCEPTED MANUSCRIPT
1.2 equiv) solution in toluene was added dropwise. Afterwards, the mixture solution
was recovered to room temperature and stirred for 3 h. At the end of the reaction, the
solution was diluted with 100 mL saturated brine, and then extracted with 80 mL ethyl
acetate. The organic phase was washed twice with 50 mL distilled water and dried
over MgSO4. The solvent was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography using CH2Cl2/ethyl acetate (8: 2 v/v) as
eluent to give compounds 16-20.
4.1.4.1. N-Butyl-2-(2-chlorophenyl)oxazolo[5,4-d]pyrimidin-7-amine (17)
The title compound was obtained as a white solid, mp 104-105 °C, purity
1
(HPLC): 98.8%. H NMR (300 MHz, DMSO-d6) δ 8.46 – 8.19 (m, 2H), 8.09 (d, J =
7.8 Hz, 1H), 7.72 (dd, J = 7.9, 1.6 Hz, 1H), 7.70 – 7.52 (m, 2H), 3.90 – 3.71 (m,
0.5H), 3.62 – 3.41 (m, 1.5H), 1.73 – 1.50 (m, 2H), 1.48 – 1.27 (m, 2H), 0.92 (t, J =
7.3 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.1 (C), 156.2 (C), 155.5 (C), 155.2
(CHA), 154.8 (CHB), 133.3 (CH), 132.4 (C), 132.2 (CH), 131.7 (CH), 128.2 (CH),
125.8 (C), 116.1 (C), 42.7 (CH2B), 40.3 (CH2A), 32.5 (CH2B), 31.4 (CH2A), 20.1 (CH2),
14.2 (CH3). HRMS (ESI) calcd. for C15H15ON4Cl ([M+H]+) 303.10072, found
303.10004.
4.1.4.2.
2-(2-Chlorophenyl)-N-(2-methoxyethyl)oxazolo[5,4-d]pyrimidin-7-amine
(18)
The title compound was obtained as a white solid, mp 124-125 °C, purity
(HPLC): 95.1%. 1H NMR (300 MHz, DMSO-d6) δ 8.43 – 8.20 (m, 2H), 8.10 (dd, J =

ACCEPTED MANUSCRIPT
7.6, 2.0 Hz, 1H), 7.72 (dd, J = 7.8, 1.6 Hz, 1H), 7.70 – 7.52 (m, 2H), 4.09 – 3.88 (m,
13
0.5H), 3.76 – 3.64 (m, 1.5H), 3.63 – 3.51 (m, 2H), 3.28 (s, 3H). C NMR (75 MHz,
DMSO-d6) δ 164.1 (C), 156.3 (C), 155.5 (C), 155.1 (CH), 133.3 (CH), 132.4 (C),
132.2 (CH), 131.7 (CH), 128.2 (CH), 125.7 (C), 116.3 (C), 71.4 (CH2B), 70.5 (CH2A),
58.4 (CH3), 42.6 (CH2B), 40.1 (CH2A). HRMS (ESI) calcd. for C14H13O2N4Cl
([M+H]+) 305.07998, found 305.07927.
4.1.4.3. 2-(2-Chlorophenyl)-N-pentyloxazolo[5,4-d]pyrimidin-7-amine (19)
The title compound was obtained as a white solid, mp 122-123 °C, purity
(HPLC): 98.4%. 1H NMR (300 MHz, DMSO-d6) δ 8.43 – 8.19 (m, 2H), 8.08 (dd, J =
7.5, 1.9 Hz, 1H), 7.70 (dd, J = 7.9, 1.5 Hz, 1H), 7.68 – 7.50 (m, 2H), 3.86 – 3.68 (m,
0.5H), 3.58 – 3.41 (m, 1.5H), 1.72 – 1.49 (m, 2H), 1.38 – 1.25 (m, 4H), 0.86 (t, J =
6.7 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.1 (C), 156.2 (C), 155.5 (C), 155.2
(CHA), 154.8 (CHB), 133.3 (CH), 132.4 (C), 132.2 (CH), 131.7 (CH), 128.2 (CH),
125.8 (C), 116.1 (C), 43.0 (CH2B), 40.8 (CH2A), 30.0 (CH2B), 29.0 (CH2A × 2), 22.4
(CH2), 14.4 (CH3). HRMS (ESI) calcd. for C16H17ON4Cl ([M+H]+) 317.11637, found
317.11586.
4.1.4.4. 2-(2-Chlorophenyl)-N-(cyclohexylmethyl)oxazolo[5,4-d]pyrimidin-7-amine
(20)
The title compound was obtained as a solid, mp 140-141 °C, purity (HPLC):
1
97.6%. H NMR (300 MHz, DMSO-d6) δ 8.45 – 8.20 (m, 2H), 8.08 (d, J = 7.7 Hz,
1H), 7.76 – 7.66 (m, 1H), 7.69 – 7.51 (m, 2H), 3.70 – 3.57 (m, 0.5H), 3.36 (t, J = 6.4

ACCEPTED MANUSCRIPT
13
Hz, 1.5H), 1.83 – 1.55 (m, 6H), 1.28 – 1.11 (m, 3H), 1.06 – 0.86 (m, 2H). C NMR
(75 MHz, DMSO-d6) δ 164.1 (C), 156.2 (C), 155.7 (C), 155.1 (CHA), 154.8 (CHB),
133.3 (CH), 132.4 (C), 132.3 (CH), 131.7 (CH), 128.2 (CH), 125.8 (C), 116.1 (C),
48.9 (CH2B), 46.7 (CH2A), 38.6 (CHB), 37.6 (CHA), 30.9 (CH2 × 2), 26.6 (CH2), 25.8
(CH2 × 2). HRMS (ESI) calcd. for C18H20ON4Cl ([M+H]+) 343.13202, found
343.13139.
4.1.5. Synthesis of 2-(2-chlorophenyl)-N-(piperidin-4-ylmethyl)oxazolo[5,4-d]
pyrimidin-7-amine (21)
To a solution of compound 16 (0.2 mmol, 1 equiv) in 50 mL CH2Cl2 was added
TFA (2 mmol, 10 equiv). The mixture was stirred at room temperature for 3 h. At the
end of the reaction, the solvent was removed under reduced pressure and the residue
was dissolved in water and the solution was neutralised to pH 9 using 30% NaOH,
then the solution was extracted with ethyl acetate. The organic layer was washed with
brine and distilled water successively, dried over MgSO4 and evaporated under
reduced pressure to give compound 21. The title compound was obtained as a white
1
solid, mp > 270 °C, purity (HPLC): 96.8%. H NMR (300 MHz, DMSO-d6) δ 8.53 –
8.19 (m, 2H), 8.09 (d, J = 7.6 Hz, 1H), 7.71 (dd, J = 7.8, 1.6 Hz, 1H), 7.70 – 7.51 (m,
2H), 3.71 – 3.61 (m, 0.5H), 3.44 – 3.33 (m, 1.5H), 3.00 – 2.84 (m, 2H), 2.46 – 2.31
(m, 2H), 1.88 – 1.52 (m, 3H), 1.20 – 0.92 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ
164.1 (C), 156.2 (C), 155.7 (C), 155.1 (CHA), 154.8 (CHB), 133.3 (CH), 132.4 (C),
132.2 (CH), 131.7 (CH), 128.2 (CH), 125.8 (C), 116.1 (C), 48.9 (CH2B), 46.7 (CH2A),

ACCEPTED MANUSCRIPT
46.3 (CH2 × 2), 37.7 (CHB), 36.6 (CHA), 31.5 (CH2), 31.2 (CH2). HRMS (ESI) calcd.
for C17H19ON5Cl ([M+H]+) 344.12726, found 344.12650.
4.1.6. Synthesis of 2-(2-substituted phenyl)oxazolo[5,4-d]pyrimidin-7-ols (22-25)
To a solution of compound 11, 12, 13, or 14 (1 mmol) in 20 mL formic acid was
refluxed overnight. At the end of the reaction, after cooling, formic acid was removed
under reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2/ethyl acetate (6: 4 v/v) as eluent to give compound 22,
23, 24, or 25, respectively.
4.1.7. Synthesis of 7-chloro-2-(2-substituted phenyl)oxazolo[5,4-d]pyrimidines
(26-29)
To a solution of compound 22, 23, 24, or 25 (0.5 mmol, 1 equiv) in 30 mL
toluene were added DIPEA (0.75 mmol, 1.5 equiv) and POCl3 (1.5 mmol, 3 equiv)
successively at 0 °C. The mixture was stirred for 2 h. At the end of the reaction, the
solvent was removed under reduced pressure. The residue was purified by silica gel
column chromatography using CH2Cl2/ethyl acetate (95: 5 v/v) as eluent to give
compound 26, 27, 28, or 29, respectively.
4.1.8. Synthesis of 2-(2-substituted phenyl)-7-(4-substituted piperazinyl or
morpholine)oxazolo[5,4-d]pyrimidines (30, 32-42)
To a solution of compound 26, 27, 28, or 29 (0.15 mmol, 1 equiv) in 15 mL
DMF were added Cs2CO3 (0.3 mmol, 2 equiv) and piperazine derivative/morpholine

ACCEPTED MANUSCRIPT
(0.18 mmol, 1.2 equiv). The mixture was stirred and heated at 100 °C for 30 min. At
the end of the reaction, the solution was diluted with 50 mL saturated brine, and then
extracted twice with 30 mL ethyl acetate. The organic phase was washed twice with
50 mL distilled water and dried over MgSO4. The solvent was evaporated under
reduced pressure to give pure target compound directly or crude compound, which
was purified by silica gel column chromatography using ethyl acetate/methanol (98: 2
v/v) as eluent.
4.1.8.1. 7-(4-Methylpiperazin-1-yl)-2-phenyloxazolo[5,4-d]pyrimidine (32)
The title compound was obtained as a yellow solid, mp 136-137 °C, purity
1
(HPLC): 99.3%. H NMR (300 MHz, CDCl3) δ 8.36 (s, 1H), 8.24 – 8.13 (m, 2H),
7.56 – 7.46 (m, 3H), 4.55 – 4.06 (m, 4H), 2.64 – 2.52 (m, 4H), 2.38 (s, 3H). 13C NMR
(75 MHz, CDCl3) δ 165.5 (C), 157.5 (C), 154.1 (C), 153.7 (CH), 131.6 (CH), 128.9
(CH × 2), 127.3 (CH × 2), 126.5 (C), 116.9 (C), 55.0 (CH2 × 2), 46.2 (CH3), 45.1
(CH2 × 2). HRMS (ESI) calcd. for C16H18ON5 ([M+H]+) 296.15059, found 296.14967.
4.1.8.2. 7-(4-Ethylpiperazin-1-yl)-2-phenyloxazolo[5,4-d]pyrimidine (33)
The title compound was obtained as a yellow solid, mp 120-121 °C, purity
1
(HPLC): 99.3%. H NMR (300 MHz, CDCl3) δ 8.36 (s, 1H), 8.25 – 8.12 (m, 2H),
7.56 – 7.47 (m, 3H), 4.57 – 4.00 (m, 4H), 2.70 – 2.57 (m, 4H), 2.50 (q, J = 7.1 Hz,
2H), 1.15 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 165.5 (C), 157.5 (C),
154.1 (C), 153.7 (CH), 131.5 (CH), 128.9 (CH × 2), 127.3 (CH × 2), 126.5 (C), 116.8

ACCEPTED MANUSCRIPT
(C), 52.8 (CH2 × 2), 52.4 (CH2), 44.9 (CH2 × 2), 11.9 (CH3). HRMS (ESI) calcd. for
C17H20ON5 ([M+H]+) 310.16624, found 310.16501.
4.1.8.3. 2-(2-Fluorophenyl)-7-(4-methylpiperazin-1-yl)oxazolo[5,4-d]pyrimidine (34)
The title compound was obtained as a yellow solid, mp 142-143 °C, purity
1
(HPLC): 98.1%. H NMR (300 MHz, CDCl3) δ 8.38 (s, 1H), 8.19 – 8.11 (m, 1H),
7.56 – 7.46 (m, 1H), 7.33 – 7.27 (m, 1H), 7.25 – 7.19 (m, 1H), 4.54 – 4.09 (m, 4H),
2.57 (t, J = 5.0 Hz, 4H), 2.37 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 165.3 (C), 160.67
(C, d, J = 259.8 Hz), 154.2 (C), 154.0 (CH), 153.8 (C, d, J = 5.7 Hz), 133.1 (CH, d, J
= 8.5 Hz), 130.1 (CH), 124.5 (CH, d, J = 3.8 Hz), 117.1 (CH, d, J = 21.0 Hz), 116.6
(C), 115.0 (C, d, J = 10.6 Hz), 55.0 (CH2 × 2), 46.2 (CH3), 45.1 (CH2 × 2). HRMS
(ESI) calcd. for C16H17ON5F ([M+H]+) 314.14116, found 314.14037.
4.1.8.4. 7-(4-Ethylpiperazin-1-yl)-2-(2-fluorophenyl)oxazolo[5,4-d]pyrimidine (35)
The title compound was obtained as a yellow solid, mp 127-128 °C, purity
1
(HPLC): 99.4%. H NMR (300 MHz, CDCl3) δ 8.38 (s, 1H), 8.19 – 8.11 (m, 1H),
7.56 – 7.46 (m, 1H), 7.33 – 7.27 (m, 1H), 7.25 – 7.19 (m, 1H), 4.54 – 4.07 (m, 4H),
2.61 (t, J = 5.1 Hz, 4H), 2.50 (q, J = 7.2 Hz, 2H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ 165.3 (C), 160.7 (C, d, J = 259.9 Hz), 154.1 (C), 154.0 (CH),
153.8 (C, d, J = 5.6 Hz), 133.1 (CH, d, J = 8.5 Hz), 130.1 (CH), 124.5 (CH, d, J = 3.9
Hz), 117.1 (CH, d, J = 21.0 Hz), 116.6 (C), 115.0 (C, d, J = 10.4 Hz), 52.8 (CH2 × 2),
52.4 (CH2), 45.1 (CH2 × 2), 11.9 (CH3). HRMS (ESI) calcd. for C17H19ON5F ([M+H]+)
328.15681, found 328.15533.

ACCEPTED MANUSCRIPT
4.1.8.5.
7-(4-Methylpiperazin-1-yl)-2-(2-(trifluoromethyl)phenyl)oxazolo[5,4-d]pyrimidine
(36)
The title compound was obtained as a yellow solid, mp 137-138 °C, purity
1
(HPLC): 99.3%. H NMR (300 MHz, CDCl3) δ 8.39 (s, 1H), 8.19 – 8.09 (m, 1H),
7.93 – 7.83 (m, 1H), 7.77 – 7.59 (m, 2H), 4.57 – 3.99 (m, 4H), 2.58 (t, J = 5.0 Hz, 4H),
2.37 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 165.6 (C), 154.7 (C), 154.3 (CH, C),
132.0 (CH), 131.6 (CH), 131.1 (CH), 129.0 (C, q, J = 32.2 Hz), 127.4 (CH, q, J = 5.8
Hz), 125.3 (C), 123.3 (CF3, q, J = 273.7 Hz), 116.6 (C), 54.9 (CH2 × 2), 46.1 (CH3),
45.1 (CH2 × 2). HRMS (ESI) calcd. for C17H17ON5F3 ([M+H]+) 364.13797, found
364.13656.
4.1.8.6.
7-(4-Ethylpiperazin-1-yl)-2-(2-(trifluoromethyl)phenyl)oxazolo[5,4-d]pyrimidine (37)
The title compound was obtained as a yellow solid, mp 104-105 °C, purity
1
(HPLC): 98.1%. H NMR (300 MHz, DMSO-d6) δ 8.39 (s, 1H), 8.18 (d, J = 7.3 Hz,
1H), 8.02 (d, J = 7.3 Hz, 1H), 7.96 – 7.80 (m, 2H), 4.51 – 3.86 (m, 4H), 2.59 – 2.50
(m, 4H), 2.38 (q, J = 7.1 Hz, 2H), 1.03 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ 165.6 (C), 154.7 (C), 154.3 (CH, C), 132.0 (CH), 131.6 (CH), 131.1 (CH), 129.0 (C,
q, J = 32.2 Hz), 127.4 (CH, q, J = 5.8 Hz), 125.3 (C), 123.3 (CF3, q, J = 271.9 Hz),
116.6 (C), 52.7 (CH2 × 2), 52.4 (CH2), 45.2 (CH2 × 2), 11.9 (CH3). HRMS (ESI)
calcd. for C18H19ON5F3 ([M+H]+) 378.15362, found 378.15199.

ACCEPTED MANUSCRIPT
4.1.8.7.
1-(4-(2-(2-(Trifluoromethyl)phenyl)oxazolo[5,4-d]pyrimidin-7-yl)piperazin-1-yl)etha
none (38)
The title compound was obtained as a yellow solid, mp 145-146 °C, purity
1
(HPLC): 96.3%. H NMR (300 MHz, DMSO-d6) δ 8.42 (s, 1H), 8.19 (d, J = 6.7 Hz,
1H), 8.07 – 7.98 (m, 1H), 7.96 – 7.83 (m, 2H), 4.45 – 3.89 (m, 4H), 3.71 – 3.55 (m,
13
4H), 2.07 (s, 3H). C NMR (75 MHz, DMSO-d6) δ 169.0 (C=O), 165.7 (C), 155.1
(C), 154.6 (CH), 154.2 (C), 133.6 (CH), 132.7 (CH), 132.6 (CH), 127.7 (C, q, J =
31.9 Hz), 127.9 (CH, q, J = 5.5 Hz), 124.7 (C), 123.9 (CF3, q, J = 273.3 Hz), 116.2
(C), 45.7 (CH2), 44.9 (CH2 × 2), 41.0 (CH2), 21.7 (CH3). HRMS (ESI) calcd. for
C18H17O2N5F3 ([M+H]+) 392.13289, found 392.13206.
4.1.8.8. 2-(2-Chlorophenyl)-7-morpholinooxazolo[5,4-d]pyrimidine (39)
The title compound was obtained as a yellow solid, mp 155-156 °C, purity
(HPLC): 98.2%. 1H NMR (300 MHz, DMSO-d6) δ 8.40 (s, 1H), 8.12 (dd, J = 7.6, 1.9
Hz, 1H), 7.71 (dd, J = 7.9, 1.4 Hz, 1H), 7.67 – 7.53 (m, 2H), 4.50 – 3.88 (m, 4H),
3.76 (t, J = 4.8 Hz, 4H). 13C NMR (75 MHz, DMSO-d6) δ 165.4 (C), 155.2 (C), 154.5
(CH), 154.2 (C), 133.5 (CH), 132.5 (C), 132.1 (CH), 131.9 (CH), 128.3 (CH), 125.1
(C), 116.1 (C), 66.5 (CH2 × 2), 45.8 (CH2 × 2). HRMS (ESI) calcd. for C15H14O2N4Cl
([M+H]+) 317.07998, found 317.07944.
4.1.8.9. 2-(2-Chlorophenyl)-7-(4-methylpiperazin-1-yl)oxazolo[5,4-d]pyrimidine (40)