Design, synthesis and anti-acetylcholinesterase evaluation of some new pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives

Article abstract of DOI:10.1007/s00044-016-1576-0

The target new hybrid molecule types pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines phosphonates 4 and 2-(coumarin-3’’-yl)-7-phenylpyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines 5 were prepared via Michaelis–Arbuzov rearrangement (Arbuzov reaction) of pyrazolotriazolopyrimidines chloride 3a–c, with trialkyl phosphate and Knoevenagel reaction of 2-cyanomethyl derivatives 3d–f with salicylic aldehyde, respectively. The precursors 3 were obtained in two steps starting from aminopyrazole 1. Target compounds 4 and 5 were completely characterized by ¹H NMR, ¹³C NMR, ³¹P NMR, IR and HRMS. The anti-acetylcholinesterase activity of compounds 4 and 5 was evaluated, and results found indicated that they have possessed significant activities (IC₅₀?=?1.73–39.86?μM), and the preliminary SAR of these compounds was investigated.

Full text of DOI:10.1007/s00044-016-1576-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1576-0
ORIGINAL RESEARCH
Design, synthesis and anti-acetylcholinesterase evaluation of some
new pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives
Anis Romdhane1 Abderrahim Ben Said1 Maher Cherif1 Hichem Ben Jannet1
•
•
•
Received: 26 March 2015 / Accepted: 7 April 2016
Ó Springer Science+Business Media New York 2016
Abstract The target new hybrid molecule types pyra-
zolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines phosphonates
4 and 2-(coumarin-3’’-yl)-7-phenylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidines 5 were prepared via Michaelis–
Arbuzov rearrangement (Arbuzov reaction) of pyrazolo-
triazolopyrimidines chloride 3a–c, with trialkyl phosphate
and Knoevenagel reaction of 2-cyanomethyl derivatives
3d–f with salicylic aldehyde, respectively. The precursors
3 were obtained in two steps starting from aminopyrazole
1. Target compounds 4 and 5 were completely character-
ized by 1H NMR, 13C NMR, 31P NMR, IR and HRMS. The
anti-acetylcholinesterase activity of compounds 4 and 5
was evaluated, and results found indicated that they have
possessed significant activities (IC50 = 1.73–39.86 lM),
and the preliminary SAR of these compounds was
investigated.
triazoles and pyrimidines, as well as their condensed
derivatives, are very attractive targets from both a theo-
retical and a synthetic point of view. Moreover, they have
been the subject of many chemical and biological studies
on account of their pharmacological activity, such as anti-
inflammatory, analgesic (Amin et al., 2009), cytotoxicity,
antitumor (Rashad et al., 2010) and adenosine receptor
antagonists (Baraldi et al., 2012). In addition, some of them
have shown anti-acetylcholinesterase activity (Fig. 1a–c)
(Zhi et al., 2008).
On the other hand, diverse biological activity (Weiqin
et al., 2006; Mado et al., 2014) of heterocyclic phospho-
nates containing the P–C bond has for a long time attracted
considerable synthetic and pharmacological interest (Ali
et al., 2001). Some of them exert their biological action on
arthropods by attacking the system of neural transmission
and inhibiting the function of acetylcholinesterase (Fig. 1d)
(Fest and Schmidt 1973; Troev 2006). Thus, a large
number of new phosphonate derivatives have been pre-
pared hitherto with special attention to nitrogen hetero-
cyclic compounds (Francesca et al., 2001; Yanchang et al.,
2002; Francisco et al., 2003; Lise et al., 2007).
Keywords Aminopyrazole Á Phosphoric compounds Á
Coumarinic compounds Á Anti-acetylcholinesterase activity
Introduction
Furthermore, coumarins are a class of compounds,
which occupies a special role in nature. They have attracted
intense interest in recent years because of their diverse
pharmacological properties like anti-HIV (Ma et al., 2008),
anticoagulant (Kidane et al., 2004), antibacterial (Ap-
pendino et al., 2004), antioxidant (Kontogiorgis and Had-
jipavlou-Litina 2004), cytotoxic (Musa et al., 2008) and
particularly acetylcholinesterase inhibitors (Fig. 1e)
(Razavi et al., 2013; Nam et al., 2014).
The study of heterocyclic compounds such as five- and six-
membered rings containing nitrogen has a known consid-
erable development due to the revealing of their varied
effects in diverse domains. In this framework, pyrazoles,
& Anis Romdhane
anis_romdhane@yahoo.fr
1
´ ´
Laboratoire de Chimie Heterocyclique, Produits Naturels et
In addition, the synthesis of new hybrid molecules is a
new concept in the field of drug development based on the
association between pharmacophoric fragments of different
bioactive substances to produce new hybrid compounds
´
´
´
Reactivite (LR11SE39), Equipe: Chimie Medicinale et
Produits Naturels, Faculte des Sciences de Monastir,
´
´
Universite de Monastir, Avenue de l’Environnement,
5019 Monastir, Tunisia
123

Med Chem Res
Fig. 1 Previously reported
AChEI compounds
with improved efficiency and affinity. In this context and as
a continuation of our previous work on the synthesis of
new fused pyrimidine scaffolds (Rahmouni et al., 2014a,
b), we report here the synthesis of some new hybrid
molecules 4 and 5 bearing in their structures fragments
described as anti-acetylcholinesterase agents, as indicated
above, such as pyrazole, pyrimidine, triazole and phos-
phoric or coumarinic systems. Compounds 4 and 5 were
evaluated for their anti-acetylcholinesterase activity, and
preliminary SAR of these compounds was investigated
(Table 1).
Scheme 1 Synthetic route of compounds 2
dehydrocyclization to give triazolopyrazolopyrimidines
3a–f (Scheme 2).
We note that triazolopyrimidine derivatives, such com-
pounds 3, have generally been prepared, in two steps, via
reaction of a-functionalized iminoethers with hydrazine
followed by cyclocondensation reaction with one-carbon
cyclizing reagents, namely acids, acid chlorides, orthoe-
sters, CSI and DMFDMA (Romdhane et al., 2003, 2008;
Scheme 3).
Results and discussion
Chemistry
Our key intermediate was the a-functionalized iminoethers
type 2, which were synthesized from aminopyrazole 1
(Scheme 1), since such system constitutes a commonly
used building block for the construction of a variety of
polyheterocycles (Zaki 1998; El-Agordy et al., 2001).
For this purpose and as described in schemes 2, 4 and
5, our approach to the target systems 4 and 5 was firstly
started by the construction of the pyrazolotriazolopyrim-
idine skeleton type 3 via the intramolecular cyclocon-
densation reaction of 2. In fact, these intermediates, which
possess two reactive sites: a cyano group and an imidic
carbon, were made to react with appropriate acid hydra-
zide under ethanol reflux to give desired fused tricyclic
triazolopyrimidines. Plausible pathway involves two
successive nucleophilic additions of -NH2 group on the
imidic carbon and on the cyano function followed by
To access new pyrazolotriazolopyrimidine phosphonates
containing the P–C bond 4a–f, we thought about Michaelis–
Arbuzov rearrangement (Arbuzov reaction) which is known
as one of the most extensively investigated methods and is
widely used to prepare phosphonates, by formation of P–C
bond through reaction of an aryl/alkyl halide and trialkyl
phosphite (Kosolapov et al., 1950). In fact, formation of
new phosphonated compounds 4a–f in good yields
(Table 2) was carried out via the reaction of pyrazolotria-
zolopyrimidines chloride 3a–c, with trialkyl phosphite.
In order to determine the best conditions for the
preparation of pyrazolotriazolopyrimidines phosphonates
4, we examined the reaction under various conditions by
changing the solvent (toluene, xylene), and we found that
Table 1 Yields of pyrazolotriazolopyrimidines 3a–f
Compounds
R
R1
Yields (%)
3a
3b
3c
3d
3e
3f
H
Cl
76
82
74
68
80
82
Me
Et
H
Cl
Cl
CN
CN
CN
Me
Et
123

Med Chem Res
Scheme 2 Synthetic route of
compounds 3a–f
R1
O
R
CN
N
NH
NH
R
C
EtOH / Reflux
- EtOH
H2NHN
+
OEt
N
N
R1
N
N
O
N
N
2
2'
10
R1
R1
HO
1
2
N
9b
N
H
N 3
N 4
N
N
R
9
R
9a
5
8 N
N
N 6
N
6a
7 N
N
- H2O
1'
R = H, Me, Et
R1 = CN, Cl
2'
3'
6'
5'
3a-f
2''
4'
Scheme 3 Synthesis of
triazolopyrimidine derivatives,
in two steps
N
NH
N
CN
N
NH2
One - carbon
cyclising reagents
N
H2NNH2
N
Het.
Het.
Het.
OEt
N
N
Iminoether
Triazolopyrimidine
derivatives
Table 2 Yields of pyrazolotriazolopyrimidines dialkylphosphonates 4a–f
R
R2
Toluene/reflux
Time (h)/yield (%)
Xylene/reflux
Time (h)/yield (%)
P(OR2)3/reflux
Time (h)/yield (%)
4a
4b
4c
4d
4e
4f
H
Me
Et
48/35
48/30
48/38
48/36
48/40
48/31
24/42
24/47
24/50
24/46
24/52
24/45
6/88
7/90
6/92
7/98
6/96
7/94
H
Me
Me
Et
Et
Me
Et
Me
Et
the best yields (Table 2) were obtained when compounds
3a–c were reacted under reflux with an excess of trialkyl
phosphite used at the same time as a solvent. The reaction
was conducted until TLC indicated that the starting mate-
rials have been completely converted to products 4.
(J = 12.3–12.4 Hz), when R2 = Me, as a result of protons
of the methyl group coupled to the phosphorus atom and a
multiplet at 4.07–4.11 ppm, when R2 = Et, due to the cou-
pling of protons of the ethyl group to the phosphorus atom.
The 13C NMR spectra of these compounds were also in
agreement with the proposed structures. In fact, in addition
to the signals corresponding to the carbons introduced by the
intermediate 3, we observed a doublet at 27.5–28.2 ppm
with a very large coupling constant (161.3–161.5 Hz) as a
result of being directly bonded of C-10 to the phosphorus
atom and two doublets at 62.1–62.7 ppm (J = 24.3–24.4
Hz) and 161.1–162.2 ppm (J = 33.3–33.5 Hz) due to the
coupling of the phosphorus atom with the methoxy carbon
and C-2, respectively. For compounds 4b,d,e a new doublet
at 16.5–16.8 ppm (J = 23.4–23.6 Hz) was observed
The structures of compounds 4 have been assigned from
their analytical data, IR, 1H NMR, 13C NMR, 31P NMR and
mass spectrometry (ES-HRMS). In fact, the 1H NMR spectra
of compounds 4 showed, in addition to the signals corre-
sponding to the protons introduced by compounds 3,
a new doublet (J = 21.3–21.6 Hz) at 3.58–3.66 ppm
attributable to the methylene group (H-10) bearing the
phosphoryl moiety. The large splitting was a result of a
2J coupling of being directly bonded to the phosphorus. We
also detected the presence of a doublet at 3.82–3.86 ppm
123

Med Chem Res
resulting of the coupling between the methyl carbon of the
ethoxy group and the phosphorus atom. The 31P NMR
spectra of compounds 4a–f showed a signal corresponding
to the phosphonyl group at 21.6–24.6 ppm. The infrared
spectra also revealed the presence of an absorbance due to
the P = O band at 1230–1238 cm-1 (Scheme 4).
The reaction was conducted until TLC indicated that the
starting materials have been completely converted to products
5. The IR spectra of these compounds revealed the presence
of an absorbance band due to C = O at 1740–1747 cm-1 and
the disappearance of any absorption frequency in the CN
region. Further, the 1H NMR spectra of compounds 5 showed
the disappearance of the singlet relative to the methylene
protons (H-10) of compounds 3d–f (4,08–4,10 ppm) and the
appearance of new signals, attributable to protons of the
coumarin moiety, of which chemical shifts and multiplicities
In the second part of this study, we have converted the
intermediates 2 into pyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidine-2-acetonitriles 3d–f by reaction with cya-
noacetic acid hydrazide, which was obtained by addition of
hydrazine hydrate to ethyl cyanoacetate in ethanol with
stirring at 0 °C, under reflux of toluene (Gorobets et al.,
2004) (Scheme 2), in order to subsequently treat com-
pounds 3d–f with salicylaldehyde via Knoevenagel con-
densation reaction, to access to the coumarin derivatives 5
(Scheme 5).
are in agreement with the proposed structure. Analysis of 13
C
NMR spectra of these compounds showed, in addition to the
signals relative to the coumarin moiety carbons, the disap-
pearance of two signals in the region of 18.8–19.1 ppm and
116.8–117.5 ppm attributable to –CH2–CN and –CN in
compounds 3d–f, respectively.
The formed triazolopyrimidines 3d–f were characterized
1
The ES-HRMS showed essentially the correct proto-
nated molecular peak [M ? H]? for all examined com-
pounds 3–5.
by their IR, H and 13C spectra. In fact, the IR spectra of
compounds 3d–f showed an absorbance band at
1
2225–2230 cm-1 due to the cyano group. The H NMR
spectra of compounds 3d–f showed, in addition to the
signals of protons introduced by the intermediate 2, the
appearance of a new signal due to the methylene group
(–CH2–CN) at 4.08–4.10 ppm. The 13C NMR of 3d–f
showed the appearance of new signals at 18.8–19.1,
116.8–117.5 and 164.5–165.1 ppm relative to –CH2–CN,
–CN and C2, respectively.
Biological activity
Anti-acetylcholinesterase activity
Inhibition of acetylcholinesterase (AChE), the key enzyme
in the breakdown of acetylcholine, is considered one of the
treatment strategies against several neurological disorders
such as Alzheimer’s disease, senile dementia, ataxia and
myasthenia gravis (Orhan et al., 2006; Howes et al., 2003).
Only compounds 4a-f and 5a-c were analysed on what
concerns their acetylcholinesterase inhibition activity
(Table 3) using an adaptation of the method described in
the literature (Ferreira et al., 2006).
The 2-cyanomethyl derivatives 3d–f were treated with
salicylic aldehyde, via Knoevenagel reaction (Bylov et al.,
1999; Kovalenko et al., 2000); under reflux of ethanol, an
addition product formed 30, from which reaction of an
aqueous solution of hydrochloric acid gave the 2-
(coumarin-300-yl)-14(Aryl)-14H-naphto[2,1-b]pyrano[3,2-
e][1,2,4]triazolo[1,5-c] pyrimidines 5a-c (scheme 5).
The results indicated in Table 3 showed that all the
tested heterocycles gave significant activity. Compared to
those given in the literature for crude pure products (Mata
et al., 2007), we can say that the prepared derivatives 4 and
5 are considered good acetylcholinesterase inhibitors.
Moreover, it has been found that the methylated phos-
phonate derivatives 4a,c,e were more active than their
ethylated analogous 4b,d,f.
The greatest inhibitory activity was exhibited by the
phosphonated compound 4a (R = H, R2 = Me, IC50
=
1.73 0.05 lM), which was found almost twenty-three
times more active than (E)-anethole (IC50 = 39.86
0.1 lM) used as a standard compound, but it is about two
times less active than tacrine (IC50 = 0.86 0.02 lM), a
drag used to treat Alzheimer.
On the other hand, it has been shown that the activity of
these derivatives depends on the nature of R. In 4a,c,e
(R2 = Me), the acetylcholinesterase inhibition decreases
from R = H (4a) to R = Et (4e). The same phenomena has
been observed with 4b,d,f (R2 = Et).
Scheme 4 Synthetic route of pyrazolotriazolopyrimidine phospho-
nates 4a–f
123

Med Chem Res
Scheme 5 Synthetic route of
coumarinic
pyrazolotriazolopyrimidine
derivatives 5a–c
CN
N
O
HO
R
N
O
N
N
N
N
NH
EtOH / piperidine
Reflux
N
R
N
N
3
d-
f
N
6"
7"
N
N
5"
4"a
8"
8"a
R = H, Me, Et
3'
d
-f
O1"
4"
2"
3"
N1
O
2
9b
R
9
N3
9a
HCl, H2O
N 4
8 N
5
5a (R=H), Rdt:78%
5b (R=Me), Rdt:86%
5c (R=Et), Rdt:82%
7N
N 6
6a
1'
2'
3'
6'
5'
5a-c
4'
attached to the phosphorus atom (Ahmed et al., 2013; Gon-
c¸alves et al., 2011a; Bajqar 2004;), is no longer able to par-
ticipate in the hydrolysis of ACh. The anti-AChE potential of
phosphonate compounds 4a–f can be explained by the pos-
sible intervention of the serine from the AChE via its hydroxy
group as indicated in Scheme 6.
Table 3 Acetylcholinesterase inhibition capacity represented by IC50
(lM)a of compounds 4, 5 and standard compounds ((E)-anethole and
tacrine)
Compound
Acetylcholinesterase inhibition
capacity represented by IC50 (lM)
4a
1.73 0.05
11.71 0.18
4.65 0.20
15.32 0.22
6.50 0.20
19.03 0.13
10.31 0.17
26.54 0.21
36.54 0.20
39.86 0.10
0.86 0.02
This proposed mechanism in addition to the probable
steric hindrance due to the ethyl group (R2 = Et) in the
phosphonate moiety, compared to that induced by the
methyl group, may explain the relatively high activity of
the compounds 4a,c,e (R2 = Me).
4b
4c
4d
4e
4f
On the other hand, and according to some previous
studies (Alipour et al., 2012; Razavi et al., 2013), we can
explain the inhibition activity of coumarin derivatives 5a–
c, by formation of an additional p-p interaction between
the coumarin moiety and some actives sites of the AChE
resulting more stability of the formed ligand which pre-
vents more hydrolysis of ACh.
5a
5b
5c
(E)-Anethole
Tacrine
a
Averages SD were obtained from three different experiments
IC50 values represent the concentration of inhibitor required to
decrease enzyme activity by 50 % and are the mean of two inde-
pendent measurements, each performed in triplicate
All these findings allowed to note that the covalent bond
formation between the Ser200 hydroxyl group of AChE
and the phosphorus atom in compounds 4a–f is more
efficient than the p–p interaction between AChE and the
coumarin system in derivatives 5a–c. However, the nature
of coumarin attached to the triazole may also explain this
difference in activity.
For coumarinic compounds, it has been shown also that
the activity of these derivatives depends on the nature of R;
in fact, the acetylcholinesterase inhibition decreases from
R = H (5a) to R = Et (5c). Compound 5c (R = Et) was
found to be the less inhibitor (IC50 = 36.54 0.2 lM).
Inhibition of AChE by compounds 4a–f can be explained
by forming covalent adducts with the reactive Ser200
hydroxyl group, which prevents ACh hydrolysis. In fact, the
serine hydroxyl group, deactivated by the phosphorylated
moiety of varying degrees that depends on the groups
Conclusion
In conclusion, this work reports the synthesis of new hybrid
compounds
types
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidines phosphonate 4 and coumarinic pyrazolo[4,3-
123

Med Chem Res
Ethyl N-(4-cyano-1-phenyl-1H-pyrazol-5-
yl)formimidate (2a)
e]-1,2,4-triazolo[1,5-c]pyrimidines 5 via Michaelis–Arbu-
zov rearrangement (Arbuzov reaction) of pyrazolotria-
zolopyrimidines chloride 3a–c, with trialkyl phosphate and
Knoevenagel reaction of 2-cyanomethyl derivatives 3d–f
with salicylic aldehyde, respectively. Precursors 3 were
obtained in two steps starting from aminopyrazole 1. The
anti-acetylcholinesterase activity of the synthesized hybrid
compounds 4 and 5 was evaluated. It has been found that
the methylated phosphonates 4a,c,e were the most active
ones (IC50 = 1.73–6.50 lM).
White solid; Yield: 65 %; m.p.: 120–122 °C (ethanol); IR
(KBr, cm-1) m: 1620 (–C = N), 2228 (–CN); H NMR
1
(CDCl3, 300 MHz): d 1.34 (t, 3H, J = 7.6 Hz), 4.31 (q,
2H, J = 7.6 Hz),7.33–7.60 (m, 5H, Harom), 8.23 (s, 1H,
H4), 8.72 (s, 1H, H7); 13C NMR (DMSO-d6, 75 MHz): d
0
13.5 (C8), 64.3 (C9), 82.4 (C4), 115.3 (CN), 124.6 (C2 ,6 ),
0
0
0
0
0
127.3 (C4 ), 129.7 (C3 ,5 ), 138.3 (C1 ), 150.1 (C3), 151.4
(C5), 160.6 (C7).
Finally, we can say that the prepared derivatives 4 and 5
are considered good acetylcholinesterase inhibitors; this
encouraged us to pursue this study in order to more explain
the preliminary structure–activity relationship (SAR) of
these compounds.
Ethyl N-(4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-
yl)formimidate (2b)
White solid; Yield: 65 %; m.p.: 120–122 °C (ethanol); IR
1
(KBr, cm-1) m: 1625 (–C = N), 2225 (–CN); H NMR
(CDCl3, 300 MHz): d 1.35 (t, 3H, J = 7.5 Hz), 2.39 (s,
3H), 4.31 (q, 2H, J = 7.5 Hz),7.31–7.61 (m, 5H, Harom),
8.36 (s, 1H, H7); 13C NMR (DMSO-d6, 75 MHz): d 13.1
Experimental section
Chemistry
0
(C8), 13.9, 64.2 (C9), 81.7 (C4), 114.7 (CN), 123.9 (C2 ,6 ),
0
0
127.6 (C4 ), 128.8 (C3 ,5 ), 138.1 (C1 ), 150.0 (C3), 151.7
0
0
0
All reactions were monitored by TLC using aluminium
sheets of SDS silica gel 60 F254, 0.2 mm. Melting tem-
peratures were determined on an electrothermal 9002
apparatus and were reported uncorrected. NMR spectra
were recorded on a Bruker AC-300 spectrometer at
300 MHz (1H), 75 MHz (13C) and 120 MHz (31P). All
chemical shifts were reported as d values (ppm) relative to
residual non-deuterated solvent. IR spectra were recorded
on FTS-6000 BIO-RAD apparatus. Mass spectra were
obtained with ESI-TOF (LCT, Waters) using the reflectron
mode in the positive ion mode. The starting materials 1 and
2 were prepared according to the literature (Rashad et al.,
2005; Al-Afaleq et al., 2001).
(C5), 160.3 (C7).
Ethyl N-(4-cyano-3-ethyl-1-phenyl-1H-pyrazol-5-
yl)formimidate (2c)
White solid; Yield: 65 %; m.p.: 120–122 °C (ethanol); IR
1
(KBr, cm-1) m: 1625 (–C = N), 2228 (–CN); H NMR
(CDCl3, 300 MHz): d 1.32 (t, 3H, J = 7.3 Hz), 1.34 (t, 3H,
J = 7.6 Hz), 2.70 (q, 2H, J = 7.3 Hz), 4.45 (q, 2H,
J = 7.6 Hz),7.35–7.63 (m, 5H, Harom), 8.46 (s, 1H, H7);
13C NMR (DMSO-d6, 75 MHz): d 14.2 (C9), 14.8, 64.5,
0
65.1 (C8), 82.4 (C4), 116.3 (CN), 124.7 (C2 ,6 ), 127.6 (C4 ),
0
0
0
0
0
129.5 (C3 ,5 ), 138.5 (C1 ), 150.3 (C3), 151.1 (C5), 160.2
(C7).
General procedure for the synthesis of ethyl N-(4-
cyano-1-phenyl-1H-pyrazol-5-yl) formimidate 2a–c
General procedure for the synthesis of 2-
chloromethyl-7-phenylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidines 3a–c
A mixture of compound 1 (10 mmol) and triethylortho-
formate (3 mL) in acetic anhydride (25 mL) was refluxed
for 1 h. After cooling, the precipitated product was filtered
off and washed thoroughly with ethanol and recrystallized
from ethanol to yield 2 as white solid.
To a solution of iminoethers 2 (1 mmol) in ethanol (30 mL),
2-chloroacetohydrazide hydrochloride (1.1 mmol) and
Scheme 6 Blocking-up
reaction of serine hydroxyl by
the phosphorylated moiety
O O R2
P
O O R2
P
H2
NH
CH
O
N
O
R2
N
R
C
R
O
N
N
+
R2OH
N
C
N
HN CHC NH
+
O
N
N
HN
CH2
OH
N
N
N
N
Ph
Ph
123

Med Chem Res
7-Phenylpyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidine-2-acetonitrile (3d)
triethylamine (1.1 mmol) were added. The reaction mixture
was boiled at reflux for 4 h, and after cooling, it is poured
into ice water (50 mL) to give a white solid which was
crystallized from ethanol.
White solid; Yield: 68 %; m.p.: 172–174 °C (ethanol); IR
(KBr, cm-1) m: 2225 (–CN); 1H NMR (CDCl3, 300 MHz):
d 4.08 (s, 2H, -CH2-CN), 7.42–8.13 (m, 5H, Harom), 8.60 (s,
1H, H9), 9.13 (s, 1H, H5); 13C NMR (DMSO-d6, 75 MHz):
d 18.8 (–CH2–CN), 104.4 (C9a), 117.3 (–CN), 122.2
2-chloromethyl-7-phenylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine (3a)
0
0
0
0
0
0
(C2 ,6 ), 127.4 (C4 ), 129.5 (C3 ,5 ), 138.1 (C1 ), 138.5 (C5),
146.5 (C9b), 149.0 (C6a), 149.3 (C9), 164.6 (C2), ES-HRMS
[M ? H]? calcd for (C14H10N7)?: 276.0916, found:
276.0908.
White solid; Yield: 76 %; m.p.: 184–186 °C (ethanol); IR
(KBr, cm-1) m: 1620 (–C = N); 1H NMR (CDCl3,
300 MHz): d 4.85 (s, 2H, –CH2Cl), 7.45–8.15 (m, 5H,
H
arom), 8.58 (s, 1H, H9), 9.15 (s, 1H, H5); 13C NMR
(DMSO-d6, 75 MHz): d 37.8 (–CH2Cl), 104.4 (C9a), 122.4
0
0
0
0
0
0
9-Methyl-7-phenylpyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidine-2-acetonitrile (3e)
(C2 ,6 ), 127.4 (C4 ), 129.5 (C3 ,5 ), 138.1 (C1 ), 138.5 (C5),
146.5 (C9b), 149.0 (C6a), 149.3 (C9),164.6 (C2), ES-HRMS
[M ? H]? calcd for (C13H10ClN6)?: 285.0577, found:
285.0569.
White solid; Yield: 80 %; m.p.: 176–178 °C (ethanol); IR
1
(KBr, cm-1) m: 2228 (-CN); H NMR (CDCl3, 300 MHz):
d 2.91 (s, 3H, –CH3), 4.10 (s, 2H, –CH2–CN), 7.47–8.21
(m, 5H, Harom), 9.20 (s, 1H, H5); 13C NMR (DMSO-d6,
75 MHz): d 14.3 (–CH3), 18.8 (–CH2–CN), 104.2 (C9a),
2-chloromethyl-9-methyl-7-phenylpyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidine (3b)
0
117.5 (–CN), 122.3 (C2 ,6 ), 126.5 (C4 ), 129.2 (C3 ,5 ),
0
0
0
0
White solid; Yield: 82 %; m.p.: 192-194 °C (ethanol); IR
(KBr, cm-1) m: 1615 (–C = N); 1H NMR (CDCl3,
300 MHz): d 2.91 (s, 3H, –CH3), 4.83 (s, 2H, –CH2Cl),
7.47–8.21 (m, 5H, Harom), 9.20 (s, 1H, H5); 13C NMR
(DMSO-d6, 75 MHz): d 14.1 (–CH3), 37.6 (–CH2Cl), 103.9
0
138.4 (C1 ), 139.3 (C5), 145.3 (C9b), 148.3 (C6a), 149.7
(C9), 164.4 (C2), ES-HRMS [M ? H]? calcd for
(C15H12N7)?: 290.1074, found: 290.1066.
0
0
0
0
0
0
9-Ethyl-7-phenylpyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidine-2-acetonitrile (3f)
(C9a), 121.9 (C2 ,6 ), 126.8 (C3 ), 128.9 (C3 ,5 ), 138.2 (C1 ),
138.8 (C5), 145.9 (C9b), 148.9 (C6a), 149.1 (C9), 165.1 (C2),
ES-HRMS [M ? H]? calcd for (C14H12ClN6)?: 299.0734,
found: 299.0740.
White solid; Yield: 82 %; m.p.: 180–182 °C (ethanol); IR
(KBr, cm-1) m: 2230 (–CN); 1H NMR (CDCl3, 300 MHz):
d 1.54 (t, 3H, J = 7.5 Hz), 3.24 (q, 2H, J = 7.5 Hz), 4.10
(s, 2H, –CH2–CN), 7.17-8.21 (m, 5H, Harom), 9.17 (s, 1H,
H5); 13C NMR (DMSO-d6, 75 MHz): d 12.9 (–CH2CH3),
18.8 (–CH2–CN), 22.3 (–CH2CH3), 102.3 (C9a), 116.8
2-chloromethyl-9-ethyl-7-phenylpyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidine (3c)
White solid; Yield: 74 %; m.p.: 196–198 °C (ethanol); IR
(KBr, cm-1) m: 1617 (–C = N); 1H NMR (CDCl3,
300 MHz): d 1.54 (t, 3H, J = 7.5 Hz), 3.24 (q, 2H,
J = 7.5 Hz), 4.87 (s, 2H, –CH2Cl), 7.36–8.10 (m, 5H,
0
0
0
0
0
0
(–CN), 122.6 (C2 ,6 ), 128.1 (C4 ), 129.8 (C3 ,5 ), 136.5 (C1 ),
137.3 (C5), 146.7 (C9b), 148.7 (C6a), 150.7 (C9), 165.7(C2),
ES-HRMS [M ? H]? calcd for (C16H14N7)?: 304.1563,
found: 304.1557.
H
arom), 9.14 (s, 1H, H5); 13C NMR (DMSO-d6, 75 MHz): d
0
12.9, 22.0, 37.4 (–CH2Cl), 102.5 (C9a), 122.3 (C2 ,6 ), 127.5
0
0
0
0
0
General procedure for the synthesis
of pyrazolotriazolopyrimidine dialkyl phosphonates
4a–f
(C4 ), 129.3 (C3 ,5 ), 138.2 (C1 ), 138.4 (C5), 146.4 (C9b),
149.1 (C6a), 149.2 (C9),164.5 (C2), ES-HRMS [M ? H]?
calcd for (C15H14ClN6)?: 313.0890, found: 313.0899.
In a typical procedure, the solution of pyrazolotriazolopy-
rimidine 3a–c (1 mmol) and an excess of trialkylphosphite
(10 mL) was refluxed for 6 h. The reaction evolution was
checked by TLC. When all the starting material was con-
sumed, the mixture was cooled to room temperature, and
then, the precipitate formed was filtered, dried and purified
by silica gel column chromatography eluted with petro-
leum ether–ethyl acetate (7:3).
General procedure for the synthesis of 7-
phenylpyrazolo[3,2-e][1,2,4]triazolo[1,5-
c]pyrimidine-2-acetonitriles 3d–f
A mixture of 2 (1 mmol) and cyanoacetic hydrazide
(1.2 mmol) in absolute ethanol (20 mL) was refluxed for
10 h; after cooling, the precipitated product was filtered off
and crystallized from ethanol to give 3d–f.
123

Med Chem Res
Dimethyl ((7-phenyl-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)methyl)phosphonate (4a)
Diethyl ((9-methyl-7-phenyl-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)methyl)phosphonate (4d)
White solid; Yield: 88 %; mp 248–250 °C; IR (KBr, cm-1
)
White solid; Yield: 98 %; mp 258-260 °C; IR (KBr, cm-1
m: 1622 (C = N), 1235 (P = O), 975 (P–O–C); H NMR
)
1
1
m: 1625 (C = N), 1235 (P = O), 975 (P–O–C); H NMR
2
(300 MHz, DMSO-d6): d 3.62 (d, JP–H = 21.3 Hz, 2H,
(300 MHz, DMSO-d6): d 1.24 (t, JH–H = 7.3 Hz, 6H, 2 P–
O–CH2–CH3), 2.91 (s, 3H, –CH3), 3.66 (d, JP–H
2
H10), 3.86 (d, JP–H = 12.3 Hz, 6H,
2
P–O–CH3),
7.48–8.09 (m, 5Harom.), 8.72 (s, 1H, H9), 9.65 (s, 1H, H5);
= 21.6 Hz, 2H, H10), 4.11 (m, 4H, 2 P–O–CH2–CH3),
7.44–8.09 (m, 5Harom.), 9.68 (s, 1H, H5); 13C NMR
13C NMR (75 MHz, DMSO-d6): d 27.6 (d, JP–C
161.3 Hz, C10), 62.4 (d, JP–C = 24.3 Hz, P–O–CH3),
=
1
2
3
(75 MHz, DMSO-d6): d 14.7 (–CH3), 16.5 (d, JP–
= 23.6 Hz, P–O–CH2–CH3), 27.5 (d, 1JP–C = 161.5 Hz,
0
0
0
0
0
103.5 (C9a), 122.6 (C2 ,6 ), 128.1 (C4 ), 129.7 (C3 ,5 ), 133.7
C
2
C10), 62.7 (d, JP–C = 24.4 Hz, P–O–CH2–CH3), 104.1
0
(C1 ), 138.4 (C5), 140.6 (C9b), 146.2 (C6a), 148.1 (C9),
2
161.2 (d, JP–C = 33.3 Hz, C2); 31P NMR (121 MHz,
(C9a), 122.3 (C2 ,6 ), 128.1 (C4 ), 129.7 (C3 ,5 ), 133.6 (C1 ),
138.7 (C5), 141.0 (C9b), 146.2 (C6a), 148.3 (C9), 161.6 (d,
2JP–C = 33.5 Hz, C2); 31P NMR (121 MHz, CDCl3): d
22,0; ES-HRMS [M ? H]? calcd. For (C18H22N6O3P)?:
401.1413; found: 401.1406.
0
0
0
0
0
0
CDCl3): d 24,6; ES-HRMS [M ? H]? calcd. For (C15
H16N6O3P)?: 359.0943; found: 359.0937.
Diethyl ((7-phenyl-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)methyl)phosphonate (4b)
Diethyl ((9-methyl-7-phenyl-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)methyl)phosphonate (4e)
White solid; Yield: 90 %; mp 253–255 °C; IR (KBr, cm-1
)
1
m: 1620 (C = N), 1238 (P = O), 978 (P–O–C); H NMR
(300 MHz, DMSO-d6): d 1.23 (t, JH–H = 7.2 Hz, 6H, 2 P–
O–CH2–CH3), 3.64 (d, JP–H = 21.3 Hz, 2H, H10), 4.07
White solid; Yield: 96 %; mp 268-270 °C; IR (KBr, cm-1
)
2
1
m: 1620 (C = N), 1232 (P = O), 977 (P–O–C); H NMR
(300 MHz, DMSO-d6): d 1.51 (t, 3H, J = 7.5 Hz), 3.18 (q,
2H, J = 7.5 Hz), 3.64 (d, 2JP–H = 21.6 Hz, 2H, H10), 3.82
(d, JP–H = 12.3 Hz, 6H, 2 P–O–CH3), 7.52–8.11 (m,
5Harom.), 9.62 (s, 1H, H5); 13C NMR (75 MHz, DMSO-d6):
d 11.7, 21.7, 28.2 (d, 1JP–C = 161.3 Hz, C10), 62.2 (d, 2JP–
(m, 4H, 2 P–O–CH2–CH3), 7.41–8.11 (m, 5Harom.), 8.73 (s,
1H, H9), 9.70 (s, 1H, H5); 13C NMR (75 MHz, DMSO-d6):
d 16.7 (d, 3JP–C = 23.4 Hz,P–O–CH2–CH3), 28.1 (d, 1JP–C
2
= 161.5 Hz, C10), 62.8 (d, JP–C = 24.4 Hz, P–O–CH2–
0
CH3), 103.9 (C9a), 123.5 (C2 ,6 ), 128.0 (C4 ), 130.1 (C3 ,5 ),
0
0
0
0
0
0 0
= 24.3 Hz, P–O–CH3), 103.4 (C9a), 122.8 (C2 ,6 ), 127.9
(C4 ), 130.1 (C3 ,5 ), 133.2 (C1 ), 137.9 (C5), 141.1 (C9b),
134.7 (C1 ), 138.8 (C5), 141.3 (C9b), 146.4 (C6a), 149.3
C
(C9), 161.4 (d, 2JP–C = 33.3 Hz, C2); 31P NMR (121 MHz,
CDCl3): d 21,8; ES-HRMS [M ? H]? calcd. For (C17
H20N6O3P)?: 387.1256; found: 387.1248.
0
0
0
0
2
146.4 (C6a), 147.9 (C9), 161.4 (d, JP–C = 33.3 Hz, C2);
31P NMR (121 MHz, CDCl3):
d 24,3; ES-HRMS
[M ? H]? calcd. For (C17H20N6O3P)?: 387.1256; found:
Dimethyl ((9-methyl-7-phenyl-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)methyl)phosphonate (4c)
3871248.
Diethyl ((9-ethyl-7-phenyl-7H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)methyl)phosphonate (4f)
White solid; Yield: 92 %; mp 260-262 °C; IR (KBr, cm-1
)
1
m: 1618 (C = N), 1234 (P = O), 973 (P–O–C); H NMR
(300 MHz, DMSO-d6): d 2.87 (s, 3H, –CH3), 3.61 (d, 2JP–H
= 21.4 Hz, 2H, H10), 3.84 (d, JP–H = 12.4 Hz, 6H, 2 P–O–
White solid; Yield: 94 %; mp 266–268 °C; IR (KBr, cm-1
)
1
m: 1625 (C = N), 1230 (P = O), 975 (P–O–C); H NMR
(300 MHz, DMSO-d6): d 1.25 (t, JH–H = 7.3 Hz, 6H, 2 P–
O–CH2–CH3), 1.46 (t, 3H, J = 7.5 Hz), 3.10 (q, 2H,
J = 7.5 Hz), 3.63 (d, 2JP–H = 21.6 Hz, 2H, H10), 4.10 (m,
4H, 2 P–O–CH2–CH3), 7.30–8.10 (m, 5Harom.), 9.61 (s,
1H, H5); 13C NMR (75 MHz, DMSO-d6): d 13.8, 16.6 (d,
CH3), 7.43-8.12 (m, 5Harom.), 9.63 (s, 1H, H5); 13C NMR
1
(75 MHz, DMSO-d6): d 14.3 (-CH3), 28.1 (d, JP–C
161.4 Hz, C10), 62.2 (d, JP–C = 24.5 Hz, P-O-CH3),
=
2
0
103.8 (C9a), 122.8 (C2 ,6 ), 128.3 (C4 ), 129.5 (C3 ,5 ), 133.4
0
0
0
0
0
(C1 ), 138.6 (C5), 141.1 (C9b), 146.3 (C6a), 149.2 (C9),
2
162.1 (d, JP–C = 33.4 Hz, C2); 31P NMR (121 MHz,
3JP–C = 23.6 Hz, P–O–CH2–CH3), 26.3, 28.1 (d, JP–
1
CDCl3): d 24,5; ES-HRMS [M ? H]? calcd. For (C16
= 161.3 Hz, C10), 62.5 (d, 2JP–C = 24.3 Hz, P–O–CH2–
C
H18N6O3P)?: 373.1100; found: 373.1108.
0
CH3), 103.1 (C9a), 122.0 (C2 ,6 ), 127.5 (C4 ), 129.7 (C3 ,5 ),
0
0
0
0
123

Med Chem Res
0
138.5 (C1 ), 140.4 (C5), 142.9 (C9b), 146.5 (C6a), 148.5
1.52 (t, 3H, J = 7.5 Hz), 3.22 (q, 2H, J = 7.5 Hz),
7.43–8.13 (m, 8H, Harom), 8.41 (d, 1H, Harom., J = 8.1 Hz),
(C9), 161.4 (d, 2JP–C = 33.3 Hz, C2); 31P NMR (121 MHz,
CDCl3): d 23,3; ES-HRMS [M ? H]? calcd. For (C19
H24N6O3P)?: 415.1569; found: 415.1563.
8.71 (s, 1H, H9), 9.09 (s, 1H, H5), 9.31 (s, 1H, H4 ); 13C
0
NMR (DMSO-d6, 75 MHz): d 12.4, 22.3, 104.8 (C9a),
00
0
0
00
00
121.1 (C8 ), 122.5 (C2 ,6 ), 123.1 (C4a ), 124.7 (C6 ), 127.1
00 0 00 00 0 0
(C5 ), 127.8 (C4 ), 128.5 (C7 ), 129.8 (C3 ), 130.3 (C3 ,5 ),
General procedure for the synthesis of 2-(coumarin-
300-yl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine
5a-c
0
138.6 (C1 ), 139.3 (C5), 146.3 (C4 ), 146.6 (C9b), 150.2
00
0
(C6a), 151.2 (C9), 152.8 (C8a ), 165.3 (C2), 166.8 (CO). ES-
HRMS [M ? H]? calcd for (C23H17N6O2)?: 409.1327,
A mixture of 2-cyanomethyl derivatives 3d–f (1 mmol)
and salicylic aldehyde (1 mmol) in ethanol (20 mL) was
refluxed for 1 h in the presence of few drops of piperidine.
The intermediate 30d–f obtained after filtration was reacted
with a mixture of ethanol/water/concentrated hydrochloric
acid (20:2:2, v:v:v) at reflux for 3 h, and after cooling to
room temperature, the precipitated product was filtered off
and recrystallized from ethanol to give 5a-c.
found: 409.1321.
Biological activity
Determination of AChE inhibitory activity
Cholinesterase (ChE) inhibitory activity was measured
using Ellman’s method (Ellman et al., 1961), with modi-
fications (Moyo et al., 2010). In this study, 50 lL of 0.1 M
sodium phosphate buffer (pH 8.0), 25 lL of AChE solu-
tion, 25 lL of each compound, and 125 lL of DTNB
[5,50—dithiobis (2-nitrobenzoic acid)] were added in a
96-well microplate and incubated for 15 min at 25 C. All
compounds were re-suspended in the DMSO followed by
dilution in the buffer so that the DMSO does not exceed
1 %. 25 lL of a solution of acetylthiocholine iodide was
added, and the final mixture incubated, for 15 min, at 25 C.
The hydrolysis of acetylthiocholine iodide was monitored
by the formation of the yellow 5-thio-2-nitrobenzoate
anion as a result of the reaction of DTNB with thiocholi-
nes, catalysed by enzymes at a wavelength of 412 nm. The
concentration of the compounds which caused 50 % inhi-
bition of the AChE activity (IC50) was calculated by
nonlinear regression analysis. The percentage of inhibition
was calculated from (1 to S/E) 9 100, where E and S were
the respective enzyme activity without and with the test
samples, respectively.
2-(Coumarin-300-yl)-7-phenylpyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine (5a)
White solid; Yield: 78 %; m.p.: 272–274 °C (ethanol); IR
(KBr, cm-1) m: 1740 (CO); 1H NMR (CDCl3, 300 MHz): d
7.40–8.10 (m, 8H, Harom), 8.21 (d, 1H, Harom., J = 8.1 Hz),
8.62 (s, 1H, H9), 9.11 (s, 1H, H5), 9.21 (s, 1H, H4 ); 13C
00
00
NMR (DMSO-d6, 75 MHz): d 103.7 (C9a), 121.7 (C8 ),
0
121.8 (C2 ,6 ), 122.7 (C4a’’), 125.7 (C6 ), 126.8 (C5 ), 127.1
0
00
00
0
00
00
0
0
0
(C4 ), 128.4 (C7 ), 129.3 (C3 ), 130.3 (C3 ,5 ), 137.8 (C1 ),
00
139.2 (C5), 146.1 (C4 ), 147.2 (C9b), 149.2 (C6a), 149.5
(C9), 150.1 (C8a’), 164.2 (C2), 165.3 (CO). ES-HRMS
[M ? H]? calcd for (C21H13N6O2)?: 381.1022, found:
381.1015.
2-(Coumarin-300-yl)-9-methyl-7-phenylpyrazolo[4,3-
e]-1,2,4-triazolo[1,5-c] pyrimidine (5b)
White solid; Yield: 86 %; m.p.: 280-282 °C (ethanol); IR
(KBr, cm-1) m: 1745 (CO); 1H NMR (CDCl3, 300 MHz): d
2.90 (s, 3H, –CH3), 7.51–8.12 (m, 8H, Harom), 8.22 (d, 1H,
References
H
arom., J = 8.1 Hz), 8.63 (s, 1H, H9), 9.15 (s, 1H, H5), 9.24
(s, 1H, H4 ); 13C NMR (DMSO-d6, 75 MHz): d 14.2
Ahmed SK, Belabassi Y, Sankaranarayanan L, Chao CK, Gerdes JM,
Thompson CM (2013) Synthesis and anti-acetylcholinesterase
properties of novel b-and c-substituted alkoxy organophospho-
nates. Bioorg Med Chem Lett 23(7):2048–2051
0
00
(–CH3), 104.5 (C9a), 121.8 (C8 ), 122.8 (C2 ,6 ), 123.3
0
0
00
00
00
0
00
(C4a ), 124.8 (C6 ), 126.1 (C5 ), 127.6 (C4 ), 128.7 (C7 ),
00
129.1 (C3 ), 129.7 (C3 ,5 ), 138.2 (C1 ), 139.1 (C5), 146.7
0
0
0
Al-Afaleq EI, Abubshait SA (2001) Heterocyclic o-aminonitriles:
preparation of pyrazolo[3,4-d]pyrimidine with modification of
the susbstituents at the 1-position. Molecules 6(7):621–638
Ali MM, Tasneem Rajanna KC, Prakash PKS (2001) An efficient and
facile synthesis of 2-chloro-3-formyl quinolines from acetani-
lides in micellar media by Vilsmeier-Haack cyclisation. Synlett
251–253
Alipour M, Khobbi M, Foroumadi A, Nadri H, Moradi A, Sakhteman
A, Ghandi M, Shafiee A (2012) Novel coumarin derivatives
bearing N-benzyl pyridinium moiety: potent and dual binding
site acetylcholinesterase inhibitors. Bioorg Med Chem
20:7214–7222
00
0
(C4 ), 147.2 (C9b), 149.3 (C6a), 150.1 (C9), 150.7 (C8a ),
163.2 (C2), 164.2 (CO). ES-HRMS [M ? H]? calcd for
(C22H15N6O2)?: 395.1172, found: 395.1178.
2-(Coumarin-300-yl)-9-ethyl-7-phenylpyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c] pyrimidine (5c)
White solid; Yield: 82 %; m.p.: 284–286 °C (ethanol); IR
(KBr, cm-1) m: 1747 (CO); 1H NMR (CDCl3, 300 MHz): d
123

Med Chem Res
Amin KM, Hanna MM, Abo-Youssef HE, George RF (2009)
Synthesis, analgesic and anti-inflammatory activities evaluation
of some bi-, tri- and tetracyclic condensed pyrimidines. Eur J
Med Chem 44(11):4572–4584
Appendino G, Mercalli E, Fuzzati N, Arnoldi L, Stavri M, Gibbons S,
Ballero M, Maxia A (2004) Antimycobacterial coumarins from
the Sardinian giant fennel (Ferula communis). J Nat Prod
67:2108–2110
the proposed mechanisms for the reaction of 3-carbamoyl-2-
iminochromenes with dinucleophiles. Molecules 5(10):
1146–1165
Lise DB, Didier V, Jean-Francois L, Jane S, Paul-Alin J (2007)
Synthesis of triazolylalkylphosphonate starting from x-azi-
doalkylphosphonates or x-alkynylphosphonates. Tetrahedron
63:9677–9684
Ma T, Liu L, Xue H, Li L, Han C, Wang L, Chen Z, Liu G (2008) The
chemical library and structure-activity relationship of Calano-
lide-A analogues against HIV-1. J Med Chem 51:1432–1446
Mado N, Bunta W, Liyou H, Bun-ichi S, Kei W, Keiichi F, Hideyuki
S, Jun H (2014) Glutathione-analogous peptidyl phosphorus
esters as mechanism-based inhibitors of c-glutamyl transpepti-
dase for probing cysteinyl-glycine binding site. Bioorg Med
Chem 22:1176–1194
Bajqar J (2004) Organophosphates/nerve agent poisoning: mechanism
of action, diagnosis, prophylaxis, and treatment. Adv Clin Chem
38:151–216
Baraldi PG, Saponaro G, Tabrizi MA, Baraldi S, Romagnoli R,
Moorman AR, Varani K, Borea PA, Preti (2012) Pyrrolo- and
pyrazolo-[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as adenosine
receptor antagonists. Bioorg Med Chem 20:1046–1059
Bylov IE, Vasylyev MV, Bilokin YV (1999) Synthesis and anti-
inflammatory activity of N-substituted 2-oxo-2H-1-benzopyran-
3-carboxamides and their 2-iminoanalogues. Eur J Med Chem
11:997–1001
Mata AT, Proenc¸a C, Ferreira AR, Serralheiro MLM, Nogueira JMF,
´
Araujo MEM (2007) Antioxidant and antiacetylcholinesterase
activities of five plants used as Portuguese food spices. Food
Chem 103:778–786
El-Agordy AM, Abd El-Latif MS, El-Hady NA, Fakery AH, Bedair
AH (2001) Heteroaromatization with 4- Hydroxycoumarin Part
II: synthesis of Some New Pyrano[2,3-d]pyrimidines[1,2,4]tria-
Meth-Cohn O, Narine B (1987) Versatile new synthesis of quinolines,
thienopyridines and related fused pyridines. Tetrahedron Lett
19:2045–2048
zolo[1,5-c]pyrimidines
Derivatives. Molecules 6:519–527
Ellman GL, Courtney KD, Andres VJ, Featherstone RM (1961) A
new and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol 7:88–95
Ferreira A, Proenc¸a C, Serralheiro MLM, Araujo MEM (2006) The
in vitro screening for acetylcholinesterase inhibition and antiox-
idant activity of medicinal plants from Portugal J Eth Pharmacol
108:31–37
Fest C, Schmidt KJ (1973) Chemistry of Organophosphorus Com-
pounds. Springer-Verlag, Berlin, pp 122–124
and
Pyrimido[1,6-b][1,2,4]triazine
Moyo M, Ndhlala AR, Finnie JF, Staden JV (2010) Phenolic
Composition, antioxidant and acetylcholinesterase inhibitory
activities of Sclerocarya birrea and Harpephyllum caffrum
(Anacardiaceae) extracts. Food Chem 123:69–76
Musa MA, Cooperwood JS, Khan MO (2008) A review of coumarin
derivatives in pharmacotherapy of breast cancer. Curr Med
Chem 15(26):2664–2679
Nam SO, Park DH, Lee YH, Ryu JH, Lee YS (2014) Synthesis of
aminoalkylsubstituted coumarin derivatives as acetyl-
cholinesterase inhibitors. Bioorg Med Chem 22:1262–1267
Orhan G, Orhan I, Sener B (2006) Recent developments in natural
and synthetic drug research for Alzheimer’s disease. Lett Drug
Des Discov 3(4):268–274
Francesca C, Maria LG, Elena P, Marinella V (2001) Isothiazoles.
Part 12: isothiazolylphosphonates, a new class of isothiazole
dioxides. Tetrahedron 57:5455–5459
¨
Rahmouni A, Romdhane A, Ben Saıd A, Majouli K, Ben Jannet H
Francisco P, Ana Maria OR, Eduardo MDM, Marta R, Jaione P
(2003) Aza-Wittig reaction of N-phosphazenes with carbonyl
compounds and phenylisocyanate. Synthesis of 4-amino-3-
phosphoryl-2-azadienes and pyrazine-phosphonates. Tetrahe-
dron 59:2617–2623
Gonc¸alves ADS, Franc¸a TCC, Figueroa-Villarb JD, Pascutti PG
(2011a) Molecular Dynamics Simulations and QM/MM Studies
of the Reactivation by 2-PAM of Tabun Inhibited Human
Acethylcolinesterase. J Braz Chem Soc 22(1):155–165
Gonc¸alves ADS, Franc¸a TCC, Figueroa-Villarb JD, Pascutti PG
(2011b) Molecular dynamics simulations and QM/MM studies
of the reactivation by 2-PAM of tabun inhibited human
acethylcolinesterase. J Braz Chem Soc 22(1):155–165
Gorobets NY, Yousefi BH, Belaj F, Kappe CO (2004) Rapid
microwave-assisted solution phase synthesis of substituted
2-pyridone libraries. Tetrahedron 60:8633–8644
Howes MR, Houghton P (2003) Plants used in Chinese and Indian
traditional medicine for improvement of memory and cognitive
function. J Pharmacol Biochem Behav 75:513–527
Kidane AG, Salacinski H, Tiwari A, Bruckdorfer KR, Seifalian AM
(2004) Anticoagulant and antiplatelet agents: their clinical and
device application(s) together with usages to engineer surfaces.
Biomacromolecules 5:798–813
(2014a) Synthesis of new pyrazole and antibacterial pyrazolopy-
rimidine derivatives. Turk J Chem 38:210–221
Rahmouni A, Romdhane A, Besbes M, Elie N, Touboul D, Ben
Jannet H (2014b) Synthesis of novel pyrazolo[3, 4d]pyrimidi-
none derivatives as cytotoxic inhibitors. Mediterr J Chem
2:679–690
Rashad AE, Shamroukh AH, Hegab MI, Awad HM (2005) Synthesis
of some biologically active pyrazoles and C-nucleosides. Acta
Chim Slov 52:429–434
Rashad AE, Hegab MI, Abdel-Megeid RE, Ali MM, Abdel-Megeid
FME (2010) Synthesis and antitumor evaluation of some newly
synthesized pyrazolopyrimidine and pyrazolotriazolopyrimidine
derivatives. Phosphor Sulfur Silicon 185:74–83
Razavi SF, Khoobi M, Nadri H, Sakhteman A, Moradi A, Emami S,
Foroumadi A, Shafiee A (2013) Synthesis and evaluation of
4-substituted coumarins as novel acetylcholinesterase inhibitors.
Eur J Med Chem 64:252–259
Romdhane A, Gharbi R, Mighri Z (2003) Condensation of imi-
noethers with diarylnitrilimines. Novel synthesis of 1,2,4-
triazoles. J Soc Chem Tunisie. 5:189–192
Romdhane A, Hammami S, Trimeche B, Gharbi R, Hamza MA,
Mighri Z (2008) Novel synthesis of 1,2,4-oxadiazoles by
condensation of arylamidoximes with N-substituted iminoethers.
Heterocycl Commun 14:45–49
Troev KD (2006) Chemistry and applications of H-phosphonate.
Elsevier, Amsterdam, pp 253–261
Kontogiorgis CA, Hadjipavlou-Litina DJ (2004) Synthesis and
biological evaluation of novel coumarin derivatives with a
7-azomethine linkage. Bioorg Med Chem Lett 14:611–614
Kosolapov G (1950) Organophosphorus compounds. Wiley: New
York, Chapter 7
Weiqin J, George A, James JF, Olivia L, Pamela T, Jun X, Peifang Z,
Joseph G, Keith D, Scott L, Zhihua S (2006) New progesterone
receptor antagonists: phosphorus-containing 11b-aryl-substituted
steroids. Bioorg Med Chem 14:6726–6736
Kovalenko SM, Bylov IE, Sytnik KM, Chernykh VP, Bilokhin YV
(2000) A new pathway to 3-hertarly-2-oxo-2H-chromenes: on
123

Med Chem Res
Yanchang S, Yuming Z, Jei S (2002) Regiospecific synthesis of
perfluoroalkylated pyrrolo[2,1-a]isoquinolinyl phosphonates.
J Fluor Chem 116:157–161
Zhi H, Chen L, Zhang L, Liu S, Wan DCC, Lin H, Hu C (2008) 5H-
thiazolo[3,2a]pyrimidine derivatives as a new type of acetyl-
cholinesterase inhibitors. Arkivoc, xiii: 266–227
Zaki MEA (1998) Synthesis of novel fused heterocycles based on
pyrono [2,3-c]pyrazole. Molecules 3:71–79
123