Synthesis, antibacterial activity and cytotoxicity of new fused pyrazolo[1,5-a]pyrimidine and pyrazolo[5,1-c][1,2,4]triazine derivatives from new 5-aminopyrazoles

Article abstract of DOI:10.1016/j.ejmech.2013.04.029

New 5-aminopyrazoles 2a-c were prepared in high yields from the reaction of known α,α-dicyanoketene-N,S-acetals 1a-c with hydrazine hydrate under reflux in ethanol. These compounds were utilized as intermediates to synthesize pyrazolo[1,5-a]-pyrimidines 3a-c, 4a-d, 5a-c, and 6a-c, as well as pyrazolo[5,1-c][1,2,4]triazines 7a-c and 8a-c, by the reaction of 2-[bis(methylthio)methylene]malononitrile, α,α-dicyanoketene-N,S- acetals 1a-b, acetylacetone, acetoacetanilide as well as acetylacetone, and malononitrile, respectively. Furthermore, cyclization of 2a-c with pentan-2,5-dione yielded the corresponding 5-pyrrolylpyrazoles 9a-c. Moreover, fusion of 2a-c with acetic anhydride resulted in the corresponding 1-acetyl-1H-pyrazoles 10a-c. The antibacterial activity and cytotoxicity against Vero cells of several selected compounds are also reported.

Full text of DOI:10.1016/j.ejmech.2013.04.029

European Journal of Medicinal Chemistry 64 (2013) 464e476
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antibacterial activity and cytotoxicity of new fused pyrazolo
[1,5-a]pyrimidine and pyrazolo[5,1-c][1,2,4]triazine derivatives from
new 5-aminopyrazoles
,
b
c
c
Wedad M. Al-Adiwish a , M.I.M. Tahir , A. Siti-Noor-Adnalizawati , Siti Farah Hashim ,
*
Nazlina Ibrahim c, W.A. Yaacob a
a School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
b Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
New 5-aminopyrazoles 2aec were prepared in high yields from the reaction of known a,a-dicyanoke-
tene-N,S-acetals 1aec with hydrazine hydrate under reflux in ethanol. These compounds were utilized as
intermediates to synthesize pyrazolo[1,5-a]-pyrimidines 3aec, 4aed, 5aec, and 6aec, as well as pyrazolo
Received 22 January 2013
Received in revised form
9 April 2013
Accepted 15 April 2013
Available online 23 April 2013
[5,1-c][1,2,4]triazines 7aec and 8aec, by the reaction of 2-[bis(methylthio)methylene]malononitrile, a,a-
dicyanoketene-N,S-acetals 1aeb, acetylacetone, acetoacetanilide as well as acetylacetone, and malono-
nitrile, respectively. Furthermore, cyclization of 2aec with pentan-2,5-dione yielded the corresponding
5-pyrrolylpyrazoles 9aec. Moreover, fusion of 2aec with acetic anhydride resulted in the corresponding
1-acetyl-1H-pyrazoles 10aec. The antibacterial activity and cytotoxicity against Vero cells of several
selected compounds are also reported.
Keywords:
5-Aminopyrazoles
Pyrazolo[1,5-a]pyrimidines
Pyrazolo[5,1-c][1,2,4]triazines
Ó 2013 Elsevier Masson SAS. All rights reserved.
a,a-Dicyanoketene-S,S- and N,S-acetals
Antibacterial activity
Cytotoxicity
1. Introduction
for the synthesis of pyrazole derivatives by displacing the methyl-
thio group with bifunctionalized amines, such as hydrazine [14e
Pyrazolopyrimidine and pyrazolotriazine derivatives constitute
an interesting class of heterocycles because of their synthetic
versatility, effective biological activities, and pharmacological
importance as purine analogs [1e5]. Several derivatives such as 4-
hydroxypyrazolopyrimidine (allopurin), which are used in the
treatment of hyperuricemia and gout, inhibit de novo purine
biosynthesis and xanthine oxidase [6]. Various related pyr-
azolopyrimidine compounds were reported to possess anti-tumor
and anti-leukemic activities [7,8]. Substituted pyrazolotriazines
are often used in medicine because of their remarkable bactericidal,
fungicidal, and antiviral effects [9,10]. Ketene-S,S- and -N,S-acetals
and related compounds are versatile reagents in the synthesis of
polyfunctionalized heterocycles [11e13]. Ketene-S,S and N,S-acetals
have been paid much attention because they are extensively used
16]. Accordingly, we report in this paper a novel synthesis of
functionalized pyrazolo[1,5-a]pyrimidines 3aec, 4aed, 5aec, and
6aec, as well as pyrazolo[5,1-c][1,2,4]triazines 7aec and 8aec, by
the reaction of respective 2-[bis(methylthio)methylene]malononi-
trile,
a,a-dicyanoketene-N,S-acetals 1a and b, acetylacetone, and
acetoacetanilide as well as acetylacetone and malononitrile with 5-
aminopyrazoles 2aec. The antibacterial activity and toxicity in Vero
cells of several selected compounds are also reported.
2. Chemistry
The 5-aminopyrazole intermediates 2aec were prepared from
the cyclocondensation of respective a,a-dicyanoketene-N,S-acetals
2-[methylthio(morpholino)methylene]malononitrile 1a, 2-[meth-
ylthio(piperidin-1-yl)methylene]malononitrile 1b, and 2-[methyl-
thio(pyrrolidin-1-yl)methylene]malononitrile 1c with hydrazine
hydrate under reflux in ethanol (Scheme 1). The starting materials
1aec were prepared via the reaction of 2-[bis(methylthio)methy-
lene]malononitrile with an appropriate cyclic secondary amines of
morpholine, piperidine, and pyrrolidine in refluxing ethanol
* Corresponding author. Tel.: þ60 129 427 918.
E-mail addresses: we80dad@yahoo.com (W.M. Al-Adiwish), mibrahim@
science.upm.edu.my (M.I.M. Tahir), adnaliza@yahoo.com (A. Siti-Noor-
Adnalizawati), farah_samura@yahoo.com (S.F. Hashim), nazlina@ukm.my
(N. Ibrahim), wanyaa@ukm.my (W.A. Yaacob).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.029

W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
465
Fig. 1, which confirmed that the NeH in the pyrazole ring is adja-
cent to the carbon attached to the amino group. Suitable crystals of
2a were grown by slow evaporation of MeOH solution of the
compound for 48 h. The crystal data and structure refinement re-
sults for 2a are given in Table 1. Compound 2a crystallized in a
triclinic system with space group of Pꢀ1 (No. 2). The symmetric
unit of 2a contains two molecules, A and B. Selected bond distances
and bond angles are listed in Table 2. The bond lengths and angles
in molecules A and B of 2a are within the normal ranges [18]. The
morpholine rings adopt chair conformation in molecules A and B.
The mean planes for 2a in the molecule A were observed in the
pyrazole ring N2/N3/C1/C4/C5 and morpholine ring O11/N8/C9/
C10/C12/C13, with a maximum deviation of ꢀ0.251(1)ꢁ at the O11
atom. The mean planes in the molecular B were observed in the
pyrazole ring N16/N17/C15/C18/C19 and morpholine ring O25/N22/
C23/C24/C26/C27, with a maximum deviation of 0.253(1)ꢁ at the
O25 atom. The dihedral angles between the mean planes of the
morpholine and pyrazole rings in molecules A and B were 10.55(9)ꢁ
and 18.82(10)ꢁ, respectively (see Electronic Supplementary
information [19]).
Scheme 1. Synthesis of 5-aminopyrazoles 2aec.
according to a previously reported procedure [17]. The chemical
structures of 2aec were established based on their elemental
analysis and spectral data. The IR spectra of 2aec showed bands
between 3222 cmꢀ1 and 3453 cmꢀ1 for the NH and NH2 groups,
respectively. Their 1H NMR spectra showed singlet signals with
d
values between 4.93 ppm and 6.11 ppm corresponding to the NH2
protons, whereas the singlet singles with values from 10.74 ppm
to 11.00 ppm, which can be assigned to the NH protons. The 13C
NMR spectra were characterized by signals at values of 154.5e
d
d
The above mentioned 5-aminopyrazoles 2aec were used as
intermediates for the synthesis of new pyrazolo[1,5-a]pyrimidines
and pyrazolo[5,1-c][1,2,4]triazines. Thus, condensation of 2aec
154.6 and 158.0e158.6 ppm assigned to respective carbons of C]N
and CeNH2. The structures of 2aec were supported by their direct
infusion mass spectrometry (DIMS) results, which showed molec-
ular ions corresponding to the molecular formulas. For example,
the DIMS of 5-amino-3-morpholino-1H-pyrazole-4-carbonitrile 2a
showed a molecular ion at m/z ¼ 193.25, which corresponds to the
molecular formula C8H11N5O (193.21).
with
a,a-dicyanoketene-S,S-acetal 2-[bis(methylthio)methylene]
malononitrile in refluxing ethanol containing a catalytic amount of
triethylamine yielded the corresponding pyrazolo[1,5-a]pyrimi-
dines 3aec. The reaction of 5-aminopyrazoles 2a and 2c with a,a-
dicyanoketene-N,S-acetals 1a and 1b under similar conditions
yielded pyrazolo[1,5-a]pyrimidines 4aed as demonstrated in
Scheme 3. The proposed structures of 3aec and 4aed were
established based on their elemental analysis and spectral data (see
Experimental).
The proposed mechanism for the formation of pyrazolo[1,5-a]
pyrimidines 3aec and 4aed is similar to that for the formation of 5-
aminopyrazoles 2aec, except that the final step in the former in-
volves 1,3-hydrogen migration contrary to the 1,5-hydrogen
migration in the latter (Scheme 4).
We also attempted to synthesize directly pyrazolo[1,5-a]pyrimi-
dines 5aec and 6aec by treating 5-aminopyrazoles 2aec with cor-
responding 1,3-dicarbonyl compounds, namely, pentane-2,4-dione
and acetoacetanilide, in refluxing dimethylformamide containing a
catalytic amount of glacial acetic acid as shown in Scheme 5.
The possible formation of 5-aminopyrazoles is shown in
Scheme 2. First, Michael addition to a,a-dicyanoketene-N,S-ace-
tals 1aec occurs with lone pair of the NH2 group in hydrazine to
form an intermediate adduct A. Then, the methylthio ion is
removed, which results in the formation of intermediate B. This
methylthio ion abstracts the proton of the ammonium ion to
produce an intermediate C. Subsequently, intermolecular cycli-
zation occurs by the lone pair on the NH2 group attacking the
cyano group to produce intermediate D. The ammonium proton
abstraction in D occurs to form E, followed by aromatic-driven 1,5-
hydrogen migration to yield products 2aec.
The structure of 5-amino-3-morpholino-1H-pyrazole-4-
carbonitrile 2a was further identified by X-ray diffraction (XRD).
The ORTEP plot of 2a and the numbering scheme are illustrated in
Scheme 2. Mechanism for the formation of 5-aminopyrazoles 2aec.

466
W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
Fig. 1. Single-crystal X-ray analysis. ORTEP view of 2a showing the atom numbering scheme.
The mechanism for the formation of 5aec and 6aec is shown in
The diazotized 5-aminopyrazole is an excellent building block
for the synthesis of the pyrazolo[5,1-c][1,2,4]triazine derivatives
[20,21]. Thus, diazotization of 5-aminopyrazoles 2aec with sodium
nitrite and concentrated HCl gave the corresponding diazonium
chloride [A], which was coupled with different active methylene
compounds, namely, acetylacetone and malononitrile in pyridine to
afford the corresponding hydrazono derivatives B and C. When
intermediates B and C were refluxed in glacial acetic acid, the target
pyrazolo[5,1-c][1,2,4]triazine derivatives 7aec and 8aec were ob-
tained (Scheme 7).
Scheme 6. The conversion involves three major steps, as follows: (i)
nucleophilic attack of the NH2 group from 5-aminopyrazoles on the
carbonyl group of acetylacetone and acetoacetanilide, by releasing
water molecule, to form intermediate imine A; (ii) intramolecular
cyclization occurs by a nucleophilic attack of the NH of pyrazole on
the other carbonyl group to form an intermediate adduct B; and (iii)
dehydration in B when X ¼ methyl produces 5aec and elimination
of aniline when X ¼ NHPh yields 6aec.
The structures of the pyrazolo[1,5-a]pyrimidines 5aec and 6aec
were elucidated based on their elemental analyses and spectral data.
The mechanism for the formation of pyrazolo[5,1-c][1,2,4]tri-
azines 7aec and 8aec is presented in Scheme 8. The first step in-
volves the formation of diazotized 5-aminopyrazoles 20aec. The
second step involves the coupling off the 20aec with active meth-
ylenes in acetylacetone and malononitrile, in pyridine from 0 ꢁC to
The characteristic band in the IR spectra of 6aec is at
3146) cmꢀ1 for OH stretching vibrations. The 1H NMR spectra of 5aec
displayed singlet signals at
¼ (2.42e2.63) ppm, which correspond to
six protons of two methyl groups. The 1H NMR spectra of 6aec dis-
played singlet signals between
¼ (12.89e13.07) ppm because of the
n
¼ (3308e
d
d
5
ꢁC to yield the corresponding hydrazono intermediates A and B.
OH group. Results from DIMS of 5aec and 6aec showed molecular
ionscorrespondingtothemolecularformula.Forexample, theDIMSof
5,7-dimethyl-2-morpholinopyrazolo[1,5-a]pyrimidine-3-carbonitrile
5a showed a molecular ion at m/z ¼ 257.30, which corresponds to the
molecular formula C13H15N5O (257.29). The DIMS of 7-hydroxy-
5-methyl-2-(piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
6b showed a molecular ion at m/z ¼ 257.20, which corresponds to the
molecular formula C13H15N5O (257.29).
During the third step, intramolecular cyclization by nucleophilic
attack of nitrogen occurs in the pyrazole ring on the acetyl and
cyano groups to form intermediates A0 and B0, followed by proton
abstraction to generate intermediate adducts A00 and B00. Finally,
aromatic-driven 1,4-dehydration in A00 forms 7aec; and 1,5-
hydrogen migration in B00 yields 8aec.
The structures of 7aec and 8aec were elucidated based on their
elemental analyses and spectral data. The characteristic band in the
Table 1
Crystal data and structure refinement for 2a.
Chemical formula
Formula weight
Color
Crystal shape
Size
Temperature
Wavelength
Crystal system
C8H11N5O
Absorption coefficient
F(000)
0.83 mmꢀ1
408
4e71 (ꢁ)
193.22 g molꢀ1
Colorless
Theta range for data collection
Reflections collected/unique
Completeness to theta ¼ 25.00
Max. and min. transmission
Refinement method
Block
3337, 3002, Rint ¼ 0.023
0.08 ꢂ 0.18 ꢂ 0.20 mm
71.023
293 K
1.54180 A
Tmin ¼ 0.862, Tmax ¼ 0.936
Full-matrix least-squares on F2
3322/0/253
ꢀ
Triclinic
Pꢀ1 (No. 2)
Data/restraints/parameters
Space group
Goodness-of-fit on F2
1.11 Full-matrix least-squares on F2
R ¼ 0.042, wR ¼ 0.119
ꢀ
a, b, c (A);
a,
b
,
g
(ꢁ)
8.5155(4), 10.1546(8),
11.8235(9); 81.712(6),
78.698(5), 66.513(6)
917.02(12)
Final R indices [I > 2s (I)]
ꢀꢀ3
Cell volume
Z
Largest diff. peak and hole
Calculated density
0.24 & ꢀ0.28 e A
4
1.400 g cmꢀ3

W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
467
Table 2
refinement data for 8a are listed in Table 3. Compound 8a crystal-
lized in a monoclinic system with space group P21/c (No. 14). The
asymmetric unit of 8a contains two molecules, A and B, of the
compound and two DMSO solvent molecules. The sulfur atom of
one of the DMSO molecules is disordered over two positions, S401
and S402, with equal occupancies. Selected bond distances and
bond angles are provided in Table 4. The N112eN113 bond length is
ꢁ
ꢀ
Selected bond lengths (A) and bond angles ( ) for 2a.
Bond angle (ꢁ)
ꢀ
Bond
Bond length (A)
Bond
N2eN3
N3eC4
C1eC5
C5eC6
N3eC4
N2eC1
N7eC6
C4eC5
1.3913(18)
1.3253(18)
1.4137(19)
1.416(2)
1.3253(18)
1.333(2)
1.154(2)
1.424(2)
1.459(2)
1.514(2)
1.3914(17)
1.322(2)
1.3840(19)
1.414(2)
1.397(2)
1.3411(19)
1.466(2)
1.459(2)
1.423(2)
1.514(2)
N21eC20eC19
N16eN17eC18
N16eC15eN28
N17eC18eC19
C15eC19eC18
C18eC19eC20
N17eN16eC15
N2eN3eC4
N3eC4eC5
N3eC4eN8
C5eC4eN8
C4eC5eC1
C4eC5eC6
C1eC5eC6
C5eC6eN7
C4eN8eC9
N17eN16eC15
N16eC15eC19
N17eC18eN22
N22eC18eC19
178.64(17)
104.70(12)
124.17(14)
111.34(13)
104.81(13)
130.12(15)
112.49(12)
104.48 (12)
111.59 (13)
119.98(14)
128.42 (13)
104.64 (13)
132.06(13)
123.11(14)
177.83 (16)
113.79 (12)
112.49(12)
106.64(13)
122.64(13)
125.99(14)
ꢀ
1.3245(19) A in molecule A and the analog N212eN213 bond length
ꢀ
is ꢀ1.3219(19) A in the molecule B, which are longer than the
ꢀ
normal N]N bond (1.255 A). The C110eN109 and C111eN112 bond
ꢀ
N8eC9
lengths in molecule A are 1.3515(19) and 1.350(2) A, respectively.
C9eC10
N16eN17
N17eC18
N22eC18
C19eC20
C15eC19
N16eC15
N8eC13
N8eC9
The analog C210eN209 and C211eN212 bond lengths in molecule B
ꢀ
are 1.3522(19) and 1.344(2) A, respectively. These bonds are shorter
ꢀ
than the normal CeN bond (1.45 A). In addition, the C111eC110
ꢀ
bond length in the molecule A is 1.423(2) A, and the analog C211e
C210 bond length in the molecule B is 1.421(2), which are longer
ꢀ
than the normal CeC bond (1.34 A) and close to the CeC single
ꢀ
bond (1.45 A). In addition, the C110eN118 bond length in molecule
ꢀ
A is 1.308(2) A and the analog C210eN218 bond length in molecule
O11eC12
C12eC13
ꢀ
B is 1.313(2) A, which are slightly shorter than the normal CeN
ꢀ
bond (1.394 A) [18]. This condition is due to the electron-donating
effect of nitrogen N118 and N218 attached to C110 and C210,
respectively, of the pyrimidine rings. The mean planes for 8a were
observed in the morpholines and pyrazolopyrimidine rings in
molecules A and B, with a maximum deviation of ꢀ0.259(1)ꢁ and
0.258(1)ꢁ at the O104 and O204 atoms, respectively. The morpho-
line rings adopted chair conformation. They differed significantly in
conformation, as shown by the dihedral angle in the mean planes of
the morpholines and pyrazolopyrimidines. The dihedral angles
between the mean planes of the morpholines and pyrazolopyr-
imidines in molecules A and B were 32.32(6)ꢁ and 27.54(6)ꢁ,
respectively (see Electronic Supplementary information [22]).
Cyclocondensation reaction of 2aec with 2,5-hexadione in
boiling glacial acetic acid for 5 h produce the corresponding 5-
pyrrolylpyrazoles 9aec as shown in (Scheme 9).
Presumably, the reaction mechanism for 9aec formation is
illustrated in Scheme 10. It involves a nucleophilic attack of the NH2
group in 2aec on the carbonyl carbon in 2,5-hexandione, elimi-
nating water, to generate intermediate enamine A. Then, intra-
molecular cyclization by a nucleophilic attack of the incipient NH
onto the other carbonyl group in A forms an intermediate adduct B,
followed by dehydration to afford 9aec.
IR spectra of 7aec and 8aec is at
n
¼ (1586e1591) and (1585e1598)
cmꢀ1 for N]N stretching vibrations. Furthermore, displayed bands
at
n
¼ (1695e1698) cmꢀ1 in 7aec revealed the carbonyl group,
whereas the bands at
n
¼ (3378e3148) cmꢀ1 in 8aec revealed the
NH2 group. The 1H NMR spectra of 7aec displayed broad signals at
d
¼ (2.77e3.09) ppm, which corresponds to six protons of two
methyl groups. The 1H NMR spectra of the 8aec displayed broad
signals at
¼ (9.16e10.12) ppm caused by the NH2 group. Results
d
from DIMS of 7aec showed molecular ions corresponding to the
molecular formulas. For examples, the DIMS of 3-acetyl-4-methyl-7-
morpholinopyrazolo[5,1-c][1,2,4]triazine-8-carbonitrile 7a showed
a molecular ion at m/z ¼ 286.15, which corresponds to the molecular
formula C13H14N6O2 (286.29). The intensity peak at m/z ¼ 200 is
due to 3-acetyl-4-methylpyrazolo[5,1-c][1,2,4]triazine-8-carbonitrile
moiety. The DIMS of 4-amino-7-morpholinopyrazolo[5,1-c][1,2,4]
triazine-3,8-dicarbonitrile 8a showed
a molecular ion at m/
z ¼ 270.15, which corresponds to the molecular formula C11H10N8O
(270.25). The intensity peak at m/z ¼ 212 is due to 4-amino-7-
(methyleneamino)pyrazolo[5,1-c][1,2,4]triazine-3,8-dicarbonitrile
moiety.
The structures of 9aec were established based on their
elemental analyses and spectral data. The IR spectra showed the
absence of amino group (NH2) bands, whereas the 1H NMR spectra
The structure of 4-amino-7-morpholinopyrazolo[5,1-c][1,2,4]
triazine-3,8-dicarbonitrile 8a was further identified by XRD. The
ORTEP plot of 8a and the numbering scheme are presented in Fig. 2.
A suitable crystal of 8a was grown by slow evaporation of DMSO
solution of the compound for 72 h. The crystal and structure
showed characteristic signals between
d values of 2.11 ppme
2.12 ppm caused by the two methyl groups [(CH3eC])2]. Results
Scheme 3. Synthesis of pyrazolo[1,5-a]pyrimidines 3aec and 4aed.

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W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
Scheme 4. Mechanism for the formation of pyrazolo[1,5-a]pyrimidines 3aec and 4aed.
from DIMS of 5-pyrrolylpyrazoles 9aec showed molecular ions
corresponding to the molecular formulas. For example, the DIMS
spectrum of 5-(2,5-dimethyl-1H-pyrrol-1-yl)-3-morpholino-1H-
pyrazole-4-carbonitrile 9a showed a molecular ion at m/z ¼ 271.20,
which corresponds to a molecular formula C14H17N5O (271.32).
Fusion of 2aec with acetic anhydride under reflux for 15 min
furnished the corresponding 1-acetyl-5-amino-3-morpholino-1H-
pyrazole-4-carbonitrile 10a, 1-acetyl-5-amino-3-(piperidin-1-yl)-
1H-pyrazole-4-carbonitrile 10b, and 1-acetyl-5-amino-3-(pyrrolidin-
1-yl)-1H-pyrazole-4-carbonitrile 10c, as shown in (Scheme 11).
The structures of 10aec were established based on the elemental
analyses and spectral data. The IR spectra showed the absence of NH
2.41 ppm caused by the methyl group attached to carbonyl group
(CH3eCO). Results from the DIMS of 10a‒c showed molecular ions
corresponding to the molecular formulas. For example, the DIMS
spectrum
of
1-acetyl-5-amino-3-morpholino-1H-pyrazole-4-
carbonitrile 10a showed a molecular ion at m/z ¼ 235.25, which
corresponds to a molecular formula C10H13N5O2 (235.24).
In general, the electronic spectra of all the new synthesized
compounds were recorded. The wavelength maxima of the prin-
ciple absorption peaks in the electronic spectra for compounds
with non-fused aromatic parent structure (2aec, 9aec, and 10aec)
occur between 205 and 281 nm whereas their fused aromatic
parent structure counter parents (3a‒c, 4a‒d, 5a‒c, 6a‒c, 7a‒c and
8a‒c) occur between 209 and 306 nm.
bands and displayed bands at
1707 cmꢀ1 caused by the carbonyl group. The 1H NMR spectra
showed characteristic signals at values between 1.94 ppm and
n
values between 1702 cmꢀ1 and
d
3. Antibacterial evaluation and cytotoxicity assay
3.1. Antibacterial evaluation
The antibacterial activity of the 27 new synthesized compounds
wasevaluatedusingtheagardiffusiontechnique[23]. Thecompounds
were prepared and tested at 1 mg/ml concentration in dimethylsulf-
oxide (DMSO). The tested organisms were Gram-positive bacteria
[Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 11774,
Methicillin-resistant S. aureus (MRSA) ATCC 43300, Staphylococcus
epidermidis ATCC 12228, and Enterococcus faecalis ATCC 14506] and
Scheme 5. Synthesis of pyrazolo[1,5-a]pyrimidines 5aec and 6aec.
Scheme 6. Mechanism for the formation of pyrazolo[1,5-a]pyrimidines 5aec and 6aec.

W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
469
Scheme 7. Synthesis of pyrazolo[5,1-c][1,2,4]triazines 7aec and 8aec.
Gram-negative bacteria [Escherichia coli ATCC 10536, Pseudomonas
aeruginosa ATCC 10145, Serratia marcescens ATCC 13880, and Salmo-
nella typhimurium ATCC 51812]. The bacteria were maintained in
nutrient agar and MuellereHinton agar media, respectively. After 24 h
incubation at 37 ꢁC, the diameter (mm) of the inhibition zones was
measured. DMSO showed no inhibition zones. Streptomycin (ST) in a
butlowerthanthereferencedrugstreptomycin.For1-acetyl-5-amino-
3-(piperidin-1-yl)-1H-pyrazole-4-carbonitrile 10b, greater inhibitory
activity toward B. subtilis, S. aureus, and E. coli was shown
comparedwith1-acetyl-5-amino-3-(pyrrolidin-1-yl)-1H-pyrazole-4-
carbonitrile 10c. The antibacterial activity may be attributed to the
aromaticparent structures of thetested compounds. Compounds2ae
c, 9aec, and 10aec with non-fused aromatic parent structure dis-
played evidently higher antibacterial activity than those with fused
aromaticparentstructure(3aeb, 4aed, 5aeb, 6aeb, 7aec, and8aec).
The presence of piperidine ring in a structure has been reported to
increase antibacterial capabilityof thecompounds[25]and have been
demonstrated in this study in 2b, 3b, 9b, 5b, and 10b.
The compounds were inactive against several tested bacteria
including MRSA, S. epidermidis, E. faecalis, and S. typhimurium by agar
diffusion method. The activity of the compounds varies with the
type of bacteria tested and nature of the compounds. Some of the
compounds showed limited inhibition as a result of their inability to
diffuse through the agar. Thus, they must be tested further by broth
dilution method to determine the MIC. The antibacterial activity in
this study is also probably influenced by the polarity of the com-
pounds. Derivatizations to less polar compounds usually increase
the antimicrobial and cytotoxic effects and reduce mutagenicity, in
contrast to the effect of introducing hydroxyl group [26].
concentration of 10 mg was used as reference for antibacterial activ-
ities. Minimum inhibitory concentration (MIC) and minimum bacte-
riocidal concentration (MBC) were determined according to NCCLS
(2000) [24] for compounds that showed inhibitory zones in the agar
diffusion assay. MIC values were determined as the minimum con-
centrations that inhibited visible growth of the bacteria, and MBC
values were recorded as the lowest concentrations that showed no
growth on plate. Only gentamicin was tested for the MIC.
The antibacterial activities are listed in Table 5. The results for 5-
aminopyrazoles 2aec showed 3-amino-5-(piperidin-1-yl)-1H-
pyrazole-4-carbonitrile 2b exhibited higher antibacterial activity
against S. aureus and E. coli compared with the other 5-
aminopyrazoles 2a and 2c. For antibacterial activity in pyrazolo
[1,5-a]pyrimidines (3aec, 4aed, 5aec, and 6aec) and pyrazolo[5,1-
c][1,2,4]triazines (7aec and 8aec), only 3b, 5b, 6a, 7b, and 8b
exhibited moderate activities against some of the bacteria tested.
Compounds 9aec and 10aec also exhibited good antibacterial
activity.Compounds3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-morpholino-
1H-pyrazole-4-carbonitrile 9a and 3-(2,5-dimethyl-1H-pyrrol-1-yl)-
5-(piperidin-1-yl)-1H-pyrazole-4-carbonitrile 9b showed pro-
nounced antibacterial activityagainstB. subtilis than 5-(2,5-dimethyl-
1H-pyrrol-1-yl)-3-(pyrrolidin-1-yl)-1H-pyrazole-4-carbonitrile 9c,
However, in this study, the MIC values for all compounds were
greater than 1 mg/ml. Thus, the compounds cannot be considered
as good lead compounds for antibacterial drug compared with the
MIC value for positive control (gentamicin, 1 mg/mL) and MBC (2 mg/
mL) against P. aeruginosa.
Scheme 8. Mechanism for the formation of pyrazolo[5,1-c][1,2,4]triazines 7aec and 8aec.

470
W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
Fig. 2. Single-crystal X-ray analysis. ORTEP view of 8a showing the atom numbering scheme.
3.2. Cytotoxicity assay
4. Experimental section
The isolated compounds were tested for in vitro cytotoxicity using
Vero cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay [27]. The concentration of stock compound solution
4.1. Instrumentation
All melting points were determined using a hot stage Gallen-
kamp melting point apparatus. Infrared spectra were recorded
from KBr discs on FT-IR 8300 Shimadzu spectrometer. 1H NMR
and 13C NMR spectra were recorded on FT-NMR 400 MHz Joel,
ECP, JNM-ECX 500 MHz, Joel and FT-NMR 600 MHz Bruker,
AVANCE III spectrometer operating at 400 MHz, 500 MHz and
600 MHz for 1H NMR and at 100 MHz, 125 MHz, 150 MHz for 13C
NMR in DMSO-d6, CD3CL and AcOH as solvents and using TMS as
internal standard. DIMS spectra were recorded on QP5050A Shi-
madzu apparatus. Elemental analyses of all new compounds were
performed on Thermo Finnigan EA1112. Electronic spectra were
recorded on a UV-2450 version UVeVIS spectrophotometer and
inflections are identified by the abbreviation ‘inf’. Single-crystal X-
ray diffraction data (XRD) were collected at room temperature
with Bruker APEXII CCD a spectrometer. General purpose silica gel
of Merck No. 5545 with UV indicator were used in TLC experi-
ments to monitor completion of reactions, in which DCM was
used as eluent.
was1.0mg/ml preparedby dissolving1 mg compoundin 50
mL DMSO
and 950 L 5% FBS-DMEM. A sonicator was used to emulsify the
m
compounds for 40 min before the two-fold dilutions made in 5% FBS-
DMEM to produce a compound solution with concentrations of 0.5,
0.25, 0125 and 0.0625 mg/ml. The absorbance for each well was
measured at 540 nm in a micro-titer plate reader and percentage of
cell viability (CV) was calculated manually using the formula: CV ¼
[(Abssample ꢀ Absnegative control)/(Abscell ꢀ Absnegative control)] ꢂ 100.
A doseeresponse curve was plotted to enable the calculation of the
concentration thatkills 50% of theVero cells (CC50). Selectivityindices
were calculated as SI ¼ IC50/MIC.
The cytotoxicity evaluation results of the tested compounds as
CC50 values are shown in Table 6. The compounds were considered
safe when their CC50s are higher than 20 mg/ml [28]. However, in
terms of selectivity, all the tested compounds have SI less than 10. SI
values greater than 10 indicate the therapeutic safety and effec-
tiveness of these drugs as good candidates as antibacterial agents.
Table 3
Crystal data and structure refinement for 8a.
Chemical formula
Formula weight
Color
Crystal shape
Size
Temperature
Wavelength
Crystal system
C
26H32N16O4S1
Absorption coefficient
F(000)
2.00 mmꢀ1
1454.110
71.5e3.5 (ꢁ)
695.83 g molꢀ1
Colorless
Theta range for data collection
Reflections collected/unique
Completeness to theta ¼ 25.00
Max. and min. transmission
Refinement method
Plate-like
16,257, Rint ¼ 0.000
0.09 ꢂ 0.14 ꢂ 0.18 (mm)
0.99
150 K
Tmin ¼ 0.76, Tmax ¼ 0.84
Full-matrix least-squares on F2
6227/0/444
ꢀ
1.54180 A
Monoclinic
P21/c (No. 14)
8.92233(15), 14.9672(2),
24.4778(4); 90, 98.9113(15), 90
Data/restraints/parameters
Space group
Goodness-of-fit on F2
0.98
ꢀ
a, b, c (A);
a,
b
,
g
(ꢁ)
Final R indices [I > 2
s
(I)]
R ¼ 0.038, wR ¼ 0.101
3
ꢀ
ꢀꢀ3
Cell volume
Z
3229.37(9) A
4
Largest diff. peak and hole
Calculated density
0.44e0.31 e A
1.431 g cmꢀ3

W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
471
Table 4
ꢁ
ꢀ
Selected bond lengths (A) and bond angles ( ) for 8a.
Bond angle (ꢁ)
ꢀ
ꢀ
Bond
Bond length (A)
Bond
Bond length (A)
Bond
N101eC102
N101eC106
N101eC107
C102eC103
C103eO104
O104eC105
C105eC106
C107eN108
C107eC115
N108eN109
C110eN109
N109eC114
C111eC110
C110eN118
C111eN112
C111eC119
N112eN113
N113eC114
C114eC115
C115eC116
C116eN117
C119eN120
1.464(2)
1.456(2)
1.344(2)
1.509(2)
1.428(2)
1.425(2)
1.518(2)
1.3461(19)
1.437(2)
1.3626(17)
1.3515(19)
1.3747(19)
1.423(2)
1.308(2)
1.350(2)
1.436(2)
1.3245(19)
1.354(2)
1.396(2)
1.416(2)
1.147(2)
1.145(2)
N201eC202
N201eC206
N201eC207
C202eC203
C203eO204
O204eC205
C205eC206
C207eN208
C207eC215
N208eN209
C210eN209
N209eC214
C211eC210
C210eN218
C211eN212
C211eC219
N212eN213
N213eC214
C214eC215
C215eC216
C216eN217
C219eN220
1.455(2)
1.466(2)
1.344(2)
1.512(2
N101eC107eN108
N101eC107eC115
N108eC107eC115
C107eN108eN109
N108eN109eC110
N108eN109eC114
C110eN109eC114
N109eC110eC111
N109eC110eN118
N101eC107eN108
N101eC107eC115
N201eC207eN208
N201eC207eC215
N208eC207eC215
C207eN208eN209
N208eN209eC210
N208eN209eC214
C210eN209eC214
N209eC210eC211
N209eC210eN218
C211eC210eN218
C210eC211eN212
119.15(13)
129.46(14)
111.39(13)
104.11(12)
123.32(12)
114.02(12)
122.66(13)
111.96(13)
119.15(14)
119.15(13)
129.46(14)
119.21(14)
129.33(14)
111.45(13)
104.09(12)
123.90(12)
113.78(12)
122.22(13)
111.99(13)
119.56(14)
128.45(14)
124.76(14)
1.420(2)
1.432(2)
1.512(2)
1.3460(19)
1.437(2)
1.3636(17)
1.3522(19)
1.3729(19)
1.421(2)
1.313(2)
1.344(2)
1.436(2)
ꢀ1.3219(19)
1.351(2)
1.393(2)
1.419(2)
1.149(2)
1.142(2)
4.2. General procedure for the synthesis of 5-aminopyrazoles 2aec
A mixture of -dicyanoketene-N,S-acetals 1aec (4 mmol) and
(C]C/C]N); 1H NMR (500 MHz, DMSO-d6)
d
: 3.05 (br, 4H,
N(CH2)2(CH2)2O), 3.59 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.6 Hz), 6.11 (s,
a,a
2H, NH2), 11.00 (s, 1H, pyrazole, NH); 13C NMR (125 MHz, DMSO-d6)
hydrazine hydrate (1 g, 20 mmol) was refluxed on water-bath for
2 h. Then, 20 mL of ethanol was added, and the reaction mixture
was refluxed for further 2 h. The solvent was evaporated and the
product was collected, washed with ethanol, dried and recrystal-
lized from methanol to give pure products 2aec.
d: 48.6 N(CH2)2(CH2)2O, 62.6 (CeCN), 66.1 N(CH2)2(CH2)2O, 116.9
(CN), 154.6 (C]N), 158.0 (CeNH2); lmax (MeOH)/nm 211 (log ε
4.33), 244 inf (4.05), 264 inf (3.61); DIMS found m/z: 193.25 (calc.
for C8H11N5O Mþ requires 193.21); Anal. Calcd. for C8H11N5O: C
49.73, H 5.74, N 36.25%; found: C 50.06, H 5.46, N 36.34%.
4.2.1. 5-Amino-3-morpholino-1H-pyrazole-4-carbonitrile (2a)
4.2.2. 5-Amino-3-(piperidin-1-yl)-1H-pyrazole-4-carbonitrile (2bꢀ) 1
)
Yield, 97%; colorless crystals; mp 191e192 ꢁC; FT-IR (KBr, cmꢀ1
)
Yield, 88%; colorless crystals; mp 140e142 ꢁC; FT-IR (KBr, cm
n
: 3442, 3394, 3247 (NH2/NH), 2196 (CN), 1641, 1600, 1548
n
: 3435, 3344, 3225 (NH2/NH), 2193 (CN), 1637 (C]C), 1600 (C]N);
1H NMR (500 MHz, DMSO-d6)
: 1.50 (br. s, 6H, N(CH2)2(CH2)3), 3.12
(br. s, 4H, N(CH2)2(CH2)3), 6.11 (s, 2H, NH2), 10.97 (s, 1H, pyrazole,
NH); 13C NMR (125 MHz, DMSO-d6)
: 24.4 (N(CH2)2CH2(CH2)2),
d
d
25.3 (N(CH2)2CH2(CH2)2), 49.1 (N(CH2)2CH2(CH2)2), 62.7 (CeCN),
117.2 (CN), 154.5 (C]N), 158.5 (CeNH2); lmax (MeOH)/nm 212 (log ε
4.36), 242 inf (4.10), 263 inf (3.62); DIMS found m/z: 191.20 (calc. for
C9H13N5 Mþ requires 191.23); Anal. Calcd. for C9H13N5: C 56.53, H
6.85, N 36.62%; found: C 56.53, H 6.52, N 36.96%.
4.2.3. 5-Amino-3-(pyrrolidin-1-yl)-1H-pyrazole-4-carbonitrile (2c)
Yield, 87%; colorless crystals; mp 211e212 ꢁC; FT-IR (KBr, cmꢀ1
)
n
: 3440, 3356, 3222 (NH2/NH), 2189 (CN), 1665, 1613, 1565 (C]C/
C]N); 1H NMR (500 MHz, DMSO-d6)
d:
1.76 (s br, 4H,
Scheme 9. Synthesis of 5-pyrrolylpyrazoles 9aec.
Scheme 10. Mechanism for the formation of 5-pyrrolylpyrazoles 9aec.

472
W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
(CeCN), 66.0 (N(CH2)2(CH2)2O), 93.1 (CeCN), 117.0, 117.6 (2CN),
151.0 (]CeN), 156.9 (NeC]N), 160.4 (CeNH2), 164.8 (CeS); lmax
(MeOH)/nm 266 (log ε 3.96), 271 (4.60), 346 (3.82); DIMS found m/
z: 315.20 (calc. for C13H13N7OS Mþ requires 315.35); Anal. Calcd. for
C13H13N7OS: C 49.51, H 4.16, N, 31.09%; found: C 49.34, H 4.47, N
31.05%.
4.3.2. 5-Amino-7-(methylthio)-2-(piperidin-1-yl)pyrazolo[1,5-a]
pyrimidine-3,6-dicarbonitrile (3b)
Yield, 34%; green solid; mp 290e291 ꢁC; IR (KBr, cmꢀ1
)
n
: 3457,
3325, 3150 (NH2), 2208 (CN), 1597 (C]C/C]N); 1H NMR (600 MHz,
DMSO-d6) : 1.60 (s, 6H, N(CH2)2(CH2)3), 2.89 (s, 3H, SCH3), 3.51 (s,
4H, N(CH2)2(CH2)3), 7.76 (br, 2H, NH2); 13C NMR (150 MHz, DMSO-
d6) 16.5 (SCH3), 23.8, 24.8 (N(CH2)2(CH2)2CH2), 47.3
Scheme 11. Synthesis of 1-acetyl-5-amino-1H-pyrazoles 10aec.
d
N(CH2)2(CH2)2), 3.20 (s br, 4H, N(CH2)2(CH2)2), 5.93 (br, 2H, NH2),
10.74 (s, 1H, pyrazole, NH); 13C NMR (125 MHz, DMSO-d6)
: 24.8
d
d
:
(N(CH2)2(CH2)2), 47.9 (N(CH2)2(CH2)2), 61.1 (CeCN), 117.1 (CN),
154.5 (C]N), 158.6 (CeNH2); lmax (MeOH)/nm 205 (log ε 4.15), 245
(4.01), 281 (4.20); DIMS found m/z: 177.20 (calc. for C8H11N5 Mþ
requires 177.21); Anal. Calcd. for C8H11N5: C 54.22, H 6.26, N 39.52%;
found: C 54.35, H 5.89, N 39.67%.
(N(CH2)2(CH2)3), 65.2 (CeCN), 83.4 (CeCN),114.4,115.3 (2CN),152.7
(NeCeN), 153.7 (C]N), 157.1 (CeNH2), 160.0 (CeSCH3); lmax
(MeOH)/nm 230 (log ε 3.97), 274 (4.54); DIMS found m/z: 313.15
(calc. for C14H15N7S Mþ requires 313.38); Anal. Calcd. for C14H15N7S:
C 53.66, H 4.82, N 31.29%; found: C 53.42, H 4.74, N 30.98%.
4.3.3. 5-Amino-7-(methylthio)-2-(pyrrolidin-1-yl)pyrazolo[1,5-a]
pyrimidine-3,6-dicarbonitrile (3c)
4.3. General procedure for the preparation of pyrazolo[1,5-a]
pyrimidines 3aec from the reaction of 5-aminopyrazoles 2aec with
2-[bis(methylthio)methylene]malononitrile
Yield, 32%; green solid; mp >320 ꢁC; IR (KBr, cmꢀ1
)
n
: 3455, 3416,
3317, 3127 (NH2), 2206 (CN), 1587 (C]C/C]N); 1H NMR (400 MHz,
DMSO-d6) : 1.92 (s, 4H, N(CH2)2(CH2)2), 2.88 (s, 3H, SCH3), 3.43 (s,
4H, N(CH2)2(CH2)2, DMSO exchangeable), 7.66 (s, 2H, NH2); 13C NMR
(100 MHz, DMSO-d6) : 16.3 (SCH3), 25.0 (3CH2), 47.7 (CH2eN), 64.4
d
Pyrazolo[1,5-a]pyrimidines 3aec were prepared according to
the literature procedure of Zaharan et al. 2001 [29] as follows: to a
solution of 5-aminopyrazoles 2aec (4 mmol) in absolute ethanol
(20 mL), 2-[bis(methylthio)methylene]malononitrile (0.86 g;
4 mmol) and three drops of triethylamine were added. The reaction
mixture was refluxed for 5 h. The resulting precipitate was filtered off,
dried and crystallized from EtOH:DMF to give pure products 3aec.
d
(CeCN), 82.7 (CeCN), 114.5, 115.4 (2CN), 152.1 (NeCeN), 153.4 (C]
N), 157.1 (CeNH2),158.0 (CeSCH3); lmax (MeOH)/nm 213 (log ε 4.14),
230 (4.61), 269 (4.17); DIMS found m/z: 299.10 (calc. for C13H13N7S
Mþ requires: 299.10); Anal. Calcd. for C13H13N7S: C 52.16, H 4.38, N
32.75%; found: C 52.19, H 4.29, N 32.71%.
4.3.1. 5-Amino-7-(methylthio)-2-morpholinopyrazolo[1,5-a]
pyrimidine-3,6-dicarbonitrile (3a)
4.4. General procedure for the preparation of pyrazolo[1,5-a]
pyrimidines 4aed from the reaction of 5-aminopyrazoles 2a,c with
Yield, 43%; yellow solid; mp >315 ꢁC; IR (KBr, cmꢀ1
)
n
: 3322
(NH2), 2216 (CN), 1506 (C]C/C]N); 1H NMR (600 MHz, DMSO-d6)
a,
a-dicyanoketene-N,S-acetals 1a,b
d
: 2.89 (s, 3H, SCH3), 3.48 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.5 Hz), 3.73 (t,
4H, N(CH2)2(CH2)2O, J ¼ 4.5 Hz), 7.80 (br, 2H, NH2); 13C NMR
Pyrazolo[1,5-a]pyrimidines 4aed were prepared according to
the literature procedure of Zaharan et al. 2001 [29] as follows: to a
(150 MHz, DMSO-d6)
d: 16.6 (SCH3), 47.2 (N(CH2)2(CH2)2O), 62.4
Table 5
Inhibition zone (mean diameter of inhibition in mm), minimum inhibition concentration (MIC), and minimum bactericidal concentration (MBC) (in mg/ml) of some newly
synthesized compounds.
Compound
Gram-positive bacteria
Gram-negative bacteria
Bacillus subtilis
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Serratia marcescens
Inhibition
MIC
MBC
Inhibition
MIC
MBC
Inhibition
MIC
MBC
Inhibition
MIC
MBC
Inhibition
MIC
MBC
zone (mm)
zone (mm)
zone (mm)
zone (mm)
zone (mm)
2a
2b
2c
3b
5b
6a
7b
8b
9a
9b
9c
10b
6 ꢃ 0.56a
9 ꢃ 0.55
16
4
8
16
16
8
16
16
2
4
4
16
16
16
8
8
16
16
16
16
16
4
8
8
16
16
11 ꢃ 0.57
15 ꢃ 0.58
10 ꢃ 0.57
11 ꢃ 0.57
12 ꢃ 0.58
9 ꢃ 0.55
16
16
16
16
16
16
16
16
2
8
8
16
16
>16
16
16
16
16
16
16
16
4
8
8
>16
>16
11 ꢃ 0.57
15 ꢃ 0.56
9 ꢃ 0.55
8
4
8
16
16
16
16
16
4
8
8
16
8
16
8
7 ꢃ 0.57
7 ꢃ 0.57
7 ꢃ 0.57
7 ꢃ 1.15
6 ꢃ 0.00
7 ꢃ 0.57
6 ꢃ 0.00
7 ꢃ 1.15
6 ꢃ 0.00
7 ꢃ 0.57
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
14 ꢃ 0.57c
8
8
8
8
NT
8
NT
16
NT
4
NT
NT
NT
16
8
6 ꢃ 0.57
6 ꢃ 0.57
7 ꢃ 0.00
7 ꢃ 1.00
7 ꢃ 1.15
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
6 ꢃ 0.00
20 ꢃ 0.57a
8
2
8
8
4
8
9 ꢃ 0.57
16
16
16
>16
16
16
8
16
16
NT
>16
NT
16
NT
8
10 ꢃ 0.57
10 ꢃ 0.58
9 ꢃ 0.58
10 ꢃ 0.55
11 ꢃ 0.58
10 ꢃ 0.57
8 ꢃ 0.57
16
16
NT
NT
NT
NT
NT
NT
NT
NT
16
16
NT
NT
NT
NT
NT
NT
NT
NT
10 ꢃ 0.57
11 ꢃ 0.58
14 ꢃ 0.58
16 ꢃ 0.58
8 ꢃ 0.58
9 ꢃ 0.62
11 ꢃ 0.58
10 ꢃ 0.58
12 ꢃ 0.58
7 ꢃ 0.58
10 ꢃ 0.58
10 ꢃ 0.58
7 ꢃ 0.58
8
13 ꢃ 0.58
10 ꢃ 0.58
16 ꢃ 0.58
13 ꢃ 0.58c
16
16
16
NT
NT
NT
6 ꢃ 0.57
10 ꢃ 0.58
14 ꢃ 0.58
25 ꢃ 0.28b
10c
14 ꢃ 0.58
Reference drug
25 ꢃ 0.28b
6 mm is the diameter of the disc.
NT Not tested.
MIC value for positive control (Gentamicin) was 1 mg/mL and MBC was 2 mg/mL against Pseudomonas aeruginosa.
a
Values are mean inhibition zone (mm) ꢃ S.D of results done in triplicate.
b
c
Chloramphenicol is the antibiotic used as reference drug against Gram-positive bacteria.
Streptomycin is the antibiotic used as reference drug against Gram-negative bacteria.

W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
473
Table 6
CC50 values and selective index (SI) of tested compounds. ND ¼ not determined as the MIC is not tested.
Compound
CC50 (mg/ml)
Selective index
Bacillus subtilis
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Serratia marcescens
2a
2b
2c
2.34
0.55
2.5
1.75
1.25
0.063
0.3125
1.4
0.254
1.4
0.55
1.015
2.812
0.15
0.14
0.31
0.11
0.08
0.01
0.02
0.09
0.13
0.35
0.14
0.06
0.18
0.29
0.14
0.31
0.11
0.08
0.00
0.02
0.09
0.06
0.18
0.07
0.13
0.18
0.29
0.14
0.31
0.11
0.08
0.00
0.02
0.09
0.06
0.18
0.07
0.13
0.18
0.29
0.07
0.31
0.22
ND
0.01
ND
0.09
ND
0.29
0.28
0.31
0.11
0.08
ND
ND
ND
ND
ND
3b
5b
6a
7b
8b
9a
9b
9c
0.35
ND
ND
ND
ND
ND
10b
10c
ND
solution of 5-aminopyrazoles 2a and 2c (4 mmol) in absolute
ethanol (20 mL), 2-[methylthio(morpholino)methylene]malononi-
trile 1a (0.84 g; 4 mmol) or 2-[methylthio(piperidin-1-yl)methy-
lene]malononitrile 1b (0.83 g; 4 mmol) and three drops of
triethylamine were added. The reaction mixture was refluxed for
48 h. The resulting precipitate was filtered off, dried and crystal-
lized from EtOH:DMF to give pure products 4aed.
225 (log
16H18N8O2: C 54.23, H 5.12, N 31.62%; found: C 53.99, H 5.16, N
31.51%.
ε 4.08), 270 (4.34), 305 (3.68); Anal. Calcd. for
C
4.4.4. 5-Amino-2-morpholino-7-(piperidin-1-yl)pyrazolo[1,5-a]
pyrimidine-3,6-dicarbonitrile (4d)
Yield, 30%; white crystals; mp >300 ꢁC; IR (KBr, cmꢀ1
)
n
: 3317,
3236, 3165 (NH2), 2203 (CN), 1558 (C]C/C]N); 1H NMR
(400 MHz, DMSO-d6) : 1.67 (br, 6H, N(CH2)2(CH2)3), 3.41 (br, 4H,
4.4.1. 5-Amino-7-morpholino-2-(pyrrolidin-1-yl)pyrazolo[1,5-a]
d
pyrimidine-3,6-dicarbonitrile (4a)
N(CH2)2(CH2)3), 3.68, 3.71 (br, 8H, N(CH2)2(CH2)2O), 3.71 (br, 8H,
Yield, 47%; green solid; mp >272e270 ꢁC; IR (KBr, cmꢀ1
)
n
: 3432
(br, NH2), 2176 (CN), 1621, 1567 (C]C/C]N); 1H NMR (600 MHz,
DMSO-d6)
N(CH2)2(CH2)2O), 7.38 (s, 2H, NH2); 13C NMR (100 MHz, DMSO-
d6)
d: 23.6 (N(CH2)2(CH2)CH2), 26.4 (N(CH2)2(CH2)CH2), 47.1
d
: 1.91 (t, 4H, N(CH2)2(CH2)2, J ¼ 5.6 Hz), 3.39 (t, 4H,
(N(CH2)2(CH2)2O), 51.8 (N(CH2)2(CH2)3), 65.4 (CeCN), 66.0
(N(CH2)2(CH2)2O), 69.3 (CeCN), 116.1, 116.6 (2CN), 152.2 (NeCe
N), 156.1 (NeC]N), 159.7 (CeNH2), 160.5 (]CeN); lmax (MeOH)/
nm 226 (log ε 3.37), 270 (3.57), 3.08 (2.74); Anal. Calcd. for
N(CH2)2(CH2)2, J ¼ 5.6 Hz), 3.45 (t, 4H, N(CH2)2(CH2)2O, J ¼ 5.3 Hz),
3.66 (t, 4H, N(CH2)2(CH2)2O, J ¼ 5.3 Hz), 7.38 (s, 2H, NH2); 13C NMR
(150 MHz, DMSO-d6) d: 25.4 (N(CH2)2(CH2)2), 47.9 (N(CH2)2(CH2)2),
50.4, 50.7 (N(CH2)2(CH2)2O), 66.2 (N(CH2)2(CH2)2O), 66.8 (CeCN),
68.8 (CeCN), 116.6, 119.2 (2CN), 151.3 (NeCeN), 155.7 (C]N), 157.4
(CeNH2), 160.2 (]CeN); lmax (MeOH)/nm 226 (log ε 2.99), 276
(2.04); Anal. Calcd. for C16H18N8O: C 56.79, H 5.36, N 33.12%; found:
C 57.2, H 5.29, N 33.50%.
C16H18N8O2: C 57.94, H 5.72, N 31.80%; found: C 57.89, H 5.78, N
31.82%.
4.5. General procedure for the preparation of pyrazolo[1,5-a]
pyrimidines 5aec and 6aec from the reaction of 5-aminopyrazoles
2aec with acetylacetone and acetoacetanilide
4.4.2. 5-Amino-7-(piperidin-1-yl)-2-(pyrrolidin-1-yl)pyrazolo[1,5-
a]pyrimidine-3,6-dicarbonitrile (4b)
Pyrazolo[1,5-a]pyrimidines 5aec and 6aec were prepared ac-
cording to the literature procedure Gouda et al. 2010 [20,21] as
follows: to a mixture of 5-aminopyrazoles 2aec (4 mmol) and
acetylacetone (0.45 g, 4 mmol) or acetoacetanilide (0.708 g;
4 mmol) in DMF (20 mL) and three drops of glacial acetic acid were
added. The resulting mixture was refluxed for 3 h, and allowed to
cool at room temperature and then the reaction mixture was
poured into crushed-ice, and the separated solid was filtered
off, dried well and crystallized from EtOH:DMF to give compounds
5aec and 6aec.
Yield, 46%; green solid; mp >300 ꢁC; IR (KBr, cmꢀ1
)
n
: 3467, 3305,
3148 (NH2), 2945, 2863, 2207 (CN), 1642, 1600, 1582 (C]C/C]N); 1H
NMR (600 MHz, DMSO-d6) : 1.66 (br. d, 4H, N(CH2)2(CH2)2CH2,
d
J ¼ 4.5 Hz), 1.69 (br. d, 2H, N(CH2)2(CH2)2CH2, J ¼ 4.5 Hz), 1.91 (t, 4H,
N(CH2)2(CH2)2, J ¼ 6.5 Hz), 3.46 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.5 Hz), 3.68
(br, 4H, N(CH2)2(CH2)3,J¼ 4.5Hz),7.31(s,2H,NH2); 13CNMR(150MHz,
DMSO-d6) d: 23.7 (N(CH2)2(CH2)2CH2), 25.3 (N(CH2)2(CH2)2CH2), 26.3
(N(CH2)2(CH2)2), 47.9 (N(CH2)2(CH2)3), 51.5 (N(CH2)2(CH2)2), 64.7 (Ce
CN), 68.7 (CeCN),116.3,116.7 (2CN),151.7 (NeCeN),155.8 (C]N),157.4
(CeNH2),160.3 (]CeN); lmax (MeOH)/nm 226 (log ε 2.99), 284 (1.97);
Anal. Calcd. for C17H20N8: C 60.70, H 5.99, N 33.31%; found: C 61.04, H
5.79, N 33.51%.
4.5.1. 5,7-Dimethyl-2-morpholinopyrazolo[1,5-a]pyrimidine-3-
carbonitrile (5a)
Yield, 71%; white crystals; mp 212e214 ꢁC; IR (KBr, cmꢀ1
)
n
d
:
:
2210 (CN), 1621, 1571 (C]C/C]N); 1H NMR (600 MHz, DMSO-d6)
4.4.3. 5-Amino-2,7-dimorpholinopyrazolo[1,5-a]pyrimidine-3,6-
dicarbonitrile (4c)
2.44 (s, 3H, C]CeCH3), 2.63 (s, 3H, N]CeCH3), 3.47 (s, 4H,
N(CH2)2(CH2)2O), 3.69 (s, 4H, N(CH2)2(CH2)2O), 6.94 (s, 1H, CH); 13
NMR (100 MHz, DMSO-d6): 16.8 (C]CeCH3), 24.3 (N]CeCH3),
Yield, 34%; white solid; mp 332 ꢁC; IR (KBr, cmꢀ1
)
n
: 3381,
3287, 3203 (NH2), 2211 (CN), 1659 (C]C/C]N); 1H NMR
(400 MHz, DMSO-d6) : 4.41 (br, 4H, N(CH2)2(CH2)2O), 3.72 (br, 4H,
N(CH2)2(CH2)2O), 3.77 (br, 8H, 2[N(CH2)2(CH2)2O]), 7.45 (s, 2H,
NH2); 13C NMR (100 MHz, DMSO-d6)
: 47.1 (N(CH2)2(CH2)2O), 51.0
C
d
d
48.0 (N(CH2)2(CH2)2O), 65.8 (N(CH2)2(CH2)2O), 66.1 (CeCN), 110.8
(C]CH), 115.7 (CN), 146.6 (C]CeCH3), 152.1 (C]CeN), 161.0 (Ne
C]N), 162.2 (N]CeCH3); DIMS found m/z: 257.30 (calc. for
d
C
13H15N5O Mþ requires: 257.29); lmax (MeOH)/nm 209 (log ε 4.30),
(N(CH2)2(CH2)2O), 65.4 (CeCN), 66.0 (N(CH2)2(CH2)2O), 66.9
(N(CH2)2(CH2)2O), 69.5 (CeCN), 116.0, 116.5 (2CN), 151.8 (NeCeN),
156.0 (NeC]N), 159.7 (CeNH2), 160.4 (]CeN); lmax (MeOH)/nm
251 (4.51), 316 (3.74); Anal. Calcd. for C13H15N5O: C 60.69, H 5.88, N
27.22%; found: C 60.60, H 5.82, N 27.16%.

474
W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
4.5.2. 5,7-Dimethyl-2-(piperidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-
CN), 99.7 (]CH), 114.7 (CN), 147.0 (C]CeN), 149.9 (NeC]N), 154.8
(N]CeCH3), 156.5 (CeOH); lmax (MeOH)/nm 216 (log ε 4.16), 251
(4.37), 284 inf (3.85); DIMS found m/z: 243.10 (calc. for C12H13N5O
Mþ requires: 243.26); Anal. Calcd. for C12H13N5O: C 59.25, H 5.39, N
28.79%; found: C 59.30, H 5.68, N 28.60%.
carbonitrile (5b)
Yield, 47%; white crystals; mp 209e211 ꢁC; IR (KBr, cmꢀ1
) n:
2199 (CN), 1594, 1580, 1553 (C]C/C]N); 1H NMR (600 MHz,
DMSO-d6) : 1.55 (br. s, 2H, N(CH2)2(CH2)2CH2), 1.89 (br. s, 4H,
d
N(CH2)2(CH2)2CH2), 2.42 (s, 3H, C]CeCH3), 2.51 (s, 3H, N]CeCH3),
3.47 (br, 4H, N(CH2)2(CH2)2O), 6.87 (s, 1H, C]CH); 13C NMR
(150 MHz, DMSO-d6) d: 16.6 (C]CeCH3), 24.0 (N]CeCH3), 25.1
4.6. General procedure for the preparation of pyrazolo[5,1-c][1,2,4]
triazines 7aec and 8aec from the reaction of 5-aminopyrazoles
2aec with acetylacetone and malononitrile
(N(CH2)2(CH2)3), 47.8 (N(CH2)2(CH2)2O), 64.9 (CeCN), 109.8 (C]
CH), 115.8 (CN), 145.6 (C]CeCH3), 151.8 (C]CeN), 158.4 (NeC]N),
160.7 (N]CeCH3); DIMS found m/z: 255.15 (calc. for C14H17N5 Mþ
requires: 255.15); lmax (MeOH)/nm 215 (log ε 4.33), 255 (4.48), 339
(3.78); Anal. Calcd. for C14H17N5 C 65.86, H 6.71, N 27.43%; found: C
65.57, H 6.89, N 27.39%.
Pyrazolo[5,1-c][1,2,4]triazines 7aec and 8aec were prepared
according to the literature procedure Gouda et al. 2010 [21] as
follows: Preparation of the diazonium salt: a solution of sodium
nitrite (0.32 g, 4 mmol; in 2 mL water) was gradually added to a
well cooled solution of 5-aminopyrazoles 7aec (0.84 g, 4 mmol) in
a mixture of acetic acid and concentrated HCl [(8:2) 10 mL (1/
4) Vol.]. The diazonium salt solution was added dropwise with
continuous stirring to cold solution of acetylacetone (0.40 g,
4 mmol) or malononitrile (0.26 g, 4 mmol) in pyridine (10 mL) the
reaction mixtures were stirred at 0e5 ꢁC for 2 h and left to stand at
room temperature. The separated solid products that obtained
were filtered off, dried and crystallized from EtOH:DMF to give
products 7aec and 8aec.
4.5.3. 5,7-Dimethyl-2-(pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-
3-carbonitrile (5c)
Yield, 66%; white crystals; mp 209e210 ꢁC; IR (KBr)
n
: 2198 (CN),
1578 (C]C/C]N, br), 1H NMR (600 MHz, DMSO-d6)
d
: 1.94 (br. s,
4H, N(CH2)2(CH2)2), 2.46 (s, 3H, C]CeCH3), 2.56 (s, 3H, N]CeCH3),
3.52 (br. s, 4H, N(CH2)2(CH2)2), 6.92 (s, 1H, C]CH); 13C NMR
(150 MHz, DMSO-d6) d: 16.6 (C]CeCH3), 24.0 (N]CeCH3), 25.0
(N(CH2)2(CH2)2), 47.8 (N(CH2)2(CH2)2), 64.9 (CeCN), 109.8 (C]CH),
115.8 (CN), 145.7 (C]CeCH3), 151.4 (C]CeN), 158.5 (NeC]N),
160.7 (N]CeCH3); DIMS found m/z: 241.20 (calc. for C13H15N5 Mþ
requires: 241.29); lmax (MeOH)/nm 215 (log ε 4.27), 255 (4.41), 339
(3.71); Anal. Calcd. for C13H15N5: C 64.71, H 6.27, N 29.02%; found: C
64.47, H 6.15, N 29.02%.
4.6.1. 3-Acetyl-4-methyl-7-morpholinopyrazolo[5,1-c][1,2,4]triazine-
8-carbonitrile (7a)
Yield, 65%; yellow crystals; mp 193e194 ꢁC; IR (KBr, cmꢀ1
) n:
2216 (CN), 1698 (C]O), 1586, 1559, 1504 (C]C/N]N/C]N); 1H
NMR (600 MHz, AcOD) : 2.91 (s, 3H, COeCH3), 3.09 (s, 3H, C]Ce
d
4.5.4. 7-Hydroxy-5-methyl-2-morpholinopyrazolo[1,5-a]pyrimidine-
CH3), 3.90 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.7 Hz); 3.95 (t, 4H,
3-carbonitrile (6a)
N(CH2)2(CH2)2O, J ¼ 4. 7 Hz); 13C NMR (150 MHz, DMSO-d6)
d:
Yield, 40%; yellow crystals; mp >315 ꢁC; IR (KBr, cmꢀ1
)
n
: 3638,
3308 (OH), 2216 (CN), 1681, 1644, 1522 (C]C/C]N); 1H NMR
(400 MHz, DMSO-d6) : 2.26 (s, 3H, N]CeCH3), 3.38 (t, 4H,
13.8 (C]CeCH3), 28.8 (COeCH3), 46.6 (N(CH2)2(CH2)2O), 65.9
(CeCN), 66.9 (N(CH2)2(CH2)2O), 114.2 (CN), 138.8 (CeN]N), 142.4
(CeCO), 152.7 (NeC]N), 161.6 (CeCH3), 198.4 (C]O); lmax
(MeOH)/nm 210 (log ε 4.10), 230 (4.11), 289 (4.54); DIMS found
m/z: 286.15 (calc. for C13H14N6O2 Mþ requires: 286.29); Anal.
Calcd. for C13H14N6O2: C 54.54, H 4.93, N 29.35%; found: C 54.49,
H 4.89, N 29.15%.
d
N(CH2)2(CH2)2O,
J
¼
4.8 Hz), 3.72 (t, 4H, N(CH2)2(CH2)2O,
J ¼ 4.8 Hz), 5.75 (s, 1H, C]CH), 13.07 (s, 1H, OH); 13C NMR
(150 MHz, DMSO-d6) d: 18.7 (N]CeCH3), 47.5 (N(CH2)2(CH2)2O),
63.7 (CeCN), 65.8 (N(CH2)2(CH2)2O), 99.4 (]CH), 114.1 (CN), 147.0
(C]CeN), 150.9 (NeC]N), 155.0 (CeCH3), 158.9 (CeOH); lmax
(MeOH)/nm 210 inf (log ε 3.98), 247 (4.11), 284 (3.42); Anal. Calcd.
for C12H13N5O2: C 55.59, H 5.05, N 27.01%; found: C 55.51, H 4.95, N
26.90%.
4.6.2. 3-Acetyl-4-methyl-7-(piperidin-1-yl)pyrazolo[5,1-c][1,2,4]
triazine-8-carbonitrile (7b)
Yield, 75%; colorless; mp 179e180 ꢁC; IR (KBr, cmꢀ1
)
n
: 2217
(CN), 1691 (C]O), 1589, 1504 (C]C/N]N/C]N); 1H NMR
(600 MHz, DMSO-d6) : 1.80e2.02 (br t, 6H, N(CH2)2(CH2)3,
4.5.5. 7-Hydroxy-5-methyl-2-(piperidin-1-yl)pyrazolo[1,5-a]
d
pyrimidine-3-carbonitrile (6b)
J ¼ 7.0 Hz), 2.77 (s, 3H, CH3eCO), 2.90 (s, 3H, C]CeCH3), 3.63 (br t,
Yield, 56%; white crystals; mp >301 ꢁC; IR (KBr, cmꢀ1
)
n
: 3290,
3139 (OH), 2199 (CN), 1659, 1628, 1600, 1520 (C]C/C]N); 1H NMR
(600MHz,DMSO-d6) :1.58(s, 6H, N(CH2)2(CH2)3), 2.27(s, 3H,N]Ce
CH3), 3.42 (s, 4H, N(CH2)2(CH2)3), 5.71 (s,1H, ¼CH),12.92 (s,1H, OH);
13C NMR (150MHz, DMSO-d6)
: 18.5 (CH3), 24.9 (N(CH2)2(CH2)2CH2),
4H, N(CH2)2(CH2)3, J ¼ 7.0 Hz); 13C NMR (150 MHz, DMSO-d6)
d:
13.9 (C]CeCH3), 25.5 (N(CH2)2(CH2)3), 28.9 (COeCH3), 48.6
(N(CH2)2(CH2)3), 66.3 (CeCN), 114.4 (CN), 138.1 (CeN]N), 142.1
(CeCO), 152.5 (NeC]N), 159.7 (CeCH3), 198.5 (C]O); Anal. Calcd
for C14H16N6O: C 59.14; H 5.67; N 29.56%; found: C 59.22; H 5.77; N
29.60%.
d
d
25.3 (N(CH2)2(CH2)2CH2), 47.2 (N(CH2)2(CH2)2), 63.7 (CeCN), 100.1
(]CH), 114.5 (CN), 147.2 (C]CeN), 150.3 (NeC]N), 154.8 (CeCH3),
158.9 (CeOH); lmax (MeOH)/nm 210 (log ε 4.24), 247 (4.44), 284
(3.80); DIMS found m/z: 257.20 (calc. for C13H15N5O Mþ requires:
257.29); Anal. Calcd. for C13H15N5O: C 60.69, H 5.88, N 27.22%; found:
C 60.89, H 5.69, N 27.26%.
4.6.3. 3-Acetyl-4-methyl-7-(pyrrolidin-1-yl)pyrazolo[5,1-c][1,2,4]
triazine-8-carbonitrile (7c)
Yield, 78%; red pale; mp 169e170 ꢁC; IR (KBr, cmꢀ1
)
n
: 2217 (CN),
1695 (C]O), 1591, 1502, (C]C/N]N/C]N); 1H NMR (600 MHz,
DMSO-d6)
: 2.01 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.6 Hz), 2.78 (s, 3H,
d
4.5.6. 7-Hydroxy-5-methyl-2-(pyrrolidin-1-yl)pyrazolo[1,5-a]
CH3eCO), 2.92 (s, 3H, C]CeCH3), 3.65 (t, 4H, N(CH2)2(CH2)2,
pyrimidine-3-carbonitrile (6c)
J ¼ 6.6 Hz); 13C NMR (150 MHz, DMSO-d6)
d: 13.8 (C]CeCH3), 25.4
Yield, 47%; white crystals; mp >315 ꢁC; IR (KBr, cmꢀ1
)
n
: 3469,
3146 (OH), 2203, 2209 (CN), 1663, 1601 (C]C/C]N); 1H NMR
(600 MHz, DMSO-d6)
: 1.93 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.3 Hz), 2.27
(s, 3H, N]CeCH3), 3.47 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.3 Hz), 5.71 (s,
1H, CH), 12.89 (s, 1H, OH); 13C NMR (150 MHz, DMSO-d6)
: 19.4
(N]CeCH3), 25.4 (N(CH2)2(CH2)2), 48.5 (N(CH2)2(CH2)2), 62.7 (Ce
(N(CH2)2(CH2)2), 28.8 (CH3eCO), 48.5 (N(CH2)2(CH2)2), 66.3 (Ce
CN), 114.3 (CN), 138.1 (CeN]N), 142.1 (CeCO), 152.4 (NeC]N),
159.7 (CeCH3), 198.4 (C]O); lmax (MeOH)/nm 210 (log ε 4.08), 231
(4.06), 294 (4.51); DIMS m/z found: 270.20 (calc. for C13H14N6O Mþ
requires: 270.29); Anal. Calcd for C13H14N6O: C 57.77, H 5.22, N
31.09%; found: C 57.76, H 5.32, N 31.14%.
d
d

W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
475
4.6.4. 4-Amino-7-morpholinopyrazolo[5,1-c][1,2,4]triazine-3,8-
4.7.2. 5-(2,5-Dimethyl-1H-pyrrol-1-yl)-3-(piperidin-1-yl)-1H-
dicarbonitrile (8a)
pyrazole-4-carbonitrile (9b)
Yield, 88%, colorless; mp >313 ꢁC (decomposed); IR (KBr, cmꢀ1
)
Yield, 28%; solid brown; mp 120e122 ꢁC; IR (KBr, cmꢀ1
)
n
: 3232
n
: 3331, 3184 (NH2), 2230 (CN), 1656, 1585, 1538 (C]C/C]N/C]C);
(NH/br), 2217 (CN), 1607, 1599, 1515 (C]C/C]N); 1H NMR
1H NMR (600 MHz, DMSO-d6)
d
: 3.64 (t, 4H, N(CH2)2(CH2)2O,
(600 MHz, CD3CL)
6H, 2CH3), 3.31 (t, 4H, N(CH2)2(CH2)3, J ¼ 4.8 Hz), 5.85 (s, 2H,
2CH]), 11.05 (br, 1H, NH); 13C NMR (150 MHz, CD3CL)
: 12.4
d: 1.61 (t, 6H, N(CH2)2(CH2)3, J ¼ 4.8 Hz), 2.11 (s,
J ¼ 4.5 Hz), 3.76 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.5 Hz), 9.29 (br, 2H,
NH2); 13C NMR (150 MHz, DMSO-d6)
d: 46.8 (N(CH2)2(CH2)2O), 65.9
d
(N(CH2)2(CH2)2O), 67.8 (CeCN), 111.1 (CeCN), 114.3, 115.2 (2CN),
141.4 (NeCeN), 152.2 (CeNH2), 160.6 (NeC]N); lmax (MeOH)/nm
230 (log ε 3.53), 278 (4.06), 360 (3.50); DIMS found m/z: 270.15
(2CH3), 23.5 (N(CH2)2CH2(CH2)2), 24.9 (N(CH2)2CH2(CH2)3), 48.2
(N(CH2)2CH2(CH2)3), 73.4 (CeCN), 107.4 (CH]CH), 114.5 (CN), 129.2
(2CeCH3), 149.2 (NeCeNH), 154.6 (C]N); lmax (CH3CN)/nm 230
(log ε 4.18), 246 (4.12); DIMS found: m/z 269.20 (calc. for C15H19N5
Mþ requires 269.34); Anal. Calcd. for C15H19N5: C 66.89, H 7.11, N
26.00%; found: C 66.92, H 6.98, N 26.22%.
(calc. for
11H10N8O: C 48.89, H 3.73, N 41.46%; found C 48.82, H 3.76, N
41.39%.
C
11H10N8O Mþ requires 270.25); Anal. Calcd. for
C
4.6.5. 4-Amino-7-(piperidin-1-yl)pyrazolo[5,1-c][1,2,4]triazine-3,8-
dicarbonitrile (8b)
4.7.3. 5-(2,5-Dimethyl-1H-pyrrol-1-yl)-3-(pyrrolidin-1-yl)-1H-
pyrazole-4-carbonitrile (9c)
Yield, 75%; yellow crystals; mp >286 ꢁC (decomposed); IR (KBr,
Brown solid; yield, 28%; mp 112e113 ꢁC; IR (KBr, cmꢀ1
)
n
: 3172
(NH, br), 2215 (CN), 1611, 1529 (C]C/C]N); 1H NMR (400 MHz,
CD3CL)
: 1.92 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.4 Hz), 2.11 (s, 6H,
2CH3), 3.30 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.4 Hz), 5.83 (s, 2H, CH]
CH), 11.55 (br, 1H, NH); 13C NMR (100 MHz, CD3CL)
: 12.6 (2CH3),
cmꢀ1
)
n
: 3305, 3148 (NH2), 2222 (CN), 1649, 1586 (C]C/C]N/C]
C); 1H NMR (400 MHz, DMSO-d6)
: 2.40 (s, 6H, N(CH2)2(CH2)3),
4.44 (s, 4H, N(CH2)2(CH2)3), 10.02 (br, 2H, NH2); 13C NMR (100 MHz,
DMSO-d6) : 33.3 (N(CH2)2(CH2)2CH2), 34.5 (N(CH2)2(CH2)2CH2),
d
d
d
d
56.8 (N(CH2)2(CH2)3), 76.7 (CeCN), 120.2 (CeCN), 124.0, 124.6
(2CN), 150.4 (NeCeN), 161.7 (CeNH2), 169.6 (NeC]N); lmax
(MeOH)/nm 232 (log ε 4.07), 283 (4.51), 360 (4.04); DIMS found m/
z: 268.15 (calc. for C12H12N8 Mþ requires 268.28); Anal. Calcd. for
25.7 (N(CH2)2(CH2)2), 48.2 (N(CH2)2(CH2)2), 71.0 (CeCN), 107.4
(2CH]), 115.2 (CN), 129.2 (CeCH3), 149.1 (NeCeNH), 152.0 (C]
N); lmax (CH3CN)/nm 230 (log ε 4.18), 245 (4.15); DIMS found m/z:
255.15 (calc. for C14H17N5 Mþ requires 255.32); Anal. Calcd for
C
12H12N8: C 53.72, H 4.51, N 41.77%; found: C 53.52, H 4.38, N
C14H17N5: C 65.86, H 6.71, N, 27.43%; found: C 65.91, H 6.79, N
41.66%.
27.61%.
4.6.6. 4-Amino-7-(pyrrolidin-1-yl)pyrazolo[5,1-c][1,2,4]triazine-
3,8-dicarbonitrile (8c)
4.8. General procedure for the reaction of 5-aminopyrazoles 2aec
with acetic anhydride
Yield, 70%; yellow crystals; mp >310 (decomposed); IR (KBr,
cmꢀ1
)
n
: 3378, 3107 (NH2), 2224 (CN), 2211 (CN), 1648, 1598 (C]
C/C]N); 1H NMR (400 MHz, DMSO-d6)
1.98 (s br, 4H,
N(CH2)2(CH2)2), 3.59 (br. s, 4H, N(CH2)2(CH2)2), 9.16 (br, 2H,
NH2); 13C NMR (100 MHz, DMSO-d6)
: 25.0 (N(CH2)2(CH2)2),
The 1-acetyl-5-amino-1H-pyrazoles were prepared according to
the literature procedure of Zaharan et al. [29] as follows: a solution
of 5-aminopyrazoles 2aec (2 mmol) in acetic anhydride (10 mL)
was heated under reflux for 15 min. The solid products so formed
was collected by filtration and recrystallized from DMF:EtOH to
give 10aec.
d
:
d
47.9 (N(CH2)2(CH2)2), 66.8 (CeCN), 110.3 (CeCN), 114.2, 114.9
(2CN), 140.6 (NeCeN), 151.6 (CeNH2), 158.2 (NeC]N); lmax
(MeOH)/nm 232 (log ε 4.19), 280 (4.55), 358 (4.04); DIMS found:
m/z 254.20 (calc. for C11H10N8 Mþ requires 254.25); Anal. Calcd
for C11H10N8: C 51.96, H 3.96, N 44.07%; found: C 51.95, H 3.84, N
43.86%.
4.8.1. 1-Acetyl-5-amino-3-morpholino-1H-pyrazole-4-carbonitrile
(10a)
Yield, 95%; white solid; mp 253 ꢁC; IR (KBr, cmꢀ1
)
n
: 3422, 3172
(NH2), 2209 (CN), 1703 (CO), 1633 (C]C/C]N); 1H NMR (600 MHz,
AcOD) : 1.96 (s, 3H, CH3eCO), 2.82 (t, 4H, N(CH2)2(CH2)2O,
J ¼ 4.8 Hz), 3.22 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.8 Hz), 7.47 (s, 2H,
NH2); 13C NMR (150 MHz, AcOD)
23.1 (CH3eCO), 46.9
4.7. General procedure for the reaction of 5-aminopyrazoles 2aec
with 2,5-hexan-di-one
d
d
:
The 5-pyrrolylpyrazoles were prepared according to the litera-
ture procedure Gouda et al. 2010 [20,21] as follows: a mixture of 5-
aminopyrazoles 2aec (4 mmol) and 2,5-hexanedione (0.457 g;
4 mmol) in glacial acetic acid was refluxed for 5 h, then the reaction
mixtures was poured into crushed ice, and the separated solid was
filtered off and dried well to give 9aec.
(N(CH2)2(CH2)2O), 63.8 (CeCN), 65.4 (2 N(CH2)2(CH2)2O), 114.6
(CN), 156.2 (CeNH2), 156.3 (NeCeN]), 172.5 (C]O); lmax
(MeOH)/nm 228 (log ε 3.81), 265 (3.67); DIMS found m/z: 235.25
(calc. for
10H13N5O2: C 51.06, H 5.57, N 29.77%; found: C 51.32, H 5.69, N
29.65%.
C
10H13N5O2 Mþ requires 235.24); Anal. Calcd. for
C
4.7.1. 5-(2,5-Dimethyl-1H-pyrrol-1-yl)-3-morpholino-1H-
4.8.2. 1-Acetyl-5-amino-3-(piperidin-1-yl)-1H-pyrazole-4-
carbonitrile (10b)
pyrazole-4-carbonitrile (9a)
Yield, 27%; pale brown; mp 117e119 ꢁC; IR (KBr, cmꢀ1
)
n
: 3228
(NH/br), 2219 (CN), 1591, 1501 (C]C/C]N); 1H NMR (600 MHz,
CD3CL)
: 2.12 (s, 6H, 2CH3), 3.37 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.4 Hz),
3.78 (t, 4H, N(CH2)2(CH2)2O, J ¼ 4.4 Hz), 5.87 (s, 2H, CH]CH), 10.79
(br, 1H, NH); 13C NMR (150 MHz, AcOD)
: 11.2 (2CH3), 47.0
Yield, 99.43%; colorless crystals; mp 216e218 ꢁC; IR (KBr, cmꢀ1
)
n
: 3418, 3299 (NH2), 2213 (CN), 1702 (CO), 1633, 1536 (C]C/C]N);
d
1H NMR (400 MHz, DMSO-d6)
d
: 1.55 (s, 6H, N(CH2)2(CH2)3), 2.41 (s,
3H, COCH3), 3.30 (s, 4H, N(CH2)2(CH2)3), 7.86 (s, 2H, NH2); 13C NMR
d
(100 MHz, DMSO-d6) d: 23.2 (COCH3), 23.8 (N(CH2)2CH2(CH2)2),
(N(CH2)2(CH2)2O), 65.5 (N(CH2)2(CH2)2O), 74.5 (CeCN), 107.2, 107.4
(CH]CH), 113.5 (CN), 129.0 (CeCH3), 148.0 (NeCeNH), 154.8 (Ne
C]N); lmax (CH3CN)/nm 228 (log ε 3.80), 244 (3.73); DIMS found
m/z: 271.20 (calc. for C14H17N5O Mþ requires 271.32); Anal. Calcd.
for C14H17N5O: C 61.98, H 6.32, N 25.81%; found C 61.71, H 6.49, N
25.93%.
24.7 (N(CH2)2CH2(CH2)2), 47.5 (N(CH2)2CH2(CH2)2), 63.8 (CeCN),
114.9 (CN), 156.3 (CeNH2), 156.4 (NeCeN), 172.5 (C]O); lmax
(MeOH)/nm 229 (log ε 4.35), 270 (4.17), 306 (3.67); DIMS found m/
z: 233.15 (calc. for C11H15N5O Mþ requires 233.27); Anal. Calcd. for
C11H15N5O: C 56.64, H 6.48, N 30.02%; found: C 56.66, H 6.47, N
29.82%.

476
W.M. Al-Adiwish et al. / European Journal of Medicinal Chemistry 64 (2013) 464e476
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4.8.3. 1-Acetyl-5-amino-3-(pyrrolidin-1-yl)-1H-pyrazole-4-
carbonitrile (10c)
Yield, 99%; colorless crystals; mp >216e218 ꢁC; IR (KBr, cmꢀ1
) n:
3421, 3311 (NH2), 2210 (CN), 1707 (CO), 1638, 1579 (C]C/C]N); 1H
NMR (600 MHz, AcOD)
(s, 3H, COCH3), 2.91 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.6 Hz), 7.40 (s, 2H,
NH2); 13C NMR (150 MHz AcOD)
23.1 (COCH3), 24.8
d
: 1.40 (t, 4H, N(CH2)2(CH2)2, J ¼ 6.6 Hz),1.94
d
:
(N(CH2)2(CH2)2), 47.4 (N(CH2)2(CH2)2), 63.3 (CeCN), 114.9 (CN),
154.1 (CeNH2), 156.1 (NeCeN), 172.3 (C]O); lmax (MeOH)/nm 230
(log ε 3.35), 270 (3.47); DIMS found m/z: 219.25 (calc. for C10H13N5O
Mþ requires 219.24); Anal. Calcd. for C10H13N5O: C 54.78, H 5.98, N
31.94%; found: C 54.85, H 5.94, N 31.82%.
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5. Conclusions
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In this paper, the synthesis of new pyrazolo[1,5-a]-pyrimidines
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8aec by the reaction of 5-aminopyrazoles 2aec with respective
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2-[bis(methylthio)methylene]malononitrile,
a,a-dicyanoketene-
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N,S-acetals 1aeb, acetylacetone, acetoacetanilide as well as
acetylacetone, and malononitrile are reported. Also, the reaction of
2aec with 2,5-hexanedione afforded the corresponding 5-
pyrrolylpyrazoles 9aec. Also, 2aec reacted with acetic anhydride
to give the corresponding 1-acetyl-1H-pyrazoles 10aec. All pro-
cedures for the synthesis of these compounds are very convenient
due to the simple procedures, mild conditions, and moderate to
high yields. Another advantage is that 5-aminopyrazoles 2aec was
of the same starting material used for the preparation of all those
compounds. Some of the prepared compounds showed low to
moderate antibacterial activity and not cytotoxic to Vero cells.
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[19] CCDC 917656 contains the supplementary crystallographic data for this paper.
These data can be, obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the CCDC, 12, Union Road, Cambridge CB2 1EZ, UK,
fax: þ44 1223 336033, e-mail: deposit@ccdc.cam.ac.uk).
Acknowledgments
[20] M.A. Gouda, M.A. Berghot, A.I. Shoeib, A.M. Khalil, Synthesis and antimicrobial
activity of new anthraquinone derivatives incorporating pyrazole moiety, Eur.
J. Med. Chem. 45 (2010) 1843e1848.
The authors thank the Ministry of Higher Education Malaysia
and Universiti Kebangsaan Malaysia for research grant UKM-
GGPM-KPB-098-2010. A scholarship from the Libyan government
to WMA is greatly appreciated.
[21] M.A. Gouda, A.I. Berghot, E.A. Ghada, A.M. Khalil, Synthesis and antimicrobial
activities of some new thiazole and pyrazole derivatives based on 4,5,6,7-
tetrahydrobenzothiophene moiety, Eur. J. Med. Chem. 45 (2010) 1338e1345.
[22] CCDC 917657 contains the supplementary crystallographic data for this paper.
These data can be, obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK,
fax: þ44 1223 336033, e-mail: deposit@ccdc.cam.ac.uk).
Appendix A. Supplementary data
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microbial Disk Susceptibility Test. Approved Standard, ninth ed., CLSI, Wayne,
PA, USA, 2006.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.029.
[24] National Committee for Clinical Laboratory Standards, Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically.
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