Design, synthesis, anticancer evaluation, molecular docking and cell cycle analysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine derivatives as potent histone lysine demethylases (KDM) inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2019.102929

A novel series of pyrazolo[1,5-a]pyrimidines were synthesized and proved by their spectral and elemental analysis, some elected of the newly synthesized compounds were examined for their cytotoxic activity employing MTT assay on two cancer cell lines (Breast and Hela cancers). Compounds 5, 7e and 7i showed the higher cytotoxicity against two cancer cell lines with (IC₅₀ = 13.91 ± 1.4 and 22.37 ± 1.8 μM/L), (IC₅₀ = 6.56 ± 0.5 and 8.72 ± 0.9 μM/L) and (IC₅₀ = 4.17 ± 0.2 and 5.57 ± 0.4 μM/L) for two cancer cell lines breast and hela respectively, using doxorubicin as a reference drug. The most potent cytotoxic active compounds 5, 7e and 7i presented inhibitory activity against KDM (histone lysine demethylases) with IC₅₀ = 4.05, 1.91 and 2.31 μM, respectively. The most potent KDM inhibitor 7e (IC₅₀ = 1.91 μM) showed to cause cell cycle arrest at G2/M phase by 4 folds than control and induce total apoptotic effect by 10 folds more than control. In silico studies performed on the more potent cytotoxic active compounds 5, 7e and 7i included lipinisk's rule of five. Moreover, molecular docking study was utilized to explore the binding mode of the most active compounds to the target enzyme (PDB-ID: 5IVE). Also, some bioinformatics studies were carried out for compounds 7e and 7i using Swiss ADME (Swiss Institute of bioinformatics 2018).

Full text of DOI:10.1016/j.bioorg.2019.102929

Accepted Manuscript
Design, synthesis, anticancer evaluation, molecular docking and cell cycle anal-
ysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine derivatives as potent
histone lysine demethylases (KDM) inhibitors and apoptosis inducers
Nadia Hanafy Metwally, Mona Said Mohamed, Eman Ali Ragb
PII:
S0045-2068(18)31485-8
https://doi.org/10.1016/j.bioorg.2019.102929
102929
DOI:
Article Number:
Reference:
YBIOO 102929
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 December 2018
11 April 2019
15 April 2019
Please cite this article as: N. Hanafy Metwally, M. Said Mohamed, E. Ali Ragb, Design, synthesis, anticancer
evaluation, molecular docking and cell cycle analysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine
derivatives as potent histone lysine demethylases (KDM) inhibitors and apoptosis inducers, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102929
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Design, synthesis, anticancer evaluation, molecular docking and cell
cycle analysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine
derivatives as potent histone lysine demethylases (KDM) inhibitors
and apoptosis inducers
a
a
a
Nadia Hanafy Metwally *, Mona Said Mohamed and Eman Ali Ragb
a
Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
E-mail: nhmmohamed@yahoo.com
ABSTRACT A novel series of pyrazolo[1,5-a]pyrimidines were synthesized and
proved by their spectral and elemental analysis, some elected of the newly synthesized
compounds were examined for their cytotoxic activity employing MTT assay on two
cancer cell lines (Breast and Hela cancers). Compounds 5,7e and 7i showed the higher
cytotoxicity against two cancer cell lines with (IC50 = 13.91± 1.4 and 22.37 ±
1
0
.8μM/L), (IC = 6.56± 0.5 and 8.72± 0.9μM/L) and (IC = 4.17± 0.2 and 5.57±
50 50
.4μM/L) for two cancer cell lines breast and hela respectively, using doxorubicin as a
reference drug. The most potent cytotoxic active compounds 5, 7e and 7i presented
inhibitory activity against KDM (histone lysine demethylases) with IC = 4.05, 1.91
5
0
and 2.31μM, respectively. The most potent KDM inhibitor 7e (IC = 1.91μM) showed
5
0
to cause cell cycle arrest at G2/M phase by 4 folds than control and induce total
apoptotic effect by 10 folds more than control. In silico studies performed on the more
potent cytotoxic active compounds 5, 7e and 7i included lipinisk's rule of five.
Moreover, molecular docking study was utilized to explore the binding mode of the
most active compounds to the target enzyme (PDB-ID: 5IVE). Also, some
bioinformatics studies were carried out for compounds 7e and 7i using Swiss ADME
(Swiss Institute of bioinformatics 2018).
KEYWORDS: Pyrazolo[1,5-a]pyrimidines; Cytotoxic activity; Histone lysine
demethylases (KDM) inhibitor; Cell cycle; Apoptotic effect; Lipinsk's rule; Molecular
docking and bioinformatics studies.
1
. Introduction
Cancer, the rapid uncontrolled growth of abnormal cells [1] and is the second
leading cause of death after cardiovascular diseases in the terms of morbidity and
1

mortality according to world health organization (WHO) [2]. Cancers that affect
women are divided into six types such as cervical, endometrial, fallopian tube, breast,
ovarian and vaginal cancers: breast and cervical cancers are the most common types
occurring, considered as the second and fourth leading cause of cancer death. Several
agents directed against critical breast cancer enhancing proteins are currently being
developed such as inhibitors against the poly(ADP-ribose) polymerase [3], cyclin
dependent kinases 4 and 6 (CDK4/6) [4], as well as against the KDM4 enzymes [5].
Moreover, some inhibitors used against cervical cancer such as dynamin 2 which plays
an important role in vascular endothelial growth factor (VEGF)-mediated angiogenesis
[
6].
Histones are highly alkaline proteins in eukaryotic cells; DNA is packed
together with histone proteins in a highly organized form called chromatin.
Methylation of histone lysine plays an important role in maintaining the structure of
chromatin and regulation of transcription. Disturbance of histone methylation is a good
factor for cancer development and progression [7, 8]. The maintaining of histone
lysine methylation depend on two enzyme families histone lysine methyl transferase
(KMTs) and histone lysine demethylases (KDMs) [9]. Shi, Y et al discovered two
families of KDM [10]. One is specific-lysine demethylase (LSD) that belongs to flavin
adenine dinucleotide (FAD)-dependent monoamine oxidase and the other KDMs
belong to Jumonji domain containing lysine demethylases (JmjC KDMs) which
divided into six subfamilies (KDM2-7) with different demethylated catalysis activities.
KDM4A and other members in KDM4 subfamily are catalyze the demethylation of
histone H3 subunit lysine9 tri-/di-methylated mark (H3K9me ) [11]. Additionally,
3
/2
histone lysine demethylases (KDM5A-D) family members are responsible for
removing methyl group of (tri-methylated) lysine 4 on histone 3(H3K4me3). In
addition, KDM5 family found to play a role as oncogenic driver (molecules which
cause the formation or support the progression of cancer). It has been shown that
KDM5A and KDM5B are required for cancer cell survival, this is due to an increase in
gene expression in a number of human cancer cells such as breast (MCF-7) [12].
Therefore, KDM5 inhibitors considered as drug used in cancer treatment.
On the other hand, pyrazolo[1,5-a]pyrimidine scaffold considered as an
important class of heterocyclic compounds in the area of drug design as they are
analogs to purine ring with a variety of medicinal applications as anticancer [13], HCV
2

inhibitors [14], anxiolytic [15], positron emission tomography (PET) tumor imaging
agents [16], HIV reverse transcriptase inhibitors [17] and kinase inhibitors [18].
Pyrazolo[1,5-a]pyrimidine (core moiety) considered as important marketed drugs like
lorediplon and pyrazophos [19], ocinaplon [20], dorsomorphin [21], anagliptin [22],
zaleplon and indiplon [23] as presented in figure 1.
Fig.1. Marketed drug based on pyrazolo[1,5-a]pyrimidine
Moreover, it has been reported that 6-isopropyl-7-oxo-5-phenyl-4,7-
dihydropyrazolo[1,5-a]pyrimidine-2-carbonitrile (CPI-455) is identified as an inhibitor
of KDM5A through a high-throughput screening (HTS) assay against KDM4C JmjC
domain and subsequent structural modification work [24,25]. It exhibited good
potency toward KDM5A with IC50 = 0.02 µM. Also, compound CPI-455 could
3

specifically increase H3K4me3/2 in a dose-dependent manner in PC9, M14 and SKBR3
cells (Fig. 2).
CPI-455
IC = 0.01 M
5
0
KDM5A
Fig. 2
Thus, in view of the above mention and in continuation of our work to
synthesize bioactive heterocyclic ring systems of interest biological activity [26-30],
we report herein the synthesize of some novel pyrazolo[1,5-a]pyrimidines (Fig. 4)
which investigated in vitro cytotoxic activity against two human cancer cell lines
breast (MCF-7) and HeL a promising to be KDM5A (Jumonji domain) histone lysine
demethylase inhibitors and used in treatment of breast and HeLa cancer cells which
their structure like the native ligand of protein active site 5IVE as shown in figure 3.
Fig. 3. The native ligand of the protein active site (5IVE)
4

Fig. 4. Our target compounds
2
. Results and discussion
2
.1. Chemistry
Cyclocondesation of 5-amino-3-cyanomethyl-1H-pyrazole-4-carbonitrile (1)
with acetoacetanilide (2) in N,N-dimethylformamide containing a few drops of glacial
acetic acid produced the isolated product 5 (Scheme 1). The IR spectrum of the
-1
isolated product 5 presented absorption bands at νmax 3152, 2227 and 1671 cm
1
corresponding to NH, 2CN and CO groups, respectively. The H NMR spectrum of 5
not appeared a multiplet signals at δ near ~7.0-8.0 ppm corresponding to aryl protons
and appeared three singlet signals at δ = 2.38, 4.36 and 5.87 ppm owing to methyl
(
CH ), methylene (CH CN), and pyrimidine protons along with a D O exchangeable
3
2
2
signal at δ = 13.35 ppm due to NH proton. In addition, mass spectrum of 5 indicated a
+
correct molecular ion peak at m/z = 213 (M ) accordance with the molecular formula
C H N O. According to our results, we have postulated a route for the formation of
10
7
5
compound 5. The reaction mechanism proceeds through the nucleophilic attack of the
exo-NH function from 5-aminopyrazoles 1 on the carbonyl group of acetoacetanilide
2
2
, by releasing water molecule, to form intermediate imine A, followed by
intramolecular cyclization occurs by a nucleophilic attack of the NH pyrazole ring on
the other carbonyl group to form an intermediate adduct B followed by the elimination
of aniline yields 5 and ruled out the formation of each of compound 3 and 4 (Scheme
1
).
5

Scheme 1. Synthetic route to 2-(cyanomethyl)-5-methyl-7-oxo-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-3-carbonitrile 5.
Condensation of compound 5 with various aromatic aldehydes 6a-i in refluxing
N,N-dimethylformamide containing few drops of piperidine afforded
the
corresponding arylidene derivatives 7a-i (Scheme 2). The IR spectrum of the isolated
product 7e as representative example of the prepared series showed absorption bands
-1
at νmax 3410, 2214 and 1628cm corresponding to NH, two CN and CO groups,
1
respectively. The H NMR spectrum of 7e revealed the presence of a singlet signal at δ
=
5.56 ppm assigned for pyrimidine proton, in addition to aryl protons at δ = 6.81-7.95
ppm. Also, a singlet signal at δ = 8.09 ppm attributable to vinylic proton. The
elemental analyses with mass spectrum of 7e showed a correct molecular ion peak at
+
m/z = 291 (M , 1.3%) which affirmed the molecular formula C H N O (Scheme 2).
1
5
9
5
2
6

Scheme 2.Synthetic route to 2-(1-cyano-2-arylvinyl)-5-methyl-7-oxo-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-3-carbonitrile7a-i.
Moreover, compounds 5 condensed with o-hydroxybenzaldehydes 8a,b under
the same reaction conditions afforded in each case, red solid of melting point above
o
3
00 C 10a,b (Scheme 3). The IR spectrum of compound 10a exhibited absorption
-1
bands at νmax 3431, 2212 and 1628 cm due to NH, CN and CO groups, respectively.
1
It`s H NMR spectrum clarified the presence of two D O-exchangeable signals at δ =
2
9
.11 and 10.21 ppm assigned to two NH protons. Refluxing the compounds 10a,b in
sodium ethoxide solution gave products 11a,b. The structure of the isolated products
1a,b was deduced from correct analytical and spectral data. The IR spectrum of 11a
showed disappearance of an absorption band corresponding to nitrile group near to νmax
1
-1
-1
~
2200 cm and instead revealed an absorption band at ν
3209 cm due to NH
max
group.
7

Scheme 3. Synthetic pathway to 2-(2-imino-2H-chromen-3-yl)-5-methyl-7-oxo-4,7-
dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrilederivatives
pyrazolo[1,5-a]pyrimidine 11a,b.
10a,b
and
fused
On the other hand, refluxing of compounds 5 with indoline-2,3-dione 12 under
o
the same reaction conditions afforded a brown solid 14 of melting point above 300 C
(Scheme 4). The IR spectrum of compound 14 appeared absorption bands at νmax 3418,
-1
1
3
209, 2214 and 1656 cm relative to 2NH, CN and CO groups, respectively. It`s H
NMR also, indicated the presence of two D O-exchangeable signals at δ = 8.42 and
2
8
.62 ppm owing to the two NH protons. The elemental analyses are in agreement with
the structures 14. Refluxing the latter compound in sodium ethoxide solution gave 15.
The chemical structure of the isolated product 15 was proved from correct analytical
and spectral data (see Scheme 4 and experimental).
8

Scheme 4. Synthetic pathway to pyrano[2,3-b]indol-3-yl)-5-methyl-7-oxo-4,7-
dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 14 and fused pyrazolo[1,5-a]-
pyrimidine 15.
In the same manner, compounds 5 condensed with azosalicylaldehyde
derivatives 16a-d in N,N-dimethylformamide (DMF) in the presence of few drops of
o
piperidine afforded in each case, brown solid 18a-d of melting point above 300 C
(Scheme 5). The structure of the isolated products 18a-d was confirmed by elemental
analyses and spectral data (see Scheme 5 and experimental). The latter compounds
were converted into azopolyheterocyclic compounds through refluxing in sodium
ethoxide solution to give products 19a-d. The structure of the isolated products 19a-d
was proved from correct analytical and spectral data. As typical example, the IR
spectrum of 19c showed disappearance of an absorption band corresponding to nitrile
-1
-
group near to νmax ~2200 cm and instead revealed an absorption band at ν 3180 cm
max
1
1
due to NH group. The H NMR spectrum of 19c revealed a D O-exchangeable signal
2
at δ = 9.02 ppm attributable to NH proton.
9

Scheme 5. Synthetic pathway to 2-(2-imino-6-(aryldiazenyl)-2H-chromen-3-yl)-5-
methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile derivatives 18a-d
and fused pyrazolo[1,5-a]pyrimidine 19a-d.
Finally, we extended our presented study to synthesize new azodye derivatives.
Thus, compound 5 was coupled with the diazotized aromatic amines 20a-d in N,N-
o
dimethylformamide (DMF) in the presence of sodium hydroxide at 0-5 C leading to
red colored compounds 21a-d (Scheme 6). The IR spectrum of the isolated compound
2
1d taken as a typical example of the prepared series, showed absorption bands at νmax
-1
3
424, 2216 and 1635 cm corresponding to the 2NH, 2CN and CO groups,
1
respectively. The H NMR spectrum of 21d revealed a singlet signal at δ = 5.76 ppm
due to pyrimidine proton, in addition, a D O-exchangeable signal appeared at δ =
2
1
2.81 ppm assigned to the NH proton, beside the other expected signals for aryl
protons. The mass spectrum of 21d exhibited a correct molecular ion peak at m/z =
+
3
53 (M +2).
The electronic absorption spectra of the products 21a-d in DMF revealed, in
each case, two absorption bands in the regions λmax 447-418 and 305-263 nm. This
absorption pattern seems to indicate that the studied compounds 21a-d exist
predominantly in solution as hydrazone form A. This is because such an absorption
1
0

1
pattern is similar to that of typical hydrazones[31,32]. H NMR spectra provide an
additional evidence that they have the hydrazone form A rather than the azo-forms B.
1
For example, the H NMR spectrum of compounds 15 in DMSO-d exhibit, in each
6
case, one singlet signal near δ 12.0 ppm due to the =NNH-proton and this is
substantiated by the literature data which indicate that the chemical shift (δ) of
hydrazone NH resonance is usually observed near 13.0 ppm [33] (see Table 1).
Scheme 6. Synthetic pathway to 3-cyano-5-methyl-7-oxo-N-phenyl-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-2-carbohydrazonoyl cyanide derivatives 21a-d.
Table 1. Electronic absorption spectral data of compounds 21a-d in N,N-
dimethylformamide (DMF).
Compound No.
1a
1b
1c
2d
λmax(nm) DMF
390-303
2
2
445-305
2
304-261
1
447-263
2
2
.2. Biology
.2.1. Anticancer evaluation
The in vitro cytotoxic activity of some newly synthesized pyrazolo[1,5-a]-
pyrimidine derivatives 3, 5a, 5b, 5c, 5e, 5h, 5i, 15, 21a and 21c,d was determined by
MTT assay (a colorimetric method) against two human cancer cell lines: human breast
adenocarcinoma (MCF-7) and human epithelial cervical carcinoma (Hela) (Table 2)
using Doxorubicin as a reference drug.
The results of cytotoxic activity are expressed as concentration IC (μM/ml).
5
0
IC define as the concentration of drug required to inhibit 50% of cell growth.
5
0
1
1

Table 2. IC of the tested compounds against two human cell lines using Doxorubicin
5
0
as a reference drug.
Tested
Human cancer cell lines
compounds
5
MCF-7
HeLa
IC (μM/ml)
IC (μM/ml)
5
0
50
13.91± 1.4
19.70± 1.8
64.14± 3.6
74.26± 4.2
6.56± 0.5
22.37 ± 1.8
28.58 ± 2.6
71.62± 4.3
88.16± 5.1
8.72± 0.9
7
a
7
b
7
7
c
e
7
h
34.22± 2.7
9.48 ± 0.8
26.08± 2.2
56.28± 3.5
64.87± 3.7
68.14± 4.0
4.17± 0.2
45.01± 3.5
10.63± 1.0
37.49± 3.1
32.74± 2.8
41.16± 3.4
60.93± 4.3
5.57± 0.4
7
i
1
5
2
1a
1c
1d
Doxorubicin
2
2
IC (μM/ml) are expressed as mean ±SD
5
0
1
2

Fig. 5. Cytotoxic activity of the target compounds (5, 7a-c, 7e, 7h, 7i, 15, 21a, 21c and
1d) against the tumor MCF-7 and hela cell lines.
2
According to the previous presented results in (Table 2), it was concluded that the
parent compound 5 has strong and moderate cytotoxic activity against both breast and
hela cancer cell lines with IC = 13.91 ± 1.4 and 22.37±1.8 μM/L, respectively. When
5
0
phenyl group was introduced in the parent compound, the cytotoxic activity decreases
with respect to both human cancer cell lines with IC = 19.70 ± 1.8 and 28.58 ± 2.6
5
0
μM/L, respectively. In case of introducing substituted phenyl group to parent
compound 5 the cytotoxic activity highly decreased in case of electron donating and
electron withdrawing such as 4-methoxyphenyl and 4-cholorophenyl groups but still
electron donating has higher cytotoxic activity than electron withdrawing with respect
to both human cancer cell lines (for 4-methoxy: IC = 64.14 ± 3.6 and 71.62 ± 4.3μ
5
0
M/L) and (for 4-chloro: IC = 74.26 ± 4.2 and 88.16 ± 5.1 μM/L), respectively. On the
50
other hand, when introduced phenyl group bearing more than one electron donating
groups such as trimethoxyphenyl which carry the same electron donating species the
cytotoxic activity (IC = 34.22 ± 2.7 and 45.01 ± 3.5 μM/L) improved than mono-
5
0
methoxyphenyl group with breast and hela cell lines, respectively. But when the
electron donating groups introduced are different species as hydroxyl and methoxy
groups, the cytotoxic activity become very strong with both breast and hela with IC =
5
0
9
.48 ± 0.8 and 10.63 ± 1.0 μM/L, respectively near standard drug Doxorubicin.
1
3

In contrast to phenyl and substituted phenyl group, introducing hetero- moiety
like furan to the parent compound which gave the highest cytotoxic activity among the
all tested compounds against both breast and hela cancer cell lines with IC = 6.56 ±
50
0
4
.5 and 8.72 ± 0.9 μM/L, respectively and this result close to Doxorubicin with IC =
50
.17 ± 0.2 and 5.57 ± 0.4 μM/L.
Depending on the above data and the structure-activity relationship (SAR) for
the tested compounds against breast and hela cancer cell lines indicated that the
pyrazolo[1,5-a]pyrimidine derivatives that bearing hetero- moiety have the highest
cytotoxic activity as compound 7e with IC = 6.56 ± 0.5 and 8.72 ± 0.9μM/L than
5
0
those bearing phenyl and substituted phenyl group. In spite of, substituted phenyl
group with electron withdrawing group such as 4-choloro phenyl gave the lowest
cytotoxic activity with IC50 = 74.26 ± 4.2 and 88.16± 5.1 μM/L of all tested
compounds against two human cancer cell lines (breast and hela).
2
.2.2. KDM inhibitory assay
KDM inhibition activity was investigated by ELISA assay for the most potent
cytotoxic active compounds 5, 7e and 7i using MCF-7 cancer cell, the results deduced
from Table 3 showing that all tested compounds 5, 7e and 7i exhibited potent
inhibitory effect toward KDM with IC50 = 4.05, IC50 = 1.91 and IC50 = 2.31 μM,
respectively. This mean that the tested compounds may induce their inhibitory effect
by binding to KDM enzyme active site and inhibition of its activity cause cancer cell
death.
Table 3. Inhibitory activity in vitro for compounds 5, 7e and 7i against KDM
Compound number
5
KDM IC (μM)
50
4.05± 0.29
1.91± 0.12
2.31± 0.17
7
e
7
i
2
.2. 3. In vitro DNA- flow cytometric (cell cycle) analysis
Also, the present work extending by electing the more potent inhibitory active
compound 7e and investigate its effect on cell cycle progression and apoptosis
induction in MCF-7 cell line. Cell cycle phases were determined by flow cytometry
after propidium iodide (PI) staining. The MCF-7 cells were incubated with 10 μM of
compound 7e for 48h, and then its effect on cell cycle phases was analyzed, DMSO
1
4

used as a negative control. The results obtained from exposure of MCF-7 cell to
compound 7e refer that this compound cause a significant increase in the percentage of
cells at phases of preG1 (which could be indicative of apoptosis) and G2/M (DNA
repair and mitosis) by 10 and 4 folds respectively, compared to control. These results
indicated that compound 7e arrest MCF-7 cancer cells at G2/M phase of cell cycle
(Table 4 and Figures 6, 7).
Table 4. Results of cell cycle analysis in MCF-7 expressed by (%) of cell in each
phase when treated with compound 7e.
Compound number %G0-G1
%S
%G2-M %Pre-G1
Comment
PreG1apoptosis&
Cell growth arrest
at G2/M
7
e/MCF7
30.93
55.21
14.16
29.81
54.91
14.98
19.22
2.02
MCF7
Cell cycle analysis
100
9
8
7
6
5
0
0
0
0
0
%
%
%
%
G0-G1
S
G2-M
Pre-G1
40
30
20
10
0
1
/MCF7
MCF7
Fig. 6. Cell cycle and apoptosis effect in MCF-7 cell line treated with compound 7e.
1
5

I. Control MCF-7
II. 7e/ MCF-7
Fig. 7. Cell cycle: I. control MCF-7, II. Compound 7e by flow cytometery using PI
staining method
2
.2.4. Annexin V-FITC apoptosis assay
Annexin V binding study was performed to determine early and late apoptosis
employing flow cytometer where Annexin V conjugated with FITC is used to stain
cells in combination with propidium iodide (PI). The cells in the late apoptotic stage
that have loss membrane integrity [34] are represented as a stained positive for
Annexin V/ PI. The apoptotic value of compound 7e on MCF-7 cells was determined
via flow cytometry detection using Annexin V/ propidium iodide (PI) double staining
assay when MCF-7 cells treated with 10 μM of compound 7e for 48h. The results was
obtained indicated that early apoptosis ratio (lower right quadrant of the cytogram)
increases by about 13 fold and the late apoptosis ratio (higher right quadrant of the
1
6

cytogram) increases by about 34 fold, and this mean that compound 7e proved to
induce apoptosis by 13.94% (Table 5 and Figure 8).
Table 5. Percent of cell death induced by compound 7e on MCF-7 cells
Compound number
Apoptosis %
Early
Necrosis
Total
19.22
2.02
Late
9.38
0.28
7
e/MCF7
4.56
5.28
1.38
Cont.MCF7
0.36
Fig. 8. Percent of cell death induced by compound 7e on MCF-7 cells
2
.2.5. In silico studies of anticancer activity
Lipiniski's rule of five (the effect of lipophilic and steric parameters)
Computational studies were carried out for the more potent cytotoxic
compounds; lipiniski's parameters and topological polar surface area (TPSA) were
predicted from Swiss ADME (absorption, distribution, metabolism and excretion).
Lipinski's rule states that for molecules to be taken orally should satisfied these rules:
(i) Not more than 5 hydrogen donors (ii) Not more than 10 hydrogen acceptors (iii)
Molecular weight ˂ 500 (iv) calculated logP ˂ 5. To be considered as an orally active
drug it should has no more than one violation, if more than one violation it might have
1
7

problems in bioavailability [35]. As shown in Table 6, compound 5, 7e and 7i are in
agreement with the parameters of lipinisk's rule and this indicate that these compounds
have promising drug like properties. Topological polar surface area (TPSA) is defined
as the surface sum over all polar atoms or molecules, usually oxygen and nitrogen,
including their attached hydrogen atoms. Polar surface area (PSA) used for
optimization of drug's ability to permeate cell, thus molecule with PSA greater than
2
1
40Ǻ have poor ability to permeate cell membranes [36]. The new prepared
2
compounds 5, 7e and 7i have TPSA equals 97.74, 110.88 and 127.20 Ǻ , respectively.
These values mean that the target compounds 5, 7e and 7i can permeate cell
membranes (Table 6).
Table 6. Lipiniski's parameters and TPSA of the more potent cytotoxic compounds
Lipinisk's Parameters
2
Comp.
No.
5
TPSA (Ǻ ) Log P M. W. nHBD (OHNH) nHBA (NO) No. of viol
97.74
0.52
1.48
1.76
213.20 1
291.26 1
347.33 2
4
5
6
0
0
0
7
e
110.88
127.20
7
i
nHBD = number of hydrogen bond donor
nHBA = number of hydrogen bond acceptor
2
.2.6. Molecular modeling and docking study
Molecular docking study was performed to gain insight in to the most preferred
binding mode of compound into the enzyme binding active site. The binding affinity of
the ligand with the active site of enzyme was determined by energy score (S,
Kcal/mol), low energy score indicates good affinity. Hydrogen bond, arene-arene and
arene-cation interaction are the mode of interaction presented in molecular docking.
The target-ligand interaction determined by molecular docking of both protein as a
target and some tested compounds as a ligand. Molecular docking was performed
through using crystal structure of KDM5A protein that deduced from protein data bank
(PDB- ID: 5IVE) and the most stable conformation of some tested compounds. Target-
ligand stimulation for some newly synthesized compounds 5, 7c, 7e and 7i was carried
out using the Molecular Operating Environment (MOE 2009.10) software.
1
8

Self docking of the native ligand with the active site of the selected protein
showed two arene-arene interactions along with two for hydrogen bonds. The first
hydrogen bond formed between oxygen linked to C of ring A and NH of Lys501(1.93
4
2
Å), and the second between CN linked to C of ring B and nitrogen (N) of His483
9
(3.18Å) with binding energy (S = -10.6852) Kcal/mol (Fig. 9). The mode of interactions
for compounds 7e and 7i which appeared the highest potency against KDM5A with
both human cancer cell lines (breast and hela) was illustrated. Thus compound 7e
showed five interactions three of them for arene-arene interactions and rest two for
hydrogen bond. One of the hydrogen bond is between CN linked to C of ring B
9
(pyrazole ring) and OH of Tyr 472(2.31Ǻ), the other hydrogen bond is between CN
linked to C attached to C of ring B (pyrazole ring) and OH of Tyr 409(3.68Å) with
10
8
binding energy (S = -10.8091) Kcal/mol (Fig. 10). Also, compound 7i exhibited four
interactions one of them for arene-arene interaction and the other three for hydrogen
bonds. The first hydrogen bond is between oxygen linked to C of ring A and NH of
4
2
Arg73(2.16 Å), and the other two hydrogen bonds between OCH linked to ring C and
3
Asn 575 (1.56 Å), Lys 501 (2.29Å) with binding energy (S = -12.2875) Kcal/mol (Fig.
1
1). Moreover, the molecular docking of the parent compound 5 with active site of
enzyme; presented three interactions two of them for arene-arene interactions and last
for hydrogen bond between CN linked to C of ring B (pyrazole ring) and NH of Asn
9
2
4
93 (2.77 Å) with binding energy (S = -9.1748) Kcal/mol (Fig. 12). Molecular docking
of compound 5c which has the lowest cytotoxic activity showed only one interaction for
hydrogen bond between CN linked to C of ring B (pyrazole ring) and NH of His
9
2
4
83(2.96 Å) with binding energy (S = -8.2989) Kcal/mol (Fig. 13).
1
9

Table 7. Protein-ligand interactions of some tested compounds with KDM5A active
site pocket
Compound
number
Binding
energy (S)
Kcal/mol
Groups of the
ligand involved
in the
Amino
acids
Type of the
Bond and length of
Hydrogen bonds
Ǻ
involved in
the
interaction
interaction
Tyr 472
Tyr 409
His 483
5
e
-10.8091
Cyano (CN)
Cyano (CN)
Pyrazole ring
Polar sidechain acceptor 2.13
Ǻ
Polar sidechain acceptor 3.68
Ǻ
Furan ring
Furan ring
Tyr 472
Phe 480
Basic backbone acceptor
(
(
(
Arene-Arene interaction)
Polar sidechain acceptor
Arene-Arene interaction)
Greasy backbone donor
Arene-Arene interaction)
5
i
-9.6461
Carbonyl (CO)
Methoxy
Arg 73
Lys 501
Asn 575
Tyr 409
Basic backbone acceptor 2.16
Ǻ
(
OCH3)
Methoxy
OCH3)
Basic backbone acceptor 2.29
Ǻ
(
Polar sidechain acceptor 1.56
Ǻ
Phenyl ring
Polar sidechain acceptor
(Arene-Arene interaction)
3
-9.1748
Cyano (CN)
Pyrazole ring
Asn493
Tyr 472
Polar sidechain acceptor 2.77
Ǻ
Polar sidechain acceptor
(Arene-Arene interaction)
Greasy backbone donor
Pyrazole ring
Phe 480
2
0

(Arene-Arene interaction)
5
c
-8.2989
Cyano (CN)
His 483
Basic backbone acceptor 2.96
Ǻ
Validation
-10.6852
Cyano (CN)
Cyano (CN)
Pyrazole ring
His 483
Lys 501
Tyr 472
Basic backbone acceptor
3.18Ǻ
Basic backbone acceptor 1.93
Ǻ
Pyrazole ring
Phe 480
Polar sidechain acceptor
(
Arene-Arene interaction)
Greasy backbone donor
Arene-Arene interaction)
(
2
D structure
3D structure
Fig. 9. Binding pattern of the native ligand (2D and 3D) structure with its active site of
the protein KDM5A (PDB: 5IVE).
2
1

2
D structure
3D structure
Fig. 10. Binding pattern of compound 7e (2D and 3D) structure with the active site of
the selected protein KDM5A (PDB: 5IVE).
2
D structure
3D structure
Fig. 11. Binding pattern of compound 7i (2D and 3D) structure with the active site of
the selected protein KDM5A (PDB: 5IVE).
2
2

2
D structure
3D structure
Fig. 12. Binding pattern of compound 5 (2D and 3D) structure with the active site of
the selected protein KDM5A (PDB: 5IVE).
2
D structure
3D structure
Fig. 13. Binding pattern of compound 7c (2D and 3D) structure with the active site of
the selected protein KDM5A (PDB: 5IVE).
2
.2.7. Bioinformatics studies
Drug discovery was supported by study physicochemical descriptors, ADME
parameters, pharmacokinetic properties, drug like nature and medicinal chemistry for
one or multiple small molecules. The molecule is first described by its 2D chemical
structure and canonical SMILES together with the bioavailability radar to estimate oral
bioavailability at first glance.
2
3

The various models and data are grouped in different sections of the one-panel-
per-molecule output (physicochemical properties, lipophilicity, pharmacokinetics, drug
likeness and medicinal chemistry). Apart from efficacy and toxicity, many drug
development failures are imputable to poor pharmacokinetics and bioavailability.
Gastrointestinal absorption and brain access are two pharmacokinetic behaviors crucial
to estimate at various stages of the drug discovery processes.
Bioinformatics studies for compounds 7e and 7i are performed on Swiss ADME
(Swiss Institute of bioinformatics 2018).
Table 8. Bioactivity and ADME toxicity according to Lipinski's rule five.
Compounds
Molecular weight
Num. rotatable bonds
Num. H-bond acceptors
Num. H-bond donors
Molar refractometry
TPSA
7e
7i
291.26 g/mol
347.33 g/mol
2
5
3
6
1
2
77.69
93.93
2
2
110.88 Å
127.20 Å
Consensus Log Po/w
GI absorption
1.48
1.76
High
High
BBB permeant
No
No
No
No
P-gy substrate
Cytochrome P450
Log Kp (skin permeation)
Lipinski
CYP1A2 inhibitor
-7.14 cm/s
Yes; 0 violation
0.55
CYP2C9 inhibitor
-7.12 cm/s
Yes; 0 violation
0.55
Bioavaliability score
PAINS
0 alert
0 alert
Leadlikeness
Yes
Yes
Synthetic accessibility
3.06
3.17
Absorption, distribution, metabolism and excretion (ADME), Topological polar
surface area (TPSA), gastrointestinal absorption (GI absorption), blood brain barrier
(BBB) permeant, P-glycoprotein substrate (P-gp substrate), pan-assay interference
structure (PAINS).
2
4

Firstly, TPSA (topological polar surface area) defined as the surface sum of all
polar atom, primarily oxygen and nitrogen also including their attached hydrogen
atom. PSA used for optimization of drug's ability to permeate cell, molecule with PSA
2
greater than 140Å have poor permeating cell membranes [36]. Thus, from data
depicted in Table 8 showed physicochemical properties of compounds 7e and 7i which
2
appeared that compounds 7e and 7i have TPSA 110.88 and 127.20 Å , respectively.
These values mean that the compounds 7e and 7i can permeate cell membranes.
Consensus lipophilicity of compounds 7e and 7i is 1.48 and 1.76, respectively, that
showed low lipophilicity so that these compounds cannot permeate BBB (blood brain
barrier).
Pharmacokinetics studies are important in drug discovery process,
gastrointestinal absorption and brain access are two pharmacokinetics behavior, thus
the two studied compounds 7e and 7i are highly absorbed in gastrointestinal but can't
pass BBB or be P-glycoprotein substrate. From this collective data, it can deduced the
method of administration include oral and sublingual (dissolving the drug under the
tongue). In addition, in pharmacokinetics, the permeability can be estimated via skin
by permeability coefficient (Kp), the permeability coefficient of compounds 7e and 7i
is -7.14 and -7.12 Cm/s, respectively. Additionally, medicinal chemistry showed that
synthetic accessibility with 3.06 and 3.17 for compounds 7e and 7i, respectively. These
values mean that two compounds easily synthesized.
2
.2.8. BOILED-Egg (Brain Or IntestinaL EstimateD penetration) model
(BOILED-Egg) is proposed as an accurate predictive model that worked by
computing the lipophilicity and polarity of small molecule.
2
5

Fig. 14. (BOILED-Egg) model of compounds 7e (molecule 1) and 7i (molecule 2).



White ellipse represents compounds with high probability to be passively
absorbed by the gastrointestinal tract.
Yellow ellipse represents compounds with high probability to permeate through
BBB to access CNS.
Grey zone represents molecules that not predicted to be well absorbed nor BBB
permeate.


Blue point: molecule predicted to be substrate of the p-glycoprotein (PGP+).
Red point: molecule predicted to be non substrate of the p-glycoprotein (PGP-).
The (BOILED-Egg) model of compounds 7e (molecule 1) and 7i (molecule 2) showed
that the two compounds appear in the white ellipse that mean compounds absorbed by
the gastrointestinal tract and appear as red point that mean compounds not substrate to
P-glycoprotein.
3
. Conclusion
Some novel pyrazolo[1,5-a]pyrimidine derivatives were synthesized and
evaluated in vitro against breast and hela cancer cell lines. Furthermore, the most
potent KDM inhibitor 7e (IC = 1.91μM) showed to cause cell cycle arrest at G2/M
5
0
phase by 4 folds than control and induce total apoptotic effect by 10 folds more than
control.
2
6

4
4
4
. Experimental
.1. Chemistry
.1.1. General
Melting points were determined on an Electrothermal (9100) apparatus and are
incorrect. The IR spectra were recorded as KBr pellets on a Perkin Elmer 1430
1
13
spectrophotometer. H NMR and C NMR spectra were recorded in deuterated
dimethylsulfoxide at 300 and 75 MHz, respectively, on a Varian Gemini NMR
spectrometer using tetramethylsilane as internal reference and the results are expected
as δ value. Mass spectra were taken on a Shimadzu GCMS-QP 1000 Ex mass
spectrometer at 70 eV. UV spectra and elemental analyses were carried out at the
Microanalyses Center of Cairo University, Giza, Egypt.
4
.1.2. 5-Amino-3-cyanomethyl-1H-pyrazole-4-carbonitrile 1
To a solution of (2.64g, 0.02 mol) of 2-aminoprop-1-ene-1,1,3-tricabonitrile in
0 ml of boiling ethanol was added (1.1 g, 0.022 mol) 85% hydrazine hydrate at such a
2
rate that the reaction mixture continued to boil without external heating. The strongly
exothermic reaction was accompanied by a vigorous evolution of ammonia. After
addition of the hydrazine hydrate was completed, the reaction mixture was allowed to
cool slowly to room temperature. The solid so formed was filtered off and
recrystallized from glacial acetic acid to give compound 1. Yield: (74%), mp. = 198°C
-1
1
[
lit. 197-199°C][37], vmax / cm (KBr) 2261 (CN), 2218 (CN); H NMR (DMSO) δ =
4
4
.36 (2H, s, CH ); 6.38 (2H, s, NH ); 11.41 (1H, s, NH). Anal. Calcd for C H N : C,
2 2 6 5 5
8.98; H, 3.43; N, 47.60. Found: C, 48.79; H, 3.61; N, 47.84 %.
4
.1.3. 2-(Cyanomethyl)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-
carbonitrile5
A mixture of 5-amino-2-cyanomethyl-1H-pyrazole-4-carbonitrile 1 (0.01 mol)
and acetoacetanilide 2 (0.01 mol) in N,N-dimethylformamide (20ml) in the presence of
few drops of glacial acetic acid was refluxed for 6 h, then poured on cold water. The
solid was collected by filtration and recrystallized from N,N-dimethylformamide.
-1
Brown crystals, yield 53%, m.p >300 °C, vmax / cm (KBr) 3152 (NH), 2227 (2CN),
1
1
671 (CO); H NMR (DMSO) δ = 2.38 (s, 3H, CH ), 4.36 (s, 2H, CH ), 5.87 (s, 1H,
3
2
+
pyrimidine-H), 13.35 (s, 1H, NH); m/z = 213 (M , 100%), 184 (17.4%), 172 (15.7%),
1
5
56 (11.9%), 145 (12.7%), 77 (16.6%), 67 (99.2%); Anal. Calcd for C H N O: C,
10 7 5
6.34; H, 3.31; N, 32.85. Found: C, 56.51; H, 3.49; N, 32.66%.
2
7

4
.1.4. General procedure for preparation of compounds7a-i
A solution of compound 5 (0.01 mol) in N,N-dimethylformamide (10 ml) containing
few drops of piperidine was added to the appropriate aldehyde 6a-i (0.01 mol). The
mixture was heated under reflux for 4 h and the solid product that precipitated by
cooling was filtered off and recrystallized from N,N-dimethylformamide to give the
respective products 7a-i. The physical constants and spectral data are shown below:
5
.1.4.1.
2-(1-Cyano-2-phenylvinyl)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]-
-1
pyrimidine-3-carbonitrile 7a. Yellow crystals, yield 65%, m.p = 280°C, vmax / cm
1
(
KBr) 3146 (NH), 2218 (2CN), 1628 (CO); H NMR (DMSO) δ = 2.20 (s, 3H, CH ),
3
5
.56 (s, 1H, pyrimidine-H), 7.56-7.96(m, 5H, Ar), 8.14 (s, 1H, CH), 13.21 (s, 1H, NH);
Anal. Calcd for C H N O: C, 67.77; H, 3.68; N, 23.24. Found: C, 67.94; H, 3.52; N,
1
7
11
5
2
3.44%.
.1.4.2. 2-(1-Cyano-2-(4-methoxyphenyl)vinyl)-5-methyl-7-oxo-4,7-dihydropyrazolo-
4
[1,5-a]pyrimidine-3-carbonitrile 7b. Brown crystals, yield 74%, m.p >300 °C, vmax /
-
1
1
cm (KBr) 3190 (NH), 2222 (2CN), 1650 (CO); H NMR (DMSO) δ = 2.50 (s, 3H,
CH ), 3.86 (s, 3H, OCH ), 5.81 (s, 1H, pyrimidine-H), 7.85 (d, 2H, J = 8.4 Hz, Ar),
3
3
7
.94 (d, 2H, J = 8.4 Hz, Ar), 8.06 (s,1H, CH), 9.95 (s, 1H, NH); Anal. Calcd for
C H N O : C, 65.25; H, 3.95; N, 21.14. Found: C, 65.42; H, 3.77; N, 21.38%.
18
13
5
2
4
.1.4.3.
2-(2-(4-Chlorophenyl)-1-cyanovinyl)-5-methyl-7-oxo-4,7-dihydropyrazolo-
[1,5-a]pyrimidine-3-carbonitrile 7c. Brown crystals, yield 66%, m.p >300 °C, vmax /
-1
1
cm (KBr) 3406 (br., NH), 2216 (2CN), 1635 (CO); H NMR (DMSO) δ = 2.49 (s, 3H,
CH ), 5.74 (s, 1H, pyrimidine-H), 7.59 (d, 2H, J = 8.7 Hz, Ar), 7.92 (d, 2H, J = 7.5 Hz,
3
+
Ar), 7.63 (s,1H, CH), 10.31 (s, 1H, NH); m/z = 335 (M , 0.3%), 317 (0.9%), 305
(2.5%), 213 (1.4%), 189 (5.8%), 143 (5.2%), 113 (35.0%), 101 (22.8%), 87 (30.5%),
5
9 (100%); Anal. Calcd for C H ClN O: C, 60.82; H, 3.00; Cl, 10.56; N, 20.86.
1
7
10
5
Found: C, 60.67; H, 3.15; N, 20.64%.
4
.1.4.4.2-(2-(Benzo[d][1,3]dioxol-4-yl)-1-cyanovinyl)-5-methyl-7-oxo-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-3-carbonitrile 7d. Brown crystals, yield 70%, m.p >300 °C,
-
1
1
vmax / cm (KBr) 3152 (NH), 2215 (2CN), 1671 (CO); H NMR (DMSO) δ = 2.29 (s,
H, CH ), 5.77 (s, 1H, pyrimidine-H), 6.16 (s, 2H, CH ), 7.11-8.03 (m, 3H, Ar), 8.26
3
3
2
(
s,1H, CH), 9.97 (s, 1H, NH); Anal. Calcd for C H N O : C, 62.61; H, 3.21; N,
18 11 5 3
2
0.28. Found: C, 62.78; H, 3.06; N, 20.56%.
2
8

4
.1.4.5. 2-(1-Cyano-2-(furan-2-yl)vinyl)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-a]-
-1
pyrimidine-3-carbonitrile 7e. Brown crystals, yield 84%, m.p >300 °C, vmax / cm
1
(
KBr) 3140 (br., NH), 2214 (2CN), 1628 (CO); H NMR (DMSO) δ = 2.50 (s, 3H,
CH ), 5.82 (s, 1H, pyrimidine-H), 6.81-7.95 (m, 3H, Ar), 8.09 (s, 1H, CH), 10.62 (s,
3
+
1
H, NH); m/z = 291 (M , 1.3%), 279 (2.9%), 239 (1.8%), 211 (1.5%), 183 (2.3%), 149
(
53.2%), 85 (55.6%), 71 (82.0%), 69 (47.1%), 57 (100%); Anal. Calcd for C H N O :
15 9 5 2
C, 61.85; H, 3.11; N, 24.04. Found: C, 61.71; H, 3.28; N, 24.26%.
4
.1.4.6. 2-(1-Cyano-2-(thiophen-2-yl)vinyl)-5-methyl-7-oxo-4,7-dihydropyrazolo[1,5-
-1
a]-pyrimidine-3-carbonitrile 7f. Brown crystals, yield 91%, m.p >300 °C, vmax / cm
1
(
KBr) 3422 (br., NH), 2207 (2CN), 1630 (CO); H NMR (DMSO) δ = 2.19 (s, 3H,
CH ), 5.54 (s, 1H, pyrimidine-H), 7.28 (d, 1H, J = 5.1 Hz, Ar), 7.82 (dd, 1H, J = 3 Hz,
3
+
Ar), 8.00 (d, 1H, J = 5.1 Hz, Ar), 8.32 (s, 1H, CH), 10.84 (s, 1H, NH); m/z = 307 (M ,
6
8
2
4.8%), 278 (15.7%), 251 (8.4%), 213 (8.4%), 149 (4.5%), 113 (17.8%), 98 (19.0%),
4 (93.1%), 71 (36.3%), 57 (100%); Anal. Calcd for C H N OS: C, 58.62; H, 2.95; N,
15
9
5
2.79; S, 10.43. Found: C, 58.82; H, 2.77; N, 22.49; S, 10.63%.
4
.1.4.7. 2-(1-Cyano-2-(2,5-dimethoxyphenyl)vinyl)-5-methyl-7-oxo-4,7-dihydropyra-
zolo[1,5-a]pyrimidine-3-carbonitrile 7j. Brown crystals, yield 64%, m.p >300 °C, vmax
-1
1
cm (KBr) 3414 (br., NH), 2214 (2CN), 1630 (CO); H NMR (DMSO) δ = 2.49 (s,
/
3
7
H, CH ), 3.84 (s, 3H, OCH ), 3.87 (s, 3H, OCH ), 5.74 (s, 1H, pyrimidine-H), 7.14-
3 3 3
+
.71 (m, 3H, Ar), 8.44 (s, 1H, CH), 10.48 (s, 1H, NH); m/z = 362 (M +1, 0.3%), 305
(2.1%), 279 (1.8%), 259 (4.0%), 247 (3.6%), 213 (1.5%), 189 (4.3%), 149 (16.3%),
1
6
13 (28.9%), 87 (26.6%), 71 (29.8%), 59 (100%); Anal. Calcd for C H N O : C,
19 15 5 3
3.15; H, 4.18; N, 19.38. Found: C, 63.32; H, 4.05; N, 19.59%.
.1.4.8. 2-(1-Cyano-2-(3,4,5-trimethoxyphenyl)vinyl)-5-methyl-7-oxo-4,7-dihydropyr-
4
azolo[1,5-a]pyrimidine-3-carbonitrile 7h. Brown crystals, yield 57%, m.p >300 °C,
-
1
1
vmax / cm (KBr) 3190 (NH), 2222 (2CN), 1635 (CO); H NMR (DMSO) δ = 2.49 (s,
H, CH ), 3.76 (s, 3H, OCH ), 3.82 (s, 6H, 2OCH ), 5.78 (s, 1H, pyrimidine-H), 7.29
3
3
3
3
(
s, 2H, Ar), 8.02 (s, 1H, CH), 9.97 (s, 1H, NH); Anal. Calcd for C H N O : C, 61.38;
20 17 5 4
H, 4.38; N, 17.89. Found: C, 61.57; H, 4.55; N, 17.64%.
4
.1.4.9.
2-(1-Cyano-2-(4-hydroxy-3-methoxyphenyl)vinyl)-5-methyl-7-oxo-4,7-
dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 7i. Brown crystals, yield 61%, m.p
-1
1
>
300 °C, vmax / cm (KBr) 3170 (NH), 2211 (2CN), 1629 (CO); H NMR (DMSO) δ =
2
9