A new synthetic approach for pyrazolo[1,5-a]pyrazine-4(5H)-one derivatives and their antiproliferative effects on lung adenocarcinoma cell line

Article abstract of DOI:10.1007/s11030-020-10161-8

Abstract: Starting from the 3,5-dimethyl pyrazole ring and acetophenone derivatives, five different N-propargylated C-3 substituted pyrazoles were obtained. These derivatives were reacted with different amine derivatives using Cs₂CO₃ in methanol and 11 different pyrazolo [1,5-a] pyrazine-4(5H)-one derivatives were obtained, which are not found in the literature. The cytotoxic effects of these derivatives in the A549 cell line were investigated. The 160?μM concentration of two derivatives was found to increase cell death rate to 50%, and two derivatives increased cell death rate by up to 40%. The structure–activity relationship (SAR) study revealed an amide group with a long alkyl chain and benzene ring with a p-CF₃ group could be important for efficiency. With theoretical ADMET studies of pyrazolopyrazine derivatives, pharmacokinetic phases were predicted to be suitable. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-020-10161-8

Molecular Diversity
https://doi.org/10.1007/s11030-020-10161-8
ORIGINAL ARTICLE
A new synthetic approach for pyrazolo[1,5‑a]pyrazine‑4(5H)‑one
derivatives and their antiproliferative efects on lung adenocarcinoma
cell line
Meltem Tan Uygun1,2 · Karina Amudi1,2 · İrem Doğan Turaçlı3 · Nurettin Menges1,2
Received: 2 August 2020 / Accepted: 20 November 2020
© Springer Nature Switzerland AG 2021
Abstract
Starting from the 3,5-dimethyl pyrazole ring and acetophenone derivatives, fve diferent N-propargylated C-3 substituted
pyrazoles were obtained. These derivatives were reacted with diferent amine derivatives using Cs2CO3 in methanol and
11 diferent pyrazolo [1,5-a] pyrazine-4(5H)-one derivatives were obtained, which are not found in the literature. The cyto-
toxic efects of these derivatives in the A549 cell line were investigated. The 160 µM concentration of two derivatives was
found to increase cell death rate to 50%, and two derivatives increased cell death rate by up to 40%. The structure–activity
relationship (SAR) study revealed an amide group with a long alkyl chain and benzene ring with a p-CF3 group could be
important for efciency. With theoretical ADMET studies of pyrazolopyrazine derivatives, pharmacokinetic phases were
predicted to be suitable.
Graphic abstract
Keywords Alkyne cyclization · Allene · Pyrazole · Lung adenocarcinoma · KRAS mutant lung cancer · ADMET
Introduction
Electronic supplementary material The online version of this
Pyrazole derivatives are appreciated to manifest a wide
article (https://doi.org/10.1007/s11030-020-10161-8) contains
range of biological qualities such as cannabinoid hCB1
and hCB2 receptor, anti-infammatory, inhibitors of p38
Kinase, CB1 receptor antagonists, antimicrobial activity
[1–4]. Incorporating the heterocyclic ring into prospective
pharmaceutical candidates is a signifcant strategy to obtain
activity and safety benefts. As a consequence, much atten-
tion has been paid to the design and synthesis of pyrazo-
lopyrazinones, which reveal the HIV-1 integrase inhibitory
efect (Fig. 1, compound 2) [5] and dipeptidyl peptidase-IV
inhibitory efect (Fig. 1, compound 1) [6]. Besides, Miao
and his co-workers have announced that pyrazolopyrazinone
derivatives showed a vital efect with 22.3 uM on the A549
supplementary material, which is available to authorized users.
* Nurettin Menges
nurettinmenges@yyu.edu.tr
Meltem Tan Uygun
meltemtan@yyu.edu.tr
1
Pharmaceutical Chemistry Section, Van Yuzuncu Yil
University, 65080 Van, Turkey
2
SAFF Chemical Reagent RδD Laboratory,
VAN-TEKNOKENT, 65080 Van, Turkey
3
Department of Medical Biology, Faculty of Medicine, Ufuk
University, 06520 Ankara, Turkey
Vol.:(0123456789)
1 3

Molecular Diversity
Fig. 1 Representation of some
important C-3-substituted
pyrazolopyrazinones and our
designed molecule
cell line [7]. Surase and co-workers have emerged novel
inhibitors of mycobacterium ATP synthase in which the
central skeleton of found novel inhibitors is pyrazolo [1,5-a]
pyrazin-4(5H)-one (Fig. 1, compound 4) [12]. They have
pointed out that the skeleton was revealed after the screen-
ing of chemically diverse compounds (10.000 compounds)
from the compound library. Their synthetic strategy includes
alkylation and azidation of diethyl pyrazole-3,5-dicarboxy-
late followed the reduction of azide functional group using
triphenylphosphine. These reagents might give some dis-
advantages due to their toxicities [13] and the poisoning of
catalysts in need [14].
a personalized treatment by choosing a treatment accord-
ing to what type of cancer driver mutation is present in the
patient’s tumor tissue. One of the most common mutations
in adenocarcinoma patients, with a 25% rate, is Kirsten rat
sarcoma viral oncogene homologous (KRAS) gene muta-
tion [16]. Activated when guaccine triphosphate (GTP) is
bound, KRAS stimulates many oncogenic signal transduc-
tion pathways in the cell, such as proliferation and survival.
KRAS mutations, usually seen in codons 12, 13, and 61,
change the protein’s conformation and cause it to remain
permanently GTP-bound. These mutations are also associ-
ated with smoking [17]. One of the conditions that make
treatment difcult in patients with KRAS mutant NSCLC
is biological heterogeneity observed due to co-mutations.
Besides the inadequate chemotherapy response, there is no
direct or indirect KRAS inhibitor approved by the FDA. For
this reason, the development of inhibitors is of great impor-
tance for KRAS mutant lung adenocarcinoma.
Lung cancer is the most common cancer type all over
the World. Lung cancer is classifed into three main groups:
non-small cell lung cancer (NSCLC), small cell lung can-
cer (SCLC), and carcinoids. NSCLC is further partitioned
into adenocarcinoma, squamous cell carcinoma, and large-
cell carcinoma. Among them, adenocarcinoma accounts
for 40% of all NSCLC patients [15]. For lung adenocarci-
noma, a more appropriate treatment process is started with
Although there are some reported studies about the pyra-
zolopyrazinone framework, it is barely known. Therefore,
Scheme 1 Synthesis of starting
materials. a compound 9; b
compounds 14a–d
A
B
1 3

Molecular Diversity
we have focused on C-3 substituted pyrazolopyrazinone due
to their pharmaceutical infuence (Fig. 1, compounds 5 and
15).
atom’s propargylation of the pyrazole ring under the basic
medium.
We have studied optimum reaction condition using com-
pound 9, and it was reacted with ethylamine under diferent
reaction conditions (Table 1).
In this study, we would like to introduce the synthesis
and structural characterization of novel C-3 substituted
pyrazolo[1,5-a]pyrazin-4(5H)-ones and their antiprolifera-
tive efects on A549, which is KRAS mutant lung adenocar-
cinoma cell line.
We did some experiments to achieve optimum conditions
and found that the reaction was going too slowly to obtain
the cyclic product without base (Entry 3, Table 1). The use
of excess amines had a high efect on the cyclic product
yield, resulting from the initiation of allene isomerization.
The use of an inorganic base has been critical for cycliza-
tion (Entries 4–5, Table 1). When Na2CO3 was used, it was
observed that the cyclization product was 75%, and the
amide derivative remained in the medium as 25%. This can
be concluded that Na2CO3 does not quantitatively convert
the propargyl group in the amide molecule to its allen isomer
(crucial for cyclization) in the given time. It was, therefore,
remarkable to say that Cs2CO3 is the right choice.
Result and discussion
Chemistry
The synthesis of starting materials for pyrazolo[1,5-a]
pyrazin-4(5H)-one derivatives was fulflled as displayed in
Scheme 1.
We have followed two diferent reaction pathways to get
starting materials (9 and 14a–d). In the frst way, drawn in
Scheme 1A, the commercially available 3,5-dimethyl pyra-
zole (6) was oxidized with KMnO4 to obtain the pyrazole-
carboxylic acid 7 derivative. The second stage was a Schot-
ten–Baumann type reaction using methanol and thionyl
chloride, where the 3,5-dicarboxylate-pyrazole derivative
8 was formed.
With optimum conditions in hand, we want to extend the
scope of cyclization reaction. Primary amines which bear
long alkyl chain, steric-hindered unit, aromatic ring, and
allyl unit were utilized. We have heeded that the cyclization
protocol can tolerate the long alkyl chain up to heptyl amine
and allyl unit resulting in different pyrazolopyrazinone
derivatives 5a–f (Scheme 2A). On the other hand, the start-
ing compound containing benzene ring in the pyrazole ring’s
C-3 position was reacted with allyl and butylamine to obtain
fve diferent pyrazolopyrazinone derivatives 15a–e in good
to high yields (Scheme 2B).
In the second reaction we carried out to obtain the other
C-3 substituted pyrazole ring, the ketoester derivative
(12a–d) was obtained by the reaction of acetophenone deriv-
atives (10a–d) with diethyl oxalate (11) in basic medium.
Then the cyclization reaction of this ketoester with hydra-
zine hydrate was used (Scheme 1B). The fnal step of both
reactions to reach our starting materials was the nitrogen
We could not obtain any product other than the starting
material and the allene isomer when t-butyl amine and ani-
line were used as an amine source. Although the reaction
Table 1 Preliminary tests for the fnding of optimum reaction conditions
Entry
Equiv. of amine
Base (1 equiv.)
Time (h)
Yield
5a (%)
16a (%)
1
2
3
4
5
1
5
10
5
–
–
–
6
6
24
6
–
–
30
99
75
99
99
70
–
Cs2CO3
Na2CO3
5
6
25
1 3

Molecular Diversity
was refuxed for 24 h, we received no further reactions.
When we evaluated the reaction medium with the 1H-NMR
spectrum, we witnessed the allene isomer of the N-propar-
gyl-substituted pyrazole (compound 17).
of EtNH2 to the reaction medium without isolating the allene
isomer formed in the medium. The reaction medium was
refuxed for a further 6 h, and we isolated compound 5a as
the sole product (Scheme 3). Another control experiment
included the reaction of compound 16a–b and Cs2CO3. We
know that the primary amine leads to amide functionality
but did not provide allene isomerization quickly. When the
We have noticed that t-butyl amine cannot form amide
functional groups due to its steric hindrance but only causes
allene isomerization (compound 17). We added 5 equivalents
Scheme 2 Derivatization
of pyrazolopyrazinones. a
compounds 5a–f; b compounds
15a–e
A
B
Scheme 3 Allene isomeriza-
tion and failure attempt for
cyclization
Scheme 4 Proposed reaction
mechanism
1 3

Molecular Diversity
isolated N-propargyl amide molecules (16a–b) reacted with
the base, the reaction gave cyclic products quickly (5a–b).
Allene isomerization reaction provided a clue for the
assumption of reaction mechanism (Scheme 4). We have
concluded that the propargyl-allene isomerization was
prompted by the base, which has already been reported by
us [8, 9]. Propargyl-allene isomerization followed amidation
of compound 9 in which nitrogen atom of amide group can
attack to middle carbon of allene unit [10, 11]. Attack com-
ing from nitrogen atom resulted in cyclization intermediate,
which was fnalized with proton transfer. We have remarked
that amine with an alkyl chain can aford to make an amide
functional group. However, their basicity is not as good as
to make an isomerization in a short time. In this manner,
Cs2CO3 is a valuable base to accomplish isomerization in
a short time.
been obtained dealing with functional groups that should
use. The presence of long-chain alkyl group on amide group
(5e), presence of allyl group instead of propyl group (5f)
has emerged as important structural groups in derivatives
containing amide group (5a–f). On the other hand, almost
no activity was observed in derivatives containing aromatic
rings at the C-3 position of the pyrazolopyrazinone nucleus,
except for derivatives containing fuorine atoms (15c and
15e). Compound 15c has a fuorine atom on the para position
of the benzene ring. This partially reduced cell viability by
up to 89%. The presence of OMe in the same position did not
cause any efects. Replacing the fuorine atom with the CF3
group (15e) decreased the cell viability by up to 62.8%. This
suggests that fuorine atoms can make halogen bonds and/
or hydrogen bonds at the relevant position. Thanks to these
derivatives, it can be said that the efects of some functional
groups on the A549 cell line were revealed at the initial level
by keeping the pyrazolopyrazinone skeleton fxed.
Evaluation of biological activity
The importance of bicyclic pyrazole derivatives was also
noted in a recently published study [21]. Nasr et al. designed
and synthesized pyrazolo[3,4-d] [1–3] triazine deriva-
tives and evaluated for their anticancer activity in Huh-7,
Panc-1 and CCRF cells. They showed signifcant changes
in Caspase 3/activity with some compounds in Huh-7 cells.
According to the microarray analysis, one compound,
6c, afected apoptosis, metabolism, cell cycle, and tumor
growth-related gene expressions in Huh-7 cells.
Cytotoxicity assay
Some pyrazolopyrazinones were active molecules in the
KRAS mutant lung adenocarcinoma cell line, A549. There-
fore, the antiproliferative efect of eleven diferent pyra-
zolopyrazinone derivatives synthesized in this study was
investigated. The antiproliferative efects of molecules on
the A549 cell line were examined using the MTT method at
24 and 48 h, and the results are given in Table 2.
In this study, the antiproliferative efect of 11 diferent
molecules with pyrazolopyrazinone core (5a–f and 15a–e)
was done (Table 2). Among the molecules, 5c and 5e, which
cause 50% death of cells, and 5f and 15e, which cause
61–62% cell survival, have been identifed. Considering
the activities of these molecules, valuable information has
Theoretical pharmacokinetic study for synthesized
molecules
We also wanted to calculate the theoretical ADMET knowl-
edge of the molecule skeleton whose antiproliferative efect
was determined. For this purpose, some important ADMET
parameters were calculated with the pkCSM program
(Table 3) [18]. For this purpose, Caco-2 permeability and
small intestinal absorption (Intest. Ab., Table 3), which can
be used in the absorption parameter, were calculated. It has
been observed that all synthesized molecules have proper
Caco-2 permeability. Small intestinal absorption was also
calculated to be high, between 86 and 97% (Table 3). The
VDss values of the synthesized molecules are not very high.
According to this theoretical calculation, it can be stated that
the molecules obtained in this study will be found in plasma
rather than tissues. The Fu value decreases with the alkyl
group’s extension on the nitrogen atom, which indicates that
the molecule’s binding rates to plasma proteins increase.
The pyrazolopyrazinone derivative with the heptyl chain
(compound 5e) has the lowest Fu value (Fu:0.08; Table 3).
Total clearance of synthesized molecules is varied between
0.40 and 1.60 log(ml/min). Our synthesized molecules are
not OCT2 substrate means that they have no potential for
Table 2 Antiproliferative efect of synthesized molecules on A549 at
24 h
Molecules
Cell viability (%)
5a
76 10.5
75 8.4
51 5.5
57 4.3
51 11.6
61 5.3
97.4 7.8
124 6.3
89 17.9
99 3.1
62.8 12.2
100
5b
5c
5d
5e
5f
15a
15b
15c
15d
15e
Control (DMSO)
Concentration of molecules were 160 µM in DMSO
1 3

Molecular Diversity
Table 3 Some of ADMET parameters for synthesized molecules (For explanation of abbreviation in Table, please see supporting information)
Entry Log P Caco2 Intest. Ab.% VDss
Fu
CYPs
Total C. OCT2 hERG I-II Skin sensitive
5a
5b
5c
5d
5e
5f
15a
0.57
1.35
1.83
2.91
4.48
0.91
2.66
1.40
1.36
1.32
1.35
1.32
1.38
1.67
89
97
96
93
92
86
99.8
−0.35 0.51 CYP1A2
−0.26 0.43 CYP1A2
−0.11 0.35 CYP1A2
0.058 0.25 CYP3A4, CYP1A2, CYP2C19 1.50
0.41 0.08 CYP3A4, CYP2C19, CYP2C9 1.60
0.89
0.77
1.46
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
−0.28 0.45 CYP1A2
0.8
0.61
0.11 0.28 CYP1A2
CYP3A4
CYP2C19
15b
15c
3.27
3.41
1.67
1.46
98.7
98.2
0.24 0.24 CYP3A4
0.62
0.85
No
No
No
No
No
No
CYP1A2
CYP2C19
0.28 0.28 CYP3A4
CYP1A2
CYP2C19
CYP2C9
15d
15e
3.28
4.29
1.37
1.59
99.7
96.2
0.36 0.25 CYP3A4
0.64
0.40
No
No
No
No
No
No
CYP1A2
CYP2C19
CYP2C9
0.27 0.20 CYP3A4
CYP1A2
CYP2C19
CYP2C9
disposition and clearance efect. One of the most critical
parameters for drugs is to be no inhibition on HERGI/II.
Calculations show that molecules 5a–f and 15a–e do not
inhibit hERGI/II and molecules do not occur allergic con-
tact when encountered skin. Theoretical ADMET informa-
tion was calculated, and the pharmacokinetic information
obtained shows us that the molecules synthesized are likely
to have suitable pharmacokinetic parameters.
Conclusion
To sum up, we have found a useful, applicable synthetic
approach to yield C-3 substituted pyrazolo[1,5-a]pyrazin-
4(5H)-one derivatives. Our protocol includes the excess of
corresponding amine and Cs2CO3, which makes the protocol
easier. Primary amine with long alkyl chain and allylamine
succeeded the cyclization reaction successively. However,
t-butyl amine and aniline did not return the cyclic product,
which might be due to steric hindrance and poor nucleophi-
licity. Eleven diferent pyrazolo[1,5-a]pyrazin-4(5H)-one
derivatives were formed, and the reaction mechanism was
proposed according to analyzing of reaction media using
1H-NMR. The antiproliferative efect of all synthesized mol-
ecules was tested on A549. Compound 5c, 5e, 5f, and 15e
have shown an antiproliferative efect at a concentration of
160 uM. Theoretical ADMET parameters showed that com-
pounds have good pharmacokinetic parameters, which are
considered when a drug candidate was applied to further
phase. This approach might be a starting point for extending
the synthesis of these types of bicyclic pyrazole derivatives
and investigating their further pharmacological efects.
Considering the ADMET and biological activity studies,
derivatives that signifcantly reduce cell viability were 5c,
5e, 5f and 15e. Some comments can be made considering
the important ADMET parameters of these derivatives given
in Table 3.
1. None of these derivatives are hERG inhibitors as cared
for in a candidate drug molecule.
2. Log P values were found to be between a suitable range
when Lipinski rules were considered.
3. Considering the Caco2 and Intest Ab. values, it can be
said that the absorption will be suitable.
4. The theoretical values of the rate of binding of mol-
ecules to plasma proteins (Fu) seem appropriate.
5. The selected molecules also appear not to be skin sensi-
tive.
1 3

Molecular Diversity
Pyrazole-3,5-dicarboxylic acid 7, (0.56 g, 3.6 mmol)
in MeOH (10 mL) at 4 °C was added SOCl2 (1.68 mL,
18 mmol) over 1 h. At the end of the addition, the solution
was heated to 60–65 °C for 10 h and then cooled to room
temperature. The solution’s pH was adjusted to 7.5 with a
cold aqueous solution of NaOH, and diester 8 crystallized
(0.6 g, 90% yield) as white solid.
Experimental section
General information
All reagents used were commercially available unless oth-
erwise stated, and all solvents were used as received. Var-
1
ian 400 spectrometer was used to acquire 400 MHz H-
NMR and 100 MHz 13C-NMR spectra. Chemical shifts are
reported in ppm relative to tetramethylsilane (TMS) and
chloroform-d or d6-DMSO. HRMS spectra were recorded
using by THERMO ITQ900 LC/MS spectrometer with ESI
method. All spectroscopic analyses were conducted at the
Science Research and Application Center of Van Yuzuncu
Yil University.
Compound 8: White solid, m.p: 138–140 °C. (Lit [19]:
1
142–143 °C). H NMR (400 MHz, CDCl3) δ 7.34 (s, 1H,
Ar–H), 3.95 (s, 6H, OCH3). 13C NMR (100 MHz, CDCl3) δ
160.7, 140.2, 111.5, 52.6.
Experimental procedure for the synthesis of dimethyl
1‑(prop‑2‑yn‑1‑yl)‑1H‑pyrazole‑3,5‑dicarboxylate (9)
Synthesis of pyrazole derivatives
Synthesis of starting materials and bicyclic pyrazole deriva-
tives are discussed below.
Synthesis of 1H‑pyrazole‑3,5‑dicarboxylic acid [19]
Compound 8 (0.4 g, 2.17 mmol) was dissolved in dimeth-
ylformamide (10 mL). NaH (0.09 g, 3.48 mmol, 60% oil
suspension) was added to the solution, and the mixture
was stirred in an ice bath for 30 min. After completing the
reaction, propargyl bromide (0.25 mL, 2.82 mmol) in DMF
(5 mL) was added dropwise at room temperature, and the
solution was stirred for 16 h. Water was (30 mL) added, and
the solution was extracted with ethyl acetate (3× 15 mL).
The combined organic extracts were washed with brine and
dried over MgSO4. The solvent was evaporated to give a
crude product 9 (0.398 g, 83% yield) as light yellow solid
without purifcation.
Compound 6, 0.96 g (10 mmol), was dissolved in 20 mL of
water heated to 70 °C, and 7.9 g (50 mmol) of potassium
permanganate was added to the hot solution, maintaining the
temperature no higher than 90 °C. The mixture was refuxed
for 4 h and cooled to room temperature, the precipitate of
MnO2 was fltered of and washed with water, and the fltrate
was acidifed with aqueous HCl to pH 2 and left overnight.
The precipitate was fltered of and washed with water to
obtain compound 7 (1 g, 64% yield) as white crystals.
Compound 7: White crystal, m.p: 286–289 °C (Lit [19].:
289–290 °C).
Compound 9: Light yellow solid, m.p: 60–62 °C.
1H NMR (400 MHz, CDCl3) δ 7.36 (s, 1H, Ar–H), 5.41
(d, J = 2.5 Hz, 2H, CH2), 3.92 (bs, 6H, OCH3), 2.38 (t,
J=2.5 Hz, 1H, CH). 13C NMR (100 MHz, CDCl3) δ 161.8,
159.3, 142.8, 133.4, 114.5, 76.7, 74.1, 52.6, 52.4, 42.7. LC/
MS: Calculated for [M+H]+ C10H11N2O4+ 223.0713, Found
223.0722.
Experimental procedure for the synthesis of dimethyl
1H‑pyrazole‑3,5‑dicarboxylate [20]
1 3

Molecular Diversity
Experimental procedure for the synthesis of pyrazolo[1,5‑a]
pyrazine‑2‑carboxamide derivatives (5a–f)
Compound 5c White solid (122 mg, 89%), m.p: 174–
176 °C. 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J=0.7 Hz,
1H, Ar–H), 7.19 (quasi t, J = 0.9 Hz, 1H, CH), 6.89 (t,
J=5.5 Hz, 1H, NH), 3.99–3.95 (m, 2H, CH2), 3.48–3.43 (m,
2H, CH2), 2.34 (d, J=1.1 Hz, 3H, CH3), 1.69–1.59 (m, 4H,
CH2), 1.47–1.37 (m, 4H, CH2), 0.98 (t, J=7.4 Hz, 3H, CH3),
0.95 (t, J=7.4 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3)
δ 161.2, 155.8, 147.2, 133.6, 128.3, 108.5, 105.9, 43.5, 39.2,
31.9, 31.2, 29.9, 29.8, 20.4, 20.3, 17.4. HRMS Calculated
for [M+H]+ C16H25N4O2+ 305.1972, Found 305.1983.
Compound 9 (0.1 g, 0.45 mmol, 1 eq) was dissolved in
MeOH (20 mL). Cesium carbonate (0.146 g, 0.45 mmol,
1 eq) and an excess amount of corresponding amines (5 eq)
were added to the solution. The mixture was refuxed for 4 h.
Water was (20 mL) added, and the solution was extracted
with ethyl acetate (3 × 10 mL). The combined organic
extracts were dried over MgSO4. The solvent was evaporated
to give a crude product. The crude product was chromato-
graphed on silica gel, eluting with ethyl acetate/hexane (1:1)
to give pyrazolo[1,5-a]pyrazine-2-carboxamide derivatives.
Compound 5a White solid (99 mg, 90% yield), m.p: 192–
194 °C. 1H NMR (400 MHz, CDCl3) δ 7.48 (bs, 1H, Ar–H),
7.19 (quasi t, J=0.9 Hz, 1H, CH), 6.88 (bs, 1H, NH), 4.05
(q, J=7.1 Hz, 2H, CH2), 3.52–3.46 (m, 2H, CH2), 2.35 (d,
J=1.1 Hz, 3H, CH3), 1.31 (t, J=7.1 Hz, 3H, CH3), 1.25 (t,
J=7.3 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 161.1,
155.7, 147.1, 133.6, 128.2, 108.5, 105.8, 38.6, 34.3, 17.2,
+
15.0, 14.3. HRMS Calculated for [M + H]+ C12H17N4O2
249.1346, Found 249.1356.
Compound 5d White solid (125.6 mg, 84%), m.p:
1
132–135 °C). H NMR (400 MHz, CDCl3) δ 7.47 (d,
J=0.6 Hz, 1H, Ar–H), 7.19 (quasi t, J=0.9 Hz, 1H, CH),
6.91 (t, J = 5.7 Hz, 1H, NH), 3.96–3.92 (m, 2H, N–CH2),
3.46–3.40 (m, 2H, N–CH2), 2.33 (d, J=1.1 Hz, 3H, CH3),
1.38–1.27 (m, 12H, CH2), 0.91–0.85 (m, 6H, CH3). 13C
NMR (100 MHz, CDCl3) δ 161.2, 155.8, 147.0, 133.6,
128.4, 108.5, 105.9, 43.7, 39.4, 29.5, 29.2, 29.1, 28.8, 22.5,
22.4, 17.4, 14.1, 14.0. HRMS Calculated for [M + H]+
C18H29N4O2+ 333.2285, Found 333.2297.
Compound 5b White solid (103 mg, 83%), m.p: 176–
178 °C. 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J=0.7 Hz,
1H, Ar–H), 7.18 (quasi t, J = 0.8 Hz, 1H, CH), 6.94 (t,
J=4.9 Hz, 1H, NH), 3.93–3.89 (m, 2H, CH2), 3.43–3.38 (m,
2H, CH2), 2.32 (d, J=1.1 Hz, 3H, CH3), 1.73–1.60 (m, 4H,
CH2), 0.98 (t, J=7.5 Hz, 3H, CH3), 0.97 (t, J=7.4 Hz, 3H,
CH3). 13C NMR (100 MHz, CDCl3) δ 161.2, 155.8, 147.1,
133.5, 128.4, 108.4, 105.8, 45.0, 41.1, 23.0, 22.4, 17.3,
+
11.5, 11.3. HRMS Calculated for [M + H]+ C14H21N4O2
277.1659, Found 277.1671.
1 3

Molecular Diversity
Compound 5e White solid (150.3 mg, 86%), m.p: 143–
145 °C. 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J=0.7 Hz,
1H, Ar–H), 7.19 (quasi t, J = 0.9 Hz, 1H, CH), 6.91 (t,
J=5.9 Hz, 1H, NH), 3.96–3.92 (m, 2H, N–CH2), 3.46–3.40
(m, 2H, N–CH2), 2.33 (d, J=1.1 Hz, 3H, CH3), 1.70–1.57
(m, 4H, CH2), 1.38–1.24 (m, 16H, CH2), 0.88–0.85 (m, 6H,
CH3). 13C NMR (100 MHz, CDCl3) δ 161.1, 155.8, 147.1,
133.6, 128.3, 108.5, 105.9, 43.7, 39.5, 31.9, 31.8, 29.8, 29.1,
29.1, 27.1, 27.0, 22.7, 22.6, 17.4, 14.2, 14.1. HRMS Calcu-
lated for [M+H]+ C22H37N4O2+ 389.2911, Found 389.2927.
Compound 10a White solid (71 mg, 70% yield), m.p: 154–
156 °C. 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 1H, Ar–H),
7.05 (quasi t, J=3.7 Hz, 1H, NH), 6.94 (quasi t, J=4.4 Hz,
1H, NH), 5.46 (d, J=2.5 Hz, 2H, CH2), 3.51–3.42 (m, 4H,
CH2), 2.39 (t, J=2.5 Hz, 1H, CH), 1.25 (t, J=7.2 Hz, 3H,
CH3), 1.25 (t, J=7.3 Hz, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 161.3, 159.0, 145.7, 137.0, 107.7, 77.7, 73.5,
41.8, 34.8, 34.3, 15.0, 14.9. HRMS Calculated for [M+H]+
C12H17N4O2+ 249.1346, Found 249.1356.
Compound 10b White solid (71.5 mg, 65%), m.p: 107–
109 °C. 1H NMR (400 MHz, CDCl3) δ 7.64 (t, J=5.7 Hz,
1H, NH), 7.54 (s, 1H, Ar–H), 7.02 (t, J=5.8 Hz, 1H, NH),
5.47 (d, J=2.5 Hz, 2H, CH2), 3.40–3.33 (m, 4H, CH2), 2.38
(t, J = 2.5 Hz, 1H, CH), 1.66–1.60 (m, 4H, CH2), 0.97 (t,
J = 7.4 Hz, 3H, CH3), 0.95 (t, J = 7.4 Hz, 3H, CH3). 13C
NMR (101 MHz, CDCl3) δ 161.4, 159.1, 145.7, 137.0,
107.7, 77.6, 73.6, 41.8, 41.5, 41.1, 23.0, 11.6, 11.5. HRMS
Compound 5f White solid (106 mg, 87%), m.p: 161–
164 °C. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J=0.7 Hz,
1H, Ar–H), 7.22 (quasi t, J = 0.9 Hz, 1H, CH), 7.02 (t,
J=5.5 Hz, 1H, NH), 5.98–5.88 (m, 2H, =CH), 5.30–5.21
(m, 2H, =CH2), 5.18 (dq, J=1.4 and 10.2 Hz 1H, =CH2),
5.10 (dtd, J = 0.8, 1.8 and 17.2 Hz 1H, = CH2), 4.66 (dt,
J=1.7, 4.9 Hz, 2H, N–CH2), 4.09 (tt, J=1.6, 5.9 Hz, 2H,
N–CH2), 2.33 (d, J=1.1 Hz, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 161.0, 155.6, 146.8, 134.1, 133.4, 132.1, 128.8,
117.2, 116.7, 108.5, 106.4, 45.2, 41.8, 17.1. HRMS Calcu-
lated for [M+H]+ C14H17N4O2+ 273.1346, Found 273.1356.
+
Calculated for [M + H]+ C14H21N4O2 277.1659, Found
277.1670.
Experimental procedure for the synthesis of ethyl
3‑phenyl‑1‑(prop‑2‑yn‑1‑yl)‑1H‑pyrazole‑5‑carboxylate
and ethyl
5‑phenyl‑1‑(prop‑2‑yn‑1‑yl)‑1H‑pyrazole‑3‑carboxylate
(14a–d)
(1)
1 3

Molecular Diversity
(2)
Experimental procedure for the synthesis of pyrazolo[1,5‑a]
pyrazin‑4(5H)‑one derivatives (15a–e)
Reaction 1 [22]: A solution of t-BuOK (36 mmol) in
THF (30 mL) was added to a mixture of acetophenone deriv-
atives 10a–d (26 mmol) and diethyl oxalate (11) (28 mmol).
The fnal mixture was stirred at room temperature for 15 h
to form 12a–d. Thereafter, AcOH (64 mmol) and hydrazine
hydrate (48 mmol) were added. The mixture was heated
under refux for 2 h. The mixture was evaporated in a vac-
uum after being cooled, and the residue was extracted with
ethyl acetate (50 mL). The organic phase was washed with
saturated NaHCO3 and brine, dried over MgSO4, and con-
centrated with reduced pressure.
Reaction 2: Compound 13a–d (1 g, 4.63 mmol) was
dissolved in dimethylformamide (10 mL). NaH (0.22 g,
9.26 mmol, 60% oil suspension) was added to the solu-
tion, and the mixture was stirred in an ice bath for 30 min.
After completing the reaction, propargyl bromide (0.56 ml,
6.95 mmol) in DMF (5 mL) was added dropwise at room
temperature, and the solution was stirred for 16 h. Water was
(30 mL) added, and the solution was extracted with ethyl
acetate (3 × 15 mL). The combined organic extracts were
washed with brine and dried over MgSO4. The solvent was
evaporated to give crude products 14a–d and 14a′–d′ as a
compound mixture.
The mixture of Compound 14a–d and 14a′–d′ (4 mmol)
was dissolved in MeOH (20 mL). Cesium carbonate
(4 mmol) and an excess amount of corresponding amines
(20 mmol) were added to the solution. The mixture was
refuxed for 4 h. Water was (20 mL) added, and the solution
was extracted with ethyl acetate (3×10 mL). The combined
organic extracts were dried over MgSO4. The solvent was
evaporated to give a crude product. The crude product was
purifed with column chromatography on silica gel, elut-
ing with ethyl acetate/hexane (1:3) to give pyrazolo[1,5-a]
pyrazin-4(5H)-one derivatives (15a–e).
Note: Propargylation reaction gave two diferent isomers.
One of these isomers cannot be used for cyclization (14a′-
d′). However, to see the exact propargylated molecules, our
target starting materials, only one mixture was purifed.
Therefore, the others were used as a mixture of two isomers,
and the purifcation was done after cyclization reaction.
Compound 14a: Light yellow solid, m.p: 75–77 °C, 65%.
1H-NMR (400 MHz, CDCl3) δ 7.83–7.80 (m, 2H, Ar–H),
7.43–7.39 (m, 2H, Ar–H), 7.35–7.31 (m, 1H, Ar–H), 7.17
(s, 1H, Pyr-H), 5.41 (d, J = 2.5 Hz, 2H, -NCH2), 4.40 (q,
J=7.2 Hz, 2H, -OCH2), 2.38 (t, J=2.5 Hz, 1H, –CH), 1.42
(t, J=7.2 Hz, 3H, –CH3). 13C-NMR (100 MHz, CDCl3) δ
159.4, 150.8, 133.3, 132.2, 128.7, 128.2, 125.7, 108.6, 77.7,
73.1, 61.4, 41.6, 14.2.
Allyl amine was used. Compound 15a White solid (0.55 g,
88% yield), m.p: 120–122 °C. 1H-NMR (400 MHz, CDCl3)
δ 7.90–7.87 (m, 2H, Ar–H), 7.46–7.42 (m, 2H, Ar–H),
7.38–7.34 (m, 1H, Ar–H), 7.33 (bs, 1H,=CH), 7.31 (s, 1H,
Ar–H), 5.95 (ddt, J=4.9, 9.9, 15.3 Hz, 1H,=CH), 5.25–5.21
(m, 1H,=CH2), 5.13–5.08 (m, 1H,=CH2), 4.68 (dt, J=1.7,
4.9 Hz, 2H, N–CH2), 2.32 (d, J=1.0 Hz, 3H, –CH3). 13C-
NMR (100 MHz, CDCl3) δ 155.7, 152.4, 133.2, 132.3, 132.2,
128.8, 128.5, 126.4, 126.1, 116.7, 108.9, 101.8, 44.9, 16.9.
Compound 14a′: Yellow crystal, m.p: 93–95 °C, 45%.
1H-NMR (400 MHz, CDCl3) δ 7.50–7.47 (m, 2H, Ar–H),
7.46–7.40 (m, 3H, Ar–H), 6.82 (s, 1H, Pyr-H), 4.90 (d,
J = 2.5 Hz, 2H, -NCH2), 4.37 (q, J = 7.1 Hz, 2H, -OCH2),
2.42 (t, J=2.5 Hz, 1H, –CH), 1.35 (t, J=7.1 Hz, 3H, –CH3).
13C-NMR (100 MHz, CDCl3) δ 162.2, 145.1, 143.7, 129.3,
129.1, 129.0, 128.8, 109.1, 77.4, 74.4, 61.1, 40.6, 14.4.
1 3

Molecular Diversity
Butyl amine was used. Compound 15b White solid (0.57 g,
13C-NMR (100 MHz, CDCl3) δ 159.9, 155.8, 152.2, 133.4,
127.3, 125.7, 125.1, 114.2, 108.8, 100.8, 55.3, 43.2, 31.1,
20.2, 17.2, 13.8.
1
86% yield), m.p: 86–88 °C. H-NMR (400 MHz, CDCl3)
δ 7.89–7.86 (m, 2H, Ar–H), 7.45–7.40 (m, 2H, Ar–H),
7.37–7.32 (m, 1H, Ar–H), 7.30–7.29 (m, 1H, = CH), 7.27
(d, J = 0.7 Hz, 1H, Ar–H), 4.00–3.96 (m, 2H, N–CH2),
2.32 (d, J=1.0 Hz, 3H, –CH3), 1.72–1.64 (m, 2H, –CH2),
1.48–1.39 (m, 2H, –CH2), 0.98 (t, J = 7.4 Hz, 3H, –CH3).
13C-NMR (100 MHz, CDCl3) δ 155.9, 152.3, 133.4, 132.4,
128.8, 128.4, 126.1, 126.0, 108.8, 101.4, 43.2, 31.1, 30.9,
20.2, 17.2, 13.8.
Butyl amine was used. Compound 15e White solid (0.585 g,
90% yield), m.p: 171–173 °C. 1H-NMR (400 MHz, CDCl3)
δ 8.00–7.98 (AA′BB′, 2H, Ar–H), 7.70–7.67 (AA′BB′,
2H, Ar–H), 7.32 (s, 1H, Ar–H), 7.32–7.31(m, 1H, = CH),
4.02–3.98 (m, 2H, N–CH2), 2.36 (d, J=1.0 Hz, 3H, –CH3),
1.73–1.65 (m, 2H, –CH2), 1.49–1.40 (m, 2H, –CH2), 0.99
(t, J=7.4 Hz, 3H, –CH3). 13C-NMR (100 MHz, CDCl3) δ
155.8, 150.7, 135.8, 133.6, 130.2, 130.0, 126.8, 126.2, 125.7
(q, J=3.8 Hz, –CF3), 108.7, 102.0, 43.2, 31.1, 20.2, 17.2,
13.8.
Butyl amine was used. Compound 15c White solid (0.62 g,
94% yield), m.p: 159–160 °C. 1H-NMR (400 MHz, CDCl3)
δ 7.86–7.82 (m, 2H, Ar–H), 7.29–7.28 (m, 1H,=CH), 7.21
(s, 1H, Ar–H), 7.14–7.09 (m, 2H, Ar–H), 4.01–3.97 (m,
2H, N–CH2), 2.34 (d, J=0.9 Hz, 3H, –CH3), 1.72–1.64 (m,
2H, –CH2), 1.48–1.39 (m, 2H, –CH2), 0.98 (t, J = 7.4 Hz,
3H, –CH3). 13C-NMR (100 MHz, CDCl3) δ 162.9 (d,
J = 247.7 Hz), 155.8, 151.4, 133.5, 128.6 (d, J = 3.1 Hz),
127.8 (d, J=8.2 Hz), 126.2, 115.7 (d, J=21.7 Hz), 108.8,
101.2, 43.2, 31.1, 20.2, 17.2, 13.8.
Cell viability assay (MTT)
Cell viability was determined by 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells
were seeded in DMEM at 5 × 103 cells/200 µl per well in
96-well plates for 24 h at 37 °C. When cells were attached
after 24 h, cells were treated with DMSO (control group)
and Pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives (5 µM,
10 µM, 20 µM, 40 µM, 80 µM, and 160 µM) for 24 and
48 h. After incubation time, 10 µL MTT solution (5 mg/
mL) was added to each well. After 4 h of MTT incubation at
Butyl amine was used. Compound 15d White solid (0.55 mg,
84% yield), m.p: 128–130 °C. 1H-NMR (400 MHz, CDCl3)
δ 7.82–7.79 (AA′BB′, 2H, Ar–H), 7.29–7.28 (m, 1H,=CH),
7.19 (d, J = 0.7 Hz, 1H, Ar–H), 6.98–6.94 (AA′BB′, 2H,
Ar–H), 4.00–3.96 (m, 2H, N–CH2), 3.84 (s, 3H, -OCH3),
2.33 (d, J=1.1 Hz, 3H, –CH3), 1.72–1.64 (m, 2H, –CH2),
1.48–1.39 (m, 2H, –CH2), 0.98 (t, J = 7.4 Hz, 3H, –CH3).
1 3

Molecular Diversity
cyclization. Tetrahedron Lett 55:6698. https://doi.org/10.1016/j.
tetlet.2014.10.044
37 °C, 100 µL crystal dissolving bufer was added, and the
plates were gently shaken on an orbital shaker for 5 min. The
absorbance at 570 nm was measured with the SpectraMax
ID3 microplate reader. Each treatment was repeated 4 times.
The mean absorbance of four wells was used as an indicator
of relative cell growth.
11. Sari O, Seybek AF, Kaya S, Menges N, Erdem SS, Balci M (2019)
Mechanistic ınsights into the reaction of N-propargylated pyr-
role- and ındole-carbaldehyde with ammonia, alkyl amines, and
branched amines: a synthetic and theoretical ınvestigation. Eur J
Org Chem. https://doi.org/10.1002/ejoc.201900084
12. Surase YB, Samby K, Amale SR, Sood R, Purnapatre KP, Pareek
PK, Das B, Nanda K, Kumar S, Verma AK (2017) Identifcation
and synthesis of novel inhibitors of mycobacterium ATP syn-
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Acknowledgements Authors thank The Scientifc And Technologi-
cal Research Council Of Turkey (TÜBİTAK- 119Z011) for fnancial
support.
13. For the toxicity of triphenylphosphine, see the relevant datasheet.
https://www.nwmissouri.edu/naturalsciences/sds/t/Triphenylp
hosphine.pdf
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1 3