Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]pyrimidine-3-carboxamides as potent Aurora kinase inhibitors

Article abstract of DOI:10.1007/s00706-017-1943-7

Abstract: The Aurora kinases play a key role in mitosis and are overexpressed in multiple human tumor types; there has been considerable interest in developing Aurora kinase inhibitors as antitumor agents, particularly Aurora A and Aurora B kinases. A series of novel hydrazide hydrochlorides of pyrazolo[1,5-a]pyrimidine carboxamides were designed and synthesized and their inhibitory activities against Aurora kinases were evaluated. Some of the tested compounds exhibited low micromolar to nanomolar activity with respect to the inhibition of Aurora A kinase. The most potent compound in this series was found to be a potent inhibitor of Aurora A in an HTRF enzymatic assay with an IC₅₀ as low as 23?nM. A structure–activity relationship study indicated that halogen substitution in the benzene ring of amide plays an important role in kinase inhibitory potency. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-017-1943-7

Monatsh Chem
DOI 10.1007/s00706-017-1943-7
ORIGINAL PAPER
Design, synthesis, and evaluation of novel hydrazide
hydrochlorides of 6-aminopyrazolo[1,5-a]pyrimidine-3-
carboxamides as potent Aurora kinase inhibitors
1
2
1
1
A. K. Ajeesh Kumar
•
Yadav D. Bodke
•
Ganesh Sambasivam
•
Peter Serjious Lakra
Received: 14 October 2016 / Accepted: 19 February 2017
Ó Springer-Verlag Wien 2017
Abstract The Aurora kinases play a key role in mitosis
and are overexpressed in multiple human tumor types;
there has been considerable interest in developing Aurora
kinase inhibitors as antitumor agents, particularly Aurora A
and Aurora B kinases. A series of novel hydrazide
hydrochlorides of pyrazolo[1,5-a]pyrimidine carboxamides
were designed and synthesized and their inhibitory activi-
ties against Aurora kinases were evaluated. Some of the
tested compounds exhibited low micromolar to nanomolar
activity with respect to the inhibition of Aurora A kinase.
The most potent compound in this series was found to be a
potent inhibitor of Aurora A in an HTRF enzymatic assay
with an IC50 as low as 23 nM. A structure–activity rela-
tionship study indicated that halogen substitution in the
benzene ring of amide plays an important role in kinase
inhibitory potency.
Keywords Carboxamides Á Hydrazide hydrochloride Á
Pyrazolo[1,5-a]pyrimidine Á Anticancer activity Á
Aurora A Á Aurora B
Introduction
Aurora kinases are the members of serine/threonine kinases
and they are strongly associated with human cancer [1, 2].
Humans feature a family of Aurora kinases with three
members: isoforms A, B, and C. Both Aurora A and Aurora
B are significantly overexpressed in a wide range of human
cancer cell lines. The overexpression of Aurora A causes
aberrant phosphorylation of normal cell cycle targets and
cytoplasmic targets, leading to chromosomal instability,
oncogenic transformation, tumor progression, and devel-
opment of chemoresistance [3]. Similarly, the
overexpression of Aurora B increases the phosphorylation
of histone H3, forming more aggressive tumors in trans-
genic mouse models [4, 5]. Mechanistically, Aurora
kinases (A, B, and C) are regulatory proteins and play key
roles in the mitotic events of cell division [6, 7].
Graphical abstract
Over the past decade, extensive research has been car-
ried out towards the discovery of Aurora-selective small-
molecule inhibitors. As a result, a handful of small-mole-
cule Aurora inhibitors have been identified. Among them,
VX-680 [8] and SNS-314 [9, 10] have entered human
clinical trials as pan-Aurora kinase inhibitors; MLN8237
[11] and MK-5108 [12] are undergoing clinical assessment
as Aurora A-specific inhibitors as shown in Fig. 1.
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-017-1943-7) contains supplementary
material, which is available to authorized users.
&
A. K. Ajeesh Kumar
ajuedat@gmail.com
1
2
Anthem Biosciences Pvt. Ltd., # 49, Bommasandra Industrial
Area, Bommasandra, Bangalore, Karnataka 560 099, India
Recently, selective Aurora
B
inhibitor barasertib
(AZD1152) [13] and the pan-aurora kinase inhibitor
Department of PG Studies and Research in Industrial
Chemistry, School of Chemical Sciences, Kuvempu
University, Shankaraghatta, Shimoga, Karnataka 577 451,
India
danusertib (PHA-739358) [14] have been utilized in clin-
ical trials for the treatment of leukemia, myeloma, and
other solid tumors [15, 16].
123

A. K. A. Kumar et al.
Fig. 1 The structures of Aurora kinase inhibitors
In addition, a number of several heterocyclic amide
derivatives have been detailed as potent Aurora kinase
inhibitors. We envisioned that there are less reports for
amides from pyrazolo[1,5-a]pyrimidine pharmacophore
in this category and it was thought it would be worth-
while to synthesize some new pyrazolo[1,5-
a]pyrimidine-bearing amides. In this present article, we
describe the design, synthesis, and biochemical evalua-
tion of a novel series of pyrazolo[1,5-a]pyrimidine
analogs with representative example as shown in Fig. 2
showing good antiproliferative activity against both
Aurora A and Aurora B.
Results and discussion
Chemistry
The novel hydrazide hydrochloride derivatives of 6-amino-
pyrazolo[1,5-a]pyrimidine-3-carboxamides described
herein were synthesized over eight consecutive steps. The
first four steps involved the synthesis of novel compound
6-aminopyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
methyl ester 5 as shown in Scheme 1. Previously described
methods were used for the synthesis of both 5-amino-1H-
pyrazole-4-carboxylic acid methyl ester (2) [17–19] and
6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
methyl ester 3 [20]. The treatment of ester 3 with ben-
zophenone imine in presence of cesium carbonate,
xantphos, and Pd (dba) in 1,4-dioxane at 110 °C for 10 h
2
3
gave methyl 6-[(diphenylmethylidine)amino] pyrazolo[1,5-
a]pyrimidine-3-carboxylate 4 [21–23]. The treatment of
imine with HCl in MTBE gave 6-aminopyrazolo[1,5-
a]pyrimidine-3-carboxylic acid methyl ester 5.
The further design involved synthesis of substituted
benzoylamino-pyrazolo[1,5-a]pyrimidine-3-carboxylic
acids 8a–8h in two steps, as shown in Scheme 2. Step 5
involved the synthesis of carboxylic acid methyl esters 7a–
7h which were obtained by the treatment of 6-aminopy-
razolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester 5
with the corresponding acid chlorides 6a–6h in the pres-
ence of DIPEA at room temperature. The above carboxylic
acid methyl esters 7a–7h were subsequently treated with
lithium hydroxide monohydrate at 50 °C in THF, MeOH,
Fig. 2 Chemical structure of compound 11f which showed good
aurora kinase activity
1
23

Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]…
Scheme 1
Scheme 2
and H O for 2 h to give the corresponding benzoyl-
2
evaluate the Aurora kinase inhibitory activity of the syn-
thesized pyrazolo[1,5-a]pyrimidine carboxamide
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acids 8a–8h
via step 6.
compounds 10a–10h and 11a–11h. Table 1 shows the
percentage of inhibition of kinase activity as an average of
duplicate assay compared to the known non-selective
kinase inhibitor, staurosporine, as a positive control. Out of
these 16 derivatives, 4 analogs (11b, 11e, 11f, and 11g)
showed high potency over 90% inhibitions against Aurora
A, while the mean inhibitions were moderate in the rest of
the compounds. Interestingly, most of these analogs were
relatively less inhibitive against Aurora B (20.3–80.1%
inhibition at 10 lM). Meanwhile, it is observed that analog
11f showed good highest inhibition against both Aurora A
and Aurora B, while, analog 11a showed almost a lack of
the mean inhibition against both Aurora A and Aurora B.
After a single-dose preliminary screening, compounds
11a–11h were tested for further activity study (Table 2).
These compounds were tested against Aurora A and Aurora
The final step involved the synthesis of the target
compounds which involved the synthesis of corresponding
hydrochloride salts of hydrazides 11a–11h in two steps, as
shown in Scheme 3. Step 7 involved the synthesis of
respective hydrazine carboxylic acid tert-butyl esters 10a–
1
0h from 8a–8h using tert-butyl carbazate (9) in the
presence of DIPEA, HOAT, EDC.HCl in DMF at room
temperature. The above hydrazine carboxylic acid tert-
butyl esters 10a–10h were reacted with HCl in 1,4-dioxane
to obtain the target molecules 11a–11h via step 8.
Biological screening
An in vitro kinase assay was performed at a single-dose
concentration of 10 lM over Aurora A and Aurora B to
123

A. K. A. Kumar et al.
Scheme 3
Table 1 Inhibitory activity of compounds 10a–10h and 11a–11h
against Aur A and Aur B
Table 2 IC50 of compounds 11a–11h over Aur A and Aur B
1
a
a
Compound R
Aur A
Aur B
1
R
a
Compound
% Inhibition
Aur A
Enzymatic, IC50/lM Enzymatic, IC50/lM
Aur B
11a
11b
11c
11d
11e
11f
m-CF
p-F
3
2.83 ± 0.21
0.47 ± 0.036
2.67 ± 0.24
58.21 ± 5.11
38.13 ± 3.71
43.26 ± 4.10
49.63 ± 4.84
30.56 ± 3.73
9.63 ± 0.92
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
0a
0b
0c
0d
0e
0f
m-CF
p-F
3
64.1
74.4
50.2
41.6
67.2
79.3
71.2
42.8
81.6
90.4
86.5
82.8
92.4
94.5
93.3
80.2
20.3
36.2
51.9
49.8
58.2
53.8
46.3
35.4
42.1
56.7
37.9
32.8
55.8
80.1
65.8
34.3
p-Cl
p-Br
m-F
3,5-
1.76 ± 0.015
0.071 ± 0.006
0.023 ± 0.002
p-Cl
p-Br
m-F
Difluoro
o-F
p-OMe
3,5-Difluoro
o-F
1
1
a
1g
0.048 ± 0.004
2.65 ± 0.231
22.82 ± 2.67
66.39 ± 6.10
0g
0h
1a
1b
1c
1d
1e
1f
1h
p-OMe
IC50 values were determined from logarithmic concentration-in-
m-CF
p-F
3
hibition curves and are represented as means of at least two separate
experiments
p-Cl
p-Br
m-F
against Aurora B. Only the most potent compound 11f
showed the highest Aurora B activity with IC50 value
reaching 9.63 lM in the enzymatic assay, whereas the
internal standard staurosporine which showed a potency of
IC50 = 9 nM in the internal enzymatic Aurora assay.
3,5-Difluoro
o-F
1g
1h
p-OMe
Values are expressed as the means of three independent determi-
Structure–activity relationship
nations and are within plus or minus 10%
As can be seen from Table 1, a number of compounds
exhibited a robust inhibitory activity against isolated Aur-
ora A enzyme. With varied halogen, methoxy, and
trihalomethyl substituents in benzamide position, it was
observed that 3,5-difluoro, o-fluoro, m-fluoro, and p-fluoro
substituted benzamide hydrazine hydrochlorides (10c, 10f,
B by measuring the ability to inhibit phosphorylation of a
biotinylated-polypeptide substrate (p-GAT, CIS Bio Inter-
national) in a homogeneous time-resolved fluorescence
(HTRF) assay at an ATP concentration of 30 lM. The
results were reported as a 50% inhibition concentration
value (IC ). The IC values for these selected compounds
5
0
50
1
0i, and 10m) gave better enzymatic activity. Amongst
these derivatives 11f gave the highest activity
IC50 = 23 nM) against isolated Aurora A enzyme, which
were determined in a 10-dose IC50 mode with threefold
serial dilution starting at 30 lM as listed in Table 2. Most
of the compounds inhibited Aurora A with IC50 values in
the nanomolar to low micromolar range. Especially, com-
pounds 11e, 11f, and 11g revealed nanomolar IC50 values.
These results were encouraging with respect to the internal
standard staurosporine which showed a potency of
IC50 = 1.5 nM in the internal enzymatic Aurora A assay.
Once again, these analogs were relatively less active
(
contains 3,5-difluoro in the benzamide group as shown in
Table 2. The activity order came in the following sequence
3
,5-difluoro [ o-F [ m-F [ p-F [ m-CF . More or less
3
the same activity sequence was observed against Aurora B
enzyme. Compound 11f showed highest Aurora B activity
(
IC50 = 9.63 lM) in the enzymatic assay.
1
23

Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]…
In general, the structure–activity relationship study
indicated that halogen substitution plays important roles in
kinase inhibitory potency. It is worth noting that compound
5-Amino-1H-pyrazole-4-carboxylic acid methyl ester
(2) [17–19] and 6-bromopyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid methyl ester 3 [20] were synthesized
according to previously described methods.
1
1a showed almost a lack of the mean inhibition against
both Aurora A and Aurora B, suggesting that the—CF3
group of the benzamide played a key role in the binding of
inhibitors to Aurora kinase. Interestingly, compound 11f
showed good inhibition against both Aurora A and Aurora
B, due to the presence of 3,5-difluoro group in benzamide.
Methyl
a]pyrimidine-3-carboxylic acid methyl ester
4, C H N O )
6-[(Diphenylmethylidene)amino]pyrazolo[1,5-
(
2
1 16 4 2
To a degassed suspension of 12.00 g of 6-bromopyra-
zolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester (3,
46.86 mmol) and 21.37 g of cesium carbonate
3
(
65.60 mmol) in 180 cm of 1,4-dioxane was added
.44 g of 4,5-bis(diphenylphosphino)-9,9-dimethylanthene
4.21 mmol) and 1.40 g of tris(dibenzylideneacetone)di-
2
Conclusion
(
palladium(0) dichloromethane complex (1.40 mmol),
followed by the drop wise addition of 12.73 g of
benzophenone imine (70.29 mmol). The resultant reaction
mixture was heated to 110 °C for 10 h under argon
atmosphere. The completion of reaction was monitored by
TLC. The reaction mixture was filtered through Celite bed
A
new 6-aminopyrazolo[1,5-a]pyrimidine-3-carboxylic
acid scaffold, useful for kinase inhibition, has been
designed and synthesized. We have disclosed the synthesis
of a novel series of carboxamide containing hydrazide
hydrochloride derivatives of the above scaffold as potent
inhibitors of Aurora kinase. The compounds also provide
an opportunity of laying the foundation for promising
molecules of anticancer potency.
3
and was washed with 50 cm of ethyl acetate; the filtrate
was concentrated to dryness under reduced pressure to
afford the crude product. The crude product was purified by
column chromatography over silica gel with chloroform/
methanol (100:2, v/v) to afford methyl 6-[(diphenyl-
methylidene)amino]pyrazolo[1,5-a]pyrimidine-3-carboxy-
late (4) as a pale yellow solid (12.52 g, 75%). M.p.: 211.6–
Experimental
2
13.1 °C; TLC: R = 0.22 (EtOAc-hexane 2:8); IR (ATR):
f
Chemicals were of analytical grade and obtained from
Sigma-Aldrich Co. The purification of all intermediates
and final compounds was carried out by column chro-
matography using Merck silica gel with 230–400 mesh
size. The purity of synthesized compounds was determined
by thin layer chromatography experiments, performed on
alumina-backed silica gel 40 F254 plates (Merck, Darm-
stadt, Germany). TLC observation of these plates were
carried out by illuminating under UV (254 nm) lamp and
mꢀ = 1240 (C–N stretch), 1704 (C=N stretch), 1751 (C=O
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 3.77
-
1 1
6
(3H, s, ester CH ), 7.30–7.32 (3H, m, ArH), 7.37–7.47 (2H,
3
m, ArH), 7.52 (2H, dd, J = 7.6 Hz, J = 1.6 Hz, ArH),
1
2
7.59–7.63 (1H, m, ArH), 7.73 (2H, dd, J = 7.2 Hz,
1
J = 1.6 Hz, ArH), 8.38 (1H, d, J = 2.4 Hz, ArH), 8.51
2
1
3
(1H, s, ArH), 8.84 (1H, d, J = 2.0 Hz, ArH) ppm;
C
NMR (100 MHz, DMSO-d ): d = 51.5, 101.8, 127.4,129.0
6
(3 peaks), 129.4 (2 peaks),129.6 (2 peaks), 129.8 (2 peaks),
KMnO stain solution. Melting points were determined
4
1
1
32.4, 135.2, 136.4, 138.3, 144.2, 147.5, 149.5, 162.4,
?
73.4 ppm; LC–MS (ESI): m/z = 357.2 ([M?H] ).
1
13
using B u¨ chi B-540 instrument. All H NMR and C NMR
spectra were recorded on Bruker AM-300 (300 MHz for
1
13
H NMR and 75 MHz for C NMR) and Bruker AM-400
6-Aminopyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
1 13
400 MHz for H NMR and 100 MHz for C NMR),
(
methyl ester (5, C H N O )
8
8 4 2
Bruker BioSpin Corp., Germany. The chemical shifts are
reported in ppm (d) with reference to TMS as an internal
standard. The signals are designed as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; brs, broad singlet. IR
spectra for all the compounds were recorded using a Bruker
Alpha FTIR spectrometer using a diamond ATR single
reflectance module (24 scans). Molecular weights for all
the synthesized compounds were checked by LC–MS 6200
series Agilent Technology. Elemental analysis was carried
out with a Perkin-Elmer model 240-C apparatus. The
results of elemental analysis (C, H, and N) were within
To a suspension of 12.00 g of methyl 6-[(diphenylmethyli-
dene)amino]pyrazolo[1,5-a]pyrimidine-3-carboxylate (4,
3
133.67 mmol) in 240 cm of methyl tert-butyl ether was
3
added 30 cm of 4.5 M hydrochloric acid in methyl tert-
butyl ether maintaining the temperature between 10 and
15 °C over a period of 20 min under nitrogen atmosphere.
The resultant reaction mixture was stirred at room
temperature (28 °C) for 8 h. The completion of reaction
was monitored by TLC. The precipitate from the reaction
3
mixture was filtered, washed with 40 cm of methyl tert-
butyl ether and dried under vacuum at room temperature
±
0.4% of the calculated amounts.
(28 °C) for 6 h to afford hydrochloric salt of 6-
123

A. K. A. Kumar et al.
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
(400 MHz, DMSO-d ): d = 3.82 (3H, s, ester CH ), 7.84
6
3
ester. The suspension of hydrochloric salt of amine in
3
(1H, t, J = 7.0 Hz, ArH), 8.03 (1H, d, J = 7.6 Hz, ArH),
6
0 cm of water was basified (pH *10–12) with 10%
aqueous potassium carbonate and the product was
8.31–8.36 (2H, m, ArH), 8.61 (1H, s, ArH), 9.04 (1H, d,
J = 2.4 Hz, ArH), 9.72 (1H, d, J = 2.0 Hz, ArH), 11.07
13
3
extracted to 2 9 60 cm of n-butanol. The combined n-
(1H, s, amide NH) ppm; C NMR (100 MHz, DMSO-d ):
1
6
butanol layer was dried over anhydrous sodium sulfate and
concentrated to dryness under reduced pressure to afford 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester (5) as a pale yellow solid (5.95 g, 93%). M.p.: 197.2–
d = 51.6, 102.2, 120.3, 123.0, 125.7, 128.4 (q, JC-
= 271.0 Hz), 124.7, 124.8, 125.2, 127.4, 129.3, 129.3
F
3
(d, JC-F = 3.0 Hz), 129.6, 129.9, 130.3 (q, JC-
2
= 31.0 Hz), 130.4, 132.4, 134.7, 144.4, 147.7, 149.0,
F
-
162.5, 165.1 ppm; LC–MS (ESI): m/z = 363.0 ([M-H] ).
1
98.6 °C; TLC: R = 0.31 (MeOH–CHCl 1:9); IR (ATR):
f
3
mꢀ = 1255 (C–N stretch), 1550 (N–H bend), 3232 and 3399
6-(4-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
-
1
1
; H NMR (400 MHz, DMSO-d6):
(
N–H stretch) cm
carboxylic acid methyl ester (7b, C H FN O )
1
5
11
4 3
d = 3.76 (3H, s, ester CH ), 5.52 (2H, s, amine NH ), 8.28
3
2
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester (5) with 0.50 g of 4-fluorobenzoyl chloride (6b,
(
1H, d, J = 2.4 Hz, ArH), 8.33 (1H s, ArH), 8.48 (1H, d,
1
J = 2.4 Hz, ArH) ppm; C NMR (400 MHz, DMSO-d6):
3
d = 51.2, 101.0, 117.0, 135.5, 141.7, 145.6, 147.2,
3.14 mmol) in the presence of N,N-diisopropylethylamine
?
62.7 ppm; LC–MS (ESI): m/z = 192.9 ([M?H] ).
1
in N,N-dimethylformamide. The crude product was
obtained as a yellow solid, which on crystallization
afforded 7b as a pale yellow solid (0.77 g, 86%). M.p.:
General procedure for the synthesis of substituted car-
boxylic acid methyl esters 7a–7h
6
-Aminopyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
232.5–234.4 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
f
methyl ester (5, 0.55 g, 2.86 mmol) was dissolved in
5
(ATR): mꢀ = 1560 (N–H bend), 1659 (amidic C=O), 1708
3
.50 cm of N,N-dimethylformamide and 1.10 g of N,N-
-1
1
(ester C=O), 3306 (N–H stretch) cm
;
H NMR
diisopropylethylamine (8.58 mmol) and corresponding
acid chlorides 6a–6h (3.14 mmol) were added drop wise
under nitrogen atmosphere. The reaction mixture was
gradually allowed to attain room temperature (28 °C) and
stirred for 2–3 h. The completion of reaction was
(400 MHz, DMSO-d ): d = 3.82 (3H, s, ester CH ), 7.44
(2H, t, J = 8.8 Hz, ArH), 8.11 (2H, dd, J = 5.4 Hz,
1
6
3
J2 = 9.2 Hz, ArH), 8.60 (1H, s, ArH), 9.05 (1H, d,
J = 2.4 Hz, ArH), 9.72 (1H, d, J = 2.4 Hz, ArH), 10.89
1
(1H, s, amide NH) ppm; C NMR (100 MHz, DMSO-d ):
3
6
2
monitored by TLC. The reaction mixture was poured to
3
0 cm of ice cold water, stirred for 1 h and the
d = 51.6, 102.1, 116.0, 116.2 (d, J = 22.0 Hz), 125.5,
C-F
4
127.1, 130.3, 130.3 (d, J = 2.0 Hz), 131.0, 131.1 (d,
6
C-F
3
3
precipitate formed was filtered, washed with 5 cm of
water and dried under vacuum at room temperature
JC-F = 10.0 Hz), 144.4, 147.7, 149.1, 162.5, 163.7, 166.2
1
(d, JC-F = 249.0 Hz), 165.5 ppm; LC–MS (ESI): m/
?
z = 315.2 ([M?H] ).
(
28 °C) for 12 h to afford the crude product. The crude
product was crystallized by digesting in 5 cm of ethyl
3
6-(4-Chlorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
3
acetate and 25 cm of n-heptane at room temperature
carboxylic acid methyl ester (7c, C H ClN O )
15 11
3
4 3
(
28 °C), stirred for 1 h, filtered, washed with 5 cm of n-
This compound was prepared by the reaction of 6-aminopy-
razolo[1,5-a]pyrimidine-3-carboxylic acid methyl ester 5
with 0.55 g of 4-chlorobenzoyl chloride (6c, 3.14 mmol) in
the presence of N,N-diisopropylethylamine in N,N-
dimethylformamide. The crude product was obtained as a
yellow solid, which on crystallization afforded 7c as a pale
yellow solid (0.78 g, 83%). M.p.: 231.4–233.1 °C; TLC:
heptane and dried under vacuum at room temperature
28 °C) to afford the corresponding substituted carboxylic
acid methyl esters 7a–7h.
(
6
-[3-(Trifluoromethyl)benzoylamino]pyrazolo[1,5-
a]pyrimidine-3-carboxylic acid methyl ester
7a, C H F N O )
(
1
6 11 3 4 3
Rf = 0.41 (EtOAc-hexane 4:6); IR (ATR): mꢀ = 1563 (N–H
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester (5) with 0.65 g of 3-(trifluoromethyl)benzoyl chloride
bend), 1661 (amidic C=O), 1710 (ester C=O), 3307 (N–H
1 1
-
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 3.82
6
(3H, s, ester CH ), 7.68 (2H,d, J = 8.4 Hz, ArH), 8.05 (2H,
3
(
6a, 3.14 mmol) in the presence of N,N-diisopropylethy-
d, J = 8.8 Hz, ArH), 8.61 (1H, s, ArH), 9.04 (1H, d,
J = 2.4 Hz, ArH), 9.73 (1H, d, J = 2.4 Hz, ArH), 10.93
lamine in N,N-dimethylformamide. The crude product was
obtained as a yellow solid, which on crystallization
afforded 7a as a pale yellow solid (0.88 g, 85%). M.p.:
1
3
1H, s, amide NH) ppm; C NMR (100 MHz, DMSO-d6):
(
d = 51.6, 102.1, 125.4, 127.2, 129.2 (2 peaks), 130.2 (2
peaks), 132.5, 137.7, 144.4, 147.7, 149.1, 162.5, 165.5 ppm;
-
LC–MS (ESI): m/z = 329.6 ([M-H] ).
2
23.1–224.9 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
f
(
(
ATR): mꢀ = 1572 (N–H bend), 1660 (amidic C=O), 1701
-
1
1
ester C=O), 3311 (N–H stretch) cm
;
H NMR
1
23

Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]…
6
-(4-Bromobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
afforded 7f as a pale yellow solid (0.79 g, 83%). M.p.:
228.9–230.8 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
carboxylic acid methyl ester (7d, C H BrN O )
3
1
5
11
4
f
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester (5) with 0.69 g of 4-bromobenzoyl chloride (6d,
(ATR): mꢀ = 1562 (N–H bend), 1680 (amidic C=O), 1697
-
1
1
(ester C=O), 3360 (N–H stretch) cm
;
H NMR
(400 MHz, DMSO-d ): d = 3.82 (3H, s, ester CH ), 7.61
6
3
3
.14 mmol) in the presence of N,N-diisopropylethylamine
(1H, dt, J = 6.4 Hz, J = 11.6 Hz, ArH), 7.74 (2H, dd,
1 2
in N,N-dimethylformamide. The crude product was
obtained as a yellow solid, which on crystallization
afforded 7d as a pale yellow solid (0.85 g, 80%). M.p.:
J = 2.0 Hz, J = 8.0 Hz, ArH), 8.63 (1H, s, ArH), 9.03
1 2
(1H, d, J = 2.0 Hz, ArH), 9.71 (1H, d, J = 2.4 Hz, ArH),
1
10.99 (1H, s, amide NH) ppm; C NMR (100 MHz,
3
2
2
29.4–232.0 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
DMSO-d ): d = 51.6, 102.2, 108.0, 108.3, 108.5 (t, J
f
6
C-
2
(
(
(
(
ATR): mꢀ = 1560 (N–H bend), 1657 (amidic C=O), 1709
= 26.0 Hz), 111.6, 111.8 (d, J = 19.0 Hz), 111.7,
F C-F
-
1
1
2
111.9 (d, JC-F = 20.0 Hz), 125.1, 127.5, 137.1, 137.2,
ester C=O), 3305 (N–H stretch) cm
;
H NMR
3
137.3 (t, J = 9.0 Hz), 144.5, 147.8, 149.0, 161.4, 163.9
400 MHz, DMSO-d ): d = 3.82 (3H, s, ester CH ), 7.82
6
3
C-F
1
1
2H, dd, J = 2.0 Hz, J = 6.8 Hz, ArH), 7.97 (2H, dd,
1
(d, J = 246.0 Hz), 161.6, 164.0 (d, J = 246.0 Hz),
C-F C-F
2
-
J = 1.8 Hz, J = 6.6 Hz, ArH), 8.62 (1H, s, ArH), 9.04
1
162.5, 164.2 ppm; LC–MS (ESI): m/z = 331.1 ([M-H] ).
-(2-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid methyl ester (7g, C H FN O )
2
(
1H, d, J = 2.4 Hz, ArH), 9.72 (1H, d, J = 2.4 Hz, ArH),
6
1
0.93 (1H, s, amide NH) ppm; C NMR (100 MHz,
3
1
1
5
11
4 3
DMSO-d ): d = 51.6, 102.1, 125.4, 126.7, 127.2, 130.3 (2
6
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester 5 with 0.50 g of 2-fluorobenzoyl chloride (6g,
3.14 mmol) in the presence of N,N-diisopropylethylamine
in N,N-dimethylformamide. The crude product was
obtained as a yellow solid, which on crystallization
afforded 7g as a pale yellow solid (0.78 g, 87%). M.p.:
peaks), 132.1 (2 peaks), 132.9, 144.4, 147.7, 149.1, 162.5,
?
65.6 ppm; LC–MS (ESI): m/z = 376.7 ([M?H] ).
1
6
-(3-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid methyl ester (7e, C H FN O )
4 3
1
5
11
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester 5 with 0.50 g of 3-fluorobenzoyl chloride (6e,
236.7–238.5 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
f
3
.14 mmol) in the presence of N,N-diisopropylethylamine
(ATR): mꢀ = 1560 (N–H bend), 1658 (amidic C=O), 1709
-
1
1
in N,N-dimethylformamide. The crude product was
obtained as a yellow solid, which on crystallization
afforded 7e as a pale yellow solid (0.77 g, 86%). M.p.:
(ester C=O), 3305 (N–H stretch) cm
;
H NMR
(400 MHz, DMSO-d ): d = 3.82 (3H, s, ester CH ),
6
3
7.37–7.44 (2H, m, ArH), 7.62–7.68 (1H, m, ArH), 7.77
2
43.4–244.9 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
(1H, dt, J = 6.0 Hz, J = 13.6 Hz, ArH), 8.61 (1H, s,
1
f
2
(
(
(
(
ATR): mꢀ = 1560 (N–H bend), 1658 (amidic C=O), 1709
ester C=O), 3305 (N–H stretch) cm
ArH), 8.94 (1H, d, J = 2.0 Hz, ArH), 9.71 (1H, d,
13
-
1
1
;
H NMR
J = 2.4 Hz, ArH), 11.05 (1H, s, amide NH) ppm;
C
400 MHz, DMSO-d ); d = 3.82 (3H, s, ester CH ), 7.52
1H, dt, J = 6.4 Hz, J = 10.8 Hz, ArH), 7.64 (1H, td,
1
NMR (100 MHz, DMSO-d ): d = 51.6, 102.2, 116.7,
6
3
6
2
117.0 (d, JC-F = 22.0 Hz), 123.6, 123.8 (d, JC-
2
2
3
= 14.0 Hz), 125.2, 125.2 (d, JC-F = 7.0 Hz), 125.2,
J = 5.6 Hz, J = 8.0 Hz, ArH), 7.81–7.88 (2H, m, ArH),
1
2
F
4
126.7, 130.6, 130.6 (d, J = 3.0 Hz), 133.9, 134.0 (d,
3
8
.60 (1H, s, ArH), 9.04 (1H, d, J = 2.4 Hz, ArH), 9.71
C-F
1
(1H, d, J = 2.4 Hz, ArH), 10.92 (1H, s, amide NH) ppm;
13
JC-F = 9.0 Hz), 144.4, 147.7, 148.5, 158.3, 160.8 (d, J
C-
C NMR (100 MHz, DMSO-d ): d = 51.6, 102.1, 114.9,
= 249.0 Hz), 162.5, 163.9 ppm; LC–MS (ESI): m/
6
F
2
15.2 (d, JC-F = 23.0 Hz), 119.6, 119.8 (d, JC-
2
-
z = 313.0 ([M-H] ).
1
4
21.0 Hz), 124.5, 124.5 (d, JC-F = 2.0 Hz), 125.3,
=
F
3
1
27.2, 131.3, 131.4 (d, J = 8.0 Hz), 136.0, 136.1 (d,
C-F
6-(4-Methoxybenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
3
1
JC-F = 7.0 Hz), 144.4, 147.7, 149.0, 161.2, 163.6 (d, J
C-
carboxylic acid methyl ester (7h, C H N O4)
6 14 4
1
=
243.0 Hz), 162.5, 165.2, 165.2 ppm; LC–MS (ESI):
F
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester 5 with 0.53 g of 4-methoxybenzoyl chloride (6h,
3.14 mmol) in the presence of N,N-diisopropylethylamine
in N,N-dimethylformamide. The crude product was
obtained as a yellow solid, which on crystallization
afforded 7h as a pale yellow solid (0.82 g, 88%). M.p.:
-
m/z = 313.1 ([M-H] ).
6
3
-(3,5-Difluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-
-carboxylic acid methyl ester (7f, C H F N O )
5 10 2 4 3
1
This compound was prepared by the reaction of 6-
aminopyrazolo[1,5-a]pyrimidine-3-carboxylic acid methyl
ester 5 with 0.55 g of 3,5-difluorobenzoyl chloride (6f,
3
.14 mmol) in the presence of N,N-diisopropylethylamine
241.6–243.5 °C; TLC: R = 0.41 (EtOAc-hexane 4:6); IR
f
in N,N-dimethylformamide. The crude product was
(ATR): mꢀ = 1560 (N–H bend), 1658 (amidic C=O), 1709
-
1
1
obtained as a yellow solid, which on crystallization
(ester C=O), 3305 (N–H stretch) cm
;
H NMR
123

A. K. A. Kumar et al.
(
(
(
400 MHz, DMSO-d ): d = 3.82 (3H, s, ester CH ), 3.85
3H, s, methoxy CH ), 7.12 (2H, d, J = 8.8 Hz, ArH), 8.03
3
6-(4-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
6
3
carboxylic acid (8b, C H FN O )
4 3
14
9
2H, d, J = 8.8 Hz, ArH), 8.59 (1H, s, ArH), 9.06 (1H, d,
This compound was prepared by the reaction of 0.75 g of
6-(4-fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid methyl ester (7b, 2.38 mmol) with 0.30 g of
lithium hydroxide monohydrate (7.16 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8b as a pale yellow solid (0.56 g, 78%). M.p.:
J = 2.4 Hz, ArH), 9.73 (1H, d, J = 2.4 Hz, ArH), 10.70
1
1H, s, amide NH) ppm; C NMR (100 MHz, DMSO-d6):
3
(
d = 51.5, 53.9, 102.0, 114.3 (2 peaks), 125.8, 125.8, 126.8,
1
1
30.3 (2 peaks), 144.3, 147.59, 149.2, 162.5, 162.9,
-
65.9 ppm; LC–MS (ESI): m/z = 325.2 ([M-H] ).
General procedure for the synthesis of substituted car-
2
74.8–276.9 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
f
3
boxylic acids 8a–8h
To a suspension of substituted carboxylic acid methyl ester
(
(
ATR): mꢀ = 1562 (N–H bend), 1633 (amidic C=O), 1674
acid C=O), 3104 (N–H stretch), 3436 (O–H) cm ; H
-
1 1
3
a–7h (1.00 mmol) in 2.5 cm of tetrahydrofuran,
7
NMR (400 MHz, DMSO-d ): d = 7.44 (2H, t, J = 8.6 Hz,
3
3
.5 cm of methanol, and 2.2 cm of water was added
6
2
ArH), 8.11 (2H, dd, J = 5.4 Hz, J = 8.6 Hz, ArH), 8.51
1
2
lithium hydroxide monohydrate (3.00 mmol) and heated
to 72 °C under nitrogen atmosphere for 3–4 h. The
completion of reaction was monitored by TLC. The
reaction mixture was gradually allowed to attain room
temperature (28 °C) and concentrated to dryness under
(
1H, s, ArH), 9.01 (1H, d, J = 2.0 Hz, ArH), 9.70 (1H, d,
J = 2.4 Hz, ArH), 10.85 (1H, s, amide NH), 12.43 (1H, s,
3
acid-OH) ppm;
1
C
NMR (100 MHz, DMSO-d6):
2
d = 103.1, 116.0, 116.2 (d, JC-F = 22.0 Hz), 125.3,
1
3
4
26.9, 130.3, 130.3 (d, J = 2.0 Hz), 131.0, 131.1 (d,
3
C-F
reduced pressure. The residue was diluted with 10 cm of
1
JC-F = 9.0 Hz), 144.5, 147.8, 148.7, 163.6, 166.1 (d, J
C-
ice cold water, acidified (pH *5–6) with concentrated
hydrochloric acid and stirred for 1 h. The precipitate
3
formed was filtered, washed with 2 cm of water and
=
249.0 Hz), 163.5, 165.4 ppm; LC–MS (ESI): m/
F
?
z = 300.9 ([M?H] ).
dried at room temperature (28 °C) for 12 h to afford the
crude product. The crude product was crystallized by
6-(4-Chlorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (8c, C H ClN O )
9
1
4
4 3
3
digesting in 10 cm of methanol for 1 h at room
This compound was prepared by the reaction of 0.75 g of
6-(4-chlorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid methyl ester (7c, 2.26 mmol) with 0.28 g
of lithium hydroxide monohydrate (6.80 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8c as a pale yellow solid (0.55 g, 77%). M.p.:
3
temperature (28 °C), filtered, washed with 2 cm of
methanol, and dried under vacuum at room temperature
(28 °C) for 4 h to afford the corresponding substituted
carboxylic acids 8a–8h.
6
-[3-(Trifluoromethyl)benzoylamino]pyrazolo[1,5-
a]pyrimidine-3-carboxylic acid (8a, C H F N O )
1
5 9 3 4 3
2
70.3–272.5 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
f
3
This compound was prepared by the reaction of 0.75 g of
-[3-(trifluoromethyl)benzoylamino]pyrazolo[1,5-a]pyrim-
idine-3-carboxylic acid methyl ester (7a, 2.04 mmol) with
.26 g of lithium hydroxide monohydrate (6.17 mmol) in
(
(
ATR): mꢀ = 1632 (amidic C=O), 1677 (acid C=O), 3120
N–H stretch), 3322 (O–H) cm ; H NMR (400 MHz,
6
-
1
1
DMSO-d ): d = 7.64 (2H, d, J = 6.0 Hz, ArH), 8.08 (2H,
6
0
d, J = 8.4 Hz, ArH), 8.45 (1H, s, ArH), 8.99 (1H, s, ArH),
1
tetrahydrofuran, methanol, and water. The crude product
was obtained as a yellow solid, which on crystallization
afforded 8a as a pale yellow solid (0.54 g, 76%). M.p.:
3
9.69 (1H, s, amide NH), 11.13 (1H, s, acid-OH) ppm;
C
NMR (100 MHz, DMSO-d ): d = 105.9, 124.8, 127.0,
6
1
1
29.1 (2 peaks), 130.2 (2 peaks), 132.6, 137.5, 144.2,
2
62.3–264.1 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
f
3
47.6, 148.1, 164.4, 165.4 ppm; LC–MS (ESI): m/
-
(
(
ATR): mꢀ = 1561 (N–H bend), 1633 (amidic C=O), 1671
acid C=O), 3105 (N–H stretch), 3435 (O–H) cm ; H
z = 316.0 ([M-H] ).
-
1 1
NMR (400 MHz, DMSO-d ): d = 7.85 (1H, t, J = 8.0 Hz,
6-(4-Bromobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
6
ArH), 8.04 (1H, d, J = 7.6 Hz, ArH), 8.26–8.37 (1H, m,
carboxylic acid (8d, C H BrN O )
1
4
9
4 3
ArH), 8.45 (1H, d, J = 2.8 Hz, ArH), 8.56 (1H, s, ArH),
This compound was prepared by the reaction of 0.75 g of
6-(4-bromobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid methyl ester (7d, 2.00 mmol) with 0.25 g
of lithium hydroxide monohydrate (6.00 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8d as a pale yellow solid (0.57 g, 79%). M.p.:
268.2–269.9 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
9
.02 (1H, d, J = 2.4 Hz, ArH), 9.71 (1H, d, J = 2.0 Hz,
ArH), 11.07 (1H, s, amide NH), 12.44 (1H, s, acid-OH)
1
3
ppm; C NMR (100 MHz, DMSO-d ): d = 103.3, 120.,
6
1
23.0, 125.7, 128.4 (q, J = 271.0 Hz), 124.7, 124.8,
1
1
1
1
C-F
3
25.0, 127.3, 129.2, 129.3 (d, JC-F = 4.0 Hz), 129.3,
2
29.6, 129.9, 130.3 (q, JC-F = 32.0 Hz), 130.5, 132.4,
34.8, 144.6, 147.9, 148.7, 163.5, 163.7, 165.1 ppm; LC–
-
MS (ESI): m/z = 348.9 ([M-H] ).
f
3
(ATR): mꢀ = 1566 (N–H bend), 1633 (amidic C=O), 1678
1
23

Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]…
-1 1
acid C=O), 3119 (N–H stretch), 3323 (O–H) cm ; H
(
(100 MHz, DMSO-d ): d = 103.3, 108.0, 108.2, 108.5 (t,
6
2
2
JC-F = 26.0 Hz), 116.6, 111.8 (d, JC-F = 19.0 Hz),
NMR (400 MHz, DMSO-d6): d = 7.81 (2H, d,
J = 8.4 Hz, ArH), 8.00 (2H, d, J = 8.4 Hz, ArH), 8.47
2
111.6, 111.8 (d, JC-F = 20.0 Hz), 124.8, 127.2, 137.1,
3
137.2, 137.3 (t, JC-F = 8.0 Hz), 144.6, 147.9, 148.6,
(
1H, s, A–H), 9.00 (1H, d, J = 2.0 Hz, ArH), 9.69 (1H, s,
1
amide NH), 11.07 (1H, s, acid-OH) ppm; C NMR
3
1
1
161.4, 163.9 (d, JC-F = 248.0 Hz), 161.5, 164.0 ( J
C-
(
100 MHz, DMSO-d ): d = 105.4, 124.9, 126.5, 127.0,
30.4 (2 peaks), 132.1 (2 peaks), 133.0, 144.3, 147.7,
= 246.0 Hz), 163.4, 163.9 ppm; LC–MS (ESI): m/
F
6
-
1
1
z = 317.1 ([M-H] ).
-(2-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (8g, C H FN O )
48.2, 164.1, 165.6 ppm; LC–MS (ESI): m/z = 359.0
-
6
(
[M-2H] ).
1
4
9
4 3
This compound was prepared by the reaction of 0.75 g of
6-(2-fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid methyl ester (7g, 2.38 mmol) with 0.30 g of
lithium hydroxide monohydrate (7.16 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8g as a pale yellow solid (0.55 g, 77%). M.p.:
269.9–271.6 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
6
-(3-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (8e, C H9FN O )
1
4
4 3
This compound was prepared by the reaction of 0.75 g of
-(3-fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
6
boxylic acid methyl ester (7e, 2.38 mmol) with 0.30 g of
lithium hydroxide monohydrate (7.15 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8e as a pale yellow solid (0.54 g, 76%). M.p.:
f
3
(ATR): mꢀ = 1563 (N–H bend), 1635 (amidic C=O), 1673
-
1 1
(acid C=O), 3104 (N–H stretch), 3436 (O–H) cm ; H
2
68.3–270.1 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
NMR (400 MHz, DMSO-d ): d = 7.37–7.44 (2H, m,
f
3
6
(
(
ATR): mꢀ = 1562 (N–H bend), 1635 (amidic C=O), 1673
acid C=O), 3104 (N–H stretch), 3436 (O–H) cm ; H
ArH), 7.62–7.68 (1H, m, ArH), 7.78 (1H, dt, J = 6.0,
1
-
1 1
J2 = 13.2 Hz, ArH), 8.55 (1H, s, ArH), 8.92 (1H, d,
J = 2.4 Hz, ArH), 9.69 (1H, d, J = 2.4 Hz, ArH), 11.02
NMR (400 MHz, DMSO-d6): d = 7.51 (1H, dt,
J1 = 5.6 Hz, J2 = 13.6 Hz, ArH), 7.65 (1H, td,
J = 6.0 Hz, J = 8.0 Hz, ArH), 7.82-7.89 (2H, m,
1
3
(1H, s, amide NH), 12.43 (1H, s, acid-OH) ppm;
C
1
2
NMR (100 MHz, DMSO-d ): d = 103.2, 116.7, 117.0 (d,
6
2
3
JC-F = 21.0 Hz), 123.7, 123.8 (d, JC-F = 14.0 Hz),
ArH), 8.55 (1H, s, ArH), 9.02 (1H, d, J = 2.4 Hz, ArH),
2
125.0, 125.2 (d, J = 21.0 Hz), 126.6, 130.6, 130.64
9
1
.71 (1H, d, J = 2.4 Hz, ArH), 10.89 (1H, s, amide NH),
C-F
1
2.44 (1H, s, acid-OH) ppm; C NMR (100 MHz, DMSO-
3
4
(d, JC-F = 2.0 Hz), 133.9, 134.0 (d, JC-F = 9.0 Hz),
3
2
1
144.6, 147.9, 148.2, 158.3, 160.8 (d, J = 249.0 Hz),
d ): d = 103.2, 114.9, 115.2 (d, J = 23.0 Hz), 119.5,
6
C-F
C-F
2
19.8 (d, JC-F = 21.0 Hz), 124.4, 124.4 (d, JC-
4
1
163.52, 163.9 ppm; LC–MS (ESI): m/z = 299.2
-
([M-H] ).
3
3.0 Hz), 125.1, 127.0, 131.3, 131.3 (d, JC-
=
=
F
3
7.0 Hz), 136.0, 136.1 (d, JC-F = 7.0 Hz), 144.5,
F
1
6-(4-Methoxybenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
1
47.8, 148.6, 161.2, 163.6 (d, JC-F = 243.0 Hz), 163.5,
-
65.1, 165.1 ppm; LC–MS (ESI): m/z = 299.1 ([M-H] ).
carboxylic acid (8h, C15
This compound was prepared by the reaction of 0.75 g of
-(4-methoxybenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
H N O )
12 4 3
1
6
6
3
-(3,5-Difluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-
carboxylic acid methyl ester (7h, 2.29 mmol) with 0.29 g
of lithium hydroxide monohydrate (6.89 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8h as a pale yellow solid (0.56 g, 78%). M.p.:
-carboxylic acid (8f, C H F N O )
4 8 2 4 3
1
This compound was prepared by the reaction of 0.75 g of
-(3,5-difluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
6
carboxylic acid methyl ester (7f, 72.25 mmol) with 0.28 g
lithium hydroxide monohydrate (6.77 mmol) in tetrahy-
drofuran, methanol, and water. The crude product was
obtained as a yellow solid, which on crystallization
afforded 8f as a pale yellow solid (0.54 g, 76%). M.p.:
2
63.4–265.3 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
f
3
(
(
ATR): mꢀ = 1563 (N–H bend), 1635 (amidic C=O), 1673
acid C=O), 3104 (N–H stretch), 3436 (O–H) cm ; H
-
1 1
NMR (400 MHz, DMSO-d ): d = 3.85 (3H, s, methoxy
6
2
59.6–261.4 °C; TLC: R = 0.10 (MeOH–CHCl 1:9); IR
f
3
CH ), 7.11 (2H, d, J = 8.8 Hz, ArH), 8.03 (2H, d,
3
(
(
ATR): mꢀ = 1556 (N–H bend), 1631 (amidic C=O), 1668
acid C=O), 3128 (N–H stretch), 3363 (O–H) cm ; H
-
1
1
J = 9.2 Hz, ArH), 8.52 (1H, s, Ar–H), 9.03 (1H, d,
J = 2.4 Hz, ArH), 9.70 (1H, d, J = 2.0 Hz, ArH), 10.65
NMR (400 MHz, DMSO-d6): d = 7.60 (1H, dt,
J1 = 7.2 Hz, J2 = 16.0 Hz, ArH), 7.74 (2H, t,
J = 6.4 Hz, ArH), 8.55 (1H, s, ArH), 8.99 (1H, d,
J = 2.4 Hz, ArH), 9.68 (1H, d, J = 2.4 Hz, ArH), 10.93
13
1H, s, amide NH), 12.41 (1H, s, acid-OH) ppm; C NMR
(
(
100 MHz, DMSO-d ): d = 55.9, 103.0, 114.3, 125.6,
6
1
25.8, 126.6, 130.2, 144.4, 147.7, 148.7, 162.9, 163.5,
-
165.9 ppm; LC–MS (ESI): m/z = 311.1 ([M-H] ).
1
3
(
1H, s, amide NH), 12.45 (1H, s, acid-OH) ppm; C NMR
123

A. K. A. Kumar et al.
General procedure for the synthesis of compounds 10a–
0h
To a suspension of substituted carboxylic acid 8a–8h
carbazate (9, 1.83 mmol) in presence of 0.38 g of ethyl
carbodiimide hydrochloride (2.49 mmol), 0.27 g of 1-
hydroxy-7-azabenzotriazole (2.00 mmol), and 0.64 g of
N,N-diisopropylethylamine (5.00 mmol) in N,N-dimethyl-
formamide. The crude product was obtained as a brown
solid, which on column chromatography purification
afforded 10b as a beige solid (0.58 g, 84%). M.p.: 208.5–
210.2 °C; TLC: R = 0.71 (MeOH–CHCl 0.5:9.5); IR
1
3
(
1.00 mmol) in 2.5 cm of N,N-dimethylformamide was
added tert-butyl carbazate (9, 1.10 mmol), ethyl carbodi-
imide hydrochloride (1.50 mmol), 1-hydroxy-7-
azabenzotriazole (1.20 mmol), followed by the drop wise
addition of N,N-diisopropylethylamine (3.00 mmol) and
stirred at room temperature (28 °C) under nitrogen atmo-
sphere for 12 h. The completion of reaction was monitored
f
3
(ATR): mꢀ = 1161 (alkoxy C–O), 1510, 1564, 1602 (N–H
bend), 1666, 1687, 1708 (C=O), 3078, 3221, 3311 (N–H
-1 1
3
by TLC. The reaction mixture was poured into 25 cm of
3
ice cold water, stirred for 1 h, filtered, washed with 5 cm
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 1.43
6
(9H, s, boc CH ), 7.44 (2H, t, J = 8.8 Hz, ArH), 8.11 (2H,
3
of water, and dried under vacuum at room temperature
dt, J = 3.6 Hz, J = 10.4 Hz, ArH), 8.60 (1H, s, ArH),
1
2
(
28 °C) for 8 h to afford the crude product. The crude
9.02 (1H, d, J = 6.4 Hz, amide NH), 9.04 (1H, s, ArH),
9.32 (1H, s, ArH), 9.75 (1H, d, J = 2.0 Hz, amide NH),
product was purified by column chromatography over silica
gel with chloroform/methanol (100:2 v/v) to afford the
compounds 10a–10h.
1
10.87 (1H, s, amide NH) ppm; C NMR (100 MHz,
3
DMSO-d ): d = 28.5 (3 peaks), 79.8, 104.0, 116.0, 116.2
6
2
d, J = 22.0 Hz), 125.2, 127.4, 130.3, 130.3 (d, J
4
(
0
C-F C-
3
3.0 Hz), 131.0, 131.1 (d, JC-F = 9.0 Hz), 143.2,
N -[6-[3-(Trifluoromethyl)benzoylamino]pyrazolo[1,5-
=
F
a]pyrimidine-3-carbonyl]hydrazinecarboxylic acid tert-
1
46.1, 148.5, 155.9, 161.4, 163.7, 166.2 (d, JC-
1
butyl ester (10a, C H F N O )
2
0 19 3 6 4
=
249.0 Hz), 165.5 ppm; LC–MS (ESI): m/z = 413.2
F
This compound was prepared by the reaction of 0.50 g of
-[3-(trifluoromethyl)benzoylamino]pyrazolo[1,5-a]pyrim-
-
[M-H] ).
(
6
0
idine-3-carboxylic acid (8a, 1.42 mmol) with 0.20 g of
tert-butyl carbazate (9, 1.57 mmol) in presence of 0.33 g of
ethyl carbodiimide hydrochloride (2.14 mmol), 0.40 g of
N -[6-(4-Chlorobenzoylamino)pyrazolo[1,5-a]pyrimidine-
3-carbonyl]hydrazinecarboxylic acid tert-butyl ester
(10c, C H ClN O )
1
9
19
6 4
1
-hydroxy-7-azabenzotriazole (1.71 mmol), and 0.55 g of
This compound was prepared by the reaction of 0.5 g of 6-(4-
chlorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid (8c, 1.57 mmol) with 0.23 g of tert-butyl
carbazate (9, 1.73 mmol) in presence of 0.36 g of ethyl
carbodiimide hydrochloride (2.36 mmol), 0.25 g of 1-hy-
droxy-7-azabenzotriazole (1.89 mmol), and 0.61 g of N,N-
diisopropylethylamine (4.73 mmol) in N,N-dimethylfor-
mamide. The crude product was obtained as a brown solid,
which on column chromatography purification afforded 10c
as a beige solid (0.60 g, 89%). M.p.: 212.8–214.1 °C; TLC:
R = 0.71 (MeOH–CHCl 0.5:9.5); IR (ATR): mꢀ = 1157
N,N-diisopropylethylamine (4.28 mmol) in N,N-dimethyl-
formamide. The crude product was obtained as a brown
solid, which on column chromatography purification
afforded 10a as a beige solid (0.55 g, 83%). M.p.: 198.5–
2
00.4 °C; TLC: R = 0.71 (MeOH–CHCl 0.5:9.5); IR
f
3
(
ATR): mꢀ = 1161 (alkoxy C–O), 1535, 1556, 1618 (N–H
bend), 1666, 1687, 1722 (C=O), 3097, 3215, 3284 (N–H
-
1 1
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 1.43
6
(
9H, s, boc CH ), 7.85 (1H, t, J = 8.0 Hz, ArH), 8.05 (1H,
3
d, J = 7.6 Hz, ArH), 8.33 (1H, d, J = 7.6 Hz, ArH), 8.37
1H, s, ArH), 8.61 (1H, s, ArH), 9.05 (1H, d, J = 5.6 Hz,
f
3
(
(alkoxy C–O), 1521, 1560, 1591 (N–H bend), 1666, 1687,
-1 1
amide NH), 9.04 (1H, s, ArH), 9.33 (1H, s, ArH), 9.75 (1H,
1708 (C=O), 3103, 3221, 3294 (N–H stretch) cm ; H NMR
d, J = 1.6 Hz, amide NH), 11.07 (1H, s, amide NH) ppm;
1
(400 MHz, DMSO-d ): d = 1.43 (9H, s, boc CH ), 7.68 (2H,
6
3
3
C NMR (100 MHz, DMSO-d ): d = 28.5 (3 peaks),
d, J = 8.4 Hz, ArH), 8.06 (2H, d, J = 8.4 Hz, ArH), 8.60
(1H, s, ArH), 9.02 (1H, d, J = 6.4 Hz, amide NH), 9.03 (1H,
s, ArH), 9.32 (1H, s, ArH), 9.75 (1H, d, J = 2.0 Hz, amide
6
1
9.8, 104.1, 120.3, 123.0, 125.7, 128.4 (q, JC-
7
3
271.0 Hz), 124.7, 124.8, 124.9 (t, JC-F = 4.0 Hz),
=
F
3
27.7, 127.8, 127.9 (t, JC-F = 4.0 Hz), 129.3, 129.6,
2
30.0, 130.3 (q, JC-F = 32.0 Hz), 130.5, 132.4, 134.8,
13
1
NH), 10.92 (1H, s, amide NH) ppm; C NMR (100 MHz,
1
1
DMSO-d ): d = 28.5 (3 peaks), 79.8, 104.0, 125.1, 127.4,
6
43.3, 146.2, 148.5, 155.9, 161.4, 165.1 ppm; LC–MS
-
ESI): m/z = 463.2 ([M-H] ).
129.1 (2 peaks), 130.1 (2 peaks), 132.5, 137.6, 143.2, 146.1,
(
148.4, 155.9, 161.4, 165.4 ppm; LC–MS (ESI): m/z = 429.3
-
[M-H] ).
(
N’-[6-(4-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-
-carbonyl]hydrazinecarboxylic acid tert-butyl ester
10b, C H FN6O )
3
N’-[6-(4-Bromobenzoylamino)pyrazolo[1,5-a]pyrimidine-
3-carbonyl]hydrazinecarboxylic acid tert-butyl ester
(10d, C H BrN O )
(
1
9
19
4
This compound was prepared by the reaction of 0.5 g of 6-
4-fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid (8b, 1.66 mmol) with 0.24 g of tert-butyl
1
9
19
6 4
(
This compound was prepared by the reaction of 0.50 g
of 6-(4-bromobenzoylamino)pyrazolo[1,5-a]pyrimidine-
1
23

Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]…
0
3-carboxylic acid (8d, 1.38 mmol) with 0.20 g of tert-
butyl carbazate (9, 1.52 mmol) in presence of 0.32 g of
N -[6-(3,5-Difluorobenzoylamino)pyrazolo[1,5-a]pyrim-
idine-3-carbonyl]hydrazinecarboxylic acid tert-butyl ester
(10f, C H F N O )
ethyl carbodiimide hydrochloride (2.07 mmol), 0.22 g of
1
9 18 2 6 4
1
-hydroxy-7-azabenzotriazole (1.66 mmol), and 0.53 g
This compound was prepared by the reaction of 0.50 g of
6-(3,5-difluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (8f, 1.57 mmol) with 0.22 g of tert-butyl
carbazate (9, 1.72 mmol) in presence of 0.36 g of ethyl
carbodiimide hydrochloride (2.35 mmol), 0.25 g of 1-
hydroxy-7-azabenzotriazole (1.88 mmol), and 0.60 g of
N,N-diisopropylethylamine (4.71 mmol) in N,N-dimethyl-
formamide. The crude product was obtained as a brown
solid, which on column chromatography purification
afforded 10f as a beige solid (0.58 g, 86%). M.p.: 203.8–
205.7 °C; TLC: R = 0.71 (MeOH–CHCl 0.5:9.5); IR
of N,N-diisopropylethylamine (4.14 mmol) in N,N-
dimethylformamide. The crude product was obtained as
a brown solid, which on column chromatography purifi-
cation afforded 10d as a beige solid 0.56 g, 86%). M.p.:
2
0
1
3
15.6–217.7 °C; TLC: Rf = 0.71 (MeOH–CHCl3
.5:9.5); IR (ATR): mꢀ = 1157 (alkoxy C–O), 1525,
562, 1589 (N–H bend), 1666, 1687, 1708 (C=O),
-
1
1
099, 3221, 3254 (N–H stretch) cm
;
H NMR
(
400 MHz, DMSO-d ): d = 1.43 (9H, s, boc CH ),
6
3
7
.82 (2H, d, J = 8.4 Hz, ArH), 7.98 (2H, d,
f
3
J = 8.4 Hz, ArH), 8.60 (1H, s, ArH), 9.02 (1H, d,
J = 6.8 Hz, amide NH), 9.03 (1H, s, ArH), 9.32 (1H, s,
ArH), 9.74 (1H, d, J = 2.0 Hz, amide NH), 10.92 (1H, s,
(ATR): mꢀ = 1159 (alkoxy C–O), 1511, 1565, 1609 (N–H
bend), 1667, 1667, 1709 (C=O), 3077, 3220, 3311 (N–H
-
1 1
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 1.43
6
1
3
amide NH) ppm;
C NMR (100 MHz, DMSO-d6):
(9H, s, boc CH ), 7.62 (1H, dt, J = 4.8 Hz, J = 13.6 Hz,
3
1
2
d = 28.5 (3 peaks), 79.8, 104.0, 125.1, 126.6, 127.4,
ArH), 7.75 (2H, dt, J = 4.8 Hz, J = 6.4 Hz, ArH), 8.61
1
2
1
1
30.3 (2 peaks), 132.1 (2 peaks), 132.8, 143.1, 146.1,
(1H, s, ArH), 9.01 (2H, d, J = 2.4 Hz, amide NH, ArH),
9.33 (1H, s, ArH), 9.74 (1H, d, J = 2.0 Hz, amide NH),
13
48.4, 155.9, 161.4, 165.6 ppm; LC–MS (ESI): m/
-
z = 474.9 ([M-H] ).
10.98 (1H, s, amide NH) ppm; C NMR (100 MHz,
DMSO-d ): d = 28.5 (3 peaks), 79.8, 104.1, 108.0, 108.2,
0
6
N -[3-(2-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-
2
08.5 (t, JC-F = 26.0 Hz), 111.6, 111.8 (d, JC-
2
1
3
-carbonyl]hydrazinecarboxylic acid tert-butyl ester
2
=
19.0 Hz), 111.7, 111.8 (d, J = 19.0 Hz), 124.7,
C-F
F
(
10e, C H FN O )
1
9
19
6
4
3
27.6, 137.1, 137.2, 137.3 (t, JC-F = 8.0 Hz), 143.2,
1
Thiscompoundwaspreparedbythereactionof0.50 gof6-(3-
fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
1
46.2, 148.3, 155.9, 161.4, 163.9 (d, J = 252.0 Hz)
1
1
1
C-F
1
61.5, 164.0 (d, JC-F = 252.0 Hz), 161.5, 163.9,
boxylic acid (8e, 1.66 mmol) with 0.24 g of tert-butyl
carbazate (9, 1.83 mmol) in presence of 0.38 g of ethyl
carbodiimide hydrochloride (2.49 mmol), 0.27 g of 1-
hydroxy-7-azabenzotriazole (2.00 mmol), and 0.64 g of
N,N-diisopropylethylamine (5.00 mmol) in N,N-dimethyl-
formamide. The crude product was obtained as a brown
solid, which on column chromatography purification
afforded 10e as a beige solid (0.60 g, 87%). M.p.: 206.1–
-
64.0 ppm; LC–MS (ESI): m/z = 432.1 ([M-H] ).
0
N -[6-(2-Fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-
3-carbonyl]hydrazinecarboxylic acid tert-butyl ester
(10g, C H FN O )
1
9
19
6 4
This compound was prepared by the reaction of 0.50 g of 6-
(2-fluorobenzoylamino)pyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid (8g, 1.66 mmol) with 0.024 g of tert-butyl
carbazate (9, 1.83 mmol) in presence of 0.38 g of ethyl
carbodiimide hydrochloride (2.49 mmol), 0.27 g of 1-
hydroxy-7-azabenzotriazole (2.00 mmol), and 0.64 g of
N,N-diisopropylethylamine (5.00 mmol) in N,N-dimethyl-
formamide. The crude product was obtained as a brown
solid, which on column chromatography purification
afforded 10g as a beige solid (0.61 g, 89%). M.p.: 200.5–
202.6 °C; TLC: R = 0.71 (MeOH–CHCl 0.5:9.5); IR
2
07.9 °C; TLC: R = 0.71 (MeOH–CHCl3 0.5:9.5); IR
ATR): mꢀ = 1160 (alkoxy C–O), 1512, 1563, 1601 (N–H
bend), 1666, 1687, 1705 (C=O), 3078, 3221, 3311 (N–H
f
(
-1 1
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 1.43
9H, s, boc CH ), 7.52 (1H, dt, J = 6.4 Hz, J = 15.2 Hz,
3 1 2
6
(
ArH), 7.65 (1H, dt, J = 6.0 Hz, J = 10.0 Hz, ArH),
2
1
7
.83-7.90 (2H, m, ArH), 8.61 (1H, s, ArH), 9.03 (1H, d,
J = 6.8 Hz, amide NH), 9.04 (1H, s, ArH), 9.32 (1H, s,
ArH), 9.75 (1H, d, J = 2.0 Hz, amide NH), 10.93 (1H, s,
f
3
(ATR): mꢀ = 1159 (alkoxy C–O), 1511, 1565, 1601 (N–H
1
3
amide NH) ppm;
C NMR (100 MHz, DMSO-d ):
6
bend), 1666, 1687, 1708 (C=O), 3075, 3220, 3310 (N–H
-1 1
2
d = 28.5 (3 peaks), 79.8, 104.1, 115.0, 115.2 (d, JC-
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 1.43
6
2
22.0 Hz), 119.6, 119.8 (d, JC-F = 21.0 Hz), 124.5,
=
(9H, s, boc CH ), 7.37–7.44 (2H, m, ArH), 7.63–7.80 (1H,
3
F
4
24.5 (d, J = 2.0 Hz), 125.0, 127.5, 131.3, 131.4 (d,
1
3
m, ArH), 7.78 (1H, t, J = 6.8 Hz, ArH), 8.61 (1H, s, ArH),
8.94 (1H, d, J = 2.0 Hz, amide NH), 9.01 (1H, s, ArH), 9.32
(1H, s, ArH), 9.74 (1H, d, J = 1.6 Hz, amide NH), 11.04
C-F
3
JC-F = 8.0 Hz), 136.1, 136.1 (d, J = 7.0 Hz), 143.2,
C-F
1
1
46.1, 148.5, 155.9, 161.2, 161.4, 163.6, 165.2 (d, J
C-
1
3
=
258.0 Hz) ppm; LC–MS (ESI): m/z = 413.2
(1H, s, amide NH) ppm; C NMR (100 MHz, DMSO-d ):
6
F
-
[M-H] ).
2
(
d = 27.4 (3 peaks), 78.7, 103.1, 115.7, 115.9 (d, JC-
123

A. K. A. Kumar et al.
3
22.0 Hz), 122.6, 122.7 (d, JC-F = 14.0 Hz), 123.8,
=
1689 (C=O), 3050, 3147 (N–H stretch), 3235, 3412 (1°
F
2
24.1 (d, J = 26.0 Hz), 124.1, 125.9, 129.5, 129.5 (d,
-1 1
1
4
amine, N–H stretch) cm ; H NMR (400 MHz, DMSO-
d6): d = 7.82 (1H, t, J = 8.0 Hz, ArH), 8.01 (1H, d,
J = 7.6 Hz, ArH), 8.45 (2H, d, J = 6.4 Hz, ArH), 8.72
(1H, s, ArH), 9.31 (1H, d, J = 2.4 Hz, ArH), 9.80 (1H, d,
J = 2.0 Hz, ArH), 10.48 (2H, s, amide NH), 11.53 (1H, s,
C-F
3
JC-F = 2.0 Hz), 132.8, 132.9 (d, J = 8.0 Hz), 142.1,
C-F
1
45.1,146.9, 154.8, 157.2, 159.7 (d, JC-F = 249.0 Hz),
1
-
60.3, 162.8 ppm; LC–MS (ESI): m/z = 413.2 ([M-H] ).
1
0
N -[6-(4-Methoxybenzoylamino)pyrazolo[1,5-a]pyrim-
idine-3-carbonyl]hydrazinecarboxylic acid tert-butyl ester
1
3
amide NH) ppm;
C NMR (75 MHz, DMSO-d ):
6
1
d = 102.2, 118.8, 122.5, 126.1, 129.7 (q,
JC-
270.9 Hz), 125.0, 125.1, 125.2 (t, JC-F = 4.0 Hz),
(
10h, C H N O )
20 22 6 5
3
=
F
This compound was prepared by the reaction of 0.50 g of
-(4-methoxybenzoylamino)pyrazolo[1,5-a]pyrimidine-3-
3
25.5, 127.5, 127.6, 127.7 (t, JC-F = 4.0 Hz), 129.2,
1
6
2
29.5, 129.9, 130.2 (q, JC-F = 23.5 Hz), 132.5, 134.4,
1
1
carboxylic acid (8h, 1.60 mmol) with 0.23 g of tert-butyl
carbazate (9, 1.76 mmol) in presence of 0.37 g of ethyl
carbodiimide hydrochloride (2.40 mmol), 0.26 g of 1-
hydroxy-7-azabenzotriazole (1.92 mmol), and 0.62 g of
N,N-diisopropylethylamine (4.80 mmol) in N,N-dimethyl-
formamide. The crude product was obtained as a brown
solid, which on column chromatography purification
afforded 10h as a beige solid (0.56 g, 83%). M.p.: 213.8–
43.4, 146.3, 149.0, 160.7, 165.0 ppm; LC–MS (ESI): m/
z = 365.1 ([M?H-HCl]).
4-Fluoro-N-[3-(hydrazinocarbonyl)pyrazolo[1,5-a]pyrim-
idin-6-yl]benzamide hydrochloride
(11b, C H ClFN O )
6 2
1
4
12
From the reaction of 0.40 g of 10b (0.96 mmol) with
4.5 M hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11b as a pale yellow solid (0.29 g,
2
15.5 °C; TLC: R = 0.71 (MeOH–CHCl3 0.5:9.5); IR
f
(
ATR): mꢀ = 1157 (alkoxy C–O), 1523, 1563, 1595 (N–H
bend), 1666, 1687, 1708 (C=O), 3100, 3221, 3260 (N–H
87%). M.p.: 229.6–231.4 °C; TLC: R = 0.10 (MeOH–
f
CHCl 1:9); IR (ATR): mꢀ = 1522, 1596 (N–H bend), 1655,
3
-
1 1
stretch) cm ; H NMR (400 MHz, DMSO-d ): d = 1.43
1687 (C=O), 3053, 3154 (2° N–H stretch), 3238, 3417 (1°
N–H2 stretch) cm
6
-
1
1
; H NMR (400 MHz, DMSO-d6):
(
9H, s, boc CH ), 3.85 (3H, s, methoxy CH ), 7.11 (2H, d,
3
3
J = 8.88 Hz, ArH), 8.03 (2H, d, J = 8.8 Hz, ArH), 8.58
1H, s, ArH), 9.01 (1H, s, ArH), 9.05 (1H, d, J = 2.4 Hz,
amide NH), 9.31 (1H, s, ArH), 9.74 (1H, d, J = 2.0 Hz,
d = 7.42 (2H, dt, J = 6.8 Hz, J = 15.6 Hz, ArH), 8.22
(2H, dt, J = 3.2 Hz, J = 10.0 Hz, ArH), 8.71 (1H, s,
1 2
1
2
(
ArH), 9.30 (1H, d, J = 2.0 Hz, ArH), 9.80 (1H, d,
J = 2.4 Hz, ArH), 10.48 (1H, s, amide NH), 11.28 (1H,
1
3
amide NH), 10.68 (1H, s, amide NH) ppm; C NMR
1
s, amide NH) ppm; C NMR (100 MHz, DMSO-d ):
3
(
100 MHz, DMSO-d ): d = 27.4 (3 peaks), 54.9, 78.7,
6
6
2
1
02.9, 113.2 (2 peaks), 124.4, 124.7, 126.0, 129.2 (2
d = 102.2, 115.9, 116.1 (d, JC-F = 22.0 Hz), 125.8,
127.4, 130.1, 130.1 (d, J = 3.0 Hz), 131.3, 131.3 (d,
4
peaks), 142.0, 144.9, 147.45 154.9, 160.4, 161.8,
C-F
-
1
64.8 ppm; LC–MS (ESI): m/z = 425.2 ([M-H] ).
General procedure for the synthesis of compounds 11a–
1h
To a suspension of compounds 10a–10h (1.00 mmol) in
3JC-F = 9.0 Hz), 143.4, 146.3, 149.1, 160.8, 163.7, 166.2
1
d, JC-F = 249.0 Hz), 165.4 ppm; LC–MS (ESI): m/
(
z = 315.1 ([M?H-HCl]).
1
4-Chloro-N-[3-(hydrazinocarbonyl)pyrazolo[1,5-a]pyrim-
idin-6-yl]benzamide hydrochloride
3
3
4
cm of dichloromethane was added 1 cm of 4.5 M
hydrochloric acid in 1,4-dioxane at 0 °C. The temperature
was gradually allowed to attain room temperature (28 °C)
and stirred for 12 h under nitrogen atmosphere. The
completion of reaction was monitored by TLC. The
precipitate from the reaction mixture was filtered, washed
(11c, C H Cl N O )
1
4
12
2 6 2
From the reaction of 0.40 g of 10c (0.92 mmol) with 4.5 M
hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11c as a pale yellow solid (0.31 g,
91%). M.p.: 238.1–239.9 °C; TLC: R = 0.10 (MeOH–
f
3
with 1 cm of dichloromethane and dried under vacuum at
CHCl 1:9); IR (ATR): mꢀ = 1523, 1597 (N–H bend), 1656,
3
room temperature (28 °C) for 4 h to afford the compounds
1687 (C=O), 3053, 3155 (2° N–H stretch), 3238, 3417 (1°
N–H2 stretch) cm ; H NMR (400 MHz, DMSO-d6):
-
1
1
1
1a–11h.
N-[3-(Hydrazinocarbonyl)pyrazolo[1,5-a]pyrimidin-6-yl]-
-(trifluoromethyl)benzamide hydrochloride
11a, C H ClF N O )
d = 7.95 (2H, d, J = 8.4 Hz, ArH), 8.16 (2H, d,
J = 8.4 Hz, ArH), 8.72 (1H, s, ArH), 9.30 (1H, d,
3
J = 2.4 Hz, ArH), 9.80 (1H, d, J = 2.4 Hz, ArH), 10.48
13
(
1
5
12
3
6
2
(1H, s, amide NH), 11.36 (1H, s, amide NH) ppm;
C
From the reaction of 0.40 g of 10a (0.86 mmol) with 4.5 M
hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11a as a pale yellow solid (0.29 g,
NMR (100 MHz, DMSO-d ): d = 102.2, 125.7, 127.4,
6
129.1 (2 peaks), 130.4 (2 peaks), 132.3, 137.6, 143.4,
146.3, 149.0, 160.8, 165.5 ppm; LC–MS (ESI): m/z = 329
8
5%). M.p.: 236.7–238.8 °C; TLC: R = 0.10 (MeOH–
f
([M–H-HCl]).
CHCl3 1:9); IR (ATR): mꢀ = 1521, 1598 (N–H bend), 1657,
1
23

Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]…
-
N-H2 stretch) cm ; H NMR (400 MHz, DMSO-d6):
1
1
4
-Bromo-N-[3-(hydrazinocarbonyl)pyrazolo[1,5-a]pyrim-
idin-6-yl]benzamide hydrochloride
11d, C H ClBrN O )
d = 7.60 (1H, d, J = 6.0 Hz, ArH), 7.87 (2H, s, ArH),
8.83 (1H, s, ArH), 9.29–9.33 (1H, m, ArH), 10.55 (1H, s,
(
1
4
12
6 2
1
amide NH), 11.45 (1H, s, amide NH) ppm; C NMR
3
From the reaction of 0.40 g of 10d (0.84 mmol) with
.5 M hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11d as a pale yellow solid (0.30 g,
9%). M.p.: 239.7–242.0 °C; TLC: R = 0.10 (MeOH–
4
(100 MHz, DMSO-d ): d = 102.3, 108.0, 108.3, 108.5 (t,
6
2
2
JC-F = 26.0 Hz), 111.8, 112.0 (d, JC-F = 20.0 Hz),
2
111.9, 112.1 (d, JC-F = 19.0 Hz), 125.4, 127.7, 136.9,
8
f
3
137.0, 137.1 (t, JC-F = 9.0 Hz), 143.5, 146.4, 149.0,
CHCl 1:9); IR (ATR): mꢀ = 1520, 1595 (N–H bend), 1656,
1
3
1
160.8, 161.4, 163.8 (d, J = 245.0 Hz), 161.5, 163.9,
687 (C=O), 3054, 3157 (2° N–H stretch), 3238, 3415 (1°
C-F
-
1
;
1
N-H2 stretch) cm
H NMR (400 MHz, DMSO-d6):
164.0 ppm; LC–MS (ESI): m/z = 331.1 ([M-H-HCl]).
d = 7.78 (2H, d, J = 8.4 Hz, ArH), 8.08 (2H, d,
J = 8.4 Hz, ArH), 8.71 (1H, s, ArH), 9.29 (1H, d,
J = 2.4 Hz, ArH), 9.79 (1H, d, J = 2.0 Hz, ArH), 10.48
2
-Fluoro-N-[3-(hydrazinocarbonyl)pyrazolo[1,5-a]pyrim-
idin-6-yl]benzamide hydrochloride
11g, C H ClFN O )
(
1
3
14 12
6 2
(
1H, s, amide NH), 11.34 (1H, s, amide NH) ppm;
C
From the reaction of 0.40 g of 10g (0.96 mmol) with
.5 M hydrochloric acid in 1,4-dioxane in dichloro-
NMR (100 MHz, DMSO-d ): d = 102.3, 125.7, 126.7,
6
4
1
1
27.5, 130.5 (2 peaks), 132.0 (2 peaks), 132.7, 143.4,
methane medium was obtained 11g as a pale yellow
solid (0.29 g, 87%). M.p.: 233.5–235.6 °C; TLC:
Rf = 0.10 (MeOH–CHCl3 1:9); IR (ATR): mꢀ = 1556,
46.3, 149.0, 160.8, 165.6 ppm; LC–MS (ESI): m/
z = 377.3 ([M?2H-HCl]).
1
579 (N–H bend), 1629, 1685 (C=O), 3050, 3152 (2° N–
-1 1
3
-Fluoro-N-[3-(hydrazinocarbonyl)pyrazolo[1,5-a]pyrim-
idin-6-yl]benzamide hydrochloride
11e, C H ClFN O )
H stretch), 3235, 3414 (1° N-H stretch) cm ; H NMR
2
(400 MHz, DMSO-d ): d = 7.38-7.45 (2H, m, ArH),
6
(
7.64–7.69 (1H, m, ArH), 7.70 (1H, dt, J = 6.0 Hz,
1
1
4
12
6
2
From the reaction of 0.40 g of 10e (0.96 mmol) with 4.5 M
hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11e as a pale yellow solid (0.29 g,
J2 = 13.2 Hz, ArH), 8.72 (1H, s, ArH), 9.03 (1H, d,
J = 2.0 Hz, ArH), 9.75 (1H, d, J = 2.4 Hz, ArH), 10.37
1
3
(1H, s, amide NH), 11.09 (1H, s, amide NH) ppm;
C
8
8%). M.p.: 234.5–236.4 °C; TLC: R = 0.10 (MeOH–
NMR (100 MHz, DMSO-d ): d = 102.4, 116.8, 117.0 (d,
f
6
2
3
JC-F = 21.0 Hz), 123.5, 123.7 (d, JC-F = 13.0 Hz),
CHCl 1:9); IR (ATR): mꢀ = 1523, 1597 (N–H bend), 1656,
3
2
125.1, 125.2 (d, JC-F = 21.0 Hz), 125.4, 127.1, 130.6,
1
687 (C=O), 3053, 3155 (2° N–H stretch), 3238, 3417 (1°
-
1
1
4
130.6 (d, JC-F = 2.0 Hz), 133.9, 134.0 (d, JC-
3
N–H2 stretch) cm
;
H NMR (400 MHz, DMSO-d6):
1
= 9.0 Hz), 143.5, 146.3, 148.5, 158.3, 160.8 (d, J
d = 7.48 (1H, dt, J = 6.4 Hz, J = 14.8 Hz, ArH), 7.61
1
2
F
F
C-
(
1H, dt, J = 5.6 Hz, J = 10.0 Hz, ArH), 7.97–8.01 (2H,
1
= 243.0 Hz), 160.8, 163.9 ppm; LC–MS (ESI): m/
2
m, ArH), 8.71 (1H, s, ArH), 9.34 (1H, d, J = 2.4 Hz, ArH),
z = 313.0 ([M–H-HCl]).
9
1
.80 (1H, d, J = 2.4 Hz, ArH), 10.49 (1H, s, amide NH),
N-[3-(Hydrazinocarbonyl)pyrazolo[1,5-a]pyrimidin-6-yl]-
1
1.44 (1H, s, amide NH) ppm; C NMR (100 MHz,
3
4-methoxybenzamide hydrochloride
2
DMSO-d ): d = 102.3, 115.2, 115.4 (d, J = 23.0 Hz),
1
6
C-F
(
11h, C H ClN O )
15 15
2
4
6 3
19.6, 119.8 (d, J = 21.0 Hz), 124.7, 124.7 (d, J
C-F C-
From the reaction of 0.40 g of 10h (0.93 mmol) with
.5 M hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11h as a pale yellow solid (0.30 g,
0%). M.p.: 248.5–250.3 °C; TLC: R = 0.10 (MeOH–
3
=
=
2.0 Hz), 125.7, 127.6, 131.2, 131.3 (d,
3
8.0 Hz), 135.8, 135.9 (d, JC-F = 7.0 Hz), 143.5,
JC-
F
4
F
1
1
46.3, 149.1, 160.8, 161.2, 163.6 (d, J = 243.0 Hz),
C-F
9
f
1
65.2,
165.2 ppm;
LC–MS (ESI): m/z = 313.0
CHCl 1:9); IR (ATR): mꢀ = 1120 (alkoxy C–O), 1558,
3
(
[M-H-HCl]).
1
606 (N–H bend), 1653, 1670 (C=O), 3202, 3304 (2° N–H
-1 1
stretch), 3335, 3400 (1° N–H stretch) cm ; H NMR
2
3
,5-Difluoro-N-[3-(hydrazinocarbonyl)pyrazolo[1,5-
(400 MHz, DMSO-d ): d = 3.95 (3H, s, methoxy CH ),
6
3
a]pyrimidin-6-yl]benzamide hydrochloride
11f, C H ClF N O )
7.13 (2H, d, J = 8.8 Hz, ArH), 8.8 (2H, d, J = 8.8 Hz,
ArH), 8.70 (1H, d, J = 1.2 Hz, ArH), 9.17 (1H, s,
ArH), 9.77 (1H, d, J = 2.0 Hz, ArH), 10.39 (1H, s,
(
1
4
11
2 6 2
From the reaction of 0.40 g of 10f (0.92 mmol) with 4.5 M
hydrochloric acid in 1,4-dioxane in dichloromethane
medium was obtained 11f as a pale yellow solid (0.31 g,
1
3
amide NH), 10.80 (1H, s, amide NH) ppm; C NMR
(100 MHz, DMSO-d ): d = 55.9, 102.1, 114.2 (2 peaks),
6
9
3%). M.p.: 228.8–230.5 °C; TLC: R = 0.10 (MeOH–
125.7, 126.1, 127.2, 130.4 (2 peaks), 143.3, 146.2, 149.1,
160.8, 162.9, 166.0 ppm; LC–MS (ESI): m/z = 327.3
([M?H-HCl]).
f
CHCl 1:9); IR (ATR): mꢀ = 1568, 1597 (N–H bend), 1622,
1
3
678 (C=O), 3088, 3190 (2° N–H stretch), 3273, 3457 (1°
123

A. K. A. Kumar et al.
The kinase assay
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platform. The following base reaction buffer was used for
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2
3
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.1 mM Na VO , and 1% DMSO.
3 4
The reaction procedure is used as follows: the required
cofactor for the enzymatic reaction was added to a freshly
prepared buffer solution, followed by the addition of the
Aurora kinase at a concentration of 20 lM. The contents
were mixed gently and the compound dissolved in DMSO
was added to the reaction mixture at 10 lM concentration.
Compounds were evaluated in a 10-dose IC50 mode with
threefold serial dilution starting at 30 lM for IC deter-
1
50
1
3
3
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reaction, and the mixture was incubated at room tempera-
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compound in a five-dose IC50 mode with tenfold serial
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thank Analytical chemistry team, Department of Analytical Chem-
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the analytical work. Also, we would like to thank molecular biology
team, Department of Molecular Biology, Anthem Biosciences, Ban-
galore, India, for executing the Aurora Kinase activity studies.
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