Synthesis, cytotoxic characterization, and SAR study of imidazo[1,2-b]pyrazole-7-carboxamides

Article abstract of DOI:10.1002/ardp.201800062

The synthesis and in vitro cytotoxic characteristics of new imidazo[1,2-b]pyrazole-7-carboxamides were investigated. Following a hit-to-lead optimization exploiting 2D and 3D cultures of MCF-7 human breast, 4T1 mammary gland, and HL-60 human promyelocytic leukemia cancer cell lines, a 67-membered library was constructed and the structure–activity relationship (SAR) was determined. Seven synthesized analogues exhibited sub-micromolar activities, from which compound 63 exerted the most significant potency with a remarkable HL-60 sensitivity (IC₅₀ = 0.183 μM).

Full text of DOI:10.1002/ardp.201800062

Received: 2 March 2018
Revised: 25 April 2018
Accepted: 2 May 2018
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DOI: 10.1002/ardp.201800062
FULL PAPER
Synthesis, cytotoxic characterization, and SAR study of
imidazo[1,2-b]pyrazole-7-carboxamides
András Demjén1,2
László Hackler Jr.1
Iván Kanizsai1
Róbert Alföldi1
Anikó Angyal1,2
Márió Gyuris1
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Gábor J. Szebeni3
János Wölfling2
László G. Puskás1,3
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1 AVIDIN Ltd., Szeged, Hungary
Abstract
2 Department of Organic Chemistry,
University of Szeged, Szeged, Hungary
The synthesis and in vitro cytotoxic characteristics of new imidazo[1,2-b]pyrazole-7-
carboxamides were investigated. Following a hit-to-lead optimization exploiting 2D
and 3D cultures of MCF-7 human breast, 4T1 mammary gland, and HL-60 human
promyelocytic leukemia cancer cell lines, a 67-membered library was constructed and
the structure–activity relationship (SAR) was determined. Seven synthesized
analogues exhibited sub-micromolar activities, from which compound 63 exerted
the most significant potency with a remarkable HL-60 sensitivity (IC50 = 0.183 μM).
3 Laboratory of Functional Genomics, Institute
of Genetics, Biological Research Centre,
Hungarian Academy of Sciences, Szeged,
Hungary
Correspondence
Dr. Iván Kanizsai, AVIDIN Ltd., Alsó kikötő sor
11/D, Szeged H-6726, Hungary.
Email: i.kanizsai@avidinbiotech.com
Funding information
K E Y W O R D S
National Research, Development and
Innovation Office (NKFI), Grant numbers:
GINOP-2.3.2-15-2016-00030, GINOP-2.3.2-
15-2016-00001; Hungarian Academy of
Sciences, Grant number: BO/00139/17/8
anticancer agents, cytotoxic, Groebke–Blackburn–Bienaymé reaction, imidazo[1,2-b]pyrazole,
multicomponent reaction
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1
INTRODUCTION
pharmacological properties are barely studied, and only a limited
number of reports focused on their antitumor potential.[43–49] In terms
Pyrazoles are versatile pharmacophore scaffolds possessing a wide
range of biological activities including anticancer property.[1–5] In
particular, their amino-substituted derivatives have attracted consid-
erable attention as extensively studied templates for antitumor
agents.[6–12] The related aminopyrazole moiety is featured in several
promising drug candidates, such as PHA-533533 (cyclin-dependent
kinase inhibitor),[13,14] tozasertib (Aurora kinase A inhibitor),[15,16] and
barasertib (Aurora kinase B inhibitor).[17]
of anticancer activity, 3-aminoimidazo[1,2-b]pyrazole-7-carbonitriles
1 were shown to inhibit spleen tyrosine kinase (Syk) with IC50 in sub-
micromolar range,[50] while ethyl ester analogues 2 acted as potent
topoisomerase IIα catalytic inhibitors (Figure 1).[51] Recently, some
imidazo[1,2-b]pyrazole-7-carboxamides 3 were identified as Bruton's
tyrosine kinase inhibitors (Btk; useful in the treatment of hematological
malignancies),[52,53] while a series of C-7 aminomethylated derivatives
4 was synthesized and showed considerable antitumor activity against
five human (A549, Hs683, MCF-7, SKMEL28, U373) and a murine
(B16F10) cancer cell types.[54]
Furthermore, the incorporation of the aminopyrazole scaffold into
condensed heterocycles has emerged as a powerful strategy for novel
anticancer drug development. Numerous pyrazolo[1,5-a]pyrimi-
dines,[18–23] pyrazolo[3,4-d]pyrimidines,[24–31] pyrazolo[1,5-a][1,3,5]-
triazines,[32,33] pyrazolo[5,1-c][1,2,4]triazoles,[34,35] and other amino-
pyrazole-fused bicycles[36–42] display remarkable cancer-related
enzyme inhibitory activities. Despite several synthetic routes are
available for the construction of imidazo[1,2-b]pyrazoles, their
The most facile approach for the assembly of the imidazo[1,2-b]-
pyrazole framework is the Groebke–Blackburn–Bienaymé three-
component (GBB-3CR) reaction.[55–57] By employing this one-step
operation, highly functionalized imidazo[1,2-b]pyrazoles can be
accessed from 3-aminopyrazoles, aldehydes, and isocyanides as
starting materials in the presence of Brønsted or Lewis acid catalysts.
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Arch Pharm Chem Life Sci. 2018;e1800062.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800062

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FIGURE 1 Previously reported imidazo[1,2-b]pyrazoles with anticancer activity and design strategy
Cell-based phenotypic screening has emerged as a promising
approach for the discovery of new drug candidates.[58] In vitro cellular
screening protocols to identify cytoprotective or cytotoxic compounds
are commonly based on two-dimensional (2D) assays.[59–61] However,
cells grown on 2D plastic surfaces may result in higher drug sensitivity
compared to three-dimensional (3D) culturing methods.[62,63] To apply
a more accurate model system, the 2D plastic surfaces can be
supplemented by different 3D culturing techniques for better read-
outs and more reliable microenvironment modeling.
Herein we report the synthesis and in vitro cytotoxic activity of a
novel 67-membered imidazo[1,2-b]pyrazole-7-carboxamide library.
Cytotoxicity of imidazo[1,2-b]pyrazole-7-carboxamides was tested on
non-adherent acute promyelocytic cell line HL-60 and on adherent
breast cancer cell lines 4T1 and MCF-7. In this study, we supplemented
the conventional 2D cytotoxicity test with a scaffold-free 3D culturing
method using MCF-7 human breast cancer cells and 4T1 mouse
mammary gland tumor cells.
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
SCHEME 1 Synthesis of the imidazo[1,2-b]pyrazole-7-
carboxamide library and pyrazole precursors. Reagents and
conditions: (a) R1R2NH, toluene or DMF, 80°C, 6–12 h, 25–97%; (b)
N,N-dimethylformamide dimethyl acetal (R3 = H) or N,N-
dimethylacetamide dimethyl acetal (R3 = Me), toluene, 80°C, 6 h,
49–98%; (c) N2H4 · H2O, EtOH, reflux, 12 h, 35–91%; (d) HClO4 (20
mol%), MeCN, rt, 6 h, 23–85%
The synthesis of the imidazo[1,2-b]pyrazole-7-carboxamide library
was accomplished by the general synthetic route outlined in Scheme 1.
First, the reaction of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropane-
nitrile (5) with a series of alicyclic-, alkyl- or (hetero)aryl-substituted
amines in toluene or DMF resulted in cyanoacetamide intermediates 6,

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from which condensation reaction with the appropriate
N,N-dimethylamide dimethyl acetal in toluene led to synthones 7.
The successive cyclization step performed with hydrazine monohy-
drate in refluxing EtOH furnished secondary and tertiary 3-amino-
pyrazole-4-carboxamides 8. The 3-aminopyrazole-4-carboxamide (9)
precursor (R1 = R2 = R3 = H) was obtained by a known procedure.[64]
Target compounds 12–77 were prepared through GBB-type
one-pot three-component assembly of pyrazoles 8 or 9, aldehydes 10,
and isocyanides 11. Reactions were performed under a common GBB
condition (MeCN as solvent, HClO4 as catalyst). Since an oxidative
minor side reaction yielding dehydrogenated 3-imino derivatives of the
target products was observed, argon atmosphere was employed.
Derivative 78 was accessed by Eschweiler–Clarke reductive methyl-
ation of 63. NMR analysis of 12–78 confirmed the exclusive presence
of the tautomeric form shown in Scheme 1b.
analogue 28 was proved to be inactive in the tested range. Gratifyingly,
by introducing tert-butyl group in R4, appreciable IC50 values (<10 μM)
could be achieved in all cell lines (compound 29). Therefore, we turned
our attention to the optimization of R5 keeping tert-butyl for R4. It was
found that the replacement of the flexible cyclohexyl with the more
compact tert-butyl group (homologue 30) had negative influence on
the activity against MCF-7 and 4T1 cells, while compounds with
aromatic substituents (31: R5 = 4-MeO-C6H4 and 32: R5 = 4-F-C6H4)
completely lost the cytotoxic potency. Interestingly, compound 33
bearing the longer and sterically more demanding tert-octyl moiety in
R5 provided better growth inhibitory effects in terms of both the 2D
and 3D culture cells than compound 29. Afterwards, a series of other
aliphatic substituents were used as R4, while R5 was maintained as tert-
octyl. Note that the incorporation of smaller cyclopropyl (34) or ethyl
(35) groups led to the dramatic loss of the the overall activity compared
to that of 33, while isopropyl-substituted analogue 36 exhibited
moderate, but still lower efficacies than 33. On the other hand, either
the elongation of R4 in 33 by a vinyl group (compound 37) or the
application of a more bulky substituent (compound 38) resulted in no
improvements in the antitumor potency. From these SAR results,
product 33 (R4 = t-Bu, R5 = t-octyl) provided the best bioactivity and,
therefore, it was selected for the basis of further hit-to-lead structure
optimization.
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2.2 Biological evaluation and structure–activity
relationship (SAR)
The in vitro antitumor activity of the synthesized imidazo[1,2-b]-
pyrazole-7-carboxamide library was evaluated against two human
(acute promyelocytic leukemia HL-60 and breast adenocarcinoma
MCF-7) and a murine (mammary carcinoma 4T1) cancer cell lines using
resazurin cell viability colometric assay with doxorubicin as positive
control. For a more relevant comparison and hit identification, the
traditional 2D MCF-7 and 4T1 screens were supplemented with
in vivo-like 3D culture models. The reported IC50 values are averages of
at least two independent experiments and represent the concentration
that is required for 50% growth inhibition after an exposure time of
72 h. Of the 67 compound tested 7 possessed sub-micromolar
anticancer activities against HL-60 cells, while 14 displayed an IC50
in the single-digit μM range in all cell lines. The development of the 67-
membered chemical library was divided into two stages: First C-7
primary carboxamides (group 1; modification of R4 and R5) were
evaluated, followed by C-7 secondary/tertiary carboxamides (group 2;
modification of R1 and R2).
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2.2.2 SAR of C-7 secondary/tertiary carboxamides
(group 2)
In this group, the C-7 carboxamide functionality as a new diversity
point of the imidazo[1,2-b]pyrazole scaffold was exploited. By the
variation of R1 and R2, a series of simple alkyl-, alicyclic-, benzylic-,
and (hetero)aryl-substituted carboxamides were designed and tested
for
in vitro antitumor activity and compared with hit compound 33
(Table 3). Small aliphatic substituents such as methyl or cyclopropyl
were well tolerated (compounds 39 and 42), and only slight
decreases in IC50 values were observed compared to those of 33.
In contrast, introduction of the larger n-butyl or bulky t-butyl
moieties led to an apparent reduction in the overall potency
(analogues 40 and 41). Further increase in the size of R1/R2 resulted
in compounds exhibiting limited solubility or no activity (saturated
cyclic derivatives 43–45). Introducing the benzyl group was also
detrimental for cytotoxicity (compound 46). However, when a
phenyl group was attached to the carboxamide functionality, a
nanomolar HL-60 growth inhibitory activity was established (47:
R1 = H, R2 = C6H5, IC50 = 0.604 μM). Note that 47 displayed only
moderate efficacy against 4T1 and MCF-7 cells (IC50 = 28.5 and
18.3 μM, respectively). Incorporation of 2-pyridyl moiety provided
analogue 48 with two to fivefold lower activities, while thiazole-,
isoxazole-, or other pyridine-substituted carboxamides (49–52)
exhibited similar IC50 values than hit compound 33.
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2.2.1 SAR of C-7 primary carboxamides (group 1)
At the outset of the study, the substituent effect of R5 was investigated
with a fixed R4 (phenyl) group. However, no outstanding in vitro
cytotoxic activities were established (Table 1, compounds 12–16).
Only compound 14 (R5 = CH2COOMe) displayed a moderate efficacy
against HL-60 cell line (IC50 = 9.73 μM). Next we modified R4 with
various (hetero)aryl groups bearing electron-donating and electron-
withdrawing substituents (Table 1, compounds 17–26). Most of the
tested analogues, independently from the electronic nature of the
aromatic ring, showed insignificant or no antitumor activities. The best,
however, still unsatisfactory IC50 values were obtained for tert-
octylamino analogues 17 (R4 = 4-Me-C6H4) and 23 (R4 = 4-F3C-C6H4).
Replacing the R4 phenyl group to aliphatic substituents, such as
the cyclohexyl moiety, was beneficial in terms of cytotoxicity (Table 2,
compound 27). In contrast, the unbranched n-heptyl-containing
Since all synthesized derivatives 39–52 displayed lower potencies
against 4T1 and MCF-7 cancer cell lines than reference hit compound
33, hereafter we performed only 2D screens and focused mainly on

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TABLE 1 Structure and in vitro growth inhibitory concentrations of R4 = aryl imidazo[1,2-b]pyrazole C-7 primary carboxamides 12–26
(group 1)
Compound
12
R4
R5
4T1a
3D 4T1a
MCF-7a
3D MCF-7a
HL-60a
>50
>50
>50
>50
>50
13
14
15
16
17
18
19
>50
>50
>50
>50
12.3
25.2
34.1
>50
>50
>50
>50
11.4
38.0
37.5
>50
>50
>50
>50
17.0
18.6
40.6
>50
>50
47.6
>50
11.3
29.5
36.0
50.5
9.73
22.7
>50
12.6
19.0
26.6
20
>50
>50
>50
>50
>50
21
22
23
24
>50
>50
20.9
>50
>50
>50
17.1
>50
>50
>50
12.7
>50
>50
>50
10.9
>50
17.5
15.9
13.2
>50
25
26
>50
>50
>50
37.5
>50
>50
>50
19.5
24.6
29.2
Doxorubicin
0.057
0.141
0.024
0.063
0.052
aIC50 values in μM.
improving the activity against myelocytic leukemia. Encouraged by the
significant HL-60 selectivity and cytotoxicity observed for 47 (R1 = H,
R2 = C6H5), we generated several phenyl-substituted carboxamide
analogues and examined the effect of the electronic nature of the
benzene ring on the anticancer potency (Table 4, compounds
53–72). In comparison with unsubstituted 47, the introduction of
electron-donating alkyl or methoxy groups had a negative influence on
activity. Notably, alkyl substituents were better tolerated at ortho,
while the methoxy group at para position (compound 53 vs. 54/55 and
compound 56 vs. 57). Incorporation of the electron-withdrawing

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TABLE 2 Structure and in vitro growth inhibitory concentrations of R4 = aliphatic imidazo[1,2-b]pyrazole C-7 primary carboxamides 27–38
(group 1)
Compound
27
R4
R5
4T1a
3D 4T1a
MCF-7a
3D MCF-7a
HL-60a
12.3
11.5
11.1
13.1
9.35
28
29
30
31
32
33
>50
4.24
23.9
>50
>50
1.88
>50
9.09
13.8
>50
>50
2.97
>50
4.07
27.9
>50
40.5
1.49
>50
8.91
5.11
>50
26.5
3.79
>50
2.95
4.99
8.80
24.4
1.24
34
35
36
>50
>50
3.63
>50
>50
15.6
>50
>50
10.6
>50
>50
31.3
5.49
27.9
7.79
37
38
9.95
4.94
8.29
5.12
1.37
11.6
10.1
18.0
11.8
14.8
Doxorubicin
0.057
0.141
0.024
0.063
0.052
aIC50 values in μM. Bold values represent the compounds displaying an IC50 < 10 μM in all cell lines tested.
trifluoromethyl moiety also led to a dramatic loss of efficacy, regardless
of its position (isomers 58–60). To our surprise, systematic positioning
of a fluorine atom on the phenyl ring provided HL-60 sensitive para-
fluorophenyl derivative 63 with an excellent IC50 value of 0.183 μM
(R1 = H, R2 = 4-F-C6H5). It should be highlighted that ortho 61
(IC50 = 2.04 μM) and mostly meta 62 (IC50 = 4.47 μM) regioisomers
displayed markedly lower levels of cytotoxic activity from a HL-60
point of view. Replacing para-fluorine by other halogens, such as
chlorine and bromine-induced activities lower by one and two orders
of magnitude, respectively (64: IC50 = 3.05 μM, 65: IC50 = 19.7 μM).
Replacement by electron-withdrawing nitro (66), cyano (67), and ethyl
ester (68) or electron-donating methylthio (69) functionalities was also
detrimental for the efficacy against HL-60 cells (IC50 = 1.50–8.50 μM).
Derivative 70 bearing the para-dimethylamino group exhibited a
potency and selectivity toward myelocytic leukemia cells similar to that
of 63. Introducing an additional fluorine atom into ortho position
yielded 2,4-difluorophenyl analogue 71 with an excellent selectivity
for HL-60 cancer cells in conjunction with sub-micromolar activity
(IC50 = 0.628 μM). As expected from the tendency of cytotoxicity
observed for mono-fluorinated derivatives 61–63 (4-F > 2-F > 3-F), an
additional fluorine atom at meta position was less tolerated (72: R1 = H,
R2 = 3,4-F-C6H4, IC50 = 2.41 μM).
Afterwards, several modifications were performed on com-
pound 63 to gain further insight into SARs (Table 5). For example,
insertion of a methylene spacer between the carboxamide and the 4-
fluorophenyl units resulted in an efficacy against HL-60 cells lower

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TABLE 3 Structure and in vitro growth inhibitory concentrations of imidazo[1,2-b]pyrazole C-7 secondary/tertiary carboxamides 39–52
(group 2)
Comp.
39
R1
H
R2
4T1a
4.66
>50
>50
3D 4T1a
3.25
MCF-7a
4.46
3D MCF-7a
3.52
HL-60a
1.52
Me
40
H
>50
34.1
31.3
2.72
41
H
>50
>50
>50
>50
42
43
H
H
4.01
NDb
4.39
NDb
1.69
NDb
3.50
NDb
1.61
NDb
44
45
46
NDb
>50
9.31
NDb
>50
8.72
NDb
>50
8.57
NDb
>50
7.38
NDb
>50
6.26
H
47
48
H
H
28.5
6.91
34.7
6.34
18.3
8.35
15.2
5.09
0.604
4.38
49
H
2.59
2.22
1.90
1.81
0.934
50
51
H
H
4.34
2.41
4.32
3.62
2.55
1.95
4.20
2.81
2.71
2.86
52
H
2.85
2.21
1.70
2.07
0.935
0.052
Doxorubicin
0.057
0.141
0.024
0.063
aIC50 values in μM. Bold values represent compounds displaying an IC50 < 1 μM in HL-60 cell line.
bND: not determined because of solubility issues.
by one order of magnitude (73, IC50 = 8.70 μM). Incorporation of a
nitrogen atom at meta position (compound 75) was advantageous for
the growth inhibitory activity against 4T1 cells. At the same time,
however, it reduced the efficacy in myelocytic leukemia cells
(IC50 = 0.501 μM). As expected from earlier observations (see
Table 3), 2-pyridyl analogue 74 proved to be less effective. N-
Methylation on the nitrogen of carboxamide or the tert-octylamino
moiety revealed the importance of the hydrogen-bond donor NH
functionalities (compounds 76 and 78). Interestingly, while tertiary
carboxamide 76 gave rise to a lower but moderate overall activity
(IC50 = 2.50–9.06 μM), the presence of methyl group on the tert-
octylamino side chain led to dramatic loss of potency, especially from
the HL-60 point of view. Installing a methyl substituent at the C-6
position of the imidazo[1,2-b]pyrazole scaffold was also detrimental
for the overall cytotoxicity (compound 77).
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2.2.3 Real-time in vitro toxicity measurements
Using a cell microelectronic sensing technique, we have deter-
mined the kinetics of cell growth inhibition of two selected
compounds on MCF-7 cells (Figure 2). A sudden decrease in signal
was observed at the highest applied concentrations, while

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TABLE 4 Structure and in vitro growth inhibitory concentrations of phenyl substituted C-7 carboxamides 53–72 (group 2)
Compound
R
4T1a
>50
>50
>50
24.7
>50
>50
35.4
>50
>50
23.5
7.43
30.7
>50
27.1
>50
>50
>50
11.8
>50
19.0
0.057
MCF-7a
30.6
>50
HL-60a
2.12
>50
53
2-Me
3,5-Me
4-iPr
54
55
>50
>50
56
4-OMe
2,4-OMe
2-CF3
3-CF3
4-CF3
2-F
17.8
>50
1.11
>50
57
58
>50
>50
59
>50
11.4
>50
60
29.0
>50
61
2.04
4.47
0.183
3.05
19.7
7.64
1.50
8.50
3.93
0.297
0.628
2.41
0.052
62
3-F
17.3
2.35
29.3
>50
63
4-F
64
4-Cl
65
4-Br
66
4-NO2
4-CN
4-COOEt
4-SMe
4-NMe2
2,4-F
3,4-F
9.94
9.71
>50
67
68
69
21.9
10.0
>50
70
71
72
10.3
0.024
Doxorubicin
aIC50 values in μM. Bold values represent compounds displaying an IC50 < 1 μM in HL-60 cell line.
treatments at a lower concentration delayed the effect for several
hours after treatment. IC50 values determined for the 72-h time
points were comparable to values determined by the fluorimetric
endpoint assay (Tables 2 and 3).
dependent increase in Annexin
V
staining proportional to
phosphatidylserine exposure, an early marker of apoptotic cell
death. Upon treatment by the selected compounds for 24 h both
the early (AnnV+/PI−) and late apoptotic (AnnV+/PI+) populations
increased without the appearance of the only PI positive necrotic
population. Tertiary carboxamide 76 (N-methylated derivative of
63) showed less apoptotic activity, hardly detected at 5 µM, while
cyclopropyl-substituted secondary carboxamide analogue 42
induced apoptotosis in 20 and 40% of the treated cells at 2.5
and 5 µM, respectively. On the other hand, compound 33 was more
effective inducing apoptosis at 2.5 and 5 µM (40 and 55%,
respectively). Superior apoptotic activity was observed for hit
compound 63: 38% of the treated cells were apoptotic at
0.625 µM, which went to saturation at 1.25 µM.
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2.2.4 Imidazo[1,2-b]pyrazole-7-carboxamides induce
apoptotic cell death of HL-60 cells
In order to reveal the type of cytotoxic effect and distinguish
apoptosis from necrosis we performed Annexin V and propidium
iodide (PI) staining on HL-60 cells with hit compounds 33 and 63
(Figure 3). For comparison, two moderately active derivatives
(42: IC50 = 1.61, 76: IC50 = 2.50 µM) were also selected for Annexin
V assay. Imidazo[1,2-b]-pyrazole-7-carboxamides caused dose-

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TABLE 5 Structural analogues of compound 63
Compound
4T1a
MCF-7a
10.1
HL-60a
8.70
73
15.6
14.6
3.21
9.06
>50
74
11.8
2.38
75
2.34
0.501
2.50
76
8.44
77
>50
16.6
78
43.8
0.057
11.8
>50
Doxorubicin
0.024
0.052
aIC50 values in μM. Bold values represent compounds displaying an IC50 < 1 μM in HL-60 cell line.
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3
CONCLUSION
elucidate the molecular targets and understand the detailed mecha-
nism of action are currently in progress.
A 67-membered library of novel imidazo[1,2-b]pyrazole-7-carboxa-
mides has been synthesized and tested in vitro against MCF-7 human
breast, 4T1 mammary gland, and HL-60 human promyelocytic
leukemia cancer cell lines using 2D and 3D culture models. SARs
were established and the crucial moieties on the imidazo[1,2-b]-
pyrazole framework were defined. Among primary carboxamides
(group 1, 27 compounds), compound 33 exerted the most significant
overall cytotoxic activity with IC50 values of 1.24–3.79 μM. From
secondary/tertiary carboxamides (group 2, 40 compounds), seven
compounds exhibited sub-micromolar activity against HL-60 cells, of
which derivative 63 demonstrated the highest potency
(IC50 = 0.183 μM) with a remarkable HL-60 sensitivity. Annexin V PI
assay revealed that the most potent derivatives 33 and 63 induced
apoptosis in HL-60 cells. Kinetics of cell growth inhibition induced by
33 on MCF-7 cells were also determined. Further investigations to
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 General
All NMR spectra were recorded in deuterated solvents at 298 K on a
Bruker Avance 500 or Bruker Avance Neo 500 spectrometer. All
chemical shifts (δ) are reported in ppm relative to the internal standard
(TMS) or the residual solvent signal. The following abbreviations are
used for NMR multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; p,
pentet; sx, sextet; m, multiplet; br, broad, with coupling constants (J)
given in Hertz (Hz). Melting points were determined on a Stuart SMP10
apparatus and are uncorrected. MS spectra were recorded on an

DEMJÉN ET AL.
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phenyl isocyanide) were obtained according to a known procedure.[65]
All other reagents and solvents were commercially available and used
without further purification.
The NMR spectra and the InChI codes of the investigated
compounds together with some biological activity data are provided as
Supporting Information.
|
4.1.2 General procedure for the synthesis of
aminopyrazoles 8 through intermediates 6 and 7
Pyrazoles 8 were synthesized from cyanoacetic acid derivative 5
through intermediates 6 and 7 by adopting previously published
protocols.[66–70]
Step 1: A mixture of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopro-
panenitrile (5) (4.00 mmol, 653 mg) and the corresponding amine
(4.80 mmol) in toluene (2 mL) or DMF (if the amine was poorly soluble
in toluene; 2 mL) was heated at 80°C for 6–12 h. After the reaction was
completed (monitored by TLC), the reaction mixture was poured into
aqueous HCl solution (30 mL, 0.5 M) and extracted with EtOAc
(3 × 50 mL). The organic layers were combined, washed with saturated
Na2CO3 followed by brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The obtained crude intermediate 6 was purified
either by recrystallization or column chromatography on silica gel
(eluent: hexane/EtOAc gradient), or used without further purification
in the subsequent reaction. Pyridine derivatives were purified by
column chromatography with the omission of the acidic extraction
step.
FIGURE 2 Kinetics of cell growth inhibition induced by
imidazo[1,2-b]pyrazole-7-carboxamides in MCF-7 cells. Red, orange,
cyan, magenta, blue, and green traces correspond to 0, 0.37, 1.1,
3.3, 10, and 30 μM concentrations, respectively. Inserts on panels
show values determined at 72 h after treatment and the
corresponding IC50 values for 33 (panel A) and 42 (panel B)
Agilent G1946D mass spectrometer equipped with Multimode Source
in positive ESI or APCI mode. TLC was performed on aluminum sheets
coated with silica gel 60 F254 (Merck, 1.05554). Visualization was
done under UV light (254 nm) or by using potassium permanganate dip.
Column chromatography was carried out using silica gel (Merck, 60 Å,
0.063–0.200 mm). Flash column chromatography was performed on a
Teledyne Isco CombiFlash Rf system using RediSep Rf columns.
Aromatic isocyanides (4-fluorophenyl isocyanide and 4-methoxy-
Step 2: Afterwards, N,N-dimethylformamide dimethyl acetal (for
R3 = H; 3.9 mmol) or N,N-dimethylacetamide dimethyl acetal (for
R3 = Me; 3.90 mmol) was added to a suspension of cyanoacetamide 6
(3.00 mmol) in toluene (9 mL) and stirred at 80°C for 6 h. The
precipitated solid was filtered, washed with toluene, and used directly
in the subsequent reaction without characterization. In some cases,
further purification by column chromatography on silica gel was
necessary.
Step 3: To a solution of crude intermediate 7 (2.00 mmol) in EtOH
(4 mL) hydrazine monohydrate (2.20 mmol) was added and stirred at
reflux temperature for 12 h. The solvent was removed under vacuum
and the residue was purified by column chromatography on silica gel
(eluent: chloroform/methanol gradient) to afford pure pyrazoles 8.
3-Amino-N-methyl-1H-pyrazole-4-carboxamide (pyrazole 8
leading to product 39)
White solid; yield: 69% (calcd. for Step 3); m.p. 178–179°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.74 (s, 1H), 7.68 (s, 1H), 7.63 (s, 1H), 5.71 (bs,
2H), 2.67 (d, J = 4.6 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.4,
25.6; ESI-MS (m/z): 141.0 (M+H+).
FIGURE 3 Imidazo[1,2-b]pyrazole-7-carboxamides induced
phosphatidylserine exposure on HL-60 cells after 24 h. Cells were
incubated with imidazo[1,2-b]pyrazole-7-carboxamides with the
indicated concentrations (µM). The results are shown as arithmetic
mean values of the summary of early (AnnV+/PI−, gray column) and
late apoptosis (AnnV+/PI+, white column) of two samples SD
3-Amino-N-butyl-1H-pyrazole-4-carboxamide (pyrazole 8
leading to product 40)
Light beige solid; yield: 63% (calcd. for Step 3); m.p. 138–139°C;
1H NMR (500 MHz, DMSO-d6) δ 11.73 (s, 1H), 7.73 (s, 1H), 7.62 (s, 1H),
5.72 (bs, 2H), 3.15 (q, J = 6.6 Hz, 2H), 1.44 (p, J = 7.3 Hz, 2H), 1.30 (sx,

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J = 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6)
δ 164.8, 38.2, 32.2, 20.1, 14.2. ESI-MS (m/z): 182.9 (M+H+).
9.38 (s, 0.7H), 8.23 (s, 0.3H), 7.91 (s, 0.7H), 7.68 (d, J = 7.9 Hz, 2H), 7.30
(t, J = 7.7 Hz, 2H), 7.02 (t, J = 7.3 Hz, 1H), 6.09 (s, 1.4H), 5.42 (s, 0.6H).
13C NMR (126 MHz, DMSO-d6) δ 163.5, 151.8, 140.0, 138.2, 129.0,
123.0, 120.2. ESI-MS (m/z): 203.0 (M+H+).
3-Amino-N-(tert-butyl)-1H-pyrazole-4-carboxamide (pyrazole 8
leading to product 41)
Gray solid; yield: 51% (calcd. for Step 3); m.p. 199°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.76 (s, 0.4H), 11.63 (s, 0.6H), 8.01 (s, 0.4H), 7.71 (s, 0.6H),
6.90 (s, 1H), 5.85 (s, 1.2H), 5.25 (s, 0.8H), 1.34 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 165.1, 50.6, 29.5. ESI-MS (m/z): 183.1 (M+H+).
3-Amino-N-(pyridin-2-yl)-1H-pyrazole-4-carboxamide (pyrazole
8 leading to product 48)
Yellow solid; yield: 74% (calcd. for Step 3); m.p. 216°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.09 (s, 0.35H), 11.82 (s, 0.65H), 10.04
(bs, 1H), 8.44 (s, 0.35H), 8.32 (dd, J = 4.9, 1.9 Hz, 1H), 8.16 (d,
J = 8.4 Hz, 1H), 8.10 (s, 0.65H), 7.84–7.58 (m, 1H), 7.07 (dd, J = 7.3,
4.9 Hz, 1H), 6.17 (s, 1.3H), 5.48 (s, 0.7H). 13C NMR (126 MHz,
DMSO-d6) δ 163.8, 153.1, 148.1, 138.3, 119.2, 114.5. ESI-MS
(m/z): 204.1 (M+H+).
3-Amino-N-cyclopropyl-1H-pyrazole-4-carboxamide (pyrazole
8 leading to product 42)
White solid; yield: 68% (calcd. for Step 3); m.p. 92°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.74 (s, 1H), 7.68 (bs, 2H), 5.83 (bs, 2H), 2.74–
2.60 (m, 1H), 0.63 (s, 2H), 0.46 (s, 2H). 13C NMR (126 MHz, DMSO-d6)
δ 166.1, 22.5, 6.3. ESI-MS (m/z): 167.0 (M+H+).
3-Amino-N-(pyridin-3-yl)-1H-pyrazole-4-carboxamide (pyrazole
8 leading to product 49)
3-Amino-N-cyclopentyl-1H-pyrazole-4-carboxamide (pyrazole
8 leading to product 43)
Pale yellow solid; yield: 74% (calcd. for Step 3); m.p. 196–198°C;
1H NMR (500 MHz, DMSO-d6) δ 12.08 (s, 0.3H), 11.86 (s, 0.7H), 9.60
(s, 1H), 8.84 (d, J = 2.6 Hz, 1H), 8.23 (dd, J = 4.7, 1.5 Hz, 1H), 8.10 (dt,
J = 8.3, 1.9 Hz, 1H), 7.91 (s, 0.7H), 7.34 (dd, J = 8.3, 4.7 Hz, 1H), 6.12 (s,
1.4H), 5.45 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 163.7, 144.1,
141.8, 136.7, 127.0, 123.9. ESI-MS (m/z): 204.1 (M+H+).
Gray solid; yield: 48% (calcd. for Step 3); m.p. 194°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.76 (s, 0.4H), 11.68 (s, 0.6H), 7.95 (s, 0.4H), 7.70 (s, 0.6H),
7.45 (s, 1H), 5.87 (s, 1.2H), 5.29 (s, 0.8H), 4.14 (q, J = 7.2 Hz, 1H), 1.91–
1.78 (m, 2H), 1.73–1.61 (m, 2H), 1.58–1.38 (m, 4H). 13C NMR(126 MHz,
DMSO-d6) δ 164.5, 50.3, 32.8, 24.0. ESI-MS (m/z): 195.1 (M+H+).
3-Amino-N-(pyridin-4-yl)-1H-pyrazole-4-carboxamide (pyrazole
8 leading to product 50)
(3-Amino-1H-pyrazol-4-yl)(piperidin-1-yl)methanone (pyrazole
8 leading to product 44)
Yellow solid; yield: 82% (calcd. for Step 3); m.p. 248–250°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.10 (s, 0.3H), 11.89 (s, 0.7H), 9.72 (s, 1H),
8.40 (d, J = 6.5 Hz, 2H), 7.94 (s, 1H), 7.69 (d, J = 6.6 Hz, 2H), 6.18 (s,
1.4H), 5.51 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 164.0, 150.5,
146.8, 113.8. ESI-MS (m/z): 204.1 (M+H+).
Light beige solid; yield: 37% (calcd. for Step 3); m.p. 168°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.94 (s, 0.4H), 11.77 (s, 0.6H), 7.63 (s, 0.4H),
7.39 (s, 0.6H), 5.78 (s, 1.2H), 5.07 (s, 0.8H), 3.59–3.49 (m, 4H), 1.66–
1.57 (m, 2H), 1.54–1.43 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ
165.0, 26.3, 24.7. ESI-MS (m/z): 195.1 (M+H+).
(3-Amino-1H-pyrazol-4-yl)(4-phenylpiperazin-1-yl)methanone
(pyrazole 8 leading to product 45)
3-Amino-N-(thiazol-2-yl)-1H-pyrazole-4-carboxamide (pyrazole
8 leading to product 51)
Light brown; yield: 75% (calcd. for Step 3); m.p. 212°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.83 (bs, 2H), 8.17 (s, 1H), 7.47 (d, J = 3.6 Hz,
1H), 7.15 (d, J = 3.6 Hz, 1H), 6.03 (s, 2H). 13C NMR (126 MHz, DMSO-
d6) δ 162.3, 159.1, 137.8, 113.3. APCI-MS (m/z): 209.9 (M+H+).
Gray solid; yield: 35% (calcd. for Step 3); m.p. 197–198°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.02 (s, 0.4H), 11.82 (s, 0.6H), 7.74 (s, 0.4H),
7.51 (s, 0.6H), 7.24 (t, J = 7.9 Hz, 2H), 6.96 (d, J = 8.1 Hz, 2H), 6.81 (t,
J = 7.2 Hz, 1H), 5.86 (s, 1.2H), 5.16 (s, 0.8H), 3.74 (t, J = 5.1 Hz, 4H),
3.18 (t, J = 5.2 Hz, 4H). 13C NMR (126 MHz, DMSO-d6) δ 165.3, 151.3,
129.5, 119.6, 116.1, 49.0. ESI-MS (m/z): 272.2 (M+H+).
3-Amino-N-(isoxazol-3-yl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 52)
3-Amino-N-benzyl-1H-pyrazole-4-carboxamide (pyrazole 8
leading to product 46)
Light yellow solid; yield: 81% (calcd. for Step 3); m.p. 198°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.08 (s, 0.3H), 11.86 (s, 0.7H), 10.68 (s, 1H),
8.76 (d, J = 1.7 Hz, 1H), 8.04 (s, 1H), 7.00 (d, J = 1.8 Hz, 1H), 6.16 (s,
1.4H), 5.55 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 162.9, 160.0,
158.6, 100.0. APCI-MS (m/z): 194.2 (M+H+).
Gray solid; yield: 57% (calcd. for Step 3); m.p. 146°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.76 (bs, 1H), 8.24 (s, 1H), 7.76 (bs, 1H), 7.36–
7.27 (m, 4H), 7.23 (t, J = 7.0 Hz, 1H), 5.89 (s, 1.2H), 5.39 (s, 0.8H), 4.39
(d, J = 6.0 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 164.9, 140.8,
128.7, 127.6, 127.1, 41.9. ESI-MS (m/z): 217.1 (M+H+).
3-Amino-N-(o-tolyl)-1H-pyrazole-4-carboxamide (pyrazole 8
leading to product 53)
3-Amino-N-phenyl-1H-pyrazole-4-carboxamide (pyrazole 8
leading to product 47)
White solid; yield: 63% (calcd. for Step 3); m.p. 206°C; 1H NMR
Beige solid; yield: 64% (calcd. for Step 3); m.p. 190–191°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.83 (bs, 1H), 9.07 (s, 1H), 7.90 (bs, 1H), 7.33
(d, J = 7.8 Hz, 1H), 7.24 (d, J = 7.4 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.11
(t, J = 7.1 Hz, 1H), 5.96 (s, 1.4H), 5.41 (s, 0.6H), 2.23 (s, 3H). 13C NMR
(500 MHz, DMSO-d6) δ 12.04 (s, 0.3H), 11.83 (s, 0.7H), 9.49 (s, 0.3H),

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(126 MHz, DMSO-d6) δ 163.5, 136.9, 133.6, 130.7, 126.8, 126.3,
125.7, 18.4. APCI-MS (m/z): 217.1 (M+H+).
1H), 6.14 (s, 1.4H), 5.47 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 163.7,
140.9, 130.2, 129.7 (q, J = 31.2 Hz), 124.7 (q, J = 272.2 Hz), 123.4, 119.2,
116.0 (q, J = 4.1 Hz). ESI-MS (m/z): 271.0 (M+H+).
3-Amino-N-(3,5-dimethylphenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 54)
3-Amino-N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-4-
carboxamide (pyrazole 8 leading to product 60)
Light beige solid; yield: 80% (calcd. for Step 3); m.p. 103°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.01 (s, 0.35H), 11.81 (s, 0.65H), 9.32 (s,
0.35H), 9.22 (s, 0.65H), 8.21 (s, 0.35H), 7.90 (s, 0.65H), 7.32 (s, 2H),
6.67 (s, 1H), 6.08 (s, 1.3H), 5.41 (s, 0.7H), 2.25 (s, 6H). 13C NMR
(126 MHz, DMSO-d6) δ 163.5, 151.7, 139.9, 138.2, 137.8, 124.6,
118.0, 21.6. APCI-MS (m/z): 231.2 (M+H+).
White solid; yield: 78% (calcd. for Step 3); m.p. 196°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.12 (s, 0.3H), 11.88 (s, 0.7H), 9.80 (s, 0.3H),
9.71 (s, 0.7H), 8.25 (s, 0.3H), 7.92 (d, J = 8.1 Hz, 2.7H), 7.66 (d,
J = 8.4 Hz, 2H), 6.17 (s, 1.4H), 5.47 (s, 0.6H). 13C NMR (126 MHz,
DMSO-d6) δ 163.67, 152.10, 138.24, 126.28, 125.0 (q, J = 271.2 Hz),
119.72. ESI-MS (m/z): 271.0 (M+H+).
3-Amino-N-(4-isopropylphenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 55)
3-Amino-N-(2-fluorophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 61)
Gray solid; yield: 61% (calcd. for Step 3); m.p. 196°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.99 (s, 0.3H), 11.81 (s, 0.7H), 9.34 (s, 1H),
8.15 (s, 0.3H), 7.90 (s, 0.7H), 7.58 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.3 Hz,
2H), 6.05 (s, 1.4H), 5.41 (s, 0.6H), 2.84 (p, J = 6.9 Hz, 1H), 1.19 (d,
J = 6.9 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ 163.4, 143.1, 137.7,
126.7, 120.3, 33.3, 24.5. ESI-MS (m/z): 245.1 (M+H+).
White solid; yield: 44% (calcd. for Step 3); m.p. 186°C; 1H NMR (500 MHz,
DMSO-d6) δ 12.08 (s, 0.3H), 11.84 (s, 0.7H), 9.29 (bs, 1H), 8.21 (s, 0.3H),
7.89 (s, 0.7H), 7.62 (d, J = 8.8 Hz, 1H), 7.30–7.22 (m, 1H), 7.23–7.11 (m,
2H), 6.06 (s, 1.4H), 5.40 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 163.4,
155.8 (d, J = 244.1 Hz), 127.2, 126.4, 126.3 (d, J = 7.6 Hz), 124.6 (d,
J = 3.5 Hz), 116.1 (d, J = 19.6 Hz). ESI-MS (m/z): 221.0 (M+H+).
3-Amino-N-(4-methoxyphenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 56)
3-Amino-N-(3-fluorophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 62)
White solid; yield: 58% (calcd. for Step 3); m.p. 207°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.96 (s, 0.3H), 11.82 (s, 0.7H), 9.31 (s, 1H), 8.11
(bs, 0.3H), 7.89 (bs, 0.7H), 7.76–7.38 (m, 2H), 7.21–6.65 (m, 2H), 6.03
(bs, 1.4H), 5.42 (bs, 0.6H), 3.73 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
163.3, 155.4, 133.0, 121.9, 114.1, 55.6. ESI-MS (m/z): 233.1 (M+H+).
White solid; yield: 82% (calcd. for Step 3); m.p. 189–190°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.04 (s, 0.3H), 11.86 (s, 0.7H), 9.58 (s, 1H), 7.92
(bs, 1H), 7.70 (d, J = 12.2 Hz, 1H), 7.43 (dd, J = 8.2, 1.8 Hz, 1H), 7.33 (q,
J = 7.8 Hz, 1H), 6.84 (td, J = 8.5, 2.6 Hz, 1H), 6.11 (s, 1.4H), 5.47 (s, 0.6H).
13C NMR (126 MHz, DMSO-d6) δ 163.6, 162.6 (d, J = 240.6 Hz), 141.9
(d, J = 11.2 Hz), 130.5 (d, J = 9.4 Hz), 115.7, 109.4 (d, J = 21.1 Hz), 106.7
(d, J = 26.5 Hz). ESI-MS (m/z): 221.1 (M+H+).
3-Amino-N-(2,4-dimethoxyphenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 57)
White solid; yield: 63% (calcd. for Step 3); m.p. 103°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.00 (s, 0.3H), 11.79 (s, 0.7H), 8.74 (s, 0.3H),
8.61 (s, 0.7H), 8.16 (s, 0.3H), 7.82 (s, 0.7H), 7.47 (d, J = 8.6 Hz, 1H), 6.62
(d, J = 2.7 Hz, 1H), 6.50 (dd, J = 8.7, 2.7 Hz, 1H), 5.97 (s, 1.4H), 5.32 (s,
0.6H), 3.80 (s, 3H), 3.76 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
163.5, 157.7, 153.4, 126.5, 120.4, 104.5, 99.2, 56.1, 55.8. ESI-MS
(m/z): 263.1 (M+H+).
3-Amino-N-(4-fluorophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 63)
White solid; yield: 69% (calcd. for Step 3); m.p. 113°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.03 (s, 0.3H), 11.84 (s, 0.7H), 9.47 (s, 1H),
8.16 (s, 0.3H), 7.89 (s, 0.7H), 7.68 (dd, J = 8.9, 5.1 Hz, 2H), 7.14 (t,
J = 8.8 Hz, 2H), 6.08 (s, 1.4H), 5.43 (s, 0.6H). 13C NMR (126 MHz,
DMSO-d6) δ 163.4, 158.21 (d, J = 239.0 Hz), 136.3, 121.96 (d,
J = 7.6 Hz), 115.49 (d, J = 22.0 Hz). ESI-MS (m/z): 221.1 (M+H+).
3-Amino-N-(2-(trifluoromethyl)phenyl)-1H-pyrazole-4-
carboxamide (pyrazole 8 leading to product 58)
White solid; yield: 53% (calcd. for Step 3); m.p. 171°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.08 (s, 0.3H), 11.85 (s, 0.7H), 9.35 (s, 0.3H),
9.21 (s, 0.7H), 8.15 (s, 0.3H), 7.81 (s, 0.7H), 7.76 (d, J = 7.9 Hz, 1H), 7.69
(t, J = 7.7 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 6.01
(s, 1.4H), 5.33 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 164.1,
151.62, 138.4, 136.3, 133.3, 131.4, 127.1, 126.8 (q, J = 5.1 Hz), 126.2
(q, J = 28.9 Hz), 124.2 (q, J = 273.3 Hz). ESI-MS (m/z): 271.1 (M+H+).
3-Amino-N-(4-chlorophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 64)
Light beige solid; yield: 83% (calcd. for Step 3); m.p. 221°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.04 (s, 0.3H), 11.86 (s, 0.7H), 9.54 (s, 1H),
7.92 (bs, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.9 Hz, 2H), 6.09 (s,
1.4H), 5.47 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 163.5, 139.0,
128.9, 126.6, 121.6. ESI-MS (m/z): 237.0 and 239.0 (M+H+).
3-Amino-N-(3-(trifluoromethyl)phenyl)-1H-pyrazole-4-
carboxamide (pyrazole 8 leading to product 59)
White solid; yield: 85% (calcd. for Step 3); m.p. 168–169°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.08 (s, 0.3H), 11.87 (s, 0.7H), 9.70 (s, 1H), 8.19 (s,
1H), 7.93 (dd, J = 8.2, 2.0 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 7.5 Hz,
3-Amino-N-(4-bromophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 65)
Gray solid; yield: 63% (calcd. for Step 3); m.p. 235°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.04 (s, 0.3H), 11.84 (s, 0.7H), 9.52 (s, 1H),

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8.16 (s, 0.3H), 7.90 (s, 0.7H), 7.67 (d, J = 8.9 Hz, 2H), 7.48 (d, J = 8.8 Hz,
2H), 6.10 (s, 1.4H), 5.43 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ
163.5, 139.4, 131.8, 122.0. ESI-MS (m/z): 281.0 and 283.0 (M+H+).
3-Amino-N-(3,4-difluorophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 72)
Beige solid; yield: 69% (calcd. for Step 3); m.p. 137°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.04 (s, 0.3H), 11.87 (s, 0.7H), 9.61 (s, 1H),
7.88 (ddd, J = 13.8, 7.6, 2.1 Hz, 1H), 7.45–7.30 (m, 2H), 6.11 (s, 1.4H),
5.47 (s, 0.6H). 13C NMR (126 MHz, DMSO-d6) δ 163.5, 149.3 (dd,
J = 242.1, 13.1 Hz), 145.3 (dd, J = 240.3, 12.9 Hz), 137.1 (d, J = 8.5 Hz),
117.6 (d, J = 17.8 Hz), 116.1 (dd, J = 3.7, 3.2 Hz), 108.9 (d, J = 21.9 Hz).
ESI-MS (m/z): 238.8 (M+H+).
3-Amino-N-(4-nitrophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 66)
Yellow solid; yield: 57% (calcd. for Step 3); m.p. 270°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.94 (bs, 1H), 9.97 (s, 1H), 8.22 (d, J = 8.9 Hz, 2H), 7.97 (d,
J = 8.9 Hz, 2H), 6.21 (bs, 1.4H), 5.64 (bs, 0.6H). 13C NMR (126 MHz, DMSO-
d6) δ 163.7, 146.7, 142.0, 125.3, 119.3. APCI-MS (m/z): 248.1 (M+H+).
3-Amino-N-(4-fluorobenzyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 73)
3-Amino-N-(4-cyanophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 67)
White solid; yield: 61% (calcd. for Step 3); m.p. 149°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.76 (bs, 1H), 8.26 (s, 1H), 7.72 (bs, 1H), 7.32
(dd, J = 8.4, 5.6 Hz, 2H), 7.15 (t, J = 8.7 Hz, 2H), 5.90 (s, 1.4H), 5.37 (s,
0.6H), 4.36 (d, J = 5.9 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 163.7
(d, J = 295.0 Hz), 160.6, 137.0, 129.6 (d, J = 8.2 Hz), 115.4 (d,
J = 21.2 Hz). ESI-MS (m/z): 235.1 (M+H+).
Gray solid; yield: 59% (calcd. for Step 3); m.p. 268°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.08 (bs, 0.3H), 11.91 (bs, 0.7H), 9.79 (s, 1H),
7.90 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 6.17 (s, 1.4H), 5.56 (s,
0.6H). 13C NMR (126 MHz, DMSO-d6) δ 163.7, 144.5, 133.5, 119.8,
119.8, 104.5. APCI-MS (m/z): 228.1 (M+H+).
Ethyl 4-(3-amino-1H-pyrazole-4-carboxamido)benzoate
(pyrazole 8 leading to product 68)
3-Amino-N-(5-fluoropyridin-2-yl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 74)
Gray solid; yield: 55% (calcd. for Step 3); m.p. 213°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.11 (s, 0.3H), 11.87 (s, 0.7H), 9.70 (s, 1H),
8.25 (s, 0.3H), 7.95 (s, 0.7H), 7.92 (d, J = 8.6 Hz, 2H), 7.85 (d, J = 8.5 Hz,
2H), 6.17 (s, 1,4H), 5.47 (s, 0.6H), 4.29 (q, J = 7.1 Hz, 2H), 1.32 (t,
J = 7.1 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.9, 163.6, 144.7,
130.5, 123.8, 119.2, 60.8, 14.7. ESI-MS (m/z): 275.1 (M+H+).
Light beige solid; yield: 88% (calcd. for Step 3); m.p. 233°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.10 (bs, 0.35H), 11.83 (bs, 0.65H), 10.27 (bs,
0.35H), 10.17 (bs, 0.65H), 8.41 (bs, 0.35H), 8.36–8.30 (m, 1H), 8.24–
8.15 (m, 1H), 8.08 (bs, 0.65H), 7.80–7.67 (m, 1H), 6.17 (bs, 1.4H), 5.48
(bs, 0.7H). 13C NMR (126 MHz, DMSO-d6) δ 163.6, 155.8 (d,
J = 247.2 Hz), 152.2, 149.6, 138.7, 135.3 (d, J = 24.8 Hz), 125.5 (d,
J = 19.3 Hz), 115.6 (d, J = 4.4 Hz). ESI-MS (m/z): 222.1 (M+H+).
3-Amino-N-(4-(methylthio)phenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 69)
3-Amino-N-(6-fluoropyridin-3-yl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 75)
Light beige solid; yield: 69% (calcd. for Step 3); m.p. 230°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.02 (s, 0.3H), 11.83 (s, 0.7H), 9.42 (s, 1H), 8.17
(s, 0.3H), 7.90 (s, 0.7H), 7.65 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H),
6.08(s, 1.4H), 5.43(s, 0.6H), 2.45(s, 3H). 13CNMR(126 MHz, DMSO-d6)
δ 163.4, 137.6, 131.4, 127.6, 120.9, 16.1. APCI-MS (m/z): 249.1 (M+H+).
Beige solid; yield: 52% (calcd. for Step 3); m.p. 149°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.07 (bs, 0.3H), 11.88 (bs, 0.7H), 9.70 (bs, 1H),
8.49 (d, J = 2.7 Hz, 1H), 8.22 (ddd, J = 9.6, 7.6, 2.8 Hz, 1H), 7.89 (bs, 1H),
7.15 (dd, J = 8.9, 3.1 Hz, 1H), 6.12 (s, 1.4H), 5.47 (s, 0.6H). 13C NMR
(126 MHz, DMSO-d6) δ 163.5, 158.8 (d, J = 231.6 Hz), 138.5 (d,
J = 15.5 Hz), 135.0, 133.7 (d, J = 7.6 Hz), 109.5 (d, J = 39.2 Hz). ESI-MS
(m/z): 222.0 (M+H+).
3-Amino-N-(4-(dimethylamino)phenyl)-1H-pyrazole-4-
carboxamide (pyrazole 8 leading to product 70)
Gray solid; yield: 53% (calcd. for Step 3); m.p. 270°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.82 (s, 1H), 9.20 (s, 1H), 7.95 (s, 1H), 7.46 (d,
J = 8.7 Hz, 2H), 6.70 (d, J = 8.7 Hz, 2H), 5.92 (bs, 2H), 2.85 (s, 6H).
13C NMR (126 MHz, DMSO-d6) δ 163.1, 147.3, 129.7, 122.0, 113.1,
41.1. APCI-MS (m/z): 246.2 (M+H+).
3-Amino-N-(4-fluorophenyl)-N-methyl-1H-pyrazole-4-
carboxamide (pyrazole 8 leading to product 76)
Gray solid; yield: 37% (calcd. for Step 3); m.p. 198°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.66 (s, 1H), 7.40–7.34 (m, 2H), 7.35–7.27 (m,
2H), 6.20 (s, 1.6H), 5.77 (s, 0.4H), 5.53 (s, 1H), 3.22 (s, 3H). 13C NMR
(126 MHz, DMSO-d6) δ 165.1, 161.5 (d, J = 244.8 Hz), 141.7, 130.7 (d,
J = 8.8 Hz), 117.0 (d, J = 22.3 Hz), 37.9. ESI-MS (m/z): 235.1 (M+H+).
3-Amino-N-(2,4-difluorophenyl)-1H-pyrazole-4-carboxamide
(pyrazole 8 leading to product 71)
Beige solid; yield: 91% (calcd. for Step 3); m.p. 196°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.07 (s, 0.3H), 11.84 (s, 0.7H), 9.31 (bs, 1H),
8.17 (s, 0.3H), 7.85 (s, 0.7H), 7.57 (q, J = 8.1 Hz, 1H), 7.32 (ddd, J = 11.2,
9.2, 2.8 Hz, 1H), 7.12–7.03 (m, 1H), 6.04 (s, 1.4H), 5.38 (s, 0.6H).
13C NMR (126 MHz, DMSO-d6) δ 163.5, 159.6 (d, J = 243.7 Hz), 159.5
(d, J = 243.6 Hz), 128.6, 122.9 (d, J = 10.6 Hz), 111.4 (dd, J = 21.8,
3.7 Hz), 104.6 (t, J = 25.7 Hz). ESI-MS (m/z): 238.8 (M+H+).
3-Amino-N-(4-fluorophenyl)-5-methyl-1H-pyrazole-4-
carboxamide (pyrazole 8 leading to product 77)
Light beige solid; yield: 69% (calcd. for Step 3); m.p. 201°C; 1H NMR
(500 MHz, DMSO-d6) δ 11.84 (bs, 0.6H), 11.53 (bs, 0.4H), 9.05 (bs, 1H),
7.70–7.58 (m, 2H), 7.14 (t, J = 8.8 Hz, 2H), 5.76 (bs, 0.8H), 5.09 (bs,
1.2H), 2.37 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 163.9, 158.3 (d,

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J = 239.3 Hz), 136.2, 122.2 (d, J = 7.9 Hz), 115.5 (d, J = 22.1 Hz). ESI-
MS (m/z): 235.2 (M+H+).
6.85 (s, 2H), 6.71 (s, 2H), 6.51 (s, 2H), 3.61 (s, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 163.9, 152.3, 142.5, 139.5, 137.7, 129.4, 128.5, 127.5,
126.0, 124.9, 117.6, 114.7, 114.3, 94.7, 55.3. ESI-MS (m/z): 348.2
(M+H+).
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4.1.3 General procedure for the synthesis of
imidazo[1,2-b]pyrazoles 12–77
2-(p-Tolyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (17)
To a suspension of pyrazole 8or 9(0.50 mmol) in MeCN (0.5 mL) aldehyde
10 (0.55 mmol), HClO4 (20 mol%), and isocyanide 11 (0.55 mmol) were
added and stirred at room temperature for 6 h. Then the crude mixture
was purified by filtration followed by washing with cold MeCN or by
column chromatography on silica gel (eluent: hexane/EtOAc or chloro-
form/methanol gradient) to afford pure products 12–77.
White solid; yield: 66%; m.p. 218–219°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.54 (s, 1H), 7.92 (s, 1H), 7.81 (d, J = 7.8 Hz, 2H), 7.20
(d, J = 7.7 Hz, 2H), 6.82 (bs, 1H), 3.95 (bs, 1H), 3.49 (bs, 1H), 2.30 (s,
3H), 1.54 (s, 2H), 0.98 (s, 6H), 0.97 (s, 9H). 13C NMR (126 MHz,
DMSO-d6) δ 163.9, 142.2, 136.9, 136.5, 128.7, 127.8, 127.1, 124.4,
121.2, 93.9, 58.8, 55.5, 31.7, 31.3, 29.1, 20.9. ESI-MS (m/z): 368.3
(M+H+).
3-(tert-Butylamino)-2-phenyl-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (12)
White solid; yield: 69%; m.p. 246–248°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.61 (s, 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.94 (s, 1H), 7.39 (t, J = 7.6 Hz, 2H),
7.26 (t, J = 7.4 Hz, 1H), 7.08 (bs, 1H), 6.87 (bs, 1H), 4.04 (bs, 1H), 1.04 (s,
9H). 13C NMR (126 MHz, DMSO-d6) δ 163.9, 142.4, 137.1, 130.6, 128.2,
127.1, 126.7, 124.1, 121.8, 94.0, 54.7, 30.1; ESI-MS (m/z): 298.2 (M+H+).
2-(4-Methoxyphenyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (18)
Pale yellow solid; yield: 85%; m.p. 124–125°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.43 (s, 1H), 7.91 (s, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.00
(d, J = 8.3 Hz, 2H), 6.91–6.74 (bs, 2H), 3.87 (s, 1H), 3.81 (s, 3H), 1.58
(s, 2H), 1.03 (s, 6H), 1.00 (s, 9H). 13C NMR (126 MHz, DMSO-d6)
δ 164.4, 159.0, 142.5, 137.3, 129.1, 124.8, 123.6, 121.2, 114.1,
94.4, 59.1, 56.1, 55.7, 32.1, 31.8, 29.6. ESI-MS (m/z): 384.3
(M+H+).
2-Phenyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (13)
Pale yellow solid; yield: 51%; m.p. 154–156°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.64 (s, 1H), 7.99 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.43 (t,
J = 7.7 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 1.57 (s, 2H), 1.03 (s, 6H), 0.99 (s,
9H). 13C NMR (126 MHz, DMSO-d6) δ 164.3, 142.6, 137.5, 131.0,
128.6, 127.8, 127.6, 125.0, 122.1, 94.4, 59.3, 56.0, 32.1, 31.7, 29.5.
ESI-MS (m/z): 354.2 (M+H+).
4-(7-Carbamoyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazol-2-yl)-2-methoxyphenyl acetate (19)
White solid; yield: 50%; m.p. 190–191°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.65 (s, 1H), 7.95 (s, 1H), 7.68 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H),
7.34–6.97 (m, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.76 (s, 1H), 4.08 (s, 1H),
3.84 (s, 3H), 2.24 (s, 3H), 1.57 (s, 2H), 1.03 (s, 6H), 0.96 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 168.5, 164.0, 150.5, 142.1, 138.3, 137.3,
129.4, 123.8, 122.5, 121.6, 119.5, 111.7, 94.0, 58.7, 56.0, 55.6, 31.6,
31.3, 29.1, 20.4. ESI-MS (m/z): 442.3 (M+H+).
Methyl 2-((7-carbamoyl-2-phenyl-1H-imidazo[1,2-b]pyrazol-3-
yl)amino)acetate (14)
Gray solid; yield: 46%; m.p. 209–210°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.45 (s, 1H), 7.95 (s, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.41 (t,
J = 7.8 Hz, 2H), 7.25 (t, J = 7.4 Hz, 1H), 7.12 (bs, 1H), 6.82 (bs, 1H), 5.59
(s, 1H), 4.22 (s, 2H), 3.55 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
171.9, 163.8, 143.0, 137.3, 130.3, 128.5, 126.4, 126.0, 124.2, 115.3,
93.9, 51.5, 46.0. ESI-MS (m/z): 314.1 (M+H+).
Methyl 2-((7-carbamoyl-2-(2,4,6-trimethoxyphenyl)-1H-
imidazo[1,2-b]pyrazol-3-yl)amino)acetate (20)
Yellow solid; yield: 74%; m.p. 226–227°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.90 (s, 1H), 7.87 (s, 1H), 6.91 (s, 1H), 6.71 (s, 1H), 6.28 (s, 2H),
4.68 (t, J = 7.1 Hz, 1H), 3.82 (s, 5H), 3.71 (s, 6H), 3.45 (s, 3H). 13C NMR
(126 MHz, DMSO-d6) δ 171.4, 163.9, 161.8, 159.6, 142.1, 136.5,
124.9, 105.8, 99.3, 93.4, 90.7, 55.7, 55.4, 51.3, 45.9. ESI-MS (m/z):
404.1 (M+H+).
3-(Cyclohexylamino)-2-phenyl-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (15)
White solid; yield: 51%; m.p. 240–241°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.49 (s, 1H), 7.95 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.46–7.35 (m,
2H), 7.28–7.19 (m, 1H), 7.12 (bs, 1H), 6.82 (bs, 1H), 4.53 (bs, 1H), 1.81–
1.69 (m, 2H), 1.64–1.55 (m, 2H), 1.50–1.40 (m, 1H), 1.24–1.03 (m, 5H).
13C NMR (126 MHz, DMSO-d6) δ 163.8, 142.8, 137.1, 130.4, 128.4,
126.6, 125.8, 123.5, 119.9, 94.0, 54.2, 33.2, 25.5, 24.3. ESI-MS (m/z):
324.1 (M+H+).
2-(4-Fluorophenyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (21)
Pale yellow solid; yield: 69%; m.p. 229–230°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.66 (s, 1H), 8.08–7.78 (m, 3H), 7.23 (t, J = 8.7 Hz, 2H),
6.84 (bs, 2H), 3.97 (s, 1H), 1.52 (s, 2H), 0.98 (s, 6H), 0.95 (s, 9H).
13C NMR (126 MHz, DMSO-d6) δ 164.0, 161.3 (d, J = 244.7 Hz),
142.1, 137.2, 129.5 (d, J = 8.1 Hz), 127.2, 123.6, 121.4, 115.0 (d,
J = 21.4 Hz), 94.0, 58.7, 55.5, 31.6, 31.3, 29.0. ESI-MS (m/z): 372.3
(M+H+).
3-((4-Methoxyphenyl)amino)-2-phenyl-1H-imidazo[1,2-b]pyrazole-
7-carboxamide (16)
Gray solid; yield: 48%; m.p. 229–231°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.97 (s, 1H), 7.93 (s, 1H), 7.79 (s, 3H), 7.37 (s, 2H), 7.26 (s, 1H),

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Methyl 2-((7-carbamoyl-2-(4-fluorophenyl)-1H-
2H), 1.32–1.19 (m, 3H), 1.12 (s, 6H), 1.00 (s, 9H). 13C NMR (126 MHz,
DMSO-d6) δ 164.3, 140.8, 136.9, 130.1, 119.3, 93.6, 57.2, 55.1, 33.9,
31.7, 31.5, 31.3, 29.0, 26.4, 25.3. ESI-MS (m/z): 360.3 (M+H+).
imidazo[1,2-b]pyrazol-3-yl)amino)acetate (22)
White solid; yield: 53%; m.p. 229–230°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.49 (s, 1H), 7.95 (s, 1H), 7.88 (dd, J = 8.6, 5.3 Hz, 2H), 7.25 (t,
J = 8.7 Hz, 2H), 7.11 (bs, 1H), 6.79 (bs, 1H), 5.55 (t, J = 6.2 Hz, 1H), 4.19
(d, J = 6.2 Hz, 2H), 3.55 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 171.9,
163.8, 160.9 (d, J = 244.5 Hz), 142.8, 137.3, 128.3 (d, J = 5.0 Hz),
126.8, 123.8, 115.3 (d, J = 21.3 Hz), 115.1, 93.9, 51.5, 46.0. ESI-MS
(m/z): 332.1 (M+H+).
3-(tert-Butylamino)-2-heptyl-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (28)
White solid; yield: 44%; m.p. 203–204°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.18 (s, 1H), 7.82 (s, 1H), 7.00 (bs, 1H), 6.69 (bs, 1H), 3.84 (bs,
1H), 1.60 (t, J = 7.7 Hz, 2H), 1.32–1.18 (m, 10H), 1.09 (s, 9H), 0.88–0.78
(m, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.4, 140.5, 137.2, 125.5,
120.7, 93.7, 53.6, 31.2, 30.1, 28.8, 28.6, 28.4, 24.2, 22.1, 14.0. ESI-MS
(m/z): 320.3 (M+H+).
2-(4-(Trifluoromethyl)phenyl)-3-((2,4,4-trimethylpentan-2-
yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (23)
White solid; yield: 69%; m.p. 192–193°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.78 (s, 1H), 8.19 (d, J = 8.2 Hz, 2H), 8.01 (s, 1H), 7.77 (d,
J = 8.2 Hz, 2H), 1.60 (s, 2H), 1.06 (s, 6H), 0.99 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 164.3, 143.0, 138.3, 135.2, 128.0, 127.7 (q,
J = 32.0 Hz), 125.4 (q, J = 3.5 Hz), 124.8 (q, J = 271.7 Hz), 123.4, 94.5,
59.5, 56.0, 32.0, 31.7, 29.5. ESI-MS (m/z): 422.3 (M+H+).
2-(tert-Butyl)-3-(cyclohexylamino)-1H-imidazo[1,2-b]pyrazole-
7-carboxamide (29)
White solid; yield: 43%; m.p. 148–149°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.67 (s, 1H), 7.79 (s, 1H), 6.99 (bs, 1H), 6.63 (bs, 1H), 3.80 (s, 1H),
3.25 (s, 1H), 1.76–1.57 (m, 4H), 1.49 (s, 1H), 1.33 (s, 9H), 1.10 (s, 4H),
0.94 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 163.9, 141.8, 135.7,
129.6, 120.6, 93.6, 54.4, 33.3, 31.8, 30.0, 25.6, 24.5. ESI-MS (m/z):
304.3 (M+H+).
3-(tert-Butylamino)-2-(3,4-difluorophenyl)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (24)
White solid; yield: 80%; m.p. 256–258°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.73 (s, 1H), 8.12 (ddd, J = 13.0, 7.8, 2.2 Hz, 1H), 7.97 (s, 1H),
7.92–7.84 (m, 1H), 7.46 (dt, J = 10.7, 8.7 Hz, 1H), 7.13 (bs, 1H), 6.85 (bs,
1H), 4.25 (bs, 1H), 1.05 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 164.0,
149.2 (dd, J = 243.6, 12.7 Hz), 148.3 (dd, J = 246.6, 12.4 Hz), 142.3,
137.6, 128.2, 123.6, 122.3, 117.34 (d, J = 17.1 Hz), 115.51 (d,
J = 19.5 Hz), 94.1, 54.8, 30.1. ESI-MS (m/z): 334.4 (M+H+).
2-(tert-Butyl)-3-(tert-butylamino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (30)
White solid; yield: 49%; m.p. 221°C; 1H NMR (500 MHz, DMSO-d6) δ
10.72 (s, 1H), 7.79 (s, 1H), 7.01 (bs, 1H), 6.71 (bs, 1H), 3.59 (s, 1H), 1.36
(s, 9H), 1.17 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 164.0, 141.5,
135.9, 132.4, 119.7, 93.5, 52.4, 32.0, 30.7, 30.2. ESI-MS (m/z): 278.2
(M+H+).
2-(Pyridin-3-yl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (25)
2-(tert-Butyl)-3-((4-methoxyphenyl)amino)-1H-imidazo[1,2-b]-
pyrazole-7-carboxamide (31)
White solid; yield: 82%; m.p. 226–228°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.82 (s, 1H), 9.11 (s, 1H), 8.46 (s, 1H), 8.27 (d, J = 8.1 Hz, 1H),
7.97 (s, 1H), 7.43 (t, J = 6.6 Hz, 1H), 7.14 (bs, 1H), 6.80 (bs, 1H), 4.18 (s,
1H), 1.51 (s, 2H), 0.98 (s, 6H), 0.94 (s, 9H). 13C NMR (126 MHz,
DMSO-d6) δ 164.0, 147.9, 147.6, 142.2, 138.0, 134.6, 126.9, 123.2,
122.4, 121.7, 94.1, 58.7, 55.5, 31.6, 31.3, 29.1. ESI-MS (m/z): 355.3
(M+H+).
Pale yellow solid; yield: 41%; m.p. 260–262°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.19 (s, 1H), 7.79 (s, 1H), 7.22 (s, 1H), 7.11 (bs, 1H), 6.83
(bs, 1H), 6.68 (d, J = 8.3 Hz, 2H), 6.38 (d, J = 8.4 Hz, 2H), 3.60 (s, 3H),
1.31 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 163.9, 151.8, 141.6,
141.0, 136.5, 133.9, 115.4, 114.6, 113.7, 94.3, 55.3, 32.0, 29.4. ESI-
MS (m/z): 328.2 (M+H+).
(E)-3-(tert-Butylamino)-2-(1-phenylprop-1-en-2-yl)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (26)
2-(tert-Butyl)-3-((4-fluorophenyl)amino)-1H-imidazo[1,2-b]
pyrazole-7-carboxamide (32)
White solid; yield: 65%; m.p. 190–191°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.33 (s, 1H), 7.92 (s, 1H), 7.40–7.34 (m, 4H), 7.28–7.19 (m, 1H),
7.02 (s, 1H), 6.77 (bs, 1H), 4.15 (bs, 1H), 3.40 (bs, 1H), 2.29 (s, 3H), 1.11
(s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 164.0, 142.2, 137.2, 136.7,
128.9, 128.5, 128.3, 127.2, 127.0, 126.6, 121.9, 93.8, 54.5, 30.0, 16.7.
ESI-MS (m/z): 338.2 (M+H+).
Gray solid; yield: 54%; m.p. 249–250°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.28 (s, 1H), 7.82 (s, 1H), 7.55 (s, 1H), 7.14 (bs, 1H), 6.91 (t,
J = 8.6 Hz, 2H), 6.77 (bs, 1H), 6.43 (dd, J = 8.5, 4.5 Hz, 2H), 1.32 (s, 9H).
13C NMR (126 MHz, DMSO-d6) δ 163.9, 155.3 (d, J = 232.6 Hz), 143.6,
141.7, 136.5, 134.2, 115.4 (d, J = 22.3 Hz), 114.8, 113.8 (d, J = 7.2 Hz),
94.5, 32.0, 29.4. ESI-MS (m/z): 316.1 (M+H+).
2-Cyclohexyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (27)
White solid; yield: 39%; m.p. 190–192°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.15 (s, 1H), 7.81 (s, 1H), 6.96 (bs, 1H), 6.70 (bs, 1H), 3.75 (s, 1H),
2.71 (t, J = 12.2 Hz, 1H), 1.79–1.68 (m, 4H), 1.67–1.57 (m, 3H), 1.54 (s,
2-(tert-Butyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (33)
White solid; yield: 54%; m.p. 155–156°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.73 (s, 1H), 7.81 (s, 1H), 7.01 (s, 1H), 6.72 (s, 1H), 3.42 (s, 1H),
1.66 (s, 2H), 1.38 (s, 9H), 1.21 (s, 6H), 1.00 (s, 9H). 13C NMR (126 MHz,

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DMSO-d6) δ 164.0, 141.5, 135.9, 132.4, 119.5, 93.5, 56.5, 56.1, 32.0,
31.8, 31.4, 30.2, 29.6. ESI-MS (m/z): 334.3 (M+H+).
J = 4.4 Hz, 3H), 1.68 (s, 2H), 1.41 (s, 9H), 1.23 (s, 6H), 1.03 (s, 9H). 13CNMR
(126 MHz, DMSO-d6) δ 163.2, 141.1, 136.2, 132.8, 119.9, 94.0, 56.9,
56.5, 32.5, 32.2, 31.9, 30.7, 30.1, 25.8. ESI-MS (m/z): 348.4 (M+H+).
2-Cyclopropyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (34)
2-(tert-Butyl)-N-butyl-3-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[1,2-b]pyrazole-7-carboxamide (40)
White solid; yield: 56%; m.p. 205–207°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.80 (s, 1H), 7.81 (s, 1H), 6.90 (bs, 1H), 6.73 (bs, 1H), 3.60 (bs, 1H,
overlap with water), 2.00–1.92 (m, 1H), 1.59 (s, 2H), 1.14 (s, 6H), 1.02
(s, 9H), 0.91–0.87 (m, 2H), 0.85–0.80 (m, 2H). 13C NMR (126 MHz,
DMSO-d6) δ 164.1, 141.1, 136.2, 126.1, 121.5, 93.8, 58.1, 55.3, 31.8,
31.4, 29.2, 7.1, 6.6. ESI-MS (m/z): 318.2 (M+H+).
White solid; yield: 43%; m.p. 147–148°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.73 (s, 1H), 7.84 (s, 1H), 7.52 (t, J = 5.9 Hz, 1H), 3.45 (s, 1H), 3.22
(q, J = 6.7 Hz, 2H), 1.68 (s, 2H), 1.48 (q, J = 7.2 Hz, 2H), 1.41 (s, 9H), 1.33
(q, J = 7.5 Hz, 2H), 1.23 (s, 6H), 1.03 (s, 9H), 0.91 (t, J = 7.3 Hz, 3H).
13C NMR (126 MHz, DMSO-d6) δ 162.7, 140.8, 136.5, 132.9, 119.9,
94.2, 56.9, 56.5, 38.4, 32.5, 32.2, 31.9, 30.7, 30.1, 20.1, 14.3. ESI-MS
(m/z): 390.3 (M+H+).
2-Ethyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-imidazo[1,2-
b]pyrazole-7-carboxamide (35)
White solid; yield: 51%; m.p. 207–209°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.19 (s, 1H), 7.82 (s, 1H), 7.01 (bs, 1H), 6.65 (bs, 1H), 3.68 (s, 1H),
2.55–2.50 (m, 2H), 1.54 (s, 2H), 1.20–1.13 (m, 3H), 1.10 (s, 6H), 1.00 (s,
9H). 13C NMR (126 MHz, DMSO-d6) δ 164.4, 140.5, 137.3, 126.6,
120.1, 93.6, 57.4, 55.2, 31.8, 31.4, 29.1, 17.7, 13.8. ESI-MS (m/z):
306.3 (M+H+).
N,2-Di-tert-butyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazole-7-carboxamide (41)
White solid; yield: 66%; m.p. 177–178°C; 1H NMR (500 MHz, DMSO-d6) δ
10.74 (s, 1H), 7.93 (s, 1H), 6.94 (s, 1H), 3.43 (s, 1H), 1.67 (s, 2H), 1.40 (s, 9H),
1.38 (s, 9H), 1.23 (s, 6H), 1.03 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ
13C NMR (126 MHz, DMSO) δ163.1, 140.2, 137.6, 132.9, 119.7, 95.1, 57.0,
56.5, 50.6, 32.5, 32.2, 31.8, 30.7, 30.1, 29.7. ESI-MS (m/z): 390.4 (M+H+).
2-Isopropyl-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-imidazo
[1,2-b]pyrazole-7-carboxamide (36)
2-(tert-Butyl)-N-cyclopropyl-3-((2,4,4-trimethylpentan-2-yl)-
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (42)
White solid; yield: 57%; m.p. 161°C; 1H NMR (500 MHz, DMSO-d6) δ
10.76 (s, 1H), 7.82 (s, 1H), 7.64–7.55 (m, 1H), 2.75–2.68 (m, 1H), 1.67
(s, 2H), 1.40 (s, 9H), 1.23 (s, 6H), 1.03 (s, 9H), 0.69–0.64 (m, 2H),
0.52–0.48 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 163.9, 140.9,
136.6, 132.9, 119.9, 94.0, 56.9, 56.5, 32.5, 32.2, 31.9, 30.7, 30.1, 22.7,
6.5. ESI-MS (m/z): 374.4 (M+H+).
White solid; yield: 42%; m.p. 132–134°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.17 (s, 1H), 7.82 (s, 1H), 6.97 (bs, 1H), 6.70 (bs, 1H), 3.70 (bs,
1H), 3.12–3.02 (m, 1H), 1.55 (s, 2H), 1.22 (d, J = 7.1 Hz, 6H), 1.12 (s,
6H), 1.00 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 164.3, 140.8,
137.2, 130.7, 119.0, 93.7, 57.2, 55.2, 31.8, 31.4, 29.1, 24.0, 21.8. ESI-
MS (m/z): 320.4 (M+H+).
2-(2-Methylpent-4-en-2-yl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (37)
White solid; yield: 45%; m.p. 161–163°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.72 (s, 1H), 7.81 (s, 1H), 7.04 (s, 1H), 6.72 (s, 1H), 5.67–5.55 (m,
1H), 5.05–4.89 (m, 2H), 3.43 (s, 1H), 1.65 (s, 2H), 1.35 (s, 6H), 1.22 (s,
6H), 0.99 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 164.0, 141.5,
136.0, 135.5, 131.0, 120.5, 117.3, 93.5, 56.4, 56.1, 46.2, 35.2, 31.8,
31.4, 29.6, 27.7. ESI-MS (m/z): 360.3 (M+H+).
2-(tert-Butyl)-N-cyclopentyl-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (43)
White solid; yield: 38%; m.p. 182–183°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.75 (s, 1H), 7.90 (s, 1H), 7.40 (s, 1H), 4.19 (s, 1H), 3.44 (s, 1H),
1.89 (s, 2H), 1.69 (s, 4H), 1.51 (s, 4H), 1.41 (s, 9H), 1.24 (s, 6H), 1.04 (s,
9H). 13C NMR (126 MHz, DMSO-d6) δ 164.3, 140.5, 136.6, 133.2,
120.0, 93.4, 57.2, 56.5, 50.9, 32.2, 31.9, 31.2, 31.0, 29.4, 29.1, 23.5.
ESI-MS (m/z): 402.4 (M+H+).
2-(1-Cyano-3-ethylpentan-3-yl)-3-((2,4,4-trimethylpentan-2-yl)-
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (38)
White solid; yield: 35%; m.p. 184–186°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.61 (s, 1H), 7.83 (s, 1H), 7.05 (bs, 1H), 6.74 (bs, 1H), 3.56
(d, J = 2.1 Hz, 1H), 2.32–2.22 (m, 2H), 2.19–2.13 (m, 2H), 1.76
(q, J = 7.2 Hz, 4H), 1.66 (s, 2H), 1.27 (s, 6H), 1.00 (s, 9H), 0.68 (t,
J = 6.9 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ 163.9, 141.7, 136.2,
128.2, 122.8, 121.0, 93.7, 56.2, 56.1, 42.4, 31.8, 31.4, 29.6, 26.6, 11.5,
7.9. ESI-MS (m/z): 401.4 (M+H+).
(2-(tert-Butyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazol-7-yl)(piperidin-1-yl)methanone (44)
White solid; yield: 60%; m.p. 164–165°C; 1H NMR (500 MHz, DMSO-d6)
δ 10.69 (s, 1H), 7.63 (s, 1H), 3.80–3.56 (m, 4H), 1.70 (s, 2H), 1.67–1.59
(m, 2H), 1.56–1.47 (m, 4H), 1.40 (s, 9H), 1.24 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 163.4, 140.4, 138.5, 133.0, 119.7, 92.7, 56.9,
56.5, 32.5, 32.3, 31.9, 30.7, 30.1, 26.4, 24.8. ESI-MS (m/z): 402.4 (M+H+).
(2-(tert-Butyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-1H-
imidazo[1,2-b]pyrazol-7-yl)(4-phenylpiperazin-1-yl)methanone
(45)
2-(tert-Butyl)-N-methyl-3-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[1,2-b]pyrazole-7-carboxamide (39)
White solid; yield: 31%; m.p. 173–174°C; 1H NMR (500 MHz, DMSO-d6)
White solid; yield: 59%; m.p. 170°C; 1H NMR (500 MHz, DMSO-d6) δ
δ 10.75 (s, 1H), 7.81 (s, 1H), 7.46 (d, J = 6.2 Hz, 1H), 3.46 (s, 1H), 2.75 (d,
10.77 (s, 1H), 7.74 (s, 1H), 7.24 (t, J = 7.9 Hz, 2H), 6.97 (d, J = 8.1 Hz,

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2H), 6.81 (t, J = 7.2 Hz, 1H), 3.83–3.79 (m, 4H), 3.49 (s, 1H), 3.21–3.17
(m, 4H), 1.72 (s, 2H), 1.41 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 163.6, 151.4, 140.7, 138.5, 133.1, 129.5,
119.8, 119.7, 116.2, 92.4, 56.9, 56.5, 49.1, 32.5, 32.3, 31.9, 30.7, 30.1.
ESI-MS (m/z): 479.4 (M+H+).
2-(tert-Butyl)-N-(thiazol-2-yl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (51)
White solid; yield: 44%; m.p. 125–127°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.86 (s, 1H), 11.35 (s, 1H), 8.31 (s, 1H), 7.48 (d, J = 3.6 Hz, 1H), 7.14
(d, J = 3.5 Hz, 1H), 3.55 (s, 1H), 1.69 (s, 2H), 1.42 (s, 9H), 1.24 (s, 6H), 1.04
(s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 160.1, 159.5, 141.8, 137.8,
137.3, 133.4, 120.4, 113.1, 92.5, 57.0, 56.5, 32.6, 32.2, 31.9, 30.7, 30.1.
ESI-MS (m/z): 417.3 (M+H+).
N-Benzyl-2-(tert-butyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[1,2-b]pyrazole-7-carboxamide (46)
White solid; yield: 49%; m.p. 118–119°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.81 (s, 1H), 8.14 (t, J = 6.1 Hz, 1H), 7.91 (s, 1H), 7.35–7.30 (m,
4H), 7.28–7.20 (m, 1H), 4.46 (d, J = 6.0 Hz, 2H), 3.48 (s, 1H), 1.69 (s,
2H), 1.41 (s, 9H), 1.24 (s, 6H), 1.03 (s, 9H). 13C NMR (126 MHz, DMSO-
d6) δ 162.8, 141.1, 141.1, 136.6, 132.9, 128.7, 127.6, 127.0, 120.0,
93.9, 56.9, 56.5, 42.2, 32.5, 32.3, 31.9, 30.7, 30.1. ESI-MS (m/z): 424.3
(M+H+).
2-(tert-Butyl)-N-(isoxazol-3-yl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (52)
Gray solid; yield: 40%; m.p. 185–186°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.21 (s, 1H), 10.66 (s, 1H), 8.77 (d, J = 1.8 Hz, 1H), 8.23 (s, 1H), 7.05
(d, J = 1.7 Hz, 1H), 3.53 (s, 1H), 1.68 (s, 2H), 1.42 (s, 9H), 1.24 (s, 6H), 1.03
(s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 160.7, 159.9, 158.9, 141.8,
137.3, 133.3, 120.3, 99.9, 93.4, 57.0, 56.5, 32.5, 32.2, 31.9, 30.7, 30.1.
ESI-MS (m/z): 401.3 (M+H+).
2-(tert-Butyl)-N-phenyl-3-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[1,2-b]pyrazole-7-carboxamide (47)
White solid; yield: 55%; m.p. 147°C; 1H NMR (500 MHz, DMSO-d6) δ
11.06 (s, 1H), 9.42 (s, 1H), 8.13 (s, 1H), 7.73 (d, J = 7.2 Hz, 2H), 7.31 (t,
J = 7.9 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 3.52 (s, 1H), 1.70 (s, 2H), 1.43 (s,
9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.5,
140.8, 140.4, 137.5, 133.3, 129.0, 122.9, 120.1, 120.1, 94.4, 57.0,
56.5, 32.6, 32.2, 31.9, 30.7, 30.1. ESI-MS (m/z): 410.3 (M+H+).
2-(tert-Butyl)-N-(o-tolyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[1,2-b]pyrazole-7-carboxamide (53)
White solid; yield: 30%; m.p. 172°C; 1H NMR (500 MHz, DMSO-d6) δ
10.96 (s, 1H), 8.93 (s, 1H), 8.04 (s, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.26
(d, J = 7.4 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 3.53
(s, 1H), 2.26 (s, 3H), 1.71 (s, 2H), 1.42 (s, 9H), 1.26 (s, 6H), 1.05 (s, 9H).
13C NMR (126 MHz, DMSO-d6) δ 161.2, 141.5, 137.3, 136.8, 133.8,
133.1, 130.7, 126.8, 126.3, 125.7, 120.1, 94.0, 57.0, 56.5, 32.5, 32.3,
31.9, 30.7, 30.1, 18.6. ESI-MS (m/z): 424.3 (M+H+).
2-(tert-Butyl)-N-(pyridin-2-yl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (48)
White solid; yield: 29%; m.p. 144–145°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.17 (s, 1H), 10.05 (s, 1H), 8.33 (d, J = 4.8 Hz, 1H), 8.29 (s, 1H), 8.22
(d, J = 8.5 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.07 (t, J = 6.1 Hz, 1H), 3.53
(s, 1H), 1.69 (s, 2H), 1.43 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 161.6, 153.5, 148.1, 141.9, 138.2, 137.2, 133.2,
120.2, 119.0, 114.3, 94.0, 57.0, 56.5, 32.6, 32.3, 31.9, 30.7, 30.1. ESI-MS
(m/z): 411.3 (M+H+).
2-(tert-Butyl)-N-(3,5-dimethylphenyl)-3-((2,4,4-trimethylpentan-
2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (54)
Gray solid; yield: 51%; m.p. 180–181°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.02 (s, 1H), 9.28 (s, 1H), 8.11 (s, 1H), 7.38 (d, J = 1.6 Hz, 2H), 6.67
(s, 1H), 3.51 (s, 1H), 2.25 (s, 6H), 1.70 (s, 2H), 1.42 (s, 9H), 1.25 (s, 6H),
1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.4, 140.7, 140.2,
137.8, 137.5, 133.2, 124.4, 120.1, 117.9, 94.5, 57.0, 56.5, 32.5, 32.2,
31.9, 30.7, 30.1, 21.6. ESI-MS (m/z): 438.4 (M+H+).
2-(tert-Butyl)-N-(pyridin-3-yl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (49)
White solid; yield: 31%; m.p. 186–187°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.14 (s, 1H), 9.63 (s, 1H), 8.93 (d, J = 2.4 Hz, 1H), 8.23 (dd, J = 4.7,
1.5 Hz, 1H), 8.15 (s, 1H), 8.13–8.10 (m, 1H), 7.35 (dd, J = 8.3, 4.7 Hz, 1H),
3.54 (s, 1H), 1.70 (s, 2H), 1.43 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 161.6, 143.8, 141.7, 140.9, 137.5, 137.1, 133.4,
126.8, 123.9, 120.2, 94.0, 57.0, 56.5, 32.6, 32.2, 31.9, 30.7, 30.1. ESI-MS
(m/z): 411.3 (M+H+).
2-(tert-Butyl)-N-(4-isopropylphenyl)-3-((2,4,4-trimethylpentan-
2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (55)
White solid; yield: 49%; m.p. 155–156°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.04 (s, 1H), 9.35 (s, 1H), 8.12 (s, 1H), 7.64 (d, J = 8.1 Hz, 2H),
7.18 (d, J = 8.2 Hz, 2H), 3.51 (s, 1H), 2.85 (hept, J = 7.0 Hz, 1H), 1.70 (s,
2H), 1.43 (s, 9H), 1.25 (s, 6H), 1.20 (d, J = 6.9 Hz, 6H), 1.04 (s, 9H).
13C NMR (126 MHz, DMSO-d6) δ 161.3, 142.8, 140.8, 138.1, 137.5,
133.2, 126.7, 120.1, 120.0, 94.4, 57.0, 56.5, 33.3, 32.6, 32.2, 31.9,
30.7, 30.1, 24.5. ESI-MS (m/z): 452.4 (M+H+).
2-(tert-Butyl)-N-(pyridin-4-yl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (50)
Pale yellow solid; yield: 23%; m.p. 185–186°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.24 (s, 1H), 9.80 (s, 1H), 8.40 (d, J = 5.5 Hz, 2H), 8.17 (s,
1H), 7.73 (d, J = 5.4 Hz, 2H), 3.56 (s, 1H), 1.69 (s, 2H), 1.42 (s, 9H), 1.24
(s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.9, 150.5,
147.2, 141.0, 137.6, 133.5, 120.2, 113.7, 94.1, 57.0, 56.4, 32.6, 32.2,
31.9, 30.7, 30.1. ESI-MS (m/z): 411.3 (M+H+).
2-(tert-Butyl)-N-(4-methoxyphenyl)-3-((2,4,4-trimethylpentan-
2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (56)
White solid; yield: 59%; m.p. 187–188°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.00 (s, 1H), 9.29 (s, 1H), 8.08 (s, 1H), 7.61 (d, J = 8.7 Hz, 2H),
6.90 (d, J = 8.6 Hz, 2H), 3.51 (s, 1H), 1.70 (s, 2H), 1.43 (s, 9H), 1.25 (s,
6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.2, 155.3,

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140.8, 137.3, 133.4, 133.2, 121.9, 120.0, 114.1, 94.3, 57.0, 56.5, 55.6,
32.5, 32.2, 31.9, 30.7, 30.1. ESI-MS (m/z): 440.4 (M+H+).
(m, 1H), 7.22–7.16 (m, 2H), 3.46 (s, 1H), 1.73 (s, 2H), 1.44 (s, 9H), 1.27 (s,
6H), 1.06 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.2, 155.8 (d,
J = 245.3 Hz), 141.6, 137.1, 133.2, 127.0, 126.9, 126.0 (d, J = 7.5 Hz),
124.5 (d, J = 3.3 Hz), 120.3, 116.0(d, J = 20.2 Hz), 93.8, 57.1, 56.6, 32.5,
32.2, 31.8, 30.7, 30.1. ESI-MS (m/z): 428.3 (M+H+).
2-(tert-Butyl)-N-(2,4-dimethoxyphenyl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (57)
Light beige solid; yield: 39%; m.p. 127°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.95 (s, 1H), 8.51 (s, 1H), 8.00 (s, 1H), 7.50 (d, J = 8.7 Hz, 1H),
6.64 (s, 1H), 6.52 (d, J = 8.8 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.52 (s,
1H), 1.70 (s, 2H), 1.42 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 161.3, 157.7, 153.5, 141.3, 136.7, 133.0,
126.5, 120.7, 120.1, 104.5, 99.2, 94.1, 56.9, 56.5, 56.1, 55.8, 32.5,
32.3, 31.9, 30.7, 30.1. ESI-MS (m/z): 470.4 (M+H+).
2-(tert-Butyl)-N-(3-fluorophenyl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (62)
White solid; yield: 47%; m.p. 159°C; 1H NMR (500 MHz, DMSO-d6) δ
11.13 (s, 1H), 9.62 (s, 1H), 8.14 (s, 1H), 7.78 (dt, J = 12.3, 2.4 Hz, 1H),
7.56–7.42 (m, 1H), 7.39–7.26 (m, 1H), 6.83 (td, J = 8.4, 2.6 Hz, 1H),
3.53 (s, 1H), 1.70 (s, 2H), 1.43 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 163.6, 162.6 (d, J = 240.2 Hz), 142.3 (d,
J = 11.2 Hz), 140.8, 137.6, 133.3, 130.5 (d, J = 9.7 Hz), 120.1, 115.5,
109.1 (d, J = 21.2 Hz), 106.5 (d, J = 26.5 Hz), 94.2, 57.0, 56.5, 32.6,
32.2, 31.9, 30.7, 30.1. ESI-MS (m/z): 428.4 (M+H+).
2-(tert-Butyl)-N-(2-(trifluoromethyl)phenyl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (58)
White solid; yield: 34%; m.p. 106°C; 1H NMR (500 MHz, DMSO-d6) δ
11.01 (s, 1H), 9.11 (s, 1H), 8.02 (s, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.71
(t, J = 7.6 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 3.54
(s, 1H), 1.71 (s, 2H), 1.42 (s, 9H), 1.26 (s, 6H), 1.05 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 161.9, 141.5, 136.9, 136.7, 133.3, 133.1,
131.4, 126.9, 126.8 (q, J = 4.6 Hz), 126.2 (q, J = 29.1 Hz), 124.3
(d, J = 273.6 Hz), 120.2, 93.6, 57.0, 56.5, 32.5, 32.3, 31.9, 30.7, 30.1.
ESI-MS (m/z): 478.3 (M+H+).
2-(tert-Butyl)-N-(4-fluorophenyl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (63)
White solid; yield: 49%; m.p. 188°C; 1H NMR (500 MHz, DMSO-d6) δ
11.06 (s, 1H), 9.48 (s, 1H), 8.10 (s, 1H), 7.80–7.67 (m, 2H), 7.15 (t,
J = 8.9 Hz, 2H), 3.52 (s, 1H), 1.70 (s, 2H), 1.42 (s, 9H), 1.25 (s, 6H), 1.04
(s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.4, 158.1 (d, J = 238.7 Hz),
140.8, 137.4, 136.7, 133.3, 121.8 (d, J = 7.9 Hz), 120.1, 115.5 (d,
J = 22.0 Hz), 94.2, 57.0, 56.5, 32.6, 32.2, 31.9, 30.7, 30.1. ESI-MS (m/z):
428.4 (M+H+).
2-(tert-Butyl)-N-(3-(trifluoromethyl)phenyl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (59)
2-(tert-Butyl)-N-(4-chlorophenyl)-3-((2,4,4-trimethylpentan-2-
yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (64)
White solid; yield: 50%; m.p. 188°C; 1H NMR (500 MHz, DMSO-d6) δ
11.09 (s, 1H), 9.55 (s, 1H), 8.12 (s, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.36 (d,
J = 8.7 Hz, 2H), 3.52 (s, 1H), 1.70 (s, 2H), 1.42 (s, 9H), 1.25 (s, 6H), 1.04
(s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.4, 140.8, 139.4, 137.5,
133.3, 128.9, 126.4, 121.5, 120.1, 94.19, 57.0, 56.5, 32.6, 32.2, 31.9,
30.7, 30.1. ESI-MS (m/z): 444.3 and 446.3 (M+H+).
White solid; yield: 54%; m.p. 180°C; 1H NMR (500 MHz, DMSO-d6) δ
11.15 (s, 1H), 9.78 (s, 1H, 8.34 (s, 1H), 8.16 (s, 1H), 7.91 (d, J = 8.1 Hz,
1H), 7.55 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 7.6 Hz), 3.54 (s, 1H), 1.70 (s, 2H),
1.43 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ
161.7, 141.3, 140.7, 137.7, 133.4, 130.1, 129.7 (q, J = 31.3 Hz), 124.8
(q, J = 272.3 Hz), 123.2, 120.2, 119.0 (q, J = 3.7 Hz), 115.9 (q,
J = 4.3 Hz), 94.1, 57.0, 56.4, 32.6, 32.2, 31.9, 30.7, 30.1. ESI-MS
(m/z): 478.4 (M+H+).
N-(4-Bromophenyl)-2-(tert-butyl)-3-((2,4,4-trimethylpentan-2-
yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (65)
Pale yellow solid; yield: 42%; m.p. 153–154°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.10 (s, 1H), 9.55 (s, 1H), 8.12 (s, 1H), 7.72 (d, J = 8.5 Hz,
2H), 7.49 (d, J = 8.5 Hz, 2H), 3.53 (s, 1H), 1.70 (s, 2H), 1.42 (s, 9H), 1.25
(s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.4, 140.8,
139.8, 137.5, 133.3, 131.8, 121.9, 120.1, 114.3, 94.2, 57.0, 56.5, 32.6,
32.2, 31.9, 30.7, 30.1. ESI-MS (m/z): 488.2 and 490.3 (M+H+).
2-(tert-Butyl)-N-(4-(trifluoromethyl)phenyl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (60)
White solid; yield: 39%; m.p. 155–156°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.17 (s, 1H), 9.78 (s, 1H), 8.17 (s, 1H), 7.97 (d, J = 8.5 Hz, 2H),
7.67 (d, J = 8.5 Hz, 2H), 3.54 (s, 1H), 1.70 (s, 2H), 1.43 (s, 9H), 1.25 (s,
6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.6, 144.2,
140.9, 137.6, 133.4, 126.3 (d, J = 3.5 Hz), 125.1 (q, J = 271.0 Hz), 122.7
(q, J = 31.7 Hz), 120.2, 119.6, 94.2, 57.0, 56.5, 32.6, 32.2, 31.9, 30.7,
30.1. ESI-MS (m/z): 478.4 (M+H+).
2-(tert-Butyl)-N-(4-nitrophenyl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (66)
Yellow solid; yield: 38%; m.p. 199°C; 1H NMR (500 MHz, DMSO-d6) δ
11.27 (s, 1H), 10.06 (s, 1H), 8.24 (d, J = 9.2 Hz, 2H), 8.19 (s, 1H), 8.01
(d, J = 9.1 Hz, 2H), 3.57 (s, 1H), 1.69 (s, 2H), 1.43 (s, 9H), 1.25 (s, 6H),
1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.6, 147.1, 141.9,
141.2, 137.7, 133.5, 125.2, 120.4, 119.2, 94.1, 57.1, 56.5, 32.6, 32.2,
31.8, 30.7, 30.1. ESI-MS (m/z): 455.3 (M+H+).
2-(tert-Butyl)-N-(2-fluorophenyl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (61)
White solid; yield: 39%; m.p. 153–154°C; 1H NMR (500 MHz, DMSO-d6)
δ 10.91 (s, 1H), 9.08 (s, 1H), 8.09 (s, 1H), 7.74–7.65 (m, 1H), 7.29–7.22

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2-(tert-Butyl)-N-(4-cyanophenyl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (67)
J = 13.8, 7.6, 2.4 Hz, 1H), 7.47–7.33 (m, 2H), 3.53 (s, 1H), 1.69 (s, 2H),
1.42 (s, 9H), 1.24 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6)
δ 161.5, 149.31 (dd, J = 242.0, 13.0 Hz), 145.16 (dd, J = 240.2,
12.7 Hz), 140.8, 137.58 (dd, J = 9.3, 2.5 Hz), 137.5, 133.3, 120.1,
117.61 (d, J = 17.6 Hz), 115.93 (dd, J = 5.0, 3.1 Hz), 108.68 (d,
J = 21.9 Hz), 94.0, 57.0, 56.5, 32.6, 32.2, 31.9, 30.7, 30.1. ESI-MS
(m/z): 446.3 (M+H+).
White solid; yield: 48%; m.p. 199°C; 1H NMR (500 MHz, DMSO-d6) δ
11.20 (s, 1H), 9.85 (s, 1H), 8.16 (s, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.76 (d,
J = 8.7 Hz, 2H), 3.55 (s, 1H), 1.70 (s, 2H), 1.43 (s, 9H), 1.25 (s, 6H), 1.04
(s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.6, 144.9, 141.0, 137.6,
133.5, 133.4, 120.2, 119.8, 119.7, 104.2, 94.1, 57.0, 56.5, 32.6, 32.2,
31.9, 30.7, 30.1. ESI-MS (m/z): 435.3 (M+H+).
2-(tert-Butyl)-N-(4-fluorobenzyl)-3-((2,4,4-trimethylpentan-2-yl)
amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (73)
White solid; yield: 33%; m.p. 163–164°C; 1H NMR (500 MHz,
DMSO-d6) δ 10.82 (s, 1H), 8.16 (t, J = 6.1 Hz, 1H), 7.90 (s, 1H),
7.46–7.25 (m, 2H), 7.21–7.01 (m, 2H), 4.43 (d, J = 6.0 Hz, 2H), 3.48
(s, 1H), 1.68 (s, 2H), 1.40 (s, 9H), 1.24 (s, 6H), 1.03 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 162.8, 161.5 (d, J = 241.7 Hz), 141.0, 137.3,
136.6, 132.9, 129.5 (d, J = 8.1 Hz), 120.0, 115.4 (d, J = 21.2 Hz),
93.8, 56.9, 56.5, 41.5, 32.5, 32.2, 31.9, 30.7, 30.1. ESI-MS (m/z):
442.4 (M+H+).
Ethyl 4-(2-(tert-butyl)-3-((2,4,4-trimethylpentan-2-yl)amino)-
1H-imidazo[1,2-b]pyrazole-7-carboxamido)benzoate (68)
White solid; yield: 60%; m.p. 127–128°C; 1H NMR (500 MHz, DMSO-d6)
δ 11.17 (s, 1H), 9.78 (s, 1H), 8.17 (s, 1H), 7.97–7.83 (m, 4H), 4.29
(q, J = 7.1 Hz, 2H), 3.54 (s, 1H), 1.70 (s, 2H), 1.43 (s, 9H), 1.32 (t, J = 7.1 Hz,
3H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 165.9,
161.6, 145.1, 140.9, 137.6, 133.4, 130.5, 123.6, 120.2, 119.1, 94.3, 60.8,
57.0, 56.5, 32.6, 32.2, 31.9, 30.7, 30.1, 14.7. ESI-MS (m/z): 482.4 (M+H+).
2-(tert-Butyl)-N-(4-(methylthio)phenyl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (69)
2-(tert-Butyl)-N-(5-fluoropyridin-2-yl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (74)
White solid; yield: 28%; m.p. 169–170°C; 1H NMR (500 MHz, DMSO-
d6) δ 11.06 (s, 1H), 9.44 (s, 1H), 8.11 (s, 1H), 7.70 (d, J = 8.3 Hz, 2H), 7.25
(d, J = 8.3 Hz, 2H), 3.52 (s, 1H), 2.46 (s, 3H), 1.70 (s, 2H), 1.43
(s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR(126 MHz, DMSO-d6) δ 161.4,
140.8, 138.0, 137.5, 133.3, 131.1, 127.7, 120.8, 120.1, 94.3, 57.0, 56.5,
32.6, 32.2, 31.9, 30.7, 30.1, 16.2. ESI-MS (m/z): 456.3 (M+H+).
White solid; yield: 34%; m.p. 140°C; 1H NMR (500 MHz, DMSO-d6) δ
11.16 (s, 1H), 10.17 (s, 1H), 8.34 (d, J = 3.1 Hz, 1H), 8.28 (s, 1H), 8.25
(dd, J = 9.2, 4.2 Hz, 1H), 7.74 (td, J = 8.8, 3.1 Hz, 1H), 3.53 (s, 1H),
1.69 (s, 2H), 1.43 (s, 9H), 1.24 (s, 6H), 1.04 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 161.4, 155.7 (d, J = 247.1 Hz), 150.0, 141.9,
137.2, 135.3 (d, J = 24.7 Hz), 133.2, 125.5 (d, J = 19.4 Hz), 120.2,
115.4 (d, J = 4.0 Hz), 93.8, 57.0, 56.5, 32.6, 32.2, 31.9, 30.7, 30.1.
ESI-MS (m/z): 429.2 (M+H+).
2-(tert-Butyl)-N-(4-(dimethylamino)phenyl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (70)
Gray solid; yield: 37%; m.p. 176–177°C; 1H NMR (500 MHz, DMSO-
d6) δ 10.96 (s, 1H), 9.16 (s, 1H), 8.06 (s, 1H), 7.51 (d, J = 8.6 Hz, 2H),
6.72 (d, J = 8.5 Hz, 2H), 3.50 (s, 1H), 3.35 (s, 6H), 1.70 (s, 2H), 1.43 (s,
9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.1,
147.2, 140.8, 137.2, 133.1, 130.1, 121.9, 120.0, 113.2, 94.5, 57.0,
56.5, 41.1, 32.5, 32.3, 31.9, 30.7, 30.1. APCI-MS (m/z): 453.3 (M+H+).
2-(tert-Butyl)-N-(6-fluoropyridin-3-yl)-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (75)
White solid; yield: 25%; m.p. 177–178°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.15 (s, 1H), 9.72 (s, 1H), 8.58 (s, 1H), 8.29–8.19
(m, 1H), 8.12 (s, 1H), 7.17 (dd, J = 9.0, 3.0 Hz, 1H), 3.54 (s, 1H), 1.70
(s, 2H), 1.43 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz,
2-(tert-Butyl)-N-(2,4-difluorophenyl)-3-((2,4,4-trimethylpentan-
2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (71)
White solid; yield: 45%; m.p. 174°C; 1H NMR (500 MHz, DMSO-d6) δ
11.03 (s, 1H), 9.22 (s, 1H), 8.08 (s, 1H), 7.62 (td, J = 8.9, 6.2 Hz, 1H), 7.33
(ddd, J = 10.6, 9.1, 2.9 Hz, 1H), 7.18–7.03 (m, 1H), 3.54 (s, 1H), 1.70 (s, 2H),
1.42 (s, 9H), 1.25 (s, 6H), 1.04 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ
161.2, 159.4 (dd, J = 243.6, 11.5 Hz), 156.2 (dd, J = 248.5, 12.5 Hz), 141.5,
137.0, 133.2, 128.47 (dd, J = 9.6, 3.0 Hz), 123.28 (dd, J = 12.1, 3.5 Hz),
120.2, 111.42 (dd, J = 21.7, 3.3 Hz), 104.6 (dd, J = 26.1, 24.9 Hz), 93.5,
57.0, 56.5, 32.5, 32.2, 31.9, 30.7, 30.1. ESI-MS (m/z): 446.3 (M+H+).
DMSO-d6) δ 161.48, 158.64 (d, J = 231.2 Hz), 140.91, 138.29
(d, J = 15.4 Hz), 137.43, 135.37, 133.37, 120.17, 109.53 (d,
J = 39.4 Hz), 93.77, 56.99, 56.46, 32.57, 32.23, 31.86, 30.69,
30.10. ESI-MS (m/z): 429.2 (M+H+).
2-(tert-Butyl)-N-(4-fluorophenyl)-N-methyl-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (76)
White solid; yield: 42%; m.p. 102°C; 1H NMR (500 MHz, DMSO-d6) δ
10.67 (s, 1H), 7.41 (dd, J = 8.7, 4.9 Hz, 2H), 7.31 (t, J = 8.5 Hz, 2H), 5.95
(s, 1H), 3.39 (s, 1H), 3.29 (s, 3H), 1.62 (s, 2H), 1.39 (s, 9H), 1.16 (s, 6H),
0.99 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 163.3, 161.4 (d,
J = 244.7 Hz), 141.7, 140.3, 139.1, 133.0, 130.7 (d, J = 8.6 Hz), 119.7,
116.9 (d, J = 22.5 Hz), 93.8, 56.8, 56.4, 37.8, 32.5, 32.2, 31.8, 30.6,
30.0. ESI-MS (m/z): 442.4 (M+H+).
2-(tert-Butyl)-N-(3,4-difluorophenyl)-3-((2,4,4-trimethylpentan-
2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-carboxamide (72)
White solid; yield: 34%; m.p. 162–164°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.12 (s, 1H), 9.64 (s, 1H), 8.11 (s, 1H), 7.95 (ddd,

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2-(tert-Butyl)-N-(4-fluorophenyl)-6-methyl-3-((2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (77)
4.2.2 3D spheroid production, pellet culture system
The spheroid production using pellet culture system was previously
described.[71] Briefly, MCF-7 and 4T1 cells were gently digested
from the flasks by trypsin, washed with PBS, counted and then
resuspended for a final density of 6000 cells per well. Cellular
suspensions (80 μL/well) were dispensed into U-bottom CellStar® 96
microplates (Cellstar® Cell-Repellent Microplate, Greiner Bio-One
Hungary, Mosonmagyaróvár) and centrifuged at 1200×g for 10 min.
Plates were incubated at 37°C, 5% CO2 for 24 h before drug
treatments. The obtained spherical aggregates (one spheroid/well)
were directly used for the cytotoxicity assay.
White solid; yield: 52%; m.p. 179°C; 1H NMR (500 MHz, DMSO-d6) δ
10.68 (s, 1H), 8.82 (s, 1H), 7.63 (dd, J = 8.9, 5.1 Hz, 2H), 7.16
(t, J = 8.7 Hz, 2H), 3.49 (s, 1H), 2.44 (s, 3H), 1.73 (s, 2H), 1.43 (s, 9H),
1.25 (s, 6H), 1.05 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 161.9,
158.4(d, J = 239.0 Hz), 151.9, 136.3, 136.3, 131.8, 123.3 (d, J = 7.5 Hz),
120.0, 115.3 (d, J = 22.0 Hz), 91.4, 56.9, 56.5, 32.4, 32.2, 31.9, 30.8,
30.1, 15.7. ESI-MS (m/z): 442.3 (M+H+).
|
4.1.4 Synthesis of imidazo[1,2-b]pyrazole 78
|
To a solution of 63 (0.25 mmol) in a MeCN/AcOH mixture (1:1,
1.5 mL) formaldehyde (37 wt.% in H2O, 0.375 mmol) was added and
stirred at room temperature for 15 min. Then NaCNBH3
(0.375 mmol) was added in portions and stirred further for 1 h.
The reaction mixture was poured slowly into saturated aqueous
Na2CO3 solution (30 mL, 0.5 M) and extracted with EtOAc
(3 × 50 mL). The organic layers were combined, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (eluent: hexane/EtOAc
gradient) to afford pure 78.
4.2.3 In vitro toxicity
In vitro toxicity of the sythesized molecules was determined on MCF-7,
4T1, and HL-60 cells using the fluorescent Resazurin assay as
described previously.[72]
Briefly, cells (MCF-7 and 4T1: 6000, HL-60: 120.000 cells/well)
were seeded into 96-well plates (Corning Life Sciences, Tewksbury,
MA, USA) in media and incubated overnight. Test compounds were
dissolved in dimethyl sulfoxide (DMSO). Cells were treated with an
increasing concentration of test compounds (156 nM–100 μM). The
highest applied DMSO content of the treated cells was 0.4%. Cell
viability was determined after 72 h incubation. Resazurin reagent
(Sigma–Aldrich, Budapest, Hungary) was added at a final concentration
of 25 μg/mL. After a 2-h incubation for 2D or 12 h for 3D cultures at
37°C 5%, CO2 fluorescence (530 nm excitation/580 nm emission)
was recorded on a multimode microplate reader (Cytofluor4000,
PerSeptive Biosytems, Framingham, MA, USA). Viability was calcu-
lated with relation to untreated control cells and blank wells containing
media without cells. IC50 values (50% inhibiting concentration) were
calculated by GraphPad Prism® 5 (La Jolla, CA, USA).
2-(tert-Butyl)-N-(4-fluorophenyl)-3-(methyl(2,4,4-
trimethylpentan-2-yl)amino)-1H-imidazo[1,2-b]pyrazole-7-
carboxamide (78)
White solid; yield: 25%; m.p. 212°C; 1H NMR (500 MHz, DMSO-d6) δ
11.18 (s, 1H), 9.52 (s, 1H), 8.11 (s, 1H), 7.73 (dd, J = 8.8, 5.0 Hz, 2H),
7.15 (t, J = 8.7 Hz, 2H), 2.71 (s, 3H), 2.08 (d, J = 14.4 Hz, 1H), 1.54 (d,
J = 14.3 Hz, 1H), 1.41 (s, 12H), 1.15 (s, 3H), 0.98 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 161.3, 158.1 (d, J = 238.9 Hz), 141.0, 137.8,
136.7, 133.7, 124.2, 121.8 (d, J = 7.6 Hz), 115.5 (d, J = 22.0 Hz), 94.4,
59.2, 52.7, 35.3, 32.6, 32.2, 31.4, 30.3, 27.3, 26.5. ESI-MS (m/z): 442.4
(M+H+).
|
4.2.4 Real-time cell electronic sensing (RT-CES)
in vitro toxicity measurement
RT-CES protocol was performed as described previously[73] with slight
modifications. RT-CES 96-well E-plate (Roche, Budapest, Hungary)
was coated with gelatin solution (0.2% in PBS) for 20 min at 37°C, then
gelatin was washed twice with PBS solution. A total of 50 µL growth
media was then gently dispensed into each well of the 96-well E-plate
for background readings by the RT-CES system prior to addition of
50 µL of MCF-7 cell suspension for a final density of 4000 cells/well.
Devices containing the cell suspension were kept at room temperature
in a tissue culture hood for 30 min prior to insertion into the RT-CES
device in the incubator to allow cells to settle and, after 20 h, they were
treated with test compounds. Treated and control wells were
dynamically monitored before and after treatment over 92 h by
measurements of electrical impedance every 15 min. Continuous
recording of impedance in cells was reflected by cell index value. The
raw plate reads for each titration point were normalized relative to the
cell index status right before treatment. Each treatment was repeated
|
4.2 Biological evaluation
|
4.2.1 Cell culture
Cells were purchased from the American Type Culture Collection
(ATCC, Manassas, Virginia, USA). The human breast adenocarcinoma
cell line, MCF-7 cells were maintained in Dulbecco's Modified Eagle
Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco, Paisley, UK) 10%
fetal calf serum (FCS, Gibco, Paisley, UK). Mouse mammary carcinoma
4T1 and human promyelocytic leukemia HL-60 cells were maintained
in Roswell Park Memorial Institute 1640 medium (RPMI-1640, Gibco,
Paisley, UK) 10% FCS. Media were supplemented with 2 mM
GlutaMAX, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life
Technologies, Carlsbad, CA, USA). Cell cultures were maintained at
37°C in a humidified incubator in an atmosphere of 5% CO2 (Sanyo,
Osaka, Japan).

20 of 21
DEMJÉN ET AL.
|
in 4 wells per during the experiment. Viability of cells was calculated
relative to wells containing non-treated cells. IC50 values were
calculated by GraphPad Prism® 5.
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4.2.5 Detection of phosphatidylserine exposure
Apoptosis was assayed as described previously.[72] Briefly, HL-60 cells
(200000) were plated in 24-well tissue culture plates (Corning Life
Sciences) andtreated withtheindicated compoundsandconcentrations
in the figures in 500 µL media. After 24 h the supernatants were
harvested. Cells were harvested with the corresponding supernatant
andfugeddown (2000 rpm, 5 min, Eppendorf, Wien, Austria). Pellet was
resuspended in Annexin V binding buffer (0.01 M HEPES, 0.14 M NaCl,
and 2.5 mM CaCl2). Annexin V-Alexa Fluor® 488 (Life Technologies,
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dilute Annexin V-Alexa Fluor® 488 5x. Cells (10000 events) were
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(Becton Dickinson, Franklin Lakes, NJ, USA). The percentage of the FL1
(530/30 nm filter, Annexin V-Alexa Fluor® 488) positive and FL3
(670 nm filter, propidium iodide) negative early apoptotic cells and FL1
positive and FL3 positive late apoptotic cells were determined. The total
apoptotic population includes both early and late apoptotic cells.
Column charts were created by GraphPad Prism® 5.
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ACKNOWLEDGMENTS
This work was partly supported by the following grants: GINOP-2.3.2-
15-2016-00030 and GINOP-2.3.2-15-2016-00001 from the National
Research, Development and Innovation Office (NKFI), Hungary. Gábor
J. Szebeni was supported by János Bolyai Research Scholarship of the
Hungarian Academy of Sciences (BO/00139/17/8).
[19] M. P. Dwyer, K. Paruch, M. Labroli, C. Alvarez, K. M. Keertikar, C.
Poker, R. Rossman, T. O. Fischmann, J. S. Duca, V. Madison, D. Parry,
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CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
[21] L. Ren, E. R. Laird, A. J. Buckmelter, V. Dinkel, S. L. Gloor, J. Grina, B.
Newhouse, K. Rasor, G. Hastings, S. N. Gradl, J. Rudolph, Bioorg. Med.
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ORCID
Iván Kanizsai
http://orcid.org/0000-0003-0109-7097
[22] T. Kosugi, D. R. Mitchell, A. Fujino, M. Imai, M. Kambe, S.
Kobayashi, H. Makino, Y. Matsueda, Y. Oue, K. Komatsu, K.
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