Synthesis and anti-mitotic activity of 2,4- or 2,6-disubstituted- and 2,4,6-trisubstituted-2H-pyrazolo[4,3-c]pyridines

Article abstract of DOI:10.1016/j.ejmech.2018.03.037

An efficient synthetic route for the synthesis of 2H-pyrazolo[4,3-c]pyridines, primarily varying by the substituents at the 2-, 4- and 6-positions, is described here. A Sonogashira-type cross-coupling reaction was employed to yield 3-alkynyl-1H-pyrazole-4-carbaldehydes, ethanones and propanones from the corresponding 1H-pyrazol-3-yl trifluoromethanesulfonates. Subsequent treatment of the coupling products with dry ammonia afforded a versatile library of 2H-pyrazolo[4,3-c]pyridines, which were then evaluated for their cytotoxicity against K562 and MCF-7 cancer cell lines. The most potent of these compounds displayed low micromolar GI₅₀ values in both cell lines. Active compounds induced dose-dependent cell-cycle arrest in mitosis, as shown by flow cytometric analysis of DNA content and phosphorylation of histone H3 at serine-10. Moreover, biochemical assays revealed increased activities of caspases-3/7 in treated cells, specific fragmentation of PARP-1, and phosphorylation of Bcl-2, collectively confirming apoptosis as the mechanism of cell death.

Full text of DOI:10.1016/j.ejmech.2018.03.037

Accepted Manuscript
Synthesis and anti-mitotic activity of 2,4- or 2,6-disubstituted- and 2,4,6-
trisubstituted-2H-pyrazolo[4,3-c]pyridines
Vaida Milišiūnaitė, Eglė Arbačiauskienė, Eva Řezníčková, Radek Jorda, Veronika
Malínková, Asta Žukauskaitė, Wolfgang Holzer, Algirdas Šačkus, Vladimír Kryštof
PII:
S0223-5234(18)30280-0
10.1016/j.ejmech.2018.03.037
EJMECH 10302
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2017
Revised Date: 10 March 2018
Accepted Date: 13 March 2018
Please cite this article as: V. Milišiūnaitė, Eglė. Arbačiauskienė, E. Řezníčková, R. Jorda, V. Malínková,
A. Žukauskaitė, W. Holzer, A. Šačkus, Vladimí. Kryštof, Synthesis and anti-mitotic activity of 2,4- or
2,6-disubstituted- and 2,4,6-trisubstituted-2H-pyrazolo[4,3-c]pyridines, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.037.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and anti-mitotic activity of 2,4- or 2,6-disubstituted- and 2,4,6-trisubstituted-2H-
pyrazolo[4,3-c]pyridines
Vaida Milišiūnaitė1, Eglė Arbačiauskienė1, Eva Řezníčková2, Radek Jorda2, Veronika
Malínková3, Asta Žukauskaitė2,3, Wolfgang Holzer4, Algirdas Šačkus1,*, Vladimír Kryštof2,**
1
Department of Organic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19, LT-
50254, Kaunas, Lithuania
2
Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and
Agricultural Research, Palacký University & Institute of Experimental Botany ASCR, Šlechtitelů
27, CZ-78371 Olomouc, Czech Republic
3
Department of Chemical Biology and Genetics, Centre of the Region Haná for
Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů
27, CZ-78371 Olomouc, Czech Republic
4
Division of Drug Synthesis, Department of Pharmaceutical Chemistry, University of Vienna,
Althanstrasse 14, A-1090 Vienna, Austria
* Corresponding author
** Corresponding author
E-mail addresses: algirdas.sackus@ktu.lt (A. Šačkus), vladimir.krystof@upol.cz (V. Kryštof)
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Abstract
An efficient synthetic route for the synthesis of 2H-pyrazolo[4,3-c]pyridines, primarily varying
by the substituents at the 2-, 4- and 6-positions, is described here. A Sonogashira-type cross-
coupling reaction was employed to yield 3-alkynyl-1H-pyrazole-4-carbaldehydes, ethanones and
propanones from the corresponding 1H-pyrazol-3-yl trifluoromethanesulfonates. Subsequent
treatment of the coupling products with dry ammonia afforded a versatile library of 2H-
pyrazolo[4,3-c]pyridines, which were then evaluated for their cytotoxicity against K562 and
MCF-7 cancer cell lines. The most potent of these compounds displayed low micromolar GI50
values in both cell lines. Active compounds induced dose-dependent cell-cycle arrest in mitosis,
as shown by flow cytometric analysis of DNA content and phosphorylation of histone H3 at
serine-10. Moreover, biochemical assays revealed increased activities of caspases-3/7 in treated
cells, specific fragmentation of PARP-1, and phosphorylation of Bcl-2, collectively confirming
apoptosis as the mechanism of cell death.
Keywords:
apoptosis; G2/M cell cycle arrest; pyrazole; structure-activity relationships
1. Introduction
Due to its wide spectrum of biological activities, pyrazole is a common structural unit in many
pharmaceuticals [1] and a central axis of numerous ongoing studies devoted to the synthesis and
biological evaluation of novel pyrazole moiety-bearing molecules [2-9]. Among the vast variety
of biologically active annelated pyrazole derivatives [10, 11], synthetically demanding 2H-
pyrazolo[4,3-c]pyridines are relatively understudied. Scarce examples of such biologically active
compounds (Fig. 1) include 3-amino-2-phenyl-2H-pyrazolo[4,3-c]pyridine-4,6-diol, which has
been revealed to act as a p90 ribosomal S6 kinase 2 (RSK2) inhibitor [12], or 2,6-diphenyl-2H-
pyrazolo[4,3-c]pyridin-3-amine,
N4-(2-methoxyethyl)-6-methyl-2-phenyl-2H-pyrazolo[4,3-
c]pyridine-3,4-diamine and 6-methyl-2-phenyl-4-(piperidin-1-yl)-2H-pyrazolo[4,3-c]pyridin-3-
amine, which have been evaluated as inhibitors against a wide range of kinases, revealing their
moderate potency against p38α, aurora A and CK1δ, respectively [13].
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Fig. 1. Examples of biologically relevant 2H-pyrazolo[4,3-c]pyridines
The synthesis of 2H-pyrazolo[4,3-c]pyridines is usually accomplished by the treatment of 4-
chloro or 4-iodopyridines possessing a carbonyl moiety at the 3-position with various hydrazines
[14-19] at room or elevated temperatures. Similarly, 4-chloronicotinonitriles react with
hydrazines in the presence of organic bases forming 2-substituted-2H-pyrazolo[4,3-c]pyridines
[18, 20]. A series of 2-phenyl-2H-pyrazolo[4,3-c]pyridines were also obtained by heating
mixtures of (E)-1-(4-azidopyridin-3-yl)-N-phenylmethanimines in toluene at 105 °C [21]. On the
other hand, we have recently demonstrated that 2H-pyrazolo[4,3-c]pyridines can also be easily
accessed from 3-hydroxy-1-phenyl-1H-pyrazole-4-carbaldehyde and the corresponding ethanone
via intermediate triflates making use of a Sonogashira cross-coupling and dry ammonia induced
cyclization reactions [22]. Therefore, in this study, we further examined the applicability of our
synthetic approach and prepared a library of various 2H-pyrazolo[4,3-c]pyridines, primarily
varying by the substituents at the 2-, 4- and 6-positions in order to assess their biological activity
and to formulate possible structure-activity relationships.
2. Results and Discussion
2.1. Synthesis
In a series of recent publications we have demonstrated that pyrazole-4-carbaldehydes carrying
an alkynyl function adjacent to the formyl moiety are valuable starting materials for the
construction of condensed pyrazole systems, for instance: dipyrazolo[1,5-a:4,3-c]pyridines [23],
2H- and 3H-pyrazolo[4’,3’:3,4]pyrido[1,2-a]benzimidazoles, 3H-pyrazolo[4,3-c]imidazo[1,2-
a:5,4-b’]dipyridines and 13,13a-dihydro-3H-pyrazolo[4’,3’:3,4]pyrido[1,2-a]pyrimidines [24]
and 2,6-diphenyl-2H-pyrazolo[4,3-c]pyridines [25, 26].
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In this study, we further elaborated the synthetic potential of 1-substituted-1H-pyrazoles carrying
an alkynyl function adjacent to the carbonyl moiety. The synthesis of starting triflates 9, 10, 15,
and 16 for the Sonogashira cross-coupling reaction is presented in Schemes 1 and 2.
Commercially available but otherwise undescribed 3-hydroxy-1-methyl-1H-pyrazole-4-
carbaldehyde 7, the precursor for the preparation of the triflate 9, was synthesized following an
analogous approach, which we have previously described for its analog 3-hydroxy-1-phenyl-1H-
pyrazole-4-carbaldehyde 8 [22] (Scheme 1). In short, we applied the Vilsmeier-Haack reaction
conditions for the synthesis of 4-benzyloxy-1-methyl-1H-pyrazole 3, which was obtained by
benzylation of the readily available starting compound 1 [25] under standard conditions with
benzyl chloride, similarly to the described procedure [27, 28]. Upon heating compound 3 with
DMF/POCl3 at 70 °C for half an hour the target carbaldehyde 5 was formed in 60% yield.
Alternatively, the preparation of carbaldehyde 5 can also be achieved by the oxidation of (3-
(benzyloxy)-1-methyl-1H-pyrazol-4-yl)methanol with manganese dioxide [29]. Debenzylation of
compound 5 was accomplished by treatment with TFA in toluene, conditions typically used for
the selective deprotection of O-benzylsalicylaldehydes [30], furnishing the target carbaldehyde 7
in 90% yield. Having precursor 7 in hand, we further treated it with triflic anhydride in the
presence of TEA in DCM [31] to obtain the corresponding triflate 9 in 85% yield. The same
synthetic pathway was applied to obtain 3-triflyloxy-1-phenyl-1H-pyrazole-4-carbaldehyde 10
from 3-hydroxy-1-phenyl-1H-pyrazole 2, as we have described previously [22].
Scheme 1. Reagents and conditions: (i) BnCl, NaH, DMF, argon atmosphere, 0-60 °C, 1 h, 90%
(for 3), 85% (for 4); (ii) POCl3, DMF, 0-60 °C, 0.5 h, 85% (for 5), 81% (for 6); (iii) TFA,
toluene, rt, 15 h, 90% (for 7), 85% (for 8); (iv) Tf2O, TEA, DCM, rt, 1 h, 85% (for 9), 83% (for
10).
Triflates 15 and 16 were synthesized similarly to the previously described approach [22]
(Scheme 2). Fries rearrangement conditions (AlCl3, SC2) were applied to 1-phenyl-1H-pyrazol-
3-yl acetate 11 [32] and the corresponding pivalate 12, which were obtained from readily
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available 3-hydroxy-1-phenyl-1H-pyrazole 2 [33] and acetic anhydride or isobutylchloride,
respectively. Triflation of 1-phenyl-1H-pyrazol-3-ols 13 [34] and 14 afforded 1-phenyl-1H-
pyrazol-3-yl trifluoromethanesulfonates 15 and 16 in 85% and 96% yield, respectively.
Scheme 2. Reagents and conditions: (i) Ac2O, 100 °C, 0.5 h, 79% (for 11); isobutylchloride,
pyridine, DCM, rt, 1 h, 90% (for 12); (ii) AlCl3, CS2, reflux, 3 h, 74% (for 13), 89% (for 14); (iii)
Tf2O, TEA, DCM, rt, 1 h, 85% (for 15), 96% (for 16).
The construction of the target pyrazolopyridines from 3-alkynylpyrazole-4-carbaldehydes,
ethanones and propanones is represented in Scheme 3. The prepared triflates 9, 10, 15 and 16
were successfully coupled with various alkyl and aryl acetylenes under standard Sonogashira
cross-coupling reaction conditions (Pd(PPh3)2Cl2, CuI, TEA, DMF) to give the corresponding 3-
alkynyl-1-phenyl-1H-pyrazoles 17-34 in good to very good yields, in most cases (Scheme 3).
Couplings of 4-acetyl-1-phenyl-1H-pyrazol-3-yl trifluoromethanesulfonate (15) with phenyl-, 3-
thienyl- and n-pentylacetylenes gave corresponding products in fair yields of less than 70%.
Notably, 1-phenyl-3-triflyl-1H-pyrazole-4-carbaldehydes resulted in better yields of the
Sonogashira cross-coupling reaction in comparison with the corresponding 4-ethanones. Finally,
compounds 17-34 bearing the alkynyl group moiety adjacent to the carbonyl group were treated
with dry ammonia under elevated temperature and pressure, allowing the direct formation of
pyrazolo[4,3-c]pyridines 35-52 generally in very good to excellent yields.
It is noteworthy that this methodology proved to be applicable for the synthesis of 2-phenyl-2H-
pyrazolo[4,3-c]pyridine 42 and its 4-methyl and 4-isopropyl derivatives, 49 and 52, respectively.
Synthesis of the aforementioned 42 has been accomplished before, employing inconvenient
highly toxic and unstable reactants, i.e., by cycloaddition of 3-pyridyne, which is accessible by
lead tetraacetate oxidation of 1-aminotriazolo[4,5-c]pyridine to N-phenylsydnone [35]. In
contrast, our synthetic approach makes use of the convenient TMS-protected, commercially
available, trimethylsilylacetylene, which efficiently undergoes Sonogashira cross-coupling with
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corresponding triflates 10, 15, and 16, furnishing 24, 31, and 34 in 78-87% yield. The latter, due
to the convenient lability of TMS protecting group, upon treatment with dry ammonia under
elevated temperature and pressure directly gives rise to the target TMS-deprotected 2-phenyl-
2H-pyrazolo[4,3-c]pyridines 42, 49 and 52 in 88-95% yield (Scheme 3).
Scheme 3. Reagents and conditions: (i) R3-C≡CH, Pd(PPh3)2Cl2, TEA, CuI, DMF, 60 °C, 1-38
h; (ii) NH3, MeOH, 120 °C, 15 h.
2.2. Anticancer activity in vitro
Prepared compounds 35-52 were evaluated for their cytotoxicity against two human cancer cell
lines: K562 (chronic myeloid leukemia cells) and MCF-7 (breast cancer cells). In general, most
tested compounds exhibited moderate cytotoxicity, with GI50 values in the micromolar range
(Table 1). The most potent derivatives bear a phenyl moiety at the 2-position, with no substituent
at the 4-position and either an aryl or alkyl at the 6-position (compounds 36, 37, 39, 40). It is
noteworthy that replacing a phenyl substituent at the 2-position (compound 36) with a methyl
group resulted in a complete loss of the activity (compound 35). Increasing the bulkiness of the
substituents at the 4-position reduced cytotoxicity of the derivatives, i.e., introducing methyl
(compounds 43, 47) and isopropyl (compound 50) substituents resulted in a gradual decrease in
potency of the compounds compared to unsubstituted derivatives (compounds 36, 40).
Replacement of the substituents at the 6-position enabled the fine-tuning of cytotoxicity of the
compounds. Surprisingly, a lack of the substituent at this position or introduction of a polar
ethylhydroxy group resulted in completely inactive derivatives (compounds 41, 42, 48, 49),
while various other substituents were relatively well tolerated. The most effective substituents at
6-position were alkyl chains and aromatic rings. Derivatives bearing a phenyl ring at the 7-
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position exhibited good cytotoxic values, with GI50 values for compound 36 reaching 3.4 µM for
K562 and 4.8 µM for MCF-7 cells. Unfortunately, attempts to replace the phenyl ring with a
slightly less aromatic thiophene or aliphatic cyclopropyl rings resulted in less active derivatives;
phenyl derivatives 36 and 43 were twofold more potent than analogous thiophen-3-yl derivatives
37 and 44. Interestingly, replacing a cyclopropyl ring with linear aliphatic substituents resulted in
an increased cytotoxicity of the compounds. Thus, out of the prepared derivatives, compound 40,
bearing a pentyl substituent at the 6-position, a phenyl ring at the 2-position and lacking a
substituent at the 4-position, proved to be the most cytotoxic, reaching a GI50 value of 2.1 µM for
K562 cells.
Table 1. In vitro cytotoxicity of previously and newly synthesized 2H-pyrazolo[4,3-c]pyridines
35-52.
GI50 (µM)*
MCF-7
General
Compound
R1
R2
R3
structure
K562
Me
H
>100
>100
4.8
35
36
37
38
39
40
41
42
43
44
H
3.4
H
8.3
9.8
H
60.5
6.7
69.3
13.3
11.8
>100
>100
11.0
22.2
H
H
2.1
H
>100
>100
6.3
H
H
Me
Me
10.0
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Me
Me
Me
Me
25.9
8.5
63.5
11.1
17.0
>100
>100
91.0
33.3
98.8
45
46
47
48
49
50
51
52
6.9
>100
>100
36.8
12.9
>100
Me
i-Pr
i-Pr
i-Pr
H
H
* Data are means of at least two independent measurements.
2.3. Effect on the cell cycle and apoptosis
All compounds displaying a GI50 lower than 80 µM in at least one cell line underwent cell-cycle
investigation in K562 and MCF-7 cell lines to gain preliminary information about their
mechanism of action. The cells were treated with the tested compounds for 24 hours at 10 µM
concentrations. Tubuline-interfering agent nocodazole and cyclin-dependent kinase 4 inhibitor
palbociclib were used as positive controls, causing dominant mitotic and G1 arrest, respectively.
The majority of compounds exhibited a clear effect on the cell cycle in both cell lines; with
increased cell populations in G2/M phases and corresponding decreases in G1 and S phase
populations observed post treatment (Table 2). The highest percentages of a G2/M population
were found in cultures treated with compounds 36, 39, 40, 43 and 47, all of which also displayed
the strongest cytotoxicities (Table 1). In addition, a substantial increase of sub-G1 populations
was observed in cultures treated with the most potent compounds 36, 37, 39, 40, 43, 44, 46 and
51, indicating ongoing apoptosis. Sub-G1 populations were usually higher in K562 cells,
probably as a consequence of their higher sensitivity to novel compounds, which was observed
in the cytotoxicity assays (Table 1).
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Table 2. Cell cycle analysis in K562 and MCF-7 cells treated with active 2-phenyl-2H-
pyrazolo[4,3-c]pyridines at a single dose of 10 µM.
Compound
K562 cell cycle phases (%)
MCF-7 cell cycle phases (%)
subG1
15.0
43.0
34.2
19.6
37.7
35.4
46.2
45.7
15.3
40.8
28.3
21.3
47.1
49.4
35.0
G1
S
G2/M
15.0
74.7
41.1
15.5
62.3
82.0
50.2
44.6
19.6
41.4
49.9
18.3
29.0
69.7
15.9
subG1
7.1
G1
S
G2/M
5.3
Untreated
34.6
19.8
21.7
32.3
24.8
14.8
13.7
26.2
34.7
28.2
21.8
35.1
26.7
3.6
50.3
5.5
60.7
56.8
39.5
64.7
36.0
18.1
65.5
37.2
63.6
48.5
50.2
62.6
66.1
14.4
90.0
34.1
26.2
29.1
29.4
30.3
26.3
20.5
31.2
30.2
24.0
18.1
30.6
24.5
49.9
5.5
14.3
24.2
7.5
17.0
31.4
5.9
36
37.3
52.2
12.8
3.3
37
38
31.1
50.3
11.9
22.7
9.0
33.7
55.6
14.1
31.6
6.3
39
40
36.1
29.2
45.8
30.5
28.3
46.6
44.2
26.8
30.1
43
44
45
19.3
31.1
6.7
27.5
31.7
6.9
46
47
50
7.8
9.4
51
nocodazole (25 ng/mL)
palbociclib (0.25 µM)
32.1
7.5
35.7
4.6
54.0
Compound 40, with the strongest effect on the cell cycle, was further assayed at several
concentrations (Fig. 2a). DNA histograms revealed a clear dose-dependent arrest in G2/M
phases. To discriminate between arrest in G2 and M, we quantified the phosphorylation of
histone H3 at serine-10, a common mitotic marker. Flow cytometric analysis revealed a strong
accumulation of cells with phosphorylated histone, confirming arrest in mitosis (Fig. 2b).
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Fig. 2. Cell cycle arrest in K562 cells treated with compound 40 for 24 h. (a) DNA histograms of
cells treated with different doses of the compound 40. (b) Phosphorylation of histone H3 at
serine-10 in cells treated with 5 µM dose of compound 40. Nocodazole was used as a positive
control.
Due to the strong cytotoxicity of 40 in the K562 cell line, we sought to identify the type of cell
death that occurs. Caspase activation in treated cells was measured by an enzymatic assay, using
the fluorescently labeled peptide substrate Ac-DEVD-AMC of caspases 3 and 7 (Fig. 3a) which
revealed a clear dose-dependent responses in the micromolar range. In parallel, lysates of treated
K562 and MCF-7 cells were subjected to immunoblotting, the analysis of which revealed a dose-
dependent increase in the 89 kDa fragment of PARP-1, a known caspase substrate (Fig. 3b).
Phosphorylation of histone H2AX at Ser-139 (γH2AX), a modification required for DNA
fragmentation during apoptosis [36], was also detected. Additionally, we observed the
appearance of a slowly migrating, phosphorylated form of Bcl-2 connected with G2/M arrest of
the cell cycle and apoptosis [37]. In summary, the analyses performed confirmed that compound
40 can activate apoptotic machinery in a dose-dependent manner.
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Fig. 3. Induction of apoptosis in cells treated with different doses of compound 40 for 24 h. (a)
Caspase-3/7 activity was measured in lysates prepared from treated K562 cells using the
fluorogenic substrate Ac-DEVD-AMC and normalized against untreated control cell lysates. (b)
Immunoblotting analysis of apoptosis-related proteins in treated K562 and MCF-7 cells.
Doxorubucin (DOX, 1 µM) was used as a control. Actin levels were detected to verify equal
loading.
To describe the mechanism of antimitotic action of the prepared compounds, we first explored
CDK1/cyclin B, a well-known mitotic regulator, as a possible target, especially due to the
structural similarity to 2,3,4,6-substituted pyrazolo[4,3-c]pyridines which have been identified as
kinase inhibitors [12, 13]. The synthesized compounds 35-52 were screened for their inhibitory
activity against CDK1, but the assay did not reveal any inhibition (data not shown). Further
profiling of compound 40 against other mitotic kinases, including DAPK3, CDK1, CHK1,
NIM1, AURKA, NDR1, NEK2/4/6, PIM2, PLK2/3, TTK and WEE1, did not point to any of
these kinases as a target of compound 40 (data not shown). This finding was expected given that
these compounds lack an H-bond donor-acceptor motif which is common in most kinase
inhibitors, including the previously mentioned pyrazolo[4,3-c]pyridines [12, 13].
3. Conclusion
We detail the development of an efficient approach for the synthesis of variously substituted 2H-
pyrazolo[4,3-c]pyridines, employing Sonogashira cross-coupling and a subsequent substituent-
tolerant annulation reaction in the presence of ammonia. As a result of this, a library of 2H-
pyrazolo[4,3-c]pyridines, varying the substituents at the 2-, 4-, and 6-positions, was synthesized.
These compounds were also evaluated for their cytotoxicity against K562 and MCF-7 cancer cell
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lines. The tested compounds exhibited anticancer activity in vitro through arresting cell cycle in
mitosis and induction of apoptosis, although, the mechanism of cellular action remains unclear.
4. Experimental Section
4.1. Chemistry
All starting materials were obtained from commercial suppliers and used without further
purification. Melting points were determined on a Büchi M-565 melting point apparatus and
were uncorrected. Mass spectra were obtained on a Shimadzu LCMS 2020 Single Quadrupole
Liquid Chromatograph Mass Spectrometer. IR spectra in KBr pellets were recorded on a Bruker
Tensor 27 spectrometer and are reported in wave numbers (cm–1). High-resolution ESI-TOF
mass spectra were measured on a Bruker maXis spectrometer. 1H NMR, 13C NMR and 15N NMR
spectra were recorded from CDCl3 or DMSO-d6 solutions at 25 °C on a Bruker Avance III 700
1
1
instrument (700 MHz for H, 176 MHz for 13C) equipped with a 5 mm TCI H-13C/15N/D z-
gradient cryoprobe. The solvent (residual) signals were used as internal standards and were
related to TMS, with δ 7.26 ppm (1H) and δ 77.00 ppm (13C) for CDCl3 and with 2.50 ppm (1H)
and δ 39.52 ppm (13C) for DMSO-d6. 15N NMR spectra were referenced against neat, external
15
nitromethane. The full and unambiguous assignments of 1H, 13C and N NMR resonances were
achieved using combined applications of standard NMR spectroscopic techniques such as APT,
COSY, TOCSY, NOESY, gs-HSQC and gs-HMBC. For chromatographic separations, silica gel
60 (230–400 mesh, Merck) was used.
4.1.1. 3-(Benzyloxy)-1-methyl-1H-pyrazole (3) [27, 28]. A solution of 3-hydroxy-1-methyl-1H-
pyrazole (1) [38] (710 mg, 7.2 mmol) in dry DMF (20 mL) was cooled to 0 ºC under inert
atmosphere and NaH (60% dispersion in mineral oil, 290 mg, 7.2 mmol) was added portion wise.
After stirring mixture for 15 min benzyl chloride (0.82 mL, 7.2 mmol) was added drop wise. The
mixture was stirred at 60 ºC for 1 hour, then poured into water and extracted with ethyl acetate.
The organic layers were combined, washed with brine, dried over Na2SO4, filtrated, and the
solvent was evaporated. The residue was purified by column chromatography (SiO2, eluent: ethyl
acetate/n-hexane, 1:7, v/v) to give pure 3 as a brown liquid. Yield 90%, 1205 mg. IR (νmax, cm-1):
3118, 3089, 3064, 3032, 3008 (CHarom), 2931 (CHaliph), 1537, 1491, 1429, 1360, 1223, 1052,
1
1018 (C=C, C–N), 731, 696, 658, 457 (CH=CH of monosubstituted benzene). H NMR (700
3
MHz, CDCl3): δ 3.75 (s, 3H, CH3), 5.20 (s, 2H, CH2Ph), 5.66 (d, J(4-H,5-H) = 2.4 Hz, 1H, 4-
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3
H), 7.13 (d, J(5-H,4-H) = 2.4 Hz, 1H, 5-H), 7.30-7.33 (m, 1H, Ph 4-H), 7.36-7.39 (m, 2H, Ph
3,5-H), 7.45-7.48 (m, 2H, Ph 2,6-H). 13C NMR (176 MHz, CDCl3): δ 39.0 (CH3), 70.9 (CH2Ph),
90.5 (C-4), 127.8 (Ph C-2,6), 128.0 (Ph C-4), 128.5 (Ph C-3.5), 131.4 (C-5), 137.3 (Ph C-1),
163.3 (C-3). MS m/z (%): 189 ([M+H]+, 100). HRMS (ESI) for C11H12N2ONa ([M+Na]+) calcd
211.0842, found 211.0842.
4.1.2. 3-(Benzyloxy)-1-methyl-1H-pyrazole-4-carbaldehyde (5). Phosphorus oxychloride (1.59
mL, 17 mmol) was added dropwise to DMF (1.32 mL, 17 mmol) at –10 °C. Then 3 (800 mg,
4.25 mmol) was added to the Vilsmeier-Haack complex and the reaction mixture was heated at
70 °C for 12 hours. After neutralization with 10% aq NaHCO3, the preticipate was filtered off
and recrystallized from DCM to give pure 5 as a white solid. Yield 85%, 775 mg, mp 54-56 °C.
IR (νmax, cm-1): 3121, 3099, 3045 (CHarom), 2992, 2947 (CHaliph), 1666 (C=O), 1588, 1577, 1541,
1
1510, 1315, 1181 (C=C, C–N), 894, 883, 706 (CH=CH of monosubstituted benzene). H NMR
(700 MHz, CDCl3): δ 3.77 (s, 3H, CH3), 5.32 (s, 2H, CH2Ph), 7.32-7.36 (m, 1H, Ph 4-H), 7.36-
7.40 (m, 2H, Ph 3,5-H), 7.46-7.47 (m, 2H, Ph 2,6-H), 7.69 (s, 1H, 5-H), 9.76 (s, 1H, CHO). 13C
NMR (176 MHz, CDCl3): δ 39.8 (CH3), 71.0 (CH2Ph), 109.6 (C-4), 128.0 (Ph C-2,6), 128.3 (Ph
C-4), 128.6 (Ph C-3.5), 133.4 (C-5), 136.4 (Ph C-1), 163.3 (C-3), 183.1 (CHO). MS m/z (%):
217 ([M+H]+, 100). HRMS (ESI) for C11H12N2ONa ([M+Na]+) calcd 239.0791, found 239.0791.
4.1.3. 3-Hydroxy-1-methyl-1H-pyrazole-4-carbaldehyde (7). To a solution of 5 (570 mg, 2.6
mmol) in toluene (10 mL) TFA (10 mL) was added. The mixture was stirred at room temperature
for 48 hours. Toluene and TFA were evaporated. The residue was purified by column
chromatography (SiO2, eluent: ethyl acetate/n-hexane, 1:1, v/v) to give pure 7 as a colorless
solid. Yield 90%, 295 mg, mp 201-202 °C. IR (νmax, cm-1): 3291 (OH), 3099 (CHarom), 2992,
1
2947 (CHaliph), 1666 (C=O), 1541, 1510, 1315, 1181 (C=C, C–N). H NMR (700 MHz, DMSO-
d6): δ 3.67 (s, 3H, CH3), 8.04 (s, 1H, 5-H), 9.60 (s, 1H, CHO), 10.90 (s, 1H, OH). 13C NMR (176
MHz, DMSO-d6): δ 38.9 (CH3), 108.4 (C-4), 134.0 (C-5), 161.9 (C-3), 182.6 (CHO). MS m/z
(%): 127 ([M+H]+, 100). HRMS (ESI) for C5H6N2O2Na ([M+Na]+) calcd 149.0322, found
149.0321.
4.1.4. 4-Formyl-1-methyl-1H-pyrazol-3-yl trifluoromethanesulfonate (9). Pyrazole 7 (250
mg, 2 mmol), trifluormethansulfonic anhydride (1 mL, 6 mmol) and TEA (1 mL, 7.2 mmol)
were dissolved in DCM (20 mL) and the mixture was stirred at room temperature for 1 hour. The
reaction mixture was poured into water and extracted with ethyl acetate. Combined organic
13

ACCEPTED MANUSCRIPT
layers were washed with brine and dried over Na2SO4, the solvent was evaporated. The residue
was purified by flash chromatography (SiO2, eluent: ethyl acetate/n-hexane, 1:4, v/v) to give 9 as
a brown solid. Yield 85%, 435 mg, mp 54-55 °C. IR (νmax, cm-1): 3091, 3043, 3017 (CHarom),
2958, 2918 (CHaliph), 1675 (C=O), 1552, 1429, 1205, 1135, 884, 875, 794, 744, 601, 511 (C-C,
1
C-N, C-F). H NMR (700 MHz, CDCl3): δ 3.93 (s, 3H, CH3), 7.87 (s, 1H, 5-H), 9.80 (s, 1H,
CHO). 13C NMR (176 MHz, CDCl3): δ 40.7 (CH3), 113.6 (C-4), 116.0, 117.8, 119.6, 121.5 (CF3
1J = 321.2 Hz), 134.7 (C-5), 151.2 (C-3), 181.0 (CHO). MS m/z (%): 259 ([M+H]+, 100). HRMS
(ESI) for C6H5F3N2O4SNa ([M+Na]+) calcd 280.9814, found 280.9814.
4.1.5. 1-Phenyl-1H-pyrazol-3-yl isobutyrate (12). To a solution of 3-hydroxy-1-phenyl-1H-
pyrazole (2) [33] (470 mg, 2.9 mmol) in DCM (10 mL) pyridine (2.3 mL) and isobutyrylchloride
(0.33 mL, 3.2 mmol) were added. The mixture was stirred at room temperature for 30 min then
poured into water and extracted with DCM. The organic layers were combined, washed with
brine, dried over Na2SO4, filtrated, the solvent was evaporated. The residue was purified by
column chromatography (SiO2, eluent: ethyl acetate/n-hexane, 1:4, v/v) to give pure 12 as a
brown liquid. Yield 90%, 601 mg. IR (νmax, cm-1): 3134, 3064, 3050 (CHarom), 2977, 2938, 2912
(CHaliph), 1761 (C=O), 1599, 1532, 1453, 1391, 1124, 1113, 1092 (C=C, C–N), 752, 688
1
(CH=CH of monosubstituted benzene). H NMR (700 MHz, CDCl3): δ 1.36 (d, J = 7.1 Hz, 6H,
3
CH(CH3)2), 2.85-2.90 (m, 1H, CH(CH3)2), 6.41 (d, J(4-H,5-H) = 2.6 Hz, 1H, 4-H), 7.28-7.30
(m, 1H, Ph 4-H), 7.44-7.46 (m, 2H, Ph 3,5-H), 7.64-7.66 (m, 2H, Ph 2,6-H), 7.86 (d, 3J(5-H,4-H)
= 2.6 Hz, 1H, 5-H). 13C NMR (176 MHz, CDCl3): δ 18.8 (CH(CH3)2), 34.1 (CH(CH3)2), 98.8 (C-
4), 118.7 (Ph C-2,6), 126.5 (C-5), 127.7 (Ph C-4), 129.4 (Ph C-3,5), 139.7 (Ph C-1), 156.8 (C-3),
174.2 (O-C=O). MS m/z (%): 231 ([M+H]+, 100). HRMS (ESI) for C13H14N2O2Na ([M+Na]+)
calcd 253.0947, found 253.0947.
4.1.6. 1-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-2-methylpropan-1-one (14). To a solution of
AlCl3 (2.453 g, 18.4 mmol) in CS2 (8 mL) a solution of 12 (350 mg, 1.5 mmol) in CS2 (28 mL)
was added drop wise at 0 °C. The mixture was stirred at 55 °C for 3 hours. After neutralization
with ice-cold water (33 mL) and 6N HCl (15 mL), the preticipate was filtered off and purified by
flash chromatography (SiO2, eluent: ethyl acetate/n-hexane, 1:4, v/v) to give pure 10 as a white
solid. Yield 89%, 310 mg, mp 97-98 °C. IR (νmax, cm-1): 3309 (OH), 3122, 3068, 3052 (CHarom),
2965, 2932 (CHaliph), 1656 (C=O), 1559, 1530, 1508, 1459, 1319, 1233, 1217 (C=C, C–N), 747,
685, 671 (CH=CH of monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 1.07 (d, J = 6.9
14

ACCEPTED MANUSCRIPT
Hz, 6H, CH(CH3)2), 3.33-3.36 (m, 1H, CH(CH3)2), 7.29-7.31 (m, 1H, Ph 4-H), 7.47-7.49 (m, 2H,
Ph 3,5-H), 7.80-7.81 (m, 2H, Ph 2,6-H), 8.87 (s, 1H, 5-H), 11.12 (s, 1H, OH). 13C NMR (176
MHz, CDCl3): δ 18.7 (CH(CH3)2), 36.9 (CH(CH3)2), 109.4 (C-4), 118.1 (Ph C-2,6), 126.5 (Ph C-
4), 129.6 (Ph C-3,5), 131.4 (C-5), 138.9 (Ph C-1), 161.4 (C-3), 198.6 (C=O). MS m/z (%): 231
([M+H]+, 100). HRMS (ESI) for C13H14N2O2Na ([M+Na]+) calcd 253.0947, found 253.0947.
4.1.7. 4-Isobutyryl-1-phenyl-1H-pyrazol-3-yltrifluoromethanesulfonate (16). This compound
was synthesized in analogy to compound 9 from pyrazole 14 (252 mg, 2 mmol). 16 was obtained
as a white solid. Yield 96%, 495 mg, mp 96-97 °C. IR (νmax, cm-1): 3094, 3061 (CHarom), 2980,
2937, 2918 (CHaliph), 1555, 1452, 1427, 1231, 1213, 1201, 1138 (C=C, C–N, C-F), 973, 881,
1
760, 740, 607, 599, 505 (CH=CH of monosubstituted benzene). H NMR (700 MHz, CDCl3): δ
1.23-1.24 (m, 6H, CH(CH3)2), 3.15-3.18 (m, 1H, CH(CH3)2), 7.39-7.41 (m, 1H, Ph 4-H), 7.49-
7.51 (m, 2H, Ph 3,5-H), 7.66-7.67 (m, 2H, Ph 2,6-H), 8.35 (s, 1H, 5-H). 13C NMR (176 MHz,
CDCl3): δ 18.7 (CH(CH3)2), 38.8 (CH(CH3)2), 113.6 (C-4), 116.0, 117.8, 119.5 (Ph C-2,6),
1
119.6, 121.5 (CF3 J = 322.1 Hz), 128.5 (Ph C-4), 129.9 (Ph C-3,5), 130.8 (C-5), 138.5 (Ph C-1),
151.9 (C-3), 196.6 (C=O). MS m/z (%): 363 ([M+H]+, 100). HRMS (ESI) for C6H5F3N2O4SNa
([M+Na]+) calcd 385.0440, found 385.0440.
4.1.8. General procedure for the preparation of 4-alkynyl-1-phenyl-1H-pyrazole-4-
carbaldehydes, ethanones and propanones by Sonogashira cross-coupling reaction.
To a solution of appropriate pyrazole 9, 10, 15 or 16 (0.5 mmol) in dry DMF (1 mL) under argon
atmosphere TEA (4.0 mL, 2.5 mmol), appropriate acetylene (0.75 mmol), Pd(PPh3)2Cl2 (35 mg,
0.05 mmol) and CuI (18 mg, 0.1 mmol) were added. The mixture was stirred for the given time
under argon atmosphere at 70 °C, diluted with water, and the extraction was done with ethyl
acetate. The combined organic layers were washed with brine, dried over Na2SO4, and the
solvent was evaporated. The residue was purified by flash chromatography (SiO2, eluent: ethyl
acetate/n-hexane, 1:8, v/v) to yield compounds 17-34.
4.1.8.1. 1-Methyl-3-(phenylethynyl)-1H-pyrazole-4-carbaldehyde (17). The reaction mixture
was stirred for 8 hours. Brown amorphous solid, yield 75%, 105 mg, mp 123-124 °C. IR (νmax
,
cm-1): 3100, 3057, 3042 (CHarom), 2954, 2925 (CHaliph), 2221 (C≡C), 1681 (CHO), 1598, 1560,
1533, 1497, 1441, 1181 (C=C, C–N), 909, 880, 775, 755, 690 (CH=CH of monosubstituted
benzene). 1H NMR (700 MHz, CDCl3): δ 3.85 (s, 3H, CH3), 7.24-7.7(m, 3H, Ph 3,4,5-H), 7.46-
13
7.47 (m, 2H, Ph 2,6-H), 7.90 (s, 1H, 5-H), 9.88 (s, 1H, CHO). C NMR (176 MHz, CDCl3): δ
15

ACCEPTED MANUSCRIPT
40.1 (CH3), 79.0 (C≡C-Ph), 94.1 (C≡C-Ph), 113.6 (C-3), 122.0 (Ph C-1), 125.0 (C-4), 128.6 (Ph
C-2,6), 134.8 (C-5), 184.3 (CHO). MS m/z (%): 211 ([M+H]+, 100). HRMS (ESI) for
C16H10N2OSNa ([M+Na]+) calcd 233.0685, found 233.0685.
4.1.8.2. 1-Phenyl-3-(thiophen-3-ylethynyl)-1H-pyrazole-4-carbaldehyde (19). The reaction
mixture was stirred for 8 hours. White solid, yield 75%, 105 mg, mp 123-124 °C. IR (νmax, cm-1):
3126, 3036 (CHarom), 2834, 2788 (CHaliph), 2229 (C≡C), 1676 (C=O), 1596, 1561, 1462, 1409,
1297, 1125 (C=C, C–N), 909, 873, 815, 621, 598, 512 (CH=CH of monosubstituted benzene). 1H
NMR (700 MHz, CDCl3): δ 7.28 (d, 3J(4-H,5-H) = 4.9 Hz, 1H, Th 4-H), 7.34 (dd, 3J(5-H,4-H) =
4
4
4.9 Hz, J(5-H,2-H) = 2.9 Hz, 1H, Th 5-H), 7.40-7.42 (m, 1H, Ph 4-H), 7.68 (d, J(2-H,5-H) =
2.9 Hz, 1H, Th 2-H), 7.50-7.52 (m, 2H, Ph 3,5-H), 7.74-7.76 (m, 2H, Ph 2,6-H), 8.44 (s, 1H, 5-
13
H), 10.09 (s, 1H, CHO). C NMR (176 MHz, CDCl3): δ 78.6 (C≡C-Th), 90.3 (C≡C-Th), 120.0
(Ph C-2,6), 120.9 (Th C-3), 125.91 (C-4), 125.95 (Th C-5), 128.5 (Ph C-4), 128.7 (C-5), 129.9
(Ph C-3,5), 130.0 (Th C-4), 130.8 (Th C-2), 138.7 (C-3), 138.9 (Ph C-1), 182.7 (CHO). 15N
NMR (71 MHz, CDCl3): δ −158.2 (N-1), −72.0 (N-2). MS m/z (%): 279 ([M+H]+, 100). HRMS
(ESI) for C16H10N2OSNa ([M+Na]+) calcd 301.0406, found 301.0406.
4.1.8.3. 3-(Cyclopropylethynyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (20). The reaction
mixture was stirred for 1 hour. White solid, yield 86%, 103 mg, mp 90-91 °C. IR (νmax, cm-1):
3121, 3065, 3014 (CHarom), 2881, 2782 (CHaliph), 2232 (C≡C), 1677 (C=O), 1599, 1530, 1504,
1361, 1226 (C=C, C–N), 865, 849, 801, 757, 704, 685, 506 (CH=CH of monosubstituted
1
benzene). H NMR (700 MHz, CDCl3): δ 0.92-0.98 (m, 4H, CH(CH2)2, 1.52-1.57 (m, 1H,
CH(CH2)2), 7.37-7.41 (m, 1H, Ph 4-H), 7.47-7.52 (m, 2H, Ph 3,5-H), 7.70-7.73 (m, 2H, Ph 2,6-
13
H), 8.37 (s, 1H, 5-H), 9.97 (s, 1H, CHO). C NMR (176 MHz, CDCl3): δ 0.4 (CH(CH2)2), 9.1
(CH(CH2)2), 65.5 (C≡C(CH(CH2)2), 100.2 (C≡C(CH(CH2)2), 119.9 (Ph C-2,6), 125.9 (C-4),
128.3 (Ph C-4), 128.4 (C-5), 129.8 (Ph C-3,5), 138.9 (C-3), 139.3 (Ph C-1), 184.8 (CHO). 15N
NMR (71 MHz, CDCl3): δ −157.6 (N-1), N-2 was not found. MS m/z (%): 237 ([M+H]+, 100).
HRMS (ESI) for C15H12N2ONa ([M+Na]+) calcd 359.0842, found 359.0844.
4.1.8.4. 3-(Hept-1-yn-1-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (22). The reaction mixture
was stirred for 4 hours. Brown liquid, yield 92%, 128 mg. IR (νmax, cm-1): 3124, 3053 (CHarom),
2956, 2931, 2860 (CHaliph), 2243 (C≡C), 1684 (C=O), 1599, 1531, 1504, 1464, 1398, 1296
1
(C=C, C–N), 956, 757, 689, 509 (CH=CH of monosubstituted benzene). H NMR (700 MHz,
CDCl3): δ 0.89-0.93 (m, 3H, C4H8CH3), 1.34-1.39 (m, 2H, C3H6CH2CH3), 1.44-1.48 (m, 2H,
16

ACCEPTED MANUSCRIPT
C2H4CH2C2H5), 1.65-1.69 (m, 2H, CH2CH2C3H7), 2.48-2.51 (m, 2H, CH2C4H9), 7.36-7.39 (m,
1H, Ph 4-H), 7.46-7.49 (m, 2H, Ph 3,5-H), 7.69-7.72 (m, 2H, Ph 2,6-H), 8.38 (s, 1H, 5-H), 9.99
(s, 1H, CHO). 13C NMR (176 MHz, CDCl3): δ 14.0 (C4H8CH3), 19.5 (CH2C4H9), 22.2
(C3H6CH2CH3), 28.0 (CH2CH2C3H7), 31.1 (C2H4CH2C2H5), 70.3 (C5H11C≡C) 97.2 (C5H11C≡C),
119.8 (Ph C-2,6), 125.7 (C-4), 128.2 (Ph C-4), 128.3 (C-5), 129.7 (Ph C-3,5), 138.8 (Ph C-1),
139.3 (C-3), 184.8 (CHO). 15N NMR (71 MHz, CDCl3): δ –158.6 (N-1), –72.4 (N-2). MS m/z
(%): 267 ([M+H]+, 100). HRMS (ESI) for C17H18N2ONa ([M+H]+) calcd 289.1306, found
289.1311.
4.1.8.5. 3-(4-Hydroxybut-1-yn-1-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (23). The
reaction mixture was stirred for 4 hours. White solid, yield 90%, 108 mg, mp 111-112 °C. IR
(νmax, cm-1): 3454 (OH), 3126, 30965 (CHarom), 2929 (CHaliph), 2240 (C≡C), 1679 (C=O), 1598,
1531, 1504, 1462, 1402, 1362, 1315, 1226, 1051 (C=C, C–N), 866, 812, 796, 755, 685 (CH=CH
of monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 2.74-2.76 (m, 2H,
C≡CCH2CH2OH), 3.87-3.88 (m, 2H, C≡CCH2CH2OH), 7.35-7.37 (m, 1H, Ph 4-H), 7.44-7.47
(m, 2H, Ph 3,5-H), 7.66-7.68 (m, 2H, Ph 2,6-H), 8.37 (s, 1H, 5-H), 9.95 (CHO). 13C NMR (176
MHz, CDCl3): δ 24.1(C≡CCH2CH2OH), 60.8 (C≡CCH2CH2OH), 72.4 (C≡CCH2CH2OH), 94.0
(C≡CCH2CH2OH), 119.8 (NPh C-2,6), 125.8 (C-4), 128.4 (Ph C-4), 129.5 (Ph C-3,5), 129.8 (C-
5), 138.1 (C-3), 138.7 (Ph C-1), 184.8 (CHO). MS m/z (%): 241 ([M+H]+, 100), 263 ([M+Na]+,
30). HRMS (ESI) for C14H12N2ONa ([M+Na]+) calcd 263.0792, found 263.0791.
4.1.8.6. 1-Phenyl-3-((trimethylsilyl)ethynyl)-1H-pyrazole-4-carbaldehyde (24). The reaction
mixture was stirred for 10 hours. White solid, yield 78%, 105 mg, mp 97-98 °C. IR (νmax, cm-1):
3128, 3067 (CHarom), 2960, 2854 (CHaliph), 2167 (C≡C), 1678 (C=O), 1598, 1531, 1503, 1363,
1
1251, 1223 (C=C, C–N), 956, 838, 751, 726, 683 (CH=CH of monosubstituted benzene). H
NMR (700 MHz, CDCl3): δ 0.30 (s, 9H, TMS), 7.37-7.41 (m, 1H, Ph 4-H), 7.47-7.51 (m, 2H, Ph
3,5-H), 7.70-7.72 (m, 2H, Ph 2,6-H), 8.39 (s, 1H, 5-H), 10.01 (s, 1H, CHO). 13C NMR (176
MHz, CDCl3): δ 0.2 (TMS), 93.6 (TMSC≡C), 102.0 (TMSC≡C), 120.1 (Ph C-2,6), 126.2 (C-4),
128.3 (C-5), 128.5 (Ph C-4), 129.9 (Ph C-3,5), 138.7 (C-3), 138.8 (Ph C-1), 184.7 (CHO). 15N
NMR (71 MHz, CDCl3): δ –157 (N-1), N-2 was not found. MS m/z (%): 269 ([M+H]+, 100).
HRMS (ESI) for C15H16N2OSiNa ([M+Na]+) calcd 291.0924, found 291.0928.
4.1.8.7. 1-(1-Phenyl-3-(thiophen-3-ylethynyl)-1H-pyrazol-4-yl)ethanone (26). The reaction
mixture was stirred for 8 hours. White solid, yield 66%, 97 mg, mp 116-117 °C. IR (νmax, cm-1):
17

ACCEPTED MANUSCRIPT
3117, 3054, 3022 (CHarom), 2996, 2955, 2917 (CHaliph), 2232 (C≡C), 1655 (C=O), 1597, 1514,
1508, 1351, 1264, 1232, 1058 (C=C, C–N), 872, 774, 761, 708, 691, 680, 620, 503 (CH=CH of
1
monosubstituted benzene). H NMR (700 MHz, CDCl3): δ 2.73 (s, 3H, CH3), 7.28 (d, 3J(4-H,5-
H) = 5.2 Hz, 1H, Th 4-H), 7.35-7.37 (m, 1H, Th 5-H), 7.39-7.42 (m, 1H, Ph 4-H), 7.50-7.52 (m,
2H, Ph 3,5-H), 7.68 (d, 4J(2-H,5-H) = 2.9 Hz, 1H, Th 2-H), 7.75-7.77 (m, 2H, Ph 2,6-H), 8.47 (s,
13
1H, 5-H). C NMR (100 MHz, CDCl3): δ 29.1 (CH3), 80.8 (ThC≡C), 90.1 (ThC≡C), 119.7 (Ph
C-2,6), 121.1 (Th C-3), 125.8 (Th C-5), 126.9 (C-4), 128.1 (Ph C-4), 129.7 (Th C-4), 129.7 (Ph
C-3,5), 129.9 (C-5), 130.2 (Th C-2), 136.2 (C-3), 138.8 (Ph C-1), 192.2 (CO). 15N NMR (71
MHz, CDCl3): δ −160.4 (N-1), −72.5 (N-2). MS m/z (%): 293 ([M+H]+, 100). HRMS (ESI) for
C17H12N2OSiNa ([M+Na]+) calcd 315.0563, found 315.0561.
4.1.8.8. 1-(3-(Cyclopropylethynyl)-1-phenyl-1H-pyrazol-4-yl)ethanone (27). The reaction
mixture was stirred for 1 hour. White solid, yield 80%, 100 mg, mp 127-128 °C. IR (νmax, cm-1):
3129, 3078 (CHarom), 2993, 2956, 2923 (CHaliph), 2233 (C≡C), 1670 (C=O), 1599, 1521, 1448,
1362, 1260, 1241, 1221 (C=C, C–N), 977, 940, 863, 751, 705, 683 (CH=CH of monosubstituted
1
benzene). H NMR (700 MHz, CDCl3): δ 0.90-0.96 (m, 4H, CH(CH2)2), 1.53-1.57 (m, 1H,
CH(CH2)2), 2.62 (s, 3H, COCH3), 7.34-7.36 (m, 1H, Ph 4-H), 7.45-7.47 (m, 2H, Ph 3,5-H), 7.69
(m, 2H, Ph 2,6-H), 8.38 (s, 1H, 5-H). 13C NMR (176 MHz, CDCl3): δ 0.4 (CH(CH2)2), 8.8
(CH(CH2)2), 29.1 (COCH3), 67.8 ((CH2)2CHC≡C), 99.9 ((CH2)2CHC≡C), 119.8 (Ph C-2,6),
126.9 (C-4), 128.0 (Ph C-4), 129.7 (C-5), 129.8 (Ph C-3,5), 136.7 (C-3), 139.0 (Ph C-1), 192.6
(C=O). 15N NMR (71 MHz, CDCl3): δ –160.2 (N-1), N-2 was not found. MS m/z (%): 251
([M+H]+, 100). HRMS (ESI) for C16H14N2O ([M+H]+) calcd 273.0998, found 273.0998.
4.1.8.9. 1-(3-(Hex-1-yn-1-yl)-1-phenyl-1H-pyrazol-4-yl)ethanone (28). The reaction mixture
was stirred for 1 hour. Yellow liquid, yield 75%, 106 mg. IR (νmax, cm-1): 3130, 3064 (CHarom),
2957, 2928, 2860 (CHaliph), 2240 (C≡C), 1666 (C=O), 1522, 1457, 1362, 1262, 1245, 1217
(C=C, C–N), 751, 706, 685 (CH=CH of monosubstituted benzene). 1H NMR (700 MHz, CDCl3):
δ 0.94 (t, J = 7.4 Hz, 3H, C3H6CH3), 1.48-1.51 (m, 2H, C2H4CH2CH3), 1.62-1.66 (m, 2H,
CH2CH2C2H5), 2.49-2.51 (m, 2H, CH2C3H7), 2.51 (s, 1H, COCH3), 7.32-7.34 (m, 1H, Ph 4-H),
7.43-7.46 (m, 2H, Ph 3,5-H), 7.69-7.70 (m, 2H, Ph 2,6-H), 8.38 (s, 1H, 5-H). 13C NMR (176
MHz, CDCl3): δ 13.7 (C3H6CH3), 19.4 (CH2C3H7), 22.2 (C2H4CH2CH3), 29.0 (CH3), 30.3
(CH2CH2C2H5), 72.7 (C4H9C≡C), 96.9 (C4H9C≡C), 119.7 (Ph C-2,6), 126.7 (C-4), 128.0 (Ph C-
4), 129.7 (C-5), 129.8 (Ph C-3,5), 136.7 (C-3), 138.9 (Ph C-1), 192.5 (C=O). 15N NMR (71
18

ACCEPTED MANUSCRIPT
MHz, CDCl3): δ –160.1 (N-1), –70.8 (N-2). MS m/z (%): 267 ([M+H]+, 100). HRMS (ESI) for
C17H18N2ONa ([M+Na]+) calcd 289.1311, found 289.1312.
4.1.8.10. 1-(3-(Hept-1-yn-1-yl)-1-phenyl-1H-pyrazol-4-yl)ethanone (29). The reaction mixture
was stirred for 4 hours. Brown liquid, yield 65%, 91 mg. IR (νmax, cm-1): 3130, 3064 (CHarom),
2957, 2928, 2860 (CHaliph), 2240 (C≡C), 1666 (C=O), 1522, 1457, 1362, 1262, 1245, 1217
(C=C, C–N), 751, 706, 685 (CH=CH of monosubstituted benzene). 1H NMR (700 MHz, CDCl3):
δ 0.91 (t, J = 7.3 Hz, 3H, C4H8CH3), 1.33-1.38 (m, 2H, C3H6CH2CH3), 1.43-1.47 (m, 2H,
C2H4CH2C2H5), 1.64-1.69 (m, 2H, CH2CH2C3H7), 2.50 (t, J = 7.2 Hz, 2H, CH2C4H9), 2.63
(CH3), 7.31-7.36 (m, 1H, Ph 4-H), 7.44-7.46 (m, 2H, Ph 3,5-H), 7.69-7.70 (m, 2H, Ph 2,6-H),
8.39 (s, 1H, 5-H). 13C NMR (176 MHz, CDCl3): δ 14.0 (C4H8CH3), 19.7 (CH2C4H9), 22.3
(C3H6CH2CH3), 28.0 (CH2CH2C3H7), 29.0 (CH3), 31.3 (C2H4CH2C2H5), 72.7 (C5H11C≡C), 97.0
(C5H11C≡C), 119.7 (Ph C-2,6), 126.7 (C-4), 128.0 (Ph C-4), 128.6 (C-5), 129.7 (Ph C-3,5), 136.7
15
(C-3), 138.9 (Ph C-1), 192.5 (C=O). N NMR (71 MHz, CDCl3): δ –161.8 (N-1), N-2 was not
found. MS m/z (%): 281 ([M+H]+, 100). HRMS (ESI) for C18H20N2ONa ([M+Na]+) calcd
303.1468, found 303.1468.
4.1.8.11. 1-(3-(4-Hydroxybut-1-yn-1-yl)-1-phenyl-1H-pyrazol-4-yl)ethanone (30). The
reaction mixture was stirred for 4 hours. White solid, yield 86%, 109 mg, mp 121-122 °C. IR
(νmax, cm-1): 3130, 3068, 3026 (CHarom), 2995, 2961, 2945 (CHaliph), 2240 (C≡C), 1666 (C=O),
1521, 1460, 1350, 1262, 1249, 1217 (C=C, C–N), 755, 731, 685, 577, 468 (CH=CH of
1
monosubstituted benzene). H NMR (700 MHz, CDCl3): δ 2.59 (CH3), 2.75-2.77 (m, 2H,
C≡CCH2CH2OH), 3.89-3.91 (m, 2H, C≡CCH2CH2OH), 7.36-7.38 (m, 1H, Ph 4-H), 7.47-7.49
(m, 2H, Ph 3,5-H), 7.69-7.71 (m, 2H, Ph 2,6-H), 8.36 (s, 1H, 5-H). 13C NMR (176 MHz, CDCl3):
δ 24.3(C≡CCH2CH2OH), 28.8(COCH3), 60.8 (C≡CCH2CH2OH), 74.7 (C≡CCH2CH2OH), 93.7
(C≡CCH2CH2OH), 119.7 (NPh C-2,6), 126.4 (C-4), 128.2 (Ph C-4), 129.8 (Ph C-3,5), 130.1 (C-
5), 136.3 (C-3), 138.9 (Ph C-1), 192.0 (COCH3). 15N NMR (71 MHz, CDCl3): δ –160.4 (N-1), –
70.5 (N-2). MS m/z (%): 255 ([M+H]+, 100). HRMS (ESI) for C15H14N2ONa ([M+Na]+) calcd
277.0947, found 277.0947.
4.1.8.12. 1-(1-Phenyl-3-((trimethylsilyl)ethynyl)-1H-pyrazol-4-yl)ethanone (31). The reaction
mixture was stirred for 12 hours. White solid, yield 83%, 117 mg, mp 89-90 °C. IR (νmax, cm-1):
3129, 3065 (CHarom), 2959, 2899 (CHaliph), 2167 (C≡C), 1666 (C=O), 1523, 1507, 1442, 1363,
1
1261, 1206 (C=C, C–N), 858, 847, 755, 690 (CH=CH of monosubstituted benzene). H NMR
19

ACCEPTED MANUSCRIPT
(700 MHz, CDCl3): δ 0.30 (s, 9H, TMS), 2.66 (s, 3H, CH3), 7.36-7.38 (m, 1H, Ph 4-H), 7.46-
7.48 (m, 2H, Ph 3,5-H), 7.70-7.71 (m, 2H, Ph 2,6-H), 8.40 (s, 1H, 5-H). 13C NMR (176 MHz,
CDCl3): δ 0.3 (TMS), 29.1 (CH3), 96.3 (TMSC≡C), 101.9 (TMSC≡C), 119.9 (Ph C-2,6), 127.5
(C-4), 128.2 (C-5), 129.8 (Ph C-4), 129.9 (Ph C-3,5), 135.9 (C-3), 138.9 (Ph C-1), 192.5
(COCH3). 15N NMR (71 MHz, CDCl3): δ –158.7 (N-1), N-2 was not found. MS m/z (%): 283
([M+H]+, 100). HRMS (ESI) for C16H18N2OSiNa ([M+Na]+) calcd 305.1081, found 305.1081.
4.1.8.13. 2-Methyl-1-(1-phenyl-3-(phenylethynyl)-1H-pyrazol-4-yl)propan-1-one (32). The
reaction mixture was stirred for 1 hour. White solid, yield 90%, 141 mg, mp 105-106 °C. IR
(νmax, cm-1): 3121, 3061 (CHarom), 2968, 2869 (CHaliph), 2265 (C≡C), 1664 (C=O), 1521, 1443,
1
1350, 1227 (C=C, C–N), 989, 875, 751, 685 (CH=CH of monosubstituted benzene). H NMR
(700 MHz, CDCl3): δ 1.29 (d, J = 6.9 Hz, 6H, CH(CH3)2), 3.72-3.76 (m, 1H, CH(CH3)2, 7.37-
7.40 (m, 4H, NPh 4-H, CPh 3,4,5-H), 7.48-7.50 (m, 2H, NPh 3,5-H), 7.61-7.62 (m, 2H, CPh 2,6-
H), 7.75-7.76 (m, 2H, NPh 2,6-H), 8.47 (s, 1H, 5-H). 13C NMR (100 MHz, CDCl3): δ 18.9
(CH(CH3)2), 38.0 (CH(CH3)2), 81.3 (PhC≡C), 94.0 (PhC≡C), 119.9 (NPh C-2,6), 122.2 (CPh C-
1), 125.8 (C-4), 128.1 (NPh C-4), 128.6 (CPh C-3,5), 129.3 (CPh C-4), 129.8 (NPh C-3,5), 130.6
(C-5), 131.9 (CPh C-2,6), 135.7 (C-3), 139.0 (NPh C-1), 198.9 (C=O). 15N NMR (71 MHz,
CDCl3): δ –158.6 (N-1), N-2 was not found. MS m/z (%): 315 ([M+H]+, 100). HRMS (ESI) for
C21H18N2ONa ([M+Na]+) calcd 337.1311, found 337.1311.
4.1.8.14. 1-(3-(Hex-1-yn-1-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methylpropan-1-one (33). The
reaction mixture was stirred for 1 hour. Colorless liquid, yield 81%, 119 mg. IR (νmax, cm-1):
3122, 3056 (CHarom), 2961, 2929, 2871, 2855 (CHaliph), 2237 (C≡C), 1665 (C=O), 1519, 1436,
1
1352, 1228 (C=C, C–N), 959, 879, 857, 756, 686 (CH=CH of monosubstituted benzene). H
NMR (700 MHz, CDCl3): δ 0.93-0.95 (m, 3H, C3H6CH3), 1.22 (d, J = 7.0 Hz, 6H, CH(CH3)2),
1.47-1.52 (m, 2H, C2H4CH2CH3), 1.62-1.66 (m, 2H, CH2CH2C2H5), 2.49-2.51 (m, 2H,
CH2C3H7), 3.64-3.70 (m, 1H, CH(CH3)2, 7.31-7.36 (m, 1H, NPh 4-H), 7.44-7.48 (m, 2H, NPh
13
3,5-H), 7.68-7.72 (m, 2H, NPh 2,6-H), 8.41 (s, 1H, 5-H). C NMR (100 MHz, CDCl3): δ 13.7
(C3H6CH3), 18.8 (CH(CH3)2), 19.4 (CH2C3H7) 22.2 (C2H4CH2CH3), 30.4 (CH2CH2C2H5), 37.6
(CH(CH3)2), 72.6 (C4H9C≡C), 96.0 (C4H9C≡C), 119.7 (NPh C-2,6), 125.3 (C-4), 127.9 (NPh C-
15
4), 129.6 (NPh C-3,5), 130.3 (C-5), 136.1 (C-3), 139.0 (NPh C-1), 199.2 (C=O). N NMR (71
MHz, CDCl3): δ –160.5 (N-1), –72.2 (N-2). MS m/z (%): 295 ([M+H]+, 100). HRMS (ESI) for
C19H24N2O2Na ([M+Na+H2O]+) calcd 335.1730, found 335.1723.
20

ACCEPTED MANUSCRIPT
4.1.8.15.
2-Methyl-1-(1-phenyl-3-((trimethylsilyl)ethynyl)-1H-pyrazol-4-yl)propan-1-one
(34). The reaction mixture was stirred for 12 hours. White solid, yield 87%, 135 mg, mp 91-92
°C. IR (νmax, cm-1): 3124, 3060 (CHarom), 2987, 2971, 2933, 2903 (CHaliph), 2166 (C≡C), 1665
(C=O), 1518, 1435, 1354, 1247, 1226 (C=C, C–N), 873, 848, 757, 705 (CH=CH of
monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 0.30 (s, 9H, TMS), 1.20-1.26 (m, 6H,
CH(CH3)2), 3.72-3.76 (m, 1H, CH(CH3)2, 7.36-7.39 (m, 1H, Ph 4-H), 7.45-7.49 (m, 2H, Ph 3,5-
H), 7.71-7.72 (m, 2H, Ph 2,6-H), 8.42 (s, 1H, 5-H). 13C NMR (176 MHz, CDCl3): δ 0.32 (TMS),
18.7 (CH(CH3)2), 37.7 (CH(CH3)2), 96.1 (C4H9C≡C), 100.9 (C4H9C≡C), 119.9 (Ph C-2,6), 126.2
(C-4), 128.2 (Ph C-4), 129.7 (Ph C-3,5), 130.5 (C-5), 135.3 (C-3), 139.0 (Ph C-1), 199.2 (C=O).
15N NMR (71 MHz, CDCl3): δ –158.9 (N-1), N-2 was not found. MS m/z (%): 311 ([M+H]+,
100). HRMS (ESI) for C18H22N2ONa ([M+Na]+) calcd 333.1394, found 333.1394.
4.1.9. General procedure for the cyclization of 3-ethynyl-1-phenyl-1H-pyrazole-4-
carbaldehydes (17-24), ethanones (25-31) and propanones (32-34).
A solution of compound 17-34 (0.5 mmol) in dry ammonia and methanol (NH3/MeOH 2 M, 8
mL) was heated at 120 °C for 15 h in a steel reactor. The solvent was evaporated and the crude
was purified by flash chromatography (SiO2, eluent: ethyl acetate/n-hexane, 1:4, v/v) to yield
compounds 35-51.
4.1.9.1. 2-Methyl-6-phenyl-2H-pyrazolo[4,3-c]pyridine (35). White solid, yield 79%, 83 mg,
mp 159-160 °C. IR (νmax, cm-1): 3106, 3062, 3030 (CHarom), 2997, 2943 (CHaliph), 1615, 1471,
1367, 1240, 1153 (C=C, C–N), 926, 858, 831, 755, 688, 677 (CH=CH of monosubstituted
1
benzene). H NMR (700 MHz, CDCl3): δ 4.27 (s, 3H, CH3), 7.38-7.40 (m, 1H, Ph 4-H), 7.47-
7.50 (m, 2H, Ph 3,5-H), 7.93 (s, 1H, 7-H), 8.05-8.09 (m, 2H, Ph 2,6-H), 8.08 (s, 1H, 3-H), 9.28
(s, 1H. 4-H). 13C NMR (176 MHz, CDCl3): δ 40.9 (CH3), 107.0 (C-7), 119.4 (C-3a), 125.1 (C-3),
127.2 (Ph C-2,6), 128.4 (Ph C-4), 128.8 (Ph C-3,5), 140.3 (Ph C-1), 146.3 (C-4), 151.1 (C-6),
151.6 (C-7a). 15N NMR (71 MHz, CDCl3): δ –160.6 (N-2), –97.6 (N-1), –87.7 (N-5). MS m/z
(%): 210 ([M+H]+, 100). HRMS (ESI) for C13H12N3 ([M+H]+) calcd 210.1027, found 210.1026.
4.1.9.2. 2-Phenyl-6-(thiophen-3-yl)-2H-pyrazolo[4,3-c]pyridine (37). White solid, yield 94%,
130 mg, mp 129-130 °C. IR (νmax, cm-1): 3132, 3113, 3064, 3041 (CHarom), 2920, 2851 (CHaliph),
1506, 1362, 1254, 1202, 1039 (C=C, C–N), 862, 803, 760, 750, 687 (CH=CH of
monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 7.42-7.43 (m, 1H, Th 4-H), 7.47-7.49
(m, 1H, Ph 4-H), 7.56-7.59 (m, 2H, Ph 3,5-H), 7.70-7.71 m, 1H, Th 5-H), 7.91 (s, 1H, 7-H),
21

ACCEPTED MANUSCRIPT
7.92-7.94 (m, 2H, Ph 2,6-H), 8.00-8.01 (m, 1H, Th 2-H), 8.58 (d, 4J(3-H,4-H) = 0.8 Hz, 1H, 3-
4
H), 9.28 (d, J(4-H,3-H) = 1.2 Hz 1H, 4-H). 13C NMR (176 MHz, CDCl3): δ 106.4 (C-7), 119.8
(C-3a), 121.4 (Ph C-2,6), 122.1 (C-3), 123.3 (Th C-2), 126.2 (Th C-5), 126.4 (Th C-4), 129.0 (Ph
C-4), 130.0 (Ph C-3,5), 140.1 (Ph C-1), 142.7 (Th C-3), 147.4 (C-4), 147.6 (C-6), 151.7 (C-7a).
15N NMR (71 MHz, CDCl3): δ −145.1 (N-2), −99.7 (N-1). MS m/z (%): 278 ([M+H]+, 100).
HRMS (ESI) for C16H12N3S ([M+H]+) calcd 278.0746, found 278.0747.
4.1.9.3. 6-Cyclopropyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (38). White solid, yield 98%,
115 mg, mp 143-144 °C. IR (νmax, cm-1): 3116, 3090, 3036, 3006 (CHarom), 2923, 2850 (CHaliph),
1597, 1497, 1377, 1318, 1201, 1053, 1038 (C=C, C–N), 769, 761, 691 (CH=CH of
monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 0.99-1.01 (m, 2H, CH2CHCH2), 1.05-
1.07 (m, 2H, CH2CHCH2), 2.14-2.18 (m, 2H, CH2CHCH2), 7.40 (s, 1H, 7-H), 7.42-7.45 (m, 1H,
Ph 4-H), 7.52-7.54 (m, 2H, Ph 3,5-H), 7.87-7.89 (m, 2H, Ph 2,6-H), 8.52 (s, 1H, 3-H), 9.12 (s,
1H, 4-H). 13C NMR (176 MHz, CDCl3): δ 8.9 (CH2CHCH2), 17.5 (CH2CHCH2), 106.6 (C-7),
119.3 (C-3a), 121.4 (Ph C-2,6), 122.0 (C-3), 128.8 (Ph C-4), 129.8 (Ph C-3,5), 140.1 (Ph C-1),
15
146.9 (C-4), 151.6 (C-7a), 155.7 (C-6). N NMR (71 MHz, CDCl3): δ −145.3 (N-2), −91.0 (N-
5), N-1 was not found. MS m/z (%): 236 ([M+H]+, 100). HRMS (ESI) for C15H14N3 ([M+H]+)
calcd 236.1182 , found 236.1184.
4.1.9.4. 6-Butyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (39). Brown amorphous solid, yield
84%, 105 mg. IR (νmax, cm-1): 3138, 3013 (CHarom), 2949, 2930, 2867 (CHaliph), 1507, 1468,
1372, 1320, 1208, 1053, 1037 (C=C, C–N), 763, 751, 689 (CH=CH of monosubstituted
1
benzene). H NMR (700 MHz, CDCl3): δ 0.95-0.97 (m, 3H, C3H6CH3), 1.40-1.45 (m, 2H,
(C2H4CH2CH3), 1.76-1.80 (m, 2H, CH2CH2C2H5), 2.88-2.90 (m, 2H, CH2C3H7), 7.40 (s, 1H, 7-
H), 7.43-7.45 (m, 1H, Ph 4-H), 7.53-7.55 (m, 2H, Ph 3,5-H), 7.88-7.89 (m, 2H, Ph 2,6-H), 8.52
(s, 1H, 3-H), 9.18 (s, 1H, 4-H). 13C NMR (176 MHz, CDCl3): δ 14.1 (C3H6CH3), 22.6
(C2H4CH2CH3), 32.0 (CH2CH2C2H5), 38.3 (CH2(C3H7)), 108.3 (C-7), 119.2 (C-3a), 121.4 (Ph C-
2,6), 121.8 (C-3), 128.8 (Ph C-4), 129.9 (Ph C-3,5), 140.2 (Ph C-1), 147.0 (C-4), 151.7 (C-7a),
155.7 (C-6). 15N NMR (71 MHz, CDCl3): −145.5 (N-2), −78.9 (N-5), N-1 was not found. MS
m/z (%): 252 ([M+H]+, 100). HRMS (ESI) for C16H18N3 ([M+H]+) calcd 252.1495 , found
252.1497.
4.1.9.5. 6-Pentyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (40). Brown amorphous solid, yield
89%, 118 mg. IR (νmax, cm-1): 3122, 3063, 3040 (CHarom), 2949, 2925, 2856 (CHaliph), 1466,
22

ACCEPTED MANUSCRIPT
1
1324, 1206, 1037 (C=C, C–N), 862, 759, 750, 684 (CH=CH of monosubstituted benzene). H
NMR (700 MHz, CDCl3): δ 0.89-0.90 (m, 2H, CH2(CH2)3CH3), 1.37-1.39 (m, 4H,
(CH2)2(CH2)2CH3), 1.78-1.81 (m, 2H, CH2CH2 (CH2)2CH3), 2.86-2.89 (m, 2H, CH2(CH2)3CH3),
7.40 (s, 1H, 7-H), 7.43-7.44 (m, 1H, Ph 4-H), 7.52-7.55 (m, 2H, Ph 3,5-H), 7.88-7.89 (m, 2H, Ph
2,6-H), 8.52 (s, 1H, 3-H), 9.18 (s, 1H, 4-H). 13C NMR (176 MHz, CDCl3): δ 14.2 (C4H8CH3),
22.7 (C3H6CH2CH3), 29.6 (CH2CH2C3H7), 31.7 (C2H4CH2C2H5), 38.6 (CH2C4H11), 108.3 (C-7),
119.1 (C-3a), 121.4 (Ph C-2,6), 121.8 (C-3), 128.8 (Ph C-4), 129.8 (Ph C-3,5), 140.2 (Ph C-1),
15
146.9 (C-6), 151.7 (C-4), 155.8 (C-7a). N NMR (71 MHz, CDCl3): δ −145.3 (N-2), −79.8 (N-
5), N-1 was not found. MS m/z (%): 266 ([M+H]+, 100). HRMS (ESI) for C17H20N3 ([M+H]+)
calcd 266.1652 , found 266.1652.
4.1.9.6. 2-(2-phenyl-2H-pyrazolo[4,3-c]pyridin-6-yl)ethanol (41). Brown solid, yield 86%,
103 mg, mp 215-216 °C. IR (νmax, cm-1): 3187 (OH), 3108, 3067, 3022 (CHarom), 2961, 2935,
2916 (CHaliph), 1503, 1325, 1227, 1213, 1056 (C=C, C–N), 765, 752, 677 (CH=CH of
monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 3.11-3.12 (m, 2H, CH2CH2OH), 4.05-
4.06 (m, 2H, CH2CH2OH),), 7.45-7.48 (m, 2H, H-7, Ph 4-H), 7.55-7.58 (m, 2H, Ph 3,5-H), 7.89-
7.90 (m, 2H, Ph 2,6-H), 8.58 (s, 1H, 3-H), 9.16 (s, 1H, 4-H). 13C NMR (176 MHz, CDCl3): δ
39.6 (CH2CH2OH) 62.7 (CH2CH2OH), 109.5 (C-7), 119.4 (C-3a), 121.5 (Ph C-2,6), 122.3 (C-3),
129.1 (Ph C-4), 129.9 (Ph C-3,5), 140.0 (Ph C-1), 151.3 (C-4), 153.6 (C-7a). 15N NMR (71 MHz,
CDCl3): δ −144.3 (N-2), −100.5 (N-1), −88.3 (N-5). MS m/z (%): 240 ([M+H]+, 100). HRMS
(ESI) for C14H14N3O ([M+H]+) calcd 240.1131, found 240.1131.
4.1.9.7. 2-Phenyl-2H-pyrazolo[4,3-c]pyridine (42). White solid, yield 95%, 193 mg, mp 132
°C (lit mp 132-133 °C [35]). IR (νmax, cm-1): 3129, 3094, 3064 (CHarom), 1614, 1490, 1466, 1365,
1349, 1315, 1210, 1178 (C=C, C–N), 916, 827, 760, 750, 684, 587, 508, 431 (CH=CH of
monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 7.45-7.48 (m, 1H, Ph 4-H), 7.54-7.56
(m, 2H, Ph 3,5-H), 7.62-7.64 (m, 1H, 7-H), 7.90-7.91 (m, 2H, Ph 2,6-H), 8.31-8.32 (m, 1H, 6-H),
8.63 (s, 1H, 3-H), 9.27 (4-H). 13C NMR (176 MHz, CDCl3): δ 111.8 (C-7), 120.5 (C-3a), 121.6
(Ph C-2,6), 122.4 (C-3), 129.1 (Ph C-4), 129.9 (Ph C-3,5), 140.0 (Ph C-1), 142.1 (C-6), 147.5
(C-4), 150.4 (C-7a). 15N NMR (71 MHz, CDCl3): δ −147.4 (N-2), –99.6 (N-1), –89.8 (N-5). MS
m/z (%): 196 ([M+H]+, 100). HRMS (ESI) for C12H10N3 ([M+H]+) calcd 196.0869, found
196.0869.
23

ACCEPTED MANUSCRIPT
4.1.9.8. 4-Methyl-2-phenyl-6-(thiophen-3-yl)-2H-pyrazolo[4,3-c]pyridine (44). White solid,
yield 81%, 118 mg, mp 92-93 °C. IR (νmax, cm-1): 3129, 3104, 3072 (CHarom), 2982, 2948, 2911
(CHaliph), 1546, 1507, 1374, 1202, 1045 (C=C, C–N), 849, 796, 763, 744, 690 (CH=CH of
1
monosubstituted benzene). H NMR (700 MHz, CDCl3): δ 2.86 (s, 3H, CH3), 7.39-7.40 (m, 1H,
Th 4-H), 7.43-7.45 (m, 1H, Ph 4-H), 7.53-7.55 (m, 2H, Ph 3,5-H), 7.67-7.68 (m, 1H, Th 5-H),
7.72 (s, 1H, 7-H), 7.90-7.91 (m, 2H, Ph 2,6-H), 8.01-8.02 (m, 1H, Th 2-H), 8.49 (s, 1H, 3-H).
13C NMR (176 MHz, CDCl3): δ 23.3 (CH3), 104.3 (C-7), 119.7 (C-3a), 121.2 (Ph C-2,6), 121.9
(C-3), 123.2 (Th C-2), 126.2 (Th C-4,5), 128.7 (Ph C-4), 129.8 (Ph C-3,5), 140.1 (Ph C-1), 142.8
(Th C-3), 147.3 (C-4), 152.0 (C-7a), 156.2 (C-6). 15N NMR (71 MHz, CDCl3): δ −147.4 (N-2), –
99.6 (N-1), –89.8 (N-5). MS m/z (%): 292 ([M+H]+, 100). HRMS (ESI) for C17H14N3S ([M+H]+)
calcd 292.0903, found 292.0903.
4.1.9.9. 6-Cyclopropyl-4-methyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (45). White solid,
yield 80%, 99 mg, mp 85-86 °C. IR (νmax, cm-1): 3129, 3078, 3044 (CHarom), 2996, 2911, 2850
(CHaliph), 1500, 1401, 1300, 1202, 1071, 1041 (C=C, C–N), 821, 760, 750, 685, 538 (CH=CH of
monosubstituted benzene). 1H NMR (700 MHz, CDCl3): δ 0.95-0.98 (m, 2H, CH2CHCH2), 0.99-
1.01 (m, 2H, CH2CHCH2), 2.14-2.16 (m, 2H, CH2CHCH2), 2.75 (s, 3H, CH3), 7.16 (s, 1H, 7-H),
7.40-7.42 (m, 1H, Ph 4-H), 7.50-7.53 (m, 2H, Ph 3,5-H), 7.86-7.87 (m, 2H, Ph 2,6-H), 8.44 (s,
1H, 3-H). 13C NMR (176 MHz, CDCl3): δ 8.7 (CH2CHCH2), 17.6 (CH2CHCH2), 23.0 (CH3),
103.6 (C-7), 119.4 (C-3a), 121.2 (Ph C-2,6), 121.7 (C-3), 128.5 (Ph C-4), 129.8 (Ph C-3,5),
15
140.2 (Ph C-1), 152.0 (C-7a), 155.6 (C-4), 155.9 (C-6). N NMR (71 MHz, CDCl3): δ −147.9
(N-2), −90.4 (N-5), N-1 was not found. MS m/z (%): 250 ([M+H]+, 100). HRMS (ESI) for
C16H16N3 ([M+H]+) calcd 250.1339, found 250.1337.
4.1.9.10. 6-Butyl-4-methyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (46). Brown liquid, yield
88%, 116 mg. IR (νmax, cm-1): 3070 (CHarom), 2955, 2928, 2870 (CHaliph), 1618, 1597, 1544,
1510, 1374, 1165, 1043 (C=C, C–N), 758, 688 (CH=CH of monosubstituted benzene). 1H NMR
(700 MHz, CDCl3): δ 0.94-0.97 (m, 3H, C3H6CH3), 1.40-1.45 (m, 2H, (C2H4CH2CH3), 1.71-1.79
(m, 2H, CH2CH2C2H5), 2.81 (s, 3H, CH3) 2.81-2.86 (m, 2H, CH2C3H7), 7.24 (s, 1H, 7-H), 7.42-
7.44 (m, 1H, H-4), 7.52-7.54 (m, 2H, Ph 3,5-H), 7.88-7.89 (m, 2H, Ph 2,6-H), 8.49 (s, 1H, 3-H).
13C NMR (176 MHz, CDCl3): δ 14.2 (C3H6CH3), 22.6 (C2H4CH2CH3), 23.1 (CH3), 32.1
(CH2CH2C2H5), 38.3 (CH2(C3H7), 106.0 (C-7), 119.2 (C-3a), 121.3 (Ph C-2,6), 121.8 (C-3),
128.6 (Ph C-4), 129.8 (Ph C-3,5), 140.2 (Ph C-1), 152.0 (C-7a), 155.5 (C-4), 155.6 (C-6). 15N
24

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NMR (71 MHz, CDCl3): δ −147.6 (N-2), −85.2 (N-5), N-1 was not found. MS m/z (%): 266
([M+H]+, 100). HRMS (ESI) for C17H20N3 ([M+H]+) calcd 266.1652, found 266.1654.
4.1.9.11. 4-Methyl-6-pentyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (47). Brown liquid, yield
88%, 123 mg. IR (νmax, cm-1): 3100, 3071 (CHarom), 2954, 2928, 2870, 2857 (CHaliph), 1619,
1597, 1544, 1510, 1374, 1309, 1043 (C=C, C–N), 758, 688 (CH=CH of monosubstituted
1
benzene). H NMR (700 MHz, CDCl3): δ 0.82-0.91 (m, 2H, C4H11CH3), 1.35-1.41 (m, 4H,
C2H4(CH2)2CH3), 1.76-1.80 (m, 2H, CH2CH2C3H7), 2.82 (CH3), 2.84-2.86 (m, 2H, CH2C4H9),
7.24 (s, 1H, H-7), 7.42-7.45 (m, 1H, Ph 4-H), 7.53-7.55 (m, 2H, Ph 3,5-H), 7.88-7.89 (m, 2H, Ph
2,6-H), 8.50 (s, 1H, 3-H). 13C NMR (176 MHz, CDCl3): δ 14.2 (C4H8CH3), 22.8 (C3H6CH2CH3),
23.0 (CH3), 29.6 (CH2CH2C3H7), 31.7 (C2H4CH2C2H5), 38.4 (CH2C4H11), 106.1 (C-7), 119.2 (C-
3a), 121.3 (Ph C-2,6), 121.9 (C-3), 128.7 (Ph C-4), 129.8 (Ph C-3,5), 140.2 (Ph C-1), 152.0 (C-
7a), 155.4 (C-4), 155.6 (C-6). 15N NMR (71 MHz, CDCl3): δ −148.9 (N-2), −85.0 (N-5), N-1
was not found. MS m/z (%): 280 ([M+H]+, 100). HRMS (ESI) for C18H22N3 ([M+H]+) calcd
280.1808, found 280.1808.
4.1.9.12. 2-(4-Methyl-2-phenyl-2H-pyrazolo[4,3-c]pyridin-6-yl)ethanol (48). White solid,
yield 86%, 109 mg, mp 126-127 °C. IR (νmax, cm-1): 3227 (OH), 3149, 3072, 3018 (CHarom),
2945, 2919, 2870 (CHaliph), 1622, 1501, 1389, 1374, 1306, 1212, 1164, 1053 (C=C, C–N), 844,
1
756, 741, 682, 571 (CH=CH of monosubstituted benzene). H NMR (700 MHz, CDCl3): δ 2.74
(s, 3H, CH3), 3.03 (t, J=5.5 Hz, 2H, CH2CH2OH), 4.00-4.02 (m, 2H, CH2CH2OH), 7.22 (s, 1H,
7-H), 7.40-7.42 (m, 2H, H-7, Ph 4-H), 7.49-7.51 (m, 2H, Ph 3,5-H), 7.84-7.85 (m, 2H, Ph 2,6-
H), 8.48 (s, 1H, 3-H). 13C NMR (176 MHz, CDCl3): δ 23.0 (CH3), 39.4 (CH2CH2OH) 62.7
(CH2CH2OH), 107.0 (C-7), 119.2 (C-3a), 121.2 (Ph C-2,6), 122.0 (C-3), 128.7 (Ph C-4), 129.8
(Ph C-3,5), 140.0 (Ph C-1), 151.5 (C-7a), 153.4 (C-6), 155.7 (C-4). 15N NMR (71 MHz, CDCl3):
δ −146.8 (N-2), −92.3 (N-5), N-1 was not found. MS m/z (%): 254 ([M+H]+, 100). HRMS (ESI)
for C15H16N3O ([M+H]+) calcd 254.1288, found 254.1287.
4.1.9.13. 4-Methyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (49). Brown amorphous solid, yield
88%, 92 mg. IR (νmax, cm-1): 3066 (CHarom), 2995 (CHaliph), 1596, 1498, 1416, 1370, 1345, 1237,
1190, 1031 (C=C, C–N), 807, 756, 746, 687, 638, 529 (CH=CH of monosubstituted benzene). 1H
NMR (700 MHz, CDCl3): δ 2.85 (s, 3H, CH3), 7.46-7.48 (m, 2H, 7-H, Ph 4-H), 7.55-7.58 (m,
3
2H, Ph 3,5-H), 7.91-7.93 (m, 2H, Ph 2,6-H), 8.20 (d, J(6-H,7-H) = 6.4 Hz, 1H, 6-H), 8.58 (s,
1H, 3-H). 13C NMR (176 MHz, CDCl3): δ 23.0 (CH3) 109.5 (C-7), 120.6 (C-3a), 121.5 (Ph C-
25

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2,6), 122.0 (C-3), 128.9 (Ph C-4), 129.9 (Ph C-3,5), 140.1 (Ph C-1), 142.1 (C-6), 150.9 (C-4),
15
156.4 (C-7a). N NMR (71 MHz, CDCl3): δ −146.9 (N-2), −98.48 (N-1), −91.0 (N-5). MS m/z
(%): 210 ([M+H]+, 100). HRMS (ESI) for C12H13N3 ([M+H]+) calcd 210.1026, found 210.1026.
4.1.9.14. 4-Isopropyl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine (50). White solid, yield 92%,
143 mg, mp 97-98 °C. IR (νmax, cm-1): 3126, 3064, 3023 (CHarom), 2960, 2924, 2898 (CHaliph),
1600, 1510, 1466, 1399, 1314, 1277, 1203, 1046 (C=C, C–N), 767, 760, 756, 696, 684, 635
(CH=CH of monosubstituted benzenes). 1H NMR (700 MHz, CDCl3): δ 1.54 (d, J = 7.0 Hz, 6H,
CH(CH3)2), 3.52-3.56 (m, 1H, CH(CH3)2), 7.38-7.40 (m, 1H, CPh 4-H), 7.44-7.47 (m, 1H, NPh
4-H), 7.48-7.52 (m, 2H, CPh 3,5-H), 7.55-7.58 (m, 2H, NPh 3,5-H), 7.89 (s, 1H, 7-H), 7.93-7.96
(m, 2H, NPh 2,6-H), 8.18-8.19 (m, 2H, CPh 2,6-H), 8.57 (s, 1H, 3-H). 13C NMR (176 MHz,
CDCl3): δ 21.9 (CH(CH3)2), 35.8 (CH(CH3)2), 104.5 (C-7), 118.0 (C-3), 121.2 (NPh C-2,6),
127.0 (CPh C-2,6), 128.2 (CPh C-4), 128.56 (CPh C-3,5), 128.62 (NPh C-4), 129.7 (NPh C-3,5),
15
140.1 (CPh C-1), 140.4 (NPh C-1), 150.5 (C-6), 152.8 (C-7a), 164.1 (C-4). N NMR (71 MHz,
CDCl3): δ −148.9 (N-2), −86.5 (N-1), −90.8 (N-5). MS m/z (%): 314 ([M+H]+, 100). HRMS
(ESI) for C21H20N3 ([M+H]+) calcd 314.1652, found 314.1652.
4.1.9.15. 6-Butyl-4-isopropyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (51). Brown liquid, yield
84%, 123 mg. IR (νmax, cm-1): 3111, 3065, 3047 (CHarom), 2959, 2929 (CHaliph), 1615, 1598,
1541, 1510, 1467, 1376, 1306, 1275, 1211, 1196, 1161, 1044 (C=C, C–N), 848, 757, 688
1
(CH=CH of monosubstituted benzene). H NMR (700 MHz, CDCl3): δ 0.95-0.97 (m, 3H,
C3H6CH3), 1.40-1.44 (m, 2H, (C2H4CH2CH3), 1.44-1.47 (m, 6H, CH(CH3)2), 1.75-1.80 (m, 2H,
CH2CH2C2H5), 2.85-2.90 (m, 2H, CH2C3H7), 3.41-3.45 (s, 1H, CH(CH3)2), 7.23 (s, 1H, 7-H),
7.42-7.46 (m, 1H, H-7), 7.53-7.54 (m, 2H, Ph 3,5-H), 7-89-7.90 (m, 2H, Ph 2,6-H), 8.53 (s, 1H,
13
3-H). C NMR (176 MHz, CDCl3): δ 14.2 (C3H6CH3), 22.1 (CH(CH3)2), 22.6 (C2H4CH2CH3),
31.8 (CH2CH2C2H5), 36.3 (CH(CH3)2), 38.3 (CH2(C3H7), 105.8 (C-7), 116.9 (C-3a), 121.3 (C-3),
121.4 (Ph C-2,6), 128.5 (Ph C-4), 129.8 (Ph C-3,5), 140.3 (Ph C-1), 152.8 (C-7a), 155.4 (C-6),
164.1 (C-4). 15N NMR (71 MHz, CDCl3): δ −147.4 (N-2), −99.6 (N-1), −90.8 (N-5). MS m/z
(%): 294 ([M+H]+, 100). HRMS (ESI) for C19H24N3 ([M+H]+) calcd 294.1965 , found 294.1965.
4.1.9.16. 4-Isopropyl-2-phenyl-2H-pyrazolo[4,3-c]pyridine (52). Brown liquid, yield 92%,
109 mg. IR (νmax, cm-1): 3061 (CHarom), 2966, 2927, 2869 (CHalif), 1609, 1510, 1498, 1369, 1276,
1
1238, 1193, 1026 (C=C, C–N), 806, 757, 688 (CH=CH of monosubstituted benzene). H NMR
(700 MHz, CDCl3): δ 1.47 (d, J = 7.1 Hz, 6H, CH(CH3)2), 3.46-3.50 (m, 1H, CH(CH3)2), 7.44-
26

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7.47 (m, 2H, 7-H, Ph 4-H,), 7.54-7.56 (m, 2H, Ph 3,5-H), 7.90-7.92 (m, 2H, Ph 2,6-H), 8.25 (d,
3J(6-H,7-H) = 6.4 Hz, 1H, 6-H), 8.60 (s, 1H, 3-H). 13C NMR (176 MHz, CDCl3): δ 21.8
(CH(CH3)2, 35.4 (CH(CH3)2), 109.4 (C-7), 118.7 (C-3a), 121.4 (Ph C-2,6), 121.5 (C-3), 128.7
(Ph C-4), 129.7 (Ph C-3,5), 140.0 (C-6), 142.1 (Ph C-1), 151.2 (C-7a), 165.0 (C-4). 15N NMR
(71 MHz, CDCl3): δ −147.4 (N-2), −100.6 (N-1), −95.3 (N-5). MS m/z (%): 238 ([M+H]+, 100).
HRMS (ESI) for C15H16N3 ([M+H]+) calcd 238.1339 , found 238.1339.
4.2. Cancer cell lines and cytotoxicity assay
Human cancer cell lines were obtained from the American Type Culture Collection and were
cultivated according to the provider’s instructions. In brief, MCF-7 and K562 cell lines were
maintained in DMEM medium supplemented with 10% fetal bovine serum, penicillin (100
U/mL), and streptomycin (100 µg/mL) at 37 °C in 5% CO2. For the cytotoxicity assays, cells
were treated in triplicate with six different doses of each compound for 72 hours. After
treatment, Calcein AM solution was added for 1 hour, and the fluorescence from live cells was
measured at 485 nm/538 nm (excitation/emission) using a Fluoroskan Ascent microplate reader
(Labsystems). The GI50 value, the drug concentration lethal to 50% of the cells, was calculated
from the dose-response curves that resulted from the assays.
4.3. Flow cytometry
Asynchronous cells were seeded into a 96 well plate and then, after a preincubation period,
treated with the tested compounds for 24 hours at a single dose of 10 µM. MCF-7 cells were first
washed with PBS, trypsinized, and finally treated with a solution of trypsin inhibitor (0.1 %).
After incubation, 5× staining solution (17 m
M
trisodium citrate dihydrate, 0.5% IGEPAL® CA-
Tris; pH 7.6 containing 50 µg/mL propidium
630, 7.5 m spermine tetrahydrochloride, 2.5 m
M
M
iodide) was added. K-562 cells were stained directly with the 5x staining solution (i.e. without
trypsinization). The cells’ DNA content was analyzed by flow cytometry using a 488 nm laser
(BD FACS Verse with software BD FACSuiteTM, version 1.0.6.). Cell cycle distribution was
analyzed using ModFit LT (Verity Software House, version 4.1.7).
For quantification of histone H3 phosphorylation, the cells were harvested by trypsinization,
washed in PBS, fixed with ice-cold 90% methanol, incubated on ice for 30 min and washed with
PBS/BSA containing 0.5% Tween-20. Then, the cells were incubated with the primary antibody
27

ACCEPTED MANUSCRIPT
raised against histone H3 phosphorylated at Ser10 (Millipore) for 1 hour at room temperature,
washed with PBS containing 1% BSA and incubated with the secondary antibody (goat-anti-
rabbit-Alexa Fluor 488, Invitrogen) for 1 hour in the dark. After washing with PBS/BSA, each
sample was incubated with propidium iodide (final concentration 10 µg/mL) and RNAse A (final
concentration 200 µg/mL) for 30 minutes at room temperature in the dark. The cells were then
analyzed by flow cytometry using a 488 nm laser (BD FACS Verse with software BD
FACSuiteTM, version 1.0.6.).
4.4. Caspase-3/7 assay
Cellular caspase-3/7 activity was measured according to previously published procedure [39].
K562 cells were cultivated in a 96-well plate overnight. Next day, the cells were treated with
increasing concentrations of compound 5 for next 24 hours. After incubation, 3x caspase-3/7
assay buffer (150 mM HEPES pH 7.4, 450 mM NaCl, 150 mM KCl, 30 mM MgCl2, 1.2 mM
EGTA, 1.5% Nonidet P40, 0.3% CHAPS, 30% sucrose, 30 mM DTT, 3 mM PMSF) containing
150 µM peptide substrate Ac-DEVD-AMC (Enzo Life Sciences) was added and after 2 hours
incubation, the caspase-3/7 activity was measured using Fluoroskan Ascent microplate reader
(Labsystems) at 346 nm/442 nm (excitation/emission). The activity was normalized to an
untreated control.
4.5. Immunoblotting
Immunoblotting was performed as described earlier [40]. In brief, cellular lysates were prepared
by harvesting cells in Laemmli sample buffer. Proteins were separated on SDS-polyacrylamide
gels and electroblotted onto nitrocellulose membranes. After blocking, the membranes were
incubated with specific primary antibodies overnight, washed and then incubated with
peroxidase-conjugated secondary antibodies. Finally, peroxidase activity was detected with
ECL+ reagents (AP Biotech) using a CCD camera LAS-4000 (Fujifilm). Specific antibodies
were purchased from Santa Cruz Biotechnology (PARP, β-actin), Cell Signaling (Ser-139
phosphorylated H2AX, Bcl-2) and Sigma Aldrich (peroxidase-labeled secondary antibodies).
28