New bioactive 5-arylcarboximidamidopyrazolo[3,4-c]pyridines: Synthesis, cytotoxic activity, mechanistic investigation and structure-activity relationships

Article abstract of DOI:10.1016/j.ejmech.2021.113387

In this study, a series of novel substituted pyrazolo[3,4-c]pyridin-5-ylamidines was synthesized and their cytotoxicity against three cancer cell lines (MDA-MB-231, HT-1080, PC-3), as well as a human normal cell line (AG01523) was evaluated. A number of derivatives could strongly reduce cancer cells proliferation and exhibit apoptotic induction capability, while reasonable structure-activity relationships could be extracted. Certain analogues were endowed with low toxicity against normal cells. Cell cycle analysis revealed that most of the active compounds induced a G₀/G₁ arrest of HT-1080 cells. Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds were investigated in HT-1080 cells, upon study of their effects on the phosphorylation of Akt, ERK and p38 MAPK. Most of the active derivatives inhibit phosphorylation of Akt and ERK and/or induce p38 MAPK phosphorylation, providing a potential indication on the mode of action of this class.

Full text of DOI:10.1016/j.ejmech.2021.113387

European Journal of Medicinal Chemistry 218 (2021) 113387
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
New bioactive 5-arylcarboximidamidopyrazolo[3,4-c]pyridines:
Synthesis, cytotoxic activity, mechanistic investigation and structure-
activity relationships
Athanasios Papastathopoulos a, Nikolaos Lougiakis a, Ioannis K. Kostakis a,
,
Panagiotis Marakos a *, Nicole Pouli a, Harris Pratsinis b, Dimitris Kletsas b
a Division of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens,
Panepistimiopolis Zografou, 15771, Athens, Greece
b Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, NCSR ‘‘Demokritos’’, 15310, Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of novel substituted pyrazolo[3,4-c]pyridin-5-ylamidines was synthesized and their
cytotoxicity against three cancer cell lines (MDA-MB-231, HT-1080, PC-3), as well as a human normal cell
line (AG01523) was evaluated. A number of derivatives could strongly reduce cancer cells proliferation
and exhibit apoptotic induction capability, while reasonable structure-activity relationships could be
extracted. Certain analogues were endowed with low toxicity against normal cells. Cell cycle analysis
revealed that most of the active compounds induced a G0/G1 arrest of HT-1080 cells. Moreover, the
potential mechanisms of the cytotoxic activity of the promising compounds were investigated in HT-
1080 cells, upon study of their effects on the phosphorylation of Akt, ERK and p38 MAPK. Most of the
active derivatives inhibit phosphorylation of Akt and ERK and/or induce p38 MAPK phosphorylation,
providing a potential indication on the mode of action of this class.
Received 10 January 2021
Received in revised form
11 March 2021
Accepted 14 March 2021
Available online 19 March 2021
Keywords:
Purine analogues
Pyrazolopyridine
Cytotoxicity
Apoptosis
© 2021 Elsevier Masson SAS. All rights reserved.
Cell cycle analysis
Akt
ERK
p38 MAPK
1. Introduction
Apoptosis is typically characterized by the activation of caspases,
which integrate signals at different cellular components, to induce
Based on incidence and mortality, cancer remains a substantial
health problem and a leading cause of mortality worldwide. Recent
advances towards the discovery of effective treatment options
focus in the development of molecularly targeted therapies, which
are selectively cytotoxic for cancer cells [1]. Among the anti-cancer
agents that undergo clinical trials, or are in clinical use, purine
isosters are of great importance, possessing multiple biological
activities, mainly acting as protein kinase inhibitors, mimicking ATP
in the kinase binding pocket. Interestingly, many of these com-
pounds target cell survival-related kinases and induce apoptosis in
tumor cells [2]. Escape of apoptosis has long been recognized as a
hallmark of cancer and targeting of cell death machinery is asso-
ciated to drug efficacy and reduced resistance to chemotherapy [3].
proteolytic cleavage of substrates, such as poly(ADP-ribose) poly-
merase (PARP), and a number of characteristic morphological cell
changes [4]. These catabolic enzymes can be activated by extrinsic
signals, as the binding of specific ligands to cell surface death re-
ceptors, or by intrising signals, as hypoxia, reactive oxygen species
production or inhibition of growth factor signaling [5]. The loss of
balance between cell division and cell death leads to the develop-
ment of various acute and chronic degenerative diseases, including
cancer [6]. Therefore, the successful pharmacological targeting of
specific survival-associated apoptotic signaling cascades in tumor
cells has long been considered as a promising strategy for anti-
cancer drug discovery [7]. Although studies concerning conven-
tional or targeted anticancer drug-induced apoptosis resulted in
somehow controversial results, strong evidence suggests the
implication of oncogenic mutations-driven dysregulations of PI3K/
Akt and MAPKs pathways in cell survival [8]. The existence of
* Corresponding author.
E-mail address: marakos@pharm.uoa.gr (P. Marakos).
feedback loops and
a cross-talk between these two signal
https://doi.org/10.1016/j.ejmech.2021.113387
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
transduction cascades, suggests the simultaneous inhibition of both
pathways for effective cancer chemotherapy [9]. Regarding the
transmission of anti-apoptotic signals, the PI3K/Akt cascade is
probably the best characterized and most prominent pathway [10].
The Ser/Thr kinase Akt, (also known as PKB), is overexpressed or
constitutively activated in a wide spectrum of human cancers. Its
full activation is dependent upon signaling of phosphatidylinositol-
3 kinase (PI3K), which recruits Akt/PKB to the inner surface of the
cell membrane, to be phosphorylated at two regulatory sites, Thr
308 and Ser 473 [11]. On the other hand, the mitogen-activated
protein kinases (MAPKs) are also known to play fundamental
roles in converting extracellular stimuli into multiple cell re-
sponses, including cell growth, cell division, migration, prolifera-
tion, differentiation and apoptosis [12]. In most of these processes,
MAPKs have a dual role, since they can act as activators, or in-
hibitors, depending on the stimulus and the cell type. Among
MAPKs, c-Jun N-terminal kinase JNK and p38 MAPK subgroups are
activated most notably following cell exposure to stress stimuli,
whereas extracellular signal regulated protein kinase (ERK 1 and 2)
cascades typically process cell growth factor-stimulated stimuli
[13]. MAPKs regulate apoptosis through multiple transcriptional
and post-transcriptional mechanisms and alterations of MAPK
signaling influence apoptotic responses to anti-cancer drugs
exposure [14]. Activation of p38 MAPK and JNK is usually associated
with pro-apoptotic effects, while ERK activation with anti-
apoptotic effects. However, the opposite effects cannot be
excluded, since the roles played by MAPKs in apoptosis tend to be
strongly dependent on context and profoundly influenced by cell
type, drug concentration and duration of exposure, as well as on the
type of assay employed to monitor apoptosis or cell survival [15].
In the course of an on-going research project concerning the
synthesis of purine isosters with potential cytotoxic and/or kinase
inhibitory activity, we have previously identified a pyrazolo[3,4-c]
pyridine derivative bearing a 5-(N-phenyl)carboxamidine substi-
tution. This compound resulted unexpectedly as a synthesis side
product, but, surprisingly it was endowed with very potent cyto-
toxic activity, possessing IC50 values in the nM range against a panel
of cancer cell lines that were used (pancreatic, ovarian and prostatic
cell lines) [16]. We also found that this compound causes a statis-
tically significant accumulation of PC-3 cells at G0/G1 phase and a
high percentage of apoptosis, but it also presented a certain degree
of cytotoxicity against normal human fibroblasts (WI-38). However,
the possible relationship between the observed cytotoxic activity
and apoptosis remains to be elucidated. In view of these findings,
we decided that this scaffold was worth to be further investigated
with the aim to generate more effective compounds. We used the
above-mentioned derivative as a lead, focusing on the unprece-
dented 5-arylcarboximidamido substitution and have decorated
the central pyrazolo[3,4-c]pyridine scaffold inserting denotative
substituents on the majority of the available positions of the het-
erocyclic system. We have thus introduced a small alkyl (methyl) or
a bulkier arylalkyl (p-methoxybenzyl) substituent on the N1 or N2 of
the pyrazole ring. We prepared analogues with a vacant position 3,
together with derivatives bearing a 3-phenyl group, which was
present in the lead compound. At position 7 we preserved the
arylamino substituent, also present in the lead compound and
alternatively we replaced it with cyclohexylamine or morpholine to
result in secondary or tertiary alkylamine analogues. Concerning
position 5, we took advantage of the carboxamidine phenyl group
in order to substitute it with suitable pharmacophores (fluorine or
piperazine) as well (Fig. 1).
contribute in mechanistic and SAR studies of this new class of
compounds.
2. Results and discussion
2.1. Chemistry
We have first synthesized groups of mono- or disubstituted
pyrazolopyridines using the chloride 1 (Scheme 1), which was
prepared according to a published procedure [17]. Compound 1 was
treated with either methyliodide, or 4-methoxybenzylchloride
(PMB chloride) to provide the N1 and N2 isomeric pyrazolo[3,4-c]
pyridines 2 [18], 3 (new compound) and 4, 5 [19], respectively.
These isomers were chromatographically separated and charac-
terized using 1D and 2D-NMR spectral data. Then, each isomer was
subjected to a cross-coupling reaction in the presence of Zn(CN)2
and Zn dust, using tris(dibenzylideneacetone)dipalladium(0)
[Pd2(dba)3] as catalyst and 1,10-ferrocenediyl-bis(diphenylphos-
phine) (dppf) as ligand and was converted to the corresponding 5-
carbonitrile (6, 7, 17 and 18). Suitable anilines subsequently reacted
with the carbonitriles to result in the carboxamidines 8e13 and
19e24. For the preparation of carboxamidines, we have investi-
gated a variety of experimental conditions in order to result in the
optimal methodology. Thus, we have tried the direct reaction of the
carbonitrile with aniline in reflux or under microwave irradiation,
or in the presence of AlCl3. Alternatively, we attempted the corre-
sponding reaction involving the attack of a generated aniline anion,
upon addition of NaH or potassiumbis(trimethylsilyl)amide in DMF
or THF solution, to the carbonitrile or to a corresponding imi-
noether, but all these methods gave rather poor results. We
concluded that the highest yield in carboxamidine formation (an
average of 60%) was effected using NaH as the base in DMSO so-
lution. Finally, the 4-methoxybenzyl- group of derivatives 11e13
was removed upon treatment with trifluoroacetic acid and pro-
vided the monosubstituted compounds 14e16.
The synthesis of the corresponding 3-phenyl-substituted ana-
logues 32e40 (Scheme 2), was accomplished through the iodin-
ation of 5-chloropyrazolopyridine 1. It is worth mentioning that
only the N1-regioisomer was obtained upon alkylation of the in-
termediate iodide 25 [20], obviously due to the steric hindrance of
the bulky 3-iodine. The resulting iodide 26 was then converted into
the corresponding 3-phenylsubstituted chloride 28 through a
Suzuki-type coupling using phenylboronic acid in the presence of
Pd(PPh3)4. Using an analogous methodology, we have also prepared
the iodide 27 [20] and the chloride 29 [19]. The chlorides 28 and 29
were then used for the synthesis of the target compounds 32e40,
following the already described procedure concerning the prepa-
ration of compounds 8e16.
Finally, the chloride 29 was used for the insertion of an addi-
tional substituent into the pyrazolopyridine scaffold. Hence, 29 was
converted to the N-oxide 41 using m-CPBA as oxidizing agent
(Scheme 3) [21]. The rearrangement of the N-oxide in the presence
of
phosphorous
oxychloride,
resulted
in
the
5,7-
dichloropyrazolopyridine 42 [21] which was used for the nucleo-
philic substitution of the 7-chloro group by 3,4,5-
trimethoxyaniline, cyclohexylamine or morpholine in order to
provide compounds 43 [21], 44 and 45 respectively. These were
first converted into the carbonitriles 46e48 and subsequently to
the carboxamidines 49e66, following the above-mentioned
methodology.
Therefore, we present herein the synthesis and biological ac-
tivity evaluation of these derivatives, through the examination of
their effects on the viability, cell-cycle regulation and pivotal kinase
activity of three representative cancer cell lines, aiming to
2.2. Assessment of cytotoxicity
The target compounds, as well as the corresponding carbon-
itriles, were tested for their cytotoxic activity on three human
2

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
Fig. 1. Design of the new pyrazolo[3,4-c]pyridines.
cancer cell lines (all from American Type Culture Collection; ATCC,
Rockville, MD, USA), the mammary adenocarcinoma MDA-MB-231,
the fibrosarcoma HT-1080 and the prostate adenocarcinoma PC-3,
using doxorubicin hydrochloride as positive control. The most
potent derivatives were also tested on normal human foreskin fi-
broblasts (AG01523 from Coriell Institute for Medical Research,
Camden, NJ, USA) for reasons of comparison. Results are presented
in Table 1.
Among the synthesized derivatives, the intermediate carbon-
itriles (6, 7, 17, 18, 30, 31, 46e48) were devoid of cytotoxic activity,
irrespective of the number or the nature of the substituents present
in the scaffold. The only deviation concerns compound 30, which
1-ylphenyl group, presents low IC50 values, ranging from 1.4 to 5.9
m
4-methoxybenzyl-substituted derivatives 35e37, the 5-[N-(3-
trifluoromethyl)phenyl] carboxamidine 36 is devoid of activity,
while the corresponding N-methylpiperazin-1-ylphenyl compound
37 is also endowed with strong cytotoxicity, presenting IC50 values
М in the three cell lines tested. On the other hand, among the N1-
in the low
mМ range. Compounds 38e40, that derived upon
removal of the p-methoxybenzyl group of 35e37, possess poor to
moderate cytotoxicity, in contrast to their inactive counterparts
14e16, which lack the 3-phenyl group.
Within our attempts to get insight in SARs of this series, we have
also inserted additional, selected substituents, namely 3,4,5-
trimethoxyphenylamino-, cyclohexylamino-as well as morpholin-
1-ylο-, at the vacant 7-position of the scaffold. By examining the
in vitro results, we can resume that the less interesting compounds
were the 5-[N-(3-trifluoromethyl)phenyl] carboxamidines 50, 53,
56, 59, 62 and 65. Indeed, the lack of activity was a common remark
for the majority of the analogously substituted derivatives syn-
thesized in this work, with the exception of 59 and 65, which
present a certain degree of activity. The 5-(N-phenyl)carbox-
amidines 49, 52, 55, 58, 61 and 64, showed a variety of IC50 values
with moderate (49, 55), strong (52, 58, 64), or very interesting (61)
cytotoxicity. Concerning the 5-[N-methylpiperazin-1-ylphenyl]
substituted compounds of this series, in analogy with the above-
mentioned presented remarks, the vast majority of them, namely
51, 54, 57 and 63, present notable cytotoxic properties, with IC50
showed marginal activity against HT-1080 cell line (IC50: 37.4 mM).
Similarly, the majority of the N1- or N2-methyl-5-
carboxamidines 8e10 and 19e21 respectively, do not possess
interesting cytotoxicity. However, even if lack of activity is obvious
for compounds 8, 9, 19 and 20 the 5-(N-methylpiperazin-1-
ylphenyl)carboxamidines 10 and 21, show a noteworthy degree of
activity, with IC50 values in the range of 10.9e38.0 mM against all
three cell lines tested. On the other hand, the N1-4-methoxybenzyl-
substituted derivatives 11e13 and in particular their N2-substituted
counterparts 22e24, possess marginal (11, 12) to strong cytotoxic
activity, with IC50 values ranging from 1.13 to 13.3 mM. Among these
analogues, 22 possesses the most interesting profile, exhibiting low
micromolar IC50’s against all tested cell lines. It is of interest that
compounds that do not possess a N-pyrazole ring substituent
(14e16) are almost inactive.
Upon the insertion of the 3-phenyl ring, we could extract the
following remarks concerning the pairs of analogously N1-
substituted compounds. The N1-methyl-substituted derivatives 32
values in the nM, or low
the similarly substituted compounds 60 and 66 appear slightly less
active (IC50’s: 3.4e8.3 M). A noticeable structural feature of the
mМ range (IC50’s: 0.64e4.63 mM). However,
m
most active derivatives (51, 52, 54, 57, 61, 63) is that, except for 51,
and 33 showed moderate cytotoxicity (IC50’s: 11e22
mМ), whereas
they all bear a 7-alkylamino group.
compound 34, which bears at position 5-, the N-methylpiperazin-
3

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
Scheme 1. a: for 2, 3: MeI, KOH, acetone, Ar, rt, 1 h; for 4, 5: NaH, 4-methoxybenzylchloride, DMF, Ar, rt, 30min; b: Zn(CN)2, Zn, Pd2(dba)3, dppf, N,N-dimethylacetamide (DMA), Ar,
reflux, 2 h; c: NaH, suitable aniline, DMSO, Ar, rt, 3 h; d: CF3CO2H, Ar, 70 ꢀC, 5 h.
2.3. Cell cycle analysis
treated with these compounds for 24 h.
In accordancck that indicates DNA fragmentation, all seven
compounds were found to induce apoptosis. The latter was also
confirmed by Western analysis of the expression of poly (ADP-
ribose) polymerase (PARP). As shown in Fig. 2, all seven compounds
were found to induce the cleaved 89-kDa carboxy-terminal form of
Seven compounds exhibiting the lowest IC50’s, i.e. 22, 37, 51, 54,
57, 61, and 63 were further studied regarding their effects on cell
cycle phase distribution of HT-1080 cells. Table 2 summarizes the
results obtained by flow cytometric analysis of DNA content of cells
4

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
Scheme 2. a: NIS, MeOH, Ar, rt, 3 h; b: for 26: MeI, KOH, acetone, Ar, rt, 16 h; for 27: NaH, 4-methoxybenzylchloride, DMF, Ar, rt, 2 h; c: phenylboronic acid, Pd(PPh3)4, toluene/EtOH,
NaHCO3/H2O, Ar, reflux, 15e30 h; d: Zn(CN)2, Zn, Pd2(dba)3, dppf, N,N-dimethylacetamide, Ar, reflux, 3 h; e: NaH, suitable aniline, DMSO, Ar, rt, 3 h; f: CF3CO2H, Ar, 70 ꢀC, 5 h.
PARP, indicating that the cells were undergoing apoptosis.
may provide a clue on the mechanism underlying the cytotoxic
effects of the tested compounds [22]. Induction of p38 MAPK
phosphorylation has been also associated with apoptosis in a va-
riety of cell types [23e25].
2.4. Effects of the compounds on kinase activation
In an attempt to gain insight into the molecular mode of action
of the new derivatives, nine compounds (22, 34, 37, 51, 52, 54, 57, 61
and 63), with potent cytotoxic activities were selected and tested in
a cell culture setting (HT-1080 cells) for their effects on the phos-
phorylation of pivotal kinases, such as Akt/PKB, extracellular signal-
regulated kinase (ERK) and p38 mitogen-activated protein kinase.
As shown in Fig. 3, 37, 57, and 63 completely blocked phosphory-
lation of Akt at serine 473, while 51 exhibited a modest inhibitory
effect.
Moreover, 37, 51, 54, 57 and to a lesser extent 63 inhibited also
phosphorylation of ERK. All these derivatives possess the 5-(N-
methylpiperazin-1-ylphenyl)-substitution pattern, which, together
with the 3-phenyl group seem to be favorable issues for the com-
pounds to display activity. On the other hand, some compounds
were observed to stimulate phosphorylation of the kinases tested,
more precisely, 22 of ERK and 52 of Akt. It is noticeable that both
derivatives are 5-(N-phenyl)carboxamidines and in addition, 22
lacks the 3-phenyl group. The phosphorylation stimulation is more
profound in the case of p38 MAPK, most notably with the tetra-
substitued compound 51 and the trisubstituted analogues 61 and
63.
2.5. Cytotoxicity against normal human fibroblasts
The most active of the synthesized compounds were examined
for cytotoxicity against normal human foreskin fibroblasts
(AG01523). Compounds 22 and 52 present very low toxicity,
whereas 34, 37, 61 and 63 are 2e3 fold less toxic on normal cells.
Finally, three compounds, namely 51, 54 and 57 are equally toxic
against normal and cancer cells (Table 1). The N2 substituted
compound 22 presents a quite interesting cytotoxicity profile, be-
ing active against the three cancer cell lines tested and not cytotoxic
against the normal cells. On the other hand, the majority of the
active analogues bear a 3-phenyl substituent. The most effective
compounds are characterized by a vacant 7-position or a 7-
alkylamino substituent, since the 7-arylamino substituted deriva-
tive 51 appears cytotoxic for normal cells. Furthermore, it seems
that the most decorated derivatives 51, 54 and 57 albeit endowed
with strong cytotoxic properties, appear rather toxic against the
normal cell line. Overall, considering both cytotoxicity and selec-
tivity, compounds 52, its respective N1-unsubstituted derivative 61
and 63 possess the most promising profile.
The inhibitory effects on the phosphorylation of Akt and ERK
5

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
Scheme 3. a: m-CPBA, CHCl3, rt, 3d; b: POCl3, THF, rt, 12 h; c: for 43: NaH, 3,4,5-trimethoxyaniline, DMF, Ar, 100 ꢀC, 2 h; for 44: cyclohexylamine, 2-ethoxyethanol, Ar, reflux, 12 h;
for 45: morpholine, Ar, reflux, 1 h; d: Zn(CN)2, Zn, Pd2(dba)3, dppf, N,N-dimethylacetamide, Ar, reflux, 2 h; e: NaH, suitable aniline, DMSO, Ar, rt, 3 h; f: CF3CO2H, Ar, 70 ꢀC, 5 h.
3. Conclusion
Bruker Avance III 600 or a Bruker Avance DRX 400 instrument,
whereas 13C NMR spectra were recorded on a Bruker Avance III 600
or a Bruker AC 200 spectrometer in deuterated solvents and were
In conclusion, among the new pyrazolopyridine carboxamidines
presented in this work, a number of biologically interesting ana-
logues have been emerged, with reasonable SARs. Some in-
termediates, possessing the p-methoxybenzyl group on the
pyrazole nitrogen were also found to possess strong cytotoxic
properties. A striking structural feature in the whole series is the
referenced to TMS (d
scale). The signals of 1H and 13C spectra were
unambiguously assigned by using 2D NMR techniques: 1H1H COSY,
NOESY, HMQC, and HMBC. For the interpretation of the NMR data
throughout the experimental part, the following abbreviation are
used concerning the central scaffold substituents: TMP: trime-
thoxyphenyl, PMB: p-methoxybenzyl. Mass spectra were recorded
with a LTQ Orbitrap Discovery instrument, possessing an Ionmax
ionization source. The purity of the key compounds (>95%) was
determined on Thermo Scientific liquid chromatography (LC) sys-
presence of
a 5-[N-methylpiperazin-1-ylphenyl] substituent,
conferring profound cytotoxic activity in this class of compounds.
However, this substitution reduces the selectivity, resulting in
medium cytotoxicity against normal cells. Overall, the 7-
cyclohexylamino-substituted derivatives 52, 61 and 63 present
the most promising profile, in terms of cytotoxic activity and
selectivity against normal cells. Most compounds seemed to pro-
voke a G0/G1 arrest of HT-1080 cells and induce apoptosis. From a
mechanistic point of view, the majority of the active derivatives
inhibit phosphorylation of Akt and ERK kinases and/or induce p38
MAPK phosphorylation and this may contribute to indicate a po-
tential mode of action of this class of compounds.
tem (Discovery HS-C18 column 250 ꢁ 4.6 mm, 5
mm; mobile phase
CH3CN/H2O; UV detector; absorbance at 254 nm). Flash chroma-
tography was performed on Merck silica gel 60 (0.035e0.070 mm).
Analytical thin layer chromatography (TLC) was carried out on
precoated (0.25 mm) silica gel F-254 plates.
4.1.1. 5-Chloro-1-methyl-1Н-pyrazolo[3,4-c]pyridine (2) and 5-
chloro-2-methyl-2Н-pyrazolo[3,4-c]pyridine (3)
Compound 2 has been reported to a patent [18]. Thus, we pro-
vide here a brief synthetic procedure as well as the identification
data of both derivatives.
4. Experimental section
4.1. Chemistry
To a cold solution of 1 (800 mg, 5.21 mmol) [17] in acetone
(55 ml), was added under argon potassium hydroxide (440 mg,
7.84 mmol) and the mixture was stirred for 30 min. Then, meth-
yliodide was added (0.35 ml, 5.21 mmol) and stirring continued for
an additional hour. The solvent was then vacuum-evaporated, the
Melting points were determined on a Büchi apparatus and are
uncorrected. FT-IR spectra were recorded on a PerkinElmer RX1 FT-
IR spectrometer. 1H NMR spectra and 2D spectra were recorded on a
6

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
Table 1
Cytotoxic effect of the compounds against MDA-MB-231, HT1080, PC-3 and AG01523 cell linesa.
Cmpd
MDA-MB-231
HT-1080
PC-3
AG01523
R1
R2
R3
R4
IC50 (mM)
8
9
10
N1-(CH3)
N1-(CH3)
N1-(CH3)
H
3-CF3
H
H
H
H
H
H
>50
>50
30.2 4.7
N.T.
33.8 1.3
24.0 5.9
N.T.
>50
32.3 0.1
N.T.
N.T.
N.T.
11
12
13
N1- PMB
N1- PMB
N1- PMB
H
3-CF3
H
H
H
H
H
H
34.7 7.9
29.8 3.4
5.70 0.47
25.7 1.2
24.9 4.3
5.30 0.28
36.0 2.8
27.6 3.5
6.10 0.81
N.T.
N.T.
N.T.
14
15
16
H
H
H
H
3-CF3
H
H
H
H
H
H
>50
>50
47.5 1.2
N.T.
36.5 6.4
31.3 0.5
N.T.
>50
45.4 3.2
N.T.
N.T.
N.T.
19
20
21
N2-(CH3)
N2-(CH3)
N2-(CH3)
H
3-CF3
H
H
H
H
H
H
>50
>50
20.5 9.6
N.T.
N.T.
21.0 9.0
N.T.
N.T.
24.8 5.2
N.T.
N.T.
N.T.
22
23
24
N2- PMB
N2- PMB
N2- PMB
H
3-CF3
H
H
H
H
H
H
1.52 0.01
15.5 7.9
8.00 0.98
1.13 0.34
13.0 8.1
3.20 1.74
2.04 0.91
12.8 6.7
9.90 3.37
32.40 10.60
N.T.
N.T.
32
33
34
N1-(CH3)
N1-(CH3)
N1-(CH3)
H
3-CF3
C6H5
C6H5
C6H5
H
H
H
11.1 4.7
21.4 0.1
4.9
17.4 2.7
17.8 1.3
1.4
17.3 1.6
20.9 6.4
5.9
N.T.
N.T.
10.40 0.20
0.13
0.18
0.63
35
36
37
N1- PMB
N1- PMB
N1- PMB
H
3-CF3
C6H5
C6H5
C6H5
H
H
H
5.40 0.85
>50
1.76 1.01
4.70 0.20
46.7 2.9
1.09 0.38
6.30 1.14
>50
3.99 1.13
N.T.
N.T.
10.20 4.30
38
39
40
H
H
H
H
3-CF3
C6H5
C6H5
C6H5
H
H
H
11.0 4.8
22.3 2.2
15.9 3.2
22.0 4.0
24.6 0.8
11.4 6.8
23.0 2.1
19.9 0.4
17.1 2.7
N.T.
N.T.
N.T.
49
50
51
N1- PMB
N1- PMB
N1- PMB
H
3-CF3
C6H5
C6H5
C6H5
TMPNH
TMPNH
TMPNH
42.1 4.1
>50
0.89 0.00
11.1 5.6
N.T.
1.20 0.64
>50
N.T.
1.24 0.35
N.T.
N.T.
1.81 0.18
52
N1- PMB
H
C6H5
C6H11NH
3.4
3.4
1.1
26.80
0.27
>50
1.29 0.37
0.17
N.T.
0.64 0.07
0.22
N.T.
3.54 0.70
6.20
N.T.
3.33 1.38
53
54
N1- PMB
N1- PMB
3-CF3
C6H5
C6H5
C6H11NH
C6H11NH
55
56
57
N1- PMB
N1- PMB
N1- PMB
H
C6H5
C6H5
C6H5
21.2 2.7
>50
6.20 2.83
N.T.
16.4 3.5
N.T.
N.T.
3-CF3
N.T.
1.13 0.18
0.70 0.02
4.58 0.43
2.19 0.44
58
59
60
H
H
H
H
3-CF3
C6H5
C6H5
C6H5
TMPNH
TMPNH
TMPNH
4.50 1.37
7.60 1.66
4.60 0.14
3.90 1.20
6.70 2.49
3.40 0.51
5.80 1.37
13.90 0.28
4.30 0.73
N.T.
N.T.
N.T.
61
62
63
H
H
H
H
3-CF3
C6H5
C6H5
C6H5
C6H11NH
C6H11NH
C6H11NH
1.71 1.09
24.6 1.5
1.50 0.27
0.85 0.25
21.2 6.4
0.65 0.08
1.73 0.94
25.0 5.7
4.63 0.70
4.19 1.97
N.T.
12.30 5.03
(continued on next page)
7

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
Table 1 (continued)
Cmpd
MDA-MB-231
HT-1080
PC-3
AG01523
R1
H
R2
H
R3
R4
IC50 (mM)
64
65
66
C6H5
6.80 0.56
13.3 6.8
3.90 0.94
4.80 1.71
6.8 2.7
6.80 2.07
17.3 3.4
8.30 0.58
N.T.
N.T.
N.T.
H
H
3-CF3
C6H5
C6H5
5.60 2.52
Dox
0.19 0.23
0.008 0.003
0.38 0.20
0.40 0.16
PMB: 4-methoxybenzyl, TMP: 3,4,5-trimethoxyphenyl, N.Τ.: Not Tested, Dox: doxorubicin hydrochloride.
a
Еxperiments were performed in triplicate and the mean values standard deviation are indicated.
cyclohexane/ethyl acetate 8/2 as the eluent to provide both isomers
2 (440 mg, 50%) and 3 (410 mg, 47%).
Table 2
Cell cycle phase distribution (HT-1080 cells).
5-Chloro-1-methyl-1Н-pyrazolo[3,4-c]pyridine
(2):
d 4.15 (s, 3H,
Mp:
103e105 ꢀC (CH2Cl2/Et2O). 1H NMR (600 MHz, CDCl3)
Cmpd
G0/G1
S
G2/M
Sub-diploid
CH3), 7.59 (s, 1H, H-4), 7.94 (s, 1H, H-3), 8.69 (s, 1H, H-7). 13C NMR
22
37
51
54
57
61
63
control
56.57
46.75
26.67
38.53
34.31
40.23
43.78
32.86
7.35
36.07
35.98
9.29
48.57
31.48
43.29
41.85
47.55
24.06
17.77
27.79
14.34
21.09
8.27
17.27
64.04
12.90
34.21
16.48
14.37
19.59
(151 MHz, CDCl3)
d 36.41 (CH3), 114.27 (C-4), 130.63 (C-3a), 131.68
(C-3), 132.79 (C-7), 136.05 (C-7a), 140.80 (C-5). ESI-HRMS m/z:
calculated for C7H7ClN3 [MþH]þ: 168.0323, Found: 168.0337.
5-Chloro-2-methyl-2Н-pyrazolo[3,4-c]pyridine
(3):
Mp:
142e144 ꢀC (CH2Cl2/Et2O). 1H NMR (600 MHz, CDCl3)
d 4.26 (s, 3H,
13.49
5.36
CH3), 7.50 (s, 1H, H-4), 7.90 (s, 1H, H-3), 8.99 (s, 1H, H-7). 13C NMR
(151 MHz, CDCl3) d 41.27 (CH3), 112.80 (C-4), 123.17 (C-3), 126.75 (C-
3a), 140.35 (C-5), 143.60 (C-7), 144.73 (C-7a). ESI-HRMS m/z:
calculated for C7H6ClN3Na [MþNa]þ: 190.0143, found: 190.0159.
4.1.2. 1-Methyl-1Н-pyrazolo[3,4-c]pyridine-5-carbonitrile (6)
To a solution of 2 (440 mg, 2.61 mmol) in dry N,N-dimethyla-
cetamide (3 ml) were added under argon at rt zinc cyanide (183 mg,
1.54 mmol) and zinc dust (19 mg, 2.91 mmol), followed by the
addition of tris(dibenzylideneacetone)dipalladium (0) (48 mg,
0.02 mmol) and 1,10-bis(diphenylphosphino)ferrocene (58 mg,
0.11 mmol). The resulting mixture was refluxed for 2 h and upon
cooling, water was added and it was extracted with ethyl acetate.
The organic phase was washed with brine, dried (Na2SO4) and
concentrated to dryness. The crude product was purified by column
chromatography (silica gel) using a mixture of cyclohexane/ethyl
acetate 8/2e5/5 as the eluent, to obtain 6 (280 mg, 68%). Mp:
Fig. 2. Induction of PARP cleavage by the test compounds compared to negative
control cultures (C) and cultures treated with staurosporine (S) as positive control.
168e169 ꢀC (CH2Cl2/Et2O). IR (Nujol)
(600 MHz, CDCl3) 4.21 (s, 3H, CH3), 8.07 (s, 1H, H-4), 8.12 (s, 1H, H-
3), 8.96 (s, 1H, H-7). 13C NMR (151 MHz, CDCl3)
36.61 (CH3), 118.41
n
(C^N) 2227 cmꢂ1.1H NMR
d
d
(CN), 122.24 (C-4), 122.68 (C-3a), 126.96 (C-5), 133.52 (C-3), 135.33
(C-7), 136.72 (C-7a). ESI-HRMS m/z: calculated C8H6N4Na [MþNa]þ:
181.0485, found: 181.0499.
4.1.3. 1-(4-Methoxybenzyl)-1Н-pyrazolo[3,4-c]pyridine-5-
carbonitrile (7)
This compound was prepared following a method analogous to
that of 6, starting from 4. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 9/1 as the
eluent. Yield 60%. Mp: 137e139 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
(C^N) 2360 cmꢂ1.1H NMR (600 MHz, CDCl3)
d 3.75 (s, 3H, OCH3),
Fig. 3. Phosphorylation of Akt, ERK and p38 MAPK by the test compounds compared
to negative control cultures.
5.63 (s, 2H, CH2), 6.84 (d, 2H, PMB-H3,5, J ¼ 8.4 Hz), 7.20 (d, 2H,
PMB-H2,6, J ¼ 8.4 Hz), 8.08 (s, 1H, H-4), 8.16 (s, 1H, H-3), 8.85 (s, 1H,
H-7).13C NMR (151 MHz, CDCl3)
d 54.20 (CH2), 55.50 (OCH3), 114.71
residue was extracted with dichloromethane, the organic phase
was dried (Na2SO4) and concentrated to dryness. The residue was
purified by column chromatography (silica gel) using a mixture of
(PMB-C3,5), 118.41 (CN), 122.36 (C-4), 123.02 (C-3a), 127.07 (PMB-
C1), 127.64 (C-5), 129.31 (PMB-C2,6), 133.82 (C-3), 135.76 (C-7),
136.28 (C-7a), 160.07 (PMB-C4). ESI-HRMS m/z: calculated
8

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
C15H12N4NaO [MþNa]þ: 287.0904, found: 287.0900.
2H, C6H5NeH2,6, J ¼ 7.5 Hz), 7.12 (t, 1H, C6H5NeH4, J ¼ 7.5 Hz), 7.25
(d, 2H, PMB-H2,6, J ¼ 8.8 Hz), 7.39 (t, 2H, C6H5NeH3,5, J ¼ 7.8 Hz),
8.27 (s, 1H, H-3), 8.61 (brs, 1H, H-4), 8.99 (brs, 1H, H-7). 13C NMR
4.1.4. N-phenyl-1-methyl-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (8)
(151 MHz, CD3OD) d 54.31 (CH2), 55.87 (OCH3), 115.25 (C-4), 115.39
To a cooled solution of aniline (0.60 ml, 6.30 mmol) in dry DMSO
(5 ml), was added under argon NaH (60% in hexanes, 165 mg,
4.20 mmol) and the mixture was stirred at rt for 30 min. The nitrile
6 (60 mg, 0.39 mmol) was then added and the resulting mixture
was stirred at rt for 3 h. Water was then added followed by
extraction with ethyl acetate. The organic layer was washed suc-
cessively with water, brine and a 4% HCl solution and then dried
(Na2SO4) and concentrated to dryness. The crude product was pu-
rified by column chromatography (silica gel) using a mixture of
dichloromethane/methanol 9/1 as the eluent, to give compound 8
(90 mg, 96%). Mp: 133e135 ꢀC (CH2Cl2/Et2O). 1H NMR (600 MHz,
(PMB-C3,5), 124.01 (C6H5NeC2,6), 125.16 (C6H5NeC4), 129.72
(PMB-C1), 130.30 (PMB-C2,6), 130.33 (C-3a), 130.81 (C6H5NeC3,5),
134.23 (C-3), 135.31 (C-7), 138.20 (C-7a), 142.29 (C-5), 149.37
(C6H5NeC1), 157.46 (CNH), 161.26 (PMB-C4). ESI-HRMS m/z:
calculated C21H20N5O [MþH]þ: 358.1663. Found: 358.1646.
4.1.8. N-(3-Trifluoromethylphenyl)-1-(4-methoxybenzyl)-1Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (12)
This compound was prepared following a method analogous to
that of 8, starting from 7. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 3/7 as the
CD3OD)
(t, 2H, C6H5N-Н3,5, J ¼ 7.6 Hz), 8.34 (s, 1H, H-3), 8.71 (brs, 1H, H-4),
9.23 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
36.85 (CH3), 117.32
d
4.29 (s, 3H, CH3), 7.30e7.45 (m, 3H, C6H5NeH2,6,4), 7.55
eluent. Yield 80%. Oil. 1H NMR (600 MHz, CD3OD)
d 3.72 (s, 3H,
OCH3), 5.67 (s, 2H, CH2), 6.85 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz), 7.24 (d,
2H, PMB-H2,6, J ¼ 8.7 Hz), 7.27 (d, 1H, C6H4FN-H6, J ¼ 8.0 Hz), 7.30
(brs, 1H, C6H4FN-H2), 7.36 (d,1H, C6H4FN-H4, J ¼ 7.8 Hz), 7.52 (t,1H,
C6H4FN-H5, J ¼ 7.9 Hz), 8.24 (s, 1H, H-3), 8.62 (brs, 1H, H-4), 8.95
d
(C-4), 126.19 (C6H5NeC2,6), 128.86 (C6H5NeC4), 129.22 (C-3a),
129.36 (C-3),131.40 (C6H5NeC3,5),135.55 (C-7),137.12 (C-5),139.23
(C-7a), 140.00 (C6H5NeC1), 160.89 (CNH). ESI-HRMS m/z: calcu-
lated C14H14Nþ5 [MþH]þ: 252.1244. Found: 252.1237.
(brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 54.29 (CH2), 55.85
(OCH3), 115.18 (C-4), 115.38 (PMB-C3,5),120.54 (C6H4FN-C2),120.90
(C6H4FN-C6), 123.17, 124.97, 126.77, 128.57 (CF3), 127.50 (C6H4FN-
C4), 129.72 (PMB-C1), 130.28 (PMB-C2,6), 130.37 (C-3a), 131.48
(C6H4FN-C5), 132.66, 132.88, 133.09, 133.30 (C6H4FN-C3), 134.05 (C-
7), 135.27 (C-3), 138.17 (C-7a), 142.67 (C-5), 152.00 (C6H4FN-C1),
157.24 (CNH), 161.23 (PMB-C4). ESI-HRMS m/z: calculated
4.1.5. N-(3-Trifluoromethylphenyl)-1-methyl-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (9)
This compound was prepared following a method analogous to
that of 8, starting from 6. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 98/2 as
the eluent. Yield 81%. Mp: 123e125 ꢀC (CH2Cl2/Et2O). 1H NMR
C
22H19F3N5O [MþH]þ: 426.1537, found: 426.1521.
(600 MHz, CD3OD)
d
4.23 (s, 3H, CH3), 7.28e7.32 (m, 2H, C6H4FN-
4.1.9. N-[4-(4-methylpiperazin-1-yl)phenyl]-1-(4-methoxybenzyl)-
1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (13)
This compound was prepared following a method analogous to
that of 8, starting from 7. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol/triethyl-
amine 8/2/0.01 as the eluent. Yield 62%. Amorphous solid. 1H NMR
Н2,6), 7.37 (d, 1H, C6H4FN-H4, J ¼ 7.8 Hz), 7.55 (t, 1H, C6H4FN-H5,
J ¼ 7.8 Hz), 8.22 (s, 1H, H-3), 8.62 (brs, 1H, H-4), 9.05 (brs, 1H, H-7).
13C NMR (151 MHz, CD3OD)
d 36.59 (CH3), 115.03 (C-4), 120.55
(C6H4FN-C2), 120.90 (C6H4FN-C4), 123.18, 124.98, 126.78, 128.58
(CF3), 127.55 (C6H4FN-C6), 129.81 (C-3a), 131.51 (C6H4FN-C5),
132.69, 132.90, 133.11, 133.32 (C6H4FN-C3), 133.88 (C-7), 134.90 (C-
3), 138.78 (C-7a), 142.54 (C-5), 152.03 (C6H4FN-C1), 157.38 (CNH).
ESI-HRMS m/z: calculated C15H13N5Fþ3 [MþH]þ: 320.1118. Found:
320.1119.
(600 MHz, CD3OD)
d 2.37 (s, 3H, CH3), 2.64 (t, 4H, piperazine-H3,5,
J ¼ 4.9 Hz), 3.26 (t, 4H, piperazine-H2,6, J ¼ 4.9 Hz), 3.76 (s, 3H,
OCH3), 5.76 (s, 2H, CH2), 6.89 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz), 7.11 (d,
2H, C6H4NeH3,5, J ¼ 8.9 Hz), 7.22 (d, 2H, C6H4NeH2,6, J ¼ 8.9 Hz),
7.29 (d, 2H, PMB-H2,6, J ¼ 8.7 Hz), 8.37 (s, 1H, H-3), 8.67 (brs, 1H, H-
4.1.6. N-[4-(4-methylpiperazin-1-yl)phenyl]-1-methyl-1Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (10)
4), 9.14 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 46.23 (CH3),
49.88 (piperazine-C2,6), 54.51 (CH2), 55.90 (OCH3), 56.06 (pipera-
zine-C3,5), 115.45 (PMB-C3,5), 117.13 (C-4), 118.48 (C6H4NeC3,5),
126.75 (C6H4NeC2,6), 129.53 (PMB-C1), 129.96 (C-3a), 130.42
(PMB-C2,6), 131.92 (C6H4NeC4), 135.56 (C-7), 135.83 (C-3), 137.70
(C-7a), 138.54 (C-5), 151.81 (C6H4NeC1), 160.66 (CNH), 161.37
This compound was prepared following a method analogous to
that of 8, starting from 6.4-(4-Methylpiperazin-1-yl)aniline was
prepared following reported procedures [26]. Purification by col-
umn chromatography (silica gel) using a mixture of dichloro-
methane/methanol/triethylamine 8/2/0.01 as the eluent. Yield 60%.
(PMB-C4). ESI-HRMS m/z: calculated
456.2507, found: 456.2497.
C
26H30N7O [MþH]þ:
Mp: 190e192 ꢀC (CH2Cl2/Et2O). 1H NMR (600 MHz, CD3OD)
d
2.36
(s, 3H, piperazine-CH3), 2.63 (t, 4H, piperazine-Н3,5, J ¼ 4.9 Hz),
3.21 (t, 4H, piperazine-Н2,6, J ¼ 4.9 Hz), 4.25 (s, 3H pyrazole-CH3),
7.05e7.11 (m, 4H, C6H4N), 8.26 (s, 1H, H-3), 8.61 (brs, 1H, H-4), 9.12
4.1.10. N-phenyl-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (14)
A mixture of compound 11 (110 mg, 0.31 mmol) and trifluor-
acetic acid (5 ml) was heated under argon at 70 ꢀC for 5 h. Upon
vacuum evaporation, water was added to the residue, the pH was
adjusted to 10 using Na2CO3 and it was extracted with ethyl acetate.
The solvent was dried (Na2SO4) and evaporated to dryness and the
residue was purified by column chromatography (silica gel) using a
mixture of dichloromethane/methanol 9/1 as the eluent, to provide
14 (50 mg, 70%). Mp: 153e155 ꢀC (EtOAc/Et2O). 1H NMR (600 MHz,
(brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 36.69 (pyrazole-CH3),
46.26 (piperazine-CH3), 50.49 (piperazine-C2,6), 56.15 (piperazine-
C3,5), 115.73 (C-4), 118.92 (C6H4NeC3,5), 125.49 (C6H4NeC2,6),
129.61 (C-3a),134.56 (C-7), 135.12 (C-3), 138.02 (C6H4NeC4),138.90
(C-7a), 140.42 (C-5), 150.34 (C6H4NeC1), 158.97 (CNH). ESI-HRMS
m/z: calculated C19H24Nþ7 [MþH]þ: 350.2088. Found: 350.2073.
4.1.7. N-phenyl-1-(4-methoxybenzyl)-1Н-pyrazolo[3,4-c]pyridine-
5-carboxamidine (11)
CD3OD)
Н3,5, J ¼ 7.8 Hz), 8.32 (s, 1H, H-3), 8.69 (brs, 1H, H-4), 9.10 (brs, 1H,
H-7). 13C NMR (151 MHz, CD3OD)
114.89 (C-4), 123.85
d 7.20e7.27 (m, 3H, C6H5NeH2,6,4), 7.45 (t, 2H, C6H5N-
This compound was prepared following a method analogous to
that of 8, starting from 7. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
d
(C6H5NeC2,6), 124.83 (C6H5NeC4), 128.91 (C-3a), 130.77
(C6H5NeC3,5), 134.74 (C-7), 135.75 (C-3), 139.42 (C-7a), 142.44 (C-
5), 150.24 (C6H5NeC1), 157.37 (CNH). ESI-HRMS m/z: calculated
eluent. Yield 91%. Oil. 1H NMR (600 MHz, CD3OD)
d
3.74 (s, 3H,
OCH3), 5.70 (s, 2H, CH2), 6.87 (d, 2H, PMB-H3,5, J ¼ 8.8 Hz), 7.08 (d,
C
13H12N5 [MþH]þ: 238.1088, found: 238.1081.
9

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
4.1.11. N-(3-Trifluoromethylphenyl)-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (15)
(600 MHz, CD3OD) d 4.33 (s, 3H, CH3), 7.10 (d, 2H, C6H5NeH2,6,
J ¼ 7.8 Hz), 7.14 (t, 1H, C6H5NeH4, J ¼ 7.4 Hz), 7.41 (t, 2H,
This compound was prepared following a method analogous to
that of 14, starting from 12. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
C6H5NeH3,5, J ¼ 7.7 Hz), 8.49 (s, 1H, H-3), 8.57 (brs, 1H, H-4), 9.13
(brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 41.38 (CH3), 115.04 (C-
4), 124.15 (C6H5NeC2,6), 125.22 (C6H5NeC4), 126.19 (C-3a), 128.26
(C-3), 130.83 (C6H5NeC3,5), 141.14 (C-5), 142.97 (C-7),147.40 (C-7a),
149.26 (C6H5NeC1), 158.00 (CNH). ESI-HRMS m/z: calculated
eluent. Yield 70%. Oil. 1H NMR (600 MHz, CD3OD)
d 7.29e7.34 (m,
2H, C6H4FN-H2,6), 7.38 (d, 1H, C6H4FN-H4, J ¼ 7.8 Hz), 7.55 (t, 1H,
C6H4FN-H5, J ¼ 7.8 Hz), 8.30 (s, 1H, H-3), 8.68 (brs, 1H, H-4), 9.03
C
14H14N5 [MþH]þ: 252.1244, found: 252.1235.
(brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 115.11 (C-4), 120.63
(C6H4FN-C2), 121.02 (C6H4FN-C4), 123.17, 124.97, 126.77, 128.57
(CF3), 127.63 (C6H4FN-C6), 128.93 (C-3a), 131.53 (C6H4FN-C5),
132.71, 132.93, 133.14, 133.35 (C6H4FN-C3), 134.74 (C-7), 135.81 (C-
3), 139.42 (C-7a), 142.41 (C-5), 151.75 (C6H4FN-C1), 157.62 (CNH).
ESI-HRMS m/z: calculated C14H11F3N5 [MþH]þ: 306.0962, found:
306.0950.
4.1.16. N-(3-Trifluoromethylphenyl)-2-methyl-2Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (20)
This compound was prepared following a method analogous to
that of 8, starting from 17. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 97/3 as
the eluent. Yield 81%. Mp: 147e149 ꢀC (CH2Cl2/Et2O). 1H NMR
(600 MHz, CD3OD)
d 4.33 (s, 3H, CH3), 7.28e7.32 (m, 2H, C6H4FN-
4.1.12. N-[4-(4-methylpiperazin-1-yl)phenyl]-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (16)
Н-2,6), 7.37 (d, 1H, C6H4FN-H4, J ¼ 7.8 Hz), 7.55 (t, 1H, C6H4FN-H5,
J ¼ 7.8 Hz), 8.48 (s, 1H, H-3), 8.58 (brs, 1H, H-4), 9.13 (brs, 1H, H-7).
This compound was prepared following a method analogous to
that of 14, starting from 13. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol/triethyl-
amine 8/2/0.01 as the eluent. Yield 75%. Mp: 228e230 ꢀC (EtOAc/
13C NMR (151 MHz, CD3OD)
d 41.37 (CH3), 114.94 (C-4), 120.67
(C6H4FN-C2), 120.95 (C6H4FN-C4), 123.19, 124.99, 126.79, 128,59
(CF3), 126.24 (C-3a), 127.66 (C6H4FN-C6), 128.20 (C-3), 131.52
(C6H4FN-C5), 132.70, 132.92, 133.13, 133.34 (C6H4FN-C3), 141.58 (C-
5), 142.83 (C-7), 147.41 (C-7a), 151.95 (C6H4FN-C1), 157.88 (CNH).
ESI-HRMS m/z: calculated C15H13F3N5 [MþH]þ: 320.1118, found:
320.1132.
Et2O). 1H NMR (600 MHz, CD3OD)
piperazine-H3,5,
J ¼ 4.6 Hz), 7.05e7.09 (m, 4H, C6H4N), 8.32 (s, 1H, H-3), 8.65 (brs,
1H, H-4), 9.07 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
46.26
d
2.36 (s, 3H, CH3), 2.63 (t, 4H,
J
¼
4.6 Hz), 3.20 (t, 4H, piperazine-H2,6,
d
(CH3), 50.45 (piperazine-C-2,6), 56.15 (piperazine-C-3,5), 115.91 (C-
4), 118.90 (C6H4NeC3,5), 125.67 (C6H4NeC2,6), 128.70 (C-3a),
135.57 (C-7), 135.96 (C-3), 137.33 (C6H4NeC4), 139.82 (C-7a), 140.02
(C-5), 150.55 (C6H4NeC1), 159.34 (eCNHe). ESI-HRMS m/z: calcu-
lated C18H22N7 [MþH]þ: 336.1932, found: 336.1935.
4.1.17. N-[4-(4-methylpiperazin-1-yl)phenyl]-2-methyl-2Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (21)
This compound was prepared following a method analogous to
that of 8, starting from 17. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol/triethyl-
amine 8/2/0.01 as the eluent. Yield 58%. Mp: 215e217 ꢀC (CH2Cl2/
4.1.13. 2-Methyl-2Н-pyrazolo[3,4-c]pyridine-5-carbonitrile (17)
This compound was prepared following a method analogous to
that of 6, starting from 3. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 6/4e3/7 as
Et2O). 1H NMR (600 MHz, CD3OD)
d 2.35 (s, 3H, piperazine-CH3),
2.62 (t, 4H, piperazine-Н3,5, J ¼ 4.9 Hz), 3.18 (t, 4H, piperazine-Н2,6,
J ¼ 4.9 Hz), 4.32 (s, 3H, pyrazole-CH3), 7.00e7.08 (m, 4H, C6H4N),
8.47 (s, 1H, H-3), 8.54 (s, 1H, H-4), 9.12 (s, 1H, H-7). 13C NMR
the eluent. Yield 50%. Mp: 220e221 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
(151 MHz, CD3OD) d 41.47 (pyrazole-CH3), 46.26 (piperazine-CH3),
(C^N) 2226 cmꢂ1.1H NMR (600 MHz, CDCl3)
8.03 (s, 1H, H-4), 8.13 (s, 1H, H-3), 9.19 (s, 1H, H-7). 13C NMR
(151 MHz, CDCl3) 41.66 (CH3), 118.72 (CN), 122.28 (C-3a), 122.42
d
4.32 (s, 3H, CH3),
50.43 (piperazine-C2,6), 56.14 (piperazine-C3,5), 115.97 (C-4),
118.87 (C6H4NeC3,5), 125.68 (C6H4NeC2,6), 125.95 (C-3a), 128.61
(C-3), 139.16 (C6H4NeC4), 139.18 (C-5), 143.48 (C-7), 147.42 (C-7a),
150.50 (C6H4NeC1), 159.46 (CNH). ESI-HRMS m/z: calculated
d
(C-4), 123.45 (C-5), 125.72 (C-3), 145.40 (C-7a), 145.53 (C-7). ESI-
HRMS ESI-HRMS m/z: calculated C8H6N4Na [MþNa]þ: 181.0485,
found: 181.0482.
C
19H24N7 [MþH]þ: 350.2088, found: 350.2076.
4.1.18. N-phenyl-2-(4-methoxybenzyl)-2Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (22)
This compound was prepared following a method analogous to
that of 8, starting from 18. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 95/5 as
the eluent. Yield 90%. Mp: 112e114 ꢀC (CH2Cl2/Et2O). 1H NMR
4.1.14. 2-(4-Methoxybenzyl)-2Н-pyrazolo[3,4-c]pyridine-5-
carbonitrile (18)
This compound was prepared following a method analogous to
that of 6, starting from 5. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 1/1 as the
eluent. Yield 66%. Mp: 137e138 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
(600 MHz, CD3OD) d 3.77 (s, 3H, OCH3), 5.66 (s, 2H, CH2), 6.92 (d,
(C^N) 2359 cmꢂ1.1H NMR (600 MHz, CDCl3)
d
3.80 (s, 3H, OCH3),
2H, PMB-H3,5, J ¼ 8.7 Hz), 7.10 (d, 2H, C6H5NeH2,6, J ¼ 7.5 Hz), 7.14
(t, 1H, C6H5NeH4, J ¼ 7.4 Hz), 7.36 (d, 2H, PMB-H2,6, J ¼ 8.7 Hz),
7.40 (t, 2H, C6H5NeH3,5, J ¼ 7.9 Hz), 8.51 (s, 1H, H-3), 8.55 (brs, 1H,
5.65 (s, 2H, CH2), 6.93 (d, 2H, PMB-H3,5, J ¼ 8.2 Hz), 7.34 (d, 2H,
PMB-H2,6, J ¼ 8.2 Hz), 8.03 (s, 1H, H-4), 8.18 (s, 1H, H-3), 9.21 (s, 1H,
H-7). 13C NMR (151 MHz, CDCl3)
d
55.33 (OCH3), 58.07 (CH2), 114.58
H-4), 9.14 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 55.91 (OCH3),
(PMB-C3,5), 118.77 (CN), 121.81 (C-3a), 122.54 (C-4), 123.01 (C-5),
124.65 (C-3), 126.08 (PMB-C1), 130.09 (PMB-C2,6), 145.07 (C-7a),
145.52 (C-7), 160.15 (PMB-C4). ESI-HRMS m/z: calculated
58.66 (CH2), 115.28 (C-4), 115.49 (PMB-C3,5), 124.22 (C6H5NeC2,6),
125.37 (C6H5NeC4), 126.04 (C-3a), 127.23 (C-3), 128.89 (PMB-C1),
130.85 (C6H5NeC3,5), 131.14 (PMB-C2,6), 140.92 (C-5), 143.34 (C-7),
147.38 (C-7a), 148.80 (C6H5NeC1), 158.09 (CNH), 161.63 (PMB-C4).
ESI-HRMS m/z: calculated C21H20N5O [MþH]þ: 358.1663, found:
358.1666.
C
15H12N4NaO [MþNa]þ: 287.0904, found: 287.0898.
4.1.15. N-phenyl-2-methyl-2Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (19)
This compound was prepared following a method analogous to
that of 8, starting from 17. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
eluent. Yield 93%. Mp: 157e159 ꢀC (CH2Cl2/Et2O). 1H NMR
4.1.19. N-(3-Trifluoromethylphenyl)-2-(4-methoxybenzyl)-2Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (23)
This compound was prepared following a method analogous to
that of 8, starting from 18. Purification by column chromatography
10

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
(silica gel) using a mixture of dichloromethane/ethyl acetate 6/4 as
(C^N) 2230 cmꢂ1.1H NMR (600 MHz, CDCl3)
d 4.22 (s, 3H, CH3), 7.41
the eluent. Yield 81%. Oil. 1H NMR (600 MHz, CD3OD)
d
3.73 (s, 3H,
(t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.47 (t, 2H, C6H5eH3,5, J ¼ 7.4 Hz), 7.81
OCH3), 5.60 (s, 2H, CH2), 6.88 (d, 2H, PMB-H3,5, J ¼ 8.5 Hz), 7.27 (d,
1H, C6H4FN-Н6, J ¼ 7.7 Hz), 7.29e7.37 (m, 4H, C6H4FN-H2,4, PMB-
Н2,6), 7.51 (t, 1H, C6H4FN-H5, J ¼ 7.8 Hz), 8.44 (s, 1H, H-3), 8.54 (brs,
(d, 2H, C6H5eH2,6, J ¼ 7.4 Hz), 8.22 (s, 1H, H-4), 8.94 (s, 1H, H-7). 13
C
NMR (151 MHz, CDCl3)
d 36.61 (CH3), 118.44 (CN), 122.44 (C-4),
123.02 (C-3a), 124.48 (C-5), 127.21 (C6H5eC2,6), 129.24 (C6H5eC4),
129.28 (C6H5eC3,5), 131.33 (C6H5eC1), 135.59 (C-7), 138.03 (C-7a),
144.88 (C-3). ESI-HRMS m/z: calculated C14H10N4Na [MþNa]þ:
257.0798, found: 257.0799.
1H, H-4), 9.09 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 55.94
(OCH3), 58.67 (CH2), 115.27 (C-4), 115.51 (PMB-C3,5), 120.82
(C6H4FN-C2), 121.23 (C6H4FN-C4), 123.20, 124.95, 126.75, 128.55
(CF3), 126.11 (C-3a), 127.27 (C-3), 127.81 (C6H4FN-C6), 128.91 (PMB-
C1), 131.15 (PMB-C2,6), 131.60 (C6H4FN-C5), 132.74, 132.95, 133.16,
133.38 (C6H4FN-C3), 141.29 (C-5), 143.24 (C-7), 147.40 (C-7a), 151.28
(C6H4FN-C1), 158.12 (CNH), 161.64 (PMB-C4). ESI-HRMS m/z:
calculated C22H19F3N5O [MþH]þ: 426.1537, found: 426.1522.
4.1.24. 1-(4-Methoxybenzyl)-3-phenyl-1Н-pyrazolo[3,4-c]
pyridine-5-carbonitrile (31)
This compound was prepared following a method analogous to
that of 6, starting from 29 [19]. Purification by column chroma-
tography (silica gel) using a mixture of cyclohexane/ethyl acetate 8/
4.1.20. N-[4-(4-methylpiperazin-1-yl)phenyl]-2-(4-
2 as the eluent. Yield 72%. Mp: 88e90 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
methoxybenzyl)-2Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (24)
This compound was prepared following a method analogous to
that of 8, starting from 18. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol/triethyl-
amine 8/2/0.01 as the eluent. Yield 58%. Amorphous solid. 1H NMR
(C^N) 2230 cmꢂ1.1H NMR (600 MHz, CDCl3)
d 3.75 (s, 3H, OCH3),
5.67 (s, 2H, CH2), 6.85 (d, 2H, PMB-H3,5, J ¼ 8.2 Hz), 7.26 (d, 2H,
PMB-H2,6, J ¼ 8.2 Hz), 7.45 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.51 (t, 2H,
C6H5eH3,5, J ¼ 7.5 Hz), 7.90 (d, 2H, C6H5eH2,6, J ¼ 7.5 Hz), 8.31 (s,
1H, H-4), 8.85 (brs, 1H, H-7). 13C NMR (151 MHz, CDCl3)
d 54.26
(600 MHz, CD3OD)
d
2.36 (s, 3H, CH3), 2.63 (t, 4H, piperazine-H3,5,
(CH2), 55.47 (OCH3), 114.66 (PMB-C3,5), 118.41 (CN), 122.72 (C-4),
123.20 (C-3a), 125.34 (C-5), 127.06 (PMB-C1), 127.49 (C6H5eC2,6),
129.31 (PMB-C2,6), 129.38 (C6H5eC3,5,4), 131.46 (C6H5eC1), 136.07
(C-7), 137.58 (C-7a), 145.22 (C-3), 160.01 (PMB-C4). ESI-HRMS m/z:
calculated C21H16N4NaO [MþNa]þ: 363.1217, found: 363.1215.
J ¼ 4.7 Hz), 3.21 (t, 4H, piperazine-H2,6, J ¼ 4.7 Hz), 3.78 (s, 3H,
OCH3), 5.67 (s, 2H, CH2), 6.93 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz),
7.04e7.08 (m, 4H, C6H4N), 7.37 (d, 2H, PMB-H2,6, J ¼ 8.7 Hz), 8.54 (s,
1H, H-3), 8.55 (brs, 1H, H-4), 9.16 (brs, 1H, H-7). 13C NMR (151 MHz,
CD3OD) d 46.26 (CH3), 50.68 (piperazine-C2,6), 55.93 (OCH3), 56.18
(piperazine-C3,5), 58.70 (CH2), 115.50 (C-4, PMB-C3,5), 118.06
(C6H4NeC3,5), 125.22 (C6H4NeC2,6), 125.99 (C-3a), 127.36 (C-3),
128.88 (PMB-C1), 131.16 (PMB-C2,6), 140.30 (C6H4NeC4), 141.13 (C-
5), 143.50 (C-7), 147.31 (C-7a), 149.98 (C6H4NeC1), 158.88 (CNH),
161.66 (PMB-C4). ESI-HRMS m/z: calculated C26H30N7O [MþH]þ:
456.2507, found: 456.2504.
4.1.25. N,3-diphenyl-1-methyl-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (32)
This compound was prepared following a method analogous to
that of 8, starting from 30. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 92/8 as
the eluent. Yield 96%. Mp: 88e90 ꢀC (CH2Cl2/Et2O). 1H NMR
(600 MHz, CD3OD)
d 4.25 (s, 3H, CH3), 7.08e7.17 (m, 3H, C6H5N-
4.1.21. 5-Chloro-3-iodo-1-methyl-1Н-pyrazolo[3,4-c]pyridine (26)
Н2,6,4), 7.39e7.45 (m, 3H, C6H5N-Н3,5, C6H5-Н4), 7.52 (t, 2H, C6H5-
Н3,5, J ¼ 7.7 Hz), 8.05 (d, 2H, C6H5eH2,6, J ¼ 7.4 Hz), 8.93 (brs, 1H,
This compound was prepared from 25 [20], following
a
methylation procedure previously described for pyrazolo[3,4-c]
pyridines [19]. Purification by column chromatography (silica gel)
using a mixture of dichloromethane/ethyl acetate 98/2 as the
H-4), 9.05 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 36.70 (CH3),
115.35 (C-4), 124.11 (C6H5NeC2,6), 125.28 (C6H5NeC4), 126.92 (C-
3a), 128.38 (C6H5eC2,6), 129.87 (C6H5eC4), 130.22 (C6H5eC3,5),
130.86 (C6H5NeC3,5), 133.59 (C6H5eC1), 134.41 (C-7), 140.23 (C-
7a), 142.34 (C-5), 146.00 (C-3), 149.17 (C6H5NeC1), 157.55 (CNH).
ESI-HRMS m/z: calculated C20H18N5 [MþH]þ: 328.1557, found:
328.1558.
ο
eluent. Yield: 68%. Mp: 144 C (EtOAc). 1H NMR (400 MHz, CDCl3)
d
4.18 (s, 3H, CH3), 7.33 (s, 1H, H-4), 8.64 (s, 1H, H-7). 13C NMR
(50 MHz, CDCl3)
d 36.99 (CH3), 88.31 (C-3), 114.56 (C-4), 133.11 (C-
7), 135.09 (C-3a), 136.85 (C-7a), 141.43 (C-5). ESI-HRMS m/z:
calculated C7H6ClIN3 [MþH]þ: 293.9290, found: 293.9276.
4.1.26. N-(3-Trifluoromethylphenyl)-3-phenyl-1-methyl-1Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (33)
This compound was prepared following a method analogous to
that of 8, starting from 30. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 98/2 as
the eluent. Yield 84%. Mp: 154e156 ꢀC (CH2Cl2/Et2O). 1H NMR
4.1.22. 5-Chloro-1-methyl-3-phenyl-1Н-pyrazolo[3,4-c]pyridine
(28)
The synthesis of this compound was effected following a pro-
cedure analogous to a previously reported one [19], starting from
26. Purification by column chromatography (silica gel) using a
mixture of dichloromethane/ethyl acetate 98/2 as the eluent. Yield
(600 MHz, CD3OD)
d 4.21 (s, 3H, CH3), 7.30 (d, 1H, C6H4FN-H6,
95%. Mp: 141 ꢀC (CH2Cl2/Et2O). 1H NMR (400 MHz, CDCl3)
d
4.14 (s,
J ¼ 7.8 Hz), 7.34 (brs, 1H, C6H4FN-H2), 7.36e7.43 (m, 2H, C6H4FN-
H4, C6H5-Н4), 7.50 (t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 7.55 (t, 1H,
C6H4FN-H5, J ¼ 7.8 Hz), 8.02 (d, 2H, C6H5eH2,6, J ¼ 7.7 Hz), 8.87
(brs, 1H, H-4), 8.99 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
3H, CH3), 7.39 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.47 (t, 2H, C6H5-Н3,5,
J ¼ 7.7 Hz), 7.77 (s, 1H, H-4), 7.82 (d, 2H, C6H5eH2,6, J ¼ 7.5 Hz), 8.64
(s, 1H, H-7). 13C NMR (50 MHz, CDCl3)
d 36.30 (CH3), 114.42 (C-4),
126.86 (C6H5eC2,6), 127.85 (C-3a), 128.55 (C6H5eC4), 129.03
(C6H5eC3,5), 131.93 (C6H5eC1), 132.97 (C-7), 137.24 (C-7a), 141.12
(C-5), 142.80 (C-3). ESI-HRMS m/z: calculated C13H11ClN3 [MþH]þ:
244.0637, found: 244.0642.
d 36.69 (CH3),115.26 (C-4),120.70 (C6H4FN-C2),121.12 (C6H4FN-C4),
123.19, 124.99, 126.78, 128.96 (CF3), 126.99 (C-3a), 127.67 (C6H4FN-
C6), 128.39 (C6H5eC2,6), 129.87 (C6H5eC4), 130.23 (C6H5eC3,5),
131.58 (C6H4FN-C5), 132.76, 132.97, 133.17, 133.35 (C6H4FN-C3),
133.63 (C6H5eC1), 134.31 (C-7), 140.27 (C-7a), 142.73 (C-5), 146.00
(C-3), 151.70 (C6H4FN-C1), 157.54 (CNH). ESI-HRMS m/z: calculated
4.1.23. 1-Methyl-3-phenyl-1Н-pyrazolo[3,4-c]pyridine-5-
carbonitrile (30)
C
21H17F3N5 [MþH]þ: 396.1431, found: 396.1432.
This compound was prepared following a method analogous to
that of 6, starting from 28. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 8/2e5/5 as
4.1.27. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-1-methyl-
1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (34)
the eluent. Yield 94%. Mp: 160e162 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
This compound was prepared following a method analogous to
11

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
that of 8, starting from 30. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 63%. Amorphous solid. 1H NMR (600 MHz, CD3OD)
(600 MHz, CD3OD)
d
2.38 (s, 3H, CH3), 2.65 (t, 4H, piperazine-Н3,5,
J ¼ 4.6 Hz), 3.28 (t, 4H, piperazine Н2,6, J ¼ 4.6 Hz), 3.76 (s, 3H,
OCH3), 5.80 (s, 2H, CH2), 6.91 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz), 7.12 (d,
2H, H-3000, Н-5000, J ¼ 7.7 Hz), 7.25 (d, 2H, H-2000, Н-6000, J ¼ 7.7 Hz), 7.36
(d, 2H, PMB-H2,6, J ¼ 8.7 Hz), 7.47 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.56
(t, 2H, C6H5eH3,5, J ¼ 7.7 Hz), 8.10 (d, 2H, C6H5eH2,6, J ¼ 7.3 Hz),
9.03 (brs, 1H, H-4), 9.17 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d
2.37 (s, 3H, piperazine-CH3), 2.64 (t, 4H, piperazine-Н3,5,
J ¼ 4.7 Hz), 3.24 (t, 4H, piperazine-Н2,6, J ¼ 4.7 Hz), 4.28 (s, 3H,
pyrazole-CH3), 7.09 (d, 2H, C6H4NeH3,5, J ¼ 8.1 Hz), 7.17 (d, 2H,
C6H4N-Н2,6, J ¼ 8.1 Hz), 7.45 (t,1H, C6H5eH4, J ¼ 7.4 Hz), 7.53 (t, 2H,
C6H5eH3,5, J ¼ 7.6 Hz), 8.06 (d, 2H, C6H5eH2,6, J ¼ 7.5 Hz), 8.97
(brs, 1H, H-4), 9.15 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d
46.20 (CH3), 49.69 (piperazine-C2,6), 54.64 (CH2), 55.91 (OCH3),
56.02 (piperazine-C3,5), 115.51 (PMB-C3,5), 117.71 (C-4), 118.37
(C6H4NeC3,5), 127.10 (C6H4NeC2,6), 127.17 (C-3a), 128.69
(C6H5eC2,6), 129.44 (PMB-C1), 130.31 (C6H5eC4, C6H4NeC4),
130.34 (C6H5eC3,5), 130.50 (PMB-C2,6), 133.13 (C6H5eC1), 136.10
(C-7), 137.22 (C-5), 140.02 (C-7a), 147.08 (C-3), 152.19 (C6H4NeC1),
160.98 (eCNHe), 161.42 (PMB-C4). ESI-HRMS m/z: calculated
d
36.89 (pyrazole-CH3), 46.24 (piperazine-CH3), 50.03 (piperazine-
C2,6), 56.07 (piperazine-C3,5), 116.87 (C-4), 118.60 (C6H4NeC3,5),
126.44 (C6H4NeC2,6), 126.57 (C-3a), 128.49 (C6H5eC2,6), 130.12
(C6H5eC4), 130.29 (C6H5eC3,5), 133.26 (C6H5eC1), 135.51 (C-7),
135.66 (C6H4NeC4), 138.50 (C-7a), 140.47 (C-5), 146.48 (C-3), 151.42
(C6H4NeC1), 160.06 (CNH). ESI-HRMS m/z: calculated C25H28N7
[MþH]þ: 426.2401, found: 426.2397.
C
32H34N7O [MþH]þ: 532.2820, found: 532.2810.
4.1.31. N,3-diphenyl-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine
(38)
This compound was prepared following a method analogous to
that of 14, starting from 35. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
eluent. Yield 75%. Mp: 163e165 ꢀC (EtOAc/Et2O). 1H NMR (600 MHz,
4.1.28. N,3-diphenyl-1-(4-methoxybenzyl)-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (35)
This compound was prepared following a method analogous to
that of 8, starting from 31. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 9/1 as
the eluent. Yield 94%. Mp: 170e172 ꢀC (CH2Cl2/Et2O). 1H NMR
CD3OD)
d 7.14e7.23 (m, 3H, C6H5NeH2,6,4), 7.42e7.48 (m, 3H,
(600 MHz, CD3OD)
d
3.76 (s, 3H, OCH3), 5.76 (s, 2H, CH2), 6.90 (d,
C6H5NeH3,5, C6H5-Н4), 7.53 (t, 2H, C6H5eH3,5, J ¼ 7.7 Hz), 8.07 (d,
2H, PMB-H3,5, J ¼ 8.7 Hz), 7.07 (d, 2H, C6H5NeH2,6, J ¼ 7.4 Hz), 7.12
(t, 1H, C6H5NeH4, J ¼ 7.5 Hz), 7.34 (d, 2H, PMB-H2,6, J ¼ 8.7 Hz),
7.39 (t, 2H, C6H5NeH3,5, J ¼ 7.8 Hz), 7.44 (t, 1H, C6H5eH4,
J ¼ 7.4 Hz), 7.54 (t, 2H, C6H5eH3,5, J ¼ 7.7 Hz), 8.10 (d, 2H,
C6H5eH2,6, J ¼ 7.3 Hz), 8.97 (s, 1H, H-4), 9.01 (s, 1H, H-7). 13C NMR
2H, C6H5eH2,6, J ¼ 7.4 Hz), 8.95e9.10 (m, 2H, H-4, H-7). 13C NMR
(151 MHz, CD3OD)
d 115.95 (C-4), 124.63 (C6H5NeC2,6), 125.91 (C-
3a), 126.10 (C6H5NeC4), 128.55 (C6H5eC2,6), 129.95 (C6H5eC4),
130.24 (C6H5eC3,5), 130.95 (C6H5NeC3,5), 133.70 (C6H5eC1),
135.60 (C-7), 140.81 (C-7a), 141.16 (C-5), 147.02 (C-3), 147.25
(C6H5NeC1), 158.38 (CNH). ESI-HRMS m/z: calculated C19H16N5
[MþH]þ: 314.1401, found: 314.1406.
(151 MHz, CD3OD) d 54.42 (CH2), 55.88 (OCH3), 115.26 (C-4), 115.46
(PMB-C3,5), 123.82 (C6H5NeC2,6), 124.85 (C6H5NeC4), 127.71 (C-
3a), 128.52 (C6H5eC2,6), 129.76 (PMB-C1), 129.94 (C6H5eC4),
130.25 (C6H5eC3,5), 130.35 (PMB-C2,6), 130.81 (C6H5NeC3,5),
133.65 (C6H5eC1), 134.52 (C-7), 139.69 (C-7a), 143.22 (C-5), 146.34
(C-3), 150.34 (C6H5NeC1), 157.20 (CNH), 161.33 (PMB-C4). ESI-
4.1.32. N-(3-Trifluoromethylphenyl)-3-phenyl-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (39)
This compound was prepared following a method analogous to
that of 14, starting from 36. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 9/1e5/
5 as the eluent. Yield 75%. Oil. 1H NMR (600 MHz, CD3OD)
HRMS m/z: calculated
434.1971.
C
27H24N5O [MþH]þ: 434.1976, found:
4.1.29. N-(3-Trifluoromethylphenyl)-3-phenyl-1-(4-
d 7.31e7.36 (m, 2H, C6H4FN-H2,6), 7.39 (d, 1H, C6H4FN-H4,
methoxybenzyl)-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (36)
This compound was prepared following a method analogous to
that of 8, starting from 31. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 9/1 as
J ¼ 7.8 Hz), 7.44 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.52e7.59 (m, 3H,
C6H5eH3,5, C6H4FN-Н5), 8.08 (d, 2H, C6H5eH2,6, J ¼ 7.6 Hz), 8.98
(brs, 1H, H-4), 9.04 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d
115.37 (C-4), 120.75 (C6H4FN-C2), 121.18 (C6H4FN-C4), 123.17,
the eluent. Yield 84%. Oil. 1H NMR (600 MHz, CD3OD)
d
3.71 (s, 3H,
124.97, 126.77, 128.67 (CF3), 126.26 (C-3a), 127.72 (C6H4FN-C6),
128.52 (C6H5eC2,6), 129.92 (C6H5eC4), 130.24 (C6H5eC3,5), 131.58
(C6H4FN-C5), 132.76, 132.97, 133.18, 133.39 (C6H4FN-C3), 133.78
(C6H5eC1), 135.10 (C-7), 140.97 (C-7a), 142.64 (C-5), 147.12 (C-3),
151.55 (C6H4FN-C1), 157.73 (eCNHe). ESI-HRMS m/z: calculated
OCH3), 5.67 (s, 2H, CH2), 6.85 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz),
7.25e7.30 (m, 3H, PMB-H2,6, C6H4FN-Н6), 7.32 (brs, 1H, C6H4FN-
H2), 7.37 (d, 1H, C6H4FN-H4, J ¼ 7.7 Hz), 7.40 (t, 1H, C6H5eH4,
J ¼ 7.4 Hz), 7.48e7.55 (m, 3H, C6H5eH3,5, C6H4FN-Н5), 8.05 (d, 2H,
C6H5eH2,6, J ¼ 7.4 Hz), 8.92 (brs, 2H, H-4, H-7). 13C NMR (151 MHz,
C
20H15F3N5 [MþH]þ: 382.1275, found: 382.1279.
CD3OD) d 54.39 (CH2), 55.84 (OCH3), 115.42 (PMB-C3,5, C-4), 120.61
(C6H4FN-C2), 121.00 (C6H4FN-C4), 123.18, 124.98, 126.78, 128.58
(CF3), 127.61 (C6H4FN-C6, C-3a), 128.47 (C6H5eC2,6), 129.64 (PMB-
C1), 129.91 (C6H5eC4), 130.21 (C6H5eC3,5), 130.32 (PMB-C2,6),
131.52 (C6H4FN-C5), 132.69, 132.90, 133.11, 133.32 (C6H4FN-C3),
133.58 (C6H5eC1), 134.39 (C-7), 139.61 (C-7a), 142.94 (C-5), 146.25
(C-3), 151.86 (C6H4FN-C1), 157.29 (CNH), 161.26 (PMB-C4). ESI-
HRMS m/z: calculated C28H23F3N5O [MþH]þ: 502.1850, found:
502.1841.
4.1.33. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-1Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (40)
This compound was prepared following a method analogous to
that of 14, starting from 37. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol/triethyl-
amine 8/2/0.01 as the eluent. Yield 75%. Mp: 148e150 ꢀC (EtOAc/
Et2O). 1H NMR (600 MHz, CD3OD)
piperazine-Н3,5,
d
2.37 (s, 3H, CH3), 2.65 (t, 4H,
J
¼
4.9 Hz), 3.28 (t, 4H, piperazine-Н2,6,
J ¼ 4.9 Hz), 7.12 (d, 2H, C6H4NeH3,5, J ¼ 8.7 Hz), 7.25 (d, 2H, C6H4N-
Н2,6, J ¼ 8.7 Hz), 7.48 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.57 (t, 2H,
C6H5eH3,5, J ¼ 7.7 Hz), 8.10 (d, 2H, C6H5eH2,6, J ¼ 7.1 Hz), 9.06 (brs,
4.1.30. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-1-(4-
methoxybenzyl)-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (37)
This compound was prepared following a method analogous to
that of 8, starting from 31. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 63%. Mp: 188e190 ꢀC (CH2Cl2/Et2O). 1H NMR
1H, H-4), 9.18 (brs, 1H, H-7). 13C NMR (151 MHz, CD3OD)
d 46.25
(CH3), 49.88 (piperazine-C2,6), 56.08 (piperazine-C-3,5), 117.38 (C-
4), 118.50 (C6H4NeC3,5), 125.84 (C-3a), 126.86 (C6H4NeC2,6),
128.66 (C6H5eC2,6), 130.18 (C6H5eC4), 130.33 (C6H5eC3,5), 133.46
12

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
(C6H5eC1), 136.66 (C-7, C6H4NeC4), 137.60 (C-7a), 140.45 (C-5),
147.65 (C-3), 151.93 (C6H4NeC1), 160.82 (eCNHe). ESI-HRMS m/z:
calculated C24H26N7 [MþH]þ: 412.2245, found: 412.2239.
126.64 (C-3a), 127.28 (C6H5eC-2,6), 127.71 (PMB-C1), 127.82 (PMB-
C2,6), 128.94 (C-7a), 129.02 (C6H5eC4), 129.13 (C6H5eC3,5), 131.40
(C6H5eC1), 133.77 (TMP-C4), 134.72 (TMP-C1), 142.56 (C-7), 144.91
(C-3), 153.03 (TMP-C3,5), 160.22 (PMB-C4). ESI-HRMS m/z: calcu-
lated C30H28N5O4 [MþH]þ: 522.2136, found: 522.2141.
4.1.34. N-cyclohexyl-5-chloro-3-phenyl-1-(4-methoxybenzyl)-1Н-
pyrazolo[3,4-c]pyridin-7-amine (44)
Cyclohexylamine (0.45 ml, 3.90 mmol) was added at rt, under Ar
to a solution of 42 (150 mg, 0.39 mmol) [21] in 2-ethoxyethanol
(2 ml) and the resulting solution was refluxed for 12 h. The
mixture was then concentrated in vacuo, water was added and
extracted with ethyl acetate. The organic phase was dried (Na2SO4)
and concentrated to dryness. The residue was purified by column
chromatography (silica gel) using a mixture of cyclohexane/ethyl
acetate 9/1 as the eluent, to result in 44 (150 mg, 86%) as an oil. 1H
4.1.37. 1-(4-Methoxybenzyl)-3-phenyl-7-(cyclohexylamino)-1Н-
pyrazolo[3,4-c]pyridine-5-carbonitrile (47)
This compound was prepared following a method analogous to
that of 6, starting from 44. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 9/1 as the
eluent. Yield 95%. Mp: 155e157 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
(NH)
3428, (C^N) 2228 cmꢂ1.1H NMR (600 MHz, CDCl3)
d 0.87e0.96 (m,
2H, cyclohexyl-H), 1.09e1.18 (m, 1H, cyclohexyl-H), 1.31e1.40 (m,
2H, cyclohexyl-H), 1.56e1.65 (m, 3H, cyclohexyl-H), 1.82e1.90 (m,
2H, cyclohexyl-H), 3.79 (s, 3H, OCH3), 3.89e3.99 (m,1H, cyclohexyl-
H1), 4.78 (d, 1H, D2O exch., NH, J ¼ 6.2 Hz), 5.80 (s, 2H, CH2), 6.92 (d,
2H, PMB-H3,5, J ¼ 8.7 Hz), 7.09 (d, 2H, PMB-H2,6, J ¼ 8.7 Hz), 7.43 (t,
1H, C6H5eH4, J ¼ 7.4 Hz), 7.52 (t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 7.62 (s,
1H, H-4), 7.88 (d, 2H, C6H5eH2,6, J ¼ 7.4 Hz). 13C NMR (151 MHz,
NMR (600 MHz, CDCl3)
d 0.84e0.95 (m, 2H, cyclohexyl-H),
1.08e1.18 (m, 1H, cyclohexyl-H), 1.31e1.41 (m, 2H, cyclohexyl-H),
1.54e1.62 (m, 3H, cyclohexyl-H), 1.82e1.89 (m, 2H, cyclohexyl-H),
3.78 (s, 3H, OCH3), 3.91e3.98 (m, 1H, cyclohexyl-H1), 4.65 (d, 1H,
D2O exch., NH, J ¼ 7.2 Hz), 5.75 (s, 2H, CH2), 6.91 (d, 2H, PMB-H3,5,
J ¼ 8.7 Hz), 7.08 (d, 2H, PMB-H2,6, J ¼ 8.7 Hz), 7.14 (s, 1H, H-4), 7.40
(t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.50 (t, 2H, C6H5eH3,5, J ¼ 7.8 Hz), 7.91
CDCl3)
d 24.76 (cyclohexyl-C3,5), 25.85 (cyclohexyl-C4), 32.71
(d, 2H, C6H5eH2,6, J ¼ 7.2 Hz). 13C NMR (151 MHz, CDCl3)
d
24.65
(cyclohexyl-C2,6), 49.54 (cyclohexyl-C1), 55.57 (OCH3), 55.75
(CH2), 112.89 (C-4), 115.13 (PMB-C3,5), 119.46 (CN), 122.19 (C-5),
125.87 (C-7a), 127.42 (C6H5eC2,6),127.59 (PMB-C2,6), 128.19 (PMB-
C1), 128.83 (C6H5eC4), 128.98 (C-3a), 129.17 (C6H5eC3,5), 132.09
(C6H5eC1), 144.64 (C-7), 145.61 (C-3), 160.17 (PMB-C4). ESI-HRMS
m/z: calculated C27H28N5O [MþH]þ: 438.2289, found: 438.2286.
(cyclohexyl-C3,5), 25.86 (cyclohexyl-C4), 32.76 (cyclohexyl-C2,6),
49.30 (cyclohexyl-C1), 55.53 (OCH3, CH2), 102.53 (C-4), 115.01
(PMB-C3,5), 127.22 (C6H5eC2,6, PMB-C1), 127.54 (PMB-C2,6),
128.26 (C6H5eC4), 128.62 (C-7a), 128.99 (C6H5eC3,5), 129.35 (C-
3a), 132.79 (C6H5eC1), 140.42 (C-5), 142.99 (C-7), 144.38 (C-3),
160.02 (PMB-C4). ESI-HRMS m/z: calculated C26H26ClN4O [M-H]-:
445.1800, found: 445.1787.
4.1.38. 1-(4-Methoxybenzyl)-3-phenyl-7-(morpholin-4-yl)-1Н-
pyrazolo[3,4-c]pyridine-5-carbonitrile (48)
4.1.35. 5-Chloro-3-phenyl-1-(4-methoxybenzyl)-7-(morpholin-4-
yl)-1Н-pyrazolo[3,4-c]pyridine (45)
This compound was prepared following a method analogous to
that of 6, starting from 45. Purification by column chromatography
(silica gel) using a mixture of cyclohexane/ethyl acetate 7/3 as the
This compound was prepared following a method analogous to
that of 44, starting from 42 [21], using morpholine as solvent and
reflux for 1 h. Purification by column chromatography (silica gel)
using a mixture of cyclohexane/ethyl acetate 7/3 as the eluent.
eluent. Yield 86%. Mp: 153e155 ꢀC (CH2Cl2/Et2O). IR (Nujol)
n
(C^N) 2226 cmꢂ1.1H NMR (600 MHz, CDCl3)
d 3.33 (brs, 4H, mor-
pholine-Н3,5), 3.74 (s, 3H, OCH3), 3.93 (brs, 4H, morpholine-Н2,6),
5.82 (s, 2H, CH2), 6.81 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz), 7.21 (d, 2H,
PMB-H2,6, J ¼ 8.7 Hz), 7.44 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.50 (t, 2H,
C6H5eH3,5, J ¼ 7.6 Hz), 7.86 (d, 2H, C6H5eH2,6, J ¼ 7.3 Hz), 8.01 (s,
Yield 78%. Oil. 1H NMR (600 MHz, CDCl3)
d 3.31 (t, 4H, morpholine-
Н3,5, J ¼ 4.5 Hz), 3.71 (s, 3H, OCH3), 3.91 (t, 4H, morpholine-Н2,6,
J ¼ 4.5 Hz), 5.76 (s, 2H, CH2), 6.80 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz), 7.20
(d, 2H, PMB-H2,6, J ¼ 8.7 Hz), 7.39 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.48
(t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 7.58 (s, 1H, H-4), 7.89 (d, 2H, C6H5eH-
1H, H-4). 13C NMR (151 MHz, CDCl3)
d 51.12 (morpholine-C3,5),
53.70 (CH2), 55.31 (OCH3), 66.51 (morpholine-C2,6), 114.22 (PMB-
C3,5), 118.57 (CN), 118.75 (C-4), 121.46 (C-5), 127.54 (C6H5eC2,6),
127.63 (C-7a), 128.62 (PMB-C2,6), 128.70 (PMB-C1), 129.19
(C6H5eC4,3,5), 131.60 (C6H5eC1), 132.38 (C-3a), 146.59 (C-3),
150.56 (C-7), 159.30 (PMB-C4). ESI-HRMS m/z: calculated
2,6, J ¼ 7.3 Hz). 13C NMR (151 MHz, CDCl3)
d 51.03 (morpholine-
C3,5), 53.23 (CH2), 55.10 (OCH3), 66.46 (morpholine-C2,6), 109.89
(C-4), 113.96 (PMB-C3,5), 127.21 (C6H5eC2,6), 128.39 (PMB-C2,6),
128.51 (C6H5eC4), 128.89 (C6H5eC3,5), 129.07 (PMB-C1, C-7a),
130.85 (C-3a), 132.18 (C6H5eC1), 139.19 (C-5), 144.85 (C-3), 148.71
(C-7), 159.07 (PMB-C4). ESI-HRMS m/z: calculated C24H24ClN4O2
[MþH]þ: 435.1583, found: 435.1564.
C
25H24N5O2 [MþH]þ: 426.1925, found: 426.1926.
4.1.39. N,3-diphenyl-1-(4-methoxybenzyl)-7-(3,4,5-
trimethoxyphenylamino)-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (49)
4.1.36. 1-(4-Methoxybenzyl)-3-phenyl-7-(3,4,5-
trimethoxyphenylamino)-1Н-pyrazolo[3,4-c]pyridine-5-
carbonitrile (46)
This compound was prepared following a method analogous to
that of 8, starting from 46. Purification by column chromatography
(silica gel) using a mixture of ethyl acetate/methanol 95/5 as the
eluent. Yield 40%. Mp: 139e141 ꢀC (CH2Cl2/Et2O). 1H NMR
This compound was prepared following a method analogous to
that of 6, starting from 43 [21]. Purification by column chroma-
tography (silica gel) using a mixture of cyclohexane/ethyl acetate 2/
1 as the eluent. Yield 90%. Mp: 188e190 οC (EtOAc/cyclohexane). IR
(600 MHz, Acetone-d6) d 3.66 (s, 3H, PMB-OCH3), 3.69 (s, 3H, TMP-
4-OCH3), 3.73 (s, 6H, TMP-3,5-OCH3), 6.25 (brs, 2H, CH2), 6.79 (d,
2H, PMB-H3,5, J ¼ 8.4 Hz), 6.95 (brs, 2H, TMP-H2,6), 7.20 (d, 2H,
PMB-H2,6, J ¼ 8.4 Hz), 7.27 (t, 1H, C6H5eH4, J ¼ 6.8 Hz), 7.32 (t, 1H,
(Nujol)
3.77 (s, 3H, PMB-OCH3), 3.78 (s, 3H, TMP-4-OCH3), 3.79 (s, 6H,
n
(NH) 3420, (C^N) 2225 cmꢂ1.1H NMR (600 MHz, CDCl3)
d
TMP-3,5-OCH3), 5.89 (s, 2H, CH2), 6.47 (s, 2H, TMP-H2,6), 6.78 (brs,
1H, D2O exch., NH), 6.98 (d, 2H, PMB-H3,5, J ¼ 8.0 Hz), 7.22 (d, 2H,
PMB-H2,6, J ¼ 8.0 Hz), 7.45 (t, 1H, C6H5eH4, J ¼ 8.0 Hz), 7.52 (t, 2H,
C6H5eH3,5, J ¼ 8.0 Hz), 7.79 (s, 1H, H-4), 7.89 (d, 2H, C6H5eH2,6,
C6H5NeH4,
J
¼
6.9 Hz), 7.36e7.43 (m, 4H, C6H5eH3,5,
C6H5NeH2,6), 7.47 (t, 2H, C6H5NeH3,5, J ¼ 6.9 Hz), 8.17 (d, 2H,
C6H5eH2,6, J ¼ 6.5 Hz), 8.95 (brs, 1H, D2O exch., NH), 9.06 (brs, 1H,
H-4). 13C NMR (151 MHz, Acetone-d6)
d 55.99 (PMB-OCH3), 56.57
J ¼ 8.0 Hz). 13C NMR (151 MHz, CDCl3)
d
52.32 (PMB-OCH3), 55.61
(CH2, TMP-3,5-OCH3), 60.73 (TMP-4-OCH3), 98.99 (TMP-C2,6),
111.28 (C-4), 115.08 (PMB-C3,5), 125.67 (C6H5NeC2,6), 128.10
(C6H5NeC4), 128.35 (C6H5eC-2,6), 129.22 (C6H5eC4), 129.53 (PMB-
(CH2), 55.90 (TMP-3,5-OCH3), 60.79 (TMP-4-OCH3), 97.43 (TMP-
C2,6), 114.88 (C-4), 115.18 (PMB-C3,5), 118.61 (CN), 121.22 (C-5),
13

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
C-2,6), 129.70 (C6H5eC-3,5, C-7a), 130.35 (PMB-C1), 130.86
(C6H5NeC3,5),131.54 (C-3a),132.58 (C6H5eC1),133.76 (C-5),134.66
(TMP-C4), 137.22 (TMP-C1), 138.99 (C6H5NeC1), 142.32 (C-7),
146.21 (C-3), 154.18 (TMP-C3,5), 158.64 (CNH), 160.46 (PMB-C4).
ESI-HRMS m/z: calculated C36H35N6O4 [MþH]þ: 615.2715, found:
615.2698.
(m, 1H, cyclohexyl-H1), 5.36 (d, 1H, D2O exch., cyclohexyl-NH,
J ¼ 6.5 Hz), 5.98 (s, 2H, CH2), 6.96 (d, 2H, PMB-H3,5, J ¼ 8.4 Hz),
7.03e7.09 (m, 3H, C6H5NeH2,6,4), 7.21 (d, 2H, PMB-H2,6,
J ¼ 8.4 Hz), 7.34 (t, 2H, C6H5NeH3,5, J ¼ 7.7 Hz), 7.43 (t, 1H,
C6H5eH4, J ¼ 7.4 Hz), 7.55 (t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 8.10 (d, 2H,
C6H5eH2,6, J ¼ 7.7 Hz), 8.47 (brs, 1H, H-4). 13C NMR (151 MHz,
Acetone-d6) d 25.56 (cyclohexyl-C3,5), 26.67 (cyclohexyl-C4), 33.31
4.1.40. N-(3-Trifluoromethylphenyl)-3-phenyl-1-(4-
methoxybenzyl)-7-(3,4,5-trimethoxyphenylamino)-1Н-pyrazolo
[3,4-c]pyridine-5-carboxamidine (50)
This compound was prepared following a method analogous to
that of 8, starting from 46. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 7/3 as
the eluent. Yield 36%. Mp: 128e130 ꢀC (CH2Cl2/Et2O). 1H NMR
(cyclohexyl-C2,6), 50.60 (cyclohexyl-C1), 55.77 (OCH3), 56.04
(CH2),104.42 (C-4),115.53 (PMB-C3,5),123.19 (C6H5NeC2,6),123.66
(C6H5NeC4), 128.12 (C6H5eC2,6), 128.55 (C-3a), 128.83 (PMB-C2,6),
129.11 (C6H5eC4), 129.66 (C-7a), 129.87 (C6H5eC3,5), 130.21
(C6H5NeC3,5), 130.37 (PMB-C1), 133.97 (C6H5eC1), 141.47 (C-5),
144.66 (C-7), 145.07 (C-3), 150.13 (C6H5NeC1), 154.86 (CNH), 160.82
(PMB-C4). ESI-HRMS m/z: calculated C33H35N6O [MþH]þ: 531.2867,
found: 531.2866.
(600 MHz, Acetone-d6)
d 3.69 (s, 3H, TMP-4-OCH3), 3.77 (s, 9H,
PMB-4-OCH3, TMP-3,5-OCH3), 6.07 (brs, 2H, CH2), 6.70 (brs, 2H,
TMP-H-2,6), 7.00 (brs, 2H, PMB-H3,5), 7.22e7.36 (m, 4H, PMB-H-
2,6, C6H4FN-Н2,6), 7.42e7.54 (m, 3H, C6H5eH4, C6H4FN-H4,5),
7.57 (t, 2H, C6H5eH3,5, J ¼ 7.5 Hz), 8.06 (d, 2H, C6H5eH2,6,
J ¼ 7.6 Hz), 8.56 (brs, 1H, H-4). 13C NMR (151 MHz, Acetone-d6)
4.1.43. N-(3-Trifluoromethylphenyl)-1-(4-methoxybenzyl)-3-
phenyl-7-(cyclohexylamino)-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (53)
This compound was prepared following a method analogous to
that of 8, starting from 47. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 9/1 as
the eluent. Yield 32%. Oil. 1H NMR (600 MHz, Acetone-d6)
d
55.71 (PMB-4-OCH3), 56.03 (CH2), 56.52 (TMP-3,5-OCH3), 60.75
(TMP-4-OCH3), 99.14 (TMP-C2,6), 107.28 (C-4), 115.60 (PMB-C3,5),
119.69 (C6H4FN-C2), 126.84 (C6H4FN-C6), 126.92 (C-3a), 127.13
(C6H5eC1), 128.19 (C6H5eC2,6), 128.36 (PMB-C1), 129.29 (PMB-
C2,6), 129.38 (C6H4FN-C4), 129.72 (C-7a), 129.96 (C6H5eC3,5),
131.10 (C6H5eC4), 132.10 (C6H4FN-C5), 133.56 (C-5), 133.75 (TMP-
C1), 134.97 (TMP-C4), 135.44 (C6H4FN-C1), 141.91 (C-7), 145.52 (C-
3), 154.50 (TMP-C3,5), 160.86 (PMB-C4),168.11 (CNH). ESI-HRMS m/
z: calculated C37H34F3N6O4 [MþH]þ: 683.2589, found: 683.2575.
d
1.16e1.26 (m, 3H, cyclohexyl-H), 1.27e1.35 (m, 2H, cyclohexyl-H),
1.52e1.57 (m, 1H, cyclohexyl-H), 1.58e1.64 (m, 2H, cyclohexyl-H),
1.90e1.96 (m, 2H, cyclohexyl-H), 3.72 (s, 3H, OCH3), 3.77e3.79
(m, 1H, cyclohexyl-H-1), 5.87 (s, 2H, CH2), 5.96 (d, 1H, D2O exch.,
cyclohexyl-NH, J ¼ 9.0 Hz), 6.82 (d, 2H, PMB-H3,5, J ¼ 8.7 Hz),
7.20e7.28 (m, 4H, PMB-H2,6, C6H4FN-H2,6), 7.30 (d, 1H, C6H4FN-
H4, J ¼ 7.6 Hz), 7.45 (t, 1H, C6H5eH-4, J ¼ 7.4 Hz), 7.52 (t, 1H,
C6H4FN-H5, J ¼ 7.8 Hz), 7.56 (t, 2H, C6H5eH3,5, J ¼ 7.7 Hz), 8.03 (d,
2H, C6H5eH2,6, J ¼ 8.0 Hz), 8.67 (brs, 1H, H-4). 13C NMR (151 MHz,
4.1.41. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-1-(4-
methoxybenzyl)-7-(3,4,5-trimethoxyphenylamino)-1Н-pyrazolo
[3,4-c]pyridine-5-carboxamidine (51)
Acetone-d6) d 25.58 (cyclohexyl-C3,5), 26.69 (cyclohexyl-C4), 33.35
This compound was prepared following a method analogous to
that of 8, starting from 46. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 40%. Mp: 136e138 ꢀC (CH2Cl2/Et2O). 1H NMR
(cyclohexyl-C2,6), 54.58 (CH2), 55.53 (OCH3), 56.06 (cyclohexyl-
C1), 109.47 (C-4), 114.72 (PMB-C3,5), 119.37 (C6H4FN-C4), 119.51
(C6H4FN-C2), 122.87, 124.71, 126.51, 128.30 (CF3), 126.70 (C6H4FN-
C6), 128.15 (C6H5eC2,6), 128.84 (C-3a), 129.43 (C6H5eC-4), 129.90
(C-7a), 129.96 (C6H5eC3,5), 130.12 (PMB-C2,6), 130.70 (PMB-C1),
131.05 (C6H4FN-C5), 131.85, 132.06, 132.25 (C6H4FN-C3), 133.25 (C-
5), 133.76 (C6H5eC1), 146.71 (C-3), 150.60 (C-7), 153.03 (C6H4FN-
C1), 154.24 (CNH), 160.28 (PMB-C4). ESI-HRMS m/z: calculated
(600 MHz, Acetone-d6)
d 2.26 (s, 3H, CH3), 2.49 (t, 4H, piperazine-
Н3,5, J ¼ 4.7 Hz), 3.14 (t, 4H, piperazine-Н2,6, J ¼ 4.7 Hz), 3.69 (s, 3H,
TMP-4-OCH3), 3.70 (s, 3H, PMB-4-OCH3), 3.74 (s, 6H, TMP-3,5-
OCH3), 6.15 (s, 2H, CH2), 6.83e6.90 (m, 4H, TMP-H-2,6,
C6H4NeH3,5), 6.93 (d, 2H, C6H4NeH2,6, J ¼ 8.7 Hz), 7.05 (d, 2H,
PMB-H3,5, J ¼ 8.4 Hz), 7.25 (d, 2H, PMB-H2,6, J ¼ 8.4 Hz), 7.33 (t, 1H,
C6H5eH4, J ¼ 7.4 Hz), 7.45 (t, 2H, C6H5eH-3,5, J ¼ 7.6 Hz), 8.10 (d,
2H, C6H5eH-2,6, J ¼ 7.6 Hz), 8.37 (brs, 1H, D2O exch., NH), 8.80 (brs,
C
34H34F3N6O [MþH]þ: 599.2741, found: 599.2753.
4.1.44. N-[4-(4-methylpiperazin-1-yl)phenyl]-1-(4-
methoxybenzyl)-3-phenyl-7-(cyclohexylamino)-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (54)
1H, H-4). 13C NMR (151 MHz, Acetone-d6)
d 46.39 (CH3), 50.27
(piperazine-C2,6), 55.70 (PMB-4-OCH3), 56.04 (piperazine-C3,5,
CH2), 56.50 (TMP-3,5-OCH3), 60.74 (TMP-4-OCH3), 98.87 (TMP-
C2,6), 107.39 (C-4), 115.53 (PMB-C3,5), 117.89 (C6H4NeC3,5), 123.81
(C6H4NeC2,6), 128.19 (C6H5eC2,6,1), 129.28 (PMB-C2,6,1), 129.44
(C6H5eC4), 129.88 (C6H5eC3,5, C-7a), 130.25 (C-3a), 133.58 (C-5),
134.75 (TMP-C4),137.20 (TMP-C1), 141.02 (C6H4NeC4),141.74 (C-7),
145.48 (C-3), 148.66 (C6H4NeC1), 154.42 (TMP-C3,5), 154.65 (CNH),
160.80 (PMB-C4). ESI-HRMS m/z: calculated C41H45N8O4 [MþH]þ:
713.3559, found: 713.3514.
This compound was prepared following a method analogous to
that of 8, starting from 47. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 36%. Mp: 133e135 ꢀC (CH2Cl2/Et2O). 1H NMR
(600 MHz, Acetone-d6) d 1.21e1.26 (m, 3H, cyclohexyl-H), 1.27e1.35
(m, 2H, cyclohexyl-H), 1.52e1.57 (m, 1H, cyclohexyl-H), 1.58e1.64
(m, 2H, cyclohexyl-H), 1.90e1.96 (m, 2H, cyclohexyl-H), 2.27 (s, 3H,
CH3), 2.51 (t, 4H, piperazine-Н3,5, J ¼ 4.5 Hz), 3.15 (t, 4H, pipera-
zine-Н2,6, J ¼ 4.5 Hz), 3.75 (s, 3H, OCH3), 3.88e3.98 (m, 1H,
cyclohexyl-H-1), 5.67 (brs, 1H, D2O exch., cyclohexyl-NH), 6.03 (s,
2H, CH2), 6.88e6.99 (m, 4H, PMB-H3,5, C6H4NeH2,6), 7.11 (brs, 2H,
C6H4NeH3,5), 7.19 (d, 2H, PMB-H2,6, J ¼ 8.5 Hz), 7.39 (t, 1H,
C6H5eH4, J ¼ 7.2 Hz), 7.51 (t, 2H, C6H5eH3,5, J ¼ 7.5 Hz), 8.21 (m,
2H, C6H5eH2,6), 8.71 (brs,1H, H-4). 13C NMR (151 MHz, Acetone-d6)
4.1.42. N,3-diphenyl-1-(4-methoxybenzyl)-7-(cyclohexylamino)-
1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (52)
This compound was prepared following a method analogous to
that of 8, starting from 47. Purification by column chromatography
(silica gel) using a mixture of ethyl acetate/methanol 9/1 as the
eluent. Yield 36%. Amorphous solid. 1H NMR (600 MHz, Acetone-d6)
d
25.59 (cyclohexyl-C3,5), 26.69 (cyclohexyl-C4), 33.34 (cyclohexyl-
C2,6), 46.44 (CH3), 50.45 (cyclohexyl-C1, piperazine-C2,6), 55.77
(OCH3), 56.03 (CH2), 56.13 (piperazine-C3,5), 104.84 (C-4), 115.54
(C6H4NeC3,5), 117.97 (PMB-C3,5), 123.66 (C6H4NeC2,6), 128.11
(C6H5eC2,6), 128.63 (C-3a), 128.84 (PMB-C2,6), 129.09 (C6H5eC4),
d
1.18e1.26 (m, 3H, cyclohexyl-H), 1.32e1.42 (m, 2H, cyclohexyl-H),
1.55e1.61 (m, 1H, cyclohexyl-H), 1.62e1.69 (m, 2H, cyclohexyl-H),
1.93e1.99 (m, 2H, cyclohexyl-H), 3.78 (s, 3H, OCH3), 4.01e4.09
14

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
129.74 (C-7a), 129.87 (C6H5eC3,5), 130.40 (PMB-C1), 133.53
(C6H4NeC4), 134.06 (C6H5eC1), 142.06 (C-5), 144.56 (C-7), 145.01
(C-3), 148.68 (C6H4NeC1), 155.89 (CNH), 160.83 (PMB-C4). ESI-
(151 MHz, Acetone-d6) d 46.08 (CH3), 49.82 (piperazine-C2,6), 52.13
(morpholine-C-3,5), 54.49 (CH2), 55.56 (OCH3), 55.78 (piperazine
C3,5), 67.20 (morpholine-C2,6), 111.75 (C-4), 114.83 (PMB-C3,5),
117.80 (C6H4NeC3,5), 124.64 (C6H4NeC2,6), 128.29 (C6H5eC2,6, C-
3a), 129.53 (C6H5eC4), 129.77 (PMB-C2, 6), 129.85 (C-7a), 129.97
(C6H5eC3,5), 130.59 (PMB-C1), 133.34 (C6H5eC1), 133.19 (C-5),
139.96 (C6H4NeC4), 146.96 (C-3), 149.27 (C6H4NeC1), 149.66 (C-7),
155.81 (CNH), 160.28 (PMB-C4). ESI-HRMS m/z: calculated
HRMS m/z: calculated
C
38H45N8O [MþH]þ: 629.3711, found:
629.3730.
4.1.45. N,3-diphenyl-1-(4-methoxybenzyl)-7-(morpholin-4-yl)-1Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (55)
This compound was prepared following a method analogous to
that of 8, starting from 48. Purification by column chromatography
(silica gel) using a mixture of ethyl acetate/methanol 8/2 as the
eluent. Yield 96%. Mp: 150e152 ꢀC (CH2Cl2/Et2O). 1H NMR
C
36H41N8O2 [MþH]þ: 617.3347, found: 617.3340.
4.1.48. N,3-diphenyl-7-(3,4,5-trimethoxyphenylamino)-1Н-
pyrazolo[3,4-c]pyridine-5-carboxamidine (58)
(600 MHz, Acetone-d6)
d
3.40 (m, 4H, morpholine-H3,5), 3.72 (s,
This compound was prepared following a method analogous to
that of 14, starting from 49. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
eluent. Yield 90%. Mp: 230e232 ꢀC (EtOAc/Et2O). 1H NMR
3H, OCH3), 3.91 (m, 4H, morpholine-H-2,6), 5.58 (s, 2H, CH2), 6.85
(d, 2H, PMB-H3,5, J ¼ 8.7 Hz), 7.17 (t, 1H, C6H5NeH4, J ¼ 6.9 Hz),
7.24e7.39 (m, 7H, C6H5NeH2,3,5,6, PMB-H2,6, C6H5eH4), 7.47 (t,
2H, C6H5eH3,5, J ¼ 7.6 Hz), 8.17 (d, 2H, C6H5eH2,6, J ¼ 6.7 Hz), 9.14
(600 MHz, CD3OD)
d 3.74 (s, 3H, TMP-4-OCH3), 3.80 (s, 6H, TMP-
(brs,1H, H-4). 13C NMR (151 MHz, Acetone-d6)
d
52.08 (morpholine-
3,5-OCH3), 7.33 (s, 2H, TMP-H2,6), 7.46e7.53 (m, 4H, C6H5-Н4,
C6H5N-Н2,6,4), 7.56 (t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 7.61 (t, 2H,
C6H5NeH3,5, J ¼ 7.6 Hz), 7.99 (d, 2H, C6H5eH2,6, J ¼ 7.5 Hz), 8.36
C3,5), 54.57 (CH2), 55.60 (OCH3), 67.12 (morpholine-C2,6), 114.23
(C-4), 114.87 (PMB-C3,5), 125.52 (C6H5NeC2,6), 127.27 (C6H5NeC4),
128.51 (C6H5eC2,6), 129.32 (C-3a), 129.57 (C6H5eC4), 129.80 (PMB-
C2,6), 129.94 (C6H5eC3,5), 130.36 (PMB-C1), 130.66 (C6H5NeC3,5),
132.72 (C6H5eC1), 133.82 (C-5), 136.14 (C-7a), 140.82 (C6H5NeC1),
147.41 (C-3), 150.07 (C-7), 158.15 (CNH), 160.31 (PMB-C4). ESI-
HRMS m/z: calculated C31H31N6O2 [MþH]þ: 519.2503, found:
519.2498.
(brs, 1H, H-4). 13C NMR (151 MHz, CD3OD)
d 56.72 (TMP-3,5-OCH3),
61.42 (TMP-4-OCH3), 98.73 (TMP-C2,6), 110.63 (C-4), 125.93 (C-3a),
126.68 (C6H5NeC4,2,6), 128.52 (C6H5eC2,6), 129.97 (C6H5eC1),
130.20 (C-7a), 130.33 (C6H5eC4), 130.45 (C6H5eC3,5), 131.67
(C6H5NeC3,5), 134.64 (TMP-C4), 134.90 (C-5), 136.29 (C6H5NeC1),
137.67 (TMP-C1), 140.25 (C-7), 146.22 (C-3), 154.68 (TMP-C3,5),
163.33 (CNH). ESI-HRMS m/z: calculated C28H27N6O3 [MþH]þ:
495.2140, found: 495.2134.
4.1.46. N-(3-Trifluoromethylphenyl)-1-(4-methoxybenzyl)-3-
phenyl-7-(morpholin-4-yl)-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (56)
This compound was prepared following a method analogous to
that of 8, starting from 48. Purification by column chromatography
(silica gel) using a mixture of ethyl acetate/methanol 9/1 as the
eluent. Yield 81%. Mp: 114e116 ꢀC (CH2Cl2/Et2O). 1H NMR
4.1.49. N-(3-Trifluoromethylphenyl)-3-phenyl-7-(3,4,5-
trimethoxyphenylamino)-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (59)
This compound was prepared following a method analogous to
that of 14, starting from 50. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 8/2 as
the eluent. Yield 90%. Mp: 142e144 ꢀC (EtOAc/Et2O). 1H NMR
(600 MHz, Acetone-d6)
d 3.35 (m, 4H, morpholine-H3,5), 3.71 (s,
3H, OCH3), 3.91 (m, 4H, morpholine-H-2,6), 5.86 (s, 2H, CH2), 6.84
(d, 2H, PMB-H3,5, J ¼ 8.4 Hz), 7.28 (d, 2H, PMB-H2,6, J ¼ 8.4 Hz),
7.35e7.60 (m, 7H, C6H5eH3,4,5, C6H4FN-Н2,4,5,6), 8.13 (d, 2H,
C6H5eH2,6, J ¼ 7.2 Hz), 9.00 (brs, 1H, H-4). 13C NMR (151 MHz,
(600 MHz, CD3OD)
d 3.74 (s, 3H, TMP-4-OCH3), 3.82 (s, 6H, TMP-
3,5-OCH3), 7.19 (s, 2H, TMP-H2,6), 7.37e7.47 (m, 4H, C6H4FN-H2,6,4,
C6H5-Н4), 7.50 (t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 7.59 (t, 1H, C6H4FN-
Н5, J ¼ 7.8 Hz), 7.97 (d, 2H, C6H5eH2,6, J ¼ 7.5 Hz), 8.31 (brs, 1H, H-
Acetone-d6)
d 52.06 (morpholine-C3,5), 54.43 (CH2), 55.56 (OCH3),
67.13 (morpholine-C2,6), 112.64 (C-4), 114.80 (PMB-C3,5), 121.05
(C6H4FN-C4), 121.54 (C6H4FN-C2), 122.58, 124.38, 126.18, 127.98
(CF3), 128.15 (C6H4FN-C6), 128.32 (C6H5eC2,6), 129.50 (C6H5eC4, C-
3a), 129.60 (C-7a), 129.69 (PMB-C2,6), 129.89 (C6H5eC3,5), 130.42
(PMB-C1), 131.35 (C6H4FN-C5), 131.71, 131.92, 132.13, 132.34
(C6H4FN-C3), 133.07 (C-5), 133.47 (C6H5eC1), 138.89 (C6H4FN-C1),
147.03 (C-3), 149.63 (C-7), 155.54 (CNH), 160.21 (PMB-C4). ESI-
HRMS m/z: calculated C32H30F3N6O2 [MþH]þ: 587.2377, found:
587.2377.
4). 13C NMR (151 MHz, CD3OD)
d 56.72 (TMP-3,5-OCH3), 61.43
(TMP-4-OCH3), 98.25 (TMP-C2,6), 107.78 (C-4), 121.43 (C6H4FN-C2),
122.18 (C6H4FN-C4), 124.85, 126.65 (CF3), 125.02 (C-3a), 128.32
(C6H5eC2,6, C6H4FN-C6), 129.90 (C6H5eC4), 130.26 (C6H5eC3,5),
130.77 (C-7a), 131.75 (C6H4FN-C5), 132.75 (C6H5eC1), 133.06,
133.27 (C6H4FN-C3, C-5), 134.51 (TMP-C4), 138.07 (TMP-C1), 139.68
(C-7), 143.91 (C-3), 148.79 (C6H4FN-C1), 154.70 (TMP-C3,5), 159.47
(CNH). ESI-HRMS m/z: calculated C29H26F3N6O3 [MþH]þ: 563.2013,
found: 563.2010.
4.1.47. N-[4-(4-methylpiperazin-1-yl)phenyl]-1-(4-
methoxybenzyl)-3-phenyl-7-(morpholin-4-yl)-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (57)
4.1.50. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-7-(3,4,5-
trimethoxyphenylamino)-1Н-pyrazolo[3,4-c]pyridine-5-
carboxamidine (60)
This compound was prepared following a method analogous to
that of 8, starting from 48. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 60%. Mp: 103e105 ꢀC (CH2Cl2/Et2O). 1H NMR
This compound was prepared following a method analogous to
that of 14, starting from 51. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 90%. Mp: 165e167 ꢀC (EtOAc/Et2O). 1H NMR (600 MHz,
(600 MHz, Acetone-d6)
d
2.32 (s, 3H, CH3), 2.59 (t, 4H, piperazine-
CD3OD)
d
2.49 (s, 3H, CH3), 2.79 (t, 4H, piperazine-Н3,5, J ¼ 4.0 Hz),
Н3,5, J ¼ 4.9 Hz), 3.18 (t, 4H, piperazine-Н2,6, J ¼ 4.9 Hz), 3.39 (t, 4H,
morpholine-H3,5, J ¼ 4.6 Hz), 3.72 (s, 3H, OCH3), 3.95 (t, 4H,
morpholine-H2,6, J ¼ 4.6 Hz), 5.90 (s, 2H, CH2), 6.84 (d, 2H, PMB-
H3,5, J ¼ 8.8 Hz), 6.96 (d, 2H, C6H4NeH3,5, J ¼ 8.8 Hz), 7.02 (d,
2H, C6H4NeH2,6, J ¼ 8.8 Hz), 7.28 (d, 2H, PMB-H2,6, J ¼ 8.8 Hz), 7.43
(t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.54 (t, 2H, C6H5eH3,5, J ¼ 7.7 Hz), 8.07
(d, 2H, C6H5eH2,6, J ¼ 7.6 Hz), 8.89 (brs, 1H, H-4). 13C NMR
3.35 (t, 4H, piperazine-Н2,6, J ¼ 4.0 Hz), 3.74 (s, 3H, TMP-4-OCH3),
3.78 (s, 6H, TMP-3,5-OCH3), 7.14 (d, 2H, C6H4NeH3,5, J ¼ 8.7 Hz),
7.28 (s, 2H, TMP-H2,6), 7.33 (d, 2H, C6H4NeH2,6, J ¼ 8.7 Hz), 7.46 (t,
1H, C6H5eH4, J ¼ 7.4 Hz), 7.54 (t, 2H, C6H5eH3,5, J ¼ 7.6 Hz), 7.97 (d,
2H, C6H5eH2,6, J ¼ 7.5 Hz), 8.31 (brs, 1H, H-4). 13C NMR (151 MHz,
CD3OD)
d 45.71 (CH3), 49.01 (piperazine-C2,6 - overlapping with
CD3OD), 55.70 (piperazine-C3,5), 56.72 (TMP-3,5-OCH3), 61.43
15

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
(TMP-4-OCH3), 98.61 (TMP-C2,6), 110.35 (C-4), 118.31
(C6H4NeC3,5), 124.04 (C-3a), 127.14 (C6H4NeC4), 127.61
(C6H4NeC2,6), 128.46 (C6H5eC2,6), 130.27 (C6H5eC4), 130.41
(C6H5eC3,5, C-7a), 132.40 (C6H5eC1), 134.34 (C-5), 134.80 (TMP-
C4), 137.67 (TMP-C1), 141.28 (C-7), 146.19 (C-3), 152.65 (C6H4NeC1),
154.64 (TMP-C3,5), 163.18 (CNH). ESI-HRMS m/z: calculated
7.15 (d, 2H, C6H4NeH3,5, J ¼ 8.5 Hz), 7.35 (d, 2H, C6H4NeH2,6,
J ¼ 8.5 Hz), 7.45 (t, 1H, C6H5eH4, J ¼ 7.4 Hz), 7.54 (t, 2H, C6H5eH3,5,
J ¼ 7.5 Hz), 7.97 (d, 2H, C6H5eH2,6, J ¼ 7.3 Hz), 8.25 (brs, 1H, H-4).
13C NMR (151 MHz, CD3OD)
d 26.08 (cyclohexyl-C3,5), 27.05
(cyclohexyl-C4), 34.10 (cyclohexyl-C2,6), 45.36 (CH3), 48.78
(piperazine-C2,6 - overlapping with CD3OD), 50.40 (cyclohexyl-C1),
55.51 (piperazine-C3,5), 107.50 (C-4), 118.44 (C6H4NeC3,5), 123.73
(C-3a), 127.34 (C6H4NeC4), 127.90 (C6H4NeC2,6), 128.45
(C6H5eC2,6), 130.10 (C6H5eC4, C-7a), 130.33 (C6H5eC3,5), 132.77
(C6H5eC1), 134.23 (C-5), 146.67 (C-3), 148.38 (C-7), 152.46
(C6H4NeC1), 162.72 (CNH). ESI-HRMS m/z: calculated C30H37N8
[MþH]þ: 509.3136, found: 509.3128.
C
33H37N8O3 [MþH]þ: 593.2984, found: 593.2960.
4.1.51. N,3-diphenyl-7-(cyclohexylamino)-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (61)
This compound was prepared following a method analogous to
that of 14, starting from 52. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
eluent. Yield 97%. Mp: 165e167 ꢀC (EtOAc/Et2O). 1H NMR (600 MHz,
4.1.54. N,3-diphenyl-7-(morpholin-4-yl)-1Н-pyrazolo[3,4-c]
pyridine-5-carboxamidine (64)
CD3OD)
d 1.30e1.38 (m, 1H, cyclohexyl-H), 1.39e1.47 (m, 2H,
cyclohexyl-H), 1.48e1.57 (m, 2H, cyclohexyl-H), 1.67e1.73 (m, 1H,
cyclohexyl-H), 1.80e1.87 (m, 2H, cyclohexyl-H), 2.13e2.19 (m, 2H,
cyclohexyl-H), 4.31e4.38 (m, 1H, cyclohexyl-H1), 7.42e7.50 (m, 4H,
C6H5-Н4, C6H5N-Н2,6,4), 7.53 (t, 2H, C6H5eH3,5, J ¼ 7.7 Hz), 7.59 (t,
2H, C6H5NeH3,5, J ¼ 7.8 Hz), 7.98 (d, 2H, C6H5eH2,6, J ¼ 7.4 Hz),
This compound was prepared following a method analogous to
that of 14, starting from 55. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
eluent. Yield 99%. Mp: 171e173 ꢀC (EtOAc/Et2O). 1H NMR (600 MHz,
CD3OD)
d
3.86 (t, 4H, morpholine-H2,6, J ¼ 4.5 Hz), 4.01 (t, 4H,
8.26 (brs, 1H, H-4). 13C NMR (151 MHz, CD3OD)
d
26.10 (cyclohexyl-
morpholine-H3,5, J ¼ 4.5 Hz), 7.28 (d, 2H, C6H5NeH2,6, J ¼ 7.6 Hz),
7.32 (t, 1H, C6H5eH4, J ¼ 7.5 Hz), 7.42 (t, 1H, C6H5N-Н4, J ¼ 7.5 Hz),
7.47e7.54 (m, 4H, C6H5eH3,5, C6H5N-Н3,5), 7.88 (d, 2H, C6H5eH2,6,
C3,5), 27.07 (cyclohexyl-C4), 34.13 (cyclohexyl-C2,6), 50.40 (cyclo-
hexyl-C1), 107.56 (C-4), 126.32 (C-3a), 126.86 (C6H5NeC4,2,6),
128.42 (C6H5eC2,6), 129.95 (C6H5eC4), 130.09 (C-7a), 130.31
(C6H5eC3,5), 131.54 (C6H5NeC3,5), 132.84 (C6H5eC1), 134.56 (C-5),
136.72 (C6H5NeC1), 146.53 (C-3), 147.23 (C-7), 162.48 (CNH). ESI-
J ¼ 7.8 Hz), 8.28 (brs, 1H, H-4). 13C NMR (151 MHz, CD3OD)
d 48.63
(morpholine-C3,5 - overlapping with CD3OD), 68.12 (morpholine-
C2,6), 108.69 (C-4), 125.13 (C-3a), 125.85 (C6H5NeC2,6), 128.03
(C6H5NeC4), 128.36 (C6H5eC2,6), 130.09 (C6H5eC4), 130.43
(C6H5eC3,5), 131.20 (C6H5NeC3,5), 131.47 (C6H5eC1), 136.18
(C6H5NeC1), 137.85 (C-5), 141.56 (C-7a), 142.60 (C-3), 150.52 (C-7),
160.79 (CNH). ESI-HRMS m/z: calculated C23H23N6O [MþH]þ:
399.1928, found: 399.1949.
HRMS m/z: calculated
411.2299.
C
25H27N6 [MþH]þ: 411.2292, found:
4.1.52. N-(3-Trifluoromethylphenyl)-3-phenyl-7-
(cyclohexylamino)-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine
(62)
This compound was prepared following a method analogous to
that of 14, starting from 53. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/ethyl acetate 7/3 as
the eluent. Yield 97%. Mp: 102e104 ꢀC (EtOAc/Et2O). 1H NMR
4.1.55. N-(3-Trifluoromethylphenyl)-3-phenyl-7-(morpholin-4-yl)-
1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine (65)
This compound was prepared following a method analogous to
that of 14, starting from 56. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 9/1 as the
eluent. Yield 99%. Mp: 149e151 ꢀC (EtOAc/Et2O). 1H NMR (600 MHz,
(600 MHz, CD3OD) d 1.31e1.37 (m, 1H, cyclohexyl-H), 1.38e1.47 (m,
2H, cyclohexyl-H), 1.48e1.57 (m, 2H, cyclohexyl-H), 1.66e1.73 (m,
1H, cyclohexyl-H), 1.80e1.87 (m, 2H, cyclohexyl-H), 2.11e2.18 (m,
2H, cyclohexyl-H), 4.23 (brs, 1H, cyclohexyl-H1), 7.33 (d, 1H,
C6H4FN-H2, J ¼ 7.5 Hz), 7.35e7.40 (m, 2H, C6H4FN-Н6,4), 7.43e7.47
(m, 1H, C6H5eH4), 7.52e7.60 (m, 3H, C6H5-Н3,5, C6H4FN-Н5), 8.03
(d, 2H, C6H5eH2,6, J ¼ 7.0 Hz), 8.47 (brs, 1H, H-4). 13C NMR
CD3OD)
d 3.85 (m, 8H, morpholine-H), 7.33 (d, 1H, C6H4FN-H6,
J ¼ 7.8 Hz), 7.36e7.43 (m, 3H, C6H5eH4, C6H4FN-Н2,4), 7.45e7.50
(m, 2H, C6H5eH3,5), 7.54 (t, 1H, C6H4FN-H5, J ¼ 7.8 Hz), 7.88 (d, 2H,
C6H5eH2,6, J ¼ 7.5 Hz), 8.27 (brs, 1H, H-4). 13C NMR (151 MHz,
CD3OD)
d 48.72 (morpholine-C3,5 - overlapping with CD3OD),
(151 MHz, CD3OD)
d
26.10 (cyclohexyl-C3,5), 26.80 (cyclohexyl-C4),
68.07 (morpholine-C2,6), 107.66 (C-4), 121.30 (C6H4FN-C2), 121.77
(C6H4FN-C4), 123.04, 124.84, 126.64, 128.44 (CF3), 126.01 (C-3a),
128.23 (C6H4FN-C6), 128.30 (C6H5eC2,6), 129.81 (C6H5eC4), 130.29
(C6H5eC3,5), 131.62 (C6H4FN-C5), 132.16 (C6H5eC1), 132.73, 132.95,
133.16, 133.37 (C6H4FN-C3), 136.46 (C-5), 139.38 (C-7a), 143.15 (C-
3), 149.59 (C-7), 149.75 (C6H4FN-C1), 159.02 (CNH). ESI-HRMS m/z:
calculated C24H22F3N6O [MþH]þ: 467.1802, found: 467.1802.
34.81 (cyclohexyl-C2,6), 54.27 (cyclohexyl-C1), 108.18 (C-4), 119.72
(C6H4FN-C2), 121.35 (C6H4FN-C4), 124.87 (C-3a), 125.62, 126.68
(CF3), 127.80 (C6H4FN-C6), 128.29 (C6H5eC2,6), 129.88 (C6H5eC4),
130.11 (C-7a), 130.24 (C6H5eC3,5), 131.51 (C6H4FN-C5), 132.05
(C6H5eC1), 132.71, 132.88, 133.10, 133.20 (C6H4FN-C3), 134.41 (C-5),
146.25 (C-3), 150.12 (C6H4FN-C1), 151.15 (C-7), 158.48 (CNH). ESI-
HRMS m/z: calculated
479.2140.
C
26H26F3N6 [MþH]þ: 479.2166, found:
4.1.56. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-7-
(morpholin-4-yl)-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine
(66)
4.1.53. N-[4-(4-methylpiperazin-1-yl)phenyl]-3-phenyl-7-
(cyclohexylamino)-1Н-pyrazolo[3,4-c]pyridine-5-carboxamidine
(63)
This compound was prepared following a method analogous to
that of 14, starting from 54. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol 8/2 as the
eluent. Yield 96%. Amorphous solid. 1H NMR (600 MHz, CD3OD)
This compound was prepared following a method analogous to
that of 14, starting from 57. Purification by column chromatography
(silica gel) using a mixture of dichloromethane/methanol/triethyl-
amine 8/2/0.01 as the eluent. Yield 99%. Mp: 178e180 ꢀC (EtOAc/
Et2O). 1H NMR (600 MHz, CD3OD)
d 2.38 (s, 3H, CH3), 2.65 (m, 4H,
piperazine-Н3,5), 3.35 (m, 4H, piperazine-Н2,6 - overlapping with
CD3OD), 3.89 (m, 4H, morpholine-H2,6), 4.17 (m, 4H, H3,5), 7.13 (d,
2H, C6H4NeH3,5, J ¼ 8.0 Hz), 7.31 (d, 2H, C6H4NeH2,6, J ¼ 8.0 Hz),
7.49 (t, 1H, C6H5eH4, J ¼ 7.2 Hz), 7.58 (t, 2H, C6H5eH3,5, J ¼ 7.2 Hz),
7.92 (d, 2H, C6H5eH2,6, J ¼ 6.8 Hz), 8.33 (brs, 1H, H-4). 13C NMR
d
1.31e1.37 (m, 1H, cyclohexyl-H), 1.38e1.47 (m, 2H, cyclohexyl-H),
1.48e1.57 (m, 2H, cyclohexyl-H), 1.66e1.73 (m, 1H, cyclohexyl-H),
1.80e1.87 (m, 2H, cyclohexyl-H), 2.11e2.18 (m, 2H, cyclohexyl-H),
2.54 (s, 3H, CH3), 2.87 (t, 4H, piperazine-H3,5, J ¼ 4.5 Hz), 3.37 (t,
4H, piperazine-Н2,6, J ¼ 4.5 Hz), 4.30e4.38 (m, 1H, cyclohexyl-H1),
(151 MHz, CD3OD)
d 46.20 (CH3), 48.51 (morpholine-C3,5), 49.58
16

A. Papastathopoulos, N. Lougiakis, I.K. Kostakis et al.
European Journal of Medicinal Chemistry 218 (2021) 113387
(piperazine-C2,6 - overlapping with CD3OD), 55.98 (piperazine-
C3,5), 68.21 (morpholine-C2,6), 109.43 (C-4), 118.15 (C6H4NeC3,5),
124.56 (C-3a), 127.54 (C6H4NeC4), 127.82 (C6H4NeC2,6), 128.52
(C6H5eC2,6), 130.43 (C6H5eC4), 130.61 (C6H5eC3,5), 133.90
(C6H5eC1), 138.58 (C-5), 140.00 (C-7a), 142.32 (C-3), 151.29 (C-7),
152.82 (C6H4NeC1), 162.66 (CNH). ESI-HRMS m/z: calculated
centrifugation and stored at ꢂ80 ꢀC until use. They were separated
on 10% SDS-PAGE and the proteins were transferred to Polyscreen
PVDF membranes (PerkinElmer, Thessaloniki, Greece). After
blocking with 5% (w/v) non-fat dried milk in 10 mMTris-HCl, pH 7.4,
150 mMNaCl, and 0.05% Tween-20 (TTBS) buffer, membranes were
incubated with the primary antibody. The following primary anti-
bodies were used in the present study: anti-phospho-Akt (Ser 431),
anti-Akt, anti-phospho-p38-MAPK (Thr180/Tyr182), anti-p38-
MAPK, and anti-poly-(ADP-ribose)-polymerase (PARP) (all rabbit
polyclonal from Cell Signaling Technology, Hertfordshire, UK), as
well as, anti-phospho-ERK (Thr202/Tyr204) and anti-ERK (both
mouse monoclonal from BD Biosciences, San Jose, CA, USA). Then,
the membranes were washed with TTBS, incubated with the
appropriate horseradish peroxidase-conjugated secondary anti-
body (either anti-rabbit or anti-mouse, both form Sigma), washed
again with TTBS and the immunoreactive bands were visualized by
chemiluminescence (LumiSensor HRP Substrate Kit, GenScript,
Piscataway, NJ, USA) according to the manufacturer’s instructions
on a Fujifilm LAS-4000 luminescent image analyzer (Fujifilm
Manufacturing, Greenwood, SC, USA).
C
28H33N8O [MþH]þ: 497.2772, found: 497.2746.
4.2. Cell culture and assessment of cytotoxicity
Compounds were tested for their cytotoxic activity on three
well-established human cancer cell lines representing various tu-
mor types (all from American Type Culture Collection; ATCC,
Rockville, MD, USA), the mammary adenocarcinoma MDA-MB-231,
the fibrosarcoma HT-1080, and the prostate adenocarcinoma PC-3.
Cells were cultured as described previously [27], in an environment
of 5% CO2, 85% humidity, and 37 ꢀC, using Dulbecco’s minimal
essential medium (DMEM) supplemented with penicillin (100 U/
ml), streptomycin (100 mg/ml), and 10% (v/v) fetal bovine serum (all
media from Life Technologies Europe BV, Thessaloniki, Greece).
Cells were subcultured using a trypsin (0.25%; Life Technologies
Europe BV) - citrate (0.30%; Sigma, St. Louis, MO, USA) solution. For
cytotoxicity assessment, the cells were plated at a density of
approximately 5000 cells/well in 96-well flat-bottomed micro-
plates, they wer left overnight to adhere, and then serial dilutions of
Authors declaration
All authors have participated significantly in the completion of
the manuscript. PM, NP, HP, DK: conceived and designed the ex-
periments; AP, NL, HP, performed the experiments; NL, IKK, PM, NP,
HP, DK: analysed the data; NL, PM, NP, HP, DK: wrote the
manuscript.
the test compounds (ranging from 50 mM to 80 nM) were added for
72 h. Corresponding dilutions of dimethylsulfoxide (DMSO) ranging
from 0.5% v/v to 0.0008% v/v served as controls. At the end of the
incubation, the medium was replaced with 3-(4,5-Dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma)
dissolved at a final concentration of 1 mg/ml in serum-free, phenol-
red-free DMEM for a further 4-h incubation. The MTT formazan was
solubilized in 2-propanol, and the optical density was measured
with a FLUOstar Optima (BMG Labtech, Ortenberg, Germany)
microplate analyzer at a wavelength of 550 nm (reference wave-
length 690 nm). Doxorubicin hydrochloride (Sigma) was included
in the experiments as a positive control. The results represent the
Declaration of competing interest
The authors declare that they have no conflict of interest to
disclosure.
Acknowledgements
This work was supported by the project “An Open-Access
Research Infrastructure of Chemical Biology and Target-Based
Screening Technologies for Human and Animal Health, Agricul-
ture and the Environment (OPENSCREEN-GR)” (MIS 5002691)
which is implemented under the Action “Reinforcement of the
Research and Innovation Infrastructure”, funded by the Operational
Programme “Competitiveness, Entrepreneurship and Innovation”
(NSRF 2014e2020) and co-financed by Greece and the European
Union (European Regional Development Fund).
mean of three independent experiments and are expressed as IC50
,
the concentration that reduced by 50% the optical density of treated
cells with respect to untreated controls.
4.3. Cell cycle analysis
Exponentially growing HT-1080 cells were treated with the test
compounds at a concentration of 2 mM for 24 h. They were then
washed in PBS, fixed in 50% ethanol, and stained with an RNAse-
containing propidium iodide solution (all reagents from Sigma),
as described [27]. Fluorescence was measured on a FACS Calibur
(Becton Dickinson, San Jose, CA, USA) flow cytometer, and DNA
content was estimated using the ModFit software (Verity Software
House, Topsham, ME, USA).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113387.
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