Synthesis, molecular docking, antimicrobial, antiquorum-sensing and antiproliferative activities of new series of pyrazolo[3,4-b]pyridine analogs

Article abstract of DOI:10.1080/00397911.2019.1618873

New series of pyrazolo[3,4-b]pyridines were prepared and evaluated for antimicrobial activity toward six selected microorganisms. Compounds 2a, 3b, 3d and 3e exhibited good activity toward B. cereus. On the other hand, 2a and 3b evinced interesting activi

Full text of DOI:10.1080/00397911.2019.1618873

Synthetic Communications
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Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis, molecular docking, antimicrobial,
antiquorum-sensing and antiproliferative activities
of new series of pyrazolo[3,4-b]pyridine analogs
Samia S. Hawas, Nadia S. El-Gohary, Moustafa T. Gabr, Mona I. Shaaban &
Mahmoud B. El-Ashmawy
To cite this article: Samia S. Hawas, Nadia S. El-Gohary, Moustafa T. Gabr, Mona I. Shaaban
& Mahmoud B. El-Ashmawy (2019): Synthesis, molecular docking, antimicrobial, antiquorum-
sensing and antiproliferative activities of new series of pyrazolo[3,4-b]pyridine analogs, Synthetic
Communications, DOI: 10.1080/00397911.2019.1618873
To link to this article: https://doi.org/10.1080/00397911.2019.1618873
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https://doi.org/10.1080/00397911.2019.1618873
Synthesis, molecular docking, antimicrobial, antiquorum-
sensing and antiproliferative activities of new series of
pyrazolo[3,4-b]pyridine analogs
Samia S. Hawasa,b, Nadia S. El-Goharya, Moustafa T. Gabra,c, Mona I. Shaaband, and
Mahmoud B. El-Ashmawya
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt;
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University, New Damietta, Egypt;
d
cDepartment of Chemistry, University of Iowa, Iowa City, Iowa, USA; Department of Microbiology,
Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
ABSTRACT
ARTICLE HISTORY
Received 11 March 2019
New series of pyrazolo[3,4-b]pyridines were prepared and evaluated
for antimicrobial activity toward six selected microorganisms.
Compounds 2a, 3b, 3d and 3e exhibited good activity toward B. cer-
eus. On the other hand, 2a and 3b evinced interesting activity over
C. albicans, whereas 2a, 3b and 3e displayed promising activity over
A. fumigatus. Antiquorum-sensing effectiveness of the new members
over C. violaceum was also assessed, where compounds 2a and 3b
exhibited higher activity than that of the reference compound, indole.
In vitro antiproliferative assessment toward HepG2, HCT-116 and MCF-
7 cancer cells evidenced that 2f has notable effectiveness on all exam-
ined cell lines, whereas 3a–c were active but to a lower extent. In vivo
antitumor activity of 2f and 3a–c against EAC cells was also esteemed,
where 2f and 3c showed considerable activity comparable to that of
doxorubicin. Cytotoxicity screening over WI38 and WISH normal cells
evinced that 2f and 3a–c are less cytotoxic than doxorubicin.
Compounds 2a, 2f, 3a–c and 3e were evaluated for DNA-binding
affinity and topoisomerase IIb inhibitory activity. Analogs 2a, 2f, 3a
and 3b illustrated strong DNA-binding affinity, whereas 2a, 2f and 3a
exhibited interesting topoisomerase IIb inhibitory activity. Compounds
2a and 2f were docked into topoisomerase IIb, where 2f showed pref-
erential binding to topoisomerase IIb. Computational studies articu-
lated that the new members are in compliance with Veber’s standards
and Lipinski’s rule.
KEYWORDS
Pyrazolopyridines;
antimicrobial; antiprolifera-
tive; DNA-binding;
topoisomerase IIb;
computational studies
GRAPHICAL ABSTRACT
CONTACT Nadia S. El-Gohary
dr.nadiaelgohary@yahoo.com
Department of Medicinal Chemistry, Faculty of
Pharmacy, Mansoura University, Mansoura 35516, Egypt.
Supplemental data for this article is available online at on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

2
S. S. HAWAS ET AL.
Introduction
The adequate treatment of bacterial, fungal, viral and parasitic infections is prohibited
by the development of antimicrobial resistance (AMR). AMR is rising perilously all over
the world, and it became a serious public health problem.[1] Subsequently, new
approaches are desired to contend the infection caused by multidrug-resistant microor-
ganisms. Quorum sensing (QS) inhibition is one of such new approaches. QS is a mech-
anism through which bacterial cells communicate with each other, and it governs
diverse processes as biofilm formation and virulence;[2] consequently, it has turned into
a prominent target for new antimicrobial agents. However, antibiotics kill or retard the
growth of bacteria, antiquorum-sensing agents (anti-QS) mitigate bacterial virulence.[3,4]
Thus, the present research is steered toward the synthesis of new antimicrobial agents,
and also considered the evolution of new QS inhibitors.
On the other hand, cancer is a leading health hazard, since cancer cells can grow and
spread to nearby tissues via lymph system or bloodstream. Metastasis is the principal
cause of 90% of cancer deaths.[5] A typical anticancer drug should be cytotoxic to can-
cer cells without damaging healthy ones. Currently, cancer is a master theme of
research, and scientists are inspired in design and invention of new efficacious and
selective anticancer agents with various chemical scaffolds, including pyrazolo[3,4-
b]pyridine nucleus. A variety of antitumor and antimicrobial agents act as DNA minor
groove binders.[6] Likewise, DNA topoisomerases (TOP I, TOP IIa and TOP IIb) are
molecular targets for lots of antibacterial and anticancer drugs.[7,8] Topoisomerase II
(TOP II) plays pivotal roles, including DNA replication and transcription, and hence
assuring genomic integrity. The capability to disrupt TOP II and produce enzyme-medi-
ated DNA injury is an efficient approach in cancer therapeutics.[9]
Pyrazolo[3,4-b]pyridine system is a fundamental skeleton in various antidepres-
sant-anxiolytic drugs as cartazolate,[10] tracazolate[11] and etazolate[12] (Figure 1).
Additionally, pyrazolo[3,4-b]pyridines were proved to possess diverse pharmaco-
logical efficacies as antibacterial,[13–15] antifungal,[16] antimicrobial[17–27] and antitu-
mor[15,17,18,22,24–37] activities. For example, pyrazolo[3,4-b]pyridines A, B and C[32]
(Figure 2) were assured to have DNA-binding affinity with distinguished antitumor
effectiveness. Also, pyrazolo[3,4-b]pyridines D, E and F[17] (Figure 2) were character-
ized as DNA-binding agents with interesting antimicrobial and antitumor efficacies.
Figure 1. Examples of marketed pyrazolopyridine drugs.

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3
Figure 2. Examples of pyrazolo[3,4-b]pyridines with declared DNA-binding affinity (A–F), antitumor
(A–G), antimicrobial (D–F) and TOP II inhibitory activities (G).
Figure 3. The suggested target compounds carrying (un)substituted benzoylamino 2a-f and N-(aryla-
minomethylene)amino moieties 3a-e.
Moreover, pyrazolo[3,4-b]pyridines G[36] (Figure 2) were described as TOP II inhibi-
tors with eminent antitumor activity. Motivated by these literature findings and as a
continuation to our preceding studies,[17,18] new series of pyrazolo[3,4-b]pyridines
carrying (un)substituted benzoylamino 2a–f and N-(arylaminomethylene)amino moi-
eties 3a–e (Figure 3) were prepared and evaluated as antimicrobial, anti-QS and anti-
proliferative agents. DNA-binding affinity and TOP IIb inhibitory activity of 2a, 2f,
3a–c and 3e were assessed to detect their potential mode of action. A thorough study
of the structure-activity relationship of the new pyrazolopyridines will flatten the
road for design and development of new chemotherapeutic agents.

4
S. S. HAWAS ET AL.
Results and discussion
Chemistry
The synthetic pathways for the new pyrazolo[3,4-b]pyridines 2a–f and 3a–e starting with
6-aminopyrazolo[3,4-b]pyridine-5-carbonitriles 1a–c[37] are illustrated in Scheme 1.
Heating ortho aminonitriles 1a–c with benzoyl chloride or 4-nitrobenzoyl chloride in
glacial acetic acid at reflux temperature gave the benzamides 2a–f in 62-77% yields.
Comp. No.
2a
R
H
R1
H
Comp. No.
3a
R
H
Het.
2b
2c
Cl
N(CH3)2
H
H
H
3b
3c
Cl
N(CH3)2
N(CH3)2
2d
NO2
3d
2e
2f
Cl
NO2
NO2
3e
N(CH3)2
---------
N(CH3)2
---------
---------
Scheme 1. Synthesis of compounds 2a-f and 3a-e

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5
Preparation of 3a–e was accomplished through refluxing 1a–c with ethyl N-(heteroaryl)-
formimidates[38,39] in glacial acetic acid. Spectral and elemental analyses confirmed the
structures of the new members.
Biological evaluation
Antimicrobial and antiquorum–sensing screening
The new members, 2a–f and 3a–e were evaluated for antimicrobial activity toward
Gram-positive bacteria (Staphylococcus aureus ATCC 29213 and Bacillus cereus UW 85)
and Gram-negative bacteria (Escherichia coli ATCC 12435 and Pseudomonas aerugi-
nosa).[40–42] Further, antifungal assay over Candida albicans (clinical isolate) and
Aspergillus fumigatus 293 was implemented.[40,41,43,44] Ampicillin (antibacterial) and flu-
conazole (antifungal) were employed as reference drugs. Inhibition zone diameters
(mm) were measured (Table 1). Minimal inhibitory concentrations (MICs, lg/mL and
lM) of 2a, 2d, 3b, 3d and 3e (the effective compounds) over the chosen microbial
strains were set (Table 2). Results disclosed that 2a and 3b have promising activity over
Table 1. Results of antimicrobial and antiquorum-sensing assessment.
Inhibition zone diameter (mm)a
QS inhibition (mm)a
Comp. No.
S. aureus B. cereus E. coli P. aeruginosa C. albicans A. fumigatus
C. violaceum
2a
2b
19
6
21
8
17
–
17
–
19
–
15
–
19
–
2c
2d
3
16
2
9
–
9
–
7
–
–
–
–
–
–
2e
2f
3a
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11
14
–
3b
3c
20
–
21
–
19
–
17
–
21
–
23
–
17
–
3d
3e
17
17
18
Nd
Nd
21
19
15
Nd
Nd
16
–
23
Nd
Nd
11
–
18
Nd
Nd
–
–
Nd
11
Nd
–
–
–
Nd
Nd
15
19
Nd
11
Nd
Ampicillin
Fluconazole
Indole
aInhibition zone diameter (mm): (-, <2, no activity); (2-9, weak activity); (10-15, moderate activity); (16-25, strong activ-
ity); (26-35, very strong activity).
Nd, not defined.
Bold values show the favored data.
Table 2. MICs of the active compounds.
MIC, mg/mL (mM)a
Comp. No.
S. aureus
156.25 (611.8) 156.25 (611.8)
625 (1560.9) 1250 (3121.8)
156.25 (400.8) 156.25 (400.8) 156.25 (400.8)
B. cereus
E. coli
312.5 (1223.6) 312.5 (1223.6) 156.25 (611.8)
1250 (3121.8) 1250 (3121.8) Nd
P. aeruginosa
C. albicans
A. fumigatus
2a
2d
3b
312.5 (1223.6)
Nd
312.5 (801.6) 156.25 (400.8) 156.25 (400.8)
3d
3e
312.5 (721.8) 156.25 (360.9)
312.5 (650.25) 156.25 (325.1)
625 (1443.7)
Nd
9.76 (26.71)
1250 (2887.4)
Nd
Nd
Nd
Nd
156.25 (325.1)
Nd
Nd
156.25 (427.61)
Nd
Ampicillin
Fluconazole
78.125 (213.81)
Nd
312.5 (855.23)
Nd
Nd
1250 (4081.35) 2500 (8162.70)
aMICs (mM) are illustrated between brackets.
Nd, not defined.
Bold values show the favored data.

6
S. S. HAWAS ET AL.
all tested microorganisms, their activity was two times that of ampicillin over B. cereus,
eight times that of fluconazole over C. albicans, and eight times and sixteen times that
of fluconazole over A. fumigatus for 2a and 3b, respectively. Analogs 3d and 3e exhib-
ited good activity on B. cereus and considerable activity toward S. aureus. On the other
hand, 3e displayed interesting efficacy on A. fumigatus. The rest of the screened com-
pounds revealed either weak or no activity toward the selected microrganisms.
Antiquorum-sensing (anti-QS) effectiveness of the same members toward
Chromobacterium violaceum ATCC 12472 was esteemed,[40,41,45] and indole was utilized
as a comparative agent.
QS system of C. violaceum liberates signals that adjust the release of a violet pigment
(violacein) that regulates the interspecies cooperative interactions.[46,47] Consequently,
QS inhibition in C. violaceum will stop violacein production. Anti-QS efficacy toward
C. violaceum was estimated: QS inhibition ¼ (r2–r1, mm), where r1 is the radius of
growth inhibition and r2 is the overall radius of growth and pigment inhibition.
Compounds 2a and 3b exhibited strong anti-QS efficacy compared to that of indole,
whereas 2e and 2f were moderately effective (Table 1).
SAR of analogs 2a–f. The N-(4-phenylpyrazolopyridin-6-yl)benzamide 2a displayed good
effectiveness toward Gram-positive bacteria and C. albicans, and considerable effectiveness
toward Gram-negative bacteriaand A. fumigatus. Replacing 4-phenyl and/or benzamide in
2a with 4-(4-chlorophenyl/4-dimethylaminophenyl) and/or 4-nitrobenzamide counterparts
led to diminished activity against all screened microorganisms (2b–f versus 2a). On the
other hand, the N-(4-phenylpyrazolopyridin-6-yl)-4-nitrobenzamide 2d displayed moder-
ate activity over S. aureus, and weak activity toward B. cereus, E. coli and P. aeruginosa.
Also, substitution of the phenyl moiety in 2d with 4-chloro or 4-dimethylamino led to
repealed activity toward all screened bacteria and fungi (compounds 2e and 2f).
SAR of compounds 3a–e. The 4-(4-chlorophenyl)-6-((pyridin-2-yl)aminomethyleneamino)-
pyrazolopyridine analog 3b showed the highest antimicrobial effectiveness on all tested
strains. Replacing 4-(4-chlorophenyl) in 3b with 4-(unsubstituted phenyl) or 4-(4-(dimethy-
lamino)phenyl) led to diminished activity on all investigated microorganisms (compounds
3a and 3c, respectively). Otherwise, exchanging 6-(pyridin-2-yl)aminomethyleneamino sub-
stituent in 3c with 6-(3-chloropyridin-2-yl)aminomethyleneamino led to improved efficacy
on the four tested bacteria (compound 3d), and this might be pertaining to the high lipophi-
licity of 3d (logP ¼ 2.35) compared to 3c (logP ¼ 1.73). On the other hand, exchanging
6-(3-chloropyridin-2-yl)aminomethyleneamino in 3d with 6-(4-phenylthiazol-2-yl)amino-
methyleneamino led to boosted activity over A. fumigatus, retained activity over Gram-posi-
tive bacteria, and reduced activity over Gram-negative bacteria (compound 3e).
Antitumor screening
In vitro antiproliferative screening. Antiproliferative effectiveness of the new members
was assessed over liver (HepG2), colon (HCT-116) and breast (MCF-7) cancer cells fol-
lowing MTT assay and employing doxorubicin as a positive control.[48,49] The concen-
tration of compound that prohibits the proliferation of cell viability by 50% (IC50, mM)

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Table 3. In vitro antiproliferative assay results.
IC50 (mM)a,b
Comp. No.
HepG2
HCT-116
MCF-7
2a
2b
2c
2d
2e
2f
3a
3b
>100.00
86.92 4.8
75.18 4.1
29.13 2.2
46.35 2.9
7.18 0.8
11.13 1.2
20.07 2.0
9.29 1.2
60.36 4.0
78.26 4.5
4.50 0.2
89.16 5.3
80.28 4.9
71.39 4.2
28.37 3.2
30.27 2.5
5.75 0.7
9.38 1.0
17.89 1.6
7.96 0.4
55.78 3.8
72.91 4.3
5.23 0.3
87.65 4.7
77.68 4.3
62.50 3.7
20.54 1.6
31.87 2.1
5.28 0.6
12.43 1.5
16.85 1.5
8.97 1.0
48.04 3.5
68.91 3.8
4.17 0.2
3c
3d
3e
Doxorubicin
aIC50 values are the mean SD of three readings.
bIC50 (mM): (1–20, strong activity); (21–50, moderate activity); (51–100, weak activ-
ity); (>100, no activity).
Bold values show the favored data.
was set (Table 3). Compound 2f has notable efficacy toward all chosen cell lines.
Likewise, compounds 3a and 3c demonstrated interesting effectiveness on all examined
cell lines; in addition, 3b demonstrated considerable activity over the three chosen can-
cer cells. The other analogs evidenced moderate to weak effect on the examined can-
cer cells.
SAR of analogs 2a–f. Presence of 4-(unsubstituted phenyl) and 6-(4-nitrobenzoylamino)
substituents on the pyrazolopyridine nucleus led to distinguished effectiveness on MCF-
7 cells, and feasible effectiveness over HepG2 and HCT-116 cells (analog 2d). The
4-nitrobenzamide analogs 2d–f were evinced to be more effective toward all examined
cancer cells compared to the unsubstituted benzamide analogs 2a–c, and this might be
ascribed to presence of extra sites of hydrogen bonding in 2d-f. The N-(4-(4-(dimethy-
lamino)phenyl)pyrazolopyridin-6-yl)-4-nitrobenzamide analog 2f exhibited notable effi-
cacy on the three cancer cells. Replacing 4-(4-(dimethylamino)phenyl) in 2f with
4-(unsubstituted phenyl) or 4-(4-chlorophenyl) resulted in lower activity on all exam-
ined cell lines (compounds 2d and 2e), and this might be ascribed to presence of an
extra site of hydrogen bonding in 2f. Contrariwise, replacing 4-(4-chlorophenyl) with
4-(unsubstituted phenyl) led to improved efficacy on all cell lines (2d versus 2e), and this
might be pertaining to the reduced lipophilicity of 2d (logP ¼ 1.96) compared to 2e (logP
¼ 2.64). The N-(4-(4-(dimethylamino)phenyl)pyrazolopyridin-6-yl)benzamide 2c displayed
weak activity on the three cancer cells. Exchanging 4-(4-(dimethylamino)phenyl) substitu-
ent in 2c with 4-(unsubstituted phenyl) or 4-(4-chlorophenyl) led to weakened activity on
all cell lines (compounds 2a and 2b).
SAR of compounds 3a–e. The 4-(4-(dimethylamino)phenyl)-6-(pyridin-2-yl)aminomethy-
leneaminopyrazolopyridine analog 3c showed superior efficacy on all screened cell lines.
Replacing 4-(4-(dimethylamino)phenyl) substituent with 4-(unsubstituted phenyl) or 4-
(4-chlorophenyl) led to mild decrease in activity over the three cell lines (3a and 3b ver-
sus 3c). On the other hand, replacing 4-(4-chlorophenyl) in 3b with 4-(unsubstituted
phenyl) led to slight improvement in activity toward all inspected cell lines (analog 3a),

8
S. S. HAWAS ET AL.
and this might be pertaining to the reduced lipophilicity of 3a (logP ¼ 1.62) compared
to 3b (logP ¼ 2.30). On contrary, replacing 6-(pyridin-2-yl)aminomethyleneamino sub-
stituent in 3c with 6-(3-chloropyridin-2-yl)aminomethyleneamino or 6-(5-phenylthiazol-
2-yl)aminomethyleneamino counterparts gave rise to minimized activity toward all
screened cell lines (3d and 3e versus 3c), and this might be pertaining to the
increased lipophilicity of 3d and 3e (logP ¼ 2.35 and 3.35, respectively) compared to 3c
(logP ¼ 1.73). The activity of compounds 3a, 3b, 3d and 3e is relying on the
compound’s lipophilicity, the decrease in lipophilicity led to improvement in activity,
the order of activity is 3a > 3b > 3d > 3e.
In vivo antitumor screening. Compounds 2f and 3a–c (with significant in vitro antiproli-
ferative activity) were screened for in vivo antitumor efficacy toward Ehrlich ascites car-
cinoma (EAC) in mice,[50–52] and doxorubicin was utilized as a comparative drug.
Mean survival time (MST) and % increase in lifespan (%ILS) of mice injected with EAC
were set (Table 4), where 2f and 3c exhibited the greatest activity. Tumor size parame-
ters were esteemed (Table 5), where 2f and 3c demonstrated a marked reduction in
tumor volume and tumor cell count. Hemoglobin (Hb), red blood cells (RBCs) and
white blood cells (WBCs) count were estimated (Table 6), where 2f and 3c manifested
higher Hb and RBCs levels and lower WBCs count than doxorubicin.
In vitro cytotoxicity screening against normal cell lines. In vitro cytotoxicity of 2f and
3a–c over lung fibroblast (WI38) and amnion epithelial (WISH) normal cells[48,49] was
assessed. IC50 values (mM) of the investigated analogs were calculated (Table 7).
Table 4. Effect of 2f and 3a-c on MST and % ILS of mice bear-
ing EAC.
Group
MST (day)a
% ILS
Normal
EAC only
2f
3a
3b
3c
Nd
Nd
Nd
200.00
87.50
106.25
216.75
256.25
16.0
48.0
30.0
33.0
51.0
57.0
Doxorubicin
aResults are the mean of three readings.
Nd, not defined.
Bold values show the favored data.
Table 5. Effect of 2f and 3a-c on tumor size parameters of mice
bearing EAC.
Group
Tumor volume (mL)a
Viable tumor cell count (106/mL)a
Normal
EAC only
2f
3a
3b
3c
Nd
Nd
9.60
2.70
4.60
4.00
2.40
2.00
72.76
21.49
32.28
30.78
19.60
15.25
Doxorubicin
aResults are the mean of three readings.
Nd, not defined.
Bold values show the favored data.

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Table 6. Effect of 2f and 3a-c on hematological count of mice bear-
ing EAC.
Group
Hb (g/dl)a
RBCs (106/mm3)a
WBCs (103/mm3)a
Normal
EAC only
2f
3a
3b
3c
13.16
7.86
12.31
10.95
11.27
12.49
12.25
6.14
3.57
5.58
5.05
5.18
5.67
5.56
5.49
20.25
8.66
13.64
10.68
8.62
Doxorubicin
8.79
aResults are the mean of three readings.
Bold values show the favored data.
Table 7. Cytotoxicity of 2f and 3a-c against WI38 and WISH nor-
mal cells.
IC50 (mM)a,b
Comp. No.
WI38
WISH
2f
3a
3b
3c
46.85 2.7
35.42 2.0
52.91 2.9
61.60 3.4
6.68 0.5
39.27 2.5
28.96 1.9
37.11 2.3
45.92 2.8
8.14 0.9
Doxorubicin
aIC50 values are the mean SD of three readings.
bIC50 (mM): (1–20, strong activity); (21–50, moderate activity); (51–100, weak activ-
ity); (>100, no cytotoxicity).
The four analogs showed lower cytotoxicity than doxorubicin (standard drug), and they
were proved to be safe against the tested normal cell lines at their cytotoxic concentra-
tions over the selected cancer cells.
Mechanistic study
DNA-binding assay. DNA-binding interaction is the mode of action of diverse antimicro-
bial and anticancer agents,[6] pyrazolo[3,4-b]pyridines are amongst these agents.[17,18,32]
Pyrazolo[3,4-b]pyridine ring is isosteric to purine, and its derivatives might contest with
purines and prohibit DNA synthesis. Accordingly, methyl green/DNA displacement assay[53]
was embraced to assess the potentiality of the efficacious compounds to bind to DNA.
Methyl green/DNA displacement assay is a colorimetric method[53] for estimation of
the displacement of methyl green from DNA by agents with DNA-binding affinity.
Concentrations of 2a, 2f, 3a–c, 3e, and doxorubicin (DNA-binding agent) that reduce
absorbance of methyl green/DNA complex by 50% (IC50, mM) were set (Table 8).
Results indicated that 2a, 2f, 3a and 3b have strong binding affinity analogous to that
of doxorubicin, whereas 3c and 3e displayed moderate affinity. The six members are
predicted to act via binding to DNA.
Topoisomerase IIß inhibition assay. DNA topoisomerase II (TOP II) is a prime target
for certain antibacterial and anticancer agents.[7,8] Quinolones are vastly utilized antibi-
otics that inhibit DNA gyrase (bacterial form of TOP II),[54] and they are studied as
anticancer agents.[55,56] It is believed that TOP II might be the prime target for the new

10
S. S. HAWAS ET AL.
Table 8. DNA-binding testing results.
Comp. No.
IC50 (mM)a
2a
2f
3a
3b
3c
3e
32.79 1.9
29.38 1.8
35.43 2.3
35.81 2.2
41.42 2.5
51.26 3.0
31.27 1.8
Doxorubicin
aIC50 values are the mean SD of three readings.
Bold values show the favored data.
Table 9. Results of topoisomerase IIb inhibition.
Comp. No.
IC50 (mM)a
2a
2f
3a
3b
0.432
0.613
0.933
2.89
3c
3e
1.96
3.15
Doxorubicin
0.727
aIC50 values are the mean of three readings.
Bold values show the favored data.
pyrazolopyridines which are structurally related to the well-known class of quinolone
antibiotics. Thus, TOP IIb inhibitory activity of the most effective antimicrobial and/or
antitumor pyrazolopyridines 2a, 2f, 3a–c and 3e was evaluated,[57] and compared to
that of doxorubicin (positive control). Results (Table 9) indicated that analogs 2a, 2f
and 3a have the highest efficacy in the enzymatic assay.
Computational studies
Molecular docking
For the sake of getting an insight into the binding mode of compounds 2a and 2f
to TOP IIb, a molecular docking study relied on the crystal structure of TOP IIb in
complex with DNA and etoposide (PDB code: 3QX3, https://www.rcsb.org/3d-view/
3QX3/1) was accomplished. The two investigated compounds 2a and 2f possessed
estimated binding energy to TOP IIb of ꢀ18.1 and ꢀ19.7 kcal/mol, respectively, in
comparison to doxorubicin that revealed binding energy of ꢀ20.6 kcal/mol. 3D
Interactions of 2a (one of the most active compounds in the antimicrobial screening)
and 2f (the most active compound in the antitumor screening) with TOP iib bind-
ing site were generated by Molegro 2.5 software[58] and they are shown in Figures
4A and 5A, respectively. The itemized analyses of the binding interactions of 2a and
2f to TOP IIb are displayed in their 2D binding modes (generated by Lead IT 2.3.2
software[59]) featuring key hydrogen bonding interactions with Asp479 residue
(Figures 4B and 5B, respectively) similarly to the binding pose of etoposide to TOP
IIb. The 2D binding modes of etoposide and doxorubicin to TOP IIb are shown in
Figure 6A,B, respectively.

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Figure 4. (A) 3D Interaction of 2a with TOP iib binding site. The atoms are colored as follows: blue
for nitrogen atoms, red for oxygen atoms, white for hydrogen atoms and cyan for carbon atoms. (B)
2D Interaction of 2a with TOP iib binding site. Dashed lines show hydrogen bonds.
3D and 2D pharmacophoric maps of the structural features of 2a (Supplementary
Figure S1A,B) and 2f (Figure 7A,B) were established using LigandScout 4.1 software.[60]
The exact pharmacophoric features include hydrogen bond acceptors and donors as
directed vectors along with lipophilic areas which are expressed by spheres. The overlay
of pharmacophoric map of 2f to pharmacophoric map of the binding of etoposide to
TOP IIb revealed satisfactory alignment (Figure 7C). These results come in good agree-
ment with the results of in vitro antiproliferative screening which demonstrated that 2f

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S. S. HAWAS ET AL.
Figure 5. (A) 3D Interaction of 2f with TOP iib binding site. The atoms are colored as follows: blue
for nitrogen atoms, red for oxygen atoms, white for hydrogen atoms and cyan for carbon atoms. (B)
2D Interaction of 2f with TOP iib binding site. Dashed lines show hydrogen bonds.
is the most active analog in this study against the three opted cancer cell lines
(Table 3). The overlay of pharmacophoric map of 2a to pharmacophoric map of the
binding of etoposide to TOP IIb is shown in Supplementary Figure S1C. Ultimately,
molecular docking studies revealed a preliminary conception of the binding mode of
the new analogs.
Molinspiration, preadmet and molsoft calculations
Computational analyses are helpful in the speculation of physicochemical properties,
pharmacokinetics, and toxicity of compounds.[61] Consequently, the new members were

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Figure 6. (A) 2D Interaction of etoposide with TOP iib binding site. (B) 2D Interaction of doxorubicin
with TOP iib binding site. Dashed lines show hydrogen bonds.
Figure 7. (A) 3D Pharmacophoric map of 2f; pharmacophore color coding is yellow for hydrophobic
regions, red for hydrogen acceptors and green for hydrogen donors. (B) 2D Pharmacophoric map of
2f; H is hydrophobic center, AR is aryl, HBA is hydrogen bond acceptor and HBD is hydrogen bond
donor. (C) The overlay of pharmacophoric map of 2f to pharmacophoric map of the binding of etopo-
side to TOP IIb.

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S. S. HAWAS ET AL.
studied for the prediction of Lipinski’s rule[62,63] and Veber’s standards.[63,64] As well,
their carcinogenicity[65] and drug score values[66] were prophesied. Results are discussed
in the supplemental data.
Conclusion
Results of antimicrobial testing proved that 2a and 3b have interesting efficacy over
all examined microorganisms as well as strong QS inhibitory activity. Consequently,
2a and 3b might be regarded as efficacious antimicrobial agents with reduced danger
of AMR. Turning into results of in vitro antiproliferative testing, 2f displayed the
greatest effectiveness on the three screened cell lines; moreover, 3c manifested expli-
cit activity on the three cell lines. As well, analogs 2f and 3c evinced excellent in
vivo antitumor effectiveness over EAC cells, and they were evidenced to be safe
against both normal cells at their cytotoxic concentrations over the tested cancer
cells. Results of DNA-binding and TOP IIb assays revealed that 2a, 2f, 3a and 3b
have strong DNA-binding affinity, whereas 2a, 2f and 3a exhibited interesting TOP
IIb inhibitory activity. Collectively referred, 2a is regarded as an efficacious anti-
microbial agent with powerful DNA-binding affinity and TOP IIb inhibitory activity.
As well, 2f and 3c are considered as efficacious and selective antiproliferative agents
through targeting DNA and TOP IIb. Further, docking studies fostered the efficient
binding interactions of 2a and 2f to TOP IIb. Computational studies clarified that
all of the checked compounds are in conformity with Veber’s and Lipinski’s rule
standards, and they are thought to display interesting oral absorption. The full
attained results corroborated that rational design of the new analogs as antimicrobial
and antiproliferative agents was competent, and the efficacious members in the pre-
sent research will be subjugated to extra structural amendments to get new more
efficacious derivatives.
Experimental
All melting points (ꢁC) were measured using Stuart melting point apparatus SMP30.
Unicam SP 1000 IR spectrometer (v in cmꢀ1) was employed for recording IR spectra (KBr
disc). 1H and 13C-NMR spectral analyses were accomplished on Bruker 500 MHz spectrom-
eter using DMSO-d6 as solvent. Mass spectrometer (JEOL JMS-600H, 70 eV) was utilized to
record mass spectra. Elemental analyses (% C, H, N) were performed, and they were in con-
gruence with the proposed structures. TLC, silica gel plates 60 F254 were used to monitor
reaction times, and UV (366, 245 nm) was employed for visualization of the spots. Elution
was done utilizing chloroform/methanol (9:1). Ortho aminonitriles 1a–c[37] and ethyl
N-(heteroaryl)formimidates[38,39] were prepared following the published procedures.
Chemistry
Synthesis of benzamide analogs 2a–f
A mixture of 1a–c (0.002 mol) and benzoyl chloride (0.281 g, 0.002 mol) or 4-nitroben-
zoyl chloride (0.371 g, 0.002 mol) was refluxed in glacial acetic acid (10 mL) for 8–15 h.

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The solution was concentrated and the solid obtained was filtered and crystallized from
ethanol/water (2:1) to produce 2a–f.
N-(5-Cyano-3-methyl-4-phenyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridin-6-yl)benza-
mide (2a)
Yield 75%, m.p. 130–132 ꢁC. IR: 3405, 3278 (3NH), 2210 (CꢂN), 1652 (C ¼ O).
1H-NMR d: 2.03 (s, 3H, CH3), 4.15 (s, 1H, C4–H), 5.92 (s, 1H, NH), 7.09–7.95 (m,
11H, Ar–H, NH), 11.35 (s, 1H, NH). 13C-NMR d: 10.0, 29.8, 55.8, 103.4, 115.2, 126.0,
127.8, 128.4, 128.8, 129.7, 133.0, 136.2, 137.8, 145.7, 160.1, 161.8, 173.6. MS m/z (%):
357 (0.68, Mþþ2), 356 (0.63, Mþþ1), 355 (0.79, Mþ), 57 (100.00). Anal. Calcd (Found)
for C21H17N5O (355.40): C, 70.97 (70.67); H, 4.82 (4.61); N, 19.71 (19.46) %.
N-(4-(4-Chlorophenyl)-5-cyano-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridin-6-
yl)benzamide (2b)
Yield 62%, m.p. 155–156 ꢁC. IR: 3372, 3311, 3169 (3NH), 2191 (CꢂN), 1648 (C ¼ O).
1H-NMR d: 2.28 (s, 3H, CH3), 4.28 (s, 1H, C4–H), 5.89 (s, 1H, NH), 7.47 (s, 1H, NH),
7.48–7.51 (m, 4H, Ar–H), 7.59–7.63 (m, 2H, Ar–H), 7.93–7.95 (m, 3H, Ar–H), 9.35
(s, 1H, NH). 13C-NMR d: 9.9, 33.8, 79.7, 104.2, 116.3, 127.7, 128.5, 128.7, 129.2, 130.2,
130.7, 132.8, 133.9, 134.7, 158.6, 160.9, 167.3. MS m/z (%): 389 (0.99, Mþ-1), 269
(100.00). Anal. Calcd (Found) for C21H16ClN5O (389.84): C, 64.70 (64.47); H, 4.14
(4.32); N, 17.96 (17.62) %.
N-(5-Cyano-4-(4-(dimethylamino)phenyl)-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyri-
din-6-yl)benzamide (2c)
Yield 70%, m.p. 268–269 ꢁC. IR: 3483, 3423, 3384 (3NH), 2205 (CꢂN), 1685 (C ¼ O).
1H-NMR d: 2.07 (s, 3H, CH3), 3.10 (s, 6H, N(CH3)2), 4.46 (s, 1H, C4–H), 7.29 (s, 1H,
NH), 7.41 (d, 2H, Ar–H, J ¼ 7.5 Hz), 7.50 (t, 2H, Ar–H, J ¼ 8.0 Hz), 7.60 (d, 2H, Ar-H,
J ¼ 7.5 Hz), 7.65 (t, 1H, Ar–H, J ¼ 7.5 Hz), 7.79 (s, 1H, NH), 7.81 (s, 1H, NH), 7.93 (d,
2H, Ar–H, J ¼ 7.5 Hz). 13C-NMR d: 12.1, 24.3, 41.9, 79.7, 116.4, 118.5, 123.5, 123.7,
125.0, 128.7, 130.8, 133.9, 134.7, 136.4, 150.0, 158.6, 160.9, 165.8. MS m/z (%): 396
(0.78, Mþ-2), 105 (100.00). Anal. Calcd (Found) for C23H22N6O (398.47): C, 69.33
(69.17); H, 5.57 (5.89); N, 21.09 (21.31) %.
N-(5-Cyano-3-methyl-4-phenyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridin-6-yl)-4-nitroben-
zamide (2d)
1
Yield 76%, m.p. 135–136 ꢁC. IR: 3406 (3NH), 2265 (CꢂN), 1694 (C ¼ O). H-NMR d:
2.03 (s, 3H, CH3), 2.93 (s, 2H, 2NH), 4.11 (s, 1H, NH), 4.45 (s, 1H, C4–H), 7.09–7.34
(m, 5H, Ar–H), 8.15 (d, 2H, Ar–H, J ¼ 8.0 Hz), 8.30 (d, 2H, Ar–H, J ¼ 8.0 Hz).
13C-NMR d: 10.2, 36.2, 55.9, 102.9, 117.9, 123.7, 125.7, 127.3, 128.1, 130.7, 136.5, 136.8,
144.9, 149.9, 159.3, 165.9, 173.2. MS m/z (%): 402 (0.18, Mþþ2), 401 (0.12, Mþþ1), 400

16
S. S. HAWAS ET AL.
(0.13, Mþ), 185 (100.00). Anal. Calcd (Found) for C21H16N6O3 (400.40): C, 63.00
(63.32); H, 4.03 (4.29); N, 20.99 (20.66) %.
N-(4-(4-Chlorophenyl)-5-cyano-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridin-6-yl)-4-
nitrobenzamide (2e)
1
Yield 77%, m.p. 180–182 ꢁC. H-NMR d: 2.10 (s, 3H, CH3), 4.62 (s, 1H, C4–H), 7.46
(s, 1H, NH), 7.50 (d, 2H, Ar–H, J ¼ 7.0 Hz), 7.60 (d, 2H, Ar–H, J ¼ 7.0 Hz), 8.16 (d, 2H,
Ar–H, J ¼ 6.5 Hz), 8.19 (s, 1H, NH), 8.31 (d, 2H, Ar–H, J ¼ 7.0 Hz), 10.55 (s, 1H, NH).
13C-NMR d: 12.1, 24.3, 79.7, 116.4, 118.5, 123.8, 125.0, 128.7, 130.3, 130.7, 134.7, 136.4,
141.9, 150.0, 158.6, 160.9, 165.8. MS m/z (%): 436 (0.3, Mþþ1), 435 (0.31, Mþ), 434
(0.41, Mþ-1), 57 (100.00). Anal. Calcd (Found) for C21H15ClN6O3 (434.84): C, 58.01
(57.76); H, 3.48 (3.16); N, 19.33 (19.64) %.
N-(5-Cyano-4-(4-(dimethylamino)phenyl)-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyri-
din-6-yl)-4-nitrobenzamide (2f)
Yield 67%, m.p. 295–296 ꢁC. IR: 3406, 3420, 3363 (3NH), 2206 (CꢂN), 1693 (C ¼ O).
1H-NMR d: 2.07 (s, 3H, CH3), 3.03 (s, 6H, N(CH3)2), 4.38 (s, 1H, C4–H), 7.10 (d, 2H,
Ar–H, J ¼ 6.5 Hz), 7.35 (d, 2H, Ar–H, J ¼ 7.5 Hz), 7.70 (d, 2H, Ar–H, J ¼ 7.5 Hz), 7.96
(d, 2H, Ar–H, J ¼ 7.5 Hz), 8.36 (s, 1H, NH), 8.42 (s, 1H, NH), 8.75 (s, 1H, NH).
13C-NMR d: 10.6, 30.1, 42.2, 75.0, 106.4, 118.7, 123.8, 130.6, 131.2, 131.9, 132.1, 133.4,
133.9, 148.4, 148.9, 158.4, 189.9. Anal. Calcd (Found) for C23H21N7O3 (443.47): C, 62.29
(62.08); H, 4.77 (4.49); N, 22.11 (22.43) %.
Synthesis of N-(5-cyano-3-methyl-4-((un)substituted phenyl)-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridin-6-yl)-N’-(heteroaryl)formamidines 3a–e
A mixture of 1a–c (0.002 mol) and ethyl N-(heteroaryl)formimidate (0.002 mol) in gla-
cial acetic acid (10 mL) was refluxed for 16–24 h. The solution was cooled and the solid
obtained was filtered and crystallized from acetic acid to give 3a-e.
N-(5-Cyano-3-methyl-4-phenyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridin-6-yl)-N’-(pyridin-
2-yl)formamidine (3a)
1
Yield 70%, m.p. 145–146 ꢁC. IR: 3426 (3NH), 2199 (CꢂN). H-NMR d: 2.02 (s, 3H,
CH3), 3.82 (s, 1H, NH), 4.11 (s, 1H, C4–H), 5.85 (s, 1H, NH), 6.41 (s, 1H, NH),
7.22–7.30 (m, 9 H, Ar–H), 7.87 (s, 1H, CH ¼ N). 13C-NMR d: 10.2, 36.2, 55.7, 102.9,
107.9, 111.8, 125.7, 127.3, 128.1, 128.2, 136.3, 136.9, 144.9, 147.6, 159.3, 159.7, 161.9,
173.2. MS m/z (%): 357 (0.89, Mþþ2), 356 (0.84, Mþþ1), 355 (0.96, Mþ), 185 (100.00).
Anal. Calcd (Found) for C20H17N7 (355.41): C, 67.59 (67.27); H, 4.82 (4.97); N, 27.59
(27.78) %.

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N-(4-(4-Chlorophenyl)-5-cyano-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridin-6-yl)-
N’-(pyridin-2-yl)formamidine (3b)
1
Yield 73%, m.p. 140–141 ꢁC. IR: 3380, 3206 (3NH), 2207 (CꢂN). H-NMR d: 2.06
(s, 3H, CH3), 4.79 (s, 1H, C4–H), 5.84 (s, 1H, NH), 6.39–6.41 (m, 2H, Ar–H), 7.10
(d, 2H, Ar–H, J ¼ 8.0 Hz), 7.25 (d, 2H, Ar–H, J ¼ 8.5 Hz), 7.22 (s, 1H, NH), 7.30–7.34
(m, 2H, Ar–H), 7.87 (s, 1H, CH¼ N), 11.33 (s, 1H, NH). 13C-NMR d: 10.4, 32.2, 79.7,
103.9, 108.0, 111.8, 116.4, 127.6, 129.4, 130.0, 137.0, 139.7, 142.4, 147.5, 158.6, 159.7, 160.9,
172.1. MS m/z (%): 390 (0.19, Mþ), 389 (0.17, Mþ-1), 57 (100.00). Anal. Calcd (Found)
for C20H16ClN7 (389.85): C, 61.62 (61.33); H, 4.14 (4.42); N, 25.15 (25.27) %.
N-(5-Cyano-4-(4-(dimethylamino)phenyl)-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyri-
din-6-yl)-N’-(pyridin-2-yl)formamidine (3c)
1
Yield 66%, m.p. 165–166 ꢁC. IR: 3423 (3NH), 2205 (CꢂN). H-NMR d: 1.97 (s, 3H,
CH3), 2.98 (s, 6H, N(CH3)2), 4.02 (s, 1H, C4–H), 5.88 (s, 1H, NH), 6.40 (d, 1H, Ar–H,
J ¼ 8.0 Hz), 6.59 (d, 2H, Ar–H, J ¼ 8.5 Hz), 7.09 (d, 2H, Ar–H, J ¼ 8.5 Hz), 7.31–7.35 (t,
1H, Ar–H, J ¼ 8.5 Hz), 7.67 (d, 1H, Ar–H, J ¼ 8.5 Hz), 7.82–7.87 (m, 1H, Ar–H), 8.04 (s,
1H, CH ¼ N), 9.65 (s, 1H, NH), 11.20 (s, 1H, NH). 13C-NMR d: 10.2, 35.2, 41.9, 68.6,
103.9, 108.0, 111.8, 112.5, 119.6, 127.7, 129.6, 133.6, 137.0, 147.5, 148.8, 154.4, 158.9,
159.7, 173.4. MS m/z (%): 396 (1.0, Mþ-2), 57 (100.00). Anal. Calcd (Found) for
C22H22N8 (398.47): C, 66.31 (66.62); H, 5.57 (5.31); N, 28.12 (27.83) %.
N-(5-Cyano-4-(4-(dimethylamino)phenyl)-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyri-
din-6-yl)-N’-(3-chloropyridin-2-yl)formamidine (3d)
1
Yield 69%, m.p. 295–296 ꢁC. IR: 3422 (3NH), 2205 (CꢂN). H-NMR d: 1.89 (s, 3H,
CH3), 3.03 (s, 6H, N(CH3)2), 4.88 (s, 1H, C4–H), 6.58 (d, 2H, Ar–H, J ¼ 8.5 Hz), 7.16
(d, 2H, Ar–H, J ¼ 8.5 Hz), 7.62–7.75 (m, 3H, Ar–H), 8.20 (s, 1H, CH ¼ N), 10.57 (s, 1H,
NH), 12.12 (s, 2H, 2NH). 13C-NMR d: 9.8, 36.6, 41.1, 56.3, 97.4, 111.2, 112.4, 119.9,
121.9, 128.1, 129.6, 136.5, 136.7, 146.2, 149.6, 155.6, 156.2, 163.5, 165.9. MS m/z (%):
435 (0.13, Mþþ2), 434 (0.30, Mþþ1), 433 (2.52, Mþ), 57 (100.00). Anal. Calcd (Found)
for C22H21ClN8 (432.92): C, 61.04 (61.37); H, 4.89 (4.66); N, 25.88 (25.51) %.
N-(5-Cyano-4-(4-(dimethylamino)phenyl)-3-methyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyri-
din-6-yl)-N’-(4-phenylthiazol-2-yl)formamidine (3e)
1
ꢁ
Yield 77%, m.p. 151–153 C. IR: 3420 (3NH), 2209 (CꢂN). H-NMR d: 1.92 (s, 3H,
CH3), 2.86 (s, 6H, N(CH3)2), 4.01 (s, 1H, C4–H), 6.59 (d, 2H, Ar–H, J ¼ 8.5 Hz), 7.03
(s, 1H, Thiazole-H), 7.07 (d, 2H, Ar–H, J ¼ 8.5 Hz), 7.29–7.51 (m, 5H, Ar–H), 7.70 (s,
1H, CH ¼ N), 8.02 (s, 1H, NH), 8.09 (s, 1H, NH), 8.95 (s, 1H, NH). 13C-NMR d: 10.8,
35.8, 40.4, 80.3, 101.9, 104.0, 108.4, 112.9, 116.8, 117.8, 126.1, 128.2, 129.2, 129.8, 130.8,
149.3, 150.4, 159.1, 160.3, 161.5, 172.6. MS m/z (%): 482 (0.42, Mþþ1), 481 (0.87, Mþ),
269 (100.00). Anal. Calcd (Found) for C26H24N8S (480.59): C, 64.98 (64.73); H, 5.03
(5.31); N, 23.32 (23.11) %.

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S. S. HAWAS ET AL.
Biology
Full biological assay methods are presented in the supplemental data.
Antimicrobial and antiquorum-sensing evaluation
Antibacterial assay[40–42]
Antifungal assay[40,41,43,44]
Antiquorum-sensing assay[40,41,45]
Antitumor evaluation
In vitro antiproliferative assay[48,49]
In vivo antitumor assay[50–52]
Cytotoxicity assay[48,49]
Mechanistic study
DNA-binding assay[53]
Topoisomerase IIb assay[57]
Supporting information
1
Results of computational studies, full experimental details, IR, H-NMR, 13C-NMR and
mass spectra are described in the supplemental data.
Acknowledgments
Appreciation to Holding Company for Biological Products and Vaccines (VACSERA), Egypt, for
accomplishing the in vitro antiproliferative, in vivo antitumor, cytotoxicity, and topoisomerase
IIb assays.
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