lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth of colon cancer cells via apoptosis and sub G1 cell cycle arrest

Article abstract of DOI:10.1016/j.bmcl.2018.05.045

A series of lH-pyrazolo[3,4-b]quinolin-3-amine derivatives were synthesized and evaluated for anticancer efficacy in a panel of ten cancer cell lines, including breast (MDAMB-231 and MCF-7), colon (HCT-116, HCT-15, HT-29 and LOVO), prostate (DU-145 and PC3), brain (LN-229), ovarian (A2780), and human embryonic kidney (HEK293) cells, a non-cancerous cell line. Among the eight derivatives screened, compound QTZ05 had the most potent and selective antitumor efficacy in the four colon cancer cell lines, with IC₅₀ values ranging from 2.3 to 10.2 μM. Furthermore, QTZ05 inhibited colony formation in HCT-116 cells in a concentration-dependent manner. Cell cycle analysis data indicated that QTZ05 caused an arrest in the sub G1 cell cycle in HCT-116 cells. QTZ05 induced apoptosis in HCT-116 cells in a concentration-dependent manner that was characterized by chromatin condensation and increase in the fluorescence of fluorochrome-conjugated Annexin V. The findings from our study suggest that QTZ05 may be a valuable prototype for the development of chemotherapeutics targeting apoptotic pathways in colorectal cancer cells.

Full text of DOI:10.1016/j.bmcl.2018.05.045

Accepted Manuscript
lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth of colon cancer
cells via apoptosis and sub G1 cell cycle arrest
Chandrabose Karthikeyan, Haneen Amawi, Arabela Guedes Viana, Leticia
Sanglard, Noor Hussein, Maria Saddler, Charles R. Ashby Jr., N.S. Hari
Narayana Moorthy, Piyush Trivedi, Amit K. Tiwari
PII:
S0960-894X(18)30457-8
https://doi.org/10.1016/j.bmcl.2018.05.045
BMCL 25862
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 April 2018
5 May 2018
23 May 2018
Please cite this article as: Karthikeyan, C., Amawi, H., Viana, A.G., Sanglard, L., Hussein, N., Saddler, M., Ashby,
C.R. Jr., Moorthy, S.H.N., Trivedi, P., Tiwari, A.K., lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth
of colon cancer cells via apoptosis and sub G1 cell cycle arrest, Bioorganic & Medicinal Chemistry Letters (2018),
doi: https://doi.org/10.1016/j.bmcl.2018.05.045
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lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth of colon cancer cells via
apoptosis and sub G1 cell cycle arrest.
Chandrabose Karthikeyana,e#*, Haneen Amawib#, Arabela Guedes Vianac, Leticia Sanglardc,
Noor Husseinb, Maria Saddlerc, Charles R. Ashby, Jr.d, N. S. Hari Narayana Moorthya,
Piyush Trivedie, Amit K. Tiwarib,c,*
aDepartment of Pharmacy, Indira Gandhi National Tribal University, Amarkantak-484887,
India.
bDepartment of Pharmacology and Experimental Therapeutics, College of Pharmacy &
Pharmaceutical Sciences, University of Toledo, OH, USA
cDepartment of Biomedical Sciences, Tuskegee University, Tuskegee, AL.
dCollege of Pharmacy and Pharmaceutical Sciences, St. John’s University, NY
eSchool of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya,Bhopal-
462036, India
Running title: Anticancer activity of lH-Pyrazolo[3,4-b]quinolin-3-amines
#Note: First two authors contributed equally to this work.
*Corresponding Authors:
Amit K. Tiwari, PhD
Principal Investigator,
Department of Pharmacology and Experimental Therapeutics,
College of Pharmacy & Pharmaceutical Sciences,
University of Toledo, Ohio 43614
Tel: 419-383-1913
Fax: 419-383-1909
Email: amit.tiwari@utoledo.edu
ChandraboseKarthikeyan, PhD
Department of Pharmacy,
Indira Gandhi National Tribal University,
Amarkantak - 484887.
Tel: +917629-269869
Email: karthikeyanchandrabose@gmail.com

Abstract
A series of lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives were synthesized and evaluated
for anticancer efficacy in a panel of ten cancer cell lines, including breast (MDAMB-231 and
MCF-7), colon (HCT-116, HCT-15, HT-29 and LOVO), prostate (DU-145 and PC3), brain
(LN-229), ovarian (A2780), and human embryonic kidney (HEK293) cells, a non-cancerous
cell line. Among the eight derivatives screened, compound QTZ05 had most potent and
selective antitumor activity in the four colon cancer cell lines with IC50 values ranging from
2.3 to 10.2 µM. Furthermore, QTZ05 inhibited colony formation in HCT-116 cells in a
concentration-dependent manner. Cell cycle analysis data indicated that QTZ05 caused an
arrest in the sub G1 cell cycle in HCT-116 cells. QTZ05 induced apoptosis in HCT-116 cells
in a concentration-dependent manner that was characterized by chromatin condensation and a
reduction in mitochondrial membrane potential. The findings our study suggest that QTZ05
may be a valuable prototype for the development of chemotherapeutics targeting apoptotic
pathways in colorectal cancer cells.
Keywords: lH-Pyrazolo[3,4-b]quinolin-3-amines; Cytotoxicity; colorectal cancer; Apoptosis

Cancer still remains the leading cause of human deaths worldwide, with 8.2 million reported
deaths (around 13% of all deaths) in 20121. Despite an increasing understanding of the
molecular biology of cancer and the consequent increase in the development of anticancer
compounds, most of the chemotherapeutic drugs currently used for treating many types of
cancer are often rendered ineffective due to the development of drug resistance during
treatment [2]. Furthermore, treatment may be discontinued as a result of drug-related
toxicities2. Therefore, there is a continuous need for the development of novel
chemotherapeutic drugs for cancer therapy that can surmount these aforementioned
limitations.
Figure 1: Design Strategy and Eight 1H-pyrazolo[3,4-b]quinolin-3-amines reported in this
study.
Functionalized quinolines and their hetero-fused analogues represent an important class of
organic molecules and they are present in numerous natural products 3, 4. Pyrazoloquinolines,

which have quinoline and pyrazole moieties in their molecular framework, is an excellent
example of this class of compounds that have abroad spectrum pharmacological efficacies,5, 6,
7,8, 9, 10-13. Notably, we are interested in the antitumor efficacy of pyrazoloquinolines, which
is elicited through inhibition of topoisomerase II7, CHK1 kinase14, DNA-dependent protein
kinase15, Oncogenic Ras protein16 and by induction of apoptosis17.
During the course of our investigations on polycyclic quinolines with antitumor efficacy, we
reported the discovery of IND-2, a pyrimido[1”,2”:1,5]-pyrazolo[3,4-b]quinoline (Figure 1)
with potent cytotoxic and apoptosis-inducing properties in colon cancer cells18. Thus, in this
study, we chose to evaluate 1H-pyrazolo[3,4-b]quinolin-3-amines, the major substructural
unit and synthetic precursor of IND-2, for their anticancer efficacy (Figure 1). Eight
pyrazolo[3,4-b]quinolin-3-amine derivatives (Figure 1), bearing different substituents on the
fused benzo ring, were synthesized and their anticancer efficacy determined in a panel of 10
cancer cell lines.
Scheme 1: Synthesis of lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives.

The synthesis of 1H-pyrazolo [3,4-b]quinolin-3-amines was accomplished, using a three-step
protocol, as shown in scheme 1. The first two steps involved the synthesis of the starting
material, 2-chloroquinoline-3-carbaldehydes (2a-2h), from suitable acetanilides (1a-1h).
This was followed by subsequent conversion to the corresponding 2-chloroquinoline-3-
carbonitriles (3a-3h), using well-established procedures as described previously18-20. The
obtained 2-chloroquinoline-3-carbonitriles on addition with hydrazine hydrate in under reflux
conditions produced 1H-pyrazolo[3,4-b]quinolin-3-amines in good yields (QTZ01-QTZ08)8.
The IR spectra of the synthesized 1H-pyrazolo[3,4-b]quinolin-3-amines displayed two
characteristic peaks for the NH and NH2 groups in the region 3424-3325 cm-1 and 3300-3256
cm-1, respectively. In the 1H NMR spectra, the signal for NH and NH2 appeared as singlets at
δ 11.24-12.5 ppm (D2O exchangeable) and δ 5.52-5.96 ppm (D2O exchangeable),
respectively. The 1H NMR spectra of compounds also showed a singlet in the region δ 8.28-
8.72 ppm due to the presence of the C13 proton of the 1H-pyrazolo[3,4-b]quinoline ring.
Further, the presence of singlet, doublet, triplet and multiplets at around δ 6.77-8.03 ppm in
the compounds indicated aromatic protons in the benzo ring of the pyrazolo[3,4-b]quinolinyl
moiety. The structures assigned for the compounds were also supported by the elemental
analysis and mass spectral data.
The cytotoxicity of synthesized compounds was determined in a panel of ten cancer cell
lines, including breast (MDAMB-231 and MCF-7), colon (HCT-116, HCT-15, HT-29 and
LOVO), prostate (DU-145 and PC3), brain (LN-229), ovarian (A2780), and a non-cancerous
cell line, human embryonic kidney (HEK293) (Table 1). The cytotoxicity was determined
using the MTT assay with a series of different concentrations for each compound was used
(0, 0.1, 03, 1, 3, 10, 30, and 100 µM). The results are expressed as IC50 and are summarized
in Table 1.

Table 1: Activity of 1H-pyrazolo[3,4-b]quinolin-3-amine derivatives against various cell
lines (cancerous and non-cancerous).
IC50 ± SD, µM
Comp. code
Breast
MDAMB-231
Prostate
Brain
Ovarian
MCF-7
81.5 ± 28.4
>100 ± NA
78.8 ± 13.0
49.5 ± 8.4
59.2 ± 11.9
72.1 ± 7.7
>100 ± NA
73.6 ± 12.6
DU-145
PC3
LN-229
A2780
QTZ-1
QTZ-2
QTZ-3
QTZ-4
QTZ-5
QTZ-6
QTZ-7
QTZ-8
>100 ± NA
>100 ± NA
>100 ± NA
62.3 ± 5.9
50.6 ± 8.9
77.9 ± 5.6
89.9 ± 19.6
>100 ± NA
>100 ± NA
>100 ± NA
>100 ± NA
76.5 ± 12.6
53.4 ± 11.5
73.4 ± 16.7
>100 ± NA
>100 ± NA
IC50 ± SD, µM
>100 ± NA
>100 ± NA
>100 ± NA
>100 ± NA
87.1 ± 25.1
7.8 ± 5.5
83.9 ± 6.5
61.2 ± 12.4 98.3 ± 13.7
38.9 ± 17.0 >100 ± NA
93.7 ± 25.3 35.8 ± 10.6
7.8 ± 2.3
56.6 ± 12.6
19.6 ± 25.1
59.9 ± 10.5
13.5 ± 8.5
65.2 ± 15.5 26.0 ± 5.3
>100 ± NA 58.6 ± 4.9
59.7 ± 16.9 54.3 ± 8.2
Colon
Normal
HEK 293/pcDNA.3.1
Comp. code
HCT-116
67.7 ± 7.4
>100 ± NA
>100 ± NA
57.0 ± 5.8
2.3 ± 0.9
HCT-15
58.6 ± 6.2
>100 ± NA
>100 ± NA
69.3 ± 8.6
9.6 ± 3.6
HT-29
LOVO
88.6 ± 16.1
89.6 ± 15.4
>100 ± NA
55.6 ± 8.6
10.2 ± 11.0
33.4 ± 12.3
77.5 ± 20.3
80.6 ± 23.5
>100
>100
>100
>100
QTZ-1
QTZ-2
QTZ-3
QTZ-4
QTZ-5
QTZ-6
QTZ-7
QTZ-8
95.7 ± 22.4
32.9 ± 5.9
>100 ± NA
85.2 ± 13.6
3.9 ± 0.5
±
±
±
±
NA
NA
NA
NA
45.8 ± 12.2
81.1
36.6
40.8 ± 14.6
56.6 ± 4.6
76.1 ± 21.4
12.8 ± 3.9
44.8 ± 8.6
68.6 ± 13.3
19.4 ± 8.3
42.3 ± 10.8
98.7 ± 14.9
±
±
15.4
4.8
>100 ± NA
Cell survival assay was determined by the MTT assay. IC50 values are represented as means
of three independent experiments performed in triplicate. A mean IC50 value of 100 μM was
the cut off. The compounds were screened on breast (MDAMB-231 and MCF-7), colon
(HCT-116, HCT-15, HT-29, and LOVO), prostate (DU145), brain (LN-229), and ovarian
(A2780) cancer cell lines. Human embryonic kidney cells (HEK293/pcDNA 3.1) is a non-
cancerous cell line.
According to the cytotoxicity data in Table 1, the compounds QTZ01, QTZ05, QTZ06 and
QTZ08 displayed variable efficacy on the inhibition of the growth of the human tumor cell
lines HCT-116, HCT-15, HT-29, LOVO, LN-229 and A2780. Compound QTZ05, with a 7-
OCH3 substituent in the benzo ring of the 1H-pyrazolo[3,4-i]quinolin-3-amine moiety, was
the most potent in the series. QTZ05 had broad spectrum cytotoxic efficacy in colon cancer
cells, with IC50 values ranging from 2.3 to 10.2 µM. QTZ05 also displayed excellent
selectivity for colon cancer cells compared to non-cancerous HEK293 cells, with 20, 5, 11
and 4-fold greater selectivity for HCT-116, HCT-15, HT-29 and LOVO cells, respectively
(Table 1).

Figure 2: Morphological analysis of the cytotoxic effects of QTZ05 (1, 10 and 30 µM) or
vehicle on the colon cancer cells, HCT-116, HCT-15, and LOVO and human embryonic
kidney cells HEK293, incubated for 72 h. The cells were photographed for each triplicate
incubation using an inverted microscope (Olympus, BX53F) with digital cameras. A
representative image is shown for each treatment. The data were acquired and analyzed with
CellSens software.
Figure 2 illustrates the cytotoxicity of QTZ05 in colon cancer cells compared to normal
cells. Furthermore, QTZ05, at 7.6 µM, also significantly inhibited the growth of the brain
cancer cell line. QTZ01, the unsubstituted derivative, also inhibited LN-229 cells at
concentration similar to that of QTZ05. However, neither QTZ05 nor QTZ01 had significant
cytotoxic efficacy in other cancer cell lines (Table1). Compound QTZ06, with a 6,7-
dimethoxy substitution, had moderate cytotoxic efficacy against the colon cancer cell lines
(HCT-15 and HT-29 (IC50 < 20 µM) and A2780 ovarian cancer cells (IC50 = 19.6 µM) (Table
1). In contrast, compound QTZ08, a benzo fused derivative, had selective and moderate
growth inhibitory efficacy in A2780 cancer cells (IC50 = 13.6 µM) (Table 1).

Overall, the results of the MTT assay establishes compound QTZ05 to be the most
interesting candidate for further mechanistic investigations in terms of its potent cytotoxicity
on colon cancer cell lines.
Figure 3: The cytotoxicity of QTZ05 on HCT116 cells over time. The cells were incubated
with, 10, 20 and 30 µM of QTZ05 or vehicle for 72 h. The pictures were taken every 15 min
from the same spot by a CytoSMART™ Lux 10X system for live cell imaging. The
confluence at each time point were used to generate confluence curves over time for each
compound or vehicle.

First, we investigated the cytotoxicity of QTZ05 on HCT116 cells over time. The
cytotoxicity of different concentrations of QTZ05 (0, 10, 20, and 30 µM) at different times
(every 15 minutes up to 72 h) was determined. Figure 3 shows representative pictures of the
cell confluence for the different concentrations (0, 10, 20, and 30 µM) at 0, 12, 24, 36, 48, 60,
and 72 h. Clearly, control cells were able to grow and replicate over time, and they reached
high confluence after 3 days of incubation (≈ 60%, Figure 3). However, the cells incubated
with QTZ05 grew significantly slower over time and they only reached a very low confluence
at the experiment (≈ or < that 20% confluence for all 3 concentrations of QTZ05, Figure 3).
These results also indicated that QTZ05 produces cytotoxicity at relatively early time points
(≈20 h of incubation). A detailed graph(Figure 3) showing the results for each compound and
each time point was generated to clarify the cytotoxicity over time.
Figure 4: QTZ05 inhibits the colony formation rate of HCT 116 cells. The cells were
incubated with different concentrations QTZ05 (5, 10 or 20 uM) or vehicle. The pictures
show the effect of QTZ05 colonies density and colony size. Each experiment was repeated
independently three times.

Next, we investigated the efficacy of QTZ05 (5, 10 or 20 µM) to inhibit growth of HCT116
cells colonies. Figure 4 shows the number of colonies formed, as well as a representation of
the colony sizes in the presence of QTZ05 (0, 5, 10, and 20 µM). QTZ05 significantly
inhibits colony formation abilities in HCT116 cells (Figure 4). In addition, the HCT116 cells
that survive incubation with QTZ05 are unable to replicate (Figure 4). The mean colony
formation rate was decreased significantly in HCT116 cells incubated with 5, 10, and 20 µM,
respectively, of compound QTZ05, compared to control cells (p < 0.001 for all
concentrations, Figure 4).
Figure 5: QTZ05 induces sub G1 cell cycle arrest in HCT116 cells. HCT116 cells were
incubated with different concentrations (5, 10, 20 µM) of QTZ05 or vehicle for 24 h and
subjected to cell cycle analysis by flow cytometry of PI (X axis)/cell counts (Y axis). The
data represents means ± SD of three independent experiments performed in triplicate.
Further, the effect of QTZ05 on the cell cycle was analyzed using flow cytometry cell cycle
analysis with propidium iodide (PI)). QTZ05 produced a significant concentration-dependent

increase in the number of cells in the subG1phase in cells treated with QTZ05 compared to
cells incubated with vehicle (control cells, Figure 5). The percentage of cells in the sub G1
phase increased from 3.8% in control cells, to 22.4%, 32.0%, and 55.9% in cells incubated
with5, 10, or 20 µM of QTZ05, respectively. QTZ05 also significantly shifted the cells from
the G1 phase, where incubation with 5, 10 or 20 µM of QTZ05 decreased the number of cells
in the G1 phase to 59.3%, 45.9% and 33.5%, respectively, compared to 78.7% in cells
incubated with vehicle (Figure 5). In contrast, QTZ05 (5, 10 or 20 uM) did not significantly
alter the number of cells in the S or G2 phases (Figure 5). Our results indicate that QTZ05
produces a concentration-dependent increase in the number of cells in the sub G1 phase of
the cell cycle.
Figure 6: QTZ05 induces
apoptosis in HCT116 cells.
HCT116 cells in complete
medium were incubated with
QTZ05 at 5, 10, 20, or 30 µM
or vehicle for 24 h. Cells were
then incubated with the
reagents of the MitoTracker
Red and Alexa Fluor 488
annexin
V
kits for flow
Representative
cytometry.
results of HCT116 cells from at
least two independent
experiments, each performed in
triplicate, are shown.
The induction of apoptosis is one of the main mechanisms by which many clinically
approved anticancer drugs, despite differences in their structures and interaction with cellular

targets, produce cytotoxicity in cancer cells21. Therefore, we used the annexin mitotracker
red kit to measure the mitochondrial membrane potential and the fluorescence of
fluorochrome-conjugated annexin V as indicators for apoptosis levels. Annexin V can only
bind to exposed phosphatidylserine (PS) that translocates from the inner to the outer side of
the cell membrane during apoptosis22. As shown in Figure 6, about 92.5% of the control
HCT116 cells are in quadrant I, indicating that most of the cells are viable and intact (Figure
6), whereas only a small portion of the cells express high annexin fluorescence, indicating
apoptosis (7.48%) in quadrant II (Figure 6). However, more cells were shifted towards
quadrant II after incubation with 5, 10, 20, or 30 μM of QTZ05 (Figure 6). QTZ-05 at 5, 10,
20 or 30 μM, produced a non-significant reduction (77%, 68.1%, 66%, and 46.7%
respectively), in the percent of viable cells located in quadrant I (Figure 6). Furthermore,
QTZ05 produced a significant accumulation of cells in quadrant II in a concentration-
depended manner (5 µM: 22.9%, 10 µM:31.8%, 20 µM: 34%, and 30 μM:53.3 %
respectively, Figure 6), indicating that QTZ05 induces apoptosis.
Nuclear condensation is one of the hallmarks of cellular apoptosis23. Therefore, we used
DAPI staining to visualize nuclear changes after incubating HCT116 cells with different
concentrations of QTZ05 (0, 5 or 10 µM) for 24 and 48 h. As shown in Figure 7, control
cells had typical oval nuclei (indicative of viable cells) with non-condensed, low bright - blue
staining. Overall, HCT116 cells incubated with vehicle did not undergo a significant
magnitude of apoptosis as virtually all of the cells had no nuclear condensation. However, the
incubation of cells with 5 or 10 µM of QTZ05 for 24 h significantly increased the number of
cells with condensed, fragmented nuclei, (Figure 7). Furthermore, the incubation of cell with
5 or 10 µM of QTZ05 for 48 h resulted in a higher chromatin condensation and brighter
staining compared to cells incubated for 24 h (Figure 7). Overall, the above results indicate
that QTZ05-induced cytotoxicity in HCT116 cells is due to apoptosis.

Figure 7: The effect of QTZ05 on the nuclear morphology of HCT116 cells. The changes in
the nuclear morphology of HCT116 cells upon incubation with different concentrations (5, or
10 μM) of QTZ05 or vehicle for 24 or 48 h, respectively, was detected using DAPI staining.
The cells were fixed and stained with the DAPI. Condensed and fragmented nuclei were
observed under an EVOS fluorescent microscope at 40X.
In conclusion, a series of eight lH-Pyrazolo[3,4-b]quinolin-3-amineswere synthesized and
their cytotoxic efficacy was determined in ten cancer cell lines. The compound QTZ05, with
a 7-OCH3 substituent in the benzo ring of 1H-pyrazolo[3,4-b]quinolin-3-amine moiety, had
significant and relatively selective cytotoxic efficacy in four colon cancer cell lines, with IC50
values ranging from 2.3 to 10.2 µM. This cytotoxicity of QTZ05 on colon cancer cells is not
strong as our previously reported compound (IND-2), but the cytotoxicity on the other cells
lines is also lowered. QTZ05’s cytotoxic efficacy in colon cancer cells is mediated by cell
cycle arrest at the sub G1 phase and induction of apoptosis, as indicated by chromatin
condensation and translocation of PS detected by Annexin V in HCT-116 cells. Overall, the
findings our study suggest that lH-Pyrazolo[3,4-b]quinolin-3-amines,in particular, QTZ05,

may be an important prototype for the development of compounds targeting apoptotic
pathways in colorectal cancer cells.
Acknowledgements
This research was funded by University of Toledo start-up funds to A.K.T. The authors
gratefully acknowledge IISER-Bhopal for the NMR spectral analysis of the compounds used
in this study. We thank Ms. Charisse Montgomery, University of Toledo, Ohio, for editing
this manuscript. We also thank the Council of Scientific and Industrial Research (CSIR),
New Delhi, India for a Senior Research Fellowship (C. K.).
Conflict of Interest: Authors declared no conflict of interest.

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In Vitro Antitumor Activity
IC50: 2.3 to 10 µM against 4
colon cancer cell lines
Sub G1 Cell Cycle Arrest
Apoptosis

Highlights:
 Eight lH-Pyrazolo[3,4-b]quinolin-3-amine (QTZ) derivatives were synthesized
 The QTZ05 is the most potent compounds against colon cancer cells
 The QTZ05 acts via apoptosis and sub G1 cell cycle arrest.