Synthesis and biological evaluation of 4-aza-2,3- dihydropyridophenanthrolines as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.096

A series of novel 4-aza-2,3-dihydropyridophenanthrolines 12(a-t) were synthesized by a one-step three component condensation of 1,10-phenanthroline amine, tetronic acid and various aromatic aldehydes. These were evaluated for their antiproliferative activity against three human cancer cell lines (MIAPACA, MCF-7 and HeLa) using SRB assay. Majority of the tested compounds exhibited significant anticancer activity on these cell lines and interestingly compounds 12h and 12i were more potent than etoposide and podophyllotoxin against all three tested cancer cell lines with GI₅₀ values in the range of 0.01-0.5 μM. Furthermore, these compounds showed significant inhibition of tubulin polymerization which is comparable to that of podophyllotoxin and disrupted microtubule network by accumulating tubulin in the soluble fraction. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G2/M phase. Moreover, the structure activity relationship studies in this series are also discussed.

Full text of DOI:10.1016/j.bmcl.2014.05.096

Accepted Manuscript
Synthesis and biological evaluation of 4-aza-2,3-dihydropyridophenanthrolines
as tubulin polymerization inhibitors
Ahmed Kamal, T. Srinivasa Reddy, Sowjanya Polepalli, Suresh Paidakula,
Vunnam Srinivasulu, V. Ganga Reddy, Nishant Jain, Nagula Shankaraiah
PII:
S0960-894X(14)00605-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.096
BMCL 21710
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 March 2014
19 May 2014
29 May 2014
Please cite this article as: Kamal, A., Srinivasa Reddy, T., Polepalli, S., Paidakula, S., Srinivasulu, V., Ganga Reddy,
V., Jain, N., Shankaraiah, N., Synthesis and biological evaluation of 4-aza-2,3-dihydropyridophenanthrolines as
tubulin polymerization inhibitors, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/
j.bmcl.2014.05.096
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Synthesis and biological evaluation of 4-aza-2,3-
dihydropyridophenanthrolines as tubulin polymerization inhibitors
Ahmed Kamal,*a,d T. Srinivasa Reddy,a,b Sowjanya Polepalli,c Suresh Paidakula,a Vunnam
Srinivasulu,a V. Ganga Reddy,a Nishant Jain,c Nagula Shankaraiahd
aMedicinal Chemistry and Pharmacology, bIICT-RMIT Research Centre,
cChemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
dDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
Abstract:
A series of novel 4-aza-2,3-dihydropyridophenanthrolines 12(a–t) were synthesized by a one-
step three component condensation of 1,10-phenanthroline amine, tetronic acid and various
aromatic aldehydes. These were evaluated for their antiproliferative activity against three human
cancer cell lines (MIAPACA, MCF-7 and HeLa) using SRB assay. Majority of the tested
compounds exhibited significant anticancer activity on these cell lines and interestingly
compounds 12h and 12i were more potent than etoposide and podophyllotoxin against all three
tested cancer cell lines with GI50 values in the range of 0.01-0.5 µM. Furthermore, these
compounds showed significant inhibition of tubulin polymerization which is comparable to that
of podophyllotoxin and disrupted microtubule network by accumulating tubulin in the soluble
fraction. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle
arrest at the G2/M phase. Moreover, the structure activity relationship studies in this series are
also discussed.
Keywords: 4-azapodophyllotoxin, 1,10-phenanthroline, multicomponent reaction, cytotoxicity,
tubulin polymerization.
∗,a,dCorresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189 (A.K.); e-mail:
ahmedkamal@iict.res.in (A. Kamal).

Podophyllotoxin (1), is the main component of podophyllum resin, a naturally occurring
antimitotic cyclolignan which shows strong anticancer activity against various cancer cell lines.1-
2 Interest in podophyllotoxin has been heightened by its potent antimitotic activity and it inhibits
the assembly of tubulin protein into microtubules through tubulin binding at the colchicine site.3
Attempts to use podophyllotoxin (1) for the treatment of cancer were mostly unsuccessful and
complicated by severe gastrointestinal side effects.4 However, its use as a lead in anticancer drug
design has resulted in several clinically useful compounds like etoposide (2), teniposide (3) and
etopophos (4).5-7 These compounds inhibit DNA topoisomerase-II (topo-II) by stabilizing the
covalent topo-II DNA cleavable complex8 and are used against different types of cancers,
including small-cell lung cancer, non-Hodgkin’s lymphoma, leukemia, neuroblastoma, kaposi’s
sarcoma and soft tissue sarcoma.9 In spite of their wide clinical applications, they have several
limitations, such as development of drug resistance, poor water solubility, metabolic inactivation,
and toxic effects like myelosupression.10 Extensive structural modifications have been carried
out by a number of researchers to overcome such limitations, which led to the development of
NK-611 (5)11 and GL-331 (6),12 which are in different stages of clinical studies and exhibit
improved cytotoxicity as well as DNA topo-II inhibition, compared to etoposide.
The presence of four stereogenic carbons in ring C is responsible for the structural
complexcity of podophyllotoxin (Fig 1), thus preventing the generation of its analogues from
commercially available materials.13 Furthermore, stereochemical complexity of podophyllotoxin
can be simplified by the removal of the stereo centers at C-2 and C-3, which also solves the
problem of epimerization at C-2 that afflicts the clinical usage podophyllotoxin, because the
rapidly formed cis-lactone metabolite (in vivo) is significantly less potent.14 In this connection,
many researchers made an important contribution for the synthesis of 4-azapodophyllotoxin (7)
and its derivatives (7a,7b) which retains cytotoxicity as well as inhibition of tubulin
polymerization comparable to that of podophyllotoxin.15-16
<Please Insert Figure 1>
1,10-Phenanthroline (8) and their derivatives have recently attracted much attention,
because of their potential activity against cancer as well as viral, bacterial and fungal
infections.17-20 1,10-phenanthroline (8) itself shows potential activity towards neoplastic cell

lines.21 Based on the promising anticancer activity of 4-azapodophyllotoxins and 1,10-
phenanthrolines, we have designed some new analogues by replacing A and B rings of 4-
azapodophyllotoxin scaffold with 1,10-phenanthroline moiety with a view to combine the
pharmacological characteristics of both these chromophores.
<Please Insert Figure 2>
Multicomponent reactions (MCRs) carry a privileged position because they provide a
powerful tool towards the one-pot synthesis of diverse and complex compounds as well as small
and drug-like heterocycles required for drug discovery programmes.22,23 Such processes avoid
time consuming protection/deprotection steps, as well as purification processes, and allows eco-
friendly synthesis of molecules.24-26 Recently, we have been involved in the design and synthesis
of new analogs of podophyllotoxin as potential anticancer agents.27-29 In continuation, our
interest in the structural modifications of the podophyllotoxin (1), we herein report the synthesis
of new 4-azapodophyllotoxin analogs containing phenanthroline skeleton via one step three-
component reactions of 1,10-phenanthroline amine (9), tetronic acid (10) and different aromatic
aldehydes (11 a-t) as shown in scheme 1, and the evaluation of their antiproliferative activity
including mechanistic studies of some potent compounds.
<Please Insert Scheme 1>
The 4-aza-2,3-dihydropyridophenanthroline congeners (12 a–t) were synthesized by three
component one-step synthesis involving the condensation of 1,10-phenanthroline amine (9),
tetronic acid (10), and various substituted aromatic aldehydes (11 a-t) that proceeds smoothly in
refluxing ethanol (Scheme 1).16 In all cases, the resulting compounds were precipitated as the
reaction mixtures were allowed to cool to room temperature and were isolated by simple
filtration followed by recrystallization from ethanol to afford the pure compounds. All the
synthesized compounds were characterized by 1H NMR, 13C NMR as well as HRMS.
All the synthesized 4-aza-2,3-dihydropyridophenanthroline derivatives (12 a–t) were
evaluated for their antiproliferative activity in three human cancer cell lines of pancreatic
(MIAPACA), breast (MCF-7) and cervical (HeLa) origin by using sulforhodamine B (SRB)

method. Podophyllotoxin and etoposide were used as the positive controls and the results are
summarized in Table 1. It is observed that all the synthesized compounds showed significant
antiproliferative activity with GI50 values ranging from <0.01 to 13.6 µM, while the positive
controls, etoposide and podophyllotoxin demonstrated the GI50 in the range of 0.23 to 0.71 µM
and 0.25 to 1.3 µM, respectively. The majority of the tested compounds displayed potent growth
inhibition on cervical (HeLa) and pancreatic (MIAPACA) cancer cell lines as compared to the
breast cancer cell lines (MCF-7). Some of the compounds like 12d, 12e, 12f, 12h, 12i, 12j, 12k
and 12t were distinctly more potent than podophyllotoxin and etoposide, with GI50
concentrations in the submicromolar level on certain cell lines. However, compounds 12h and
12i were the only compounds that were more active than etoposide and podophyllotoxin against
all the three tested cancer cell lines with GI50 values in the range of 0.01- 0.5 µM.
<Please Insert Table 1>
Based on the cytotoxicity data, the structure activity relationship (SAR) for these new 4-
aza-2,3-didehydropodophyllotoxin derivatives (12 a-t) was examined. It is interesting to observe
that the compounds with flouro substitution at para-position (12a) and 3,4,5-triflouro
substitution (12s) on the phenyl ring showed similar or slightly reduced activity on HeLa cancer
cell line in comparison to the 3,4-diflouro substitution (12i), which displayed potent cytotoxic
activity against all the tested cell lines. Similarly, methoxy substitution on the phenyl ring also
plays an important role in the activity, and this preference remains unchanged whether there are
other substituents on ring E in combination with methoxy substitution. For instance, 3,4-
dimethoxy (12e), 2,5-dimethoxy (12f), 3-flouro-4-methoxy (12g), 2-flouro-5-methoxy (12h)
groups are well tolerated and showed significant growth inhibition on certain cell lines, while
compounds having 4-methoxy (12c) 2-hydroxy-4-methoxy (12p) substituents, displayed
moderate to low activity. Moreover, the compounds having substitution of 3,4,5-trimethoxy
phenyl E ring (12r) as incase of podophyllotoxin and 3,5-dimethoxy-4-hydroxy (12t)
substitution as in case of etoposide, maintained cytotoxicity similar or equal to the
podophyllotoxin. The combination of hydroxyl group at 2 or 4 position of E ring with methoxy,
methyl, bromo and chloro produces compounds 12j-12p, showed selective inhibition on HeLa
cancer cells with GI50 values in the range of 0.01-1.76 µM. In view of the promising anti

proliferative activity of some leads like 12f, 12h and 12i, it was considered of interest to pursue
detailed biological studies on these compounds.
Since these new compounds have structural resemblance to podophyllotoxin, it was
considered of pertinent to investigate their effect on the inhibition of tubulin polymerization. To
examine this, varying concentrations of 12f, 12h and 12i were incubated with the tubulin protein
along with podophyllotoxin as a positive control. Table 2 shows that the compounds 12h and 12i
significantly decreased tubulin assembly with IC50 values of 0.83 µM and 0.92 µM, which are
comparable to that of podophyllotoxin. In contrast, compound 12f showed moderate inhibition of
tubulin polymerization with IC50 values 2.0 µM. These results are in correlation to the
corresponding cytotoxicity values and the order of inhibition of tubulin polymerization is Podo >
12h> 12i > 12f.
<Please Insert Table 2>
The inhibition of tubulin assembly is often associated G2/M-phase arrest of cell cycle in
various cancer cell lines.30 Therefore effect of these compounds (12f, 12h and 12i) were
determined on the cell cycle progression in HeLa cells by employing at 5 µM concentration for
24 h duration. It was observed that, these compounds markedly arrested the progression of cell
cycle at G2/M. Among them, 12h exhibited 82% of cells in G2/M phase, whereas 12i and 12f
accumulated cells to 76.44% and 73.77% respectively in G2/M phase. Thus these results suggest
that compounds induce cytotoxicity in HeLa cells through cell cycle arrest at the mitosis phase
(Figure 3).
<Please Insert Figure 3>
Microtubule depolymerizing agents prevent the organization of the mitotic spindle and
result in improper chromosome separation.31 As 12h and 12i significantly inhibit the tubulin
assembly, we analyzed their effect on the cellular microtubule network by
immunohistochemistry. Human cervical cancer cells were treated with the compounds at 5 µM
concentrations for 24 h and the cells were stained with antitubulin antibody. As expected,

treatment by these compounds disrupted microtubule organization and cells exemplify a typical
rounded phenotype, which is a hallmark of arrest at mitosis.
<Please Insert Figure 4>
Microtubule depolymerization agents disrupt the cellular tubulin protein levels by
increasing the fraction of free tubulin.32 Since the compounds (12h and 12i) efficiently arrest
cells at G2/M phase and inhibit tubulin polymerization, it was considered of interest to analyze
the effect of these compounds on intracellular tubulin. To examine this, HeLa cells were treated
with compounds 12h and 12i at 5 µM concentrations for 24 h. Subsequently, cells were
permeabilized, soluble and polymerized fractions of tubulin were collected. The DMSO treated
cells showed equal amounts of tubulin in both the fractions indicating normal equilibrium among
free and polymerized tubulin. In contrast, podophyllotoxin, 12h and 12i treated cells showed
tubulin in the soluble fraction. In comparison, paclitaxel a microtubule polymerization agent
showed more amount of tubulin in the polymerized fraction. Therefore, these results suggest that
12h and 12i robustly inhibit tubulin polymerization (in vitro) by increasing the soluble fraction
of cellular tubulin.
<Please Insert Figure 5>
In
summary,
a
library
of
new
heterocyclic
compounds,
4-aza-2,3-
dihydropyridophenanthroline derivatives (12 a–t) were synthesized by a straightforward one-step
multicomponent synthesis, which involves simple isolation of the products by filtration and
requires no further purification. These new compounds exhibited remarkable antiproliferative
activities on different cancer cell lines, which are comparable to that of podophyllotoxin and
etoposide. Compounds 12h and 12i showed potent inhibition of the tubulin polymerization and
arrested the cells at G2/M phase of the cell cycle in HeLa treated cancer cells. Moreover, these
compounds cause disruption of microtubule network and accumulated tubulin in the soluble
fraction in a manner similar to podophyllotoxin. The ease of synthesis and encouraging
biological activities make these dihydropyridophenanthroline derivatives as promising leads in
the development of anticancer agents.

Supplementary data
Experimental procedure and spectroscopic characterizations of the compounds associated with
this article could be found in the Supplementary data.
Acknowledgment
T.S.R is thankful to IICT-RMIT Research centre for the award of research fellowship. S.P, V.S,
V.G.R are thankful to CSIR-New Delhi for the award of the fellowship. We also acknowledge
CSIR for financial support under the 12th Five Year plan project “Affordable Cancer
Therapeutics (ACT)” (CSC0301)
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Figure 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), Etopophos (4), NK-
611 (5), GL-331 (6) and 4-azapodophyllotoxin (7)

Figure 2. Selected structures of 4-azapodophyllotoxin analogues (7a, 7b) and 1,10-
phenanthroline (8) as well as proposed dihydropyridophenanthrolines (12 a-t).
Table 1. Antiproliferative activity (aGI50 µM) of compounds (12 a-t)
MIAPACAb
NA
MCF-7c
NA
HeLad
0.15 0.1
NA
Compound
12a
13.6 1.9
3.4 1.0
0.7 0.3
5.9 2.1
0.1 0.04
1.2 0.7
0.02 0.01
0.5 0.04
0.9 0.07
2.0 0.18
NA
NA
12b
12c
4.6 1.1
0.06 0.02
4.6 0.6
8.4 0.7
6.4 0.6
0.01
4.2 0.5
2.6 0.43
0.04 0.04
<0.01
12d
12e
12f
0.8 0.6
<0.01
12g
12h
12i
<0.01
0.03 0.01
0.3 0.02
<0.01
7.7 1.3
7.6 1.2
NA
12j
12k
12l
0.3 0.1
1.76 0.6
0.6 0.09
0.2 0.1
7.0 1.6
5.4 1.5
1.9 1.6
0.5 0.4
9.8 1.3
9.2 2.2
NA
12m
12n
12o
10.3 2.4
9.7 1.9
12p

3.7 0.9
2.8 0.5
NA
8.2 0.2
1.7 0.2
NA
0.16 0.01
0.1 0.03
2.5 0.2
12q
12r
12s
12t
1
0.1 0.03
0.5 0.1
1.3 0.03
2.1 0.5
0.23 0.09
0.25 0.1
1.7 0.3
0.71 0.2
0.64 0.4
2
b
a50% Growth inhibition and the values are mean of three independent experiments; pancreatic
cancer, cbreast cancer, dcervix cancer, 1podophyllotoxin, 2etoposide, NA = not active
Table 2. Inhibition of tubulin polymerization of 12f, 12h and 12i.
S.No
Compound
IC50 (µM)
2.0
1
2
3
4
12f
12h
12i
1
0.83
0.92
0.81
Effect of compounds on tubulin polymerization. IC50 values for 12f, 12h and 12i were
determined from the tubulin polymerization assays.

Figure 3. Antimitotic effects of 12f, 12h and 12i by FACS analysis:
Induction of cell cycle G2/M arrest by compounds 12f, 12h and 12i. HeLa cells were harvested
after treatment at 5 µM for 24h. Untreated cells and DMSO treated cells served as controls. The
percentage of cells in each phase of cell cycle was quantified by flow cytometry.

Figure 4. Effect of 12h and 12i on microtubules and nuclear condensation
HeLa cells were independently treated with 12h and 12i at 5 µM concentrations for 24h.
Following the termination of experiment, cells were fixed and stained for tubulin. DAPI was
used as counter stain. The merged images of cells stained for tubulin and DAPI are represented.
Figure 5. Distribution of tubulin in polymerized vs soluble fractions as analyzed by
immunobloting in 12h and 12i treated HeLa cells: the cells were treated with 5 µM of 12h and
12i for 24h. Tubulin was detected by Immunoblot analysis.

N
N
NH2
O
H
N
N
N
O
EtOH, reflux
1-3 h
O
CHO
O
9
10
O
R
R
11
12(a-t)
12k
12l
= 3-bromo-4-hydroxy
= 2-hydroxy-5-chloro
= 2-hydroxy-5-bromo
= 2-hydroxy-5-methyl
= 2-hydroxy-3-methyl
= 2-hydroxy-4-methoxy
= 2,3,4-trimethoxy
= 3,4,5-trimethoxy
= 3,4,5-triflouro
= 3,5-dimethoxy-4-hydroxy
12a
= 4-flouro
= 3-chloro
= 4-methoxy
= 4-triflouromethyl
= 3,4-dimethoxy
= 2,5-dimethoxy
= 3-flouro-4-methoxy
= 2-flouro-5-methoxy
= 3,4-diflouro
12b
12c
12d
12e
12f
12g
12h
12i
12m
12n
12o
12p
12q
12r
12s
12t
12j
= 3-chloro-4-hydroxy
Scheme 1. Synthesis of 4-aza-2,3-dihydropyridophenanthrolines (12 a–t)

Graphical Abstract
Synthesis and biological evaluation of 4-aza-2,3dihydropyrido
phenanthrolines as tubulin polymerization inhibitors
Ahmed Kamal,*a,d T. Srinivasa Reddy,a,b Sowjanya Polepalli,c Suresh Paidakula,a
Vunnam Srinivasulu,a V. Ganga Reddy,a Nishant Jain,c Nagula Shankaraiahd
N
N
NH2
O
H
12h
12i
N
N
N
O
O
O
CHO
O
R
R
12(a-t)
tubulin polymerisation inhibitors
12h = 2-flouro-5-methoxy (GI50 0.01-0.02 µM)
12i
µ
= 3,4-diflouro (GI50 0.01-0.5 M)