Synthesis and Biological Evaluation of Calothrixins B and their Deoxygenated Analogues

Article abstract of DOI:10.1021/acs.jmedchem.7b01797

A series of calothrixin B (2) analogues bearing substituents at the 'E' ring and their corresponding deoxygenated quinocarbazoles lacking quinone unit were synthesized. The cytotoxicities of calothrixins 1, 2, and 15b-p and quinocarbazole analogues were i

Full text of DOI:10.1021/acs.jmedchem.7b01797

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Article
Synthesis and Biological Evaluation of
Calothrixins B and their Deoxygenated Analogues
Bose Muthu Ramalingam, Nachiappan Dhatchana Moorthy, Somenath Roy Chowdhury,
Thiyagarajan Mageshwaran, Elangovan Vellaichamy, Sourav Saha, Karthikeyan
Ganesan, B. Navin Rajesh, Saleem Iqbal, Hemanta Kumar Majumder, Krishnasamy
Gunasekaran, Ramamoorthy Siva, and Arasambattu Kannan Mohanakrishnan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01797 • Publication Date (Web): 09 Jan 2018
Downloaded from http://pubs.acs.org on January 11, 2018
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Synthesis and Biological Evaluation of Calothrixins B and their
Deoxygenated Analogues
Authors List:
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Bose Muthu Ramalingam, Nachiappan Dhatchana Moorthy, Somenath Roy Chowdhury,
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Thiyagarajan Mageshwaran, Elangovan Vellaichamy, Sourav Saha, Karthikeyan Ganesan,
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B. Navin Rajesh, Saleem Iqbal, Hemanta K. Majumder, Krishnasamy Gunasekaran,
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1*
Ramamoorthy Siva, and Arasambattu K. Mohanakrishnan
1
Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025,
India
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Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India
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Division of Infectious Diseases & Immunology, Indian Institute of Chemical Biology, 4 Raja
S. C. Mullick Road, Jadavpur, Kolkata 700032, India.
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Research and Development Centre, Orchid Pharma Ltd, Sholinganallur, Chennai 600 119,
India
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CAS in Crystallography & Biophysics, University of Madras, Chennai 600 025, India
School of Bio Sciences and Technology, VIT University, Vellore 632 014, India
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Abstract
A series of calothrixin B (2) analogues bearing substituents at the ‘E’ ring and their
corresponding deoxygenated quinocarbazoles lacking quinone unit were synthesized. The
cytotoxicities of calothrixins 1, 2, 15b-p and quinocarbazole analogues were investigated
against nine cancer cell lines. The quinocarbazoles 21a and 25a inhibited the catalytic activity
of human topoisomerase II. The plasmid DNA cleavage abilities of calothrixins 1, 2, 15b-p
identified compound 15h causing DNA cleavage comparable to that of calothrixin A (1).
Calothrixin A (1), 3-fluorocalothrixin 15h and 4-fluoroquinocarbazole 21b induced extensive
DNA damage followed by apoptotic cell death. Spectral and plasmid unwinding studies
demonstrated an intercalative mode of binding for quinocarbazoles. We identified two
promising drug candidates, the 3-fluorocalothrixin B 15h with low toxicity in animal model and
its deoxygenated derivative 4-fluoroquinocarbazole 21b as having potent cytotoxicity against
NCI-H460 cell line with a GI of 1 nM.
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Introduction
Quinones consist an important class of antitumor agents and are known to act as DNA
intercalators, reductive alkylators of biomolecules and also as generators of reactive oxygen
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species. The biological redox activity of these compounds is recognized to play a key role in
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their cytotoxicity. However, the biological mode of action of quinones is highly dependent on
their structural motif. The structurally complex quinones such as doxorubicin are known to
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exhibit cytotoxicity through polymodal action. Among indole based quinone alkaloids,
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murrayaquinone A and mitomycin C are known to exhibit cytotoxicity. Calothrixin A (1) and
B 2 (Chart 1) are carbazole-1, 4-quinone alkaloids isolated from Calothrix cyanobacteria with
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unique indolo [3,2-j] phenanthridine framework.
Chart 1. Calothrixin A (1), Calothrixin B (2) and its Analogues 3-6
Calothrixins inhibit the growth of human HeLa cancer cells and chloroquine resistant strain of
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Plasmodium falciparum within the nanomolar range. They inhibit the bacterial RNA
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polymerase and also poisons the DNA topoisomerase I. In addition, calothrixin A (1) is also
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known to induce the intracellular formation of reactive oxygen species.
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Quinocarbazole 3 (Chart 1) was first synthesized from this laboratory via thermal
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electrocyclization of 1-phenylsulfonyl-2,3-divinylindole as a key step. Five years later,
oxidized form of quinocarbazole, i.e. calothrixin A (1) and B (2) were isolated by Rickards and
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coworkers. To date, twenty different syntheses for calothrixin have been reported. In 2000,
Kelly et al reported the first synthesis of calothrixin B (2) involving double lithiation strategy
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from N,N-diethyl-quinoline-4-carboxamide and N-MOM-protected indole-3-carboxaldehyde.
Chai and coworkers reported a simple and concise route to calothrixin B (2) via Friedel-Crafts
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acylation followed by lithiation mediated cyclization. Hibino and coworkers employed an
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allene-mediated electrocyclization as a key step for calothrixin B (2). The same group also
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achieved the biomimetic synthesis of calothrixin B (2). Syntheses of calothrixin were also
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established using hetero Diels-Alder reaction as well as radical cyclization protocols.
Moody and coworkers reported the biomimetic synthesis of calothrixin from indolo[2,3-
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a]carbazole. Synthesis of calothrixin B (2) was achieved by Abe et al as well as Kumar et
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al, involving Pd-catalyzed tandem cyclization/cross coupling reactions. The Pd-mediated
cross coupling strategies for indolophenanthridine system were also achieved independently by
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Kusurkar et al as well as Nagarajan and coworkers. A linear synthesis of calothrixin B and its
analogues were achieved from this laboratory involving thermal electrocyclization of 2-
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nitroarylvinyl-3-phenylsulfonylvinyl indoles. Total synthesis of calothrixins A (1) and B (2)
was achieved via Mn(OAc) -mediated oxidative radical reaction of cyclohexenone with amino-
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phenanthridinedione. Mal and coworkers outlined a short synthesis of calothrixin B unit via
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anionic annulation of furoindolone with 4-bromoquinoline. Dethe and coworkers reported a
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concise total synthesis of calothrixin B using LTA-mediated rearrangement as the key step.
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Despite, several synthetic routes explored for calothrixins, there are only few reports on
cytotoxicity and antimalarial activity of these compounds. An assembly of three
pharmacophores in calothrixins namely indole, quinone and quinoline spanning five rings (A-E)
provides a unique possibility for structural modification so as to produce analogues of
calothrixin with better cytotoxic profile. Earlier attempts to study the contribution of different
structural features of calothrixins towards anti-cancer potency indicate that the introduction of
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MOM group on the indole nitrogen (N-MOM-calothrixin B) or methyl group to the D-ring
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nitrogen (N-methycalothrixin B) decrease the cytotoxic potency in HeLa and CEM leukemic
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cell lines, respectively. The removal of D-ring nitrogen atom (indolophenanthrene-7,13-dione)
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or both the E and D rings (2-methylcarbazoledione) of calothrixin B (2) decreased anti-
proliferative potency in HeLa cells with the exception of ellipticine quinone which lack the E-
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ring is equally potent as calothrixin B (2). The quinones containing bi- and tricylic systems
(murrayaquinone, 2-methylcarbazoledione, and isoquinoline-5,8-dione) are less active than the
pentacyclic calothrixin B (2) in HeLa cells but not in case of murine P388 macrophage cancer
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cells.
To date, the anti-cancer SAR studies carried out with calothrixin analogues highlight the
involvement of ring structure (A-E) and importance of indole or D-ring nitrogen atom on the
inhibitory action, mainly in HeLa cells. However, the contribution of the quinone unit to the
cytotoxicity of calothrixin in cancer cells is not studied. Most importantly, the spectrum of
anti-cancer activity of calothrixins in other cancer cell lines is not reported. The ring structure
of calothrixins might favor DNA intercalation which can result in cellular cytotoxicity, however
less is known about its interaction with DNA. The precise mechanism on the effect of
calothrixins on cell cycle is not clear. When administered at low concentration (0.1 µM) to p53
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proficient CEM leukemic cells, calothrixin B (2) but not calothrixin A (1) caused arrest of cells
in G1-phase. Whereas higher concentration of calothrixin A (1) and N-methyl calothrixin B
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caused G2-M arrest although in a reversible manner. The calothrixins A (1), B (2) and N-
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methyl calothrixin B are shown to stabilize cleavable complexes of topo I-DNA, though at one
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fifth of the potency of camptothecin. Along with the cell cycle arrest at G2-M phase,
calothrixin A (1) was observed to generate reactive oxygen species (ROS) and induce cell death
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through apoptosis in Jurkat cell line. Recently, Velu and coworkers reviewed the synthesis and
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biological evaluation of calothrixin B (2).
In the present study, we planned to address the above mentioned lacunae and to get further
insight on the biological effects of calothrixins. Another objective is to delineate the role of
quinone moiety towards cellular cytotoxicity by synthesizing quinocarbazole analogues which
lack the quinone unit of calothrixin. The quinocarbazole ring system is of interest because of its
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close relationship with ellipticine, a well-known anti-cancer alkaloid. The presence of quinone
functionality in calothrixins may lead to the generation of toxicological intermediates in cellular
milieu which may result in variety of harmful effects in vivo, including acute cytotoxicity,
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immuno and genotoxicity. On the other hand, the quinone lacking pyridocarbazole namely the
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ellipticine being structurally related to calothrixin possess fewer adverse effects. Thus the role
of quinone in mediating the adverse effects of calothrixin was investigated in an acute
toxicological study in mice following oral administration of promising calothrixin analogue. A
schematic view of overall research work carried out is represented in the form of a flow chart
(Fig. 1).
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Fig. 1. Schematic representation of synthetic and biological studies carried on calothrixin and
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Results and Discussion
Synthesis
Recently, a facile synthesis of calothrixin B and its analogues were achieved from this lab
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involving FeCl -mediated domino reaction of enamines. Among the various methodologies
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reported for calothrixin,
this method provides calothrixin B and its analogues in very good
overall yields involving minimum number of steps. Hence, we decided to explore the synthesis
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of diverse calothrixin analogues for biological screening using this FeCl -mediated domino
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reaction strategy.
a
Scheme 1
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O
O
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(b)
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Me
Me
SO Ph
N
N
H
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H
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9
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O
O
Me
PPh3
(c)
Me
(d)
N
CH2Br
N
SO Ph
SO Ph
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11
2
10
COCH3 R4
R3
(e)
R4
R3
R2
N
SO Ph O2N
OHC
R2
2
R1
13a-p
O N
2
R1
12a-p
R1
H
R2 R3
13
a
b
c
d
e
f
R4
H
H
yield (%)
H
H
H
9
2
5
OMe H
8
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
92
OMe OMe
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-OCH O-
90
2
Br
Cl
F
H
90
94
93
g
h
i
H
H
H
H
H
Cl
H
H
H
Cl
H
H
H
H
F
Br
Cl
H
90
91
80
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90
93
90
j
k
l
Cl
H
H
H
H
H
H
m
Br
Cl
Cl
Cl
n
o
p
F
Cl
H
a
Reagents and conditions : (a) DMA, POCl ; (b) PhSO Cl, PTC, Benzene,
3
2
5
0% NaOH, rt,1 h; (c) NBS, AIBN, CCl4, reflux, 3 h; (d)PPh , THF, reflux, 2 h
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followed by K CO DCM rt, 12 h; (e) 12a-p, DCM/DCE, reflux, 6-12 h.
2
3,
Our synthetic work began with acetylation of 2-methylindole 7 under Vilsmeier condition.
The resulting 3-acetylindole 8 upon protection of indole nitrogen by employing PTC conditions
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furnished the required 1-phenylsulfonyl-2-methyl-3-acetylindole 9. Subsequently, an allylic
bromination at 2-methyl position of 9 using NBS and a catalytic amount of AIBN in CCl at
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reflux
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corresponding
2-bromomethylindole
10.
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the
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-bromomethylindole 10 was then smoothly transformed into phosphorous ylide 11 as a stable
yellow solid. The ylide 11 underwent a facile Wittig reaction with 2-nitroarylaldehydes 12a-p
under refluxing conditions in DCM/1,2-DCE to afford the respective 2-nitroarylvinylindoles
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3a-p in 80-94% yields (Scheme 1).
a
Scheme 2
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The 3-acetyl-2-nitroarylvinylindoles 13a-p upon heating with dimethylformamide dimethyl
o
acetal (DMF.DMA) in the presence of 50 mol% glycocyamine at 100 C for 3-5 h produced
push-pull type enamines 14a-p (Scheme 2). Most of these enamines were isolated and well
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characterized by H and C NMR spectral studies. However, it should be noted that some of
the enamines 14d, 14f and 14j were used as such for next step without any further
characterization.
The enamines 14a-p upon refluxing with 3 equiv of anhydrous FeCl in DMF underwent
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electrocyclization, reductive cyclization and oxidation followed by cleavage of phenylsulfonyl
group to afford calothrixin B 2 and its analogues 15b-p in 45-67% yields (Scheme 3).
a
Scheme 3
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As expected calothrixin B 2 upon oxidation using m-CPBA in DCM at reflux furnished
calothrixin A 1. However, all our attempts to transform representative calothrixin B analogues
1
1,18
into their respective calothrixin A analogues using reported conditions
unsuccessful (Scheme 4). The reason for failure to oxidize the calothrixin B analogues (15c,
5d, 15g and 15h) containing electron releasing as well as electron withdrawing substituents is
were found to be
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
highly surprising. For some unclarified reasons, these calothrixin B analogues were found to be
highly resistant for oxidation.
a
Scheme 4
In order to understand the importance of quinone unit on structure activity relationship of
calothrixin B, synthesis of benzoellipticine analogues were planned. Accordingly, the known
phosphonate ester 16 was reacted with 2-nitroarylaldehydes 12a/12h/12g using K CO as a
2
3
base in DMF at room temperature for 12 h to afford 2,3-divinylindoles 17a-c. Refluxing, a
xylene solution of 2,3-divinylindoles 17a-c in the presence of 10% Pd/C underwent smooth
electrocyclization followed by aromatization to furnish the respective 2-nitroarylcarbazoles
18a-c. After several attempts, the chemo-selective reduction of ester group without affecting the
nitro group was carried out by DIBAL-H. Thus, the reduction of carbazoles 18a-c with DIBAL-
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
H (20% in toluene) in dry DCM at 0 C afforded the elusive benzylic alcohols 19a-c.
Transformation of benzylic alcohols 19a-c into corresponding aldehydes 20a-c was performed
using PCC in dry DCM. Subsequent reductive cyclization of carbazole-3-carbaldehydes 20a-c
using Ra-Ni in THF at room temperature for 3 h followed by hydrolysis using 50% NaOH
afforded quinocarbazoles 21a-c in good yields.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 5
Next, synthesis of the similar structural motiff of quinocarbazole having a methyl group at
4-position of carbazole was planned. As expected, reduction of ester unit of known carbazole
33
2
2a/22b using DIBAL-H (20% in toluene) followed by the oxidation of crude carbazole-3-
methanol 23a/23b using PCC in dry DCM furnished corresponding aldehyde 24a/24b.
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Reductive cyclization of carbazole aldehydes 24a/24b employing Ra-Ni followed by cleavage
of the phenylsulfonyl unit using 50% NaOH afforded quinocarbazole 25a/25b.
a
Scheme 6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Using the similar sequence of reactions, isomeric quinocarbazole 29 was smoothly prepared
3
4
from the corresponding 3-2'-nitrophenylcarbazole 26.
a
Scheme 7
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Towards enhancing the solubility of quinocarbazole, an incorporation of hydrophilic
substituent, amino group was planned (Scheme 8). Accordingly, Wittig reaction of aldehyde 30
with (cyanomethylene)triphenylphosphorane in xylenes at reflux afforded vinyl compound 31.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Benzylic bromination of 31 using NBS and a catalytic AIBN in CCl at reflux for 45 min
4
afforded bromo compound 32. Subsequent reaction of the bromo compound 32 with PPh3
followed by Wittig reaction of resulting phosphonium salt with 2-nitroaryl aldehydes
1
2a/12h/12g using K CO as a base in DCM at room temperature for 8 h led to divinyl
2 3
compounds 33a-c.
a
Scheme 8
CN
CN
CHO
a
b
c
CH3
N
CH Br
CH3
2
N
N
SO Ph
SO Ph
2
2
SO Ph
31
2
30
32
H N
CN
CN
2
N
d
e
R1
R1
N
N
SO Ph
34a-c
Compound
R1
O N
O N
N
SO Ph
2
2
2
2
H
35a-c
33a-c
R1
33-35a
33-35b
33-35c
H
F
Cl
CH3
CHO
CH3
CH2Br
a
b
c
CN
CN
N
N
N
SO Ph
SO Ph
SO Ph
2
2
2
36
37
38
O N
O N
2
2
d
e
N
CN
CN
N
N
;
N
NH2
;
SO Ph
SO Ph
2
H
2
39
41
40
a
Reagents and conditions : (a) Ph P=CHCN, xylenes, reflux, 8 h, 84-86%;(b) NBS, AIBN, CCl4, reflux, 45 min, 89-91%;
3
(
c) PPh , THF, reflux, 3 h, followed by K CO DCM rt, 8 h, 74-83%; (d) 10% Pd-C, xylenes, reflux, 24 h, 81-85%;
3
2
3,
(
e) i) Ra-Ni, THF, reflux, 30 min; ii) 50% NaOH DMSO, rt, 3 h, 74-78% (two steps).
,
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Thermal electrocyclization of the divinyl indoles 33a-c using 10% Pd/C in xylenes at reflux
furnished 3-cyanocarbazoles 34a-c. The reductive cyclization of the 3-cyanocarbazoles 34a-c
using Ra-Ni followed by cleavage of phenylsulfonyl group led to the expected
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
-aminoquinocarbazoles 35a-c. By using the similar sequence of reactions, 3-methyl-2-indole
aldehyde 26 was smoothly transformed into an isomeric 1-aminoquinocarbazole 41 (Scheme 8).
Next, reductive cyclization of carbazole ester 18a with Ra-Ni followed by hydrolysis using
5
1
0% NaOH afforded amide compound 42. The amide 42 upon refluxing with POCl furnished
3
-chloroquinocarbazole 43, which upon refluxing with 3-(N,N-dimethylamino)-l-propylamine
furnished quinocarbazole 44 (Scheme 9).
a
Scheme 9
3
5
Finally, the known 1-phenylsulfonyl napthocarbazole 45 upon hydrolysis using 50% NaOH
afforded 12H-naphtho[1,2-b]carbazole 46.
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1
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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5
5
5
5
5
5
5
5
5
6
Cell Proliferation Inhibitory Activities of calothrixins B and quinocarbazole analogues
In order to gain information about the structure-activity relationships of the different
calothrixin B and quinocarbazole analogues, chemosensitivity studies were performed and GI50
values were determined against nine different human cancer cell lines of diverse tumour origin.
The in vitro GI50 (growth inhibition) values of calothrixins against individual cell lines are
presented in Table 1, which also includes values of parent calothrixin A (1), calothrixin B (2)
and the cytotoxic quinoline alkaloid, camptothecin.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
It should be noted that for the first time, antiproliferative potential of calothrixins 1, 2 and
6
1
5b-p were evaluated against various cancer cell lines. As observed earlier, calothrixin A (1)
displayed comparatively better inhibitory effects than calothrixin B (2) against all the different
cell lines tested. The calothrixin 15b having a methoxy substituent at 4-position more or less
maintains the growth inhibitory potential with exception of Jurkat, SiHa, HCT116 p53-/- and
NCI-H460 cell lines. However, the presence of a 2-methoxy substituent on calothrixin B 15c
diminishes growth inhibitory effect in most of the cell lines except HCT116. The calothrixins
1
5d and 15e containing dimethoxy and methylenedioxy substituent at the 2- and 3-positions
displayed negligible inhibitory effects in majority of the cell lines evaluated. However, these
compounds showed greatest selectivity with GI values of 0.36 µM and 0.11 µM, respectively,
5
0
against MCF-7 cancer cell line.
The presence of electron withdrawing halogen atoms at 3-position of calothrixin B (2)
maintains or even improve the GI50 values. For 3-halo substituted calothrixins 15f-h, the
observed GI values are in the order of F > Cl ≥ Br. Compared to the parent compound 2, the
5
0
presence of chlorine/bromine at the 2-position of calothrixin B 15i/15j enhances the GI values
5
0
in many cell lines. It should be noted that the presence of a chlorine or bromine atom at
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Table 1. In vitro cytotoxicity data for calothrixins 1, 2 and 15b-p against nine human tumor cell lines
a
GI Average ± S.D. (µM)
50
Compound
Jurkat
HeLa
SiHa
MCF7
HCT116
HCT116 p53-/-
MDA-MB 231
U251
± 0.01
NCI-H460
1
0.07 ± 0.01
0.08
0.47
0.39
>4
± 0.00 0.05 ± 0.05 0.03 ± 0.0
± 0.09 3.50 ± 0.71 0.26 ± 0.01
0.11 ± 0.05 0.08
0.65 ± 0.07 2.55
0.33 ± 0.06 >4
0.63 ± 0.11 >4
± 0.01 0.04
± 0.01 0.08
± 0.02 2.25
± 0.01 1.83
>4
0.22
± 0.02
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
>4
± 0.92 0.16
± 0.35
± 0.32
>4
1
5b
>4
± 0.20 >4
0.60 ± 0.42
>4
0.31
>4
1
5c
5d
15e
5f
5g
15h
>4
>4
>4
>4
>4
>4
1
>4
>4
0.36 ± 0.34
0.11 ± 0.01
0.60 ± 0.28
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
1
1.50 ± 0.57
3.55 ± 0.64
1.15 ± 0.07
1.50 ± 0.71
1.40 ± 0.28
>4
0.21
0.07
0.05
0.29
0.50
>4
± 0.13 >4
1.50 ± 0.42 2.60
1.50 ± 0.28 2.60
0.83 ± 0.06 1.10
0.70 ± 0.00 1.13
0.42 ± 0.17 1.50
3.50 ± 0.71 >4
± 0.57 1.00
± 0.71 0.36
± 0.14 0.30
± 0.15 0.27
± 0.42 0.17
>4
± 0.00 2.00
± 0.04 2.83
± 0.12 1.20
± 0.05 0.60
± 0.00 0.50
>4
± 0.42
± 0.29
± 0.22
± 0.14
± 0.00
1.27
1.40
1.18
1.10
0.70
>4
± 0.06
± 0.14
± 0.13
± 0.14
± 0.42
1
± 0.02 1.60 ± 0.57 0.53 ± 0.38
± 0.02 0.93 ± 0.12 0.85 ± 0.07
± 0.05 1.40 ± 0.14 0.22 ± 0.05
± 0.14 1.67 ± 0.19 0.35 ± 0.22
1
5i
5j
1
15k
5l
5m
15n
5o
5p
CPT
>4
>4
0.80 ± 0.42
>4
1
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
1
>4
0.27
2.00
1.80
0.50
0.19
± 0.11 >4
± 0.00 >4
± 0.00 >4
0.66 ± 0.76
1.35 ± 0.35
>4
0.4
± 0.14 >4
>4
2.25
>4
± 0.78
>4
>4
1
>4
1.80
± 0.28 >4
± 0.00 1.50
± 0.01 0.01
>4
1
2.60 ± 0.57
± 0.00 1.80 ± 0.00 0.50 ± 0.00
± 0.03 0.55 ± 0.00 0.06 ± 0.00
1.20 ± 0.14 2.00
± 0.00 0.2
± 0.28
0.73
±0.25
0.06 ± 0.01
0.05 ± 0.01 0.195 ± 0.01 0.4
±0.0071 0.0057 ±0.0029
a
Values represents mean from at least two independent experiment
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
-position has almost identical influence on its GI values. The 1-chlorocalothrixin B 15k as
5
0
well as 4-chlorocalothrixin B 15l displayed reduced antiproliferative potential when compared
to calothrixin B (2). In general, the mono-substitution of halogens at 3- or 2-positions of
calothrixin B maintains or even improve the GI50 values whereas a similar substitution of
halogens at 1- or 4-position is detrimental. Among the seven mono halo-substituted calothrixins
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5f-l, 3-fluorocalothrixin B 15h showed higher GI50 value which is better than the parent
calothrixin B (2) inhibition profile.
Addition of a second halogen atom (Br or Cl) at 2-position of highly active 3-fluoro
calothrixin B 15h greatly diminish the GI values. The resulting analogues 15m and 15n did
5
0
not inhibit the growth of majority of cancer cell lines taken for the study with the exception of
HeLa and MCF7. The presence of chlorine atoms at 2- and 3-positions of calothrixin B 15o also
reduced its antiproliferative potential. However, the presence of chlorine atoms at
2- and 4-positions (15p) more or less maintains the growth inhibition potency in comparison to
the parent calothrixin B (2).
Calothrixin A (1) is more potent than calothrixin B (2) as well as its analogues 15b-p.
Calothrixin A (1) is equipotent in inhibiting the growth of all cell lines tested with the GI50
values ranging in sub-nanomolar concentration (30-220 nM). Among the analogues of
calothrixin B 15b-p, the 3-fluoro derivative 15h is more potent followed by other 3- or 2-mono
halo substituted calothrixin B analogues 15f, 15g, 15i and 15j.
Next, the synthesized quinocarbazoles were evaluated for their cytotoxicity against different
human cancer cell lines and their in vitro GI (growth inhibition) values against individual cell
5
0
lines are presented in Table 2.
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Table 2. In vitro cytotoxicity data for quinocarbazoles 21a-c, 25a, b and 29, naphthocarbazole 31 against ten human tumor cell lines
a
GI Average ± S.D. (µM)
5
0
Name
Jurkat
HeLa
SiHa
MCF7
HCT116
MDA-MB 231
U251
>4
NCI-H460
5.5 ± 0.70
HEK293
A549
>4
2
1a
4.00 ± 0.00 0.41 ± 0.08 >4
>4
>4
11.5 ± 0.07
>4
>4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
2
2
1b
>4
>4
>4
>4
>4
>4
0.02 ± 0.01 >4
0.43 ± 0.18 >4
0.36 ± 0.06 >4
0.05 ± 0.01 >4
1.40 ± 0.14 >4
0.02 ± 0.01
6
± 0.05 0.95
± 0.07
>4
0.0010 ±0.0002 0.0052 ±0.0021
>4
>4
>4
>4
>4
>4
1c
5a
>4
>4
>4
>4
28
± 0.23
± 0.00
± 0.12
>4
>4
10
± 0.00
± 0.70
± 0.04
± 0.05
>4
>4
>4
>4
>4
15
>4
>4
10.5
0.04
10
2
5b
0.27 ± 0.02
30
>4
>4
29
>4
>4
>50
>50
>4
>4
4
6
>4
>4
>4
>4
>50
a
Values represents mean from at least two independent experiments
Table 3. In vitro cytotoxicity data for amino quinocarbazoles 35a-c, 41 and 44 against seven human tumor cell lines
a
GI Average ± S.D. (µM)
5
0
Name
HeLa
SiHa
MCF7
HCT116
MDA-MB 231
U251
NCI-H460
3
5a
35b
5c
41
0.80 ± 0.14 1.05
± 0.07
1.00 ± 0.14 1.00 ± 0.09 1.00
± 0.1
1.05 ± 0.09 1.00
± 0.05
± 0.04
± 0.07
± 0.12
± 0.70
0.01 ± 0.008 1.35
0.53 ± 0.10 1.10
1.40 ± 0.14 1.30
1.10 ± 0.00 1.15
± 0.21
± 0.00
± 0.28
± 0.07
0.13 ± 0.07 1.20
0.95 ± 0.07 1.00
± 0.0
± 0.0
1.25 ± 0.35 1.20 ± 0.14 0.14
0.90 ± 0.28 1.20 ± 0.00 1.05
3
1.00 ± 0.00 1.05 ± 0.07 1.10 ± 0.00 0.65 ± 0.21 1.05
2.75 ± 0.35 1.75 ± 0.35 1.00 ± 0.14 1.35 ± 0.21 1.50
4
4
a
Values represents mean from at least two independent experiments
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In the case of quinocarbazole 21a, which lacks the quinone unit of calothrixin B there is a
reduction in cytotoxic potential. However, introduction of the fluorine atom to the 4-position of
21a resulted in quinocarbazole 21b with greater enhancement of cytotoxicity with respect to
HeLa, MCF7, MDA-MB-231 and NCI-H460 cells. With a GI value of 1 nM for NCI-H460
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and 5 nM for transformed HEK293 cells, the compound 21b appears to be the most active
among the quinocarbazoles reported in this work. Also, 21b is more active than the naturally
occurring calothrixins or synthetic analogues of calothrixin B. However, the introduction of
chlorine atom at the 4-position of 21a produced only an inactive quinocarbazole 21c. The
introduction of electron releasing methyl group at 13-position (21a→25a) reduced its anti-
proliferative potential. But with simultaneous introduction of fluorine and methyl groups at 4-
and 13-positions (21a→25b) led to the marginal increase in cytotoxicity against three of the ten
cell lines taken for the study. Unlike NCI-H460 cells, the other lung adenocarcinoma cell line
A549 was insensitive to 21b. Both the isomeric quinocarbazole 29 and naphthocarbazole 31
were inactive confirming that the presence of nitrogen atom and also its position are essential
for the anticancer properties of quinocarbazoles. It is worth to mention that the introduction of
fluorine atom to the 4-position of quinocarbazole imparts favorable cytototoxic potency to 21b
and 25b which are otherwise less active (as in the case of 21a and 25a).
On the other hand, synthesized amino quinocarbazoles displayed significant cytotoxic profile
with the GI values ranging between 0.01 µM to 2.75 µM (Table 3), which is far superior to
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the quinocarbazoles reported in this work (Table 2). Although, incremental benefit with respect
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resulted in ten fold or greater increase in cytotoxicity in comparison to its parent compound 35a
in NCI-H460, MCF7 and HeLa cell lines.
Only minor differences in the growth inhibition pattern were observed between cell lines,
where the p53 gene is functionally active (MCF7, HCT116, NCI-H460), mutated (Jurkat,
MDA-MB 231 and U251), inactive (HeLa and SiHa due to Human Papilloma Viral (HPV)
proteins) or absent (HCT116 p53-/-). This clearly indicates that the cellular cytotoxicity of
calothrixins and its analogues/quinocarbazoles were not mediated by p53. Among the cell lines
tested, HeLa (a HPV-18 positive cervical cancer cell line) is the more sensitive to calothrixin A
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(1), calothrixin B (2) and its analogues 15b-p as well as quinocarbazoles or its amino
derivatives. However, another HPV positive cell line (SiHa) was found to be less sensitive to
calothrixin B and its analogues/quinocarbazoles in comparison to HeLa cells. Obviously, the
extreme sensitivity of HeLa cells to the above calothrixin B analogues/quinocarbazoles is not
attributed to HPV infection but rather by different mechanisms.
The cytotoxicity data indicate that the p-benzoquinone unit in calothrixin can be spared as
the corresponding quinocarbazoles too exhibited antiproliferative activity and elimination of the
quinoline nitrogen atom resulted in inactive analogues. Thus, the preliminary in vitro screening
of the synthesized analogues indicated that the calothrixin B/quinocarbazole with a fluoro
substituent to the E-ring of indolophenanthridine framework were highly potent against the cell
lines tested (Table 1 & 2), and these analogues 15h, 21b along with their parent compounds 2
and 21a were taken for further functional studies.
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Inhibition of clonogenic activity by E-ring fluoro analogue of calothrixin
B/quinocarbazole
The cytotoxicity data highlighted the fact that E-ring fluoro analogues of calothrixin
B/quinocarbazole were the most promising derivatives of the synthesized compounds tested in
this work. Therefore, it was used in clonogenic cell survival assay to assess the long term
effects of these analogues on colony forming properties of HCT116 and NCI-H460 cells and
the results were compared to natural calothrixins, parent quinocarbazole 21a and camptothecin.
As shown in Fig. 2, the clonal growth was inhibited by calothrixin A (1), calothrixins B (2),
15h and 21b in a dose dependent manner in both the cell lines tested. The quinocarbazole 21a
does not abolish colony formation even at the highest concentration (5 µM) taken for the study.
More importantly, the clonal potency of NCI-H460 cells were severely affected by treatment
with as low as 1 nM of 4-fluoroquinocarbazole 21b thus corroborating the findings from
cytotoxicity assay (GI50 Value = 1 nM) and the other cell line HCT116 also shows extreme
sensitivity to 21b analogue where the clonogenic growth is inhibited above 0.1 µM although the
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GI value from SRB assay was high at 6 µM. However, the colonies in the 21b treated plates
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of HCT116 cells were smaller when compared to the control colonies indicating arrest in the
growth of surviving colonies. Consistent with their enhanced activity in preliminary cytotoxic
studies, the E-ring fluoro analogues of calothrixin B/quinocarbazole effectively attenuated the
clonogenic activity of HCT116 and NCI-H460 cells.
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Fig. 2. Effects of calothrixin A1, B2, 15h and Quinocarbazoles 21a, 21b on the clonogenic growth of colon
adenocarcinoma (HCT116) and Lung adenocarcinoma (NCI-H460) cell lines.
Cells were seeded in a six well plate and after over-night adherence, treated with different concentrations (0.1 – 5
µM) of calothrixin A1, B2, 15h and quinocarbazoles 21a, 21b (HCT116), 0.001 – 0.05 µM of quinocarbazole 21b
in case of NCI-H460 cells, for 48 h. After drug treatment, the cells were washed with Dulbecco’s phosphate
buffered saline and let grow up to 14 days in drug-free medium. Cell colonies were stained with crystal violet and
photographed.
Calothrixins and quninocarbazoles exhibits differential cell cycle effects in HCT116 cells
With the aim of investigating the cellular effects that are induced by
calothrixin/quinocarbazole analogues in terms of cell cycle modifications, a time-dependent
evaluation of the cell cycle profile was performed by FACS in HCT116 cancer cells treated
with these analogues (Fig. 3). The cell cycle analysis with 5 µM concentrations of calothrixins
1
, 2, 15g and 15h for 20 h in HCT116 cells caused an accumulation of cells in G0/G1 phase and
S-phase in case of 2 and 1, 15h, respectively. However, 21b treatment to HCT116 cells caused
accumulation of cells in G2/M phase, whereas 21a treatment hardly caused any perturbation in
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cell cycle. In order to determine the population of cells which are most affected by the
calothrixins and maintenance of cell cycle arrest, nocodazole (a mitotic inhibitor which arrests
all the cycling cells at G2/M phase) was added following treatment with
calothrixins/quinocarbazoles. As seen in Fig. 3, calothrixin A (1) or 3-fluorocalothrixin B 15h
pre-treatment to HCT116 cells caused arrest of cells in S-phase which is maintained even after
the addition of the nocodazole. Whereas calothrixin B (2) was unable to maintain the arrest of
cells in G1 phase following nocodazole treatment, which pulled all the cells and arrests in
G2/M phase. In a striking difference, addition of nocodazole to 21b pre-treated cells reinforced
accumulation of cells in G2/M phase of cell cycle. In other words, following treatment with
nocodazole the cycling cells in G1-S phase adds on to the population of cells (G2/M) that were
truly arrested by 21b. The quinocarbazole 21a behaved similar to that of its oxygenated
congener, calothrixin B (2).
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These results from cell cycle analysis clearly indicate that 15h causes G1-S phase arrest
whereas its corresponding quinocarbazole 21b arrests cells at G2/M phase of cell cycle. It can
be inferred that presence or absence of quinone functionality as in case of calothrixins or
quinocarbazoles determines differential cell cycle effects by targeting diverse cellular target.
The presence of fluorine atom at the 3-position of 15h or 4-position of 21b confers unique cell
cycle signature with better cytotoxicity compared to their counterpart calothrixin B (2) or
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Fig. 3. Cell cycle effects of calothrixin and its analogues. Cell cycle perturbation by calothrixins (1, 2, and
5h) or Quinocarbazoles (21a and 21b) in the presence or absence of nocodazole. HCT116 cells were treated with
5 µM of calothrixin and its analogues for 20 h or 3 h pretreatment with 17 h in the presence of nocodazole (0.3
µM) followed by propidium iodide staining. Population of cells in different phases of cell cycle were analysed by
flow cytometry. Figure is representative of other two experiments. (B) Percentages of HCT116 cells in the
different phases of the cell cycle
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quinocarbazole 21a. Similar results were obtained from experiments with 1 µM treatment of
calothrixins to HeLa cells followed by nocodazole treatment (SI: Fig. 1).
Fig. 4. (A) Cell cycle analysis of human colon HCT116 cells treated with the calothrixins (1, 2, and 15h) or
quinocarbazoles (21a and 21b) for 48 h. (B) Lung NCI-H460 cells treated with increasing concentrations of
quinocarbazoles 21b for 48 h. Figures are representative of other two experiments. (C&D) Percentages of HCT116
(
A) or NCI-H460 (B) cells in the different phases of the cell cycle
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Cell cycle analysis with HCT116 cells treated with calothrixin A (1) (2 µM), 3-
fluorocalothrixin B 15h (5 µM) for 48 h showed maintenance of G1-S phase arrest with
concomitant increase in sub-G1 population (Fig. 4 A, C). This indicates that these cells after
DNA damage gets arrested in G1-S phase, followed by cell death at later time point (48 h) due
to failure in the repair of damaged DNA and/or cellular stress. However, treatment with the
parent calothrixin B (2) for 48 h showed G1 arrest with a marginal increase in sub-G1
population (cell death). Whereas quinocarbazole 21b (5 µM) treatment to HCT116 cells for 48
h showed profound G2-M arrest, which was not seen with the parent quinocarbazole 21a. In
contrast, the quinocarbazole 21b caused extensive cell death even with 0.1 µM treatment to
NCI-H460 cells (Fig. 4 B, D) with subsequent dose dependent accumulation of cells in G1
phase indicating altogether different mechanistic action of 21b in this cell line.
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Calothrixins as weak topo I inhibitor and quinocarbazoles as catalytic topo II inhibitor
To elucidate the possible mechanistic action of calothrixin B/quinocarbazole analogues, topo
inhibition studies of these compounds are explored due to their structural similarities with the
natural calothrixins/ellipticine which were reported to possess topoisomerase inhibitory
8, 36
activity.
In general, topoisomerase I (hTopI) generates transient single stranded break and
religates, thus relieving torsional stress, whereas topoisomerase II (hTopII) is able to cleave and
religate double-stranded DNA, allowing strand passage. The calothrixin A (1), calothrixins B
(2), 3-fluorocalothrixin B 15h and quinocarbazole analogues 21a, 21b and 25a were
investigated for hTopI and hTopII inhibition using a relaxation assay by simultaneous
incubation of supercoiled plasmid DNA, enzyme, and drug. The results from hTopI inhibition
studies (Fig. 5) showed that the tested analogues did not have any significant effect even at the
concentrations as high as 200 µM (Fig. 5, lanes 5 -10). Next, the hTopII inhibition capacity of
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these selected calothrixin and quinocarbazole analogues were evaluated. The Fig. 6 shows the
relaxation of supercoiled pBS (SK+) plasmid DNA catalyzed by topo II (lane 2) and the effect
of calothrixin A (1), calothrixins B (2), 15h and quinocarbazole analogues (21a, 21b & 25a) at
a concentration of 200 µM. The hTopII inhibition by 25 µM of etoposide was used as a positive
control (Fig. 6, lane 4). Interestingly, among the quinone containing calothrixins, only
calothrixin A (1) (Fig. 6, lane 5) showed effective inhibition of hTopII whereas in case of
quinocarbazoles, only 21a and 25a showed complete inhibition (Fig. 6, lanes 8 and 10).
Subsequent dose-dependent relaxation studies with these three analogues revealed that
calothrixin A (1) could not inhibit the enzyme activity at a concentration <100 µM (data not
shown). However, quinocarbazoles 21a and 25a exhibited almost 97% and 98% inhibitions,
respectively, at 40 µM concentration (Fig. 7, 8). Their IC and IC values have been reported
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in Table 4. It is surprising to note that the 4-fluoroquinocarbazole analogue 21b does not have
the capacity to interfere with the relaxation activity of hTopII which is seen with its congener
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1a, although both these analogues intercalate with DNA.
Fig. 5. Effects of calothrixin and quinocarbazole analogues on recombinant human topoisomerase I: Relaxation of
negatively supercoiled pBS (SK+) DNA with purified hTopI at a molar ratio of 3:1 in simultaneous assay
condition. Lane 1, 90 fmol of pBS (SK+) DNA; Lane 2, same as lane 1, but simultaneously incubated with 30 fmol
o
of hTopI for 30 mins at 37 C; Lane 3, same as lane 2 but in presence of 2% v/v DMSO; Lane 4, same as lane 2 but
in presence of 25 µM camptothecin as positive control; Lanes 5-10, same as lane 2 but in presence of 200 µM
concentration of calothrixin A 1, calothrixins B 2, 15h, quinocarbazoles21a, 21b and 25a, respectively. Positions
of supercoiled monomer (SM) and relaxed and nicked monomer (RL/NM) are indicated.All results depicted were
performed three times and representative data are from one set of these experiments.
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Fig. 6. Effects of calothrixin and quinocarbazole analogues on recombinant human topoisomerase II: Relaxation of
negatively supercoiled pBS (SK+) DNA with purified hTopII at a molar ratio of 3:1 in simultaneous assay
condition. Lane 1, 90 fmol of pBS (SK+) DNA; Lane 2, same as lane 1, but simultaneously incubated with 30 fmol
o
of hTopII for 30 mins at 37 C; Lane 3, same as lane 2 but in presence of 2% v/v DMSO; Lane 4, same as lane 2
but in presence of 25 µM etoposide as positive control; Lanes 5-10, same as lane 2 but in presence of 200 µM
concentration of calothrixin A 1, calothrixins B 2, 15h, quinocarbazoles 21a, 21b and 25a, respectively. Positions
of supercoiled monomer (SM) and relaxed and nicked monomer (RL/NM) are indicated. All results depicted were
performed three times and representative data are from one set of these experiments.
Fig. 7. Dose-dependent inhibition of recombinant human topoisomerase II by quinocarbazole analogues 21a and
2
5a: Relaxation of negatively supercoiled pBS (SK+) DNA with purified hTopII at a molar ratio of 3:1 in
simultaneous assay condition. Lane 1, 90 fmol of pBS (SK+) DNA; Lane 2, same as lane 1, but simultaneously
o
incubated with 30 fmol of hTopII for 30 mins at 37 C; Lane 3, same as lane 2 but in presence of 2% v/v DMSO;
Lane 4, same as lane 2 but in presence of 25 µM etoposide as positive control; Lanes 5-10, same as lane 2 but in
presence of increasing concentrations (10, 20, 30, 40, 50, and 60 µM) of quinocarbazole 21a and Lanes 11-16,
same as lane 2 but in presence of increasing concentrations(10, 20, 30, 40, 50, and 60 µM) of quinocarbazole 25a.
Positions of supercoiled monomer (SM) and relaxed and nicked monomer (RL/NM) are indicated. All results
depicted were performed three times and representative data are from one set of these experiments.
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