Synthesis and biological evaluation of novel analogues of batracylin with synthetic amino acids and adenosine: An unexpected effect on centromere segregation in tumor cells through a dual inhibition of topoisomerase IIα and Aurora B

Article abstract of DOI:10.1039/c6ra04957e

In the search for new anticancer agents we designed and synthesized batracylin derivatives with linking synthetic amino acid side chains of different lengths and adenosine. Unexpectedly, we have found that in water and the culture media adenosine-amino acid-BAT conjugates form supramolecular structures and this prevents these compounds from entering cells. Consequently, these compounds exerted no biological activity when tested towards two human cell lines, lung adenocarcinoma (A549) and human leukemia (HL-60). In contrast, several amino acid-BAT precursors showed up to 25-fold enhanced cytotoxic activity compared to BAT and these compounds strongly interfered with DNA topoisomerase II activity and its cellular functions. In particular, these conjugates inhibited centromere segregation during mitosis in drug-treated tumor cells by preventing topoisomerase II-dependent Aurora B activation.

Full text of DOI:10.1039/c6ra04957e

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: W. Januchta, M.
Serocki, K. Dzierzbicka, G. Cholewinski, M. Gensicka and A. Skadanowski, RSC Adv., 2016, DOI:
10.1039/C6RA04957E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
Synthesis and biological evaluation of novel analogues of batracylin with
synthetic amino acids and adenosine: an unexpected effect on centromere
segregation in tumor cells through a dual inhibition of topoisomerase IIα and
Aurora B
Wioleta Januchta,+a Marcin Serocki,+b Krystyna Dzierzbicka,*a Grzegorz
Cholewinski,a Monika Gensickaa and Andrzej Skladanowskib
aDepartment of Organic Chemistry, Chemical Faculty, Gdansk University of
Technology, G. Narutowicza 11/12, 80ꢀ233 Gdansk, Poland. Eꢀmail: krydzier@pg.gda.pl
bDepartment of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk
University of Technology, 11/12 G. Narutowicza St, 80ꢀ233 Gdansk, Poland
+These authors contributed equally to this work.
Electronic supplementary information (ESI) available: analytical and spectroscopic data for
synthesized compounds as well as additional figures. See DOI:

RSC Advances
Page 2 of 30
View Article Online
DOI: 10.1039/C6RA04957E
Table of Contents Graphic:
Abstract
In search of new anticancer agents we designed and synthesized batracylin
derivatives with linking synthetic amino acid side chains of different length and
adenosine. Unexpectedly, we have found that in water and culture media adenosineꢀ
amino acidꢀBAT conjugates form supramolecular structures and that prevents these
compounds from entering cells. Consequently, these compounds exerted no
biological activity when tested toward two human cell lines lung adenocarcinoma
(A549) and human leukemia (HLꢀ60). In contrast, several amino acidꢀBAT precursors
showed up to 25ꢀfold enhanced cytotoxic activity compared to BAT and these
compounds strongly interfered with DNA topoisomerase II activity and its cellular
functions. In particular, these conjugates inhibited centromere segregation during
mitosis in drugꢀtreated tumor cells by preventing a topoisomerase IIꢀdependent
Aurora B activation.
Introduction
Medicinal chemists continue to develop novel cytotoxic agents with new and unique
mechanisms of action, however, many of these compounds still lack tumor selectivity

Page 3 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
or have not been therapeutically useful due to undesired toxic effects or low
bioavailability in vivo. Apart from this classical approach to design and synthesize
new molecules with antitumor activity, there is yet another line of activity in medicinal
chemistry to combine different molecules with defined functions and activities to
produce the soꢀcalled ‘functionalized’ molecules or drugs. In this approach, one can
conjugate cytotoxic drugs with other molecules such as e.g. monoclonal antibodies
which bind to specific cellular markers on the surface of tumor cells and offer an
alternative therapy that is tumorꢀspecific and/or less toxic. This direction has recently
attracted a great deal of attention (for review see Chari 2008).1 Another example can
be to synthesize ‘functionalized’ cytotoxic molecules by combination of with other
small molecules, such as proteins, peptides or sugars.
Batracylin
(8ꢀaminoisoindolo[1,2ꢀb]quinazolinꢀ12(10H)ꢀone,
BAT)
exhibits
excellent antitumor properties2ꢀ5 and inhibits the catalytic activity of DNA
topoisomerases that leads to production of DNA breaks and induction of the
unscheduled DNA synthesis (UDS) in nonproliferating cells.6,7 At the same time, in
preꢀclinical studies BAT produced severe toxicity in rats and mice. In addition, even
between rodents different toxicity of BAT was observed and oral administration
batracylin to rats was found to significantly more toxic, compared to mice. The BAT
toxicity observed across all studied species was caused by presence of its toxic
metabolite, Nꢀacetylꢀbatracylin (ABAT), following metabolism mediated by Nꢀacetylꢀ
transferase 2 (NAT2).8ꢀ10 Firstꢀinꢀhuman study was conducted in pharmacogenetically
selected thirtyꢀone patients with advanced refractory solid tumors and lymphomas
and a slow acetylator NAT2 genotype.11 The objectives included determination of the
safety, tolerability, maximum tolerated dose (MTD) and pharmacokinetics (PK) of
batracylin and its metabolites. Although no objective responses were observed, even
at doses of BAT as high as 400 mg/day for 7 days (in a 28ꢀday cycles), patients
tolerated doses which were about 20ꢀfold higher than the MTD in rats and 70% of the
MTD in mice.
The low water solubility of BAT, its high toxicity as well as large doses required for
anticancer activity, are the causes that drawn attention to the synthesis of its new
analogues.12,13 The synthesis of different types of BAT derivatives was described in
the 1990s.14ꢀ16 The new compounds were prepared by reacting the batracylin with
natural amino acids or short peptides containing additional amino or carboxyl groups.
These derivatives were sufficiently soluble in water and reconverted into batracylin by

RSC Advances
Page 4 of 30
View Article Online
DOI: 10.1039/C6RA04957E
the action of enzymes like peptidases which are present in blood plasma and other
organs. Accordingly, these derivatives have been shown to exhibit the cytotoxic
activity that is comparable to that of batracylin.14,15 In 1992, the synthesis of βꢀ
fucose derivative of batracylin was published.16 The linkage of βꢀ
ꢀfucose derivatives
Lꢀ
L
to peptide conjugates of batracylin led to an active compound. The procedure was
also reported of the synthesis of batracylin analogues modified by carbonylation and
based on the use 1,2ꢀdibromobenzenes and 2ꢀaminobenzyl amine as substrates and
palladium as the catalyst.17 The desired products were isolated in moderate to good
yields (36–80%) with the installation of two molecules of carbon monoxide. The
synthesis of Nꢀsulfonoamido analogues was also presented, which contain alkyl and
aromatic groups on the batracylin pharmacophore in the presence of pyridine in
[bꢀ3Cꢀim][NTf2] ionic liquid and were further assayed for biological activity. Some of
synthesized compounds exhibited potent inhibitory activity against human type I DNA
topoisomerase.18
We propose that yet another group of the compounds that can be conjugated with
BAT is adenosine (6ꢀaminoꢀ9ꢀβꢀDꢀribofuranosylꢀ9ꢀHꢀpurine, ADO), which is an
endogenous purine nucleoside. Adenosine is released from cells or is formed outside
the cell in the organism, and binds specifically to its receptors, which are present on
the cellular membrane.19,20 There are four types of adenosine receptors: A1, A2A, A2B
and A3, which belong to the Gꢀproteinꢀcoupled family of receptors and frequently
have opposing effects on cell signaling pathways.21,22 For example, A1 receptors
stimulate formation of giant multinucleated cells from monocytes, whereas A2
receptors inhibit this process. Similarly, A2A adenosine receptors are generally antiꢀ
inflammatory, whereas A2B and A3 receptors are implicated in proinflammatory action
of adenosine.23 Even more importantly, adenosine by its binding to membrane
receptors can induce cell death by apoptosis. The molecular mechanism of this proꢀ
apoptotic activity of adenosine involves modulation gene expression of genes such
as Bax, Bad, and Puma, that leads to disruption of the mitochondrial transmembrane
potential and activation of caspaseꢀ9 and caspaseꢀ3.24 In this way, combined effect
of cytotoxic drug and adenosine can increase cell killing effect induced by the
conjugate. However, adenosine is quickly metabolized by adenosine deaminase25
that limits its clinical usefulness as a part of combination therapies.
We described previously the synthesis of muramyl dipeptide (D,D) (MDP(D,D)) and
norꢀmuramyl dipeptide (D,D) (norꢀMDP(D,D)) derivatives conjugated with adenosine

Page 5 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
through amino acid spacers as potential immunosuppressants.26 Some of the
synthesized compounds exhibited moderate cytotoxic activity against murine
leukemia L1210 cells.27 In this study, we report the synthesis of a new series of
batracylin derivatives conjugated with adenosine as well as their precursors Nꢀ(Nωꢀ
amino acid)ꢀBAT with linking synthetic amino acid side chains of different length. In
our method, synthesis of new conjugates is based on the reaction of batracylin
derivatives with linking synthetic amino acids and 6ꢀchloropurine riboside. We
hypothesized that combination of adenosine and batracylin derivatives will result in
analogues with improved activity and/or selectivity toward tumor cells. In this paper,
we present the method of synthesis of new these analogues and evaluation of their
cytotoxic activity as well as biological effects induced by these compounds in tumor
cells. Unexpectedly, we have found that at physiological pH in water and media
adenosineꢀamino acidꢀBAT conjugates form supramolecular structures and that
prevents these compounds from entering cells. Consequently, these compounds
exert no biological activity. In contrast, several their amino acidꢀBAT precursors
showed greatly enhanced cytotoxic activity compared to BAT and these compounds
strongly interfered with DNA topoisomerase II activity and its cellular functions, in
particular inhibited centromere segregation during mitosis in drugꢀtreated tumor cells
by preventing a topoisomerase IIꢀdependent Aurora B activation.
Results and discussion
Chemistry
We synthesized new analogues of batracylin combined with adenosine as potential
anticancer agents. The synthesis of this compounds were carried out according to
Scheme 1 and 2. The batracylin (4) was synthesized by our modified method based
on the CzerniakꢀEinhorn reaction. In this method, a symmetrically protected 1,4ꢀ
phenylenediamine derivative has undergone the CzerniakꢀEinhorn reaction, and after
hydrolysis of the protecting groups, BAT (4) was obtained.28 We synthesized also the
batracylin
derivatives
Nꢀ(NωꢀBocꢀamino
acid)ꢀBAT
6a–h,
using
N,N’ꢀ
dicyclohexylcarbodiimide (DCC) and 4ꢀdimethylaminopyridine (DMAP) in anhydrous
dichloromethane (DCM).28 We obtained derivatives 6a–h in yields 49–65%. The
compounds 6b, 6c, 6e have been published previously.28,29 The compounds 6a, 6d
and 6f–h were prepared by the same procedure.28

RSC Advances
Page 6 of 30
View Article Online
DOI: 10.1039/C6RA04957E
The tertꢀbutyloxycarbonyl (Boc) protecting group was removed by treatment with 90%
trifluoroacetic acid (TFA) to give the products 2a–h (Scheme 1).30 Reaction of
nucleophilic substituted between 6ꢀchloropurine riboside 1 and an amine group of
batracylin derivatives 2a–h and N,Nꢀdiisopropylethylamine (DIEA) were achieved
during reaction in anhydrous ethanol under N2 for 4h. The reaction was carried out in
the boiling solvent. After 4h EtOH was removed from a reaction by evaporation under
vacuum (Scheme 2). The final products 3a–h were purified with preparative TLC, and
1
their identities were confirmed by high resolution H NMR (500 MHz), 13C NMR
spectroscopy and MALDIꢀTOF mass spectrometry analysis. The purity of all
compounds was evaluated by HPLC and was found to be higher than 95% (see
Supplementary Fig. 1 and 2).
O
O
H2N
Boc
DCC, DMAP
NH
N
N
n
NH
OH
Boc
n
NH
O
+
N
N
CH2Cl2
O
5
4
6
5a: n = 1
5b: n = 2
5c: n = 3
5d: n = 4
5e: n = 5
5f: n = 6
5g: n = 7
6a: n = 1
6b: n = 2
6c: n = 3
6d: n = 4
6e: n = 5
6f: n = 6
6g: n = 7
5h: n = 11
6h: n = 11
O
O
F
F
HN
TFA
O
H3N
N
n
6
F
O
N
2
2a: n = 1
2b: n = 2
2c: n = 3
2d: n = 4
2e: n = 5
2f: n = 6
2g: n = 7
2h: n = 11
Scheme 1 Synthesis of amino acidꢀBAT derivatives 2a–h.

Page 7 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
Cl
N
N
N
O
N
O
F
F
HN
O
HO
O
DIEA, EtOH
4hr, b.p.
H3N
N
n
+
F
O
N
HO
OH
2
1
2a: n = 1
2b: n = 2
2c: n = 3
2d: n = 4
2e: n = 5
2f: n = 6
2g: n = 7
2h: n = 11
O
HN
HN
N
n
O
N
N
N
N
HO
O
N
3
3a: n = 1
3b: n = 2
3c: n = 3
3d: n = 4
3e: n = 5
3f: n = 6
3g: n = 7
3h: n = 11
HO
OH
Scheme 2 Synthesis of adenosineꢀamino acidꢀbatracylin analogues 3a–h.
Our synthesized analogues 2a–h and 3a–h were tested for their cytotoxicity and
evaluation of their mechanisms of actions toward tumor cells.
Biological Assays
Cytotoxic properties toward tumor cells
Cytotoxic activity of studied compounds was evaluated using two human
cancer cell lines, A549 lung adenocarcinoma and HLꢀ60 myeloid leukemia cells. We
established that these two types of tumor cells were the most sensitive to
batracylin.31 Additionally, it was previously shown that both HLꢀ60 and A549 cells
express adenosine receptors.32,33 Riboside alone showed low cytoxicity toward both
tumor cell models (Table 1). Surprisingly, the majority of amino acidꢀadenosineꢀBAT
conjugates were completely biologically inactive toward both A549 as well as HLꢀ60
cells. Only compound 3e was specifically cytotoxic toward A549 cells but showed
comparable activity to BAT (Table 1). Our further studies showed that adenosineꢀ
BAT conjugates when added to water or culture media form supramolecular
structures (data not shown) that most certainly prevents them from entering tumor
cells and therefore all these compounds were devoid of any biological activity i.e. no

RSC Advances
Page 8 of 30
View Article Online
DOI: 10.1039/C6RA04957E
cytotoxic effect was observed even at the highest concentration tested (100 ꢀM).
Interestingly, only compound 3e did not form these structures and it remained
biologically active, specifically toward A549 cells, although it was similarly cytotoxic
as its parent compound BAT (Table 1). The molecular mechanism responsible for
this unexpected formation of supramolecular structures in water solutions by the
majority of adenosineꢀamino acidꢀBAT analogs is not clear at the moment and
requires further studies.
Even more unexpectedly, several of the amino acidꢀBAT precursors showed a
greatly increased cytotoxicity toward both tumor cell types compared to BAT (Table
2). Sensitivity toward both A549 and HLꢀ60 cells was higher from about 2ꢀ to more
than 25ꢀfold compared to the parent compound. Sensitivity profile for all tested
compounds was strikingly comparable in both types of tumor cells e.g. compound 2c
was about 17 times more cytotoxic toward A549 cells than BAT and, at the same
time, was about 16 times more active toward HLꢀ60 cells than BAT. Similarly, we
found that compound 2g was slightly less active than BAT toward both types of tumor
cells tested (Table 2).
We hypothesize that observed differences in cytotoxicity of amino acidꢀBAT
conjugates are resulting from the similarity to the natural amino acids side chain
structure. Structure of amino acid chain in compound 2a imitate the smallest amino
acid ꢀ glycine, therefore its mechanism of action relay more on drugꢀDNA
intercalation (Supplementary Fig. 7) than drugꢀprotein interactions. Decreased
activity of 2b compound can be explain, that there is no natural amino acid with two
carbon chain and positively charged group. Both serine and cysteine contain
negatively charged groups. Also decreased intercalation to DNA is observed for
compound 2b (Supplementary Fig. 7) due to larger distortion of flat structure than in
2a derivative. Structure of side chain of lysine can be imitate by the structure of
amino acids in compounds from 2c to 2f. Those four compounds have moderate
cytotoxic properties and do not strongly intercalate to the DNA, however mimic of the
lysine side chain may suggest that derivatives from 2c to 2f interact with enzymes.
This hypothesis however, need to be support by further study using molecular
modeling methods. Lack of activity of derivative 2g is caused probably by poor
lipophilic efficiency (logP=2.08±0.90 for 2g and logP 0.69±0.90 for BAT) and for this
reason 2g interact forcefully with membrane lipids molecules than proteins or DNA
molecules. Compound 2h is a exception because this compounds have very different

Page 9 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
logD distribution curves than other compounds (Supplementary Fig. 8). Derivative 2h
is more water than lipid soluble only in acidic pH. Therefore we observed very
significant accumulation of this compounds in acidic vesicles (Supplementary Fig. 1)
and as mentioned later for this reasons we excluded 2h compound from extended
studies.
Table 1 Cytotoxic activity of amino acidꢀadenosineꢀBAT conjugates after 120h
continuous exposure of tumor cells as revealed by the MTT assay.
IC50 value [ꢁM]
Compound
A549 cells
46.3 ± 7.9
>100
HLꢀ60 cells
66.6 ± 4.4
>100
BAT
3a
3b
>100
>100
3c
>100
>100
3d
>100
>100
3e
40.6 ± 3.2
>100
>100
3f
>100
3g
>100
>100
3h
>100
>100
riboside
29.3 ± 1.5
48.5 ± 2.5
Table 2 Cytotoxic activity of amino acidꢀBAT conjugates after a 120h continuous
exposure of tumor cells as revealed by the MTT assay.
IC50 value [ꢁM]
Compound
A549 cells
46.3 ± 7.9
2.7 ± 0.4
HLꢀ60 cells
66.6 ± 4.4
4.2 ± 0.5
BAT
2a
2b
2c
2d
2e
2f
35.0 ± 5.9
17.3 ± 3.1
6.3 ± 1.2
18.6 ± 3.4
15.9 ± 2.7
6.0 ± 0.9
7.9 ± 0.5
15.9 ± 3.1
26.2 ± 2.4
81.1 ± 5.6
2.4 ± 0.4
15.3 ± 1.3
69.7 ± 6.9
1.8 ± 0.2
2g
2h

RSC Advances
Page 10 of 30
View Article Online
DOI: 10.1039/C6RA04957E
Inhibition of DNA topoisomerases in vitro by BAT and amino acidꢀBAT
conjugates
BAT was earlier described as a dual inhibitor of both type I and II DNA
topoisomerases.7,34 We showed that amino acidꢀBAT conjugates inhibited human
DNA topoisomerase Ib and topoisomerase IIα at concentrations much lower that BAT
itself and two standard inhibitors of these two enzymes, active metabolite of
topotecan, compound SNꢀ38, or VPꢀ16 (etoposide) (Fig. 1). Interestingly, this effect
directly correlated with increased cytotoxicity exerted by these conjugates toward
tumor cells i.e. for compounds 2a, 2d and 2h which were from 7 to about 26ꢀfold
more cytotoxic toward A549 cells (or 11 to 28ꢀfold toward HLꢀ60 cells) than BAT and
showed inhibitory effect toward DNA topoisomerase IIα in vitro at concentrations at
least 100ꢀtimes lower than BAT itself (Fig. 1, lower panel).
Fig. 1 Effect of selected batracylin (BAT) and BATꢀconjugates on the catalytic activity
of purified type I (upper panel) and type II (lower panel) DNA topoisomerases.
Effect of BAT and its synthetic amino acid conjugates on cell cycle
progression and cell death of drugꢀtreated tumor cells
For further studies we chose BAT and its two derivatives 2a and 2d, which are much
more cytotoxic than parental BAT. Unfortunately, the most cytotoxic compound 2h is
strongly accumulated mostly in acidic vesicles (compare intracellular localization of
BAT and compound 2h in Supplementary Fig. 3). This was associated with a greatly
enhanced protonation in acidic pH of compound 2h compared to BAT and other
amino acidꢀBAT conjugates (compare protonation profiles for BAT and 2a, 2d, and
2h compounds in Supplementary Fig. 4AꢀD). Since biological activity of compound

Page 11 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
2h strongly depended on the initial cell number (i.e. drug to number of cells ratio), this
compound was excluded from further mechanistic studies.
HLꢀ60 and A549 cells were treated with BAT and two amino acidꢀBAT
conjugates (compounds 2a and 2d) at doses corresponding to IC90 concentrations
and showed significant changes in cell cycle progression (Fig. 2).
Fig. 2 Effect of BAT and its synthetic amino acid conjugates on the cell cycle
distribution of tumor cells. Cells were exposed to specified drug doses for the time
indicated and analyzed by flow cytometry. * ꢀ measured as a fraction of living cells;
**ꢀ measured as a fraction of subꢀG1 of the total counted events.
After 24h most of the cells arrested their cell cycle progression in G2/M phases. This
block was stable for next 120h of incubation of A549 cells. However, for HLꢀ60 after
long term 120h exposure a significant increase of cell debris fraction (with
hypodiploid DNA content) was observed. When this fraction was reꢀcalculated as a
percentage of total events, the fraction changed from less than 1% in control samples
to more than 25% for BAT and more than 75% for 2a and 2d derivatives. These

RSC Advances
Page 12 of 30
View Article Online
DOI: 10.1039/C6RA04957E
results suggested that the prolonged arrest of HLꢀ60 cells in G2/M phases was
sufficient to induce apoptosis, that resulted in fragmentation of drugꢀtreated leukemia
cells and increased cell debris fraction. Analysis of nuclear morphology of drugꢀ
treated HLꢀ60 cells showed that these cells after 24h of treatment entered mitosis.
However, most of these mitoses were abnormal with many aberrant features like
multipolar spindle or lagging chromosomes during metaphase to anaphase transition
(see Supplementary Fig. 5). At later incubation times, a small fraction of drugꢀtreated
cells become polyploid (Fig. 2). In contrast to HLꢀ60 cell line, treatment of A549 cells
with studied compounds led to G2/M arrest at 24h followed by increasing fraction of
polyploid cells at longer incubation time (120h) with a marginal fraction of dying cells
(Fig. 2 and Supplementary Fig. 5).
Inhibition of centromere segregation in tumor cells treated with BAT and BATꢀ
amino acid conjugates
In agreement with the data on the effect of studied compounds on cell cycle
progression, treatment of A549 cells with BAT and its analogs induced p53 and cyclin
B1 expression (Fig. 3), that can be related to the induction of DNA double strand
breaks that appears as a result of topo IIα inhibition by investigated compounds
during S phase of cell cycle. Induction of DNA damage was further confirmed by
immunofluorescence microscopy after staining tumor cells with antibodies directed
toward phospho Ser 139 H2AX histone (Fig. 4), a wellꢀknown marker of doubleꢀ
stranded DNA breaks.35 Cells which were in G1 and S phase during drug treatment,
accumulated a sufficient number of DNA breaks to activate the G2 checkpoint and
arrested before cell division. Intriguingly, the level of double strand DNA breaks
induced by studied compounds in cells that were in G2 and M phases when the
treatment began were able to enter mitosis. At the same time, a strong reduction of
histone H3 phosphorylation at serine 10, that is mediated in mitosis by Aurora B
kinase, was observed already after 3h of treatment (Fig. 3).

Page 13 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
Fig. 3 Effect of BAT and its conjugates on the expression of proteins involved in the
regulation of cells proliferation and mitotic transit. Cells were exposed to specified
drug doses for the time indicated and analyzed by Western blotting.
Fig. 4 Induction of doubleꢀstranded DNA breaks in A549 cells by BAT and its amino
acid conjugates. Cells were exposed to specified drug doses for the time indicated
and analyzed by fluorescence microscopy after staining with DAPi (blue) γꢀH2AX

RSC Advances
Page 14 of 30
View Article Online
DOI: 10.1039/C6RA04957E
antibodies (green). Thick arrow shows cell nucleus containing DNA breaks, thin
arrow points to cell nucleus of a daughter cell with apparently no DNA breaks.
It was shown previously by Coelho et al. that inhibition of Topo II activity by its
chemical inhibitor ICRFꢀ187 or silencing Topo II expression with siRNA can
significantly attenuate Aurora B activity, however this inhibition is only moderate
when using ICRFꢀ187 and requires prolonged time (72h) when using siRNA.36 Strong
reduction of histone H3 phosphorylation during whole incubation time, strongly
supports the idea that BAT and its amino acid derivatives directly inhibited Aurora B
activity or its Topo IIαꢀdependent activation. When the activity of Topo IIα and Aurora
B is inhibited in cells entering mitosis, mitotic division cannot properly proceed. As it
was shown previously, inhibition of Aurora B activity leads to increased number of
syntelic as well as meroꢀamphitelic microtubule attachments.37 A549 cells treated
with our compounds arrested during cytokinesis and formed dropletꢀlike structures
with lagging DNA between dividing cell nuclei that frequently was associated with an
unequal DNA distribution between daughter cells. More importantly, these lagging
structures contained centromeric but not telomeric DNA as revealed using FISH
analysis with appropriate molecular probes (Fig. 5). Unusual DNA structures were
quite stable and were still observed after 72ꢀ120h of drug treatment and followed by
accumulation of tumor cells with features of senescent cells, such as enlarged cells
wit flat morphology and increased activity of senescenceꢀassociated βꢀgalactosidase
(see Supplementary Fig. 6 and 7). Spindle assembly checkpoint requires Aurora B
activity to arrest the cell cycle in mitosis,36 and since activity of Aurora B is inhibited
by our compounds, spindle assembly checkpoint cannot be maintained and mitotic
division is allowed, even in the presence of DNA damage. Drugꢀtreated tumour cells
undergo aberrant cytokinesis at least in part because midꢀbody complex containing
Aurora B protein had abnormal structure (see Supplementary Fig. 8). As a result,
cytokinesis could not be completed, which explains increased fraction of polyploid
cells after 120h of treatment. Taken together cells treated with our compounds arrest
their cell cycle progression in G2 phase and undergo drug induced premature
senescence or enter abnormal mitosis followed by cell polyploidization or nonꢀequal
DNA distribution between daughter cells associated with the presence of lagging
DNA structures. Nonꢀproliferative status of cells treated with studied compounds was

Page 15 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
evident from Western Blot analysis since expression of proteins (Topo IIα and Aurora
B – proliferative markers, cyclin B1 – marker of mitotic exit) involved in cell cycle
progression was strongly reduced after 72h of treatment (Fig. 3). Loss of Aurora B
kinase activity by batracylin and its amino acid conjugates may be due to a direct
inhibition on as another author says by indirect interactions with topoisomerase IIα.
Together, our study led to the synthesis of original group of BAT analogues 2a–h
and 3a–h with potential increased antitumor activity and improved pharmacological
properties, including potentially lower general toxicity. After evaluation in further preꢀ
clinical studies in vivo can be proposed as new antitumor agents. Unfortunately,
functionalization of BAT by addition of adenosine with a synthetic amino acid linker
led to conjugates which showed no biological activity due to formation of
supramolecular structures. However, BATꢀamino acid derivatives were up to 25ꢀfold
more cytotoxic toward tumor cells compared to the parent BAT. Our result showed
that BAT and its new BATꢀamino acid derivatives produced DNA damage and
strongly interfered with centromere separation in tumor cells as a result of the
simultaneous inhibition of DNA topoisomerase IIα and Aurora B activation.

RSC Advances
Page 16 of 30
View Article Online
DOI: 10.1039/C6RA04957E
Fig. 5 Nuclear morphology of cells treated with BAT and its amino acid conjugates.
Celle were exposed for 3h to studied compounds and analysed with FISH as
described in Materials and Methods followed by fluorescence confocal microscopy.
Blue: DAPI staining of DNA; green – telomeric DNA probe and red – centromeric
DNA probe.
Conclusions
A series of non-protein amino acidꢀbatracylin derivatives 2a–h (Scheme 1) and its
conjugates with adenosine 3a–h (Scheme 2) were synthesized as potential
anticancer compounds. These compounds were tested for their cytotoxicity and
evaluation of their mechanisms of actions toward two human cell lines lung
adenocarcinoma (A549) and human leukemia (HLꢀ60).
Several of the amino acidꢀBAT derivatives showed greatly increased cytotoxicity
against both tumor cell types compared to BAT. Sensitivity toward both A549 and HLꢀ
60 cells was higher from about 2ꢀ to more than 25ꢀfold compared to the BAT e.g.

Page 17 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
compound containing 12ꢀaminododecanoic acid was about 30 times more cytotoxic
against A549 and HLꢀ60 cells than BAT. These compounds strongly interfered with
DNA topoisomerase II activity and its cellular functions. In particular, these
conjugates inhibited centromere segregation during mitosis in drugꢀtreated tumor
cells by preventing a topoisomerase IIꢀdependent Aurora B activation.
The majority of BATꢀamino acidꢀadenosine conjugates were biologically inactive
toward both A549 as well as HLꢀ60 cells. Only compound containing
6ꢀaminohexanoyl linker was specifically cytotoxic against A549 cells but indicated
comparable activity to BAT. Unexpectedly, we have found that in water and culture
media adenosineꢀamino acidꢀBAT conjugates form supramolecular structures and
that prevents these compounds from entering cells. The molecular mechanism
responsible for this unexpected formation of supramolecular structures in water
solutions is not clear at the moment and requires further studies.
Several received under the paper compounds characterized by promising biological
properties, requiring further research and have potential application.
Experimental
Materials and methods
Melting points (mp uncorrected) were determined on the Koflerꢀblock apparatus. All
chemicals and solvents were of reagent grade and were used without further
purification. The reactions were monitored by TLC on Merck F254 silica gel preꢀ
coated plates. The following solvent systems (by vol.) were used for TLC
development: CH2Cl2ꢀMeOH (7:1, v/v) (A), CH2Cl2ꢀMeOH (10:1, v/v) (B), CH2Cl2ꢀ
MeOH (20:1, v/v) (C). CH2Cl2ꢀMeOH (50:1, v/v) (D), CH2Cl2ꢀ(CH3)2CO (1:1, v/v) (E),
CH2Cl2ꢀMeOHꢀTEA (40:1:0.1 v/v) (F).
MS spectra were recorded on matrixꢀassisted laser desorption/ionizationꢀtime on
flight mass spectrometry (MALDIꢀTOF MS, Biflex III Bruker) (samples: 3a, 3b, 3d, 3f–
h) and with a 6540 UHD AccurateꢀMass QꢀTOF spectrometer using the negative ESI
mode (samples: 3c, 3e, 6a, 6d, 6f–h).
Proton and carbon NMR spectra were recorded in dimethyl sulfoxideꢀd6 (DMSOꢀ
d6) (isotopic enrichment 99.95%) solutions at 293K and 300K (sample 3h) using a
1
Varian Unity 500 Plus spectrometer (500.13 MHz for H, 125.76 MHz for 13C) with a
sample concentrations collected in Table 1, using 5 mm inverse detection broadband
1
probes and deuterium lock. The central peak of DMSOꢀd6 signals (2.49 ppm for H

RSC Advances
Page 18 of 30
View Article Online
DOI: 10.1039/C6RA04957E
and 39.50 ppm for 13C) was used as the internal reference standard. Twoꢀ
1
dimensional H spectra were measured in the phaseꢀsensitive mode with a spectral
width of 8000 Hz and 30272 data points providing a digital resolution of 0.529 Hz per
point. The 13C spectra were measured with the spectral width of 28777 Hz and 74816
data points providing a digital resolution of 0.769 Hz per point, relaxation delay 1.5 s.
The structures of compounds 3d and 3h were confirmed additionally by 2D gꢀCOSY
1
and ROESY H NMR (see Supplementary Table 1). The gꢀCOSY spectrum was
acquired in 4096
processed in a 4K
x
320 matrix with eight accumulations per increment and was
1K matrix. The ROESY spectrum was acquired with a mix time of
x
350 ms in a 2560
x 300 matrix with 8 accumulations per increment in a 4K x 1K
matrix. HSQC and HMBC experiments were performer with pulse field gradients. The
HSQC spectrum was acquired in the phaseꢀsensitive mode. The spectral windows for
1H and 13C axes were 5.169 Hz and 18.853 Hz, respectively. The data were collected
in a 2048
x
187 matrix and processed in a 2K
x
1K matrix. The HMBC spectrum was
1
acquired in absolute value mode. The spectral windows for H and 13C axes were
5.169 Hz and 21.995 Hz, respectively. The data were collected in a 2560
matrix and processed in a 2K 1K matrix.
x 210
x
The purity of all compounds was evaluated by HPLC using Agilent liquid
chromatograph series 1290 (Agilent Technology, Waldbronn, Germany) consisting of
binary pump G4220A, autosampler G4226A, thermostated column compartment
G1316C, diodeꢀarray detector G1315C. Chromatographic column: Supelcosil Cꢀ18,
(4.6 x 150 mm), 3 ꢀm, Supelco. Analytes were dissolved in a mixture of DMSO/
MeOH (1:1; v/v) and 2 µL of the solution were injected onto the chromatographic
column. A mixture of A: 0.1% HCOOH in water B: 0.1% HCOOH in AcCN/MeOH
(1:1; v/v) was used as a mobile phase in gradient mode at flow rate of 2.0 mL minꢀ1.
Gradient: at 5 min 20% B; at 15 min 100% B for analyzed compounds. The UVꢀVis
detector was operated at 254, 210 and 580 nm (DAD in single wavelength mode). All
analyses were done at 40 ºC.
8ꢀAminoisoindolo[1,2ꢀb]quinazolinꢀ12(10H)ꢀone (BAT) 4
This was prepared according to the method described from Nꢀ[2ꢀ(phthalimidomethyl)ꢀ
1,4ꢀacetyl]phenylenediamine (8) and recrystallized from N,Nꢀdimethylformamide
(DMF).28
Batracylin derivatives [Nꢀ(NωꢀBocꢀamino acid)ꢀBAT] 6a–h

Page 19 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
The procedure for the synthesis of compounds 6b, 6c, 6e have been published
previously.28 The compounds 6a, 6d and 6f–h were prepared by the same
procedure. The Bocꢀprotecting groups were removed by treatment with TFA and then
coupling gave compounds 2a–h.
2ꢀ[(tertꢀbutoxycarbonyl)amino]ꢀNꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀ
1
b]quinazolinꢀ8ꢀyl)ethanamide 6a. Yield 49% (yellow solid), mp 204−208 ºC; H
NMR (DMSO, 500 MHz) δ ppm: 1.38 (s, 9H, (CH3)3), 2.48 (m, 2H, CH2), 4.91 (s, 2H,
10), 7.0 (brs, 1H, NHBoc), 7.35 (d, J = 8.3 Hz, 1H, 6), 7.52 (d, J = 8.2 Hz, 1H, 7),
7.54 (s, 1H, 9), 7.75 (t, J = 7.33 Hz, 1H, 3), 7.87 (t, J = 7.32 Hz, 1H, 2), 7.88 (d, J =
7.3 Hz, 1H, 4), 7.98 (d, J = 7.3 Hz, 1H, 1), 10.16 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO,
125 MHz, 50.0 ºC): 171.97 (Cꢀ12), 166.82 (Cꢀ12”), 156.31 (CꢀBoc), 148.10 (Cꢀ4b”),
139.36 (Cꢀ5a”), 136.08 (Cꢀ8”), 134.86 (Cꢀ4a”), 133.81 (Cꢀ2”), 132.83 (Cꢀ3”), 130.78
(Cꢀ12a”), 128.58 (Cꢀ6”), 123.48 (Cꢀ4”), 123.02 (Cꢀ9a”), 122.43 (Cꢀ1”), 119.53 (Cꢀ7”),
118.18 (Cꢀ9”), 78.08 (CꢀBoc), 41.11 (Cꢀ10”), 39.67 (Cꢀ11), 29.0 (CꢀBoc); MS [M+H]+
m/z calcd for C22H22N4O4 406.43, found 407.18; Rf = 0.78 (B).
5ꢀ[(tertꢀbutoxycarbonyl)amino]ꢀNꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]
quinazolinꢀ8ꢀyl)pentanamide 6d. Yield 65% (yellow solid), mp 226−229 ºC; 1H NMR
(DMSO, 500 MHz) δ ppm: 1.36 (s, 9H, (CH3)3), 1.50 (m, 4H, CH2), 2.30 (t, J = 7.3 Hz,
2H, CH2CO), 2.90 ( m, 2H, CH2N), 4.90 (s, 2H, 10), 6.8 (brs, 1H, NHBoc), 7.35 (d, J
= 8.8 Hz, 1H, 6), 7.50 (d, J = 8.3 Hz, 1H, 7), 7.55 (s, 1H, 9), 7.75 (t, J = 7.3, Hz, 1H,
3), 7.87 (t, J = 7.3 Hz, 1H, 2), 7.88 (d, J = 6.8 Hz, 1H, 4), 8.0 (d, J = 7.3 Hz, 1H, 1),
10.0 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 50.0 ºC): 171.99 (Cꢀ15), 166.85
(Cꢀ12”), 156.31 (CꢀBoc), 148.16 (Cꢀ4b”), 139.37 (Cꢀ5a”), 136.11 (Cꢀ8”), 134.88 (Cꢀ
4a”), 133.87 (Cꢀ2”), 132.89 (Cꢀ3”), 130.81 (Cꢀ12a”), 128.59 (Cꢀ6”), 123.52 (Cꢀ4”),
123.08 (Cꢀ9a”), 122.47 (Cꢀ1”), 119.56 (Cꢀ7”), 118.22 (Cꢀ9”), 78.07 (CꢀBoc), 41.14 (Cꢀ
10”), 39.90 (Cꢀ11), 36.81 (Cꢀ14), 29.86 (Cꢀ12), 29.0 (CꢀBoc), 23.16 (Cꢀ13); MS
[M+H]+ m/z calcd for C25H28N4O4 448.51, found 449.22; Rf = 0.80 (B).
7ꢀ[(tertꢀbutoxycarbonyl)amino]ꢀNꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀ
1
b]quinazolinꢀ8ꢀyl)heptanamide 6f. Yield 48% (yellow solid), mp 217−220 °C; H
NMR (DMSO, 500 MHz) δ ppm: 1.26 (m, 4H, CH2), 1.35 (s, 11H, (CH3)3, CH2), 1.57
(m, 2H, CH2), 2.30 (t, J = 7.4 Hz, 2H, CH2CO), 2.88 ( m, 2H, CH2N), 4.90 (s, 2H, 10),
6.77 (brs, 1H, NHBoc), 7.34 (d, J = 8.5 Hz, 1H, 6), 7.50 (d, J = 8.5 Hz, 1H, 7), 7.56 (s,
1H, 9), 7.75 (t, J = 7.4, Hz, 1H, 3), 7.80 (t, J = 7.4 Hz, 1H, 2), 7.88 (d, J = 7.1 Hz, 1H,
4), 7.99 (d, J = 7.4 Hz, 1H, 1), 10.0 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz,

RSC Advances
Page 20 of 30
View Article Online
DOI: 10.1039/C6RA04957E
50.0 ºC): 172.09 (Cꢀ17), 166.84 (Cꢀ12”), 156.31 (CꢀBoc), 148.12 (Cꢀ4b”), 139.39 (Cꢀ
5a”), 136.09 (Cꢀ8”), 134.88 (Cꢀ4a”), 133.85 (Cꢀ2”), 132.86 (Cꢀ3”), 130.80 (Cꢀ12a”),
128.58 (Cꢀ6”), 123.51 (Cꢀ4”), 123.06 (Cꢀ9a”), 122.46 (Cꢀ1”), 119.55 (Cꢀ7”), 118.21 (Cꢀ
9”), 78.01 (CꢀBoc), 41.13 (Cꢀ10”), 39.89 (Cꢀ11), 37.12 (Cꢀ16), 30.06 (Cꢀ12), 29.08 (Cꢀ
13), 29.0 (CꢀBoc), 26.77 (Cꢀ14), 25.75 (Cꢀ15); MS [M+H]+ m/z calcd for C27H32N4O4
476.57, found 477.25; Rf = 0.84 (B).
8ꢀ[(tertꢀbutoxycarbonyl)amino]ꢀNꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀ
1
b]quinazolinꢀ8ꢀyl)octanamide 6g. Yield 52% (yellow solid), mp 214−218 ºC; H
NMR (DMSO, 500 MHz) δ ppm: 1.22 (m, 6H, CH2), 1.28 (s, 11H, (CH3)3, CH2), 1.58
(m, 2H, CH2), 2.31 (t, J =7.3 Hz, 2H, CH2CO), 2.88 ( m, 2H, CH2N), 4.90 (s, 2H,
C10), 7.16 (brs, 1H, NHBoc), 7.33 (d, J = 8.3 Hz, 1H, C6), 7.52 (d, J = 8.7 Hz, 1H, 7),
7.54 (s, 1H, C9), 7.75 (t, J = 7.3 Hz, 1H, C3), 7.79 (t, J = 7.3 Hz, 1H, C2), 7.87 (d, J =
7.3 Hz, 1H, C4), 7.98 (d, J = 7.3 Hz, 1H, C1), 10.11 (s, 1H, 8”ꢀCONH); 13C NMR
(DMSO, 125 MHz, 50.0 ºC): 172.16 (Cꢀ18), 166.79 (Cꢀ12”), 156.34 (CꢀBoc), 148.02
(Cꢀ4b”), 139.46 (Cꢀ5a”), 136.01 (Cꢀ8”), 134.88 (Cꢀ4a”), 133.77 (Cꢀ2”), 132.82 (Cꢀ3”),
130.78 (Cꢀ12a”), 128.57 (Cꢀ6”), 123.46 (Cꢀ4”), 122.99 (Cꢀ9a”), 122.43 (Cꢀ1”), 119.51
(Cꢀ7”), 118.18 (Cꢀ9”), 77.98 (CꢀBoc), 41.11 (Cꢀ10”), 39.89 (Cꢀ11), 37.14 (Cꢀ17), 30.13
(Cꢀ12), 29.65, 29.34, 28.99 (CꢀBoc), 26.91, 25.75; MS [M+H]+ m/z calcd 490.59 for
C28H34N4O4, found 491.42; Rf = 0.88 (B).
12ꢀ[(tertꢀbutoxycarbonyl)amino]ꢀNꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀ
1
b]quinazolinꢀ8ꢀyl)dodecanamide 6h. Yield 57% (yellow solid), mp 205−210 °C; H
NMR (DMSO, 500 MHz) δ ppm: 1.20 (m, 14H, CH2), 1.35 (s, 11H, (CH3)3, CH2), 1.57
(m, 2H, CH2), 2.30 (t, J = 7.4 Hz, 2H, CH2CO), 2.85 ( m, J = 6.8 Hz, 2H, CH2N), 4.90
(s, 2H, C10), 6.75 (brs, 1H, NHBoc), 7.34 (d, J = 8.3 Hz, 1H, 6), 7.50 (d, J = 8.8 Hz,
1H, 7), 7.56 (s, 1H, 9), 7.75 (t, J = 6.8, Hz, 1H, 3), 7.80 (t, J = 7.3 Hz, 1H, 2), 7.88 (d,
J = 7.3 Hz, 1H, 4), 7.99 (d, J = 7.8 Hz, 1H, C1), 10.0 (s, 1H, 8”ꢀCONH); 13C NMR
(DMSO, 125 MHz, 50.0 ºC): 172.14 (Cꢀ22), 166.82 (Cꢀ12”), 156.31 (CꢀBoc), 148.06
(Cꢀ4b”), 139.48 (Cꢀ5a”), 136.03 (Cꢀ8”), 134.87 (Cꢀ4a”), 133.82 (Cꢀ2”), 132.83 (Cꢀ3”),
130.79 (Cꢀ12a”), 128.55 (Cꢀ6”), 123.48 (Cꢀ4”), 122.98 (Cꢀ9a”), 122.44 (Cꢀ1”), 119.54
(Cꢀ7”), 118.17 (Cꢀ9”), 77.95 (CꢀBoc), 41.12 (Cꢀ10”), 39.89 (Cꢀ11), 37.14 (Cꢀ21), 30.16
(Cꢀ12), 29.66, 29.61, 29.60, 29.48, 29.39, 29.37, 28.98 (CꢀBoc), 26.94, 25.78; MS
[M+H]+ m/z calcd for C32H42N4O4 546.70, found 547.33; Rf = 0.93 (B).
General procedure for synthesis of adenosine analogues (3a–h). Under a nitrogen
atmosphere, a mixture of 6ꢀchloropurine riboside (1) (1 mmol), batracylin derivatives

Page 21 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
(2a–h) (1 mmol) and DIPEA (10 mmol) in anhydrous EtOH (4 mL) was stirred at
boiling point of solvent for 4 hr. The reaction was monitored with TLC in system
solvent A. After evaporating the solvent in vacuo, product was purified by TLC in
solvent systems B and C.
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)ethanamido]
adenosine 3a. Yield 55% (yellow solid), mp 225−227 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 3.49 (bs, 2H, 11), 3.53 (dd, J = 12.0 Hz, 3.2 Hz, 1H, 5’b), 3.64 (dd, J = 12.0
Hz, 3.2 Hz, 1H, 5’a), 3.95 (dd, J = 4.4 Hz, 3.2 Hz, 1H, 4’), 4.12 (t, J = 3.0 Hz, 1H. 3’),
4.60 (t, J = 6.0 Hz, 1H, 2’), 4.90 (s, 2H, 10”), 5.21 (bs, 1H, 3’OH), 5.40 (bs, 1H,
5’OH), 5.46 (bs, 1H, 2’OH), 5.87 (d, J = 6.0 Hz, 1H, 1’), 7.36 (bd, J = 8.0 Hz, 1H, 6”),
7.53 (bd, J = 8.0 Hz, 1H, 7”), 7.57 (bs, 1H, 9”), 7.75 (t, J = 6.0 Hz, 1H, 3”), 7.78 (t, J =
6.0 Hz, 1H, 2”), 7.87 (d, J = 7.0 Hz, 1H, 4”), 7.93 (bs, 1H, 10ꢀNH), 7.98 (d, J = 7.0 Hz,
1H, 1”), 8.24 (bs, 1H, 2), 8.35 (bs, 1H, 8), 10.18 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO,
125 MHz, 30.0 ºC): 168.91 (Cꢀ12),166.88 (Cꢀ12”), 155.38 (Cꢀ6), 152.87 (Cꢀ2), 148.21
(Cꢀ4), 148.07 (Cꢀ4b”), 140.35 (Cꢀ8), 139.24 (Cꢀ5a”), 136.12 (Cꢀ8”), 134.78 (Cꢀ4a”),
133.93 (Cꢀ2”), 132.96 (Cꢀ3”), 130.75 (Cꢀ12a”), 128.65 (Cꢀ6”), 123.56 (Cꢀ4”), 123.23
(Cꢀ9a”), 122.52 (Cꢀ1”), 120.68 (Cꢀ5), 119.49 (Cꢀ7”), 118.15 (Cꢀ9”), 88.63 (Cꢀ1’), 86.55
(Cꢀ4’), 74.20 (Cꢀ2’), 71.30 (Cꢀ3’), 62.29 (Cꢀ5’), 41.10 (Cꢀ10”), 39.67 (Cꢀ11): MS
[M+H]+ m/z calcd for C27H24N8O6 556.53, found 557.20; Rf = 0.62 (A).
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)propanamido]
adenosine 3b. Yield 71% (yellow solid), mp 224−228 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.21 (m, 2H, 12), 3.49 (bs, 2H, 11), 3.54 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’b),
3.65 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’a), 3.95 (t, J = 5.0 Hz, 1H, 4’), 4.14 (t, J = 5.0 Hz,
1H, 3’), 4.60 (t, J = 5.0 Hz, 1H, 2’), 4.90 (s, 2H, 10”), 5.23 (bs, 1H, 3’OH), 5.38 (bs,
1H, 5’OH), 5.51 (bs, 1H, 2’OH), 5.89 (d, J = 4.0 Hz, 1H, 1’), 7.36 (bd, J = 8.0 Hz, 1H,
6”), 7.55 (bd, J = 8.0 Hz, 1H, 7”), 7.57 (bs, 1H, 9”), 7.75 (t, J = 7.0 Hz, 1H, 3”), 7.79 (t,
J = 7.0 Hz, 1H, 2”), 7.87 (d, J = 7.0 Hz, 1H, 4”), 7.93 (bs, 1H, 10ꢀNH), 7.98 (d, J = 7.0
Hz, 1H, 1”), 8.21 (bs, 1H, 2), 8.40 (bs, 1H, 8), 10.36 (s, 1H, 8”ꢀCONH); 13C NMR
(DMSO, 125 MHz, 30.0 ºC): 170.47 (Cꢀ13), 166.87 (Cꢀ12”), 155.49 (Cꢀ6), 153.04 (Cꢀ
2), 148.15 (Cꢀ4), 148.12 (Cꢀ4b”), 140.51 (Cꢀ8), 139.20 (Cꢀ5a”), 136.10 (Cꢀ8”), 134.81
(Cꢀ4a”), 133.90 (Cꢀ2”), 132.93 (Cꢀ3”), 130.76 (Cꢀ12a”), 128.59 (Cꢀ6”), 123.55 (Cꢀ4”),
123.12 (Cꢀ9a”), 122.50 (Cꢀ1”), 120.58 (Cꢀ5), 119.54 (Cꢀ7”), 118.22 (Cꢀ9”), 88.61 (Cꢀ
1’), 86.57 (Cꢀ4’), 74.22 (Cꢀ2’), 71.30 (Cꢀ3’), 62.34 (Cꢀ5’), 41.12 (Cꢀ10”), 39.74 (Cꢀ11),

RSC Advances
Page 22 of 30
View Article Online
DOI: 10.1039/C6RA04957E
37.91 (Cꢀ12); MS [M+H]+ m/z calcd for C28H26N8O6 570.56, found 571.20; Rf = 0.62
(A).
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)butanamido]
adenosine 3c. Yield 58% (yellow solid), mp 215−219 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.24 (m, 2H, 12), 2.38 (t, J =7.0 Hz, 2H, 13), 3.52 (bs, 2H, 11), 3.55 (dd, J =
12.0 Hz, 3.5 Hz, 1H, 5’b), 3.64 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’a), 3.95 (dd, J = 5.0
Hz, 3.5 Hz, 1H, 4’), 4.13 (t, J = 5.0, 1H, 3’), 4.59 (t, J = 5.0 Hz, 1H, 2’), 4.90 (s, 2H,
10”), 5.26 (bs, 1H, 3’OH), 5.44 (bs, 1H, 5’OH), 5.58 (bs, 1H, 2’OH), 5.86 (d, J = 5.0
Hz, 1H, 1’), 7.33 (bd, J = 8.0 Hz, 1H, 6”), 7.52 (bd, J = 8.0, 1H, 7”), 7.56 (bs, 1H, 9”),
7.75 (t, J =7.0 Hz, 1H, 3”), 7.78 (t, J =7.0 Hz, 1H, 2”), 7.87 (d, J =7.0 Hz, 1H, 4”), 7.89
(bs, 1H, 10ꢀNH), 7.98 (d, J =7.0 Hz, 1H, 1”), 8.19 (bs, 1H, 2), 8.34 (bs, 1H, 8), 10.09
(s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 30.0 ºC): 172.00 (Cꢀ14), 167.00 (Cꢀ
12”), 155.31 (Cꢀ6), 153.05 (Cꢀ2), 148.83 (Cꢀ4), 148.27 (Cꢀ4b”), 140.41 (Cꢀ8), 139.19
(Cꢀ5a”), 135.86 (Cꢀ8”), 134.60 (Cꢀ4a”), 133.04 (Cꢀ2”), 132.91 (Cꢀ3”), 130.61 (Cꢀ12a”),
128.48 (Cꢀ6”), 123.15 (Cꢀ4”), 123.05 (Cꢀ9a”), 122.55 (Cꢀ1”), 120.37 (Cꢀ5), 119.53 (Cꢀ
7”), 118.18 (Cꢀ9”), 88.61 (Cꢀ1’), 86.60 (Cꢀ4’), 74.17 (Cꢀ2’), 71.30 (Cꢀ3’), 62.30 (Cꢀ5’),
41.02 (Cꢀ10”), 39.71 (Cꢀ11), 34.51 (Cꢀ13), 25.77 (Cꢀ12); MS [M+H]+ m/z calcd for
C29H28N4O4 584.58, found 585.22; Rf = 0.62 (A).
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)pentanamido]
adenosine 3d. Yield 51% (yellow solid), mp 220−224 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.62 (m, 4H, 12/13), 2.37 (t, J =7.0 Hz, 2H, 14), 3.49 (bs, 2H, 11), 3.55 (dd, J
= 12.0 Hz, 3.5 Hz, 1H, 5’b), 3.65 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’a), 3.95 (dd, J = 5.0
Hz, 3.5 Hz, 1H, 4’), 4.14 (t, J = 5.0, 1H, 3’), 4.59 (t, J = 5.0 Hz, 1H, 2’), 4.89 (s, 2H,
10”), 5.26 (bs, 1H, 3’OH), 5.43 (bs, 1H, 5’OH), 5.51 (bs, 1H, 2’OH), 5.86 (d, J = 5.0
Hz, 1H, 1’), 7.33 (bd, J = 8.0 Hz, 1H, 6”), 7.53 (bd, J = 8.0, 1H, 7”), 7.57 (bs, 1H, 9”),
7.74 (t, J =7.0 Hz, 1H, 3”), 7.79 (t, J =7.0 Hz, 1H, 2”), 7.87 (d, J =7.0 Hz, 1H, 4”), 7.93
(bs, 1H, 10ꢀNH), 7.98 (d, J =7.0 Hz, 1H, 1”), 8.19 (bs, 1H, 2), 8.34 (bs, 1H, 8), 10.15
(s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 30.0 ºC): 172.07 (Cꢀ15), 166.87 (Cꢀ
12”), 155.34 (Cꢀ6), 153.07 (Cꢀ2), 148.93 (Cꢀ4), 148.10 (Cꢀ4b”), 140.34 (Cꢀ8), 139.37
(Cꢀ5a”), 135.96 (Cꢀ8”), 134.79 (Cꢀ4a”), 133.90 (Cꢀ2”), 132.92 (Cꢀ3”), 130.73 (Cꢀ12a”),
128.59 (Cꢀ6”), 123.54 (Cꢀ4”), 123.10 (Cꢀ9a”), 122.49 (Cꢀ1”), 120.43 (Cꢀ5), 119.41 (Cꢀ
7”), 118.80 (Cꢀ9”), 88.64 (Cꢀ1’), 86.60 (Cꢀ4’), 74.18 (Cꢀ2’), 71.35 (Cꢀ3’), 62.36 (Cꢀ5’),
41.10 (Cꢀ10”), 39.70 (Cꢀ11), 36.89 (Cꢀ14), 29.40 (Cꢀ12), 23.30 (Cꢀ13); MS [M+H]+
m/z calcd for C30H30N8O6 598.61, found 599.20; Rf = 0.63 (A).

Page 23 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)hexanamido]
adenosine 3e. Yield 60% (yellow solid), mp 218−221 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.15 (m, 2H, 13), 1.61 (m, 4H, 12/14), 2.38 (t, J =7.0 Hz, 2H, 15), 3.53 (bs,
2H, 11), 3.54 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’b), 3.64 (dd, J = 12.0 Hz, 3.5 Hz, 1H,
5’a), 3.94 (dd, J = 5.0 Hz, 3.5 Hz, 1H, 4’), 4.12 (t, J = 5.0, 1H, 3’), 4.59 (t, J = 5.0 Hz,
1H, 2’), 4.90 (s, 2H, 10”), 5.19 (bs, 1H, 3’OH), 5.43 (bs, 1H, 5’OH), 5.44 (bs, 1H,
2’OH), 5.86 (d, J = 5.0 Hz, 1H, 1’), 7.33 (bd, J = 8.0 Hz, 1H, 6”), 7.51 (bd, J = 8.0, 1H,
7”), 7.56 (bs, 1H, 9”), 7.75 (t, J =7.0 Hz, 1H, 3”), 7.76 (t, J =7.0 Hz, 1H, 2”), 7.87 (d, J
=7.0 Hz, 1H, 4”), 7.89 (bs, 1H, 10ꢀNH), 7.98 (d, J =7.0 Hz, 1H, 1”), 8.19 (bs, 1H, 2),
8.34 (bs, 1H, 8), 10.11 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 30.0 ºC):
172.17 (Cꢀ18), 166.88 (Cꢀ12”), 155.31 (Cꢀ6), 153.10 (Cꢀ2), 148.84 (Cꢀ4), 148.10 (Cꢀ
4b”), 140.32 (Cꢀ8), 139.37 (Cꢀ5a”), 135.93 (Cꢀ8”), 134.77 (Cꢀ4a”), 133.91 (Cꢀ2”),
132.93 (Cꢀ3”), 130.73 (Cꢀ12a”), 128.58 (Cꢀ6”), 123.55 (Cꢀ4”), 123.11 (Cꢀ9a”), 122.50
(Cꢀ1”), 120.41 (Cꢀ5), 119.38 (Cꢀ7”), 118.06 (Cꢀ9”), 88.61 (Cꢀ1’), 86.60 (Cꢀ4’), 74.12
(Cꢀ2’), 71.35 (Cꢀ3’), 62.36 (Cꢀ5’), 41.10 (Cꢀ10”), 39.82 (Cꢀ11), 37.09 (Cꢀ17), 29.72 (Cꢀ
12), 27.03ꢀ29.37 (Cꢀ13ꢀ15), 25.77 (C16); MS [M+H]+ m/z calcd for C31H32N8O6
612.24, found 613.25; Rf = 0.67 (A).
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)heptanamido]
adenosine 3f. Yield 61% (yellow solid), mp 223−226 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.22 (m, 4H, 13ꢀ14), 1.58 (m, 4H, 12/15), 2.32 (t, J =7.0 Hz, 2H, 16), 3.53 (bs,
2H, 11), 3.55 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’b), 3.64 (dd, J = 12.0 Hz, 3.5 Hz, 1H,
5’a), 3.94 (dd, J = 5.0 Hz, 3.5 Hz, 1H, 4’), 4.12 (t, J = 5.0, 1H, 3’), 4.58 (t, J = 5.0 Hz,
1H, 2’), 4.90 (s, 2H, 10”), 5.19 (bs, 1H, 3’OH), 5.43 (bs, 1H, 5’OH), 5.44 (bs, 1H,
2’OH), 5.86 (d, J = 5.0 Hz, 1H, 1’), 7.33 (bd, J = 8.0 Hz, 1H, 6”), 7.52 (bd, J = 8.0, 1H,
7”), 7.57 (bs, 1H, 9”), 7.74 (t, J =7.0 Hz, 1H, 3”), 7.76 (t, J =7.0 Hz, 1H, 2”), 7.87 (d, J
=7.0 Hz, 1H, 4”), 7.89 (bs, 1H, 10ꢀNH), 7.98 (d, J =7.0 Hz, 1H, 1”), 8.18 (bs, 1H, 2),
8.32 (bs, 1H, 8), 10.10 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 30.0ºC):
172.16 (Cꢀ17), 166.86 (Cꢀ12”), 155.30 (Cꢀ6), 153.06 (Cꢀ2), 148.09 (Cꢀ4), 148.04 (Cꢀ
4b”), 140.27 (Cꢀ8), 139.40 (Cꢀ5a”), 135.98 (Cꢀ8”), 134.80 (Cꢀ4a”), 133.88 (Cꢀ2”),
132.90 (Cꢀ3”), 130.75 (Cꢀ12a”), 128.57 (Cꢀ6”), 123.53 (Cꢀ4”), 123.07 (Cꢀ9a”), 122.48
(Cꢀ1”), 120.44 (Cꢀ5), 119.44 (Cꢀ7”), 118.10 (Cꢀ9”), 88.65 (Cꢀ1’), 86.59 (Cꢀ4’), 74.19
(Cꢀ2’), 71.34 (Cꢀ3’), 62.37 (Cꢀ5’), 41.10 (Cꢀ10”), 39.72 (Cꢀ11), 37.07 (Cꢀ16), 29.65 (Cꢀ
12), 29.16 (Cꢀ13), 26.90 (Cꢀ14), 25.79 (Cꢀ15); MS [M+H]+ m/z calcd for C32H34N8O6
626.66, found 627.30; Rf = 0.71 (A).

RSC Advances
Page 24 of 30
View Article Online
DOI: 10.1039/C6RA04957E
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)octanamido]
adenosine 3g. Yield 65% (yellow solid), mp 225−229 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.30 (m, 6H, 13ꢀ15), 1.57 (m, 4H, 12/16), 2.29 (t, J =7.0 Hz, 2H, 17), 3.49 (bs,
2H, 11), 3.54 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’b), 3.63 (dd, J = 12.0 Hz, 3.5 Hz, 1H,
5’a), 3.94 (dd, J = 5.0 Hz, 3.5 Hz, 1H, 4’), 4.12 (t, J = 5.0, 1H, 3’), 4.58 (t, J = 5.0 Hz,
1H, 2’), 4.90 (s, 2H, 10”), 5.18 (bs, 1H, 3’OH), 5.42 (bs, 1H, 5’OH), 5.44 (bs, 1H,
2’OH), 5.86 (d, J = 5.0 Hz, 1H, 1’), 7.33 (bd, J = 8.0 Hz, 1H, 6”), 7.53 (bd, J = 8.0, 1H,
7”), 7.56 (bs, 1H, 9”), 7.73 (t, J =7.0 Hz, 1H, 3”), 7.78 (t, J =7.0 Hz, 1H, 2”), 7.87 (d, J
=7.0 Hz, 1H, 4”), 7.89 (bs, 1H, 10ꢀNH), 7.98 (d, J =7.0 Hz, 1H, 1”), 8.18 (bs, 1H, 2),
8.31 (bs, 1H, 8), 10.06 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 30.0 ºC):
172.17 (Cꢀ22), 166.92 (Cꢀ12”), 155.24 (Cꢀ6), 153.16 (Cꢀ2), 148.83 (Cꢀ4), 148.09 (Cꢀ
4b”), 140.22 (Cꢀ8), 139.43 (Cꢀ5a”), 135.88 (Cꢀ8”), 134.77 (Cꢀ4a”), 133.84 (Cꢀ2”),
132.90 (Cꢀ3”), 130.73 (Cꢀ12a”), 128.56 (Cꢀ6”), 123.51 (Cꢀ4”), 123.13 (Cꢀ9a”), 122.46
(Cꢀ1”), 120.37 (Cꢀ5), 119.36 (Cꢀ7”), 118.01 (Cꢀ9”), 88.56 (Cꢀ1’), 86.58 (Cꢀ4’), 74.15
(Cꢀ2’), 71.35 (Cꢀ3’), 62.34 (Cꢀ5’), 41.10 (Cꢀ10”), 39.66 (Cꢀ11), 37.08 (Cꢀ17), 29.69 (Cꢀ
12), 22.3ꢀ27.1 (Cꢀ13ꢀ16); MS [M+H]+ m/z calcd for C33H36N8O6 640.69, found 641.20;
Rf = 0.78 (A).
N6ꢀ[Nꢀ(12ꢀoxoꢀ10,12ꢀdihydroisoindolo[1,2ꢀb]quinazolinꢀ8ꢀyl)dodecanamido]
adenosine 3h. Yield 57% (yellow solid), mp 225−228 ºC; 1H NMR (DMSO, 500 MHz)
δ ppm: 1.25 (m, 14H, 13ꢀ19), 1.56 (m, 4H, 12/20), 2.30 (t, J = 7.0 Hz, 2H, 21), 3.49
(bs, 2H, 11), 3.55 (dd, J = 12.0 Hz, 3.5 Hz, 1H, 5’b), 3.65 (dd, J = 12.0 Hz, 3.5 Hz,
1H, 5’a), 3.95 (dd, J = 5.0 Hz, 3.5 Hz, 1H, 4’), 4.12 (t, J = 5.0, 1H, 3’), 4.58 (t, J = 5.0
Hz, 1H, 2’), 4.89 (s, 2H, 10”), 5.31 (bs, 1H, 3’OH), 5.47 (bs, 1H, 5’OH), 5.56 (bs, 1H,
2’OH), 5.86 (d, J = 5.0 Hz, 1H, 1’), 7.33 (bd, J = 8.0 Hz, 1H, 6”), 7.54 (bd, J = 8.0, 1H,
7”), 7.58 (bs, 1H, 9”), 7.73 (t, J =7.0 Hz, 1H, 3”), 7.78 (t, J =7.0 Hz, 1H, 2”), 7.87 (d, J
=7.0 Hz, 1H, 4”), 7.90 (bs, 1H, 10ꢀNH), 7.98 (d, J =7.0 Hz, 1H, 1”), 8.18 (bs, 1H, 2),
8.33 (bs, 1H, 8), 10.17 (s, 1H, 8”ꢀCONH); 13C NMR (DMSO, 125 MHz, 30.0 ºC):
172.19 (Cꢀ22), 166.87 (Cꢀ12”), 155.31 (Cꢀ6), 153.07 (Cꢀ2), 148.84 (Cꢀ4), 148.07 (Cꢀ
4b”), 140.30 (Cꢀ8), 139.42 (Cꢀ5a”), 135.91 (Cꢀ8”), 134.78 (Cꢀ4a”), 133.89 (Cꢀ2”),
132.91 (Cꢀ3”), 130.73 (Cꢀ12a”), 128.58 (Cꢀ6”), 123.54 (Cꢀ4”), 123.07 (Cꢀ9a”), 122.48
(Cꢀ1”), 120.39 (Cꢀ5), 119.38 (Cꢀ7”), 118.04 (Cꢀ9”), 88.63 (Cꢀ1’), 86.59 (Cꢀ4’), 74.17
(Cꢀ2’), 71.34 (Cꢀ3’), 62.36 (Cꢀ5’), 41.10 (Cꢀ10”), 39.67 (Cꢀ11), 37.09 (Cꢀ21), 29.70 (Cꢀ
12), 20.0ꢀ32.0 (Cꢀ13ꢀ19), 25.80 (Cꢀ20); MS [M+H]+ m/z calcd for C37H44N8O6 696.80,
found 697.20; Rf = 0.87 (A).

Page 25 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
Cell culture
Cell lines were purchased from American Type Culture Collection (Rockville, MD).
The cells A549 and HLꢀ60 were maintained in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 2 mM
Lꢀglutamine, and antibiotics (100 units/mL
penicillin and 100 ꢁg/mL streptomycin) and cells were cultivated at 37°C in 5%
CO2/95% air atmosphere. Cell lines were screened routinely for Mycoplasma by the
PCR method with Mycoplasma Plus PCR primer set (Stratagene, La Jolla, CA).
Cytotoxicity assay
The cytotoxicity was determined by the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) assay. Briefly, exponentially growing cells were
continuously exposed to different drug concentrations and the cellular viability was
evaluated after 120h. Cells were exposed to the MTT tetrazolium salt for 4h at 37°C,
and the formation of formazan was measured after solubilization in 1 mL DMSO by a
ASYS UVM340 microplate reader (Biochrom Ltd.). The concentrations required to
inhibit cell growth by 50% compared to untreated controls were determined from the
curves plotting survival as a function of dose by use of the GraphPad Prism 5
program. All values are averages of at least three independent experiments, each
done in duplicate.
Protonation profiles, LogD and LogP of studied compounds. The protonation at
different pH was calculated for BAT and its derivatives using the SPARC online
calculator
(ARChem
–
Automated
Reasoning
in
Chemistry)
at
http://archemcalc.com/sparc/. The LogD curves were created using ChemAxon
online calculator (at https://www.chemaxon.com). The LogP values were calculated
using ACD/LogP Freeware (component of ACD/ChemSketch Freeware program).
Flow cytometry analysis of cell cycle distribution
Distribution in different phases of the cell cycle was analyzed by flow cytometry after
treatment of tumor cells with studied drugs. Briefly, following drug treatment with a
dose corresponding to IC90, cells were washed in iceꢀcold phosphate buffered saline
(PBS) and fixed in 70% ethanol at ꢀ20°C. Cells were stained in PBS containing 20

RSC Advances
Page 26 of 30
View Article Online
DOI: 10.1039/C6RA04957E
ꢁg/mL propidium iodide and 100 ꢁg/mL ribonuclease A for 30 min at room
temperature. Samples were analyzed by a Guava EasyCyte™ 8 flow cytometer
(MerckꢀMillipore) equipped with a 488 nm laser, and the distribution of cells in the cell
cycle was calculated using MultiCycle software (Phoenix Flow Systems).
Inhibition of DNA relaxation mediated by DNA topoisomerases
Supercoiled DNA plasmid pBR322 (>95% form I) was purchased from Thermo
Scientific, human type I topoisomerase was purchased from MoBiTec and human
type IIα topoisomerase was purchased from TopoGen. The reaction mixture
contained 200 ng of pBR322 DNA in reaction buffer (20 mM TrisꢀHCl, pH 7.5, 7.5 mM
MgCl2, 0.5 mM DTT, 150 mM KCl, 1 mM ATP) as well as studied compounds. The
reaction was initiated by the addition of topoisomerase IIα enzyme and allowed to
proceed at 37°C for 30min. Reactions were terminated by addition of a loading buffer
(0.1% SDS, 0.05% bromophenol blue, 2.5 mM EDTA, 10% sucrose, final
concentrations). The samples were separated in 1% agarose gels at 0.5 V/cm for 18h
in TBE buffer (90 mM Trisꢀbase, 70 mM boric acid, 1 mM EDTA, pH 8). Gels were
stained with 0.5 ꢁg/mL ethidium bromide to visualize DNA and photographed under
UV illumination. The conditions for DNA relaxation assay for type I topoisomerase
were the same except different reaction buffer was used (10 mM TrisꢀHCl, pH 7.5,
7.5 mM MgCl2, 60 mM KCl).
Nuclear and cellular morphology of tumor cells
Cells were attached to cover slides in 35 mm Petri dishes and treated with studied
compounds for different time periods. Following drug treatment, cells were stained
with 1 ꢁg/mL Hoechst 33342 alone or with 1 ꢁg/mL Hoechst 33342 and 7.5 ꢁg/mL
fluorescein diacetate dyes for 15 min and analyzed by Olympus BXꢀ60 fluorescent
microscope equipped with respective optical filters. Images were recorded using
XC50 digital camera and image acquisition software CellSens.
Immunofluorescence
Cells were fixed with ice cold methanol, blocked with 5% BSA/0.05% Tweenꢀ20/PBS
for 1h at 37°C, and incubated with antiꢀAurora B diluted 1:000 in 5%BSA/PBS
(Abcam, ab2254) overnight at 4°C. An antiꢀrabbit AlexaFluor 488 (BioLegend,
406416) was used as the secondary antibody. For detection of H2AX

Page 27 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
phosphorylation, cells were fixed with 3.7% PFA, permeabilized with 0.2% Triton Xꢀ
100 and blocked with 5%BSA/PBS for 10 min/RT. Slides were incubated overnight at
4°C with antiꢀphosphoꢀ(S139)ꢀH2AX Alexa Fluor 488 conjugated antibody diluted
1:200 in 5%BSA/PBS (BioLegend, 613406). DAPI was used to stain the cell nuclei at
a concentration 0.5 µg/mL. Images were analyzed using LSM 700 confocal
microscope (Zeiss).
Fluorescence in situ hybridization (FISH) assay
The FISH assay was performed according to manufacturer instructions. Cells where
fixed in methanol:acetic acid solution 3:1 and dehydrated through a series of ethanol
solutions (70, 90 and 100%) and airꢀdried. Slides were then baked at 65°C and after
cooling, washed with acetone for 10 min and airꢀdried. RNA was digested by
incubation of samples with RNase A (100 µg/mL in 2xSSC) at 37°C for 1h. After 2
washes with PBS and 2xSSC buffer, slides were incubated with pepsin (0.5 µg/ml in
10 mM HCl) for 3min at RT, then washed with PBS and dehydrated in ethanol as
described above. DNA was denatured by incubation of slides in 70%
formamide/2XSSC solution at 70°C for 2min and instantly cooled to ꢀ20°C by
immersion in a cold 70% ethanol/water solution. FITCꢀconjugated panꢀtelomeric
chromosome (starFISH, 1696ꢀFꢀ02) and biotinꢀconjugated panꢀcentromeric
chromosome (starFISH, 1695ꢀBꢀ02) probes were used (Cambio). Probes for
centromeric and telomeric DNA were diluted in a buffer provided by the
manufacturer, denatured at 85°C for 10min and rapidly cooled on ice. Hybridization
under coverslips was carried out at 37°C overnight in a humidified chamber. Cover
glasses were then removed by immersion in 2xSSC at 37°C and nonꢀhybridized
probes were washed off by incubation in 50% formamide/2xSSC at 37°C. Blocking
step with 5% BSA/0.05% Tweenꢀ20/PBS was performed and then slides were
incubated with rabbit antiꢀFITC antibodies (1:200, Invitrogen, 71ꢀ1900) and mouseꢀ
antiꢀbiotin Alexa Fluor 594ꢀconjugated antibodies (1:100, Jackson ImmunoResearch
Labs, 200ꢀ582ꢀ211) for 1h at 37°C. Samples were washed three times in 0.05%
Tween 20/PBS and incubated 1h at 37°C with donkey antiꢀrabbit Alexa Fluor 488ꢀ
conjugated antibodies (1:100, Thermo Fisher Scientific, SA5ꢀ10038). DAPI was used
to stain the cell nuclei at a concentration 0.5 µg/mL in water. Images were analyzed
by Leica confocal microscope TCS SP8 X equipped with the white light laser and
appropriate filters (Leica Microsystems).

RSC Advances
Page 28 of 30
View Article Online
DOI: 10.1039/C6RA04957E
SAꢀβꢀGalactosidase staining
Cells were treated with studied compounds with the IC90 doses for 120h and washed
in PBS, fixed for 3ꢀ5 min (room temperature) in 3.7% formaldehyde, washed, and
incubated at 37°C (no CO2) with freshly prepared staining solution: 1 mg/mL of
5ꢀbromoꢀ4ꢀchloroꢀ3ꢀindolyl βꢀDꢀgalactoside (XꢀGal) in 40 mM citric acid/sodium
phosphate, pH 6.0, (containing 5 mM potassium ferrocyanide; 5 mM potassium
ferricyanide; 150 mM NaCl; 2 mM MgCl2) for 12ꢀ16h. Images were analyzed by
Olympus BXꢀ60 microscope and recorded using XC50 digital camera as well as
image acquisition software CellSens.
Western blot analysis
Cells were harvested on ice by scrubbing with RIPA buffer supplemented with
protease and phosphatase inhibitors. Protein concentration was determined using
BCA method (Price) and 50 ꢁg of total protein was loaded onto SDSꢀPAGE gel and
blotted on the PVDF membrane. Following blocking step in 5% BSA/TBST
membranes where incubated with primary antibodies toward topoisomerase IIα
(BioTrend), Cyclin B1 (Santa Cruz), p53 (Santa Cruz), Aurora B (Abcam), pꢀSer10ꢀ
H3 (Sigma), Histone H3 (Cell Signaling), Actin (Santa Cruz) overnight at 4°C.
Membranes were washed tree times with TBST and incubated with indicated
secondary antibody antiꢀmouseꢀHRP, antiꢀrabbitꢀHRP, antiꢀgoatꢀHRP (Jacson
ImmunoResearch) for 1h at RT. Membranes where visualized by ECL (Thermo) and
developed using Xꢀray films and developing machine (Agfa).
Acknowledgements
This work was financially supported by the National Science Center (Poland), grant
no 2012/05/B/NZ7/02461. The authors would like to thank Dr Michał Rychłowski
(Laboratory of Viral Molecular Biology, Intercollegiate Faculty of Biotechnology,
Gdansk) for help with confocal microscope analyses.
References
1 R. V. Chari, Acc. Chem. Res., 2008, 41, 98.
2 J. Plowman, K. D. Paull, G. Atassi, S. D. Harrison, D. J. Dykes, H. J. Kabbe,
V. L. Narayanan and O. C. Yoder, Invest. New Drugs, 1988, 6, 147.

Page 29 of 30
RSC Advances
View Article Online
DOI: 10.1039/C6RA04957E
3 W. R. Waud, S. D. Harrison, K. S. Gilbert, W. R. Laster and D. P. Griswold, Cancer
Chemother. Pharmacol., 1991, 27, 456.
4 P. MucciꢀLoRusso, L. Polin, M. C. Bissery, F. Valeriote, J. Plowman, G. D. Luk and
T. H. Corbett, Invest. New Drugs, 1989, 7, 295.
5 G. Atassi, P. Dumont, H. J. Kabbe and O. Yoder, Drugs Exp. Clin. Res., 1988, 14,
571.
6 Y. Luo, Y. F. Ren, T. C. Chou, A. Y. Chen, C. Yu, L. F. Liu and C. C. Cheng,
Pharm. Res., 1993, 10, 918.
7 V. A. Rao, K. Agama, S. Holbeck and Y. Pommier, Cancer Res., 2007, 67, 9971.
8 M. M. Ames, D. A. Mathiesen and J. M. Reid, Invest. New Drugs, 1991, 9, 219.
9 G. J. Stevens, J. L. Burkey and C. A. McQueen, Cell Biol. Toxicol., 2000, 16, 31.
10 L. W. Wormhoudt, J. N. M. Commandeur and N. P. E. Vermeulen, Crit. Rev.
Toxicol., 1999, 29, 59.
11 S. Kummar, M. E. Gutierrez, L. W. Andrson, R. W. Klecker, A. Chen, A. J. Murgo,
J. H. Doroshow and J. M. Collins, Cancer Chemother. Pharmacol., 2013, 72, 917.
12 K. Dzierzbicka, W. Januchta and A. Skladanowski, Curr. Med. Chem., 2012, 19,
4475.
13 J. Roservear and F. K. Wilshire, Aut. J. Chem., 1990, 43, 339.
14 V. Stella, L. A. AlꢀRazzak and H. J. Kabbe, U.S. Patent 4.980.343, 1990.
15 H. G. Larchen, N. Piel, J. P. Lorentzen, H. P. Antonicek and H. J. Kabbe,
European Patent Office No. 0 501 250 A1, 1992. Chem. Abstr., 1993, 118, 60133k.
16 V. Stella, L. A. AlꢀRazzak and H. J. Kabbe, C.A. Patent 2.001.494, 1990.
17 J. Chen, H. Neumann, M. Beller and X. Wu, Org. Biomol. Chem., 2014, 12, 5835.
18 M. Ch. Tseng, P. Y. Lai, L. Shi, H. Y. Li, M. J. Tseng and Y. Ch. Chu,
Tetrahedron, 2014, 70, 2629.
19 V. Ralevic and G. Burnstock, Pharmacol. Rev., 1998, 50, 413.
20 B. B. Fredholm, A. P. Jzerman, K. A. Jacobson, K. N. Klotz and J. Linden,
Pharmacol. Rev., 2001, 53, 527.
21 S. Gessi, K. Varani, S. Merighi, E. Fogli, V. Sacchetto, A. Bennini, E. Leung, S.
MacꢀLennen and P. A. Borea, Purinergic Signal., 2007, 3, 109.
22 M. Samsel and K. Dzierzbicka, Pharmacol. Rep., 2011, 63, 601.
23 J. T. Merrill, C. Shen and D. Schreibmab, Arthritis Rheum., 1997, 40, 1308.
24 H. Kamiya, T. Kanno, Y. Fujita, A. Gotoh, T. Nakano and T. Nishizaki, Cell
Physiol. Biochem., 2012, 29, 687.