In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

019, VOL. 34, NO. 1, 877–897

2

https://doi.org/10.1080/14756366.2019.1593159

RESEARCH ARTICLE

In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

a,b

b,c

a,d

b,c

a

ꢀ

Jana Janockova

, Jan Korabecny

, Jana Plsikova , Katerina Babkova , Eva Konkolova ,

e

f

Dana Kucerova , Jana Vargova , Jan Koval , Rastislav Jendzelovsky , Peter Fedorocko , Jana Kasparkova ,

f

d

b,c

g

b

a,b

Viktor Brabec , Jan Rosocha , Ondrej Soukup , Slavka Hamulakova , Kamil Kuca and Maria Kozurkova

a

b

ꢀ

Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Saf ꢁa rik University, Kosice, Slovak Republic; Biomedical Research

c

Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic; Department of Toxicology and Military Pharmacy, Faculty of

d

ꢀ

Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic; Associated Tissue Bank, Faculty of Medicine, P.J. Saf ꢁa rik

e

ꢀ

University, Kosice, Slovak Republic; Department of Cellular Biology, Institute of Biology and Ecology, Faculty of Science, P. J. Saf ꢁa rik University,

f

g

Kosice, Slovak Republic; Department of Biophysics, Faculty of Science, Palacke University, Olomouc, Czech Republic; Department of Organic

Chemistry, Institute of Chemistry, Faculty of Science, P. J. Saf ꢁa rik University, Kosice, Slovak Republic

ꢀ

ABSTRACT

ARTICLE HISTORY

A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA

interaction with newly synthesized 7-MEOTA-tacrine thiourea 12–17 and urea heterodimers 18–22, and to

measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro

on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mito-

chondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds

exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topo-

isomerase at 1 mM concentration). The treatment of HL-60 cells with the studied compounds showed

inhibition of cell growth especially with hybrids containing thiourea (14–17) and urea moieties (21 and

Received 30 October 2018

Revised 11 February 2019

Accepted 27 February 2019

KEYWORDS

7

-MEOTA-tacrine hetero-

dimers; calf thymus DNA;

topoisomerases; HL-60;

human dermal fibroblasts

2

2). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate sig-

nificant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential

drugs to target cancer cells.

Introduction

THA is currently used as a versatile scaffold in medicinal chemis-

try for designing novel hybrid compounds with improved pharma-

cological and toxicological profiles affecting several pathological

Tacrine (9-amino-1,2,3,4-tetrahydroacridine, THA, Figure 1) was

first described as an analeptic able to cause rapid arousal of

morphinized dogs and cats . Later, THA was found to be a

potent cholinesterase inhibitor of both acetylcholinesterase

1

4–18

1

,2

mechanisms, e.g. in Alzheimer’s disease pathophysiology

. The

THA derivative 7-MEOTA (7-methoxy-1,2,3,4-tetrahydroacridin-9-

amine, Figure 1), which was developed in our laboratory and pri-

marily tested to antagonize anticholinergic syndrome evoked by

(

3

AChE, E.C. 3.1.1.7) and butyrylcholinesterase (BuChE, E.C.

.1.1.8) . Notably, a potential crosstalk between some types of

3

1

9,20

scopolamine, ditrane and 3-quinuclidinyl benzilate

and also as

cancer and modulation of AChE activity has been proposed.

Accordingly, the inhibition of AChE affecting cholinergic signal-

ing has been associated with some potential benefits in e.g. can-

cerous lung tissue . The effect of THA has been well-described

in neurological diseases . Indeed, THA was licensed in USA and

Canada as the first symptomatic treatment for cognitive symp-

toms associated with Alzheimer’s disease (the drug Cognex) .

Clinical use of THA was limited due to its side effects, mainly

hepatotoxicity and gastrointestinal symptoms . The precise

2

1

a prophylactic agent against organophosphate poisoning , dis-

played a better toxicological profile than THA . Mansouri et al.

5

22

4

examined the mechanism of mitochondrial function damage dur-

ing treatment with THA. Based on the chemical structure, it was

postulated that THA might have a similar mechanism of action as

the antitumor agent acridine (Figure 1) and related derivatives (e.g.

5

6

2

mAMSA, imidazoacridinones, bis-tacrine, Figure 1) . These acri-

7

dines are able to intercalate between the planar bases of DNA and

8

23–26

mechanism of hepatotoxicity still remains unclear . Some to inhibit nuclear type II topoisomerase (Topo II)

. In addition, a

researchers associate the THA-associated hepatotoxicity with oxi- variety of bis-acridines have been developed with anticancer activ-

2

7,28

dative bio-activation and the formation of chemically highly ity

. Further research also revealed that THA derivatives might

9

reactive metabolites ; however, the cell-killing effect is more be engaged also in interaction with type I topoisomerase (Topo

1

0–12

29

probably mediated by membrane fluidity alterations

. Apart I) . THA itself was found to be a relatively weak catalytic inhibitor

and necrosis of liver of Topo II implying inhibition of DNA synthesis. The latter led to

1

3

from that, mitochondrial dysfunction

7

22,29

cells also emerged as other routes for THA toxicity.

depletion of mitochondrial DNA and ultimately to apoptosis

.

ꢀ

CONTACT Maria Kozurkova

maria.kozurkova@upjs.sk

Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Saf ꢁa rik University, Kosice,

Slovak Republic

ꢀ

These authors contributed equally to this work.

Supplemental data for this article can be accessed here .

ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

8

78

J. JANOCKOVA ET AL.

NH₂

N

O

N

A

B

C

N

H

O

N

O

N

S

H

N

HN

N

S

N

O

N

Cl

D

E

F

5

Figure 1. Chemical structures of acridine (A), THA (B) and their derivatives: 7-MEOTA (C) , mAMSA (D), 6-chloro-2-methoxy-N-{2-[4-(1,2,3,4-tetrahydroacridin-9-yl)pipera-

35

zin-1-yl]ethyl}acridin-9-ylamine (E) , 1-(1,2,3,4-tetrahydro-acridin-9-yl)-3-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl]thiourea (F) .

Topo I and II are nuclear enzymes which play an important role by 1,x-diamines in the presence of phenol then provided the desired

formatting superhelical DNA structures and thus ensuring essential intermediates N-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)alkane-

cell functions. The topology of DNA can be regulated by certain 1,x-diamines 3–8 (71–93%). 1,2,3,4-Tetrahydroacridin-10H-9-one 9

enzymes leading to mutual transformation of its topological iso- was formed directly from the neat reaction of cyclohexanone with

mers and to the successive relaxation. Catalytic activity of topoiso- N-methylanthranilate (65%). Chlorination of 9 was carried out by

merases lies in introduction DNA of single (Topo I) or double (Topo refluxing with phosphorus oxychloride to obtain 9-chloro-1,2,3,4-

II) strand breaks. More recently, topoisomerases emerged as target tetrahydroacridine 10 in quantitative yield. 9-Isothiocyanato-

of great interest in the development of novel antibacterial and 1,2,3,4-tetrahydroacridine 11 was obtained by refluxing 10 in the

3

0,31

41

anticancer drugs

.

dark with silver thiocyanate in anhydrous toluene . The desired

Apoptosis is a critical cellular process of programed cell death 7-MEOTA-THA thioureas 12–17 were formed by reaction of the

which features changes in cell morphology such as membrane

blebbing, nuclear fragmentation, chromatin condensation and

chromosomal DNA fragmentation. Every unwanted regulation of

the apoptotic pathway has been associated with either the onset

two synthons 11 and diamines 3–8 (65–78%). The oxo-analogs

were obtained using 2,4,6-trimethylbenzonitrile N-oxide to afford

7

-MEOTA-THA ureas (18–23; 88–95%). Structural determination

and signal assignments of thiourea hybrids 12–17 and urea

hybrids 18–23 were accomplished by application of the usual

3

2–34

or progression of different diseases including cancer

. These

1

13

findings spurred cancer drug discovery in the medicinal chemistry

combination of H and C NMR spectra. Unequivocal assignments

were performed by homo- and hetero-correlated two-dimensional

NMR experiments (H,H-COSY, H,C-HSQC, H,C-HMBC). The infrared

spectrum was obtained only for molecule 11 to observe isothio-

cyanate group vibrations.

3

5

field . However, tetrahydroacridines with their non-planar scaffold

3

6

can bind to DNA with much lower affinity than acridines .

In this work, new 12 analogs were synthesized by amalgamat-

ing THA with the less toxic THA derivative 7-MEOTA via an alkyl

chain containing either urea or thiourea moieties. These two par-

ent compounds were formerly anti-Alzheimer’s disease (AD)

agents structurally resembling amsacrine or the imidazoacridi-

nones, both being developed as anticancer agents capable to

In this work, we have studied DNA binding of novel THA-7-

MEOTA dimers 12–22, their influence on the catalytic activity of

Topo I/II, their ability to affect cell cycle distribution, and their

effect on the mitochondrial membrane potential (MMP) and the

metabolic activity and viability of the HL-60 cell line. The results

acquired from these in vitro studies should enhance understand-

ing of the effects pertinent for drugs targeting cancer. The urea

heterodimer 23 was not tested due to its low solubility.

3

7,38

intercalate between DNA-bases and to target Topo II

. Our

study disclosed their antiproliferative effect against human acute

promyelocytic leukemia cell line HL-60 in contrast to their effect

on dermal fibroblasts. In summary, herein we provide deep insight

into the mode of action of compounds 12–22 that might lead to

development of novel DNA topoisomerase inhibitors as chemo-

therapeutic agents.

Biological evaluation

For biological effect analysis of novel compounds 12–22, we pre-

fer to use multiple assays that reflect real physiological/patho-

logical changes in more detail and on single cell level (FACS

analysis). It is generally known that most of the chemotherapeutic

Results and discussion

Chemistry

Scheme 1 illustrates the general synthetic procedure for 7-MEOTA- agents exert their cytotoxic effect either by induction of cancer

4

2

THA hybrids. The starting fused ring of 7-MEOTA, 7-methoxy- cell apoptosis or by cell cycle arrest at a specific point .

1

,2,3,4-tetrahydroacridin-10H-9-one , was prepared by p-toluene- Cumulative evidence has shown that opening of MMP transition

sulfonic acid-catalysed condensation reaction of 4-methoxyaniline pores results in the dissipation of MMP (Dw ) followed by release

with ethyl-2-oxocyclohexanecarboxylate in refluxing toluene in of pro-apoptotic molecules into the cytoplasm, which leads to

m

4

3

good yield (74%). In the next step, 1 was treated with phosphorus programed cell death . To examine whether 12–22 affect mito-

oxychloride to give a quantitative yield of 9-chloro-7-methoxy- chondrial physiological processes, HL-60 cells were treated with

39,40

1

,2,3,4-tetrahydroacridine 2

. Treatment of 2 with appropriate the studied compounds for 24, 48 and 72 h and labeled with

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

879

O

NH₂

O

H C

3

+

a

b

N

H

OCH₃

1

O

Cl

O

H C

H

C

3

N

H

2

Cl

N

NH

2

HN

NH

2

n

O

H C

H

2

N

c

H C

3

n

+

N

n = 2-7

- 8

3

O

NH

2

O

d

O

+

N

H

9

e

Cl

NCS

N

f

N

11

10

S

N

HN

NH₂

HN

N

n

H

O

g

H C

3

N

n = 2-7

- 8

n = 2-7

12 - 17

3

S

N

O

N

HN

N

H

N

HN

N

n

H

O

h

O

H C

3

N

n = 2-7

18 - 23

1

2 - 17

ꢁ

3

Scheme 1. Synthesis of 7-MEOTA-THA thioureas 12–17 and ureas 18–23. Conditions: (a) Dean–Stark trap, toluene, reflux 8 h; diphenylether, 220 C 30 min; (b) POCl ,

ꢁ

reflux 1 h; (c) phenol, 130 C, 2–4 h; (d) P

2

O

5

, N,N-dimethylcyclohexylamine, 170 C, 4 h; (e) POCl

Cl , RT, 48h.

3 2 2

, reflux 1 h; (f) AgSCN, dry toluene, reflux overnight; (g) 11, CH Cl , RT,

4

8 h; (h) 2,4,6-trimethylbenzonitrile N-oxide, CH

2 2

TMRE. In general, mitochondria with normal MMP retain the dye, 80 and 100% at 15 mM for urea analogs and 25 mM for thioureas

resulting in strong fluorescence. Compounds inducing a collapse after 24, 48 and 72 h (Figure 2(A)). Their effect is comparable to

in MMP allow flow of the dye from mitochondria to cytoplasm that recorded for the acridine which have been explored as a

44

leading to depolarization of the mitochondrial membrane and potential therapeutic agent for the treatment of cancer (Table

fluorescence decrease. The results from MMP analysis clearly show 1). The other tested heterodimers 12, 13, 18–20 and parent THA

that 14–17 and 21 at 15 mM and 22 at 5 mM were able to evoke induced a negligible effect on MMP and a weak cytotoxic effect

MMP dissipation in more than approximately 60% of cells after (data not shown). A simultaneous analysis of viability (staining

2

4 h treatment and caused cell death with an efficiency between with PI) and the activity of cellular metabolism showed a very

8

80

J. JANOCKOVA ET AL.

similar trend to acquired changes in MMP analysis identifying the compounds within both subseries, the most toxic being 17 and

effectivity of compounds 14–17 and 21, 22 to kill cancer cells 22 from the urea and thiourea families, respectively. The most

(Figure 2(B)). IC50 from both techniques over different time peri- pronounced cytotoxic effect was associated with tether lengthen-

ods are displayed in Table 1. The cytotoxic effects on HL-60 cells ing, with urea derivatives 21 (at 15 mM) and 22 (at 5 mM) having

go hand-in-hand with the lipophilicity of the individual five and six methylenes in the linker being found to exert the

Figure 2. Effect of 7-MEOTA-THA thioureas 14–17 (5–25 lM), ureas 20–22 (1–15lM) and THA (5–10lM) on changes in A: MMP and B: viability in HL-60 cells. Cells

were analyzed 24, 48 and 72 h after treatment with studied compounds. The results were calculated as mean ± SD from three independent experiments. Statistical sig-

ꢀ

ꢀꢀ

ꢀꢀꢀ

nificance p < .05 ( ), .01 ( ), and .001 ( ) for the particular experimental group compared to untreated control (C).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

881

Figure 2. Continued

4

5

most cytotoxic profile. The higher cytotoxic effect presumably apoptosis . Cryptolepine, a plant-derived alkaloid, has recently

reflects easier cell membrane penetration. Interestingly, the cyto- been considered as a promising anticancer agent due to a dem-

toxic effect against HL-60 also correlated well with the ability to onstrated effect of Topo II, inhibition of DNA-synthesis, inhibitory

inhibit activity of topoisomerases. It is known that inhibitors of effects on the cell cycle, and potent toxicity to a number of cancer

4

6

47

Topo I, such as camptothecin, exhibit cell cycle arrest induced cells . Hu

demonstrated that some bis-tacrine derivatives

8

82

J. JANOCKOVA ET AL.

Table 1. IC50 values for MMP dissipation (TMRE) and viability (FDA/PI) of HL-60 concentration of THA, it did not exert any effect in this experi-

cells after 24, 48 and 72 h incubation with studied 7-MEOTA-tacrine thio-/

ureas 12–22.

ment (48 h incubations with THA is shown Supplementary

Figure S2(B)).

IC50 (lM)±SEM

The intracellular distribution of 12–22 was analyzed on the

Compound

Time (h)

24

TMRE

FDA/PI

model of A549 adherent cancer cells and human dermal fibro-

43.94 ± 1.04 blasts by a simple approach. After 24 h incubation with the deriva-

33.39 ± 1.02 tives, the cells were washed in order to dispose of unbound

1

2

3

4

5

6

7

1

2

47.29 ± 2.15

30.46 ± 4.78

38.00 ± 4.68

43.95 ± 1.98

29.90 ± 3.95

26.97 ± 6.37

17.89 ± 3.04

15.48 ± 4.13

14.56 ± 5.04

15.81 ± 3.47

13.54 ± 2.08

13.33 ± 4.13

15.88 ± 1.23

16.54 ± 2.16

14.33 ± 2.94

10.18 ± 3.22

7.59 ± 1.62

7.36 ± 2.82

9.19 ± 5.39

7.11 ± 5.47

9.18 ± 2.75

3.95 ± 2.23

3.76 ± 1.17

3.94 ± 2.79

n.d

4

7

8

2

47.82 ± 2.83

40.98 ± 0.95

30.16 ± 3.41

29.75 ± 1.49

compounds, so that only compounds that were able to permeate

into the cells could be visualized. The fluorescence of each sample

was examined in each channel but images were captured using

24

4

7

8

2

24

18.11 ± 6.61 only two channels: (1) blue excitation range [BP excitation filter

4

7

8

2

15.87 ± 3.75

4

50–490 nm (cube I3) and LP suppression filter 515 nm]—when

15.12 ± 3.74

19.51 ± 4.33

15.74 ± 2.49

heterodimers were present in the samples they exhibited positiv-

ity only in this channel; (0) brightfield mode was also applied in

24

4

8

72

13.57 ± 4.01 order to capture the cells themselves, visualizing single cells

24

16.64 ± 3.01

15.71 ± 5.31

15.12 ± 2.69

9.58 ± 1.27

regardless of fluorescence of the studied compounds. According

to the signal in channel 1 (I3 filter cube), compounds 14–17

4

7

8

2

(5 mM) showed abundant accumulation after 24 h incubation and

24

4

7

8

2

7.64 ± 1.69 a strong fluorescence signal in the A549 cells. All these hetero-

7.24 ± 2.71 dimers were visible as dark spots even in brightfield mode (Figure

9.80 ± 4.13

24

3

(A)). Phase contrast microscopy was used to visualize the intracel-

4

7

8

2

6.87 ± 3.04

6.85 ± 1.92

4.19 ± 2.64

lular distribution of 12–22 in human dermal fibroblasts as well.

Thiourea hybrids 14–17 displayed accumulation also in fibroblasts

24

4

8

3.78 ± 2.74 at higher concentrations (25 mM, Figure 3(B)), whereas in the case

72

3.67 ± 2.40

n.d

n.d.

6.40 ± 2.09

5.04 ± 4.96

of A549 cells, such concentrations were lethal. Hybrids 12, 13, 18

and 19 exhibited only selective accumulation in A549 cells.

Tacrine

24

4

7

8

2

n.d

n.d.

6.37 ± 2.51

5.23 ± 3.18

Interestingly, heterodimers 12 and 13 accumulated in fibroblast

cytoplasm (similarly to 14–17) whereas urea heterodimers 18, 19

did not (data not shown). It was also observed that ureas 20–22

accumulated neither in A549 cells nor in fibroblasts (Figure 4).

Taking all these facts together, it was noticed that the thiourea

hybrids with longer chains permeate through the cell membrane

more easily than their urea counterparts. This phenomenon might

be sufficiently explained by the higher lipophilicity of thiourea

hybrids compared to urea derivatives, as has been experimentally

Acridine

24

4

7

8

2

n.d.: not defined; TMRE: tetramethylrhodamine ethyl ester perchlorate; FDA:

fluorescein diacetate; PI: propidium iodide; 23 was not tested due to its

low solubility.

exhibited moderate selectivity toward colon and melanoma cells

and interestingly, butyl-linked bis-tacrine showed strong cytotox-

icity against non-small cell lung, colon cancers and melanoma. A

variety of dimeric compounds have been shown a broad spectrum

activity against different human tumor cells [e.g. bis(phenazine-1-

carboxamides) act as dual Topo I/II-directed anticancer drugs and

were evaluated for growth inhibitory activity in cancer cell lines

including P388 leukemia and Lewis lung carcinoma ] and also

dimeric naphthalimides and bis-acridines have been recognized

for clinical trials as anticancer candidates .

50

determined in many studies . Interestingly, changes in morph-

ology were observed in both cancer and non-cancer cells treated

with 14–17. In the case of A549, treatment resulted in a more

rounded shape of the cells, suggesting that the hybrids impaired

cell viability. These changes in morphology confirmed our previ-

ous results in which the same concentration of derivatives

decreased the viability and MMP of A549 cells after 24 h of incu-

bation. The size of human dermal fibroblasts was increased after

permeation of compounds 14–17 into the cells. In general, this

led to a loss of typical fibroblast-like morphology. However, pene-

tration of the derivatives into fibroblasts did not affect MMP, in

contrast to A549 cells. These findings cannot be sufficiently

48

49

47

The cytotoxicity of 12–22 was also determined on cultured

human dermal fibroblasts by measuring MMP. As shown in

Supplementary Figure S1 , all the studied compounds were less

toxic to non-cancer cells compared to cancer cells at given con- explained due to the unclear mode of action at the intracellular

centrations after 72 h treatment, which underlies their advantage level. Moreover, hybrids 14–17 seem to be good candidates for

in development of anticancer drugs.

further studies of the exact localization of these compounds inside

In order to address the effect of compounds 12–22 to impair the cells, using confocal microscopy and other techniques.

cell proliferation, flow cytometry analysis of the cell cycle distribu-

tion was hence used. Based on the results from MMP dissipation

and viability analyzes, HL-60 cells were treated with different con-

Activity of Topo I and II

centrations of 12–17 (5–25 mM) and 18–22 (1–15 mM) for 24, 48

and 72 h. After 48 h incubation, results for urea derivatives 21

Given that the studied 7-MEOTA-THA thio-/urea hybrids 12–22 are

able to interact with ctDNA (see section below), it is important to

consider whether they are also able to inhibit some of the cell

(15 mM) and 22 (10 mM) clearly demonstrated a significant accumu-

lation of cells in G1 phase (Supplementary Figure S2(B)). In com- nucleus enzymes involved in DNA operations, and which alter its

parison, thiourea analogs 14 –17 revealed a concentration effect topology through decatenation and relaxation of supercoiled

(Supplementary Figure S2(A)). These compounds significantly DNA, as for example Topo I/II. Moreover, Topo I/II are also

altered cell cycle progression, inducing block of the cell cycle in S employed in other cell functions, such as replication, transcription,

5

1–53

phase after just 24 h treatment at 25 mM concentration. THA and recombination and chromosomal segregation

. In recent years,

acridine were used as controls. Despite the relatively high Topo I/II have been under increasing study as a cell target for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

883

Figure 3. Intracellular accumulation after 24h incubation of 14–17 in A549 cells (5 mM, A) visualized by fluorescence microscopy, and in fibroblasts (25 mM, B) visual-

ized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0ꢂ 0.40 DRY UV objective) and Leica

DMI3000B. Presented images were obtained from three independent experiments.

anticancer drugs capable of targeting their catalytic cycle. Some established Topo I inhibitor camptothecin and DNA intercalator

clinical studies showed that such inhibition ability could be associ- EtBr, both applied at 5 mM were used as positive controls. Results

54

ated with the cytotoxicity effect of inhibitors . Compounds inhib- were compared with standards THA (Topo I inhibition at 60 mM,

iting the catalytic functions of Topo I/II can cause permanent not shown) and 7-MEOTA (Topo inhibition at 30 mM,

I

5

9

breaks in DNA structure which can lead to cell death, but the not shown) .

55–57

detailed mechanism remains unknown

. Topo I/II inhibitors are

classified as catalytic inhibitors (they decrease enzyme activity) or

poisons (they stabilize the DNA-cleavage complex and protect

repeated DNA religation) .

Effect on Topo II decatenation activity and Topo II drug screening

It is well-established that catalytic Topo II inhibitors interfere with

the binding between Topo II and DNA (e.g. suramin, aclarubicin),

inhibit ATP binding (e.g. novobiocin), or stabilize the noncovalent

DNA-Topo II complex (e.g. dexrazoxane, merbarone). Topo II poi-

58

Effect on Topo I relaxation activity

In this study, we examined the effect of 7-MEOTA-THA thio-/urea sons are able to stabilize the covalent DNA-Topo II cleavage com-

derivatives 12–22 on the catalytic activity of Topo I by measuring plex and are clinically used for their antitumor activity (e.g.

6

0

the Topo I-mediated relaxation of supercoiled plasmid pUC19. etoposide, teniposide, doxorubicin) .

Due to significant and dose-dependent inhibition of Topo I cata-

The ability of 12–22 to affect the decatenation of catenated

lytic activity all the compounds under study reduced the amount kinetoplast DNA (kDNA) from the insect trypanosome Crithidia fas-

of relaxed DNA, while simultaneously increasing the amount of ciculata by Topo II was examined (Figure 5 and Supplementary

supercoiled DNA. 7-MEOTA-THA thioureas 12 –17 inhibited the Figure S5 ). ATP-dependent decatenation assay is a specific assay

catalytic activity of Topo I at 60 mM (Supplementary Figure S3 ). for detection of compounds with potential Topo II inhibition activ-

Compounds 14–17 were able to inhibit the catalytic relaxation ity. Topo II-targeted compounds are able to interact with (at least)

ability of Topo I on pUC19 already at 5 mM, and hence further test- one step of the topoisomerase catalytic cycle. The decatenation of

ing was carried out at concentrations of 1–10 mM (Supplementary kDNA by Topo II generates different products (nicked, relaxed or

Figure S4 ). The most effective from this subset was compound 17 supercoiled form of DNA) which move easily into the agarose gel

6

1

due to strong inhibition at 1 mM (Figure 5(A)). On the other hand, compared with the larger-sized catenated kDNA . Interestingly,

the urea analogs 18–22 showed strong inhibition potency at slight evidence of decatenated products was observed in the pres-

3

0 mM except for 20 which inhibited Topo I partially at that con- ence of the studied compounds 12–22 at 5 mM compared to

centration (Supplementary Figure S3 ). Intriguingly, compound 22 200 mM etoposide, 100 mM ellipticine and 100 mM mAMSA (Topo II

was effective at 1 mM concentration (Figure 5(A)). The well- poisons/inhibitors used as standards). All the compounds revealed

8

84

J. JANOCKOVA ET AL.

Figure 4. Intracellular accumulation after 24h incubation of 20–22 in A549 cells (5 mM, A) visualized by fluorescence microscopy, and in fibroblasts (5 mM, B) visual-

ized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0ꢂ 0.40 DRY UV objective) and Leica

DMI3000B. Presented images were obtained from three independent experiments.

the same pattern of inhibition of catalytic activity of Topo II at 50 nowadays used in combination with chemotherapeutic agents (e.

62

66–68

and 100 mM concentrations. Vispe et al. studied a series of novel g. aclarubicin, suramin, MST-16)

bis- and tetra-acridine anticancer drugs, which were found not

.

only as DNA interacting agents but also as Topo II-mediated DNA

decatenation inhibitors. These acridines inhibited Topo II activity Spectroscopic properties

at 32 mM, which is comparable with our obtained results. Given

our findings, we propose that Topo I and II both represent an

important target for our studied heterodimers.

Several binding modes have been identified between native and/

or synthetic double-stranded DNA (dsDNA) and low molecular-

weight compounds acting as chemotherapeutic agents or gene

regulators. The non-covalent DNA interactions are represented

mainly by intercalation between the base pairs of dsDNA; and

binding to minor/major grooves of dsDNA, stabilized by a mix of

electrostatic, hydrophobic, hydrogen-bonding interactions or out-

side random binding . The interactions of 7-MEOTA-THA thio-/

ureas 12–22 with ctDNA were studied in order to investigate their

possible binding modes. Representative UV–vis absorption titra-

tion spectra for thiourea hybrid 17 and urea analog 22 are dis-

played in Figure 6. The addition of increasing amounts of ctDNA

Topo II poisons (such as etoposide or mAMSA) are able to sta-

bilize the DNA-Topo II complex and thus form linear DNA due to

the simultaneous cleavage of both strands of double-stranded

DNA. Plasmid DNA pHOT1 (the DNA substrate) used in the cleav-

6

3

age assay contains a single high affinity Topo II cleavage site . In

our study, the linear form of DNA was found when 100 mM of eto-

poside or 100 m M of mAMSA was applied, but not for 12–22

6

9

(Supplementary Figure S6 ). These results indicated that 12–22 are

characterized with Topo II catalytic inhibition, but not as Topo II

poisons. Topo II poisons were found to be important for activating

secondary leukemias involving certain chromosomal transloca- to a constant concentration of the compounds (UV–vis spectro-

tions. On the other hand, Topo II catalytic inhibitors are able to scopic titration) revealed a hypochromic shift ranging from 16.31

change the cytotoxic effect of poisons and overcome multidrug to 46.10%. A mild bathochromic shift to higher wavelengths was

6

0,64,65

resistance

.

Numerous Topo II catalytic inhibitors are observed only for compounds 17, 18, 20 and 21 (Dk ¼ 1 nm). The

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

885

Figure 5. Effect of 17 and 22 on A: relaxation of supercoiled plasmid DNA by calf thymus Topo I. Supercoiled plasmid DNA pUC19 (lane pUC19) was incubated with

calf thymus Topo I in the absence (lane pUC19þ Topo I) and in the presence of various compound concentrations (0–10 lM); B: decatenation of kDNA (0.16lg) by

human Topo IIa. Catenated kinetoplast DNA (lane kDNA) was incubated with human Topo IIa in the absence (lane kDNA þ Topo II) and in the presence of various

compounds concentration (0–100lM).

ꢁ

observed changes suggested that studied compounds 12–22 are which exhibited a profound increase of T

m

of more than 10 C.

able to interact with ctDNA, although from the small bathochro- Thermal characterization of the studied compounds 12–22 and

mic shifts we can rather point out to groove binding or electro- the first derivative of ctDNA denaturation curves in the presence

static interaction, rather than intercalation. The DNA-binding of the studied compounds are shown in Supplementary Figure

characteristics of the studied 7-MEOTA-THA thio-/ureas 12–22 are S7. Usually, ligands which could bind to DNA through intercal-

summarized in Table 2. The binding constants K of the studied ation significantly increase T

compounds in complex with ctDNA ranged between 0.5 and around 5–8 C, due to stabilization of dsDNA. Groove binding and

m m

of DNA, and D T is typically

ꢁ

6

ꢃ1

8

.0 ꢂ 10 M and were comparable with the measured K of acrid- electrostatic binding ligands produce mostly negligible effects on

7

2

73

ine and greater than the literature-cited K for acridin-3,6-diyl

T

m

of DNA . Tao et al. revealed that a subtle decrease of T

m

(0.

5

ꢃ1 70

ꢁ

dithiourea hydrochlorides (2.9–7.6 ꢂ 10 M ) , proflavine diureas 9 C) in melting temperature analysis with ctDNA for resmethrin

5

ꢃ1 71

ꢃ1 29

(

0.9–4.2 ꢂ 10 M

)

and tacrine derivatives with ctDNA characterizes this compound as a groove binder. Results from our

5

0.16–4.0 ꢂ 10 M ) . In addition, it is shown that linking of THA study indicated that only compound 22 could possibly bind

and 7-MEOTA with thio-/urea moieties positively influenced the strongly to ctDNA in intercalative mode, but it is not excluded

interaction power. Binding constants typical for DNA intercalators that 7-MEOTA thio-/ureas 12–21 are able to insert between the

4

6

ꢃ1

lie in the range 10 –10

M

and they are usually lower than planar bases of ctDNA.

5

binding constants typical for groove binders (range from 10

A more sensitive evaluation of the interaction of studied 7-

MEOTA-THA thio-/ureas 12–22 with ctDNA was accomplished

9

ꢃ1

to 10 M ).

DsDNA tertiary structure is stabilized via hydrogen bonds and using steady-state fluorescence measurements. Fluorescence spec-

base stacking interactions. When a solution of dsDNA is exposed tra of studied heterodimers at fixed concentration with increasing

to extreme heat, the double helix dissociates to the single- concentration of ctDNA showed a progressive quenching in the

stranded form (known as denaturated DNA). This leads to disrup- fluorescence intensity which was directly proportional to the

tion of intermolecular forces and hydrogen bonding interactions ctDNA concentration. Representative results are displayed in

72

between the DNA base pairs . The stability of the dsDNA struc- Figure 7 (for hybrids 17 and 22) and suggest interaction between

ture is determined by the melting temperature (T ). DNA the compounds and ctDNA. Data from these fluorescence quench-

m

denaturation is usually measured spectrophotometrically at the ing experiments were also used for determination of K_S_Vthat pro-

temperature-dependent excitation point of 260 nm. In this work, vides a measure of the quenching ability of ctDNA. The inserted

ꢁ

T

m

of ctDNA was found to be 68 C whereas in the presence of graphs in Figure 7 present the Stern–Volmer plots obtained as a

7

-MEOTA-THA thioureas 12–16 slightly increased (DT_mwas function of F /F versus [Q]. These plots are characterized by an

0

ꢁ

1–2 C). In the presence of 7-MEOTA-THA ureas 18–22, no intercept of one on the y-axis and the slopes are equal to K_S_V, as

of ctDNA was observed, except for compound 22 summarized in Table 3.

increase of T

m

8

86

J. JANOCKOVA ET AL.

Figure 7. Steady-state fluorescence spectra of studied 7-MEOTA-THA thiourea 17

and urea 22 (5 mM, upper lines) in 10mM Tris–HCl buffer (pH 7.4) with increasing

concentration of ctDNA (0–280 mM, color lines). Arrows show the changes in

emission intensity upon increasing ctDNA concentration. The inserted graphs rep-

0

resent the Stern–Volmer plot (plot F /F versus [Q]) at three different temperatures

(

288, 298 and 310K).

Figure 6. UV–vis absorption titration spectra of studied 7-MEOTA-THA thiourea

7 (49 mM) and urea 22 (49 mM) in 10 mM Tris–HCl buffer (pH 7.4; RT) with

1

increasing concentration of ctDNA (0–1.24 mM). Arrows indicate the changes after

addition of increasing concentration of ctDNA. Inserted graphs represent the

non-linear fitting of absorption titration, the plot of [DNA] versus appropri-

ate absorbance.

Fluorescence quenching is observed in two processes, as

dynamic or static quenching. Static quenching is based on the

fact that the fluorophore and added quencher form a stable non-

fluorescent complex in the ground state which promptly goes

back to the ground state without photon emission after light

absorption. For dynamic (collisional) quenching, quencher diffu-

sion to the fluorophore is typical during the lifetime of an excited

state and the rate of this quenching is dependent upon tempera-

ture, viscosity and diffusion. The decrease of fluorescence in the

case of dynamic quenching is due to random collision between

Table 2. DNA-binding characteristics of 7-MEOTA-THA thioureas 12–17 and

ureas 18–22.

k

max

Bathochromic

shift (nm)

a

ꢃ1

Compound

Free Bound

Hypochromicity (%)

K (M )

6

4

6

4

1

2

3

4

5

6

7

8

9

0

1

2

312

313

311

310

311

309

324

400

334

312

311

312

309

329

407

336

0

1

0

1

0

5

7

2

16.31

21.83

32.40

33.52

38.99

46.10

31.33

30.10

30.19

29.06

38.06

40.25

50.02

13.10

3.8 ꢂ 10

5.5 ꢂ 10

6.5 ꢂ 10

8.0 ꢂ 10

4.0 ꢂ 10

4.3 ꢂ 10

4.5 ꢂ 10

0.5 ꢂ 10

0.6 ꢂ 10

3.0 ꢂ 10

6.0 ꢂ 10

3.8 ꢂ 10

4.5 ꢂ 10

7.9 ꢂ 10

74

the fluorophore and quencher . A Stern–Volmer plot can depend

on the mechanism of quenching, and can be non-linear or linear,

and upward or downward curving. In this work, we observed lin-

ear Stern–Volmer plots indicating that only one type of quenching

process is possible, either dynamic or static quenching. Both static

and dynamic quenching can be recognized with different temper-

atures. Higher temperature of solution leads to a higher diffusion

coefficient, and an increase of KSV (by increasing the temperature

random collision is more probable). On the other hand, increased

THA

Acridine

temperature goes hand in hand with decreased complex stability

75,76

7

a

-MEOTA

and thus the static quenching constant is lower

. With increas-

The concentrations of studied compounds were as follows: compounds 12–17:

ⁱⁿ^g^t^e^m^p^e^r^a^t^u^r^e^,^f^r^o^m^t^h^e^o^b^t^aⁱⁿ^e^d^d^a^t^a^s^h^o^wⁱⁿ^g^d^e^c^r^e^a^sⁱⁿ^g^KSV

4

9 mM; 18–20: 99 mM; 21, 22: 49 mM; 23 was not tested due to its low solubil- values it is clear that ctDNA can quench fluorescence of the

ity, THA and acridine: 50 mM; 7-MEOTA: 25 mM.

studied compounds as a static quencher. To further confirm the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

887

Table 3. Stern–Volmer (KSV) and quenching constants (k

MEOTA-THA thio-/ureas 12–22 at three different temperatures and pH 7.4.

q

) of complex ctDNA: 7-

Table 4. Binding constants and various thermodynamic parameters for studied

7-MEOTA-THA heterodimers 12–22 in complex with DNA at three different

temperatures.

ꢀ

3

ꢃ1

11 ꢃ1 ꢃ1

2

Compound

Temperature (K)

288

K

SV (ꢂ10 M

)

k

q

(ꢂ10

M

s

)

R

ꢀ

4

ꢃ1

2

Compound

Temperature (K)

K

b

(ꢂ10 M

)

n

R

1

2

ꢀ

2

3

4

5

6

7

8

9

0

1

2

5.58

4.83

3.77

7.18

6.69

5.66

10.70

8.92

7.81

7.47

7.33

6.16

8.75

7.09

5.97

10.08

8.98

7.69

6.72

6.18

5.26

9.54

8.76

7.34

5.31

5.26

4.77

8.05

7.30

6.56

5.48

5.00

4.81

5.58

4.83

3.77

7.18

6.69

5.66

0.991

0.989 12

0.994

0.986

0.995 13

0.995

0.999

2

98

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

288

298

310

0.36

0.48

0.69

1.13

1.39

2.16

0.77

2.34

3.46

1.32

2.11

2.70

2.61

1.83

1.06

2.12

3.02

4.02

1.90

2.31

2.51

2.31

4.02

5.92

0.69

0.49

0.13

1.65

1.83

2.03

0.49

1.59

2.45

0.95

1.00

1.07

1.05

1.08

1.15

0.96

1.11

1.17

1.07

1.12

1.17

1.13

1.11

1.07

1.09

1.14

1.19

1.12

1.15

1.18

1.10

1.18

1.24

1.03

0.99

0.83

1.08

1.10

1.13

0.99

1.13

1.19

0.995

0.993

0.996

0.989

0.997

0.999

1.000

0.999

0.997

0.999

0.998

0.999

0.998

0.997

0.999

0.997

0.999

0.995

0.999

0.996

1.000

0.999

0.997

0.999

310

288

2

3

98

10

288

10.70

8.92

7.81

7.47

7.33

6.16

8.75

7.09

5.97

10.08

8.98

7.69

6.72

6.18

5.26

9.54

8.76

7.34

5.31

5.26

4.77

8.05

7.30

6.56

5.48

5.00

4.81

2

3

98

10

0.996 14

0.992

0.999

0.995 15

0.993

0.997

0.998 16

0.998

0.997

0.994 17

0.993

0.992

0.995 18

0.993

0.993 19

0.987

0.991

0.998 20

0.993

0.997

0.997 21

0.996

0.993

288

2

3

98

10

288

2

98

310

288

2

3

98

10

288

2

3

98

10

288

2

3

98

10

288

2

98

310

288

2

3

98

10

288

2

3

98

10

0.998 22

0.991

The concentration of studied compounds 12–22 was 5 mM, 23 was not tested

due to its low solubility, R : correlation coefficient.

2

ꢀ

The concentration of studied compounds 12–22 was 5 mM, 23 was not tested

due to its low solubility, R : correlation coefficient.

2

quenching process k was calculated; this indicates the accessibil-

ity of a fluorophore to a quencher, or the efficiency of quenching.

q

7

-MEOTA-THA thio-/ureas 12–22 with ctDNA. Based on the nega-

tive DH and DS for 16 and 20, we can assume that interaction in

these ligand-ctDNA complexes is mediated by van der Waals

forces (Table 5).

Polarized light spectroscopy is a suitable technique for fast

characterization of nucleic acids and their complexes with small

molecules. From linear dichroism (LD) can be deduced structure

information about the relative conformation between the DNA

axis and the ligand. Circular dichroism (CD) is another powerful

tool to gain insight into the structural determinants characterizing

Calculated k

.77–10.70 ꢂ 10

diffusion rate constant of biomolecules in the case of dynamic

q

are listed in Table 3 and were found to be

1

ꢃ1 ꢃ1

3

M

s

at different temperatures. The limiting

10

ꢃ1 ꢃ1

quenching was found to be 2.00 ꢂ 10

M

s

. However, there

77,78

is no limit for static quenching

. Since the obtained values of

k_qwere higher than the limiting diffusion rate constant, it is sug-

gested that the quenching process in this study was static rather

than dynamic.

The modified Stern–Volmer equation gives the relationship

between fluorescence emission intensity and the concentration of such complexes. Whereas LD can be used to monitor the angles

quencher and was used to determine K

of the forming complex between DNA-base transition states relative to the axis orientation

b

between 7-MEOTA-THA thio-/urea hybrids and ctDNA. The calcu- which are identical with the helix axis in flow or electric field-ori-

lated K and n are recorded in Table 4. The nature of the forces ented DNA, the induced CD of the same transition could provide

b

useful for the formation of the complex between the studied com- information about molecular orientation relative to the surrounding

81

pounds and ctDNA can be identified by the thermodynamic nucleobases . CD is the difference in the absorption of left-handed

parameters, DH, DS and DG. Values of all thermodynamic parame- circularly polarized light (L-CPL) and right-handed circularly polar-

8

2

ters are summarized in Table 5. The negative values of DG are ized light (R-CPL): CD ¼ AL-CPL—

AR-CPL . CD spectra of ctDNA were

characteristic of a favorable and spontaneous binding reaction. modified by addition of 7-MEOTA-THA thio-/ureas 12–22 to study

Positive values of both DS and DH are typical for hydrophobic the orientation mode and overall conformation changes produced

interaction, while negative values of both can be regarded as indi- due to the influence of conjugates. Representative CD spectra for

cating van der Waals forces and hydrogen bonds. However, posi- hybrids 12–22 can be found in Figure 8. The studied derivatives

tive values of DH and negative values of DS describe electrostatic are achiral and thus do not exhibit any CD signal of their own. The

7

9,80

interaction between ionic species in an aqueous solution

.

B-DNA conformation is characteristic with the CD spectrum in the

Hence, the obtained results indicate that binding between the region 220–300 nm. The positive band of ctDNA at approximately

studied 7-MEOTA-THA thio-/ureas 12–22 and ctDNA was spontan- 275 nm is caused by base stacking, whereas the negative band

83

eous due to negative DG values at all three temperatures, and the around 245 nm is indicative of the helicity of the B-DNA .

positive values of DH and DS showed that hydrophobic Incubation of 12–17 with ctDNA resulted in CD spectral shifts in

interactions play a central role in the binding of the studied both positive and negative CD bands. The positive band of ctDNA

8

88

J. JANOCKOVA ET AL.

Table 5. Various thermodynamic parameters for studied 7-MEOTA-THA hetero-

dimers 12–22 in complex with DNA at three different temperatures.

4

2

ꢀ _T_e_m_p_e_r_a_t_u_r_e₍_K₎_D_H₍_k_J_m_o_lꢃ1

ꢃ1

ctDNA

Compound

) DS (J K mol ) DG (kJ mol

ꢃ64.85

)

12

13

14

15

16

17

18

19

20

21

22

ꢀ

288

22.64

22.11

50.49

23.98

ꢃ30.59

21.56

9.43

146.75

154.01

250.77

162.50

ꢃ21.39

157.81

114.84

193.90

ꢃ125.09

104.68

259.46

ctDNA + 12

ctDNA + 13

ctDNA + 14

ctDNA + 15

ctDNA + 16

ctDNA + 17

2

98

ꢃ66.31

ꢃ68.07

ꢃ66.49

ꢃ68.03

ꢃ69.87

ꢃ122.75

ꢃ125.26

ꢃ128.27

ꢃ70.80

ꢃ72.43

ꢃ74.38

36.76

310

0

288

2

3

98

10

−2

−4

288

2

3

98

10

288

2

3

98

10

3

00

400

500

288

Wavelength [nm]

98

36.97

37.23

4

2

0

2

4

288

ꢃ67.03

ꢃ68.61

ꢃ70.50

ꢃ42.52

ꢃ43.67

ꢃ45.05

ꢃ87.59

ꢃ89.52

ꢃ91.85

93.65

ctDNA

2

3

98

10

ctDNA + 18

ctDNA + 19

ctDNA + 20

ctDNA + 21

ctDNA + 22

288

2

3

98

10

288

31.71

ꢃ57.61

6.88

2

3

98

10

−

288

2

98

94.91

96.41

310

288

ꢃ37.05

ꢃ38.10

ꢃ39.35

ꢃ128.83

ꢃ131.43

ꢃ134.54

2

3

288

2

3

98

10

300

400

500

Wavelength [nm]

54.07

98

10

Figure 8. CD spectrum of ctDNA (black line, 7.46 mM) in the absence and in the

presence of studied 7-MEOTA-THA heterodimers 12–22 (color lines, 0.3mM) in

0 mM Tris–HCl buffer (pH 7.4).

1

The concentration of studied compounds 12–22 was 5 mM, 23 was not tested

due to its low solubility.

the LD signal of DNA (at 258 nm) increases in magnitude upon

addition of each of the compounds 12–22 indicating either

at 280 nm showed a visible decrease of molar ellipticity and the

negative band at 246 nm exhibited an apparent increase of molar lengthening and/or an increase in rigidity of double-helical DNA,

7

1

ellipticity after modulation with thioureas 12–17. Kozurkova et al.

reported previously that proflavine compounds in complex with ation

which can usually be related to unwinding due to intercal-

8

5,86

. The observed changes upon addition of the hetero-

ctDNA resulted in changes in both CD bands, and in our obtained dimers to DNA were not clearly correlated with their chemical

results the decrease of peak ellipticity at 280 nm may be explained structures. However, neither the perpendicular orientation of tran-

as disruption of stacked bases due to possible intercalation of thio- sition moments of the compounds to the helix axis nor increased

urea analogs to ctDNA in order to optimize the binding interaction. rigidity of the DNA helix represents an unequivocal proof for DNA

8

4

Accordingly, the changes of peak ellipticity at 246 nm came about intercalation . Therefore, the data are interpreted also with

due to hydration binding of DNA which affects its helicity. When respect to the results of other experiments.

modulated by urea derivatives 18–22 under the same conditions,

no significant changes in the subset were recorded within ctDNA,

which indicates that the nature of binding of the urea hybrids to

ctDNA is non-intercalative. However, these small visible changes at

both bands may be attributed to certain conformational changes.

Experimental

Materials

LD is defined as LD ¼ A//ꢃA

? ?

, where A//and A are absorbance

All chemicals and reagents purchased were of reagent grade

and used without further purification. The applied material and

cell lines are listed in Supplementary data (see “Experimental,

Materials” section).

values measured with the light plane polarized parallel and per-

pendicular, respectively, to the oriented ctDNA in this study. The

orientation of measured ctDNA in the absence and in the pres-

ence of the studied 7-MEOTA-THA thio-/ureas 12–22 was obtained

by flow using a cuvette cell. Representative spectra are shown in

Figure 9. The appearances of the LD spectra for all complexes of

ctDNA with the studied compounds were almost identical, charac-

terized by a negative signal in both the DNA absorption region

Methods

DNA and tested sample solutions

CtDNA was dissolved in TE buffer (Tris–EDTA) overnight at 4 C to

ꢁ

(

220–300 nm) and in the Soret region (320–450 nm). The existence

of LD signals in these regions proves that 12–22 interact with completely solubilize. The concentration of ctDNA was determined

DNA and become oriented after anchoring to DNA. The negative from its absorbance at 260 nm using extinction coefficient

ꢃ1

values of these LD signals are consistent with perpendicular orien- e ¼ 6600 M cm . The purity of ctDNA was identified by moni-

tations of the chromophoric molecules 12–22 relative to the DNA toring the value A260/A280. Stock solutions of tested 7-MEOTA-THA

84

helix axis as expected for intercalative binding mode . Moreover, thio-/ureas 12–22 were prepared in DMSO (stock concentration of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

889

0,02

17

0

0,0004

0

−

0,0004

0,0008

0,0012

0,0016

−

0,02

0,04

−

0,002

300

400

500

Wavelength [nm]

200

300

Wavelength [nm]

400

500

0,02

22

0

0,0004

0

−

0,0004

0,0008

0,0012

0,0016

−

0,02

−

0,002

0,04

300

400

500

Wavelength [nm]

2

00

300

Wavelength [nm]

Figure 9. LD spectrum of ctDNA (black line, 310 mM) in the absence and in the presence of studied 7-MEOTA-THA thio-/ureas 17 and 22 (color lines, 0–0.1 mM) in

400

500

1

0 mM Tris–HCl buffer (pH 7.4).

samples was 30 mM). Further dilutions were prepared in the GmbH, Bergisch Gladbach, Germany) for 10 min, washed with

appropriate aqueous buffer.

1–2 mL of PBS and centrifuged at 3000g for 10 min. The resus-

pended cell pellet was analyzed in Becton Dickinson

a

FACSCalibur using CellQuest software (Becton Dickinson).

Cell cycle distribution. HL-60 cells were treated with various con-

centrations of the studied seven MEOTA-THA thio-/ureas (12–17:

Flow cytometry analysis

For the flow cytometry analysis, HL-60 cells were seeded in six-

6

well plates (TPP, Trasadingen, Switzerland) at 1 ꢂ 10 per well and

5

–25 mM; 18–22: 1–15 mM) for 24, 48 and 72 h. Untreated cells

changes in the total cell number, metabolic activity, MMP, cell

cycle distribution and cell death were analyzed at 24, 48 and 72 h

after treatment with the studied 7-MEOTA-THA thio-/ureas 12–22,

were also included in this test for comparison. The method used

8

9

to perform the experiment has been published previously .

ModFit 3.0 (Verity Software House, Topsham, ME) software was

used to generate DNA content frequency histograms and to quan-

87,88

4

as described previously

. Human dermal fibroblasts (5 ꢂ 10 )

were seeded into a 12-well plate (Sarstedt, Deutschland) and tify the number of cells in the individual cell cycle phases.

treated with the studied 7-MEOTA-THA thio-/ureas 12–22 for 72 h,

Analysis of MMP (DW ). For detection of DW , HL-60 cells/

m m

and changes in MMP were analyzed.

fibroblasts were treated with various concentrations of the studied

Immunophenotype characterization of human fibroblasts. 7-MEOTA-THA thio-/ureas (HL-60: 12–17: 5–25 mM, 18–22:

Isolated dermal fibroblasts were characterized using flow cytome- 1–15 mM; fibroblasts: 12–17: 15 and 25 mM, 18–22: 2.5 and 5 mM)

try. After dissociation with trypsin–EDTA (Gibco, Waltham, MA), for 24, 48 and 72 h (for fibroblasts only 72 h). The method used to

the cells were washed twice with PBS (Gibco, Life Technologies, perform the experiment has been published according to a previ-

8

7,89

Carlsbad, CA) supplemented with 2% (v/v) FBS. Aliquots of ously described protocol

.

5

2

.0 ꢂ 10 cells were incubated with mouse anti-human CD90/FITC,

Analysis of metabolic activity and viability. Analysis of metabolic

CD105/PE, CD73/APC, CD26/PE, and with a cocktail of hematopoi- activity and viability of HL-60 cells was performed using fluores-

etic markers: CD14-/CD20-/CD34-/CD45/PerCP (Miltenyi Biotec cein diacetate (FDA; BD Pharmingen, San Diego, CA) and PI

8

90

J. JANOCKOVA ET AL.

double-staining. HL-60 cells were treated with various concentra- Topoisomerase II-mediated DNA cleavage assay

tions of the studied 7-MEOTA-THA thio-/ureas (12–17: 5–25 mM; DNA cleavage reactions were carried out in a 20 mL final volume

1

8–22: 1–15 mM) for 24, 48 and 72 h and subsequently harvested containing freshly prepared 5ꢂ complete assay buffer, 0.2 mg

by centrifugation, washed with HBSS and stained with FDA supercoiled pHOT-1 and 5U human Topo IIa. Reactions were incu-

(

ꢃ1

100 ng mL ) in HBSS buffer for 20 min in darkness at RT. Prior to bated in the absence or in the presence of the studied 7-MEOTA-

ꢃ3

ꢁ

measurements, cells were stained with PI (1 mg cm ) and ana- THA thio-/ureas (12–22: 10 and 100 mM) at 37 C for 30 min. The

lyzed using a BD FACSCalibur flow cytometer. Fluorescence of method used to perform the experiment has been published

2

9,90

FDA was detected via a 530/30 nm band pass filter (FL-1) and PI previously

via a 670 nm long-pass filter (FL-3). Results were analyzed using

FlowJo software (TreeStar Inc.).

.

UV–vis absorption spectroscopy and determination of bind-

ing constants

Statistical analysis

UV–vis spectroscopic analyses were realized in 10 mM Tris–HCl

Results were presented as the mean ± SD (standard deviation) of buffer (pH 7.4; RT). UV–vis absorption titration spectra were meas-

at least three independent experiments. Statistical significance ured on a Varian Cary 100 spectrophotometer in a 100-QS quartz

was determined by Student’s t-test and results were considered cuvette with optic length 1 cm. UV–Vis spectra of newly devel-

ꢀ

^ꢀ^ꢀand p<.001^ꢀ^ꢀ^ꢀ.

significant if p<.05 , p<.01

oped 7-MEOTA-THA thioureas (12–17: 49 mM) and ureas (18–20:

9 mM; 21, 22: 49 mM) in the absence or in the presence of an

9

increasing concentration of ctDNA (0–1.24 mM) were recorded in

the wavelength range 230–450 nm.

Intracellular accumulation

3

A549 cells (6 ꢂ 10 per well) were seeded onto microscopy glass

slides with mounted 12-well silicone chamber (Ibidi GmbH,

Planegg, Germany) and left to settle for 24 h. They were then

treated with the studied 7-MEOTA-THA thio-/ureas 12–22 (5 mM)

The absorbance A measured at any wavelength indicates both

the free (Af) and DNA-bound (A

b

) types:

A ¼ Af þ Ab ¼ ef: Cf þ eb: Cb

(1)

or with the medium alone (control group) for 24 h and washed where C is the constant compound concentration (C ¼ C þC )

f

b

with pre-warmed PBS in order to remove the unbound fraction. and

e

f

and

e

b

are the appropriate extinction coefficients

Intracellular accumulation of studied heterodimers in A549 cells (Equation (1)). For determination of C and r was used the equa-

f

was evaluated by fluorescence microscope Leica DMI6000B (Leica tion (Equation (2)):

Microsystems GmbH, Wetzlar, Germany) using two channels as

Cb

1ꢃCf

AfꢃA

stated in Supplementary Table S1 . Every sample was captured

using the same settings for HCX PL APO CS 10.0 ꢂ 0.40 DRY UV

objective. Results were analyzed using Leica Application Suite Lite

a ¼ _C

¼

(2)

C

Af ꢃ Ab

where a is the fraction of binding compound, and A

f

and A

b

are

the absorptions for free and fully bound compound at the check-

ing wavelength. Subsequently, r ¼ aC/C and C ¼ (1–a)C, where

(

LAS AF Lite) software. Each presented image represents a single

sample and consists of separated images from a channel and their

mutual overlap.

DNA

f

9

1

C

DNA is the total DNA concentration . Data from spectroscopic

4

titration were used for determination of binding constants K of

the derivative-ctDNA complexes using a Scatchard plot in the

Fibroblasts (4 ꢂ 10 per well) were seeded into a 12-well plate,

left to settle for 24 h and treated with the studied 7-MEOTA-THA

thio-/ureas 12–22 (12–17: 15 and 25 mM, 18–22: 2.5 and 5 mM)

or with the medium alone (control group). After 24 h of incuba-

tion with the studied derivatives, the cells were analyzed with

an inverted fluorescence microscope Leica DMi3000B (Leica

Microsystems).

form r/C (where r is the number of ligand molecules which bind

f

to one mol of nucleotide as a function of added titrant, and C is

f

the molar concentration of free ligand), the McGhee and von

9

2

Hippel equation (Equation (3) , and then using a fitting function

incorporated into graphic software GnuOctave 2.1.73:

ꢀ

ꢁ_n_ꢃ₁

r

1ꢃnr

¼

Kð1 ꢃ nrÞ

(3)

Cf

1 ꢃ ðnꢃ1Þr

Topoisomerase I-mediated supercoiled pUC19 relaxation

The influence of the studied 7-MEOTA-THA thio-/urea hybrids on

In this equation, n is the number of binding sites for binding

the catalytic activity of Topo I was identified according to a previ- of one ligand molecule to DNA, r is the molar ratio of the bound

2

9,90

ously described protocol

. Reaction mixtures were incubated ligand to the polynucleotide and C is the molar concentration of

f

ꢁ

for 45 min at 37 C in the absence or in the presence of the free ligand.

studied 7-MEOTA-THA thio-/urea derivatives 12–22 (1–60 mM).

The gel was photographed under UV and gel images were

obtained using a photogel documentation system (Syngene,

Cambridge, UK).

^Tm measurements

DNA melting temperature measurements were performed in a

quartz cuvette (1 cm path length) on a Varian Cary 100 spectro-

photometer equipped with a heating multiple cell block appar-

atus. Measurements were realized in BPE buffer (pH 7.1; 1 mM

Influence of derivatives on topoisomerase II decatenation

Freshly prepared 5ꢂ complete assay buffer for Topo II decatena- EDTA, 2 mM NaH

2

PO

4

, 6 mM Na

2

HPO

4

). The temperature was

ꢁ

ꢃ1

ꢁ

tion containing 0.16 mg catenated kinetoplast DNA (kDNA) and 2 U increased at a rate of 1 C min over the range 40–90 C. The

of human Topo IIa in the absence or presence of the studied 7- absorbance was measured at 260 nm for ctDNA alone (concentra-

MEOTA-THA thio-/ureas (12–22: 5, 50 and 100 mM) was incubated tion of ctDNA in measurements with 12–17: 93.2 mM and with

ꢁ

for 45 min at 37 C. The method used to perform the experiment 18–22: 0.32 mM) and for ctDNA in complex with the studied deriv-

29,90

has been published previously

.

atives (concentration of 12–17: 37.5 mM and 18–22: 50 mM). The

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

891

ꢁ

m

thermal melting points (T , C) were determined as the maxima Circular and LD spectroscopy

of the first derivative plots of the melting curves.

Spectral measurements of CD were realized using a J-810 Jasco

spectropolarimeter in a quartz cuvette with optic length 1 mm. CD

spectra of ctDNA (7.46 mM) in the absence or in the presence of

the newly developed 7-MEOTA-THA thio-/urea hybrids (12–22:

Fluorescence spectroscopy

Fluorescence spectra of the studied 7-MEOTA-THA thio-/ureas

were recorded in a quartz cuvette (1 cm path length) on a

0

.3 mM) were recorded in 10 mM Tris–HCl buffer (pH 7.4; RT) in

the wavelength range of 200–500 nm. The results are presented as

Varian Cary Eclipse spectrofluorimeter. The widths of both the the mean of at least three repeated measurements and obtained

excitation and emission slit were set at 10 nm. The excitation data were transferred to Grafit 7.0 (Erithacus Software, West

wavelengths were 340 nm for compounds 13–21 and 335 nm for Sussex, UK) for analysis.

compounds 12 and 22. The emission spectra were recorded in

Flow LD spectra were measured using a Jasco J-720 spectropo-

the range 350–550 nm with maximum observed at 376–396 nm. larimeter in a flow Couette cell adapted for LD measurements. The

Fluorescence titrations were realized in 10 mM Tris–HCl buffer flow cell includes a fixed outer cylinder and a solid rotating quartz

(

pH 7.4; RT) containing a constant concentration of the studied inner cylinder. These two cylinders are separated with a 0.5 mm

derivative (12–22: 5 mM), and to which were added equivalent gap (total path length was 1 mm). LD spectra of ctDNA (310 mM)

in the absence or in the presence of the newly synthesized 7-

MEOTA-THA thio-/urea derivatives (12–22: 0–0.1 mM) were

recorded in 10 mM Tris–HCl buffer (pH 7.4; RT) in the wavelength

range of 200–500 nm. The baseline (10 mM Tris–HCl) was also

recorded for each measurement. The results are presented as the

mean of at least three independent measurements and obtained

data were transferred to Grafit 7.0 (Erithacus Software) for analysis.

increased amounts of ctDNA (0–280 mM). The steady state fluor-

escence measurements were performed at three different tem-

peratures (288, 298 and 310 K) for the evaluation of various

thermodynamic parameters important in the detection of com-

plex formation between DNA and 7-MEOTA-THA heterodimers.

The obtained data were used for determination of

Stern–Volmer quenching constants (K_S_V) by applying the equation

7

3

(Equation (4) :

Chemistry

F0

F

¼

1 þ KSV½Qꢄ

(4)

7

-MEOTA was prepared at the University of Defense (Faculty of

Military Health Sciences, Hradec Kralove, Czech Republic) by the

0

where F is the fluorescence intensity of the studied compound

3

9,97,98

previously described method

. Other reagents were obtained

alone, F is the fluorescence intensity of the studied 7-MEOTA-THA

thio-/urea in the presence of ctDNA as a quencher and [Q] is the

molar concentration of ctDNA. To further confirm the quenching

from Sigma-Aldrich (Prague, Czech Republic) in reagent grade

quality. All experiments were carried out under nitrogen atmos-

phere. Thin-layer chromatography (TLC) was performed on alumi-

num sheets with pre-coated silica gel 60 F254 (Merck, Czech

Republic). Column chromatography was performed at normal

pressure on silica gel 100 (particle size 0.063–0.200 mm,

70–230 mesh ASTM; Fluka, Bucharest, Romania). Elemental analysis

was measured on a Perkin-Elmer CHN Analyzer 2400 Series II

apparatus (PerkinElmer, Waltham, MA). Mass spectra were

recorded using combined high-performance liquid chromatog-

raphy (HPLC) and mass spectrometry (MS). The HP1100 HPLC sys-

tem was obtained from Agilent Technologies (Waldbronn,

Germany). It consisted of vacuum degasser G1322A, quaternary

pump G1311A, autosampler G1313A and quadrupole mass spec-

(6) trometer MSD1456 VL equipped with electrospray ionization

process were calculated biomolecular quenching constants k

using the equation (Equation (5)):

q

KSV

kq ¼

(5)

s0

where s₀is the fluorescence lifetime of the biomolecule in the

ꢃ8 93

absence of quencher, which is around 10 s .

9

4

The modified Stern–Volmer equation (Equation (6) was used

to determine binding constants (K ) of the forming complex

b

between 7-MEOTA-THA thio-/urea hybrids and ctDNA:

F0ꢃF

log

¼ logKb þ n log½Qꢄ

F

source. Nitrogen for MS was supplied by a Whatman 75–720 nitro-

where n is the number of binding sites. K and n were calculated gen generator. Data were collected in positive ion mode with an

b

at three different temperatures from double logarithm regression ESI probe voltage of 4000 V. The pressure of nebulizer gas was

ꢁ

curves of log(F ꢃF)/F versus log[Q] and they were obtained from set to 35 psig. Drying gas temperature was operated at 335 C

0

1

13

intercept and slope, respectively.

and flow at 13 L/min. H NMR and C NMR spectra were recorded

The van’t Hoff equation (Equation (7)) was used to calculate on a Varian Mercury Plus 400 spectrometer operating at 400 and

thermodynamic parameters, namely change of enthalpy (DH), 100 MHz, respectively, in deuterochloroform [CDCl ; 7.27 (D), 77.2

(C) ppm] or hexadeuterodimethylsulfoxide [DMSO-d ; 2.50 (D),

9.7 (C) ppm] using tetramethylsilane (TMS) as internal reference

¼0 ppm for both nuclei). Chemical shifts are reported in parts per

million (ppm, d) relative to TMS. The assignment of chemical shifts

3

9

5

change of entropy (DS) and change of free energy (DG) :

DH DS

:303 RT 2:303 R

where T is the temperature in kelvin and R is the universal gas

6

3

(

logKb ¼ ꢃ

þ

(7)

2

1

13

1

is based on standard NMR experiments ( H, C, Hꢃ H COSY,

1

13

ꢃ1

Hꢃ C HSQC, HMBC, DEPT). Uncalibrated purity was ascertained

constant equal to 8.314 kJ K

mined as the slope and intercept respectively of the van’t Hoff

plot (log K against 1/T). The DG values at three different concen-

mol . DH and DS were deter-

by LC ꢃ UV using a reverse phase C18 chromatographic column.

All of the biologically tested compounds exhibited purity

96 ꢃ 99% at wavelength 254 nm. Melting points were measured

on a micro heating stage PHMK 05 (VEB Kombinat Nagema,

(8) Germany) and were uncorrected.

b

96

trations were calculated using equation (Equation (8)) :

DG ¼ DH ꢃ TDS

.

8

92

J. JANOCKOVA ET AL.

General procedure for synthesis of novel 7-MEOTA-THA hetero- (C¼S); ESI-MS: m/z 540.2 [M]^þ(calculated for: [C32H38N5OS]

þ

dimers 12–23

540.3); Anal. Calcd. for C32H37N5OS: C, 71.21; H, 6.91; N, 12.98; S,

Intermediates 3–8 (1.7 mmol) and 11 (0.5 g, 2.1 mmol) were added 5.94. Found: C, 71.19; H, 6.55; N, 13.26; S, 5.80.

to 20 ml of dichloromethane and stirred for 48 h at room tempera-

1-(5-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)pentyl)-3-

ture (RT). The mixture was concentrated under reduced pressure (1,2,3,4-tetrahydroacridin-9-yl)thiourea 15. Yellow solid (0.78 g,

ꢁ

1

0

to give the crude product. Purification was performed by flash 81%): m.p.¼93.2–94.1 C; H NMR (CDCl

) d 1.43 (m, 2H, CH

, H-2 ,4 ), 1.83 (m, 8H, 4 ꢂ CH

, H-3 ),

3

2

0

chromatography (eluent CHCl /MeOH, 9:1) to give 12–17 as a yel- 1.59 and 1.71 (m, 4H, 2 ꢂ CH

2

, H-

3

0

00

00 00

, H-4,1 ,4 ),

2

low solid. In a further step, the appropriate 7-MEOTA-THA product 2,3,2 ,3 ), 2.63 (m, 2H, CH

, H-1), 2.95 (m, 6H, 3 ꢂ CH

2

0

containing the thiourea moiety (12–17, 1.1 mmol) was stirred in 3.57 (m, 4H, 2 ꢂ CH

2

, H-1 ,5 ), 3.87 (s, 3H, OCH3), 7.18 (dd, 1H, CH,

0

dry dichloromethane (25 ml) with 2,4,6-trimethylbenzonitrile N- H-6, J ¼ 9.2, 2.4 Hz), 7.27 (m, 1H, CH, H-8), 7.33 (m, 1H, CH, H-7 ),

oxide (0.2 g, 1.2 mmol) for 48 h. The resulting mixture was subse- 7.54 (m, 1H, CH, H-6 ), 7.82 (m, 2H, 2 ꢂ CH, H-5,8 ), 7.90 (d, 1H,

) d 21.8, 22.2, 22.5, (C-

0

00

0

quently evaporated under reduced pressure and purified by flash CH, H-5 , J ¼ 8.4 Hz); 13C NMR (CDCl

3

0

00

0

00

0

chromatography using CHCl

as white solids.

3

/MeOH (9:1) as eluent to yield 18–23 2,3,2 ,3 ), 24.0 (C-3 ), 24.6 and 25.3 (C-1,1 ), 28.6 and 30.9 (C-2 ,4 ),

0

3

31.4 and 33.8 (C-4,4 ), 44.8 and 48.3 (C-1 ,5 ), 55.7 (OCH ), 102.3

-(2-(7-Methoxy-1,2,3,4-tetrahydroacridin-9ylamino)ethyl)-3-(1,2,3,4- (C-8), 117.0 (C-9a), 119.6 (C-8a), 121.6 (C-6), 122.6 (C-8 ), 124.3 (C-

tetrahydroacridin-9-yl)thiourea 12. Yellow solid (0.61 g, 69%): 8a ), 126.4 (C-7 ), 128.6 and 128.7 (C-5,5 ), 129.2 (C-6 ,9a ), 139.8

0

1

0

00

ꢁ

1

00

m.p.¼142.1–143.2 C; H NMR (CDCl ) d 1.83 (m, 8H, 4 ꢂ CH , H- (C-9 ), 143.5 (C-10a), 147.1 (C-10a ), 151.8 (C-9), 156.3 (C-7,4a),

3

2

00

þ

0

00

2

4

2

,3,2 ,3 ), 2.63 and 2.88 (m, 4H, 2 ꢂ CH , H-1,1 ), 2.95 and 3.05 (m, 160.4 (C-4a ), 181.5 (C ¼ S); ESI-MS: m/z 554.3 [M] (calculated for:

2

þ

0

H, 2 ꢂ CH , H-4,4 ), 3.87 (s, 3H, OCH3), 3.60 and 3.66 (m, 4H, [C33H40N5OS] 554.3); Anal. Calcd. for C33H39N5OS: C, 71.57; H,

2

0

00

ꢂ CH

, H-1 ,2 ), 7.33 (t, 1H, CH, H-7 , J ¼ 7.6 Hz), 7.20 (dd, 1H, CH, 7.10; N, 12.65; S, 5.79. Found: C, 71.23; H, 6.95; N, 12.88; S, 5.93.

2

H-6, J ¼ 9.2, 2.4 Hz), 7.27 (d, 1H, CH, H-8, J ¼ 2.8 Hz), 7.56 (d, 1H, CH,

1-(6-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)hexyl)-3-

0

00

H-6 , J ¼ 7.6 Hz), 7.74 (m, 1H, CH, H-8 ), 7.82 (d, 1H, CH, H-5, (1,2,3,4-tetrahydroacridin-9-yl)thiourea 16. Yellow solid (0.92 g,

1

0

ꢁ

J ¼ 9.2 Hz), 7.90 (d, 1H, CH, H-5 , J ¼ 8.8 Hz), 6.80 (bs, 1H, NH); 93%): m.p.¼74.2–74.8 C; H NMR (CDCl

3

) d 1.25 and 1.36 (m, 4H,

1

3

00 00

0

C NMR (CDCl ) d 21.8, 22.0, 22.2, 22.4 (C-2,3,2 ,3 ), 24.6 and 25.3 2 ꢂ CH

, H-3 ,4 ), 1.50 and 1.61 (m, 4H, 2 ꢂ CH

, H-2 ,5 ), 1.89 (m,

3

2

0

00

0

00 00

00

(

C-1,1 ), 32.2 and 33.6 (C-4,4 ), 42.5 and 46.8 (C-1 ,2 ), 55.7 (OCH3), 8H, 4 ꢂ CH

2

, H-2,3,2 ,3 ), 2.65 and 2.88 (m, 4H, 2 ꢂ CH

2

, H-1,1 ),

0

00

1

6

01.9 (C-8), 116.2 (C-9a), 119.6 (C-8a), 121.6 (C-6), 122.6 (C-8 ), 2.97 and 3.07 (m, 4H, 2 ꢂ CH2, H-4,4 ), 3.44 and 3.58 (m, 4H,

0

00

0

24.2 (C-8a ), 126.5 (C-7 ), 128.8 (C-5,5 ), 129.1 (C-9a ), 129.3 (C- 2 ꢂ CH

2

, H-1 ,6 ), 3.88 (s, 3H, OCH

3

), 7.20 (m, 1H, CH, H-6), 7.27 (m,

0

00

), 139.8 (C-9 ), 143.2 (C-10a), 147.2 (C-10a ), 150.2 (C-9), 156.2 1H, CH, H-8), 7.41 (dd, 1H, CH, H-7 , J ¼ 8.4, 6.8 Hz), 7.59 (dd, 1H,

0

þ

00

(

C-7,4a), 160.5 (C-4a ), 181.5 (C ¼ S); ESI-MS: m/z 512.2 [M] (calcu- CH, H-6 , J ¼ 8.4, 6.8 Hz), 7.82 (m, 2H, 2 ꢂ CH, H-5,8 ), 7.94 (d, 1H,

þ

0

13

lated for: [C30H34N5OS] 512.2); Anal. Calcd. for C30H33N5OS: C, CH, H-5 , J ¼ 8.4 Hz); C NMR (CDCl

3

) d 22.2, 22.3, 22.5, 22.7 (C-

0

00

0

7

0.42; H, 6.50; N, 13.69; S, 6.27. Found: C, 70.37; H, 6.81; N, 13.78; 2,3,2 ,3 ), 24.6 and 25.3 (C-1,1 ), 26.3 and 26.4 (C-3 ,4 ), 28.8 and

0

00

0

S, 6.05.

31.4 (C-2 ,5 ), 32.6 and 33.8 (C-4,4 ), 45.1 and 48.6 (C-1 ,6 ), 55.5

1

-(3-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)propyl)-3-(1,2,

(OCH ), 102.0 (C-8), 116.1 (C-9a), 120.4 (C-8a), 120.8 (C-6), 122.5 (C-

,4-tetrahydroacridin-9-yl)thiourea 13. Yellow solid (0.71 g, 78%): 8 ),124.2 (C-8a ), 126.5 (C-7 ), 128.3 (C-5), 128.7 (C-5 ), 129.0 (C-

3

0

00

3

ꢁ

1

00

m.p.¼106.3–107.5 C; H NMR (CDCl ) d 1.81 (m, 10H, 5 ꢂ CH , H- 9a ), 129.3 (C-6 ), 138.2 (C-9 ), 142.0 (C-10a), 147.3 (C-10a ), 150.7

3

2

0

00 00

00

2

3

1

(

2

3

(

1

(

,3,3 ,2 ,3 ), 2.50 and 2.70 (m, 4H, 2 ꢂ CH , H-1), 2.94 (m, 4H, (C-9), 156.0 (C-7), 154.8 (C-4a), 160.5 (C-4a ), 181.2 (C ¼ S); ESI-MS:

2

0

þ

ꢂ CH , H-4,4 ), 3.24 and 3.48 (m, 4H, 2 ꢂ CH , H-1 ,3 ), 3.85 (m, m/z 568.3 [M] (calculated for: [C34H42N5OS] 554.3); Anal. Calcd.

2

H, OCH

3

), 5.65 (bs, 1H, NH), 7.17 (m, 2H, 2 ꢂ CH, H-6,8), 7.33 (m, for C33H39N5OS: C, 71.57; H, 7.10; N, 12.65; S, 5.79. Found: C,

0

00

H, CH, H-7 ), 7.51 (m, 1H, CH, H-6 ), 7.81 (m, 1H, CH, H-8 ), 7.83 71.23; H, 6.95; N, 12.88; S, 5.93.

0

13

m, 1H, CH, H-5), 7.94 (m, 1H, CH, H-5 ); C NMR (CDCl

3

) d 22.0,

1-(7-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)heptyl)-3-

2.2, 22.4, 22.6 (C-2,3,2 ,3 ), 24.8 and 25.3 (C-1,1 ), 30.6 (C-2 ), (1,2,3,4-tetrahydroacridin-9-yl)thiourea 17. Yellow solid (0.86 g,

0

00

0

ꢁ

1

3.6 (C-4,4 ), 43.3 and 42.3 (C-1 ,3 ), 55.7 (OCH3), 101.5 (C-8), 116.4 85%): m.p.¼80.8–81.9 C; H NMR (CDCl

) d 1.32 (m, 6H, 3 ꢂ CH

,

3

2

0

00

0

C-9a), 120.6 (C-8a), 121.6 (C-6), 122.6 (C-8 ), 124.4 (C-8a ), 126.3 H-3 ,4 ,5 ), 1.43 and 1.63 (m, 4H, 2 ꢂ CH

2

, H-2 ,6 ), 1.85 (m, 8H,

0

00

00 00

00

C-7 ), 128.5 (C-5,5 ), 129.1 (C-6 ,9a ), 139.0 (C-9 ), 143.0 (C-10a), 4 ꢂ CH

, H-2,3,2 ,3 ), 2.64 and 2.90 (m, 4H, 2 ꢂ CH

, H-1,1 ), 2.97

2

0

00

0 0

47.0 (C-10a ), 150.5 (C-9), 156.3 (C-7,4a), 160.3 (C-4a ), 181.5 and 3.10 (m, 4H, 2 ꢂ CH

2

, H-4,4 ), 3.52 (m, 4H, 2 ꢂ CH

2

, H-1 ,7 ),

þ

C ¼ S); ESI-MS: m/z 526.2 [M] (calculated for: [C31H36N5OS]

3.88 (s, 3H, OCH

3

), 7.20 (dd, 1H, CH, H-6, J ¼ 8.8, 2.0 Hz), 7.27 (m,

0

00

5

6

26.3); Anal. Calcd. for C31H35N5OS: C, 70.82; H, 6.71; N, 13.32; S, 1H, CH, H-8), 7.40 (m, 1H, CH, H-7 ), 7.58 (t, 1H, CH, H-6 ,

0

.10. Found: C, 70.65; H, 6.67; N, 13.45; S, 6.01.

-(4-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)butyl)-3-(1,2,3,4- J ¼ 8.4 Hz); C NMR (CDCl

tetrahydroacridin-9-yl)thiourea 14. Yellow solid (0.54 g, 58%): 24.5 and 25.3 (C-1,1 ), 26.5 (C-4 ), 28.6 and 28.7 (C-3 ,5 ), 30.3 and

J ¼ 7.6 Hz), 7.84 (m, 2H, 2 ꢂ CH, H-5,8 ), 7.94 (d, 1H, CH, H-5 ,

1

3

00 00

) d 22.0, 22.2, 22.3, 22.6 (C-2,3,2 ,3 ),

3

1

0

ꢁ

1

0 0

0 0 00 0 0

, H-2 ,3 ), 31.3 (C-2 ,6 ), 31.7 and 33.9 (C-4,4 ), 45.2 and 48.6 (C-1 ,7 ), 55.6

2

m.p.¼95.6–96.4 C; H NMR (CDCl

3

) d 1.64 (m, 4H, 2ꢂ CH

0

00

1

2

.85 (m, 8H, 4ꢂ CH

2

, H-2,3,2 ,3 ), 2.64 and 2.87 (m, 4H, 2ꢂ CH

2

, H- (OCH3), 102.3 (C-8), 115.5 (C-9a), 120.0 (C-8a), 121.4 (C-6), 122.6

0

00

,1 ), 2.98 and 3.04 (m, 4H, 2 ꢂ CH , H-4, 4 ), 3.43 and 3.64 (m, 4H, (C-8 ), 124.8 (C-8a ), 126.4 (C-7 ), 128.7 (C-5,5 ), 129.2 (C-6 ,9a ),

2

0

00

ꢂ CH , H-1 ,4 ), 3.87 (s, 3H, OCH ), 5.46 (bs, 1H, NH), 7.17 (d, 1H, 139.7 (C-9 ), 145.2 (C-10a), 147.3 (C-10a ), 151.3 (C-9), 156.2 (C-

2

3

0

þ

CH, H-8, J ¼ 2.8 Hz), 7.20 (dd, 1H, CH, H-6, J ¼ 8.8, 2.8 Hz), 7.40 (t, 7,4a), 160.5 (C-4a ), 181.3 (C ¼ S); ESI-MS: m/z 582.3 [M] (calcu-

0

00

þ

1

H, CH, H-7 , J ¼ 7.6Hz), 7.60 (t, 1H, CH, H-6 , J ¼ 7.6 Hz), 7.81 (m, lated for: [C35H44N5OS] 582.3); Anal. Calcd. for C35H43N5OS: C,

H, CH, H-8 ), 7.83 (m, 1H, CH, H-5), 7.94 (d, 1H, CH, H-5 , 72.25; H, 7.45; N, 12.04; S, 5.51. Found: C, 72.43; H, 7.58; N, 12.12;

0

3

0

00

00 00

1

J ¼ 8.8 Hz); C NMR (CDCl3) d 22.2, 22.4, 22.6, 22.8 (C-2,3,2 ,3 ), S, 5.43.

0

2

4

1

4.7 and 25.3 (C-1,1 ), 26.5 and 28.6 (C-2 ,3 ), 32.8 and 33.8 (C-4,

1-(2-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)ethyl)-3-

0

), 44.8 and 48.1 (C-1 ,4 ), 55.6 (OCH3), 101.7 (C-8), 117.0 (C-9a), (1,2,3,4-tetrahydroacridin-9-yl)urea 18. White solid (0.53 g, 95%):

0

00

ꢁ

1

20.7 (C-8a), 120.9 (C-6), 122.5 (C-8 ), 124.1 (C-8a ), 126.7 (C-7 ), m.p.¼103.7–104.8 C; H NMR (CDCl

3

) d1.70 (m, 8H, 4 ꢂ CH

2

, H-

0

00

00 00

00

28.8 (C-5,5 ), 129.1 (C-9a ), 129.4 (C-6 ), 139.0 (C-9 ), 142.5 (C- 2,3,2 ,3 ), 2.66 and 2.81 (m, 4H, 2 ꢂ CH

, H-1,1 ), 3.0 (m, 4H,

2

0

00

0 0

0a), 147.3 (C-10a ), 150.3 (C-9), 156.2 (C-7,4a), 160.6 (C-4a ), 181.4 2 ꢂ CH2, H-4,4 ), 3.66 and 3.93 (m, 4H, 2 ꢂ CH

2

, H-1 ,2 ), 3.85 (s,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

893

0

þ

3

1

H, OCH

3

), 7.20 (m, 1H, CH, H-6), 7.26 (m, 1H, CH, H-7 ), 7.31 (m, [M] (calculated for: [C33H40N5O2] 538.3); Anal. Calcd for

0

H, CH, H-8), 7.42 (t, 1H, CH, H-6 , J ¼ 7.6 Hz), 7.83 (m, 2H, 2 ꢂ CH, C33H39N5O2: C, 73.71; H, 7.31; N, 13.02. Found: C, 73.39; H, 7.35;

0

00

H-5 ,8 ), 7.91 (bs, 1H, NH), 8.0 (d, 1H, CH, H-5, J ¼ 8.8 Hz), 9.05 (bs, N, 13.33.

1

3

00 00

1

(

1

H, NH); C NMR (CDCl

3

) d 22.2, 22.4, 22.5, 22.6 (C-2,3,2 ,3 ), 25.4

1-(6-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)hexyl)-3-

0

00

0

C-1,1 ), 28.4 and 33.8 (C-4,4 ), 40.1 and 51.5 (C-1 ,2 ), 55.2 (OCH ), (1,2,3,4-tetrahydroacridin-9-yl)urea 22. White solid (0.58 g, 93%):

3

0

ꢁ

1

0

02.1 (C-8), 116.5 (C-9a), 119.8 (C-8a), 121.1 (C-6), 123.2 (C-8 ), m.p.¼91.4–92.1 C; H NMR (CDCl

3

) d 1.32 (m, 4H, 2 ꢂ CH

2

, H-3 ,4 ),

0

00

0

00 00

25.3 (C-7 ), 124.5 (C-8a ), 127.2 (C-5), 128.0 (C-5 ), 128.3 (C-6 ), 1.57 (m, 4H, 2 ꢂ CH

2

, H-2 ,5 ), 1.80 (m, 8H, 4 ꢂ CH

2

, H-2,3,2 ,3 ),

0

00

28.7 (C-9a ), 139.9 (C-9 ), 146.6 (C-10a,10a ), 150.0 (C-9), 155.4 2.57 and 2.77 (m, 4H, 2 ꢂ CH

2

, H-1,1 ), 2.90 and 2.97 (m, 4H,

0

00

0 0

, H-1 ,6 ), 3.85 (s, 3H,

2

(

[

C-4a), 156.3 (C-7), 157.1 (C¼O), 159.5 (C-4a ); ESI-MS: m/z 496.2 2 ꢂ CH

2

, H-4,4 ), 3.10 and 3.40 (m, 4H, 2 ꢂ CH

), 4.52 (bs, 1H, NH), 5.82 (bs, 1H, NH), 7.15 (dd, 1H, CH, H-6,

3

þ

M] (calculated for: [C30H34N5O2] 496.3); Anal. Calcd. for OCH

0

00

C30H33N5O2: C, 72.70; H, 6.71; N, 14.13. Found: C, 72.43; H, 6.35; J ¼ 9.2, 2.8 Hz), 7.23 (m, 2H, 2 ꢂ CH, H-8,7 ), 7.43 (dd, 1H, CH, H-6 ,

N, 13.69.

J ¼ 8.4, 7.2 Hz), 7.68 (bs, 1H, NH), 7.74 (d, 1H, CH, H-5, J ¼ 9.2 Hz),

-(3-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)propyl)-3-

1,2,3,4-tetrahydroacridin-9-yl)urea 19. White solid (0.52 g, 91%): (C-2,3,2 ,3 ), 24.6 and 25.3 (C-1,1 ), 26.2 (C-3 ,4 ), 29.9 and 31.3 (C-

0

00 13

1

7.82 (m, 2H, 2 ꢂ CH, H-5 ,8 ); C-NMR (CDCl

3

0

) d 22.1, 22.4, 22.7

0

00

0

(

ꢁ

1

0

00

0

m.p.¼104.5–105.4 C; H NMR (CDCl

3

) d 1.72 (m, 10H, 5 ꢂ CH

2

3

, H- 2 ,5 ), 32.1 and 33.8 (C-4,4 ), 39.8 and 48.2 (C-1 ,6 ), 55.6 (OCH ),

0

00 00

00

2

(

3

,3,2 ,2 ,3 ), 2.56 and 2.77 (m, 4H, 2 ꢂ CH

2

, H-1,1 ), 2.84 and 2.95 102.2 (C-8), 115.5 (C-9a), 120.1 (C-8a), 121.1 (C-6), 122.8 (C-8 ),

0

00

m, 4H, 2 ꢂ CH , H-4,4 ), 3.34 and 3.50 (m, 4H, 2 ꢂ CH , H-1 ,3 ), 124.5 (C-8a ), 125.4 (C-7 ), 127.4 (C-9a ), 127.5 (C-5), 128.2 (C-5 ),

2

0

00

.85 (m, 3H, OCH

3

), 6.01 (bs, 1H, NH), 7.20 (dd, 1H, CH, H-6, 128.5 (C-6 ), 139.6 (C-9 ), 140.5 (C-10a), 146.8 (C-10a ), 151.2 (C-9),

0

00

J ¼ 8.8, 2.4 Hz), 7.26 (t, 1H, CH, H-7 , J ¼ 7.2 Hz), 7.30 (m, 1H, CH, 154.2 (C-4a), 156.1 (C-7), 156.4 (C¼O), 159.7 (C-4a ); ESI-MS: m/z

þ

0

H-8), 7.43 (dd, 1H, CH, H-6 , J ¼ 8.4, 7.2 Hz), 7.70 (d, 1H, CH, H-5, 552.3 [M] (calculated for: [C34H42N5O2] þ 552.3); Anal. Calcd for

J ¼ 8.8 Hz), 7.82 (m, 2H, 2 ꢂ CH, H-5 ,8 ), 8.35 (bs, 1H, NH); C34H41N5O2: C, 74.02; H, 7.49; N, 12.69. Found: C, 74.48; H, 7.15;

C NMR (CDCl

) d 22.4, 22.6 (C-2,3,2 ,3 ), 25.3 (C-1,1 ), 31.2 (C-2 ), N, 12.88.

2.1 and 33.8 (C-4,4 ), 36.7 and 44.0 (C-1 ,3 ), 55.4 (OCH ), 101.5

0

00

1

3

00 00

00

0

3

0

3

(

8

(

1

[

6

1-(7-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)heptyl)-3-

C-8), 114.6 (C-9a), 119.9 (C-8a), 122.0 (C-6), 122.9 (C-8 ), 124.6 (C- (1,2,3,4-tetrahydroacridin-9-yl)urea 23. White solid (0.61 g, 96%):

3

0

00

0

00

ꢁ

1

a ), 125.3 (C-7 ), 127.3 (C-5), 128.2 (C-5 ), 128.4 (C-6 ,9a ), 139.8 m.p.¼66.4–67.4 C; H NMR (CDCl

3

) d 1.25 (m, 6H, 3 ꢂ CH

2

, H-

0

00

0

C-9 ), 146.8 (C-10a,10a ), 152.1 (C-9), 153.3 (C-4a), 156.1 (C-7), 3 ,4 ,5 ), 1.58 and 1.74 (m, 4H, 2 ꢂ CH

, H-2 ,6 ), 1.80 (m, 8H,

2

0

þ

00 00

00

57.6 (C¼O), 159.7 (C-4a ); ESI-MS: m/z 510.2 [M] (calculated for: 4 ꢂ CH

, H-2,3,2 ,3 ), 2.62 and 2.77 (m, 4H, 2 ꢂ CH

, H-1,1 ), 2.93

, H-7 ), 3.41 (t,

3

), 5.62 (bs, 1H, NH), 7.18

2

þ

00

0

C31H36N5O2] 510.3); Anal. Calcd. for C31H35N5O2: C, 73.06; H, and 3.0 (m, 4H, 2 ꢂ CH

2

, H-4,4 ), 3.08 (m, 2H, CH

2

0

.92; N, 13.74. Found: C, 73.42; H, 6.86; N, 13.95.

1

2H, CH

2

, H-1 , J ¼ 7.2 Hz), 3.86 (s, 3H, OCH

0

-(4-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)butyl)-3-

(dd, 1H, CH, H-6, J ¼ 9.2, 2.4 Hz), 7.26 (m, 2H, 2 ꢂ CH, H-8,7 ), 7.46

0

(

1,2,3,4-tetrahydroacridin-9-yl)urea 20. White solid (0.56 g, 94%): (dd, 1H, CH, H-6 , J ¼ 6.8, 1.2 Hz), 7.57 (bs, 1H, NH), 7.77 (d, 2H,

1

0

00 00

1

3

ꢁ

m.p.¼69.2–69.8 C; H NMR (CDCl

3

) d 1.70 (m, 4H, 2 ꢂ CH

2

, H-2 ,3 ), CH, H-5, J ¼ 9.2 Hz), 7.83 (m, 2H, 2 ꢂ CH, H-5 ,8 ); C NMR (CDCl

)

3

0

00

00 00

00

1

.80 (m, 8H, 4 ꢂ CH , H-2,3,2 ,3 ), 2.57 and 2.80 (m, 4H, 2 ꢂ CH , d 22.3, 22.4, 22.6, 22.7 (C-2,3,2 ,3 ), 24.5 and 25.3 (C-1,1 ), 26.6 (C-

2

0

00

, H-4,4 ), 3.40 and 3.50 (m, 4 ), 28.6 and 28.8 (C-3 ,5 ), 30.3 and 31.3 (C-2 ,6 ), 32.4 and 33.8 (C-

2

0

H-1,1 ), 2.90 and 3.0 (m, 4H, 2 ꢂ CH

H, 2 ꢂ CH , H-1 ,4 ), 3.85 (m, 3H, OCH ), 4.50 (bs, 1H, NH), 6.15 4,4 ), 40.0 and 48.6 (C-1 ,7 ), 55.5 (OCH

0

00

0

4

(

3

), 102.1 (C-8), 115.8 (C-9a),

2

3

0

00

bs, 1H, NH), 7.20 (dd, 1H, CH, H-6, J ¼ 8.8, 2.4 Hz), 7.26 (t, 1H, CH, 120.3 (C-8a), 120.9 (C-6), 122.8 (C-8 ), 124.5 (C-8a ), 125.4 (C-7 ),

H-7 , J ¼ 7.2 Hz), 7.30 (m, 1H, CH, H-8), 7.43 (dd, 1H, CH, H-6 , 127.4 (C-5), 128.1 (C-9a ), 128.5 (C-6 ,5 ), 139.5 (C-9 ), 141.1 (C-

0a), 146.8 (C-10a ), 150.9 (C-9), 154.6 (C-4a), 156.0 (C-7), 156.3

0

00 00

00

0

00

0

1

(

[

J ¼ 8.4, 7.2 Hz), 7.75 (d, 1H, CH, H-5, J ¼ 8.8 Hz), 7.84 (m, 2H,

þ

0

00

13

0

C¼O), 159.7 (C-4a ); ESI-MS: m/z 566.3 [M] (calculated for:

2

3

(

1

(

[

ꢂ CH, H-5 ,8 ); C NMR (CDCl ) d 22.1, 22.4, 22.5, 22.6 (C-

3

þ

0

00

0

C35H44N5O2] 566.4); Anal. Calcd for C35H43N5O2: C, 74.30; H,

,3,2 ,3 ), 24.6 and 25.3 (C-1,1 ), 27.5 and 28.5 (C-2 ,3 ), 32.0 and

0

7

.66; N, 12.38. Found: C, 74.55; H, 7.95; N, 12.36.

3.8 (C-4,4 ), 39.5 and 48.1 (C-1 ,4 ), 55.5 (OCH3), 102.1 (C-8), 115.6

00 00

C-9a), 120.1 (C-8a), 121.1 (C-6), 122.7 (C-8 ), 124.4 (C-8a ), 125.4

00 00 00 00 00

C-7 ), 127.4 (C-9a ), 128.2 (C-5), 128.5 (C-5 ,6 ), 139.5 (C-9 ),

00

46.8 (C-10a,10a ), 151.1 (C-9), 154.2 (C-4a), 156.1 (C-7), 156.4

Conclusion

0

þ

C¼O), 159.7 (C-4a ); ESI-MS: m/z 524.3 [M] (calculated for: In this study, we developed novel 7-MEOTA-THA heterodimers

þ

C32H38N5O2] 524.3); Anal. Calcd for C32H37N5O2: C, 73.39; H, 12–22 combining the two fragments via a linker of varying length

7

.12; N, 13.37. Found: C, 73.68; H, 7.34; N, 13.48.

and containing either urea or thiourea moieties. In conclusion,

1

-(5-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-ylamino)pentyl)-3-

extensive comparative biochemical, biophysical and biological

(

1,2,3,4-tetrahydroacridin-9-yl)urea 21. White solid (0.58 g, 95%): measurements consistently indicated that 12–22 acted as DNA-

ꢁ

1

0

m.p.¼114.2–114.9 C; H NMR (CDCl

3

) d 1.43 (m, 2H, CH

2

, H-3 ), binders and inhibitors of Topo I/II, and were able to affect the

2

0

1

2

4

OCH

7

.55 and 1.66 (m, 4H, 2 ꢂ CH , H-2 ,4 ), 1.80 (m, 8H, 4 ꢂ CH , H- MMP and viability of HL-60 cells and suppress selectively cancer

2

0

00

,3,2 ,3 ), 2.56 and 2.73 (m, 4H, 2 ꢂ CH

2

, H-1,1 ), 2.98 and 3.02 (m, cell proliferation.

0

H, 2 ꢂ CH

2

, H-4,4 ), 3.30 (m, 4H, 2 ꢂ CH

2

, H-1 ,5 ), 3.85 (s, 3H,

In summary, spectroscopic data proved that linking of THA and

3

), 5.62 (bs, 1H, NH), 7.20 (m, 1H, CH, H-6), 7.26 (m, 1H, CH, H- 7-MEOTA with an alkyl linker containing thio-/urea moieties posi-

), 7.27 (m, 1H, CH, H-8), 7.43 (m, 1H, CH, H-6 ), 7.67 (bs, 1H, tively influenced the DNA interaction power. From UV–vis absorp-

0

00

0

NH), 7.77 (d, 1H, CH, H-5, J ¼ 8.4 Hz), 7.82 (m, 1H, CH, H-8 ), 7.84 tion spectroscopic titration were calculated DNA-binding

0

13

6

ꢃ1

(

2

3

(

8

6

d, 1H, CH, H-5 , J ¼ 8.8 Hz); C NMR (CDCl

3

) d 22.9, 23.1 (C- constants K ¼ 0.5–8.0 ꢂ 10

M . The obtained K for the hetero-

0

00

0

00

,3,2 ,3 ), 24.8 (C-3 ), 24.7 and 25.4 (C-1,1 ), 29.0 and 33.4 (C-4,4 ), dimers complexed with ctDNA were higher than K for THA

0

4

ꢃ1

4

ꢃ1

0.7 and 31.2 (C-2 ,4 ), 40.4 and 48.5 (C-1 ,5 ), 56.4 (OCH

3

), 102.2 (K ¼ 3.8 ꢂ 10

M

) and 7-MEOTA (K ¼ 7.9 ꢂ 10

M ) complexed

00

C-8), 115.7 (C-9a), 120.3 (C-8a), 121.0 (C-6), 122.8 (C-8 ), 124.5 (C- with ctDNA, and moreover were comparable with K for the

0

00

6

ꢃ1

a ), 125.3 (C-7 ), 127.3 (C-5), 127.8 (C-9a ), 128.2 (C-5 ), 128.5 (C- acridine–ctDNA complex (K ¼ 4.5 ꢂ 10

M ). Data from fluores-

0

00

), 139.2 (C-9 ), 143.5 (C-10a), 145.8 (C-10a ), 150.5 (C-9), 154.9 cence quenching experiments clearly demonstrated that ctDNA

0

(

C-4a), 156.1 (C-7), 156.3 (C¼O), 160.1 (C-4a ); ESI-MS: m/z 538.3 can quench fluorescence of the studied heterodimers, by static

8

94

J. JANOCKOVA ET AL.

quenching. The latter correlates well with decreasing KSV values

and increasing temperatures and was further confirmed by

2. Shaw FH, Bentley G. Morphine antagonism. Nature 1952;169:

712–3.

3. Hunter AJ, Murray TK, Jones JA, et al. The cholinergic

pharmacology of tetrahydroaminoacridine in vivo and

in vitro. Br J Pharm 1989;98:79–86.

4. Zovko A, Sepcic K, Turk T, et al. New aspects of the relation-

ship between acetylcholinesterase activity and cancer I.

Poly-Aps Exp 2009;6:58–69.

11

ꢃ1 ꢃ1

obtained values of k

q

(4.77–10.69 ꢂ 10

M

s

) which were

higher than the limiting diffusion rate constant for dynamic

quenching. Thermodynamic parameters (DH > 0, DS > 0) indicated

that hydrophobic interactions play the key role in the binding of

novel compounds to ctDNA, except in the case of heterodimers

16 and 20 which revealed a different pattern of negative DH and

DS, pointing to van der Waals associations with ctDNA. The exist-

ence of LD signals also demonstrated that 12–22 interact with

DNA and become oriented after attachment to DNA.

5. Soukup O, Jun D, Zdarova-Karasova J, et al. A resurrection

of 7-MEOTA: a comparison with tacrine. Curr Alzh Res 2013;

1

0:893–906.

Based on all the obtained biological positive outcomes, 7-

MEOTA-THA thio-/ureas can be considered as interesting and poten-

tial drug candidates as anticancer agents. This assertion is supported

by the fact that they are able to affect cancer cell lines, whereas

they remained unattached in healthy non-cancer fibroblasts at the

tested concentration. The more effective heterodimers are those

which contain longer alkyl chains. The higher antiproliferative effect

of the more lipophilic agents containing either pentyl or hexyl

chains could be ascribed to better cell membrane penetration. In

this regard, thiourea hybrid 17 and urea hybrid 22 can be high-

lighted as the most potent. The studied heterodimers 14–17 and

6

7

8

.

Summers WK, Majovski LV, Marsh GM, et al. Oral tetrahy-

droaminoacridine in long-term treatment of senile dementia,

Alzheimer type. N Engl J Med 1986;315:1241–5.

Watkins PB, Zimmerman HJ, Knapp MJ, et al. Hepatotoxic

effects of tacrine administration in patients with Alzheimers

disease. J Am Med Assoc 1994;271:992–8.

Kozurkova M, Hamulakova S, Gazova Z, et al. Neuroactive

multifunctional tacrine congeners with cholinesterase, anti-

amyloid aggregation and neuroprotective properties. Pharm

2

011;4:382–418.

9

.

Patocka J, Jun D, Kuca K. Possible role of hydroxylated

metabolites of tacrine in drug toxicity and therapy of

Alzheimer’s disease. Curr Drug Metab 2008;9:332–5.

21, 22 were able to evoke MMP dissipation after 24 h treatment and

caused cell death with an efficiency between 80 and 100%. In add-

ition, thioureas 14–17 significantly altered cell cycle progression and

induced block of the cell cycle in S phase, and ureas 21, 22 demon-

strated an increase of cells in G1 phase.

We believe that our study can contribute towards further

development of novel, more potent and highly selective antiproli-

ferative agents with intercalating properties.

1

0. Ezoulin MJM, Dong CZ, Liu Z, et al. Study of PMS777, a new

type of acetylcholinesterase inhibitor, in human HepG2 cells.

Comparison with tacrine and galanthamine on oxidative

stress and mitochondrial impairment. Toxicol In Vitro 2006;

2

0:824–31.

1

1. Galisteo M, Rissel M, Sergent O, et al. Hepatotoxicity of tac-

rine: occurrence of membrane fluidity alterations without

involvement of lipid peroxidation. J Pharmacol Exp Ther

Acknowledgments

2

000;294:160–7.

The authors are grateful to Ian McColl MD, PhD for assistance

with the article.

2. Osseni RA, Debbasch C, Christen MO, et al. Tacrine-induced

reactive oxygen species in a human liver cell line: the role

of anethole dithiolethione as a scavenger. Toxicol In Vitro

1

999;13:683–8.

Disclosure statement

3. Robertson DG, Braden TK, Urda ER, et al. Elucidation of mito-

chondrial effects by tetrahydroaminoacridine (tacrine) in rat,

dog, monkey and human hepatic parenchymal cells. Arch

Toxicol 1998;72:362–71.

4. Gazova Z, Soukup O, Sepsova V, et al. Multi-target-directed

therapeutic potential of 7-methoxytacrine-adamantylamine

heterodimers in the Alzheimer’s disease treatment. Biochim

Biophys Acta Mol Basis Dis 2017;1863:607–19.

5. Nepovimova E, Korabecny J, Dolezal R, et al. Tacrine-trolox

hybrids: a novel class of centrally active, nonhepatotoxic

multi-target-directed ligands exerting anticholinesterase and

antioxidant activities with low in vivo toxicity. J Med Chem

No potential conflict of interest was reported by the authors.

Funding

This study was supported by the University of Defense (Long

Term Development Plan), by MH CZ-DRO [UHHK, 00179906], VEGA

1

/0016/18, VVGS 2016–277 and by Operational Program Research

and Innovations for project Medical University Scientific Park in

Kosice (MediPark, Kosice–Phase II), ITMS2014 þ 313011D103, co-

financed from the European fund of regional progress.

2

015;58:8985–9003.

ORCID

1

6. Nepovimova E, Uliassi E, Korabecny J, et al. Multitarget drug

design strategy: quinone-tacrine hybrids designed to block

amyloid-b aggregation and to exert anticholinesterase and

antioxidant effects. J Med Chem 2014;57:8576–89.

Jana Janockova

Jan Korabecny

Maria Kozurkova

http://orcid.org/0000-0002-2034-1860

http://orcid.org/0000-0001-6977-7596

http://orcid.org/0000-0002-2789-8397

1

7. Spilovska K, Korabecny J, Nepovimova E, et al. Multitarget

tacrine hybrids with neuroprotective properties to confront

Alzheimer’s disease. Curr Top Med Chem 2017;17:1006–26.

8. Tumiatti V, Minarini A, Bolognesi ML, et al. Tacrine deriva-

tives and Alzheimer’s disease. Curr Med Chem 2010;17:

1825–38.

References

1

.

Giacobini E. Invited review cholinesterase inhibitors for

Alzheimer’s disease therapy: from tacrine to future applica-

tions. Neurochem Int 1998;32:413–9.

19. Dejmek L. 7-MEOTA. Drugs Future 1990;15:126–9.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

895

2

0. Koupilova M, Hrdina V, Fusek J. Exploitation of the antagon-

istic properties of some drugs against poisoning with psy-

chochemicals. Activitas Nervosa Superior 1981;23:292–3.

pharmacological evaluation, molecular modeling and QSAR

studies. Eur J Med Chem 2014;82:426–38.

40. Spilovska K, Korabecny J, Kral J, et al. 7-Methoxytacrine-ada-

mantylamine heterodimers as cholinesterase inhibitors in

Alzheimer’s disease treatment – synthesis, biological evalu-

ation and molecular modeling studies. Molecules 2013;18:

2397–418.

1. Bajgar J, Fusek J, Patoka J, Hrdina V. Protective effect of

9-amino-7-methoxy-1,2,3,4-tetra-hydroacridine against inhib-

ition of acetylcholinesterase by o-ethyl s-(2-dimethylami-

noethyl) methylphosphonotioate in vivo. Arch Toxicol 1983;

5

4:163–6.

41. Hamulakova S, Janovec L, Hrabinova M, et al. Synthesis,

design and biological evaluation of novel highly potent tac-

rine congeners for the treatment of Alzheimer’s disease. Eur

J Med Chem 2012;55:23–31.

2

2. Mansouri A, Haouzi D, Descatoire V, et al. Tacrine inhibits

topoisomerases and DNA synthesis to cause mitochondrial

DNA depletion and apoptosis in mouse liver. Hepatology

2

003;38:715–25.

42. Los M, Burek CJ, Stroh C, et al. Anticancer drugs of tomor-

row: apoptotic pathways as targets for drug design. Drug

Discov Today 2003;8:67–77.

43. Gottlieb E, Armour SM, Harris MH, Thompson CB.

Mitochondrial membrane potential regulates matrix config-

uration and cytochrome c release during apoptosis. Cell

Death Different 2003;10:709–17.

2

3. Freudenreich CH, Kreuzer KN. Localization of an aminoacri-

dine antitumor agent in a type-ii topoisomerase DNA com-

plex. Proc Natl Acad Sci USA 1994;91:11007–11.

4. Li TK, Liu LF. Tumor cell death induced by topoisomerase-

targeting drugs. Ann Rev Pharm Toxicol 2001;41:53–77.

5. Lin JH, Castora FJ. DNA topoisomerase II from mammalian

2

mitochondria is inhibited by the antitumor drugs, m-AMSA 44. Zhang B, Li X, Li B, et al. Acridine and its derivatives: a

and VM-26. Biochem Biophys Res Commun 1991;176:690–7.

6. Skladanowski A, Plisov SY, Konopa J, Larsen AK. Inhibition of

patent review (2009–2013). Exp Opin Therap Patents 2014;

24:647–64.

2

DNA topoisomerase II by imidazoacridinones, new antineo- 45. Hsiang YH, Lihou MG, Liu LF. Arrest of replication forks by

plastic agents with strong activity against solid tumors. Mol

Pharmacol 1996;49:772–80.

7. Chen TK, Fico R, Canellakis ES. Diacridines, bifunctional inter-

drug-stabilized topoisomerase i-DNA cleavable complexes as

a mechanism of cell killing by camptothecin. Cancer Res

1989;49:5077–82.

2

3

calators. Chemistry and antitumor activity. J Med Chem 46. Zhu HJ, Gooderham NJ. Mechanisms of induction of cell

1978;21:868–74.

cycle arrest and cell death by cryptolepine in human lung

adenocarcinoma A549 cells. Toxicol Sci 2006;91:132–9.

prospects for future design. Anti-Cancer Drug Design 1989; 47. Hu MK. Synthesis and in-vitro anticancer evaluation of bis-

:241–63.

tacrine congeners. J Pharm Pharmacol 2001;53:83–8.

9. Jano ꢀc kov ꢁa J, Pl ꢀs ꢁı kov ꢁa J, Koval’ J, et al. Tacrine derivatives as 48. Spicer JA, Gamage SA, Rewcastle GW, et al. Bis(phenazine-1-

8. Denny WA. DNA-intercalating ligands as anti-cancer drugs:

4

dual topoisomerase I and II catalytic inhibitors. Bioorg Chem

015;59:168–76.

0. Bailly C. Homocamptothecins: potent topoisomerase I inhibi-

carboxamides): structure ꢃ activity relationships for a new

class of dual topoisomerase I/II-directed anticancer drugs.

J Med Chem 2000;43:1350–8.

2

tors and promising anticancer drugs. Crit Rev Oncol 49. Bra n~ a MF, Castellano JM, Perron D, et al. Chromophore-

Hematol 2003;45:91–108.

modified bis-naphthalimides: synthesis and antitumor activ-

ity of bis-dibenz[de,h]isoquinoline-1,3-diones. J Med Chem

1997;40:449–54.

1. Salerno S, Da Settimo F, Taliani S, et al. Recent advances in

the development of dual topoisomerase I and II inhibitors

as anticancer drugs. Curr Med Chem 2010;17:4270–90.

2. Elmore S. Apoptosis: a review of programmed cell death.

Toxicol Pathol 2007;35:495–516.

50. Hallgas B, Patonay T, Kiss-Szikszai A, et al. Comparison of

measured and calculated lipophilicity of substituted aurones

and related compounds. J Chromatogr B Anal Technol

Biomed Life Sci 2004;801:229–35.

3

3. Fox JL, MacFarlane M. Targeting cell death signalling in can-

cer: minimising ‘collateral damage’. Br J Cancer 2016;115: 51. Alonso C, Fuertes M, Gonzalez M, et al. Synthesis and bio-

5

–11.

logical evaluation of indeno[1,5]naphthyridines as topoisom-

erase I (TopI) inhibitors with antiproliferative activity. Eur J

Med Chem 2016;115:179–90.

3

4. Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis path-

ways in cancer therapy. Cancer J Clin 2005;55:178–94.

5. Andrs M, Korabecny J, Jun D, et al. Phosphatidylinositol 3- 52. Goodell JR, Madhok AA, Hiasa H, Ferguson DM. Synthesis

kinase (PI3K) and phosphatidylinositol 3-kinase-related kin-

ase (PIKK) inhibitors: importance of the morpholine ring.

J Med Chem 2015;58:41–71.

and evaluation of acridine- and acridone-based anti-herpes

agents with topoisomerase activity. Bioorg Med Chem 2006;

14:5467–80.

3

6. Papaphilis AD, Shaw YH. Interaction of acridines and tetrahy- 53. Kumar A, Ehrenshaft M, Tokar EJ, et al. Nitric oxide inhibits

droacridines with DNA at low DNA/dye ratios. Biochim

Biophys Acta 1977;476:122–30.

7. Crenshaw JM, Graves DE, Denny WA. Interactions of acridine

topoisomerase II activity and induces resistance to topo-

isomerase II-poisons in human tumor cells. Biochim Biophys

Acta General Subjects 2016;1860:1519–27.

antitumor agents with DNA: binding energies and groove 54. Wethington SL, Wright JD, Herzog TJ. Key role of topoisom-

preferences. Biochemistry 1995;34:13682–7.

8. Nelson EM, Tewey KM, Liu LF. Mechanism of antitumor drug

action: poisoning of mammalian DNA topoisomerase II on

erase I inhibitors in the treatment of recurrent and refrac-

tory epithelial ovarian carcinoma. Exp Rev Anticancer Ther

2008;8:819–31.

DNA by 4’-(9-acridinylamino)-methanesulfon-m-anisidide. 55. Ikeguchi M, Arai Y, Maeta Y, et al. Topoisomerase I expres-

Proc Natl Acad Sci USA 1984;81:1361–5.

9. Korabecny J, Dolezal R, Cabelova P, et al. 7-MEOTA-donepe-

zil like compounds as cholinesterase inhibitors: synthesis,

sion in tumors as a biological marker for CPT-11 chemosen-

sitivity in patients with colorectal cancer. Surgery Today

2011;41:1196–9.

3

8

96

J. JANOCKOVA ET AL.

5

6. McClendon AK, Osheroff N. DNA topoisomerase II, genotox-

icity, and cancer. Mutat Res Fund Mol Mechan Mutag 2007;

23:83–97.

electrophoretic determination of their effect on topoisomer-

ase I and II activity. Mol Biosys 2016;12:2910–20.

75. Lakowicz JR. Principles of fluorescence spectroscopy. In:

Lakowicz JR, ed. Quenching of fluorescence. Boston, MA:

Springer US; 2006:277–330.

6

5

7. Topcu Z. DNA topoisomerases as targets for anticancer

drugs. J Clin Pharm Therap 2001;26:405–16.

5

8. Vos SM, Tretter EM, Schmidt BH, Berger JM. All tangled up: 76. Kashanian S, Khodaei MM, Pakravan P. Spectroscopic studies

how cells direct, manage and exploit topoisomerase func-

on the interaction of isatin with calf thymus DNA. DNA Cell

tion. Nat Rev Mol Cell Biol 2011;12:827–41.

Biol 2010;29:639–46.

5

6

9. Janockova J, Gulasova Z, Plsikova J, et al. Interaction of cho- 77. Qiao CY, Bi SY, Sun Y, et al. Study of interactions of anthra-

linesterase modulators with DNA and their cytotoxic activity.

Int J Biol Macrom 2014;64:53–62.

0. Larsen AK, Escargueil AE, Skladanowski A. Catalytic topo-

quinones with DNA using ethidium bromide as a fluores-

cence probe. Spectrochim Acta A Mol Biomol Spectrosc

2008;70:136–43.

isomerase II inhibitors in cancer therapy. Pharmacol Ther 78. Tian FF, Jiang FL, Han XL, et al. Synthesis of a novel hydra-

2

003;99:167–81.

zone derivative and biophysical studies of its interactions

with bovine serum albumin by spectroscopic, electrochem-

ical, and molecular docking methods. J Phys Chem B 2010;

114:14842–53.

1. Baviskar AT, Amrutkar SM, Trivedi N, et al. Switch in site of

inhibition: a strategy for structure-based discovery of human

topoisomerase IIalpha catalytic inhibitors. ACS Med Chem

Lett 2015;6:481–5.

2. Vispe S, Vandenberghe I, Robin M, et al. Novel tetra-acridine

derivatives as dual inhibitors of topoisomerase II and the

human proteasome. Biochem Pharmacol 2007;73:1863–72.

3. Nitiss JL. Targeting DNA topoisomerase II in cancer chemo-

therapy. Nat Rev Cancer 2009;9:338–50.

79. Khan SN, Danishuddin M, Khan AU. Inhibition of transcrip-

tion factor assembly and structural stability on mitoxantrone

binding with DNA. Biosci Rep 2010;30:331–40.

80. Shahabadi N, Fili SM, Kheirdoosh F. Study on the interaction

of the drug mesalamine with calf thymus DNA using

6

molecular docking and spectroscopic techniques.

J

4. Denny WA. Emerging DNA topoisomerase inhibitors as anti-

cancer drugs. Exp Opin Emerg Drugs 2004;9:105–33.

Photochem Photobiol B Biol 2013;128:20–6.

81. Rodger A. Circular dichroism and linear dichroism.

Encyclopedia of analytical chemistry. New York: John Wiley

& Sons, Ltd; 2006.

82. Ranjbar B, Gill P. Circular dichroism techniques: biomolecular

and nanostructural analyses – a review. Chem Biol Drug

Design 2009;74:101–20.

5. Jensen PB, Sehested M. DNA topoisomerase II rescue by

catalytic inhibitors: a new strategy to improve the antitumor

selectivity of etoposide. Biochem Pharmacol 1997;54:755–9.

6. Koceva-Chyla A, Jedrzejczak M, Skierski J, et al. Mechanisms

of induction of apoptosis by anthraquinone anticancer

6

drugs aclarubicin and mitoxantrone in comparison with 83. Pabbathi A, Samanta A. Spectroscopic and molecular dock-

doxorubicin: relation to drug cytotoxicity and caspase-3 acti-

ing study of the interaction of DNA with a morpholinium

vation. Apoptosis 2005;10:1497–514.

ionic liquid. J Phys Chem B 2015;119:11099–105.

6

7. Lu Z, Wientjes TSS, Au JS. Nontoxic suramin treatments 84. Nord ꢁe n B, Kurucsev T. Analysing DNA complexes by circular

enhance docetaxel activity in chemotherapy-pretreated non-

and linear dichroism. J Mol Recognit 1994;7:141–55.

small cell lung xenograft tumors. Pharm Res 2005;22: 85. Galindo MA, Olea D, Romero MA, et al. Design and non-

1069–78.

covalent DNA binding of platinum(II) metallacalix[4]arenes.

8. Yoshida M, Maehara Y, Sugimachi K. MST-16, a novel bis-

Chem Eur J 2007;13:5075–81.

dioxopiperazine anticancer agent, ameliorates doxorubicin- 86. Pascu GI, Hotze ACG, Sanchez-Cano C, et al. Dinuclear

induced acute toxicity while maintaining antitumor efficacy.

Clin Cancer Res 1999;5:4295–300.

ruthenium(II) triple-stranded helicates: luminescent supra-

molecular cylinders that bind and coil DNA and exhibit

activity against cancer cell lines. Angew Chem Int Ed 2007;

46:4374–8.

6

9. Sarwar T, Rehman SU, Husain MA, et al. Interaction of cou-

marin with calf thymus DNA: deciphering the mode of bind-

ing by in vitro studies. Int J Biol Macrom 2015;73:9–16.

0. Vantova Z, Paulikova H, Sabolova D, et al. Cytotoxic activity

of acridin-3,6-diyl dithiourea hydrochlorides in human leuke-

mia line HL-60 and resistant subline HL-60/ADR. Int J Biol

Macromol 2009;45:174–80.

87. Hilovska L, Jendzelovsky R, Jendzelovska Z, et al.

Downregulation of BCRP and anti-apoptotic proteins by

proadifen (SKF-525A) is responsible for the enhanced mitox-

antrone accumulation and toxicity in mitoxantrone-resistant

human promyelocytic leukemia cells. Int J Oncol 2015;47:

1572–84.

7

1. Kozurkova M, Sabolova D, Janovec L, et al. Cytotoxic activity

of proflavine diureas: synthesis, antitumor, evaluation and 88. Jendzelovsky R, Jendzelovska Z, Hilovska L, et al. Proadifen

DNA binding properties of 1’,1’’-(acridin-3,6-diyl)-3’,3’’-dialkyl-

diureas. Bioorg Med Chem 2008;16:3976–84.

sensitizes resistant ovarian adenocarcinoma cells to cisplatin.

Toxicol Lett 2016;243:56–66.

2. Chaveerach U, Meenongwa A, Trongpanich Y, et al. DNA 89. Janockova J, Plsikova J, Kasparkova J, et al. Inhibition of

binding and cleavage behaviors of copper(II) complexes

with amidino-O-methylurea and N-methylphenyl-amidino-O-

methylurea, and their antibacterial activities. Polyhedron

DNA topoisomerases I and II and growth inhibition of HL-60

cells by novel acridine-based compounds. Eur J Pharm Sci

2015;76:192–202.

2

010;29:731–8.

90. Plsikova J, Janovec L, Koval J, et al. 3,6-Bis(3-alkylguanidi-

no)acridines as DNA-intercalating antitumor agents. Eur J

Med Chem 2012;57:283–95.

91. Jenkins TC. Optical absorbance and fluorescence techniques

for measuring DNA-drug interactions. Meth Mol Biol 1997;

90:195–218.

7

3. Tao M, Zhang GW, Xiong CH, Pan JH. Characterization of the

interaction between resmethrin and calf thymus DNA in

vitro. New J Chem 2015;39:3665–74.

4. Janockova J, Zilecka E, Kasparkova J, et al. Assessment of

DNA-binding affinity of cholinesterase reactivators and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

897

9

2. McGhee JD, von Hippel PH. Theoretical aspects of DNA-

protein interactions: co-operative and non-co-operative

binding of large ligands to a one-dimensional homogeneous

lattice. J Mol Biol 1974;86:469–89.

3. Lerman LS. Structural considerations in the interaction of

DNA and acridines. J Mol Biol 1961;3:18–IN4.

albumin: fluorescence and circular dichroism studies.

Spectrochim Acta

A Mol Biomol Spectrosc 2008;71:

1617–22.

96. Rehman SU, Sarwar T, Husain MA, et al. Studying non-

covalent drug-DNA interactions. Arch Biochem Biophys

2015;576:49–60.

9

4. Haris P, Mary V, Aparna P, et al. A comprehensive approach 97. Bielavsky J. Analogs of 9-amino-1,2,3,4-tetrahydroacridine.

to ascertain the binding mode of curcumin with DNA.

Spectrochim Acta A Mol Biomol Spectrosc 2017;175:155–63.

5. Gharagozlou M, Boghaei DM. Interaction of water-soluble

amino acid Schiff base complexes with bovine serum

Collect Czechoslovak Chem Commun 1977;42:2802–8.

98. Mezeiova E, Korabecny J, Sepsova V, et al. Development of

2-methoxyhuprine as novel lead for Alzheimer’s disease

therapy. Molecules 2017;22:1–19.

9

Article Doi

In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

DOI: 10.1080/14756366.2019.1593159

Source and publish data:

Authors:

Article abstract of DOI:10.1080/14756366.2019.1593159

Full text of DOI:10.1080/14756366.2019.1593159

Products guided by the article

R&D Labs maybe for 13161-85-8

Relevant to this article

Hot Product