49 

 

CHAPTER 3

50 

 

SYNTHESIS OF MITOXANTRONE ANALOGUES AND THEIR CYTOTOXIC ACTIVITIES.

3.1.1 Introduction;

For well over a century substituted anthraquinone or anthracene-9,10-diones such as

those 1,4-bis(amino)anthracene-9,10-dione and 1,4-bis(amino) 5,8- dihydroxy

anthracene9,10-dione,have been used as dyes or pigments because of their high chemical,

photochemical and thermal stability.1-5 In the 1970s, new application emerged for these

derivatives and Murdock and Child were the first to show that the N-alkyl derivatives of

anthraquinone, were chemotherapeutically active as anti-cancer agents.6 Subsequently it

was shown that functionalized N-alkyl derivatives such as Mitoxantrone(1) and

Ametantrone (2) (figure 3.1) were highly effective anti-cancer drugs with an efficacy and

therapeutic index exceeding doxorubicin (3), methotrexate, (4) or 5-fluorouracil (5).7-13

Mitoxantrone13a is anti-neoplastic agent, is active against breast cancer, acute leukemia,

lymphoma, cervix carcinoma, and liver cell cancer. Unlike the anthracyclines that have a

red color, the amino anthracene diones are deep blue color.

O

O

X

X

NH(CH2)2NH(CH2)2OH

NH(CH2)2NH(CH2)2OH

Mitoxantrone (X = OH)

Ametantrone (X = H)

(1)

(2)

Figure 3.1

51 

 

OO

O

OCH3

OH

OH

O

OHO

R

CH3

OH

NH2

DOXORUBICIN ( R = OH)

DAUNORUBICIN ( R = H) (6)

(3)

OO

O

OCH3

OH

OH

O

OHO

R

CH3

OH

NH2OO

O

OH

OH

O

OHO

CH3

CH3

OH

NH2

Epirubicin (7) Idarubicin (8)

N

NN

NNH2

NH2

N

NH

HOOC

COOH

O

methotrexate (4)

52 

 

NH

NH

O

O

F

5 - flurouracil (5)

However, while the additional hydroxyl groups present at 5-and 8-positions of

mitoxantrone lead to tenfold increase in its antineoplastic activity over ametantrone, this

beneficial enhancement is unfortunately countered by a tenfold increase in its

cardiotoxicity.14 Anthracyclins 14a,b, are derivatives of 5,12-napthacene and these are

antibiotics, which are used to treat to wide range of cancers, including (but not limited to)

leukemias, lymphomas, breast, ovarian, and lung cancers. Anthracyclines inhibit DNA

and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, thus

prevent the replication of rapidly growing cancer cells 15,16. Important anthracyclin drugs

are doxorubicin (3) daunorubicin (6), epirubicin (7) and idarubicin (8) The mechanism of

the cytotoxicity of anthracyclines is pleiotropic and its significance in cell growth

inhibition seems to be highly specific and dependent on cell type. The anthracycline

antibiotics daunorubicin, and doxorubicin have been used widely as anticancer drugs for

more than 30 years, but their cardiotoxicity limits their clinical use. The clinical use of

anthracyclines like doxorubicin and daunorubicin can be viewed as a sort of double-

edged sword. Doxorubicin and its analogues function as topoisomerase inhibitor, poisons

through stabilization of the cleavable complex that forms as part of the catalytic cycle of

enzyme. The detailed mechanism of how these compounds, all of which contain an

anthraquinone core that forms between the DNA base pairs, the drug, and the enzyme in

the minor groove has remained elusive. Anthracyclines and their anthraquinone

derivatives remain evergreen drugs with broad clinical indications but have an

improvable pharmacological index.

Mitoxantrone (1) demonstrate potent antitumor activity and has been used widely in

clinic since 1980s17,18, In addition, mitoxantrone has been the only drug approved by the

FDA for the treatment of worsening relapsing-remitting multiple sclerosis (MS),

secondary progressive MS, and progressive-relapsing MS since 2000.19,20, However,

53 

 

these clinical applications are limited due to the accumulative and irreversible

cardiotoxicity.18-20, Recently, Mitoxantrone was found to induce a progressive increase in

mitochondrial mass in the cancer cells but not in the cardiac cells.21 This suggests the

opportunities to develop novel anthraquinones with reduced cardiotoxicity.The planar

tricyclic structure of antraquinone is essentional for intercalating into DNA base pairs.

The two sidechains of 1and 2 could be used to connect with a variety of substituent’s,

which may form additional interactions with the double-strand DNA (ds-DNA) or DNA-

topoisomerase II (TOP2) cleavable complex to increase their binding affinities and

selectivities. It is well known that precise recognition of defined DNA sequences in

biological systems is mediated by enzymes and proteins having appropriate structure

motifs22 In the literature, various anthraquinone-peptide conjugates23-27, were reported to

demonstrate remarkable DNA binding and exhibit cytostatic or cytotoxic activities. In

addition, mono-amino acid-substituted anthraquinone conjugates could inhibit the

catalytic activity of TOP1 and TOP2.28-29 Theoretical design of anthraquinone-

oligopeptide conjugates to selectively target the double-stranded oligonucleotides has

also been published.30-31

With the aim to discover more specific chemotherapeutic agents , a focused library of

1,4-bis(2-amino-ethylamino)anthraquinone-amino acid conjugates (BACs) was designed

and synthesized32. The alpha amino acids possessing a variety of side chains, which could

be used to create specific interactions by one of the following mechanisms, were chosen

for construction of BACs.

(a) Metal ions, such as magnesium and zinc, are involved in DNA synthesis and

repair processes. Attaching the amino acids containing potential metal ion-

chelating side chain(i.e., the methylsulfanyl group of methionine) to the core

structure may be useful to interrupt DNA synthesis by stabilization of the DNA-

TOP2 cleavable complex.

(b) The hydroxyl group of tyrosine residue in TOP2 plays an essential role in TOP2-

mediated DNA strand cleavage.33,34 Furthermore, the side chains of 2 and 1 also

contain the hydroxyl groups. Therefore, introducing hydroxyl group-containing

54 

 

amino acids (i.e., serine and tyrosine) to anthraquinone may provide the

opportunity to enhance the cytotoxicity of this series of BACs.

(c) The €-amino group of lysine is protonated under physiological conditions, which

would be expected to produce strong electrostatic interactions with the negatively

charged phosphate groups on DNA skeleton.35,36,

3.1.2 Synthetic mitoxantrone analogues and their application;

Pixantrone (9) (6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione; is an

experimental antineoplastic drug, an analogue of mitoxantrone with fewer toxic effects

on cardiac tissue. It acts as a topoisomerase II poison and intercalating agent, the code

name BBR 2778 refers to pixantrone dimaleate, the actual substance commonly used in

clinical trials36a.

N

O

O

NH(CH2)2NH2

NH(CH2)2NH2

Pixantrone(9)

In an attempt to identify new molecular or pharmacophore targets for anticancer drugs,

Hsu-Shan Huang et al37 attention has been focused on cytotoxicity and telomerase.

Telomerase activity is thought to be required for the development of cellular immortality

and oncogenesis. Thus, considerable interest is focused on telomerase because of its

potential uses in assays for cancer diagnosis, research into cell biology and for anti-

telomerase drugs as a strategy for cancer chemotherapy. At present, among different

strategies pursued telomerase inhibitors in chemotherapy, the development of G-

quadruplex stabilizers has emerged as a highly promising approach. Herein, Hsu-Shan

Huang reported the design and synthesis of a series of non-nucleoside telomerase

inhibitors, in an attempt to identify potent enzyme A series of 1,2-heteroannelated

anthraquinones and anthrax[1,2-d]imidazole-6,11-dione tetracyclic analogues (10, 11, 12,

55 

 

13, 14) with different side chain were prepared using various synthetic route via

acylation, cyclization, condensation, and intramolecular heterocyclization. Tetracyclic

system containing alkyl and aryl, aromatic and heterocyclic, linear and cyclic polar and

apolar, and basic and acids residues were incorporated. They were evaluated for their

effects on telomerase activity, human telomerase reverse transcriptase (htert) expression,

cell proliferations, and in vitro cytotoxicity against NCI’s 60 cell line human

tumorscreen. It appeared that addition of a fourth planar aromatic system to a tricyclic

chromophore might enhances potent cytotoxic agents, at a level equivalent to a second

side chain in one of the tricyclic series.

O

O

NNH

CH3

O

O

NHNH

CH3CH3

N

NO

O

NH

NHO

O

O

O

Figure 3.2

O

O

N

SN

10 1112

13 14

Gouda, M. A. et al synthesized anthraquinone compounds (Figure 3.3) and were screened

in vitro for their antimicrobial activity. The diameter of inhibition zone was measured as

an indicator for the activity of the compounds. The results for antibacterial activities

depicted below revealed that few compounds have exhibited good activities38.

56 

 

O

O

NH

O

CN

PhHN NH

NN

CH3

CH3Ph

O

O

O

NH

O

PhHN NN

N

O

O

NH

O

PhHN NN

N

O

CH3

O

O

NH

O

N

CN

NH

N

NHN

CH3

CH3

Figure 3.3

15 16

17

18

Telomerase inhibitors have been touted as a novel cancer specific therapy, as most tumor

cells have high expression of telomerase, whereas most normal somantic cells express

low or undetectable levels of telomerase. Telomerase is a reverse transcriptase that

prevents the mortality checkpoints evoked by programmed telomere shortening during

each round of cell division. Expression hTERT, the catalytic subunit of telomerase,

appears to be a key determinant for telomerase activity. Insufficient hTERT expression in

most mortal somatic cells cannot produce enough telomerase to maintain telomere length

during cycles of chromosome replication. The hTERT is highly expressed approximately

90% in stem cells, germ cell lines, and most human tumors, and its inhibition represents a

strategy for the development of selective anti-cancer drugs. It is also well accepted that

human cancer cells achieve immortalization in large part through the illegitimate

activation of telomerase expression. The reactivation of telomerase activity in most

57 

 

cancer calls supports the concept that telomerase is a relevant target in oncology, and

telomerase inhibitors have been proposed as new potential anti-cancer agents. The

important roles of telomerase in tumor initiation and cellular immortalization have led to

the identification of telomerase as a potentially important molecular target in cancer

therapeutics. The development of telomerase inhibitors and G-quadruplex stabilizers has

emerged as a highly promising approach. Telomerase is important in tumor initiation and

cellular immortalization. Given the striking correlations between telomerase activity and

proliferation capacity in tumor cells, telomerase had been considered as a potentially

important molecular target in cancer therapeutics. A series of 2,7-diamidoanthraquinone

were designed and synthesized. They were evaluated for their effects on telomerase

activity, hTERT expression, cell proliferations and cytotoxicity. Few compounds shown

bellow (19, 20, 21), potent telomerase inhibitory activity, while compounds activated

hTERT expression in normal human fibroblasts. The results indicated that 2,7-

diamidoanthraquinones represent an important class of compounds for telomerase-related

drug developments.

O

O

NHCO(CH 2)2NHCH 2N(CH 3)2(H3C)2NH 2CHN(H 2C)2OCHN

O

O

NHCO(CH 2)2N(CH 2CH 3)2(H 3CH 2C) 2N(H 2C) 2OCHN

O

O

NH

N

NH

N OO

Figure 3.4

19

20

21

58 

 

Hsu-Shan Huang et al39 has designed and synthesized a series of 2,7-disubstituted

amidoanthraquinone derivatives (figure 3.4) and evaluated their effects to telomerase

activity as inferred from the TRAP assay and collated some selected compounds growth-

percent against tumor cell lines in vitro (NC160 assays). The antiproliferative effect and

hTERT repressing activity of these synthesized compounds were also determined results

indicated that the 2,7-disubstituted amido-anthraquinones are potent telomerase

inhibitors. Results indicated that only few compounds showed significant cytotoxic

activity. The growth percent values at 10 -5 molar for 60 cancer cell lines, it is especially

notable that compound with side chain NHCO-CH2N(CH3)2 (19) displayed relatively

potent and differential cytotoxic activity.

Telomerase is a ribonucleoprotein for telomere maintenance which utilizes its RNA

component as the template to extend telomeric DNA length. The activity of telomerase

could be detected in about 85-90% of tumor cells, (where as it is low or not present in

most somantic cells). Thus, the maintenance of telomere length is considered as a

biological marker for determining the proliferation of cancer cells. Chia-Chung Lee et

al40 group have reported a series of amidoanthraquinones at 1,4-, 1,5-, 2,6- and 2,7-

position that showed diversely in vitro antitumor activity and telomerase inhibitory

activities. They also reported the design and synthesis of a series of novel asymmetrical

mono- or disubstituted 1,2-diamidoanthraquinone derivatives. These compounds were

evaluated for cytotoxicity and telomerase inhibitory activity using Cell Cultures and

Sulfornodamine B(SRB) assay, and repressing hTERT-expression, respectively. Among

these derivatives, few compounds showed the highest potency against PC-3 (prostate

cancer) with IC50 from 0.95 mM to 2.64 mM (figure 3.5)

.

O

O

NHCOC6H5

NH

O

Cl

Figure 3.5

22

59 

 

In the course of their continuous search for new antitumor agents from anthraquinone

moiety, Chia-Chung Lee et al described a method of synthesizing diversely symmetrical

or asymmetrical substituted 1,2-diamidoanthraquinone derivatives and comparing to their

cytotoxicity and telomerase activity. Focused attention on the role of systematic

synthesized tricyclic pharmacophore bearing the symmetrical or asymmetrical side chain

linked to the planar anthraquinone moiety and to understand the basis of tricyclic system

selectivity. Forty new compounds have been prepared among which twenty-seven

displayed a broad spectrum of antitumor activity below 10 mM range.

A single molecule containing features of both Intercalating (example; Mitoxantrone) and

alkylating agent (example; Cyclophosphamide,23) 1,4-Bis-(2,3-epoxypropylamino)-9,10-

anthracenedione (25) was synthesized and was found to be a potent antitumor agent.

Derivatives of this compound containing planar skeleton and diamino side chain

substitution pattern of mitoxantrone and the alkylating epoxide moiety of teroxirone

(24)41 and evaluated for in vitro cytotoxic activity in several cell lines. ED50 of less than

40ng/ml against human epidermoind carciroma (KB cells)42 as shown in figure 3.6.

              

N

N

N

O O

O

O

O

O

(24)O

O

NH

N H

O

O

(25)

Figure 3.6

P

N H

O NO

C l

C l

23

60 

 

A red pigment that accumulates in cultures of a Drechslra avenae pathotype with

specificity for Avena sterilis was isolated and identified as the anthraquinone cynodontin

(3-methyl-1,4,5,8-tetrahydroxyanthraquinone)(26) As shown in figure 3.7, satisfactory

yield of compound was obtained with 20-60 day incubations at temperatures between 20

and 27oC. Cynodontin (26) was tested in vitro for fungitoxicity and was found to be a

potent inhibitor of the growth of sclerotinia minor, Sclerotinia sclerotiorum and to a

lesser extent, of Verticillium dahliae. The ED50 values obtained with these fungi were of

the same order of magnitude as those of the commercial fungicides dicloran and

carbendazim, which were used as reference chemicals. In contrast, the growth of a

number of other fungi was not significantly inhibited by cynodontin. Anthraquinone and

two other anthraquinone derivatives, emodin and chrysophanol, which were also included

in the tests, did not affect the growth of the cynodontin-sensitive fungi. It thus appears

that the type and position of the substitution at the C-ring play a role in the expression of

antifungal activity.

O

O

OH

OH

OH

OH

CH3

(26)

Figure 3.7

The growth of the majority of the fungi used in the fungitoxicity tests was not affected by

cynodontin at concentrations of up to 100 µg/ml. Antifungal activity would probably

have not been detected if the three members of the family Sclerotiniaceae of the cup

fungi (Discomycetes) had not been included in these tests. Cynodontin was recognized as

a potent inhibitor of the mycelial growth of B. cinerea, S. minor, and S. sclerotiorum. The

ED50 values of 5.25, 4.31, and 5.52, respectively, were comparable to those obtained

with dicloran, which is used commercially to control diseases caused by these

pathogens43.

61 

 

3.1.3 Anthraquinones in photovoltaics and in electric devises;

The rapidly growing global energy demand and the increase of CO2 emissions associated

with the burning of fossil fuels are the key driving forces for the development of

inexpensive renewable energy sources. The sun is the primary source of most forms of

energy found on the earth and can give out a large amount of solar energy. Solar energy

is one of most important renewable sources of energy in this century, which can be used

over and over again. Besides, it causes little pollution and does not contribute to the

greenhouse effect. In this context, polymeric solar cells (PSCs) have attracted

considerable attention for their great advantages over the existing inorganic solar cells,

such as ease of processing, light weight, flexibility, low cost, and so forth. The bulk

heterojunction) concept has improved the power conversion efficiency (PCE) of the PSCs

significantly by forming a donor acceptor bicontinuous interpenetrated network, which

creates large interfacial areas between the polymers and electron acceptors (e.g. fullerene

derivatives), thus leading to efficient photoinduced charge separation in a device. In order

to capture a larger portion of solar energy, various types of low-bandgap polymers have

been designed and investigated as a new donor structure in PSCs. Although recent

advances in organic photovoltaics are largely based on conjugated organic polymers, a

possible alternative approach to harvest sunlight for generating electrical power involves

adding metals into organic-based polymers. This interest derives from the fact that

incorporation of heavy metals into an organic framework can have a significant influence

on their electronic and optical properties. Among these, metal-containing polymers stand

out to be particularly interesting candidates in this area. Li Li a, Wing-Cheong et al44

reported the synthesis, characterization and photovoltaic properties of some donor-

acceptor-based metal acetylide polymers containing electron-deficient 9,10-

anthraquinone (figure 3.8), A class of soluble, solution-processable platinum(II) acetylide

polymers functionalized with electron-deficient 9,10-anthraquinone spacer and their

corresponding diplatinum model complexes were synthesized and characterized. The

organometallic polymers exhibit good thermal stability and show low-energy broad

absorption bands in the visible region. The effect of the presence of thiophene rings along

62 

 

the polymer chain on the optical and photovoltaic properties of these metallated materials

was examined. The low-bandgap polymer with thiophene, anthraquinone, thiophene

(donor, acceptor, donor) fragment can serve as a good electron donor for fabricating bulk

heterojunction polymer solar cells by blending with a methanofullerene electron acceptor.

At the same donor:acceptor blend ratio of 1:4, the light-harvesting ability and solar cell

efficiency notably increase when the anthraquinone ring is sandwiched by two thiophene

units. Photoexcitation of such polymer solar cells results in a photoinduced electron

transfer from the p-conjugated metallopolymer to [6,6]-phenyl C61-butyric acid methyl

ester with power conversion efficiency up to w 0.35%. For safety concern, these

metallopolymers were also tested for possible cytotoxicity and they do not show

significant cytotoxic activity on human liver derived cells and skin keratinocytes at

reasonable doses, rendering these functional materials safe to use in practical devices.

O

O

SS

C6H13H13C6

Pt-

Pt-PBu3

Bu3P

PBu3

PBu3

m

n

m

n

Figure 3.8

27

Discotic liquid crystals have been attracting growing interest not only because of

fundamental importance as model systems for the studay of charge and energy transport

but also due their potential application in organic electronic devices. The 1,2,3,5,6,7-

hexahydroxy-9,10anthraquinone (28), commonly known as Rufigallol, (figure. 3.9) is one

of the earliest systems reported to form columnar mesophases. Over the past 25 years,

more than 100 discotic liquid crystals based on this core have been realized and studied

for various physical properties45.

63 

 

O

OOH

OH

OH

OH OH

OH

(28)

Figure 3.9 3.2 Importance of Present work;

N-Alkyl derivatives of anthraquinone-9,10-diones exemplified by drugs ametantrone(2)

and mitoxantrone(1)46,47,48 are potent anti-cancer agents, Worldwide, cancer remains a

leading cause of death. According to world Health Organiszation (WHO) of 58 million

deaths in 2005, cancer accounted for 7.6 million (or 13%). Deaths from cancer in the

world are projected to continue rising, with an estimated 9 million people dying from

cancer in 2015 and 11.4 million dying in 2030. WHO also states that 40% of cancer can

be prevented by a healthy diet, physical activity, and not using tobacco. The lung cancer

is the most common cause of cancer death for men and women49,

Chemotheraphy is the treatment of diseases such as cancer using chemicals. Since 1960’s

the development and use of drugs has significantly improved the prognosis for some

types of cancer. Most types of cancer chemotherapy will consist of a number of different

drugs, this is known as combination chemotherapy. Chemotherapy may be given in a

variety of ways; intravenously, intramuscularly, orally, subcutaneously, intralesionally i.e

directly into a cancerous area, intrathecally i.e. into the fluid around the spine, or

Topically-medication will be applied onto the skin. Since the drugs used to kill cancer

cells also damage normal cells, the searching for antineoplastic agents with improved

selectivity to malignant cells remains the central task for drug discovery and development 49,50. According to the survey published in 1997 more than 315 drugs were under

development in the United states for the treatment of cancer 51a-c. According to the review 52 of ca. 90 approved cancer drugs, more than 60% are of natural origin or modeled on

natural products.

However, while the additional hydroxyl groups present at 5-and 8-positions of

mitoxantrone lead to tenfold increase in its antineoplastic activity over ametantrone. This

64 

 

beneficial enhancement is unfortunately countered by a ten fold increase in its

cardiotoxicity. Mitoxantrone is drug of choice for the treatment of worsening relapsing-

remitting multiple sclerosis (ms), secondary progressive MS.51,52 However, these clinical

applications are limited due to the accumulative and irreversible cardiotoxicity. Recently.

Mitoxantrone was found to induce a progressive increase in mitochondrial mass in the

cancer cells but not in the cardiac cells 53.

This suggests the opportunities to look for novel anthraquinones with reduced

cardiotoxicity. The planar tricyclic structure of anthraquinone is essential for interacting

with DNAbase pairs. Perhaps by introducing different side chains at 1- and 4-positions in

anthraquinone skeleton with a variety of substituents, which may form additional

interactions with the double-strand DNA (ds-DNA) or DNA-topoisomerase II (TOP II)

cleavable complex to increase their binding affinities and selectivity

3.3 Results and Discussion;

Synthesis of 1,8-Dihydroxy-4,5-diaminoanthraquinone (31) involves three steps starting

from chrysazin (1,8-Dihydroxy anthraquinone) using Diethyl sulphate in DMF in

presence of Potassium carbonate to obtain 1,8-Diethoxy anthraquinone(29) which when

nitrated with nitrating mixture in presence of Boric acid yielded high pure 1,8-Diethoxy-

4,5-dinitroanthraquine(30).This on treatment with Hydroiodic acid resulted not only in O-

deethylation, but also in reduction of nitro groups with more than 95% yield of 4,5-

Diaminochrysazin(31)54 as described in chapter 2. Treatment of (31) with sodium

hydrosulfite in alkaline solution led55 to leuco-1,4,5,8-tetrahydroxyanthraquinone (32),

this on condensation with appropriate amine in an inert gas atmosphere followed by

oxidation of the resulting intermediate with oxygen gave 1,4-diamino substituted

anthraquinones (33-37) as shown in Scheme 3.1.

All compounds were fully characterized by IR, NMR and Mass spectroscopy

65 

 

O

O

OH OH O O

O

CH3

O

CH3

O

NO2

O

O NO2

O

CH3 CH3

OH O

O

OH

NH2NH2

OH

OH OH

OHO

O

31

32

33 - 37

Scheme 3.1

29

30

a

b

c

d

e

Scheme 3.1; reagents and conditions; (a) Diethyl sulphate, K2CO3, DMF, 1000C, (b) H2SO4, HNO3, Boric

acid (c) Hydroiodic acid, (d) Aq. N-buanol, Sodium hydrosulfite, 600C, (e) beta-alanine , Amine(benzyl,

cyclohexyl, cyclopentyl, cyclopropyl), ethanol, O2, 500C,

66 

 

OH

OH

O

O

NH

OH

33

OH

OH

O

O

NH

NH

34

OH

OH

O

O

NH

NH

35

OH

OH

O

O

NH(CH2)2COOH

NH(CH2)2COOH

36

OH

OH

O

O

NH

NH

37

Biological Activity; Anti-neoplastic :

Compounds 33-37 subjected to in vitro testing using breast carcinoma MCF-7 & cervical

cancer Hela cell lines for their cytotoxicity (Figure 3.1and table 1) in comparison with

mitoxantrone, it is observed that all compounds 33 – 37 exhibited very good inhibitory

activity in both cell lines in comparision with mitoxantrone. while IC50 values in MCF-7

Breast cancer cell line for all compounds ranged between 100 and 120 nM, but in Hela

cervical cancer cell line still lower concentrations ranging 70 to 80 nM were effective.

1,4-biscyclohexylamino-5,8-dihydroxyanthraquinone (34) exhibited relatively better

inhibitory activity while bisbeta-alanino-5,8-dihydroxyanthraquinone (36) showed

67 

 

relatively less inhibitory activity against both cell lines. Relatively better inhibitory

activity exhibited by compound (34) may be due to flexible nature of cyclohexyl moiety

which undergoes different conformations under different conditions and also due to

lipophilicity conferred to the molecule by the cyclohexyl group and which may increase

the affinity for the cell membrane 56. Since these molecules showed very good in-vitro

activity we plan to carry out in-vivo testing in future to find out the cardiotoxicity.

Fig.3.1: Cytotoxic activity : MXPHK 1 ( Compound 33); MXPHK 2 ( Compound 34);

MXPHK 3 ( Compound 35); MXPHK 4 ( Compound 36); MXPHK 5 ( Compound 37);

68 

 

Conclusion;

Synthesiszed five analogues of mitoxantrone starting from 4,5-Diaminochrysazin (31) in

two steps. Treatment of (31) with sodium hydrosulfite in alkaline solution led55 to leuco-

1,4,5,8-tetrahydroxyanthraquinone (32), this on condensation with appropriate

amine(benzylamine, cyclohexyl amine, cyclopentyl amine, beta-alanino and

cyclopentylamine) in an inert gas atmosphere followed by oxidation of the resulting

intermediate with oxygen gave 1,4-diamino substituted anthraquinones (33-37).

Compounds 33-37 subjected to in vitro testing using breast carcinoma MCF-7 & cervical

cancer Hela cell lines for their cytotoxicity in comparison with mitoxantrone. It is

observed that all compounds 33 – 37 exhibited very good inhibitory activity in both cell

lines in comparision with mitoxantrone, 1,4-biscyclohexylamino-5,8-

dihydroxyanthraquinone (34) exhibited relatively better inhibitory activity while bisbeta-

alanino-5,8-dihydroxyanthraquinone (36) showed relatively less inhibitory activity

against both cell lines,

69 

 

3.4 Expeimental;

3.4.1 General methods/ instrument

Chemicals and reagents of laboratory grade were obtained from local dealers and were

used without further purification IR spectra were recorded on Nicolet avatar 320 FT-IR

spectrometer, 1H and 13C NMR spectra were recorded in CDCl3 / DMSO-d6 at 200MHz

on Bruker A G spectrometer Chemical shifts are reported in ∂ units down field from T M

S as internal standard Mass spectra were recorded using GC-MS-QP2010S (direct probe)

and Q-TOF microTM AMPSMAX10/6A system.

3.4.2. Preparation of leuco-1, 4, 5, 8-tetrahydroxyanthraquinone (32);

To a sodium hydroxide aqueous solution (10% 1L) containing n-butanol (50ml) was

added 4,5-diaminochrysazin(31)[preparation as described in chapter 2](38.3g, 142mmol)

with stirring and the resulting dark-blue suspension was de-aerated by stirring for 15min

with a stream of N2 which was bubbled through it. Sodium hydrosulfite(22.5g, 123mmol)

was gradually added with stirring, while the reaction mixture was heated and maintained

at 600c for 30 min. Then the reaction mass was cooled to room temperature, neutralized

with HCl(4N) and allowed to stand. The resulting precipitate was collected by filtration,

washed with water and dried in vaccuo at 500c to give leuco-1,4,5,8-

tetrahydroxyanthraquinone(32) 35g, 90% as a brown flake, MP.230-2350c

(decomposition); 1H NMR (DMSO-d6); ∂ 3.0 (s, 4H), 7.15 (s, 2H), 9.8 (s, OH), GC-MS

(DI); 274 (M+)

3.4.3.0 General procedure for the preparation of final compounds (33-37);

Condensation of leuco-1, 4, 5, 8-tetrahydroxyanthraquinone (32) with large excess of

appropriate Amine in ethanol in an inert gas atmosphere followed by oxidation of

resulting intermediate with oxygen at 500c for 15hrs gave 1,4-disubstituted

anthraquinone. These compounds were purified by subjecting to silica column

chromatography.

70 

 

3.4.3.1. 1-benzylamino-4, 5, 8-trihydroxyanthraquinone-9,10-dione(33);

Using compound 32(1g, 3.64mmol) and benzylamine (5g, 46.7mmol) as starting

materials the Title compound 33 was obtained as a blue-Brown solid (0.3g, yield=23%);

MP194oC (decomposition); IR (K Br); 2900, 2800,1569, 1496, 1454, 1392, 1350, 1172,

1087, 968, 790, 744, 698, 551, 466cm-1 ; 1H NMR (CDCl3); ∂ 4.64(d, 2H), 7.16-7.27(m,

9H), 10.34(b, 1H), 12.39(s, 1H), 13.01(s,1H), 13.32(s, 1H); 13C NMR(CDCL3, 50MHz);

∂ 46.9, 124.2, 126.3, 126.8, 127.6, 128.9, 129.0,156.8, 183; HRMS: 362.2043(M+H)

3.4.3.2. 1, 4-bis (cyclohexylamino)-5, 8-dihydroxyanthraquinone-9,10-dione(34);

Using compound 32 (1g, 3.64mmol) and cyclohexylamine(5g, 50mmol) as starting

materials the compound 34 was obtained as a blue-Brown solid (1.0g, yield=63%); MP

220oC (decomposition); IR (K Br); 2927, 2854, 1566, 1446, 1392, 1149, 1080, 956, 825,

729, 675, 551, 482, 428cm-1; 1H NMR (CDCl3, 200MHz); ∂ 1.35(m,20H), 3.8 (m, 2H),

7.1(s,2H), 7.26(s, 2H), 10.8(b, 2H), 13.8(s, 2H); 13C NMR (CDCl3, 50MHz); ∂ 24.3,

25.4, 33.2, 50.9, 108.6, 115.5, 124.3, 129.2, 145.7, 155.2, 184.6; HRMS :434.320(M+)

3.4.3.3. 1, 4-bis (cyclopentylamino)-5, 8-dihydroxyanthraquinone-9, 10-dione(35);

Using compound 32 (1g, 3.64mmol) and cyclopentylamine(5g, 58.8mmol) as starting

materials the Title compound 35 was obtained as a blue-Broun solid (0.3g, yield=20%);

MP 240oC (decomposition); IR (K Br); 2954, 2866, 1604, 1558, 1500, 1450, 1396, 1353,

1164, 1080, 972, 825, 663, 547, 458cm-1; 1H NMR (CDCl3, 200MHz); ∂ 1.58(m,16H),

4.1(m,2H), 7.1(s, 2H), 7.26(s, 2H), 10.8(b, 2H), 13.8(s, 2H); 13C NMR (CDCl3, 50MHz);

∂ 24.5, 34.5, 54.4, 109.2, 115.9, 124.9, 125, 146.5, 155.7, 185.2; HRMS(ES+) =

406.2104(M+).

3.4.3.4. 1, 4-bis (beta-alanino-5, 8-dihydroxyanthraquinone-9, 10-dione (36);

Using compound 32 (1g, 3.64mmol) and beta-alanine(5g, 56.1mmol) as starting materials

the Title compound 36 was obtained as a blue-Brown solid (0.3g, yield=20%); MP

>280oC; IR (K Br); 3400, 2900, 2657, 2333, 1743, 1693, 1608, 1562, 1454, 1350, 1172,

1076, 972, 825, 628, 547, 470 cm-1; 1H NMR (DMSO-d6, 200MHz); ∂ 2.65(t, 4H),

71 

 

3.69(t, 4H), 7.13(s, 2H), 7.53(s, 2H), 10.57(b, 2H), 13.46(s, 2H); 13C NMR (DMSO-d6,

50MHz); ∂ 34.6, 38.7, 107.7, 115.7, 124.7, 125.5, 147.0, 154.9, 173.1, 183.7; HRMS

(ES+)= 414.2769(M+).

3.4.3.5. 1, 4-bis (cyclopropylamino)-5, 8-dihydroxyanthraquinone-9, 10-dione (37);

Using compound 32 (1g, 3.64mmol) and cyclopropylamine(5g, 87.8mmol) as starting

materials The compound 37 was obtained as a blue-Broun solid (0.3g, yield=20%); MP

>280oC; IR (K Br); 2900, 2800, 1608, 1569, 1454, 1404, 1342, 1299, 1211, 1164, 1022,

960, 806, 628, 474 cm-1; 1H NMR (CDCl3, 200MHz); ∂ 0.83(m, 8H), 2.5(m, 2H), 7.13(s,

2H), 7.72(s, 2H), 10.2(b, 2H), 13.4(s, 2H); 13C NMR (CDCl3, 50MHz); ∂ 7.9, 24.3, 29.6,

110, 115, 124, 125, 147, 155, 186; HRMS(ES+)= 373.1162(M+ Na).

3.4.4 Biological activity;

3.4.4.1 In vitro growth inhibition assay;

The cells were maintained in Dulbecco̒s Modified Eagle̒s Medium (Sigma-Aldrich Inc.,

USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich Inc., USA) in a CO2

incubator. The cytotoxicity of the compounds was measured by MTT assay57. The cells

were planted in a 96-well plate at the density of 10,000 cells per well (Hela) and 10,000

cells per well (MCF-7). After 24 hours, the cells were treated with different

concentrations of analogues of mitoxantrone (25 nM to 400 nM). The cells were further

incubated for 24 hours. The cytotoxicity was measured by adding 5mg/ml of MTT

(Sigma-Aldrich Inc., USA) TO each well and incubated for another three hours. The

purple formazan crystals were dissolved by adding 100μl of DMSO to each well. The

absorbance was read at 570 nm in a spectrophotometer (spectra Max 340). The cell death

was calculated as follows;

Cell death = 100- [(test absorbance / control absorbance) X 100]

The test result is expressed as concentration of a test compound which inhibits the cell

grouth by 50% (IC50).

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Table 1: Cytotoxic activity of compounds 33-37 on HeLa cell line (cervical epithelial

adenocarcinoma cell line) and MCF-7 (Breast carcinoma cell line).

Compound IC50 (nM) + SD] *HeLa IC50 (nM) + SD] *MCF-7

Mitoxantrone 59.36 ± 5.24 101.16±18.49

33 73.82 ± 5.23 107.93±3.9

34 73.7 ± 0.89 105.78±5.18

35 75.66 ±13.61 112.65±5.69

36 79.51±7.28 115.2±10.53

37 76.88±3.49 104.48±7.86

* Values are average of three independent experiments ± standard deviation, conducted in

triplicate for each concentration.

73 

 

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