Synthesis, characterization and biological applications of selenoureas having ferrocene and substituted benzoyl functionalities

Article abstract of DOI:10.1016/j.poly.2019.05.027

In this article we have comprehensively reported the synthesis, complete chemical characterization (FTIR, multinuclear NMR, single crystal XRD, CHNS, AAS) and biological applications of seventeen ferrocene incorporated selenoureas. Biological applications

Full text of DOI:10.1016/j.poly.2019.05.027

Accepted Manuscript
Synthesis, characterization and biological applications of selenoureas having
ferrocene and substituted benzoyl functionalities
Raja Azadar Hussain, Amin Badshah, Nadeem Ahmed, John M. Pezzuto,
Tammara P. Kondratyuk, Eun-Jung Park, Iqtadar Hussain
PII:
S0277-5387(19)30357-2
https://doi.org/10.1016/j.poly.2019.05.027
POLY 13952
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 April 2019
11 May 2019
13 May 2019
Please cite this article as: R.A. Hussain, A. Badshah, N. Ahmed, J.M. Pezzuto, T.P. Kondratyuk, E-J. Park, I. Hussain,
Synthesis, characterization and biological applications of selenoureas having ferrocene and substituted benzoyl
functionalities, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.05.027
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Biological applications of selenoureas. . .
Synthesis, characterization and biological applications of selenoureas having ferrocene and
substituted benzoyl functionalities
Raja Azadar Hussain*1, Amin Badshah*1, Nadeem Ahmed2, 3, John M. Pezzuto4, Tammara P.
Kondratyuk4, Eun-Jung Park4, Iqtadar Hussain5
1. Co-ordination Chemistry Laboratory, Department of Chemistry, Quaid-i-Azam
University, Islamabad (45320), Pakistan.
2. College of Life Sciences, Northwest A & F University, Yangling, Shaanxi, China.
3. Department of Botany, Mohi-ud-Din Islamic University, Nerian Sharif, AJ & K.
4. University of Hawaii at Hilo, United States.
5. Department of Mathematics, Statistics and Physics, Qatar University, Doha (2713),
Qatar.
Corresponding Email: hussainazadar@yahoo.com , aminbadshah@yahoo.com , +923009870170
Abstract:
In this article we have comprehensively reported the synthesis, complete chemical
characterization (FTIR, multinuclear NMR, single crystal XRD, CHNS, AAS) and biological
applications of seventeen ferrocene incorporated selenoureas. Biological applications include
comparative DNA binding studies (with, cyclic voltammetry), antioxidant activities (DPPH (2,2-
diphenyl-1-picrylhydrazyl) scavenging), anti-inflammatory activities (nitrite inhibition), phase I
enzyme inhibition activities (aromatase inhibition), phase II enzyme induction studies (quinone
reductase induction) and cytotoxic studies against neuroblastoma (MYCN2 and SK-N-SH), liver
1

cancer (Hepa 1c1c7) breast cancer (MCF-7) and human cervical carcinoma HeLa cells (ATCC®
CCL-2). All the compounds showed decent structure dependent biological applications.
Keywords: Selenourea, ferrocene, enzymology, cytotoxicity, free radicals
1. Introduction
Chemotherapy and radiotherapy are two main routes for treatment of cancer which
destroy the cancer cells by apoptosis, mitotic catastrophy, atrophy or terminal growth arrest [1].
However, combination of different methodologies is adopted for treatment of cancer, depending
upon the condition of patients, for example in case of advanced stage ovarian cancer (FIGO stage
IC-IV), initial debulking surgery is followed by carboplatin-paclitaxel combination
chemotherapy. Chemotherapeutic drugs such as melphalan and cyclophosphamide (alkylating
agents) doxorubicin and epirubicin (anthracyclines) podophyllin derivatives (etoposide),
cisplatin, carboplatin and camptothecins follow a mechanism which is mainly dependent upon
the formation of reactive oxygen species (ROS) or free radicals [2]. These free radicals are too
much reactive and have the side effects of peripheral neuropathy, nephrotoxicity, ototoxicity, and
cardiotoxicity. Therefore it is need of the time to synthesize the drugs which should be more
active and less toxic.
Organoselenium derivatives have shown decent activities as anti-infective drugs
photochemotherapeutic agents, antitumor agents, cytokine inducers/immunomodulators,
antioxidant defense enzymes, enzyme inhibition, antihypertensive and carditonic agents and
DNA binders [3-8]. Selenoureas specifically are important enzyme inhibitors, free radical
scavengers and anticancer agents [9-11]. Inorganic selenium species (selenium and selenite) are
more toxic than organoselenium moieties (selenomethionine and methylseleninic acid) but have
2

their own importance. So we came up with an idea to synthesize a class of hybrid compounds i.e.
organometallic ferrocene incorporated N,N-disubstituted selenoureas which should be active like
inorganic selenium species and less toxic like orgaoselenium derivatives [12, 13]. Incorporation
of ferrocene into selenourea is important because it is not only a good spectroscopic and
electrochemical marker but its presence within the structure of compound has a positive effect on
the biological applications of the compound. For example if tamoxifen (active against breast
cancer) is incorporated with ferrocene then the development of resistances against it can be
reduced with higher activities [14]. In another attempt it has been reported that Illudin M
(derived from mushrooms) which is active against prostate, ovarian, pancreatic, renal and breast
cancer when connected with ferrocene shows less toxicity for nonmalignant fibroblasts while
more cancer selectivity and more cell line specificity than parent Illudin M [15]. Ferrocene
incorporated (FI) raloxifens [16], FI nilutamides[17, 18] and FI testosterones [19-22] have shown
better results than their parent compounds. Moreover ferrocene is nontoxic and lipophilic which
increases the compatibility of the compounds with the cellular media [23]. We have recently
reviewed the biological applications of selenoureas [23] and to the best of our knowledge
detailed sequential anticancer activities for selenoureas which should start from activities against
cancer initiation, proceed through cancer propagation and end up with cancer termination have
not been carried out.
In this article we have reported the synthesis of a new class of hybrid (between pure
organic and pure inorganic) organometallic compounds in the form of seventeen ferrocene
incorporated N,N-disubstituted selenoureas (FIS) and have scanned them for DNA binding
studies (with, cyclic voltammetry), antioxidant activities (DPPH (2,2-diphenyl-1-picrylhydrazyl)
scavenging), anti-inflammatory activities (nitrite inhibition), phase I enzyme inhibition activities
3

(aromatase inhibition), phase II enzyme induction studies (quinone reductase induction) and
cytotoxic studies against neuroblastoma (MYCN2 and SK-N-SH), liver cancer (Hepa 1c1c7)
breast cancer (MCF-7) and human cervical carcinoma HeLa cells (ATCC® CCL-2).
2. Materials and Methods
2.1 General instrumentation
Melting points were determined in a capillary tube using Gallenkamp (U.K) electrothermal
melting point apparatus. Infrared spectra were taken on Thermoscientific NICOLET 6700 FTIR
1
between 4000-400 cm-1. H and 13C spectra were recorded between 0-13 ppm and 0-210 ppm
respectively on Jeol JNM-LA 500 FT-NMR. Si(CH3)4 was used as internal reference. Elemental
analysis was performed using a LECO-932 CHNS analyzer while the Fe concentrations were
determined on an Atomic Absorption Spectrophotometer Perkin Elmer 2380.
2.2 DNA binding studies
Commercial salmon DNA was solubalized in doubly distilled water to prepare a stock solution of
5.88 x 10-4 M from which working concentrations of DNA were prepared. Concentration of
stock solution was measured by UV absorbance at 260 nm using an epsilon value of 6600 M-
1cm-1. This DNA was protein free because A260/A280 > 1.8. Working solutions for DNA binding
studies were prepared by our previously reported method [6].
2.3 Cyclic voltammetry (CV)
In CV studies a three electrode system was used consisting of working (platinum disc electrode
with a geometric area of 0.071 cm2) reference (Ag/AgCl) and auxiliary electrodes (platinum
electrode with geometric area much greater than working electrode). Changes in peak current
and peak potential provided the information about drug-DNA binding constant and mode of
4

interaction respectively. Changes in peak current of the free drug (test compounds in absence of
DNA) by the addition of varying concentrations of DNA were used to evaluate the drug-DNA
binding constant with the help of following equation [24]:
log (1/[DNA]) = log K + log (I/Iₒ - I)
Eq 1
Where K is the binding constant and Iₒ and I are the peak currents of free drug and DNA bound
drug respectively.
For the measurement of binding site size (s) following equation was used [25]:
Cb/Cf = K[(free base pairs)/s]
Eq 2
Where s is the binding site size in terms of base pair, K is the binding constant, Cf is the
concentration of free species and Cb represents concentration of drug-DNA bound species.
Considering the concentration of DNA in terms of nucleotide phosphate, the concentration of
DNA base pair will be taken as [DNA base pair]/2 and Eq 2 will be written as [26]:
Cb/Cf = K[(DNA base pair)/2s]
Eq 3
The value of Cb/Cf is equal to (Iₒ - I/I) which are the values of experimental peak currents.
For the determination of diffusion coefficient of free and DNA bound drug following form of
Randles-Sevcik Equation was used [27]:
Ipa = 2.69 x 105 n3/2 A Cₒ* Dₒ1/2 ʋ1/2
Eq 4
Where Ipa is the anodic peak current in ampere, Cₒ* is the reductants’s concentration in molcm-3,
ʋ is the scan rate in volts-1, A is the geometric area of the electrode in cm2, n is the number of
5

electrons involved in the process and Dₒ is the diffusion coefficient in cm2s-1. Same procedure
was followed for the interaction of DNA with FIS.
2.4 Free radical scavenging activity
Reducing abilities of all the FIS were determined by monitoring the decrease in the absorbance
(with micropipette reader or UV-vis spectrophotometer) at 517 nm of 1,1-diphenylpicrylhydrazyl
radical to form 1,1-diphenylpicrylhydrazine. Scavenging activity values were calculated by
following equation:
Scavenging activity (%) = Aₒ-A/Aₒ x 100
Eq 5
Where Aₒ is the absorbance of DPPH alone and A is the absorbance of DPPH with different FIS.
2.5 Nitrite inhibition activity
Nitrite inhibition assay was performed according to a previously reported method [28].
According to this method, inside a 96-well plate, 10 x 104 cells/plate of RAW 264.7 cells
(murine leukemia) were seeded in 10% FBS containing DMEM (Dulbecco's Modified Eagle
Medium) for 24 h. Then the media was replaced with 1% FBS-containing (190 μL) phenol red
free DMEM and the cultured cells were treated with 10 μL of test samples in 10% DMSO for 15
min. After this step each plate was treated with 1 μgmL-1 of LPS for 20 h. Quantitative
estimation of nitrite (which is the major oxidation product of NO) was carried out by reacting the
incubation media with Griess reagent (90 μL of 1% sulfanilamide in 5% phosphoric acid, and 90
μL of N-(1-Naphthyl)ethylenediamine). Absorbance was scanned at 540 nm according to Eq 5
for percentage inhibition values.
2.6 Aromatase inhibition activity
6

For aromatase inhibition activity [28], test samples of synthesized compounds were preincubated
with NADPH generating system at 37 °C for 10 min. Further incubation for 30 min at 37 °C was
carried out before quenching with NaOH after adding the enzyme and substrate to the plates
which contained NADPH generating system. Addition of NaOH terminates the reaction and after
complete termination the plates were incubated for further 2 h to improve the signal to noise
ratio. Fluorescence measurements were carried out at 485 and 530 nm for triplicate readings of
five different concentrations of test samples using Letrozole as a positive control.
2.7 Quinone reductase assay
QR (quinone reductase) assay was carried out with hepa 1c1c7 (murine hepatoma) cells which
were plated with 200 μL per well with a cell density of 0.5 x 104 cells/mL in MEM-α (minimum
essential medium) without ribonucleosides or deoxyribonucleosides, supplemented with
antimycotic/antibiotic and 10% FBS for 24 h in a carbon dioxide incubator. After 24 h medium
was replaced with 190 μL of fresh medium and 10 μL of test samples were added for a final
concentration of 4 ppm. After 48 h of incubation, cell membrane was permeabilized with
digitonin and enzyme activity was measured by the reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to a blue formazan. 4-bromoflavone was used as a positive
control and production was measured by absorption at 595 nm [29] with a total protein assay
using crystal violet staining run, in parallel [30]. Test samples with an induction ratio > 2 were
tested in a 5 fold serial dilutions to determine the CD values [28] .
2.8 Cytotoxic activities
The cytotoxic activities of the test compounds were carried out against neuroblastoma cell lines
MYCN and SK-N-SH, hepa 1c1c7 liver carcinoma cell line, human cervical
7

carcinoma HeLa cells (ATCC® CCL-2) and human breast cancer cell line MCF-7. Briefly, 10
μL of test compounds in all the three series were filled in 96-well plate using 10% DMSO in
phosphate buffered saline along with 190 μL of cells (5 x 104 cells/mL) and incubated at 37 °C
for 72 h in a carbon dioxide incubator. After quenching the incubation with 50 μL of cold 20%
trichloroacetic acid, the cells were washed, air dried and stained with 0.4% SRB in 1% acetic
acid for 30 min at room temperature. Wells were then washed 4 times with 1% acetic acid
solution and plates were dried for 12 h. Bound dye was solubalized in trisbase (200 μL, 10 mM,
pH 10) for 10 min on gyratory shaker. For the measurement of optical density at 515 nm,
microplate reader (bio-tek) was used which gave information about percent survival. Zero day
control was performed in each case with all the procedure mentioned above [28] .
Ferrocene, anilines, sodium nitrite, diethyl ether, acetone, DMSO, Pd-charcoal,
carboxylic acid chlorides and hydrazine were purchased from Sigma Aldrich. Ferrocenyl anilines
were synthesized by a procedure reported by our group previously [31].
3. Experimental
For the synthesis of FIS, respective carboxylic acid chloride was reacted with potassium
selenocyanate in 1:1 using dried acetone as a solvent inside a two necked round bottom flask
under constant magnetic stirring. This resulted in the formation of white precipitates of
potassium chloride with yellow isoselenocyanate in the solution. The reaction was carried out for
3 h to completion and then 3-methyl-4-ferrocenylphenylaniline was added (1:1 with acid
chloride and KSeCN). The reaction took almost 4 h for completion and the progress of reaction
was monitored with the help of thin layer chromatography. The resulting orange colored solution
was mixed with cold water under constant magnetic stirring. This removed the potassium
8

chloride suspension and precipitated the product which was then filtered, dried and washed with
n-hexane and methanol.
Seventeen new FIS (a1 to a17) were synthesized by the reaction of 3-methyl-4-
ferrocenylphenylaniline with different isoselenocyanates according to Scheme 1. 3-methyl-4-
ferrocenylphenylaniline was synthesized by using our previously reported procedure [4-7, 23,
32-37]. Structural elucidation of compounds has been given below with structural formula:
3.1 1-acetyl-3-(3-methyl-4-ferrocenylphenyl)selenourea (a1)
Yield: 69%; Decomposition temperature: 136-137 °C; FT-IR (cm-1): 3400-3100 (NH
stretching broad), 3031 (Aromatic-H stretching), 2956, 2816 (Aliphatic-H stretching), 1659
(C=O stretching), 1573, 1551, 1508 (C=C stretching of aromatic ring), 1382, 1370 (CH3
1
bending) 1243 (C=Se stretching); H-NMR (300 MHz, Acetone, δ (ppm): 13.06 (s, 1H,
CSeNH), 10.62 (s, 1H, CONH), 8.14-7.30 (m, 3H, Aromatic), 4.73 (apparent t, 2H, J 2.1 Hz,
ortho on substituted Cp), 4.29 (apparent t, 2H, J 1.8 Hz, meta on substituted Cp), 4.11 (s, 5H,
13
Cp-C1), 2.98 (s, 3H, C5), 2.44 (s, 3H, C8); C-NMR (75 MHz, Acetone, δ (ppm): 178.0 (C6),
167.3 (C7), 136.4 (1C-Ar), 135.6 (1C-Ar), 129.3(1C-Ar), 128.7 (1C-Ar), 125.2 (1C-Ar), 112.1
(1C-Ar) 86.3 (C4), 70.0 (C1), 69.4 (C2), 68.2 (C3), 23.1 (C8), 21.1 (C5); Anal. Calcd. for
C20H20N2OFeSe: C 54.66, N 6.37, H 4.55, Fe 12.75; Found: C 53.43, N 6.21, H 4.51, Fe
12.40%.
9

3.2 1-benzoyl-3-(3-methyl-4-ferrocenylphenyl)selenourea (a2)
Yield: 63%; Decomposition temperature: 134-135 °C; FT-IR (cm-1): 3400-3100 (NH
stretching broad), 3088 (Aromatic-H stretching), 2921, 2849 (Aliphatic-H stretching) 1667 (C=O
stretching), 1584, 1539, 1507 (C=C stretching of aromatic ring), 1361 (CH3 bending) 1251
(C=Se stretching); 1H-NMR (300 MHz, Benzene, δ (ppm): 13.41 (s, 1H, CSeNH), 10.50 (s, 1H,
CONH), 7.92-6.53 (m, 8H, Aromatic), 4.75 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp),
4.36 (apparent t, 2H, J 2.1 Hz, meta on substituted Cp), 4.01 (s, 5H, Cp-C1), 2.44 (s, 3H, C5);
13C-NMR (75 MHz, Benzene, δ (ppm): 179.0 (C6), 168.6 (C7), 137.5 (1C-Ar), 136.2 (1C-Ar),
133.1 (1C-Ar), 132.0 (1C-Ar), 129.1(1C-Ar), 128.9 (2C-Ar), 126.3 (2C-Ar), 124.4 (1C-Ar),
120.1 (1C-Ar), 112.4 (1C-Ar), 86.4 (C4), 70.0 (C1), 69.7 (C2), 68.5 (C3), 21.5 (C5); Anal.
Calcd. for C22H22N2OFeSe: C 59.88, N 5.50, H 4.39, Fe 11.11; Found: C 59.01, N 5.39, H 4.27,
Fe 10.98%.
3.3 1-(2-bromobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a3)
10

Yield: 62%; Decomposition temperature: 163-165 °C; FT-IR (cm-1): 3410-3296 (NH
stretching broad), 3101 (Aromatic-H stretching), 2929 (Aliphatic-H stretching), 1664 (C=O
stretching), 1590, 1552, 1500 (C=C stretching of aromatic ring), 1358 (CH3 bending), 1245
1
(C=Se stretching); H-NMR (300 MHz, Acetone, δ (ppm): 13.39 (s, 1H, CSeNH), 9.10 (s, 1H,
CONH), 8.15-7.35 (m, 7H, Aromatic), 4.58 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp),
4.33 (apparent t, 2H, J 1.8 Hz, meta on substituted Cp), 4.14 (s, 5H, Cp-C1), 2.44 (s, 3H, C5);
13C-NMR (75 MHz, Acetone, δ (ppm): 177.9 (C6), 164.7 (C7), 145.1 (1C-Ar), 136.7 (1C-Ar),
136.0 (1C-Ar), 133.6 (1C-Ar), 133.1 (1C-Ar), 132.4 (1C-Ar), 131.5 (1C-Ar), 128.6 (1C-Ar)
126.3 (1C-Ar), 126.2 (1C-Ar), 124.0 (1C-Ar), 112.3 (1C-Ar), 86.4 (C4), 69.6 (C1), 69.3 (C2),
68.0 (C3), 21.1 (C5); Anal. Calcd. for C25H21N2OFeSeBr: C 51.72, N 4.82, H 3.62, Fe 9.65;
Found: C 51.31, N 4.19, H 3.28, Fe 9.09%.
3.4 1-(3-bromobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a4)
Yield: 67%; Decomposition temperature: 172-173 °C; FT-IR (cm-1): 3417-3312 (NH
stretching broad), 3091 (Aromatic-H stretching), 2948 (Aliphatic-H stretching), 1660 (C=O
stretching), 1586, 1557, 1510 (C=C stretching of aromatic ring), 1370 (CH3 bending), 1244
1
(C=Se stretching); H-NMR (300 MHz, DMSO, δ (ppm): 12.91 (s, 1H, CSeNH), 9.60 (s, 1H,
CONH), 8.44-7.14 (m, 7H, Aromatic), 4.58 (s, 2H, broad, ortho on substituted Cp), 4.33 (s, 2H,
broad, meta on substituted Cp), 4.16 (s, 5H, Cp-C1), 2.45 (s, 3H, C5); 13C-NMR (75 MHz,
11

DMSO, δ (ppm): 175.9 (C6), 164.7 (C7), 145.0 (1C-Ar), 136.2 (1C-Ar), 135.4 (1C-Ar), 133.1
(1C-Ar), 133.0 (1C-Ar), 132.8 (1C-Ar), 131.5 (1C-Ar), 127.1 (1C-Ar), 126.0 (1C-Ar), 125.6
(1C-Ar), 124.0 (1C-Ar), 112.1 (1C-Ar), 85.1 (C4), 69.1 (C1), 69.0 (C2), 66.0 (C3), 20.9 (C5);
Anal. Calcd. for C25H21N2OFeSeBr: C 51.72, N 4.82, H 3.62, Fe 9.65; Found: C 51.09, N 4.63,
H 3.58, Fe 9.48%.
3.5 1-(4-bromobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a5)
Yield: 53%; Decomposition temperature: 189-190 °C; FT-IR (cm-1): 3400-3200 (NH
stretching broad), 3061 (Aromatic-H stretching), 2938 (Aliphatic-H stretching), 1663 (C=O
stretching), 1576, 1561, 1490 (C=C stretching of aromatic ring), 1375 (CH3 bending), 1256
(C=Se stretching); 1H-NMR (300 MHz, Acetone, δ (ppm): 13.21 (s, 1H, CSeNH), 10.60 (s, 1H,
CONH), 8.20-7.00 (m, 7H, Aromatic), 4.63 (apparent t, 2H, J 2.1, ortho on substituted Cp), 4.33
(apparent t, 2H, J 1.8, meta on substituted Cp), 4.07 (s, 5H, Cp-C1), 2.46 (s, 3H, C5); 13C-NMR
(75 MHz, Acetone, δ (ppm): 177.9 (C6), 166.2 (C7), 144.3 (1C-Ar), 136.1 (1C-Ar), 135.0 (1C-
Ar), 133.4 (1C-Ar), 132.8 (2C-Ar), 131.5 (2C-Ar), 126.3 (1C-Ar), 125.4 (1C-Ar), 124.1 (1C-Ar),
112.9 (1C-Ar), 85.2 (C4), 69.9 (C1), 69.1 (C2), 68.3 (C3), 20.8 (C5); Anal. Calcd. for
C25H21N2OFeSeBr: C 51.72, N 4.82, H 3.62, Fe 9.65; Found: C 51.69, N 4.59, H 3.41, Fe
9.37%.
3.6 1-(2-fluorobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a6)
12

Yield: 78%; Decomposition temperature: 154-155 °C; FT-IR (cm-1): 3410-3296 (NH
stretching broad), 3101 (Aromatic-H stretching), 2929 (Aliphatic-H stretching), 1664 (C=O
stretching), 1590, 1552, 1500 (C=C stretching of aromatic ring), 1358 (CH3 bending), 1245
1
(C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 13.41 (s, 1H, CSeNH), 9.15 (s, 1H,
CONH), 8.80-6.54 (m, 7H, Aromatic), 4.49 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp),
4.44 (apparent t, 2H, J 1.8 Hz, meta on substituted Cp), 4.10 (s, 5H, Cp-C1), 2.41 (s, 3H, C5);
13C-NMR (75 MHz, Benzene, δ (ppm): 178.5 (C6), 168.2 (C7), 158.6 (1C(F)-Ar), 136.9 (1C-
Ar), 136.5 (1C-Ar), 135.3 (1C-Ar), 133.6 (1C-Ar), 132.2 (1C-Ar), 130.8 (1C-Ar), 130.4 (1C-Ar),
130.0 (1C-Ar), 129.8 (1C-Ar), 129.6 (1C-Ar), 112.3 (1C-Ar), 87.0 (C4), 69.9 (C1), 69.7 (C2),
68.1 (C3), 21.4 (C5); Anal. Calcd. for C25H21N2OFeSeF: C 57.83, N 5.39, H 4.04, Fe 10.75;
Found: C 57.82, N 5.37, H 3.98, Fe 10.72%.
3.7 1-(3-fluorobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a7)
13

Yield: 61%; Decomposition temperature: 149-150 °C; FT-IR (cm-1): 3400-3100 (NH
stretching broad), 3029 (Aromatic-H stretching), 2953, 2929 (Aliphatic-H stretching), 1657
(C=O stretching), 1561, 1541, 1499 (C=C stretching of aromatic ring), 1377 (CH3 bending),
1250 (C=Se stretching); 1H-NMR (300 MHz, Benzene, δ (ppm): 13.40 (s, 1H, CSeNH), 9.08 (s,
1H, CONH), 8.80-6.54 (m, 7H, Aromatic), 4.45 (apparent t, 2H, J 2.1 Hz, ortho on substituted
Cp), 4.41 (apparent t, 2H, J 1.8 Hz, meta on substituted Cp), 4.12 (s, 5H,Cp-C1), 2.44 (s, 3H,
13
C5); C-NMR (75 MHz, Benzene, δ (ppm): 178.3 (C6), 166.3 (C7), 161.0 (1C(F)-Ar), 136.7
(1C-Ar), 136.6 (1C-Ar), 136.5 (1C-Ar), 134.8 (1C-Ar), 133.1 (1C-Ar), 131.7 (1C-Ar), 130.5
(1C-Ar) 130.0 (1C-Ar), 129.6 (1C-Ar), 129.5 (1C-Ar), 112.5 (1C-Ar), 87.4 (C4), 69.1 (C1), 68.7
(C2), 68.1 (C3), 21.3 (C5); Anal. Calcd. for C25H21N2OFeSeF: C 57.83, N 5.39, H 4.04, Fe
10.75; Found: C 57.79, N 5.36, H 4.01, Fe 10.70%.[7]
3.8 1-(4-fluorobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a8)
Yield: 74%; Decomposition temperature: 165-166 °C; FT-IR (cm-1): 3400-3100 (NH
stretching broad), 3045 (Aromatic-H stretching), 2951, 2933 (Aliphatic-H stretching), 1663
(C=O stretching), 1569, 1545, 1497 (C=C stretching of aromatic ring), 1377 (CH3 bending),
1250 (C=Se stretching); 1H-NMR (300 MHz, Benzene, δ (ppm): 13.41 (s, 1H, CSeNH), 9.13 (s,
1H, CONH), 8.80-6.54 (m, 7H, Aromatic), 4.48 (apparent t, 2H, J 2.1 Hz, ortho on substituted
Cp), 4.43 (apparent t, 2H, J 2.1 Hz, meta on substituted Cp), 4.10 (s, 5H, C1), 2.40 (s, 3H, C5);
13C-NMR (75 MHz, Benzene, δ (ppm): 178.5 (C6), 166.3 (C7), 158.2 (1C(F)-Ar), 136.9 (1C-
14

Ar), 136.5 (1C-Ar), 136.3 (1C-Ar), 133.8 (1C-Ar), 132.1 (1C-Ar), 130.9 (1C-Ar), 130.0 (2C-Ar),
129.9 (2C-Ar), 112.5 (1C-Ar), 87.4 (C4), 69.1 (C1), 68.7 (C2), 68.1 (C2), 21.3 (C5); Anal.
Calcd. for C25H21N2OFeSeF: C 57.83, N 5.39, H 4.04, Fe 10.75; Found: C 57.80, N 5.37, H
4.00, Fe 10.74%.[7]
3.9 1-(2-methoxybenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a9)
Yield: 49%; Decomposition temperature: 181-182 °C; FT-IR (cm-1): 3400-3200 (NH
stretching broad), 3090 (Aromatic-H stretching), 2944, 2928 (Aliphatic-H stretching), 1659
(C=O stretching), 1599, 1572, 1501 (C=C stretching of aromatic ring), 1363, 1357 (CH3
1
bending), 1266 (C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 13.33 (s, 1H,
CSeNH), 9.05 (s, 1H, CONH), 8.02-6.03 (m, 7H, Aromatic), 4.83 (apparent t, 2H, J 2.1 Hz,
ortho on substituted Cp), 4.36 (apparent t, 2H, J 1.8 Hz, meta on substituted Cp), 4.10 (s, 5H,
13
Cp-C1), 3.21 (s, 3H, C8), 2.41 (s, 3H, C5); C-NMR (75 MHz, Benzene, δ (ppm): 178.4 (C6),
167.3 (C7), 137.1 (1C-Ar), 136.2 (1C-Ar), 135.0 (1C-Ar), 132.6 (1C-Ar), 132.0 (1C-Ar), 130.0
(1C-Ar), 129.9 (1C-Ar), 129.8 (1C-Ar), 129.7 (1C-Ar), 129.6 (1C-Ar), 119.1 (1C-Ar), 116.2
(1C-Ar), 84.0 (C4), 69.8 (C1), 69.7 (C2), 67.1 (C3), 53.1 (C8), 20.3 (C5); Anal. Calcd. for
C26H24N2O2FeSe: C 58.75, N 5.27, H 4.51, Fe 10.54; Found: C 57.98, N 5.20, H 4.40, Fe
10.52%.
3.10 1-(3-methoxybenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a10)
15

Yield: 55%; Decomposition temperature: 170-712 °C; FT-IR (cm-1): 3400-3200 (NH
stretching broad), 3040 (Aromatic-H stretching), 2941, 2921 (Aliphatic-H stretching), 1670
(C=O stretching), 1588, 1571, 1500 (C=C stretching of aromatic ring), 1359, 1351 (CH3
1
bending), 1257 (C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 13.13 (s, 1H,
CSeNH), 9.07 (s, 1H, CONH), 8.31-6.59 (m, 7H, Aromatic), 4.81 (apparent t, 2H, J 2.1 Hz,
ortho on substituted Cp), 4.41 (apparent t, 2H, J 2.1 Hz, meta on substituted Cp), 4.15 (s, 5H,
13
Cp-C1), 3.40 (s, 3H, C8), 2.40 (s, 3H, C5); C-NMR (75 MHz, Benzene, δ (ppm): 177.6 (C6),
164.3 (C7), 137.4 (1C-Ar), 135.2 (1C-Ar), 135.0 (1C-Ar), 132.0 (1C-Ar), 131.8 (1C-Ar), 130.0
(1C-Ar), 129.7 (1C-Ar), 129.4 (1C-Ar), 128.9 (1C-Ar), 128.6 (1C-Ar), 118.9 (1C-Ar), 116.4
(1C-Ar), 84.2 (C4), 69.7 (C1), 69.6 (C2), 67.0 (C3), 53.0 (C8), 20.6 (C5); Anal. Calcd. for
C26H24N2O2FeSe: C 58.75, N 5.27, H 4.51, Fe 10.54; Found: C 57.23, N 5.16, H 4.40, Fe
10.42%.
3.11 1-(4-methoxybenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a11)
16

Yield: 53%; Decomposition temperature: 156-157 °C; FT-IR (cm-1): 3400-3200 (NH
stretching broad), 3085 (Aromatic-H stretching), 2967, 2923 (Aliphatic-H stretching), 1645
(C=O stretching), 1600, 1571, 1506 (C=C stretching of aromatic ring), 1379, 1361 (CH3
1
bending), 1229 (C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 13.03 (s, 1H,
CSeNH), 9.54 (s, 1H, CONH), 8.43-6.33 (m, 7H, Aromatic), 4.73 (apparent t, 2H, J 2.1 Hz,
ortho on substituted Cp), 4.33 (apparent t, 2H, J 1.8 Hz, meta on substituted Cp), 4.00 (s, 5H,
13
Cp-C1), 3.45 (s, 3H, C8), 2.41 (s, 3H, C5); C-NMR (75 MHz, Benzene, δ (ppm): 178.4 (C6),
167.0 (C7), 136.9 (1C-Ar), 135.0 (1C-Ar), 132.0 (1C-Ar), 130.0 (1C-Ar), 129.8 (1C-Ar), 129.5
(1C-Ar), 128.7 (2C-Ar), 128.6 (2C-Ar), 116.4 (1C-Ar), 115.3 (1C-Ar), 84.0 (C4), 69.6 (C1),
69.0 (C2), 68.1 (C3), 54.2 (C8), 20.1 (C5); Anal. Calcd. for C26H24N2O2FeSe: C 58.75, N 5.27,
H 4.51, Fe 10.54; Found: C 57.30, N 5.20, H 4.30, Fe 10.05%.
3.12 1-(2-methylbenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a12)
Yield: 73%; Decomposition temperature: 146-147 °C; FT-IR (cm-1): 3400-3200 (NH
stretching broad), 3037 (Aromatic-H stretching), 2936 (Aliphatic-H stretching), 1657 (C=O
stretching), 1582, 1544, 1500 (C=C stretching of aromatic ring), 1375 (CH3 bending), 1264
1
(C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 12.95 (s, 1H, CSeNH), 9.99 (s, 1H,
CONH), 8.42-7.64 (m, 7H, Aromatic), 4.77 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp),
4.29 (apparent t, 2H, J 2.1 Hz, meta on substituted Cp), 4.08 (s, 5H, Cp-C1), 2.46 (s, 3H, CH3),
13
2.43 (s, 3H, CH3); C-NMR (75 MHz, Benzene, δ (ppm): 179.3 (C6), 167.4 (C7), 144.3 (1C-
17

Ar), 136.5 (1C-Ar), 136.1 (1C-Ar), 135.1 (1C-Ar), 132.4 (1C-Ar), 132.1 (1C-Ar), 130.0 (1C-Ar),
129.9 (1C-Ar), 129.7 (1C-Ar), 128.7 (1C-Ar), 128.6 (1C-Ar), 86.5 (C4), 69.6 (C1), 69.7 (C2),
67.7 (C3), 20.7 (1C), 20.3 (1C); Anal. Calcd. for C26H24N2OFeSe: C 60.58, N 5.43, H 4.66, Fe
10.87; Found: C 60.50, N 5.30, H 4.32, Fe 10.39%.
3.13 1-(3-methylbenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a13)
Yield: 68%; Decomposition temperature: 143-144 °C; FT-IR (cm-1): 3400-3100 (NH
stretching broad), 3039 (Aromatic-H stretching), 2939, 2932 (Aliphatic-H stretching), 1668
(C=O stretching), 1582, 1543, 1498 (C=C stretching of aromatic ring), 1371, 1360 (CH3
1
bending), 1265 (C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 13.41 (s, 1H,
CSeNH), 10.02 (s, 1H, CONH), 8.39-7.09 (m, 7H, Aromatic), 4.76 (apparent t, 2H, J 2.1 Hz,
ortho on substituted Cp), 4.26 (apparent t, 2H, J 2.1 Hz, meta on substituted Cp), 4.18 (s, 5H,
13
Cp-C1), 2.46 (s, 3H, CH3), 2.45 (s, 3H, CH3); C-NMR (75 MHz, Benzene, δ (ppm): 179.5
(C6), 166.3 (C7), 143.2 (1C-Ar), 136.3 (1C-Ar), 136.0 (1C-Ar), 135.1 (1C-Ar), 132.0 (1C-Ar),
131.1 (1C-Ar), 130.0 (1C-Ar), 129.6 (1C-Ar), 129.5 (1C-Ar), 128.6 (1C-Ar), 128.5 (1C-Ar),
121.3 (1C-Ar), 86.3 (C4), 69.5 (C1), 69.4 (C2), 67.0 (C3), 20.6 (1C), 20.2 (1C); Anal. Calcd. for
C26H24N2OFeSe: C 60.58, N 5.43, H 4.66, Fe 10.87; Found: C 60.43, N 5.39, H 4.31, Fe
10.46%.
3.14 1-(4-methylbenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a14)
18

Yield: 79%; Decomposition temperature: 155-157 °C; FT-IR (cm-1): 3400-3200 (NH
stretching broad), 3041 (Aromatic-H stretching), 2939, 2929 (Aliphatic-H stretching), 1660
(C=O stretching), 1592, 1543, 1499 (C=C stretching of aromatic ring), 1371, 1365 (CH3
1
bending), 1244 (C=Se stretching); H-NMR (300 MHz, Benzene, δ (ppm): 13.28 (s, 1H,
CSeNH), 10.34 (s, 1H, CONH), 8.02-7.42 (m, 7H, Aromatic), 4.60 (apparent t, 2H, J 2.1 Hz,
ortho on substituted Cp), 4.36 (apparent t, 2H, J 2.1 Hz, meta on substituted Cp), 4.17 (s, 5H,
Cp-C1), 2.46 (s, 3H, CH3), 2.44 (s, 3H, CH3); 13C-NMR (75 MHz, Benzene, δ (ppm): 179.5
(C6), 167.7 (C7), 144.5 (1C-Ar), 136.0 (1C-Ar), 130.5 (1C-Ar), 129.4 (1C-Ar), 129.3 (1C-Ar),
129.0 (1C-Ar), 128.3 (2C-Ar), 127.4 (2C-Ar), 125.6 (1C-Ar), 121.2 (1C-Ar), 86.5 (C4), 69.6
(C1), 69.3 (C2), 69.2 (C3), 20.7 (1C), 20.5 (1C); Anal. Calcd. for C26H24N2OFeSe: C 60.58, N
5.43, H 4.66, Fe 10.87; Found: C 60.51, N 5.38, H 4.29, Fe 10.61%.
3.15 1-(2-chlorobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a15)
Yield: 63%; Decomposition temperature: 167-169 °C; FT-IR (cm-1): 3420-3390 (NH
stretching broad), 3066 (Aromatic-H stretching), 1659 (C=O stretching), 1599, 1544, 1508 (C=C
19

1
stretching of aromatic ring), 1369 (CH3 bending), 1257 (C=Se stretching); H-NMR (300 MHz,
Acetone, δ (ppm): 13.60 (s, 1H, CSeNH), 10.32 (s, 1H, CONH), 8.01-7.10 (m, 7H, Aromatic),
4.65 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp), 4.30 (apparent t, 2H, J 2.1 Hz, meta on
13
substituted Cp), 4.07 (s, 5H, Cp-C1), 2.43 (s, 3H, C5); C-NMR (75 MHz, Acetone, δ (ppm):
179.9 (C6), 167.5 (C7), 136.9 (1C-Ar), 136.5 (1C-Ar), 134.1 (1C-Ar), 133.6 (1C-Ar), 133.4 (1C-
Ar), 133.1 (1C-Ar), 132.4 (1C-Ar), 126.9 (1C-Ar), 126.0 (1C-Ar), 123.4 (1C-Ar), 120.2 (1C-Ar).
118.6 (1C-Ar), 85.7 (C4), 70.1 (C1), 69.9 (C2), 68.5 (C3), 21.1 (C5); Anal. Calcd. for
C25H21N2OFeSeCl: C 56.02, N 5.22, H 3.92, Fe 10.45; Found: C 55.67, N 5.11, H 3.74, Fe
10.31%.
3.16 1-(3-chlorobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a16)
Yield: 67%; Decomposition temperature: 177-178 °C; FT-IR (cm-1): 3400-3300 (NH
stretching broad), 3065 (Aromatic-H stretching), 1658 (C=O stretching), 1598, 1546, 1507 (C=C
1
stretching of aromatic ring), 1368 (CH3 bending), 1257 (C=Se stretching); H-NMR (300 MHz,
Acetone, δ (ppm): 13.60 (s, 1H, CSeNH), 10.30 (s, 1H, CONH), 8.11-7.16 (m, 7H, Aromatic),
4.64 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp), 4.36 (apparent t, 2H, J 1.8 Hz, meta on
13
substituted Cp), 4.06 (s, 5H, Cp-C1), 2.43 (s, 3H, C5); C-NMR (75 MHz, Acetone, δ (ppm):
179.8 (C6), 166.4 (C7), 136.9 (1C-Ar), 136.4 (1C-Ar), 134.0 (1C-Ar), 133.5 (1C-Ar), 133.4 (1C-
Ar), 133.0 (1C-Ar), 132.1 (1C-Ar), 126.5 (1C-Ar), 126.0 (1C-Ar), 123.1 (1C-Ar), 120.1 (1C-Ar).
20

119.9 (1C-Ar), 85.6 (C4), 69.9 (C1), 69.7 (C2), 66.9 (C3), 21.3 (C5); Anal. Calcd. for
C25H21N2OFeSeCl: C 56.02, N 5.22, H 3.92, Fe 10.45; Found: C 55.77, N 5.18, H 3.22, Fe
10.02%.
3.17 1-(4-chlorobenzoyl)-3-(3-methyl-4-ferrocenylphenyl)selenourea (a17)
Yield: 77%; Decomposition temperature: 182-184 °C; FT-IR (cm-1): 3400-3300 (NH
stretching broad), 3061 (Aromatic-H stretching), 1661 (C=O stretching), 1597, 1546, 1506 (C=C
1
stretching of aromatic ring), 1371 (CH3 bending), 1256 (C=Se stretching); H-NMR (300 MHz,
Acetone, δ (ppm): 13.60 (s, 1H, CSeNH), 9.91 (s, 1H, CONH), 8.10-7.19 (m, 7H, Aromatic),
4.66 (apparent t, 2H, J 2.1 Hz, ortho on substituted Cp), 4.33 (apparent t, 2H, J 1.8 Hz, meta on
13
substituted Cp), 4.17 (s, 5H, Cp-C1), 2.44 (s, 3H, C5); C-NMR (75 MHz, Acetone, δ (ppm):
179.7 (C6), 166.0 (C7), 136.8 (1C-Ar), 136.3 (1C-Ar), 133.5 (1C-Ar), 133.3 (1C-Ar), 132.0 (1C-
Ar), 128.5 (2C-Ar), 126.9 (2C-Ar), 123.4 (1C-Ar), 120.6 (1C-Ar). 120.0 (1C-Ar), 85.6 (C4),
69.9 (C1), 69.4 (C2), 66.8 (C3), 20.9 (C5); Anal. Calcd. for C25H21N2OFeSeCl: C 56.02, N
5.22, H 3.92, Fe 10.45; Found: C 55.39, N 5.10, H 3.81, Fe 10.33%.
4. Results and Discussion
All the FIS were synthesized in good yield having solid state and orange color. In FTIR, -C=Se
in all the FIS provided a weak band between 1229-1265 cm-1 whereas –CH3 bending vibration
was available at ~ 1360 cm-1. Carbonyl stretch in all the synthesized FIS was available above
21

1650 cm-1 whereas –NH stretchings were present above 3200 cm-1 as a broad signal. In proton
NMR (Figure S-1, supplementary information) the characteristic signals for –NH protons were
available above 9 ppm for all the compounds in a way that the –NH proton which was between
the phenyl ring attached with ferrocene and –C=Se was comparatively more downfield than the –
NH proton which was present between –C=Se and –C=O. The reason may be the intramolecular
hydrogen bonding between the oxygen of carbonyl and –NH proton which was present between
the phenyl ring and –C=Se. This intramolecular hydrogen bonding was evident in the crystal
structure of the representative compound in the series (Figure 1). Unsubstituted cycopentadienyl
(Cp) ring yields a singlet at ~ 4 ppm whereas the two protons which are ortho on substituted Cp
ring provide a signal which appears as an apparent triplet due to two equivalent protons on meta
position. Similarly two protons which are meta on substituted Cp ring provide a signal which
appears as an apparent triplets due to two equivalent protons on ortho position with a coupling
constant value of ~ 2 Hz. Methyl group present on the phenyl ring (which is attached with
ferrocene) gives a singlet for its three protons in all the compounds at ~ 2 ppm. In C-13 NMR
(Figure S2, supplementary materials), maximum downfield carbon in all the compounds was the
carbon attached with selenium which appeared at ~ 178 ppm whereas carbonyl carbon in all the
synthesized FIS was available at ~ 165 ppm. Aromatic carbons were available between 120-140
ppm which is their usual region whereas five carbons of substituted Cp ring provided a singlet
and unsubstituted Cp ring gave three peaks in which ipso carbon appeared at ~ 84 ppm due to
electron withdrawing effect of phenyl ring with which it is attached. Methyl and metoxy protons
whenever available gave their signals at ~ 20 ppm and ~ 51 ppm respectively. Calculated and
found percentages of carbon, hydrogen, nitrogen and iron are in close agreement with each other
which confirms the purity of the synthesized compounds [34].
22

4.1 Carcinogenesis
Cancer is the unmanaged growth of cells which may be initiated by the change in genetic
makeup or DNA repair mechanism, propagated by further growing of these cells which have
wrong growth information to lead the final stage of carcinogenesis which is called tumor. Best
chemopreventive agent (CP) will be the one which is active against all the three stages and for
that one of the preliminary requirements is that it should bind or interact with DNA. Depending
upon the structure and functional groups available on the compounds they can interact via
noncovalent and covalent interactions with DNA. Covalent binding is irreversible i.e. it causes
complete inhibition of the cellular processes. Alkylating agents and intrastrand linkers generally
exhibit covalent interactions with DNA. Noncovalent interactions may be characterized as
groove binding, intercalation and external binding. Different techniques are used for the
evaluation of DNA binding capabilities of certain possible drugs but we have used cyclic
volammetry (CV).
4.1.1 DNA binding studies
DNA binding studies were carried out with the help of CV, mainly because of the best
electrochemical signals of ferrocene attached with selenoureas of all the synthesized compounds.
DNA binding studies of a representative FIS namely 1-(2-methylbenzoyl)-3-(3-methyl-4-
ferrocenylphenyl)selenourea (a12) has been presented in Figure 2. This representative compound
gives a couple of well defined redox peaks in a region between 0.6 V to 0.8 V in a diffusion
controlled 100% reversible electrochemical process. Reversibility of the process is confirmed by
observing three facts i) the difference between oxidation maxima and reduction maxima is
almost equal to 60 mV, ii) amplitude of the oxidation peak current and reduction peak current is
23

~1 and iii) values of current at different scan rates is constant (Figure 2a)) [6]. There is a negative
shift in the peak potential of a12-1DNA relative to free a12 which indicates the electrostatic
interaction between the phosphate backbone of the DNA and ferrocenium moiety of a12.
Because of this electrostatic interaction electrons become available to a12 from DNA which
make its oxidation easy and as a result different DNA adducts with a12 provide their oxidation
and reduction maxima towards the negative potential side relative to those peaks which are
available for free a12 (Figure 2b). Diffusion coefficients were calculated with the help of Eq 4 by
a plot of current vs. scan rate. Free a12 has a diffusion coefficient of 1.34 x 10-6 cm2s-1 whereas
a12-1DNA adduct has a diffusion coefficient value of 4.86 x 10-7 cm2s-1 and the same
phenomena were true for all the other compounds reported in this article. Lower diffusion
coefficients of drug-DNA adducts relative to free drugs confirm the formation of adducts
between the drugs and DNA which are of higher molecular weight than free drugs. This
increased molecular weight hinders the swift approach of adducts towards the electrode and
hence reduces the magnitude of diffusion coefficients. Drug-DNA binding constant of 3.283 x
105 M-1 was calculated for a12 by using Eq 1. A lower value of the binding site size (0.44 base
pair) is also in favor of electrostatic interactions. Drug-DNA binding parameters for all the other
compounds have been presented in Table 1 which show that mostly the binding constants are in
the range of 103-105 M-1 which are better than DNA binding constants of simple ferrocene [26],
nitrophenyl ferrocene [27], ferrocene incorporated thioureas (FIT) [37, 38] and cis-platin.
However surface electrostatic interactions generally have inferior binding magnitudes than the
universal intercalators ethidium [39] and proflavin [40]. These results gave us very good
preliminary information about the possibility of using these FIS as anticancer agents. Our
previous findings with molecular docking prove the electrostatic interaction of ferrocenyl moiety
24

with oxygen (located at DC3 and DG2) of phosphate backbone whereas oxygen of carbonyl
group has a tendency to form hydrogen bonds with nitrogenous base pairs [4]. We expect the
same behavior of all the FIS presented in this article. If we compare the DNA binding behavior
of FIS presented in Table 1 with literature reported FIT [37, 38], first difference is the mode of
interaction. FIT-DNA adducts show a positive shift in the peak potential relative to free FIT in
CV, favoring intercalative mode of interaction whereas FIS-DNA adducts of FIS presented in
Table 1 mostly show negative shifts in the peak potentials relative to free FIS, favoring surface
electrostatic mode of interaction of these compounds with DNA. This difference in binding
behavior of selenoureas and thioureas may be due to the difference in the reactivities of sulphur
and selenium in these two classes of compounds. Because of low ionization potential and high
electron density of selenium relative to sulphur it is more susceptible to interact with DNA base
pairs [41].
Methyl substitution of phenyl ring attached with ferrocene moiety decreases the
magnitude of binding constant by a factor of 10 M-1 in general (this article) relative to the scheme
in which this ring was not substituted with methyl group in our previous publication [42]. This
difference may be because of electron donating nature of methyl group which in turn decreases
the electrostatic affinity of FIS with negatively charged phosphate backbone of DNA. Steric
factor may also play its role but exact mechanism is not yet clear. In this article 3-chlorophenyl
derivative (a16) has shown a binding constant of 2.66 x 105 M-1 which is ~ 100 times more than
3-chlorophenyl derivative in our previously reported results [42]. Interaction of DNA with drug
is very complex and involves different unidentified kinetic parameters because of which
structure and activity relation in terms of binding constant, diffusion coefficients and binding site
size is not 100% possible.
25

4.1.2 Free radical scavenging activity
Free radicals are highly reactive and cause diseases such as Alzheimer’s disease, myocardial
infarction, atherosclerosis, Parkinson’s disease, autoimmune diseases, radiation injury,
emphysema, sunburns and cancer initiation [23]. In cancer patients free radicals are present at
higher concentration than the healthy organisms. It is not yet clear to use the antioxidants during
cancer therapy or not. Generally their use is stopped a week before the start of cancer treatment
[1]. If an anticancer agent is an efficient free radical scavenger than its importance increases
because it can stop cancer initiation as well. This article presents DPPH scavenging activity of
synthesized FIS using ascorbic acid as a positive control.
Figure 3 displays the results of DPPH scavenging for synthesized FIS. Generally, ortho
substituted derivatives have higher scavenging activities than meta and para substituted
derivatives i.e. 2-fluorophenyl derivative has higher scavenging than 3-fluorophenyl and 4-
fluorophenyl derivatives and same is the case for methylphenyl derivatives. However this
generalization is not followed for other derivatives because there are three different positions in
the synthesized FIS which may be involved at the same time in DPPH scavenging i.e. two are the
NH groups and the 3rd one is the selenium atom (Figure 4). FIS which showed more than 50%
scavenging were evaluated for IC50 values which were 152 ± 11.6 µM, 385.1 ± 54.5 µM, 165.6 ±
24.5 µM, 84.3 ± 8.7 µM for a7, a17, a12 and a14 respectively relative to 54.7 ± 2.6 µM of
ascorbic acid.
4.1.3 Anti-inflammatory activity
Nitric oxide (NO) is an important signaling molecule but its higher levels are considered to be
responsible for ischemic damage, reperfusion injury, obesity, cancer and diabetes [43].
26

Cancerous cells have been reported to have higher levels of NO [44]. Tumor tissues of male
mouse with murine leukemia (Raw 264.7) cells were used to determine the NO inhibition of
synthesized FIS using LNMMA (L-N-monomethyl arginine citrate) as a positive control. Figure
5 portraits the NO production inhibition activity of the synthesized FIS which shows that all the
compounds displayed the inhibition of NO. Four compounds have better activities than the
positive control out of which three are the ortho substituted derivatives. Higher activities of those
derivatives which have halogen on ortho position may be due to the formation of an intermediate
specie (Figure 6) but higher activities of methyl substituted derivatives is attributed to the higher
permeability of these derivatives through the membrane. FIS which have more than 50%
inhibition of NO were further evaluated for IC50 values which were 10.9 ± 1.4 µM, 35.6 ± 1.0
µM, 24.1 ± 0.4 µM, 14.4 ± 0.7 µM for a7, a17, a12 and a14 respectively relative to the 17.6 ±
5.4 µM of LNMMA.
4.1.4 Enzymatic activities
Cancer initiation in the body is caused by the phase I enzymes (cytochrome P450) because they
are responsible for the production of reactive oxygen species (ROS). In response to the higher
concentration of ROS, body produces phase II enzymes (glutathione S-transferases (GSTs),
UDP-glucuronosyl transferases, and quinone reductases) as a self defense mechanism. Now if an
anticancer drug has the ability to inhibit phase I enzyme and induce phase II enzymes in the body
it is very useful. In this article we have reported aromatse inhibition (phase I enzyme) and
quinone reductase induction (phae II enzyme) activities of the FIS.
4.1.4.1 Aromatase inhibition activity
27

Breast cancer, ovarian cancer and lungs cancer patients have higher concentrations of estrogen.
Biosynthesis of estrogen is controlled by aromatase and it has been reported that aromatse
inhibitors are better than tamoxifen for the treatment of breast cancer [45]. Aromatase inhibition
activities of the FIS are inferior to the positive control.
4.1.4.2 Quinone reductase (QR) induction activity
Hepa 1c1c7 (murine hepatoma) cells were used for QR activity. For this activity induction ratio
of QR1 (IR of QR1) is the specific enzyme induction activity of agent treated cell with reference
to DMSO (dimethyl sulfoxide) treated control. Compounds with a QR1 value of 2 or more than 2
are further evaluated for concentration dose (CD) experimentation. CD is that concentration of a
chemical which is required to double the QR activity. Figure 7 shows that there are six
compounds in the series with QR1 values of more than 2 which were further evaluated for CD
values (Figure 8). Quinone reductase induction activities of compounds reported in this article
are better than previously reported unsymmetrical diphenyl selenourea and unsymmetrical
dimethyl selenourea [23]. Better QR values of compounds with R = methyl, 2-methylphenyl, 3-
methylphenyl and 4-methylphenyl are attributed to their easy permeability through cell
membrane as they do not contain electronegative substituent which can hinder the permeation.
Our hypothesis is further strengthened by highest CD values of methyl substituted derivative
(a1). Previously, lower activities of unsymmetric dimethyl selenourea and unsymmetric diphenyl
selenourea were attributed to their structural un-similarity with methyl selenol which is
considered to be responsible for better chemopreventive action in comparison to the elemental
selenium [23]. Our compounds (a1-a17) are not structurally similar to methyl selenol but have
shown better activities.
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Better performance of Se derivatives (this article) over sulphur derivatives (previous
literature) [37, 38] is because of the ability of selenium to attain zwitterionic form via formation
of dimeric two cantered three electron bonds which gives selenium an edge over sulphur during
different enzymatic studies [23]. These enzymatic activities show that these compounds have the
ability to stop the cancer initiation and cancer propagation. In comparison to cis-platin, these
compounds are more impressive because cis-platin only kills the cancerous cells but have no
effect on cancer initiation and propagation [46].
4.1.5 Activities against the cancer cell lines
Cytotoxic activities have been carried out against five cancerous cell lines namely neuroblastoma
cell lines (MYCN and SK-N-SH), lungs cancer cell line (hepa 1c1c7), human cervical
carcinoma HeLa cells (ATCC® CCL-2) and breast cancer cell line MCF-7 to further investigate
the CP nature of the synthesized FIS.
Neuroblastoma is the cancer of childhood and is extra-cranial malignant in nature [47].
Although the frequency is low in USA (650 cases/year) and UK (100 cases/year) but the problem
is serious due to its existence in children. Over expression of a protein NYMc is the reason of
this cancer. NYMc is in turn controlled by a gene known as MYCN (neuroblastoma
myelocytomatosis). If over expression of NYMc is controlled by controlling or inhibiting
MYCN then this cancer can be controlled. Table 2 presents the data of FIS scanned against
MYCN. Another neuroblastoma cell line SK-N-SH was also scanned but FIS synthesized are
comparatively less active against this cell line. All the compounds have shown activities against
MYCN but orthosubstituted compounds (2-fluorophenyl, 2-chlorophenyl, 2-methylphenyl) are
the most active and these results are in accordance with our previously published findings [8].
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