New supramolecular ferrocenyl phenylguanidines as potent antimicrobial and DNA-binding agents

Article abstract of DOI:10.1080/00958972.2013.796371

Six new ferrocenyl phenylguanidines have been synthesized and characterized by elemental analysis, FT-IR, multinuclear (1H and 13C) NMR, and single crystal analysis. The latter showed a supramolecular structure for 2 mediated by O H and π H interactions. A subsequent DNA-binding study of these complexes proved them to be good DNA binders with the binding constant varying in the range of 1.2-5.6×105M-1. These compounds were found to have moderate antibacterial and significant antifungal activities, especially for compounds having a chlorophenyl. These compounds may emerge as a new class of anticancer and antifungal agents alone or in combination with other drugs.

Full text of DOI:10.1080/00958972.2013.796371

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New supramolecular ferrocenyl
phenylguanidines as potent
antimicrobial and DNA-binding agents
a b
b
a
Rukhsana Gul
, Azim Khan , Amin Badshah , Muhammad
a
a
b
c
Khawar Rauf , Afzal Shah , Zia-ur-Rehman , Asghari Bano ,
c
d
Rabia Naz & Muhammad Nawaz Tahir
a
Department of Chemistry , Quaid-i-Azam University ,
Islamabad , Pakistan
b
Department of Chemistry , Gomal University , Dera Ismail Khan ,
Pakistan
c
Department of Plant Sciences , Quaid-i-Azam University ,
Islamabad , Pakistan
d
Department of Physics , Sargodha University , Sargodha ,
Pakistan
Accepted author version posted online: 16 Apr 2013.Published
online: 14 May 2013.
To cite this article: Rukhsana Gul , Azim Khan , Amin Badshah , Muhammad Khawar Rauf ,
Afzal Shah , Zia-ur-Rehman , Asghari Bano , Rabia Naz & Muhammad Nawaz Tahir (2013) New
supramolecular ferrocenyl phenylguanidines as potent antimicrobial and DNA-binding agents,
Journal of Coordination Chemistry, 66:11, 1959-1973, DOI: 10.1080/00958972.2013.796371
To link to this article: http://dx.doi.org/10.1080/00958972.2013.796371
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Journal of Coordination Chemistry, 2013
Vol. 66, No. 11, 1959–1973, http://dx.doi.org/10.1080/00958972.2013.796371
New supramolecular ferrocenyl phenylguanidines as potent
antimicrobial and DNA-binding agents
RUKHSANA GULyz, AZIM KHANz, AMIN BADSHAH*y, MUHAMMAD KHAWAR
RAUFy, AFZAL SHAHy, ZIA-UR-REHMANz, ASGHARI BANOx, RABIA NAZx and
MUHAMMAD NAWAZ TAHIR{
yDepartment of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan
zDepartment of Chemistry, Gomal University, Dera Ismail Khan, Pakistan
xDepartment of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan
{Department of Physics, Sargodha University, Sargodha, Pakistan
(Received 13 November 2012; final version received 14 February 2013)
Six new ferrocenyl phenylguanidines have been synthesized and characterized by elemental
1
13
analysis, FT-IR, multinuclear ( H and C) NMR, and single crystal analysis. The latter showed a
supramolecular structure for subsequent
mediated by Oꢀ ꢀ ꢀH and πꢀ ꢀ ꢀH interactions.
DNA-binding study of these complexes proved them to be good DNA binders with the binding
2
A
5
ꢂ1
constant varying in the range of 1.2–5.6 ꢁ 10 M . These compounds were found to have
moderate antibacterial and significant antifungal activities, especially for compounds having a chlo-
rophenyl. These compounds may emerge as a new class of anticancer and antifungal agents alone
or in combination with other drugs.
Keywords: Ferrocenyl phenylguanidine; DNA binding; Antibacterial; Antifungal activity
1. Introduction
Compounds containing CN named guanidines have a rich history in biological and bio-
3
inspired molecular recognition due to their amphoteric nature [1–3]. For example, a
guanidine is present in arginine side chains which not only involve in binding and recogni-
tion of ionic substrates, but also maintain a protein tertiary structure. The guanidinium
within a variety of molecular architectures forms strong noncovalent interactions with anio-
nic groups through hydrogen-bonding and charge-pairing interactions. Guanidine
derivatives are being used as neurotransmitters (neuropeptide Y) [4], cardiovascular or
antihypertensive drugs (amiloride, triamterene, doxazosin mesylate, etc.) [5], and as antibi-
otics (streptomycin, trimethoprim, and chlorhexidine) [6]. Some naturally occurring
guanidines were screened for their nuclease activity and exhibited cytotoxic properties [7].
Guanidine is an inhibitor of urokinase, which is responsible for a large number of malig-
nancies including breast, lung, bladder, stomach, cervix, kidney, and brain cancers [8–10].
*
Corresponding author. Email: aminbadshah@yahoo.com
Ó 2013 Taylor & Francis

1
960
R. Gul et al.
It has been established that polyaromatic guanidinium derivatives offer cytotoxic activities
by forming stronger adducts with DNA [11].
Incorporation of ferrocenyl into an organic compound often enhances the biological
action of the resulting products [12]. Various researchers have reported ferrocenic analogs
of chloroquine, mefloquine, and quinine, with enhanced antimalarial activities [13, 14].
The substitution of the aromatic ring of the well-known anticancer drug tamoxifen with
ferrocenyl group (called ferrocifen) showed pronounced activity against tamoxifen resistive
breast cancer cells [15]. Similarly, the antibiotic activity of penicillin and cephalosporin
was enhanced many fold on incorporation of the ferrocenyl moiety [16]. Low cytotoxicity
in biological systems, lipophilicity, cytotoxicity of metabolites towards tumors, the pi-con-
jugated system, and the resulting electron-transfer ability of ferrocene derivatives make
them candidates for investigation of their biological applications [17–21].
Here, we combine a ferrocenyl with phenyl guanidines to get electrochemistry and
improved biological action. Study including DNA interaction and antimicrobial activity is
not reported for ferrocenyl guanidine, and this contribution will provide a foundation for
future work.
2
2
. Experimental
.1. Materials
Ferrocene, 3- and 4-nitroaniline, sodium nitrate, hexadecyltrimethyl ammonium bromide,
palladium on charcoal and hydrazine (for the synthesis of para and meta ferrocenyl
aniline), benzoic acid, thionyl chloride, KSCN/NH SCN, simple and chloro substituted
4
anilines (for the synthesis of thioureas), and mercuric chloride were obtained from Fluka,
Switzerland. Solvents like diethyl ether, triethylamine, dimethylformamide (DMF), acetone,
ethanol, and dimethyl sulfoxide (DMSO) were obtained from Merck, Germany and freshly
dried using standard methods. Elemental analysis was performed using Fisons EA1108
CHNS analyzer and LECO-183 CHNS analyzer. Melting points were determined on a Bio
Cote SMP10-UK using open capillary tubes. The solid state Fourier transform spectrum
was recorded on Bio-Rad Excalibur FT-IR Model FTS 3000 MX and Bruker Tensor 37
1
13
spectrophotometers. H and C-NMR spectra were obtained on a Bruker 300 MHz NMR
spectrophotometer in CDCl using tetramethylsilane as internal reference. The data for the
3
crystal structure analysis were collected on a Bruker Kappa APEX II CCD Diffractometer.
2
2
.2. Apparatus and procedure
.2.1. Synthesis of ferrocenylaniline and thioureas. Two anilines (p-ferrocenylaniline
and m-ferrocenylaniline) and three thioureas–(1-benzoyl-3-phenyl) thiourea, 1-benzoyl-3-
2-chlorophenyl) thiourea, and 1-benzoyl-3-(2,5-dichlorophenyl) thiourea, were synthesized
by the reported method [22, 23].
(
2
.2.2. Synthesis of ferrocenyl phenylguanidine. All guanidines were synthesized by
the reported method [24]. The thioureas were mixed with ferrocenylaniline in DMF in

Ferrocenyl phenylguanidines
1961
Scheme 1. Synthesis of ferrocenyl phenylguanidine.
equimolar ratios with two equivalents of triethylamine. The temperature was maintained
below 5 °C using an ice bath and one equivalent of mercuric chloride was added to the
reaction mixture with vigorous stirring. The ice bath was removed after 30 min and the
mixture was stirred overnight. Progress of the reaction was monitored by TLC till comple-
tion. Chloroform (20 mL) was added to the reaction mixture and the suspension was
filtered through a sintered glass funnel to remove HgS. The solvent from the filtrate was
evaporated under reduced pressure and the residue was redissolved in CH Cl (20 mL),
2
2
washed with water (4 ꢁ 50 mL), and the organic phase, dried over anhydrous MgSO . The
4
solvent was evaporated; the residue was purified by column chromatography and recrystal-
lized in ethanol (scheme 1). Crystals were obtained for 2 only.
2
.3. X-ray crystallography
For 2, X-ray data were collected on a Bruker kappa APEXII CCD diffractometer using
graphite-monochromated Mo-Kα radiation (wavelength = 71,073 Å). The structure was
2
solved using SHELXS-97. Final refinement on F was carried out by full-matrix least-
squares using SHELXL-97. The disordered cyclopentadienyl was refined in two groups as
regular pentagons of 1.39 and 1.44 Å. The anisotropic temperature factors of the disordered
carbons were restrained to be nearly isotropic.
2
.4. Cyclic voltammeter and UV–Vis spectrophotometer
Cyclic voltammetric experiments were performed using a Bio-Logic SP-300 voltameter. Tet-
rabutylammonium perchlorate (TBAP) (Fluka, 99% purity) was further purified by recrystal-
lization using methanol. Measurements were carried out in a conventional three electrode

1
962
R. Gul et al.
cell consisting of Ag/AgCl as a reference electrode, a thin Pt wire of thickness 0.5 mm with
an exposed end of 10 mm as the counter electrode, and a platinum disk as the working elec-
trode. For electrochemical measurements, a known concentration of the test solution was
kept in an electrochemical cell and the voltammogram was recorded in the absence of DNA.
The procedure was then repeated for systems with a constant concentration of the drug and
varying concentration of DNA. The working electrode was cleaned after every electrochemi-
cal assay. Ct-DNA stock solution was prepared in doubly-distilled water and stored at 4 °C.
The concentration of the stock solution of DNA was determined by UV absorbance at
ꢂ1
ꢂ1
2
60 nm using the molar extinction coefficient (ɛ) of 6600 M cm [25].
Absorption spectra were measured with a Shimadzu 1800 UV–Vis spectrometer. The
electronic absorption spectrum of a known concentration of the drug was obtained without
DNA. The spectroscopic response of the same amount of the drug was then monitored by
addition of small aliquots of DNA solution. All samples were allowed to equilibrate for
1
2
2
5 min prior to every spectroscopic measurement.
.5. Biological studies
.5.1. Antibacterial assay. All six guanidines were studied against five bacterial strains,
Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella pneumo-
neae, and Escherchia coli. The agar diffusion method [26, 27] was used for determination
of the inhibition zone. A single colony from each bacterial culture plate was transferred to
nutrient broth (pH 7) and incubated at 37 °C for 24 h. About 2 mg/mL concentration of
6
each compound was prepared. Briefly, 0.75 mL of broth culture containing 10 colony
forming units per millimeter of test strain was added to the 75 mL of nutrient agar
medium, mixed well, and then poured into a 14 cm sterile agar plate. The reaction was
performed in triplicate. Wells were prepared using an 8 mm sterilized metallic borer, sealed
with media, and filled with 100 μL of respective concentration of each compound. Penicil-
lin (1 mg/mL) was used as standard drug, while DMSO was used as negative control.
Plates were incubated at 37 °C aerobically and the zone of inhibition was measured after
2
4 h. Experiments were run in triplicates.
2
.5.2. Antifungal assay. Activity was tested against three different fungal strains,
Fusarium moniliforme, Aspergillus fumigates, and Aspergillus flavus, according to the
reported method [26, 27]. The samples for antifungal assay were prepared from an initial
stock of 2 mg of the compound in 1 mL of DMSO. Culture media were prepared by dis-
solving 6.5 mg of Sabouraud dextrose agar per 100 mL distilled water and pH was adjusted
to 5.6. The Sabouraud dextrose agar (MERCK) was dispensed as 10 mL volume into
screw capped tubes or cotton plugged test tubes and was autoclaved at 121 °C for 21 min.
Tubes were allowed to cool to 50 °C and Sabouraud dextrose agar was loaded with 67 μL
from the stock solution. Tubes were then allowed to solidify in a slanting position at room
temperature. Three slants of the test sample were prepared for each fungus. Tubes contain-
ing solidified media and test compounds were inoculated with a 4 mm diameter piece of
inoculum, taken from a seven day old culture of fungus. One sample of each compound
was prepared, which was used for positive control. Slants without test sample were used
for negative control. The test tubes were incubated at 28 °C for seven days. Cultures were
examined twice weekly during the incubation. Reading was taken by measuring the linear
length of fungus in slant by measuring growth (mm) and growth inhibition was calculated

Ferrocenyl phenylguanidines
1963
with reference to the negative control. Percentage inhibition of fungal growth for each
concentration of the compound was determined as:
1
ꢂ Linear growth (cm) in test sample
Percentage inhibition of fungal growth ¼
ꢁ 100
Linear growth (cm) in control
2
2
1
4
.5.2.1. Synthesis of N-(4-ferrocenylphenyl-N'-phenyl-N″-benzoylguanidine) (1). Yield:
.59 g, 72%; m.p. 151-152 °C; FT-IR (KBr, cm ): 3340, 3278, 3117, 2995, 2950, 2917,
ꢂ1
673, 1583, 1517, 1465, 1449, 1394, 1331, 1243, 1102, 1078, 1033, 877, 860, 800, 730,
1
80, 448, 420; H NMR (300 MHz, CDCl , 25 °C): δ 4.8 (s, 5H), 4.91 (t, 2H, J = 1.9 Hz,
3
C H ), 5.2 (t, 2H, J = 1.9 Hz, C H ), 7.2 (d, 2H, J = 8.2 Hz), 7.3 (d, 2H, J = 8.1 Hz), 7.4–
5
4
5 4
1
3
7
.91 (m, 10H), 10.3 (s, H, N–H), 11.3 (s, H, N–H); C NMR (75.47 MHz, CDCl , 25 °
3
C): δ 66.1, 67.01, 67.5, 83.1, 121.2, 122.3, 122.9, 129.2, 129.8, 130.2, 130.6, 134.1,
34.5, 135.5, 140.1, 140.4, 156.8 (CN ), 176 (C=O); Anal. Calcd for C H N FeO
1
3
30 25 3
(499.13): C, 72.15; H, 5.05; N, 8.41. Found: C, 72.4; H, 5.13; N, 7.9%.
2
2
1
7
.5.2.2. Synthesis of N-(3-ferrocenylphenyl-N'-phenyl -N″-benzoylguanidine) (1a). Yield:
.1 g (71%), m.p. 151–152 °C; FT-IR (KBr, cm ): 3342, 3279, 3118, 2991, 2952, 2917,
ꢂ1
675, 1610, 1583, 1515, 1465, 1448, 1394, 1333, 1243, 1102, 1078, 1033, 876, 860, 801,
1
30, 497, 482, 455, 422; H NMR (300 MHz, CDCl , 25 °C): δ 4.8 (s, 5H), 4.9 (t, 2H,
3
J = 1.9 Hz, C H ), 5.2 (t, 2H, J = 1.9 Hz, C H ), 7.25–7.35 (m, 3H), 7.38 (s, H), 7.4–7.9
5
4
5 4
1
3
(
m, 10H), 10.2 (s, H, N–H), 11.3 (s, H, N–H); C NMR (75.47 MHz, CDCl , 25 °C): δ
3
6
1
6.0, 67.2, 67.6, 83.1, 121.4, 122, 122.3, 122.9, 124, 129.1, 129.8, 130.4, 130.6, 134.2,
34.5, 135.5, 140.1, 140.6, 156.8 (CN ), 176 (C=O); Anal. Calcd for C H N FeO
3
30 25 3
(499.13): C, 72.15; H, 5.05; N, 8.41. Found: C, 71.5; H, 5.2; N, 7.1%.
2
.5.2.3. Synthesis of N-(4-ferrocenylphenyl-N'-(2-chlorophenyl)-N″-benzoylguanidine) (2).
ꢂ1
ꢂ1
Yield: 2.6 g, 74%; m.p. 157–158 °C; FT-IR (KBr, cm ): FT-IR (KBr, cm ): 3315, 3158,
3
1
059, 2953, 1670, 1596, 1559, 1509, 1455, 1398, 1382, 1307, 1267, 1230, 1185, 1101,
075, 1012, 833, 725, 625, 609, 588, 569, 520, 505, 476, 447, 407; H NMR (300 MHz,
1
CDCl , 25 °C): δ 4.9 (s, 5H), 4.97 (t, 2H, J = 1.9 Hz, C H ), 5.3 (t, 2H, J = 1.9 Hz, C H ),
3
5
4
5 4
7
.26 (d, 2H, J = 8.1 Hz), 7.35 (d, 2H, J = 8.1 Hz), 7.4–8.0 (m, 9H), 10.3 (s, H, N–H), 11.4
1
3
(
s, H, N–H); C NMR (75.47 MHz, CDCl , 25 °C): δ 66.8, 67.4, 67.5, 83.6, 121.5, 122.7,
3
1
24.5, 124.8, 129.6, 130.2, 131, 133.7, 134.7, 135.5, 136.4, 139.7, 141.2, 141.9, 160.5
(
CN ), 179.3 (C=O); Anal. Calcd for C H N ClFeO (533.8): C, 67.58; H, 4.53; N, 7.87.
3
30 24 3
Found: C, 67.2; H, 4.1; N, 7.2%.
2
.5.2.4. Synthesis of N-(3-ferrocenylphenyl-N'-(2-chlorophenyl)-N″-benzoylguanidine) (2a).
ꢂ1
Yield: 2.3 g, 73%; m.p. 157–158 °C; FT-IR (KBr, cm ): 3308, 3158, 3060, 2954, 1670,
1
8
624, 1594, 1558, 1506, 1456, 1398, 1385, 1307, 1263, 1230, 1182, 1101, 1070, 1012,
33, 725, 628, 609, 584, 569, 543, 520, 505, 476, 447, 433, 412, 403; H NMR
1
(
300 MHz, CDCl , 25 °C): δ 4.89 (s, 5H), 4.95 (t, 2H, J = 1.9 Hz, C H ), 5.25 (t, 2H,
3
5
4
J = 1.9 Hz, C H ), 7.2–7.3 (m, 3H), 7.4 (s, H), 7.6–8.0 (m, 9H), 10.3 (s, H, N–H), 11.4 (s,
5
4
1
3
H, N–H); C NMR (75.47 MHz, CDCl , 25 °C): δ 66.6, 67.3, 67.5, 83.4, 121.3 122.7,
3
1
23.1, 124.2, 124.7, 129.1, 129.7, 130.2, 130.8, 133.5, 134.7, 135, 136.5, 139.2, 141.1,

1
964
R. Gul et al.
1
4
42, 160.8 (CN ), 179.4 (C=O); Anal. Calcd. for C H N ClFeO (533.8): C, 67.58; H,
.53; N, 7.87. Found: C, 67.01; H, 4.30; N, 7.6%.
3
30 24 3
2
.5.2.5. Synthesis of N-(4-ferrocenylphenyl-N'-(2,5-dichlorophenyl)-N″-benzoylguanidine)
ꢂ1
(
3). Yield: 2.6 g (74%), m.p. 157–158 °C, FT-IR (KBr, cm ): 3305, 3162, 3091, 2960,
2
925, 2855, 1670, 1615, 1594, 1555, 1506, 1458, 1398, 1389, 1307, 1263, 1236, 1187,
1
101, 1077, 1012, 833, 726, 628, 607, 586, 569, 545, 520, 508, 476, 446, 438, 412, 408;
1
H NMR (300 MHz, CDCl , 25 °C): δ 4.8 (s, 5H), 4.9 (t, 2H, J = 1.9 Hz, C H ), 5.2 (t,
3
5 4
2
H, J = 1.9 Hz, C H ), 7.25 (d, H, J = 8.1 Hz, Ar–H), 7.35 (d, H, J = 8.2 Hz, Ar–H), 7.41
5 4
(
1
s, H), 7.63 (d, H, J = 8.2 Hz), 7.71 (d, H, J = 8.2 Hz), 7.8–8.0 (m, 5H), 10.4 (s, H, N–H),
1
3
1.5 (s, H, N–H); C NMR (75.47 MHz, CDCl , 25 °C): δ 66.8, 67.6, 67.7, 83.3 121.1,
3
1
21.7, 122.7, 124.8, 129.6, 130.4, 131.1, 133.7, 134.7, 135.5, 136.1, 139.7, 141.2, 160.7
(
CN ), 179.1 (C=O); Anal. Calcd for C H N Cl FeO (568.2): C, 63.4; H, 4.08; N, 7.39.
3 30 23 3 2
Found: C, 62.5; H, 3.7; N, 7.2%.
2
.5.2.6. Synthesis of N-(3-ferrocenylphenyl-N'-(2,5-dichlorophenyl)-N″-benzoylguanidine)
ꢂ1
(
3a). Yield: 2.2 g (72%), m.p. 157–158 °C, FT-IR (KBr, cm ): 3301, 3156, 3092, 2955,
1
1
966, 1673, 1605, 1594, 1558, 1506, 1456, 1398, 1385, 1310, 1263, 1230, 1185, 1105,
073, 1012, 833, 726, 627, 607, 586, 572, 545, 520, 508, 477, 445, 417, 405; H NMR
1
(
300 MHz, CDCl , 25 °C): δ 4.89 (s, 5H), 4.99 (t, 2H, J = 1.9 Hz, C H ), 5.3 (t, 2H,
3
5
4
J = 1.9 Hz, C H ), 7.28–7.38 (m, 3H), 7.4 (s, H), 7.55 (s, H), 7.6 (d, H, J = 8.2 Hz), 7.7 (d,
5
4
1
3
H, J = 8.2 Hz), 7.8–8.0 (m, 5H), 10.4 (s, H, N–H), 11.51 (s, H, N–H); C NMR
75.47 MHz, CDCl , 25 °C): δ 66.8, 67.6, 67.7, 83.2, 121.5, 121.6, 122.7, 124.5, 124.8,
(
3
1
1
29.2, 129.7, 130.3, 131.1, 133.7, 134.7, 135.5, 136, 139.7, 141.5, 142.1, 160.8 (CN3),
79.3 (C=O); Anal. Calcd for C H N Cl FeO (568.2): C, 63.4; H, 4.08; N, 7.39. Found:
3
0
23
3
2
C, 62.8; H, 4.50; N, 7.1%.
3
. Results and discussion
All ferrocenyl phenylguanidines were synthesized in good yield and characterized by
different techniques. Elemental analyses of all the compounds were in agreement with cal-
culated values. In FT-IR spectra two peaks, a sharp and a weak, were observed for N–H
ꢂ1
bonds at 3392-3245 cm . The C=N stretch in all compounds was observed at 1544-
ꢂ1
1
586 cm , intermediate between single and double bond, an indication of conjugation
between all three nitrogens of the guanidine [28]. A sharp C=O stretch was also observed
ꢂ1
1
13
at 1601–1665 cm
relative to TMS as reference in chloroform. Both N–H protons appeared downfield at
.2–9 ppm due to the presence of aromatic groups on nitrogen. Aromatic protons were not
for all the compounds. H and C-NMR spectra were recorded
8
1
3
well resolved and appeared as complex patterns. In C-NMR, the carbonyl carbon was at
1
76–178 ppm while CN carbon was at 156–158 ppm as expected for guanidine [29].
3
3
.1. Single crystal X-ray analysis of 2
Crystals of 2, suitable for crystallographic analysis, were grown from ethanol by slow
evaporation. The molecular structure determination was carried out using X-ray crystallo-
graphic analyses (figure 1). Crystals were monoclinic P2(1)/c space group. The C–O bond

Ferrocenyl phenylguanidines
1965
length indicates full double bond character while the C–N bond length showed partial dou-
ble bond character, an indication of resonance in the molecule. Geometrical parameters are
summarized in table 1 while selected bond lengths and angles are given in table 2.
The packing diagram showed supramolecular structure for 2 mediated by Oꢀ ꢀ ꢀH and
πꢀ ꢀ ꢀH interactions, connecting each molecule with five other molecules resulting in a
supramolecular structure (figure 2). The Oꢀ ꢀ ꢀH interaction involving O-1 and H-13 is
2
.519 Å connecting two molecules resulted in a cyclic structure with 18-membered ring of
composition C N O .
8
4 2
Ferrocene moeities are in opposite directions to minimize repulsion; 2-chlorophenyl of
the dimer moves upward and downward for the same reason. H-27 of benzoyl of the dimer
is involved in noncovalent interaction with the chloro substituted phenyl ring (2.519 Å).
Ferrocene is also involved in H-bonding with H-1a of the guanidine (2.857 Å). These
noncovalent interactions result in the supramolecular structure as depicted in figure 3 with
cavities along the b-axis (figure 4). These compounds may find applications in separation,
storage, and transport and also in catalysis [30].
3
3
.2. DNA binding studies of ferrocenyl phenylguanidines
.2.1. By cyclic voltammetry. Cyclic voltammogram of 1 is shown in figure 5 and
comparison of 1, 2, and 3 at different concentration is given in Supplementary mate-
rial. All ferrocenyl phenylguanidines show similar voltammetric behavior, with a
couple of well-defined and stable redox peaks in the potential range of ± 1.0 V. As
no other prominent peaks were observed, all compounds were further studied from
0
.0–0.8 V.
The anodic peaks of 1, 2, and 3 are at 0.491, 0.551, and 0.521 V with corre-
sponding cathodic peaks at 0.363, 0.397, and 0.391 V, respectively. For simple ferro-
cene, oxidation was observed at 0.518 V under the same conditions. The
electrochemical behavior of the oxidizing moiety of ferrocene can be modulated by
Figure 1. Crystal structure of 2.

1
966
R. Gul et al.
Table 1. Crystallographic data for 2.
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
30 3
C H24ClFeN O
533.82
296(2)
1.54178
Monoclinic
P2(1)/c
11.1555(6)
9.4859(4)
23.1419(12)
90.00
b (Å)
c (Å)
α (°)
β (°)
γ (°)
92.978(2)
90.00
Cell volume
Density (calcd) (Mg/m )
Crystal size (mm )
Index ranges
2445.6(2)
1.450
3
3
0.40 ꢁ 0.30 ꢁ 0.20
ꢂ15 < =h < = 15
ꢂ13 < = k < = 13
ꢂ12 < = l < = 12
1104
F (0 0 0)
Total reflections
19,031
2
Refinement method
Independent reflections
R indices (all data)
Final R indices [I > 2σ(I)]
Goodness-of-fit
Full-matrix least-squares on F
1544 [R (int) = 0.0607]
R1 = 0.0691, wR2 = 0.2367
R1 = 0.0735, wR2 = 0.2311
1.060
h Range for data collection (°)
1.83 – 25.25
Table 2. Selected bond lengths (Å) and angles (°) for 2.
Fe1–C1
O1–C24
N1–C14
N1–C17
N2–C17
N2–C18
N3–C17
N3–C24
C24–C25
C23–Cl1
2.022(4)
1.239(3)
1.433(3)
1.245(3)
1.359(3)
1.399(3)
1.319(3)
1.366(3)
1.494(4)
1.738(3)
C1–Fe1–C2
C1–Fe1–C3
N1–C17–N2
N1–C17–N3
N2–C17–N3
O1–C24–N3
O1–C24–C25
Cl1–C23–C22
Cl1–C23–C18
N3–C24–C25
39.8(2)
67.1(2)
114.7(2)
125.7(2)
119.6(2)
126.6(2)
118.5(2)
119.0(2)
119.6(2)
114.9(2)
changing the electronic properties of the cyclopentadienyl ring. The slight change in
the redox behavior of the compounds to pure ferrocene is attributed to the electron
donating effect of NH on ring-A (scheme 2(a)). This group makes oxidation slightly
easier than ferrocene as evidenced by a negative shift for 1. For electron withdrawing
chloro at ring-B, a slight positive shift was observed due to a decrease in the donat-
ing effect of NH of ring-A. In general, the inductive effect works up to three or four
bonds, However, the minor change in the position of redox peak due to chloro (2,
2
a, 3, and 3a) suggests that the three nitrogens are in conjugation with each other
(scheme 2(b)) [28]. The effect is minimized when a second chloro on ring-B is pres-
ent at para position to the first one (3, 3a) and as a consequence, the two chloro
groups cancel the inductive effect of each other (scheme 2(c)).

Ferrocenyl phenylguanidines
1967
Figure 2. Connection of a reference molecule (light green) with five other molecules (2) (see http://dx.doi.org/
0.1080/00206814.2013.796371 for color version).
1
Figure 3. Supramolecular structure of 2.
Figure 4. Space filling diagram of 2 showing cavities along the b-axis.

1
968
R. Gul et al.
Figure 5. Cyclic voltammogram of 1.
Scheme 2. (a) Ferrocenyl phenylguanidine skeleton. (b) Resonating structures of guanidine and (c) effect of
electronegative substituent on basicity.
In the presence of DNA, the anodic peak potential of the compound was slightly shifted
cathodically (Supplementary material) along with decrease in I . The substantial diminu-
pa
tion in peak current is attributed to the formation of slowly diffusing compound-DNA
supramolecular complex, diminishing free compound (mainly responsible for the transfer
of current) is lowered. The mode of drug–DNA interaction can be judged from variation
in formal potential. Positive peak potential shift indicates intercalation of the drug into
DNA, negative peak potential shift is suggestive of electrostatic interaction and groove
binding is witnessed by decrease in peak current accompanied by no shift in peak position.
The negative shift in signal remaining at the same position demonstrates the interaction of
ferrocenyl phenylguanidines with DNA by electrostatic followed by groove binding.
The cyclic voltammetric behavior of guanidine without ferrocene, N-N'-diphenyl-N''-
benzoyl guanidine in the absence and presence of 30-90 μM DNA was also studied. The
voltammogram without DNA featured two irreversible oxidation peaks and no reduction
peak in the negative potential domain. By addition of DNA, a shift to less positive poten-
tials and a decrease in peak current indicates the same mode of interaction with DNA.

Ferrocenyl phenylguanidines
1969
Based upon the decay in peak current of all compounds by addition of different concen-
trations of DNA (30–60 μM, B-E), the binding constant values were calculated according
to the equation [31]:
1
Kð1 ꢂ AÞ
¼
½
DNAꢃ 1 ꢂ ðI=I Þ
0
where K is binding constant, I and I are the peak currents with and without DNA and A
0
is proportionality constant. The plot of 1/[DNA] versus 1/(1 ꢂ I/I ) yielded binding con-
0
stant values.
3
.2.2. By UV–Vis spectrophotometer. The interaction of ferrocenyl phenylguanidines
with DNA was also probed by UV–Vis spectrophotometry. The representative spectrum of
1
is given in Supplementary material. All the compounds had three strong bands at 205–
2
85 nm. In the visible region, a weak signal at 450 nm was noticed. The strong bands in the
⁄
UV–region of the spectrum can be assigned to π–π transition of aromatic phenyl ring and
C=N. It has been reported that UV–Vis spectra of ferrocene derivatives give two absorp-
tions originating from ferrocene. The following three spin-allowed ligand field transitions
1
1
1
1
1
1
are expected: A →a E , A → E , and A →b E . The first two transitions are unre-
1
g
1g
1g
2g
1g
1g
solved and give the band at 450 nm and the third transition is responsible for the signal at
25 nm. Both bands are weak owing to the Laporte-forbidden d–d character of ligand field
3
transitions [32]. Thus, the weak bands in the compounds may be assigned to ferrocene
based d–d transitions. The UV–visible spectrum of only guanidine having no ferrocene pos-
sesses three peaks and the peak corresponding to d–d transition of ferrocene was missing.
Peaks 1 and 4 were not affected by addition of DNA. Peak 2 showed a slight red shift
and 3 exhibited a blue shift. Both peaks showed pronounced hyperchromic effect (figure 6)
by the incremental addition of DNA. A small bathochromic effect is associated with a
decrease in the energy gap between the highest unoccupied and lowest occupied molecular
orbitals after interaction with DNA via groove binding mode. Minor groove binders should
be crescent in shape; the L-shaped structure of our compounds indicates that they can bind
in major grooves [33]. The hyperchromic effect and slight blue shift of 3 suggest electro-
static interaction with DNA [34]. Thus, the compounds interact with DNA by mixed binding
mode. The guanidines without ferrocene showed the same behavior on addition of DNA.
The binding constants for all compounds were calculated by the following equation [33]:
A0
eG
eG
1
¼
þ
A ꢂ A0 eH–G ꢂ eG eH–G ꢂ e K½DNAꢃ
G
where K is the binding constant, A0 and A are absorbance of the free drug and the
apparent drug-DNA adduct, while ɛG and ɛH–G are their corresponding absorption coef-
ficients. The slope to intercept ratio of the plot between A /(A ꢂ A ) vs. 1/[DNA] gave
0
0
the binding constant with values very close to those obtained from cyclic voltammetry,
5
5
ꢂ1
1
.1 ꢁ10 –5.6 ꢁ 10 M
(table 3). The binding constant values calculated for the
ferrocenyl phenylguanidines were greater with the guanidines having no ferrocene.
Hence, the presence of ferrocene enhances the DNA binding activity of guanidines.

1
970
R. Gul et al.
Figure 6. UV–Vis absorption spectra of 25 μM of 1 (a), 2 (b) and 3 (c) in the absence of DNA (A) and
presence of 30–60 μM DNA (B-E) in 20% aqueous ethanol.
3.3. Biological studies
All six compounds were screened for antifungal and antibacterial activities (tables 4 and 5),
showing moderate to significant antibacterial and antifungal activities, better than phenyl-
guanidines having no ferrocene [24]. Compounds 2, 2a, 3, and 3a having chlorophenyl
showed slightly better antibacterial and significantly better antifungal activity than 1 and
1
a, especially against Aspergillusflavus and Fusariummoniliforme fungi. Antifungal activity
of the latter class was more than the standard drug, attributed to the presence of chloro on
the phenyl ring (ring-B, scheme 2(a)). Due to its electronegativity, a decrease in the basicity
Table 3. Binding constant values.
ꢂ1
Compound name
K (M
)
5
5
1
2
3
4
5
6
A
B
N-(4-ferrocenylphenyl-N'-phenyl-N″-benzoylguanidine) 1
N-(3-ferrocenylphenyl-N'-phenyl-N″-benzoylguanidine) 1a
N-(4-ferrocenylphenyl-N'-(2-chlorophenyl)-N″-benzoylguanidine) 2
N-(3-ferrocenylphenyl-N'-(2-chlorophenyl)-N″-benzoylguanidine) 2a
N-(4-ferrocenylphenyl-N'-(2,5-dichlorophenyl-N″-benzoylguanidine) 3
N-(3-ferrocenylphenyl-N'-(2,5-dichlorophenyl)-N″-benzoylguanidine) 3a
N,N'-diphenyl-N″-benzoylguanidine
1.22 ꢁ 10
1.25 ꢁ 10
5
2.51 ꢁ10
5
2.11 ꢁ10
5
5.60 ꢁ 10
5.00 ꢁ 10
5
3
1.6 ꢁ 10
3
N-phenyl-N'-(2-chlorophenyl)-N″-benzoylguanidine
6.3 ꢁ 10

Ferrocenyl phenylguanidines
1971
Table 4. In vitro antibacterial assay of ferrocenyl phenylguanidines.
Staphylococcus
aureus
Pseudomonas
aeruginosa
Bacilus
subtilis
Klebsiella
pneumonie
Escherchia
coli
⁄
1
1
2
2
3
3
A
B
PC
13.3 ± 0.3
10.3 ± 0.3
12.0 ± 0.6
14.0 ± 0.6
14.3 ± 0.3
15.7 ± 0.3
14.3 ± 0.3
7.0 ± 0.0
13.3 ± 0.3
12.7 ± 0.3
14.3 ± 0.3
15.3 ± 0.7
15.7 ± 0.3
14.3 ± 0.3
7.70 ± 0.6
8.7 ± 0.6
12.0 ± 0.6
11.3 ± 0.3
11.6 ± 0.3
12.3 ± 0.3
13.0 ± 0.0
12.7 ± 0.3
9.91 ± 0. 6
10.0 ± 0.0
23.0 ± 0.6
10.7 ± 0.3
10.0 ± 0.0
11.7 ± 0.3
11.3 ± 0.3
11.7 ± 0.9
11.0 ± 0.6
–
a
a
a
13.3 ± 0.3
15.0 ± 0.6
15.7 ± 0.3
18.3 ± 0.7
16.7 ± 0.3
–
10.7 ± 0.6
26.7 ± 0.3
7.70 ± 0.6
26.3 ± 0.3
–
27.3 ± 0.3
20.3 ± 0.7
⁄
PC = Positive control, zone of inhibition (mm) , (ꢂ) no zone of inhibition observed, penicillin (1 mg/mL) was
'
used as standard drugs, while DMSO was used as negative control. A= N,N'-diphenyl-N″-benzoylguanidine,
B = N-phenyl-N'-(2-chlorophenyl)-N″-benzoylguanidine.
Table 5. In vitro antifungal assay of ferrocenyl phenylguanidines.
Fusarium moniliforme
Aspergillus fumigates
Aspergillus flavus
⁄
#
1
1
2
2
3
3
A
B
PC
NC
7.43 ± 0.03
7.43 ± 0.03
2.37 ± 0.33
2.47 ± 0.03
4.57 ± 0.07
3.63 ± 0.07
–
28
7.87 ± 0.09
6.8 ± 0.06
6.93 ± 0.09
2.47 ± 0.03
9.33 ± 0.03
8.4 ± 0.1
7.43 ± 0.03
5.33 ± 0.03
0.89 ± 0.03
10.7 ± 0.03
26
6.5 ± 0.1
5.33 ± 0.07
0.53 ± 0.03
0.37 ± 0.03
0.7 ± 0.1
0.37 ± 0.07
–
6.5 ± 0.03
0.83 ± 0.01
10.3 ± 0.3
33
46
95
96
93
96
a
a
a
28
77
77
56
65
35
36
77
13
21
28
46
92
0
5.57 ± 0.03
0.77 ± 0.07
10.3 ± 0.3
49
92
0
33
92
0
PC = Positive control (Terbinafin), NC = Negative control, ± shows the standard errors of mean values, (–) no
⁄
#
zone of inhibition observed, fungal growth (cm) , growth inhibition (%) .
Scheme 3. (a) Hydrophobicity of guanidine. (b) Decrease in hydrophobicity and increase in lipophilicity by
chloro group.
of NH (scheme 2(c)) and increase in lipophilicity may occur. The decrease in basicity is
due to less availability of the lone pair of electron to H-bond with water in the living cell
(
scheme 3(a) and (b)).

1
972
. Conclusion
R. Gul et al.
4
This paper describes the synthesis of six new ferrocenyl phenylguanidines and their struc-
tural characterization. They are good DNA binders with binding constant varying in the
sequence 3 ꢄ 3a > 2 ꢄ 2a > 1 ꢄ 1a. Antimicrobial study, proved them to be good anti-
fungal agents and they may emerge as environmental friendly fungicides after clinical tri-
als. Compound 2 has a supramolecular structure with channels or cavities when viewed
along the b-axis. These compounds may find applications in selective trapping of reactive
species and for conducting chemical reactions by using the cavities as reaction vessels.
Supplementary material
Single crystal X-ray diffraction data for the structural analysis have been deposited with the
Cambridge Crystallographic Data Center, CCDC No. 901,848 (2). The copy of this
information may be obtained free of charge from the Director, CCDC, 12 Union Road,
Cambridge, CBZ 1EZ, UK (Fax:+44 1223 336,033; E-mail: deposit@ccdc.cam.ac.uk or
www: http://www.ccdc.cam.ac.uk). DNA binding study for N,N'-diphenyl-N''-benzoylguani-
dine by UV–Vis spectrophotometer and cyclic voltammeter (UV–Vis spectrum and voltam-
mogram).
Acknowledgments
We are grateful to Higher Education Commission (HEC) of Pakistan for financial support.
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