Synthesis of symmetrical and asymmetrical azines from hydrazones and/or ferrocenecarboxaldehyde as potential antimicrobial–antitumor agents

Article abstract of DOI:10.1016/j.molstruc.2018.03.030

9-Fluorenone azine 2a and benzophenone azine 2b were synthesized, respectively, by treatment of 9-fluorenone hydrazone 1a or benzophenone hydrazone 1b with FeCl₃ in chloroform. Ferrocenecarboxaldehyde 3 reacts with 1a or 1b, in ethanol, to furn

Full text of DOI:10.1016/j.molstruc.2018.03.030

Accepted Manuscript
Synthesis of symmetrical and asymmetrical azines from hydrazones and/or
ferrocenecarboxaldehyde as potential antimicrobial–antitumor agents
Jamal Lasri, Magda M. Aly, Naser Eltaher Eltayeb, Bandar A. Babgi
PII:
S0022-2860(18)30319-3
10.1016/j.molstruc.2018.03.030
MOLSTR 24969
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 6 January 2018
Revised Date: 9 February 2018
Accepted Date: 11 March 2018
Please cite this article as: J. Lasri, M.M. Aly, N.E. Eltayeb, B.A. Babgi, Synthesis of symmetrical and
asymmetrical azines from hydrazones and/or ferrocenecarboxaldehyde as potential antimicrobial–
antitumor agents, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.03.030.
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ACCEPTED MANUSCRIPT
Synthesis of symmetrical and asymmetrical azines from hydrazones and/or
ferrocenecarboxaldehyde as potential antimicrobial–antitumor agents
*
Jamal Lasri , Magda M. Aly, Naser Eltaher Eltayeb, Bandar A. Babgi
GRAPHICAL ABSTRACT
O
H
Fe
NH2
FeCl3
N
R
R

ACCEPTED MANUSCRIPT
Synthesis of symmetrical and asymmetrical azines from hydrazones and/or
ferrocenecarboxaldehyde as potential antimicrobial–antitumor agents
a,*
b
a
a
Jamal Lasri , Magda M. Aly , Naser Eltaher Eltayeb , Bandar A. Babgi
a
Department of Chemistry, Rabigh College of Science and Arts, P.O. Box 344, King Abdulaziz University,
Jeddah, Saudi Arabia
b
Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
*
Corresponding author.
E-mail address: jlasri@kau.edu.sa (J. Lasri).
ABSTRACT
9
-Fluorenone azine 2a and benzophenone azine 2b were synthesized, respectively, by treatment of 9-fluorenone
hydrazone 1a or benzophenone hydrazone 1b with FeCl in chloroform. Ferrocenecarboxaldehyde 3 reacts with
a or 1b, in ethanol, to furnish novel asymmetrical azine products 1-((ferrocenyl)methylene)-2-(9H-fluoren-9-
ylidene)hydrazine 3a or 1-((ferrocenyl)methylene)-2-(diphenylmethylene)hydrazine 3b, respectively. The
3
1
1
13
+
compounds were characterized by IR, H, C and DEPT-135 NMR spectroscopy, high resolution ESI -MS or
EI, and also by single crystal X-ray diffraction analysis (in the case of 2b and 3b). The contribution of the azine
functional group (3a) in the LUMO orbital was justified by observing a red shift in the MLCT upon its
protonation. The antimicrobial activities of 2a, 2b, 3a and 3b were determined against some Gram-positive and
Gram-negative bacteria in addition to Candida albicans and Aspergillus niger using paper disc diffusion
method. Moderate antibacterial activities were found for 3a and 3b while weak activities were recorded for 2a
and 2b compared to Ampicillin, positive control. No antifungal or antitumor activities were found for all the
tested compounds, except 3a which showed antitumor activity. Low toxicity was recorded for 3a and 3b using
Artemia salina as test organism. Hence, the prepared products 3a and 3b can be used as antimicrobial agents
due to their antibacterial activities and low cell toxicity.
Keywords: Hydrazones, Ferrocenes, Azines, NMR, Single crystal X-ray, Biological studies.
1
.
Introduction
Azines, (R C=N–N=CR
and unusual reactivity. Azines showed non-linear optical (NLO) characteristics [1]. Symmetrical and
2
2
), have attracted growing attention due to their spectral properties, biological activity

ACCEPTED MANUSCRIPT
asymmetrical azines containing naphtho[2,1-b]furan showed moderate antibacterial and antifungal activities [2].
Azines were also used as ligands in coordination chemistry [3], they are 2,3-diaza compounds, therefore, they
participate in [2 + 3] criss-cross cycloaddition reactions with dienophiles affording five membered heterocycles
[
4]. Also, azines undergo [2 + 3] cycloadditions as an ene-fragment [5]. Acetophenone azines were investigated
6], using crystallographic [7], NMR measurements [8], and theoretical calculations [9]. There are several
[
methods for the synthesis of azines. They can be synthetized by reaction of aldehydes and ketones with
hydrazine sulfate, in the presence of CH CO Na/CaCl , under focused microwave irradiation [10], by refluxing
3
2
2
ketones with hydrazine hydrate in acidic ethanolic solution [11], or by reaction of carbonyl compounds with
hydrazine in the presence of molecular iodine [12]. Azine can also be synthesized by oxidation of hydrazines
using in situ generated Et NO [13], or by reaction of hydrazones with N-Bromosuccinimide (NBS) [14].
4
2
Azines with push–pull moieties were synthetized by combination of diazoalkanes with N-heterocyclic carbenes
15] which showed structural trends consistent with delocalization within the azine framework [16].
[
On the other hand, ferrocene have been of considerable interest since its discovery in 1951. The well-
established chemistry of ferrocene and its derivatives along with their remarkable stability are some of the
features that encouraged their incorporation to produce materials with non-linear optical properties [17],
reversible redox properties [18] and/or catalytic activities [19]. In addition, biologically active material
containing ferrocene have been reported recently. Jaouen et al. reported a modified Tamoxifen drug by
replacing one π-π of the aromatic rings with ferrocene fragment. The modified Tamoxifen have more effective
anticancer activity than the original drug [20]. Ferroquine and some of its derivatives have been synthesized as
an analogue to chloroquine (an antimalarial drug). Reasonable antimalarial activity was noted against
Plasmodium falciparum, even against those who are chloroquine resistant [21]. Moreover, out of fourteen
synthesized ferrocenyl amino-hydroxy-naphthoquinones compounds, three were found to be active against
Malaria and Toxoplasmosis infections, caused by Toxoplasma gondii and Plasmodium falciparum [22].
Ferrocene-based Schiff base (containing 4-aminoantipyrine moiety) and ferrocene Schiff base (containing
benzenamine fragment) were tested for their antibacterial activities using Staphylococcus aureus, Escherichia
coli and Bacillus subtilis as test organisms. Both compounds showed antibacterial activity against the three
tested bacteria [23].
The current study aimed i) to synthetize symmetrical and asymmetrical azines from hydrazones and/or
ferrocenecarboxaldehyde, ii) to characterize the prepared compounds, and, iii) to study their antimicrobial and
antitumor activities.
2
.
Experimental Section
2
2
.1. Chemistry
.1.1. Material and instrumentation

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Solvents and reagents were obtained from commercial sources (Aldrich) and used as received. For TLC, Silica
1
13
gel plates with F-254 indicator were used. H, C and DEPT-135 NMR spectra (in CDCl ) were measured
3
TM
1
13
using Bruker Avance III HD 600 MHz (Ascend Magnet) spectrometer at ambient temperature. H, C and
DEPT-135 chemical shifts (δ) are expressed in ppm relative to TMS. Fourier Transform Infrared spectra (400–
-1
4
000 cm ) was made using Alpha Bruker FTIR instrument by fusing the compounds in KBr disc. High
+
resolution electrospray ionization mass spectrometry (ESI -MS) or high resolution electron ionization (EI) mass
spectra were recorded using a MicrOTOF II mass spectrometer from Bruker or Agilent Q-TOF 6520
instrument, respectively; all mass spectrometry are reported as m/z.
2
.1.2. Synthesis of symmetrical azines, 2a and 2b
To a solution of 9-fluorenone hydrazone 1a (200 mg, 1.029 mmol) or benzophenone hydrazone 1b (202 mg,
.029 mmol) in chloroform (5 mL) was added iron(III) chloride (82.7 mg, 0.511 mmol) and the mixture was
1
refluxed for 3 h. The compound was then purified by silica-gel column chromatography using chloroform as an
eluent. The product was identified as 9-fluorenone azine 2a or benzophenone azine 2b, and obtained in 97% or
9
9% yield, respectively.
o
-1
1
9
-Fluorenone azine, 2a [24]: Mp. 265 C. IR (cm ): 3453, 3050, 1623, 1598, 1445, 1428, 1305, 1098, 940. H
NMR (CDCl ), δ: 7.27 (t, 2H, J = 7.6 Hz, CH ), 7.42 (t, 2H, J = 7.5 Hz, CH ), 7.43 (t, 2H, J = 7.4 Hz,
3
HH
ar
HH
ar
HH
CHar), 7.49 (t, 2H, JHH = 7.5 Hz, CHar), 7.68 (dd, 4H, JHH = 3.9 and 7.4 Hz, CHar), 8.08 (d, 2H, JHH = 7.4 Hz,
13
CH ), 8.17 (d, 2H, J = 7.6 Hz, CH ). C NMR (CDCl ), δ: 120.0, 120.1, 122.9, 128.2, 128.3, 129.9, 131.0,
ar
HH
ar
3
1
1
31.4 (CH ), 131.3, 136.5, 141.3, 142.3 (C ), 154.9 (C=N). DEPT-135 NMR (CDCl ), δ: 120.0, 120.1, 122.9,
ar ar 3
+
+
28.2, 128.3, 129.9, 131.0, 131.4 (CHar). ESI -MS: m/z 357.1373 [M+H] .
o
-1
1
Benzophenone azine, 2b [25]: Mp. 162 C. IR (cm ): 3442, 1637, 1628, 1561, 1489, 1444, 1320. H NMR
(
CDCl ), δ: 7.32 (t, 4H, J = 7.6 Hz, CH ), 7.37 (t, 6H, J = 7.6 Hz, CH ), 7.42 (d, 6H, J = 7.3 Hz, CHar),
3 HH ar HH ar HH
1
3
7
1
1
.50 (d, 4H, JHH = 7.5 Hz, CH ). C NMR (CDCl ), δ: 127.9, 128.0, 128.3, 128.6, 128.7, 129.3, 129.6, 130.0,
ar 3
3
32.4 (CHar), 135.5, 138.1 (Car), 159.0 (C=N). DEPT-135 NMR (CDCl ), δ: 127.9, 128.0, 128.3, 128.6, 128.7,
+
+
29.3, 129.6, 130.0, 132.4 (CH ). ESI -MS: m/z 361.1700 [M+H] .
ar
2
.1.3. Synthesis of asymmetrical azines, 3a and 3b
To a solution of ferrocenecarboxaldehyde 3 (100 mg, 0.467 mmol) in ethanol (5 mL) was added 9-fluorenone
hydrazone 1a (90.5 mg, 0.467mmol) or benzophenone hydrazone 1b (91.4 mg, 0.467mmol) and the mixture

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was refluxed for 2 h. After evaporation of the solvent in vacuo the compound was purified by silica-gel column
chromatography using chloroform as an eluent, then was characterized by IR, NMR spectroscopy, EI, and
identified as 1-((ferrocenyl)methylene)-2-(9H-fluoren-9-ylidene)hydrazine 3a (89% yield) or 1-
((ferrocenyl)methylene)-2-(diphenylmethylene)hydrazine 3b (90% yield), respectively.
-1
1
1
-((Ferrocenyl)methylene)-2-(9H-fluoren-9-ylidene)hydrazine, 3a: IR (cm ): 1625 (C=N). H NMR
(
CDCl ), δ: 4.28 (s, 5H, CH ), 4.58 (s, 2H, CH ), 4.88 (s, 2H, CH ), 7.33-7.37 (m, 2H, CH ), 7.43-7.47 (q,
3 Fc Fc Fc ar
2
7
H, JHH = 6.6 Hz, CH ), 7.64 (d, 1H, JHH = 7.4 Hz, CH ), 7.67 (d, 1H, J = 7.4 Hz, CH ), 7.93 (d, 1H, J
=
ar
ar
HH
ar
HH
1
3
3
.4 Hz, CHar), 8.54 (s, 1H, C(H)=N), 8.59 (d, 1H, JHH = 7.4 Hz, CHar). C NMR (CDCl ), δ: 69.1 (CHFc), 69.6
(CH ), 71.5 (CH ), 78.2 (C ), 119.8 (CH ), 122.6 (CH ), 128.0 (CH ), 128.1 (CH ), 130.6 (CH ), 130.7
Fc Fc Fc ar ar ar ar ar
(
CHar), 131.1 (CHar), 131.9 (Car), 137.1 (Car), 141.5 (Car), 142.2 (Car), 159.2 (C=N), 161.6 (C(H)=N). DEPT-
35 NMR (CDCl ), δ: 69.1 (CH ), 69.6 (CH ), 71.5 (CH ), 119.8 (CH ), 122.6 (CH ), 128.0 (CH ), 128.1
1
3
Fc
Fc
Fc
ar
ar
ar
+
(
CH ), 130.6 (CH ), 130.7 (CH ), 131.1 (CH ). EI: m/z 390.1 [M] .
ar ar ar ar
-1
1
-((Ferrocenyl)methylene)-2-(diphenylmethylene)hydrazine, 3b: IR (cm ): 1679 and 1656 (C=N), 1602
1
(
C=C). H NMR (CDCl
3
), δ: 4.22 (s, 5H, CHFc), 4.43 (s, 2H, CHFc), 4.62 (s, 2H, CHFc), 7.29-7.84 (m, 10H,
1
3
CH ), 8.53 (s, 1H, C(H)=N). C NMR (CDCl ), δ: 68.8 (CH ), 69.3 (CH ), 69.7 (CH ), 70.9 (CH ), 73.2
ar
3
Fc
Fc
Fc
Fc
(
(
(
(
(
(
CH ), 77.3 (CH ), 78.7 (C ), 127.5 (CH ), 127.9 (CH ), 128.0 (CH ), 128.2 (CH ), 128.3 (CH ), 128.7
Fc Fc Fc ar ar ar ar ar
CHar), 128.9 (CHar), 129.0 (CHar), 129.3 (CHar), 129.9 (CHar), 130.1 (CHar), 130.4 (CHar), 132.4 (CHar), 135.7
C ), 138.6 (C ), 159.6 (C=N), 164.2 (C(H)=N). DEPT-135 NMR (CDCl ), δ: 68.8 (CH ), 69.3 (CH ), 69.7
ar
ar
3
Fc
Fc
CHFc), 70.9 (CHFc), 73.2 (CHFc), 77.3 (CHFc), 127.5 (CHar), 127.9 (CHar), 128.0 (CHar), 128.2 (CHar), 128.3
CH ), 128.7 (CH ), 128.9 (CH ), 129.0 (CH ), 129.3 (CH ), 129.9 (CH ), 130.1 (CH ), 130.4 (CH ), 132.4
ar
ar
ar
ar
ar
ar
ar
ar
+
CH ). EI: m/z 392.1 [M] .
ar
2
2
.2. Biology
.2.1. Antimicrobial activities
The antimicrobial activities of 2a, 2b, 3a and 3b were determined against some Gram-positive and Gram-
negative bacteria using paper disc diffusion method [26].
The tested Gram-positive bacteria were Staphylococcus aureus, S. saprophyteus and Streptococcus pyogenes
while, the tested Gram-negative were Acinetobacter baumannii, Escherichia coli, Klebsilla pneumonia,
Pseudomonas earuginosa, Salmonella sp. and Serrratia marcescens. All the previous bacteria were obtained
from KFG hospital, Jeddah, Saudi Arabia. Mueller Hinton agar (MHA) medium was used for bacteria, while
MHA supplemented with 2% dextrose was used for Candida albicans and Aspergillus niger. Each plat was
6
inoculated with 100 µL of the tested microbe (4x10 cfu/mL) and using sterile glass rod, the inoculum was

ACCEPTED MANUSCRIPT
distributed on the surfaces of the agar plates. A sterile paper disk (7 mm in diameter) impregnated with 10 µL
(50 µg/disk) of the tested material, was put on the surface of each agar plate. All Petri dishes were incubated at
3
7 °C for bacteria and C. albicans and 25 °C for A. niger for 24 and 48 h, respectively. Each experiment was
carried out in triplicate and the mean diameters of the inhibition zones were measured in mm. A disk
impregnated with dimethyl sulfoxide (DMSO) at 2% was used as negative control. Minimum inhibitory
concentrations (MICs) were determined using microtiter plate following the method described by Chand et al.
[27] and modified by Aly et al. [28]. Solution of fluorescein diacetate (0.2%, w/v) in acetone was added to the
incubated bacterial cells and the resulting green color was measured at 490 nm.
2
.2.2. Toxicity test
Toxicity was determined using brine shrimp lethality test [29]. The tested material in varying concentrations
was used, the average number of larvae that survived was determined and the concentration killing fifty percent
of the larvae (LD50) was determined [29].
2
.2.3. Antitumor activity
The antitumor activity against Ehrlich carcinoma and Lymphoma cell line were determined in RPMI 1640
medium (Sigma, USA) with 10% fetal calf serum (Gibco, USA) at 37 °C under a humidified atmosphere
consisting of 95% air and 5% CO for 48 h. Cell viability percentage was determined and LD was calculated
2
50
[30].
2
.3. X-Ray crystallography
The crystallographic data and structure refinement parameters for the compounds 2b and 3b are given in Table
. The data were collected using a Bruker D8 Quest diffractometer [31] equipped with graphite monochromated
1
Mo Kα (λ = 0.71073 Å) radiation. The structure was solved using APEX3 [31] and refinement was carried out
by full-matrix least squares on F2 [32]. Molecular graphics employed was SHELXTL [31] and PLATON [33].
Hydrogen atoms were placed in calculated positions.
A specimen of 2b was used for the X-ray crystallographic analysis. The X-ray intensity data were measured.
The integration of the data using a monoclinic unit cell yielded a total of 11463 reflections to a maximum θ
angle of 25.37° (0.83 Å resolution), of which 1787 were independent (average redundancy 6.415, completeness
2
=
=
99.1%, R = 6.47%, R = 3.59%) and 1227 (68.66%) were greater than 2σ(F ). The final cell constants of a
int
sig
3
21.9160 Å, b = 5.4740 Å, c = 16.2410 Å, α = 90.000°, β = 94.410°, γ = 90.000°, volume = 1942.6 Å , are
based upon the refinement of the XYZ-centroids of reflections above 20 σ(I).

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A specimen of 3b was used for the X-ray crystallographic analysis. The X-ray intensity data were measured.
The integration of the data using a monoclinic unit cell yielded a total of 12859 reflections to a maximum θ
angle of 25.34° (0.83 Å resolution), of which 3452 were independent (average redundancy 3.725, completeness
2
=
=
99.8%, Rint = 4.31%, Rsig = 3.83%) and 2680 (77.64%) were greater than 2σ(F ). The final cell constants of a
3
16.4335(17) Å, b = 10.2203(9) Å, c = 11.7244(12) Å, β = 106.483(3)°, volume = 1888.2(3) Å , are based
upon the refinement of the XYZ-centroids of reflections above 20 σ(I). Data were corrected for absorption
effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was
0
.876.
The CIF files are obtainable free of charge from Cambridge Crystallographic Data Center (CCDC) under
CCDC number 1812976 for 2b, and 1812978 for 3b.
Table 1.Crystal data and structure refinement for 2b and 3b.
Identification code
Empirical formula
Formula weight
Temperature
2b
3b
C H N
C H FeN2
1
3
10
24 20
180.22 g/mol
293(2) K
392.27 g/mol
273(2) K
Wavelength
0.71073 Å
0.71073 Å
Crystal system
Space group
Monoclinic
C 1 2/c 1
Monoclinic
P 1 21/c 1
Unit cell dimensions
a = 21.9160 Å; α = 90°
b = 5.4740 Å; β = 94°
c = 16.2410 Å; γ = 90°
a = 16.4335(17) Å;
α
= 90°
= 106.483(3)°
γ = 90°
b = 10.2203(9) Å;
β
c = 11.7244(12) Å;
3
3
Volume
1942.6 Å
1888.2(3) Å
Z
8
4
3
3
Density (calculated)
Absorption coefficient
F(000)
1.232 g/cm
0.072 mm
760
1.380 g/cm
0.809 mm
816
-
1
-1
Theta range for data collection 2.52 to 25.37°
2.58 to 25.34°
Index ranges
-26<=h<=26, -6<=k<=6, -19<=l<=19 -19<=h<=19, -12<=k<=12, -14<=l<=14
Reflections collected
Independent reflections
Completeness to theta = 25.37° 99.1%
11463
1787 [R(int) = 0.0647]
12859
3452 [R(int) = 0.0431]
99.8%
Full-matrix least-squares on F
3452 / 0 / 244
1.058
2
2
Refinement method
Full-matrix least-squares on F
1787 / 0 / 128
1.079
Data / restraints / parameters
2
Goodness-of-fit on F
Final R indices [I>2sigma(I)]
R indices (all data)
Largest diff. peak and hole
R1 = 0.0483, wR2 = 0.1107
R1 = 0.0800, wR2 = 0.1254
0.145 and -0.151
R1 = 0.0336, wR2 = 0.0877
R1 = 0.0549, wR2 = 0.1034
0.454 and -0.350
3
.
Results and discussion

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3
3
.1. Chemistry
.1.1. Synthesis of symmetrical 9-fluorenone azine 2a and benzophenone azine 2b
The 9-fluorenone azine 2a was synthesized, in 97% yield, starting from 9-fluorenone hydrazone 1a and using
FeCl as Luis acid catalyst (Scheme 1) [24].
3
FeCl3
N
NH2
N
N
CHCl3
reflux, 3 h
1
a
2a
97%)
(
Scheme 1. Synthesis of 9-fluorenone azine 2a.
Similarly, benzophenone azine 2b was synthesized, in 99% yield, starting from benzophenone hydrazone 1b
and using the same reaction conditions (FeCl , chloroform, reflux for 3 h) (Scheme 2).
3
Ph
Ph
Ph
Ph
Ph
Ph
FeCl3
N
NH2
N
N
CHCl3
reflux, 3 h
1
b
2b
99%)
(
Scheme 2. Synthesis of benzophenone azine 2b.
Regarding the plausible mechanism of the above reactions (Schemes 1 and 2), the reaction is believed to
proceed by coordination of FeCl Lewis acid catalyst to the terminal nitrogen of hydrazone 1, this coordination
3
enhances the electrophilicity of the imino-carbon atom of 1 which can readily undergo nucleophilic attack by
2 3
the amino moiety NH of the uncoordinated hydrazone 1 to produce, after elimination of FeCl and hydrazine
[34], the symmetrical azine 2 (Scheme 3).

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H
R
H
N
H
R
R
N
N
H
FeCl3
H
N
FeCl3
R
R
R
NH
N
FeCl3
NH2
2
N
NH2
N
1
R
R
R
R
1
R
H
-
FeCl3
N
NH2
R
R
R
R
H
-
H NNH2
2
N
N
N
N
R
2
R
R
3
Scheme 3. Proposed mechanism for the synthesis of symmetrical azine 2 from hydrazone 1 catalysed by FeCl .
1
13
The formation of symmetrical azines 2a and 2b was confirmed by IR, H, C and DEPT-135 NMR
+
spectroscopy, ESI -MS and also by single crystal X-ray diffraction analysis (in the case of 2b).
The compound 2b (Figure 1) consists of a centrosymmetric tetraphenyl hydrazone molecule. The 1,2-
bis(diphenylmethylene) moieties adopt an anti-configuration with respect to the hydrazine N-N fragment. The
dihedral angle between the two phenyl rings in the hydrazone molecule is 77.85(11)°. The crystal of 2b is lack
of intra- and intermolecular hydrogen bonds as well as pi-pi bonds. Similar crystal was previously reported and
it showed the same feature with a nitrogen-nitrogen single bond 1.40 Å and a nitrogen-carbon multiple bonding
1
.28 Å [35].

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Figure 1.Crystal structure of benzophenone azine 2b.
3
.1.2. Synthesis of novel asymmetrical ferrocene azines 3a and 3b
On the other hand, the ferrocenecarboxaldehyde 3 reacts with 9-fluorenone hydrazone 1a, in refluxing ethanol
for 2 h, to furnish the asymmetrical azine product 1-((ferrocenyl)methylene)-2-(9H-fluoren-9-ylidene)hydrazine
3
a in 89% yield (Scheme 4, reaction a). Similarly, carboxaldehyde 3 reacts with benzophenone hydrazone 1b
under the same experimental conditions to produce 1-((ferrocenyl)methylene)-2-(diphenylmethylene)hydrazine
b in 90% yield (Scheme 4, reaction b).
3

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H
N
N
Fe
3
a
(
89%)
O
H
Fe
h
2
,
x
3
u
l
f
Ph
Ph
e
H
r
N
N
Fe
3
b
(
90%)
Scheme 4. Syntheses of 1-((ferrocenyl)methylene)-2-(9
H-fluoren-9-ylidene)hydrazine 3a (reaction a) and 1-
((ferrocenyl)methylene)-2-(diphenylmethylene)hydrazine 3b (reaction
b
).
1
13
The structure of novel compounds 3a and 3b was confirmed by IR, H, C and DEPT-135 NMR spectroscopy,
high resolution electron ionization (EI) and in the case of 3b also by single crystal X-ray diffraction analysis.
In the compound 3b (Figure 2) the ferrocenyl moiety adopts an eclipsed conformation. The average Fe—C(Cp)
bond length (Cp is cyclopentadienyl) is 2.03 Å, in agreement with that of other ferrocenes. The dihedral angle
between the two phenyl rings in the hydrazone molecule is 74.92(15)°. Apart from pi-pi interaction, 3.2883(18)
Å, of Cg(1)…Cg(2) with symmetry code: X,Y,Z [1555.01] 3.2883(18); Cg1 is centroid of the ring C15-C19 and
Cg2 is centroid of the ring C20-C24. No significant intermolecular interactions are observed in the crystal
packing.

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Figure 2. Crystal structure of 1-((ferrocenyl)methylene)-2-(diphenylmethylene)hydrazine 3b.
3
.1.3. Spectral studies
The absorption spectra of solutions of ferrocene-containing compound collected in dichloromethane are shown
in Figure 3. The UV-vis spectra of asymmetrical azine 3a show three major bands centered at 260, 345 and 510
nm. The UV-vis spectra of substituted ferrocenes have been explored recently [36]. In ferrocene containing
2
z
conjugated substituents, the HOMO orbital is assigned as d
orbital and the LUMOs are a set of ligands orbitals
in combination with the metal d and d . The absorption around 510 nm in ferrocene is ascribed as the d ꢀπ
xz
yz
transition while the energy band around 345 nm is assigned as the d
ꢀd transition [37]. The presence of the
azine functional group as a Lewis base encouraged us to test the effect of acidic medium on our system. The
solution of compound 3a was treated with one drop of diluted sulfuric acid, causing a color change from red to
green and a strong shift in the low energy band in the spectrum which suggest the strong contribution of the
azine linkage in the LUMO orbital.
Unfortunately, the absorption spectra of compound 3b were unsuccessful due to its instability and/or
decomposition in acidic solution.

ACCEPTED MANUSCRIPT
7
5
2
5000
0000
5000
0
2
30
330
430
530
630
730
Wavelength (nm)
Figure 3. The spectral change observed in the compound 3a upon the acidification process (red line: neutral,
blue line: acidified).
3
.2. Biology
The antimicrobial activity of the tested compounds was qualitatively and quantitatively assessed using paper
disk diffusion method. The diameters of the inhibition zone obtained with the different products against bacteria
were reported (Table 2). Acinetobacter baumannii was the most resistant bacterium to the synthesized
compounds 2a, 3a and 2b, while excellent growth inhibition was obtained using 3b. However, 2a and 2b have
weak inhibitory activities, 3a and 3b showed moderate activities, especially against Salmonella sp. Compound
2
a showed weak activities against all tested bacteria except Serrratia marcescens. Ampicillin was used a
positive control and it was more active against Gram-positive bacteria, diameter of inhibition zone ranged from
2-29 mm and it ranged from 10-17 mm for Gram-negative bacteria. Similarly, several studies found that
2
Gram-positive bacteria were generally more sensitive than Gram-negative [38].
The minimum inhibitory concentration (MIC) was determined for 3a and 3b using micromethod and phenol red
as an indicator. It was clear that MIC ranged from 5-12 and 10-20 µg/ml for 3a and 3b, respectively, while it
ranged from 2-10 µg/ml for Ampicillin (Table 3). Low toxicity was recorded for 3a and 3b (LD >50 µg/ml),
5
0
while antitumor activity (LD50 = 20 µg/ml) was recorded for 3a against both Erlish and Lymphoma cell lines.
No antitumor activity against the two tested cell lines was recorded for 3b. Elspar (trade name for asparaginase)
was used as control antitumor agent (Table 4).

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Table 2. Antibacterial activity of the four tested compounds 2a, 2b, 3a and 3b against some Gram-positive and
Gram-negative bacteria.
Diameter of inhibition zone (mm)
Ampicillin
(control)
Bacterial isolates
2
a
3a
2b
3b
Staphylococcus aureus
9.3 ±1.5*
9.2 ±1.8*
8.3 ±1.9*
14.3 ±1.5*
10.4 ±0.1*
11.3
22
25
29
±1.7*
Staphylococcus saprophyteus
Streptococcus pyogenes
20 ±1.7*
10.6 ±2.0*
10.1 ±0.9*
8.0±0.1*
9.3±1.6* 10.5±0.9*
9.6±1.2* 12.7±0.7*
10.4±0.2*
10.6
±1.1*
14.0±1.0*
10.5
±1.1*
Acinetobacter baumannii
Escherichia coli
Klebsilla pneumonia
Pseudomonas earuginosa
7.0±1.0*
8.3±1.4*
8.6±1.6*
8.4±1.0*
ND
19.7±1.1
10
14
14
12
11.0±1.0*
10.5±2.9*
10.0±1.0*
10.7±0.
8
*
Salmonella sp.
Serrratiamarcescens
ND
15.3±2.3
17.8±2.9
13.0±2.0*
9.7±0.7* 19.6±3.98
9.3±0.3 17.3±3.0
16
17
*
: Significant results compared to control, ND: No antibacterial activity
Table 3. Minimal inhibitory concentrations of 3a and 3b compared to control antibacterial agent.
MIC (µg/ml)
Bacterial isolates
3
a
3b
Ampicillin
(control)
Staphylococcus aureus
Staphylococcus saprophyteus
Streptococcus pyogenes
Acinetobacter baumannii
Escherichia coli
Klebsilla pneumonia
Pseudomonas earuginosa
Salmonella sp.
12
5
20
20
20
10
20
20
20
10
12
2
2
4
10
4
2
4
4
10
12
ND
20
20
20
5
Serrratia marcescens
12

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Table 4. Toxicity against Artemia salina and antitumor activities of 3a and 3b.
Tested
Toxicity
against
Artimia salina
Antitumor activity (LD µg/ml)
50,
products
Lymphoma cell
line
Erlish cell
line
(
LD µg/ml)
50,
3
a
>50
>50
>50
40
>50
30
45
>50
30
3
b
Elspar*
*: control antitumor
4
.
Conclusions
9
-Fluorenone azine 2a and benzophenone azine 2b were synthesized, respectively, by treatment of 9-fluorenone
. Additionally, ferrocenecarboxaldehyde reacts with 9-
hydrazone or benzophenone hydrazone with FeCl
3
fluorenone hydrazone or benzophenone hydrazone to afford novel asymmetrical azines 3a and 3b, in very good
yields, without the need of any acid catalyst. The protonation of the azine functional group by adding acids to
the solution of compound 3a caused a color change from red to green associated with a strong shift in the low
energy band in the spectrum, suggesting the strong contribution of the azine linkage in the LUMO orbital. On
the other hand, molecules 3a and 3b showed a promising antibacterial activity against both Gram-positive and
Gram-negative bacteria. Compound 3a showed an excelled antibacterial activity against Multidrug-resistant
(MDR) Acinetobacter baumannii which emerged in the last decades as a major cause of healthcare-associated
infections and nosocomial outbreaks. Antimicrobial resistance of many bacteria poses great limits for
therapeutic options in infected patients, especially if the isolates are resistant to the used antibiotics. Thus, the
synthesis of new antibacterial agents is urgently needed and the use of these new agents singly or in
combinations with existing antibiotics is essential to face these important hospital problems. Moreover, MIC of
3
a was lower than that calculated for 3b, indicating higher antibacterial activities of 3a. No toxicity was
recorded for the two tested compounds at 50 µg/ml, while 3a recoded antitumor activity against both
Lymphoma and Erlish cell lines (LD = 40-45µg/ml).
5
0
Acknowledgment

ACCEPTED MANUSCRIPT
This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah,
under grant no. (G-100-662-37). The authors, therefore, acknowledge with thanks DSR for technical and
financial support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/....
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Highlights
•
Symmetrical and asymmetrical azines were synthetized from hydrazones and/or
ferrocenecarboxaldehyde.
+
•
•
•
Azines were characterized by IR, NMR, ESI -MS and X-ray diffraction.
The absorption spectra of solutions of ferrocene-containing compound were investigated.
The antimicrobial and antitumor activities of azine compounds were determined.