Novel Pyrimidine-based Ferrocenyl substituted Organometallic Compounds: Synthesis, Characterization and Biological Evaluation

Article abstract of DOI:10.1002/aoc.4261

Some novel pyrimidine-based ferrocenyl substituted organometallic compounds were synthesized via multistep reactions, well characterized by different spectroscopic techniques and elemental analyses and evaluated for in vitro antiprotozoal susceptibility against HM1: IMSS strain of Entamoeba histolytica. The results of antiprotozoal susceptibility unveiled these compounds, as new leads in protozoal chemotherapy as most of the organometallics displayed an exceptionally higher antiamoebic activity (IC₅₀ = 0.055 μM - 0.815 μM) than the reference drug metronidazole which gave IC₅₀ (50% inhibitory concentration) value 1.781 μM in our experiments, concluding that newly synthesized organometallic compounds have potential to be employed as effective antiamoebic agents and these organometallics can be very useful for further optimization work on amoebic chemotherapy.

Full text of DOI:10.1002/aoc.4261

Received: 23 November 2017
DOI: 10.1002/aoc.4261
Revised: 10 December 2017
Accepted: 12 December 2017
F U L L P A P E R
Novel Pyrimidine‐based Ferrocenyl substituted
Organometallic Compounds: Synthesis, Characterization
and Biological Evaluation
1
1
1
1
Humaira Parveen
| Meshari A. Alsharif | Mohammed I. Alahmdi | Sayeed Mukhtar |
2
Amir Azam
1
Department of Chemistry, Faculty of
Science, University of Tabuk, Tabuk
Some novel pyrimidine‐based ferrocenyl substituted organometallic com-
pounds were synthesized via multistep reactions, well characterized by differ-
ent spectroscopic techniques and elemental analyses and evaluated for in vitro
antiprotozoal susceptibility against HM1: IMSS strain of Entamoeba histolytica.
The results of antiprotozoal susceptibility unveiled these compounds, as new
leads in protozoal chemotherapy as most of the organometallics displayed an
exceptionally higher antiamoebic activity (IC50 = 0.055 μM ‐ 0.815 μM) than
the reference drug metronidazole which gave IC50 (50% inhibitory concentra-
tion) value 1.781 μM in our experiments, concluding that newly synthesized
organometallic compounds have potential to be employed as effective
antiamoebic agents and these organometallics can be very useful for further
optimization work on amoebic chemotherapy.
71491, Kingdom of Saudi Arabia
2
Department of Chemistry, Jamia Millia
Islamia, Jamia Nagar, New Delhi 110025,
India
Correspondence
Humaira Parveen, Department of
Chemistry, Faculty of Science, University
of Tabuk, Tabuk‐71491, Kingdom of Saudi
Arabia.
Email: h.nabi@ut.edu.sa
Funding information
Deanship of Scientific Research (DSR),
University of Tabuk, Kingdom of Saudi
Arabia, Grant/Award Number: S‐0117‐
KEYWORDS
1438
antiprotozoal assay, ferrocenyl unit, HM1: IMSS strain of Entamoeba histolytica, new leads in amoebic
chemotherapy, novel pyrimidine‐based organometallics
1
| INTRODUCTION
activity have justified it an outstanding pharmacophore
for drug design.
[
9]
The discovery of ferrocene and ferrocene‐linked organo-
Amoebic dysentery (amoebiasis) is caused by an
anaerobic protozoan parasite Entamoeba histolytica. This
eukaryotic parasite penetrates into the intestinal mucosa
by the ingestion of food or water carrying the cysts of Ent-
amoeba histolytica and causes amoebic colitis. Entamoeba
metallic compounds have made marked influence on the
[1–3]
growth of bioorganometallic chemistry
as these
organometallics have been well established their pharma-
ceutical potential in drug discovery, for example,
ferroquine discovered as antimalarial drug and it is in
histolytica is also the causative agent of amoebic liver
[4]
[10–12]
phase II clinical trials, ferrocifen and hydroxy ferrocifen
abscess.
It has been reported that fifty million cases
[
5,6]
are discovered as anticancer drugs,
also ferrocenyl
of amoebic dysentery and one lakh deaths occur annually,
due to the infectious diseases caused by this protozoan
[7]
conjugates of antiestrogen tamoxifen and antiandrogen
[8]
[13]
nilutamide have been reported as more potent, curative
parasite (E. histolytica).
[9]
agents than the core drug.
Metronidazole (an antiprotozoal drug) is extensively
used for the treatment of amoebic dysentery but it has
been reported to clinical resistance to anaerobic proto-
The unique properties of ferrocene such as low toxic-
ity to mammals, high stability, formal oxidation potential,
lipophilicity, electron donating entity and iron redox
[
14]
zoans,
mutagenic in microbiological system and
Appl Organometal Chem. 2018;e4261.
wileyonlinelibrary.com/journal/aoc
Copyright © 2018 John Wiley & Sons, Ltd.
1 of 8
https://doi.org/10.1002/aoc.4261

2
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PARVEEN ET AL.
carcinogenic in rodents 15–17] and it has been also associ-
ated with adverse side effects such as sensory neuropa-
thies, toxicity with ataxia, metallic taste, vomiting,
[
2 | RESULTS AND DISCUSSION
2.1 | Synthesis and characterization
[
18,19]
diarrhea, vertigo, seizures and encephalopathy.
Moreover, in some cases, failure of treatment with metro-
nidazole has also been reported.
search of new effective antiprotozoal agents is urgently
needed to develop potent yet safer therapeutic agents for
amoebiasis.
The synthesis of novel pyrimidine‐based ferrocenyl
substituted organometallics (3a‐3h) was carried out as
illustrated in Scheme 1. The ferrocenyl chalcones
(1a‐1h) were synthesized according to the reported
[10,20,21]
Therefore, the
[
58]
method
by the Claisen–Schmidt condensation of ace-
Furthermore, pyrimidines are ubiquitous, being an
essential part of nucleic acids, involve in several biological
processes and are main constituents of many drugs such
as Zidovudine (anti‐HIV), Lamivudine (anti HIV‐AIDS,
Hepatitis B) and many of its derivatives display wide‐
tyl ferrocene with substituted aromatic aldehydes in the
presence of potassium hydroxide and absolute ethanol.
Morpholin‐4‐carboxamidine hydrochloride (2) was pre-
[
59]
pared according to reported procedure
by refluxing
morpholine with S‐methyl isothiourea sulfate in water.
The cyclization of ferrocenyl chalcones (1a‐1h) with
morpholin‐4‐carboxamidine hydrochloride (2) in the
presence of sodium isopropoxide led to the formation of
corresponding pyrimidine‐based ferrocenyl substituted
organometallic compounds (3a‐3h). The structures of
all compounds were established by different spectro-
[
22]
spread pharmacological properties such as anti‐cancer,
[
23]
[24]
[25]
[26,27]
antiviral,
antimalarial,
diuretic,
anti HIV‐1,
[28]
[29]
antitumor
and antiplasmodial activities.
Moreover,
extensive literature survey on nucleobase‐derived com-
pounds in modern medicinal chemistry also unveiled
the importance of ferrocenyl nucleobases and half‐sand-
[30–32]
1
13
wich nucleobase derivatives.
scopic techniques such as IR, H‐NMR and C NMR
and Mass spectrometry. The purity of compounds was
confirmed by elemental analysis which was found in
accordance with ± 0.3%.
In the recent decades, morpholine scaffold has
emerged as an outstanding pharmacophore in medicinal
[
33]
chemistry and a number of clinically approved drugs
such as linezolid, aprepitant, and gefitinib
[34,35]
possesses
The data of selected characteristic IR bands provide
significant indications of the formation of the pyrimi-
dine‐based organometallics (3a‐3h). All compounds
morpholine unit in their molecules. Morpholine deriva-
tives also have been reported as novel scaffold for new
[36]
‐1
drug discovery.
Also, a number of reports have been
showed sharp bands in the region 1571–1577 cm due
suggested the potential of morpholine derivatives as effec-
to C=N stretch which confirmed the formation of
pyrimidine ring. In addition, the absorption bands in
the region 1251–1258 cm‐ were assigned to the C–N
stretch vibrations, which also confirm the formation of
desired pyrimidine ring in all organometallics. The
structures of compounds were further confirmed by
[37–43]
tive anticancer agents.
Beside this, morpholine con-
1
taining compounds also have been reported to exhibit
[
44–49]
diverse biological properties
such as γ‐secretase
[50]
[51]
[52]
inhibitors,
antioxidant
and anti‐inflammatory.
According to mechanism of action of morpholine deriva-
tives it is clear, that these derivatives prevent the biosyn-
thesis of sterol by blocking two successive enzymatic
processes: (1) inhibiting the biotransformation of
lanosterol into zymosterol by blocking the enzyme C‐14
sterol reductase and; (2) inhibiting the synthesis of ergos-
terol from the biotransformation of fecosterol into
episterol by blocking the enzyme C‐8 sterol isomer-
1
13
H‐NMR and C NMR. The appearance of a singlet in
the range of δ 7.32‐7.38 was attributed to the C‐H pro-
ton of pyrimidine ring in all organometallics (3a‐3h).
The chemical shift values for aromatic protons, ferro-
cene ring protons and morpholine ring protons reso-
nated at their usual position, and the data are given in
the experimental section.
[53,54]
ase.
The advantage of synthesizing morpholine
Further evidence for the formation of pyrimidine‐
1
3
derivatives resides in the fact that morpholine derivatives
provide chlorhydrates that are soluble in water for phar-
based organometallics was obtained from their
C
NMR spectra. A signal in the range of 166.2–166.9 and
162.5–162.8 was attributed to C=N and C=C respectively
in all organometallics (3a‐3h). The characteristic signal
for C‐H of pyrimidine ring in all organometallics has
appeared in the range of 104.2‐104.6, which clearly
favored the formation of pyrimidine nucleus in all com-
pounds (3a‐3h). Other signals attributed to phenyl,
ferrocenyl and morpholine moieties, resonated at their
usual positions, and their values are given in the experi-
mental section.
[55]
macological assays.
In view of above observations and in continuation of
our ongoing research based on to explore biologically
[56,57]
active organometallic/heterocyclic compounds,
we
report herein synthesis and characterization of some
novel pyrimidine‐based ferrocenyl substituted organome-
tallic compounds containing morpholine unit, and evalu-
ation of their in vitro antiprotozoal studies against HM1:
IMSS strain of Entamoeba histolytica.

PARVEEN ET AL.
3 of 8
SCHEME 1 General synthesis of pyrimidine‐based ferrocenyl substituted organometallics (3a‐3h)
2
.2 | Pharmacology (In vitro antiamoebic
experiments were performed in triplicate at each concen-
tration level and repeated three times. The results of the
antiprotozoal assay are given in Table 1.
activity)
All novel organometallics (3a‐3h) were evaluated in vitro
The data have been given in terms of percent growth
inhibition relative to untreated controls and plotted as
probit values as a function of drug concentration.
antiamoebic activity against HM1: IMSS strain of
[
60]
Entamoeba histolytica by microdilution method.
All
TABLE 1 Novel pyrimidine‐based ferrocenyl substituted organometallics (3a‐3h), their antiamoebic activity against HM1: IMSS strain of
Entamoeba histolytica
Antiamoebic activity
S. No.
R
a
IC50 (μM)
S. D. (±)
± 0.011049
3
a
H
0.815
3
b
4‐OH
0.142
3.780
4.541
0.697
0.055
0.251
±0.010578
±0.003505
±0.002976
±0.002659
±0.004103
±0.004257
3
c
4‐NO
2
3
d
4‐CH
3
3
e
3,4‐DiCH
3
3
f
4‐OCH
3
2,5‐Di OCH
3
3
g
3
h
4‐Cl
7.717
1.781
±0.001923
±0.003151
Metronidazole
a
Standard Deviation. The compounds with bold font IC50 values are more active than metronidazole.

4
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PARVEEN ET AL.
Metronidazole was used as a reference drug to compare
antiamoebic effect which gave IC50 (50% inhibitory con-
centration) 1.781 μM in our experiments.
The results showed that in pyrimidine‐based organo-
metallics when the R group was a phenyl ring the
morpholine‐linked compound (3a) showed IC50
analyzer. The results were within ± 0.3% of the theoretical
values. Melting points were determined on Thomas Hoo-
ver capillary melting apparatus and are uncorrected. IR
spectra were recorded on Perkin‐Elmer model 1600 FT‐IR
1
13
RX1 spectrophotometer as KBr discs. H‐NMR and
C
NMR spectra were recorded on Bruker AVANCE 400 spec-
0
.815 μM. Substitution of the phenyl ring with hydroxyl
trometer using CDCl as a solvent with tetramethylsilane
3
group (3b), dimethyl group (3e), monomethoxy (3f) and
dimethoxy (3g) enhanced the antiamoebic activity, as all
these compounds exhibited IC50 in the range of
(TMS) as an internal standard. Splitting patterns are desig-
nated as follows; s, singlet; d, doublet; m, multiplet. Chem-
ical shift (δ) values are given in ppm. ESI‐MS was recorded
on a MICROMASS QUATTRO II triple quadrupole mass
spectrometer.
0
.055 μM‐0.815 μM, which is higher than the standard
drug metronidazole. However, nitro group (3c), methyl
group (3d) and chloro group (3h) did not affect the
activity. In general, compound (3f) emerged as the most
active antiamoebic organometallic with IC50 0.055 μM
with respect to standard drug metronidazole (IC50
4
.1 | General procedure for the synthesis
of ferrocenyl chalcones (1a‐1h)
1
.781 μM).
The results of antiamoebic activity identified these
organometallics as useful leads in amoebic chemotherapy.
Ferrocenyl chalcones (1a‐1 h) were synthesized according
[
58]
to reported method
and their data has been found in
[
58]
agreement with the reported ones.
A mixture of acetyl
ferrocene (6 mmol) and potassium hydroxide (7.14 mmol)
was dissolved in absolute ethanol (50 ml) and stirred for
15 minutes at room temperature, then a solution of
substituted aromatic aldehydes (prepared in absolute eth-
anol) was added drop wise to the reaction mixture and
was further stirred at room temperature. The reaction
was monitored by thin layer chromatography on pre-
coated silica gel sheets. After completion the reaction mix-
ture was neutralized by 2 M hydrochloric acid (HCl),
leading to the formation of a deep red precipitate which
was filtered and first washed with water alone several
times then with cold ethanol‐water mixture, dried under
reduced pressure to obtain pure respective ferrocenyl
chalcone.
3
| CONCLUSION
A series of some novel pyrimidine‐based ferrocenyl
substituted organometallic compounds were synthesized
as new leads in antiamoebic chemotherapy. The struc-
tures of all organometallics (3a‐3h) were characterized
by different analytical techniques and elemental analyses.
The in vitro antiamoebic activity was performed by
microdilution method against HM1: IMSS strain of Ent-
amoeba histolytica.
The results of antiamoebic activity revealed that most
of the organometallics exhibited exceptionally higher IC50
values ranging from 0.055 μM to 0.815 μM, than the refer-
ence drug Metronidazole (IC50
1.781 μM). 4‐(4‐
=
4
.2 | General procedure for the synthesis
methoxyphenyl)‐6‐ferrocenyl‐2‐morpholin‐1‐yl‐pyrimi-
dine (3f) was emerged as most potent antiamoebic agent
with the IC50 = 0.055 μM. The results of antiamoebic
activity concluded that these novel organometallic com-
pounds have potential to be employed as effective
antiamoebic agents and can be very useful for further
optimization work on amoebic chemotherapy.
of morpholine‐4‐carboxamidine
hydrochloride (2)
Morpholine‐4‐carboxamidine hydrochloride (2) was
synthesized according to reported procedure
refluxing morpholine with S‐methyl isothiourea sulfate
in water.
[59]
by
4
.3 | General procedure for the synthesis
4
| EXPERIMENTAL
of pyrimidine‐based ferrocenyl substituted
organometallics (3a‐3h)
Acetylferrocene,
substituted
aromatic
aldehydes,
morpholine, other chemicals, and reagents were pur-
chased from Sigma Aldrich, St. Louis, MO, USA). Thin‐
layer chromatography (TLC) was performed on precoated
aluminum sheets (silica gel 60 F254, Merck, Darmstadt,
Germany) and spots were visualized under UV light. Ele-
mental analyses were performed on Heraeus Vario EL III
To a solution of morpholine‐4‐carboxamidine hydrochlo-
ride (6.10 mmol) in isopropanol (50 ml), sodium metal
(6.71 mmol) was added. The reaction mixture was
refluxed for two hours, then substituted ferrocenyl
chalcone (1a‐1h) (6.10 mmol) was added to the reacion
mixture and further refluxed for eight hours. Progress of

PARVEEN ET AL.
5 of 8
reaction was checked by TLC, after completion of the
reaction, solvent was removed under reduced pressure,
then water was added to the reaction mixture and the
aq. layer was extracted with chloroform then washed with
phenyl, J = 8.65 Hz), 7.18 (d, 2H, H‐3,5 phenyl, J =
8.65 Hz), 7.38 (s, 1H, Pyrimidine), 4.76 (s, 2H, ferrocene),
4.55 (s, 2H, ferrocene), 4.21 (s, 5H, ferrocene), 3.79 (t, 4H,
J = 4.6 Hz, morpholine), 2.88 (t, 4H, J = 4.6 Hz,
13
1
0% NaCl solution. The organic layer was dried over
morpholine); C NMR (CDCl ) δ(ppm): 166.5 (C=N
3
anhydrous sodium sulphate, filtered and concentrated in
pyrimidine), 162.8 (C=C pyrimidine), 162.2 (N=C‐N
pyrimidine), 145.6‐ 111.9 (Ar‐C), 104.6 (C‐H pyrimidine),
66.9 (C‐O‐C, morpholine), 44.5 (C‐N‐C, morpholine).
vacuo. The crude product was crystallized from CH OH
3
or C H OH to yield, respective pure organometallic
2
5
+
compound.
ESI‐MS m/z: [M +1] 471.3.
4
.3.1 | 4‐Phenyl‐2‐morpholin‐1‐yl‐6‐
4.3.4 | 4‐Ferrocenyl‐2‐morpholin‐1‐yl‐6‐p‐
tolyl‐pyrimidine (3d)
ferrocenyl pyrimidine (3a)
Yield: 68%; m.p: 212 °C; Maroon solid. Anal. calc. for
C H FeN O: C 67.78, H 5.45, N 9.88 % . Found: C
Yield: 72%; m.p: 251 °C; Reddish brown solid. Anal. calc.
for C H FeN O: C 68.85, H 6.02, N 9.28 % . Found: C
68.81, H 6.05, N 9.25 %. IR νmax (cm ): 3047 (Ar–H),
24
23
3
25 25
3
‐1
‐1
6
2
7.72, H 5.47, N 9.79 %. IR νmax (cm ): 3048 (Ar–H),
924 (C‐H), 1577 (C=N), 1472 (C=C), 1251 (C–N), 1120
2929 (C‐H), 1571 (C=N), 1469 (C=C), 1255 (C–N), 1116
1
1
(
C‐O‐C); H NMR (CDCl ) δ (ppm): 7.62‐7.85 (m, 5H,
(C‐O‐C); H NMR (CDCl ) δ (ppm): 7.36 (s, 1H, Pyrimi-
3
3
Ar‐H), 7.33 (s, 1H, Pyrimidine), 4.78 (s, 2H, ferrocene),
dine), 7.06 (d, 2H, H‐2,6 Phenyl, J = 8.85 Hz), 6.99 (d,
2H, H‐3,5 phenyl, J = 8.85 Hz), 4.79 (s, 2H, ferrocene),
4.55 (s, 2H, ferrocene), 4.19 (s, 5H, ferrocene), 3.76 (t,
4H, J = 4.5 Hz, morpholine), 2.88 (t, 4H, J = 4.5 Hz,
morpholine), 2.35 (s, 3H, ‐CH3); C NMR (CDCl3)
δ(ppm): 166.2 (C=N pyrimidine), 162.6 (C=C pyrimi-
dine), 162.1 (N=C‐N pyrimidine), 145.1‐ 112.7 (Ar‐C),
4
.51 (s, 2H, ferrocene), 4.20 (s, 5H, ferrocene), 3.79 (t,
4
H, J=4.5 Hz, morpholine), 2.85 (t, 4H, J=4.5 Hz,
13
morpholine); C NMR (CDCl ) δ(ppm): 166.2 (C=N
3
13
pyrimidine), 162.8 (C=C pyrimidine), 162.5 (N=C‐N
pyrimidine), 145.5‐ 118.2 (Ar‐C), 104.2 (C‐H pyrimidine),
6
6.5 (C‐O‐C, morpholine), 44.2 (C‐N‐C, morpholine),
+
ESI‐MS m/z: [M +1] 425.31.
104.2 (C‐H pyrimidine), 66.2 (C‐O‐C, morpholine), 44.5
+
(
C‐N‐C, morpholine), 22.4 (CH ), ESI‐MS m/z: [M +1]
3
4
40.34.
4
.3.2 | 4‐(4‐hydroxyphenyl)‐6‐ferrocenyl‐2‐
morpholin‐1‐yl‐pyrimidine (3b)
4
.3.5 | 4‐(3,4‐Dimethylphenyl)‐6‐
ferrocenyl‐2‐morpholin‐1‐yl‐pyrimidine
3e)
Yield: 71%; m.p: 168 °C; Dark red solid. Anal. calc. for
C H FeN O : C 65.38, H 5.22, N 9.59 % . Found: C
24
23
3 2
(
‐1
6
2
5.32, H 5.24, N 9.51 %. IR νmax (cm ): 3046 (Ar–H),
929 (C‐H), 1572 (C=N), 1469 (C=C), 1256 (C–N), 1118
Yield: 66%; m.p: 295 °C; dark red solid. Anal. calc. for
C H FeN O: C 69.41, H 6.28, N 8.96 %. Found: C
69.49, H 6.21, N 8.95 %. IR νmax (cm ): 3049 (Ar–H),
1
(C‐O‐C); H NMR (CDCl ) δ (ppm): 7.12 (d, 2H, H‐2,6
3
26 27
3
‐1
phenyl, J = 8.55 Hz), 7.05 (d, 2H, H‐3,5 phenyl, J =
8
4
.55 Hz), 7.36 (s, 1H, Pyrimidine), 4.78 (s, 2H, ferrocene),
.68 (s, 1H, OH), 4.52 (s, 2H, ferrocene), 4.18 (s, 5H, ferro-
2926 (C‐H), 1577 (C=N), 1466 (C=C), 1258 (C–N), 1115
1
(C‐O‐C); H NMR (CDCl ) δ (ppm): 7.09‐7.14 (m, 2H, phe-
3
cene), 3.77 (t, 4H, J = 4.6 Hz, morpholine), 2.86 (t, 4H, J =
nyl), 7.03 (s, 1H, phenyl), 7.32 (s, 1H, Pyrimidine), 4.76 (s,
2H, ferrocene), 4.52 (s, 2H, ferrocene), 4.21 (s, 5H, ferro-
cene), 3.78 (t, 4H, J = 4.5 Hz, morpholine), 2.86 (t, 4H, J
13
4
.6 Hz, morpholine); C NMR (CDCl ) δ(ppm): 166.9
3
(C=N pyrimidine), 162.5 (C=C pyrimidine), 162.1 (N=C‐
N pyrimidine), 145.2‐ 120.4 (Ar‐C), 104.6 (C‐H pyrimi-
dine), 66.3 (C‐O‐C, morpholine), 44.2 (C‐N‐C,
morpholine). ESI‐MS m/z: [M +1] 442.11.
= 4.5 Hz, morpholine), 2.22 (s, 3H, ‐CH ), 2.16 (s, 3H, ‐
3
13
CH3); C NMR (CDCl ) δ(ppm): 166.2 (C=N pyrimidine),
3
+
162.8 (C=C pyrimidine), 162.5 (N=C‐N pyrimidine),
145.1‐ 118.7 (Ar‐C), 104.4 (C‐H pyrimidine), 66.5 (C‐O‐
C, morpholine), 44.6 (C‐N‐C, morpholine), 22.4
4
.3.3 | 4‐(4‐nitrophenyl)‐6‐ferrocenyl‐2‐
+
(
2×CH ), ESI‐MS m/z: [M +1] 454.37.
3
morpholin‐1‐yl‐pyrimidine (3c)
Yield: 69%; m.p: 195 °C; Reddish brown solid. Anal. calc.
for C H FeN O : C 61.32, H 4.72, N 11.94 %. Found: C
4
2
.3.6 | 4‐(4‐methoxyphenyl)‐6‐ferrocenyl‐
‐morpholin‐1‐yl‐pyrimidine (3f)
24
22
4 3
‐
1
6
2
1.35, H 4.75, N 11.88 %. IR νmax (cm ): 3048 (Ar–H),
926 (C‐H), 1577 (C=N), 1466 (C=C), 1254 (C–N), 1120
Yield: 71%; m.p: 168 °C; dark red solid. Anal. calc. for
C H FeN O : C 65.91, H 5.54, N 9.26 % . Found: C
1
(
C‐O‐C); H NMR (CDCl ) δ (ppm): 7.26 (d, 2H, H‐2,6
3
25 25
3 2

6
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PARVEEN ET AL.
‐
1
6
2
(
5.95, H 5.58, N 9.29 %. IR νmax (cm ): 3048 (Ar–H),
4.4 | In vitro antiamoebic assay
929 (C‐H), 1572 (C=N), 1468 (C=C), 1256 (C–N), 1120
C‐O‐C); H NMR (CDCl ) δ (ppm): 7.02 (d, 2H, H‐2,6
phenyl, J = 8.65 Hz), 6.94 (d, 2H, H‐3,5 phenyl, J =
.65 Hz), 7.32 (s, 1H, Pyrimidine), 4.78 (s, 2H, ferrocene),
.55 (s, 2H, ferrocene), 4.22 (s, 5H, ferrocene), 3.85 (s,
H, OCH ), 3.77 (t, 4H, J = 4.6 Hz, morpholine), 2.88
t, 4H, J = 4.6 Hz, morpholine); C NMR (CDCl3)
1
All novel organometallic compounds (3a‐3 h) were evalu-
ated in vitro antiamoebic activity against HM1: IMSS
strain of Entamoeba histolytica by microdilution
3
8
4
3
[
60]
method.
The culture of Entamoeba histolytica tropho-
zoites was prepared in the wells of 96‐well microtiter plate
3
13
[61]
(
by Diamond TYIS‐33 growth medium.
The organome-
δ(ppm): 166.4 (C=N pyrimidine), 162.8 (C=C pyrimi-
dine), 162.5 (N=C‐N pyrimidine), 145.1‐ 116.4 (Ar‐C),
tallics (1 mg) were dissolved in dimethyl sulfoxide 40 μL,
[
62,63]
level at which no inhibition of amoeba occurs].
The
1
04.2 (C‐H pyrimidine), 66.5 (C‐O‐C, morpholine), 44.4
stock solutions of the organometallics were prepared
freshly at a concentration of 1 mg/ml two‐fold serial dilu-
tions were made in the wells of a 96‐well microtiter plate
+
(C‐N‐C, morpholine), 55.7 (‐OCH ); ESI‐MS m/z: [M
3
+
1] 456.34.
(
costar). Each test included metronidazole as a standard
drug, control wells (culture medium plus amoebae) and
a blank (culture medium only). All the experiments were
performed in triplicate at each concentration level and
repeated three times. The amoeba suspension was pre-
pared from a confluent culture by pouring off the medium
at 37 °C and adding 5 ml of fresh medium, chilling the
culture tube on ice to remove the amoebae from the side
of the flask. The number of amoeba/ml was estimated
with a haemocytometer, using trypan blue exclusion to
4
.3.7 | 4‐(2,5‐dimethoxyphenyl)‐6‐
ferrocenyl‐2‐morpholin‐1‐yl‐pyrimidine
3g)
(
Yield: 76%; m.p: 241 °C; Reddish solid. Anal. calc. for
C H FeN O : C 64.35, H 5.62, N 8.66 % . Found: C
26
27
3 3
‐
1
6
2
4.32, H 5.65, N 8.62 %. IR νmax (cm ): 3045 (Ar–H),
922 (C‐H), 1577 (C=N), 1467 (C=C), 1255 (C–N), 1120
1
(C‐O‐C); H NMR (CDCl ) δ (ppm): 6.92 (s, 1H, phenyl),
3
5
confirm the viability. The suspension was diluted to 10
6
.88‐6.84 (m, 2H, phenyl), 7.32 (s, 1H, Pyrimidine), 4.79
amoebae/mL by adding fresh medium and 170 μL of this
suspension was added to the test and control wells in the
plate so that the wells were completely filled (total vol-
(s, 2H, ferrocene), 4.56 (s, 2H, ferrocene), 4.18 (s, 5H, fer-
rocene), 3.90 (s, 3H, OCH ), 3.79 (t, 4H, J = 4.6 Hz,
morpholine), 3.76 (s, 3H, OCH ), 2.87 (t, 4H, J =
3
3
4
ume, 340 μL). An inoculum of 1.7 × 10 amoebae/well
13
4
.6 Hz, morpholine); C NMR (CDCl ) δ(ppm): 166.5
3
was chosen so that confluent, but not excessive growth,
took place in control wells. Plates were sealed and gassed
for 10 minutes with nitrogen before incubation at 37 °C
for 72 hours. After incubation, the growth of amoeba in
the plate was checked with a low power microscope.
The culture medium was removed by inverting the plate
and shaking gently. Plate was then immediately washed
with 0.9% NaCl solution at 37 °C. This procedure has been
performed quickly and the plate was not allowed to cool
to avoid detachment of amoebae. After this step, the plate
was allowed to dry at room temperature and the amoebae
(
(
C=N pyrimidine), 162.8 (C=C pyrimidine), 162.4
N=C‐N pyrimidine), 145.2‐ 118.4 (Ar‐C), 104.6 (C‐H
pyrimidine), 66.5 (C‐O‐C, morpholine), 44.2 (C‐N‐C,
morpholine), 56.8, 55.2 (2×‐OCH ); ESI‐MS m/z: [M
+
+
3
1] 486.37.
4
.3.8 | 4‐(4‐Chlorophenyl)‐6‐ferrocenyl‐2‐
morpholin‐1‐yl‐pyrimidine (3h)
Yield: 62%; m.p: 310 °C; Reddish solid. Anal. calc. for
C H ClFeN O: C 62.72, H 4.82, N 9.14 % . Found: C
6
2
1
H‐2,6 phenyl, J = 8.85 Hz), 7.25 (d, 2H, H‐3,5 phenyl,
J = 8.85 Hz), 7.38 (s, 1H, Pyrimidine), 4.78 (s, 2H,
ferrocene), 4.56 (s, 2H, ferrocene), 4.22 (s, 5H,
ferrocene), 3.78 (t, 4H, J = 4.6 Hz, morpholine), 2.88
were fixed with CH OH and dried then stained with 0.5%
2
4
22
3
3
‐
1
2.75, H 4.85, N 9.18 %. IR νmax (cm ): 3043 (Ar–H),
aq. eosin for 15 minutes. The stained plate was first
washed with tap water, then washed twice with distilled
water and was allowed to dry. A 200 μL portion of 0.1 N
sodium hydroxide solution was added to each well to dis-
solve the protein and release the dye. The optical density
of the resulting solution in each well was determined at
490 nm with a microplate reader. The percentage of inhi-
bition of amoebal growth was calculated from the optical
densities of the control and test wells and plotted against
the logarithm of the dose of the drug tested. Linear regres-
sion analysis was used to determine the best fitting line
from which the IC50 value was found. The IC50 values in
μM are reported in Table 1.
929 (C‐H), 1576 (C=N), 1468 (C=C), 1257 (C–N),
1
119 (C‐O‐C); H NMR (CDCl ) δ (ppm): 7.31 (d, 2H,
3
13
(t, 4H, J = 4.6 Hz, morpholine); C NMR (CDCl3)
δ(ppm): 166.8 (C=N pyrimidine), 162.5 (C=C
pyrimidine), 162.2 (N=C‐N pyrimidine), 145.1‐ 118.4
(
morpholine), 44.2 (C‐N‐C, morpholine). ESI‐MS m/z:
[
Ar‐C), 104.2 (C‐H pyrimidine), 66.6 (C‐O‐C,
+
M +1] 460.76.

PARVEEN ET AL.
7 of 8
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