Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-benzoquinones

Article abstract of DOI:10.1016/j.molstruc.2019.127422

Recently, amine substituted and halogen containing 1,4-benzoquinone molecules have attracted a significant attention because of the efficient biological activities. Thus, novel chlorinated/unchlorinated and piperazine substituted dimethyl-1,4-benzoquinone derivatives (4a-h and 5a-h) were designed, synthesized and characterized in the present paper. Furthermore, antibacterial and antifungal activity performances of the new products were compared and evaluated employing the MIC (Minimum Inhibitory Concentrations) values of reference antimicrobial substances. The compounds of 4a and 4b were highly potent with the MIC values of 4.88 μg/mL and 78.12 μg/mL compared to Cefuroxime (MIC = 9.8 μg/mL against S. epidermidis) and Amikacin (MIC = 128.0 μg/mL against E. faecalis), respectively. The presence of the chlorine atom in the structure appeared essential, since most of the chlorinated compounds exhibited more improved activity in comparison to those of unchlorinated products. On the other side, an opposite tendency was observed for the antifungal activity that the MIC values of the unchlorinated derivatives were lower in most cases than those of chlorinated ones. According to the obtained results, while chlorinated derivatives, in particular 4a and 4b, can be proposed as potential antibacterial agents with nearly two fold lower MIC values compared to reference drugs, unchlorinated compounds might be suggested as a relatively active antifungal agents which are needed further improvements due to the higher MIC values than those of reference antifungal materials.

Full text of DOI:10.1016/j.molstruc.2019.127422

Journal of Molecular Structure xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Design, synthesis, characterization, and antimicrobial activity of novel
piperazine substituted 1,4-benzoquinones
Mahmut Yıldız
Gebze Technical University, Faculty of Science, Department of Chemistry, 41400, Gebze, Kocaeli, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, amine substituted and halogen containing 1,4-benzoquinone molecules have attracted a sig-
nificant attention because of the efficient biological activities. Thus, novel chlorinated/unchlorinated and
piperazine substituted dimethyl-1,4-benzoquinone derivatives (4a-h and 5a-h) were designed, synthe-
sized and characterized in the present paper. Furthermore, antibacterial and antifungal activity perfor-
mances of the new products were compared and evaluated employing the MIC (Minimum Inhibitory
Concentrations) values of reference antimicrobial substances. The compounds of 4a and 4b were highly
Received 24 September 2019
Received in revised form
13 November 2019
Accepted 14 November 2019
Available online xxx
potent with the MIC values of 4.88
against S. epidermidis) and Amikacin (MIC ¼ 128.0
m
g/mL and 78.12
m
g/mL compared to Cefuroxime (MIC ¼ 9.8
mg/mL
Keywords:
1,4-Benzoquinone
m
g/mL against E. faecalis), respectively. The presence of
the chlorine atom in the structure appeared essential, since most of the chlorinated compounds
exhibited more improved activity in comparison to those of unchlorinated products. On the other side, an
opposite tendency was observed for the antifungal activity that the MIC values of the unchlorinated
derivatives were lower in most cases than those of chlorinated ones. According to the obtained results,
while chlorinated derivatives, in particular 4a and 4b, can be proposed as potential antibacterial agents
with nearly two fold lower MIC values compared to reference drugs, unchlorinated compounds might be
suggested as a relatively active antifungal agents which are needed further improvements due to the
higher MIC values than those of reference antifungal materials.
Piperazine substitution
Structure-activity relationship (SAR)
Antibacterial activity
Antifungal activity
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
anticancer [25e29], antioxidant [30], anti-inflammatory [31],
antiviral [32], antimalarial [33e35], antibiotic [36e38] and herbi-
Quinones are extensively appeared in nature. They are particu-
larly obtained from animals, plants, bacteria and fungi in a direct
way or synthesized indirectly as synthetic derivatives expecting the
utilization in many research and industrial areas [1e8]. Quinones
find a wide variety of application field in the following topics:
chemosensor, reactive oxygen species generator, redox agent for
batteries, dye, energy harvesting-storage material, catalyst and
electron transfer agent for flow batteries [9e18]. Besides of many
aforementioned properties, a great number of quinone molecules
are biologically active compounds. Therefore, natural and/or syn-
thetic benzoquinone, naphthoquinone and anthraquinone de-
rivatives are members of a prominent family of commercial or
possible drug molecules in medicinal chemistry and pharmaceu-
tical industry. Quinone-core structured compounds exhibit diverse
pharmacological properties such as antimicrobial [19e24],
cidal [39] activities.
Phylloquinone, vitamin K,
b-lapachone, lapachol, lawsone,
plumbagin, adriamycin, daunomycin, mitoxantrone, juglomycin A,
juglone, menadione and lambertellin can be given as examples
(Fig. 1) for bioactive and natural molecules which consist of
naphthoquinone or anthraquinone structures. Wellington et al.
prepared some aminonaphthoquinone compounds bearing various
electron withdrawing or donating groups (-F, -Cl, eCN, eOH, eCH3)
in different positions in the molecules. The compounds showed
antibacterial, antifungal (against C. albicans) and anticancer
(against the PC3 prostate cancer cell line) activity. They reported
that a fluoro group in the ortho position of the aminobenzene ring
structure provided an improved antifungal activity [40]. Tuyun
et al. prepared
a
series of 2-arylamino-3-chloro-1,4-
naphthoquinone derivatives and evaluated for their in vitro anti-
bacterial and antifungal activities. Two of derivatives showed
remarkable activity against both Gram-positive and Gram-negative
bacteria and against the tested fungi (C. albicans). Some compounds
E-mail addresses: yildizm@gtu.edu.tr, mahmutyildiz1981@gmail.com.
https://doi.org/10.1016/j.molstruc.2019.127422
0022-2860/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
benzoquinones, Journal of Molecular Structure, https://doi.org/10.1016/j.molstruc.2019.127422

2
M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
Fig. 1. Some examples of naturally occurring and synthetic bioactive quinone derivatives, a [47], b [48], c [49].
had moderate activity against E. faecalis with MIC values of between
312.5 and 1250 g/mL compared to the reference antibiotic of
Amikacin (128 g/mL). Benzo [b]phenazine-6,11-dione derivatives
evaluated the antibacterial activity of six 1,4-naphthoquinones
derivatives against strains of Mycobacterium tuberculosis. Every
compound was active against M. tuberculosis strains possessing
various MIC values and it was found that tetrahydrofuran fused 1,4-
m
m
were mostly active against Gram-positive bacteria. The change of
the positions of sulfonic acid group (-SO3H), sulfonamide group
(-SO2NH2), trifluoromethyl group (-CF3) and alkoxy group in the
substituted phenyl ring caused some differences in the antimicro-
bial activity [41]. Bayrak synthesized, characterized and compared
antimicrobial test results of a new family of 2-methylquinoline-5,8-
dione compounds containing methoxy- or ethoxyphenylamino
group and chlorine atom. Some of these novel molecules exhibited
strong antibacterial activity against Gram-positive bacteria,
S. epidermidis and E. faecalis. As the structure-activity relationship it
was concluded that methoxy group(s) bearing phenyl substituted
primary-amine introduction to azanaphthoquinone derivatives
improved positively the antibacterial activity against tested path-
ogens [42]. Novais et al. synthesized a series of hydroxyl naph-
thoquinone derivatives and tested them against Gram-negative and
naphthoquinones showed
a better antimycobacterial activity
against M. tuberculosis strains than that of tetrahydropyran fused
1,4-naphthoquinones. Tetrahydrofuran including compound rep-
resented also a reduced cytotoxicity [45]. Kacmaz et al. introduced
bromine containing aminonaphthoquinones and amino-thio-
substituted 1,4-naphthoquinones and investigated their electro-
chemical behavior and antifungal, antibacterial properties. The
synthesized compounds exhibited as high and moderate activity
against tested fungi (M.canis and Trichopyton sp.) in comparison to
the reference antifungal molecule of Amphotericin B and they were
also mostly bioactive against Gram negative bacteria (E. coli) [46].
1,4-benzoquinone is the simpliest member of the family of
organic quinone compounds. Recently, numerous p-benzoquinone
derivatives attract great attention due to their chemical and bio-
logical significance. Mitomycin, geldanamycin, streptonigrin,
abenquine, thymoquinone, ubiquinone and plastoquinone com-
pounds (Fig. 1) include also p-benzoquinone scaffold and they are
exploited as well-known drug and drug-candidate of quinoid de-
rivatives [50,51]. Johnson-Ajinwo et al. described the synthesis of
thymoquinone analogues including different amine, alkyl chain and
halogen substituents. The growth inhibition in three human
ovarian cancer cell lines and immortalized human ovarian epithe-
lial cell line (HOE) by determining their IC50 values using sulfo-
rhodamine B (SRB) cytotoxicity assay and antiproliferative activities
against human malaria parasite were investigated for their bio-
logical activity potential. Certain analogues showed significant
inhibitory activities that two-fold more than that of thymoquinone
and also halogen substitution in some molecules led to an
improvement against ovarian cancer cell lines [52]. Abenquine is
Gram-positive
bacteria
strains.
2-hydroxy-3-
phenylsulfanylmethyl-1,4-naphthoquinones were found prom-
ising structures against E. coli and P. aeruginosa strains and it was
suggested a correlation between phenylsulfanymethyl ring with
the antibacterial profile against Gram-negative strains [43].
Yıldırım et al. synthesized and characterized successfully sulfanyl
and trifluoromethyl containing aryl amine substituted 1,4-
naphthoquinones. The influences of the eCF3 group position in
aryl amine ring of the prepared compounds were clearly elucidated
and the molecular docking studies have also supported the exper-
imental results. A number of these compounds were reported as
promising antibacterial and antimicrobial agents and it was sug-
gested that the sec-butylthio and 2-hydroxypropylthio moieties
with the additional effect of the position of eCF3 are promising for
the exploration of new antibacterial agents [44]. Halicki et al.
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
benzoquinones, Journal of Molecular Structure, https://doi.org/10.1016/j.molstruc.2019.127422

M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
3
one of the bioactive natural quinones having anticyanobacterial
activity. Nain-Perez et al. designed and synthesized some synthetic
abenquine analogues by substitution of the acetyl group by a
benzoyl group in the quinone core and by replacement of the amino
acid moiety with ethylpyrimidinyl or ethylpyrrolidinyl groups. This
modification resulted in new analogues which exhibited 25-fold
better activity than those of the natural abenquines [53]. Blunt
et al. reported the first chemical synthesis of aulosirazole that
shows selective antitumor cytotoxicity and synthesized pronqo-
dine A analogues to be able to compare their results. Biological
evaluation of the compounds was performed by targeting indole-
amine-2,3-dioxygenase (IDO) enzyme. The isothiazoloquinone de-
rivatives generate reactive oxygen species in the intracellular
NQO1-dependent redox cycling. These compounds provide a
benefit to change the ratio of intracellular oxidized to reduced
pyridine nucleotides at lower proportions [54]. Embelin is a natural
hydroxy benzoquinone with alkyl substitution and known to
possess miscellaneous biological activities. Singh et al. carried out
the reaction of embelin with various secondary amines in order to
obtain a series of Mannich products. Antiproliferative, antimicro-
bial and cytotoxicity tests were conducted for the synthesized
products. The benzyl-piperidine linked derivative indicated better
antiproliferative activity in comparison to embelin family against a
panel of cell lines including MIAPaCa-2, HCT-116, PC-3 and MCF-7.
Besides that, dimethylamino- and piperidine-linked derivatives
exhibited antibacterial activity against Staphylococcus aureus.
While the Mannich derivatives did not show adequate solubility of
aqueous, the aqueous solubility of their hydrochloride salts became
better without any effect of biological activities [55]. Park et al.
investigated antimicrobial activities of dimethoxy-, dimethyl- and
dichloro-1,4-benzoquinones and evaluated their structure-activity
relationships of against the seven food-borne bacteria. Whereas
2,6-Dimethoxy-1,4-benzoquinone showed activity against Staphy-
lococcus intermedius, Staphlococcus epidermidis, Shigella sonnei and
Listeria monocytogenes, 2,6-dichloro-1,4-benzoquinone was not
bioactive compound against all tested food-borne bacteria. It was
pointed out that the 2,6-dimethoxy-1,4-benzoquinone and its
structural analogues can be beneficial by utilization of food sup-
plemental additives [56]. Tandon et al. demonstrated the prepara-
tion of diverse thio- and aminobenzoquinone compounds by
applying green methodology. Laundry detergent (surfactant) was
used as a catalyst and the reactions was carried out in water. Sulfur
atom containing alkyl and arylsulfanyl-p-benzoqinones gave better
antifungal activity test results than drugs of Fluconazole and Flu-
cytosine in vitro against S. schenckii, and T. mentagraphytes. A 2,5-
diaminosubstituted-p-benzoquinone compound showed better
antifungal activity compared to Fluconazole drug against
C. neoformans, S. schenckii, T. mentagraphytes and A. fumigatus. The
same molecule displayed a better antibacterial activity in compar-
ison to Ampicillin against E. coli, S. aureus and K. Pneumonia in vitro
and did not cause any toxicity towards mammalian cells L929 [57].
Plastoquinone is one of the most important naturally occurring
1,4-benzoquinone compounds and involved mostly in the electron
transport chain of the light-dependent photosynthesis reactions. Its
structural framework is consist of 2,3-dimethyl-1,4-benzoquinone
with a polyprenyl side chain at the fifth position. There are
several natural plastoquinones with side chains of different length
(containing between six and nine isoprene units). Besides that,
derivatives of plastoquinone molecule are also used for pharma-
cological purposes. Latterly, Yıldırım et al. prepared a number of
thiolated plastoquinone analogues to investigate their antimicro-
bial activity. 2-Chloro-3-(2,4-dimethylphenylthio)-5,6-dimethyl-
1,4-benzoquinone was the analog which showed the best in vitro
antibacterial activity against E. faecalis compared to that of Ami-
kacin. The same compund was also the most potent analog in the
series against C. albicans and C. tropicalis. The chlorine atom of
thiolated analogues were speculated to be essential for acceptable
inhibitory activity. Whereas the methoxy group substituted thiol
structure bearing analogues exhibited no inhibitory activity against
the most of the microorganisms, methyl groups containing ana-
logues were suggested as promising molecules as potent antimi-
crobial agents for further works [58].
The introduction of amine molecules into the 1,4-quinone
structures yields aminoquinone derivatives and improves antimi-
crobial activity substantially [59e62]. Moreover, recently it has
been pointed out prominently that presence or addition of a
halogen atom, in particular chlorine, in the pharmacophore scaffold
seems to be crucial for potential biological activity
[26,40,44,58,63e65].
The above reported mini review reveal that a 1,4-quinone core
containing quinone derivatives play an important role in diverse
biologically active processes particularly such as in antibacterial
and antifungal activities. Since antibiotic resistance has become
worldwide a critical challenge in the health of public and the
decline of the number of newly discovered and clinically approved
antibiotics, an instant demand has arisen on the research and
development of novel antimicrobial drugs in both academia and
pharmaceutical industry. Therefore, it is reported in the present
study the design, synthesis, characterization and antimicrobial
activity of halogenated and non-halogenated plastoquinone-
resembling derivatives containing variously substituted piperazine
groups (Fig. 2). The structure-activity relationship (SAR) has been
discussed for the novel dimetyhl-1,4-benzoqinone moiety bearing
compounds by means of the diversity of the secondary amine
groups, their methyl- and methoxy-group substitutions in different
positions and presence or absence of chlorine atom.
2. Materials and methods
2.1. Chemicals and apparatus
All reagents, solvents, and compounds were commercially ob-
tained from commercial supplier with a minimum purity of 95%
Fig. 2. Design of desired plastoquinone derivatives based on literature survey: i) 2,3-
dimethyl-1,4-benzoquinone moiety of plastoquinone as a core structure, ii) substitu-
tion of the side chain with diversely substituted amines, iii) chlorine atom at the
second position of the 1,4-benzoquinone moiety to increase the inhibitory activity, iv)
methyl- and methoxy-substitution at different positions and presence or absence of
chlorine atom to investigate structure-activity relationship of the novel molecules.
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
benzoquinones, Journal of Molecular Structure, https://doi.org/10.1016/j.molstruc.2019.127422

4
M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
and used without further purification unless specified otherwise. A
Stuart SMP-10 melting point apparatus was used to determine the
melting points (mp) that were uncorrected. For column chroma-
and 2,3-dimethylhydroquinone (0.100 g, 0.724 mmol) were sus-
pended in MeOH (5 mL) in a round-bottom flask. After that, sodium
iodate (2.172 mmol, 3 equiv.) was added in water (5 mL) to the
stirred solution at room temperature for 5e10 h. The reaction was
checked by TLC until the spot of starting compounds disappeared
under UV light. After the reaction was completed, the mixture of
reaction in solution was extracted by using chloroform and the
organic phase was washed with water. At the end, the organic
phase was dried with Na2SO4 and the solvent was removed in
vacuo. The residue was purified by column chromatography on
silica gel to achieve desired pure products.
tography, silica gel 60 (Merck, 63e200 mm particle sized, 60e230
mesh) was used as the stationary phase. Thin layer chromatography
(TLC) was purchased from Merck KGaA (silica gel 60 F254) based on
Merck DC-plates (aluminum based). Visualization of TLC plates was
performed by means of UV light (254 nm). Proton nuclear magnetic
resonance (1H NMR) and carbon nuclear magnetic resonance (13
C
NMR) spectra were obtained on a VarianUNITY INOVA spectrometer
(500 MHz for 1H NMR and 125 MHz for 13C NMR) in CDCl3 refer to
the solvent signal centre at d 7.19 and d 76.0 ppm. Chemical shifts (d)
are reported in parts per million (ppm) and coupling constants (J)
are reported in Hz. Multiplicities were described using the
following abbreviations: s (singlet), bs (broad singlet), d (doublet), t
(triplet), and m (multiplet). Mass spectra were obtained with a
BRUKER Microflex LT by MALDI (Matrix Assisted Laser Desorption
Ionization)-TOF technique via addition of 1,8,9-anthracenetriol
(DIT, dithranol) or 2,5-dihydroxybenzoic acid (DHB) as matrix.
Infrared spectrums were recorded as ATR on a PerkinElmer Spec-
trum 100 Optical FT-IR Spectrometer.
2.3.3. 2-Chloro-5,6-dimethyl-3-(4-(isopropyl)piperazin-1-yl)-1,4-
benzoquinone (4a)
The method A was implemented to synthesize the title com-
pound which was obtained from the reaction of 2,3-dichloro-5,6-
dimethyl-1,4-benzoquinone (2) with 1-isopropylpiperazine (3a).
The crude product was purified by column chromatography to
furnish (4a) as a brown solid. Yield: 16%, mp > 250 ꢀC. FTIR (ATR)
y
(cmꢁ1): 2964, 2922, 2848 (CHaliphatic), 1659 (>C]O). 1H NMR
(CDCl3) (ppm): 1.09e1.47 (m, 6H, CH3isopropyl), 2.01e2.07 (m, 6H,
CH3), 2.16e2.23 (m, 1H, CHisopropyl), 2.69e2.88 (m, 4H, CH2piperazine),
3.55e3.73 (m, 4H, CH2piperazine). 13C NMR (CDCl3)
(ppm): 12.6, 13.1,
d
2.2. X-ray diffraction analysis
d
17.4 (CH3), 29.6, 48.8 (CH2piperazine), 56.9 (CHisopropyl), 139.0, 141.5,
147.1 (Cquinone), 180.0, 183.7 (>C]O). MS MALDI TOF (m/z): 296
[M]þ. Anal. Calcd. for C15H21ClN2O2 (296.13).
The single-crystal data of the some compounds were obtained
with Bruker APEX II QUAZAR three-circle diffractometer. Crystal
structure validations and geometrical calculations were performed
using the Platon software [66]. Mercury software [67] was used for
visualization of the. cif files. Each of the structures has been solved
and refined using the Bruker SHELXTL Software Package [68].
Indexing was performed using APEX2 [69]. Data integration and
reduction were carried out with SAINT [70]. Absorption correction
was performed by multi-scan method implemented in SADABS
[71]. Aromatic and aliphatic H atoms bonded to C atoms were
positioned geometrically and refined using a riding mode. Details of
data collection and crystal structure determinations are given in
Table 1. The selected bond lengths and bond angles are given in
Tables 2 and 3. The crystallographic data have been deposited at the
Cambridge Crystallographic Data Centre and CCDC reference
numbers are 1941240 for the compound 5d, 1941244 for the
compound 5e, and 1941241 for the compound 5f. The data can be
obtained available free of charge from http://www.ccdc.cam.ac.uk/
conts/retrieving.html or from the Cambridge Crystallographic Data
Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ44 (0)
1223336033; email: nndeposit@ccdc.cam.ac.uk.
2.3.4. 2-Chloro-5,6-dimethyl-3-(4-(cyclohexyl)piperazin-1-yl)-1,4-
benzoquinone (4b)
The method A was implemented to synthesize the title com-
pound which was obtained from the reaction of 2,3-dichloro-5,6-
dimethyl-1,4-benzoquinone (2) with 1-cyclohexylpiperazine (3b).
The crude product was purified by column chromatography to
furnish (4b) as a dark brown solid. Yield: 37%, mp 180e182 ꢀC. FTIR
(ATR)
NMR (CDCl3)
y
(cmꢁ1): 2926, 2852, 2811 (CHaliphatic), 1659 (>C]O). 1H
(ppm): 1.04e1.30 (m, 5H, CH2cyclohexyl), 1.59e1.72
d
(bs, 1H, CH2cyclohexyl), 1.77e1.87 (m, 2H, CH2cyclohexyl), 1.87e1.97 (m,
2H, CH2cyclohexyl), 2.00 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.32e2.42 (bs,
1H, CHcyclohexyl), 2.70e2.80 (bs, 4H, CH2piperazine), 3.53e3.61 (bs, 4H,
CH2piperazine). 13C NMR (CDCl3)
d (ppm): 12.6, 13.1 (CH3), 25.7, 26.0,
28.4 (CH2cyclohexyl), 49.6, 50.7 (CH2piperazine), 64.2 (CHcyclohexyl),
138.7, 141.3, 147.7 (Cquinone), 180.0, 183.8 (>C]O). MS MALDI TOF
(m/z): 336 [M]þ. Anal. Calcd. for C18H25ClN2O2 (336.16).
2.3.5. 2,3-Dimethyl-5-(4-(isopropyl)piperazin-1-yl)-1,4-
benzoquinone (5a)
2.3. General methods for the synthesis of compounds
The method B was implemented to synthesize the title com-
pound which was obtained from the reaction of 2,3-
dimethylhydroquinone (1) with 1-isopropylpiperazine (3a). The
crude product was purified by column chromatography to furnish
2.3.1. Method A for the synthesis of the piperazine and chlorine
substituted dimethyl-1,4-benzoquinones (4a-h) [49]
To
a
suspension of the 2,3-dichloro-5,6-dimethyl-1,4-
(5a) as a dark red oil. Yield: 11%. FTIR (ATR)
y
(cmꢁ1): 2965, 2930
(ppm): 1.08 (d,
benzoquinone (0.1025 g, 0.50 mmol) in H2O (10 mL), a suspen-
sion of the appropriate piperazine (1.10 mmol, 2.2 equiv.) was
added dropwise and stirred at 50e60 οC for 5e10 h until con-
sumption of the 1,4-benzoquinone. The reaction mixture was
cooled to room temperature. After the solvent evaporation, the
crude product was dissolved in chloroform, and the solution was
washed with distilled water. The organic layer was dried over
Na2SO4, filtered, and concentrated under vacuum. Column chro-
matography on silica gel was conducted for the residue to obtain
separated and purified target compounds.
(CHaliphatic), 1659 (>C]O). 1H NMR (CDCl3)
d
J ¼ 6.5 Hz, 6H, CH3isopropyl), 1.99 (d, J ¼ 3.4, 6H, CH3), 2.64e2.69 (m,
4H, CH2piperazine), 2.72e2.78 (m, 1H, CHisopropyl), 3.36e3.42 (m, 4H,
CH2piperazine), 5.74 (s, 1H, CHquinone). 13C NMR (CDCl3)
d (ppm): 12.3,
12.4, 18.4 (CH3), 48.2, 48.9 (CH2piperazine), 54.6 (CHisopropyl), 109.1,
139.2, 141.1, 152.4 (Cquinone), 185.1, 186.2 (>C]O). MS MALDI TOF
(m/z): 262 [M]þ. Anal. Calcd. for C15H22N2O2 (262.17).
2.3.6. 2,3-Dimethyl-5-(4-(cyclohexyl)piperazin-1-yl)-1,4-
benzoquinone (5b)
The method B was implemented to synthesize the title com-
pound which was obtained from the reaction of 2,3-
dimethylhydroquinone (1) with 1-cyclohexylpiperazine (3b). The
crude product was purified by column chromatography to furnish
2.3.2. Method B for the synthesis of the piperazine substituted
dimethyl-1,4-benzoquinones (5a-h) [72]
An appropriate substituted piperazine (1.448 mmol, 2 equiv.)
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
benzoquinones, Journal of Molecular Structure, https://doi.org/10.1016/j.molstruc.2019.127422

M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
5
Table 1
Crystallographic data for compounds 5d, 5e, and 5f.
Identification code
5d
5e
5f
Chemical formula
C19H22N2O2
310.38
296 (2)
C19H22N2O2
310.38
296 (2)
C19H22N2O3
326.38
296 (2)
Formula weight (g molꢁ1
Temperature (K)
)
Radiation,
l
(Å)
0.71073
0.71073
0.71073
Crystal system
Space groups, Z
monoclinic
P1 21/c 1
orthorhombic
P b c a
orthorhombic
P b c a
Unit cell dimensions (Å)
a ¼ 10.101 (2)
b ¼ 20.911 (6)
c ¼ 8.0122 (19)
a ¼ 11.9340 (19)
b ¼ 7.7876 (19)
c ¼ 35.739 (7)
a ¼ 11.8686 (14)
b ¼ 7.9234 (10)
c ¼ 35.924 (4)
(
a
,
g
¼ 90ꢀ
b
¼ 104.075 (15)ꢀ)
(
a
,
b,
g
¼ 90ꢀ)
(a
,
b
,
g
¼ 90ꢀ)
Volume (Å3)
1641.5 (7)
3321.5 (12)
3378.3 (7)
Crystal sizes (mm)
0.125 ꢂ 0.167 ꢂ 0.269
0.140 ꢂ 0.157 ꢂ 0.324
0.098 ꢂ 0.198 ꢂ 0.273
dcalc (g cmꢁ3
)
1.256
1.241
1.283
Absorption coefficient (mmꢁ1
)
0.082
0.081
0.087
Absorption correction, Tmin, Tmax
qmax, deg
multi-scan, 0.9900, 0.9780
25.0
1.045
multi-scan, 0.9890, 0.9740
multi-scan, 0.9910, 0.9770
Goodness-of-fit on F2
Index ranges
1.030
1.028
ꢁ11 ꢃ h<¼12,
ꢁ23 ꢃ k<¼20,
ꢁ9 ꢃ l<¼9
ꢁ13 ꢃ h<¼14,
ꢁ9 ꢃ k<¼5,
ꢁ41 ꢃ l<¼42
14860
ꢁ12 ꢃ h<¼13,
ꢁ9 ꢃ k<¼9,
ꢁ42 ꢃ l<¼42
23046
Reflections collected
8171
Independent reflections
Final R indices [I > 2s(I)]
2807 [R (int) ¼ 0.0641]
2912 [R (int) ¼ 0.0963]
2898 [Rint ¼ 0.0679]
1595 data;
1592 data;
1684 data;
R1 ¼ 0.0687,
wR2 ¼ 0.1808
R1 ¼ 0.1252,
wR2 ¼ 0.2278
Full-matrix least-squares on F2
R1 ¼ 0.0960,
wR2 ¼ 0.2344
R1 ¼ 0.1587,
wR2 ¼ 0.2773
R1 ¼ 0.0546,
wR2 ¼ 0.1293
R1 ¼ 0.1035,
wR2 ¼ 0.1605
R indices (all data)
Refinement method
Scan mode
u/f
Data/restraints/parameters
2807/0/212
0.289, ꢁ0.249
2912/0/211
0.456, ꢁ0.297
2898/0/220
0.163 and ꢁ0.211
Drmax
,
Drmin (eÅꢁ3
)
Table 2
Selected bond lengths (Å) for compounds 5d, 5e, and 5f.
5d
5e
5f
O2eC14
O1eC19
C17eC15
C12eC13
C17eC19
C12eC19
N2eC12
N2eC9
1.221 (4)
1.222 (4)
1.329 (4)
1.346 (4)
1.493 (4)
1.495 (4)
1.382 (3)
1.468 (4)
1.417 (3)
1.395 (4)
1.513 (4)
C6eO2
C1eO1
C2eC4
C4eC6
C6eC7
1.221 (6)
1.220 (6)
1.323 (7)
1.496 (8)
1.446 (7)
1.339 (7)
1.376 (6)
1.459 (6)
1.499 (6)
1.414 (5)
1.380 (6)
C13eO2
C18eO3
C13eC14
C14eC16
C12eC13
C12eC19
C12eN2
C11eN2
C10eC11
C5eN1
1.219 (3)
1.234 (3)
1.478 (3)
1.333 (4)
1.490 (4)
1.346 (3)
1.376 (3)
1.458 (3)
1.508 (3)
1.427 (3)
1.386 (3)
C7eC8
C8eN1
C12eN1
C11eC12
C13eN2
C13eC19
N1eC7
C7eC1
C9eC8
C5eC6
Table 3
Selected bond angles (ꢀ) for compounds 5d, 5e, and 5f.
5d
5e
5f
N2eC12eC19
C13eC12eN2
C12eN2eC9
C12eN2eC10
C15eC17eC19
C17eC15eC14
O1eC19eC17
O1eC19eC12
C9eN2eC10
C1eC7eN1
116.5 (3)
125.3 (3)
117.4 (3)
118.5 (2)
119.8 (3)
120.6 (3)
119.6 (3)
121.3 (3)
110.3 (2)
119.0 (3)
122.5 (3)
N1eC8eC1
C7eC8eN1
C8eN1eC12
C8eN1eC9
C4eC2eC1
C2eC4eC6
O1eC1eC2
O1eC1eC8
C12eN1eC9
C19eC13eN2
C14eC13eN2
117.2 (4)
124.8 (5)
116.9 (4)
117.4 (4)
120.6 (5)
119.3 (5)
119.2 (5)
121.7 (4)
111.0 (4)
123.1 (4)
120.2 (4)
N2eC12eC13
C19eC12eN2
C12eN2eC11
C12eN2eC9
C16eC14eC13
C14eC16eC18
O2eC13eC14
O2eC13eC12
C11eN2eC9
C6eC5eN1
117.7 (2)
125.2 (2)
117.20 (19)
117.75 (19)
119.5 (2)
119.3 (2)
119.1 (2)
120.8 (2)
110.75 (18)
122.9 (2)
118.0 (2)
C6eC7eN1
C2eO1eC1
(5b) as a dark red oil. Yield: 15%. FTIR (ATR)
(CHaliphatic), 1659 (>C]O). 1H NMR (CDCl3)
y
(cmꢁ1): 2927, 2853
CH2piperazine), 3.32e3.43 (m, 4H, CH2piperazine), 5.73 (s, 1H, CHqui-
none). 13C NMR (CDCl3)
(ppm): 12.3, 12.5 (CH3), 25.8, 26.2, 28.8
d
(ppm): 1.05e1.35 (m,
d
6H, CH2cyclohexyl), 1.73e1.93 (m, 4H, CH2cyclohexyl), 1.95e2.11 (m, 6H,
CH3), 2.23e2.40 (m, 1H, CHcyclohexyl), 2.65e2.76 (m, 4H,
(CH2cyclohexyl), 48.5, 49.1 (CH2piperazine), 63.6 (CHcyclohexyl), 109.0,
139.2, 141.1, 152.4 (Cquinone), 185.1, 186.2 (>C]O). MS MALDI TOF
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
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6
M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
(m/z): 302 [M]þ. Anal. Calcd. for C18H26N2O2 (302.19).
(Scheme 2).
After preparation of chlorinated compounds, unchlorinated
In the present section, the experimental data of 4a-4b and 5a-5b
are given comprehensively. However, for all data, spectra, and
analysis results of other novel compounds (4c-4h) and (5c-5h),
please refer to the supplementary material of this publication.
compounds were also synthesized to be able to check, compare and
discuss the structure-activity relationship about the presence and
absence of chlorine atom in newly obtained quinone molecules. For
this purpose, various piperazine compounds (3a-h) mentioned
above reacted in a single step way with 2,3-dimethylhydroquinone
(1) compound according to a previously present method [72] at
room temperature in the presence of NaIO3 using mixture of water
and methanol as solvent to yield desired unchlorinated and mono
piperazine substituted 1,4-benzoquinone derivatives in yield of
11e57% assigning the codes of 5a to 5h (Scheme 3).
2.4. Antimicrobial activity
2.4.1. Determination of Minimum Inhibitory Concentrations (MIC)
Antimicrobial activities against Staphylococcus aureus ATCC
29213, Staphylococcus epidermidis ATCC 12228, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC 4352, Pseudomonas aer-
uginosa ATCC 27853, Proteus mirabilis ATCC 14153, Enterococcus
faecalis ATCC 29212, Candida albicans ATCC 10231, Candida para-
psilosis ATCC 22019, and Candida tropicalis ATCC 750 were deter-
mined by the microbroth dilution technique using the Clinical
Laboratory Standards Institute (CLSI) recommendations [73,74].
Mueller-Hinton broth for bacteria and RPMI-1640 medium for the
yeast strain were used as the test media. Serial two-fold dilutions
The novel dimethyl-1,4-benzoquinone compounds (4a-h and
5a-h) were purified by utilization of silica gel column chromatog-
raphy applying mixture of solvents as a mobile phase. The afore-
named products were elucidated on the basis of FTIR, 1H NMR, 13
C
NMR, and mass spectrometry. The FTIR spectra of the derivatives
showed a characteristic carbonyl (C]O) signals between 1645 and
1666 cmꢁ1, aliphatic (CeH) signals between 2811 and 2965 cmꢁ1
,
ranging from 2500
m
g/mL to 1.2
m
g/mL were prepared in the media.
aromatic (CeH) signals between 2976 and 3074 cmꢁ1. Whereas the
13C NMR spectra exhibited the peaks of (-CH3) carbons bonded to
(C]C)quinone around 12.3e13.1 ppm, the (-CH3) carbons of isopro-
pyl- and methylphenyl-substituted piperazines gave signals be-
tween 17.4 and 21.8 ppm. Besides that, the (-CH3) carbons which
are adjacent to oxygen atom showed peaks around 55 ppm and the
signals of (CeH)isopropyl and (CeH)cyclohexyl carbon atoms were
detected at 54.6, 56.9 ppm and 63.6, 64.2 ppm, respectively. While
the (CH2)piperazine carbon peaks were observed mostly around
48e50 ppm, the (CH2)cyclohexyl carbon signals were determined
between 25.7 and 28.8 ppm. The carbon atoms of (C]C)quinone and
(CeH)aromatic were depicted peaks between 102.7 and 160.8 ppm
and the typical signals of all (C]O) carbons were observed around
180 and 183 ppm for the chlorinated compounds (4a-h), 185 and
186 ppm for the unchlorinated compounds (5a-h). The 1H NMR
spectra showed the peaks of (-CH3) protons linked to (C]C)quinone
between 1.97 and 2.10 ppm. While the signals of (-CH3) protons
bonded to phenyl moiety were detected around 2.30 ppm, the
(-CH3) proton peaks of isopropyl group were observed between
1.09 and 1.47 ppm. On the other hand, the (-CH3) protons attached
to the oxygen atoms were shown around 3.80 ppm. The (CeH)
protons of isopropyl group gave signals between 2.72 and 2.92 ppm
and also the peaks of cyclohexyl (CeH) protons were seen around
2.32 ppm. The 1H NMR spectra exhibited the peaks of (CeH) pro-
tons of unchlorinated quinone products (5a-h) around 5.80 ppm,
whereas the aromatic (CeH) proton signals were determined be-
tween 6.47 and 7.23 ppm. The (CH2) proton peaks of cyclohexyl
group were depicted between 1.11 and 1.93 ppm. Besides that, the
(CH2) protons of piperazine moiety were characterized by the sig-
nals between 2.64 and 3.73 ppm. The structure of the new com-
pounds were also characterized and supported by mass
spectroscopy results as given in the following: 4a, 4b, 4c-e, 4f-h
(296 [M]þ), (336 [M]þ), (344 [M]þ), (360 [M]þ) and 5a, 5b, 5c-e, 5f-h
(262 [M]þ), (302 [M]þ), (310 [M]þ), (326 [M]þ), respectively.
Moreover, the structures of the 5d (1941240), 5e (1941244) and 5f
(1941241) were further approved by the diffraction analysis of a
single crystal obtained by slow evaporation of the ethanol solution
(Fig. 3). The crystallographic data of molecules (5d, 5e and 5f) are
summarized in Tables 1e3 For details about characterization re-
sults, please see the supplementary file.
The inoculum was prepared using a 4e6 h broth culture of each
bacteria and 24 h culture of yeast strains adjusted to a turbidity
equivalent of a 0.5 McFarland standard, diluted in broth media to
give a final concentration of 5 ꢂ 105 cfu/mL for bacteria and
0.5 ꢂ 103 to 2.5 ꢂ 103 cfu/mL for yeast in the test tray. The trays
were covered and placed in plastic bags to prevent evaporation. The
trays containing Mueller-Hinton broth were incubated at 35 ꢀC for
18e20 h while the trays containing RPMI-1640 medium were
incubated at 35 ꢀC for 46e50 h. The MIC was defined as the lowest
concentration of compound giving complete inhibition of visible
growth. As a control, antimicrobial effects of the solvents were
investigated against test microorganisms. The results were evalu-
ated according to the values of the controls.
3. Results and discussions
3.1. Chemical synthesis
The nucleophilic substitution reactions of 1,4-quinones with
amines, thiols and alcohols are well-recognized and fast way to
obtain possible biologically active molecules. In this study, some
amine groups linked dimethyl-1,4-benzoquinone compounds were
prepared from the reactions of various secondary amines with 1,4-
hydroquinone to achieve unchlorinated products or with 1,4-
quinone to achieve chlorinated products. Since a halogen atom
presence in quinone structure is of an important influence on
biological activity as mentioned in the introductory part, chlori-
nated target compounds were initially attempted for this purpose.
Thus, commercially available 2,3-dimethylhydroquinone (1) com-
pound was firstly oxidized and chlorinated to the corresponding
quinone structure, 2,3-dichloro-5,6-dimethyl-1,4-benzoquinone
(2), in HNO3/HCl medium at 90 ꢀC (Scheme 1) according to a
formerly published preparation method [75].
After preparation of starting compound in quinoid structure, the
reactions were carried out to achieve the target products. In order
to conduct these experiments, 2,3-dichloro-5,6-dimethyl-1,4-
benzoquinone (2) was stirred with diverse piperazine compounds
(1-isopropylpiperazine (3a), 1-cyclohexylpiperazine (3b), 1-(2-
methylphenyl)piperazine (3c), 1-(3-methylphenyl)piperazine (3d),
1-(4-methylphenyl)piperazine (3e), 1-(4-methoxyphenyl)pipera-
The bond lengths between carbonyl carbon atoms and oxygen
atoms of 5d, 5e and 5f compounds are around 1.22 Å. The average
values of CeN bond lengths of 5d, 5e and 5f are around
1.38e1.508 Å. Regarding the bond angles among N atom and
carbonyl carbon and vinylic carbon atoms, it is seen that they
support the arrangement shown in Tables 2 and 3 The CeCeC and
zine
(3f),
1-(3-methoxyphenyl)piperazine
(3g),
1-(2-
methoxyphenyl)piperazine (3h)) by applying a method from liter-
ature [49] at 50e60 ꢀC in the absence of a base using water as
solvent. As a result of that mono piperazine substituted products
were obtained in yield of 16e58% as nominated from 4a to 4h
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
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M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
7
Scheme 1. Preparation way of chlorinated dimethyl-1,4-benzoquinone adopted from Ref. [75].
Scheme 2. Synthesis of piperazine linked target products and the codes of the substituted piperazines. Synthesis method was adopted from Ref. [49].
Scheme 3. Synthesis of piperazine linked unchlorinated derivatives. Synthesis method was adopted from Ref. [72]. R groups can be seen in Scheme 2 above.
CeCeO angles of the compounds 5d, 5e, and 5f are close to 120.8ꢀ
(minimum inhibitory concentration) values were calculated by a
which proves the structures involving sp2 hybridized atoms.
comparison with standard agents. Table 4 shows the antimicrobial
test results of all newly synthesized compounds (4a-h and 5a-h).
Codes a to h stands for the groups of isopropyl, cyclohexyl, 2-
3.2. Antimicrobial activity
methylphenyl,
3-methylphenyl,
4-methylphenyl,
4-
methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl and codes 4
and 5 represent the chlorinated and unchlorinated piperazine
substituted dimethyl-1,4-benzoquinones, respectively.
Generally speaking, most chlorinated derivatives exhibited
more or less activity against tested Gram-positive and Gram-
negative bacteria. However, most of these compounds except 4c
and 4f, which represented clearly no activity, possessed activity
In vitro antimicrobial activity of the chlorinated/unchlorinated
piperazine substituted dimethyl-1,4-benzoquinone derivatives (4a-
h and 5a-h) was investigated against four Gram-negative bacteria
(P. aeruginosa ATCC 27853, E. coli ATCC 25922, K. pneumoniae ATCC
4352, and P. mirabilis ATCC 14153), three Gram-positive bacteria (E.
faecalis ATCC 29212, S. epidermidis ATCC 12228, S. aureus ATCC
29213) and two fungi (C. albicans ATCC 10231 and C. parapsilosis
ATCC 22019) by comparing them with the activity results of the
known reference antimicrobials used as drug product. The micro-
broth dilutions technique was used by applying the Clinical Labo-
ratory Standards Institute (CLSI) recommendations [73,74]. The MIC
against E. faecalis with the MIC values around 300 and 1250
mg/mL
among Gram-positive bacteria. Furthermore, compound 4b
showed an excellent activity against E. faecalis with 78.12
MIC value better than that of Amikacin (MIC ¼ 128
m
g/mL
mg/mL)
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
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8
M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
Fig. 3. ORTEP drawings of 5d, 5e and 5f.
reference material. Moreover, the antibacterial activity of com-
the MIC values of 1250 mg/mL against Gram-negative bacteria than
pound 4a against S. epidermidis was also outstanding performance
those of reference drugs, 4f and 4g were evidently not active for any
Gram-negative bacterium. On the other hand, unchlorinated com-
pounds showed hardly any activity against the Gram-negative
bacteria except 5a compound which had even very low activity
against K. pneumoniae compared to that of Ceftazidime. Besides
that, these molecules were active for Gram-positive bacteria but
once again with very low level such as around 150, 300, 600 and
at 4.88
ering the result of Cefuroxime drug (MIC ¼ 9.8
reference. Besides that, other compounds, except 4b that showed
activity in a moderate level (MIC ¼ 78.12 g/mL), displayed less-
ened performance between the MIC values of 312.5e1250 g/mL
for the S. epidermidis. For S. aureus; performances of the 4a
mg/mL MIC value which is nearly two-fold better consid-
mg/mL) used as
m
m
(19.53
(1250
m
g/mL), 4b, 4c, 4e (78.12
m
g/mL), 4d (312.5
m
g/mL) and 4f-h
1250
mg/mL of MIC values. However, 5a and 5b displayed a mod-
m
g/mL) can be classified as remarkable for the 4a-c, 4e and
erate activity against E. faecalis and S. epidermidis with the MIC
poor for the 4d, 4f-h products. Besides that, while nearly all chlo-
rinated compounds shown activity had very low performance at
values of 625 and 39.06
and Cefuroxime.
mg/mL in comparison to those of Amikacin
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
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M. Yıldız / Journal of Molecular Structure xxx (xxxx) xxx
9
Table 4
In vitro antimicrobial activity results of the chlorinated/unchlorinated piperazine substituted dimethyl-1,4-benzoquinone derivatives (4a-h and 5a-h) and determined MIC
values in
mg/mL.
Code
Microorganism
Gram-negative Bacteriaa
Gram-positive Bacteriab
Fungic
CA
PA
EC
KP
PM
EF
SE
SA
CP
4a
5a
4b
5b
4c
5c
4d
5d
4e
5e
4f
1250
e
1250
e
1250
625
1250
e
625
e
312.5
625
78.12
e
4.88
625
19.53
312,5
78.12
156,2
78.12
e
625
312,5
625
156,2
1250
e
312.5
156,2
312.5
156,2
625
1250
e
1250
e
1250
e
78.12
39,06
1250
1250
312.5
1250
156.2
1250
1250
1250
e
1250
e
1250
e
e
e
e
e
e
e
312,5
625
1250
e
1250
e
1250
e
1250
e
1250
e
312.5
e
1250
e
1250
e
1250
e
1250
e
1250
e
1250
e
78.12
1250
1250
1250
1250
1250
1250
1250
1.2
1250
625
e
625
e
e
e
e
e
e
e
5f
e
e
e
e
e
625
e
e
4g
5g
4h
5h
e
e
e
e
1250
e
e
e
e
e
e
625
312,5
e
312,5
e
1250
e
1250
e
1250
e
1250
e
1250
e
1250
1250
9.8
312,5
4.9
312,5
0.5
Reference Antimicrobial 2.4
4.9
4.9
2.4
128.0
Ceftazidime Cefuroxime-Na Cefuroxime-Na Cefuroxime-Na Amikacin Cefuroxime Cefuroxime-Na Clotrimazole Amphotericin B
a
Abbreviations and full forms of the screening Gram-negative bacteria: PA; Pseudomonas aeruginosa (ATCC 27853), EC; Escherichia coli (ATCC 25922), KP; Klebsiella
pneumoniae (ATCC 4352), PM; Proteus mirabilis (ATCC 14153).
b
Abbreviations and full forms of the screening Gram-positive bacteria: EF; Enterococcus faecalis (ATCC 29212), SE; Staphylococcus epidermidis (ATCC 12228), SA; Staphy-
lococcus aureus (ATCC 29213).
c
Abbreviations and full forms of the screening Fungi: CA; Candida albicans (ATCC 10231), CP; Candida parapsilosis (ATCC 22019).
From the point of view of the antifungal activity, the test-
cultures C. albicans and C. parapsilosis were definitely appeared
as resistant to the 4f-h and to the 5c-d, 5d-f, respectively. The MIC
The effect of chlorine atom on the antifungal activity displays a
contrary trend compared to that of the antibacterial activity. For
C. albicans, most unchlorinated compounds were superior to the
chlorinated derivatives in terms of the antifungal activity except 4c-
4d and 5c-5d analogues which maintained the same promotive
halogen influence for the 4c and 5c. Besides that, while presence of
chlorine provided a positive effect on the activity for 4be5b, 4d-5d,
4e-5e, absence of chlorine atom enhanced the activity of 4a-5a, 4c-
5c, 4ge5g and 4he5h analogues against C. parapsilosis with the
exception of completely inactive 4f-5f analogues.
Another discussion on the structure-activity relationship can be
made for 4a-h and 5a-h series by comparing the influences of the
different substitutions in piperazine groups on the activities. For
the chlorinated compounds, there was no connection between the
activity results against Gram-negative bacteria which showed
values of the rest compounds were between 156.2 and 1250
for C. albicans and 156.2e625 g/mL for C. parapsilosis which in-
dicates substantially lower inhibitory activity by taking into ac-
count the MIC values of Clotrimazole (MIC ¼ 4.9 g/mL) and
Amphotericin B (MIC ¼ 0.5 g/mL).
mg/mL
m
m
m
The main purpose of the preparation of chlorinated and un-
chlorinated dimethylbenzoquinone series was to investigate the
influence of the halogen atom in the quinoid skeletone on the
antimicrobial activity of the novel compounds. When the test re-
sults of the newly synthesized derivatives against the Gram-
negative bacteria were analyzed, it was clearly seen that unchlo-
rinated compounds (5a-h) did not show any activity against all four
tested bacteria strains. On the other hand, most of the chlorinated
compounds were active against the Gram-negative bacteria, even
in a very low level. The 4c compound did not reveal any activity
against K. pneumoniae and P. mirabilis.
mostly MIC values at 1250
mg/mL and isopropyl, cyclohexyl,
methylphenyl and methoxyphenyl substituted piperazines moi-
eties. In addition to that, 4-methoxyphenyl substituted (4f) and 3-
methoxyphenyl substituted (4g) molecules were completely inac-
tive against all Gram-negative bacteria. The isopropyl or cyclohexyl
groups containing piperazine substituted chlorinated derivatives
seemed in particular more potent against Gram-positive bacteria
than methyl or methoxyphenyl groups bearing piperazine
substituted compounds. While exactly the same MIC values were
determined against E. faecalis for methylphenyl and methox-
yphenyl substituted piperazines including quinones, methyl-
phenylpiperazine containing derivatives were comparatively more
active that methoxyphenylpiperazine linked compounds. No ac-
tivity was observed for methoxyphenylpiperazine substituted
quinones against both C. albicans and C. parapsilosis. However, the
methylphenyl containing derivatives possessed relatively worse
activity in comparison to those of isopropyl or cyclohexyl carrying
compounds. Since hardly any unchlorinated derivative of dimethyl-
1,4-benzoquinone exhibited almost no activity against Gram-
negative bacteria and E. faecalis, a detection of any correlation be-
tween different substituents and activities was indeed not possible.
Nearly the same trend about the halogen effect on the activity is
also prevailing for the Gram-positive bacteria as determined for the
Gram-negative bacteria. Chlorine atom in quinone moiety either
improve the existing poor activity or gain to the inactive molecule
an antibacterial activity. Particularly, some great improvements
were distinctly observed for the potent molecules of 4a and 4b by
addition of chlorine atom to 5a and 5b compounds. Besides that, it
is noteworthy to say that, the activities of 4c, 4f, 5c and 5f against
E. faecalis, of 4h and 5h against S. epidermidis, of 4f-h and 5f-h
against S. aureus remained constant at the MIC value of 1250 mg/mL
and were not influenced from the presence or absence of chlorine
atom. Only 5b and 5g compounds were outlier within this present
trend, because their activities were better than those of chlorinated
products against S. epidermidis. Independently from the chlorine
atom in the structure, the Gram-positive and Gram-negative bac-
teria were resistant against 4c, 4f, 4g, 5c and 5f and no activity was
detected for these compounds.
Please cite this article as: M. Yıldız, Design, synthesis, characterization, and antimicrobial activity of novel piperazine substituted 1,4-
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Furthermore, the same tendency with chlorinated derivatives in
structure-activity relationship can be seen for the unchlorinated
compounds against S epidermidis and S. aureus. Namely, the iso-
propyl or cyclohexyl bearing molecules were comparatively more
effective than methyl- or methoxyphenyl including substances. The
activity against Gram-positive bacteria was almost not influenced
from differently positioned electron donating groups (-CH3 or
eOCH3) in the phenyl ring. The unchlorinated products revealed
also either no activity or very high MIC numbers which are also
undesired against tested two different fungi. Nevertheless, the
isopropyl or cyclohexyl group containing derivatives were com-
peratively more potent compared to the other unchlorinated
compounds against fungi as determined evidently for Gram-
positive bacteria. Notwithstanding, the two methoxyphenylpiper-
azine linked quinones (5g and 5h) were relatively efficient rather
than other substituted phenyl ring bearing compounds against
C. albicans or C. parapsilosis.
effected from the substitution of phenyl ring. Eventually, isopropyl
or cyclohexyl group and chlorine atom improved the antimicrobial
activities of the 2,3-dimethyl-1,4-benzoquinone derivatives,
particularly 4a and 4b, and the evaluated results promise room for
further improvement to develop potential antimicrobial agents.
Acknowledgments
This work has been supported by Research Fund of the Gebze
Technical University (Project Number: 2018-A105-31). Emel Mat-
€
aracı Kara and Berna Ozbek Çelik from the Pharmaceutical Micro-
biology Department of Istanbul University, are sincerely
appreciated for performing the antimicrobial activity tests of the
novel compounds. Sadık Metin Ceyhan and Yunus Zorlu, from the
Chemistry Department of Gebze Technical University, are cordially
acknowledged about their helpful discussions on crystal structure
analyses.
4. Conclusions
Appendix A. Supplementary data
In
the
present
work,
2,3-dichloro-5,6-dimethyl-1,4-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127422.
benzoquinone (2) was prepared by oxidation and chlorination of
commercial 2,3-dimethylhydroquinone (1) fast but at relatively
high temperature according to a previously present method [75].
The chlorinated and piperazine substituted novel 2,3-dimethyl-1,4-
benzoquinone molecules (4a-h) were obtained from the reaction of
the compound (2) with various piperazines at moderate tempera-
ture and in water by applying a formerly reported procedure in
literature [49]. In order to examine the influence of the chlorine
atom on the activity, unchlorinated and piperazine linked novel
2,3-dimethyl-1,4-benzoquinone molecules (5a-h) were also syn-
thesized from the commercial 2,3-dimethylhydroquinone (1) in the
presence of NaIO3 and diverse piperazines via a single-step method
published in a patent [72]. The characterizations of the novel
compounds were performed by applying IR, 1H NMR, 13C NMR and
mass spectroscopic methods. Besides that, the structures of the
some new compounds (5d, 5e and 5f) were obviously confirmed via
the X-ray single crystal method. In vitro antimicrobial activity tests
of all new compounds (4a-h and 5a-h) were carried out against
microbiologic references. The hit molecules among the novel
Author contributions
Mahmut Yıldız: Literature investigation, Conceptualization,
Design, Experimental work, Writing-Original draft preparation,
Writing-Reviewing and Editing, Writing-Revision.
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