Novel plastoquinone analogs containing benzocaine and its analogs: structure‐based design, synthesis, and structural characterization

Article abstract of DOI:10.1007/s11164-020-04384-5

Introducing new biologically active organic molecules or their analogs into the pharmaceutically important molecules significantly enables the discovery and development of the medicines that improve the health of patients. Chemical synthesis has a key role in pharmaceutical research and development, aiming to design and build molecules with the essential biological activity needed in drugs. For that reason, herein, three series of novel PQ analogs (the nonhalogenated (PQ1-6) and the halogenated PQ analogs (BrPQ1-6 and ClPQ1-6)) containing benzocaine and its analogs were designed and synthesized starting from the commercially available dimethylhydroquinone (1) with benzocaine and its analogs. The structures of all synthesized analogs were characterized by using spectroscopic methods. The in vitro antibacterial and antifungal activities were evaluated for the PQ analogs.

Full text of DOI:10.1007/s11164-020-04384-5

Research on Chemical Intermediates (2021) 47:2125–2141
https://doi.org/10.1007/s11164-020-04384-5
Novel plastoquinone analogs containing benzocaine
and its analogs: structure‐based design, synthesis,
and structural characterization
Nilüfer Bayrak1 · Mahmut Yıldız2 · Hatice Yıldırım1 · Emel Mataracı‑Kara3 ·
Amaç Fatih Tuyun4
Received: 7 August 2020 / Accepted: 23 December 2020 / Published online: 3 February 2021
© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021
Abstract
Introducing new biologically active organic molecules or their analogs into the
pharmaceutically important molecules signifcantly enables the discovery and
development of the medicines that improve the health of patients. Chemical syn-
thesis has a key role in pharmaceutical research and development, aiming to design
and build molecules with the essential biological activity needed in drugs. For that
reason, herein, three series of novel PQ analogs (the nonhalogenated (PQ1-6) and
the halogenated PQ analogs (BrPQ1-6 and ClPQ1-6)) containing benzocaine and
its analogs were designed and synthesized starting from the commercially avail-
able dimethylhydroquinone (1) with benzocaine and its analogs. The structures of
all synthesized analogs were characterized by using spectroscopic methods. The
in vitro antibacterial and antifungal activities were evaluated for the PQ analogs.
Keywords Aminobenzoquinone · Benzocaine · Plastoquinones · Antimicrobial
activity · Halogen
Supplementary Information The online version of this article (https://doi.org/10.1007/s11164-020-
04384-5) contains supplementary material, which is available to authorized users.
*
Amaç Fatih Tuyun
aftuyun@gmail.com; aftuyun@istanbul.edu.tr
1
Department of Chemistry, Faculty of Engineering, Istanbul University-Cerrahpasa,
İstanbul 34320, Avcılar, Turkey
2
3
Department of Chemistry, Gebze Technical University, Kocaeli 41400, Gebze, Turkey
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Istanbul University,
İstanbul 34116, Beyazit, Turkey
4
Department of Chemistry, Faculty of Science, Istanbul University, İstanbul 34134, Fatih, Turkey
Vol.:(0123456789)
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Introduction
Local anesthetic agents are the most important drug group in numerous felds, espe-
cially in dentistry [1]. Dentists frequently ofer “-caine”-type drugs such as procaine,
lidocaine, and benzocaine to induce local anesthesia. All local anesthetic agents in
use today have the similar three main structural components (an amino group, an
aromatic group, and an intermediate chain) and could be classifed into two main
groups containing ester (–COOR) and amide (–NHCO–) as shown in Fig. 1 [2].
Among these agents, the discovery and employment of the ethyl 4-aminobenzo-
ate, commonly known as benzocaine, in 1890 by Eduard Ritsert was the beginning
of the completely new applications. The benzocaine was launched in the market in
1902 with “Anasthesin” as the trade name [3]. Indeed, benzocaine and its analogs
exhibit diverse biological activities including anticancer [4, 5], local anesthesia [6,
7], antimicrobial [8, 9], and redox activity [10]. Considering the literature evidence,
studies show the construction of benzocaine analogs with other small drug moieties
with noticeable biological activities [2].
Specifcally, structures containing quinone moiety provide the basis for the
development of the versatile biologically and/or synthetically useful compounds
[11–16]. Among the numerous structures, 1,4-quinones occupy a special place since
they exist widely in the structures of many natural products and synthetic com-
pounds possessing a large spectrum of biological activities including anticancer [17,
18], antibacterial [19, 20], antifungal [21], anti-infammatory [22], antitrypanosomal
[23], antimalarial [24], antiviral [25], and antitubercular activities [26]. Considering
the important biological properties of 1,4-quinone moiety, numerous publications,
Fig. 1 Some commonly used
local anesthetics
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including several reviews, related to 1,4-quinone chemistry have been studied over
the past decades [17, 27]. As distinguished from their efective biological properties,
making them favored structures in medicinal chemistry and pharmacology [28–30],
these structures recently also attracted attention in diferent areas such as organic
synthesis [31] and sensor applications [32, 33]. Our group speculated that incorpo-
ration of aryl amino group into the dimethylquinone skeleton in structures named as
plastoquinone analogs (I, shown in Fig. 2, PQ analogs) would change the biologi-
cal properties. Our recent reports revealed that the presence of electron-withdrawing
or electron-donating group within aryl amino group and chlorine or hydrogen atom
within dimethylquinone was considerably important factor to afect their activities.
Considering these fndings, we previously continued to develop new antimicrobial
and anticancer agents with high therapeutic value and minimal side efects based on
PQ analogs [34–39]. In our recent reports, we designed, synthesized, and discov-
ered a new family of heterocyclic amine linked PQ analogs for antimicrobial evalua-
tion. The two analogs were identifed as the hits with the strong antibacterial activi-
ties against the S. epidermidis with low MIC values (II, shown in Fig. 2) [34]. Our
another study revealed that one analog containing methoxy group at the meta posi-
tion within aryl amino group was found to be the most efective anticancer analog
against both K562 and Jurkat cell lines. In addition, the analog was non-cytotoxic to
normal peripheral blood mononuclear cells. Its further studies also showed that this
analog displayed anti-Abelson kinase 1 activity with diferent inhibitory profle than
Imatinib in the panel of eight kinases (III, shown in Fig. 2) [35]. In addition to these
important studies, our group designed and synthesized another two novel series of
PQ analogs to explore the chlorine atom efect on inhibitory efect. Among these
analogs, the analog containing chlorine atom within the quinone moiety showed a
better anticancer activity and selectivity than the reference drug Imatinib on cancer
Fig. 2 Important PQ analogs possessing interesting biological activities
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N. Bayrak et al.
cells. Therefore, this analog was selected for a further apoptosis/necrosis evalua-
tion (IV, shown in Fig. 2) [36]. By combining these two studies (methoxy group
within aryl amino group and chlorine atom within the quinone moiety), we reported
the synthesis and cytotoxic efects of novel chlorinated PQ analogs against chronic
myelogenous leukemia cell lines. One analog containing dimethoxy group exhibited
approximately twenty times higher activity against K562 cell line and three times
higher selectivity than Imatinib on CML (V, shown in Fig. 2) [37]. Twenty-four
chlorinated PQ analogs were employed for their in vitro antimicrobial potential. The
PQ analog containing methoxy group at the ortho position within aryl amino group
showed the highest growth inhibition against S. epidermidis (VI, shown in Fig. 2)
[38]. In light of these remarkable reports published by our group, herein, we further
extended to synthesize novel PQ analogs incorporating both benzocaine and its ana-
logs (Fig. 3) and the hydrogen, bromine, and chlorine atom within the quinone moi-
ety. To this end, consisting of three new series of PQ analogs were prepared, then
these analogs are structurally characterized and investigated the in vitro antimicro-
bial activity.
Results and discussion
Aminoquinones have been synthesized from the reaction between amines and qui-
nones as previously reported literature [40, 41]. As far as we know, the synthetic
procedures to aminoquinones in terms of the general routes could be summarized
by three pathways. The frst one is the nucleophilic addition reactions of 1,4-qui-
nones with amines [40, 42]. Another route is the reaction between hydroquinone and
amines in the presence of oxidizing agent [43] or some fungal laccases [44]. The
fnal one is the nucleophilic substitution reaction of the halogen or methoxy deriva-
tive of 1,4-quinones with amines in the diferent reaction media [28, 45–47].
The general synthetic route of the target molecules is stated in Scheme 1. The
reaction routes of the nonhalogenated (PQ1-6) and halogenated PQ analogs (BrPQ1-
6 and ClPQ1-6) started from commercially available dimethylhydroquinone (1).
Fig. 3 Design of the novel PQ analogs containing benzocaine and its analogs
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Initially, a library of the nonhalogenated PQ analogs (PQ1-6) was prepared from
dimethylhydroquinone (1) and the corresponding substituted amine in the presence
of oxidizing agent (NaIO3) according to the literature [43]. The key precursor (3)
was prepared by dibromination in two steps, then followed by oxidation according
to the literature [48]. The brominated PQ analogs (BrPQ1-6) were formed by the
reaction between the corresponding substituted amines and dibromobenzoquinone
(3) in ethanol. Finally, the chlorinated PQ analogs (ClPQ1-6) were synthesized in
turn by the reaction of the corresponding substituted amines in refux EtOH with the
dichlorobenzoquinone (4), prepared from the oxidation of dimethylhydroquinone
(1) with HNO3/HCl variation at elevated temperature according to the literature
[49]. We employed the substituted amines that possess ester groups (–COOCH3,
–COOCH2CH3, –COOC(CH3)3) at the ortho, meta, or para position to obtain the
target PQ analogs. Unfortunately, the synthesis of the target PQ analogs with ester
at the ortho position of the corresponding amine failed due to steric hindrance of
the bulky ortho substituent as the spatial arrangement of atoms or functional groups
near the reacting site hindered the reaction. All the analyses (FTIR, NMR (1H
NMR and 13C NMR), and HRMS spectroscopic data) performed to characterize the
Scheme 1 Synthesis of the nonhalogenated (PQ1-6) and the halogenated PQ analogs (BrPQ1-6 and
ClPQ1-6)a
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N. Bayrak et al.
structures of the nonhalogenated (PQ1-6) and halogenated PQ analogs (BrPQ1-6
and ClPQ1-6) confrmed their chemical structures. (For details, please see the Sup-
plementary fle.) In addition, the biological activity of the nonhalogenated (PQ1-6)
and the halogenated PQ analogs (BrPQ1-6 and ClPQ1-6) was carried out against
four Gram-negative bacteria, three Gram-positive bacteria for antibacterial activity,
and three fungi for antifungal activity by employing the standard drugs using the
broth microdilution method according to the Clinical Laboratory Standards Institute
(CLSI) recommendations [50, 51]. But, unfortunately, we could say that the in vitro
inhibitory activity of the nonhalogenated (PQ1-6) and the halogenated PQ analogs
(BrPQ1-6 and ClPQ1-6) does not fully correspond to the bacteria and fungi strains.
(For details, please see Tables S1 and S2 in the Supplementary fle.)
Some characteristic bands of the PQ analogs must be mentioned. The absorption
bands from vibrations of the following bands were observed at around 1660 cm−1
for C=O; 3250 cm−1 for NH; and 2900 cm−1 for CHaliphatic in the FTIR spectra.
Just before the fngerprint region at about 1700 was the carbonyl ester peak. It is
established from the data of the 1H NMR spectra of the nonhalogenated PQ analogs
(PQ1-6) that the single proton belonging to quinone moiety resonated as a singlet at
around 6.10 ppm. In the 1H NMR spectra of all PQ analogs, the singlet in the region
3.90 ppm was assigned to the protons of methoxy group, the quartet and triplet in
the region 4.35 and 1.35 ppm to the protons of ethoxy group, and the singlet in the
region 1.60 ppm was assigned to the protons of tert-butyl group. The two methyl
protons attached to the quinone moiety were observed as singlet at around 2.05 ppm
1
and 2.10 ppm. The H NMR spectra showed the signal of the amino proton signal
at around 7.40 ppm. In the 1H NMR spectra of all PQ analogs, the aromatic protons
showed typical ppm values within the around 7.00 and 8.00 ppm. Moreover, the 13
C
NMR data showed the presence of the three carbonyl carbon atoms at the appro-
priate δ values. The particular carbonyl carbon atoms belonging to quinone moiety
exhibited resonances in a range between 179 and 182 ppm, whereas the other car-
bonyl carbon atom resonating at around 165 ppm belongs to the ester group within
amine. Additionally, the signals at around 52, 62, and 81 ppm were present that is
typical of methyl, ethyl, and tert-butyl carbon atoms attached to the oxygen atom,
respectively. In the HRMS of the PQ analogs, the mass measurement of the molecu-
lar ion peaks was noticed with hydrogen adduct or not.
Protons are shielded by diferent amounts due to their diferent chemical envi-
ronments. A proton is shielded or deshielded whether the induced magnetic feld
opposes the applied magnetic feld. The efcient magnetic feld depends on the
1
amount of the induced magnetic feld. The extended H NMR spectra of PQ4,
BrPQ4, and ClPQ4 are shown in Fig. 4. To examine the diferences among the
spectra, we looked into the NH proton shift. While the NH proton of PQ4 appeared
at 7.55 ppm, that shiftings of BrPQ4 and ClPQ4 are 7.47 and 7.42 ppm, respec-
tively. For PQ analogs without halogen atom, there is less electron density around
NH proton, so this proton is afected by a higher efcient magnetic feld. However,
for BrPQ and ClPQ analogs with bromine and chlorine atom, because the halogen
atom increases the electron density around the NH proton, it increases the shielding
of that proton. Therefore, NH proton is afected by a lower efcient magnetic feld
than that of PQ analogs. It causes these NH proton shiftings to appear at the lower
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frequency [52]. Similarly, CH protons close to the NH group of the benzene ring of
BrPQ and ClPQ analogs also appear at a lower frequency.
Conclusion
In summary, three series of novel PQ analogs (the nonhalogenated (PQ1-6) and
the halogenated PQ analogs (BrPQ1-6 and ClPQ1-6)) containing benzocaine and
its analogs have been designed, synthesized, and structurally characterized (FTIR,
NMR, and HRMS), and in vitro antibacterial and antifungal activities have been
studied against a panel of microorganisms. The antimicrobial activity has been eval-
uated for these PQ, BrPQ, and ClPQ analogs against Gram-positive bacteria strains
and fungi strains. Even though it is known that benzocaine and its analogs exhibit
diverse biological activities, quinone with benzocaine, used as a substitute group,
was not found to be efective on bacterial and fungal strains. Therefore, further
extension of this important research by employing dimethylbenzoquinone is now
ongoing in our laboratory. In addition, further investigation of these PQ analogs on
their potential anticancer activity is in progress since they have been selected by the
National Cancer Institute, and the results will be reported in due course.
Fig. 4 The extended 1H NMR spectra of PQ4, BrPQ4, and ClPQ4 between 7.00 and 8.10 ppm
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Materials and methods
Apparatus and chemicals
Solvents, chemical reagents, and chemicals were purchased from various commer-
cial sources used without purifcation. Column chromatography was performed
with silica gel (63–200 µm particle sized) with appropriate solvent system as elu-
ents. All reactions were monitored by analytical thin-layer chromatography (TLC)
on Merck DC plates (aluminum-based silica gel 60 F254) purchased from Merck
KGaA. Visualization of TLC plates was performed by means of UV light (254 nm).
Melting points were measured on a Buchi B-520 melting point apparatus and were
uncorrected. The precursors, 2,3-dibromo-5,6-dimethyl-1,4-benzoquinone (3) [48]
and 2,3-dichloro-5,6-dimethyl-1,4-benzoquinone (4) [49], were synthesized using
1
the reported method in the literature. H and 13C NMR spectra were recorded on
1
a VarianUNITY INOVA 500 MHz frequency for H and 125 MHz frequency for 13C
1
NMR spectrometers at 25 °C and CDCl3 as solvent. H NMR chemical shifts (δ)
values were reported in parts per million (ppm) relative to residual solvent (CDCl3,
δ 7.26), and 13C NMR chemical shifts (δ) were reported in ppm relative to (CDCl3,
δ 77.16), and coupling constants (J) were expressed in Hertz (Hz). High-resolution
mass spectrometry (HRMS) was performed on either a Waters SYNAPT G1 MS or
an Agilent 6530 Accurate-Mass Q-TOF LC/MS. FTIR spectra were recorded with
Alpha T FTIR spectrometer with single refection diamond ATR module.
General procedure for the synthesis of the plastoquinone analogs (PQ1‑6)
To a solution of the corresponding substituted amines (4.00 mmol, 2 equiv.) and
2,3-dimethylhydroquinone (1, 0.276 g, 2.00 mmol) in methanol (12 mL), sodium
iodate (1.186 g, 6.00 mmol, 3 equiv.) in water (12 mL) was added at room tempera-
ture and stirred for 12–24 h. The progress of the reaction was monitored by TLC.
After the completion of reaction, the solution was extracted with dichloromethane
(3×50 mL), and the combined organic layers were washed with water (3×50 mL),
dried over anhydrous CaCl2, and concentrated under vacuum. Finally, the residue
was chromatographed over silica gel to provide the corresponding PQ.
Methyl 3‑((4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate (PQ1)
By using the dimethylhydroquinone (1, 0.276 g, 2.00 mmol) and methyl 3-amin-
obenzoate (0.605 g, 4.00 mmol) to the general procedure for the target analog as
a dark red solid using petroleum ether/CHCl3 (1:2) as an eluent. Yield: 47%, mp
139.5–141.2 °C. FTIR (ATR) υ (cm−1): 3260 (NH), 3074, 3048 (CHaromatic), 2956
(CHaliphatic), 1722 (>C=Oester), 1666 (>C=Oquinone), 1643, 1589, 1522, 1492, 1437,
1
1420, 1379, 1348, 1292, 1211, 1110, 1086, 1043. H NMR (500 MHz, CDCl3) δ
(ppm): 7.94–7.59 (m, 2H, CHaromatic), 7.50–7.22 (m, 3H, NH and CHaromatic), 6.14
(s, 1H, CH), 3.91 (s, 3H, OCH3), 2.04 (s, 6H, CH3). 13C NMR (125 MHz, CDCl3) δ
(ppm): 186.4, 183.7, 166.2 (>C=O), 143.8, 142.3, 138.2, 136.8, 131.7, 129.6, 126.0,
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125.9, 122.8, 101.2 (Caromatic and Cq), 52.4 (OCH3), 12.7, 12.1 (CH3). HRMS(+ESI)
m/z calcd for C16H16NO4 [M+H]+: 286.1079; found: 286.1077.
Methyl 4‑((4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate (PQ2)
By using the dimethylhydroquinone (1, 0.276 g, 2.00 mmol) and methyl 4-amin-
obenzoate (0.605 g, 4.00 mmol) to the general procedure for the target analog
as a red solid using petroleum ether/CHCl3 (1:2) as an eluent. Yield: 74%, mp
203.5–204.3 °C. FTIR (ATR) υ (cm−1): 3277 (NH), 2959, 2924, 2856 (CHaliphatic),
1699 (>C=Oester), 1663 (>C=Oquinone), 1644, 1592, 1525, 1503, 1428, 1415, 1374,
1
1274, 1239, 1176, 1108, 1030. H NMR (500 MHz, CDCl3) δ (ppm): 8.13–7.96
(m, 2H, CHaromatic), 7.53 (br s, 1H, NH), 7.33–7.15 (m, 2H, CHaromatic), 6.33 (s, 1H,
CH), 3.93 (s, 3H, OCH3), 2.09 (s, 6H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm):
186.5, 183.6, 166.2 (>C=O), 143.8, 142.3, 141.2, 137.0, 131.3, 125.8, 120.1, 102.8
(Caromatic and Cq), 52.1 (OCH3), 12.8, 12.2 (CH3). HRMS(+ESI) m/z calcd for
C16H16NO4 [M+H]+: 286.1079; found: 286.1079.
Ethyl 3‑((4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate (PQ3)
By using the dimethylhydroquinone (1, 0.276 g, 2.00 mmol) and ethyl 3-amin-
obenzoate (0.661 g, 4.00 mmol) to the general procedure for the target analog as
a dark red solid using petroleum ether/CHCl3 (1:2) as an eluent. Yield: 76%, mp
135.8–136.9 °C. FTIR (ATR) υ (cm−1): 3244 (NH), 3081 (CHaromatic), 2985, 2956,
2930, 2904 (CHaliphatic), 1713 (>C=Oester), 1662 (>C=Oquinone), 1644, 1584,
1527, 1476, 1443, 1367, 1313, 1286, 1261, 1210, 1106, 1033. 1H NMR (500 MHz,
CDCl3) δ (ppm): 7.82 (s, 1H, CHaromatic), 7.78 (d, J=6.9 Hz, 1H, CHaromatic), 7.47
(br s, 1H, NH), 7.43–7.35 (m, 2H, CHaromatic), 6.10 (s, 1H, CH), 4.35 (q, J=7.1 Hz,
2H, OCH2), 2.01 (s, 3H, CH3), 2.00 (s, 3H, CH3), 1.36 (t, J=7.1 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3) δ (ppm): 186.4, 183.7, 165.6 (>C=O), 143.7, 142.3,
138.2, 136.7, 132.0, 129.5, 125.9, 125.8, 122.9, 101.1 (Caromatic and Cq), 61.3
(OCH2), 14.3, 12.7, 12.0 (CH3). HRMS(+ESI) m/z calcd for C17H17NO4 [M]+:
299.1158; found: 299.1161.
Ethyl 4‑((4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate (PQ4)
By using the dimethylhydroquinone (1, 0.276 g, 2.00 mmol) and ethyl 4-amin-
obenzoate (0.661 g, 4.00 mmol) to the general procedure for the target analog
as a red solid using petroleum ether/CHCl3 (1:2) as an eluent. Yield: 28%, mp
156.6–157.7 °C. FTIR (ATR) υ (cm−1): 3307 (NH), 2985 (CHaliphatic), 1706
(>C=Oester), 1666 (>C=Oquinone), 1647, 1597, 1542, 1477, 1416, 1367, 1326,
1276, 1237, 1210, 1178, 1106, 1021. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.05 (d,
J=7.8 Hz, 2H, CHaromatic), 7.55 (br s, 1H, NH), 7.24 (d, J=7.8 Hz, 2H, CHaromatic),
6.31 (s, 1H, CH), 4.38 (q, J=6.2 Hz, 2H, OCH2), 2.07 (s, 6H, CH3), 1.40 (t,
J=6.7 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm): 186.5, 183.6, 165.7
(>C=O), 143.8, 142.2, 141.3, 137.0, 131.2, 126.1, 120.1, 102.7 (Caromatic and Cq),
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61.0 (OCH2), 14.3, 12.7, 12.1 (CH3). HRMS(+ESI) m/z calcd for C17H17NO4 [M]+:
299.1158; found: 299.1158.
tert‑Butyl 3‑((4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate (PQ5)
By using the dimethylhydroquinone (1, 0.276 g, 2.00 mmol) and tert-butyl 3-amin-
obenzoate (0.773 g, 4.00 mmol) to the general procedure for the target analog as
a dark red solid using petroleum ether/CHCl3 (1:2) as an eluent. Yield: 84%, mp
121.8–122.9 °C. FTIR (ATR) υ (cm−1): 3253 (NH), 3063 (CHaromatic), 2970, 2930
(CHaliphatic), 1708 (>C=Oester), 1669 (>C=Oquinone), 1648, 1588, 1531, 1488, 1424,
1
1367, 1298, 1259, 1212, 1161, 1110, 1078. H NMR (500 MHz, CDCl3) δ (ppm):
7.82–7.73 (m, 2H, CHaromatic), 7.47–7.34 (m, 3H, NH and CHaromatic), 6.12 (s, 1H,
CH), 2.04 (s, 6H, CH3), 1.59 (s, 9H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm):
186.4, 183.8, 164.7 (>C=O), 143.8, 142.4, 138.0, 136.7, 133.6, 129.4, 125.9, 125.6,
122.9, 101.0 (Caromatic and Cq), 81.6 (OC), 28.1, 12.8, 12.1 (CH3). HRMS(+ESI)
m/z calcd for C19H21NO4 [M]+: 327.1471; found: 327.1473.
tert‑Butyl 4‑((4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate (PQ6)
By using the dimethylhydroquinone (1, 0.276 g, 2.00 mmol) and tert-butyl 4-amin-
obenzoate (0.773 g, 4.00 mmol) to the general procedure for the target analog as
a dark red solid using petroleum ether/CHCl3 (1:2) as an eluent. Yield: 56%, mp
183.4–184.2 °C. FTIR (ATR) υ (cm−1): 3305 (NH), 3011 (CHaromatic), 2981, 2968,
2926 (CHaliphatic), 1701 (>C=Oester), 1668 (>C=Oquinone), 1649, 1595, 1542, 1477,
1415, 1368, 1324, 1287, 1235, 1208, 1158, 1109, 1034. 1H NMR (500 MHz, CDCl3)
δ (ppm): 8.01 (d, J=8.2 Hz, 2H, CHaromatic), 7.52 (br s, 1H, NH), 7.23 (d, J=8.1 Hz,
2H, CHaromatic), 6.30 (s, 1H, CH), 2.08 (s, 6H, CH3), 1.61 (s, 9H, CH3). 13C NMR
(125 MHz, CDCl3) δ (ppm): 186.5, 183.7, 164.9 (>C=O), 143.8, 141.8, 141.4,
136.9, 131.1, 127.8, 120.1, 102.5 (Caromatic and Cq), 81.1 (OC), 28.2, 12.8, 12.2
(CH3). HRMS(+ESI) m/z calcd for C19H21NO4 [M]+: 327.1471; found: 327.1472.
General procedure for the synthesis of the brominated plastoquinone analogs
(BrPQ1‑6)
To a solution of the 2,3-dibromo-5,6-dimethyl-1,4-benzoquinone (3, 0.100 g, 1
equiv.) in ethanol was added dropwise corresponding substituted amines (1.1 equiv.)
over 5 min, and the resulting mixture was stirred at room temperature for 8–16 h
until consumption of the dibromobenzoquinone (3). After ethanol was evaporated
under reduced pressure, CH2Cl2 (50 mL) was added, and the solution was washed
sequentially with water (3×30 mL). The organic layer was dried over CaCl2, fl-
tered, and concentrated under reduced pressure. Finally, the residue was chromato-
graphed over silica gel to provide the corresponding BrPQ.
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Methyl 3‑((2‑bromo‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(BrPQ1)
By using the dibromobenzoquinone (3, 0.100 g, 0.34 mmol) and methyl 3-amin-
obenzoate (0.057 g, 0.374 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:4) as an eluent. Yield: 23%, mp
165–166 °C. FTIR (ATR) υ (cm−1): 3225 (NH), 3010 (CHaromatic), 2989, 2941, 2844
(CHaliphatic), 1721 (>C=Oester), 1662 (>C=Oquinone), 1567, 1507, 1472, 1446, 1431,
1
1368, 1284, 1200, 1147, 1102, 1051. H NMR (500 MHz, CDCl3) δ (ppm): 7.87
(br s, 1H, NH), 7.70 (s, 1H, CHaromatic), 7.45 (s, 2H, CHaromatic), 7.32–7.19 (m, 1H,
CHaromatic), 3.93 (s, 3H, OCH3), 2.17 (s, 3H, CH3), 2.10 (s, 3H, CH3). 13C NMR
(125 MHz, CDCl3) δ (ppm): 182.1, 179.4, 166.4 (>C=O), 143.8, 141.9, 137.7,
137.1, 130.6, 128.5, 128.4, 126.4, 125.1, 104.6 (Caromatic and Cq), 52.3 (OCH3), 13.8,
12.3 (CH3). HRMS(+ESI) m/z calcd for C16H14BrNO4 [M]+: 363.0106; found:
363.0114.
Methyl 4‑((2‑bromo‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(BrPQ2)
By using the dibromobenzoquinone (3, 0.100 g, 0.34 mmol) and methyl 4-amin-
obenzoate (0.057 g, 0.374 mmol) to the general procedure for the target analog
as a red solid using petroleum ether/CHCl3 (1:4) as an eluent. Yield: 10%, mp
183–184 °C. FTIR (ATR) υ (cm−1): 3239 (NH), 2959, 2922, 2852 (CHaliphatic), 1703
(>C=Oester), 1654 (>C=Oquinone), 1575, 1518, 1490, 1437, 1412, 1374, 1311, 1279,
1253, 1198, 1171, 1147, 1101, 1053. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.02 (d,
J=8.3 Hz, 2H, CHaromatic), 7.46 (br s, 1H, NH), 7.03 (d, J=8.2 Hz, 2H, CHaromatic),
3.93 (s, 3H, OCH3), 2.17 (s, 3H, CH3), 2.11 (s, 3H, CH3). 13C NMR (125 MHz,
CDCl3) δ (ppm): 182.1, 179.4, 166.5 (>C=O), 143.7, 141.6, 141.5, 137.4, 130.0,
126.1, 122.6, 106.9 (Caromatic and Cq), 52.1 (OCH3), 13.8, 12.4 (CH3). HRMS(+ESI)
m/z calcd for C16H14BrNO4 [M]+: 363.0106; found: 363.0115.
Ethyl 3‑((2‑bromo‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(BrPQ3)
By using the dibromobenzoquinone (3, 0.100 g, 0.34 mmol) and ethyl 3-amin-
obenzoate (0.062 g, 0.374 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:4) as an eluent. Yield: 38%, mp
133–134 °C. FTIR (ATR) υ (cm−1): 3218 (NH), 2985, 2956, 2900 (CHaliphatic),
1715 (>C=Oester), 1663 (>C=Oquinone), 1568, 1505, 1470, 1442, 1366, 1273, 1197,
1
1150, 1105, 1030. H NMR (500 MHz, CDCl3) δ (ppm): 7.88 (d, J=7.1 Hz, 1H,
CHaromatic), 7.71 (br s, 1H, NH), 7.46 (s, 1H, CHaromatic), 7.41 (t, J=7.7 Hz, 1H,
CHaromatic), 7.22 (d, J=7.7 Hz, 1H, CHaromatic), 4.39 (q, J=6.8 Hz, 2H, OCH2), 2.16
(s, 3H, CH3), 2.09 (s, 3H, CH3), 1.40 (t, J=6.8 Hz, 3H, CH3). 13C NMR (125 MHz,
CDCl3) δ (ppm): 182.1, 179.4, 165.9 (>C=O), 143.8, 141.9, 137.7, 137.1, 131.0,
128.4, 126.3, 125.0, 104.6 (Caromatic and Cq), 61.3 (OCH2), 14.3, 13.8, 12.3 (CH3).
HRMS(+ESI) m/z calcd for C17H16BrNO4 [M]+: 377.0263; found: 377.0275.
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Ethyl 4‑((2‑bromo‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(BrPQ4)
By using the dibromobenzoquinone (3, 0.100 g, 0.34 mmol) and ethyl 4-amin-
obenzoate (0.062 g, 0.374 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:4) as an eluent. Yield: 13%, mp
168–169 °C. FTIR (ATR) υ (cm−1): 3251 (NH), 2981, 2963, 2926 (CHaliphatic), 1697
(>C=Oester), 1655 (>C=Oquinone), 1575, 1516, 1491, 1413, 1370, 1309, 1276, 1252,
1204, 1172, 1145, 1101, 1051, 1016. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.02 (d,
J=8.2 Hz, 2H, CHaromatic), 7.47 (br s, 1H, NH), 7.03 (d, J=8.3 Hz, 2H, CHaromatic),
4.39 (q, J=7.0 Hz, 2H, OCH2), 2.17 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.41 (t,
J=7.1 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm): 182.1, 179.4, 166.0
(>C=O), 143.7, 141.6, 141.4, 137.4, 130.0, 126.5, 122.6, 106.8 (Caromatic and Cq),
61.0 (OCH2), 14.4, 13.8, 12.4 (CH3). HRMS(+ESI) m/z calcd for C17H16BrNO4
[M]+: 377.0263; found: 377.0275.
tert‑Butyl 3‑((2‑bromo‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)
benzoate (BrPQ5)
By using the dibromobenzoquinone (3, 0.100 g, 0.34 mmol) and tert-butyl 3-amin-
obenzoate (0.072 g, 0.374 mmol) to the general procedure for the target analog
as a red solid using petroleum ether/CHCl3 (1:4) as an eluent. Yield: 49%, mp
186–187 °C. FTIR (ATR) υ (cm−1): 3223 (NH), 2978, 2930 (CHaliphatic), 1716
(>C=Oester), 1660 (>C=Oquinone), 1574, 1501, 1468, 1440, 1365, 1290, 1252, 1202,
1
1148, 1107, 1056. H NMR (500 MHz, CDCl3) δ (ppm): 7.81 (d, J=7.7 Hz, 1H,
CHaromatic), 7.66 (br s, 1H, NH), 7.46 (s, 1H, CHaromatic), 7.39 (t, J=7.5 Hz, 1H,
CHaromatic), 7.18 (d, J=7.6 Hz, 1H, CHaromatic), 2.16 (s, 3H, CH3), 2.09 (s, 3H,
CH3), 1.60 (s, 9H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm): 182.1, 179.4,
165.0 (>C=O), 143.8, 141.9, 137.5, 137.0, 132.5, 128.2, 128.0, 126.2, 124.9, 104.4
(Caromatic and Cq), 81.5 (OC), 28.2, 13.8, 12.3 (CH3). HRMS(+ESI) m/z calcd for
C19H20BrNO4 [M]+: 405.0576; found: 405.0590.
tert‑Butyl 4‑((2‑bromo‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)
benzoate (BrPQ6)
By using the dibromobenzoquinone (3, 0.100 g, 0.34 mmol) and tert-butyl 4-amin-
obenzoate (0.072 g, 0.374 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:4) as an eluent. Yield: 80%, mp
166–167 °C. FTIR (ATR) υ (cm−1): 3216 (NH), 2985, 2926, 2856 (CHaliphatic), 1707
(>C=Oester), 1657 (>C=Oquinone), 1573, 1512, 1491, 1411, 1367, 1284, 1250, 1203,
1160, 1108, 1051, 1016. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.96 (d, J=7.2 Hz,
2H, CHaromatic), 7.46 (br s, 1H, NH), 7.01 (d, J=7.2 Hz, 2H, CHaromatic), 2.17 (s, 3H,
CH3), 2.10 (s, 3H, CH3), 1.61 (s, 9H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm):
182.1, 179.4, 165.1 (>C=O), 143.7, 141.7, 141.1, 137.3, 129.8, 128.2, 122.6, 106.4
(Caromatic and Cq), 81.1 (OC), 28.2, 13.8, 12.4 (CH3). HRMS(+ESI) m/z calcd for
C19H20BrNO4 [M]+: 405.0576; found: 405.0583.
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General procedure for the synthesis of the chlorinated plastoquinone analogs
(ClPQ1‑6)
To a solution of the 2,3-dichloro-5,6-dimethyl-1,4-benzoquinone (4, 0.256 g, 1
equiv.) in ethanol was added dropwise corresponding substituted amines (1.1 equiv.)
over 5 min, and the resulting mixture was refuxed for 8–16 h until consumption
of the dichlorobenzoquinone (4). After the reaction mixture was cooled to ambi-
ent temperature, ethanol was removed under reduced pressure. Dichloromethane
(50 mL) was added to residue, and the obtained solution was washed sequentially
with water (3×30 mL). The organic layer was dried over CaCl2, fltered, and con-
centrated under reduced pressure. Finally, the residue was chromatographed over
silica gel to provide the corresponding ClPQ.
Methyl 3‑((2‑chloro‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(ClPQ1)
By using the dichlorobenzoquinone (4, 0.256 g, 1.25 mmol) and methyl 3-amin-
obenzoate (0.208 g, 1.375 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:3) as an eluent. Yield: 47%,
mp 163.1–165.2 °C. FTIR (ATR) υ (cm−1): 3221 (NH), 2941 (CHaliphatic), 1721
(>C=Oester), 1664 (>C=Oquinone), 1571, 1508, 1472, 1446, 1432, 1371, 1287, 1272,
1
1250, 1205, 1152, 1102, 1087, 1054. H NMR (500 MHz, CDCl3) δ (ppm): 7.83
(d, J=7.7 Hz, 1H, CHaromatic), 7.66 (br s, 1H, NH), 7.44 (s, 1H, CHaromatic), 7.39 (t,
J=7.9 Hz, 1H, CHaromatic), 7.19 (d, J=7.6 Hz, 1H, CHaromatic), 3.91 (s, 3H, OCH3),
2.13 (s, 3H, CH3), 2.07 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm): 182.5,
179.5, 166.4 (>C=O), 143.7, 139.1, 137.8, 137.2, 130.5, 128.4, 127.9, 126.1, 124.6,
112.3 (Caromatic and Cq), 52.3 (OCH3), 13.4, 12.3 (CH3). HRMS(+ESI) m/z calcd for
C16H13ClNO4 [M–H]+: 318.0533; found: 318.0530.
Methyl 4‑((2‑chloro‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(ClPQ2)
By using the dichlorobenzoquinone (4, 0.256 g, 1.25 mmol) and methyl 4-amin-
obenzoate (0.208 g, 1.375 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:3) as an eluent. Yield: 35%,
mp 207.5–208.6 °C. FTIR (ATR) υ (cm−1): 3233 (NH), 2953 (CHaliphatic), 1706
(>C=Oester), 1660 (>C=Oquinone), 1578, 1518, 1493, 1437, 1412, 1376, 1310, 1286,
1
1271, 1250, 1213, 1172, 1150, 1103, 1055, 1016. H NMR (500 MHz, CDCl3) δ
(ppm): 8.01 (d, J=7.6 Hz, 2H, CHaromatic), 7.42 (br s, 1H, NH), 7.00 (d, J=7.7 Hz,
2H, CHaromatic), 3.92 (s, 3H, OCH3), 2.16 (s, 3H, CH3), 2.10 (s, 3H, CH3). 13C NMR
(125 MHz, CDCl3) δ (ppm): 182.5, 179.6, 166.5 (>C=O), 143.7, 141.6, 138.7,
137.5, 130.0, 126.0, 122.2, 114.2 (Caromatic and Cq), 52.1 (OCH3), 13.5, 12.4 (CH3).
HRMS(+ESI) m/z calcd for C16H13ClNO4 [M–H]+: 318.0533; found: 318.0534.
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Ethyl 3‑((2‑chloro‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(ClPQ3)
By using the dichlorobenzoquinone (4, 0.256 g, 1.25 mmol) and ethyl 3-amin-
obenzoate (0.227 g, 1.375 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:3) as an eluent. Yield: 39%,
mp 161.5–162.2 °C. FTIR (ATR) υ (cm−1): 3310 (NH), 2908 (CHaliphatic), 1718
(>C=Oester), 1653 (>C=Oquinone), 1598, 1585, 1522, 1479, 1439, 1375, 1302,
1
1293, 1271, 1248, 1199, 1147, 1106, 1079, 1054. H NMR (500 MHz, CDCl3) δ
(ppm): 7.89–7.74 (m, 1H, CHaromatic), 7.68 (br s, 1H, NH), 7.44 (s, 1H, CHaromatic),
7.42–7.37 (m, 1H, CHaromatic), 7.22–7.07 (m, 1H, CHaromatic), 4.44–4.26 (m, 2H,
OCH2), 2.13 (s, 3H, CH3), 2.07 (s, 3H, CH3), 1.39 (t, J=7.1 Hz, 3H, CH3). 13C
NMR (125 MHz, CDCl3) δ (ppm): 182.5, 179.6, 165.9 (>C=O), 143.8, 139.1,
137.8, 137.1, 130.9, 128.3, 127.9, 126.1, 124.6, 112.2 (Caromatic and Cq), 61.2
(OCH2), 14.3, 13.4, 12.3 (CH3). HRMS(+ESI) m/z calcd for C17H15ClNO4 [M–H]+:
332.0690; found: 332.0690.
Ethyl 4‑((2‑chloro‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)benzoate
(ClPQ4)
By using the dichlorobenzoquinone (4, 0.256 g, 1.25 mmol) and ethyl 4-amin-
obenzoate (0.227 g, 1.375 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:3) as an eluent. Yield: 38%, mp
174–175.6 °C. FTIR (ATR) υ (cm−1): 3250 (NH), 2959, 2924, 2854 (CHaliphatic),
1712 (>C=Oester), 1660 (>C=Oquinone), 1578, 1515, 1496, 1464, 1412, 1377, 1366,
1
1270, 1253, 1210, 1172, 1148, 1100, 1054, 1016. H NMR (500 MHz, CDCl3) δ
(ppm): 8.02 (d, J=8.5 Hz, 2H, CHaromatic), 7.42 (br s, 1H, NH), 7.00 (d, J=8.5 Hz,
2H, CHaromatic), 4.39 (q, J=7.1 Hz, 2H, OCH2), 2.16 (s, 3H, CH3), 2.11 (s, 3H,
CH3), 1.41 (t, J=7.1 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ (ppm): 182.5,
179.6, 166.0 (>C=O), 143.7, 141.5, 138.7, 137.5, 130.0, 126.4, 122.2, 114.1
(Caromatic and Cq), 61.0 (OCH2), 14.4, 13.4, 12.3 (CH3). HRMS(+ESI) m/z calcd for
C17H16ClNO4 [M]+: 333.0768; found: 333.0772.
tert‑Butyl 3‑((2‑chloro‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)
benzoate (ClPQ5)
By using the dichlorobenzoquinone (4, 0.256 g, 1.25 mmol) and tert-butyl 3-amin-
obenzoate (0.266 g, 1.375 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:3) as an eluent. Yield: 51%, mp
172.9–174.4 °C. FTIR (ATR) υ (cm−1): 3225 (NH), 2923, 2853 (CHaliphatic), 1715
(>C=Oester), 1662 (>C=Oquinone), 1578, 1503, 1469, 1441, 1367, 1291, 1250, 1209,
1151, 1107, 1082, 1055. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.81 (d, J=7.6 Hz,
1H, CHaromatic), 7.65 (br s, 1H, NH), 7.39 (t, J=7.5 Hz, 2H, CHaromatic), 7.15 (d,
J=7.4 Hz, 1H, CHaromatic), 2.16 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.61 (s, 9H, CH3).
13C NMR (125 MHz, CDCl3) δ (ppm): 182.5, 179.6, 165.0 (>C=O), 143.8, 139.2,
137.6, 137.1, 132.5, 128.1, 127.6, 126.1, 124.5 (Caromatic and Cq), 81.5 (OC), 28.2,
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Novel plastoquinone analogs containing benzocaine and its…
2139
13.5, 12.3 (CH3). HRMS(+ESI) m/z calcd for C19H20ClNO4 [M]+: 361.1081; found:
361.1089.
tert‑Butyl 4‑((2‑chloro‑4,5‑dimethyl‑3,6‑dioxocyclohexa‑1,4‑dien‑1‑yl)amino)
benzoate (ClPQ6)
By using the dichlorobenzoquinone (4, 0.256 g, 1.25 mmol) and tert-butyl 4-amin-
obenzoate (0.266 g, 1.375 mmol) to the general procedure for the target analog
as a purple solid using petroleum ether/CHCl3 (1:3) as an eluent. Yield: 26%,
mp 168.5–169.3 °C. FTIR (ATR) υ (cm−1): 3217 (NH), 2928 (CHaliphatic), 1707
(>C=Oester), 1661 (>C=Oquinone), 1577, 1513, 1493, 1411, 1367, 1287, 1248,
1211, 1161, 1110, 1101, 1055, 1015. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.96 (d,
J=8.1 Hz, 2H, CHaromatic), 7.42 (br s, 1H, NH), 6.99 (d, J=8.1 Hz, 2H, CHaromatic),
2.16 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.61 (s, 9H, CH3). 13C NMR (125 MHz,
CDCl3) δ (ppm): 182.5, 179.6, 165.1 (>C=O), 143.7, 141.1, 138.7, 137.4,
129.8, 128.0, 122.2, 113.8 (Caromatic and Cq), 81.1 (OC), 28.2, 13.4, 12.3 (CH3).
HRMS(+ESI) m/z calcd for C19H20ClNO4 [M]+: 361.1081; found: 361.1083.
Antimicrobial activity
Determination of minimum inhibitory concentrations (MICs)
Antimicrobial activities against Staphylococcus aureus ATCC 29213, Staphylococ-
cus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Klebsiella pneumo-
niae ATCC 4352, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC
14153, Enterococcus faecalis ATCC 29212, Candida albicans ATCC 10231, Can-
dida parapsilosis ATCC 22019, and Candida tropicalis ATCC 750 were deter-
mined by the broth microdilution technique using the Clinical Laboratory Standards
Institute (CLSI) recommendations [50, 51]. Mueller–Hinton broth for bacteria and
RPMI-1640 medium for the yeast strain were used as the test media. Serial two-
fold dilutions ranging from 2500 to 1.2 µg/mL were prepared in the media. The
inoculum was prepared using a 4–6-h broth culture of each bacterium and 24-h cul-
ture of yeast strains adjusted to a turbidity equivalent of a 0.5 McFarland standard,
diluted in broth media to give a fnal concentration of 5×105 cfu/mL for bacteria
and 0.5×103–2.5×103 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 18–20 h, while the trays containing RPMI-1640
medium were incubated at 35 °C for 46–50 h. The MIC was defned as the lowest
concentration of compound giving complete inhibition of visible growth. As a con-
trol, antimicrobial efects of the solvents were investigated against test microorgan-
isms. The results were evaluated according to the values of the controls.
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N. Bayrak et al.
Acknowledgements This work was fnancially supported by the Scientifc Research Projects Coordina-
tion Unit of Istanbul University-Cerrahpasa (Project number: FBA-2018-29424) for supplying the equip-
ment and materials. The authors thank both the Central Laboratory of the Middle-East Technical Uni-
versity and Advanced Technology Laboratory of the Kütahya Dumlupınar University for high-resolution
mass spectroscopy measurements for mass analyses.
Compliance with ethical standards
Confict of interest The authors declare no confict of interest.
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