Synthesis of nitrogen-containing monoterpenoids with antibacterial activity

Article abstract of DOI:10.1080/14786419.2018.1548456

Incorporation of the Beckmann rearrangement into the presented research resulted in the formation of nitrogen-containing terpenoid derivatives originating from naturally occurring compounds. Both starting monoterpenes and obtained derivatives were subjected to estimation of their antibacterial potential. In the presented study, Staphylococcus aureus was the most sensitive to examined compounds. The Minimal Inhibitory Concentration (MIC) experiments performed on S. aureus demonstrated that the (?)-menthone oxime (?)-8 and (+)-pulegone oxime (+)-13 had the best antibacterial activity among the tested derivatives and starting compounds. Their MIC₉₀ value was 100 μg/mL. The obtained derivatives were also evaluated for their inhibitory activity against bacterial urease. Among the tested compounds, three active inhibitors were found–oxime 14 and lactams (?)-15 and 16 limited the activity of Sporosarcina pasteurii urease with K_i values of 174.3 μM, 43.0 μM and 4.6 μM, respectively. To our knowledge, derivative 16 is the most active antiureolytic lactam described to date.

Full text of DOI:10.1080/14786419.2018.1548456

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Synthesis of nitrogen-containing monoterpenoids
with antibacterial activity
Agata Kozioł, Ewa Grela, Katarzyna Macegoniuk, Agnieszka Grabowiecka &
Stanisław Lochyński
To cite this article: Agata Kozioł, Ewa Grela, Katarzyna Macegoniuk, Agnieszka Grabowiecka &
Stanisław Lochyński (2019): Synthesis of nitrogen-containing monoterpenoids with antibacterial
activity, Natural Product Research, DOI: 10.1080/14786419.2018.1548456
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1548456
Synthesis of nitrogen-containing monoterpenoids with
antibacterial activity
Agata Kozioła,b, Ewa Grelaa, Katarzyna Macegoniuka, Agnieszka Grabowieckaa
a,b
ꢀ
and Stanisław Lochynski
aDepartment of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and
b
Technology, Wrocław, Poland; Institute of Cosmetology, Wrocław College of Physiotherapy,
Wrocław, Poland
ABSTRACT
ARTICLE HISTORY
Received 16 August 2018
Accepted 12 November 2018
Incorporation of the Beckmann rearrangement into the presented
research resulted in the formation of nitrogen-containing terpenoid
derivatives originating from naturally occurring compounds. Both
starting monoterpenes and obtained derivatives were subjected to
estimation of their antibacterial potential. In the presented study,
Staphylococcus aureus was the most sensitive to examined com-
pounds. The Minimal Inhibitory Concentration (MIC) experiments
performed on S. aureus demonstrated that the (ꢀ)-menthone oxime
(2)-8 and (þ)-pulegone oxime (1)-13 had the best antibacterial
activity among the tested derivatives and starting compounds.
Their MIC90 value was 100 mg/mL. The obtained derivatives were
also evaluated for their inhibitory activity against bacterial urease.
Among the tested compounds, three active inhibitors were found –
oxime 14 and lactams (2)-15 and 16 limited the activity of
Sporosarcina pasteurii urease with Ki values of 174.3 mM, 43.0 mM
and 4.6 mM, respectively. To our knowledge, derivative 16 is the
most active antiureolytic lactam described to date.
KEYWORDS
Terpenoid; antibacterial
activity; inhibitors; bacterial
susceptibility
1. Introduction
Terpenes are the largest class of secondary plant metabolites. This group contains
approximately 30,000 compounds, including more than 400 monoterpenes. Their
ꢀ
CONTACT Stanisław Lochynski
stanislaw.lochynski@pwr.edu.pl
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1548456.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
A. KOZIOŁ ET AL.
therapeutic use is associated with the antimicrobial activity. Microorganisms show dif-
ferential susceptibility to compounds of plant origin. Gram-positive bacteria are usually
more affected by monoterpenes than Gram-negative strains (Kozioł et al. 2014; Singh
and Sharma 2015).
Further search for compounds with strong biological activity among monoterpene
derivatives is a promising research goal. It is known that incorporation of the oxime
group into naturally occurring compounds may enhance their antimicrobial activity
(Kozłowska et al. 2017). Nitrogen-containing terpene derivatives (including lactams,
oximes and amines) have also been described with diverse biological properties. To
date, a limited but promising data have been published. Within the terpenoids con-
taining an N-OH moiety, effective anticancer (Kahnt et al. 2018) and antimicrobial
agents (Hertiani et al. 2010; Grishko et al. 2014) were found. Chemical modifications of
monoterpenoids create a possibility of obtaining novel inhibitors of various enzymes
(Gajcy et al. 2010). Librowski et al. (2001) described the strong local anaesthetic activ-
ity of hydroxyamino carane derivatives. Thus, both the increase in synthesis yield and
the growing scientific knowledge of compounds substituted with nitrogen-containing
groups provide great hope for the development of new therapeutic drugs in various
medical fields (Adams and Robertson 1997).
Despite the long-established knowledge of natural monoterpenes biological activity,
the properties of their nitrogen derivatives remain vastly unknown. In this study, we
describe the chemistry of a group of nitrogen-containing monoterpene derivatives that
includes 9 oximes and 3 lactams. The research is complemented by antibacterial studies
against Gram-negative Escherichia coli, Gram-positive Bacillus subtilis and S. aureus strains.
The antiureolytic characteristics against Sporosarcina pasteurii urease are also presented, as
monoterpenoid derivatives constitute a novel group of inhibitors of this enzyme.
2. Results and discussion
2.1. Chemistry
The synthesis of oximes was carried out in an ethanolic solution of hydroxylamine
hydrochloride in the presence of pyridine as a hydrogen chloride trapping agent. This
method was chosen to synthesize crystalline oximes due to the ease of isolation of
the post-reaction products after dilution with water. Oximes were the starting com-
pounds used in the Beckmann rearrangement to create lactams (Table S1, Figure 1).
In the Beckmann method of lactam synthesis from monoterpene ketones modified
_
by Zabza et al. (1972), a strongly acidic medium is not used. We introduced the p-tol-
uenesulfonyl chloride reagent in this reaction, for the “second type” rearrangement.
Besides the starting monoterpenes 1, 3–7 (Table S1), the compound t-butylcyclohexa-
none 2 was incorporated into the presented research. It was chosen as the analogue of
monoterpenoids with a cyclohexane ring that contains 10 carbon atoms in its structure.
2.2. Biological activity
Monoterpene ketones chosen as the starting compounds in the presented study have
already been described with negligible antibacterial activity (Gallucci et al. 2009;

NATURAL PRODUCT RESEARCH
3
Figure 1. Synthesis of oxime (2)-8 and lactam (2)-15 from (ꢀ)-menthone (2)-1.
Rasoul et al. 2012; Kozioł et al. 2014). This characteristics was confirmed in our
research group (Kozioł et al. 2018), we observed that nearly all monoterpene ketones
had no bacteriostatic activity up to 250 mg/mL under standard, CLSI (2012) approved
conditions. None of compounds 1–7 affected the growth of Escherichia coli. Only
(ꢀ)-carvone (2)-4 and (þ)-dihydrocarvone (1)-5 were characterised with a Minimal
Inhibitory Concentration (MIC50) of 250 mg/mL against Staphylococcus aureus and
Bacillus subtilis.
Subsequently, all synthesized nitrogen-containing derivatives were evaluated for
their antibacterial activity (Table S2, Figure 2). Some chemical modifications of mono-
terpene ketones resulted in a significant increase in antibacterial activity. Nearly all
oximes with isopropyl and isopropene substitutions ((2)-8, (2)-11, (1)-11, (1)-13
and 16, with the exception of (1)-12) inhibited the growth of Gram-positive bacteria.
(2)-8, (2)-11 and (1)-11, namely (ꢀ)-menthone and carvone oximes, were effective
against both B. subtilis and S. aureus with MIC90 values of 150 mg/mL or lower. The
MIC90 value of (2)-8 against S. aureus was calculated as 100 mg/mL. In the presented
research, S. aureus was more susceptible to monoterpene oximes than the other two
bacterial strains. Compounds ineffective against B. subtilis and E. coli – (1)-13 and 16 –
restricted the growth of S. aureus, with MIC90 values of 100 and 150 mg/mL, respectively.
Additionally, compounds (2)-8 and (1)-13 were the most effective among tested nitro-
gen derivatives, both demonstrating a MIC50 = 25mg/mL and a MIC90 = 100 mg/mL
against S. aureus. On the other hand, carvone oximes ((2)-11, (1)-11) were the only
compounds effective against all tested bacteria, with MIC values considerably higher for
Gram-negative E. coli (250mg/mL) than for both Gram-positive strains. A similar pattern
of susceptibility to monoterpene derivatives was previously observed and described
(Cristani et al. 2007).
In addition to the antibacterial characteristics presented above, the inhibitory prop-
erties of monoterpene ketones and obtained derivatives were evaluated against
Sporosarcina pasteurii urease (Table S3, Figure 2). To date, only a limited group of
monoterpenoid urease inhibitors has been described in the literature. First, both car-
vone stereoisomers were discovered to limit the activity of soil urease, consisting of a
variety of enzyme isoforms (Efimia et al. 2014). Further experiments conducted in our
research group confirmed the effectiveness of (ꢀ)-carvone ((2)-4) against bacterial
urease (Ki = 418.6 lM). We characterized monoterpenoid ketones and their derivatives

4
A. KOZIOŁ ET AL.
Figure 2. Structures and bioactivity of the most potent compounds. Presented Minimal Inhibitory
Concentration (MIC90) was evaluated against Staphylococcus aureus cells and inhibitory constant Ki
calculated for purified Sporosarcina pasteurii urease. The characteristics of remaining compounds is
presented in the Supplementary file in the Tables S1–S3.
as reversible inhibitors with a non-competitive mechanism of activity (Kozioł et al.
2018). In the presented article, three novel urease inhibitors were found among the
tested nitrogen-containing derivatives. Compounds 14, (2)-15 and 16 effectively lim-
ited the activity of purified S. pasteurii urease. Their Ki values were calculated to be
174.3 mM, 43.0 mM and 4.6 mM, respectively. They were noticeably lower than the affin-
ity constants obtained for the monoterpene ketone substrates. The inhibitory potency
of the most active compound 16 was comparable to the standard, FDA-approved
urease inhibitor – acetohydroxamic acid (AHA) characterized with Ki = 3.4 mM
(Macegoniuk et al. 2016). Moreover, two of the most active derivatives share a lactam
moiety and are, to our knowledge, the first group of lactams that actively inhibit bac-
terial urease at micromolar concentrations. Limited information on lactam-containing
urease inhibitors can be found in the literature. In general, such compounds are
described as inactive or weak urease inhibitors. A recently published paper (Mermer
et al. 2017) describes the weak antiureolytic characteristics of a group of highly

NATURAL PRODUCT RESEARCH
5
substituted piperazine-azole hybrids against jack bean urease. Antiureolytic properties
of the examined compounds may be a promising factor for treating a variety of infec-
tions caused by pathogenic, ureolytically active microorganisms.
3. Experimental section
The general chemistry of presented research and methodology of antibacterial experi-
ments were presented in the Supplementary Material.
4. Conclusion
In the first stage of the presented reaction, nine pure oximes with a yield of 88–98%
were synthesized. In the subsequent Beckmann rearrangement, three lactams with a
yield of 96% were obtained from the selected oximes.
Incorporation of nitrogen-containing groups into the structures of monoterpenoids
allowed achieving a significant improvement in their antibacterial activity. The micro-
biological tests demonstrated that (2)-8 and (1)-13 were the best antibacterial com-
pounds among the tested derivatives. Both compounds were characterized with a
MIC50 = 25 mg/mL and a MIC90 = 100 mg/mL against S. aureus.
Obtained derivatives were also screened for their antiureolytic characteristics
against S. pasteurii urease. Compound 16 was found to be the most active of the
examined derivatives, with a Ki = 4.6 mM. To our knowledge, this is the first lactam
reported to inhibit the activity of bacterial urease with high effectiveness.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
English editing was funded by the Wroclaw Centre of Biotechnology Program, The Leading
National Research Centre (KNOW) for the years 2014–2018 (know.wroc.pl).
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