Synthesis of a pyrrolidine derivative of a carvotacetone and monoterpenes for anti-methicillin-resistant Staphylococcus aureus and anti-cryptococcal properties

Article abstract of DOI:10.1080/14786419.2020.1833201

Monoterpene derivatives are of great biological relevance in the pharmaceutical industry. In the present study, pyrrolidine derivative of a carvotacetone, 3-O-benzylcarvotacetone (1), and selected monoterpenes (3-hydroxy-2-isopropyl-5-methyl-p-benzoquinone (3) and cis-piperitol (5)) were prepared to provide (R)-1-(4-(benzyloxy)-5-isopropyl-2-methylcyclohexa-1,3-dien-1-yl)-pyrrolidine (2), 2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl acetate (4), cis-3-hydroxypiperitone (6) and carvacrol (7). Structure of 2 was determined based on NMR and HRMS spectral data. Compound 4 exhibited activity against fungi Cryptococcus neoformans with an IC₅₀ value of < 0.8μg/mL. In addition, this compound 4 had an IC₅₀ value of 14.97μg/mL against methicillin resistant Staphylococcus aureus bacteria. Previous to the current study, both compound 6 and 7 had been reported to have anti-microbial and anti-fungal activities.

Full text of DOI:10.1080/14786419.2020.1833201

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Synthesis of a pyrrolidine derivative of a
carvotacetone and monoterpenes for anti-
methicillin-resistant Staphylococcus aureus and
anti-cryptococcal properties
Veronica M. Masila , Albert J. Ndakala , Jacob O. Midiwo , Robert
Byamukama , Rahab W. Kamau , Mallika Kumarihamy & Ilias Muhammad
To cite this article: Veronica M. Masila , Albert J. Ndakala , Jacob O. Midiwo , Robert
Byamukama , Rahab W. Kamau , Mallika Kumarihamy & Ilias Muhammad (2020): Synthesis
of a pyrrolidine derivative of a carvotacetone and monoterpenes for anti-methicillin-resistant
Staphylococcusꢀaureus and anti-cryptococcal properties, Natural Product Research, DOI:
10.1080/14786419.2020.1833201
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1833201
Synthesis of a pyrrolidine derivative of a carvotacetone
and monoterpenes for anti-methicillin-resistant
Staphylococcus aureus and anti-cryptococcal properties
Veronica M. Masilaa, Albert J. Ndakalab, Jacob O. Midiwob, Robert Byamukamaa,
Rahab W. Kamauc, Mallika Kumarihamyd and Ilias Muhammadd
aDepartment of Chemistry, College of Natural Sciences, Makerere University, Kampala, Uganda;
bDepartment of Chemistry, School of Physical Sciences, University of Nairobi, Nairobi, Kenya;
cDepartment of Chemistry, College of Natural and Applied Sciences, University of Dar es Salaam, Dar
d
es Salaam, Tanzania; National Centre for Natural Products Research, Research Institute of
Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Mississippi, MS, USA
ABSTRACT
ARTICLE HISTORY
Received 14 July 2020
Accepted 16 September 2020
Monoterpene derivatives are of great biological relevance in the
pharmaceutical industry. In the present study, pyrrolidine deriva-
tive of a carvotacetone, 3-O-benzylcarvotacetone (1), and selected
monoterpenes (3-hydroxy-2-isopropyl-5-methyl-p-benzoquinone
(3) and cis-piperitol (5)) were prepared to provide (R)-1-(4-(benzy-
loxy)-5-isopropyl-2-methylcyclohexa-1,3-dien-1-yl)-pyrrolidine (2),
2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl acetate (4),
cis-3-hydroxypiperitone (6) and carvacrol (7). Structure of 2 was
determined based on NMR and HRMS spectral data. Compound 4
exhibited activity against fungi Cryptococcus neoformans with an
IC50 value of < 0.8 mg/mL. In addition, this compound 4 had an
IC50 value of 14.97 mg/mL against methicillin resistant
Staphylococcus aureus bacteria. Previous to the current study,
both compound 6 and 7 had been reported to have anti-micro-
bial and anti-fungal activities.
KEYWORDS
Monoterpenes; 2-Isopropyl-
5-methyl-3,6-dioxocyclo-
hexa-1,4-dien-1-yl acetate;
Carvacrol; Cis-3-
hydroxypiperitone; Anti-
MRSA; Anti-cryptococcal
1. Introduction
Scientists estimate that there are over 5 million fungal species worldwide with close
to 300 species causing disease to humans (Perfect et al. 2010). Several species of fungi
are known to cause invasive infections, which lead to death of about one million
CONTACT Veronica Masila
masilaveronica@gmail.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1833201.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
V. M. MASILA ET AL.
people every year worldwide (Brown et al. 2012; Krummenauer et al. 2019). One mil-
lion cases of meningitis are reported annually worldwide with over half a million
deaths estimated in endemic areas of sub-Saharan Africa (Mpoza et al. 2018; Park
et al. 2009). The key treatment agents include polyenes, azoles and flucytosine. The
emergence of drug resistance to the currently available anti-fungal drugs severely lim-
its therapy because of the few treatment options available in the market (Perlin
et al. 2017).
On the other hand, methicillin-resistant bacteria Staphylococcus aureus leads to bac-
teraemia, skin and soft tissue infections, osteomyelitis and endocarditis (Turner et al.
2019). The first medicament (1941) in the treatment of S. aureus infections involved
use of penicillin, a beta lactam antibiotic on which resistance was immediately noted
on introduction (North and Christie 1946). Emergence of resistant strains led to the
development of methicillin in 1960s, a semisynthetic analogue of penicillin and in less
than a decade, methicillin resistant strains of S. aureus (MRSA) were reported (Parker
1970). MRSA infections are majorly found in hospitals and communities, with several
anti-microbials developed and others still under clinical trials such as daptomycin
(Steenbergen et al. 2005), dalbavancin (Chen et al. 2007), linezolid (Brickner et al.
2008), ceftobiprole (Dauner et al. 2010), telavancin (Damodaran and Madhan 2011),
ceftaroline (Laudano 2011), oritavancin (Zhanel et al. 2012) and tedizolid (Zhanel et al.
2015). Resistance of these pathogens to multiple antibiotics has activated the search
and discovery of novel antibiotic agents with better activity and less side effects.
Plants contain thousands of useful secondary metabolites of medicinal value includ-
ing essential oils composite of many volatile and odourless compounds (Boye et al.
2020). The essential oils constitute of terpenes which exist as hemiterpenes, monoter-
penes or sesquiterpenes, some of which have been reported to be active against
fungi, Gram-positive and Gram-negative bacteria (Bhatti et al. 2014; Loomis and
Croteau 1980). For instance, carvacrol (7), an aromatic monoterpenoid has been
reported to demonstrate anti-fungal activities against Candida albicans hyphae and
Cryptococcus neoformans (Teixeira et al. 2020; Vale-Silva et al. 2010), anti-protozoal
properties against P. falciparum chloroquine sensitive strain (Can Baser 2008), anti-oxi-
dative properties (Prieto et al. 2007). In addition, carvacrol (7) has shown anti-microbial
efficacy against several bacteria such as methicillin resistant Staphylococcus aureus and
E. coli (Tabanca et al. 2001).
The strong anti-bacterial activities of the aerial extracts of Foeniculum vulgare
against Staphylococcus saprophyticus and Escherichia coli were attributed to the pres-
ence of several monoterpenoid metabolites such as cis-3-hydroxypiperitone (6), a
monoterpenoid carvotacetone (Zellagui et al. 2011). On the other hand, nitrogenous
derivatives including pyrrolidine, morpholine, piperazine and piperidine derivatives are
also constituents of secondary metabolites and synthetic bioactive molecules and is
deemed as an important pharmacophore in the drug industry (Bellina and Rossi 2006;
Enyedy et al. 2001).
In continuation of our previous report on synthesis of 3-O-benzyl-carvotacetone
and 3-hydroxy-2-isopropyl-5-methyl-p-benzoquinone (Masila et al. 2020), in this paper,
we describe a strategy to synthesise (R)-1-(4-(benzyloxy)-5-isopropyl-2-methylcyclo-
hexa-1,3-dien-1-yl)-pyrrolidine (2), 2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl

NATURAL PRODUCT RESEARCH
3
acetate (4), cis-3-hydroxypiperitone (6) and carvacrol (7) that could be of anti-microbial
potential. Full spectroscopic characterization and HRMS of the synthesized nitrogenous
derivative (2) is discussed together with the anti-cryptococcal and anti-MRSA activities
of 2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl acetate (4).
2. Results and discussion
2.1. Synthesised compounds
The synthesis began from 3-O-benzyl-carvotacetone (1) as synthesized in Masila et al.
(2020). Compound 1 was subjected to N-alkylation in presence of pyrrolidine to pro-
vide (R)-1-(4-(benzyloxy)-5-isopropyl-2-methylcyclohexa-1,3-dien-1-yl)-pyrrolidine (2) in
38% (w/w) yield. Success of this reaction was confirmed from both NMR (Figure S2-S7)
and HRMS (Figure S8) spectroscopic data. Compound 2 had m/z 312.2249 [M þ H] cor-
responding to a molecular formula of C21H29NO. From the 13C-NMR, the carbonyl sig-
nal at d 198.1 of the 3-O-benzyloxycarvotacetone had disappeared and additional
methylene carbons were observed at d 62.5 (C-11/14) and d 29.4 (C-12/13) as envi-
sioned in the DEPT spectrum, a clear indication that the pyrrolidine moiety had been
incorporated in the compound leading to the formation of an enamine. The
deshielded methylene carbon at d 62.5 confirmed presence of a heteroatom (N). In
1
the H-NMR spectrum, the deshielded methylene protons appeared at d 4.71 (4H, H-
11/14) and the other set resonated at d 1.28 (4H, H-12/13). The presence of a high
field deshielded olefinic proton resonating at d 8.13 (H-3) with strong HMBC correla-
tions with carbon one and two (H-3 ! C-1, C-2) confirmed formation of an enamine.
Acylation of 3-hydroxy-2-isopropyl-5-methyl-p-benzoquinone (3) in acetic anhydride
(Ranu et al. 2003), provided 4 in 60% (w/w) yield. In addition to the peaks observed in
compound 3 as described by Masila et al. (2020), a carbonyl peak of the acyl group
(C-10) resonated at d 168.1 and the methyl (C-20) carbon at d 20.4 ppm with its protons
observed at d 2.37 (H-20, s, 3H) ppm integrating for three protons appearing as a sing-
let (Figure S10 and S11).The observed values were in agreement with the literature
values as reported by Joseph-Nathan and Burgueno-Tapia (2000) (Table S9).
Riley oxidation of cis-piperitol (5) in the presence of SeO2 and 1,4-dioxane was also
carried out to obtain cis-3-hydroxypiperitone (6) in 40% (w/w) yield as a reddish-
1
brown oil. The 13C NMR and H NMR spectrum (Figure S13 and S14) had a close simi-
larity to compound 5 (Masila et al. 2020). The major difference was presence of an
oxy-methine carbon resonating at d 69.1 (C-4) with an oxy-methine proton observed
as a multiplet at d 4.38 ppm (H-4). The observed and literature values were in agree-
ment as reported by Zellagui et al. (2011) (Table S12). Cis-3-hydroxypiperitone has pre-
_
viously been obtained through biotransformation of (-)-(R-)-a-phellandrene (Is¸can et al.
2012) and previously isolated as a secondary metabolite from the aerial parts of
Foenicium vulgare (Zellagui et al. 2011).
In addition, cis-piperitol (5) synthesised from piperitone (Masila et al. 2020) was suc-
cessively subjected to Riley oxidation to yield carvacrol (7), 33% (w/w) yield. The 13C
NMR (Figure S17) spectrum had a total of 10 signals just like the starting material (5)
(Masila et al. 2020) with one signal at d 24.1 integrating for two methyl carbons (C-9/
C-10) and a methine carbon for the isopropyl unit resonating at d 33.7 (C-8) ppm. An

4
V. M. MASILA ET AL.
additional methyl carbon attached to an aromatic ring was observed at d 15.4 (C-7)
ppm. However, instead of two olefinic carbons, the compound seemed to have arom-
atized with three sp2 hybridized carbons appearing at d 113.0 (C-6), 118.8 (C-4) and
130.9 (C-3). An oxygenated sp2 hybridized quaternary carbon appeared at d 153.6 (C-
1) with other two quaternary sp2 hybridized carbons appearing at d 120.9 (C-2) and
148.5 (C-5) as supported by DEPT spectrum (Figure S18). The isopropyl unit was not-
ably observed, the methine proton appeared at d 2.86 (m, 1H, H-8) and the methyl
protons at d 1.26 (d, J ¼ 5 Hz, 6H, H-9/10). The methyl protons at position C-7 attached
to the aromatic ring appeared at d 2.26, observed as a sharp singlet. In addition, three
aromatic protons were observed at d 6.70 (s, 1H, H-6), d 6.78 (d, 1H, H-4) and d 7.08
(d, 1H, H-3) (Figure S16). These observed values were in agreement with the literature
values (Tang et al. 2011) (Table S15).
2.2. Anti-microbial activities
Compound 4, an acylated benzoquinone monoterpenoid, exhibited anti-bacterial activ-
ity against MRSA (ATCC 33591) (IC50 value of 14.97 mg/mL) with the benzoquinone (3)
showing no activity towards the same pathogen (IC50 value > 20 mg/mL). The standard
drug cefotaxime, meropenem and methicillin had IC50 values of 10.50, 2.63 and
17.57 mg/mL (Figure S19). In addition, compound 4 exhibited anti-fungal activity
towards C. neoformans (ATCC8 90113) with an IC50 value of < 0.80 mg/mL while the
standard drug amphotericin B had an IC50 value of 0.53 mg/mL (Figure S20).
Furthermore, compound 4 had an IC50 > 20 mg/mL against the yeast Candida albicans
(ATCC 90028) and Aspergillus fumigatus (ATCC 204305), and the bacteria E. coli (ATCC
35218), Pseudomonas aeruginosa (ATCC 27853) and VRE.
3. Experimental
3.1. Instrumentation and reagents
All reagents and other chemicals were purchased from Loba Chemie, Merck and Santa
Cruz and used without further purification. Reactions requiring anhydrous conditions
were performed under nitrogen. The reactions were done in glassware that were dried
in an oven (110 ꢀC) for at least 2 hours and allowed to cool under nitrogen. Solvents
for column chromatography were distilled in the laboratory in glass apparatus.
Chromatographic separation was done on silica gel (60-120 mesh). Thin layer chroma-
tography was performed on Merck silica gel (60 F254) TLC plates and visualised under
254 nm UV lamp.
1H-NMR (500 MHz) and 13C-NMR (125 MHz) were recorded on an Agilent DD2-500
NMR spectrometer (Santa Clara, CA, USA) with tetramethylsilane as an internal stand-
ard. The high-resolution mass spectrum was acquired via LCMS (Agilent).
3.2. Synthesis of compound 2, 4, 6 and 7
The compounds were synthesised as represented in Figure 1

NATURAL PRODUCT RESEARCH
5
Figure 1. Synthetic route to compounds 2, 4, 6 and 7.
3.2.1. N-alkylation of 3-O-benzyl-carvotacetone (1)
Pyrrolidine (0.1 mL) was added to a solution of 3-O-benzyl-carvotacetone 1, (160 mg,
6.2 ꢁ 10ꢂ4 mmol) in toluene (10 mL) in a 50 mL round bottomed flask. A catalytic
amount of p-toluene sulfonic acid (16 mg, 8.41 ꢁ 10ꢂ5 mmol) was added to the reac-
tion mixture with constant stirring and the mixture refluxed at 80 ꢀC for 72 hours.
Formic acid (5 mL) was then added and mixture refluxed for six hours then neutralized
with NaOH after cooling. Work up was done by concentrating the mixture on a rotary
evaporator. The residue was diluted with water (5 mL) and then extracted with CH2Cl2
(20 mL twice). The organic phase was dried (Na2SO4), filtered and concentrated under
reduced pressure and purified on column chromatography by eluting with 100%
dichloromethane to afford a reddish brown oil of 2 (60 mg, 38% (w/w) yield).
3.2.2. Acylation of 3-Hydroxy-2-isopropyl-5-methyl-p-benzoquinone (3)
A
mixture
of
3-hydroxy-2-isopropyl-5-methyl-p-benzoquinone
3,
(10 mg,
5.5 ꢁ 10ꢂ5 mmol) and acetic anhydride (approximately 0.1 mL) in a 50 mL round bot-
tomed flask was heated at 80 ꢀC for 3 hours. The acetic anhydride was evaporated and
the product extracted with diethyl ether (5 mL) then purified by column chromatog-
raphy on silica gel with 40% dichloromethane in n-hexane to provide 4 (6 mg, 60%
(w/w) yield).
3.2.3. Riley oxidation of cis-piperitol (5)
Cis-piperitol 5, (4 g, 0.026 mol) was dissolved in a mixture of formic acid (2 mL) in
dioxane (50 mL) in a 100 mL flask. Selenium dioxide (2.5 g, 0.023 mol) was added to
the reaction flask and refluxed at 80 ꢀC with constant stirring for three days. The mix-
ture was then concentrated under reduced pressure. The work up was accomplished
by adding water (30 mL) and extraction done with dichloromethane (2 ꢁ 200 mL). The
combined extract was dried over Na2SO4, filtered and then concentrated on a rotary
evaporator. The residue was purified on column chromatography with 80% dichloro-
methane in n-hexane to obtain reddish brown oil of 7 (1 g, 33% (w/w) yield). In add-
ition, using the same procedure, cis-piperitol, 5, (200 mg, 1.31 ꢁ 10ꢂ3 mmol) was also

6
V. M. MASILA ET AL.
subjected to Riley oxidation and purification carried out with dichloromethane on the
column chromatography to yield 6 (80 mg, 40% (w/w) yield).
3.3. Spectroscopic data
(R)-1-(4-(Benzyloxy)-5-isopropyl-2-methylcyclohexa-1,3-dien-1-yl)-pyrrolidine
(2):
1
Reddish brown oil. H and 13C NMR (CDCl3, 500 and 125 MHz): See Table S1, Figure
S2-S7: m/z 312.2249 [M þ H] See Figure S8.
3.4. Anti-microbial assay
All the organisms in the antimicrobial assays were obtained from the American Type
Culture Collection (Manassas, VA) and included yeasts C. albicans (ATCC 90028), the
fungi C. neoformans (ATCC 90113) and A. fumigatus (ATCC 204305), and the bacteria
MRSA (ATCC 33591), E. coli (ATCC 35218), P. aeruginosa (ATCC 27853) and VRE. Drug
control cefotaxime, meropenem and methicillin for bacteria and amphotericin B for
yeasts and fungi were included in each assay. Susceptibility testing was performed at
the National Centre for Natural Products Research, University of Mississippi, as
described (Samoylenko et al. 2009).
Acknowledgements
The authors sincerely thank Dr. Siddharth K. Tripathi, Dr. Shabana Khan and Mr. John Trott for
assistance in anti-microbial assays. This work was financially supported by International Science
Programme, Uppsala University, Sweden through (ISP)-KEN-02 project, Department of Chemistry,
University of Nairobi, and partially by the USDA ARS Specific Cooperative Agreement No. 58-
6408-1-603. Veronica M. Masila acknowledges DAAD-NAPRECA for the Ph.D. scholarship.
Disclosure statement
Authors declare no conflict of interest.
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