Pharmacodynamic Functions of Synthetic Derivatives for Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis

Article abstract of DOI:10.3389/fmicb.2020.551189

Drug resistant bacteria have emerged, so robust methods are needed to evaluate combined activities of known antibiotics as well as new synthetic compounds as novel antimicrobial agents to treatment efficacy in severe bacterial infections. Marine natural products (MNPs) have become new strong leads in the drug discovery endeavor and an effective alternative to control infections. Herein, we report the bioassay guided fractionation of marine extracts from the sponges Lendenfeldia, Ircinia, and Dysidea that led us to identify novel compounds with antimicrobial properties. Chemical synthesis of predicted compounds and their analogs has confirmed that the proposed structures may encode novel chemical structures with promising antimicrobial activity against the medically important pathogens. Several of the synthetic analogs exhibited potent and broad spectrum in vitro antibacterial activity, especially against the Methicillin-resistant Staphylococcus aureus (MRSA) (MICs to 12.5 μM), Mycobacterium tuberculosis (MICs to 0.02 μM), uropathogenic Escherichia coli (MIC o 6.2 μM), and Pseudomonas aeruginosa (MIC to 3.1 μM). Checkerboard assay (CA) and time-kill studies (TKS) experiments analyzed with the a pharmacodynamic model, have potentials for in vitro evaluation of new and existing antimicrobials. In this study, CA and TKS were used to identify the potential benefits of an antibiotic combination (i.e., synthetic compounds, vancomycin, and rifampicin) for the treatment of MRSA and M. tuberculosis infections. CA experiments indicated that the association of compounds 1a and 2a with vancomycin and compound 3 with rifampicin combination have a synergistic effect against a MRSA and M. tuberculosis infections, respectively. Furthermore, the analysis of TKS uncovered bactericidal and time-dependent properties of the synthetic compounds that may be due to variations in hydrophobicity and mechanisms of action of the molecules tested. The results of cross-referencing antimicrobial activity, and toxicity, CA, and Time-Kill experiments establish that these synthetic compounds are promising potential leads, with a favorable therapeutic index for antimicrobial drug development.

Full text of DOI:10.3389/fmicb.2020.551189

ORIGINAL RESEARCH
published: 27 November 2020
doi: 10.3389/fmicb.2020.551189
Pharmacodynamic Functions of
Synthetic Derivatives for Treatment
of Methicillin-Resistant
Staphylococcus aureus (MRSA) and
Mycobacterium tuberculosis
Mojdeh Dinarvand1,2,3 , Malcolm P. Spain1 and Fatemeh Vafaee3
*
*
Edited by:
Mirian A. F. Hayashi,
1 School of Chemistry, Faculty of Science, The University of Sydney, Sydney, NSW, Australia, 2 Department of Infectious
Diseases and Immunology, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia, 3 School of
Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW, Australia
Federal University of São Paulo, Brazil
Reviewed by:
Sophia Vourli,
Drug resistant bacteria have emerged, so robust methods are needed to evaluate
combined activities of known antibiotics as well as new synthetic compounds as novel
antimicrobial agents to treatment efficacy in severe bacterial infections. Marine natural
products (MNPs) have become new strong leads in the drug discovery endeavor and
an effective alternative to control infections. Herein, we report the bioassay guided
fractionation of marine extracts from the sponges Lendenfeldia, Ircinia, and Dysidea that
led us to identify novel compounds with antimicrobial properties. Chemical synthesis of
predicted compounds and their analogs has confirmed that the proposed structures
may encode novel chemical structures with promising antimicrobial activity against the
medically important pathogens. Several of the synthetic analogs exhibited potent and
broad spectrum in vitro antibacterial activity, especially against the Methicillin-resistant
Staphylococcus aureus (MRSA) (MICs to 12.5 µM), Mycobacterium tuberculosis (MICs
to 0.02 µM), uropathogenic Escherichia coli (MIC o 6.2 µM), and Pseudomonas
aeruginosa (MIC to 3.1 µM). Checkerboard assay (CA) and time-kill studies (TKS)
experiments analyzed with the a pharmacodynamic model, have potentials for in vitro
evaluation of new and existing antimicrobials. In this study, CA and TKS were
used to identify the potential benefits of an antibiotic combination (i.e., synthetic
compounds, vancomycin, and rifampicin) for the treatment of MRSA and M. tuberculosis
infections. CA experiments indicated that the association of compounds 1a and 2a
with vancomycin and compound 3 with rifampicin combination have a synergistic effect
against a MRSA and M. tuberculosis infections, respectively. Furthermore, the analysis of
TKS uncovered bactericidal and time-dependent properties of the synthetic compounds
University General Hospital Attikon,
Greece
Nagendran Tharmalingam,
Rhode Island Hospital, United States
*Correspondence:
Mojdeh Dinarvand
mdin7586@uni.sydney.edu.au;
m.dinarvand@unsw.edu.au
Fatemeh Vafaee
f.vafaee@unsw.edu.au
Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Received: 16 April 2020
Accepted: 05 November 2020
Published: 27 November 2020
Citation:
Dinarvand M, Spain MP and
Vafaee F (2020) Pharmacodynamic
Functions of Synthetic Derivatives
for Treatment of Methicillin-Resistant
Staphylococcus aureus (MRSA)
and Mycobacterium tuberculosis.
Front. Microbiol. 11:551189.
doi: 10.3389/fmicb.2020.551189
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November 2020 | Volume 11 | Article 551189

Dinarvand et al.
Pharmacodynamic Functions of Synthetic Derivatives
that may be due to variations in hydrophobicity and mechanisms of action of the
molecules tested. The results of cross-referencing antimicrobial activity, and toxicity,
CA, and Time-Kill experiments establish that these synthetic compounds are promising
potential leads, with a favorable therapeutic index for antimicrobial drug development.
Keywords: marine natural products, antibiotic resistance (AMR), time-kill studies, checkerboard assay,
methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis
INTRODUCTION
ziconotide (ω-conotoxin MVIIA) in 2004 to alleviate chronic
pain (McGivern, 2007) and sea squirt metabolite trabectedin in
The first isolation of methicillin-resistant Staphylococcus aureus
(MRSA) and Mycobacterium tuberculosis was reported in
1882 and 1961, respectively (Enright et al., 2002; Cambau
and Drancourt, 2014). The increasing prevalence of antibiotic
resistance and lack of clinically approved treatments has made
these infections a new challenge for infection control teams
worldwide (Enright et al., 2002; Zaman, 2010; Diallo et al.,
2018; Yang et al., 2018). This determined existing antibiotic
therapies require substantial improvements in efficacy, in order
to overcome severe chronic bacterial infections (Broussou et al.,
2019; Dinarvand and Peter Spain, 2020).
Marine ecosystems have long been a rich source of bioactive
natural products, in the search for interesting molecules and
novel therapeutic agents (Molinski et al., 2009; Hughes and
Fenical, 2010; Indraningrat et al., 2016; Mehbub et al., 2016;
Schroeder et al., 2018; El-Demerdash et al., 2019) over the last
70 years (Newman and Cragg, 2016; Blunt et al., 2017, 2018;
Pye et al., 2017). The preclinical pharmacology of seventy-five
compounds isolated from marine organisms was reported to have
biological activities (Mayer et al., 2017).
The most prolific marine organisms are sponges (Indraningrat
et al., 2016), and the oldest metazoans on earth belong the phylum
Porifera (Matobole et al., 2017). The Demospongiae, being most
abundant class of Porifera, represent 83% of described species
(Van Soest et al., 2012; Matobole et al., 2017) and have the largest
number of bioactive compounds (El-Damhougy et al., 2017).
Member of the genus Lendenfeldia are a known source of
sulfated sterols (Radwan et al., 2007). The Lendenfeldia species
metabolites have anti-HIV, anti-tumor (Liu et al., 2008), anti-
inflammatory, and antifouling (Sera et al., 1999) activities, but
they are believed to lack antimicrobial activity (Radwan et al.,
2007). Secondary metabolites of the genus Ircinia and Dysidea
have been well studied for antibacterial activity (Thakur and
Anil, 2000; Mohamed et al., 2008; Belma Konuklugil, 2015; El-
Damhougy et al., 2017).
2007 for treatment of soft-tissue sarcoma (Gordon et al., 2016).
Marine natural products (MNPs) have also displayed exceptional
potential as anticancer therapeutics (El-Damhougy et al., 2017).
Interest in MNPs has continued to grow since (Newman and
Cragg, 2016; Blunt et al., 2017, 2018), spurred in part by the
spread of antimicrobial resistant pathogens and the need for
new drugs to combat them (Indraningrat et al., 2016). In search
of novel antimicrobial agents, we previously elucidated five
active component structures using high resolution and tandem
mass spectrometry (MS) which resulted in a series of potential
structures for new, bioactive amine natural products (Dinarvand
and Peter Spain, 2020). Generally, successful antimicrobial
therapy depends on complex interactions between different
factors like the infecting agent, host and the dose-response
(Mueller et al., 2004).
To validate the proposed structures and explore the
potential of this compound class more broadly, analogs of
five general structures were synthesized and evaluated as
potential antimicrobial agents against the medically important
microorganisms: MRSA, M. tuberculosis, UroPathogenic
Escherichia coli (UPEC), and Pseudomonas aeruginosa.
Checkerboard assay (CA) (Odds, 2003) was used to measure
interaction between inhibitory agents categorized into synergy,
indifference or antagonism. Furthermore, time-kill studies (TKS)
(Broussou et al., 2019) experiment performed to study dynamic
interpretation of drug-bacteria interactions. Also, combining
existing antimicrobials has been successfully applied to optimize
dosing strategies, extend the spectrum, minimize the emergence
of resistance, enhance the bactericidal activity, manage an
infection and prevent treatment failures (Foerster et al., 2016;
Broussou et al., 2019).
MATERIALS AND METHODS
In fact, the first ‘drugs from the sea’ were only approved
in the early 2000s. They included: the cone snail peptide
General
Chemical reagents were purchased from BDH Chemicals and
Sigma Aldrich (Castle Hill, Sydney, Australia) and used as
supplied unless otherwise indicated.
Abbreviations: A549, human alveolar epithelial cells; AIMS, Australian Institute
for Marine Science; DMEM, Dulbecco’s Modified Eagle’s medium; HEK293,
human embryonic kidney cells 293; Hep-G2, human hepatocellular carcinoma
cells; HPLC, high-performance liquid chromatography; HTS, high-throughput
screening; MDCK, Madin-Darby canine kidney epithelial cells; MIC, minimum
inhibitory concentration; MRSA, methicillin resistant Staphylococcus aureus; MS,
mass spectrometry; MS/MS, tandem mass spectrometry; MTC, minimum toxic
concentration; NMR, nuclear magnetic resonance; RPMI, Roswell Park Memorial
Institute Medium; THP-1, human leukemia cells; VRSA, vancomycin-resistant
Staphylococcus aureus.
Natural Product Library
Natural product extracts were provided by the Australian
Institute of Marine Science (AIMS), Townsville, Queensland
as part of the AIMS Bioresources Library (Evans-Illidge et al.,
2013), via the Queensland Compound Library (Simpson
and Poulsen, 2014) (now called Compounds Australia
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Pharmacodynamic Functions of Synthetic Derivatives
(Compounds Australia, 2018)). Crude extracts had been
partially fractionated by AIMS/QCL to generate a library of
1,434 samples, supplied in DMSO (100%) solution and stored at
Bacterial Growth Conditions and Curves
Bacterial growth was monitored over a time-course of 60 h (0,
2, 4, 6, 8, 10, 12, 20, 22, 24, 26, 28, 30, 32, 34, 40, 44, 48, and
60 h) and 7 days (24, 48, 72, 96, 120, 144, and 168 h) for A MRSA
and M. tuberculosis H37Ra, respectively. For every sampled time
point harvest, 1 mL aliquot of the sample was isolated and
viable counts were determined. Growth curves were analyzed
by plotting the log CFU/ml against the time using previously
developed R scripts (version 3.2.0) (Foerster et al., 2016). Only
lag, log, and stationary phases were included in the analysis while
the decline phase was excluded.
−80◦C. Original concentrations as provided were 5 mg mL−1
.
Stock solutions were made by diluting these samples by a factor
of 1:10 in dH2O and stored at −80◦C.
Bacterial Strains
Pseudomonas aeruginosa PAO1 was provided by Dr. Jim Manos,
University of Sydney. E. coli EC958 was a kind gift of Professor
Mark Schembri, University of Queensland. Mycobacterial strains
(M. tuberculosis H37Rv, M. bovis BCG Pasteur, M. smegmatis
mc2155) are laboratory stocks from the Triccas group (University
of Sydney). The methicillin-resistant Staphylococcus aureus
(MRSA) strain was clinical isolation provided by Dr. John
Merlino (Concord Hospital NSW, Australia).
Antibiotics
Vancomycin and Rifampicin powders were dissolved in DMSO to
prepare antibiotic stock solutions in 2and 50 mM concentrations,
respectively. The stocks were stored at −20◦C for less than 1
month. Antibiotic solutions were thawed and diluted to the
desired concentrations just before use.
Bacterial Inhibition Assays
For screening of the AIMS library, each test sample (10 µL)
was dispensed into a separate well of a 96 well microtiter plates
(final sample concentration 0.5 mg mL−1) using sterile dH2O.
For determination of minimum inhibitory concentrations (MIC),
extracts (250 to 0.5 µg mL−1) or synthesized compounds (100
to 0.0002 µM) were serially diluted in microtiter plates. Bacterial
suspension (90 µL, OD 600 nm 0.001) was added to each well
and plates were incubated at 37◦C for either 18 h (MRSA,
P. aeruginosa PAO1, E. coli EC958) or 7 days (M. tuberculosis
H37Rv) as described previously (Harrison et al., 2017; Irene et al.,
2019; Larcombe et al., 2019; Mouton et al., 2019). Resazurin
(10 µL; 0.05% w/v) was added and plates were incubated for 3 h
or 24 h (M. tuberculosis) at 37◦C. The inhibitory activity was
calculated by visual determination of color change within wells
or detection of fluorescence at 590 nm using a FLUOstar Omega
microplate reader (BMG Labtech, Germany). Percentage survival
was calculated in comparison to the average of untreated control
wells after normalizing for background readings.
Determination of the Minimum
Bactericidal Concentration (MBC)
Bacterial solutions (100 µL) were taken from the two lowest
concentrations of the well exhibiting invisible growth (clear
well) and sub-cultured onto sterile agar plates. The MRSA and
M. tuberculosis H37Ra plates were incubated at 37◦C for 18 h
and 7 days, respectively, and then examined for bacterial growth.
MBC was taken as the concentration of antimicrobial agents that
did not exhibit any bacterial growth on the freshly inoculated agar
plates. Whole experiments were performed in triplicate, and each
experiment was repeated a minimum of three times.
Checkerboard Assays (CA)
Referring to the MICs of six synthetic compounds, CA were
designed to determine their fractional inhibitory concentrations
(FICs) in combinations against MRSA and M. tuberculosis
H37Rv. The tests were performed on 96-well plates according
to previous methods (Odds, 2003; Jacqueline et al., 2005, 2006;
Xu et al., 2018). Briefly, aliquots (10 µL) of four-fold dilutions
of the drug solutions (vancomycin and rifampicin) were added
to each plate, such that the vancomycin concentrations ranged
Evaluating Toxicity of AIMS Extract
Library
Human alveolar epithelial cells (A549), (Giard et al., 1973),
Madin-Darby canine kidney epithelial cells (MDCK), (Gaush
et al., 1966), human leukemia cells (THP-1) (Tsuchiya et al.,
1980), human hepatocellular carcinoma cells (Hep-G2), (Aden
et al., 1979), and human embryonic kidney cells 293 (HEK293)
(Graham et al., 1977) were grown and differentiated in complete
RPMI (Roswell Park Memorial Institute Medium) and DMEM
(Dulbecco’s Modified Eagle’s medium) tissue culture media
(RPMIc and DMEMc). To determine toxicity of the AIMS extract
library, 2 × 105 of each cell type were added to a 96-well plate
and left for 48 h at 37◦C to adhere. Extract samples at a final
concentration of 0.5 mg mL−1 were added to the wells, then
incubated for 7 days in a humidified 5% CO2 incubator at 37◦C.
Then resazurin (10 µL of 0.05% w/v) was added, and after 4 h,
fluorescence measured as described previously. Cell viability was
calculated as percentage fluorescence relative to untreated cells.
TABLE 1 | Biological origin of samples of interest.
Entry† AIMS Sample Biological origin
Code
QCL Sample Fraction
Number
4
20608
26051
19033
20608
Lendenfeldia sp.
Ircinia gigantea
Dysidea herbacea
Lendenfeldia sp.
SN00760947 Crude extract
SN00731005 Crude extract
SN00733110 Crude extract
6
10
13
SN00760956 75% MeOH
eluent
14
16
20608
24307
Lendenfeldia sp.
SN00760958 100% MeOH
eluent
Class Demospongiae‡ SN00730755 75% MeOH
eluent
†Corresponds to Entry Number in Table 1 above. ‡More detailed taxonomic
classification not available.
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Pharmacodynamic Functions of Synthetic Derivatives
from 100 to 25 µg mL−1, and rifampicin concentrations from
4.0 to 0.25 µm were added to each well in the plates. Then
solutions of the synthetic samples (10 µL) were added to plates,
in order to yield final drug concentrations spanning their MICs.
Finally, bacterial stock suspensions (80 µL) were added to all
wells with MIC. In addition, bacteria were quantified in each
i.
N
1a
ii.
Br
N
6
1b
iii.
N
2a
SCHEME 1 | Synthesis of amines related to natural products 1 and 2. (i) m-toluidine 7, K2CO3, KI, MeCN, 60◦C, overnight, 8%; (ii) p-toluidine 8, K2CO3, KI, MeCN,
60◦C, overnight, 6%; (iii) benzylamine 9, K2CO3, MeCN, 82◦C, overnight, 19%.
i.
I
N
10
3
ii.
Cl
N
NH
12
13
SCHEME 2 | Synthesis of amines related to natural products 3–5. (i) N,N-dihexylamine 11, K2CO3, MeCN, 82◦C, overnight, 21%; (ii) HCl in 1,4-dioxane, 5 min, 99%.
O
N
N
N
N
i.
NH
N
ii.
O
14
18
SCHEME 3 | Synthesis of naphthalimide derivative 18. (i) 4-bromo-1-butyne 15, K2CO3, KI, MeCN, 60◦C, overnight, 2%; (ii)
6-azido-2-ethyl-1H-benzo[de]isoquinoline-1,3(2H)-dione 17, CuSO4, sodium ascorbate, tBuOH/H2O, 60◦C, overnight, 1%.
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Pharmacodynamic Functions of Synthetic Derivatives
FIGURE 1 | Synthesis structures of bioactive amine natural products identified as new compounds in this study.
well to determine the MBC. FICs were calculated using below
equation:
Synthesis
N,N-Dioctyl-3-Methylaniline 1a
To a solution of 1-bromooctane 6 (6.3 mL, 36.6 mmol) in
acetonitrile (50 mL) was added potassium carbonate (25.3 g,
183 mmol), KI (6.08 g, 36.6 mmol), and m-toluidine 7 (1.96 mL,
18.3mmol) stirred at 60◦C overnight. The suspension was
filtered and washed with acetonitrile (3 × 50 mL), then
concentrated by rotary evaporation to yield the crude product
(1.50 g). Purification by automated column chromatography
(100 g cartridge, 100% petroleum benzine over 12 CV) then by
preparative TLC (10% ethyl acetate:petroleum benzine) afforded
the pure compound 1a as a yellow oil (0.46 g, 8%). 1H NMR
(500 MHz, CDCl3): δ 7.13–7.07 (m, 1 H), 6.50–6.44 (m, 3 H),
3.28–3.22 (m, 4 H), 2.32 (s, 3 H), 1.64–1.53 (m, 4 H), 1.38–1.24
FIC(X + Y) = [MICofcompoundXincombinationwithY]
[MICofXalone] − 1
The ratio of FIC to MIC is referred to as the FIC index
(Odds, 2003; Cikman et al., 2015; Guy et al., 2017). To evaluate
interaction profiles, the fractional inhibitory index (6FIC) was
calculated as a sum of both FIC drug X (antimycobacterial drug)
and FIC drug Y (synthetic amine) where 6FICs is smaller than
0.25 designated synergistic activity, 0.25–4 indifferent or additive
activity and >4 indicate antagonism (Odds, 2003; Cikman et al.,
2015; Guy et al., 2017).
TABLE 2 | Anti-bacterial activity of synthetic compounds against MRSA,
P. aeruginosa, E. coli, and M. tuberculosis.
sTKS (Constant Antibiotic
Concentrations)
Synthetic
MIC (µM)†,‡
compound
Time-kill experiments were performed by culturing MRSA
and M. tuberculosis in liquid medium in the presence of six
synthesized sample concentrations in doubling dilutions ranging
from 8 to 0.001 × MIC and 2 × MIC for both bacteria,
respectively. Growth curves were initially generated to confirm
that bacteria would reach a stable early- to mid-log phase after 2
and 48 h of pre-incubation in antimicrobial-free both medium,
respectively. Samples were taken in different incubation times
after drug exposure at 0, 2, 4, 6, 8, and 24 h for MRSA and 24,
48 h, and 4 and 6 days for M. tuberculosis and then plated onto
agar plates for CFU determination. The experiments were carried
out in duplicate and repeated three times.
MRSA
M. tuberculosis
E. coli
P. aeruginosa
1a
1b
2a
3
12.5 ± 1.0
100 ± 5.4
12.5 ± 0.7
12.5 ± 1.0
12.5 ± 2.7
12.5 ± 2.0
1.5 ± 3.0
25 ± 1.0
1.5 ± 1.0
3.1 ± 0.7
0.02 ± 1.1
12.5 ± 1.0
12.5 ± 5.0
100 ± 1.0
50 ± 1.0
6.2 ± 1.0
25 ± 2.0
1.5 ± 4.0
6.2 ± 9.0
100 ± 6.5
50 ± 1.0
3.1 ± 5.0
25 ± 4.0
12.5 ± 0.8
13
18
†Samples were diluted from 100 to 0.0002 µM and incubated with bacteria
(starting concentration OD600 = 0.001) under optimum assay conditions as
described in the Experimental section. ‡Values represent averages from three
independent repeats.
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(m, 20 H), 0.94–0.87 (m, 6 H); 13C NMR (125 MHz, CDCl3): δ [M + H]+, 100%; HRMS (ESI): m/z calculated for [C22H48N]+
148.3, 138.7, 129.0, 116.0, 112.4, 109.0, 51.0, 31.8, 29.5, 29.3, 27.3, [M + H]+ 326.3787, found 326.3776.
27.2, 22.7, 22.0, and 14.1; LRMS (ESI+): m/z 332.33 [M + H]+,
100%; HRMS (ESI): m/z calculated for [C23H42N]+, [M + H]+
N,N,N-Trioctylammonium Chloride 13
332.3317, found 332.33054.
To a solution of N,N,N-trioctylamine 12 (1.0 g, 2.80 mmol) in
1,4-dioxane (1.0 mL) was added 4 M HCl in dioxane (2.80 mL,
N,N-Dioctyl-4-Methylaniline 1b
To a solution of 1-bromooctane 6 (6.3 mL, 36.6 mmol) in
11.2 mmol) in an ice bath. Instantaneously a precipitate formed
and after 5 min this was collected by vacuum filtration to yield
acetonitrile (50 mL) was added potassium carbonate (25.3 g,
a white solid (1.08 g, 99% yield). 1H NMR (500 MHz, CDCl3)
183 mmol), KI (6.08 g, 36.6 mmol), and p-toluidine 8 (1.96 g,
δ 11.40 (br s, 1 H), 2.90 (td, J = 4.7, 12.5 Hz, 6 H), 1.74–
18.3 mmol) stirred at 60◦C overnight. The suspension was
1.66 (m, 6 H), 1.29–1.13 (m, 30 H), 0.79 (t, J = 7.0 Hz, 9 H);
filtered then concentrated by rotary evaporation to yield the
crude product (1.65 g). Purification by automated column
13C NMR (125 MHz, CDCl3) δ 52.2, 31.4, 28.8, 28.7, 26.6,
23.0, 22.3, and 13.8.
chromatography (100 g cartridge, 100% petroleum benzine over
4 CV, ramping to 100% ethyl acetate over 4 CV) gave the product
N-(But-3-yn-1-yl)-N-Octyloctan-1-Amine 16
1b as a yellow oil (0.39 g, 6%). 1H NMR (500 MHz, CDCl3): δ
To a flask charged with potassium carbonate (4.4 g, 31.8 mmol)
7.02 (d, J = 8.2 Hz, 2 H), 6.68–6.47 (m, 2 H), 3.33–3.10 (m, 4 H),
and potassium iodide (880 mg, 5.30 mmol) was added acetonitrile
2.25 (s, 3 H), 1.58–1.52 (m, 4 H), 1.40–1.20 (m, 20 H), 0.95–0.82
(72 mL) followed by dioctylamine 14 (8 mL, 26.5 mmol) and
(m, 6 H); 13C NMR (125 MHz, CDCl3): δ 146.2, 129.7, 124.3,
4-bromo-1-butyne 15 (2.74 mL, 29.2 mmol). The suspension
112.2, 51.3, 31.9, 29.5, 29.4, 27.3, 27.2, 22.7, 20.1, and 14.1; LRMS
was stirred at 60◦C for 18 h, then filtered and washed with
(ESI+): m/z 332.33 [M + H]+, 100%; HRMS (ESI): m/z calculated
acetonitrile (3 × 50 mL), and concentrated by rotary evaporation
for [C23H42N]+ [M + H]+ 332.3317, found 332.33095.
to yield the crude product. This was purified by automated
column chromatography (100 g cartridge, 0%–15% ethyl acetate
N-Benzyl-N-Octyloctan-1Amine 2a
in petroleum benzine over 10 CV) to yield the product as a yellow
To a solution of 1-bromooctane 6 (6.3 mL, 36.6 mmol) in
oil (0.15 g, 2%). 1H NMR (500 MHz, CDCl3): δ 2.64–2.57 (m,
acetonitrile (50 mL) were added potassium carbonate (25.3 g,
2 H), 2.39–2.31 (m, 4 H), 2.23 (dt, J = 2.7, 7.6 Hz, 2 H), 1.88 (t,
183 mmol) and benzylamine 9 (2 mL, 18 mmol) then stirred
J = 2.6 Hz, 1 H), 1.41–1.31 (m, 4 H), 1.27–1.14 (m, 20 H), 0.81
at 60◦C overnight. The mixture, a clear colorless solution, was
(t, J = 7.0 Hz, 6 H); 13C NMR (125 MHz, CDCl3): δ 83.3, 68.7,
concentrated using the rotary evaporator, to give a white solid.
54.0, 52.7, 31.8, 29.6, 29.3, 27.6, 27.2, 22.6, 16.7, and 14.1; LRMS
The solid was triturated with DCM (1 × 50 mL, 1 × 25 mL)
(ESI+): m/z 294.29 [M + H]+, 100%; HRMS (ESI): m/z calculated
and the DCM solution concentrated on the rotary evaporator
for C20H40N+ [MH]+ 294.3161, found 294.3157.
to yield yellow oil (5.1 g). Purification by automated column
chromatography (100 g cartridge, 0–40% ethyl acetate (EtOAc) in
6-(4-(2-(Dioctylamino) Ethyl)-1H-1,2,3-Triazol-1-yl)-2-
Ethyl-1H-Benzo[de]isoquinoline-1,3(2H)-Dione
18
petroleum benzine over 10 CV) yielded N-benzyl-N-octyloctan-
1-amine 2a as a colorless oil (1.15 g, 19%). 1H NMR (500 MHz,
CDCl3): δ 7.36–7.29 (m, 4H), 7.26–7.21 (m, 1H), 3.56 (s, 2H),
2.47–2.35 (m, 4H), 1.54–1.41 (m, 4H), 1.36–1.21 (m, 20H), 0.90 To a solution of 16 (400 mg, 1.24 mmol) and 6-azido-2-ethyl-1H-
(t, J = 7.0 Hz, 6H); 13C NMR (125 MHz, CDCl3): δ 140.3, 128.8, benzo[de]isoquinoline-1,3(2H)-dione 17 (365 mg, 1.61 mmol)
128.0, 126.6, 58.6, 53.8, 31.9, 29.6, 29.3, 27.5, 27.0, 22.7, and 14.1; in tert-butanol: water (12.4 mL) were added copper sulfate
LRMS (ESI+): m/z 332.33 [M + H]+, 100%; HRMS (ESI): m/z hexahydrate (31.0 mg, 0.12 mmol) and ascorbic acid sodium
calculated for [C23H42N]+ [M + H]+ 332.3317, found 332.3306. salt (73.8 mg, 0.37 mmol) then the solution was stirred at
60◦C overnight. Precipitants were removed by filtration and
N,N-Dihexyldecan-1-Amine 3
washed with acetonitrile (3 × 50 mL), then the filtrate was
To a solution of 1-iododecane 10 (2.75 mL, 12.9 mmol) in concentrated by rotary evaporation and purified by automated
acetonitrile (60 mL) was added potassium carbonate (16.5 g, column chromotography (100 g cartridge, 0–20% methanol in
129 mmol) and dihexylamine 11 (3 mL, 12.9 mmol), then dichloromethane over 8 CV). Purified fractions were re-purified
the mixture was stirred at reflux overnight. The suspension by automated reversed phase chromotography (30 g cartridge
was filtered to remove K2CO3 and washed with acetonitrile C18 silica (Biotage), 0–90% acetonitrile in water over 17 CV) and
(3 × 50 mL), then concentrated by rotary evaporation to yield concentrated to yield the product as a yellow solid (10 mg, 1%).
the crude product (7.8 g). The crude product was purified 1H NMR (500 MHz, CDCl3): δ 8.73 (d, J = 7.6 Hz, 2 H), 8.21 (dd,
by automated column chromatography (100 g cartridge, 100% J = 0.9, 8.5 Hz, 1 H), 8.09 (s, 1 H), 7.89–7.81 (m, 2 H), 4.29 (q,
petroleum benzene 2CV, then 0–60% ethyl acetate in petroleum J = 7.1 Hz, 2 H), 3.67–3.56 (m, 2 H), 3.49–3.37 (m, 2 H), 3.25–3.06
benzene over 10 CV) to yield the product as an oil (0.89 g, 21%). (m, 4 H), 1.87–1.70 (m, 4 H), 1.38 (t, J = 7.0 Hz, 3 H), 1.34–
1H NMR (500 MHz, CDCl3): δ 2.41–2.37 (m, 6 H), 1.38–1.46 1.23 (m, 10 H), 0.89 (t, J = 7.0 Hz, 6 H); 13C NMR (126 MHz,
(m, 6 H), 1.35–1.21 (m, 28 H), 0.91–0.86 (m, 9 H); 13C NMR CDCl3): δ 162.4, 161.9, 142.1, 136.8, 131.2, 129.6, 128.1 (128.09),
(125 MHz, CDCl3): δ 54.2,54.2, 31.9, 31.9, 29.7, 29.6, 29.6, 29.3, 128.1 (128.08), 127.7, 125.4, 124.1, 123.2, 122.6, 122.1, 51.6, 51.3,
27.7, 27.4, 26.9, 22.7, 14.1, and 14.1; LRMS (ESI+): m/z 326.41 34.8, 30.6, 28.7, 28.0, 25.7, 22.0, 21.5, 20.1, 13.0, and 12.3; LRMS
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Pharmacodynamic Functions of Synthetic Derivatives
(ESI+): m/z 560.38 [M + H]+, 100%; HRMS (ESI): m/z calculated
Synthesis of Bioactive Compounds From
Marine Extracts
for C34H50N5O2+ [MH]+ 560.3965, found 560.3950.
Five of the six bioactive components (Table 1) were isolated and
characterized from extracts as a reported previously (Dinarvand
and Peter Spain, 2020). To validate the structures proposed
for the natural products, and to explore the potential of these
a compounds as bioactive agents, a series of tertiary amine
derivatives of compounds 1–5 (Dinarvand and Peter Spain,
2020) were synthesized from 1-bromooctane 6, m-toluidine
7, p-toluidine 8, benzyl amine 9, 1-iododecane 10, N,N-
dihexylamine 11, and N,N,N-trioctylamine 12 (Schemes 1, 2).
O-Toluidine is carcinogenic and therefore was not used in
synthetic experiments.
Compounds 1a, 1b, and 2a were prepared using 1-
bromooctane 6 to alkylate m-toluidine 7, p-toluidine 8, benzyl
amine 9, respectively (Scheme 1), giving three compounds based
on the active component of AIMS sample 20608. Compound 3,
the active component of AIMS sample 19033, was prepared by
reacting 1-iododecane 10 with N,N-dihexylamine 11 (Scheme 2),
while N,N,N-trioctylamine hydrochloride 13 was prepared from
the free amine 12 as a simple and readily accessible analog of the
natural product structures 4 and 5 that had been isolated from
AIMS sample 26051.
Finally, we sought to combine the tertiary amine structures
elucidated in this study with a triazolyl naphthalimide pendant,
to result in a fluorescent label compound for visualization of
fluorescent compounds in the cells. Thus N,N-dioctylamine 14
was alkylated with 4-bromo-1-butyne 15, and the resulting
alkyne product 16 “clicked” with 6-azido-2-ethyl-1H-
benzo[de]isoquinoline-1,3(2H)-dione 17 (Yu et al., 2013) to
afford the naphthalimide derivative 18 (Scheme 3).
While the yields of many synthetic steps were low, sufficient
quantities of material were nonetheless isolated to enable
characterization, and biological evaluation, so the synthetic
reactions were not further optimized.
RESULTS AND DISCUSSION
Identification of Active Marine Extracts
To identify marine samples with biological activity, 1,434
compounds from the AIMS Bioresources Library (Evans-Illidge
et al., 2013; Simpson and Poulsen, 2014; Compounds Australia,
2018) were screened against MRSA in a resazurin cell viability
assay. Twenty-three compounds from extracts and fractions
derived from the phyla Porifera (90%), Echinodermata (5%), and
Chordata (5%) inhibited of MRSA growth by greater than 50%
compared to non-treated controls (Supplementary Table S1).
MICs for promising samples were determined (Supplementary
Table S1). The five most active samples showed MICs at
31.3 µg mL−1 (all Porifera samples), while another four samples
returned MICs of 62.5 µg mL−1 (also all Porifera). Cytotoxicity
screens against HepG2, HEK 293, A549 and THP-1 cell lines
were performed to define the cytotoxicity profile of the most
active samples (Supplementary Table S2). Pleasingly, all the
samples most active against MRSA were also non-toxic to the
cell lines tested.
TABLE 3 | Toxicity of synthetic compounds to cell lines.
Synthetic
samples
Minimum toxicity concentration of drug (MTC µM)†,‡,§
A549
THP1
HepG2
HEK 293
1a
1b
2a
3
>100 ± 4.1 50 ± 2.0 >100 ± 3.0
>100 ± 3.8 >100 ± 2.0 >100 ± 0.9
>100 ± 1.0 6.5 ± 0.8 >100 ± 0.8
>100 ± 2.0 50 ± 5.0 >100 ± 1.0
50 ± 0.9
>100 ± 6.0
50 ± 4.0
25 ± 2.0
6.5 ± 3.0
3.1 ± 8.2
13
18
50 ± 1.4
3.1 ± 0.7 >100 ± 5.2
>100 ± 6.0 >100 ± 9.0 >100 ± 5.0
Structural Comparison of Synthetic
Compounds to Natural Products
†Cells seeded at a concentration of 2 × 105 cells/well; samples diluted 100 to
0.0002 µM. ‡Cells incubated with sample under humidified incubation of 37◦C
with 5% CO2. §Values represent averages of three independent repeats.
Synthetic compounds (Figure 1) were investigated using mass
spectrometry (MS/MS and accurate mass) and analysis of
biological activity. Comparing the major ions in the mass
spectra of synthetic 1a and 1b (Supplementary Table S3), 2a
(Supplementary Table S4), 3 (Supplementary Table S5), and 13
(Supplementary Table S6) with the natural products shows good
correlation that we have previously reported (Dinarvand and
Peter Spain, 2020). Some minor differences are apparent, which
most likely arise due to differences in the amounts of material
analyzed (which are significantly greater for the synthesized
products), and differences in the instrumentation used.
Antibacterial Activity and Toxicity of
Synthetic Compounds
Synthetic compounds were assessed against MRSA, P. aeruginosa,
uropathogenic E. coli, and M. tuberculosis for antimicrobial and
selectivity’s activity. Compound selectivity is one of the key
properties that successful drugs need to deliberate simultaneously
FIGURE 2 | Growth curve of MRSA grown at 37◦C in LB for 24 h.
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so as to determine how a compound can differentially bind (Liu and Ning, 2017). However, the compound selectivity
to only the target of interest with high affinity (i.e., high and mechanism in the drug development process have to be
activity) while binding to other proteins with low affinities considered more.
A
B
C
FIGURE 3 | Checkerboard study between vancomycin 1a (A), 1b (B), and 2a (C). MRSA was incubated with varying concentrations of vancomycin (Van) together
with dilutions of the synthetic drugs 2a (A), 1a (B), and 1b (C). Graph displays the mean survival of triplicate samples and represents data from three independent
repeats.
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Interestingly, all synthetic derivatives compounds showed salt 13 proved the most effective of the synthetic compounds
similar MICs against MRSA (the organism against which against M. tuberculosis with an MIC of 0.02 µM and showed
the original natural product screening assays had been moderate inhibitory activity against the other bacteria.
conducted), typically around 12.5 µM. The simple amine Compound
3 displayed broad activity, with low MICs
A
B
C
FIGURE 4 | Checkerboard study between vancomycin and 3 (A), 13 (B), and 18 (C). MRSA was incubated with varying concentrations of vancomycin (Van)
together with dilutions of the synthetic drugs 3 (A), 13 (B), and 18 (C). Graph displays the mean survival of triplicate samples and represents data from three
independent repeats.
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against P. aeruginosa (MIC 3.1 µM), E. coli (6.2 µM), and
The potential toxicity of the synthetic compounds was also
M. tuberculosis (3.1 µM). The naphthalimide derivative evaluated, against A549, THP-1, HepG2 and HEK 293 cell lines
18 displayed good selective activity against E. coli (MIC (Table 3). None of the synthetic compounds showed significant
1.5 µM) (Table 2).
toxicity against HepG2 or A549 cells. Synthetic compounds
A
B
C
FIGURE 5 | Checkerboard study between rifampicin and 1a (A), 1a (B), and 2a (C). TB. H37Rv was incubated with varying concentrations of rifampicin (Rif)
together with dilutions of the synthetic drugs 1a (A), 2a (B), and 1b (C). Graph displays the mean survival of triplicate samples and represents data from three
independent repeats.
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1a, 2a, 3, 13 and 18 all showed some toxicity against THP1 Compound 1a showed only mild effects on all cell lines tested
and/or HEK 293 cells, with minimum toxicity concentrations (MTC 50–100 µM), while it also displayed a broad antibacterial
(MTC) as low as 3.1 µM. Compound 1b showed low toxicity profile, suggesting this compound may be a candidate for further
against all four of these cell lines, but also low activity (Table 3). investigation.
A
B
C
FIGURE 6 | Checkerboard study between rifampicin and 3 (A), 13 (B), and 18 (C). TB. H37Rv was incubated with varying concentrations of rifampicin (Rif) together
with dilutions of the synthetic drugs 3 (A), 13 (B), and 18 (C). Graph displays the mean survival of triplicate samples and represents data from three independent
repeats.
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TABLE 5 | Summary of the checkerboard study between rifampicin and
synthetic compounds.
Comparing data from the antimicrobial activity and
cytotoxicity assays shows that the active concentration ranges
for these synthetic compounds against bacteria are substantially
lower than active concentration ranges against the mammalian
cells tested, particularly HepG2 and A549 cells. THP1 cells and
the primary cells (data not shown) appeared more sensitive to
these compounds. Cross-referencing the biological activity and
toxicity data for these compounds suggest that they have some
potential for further development.
Compound 13 exhibited strong inhibitory activity against all
resistant strains with MICs as low as 0.24 µm; Compound 1a
showed moderate and variable activity against all assay strains
(MIC 6.7–20.0 µm) and 18 showed similar activity against all
resistant strains with an MIC of 2.2 µm. In this study, MRSA and
M. tuberculosis was chosen as a candidate for future investigation.
Structure Compound alone
Compound
MIC50 for
FIC
Drug–drug
interactions
name
MIC50 (µM) for
M. tuberculosis M. tuberculosis in
presence of Rif
(0.25 nM)
1a
1b
2a
3
1.5
6.25
1.5
1.5
6.25
1.5
1.1
1.1
1.1
0.4
1.1
1
Indifference
Indifference
Indifference
Synergy
1.5
0.38
0.02
3.13
13
18
0.02
6.25
Indifference
Indifference
TABLE 6 | Summary of MIC and MBC.
Structure
name
Compound (µm/mL)
Checkerboard Assays
Growth curves for the MRSA confirmed that growth was
well supported in LB medium and log phase (exponential)
growth period estimated between 30 and 60 min (Figure 2)
and TB in Middlebrook 7H9 broth between 1 and 2 days.
Previously, the combination of known drugs against MRSA
and M. tuberculosis was reported (Maltempe et al., 2017;
Bakthavatchalam et al., 2019). In this study, the CA applied in
triplicate for MRSA and M. tuberculosis to assess the bactericidal
activity of synthesized samples in combination with known
drugs (Figures 3–6). The results from this work showed that
certain combination regimens displayed improved bactericidal
activities. All treatments with combinations of commercial
antimicrobial agents at suboptimal MIC were effective to some
extent against the test pathogens, however effects were not
consistent. Vancomycin plus compounds 2a and 1a displayed
bactericidal activity against MRSA (Figure 3). On the other hand,
synthetic samples in combination with rifampicin were typically
not effective against M. tuberculosis, except for compounds 3
and 18 that showed synergistic or bactericidal effects (Figure 6).
Thus, some of our newly discovered antimicrobial agents have
the potential to work in combination with existing drugs to
combat drug resistant bacteria, a result that is in accordance with
previous studies.
MIC for
MRSA
MBC for
MRSA
MIC for
TB. H37Rv
MBC for
TB. H37Rv
1a
1b
2a
3
12.5
100
25
1.5
6.25
1.5
25
100
25
200
100
12.5
100
12.5
12.5
12.5
12.5
12.5
1.5
6.25
0.39
50
13
18
0.02
6.25
under the classical conditions of this CA, antagonism (no
interaction) was demonstrated between these two combinations.
Synergistic effects were observed with 2a and 1a when
10 µg mL−1 vancomycin was combined with 1.6 µM 2a and
3.1 µM 1a (Figure 4), which showed FIC = 0.328 and 0.45,
respectively.
Compound 3 in concentration of 1.5 µM shown synergism
effect on M. tuberculosis H37Ra, in our study when combine
with 0.25 nM rifampicin (Figure 6). The results of the “dynamic
checkerboard,” which quantifies bacterial growth when drugs are
used in combination, showed that compounds alone reduced the
bacteria by >1 to 2 log10 CFU mL−1, while in combination with
known drugs bacterial numbers were reduced at maximum by >2
to 3 log10 CFU mL−1 (Table 5).
The FIC index of vancomycin and rifampicin with four and
five synthesized compounds for each of the 2 tested strains,
ranged between 1 to 1.2, respectively (Table 4), meaning that
Time-Kill Curves
An additional important step in the drug development process
is evaluation of pharmacodynamics in vitro, a necessary
step before animal experiments. One important assay is use
of time-kill curves, which monitor antibacterial effect on
bacterial growth and death over times at a wide range of
antimicrobial concentration. In this study, we obtain and
compare in vitro pharmacodynamic parameters of synthetic
compounds and commercial drugs, opening up avenues into
understanding the effects of different concentration of synthetic
compounds alone and in combinations with commercial drugs
on resistant bacteria that describes the relationship between the
concentration of synthetic compounds and commercial drugs
with bacterial growth rate.
TABLE 4 | Summary of the checkerboard analysis between vancomycin and
synthetic compounds.
Structure
name
Compound
alone MIC50 MIC50 for MRSA
Compound
FIC
Drug–drug
interactions
(µM)
in presence of
Van (10 µg mL−1
)
1a
1b
2a
3
12.5
100
3.1
100
1.5
0.45
1.2
Synergy
Indifference
Synergy
12.5
12.5
12.5
12.5
0.33
1.2
12.5
6.25
12.5
Indifference
Indifference
Indifference
13
18
1.2
1.2
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The time-kill assay we developed worked well for different bactericidal activity was dependent on the concentration of the
synthetic compounds, including commercial drugs. The results antimicrobial and differed between synthetic compounds. MRSA
showed the MIC and MBC were time-dependent and should were killed to below the limit of detection (100 CFU/mL) at
be read within 18 to 24 h and (Table 6). Time-kill curves for the highest antimicrobial concentration (16-fold MIC). MRSA
1a, 1b, and 2a using the MRSA (≤ MIC ≥) are shown in bactericidal activity of synthetic compounds at concentration
Figure 7. Concentrations of up to 12.5 µM of these synthetic 12.5 to 50 µM decreased during the 4 to 6 h of the
compounds induced a bactericidal effect, but the onset of the assay.
FIGURE 7 | Time-kill curve study of 2a (A), 1a (B), and 1b (C) versus MRSA in log-phase at drug exposure. The curve displays the mean CFU for duplicate samples
and represents data from two independent experiments. Twelve doubling dilutions are plotted, the highest concentration (black line) corresponds to 16 × MIC.
Growth in absence of antimicrobial is drawn in red. The compound was added at time point 0 and monitored until 24 h. The limit of detection in the assay was
100 CFU mL−1
.
FIGURE 8 | Time-kill curve study of 3 (A), 13 (B), and 18 (C) versus MRSA in log-phase at drug exposure. The curve displays the mean CFU for duplicate samples
and represents data from two independent experiments. Twelve doubling dilutions are plotted, the highest concentration (black line) corresponds to 16 × MIC.
Growth in absence of antimicrobial is drawn in red. The compound was added at time point 0 and monitored until 24 h. The limit of detection in the assay was
100 CFU mL−1
.
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MRSA but compound 1b at 100 µM concentration rapidly
reduced growth by 1 h post-treatment, followed by inhibition
of growth up to 6 h (Figure 8). For vancomycin (Figure 9),
concentrations up to 50 µg mL−1 did not have an antibacterial
effect compared to untreated bacteria. At this concentration,
initial killing to 102–104 CFU mL−1 until 8 to 12 h was observed,
which was followed by regrowth to 106 CFU mL−1 after 24h.
For vancomycin ≥50 µg mL−1, persistent killing without re-
growth was observed and a bactericidal effect was obtained for
vancomycin 100 µg mL−1 at 24 h.
Based on these results, the effect of compounds 18 and
3 was slightly more rapid killing at 12.5 µg mL−1 which is
the MIC50, while for compound 13 killing was observed at
the highest concentration (>100 µg mL−1) as compared to
inhibition concentration. Since compounds 3 and 13 are related
to the same class II compounds but display different behaviors
in the killing time study, this could be explained by each having
a different mechanism of action. Interestingly, the checkerboard
and TKS illustrated decreases in the viable cell counts for these
same synthetic compounds over 4 or 6 h, but regrowth was noted
at 24 h (Figures 3–8). The in vitro pharmacodynamic parameters
suggest that there is a continuous gradient from bacteriostatic to
bactericidal effects and that compound 1b might fall in between
these two categories.
Sub-inhibitory combinations of compounds 2a, 1a, 1b, 3, 13,
18, and vancomycin substantially inhibited growth of MRSA
and displayed an increased antibacterial effect compared to
the effect of each agent alone. In combination, compound 2a
at 1.5 to 0.1 µM and vancomycin (50 µg mL−1) resulted in
protracted growth of MRSA but net-growth up to 102 CFU
mL−1 at 24 h. The addition of sub-inhibitory vancomycin at
50 µg mL−1, which displayed no effect alone, and 12.5 µM of
compound 1b substantially reduced regrowth of the MRSA that
was observed with compound 2a at 25 µM alone. From these
combinations of vancomycin at 50 µg mL−1 and compound
2a at 12.5 µM a bactericidal effect was observed in 1 h
(Figure 10). Hence, a favorable interaction between vancomycin
FIGURE 9 | Time-kill curve study of vancomycin versus MRSA in log-phase at
drug exposure. The curve displays the mean CFU for duplicate samples and
represents data from two independent experiments. Twelve doubling dilutions
are plotted, the highest concentration (black line) corresponds to 16 × MIC.
Growth in absence of antimicrobial is drawn in red. The compound was
added at time point 0 and monitored until 24 h. The limit of detection in the
assay was 100 CFU mL−1
.
Time-kill curves for three additional compounds (3, 13, and
18) were also made (Figure 8). Similar to the effect of 2a,
1a, and 1b (Figure 7) exhibited rapid killing during the first
2 h of the assay for concentrations above MIC. Compounds
up to 12.5 µM exhibited a bactericidal effect against log-phase
FIGURE 10 | Time-kill curve study of combinations of vancomycin and 2a versus MRSA at drug exposure in MIC concentrations. The curve displays the mean CFU
for duplicate samples and represents data from two independent experiments. Twelve doubling dilutions are plotted, the highest concentration (black line)
corresponds to 16 × MIC. Growth in absence of antimicrobial is drawn in red. The compound was added at time point 0 and monitored until 24 h. The limit of
detection in the assay was 100 CFU mL−1
.
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Pharmacodynamic Functions of Synthetic Derivatives
and all compounds was observed. The in vitro pharmacodynamic Our results suggest that rates of synergy for vancomycin and
parameters can provide relative comparisons across different rifampicin with synthetized compounds for the test organisms
synthesized compounds and current antimicrobials which can in this study are comparable, although antagonism was observed
be extremely valuable in preclinical studies but furthermore, more frequently with the latter.
pharmacokinetic effects need to be study. A novel synthesized
compound can be categorized and compared to mechanistically 1a, 2a and 3, which were found to be strongly bactericidal and
well-understood antibiotics. concentration dependent. More extensive work with a variety
In conclusion, that this could be the case for Compound
The activities of rifampicin alone and in combine with of combinations is needed to confirm this impression, but we
six synthesized compounds against M. tuberculosiss were believe that such work is warranted on the basis of the results
determined. Results showed that, while significant reduction in presented in this work.
bacterial viability was observed at day 6 following compounds 1a,
1b, 2a, and 3 treatments, exposure to rifampicin and compound
3, 13, and 18 achieved comparable killing effects at day 7 of
treatment (data not shown).
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
CONCLUSION
Vancomycin and rifampicin remain the main agents for the
treatment of invasive MRSA and M. tuberculosis diseases,
respectively. Yet the number of vancomycin-resistant S. aureus
(VRSA) and rifampicin-resistant M. tuberculosis strains is on
the rise. The emergence of antibiotic resistance brings a need
for novel, effective antibacterial agents that are resistant to
antimicrobial resistance.
AUTHOR CONTRIBUTIONS
MD conceived, designed the experiments, and wrote the
manuscript. MD and MS performed the experiments. MD, MS,
and FV analyzed the data. FV oversighted statistical modelling
and revised the manuscript. All authors contributed to the article
and approved the submitted version.
The pharmacodynamic parameters can be applied for the
evaluation of new antimicrobials and to study the effects of
combining antimicrobials against MRSA and M. tuberculosis.
Synthetic compounds based on the natural product structures
(Dinarvand and Peter Spain, 2020) were prepared to validate
and expand these findings. Synthetic compounds 1a, 1b,
2a, 3, and 13 showed promising bioactivity/toxicity profiles.
Naphthalimide derivative 18 was prepared as a hybrid of the
amines uncovered in this study.
The compounds uncovered in this study add to the growing
arsenal of antimicrobial agents from the sea (Hughes and Fenical,
2010; Indraningrat et al., 2016) and offer interesting new avenues
for further investigation in the quest for new, effective agents to
combat the growing scourge of multidrug resistant bacteria.
An additional important step in the drug development process
is evaluation of pharmacodynamics, a necessary step before
animal experiments. The CA and sTKS are two important
assays to monitor antibacterial effect on bacterial growth and
death overtime at a wide range of antimicrobial concentration.
ACKNOWLEDGMENTS
We thank Dr. John Merlino (Concord Hospital, Sydney) for
provision of the MRSA. This work was supported by the
National Health and Medical Research Council (NHMRC)
Project APP1084266, the NHMRC Centre of Research Excellence
in Tuberculosis Control (APP1043225), and the University of
Sydney. MD was supported by an International Postgraduate
Research Scholarship (IPRS) and Australian Postgraduate Award
(APA) from the Australian Government. The novel parts of
MD Ph.D. which are synthesis, identification, and structure
elucidation of new compounds parts have been released as a
pre-print at BioRxiv (Dinarvand and Peter Spain, 2020).
SUPPLEMENTARY MATERIAL
All treatments with combinations of commercial antimicrobial The Supplementary Material for this article can be found
agents at suboptimal MIC were effective to some extent against online at: https://www.frontiersin.org/articles/10.3389/fmicb.
the tested pathogens, however, effects were not consistent. 2020.551189/full#supplementary-material
Belma Konuklugil, B. G. (2015). Antimicrobial activity of marine samples collected
from the different coasts of Turkey. Turk. J. Pharm. Sci. 12, 116–125. doi:
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