Synthesis and antibacterial activities of marine natural product ianthelliformisamines and subereamine synthetic analogues

Article abstract of DOI:10.1016/j.bmcl.2021.127883

Marine sponges of the genus Suberea produce variety of brominated tyrosine alkaloids which display diverse range of biological activities including antiproliferative, antimicrobial and antimalarial activities. In continuation of our search for biologicall

Full text of DOI:10.1016/j.bmcl.2021.127883

Bioorg. Med. Chem. Lett. 39 (2021) 127883
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial activities of marine natural product
ianthelliformisamines and subereamine synthetic analogues
Shivaji Narayan Khadakea, Shaik Karamathullaa, Tapan Kumar Jenaa, Mohan Monishab,
,
,*
Nikhil Kumar Tutib, Faiz Ahmed Khana *, Roy Anindyab
a Department of Chemistry, Indian Institute of Technology Hyderabad, Kandi, Sangareddy 502285, India
b Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy 502285, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Marine sponges of the genus Suberea produce variety of brominated tyrosine alkaloids which display diverse
range of biological activities including antiproliferative, antimicrobial and antimalarial activities. In continua-
tion of our search for biologically active marine natural products for antibacterial compounds, we report here the
synthesis and evaluation of biological activity of panel of ianthelliformisamines and subereamine analogues
using the literature known acid-amine coupling reaction. Several derivatives of Ianthelliformisamine were
achieved by the coupling of Boc-protected polyamine chain with brominated aromatic acrylic acid derivatives by
varying the bromine substituents on aromatic acid derivatives, amine spacer as well as geometry of the double
bond, and then Boc-deprotection using TFA. Similarly, subereamine analogues were also synthesized employing
coupling reaction between various brominated phenyl acrylic acids with commercially available chiral amino
ester derivatives followed by ester hydrolysis. We screened these synthetic analogues for antibacterial activity
against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) strains. One of the
Marine sponges
Suberea
Bromotyrosine
Subereamines
Ianthelliformisamines
compound 7c showed bactericidal activity against Staphylococcus aureus with an IC50 value of 3.8
25 M).
μM (MIC =
μ
Indiscriminate use of antibiotics has led to world-wide increase in the
incidence of antibiotic-resistant bacteria. As a consequence, there is an
urgent need for new antibiotics to overcome bacterial resistance. Due to
high development cost, less return on investment and low profitability
very few pharmaceutical companies are actively pursuing antibiotics
development program. In view of growing menace of antibiotic-resistant
bacteria, identification of new compounds having antibacterial activity
is urgently required.
ianthelliformisamines A-C and 39 synthetic analogues.4 Our results
revealed that some of the synthetic analogues of ianthelliformisamines A
and B are more potent against both Gram-negative and Gram-positive
bacteria than the parent natural products. Considering the prominent
anti-bacterial activity of ianthelliformisamines analogues we continued
our effort to make more ianthelliformisamines and subereamine ana-
logues and herein, we report synthesis and biological evaluation of 28
such synthetic compounds.
Simple polyamine-containing compounds, such as spermine and
spermidine, are known to have antibacterial property but only at high
millimolar concentration.1 The search for more potent polyamine-
containing natural products with intrinsic antibacterial activity led to
the discovery of ianthelliformisamine A-C, bromotyrosine-derived me-
tabolites from the marine sponge, Suberea ianthelliformis2 and subere-
amines A-B, bromoarginine-derived metabolites from the marine
sponge, Suberea molli.3 Encouraged by these findings, attempts were
made to generate synthetic ianthelliformisamine derivatives.4,5 Previ-
ously, we reported synthesis and biological evaluation natural products
Ianthelliformisamine derivatives were prepared by varying central
amine spacers such as 1,2-trans-cyclohexyldiamine and meta-xylenedi-
amine. Initially Boc protected polyamine chain spermine or spermidine
1 was synthesized from 1,2-trans-cyclohexyldiamine according to liter-
ature methods.6–8 In the reaction sequence, 1,2-trans-cyclohexyldiamine
used as spacer group which on treatment with acrylonitrile (2.0 equiv)
gave the bis Michael adduct. Further, central amines were protected
with the Boc-group and then nitrile groups were reduced by RANEY-Ni®
to get Boc-protected amine 1. Boc-protected sperimidine 2 also syn-
thesized from the 1,2-trans-cyclohexyldiamine through selective mono-
* Corresponding authors.
E-mail address: anindya@bt.iith.ac.in (R. Anindya).
https://doi.org/10.1016/j.bmcl.2021.127883
Received 14 November 2020; Received in revised form 9 February 2021; Accepted 10 February 2021
Available online 2 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S.N. Khadake et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127883
Scheme 1. Synthesis of Boc-protected spermine.
Michael addition to acrylonitrile and then Boc-protection of primary and
secondary amine followed by reduction of the nitrile group. In the same
way Boc-protected sperimidine 3 also synthesized using meta-xylenedi-
amine as spacer group (Scheme 1).
After successfully synthesizing ianthelliformisamines analogues,
next our aim to synthesize various analogs of subereamine natural
product (9a-9s) by the coupling of compound 4 with different chiral
amino ester derivatives (8) in presence of EDC-HCl and HOBt, which on
further hydrolysis to delivered the desired final products in very good
yields (Scheme 4).
Similarly, 3-Phenylacrylic acid derivatives prepared via bromination
with Br2/AcOH9 by methylation of the hydroxyl group with MeI.10 To
access a library of analogues, 2,4,6-tribromo-3-methoxybenzaldehyde
2b and 2,4-dibromo-5-methoxybenzaldehyde 2c were prepared from
3-hydroxybenzaldehyde by treating it with Br2/H2O and Br2/CHCl3,
respectively, followed by methylation of the hydroxyl group with MeI.11
For antibacterial activity, the minimum inhibitory concentrations
(MICs) of synthetic ianthelliformisamines and subereamine compounds
were evaluated against a representative Gram-negative (Escherichia coli)
and Gram-positive bacteria (Staphylococcus aureus) bacterial strains. The
MIC was determined as the lowest concentration of a compound that
prevents visible growth of a microorganism in a broth dilution assay. As
shown in Fig. 1, when the compounds were added to log-phase culture of
The aldehydes 2a–c were converted into
α
,β-unsaturated ethyl esters
3a–d
by
Wittig
reaction
of
ethoxycarbonylmethylene-
triphenylphosphorane in THF (Scheme 2).12 We found that aldehydes 2a
and 2b gave exclusively trans products in excellent yields, while other
aldehyde 2c gave a mixture of chromatographically separable cis and
bacteria at final concentration of 50 μM and evaluated for the capability
to inhibit bacterial growth, 7a-7c showed growth inhibition against E
coli (Fig. 1A) and only 7c exhibited growth inhibition against S aureus
(Fig. 1B). We observed that all the synthetic subereamine compounds
failed to demonstrate and growth inhibition. Most of the ianthelli-
trans products in a ratio of 1:3 respectively. Further, α,β-unsaturated
ethyl esters 3a–c were hydrolyzed by lithium hydroxide monohydrate
(LiOH⋅H2O)–THF–water to get α,β-unsaturated carboxylic acid 4a–4c
(Scheme 2).13
formisamine compounds also showed MICs over 200
μ
M against E coli
Coupling of 3-Phenylacrylic acid derivatives and Boc-protected
spermine (1–3) by using EDC⋅HCl as a coupling reagent14 gave the
corresponding amide, which after Boc deprotection with trifluoroacetic
acid (TFA) in CH2Cl2 at 0 ◦C yielded products as a TFA salt with mod-
erate yield in 2 steps (Scheme 3). All the new compounds were char-
acterized by 1H NMR and 13C NMR.
except for the 7c, which showed MIC of 50
μ
M and 25 M against S
μ
aureus and E coli, respectively. The OD600 nm value being directly
proportional to the number of bacteria present in the culture, this result
suggests that the bacterial growth could be completely suppressed when
the concentration of 7c was 50 μM (55.6 μg/mL).
We have next determined the IC50 of 7c by adding gradually
2

S.N. Khadake et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127883
Scheme 2. Synthesis of 3-Phenylacrylic acid derivatives.
increasing concentration of 7c to the growing bacterial cultures. We
noted that synthetic ianthelliformisamines analogue 7c showed IC50 of
concentration of 7c. To explore the cytoxicity of 7c cell viability was
examined using Calcein-AM uptake assay. Calcein-AM is a cell-
permeable non-fluorescent compound that is hydrolysed by living cell
esterases and produces strongly fluorescent compound calcein. When
human cell line HEK293 were treated with 7c, no significant difference
in cell viability between the 7c-treated samples and untreated control,
with over 95% of the HEK293 cells remaining viable (Fig. 2B). To
examine if there is any membrane damaging effect of 7c propidium
iodide (PI) uptake assay was performed. DNA intercalating dye PI is only
permeable to cells with damaged membranes. When 7c was added at its
MIC, (Fig. 2C), bacterial cells were stained with PI but this effect was
time-dependent and maximum permeabilization occurred after longer
incubation. Approximately 80% of cells were stained with PI suggesting
37.3
μM against E coli and 13.9 μM against S aureus (Fig. 1C). For
comparison, antibiotic ampicillin exhibited IC50 of 33.6 μM against E coli
and 3.8
μM against S aureus. After determining the MIC, minimal
bactericidal concentration (MBC) was determined as the lowest con-
centration that achieved a 90% decrease in viable bacteria. As shown in
Fig. 1D, 7c showed MBC of 50 μM against E coli and 25 μM against S
aureus, which is same as MIC of 7c. We hypothesized that 7c might be a
bactericidal compound as most bactericidal drugs exhibit MBC value
same as, or very close to the MIC. To further confirm anti-bacterial effect
of 7c disc-diffusion experiments were performed for 7c and as shown in
Fig. 2A, inhibition zone diameter gradually increased with increasing
3

S.N. Khadake et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127883
Scheme 3. Syntheses of coupling products.
4

S.N. Khadake et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127883
Scheme 3. (continued).
5

S.N. Khadake et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127883
Scheme 4. Syntheses of subereamine analogous.
that 7c treatment could permeabilize bacterial cells (Fig. 2D). This result
indicates that the viable cell number decreases rapidly under 7c treat-
ment, suggesting the efficient killing of the bacterial cells.
Gram-negative E coli and Gram-positive S aureus with a MIC and MBC of
50 and 25 µM (27.8 and 55.6 μg/mL), respectively. In summary, syn-
thetic ianthelliformisamine analogue 7c showed as antibactericidal ac-
tivity without any cytotoxicity to human cells, demonstrating its
potential for development as a novel antibacterial therapeutic agent.
With expanding knowledge of antibacterial natural products in recent
years, this study demonstrates the untapped potential for discovering
In conclusion, the present study describes synthesis and biological
evaluation of a panel of synthetic analogues of ianthelliformisamines
and subereamine. Through screening of the compounds, we discovered
one ianthelliformisamine analogue, namely 7c, inhibits the growth of
6

S.N. Khadake et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127883
Fig. 1. Bacterial growth inhibition assays. (A)
Inhibition of Escherichia coli growth. All the
compounds were dissolved in DMSO at and
added to the bacterial culture at log-phase at
E coli
B
C
S aureus
DMSO
7c
A
E coli
S aureus
DMSO
7c
5a-5c
6a-6c
7a-7b
9a-9w
120
100
80
60
40
20
0
0.8
0.6
0.4
0.2
0.0
0.8
5a-5c
6a-6c
9a-9w
final concentration of (50 μM); DMSO repre-
0.6
0.4
0.2
0.0
sent the negative control. (B) Inhibition of
Staphylococcus aureus growth. (C) IC50 analysis
of 7c against E. coli and S. aureus. (D) Minimal
bactericidal concentration (MBC) of 7c in
E. coli and S. aureus. MBC was determined by
dilution of 7c treated culture and plating on
growth media at 37 ◦C for 24 h.
7a
7c
7b
7c
0
20 40 60 80 100
Concentration
0
1 2 3 4 5 6 7 8 9
0
1 2 3 4 5 6 7 8 9
µ
( M)
Time (h)
Time (h)
S aureus
E coli
D
0
6.25
12.5
7c (
25
M)
50
100
0
6.25
12.5
7c (
25
M)
50
100
µ
µ
Fig. 2. Analysis of antibacterial activity and evaluation of cytotoxicity of
7c. (A) Representative agar plates showing the zones of inhibition formed
A
B
7c (
µ
g)
25
100 50
by 7c (0, 25, 50 and 100 μg of 7c was applied) (B) Cytotoxic effect of 7c on
100
HEK293 cells. Representative images of 7c-treated HEK293 cells at 48 h
post treatment with 25 and 50 µM 7c. Cell viability is expressed as per-
centage of HEK293 cells treated with 7c at different concentrations. (C)
Effect of 7c on membrane permeability. Propidium iodide (PI) staining in
bacterial cells treated with 7c. (D) Graph showing percentage of PI positive
bacterium treated with 7c.
0
80
60
40
Control
20
0
µ
M)
µ
M)
7c (50
7c (25
0
5
S aureus
E coli
(
c
7
C
D
7c (25
µM)
7c (50
µ
M)
control
control
100
80
60
40
20
0
-
+
-
-
+
+
+
-
-
+
-
+
E coli
S aureus
7c
S aureus
E coli
novel effective synthetic antibiotic compounds, which can help to
overcome current challenges of the treatment of drug resistant bacterial
infections.
org/10.1016/j.bmcl.2021.127883.
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