Synthesis and antibacterial analysis of analogues of the marine alkaloid pseudoceratidine

Article abstract of DOI:10.3390/molecules25112713

In an effort to gain more understanding on the structure activity relationship of pseudoceratidine 1, a di-bromo pyrrole spermidine alkaloid derived from the marine sponge Pseudoceratina purpurea that has been shown to exhibit potent biofouling, anti-fungal, antibacterial, and anti-malarial activities, a large series of 65 compounds that incorporated several aspects of structural variation has been synthesised through an efficient, divergent method that allowed for a number of analogues to be generated from common precursors. Subsequently, all analogues were assessed for their antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Overall, several compounds exhibited comparable or better activity than that of pseudoceratidine 1, and it was found that this class of compounds is generally more effective against Gram-positive than Gram-negative bacteria. Furthermore, altering several structural features allowed for the establishment of a comprehensive structure activity relationship (SAR), where it was concluded that several structural features are critical for potent anti-bacterial activity, including di-halogenation (preferable bromine, but chlorine is also effective) on the pyrrole ring, two pyrrolic units in the structure and with one or more secondary amines in the chain adjoining these units, with longer chains giving rise to better activities.

Full text of DOI:10.3390/molecules25112713

molecules
Article
Synthesis and Antibacterial Analysis of Analogues of
the Marine Alkaloid Pseudoceratidine
David Barker 1,2,
*
, Stephanie Lee 1, Kyriakos G. Varnava 1, Kevin Sparrow 1,
Michelle van Rensburg 1, Rebecca C. Deed 1,3 , Melissa M. Cadelis 1 , Steven A. Li 1
Brent R. Copp 1 , Vijayalekshmi Sarojini 1,2 and Lisa I. Pilkington 1
,
1
School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand;
slee526@aucklanduni.ac.nz (S.L.); kvar749@aucklanduni.ac.nz (K.G.V.);
kevin.sparrow@auckland.ac.nz (K.S.); mvan126@aucklanduni.ac.nz (M.v.R.);
rebecca.deed@auckland.ac.nz (R.C.D.); m.cadelis@auckland.ac.nz (M.M.C.);
sli359@aucklanduni.ac.nz (S.A.L.); b.copp@auckland.ac.nz (B.R.C.); v.sarojini@auckland.ac.nz (V.S.);
lisa.pilkington@auckland.ac.nz (L.I.P.)
MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University of Wellington,
Wellington 6140, New Zealand
School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand
Correspondence: d.barker@auckland.ac.nz; Tel.: +64-9-923-9703
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Thomas J. Schmidt
Received: 21 April 2020; Accepted: 10 June 2020; Published: 11 June 2020
Abstract: In an effort to gain more understanding on the structure activity relationship of
pseudoceratidine , a di-bromo pyrrole spermidine alkaloid derived from the marine sponge
1
Pseudoceratina purpurea that has been shown to exhibit potent biofouling, anti-fungal, antibacterial,
and anti-malarial activities, a large series of 65 compounds that incorporated several aspects of
structural variation has been synthesised through an efficient, divergent method that allowed for
a number of analogues to be generated from common precursors. Subsequently, all analogues
were assessed for their antibacterial activity against both Gram-positive (Staphylococcus aureus) and
Gram-negative (Escherichia coli) bacteria. Overall, several compounds exhibited comparable or better
activity than that of pseudoceratidine 1, and it was found that this class of compounds is generally
more effective against Gram-positive than Gram-negative bacteria. Furthermore, altering several
structural features allowed for the establishment of a comprehensive structure activity relationship
(SAR), where it was concluded that several structural features are critical for potent anti-bacterial
activity, including di-halogenation (preferable bromine, but chlorine is also effective) on the pyrrole
ring, two pyrrolic units in the structure and with one or more secondary amines in the chain adjoining
these units, with longer chains giving rise to better activities.
Keywords: marine natural product; analogue synthesis; SAR; pseudoceratidine; antibacterial activity
1. Introduction
Bacteria are ubiquitous, unicellular microorganisms that occupy critical roles in maintaining life
on earth [
and desert plains, caves to Antarctic regions and existing on and in other living organisms, including
humans [ ]. The relationship between humans and bacteria is complex and while most bacteria have a
symbiotic relationship with humans, some are pathogenic and are the cause of diseases [ ]. In recent
1,2]. Bacteria are found in a range of diverse environments; from the deep sea to volcanic lakes
3
4
history, combatting diseases caused by bacterial infections has been addressed through the extensive
development and use of antibacterial agents [5].
Molecules 2020, 25, 2713; doi:10.3390/molecules25112713
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2713
2 of 13
Due to the overwhelmingly successful outcomes achieved by the discovery of early antibacterial
drugs such as penicillin and sulfa drugs, the intensive research surrounding antibacterial agents
conducted during the 1950s–70s led to the establishment of a wide range of antibacterial classes [6].
However, no new classes of antibacterial agents have been discovered since this era—this fact, coupled
with the rise in pathogenic drug-resistant bacterial species predicates the need for new antibacterial
classes and agents to be found [6,7].
In recent history, natural products have proved to be a great source of inspiration in the pursuit
of drug therapies. This is exemplified by the fact that a range of therapeutics have been inspired by
compounds of natural origin—a significant proportion (~40%) of existing drugs on the market are
either natural products or derivatives thereof [
therapeutics are developed from natural products isolated from terrestrial organisms, there is an
ever-increasing interest in the discovery and development of drugs from marine natural products [ 11].
8]. While almost all of these natural product-derived
9–
This movement can be attributed to the diverse range of novel and unique chemical structural motifs
that are found in marine organisms in conjunction with the fact that natural products of marine origins
typically have greater biological activity than terrestrial natural products [9,11,12].
Marine sponges, in particular, have been found to be a rich marine-based source of bioactive
molecules—40% of the total secondary metabolites discovered in marine organisms originate from
marine sponges [13]. The rationale for this is that marine sponges are sessile organisms that rely on the
production of secondary metabolites to act as a chemical defense mechanism against predators [14].
Pseudoceratidine
1996 in coastal regions around Japan and has instigated enthusiasm in further investigation of
its analogues due its relatively simple molecular structure, yet potent biological activity [15 19].
Structural elucidation of pseudoceratidine showed it to be an asymmetrical alkaloid with bromopyrrole
units at each end of the triamine, spermidine, chain (Figure 1) [15]. Thought to function as a natural
biofouling agent, pseudoceratidine was initially tested for its antifouling activity against the
marine larval organism Balanus amphitrite (EC50 g/mL) [15]. Subsequent to this, its anti-fungal
(against Candida albicans minimal inhibitory concentration (MIC) 32 g/mL) and antibacterial
(against Gram-positive Staphylococcus aureus MIC 4 g/mL, Listeria monocytogenes MIC 5 g/mL
and Gram-negative Escherichia coli MIC 32 g/mL, Pseudomonas aeruginosa MIC 128 g/mL) activities
were investigated [17 19]. In addition to this, pseudoceratidine was assessed for its anti-malarial
activity against the malarial protist Plasmodium falciparum EC50 M) [16]. This testing verified
1 was first extracted from the marine sponge Pseudoceratina purpurea in
–
1
1
8
µ
µ
µ
µ
µ
µ
–
1
1
µ
pseudoceratidine 1 as a promising lead compound in the search for new antibacterials.
In addition to the pseudoceratidine 1, a series of closely related analogues have been previously
prepared and assessed for their antibacterial activity (Figure 1) [17,19]. These various series have
explored the effect of omitting one of the pyrrolic units (i.e., monoacyl derivatives) and bromination
(or lack thereof) of the pyrrolic units and limited exploration of the length and symmetry of the amine
chain. It was found that analogues with the most effective biological activities were those that possessed
a di-brominated pyrrole in addition to a bis-amide structure with a polyamine chain with 1 positive
≥
charge at physiological pH. Despite these explorations and the development of an initial structure
activity relationship (SAR) for this class of compounds, thus far there are no reported pseudoceratidine
analogues that have better activity than pseudoceratidine 1 itself.
In an effort to extend the previous limited SAR and to investigate the effect of a variety
of halogenations (degree and type of halogen) as well as a large variety of polyamine chains
(both symmetric and asymmetric chains with no, one or two secondary amines), it was decided
to synthesise a large and comprehensive library of analogues with these variations and assess their
biological activity against representative Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria.

Molecules 2020, 25, 2713
3 of 13
Figure 1. Molecular structure of pseudoceratidine
activity has been investigated. On psuedoceratidine
1
and previous analogues for which that antibacterial
, the features investigated in this study are
1
indicated by colour; the type and number of halogenation on the pyrrolic ring (blue), the length
and symmetry of polyamine chain (red); the number of secondary amine groups—none, one or
two (green)—the presence of a second pyrrolic unit (orange) and, if no pyrrolic unit, an alternative
functionality (purple).
2. Results and Discussion
The general strategy for the synthesis of the proposed compounds was the base-mediated coupling
of trichloroacetyl halogenated pyrroles 2 with a large variety of polyamines (Scheme 1).
Scheme 1. General strategy and the pseudoceratidine analogues targeted.

Molecules 2020, 25, 2713
4 of 13
As such, the first major task was the generation and subsequent halogenation of 2-trichloroacetyl
pyrrole 2a to give the desired mono- and di-substituted pyrroles 2b . The synthesis of 2-trichloroacetyl
pyrrole 2a was achieved through the selective electrophilic aromatic substitution of commercially
available pyrrole with trichloroacetyl chloride following the method reported by Dubis et al.
–
g
3
(Scheme 2) [20]. Recrystallisation of the crude product with n-hexanes and a minimal amount of diethyl
ether provided the desired product 2a in a yield of 89%.
Scheme 2. Synthesis of substituted pyrroles 2a–g.
Following its synthesis, pyrrole 2a was chlorinated with one equivalent of sulfuryl chloride to
provide the C-4 mono-chloro pyrrole 2b as the major product (Table 1) [21]. The C-4 position is the
preferential site of reaction owing to the presence of the strongly electron-withdrawing properties
of the trichloroacetyl group and longer and/or more forcing conditions were required for additional
substitution at the C-5 position. As such, employing the same chlorinating reagent in a higher
concentration and longer reaction time provided the di-chloro analogue 2c in 85% yield. Following the
reaction conditions reported by Behrens et al., the synthesis of 4-bromo-2-trichloroacetyl pyrrole 2d was
accomplished using a similar reaction procedure as that employed for mono-chloro pyrrole 2b, but using
bromine (1 equiv.) for 10 min [17]. Furthermore, doubling the amount of bromine and using acetic
acid as a solvent to facilitate a higher reaction temperature allowed for the high-yielding production of
the di-brominated pyrrole 2e (91%) [22
2a with iodine monochloride provided 4-iodo-2-trichloroacetyl pyrrole 2f
,
23]. Finally, the selective iodination of 2-trichloroacetyl pyrrole
24], while diiodination was
[
achieved using a combination of iodine and silver trifluoroacetate according to previously reported
reaction conditions [22,25], giving 2g.
Table 1. Reaction conditions and outcomes for the synthesis of halogenated pyrroles 2b–g.
Conditions
Product (Yield)
SOCl2 (1.2 equiv.), CH2Cl2, 0 ◦C to r.t., 18 h
SOCl2 (5 equiv.), CH2Cl2, r.t., 4.5 d
R1 = H, R2 = Cl 2b (63%)
R1 = R2 = Cl 2c (85%)
R1 = H, R2 = Br 2d (45%)
R1 = R2 = Br 2e (91%)
R1 = H, R2 = I 2f (64%)
R1 = R2 = Br 2g (43%)
Br2 (1 equiv.), CH2Cl2, 0 ◦C to r.t., 10 min
Br2 (2 equiv.), AcOH, r.t. to 60 ◦C, 2 h
ICl (1 equiv.), CH2Cl2, r.t., 2 h
Silver trifluoro acetate (2 equiv.), I2 (2 equiv.), CH2Cl2, r.t., 36 h
Following the successful synthesis of the halogenated 2-trichloroacetylated pyrroles
, all that was required for the synthesis of the targeted dipyrrole pseudoceratidine
analogues was the coupling of these pyrroles 2a with a range of polyamines, namely
putrescine , N’-(2-aminoethyl)ethane-1,2- diamine , N’-(3-aminopropyl)propane-1,3-diamine
N’-(6-aminohexyl)hexane-1,6-diamine , N’-(2-aminoethyl)propane-1,3-diamine , spermidine
2b–g
–
g
4
5
6
9
,
,
7
8
N’,N’-(propane-1,3-diyl)bis(propane-1,3-diamine) 10 and spermine 11. The desired couplings were
achieved using two equivalents of the trichloroacetylated pyrrole and the polyamine in dry THF
2
at room temperature for 18–72 h (Scheme 3) [17]. This reaction was generally carried out with the
addition of Et3N, which was used to improve the reactivity of the pyrrole. While this addition was
necessary for almost all amines, it was not generally required for reactions involving putrescine 4,

Molecules 2020, 25, 2713
5 of 13
except for the generation of analogue 12b, and the putrescine analogues 12a
–
f
being successfully
produced in moderate to very good yields (57–79%). Reaction of amines
in the generation of a large library of di-pyrrole analogues 12 19 (including pseudoceratidine
range of linear chains and halogenation patterns on the pyrrolic units. However, it should be noted that
5–
11 with pyrroles
2
resulted
–
1) with a
all of the reactions involving diiodo-substituted pyrrole 2g gave none of the desired amides 12g–19g
possibly due to SNAr of the labile iodine substituents.
R2
R1
H2N
NH2
4
HN
4
H
H
R1
R2
N
N
(Et3N b only)
THF, r.t., 18 - 72 h,
N
4
H
O
O
12a-f
57-79%
R2
H
H2N
N
NH2
m
R1
n
CCl3
N
5 n = m = 2; 6 n = m = 3;
R2
R1
H
O
7
8
n = m = 6; n = 2, m = 3;
HN
2
9 n = 3, m = 4
Et3N, THF,
r.t., 18 - 72 h,
H
N
H
N
n
H
R1
R2
N
N
a
R1 = R2 = H
m
H
R
R
R
R
1 = H, R2 = Cl b
O
O
c
1 = R2 = Cl
1 = H, R2 = Br d
5; 13a-f
from
12-40%,
monopyrrole 20 also produced in 49% using 2a,
6; 14a-f
7; 15a-f
e
1 = R2 = Br
from
from
26-86%,
28-84%,
f
R1 = H, R2 = I
1 = R2 = I g
R
from 8; 16a-f 31-51%,
21
22
2d
,
monopyrroles
(28%) and
(35%) also produced using
monopyrroles 23 (28%) and 24 (24%) also produced using 2e
9; 17a-d,1,17f
from
21-50%
H
H
R2
R1
H2N
N
N
NH2
3
n
3
HN
H
N
H
H
H
R1
R2
10 n = 3; 11 n= 4
N
N
N
N
H
3
n
3
Et3N, THF,
r.t., 18 h,
O
O
from 10 18a-f 30-46%
11 19a-f
from
19-85%
Br
Br
H
H
H
N
H
N
3
H
N
H
N
N
N
NH2
2
NH2
2
N
NH2
3
2
N
N
H
H
2
H
O
O
O
20 49%
21 28%
also produced in the reaction
22 35%
also produced in the reaction
also produced in the reaction
of 2a and 5
of 2d and 8
of 2d and 8
Br
Br
H
N
H
H
H
Br
N
Br
N
NH2
3
N
N
NH2
2
N
2
3
H
H
O
O
23
24
28%
24%
also produced in the reaction
2e
also produced in the reaction
8
2e
8
and
of
and
of
Scheme 3. Synthesis of pseudoceratidine analogues 12
pyrrole 2g were successful.
–19a
–f
,
20–24. NB: no reactions involving diiodo
Mono-pyrrole products were seldom isolated as products of these reactions, with the exception of
mono-pyrrole 20, which was synthesised in addition to the desired dipyrrole 13a, from pyrrole 2a and
N’-(2-aminoethyl)ethane-1,2-diamine
with the asymmetric triamine N’-(2-aminoethyl)propane-1,3-diamine
of mono-pyrroles 21 24 along with their respective di-pyrrole analogues 16d and 16e (Scheme 3).
To generate further mono-pyrrole pseudoceratidine analogues, N’,N’-dimethylpropane-1,3-diamine 25
and tert-butyl (3-aminopropyl)carbamate 26 were reacted with pyrroles 2a , using the aforementioned
5. Additionally, the reaction of brominated pyrroles 2d and 2e
8
, formed appreciable amounts
–
–
g

Molecules 2020, 25, 2713
6 of 13
coupling procedure, providing mono-pyrroles 27a
–f
and 28a
–
f, respectively (Scheme 4). Once again,
it was found that di-iodo pyrrole 2g did not undergo the reaction. In total, 65 compounds were
successfully synthesised, achieving the goal of generation of a large library of pseudoceratidine
analogues which, together, it was hoped would allow for the desired SAR to be developed.
Scheme 4. Synthesis of mono-pyrrole pseudoceratidine analogues 27a
involving diiodo pyrrole 2g were successful.
–f, 28a–f. NB: no reactions
The antibacterial activity of all 65 synthesised compounds against both Gram-positive (S. aureus)
and Gram-negative (E. coli) bacteria was assessed by measuring the MIC (minimal inhibitory
concentration). The known antibiotics streptomycin and polymyxin B were used as positive controls
against S. aureus and E. coli, respectively (Table 2). The measured MIC values of pseudoceratidine
were in accordance with those previously reported in the literature [17 19] and unless otherwise stated,
M for S. aureus and 128
1
–
the highest concentration of each compound that was tested was 32
µ
µM
for coli.
Table 2. Antibacterial activity of all synthesised analogues in comparison to standard antibiotics.
MIC (µM)
Compound
S. Aureus
E. Coli
Pseudoceratine 1
Streptomycin
Polymyxin B
8–16
0.625–1.25
-
32–64
-
0.3–0.6
12a
12b
12c
12d
12e
12f
13a
13b
13c
13d
13e
13f
14a
14b
14c
14d
14e
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
8–16
>32
8–16
>128
>64 †
>64 †
>64 †
>64 †
>64 †
>128
>128
>64 †
>128
>64 †
>64 †
>128
>128
64–128
>128
32–64

Molecules 2020, 25, 2713
7 of 13
Table 2. Cont.
MIC (µM)
Compound
S. Aureus
E. Coli
14f
15a
15b
15c
15d
15e
15f
16a
16b
16c
16d
16e
16f
17a
17b
17c
17d
17f
18a
18b
18c
18d
18e
18f
19a
19b
19c
19d
19e
19f
27a
27b
27c
27d
27e
27f
28a
28b
28c
28d
28e
28f
20
>32
>32
>32
4–8
>32
4–8
>128
>128
>128
32–64
>64
16–32
>64
>32
>32
>32
16–32
>32
8–16
>32
>32
>32
>32
>32
>32
>32
>32
4–8
>128
>128
32–64
>128
64–128
>128
>128
>128
128–256
>128
>128
>128
64–128
32–64
64–128
32–64
64–128
>128
>128
128–256
64–128
32–64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16–32
2–4
16–32
>32
>32
4–8
16–32
2–4
16–32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
21
22
23
24
†
Starting concentration is lower due to the insolubility of compounds at higher concentrations.
Pleasingly, ten synthetic analogues were equally or more potent than the natural product,
pseudoceratidine —an excellent result considering no previously synthesised pseudoceratidine
1
analogue had been found to exhibit comparable activity to the lead compound (Figure 2). It was
found that no mono-pyrrole analogues showed activity of note, while the most potent analogues were
those that had di-bromo or di-chloro substitution on each of its two pyrrole moieties. This particularly
highlights both the importance of the presence of both pyrrole units, as well as the positive effects

Molecules 2020, 25, 2713
8 of 13
of dihalogenation of each pyrrole. It can also be seen that brominated analogues display more
potent activity than their chlorinated counterparts. The lack of activity exhibited by the putrescine
analogues 12a
–f attest to the necessity of at least one secondary amine functionality in the linear chain,
although it should be noted that the N’-(2-aminoethyl)ethane-1,2-diamine derived analogues 13a
–
f
also displayed no notable antibacterial activity despite possessing a secondary amine in its structure.
While the presence of secondary amines in the chain was shown to be important, the symmetry
of this chain did not seem to hold any significance on its activity, although it was observed that
analogues with longer chains seems to be more active than those with shorter chains. The more potent
activity in Gram-positive S. aureus appears to indicate that the antibacterial effects exerted by the
compounds is dictated by the difference in the membrane composition of the bacterial species with these
compounds [26]. Gram negative bacteria are known to have an additional outer-membrane barrier
which is difficult to penetrate [27,28] so it appears these compounds are far more active against bacteria
that possess a single lipid membrane. Analogues possessing a longer carbon chain are potentially able
to penetrate the bacterial cell membrane more effectively.
Figure 2. Pseudoceratidine
antibacterial activity than the natural product pseudoceratidine
1
and the analogues shown to display comparative or more potent
. Gram-positive minimal inhibitory
1
concentration (MIC) values are also given. N. B. 16c showed equivalents activity to
1 against
Gram-negative bacteria.
Furthermore, the cytotoxicity of pseudoceratidine
1 and these ten analogues with comparable
or better anti-bacterial activities was determined against HEK293 human embryonic kidney cells
(Table 3). As can be seen, many of the most analogues with potent anti-bacterial activity were also

Molecules 2020, 25, 2713
9 of 13
shown to be cytotoxic against human cells at 16
µM. Of particular note, however, were two of the
analogues, 18c and 19e, which showed almost no cytotoxic activity at concentrations 2–4 times the
MIC. It is interesting that other analogues similar to these, including 19e itself, have been tested for
their cytotoxicity and in general were found to be far less toxic versus the HepG2 liver cell line [16].
This shows that these compounds not only exhibit different activities verses various bacteria but also
against different mammalian cell lines. Whilst there is a clear SAR associated with antibacterial acitivty,
stated above, there is no obvious SAR trends for cytotoxic activity.
Table 3. Cytotoxic activity of
1 and other compounds in Figure 2 against HEK293 human embryonic
kidney cells when treated with 16 µM of the stated compound.
Compound
Relative Growth % *
Pseudoceratine 1
0.7 ± 0.8
0.5 ± 0.3
1.1 ± 0.9
1.8 ± 2.0
1.3 ± 0.8
2.5 ± 1.8
2.6 ± 2.0
98.0 ± 4.5
2.1 ± 1.5
1.6 ± 2.0
94.5 ± 5.9
14c
14e
15c
15e
16c
16e
18c
18e
19c
19e
* relative compared to a positive control with no treatment. The values are the average of triplicate
experiments ± standard deviation.
3. Experimental Procedures
3.1. General Details
All reactions were carried out under a nitrogen atmosphere in dry, freshly distilled solvents unless
otherwise noted. NMR spectra were recorded on a either a 300 or 400 MHz spectrometer. Chemical
shifts are reported relative to the solvent peak of chloroform and/or CDCl3 (
13C, respectively) or d6-DMSO ( 2.50 for 1H and 39.5 for 13C). 1H-NMR data is reported as position
), relative integral, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad peak;
qd, quartet of doublets), coupling constant (J, Hz), and the assignment of the atom. 13C-NMR data
are reported as position ( ) and the assignment of the atom. NMR assignments were performed
δ δ 77.16 for
7.26 for 1H and
δ
δ
(δ
δ
using HSQC and HMBC experiments. High-resolution mass spectroscopy (HRMS) was carried out by
electrospray ionization (ESI) on a MicroTOF-Q mass spectrometer. Unless noted, chemical reagents
were used as purchased.
3.2. General Procedures
General Procedure A: the coupling of substituted pyrroles with amine for synthesis of di-pyrrole
analogues 12–19.
To a solution of pyrrole 2 (2 equiv.) in dry THF at r.t., under an atmosphere of nitrogen, amine
(1 equiv.) and triethylamine (4 equiv.) were added dropwise. The mixture was stirred for 18–72 h and
the solvent was removed in vacuo to give the crude product, which was purified as stated.
General Procedure B: the coupling of substituted pyrroles with amine for synthesis of mono-pyrrole
analogues 27 and 28.
To a solution of pyrrole 2 (1 equiv.) in dry THF at r.t., under an atmosphere of nitrogen, amine
(1 equiv.) and triethylamine (4 equiv.) was added dropwise. The mixture was stirred for 18–72 h and
the solvent was removed in vacuo to give the crude product which was purified as stated.

Molecules 2020, 25, 2713
10 of 13
3.3. Synthesis of Compounds
The experimental procedures and characterisation data for all synthesised compounds are provided
in the supporting information file.
3.4. Antibacterial Assays
The MIC was determined using the broth microdilution method [29
broth base was prepared in milli-Q water, then autoclaved at 120 ◦C for 1 h before allowing it to cool
to room temperature. A single colony of each bacterial strain (E. coli DH5 and S. aureus 16207) was
inoculated in MHB and incubated at 37 ◦C overnight. Overnight cultures were adjusted to 106 CFU/mL
in MHB according to the McFarland Standard,100 L of which was further diluted to a total of 10 mL
in the media. The stock solutions of the compounds were prepared at 12.8 mM in purified DMSO.
The stock solutions were further diluted in milli-Q water to 64 M in 0.5% DMSO for testing against
S. aureus, and 256 M in 2% DMSO for testing against E. coli. Compounds that were not soluble in
–32]. Mueller Hinton (MH)
α
µ
µ
µ
these solvent mixtures were further diluted in half to get clear solutions for the bioassay. DMSO is
known to have weak antibacterial activity [33]. To exclude the antibacterial effect of DMSO from the
results, a solution of 2% DMSO in milli-Q water was used as an additional control in the experiments.
The compounds and three controls (the natural product pseudoceratidine
streptomycin for S. aureus, and polymyxin B for E. coli) then underwent a dilution series. The assay
was performed by adding 50 L of each compound solution at the different concentrations and 50
1 for both strains,
µ
µL
of the diluted bacterial culture to the different wells of a 96-well microtiter plate. The experiments
were performed at least thrice and in triplicates. MIC after 24 h of incubation was determined by both
visual inspection and absorbance at 600 nm using an EnSpire Multimode plate reader.
3.5. Cytotoxicity Assay
As described previously [34], cell proliferation was measured by sulforhodamine B colorimetric
assay in which 2000 HEK293 human embryonic kidney cells were seeded in 96-well plates in the
presence of varying concentrations of drugs. The sulforhodamine B colorimetric assay [35], which is
based on the measurement of cellular protein content, was used to measure cell density. After drug
treatment for 3 days, cells were fixed with 10% (w/v) trichloroacetic acid and stained with 0.057% (w/v)
sulforhodamine B for 30 min, and the excess dye was removed by washing repeatedly with 1% (v/v)
acetic acid. The protein-bound dye was dissolved in Tris base solution (10 mM) for optical density
determination at 510 nm using a microplate reader. All experiments were done in triplicate, and were
repeated at least twice. The optimal cell densities were previously determined to select initial cell
densities that ensured that the cells were in the logarithmic phase for the experiments.
4. Conclusions
Pseudoceratidine
1 and 64 closely-related analogues have been successfully synthesised,
constituting a comprehensive series of compounds that explore structural motifs, most of which have
not previously been investigated before. This synthesis utilised an efficient, divergent methodology
that allowed for a large number of final compounds to be synthesised from common starting materials.
Following the successful synthesis of this library of compounds, antibacterial testing against both
Gram-positive and Gram-negative bacteria was performed, allowing for significant insight into the
SAR of pseudoceratidine 1 and the establishment of essential structural features that are required for
its antibacterial activity. It was able to be established that these analogues are most effective against
Gram-positive bacteria and furthermore that features essential for good anti-bacterial activity include
two pyrrole units and the presence of one or more secondary amines in the linear chain between units.
It was also verified that the halogenation of the pyrrole units is essential for activity, with dichloro and
dibromo pyrroles featuring in the most active analogues. It was also shown that while the symmetry
of the chain was not important, longer chain compounds were more active. This work afforded

Molecules 2020, 25, 2713
11 of 13
analogues with more potent activity than the parent compound pseudoceratidine
1
and allowed for the
establishment and verification of a detailed antibacterial SAR (Figure 3). This work has highlighted the
ability to prepare compounds in this class with improved activity over the natural product and provides
an excellent basis for further investigation into analogues with even greater potency. Whilst some of
the most active compounds also showed mammalian cell cytotoxicity, the novel compound 18c and
19e were found to be potent antibacterial agents whilst having almost no cytotoxic activity.
Figure 3. Summary of the SAR of pseudeoceratidine analogues revealed during this investigation.
Supplementary Materials: The following are available online. The experimental procedures and characterisation
data for all synthesised compounds are provided in the supporting information file.
Author Contributions: Conceptualization, D.B., M.v.R. and L.I.P.; Data curation, S.A.L., K.G.V., K.S., B.C. and
L.I.P.; Formal analysis, R.C.D.; Funding acquisition, D.B., V.S. and L.P.; Investigation, S.L. and K.G.V.; Methodology,
D.B., K.G.V., M.v.R. and V.S.; Project administration, D.B.; Resources, D.B., B.R.C. and V.S.; Supervision, D.B., K.S.,
M.v.R., M.M.C., S.A.L. and L.I.P.; Writing—original draft, D.B., B.R.C., V.S. and L.I.P.; Writing—review & editing,
D.B., R.C.D. and L.I.P.. All authors have read and agreed to the published version of the manuscript.
Funding: The research was funded by the MacDiarmid Institute for Advanced Materials and Nanotechnology
and Lottery Health Research New Zealand.
Acknowledgments: We thank the MacDiarmid Institute for Advanced Materials and Nanotechnology for funding
K.G.V and Lottery Health Research New Zealand for funding L.I.P. We also thank the University of Auckland for
additional support.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1.
2.
3.
4.
5.
6.
7.
Carmona-Gutierrez, D.; Kroemer, G.; Madeo, F. One Cell, One Love: A Journal for Microbial Research.
Microb. Cell 2014, 1, 1–5. [CrossRef] [PubMed]
Gilbert, J.A.; Neufeld, J.D. Life in a World without Microbes. PLoS Biol. 2014, 12, e1002020. [CrossRef]
[PubMed]
Microbiology Society. About Microbiology—Bacteria. Available online: https://microbiologyonline.org/
about-microbiology/introducing-microbes/bacteria (accessed on 10 June 2019).
Wang, B.; Yao, M.; Lv, L.; Ling, Z.; Li, L. The Human Microbiota in Health and Disease. Engineering 2017, 3,
71–82. [CrossRef]
Kardos, N.; Demain, A.L. Penicillin: The Medicine with the Greatest Impact on Therapeutic Outcomes.
Appl. Microbiol. Biotechnol. 2011, 92, 677–687. [CrossRef] [PubMed]
Aminov, R.I. A Brief History of the Antibiotic Era: Lessons Learned and Challenges for the Future.
Front. Microbiol. 2010, 1, 1–7. [CrossRef]
Lobanovska, M.; Pilla, G. Penicillin’s Discovery and Antibiotic Resistance: Lessons for the Future? Yale J.
Biol. Med. 2017, 90, 135–145.

Molecules 2020, 25, 2713
12 of 13
8.
9.
Zhang, J.; Chen, J.; Liang, Z.; Zhao, C. New lignans and their biological activities. Chem. Biodivers. 2014, 11,
1–54. [CrossRef]
Montaser, R.; Luesch, H. Marine natural products: A new wave of drugs? Future Med. Chem. 2011, 12,
1475–1489. [CrossRef]
10. Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Drug development from marine natural products.
Nat. Rev. Drug Discov. 2009, 8, 69–85. [CrossRef]
11. Altmann, K.H. Drugs from the Oceans: Marine Natural Products as Leads for Drug Discovery. Chim. Int.
J. Chem. 2017, 71, 646–652. [CrossRef]
12. Munro, M.H.G.; Blunt, J.W.; Dumdei, E.J.; Hickford, S.J.H.; Lill, R.E.; Li, S.; Battershill, C.N.; Duckworth, A.R.
The discovery and development of marine compounds with pharmaceutical potential. J. Biotechnol. 1999, 70,
15–25. [CrossRef]
13. Blunt, J.W.; Copp, B.R.; Munro, M.H.G.; Northcote, P.T.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep.
2003, 20, 1–49. [CrossRef]
14. Melander, R.J.; Liu, H.; Stephens, M.D.; Bewley, C.A.; Melander, C. Marine Sponge Alkaloids as a Source of
Anti-Bacterial Adjuvants. Bioorg. Med. Chem. Lett. 2016, 26, 5863–5866. [CrossRef]
15. Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. Pseudoceratidine: A New Antifouling Spermidine Derivative
from the Marine Sponge Pseudoceratina Purpurea. Tetrahedron Lett. 1996, 37, 1439–1440. [CrossRef]
16. Parra, L.L.L.; Bertonha, A.F.; Severo, I.R.M.; Aguiar, A.C.C.; de Souza, G.E.; Oliva, G.; Guido, R.V.C.;
Grazzia, N.; Costa, T.R.; Miguel, D.C.; et al. Isolation, Derivative Synthesis, and Structure–Activity
Relationships of Antiparasitic Bromopyrrole Alkaloids from the Marine Sponge Tedania Brasiliensis.
J. Nat. Prod. 2018, 81, 188–202. [CrossRef]
17. Behrens, C.; Christoffersen, M.W.; Gram, L.; Nielsen, P.H. A Convenient Synthesis of Pseudoceratidine and
Three Analogs for Biological Evaluation. Bioorg. Med. Chem. Lett. 1997, 7, 321–326. [CrossRef]
18. Ponasik, J.A.; Conova, S.; Kinghorn, D.; Kinney, W.A.; Rittschof, D.; Ganem, B. Pseudoceratidine, a Marine
Natural Product with Antifouling Activity: Synthetic and Biological Studies. Tetrahedron 1998, 54, 6977–6986.
[CrossRef]
19. Ponasik, J.A.; Kassab, D.J.; Ganem, B. Synthesis of the Antifouling Polyamine Pseudoceratidine and Its
Analogs: Factors Influencing Biocidal Activity. Tetrahedron Lett. 1996, 37, 6041–6044. [CrossRef]
20. Dubis, A.T.; Domagała, M.; Grabowski, S.J. Spectroscopic and Theoretical Studies on Some New
Pyrrol-2-Yl-Chloromethyl Ketones. New J. Chem. 2010, 34, 556. [CrossRef]
21. Jones, P.; Kinzel, O.; Llauger Bufi, L.; Muraglia, E.; Pescatore, G.; Torrisi, C. Pyrrolo[1,2-a]Pyrazin-1(2H)-One
and Pyrrolo[1,2-d][1,2,4]Triazin-1(2H)-One Derivatives as Inhibitors of Poly(ADP-ribose)Polymerase (PARP).
WO2007138355A1. U.S. Patent 7,834,015, 6 December 2007.
22. Essa, A.H.; Lerrick, R.I.; Tuna, F.; Harrington, R.W.; Clegg, W.; Hall, M.J. Reduction of 2,2,2-Trichloro-
1-Arylethanones by RMgX: Mechanistic Investigation and the Synthesis of Substituted α,α-Dichloroketones.
Chem. Commun. 2013, 49, 2756. [CrossRef]
23. Richards, J.J.; Ballard, T.E.; Huigens, R.W.; Melander, C. Synthesis and Screening of an Oroidin Library
against Pseudomonas Aeruginosa Biofilms. ChemBioChem 2008, 9, 1267–1279. [CrossRef] [PubMed]
24. Wang, J.; Fan, C.; Zhang, L.; Guo, Z.; Li, Y.; Yang, S.; Yan, S.; Zhu, J.; Yang, Q.; Zhang, M. 4-(Substituted
Anilino)-Quinazoline Derivatives Useful as Tyrosine Kinase Inhibitors. WO2009012647A1. U.S. Patent
8,916,574, 29 January 2009.
25.
B
é
langer, P. Electrophilic substitution on 2-trichloroacetylpyrrole. Tetrahedron Lett. 1979, 20, 2505–2508.
[CrossRef]
26. Lv, Y.; Wang, J.; Gao, H.; Wang, Z.; Dong, N.; Ma, Q.; Shan, A. Antimicrobial Properties and Membrane-Active
Mechanism of a Potential -Helical Antimicrobial Derived from Cathelicidin PMAP-6. PLoS ONE 2014
9, e86364. [CrossRef] [PubMed]
α
,
27. Hartmann, M.; Berditsch, M.; Hawecker, J.; Ardakani, M.F.; Gerthsen, D.; Ulrich, A.S. Damage of the bacterial
cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning
electron microscopy. Antimicrob. Agents Chemother. 2010, 54, 3132–3142. [CrossRef]
28. Alkotaini, B.; Anuar, N.; Kadhum, A.A.H. Evaluation of Morphological Changes of Staphylococcus aureus
and Escherichia coli Induced with the Antimicrobial Peptide AN5
1868–1878. [CrossRef] [PubMed]
−
1. Appl. Biochem. Biotechnol. 2015, 175,

Molecules 2020, 25, 2713
13 of 13
29. Wiegand, I.; Hilpert, K.; Hancock, R.E.W. Agar and Broth Dilution Methods to Determine the Minimal
Inhibitory Concentration (MIC) of Antimicrobial Substances. Nat. Protoc. 2008, 3, 163–175. [CrossRef]
[PubMed]
30. De Zoysa, G.H.; Cameron, A.J.; Hegde, V.V.; Raghothama, S.; Sarojini, V. Antimicrobial Peptides with
Potential for Biofilm Eradication: Synthesis and Structure Activity Relationship Studies of Battacin Peptides.
J. Med. Chem. 2015, 58, 625–639. [CrossRef]
31. Varnava, K.G.; Reynisson, J.; Raghothama, S.; Sarojini, V. Synthesis, Antibacterial, and Antibiofilm Potential
of Human Autophagy 16 Polypeptide and Analogues. Pept. Sci. 2018, 110, e24076. [CrossRef]
32. Varnava, K.G.; Mohid, S.A.; Calligari, P.; Stella, L.; Reynison, J.; Bhunia, A.; Sarojini, V. Design, Synthesis,
Antibacterial Potential, and Structural Characterization of N-Acylated Derivatives of the Human Autophagy
16 Polypeptide. Bioconjug. Chem. 2019. [CrossRef]
33. Ansel, H.C.; Norred, W.P.; Roth, I.L. Antimicrobial Activity of Dimethyl Sulfoxide against Escherichia Coli,
Pseudomonas Aeruginosa, and Bacillus Megaterium. J. Pharm. Sci. 1969, 58, 836–839. [CrossRef]
34. Leung, E.; Kim, J.E.; Askarian-Amiri, M.; Joseph, W.R.; McKeage, M.J.; Baguley, B.C. Hormone resistance
in two MCF-7 breast cancer cell lines is associated with reduced mTOR signaling, decreased glycolysis,
and increased sensitivity to cytotoxic drugs. Front. Oncol. 2014, 4, 4. [CrossRef]
35. Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 2006, 1,
1112–1116. [CrossRef]
Sample Availability: Samples of the compoundsare available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).