Phidianidine A and Synthetic Analogues as Naturally Inspired Marine Antifoulants

Christophe Labriere, Vijayaragavan Elumalai, Jannie Sta ﬀ ansson, Gunnar Cervin, Ti ﬀ any Le Norcy,

Article

Phidianidine A and Synthetic Analogues as Naturally Inspired

Marine Antifoulants

Hugo Denardou, Karine Rehel, Lindon W. K. Moodie, Claire Hellio, Henrik Pavia, Jørn H. Hansen,

and Johan Svenson *

Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00881

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sı Supporting Information

ABSTRACT: Stationary and slow-moving marine organisms regularly

employ a natural product chemical defense to prevent being colonized by

marine micro- and macroorganisms. While these natural antifoulants can

be structurally diverse, they often display highly conserved chemistries

and physicochemical properties, suggesting a natural marine antifouling

pharmacophore. In our current report, we investigate the marine natural

product phidianidine A, which displays several chemical properties found

in highly potent marine antifoulants. Phidianidine A and synthetic

analogues were screened against the settlement and metamorphosis of

Amphibalanus improvisus cyprids, and several of the compounds displayed

inhibitory activities at low micromolar concentrations with IC₅₀values

down to 0.7 μg/mL observed. The settlement study highlights that

phidianidine A is a potent natural antifoulant and that the scaﬀold can be

tuned to generate simpler and improved synthetic analogues. The bioactivity is closely linked to the size of the compound and to its

basicity. The study also illustrates that active analogues can be prepared in the absence of the natural constrained 1,2,4-oxadiazole

ring. A synthetic lead analogue of phidianidine A was incorporated in a coating and included in antifouling ﬁeld trials, where it was

shown that the coating induced potent inhibition of marine bacteria and microalgae settlement.

arine organisms represent a rich source of novel

equipment. Methods to provide eﬀective and long-term

bioactive natural products with signiﬁcant chemical

marine surface protection without general toxicity to nontarget

marine organisms are lacking. Hence there is an unmet need

diversity and high potency. As a result, several drugs and

drug leads in clinical trials are of marine origin. While slow-

moving marine organisms are under a constant threat from

predators, sessile organisms also have to deal with the dangers

of being colonized and overgrown. In response to these threats,

several marine organisms have developed a sophisticated

chemical defense to deter predators, colonizers, and com-

for novel and environmentally friendly antifouling solutions.

Developing repelling antifouling compounds inspired by

,13

Nature represent a promising strategy.

A particular group of marine natural products (MNP) with

broad antifouling activities are brominated dipeptidic deriva-

tives often isolated from stationary marine organisms. The

MNPs in this group share several structural features such as a

brominated aromatic moiety, regularly tyrosine or tryptophan,

a constrained or heterocyclic center, and often a cationic arm

terminated with a guanidine derivative. Ianthelline, initially

petitors. Several of these natural products do not directly kill

their target organisms but instead function as repellants at low

concentrations toward colonizing marine micro- and macro-

organisms by acting as antifoulants.

discovered in the Bahamian sponge Ianthella ardis has

Being able to prevent the settlement and fouling of marine

structures by marine organisms is complicated, and biofouling

countermeasures represent a signiﬁcant challenge and cost for

recently been shown to be a potent natural antifoulant.

Ianthelline inhibits the settlement and metamorphosis of

barnacle cyprids (IC = 6 μM) and also displays a pronounced

many marine industries. Biofouling is a rapid and naturally

occurring process aﬀecting all untreated submerged surfaces

,10

Received: August 7, 2020

which results in unwanted maintenance costs.

The main

challenges are associated with the loss of structural integrity

and performance (often increased drag) by the added weight

and thickness/rugosity to marine devices and structure.

Furthermore, biofouling may promote corrosion, which

signiﬁcantly reduces the eﬀective operation life-span of marine

XXXX American Chemical Society

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 1. Chemical structure of phidianidine A (1) and a selection of natural potent marine barnacle settlement inhibitors with inhibitory

concentration (IC ) included.

eﬀect on marine bacteria (minimum inhibitory concentration

study is aimed at evaluation of the antifouling properties of 1

and synthetic analogues. In this report, 1 and a library of 10

synthesized analogues (8a−e, 9a−d, 10) were prepared and

evaluated for the ability to prevent the settlement of A.

improvisus cyprid. Furthermore, the most promising compound

was formulated into a coating and evaluated in ﬁeld studies for

3 months.

(

MIC) values 0.1−10 μg/mL). The 2,5-diketopiperazine

barettin, initially isolated from the sponge Geodia baretti in

Swedish waters together with structural analogues, prevents the

settlement of Amphibalanus improvisus at 0.9 μM concen-

trations. The high potency of barettin has spurred the

synthesis of simpliﬁed analogues with ranging bioactiv-

7−19

ities.

The pyrrole-imidazole oroidin is found in sponges

of the Agelasidae family where it is considered a likely biogenic

precursor to bromoageliferin 1. In addition to being able to

inhibit marine bacterial colonization and bioﬁlm formation,

oroidin has also been shown in analogy to both ianthelline and

barettin, to be a moderate inhibitor of barnacle settlement with

a reported IC₅₀value of 49 μM. Furthermore, the

synoxazolidinones from the cold-water colonial ascidian

3−25

Synoicum pulmonaria

and the numerous bastadin

analogues commonly isolated from Ianthella marine sponges

have also been assigned as potent inhibitors of marine micro-

,26,27

and macrofouling.

A selection of potent natural marine

inhibitors of barnacle settlement are presented in Figure 1.

Several of the reported marine antifoulants have also been

reported with other bioactivities such as antitumoral, anti-

8,28

21,23

inﬂammatory,

acetylcholinesterase.

a₉n_,₃t₀ibacterial,

and as inhibitors of

Phidianidine A (1), with an uncommon 1,2,4-oxadiazole

RESULTS AND DISCUSSION

■

ring linked to the brominated indole system, is a MNP isolated

Library Design and Synthesis. The library of compounds

was designed to probe the contribution of structure to the

activity as previously successfully demonstrated for natural

from the aeolid opisthobranch mollusk Phidiana militaris.

Compound 1 is structurally and chemically highly analogous to

ianthelline, barettin, and the synoxazolidinones and thus

displays the structural features often linked to a high

35−37

antifoulants.

With an emphasis on ease of preparation,

only 1 was prepared with the 1,2,4-oxadiazole ring motif

antifouling activity. Initial studies of 1 revealed cytotoxic

activity, and more recently, 1 and synthetic analogues showed

according to published routes. The rest of the analogues

were designed with an amide bond linkage between the

brominated indole and the cationic arm. Studies by Lin and

Snider have reported a signiﬁcant loss of activity when the

indole bromine is excluded in analogues of 1 and it was

decided to keep the 6-bromoindole motif intact. These

observations have also been reported for analogues of barettin

immunosuppressive properties. In addition, 1 was also shown

to be a selective inhibitor of the dopamine transporter and a

selective, potent ligand and partial agonist of the μ-opioid

receptor (versus δ- and κ-opioid receptors). Furthermore, 1

was identiﬁed using virtual screening and experimentally

veriﬁed as a new antagonist of CXCR4 which is a chemokine

receptor associated with several diseases like HIV, rheumatoid

and the synoxazolidinones when the halogens are removed.

Instead focus was placed on the length of the cationic arm and

the nature of the terminal cationic group, amine vs guanidine.

Two of the compounds were also N-methylated and one also

contained a quaternary ammonium group. Compound 1 and

its analogues 8−10 were all prepared in generally good yields

arthritis, and cancer.

Inspired by the structural resemblance of 1 with several

reported highly active antifouling MNPs and the straightfor-

ward synthetic tools available for modiﬁcation, the current

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 2. Synthetic outline for compounds 8−10.

with the exception of the N-methylated 8e and 9d (8 and 26%,

respectively). The synthetic scheme employed for preparing

compounds 8−10 is outlined in Figure 2.

Table 1. Potency and Toxicity of Tested Compounds

against the Barnacle A. improvisus

compound

metamorphosis (%) IC₅₀(μg/mL) toxicity (%)

Barnacle Settlement. Several MNPs displaying the

functionalities of 1 and analogues have been linked to

signiﬁcant inhibitory potency against the settlement and

subsequent metamorphosis of barnacle cyprids. To evaluate

the potential of the prepared library as settlement inhibitors,

they were tested against A. improvisus cyprid larvae. Barnacles

phidianidine A (1)

4.0

>5.0

2.2

represent a major fouling organism, and their tiny footprint

and colonization of man-made surfaces results in huge

economical implications, leading to multibillion dollar

39,40

maintenance costs.

A. improvisus originates from the east

0.7

coast of the American continent but is today spread worldwide

sea-nine

ianthelline

barretin

n.d.

>5.0

0.25

3.0

1.0

7.4

via shipping. It represents a major fouling organism and is

commonly found on rocks, jetties, and boat hulls where it

n.d

n.d.

forms dense colonies on suitable substrata. The extreme

tolerance to ranging salinity makes A. improvisus a relevant

synoxazolidinone A

model for studies on both biofouling and osmoregulation.

Reported at 5 μg/mL. Toxicity for the negative control DMSO

Most of the evaluated compounds displayed inhibitory

eﬀects on the settlement of the barnacle cyprids, and the dose

response behavior allowed evaluation of the IC₅₀values for

compounds displaying <50% settlement at 5 μg/mL. The

degree of inhibition of the compounds and also their potential

cyprid toxicity is compiled and compared to the positive

control Sea-nine and potent MNPs in Table 1.

(

0.1%, v/v) in ﬁltered seawater was 2.4%. Data from Moodie et al.

d 15 e

Not determined. Data from Hanssen et al. Data from Sjo

gren et

al. Data from Trepos et al.

concentrations. While several of the simpliﬁed synthetic

compounds were inactive at 5 μg/mL, compounds 9c and

9d displayed potent inhibitory properties with IC₅₀values

lower than those observed for 1 at 2.2 and 0.7 μg/mL

respectively. Compound 9d was the most eﬀective inhibitor

with activity superior to several potent antifouling MNPs. In

addition, the toxicity of 9d was low as illustrated in Figure 3.

Compound 1 displayed an IC₅₀of 4.0 μg/mL and thus it

compares well with other established MNPs such as ianthel-

line, barretin and synoxazolidione A. A 3% mortality of

the cyprids was observed at 5 μg/mL which is similar to the

negative DMSO control (2.4%) and illustrates that 1 exerts its

inhibitory activity via a nontoxic mechanism at the employed

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 3. Dose response analysis (0.1−5.0 μg/mL) of 9d on the settlement inhibition of A. improvisus cyprid larvae presented as percentages of

settled (black columns), free swimming (light gray columns), and dead cyprids (dark gray columns) and given as means ± standard error (n = 4).

DMSO (0.1%, v/v) in ﬁltered seawater was used as the negative control.

The current library was composed of compounds probing

activity is linked to the size of the compound and to the

basicity of the cationic group and the study demonstrates that

active analogues can be prepared in the absence of the

constrained 1,2,4-oxadiazole ring.

the eﬀects of basicity and hydrogen bonding capacity of the

cationic arm and also the distance between the brominated

indole and the cationic charge. Several structure activity

relationship studies on these speciﬁc functionalities and

Coating Development. Given the high and repelling

activity of 9d, it was decided to evaluate the ability of the

compound to inhibit fouling in ﬁeld immersion experiments.

Such an experiment would also establish the general

antifouling potential of the compound as the natural biofouling

consortium contains microfoulers such as marine bacteria,

micro- and macroalgae, and other potential macrofoulers in

addition to barnacles. Many studies are published on

antifouling experiments performed in laboratory bioassay

settings but it is often highly speculative to conclude about

the repelling potential of a compound or coating without

7,43

properties have been described for related MNPs

natural antifoulants.

and

35,37,44

In Nature, most of these MNPs

resemble a dipeptide in size and this library contain

compounds within that size span with a number of shorter

analogues. The active compounds 1, 9c, and 9d share several

structural features. They all bear guanidine functionalities, and

they all represent the longest compounds in the libraries with

either ﬁve or six methylenes in the alkane linker. 9d is the

longest compound in the study and also the most active one.

The shorter guanidine bearing compounds 9a and 9b are

inactive as are the primary amines 8a−8e, indicating that the

increased basicity and hydrogen bonding capacity is essential

access to data from ﬁeld experiments. In addition, the search

for new environmentally friendly, cost-eﬀective, and legally

acceptable ways of preventing growth of marine organisms on

for activity. This correlates well with related antifouling

MNPs which commonly display a guanidine or a guanidine

marine materials is urgent, as reviewed by Ciriminna and

5,16,22

derivative.

Compound 10, which displays a cationic

Kyei. To generate a coating suited for ﬁeld studies, a

quaternary ammonium group, is inactive. The N-methylated 8e

represents an amine version of the highly active 9d but is

inactive and illustrates how potential beneﬁts arising from the

N-methylation is not suﬃcient in the absence of a guanidine

functionality. Like all the compounds in the library, the indole

motifs of 9c and 9d are linked with the cationic arm with an

amide bond as opposed to the natural 1,2,4-oxadiazole ring

found in 1. Both 9c and 9d display higher antifouling activity

than 1, which illustrates that the 1,2,4-oxadiazole ring is not

essential for activity if suﬃcient linker length and basicity is

provided. This is an important observation and many studies

on analogues of 1 in medically relevant screens have

incorporated the synthesis of the 1,2,4-oxadiazole ring system

previously developed biodegradable poly(ε-caprolactone-co-δ-

valerolactone) (80:20) polymer for antifouling applications

was prepared and solubilized in xylene. Compound 9d was

added to the polymer, and 200 μm thick polymer coatings

were prepared on PVC panels, which were submerged for 84

days at a depth of 1 m in the port of Kernevel in Lorient,

France during the summer. The panels were regularly

investigated for growth during the 84-day study, and the

growth on the panels during the period is shown in Figure 4.

After 84 days the panels were removed from the water and

the growth was assessed using confocal laser scanning

microscopy (CLSM). On a macroscopic level no obvious

diﬀerences between the diﬀerent panels can be observed. No

pronounced “edge eﬀects”, often observed during evaluation

novel antifouling coatings, were seen on the treated panels

32,33

into the library of screened compounds.

Our studies

suggest that signiﬁcantly simpliﬁed analogues can be used to

yield bioactive compounds with even improved activity.

Collectively, the barnacle cyprid screen highlights that 1 is a

potent natural antifoulant and that the scaﬀold can be used to

generate simpler and improved synthetic analogues. The

either. Growth was observed on the treated panels after 28

days, while the control panels displayed growth after 14 days.

This small diﬀerence however disappears with time. At the end

of the experiments, the panels are seemingly covered to a

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

powerful inhibitors of the microfouling bioﬁlm with IC values

down to 10 ng/mL reported, which is 2 orders of magnitude

lower than the concentration of 9d in the coating. This

illustrates how this class of compounds are often highly

repelling against both micro- and macrofoulers. In parallel,

while studies are ongoing on evaluating extracts of marine

organisms to generate eﬀective marine antifouling coatings,

focus on single compounds of known concentrations provides

a more extensive understanding of the potency and mode of

action. Being able to generate functional and environmentally

benign coatings based on natural products or simpliﬁed

synthetic mimics has been heralded as a promising way toward

a new generation of antifouling coating technologies, and our

work sheds additional light on the performance of these

compounds and materials in vitro and in the ﬁeld.

CONCLUSIONS

■

The antifouling potential of the MNP phidianidine A (1) has

been established, and our study illustrates that 1 is a potent,

nontoxic inhibitor of barnacle cyprid metamorphosis. Struc-

ture−activity studies employing simpliﬁed synthetic analogues

illustrate that the antifouling activity against A. improvisus

barnacles can be tuned, and optimized analogues can readily be

prepared while excluding the 1,2,4-oxadiazole ring. A

promising antifouling lead was identiﬁed (9d), and it was

incorporated into an antifouling coating and evaluated in ﬁeld

trials for 84 days. A strong reduction in the settlement of

microfouling organisms was shown, illustrating that the lead

analogue also is a powerful inhibitor of microfouling. The

eﬀect on barnacle settlement in the ﬁeld was inconclusive

because of low amounts of naturally free-swimming barnacle

cyprids during the ﬁeld experiments. The current study sheds

additional light on the structural motif of MNPs governing

slow-moving or sessile marine organism protection against

colonizing organisms and illustrates how these compounds can

be signiﬁcantly structurally simpliﬁed with maintained or even

improved bioactivities.

Figure 4. Results from the ﬁeld trials illustrating the buildup of

biofouling communities. Left column: 9d containing coating applied

to PVC panel, center column: negative control coating without 9d on

PVC panel, right column: control PVC panel without any coating.

similar extent with a layer of biofouling organisms, the bulk of

which appear to be chlorophycea (Ulva intestinalis) and

phaeophycea macroalgae. It is not surprising to ﬁnd such

organisms on the panels as the formulation did not contain any

photosynthesis inhibitors such as commercial herbicides. No

barnacles were observed on any of the panels making

assessments of the potential repelling eﬀect of 9d on the

barnacle settlement inconclusive. The reproductive period of

A. improvisus is mainly from May to September and the

EXPERIMENTAL SECTION

■

IR spectra were obtained on an Agilent Technologies Cary 630 FTIR

spectrometer. H and C NMR spectra were recorded at ambient

temperature at a frequency of 400 and 101 MHz, respectively on a

Bruker spectrometer. The chemical shifts are reported in ppm and are

referenced to the relevant solvent peak: CDCl at δ 7.26 and δ_C

presence of free swimming cyprids occurs during this period.

The reason for the general low settlement of barnacles during

the experimental period is thus likely coupled with unexpected

seasonal or local variability in free swimming cyprids and not

due to speciﬁc repelling eﬀects from the diﬀerent panels and

their coatings. CLSM analysis of the bioﬁlms however revealed

diﬀerences between the coatings as displayed in Figure 5.

The diﬀerent staining methods employed allowed quantiﬁ-

cation of both marine bacteria and microalgae. Clear

diﬀerences are seen between the coating incorporation 9d

77.16; CD OD at δ 3.31 and δ 49.0; (CD ) SO at δ 2.50 and δ

39.5 ppm. High-resolution mass spectra (HRMS) were recorded

using MeOH solution on LTQ Orbitrap XL in either positive or

negative electrospray ionization (ESI) modes. TLC was performed on

Merck silica gel 60 F254 plates, using UV light at 254 nm, and PMA

staining followed by heating for detection. Flash column chromatog-

raphy was performed by using the indicated solvent system and silica

gel (40−63 μm). Microwave reactions were performed in 20 mL vials

using a Discover SP from CEM using an Anton Parr Monowave 300

instrument. All reagents and solvents were purchased commercially

and used directly without any further puriﬁcation.

and the control coating. The coating incorporating 9d

displays both lower coverage and biovolumes of bacteria (3-

fold) and microalgae (6-fold), suggesting that 9d also displays

potent antifouling activities against marine microfoulers. The

recorded reduction in bioﬁlm establishment was observed at

lower concentrations (in the coating) than those described for

Synthesis. Phidianidine A (1). A sample of phidianidine (1) was

38,52

prepared for biological testing according to literature procedures.

The substituted 1,2,4-oxadiazole ring of 1 was prepared via the route

described for the synthesis of phidianidine by Manzo et al. from N-

Boc-1-amino-5-[(E)-2-hydroxyguanidino]-pentane and 6-bromo-3-

indoleacetic acid ethyl ester and was subsequently guanidylated

with N,N′-di-Boc-1H-pyrazole-1-carboxamidine according to Lin and

dibromohemibastadin-1 in vitro and also correlates well with

in situ studies up to 35 days. Also in those recent studies, a

more pronounced eﬀect was seen on microalgal bioﬁlm

formation than for bacterial bioﬁlms. Our current study

shows that this eﬀect persists for up to 84 days for 9d. Related

Snider. Compound 1 was prepared in 28% yield (2 steps) with

spectral data matching those previously reported. H NMR (400

compounds such as the synaxozolidiones and ianthelline are

MHz, Methanol-d ) δ 7.56−7.51 (m, 1H), 7.48−7.42 (m, 1H), 7.24

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 5. Upper row: quantiﬁcation of the biovolumes (μm /μm ) of adhered bacteria and microalgae after 84 days immersion (A) and CLSM

images used to assess the volume of the bioﬁlms (B). Bottom row illustrates a top view of the formed bioﬁlm on the surfaces coated with 9d (left)

and the control varnish (right).

(

t, J = 0.9 Hz, 1H), 7.13 (dd, J = 8.5, 1.8 Hz, 1H), 4.20 (d, J = 0.8 Hz,

H), 3.19−3.09 (m, 4H), 1.66−1.54 (m, 4H), 1.48−1.37 (m,

7.42 (dd, J = 8.6, 2.0 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d ) δ

180.6, 164.8, 139, 135.5, 127.4, 126.3, 123.2, 115.5, 114.8, 111.8.

FTMS m/z 265.9455 [M-H] (calcd for C H^B^r^N^O3⁻,

−

H). C NMR (101 MHz, Methanol-d ) δ 178.98, 170.10, 158.63,

65.9458).

2-(6-Bromo-1H-indol-3-yl)acetic Acid (4). To a microwave-vial

equipped with a stir bar were added 3 (1.00 g, 3.74 mmol, 1.0 equiv),

-ethoxyethanol (10 mL), and hydrazine (0.91 mL, 18.73 mmol, 5.0

38.89, 127.16, 125.71, 123.23, 120.82, 116.18, 115.30, 108.68, 43.77,

2.35, 29.74, 29.51, 24.90, 23.90. FTMS m/z 420.1131 [M + H]

(

calcd for C H BrN O , 420.1142).

17 23 7

-(6-Bromo-1H-indol-3-yl)-2-oxoacetic Acid (3). To a solution of

equiv). The vial was sealed and stirred for 15 min at 80 °C under

microwave irradiation. Sodium methoxide (1.84 g, 34.1 mmol, 10.0

equiv) was then added, and the reaction mixture resealed and stirred

for 1 h at 160 °C under microwave irradiation before being quenched

-bromoindole (1.00 g, 5.1 mmol, 1.0 equiv) in diethyl ether (20 mL)

was added oxalyl chloride (0.89 mL, 10.2 mmol, 2.0 equiv). The

solution was stirred at room temperature (rt) for 45 min. Water (0.5

mL, 27.8 mmol, 5.0 equiv) was carefully added to the solution,

followed by diethyl ether (50 mL). The resulting mixture was stirred

for 30 min, ﬁltered, washed with diethyl ether, and dried under

vacuum to aﬀord 3 as a yellow powder (1.05 g, 77%). IR (cm ).

NMR (400 MHz, DMSO-d ) δ 13.66 (s, 1H), 12.52 (s, 1H), 8.49 (d,

J = 3.3 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H),

with 4 M HCl (20 mL), H O (20 mL), and EtOAc (50 mL);

extracted with EtOAc (2 × 20 mL); dried over Na SO ; and the

solvent removed under reduced pressure. The crude product was

puriﬁed by silica gel column chromatography [(EtOAc + 1%

AcOH:n-heptane, 4:6)] to aﬀord 4 as a pale yellow solid (0.76 g,

−

167, 3146, 1722, 1626, 1410, 1227, 1134, 888, 795, 754, 683. H

−

80%). R = 0.125 [(EtOAc:n-heptane, 1:1)]. IR (cm ). 3417, 2933,

2895, 2117, 1693, 1402, 1223, 1097, 866, 803, 773, 695. H NMR

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(

400 MHz, Methanol-d ) δ 7.68 (d, J = 1.8 Hz, 1H), 7.26 (d, J = 8.6

The crude product was puriﬁed by silica gel column chromatography

[(CH Cl : 100 to MeOH: 100)] to aﬀord 8a as a light yellow oil

Hz, 1H), 7.20−7.16 (m, 2H), 3.69 (s, 2H). C NMR (101 MHz,

Methanol-d ) δ 176.1, 136.6, 130.4, 126.2, 125.2, 122.2, 113.9, 113,

−

(0.89 g, 73%). R = 0.03 [(MeOH: 100)]. IR (cm ). 3253, 2929,

−

08.8, 31.8. FTMS m/z 251.9670 [M-H] (calcd for C H BrNO ,

2869, 2117, 1640, 1529, 1454, 1343, 1227, 1108, 884, 795. H NMR

51.9666).

(400 MHz, Methanol-d ) δ 7.71 (d, J = 1.9 Hz, 1H), 7.27 (d, J = 8.6

Monoboc Protected Diamines (6a−d). Monoboc protected

Hz, 1H), 7.21−7.16 (m, 2H), 3.59 (s, 2H), 3.23 (t, J = 6.7 Hz, 2H),

amines 6a−d were prepared using standard protocols. The synthesis

of compound 6d is provided as a representative example.

2.57 (t, J = 6.9 Hz, 2H), 1.60 (p, J = 6.8 Hz, 2H). C NMR (101

MHz, Methanol-d ) δ 174.7, 136.7, 130.3, 126.5, 125.3, 122, 114,

tert-Butyl-(6-aminohexyl)carbamate (6d). To a solution of 1,5-

113.1, 109.4, 39.6, 37.8, 33.8, 33.3. FTMS m/z 310.0567 [M + H]

diaminohexane (5.00 g, 43.03 mmol, 4.0 equiv) in dioxane:H O (9:1,

(calcd for C H BrN O , 310.0550).

13 17 3

0 mL) was added dropwise a solution of di-tert-butyl dicarbonate

N-(4-Aminobutyl)-2-(6-bromo-1H-indol-3-yl)acetamide (8b). To

a solution of 4 (0.500 g, 2 mmol, 1.0 equiv) in CH Cl (7 mL) were

(2.47 mL, 10.76 mmol, 1.0 equiv) in dioxane: H O (9:1, 50 mL) over

h. The resulting mixture was stirred at rt overnight. The solvent was

added DIPEA (0.38 mL, 2.2 mmol, 1.1 equiv) and HATU (0.760 g, 2

mmol, 1 equiv). The mixture was stirred at rt for 30 min. A solution of

6b (0.414 g, 2 mmol, 1.1 equiv) in CH Cl (7 mL) was then added,

removed under reduced pressure and the residue was suspended in

H O (50 mL) and ﬁltered. The ﬁltrate was then extracted with

CH Cl (3 × 20 mL), washed with H O, dried over Na SO , ﬁltered,

and the resulting mixture was stirred at room rt for 90 min. A solution

of CH Cl :TFA (1:1, 20 mL) was directly added to the reaction

mixture and it was stirred for 3 h at rt. The dark brown solution was

and the solvent removed under reduced pressure to aﬀord 6d as a

−

white oil (6.6 g, 71%). IR (cm ). 3361, 2977, 2929, 2858, 1696,

529, 1369, 1253, 1171. H NMR (400 MHz, Chloroform-d) δ 4.73

quenched with H O (20 mL) and the pH adjusted to 12 with 4 M

s, 1H), 3.02 (q, J = 6.8 Hz, 2H), 2.62−2.57 (m, 2H), 1.45−1.28 (m,

NaOH (35 mL), extracted with CH Cl (2 × 20 mL), dried over

3H), 1.28−1.21 (m, 4H), 1.04 (s, 2H). C NMR (101 MHz,

Na SO , ﬁltered, and the solvent removed under reduced pressure.

2 4

The crude product was puriﬁed by silica gel column [(CH Cl : 100 to

2 2

MeOH: 100)] to aﬀord 8b as a light yellow oily solid (0.28 g, 43%).

−

tert-Butyl-(6-(methylamino)hexyl)carbamate (7). To a solution

R = 0.03 [(MeOH: 100)]. IR (cm ). 3249, 2925, 2858, 2113, 1640,

1529, 1454, 1343, 1227, 1108, 884, 795. H NMR (400 MHz,

was added di-tert-butyl dicarbonate (5.2 g, 4.7 mmol). Triethylamine

3.7 mL) was added dropwise, and the reaction stirred at rt for 2 h.

Methanol-d ) δ 7.72 (d, J = 1.8 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H),

(

7.21−7.16 (m, 2H), 3.59 (s, 2H), 3.17 (t, J = 6.7 Hz, 2H), 2.57 (t, J =

The reaction mixture was then washed with H O (20 mL) and

7.0 Hz, 2H), 1.51−1.35 (m, 4H). C NMR (101 MHz, Methanol-d )

extracted with CH Cl (2 × 50 mL). The solvent was evaporated to

δ 174.5, 136.7, 130.3, 126.5, 125.2, 122.1, 114, 113.1, 109.5, 42.1,

40.3, 33.9, 30.9, 27.7. FTMS m/z 324.0713 [M + H] (calcd for

aﬀord N-(tert-butoxycarbonyl)-6-hydroxy-1-hexylamine (4.95 g, 95%)

as pale-white solid, which was used without further puriﬁcation. H

C H BrN O , 324.0706).

14 19 3

NMR (400 MHz, Chloroform-d) δ 4.56 (s, 1H), 3.61 (td, J = 6.5, 5.3

Hz, 2H), 3.09 (q, J = 6.7 Hz, 2H), 1.77 (t, J = 5.5 Hz, 1H), 1.59−1.51

N-(5-Aminopentyl)-2-(6-bromo-1H-indol-3-yl)acetamide (8c).

To a solution of 4 (1.00 g, 3.94 mmol, 1.0 equiv) in CH Cl (10

(

m, 2H), 1.50−1.44 (m, 2H), 1.42 (s, 9H), 1.35 (ddd, J = 14.9, 9.0,

mL) were added DIPEA (0.75 mL, 4.33 mmol, 1.1 equiv) and HATU

(1.497 g, 3.94 mmol, 1 equiv). The mixture was stirred at rt for 30

min. A solution of 6c (0.876 g, 4.33 mmol, 1.1 equiv) in CH Cl (10

(

.9 Hz, 4H). C NMR (101 MHz, Chloroform-d) δ 156.1, 79.1, 62.6,

6.0, 40.4, 32.6, 31.2, 30.1, 28.4, 26.4, 25.3. To a 0 °C solution of N-

tert-butoxycarbonyl)-6-hydroxy-1-hexylamine (2.0 g, 9.2 mmol, 1.0

mL) was then added, and the resulting mixture was stirred at rt for 90

min. A solution of CH Cl :TFA (1:1, 20 mL) was directly added to

equiv) in CH Cl (20 mL) was added methane sulfonyl chloride

(

0.85 mL, 9.3 mmol, 1.1 equiv). Triethylamine (1.5 mL, 9.4 mmol,

.2 equiv) was added dropwise to the 0 °C solution, and then the

the reaction mixture and it was stirred for 3 h at rt. The dark brown

solution was quenched with H O (20 mL) and the pH adjusted to 12

reaction was stirred at rt for 5 h. The solvents were removed under

with 4 M NaOH (35 mL), extracted with CH Cl (2 × 20 mL), dried

reduced pressure to aﬀord N-(tert-butoxycarbonyl)-6-

over Na SO , ﬁltered, and the solvent removed under reduced

2 4

(

(methanesulfonyl)oxy)hexylamine (2.65 g, 97%) as white crystalline

pressure. The crude product was puriﬁed by silica gel column

chromatography [(CH Cl : 100 to MeOH: 100)] to aﬀord 8c as a

solid, which was used without further puriﬁcation. H NMR (400

MHz, CDCl ) δ 4.18 (t, J = 6.5 Hz, 2H), 3.07 (t, J = 7.0 Hz, 2H),

light yellow oily solid (0.92 g, 69%). R = 0.03 [(MeOH: 100)]. IR

−

.97 (s, 3H), 1.78−1.65 (m, 2H), 1.51−1.43 (m, 2H), 1.40 (s, 9H),

(cm ). 3242, 2929, 2858, 2125, 1640, 1529, 1439, 1343, 1227, 1108,

.39−1.27 (m, 4H). C NMR (101 MHz, CDCl ) δ 156.0, 79.1,

884, 795. H NMR (400 MHz, Methanol-d ) δ 7.72 (d, J = 1.8 Hz,

0.0, 37.3, 29.9, 29.0, 28.4, 26.2, 25.1. To a solution of N-(tert-

1H), 7.27 (d, J = 8.6 Hz, 1H), 7.22−7.15 (m, 2H), 3.59 (s, 2H), 3.17

butoxycarbonyl)-6-((methanesulfonyl)oxy)hexylamine (2.6 g, 8.97

mmol, 1.0 equiv) in dioxane (20 mL) was slowly added aqueous

methylamine (20 mL, 40%, 60 equiv). The mixture was stirred at 60

(t, J = 6.9 Hz, 2H), 2.53 (t, J = 7.0 Hz, 2H), 1.52−1.34 (m, 4H),

1.30−1.18 (m, 2H). C NMR (101 MHz, Methanol-d ) δ 174.4,

136.7, 130.3, 126.5, 125.2, 122.1, 114, 113.1, 109.5, 42.4, 40.4, 33.9,

33.4, 30.2, 25.1. FTMS m/z 338.0878 [M + H] (calcd for

C H BrN O , 338.0863).

C for 1 h and monitored by TLC (CH Cl /MeOH/NH aq.,

9.0:0.9:0.1, PMA stain), concentrated under reduced pressure to

15 21 3

aﬀord the product 7 as a light beige solid (1.2 g, 58%), which was

N-(6-Aminohexyl)-2-(6-bromo-1H-indol-3-yl)acetamide (8d). To

a solution of 4 (1.00 g, 3.94 mmol, 1.0 equiv) in CH Cl (10 mL)

used without further puriﬁcation. H NMR (400 MHz, CDCl ) δ 3.06

(

t, J = 7.0 Hz, 2H), 2.97−2.85 (m, 3H), 2.65 (t, J = 5.5 Hz, 2H), 1.83

were added DIPEA (0.75 mL, 4.33 mmol, 1.1 equiv) and HATU

(1.497 g, 3.94 mmol, 1.0 equiv). The mixture was stirred at rt for 30

min. A solution of 6d (0.937 g, 4.33 mmol, 1.1 equiv) in CH Cl (10

q, J = 7.6 Hz, 2H), 1.41 (s, 15H). C NMR (101 MHz, CDCl ) δ

56.1, 79.1, 49.2, 38.6, 32.9, 29.7, 29.3, 28.4, 26.1, 26.0, 25.7. FTMS

m/z 231.2061 [M + H] (calcd for C H N O , 231.2067).

mL) was then added, and the resulting mixture was stirred at rt for 90

min. A solution of CH Cl :TFA (1:1, 20 mL) was directly added to

N-(3-Aminopropyl)-2-(6-bromo-1H-indol-3-yl)acetamide (8a).

To a solution of 4 (1.00 g, 3.94 mmol, 1.0 equiv) in CH Cl (10

the reaction mixture and was stirred for 3 h at rt. The dark brown

mL) were added DIPEA (0.75 mL, 4.33 mmol, 1.1 equiv) and HATU

1.497 g, 3.94 mmol, 1.0 equiv). The mixture was stirred at rt for 30

min. A solution 6a (0.755 g, 4.33 mmol, 1.1 equiv) in CH Cl (10

solution was quenched with H O (20 mL) and the pH adjusted to 12

(

with 4 M NaOH (35 mL), extracted with CH Cl (2 × 20 mL), dried

over Na SO , ﬁltered, and the solvent removed under reduced

mL) was then added, and the resulting mixture was stirred at rt for 90

min. A solution of CH Cl :TFA (1:1, 20 mL) was directly added to

pressure. The crude product was puriﬁed by silica gel column

chromatography [(CH Cl : 100 to MeOH: 100)] to aﬀord 8d as a

the reaction mixture and stirred for 3 h at rt. The dark brown solution

light yellow oily solid (0.23 g, 33%). R = 0.03 [(MeOH: 100)]. IR

−

was quenched with H O (20 mL) and the pH adjusted to 12 with 4

(cm ). 3238, 2929, 2858, 2180, 1644, 1529, 1439, 1361, 1227, 1108,

M NaOH (35 mL), extracted with CH Cl (2 × 20 mL), dried over

884, 795. H NMR (400 MHz, Methanol-d ) δ 7.71 (d, J = 1.9 Hz,

Na SO , ﬁltered, and the solvent removed under reduced pressure.

1H), 7.27 (d, J = 8.6 Hz, 1H), 7.21−7.16 (m, 2H), 3.59 (s, 2H), 3.16

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(

t, J = 6.9 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 1.48−1.36 (m, 4H),

(s, 13H), 1.46 (s, 9H). ¹³C NMR (101 MHz, Methanol-d ) δ 174.5,

164.5, 157.6, 154.2, 136.7, 130.3, 126.5, 125.3, 122.1, 114, 113.1,

.31−1.22 (m, 4H). C NMR (101 MHz, Methanol-d ) δ 174.4,

36.8, 130.3, 126.5, 125.2, 122.1, 114, 113.1, 109.5, 42.3, 40.4, 38.9,

109.4, 84.4, 80.3, 41.4, 40.2, 33.9, 28.6, 28.2, 27.6, 27.5. FTMS m/z

3.9, 33.4, 30.3, 27.6. FTMS m/z 352.1027 [M + H] (calcd for

566.2031 [M + H] (calcd for C H BrN O , 566.1973).

25 37 5 5

C H BrN O , 352.1019).

The intermediate (0.24 g, 0.42 mmol, 1.0 equiv) was dissolved in

CH Cl (5 mL) and a solution of TFA:CH Cl (1:1, 10 mL) was

N-(6-Aminohexyl)-2-(6-bromo-1H-indol-3-yl)-N-methylaceta-

mide (8e). To a solution of 4 (0.560 g, 2.20 mmol, 1.0 equiv) in

CH Cl (10 mL) were added DIPEA (0.45 mL, 2.44 mmol, 1.1

equiv) and HATU (0.900 g, 2.20 mmol, 1 equiv). The mixture was

stirred at rt for 30 min. A solution of 7 (0.550 g, 2.44 mmol, 1.1

equiv) in CH Cl (10 mL) was then added, and the resulting mixture

was stirred at rt for overnight. A solution of CH Cl :TFA (1:1, 20

mL) was directly added to the reaction mixture and was stirred for 3 h

at rt. The dark brown solution was quenched with H O (20 mL) and

the pH adjusted to 12 with 4 M NaOH (35 mL), extracted with

CH Cl (2 × 20 mL), dried over Na SO , ﬁltered, and the solvent

removed under reduced pressure. The crude product was puriﬁed by

silica gel column chromatography [(CH Cl : 100 to MeOH: 100)] to

aﬀord 8e as a pale-yellow oil (61 mg, 8%) H NMR (400 MHz,

added. The reaction mixture was stirred at rt for 2 h, quenched with

H O, extracted with CH Cl (2 × 10 mL), washed with H O (2 × 10

mL), and the solvent was removed under reduced pressure to aﬀord

−

9b as a green-orange oil (167 mg, 83%). IR (cm ). 3286, 3181, 2944,

2125, 1633, 1566, 1436, 1436, 1182, 1134, 1026, 888, 840, 803, 724.

H NMR (400 MHz, Methanol-d ) δ 7.70 (d, J = 1.8 Hz, 1H), 7.27

(d, J = 8.6 Hz, 1H), 7.20−7.16 (m, 2H), 3.60 (s, 2H), 3.19 (t, J = 6.1

Hz, 2H), 3.10 (t, J = 6.9 Hz, 2H), 1.53−1.47 (m, 4H). C NMR

(101 MHz, Methanol-d ) δ 174.7, 158.5, 136.7, 130.2, 126.5, 125.2,

122, 114.1, 113.1, 109.4, 42, 39.8, 33.8, 27.5, 27.0. FTMS m/z

366.0947 [M + H] (calcd for C H BrN O , 366.0924).

15 21 5

Amino-((5-(2-(6-bromo-1H-indol-3-yl)acetamido)pentyl)amino)-

methaniminium 2,2,2-triﬂuoroacetate (9c). To a solution of 8c

(0.40 g, 1.19 mmol, 1.0 equiv) and N,N′-Di-Boc-1H-pyrazole-1-

carboxamidine (0.74 g, 2.39 mmol, 2.0 equiv) in THF (8 mL) was

added DIPEA (0.42 mL, 2.39 mmol, 2.0 equiv). The mixture was

Methanol-d ) δ 7.68−7.56 (m, 1H), 7.15 (ddt, J = 8.9, 6.2, 2.8 Hz,

H), 7.11−6.99 (m, 2H), 3.80−3.59 (m, 2H), 3.31−3.21 (m, 2H),

.03−2.77 (m, 3H), 2.56−2.41 (m, 2H), 1.36−1.21 (m, 4H), 1.14−

.94 (m, 4H). ¹³C NMR (101 MHz, Methanol-d ) δ: 172.4, 135.4,

stirred at rt for 3 h, quenched with H O (10 mL), extracted with

29.0, 124.5, 123.9, 121.0, 112.61, 111.7, 107.5, 50.2, 47.4, 47.2, 47.0,

diethyl ether (2 × 10 mL), washed with sat. NaHCO and brine (10

0.6, 35.0, 32.5, 31.1, 27.7, 26.6, 26.0. FTMS m/z 366.1176 [M + H]

mL), dried over Na SO , and concentrated. The crude product was

(

calcd for C H BrN O , 366.1176).

puriﬁed on silica gel column chromatography [(EtOAc:n-heptane,

Amino-((3-(2-(6-bromo-1H-indol-3-yl)acetamido)propyl)amino)-

6:4)] to give the intermediate as a light white powder (0.576 g, 83%).

−

methaniminium 2,2,2-triﬂuoroacetate (9a). To a solution of 8a (90

mg, 0.3 mmol, 1.0 equiv) and N,N′-Di-Boc-1H-pyrazole-1-carbox-

amidine (0.18 g, 0.59 mmol, 2.0 equiv) in THF (5 mL) was added

DIPEA (0.1 mL, 0.59 mmol, 2.0 equiv). The mixture was stirred at rt

R = 0.26 [(EtOAc:n-heptane, 4:1)]. IR (cm ). 3413, 3288, 2981,

2936, 1722, 1618, 1577, 1417, 1369, 1328, 1134, 1058, 1030, 884,

799. H NMR (400 MHz, Methanol-d ) δ 7.71 (d, J = 1.8 Hz, 1H),

7.27 (d, J = 8.9 Hz, 1H), 7.21−7.17 (m, 2H), 3.60 (s, 2H), 3.27 (t, J =

for 3 h, quenched with H O (10 mL), extracted with diethyl ether (2

7.2 Hz, 2H), 3.18 (t, J = 6.8 Hz, 2H), 1.58−1.49 (m, 13H), 1.46 (s,

10 mL), washed with sat. NaHCO and brine (10 mL), dried over

9H), 1.33−1.26 (m, 2H). C NMR (101 MHz, Methanol-d ) δ

Na SO , and concentrated. The crude product was puriﬁed on silica

174.5, 164.6, 157.5, 154.2, 136.7, 130.3, 126.4, 125.3, 122.1, 114,

113.1, 109.5, 84.4, 80.3, 41.7, 40.3, 33.9, 30, 29.7, 28.6, 28.2, 25.1.

FTMS m/z 580.2158 [M + H] (calcd for C H BrN O ,

gel column chromatography [(EtOAc:Hept-n, 6:4)] to give the

intermediate as a transparent oil (0.15 g, 91%). R_f= 0.21

26 39 5 5

[

(

(EtOAc:Hept-n, 4:1)]. H NMR (400 MHz, Methanol-d ) δ 7.83

580.2129).

broad s, 1H), 7.31−7.14 (m, 3H), 3.64 (s, 2H), 3.39−3.29 (m, 4H,

The intermediate (0.55 g, 0.95 mmol, 1.0 equiv) was dissolved in

CH Cl (5 mL) and a solution of TFA:CH Cl (1:1, 10 mL) was

solvent), 3.22 (broad s, 2H), 1.60−1.36 (m, 20H). C NMR (101

MHz, Methanol-d4) δ 174.4, 164.4, 157.9, 153.9, 136.6, 130.2, 126.4,

added. The reaction mixture was stirred at rt for 2 h, quenched with

25.2, 122.2, 114, 113.1, 109.5, 84.3, 80.4, 38.8, 37.3, 34.1, 30.1, 28.6,

H O, extracted with CH Cl (2 × 10 mL), washed with H O (2 × 10

8.2. FTMS m/z 552.1878 [M + H] (calcd for C H BrN O ,

mL) and the solvent was removed under reduced pressure to aﬀord

−1

52.1816).

The intermediate (0.15 g, 0.27 mmol, 1.0 equiv) was dissolved in

CH Cl (5 mL) and a solution of TFA:CH Cl (1:1, 10 mL) was

9c as a yellow-orange oil (112 mg, 24%). IR (cm ). 3283, 3176,

2940, 2125, 1633, 1566, 1462, 1436, 1182, 1138, 1028, 888, 840, 803,

724. H NMR (400 MHz, Methanol-d ) δ 7.70 (d, J = 1.9 Hz, 1H),

added. The reaction mixture was stirred at rt for 2 h, quenched with

7.27 (d, J = 8.6 Hz, 1H), 7.20−7.16 (m, 2H), 3.60 (s, 2H), 3.17 (t, J =

H O, extracted with CH Cl (2 × 10 mL), washed with H O (2 × 10

6.9 Hz, 2H), 3.04 (t, J = 7.1 Hz, 2H), 1.54−1.44 (m, 4H), 1.32−1.25

mL), and the solvent was removed under reduced pressure to aﬀord

(m, 2H). C NMR (101 MHz, Methanol-d ) δ 174.6, 158.5, 136.7,

a as an orange oil (95 mg, 75%). H NMR (400 MHz, Methanol-d4)

130.2, 126.5, 125.2, 122.1, 114.1, 113.1, 109.4, 42.3, 40.2, 33.9, 29.9,

δ 7.78 (d, J = 1.9 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.30−7.22 (m,

29.3, 24.8. FTMS m/z 380.1092 [M + H] (calcd for

H), 3.72−3.67 (m, 2H), 3.30 (t, J = 6.8 Hz, 2H), 3.17 (t, J = 7.0 Hz,

C H BrN O , 380.1080).

16 23 5

H), 1.78 (p, J = 6.9 Hz, 2H). C NMR (101 MHz, Methanol-d4) δ

73.8, 173.7, 135.4, 128.9, 125.2, 123.9, 120. 7, 112.8, 111.8, 107.9,

7.5, 47.2, 47.0, 38.5, 36.2, 32.5, 32.4, 28.5. FTMS m/z 352.0813 [M

2-(6-Bromo-1H-indol-3-yl)-N-(6-guanidinohexyl)-N-methylace-

tamide Triﬂuoroacetate (9d). To a solution of crude 8e (0.049 g,

0.14 mmol, 1.0 equiv) and N,N′-Di-Boc-1H-pyrazole-1-carboxami-

dine (0.087 g, 0.28 mmol, 2.0 equiv) in THF (10 mL) was added

DIPEA (0.05 mL, 0.28 mmol, 2.0 equiv). The mixture was stirred at rt

H] (calcd for C H BrN O , 352.0767).

Amino-((4-(2-(6-bromo-1H-indol-3-yl)acetamido)butyl)amino)-

methaniminium 2,2,2-triﬂuoroacetate (9b). To a solution of 8b

for 3 h, quenched with H

× 20 mL), washed with sat. NaHCO

O (20 mL), extracted with diethyl ether (3

and brine, dried over Na SO

(

0.19 g, 0.59 mmol, 1.0 equiv) and N,N′-Di-Boc-1H-pyrazole-1-

carboxamidine (0.37 g, 1.17 mmol, 2.0 equiv) in THF (7 mL) was

added DIPEA (0.21 mL, 1.17 mmol, 2.0 equiv). The mixture was

and concentrated. The diboc-intermediate compound (conﬁrmed by

HRMS) was used without further puriﬁcation and dissolved in

stirred at rt for 3 h, quenched with H O (10 mL), extracted with

TFA:CH

room temperature for 2 h, quenched with H

CH Cl (2 × 10 mL), washed with H O (2 × 10 mL), and the solvent

was removed under reduced pressure to aﬀord 9d as brown oil (19

(1:1, 10 mL), and the reaction mixture was stirred at

diethyl ether (2 × 10 mL), washed with sat. NaHCO and brine (10

O, extracted with

mL), dried over Na SO , and concentrated. The crude product was

puriﬁed on silica gel column chromatography [(EtOAc:Hept-n, 6:4)]

to give the intermediate as a light white powder (0.29 g, 86%). R =

mg, 26%). H NMR (400 MHz, Methanol-d ) δ 7.63 (dd, J = 4.9, 1.9

−

.19 [(EtOAc:n-heptane, 4:1)]. IR (cm ). 3413, 3286, 2933, 2866,

Hz, 1H), 7.18 (dd, J = 8.6, 3.1 Hz, 1H), 7.13−7.01 (m, 2H), 3.72 (d, J

= 0.9 Hz, 2H), 3.30 (td, J = 7.4, 2.5 Hz, 2H), 3.02−2.96 (m, 2H),

2.97 (d, J = 11.6 Hz, 3H), 1.49−1.28 (m, 4H), 1.23−1.02 (m, 4H).

516, 2445, 2378, 2154, 1722, 1629, 1581, 1458, 1369, 1287, 1153,

052, 1030, 877, 803, 739. H NMR (400 MHz, Methanol-d ) δ 7.71

(

d, J = 1.8 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 7.21−7.16 (m, 2H),

C NMR (101 MHz, Methanol-d ) δ: 172.5, 157.2, 135.4, 128.9,

.60 (s, 2H), 3.31−3.25 (m, 2H), 3.21 (t, J = 6.6, 5.8 Hz, 2H), 1.52

124.7, 123.9, 121.0, 112.6, 111.7, 107.5, 54.4, 40.9, 34.9, 32.5, 31.1,

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

The Supporting Information is available free of charge at

Article

8.3, 26.4, 25.7. FTMS m/z 408.1406 [M + H] (calcd for

nm, λ_e_m_i_s_s_i_o_n= 638−720 nm). The extent of growth, biovolume, and

overlap percentage was determined with a JAVA program (Universite′

C H BrN O , 408.1393).

50,51

-(2-(6-Bromo-1H-indol-3-yl)acetamido)-N,N,N-trimethylpro-

de Bretagne-sud, Lorient, France).

pan-1-aminium Iodide (10). To a 0 °C solution of 8a (100 mg, 0.32

mmol, 1.0 equiv) and glacial acetic acid (72 μL, 1.28 mmol, 4.0 equiv)

in MeOH (5 mL) under argon atmosphere, was added sodium

cyanoborohydride (40 mg, 0.64 mmol, 2.0 equiv). A solution of

formaldehyde (21 μL, 0.38 mmol, 2.4 equiv) in MeOH (2 mL) was

carefully added dropwise over 10 min, and then stirred at rt for 21 h,

quenched with Na CO (2 M) until pH 8−9 and concentrated under

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00881.

■

¹H NMR and ¹³C NMR data for all new synthetic

compounds and synthesized phidianidine A together

with 2D NMR data for 8a, 9c, and 10 (PDF)

reduced pressure. The residue was taken up in CHCl , washed with

brine and dried over Na SO , ﬁltered, and concentrated to obtain the

dimethylated product. To a 0 °C solution of the dimethylated product

(

80 mg, 0.24 mmol) in CHCl (1 mL) was added iodomethane (0.05

mL, 0.72 mmol, 10 equiv) in portions (× 3). The precipitate was

isolated, coevaporated with MeOH (× 3) to aﬀord the product 10 as

AUTHOR INFORMATION

■

a light yellow oil (81 mg, 53%). H NMR (400 MHz, Methanol-d ) δ

Corresponding Author

.80 (d, J = 1.9 Hz, 1H), 7.39−7.28 (m, 2H), 7.23 (dd, J = 8.7, 1.9

Johan Svenson − Department of Chemistry, Chemical Synthesis

and Analysis Group, UiT The Arctic University of Norway, N-

9037 Tromsø, Norway; Department of Chemistry, Biomaterial

Hz, 1H), 3.68 (s, 2H), 3.29 (t, J = 6.4 Hz, 2H), 3.25−3.18 (m, 2H),

.00 (s, 9H), 2.01−1.86 (m, 2H). C NMR (101 MHz, Methanol-d )

δ 173.6, 135.4, 128.9, 125. 6, 124.0, 120.8, 113.0, 111. 8, 108.1, 64.3,

2.3, 47.7, 47. 5, 47.2, 47.0, 35.7, 32.0. FTMS m/z 352.1027 [M]

& Textile, RISE Research Institutes of Sweden, 501 15 Boras

Sweden; orcid.org/0000-0002-4729-9359 ; Phone: +64 27

72 1876; Email: Johan.svenson@cawthron.org.nz

(

calcd for C H BrN O , 352.1019).

16 23 5

Barnacle Settlement. The barnacle cyprids were reared in a

laboratory rearing system, and their settlement was evaluated

according to the methods of Berntsson at Tjarno Marine Biological

Authors

Laboratory. In brief, a total of 18−22 freshly molted A. improvisus

cyprids were added to nontreated polystyrene Petri dishes (48 mm,

Nunc #150340) containing 10 mL of ﬁltered (0.2 μm) seawater

Christophe Labriere − Department of Chemistry, Chemical

Synthesis and Analysis Group, UiT The Arctic University of

Norway, N-9037 Tromsø, Norway

Vijayaragavan Elumalai − Department of Chemistry, Chemical

Synthesis and Analysis Group, UiT The Arctic University of

Norway, N-9037 Tromsø, Norway

Jannie Staﬀansson − Department of Chemistry, Chemical

Synthesis and Analysis Group, UiT The Arctic University of

Norway, N-9037 Tromsø, Norway

(

freshly collected from a depth of 45 m from the Kosterfjord

Sweden), ﬁltered through 20 μm ﬁlter and diluted with fresh water to

5 psu prior to use), and the compounds were serially diluted with

DMSO (10 μL added) to yield the desired concentration series. The

dishes were incubated at ambient temperature (20−25 °C) for 5 days,

and at the end of the experiment, the number of metamorphosed

juvenile barnacles, as well as live and dead cyprids, was assessed under

a dissection microscope. The concentration of a compound leading to

Gunnar Cervin − Department of Marine Sciences, Tjar

Marine Laboratory, University of Gothenburg, SE-452 96

Stromstad, Sweden

̈ ̈

0% inhibition of the settlement (metamorphosed cyprids) compared

to the control was reported as the IC₅₀value. Dead cyprids are not

included in the IC₅₀value. Dishes with 10 μL of DMSO added served

as negative control. Sea-nine was used as positive control, and each

test concentration was replicated four times (n = 4).

Tiﬀany Le Norcy − Univ. Bretagne-Sud, EA 3884, LBCM,

IUEM, F-56100 Lorient, France

Coating Development. A biodegradable poly(ε-caprolactone-co-

δ-valerolactone) (80:20) polymer was prepared according to

Hugo Denardou − Department of Chemistry, Chemical

Synthesis and Analysis Group, UiT The Arctic University of

Norway, N-9037 Tromsø, Norway

Karine Rehel − Univ. Bretagne-Sud, EA 3884, LBCM, IUEM,

F-56100 Lorient, France

Lindon W. K. Moodie − Department of Medicinal Chemistry

and Uppsala Antibiotic Centre, Biomedical Centre, Uppsala

University, 75123 Uppsala, Sweden; orcid.org/0000-0002-

previously published methodology and solubilized in xylene

mixture of isomers) (1:1 w/w). The compound was dissolved in

(

MeOH (1 M ﬁnal concentration) and added to polymer solution to

yield a ﬁnal compound concentration of 0.1 mg/L. The solution was

vortexed for 1 min and subsequently ultrasonicated (3 × 15 min) to

yield a homogeneous coating solution which was applied at a

thickness of 200 μm to PVC panels (10 × 10 cm ) using a ﬁlm

500-4535

applicator. The coating was allowed to dry for 24 h at ambient

temperature before further analyses and studies.

Claire Hellio − Univ. Brest, Laboratoire des Sciences de

l’Environnement MARin (LEMAR), CNRS, IRD, IFREMER,

Brest 29285, France

Field Studies. The coated and uncoated reference PVC panels as

well as control panels coated with polymer in the absence of

compound were mounted on structures and immersed at a depth of 1

Henrik Pavia − Department of Marine Sciences, Tjar

Laboratory, University of Gothenburg, SE-452 96 Stro

Sweden

Marine

mstad,

m deep in the port of Kernev

°23′40.68″ E). Each type of panel was included in triplicate in the

el in Lorient (France, 47°42′20.78″ N,

ﬁeld study. The immersion was carried out in spring at an average

water temperature of 16 °C and a salinity of 29 mg/mL. The panels

were regularly controlled and photographed to document and assess

the settlement of organisms. After day 84, the panels were retrieved,

and the extent and type of colonization was analyzed and quantiﬁed.

Prior to quantiﬁcation of growth, the panels were gently rinsed to

remove loosely adhered material and the adhesion of cells was

observed with confocal laser scanning microscopy (Zeiss, LSM 710)

by using a 40× oil immersion objective for bacteria and 20× air

objective for diatoms. Adhered bacteria were observed with Syto9

nucleic acid stain (5 μM, λ_e_x_c_i_t_a_t_i_o_n= 488 nm, λ_e_m_i_s_s_i_o_n= 498−540 nm).

Adhered diatoms were observed by their ﬂuorescence (λ_e_x_c_i_t_a_t_i_o_n= 633

Jørn H. Hansen − Department of Chemistry, Chemical Synthesis

and Analysis Group, UiT The Arctic University of Norway, N-

037 Tromsø, Norway; orcid.org/0000-0002-3888-5217

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jnatprod.0c00881

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Biotechnol. 2007 , 9 , 776 −785.

Article

Notes

(25) Tadesse, M.; Svenson, J.; Jaspars, M.; Strøm, M. B.;

Abdelrahman, M. H.; Andersen, J. H.; Hansen, E.; Kristiansen, P.

The authors declare no competing ﬁnancial interest.

E.; Stensvag

, K.; Haug, T. Tetrahedron Lett. 2011 , 52 , 1804 −1806.

(26) Ortlepp, S.; Sjo

gren, M.; Dahlstrom, M.; Weber, H.; Ebel, R.;

ACKNOWLEDGMENTS

■

Edrada, R.; Thoms, C.; Schupp, P.; Bohlin, L.; Proksch, P. Mar.

For C.L., J.H.H., J.S., and V.E., this work was partly supported

with grants from the Norwegian Research Council (ES508288

and 275043 CasCat). L.W.K.M. acknowledges the Uppsala

Antibiotic Centre for support. H.P. and G.C. were supported

by the Centre for Marine Chemical Ecology (http://www.

cemace.science.gu.se) at the University of Gothenburg. Finally,

the authors are grateful to V. Bhave (TOC) for providing the

image of Phidiana militaris.

(

27) Bayer, M.; Hellio, C.; Marechal, J. P.; Frank, W.; Lin, W.;

Weber, H.; Proksch, P. Mar. Biotechnol. 2011 , 13 , 1148 −1158.

(28) Lind, K. F.; Hansen, E.; Østerud, B.; Eilertsen, K. E.; Bayer, A.;

Engqvist, M.; Leszczak, K.; Jørgensen, T. Ø.; Andersen, J. H. Mar.

Drugs 2013 , 11 , 2655 −2666.

(

29) Tadesse, M.; Svenson, J.; Sepci

M.; Andersen, J. H.; Jaspars, M.; Stensvag

014 , 77 , 364 −369.

30) Moodie, L. W.; Sepci

Nat. Prod. Rep. 2019 , 36 , 1053 −1092.

31) Carbone, M.; Li, Y.; Irace, C.; Mollo, E.; Castelluccio, F.; Di

K.; Trembleau, L.; Engqvist,

, K.; Haug, T. J. Nat. Prod.

Org. Lett. 2011 , 13 , 2516 −2519.

c, K.; Turk, T.; Frangez, R.; Svenson, J.

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