Design and synthesis of marine natural product-based 1H-indole-2,3-dione scaffold as a new antifouling/antibacterial agent against fouling bacteria

Article abstract of DOI:10.1016/j.bioorg.2014.05.001

Marine organisms such as seaweeds, sponges and corals protect their own surfaces from fouling by their high anesthetic, repellant, and settlement inhibition properties. Within the marine ecosystem, evolution has allowed for the development of certain antifouling properties. Isatin is a biologically active chemical produced by an Alteromonas sp. strain inhibiting the surface of embryos of the cardiean shrimp Palaemon macrodectylus, which protect them from the pathogenic fungus Lagenidium callinectes. In present study, an antibacterial activity of isatin and its synthetic analogues were evaluated against different fouling bacteria in order to explore the structure activity relationships for the first time. The synthesized compounds along with parent isatin were tested against different ecologically relevant marine microorganisms by using the Kirby-Bauer disc diffusion method. Few synthetically modified isatin exhibited potent inhibitory activity at concentration of 2 μg/disc against Planococcus donghaensis, Erythrobacter litoralis, Alivibrio salmonicida, Vibrio furnisii. Overall, the modified analogues showed stronger activity than the parent marine natural product (isatin) and hence 1H-indole-2,3-dione scaffold has immense potential as future antibacterial/antifouling candidate.

Full text of DOI:10.1016/j.bioorg.2014.05.001

Accepted Manuscript
Design and synthesis of marine natural product-based 1H-indole-2,3-dione
scaffold as a new antifouling/ antibacterial agent against fouling bacteria
Mahesh S. Majik, Cheryl Rodrigues, Stacey Mascarenhas, Lisette D’Souza
PII:
S0045-2068(14)00032-7
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.05.001
YBIOO 1719
Reference:
Bioorganic Chemistry
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Received Date:
23 January 2014
Please cite this article as: M.S. Majik, C. Rodrigues, S. Mascarenhas, L. D’Souza, Design and synthesis of marine
natural product-based 1H-indole-2,3-dione scaffold as a new antifouling/ antibacterial agent against fouling bacteria,
Bioorganic Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.05.001
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Design and synthesis of marine natural product-based 1H-indole-2,3-dione
scaffold as a new antifouling/ antibacterial agent against fouling bacteria
Mahesh S. Majik ∗, Cheryl Rodrigues, Stacey Mascarenhas and Lisette D’Souza
Bio-organic Chemistry Laboratory, CSIR-National Institute of Oceanography, Dona-Paula Goa
403 004, India;
∗Corresponding author. Tel.: +91-832-2450458; fax: +91-832-2450607;
e-mail: majikms@gmail.com, mmajik@nio.org
Abstract
Marine organisms such as seaweeds, sponges and corals protect their own surfaces from fouling
by their high anesthetic, repellant, and settlement inhibition properties. Within the marine
ecosystem, evolution has allowed for the development of certain antifouling properties. Isatin is
a biologically active chemical produced by an Alteromonas sp. strain inhibiting the surface of
embryos of the cardiean shrimp Palaemon macrodectylus, which protect them from the
pathogenic fungus Lagenidium callinectes. In present study, an antibacterial activity of isatin and
its synthetic analogues were evaluated against different fouling bacteria in order to explore the
structure activity relationships for the first time. The synthesized compounds along with parent
isatin were tested against different ecologically relevant marine microorganisms by using the
Kirby-Bauer disc diffusion method. Few synthetically modified isatin exhibited potent inhibitory
activity at concentration of 2 µg/disc against Planococcus donghaensis, Erythrobacter litoralis,
Alivibrio salmonicida, Vibrio furnisii. Overall, the modified analogues showed stronger activity
than the parent marine natural product (isatin) and hence 1H-indole-2,3-dione scaffold has
immense potential as future antibacterial/antifouling candidate.
Keywords: Biofilm, Biofouling, Secondary metabolites, Isatin, Marine natural products,
Structure activity relationships.

1. Introduction
Marine organisms are highly prolific sources of biologically active metabolites, many of which
assumed key roles in elucidation of cellular mechanisms or as lead structure for drug discovery
[1]. Marine sessile organisms such as barnacles, mussels, and hydroids also known as fouling
organisms cause serious global damages in marine systems by settling on ship bulls, fishing
equipment, aquaculture cages and also the cooling systems of power stations. And hence,
biofouling emerged as a problem many centuries ago and likely to be continued for long as
mankind will sail the oceans [2]. Apart from creating technical and economical challenges for
marine organisms, biofouling also generates many other environmental problems such as spread
of invasive species [3]. Corresponding to these facts, various antifouling technologies have been
developed over past several decades to protect submerged surfaces by deterring the settlement of
colonizing stages of fouling organisms [4]. These include UV irradiations, ultrasound, foul-
release polymeric coating and antifouling agents (biocides) [5]. In the past, the most widely used
and best performing AF-compounds such as organotin (TBT) was recently prohibited for
application to ships, due to the severely toxic biocides and lack of degradation in the natural
environment [ 6 ]. Subsequently, marine coating industries have been actively developing
alternative AF biocides including copper (Cu) supplemented by booster biocides (irgarol 1051,
diuron, zinc pyrithione, copper pyrithione, chlorothalonil and SeaNine 211) [7]. However, many
of the booster biocides used worldwide cause serious damage to the marine environment and are
found to accumulate in coastal water at levels that are deleterious for marine organism [8].
Therefore, discovery of new environmentally friendly antifouling agents is desired. It is well
recognized that marine organisms produced diverse natural products to protect themselves from
the harmful process of biofouling and protect the surface of their bodies without causing serious
environmental problems [9]. On the basis of a literature survey, various potential antifouling
marine natural products such as terpenes, acetylenes, steroids, phenols, isothiocyanates, nitrogen-
containing compounds, glycerol derivatives, and higher fatty acids are derived from sponges,
algae, and cnidarians and exhibit not only toxins but also anesthetics, growth-inhibiting,
attachment-inhibiting, metamorphosis-inhibiting, and repelling properties [10].

Fig. 1. Isatin 1 from Alteromonas sp. and chemical compounds 2 created by considering SAR in
present work.
The chemistry behind the sessile, unfouled surfaces of marine organisms is largely being
investigated. In the course of search for compounds involved in defense mechanisms of
crustacean eggs, Fenical and co-workers demonstrated that marine bacteria consistently isolated
from healthy embryos of the caridean shrimp Palaemon macrodectylus (altermonas sp.)
produced isatin and showed inhibitory activity against the phycomycetous fungus Lagenidium
callinectes (a pathogen of many crustaceans) [11] (Fig. 1). Recently, Sakata et al. had identified
isatin as an algicidal substance produced by the marine bacterium Pseudomonas sp. C55a-2
isolated from coastal sea water Kagoshima Bay, Japan. Moreover, many strains belonging to
genera Pseudomonas, Alteromonas and Pseudoalteromonas spp. were reported to use an indirect
attack in the form of extracellular agents wherein, they produce extracellular algicidal or growth
inhibiting substances [12]. These species are reported to produce isatin as a defensive compound.
A variety of natural products have been isolated from marine organisms and some may be
developed as antifouling agents. However, poor yield of natural compounds from natural source
have hampered further developments. In order to create a potent antifouling agent, structural
activity relationships must be investigated in detail with respect to inhibitory activity against
fouling bacteria [13]. Realizing the importance of these facts and our research interest in marine
natural products [14] has motivated us to undertake these studies to provide an efficient synthesis
of natural product-based synthetic analogues of isatin and to determine therein antibacterial
activities (also called as antifouling activity, as this bioassay study in conducted on fouling
bacteria) in order to understand detail SAR profile. To provide an alternative molecular scaffold

that inhibits the fouling organisms, we have turned to antifungal and ecologically defensive
marine natural product isatin as source for structural insight to guide molecular design.
2. Materials and methods
2.1. Antibacterial assay (Inhibitory studies against fouling bacteria)
Nine strains of fouling bacteria were tested to determine the antibacterial capacity of the
compounds: Gram positive bacteria (Planococcus donghaensis) & Gram negative bacteria
(Alcanivorax spp, Aeromonas hydrophila subsp hydrophila ATCC 7966, Aeromonas hydrophila
subsp. salmonicida A449, Erythrobacter litoralis, Pseudomonas mendocina, Alcanivorax
borkumensis, Allivibrio salmonicida, Pseudoalteromonas spp., Vibrio furnisii). The strains were
isolated and identified by the method of Allegrucci and Sauer [15], Dalton, et al. [15], Weisburg
et al. [15], from natural biofilms allowed to develop on steel and copper panels for 14 days,
exposed at Dona Paula, Arabian Sea (15° 27’17’’N and 73°48’17’’E). Exposure was done at a
temperature of 15-20 °C at a salinity of 4%. The cultures were preserved in 30% glycerol at -
80 °C & prior to the assay, subcultured in Zobell’s marine broth (having 1% peptone & 0.1%
yeast extract) at 28 °C till they attained turbidity comparable to 0.5 McFarland turbidity
standards containing approximately 1-2 X 108 CFU/ml for E. coli ATCC 25922.
The Kirby-Bauer disc diffusion method [16] was used to conduct the assay. The compounds to
be tested were dissolved in 5% DMSO & pipetted onto sterile paper discs (Whatman no. 1,
diameter = 6 mm) at varying concentrations of 2-10 μg/disc. Control discs with 5% DMSO &
copper sulphate were used as controls at concentrations of 2-100 μg/disc as additional controls.
The discs were dried aseptically at room temperature. For the assay, 0.1 ml of each fouling strain
suspension having approximately 108 CFU/m l was spread plated onto Mueller Hinton agar
plates and the dry paper discs with either the test compounds or the control compounds were
aseptically laid on the agar surface. The plates were then incubated at 28°C for 24 hrs. till a
bacterial matt growth was observed on the agar surface. The zones of growth inhibition
surrounding the discs were measured up to 0.5 mm. The compounds were tested in triplicates (3
times) and with different concentrations to determine the minimum concentration required to
inhibit the test bacteria.
2.2. Synthesis of compounds

General methods: Melting points (Mp.) were determined by using melting point apparatus MR-
VIS (LABINDIA). IR spectra were recorded on Shimadzu (IR affinity-1) FT/IR spectrometer on
KBr pellets. 1H NMR spectra (CDCl3) were recorded on Varian Unity Inova 300 MHz. Chemical
shifts are reported in parts per million (δ) units relative to the solvent peak. The 1H NMR data are
reported as peak multiplicities: s for singlet; d for doublet; dd for doublet of doublets; t for
triplet; br s for broad singlet; and m for multiplet. Coupling constants were reported in Hertz.
Mass spectra were recorded on Agilent Technologies 6220 Accurate-Mass TOF LC/MS
spectrometer. Reactions were checked with TLC (Merck precoated 60F254 plates) and spots
were detected by viewing under a UV light and spraying with acidic p-anisaldehyde. Column
chromatography was performed on silica gel 60-120 (mesh, Merck). Reagents were purchased
from Aldrich Chemical Company. Solvents were obtained from local suppliers. All the
anhydrous solvents were distilled over CaH2, P2O5, or Na/benzophenone prior to the reaction.
3. Experimental procedures
3.1. General method for alkylation of isatin [17]
The isatin 1 (1 equv.) was taken in anhyd DMF (2 mL per 0.1 mmol of isatin) and cooled on ice
bath with stirring. Solid K2CO3 (1.2 equv.) was added in one portion and the dark colored
suspension was stirred at room temp for 2 h. The appropriate alkyl bromide (1.2 equv.) was
o
added and reaction mixture was stirred at 60 C for 8-12 h until isatin starting material was
consumed (monitored by TLC). The reaction mixture was diluted with water (50 mL) and
extracted with EtOAc (2 × 50 mL). The combined organic extract was washed with brine (2 × 50
mL), and dried over anhyd Na2SO4 and filtered. The solvent was evaporated and the crude
product was purified by using silica gel column chromatography (EtOAc: hexanes). This
procedure is utilized in the preparation of 3a and 3b.
3.2. Bromination of isatin [18]
To a stirred solution of isatin 1 (2.00 g, 0.013 mol) in EtOH (10 mL) was added bromine (6.52 g,
o
0.041 mol) at 0 C and the reaction mixture was stirred at room temperature for 12 h. The
o
reaction was cooled to 0 C, the orange solid was filtered, washed with water (3 × 50 mL) and
dried under high vacuum to give orange color solid 4.

3.3. General procedure for Wittig reaction [19]
To a stirred solution of isatin 1 or substituted isatin (3 & 4) in EtOH (20 mL) was added
corresponding phosphoranes P1-P3 (prepared using the standard procedure). The reaction
mixture was stirred at room temperature for 12 h. The solvent was evaporated under vaccum and
the crude product was purified by using silica gel column chromatography (EtOAc: hexanes) to
give orange crystalline solid. Compounds 5, 6, 7 and 8 (a-c) were synthesized using this
procedure.
3.4. 1H-indole--2,3-dione 1
Orange crystalline solid, mp 196-198 °C. IR (ν max): 3461, 1760, 1696 cm-1. 1H NMR (CDCl3,
300 MHz) δ 6.91 (d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1 H), 7.55 (t, J = 7.7 Hz, 1 H), 7.60 (d,
J = 7.8 Hz, 1 H), 8.16 (br s, 1 H).
3.5. (E)-3-(2-Oxopropylidene)indolin-2-one 5a
Yield 70%; orange solid, mp 150-151 °C. IR (ν max): 3233, 1726, 1678, 1603, 1462, 1325, 1222,
1
746 cm-1. H NMR (CDCl3, 300 MHz) δ 2.51 (s, 3 H), 6.85 (d, J = 7.5 Hz, 1 H), 7.06 (t, J = 7.2
Hz, 1 H), 7.18 (s, 1 H), 7.33 (t, J = 7.8 Hz, 1 H), 7.75 (br s, 1 H), 8.53 (d, J = 7.8 Hz, 1 H).
HRMS: m/z [M + H]+ calcd for C11H10NO2, 118.0712; found, 118.0710.
3.6. (E)-Ethyl 2-(2-oxoindolin-3-ylidene)acetate 5b
Yield 82%; orange solid, mp 152-154 °C. IR (ν max): 3160, 1713, 1655, 1607, 1462, 1332, 1230,
1
1027, 752 cm-1. H NMR (CDCl3, 300 MHz) δ 1.39 (t, J = 7.2 Hz, 3 H), 4.35 (q, J = 7.2 Hz, 14.4
Hz, 2 H), 6.87 (d, J = 7.5 Hz, 1 H), 6.89 (s, 1 H), 7.06 (t, J = 7.8 Hz, 1 H), 7.34 (t, J = 7.5 Hz, 1
H), 8.47 (br s, 1 H). 8.56 (d, J = 7.8 Hz, 1 H). HRMS: m/z [M + H]+ calcd for C12H12NO3,
218.0817; found, 218.0811.
3.7. (E)-3-(2-Oxo-2-phenylethylidene)indolin-2-one 5c
Yield 76%; orange solid, mp 174-176 °C. IR (ν max): 3188, 1715, 1645, 1612, 1464, 1327, 1032,
791, 754 cm-1. 1H NMR (CDCl3, 300 MHz) δ 6.90 (d, J = 7.8 Hz, 1 H), 7.04 (t, J = 7.8 Hz, 1 H),
7.34 (t, J = 7.5 Hz, 1 H), 7.55-7.66 (m, 3 H), 7.89 (s, 1 H), 8.14 (d, J = 7.2 Hz, 2 H), 8.33(d, J =
8.1 Hz, 2 H). HRMS: m/z [M + H]+ calcd for C16H12NO2, 250.0868; found, 250.0852.

3.8. 1-Methylindoline-2,3-dione 3a
Yield 86%; orange solid, mp 128-129 °C [lit. 20]. IR (ν max): 1732, 1724, 1606, 1470, 1327,
1032, 864, 760 cm-1. 1H NMR (CDCl3, 300 MHz) δ 3.27 (s, 3 H), 6.91 (d, J = 8.1 Hz, 1 H), 7.15
(t, J = 7.5 Hz, 1 H), 7.61-7.65 (m, 2 H). HRMS: m/z [M + H]+ calcd for C9H8NO2, 162.0555;
found, 162.0543.
3.9. (E)-1-Methyl-3-(2-oxopropylidene)indolin-2-one 6a
Yield 71%; orange solid, mp 120-121 °C. IR (ν max): 1707, 1688, 1599, 1471, 1340, 786 cm-1 .
1H NMR (CDCl3, 300 MHz) δ 2.50 (s, 3 H), 3.25 (s, 3 H), 6.81 (d, J = 7.8 Hz, 1H), 7.07 (t, J =
7.8 Hz, 1 H), 7.21 (s, 1 H), 7.40 (t, J = 7.5 Hz, 1 H), 8.53 (d, J = 7.8 Hz, 1 H). HRMS: m/z [M +
H]+ calcd for C12H12NO2, 202.0868; found, 202.0853.
3.10. (E)-Ethyl 2-(1-methyl-2-oxoindolin-3-ylidene)acetate 6b
Yield 74%; orange solid, mp 75-76 °C. IR (ν max): 1708, 1663, 1607, 1471, 1342, 1200, 1027,
1
754 cm-1. H NMR (CDCl3, 300 MHz) δ 1.39 (t, J = 7.2 Hz, 3 H), 3.25 (s, 3 H), 4.34 (q, J = 7.2
Hz, 14.1 Hz, 2 H), 6.81 (d, J = 7.8 Hz, 1H), 6.92 (s, 1 H), 7.05 (t, J = 7.8 Hz, 1 H), 7.38 (t, J =
7.5 Hz, 1 H), 8.53 (d, J = 7.8 Hz, 1 H). HRMS: m/z [M + H]+ calcd for C13H14NO3, 232.0974;
found, 232.0964.
3.11. (E)-1-Methyl-3-(2-oxo-2-phenylethylidene)indolin-2-one 6c
Yield 79%; orange solid, mp 105-106 °C. IR (ν max): 1707, 1663, 1603, 1468, 1338, 1227, 781,
750 cm-1.1H NMR (CDCl3, 300 MHz) δ 3.30 (s, 3 H), 6.83 (d, J = 8.1 Hz, 1 H), 7.05 (t, J = 7.8
Hz, 1 H), 7.38 (t, J = 7.5 Hz, 1 H), 7.55 (t, J = 7.8 Hz, 2 H), ), 7.63 (t, J = 7.5 Hz, 1 H), 7.92 (s, 1
H), 8.12 (d, J = 7.2 Hz, 2 H). 8.53 (d, J = 7.5 Hz, 1 H). HRMS: m/z [M + H]+ calcd for
C17H14NO2, 264.1025; found, 264.1011.
3.12. 1-Butylindoline-2,3-dione 3b
Yield 90%; orange solid, mp 40-42 °C [lit. 20]. IR (ν max): 1732, 1724, 1606, 1470, 1327, 1032,
864 cm-1. 1H NMR (CDCl3, 300 MHz) δ 0.98 (t, J = 7.5 Hz, 3 H), 1.39-1.46 (m, 2 H), 1.65-1.75

(m, 2 H), 3.73 (t, J = 7.2 Hz, 2 H), 6.91 (d, J = 8.4 Hz, 1 H), 7.12 (t, J = 7.5 Hz, 1 H), 7.57-7.62
(m, 2 H). HRMS: m/z [M + H]+ calcd for C12H14NO2, 204.1025; found, 264.1013.
3.13. (E)-1-Butyl-3-(2-oxopropylidene)indolin-2-one 7a
1
Yield 71%; orange solid, mp 65-66 °C. IR (ν max): 1707, 1680, 1603, 1466, 1362, 787 cm-1. H
NMR (CDCl3, 300 MHz) δ 0.98 (t, J = 7.5 Hz, 3 H), 1.39-1.44 (m, 2 H), 1.62-1.72 (m, 2 H),
2.49 (s, 3 H); 3.74 (t, J = 7.2 Hz, 2 H), 6.81 (d, J = 7.8 Hz, 1 H), 7.05 (t, J = 7.8 Hz, 1 H), 7.20
(s, 1 H), 7.38 (t, J = 7.5 Hz, 1 H), 8.53 (d, J = 7.5 Hz, 1 H). HRMS: m/z [M + H]+ calcd for
C15H18NO2, 244.1338; found, 244.1326.
3.14. (E)-Ethyl 2-(1-butyl-2-oxoindolin-3-ylidene)acetate 7b
Yield 78%; orange solid, mp 187-189 °C. IR (ν max): 1769, 1699, 1526, 1344, 1196, 1027, 852
cm-1. 1H NMR (CDCl3, 300 MHz) δ 0.97 (t, J = 7.5 Hz, 3 H), 1.36-1.46 (m, 4 H), 1.61-1.72 (m, 3
H), 3.74 (t, J = 7.2 Hz, 2 H), 4.34 (q, J = 7.2 Hz, 14.4 Hz, 2 H), 6.82 (d, J = 7.8 Hz, 1 H), 6.92
(s, 1 H), 7.06 (t, J = 7.8 Hz, 1 H), 7.37 (t, J = 7.8 Hz, 1 H), 8.58 (d, J = 7.8 Hz, 1 H). HRMS: m/z
[M + H]+ calcd for C16H20NO3, 274.1443; found, 274.1431.
3.15. (E)-1-Butyl-3-(2-oxo-2-phenylethylidene)indolin-2-one 7c
Yield 76%; orange semi solid. IR (ν max): 1708, 1665, 1602, 1470, 1338, 1227, 784, 752 cm-1.
1H NMR (CDCl3, 300 MHz) δ 0.99 (t, J = 7.5 Hz, 3 H), 1.40-1.48 (m, 2 H), 1.69-1.74 (m, 2 H),
3.78 (t, J = 7.2 Hz, 2 H), 6.84 (d, J = 7.8 Hz, 1 H), 7.03 (t, J = 7.5 Hz, 1 H), 7.34 (d, J = 7.8 Hz,
1 H), 7.52-7.65 (m, 3 H), 7.90 (s, 1 H), 8.13 (d, J = 8.1 Hz, 2 H), 8.33 (d, J = 7.5 Hz, 1 H).
HRMS: m/z [M + H]+ calcd for C20H20NO2, 306.1494; found, 306.1480.
3.16. 5-Bromoindoline-2,3-dione 4
Yield 66%; orange crystalline solid, mp 185-187 °C. IR (ν max): 3160, 1713, 1655, 1606, 1462,
1
1333, 1230, 752 cm-1. H NMR (CDCl3, 300 MHz) δ 6.84 (d, J = 8.4 Hz, 1 H), 7.71 (d, J = 8.4
Hz, 1 H), 7.76 (s, 1 H). HRMS: m/z [M + H]+ calcd for C8H5BrNO2, 225.9504; found, 225.9502.
3.17. (E)-5-Bromo-3-(2-oxopropylidene)indolin-2-one 8a

Yield 72%; orange solid, mp 148-149 °C. IR (ν max): 3215, 1720, 1682, 1611, 1300, 816 cm-1.
1H NMR (CDCl3, 300 MHz) δ 2.52 (s, 3 H), 6.78 (d, J = 8.4 Hz, 1 H), 7.19 (s, 1 H), 7.47 (d, J =
8.1 Hz, 1 H), 8.68 (br s, 1 H), 8.70 (s, 1 H). HRMS: m/z [M + H]+ calcd for C11H9BrNO2,
265.9817; found, 265.9801.
3.18. (E)-Ethyl 2-(5-bromo-2-oxoindolin-3-ylidene)acetate 8b
Yield 80%; orange solid, mp 207-208 °C. IR (ν max): 3169, 1713, 1650, 1612, 1309, 816 cm-1.
1H NMR (CDCl3, 300 MHz) δ 1.38 (t, J = 7.2 Hz, 3H), 4.37 (q, J = 7.2 Hz, 14.1 Hz, 2 H), 6.76
(d, J = 8.1 Hz, 1 H), 6.92 (s, 1 H), 7.47 (d, J = 8.4 Hz, 1 H), 7.74 (br s, 1 H), 8.76 (s, 1 H).
HRMS: m/z [M + H]+ calcd for C12H11BrNO3, 295.9922; found, 295.9902.
3.19. (E)-5-Bromo-3-(2-oxo-2-phenylethylidene)indolin-2-one 8c
Yield 72%; orange solid, mp 205-206 °C. IR (ν max): 3194, 1721, 1663, 1599, 1448, 1312, 1230,
1
721, 694 cm-1. H NMR (CDCl3, 300 MHz) δ 6.78 (d, J = 8.4 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1 H),
7.54-7.67 (m, 3 H), 7.80 (br s, 1 H), 7.93 (s, 1 H), 8.13 (d, J = 8.4 Hz, 2 H), 8.56 (s, 1 H).
HRMS: m/z [M + H]+ calcd for C16H11BrNO2, 327.9973; found, 327.9971.
4. Results and discussion
4.1. Synthesis of isatin analogues
Isatin and its derivatives are well known compounds with pharmacological properties e.g.
anticonvulsants, antimicrobial, depressants of central nervous systems, antitumour agents etc
[ 21 ]. However, there is only one report regarding the remarkable role of isatin as an
allelochemical in protection of shrimp embryos from fungal infections [11]. In the present work,
we have modified the 3-carbonyl functionality of isatin 1 using simple synthetic manipulation, in
order to understand the importance of carbonyl function (C-3 position) of isatin for its observed
inhibitory activity. Since these compounds are Michael acceptor and therefore would be
expected to be biologically active. Towards this end, isatin 1 was treated with different
phosphoranes (P-1, P-2, P-3) to furnish corresponding olefins 5a-c (Scheme 1). The geometry
around the double bond was assumed to be trans (E) by comparing the spectroscopic data with
the reported compound [22]. Also the detailed study of NOE NMR spectrum of the related
compound having similar structure were previously investigated by Shimazawa et al. and were

found to contain trans-geometry of the double bond [23] (refer supplementary information for
detail 1D-NOE and 2D-NOESY NMR spectra of 5a). The intension of using this phosphoranes
is that the modification at C-3 position of isatin could give us library of isatin derivatives with
different substitution at terminal carbon of olefin i.e. α, β-unsaturated ketone (methyl ketone,
phenyl ketone), and α, β-unsaturated ester. The effect of this different functionality on inhibitory
activity is also evaluated in this work. Furthermore, various N-protected derivatives 3a-3b were
prepared in 86-90% yield by treating isatin 1 with different alkylating agents using potassium
carbonate as base in ethanol. The presence of bromine atom in structure of many antifouling
agents [24] have inspired us to study the impact of bromination upon antibacterial activity in
isatin derivatives by introducing bromine atom at 5-position of indole skeleton. Hence, 5-
bromoisatin 4 was synthesized by using reported method employing bromination of isatin at
room temp. With the sufficient amounts of 5-bromoisatin 4 and N-protected isatin 3a-3b in our
hands, these compounds were subjected to Wittig olefination to give corresponding alkenes 6, 7,
8 in 70-82% yield. This gives another series of compounds containing N-protected isatin
derivatives (6a-c and 7a-c) and brominated isatin derivatives (8a-c). All the synthesized
compounds were obtained in good yield and were characterized by different spectroscopic
methods like NMR and mass spectroscopy. The synthesized compounds were then screened for
their inhibitory activity against marine fouling bacteria.

R1
O
Br
O
O
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O
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N
H
N
H
1
1
1
3
2
5
O
R1
R1
O
O
Br
O
N
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3
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4
9
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1
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3
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O
O
Scheme 1. Synthesis of isatin derivatives. Reagents and condition: (a) K2CO3, DMF, methyl
bromide or n-butyl bromide, 2 h, room temp (86-90%); (b) bromine, EtOH, 0 oC to room temp, 2
h (66%); (c) Phosphorane (P1 or P2 or P3), EtOH, room temp, 12 h (70-82%).
4.2. Antibacterial activities of isatin and its synthesized derivatives against fouling bacteria
Compounds were screened initially at concentration of 100 µg/disc to investigate their effects on
fouling bacteria using E. coli, S. aureus etc. The antifouling activity was monitored under static
condition using Kirby-Bauer disc diffusion method [25]. Initially, isatin 1, 5a and 4 was
evaluated for their antifouling activity. This preliminary screen analyses [25] revealed that isatin
1 is moderately active against A. salmonicida A449 and P. donghaensis whereas, compound 5a
showed broad spectrum of antibacterial activity against Gram +ve and Gram –ve fouling bacteria
such as E. litoralis, A. salmonicida, V. furnisii etc. Also, bromoisatin 4 exhibited weak inhibitory

activity. This observation indicated that the introduction of bromine atom at C-5 position of
isatin diminishes its antibacterial property (Fig. 2).
Fig. 2. Effects of bromine substitution of isatins on antibacterial activities against A. salmonicida
A449 (std is Gentamycin). X-axis represents synthesized derivatives and Y-axis represents zone
of inhibition in mm at conc. of 100 µg/disc.
Fig. 3. Effects of N-protection of isatin derivatives against E. litoralis at conc. of 100 µg/disc.
(std is Gentamycin).

Table 1: Table showing zones of inhibition valuesa of 5a and 6a against fouling bacteria
Sr. Compounds
no.
Concentration Planococcus Erythrobacter Alivibrio
Vibrio
furnisii
(μg)
donghaensis litoralis
salmonicida
1
2
3
4
5
5a
5a
5a
5a
5a
2
4
6
8
10
9
10
11
12
14
15
10
11
11
13
13
7
9
9
9
10
13
13
13
15
6
7
8
9
6a
6a
6a
6a
6a
2
4
6
8
10
5
6
3
7
5
6
7
5
7
7
6
5
7
5
4
4
6
6
5
3
10
11 Std antibiotic
12 Std antibiotic
13 Std antibiotic
14 Std antibiotic
15 Std antibiotic
2
4
6
8
10
26
28
31
33
35
14
18
20
25
28
15
20
23
26
29
12
17
20
24
27
aThe data are expressed as the measure of inhibition zones (mm) at concentration varying from
2- 10 µg/disc; std antibiotic is Gentamycin; data given are the mean of three replicates.

Fig. 4. Graph of concentration of 5a & 6a v/s Zones of inhibition, showing the minimum
concentration required against the fouling bacterium E. litoralis.
Furthermore, we selected 5a as our most promising lead compound sought to identify potential
structure motif within 5a that were responsible for the observed antibacterial activity to further
improve its inhibitory potential. Five more derivatives of isatin were synthesized imposing
modifications at C-3 position and keeping C-1 and C-2 position intact as that of parent isatin to
give series of compounds 5b-5c and 8a-8c. Compound 5b (with ethoxy moiety) shows weak
activity whereas compound 5c (aromatic component) loses the activity. This result suggests that,
the replacement of methyl group with hydrophobic aromatic moiety led to the complete loss of
inhibitory activity. However, the introduction of bromine atom at C-5 position (8a-8c) led to
either total loss or significant reduction in activity [25] Intrigued by these results; we next
synthesized more isatin derivatives to further probe the activity of this compound and to
understand the role of free NH group of isatin. Eight more derivatives 3a, 6a-6c and 3b, 7a-7c
were prepared by imparting modification at N-substituent (N-methyl and N-butyl, instead of NH)
and maintaining C-3 positions unchanged (as that of 5a-5c). Introduction of methyl group at 1-
position of isatin led to decrease in antibacterial activity (3a) and further increase in alkyl chain
length led to the complete loss of inhibitory activity (comparison 1, 3a and 3c). This suggests

that the presence of NH group is necessary for the observed inhibitory activity of isatin family
(Fig. 3). This was further supported by observation that the antibacterial activity of derivatives
decreases in order of 5a> 6a> 7a> 8a. For the compounds showing the ability to either inhibit
fouling organisms, dose-response studies are subsequently performed to quantify activity (Table
1). Overall compounds 5a, 6a, 7a were prominently inhibitory towards 3 Gram negative fouling
bacteria E. litoralis, A. salmonicida and V. furnisii with only 5a showing good broad spectrum of
activity against Gram +ve and Gram –ve bacteria. The most potent antibacterial agent identified
overall was the 5a (2 µg/disc), which was more active than the parent marine natural product 1
(Fig. 4).
4.3. Analyses of structure activity relationship
To examine the role of different modifications that would be useful in understanding the
functional components responsible for the activities of these compounds towards fouling
organisms, isatin modifications has been undertaken in present project (Fig. 5) such as (a)
bromine substituent at C-5 carbon of isatin ring (8a-c), (b) the length of alkyl chain (6a-c & 7a-
c ) relationship, (c) N-protections in compounds (5, 6 & 7).
R1
Effects of halogen
O
substitution on
Effects of substituent
at C-3 position
X
antibacterial activity
O
N
R
Effects of alkyl
chain length on
antibactrial activity
Effects of N-protection
on antibacterial activity
Fig. 5. Different modifications on isatin ring
(a) Effect of bromine substituent at C-5 carbon of the isatin ring (4, 8a-c): From the data
presented in supplementary information [25], it has been observed that the use of
bromine substituent at C-5 carbon atom in the derivatives of isatin leads to the decrease
in antibacterial activity as compared with parent isatin (Fig. 2).

(b) Effect of alkyl chain length of N-protected isatin derivatives (3, 6, 7): N-methyl and N-
butyl derivatives were prepared in order to check the effect of hydrophobicity on
inhibitory activity of synthesized compounds i.e. [NH (less hydrophobic), N-CH3, N-
C4H10 (more hydrophobic)]. The antibacterial activity remains unchanged for
derivatives for N-methyl and N-butyl iastin derivatives but their activity decreases as
compared to isatin with free NH group. Hence, free NH moiety of isatin is necessary for
its good inhibitory property against fouling bacteria (Fig. 3).
(c) Effects of modification of C-3 positions of isatin (5-8): Isatin derivatives with the extended
conjugation at C-3 position (Michael acceptor) were prepared by maintaining C-2 carbonyl
and 1H intact (NH). The antibacterial activity increased in this class of systems wherein
acetonyl moiety is found to have better functionality compared to more extended
hydrophobic benzoyl, as well as electron donating ethoxy group. Also the series of
compounds 5a-c from this group were found to possess good inhibitory effect compared to
parent isatin as well as N-protected isatins (6a-c & 7a-c) and 5-bromoisatin derivatives 8a-c.
The presence of 3-acetonylidene group and free NH moiety is a crucial factor responsible for
enhancing antibacterial activity amongst isatin family 5a.
5. Conclusions
This report represents the first studies towards the antibacterial activity of marine natural isatin
and its modified compounds. The syntheses of compounds have been achieved by using simple
synthetic manipulation employing easily available starting materials. The 3-acetonylidene
oxindole 5a is identified as the most potent with maximum antibacterial properties against
fouling bacteria. Moreover, the modified analogues showed stronger activity than the parent
marine natural product (isatin). The compounds in present study show good activity against
bacteria that cause fouling, whereas to demonstrate actual antifouling activity, further antifouling
assays on barnacle larvae would need to be carried out. Overall, this study suggest that the
screening of natural products and its modified analogues is a promising way to find novel
antifouling agents and may potentially be used in the future for antifouling and antibiofilm
applications.

Acknowledgments
The authors thank the Director, CSIR-National Institute of Oceanography for constant
encouragement. Financial assistance provided by the OCEAN FINDER and EU-FP7-KBBE-
2009-3-245137 MAREX is highly acknowledged. Author MSM is grateful to CSIR-NIO for the
award of Scientist Fellow-QHS.

Design and synthesis of marine natural product-based 1H-indole-2,3-dione
scaffold as a new antifouling/ antibacterial agent against fouling bacteria
Mahesh S. Majik ∗, Cheryl Rodrigues, Stacey Mascarenhas and Lisette D’Souza
Bio-organic Chemistry Laboratory, CSIR-National Institute of Oceanography, Dona-Paula Goa
403 004, India;
Graphical Abstract

Highlights.
ꢀ Isatin is antifungal and ecologically defensive marine natural product tested for SAR study.
ꢀ An efficient synthesis was achieved using simple synthetic manipulations.
ꢀ 3-Acetonylidene oxindole 5a is identified as the most potent antibacterial agent.
ꢀ Report represents the first studies towards the inhibitory activity of isatin & its derivatives.
ꢀ The screening of natural products analogues is a promising way to find novel antimicrobial
agents.

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