A New Antiplasmodial Compound from the Papuan Marine Sponge Xestospongia sp

A new antiplasmodial compound, 2-(3H-diazirine-3-yl)benzaldehyde (1), has been isolated from the Papuan marine sponge Xestospongia sp. The structure elucidation of compound 1 was determined by spectroscopic evidences including UV, IR, MS, 1D and 2D-NMR analysis. Compound 1 showed moderate antiplasmodial activity against Plasmodium falciparum with IC50 value of 1.08  10 -6 M.


INTRODUCTION
In tropical countries, malaria is known as the most severe disease infected more than two billion people across the world (Bagavan et al., 2011;Poostchi et al., 2017).This disease is also responsible of the human death (Weiss et al., 2018), most of the death cases occurred in Africa, Asia, and other tropical countries (Snow et al., 2005;Nayyar et al., 2012;Kabaria et al., 2017).
Currently, Indonesia harbours twenty vectors malaria associating with four species of Malaria (Elyazar et al., 2011).In Indonesia, cases of malaria are estimated about ninety millions cases by 2011 where one people died per a hundred thousand cases (WHO, 2008).Most cases of malaria are reported in the eastern provinces of Indonesia (Papua and West Papua) (Nagesha et al., 2001;Douglas et al., 2017).In the region, malaria have infected seventy people per a hundred population, most of them are women and children (WHO, 2008).
It is estimated about 30% of antimalarial medicaments are obtained from nature herbs, including terrestrial plants and marine biota's (Sanon et al., 2013;Boampong et al., 2015).In marine drug exploration, marine sponges (Xestopongia sp. and Haliclona sp.) are intensively studied in recent decades, since its potential antiplasmodial and bacterial properties, in which dozen patents already obtained from this unique biota (Inbaneson and Ravikumar, 2012;Davis et al., 2012;Beesoo et al., 2017).Marine sponge of the genus Xestospongia is one of the world's potential antimalarial agents containing xestoquinone (Laurent et al., 2006;Nogueira and Lopes, 2011;Bottee et al., 2012).
In the present study, a new antiplasmodial compound obtained from the Papuan marine sponge is desribed, and exhibited ability against Plasmodium falciparum.

General Experimental Procedure
The IR spectra were recorded on a SHIMADZU IR Prestige-21 in KBr.The mass spectra were recorded with a Waters Xevo QTOF MS.NMR spectra were recorded on a Bruker Topspin spectrometer at 500 MHz for 1 H and 125 MHz for 13 C using TMS as an internal standard.Column chromatography was conducted on silica gel 60.TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% H 2 SO 4 in EtOH, followed by heating and under UV light at wave length at 254 and 367 nm.

Sponge Collection and Cultured Parasites
The study was based on material Porifera genus Xestospongia, collected by scuba in about 10 m depth in the south west of Kaimana, West Papua, Indonesia (GPS: 4°20.341'S-133°30.265'E).It has been identified taxonomically as Xestospongia sp., (Lamarck, 1813), the species belongs to the Xestospongia (Order Haplosclerida: Family Petrosiidae).Identification taxonomic and nomenclature were provided by the Laboratory Biology and Conservation, Jakarta Fisheries University, Ministry of Marine Affairs and Fisheries (LIN BIOVASI, No: 041/STP-V/2016, Catalogue: MS041.1-5).Subject of the study was the protozoan parasite P. falciparum strain 3D7 (chloroquine-sensitive) obtained from University of Tokyo and cultured by the Eijkman Institute for Molecular Biology in Jakarta.

Antiplasmodial Assay in Vitro
Strain 3D7 of P. falciparum was cultured and developed by Eijkman Institute for Molecular Biology in Jakarta.Antimalarial examination was used 96 wells, each well filled by parasitemia culture 1%.About 50 µL of the sponge fractions filled in to the well with the following concentration 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 μg/mL.All wells were then stored in the Laminar Airflow cabinets, until all solvents are evaporated.Further, we added 50 µL of parasitemia red blood cell into the well then were incubated at 37ºC.After 48 h, slides preparation were stained using Giemsa 20% (WHO, 2008).

RESULT AND DISCUSSION
The ethanol extract from the milled fresh of sponge of Xestospongia sp. was concentrated and extracted successively with n-hexane, ethyl acetate, and n-butanol.The ethyl acetate fraction was separated by combination of column chromatography on silica gel G60, ODS, and preparative TLC on silica gel GF 254 to afford compound 1.Compound 1 was obtained as yellow oily.QTOF-MS of 1 showed m/z 146.3106, which in line to the molecular formula of C 8 H 6 ON 2 thus requiring seven degrees of unsaturation.UV spectra in MeOH showed λ max nm (log ε) 260 (3.74), indicated the presence of benzene ring attached to electron with drawing group (Ursula and Stanley, 1987).The IR spectrum showed absorption peaks due to of aldehyde group (1720 cm -1 ), C=C aromatic ring (1515 cm -1 ), N=N group (1620 cm -1 ), and disubstituted aromatic ring (1020 and 820 cm -1 ).The 1 H-NMR spectrum of 1 showed the presence of an aldehyde proton at  H 10.03 (1H, s, H-7), aromatic protons at  H 7.50 (1H, d, J=7.8 Hz, H-3), 7.27 (1H, dd, J=7.8 Hz, H-4), 7.23 (1H, dd, J=6.8 and 7.1 Hz, H-5) and 8.21 (1H, d, J=6.8 Hz, H-6) as well as a deshielded aliphatic proton at  H 8.20 (1H, s, H-8).The 13 C NMR spectrum showed seven carbon resonances, which were classified by their chemical shift sand the DEPT spectra as one aldehyde ( C 185.4), two sp 2 quartenary carbons at  C 125.6 and 120.2, four sp 2 methine carbons at  C 113.0, 124.5, 123.0 and 122.3 as well as one sp 3 deshielded carbon at  C 138.1.These functionalities accounted for five out of the total seven degrees of unsaturation and the remaining two degrees of unsaturation were consistent with the diazirine-benzaldehyde derivative sructure (Sakurai et al., 2014).
A comparison of the NMR data of 1 with those of 2-methoxy-5-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzaldehyde (Sakurai et al., 2014), revealed that the structures of the two compounds are closely related, the main differences are the absence of methoxyl and trifluoromethyl group and the presence additional aromatic protons in 1.The gross structure of 1 was deduced from the 1 H-1 H COSY and HMBC spectra (Fig. 2).
Consequently, the structure of a new diazirine benzaldehyde (1), was established as 2-(3Hdiazirine-3-yl)-benzaldehyde and was isolated from marine sponge for the first time.
Currently, the marine sponges are pointed as one of the potential object of biomedical research due to its richness bioactive properties.In recent decades, marine sponges are known as marine drugs resources such as anticancer, antimicrobe, antivirus, and antimalaria (Sipkema et al., 2004;Kutluay et al., 2016).In the present study, we reported here the presence of a compound was recognised from derivate of benzaldehyde, which is highly capable of inhibiting Plasmodium (IC 50 1.0810 - M).The obtained value of IC 50 in the present work was far lowest compared to commonly IC 50 obtained from the same type of biota, such as sponge of Northern New Guinea IC 50 34.6µM (Murtihapsari et al., 2013), sponge of New Caledonia 3 M.(Laurent et al., 2006;Nogueira and Lopes, 2011).Our finding of antiplasmodial activity is rangked as very active compound according to norms (Rasoanaivo et al., 1992;Ringwald, 1999;Ravikumar et al., 2012a;2012b).
Malaria has been one of the most deadly disease in eastern part of Indonesia.In respond to the Indonesia and international program of reducing malarial effects, research on malaria was recently attracted much attention and has been supported by government.The eastern part of Indonesia was known as center of World Coral Triangle, a vast richness of marine landscape harboring potential marine drugs for Malaria.Recently (Tapilatu, 2015) summarized a list of newly compound from the marine life of eastern part of the country where antimalarial source remain poorly studied.We conclude that our finding exhibit a strongly signal antimalarial compound from Papuan sponge, which is need a continuation and comprehensive studied.

Figure 1 .
Figure 1.The Structure of Compound 1

Table 1 .
NMR data for Compound 1 (500 MHz for 1 H and 125 MHz for 13 C in DMSO-d 6 ) ) was isolated from the Papuan marine sponge