Natural Products Diversity of Marine Ascidians (Tunicates; Ascidiacea) and Successful Drugs in Clinical Development

This present study reviewed the chemical diversity of marine ascidians and their pharmacological applications, challenges and recent developments in marine drug discovery reported during 1994–2014, highlighting the structural activity of compounds produced by these specimens. Till date only 5% of living ascidian species were studied from <3000 species, this study represented from family didemnidae (32%), polyclinidae (22%), styelidae and polycitoridae (11–12%) exhibiting the highest number of promising MNPs. Close to 580 compound structures are here discussed in terms of their occurrence, structural type and reported biological activity. Anti-cancer drugs are the main area of interest in the screening of MNPs from ascidians (64%), followed by anti-malarial (6%) and remaining others. FDA approved ascidian compounds mechanism of action along with other compounds status of clinical trials (phase 1 to phase 3) are discussed here in. This review highlights recent developments in the area of natural products chemistry and biotechnological approaches are emphasized.


Introduction
The study of marine natural products (MNPs) is becoming ever more sophisticated and an increasingly collaborative effort between marine biologist, chemist and pharmacologist, which involves the discovery of new natural products to enter preclinical studies and clinical tests. Since the 1990s, several MNPs and their applications towards marine biotechnological and therapeutical potential were reported. Large numbers of bioactive compounds were dragged up from marine invertebrates, especially sponges, ascidians, bryozoans and molluscs in which some of them are approved by FDA and currently utilized in clinical trials [1].
Ascidians or sea squirts (Phylum: Chordata, Class: Ascidiacea) are also known as tunicates due to their external covering, found tied to rocks and high-current fields. There are approximately 3000 living species of ascidians were reported [2]. The production of chemical compounds is principally important for soft bodied ascidian species, which use secondary metabolites to deter predatory fishes, to compete for space, to control settlement and growth of microbial fauna and other fouling organisms. Ascidians represent the most highly evolved group of marine organisms commonly investigated for identification of MNPs and provide rich sources of bioactive secondary metabolite with promising potential biomedical applications [3][4][5]. So far a few novel compounds have been purified and characterized with a view of developing marine drug discovery. However, the most well known didemnins has been isolated from whole body homogenates of Caribbean ascidians belonging to the genus of Trididenium sp. [6]. More than 80% of new ascidians compounds contain nitrogen, and nearly 70% of nitrogenous metabolites are alkaloids [7][8][9]. These compounds often exhibit a range of biological activities such as cytotoxicity, antibiotic, immunosuppressive activities, inhibition of topoisomerases and cyclin kinases [10]. On the other hand, nonnitrogenous metabolites are fewer available in ascidians and also less significant. Hence, identification of the biogenetic origin of ascidian natural products is often challenging [11]. The first bioactive metabolite geranyl hydroquinone was isolated from the ascidian Aplidium sp. [12]; only 230 metabolites were isolated from ascidians during 1974-1992 [3].
At that time, a wide-ranging attention has focused on ascidians because of their biologically active metabolites and the chemical diversity of ascidians has become one of the most significant sources of MNPs. It has been demonstrated that marine ecosystems are essential producers of unusual chemical compounds and potent bioactivities [4,5,9,13]. Nonetheless, significant research in the area of marine pharmacology is a very recent origin, and also few products (or their analogues) have already reached the market as therapeutic drugs. Indeed, ascidian-derived natural products have yielded promising drug leads, among which ecteinascidin 743 (Yondelis Ò ) and dehydrodidemnin B (Aplidin Ò ) are in clinical usage for the treatment of specific cancers [14,15].
The research attempt on MNPs has not targeted all marine invertebrates equally. Ascidians are one of the most intensely studied organisms during the 21st century so that 572 secondary metabolites were reported from 1994 to 2014. This present study represented MNPs studied from family didemnidae (32%), polyclinidae (22%), styelidae and polycitoridae (11-12%) exhibiting the highest number of promising MNPs (Fig. 1). The distribution of chemistry class of ascidian MNPs are given in (Fig. 2). Close to 580 compound structures are here discussed in terms of their occurrence, structural type and reported biological activity. Anti-cancer drugs are the main area of interest in the screening of MNPs from ascidians (64%), followed by anti-malarial (6%) and remaining others (Fig. 3). The National Cancer Institute-United states estimate that approximately 1% of MNPs showing anti-tumor cytotoxicity properties as against only 0.01% amongst their terrestrial counterparts. Accordingly, finding MNPs research must being continued to progress to improve existing therapies and to develop novel cures.
This review focuses on the chemical diversity of marine ascidians. The recent research on MNPs has been surveyed at relatively frequent intervals [4,5,9,13]. Davidson [3] was published the first review on secondary metabolites derived ascidians from 1988 to 1993. Additionally, in contrast to the review of ascidian metabolites, the present study provides complete list of the compounds with biological activities; the primary focus of this article is addressed to structures and properties of promising ascidian metabolites and their biological activities. In this latter regard, anti-bacterial, antifungi, anti-diabetic, anti-viral, anti-HIV, anti-inflammatory, anti-proliferative, anti-malarial, anti-oxidant, anti-tumor, anti-cancer compounds, described in marine ascidians during 1994-2014 were reported here in. In this study, we did not aim to review proceedings of conference, scientific reports and patent literature. Wherever possible, we made attempt to focus on their potential biogenesis, chemical structure-activity relationships. Nevertheless, the present study has been mainly focused on their recent developments in the preclinical studies, biotechnology advances and future directions of ascidian secondary metabolites. Meanwhile, the possible cytotoxicity and growth inhibition of MNPs is an identifying and short-listing of a potential drug molecule.  This review is restricted to those compounds exhibiting promising in vitro activity which was reported during the above period. We listed out 327 anti-tumor/cancer, 93 antimicrobial, 16 anti-HIV, 16 Central Nervous system depression, 15 anti-inflammatory compounds and other important compounds reported during the years 1994-2014.

Natural Products from Marine Ascidians
MNPs can also be prepared by chemical synthesis method by both total synthesis and semisynthesis method and it is playing a major role in drug discovery process. In recent years' notable studies have been carried out in the area of chemical diversity from the marine ascidians. Major alkaloids were reported in ascidians of purple, blue, green, and brown morphs of Cystodes dellechiajei collected from Mediterranean Sea [16]. Lopez-Legentil et al. [16] reported two distinct chemotypes in ascidian species: the purple morph of C. dellechiajei have the pyridoacridines shermilamine B (1) and kuanoniamine D (2) in tunic and its deacetylated forms (3,4) in zooids, while the blue and green morphs comprised the C9-unsubstituted pyrridoacridines, ascididemin (5) and 11-hydroxyascididemin (6) in tunic and zooids as well. However, brown morphs consist low concentration of ascididemin. The advanced studies of the mitochondrial DNA of the distinct colour morphs of C. dellechiajei exhibited weak a correlation between the chemotypes, morphotypes (spicules), and genotypes with the clear relationship among the colour of the purple morph and the pyridoacridines. The purple morphs were found under the acidic conditions of tunic in the Cystodytes sp. 11 secondary metabolites, among which eight are indole alkaloids were reported from the ascidian Leptoclinides sp. [17]. The first group of Leptoclinidesderived indole metabolites consists of N-(1H-indolyl-3carbonyl)-D-arginine (7), N-(6-bromo-1H-indolyl-3-carbonyl)-L-arginine (8), N-(6-bromo-1H-indolyl-3-carbonyl)-L-histidine (9) and N-(6-bromo-1H-indolyl-3-carbonyl)-Lenduracididine (10) compounds. Furthermore, the other metabolites leptoclinidamines A-C (11)(12)(13) were reported from the ascidian L. durus [18] and C 2 -a-D-mannosylpyranosyl-L-tryptophan (14) was isolated from L. dubius [19].
Two novel phenylalanine-peptides styelins A, B were isolated from the hemocytes of solitary tunicate Styela clava collected in California coast [72]. Both the peptides had very similar m/z (Styelin A, 3685.8; Styelin B, 3700.6). Styelins A and B showed a significant inhibition against bacterial pathogens of humans (MIC 0.5 lM). Styelins killed marine bacteria, Psychrobacter immobilis and Planococcus citreus, in media containing 0.4 mM NaCl. Moreover, histidine rich amidated 23-residue peptide clavaspirin (FLRF IG SVIHGIGHLVHHIGVAL-NH 2 ) was identified by cloning of a peptide cDNA from the pharyngeal tissues of S. clava [72].

Anti-HIV Activity
An unusual sulfated mannose, homopolysaccharide (143) also known as kakelokelose, was reported from the mucous  Structure 4 Anti-fungal potential compounds  from Didemnum chartaceum from the Great Barrier Reef, Australia. Unusually long relaxation times were observed for certain signals in their 1 H NMR spectra [86]. During further investigation, Reddy et al. [87] reported lamellarin a 20-sulfate (149) from an unidentified ascidian, collected in Indian waters. Compound (149) showed moderate inhibition of anti-HIV activity against HIV-1 protease (IC 50 16 lM).
An anti-retro viral metabolite, cyclodidemniserinol trisulfate (150)  reported from an Indonesian Didemnum sp. [88], though few significant variations between these two chemical structures, like the presence of an additional ring containing a glycine unit and the presence of sulfate groups, were observed. Moreover, Gonzalez et al. [88] also reported didemniserinolipids B and C (152-153) from the same ascidian species. Anti-HIV activity of compound (150) showed a modest inhibition against HIV-1 protease (IC 50 60 lg/mL) and with MCV topoisomerase IC 50

Anti-inflammatory Activity
Two new tricyclic thiazine-containing quinolinequinone alkaloids, ascidiathiazones A and B (177,206) were reported from Aplidium sp. collected in New Zealand ascidian coast. Compounds (177,206) showed a potential inhibition against human neutrophils (IC 50 1.55, 0.44 lM), respectively [117]. Structure 7 Anti-diabetic and anti-oxidant potential compounds (191)(192)(193)(194)(195)(196)(197)(198)(199)(200)(201)(202)(203)(204)(205) release (IC 50 35 lM) [118]. Belmiro and co-workers reported anti-inflammatory properties of a dermatan sulfate similar to mammalian heparin from the ascidian Styela plicata, collected in Brazil coast [119,120]. The molecular characterization of this analogue have been tested by in vivo rat colitis model, showing a significantly decreased lymphocyte and macrophage recruitment as well as TNF-a, TGF-b, and VEGF production in the inflamed rat colon at concentration 8 mg/kg per day. New anti-inflammatory meroterpenoids, 2-geranyl-6methoxy-1,4-hydroquinone-4-sulfate (161) and scabellone B (163) showed a moderate potent anti-inflammatory activity (21, 125 lM), and selective ability to inhibit neutrophil respiratory burst proves that meroterpenoid sulfates may have potential for developing novel marine drugs for treatment of inflammation [96]. Biologically active amino acid derivatives, herdmanines A-D (208)(209)(210)(211) were reported from the ascidian Herdmania momus, the unusual D-form of arginine present in herdmanines A-C [121]. Both compounds (210,211) showed adequate suppressive effects on the production of NO (IC 50 96 and 9 lM) and these compounds have the potential to inhibit the mRNA expression of iNOS. Additionally, compound 211 exhibited a strong inhibition of mRNA expression of pro-inflammatory cytokines IL-6. Further investigations are needed to search for potential anti-inflammatory MNP from the same ascidian. A series of anti-diabetic amino acid derivatives new congeners herdmanines E-L (212-220) and (-)-(R)-leptoclinidamine B were reported [122,123]. Indoleglyoxylyl derivatives, herdmanine K showed strong PPAR-c activation in rat liver cells (Ac2F) 1 and known metabolites homarine and trigonelline were reported from Eusynstyela latericius collected in Great Barrier Reef, Australia [128]. The spectral data of eusynstyelamide A (226) was similar to previously reported compound eusynstyelamide from a Fijian Ascidian E. misakiensis [129], remarkably, the compound showed opposed specific rotations. This study proved that the structure of eusynstyelamide was elucidated mistaken by Swersey et al. [129] and the authors have predicted the correct chemical structure of this compound and named as eusynstyelamide A. Both compounds 227, 228 showed neuronal nitric oxide synthase inhibition IC 50 values of 4.3-5.8 lM.
Chem-biological evaluation of methanol crude extract from the ascidian Eudistoma virde showed a dose-dependent depression of locomotor activity, indicating central nervous system (CNS) depressant activity. The extract at 200 mg/kg concentration showed substantial depression activity of 90.7% which was lower compare to the positive control drug chloropromazine 99% at 4 mg/kg concentration [124]. The similar results of CNS depressant activity in the crude compounds of Distapila nathensis at concentration 100 mg/kg was reported earlier [133].

Anti tumor/Anti-cancer, Anti-proliferative Activity
The drug discovery of anti-cancer activity has been the main area of interest in the field of natural product chemistry. Tunicates are particularly successful to yielding antitumor compounds; several MNPs were under various clinical trials in Europe and USA. With the continuous searching of effective and targeted anti-cancer drugs, in this section we explore the unique perspectives, novel biomolecules isolated from marine ascidians with promising pharmacological potential source of anti tumor/anticancer and anti-proliferative activity.
During further investigation from the same ascidian A. conicum collected in Porto Cesareo, Italy: Menna and coworkers reported two new meroterpenes, the conithiaquinones A (261) and B (262), and known metabolite chromenols A, B (263,264) and conicaquinone (254) [144]. Both compounds (261, 262) exhibited significant effects on the growth and viability of cells, compound (261) shows moderate cytotoxicity inhibition against human breast cancer cells (IC 50 44.5 lM). It is noticed from all the above investigations, Mediterranean ascidian species A. conicum have been extensively studied well. A rare collection of this tunicates depends on is that habitat and the abundance of its meroterpenes apparently depend on the geographical place of collection [144].
Staurosporines group of MNPs and their analogues are frequently reported in Eudistoma sp. [148]. Staurosporines, 11-hydroxystaurosporine (272), and 11-dihydroxystaurosporine (273) were reported from Eudistoma sp. collected at Micronesian island [149], additionally; the staurosporine aglicone (K252-c) 274 was isolated from same ascidian Eudistoma sp. collected at west coast of Africa [150]. In continuing search of anti-cancer drugs, Schupp and co-authors have reported three new indolocarbazole alkaloids, 3-hydroxy-4 0 -N-methylstaurosporine (275), 3-hydroxy-4 0 -N-demethylstaurosporine (276), 3 0demethoxy-3 0 -hydroxy-4 0 -N demethylestaurosporine (277) and known metabolites staurosporine (278), 4 0 -N-methylstaurosporine (279), 3-OH-staurosporine (280), 3-OH-4 0 -Nmethylstauro (281) and their derivatives from the ascidian Eudistoma toealensis [151][152][153]. Four metabolites were tested as inhibitors of cell proliferation with twelve human leukaemia cell lines. Among these, compound (280) showed potential anti-proliferative activities against MONO-MAC-6 cell line (IC 50 13 ng/mL). Though, from all these staurosporine derivatives, excluding 3 0 -demethoxy-3 0 -hydroxy-4 0 -N demethylestaurosporine and 4 0 -Nmethylstaurosporine was potentially reduced RNA and DNA synthesis. Compare to all the derived staurosporine, 3-hydroxystaurosporine is the most potential and highly effective staurosporine-type inhibitors discovered so far in the drug discovery process. This study demonstrated structure-activity relationships that hydroxylation of staurosporine at position 3 of the indolocarbazole moiety provoked to increase the anti-proliferative activity, while hydroxylation at carbon (C) 11 lead to decrease the biological activity of these derivatives. The authors also proposed that presence and absence of hydrophilic substitutions and the alteration of position in the biomolecules are vital in the anti-proliferative properties of staurosporine derivatives. It is worth to mention here, protein kinase is one of the major targets of staurosporine and have been widely used as molecular tools, the pharmacological potential variation of the staurosporine derivatives in regulating cell growth inhibition may due to differences in their ability to inhibit certain kinases involved in cell growth and tumour promotion [154,155]. 7-hydroxystaurosporine (UCN-01) is currently in clinical phase trials II at the NCI-USA, UCN-01 strongly inhibits the growth of T cell lymphomas [147].
During the chemical investigation, Tadesse and co-authors was isolated marine alkaloid synoxazolidinone A (86) from the ascidian Synoicum pulmonaria [58]. Metabolite (86) showed modest cytotoxicity against HT-29 colon carcinoma cells (IC 50 30.5 lM) and also killed normal lung fibroblast cells (MRC-5) at the same concentration. This suggests that the additional pyrrolidine ring of compound (85) modulating cell growth and increasing cell cytotoxicity. The known rubrolide derivatives, prunolide A (138) was isolated from the Indian ascidian Synoicum sp. [83]. Compound (138) exhibited potent cytotoxicity against breast cancer cell lines at a concentration of \1 lM.
Six new bromoindole derivatives, aplicyanins A-F (517-522), were isolated from the ascidian Aplidium cyaneum collected in Antarctica [232]. The metabolites 518-522 showed significant cytotoxic and anti-mitotic activities. Compare to all the metabolites, compounds 518, 520 completely inactive at maximum concentration and compound 521 showed poor cytotoxicity. The above results demonstrating that key role for the presence of the acetyl group at N-16 in the efficacy of this group of compounds. Kehraus and co-workers reported 5 new amino acid derivatives (523-527) from the Atriolum robustum collected in Great Barrier Reef [233]. Compound (523) contains a unique 3-(4-hydroxy-phenyl)-2-methoxyacrylic acid moiety, so far only reported in ascidian Botryllus sp. [130]. Compound (526) showed moderate inhibition with cAMP accumulation in Chinese hamster ovary (CHO) cell membranes and recombinantly exhibiting the human A 3 adenosine receptor, this clearly indicates that the adenosine derivative is partially acting against A3ARs. In radioligand binding analysis, 5 0 -deoxy-5 0 -methylthioadenosine-2 0 , 3 0 -diester 4 exhibited strong affinity with A 1 and A 3 adenosine receptors (K i [ 10 lM) and with A 2A and A 2B adenosine receptors (K i 17 lM).
A series of meroterpenes derivatives reported from the ascidian A. densum by [54,55] and demonstrated chemical synthesize [235]. Compounds, cordiachromene A (76),  [54]. Similar results of cytotoxicity of epiconicol reported [236] (IC 50 10 lg/mL) against the tumour cell lines (P388, A549, HT29, CV1). In addition, primary screening of methoxyconidiol had shown an adequate anti-proliferative activity against bacterial strains. The results of this study suggesting that the mechanism of the action of methoxyconidiol could be arbitrate by interruption of microtubule dynamics [55]. This led authors to synthesize methoxyconidiol together with epiconicol and didehydroconicol [235]. Synthesized meroterpenes, chromane (532), chromene (533) and hydroquinone (534) have showed modest inhibition of egg division. Methoxyconidiol showed significant results on P. lividus eggs (IC 50 0.80 lM) and S. granularis Compounds (77,533) showed moderate inhibition of P. lividus eggs division (IC 50 9.80, 11.30 lM) and completely inactive with S. granularis eggs [235]. Authors' suggesting that anti-proliferative activity on sea urchin eggs was due to methoxyconidiol and not due to one of its degradation products. The results of this study confirmed previous research finding on sea urchin eggs. Additionally, epiconicol (77) was showed most potent activity with human carcinoma cells and had shown poor cytotoxicity against serum stimulated carcinoma cells. Authors confirmed from the study, the position alteration of compound is reducing anti-bacterial and anti-proliferative activity of meroterpenes on sea urchin eggs and on normal, immortalized and Compound (535) showed potent cytotoxicity against A2780 (IC 50 0.34 lM). Pyrrole alkaloids, Lamellarin N (538) with nine sulphate derivatives, lamellarins T, U, V and Y (539-542) and lamellarins T-X (543-547) were isolated from the unidentified ascidian collected in Arabian Sea, India [238]. Lamellarin N (538) showed cytotoxicity

Successful Ascidian Marine Natural Products in Clinical Development
Nature products chemistry research has become much more applied, targeting compounds, which exhibit pharmacologically useful biological activities. Till date there are few metabolites reported from the ascidian were approved by FDA and has reached Phase I, II clinical trials, the list are presented in (Table 1). Didemnin B (564) was reported from Trididemnum solidum (Family Didemnidae), showed strong anti-viral and invivo cytotoxicity [241,242]. Total synthesis, complete spectroscopic characterization and single-crystal X-ray structure of metabolite (564) was reported [243][244][245]. Didemnin B was the first MNPs to enter phase I and II clinical trials as an anti-cancer agent.
Previous studies demonstrated that compound (564) strongly inhibits palmitoyl protein thioesterase in a noncompetitive manner [246,247] while this low affinity target did not completely show inhibition at nano molar concentration. Compound (564) had showed potential activity against herpes simplex virus [248] and also against Ehrlich's carcinoma in mice [249]. During initial cancer trials, didemnin B exhibited modest activity and displayed the constraint for the treatment with anti-emetics [250]. Invitro test has demonstrated that compound (564) was showed potential activity against colorectal cancer cells [251], lymphatic [252] and prostate cancers [253]. In addition, compound (564) was submitted to various Phase I trials [250,254] and Phase II clinical trials against nonsmall cell lung cancer (NSClC) [255], breast cancer [256] small-cell lung cancer [257], non-Hodgkin's lymphoma [258], metastatic melanoma [259], glioblastoma multiforme [260], and CNS tumours [261]. All these trials of compound (564) caused significant neuromuscular toxicity and no objective tumour responses. However, compound (564) was exhibited activity in patients with advanced pretreated non-Hodgkin's lymphoma, but trials are on hold owing to onset of severe fatigue in patients [262]. Other trials were terminated because of high incidence of anaphylaxis [263], currently all the clinical trials of compound (564) are on hold.
The toxicities of novel drugs are depending on the concentration of dose including neutropaenia and thrombocytopaenia. Hepatotoxicity was previously observed in preclinical trails but these may be able to control by dose regulation. In recent discovery utilizing HepG2 human hepatocellular liver carcinoma cells have shown a cytochrome P450-mediated metabolism of ET-743 (565). The hepatotoxicity was demonstrated in rats that pre-treatment with metabolism modulators such as dexamethasone and bnaphthoflavone abrogates ET-743-arbitrate hepatotoxicity [264], and dexamethasone-ET-473 combination drug treatment was recommended for study in humans [265,266]. Further, Yondelis Ò discovered an antitumor agent (Ecteinascidin/trabectedin, ET-743) in a marine colonial tunicate Ecteinascidia turbinate, and now produced synthetically, received Orphan Drug designation from the European Commission (EC) and FDA for soft tissue sarcomas and ovarian cancer and its registration in 2007 in the EU for the treatment of soft tissue sarcoma.
However, all the clinical trials with didemnin B are on hold, promising simple analogue of didemnin B, aplidine (dehydrodidemnin B) was first reported from the Mediterranean tunicate A. albicans and patent by Rinehart [267]. Aplidine (566) are being at variance from didemnin B (564) only in replacement of the N-lactyl side chain with a pyruvyl group. Compound (566) showed identical levels of anti-tumour activity compare to compound (564) against several cancer cell lines [263], and also showed improved cell apoptosis by induction of oxidative stress [267], which causes the pro-apoptotic receptor Fas (CD-95) [268] and induces the mitochondrion-mediated apoptosis [269,270]. Compound (566) also activates p38 mitogen-activated protein kinases (MAPKs) and jNK [271,272], and also inhibits secretion of vascular endothelial growth factor (VEGF) [273,274]. Non-P-glycoprotein have expressing cell lines that are resistant to compound (566), it demonstrated temporary phosphorylation of jNK and p38 MAPKs upon exposure to aplidine, and the short duration of activation was insufficient to trigger apoptosis [275] relapsed-recalcitrant leukaemia cell lines, aplidine arrests the cell cycle at the G1 and G2/M phases, and induces p53independent apoptosis [276]. Significant potential cytotoxicity of compound (566) was noticed against cultured lymphocytes and in transformed cell lines; it is suggesting that during in vivo compounds (564, 564) exhibited lowest haemotoxicity [277,278]. Currently phase III clinical trials of Aplidine with stomach, prostate and bladder cancers. In July, 2003 Pharmamar Ò was discovered plitidepsin (Aplidine) and received orphan drug status for treatment of lymphoblastic leukemia from European Medicines agency.
Semisynthetic derivative of staurosporine PKC-412 midostaurin (567) was from the bacterium Streptomyces staurosporeus (isolated from the Ascidian). Compound midostaurin (567) is currently in phase III trials under Novartis Ò for acute myeloid leukemia (AML), and other cancer disease. PKC-412 is a multi-target protein kinase inhibitor being tested for the treatment of myelodysplastic syndrome (MDS), AML and showed strong activity in patients with mutations of CD135 (FMS-like tyrosine kinase 3 receptor) [279]. After successful Phase II clinical trial, a Phase III trial for AML has started in 2008. It is testing midostaurin in combination with daunorubicin and cytarabine (clinical trial number NCT00651261) and in another phase II trial, the substance was proved ineffective in metastactic melanoma [280].
An Ascdiian derivative, becatecarin (570) is a synthetic diethylaminoethyl analogue of the indolocarbazole glycoside antineoplastic antibiotic rebeccamycin. Compound (570) stimulated ATPase activity and inhibited ABCG2 arbitrated transport at concentration [10 lM and induced ABCG2 expression in lung cancer cells. In phase I trial were tested in children with solid tumours to establish the dose limiting toxicity and maximum tolerated dose [292]. In phase II trial, cecatecarin (570) intercalates into DNA and stabilizes the DNA-topoisomerase I complex, thereby interfering with the topoisomerase I-catalyzed DNA breakage-reunion reaction and initiating DNA cleavage and apoptosis [293]. Compound (570)  activity against several tumours cell (medullary thyroid carcinoma) and blocks certain proteins involved in the growth of some tumors and kill cancer cells [294]. It is also behave a type of receptor which includes the tyrosine kinase inhibitor Trk-A, Trk-B,Trk-C and platelet-derived growth factor (PDGF) [295]. CEP-2563 was evaluated Phase I clinical trial in patients with advanced stage solid tumours. Undevia (571) is an indolocarbazole kinase inhibitor originally discovered by Kyowa Hakko Kogyo in the course of a program investigation of neurotrophic properties of derivatives of the natural product K-252a [296]. These are ploycyclic aromatic compounds containing an indole fused to carbazole. CEP-1347 demonstrated that apoptosis with multiple nerve cell types from a variety of agents leading to programmed cell death which significantly increase the dopamine neurons survival prior to and afterward transplantation. CEP-1347 blocks the activation of JNKs through ATP competitive inhibition of the upstream mixed lineage kinase (MLK) family. CEP-1347 had showed prominent neurotrophic and neuroprotective properties in-vitro and in animal models of neurodegeneration [297,298]. In particular, this inhibitor was able to reduce the loss of tyrosine hydroxylase immunoreactivity and dopamine transporter density in mice and monkeys following administration of the neurotoxin 1-methyl-4phenyl-tetrahydropyridine (MPTP) [298,299]. Unfortunately, the direct effect of CEP-1347 administration on inhibition of the MLK/JNK pathway in central nervous system of human subjects could not be determined in PRECEPT trial. Therefore, the failure of the PRECEPT trial has limited utility for assessing the relationship between JNK activity and neurodegeneration [300]. Preclinical trials indicated it was a neuroprotective drug, currently all the clinical trials of CEP-1347 were terminated on 2012 (clinical trial number NCT00040404).

Modern Instrumentation and Computational Biology
Marine ecosystem forms an important source of unique compounds with high structural uniqueness and incomparable chemical properties. At the core of MNPs discovery is the identification procedure and NMR stays on the most useful tool [310]. At this time, natural products chemistry research is progressing a dynamic comeback in the modern drug discovery. Relatively more advances have taken place concerning the inherent capabilities of NMR apparatuses, able to reduce experiment times and increase sensitivity toward more efficient analyses of novel compounds present in lM level [311]. The advance of high resolution magic angle spin NMR (HR-MAS NMR) probes is most useful to analyse intact tissues. Nevertheless, while (HR-MAS NMR) is incorporated in food chemistry where both primary and secondary metabolites are of importance, it has not been yet widely introduced in natural products chemistry [312]. Queiroz Junior et al. have impressively demonstrated the significance of the synergy between NMR hardware and innovative pulse sequences. It is the first report that an ultrafast COSY pulse sequence is applied to a hyphenated LC-NMR separation of crude extracts (Ex. three natural flavonoids; naringin, epicatechin and naringenin). The detection volume was only 60 mL, while two scans have proven sufficient to get spectra with optimized resolution and sensitivity. This application portrays the generality of ultrafast methodologies in natural product chemistry, placing LC-NMR as an effective analytical tool [313,314]. For example, NMR experiments such as DOSY and JRES were also very useful routine methodology for unraveling new chemical structures. NMR spectroscopy tirelessly continues leading this procedure. Furthermore, decisive chemical structural information could be derived from statistical interpretation methods applied in metabolomics such as statistical heterospectroscopy (SHY) [315], statistical total correlation spectroscopy (STOCSY) [316], heteronuclear single quantum correlation spectroscopy (HSQC), heteronuclear multible-bond correlation Spectroscopy (HMBC), Subset optimization by reference matching (STORM) [317], cluster analysis statistical spectroscopy (CLASSY) [318], multivariate statistical analysis of natural products fragments [319].
Nearly, dereplication analysis is necessary for computational support of data handling, processing and for structure elucidation purpose. Whereas user-friendly and sophisticated software packages are easily reached for effective data mining, they are not widely used for dereplication purposes in marine natural product chemistry [320]. However, the structure elucidation is most challenging task mainly due to uniqueness of natural products and unexpected spectral patterns and the residual complexity frequently noticed. For instance, prediction and simulation software such as PERCH, in combination with 1 H iterative full spin analysis (HiFSA approach), given an accurate distinction of natural products with nearly identical NMR spectra. As, proposed by the authors as a tool for puzzling qNMR analyses, it could be an alternative source of dereplication data [321]. Moreover, computerassisted structural elucidation (CASE) is the techniques of using software that allows users to input their NMR data, and through matching algorithms to generate all possible molecular structures. For this purpose, software used are mainly the Structure Elucidator by ACD Labs, StrucEluc and CCASA [322][323][324]. Nevertheless, the success of these approaches is dependent on the quality of the spectra to be processed and the efficacy of the algorithms used. Furthermore, the software used present an inherent dependence on the databases from which data are extracted. Unfortunately, NMR databases dedicated to NPs appear as in-house, fragmented attempts, or are chemical group/  [327,328].

LC-MS and 1 H-NMR Metabolomics
Recent developments in analytical methods have resulted in many different platforms for metabolomic investigation. From these, liquid chromatography-mass spectrometry (LC-MS) [329], and nuclear magnetic resonance spectroscopy (NMR) based approach are generally preferred analytic methods because they are based on the physical properties of MNPs, which are not influenced by other external factors and easily reproducible [330]. In the recent years, NMR combined with metabolomics tool is increasingly utilized for its systematic manner of profiling chemical finger prints of individual samples, either plant or animals [329,330]. NMR-metabolomics snap shots the organism's metabolites or biomolecules that are present in a given quantity at the given time point [331,332]. Metabolomics can be used in functional genomics and to differentiate marine organism from external variation. The metabolomics of biota is compilation of all its primary and secondary metabolites using 1 H-NMR and 2D-COSY spectroscopy methods. Kim and co-authors reported the protocol NMR based metabolomics of plant species [332]. Tikunov et al. [333] carried out study of taxonomy based metabolite profiling of an oysters using NMR metabolomics along with Multivariate Statistical Analysis Approach (MSAA). Additionally, in manila clam [334], corals [335], and LC-MS based metabolomic approach in marine bacteria [336], studies utilized the same methodology for classifying biomolecules based on their taxonomy. In earlier study, Halouska and co-workers [337] predicted the in-vivo mechanism of action for drug leads against anti-tubercular activity using NMR metabolomics and orthogonal partial least square-discriminant analysis (OPLS-DA).
Mass spectrometry based metabolomics approach can provide significant information about the discrimination between the species using multivariate statistical analysis, classifying chemical groups, discriminate the metabolites with unknown biological potencies [338]. A typical metabolomics profiling requires enormous number of samples to generate the results that are statistically rigorous. Besides, highly sensitive and accurate instrumentation, powerful software tools (e.g. XCMS-METLIN) are essential to address the vast amount of data generated by these experiments [339]. The recent development in the field of natural products chemistry and LC-MS/NMR based metabolomics research on marine origin secondary metabolites exhibits diverse range of biological properties for developing new therapies to improve the health of individuals across the universe suffering from various deadly diseases such as infectious disease malaria, HIV, neurological and immunological diseases and cancer [329,330,339]. The application of LC-MS based metabolic profiling of biological systems has gained more extensive use in identifying drug metabolite, developing metabolite maps and lending clues mechanism of bioactivation [338]. However, the knowledge of the metabolite accumulates in different ascidians chemical diversity are meagre. Recently, Palanisamy et al. [340] reported the The results of this study confirmed that LC-MS based metabolomics method could be used as reliable tool for taxonomic classification of marine ascidian species and species discrimination in future studies. Ascidian, S. plicata showed significant anti-microbial activity against Burkholderia mallei (10 mg/mL) [341], and S. plicata fraction SP50 exhibited strong inhibition and induced apoptosis against cervical carcinoma (HeLa) and colon carcinoma (HT29) with IC50 (33.27 and 31.66 lmol/L).

Recent Biotechnology Advances
In a new marine drug discovery approach, structurally more complex MNPs was moved the next step from discovery to clinical trials based upon the strength of the industrial reproducibility. The discovery of novel marine drugs will continue to diversify. Research laboratories, academic entrepreneurs, and innovative biotechnology industries will play major role in the discovery of novel marine drugs. The industrial collaboration program between natural products researchers and biotechnology industries will be instrumental to the primary clinical trials and mechanism of action studies crucial to provide the compelling preclinical data to create ample interest from larger pharmaceutical companies to lead and support for drug discovery program of MNPs. Also, it is essential to identify molecular targets for strongly active biomolecules and the ability to synthetically produce novel biomolecules to progress and discover new drugs.
A recent development in biotechnological approach is revolutionizing the field of natural products chemistry. It is Structure 11 continued developments in analytical tools and molecular biological science facilitate to identify the primary producers of secondary metabolites from symbiotic assemblage, and enable researchers to further explore the marine microbial chemical diversity for drug like biomolecules. Furthermore, those advances aide the characterization of several biosynthetic gene clusters and pathways and ultimately allow for their manipulation. Marine microbial chemical diversities are now easily explored drug like compounds using effective biosynthetic genetic engineering and in vitro multi enzyme synthesis methods [342]. Remarkably, David Hopwood's group [343] has biosynthesized anti-biotic compound actinorhodin from Streptomyces coelicolor by cloning and heterologous expression of an entire biosynthetic pathway. Using genetic engineering techniques, Donia et al. [344] were prenylated anti-tumor compound trunkamide which is previously isolated in ascidian and different genera of cyanobacteria in E. coli culture. Didmnid ascidian species specificity of symbiosis and secondary metabolism in ascidian species were reported [344], collected in Florida coast. In this study, species specific and location-specific components were observed in Dideminid ascidian microbiomes and metabolomes. It is concluded that the biotechnological approaches in the field of natural products chemistry is more useful for sustainable supply of high quality marine drugs.

Conclusions
This review study represents trends in chemical diversity of marine ascidians and potential biomolecules, covering the various tunicates family, recent developments and future direction and modern biotechnology advances are highlighted. Remarkably, Genus Didmnium sp. is most studied species in this group followed by Aplidium sp., Synoicum sp., and Eudistoma sp. collected from coral reefs, intertidal regions, shallow water and mangrove ecosystem which facilitates potential bioprospecting. Several MNP isolated from ascidian that are in various phase of pre-clinical and clinical studies from that Ecteinascidia and aplidine have great potential to reach market.
Anti-cancer drugs are the main area of interest in the screening of MNPs from ascidians (64%), followed by anti-malarial (6%) and remaining others. It is worth to note here, as the major financial support for the screening of new MNPs is made in cancer research [344]. The data discovered here undoubtedly confirmed that promising value of MNPs and their derived analogs are most important candidates for further pharmaceutical studies for discover new therapeutic treatment the anti-tumor/anti-cancer Anti-HIV and various diseases drug pipeline.
The unique chemical structures and novel chemical class of ascidians and promising biological activity which make them excellent candidates for development of many first class marine drugs in the near future with current advanced sampling methods, highly advanced analytical tools, new methods for genetic, chemical dereplication, molecular biology tools, LC-MS, NMR metabolomics approach, nature bank databases, computational biology, directed biosynthesis and biosynthetic pathway and high throughput screening the efficiency of exploring MNPs to discover novel therapeutics has increased significantly. It is concluded from this study, Ascidian resources contains vast pool of novel metabolites, exploring drug-like biomolecules will provide promising biomolecules with potential therapeutic use which may serve as lead candidates for drug discovery program.