handbook of marine natural products volume 426 || enzyme inhibitors from marine invertebrates

Enzyme Inhibitors from MarineInvertebrates 23Yoichi Nakao and Nobuhiro Fusetani

Contents

23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146

23.2 Oxidoreductases (EC 1.-.-.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147

23.2.1 Aldose Reductase (EC 1.1.1.21) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147

23.2.2 Enoyl-ACP Reductase (EC 1.3.1.9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1148

23.2.3 Lipoxygenases (EC 1.13.11.12) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149

23.2.4 Indoleamine 2,3-Dioxygenases (EC 1.13.11.42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150

23.2.5 Neuronal Nitric Oxide Synthase (EC 1.14.13.39) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151

23.3 Transferases (EC 2.-.-.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152

23.3.1 1a-1,3-Fucosyltransferases (EC 2.4.1.214) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152

23.3.2 Adenosine Phosphoribosyltransferase (EC 2.4.2.7) . . . . . . . . . . . . . . . . . . . . . . . . 1153

23.3.3 Farnesyl Protein Transferase (EC 2.5.1.58) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154

23.3.4 Geranylgeranyltransferase Type I (EC 2.5.1.59) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1156

23.3.5 HIV Reverse Transcriptase (EC 2.7.7.49) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1157

23.3.6 HIV Integrase (EC 2.7.7.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1158

23.3.7 Telomerase (EC 2.7.7.49) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1160

23.3.8 Epidermal Growth Factor Receptor Kinase (EC 2.7.10.1) . . . . . . . . . . . . . . . . . 1162

23.3.9 Tyrosine Kinase pp60V-SRC (EC 2.7.10.2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163

23.3.10 Other Tyrosine Protein Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164

23.3.11 MSK1 (EC 2.7.11.1) and MAPKAPK-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164

23.3.12 Raf/MEK1/MAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164

23.3.13 Checkpoint Kinases (EC 2.7.11.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164

23.3.14 Protein Kinase C (EC 2.7.11.13) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1166

23.3.15 Cyclin-Dependent Kinases (EC 2.7.11.22) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1170

23.3.16 Glycogen Synthase Kinase-3 (2.7.11.17) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1171

23.4 Hydrolases (EC 3.-.-.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172

23.4.1 Phospholipase A2 (EC 3.1.1.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172

Y. Nakao (*)

School of Advanced Sciences and Technologies, Waseda University, 3-4-1, Okubo, Shinjuku-ku,

Tokyo, Japan

N. Fusetani

Fisheries and Oceans Hakodate, Graduate School of Fisheries Sciences, Hokkaido University,

3-1-1, Minato-cho, Hakodate, Japan

E. Fattorusso, W. H. Gerwick, O. Taglialatela-Scafati (eds.),

Handbook of Marine Natural Products, DOI 10.1007/978-90-481-3834-0_23,# Springer Science+Business Media B.V. 2012

1145

23.4.2 Acetylcholinesterase (EC 3.1.1.7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177

23.4.3 Protein Phosphatases (EC 3.1.3.16) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178

23.4.4 Protein Tyrosine Phosphatases (EC 3.1.3.48) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183

23.4.5 Phospholipase C (EC 3.1.4.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184

23.4.6 Phosphodiesterase (EC 3.1.4.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185

23.4.7 Sialidase (EC 3.2.1.18) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186

23.4.8 Chitinase (EC 3.2.1.14) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188

23.4.9 a-Glucosidases (EC 3.2.1.20) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189

23.4.10 S-Adenosylhomocysteine Hydrolase (EC 3.3.1.1) . . . . . . . . . . . . . . . . . . . . . . . . 1190

23.4.11 Serine Proteases (EC 3.4.21.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191

23.4.12 Cysteine Proteases (EC 3.4.22.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1196

23.4.13 Aspartic Proteases (EC 3.4.23.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1198

23.4.14 Metalloproteases (EC 3.4.24.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1198

23.4.15 Histone Deacetylases (EC 3.23.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200

23.4.16 ATPases (EC 3.6.1.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1202

23.5 Lyases (EC 4.-.-.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208

23.5.1 ATP Citrate Lyase (EC 4.1.3.8) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208

23.6 Isomerases (EC 5.-.-.-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209

23.6.1 Topoisomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209

23.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1213

23.8 Study Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1213

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1214

Abstract

Marine invertebrates are the rich source of small molecules with unique

chemical skeletons and potent bioactivities; however, the major biological

activities tested for marine natural products have been mainly limited to

the traditional phenotype-oriented bioactivities such as cytotoxicity or anti-

microbial activities. Enzyme inhibition is the target-oriented bioactivity and

small molecule inhibitors are quite useful for controlling biological processes

and understanding complicated biological phenomena. Recently, it has been

recognized that the natural products are superior source of small molecule

probes useful for the chemical biology. Especially, marine-derived enzyme

inhibitors still remain to be fully investigated in spite of their high potentials

as a source of unique compounds. In this chapter, selected enzyme inhibitors

from marine invertebrates are described.

23.1 Introduction

Enzymes are vital to living organisms, maintaining the homeostasis of their lives by

mediating/regulating numerous biochemical events, including metabolism, catab-

olism, cellular signal transduction, cell cycles, and development. Dysfunction,

overexpression, or hyperactivation of the enzymes are often associated with

human diseases as evidenced from the molecular-based analysis of diseases.

Umezawa’s pioneering work on enzyme inhibitors resulted in the discovery of

1146 Y. Nakao and N. Fusetani

numbers of valuable compounds from terrestrial microorganisms [1]. Thereafter,

search for small molecular enzyme inhibitors has been actively conducted in both

academia and pharmaceutical industries, and a considerable number of enzyme

inhibitors have been developed as drugs, including blockbusters such as statins.

However, only recently, the majority of the marine natural products chemists

started systematic programs for discovering enzyme inhibitors; the first systematic

investigation was done by our group for H,K-ATPase inhibitors [2]. Although

a large proportion of enzyme inhibitors reported from marine organisms were

known compounds found to inhibit enzymes in the reevaluation of activities,

these known compounds with new activities are important for the related research

areas such as chemical biology. It may be predicted that the trends of marine natural

products chemistry will move from finding merely “new structures” to finding “newactivities” of known compounds. Recent reevaluation of natural products as

a superior source of small molecule probes useful for studying biological phenom-

ena is encouraging natural products chemists to investigate compounds with new

activities.

In this chapter, we will describe the structures and activities of selected

enzyme inhibitors isolated from marine invertebrates. Some inhibitors are not

from marine invertebrates, but they are related to structures from microorgan-

isms. Synthetic enzyme inhibitors based on the marine natural products are also

described. Most of them were designed based on the crystal structures of

enzyme–inhibitor complexes and gave a detailed insight into the modes of

action. However, we excluded most of bromotyrosines, polyacetylenes, and

highly sulfated steroids which are often termed “nuisance compounds,” because

they show a variety of biological activities including inhibition of enzymes.

Inhibitors are listed according to the classification of target enzymes

(EC number).

23.2 Oxidoreductases (EC 1.-.-.-)

23.2.1 Aldose Reductase (EC 1.1.1.21)

Aldose reductase catalyzes the reduction of glucose to sorbitol with concomitant

conversion of NADPH to NAPD+. The highly activated enzyme under hypergly-

cemic environment results in unusual accumulation of sorbitol in the eye lens or

peripheral nerves that lead to retinopathy, cataract, neuropathy, nephropathy, or

cardio-vascular diseases. Three polycyclic bis-indoles (1–3) from a marine sponge

Dictyodendrilla sp. strongly inhibited bovine lens aldose reductase with IC50 values

of 49, 125, and 112 nM, respectively. Interestingly, the sulfate group in 1 and 2 didnot influence the activity (4: IC50 102 nM), while the sulfate group in 3 potentiatedthe activity (5: IC50 567 nM) [3].

23 Enzyme Inhibitors from Marine Invertebrates 1147

N

NH

O

OR

OH

OH

HO

O

HO

HO

O

N

NH

O

OR

OH

OH

HO

MeOOC

HO

1 R = SO3Na2 R = SO3H4 R = H

3 R = SO3H5 R = H

23.2.2 Enoyl-ACP Reductase (EC 1.3.1.9)

In mammals and other higher eukaryotes, all reactions in fatty acid synthesis are

catalyzed by the type I fatty acid synthase (FAS-I), while bacteria, plants, and algae

contain a type II system (FAS-II), in which each reaction is carried out by

a monofunctional enzyme. The type II fatty acid biosynthetic pathway has recently

been discovered in a number of Apicomplexan parasites, including the malaria

parasite, Plasmodium falciparum. Mycobacterium tuberculosis employs both

FAS-I and FAS-II systems. FAS-I is responsible for the synthesis of short-chain

FAs, whereas the FAS-II system extends these FAs to very long-chain

mycolic acids, important components of the mycobacterial cell wall. The central

role of FA biosynthesis and the structural differences between the human and

microbial FAS systems render FAS-II as an attractive target for antimicrobial

drug discovery.

The FabI (enoyl-ACP reductase) is a crucial enzyme of all FAS-II systems, since it

catalyzes the last NADH-dependent reduction step in each elongation cycle. Six

known metabolites (6–11) and complex fatty acid mixtures (FAMA-FAMG) iso-

lated from the Turkish marine sponge Agelas oroides were identified as active

components in the target (Pf FabI)-based antimalarial screening. Compounds 6–11showed inhibitory activity against Pf FabI with IC50 values of>100,>100, 40, 0.30,

5.0,>100, and>100 mg/mL, respectively. Interestingly, TFA salt of 8 showed betterinhibitory activity (IC50 40 mg/mL), while the activity of TFA salt of 9 became

weaker (IC50 5.0 mg/mL). These enzyme inhibitory activities were well correlated

with anti-plasmoidal activities against P. falciparum, except that 10 showed better

anti-protozoan activity (IC50 7.4 mg/mL) compared to its enzyme inhibitory activity.


Mixtures of fatty acids showed Pf FabI inhibitory activity with IC50s in the range of

0.35–20 mg/mL, while they showed anti-plasmoidal activities against P. falciparum(IC50s 3.4–40.9 mg/mL), Trypanosoma brucei rhodesiense (IC50s 5.3–42.3 mg/mL),

Trypanosoma cruzi (IC50s 16 to > 30 mg/mL), Leishmania donovani (IC50s

6.0–14.4 mg/mL), respectively. In addition, two mixtures (FAMA and FAMD)inhibited FabI of Mycobacterium tuberculosis (MtFabI, IC50s 9.4 and 8.2 mg/mL,

respectively) and Escherichia coli (EcFabI, IC50s 0.5 and 0.07 mg/mL, respectively).

FAMAwas a mixture of 5,9-dienoic fatty acids of C23(12) and C24(13), while FAMD

contained 10-methylhexadecanoic and palmitic acids as major lipids [4].

6

HOH

NH

Br

Br COOMe

7

NH

Br

Br COOH

8

NH

Br

Br

HN

ONH

N

NH2

9

H2NNH

N

NH2

10

+H3NS

O−

O

O

11

HO

O

n

12: n = 1113: n = 12

23.2.3 Lipoxygenases (EC 1.13.11.12)

Lipoxygenases (LO) mediate hydroperoxidation of polyunsaturated fatty acids

(PUFAS), leading to formation of leukotrienes and lipoxins. Selective inhibitors

of lipoxygenase isoforms can be useful for pharmacological agents, nutraceuticals,


or molecular tools. Fucosides, originally isolated from the Caribbean gorgonian

Eunicea fusca [5], selectively and irreversibly inhibited leukotriene (LT) formation

in murine models of inflammation. Further pharmacological study indicated that

fucoside B (14) inhibits conversion of arachidonic acid (AA) to leukotriene

B4 (LTB4) by inhibition of 5-LO with an IC50 of 18 mM [6]. Similarly,

didemnilactones A (15) and B (16) isolated from the tunicate Didemnum moseleyishowed inhibition against lipoxygenases of human polymorphonuclear leukocytes

with IC50 values of 9.4 and 8.5 mM (15 and 16, respectively against 5-LO), and

41 mM (15 against 15-LO) [7].

O

H H

O

OH

OH

OH

fucoside B (14)

O

O

OH

O

O

OH

didemnilactone A (15) didemnilactone B (16)

23.2.4 Indoleamine 2,3-Dioxygenases (EC 1.13.11.42)

Indoleamine-2,3-dioxygenase (IDO) catalyzes the oxidation of tryptophan to

N-formylkynurenine at the rate-limiting step in the metabolic degradation of trypto-

phan. Because T-cells are sensitive to tryptophan depletion, overexpression of IDO in

most tumors is thought to cause apoptosis of T-cells and, as the results, leads to immune

escape of solid tumors. Significant regression of tumors observed in a mouse model

system treated by a combination of relatively poor IDO inhibitor such as

1-methyltryptophan and cytotoxic agents (e.g., paclitaxel) have provided proof of

demonstration for the potential value of IDO inhibitors in cancer treatment.

Exiguamine A (17) was isolated as an IDO inhibitor from the marine sponge

Neopetrosia exigua collected at Papua New Guinea. The biogenesis of the unprec-

edented complex hexacyclic alkaloid skeleton of 17 was proposed to be starting

from DOPA, tryptophan, and N,N-dimethylhydantoin. Exiguamine A has a Ki of210 nM for inhibition of IDO in vitro [8]. Synthetic analogues of 17were preparedand evaluated their inhibitory activity against IDO. Although all of the synthetic

analogues turned out to be less active than 17 (Ki 41 nM in the modified


assay system), a simpler analogue 18 showed relatively potent noncompetitive

inhibition against IDO (Ki 200 nM) [9]. Four new polyketides annulin C (21),2-hydroxygarveatin, garveatin, and garvin C, as well as known annulins A (19) andB (20), garveatins A and C, and 2-hydroxy garvin A were isolated from the

Northeastern Pacific hydroid Garveia annulata. Among these compounds,

annulins A–C showed potent submicromolar IDO inhibition with Ki values of

0.14, 0.69, and 0.12 mM, respectively [10].

O

N

NH

O

O

H2N

OH

NN

O

O

exiguamine A (17)

NH

H2NO

O

NN

O

O

analogue (18)

O

O

OH

OO

OOR

annulin A (19) R = H

annulin C (21) R = Me

O

O

O

O

O

O OH

annulin B (20)

23.2.5 Neuronal Nitric Oxide Synthase (EC 1.14.13.39)

Neuronal nitric oxide synthase (nNOS) represents an important therapeutic target

for neuropathological disorders, such as stroke, Alzheimer’s disease, Parkinson’s

disease, AIDS, and dementia, because NO plays an important multifunctional role

in the modulation of many neurotransmitters in the brain and its overproduction has

been associated with such diseases. Eusynstyelamides A–C (22–24) isolated from

the Australian ascidian Eusynstyela latericius inhibited nNOS with IC50 values of

41.7, 4.3, and 5.8 mM, respectively, while they were found to be nontoxic toward

the three human tumor cell lines MCF-7 (breast), SF-268 (CNS), and H-460 (lung).

Eusynstyelamides A and B were mildly antibacterial against Staphylococcus aureus(IC50s 5.6 and 6.5 mM, respectively), and weakly inhibited the C4 plant regulatory

enzyme pyruvate phosphate dikinase (PPDK) (IC50 values of 19 and 20 mM,

respectively) [11].


eusynstyelamide A (22)

NH

Br

N

HN NH2

OOH

NH

HN

O

H OH

NH

Br

NH

NH2

NH

eusynstyelamide B (23)

NH

Br

N

HN NH2

OOH

NH

HN

O

H OH

NH

Br

NH

NH2

NH

eusynstyelamide C (24)

NH

Br

N

HN NH2

OOH

NH

HN

O

H OH

NH

Br

NH

NH2

NH

23.3 Transferases (EC 2.-.-.-)

23.3.1 1a-1,3-Fucosyltransferases (EC 2.4.1.214)

a-1,3-Fucosyltransferases (Fuc Ts, EC 2.4.1.214) which catalyze the transfer of

L-fucopyranoside residues from guanosine diphosphate fucose (GDP-fucose) to

glycoconjugate acceptors are known to be involved in the biosynthesis of sialyl

Lewis X (SLeX) present on the extracellular surfaces of leukocytes. The E-selectin-

SLeX interaction encourages leukocytes to move from the bloodstream into sites

of injury or infection and to cause inflammation, thus indicating that inhibitors


of a-1,3-fucosyltransferase are potential drugs for the treatment of inflammatory

diseases [12]. Octa- and nonaprenylhydroquinone sulfates 25 and 26 isolated from

an Australian marine sponge Sarcotragus sp. inhibited a-1,3-fucosyltransferase VII(Fuc TVII) with IC50 values of 3.9 and 2.4 mg/mL, respectively, while they showed

very weak activity against Fuc TVI [13].

OSO3Na

OH

n

octaprenylhydroquinone sulfate (25)

nonaprenylhydroquinone sulfate (26)

n6

7

23.3.2 Adenosine Phosphoribosyltransferase (EC 2.4.2.7)

Adenosine phosphoribosyltransferase (APRT) catalyzes the salvage pathway of

purine nucleotides. In mammals, there are both de novo and salvage pathways for

purine nucleotides synthesis, while kinetoplastid parasites such as Leishmaniaspp. lack the de novo pathway. Therefore, selective inhibitors of phosphoribosyl

transferase should compromise the parasite metabolism selectively. Three new

disulfated meroterpenoids, ilhabelanol (27), ilhabrene (28), and isoakaterpin (29)were isolated from a Brazilian sponge Callyspongia sp. as L. tarentolae adenosinephosphoribosyltransferase (L-APRT) inhibitors. Isoakaterpin (29) inhibited

L-APRT with an IC50 of 1.05 mM, while the accurate IC50 values for ilhabelanol

(27) and ilhabrene (28) could not be obtained because of the too small amount of

isolated compounds [14].

OSO3Na OSO3Na

NaO3SO NaO3SO

H

OH

H

ilhabelanol (27)

H

ilhabrene (28)


OSO3Na

NaO3SO

H

H

isoakaterpin (29)

23.3.3 Farnesyl Protein Transferase (EC 2.5.1.58)

The Ras family of guanine nucleotide binding proteins plays important roles

in signal transduction and regulation of cell differentiation and proliferation.

The Ras protein requires posttranslational processing in order to associate with

the plasma membrane and to function in signal transduction or cellular transfor-

mation. The first processing step is catalyzed by farnesyl protein transferase

(FPT) which adds a farnesyl group to a cystein residue near the carboxy terminus

of the Ras protein. Inhibition of FPT is a potential therapeutic target for novel

anticancer agents. A cembranolide diterpene 30 isolated from the soft coral

Lobophytum cristagalli showed potent inhibitory activity against FPT at an

IC50 of 0.15 mM [15]. The bioassay guided isolation of FPT inhibitors from the

marine sponge Hyrtios reticulate yielded a knowm sesterterpene, heteronemin

(31) as the active constituent which inhibited FPT with an IC50 value of 3 mM,

while its 12-epimer (32) did not show any noticeable activity [16].

Solandelactones C (33), D (34), and G (35) were cyclopropyl oxylipins isolated

from the hydroid Solanderia secunda and exhibited moderate inhibitory activity

against FPT (69, 89, and 61% inhibition at 100 mg/mL, respectively) [17].

Spongolactams A–C (36–38) were FPT inhibitors isolated from an Okinawan

marine sponge Spongia sp. by employing newly developed LC/MS-guided assay

systems. To study their structure–activities relationships, spongolactams were

semisynthesized from the known diterpene. The inhibitory activities of 36–38against FPT were confirmed as IC50 values of 23, 130, and >260 mM, respec-

tively [18]. Four new dual inhibitors, alisiaquinones A–C (39–41) and

alisiaquinol (42), against FPT and plasmoidal kinase Pfnek-1 were isolated

from an unidentified New Caledonian deepwater sponge. Compounds 39–42showed FPT inhibition at IC50 values of 2.8, 2.7, 1.9, and 4.7 mM, respectively.


Moreover, alisiaquinone A and alisiaquinol strongly inhibited plasmoidal NIMA-

like kinase Pfnek-1 at as low as 1 mM, whereas alisiaquinone C (41) showed

almost no inhibition at this concentration. However, alisiaquinone C showed

a much better inhibitory activity against P. falciparum in vitro. These results

correlated with a better activity of alisiaquinone C on FPT, as was shown by the

dramatic effects of FPT inhibitors on malaria parasites. The in vivo activity of

alisiaquinones A and C was investigated on rodent malaria and was shown that

they reduced by 50% the parasitemia at 5 mg/kg and displayed relatively high

level of toxicity with 100% and 80% mortality, respectively at 20 mg/kg, which

precluded further antimalarial development [19].

O

OAc

O

H

H O

OAcO

OAc

R

30heteronemin (31) R = β-OH

12-epi-heteronemin (32) R = α-OH

O

H

H OH

R1R2

OH

R1

OH

H

R2

H

OH

O

H

H OH

OH

OH

solandelactone C (33)

solandelactone D (34)

solandelactone G (35)

N

N

HN

COOH

X

Y

NCOOH

COOH

O

spongolactam A (36) X = H2, Y = O

spongolactam B (37) X = O, Y = H2

spongolactam C (38)


O

O

O

OOH

H

O

HO

OH

OOH

H

O

O

O

OOH

H

R SO2

HN

alisiaquinone A (39) R = H

alisiaquinone B (40) R = OMe

alisiaquinone C (41) alisiaquinol (42)

23.3.4 Geranylgeranyltransferase Type I (EC 2.5.1.59)

Geranylgeranyltransferase type I (GGTase I) catalyzes the posttranslational attach-

ment of the geranylgeranyl unit on the carboxy terminal cysteine residues of pro-

teins, to promote membrane interaction and biological activities of these proteins

[20]. Rho1p, a regulatory subunit of 1,3-b-D-glucan synthesis and the key player in

cell wall biosynthesis [21], is known as one of the targets of GGTase I and is

essential for viability of Saccharomyces cerevisiae. Since there is only 30%

sequence homology between the human and pathogenic fungus Candida albicansGGTase I [22], its inhibitors are expected to be selective antifungal agents.

Corticatic acids are the polyacetylenic GGTase I inhibitors isolated from the

marine sponge Petrosia corticata. Corticatic acids A (43), D (44), and E (45)inhibited C. albicans GGTase I with IC50 values of 1.9, 3.3, and 7.3 mM, respec-

tively, while corticatic acid A (43) also inhibited the growth of C. albicans with an

MIC value of 54 mM [23].

Massadine (46), a highly oxygenated alkaloid isolated from the marine sponge

Stylissa aff. massa as an inhibitor of GGTase I from C. albicans inhibited GGTase

I with an IC50 value of 3.9 mM [24].

OH

OH

O

Corticatic acid A (43)

OH

O

OHOH

Corticatic acid D (44)


OH

OOH

Corticatic acid E (45)

ONH

HN

HN

NH

NH

NH

NH2

H2NOH

O

O HN

HN

Br

BrBr

Br

OH

+

+

Massadine (46)

23.3.5 HIV Reverse Transcriptase (EC 2.7.7.49)

Reverse transcriptase (RT) is the key enzyme in the life cycle of human immuno-

deficiency virus (HIV). RT is a multifunctional enzyme responsible for the

transcription of viral RNA into double-stranded DNA, and exhibits both

RNA-dependent DNA polymerase (RDDP) and DNA-dependent DNA polymerase

(DDDP) activities as well as an inherent ribonuclease H (RNase H) activity. All the

catalytic functions of RT play a pivotal role in HIV replication. Taurospongin

A (47), a metabolite of an Okinawan sponge Hippospongia sp., is another example

of acetylenic compounds which possess HIV RT inhibitory activity (IC50 6.5 mM,

Ki 1.3 mM), along with inhibitory activity against DNA polymerase b (IC50 7.0 mM,

Ki 1.7 mM) and c-erbB-2 kinase (IC5028 mg/mL), but no cytotoxicity

(IC50>10 mg/mL) against L1210 and KB cells [25]. Tryptophan-derived pigments

48 and 49 and accompanying sesterterpenes 50 and 51 isolated from the Fijian

sponge Fascaplysinopsis reticulata showed weak inhibition against HIV RT [26].

Toxicols A–C (52–54) and toxiusol (55), triterpenes isolated from the Red Sea

sponge Toxiclona toxius, were reported to be inhibitory against HIV RT, but no

detailed bioactivities were available [27]. Clathsterol (56) isolated from a Red Sea

sponge Clathria sp. showed anti-HIV-1 RT at 10 mM [28]. Polycitone A (57),a general inhibitor of retroviral reverse transcriptases and cellular DNA polymer-

ases, was isolated from an ascidian Polycitor sp. It inhibited RDDP and DDDP

activity of HIV-1 RT with IC50 values of 245 and 470 nM, respectively, while it

showed general inhibition against MuLV (murine leukemia virus) RT, MMTV

(mouse mammary tumor virus) RT, calf-thymus pol a, human pol b, and KF

(E. coli DNA polymerase I) with IC50 values ranging from 73 to 600 nM [29].


HO3S

HN

O OH OAc O

O

OOR1

OR2R1

SO3Na

H

SO3Na

R2

SO3Na

H

H

taurospongin A (47)

NH

N

O

+Cl−

48

NH

N

OH

O

+

49

O

O

O

50

OH

OH

O

O51

toxicol A (52)

toxicol B (53)

toxicol C (54)

OSO3Na

NaO3SO

HH

toxiusol (55)

N

OH

Br

BrBrHO

BrBr

HO

Br

OH

Br

Br

OH

O O

O

OOH

OAc

NaO3SO

NaO3SO

O

O

clathsterol (56) polycitone A (57)

23.3.6 HIV Integrase (EC 2.7.7.-)

HIV integrase catalizes the initial DNA-breaking and DNA-joining reactions

responsible for the attachment of HIV cDNA to host DNA. Since there are no

similar proteins known to be important for normal function of cells, HIV integrase


is a promising target for less toxic anti-HIV drugs. A series of ascidian alkaloids,

the lamellarins (58)–(63) showed selective inhibition of HIV integrase with IC50

values of 16–73 mM for terminal cleavage and 14–51 mM for strand transfer

activities, respectively; lamellarin a 20-sulfate (58) showed inhibition in the early

steps of replication of HIV-1 in cell culture with an IC50 value of 8 mM [30].

Cyclodidemniserinol trisulfate (64) isolated from the Palauan ascidian Didemnumguttatum showed inhibition against HIV integrase at an IC50 of 60 mg/mL [31].

Haplosamates A (65) and B (66) isolated from two haplosclerid sponges

(Xestospongia sp. and unidentified sponge) inhibited HIV-1 integrase with IC50

values of 50 and 15 mg/mL, respectively [32]. Prenylhydroquinone sulfates 67 and

68 from a deepwater sponge Ircinia sp. showed HIV-1 integrase inhibition (65%

inhibition at 1 mg/mL and 45% inhibition at 5 mg/mL, respectively) [33].

O

N

RO O

MeO

HO

MeOYMeO

X

R

SO3Na

H

H

X

H

OMe

H

Y

OMe

OMe

OH

lamellarin α 20-sulfate

lamellarin W

lamellarin N

(58)

(59)

(60)

O

N

RO O

MeO

HO

MeOOMeMeO

X

Y R

SO3Na

SO3Na

H

X

H

OMe

OMe

Y

H

OH

H

NH

O

OO

O

O

OO

NaO3SO O

NHSO3Na

OSO3Na

cyclodidemniserinol trisulfate (64)

lamellarin U 20-sulfate

lamellarin V 20-sulfate

lamellarin T

(61)

(62)

(63)


O

NaO3SO

OH OH

OSO3NaOPO2HNa

haplosamate B (66)

68

H

OHOH

OH

6

O

H

H

H

OH

OHH

OH

NaO3SOO

P

O

OMeNaO

haplosamate A (65)

H

OH

OSO3Na

n

67: n = 5

23.3.7 Telomerase (EC 2.7.7.49)

Telomerase is a ribonucleoprotein enzyme that adds repeats of the DNA sequence,

TTAGGG, called telomere, onto the 3”-ends of chromosomes [34]. Telomerase

activity is found in about 90% of human tumors, but not in normal cells [35]. Thus,

inhibitors of telomerase are potential antitumor agents [36]. In fact, some synthetic

inhibitors based on the function of telomerase have been successful in clinical trials

[37]. From the marine sponge Dictyodendrilla verongiformis collected in southern

Japan, five telomerase inhibitors, dictyodendrins A–E (69–73) were isolated along

with the sodium salts of two known compounds (2 and 3) which were obtained as

aldose reductase inhibitors from a marine sponge of the same genus [3]. All these

seven compounds showed 100% inhibition of telomerase activity at the concentra-

tion of 50 mg/mL, while the desulfated analogue of dictyodendrin C (71) was notactive at this concentration [38]. Recently, the total synthesis of dictyodendrin

B (70), C (71), and E (73) was accomplished [39]. An unprecedented highly

sulfated lipopolysaccharide, axinelloside A (74), isolated from the marine sponge

Axinella infundibula showed a potent inhibition against human telomerase

(IC50 2.0 mg/mL) [40].


N

NH

OH

OH

OHMeO

O

HO

HO OSO3Na OSO3Na

OSO3Na

OSO3Na

OSO3Na

N

NH

OH

OH

OHO

HO

HO

N

NH

OH

OH

OO

HO

N

NH

OH

OO

HO

Dictyodendrin A (69) Dictyodendrin B (70)

Dictyodendrin C (71) Dictyodendrin D (72)

N

NH

OH

OH

O

HO

HO OSO3Na

Dictyodendrin E (73)


O

O

O

O

O

OO

OHXO

XO

MeXO

O

XO

OX

O

O

OH OH

O

OXO

Me

O

OXO

Me

OH

OO

XOO O

OH

XOO

O

MeO

XOO

XOOH

XOO

OMe

OH

O

XO

O

OH

OX

OXO

HOOH

OXO

O

OX

XO

OXHO

O

Axinelloside A (74)

X = SO3Na

23.3.8 Epidermal Growth Factor Receptor Kinase (EC 2.7.10.1)

Protein tyrosine kinases comprise a large family of enzymes that regulate cell

growth and intracellular signaling pathways; inhibitors of these enzymes may be

potential anticancer drugs [41]. There are at least four members of the type 1 growth

factor receptor gene family, including the epidermal growth factor receptors

(EGFR), erbB-1, erbB-2, erbB-3, and erbB-4. Members of this tyrosine kinase

receptor family have been implicated in the establishment or progression of

human cancer, particularly breast cancer, and enormous amounts of effort have

been made to identify specific small molecule inhibitors of EGFR tyrosine kinase

[42]. Tauroacidins A (75) and B (76), bromopyrrole metabolites of an Okinawan

spongeHymeniacidon sp., exhibited inhibitory activity against c-erbB-2 kinase withan IC50 of 20 mg/mL [43]. (+)-Aeroplysinin-1 (77) isolated from the marine sponge

Verongia aerophoba inhibited EGF receptor kinase completely at 0.5 mM [44].

Ma’edamine A (78), a cytotoxic bromotyrosine alkaloid isolated from a marine

sponge Suberea sp., inhibits c-erbB-2 kinase with an IC50 value of 6.7 mg/mL [45].

HN

OH

NH

NHHN

Br

R

O

NH

HNSO3

−

+

RBrH

OMe

Br Br

HOHO CN

tauroacidin A (75)tauroacidin B (76)

(+)-aeroplysinin-1 (77)

N

HNO

Br

MeO

O NMe

Br

Br Mema'edamine A (78)


23.3.9 Tyrosine Kinase pp60V-SRC (EC 2.7.10.2)

A tyrosine kinase, pp60V-SRC is the oncogenic protein encoded by Rous sarcoma virus.

Halenaquinone (79), halenaquinol (80), halenaquinol sulfate (81), and xestoquinone

(82) isolated from the Fijian sponge Xestospongia cf. carbonaria was found

to inhibit the kinase activity of pp60V-SRC with IC50 values of 1.5, 60, 0.55, and

28 mM, respectively [46]. Halenaquinone (79) also inhibited phosphatidylinositol

3-kinase with an IC50 value of 3 mM [47]. Two more pp60V-SRC inhibitors,

14-methoxyhalenaquinone (83) and xestoquinolide A (84) were isolated from the

same sponge (IC50 values of 5 and 80 mM, respectively) [48]. Melemeleone B (85),a sesquiterpene quinone isolated from twoDysidea sponges, showed inhibitory activityagainst pp60V-SRC with an IC50 value of 28 mM [49].

O

O O

O

O

NaO3SO

OH

O

O O

halenaquinol sulfate (81)halenaquinone (79)

HO

OH

O

O O

halenaquinol (80)

HO

OHN

SO3H

O

O O

O

O

OMe

O

O

O

O

melemeleone B (85)

14-methoxyhalenaquinone (83)

xestoquinolide B (84)

O

O O

O

O

xestoquinone (82)


23.3.10 Other Tyrosine Protein Kinase

Four prenylhydroquinone sulfates 67–68 and 90–91 obtained from a deepwater

sponge Ircinia sp. showed inhibitory activity of tyrosine phosphorylation by mem-

brane fraction prepared from HER2 overexpressing 3T3 cells with IC50 values of 8,

4, 8, and 5.9 mg/mL, respectively. Compounds 67 and 68 were also reported to be

HIV-1 integrase inhibitors [33].

23.3.11 MSK1 (EC 2.7.11.1) and MAPKAPK-2

Mitogen- and stress-activated kinase 1 (MSK1) and mitogen-activated protein

kinase–activated protein kinase 2 (MAPKAPK2) are two serine protein kinases

involved in signal transduction. Both of these enzymes are located in the nucleus

and are involved in the late stage of signal transduction pathway, therefore,

selective inhibitors of these enzymes are most likely to exhibit highly specific

cellular effects. Four cheilanthane sesterterpenoids 86–89 were isolated from

a marine sponge Ircinia sp. as inhibitors of both MSK1 (IC50s 4 mM for all

compounds) and MAPKAPK-2 (IC50 90 mM for all compounds) [50].

23.3.12 Raf/MEK1/MAPK

The Ras-MAPK-signaling cascade is found in all eukaryotic organisms and is

involved in cellular signaling processes. Since the oncogenic form of Ras is

associated with 30% of all cancers, Ras and the downstream kinases represent

attractive targets for pharmacological intervention. The Raf/MEK-1/MAPK cas-

cade inhibition assay-guided fractionation of the Philippine marine sponge Stylissamassa yielded eight known pyrrole alkaloids, aldisine (92), 2-bromoaldisine (93),10Z-debromohymenialdisine (94), a 1:1 mixture of 10E- and 10Z-hymenialdisines

(95–96), hymenin (97), oroidin (98), and 4,5-dibromopyrrole-2-carbonamide (99),among which 93–97 were active with IC50s ranging from 3 to 1288 nM; the most

active were 95 and 96 (IC50s 3 and 6 nM, respectively). In addition, all compounds

showed essentially identical IC50 values in the MEK-1 to MAPK assay, while none

of them showed activity in the Raf to MEK-1 assay [51].

23.3.13 Checkpoint Kinases (EC 2.7.11.1)

The synthetic10Z-debromohymenialdisine (94) inhibited checkpoint kinases

Chk1 and 2 with IC50 values of 3 and 3.5 mM, respectively [52]. Further investiga-

tion into its inhibitory activity toward kinases led to identification of 11 new targets

including p90RSK, KDR, c-Kit, Fes, MAPK1, PAK2, PDK1, PKCy, PKD2, Rsk1,and SGK for this class of alkaloids [53].


OHH

O

HO

HH

O

OH

O

H

86 87

HH

OO

OH

88

OHH

O

HO

OH

89

NHNH

O

O

R

R = HR = Br

aldisine (92)2-bromoaldisine (93)

NHNH

O

Br

NHN

O

NH2

10E-hymenialdisine (95)

NHNH

O

R

N

HN

H2N

hymenin (97)

H

OH

OSO3Na

n

90: n = 691: n = 7

NHNH

O

R

N

HN O

H2N

R = HR = Br

10Z-debromohymenialdisine (94)10Z-hymenialdisine (96)

HN

O

N

HN

NH

Br

Br

NH2

oroidin (98)

NH2

O

NH

Br

Br

99


23.3.14 Protein Kinase C (EC 2.7.11.13)

Protein kinase C (PKC), a phospholipid-dependent protein phosphorylating

enzyme, is a key player of cellular signal transduction and is believed to be

responsible for cancer, cardiovascular and renal disorders, and immunosuppres-

sion and autoimmune diseases, such as rheumatoid arthritis. Therefore, inhibi-

tors of protein kinase C (PKC) can be potential drug leads for the treatment of

such diseases. Staurosporine (100), isolated from Streptomyces staurosporeus, isthe most famous natural product PKC inhibitor [54]. 11-Hydroxystaurosporine

(101) isolated from a tunicate Eudistoma sp. collected in Pohnpei inhibited PKC

with an IC50 value of 2.2 nM which is about 30% more active than

staurosporine (100) [55]. Staurosporine aglycone K252-c (102) isolated from

another Eudistoma sample inhibited eight cloned PKC isoenzymes a, b I, b II,

d, e, Z, g, and z with IC50 values of 1.3, 0.6, 0.5, 1.2, 1.1, 0.8, 1.5, and

>6.4 mM, respectively [56]. Xestocyclamine A (103), bis-alkylpiperadinesfrom a marine sponge Xestospongia sp., moderately inhibited PKCe(IC50 4 mg/mL) [57]. Isoaaptamine (104), which was first reported from

a suberitid sponge in 1988, was found to be a PKC inhibitor; recent evaluation

of PKC inhibitory and antitumor activities for synthetic isoaaptamine and its

derivatives disclosed that none of them met the criteria for further evaluation

after the primary screen in the NCI tumor cell line panel [58]. Z-axinohydantoin(105) and debromo-Z-axinohydantoin (106) isolated from the marine sponge

Stylotella aurantium, moderately inhibited PKC (IC50s 9.0 and 22 mM, respec-

tively) [59].

HN

N N

O

RO

HN

OMe

MeRH

OH

HN

NH

NH

O

R

hydroxy staurosporine (100)

11-hydroxy staurosporine (101)

N

N

OH

xestocyclamine A (103)

K252-c (102)


N

OH

MeO N

NHNH

HN

HN

X

O

O O

XBr

H

isoaaptamine (104)

Z-axinohydantoin (105)

debromo-Z-axinohydantoin (106)

The mixture of corallidictyals A (107) and B (108), spirosesquiterpene alde-

hydes isolated from the marine sponge Aka (Siphonodictyon) coralliphagum,inhibited PKC with an IC50 value of 28 mM. Interestingly, the corallidictyal mixture

selectively inhibited PKCa with an IC50 value of 30 mM (IC50s 89, >300, and

>300 mM for e, Z, and z, respectively). In addition, the corallidictyal mixture

inhibited the growth of cultured Vero (African green monkey kidney) cells with an

IC50 value of 1 mM after continuous exposure (72 h) [60]. Nakijiquinones A–D

(109–112), sesquiterpene quinones isolated from an Okinawan marine sponge of

the family Spongiidae, exhibited inhibitory activity against PKC with IC50 values

of 270, 200, 23, and 220 mM, respectively, whereas they were also active against

EGF receptor kinases and c-erbB-2 kinase with IC50 values of >400, 250, 170, and

>400 mM, and 30, 95, 26, and 29 mM, respectively [61]. Recently, enantioselective

total synthesis of nakijiquinones and closely related analogues was reported [62].

The evaluation of their biological activities disclosed that C-2 epimer of

nakijiquinone (113) was a potent and selective inhibitor of tyrosine kinase

VEGFR2 (KDR) with an IC50 value of 21 mM. Incidentally, VEGFR2 is a receptor

for the vascular endothelial growth factor (VEGF) family and is responsible for

endothelial cell proliferation and blood vessel permeability, therefore inhibitors

of VEGFR2 are potential anti-angiogenic drugs [62]. Frondosines A–E (114–118),sesquiterpenoids from the sponge Dysidea frondosa, inhibited PKC with IC50

values of 1.8, 4.8, 20.9, 26, and 30.6 mM, respectively [63].

O

OHO

CHO

H

nakijiquinone A (109)nakijiquinone B (110)nakijiquinone C (111)nakijiquinone D (112)

2-epi-nakijiquinone (113)

corallidictyal B (108)

O

OHO

CHO

H

corallidictyal A (107)RGlyL-ValL-SerL-ThrL-Ser

H

O

O

HO

R

2


O

HO

frondosin B (115)

O

OH

frondosin C (116)

O

ORO

frondosin D (117)

frondosin E (118)

RH

Me

OH

HO

frondosin A (114)

Spongianolides A–E (119–123), cytotoxic sesterterpenes isolated from

a marine sponge Spongia sp., inhibited PKC at IC50 values of 20–30 mM [64].

Three secosterols 124–126 isolated from a gorgonian Pseudopterogorgia sp.

inhibited the human PKC a, bI, bII, g, d, e, Z, and z with IC50 values in the

range of 12–50 mM. The semisynthetic derivatives 127–129 also showed similar

activity [42].

O

O

O

O

OH

OH

H

H

OH

O

O

O

O

OH

OH

H

H

spongianolide A (119) spongianolide B (120)

H

H

OR

O

OH

OH

RCOCH3

COCH3

COCH(OH)CH3

spongianolide A (121) 16R

spongianolide B (122) 16S

spongianolide C (123) 16R

16


HOOC

H

O

HO

O128

HOOC

H

HOH

HO

OH129

HO

H

HO

HO

OH

126

HO

H

HO

HO127

HO

H

HO

HOH 125124

HO

H

HO

HOH

OH

Penazetidine A (130), an azetidine isolated from the Indo-Pacific sponge

Penares sollasi, inhibited PKC with an IC50 value of 1 mM [65]. BRS1 (131),a C30 bis-amino, bis-hydroxy polyunsaturated lipid from an unidentified Australian

sponge of the class Calcarea, inhibited not only PKC (EC50 98 mM) but also

radiolabeled phorbol ester binding to the enzyme (EC50 9.2 mM) [66]. Shimofuridin

A (132) isolated from an Okinawan marine tunicate Aplidium multiplicatuminhibited PKC at an IC50 of 20.0 mg/mL [67]. Bryostatin-1 (133) which was isolatedfrom the bryozoa Bugula neritina as an anticancer agent [68], is known to bind the

C1 domain of PKC and regulate its activity [69].


NH

HO

HO

NH2

OH

OH

NH2

N

NHN

OH

N

OHO

OHO

O

OH

O

O

O

O

O

BRS1 (131)

shimofuridin A (132)

penazetidine A (130)

O O

O

MeO

O O

O

O

O

OH

H

OMe

O

O OH

HO

O

bryostatin-1 (133)

23.3.15 Cyclin-Dependent Kinases (EC 2.7.11.22)

Cyclin-dependent kinases (CDK) are also a family of protein tyrosine kinases, some of

which are involved in cell cycles, and their inhibitors are expected to be potential

anticancer drugs. Konbu’acidin A (134) is a bromopyrrole alkaloid possessing inhib-

itory activity against CDK4 (IC50 20 mg/mL) isolated from a marine sponge

Hymeniacidon sp. [70]. Spongiacidins A (135) and B (136) also isolated from the

same sponge inhibited c-erbB-2 kinase (IC50s 8.5 and 6.0 mg/mL, respectively) and

cyclin-dependent kinase 4 (CDK4) (IC50s 32 and 12 mg/mL, respectively) [71].

Rhopaladins A–D isolated from an Okinawan tunicate Rhopalaea sp. are the first

example of bis-indole alkaloids possessing an imidazolinone moiety as tunicate

metabolites; rhopaladin B (137) inhibited CDK4 and c-erbB-2 kinasewith IC50 values

of 12.5 and 7.4 mg/mL, respectively [72]. Microxine (138) from an Australian marine

spongeMicroxina sp. inhibited CDK1 (cdc2) kinasewith an IC50 value of 13 mM[73].

NHN

HNHN

NH

H2N

Cl

NH

O HN

H2N

Br

OHO

Br

Br

H

H

+

+

NHNH

NHHN

NH2

O

O

Br

R

+

konbu'acidin (134) spongiacidin A (135) R = Br

spongiacidin B (136) R = H


NH

N

NH

NH

O

HO

O

N

N

NH

MeN

HN

SO3H

O

Rhopaladin B (137) microxine (138)

23.3.16 Glycogen Synthase Kinase-3 (2.7.11.17)

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase ubiquitously

expressed and involved in the regulation of many cell functions. GSK-3 phos-

phorylates glycogen synthase or the microtuble-associated protein tau, being

implicated in type-2 diabetes, Alzheimer’s disease, or different tau pathologies

such as Pick’s disease. Hymenialdisine (96) [74], indirubine (139) [75], and

meridianine (140) [76] are the known marine-derived GSK-3 inhibitors isolated

from several species of marine sponges, the mollusk Hexaplex trunculus, and the

ascidian Aplidium meridianum, respectively. Hymenialdisine (96) potently

inhibited GSK-3b (IC50 10 nM) and plant GSK-3 (ASK�g) with an IC50 value

of 80 nM as well. It inhibited in vivo phosphorylation of specific neuronal

proteins by GSK-3b and CDK5 such as AD-characteristic phosphorylation of

tau completely [74]. Indirubin inhibited GSK-3a/b with an IC50 value of 1.00 mM[75]. Studies of structure–activity relationship (SAR) in the class of manzamine

alkaloids revealed that manzamine A (141) inhibited GSK-3b at an IC50 value of

10.2 mM; 8-hydroxymanzamine A (142) being the most potent analogue

(IC50 4.8 mM) [77].

NNH

NH

I

O

NHO

139

NH

NN

H2N

OH

Br140

N

NNH

OH

R

141: R = H142: R = OH


23.4 Hydrolases (EC 3.-.-.-)

23.4.1 Phospholipase A2 (EC 3.1.1.4)

Phospholipase A2 (PLA2) cleaves the ester linkage of the b-position of phospho-

lipids to release arachidonic acid which is metabolized to prostaglandins, leukotri-

enes, and other mediators of inflammation. Prostaglandins are implicated in various

forms of pain and inflammation. In biogenesis of prostaglandins, arachidonic acid

formation is the rate-determining step, thus, PLA2 inhibitors are expected to be

potential anti-inflammatory drugs. Manoalide (143), initially isolated as an

antibacterial metabolite from the marine sponge Luffariella variabilis [78], was

later found to be a potent inhibitor of PLA2 with an IC50 value of 1.7 mM [79], which

led to synthesis and anti-inflammatory evaluation of more than 100 analogues.

Unfortunately, none of them was developed for drugs. Three congeners of

manoalide were also obtained from the same sponge [80], of which secomanoalide

(144) is more potent against bovine pancreatic PLA2 [81]. Luffariellolide (145)isolated from a Palauan sponge Luffariella sp. also inhibited the bee venom PLA2

with an IC50 of 0.23 mM. The partially reversible and weaker inhibitory activity of

145was suggesting that the g-lactol group in 143was responsible for the irreversiblereaction of 143 with Lys residues on PLA2 [82]. Luffariellins A (146) and B (147)isolated from L. variabilis are very potent against bee venom PLA2 with IC50 values

of 56 and 62 nM, respectively [83]. These sesterterpenes are known to covalently

and specifically modify PLA2 by a Schiff base formation between Lys-56 residue of

PLA2 and the hemiacetal or aldehyde functionality of these terpenoids [84].

According to this mechanism, Katsumura and coworkers designed PLA2 inhibitors,

one of which potently and selectively inhibited bovine pancreas PLA2 [85].

O

OH

O

OH

HO

O

OH

O

OH

HO

O

OH

O

OHO

O

OH

OOHO

O

OH

Oluffariellolide (145)

manoalide (143)

secomanoalide (144)

luffariellin A (146)

luffariellin B (147)


Cacospongionolides (148–150), originally isolated as antitumor compounds

from the Mediterranean sponge Fasciospongia cavernosa [86], preferentially

inhibited bee venom and human synovial PLA2 among PLA2s of various origins.

Cacospongiolide B (149) exhibited specific inhibition against human PLA2 [87],

while cacospongionolide E (150) was the most potent inhibitor toward human

synovial PLA2 (IC50 1.4 mM), showing higher potency than manoalide [88]. The

structure–activity relationship study on synthetic analogues suggested that 149 has

an enantiospecific interaction with the enzyme that is independent of the

g-hydroxybutenolide moiety [89]. Furthermore, cacospongionolide B (149) was

reported to suppresses the expression of inflammatory enzymes including

inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as

to reduce tumor necrosis factor-a (TNF-a) [90]. Cavernolide (151) isolated from

a marine sponge Fasciospongia cavernosa selectively inhibited human PLA2 with

an IC50 value of 8.8 mM [91].

O

OH

HO

OH

HO

O

cacospongionolide B (149)cacospongionolide (148)

OO

O

OH

H OH

O

cacospongionolide E (150) cavernolide (151)

Dysidiotronic acid (152), a sesquiterpenoid from a Vanuatu sponge Dysidea sp.,inhibited human synovial PLA2 with an IC50 value of 2.6 mM [92], while

bolinaquinone (153), dysidine (154), and a 1:1 mixture of dysideonones A (155)and B (156) significantly inhibited human synovial PLA2, among which 153showed the most potent activity with an IC50 value of 0.2 mM, but was not

a selective inhibitor toward this enzyme. On the contrary, dysidine (150) showedselective inhibition against this enzyme with an IC50 value of 2.0 mM. However,

153–156 did not inhibit cPLA2 [93].

H

O

O

O

OHMeO

H

O

O

OH

OH

dysidiotronic acid (152) bolinaquinone (153)


H

H

HO

O

OMe

OOMe

dysidenone A (155)

H

H

HO

O

OMe

OOMe

dysidenone B (156)

H

O

O

OH

OH

NH

SO3Hdysidine (154)

Petrosaspongiolides M–R (157–161), sesterterpens from the New Caledonian

marine sponge Petrosaspongia nigra inhibited PLA2; the most potent petrosas-

pongiolide M (157) inhibited human synovial and bee venom PLA2 with IC50

values of 1.6 and 0.6 mM, respectively, while petrosaspongiolide P (159)inhibited human synovial PLA2 (IC50 3.8 mM) more selectively than bee venom

PLA2 (IC50 >10 mM). Other petrosaspongiolides N (158), Q (160), and R (161)showed only weak activity [94]. Petrosaspongiolide M was shown to form the

covalent adduct between PLA2 and g-hydroxybutenolide which is essential for the

activity [95]. Ironically, a mild non-covalent PLA2 inhibitor, 25-acetyl-

petrosaspongiolide M, was found to be hydrolyzed with PLA2 to form the potent

inhibitor, petrosaspongiolide M [96]. Interestingly, a full characterization of the bvPLA2 adduct with petrosaspongiolide R (161), one of the least active and most

structurally different among petrosaspongiolides, using LC-MS, MSn, and compu-

tational methods, confirmed the same inhibition mechanism and covalent binding

site already found for petrosaspongiolide M (167). The correct arrangement of the

ligand in the active site, induced by non-covalent interactions, is of pivotal

importance in the inactivation process [97]. The anti-inflammatory effects of

petrosaspongiolide M was suggested to be mediated by inhibition of NF-kBsignaling pathway [98].

Aplyolide (162), a sesterterpene isolated from the marine sponge

Aplysinopsis elegans inhibited human PLA2 with an IC50 value of 10.5 mM[99], whereas the related terpenoid, luffolide (163) isolated from a Palauan

Luffariella sp., completely inhibited bee venom PLA2 at a concentration of

3.5 mM [100].


R

O

HO

O

O

OAc

HH

H

H

O

OH

HH

H

H

R

O

HO

O

RHOAc

RHOAc

petrosaspongiolide M (157)N (158)

petrosaspongiolide P (159)Q (160)

O

H

H

O

HO

O

OH

H

H

O

HO

O

OH

petrosaspongiolide R (161) aplyolide A (162)

CHO

H

H

O

HO

O

OAc

luffolide (163)

A homoscalarane sesterterpene (164) isolated from a marine sponge

Lendenfeldia frondosa showed moderate PLA2 inhibition (35% inhibition at

8 mM) [101]. A sesterterpene, 12-deacetyl-23-acetoxy-20-methyl-12-epi-

scalaradial (165), from the Pacific nudibranchs Glossodoris sedan and G. dalliinhibited mammalian cytosolic PLA2 with an IC50 value of 18.0 mM [102]. The

parent compound, scalaradial (166) is more potent (IC50 0.6 mM) [84a, 103].

HH

H

AcO

OAc

OOO

AcO

OH CHO O

164 165


OAc

CHO

CHO

scalaradial (166)

Halisulfate 1 (167) from a halichondrid sponge inhibited PLA2 completely at

16 mg/mL, while a mixture of halisulfates 2–4 (168–170) inhibited PMA-induced

inflammation in the mouse ear edema assay as well as PLA2(data not shown) [104].

Palinurin (171), originally isolated from the Mediterranean marine sponge Irciniavariabilis [105], was inhibitory against PLA2 with an IC50 value of 50 mM [2]. Two

sesterterpenes, palauolol (172) and palauolide (173) isolated from a Palauan sponge

Fascaplysinopsis sp. inhibit bee venom PLA2 (85% and 82% inhibition at

0.8 mg/mL, respectively) [106].

OSO3Na

OH

HO

OSO3NaO

halisulfate-1 (167) halisulfate-2 (168)

NaO3SOO

halisulfate-3 (169)

NaO3SOO

halisulfate-4 (170)

OO

HO

palauolide (173)

O

OHO

O

palinurin (171)

OH

O

HO

O

palaulol (172)


Two polyhydroxy sterols 174 and 175 from the Korean gorgonian Acabariaundulata inhibited PLA2 with IC50 values of 13.8 and 21.5 mM, respectively [107].

Valdivones A (176) and B (177), diterpenoids isolated from the South African soft

coral Alcyonium valdivae, strongly inhibited chemically induced inflammation in the

mouse ear assay (93% and 72% inhibition at 50 mg/ear, respectively), but their activityagainst bee venom PLA2 was not potent (43% inhibition at 16 mg/mL for both) [108].

O

OH

O

O

O

O

OH

O

O

O

valvidone A (176) valvidone B (177)

HOOH

OH

O

HOOH

OH

O

174 175

23.4.2 Acetylcholinesterase (EC 3.1.1.7)

3-Alkylpyridinium polymers 178 from a marine sponge Reniera sarai showedpotent inhibitory activity against electric eel acetylcholinesterase (AchE), human

erythrocyte AchE, insect recombinant AchE, and horse serum butylcholinesterase

(BuChE) with IC50 values of 0.06, 0.08, 0.57, and 0.14 mg/mL, respectively, while it

did not inhibit trypsin or alkaline phosphatase [109].

N+

n

MW 5520 & 18900

178


23.4.3 Protein Phosphatases (EC 3.1.3.16)

Reversible protein serine/threonine phosphorylation regulates numerous cellular

functions, including muscle contraction, neurotransmission, cell proliferation, car-

cinogenesis, and apoptosis. Although a large number of protein kinases have been

well investigated for several decades, it is quite recent that our understanding of

protein phosphatases (PPs), namely PP1, PP2A, PP2B, and PP2C, has been con-

siderably deepened by the discovery of natural product inhibitors.

23.4.3.1 PP1 and PP2Okadaic acid (179), the first natural product inhibitor of PP1 and PP2A, is

a polyether polyketide isolated as a cytotoxic substance from two sponges, Japanese

Halichondria okadai and Caribbean H. melanodocia [110]. It was succeedingly

identified as a causative agent of the diarrhetic shellfish poisoning. Later, 179 was

found to show a potent tumor promoting activity caused by potent inhibition of PP1

and PP2A with IC50 values of 3 and 0.2–1 nM, respectively, whereas it showed only

weak or no inhibition against PP2B or PP2C [111]. It has become a powerful tool for

the study of biological processes mediated by protein phosphorylation. The crystal

structure of PP1g-okadaic acid complex was elucidated in 1995 [112]. A remarkable

reduction of inhibitory activity against PP1 (IC50 465 nM) was observed for 19-epi-okadaic acid isolated from dinoflagellate Prorocentrum belizeanum, whereas the

inhibitory activity against PP2A remained as the same level (IC50 0.47 nM) for 179.It was suggested that the stereochemistry at C19 is crucial for the selectivity between

PP1 and PP2A [113]. A new okadaic acid class compound, belizeanic acid (180)with an unusual skeleton was isolated from the dinoflagellate Prorocentrumbelizeanum. Despite its opening of C/D rings, it still retains the potent inhibitory

activity against PP1 (IC50 318 nM), slightly reduced potency compared to okadaic

acid (IC50 62 nM in the same assay system) [114].

HOO

O

OHO

OH

O

O

O O

O

HOH

H HOH

H

okadaic acid (179)

HOO

O

OHOH

O O

OH

OH

HO

OH

OH

O

O

(180)

19


Calyculin A (181) was isolated from the Japanese marine sponge Discodermiacalyx as a potent antitumor agent [115]. Like okadaic acid, it was found to be

a potent tumor promoter and to inhibit PP1 and PP2A with IC50 values of 1.4 and

2.6 nM, respectively, while it showed weak inhibition against PP2B [116].

Calyculins B–H (182–188), geometrical isomers of 181, or their 32-methyl deriv-

atives which were succeedingly isolated from the same sponge, were originally

reported to have similar inhibitory activity against PP2A (IC501.0–6.0 nM) [117],

but this was later found to be not correct; the geometry of the tetraene portion

affects the enzyme inhibition significantly [119]. Furthermore, calyculin J (189),calyculinamides A (190) and F (191), des-N-methylcalyculin A (192) and

dephosphonocalyculin A (193) were isolated as PP2A inhibitors from D. calyx[118]. Later, isolation of hemicalyculin A (194) from D. calyx and structure-

activity relationship studies were reported [119]. Hemicalyculin A (194) was

equipotent as calyculin A (181) against PP1g and PP2A with IC50 values of 14

and 1.0 nM, respectively. Interestingly, 194 showed cytotoxicity over 2000 times

less than that of 181. More recently, the crystal structure of the calyculin A-PP1gcomplex was solved by X-ray crystallography [120], which showed that the

hydrophobic tail, phosphate, and 13-OH moieties are essential for binding to

PP1g. These features are the cases of the PP1c/microcystin LR [121] and PP1c/

okadaic acid complexes [112]. Clavosines A–C (195–197), glycosylated deriva-

tives of C21-epi-calyculinamide C, were isolated from the marine sponge

Myriastra clavosa. Clavosines A (195) and B (197) inhibited PP1cg (IC50s 0.5

and 13 nM, respectively), native PP1c (IC50s 0.25 and 1.0 nM, respectively), and

PP2Ac (IC50s 0.6 and 1.2 nM, respectively) [122].

R1

CNHCNHCONH

R2

HCNHCNH

R3

HHMeMeH

calyculin A (181)calyculin B (182)calyculin C (183)calyculin D (184)

calyculinamide A (190)

MeO NH

Me2N

OH

OH

O

N

O

O

OHO

O

OMeOHOH

R1

R3

R2

PHO

O

HO


HR2

OH

OH

O

N

O

O

OHO

R1

OMeOHOH

CN

R1

OPO3H2

OH

R2

NHMe

NMe2

MeO N

des-N-methylcalyculin A (192)

dephosphonocalyculin A (193)

HMe2N

OH

OH

O

N

O

O

OHO

O

OMeOHOH

R3

R2

R1 PHO

O

HO

MeO N

R1

CN

H

CN

H

H

R2

H

CN

H

CN

CONH

R3

H

H

Me

Me

H

calyculin E (185)

calyculin F (186)

calyculin G (187)

calyculin H (188)

calyculinamide F (191)

MeO NH

Me2N

OH

OH

O

N

O

O

OHO

O

OMeOH

O

CNBr

PHO

O

HO

calyculin J (189)


MeO NH

Me2N

OH

OH

O

N

O

O

OO

O

OMeOHOH

R1

R2

PHO

O

HO

OMe

OMeOMe

MeOR1

CONH2

H6Z isomer of 196

R2

HCONH2

clavosine A (195)clavosine B (196)clavosine C (197)

6

NC

O

OHO

O

OMeOHOH

CN

PHO

O

HO

hemicalyculin A (194)

Motuporin A (198), one of the most potent PP1 inhibitors known, isolated from

the Papua New Guinea sponge Theonella swinhoei inhibited PP1 at <1 nM [123].

Dragmacidins D (199) and E (200) isolated from a southern Australian deepwater

sponge Spongosorites sp. are potent serine-threonine protein phosphatase inhibi-

tors; 199 selectively inhibited PP1, while 200 inhibited both PP1 and PP2A in the

preliminary test (data not shown). Dragmacidin E (200) seemed to exist in tauto-

meric equilibrium between pyrazine and pyrazinone forms [124]. Spirastrellolide

A (201) was a novel macrolide isolated from the marine sponge Spirastrellacoccinea as an antimitotic agent [125]. Recently, its revised structure and selective

inhibitory activity against PP2A (IC50 1 nM) were reported [126]. Nagelamides are

dimeric bromopyrrole alkaloids isolated from a marine sponge Agelas sp., among

which nagelamides A–C (202–204), G (205), and H (206) were shown to inhibit

PP2A (IC50 s 13 to >50 mM) [127].


NO

O

O

NH

O NH

OHO

OH

OHN

NHOMe O

N

HN

HN

NH

O

Br

OH

NH

HN

HN

motuporin A (198)

+

TFA−

dragmacidin D (199)

HO

O

OHO

OO

OO

HO

HO

OMeOH

HO

Cl

OMe

O

O

O

spirastrellolide A (201)

N

HN

HN

HN

NHNH

O

Br

H2N

OH

+

N

N

HN

HN

NHNH

HO

Br

H2N

OH

dragmacidin E (200)

+

HN

NH

HNNH

NH

NH

NH2

O

O

HN

NH

Br

Br

Br

Br

NH2

nagelamide C (204)

HN

NH

HNNH

NH

NH

NH2

O

O

HN

NH

Br

Br

Br

Br

NH2

R

+

+

nagelamide A (202)

B (203)

R = H

R = OH


NH

HN

NH2

NH

HN

H2N

HN

O

HN

O

HN

NH

Br

Br

Br

Br

+

+

nagelamide G (205)

NH

HNNH

NH2

OHN

Br

Br

NH

HN

HN

NH

Br

Br

O

NH

HN

SO3−

+

+

nagelamide H (206)

23.4.3.2 Calcineurin (PP2B)Calcineurin (CaN), a serine-threonine protein phosphatase (PP2B) involved in signal

transduction, is recognized as one of the principal signaling molecules that regulates

the immune response [128]. Immunosuppressants such as FK506 and cyclosporin

A have been shown to exert their effect through inhibition of CaN following their

association with binding proteins [129]. Thus, inhibitors of CaN may be useful as

immunosuppressants. Secobatzellines A (207) and B (208) isolated from a Caribbean

deepwater spongeBatzella sp. inhibitedCaNwith IC50 values of 0.55 and 2.21mg/mL,

respectively. Secobatzelline A (207) also inhibited peptidase activity of CPP32

(IC50 0.02 mg/mL), a cysteine protease known as caspase-3 which play a major role

in the programmed cell death known as apoptosis [130].

HN

O

R

Cl

H2N

OH

OH

secobatzelline A (207) R = NHsecobatzelline B (208) R = O

23.4.4 Protein Tyrosine Phosphatases (EC 3.1.3.48)

Cdc25 is a dual-specificity protein tyrosine phosphatase which regulates activa-

tion of CDKs (cyclin-dependent kinases) through the dephosphorylations of

a threonine and a tyrosine residues of CDKs. Due to their unique substrate

selectivity and their essential functions in cell cycle control, cdc25 phosphatases

are attractive screening target to identify new antimitotic compounds.

Dysidiolide (209) is the first natural product inhibitor of cdc25A isolated from

the Caribbean sponge Dysidea etheria. It inhibited the dephosphorylation of

p-nitrophenol phosphate by cdc25A phosphatase with an IC50 of 9.4 mM [131].


Because of its unusual structure and interesting biological activity, a number of

synthetic chemists entered the race of total synthesis of dysidiolide, from

which some important structure-activity relationships were provided [132].

Coscinosulfate (210), a sesquiterpene sulfate isolated from the New Caledonian

sponge Coscinoderma mathewsi exhibited selective inhibition against cdc25A

phosphatase with an IC50 of 3 mM [133].

O

O

OH

H

OSO3Na

HO

OH

dysidiolide (209) coscinosulfate (210)

23.4.5 Phospholipase C (EC 3.1.4.3)

Phosphatidylinositol-specific phospholipase C (PI-PLC) is the rate-limiting enzyme

in the phosphatidylinositol turnover. PI-PLC hydrolyzes the phosphate-glycerol

linkage of phosphatidylinositol to produce two second messengers, inositol triphos-

phate and diacylglycerol which play key roles in cellular signal transduction

induced by growth factors and hormones [134].

Akaterpin (211) isolated from an Okinawan marine sponge Callyspongia sp.

showed potent inhibition against PI-PLC (IC50 0.5 mg/mL) as well as weak inhibi-

tion against neutral sphingomyelinase with an IC50 value of 30 mg/mL [135].

NaO3SO

OSO3NaH

H

akaterpin (211)


23.4.6 Phosphodiesterase (EC 3.1.4.-)

The Ca2+-calmodulin system plays an important role in the control of cellular

proliferation and tumor formation. A calmodulin antagonist, W-7, has been found

to inhibit proliferation of Chinese hamster cells and formation of mouse skin tumors.

Eudistomin A (212), a b-carboline alkaloid isolated from the Okinawan tunicate

Eudistoma glaucus, inhibited calmodulin-activated brain phosphodiesterase with an

IC50 value of 20 mM [136], while eudistomin C (213) exhibited calmodulin antag-

onistic activity (IC50 30 mM) [137]. Pseudodistomins A (214) and B (215), unsualalkylpiperazines of the Okinawan tunicate Pseudodistoma kanoko, inhibited cal-

modulin-activated brain phosphodiesterase at the same concentration (IC50 30 mM),

which is about three times more potent than W-7 [138]. Rigidin (216) isolated froman Okinawan tunicate Eudistoma cf. rigida inhibited calmodulin-activated brain

phosphodiesterase with an IC50 value of 50 mM [139]. Stellettamide A (217),an unusual indolizidine alkaloid originally isolated from a marine sponge

Stelletta sp. as an antifungal and cytotoxic compound [140], was found to inhibit

Ca2+/calmodulin-dependent phosphodiesterase and (Ca2+-Mg2+)-ATPase with IC50

values of 52 and 100 mM, respectively [141]. Related alkaloids, stellettazole A (218)[142], bistellettadines A (219), and B (220) [143], from the same sponge showed

moderate inhibitory activity against Ca2+/calmodulin-dependent phosphodiesterase

(218: 45% inhibition at 100 mM, 219 and 220: 40% inhibition at 100 mM).

NH

HN

NH

O

O

O

OH

OH

NH

N

NOH

Br

NH

N

HO

SMeHN

Br

Me

NH

HO

HO

H2N

H2NNH

pseudodistomin A (214)

pseudodistomin B (215) rigidin (216)

eudistomin A (212) eudistomin C (213)


NH

NH

HN NH2

NH2

NH2

NH2

O

HN

HN

NH

H

O NH2

NH2

+

++

+

4X−

bistellettadine A (219)

NH

NH

HN NH2

O

HN

HN

NH

NH2

NH2

NH2

O NH2

NH2

+

++

+

4X−

220

HN

O

N

H

Me+ X−

HN

O N

N NH

NH2

NH2

Me

+

+

stellettamide A (217)

2X−

stellettazole A (218)

23.4.7 Sialidase (EC 3.2.1.18)

Sialidase cleaves an a-linked terminal N-acetylneuraminic acid from glycoproteins,

glycolipids, and oligosaccharides. In several viral and bacterial infections, neur-

aminidases play important roles; influenza virus employs this enzyme to detach

itself from the infected cell in the budding stage, thus indicating requirement of

neuraminidase for replication of the virus. Therefore, selective inhibitors


of sialidase are potential therapeutic agents against ifluenza. The a-glucosidaseinhibitors, schulzeines A–C (221–223), were also inhibitory against viral neuramin-

idase with IC50 values of 60 mM. Calyceramides A–C (224–226), sulfated

ceramides isolated from the marine sponge Discodermia calyx, inhibited sialidase

from Clostridium perfringens with IC50 values of 0.4, 0.2, and 0.8 mg/mL,

respectively [144]. Another example of a marine sialidase inhibitor is nobiloside

(227), a triterpenoid saponin of the marine sponge Erylus nobilis, which inhibitedClostridium perfringens sialidase with an IC50 value of 0.46 mg/mL [145].

Asteropine A (228) is the first cystine knot of sponge origin isolated from

Asteropus simplex. Asteropine A showed potent and competitive inhibition

against bacterial sialidases (from Clostridium perfringens, Vibrio choleae,and Salmonella typhimurium) with Ki values of 36.7, 340, and 350 nM, respec-

tively [146].

N

NH

OH

HO

OO

OSO3Na

NaO3SO

OSO3Na

schulzeine A (221)

N

NH

OH

HO

OO NaO3SO

OSO3Na

OSO3Naschulzeine B (222)

N

NH

OH

HO

OO

OSO3Na

NaO3SO

OSO3Na

schulzeine C (223)


HN

NaO3SO

OH

O

HO

calyceramide C (226)

COOH

OO

HOOC

OH

OO

OO

OH

COOHHO

OHHO

OH

HO

nobiloside (227)

YCGLFGDLCTLDGTLACCIALELECIPLNDFVGICL

asteropine A (228)

HN

NaO3SO

OH

O

HO

calyceramide A (224)

HN

NaO3SO

OH

O

HO

calyceramide B (225)

23.4.8 Chitinase (EC 3.2.1.14)

Chitinase plays crucial roles in a wide variety of organisms ranging from nutrition

to defence and control of ecdysis in arthropods, thus indicating their potential for

antifungal and insecticidal agents. Styloguanidines (229–231) isolated from the


sponge Stylotella aurantium collected off Yap showed inhibitory activity against

chitinase from a bacterium Schwanella sp. at the dose of 2.5 mg/disc by using the

“squid chitin agar plate method.”[147].

N

N

NH

NH

HN

N

H2N

O

H2N

HO

Cl NH2

R1R2

R1 R2

H HBr HBr Br

styloguanidine (229)3-bromostyloguanidine (230)2,3-dibromostyloguanidine (231)

23.4.9 a-Glucosidases (EC 3.2.1.20)

a-Glucosidases are involved in glycoprotein processing and glycogenolysis and,

therefore, their inhibitors can be applied for treatment of diabetes, obesity, viral

infections, and cancer. Penarolide sulfates A1(232) and A2(233), L-proline

containing macrolide trisulfates isolated from a marine sponge Penares sp.,

inhibited a-glucosidase of yeast origin with IC50 values of 1.2 and 1.5 mg/mL,

respectively, while they showed only weak or no inhibition against b-glucosidaseor b-galactosidase. Penarolide sulfates also inhibited thrombin with IC50 values

of 3.7–4.2 mg/mL. Perhaps, the three sulfate groups are responsible for the

activity [148]. A linear type congener, penasulfate A (234), in which L-proline

was replaced by D-pipecolic acid, was isolated from the same sponge.

Penasulfate A (234) showed about ten times as potent inhibition (IC50 0.14 mg/mL) as penarolides againt the same enzyme, while the inhibitory activity against

the enzyme from the different source remained the same level [149]. The known

1,4-dideoxy-1,4-imino-D-arabinitol (235) was isolated as an a-glucosidase inhib-itor from a marine sponge Haliclona sp. collected in Western Australia.

Iminopentitol (235) strongly inhibited yeast a-glucosidase at an IC50 value of

0.16 mg/mL [150]. Callyspongynic acid (236), a polyacetylenic acid was isolated

from the Japanese marine sponge Callyspongia truncata Callyspongynic acid (236)inhibited a-glucosidase with an IC50 value of 0.25 mg/mL, but was inactive against

b-glucosidase, b-galactosidase, thrombin, and trypsin at 100 mg/mL. Both the carbox-

ylic acid and the allylic alcohol linked to an acetylene are thought to be important for

the activity [151]. The Japanese marine sponge Penares schulzei afforded three new

a-glucosidase inhibitors, schulzeines A–C (221–223), which showed potent inhibitory


activity against a-glucosidase with IC50 values of 48–170 nM. It should be noted that

desulfated schulzeines A and B still retained activity (IC50 s 2.5 and 1.1 mM, respec-

tively) [152]. Recently, the stereochemistry at C-20 of 221was revised on the basis oftotal chemical synthesis [153].

O

N

O

O

OSO3Na

OSO3NaNaO3SO

penarolide sulfate A2 (233)

O

OSO3Na

N O

O

OSO3NaNaO3SO

penarolide sulfate A1 (232)

NH

HO OH

OH

235

N

OMe

O

O

OSO3Na

OSO3Na

penasulfate A (234)

HO

O

OH

callyspongynic acid (236)

23.4.10 S-Adenosylhomocysteine Hydrolase (EC 3.3.1.1)

S-adenosylhomocysteine hydrolase that reversibly hydrolyzes

S-adenosylhomocysteine to homocysteine and adenosine has been considered

an attractive target for the design of antiviral, antiparasitic, antiarthritic and immu-

nosuppressive agents. Ilimaquinone (237) has been reported to possess antiviral,


antimitotic, antimicrobial, and anti-HIV activities. It was also shown to vesiculate the

Golgi apparatus as well as to protect cells against the toxic effects of

ricin and diphtheria. Whole-cell and cytosolic photoaffinity labeling and affinity

chromatography studies with chemical probes prepared from 237 converged on

S-adenosylhomocysteine hydrolase as a cellular target of 237. Subsequent

studies with isolated enzyme indicated that ilimaquinone is a competitive

inhibitor of S-adenosylhomocysteine hydrolase (IC50 40 mM). On the basis of

SAR studies, pharmacophore modeling, and computer docking studies have allowed

for the design and preparation of a simplified structural variant (238) possessing a

100-fold greater inhibitory activity than that of natural (�)-ilimaquinone (237) [154].

O

O OMe

HO

H

O

HO OH

N

N

NN

NH2

O

HO OH

SO

O

NH3

N

N

NN

NH2

aliphaticregion

riboseregion

baseregion

S-adenosylhomocystein

(−)-ilimaquinone (237)

IC50 40 μM

238IC50 0.4 μM

23.4.11 Serine Proteases (EC 3.4.21.-)

Proteolytic enzymes are classified into serine proteases, cysteine proteases, aspartic

proteases, and metalloproteases on the basis of their catalytic centers. Trypsin-like

enzymes, a group of serine proteases, are associated with many disease states.

The hyperproteolytic activities of this homologous family of enzymes are attractive

chemotherapeutic targets in pathways of blood coagulation, fibrinolysis, kinin forma-

tion, complement activation, digestion, reproduction, and phagocytosis. Thus,


inhibitors of specific serine proteases can be potential drug-leads for many disease

status [155]. Cyclotheonamides A (239) and B (240) from a marine sponge Theonellasp. (now known to be T. swinhoei) inhibited thrombin, trypsin and plasmin with IC50

values of 0.076, 0.2, and 0.3 mg/mL, respectively [156]. Further investigation of

T. swinhoei yielded cyclotheonamides C (241) and D (242) [157]. Cyclotheonamides

E series (244–248) were not found from this sponge, but from the other type of

T. swinhoeiwhose interior part is white (E1–E3) [158] and from an Okinawan sponge

Ircinia sp. (E4–E5) [159]. Cyclotheonamides C (241), D (142), and E1–E5 (244–248)showed potent inhibition against thrombin and trypsin with IC50 values of 2.9–68 nM

and 7.4–55 nM, respectively. Cyclotheonamide E1 (244), E4 (247), and E5 (248)strongly inhibited both mouse and human tryptases (IC50s 6.9, 23, and 84.7 nM for

human tryptase, and 17.0, 6.5, and 54.1 nM for mouse tryptase, respectively) [159].

Dihydrocyclotheonamide A (243), pseudotheonamides A1 (249), A2 (250), B2 (251),C (252), andD (253) fromT. swinhoei, inwhich a-ketohomoarginine (k-Arg) residues

are cyclized, showed only moderate activity against these enzymes with IC50 values

ranging from 0.19 to 3.0 mM (thrombin) and 3.8 mM (trypsin) [160]. Complexes

between cyclotheonamide A and human a-thrombin and between cyclotheonamide A

and trypsin were successfully crystalized and their crystal structures were solved,

disclosing that cyclotheonamide A binds to the catalytic center of these enzyme;

particularly k-Argwhich forms the hemiacetal linkagewith Ser195 of one of a catalytic

triad is important for the binding to the enzyme [161]. From the same T. swinhoei, wasisolated a thrombin inhibitory linear peptide, nazumamide A (254) which inhibited

thrombin with an IC50 value of 2.8 mg/mL, but not trypsin at 100 mg/mL [162]. Retro-

binding mode of nazumamide A to human thrombin was demonstrated by X-ray

crystallography of the nazumamide A/thrombin complex [163].

HNH

O

OHN

N

HNO

HO

NH

O

HN

HN

H2N NH

O

O

OHN

NH

O

HN

HN

H2N

HN

NH

R

N

O

O

O

O

NH

OH

O

cyclotheonamide A (239) R = Hcyclotheonamide B (240) R = Ac

cyclotheonamide C (241)

O


O

O

O

R1 R2

O

O

E1 (244)

E2 (245)

E3 (246)

E4 (247)

E5 (248)

H

H

H

H

OH

NH

O

OHN

N

HNO

NH

O

HN

HN

H2N NH

O

O

OH

O

R1

HN

R2

cyclotheonamide E's

OHN

NH

HO

HN

HN

H2N

HN

NH

H

N

O

O

O

O

NH

OH

O

dihydrocyclotheonamide A (243)

HNH

O

OHN

N

HNO

NH

O

HN

HN

H2N NH

O

O

OH

cyclotheonamide D (242)

O

HN

ONH

O

HN

OHN

NHN

N

NH

O

OH

O

NHHO

H O

H

pseudotheonamide A2 (250)

HN

ONH

O

HN

OHN

NHN

N

NH

O

OH

O

NHHO

H O

H

pseudotheonamide A1 (249)


NH

O

HN

OHN

N NH2

O

OH

H O

O

NH2NH

N

HN

H2N NH

OOH

OH

O

HN

NH

OH

O

O

O

pseudotheonamide D (253) nazumamide A (254)

HHN

ON

O

N

OHN

NHN

N

NH

O

OH

O

NHHO

H O

pseudotheonamide B2 (251)

HN

N

NH

O

OH

O

NHHO

H O

O

NH2

NH

O

HN

OHN

N

pseudotheonamide C (252)

Toxadocials A–C (255–257) and toxadocic acid A (258) isolated from a marine

sponge Toxadocia sp., are alkyl sulfate thrombin inhibitors with IC50s of 6.5, 4.6,

3.2, and 2.7 mg/mL, respectively [164]. The mode of enzyme inhibition of

toxadocials is not known.

HOOSO3Na

OSO3Na OSO3Na OSO3Na OSO3Na

OSO3NaOSO3Na

OSO3NaOSO3Na

OSO3Na OSO3Na OSO3Na

HO

OH

OHHO

OHOOSO3Na OSO3Na OSO3NaOSO3Na

Toxadocial A (255)

Toxadocial B (256)

Toxadocial C (257)

Toxadocic acid A (258)

Dysinosins A–D (259–262) are inhibitors of factor VIIa and thrombin isolated

from the Australian sponge Lamellodysidea chlorea. Dysinosins showed potent

inhibitory activity against factor VIIa and thrombin with Ki values of 0.090 to 1.32


and 0.17 to -> 23 mM, respectively [165]. The structural similarity of dysinosins to

that of aeruginosins (263) which were the serine protease inhibitors isolated from

the cyanobactera Microcystis aeruginosa [166] and Oscillatoria agardhii [167]implied that they were biosynthesized by symbiotic microbes. Based on X-ray

crystallographic data of complexes of chlorodysinosin A with thrombin, a series

of analogues were synthesized varying the nature of the P1, P2, and P3pharmacophoric sites and the central octahydroindole carboxyamide core. Intro-

duction of a hydrophobic substituent on the D-Leu amide dramatically improved

the inhibitory activity against thrombin (264: IC50 1.6 nM) [168].

N

HN

HN

HO

H

HO

O

O

HO

OH

NHNH2

HN

OHaeruginosin 298-A (263)

N

HN

HN

H

HO

O

O

HO

NH2

HN

Cl

264

N

HN

HN

R2

R3

HO

R1

H

HO

O

O

MeO

N NH2

NH2

O

OHO

HOHOH2C

HO

R3R1

OH

OH

OH

dysinosine A (259)

B (260)

C (261)

D (262)

R2

OSO3−

OSO3−

OSO3−

OH


Factor XIa (FXIa) is a trypsin-like serine protease that plays a major role in the

amplification phase of the coagulation cascade and in maintaining clot integrity.

It is a unique target where specific inhibitors of FXIa might inhibit thrombosis

without completely interrupting the function of normal hemostasis and might

prevent or minimize the risk of hemostasis complication. Clavatadines A–E

(265–269) were isolated as FXIa inhibitors from the Australian sponge Subereaclavata along with known aerophobin 1 (270), purealdin L (271), and

aplysinamisine II (272). Compounds 265 and 266 inhibited FXIa with IC50 values

of 1.3 and 27 mM, respectively, whereas they did not inhibit FIXa (< 5% inhibition

at 222 mM). The crystal structure obtained by soaking of compound 265 into a singlecrystal of FXIa showed only the carbamate side chain of 265 covalently bonded to

the active site serine residue (Ser 195) [169]. Compounds 267–272 were shown to

be weaker inhibitors (12–59% inhibition at 222 mM) [170].

23.4.12 Cysteine Proteases (EC 3.4.22.-)

23.4.12.1 Cathepsin B (EC 3.4.22.1)Cysteine proteases which have cysteine and histidine as the catalytic center are

involved in cytosolic protein metabolisms. Cathepsin B, a cysteine protease, is

known to be involved in various disease states, such as inflammation, trauma,

muscular dystrophy, and tumors. In particular, its possible roles in cancer metastasis

are of major concern in cancer chemotherapy. Two peptidic inhibitors, tokaramide

A (273) [171] and miraziridine A (274) [172] with IC50 values of 29 and 1,400 ng/

mL were isolated from the marine sponge Theonella aff. mirabilis. Miraziridine

A (274) contains three unusual amino acid residues; particularly unusual are

vinylogous arginine and aziridine-2,3-dicarboxylic acid. Secobatzelline A (207)isolated from a sponge Batzella sp. showed inhibition against peptidase activity of

CPP32, a member of caspases which are cystein proteases playing amajor role in the

programmed cell death mechanism known as apoptosis, with an IC50 value of

0.02 mg/mL as well [130]. Asteropterin (275), a uniquemolecule with a combination

of lumazine and N-methyl histamine isolated from the marine sponge Asteropussimplex, inhibited cathepsin B with an IC50 value of 1.4 mg/mL [173].

OH

Br Br

OHN

R

O

O

NH

NH2

NH

265 : R = OH266 : R = NH2

O

Br Br

O

NHN

O

NH

NH2

NH

n

267 : n = 1268 : n = 2


O

Br Br

O

NHN

O

NH

NH2

NH

Me

HO

n

271 : n = 1272 : n = 2

OH

Br

NH

NHO

OHN NH2

NH

269

O

Br Br

O

NHN

O

Me

HO

270N

NH

HONH

NH

HN

NH

HN

OH

O O

O

OH O

O

NH

HN NH2

O

miraziridine A (274)

HN

NH

HN

H

NH

NH

HO

O

O

O

O

H2Ntokaramide A (273)

NH

HN

N

N

O

O

N

Me

NH

N

asteropterin (275)


23.4.13 Aspartic Proteases (EC 3.4.23.-)

23.4.13.1 HIV Protease (EC 3.4.23.47)Replication of HIV involves the expression of several viral polyproteins that

requires the presence of a virus-specific protease for their maturation. Inhibition

of this enzyme results in immature viral particles; therefore, HIV-1 protease is an

ideal target for AIDS chemotherapeutics.

Didemnaketals A (276) and B (277), isolated from a Palauan ascidianDidemnumsp., inhibited HIV-1 protease with IC50 values of 2 and 10 mM, respectively [174].

The unusual structures and potent activity of didemnaketals indicated their possi-

bility as attractive synthetic targets [175]. Recently, simplified analogues lacking

the spiroketal portion were synthesized, some of which were as potent as

didemnaketal A. It was also proposed that didemnaketals inhibit dimerization of

HIV-1 protease monomers [176].

MeO

O O OAc

O

O

O

O

OH

O

O

O

Odidemnaketal A (276)

MeO

O O OAc

O

O

O

O

OH

O

O

O

MeO

O

didemnaketal B (277)

23.4.14 Metalloproteases (EC 3.4.24.-)

Metalloproteases contain metal ions such as Zn2+, Co2+, Mn2+, etc., in their

catalytic centers. The matrix metalloproteinases (MMPs), members of a large


subfamily of proteases which contains a catalytic zinc-binding domain, play

important roles in the remodeling and degradation of extracellular matrix (ECM).

They are linked to a range of physiological and pathological processes, including

wound healing, bone remodeling, angiogenesis, inflammation, and tumor progres-

sion and metastasis. The membrane-type matrix metalloproteinases (MT-MMPs)

are known as key enzymes in tumor metastasis. They activate progelatinase A to the

fully matured form which in turn degrades type IV collagen, the major component

of the basal membrane which prevents tumor progression. Thus, inhibitors of MT-

MMPs are believed to be potential anticancer drugs. Ancorinosides B–D (279–281)[177] were isolated as MT1-MMP inhibitors from the marine sponge Penaressollasi along with known ancorinoside A (278) [178] which was originally isolated

as an inhibitor of blastulation of starfish embryos from a marine sponge Ancorinasp. Ancorinosides A–D (278–281) inhibited MT1-MMP with IC50 values of 440,

500, 370, and 180 mg/mL, respectively. Ancorinoside B (279) also inhibited

gelatinase A (MMP2) with an IC50 value of 22 mg/mL. Haplosamate A (65) andits congener (282) were isolated from a Japanese marine sponge Cribrochalina sp.

as MT1-MMP inhibitors with IC50 values of 150 and 160 mg/mL, respectively;

[179] 65 was originally isolated as an HIV integrase inhibitor from Philippine

haplosclerid sponges [32]. Ageladine A (283) is a fluorescent alkaloid isolated

from the marine sponge Agelas nakamurai. The IC50 values of 283 against

MMPs 1, 2, 8, 9, 12, and 13 were 1.2, 2.0, 0.39, 0.79, 0.33, and 0.47 mg/mL,

respectively, while its N-methylated derivatives did not inhibit MMP2. Unlike other

MMP inhibitors, ageladine A (283) was not capable to chelate Zn2+ and not

a competitive inhibitor against MMP2 [180]. With such interesting aspects of

inhibition mode, as well as its anti-angiogenic activity, ageladine A became

a target of total synthesis [181].

OO

OO

OH

N

OH

OH

COOH

HOOH

HOHO

OH

O

O

MeO

OO

OH

N

OH

OH

COOH

HO

O

O

MeO

OO

OHHO

HOOH

OO

OH

N

OH

OH

COOH

HO

O

O

MeO

OO

OHHO

HOOH

ancorinoside A (278)

ancorinoside B (279)

ancorinoside C (280)


O

H

H

H

OH

OHH

OP

O

OMeNaOOHNaO3SO

N

NNH

NH

Br

Br

H2N

282 ageladine A (283)

OO

OO

OH

N

OH

OH

COOH

HOOH

HOHO

OH

O

O

MeO

ancorinoside D (281)

23.4.15 Histone Deacetylases (EC 3.23.-)

Histone acetylation and deacetylation are catalyzed by histone acetyltransferases

(HATs) and histone deacetylases (HDACs), respectively, which play important

roles in transcriptional regulation [182]. Inhibitors of these enzymes are known to

induce cell cycle arrest [183], p53-independent induction of the cyclin dependent

kinase inhibitor p21 [184], tumor-selective apoptosis [185], and differentiation of

normal and malignant cells [186]. HDAC inhibitors were also demonstrated to

exert anti-angiogenic effects through the alteration of vascular endothelial

growth factor (VEGF) signaling [187]. These direct and indirect effects on

tumor growth and metastasis indicate that HDAC inhibitors are potential anti-

cancer agents. Six new psammaplins E–J (288–293) were isolated from the

marine sponge Pseudoceratina purpurea collected in Papua New Guinea along

with known psammaplins A–D (284–287), and bisaprasin (294). These com-

pounds showed potent inhibitory activity against HDAC (IC50s 2.1–327 nM)

and cell-based p21 promoter activity (IC50s 0.7–15 mM), in addition to inhibitory

activity against DNA methyltransferase (DNMT) [188]. NVP-LAQ824 (295)which was developed on the basis of structures of HDAC inhibitors including

psammaplins [189] entered phase I clinical trials in patients with solid tumors or

leukemia [190]. Three new cyclostellettamines, cyclostellettamine G (297),dehydrocyclostellettamines D (298) and E (299), were isolated together with

the known cyclostellettamine A (296) from a marine sponge of the genus

Xestospongia. These compounds inhibited HDAC with IC50 values of

17–80 mM [191]. Five new cyclic tetrapeptides, azumamides A–E (300–304)were isolated from the marine sponge Mycale izuensis. Azumamides showed

inhibitory activity against human HDAC at IC50 values of 0.045–1.3 mM. In the

cell-based assay using K562 cells, azumamide A (300) inhibited deacetylation

of Ac-H3 (Lys9 and Lys 14) and Ac-H4 (Lys 8) in a dose-dependent manner


(0.19–19 mM). Furthermore, azumamide A inhibited angiogenesis in the in vitro

vascular organization model using mouse ES cells [192].

OH

Br

O

NHO

NH

Y

S S

HN

NH2

O

O

E (288) Y =

SCN

SO2NH2

S S

HN OMe

O

A (284) Y =

B (285) Y =

C (286) Y =

D (287) Y =

S S

HN

N

O

OH

Br

OH

S S

HN

OH

O

O

S S

HN

OH

O

NNH2

S S

HN OEt

OSO2CH3

S S

HN

N

OO

OH

Br

OH

F (289) Y =

G (290) Y =

H (291) Y =

I (292) Y =

J (293) Y =

psammaplin

N

NH

OH

OH

NH

O

NVP-LAQ824 (295)

N

NH

S

HO

O

Br

OH

SNH

NOH

OH

O

Br

bisaprasin (294) 2


HNHN

HN

R4

R3

O

O

O

O

R1

O

HN

R2

R1

NH2

NH2

OH

NH2

OH

R2

H

OH

OH

H

H

R3 = R4

Me

Me

Me

HMe

azumamide A (300)

B (301)

C (302)

D (303)

E (304)

NN+

CF3COO−

CF3COO−

+

cyclostellettamine A (296) : m = 1, n = 2

G (297) : m = 1, n = 1

D (298) : m = 1, n = 4, Δ13

E (299) : m = 2, n = 4, Δ13dehydrocyclostellettamine

23.4.16 ATPases (EC 3.6.1.3)

23.4.16.1 H,K-ATPaseH,K-ATPase mediates acid secretion from gastric wall cells. Thus, excess activa-

tion of this enzyme leads to hyperacidity, causing gastric ulcer. In order to discover

potential antiulcer leads, a number of Japanese marine invertebrates have been

screened for inhibition of porcine H,K-ATPase which resulted in isolation of

several inhibitors.

S-(+)-curcuphenol (305) and dehydrocurcuphenol (306), isolated from

a Japanese marine sponge Epipolasis sp., inhibited porcine H,K-ATPase with

IC50s of 8.3 and 23 mM, respectively, while hydroxycurcuphenol was inactive

[193]. It should be noted that the R-(�)-curcuphenol had already been reported

from the Caribbean gorgonian Pseudopterogorgia rigida [194]. Hexaprenylhy-

droquinone sulfate (307) obtained from a marine sponge Dysidea sp. inhibited


H,K-ATPase with an IC50 of 4.6 mM [195]. Since 307 showed promising activity,

a number of its analogues were synthesized and evaluated for inhibition of acid

secretion and antiulcer activity, however, the results were disappointing [2].

Sinulamide (308) derived from a soft coral Sinularia sp. inhibited the enzyme

activity with an IC50 of 5.5 mM [2]. The structure of 308 was recently refined by

synthetic approach [196].

OH OH

S-(+)-curcuphenol (305) dehydrocurcuphenol (306)

H

OSO3Na

OH

6

hexaprenylhydroquinone sulafte (307)

H

NN NH3O

++

+

Cl−

Cl−

Cl−

3

sinulamide (308)

23.4.16.2 Na,K-ATPaseNa,K-ATPase regulates Na+ transport through cell membranes and is directly

related to contraction and relaxation of smooth muscles, thus indicating that

inhibitors of the enzyme are potential drug leads for cardiovascular diseases.

Agelasines A–F (309–314), 9-methyladenine derivatives of diterpenes, isolated

from the Okinawan marine sponge Agelas nakamurai, were reported to inhibit

Na,K-ATPase, though their detailed activity is not available [197]. Two hypotaur-

ocyamine derivatives of diterpene, agelasidines B (315) and C (316) from the same

sponge inhibited not only Na,K-ATPase with IC50 values of 10–50 mM, but also

contraction of smooth muscle [198].


H

N N

N

NH2N

+

Cl−

agelasin A (309)

H

N N

N

NH2N

+

Cl−

agelasin B (310)

H2N

H

N N

N

N

+

Cl−

agelasin C (311)

H2N

H

H

N N

N

N

+

Cl−

agelasin D (312)

N

N

N

N

+

H2N

Cl−

agelasin E (313)

N

N

N

N

H

+

H2N

Cl−agelasin F (314)


SNH

OO NH2

NH2

Cl−+

agelasidin B (315)

NH2

NH2

S

HN

O O+Cl−agelasidin C (316)

Iantheran A (317), its diacetate, and a-hydroxy enone derivatives, isolated from

an Australian marine sponge Ianthella sp. showed moderate inhibitory activity

against Na,K-ATPase with IC50 values of 2.5, 5.0, and 10 mM, respectively [199].

This sponge also yielded ianthesins A–D (318–321), of which ianthesins B-C

inhibited dog kidney Na,K-ATPase with IC50 values of 440, 50, and 280 mM,

respectively [200].

O

NHN O

OHO

Br

OMe

Br

HO

R

O

Br

Br

ianthesin A (318)

B (319)

D (321)

RβNMe2

αNMe2

NHSO3Na

O OSO3Na

NaO3SO

Br

Br

OH

O

Br

OH

Br

iantheran A (317)

HNO

Br

OMe

HOON

HN O

Br

HN

O

Br

Br

SO3Na

O NH

Br

Br

O OH

O

ON

OH

Br

OMe

Br

ianthesin C (320)


Xestoquinone (82), a polycyclic quinone, which was isolated together with the

known antimicrobial substance halenaquinone (79) [201], from the Okinawan

marine sponge Xestospongia sapra, inhibited Na,K-ATPase. Xestoquinone was

the first example of marine natural products having parallelism between the ino-

tropic action and Na,K-ATPase inhibition. Halenaquinol (80) [202], a natural

cardioactive pentacyclic hydroquinone, from the marine sponge Petrosia seriata,was found to be a powerful inhibitor of the rat brain stem and cortex Na,K-ATPases

and the rabbit muscle sarcoplasmic reticulum Ca2+-ATPase with IC50 values of

0.70, 1.3, and 2.5 mM, respectively [203]. Sarcochromenol sulfate A (322) andsarcohydroquinone sulfates A–C (325–327) isolated from the New Zealand sponge

Sarcotragus spinulosus inhibited the rat brain Na,K-ATPase with IC50 values of

1.6, 1.6, 1.4, and 1.3 mM, respectively, while sarcochromenol sulfates B (323) andC (324) were inactive [204].

OH

NaO3SO

H

OR

OSO3Na

sarcohydroquinone sulfate A (325)

B(326)

C (327)

RH

SO3Na

H

n

sarcochromenol sulfate A (322) n = 5B (323) n = 6C (324) n = 7

n

n6

7

8

23.4.16.3 Vacuolar ATPasesVacuolar H+-ATPases, a class of proton pumps present in all eukariotic cells, are

responsible for proton transport in bone-derived membrane vesicles, and the pro-

cess of osteoclast-mediated bone resorption is directly dependent on H+-

translocating ATPases. Inhibition of osteoclast vascular H+-ATPase may be effec-

tive for reducing the rate of bone resorption in pathological conditions such as

osteoporosis. Adociasulfate 1 (328), a sesterterpenoid isolated from an Australian

marine sponge Adocia sp., reduced proton pump activity in hen bone-derived

membrane vesicles with an IC50 value of 3.6 mM and proton pumping in brain-

derived vesicles with an IC50 value of 4.69 mM, while adociasulfates 7 (329) and8 (330) were less active (IC50 30 mM and 55% inhibition at 100 mM, respectively)

[205]. Recently, antitumor polyketides, salicylihalamides A (331) and B (332)isolated from a marine sponge Haliclona sp. [206] and lobotamides A–F (333–338) from the tunicate Aplidium lobatum [207], both of which share the core

structure with YM-75518 (339), an antifungal metabolite of Pseudomonas sp.

Q38009 [208], were found to be a potent and selective inhibitors of mammalian

V-ATPases (IC50s 0.40–14.0 nM) [209]. Palmerolide A (340) isolated from the

Antarctic tunicate Synoicum adareanum is a cytotoxic macrolide. It showed 3 orders

of magnitude greater sensitivity (LC50 18 nM) against melanoma relative to other

cell lines tested in the NCI 60 cell line panel.


Palmerolide A displayed a 60-cell-line activity profile which correlated, using

the NCI COMPARE algorithm, to several vacuolar ATPase inhibitors. Indeed, it

inhibited V-ATPase with an IC50 of 2 nM and to be active in NCI’s hollow-fiber

assay [210]. Following study on its stereochemistry by degradation [211] or total

synthesis [212] revised its stereochemistry at C7, C10, and C11. The SAR study of

palmerolides using synthetic analogues showed that a phenyl substitution on side

chain and removal of the C7 hydroxy group enhanced the potency, while the

selectivity against melanoma of 340 was not reproduced [213].

R1O

OR2

HOH

R1 R2

SO3Na SO3Na

OSO3Na

NaO3SO

H SO3Na

OH

H HH H

H H

adociasulfate 1 (328)

7 (329

adociasulfate 8 (330)

O

HN

OOH O

OH

salicylihalamide A (331) Δ17 trans

salicylihalamide B (332) Δ17 cis

O

OH

O

O

CH2R

O

HN

ON

OMe

OH

lobotamide A (333) R = H(= YM-75518A)lobotamide D (336) R = OH

O

OH

O

O

CH2R

O

HN

ON OH

MeO

lobotamide B (334) R = H

(= YM-75518B)

lobotamide E (337) R = OH

O

OH

O

O

CH2R

O

HN

OOH

NMeO

lobotamide C (335) R = H

(= YM-75518C)

lobotamide F (338) R = OH


O

OH

O

O

O

HN

OOH

N

OMe

YM-75518D (339)

O

OH

HO

O NH2

O

OHN

O

palmerolide A (340)

23.5 Lyases (EC 4.-.-.-)

23.5.1 ATP Citrate Lyase (EC 4.1.3.8)

Since the very-low-density lipoproteins (VLDL) are metabolic precursors of low-

density lipoproteins (LDL) which play a key role in hyperchoresterolemia,

intervention of VLDL synthesis has provided a strategic target for hypercholes-

terolemia therapy. Inhibitors of ATP-citrate lyase (ACL) are anticipated to

reduce the production of acetyl CoA and can affect both lipogenesis and

cholesterogenesis, and as a result, expected to reduce VLDL synthesis. Purpurone

(341), a poly catecholic pyrrole isolated from a marine sponge Iotrochota sp.,

inhibits ATP-citrate lyase in a dose-dependent manner with an IC50 value of

7 mM [214].

N

OHHO

HOOH

O O

OH

OH

HO

HO

OH

purpurone (341)


23.6 Isomerases (EC 5.-.-.-)

23.6.1 Topoisomerase

DNA topoisomerases are nuclear enzymes that catalyze DNA breaking and unwind-

ing during cellular replication and RNA transcription. In eukaryotic cells, DNA

topoisomerases I (topo I) and II (topo II) play distinct roles in DNA unwinding. Topo

I catalyzes single-strand “nicking” to allow supercoiled DNA to unwind, whereas

topo II facilitates chromosomal duplication by relaxing and unwinding the DNA

duplex. Thus, inhibitors of topo I and topo II are expected to control cancer cell

proliferation and thought to be important targets for cancer chemotherapy.

23.6.1.1 Topoisomerase I (EC 5.99.1.2)Xestoquinol sulfate (342) and xestosaprols A (343) and B (344), as well as a knowncompound 345 and halenaquinol sulfate (81), were isolated as topo I inhibitors fromthe Okinawan sponge Xestospongia sapra; 342–344 inhibited DNA topo I with

MICs of 10, 12.5, and 12.5 mg/mL, respectively, while 345, xestoquinone (82), andhalenaquinone (79) were more potent with MICs of 2.5, 2, and 0.4 mg/mL, respec-

tively [215]. Interestingly, halenaquinol sulfate (81) was not active in this assay.

Makaluvamine G (346), a cytotoxic pigment which was isolated from an Indone-

sian sponge Histodermella sp., inhibited topo I with an IC50 value of 3.0 mM [216].

Two ceramide 1-sulfates 347 and 348 from the Japanese bryozoa Watersiporacucullata inhibited human topo I with IC50s of 0.4 and 0.2 mM, respectively

[217]. Similarly, amphimic acids A (349) and related long-chain fatty acids from

an Australian sponge Amphimedon sp. showed topo I inhibition with IC50s ranging

from 0.47 to 6.7 mM [218].

O

OOH

OSO3Na

N

NHN

OH

OMe

Me+

makaluvamine G (346)

xestoquinol sulfate (342)

O

OR2

R1OH

H

xestosaprol A (343)xestosaprol B (344)

(345)

R1 = H, OH; R2 = OR1 = R2 = H, OHR1 = O; R2 = H, OH


−O3SO

−O3SO

HN

OH

O

HO

HN

OH

O

HO

347

348

HO

O

amphimic acid A (349)

23.6.1.2 Topoisomerase II (EC 5.99.1.3)Wakayin (350), a cytotoxic pyrroloiminoquinone alkaloid, isolated from the ascidian

Clavelina sp., inhibited topo II at 250 mM [219]. Pyrroloiminoquinones,

makaluvamines A–F (351–356) which were isolated from Fijian sponges of the

genus Zyzzya, showed topo II inhibition with IC50s of 41, >500, 420, 320, 310, and

25 mM, respectively, while related makaluvone and damirone B were not active. The

makaluvamines showed differential toxicity toward the topo II-sensitive CHO cell line

xrs-6; makaluvamines A (351) and C (353) exhibited in vivo antitumor activity against

the human ovarian carcinoma Ovcar3 implanted in athymic mice [220]. Similarly,

makaluvamineN (357) isolated from thePhilippine sponge Zyzzya fuliginosa exhibitedgreater than 90% inhibition of topo II at 5 mg/mL [221]. OthermakaluvaminesG–H did

not show inhibitory activity against topo II [222], whereas only makaluvamine

G showedmoderate inhibition against topo I [189].Bengacarboline (358), ab-carbolinealkaloid isolated fromamarine ascidianDidemnum sp., inhibited topoII at 32mM[223].

NR2

N

O

NH2

R1

+

R1 R2

NH

N

O

NH2

Me

+

makaluvamine B (352)

NH

HN

OHN

NH

+

makaluvamine A (351) Me

Me

H

makaluvamine C (353) Hwakayin (350)


NH

N

N

S

HN

O

N

N

HN

O

O

R

RH

MeS

RHdehydrokuanoniamine B (361)

kuanoniamine D (364)

cystodytin J (363)

diplamine (365)

NH

N

HN

O

R

HN

S

O

RHshermilamine B (359)

shermilamine C (362)

N

N

N

O

ascididemin (360)

NH

N

OHN

Me

S

Br

OH

+

makaluvamine F (356)

NH

HN

OHN

OH+

makaluvamine D (354)

makaluvamine E Δ (355)

NH

HN

O

NH2

Br

+

makaluvamine N (357)

NH

NH

NH

NH

H2N

bengacarboline (358)


Two aromatic alkaloids, shermilamine B (359) [224] and ascididemin (360) [225]were reported to inhibit topo II at 30 and 75 mM, respectively [226]. Similarly,

dehydrokuanoiamine B (361), shermilamine C (362), cystodytin (363), kuanoniamine

D (364), and diplamine (365) from an ascidian Diplosoma sp., inhibited topo II with

IC50 values of 115, 138, 8.4, 127, and 9.2 mM, respectively [227]

Popolohuanone E (366), an oxidatively dimerized arenarol derivative isolated

from a Pohnpei sponge Dysidea sp., was reported to be inhibitory against topo II

with an IC50 of 400 nM and selectively cytotoxic against the A549 non-small-cell

human lung cancer cell line (IC50 2.5 mg/mL) [228]. Puupehenone (367) and

21-chloro-puupehenone (368), sesquiterpene hydroquinones isolated from

a verongid sponge, were tested for a wide variety of biological activities including

inhibitory activity against topo II, adenosine deaminase (ADA), glutathione

reductase (GR), dihydrofolate reductase (DHFR), and thymidylate synthetase

(TS), which disclosed that 367 inhibited topo II with an IC50 value of 1 mg/mL,

in addition that 367 and 368 inhibited ADA at IC50s >25 and> 25 mg/mL,

respectively, GR with an IC50 of 6 mg/mL for 368, DHFR with IC50s of 5 and

5 mg/mL, respectively, and TS with IC50s of 8 and 3 mg/mL, respectively [229].

O

O

O

OH

HO

HO

H

H

O

OH

OX

H

H

popolohuanone E (366) puupehenone (367): X = H

21-chloro-puupehenone (368): X = Cl

HO

OH

Oelenic acid (369)

HN

O

NH

Br

Br

O

HO

Br O

NOH

Br OH

Br

NOH

O

bastadin 14 (371)

H

OH

OH

O

4

370


O

NNH

NH

O

N S

virenamide A (372)

Elenic acid (369), an unusual metabolite of an Indonesian sponge Plakinastrellasp., inhibited topo II with an IC50 value of 0.1 mg/mL [230], while 3-tetraprenyl-4-

hydroxy benzylic acid (370) isolated from themarine sponge Irciniamuscarum had an

IC50 of 0.5 mg/mL [231]. Bastadin 14 (371) from the marine sponge Psammaplysillapurpurea showed inhibitory activity against topo II and dehydrofolate reductase withIC50 values of 2.0 and 2.5 mg/mL, respectively [232]. VirenamideA (372), a cytotoxiclinear peptide from the Australian ascidian Diplosoma virens, exhibited topo II

inhibitory activity with an IC50 of 2.5 mg/mL [233].

23.7 Conclusions

More than 370 metabolites derived from marine invertebrates have been shown to

inhibit various enzymes. These enzymes play pivotal roles in many aspects of the

biological processes; therefore, inhibitors are useful for understanding complicated

biological phenomena. These inhibitors fall into a wide range of biogenetic classes,

ranging from simple terpenoids or polyketides to complex polyketides and

oligopeptides. Therefore, there are still good chance to find structurally unique

new enzyme inhibitors such as okadaic acid, calyculins, and cyclotheonamides.

However, at the same time, we should make efforts to find new activities of trivial

known compounds to maximize the potentials of marine natural products. As

mentioned in the introduction, one of the trends of marine natural products will

be the discovery of new biological activities.

While this chapter was being prepared, a huge earthquake (M 9.0) occurred at the

Northeastern part of Japan. Tsunami and nuclear pollution have damaged quite

seriously the coast and marine organisms of the wide areas. Since these areas are

bountiful of marine organisms, this damage will give considerable limitation to study

marine natural products from this part. We hope the environment will be recovered in

the near future.

23.8 Study Questions

1. Is there any room for new findings in marine natural products chemistry?

2. How far have we understood the potentials of marine natural products?


3. Which should we attach more importance, to new structures or to new activities?

4. What is the status of our understanding on the mechanisms of action for marine

natural products?

5. How can marine natural products chemistry contribute to the upcoming trend of

life sciences?

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handbook of marine natural products volume 426 || enzyme inhibitors from marine invertebrates

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