1 DRUG DISCOVERY FROM MARINE CYANOBACTERIA SYMPLOCA SPP. AND PHORMIDIUM SPP.: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY METABOLITES By LILIBETH APO SALVADOR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
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1
DRUG DISCOVERY FROM MARINE CYANOBACTERIA SYMPLOCA SPP. AND PHORMIDIUM SPP.: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY
METABOLITES
By
LILIBETH APO SALVADOR
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
1 GENERAL INTRODUCTION .................................................................................. 21
Natural Products in Drug Discovery ........................................................................ 21
Drugs from the Sea ................................................................................................. 22 Marine Cyanobacteria: Source Organisms of Novel Molecules .............................. 24 Mechanism of Action of Bioactive Cyanobacterial Metabolites ............................... 25
Interference with Microtubule Dynamics ........................................................... 25 Inhibition of Histone Deacetylase ..................................................................... 26
Inhibition of Proteases ...................................................................................... 27 Objectives and Specific Aims of the Study .............................................................. 29
2 PROBING THE CHEMICAL SPACE AND ANTIPROLIFERATIVE ACTIVITIES OF CYANOBACTERIAL COLLECTIONS ............................................................... 35
Screening of Cyanobacteria Collections ................................................................. 37 Antiproliferative Assay as Preliminary Screening for Bioactivity ....................... 38
Dereplication using an HPLC-MS Approach ..................................................... 38 Prioritization of Sample Collections .................................................................. 39
Validation of the Dereplication Method ................................................................... 40
General Experimental Procedures ................................................................... 41 Biological Material ............................................................................................ 41
Isolation and Structure Elucidation ......................................................................... 50 Enzyme Inhibition ................................................................................................... 54
Molecular Basis for Elastase Inhibition by Lyngbyastatins and Symplostatins........ 55 Biological Activity Evaluation .................................................................................. 57
Cytoprotective Effects of Symplostatin 5 (1) Against Elastase-Induced Antiproliferation and Apoptosis ...................................................................... 57
Cytoprotective Effects of Symplostatin 5 (1) Against Elastase-Induced Cell Detachment and Morphological Change ....................................................... 59
Attenuation of Global Transcript Changes Induced by Elastase ....................... 62 Conclusion .............................................................................................................. 65 Experimental Methods ............................................................................................ 65
General Experimental Procedures ................................................................... 65
Biological Material ............................................................................................ 66 Extraction and Isolation .................................................................................... 66
Cell detachment and morphology change .................................................. 71 Caspase activation measurement .............................................................. 72 Measurement of sICAM-1 levels ................................................................ 72
Immunoblot analysis of mICAM-1 levels .................................................... 73 Isolation of nuclear and cytoplasmic proteins ............................................. 73
Measurement of IκBα degradation and NF-B p65 translocation............... 74 RNA isolation and reverse transcription ..................................................... 75
4 VERAGUAMIDES A–G: CYTOTOXIC CYCLIC HEXADEPSIPEPTIDES WITH A C8-POLYKETIDE-DERIVED β-HYDROXY ACID MOIETY FROM CETTI BAY, GUAM ................................................................................................................... 100
Experimental Methods .......................................................................................... 108 Biological Material .......................................................................................... 108 Extraction and Isolation .................................................................................. 109 Hydrogenation of 7 ......................................................................................... 110
Acid Hydrolysis of Veraguamides and Enantioselective Analysis ................... 111 Methanolysis of 7 ........................................................................................... 113 Preparation of MTPA Esters of 15 .................................................................. 114
Cell cycle analysis by flow cytometry ....................................................... 115
5 CAYLOBOLIDE B AND AMANTELIDES A AND B: ANTIPROLIFERATIVE POLYKETIDES FROM MARINE CYANOBACTERIA ........................................... 132
Introduction ........................................................................................................... 132 Isolation and Structure Elucidation ....................................................................... 133
Caylobolide B (18) .......................................................................................... 133 Amantelides A and B (19, 20) ......................................................................... 136
Antiproliferative Activity .................................................................................. 139 Elucidation of the Mechanism of Action of Cyanobacterial Polyketides .......... 140
General Experimental Procedures ................................................................. 142 Biological Material .......................................................................................... 143
Extraction and Isolation .................................................................................. 143 Caylobolide B (18) ................................................................................... 143 Amantelides A (19) and B (20) ................................................................. 144
Acetylation of amantelide A (19) .............................................................. 145 ESIMS/MS Fragmentation of Caylobolide B (18) and Amantelide A (19) ....... 145
6 GENERAL CONCLUSION .................................................................................... 160
APPENDIX
A CELL MORPHOLOGY AT 3 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 164
B CELL MORPHOLOGY AT 6 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 165
C CELL MORPHOLOGY AT 12 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 166
D CELL MORPHOLOGY AT 24 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 167
E ICAM1 TRANSCRIPT LEVELS AT 3 h AND 6 h .................................................. 168
F NMR SPECTRA OF ISOLATED COMPOUNDS .................................................. 169
LIST OF REFERENCES ............................................................................................. 257
3-8 Reaction conditions for protease assays ............................................................ 96
3-9 Crystallography data and refinement statistics ................................................... 99
4-1 NMR data for veraguamide A (7) in CDCl3 ....................................................... 121
4-2 NMR data for veraguamide B (8) and veraguamide C (9) in CDCl3 .................. 123
4-3 NMR data for veraguamide D (10) and veraguamide E (11) in CDCl3 .............. 125
4-4 NMR data for veraguamide F (12) in CDCl3 ..................................................... 127
4-5 NMR data for veraguamide G (13) and tetrahydroveraguamide A (14) in CDCl3................................................................................................................ 129
4-6 Antiproliferative activity (IC50, µM) of natural and semisynthetic veraguamides 131
5-1 NMR data of caylobolide B (18) in DMSO-d6 .................................................... 155
5-2 NMR data of amantelide A (19) and amantelide B (20) in DMSO-d6 ................ 157
5-3 Cytotoxic activity (IC50, µM) of the isolated cyanobacterial polyketides (18–21) .................................................................................................................... 159
11
LIST OF FIGURES
Figure page 1-1 Representative examples of natural products that influenced modern
medicine ............................................................................................................. 30
1-2 Marine natural products and analogs that have reached the clinic ..................... 31
1-3 The linear peptides symplostatin 1 and dolastatin 10 are potent antiproliferative agents that disrupt tubulin polymerization ................................. 32
1-4 Largazole is a cyclodepsipeptide prodrug that targets canonical histone deacetylases....................................................................................................... 33
1-5 Representative examples of non-cytotoxic metabolites from marine cyanobacteria that target proteases ................................................................... 34
2-1 Summary of chemical space and bioactivity profiles of Symploca spp. and Phormidium spp. collections ............................................................................... 44
2-2 Representative HPLC-MS profile of the simultaneous monitoring of largazole, dolastatin 10 and symplostatin 1 ........................................................................ 45
2-3 Prioritization scheme of cyanobacteria collections and the corresponding secondary metabolites isolated .......................................................................... 46
3-1 Elastase inhibitors from marine cyanobacteria and the clinically approved human neutrophil elastase inhibitor sivelestat .................................................... 78
3-2 Selectivity profile of Abu-containing cyclic depsipeptides from marine cyanobacteria ..................................................................................................... 79
3-4 Changes in cell viability and caspase activation mediated by elastase and effects of inhibitors .............................................................................................. 81
3-5 Elastase acts as a sheddase and promotes cell morphology change and desquamation ..................................................................................................... 82
3-6 Elastase caused a global change in transcript levels via, in part, an NF-B dependent pathway ............................................................................................ 83
4-1 Structures of veraguamides A–G (7–13) and the semisynthetic tetrahydroveraguamide A (14) .......................................................................... 117
12
4-2 MS/MS fragmentation of veraguamide A (7), veraguamide D (10), and veraguamide E (11) .......................................................................................... 118
4-3 Assignment of absolute configuration of veraguamide A (7) using methanolysis and subsequent Mosher’s analysis ............................................. 119
4-4 Cell cycle analysis of HT29 and HeLa cells treated with varying concentrations of veraguamide D (10) .............................................................. 120
5-1 Caylobolide B (18) and closely related compound caylobolide A ..................... 147
5-2 Key HSQC-TOCSY correlations for caylobolide B (18) .................................... 148
5-3 ESI-MS/MS of caylobolide B (18) ..................................................................... 149
5-4 Amantelides A and B (19, 20) and the semisynthetic derivative peracetylated amantelide A (21) ............................................................................................. 150
5-5 Partial structure of amantelide A (19) derived from NMR experiments in DMSO-d6 .......................................................................................................... 151
5-6 ESI-MS/MS fragmentation of amantelide A (19) ............................................... 152
5-7 Assignment of relative configuration of caylobolide B (18) based on Kishi’s Universal NMR Database (Database 2) ........................................................... 153
5-8 Time-course antiproliferative activities of amantelide A (19) and amphotericin B against cancer cells ....................................................................................... 154
13
LIST OF ABBREVIATIONS
Å Angstrom
[α]20D
Specific optical rotation
Abu 2-amino-2-butenoic acid
Ac Acetyl
Ahp 3-amino-6-hydroxy-2-piperidone
Ala Alanine
ANOVA Analysis of variance
Arg Arginine
APCI/ESI Atmospheric pressure chemical ionization/electrospray ionization
RT-qPCR Reverse transcription followed by quantitative polymerase chain reaction
s singlet
SAR Structure-activity relationship
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Ser Serine
sICAM-1 Soluble intercellular adhesion molecule-1
SIK2 Salt-inducible kinase 2
SV-40 Simian vacuolating virus-40
tR Retention time
TEM Temperature
Thr Threonine
TNF-α Tumor necrosis factor-α
TOCSY Total correlation spectroscopy
µM Micromolar
Val Valine
VCAM Vascular cell adhesion protein
19
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
DRUG DISCOVERY FROM MARINE CYANOBACTERIA SYMPLOCA SPP. AND
PHORMIDIUM SPP.: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY METABOLITES
By
Lilibeth Apo Salvador
May 2013
Chair: Hendrik Luesch Major: Pharmaceutical Sciences – Medicinal Chemistry
Four marine cyanobacteria collections were prioritized for the discovery of novel
secondary metabolites, based on their antiproliferative activity against HT29 human
colorectal adenocarcinoma cells and unique HPLC-MS dereplication profiles.
Bioactivity- and 1H NMR-directed purification yielded the elastase inhibitors
symplostatins 5–10 (1–6), and the antiproliferative agents veraguamides A–G (7–13),
caylobolide B (18), and amantelides A and B (19, 20). Total structure elucidation was
done using 1D and 2D NMR spectroscopy, mass spectrometry and enantioselective
analysis.
Symplostatins 5–10 (1–6) are cyclic depsipeptides bearing the modified amino
acids 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid. Comprehensive
protease profiling of 1 indicated potent and selective elastase inhibition. Structure-
activity relationship (SAR) studies on 1–6, together with the related compounds
lyngbyastatins 4 and 7, identified critical and tunable structural elements. This was
corroborated by the X-ray cocrystal structure of lyngbyastatin 7–porcine pancreatic
elastase. The effects of symplostatin 5 (1) on the downstream cellular effects of
20
elastase was probed using an epithelial lung airway model system. Compound 1
attenuated elastase-mediated receptor activation, proteolytic processing of adhesion
molecule ICAM-1, NF-B activation and global transcriptome changes, leading to
cytoprotection against elastase-induced cell death, detachment and inflammation.
Veraguamides A–G (7–13) are cyclic hexadepsipeptides bearing a C8-polyketide-
and an α-hydroxy acid. Compounds 7–13 together with the semisynthetic derivative
tetrahydroveraguamide A (14) displayed weak to moderate antiproliferative activity
against HeLa cervical carcinoma and HT29 cells, modulated by several sensitive
positions in the veraguamide scaffold. Flow cytometry indicated that veraguamide D
(10) caused a dose-dependent increase in cell populations at sub-G1 and G2.
Caylobolide B (18) and amantelides A and B (19, 20) are structurally-related
polyketides characterized by a polyhydroxylated macrolactone ring bearing an alkyl
pendant side chain. Amantelide A (19) displayed sub-micromolar IC50s against HT29
and HeLa cells, while 18 and 20 showed weaker activity. These cyanobacterial
polyketides potentially exert their cytotoxic effect through interaction with the cell
membrane.
21
CHAPTER 1 GENERAL INTRODUCTION
Natural Products in Drug Discovery
Natural products are small molecules, typically less than 2,000 Da in size,
produced by terrestrial and marine macro- and microorganisms via enzymatically-
assisted biosynthesis. Also referred to as secondary metabolites, these compounds
have indirect and specialized function in the survival of producing organisms, but are
deemed nonessential in primary metabolic pathways. Natural products have evolved out
of functional necessity and are regarded to act as chemical defenses against predators,
parasites or diseases and may also fulfill intrinsic physiological functions for the
producing organisms.1 Similar to primary metabolites, natural products are derived from
ubiquitous precursor molecules such as acetyl-CoA and proteinogenic amino acids, but
differ from the latter by being species specific, rather than prevalent across
organisms.1,2 And while primary and secondary metabolites utilize the same precursor
molecules, higher structural diversity is observed in the latter due to the involvement of
evolutionary processes in the elaboration of biosynthetic enzymes of secondary
metabolites.1 Natural products are distinguished by the presence of a large number of
ring systems, functionalized mainly by oxygen and hydrogen bonding donor moieties.3
An unprecedented feature of secondary metabolites is sterical complexity – possessing
a high number of stereocenters – as these compounds are products of and target three-
dimensional protein systems.3 Comparison of natural products and synthetics indicated
that these compounds occupy complementary chemical spaces.3 Secondary
metabolites are also able to bind to different unrelated molecular targets and are thus,
22
regarded as privileged structures.4 Hence, natural products represent structurally
diverse compounds which have been evolutionary optimized to their molecular targets.
It is for these reasons that man has relied on Nature to discover new drugs. The
earliest documented use of purified secondary metabolites as therapeutics dates back
to the early 19th century, with the discovery of morphine from opium poppy for the
alleviation of pain.5 Several centuries later, natural products continue to be recognized
as a validated source of new drugs and regarded as one of the most successful strategy
in the development of small molecule therapeutics. In a survey of agents introduced for
clinical use from 1980–2010, ~50% are derived from natural products.6 The secondary
metabolite itself may not be the final drug entity, but rather serve as template for the
design of best-in-class small molecule therapeutics. The majority of these are anti-
infectives and anticancer agents.6 Examples of these are the antibiotic penicillin,
antimalarials quinine and artemisinin, and antimitotics vinblastine and paclitaxel (Figure
1-1).
Drugs from the Sea
Terrestrial plants and microorganisms have been the traditional source of natural
products. Technological advancements in underwater exploration have paved the way
for the utilization of marine organisms as source organisms in drug discovery.7 Oceans
cover the majority of the Earth’s surface and harbor rich biodiversity. Each milliliter of
seawater is estimated to contain millions of viruses and bacteria, together with
thousands of fungi and microalgae.8,9 Complex ecological relationship also exists in
these environments, such as endosymbiosis,10 and there is intense competition for
space. These ecological factors can then be expected to impact the secondary
metabolite production in marine organisms.9
23
Six marine natural products or their derivatives have successfully reached the
clinic, and several more at different stages of clinical trials.7,11 Clinically-approved
marine natural products include ziconotide for chronic pain management, antiviral agent
vidarabine conceived based on spongouridine from the sponge Tethya crypta and the
anticancer agents cytarabine, an analog of the spongothymidine also from the sponge
Tethya crypta, ecteinascidin-743 (ET-743), eribulin mesylate inspired by the sponge
compound halichondrin B and brentuximab vedotin designed based on the sea
hare/cyanobacterial metabolite dolastatin 10 (Figure 1-2). Ziconotide is a linear
polycationic peptide ω-conotoxin from the cone snail Conus magus, characterized by 25
amino acid residues including six Cys, that forms three disulfide linkages (Figure 1-2).12
This compound is utilized by the source organism to immobilize its prey, and in
mammalian system targets N-type voltage sensitive calcium channels.12 ET-743 from
the sea squirt Ecteinascidia turbinata was approved for use in the European Union for
refractory soft-tissue sarcoma. The core structure of ET-743 (Figure 1-2) consists of
fused tetrahydroisoquinoline rings that are deemed essential in binding to and
covalently modifying DNA.13 The clinically approved agent eribulin mesylate for breast
cancer treatment is a truncated version of halichondrin B (Figure 1-2).14 Halichondrin B
was initially isolated from the sponge Halichondria okadai, and subsequently from
several more sponge species such as Axinella and Phakellia carteri.15 Halichondrin B
binds to the Vinca domain of tubulin.16 The low yield and high structural complexity of
halinchondrin B, limited its clinical development. Simplified analogs of halichondrin B, as
in the case of eribulin mesylate, showed similar bioactivities as the natural product and
tapped for drug development.14 Brentuximab vedotin is an antibody-drug conjugate
24
clinically approved for Hodgkin’s lymphoma and anaplastic large cell lymphoma.
Brentuximab vedotin consists of a CD33-targeting antibody, a cathepsin cleavable linker
and the drug monomethyl auristatin E (Figure 1-2).17 Monomethyl auristatin E is an
analog of the sea hare/cyanobacterial metabolite dolastatin 10 (Figure 1-2), which also
binds to and disrupt microtubule proteins.18
Marine Cyanobacteria: Source Organisms of Novel Molecules
Cyanobacteria or blue green algae are primitive organisms that have existed for
billions of years, despite lacking any morphological defense structures such as spines,
spicules or shell. Thus, these primitive prokaryotic organisms are thought to have
evolved an arsenal of bioweapons for chemical defense. Since the pioneering studies of
Professor Richard Moore, close to 1,000 secondary metabolites have been isolated
from these organisms.19–22 Marine cyanobacteria utilize polyketide synthases,
nonribosomal peptide synthetases and hybrids of these two biosynthetic pathways to
produce diverse secondary metabolites.23 The majority of these were isolated from the
genera Lyngbya, Oscillatoria, Phormidium and Symploca.
The complex ecological relationship among marine organisms and the production
of secondary metabolites can be observed in cyanobacteria – as the true producers of
bioactive natural products isolated from mollusks and ascidians. Sea hare-derived
dolastatins 10–15 were originally isolated from these herbivores, in low quantities.24 For
example, 1 mg of dolastatin 10 required 2 tons of sea hare.24 A comparable amount of
dolastatin 10 was isolated from a Guamanian Symploca sp., and required only 5 g of
dried cyanobacteria.25 The significantly enriched amounts of dolastatin 10 together with
the isolation of closely related compounds and other sea hare-derived metabolites from
marine cyanobacteria indicated that the true producers are marine cyanobacteria, and
25
are acquired by these herbivores through their diet.26 Cyanobacteria have also been
demonstrated to affect secondary metabolite production of other marine organisms
through endosymbiosis. For example, the production of patellamides by the Didemnidae
family of tunicate is dependent on the obligate cyanobacterial symbiont Prochloron
spp.27 It is then estimated that 35% of marine-derived anticancer agents are products of
cyanobacteria, based on structural similarity.21 The production of natural products from
microbes and microbe interaction with the host organism where the compound was
isolated has emerged as a pivotal concept in natural products discovery.6,8,10
Mechanism of Action of Bioactive Cyanobacterial Metabolites
Marine cyanobacteria are well-documented to be prolific producers of
antiproliferative agents.21 The majority of these are actin and tubulin poisons, with the
marine cyanobacteria Symploca sp. being the source organisms of the potent tubulin
poisons – dolastatin 10 and symplostatin 1.24,25,28,29 In addition, secondary metabolites
with atypical and remarkable mechanisms of action have also been isolated from this
marine cyanobacteria genus, such as largazole which inactivates histone deacetylases
(HDACs).30 Protease inhibition is perhaps the major theme among marine
cyanobacterial metabolites and are commonly encountered in various genera. 21
Interference with Microtubule Dynamics
Dolastatin 10 and symplostatin 1 are closely related linear pentapeptides
characterized by modified amino acids dolaphenine, dolaproline, and dolaisoleucine,
together with Val and a terminal N,N-dimethylated amino acid (Figure 1-3).24,25,28
Dolastatin 10 and symplostatin 1 are differentiated by their N-terminal amino acid
residue, N,N-dimethylVal and N,N-dimethylIle, respectively (Figure 1-3). These
compounds were both demonstrated to have broad spectrum cytotoxicity towards an
26
array of cancer cell lines, with pico- to nanomolar IC50s.29 A dose-dependent increase in
cell populations at G2 and concomitant formation of abnormal mitotic spindles were
observed in symplostatin 1- and dolastatin 10-treated cells. A10 and HeLa cells treated
with symplostatin 1 had disrupted cellular microtubule network as evidenced by
immunostaining using monoclonal β-tubulin antibody.29 Dolastatin 10 and symplostatin
1 were both shown to directly interact with tubulin, with the former demonstrated to
inhibit the binding of radiolabeled Vinca alkaloid.18,29 Molecular docking experiments
proposed that dolastatin 10 binds to a distinct region, close to the Vinca domain and
inhibited tubulin-dependent GTP hydrolysis and nucleotide exchange, processes that
are crucial for tubulin assembly.31
Symplostatin 1 retarded the growth of colon adenocarcinoma 38 and mammary
adenocarcinoma 16/C cells in vivo at dosages of 0.25–1.25 mg/kg.29 Symplostatin 1,
however, caused tissue damage at the site of injection and test animals showed 3–15%
body weight loss, depending on the dosing schedule.29 Dolastatin 10 reached Phase II
clinical trials for prostrate cancer treatment but was discontinued due to observed
peripheral neuropathy among patients and weak therapeutic activity as a single agent.32
Several analogs of dolastatin 10 were synthesized to improve the in vivo potency and
safety profile. On August 2011, FDA approved a dolastatin 10 analog, monomethyl
auristatin E conjugated to a CD33 targeting antibody for clinical use in Hodgkin’s
lymphoma and anaplastic large cell lymphoma treatment.17
Inhibition of Histone Deacetylase
Largazole is a cyclic depsipeptide that is characterized by several unique
structural features such as a 4R-methylthiazoline that is fused to a thiazole ring, and a
3S-hydroxy-7-mercapto-4-heptenoic acid linked to an n-octanoyl group that serves as
27
the prodrug moiety (Figure 1-4).30,33 Largazole requires a protein-assisted hydrolysis to
liberate the active species largazole thiol.34,35 Cytotoxicity testing showed potent activity
against cancer cell lines with superior selectivity index.30 Largazole is the first marine
cyanobacteria-derived agent demonstrated to target HDACs, with superior class I
isoform selectivity.34 The majority of known HDAC inhibitors were derived from
terrestrial microorganisms.36 The reported cocrystal structure of largazole and HDAC8
showed that the “warhead” thiol moiety is present as the thiolate and chelates the Zn2+
catalytic ion in a tetrahedral arrangement.37 This optimum interaction is facilitated by the
rigid depsipeptide macrocyle arising from the fused thiazole-thiazoline rings. NCI60
screening on largazole showed particular susceptibility of colon cancer cell lines to
treatment and an HCT116 xenograft mouse model was adopted.35 In this in vivo animal
model, largazole did not show significant toxic effects and was well-tolerated. Largazole
was able to retard tumor growth in test animals compared to control group, and caused
an upregulation of the cyclin-dependent kinase inhibitor p15 and pro-apoptotic effector
caspase 3, while prosurvival proteins HER2, cyclin D1, IRS-1, and pAKT were
downregulated in tumor sections.35
Inhibition of Proteases
From marine cyanobacteria, several non-cytotoxic metabolites have been
demonstrated to be potent protease inhibitors, particularly targeting the serine
proteases elastase, chymotrypsin and trypsin.21,38,39 The macrocycle of these
cyanobacterial serine protease inhibitors is distinguished by an N-methylated aromatic
amino acid residue, a small nonpolar amino acid such as Val or Ile and a characteristic
ester linkage formed by the condensation of the secondary hydroxy group of Thr. The
Thr residue is also modified on its N-terminus by one to three amino acid residues, and
28
capped by a terminal fatty or polar acid such as butanoic, hexanoic or glyceric acid,
giving rise to the pendant side chain of these cyclic depsipeptides. Lyngbyastatins 4–10
(Figure 1-5) and the related compounds somamide B and molassamide, which bear a
modified Thr residue, 2-amino-2-butenoic acid, adjacent to the Ahp residue on the N-
terminal showed potent elastase inhibition.40–44 A related compound, kempopeptin A,
from a Lyngbya sp. collection bears a Leu residue instead of Abu, and potently inhibited
elastase and chymotrypsin (Figure 1-5).45 Its analog kempopeptin B (Figure 1-5),
bearing a Lys residue, inhibited trypsin.45 Thus, it is evident that the residue on the N-
terminal side of the Ahp moiety modulates the activity of these inhibitors for different
serine proteases.38,41,46 These serine protease inhibitors have been demonstrated to
function as digestion inhibitors and feeding deterrents of herbivores, fishes and urchins
and may also possibly modulate the biosynthesis of other cyanobacterial secondary
metabolites.47–49
Statine (γ-amino-β-hydroxy acid)-containing modified linear peptides from marine
cyanobacteria on the other hand, are potent inhibitors of aspartic proteases.
Grassystatins A–C (Figure 1-5) isolated from a Floridian Lyngbya cf. confervoides
selectively inhibited the aspartic protease cathepsin E at pico- to nanomolar
concentrations and concurrently prevented cathepsin E-mediated antigen presentation
of dendritic cells.50 These compounds bear a leucine derived statine unit (4-amino-3-
hydroxy-6-methylheptanoic acid), critical for cathepsin inhibition while residues adjacent
to this moiety confer selectivity towards cathepsin E. The related linear peptide,
tasiamide B (Figure 1-5),51,52 on the other hand bears a phenylalanine-derived statine
moiety and has been demonstrated to inhibit β-site APP Cleaving Enzyme Type 1
29
(BACE1), an enzyme which has been shown to be central to the formation of amyloid
plaques and related to the progression of Alzheimer’s disease.53 Tasiamide B served as
the template in the design of new inhibitors of BACE1 with potent cellular activity and in
vivo efficacy.53
Objectives and Specific Aims of the Study
With marine cyanobacteria being validated source organisms of structurally and
pharmacologically diverse secondary metabolites, we aimed to utilize novel chemical
entities from these organisms for potential biomedical applications as antitumor agents
and modulators of elastase-mediated pathologies. This study focused on the under-
explored marine cyanobacteria genera of Symploca and Phormidium, which yielded
several of the best-in-class antitumor agents. This study aimed to:
1. Prioritize collections of Symploca and Phormidium using a preliminary profiling of bioactivity and chemical space
2. Perform a bioactivity-guided purification on cyanobacterial collections which demonstrated antiproliferative activity to isolate the bioactive constituent(s)
3. Perform a 1H NMR-guided purification to discover novel secondary metabolites from non-cytotoxic cyanobacterial collections
4. Determine the structure of isolated compounds from prioritized collections using combinations of spectroscopic techniques such as 1D and 2D NMR spectroscopy and mass spectrometry
5. Elucidate the biological activity and mechanisms of action of identified cyanobacterial secondary metabolites in mammalian cellular systems.
30
Figure 1-1. Representative examples of natural products that influenced modern
medicine.
31
Figure 1-2. Marine natural products and analogs that have reached the clinic.
32
Figure 1-3. The linear peptides symplostatin 1 and dolastatin 10 are potent
antiproliferative agents that disrupt tubulin polymerization.
33
Figure 1-4. Largazole is a cyclodepsipeptide prodrug that targets canonical histone
deacetylases.
34
Figure 1-5. Representative examples of non-cytotoxic metabolites from marine
cyanobacteria that target proteases.
35
CHAPTER 2 PROBING THE CHEMICAL SPACE AND ANTIPROLIFERATIVE ACTIVITIES OF
CYANOBACTERIAL COLLECTIONS*
Introduction
Filamentous marine cyanobacteria are a validated source of antiproliferative
agents, having yielded several of the best-in-class inhibitors of malignancies.20,21
Cytotoxins from marine cyanobacteria also display not just a variety in structure, but
mechanisms of action as well. Actin-targeting agents, with sub-nanomolar IC50s against
cancer cells, include lyngbyabellins,54–56 dolastatin 1157,58 and hectochlorin.59 The
marine cyanobacteria Lyngbya spp. afforded the cyclic depsipeptides apratoxins A–G
that are also potent cytotoxins,60–64 with apratoxin A preventing cotranslational
translocation leading to downregulation of receptors and growth factor ligands.65,66 The
marine cyanobacteria Symploca spp. and Phormidium spp. yielded several modified
linear peptides that target tubulin polymerization.25,28,29,67,68 The most potent among
these are the related dolastatin 1018 and symplostatin 1,29 with the former serving as the
template for the design of the clinically approved anti-Hodgkin’s and anaplastic large
cell lymphoma drug brentuximab vedotin. Another novel agent from Symploca sp. is the
histone deacetylase inhibitor largazole, which displayed potent activity in preclinical
evaluations.33 With the abundance of novel antitumor agents from marine
cyanobacteria, it is thus attractive to employ a primary screening of antiproliferative
activity against cancer cells for crude extracts. Measurements of cell viability can be
done using colorimetric or fluorometric reagents to measure cellular metabolism, protein
activity and interactions, membrane permeabilization and cellular respiration.36
*Reproduced with permission from J. Nat. Prod., submitted for publication. Unpublished work copyright
2013 American Chemical Society.
36
The isolation of a large number of antiproliferative agents from marine
cyanobacteria, however, also increases the possibility of reisolating known compounds
as bioactive components. Thus, it is advantageous to employ a screen of the chemical
space as well. Several dereplication methods – identification of known metabolites from
sample collections with the least effort and resources – have been developed for both
terrestrial and marine cyanobacteria, employing UV spectroscopy and mass
spectrometry.
To distinguish known bioactive compounds in a screen for phorbol debutyrate
receptor binding activity, a HPLC-UV dereplication was utilized.69 Members of the
aplysiatoxin class of compound are known to be phorbol debutyrate receptor binders,
and comparison of the retention time and UV profile of authentic debromoaplysiatoxin
allowed the identification of this compound as the active principle for several Lyngbya
majuscula collections.69 This method also accounted for debromoaplysiatoxin as the
bioactive constituent of seagrasses and macroalgae, possibly due to cyanobacterial
contamination.69 More compound-specific techniques emerged with the development of
new technologies in mass spectrometry such as MALDI-TOF and ESIMS. The initial
utilization of MALDI-TOF for dereplication was a serendipitous discovery, but
nonetheless, demonstrated the presence of microcystins, micropeptin and
anabaenopeptolin from collections of Microcystis, Anabaena and Oscillatoria.70 The
application of MALDI-TOF for dereplication has been extended to determine the spatial
distribution of secondary metabolites in cyanobacteria themselves and other marine
organisms, in addition to identification.71 Structure determination of nonribosomal
peptides have also greatly benefited from mass spectrometry, with tandem mass
37
spectrometry yielding the identity of these compounds via characteristic fragmentation
pattern. Recently introduced is comparative dereplication using tandem mass
spectrometry and spectral alignment algorithms to identify identical compounds and
related analogs.72 The requirement for minimal material to perform mass spectrometry
analysis and its amenability to high-throughput format makes this method an attractive
choice for dereplication.
Here, an HPLC-MS dereplication method utilizing multiple reaction monitoring
was developed to improve the resolution of known cytotoxins in collections of marine
cyanobacteria Symploca and Phormidium. This, together with antiproliferative screening
against HT29 colorectal adenocarcinoma cells, was utilized to prioritize cyanobacterial
collections for further studies.
Screening of Cyanobacteria Collections
A total of 38 marine cyanobacteria samples were collected in Florida, Guam and
the US Virgin Islands from 2007–2009. These collections were mainly Symploca spp.,
Phormidium spp. and several taxonomically unidentified organisms characterized by
puffy ball gross morphology characteristic for Symploca spp. Collected organisms were
lyophilized and extracted with either CH2Cl2–MeOH (1:1) or EtOAc–MeOH (1:1) to yield
the nonpolar extracts. These extracts were further subjected to a C18 solid phase
extraction (SPE) cleanup using a MeOH–H2O elution. Initial elution using 25% MeOH
removed the majority of the salts and ensured minimal non-specific bioactivity and
interference in HPLC-MS arising from these polar compounds. The fraction collected
from 100% MeOH elution was tested for antiproliferative activity against HT29 colorectal
adenocarcinoma cells and concurrently profiled by HPLC-MS.
38
Antiproliferative Assay as Preliminary Screening for Bioactivity
Antiproliferative activity was assessed based on the fractional survival of HT29
cancer cells, detected using the MTT reagent. Extracts which caused < 60% survival of
HT29 cells were considered bioactive at the specified concentration. From the 38
samples screened for antiproliferative activity, only two sample collections were inactive
at all concentrations tested (Figure 2-1A). Thirteen sample collections exhibited
moderate antiproliferative activity against HT29 cells at concentrations of 1,000 and
10,000 ng/mL (Figure 2-1A). The remaining 60% of the screened cyanobacteria
collections exhibited antiproliferative activity at concentrations of 10 and 100 ng/mL
(Figure 2-1A). With the large number of cyanobacterial collections showing
antiproliferative activity, additional information for prioritization of sample collections are
needed. Also, with potent cytotoxins such as dolastatin 10, symplostatin 1 and largazole
being produced by Symploca spp. and Phormidium spp. collections, determination of
the contribution of these known compounds to the bioactivity should be assessed at an
early stage of the discovery process.
Dereplication using an HPLC-MS Approach
The dereplication method for the known compounds largazole, dolastatin 10 and
symplostatin 1, consisted of a gradient HPLC run using CH3CN–H2O (+ 0.1% HCOOH)
and multiple reaction monitoring (MRM) as MS detection mode. This allowed for
sensitive, specific and high-throughput format for dereplication of previously isolated
metabolites from Symploca spp. and Phormidium spp. sample collections. The MRM
mode relies on the detection of both the parent ion mass (Q1) and a specific daughter
ion resulting from fragmentation (Q3), giving a significant reduction in background,
improvement in signal-to-noise ratio and limits of detection. This dereplication format
39
permitted automation, short run times per sample (< 20 min) and simultaneous
monitoring of largazole, symplostatin 1 and dolastatin 10 (Figure 2-2). This method does
not have specific structural requirements and can be done using commonly available
mass spectrometers. However, authentic standards are needed for optimization of the
HPLC-MS parameters. Since MRM is also a compound-specific detection, no
information on the presence of related congeners may be derived using this method.
Based on the HPLC-MS dereplication, the majority of the sample collections with
antiproliferative activity at 10 and 100 ng/mL contained combinations of dolastatin 10,
largazole or symplostatin 1 (Figure 2-1A, B). Except for one sample collection, all other
bioactive cyanobacterial collection at concentration of 10 ng/mL contained these three
antiproliferative agents at biologically relevant concentrations (Figure 2-1A). Extracts
containing symplostatin 1 or dolastatin 10 alone or lower concentrations of these
metabolites in combination showed activity at a higher concentration of 100 ng/mL.
Interestingly, largazole was consistently detected in combination with dolastatin 10 and
Dolastatin 10: colorless, amorphous solid; 1H NMR spectrum is identical to that of
an authentic sample,28 see Appendix F; LRESIMS m/z 785.6 [M + H]+.
44
Figure 2-1. Summary of chemical space and bioactivity profiles of Symploca spp. and
Phormidium spp. collections. (A) The majority of the cyanobacteria collections displayed antiproliferative activity against HT29 human colorectal adenocarcinoma cells as assessed using the MTT reagent. The majority of potent bioactive extracts showed combinations of dolastatin 10, largazole and symplostatin 1. (B) Distribution of the three known antiproliferative agents in profiled cyanobacterial collections.
45
Figure 2-2. Representative HPLC-MS profile of the simultaneous monitoring of
largazole, dolastatin 10 and symplostatin 1.
46
Figure 2-3. Prioritization scheme of cyanobacteria collections and the corresponding
secondary metabolites isolated.
47
Table 2-1. Antiproliferative activity (IC50, nM) of known Symploca sp. metabolitesa
amino-6-hydroxy-2-piperidone (Ahp) and a highly variable pendant side chain.21,38,39
The isolation of over 100 members of this group of cyanobacterial metabolites, together
with antiproteolytic activity data primarily against the serine proteases elastase,
chymotrypsin, and trypsin, has provided insights into the importance of the Ahp moiety
and the adjacent residue on its N-terminal side, which confer selectivity.38,46 The role of
these moieties was elegantly demonstrated through X-ray cocrystallization of A90720A–
trypsin and scyptolin–elastase complexes.73,74 Not found in terrestrial or freshwater
cyanobacteria is the 2-amino-2-butenoic acid (Abu) moiety, which is hypothesized to
contribute to higher potency.41 The majority of the marine-derived cyanobacterial
metabolites in this class bears the Abu moiety adjacent to the Ahp residue. These
compounds, which include lyngbyastatins 4–10, showed potent antiproteolytic activity
against elastase with low nanomolar IC50s, and are perhaps among the most potent
small molecule inhibitors of elastase.40–42 Therefore, these small molecules are
attractive therapeutics for elastase-mediated pathologies, as well as molecular probes
to elucidate critical interactions for effective enzyme inhibition and to interrogate specific
Reproduced with permission from Salvador, L.A.; Taori, K.; Biggs, J. S.; Jakoncic, J.; Ostrov, D. A.; Paul, V. J.; Luesch, H. J. Med. Chem. 2013, 56, 1276–1290. Copyright 2013 American Chemical Society.
49
intracellular and extracellular molecular targets of elastase. However, limited SAR and a
lack of information beyond enzymatic assay data hinder further development of these
compounds as small molecule therapeutics.
Elastase is a broad-spectrum enzyme that preferentially cleaves on the C-
terminus of small hydrophobic amino acids such as Gly, Ala, and Val and degrades
collagen, elastin, fibronectin and components of the extracellular matrix.75 Elastase has
been linked to several diseases involving chronic inflammatory conditions such as
chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, and systemic
inflammatory response syndrome, where there is a protease–antiprotease
imbalance.75,76 The canonical role of elastase in degrading the extracellular matrix has
been documented, as have the stimulating effects of elastase on signaling pathways
through direct or indirect receptor activation. The resulting changes in transcript and
protein levels have been linked to possible disease progression.77 Current therapies for
these diseases are aimed at alleviating the symptoms but not disease progression,
which may be related to the role of elastase.78 Sivelestat is the only approved drug
targeting elastase;79 however, clinical approval in the United States and Europe has
been stalled due to marginal clinical effects.80 Finding new small molecule therapeutics
for COPD is of importance since the disease has been recognized as a major public
health problem and the fourth leading cause of death worldwide.81 Intratracheal
instillation of elastase in animal models showed changes such as enlargement of
alveolar space, thickening of alveolar septae and mucus hypersecretion, comparable to
clinical observations.82 This enzyme has also been implicated in cell death,
transcriptional and translational modulation and processing of pro-inflammatory
50
cytokines, chemokines and adhesion molecules, which also dictate downstream cellular
effects.76 Development of elastase inhibitors has been particularly challenging because
of overlapping functions of elastase with those of other serine proteases, as well as
limited information on the role of elastase in the progression of disease. Here we aimed
to determine the potential utility of symplostatin 5 (1) and related compounds in
alleviating the cellular effects downstream of elastase release and compared the cellular
potency to sivelestat.
Isolation and Structure Elucidation
The lyophilized red cyanobacterium collected from Cetti Bay, Guam was
extracted with EtOAc–MeOH (1:1) to afford the nonpolar extract. Liquid-liquid
partitioning of the nonpolar extract yielded the hexanes-, n-BuOH- and H2O-soluble
fractions. The 1H NMR spectrum of the n-BuOH fraction showed characteristic
resonances for peptides and modified peptides. This fraction was further purified by
silica column chromatography and reversed-phase HPLC to give six new Ahp-
qPCR after reverse transcription (RT-qPCR) was performed on a 25 µL reaction
solution containing a 1.5 µL aliquot of cDNA, 12.5 µL TaqMan® gene expression master
mix, 1.25 µL of 20 TaqMan® gene expression assay mix and 9.25 µL RNase-free
water. qPCR was carried out on an ABI 7300 sequence detection system using the
thermocycler program: 2 min at 50 °C, 10 min at 95 °C, and 15 s at 95 °C (40 cycles)
and 1 min at 60 °C. Each experiment was performed in triplicate. IL1A
(Hs00174092_m1), IL1B (Hs01555410_m1), and IL8 (Hs00174103_m1) were used as
target genes, while GAPDH (Hs02758991_g1) was used as endogenous control.
76
Graphs and data analysis were performed using the Prism® software and analyzed
using ANOVA followed by Dunnett’s t-test.
Transcriptome profiling
RNA was analyzed using a NanoDrop Spectrophotometer and Agilent 2100
Bioanalyzer to determine the RNA concentration and quality, respectively. RNA
samples were processed using the GeneChip® 3’ IVT Express kit (Affymetrix, Santa
Clara, CA) according to the manufacturer’s instruction. In brief, 250 ng RNA were used
for cDNA synthesis by reverse transcription and the cDNA was utilized as a template for
the biotin-labeled RNA prepared by in vitro transcription reaction. The labeled RNA was
further purified, fragmented and hybridized with rotation at 45 °C for 16 h to the
Affymetrix GeneChip® Human Genome U133 plus 2.0 arrays. The arrays were washed
and stained using the GeneChip® Hybridization Wash and Stain kit on an Affymetrix
Fluidics Station 450. The chips were scanned using a GeneChip® 7G Scanner. Analysis
of the microarray data was done according to the reported method.35 Raw data was
normalized using the Robust Multichip Analysis approach and statistical analysis was
done using the Bioconductor statistical software and R program. The probe set’s
detection call was estimated using the Wilcoxon signed rank-based algorithm. Probe
sets that are absent in all of the study samples were removed from further analyses.
Differential expression analysis was performed using a linear modeling approach and
the empirical Bayes statistics as implemented in the limma package of the R software.
The P values obtained were controlled for multiple testing (false discovery rate) using
the Benjamini-Hochberg method. P value and fold induction were calculated.
Differentially expressed transcripts were ranked by P values, and P < 0.05 and fold
induction >1.5 were considered at a statistically significant level. Hierarchical clustering
77
of the data was computed on log-transformed and normalized data by using complete
linkage and Pearson correlation distances. Computation and visualization were done
with R packages. Gene ontology was performed using the DAVID Bioinformatics
Resources 6.7.118,119 The transcriptome data is deposited in NCBI’s Gene Expression
Omnibus with accession number GSE41600.
78
Figure 3-1. Elastase inhibitors from marine cyanobacteria and the clinically approved
human neutrophil elastase inhibitor sivelestat.
79
Figure 3-2. Selectivity profile of Abu-containing cyclic depsipeptides from marine
cyanobacteria. (A) Screening of lyngbyastatin 7 (10 µM) against a panel of 68 proteases. (B) Selectivity profiling for symplostatin 5 (1) on a panel of 26 serine proteases. Assays were performed by Reaction Biology, Inc.
(Fo–Fc) plot for lyngbyastatin 7. (B) Comparison of lyngbyastatin 7 (yellow, PDB ID 4GVU) and scyptolin (white, PDB ID 1OKX) binding to elastase. (C) Comparison of lyngbyastatin 7 (yellow) and FR901277 (green, PDB ID 1QR3) binding to elastase. (D) Ligplot of the lyngbyastatin 7–porcine pancreatic elastase complex. The Abu moiety serves as the key residue for elastase inhibition. Chain designations are (A) elastase, (B) lyngbyastatin 7, (C) H2O. (E) Proposed CH-π interaction between the catalytic Ser203 and the Abu moiety (F) Network of inter- and intramolecular hydrogen bonding interaction in lyngbyastatin 7 mediated by a water molecule. Data courtesy of Ms. Kanchan Taori, Dr. Jean Jakoncic and Dr. David A. Ostrov.
81
Figure 3-4. Changes in cell viability and caspase activation mediated by elastase and
effects of inhibitors. (A) Elastase displayed both time- and dose-dependent decrease in cell viability, with substantial changes at 12–24 h. (B) Symplostatin 5 (1) attenuated the antiproliferative effects of elastase. (C) Sivelestat and the caspase 3 inhibitor Z-D(OMe)E(OMe)VD(OMe)-FMK also partially protected against the antiproliferative effects of elastase. (D) Symplostatin 5 (1) did not show any significant antiproliferative effect on BEAS-2B cells at 24 h. (E) Treatment with 100 nM elastase caused a time-dependent increase in caspase activation which was abrogated by the caspase 3 inhibitor. (F) Incubation of BEAS-2B cells with elastase for 24 h caused a dose-dependent increase in caspase 3/7 activity. Symplostatin 5 (1) attenuated the potency and efficacy of elastase to activate distal caspases. Data are presented as mean ± SEM (n = 2).
82
Figure 3-5. Elastase acts as a sheddase and promotes cell morphology change and
desquamation. (A) Elastase caused cell rounding after incubation for 3 h. Cotreatment with 10 µM symplostatin 5 (1) or sivelestat prevented this effect
of elastase (10 magnification). (B) Significant increase in cell detachment was observed after 12 h of incubation with elastase, which was abrogated by both symplostatin 5 (1) and sivelestat. (C) Levels of mICAM-1 in whole cell lysates in elastase-treated and elastase-inhibitor cotreated cells as assessed by immunoblotting (D) sICAM-1 in culture supernatants of elastase-treated and elastase-inhibitor cotreated cells.Data are presented as mean + SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 compared to HNE-treated control cells using ANOVA, Dunnett’s t-test (n = 3).
83
Figure 3-6. Elastase caused a global change in transcript levels via, in part, an NF-B dependent pathway. (A) Symplostatin 5 (1) dose-dependently inhibited
elastase-induced IB degradation and p65 nuclear translocation at 3 h of cotreatment. (B) Heat map of differentially regulated transcripts by elastase with or without symplostatin 5 (1) cotreatment. Global transcriptome profiling (Affymetrix GeneChip® Human Genome U133 plus 2.0 arrays) was carried out using duplicate biological samples. (C) Validation of the microarray analysis using RT-qPCR. Data are presented as mean + SEM for A and mean + SD for C, * P < 0.05, ** P < 0.01, *** P < 0.001 compared to HNE-treated control cells using ANOVA, Dunnett’s t-test (n = 3).
84
Table 3-1. NMR data of symplostatin 5 (1) and symplostatin 8 (4) in DMSO-d6
Symplostatin 5 Symplostatin 8 unit C/H no δC
a δH (J in Hz)b COSYb HMBCb δCa δH (J in Hz)b
Ile 1 170.0,C e
2 54.0, CH 4.89, br NH 1 54.0, CH 4.87, d
(11.0) 3 37.5, CH 1.86, m H3-6 37.0, CH 1.86, m 4a 25.8, CH2 1.30, m H-4b, H3-5 2, 3 26.0, CH2 1.29, m 4b 1.11, m H-4a, H3-5 2, 3 1.12, m 5 11.2, CH3 0.92, t (7.2) H-4a, H-4b 3 11.4, CH3 0.91, t (7.3) 6 14.1, CH3 0.71, d (7.0) H-3 2, 3 14.3, CH3 0.70, d (6.8) NH 7.40, br H-2 7.40, br N-Me-Phec/ 1 172.7, C
e N-Me-Tyrd 2 60.2, CH 5.00, br H-3a, H-3b 1 60.7, CH 4.90, d
(10.6) 3a 33.4, CH2 3.23, brd
(–13.5) H-2, H-3b 4,5/9 32.7, CH2 3.11, d
(–14.2) 3b 2.84, m H-2, H-3a 5/9 2.70, dd
(–14.2, 10.6) 4 137.9, C
e 5/9 129.4, CH 7.23, d (7.5) H-6 130.3, CH 6.99, d (7.8) 6/8 128.4, CH 7.39, m H-5, H-7 4 115.2, CH 6.77, d (7.8) 7 126.5, CH 7.30, m H-6
e OH 8.13, br s N-Me 30.1, CH3 2.77, s 2, 1 (Phe) 30.3, CH3 2.75, s Phe 1 170.3, C 2 49.6, CH 4.70, dd
(11.4,4.7) H-3a, H-3b 1, 2 (Ahp) 50.0, CH 4.73, m
3a 34.8, CH2 2.84, dd (–14.7,11.4)
H-2, H-3b 4 34.6, CH2 2.87, dd (–14.2, 11.3)
3b 1.68, m H-2, H-3a 4 1.81, m 4 136.5, C
e
85
Table 3-1. Continued
Symplostatin 5 Symplostatin 8 unit C/H no δC
a δH (J in Hz)b COSYb HMBCb δCa δH (J in Hz)b
5/9 129.2, CH 6.77, d (7.5) H-6 7 129.3, CH 6.84, d (7.3) 6 127.6, CH 7.18, m H-5, H-7 4 127.7, CH 7.19, m 7 126.1, CH 7.15, m H-6 126.2, CH 7.15, m Ahp 2 168.7, C
e 3 47.8, CH 3.75, m H-4a, H-4b,
NH 2 48.0, CH 3.79, m
4a 21.7, CH2 2.38, m H-3, H-4b, H-5a
21.9, CH2 2.41, m
4b 1.56, m H-3, H-4a 1.58, m 5a 29.0, CH2 1.68, m H-4a, H-5b,
H-6 29.2, CH2 1.71, m
5b 1.50, m H-5a, H-6 1.56, m 6 73.4, CH 5.03, br s H-5a, H-5b,
OH 2 73.5, CH 5.07, m
OH 6.05, s H-6 6.07, br s NH 7.34, br H-3 7.33, br Abu 1 162.9, C
e 2 55.1, CH 4.67, br NH 55.2, CH 4.67, m 3 71.5, CH 5.52, br s H3-4
e 5.53, brs 4 17.5, CH3 1.22, d (6.5) H-3 2 17.7, CH3 1.22, d (6.2) NH 8.18, br s H-2 7.70, br s Val 1 172.2, C
e 2 56.4, CH 4.47, t (7.2) NH 1 56.7, CH 4.47, t (7.3) 3 30.7, CH 2.09, m H3-4, H3-5 30.6, CH 2.09, m
86
Table 3-1. Continued
Symplostatin 5 Symplostatin 8 unit C/H no δC
a δH (J in Hz)b COSYb HMBCb δCa δH (J in Hz)b
4 18.9, CH3 0.88, d (6.7) H-3 1 19.1, CH3 0.88, d (7.0) 5 17.5, CH3 0.83, d (6.7) H-3 1 17.5, CH3 0.83, d (7.0) NH 7.71, br s H-2 7.72, br s 2-O-CH3 1 168.9, C
e Glyceric Acid
2 79.9, CH 3.98, dd (7.4,3.4)
H-3a, H-3b 80.2, CH 3.97, dd (7.3, 3.4)
3a 66.1, CH2 3.90, dd (–10.8,3.4)
H-2, H-3b 66.2, CH2 3.89, dd (–10.7, 3.3)
3b 3.73, m H-2, H-3a 3.72, m OCH3 57.1, CH3 3.33g 2 57.3, CH3 3.32g aDeduced from HSQC and HMBC, 600 MHz. b600 MHz. cRefers to symplostatin 5 (1). dRefers to symplostatin 8 (4). eNot determined, predicted to have comparable chemical shifts based on highly homologous structures. fNo correlation observed from HMBC. gOverlapping with residual water.
87
Table 3-2. NMR data of symplostatin 6 (2) and symplostatin 9 (5) in DMSO-d6
Symplostatin 6 Symplostatin 9 unit C/H no δC
a δH (J in Hz)b δC a δH (J in Hz)b
Val 1 170.2, C e
2 56.0, CH 4.68, m 55.7, CH 4.70, m 3 30.7, CH 2.00, m 30.3, CH 2.08, m 4 19.0, CH3 0.89, d (6.8) 18.8, CH3 0.88, d (6.8) 5 17.1, CH3 0.76, d (6.8) 17.0, CH3 0.75, d (6.8) NH 7.51, d (8.8) 7.48, d (8.1) N-Me-Phec/ 1 169.3, C
e N-Me-Tyrd 2 60.3, CH 5.01, d (11.3) 60.5, CH 4.89, d (10.9) 3a 33.4, CH2 3.23, m 32.3, CH2 3.10, d (–14.2) 3b 2.85, m 2.71, m 4 137.9, C
e 5/9 129.4, CH 7.23, d (7.9) 130.1, CH 6.99, d (8.4) 6/8 128.4, CH 7.39, m 114.8, CH 6.77, d (8.4) 7 126.5, CH 7.30, m N-Me 30.2, CH3 2.79, s 29.9, CH3 2.76, s OH Phe 1 170.3, C
e 2 49.7, CH 4.71, dd (11.8,4.4) 49.8, CH 4.73, dd (11.4, 3.8) 3a 34.9, CH2 2.85, m 34.9, CH2 2.87, dd (–14.4, 11.4) 3b 1.69, m 1.81, dd (–14.4, 3.8) 4 136.5, C
e 5/9 129.1, CH 6.77, d (7.5) 129.1, CH 6.84, d (7.6) 6 127.6, CH 7.18, m 127.4, CH 7.19, m 7 126.0, CH 7.14, m 126.0, CH 7.14, m Ahp 2 168.7, C
e 3 47.9, CH 3.76, m 47.7, CH 3.78, m 4a 21.6, CH2 2.42, m 21.5, CH2 2.42, m 4b 1.56, m 1.57, m
88
Table 3-2. Continued
Symplostatin 6 Symplostatin 9 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
5a 29.1, CH2 1.70, m 29.0, CH2 1.71, m 5b 1.51, m 1.56, m 6 73.5, CH 5.04, s 73.3, CH 5.07, s OH 6.10, br s NH 7.23, br s 7.23, br s Abu 1 163.0, C
e 2 129.9, C
e 3 131.6,CH 6.51, q (7.0) 131.6, CH 6.51, q (7.1) 4 12.9, CH3 1.49, d (7.0) 12.9, CH3 1.49, d (7.1) NH 9.20, br s Thr 1
e
e 2 55.2, CH 4.65, m 54.8, CH 4.65, m 3 71.4, CH 5.54, br s 71.2, CH 5.54, br s 4 17.6, CH3 1.23, d (6.3) 17.4, CH3 1.23, d (6.4) NH 7.80, br 2 7.70, br s Val 1 171.7, C
e 2 56.5, CH 4.47, m 56.3, CH 4.46, m 3 30.4, CH 2.09, m 30.3, CH 2.09, m 4 19.0, CH3 0.89, d (6.3) 18.8, CH3 0.88, d (6.8) 5 17.5,CH3 0.82, d (6.7) 17.3, CH3 0.82, d (6.8) NH 8.13 br s 8.18, br s 2-O-CH3 1 168.9, C
e Glyceric Acid 2 80.0, CH 3.98, dd (7.3,3.4) 79.8, CH 3.98, dd (7.3, 3.4) 3a 66.0, CH2 3.90, dd (–11.1, 3.4) 65.8, CH2 3.89, dd (–10.9, 3.4) 3b 3.74, dd (–11.1, 7.3) 3.73, dd (–10.9,7.3) OCH3 57.4, CH3 3.32f 57.0, CH3 3.33f aDeduced from HSQC, 600 MHz. b600 MHz. cRefers to symplostatin 6 (2). dRefers to symplostatin 9 (5). eNot determined, predicted to have comparable chemical shifts based on highly homologous structures. fOverlapping with residual water.
89
Table 3-3. NMR data of symplostatin 7 (3) and symplostatin 10 (6) in DMSO-d6
Symplostatin 7 Symplostatin 10 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
Ile 1 e
e 2 54.1, CH 4.88, br d 53.9, CH 4.88, m 3 37.0, CH 1.88, m 36.9, CH 1.86, m 4a 26.0, CH2 1.30, m 25.8, CH2 1.29, m 4b 1.12, m 1.12, m 5 11.3, CH3 0.92, t (7.2) 11.9, CH3 0.91, t (7.4) 6 14.4, CH3 0.71, d (6.7) 14.2, CH3 0.70, d (6.8) NH 7.44, br s 7.38, br s N-Me-Phec/ 1
e e
N-Me-Tyrd 2 60.3, CH 5.01, br d 60.6, CH 4.90, m 3a 33.6,CH2 3.24, br d (–13.4) 32.5, CH2 3.11, d (–14.2) 3b 2.85, m 2.70, dd (–14.2,11.8) 4
e e
5/9 129.5, CH 7.24, d (7.6) 130.2, CH 6.99, d (8.3) 6/8 128.4, CH 7.40, m 115.1, CH 6.76, d (8.3) 7 126.6, CH 7.31, m N-Me 30.2, CH3 2.79, s 30.0, CH3 2.74, s OH 9.31, br s Phe 1
e e
2 49.9, CH 4.72,m 49.9, CH 4.72, dd (11.4,4.8) 3a 35.0, CH2 2.85, m 35.0, CH2 2.87, (–14.3,12.3) 3b 1.69, m 1.79, m 4
e e
5/9 129.2, CH 6.83, d (7.4) 129.2, CH 6.83, d (7.2) 6 127.6, CH 7.19, m 127.6, CH 7.19, m 7 126.1, CH 7.16, m 126.0, CH 7.14, m Ahp 2
e e
3 47.9, CH 3.77, m 47.9, CH 3.77, m 4a 21.8, CH2 2.39, m 21.8, CH2 2.39, m
90
Table 3-3. Continued
Symplostatin 7 Symplostatin 10 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
4b 1.57, m 1.57, m 5a 29.1, CH2 1.71, m 29.1, CH2 1.71, m 5b 1.55, m 1.55, m 6 73.5, CH 5.06, s 73.5, CH 5.06, br s OH 6.04, s 6.03, s NH 7.35, br s 7.35, br s Abu 1
e e
2 e
e 3 131.4, CH 6.48, q (7.1) 131.4, CH 6.48, q (7.2) 4 12.8, CH3 1.46, d (7.1) 12.8, CH3 1.46, d (7.2) NH 9.22, br s 9.22, br s Thr 1
e e
2 55.0, CH 4.68, m 55.0, CH 4.68, m 3 71.6, CH 5.52, br s 71.6, CH 5.52, br s 4 17.5, CH3 1.21, d (6.5) 17.5, CH3 1.21, d (6.3) NH 8.24, br s 8.19, br s Ile 1
e e
2 55.8, CH 4.48, m 55.8, CH 4.48, m 3 36.9, CH 1.86, m 36.9, CH 1.85, m 4a 23.7, CH2 1.43, m 23.7, CH2 1.43, m 4b 1.06, m 1.06, m 5 10.6, CH3 0.80, t (7.4) 10.6, CH3 0.80, t (7.4) 6 15.0, CH3 0.85, d (6.5) 15.0, CH3 0.85, d (6.8) NH 7.75, br s 7.73, br s 2-O-CH3 1
OCH3 57.1, CH3 3.31f 57.1, CH3 3.31 f aDeduced from HSQC, 600 MHz. b600 MHz. cRefers to symplostatin 7 (3). dRefers to symplostatin 10 (6). eNot determined, predicted to have comparable chemical shifts based on highly homologous structures. fOverlapping with residual water.
92
Table 3-4. Antiproteolytic activity of Abu-containing cyclic depsipeptides from marine cyanobacteriaa
1563075_s_at NA NA 2.21 59c 224917_at MIR21 MicroRNA 21 2.08 43c aRelative to control, P < 0.05. bIn response to inhibitor cotreatment. cSignificant difference with inhibitor treatment, P < 0.05.
94
Table 3-6. Relevant genes involved in NOD- and MAPK- signaling pathways significantly modulated by elastase
205207_at IL6 Interleukin 6 2.26 22 1569540_at NLK Nemo-like kinase 2.23 49 239409_at RAP1A RAP1A, member of RAS
oncogene family 2.21 53c
230337_at SOS1 Son of sevenless homolog 1 1.89 46c 210118_at IL1A Interleukin 1A 1.85 34 1565889_at TAB2 Mitogen-activated kinase
kinase 7 interacting protein 1.83 42
211506_at IL8 Interleukin 8 1.53 22 aRelative to control, P < 0.05. bIn response to inhibitor cotreatment. cSignificant difference with inhibitor treatment, P < 0.05.
95
Table 3-7. Symplostatin 5 (1)-inducible genes potentially independent of elastasea
Probe ID Symbol Annotation P-value Fold Inductionb
242824_at NFIA Nuclear factor I/A 0.03 2.02 229728_at NA NA 0.03 2.01 236545_at PPP3CA Protein phosphatase 3 (formerly
2B), catalytic subunit, alpha isoform
0.04 1.97
233037_at NA NA 0.03 1.96 242696_at NUDCD3 NudC domain containing 3 0.05 1.94 226840_at H2AFY H2A histone family, member Y 0.04 1.88 236685_at NA NA 0.04 1.79 1553145_at FLJ39653 Hypothetical FLJ39653 0.05 1.67 aThese genes were not significantly affected by elastase treatment. bRelative to control.
96
Table 3-8. Reaction conditions for protease assays
Protease Substrate [Sub] μM
Ex/Em or λmax
Buffera
ACE1 MCA-RPPGFSAFK(Dnp) 10 320/405 A Activated Protein C(H) in 50% gly
Boc-DVLR-ANSNH-C4H9 50 355/460 C
ADAM9 MCA-PLAQAV-Dpa-RSSSR-NH3 10 320/405 I ADAM10 MCA-PLAQAV-Dpa-RSSSR-NH3 10 320/405 I BACE1 MCA-SEVNLDAEFRK(Dnp)-RR-
NH2 10 320/405 J
Calpain 1 Biomol, N-Succinyl-Leu-Tyr-AMC 10 355/460 K Caspase 1 Ac-LEHD-AMC 5 355/460 G Caspase 2 Ac-LEHD-AMC 5 355/460 G Caspase 3 Ac-DEVD-AMC 5 355/460 F Caspase 4 Ac-LEHD-AMC 5 355/460 G Caspase 5 Ac-LEHD-AMC 5 355/460 G Caspase 6 Ac-LEHD-AMC 5 355/460 G Caspase 7 Ac-DEVD-AMC 5 355/460 F Caspase 8 Ac-LEHD-AMC 5 355/460 G Caspase 9 Ac-LEHD-AMC 5 355/460 G Caspase 10 Ac-LEHD-AMC 5 355/460 G Caspase 11 Ac-LEHD-AMC 5 355/460 G Caspase 14 Ac-LEHD-AMC 5 355/460 G Cathepsin B Z-FR-AMC 5 355/460 L Cathepsin C Z-FR-AMC 5 355/460 L Cathepsin D MCA-KPILFFRLK(Dnp)-D-R-NH2 10 320/405 P Cathepsin E MCA-KPILFFRLK(Dnp)-D-R-NH2 10 320/405 P Cathepsin G Suc-AAPF-AMC 10 355/460 C Cathepsin H R-AMC 10 355/460 M Cathepsin K Z-GPR-AMC 10 355/460 C Cathepsin S Z-FR-AMC 10 355/460 M Cathepsin V Z-FR-AMC 10 355/460 E Cathepsin X/Z MCA-RPPGFSAFK(Dnp) 10 320/405 D Chymase Suc-AAPF-AMC 10 355/460 C Chymotrypsin (Human Pancreatic)
Suc-AAPF-pNa 3 405 U
Chymotrypsin (Bovine Pancreatic)
Suc-GGF-pNa 1.5 405 T
Complement Component C1s (CCC1s)
Dabcyl-SLGRKIQI-EDANS 10 340/490 A
DPP IV H-GP-AMC 10 355/460 H DPP VIII H-GP-AMC 10 355/460 H DPP IX H-GP-AMC 10 355/460 H
97
Table 3-8. Continued
Protease Substrate [Sub] μM
Ex/Em or λmax
Buffera
Elastase (Human Neutrophil)
(OMeSuc)-AAPV-pNa 2 405 V
Elastase (Porcine Pancreatic)
Suc-AAA-pNa 2 405 S
Factor VIIa Z-VVR-AMC 10 355/460 A Factor Xa CH3SO2-D-CHA-Gly-Arg-AMC-
AcOH 10 355/460 N
Factor XIa (Boc-Glu(OBzl)-Ala-Arg)-MCA 10 355/460 A Granzyme B Ac-IEPD-AMC 10 355/460 A Hepatitis C virus NS3/4A protease
Kallikrein 1 Z-GPR-AMC 10 355/460 A Kallikrein 5 Z-VVR-AMC 10 355/460 A Kallikrein 8 VPR-AMC 10 380/460 E Kallikrein 12 VPR-AMC 10 380/460 B Kallikrein 13 VPR-AMC 10 380/460 A Kallikrein 14 VPR-AMC 10 380/460 A MMP1 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP2 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP3 (5-FAM/QXLTM) FRET peptide 5 485/520 H 3-7MMP7 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP8 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP9 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP10 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP11 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP12 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP13 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP14 (5-FAM/QXLTM) FRET peptide 5 485/520 H Papain Z-FR-AMC 10 355/460 M Plasma Kallikrein Z-FR-AMC 10 380/460 A Plasmin H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 A Proteinase K H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 A TACE MCA-PLAQAV-Dpa-RSSSR-NH2 10 320/405 I Thrombin alpha H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 O Tissue Plasminogen Activator
Z-GPR-AMC 10 355/460 Q
Trypsin H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 A Tryptase beta 2 Z-GPR-AMC 10 355/460 A Tryptase gamma 1 Z-GPR-AMC 10 355/460 A Urokinase Bz-b-Ala-Gly-Arg-AMC.AcOH 10 355/460 A
98
Table 3-8. Continued aBuffers A 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35 B 50 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 C 25 mM Tris pH 9, 150 mM NaCl D 25 mM Sodium Acetate pH 3.5, 5 mM DTT E 25 mM Sodium Acetate pH 5.5, 0.1 M NaCl, 5 mM DTT F 50 mM HEPES pH 7.4, 100 mM NaCl, 0.01% CHAPS, 0.1 mM
EDTA, 10 mM DTT G 50 mM HEPES pH 7.4, 1 M sodium citrate, 100 mM NaCl, 0.01%
CHAPS, 0.1 mM EDTA, 10 mM DTT H 50 mM HEPES pH 7.5, 100 mM CaCl2, 0.01% Brij35, store at 4°C,
add 0.1 mg/mL BSA before use I 25 mM Tris pH 9.0, 25 μM ZnCl2, 0.005% Brij J 0.1 M Sodium acetate, pH 4.0 K 75 mM Tris pH 7.0, 0.005% Brij35, 3 mM DTT, 0.5 mM CaCl2 L 25 mM MES pH 6.0, 50 mM NaCl, 0.005% Brij35, 5 mM DTT M 75 mM Tris pH 7.0, 1 mM EDTA, 0.005% Brij35, 3 mM DTT N 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 0.25 mg/mL BSA O 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 2.5 mM CaCl2, 1.0
mg/mL BSA P 0.1 mM Sodium Acetate pH 3.5, 0.1 M NaCl Q 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 1.0% BSA R Assay kit Buffer S 1.0 M Tris pH 8.0 T 50 mM Tris pH 7.8, 100 mM NaCl, 1 mM CaCl2 U 0.1 M Tris pH 8.3, 25 mM CaCl2 V 0.1 M Tris pH 7.5, 0.5 M NaCl
99
Table 3-9. Crystallography data and refinement statistics
PDB Title Elastase/Lyngbyastatin 7 complex
PDB ID 4GVU Data collection Space group P212121 Cell dimensions
Hmpa 1 165.9, C 2 76.1, CH 4.90, d (9.1) H-3 1, 3, 4, 5, 1 (N-Me-Val-2) 3 35.7, CH 1.97, m H-2, H3-6 4a 24.81, CH2 1.54, m H-4b, H3-5 4b 1.13, m H-4a, H3-5 5 10.5, CH3 0.87, t (7.3) H-4a, H-4b 3, 4 6 13.8, CH3 1.01, d (6.8) H-3 2, 3, 4 N-Me-Val-2 1 169.6, C 2 66.0, CH 4.15, d (9.5) H-3 1, 3, 5, N-Me, 1 (Val) 3 28.5, CH 2.27, m H-2, H3-4, H3-5 1 4 20.4, CH3 1.00, d (7.0) H-3 2 5 20.2, CH3 1.11, d (7.0) H-3 2, 3 N-Me 30.0, CH3 2.94, s 2, 1 (Val) Val 1 173.4, C 2 52.8, CH 4.70, dd (8.7, 6.5) H-3, NH 1, 3, 4, 5, 1 (Br-Hmoya) 3 32.1, CH 1.96, m H-2, H3-4, H3-5 5 4 20.3, CH3 0.95, d (6.7) H-3 2, 3, 5 5 17.5, CH3 0.88, d (6.7) H-3 2, 3 NH 6.25, d (8.7) H-2 1, 1 (Br-Hmoya) a100 MHz b600 MHz
123
Table 4-2. NMR data for veraguamide B (8) and veraguamide C (9) in CDCl3
Veraguamide B Veraguamide C unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
Br-Hmoyac/ 1 170.8, C 170.8, C Hmoyad 2 42.3, CH 3.13, br q (7.4) 42.4, CH 3.10, br q (7.2) 3 76.4, CH 4.85, d (8.7) 76.4, CH 4.86, dt (10.4, 2.5) 4a 27.5, CH2 2.07, m 27.4, CH2 2.07, m 4b 1.60, m 1.62, m 5a 24.93, CH2 1.61, m 25.2, CH2 1.62, m 5b 1.42, m 1.44, m 6 19.2, CH2 2.21, m 18.0, CH2 2.19, m 7 38.4, C 83.6, C 8 79.4, C 68.8, CH 1.93 t (2.5) 9 14.6, CH3 1.25, d (7.4) 14.5, CH3 1.25, d (7.2) N-Me-Val-1 1 170.7, C 170.7, C 2 65.0, CH 3.94, d (10.4) 65.0, CH 3.93, d (11.0) 3 28.26, CH 2.29, m 28.3, CH 2.28, m 4 19.57, CH3 0.98, d (6.5) 19.58, CH3 0.98, d (6.8) 5 19.55, CH3 0.92, d (6.5) 19.56, CH3 0.91, d (6.8) N-Me 28.7, CH3 3.00, s 28.7, CH3 3.00, s Pro 1 172.1, C 172.2, C 2 57.2, CH 4.95 dd (8.6, 4.8) 57.3, CH 4.94 dd (8.4, 5.0) 3a 29.4, CH2 2.28, m 29.5, CH2 2.28, m 3b 1.79, m 1.79, m 4a 24.99, CH2 2.04, m 24.89, CH2 2.03, m 4b 1.98, m 1.99, m 5a 47.3, CH2 3.80, dt (–16.4, 6.9) 47.3, CH2 3.84, dt (–16.8, 7.1) 5b 3.60, dt (–16.4, 6.9) 3.60, dt (–16.8, 7.1) Hivac/Hmpad 1 165.8, C 165.9, C 2 77.2, CH 4.85, d (8.7) 76.0, CH 4.89, d (9.4) 3 29.6, CH 2.17, m 35.7, CH 1.98, m
124
Table 4-2. Continued
Veraguamide B Veraguamide C unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
4 18.1, CH3 1.02, t (6.6) 24.81, CH2 1.54, m 1.13, m 5 18.5, CH3 0.93, d (6.6) 10.5, CH3 0.86, t (7.3) 6 13.8, CH3 1.01, d (6.7) N-Me-Val-2 1 169.6, C 169.6, C 2 66.1, CH 4.15, d (10.2) 66.0, CH 4.13, d (10.0) 3 28.34, CH 2.28, m 28.5, CH 2.28, m 4 20.3, CH3 0.99, d (6.8) 20.4, CH3 0.99, d (6.4) 5 20.1, CH3 1.11, d (6.8) 20.2, CH3 1.10, d (6.4) N-Me 30.0, CH3 2.94, s 30.0, CH3 2.93, s Val 1 173.5, C 173.4, C 2 52.8, CH 4.71, dd (8.6, 6.4) 52.8, CH 4.70, dd (8.6, 6.2) 3 32.2, CH 1.98, m 32.1, CH 1.96, m 4 20.3, CH3 0.94, d (6.4) 20.3, CH3 0.94, d (6.7) 5 17.5, CH3 0.88, d (6.4) 17.6, CH3 0.87, d (6.7) NH 6.26, d (8.6) 6.26, d (8.6) a100 MHz b600 MHz cRefers to veraguamide B dRefers to veraguamide C
125
Table 4-3. NMR data for veraguamide D (10) and veraguamide E (11) in CDCl3
Veraguamide D Veraguamide E unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
Hmoya 1 170.8, C 170.71, C 2 42.4, CH 3.13, br q (7.2) 42.4, CH 3.08, br q (7.4) 3 76.4, CH 4.86, dt (10.8, 2.6) 76.5, CH 4.85, d (9.0) 4a 27.5, CH2 2.07, m 27.5, CH2 2.06, m 4b 1.63, m 1.62, m 5a 25.2, CH2 1.63, m 25.2, CH2 1.61, m 5b 1.47, m 1.43, m 6 17.5, CH2 2.18, m 18.0, CH2 2.18, m 7 83.6, C 83.6, C 8 68.8, CH 1.93, t (2.5) 68.8, CH 1.93, t (2.3) 9 14.4, CH3 1.24, d (7.2) 14.5, CH3 1.23, d (7.4) N-Me-Ilec/ 1 170.7, C 170.69, C N-Me-Vald 2 64.0, CH 4.01, d (10.6) 64.9, CH 3.93, d (10.0) 3 34.6, CH 2.02, m 28.3, CH 2.29, m 4 25.7, CH2 1.46, m 19.59, CH3 0.91, d (6.4) 5 11.4, CH3 0.93, t (6.5) 19.56, CH3 0.98, d (6.4) 6 15.8, CH3 0.94, d (6.8) N-Me 28.7, CH3 2.99, s 28.6, CH3 3.00, s Pro 1 172.7, C 172.2, C 2 57.2, CH 4.94 dd (8.9, 4.8) 57.3, CH 4.94 dd (9.0, 5.3) 3a 28.8, CH2 2.26, m 29.5, CH2 2.29, m 3b 1.78, m 1.78, m 4a 24.9, CH2 2.03, m 24.89, CH2 2.01, m 4b 1.97, m 1.99, m 5a 47.2, CH2 3.82, dt (–17.0, 7.3) 47.3, CH2 3.86, dt (–17.0, 7.0) 5b 3.60, dt (–17.0, 7.3) 3.60, dt (–17.0, 7.0) Hmpa 1 165.9, C 166.0, C 2 76.0, CH 4.90, d (9.2) 76.1, CH 4.85, d (9.0)
126
Table 4-3. Continued
Veraguamide D Veraguamide E unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
3 35.7, CH 1.98, m 35.1, CH 1.98, m 4 24.8, CH2 1.53, m 24.86, CH2 1.54, m 1.12, m 1.13, m 5 10.5, CH3 0.86, t (7.6) 10.5, CH3 0.86, t (7.0) 6 13.9, CH3 0.99, d (6.9) 13.8, CH3 1.01, d (6.8) N-Me-Valc/ 1 169.6, C 169.7, C N-Me-Iled 2 66.0, CH 4.15, d (9.4) 65.2, CH 4.22, d (9.6) 3 28.4, CH 2.28, m 35.7, CH 1.98, m 4 20.3, CH3 1.10, d (6.8) 26.6, CH2 1.54, m 1.06, m 5 20.2, CH3 0.99, d (6.8) 11.7, CH3 0.96, t (7.2) 6 16.5, CH3 1.04, d (6.9) N-Me 30.0, CH3 2.92, s 30.1, CH3 2.93, s Valc/Iled 1 173.4, C 173.5, C 2 52.8, CH 4.70, dd (8.6, 6.6) 52.4, CH 4.70, dd (8.4, 6.7) 3 32.1, CH 1.98, m 38.6, CH 1.69, m 4 19.0, CH3 0.94, d (6.6) 23.9, CH2 1.47, m 5 17.6, CH3 0.87, d (6.6) 1.05, m 11.5, CH3 0.85, d (6.6) 6 16.3, CH3 0.91, d (6.6) NH 6.24, d (8.6) 6.26, d (8.4) a125 MHz b600 MHz cRefers to veraguamide D dRefers to veraguamide E
127
Table 4-4. NMR data for veraguamide F (12) in CDCl3
unit C/H no. δCa δH (J in Hz)b
Hmoya 1 170.9, C 2 42.2, CH 3.24 br q (7.3) 3 76.6, CH 4.89, dt (10.6, 2.4) 4a 27.5, CH2 2.08, m 4b 1.64, m 5a 25.2, CH2 1.65, m 5b 1.45, m 6 18.0, CH2 2.20, m 7 83.5, C 8 68.9, CH 1.93, t (2.5) 9 14.6, CH3 1.29, d (7.3) N-Me-Val-1 1 170.7, C 2 65.3, CH 3.95, d (10.4) 3 28.3, CH 2.32, m 4 19.6, CH3 1.00, d (6.5) 5 19.7, CH3 0.93, d (6.5) N-Me 28.7, CH3 3.06, s Pro 1 172.3, C 2 57.1, CH 4.97, dd (9.0, 4.5) 3a 29.1, CH2 2.27, m 3b 1.83, m 4a 25.0, CH2 2.07, m 4b 1.97, m 5a 47.0, CH2 3.62, m 5b 3.56, m Pla 1 165.4, C 2 72.8, CH 5.47, dd (9.8, 4.0) 3 36.7, CH2 3.17, m
128
Table 4-4. Continued
unit C/H no. δCa δH (J in Hz)b
2.91, m 4 136.2, C 5//9 129.3, CH 7.18, d (8.0) 6/8 128.5, CH 7.28, m 7 126.7, CH 7.20, m N-Me-Val-2 1 168.9, C 2 65.8, CH 4.05, d (10.5) 3 27.4, CH 2.08, m 4 19.8, CH3 0.89, d (6.8) 5 20.0, CH3 0.91, d (6.8) N-Me 29.0, CH3 2.60, s Val 1 173.4, C 2 52.7, CH 4.76, dd (8.5, 6.1) 3 32.3, CH 1.98, m 4 20.3, CH3 0.91, d (6.6) 5 17.7, CH3 0.88, d (6.6) NH 6.28, d (8.5) a100 MHz b600 MHz
129
Table 4-5. NMR data for veraguamide G (13) and tetrahydroveraguamide A (14) in CDCl3
Veraguamide G Tetrahydroveraguamide A unit C/H no. δC
a δH (J in Hz)b δCb,c δH (J in Hz)b
Hmoead/ 1 170.9, C 170.7, C Hmoaae 2 42.4, CH 3.10, br q (7.4) 42.1, CH 3.08, br q (7.4) 3 76.8, CH 4.85, dt (10.6, 2.4) 76.8, CH 4.86, dt (10.1, 2.1) 4a 27.9, CH2 1.98, m 31.0, CH2 1.21, m 4b 1.45, m 1.26, m 5a 25.5, CH2 1.48, m 28.2, CH2 1.39, m 5b 1.30, m 6a 33.2, CH2 2.05, m 25.8, CH2 1.39, m 6b 1.20, m 7 138.2, CH 5.74, m 22.2, CH2 1.26, m 8 114.9, CH2 4.97, m 13.6, CH3 0.85, t (6.9) 9 14.4, CH3 1.22, d (7.4) 14.0, CH3 1.23, d (7.4) N-Me-Val-1 1 170.7, C 170.7, C 2 65.0, CH 3.93, d (9.8) 64.9, CH 3.93, d (10.7) 3 28.3, CH 2.28, m 28.3, CH 2.28, m 4 19.59, CH3 0.98, d (6.4) 19.2, CH3 0.98, d (6.5) 5 19.54, CH3 0.92, d (6.4) 19.3, CH3 0.91, d (6.5) N-Me 28.6, CH3 3.01, s 28.4, CH3 3.00, s Pro 1 172.2, C 172.0, C 2 57.3, CH 4.95, dd (8.7, 5.0) 57.1, CH 4.94, dd (9.0, 5.0) 3a 29.4, CH2 2.29, m 29.1, CH2 2.28, m 3b 1.79, m 1.79, m 4a 24.9, CH2 2.03, m 24.6, CH2 2.03, m 4b 1.98, m 1.99, m 5a 47.3, CH2 3.84, dt (–16.7, 7.1) 47.0, CH2 3.84, dd (–17.0, 7.3) 5b 3.61, dt (–16.7, 7.1) 3.60, dd (–17.0, 7.3) Hmpa 1 165.9, C 165.7, C
130
Table 4-5. Continued
Veraguamide G Tetrahydroveraguamide A unit C/H no. δC
a δH (J in Hz)b δCb,c δH (J in Hz)b
2 76.0, CH 4.90, d (8.7) 76.6, CH 4.90, d (8.8) 3 35.7, CH 1.98, m 35.4, CH 1.98, m 4 24.8, CH2 1.54, m 24.5, CH2 1.54, m 1.13, m 1.13, m 5 10.5, CH3 0.86, t (7.3) 10.2, CH3 0.86, t (7.1) 6 13.8, CH3 1.00, d (6.0) 13.5, CH3 1.00, d (6.4) N-Me-Val-2 1 169.6, C 169.5, C 2 66.0, CH 4.15, d (10.2) 65.8 CH 4.14, d (9.6) 3 28.6, CH 2.28, m 28.1, CH 2.28, m 4 20.4, CH3 1.00, d (6.1) 20.0, CH3 0.99, d (6.6) 5 20.2, CH3 1.10, d (6.1) 19.9, CH3 1.10, d (6.6) N-Me 30.0, CH3 2.93, s 29.7, CH3 2.93, s Val 1 173.4, C 173.3, C 2 52.7, CH 4.70, dd (8.6, 6.7) 52.5, CH 4.70, dd (8.6, 6.2) 3 32.1, CH 1.98, m 31.7, CH 1.96, m 4 20.3, CH3 0.93, d (6.8) 19.3, CH3 0.93, d (6.3) 5 17.6, CH3 0.86, d (6.8) 17.2, CH3 0.87, d (6.3) NH 6.23, d (8.6) 6.26, d (8.6) a125 MHz b600 MHz cBased on HSQC and HMBC dRefers to veraguamide G eRefers to tetrahydroveraguamide A
131
Table 4-6. Antiproliferative activity (IC50, µM) of natural and semisynthetic veraguamidesa
Compound HT29 HeLa
Veraguamide A (7) 26 ± 3.1 21 ± 0.8 Veraguamide B (8) 30 ± 2.4 17 ± 1.0 Veraguamide C (9) 5.8 ± 0.8 6.1 ± 1.0 Veraguamide D (10) 0.84 ± 0.09 0.54 ± 0.01 Veraguamide E (11) 1.5 ± 0.09 0.83 ± 0.06 Veraguamide F (12) 49 ± 12 49 ± 1.4 Veraguamide G (13) 2.7 ± 0.7 2.3 ± 0.9 Tetrahydroveraguamide A (14) 33 ± 0.2 48 ± 2.5 aData are presented as mean ± SD (n = 2).
132
CHAPTER 5 CAYLOBOLIDE B AND AMANTELIDES A AND B: ANTIPROLIFERATIVE
POLYKETIDES FROM MARINE CYANOBACTERIA*,†
Introduction
Secondary metabolites assembled solely by polyketide synthases represent a
minor fraction of isolated compounds from the phylum Cyanobacteria. These usually
polyhydroxylated compounds are reminiscent of secondary metabolites from
dinoflagellates136 such as the cytotoxic amphidinolides, amphidinols, and luteophanols
as well as bacteria-derived antibiotics desertomycins137 and oasomycins.138 Polyketides
from marine and terrestrial cyanobacteria also possess interesting biological activities
and may be decorated with unusual moieties. Tolytoxin and the related scytophycins,
produced by terrestrial cyanobacteria are potent cytotoxins.139 Tolytoxins are
distinguished by an epoxide substituent in their backbone structure. Oscillariolide,140 a
polyketide isolated from the genus Oscillatoria, inhibited the development of fertilized
echinoderm eggs, suggestive of its effects on cell division. Phormidolide,141 a compound
related to oscillariolide, was isolated from the genus Phormidium and is also a potent
cytotoxin. Both oscillariolide and phormidolide macrocycles contain a tetrahydrofuran
ring and a terminal vinyl bromide appended to their ring structure. In addition, one
hydroxy group in phormidolide is esterified with a C-16 carboxylic acid. The well-studied
marine cyanobacterium Lyngbya majuscula afforded the polyketide caylobolide A, which
is characterized by its contiguous pentad of 1,5-diols.142
*Reproduced with permission from Salvador, L. A.; Paul V. J.; Luesch, H. J. Nat. Prod. 2010, 73, 1606–
1609. Copyright 2010 American Chemical Society. †Reproduced with permission from J. Nat. Prod., submitted for publication. Unpublished work copyright
2013 American Chemical Society.
133
The structure elucidation of polyketides is particularly challenging due to difficulty
in establishing the relative and absolute configuration of the multiple stereocenters and
substantial overlap in the methylene region. Their configurational assignment has
greatly benefited from the development of Kishi’s Universal NMR Database143–145 as
well as derivatization techniques, particularly Mosher’s analysis146 and extensions of
this method,147 although applications still have certain limitations, particularly for those
bearing 1,n-diol (n 5) moieties. Assignment of the configuration of 1,n-diols has so far
been demonstrated on model systems using exciton coupling CD after derivatization
with arylcarboxylate chromophores within liposomes.148
Here we report the isolation, structure elucidation, and antiproliferative activity of
three related polyketides characterized by a polyhydroxylated macrocyle bearing a
pendant alkyl side chain, given the names caylobolide B (18) and amantelides A and B
(19, 20), from Floridian Phormidium spp. and a Guamanian gray cyanobacterium
collections, respectively.
Isolation and Structure Elucidation
Caylobolide B (18)
A freeze-dried sample of an assemblage of Phormidium cf. dimorphum and
Phormidium inundatum from Key West, Florida was extracted with EtOAc–MeOH (1:1).
This extract was cytotoxic at a concentration of 100 ng/mL and contained symplostatin 1
based on the HPLC-MS profiling. The nonpolar extract was solvent partitioned to yield
the hexanes-, n-BuOH- and H2O- soluble fractions. The n-BuOH fraction was cytotoxic
and was subjected to a bioactivity-guided isolation using silica gel chromatography and
reversed-phase HPLC to yield caylobolide B (18) (Figure 5-1). The major cytotoxic
activity was attributed to the known compound symplostatin 1, based on comparison of
134
LRESIMS and 1H NMR with literature values. Symplostatin 1 gave an IC50 of ~1.5 nM
against HT29 cells. However, because our cyanobacterial collection was largely a
binary mixture of two different Phormidum species, it is unclear if caylobolide B (18) and
the co-isolated cytotoxin symplostatin 1 were produced by the same or both species.
Caylobolide B (18) was isolated as a colorless, amorphous solid with molecular
formula of C42H80O11 based on pseudomolecular ion peaks observed by
HRESI/APCIMS at m/z 761.5767 [M + H]+ and m/z 783.5594 [M + Na]+. Fragmentation
of the [M + H]+ peak using positive ionization showed repetitive loss of 18 amu,
corresponding to elimination of H2O typical for alcohols. The structure of 18 was
determined by NMR analysis in DMSO-d6. The presence of exchangeable hydroxy
protons was evident from the lack of HSQC correlations for nine protons which resonate
at δH 4.2–4.6 ppm. Detailed interpretation of HSQC, TOCSY, HSQC-TOCSY and HMBC
experiments with 18 (Table 5-1, Figure 5-1) established that the hydroxy groups are part
of methine carbinols that form a highly oxygenated backbone structure consisting of a
1,3-diol system (C-7, C-9), a 1,3,5-triol system (C-25, C-27, C-29) and repeating 1,5-
diol moieties. Degenerate 1H and 13C NMR chemical shifts were observed for three
oxygenated methines at δC 69.6 (C-13, C-17, C-21), seven methylenes at δC 37.3 (C-
12, C-14, C-16, C-18, C-20, C-22, C-24), and two methylenes at δC 21.6 (C-15, C-19)
that make up the contiguous chain of 1,5-diol.
The 13C NMR chemical shifts are in good agreement with reported values for 1,5-
diol units of luteophanol.149 These degenerate signals together with HSQC-TOCSY
correlations (Figure 5-2) between δC 37.3/δH 4.20 and δC 21.6/δH 4.20 supported the
1,5-diol substitution pattern. HSQC-TOCSY correlations (Figure 5-2) between C-15/9-
135
OH, C-23/25-OH suggested that the contiguous chain of 1,5-diol is flanked by the 1,3-
diol and 1,3,5-triol units. HSQC-TOCSY correlations between C-31/29-OH, C-32/29-OH,
C-32/33-OH, C-33/H-35 enabled the extension of the polyhydroxylated chain which
terminates to form an ester linkage with a carbonyl group at δC 165.4 (C-1). The low-
field chemical shift of H-35 (δH 5.00) – due to anisotropy from an unsaturated system –
and HMBC correlation between C-1/H-35 confirmed the presence of the ester linkage.
From HMBC and TOCSY correlations of C-35/H-35 (Table 5-1), it was evident that C-35
was modified by an isohexyl side chain substitution. An additional unsaturation is
present in 18 due to a carbon-carbon double bond between C-2 and C-3. HMBC
correlations (C-1/H-2, C-3/H-2) and the characteristic chemical shifts for C-2 (δC 116.5)
and C-3 (δC 159.4) were suggestive of a polarized carbon-carbon double bond,
consistent with an α,β-unsaturated ester functionality. HMBC correlations between C-
2/H3-42 and C-3/H3-42 indicated a methyl substitution at the β position.
The structure of 18 bears a close resemblance to the 36-membered
macrolactone ring present in the known compound caylobolide A142 (Figure 5-1) and
was therefore termed caylobolide B. The C-1 to C-9 portion of these compounds
presents a major difference, where an additional carbon–carbon double bond and a
different hydroxylation pattern are present in 18. The isolated 1,3-diol system (C-7 to C-
9) is a distinctive feature of 18, instead of a 1,5-diol unit from C-5 to C-9 chain in
caylobolide A. The structure of caylobolide B (18) was confirmed using ESIMS
fragmentation in the negative ionization mode (Figure 5-3). It was evident that
fragmentation occurred mainly at positions α- and β- to the hydroxy groups, similar to
fragmentation patterns observed for amphidinols.137
136
Amantelides A and B (19, 20)
A gray cyanobacterium collected at Amantes Point, Tumon Bay, Guam was
extracted with CH2Cl2–MeOH (1:1). The resulting nonpolar extract exhibited
antiproliferative activity against HT29 cells at a concentration of 10 μg/mL and did not
contain largazole, symplostatin 1 or dolastatin 10, based on the HPLC-MS profiling.
Solvent partitioning of the nonpolar extract gave the hexanes-, n-BuOH-and H2O-
soluble fractions. The antiproliferative n-BuOH fraction was further purified by silica
column chromatography, with the bioactivity concentrated in the fraction eluting from
70% i-PrOH in CH2Cl2. Reversed-phase HPLC purification, afforded two related
polyketide-derived compounds, amantelides A (19) and B (20), as bioactive
constituents.
The HRESIMS spectrum of amantelide A (19) suggested a molecular formula of
C44H84O11 based on the observed pseudomolecular [M + Na]+ ion at m/z 811.5927. The
three degrees of unsaturation was partially accounted for by an α,β-unsaturated ester
based on 1H and 13C NMR, HSQC, and HMBC spectra, suggesting the presence of one
ring system to fulfill the molecular formula requirements. HMBC correlations with the sp2
C (δC160.3) were observed for the CH3 singlet (δH 1.85) and a vinyl group (δH 5.62), with
the latter also having long-range correlations to a carbonyl group (δC 165.5), confirming
the presence of an α,β-unsaturated ester (Figure 5-4, Table 5-2). The presence of an
ester functionality was also corroborated by the presence of a low-field methine (δC/δH
76.6/4.93), which also showed HMBC correlations to C-1 (δC 165.5). In addition, two
other spin system consisting of a 1,3-methine carbinol and a tert-butyl moiety were also
deduced. Using COSY, TOCSY and HMBC correlations, a partial structure (Figure 5-5)
for amantelide A (19) was derived. This is reminiscent of the C-1 to C-9 and C-33 to C-
137
40 moieties of caylobolide B (18) (Figures 5-1, 5-5). However, instead of an isohexyl
pendant side chain, amantelide A (19) bears a tert-butyl moiety (Figures 5-4, 5-5). The
overlapping 1H and 13C NMR signals only allowed for partial assignment of the structure
of 19. Comparison of the 1H and 13C chemical shifts of 18 and 19 indicated that the
latter lacks the distinctive 1,3,5-triol system present in caylobolides A142 and B (18)
(Tables 5-1, 5-2). Based on the 1H and 13C NMR chemical shifts as well as the
remaining C27H52O6 to be accounted for from the partial structure and molecular formula
of 19, a contiguous chain of 1,5-diol is proposed to form the macrocyclic structure of
amantelide A (19). The observed degenerate 13C NMR shifts in amantelide A (19)
(Table 5-2) are in accordance with literature values for 1,5-diols in luteophanols149 and
caylobolides A142 and B (18) (Table 5-1). To verify the proposed structure, MS/MS
fragmentation of amantelide A (19) was done under negative ionization (Figure 5-6).
Fragmentations were observed at α- and β-positions to the methine carbinols (Figure 5-
6) and confirmed that amantelide A (19) has a closely related structure to the
caylobolides.
HRESIMS data for amantelide B (20) showed pseudomolecular ion [M + Na]+ at
m/z 853.6044, with a 42 amu mass difference with amantelide A (19), suggesting a
molecular formula of C46H86O12. 1H and 13C NMR, HSQC and HMBC spectra of
amantelide B (20) suggested that these compounds belong to the same structural class,
with an additional acetyl group in amantelide B (20). This was corroborated by a singlet
CH3 (δC/δH 20.7/1.97) that showed an HMBC correlation to a carbonyl group (δC170.0)
(Table 5-2). This acetyl group is proposed to modify a methine carbinol and is evident
from the appearance of a downfield shifted methine (δC/δH 73.3/4.73) that also showed
138
an HMBC correlation to the carbonyl at δC 170.0 (Table 5-2). C-7 and C-9 were
eliminated as possible sites of acetylation since the characteristic 1H and 13C NMR
shifts of this moiety can still be clearly discerned (Table 5-2). A TOCSY correlation
between δH 4.73 and δH 3.21 suggested that C-33 bears the additional acetyl group in
20. Hence, amantelide B (20) is the C-33 monoacetylated analog of amantelide A (19)
(Figure 5-4). Verification by MS/MS fragmentation was, however, not possible due to
the immediate loss of the acetyl group upon ionization, yielding a similar fragmentation
pattern as amantelide A (19).
Amantelides A and B (19, 20) showed similarities to caylobolides A and B (18),
with the presence of a polyhydroxylated macrolactone ring that is modified by a pendant
aliphatic side chain. The C-1 to C-21 portions of the macrolactone ring of 18–20 are
similar, with the characteristic 1,3-diol moiety (C-7 to C-9) flanked by a 1,5-diol moiety
(C-10 to C-24) and an α,β-unsaturated ester (C-1 to C-3). Compounds 19 and 20 are
distinguished by their 1,5-dihydroxylation pattern (C-25 to C-39), as well as a larger 40-
membered macrolactone ring instead of a 36-membered macrocycle in caylobolides.
Amantelides also possess a tert-butyl side chain instead of an isohexyl moiety as in the
caylobolides. Tert-butyl bearing-natural products are rare and present only a small
portion of secondary metabolites. Among the cyanobacterial metabolites, the cytotoxins
apratoxins,60–64 laingolides,150,151 madangolide152 and bisebromoamide153 and the Ca2+
blocker palmyrolide A,154 bear a tert-butyl moiety.
Configurational Analysis
The relative configuration of selected stereogenic centers of 18 was assigned by
independently considering the 1,3-diol and 1,3,5-triol moieties using Kishi’s Universal
NMR Database (Database 2).143–145 The 13C NMR chemical shift of C-7/C-9 was in good
139
agreement with syn arrangement of 1,3-diol model system (Figure 5-7). The 1,3,5-triol
system was assigned as either syn/anti or anti/syn between C-25/C-27, C-27/C-29
based on comparison of δC at C-27 with the characteristic δC of the central carbon of the
1,3,5-triol model system (Figure 5-7).
This method cannot differentiate between syn/anti or anti/syn orientation.
Unfortunately, Mosher’s analysis failed to give any conclusive result on the absolute
configuration and was limited by the low yield of 18. The lack of chemical shift
dispersion in the contiguous chain of 1,5-diols in caylobolide B (18) limits the
assignment of the absolute configuration of this moiety, as well as those for 19 and 20.
The absolute configuration of the stereocenters in amantelides A and B (19, 20) was not
determined. The relative configuration at C-7/C-9 of amantelides were assigned as syn,
based on comparison with caylobolide B (18) (Tables 5-1, 5-2) and also in agreement
with the Kishi’s Universal NMR Database for 1,3-diols.143
Biological Activity Studies
Antiproliferative Activity
Caylobolide B (18) exhibited moderate cytotoxic activity against HT29 colorectal
adenocarcinoma and HeLa cervical carcinoma cells with IC50 of 4.5 μM and 12.2 μM,
respectively (Table 5-3). The cytotoxic activity of 18 is comparable to that of caylobolide
A against the human colon carcinoma HCT116 cells (IC50 9.9 μM).142 Due to the limited
amount of caylobolide B (18) and its weak cytotoxic activity, it was not pursued for
further biological studies. Amantelide A (19) showed superior antiproliferative activity in
HT29 and HeLa cancer cell lines, with submicromolar IC50s, compared to the
caylobolides (Table 5-3). Monoacetylation of amantelide A (19) at C-33, however,
caused more than 10-fold decrease in antiproliferative activity, as observed for
140
amantelide B (20) (Table 5-3). This then suggested the role of acetylation and
hydroxylation in modulating the antiproliferative activity of cyanobacterial polyketides
belonging to the caylobolide class. In order to gain insight into the role of acetylation in
the antiproliferative activity of amantelides, a semisynthetic derivative of 19 was
prepared using acetic anhydride and pyridine to yield the peracetylated amantelide A
(21). Antiproliferative activity testing on 21 indicated that peracetylation caused a
dramatic decrease in potency, causing a 20-fold and 67-fold increase in IC50 in HeLa
and HT29 cells, respectively (Table 5-3). In addition to acetylation of the hydroxy
groups, the difference in antiproliferative activities of caylobolides and amantelides may
suggest that the size of the macrolide ring, hydroxylation pattern and aliphatic side
chain may contribute to the antiproliferative activity of these compounds.
Elucidation of the Mechanism of Action of Cyanobacterial Polyketides
The preliminary SAR for 18–21 suggested that the hydroxy groups of
cyanobacterial polyketides are important to the biological activity. Time-course cell
viability analysis of HeLa and HT29 cells treated with amantelide A (19) indicated that
the cellular effects of 19 are observed within 1 h post-treatment (Figure 5-8). This then
indicated that these compounds may be acting as cell membrane disrupting agents,
based on the rapid cellular effects of 19. This mechanism of action is observed for
amphotericin B, a natural product isolated from Streptomyces nodosus, where changes
in membrane permeability culminate in the leakage of mono- and divalent ions.155 Close
inspection of the structure of amphotericin B and the cyanobacterial polyketides 18–20
indicated several similarities. C-1 to C-11 of amphotericin B bears close resemblance to
C-1 to C-13 of 18–20. The C-35 to C-37 moiety of amphotericin B is homologous to the
C-37 to C-39 of amantelides A and B (19, 20) and C-33 to C-35 of caylobolide B (18)
141
(Figures 5-1, 5-4). In amphotericin B, C-35 is a methine carbinol, while C-36 bears a
methyl group, and C-37 is derivatized to an ester which forms the macrocycle. Recent
investigations on the mechanism of action of amphotericin B indicated that it binds to
ergosterol in yeast cells through the mycosamine moiety and also forms ion channels
via the polyhydroxylated portion of the molecule.155–157 The formation of ion channels by
amphotericin B is postulated to be through the formation of both monomeric and dimeric
structures, with C-1 to C-13 and C-35 to C-37 being criticial structural elements.155–157
The hydroxy group at C-35 is in particular important; suggested to bridge the
amphotericin backbone to the lipid bilayer.155 This critical structural element parallels the
observation for amantelides, where the presence of an acetyl group at C-33 caused a
decreased in activity.
In order to probe the mechanism of action of 18–20, we utilized amantelide A
(19) as the model compound since it gave the highest potency in the antiproliferative
assay and also present in sufficient amounts. To verify the proposed mechanism of
action of amantelide A (19), the antiproliferative activity and cellular phenotype of
amphotericin B- and amantelide A (19)-treated cells were compared (Figure 5-8).
Significant changes in cell viability were observed for both amantelide A (19) and
amphotericin B-treated cells after 1 h (Figure 5-8). Amphotericin B however, induced
cell death at a slower rate compared to amantelide A (19). This is in accordance with
the close to 10-fold higher potency of 19 compared to amphotericin B (Table 5-3) in
preventing the growth of HT29 and HeLa cancer cells. Based on visual inspection,
amantelide A (19) and amphotericin B also both induced rapid morphological changes
142
in HeLa cells, within 1 h of treatment. The morphology of amantelide A (19)- and
amphotericin B-treated cells were distinct from control treatments.
Conclusion
Bioactivity-guided purification of two cyanobacteria collections yielded the closely
related polyketide macrolactones caylobolide B (18) and amantelides A and B (19, 20).
The structures of 18–20 were assigned based on 1H and 13C NMR, HSQC, HMBC,
TOCSY and COSY experiments. Compounds 18–20 are characterized by a
polyhydroxylated macrocycle modified by an aliphatic pendant side chain. Caylobolide B
(18) is characterized by a 36-membered macrocycle consisting of 1,3- and 1,5-diol and
a 1,3,5,-triol systems and an isohexyl pendant side chain. Amantelides A and B (19, 20)
have a distinctive 40-membered macrocycle composed of 1,3- and 1,5-diol moieties and
a tert-butyl pendant side chain, with compound 20 additionally being acetylated at C-33.
Antiproliferative activity assays with 18–20 indicated the importance of the hydroxy
groups for bioactivity, and were verified by the loss of activity of the peracetylated
derivative of 19. Compounds 18–20 bear structural similarities with amphotericin B.
This, together with the results of time-course cell viability determination for amphotericin
B- and amantelide A (19)-treated cells suggested that the latter may also affect the
integrity of the cell membrane, similar to amphotericin B. Additional experiments to
visualize the effects of 19 on the cell membrane may be carried out.
Experimental Methods
General Experimental Procedures
Optical rotation was measured on a Perkin-Elmer 341 polarimeter. The UV
spectrum was recorded on SpectraMax M5 Molecular Devices. 1H and 2D NMR spectra
were recorded in DMSO-d6 on a Bruker Avance II 600 MHz spectrometer equipped with
143
a 5-mm triple-resonance high-temperature superconducting (HTS) cryogenic probe
using residual solvent signals (δH 2.50; δC 39.5) as internal standards. The 13C NMR
spectrum was recorded in DMSO-d6 on a Bruker 500 MHz spectrometer, operating at
125 MHz. HSQC and HMBC experiments were optimized for 1JCH = 145 and nJCH = 7
Hz, respectively. TOCSY and HSQC-TOCSY experiments were done using a mixing
time of 100 ms. HRMS data were obtained using an Agilent LC-TOF mass spectrometer
equipped with an APCI/ESI multimode ion source detector. ESIMS/MS data were
obtained on a 3200 QTRAP (Applied Biosystems) by direct injection using a syringe
driver.
Biological Material
The cyanobacteria, Phormidium spp., were hand collected on June 24, 2008, at
the breakwater at Fort Zachary Taylor State Park (Key West), Florida, by snorkeling in
shallow waters. The collection was later identified to consist primarily of P. cf.
dimorphum and P. inundatum. Voucher specimens (#VP_6_24_08_FZT1) are
maintained at Smithsonian Marine Station, Fort Pierce, FL.
The gray cyanobacterium belonging to the Family Oscilliatoriales was collected
from Amantes Point, Tumon Bay, Guam. Voucher specimens are maintained at
Smithsonian Marine Station, Fort Pierce, FL.
Extraction and Isolation
Caylobolide B (18)
The freeze-dried organism (54.2 g) was extracted with EtOAc–MeOH (1:1) to
yield 5.7 g of the nonpolar extract. Subsequent extraction of the freeze-dried material
with EtOH–H2O (1:1) gave 11.5 g of the polar extract. The nonpolar extract was further
partitioned between hexanes and 20% aqueous MeOH. The latter was concentrated
144
under reduced pressure and was further partitioned between n-BuOH and H2O. The n-
BuOH (0.56 g) fraction was concentrated and subjected to Si gel column
chromatography eluting first with CH2Cl2, followed by increasing concentrations of i-
PrOH. After 100% i-PrOH, increasing gradients of MeOH were used until 100% MeOH.
The fraction that eluted with 25% MeOH was subjected to reversed-phase HPLC
(semipreparative, Phenomenex Synergi-Hydro RP, 4 μm) using a linear gradient of
MeOH–H2O (40–100% MeOH in 40 min and then 100% MeOH for 10 min) to yield
caylobolide B (18) (tR 31.1 min, 2.1 mg). Purification of the fraction from 50% MeOH
using the same conditions yielded symplostatin 1 (tR 31.4 min, 1.5 mg).
ESIMS/MS Fragmentation of Caylobolide B (18) and Amantelide A (19)
Individual solutions of 18 and 19 in MeOH were directly infused into the mass
spectrometer using a syringe driver. MS fragmentation was obtained by positive and
negative ionization using the enhanced product ion (EPI) and MS2 scan. The [M + H]+
and [M – H]– ions were fragmented by ramping the collision energy through the possible
allowed range. Compound-dependent and source gas parameters used were as
follows: DP ±65.0, EP ±10.0, CUR 10.0, CAD High, IS ±4500, TEM 0, GS1 10, GS2 0.
146
Cell Viability Assay
HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells were
cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with
10% fetal bovine serum (FBS, Hyclone) under a humidified environment with 5% CO2 at
37 °C. HeLa (3,000) and HT29 (12,500) cells were seeded in 96-well plates. Varying
concentrations of 18–21 and amphotericin B were added to each well 24 h post-
seeding, with treatments done in duplicate. The cells were incubated for an additional 1,
3, 6, 12 and 48 h before the addition of the MTT reagent. Cell viability was measured
according to the manufacturer’s instructions (Promega). IC50 calculations were done by
GraphPad Prism® 5.03 based on duplicate experiments.
147
Figure 5-1. Caylobolide B (18) and closely related compound caylobolide A. Absolute
configuration for C-25, C-27 and C-29 is proposed by analogy to caylobolide A. Only relative configuration is shown for C-7 and C-9, which could not be related to C-25–C-29.
148
Figure 5-2. Key HSQC-TOCSY correlations for caylobolide B (18).
149
Figure 5-3. ESI-MS/MS of caylobolide B (18).
150
Figure 5-4. Amantelides A and B (19, 20) and the semisynthetic derivative peracetylated
amantelide A (21). Only the relative configurations for C-7 and C-9 are indicated.
151
Figure 5-5. Partial structure of amantelide A (19) derived from NMR experiments in
DMSO-d6. COSY correlations are indicated by solid double-headed arrows. Protons showing HMBC correlations to indicated carbons are shown by single-headed arrows.
152
Figure 5-6. ESI-MS/MS fragmentation of amantelide A (19).
153
Figure 5-7. Assignment of relative configuration of caylobolide B (18) based on Kishi’s
Universal NMR Database (Database 2). δ values between the model system and 18 are shown. The relative configuration shown is based on the best fit
with the model system. The 1,3-diol is assigned as syn. The δ values for the characteristic central carbon of the 1,3,5-triol system suggest either anti/syn or syn/anti arrangement.
154
Figure 5-8. Time-course antiproliferative activities of amantelide A (19) and
amphotericin B against cancer cells. (A) HT29 and (B) HeLa cell viability after 1, 3, 6, 12 and 48 h incubation with amantelide A (19). (C) HT29 and (D) HeLa cell viability after 1, 3, 6, 12 and 48 h incubation with amphotericin B.
155
Table 5-1. NMR data of caylobolide B (18) in DMSO-d6
Position δCa δH (J in Hz)b HMBCb TOCSYb
1 165.4, C 2 116.5, CH 5.63, s 1,3,42 H-4a, H-4b, H-42 3 159.4, C 4a 4b
5a 23.6, CH2 1.52, m H-4a,H-4b,H-7,7-OH 5b 1.40, m 7 H-4a,H-4b 6 37.1, CH2 1.37, m 7-OH 7 68.8, CH 3.58, m 9 H-4a, H-4b,H-5a,7-OH 7-OH 4.52, d (4.4) 6,7,8 H-4a, H-4b, H-5aH-6,H-7 8 44.15, CH2 1.39, m 9 69.0, CH 3.54, m 10 9-OH,13-OH 9-OH 4.47, d (4.8) 8,9,10 H-9,H-13 10 37.6, CH2 1.28, m 11 21.3, CH2 1.21, m 12 37.3, CH2 1.28, m 13 69.6, CH 3.35, m 9-OH, 13-OH 13-OH 4.20, m 12,13,14 H-9, H-13 14 37.3, CH2 1.28, m 15 21.6, CH2 1.21, m 16 37.3, CH2 1.28, m 17 69.6, CH 3.37, m 17-OH 17-OH 4.20, m 16,17,18 H-17 18 37.3, CH2 1.28, m 19 21.6, CH2 1.21, m 20 37.3, CH2 1.28, m 21 69.8, CH 3.36, m 21-OH,25-OH 21-OH 4.20, m 20,21,22 H-21,H-25 22 37.3, CH2 1.28, m 23 20.9, CH2 1.32, m
156
Table 5-1. Continued
Position δC
a δH (J in Hz)b HMBCb TOCSYb
24 37.3, CH2 1.29, m 25 68.1, CH 3.54, m 23,27 21-OH,25-OH, H-27 25-OH 4.37, d (4.4) 24,25,26 H-21,H-25,H-27 26 44.4, CH2 1.42, m 27 65.8, CH 3.79, dq (13.5, 6.4) 25,26,29 H-25,27-OH, H-29 27-OH 4.47, d (4.9) 27 H-27 28 44.08, CH2 1.39, m 29 66.6, CH 3.61, m 28,31 H-27, 29-OH 29-OH 4.28, d (5.2) 28,30 H-27,H-29 30 37.5, CH2 1.31, m 31a 21.0, CH2 1.29, m 31b 1.22, m 32 38.1, CH2 1.29, m 33 66.5, CH 3.31, m 32,34 33-OH, H-35 33-OH 4.29, d (6.03) 33,34 H-33, H-35 34 37.3, CH2 1.49, m 35 H-35 35 73.6, CH 5.00, ddd (10.2,
4.3, 2.2) 1,33,41 H-33,OH-33,H-34,H-36,H-37b,H3-
40,H3-41 36 36.0, CH 1.66, m 35 H-35,H-37b,H-41 37a 31.6, CH2 1.31, m 41 H-37b,H-38a 37b 1.06, dd (17.7, 8.9) 38a 28.8, CH2 1.31, m H-37a,H-37b 38b 1.23, m 39 22.3, CH2 1.26, m 40 H3-40 40 13.6, CH3 0.86, t (7.0) 38,39 H-35, H-39 41 14.4, CH3 0.80, d (6.9) 35,36,37 H-36 42 24.5, CH3 1.85, s 2, 3, 4 H-2 a125 MHz. b600 MHz.
157
Table 5-2. NMR data of amantelide A (19) and amantelide B (20) in DMSO-d6
Amantelide A Amantelide B Position δC
a δH (J in Hz)b δCa δH (J in Hz)b
1 165.5, C 165.8, C 2 116.2, CH 5.62, s 116.1, CH 5.67, s 3 160.3, C 160.0, C 4 32.9, CH2 2.54, m 32.4, CH2 2.56, m 5a 23.6, CH2 1.54, m 23.4, CH2 1.52, m 5b 1.40, m 1.40, m 6 37.0, CH2 1.27, m 37.0, CH2 1.23, m 7 68.7, CH 3.58, m 68.6, CH 3.58, m 7-OH 4.54, br 4.50, br 8 44.1, CH2 1.37, m 44.2, CH2 1.37, m 9 68.5, CH 3.55, m 68.8, CH 3.55, m 9-OH 4.52, br 4.55, br 10 37.0, CH2 1.22, m 37.0, CH2 1.23, m 11 21.3, CH2 1.21, m 21.2, CH2 1.22, m 12 37.0, CH2 1.31, m 36.9, CH2 1.30, m 13 69.4, CH 3.34, m 69.4, CH 3.34, m 13-OH 4.23, m 4.23, m 14 37.0, CH2 1.31, m 36.9, CH2 1.30, m 15 20.8, CH2 1.31, m 20.8, CH2 1.32, m 16 37.0, CH2 1.31, m 36.9, CH2 1.30, m 17 69.4, CH 3.34, m 69.4, CH 3.34, m 17-OH 4.23, m 4.23, m 18 37.0, CH2 1.31, m 36.9, CH2 1.30, m 19 20.8, CH2 1.31, m 20.8, CH2 1.32, m 20 37.0, CH2 1.31, m 36.9, CH2 1.30, m 21 69.4, CH 3.34, m 69.4, CH 3.34, m 21-OH 4.23, m 4.23, m 22 37.0, CH2 1.31, m 36.9, CH2 1.30, m 23 20.8, CH2 1.31, m 20.8, CH2 1.32, m
158
Table 5-2. Continued
Amantelide A Amantelide B Position δC
a δH (J in Hz)b δCa δH (J in Hz)b
24 37.0, CH2 1.31, m 36.9, CH2 1.30, m 25 69.4, CH 3.34, m 69.4, CH 3.34, m 25-OH 4.23, m 4.23, m 26 37.0, CH2 1.31, m 36.9, CH2 1.30, m 27 20.8, CH2 1.31, m 20.8, CH2 1.32, m 28 37.0, CH2 1.31, m 36.9, CH2 1.30, m 29 69.4, CH 3.34, m 69.4, CH 3.34, m 29-OH 4.23, m 4.23, m 30 37.0, CH2 1.31, m 36.9, CH2 1.30, m 31 20.8, CH2 1.31, m
c c 32 37.0, CH2 1.31, m
c c 33 69.4, CH 3.34, m 73.3, CH 4.73, m 33-OH 4.23, m 34 37.0, CH2 1.31, m
c c 35 20.8, CH2 1.31, m
c c 36 37.0, CH2 1.31, m
c c 37 66.4, CH 3.21, m 66.4, CH 3.21, m 37-OH 4.26, m 4.27, br 38 37.1, CH2 1.43, m 37.1, CH2 1.41, m 39 76.6, CH 4.93, br d 76.2, CH 4.93, br d 40 34.1, C 34.2, C 41–43 26.3, CH3 0.82, s 25.7, CH3 0.83, s 44 24.6, CH3 1.85, s 24.3, CH3 1.86, s 45 170.0, C 46 20.7, CH3 1.97, s a125 MHz. b600 MHz. cCannot be assigned due to significant overlap of signals
159
Table 5-3. Cytotoxic activity (IC50, µM) of the isolated cyanobacterial polyketides (18–21)a
aData are presented as mean ± SD (n = 2).
Compound HT29 HeLa
Caylobolide B (18) 4.5 ± 1.2 12.2 ± 1.0 Amantelide A (19) 0.87 ± 0.02 0.87 ± 0.07 Amantelide B (20) 12 ± 1.6 9.9 ± 0.05 Peracetylated amantelide A (21) 58 ± 6.7 18 ± 1.6 Amphotericin B 10 ± 2.7 10 ± 4.4
160
CHAPTER 6 GENERAL CONCLUSION
In the last 30 years, marine cyanobacteria have been utilized as a source of
small molecule therapeutics. In this study, we aimed to exploit the diverse secondary
metabolites from the marine cyanobacteria belonging mainly to the genera of
Phormidium and Symploca for drug discovery. Cyanobacteria collections from Guam,
Florida, and the US Virgin Island were extracted and profiled in an antiproliferative
assay using the HT29 human colorectal adenocarcinoma cell line and an HPLC-MS-
based dereplication as a preliminary screening of bioactivity and chemical space,
respectively. Based on the profiling results, four cyanobacteria collections were
prioritized for further purification of secondary metabolites. Bioactivity- and 1H NMR-
directed approaches for the prioritized cyanobacteria collections yielded symplostatins
5–10 (1–6), veraguamides A–G (7–13), caylobolide B (18) and amantelides A and B
(19, 20). The planar structures of purified compounds were established using a
combination of 1D and 2D NMR spectroscopy and mass spectrometry. Absolute
configurations of stereocenters were assigned by enantioselective HPLC-MS and/or
HPLC-UV analysis by comparison with authentic standards as well as derivatization
with chiral reagents and J-based analysis.
A Guamanian Symploca sp. collection yielded the cyclic depsipeptides
symplostatins 5–10 (1–6), bearing the modified amino acid residue 3-amino-6-hydroxy-
2-piperidone (Ahp) and 2-amino-2-butenoic acid (Abu). The Ahp-bearing
cyclodepsipeptides from cyanobacteria constitutes a predominant class of metabolites,
with more than 100 members isolated to date from terrestrial, marine and freshwater
origins. These cyanobacterial metabolites are serine protease inhibitors, with the Abu-
161
bearing compounds such as symplostatins 5–10 (1–6) and the related lyngbyastatins 4–
10 being potent elastase inhibitors. Using the structural diversity of these agents, the
molecular basis for potent elastase inhibition was established using structure-activity
relationship (SAR) and X-ray cocrystallization studies. Aside from the Ahp and Abu
moieties, an N-Me-Tyr residue in the macrocyle and a polar functionality in the pendant
side chain are contributors to potent elastase inhibition. This was verified from the X-ray
cocrystal structure of lyngbyastatin 7–porcine pancreatic elastase, where the hydroxy
group of the N-Me-Tyr and the terminal amide group of Gln in lyngbyastatin 7 contribute
critical hydrogen bonding interactions with the enzyme and active site water molecules.
The involvement of the pendant side chain, which is highly variable among members of
this compound class, highlights Nature’s own combinatorial chemistry. Comprehensive
serine protease profiling for symplostatin 5 (1) and lyngbyastatin 7 demonstrated
preferential inhibition of elastase by these agents. The cellular effects of symplostatin 5
(1) against the downstream cellular effects of elastase in bronchial epithelial cells were
also interrogated. Symplostatin 5 (1) attenuated the effects of elastase on cell death,
detachment, genome-wide transcript changes as well as proteolytic processing of
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BIOGRAPHICAL SKETCH
Lilibeth Apo Salvador was born in Quezon City, Philippines. She received her
Bachelor of Science in chemistry at the University of the Philippines – Diliman in 2000.
On the same year, she became a qualified chemist and joined the Marine Science
Institute at the University of Philippines – Diliman as science research specialist, under
the supervision of Professor Gisela P. Concepcion and Professor Amelia P. Guevara.
She worked with the Antibody and Molecular Oncology Research (AMOR) program and
the National Cooperative Drug Discovery Group (NCDDG) on the discovery of
anticancer therapeutics from Philippine plants and marine sponges. During this time,
she developed a strong interest on natural products-intiated drug discovery. Lilibeth
finished her Master of Science in chemistry in 2006 and subsequently served as
research and development consultant for Euro-Med Laboratories Inc. and TEDA
Pharmaceuticals Inc. She joined the Department of Medicinal Chemistry, College of
Pharmacy at the University of Florida in 2008, under the mentorship of Professor
Hendrik Luesch. Lilibeth worked on the purification and structure determination of novel
secondary metabolites from marine cyanobacteria as well as elucidation of the
mechanisms of action and pharmacokinetics of cyanobacterial-derived compounds. She
received her Ph.D. in pharmaceutical sciences – medicinal chemistry from the