Semi-synthesis of sclerotiorin derivatives for tau aggregate inhibition and antifungal activity

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Semi-synthesis of sclerotiorin derivatives for tau aggregate inhibition and antifungal activity

by

Patrick Lehman

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
PHARMACEUTICAL SCIENCES

August 2020

Copyright 2020 Patrick Lehman
ii
TABLE OF CONTENTS

List of Tables……………………………………………………………………………………iv

List of Figures…………………………..…………………………………………………………v

Abstract…………………………………………………………………………………………...vi

Introduction:
Natural products importance in medicine…………………………………………………1
Why natural products are suitable drug leads……………………………………………..1
Fungal secondary metabolites……………………………………………………………..2
Biosynthetic gene clusters ……………………………………………….………………..2
Fungal polyketide synthases……………………………………………………...……….3
Tailoring Enzymes………………………………………………………………………...3

Synthesis of diverse Sclerotiorin analogs via acylation and N-substitution
Project objectives and specific aims………………………………………………………4
Background and significance……………………………………………………………...............5
Azaphilones: A class of fungal polyketides……………………………………………….5
Sclerotiorin is a unique bioactive azaphilone……………………………………..............5
Tau aggregate inhibition by sclerotiorin analogs………………………………………....6
Significance of sclerotiorin analogs as potential tau aggregate inhibitors………...............6
Antifungal & antibacterial studies of sclerotiorin……………………………………....…7
Significance of sclerotiorin analogs as potential antifungal and antibiotic therapies……..7
Anticancer studies of sclerotiorin ………………………………………………………...8
Synthetic methodologies for producing sclerotiorin analogs…………………………...…8
Significance of semi-synthesis of (-)-sclerotiorin analogs………………………………...9

Preliminary Data
Synthesis of nitrogenated azaphilone analogs…………………………………………….9

Production of brominated sclerotiorin derivatives by Penicillium hirayamae grown on
KBr supplemented media………………...………………………………………………17

Future work………………………………………………………………………………………18

References………………………………………………………………………………………..20

Appendix……………………………………………………………………………………...….21
NMR of compound 1………………………………………………………………….…25
NMR of compound 2…………………………………………………………………….26
NMR of compound 3…………………………………………………………………….32
NMR of compound 4…………………………………………………………………….38
NMR of compound 5…………………………………………………………………….44
iii
NMR of compound 6…………………………………………………………………….46
NMR of compound 8…………………………………………………………………….50
NMR of compound 9…………………………………………………………………….55

iv

LIST OF TABLES

Table Number Title Page Number
1 1H NMR chemical shifts for
compounds 1-4
14
2 1H NMR chemical shifts for
compounds 5,6, 9
15
3 1H NMR chemical shifts for
compounds 7,8
16

v

LIST OF FIGURES

Figure Number Title Page Number
1
Visual representation of molecules
in chemical space
1
2 Schematic representation of
typical BGCs
2
3
Basic azaphilone oxygenated
bicyclic scaffold
5
4
Examples of diverse azaphilone
classes
5
6 Synthesized nitrogenated (-)-
sclerotiorin compounds
11
7
1H spectra of 3-aminobutanoic
acid sclerotioramine isomers
12
8
PS1D spectra of 3-aminobutanoic
acid sclerotioramine isomers
12
9
1H NMR spectra of racemic 2-
aminocaprylic acid by product
and 1
13
10
UV and MS data of P. hirayamae
extracts
17
11
Products from KBr-cultured P.
hirayamae
18

vi

Abstract

Natural products represent a unique and diverse area of chemical space which have yet to be
taken advantage of comprehensively. These compounds have served as a source for drug
discovery since ancient times and still represent a significant portion of modern medicines. Most
of these compounds are made by microorganisms and are secondary metabolites that have been
progressively selected by evolution. Optimization via evolutionary pressures has gifted these
compounds with properties that are commonly found in FDA approved drugs that synthetic
molecules often lack. With many diseases still without adequate treatments or medications that
are far too expensive science is turning back to natural products as a source for more economic
and environmentally safer drug lead candidates. In this work we harness the molecular
manufacturing abilities of P. hirayamae which produces biomedically relevant metabolites (-)-
sclerotiorin and rubrorotiorin. After extraction and isolation from the fungus these compounds
can be derivatized conveniently with 1-step reactions. Here we have made a chemical library by
inserting primary amines with diverse and “drug-like” substituents into the 4H-pyran ring of (-)-
sclerotiorin. These compounds were generally made in good yields and have increased water
solubility. These compounds will be used in tau aggregate inhibition assays for Alzheimer’s
disease treatment and antifungal assays against fluconazole resistant and non-fluconazole
resistant strains of C. Albicans, for which sclerotiorin and related derivatives have already shown
to be effective. In addition to semi-synthesis, growing P. hirayamae on potassium bromide
supplemented media yielded a novel compound, rubrorotiorin brominated at the C-5 position
vii
instead of the usual chlorine substituent. This compound is also medically relevant as closely
related compounds have shown to have potent bioactivities in antitumor, antifungal, tau
aggregate inhibition, and gp-120-CD4 interaction inhibition (HIV). Future work will involve
genetic engineering to produce mutant strains of P. hirayamae which make more reactive de-
acetylated (-)-sclerotiorin for derivatization.

1
Introduction
Natural products importance in medicine Natural products (NPs) account for more than one
third of all new molecular entities (NMEs) approved by the FDA.35 From 1981-2014, NP-
derived analogs were approved over 4.5 times more than NPs themselves and in the case of anti-
infectives NP-analogs gained approval over 6 times as much.36 In fact, nearly 70% of all anti-
bacterial agents are NPs and almost all are derived from microbial sources.35 Recent discovery of
new antibiotics is not meeting the challenge of antibiotic resistance.37 Unfortunately, many
organizations have been choosing to ignore the antibiotic resistance crisis for economic
reasons.38 In this same time period (1981-2014), more than half of FDA approved cancer drugs
were NPs or their derivatives.36 Among all NP drug sources, the strains Streptomyces,
Penicillium, Cephalosporium, Salix, Guaiacuum, and Digitalis contribute significantly more than
any others.35 Although much of public interest has shifted towards newer technologies over NPs
because of their drawbacks concerted discoveries in biosynthesis, semi-synthesis, and total
synthesis of NPs have established them as viable candidates for drug candidates.
Why natural products are suitable drug leads. In the search for drug leads, it is crucial to
maximize the molecular diversity of screened
molecules.1 Molecular diversity is the
distribution of compounds across an N-
dimensional space, which is determined by N
molecular descriptors.2 (Figure 1) There are
many molecular descriptors of interest, such
as molecular weight or total polar surface
area2, but bioactivity is a requisite molecular descriptor for potential drug leads. Natural products
(NPs) meet this requirement because of their inherent interactions with biomolecules, making
them an invaluable source for drug lead screening. Qualities such as more prevalent ring content,
oxygen content, high Fsp3, and stereochemical content, along with low aromaticity, distinguish
NPs from molecules found in commercial synthetic libraries.3,4 Many of these properties are
correlated with further progression in clinical trials.5,6 An explanation for NP's characteristics
and subsequent clinical success is their evolutionary origin. Along with the inherent protein
interaction, they must possess a certain degree of bioavailability. Many organisms use
metabolites as chemical messengers, and thus evolution has optimized these natural products'
Figure 1. Visual representation of molecules in
chemical space. Examples of non-diverse and diverse
leads in a molecular collection sorted by chemotype.1
2
ability to permeate cell membranes.4 These structures are "emergent" through millions of years of
evolution, making their design challenging for scientists.7 As a result of this optimization, NPs
have higher intrinsic complexity, measured by structural topology8, heteroatomic and
stereochemical content9, or more sophisticated methods10 making their total synthesis
exceptionally challenging.11 NPs circumvent this issue of synthetic complexity since they
manufacture molecules with catalytic biosynthesis.
Fungal secondary metabolites. Fungal secondary metabolites (SMs) are of interest for drug
discovery efforts because of a longstanding genetic/chemical connections to humankind and
newer technologies capable of exploiting this connection.12, 13 SMs are defined as compounds
that are made by the polymerization of primary metabolites into more complex structures, i.e.
polyketides and terpenes.12 Such compounds are ordinarily produced in response to
environmental stimuli14, often not produced under standard lab conditions, but investigators have
isolated them with testing variable growth mediums/conditions15 and extensive genetic work.16
Biosynthetic gene clusters. Most SMs are the result catalytic transformations by the gene
products of a biosynthetic gene cluster (BCG). The genes in BCGs are located nearby each other
in bacterial and fungal genomes, in contrast to primary metabolite genes which sprawl across
genomes.17 Algorithms have successfully identified BGCs in annotated genomes, but it is
necessary to confirm the boundaries of the BGC experimentally (gene deletion) as the prediction
is only an estimation.12 Almost every BGC encodes for a large multidomain core or backbone
enzyme that polymerizes primary metabolites into a conjugated product like an assembly line.12
Often there is more than one backbone (two to three) enzyme that can be separate18 or part of a
fused system19. In addition to these enzymes BGCs often contain zero to several protective and
hypothetical genes.12
Fungal polyketide synthases. Fungal polyketides (PKs) are the product of polyketide synthases
(PKSs), which are organized into groups of nonreduced (NR), partially reduced (PR), and highly
reduced (HR) based on the specific types of domains encoded.20 NR-PKSs have a starter unit
Figure 2. Schematic representation of typical BGCs.12
3
ACP transacylase (SAT), B-ketoacyl synthase (KS), acyltransferase (AT), product template (PT),
acyl carrier protein (ACP), and Claisen-cyclase/thioesterase (CLC/TE) domains. Additionally,
NR-PKS may have reductase (R) and methyltransferase (CM) domains. PR-PKSs differ in that
they have the common KS, AT, and ACP domains but additionally contain dehydratase (DH)
and ketoreductase (KR) domains. Similarly, HR-PKSs contain KS, AT, terminating ACP, and
DH domains, but the latter is often followed by CM, enoyl reductase (ER), and KR domains.21
Many PKS products are the result of one PKS enzyme (type I PKs) due to their iterative nature,
but some BGCs encode for multiple PKSs to synthesize more complex products. In this process,
the first PKS, often an HR-PKS, synthesizes a PK unit and transfers it to a second PR- or NR-
PKS for further elongation.20 These hybrid structures often react further spontaneously or with
the help of tailoring enzymes, resulting in a variety of complex structures.
Tailoring Enzymes. Tailoring enzymes are complimentary and are generally the most numerous
class of genes in any BGC.12 The intricate selectivity’s and significant chemical transformations
that tailoring enzymes perform contributing to the diversity of NPs cannot be understated.22
There are two main groups of tailoring enzymes in NP synthesis. 1) Group transfer enzymes
typically catalyze a coupling between an electrophilic moiety from a co-substrate or primary
metabolite to a nucleophilic atom, i.e. N, O, or S, contained in the NP. Examples of group
transfer reactions incorporated in antibiotic NPs include but are not limited to N-methylation23,
O-sulfonation24, and glycosylation25. Glycosylation highlights the significant impact that group
transfer enzymes have on NPs because of their ability to enhance water solubility22 and impart
specific interactions with biomolecules responsible for bioactivity26. Group transfer enzymes
may also exist as embedded enzymes in a backbone assembly line.27 2) Oxidative tailoring
enzymes utilize O2 to catalyze unfavorable O2 reductions on NP substrates via
cofactors/enzymes22. Heme28 and non-heme29 oxygenases carry out regioselective
hydroxylations30, epoxidations31, and ring formations32. Similarly, flavin-dependent oxygenases
use an unstable hydroperoxyl to install oxygens onto aromatic rings22, cleave C-C bonds regio-
and stereo-selectively to remodel polycyclic frameworks33,34.

In the following work, we explore semi-synthetic/biosynthetic approaches to generate novel
analogs of a hybrid PKS molecule, Sclerotiorin, that has been shown to possesses diverse and
potent bioactivities.
4

Synthesis of diverse Sclerotiorin analogs via acylation and N-substitution
Project objectives
Findings from previous studies suggested that the core bicyclic ring and 3,5-dimethyl-1,3-
heptadiene structural features of the NP sclerotiorin are essential for its various bioactivities.
From this we hypothesize that semi-synthetic analogs, maintaining these features, complimented
by derivatization at the tetrahedral C-7 position and N-insertions into the 4H-pyran ring will
show increased bioactivity important to neurodegenerative, antibiotic, and anticancer treatments.
To investigate the relationship between (-)-sclerotiorin derivatives and their bioactivities we will
synthesize diverse libraries with drug-like properties and test their bioactivities in tau aggregate
inhibition and antifungal activity against C. Albicans.

Specific Aims

1. Semi-synthesize derivatives of (-)-sclerotiorin, isolated from Penicillium hirayamae, with
primary amines substituted into the 4H-pyran core and substitutions at the C-7 positions
via acylations to screen for lead molecules in tau aggregate inhibition and antifungal
activity in C. Albicans.
2. Grow Penicllium hirayamae on altered growth medium to biosynthesize bromine
containing (-)-sclerotiorin and related metabolite, rubrorotiorin, to increase the diversity
of our chemical library.

5

Background and Significance
Azaphilones: A class of fungal polyketides. Azaphilones are a structurally diverse class of
fungal polyketides that are characterized by a highly oxygenated, conjugated, pyranoquinone
bicyclic core that bears a quaternary carbon which breaks aromaticity.39 (Figure 1) Azaphilones
are known for their ability to react with primary amines to form vinylogous -pyridones, which
are observed as red pigments.40, 41 Azaphilones are produced
mostly by ascomycetes and are responsible for the yellow to
green colors seen in the mycelia in these fungi.42 Many
azaphilones are made specifically by a single species and have
been used as a marker for genetic categorization.43 Azaphilones
show binding to various enzymes of interest39, including
the MDM2−p53 interaction inhibitor44 Chlorofusin which bears a nine membered cyclic peptide
linked via an ornithine side chain and novel gp-120-CD4 inhibitors45. Additionally, azaphilones
possess antimicrobial, antiviral, cytotoxic, anticancer, and anti-inflammatory activities.41,46 The
elaborate structural diversity of azaphilones makes a comprehensive chemotype categorization
beyond the scope of this proposal. However, highlighting a few broad classes displays the
number and continuum of molecular descriptors in which azaphilones encompass. (Fig 2.)
Polycyclic azaphilones come in two varieties 1) linear47 and 2) angular48. Another broad class is
that of 3) dimers which can be symmetric/asymmetric and may be linked through alkyl bonds49
or N-linked56. Finally, the last class of azaphilones can be identified as 4) those that contain a -
pyridone core from N-substitution into the 4H-pyran oxygen core (sclerotioramine analogs).50

Sclerotiorin is a unique bioactive azaphilone. Sclerotiorin and its related azaphilones are known
for their chlorine C-5 and branched 3,5-dimethyl-3,5-heptadiene C-3 substitutions on the core
azaphilone bicycle. (+)-sclerotiorin, first isolated in 1940 from Penicillium sclerotirium Van
Figure 4. Examples of diverse azaphilone classes47,
Figure 3. Basic azaphilone
oxygenated bicyclic scaffold
6
Beyma51, had its absolute stereochemistry determined X-ray crystallography of its derivative, N-
methylsclerotioramine52. They are predominantly isolated from Penicillium species39 and are
isolated along with sclerotioramine NPs frequently53,54. P. hirayamae produced (-)-sclerotiorin
along with a red pigment, rubrorotiorin in 1971.55 Unfortunately, many of sclerotiorin-related
studies are redundant. We aim to break this trend by using semi-synthesis of sclerotiorin to
diversify the structure activity relationships that sclerotiorin and related azaphilones have. While
there are overlapping and similar activities seen by both stereoisomers, (-)-sclerotiorin is the least
represented by the two. It is beneficial we contribute (-)-sclerotiorin analogs to identify the major
differences, or lack thereof, in bioactivity between the isomers. While confirming/building upon
previous knowledge we synthesizing libraries to find new lead molecules of pharmacological
interest.

Tau aggregate inhibition by sclerotiorin analogs. Alzheimer’s disease (AD) is the leading cause
of dementia and was pronounced the “global health priority” twice in the past decade by the
WHO.74,75 Targeting neurofibrillary tangles or tau aggregation has proven to be a viable
treatment option.76 Work in our lab has previously investigated fungal NPs asperbenzaldehyde77,
sclerotiorin, and related derivatives78 as lead molecules for tau aggregation inhibitors. These
studies showed that chlorinated/brominated and acetyl/deacetyl groups (at C-7) sclerotiorin
derivatives were able to inhibit tau aggregation significantly more than derivatives in which the
C-8 ketone was replaced with an ethyl acrylate group.

Significance of sclerotiorin analogs as potential tau aggregate inhibitors. Tau aggregation is
indicated in many neurological disorders and finding inhibitors to alleviate its related pathology
has become a popular therapeutic target. Inhibition of tau aggregates may treat AD by stopping
any further polymerization or by increasing the likelihood of monomers of tau being cleared by
normal cellular processes. Our work will further determine the SAR between the C-7
ester/hydroxy moiety, the substitution at the C-8 position, and the subsequent ability to inhibit
tau aggregation. Additionally, we will use altered growth media to produce brominated
sclerotiorin analogs, as well as, rubrorotiorin and Br-rubrorotiorin to investigate the relationships
between C-7 ester/C-8 ketone composition. Most importantly, our work will test the first
nitrogenated azaphilones in tau aggregate inhibition. We aim to determine if there is any
7
inhibitory activity gained from the N-substituted 4H-pyran ring core by synthesizing a
moderately diverse library.

Antifungal & antibacterial studies of sclerotiorin. In 2010, (-)-sclerotiorin was administered to
a panel of medically and agriculturally important fungal sp., where it showed moderate
antifungal activity (< 20 g/mL) for most strains.57 A synthesis based effort to generate
sclerotiorin analogs highlighted the importance of substitution of a halogen, chorine or bromine,
at the C-3 position and suggested that their C-7 hydroxy analogs has more antifungal activity
than the acetylated versions.59 Recently, antifungal and antibacterial assays suggested that the
6,8-diketone bicyclic core and 3,5-dimethyl-1,3-heptadiene moieties were critical for
sclerotiorin’s inhibitor activities.62 Screening for NP inhibitors of fatty acid synthase (FAS)
found that two related azaphilones with an unbranched heptatriene chain at C-3 and chiral 3-
hydroxypropyl groups linked to C-7 via an ester, displayed antifungal activity towards C.
albicans and selectively inhibited the Candida FAS enzyme over the human homologue.63
Interestingly, two of the azaphilones isolated in the study displayed an C-1,8a epoxy but only the
azaphilone containing an 3-hydroxypropyl-ester on C-7 had the C. albicans/FAS inhibition.
While sclerotiorin did not individually inhibit growth of M. tuberculosis, it did show moderate
inhibitory activity to a regulatory kinase and in combination with rifampicin inhibited growth.61
In 2019, a linear tricyclic, N-aniline analog of sclerotiorin was found to inhibit the growth of E.
coli, S. aureus, and C. albicans (IC50 = 2.95, 3.00, and 10.5 respectively).58 A library of N-
containing , or sclerotioramine, analogs were synthesized but tested only for their antifouling
activity. This is the largest published library of sclerotioramine analogs and it concluded the
majority of alkyl derivatives failed to show antifouling activity compared to the aromatic.60

Significance of sclerotiorin analogs as potential antifungal and antibiotic therapies
Fungal infections, e.g. C. albicans, contribute significantly to human diseases, especially among
the immunocompromised.72 The shared Eukaryotic lineage between humans and fungi
exacerbate this problem by making it difficult to find novel targets in fungal pathogens.72,73
However, derivatives similar to sclerotiorin showed selective inhibition of fungal FAS enzymes.
These derivatives contained a chiral alkyl chain at the C-7 ester position. Our efforts will explore
similar derivatives and establish an SAR, pertaining to the substitution to the C-7 ester. Synthesis
8
of derivatives with drug-like properties will be used to determine the increase or decrease in
antifungal ability. Additionally, N-aniline type derivatives will be synthesized to determine the
effect their substitution has on their antifungal activity to further the SAR for sclerotiorin and C.
Albicans. Sclerotioramine derivatives with a phenyl moiety, in lieu of aniline, will also
interrogate the SAR of the N-aromatic ring sclerotiorin derivatives.

Anticancer studies of sclerotiorin. Anti-cancer/tumor activities of sclerotiorin present important
information about the molecules bioactivity and how the NP can affect organisms at the cellular
level. (-)-sclerotiorin has been found to inhibit the maturation of activated starfish oocytes with
an IC50 of 0.5 M, suggesting that it inhibits cell cycle progression by interacting with protein
kinases/binding partners involved in cell cycle regulation.57 An in cell assay showed that O-
methylsclerotioramine, a closely related derivative, significantly inhibited binding between the
Grb-sh2 proteins.61 This is of interest because the nitrogenated sclerotiorin derivative is
structurally distinct to other inhibitors of this protein-protein interaction. In 2012, sclerotiorin
showed selective cytotoxicity for multiple cancer lines, especially in the case of HCT-116 colon
cancer cells (IC50 = 0.63 M), over normal human breast epithelium cells and induced apoptosis
via an increase in caspase-3 expression. Western blot analysis showed up and down regulation of
BAX and BCL-2 proteins in sclerotiorin treated cells.64 Clearly sclerotiorin can make significant
contacts with multiple proteins and in this way is an attractive drug candidate due to its far
reaching cellular impacts.

Synthetic methodologies for producing sclerotiorin analogs. To diversify the azaphilone
scaffold, groups have undergone total synthetic efforts to produce sclerotiorin scaffolds with
different substituents. The total synthesis of azaphilones is based on Sonogashira coupling an
alkyne with a 2-bromobenzaldehyde to generate an o-alkynylbenzaldehyde81 which undergoes
transition metal/Lewis acid mediated cycloisomerization82 to a 2-benzopyrilium salt. This salt is
then reacted with IBX to yield the desired isochromene core.79 There are a few common reaction
strategies, after the synthesis of the isochromene bicycle, that have been employed to diversify
azaphilones. Methods for preparing nitrogenated sclerotiorin analogs have been documented
before by Boger44 and Porco79 employing solvents such as triethylamine or a mixture of
DCM/ACN and DMF with an additive like K2CO3. Generally these reactions are carried out with
9
good to excellent yields. Derivatization of the C-7 hydroxy to an ester substituent has been
carried out with good results. Often these reactions use acids or acid chlorides and DMAP, again
with good to excellent yields resulting.79 Other reactions have been performed on the azaphilone
scaffold, usually in total synthetic efforts, to derivatize the C-3 and C-5 substituents. These
reactions have lower yields79,80 and often they do not improve the azaphilones potency.80

Significance of semi-synthesis of (-)-sclerotiorin analogs. While there have been
synthetic/semi-synthetic studies done before on azaphilones and sclerotiorin often these studies
do not keep the chiral C-3 3,5-dimethyl-1,3-heptadiene branch intact.79,80 By changing this
moiety the compound is likely losing key features that affect its binding macromolecules due to
elimination of sp3 hybridized atoms and rotatable bonds3,4. Our syntheses will involve acylation
at the C-7 hydroxy position and N-substitutions which have yet to be well represented in
neurodegenerative, antibiotic, and anticancer studies. By utilizing fungal cultures to produce our
core scaffold library collection is more chemically economic and greener than total synthesis.
Moreover, our semi-synthesis has the advantage of producing stereochemically pure (-)-
sclerotiorin, which in general has not shown major differences from the (+)-isomer. These
analogs will contribute to the field by confirming this trend or discovering new differences in
bioactivities between the isomers. Finally our libraries will be compromised of C-7 ester and
sclerotioramine derivatives with drug-like properties3,4, i.e. substituents with high Fsp3,
tetrahedral carbons, and rotatable bonds, in effort to identify changes that bring about new or
more potent bioactivities. The molecular diversity of our library will be complimented by
synthesis of analogs bearing differences in substitution pattern and functionality.
Preliminary Data – Synthesis of nitrogenated azaphilone analogs
Consistent with literature, reactions substituting primary amines into the 4H-pyran ring went
smoothly with good to excellent yields. Reaction mixtures used different mixtures of
dichloromethane and acetonitrile with varying amounts of DMF or DMSO (0-100%). Phenyl
amine proved to be the most efficient reaction being isolated in a 95 % yield. While the addition
of racemic 3-aminobutyric acid generated two diastereomers of the desired product (figure 4),
the reaction of 2-aminocaprylic acid only generated a one desired stereoisomer in a significant
yield. A byproduct of the reaction appeared to be the explanation for the missing isomer. This by
product showed a slightly lower molecular weight, the acidic proton shifted upfield, and the
10
appearance of additional aromatic hydrogens peaks. It may be that one stereoisomer is able to
interact with (-)-sclerotiorin in a manner that facilitates a cycloisomerization. The amino acids L-
isoleucine and L-glycine methyl ester produced the novel sclerotioramines and the observed
spectra compare well to the naturally occurring L-leucine-sclerotioramine.83 A sclerotioramine
derivative with a -butyric acid moiety had been isolated before from Penicillium sp.84 and our
obtained spectra are in agreement. Pure shift, NOESY, HSQC, HMBC, and COSY experiments
were used to help confirm the structures. Two anilic derivatives were made in good yields with
one bearing an p-carboxylic acid pattern and the other an m,p-carboxylic acid, phenol
substitution. Structures, their yields, and 1H spectra are shown in figures and tables below. NMR
spectra are attached at the end of the proposal as supplemental. We have successfully
synthesized nitrogenated (-)-sclerotiorin analogs with increased drug-like substituents that
represent a diverse distribution of chemical space by incorporating chemical moieties such as
peptide amino acids, , , and -amino acids, amino-acid ester, phenyl, and aniline structures
with varying substitution in type and pattern. Many of these structures added advantageous
properties for potential drugs such as increased water solubility, hydrogen bond
donors/acceptors, oxygen content, rotatable bonds, and high Fsp3.

Scheme 1. Reaction scheme depicting the syntheses of (-)-sclerotioramine derivative
11

Figure 6. Synthesized nitrogenated (-)-sclerotiorin compounds

12

Figure 7. Superimposed 1H spectra of two isolatable isomers of the 3-aminobutanoic acid sclerotioramine
derivatives.

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 .0 1 .1 1 .2 1 .3 1 .4 1 .5 1 .6 1 .7 1 .8 1 .9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4
f1 (ppm )
-200
0
200
400
600
800
1 000
1 200
1 400
1 600
1 800
2000
2200
2400
2600
2800
P R O T O N _ 01 — scle ro_ 1 01 7_ 1 _ fin a l_ 1 2091 9 —
0.87
0.88
0.90
1 .02
1 .03
1 .32
1 .33
1 .35
1 .36
1 .37
1 .42
1 .43
1 .44
1 .45
1 .46
1 .50
1 .51
1 .52
1 .87
2.1 4
2.45
2.46
2.48
2.49
2.50
2.80
2.81
2.83
2.84
2.90
2.91
2.92
2.94
0.6 0.7 0.8 0.9 1 .0 1 .1 1 .2 1 .3 1 .4 1 .5 1 .6 1 .7 1 .8 1 .9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0
f1 (ppm )
-2
-1
0
1
2
3
4
5
6
7
8
9
P S 1 D _ 01 — scle ro_ 1 01 7_ 1 _ fin a l_ 1 2091 9 —
0.88
1 .02
1 .52
1 .86
2.1 3
2.47
Figure 8. Superimposed PS1D spectra of the two isolatable isomers of the 3-aminobutanoic acid
sclerotioramine derivatives.
13

Figure 9. 1)1H NMR spectra of the byproduct of reacting sclerotiorin with racemic 2-aminocaprylic acid (m/z
= 517.24). 2) 1H NMR spectrum of compound 1 (m/z = 531.24)

14
Table 1. 1H NMR chemical shifts for compounds 1-4. Shift (m, J, H)

-N- position − −position − −position − −position
position 1 2 3 4
1 7.96 (s, 1H) 7.89 (s, 1H) 7.92 (s, 1H) 7.83 (s, 1H)
4 7.03 (s, 1H) 6.99 (s, 1H) 6.97 (s, 1H) 7.09 (s, 1H)
9 6.14 (d, J = 17.3 Hz, 1H) 6.42 (d, J = 15.3 Hz, 1H) 6.43 (d, J = 15.3 Hz, 1H) 6.31 (d, J = 15.3 Hz, 1H)
10 6.88 (d, J = 15.2 Hz, 1H) 6.88 (d, J = 15.3 Hz, 1H) 6.89 (d, J = 15.3 Hz, 1H) 7.01 (d, J = 15.2 Hz, 1H)
12 5.70 (d, J = 10.2 Hz, 1H) 5.69 (d, J = 9.7 Hz, 1H) 5.68 (d, J = 9.7 Hz, 1H) 5.73 (d, J = 9.7 Hz, 1H)
13 2.48 (m, 1H) 2.48 (m, 1H) 2.48 (m, 1H) 2.47 (m, 1H)b
14 1.44 (m, 1H)* 1.44 (m, 1H) 1.44 (m, 1H) 1.44 (m, 1H)

1.33(m, 1H)* 1.35 (m, 1H) 1.34 (m, 1H) 1.34 (m, 1H)
15 0.88 (t, J = 7.1 Hz, 3H)* 0.88 (t, J = 7.4 Hz, 3H) 0.88 (t, J = 7.4 Hz, 3H). 0.88 (t, J = 7.4 Hz, 3H)
16 1.02 (d, J = 6.6 Hz, 3H) 1.02 (d, J = 6.6 Hz, 3H) 1.03 (d, J = 6.6 Hz, 3H) 1.03 (d, J = 6.7 Hz, 3H)
17 1.86 (s, 3H) 1.87 (s, 3H) 1.87 (s, 3H) 1.88 (s, 3H)
18 1.56 (s, 3H) 1.52 (s, 3H) 1.55 (s, 3H) 1.55 (s, 3H)
19 2.16 (s, 3H) 2.14 (s, 3H) 2.12 (s, 3H) 2.15 (s, 3H)
1' - - - -
2' 4.85 (t, 1H) 2.92 (dd, J = 16.7, 7.6 Hz, 1H) 2.95 (dd, J = 16.9, 7.8 Hz, 1H) 2.51 (t, 2H)b

- 2.82 (dd, J = 16.4, 5.4 Hz, 1H) 2.79 (dd, J = 17.0, 6.1 Hz, 1H) -
3' 2.29 (m, 1H)* 4.91 (q, J = 6.5 Hz, 1H) 4.89 (q, J = 6.7 Hz, 1H) 2.07 (m, J = 14.2, 7.2 Hz, 2H)
2.02 (m,1H)* - - -
4’ 1.44-1.26 (m, 2H)* 1.50 (d, J = 6.4 Hz, 3H) 1.51 (d, J = 6.8 Hz, 3H) 3.99 (t, J = 7.7 Hz, 2H)
5’ 1.44-1.26 (m, 2H)* - - -
6’ 1.44-1.26 (m, 2H)* - - -
7’ 1.44-1.26 (m, 2H)* - - -
8’ 0.90-0.86 (t, 3H)* - - -

*= overlapping peaks
15
Table 2. 1H NMR chemical shifts for compounds 1-4. Shift (m, J, H)

position 5 6 9
1 8.10(s, 1H) 7.86 (s, 1H) 7.91 (s, 1H)
4 6.99 (s, 1H) 7.12 (s, 1H) 7.13 (s, 1H)
9 6.24 (d, J = 17.3 Hz, 1H) 5.97 (d, J = 15.3 Hz, 1H) 6.04 (d, J = 15.4 Hz, 1H)
10 6.89 (d, J = 15.2 Hz, 1H) 6.97 (d, J = 15.2 Hz, 1H) 6.93 (d, J = 15.3 Hz, 1H)
12 5.75 (d, J = 9.7 Hz, 1H) 5.75 (d, J = 9.8 Hz, 1H) 5.66 (d, J = 9.7 Hz, 1H)
13 2.49 (m, 1H) 2.48 (m, 1H) 2.43 (m, 1H)
14 1.46 (m, 1H) 1.48 (m, 1H) 1.41 (m, 1H)

1.35(m, 1H) 1.39 (m, 1H) 1.33 (m, 1H)
15 0.88 (t, J = 7.4 Hz, 3H) 0.87 (t, J = 7.4 Hz, 3H) 0.84 (t, J = 7.4 Hz, 3H).
16 1.03 (d, J = 6.7 Hz, 3H) 1.02 (d, J = 6.6 Hz, 3H) 1.00 (d, J = 6.6 Hz, 3H)
17 1.86 (s, 3H) 1.81 (s, 3H) 1.67 (s, 3H)
18 1.58 (s, 3H) 1.59 (s, 3H) 1.59 (s, 3H)
19 2.15 (s, 3H) 2.17 (s, 3H) 2.18 (s, 3H)
1' - - -
2' 4.49 (d, J = 10.4 Hz, 1H) 4.67 (dd, J = 10.8, 7.6 Hz, 2H) 7.18 (d, J = 7.4, 1H)

- - -
3' 2.31 (m, 1H) - 7.45-7.36 (m, 1H)
4’ 1.35 (m, 2H)* - 7.45-7.36 (m, 1H)
5’ 0.86 (m, 3H)* - 7.45-7.36 (m, 1H)
6’ 1.11 (d, J = 6.5 Hz, 3H)* - 7.18 (d, J = 7.4, 1H)
1’’ - 3.83 (s, 3H) 5.08 (s, 1H)

*= overlapping peaks
16

Table 3. 1H NMR chemical shifts for compounds 1-4. Shift (m, J, H)
position 7 8
1 7.85 (s, 1H) 7.97 (s, 1H)
4 7.21 (s, 1H) 7.32 (s, 1H)
9 5.72 (d, J = 15.5 Hz, 1H) 5.68 (d, J = 15.5 Hz, 1H)
10 7.02 (d, J = 15.5 Hz, 1H) 7.06 (d, J = 15.5 Hz, 1H)
12 5.72 (d, J = 9.6 Hz, 1H) 5.75 (d, J = 9.7 Hz, 1H)
13 2.38 (m, 1H) 2.43 (m, 1H)
14 1.40 (m, 1H) 1.42 (m, 1H)
1.29 (m, 1H) 1.32 (m, 1H)
15 0.84 (t, J = 7.4 Hz, 3H) 0.84 (t, J = 7.4 Hz, 3H)
16 0.99 (d, J = 6.6 Hz, 3H) 1.00 (d, J = 6.6 Hz, 3H)
17 1.61 (s, 3H) 1.64 (s, 3H)
18 1.52 (s, 3H) 1.56 (s, 3H)
19 2.19 (s, 3H) 2.18 (s, 3H)
1’ - -
2’ 7.44 (d, J = 8.7 Hz, 1H) 7.90 (s, 1H)
3’ 8.28 (d, J = 8.6 Hz, 1H) -
4’ - -
5’ 8.28 (d, J = 8.6 Hz, 1H) 7.15 (d, J = 8.7 Hz, 1H)
6’ 7.44 (d, J = 8.7 Hz, 1H) 7.37 (d, J = 8.1 Hz, 1H)

17
Preliminary Data – Production of brominated sclerotiorin derivatives by Penicillium
hirayamae grown on KBr supplemented media
P. hirayamae was grown on a modified Czapek-Dox medium (50g glucose, 2g NaNO3, 1g
KH2PO4, 0.5g KCl, 0.5g MgSO4 x 7H2O, 0.01g FeSO4 x 7H2O, 0.01g ZnSO4 x 7H2O, 0.005g
CuSO4 x 5H2O) with potassium bromide substituted for the normal potassium chloride.
Consistent with recent literature85, our KBr-supplemented media stimulated P. hirayamae to
produce (-)-sclerotiorin, rubrorotiorin, multipole intermediates, along with bromine containing
derivatives bromo-sclerotiorin and novel metabolite bromo-rubrorotiorin which has not been
reported in literature to our knowledge.

Figure 10. A) UV of P. hirayamae culture extracts grown on KBr supplemented Czapek-Dox media. B) TIC of P.
hirayamae culture extracts grown on KBr supplemented Czapek-Dox media. C) UV of P. hirayamae culture extracts
grown on Czapek-Dox media. D) TIC of P. hirayamae culture extracts grown on Czapek-Dox media.

C:\Users\...\p_hirayamae 19d\EA_x5 1/24/2019 1:53:57 PM
RT: 29.85 - 40.28
30 31 32 33 34 35 36 37 38 39 40
Time (min)
0
50
100
0
50
100
0
50
100
Relative Abundance
0
100000
NL: 1.94E5
Total Scan PDA EA
NL: 2.22E8
TIC F: + c ESI Full
ms [
100.00-1500.00]
MS EA
NL: 8.52E5
Total Scan PDA
EA_x5
NL: 2.45E9
TIC F: + c ESI Full
ms [
100.00-1500.00]
MS EA_x5
500 1000 1500
m/z
0
1000000
2000000
0
50
100
Relative Abundance
391.33
349.45
417.42
802.75 967.18 1390.10
435.42
391.38
349.47
497.69 784.91 1134.59 1333.54
NL: 2.64E7
EA#939 RT: 35.77 AV: 1 T: + c ESI
Full ms [ 100.00-1500.00]
NL: 2.89E6
EA#941-946 RT: 35.84-36.00 AV: 3
SB: 12 36.04-36.58 , 35.50-35.88 T:
+ c ESI Full ms [ 100.00-1500.00]
500 1000 1500
m/z
0
50
100
0
50
100
Relative Abundance
415.52
345.54
439.48 853.12
1315.73 998.69
459.48
481.32
391.51 940.90
1103.02 1417.40
NL: 1.53E7
EA#973 RT: 37.08 AV: 1 SB: 10
36.00-36.42 , 35.58-35.84 T: + c
ESI Full ms [ 100.00-1500.00]
NL: 1.21E7
EA#981 RT: 37.39 AV: 1 T: + c
ESI Full ms [ 100.00-1500.00]
C:\Users\...\p_hirayamae 19d\EA_x5 1/24/2019 1:53:57 PM
RT: 29.85 - 40.28
30 31 32 33 34 35 36 37 38 39 40
Time (min)
0
50
100
0
50
100
0
50
100
Relative Abundance
0
100000
NL: 1.94E5
Total Scan PDA EA
NL: 2.22E8
TIC F: + c ESI Full
ms [
100.00-1500.00]
MS EA
NL: 8.52E5
Total Scan PDA
EA_x5
NL: 2.45E9
TIC F: + c ESI Full
ms [
100.00-1500.00]
MS EA_x5
500 1000 1500
m/z
0
1000000
2000000
0
50
100
Relative Abundance
391.33
349.45
417.42
802.75 967.18 1390.10
435.42
391.38
349.47
497.69 784.91 1134.59 1333.54
NL: 2.64E7
EA#939 RT: 35.77 AV: 1 T: + c ESI
Full ms [ 100.00-1500.00]
NL: 2.89E6
EA#941-946 RT: 35.84-36.00 AV: 3
SB: 12 36.04-36.58 , 35.50-35.88 T:
+ c ESI Full ms [ 100.00-1500.00]
500 1000 1500
m/z
0
50
100
0
50
100
Relative Abundance
415.52
345.54
439.48 853.12
1315.73 998.69
459.48
481.32
391.51 940.90
1103.02 1417.40
NL: 1.53E7
EA#973 RT: 37.08 AV: 1 SB: 10
36.00-36.42 , 35.58-35.84 T: + c
ESI Full ms [ 100.00-1500.00]
NL: 1.21E7
EA#981 RT: 37.39 AV: 1 T: + c
ESI Full ms [ 100.00-1500.00]
A
B
C
D
E
18

Figure 11. In addition to rubrorotiorin and (-)-sclerotiorin, P. hirayamae produces Br-sclerotiorin and novel
compounds Br-rubrorotiorin.

5. Future work
The nitrogenated azaphilones are novel and possess properties which resemble FDA-approved
drugs. These molecules will be tested with a collaborator who specializes in AD and tau
aggregation assays. From the KBr supplemented cultures of P. hirayamae we will isolate
bromine-rubrorotiorin and sclerotiorin to make analogs and test for tau aggregate inhibition, as
well. In addition, the analogs will be investigated for antifungal activity against drug resistant
and non-drug resistant strains of C. Albicans. These studies will help establish nitrogenated
sclerotiorin drug leads. More analogs of (-)-sclerotiorin will be made by acylating the C-7
hydroxyl after de-acetylating initially. The analogs made will aim to have substituents with drug
like properties, i.e. high Fsp3, rotatable bonds, and high oxygen content. At the moment our
strain of P. hirayamae has been sequenced and is being subjected to assembly/ANTISMASH
algorithms to identify BGCs. With this complete information we will perform genetic
engineering methodologies like CRISPR Cas-9 system or fusion PCR to knock out the
acetyltransferase gene responsible for acetylating the C-7 hydroxy of sclerotiorin. Biosynthesis
of the more active C-7 hydroxy sclerotiorin molecule will allow for a much more efficient and
greener generation of sclerotiorin analogs. Additionally, gene deletion of a halogenase in the
19
sclerotiorin BGC will be done to shed light on the significance of the C-5 chlorine atom. In
synthetic studies it has been shown that the type of tricycle, angular or linear, formed by
azaphilones is dependent on the presence of a halogen atom at this position.87 Therefore, we will
attempt to engineer a mutant P. hirayamae strain which produces the linear form of
rubrorotiorin. The basis for this genetic work is from analyzing azaphilones closely related to
sclerotiorin that have already had their biosynthesis elucidated, like Chaetoviridin A.86 Using this
background we can identify the sclerotiorin BGC and strategically delete genes to alter its
biosynthesis to produce more reactive or pharmaceutically interesting azaphilones.

Scheme 2. Future plans for genetic engineering of P. hirayamae. A) Deletion of acetyltransferase gene to
generate (-)-C7-hydroxy-sclerotiroin which can then be easily reacted with activated acids and non-
nucleophilic bases to yield acyl derivatives. B) Deletion of a halogenase enzyme in the sclerotiorin BGC to
yield linear, rotiorin which is a bioactive relative of sclerotiorin.

20

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25

1. Supplemental NMR spectra

S1. 1H NMR of compound 1

26

S2. 1H NMR of compound 2
0.0 0.5 1 .0 1 .5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm )
P R O T O N _ 01 — scle ro_ 1 01 7_ 1 _ fin a l_ 1 2091 9 —
2.91
2.83
1 .01
1 .23
2.86
3.03
2.90
2.96
1 .01
0.97
1 .01
1 .09
1 .00
0.96
1 .02
0.93
0.89
0.87
0.88
0.90
1 .02
1 .03
1 .32
1 .33
1 .35
1 .36
1 .37
1 .42
1 .43
1 .44
1 .45
1 .46
1 .50
1 .51
1 .52
1 .87
2.1 4
2.45
2.46
2.48
2.49
2.50
2.80
2.81
2.83
2.84
2.90
2.91
2.92
2.94
4.90
4.91
4.92
5.68
5.70
6.41
6.43
6.87
6.89
6.99
7.26 C D C l3
7.89
27

S3. 13C NMR of compound 2
28

S4. PS1D spectra of compound 2

29

S.5 COSY of compound 2
30

S.6 HSQC of compound 2
31

S. 6 gHMBC of compound 2

32

S7. 1H NMR of compound 3

1 .0 1 .5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm )
0
500
1 000
1 500
2000
2500
3000
P R O T O N _ 01 — scle ro_ 1 01 7_ 2_ 1 1 251 9 —
2.97
2.80
1 .1 8
1 .21
2.98
2.75
2.72
2.79
1 .04
0.95
0.93
1 .09
1 .00
1 .05
0.92
0.90
0.87
0.88
0.89
1 .02
1 .03
1 .30
1 .31
1 .33
1 .34
1 .34
1 .35
1 .36
1 .37
1 .41
1 .42
1 .43
1 .43
1 .44
1 .45
1 .45
1 .46
1 .48
1 .51
1 .52
1 .55
1 .87
2.1 2
2.45
2.46
2.47
2.48
2.50
2.51
2.77
2.78
2.80
2.81
2.93
2.94
2.96
2.97
4.87
4.88
4.89
4.91
5.68
5.69
6.41
6.44
6.87
6.90
6.97
7.26 C D C l3
7.92
33

S8. 13C spectra of compound 3

34

S9. PS1D spectra of compound 3
35

S10. COSY of compound 3

36

S11. gHSQC of compound 3
37

S12. gHMBC of compound 3

38

S13. 1H NMR spectra of compound 4.
0.0 0.5 1 .0 1 .5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm )
P R O T O N _ 02 — P L _ scle ro _ 1 01 5_ 2D _ 1 1 041 9 —
0.86
0.88
0.89
1 .02
1 .04
1 .32
1 .33
1 .34
1 .34
1 .35
1 .37
1 .42
1 .43
1 .43
1 .44
1 .45
1 .46
1 .55
1 .88
2.02
2.04
2.05
2.06
2.08
2.09
2.1 0
2.1 1
2.1 6
2.46
2.47
2.49
2.50
2.51
2.53
3.98
3.99
4.00
5.72
5.74
6.30
6.32
6.99
7.02
7.09
7.26 C D C l3
7.83
39

S14. 13C spectra of compound 4
40

S15. PS1D spectra of compound 4
41

S16. COSY of compound 4
42

S17. gHSQC of compound 4
43

S18. gHMBC of compound 4

44

S19. 1H NMR of compound 5

45

S20. gHBMC of compound 5

46

S21. 1H NMR of compound 6
47

S22. 13C spectra of compound 6

48

S23. gHSQC of compound 6

49

S.24 gHMBC of compound 6

50

S25. 1H NMR spectra of compound 8

51

S26. 13C spectra of compound 8
52

S27. COSY of compound 8
53

S28. HSQC of compound 8
54

S29. gHMBC of compound 8
55

S25. 1H NMR of compound 9

Asset Metadata

Creator Lehman, Patrick William (author)

Core Title Semi-synthesis of sclerotiorin derivatives for tau aggregate inhibition and antifungal activity

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 08/06/2020

Defense Date 08/06/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag antifungal,fungal,OAI-PMH Harvest,sclerotiorin,Secondary metabolites,semi-synthesis,Tau

Language English

Advisor Wang, Clay (committee member)

Creator Email plehman@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-360222

Unique identifier UC11666319

Identifier etd-LehmanPatr-8895.pdf (filename),usctheses-c89-360222 (legacy record id)

Legacy Identifier etd-LehmanPatr-8895.pdf

Dmrecord 360222

Document Type Thesis

Rights Lehman, Patrick William

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract Natural products represent a unique and diverse area of chemical space which have yet to be taken advantage of comprehensively. These compounds have served as a source for drug discovery since ancient times and still represent a significant portion of modern medicines. Most of these compounds are made by microorganisms and are secondary metabolites that have been progressively selected by evolution. Optimization via evolutionary pressures has gifted these compounds with properties that are commonly found in FDA approved drugs that synthetic molecules often lack. With many diseases still without adequate treatments or medications that are far too expensive science is turning back to natural products as a source for more economic and environmentally safer drug lead candidates. In this work we harness the molecular manufacturing abilities of P. hirayamae which produces biomedically relevant metabolites (-)-sclerotiorin and rubrorotiorin. After extraction and isolation from the fungus these compounds can be derivatized conveniently with 1-step reactions. Here we have made a chemical library by inserting primary amines with diverse and “drug-like” substituents into the 4H-pyran ring of (-)-sclerotiorin. These compounds were generally made in good yields and have increased water solubility. These compounds will be used in tau aggregate inhibition assays for Alzheimer’s disease treatment and antifungal assays against fluconazole resistant and non-fluconazole resistant strains of C. Albicans, for which sclerotiorin and related derivatives have already shown to be effective. In addition to semi-synthesis, growing P. hirayamae on potassium bromide supplemented media yielded a novel compound, rubrorotiorin brominated at the C-5 position instead of the usual chlorine substituent. This compound is also medically relevant as closely related compounds have shown to have potent bioactivities in antitumor, antifungal, tau aggregate inhibition, and gp-120-CD4 interaction inhibition (HIV). Future work will involve genetic engineering to produce mutant strains of P. hirayamae which make more reactive de-acetylated (-)-sclerotiorin for derivatization.