Selective HDAC Inhibitor
Me O
Me
NH
O
S
N
Biosynthetic Studies
S
NH
O
NH2
Me
N
H
O
N
H
Marine-Derived
Aspergillus sp.
N
Terrestrial-Derived
Aspergillus versicolor
Me
Novel Drug Delivery Method
O
N
GCATG
Me
H
Me N
Me
H
Me
Me
O
H O
N
N
H
N
O
O
(+)-stephacidin A
Me
H
G
T
C
G
C
A
H
N Me Me
H
O
C
A
G
MeO
R
O
Me
NH
O H
N
N
HN
S
Me
O
O
G
T
(-)-stephacidin A
(-)-notoamide B
= cytotoxic agent + naphthyridine conjugate
Jennifer M. Finefield
Robert M. Williams, Advisor
Colorado State University
December 6, 2011
Me
H O
N
N
Me
Me
Me
H
O
O
(+)-notoamide B
Efforts Toward the Synthesis of a
Selective Histone Deacetylase Inhibitor
N
S
Me
O
Me
Me
N
NH
NH
H
N
O
O
O
NH
NH2
S
DNA Packaging
• For DNA to fit within the nucleus,
it must be condensed
• DNA is packaged into chromatin
• To begin packaging, DNA is
wound around histones
www.med.unc.edu www.christopher_vidal.com
Gene Expression: Dynamic Wrapping and
Unwrapping of DNA
•
Histone Acetyltransferase (HAT)
readies DNA for transcription
• Histone Deacetylase (HDAC)
returns DNA to the inactive state
X
• HDAC inhibitors prevent removal
of acetyl residues
O
RO
O
NHR
HAT
RO
NHR
X
HDAC
HDAC
NH3
www.med.unc.edu www.christopher_vidal.com
HN
O
Transcriptional Control
• HDAC inhibitors mimic the natural substrate
• Deacetylation is prevented, eventually leading to cell death
Zn-Dependent Histone Deacetylase Enzymes
Q u ic k T im e ™ a n d a
T I F F ( Un c o m p r e s s e d ) d e c o m p r e s s o r
a r e n e e d e d t o s e e t h is p ic t u r e .
Figure 2. Schematic domain structure of
Class I, IIA, IIB and IV Histone Deacetylases.
Deacetylase domains are depicted in color
against a silver background illustrating the
relative length of each individual enzyme.
• HDAC enzymes are divided into different classes
• Within each class, there are different isoforms
• Many known HDAC inhibitors display very little selectivity for class or
isoform
Marks, P. A., et al., Advances in Cancer Research, 2005, 137
HDAC Inhibitors and Enhancing Selectivity
O
H
N
NHOH
O
HDAC1 IC50(nM) 30
HDAC3 IC50(nM) 100
vorinostat (SAHA)
Surface
Recognition
Linker
Ph
HNO OR
Metal
Binding
NH
O O
H
RN H
N NHOH
N
H
O
O
vorinostat (SAHA)H2N
Surface
Recognition
Linker
Internal
Cavity
O
2+Zn
O
HN
2+ZnN
H
NH
O
Internal
Cavity
Internal
Metal
Binding
Cavity
• Many known HDAC inhibitors display very little selectivity for class or
isoform
Wiest, O. et al., J. Med. Chem. 2004, 47, 3409; Methot, J. L., et al., Bioorg. Med. Chem. Lett. 2008, 18, 973
Improving Selectivity of HDACi:
Targeting HDAC1 and HDAC2
S
MeO
N
H
H
N
O
O
O
NH2
N
H
H
N
MeO
NH2
HDAC1 IC50(nM) 440
HDAC2 IC50(nM) 500
MeO
NH2
MeO
MeO
MeO
N
H
H
N
HDAC1 IC50(nM) 60
HDAC2 IC50(nM) 100
HDAC1 IC50(nM) 50
HDAC2 IC50(nM) 80
O
S
S
MeO
H
N
N
H
NH2
H
N
MeO
HDAC1 IC50(nM) 50
HDAC2 IC50(nM) 70
N
H
OH
MeO
H
N
N
H
NH2
MeO
MeO
MeO
O
O
O
HDAC1 IC50(nM) 40
HDAC2 IC50(nM) 60
Moradei, O. M., et al., J. Med. Chem. 2007, 50, 5543
HDAC1 IC50(nM) >10,000
HDAC2 IC50(nM) >10,000
Improving Selectivity of HDACi:
Targeting HDAC3
N
S S
O Me
MeO
MeO
H
N
NO
H
Me
Me
N
NH2
Me
HDAC1 IC50(nM) >10,000
Increased
selectivity
HDAC2 IC
50(nM) >10,000
(possibly HDAC3)
H
N
NH
O
Me
+
O
Me
O Improved surface
O
S
recognition domain
NH
NH2
S
Finefield, J. M.; Williams, R. M.; Wiest, O.; Bradner, J. Unpublished results
NH
O
O
O
N
O
N
NH
NH
S
S
largazole
Improving Selectivity of HDACi:
Targeting HDAC3
Ser118, present in HDAC1/2 (green) » Tyr118 in HDAC3 (blue)
N
S
Me
O
N
NH
Me
Me
O
H
N
O
O
NH
NH2
S
Zn+2
Finefield, J. M.; Williams, R. M.; Wiest, O.; Bradner, J. Unpublished results
NH
Key Disconnections
N
S
Me
O
N
NH
NH
Me
Me
H
N
O
O
O
NH
NH2
S
Thiazoline-Pyridine Synthesis
Bowers, A., et al., Org. Lett., 2009, 11, 1301
Synthesis of the Amide Isostere
Bowers, A., et al., J. Am. Chem. Soc., 2009, 131, 2900
Alternate Route
N
S
Me
HO2C
N
1. DCC, DMAP
NHBoc
MeOH, DCM
N
2. TFA, DCM
52% (2 steps)
Me
MeO2C
NH
O
Me
HO2C
OH
N
NH
H
N
Me
O
Me
O
O
N
S
HATU, HOBt
i-Pr2NEt
Me
O
Me
Me
N
NH
NH
O
H
N
Me
MeO2C
Me
66%
N
NH2
Me
NH
53%
S
2. TFA, DCM
O
THF
OtBu
NHBoc
Me
LiOH (aq.)
1. LiOH (aq.), THF
MeCN
Me
NHBoc
O
S
N
PyBOP, i-Pr2NEt
Me
Me
NH2
S
O
N
N
NHBoc
H
N
O
NH
O
2-Thiophenyl Biaryl Synthesis
S
nBuLi, B(OMe)3
S
Br
B(OH)2
Et2O, HCl
NH2
NBS
NO2
HOAc
NH2
Pd(PPh3)4
52%
NH2
Br
tri-o-tolylphosphine
K2CO3, DME, H2O
NO2
S
86%
NO2
70%
O
H
N
HO
O
NO2
T3P, i-Pr2NEt
DCM
S
MeOH:DCM
1:1
57%
71%
H
N
Boc2O, NEt3
O
NHBoc
DCM
S
H
N
SnCl2
O
NH2
S
Final Steps and Future Direction
N
S
N
S
H
N
Me
+
O
NHBoc
Me
N
NH
O
Me
NH
S
Me
1. Grubbs' #2
toluene
N
NH
O
Me
NH
H
N
2. TFA, DCM
H
N
Me
O
O
O
O
O
NH
NH2
S
______________________________________________________________________________________________________________________________
N
S
O
S
S
O
N
Me
N
O
Me
NH
NH
O
O
Me
Me
O
Thiazoline-pyridine
largazole cap
O
Me
N
NH
Me
O
N
NH
O
O
Largazole cap
Me
O
Me
N
NH
Me
O
NH
O
O
Oxazoline-oxazole cap
Design and Synthesis of a Novel Drug
Delivery Method Specifically Targeted
to Multiple Myeloma Cells
GCATG
O
H3C
N
N
N
H
NH
O
H3C
N
N
N
H
O
NHOH
O
G
T
C
G
C
A
C
A
G
G
T
= cytotoxic agent + naphthyridine conjugate
Cl
N
Cl
NH2
Multiple Myeloma
• MM is a plasma cell malignancy that can lead to bone
destruction, anaemia, hypercalcaemia, and renal insufficiency
• MM is associated with older age (median age 66 years) and
is found to occur more often in men than women
• Cause of MM remains unknown
• Current treatments include a single high-dose of melphalan,
velcade, and various combination treatments
Trialx.com, Mahindra, A., et al., Blood Reviews 2010, 24, S5; Barlogie, B., et al., Blood 2004, 103, 20
Tumor Specific Oligonucleotide (MB8226)
CDR3 gene from
cell line RPMI8226
Tumor Specific
mRNA Transcript
UAGGCUACGUACUUAAGCG
GCATG
GCGAC
GTCGC
F
Q
GCATG
Complementary
sequence
C
A
G
C
C
G
A
G
C
Q
G
G
T
C
G
G
C
T
C
GF
C
Q
F
Quenched Probe MB8226
The Trojan Horse
CGUAC
GCATG
GCATG
ACGCTG
Extra guanine
(a.k.a. "G Bulge")
G
T
C
G
C
C
A
A
=
H3C
Nakatani, K. et al., J. Am. Chem. Soc. 2000, 122, 2172
N
CAG GT
C
A
G
G
G
T
T
N
N
H
DRUG
Naphthyridine Modified MM Drugs
=
H3C
N
O
H
N
N
H
N
DRUG
O
Currently undergoing clinical
trials
to be used
H3C as
N a combination
N
N
treatment for multipleHmyeloma
NHOH
O
vorinostat (SAHA)
N-vorinostat
O
OH
O
Cl
NH2
N
Cl
melphalan
H3C
N
N
N
H
Given either as a high-dose
NH
treatment or as partNH
of a
2
O the treatment
combination for
of
multiple myeloma
Cl
N
Cl
N-melphalan
NHOH
O
Naphthyridine Modified Vorinostat
O
OMe
H3PO4
+
H2N
N
OMe
NH2
H3C
89%
N
Coupling
Conditions
NH2
N
O
X
O
HO
N
N
N
H
O
1. MeOH, H2SO4
OH
H3C
HO
O
4
OMe
OMe
2. KOH, MeOH
O
O
35% (2 steps)
O
HO
(COCl)2, DMF
OMe
O
Cl
OMe
Benzene
O
N
H3C
80%
H3C
N
N
N
H
O
4
44%
O
NH2OH.HCl, KOH or NEt3
OMe
NH2
NEt3, DMAP, DCM
O
O
N
X
MeOH
0oC, rt, m.w
H3C
N
N
N
H
H
N
OH
O
Brown, E. V., J. Org. Chem. 1965, 30, 1607; Yoshida, M. et al., Synthesis 2008, 1099; Gediya, L. K. et al. J. Med. Chem. 2005,
48, 5047
Naphthyridine Modified Vorinostat
O
HO
H3C
(CH3CO)2O
OH
98%
O
O
Ethyl chloroformate
NEt3, THF
H3C
N
N
N
H
O
O
4
N
O
NH2
H3C
THF
O
O
N
N
N
H
50% NH2OH
in H2O
OH
OEt
27% (2 steps)
O
H3C
N
N
N
H
N-vorinostat
Mai, A. et al. OPPI Briefs 2001, 33, 391
4
54%
O
O
N
O
H
N
O
OH
Naphthyridine Modified Melphalan
FF
Boc
Boc22O
O
O
O
H
H22N
N
1M
1M NaOH
NaOH
dioxane
dioxane
OH
OH
C
C66FF55OH
OH
O
O
BocHN
BocHN
OH
OH
FF
FF
O
O
FF
O
O
EDCI,
EDCI, DMF
DMF
BocHN
BocHN
H
H33C
C
96%
96%
92%
92%
O
O
H
H33C
C
N
N
N
N
H
H
N
N
O
O
4M
4M HCl
HCl
NHBoc
NHBoc
CHCl
CHCl33,, EtOAC
EtOAC
H
H33C
C
N
N
N
N
N-Boc-melphalan
N
N
H
H
NH
NH22
EDCI, NMM
DCM
47%
47%
21%
O
H 3C
N
N
O
N
H
H 3C
N
N
N
H
NH
O
NH
NHBoc
HCl-saturated EtOAc
O
NH2
42%
Cl
N
Cl
Cl
N
N
N
Cl
N-melphalan
Nakatani, K. et al., Bioorg. Med. Chem. 2003, 11, 2347; Gullbo, J. et al., Oncol. Res. 2003, 14, 113
NH
NH22
i-Pr
i-Pr22NEt,
NEt, DMF
DMF
FF
53%
53%
N
N
Preliminary Test Results
O
H 3C
N
N
N
H
NH
O
Cl
N
Cl
N-melphalan
NH2
Preliminary Test Results
O
H3C
N
N
N
H
N-vorinostat
(N-SAHA)
H
N
O
OH
Studies on the Biosynthesis of
Reverse Prenylated Indole Secondary
Metabolites from Aspergillus
versicolor and Aspergillus sp. MF297-2
Aspergillus sp. MF297-2
Aspergillus versicolor
Me
Me
H
Me N
Me
H
O
Me
Me
O
H
N Me Me
H
O
H O
N
N
H
N
N
O
O
(+)-stephacidin A
Me
H
O
N
(-)-stephacidin A
MeO
O
Me
NH
HN
R
H
N
S
Me
O
O
(-)-notoamide B
Me
H O
N
N
Me
Me
Me
H
O
O
(+)-notoamide B
Reverse Prenylated Indole Secondary Metabolites
1969-2006
H Me
Me
N
H
H
Me Me N
H
H O
N
N
O
O H
N
N
HN
Me
Me
O
O
O O
H
N
O
brevianamide A
Me
Me
Me
H
O
H
N
H O
N
N
NH
H O
N
N
O
O
H
H
HO
N
N
N
N
H
O
H
Me N
Me
H
O
HN
N
H
H
O
H
O
H
N
N
brevianamide E
O
H
N
HN
N
Me H
H
Me O
H
O
Cl
Cl
Me
H
OH
Me
H
N
H
H
O
N
N
N
H
O
Me
Me
O
Me
Me
OH
3
17
N
O H
OMe
Me
O
Me O
NH
N
O H
O
O
O
Me
Me
Me
Me
N
H
norgeamide B
H
O
O
Me
O
Me
Me
N
H
Me
17
N
O
Me
Me
O
N
asperparaline B
Me
H
norgeamide C
O
N
O
N
O
O
O H
N
Me
N
O
H
O
Me
Me
H
Me
N
H
N
N
N
O
N
VM55596
Me
Me
O (+)-stephacidin A
SB203105
N
O
O
Me
Me
O
NH
O Me
Me
O
Me
Me
H
OH
O
HO
NH
O Me
Me
NH
Me
O
O
avrainvillamide (CJ-17,665)
OH
O Me
O
OH
N
N
O
paraherquamide H
Me
H
N
Me
Me
O
Me N
Me
H
Me
O
Me
20
H
O
Me
O
Me
Me
O
O
Me
stephacidin B
NH
N
21
N
Me
O Me
N
N
SB202327
O
N 55
N
Me
O
H
51
O
N
Me
O Me
Me
O
Me
O
O
Me
Me
H
H
asperparaline A
H
Me N
Me
H
Me
H
O
N
Me
H
Me
N
3 2
O
O
O Me
N
Me
O
HO
OH
Me
Me
O Me
N
N
N
H
N
H
Me
Me
H
Me
Me
H
NH
Me
O
Me
OH
Me Me N
H
O
O Me
N
N
asperparaline C
Me
O
Me
N
O
paraherquamide E Me
(VM54159)
NH
marcfortine B
norgeamide A
Me
O
Me Me
H
O Me
N
N
marcfortine A
Me
H
N
Me
Me
NH
Me
H
O
Me
O
paraherquamide D
MeO
Me
H
NH
paraherquamide C
N
Me
O H
N
N
O
N
H
O
norgeamide D
Me
H
O
Me
Me O
Me
H
N
O
Me
O
N
N
paraherquamide G
(VM54158)
O Me
N
Me
O Me
Me
Me
deoxy-12,13-dehydrobrevianamide E
O
Me Me
H
Me
NH
O Me
Me
O Me
N
N
Me
O
O
O
HO
OH
Me
N
Me
H
O
Me
paraherquamide A
(VM29919)
marcfortine C
deoxybrevianamide E
O
NH
N
NH
O H
N
N
HN
Me
Me O
O
Me
Me O
N
N
H
N
O Me
Me
malbrancheamide
brevianamide F
NH
pre-paraherquamide
brevianamide D
O
O
Me
O
Me
O
O
Me
Me
H
paraherquamide F
(VM55594)
paraherquamide B
VM55599
brevianamide C
O
Me
sclerotiamide
O Me
O H
N
N
Me
H
HO
Me
H
NH
O Me
N
N
Me
Me
MeO
Me
H
NH
H
O
H
Me Me N
H
H
Me
H O
N
N
O
N
N
O
hydratoaustamide
H Me
Me
N
H
H
N
O
Me
H
O
N
OH
O
austamide
brevianamide B
Me
HN
Me
Me
N
Me
H
O O
H
O
N
VM55597
O
Me
Me
Proposed Biosynthesis of the
Bicyclo[2.2.2]diazaoctane Ring System
Me
H
H
Me N
Me
Intramolecular
H Diels-Alder
O
N
HN
O
HN
Me
Me
O
N
H
Me
H
Me
N
O Me
H
H
N
O
N
H
N
O
O
(+)-stephacidin Aketo-premalbrancheamide
deoxybrevianamide E
Enzymatic Proposal
Diels-Alder
Reaction
Porter and Sammes Diels-Alder
(1970)
Me Me
Me Me
O
HO
N
O
Me
Me
HO
O
N
NH
N
N
N
O
OH
Me Me
H Me
Me
N
H
N
N
O
H O
O N
N
O
brevianamide A
Porter, A. E. A. et al., Chem. Commun. 1970, 1103; Williams, R. M., Chem. Pharm. Bull. 2002, 50, 711.
Me
H
O
N
Me
H
N
O
Proposed Biosynthesis of the
Bicyclo[2.2.2]diazaoctane Ring System
•
Enzyme Controlled Stereoselectivity
Me
H Me
Me
N
H
Me Me
H
19
19
19
H O
N
N
H O
N
N
O
Me Me
H
(+)-brevianamide A
O
N
O
anti-diastereomer
Me
H
Me N
O
19
O H
N
N
O
O
Me
H
H
N
O
(+)-stephacidin A
syn-diastereomer
Paraherquamides
(+)-brevianamide B
Me
H
O
N
H
Me N
H
Me Me N
HO
N
H
N
O
N
Anti-selective
O
H
N Me Me
H
[4+2]
Syn-selective
O
O
[4+2]
H
N Me Me
OH
N
H
Me N
H
N
O
H
N Me Me
H
N
H O
N
N
(+)-brevianamide A
Me
H
H O
N
N
N
O
O
VM55599
Reverse Prenylated Indole Secondary Metabolites
2007-2011
Me
H
O
N
MeO
N
OH
H
N
Me
H
O
Me
O
O
N
Me
NH
Me
O
Me
O
(-)-notoamide B
O
H
Me
Me
O
N
H
Me
H
N
N
O
O
OMe
notoamide F
O
O
H
Me
Me
HO
N
N
Me
H
Me
O
O
N
O
N
H
O
Me
notoamide C
N
H
N
H
O
Me
Me
notoamide D
OH
Me
H
Me N
Me
H
Me
O
OH
O
notoamide H
OMe
notoamide G
17
N
Me
MeO
O
O
N
H
notoamide J
HO
Me
Me
N Me
H
O
Br
HN
Me
Me
O
O H
Me
N
N
O
O
NH
O H
N
N
Me
N
O Me
Me
O
chrysogenamide A
Me
O
Me
Me
O
O
N
notoamide I
OMe
O
N
H
O
O
H O
N
N
O
HN
Me
Me
O
Me
Me
H
H O
N
N
O
(-)-versicolamide B
O
3
notoamide R
Me
H
O
N
H
N
Me
N
O H
N
17
H
O
N
H
3-epi-notoamide C
Me
OH
O
Me
H
N Me Me
H
(-)-stephacidin A
N
notoamide Q
Me
O
O
Me Me
H
O
H
N
NH
Cl
O
notoamide N
Me
H
Me N
Me
H
MeO
H
N
notoamide M
N
N
O H
H
notoamide P
Me
O
Me
O
Me Me
H
N
N
Me
OH
O
HN
N
H
O
Me
Me
notoamide L
Me
H
N
17
3
Me
Me
Me O
Me
H
N
H
O
O
N
O
HO
N
O H
notoamide K
HO
notoamide O
Me
N
Me H
O
Me
Me
Me
H2 N
O
O
O
O Me OH
O
Me
H
N
N
H
O
H
O
HO
H
O
HN
H
H
N
N
3 2
H
O
notoamide E
Me
Me
HO
Me N
Me
H
17
Me
Me
Me
Me
H
Me N
Me
H
NH
O
Me
O
(+)-notoamide B
O
HO
N
H
O
Me
N
O
O
Me
N
N
O H
3
H O
N
N
Me
H
O
Me Me
H
Me
H
HN
H
N
notoamide A
H
O
Me
MeO
Me
Me O
NH
Me
O
O
(+)-versicolamide B
Me
Isolation of the Notoamides: New Addition to
the Stephacidin Family
•
2007: Aspergillus sp. MF297-2
Me
H
MeO
N
OH
Me
H
R
O H
N
N
O
O
notoamide A
Me
O
Me
O
Me
H
N
N
O H
3
Me
N
H
O
N
H
Me
H
H
Me
Me
O
N
O
Me
Me
H
Me N
Me
H
H
N
N
H
deoxybrevianamide E
Kato, H. et al., Angew. Chem. Int. Ed. 2007, 46, 2254
Me
HO
O
sclerotiamide
O
Me
O
N
H
N
O
(+)-stephacidin A
N
O
Me
Me
notoamide D
notoamide C
NH
N
3 2
Me
Me
O
HO
H
O
Me
O
NH
O
R
O
(-)-notoamide B
H
N
NH
O H
N
N
Me
O
Me Me
H
MeO
Me
O
H
Isolation of the Notoamides: New Addition to
the Stephacidin Family
•
2008: Aspergillus versicolor NRRL 35600
Me
Me
O
O
Me
H
N Me Me
H
HN
S
H O
N
N
Me
H O
N
N
Me
O
(-)-stephacidin A
Me
H
Me
H
O
O
N
H
N
N
H
O
O
HO
H
brevianamide F
N
N
HN
O
Me
Me
N
H
O
Me
Me
norgeamide D
Greshock, T. J. et al., Angew. Chem. Int. Ed. 2008, 47, 3573
S
Me
O
O
(+)-versicolamide B
H
O
NH
H
N
O
(+)-notoamide B
Me O
OH
Me
Antipodal Natural Products
Aspergillus sp. MF297-2
Aspergillus versicolor
Me
O
Me
H
Me N
Me
H
Me
Me
O
H
N Me Me
H
O
H O
N
N
H
N
N
O
O
(+)-stephacidin A
(Aspergillus ochraceus)
Syn
Me
H
O
MeO
O
Me
NH
S
Me
O
O
Me
O
Me
R
Me
Me
O
O
Me
H
H O
N
N
(-)-versicolamide B
H O
N
N
Me
O
Me
O
(-)-notoamide B
HN
Me
H
HN
R
H
N
N
(-)-stephacidin A
(+)-notoamide B
Me
H
O
N
Me O
NH
S
H
N
Me
O
O
Anti
Me
(+)-versicolamide B
Tsukamoto, S. et al., Org. Lett. 2009, 11, 1297; Greshock, T. J. et al., Angew. Chem. Int. Ed. 2008, 47, 3573
Isolation of the Notoamides: New Addition to
the Stephacidin Family
•
2008-2010: Aspergillus sp. MF297-2
H
O
Me
N
O
H
Me
Me
N
H
O
Me
H
N
N
Me
Me
O
O
O
O
H
OMe
notoamide F
HO
N
HN
O
N
H
notoamide J
H
O
O Me OH
O
Me
Me
H
N
N
O
Me
H
Me
O
Me
MeO
N
O H
N
N
OH
O
notoamide H
OMe
notoamide G
Me
H
Me
O
OH
O
Me
Me
N Me
H
HN
notoamide O
N
Me H
O
Me
O
Br
Me
Me
N
H
H2N
Me
H
Me N
O
O H
Me
N
N
O
O
notoamide I
N
N
O Me
O
OH
O
NH
O H
N
N
N
H
O
Me
Me
notoamide L
HN
MeO
Me
H
N
17
3
Me
O
HO
O
O
Me
Me
O H
notoamide K
HO
N
Me
H
N
N
H
O
H
O
HO
O
O
HO
Me N
Me
H
notoamide E
Me
HO
O
Me
Me
H
Me N
Me
H
NH
Cl
O
notoamide N
notoamide M
Me
O
O
Me Me
H
N
N
Me H
O
Me
N
O H
O
Me
notoamide P
Me
N
H
N
OMe
O
notoamide Q
H
Me N
Me
H
O
Me
HN
O
Me
O
N
H
N
OH
O
Me
O
notoamide R
Tsukamoto, S. et al: JACS, 2009, 131, 3834; JNP 2008, 71, 2064; OL 2009, 11, 1297; JNP 2010, 73, 1438
Me
O
Me
Me
Me
H
H O
N
N
O
(-)-versicolamide B
Isolation of Notoamide E: A Potential
Biosynthetic Precursor
•
2008-2010: Aspergillus sp. MF297-2
Tsukamoto, S. et al., J. Am. Chem. Soc. 2009, 131, 3834
Proposed Biosynthetic Pathway:
Notoamide E
H
OH
O
N
N
N
NH
O
O
[ox]
H
Me
Me
Me
Me
O
Me
N
H
N
H
O
Me
Me
Me
notoamide E
achiral azadiene
[4+2]
Aspergillus sp.
Me
Me
H
O
N
MeO
NH
H
N
[ox]
Me
O
O
(-)-notoamide B
pinacol
O
Me
N
Me
HN
Me
Me
O
O
Me
H
H O
N
N
(-)-versicolamide B
O
Me
Me
O
O
[ox]
pinacol Me
H O
N
N
pinacol
O
C6-epi-stephacidin A
Greshock, T. J. et al., Angew. Chem. Int. Ed. 2008, 47, 3573
Me
Me
H
H O
N
N
O
O
O
(+)-notoamide B
(-)-stephacidin A
+
H
N Me Me
H
HN
[ox]
H O
N
N
H
N
+
Me
O
Me
H
N Me Me
H
O
(+)-stephacidin A
Me
O
Me
H
Me N
Me
H
Aspergillus versicolor
H
Me N
Me
H
O
N
Me
Me
O
H
N
[ox]
pinacol
O
C6-epi-stephacidin A
Me
H
O
N
Me O
NH
H
N
Me
O
O
(+)-versicolamide B
Me
Synthesis of [13C]2-Notoamide E
Me
HO
Cl
3 steps
Boc2O, DMAP
N
H
HO
NO2
NCS
N
H
BocO
CH3CN
DMF
75%
Cl
Me
Me
Me
Me
Me
Me
9-BBN
N
H
BocO
NEt3, THF
N
H
BocO
80%
Me
Me
TFA
CH2Cl2
HO
N
H
0.1% CuCl22H2O
DBU, CH3CN
97%
56%
81%
Me
Me
O
Me
Me
N
H
Me
Me
1,2-dichloro
benzene
95%
O
Me
Me
N
H
NMe2
aq. H2CO
aq. Me2NH
AcOH
95%
Me
Me
O
N
H
Me
Me
Tsukamoto, S. et al., JACS 2009, 131, 3834; Grubbs, A. W. et al., TL 2005, 46, 9013; Grubbs, A. W. et al., ACIE 2007, 46, 2257
Synthesis of [13C]2-Notoamide E
CO2Et
Ph
N
Ph
NMe2
Me
Me
N
H
O
1.
CO2Et
= 13C
PBu3, CH3CN,
Me
Me
2. 1 M HCl, CH2Cl2
O
Me
Me
N
H
NH2
Me
Me
O
H
1. FmocCl
10% Na2CO3
dioxane
NHFmoc
Me
Me
2. Me3SnOH
O
ClCH2CH2Cl
Me
Me
74%
76%
OH
N
H
O
EtO
OEt
O
H
N H O
Cl H
O
86%
N
morpholine
NHFmoc
Me
Me
HATU, iPr2NEt
CH3CN
N
H
O
H
THF
88%
1:1 = cis:trans
Me
Me
O
Me
Me
N
H
N 17
12
N
O
H
Me
Me
+
O
notoamide E
Aspergillus sp.
A. versicolor
Tsukamoto, S. et al., J. Am. Chem. Soc. 2009, 131, 3834
O
H
H
Me
Me
N
H
N
H
N
O
H
Me
Me
[13C]2-Notoamide E Incorporation Study with
Aspergillus sp. MF297-2
O
Me Me
H
12
HO
N
O H
O
O
H
O
Me
Me
12
N
H
O
Me
H
N
O
H
Me
Me
O
H
=13C
N 17
12
notoamide E
O
Me
Me
•
N
H
N
H
OH
OH
notoamide E2
O
Me
Me
O
Me Me
H
N
17
O
H
H
H
N
O
H
Me
Me
O
Me
Me
N
H
O
Me
Me
3-epi-notoamide C
(1.26 mg)
O
H
N
O
H
Me
Me
O
12
O
Me
Me
N
Me
Me
N
17
N
O
notoamide E4
No labeled bicyclo[2.2.2]diazaoctane containing metabolites were
produced
Tsukamoto, S. et al., J. Am. Chem. Soc. 2009, 131, 3834
H
OH
OH
notoamide E3
O
N
H
N 17
12
N 17
12
N
O H
notoamide D
notoamide C
(0.33 mg)
Aspergillus sp.
12
Me
Me
N 17
O
N
O
N
H
Me
H
N 17
H
[13C]2-Notoamide E Incorporation Study with
Aspergillus versicolor
H
O
Me Me
H
O
N
N
O H
Aspergillus versicolor
H
Me
Me
O
Me
Me
N
H
notoamide E
=13C
H
O
Me
N
H
Me
Me
Me
N
N
O
N
H
O
O
HO
NH
O
H
N
O
Me
Me
H
notoamide C
notoamide D
6.2% incorporation
6.0% incorporation
O
Me Me
H
N
N
O H
O
Me
N
H
Me
3-epi-notoamide C
trace amount detected
Finefield, J. M.; Williams, R. M. et al., Tetrahedron Lett. 2011, 52, 1987
H
O
Possible Precursors Leading to Stephacidin A
H
O
H
N
H
N
N
H
Me
Me
H
O
O
H
P450
N
H
HO
deoxybrevianamide E
O
H
N
N
H
Me
Me
H
O
DMAPP
N
H
HO
NH
O
P450
H
Me
Me
N
H
O
Me
Me
[ox; IMDA]
Me
Me
notoamide S
notoamide E
X [ox; IMDA]
[ox; IMDA]
Me
O
N
H
N
O
H
Me
Me
Enzyme
6-hydroxydeoxybrevianamide E
[ox; IMDA]
N
Me
Me
H
Me N
Me
H
O H
N
N
O
ketopremalbrancheamide
P450
H
Me N
Me
H
O H
N
N
OH
Enzyme
DMAPP
O
H
Me N
Me
H
O H
N
N
O
notoamide T
Me
OH
Me
P450
H
Me N
H
O H
N
N
O
stephacidin A
O
Synthesis of Deoxybrevianamide E and
6-Hydroxydeoxybrevianamide E
Kato, H.; Nakamura, Y.; Finefield, J. M.; Umaoka, H.; Nakahara, T.; Williams, R. M.; Tsukamoto, S., TL 2011, 52, 6923
Synthesis of Ketopremalbrancheamide
O
H
OEt
NH2
N
H
HO
HATU,
+ HO C
2
Me
Me
EtO
H
N
Boc
iPr
2NEt
CH3CN
O
O
N
H
Me
Me HO
N
H
99%
O
H
N
H
N
OH
PBu3, DEAD
N
O
H
Me
Me
N
Me
H
N
H
78%
O
2.4:1 = syn:anti
N
H
Me N
H
N
O
()-syn,
ketopremalbrancheamide
Me
H
+
2. 2-hydroxypyridine
toluene
O
N
O
H
Me
Me
CH2Cl2
94%
1. TFA, CH2Cl2
87%
1:1 = cis:trans
O
H
H
Boc
N
O
N
20% aq. KOH
H
Me N
H
N
O
()-anti
N
N
MeOH
N
H
Me
Me
OH
Biosynthetic Breakthrough:
Characterization of the ()-Notoamide Biosynthetic Gene
Cluster
notB
orf1
notA
notD
notC
notH
notF
notE
notG
notJ
notI
notL
notK
notN
notM
notO
notP
notR
notQ
Ding, Y.; de Wet, J. R.; Cavalcoli, J.; Li, S.; Greshock, T. J.; Miller, K. A.; Finefield, J. M.; Sunderhaus, J. D.; McAfoos, T. J.;
Tsukamoto, S.; Williams, R. M.; Sherman, D. H., JACS 2010, 132, 12733
Biosynthetic Breakthrough:
Identification of Two Prenyltransferases
notB
orf1
notD
notA
notE
notC
H
N
O
H
N
H
Me
Me
N
H
H
O
notH
notF
DMAPP
N
H
notI
notG
NotF
HN
DMAPP
H
O
brevianamide F
HO
N
H
notN
notK
notM
notO
notP
notR
notQ
NotC
DMAPP
ketopremalbrancheamide
deoxybrevianamide E
O
H
notL
N
O
H
Me
NH
HMe
N
O
H
H
O H Me
N
Me
NN
H
O
O
N
NotC
notJ
deoxybrevianamide E
H
Me N
Me
H
N
H
O
N
H
X
Me
Me
NotC
NotC
N
DMAPP
DMAPP
H
N
H
X
O
N
X = H or OH
6-hydroxydeoxybrevianamide E
Me
Me
O
H
N
OH
NotC
N
H
DMAPP
Me
O Me
6-hydroxyketo
premalbrancheamide
X = H or OH
Ding, Y.; de Wet, J. R.; Cavalcoli, J.; Li, S.; Greshock, T. J.; Miller, K. A.; Finefield, J. M.; Sunderhaus, J. D.; McAfoos, T. J.;
Tsukamoto, S.; Williams, R. M.; Sherman, D. H., JACS 2010, 132, 12733
Early Steps in the Biosynthetic Pathway
N
O
H
NotF
N
H
Me
Me
DMAPP
N
H
H
O
P450
NotG/H
deoxybrevianamide E
O
H
HO
N
H
O
H
N
N
H
Me
Me
H
O
6-hydroxydeoxybrevianamide E
NotC
DMAPP
N
H
HO
Me
Me
N
N
H
Me
Me
O
H
H
O
P450
N
H
O
Me
Me
N
notoamide C
or
3-epi-notoamide C
H
N
O
H
Me
Me
NotB
+
notoamide D
notoamide E
notoamide S
X
Me
Me
H
O
N
H
Me N
H
N
O
(+)-stephacidin A
Me
O
Feeding Study with [13C]2-[15N]-6Hydroxydeoxybrevianamide E
Aspergillus versicolor NRRL 35600
O
8.4% incorporation
O
H
HO
N
H
Me
Me
N
MeN
H
Me
H
HO
O
Aspergillus sp. MF297-2
H
N
12
18
N
H
H
O
O
N
H
notoamide J
= 13C
N = 15N
No incorporation into advanced metabolites
Finefield, J. M.; Williams, R. M. et al., JOC 2011, 76, 5954; Finefield, J. M.; Williams, R. M.; Tsukamoto, S. et al., TL 2011, 52,
6923
Possible Enantio-diverging Pathways from
Notoamide S
Notoamide S Incorporation Study with
Aspergillus versicolor
O
H
O
O
H
N
H
HO
Me
Me
N
N
H
Me
Me
N
H
N
O
Me Me
H
H
N
O
H
Me
Me
N
H
O
notoamide E
notoamide S
N = 15N
= 13C
N
N
N
H
O
Me
O
Me
Me
H
notoamide D
6.2% 13C incorp.
Aspergillus
versicolor
Me
Me
H
O
notoamide C
6.4% 13C incorp.
H
O
O
HO
Me
Me
Me
Me
N
N
O H
Me
H
O
H
N Me Me
H
(-)-stephacidin A
O
6.2% 13C incorp.
H O
N
N
O
Me
HN
Me
Me
O
O
Me
H
H O
N
N
HN
Me
O
(+)-notoamide B
6.4% 13C incorp.
Unlabeled synthesis of notoamide S: McAfoos, T. J. et al., Heterocycles 2010, 82, 461
Results from feeding study: Finefield, J. M.; Tsukamoto, S.; Williams, R. M. et al., unpublished results
Me
O
Me
Me
H
H O
N
N
O
(+)-versicolamide B
6.5% 13C incorp.
Notoamide S Incorporation Study with
Aspergillus sp. MF297-2
O
H
N
H
HO
O
Me Me
H
N
N
H
Me
Me
H
O
N
O H
Aspergillus sp.
Me
Me
H
O
N
H
HO
Me
O
Me Me
H
N
Me
N
O H
Me
Metabolite 2
Me
notoamide S
N = 15N
= 13C
O
H
O
Me
Me
Tsukamoto, S. et al., unpublished results
N
H
H
O
N
H
HO
Metabolite 1
N
N
H
N
O
H
Me
Me
notoamide E
Notoamide S: Additional Biosynthetic Insight
Me
Me
Me
H
Me N
Me
H
Aspergillus
sp.
O
H
N
H
HO
Me
Me
N
N
N
H
Me
Me
O
O
H
H
O
notoamide S
IMDA
N
H
HO
N
N
H
Me
Me
H
O
Me
Me
Aspergilus
notoamide
S
versicolor
Me
H
Me
H
Me N
O
[ox]
O
H
N
N
O
Me
(+)-notoamide T
Me
Aspergillus
O
versicolor
HO
Me
OH
Me
O
(+)-stephacidin A
H
N Me Me
H
HMeO
N
Me N
H
N Me Me
O
[ox] O
H (-)-stephacidin A
H O
N
N
O
(-)-notoamide T
H
N
H
N Me Me
H
H O
N
N
O
(-)-stephacidin A
Notoamide T: Precursor Incorporation Studies
Me
Me
Me
H
N Me Me
H
HO
O
Me
Me
A. versicolor
H O
N
N
H
N Me Me
H
O
= 13C
(-)-stephacidin A
O
4.7% 13C incorp.
O
()-notoamide T
HN
H O
N
N
Me
H O
N
N
O
Me
Me
H
O
(+)-notoamide B
0.6% 13C incorp.
________________________________________________________________________________________________________________________________
Me
HO
Me
Me
H
N Me Me
H
H O
N
N
O
()-notoamide T
Aspergillus sp.
=
13C
Me
H
O
N
HO
Me N
Me
H
Me O
NH
R
H
N
Me
O
Me
O
O H
N
N
O
Metabolite A
(-)-notoamide B
Me
HO
Me N
Me
H
O H
N
N
Finefield, J. M., Tsukamoto, S.; Williams, R. M. et al., unpublished results
O
O
Metabolite B
OH
Me
O
Me
O
Notoamide S: Additional Biosynthetic Insight
O
H
N
H
HO
Me
Me
N
N
H
Me
Me
Me
H
O
Aspergillus
versicolor
Me
H
N Me Me
H
O
H O
N
N
O
(-)-stephacidin A
notoamide S
?
O
H
N
H
HO
Me
Me
N
N
H
Me
Me
H
O
O
Me
Aspergillus
versicolor
HN
H O
N
N
Me
Me
Me
H
O
O
(+)-notoamide B
notoamide S
Biosynthetic Precursor Incorporation Study
Me
Me
H
N Me Me
H
O
H O
N
N
O
Me
Aspergillus
versicolor
O
(-)-stephacidin A
=13C
Me
H
Me N
Me
H
O
N
HN
Me
HN
+
H O
N
N
Me
O
Me
O
Me
Me
H
Me
Me
O
O
O
Me
H
H O
OH N N
O
(+)-sclerotiamide
5.0% incorporation
(+)-notoamide B
2.7% incorporation
H
N
O
(+)-stephacidin A
________________________________________________________________________________________________________________________________
Me
H
Me N
Me
H
O
N
H
N
O
()-stephacidin A
Me
Me
Me
O
Aspergillus sp.
H
O
=13C
N
O
NH
+
H
N
Me
O
O
(-)-notoamide B
8.1% incorporation
Finefield, J. M.; Tsukamoto, S.; Williams, R. M. et al., Org. Lett. 2011, 13, 3802
Me
Me
H
O
N
O
Me
NH
H
N
Me
HO
O
O
(-)-sclerotiamide
6.8% incorporation
Me
Characterization of the ()-Notoamide Biosynthetic
Gene Cluster
•
()-Notoamide Biosynthetic Gene Cluster
notB
orf1
notA
•
notD
notC
notH
notF
notE
notG
(+)-Notoamide Biosynthetic Gene Cluster
Li, S.; Sherman, D. H. et al., unpublished results
notJ
notI
notL
notK
notN
notM
notO
notP
notR
notQ
Current Postulated Biogenesis of the Notoamides
and Stephacidins
Summary
Me O
Me
Me
NH
O
S
N
H
Aspergillus versicolor
Me
Me
H
Me N
Me
H
N
G
T
C
G
C
A
N
N
H
O
Aspergillus sp. MF297-2
O
S
N
NH
O
NH2
GCATG
O
Me
H
N Me Me
H
O
H O
N
N
H
N
O
H3C
N
N
O
Me
NH
O H
N
N
HN
S
Me
O
O
(-)-notoamide B
O
O
H3C
N
N
N
H
NH
O
(-)-stephacidin A
MeO
R
NHOH
N
H
O
(+)-stephacidin A
Me
H
G
T
= cytotoxic agent + naphthyridine conjugate
Me
O
C
A
G
Me
H O
N
N
Me
Me
Cl
O
N
Me
H
O
(+)-notoamide B
Cl
NH2
Acknowledgements
Prof. Robert M. Williams
Prof. David H. Sherman
Prof. Sachiko Tsukamoto
Williams Research Group
Sherman Research Group
Tsukamoto Research Group
Dr. James Berenson