AN ABSTRACT OF THE DISSERTATION OF
Ana Carolina Barrios Sosa for the degree of Doctor of Philosophy in Chemistry
presented on August 21, 2001. Title: Studies on Nitrogen Containing Secondary
Metabolites from Terrestrial and Marine Origin: PART I. Enzymatic Epoxidation
of 2,5-Dihydroxyacetanilide in Streptomyces sy. PART II. Synthesis of Marine
Sponge Alkaloids: Slagenins, Axinohydantoins, and Studies Towards the
Pyrrolopiperazine System in the Agelastatin Core
Abstract approved:
Redacted for privacy
David A. Home
Kevin P. Gable
PART I. A deutenum exchange analysis of 2,5-dihydroxyacetanilide (5)
in the absence and presence of DHAE TI was performed to test the
nucleophilicity of the substrate in the absence and presence of catalyst.
In
addition, inhibition studies using 1,4-dihydroxybenzene were performed to
determine the role that the N-acetyl side chain group plays in the formation of a
stable substrate-enzyme complex.
1,4-Dihydroxybenzene was found to be a
weak inhibitor, indicating that the N-acetyl functionality may play a crucial role
in
forming
stable
enzyme-substrate interactions.
The
synthesis
of
dihydroquinoline 7 was pursued to investigate the enzyme substrate interactions
between DHAE and a substrate where the N-acetyl side chain has been fixed to a
particular orientation. Efforts towards formation of the C6-C7 bond as a key
step
in the synthesis of dihydroquinoline 7 using palladium couplings,
organocuprates, Lewis acid catalysts, and aza-Claisen reactions were pursued.
To complement the results obtained, the electron distribution in amide 21 was
calculated using Semi Empirical methods. The results revealed that the electron
density in the aromatic ring is centered around C4, suggesting that this is the
most nucleophilic carbon in the ring.
PART II. Slagenins A (1), B (2), and C (3) were synthesized by
f-
functionalization of olefin 14. The desired tetrahydrofuroimidazolidin-2-one
system was achieved by intramolecular oxidative addition of alcohol 4 to the
imidazolone ring.
When this reaction was carried out in the presence of
methanol slagenins B (2) and C (3) were obtained in good yield. Heating 2 and
3 in
aqueous
acid gave slagenin A (1)
as the sole product.
(Z)-
debromoaxinohydantoin (17) was synthesized by intramolecular cyclization of
cx-methoxy imidazolone lib under acidic conditions followed by a double
oxidation reaction to furnish the hydantoin-lactam funtionality.
These
conditions were originally developed for a practical synthesis of the related
alkaloid (Z)-debromohymenialdisine (20). A series of acid and base catalyzed
reactions of imidazoles bearing an a-3 unsaturated system or a n-halogen
functionality showed that cyclizations via an SN2 path favor formation of an
oxazoline ring system.
Preliminary studies using pyrrolocarboxamideacetals
suggest that f3-ketone 73 would be an appropriate substrate for the formation of
the pyrrolopyrazine system in the agelastatins.
©Copyright by Ana Carolina Barrios Sosa
August 21, 2001
All Rights Reserved
STUDIES ON NITROGEN CONTAINING SECONDARY METABOLITES
FROM TERRESTRIAL AND MARINE ORIGIN: PART I. ENZYMATIC
EPDXIDATION OF 2,5-DIHYDROXYACETANILIDE IN
STREPTOMYCES SP. PART II. SYNTHESIS OF MARINE SPONGE
ALKALOIDS: SLAGENINS, AXINOHYDANTOINS, AND STUDIES
TOWARDS TILE PYRROLOPIPERAZINE SYSTEM IN THE
AGELASTATIN CORE
by
Ana Carolina Barrios Sosa
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed August 21, 2001
Commencement June, 2002
Doctor of Philosophy dissertation of Ana Carolina Barrios Sosa
presented on Aigust 21, 2001
APPROVED
Redacted for privacy
Major Professor, representing Chemistry
Redacted for privacy
of the Department of Chemistry
Redacted for privacy
Dean of Gtalute School
I understand my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes the release of
my dissertation to any reader upon request.
Redacted for privacy
Ana Carolina Barrios Sosa
ACKNOWLEDGMENTS
I would like to thank professor David A. Home for giving me the
opportunity to pursue a career in organic synthesis and for his support, advice,
and guidance throughout my Ph.D. I am also thankful to past and present
members of the Home group, Fumiko Y. Miyake, Kevin Wiese, Yu Geng,
Robert Kuhn, Mike Willis and in particular to Dr. Kenichi Yakushijin.
I am indebted to Dr. Kevin Gable for his guidance during my first years
at Oregon State University as well as his support and advice throughout my
career as a PhD candidate.
I would like to express my gratitude to Dr. James White for his advice
and support during these past years.
I am thankful to Dr. Mark Zabriskie, Dr. Philip Proteau and Dr. William
Gerwick for useful discussions and for allowing me to be part of their joint
group meetings during my first years at Oregon Sate University.
I will always be indebted to my good friends Brian Marquez and Lisa
Nogle, I thank them for their support and most of all for their valuable
friendship.
To my immediate and extended family, Jaime, Ana MarIa, Juanjo,
Andrés, Marl, Robert, Kay and Sara for always being there for me. But, most
importantly, I thank my best half, Thomas, for his patience, perseverance and
unconditional support.
TABLE OF CONTENTS
Chapter 1
General Introduction. 1
PART I
Enzymatic Epoxidaton of 2,5- Dihydroxyacetanilide in
Streptomyces sp ........................................................ 3
Chapter II
Introduction ............................................................. 4
Chapter III Results and Discussion ............................................... 24
3.1 Deuterium Exchange Analysis .................................. 24
3.1.1 Control Experiments ....................................... 28
3.2 Unexpected Resistance of 2,5-Dimethoxyanilides
to Electrophilic Cyclization ....................................... 41
3.2.1 Inhibition Studies Using 1,4-Dihydroxybhenzene
andDHAEII ................................................ 44
3.2.2 Studies Towards the Synthesis of
Dihydroquinoline 7 ......................................... 48
3.3 References .......................................................... 59
Chapter IV Experimental Section .................................................. 63
PART II
Synthesis of Marine Sponge Alkaloids: Slagenins,
Axinohydantoins and Studies Towards the Pyrrolopiperazine
System in theAgelastatin Core ....................................... 86
Chapter V
Introduction ............................................................ 87
Chapter VI Results and Discussion ................................................ 98
6.1 First Total Synthesis of (±) Slagenins A, B, C ............... 98
6.2 Total Synthesis of (Z)-Debromoaxinohydantoin
and Related Alkaloids .......................................... 115
TABLE OF CONTENTS (Continuation)
6.2.1 Practical Synthesis of
(Z)-Debromohymenialdisine ............................. 118
6.2.2 Total Synthesis of (Z)-Debromoaxinohydantoin ....... 128
6.3 Studies Towards the Pyrrolopiperazine System in the
Agelastatin Core and Related Findings ....................... 146
6.3.1 Controlling Cyclizations of
2-Pyrrolecarboxamidoacetals. Facile Solvation of
13-Amido Aldehydes and Revised Structure of
Synthetic Longarnide .................................... 149
6.3.2 Intramolecular Cyclization of 3-Activated
Irnidazolones .............................................. 159
6.4 References ......................................................... 172
Chapter VII Experimental Section ................................................ 177
Chapter VIII General Conclusions .................................................. 217
Bibliography.......................................................................... 220
Appendix: 1H and 3C NMR Spectra .............................................................. 228
LIST OF FIGURES
Figure
Page
11.1
Proposed mechanism of oxidation for P450.
11.2
H Transfer prior to oxygen-oxygen bond cleavage in
P45Ocam.
7
8
11.3
Geometry of the iron center according to crystallographic data.
11
11.4
Proposed mechanism for 3,4-PCD.
12
11.5
Active site in 3,4-PDC.
13
11.6
Active site of BphC.
14
11.7
Proposed mechanisms for the reaction catalyzed by MhpB
15
11.8
MhpB catalysis using a seven member lactone analogue.
16
11.9
Epoxidations catalyzed by DHAE I and DHAE II.
17
11.10
Biosynthesis of LL-C10037o.
18
11.11
Proposed path for the biosynthesis of MM14201.
19
11.12
Formation of vitamin K oxide.
20
11.13
Proposed mechanism of epoxidation of vitamin KH2.
21
11.14
a) Proposed mechanism for the epoxidation catalyzed by
DHAE I. b) Subsequent reduction to trap the label at C4.
22
111.1
1H NMR of material used in the exchange studies.
33
111.2
Spectra acquired after enzymatic exchange...
35
111.3
Effect of the concentration of sodium hydrosulfite on
percentage conversion.
46
LIST OF FIGURES (continuation)
Figure
111.4
Page
Results obtained from the inhibition study Using 1,4dihydroxybenzene. Protein concentration of the enzyme....
47
111.5
A. Electrostatic potential map of amide 21. B. HOMO electron
distribution of amide 21. C. HOMO-1 electron density of
amide2l.
57
Logarithmic curves obtained for each aromatic hydrogen in
DHA.
64
Two-flask system used for deuterium exchange studies with
constant DHA concentration.
65
IV.3
Purification scheme of DHAE II.
68
IV.4
Results observed after purification of the extract with DE-52
column.
71
Results observed after purification using immobilized metal
affinity chromatography.
73
CDK2-hymenialdisine complex crystal structure (structure
skeleton in black)
92
IV.!
IV.2
IV.5
V.1
VI.!
VI.2
VI.3
VI.4
DPFGSE ID NOE correlations observed upon selective
excitation of HiS in slagenin A (1).
111
DPFGSE ID NOE correlations observed upon selective
excitation of H15 in slagenin B (2).
112
DPFGSE 1D NOE correlations observed upon selective
excitation of HiS in slagenin C (3).
113
Time dependent elimination of compound 33 to 34 at 60°C.
124
LIST OF FIGURES (continuation)
Figure
VI.5
Page
Determination of the Z geometry of compound 34 by NMR
spectroscopy.
125
VI.6
1H-13C HIMBC correlations observed for compound 39.
133
VI.7
DPFGSE 1D NOE correlations observed for compound 39.
134
VL8
1H-15N HN'IBC correlations observed for compound 38b.
137
VL9
'H-'5N HIvIBC correlations observed for compound 40b.
137
VI.1O
Isomerization of E-3-bromoaxinohydantoin 44 at room
temperature. T = 6d, - 30 % isomerization in DMSO-d6.
142
Heats of formation of Z and E-debromoaxinohydantoin
calculated using AM1 Semi Empirical methods.
143
VI.12
1H-'5N HMBC correlations observed for compound 58.
152
VI.13
'H-15N HMBC correlations observed for compound 57.
153
VI.14
Calculated heats of formation for isomeric N-C and C-C
products.
154
Time dependent formation of hemiacetal 27a and acetal 27b in
CD3OD at room temperature as monitored by 1H NMR
spectroscopy.
156
Time dependent formation of hemiacetal 28a and acetal 28b in
CD3OD at room temperature as monitored by 'H NMR
spectroscopy.
157
Time dependent cyclization of b-chloro amide 66 with K2CO3 at
50 °C to oxazoline 15b as monitored by 1H NMR spectroscopy.
164
'H-'3C HMBC spectrum of tetracydic compound 68.
167
VI.11
VI.15
VI.16
VI.17
VI.18
LIST OF TABLES
Table
Page
111.1
Ti values (in seconds) for hydrogens 3,4 and 6 of DHA.
29
111.2
Exchange of DHA in D20 at different pHs.
30
111.3
Integration values before and after exchange relative to the
methyl group as 3.
36
111.4
Values for the enzymatic conversion of 5 to 9 in 78 % D20.
37
Results obtained from the inhibition studies using anilides 1
through 5.
43
Summarizes the level of purification gained in each step. The
fold purification was calculated by dividing
68
IV.2
Standard used for protein quantification.
75
IV.3
Conditions used for assessing dithionite inhibition.
77
IV.4
Assay conditions for inhibition studies
78
VI.1
Improvements achieved for a practical synthesis of Z-DBH
111.5
IV.1
VI.2
(20).
128
A. Electron density distribution observed for carboxamides
lib and lie as calculated using AM1 Semi Empirical
calculations (no solvent effects)
138
This work is dedicated to the family
that I have been blessed with
Jaime Antonio Barrios Coronado
Ana MarIa Sosa Montenegro
Juan José Barrios Sosa
Andrés Barrios Sosa
MarIa Inés Barrios Sosa
They are with me everywhere I go.
STUDIES ON NITROGEN CONTAINING SECONDARY METABOLITES
FROM TERRESTRIAL AND MARINE ORIGIN: PART I. ENZYMATIC
EPDXIDATION OF 2,5-DIHYDROXYACETANILIDE IN
STREPTOMYCES SF. PART II. SYNTHESIS OF MARINE SPONGE
ALKALOIDS: SLAGEN1NS, AXINOHYDANTOINS, AND STUDIES
TOWARDS THE PYRROLOPIPERAZINE SYSTEM IN THE
AGELASTATIN CORE
CHAPTER I. GENERAL INTRODUCTION
The discipline of organic chemistry plays. a crucial role in understanding
and predicting
the
type of chemical transformations that are unique to particular
compounds. The marine and terrestrial environments represent unique sources for
new, exciting and useful chemistry that could find application in drug
development and the fight against increasing drug resistance.
Although in some cases chemical transformations can be studied in a
biological environment, this is not always possible. As an alternative, information
regarding the chemistry of a class of compounds can be obtained through
synthetic studies. These findings result in the development of new methodologies
and the expansion of the utility and scope of known methods for the production of
biologically active compounds.
In addition, it is often found that organic
synthesis represents the only means for acquiring sufficient quantities of a
particular target compound or its structural analogues.
The dissertation presented in the following pages focuses on two studies of
nitrogen containing metabolites from terrestrial and marine origin. In both cases,
2
spectroscopic methods and organic synthesis serve as important tools. Part 1 of
this thesis describes deuterium exchange analyses and efforts towards the
synthesis of a structural analogue of 2,5-dihydroxyacetanilide with the objective
to gain an understanding on the epoxidation of this substrate by unusual oxidases
in Streptomyces sp. Part II of the thesis presents the synthesis of the marine
alkaloids slagenins, axinohydantoins and an approach to the pyrrolopiperazine
system in the agelastatin core.
Our path to these alkaloids relies on the
development of methods for the synthesis and functionalization of linear
imidazolones. A practical synthesis of Z-debromohymenialdisine, a potential
drug candidate for the treatment of osteoarthritis, is also included.
3
STUDIES ON NITROGEN CONTAINING METABOLITES FROM
TERRESTRIAL AND MARINE ORIGIN. PART I. ENZYMATIC
EPDXIDATION OF 2,5-DIHYDROXYACETANILIDE IN
STREPTOMYCES SF. PART II. SYNTHESIS OF MARIN SPONGE
METABOLITES: SLAGENINS, AXINOHYDANTOINS, STUDIES
TOWARDS THE PYRROLOPIPERAZINE SYSTEM IN THE
AGELASTATIN CORE
PART I. ENZYMATIC EPDXIDATION OF 2,5DIHYDROXYACETANILIDE IN STREPTOMYCES SF.
4
ENZYMATIC EPDXIDATION OF 2,5-DIHYDROXYACETANILIDE IN
STREPTOMYCES SP.
CHAPTER II. INTRODUCTION
Enzymatic processes that catalyze the reactions of organic substrates with
dioxygen have been widely studied.' Oxygenases are the enzymes responsible for
the incorporation of oxygen atoms from dioxygen into organic substrates. The
first piece of evidence that suggested the existence of this class of enzymes was
independently collected by Mason2 and Hayaishi3in 1955. The enzyme
Pyrocatechase from Pseudomonas
sp.
was the focus of Hayaishi' s studies during
this time. Analysis of the reaction catalyzed by this enzyme using
indicated
that the 2 oxygen atoms derived from the labeled material are incorporated into the
final product; a dioxetane intermediate was proposed:
Pyrocatechase
OH
C0180H
+ 180
cIIICO18OH
OH
Parallel to this discovery,
180
labeling studies led by Mason using the
"phenolase complex" showed a similar result:
Cu
+
Protein'
"Cu2
+
Nct:i'
II
A8OH
+2 e-
18
I
OH
Cu2
OH
Protein" 1i8O
Protein'
Up until this time it was thought that molecular oxygen could only serve
as an electron acceptor in oxygen-utilizing oxidase or dehydrogenase reactions.
Therefore, evidence of incorporation of this element into the final organic product
was a discovery of great importance.
Oxygenases are currently further classified into monooxygenases and
dioxygenases:4
a) Monooxygenase: Also called "mixed function oxidases" or "mixed
function oxygenases." They require 2 electrons to reduce the second atom
of molecular oxygen to water. Electrons can come from an external source
like NADH ("external monooxygenase"), or from the substrate itself
("internal monooxygenase"):
XH+02+AH2-*X(0)H-i-H20+A
XII = substrate; All2 = electron donor
b) Dioxygenases: Do not require a reducing agent. Intermolecular
dioxygenases incorporate the oxygen atoms into two separate substrates.
Intramolecular dioxygenases incorporate both oxygen atoms into a single
molecule.
6
XH +02
X(02)H
The enzyme cytochrome P450 is one of the most abundant
monooxygenases; over 450 distinct P450 enzymes are known to exist. They are
generally membrane bound, and are found in every mammalian tissue or organ, in
plants, bacteria, yeast and insects. These enzymes catalyze a variety of reactions
like hydroxylations, reductions, dealkylations, oxidations, dehydrogenations, and
oxidative C-C bond cleavage, using principally hydrophobic substrates.th5 Steroid
biogenesis, drug activation and deactivation, pro-carcinogen activation, xenobiotic
detoxification, catabolite assimilation, and fatty acid metabolism are some
examples of the processes where P450 enzymes play a crucial role.5'6 As an
exception, it is worth mentioning that P450 is unable to use methane as a substrate
(a separate "methane monooxygenase" hasevolved to accomplish this).k
The information contained in crystal structures of camphor specific P450
(P45Ocam) has served as the basis for the study of other P450 enzymes.5
Structural information is now available for several of these enzymes and a general
mechanism of action has been proposed.59 The heme iron of the substrate-free
enzyme exists in a hexacoordinate geometry and occupies a low spin configuration
(Figure 11.1).
7
H.0H
ROH
RH
Cys
[01
Fe(IV)
Fe(III)
I
S
Cys'
H2H
Fe((fI)
e
N, cN
Fe(tI)
-_;
Cys,s
Fe(IlI)
N,I__N
Nr'N
N
N
9
,f
Cys,s
,S
Cys
FeQI)
Cys'
Cys'
IIE!
Fe(III)
Cys
Figure 11.1 Proposed mechanism of oxidation for camP45O.
In this case, a water molecule (solvent) fills the substrate binding region.
During substrate binding, stenc conflict with the substrate results in the exiting of
the aqua axial ligand. As a consequence of this process the heme iron is shifted to
a high spin configuration which increases its redox potential.
[ti
An electron is then transferred to the heme iron followed by binding of molecular
oxygen and a second electron transfer. Formation of the active iron species is
achieved by cleavage of the oxygen-oxygen bond, at which point one of the
oxygen atoms is released in the form of water.
Cleavage of the oxygen-oxygen bond is thought to proceed via a "push-
pull" mechanism which requires the presence of It donors in the active site. Site
directed mutagenesis in combination with kinetic
studies79 have
shown that for
P45Ocam amino acids Thr-252 and Asp-25 1 are crucial for bond cleavage.
b)
a)
0H
HOH---O
- /
-
Asp-251
O'
0H--O
OH
I
I
OThr-252
Asp-251
/
I
H-0Thr-252
H-i-
H-i(
(
HOH--OAsp251
0
H-_-Thr252
H-9H-"-OAsp-251
NN
0
HThr252
Figure 11.2 It Transfer prior to oxygen-oxygen bond cleavage in P4SOcam.
Mutations at these sites result in decreased or no enzymatic activity in
combination with the release of hydrogen peroxide (no 0-0 bond cleavage). The
exact role of Thr-252 is questionable. Although initial studies suggested that this
residue could act as the actual it donor (Figure ll.2a)7 it is likely that this residue
acts as an acid catalyst (Figure ll.2b).9
9
Catechol dioxygenases are enzymes involved in the biodegradation of
environmental aromatic compounds. Their mode of action involves the
incorporation of molecular oxygen into the aromatic ring of catechols followed by
a ring opening reaction. The hydroxyl substituents in the substrate are generally
ortho or para to each other, and the catalysis usually requires a tightly bound
mononuclear non-heme iron or, in rare cases, non-heme manganese ion.'°
Catechol dioxygenases are classified according to the position on the substrate at
which the ring opening step occurs:
a) Intradiol dioxygenases: Generally contain a catalytic high spin Fe(llI) ion
in the active site. They catalyze the following reaction:
180
f.CO18OH
LCO18OH
OH
OH
b) Extradiol dioxygenases: These enzymes are more abundant than the
intradiol dioxygenases; however, their sensitivity to oxidizing compounds
makes them less stable. They generally contain a catalytic high spin Fe(Il)
or a Mn(ll) ion in the active site. Extradiol dioxygenases catalyze the
following reaction:
18
CH18O
LCO18OH
OH
OH
10
Intradiol and extradiol dioxygenases are two groups of enzymes
completely different from each other. Their amino acid sequences, subunit
compositions, and the polypeptide chain foldings found in each subunit are
completely unrelated.18 For instance, although the metal centers in 2,3
dihydroxybiphenyl dioxygenase (BphC) and 3,4-dioxygenase (3,4-PCD) have
similar coordination geometry, intradiol cleavage does not occur in the extradiol
BphC and extradiol cleavage in the intradiol 3,4-PCD. It has been suggested that
while intradiol dioxygenases activate the substrate for oxygen attack, extradiol
dioxygenases activate the dioxygen bound to the ferrous ion. It is believed that the
difference in pKa of the amino acid residues bound to the metal center is
responsible for the specificity of these enzymes.
3,4-dioxygenase, isolated from Pseudomonas aeruginosa, is an intradiol
dioxygenase for which crystallographic information has been obtained.10'1' In this
case the geometry of the non-heme iron is a slightly distorted trigonal bipyramidal
Figure 11.3) where three oxygen anions are coordinated to the iron center
providing electrostatic neutrality to the complex.
Ii
Angles observed:
Y408-Fe-Y447
Y408-Fe-H460
Y408-Fe-H462
Y408-Fe-Wat827
Y447-Fe-H460
Y447-Fe-H462
Y447-Fe-Wat827
H460-Fe-H462
H460-Fe-Wat827
H462-Fe-Wat827
Tyr 447
99.4°
9330
Tyr 408
*
90.9°
123.7°
0
--
Wat 827--
167.4°
92.7°
78.6°
N
140. 8°
His 462
I
His 460
87.4°
Figure 11.3 Geometry of the iron center according to crystallographic data.
Several analyses using structural models'2'4 have demonstrated that
substrate binding activates the metal complex for oxygen intake. The synthesis of
substrate bound iron catalysts containing tripodal ligands has helped to provide
both structural clues as well as kinetic information. The following observations
were noted:
-
Increased Lewis acidity at the metal center results in the production of higher
yields of the desired anhydnde intermediate (see Figure 11.4 for the mechanism
of the reaction).
-
Enhanced covalency of the metal-c atecholate bonds results in greater
semiquinone character in the aromatic ring which increases its reactivity
towards molecular oxygen.
The catalytic cycle of the 3,4 PCD (Figure 11.4) starts with the formation of
a bidentate linkage of the substrate with the non-heme iron center. Attack of
molecular oxygen results in ketonization of the C-O bond and displacement of the
12
C3 oxygen ligand from the metal center. Cleavage of the 0-0 bond is followed
by insertion of one oxygen atom into the C3-C4 bond, resembling a Baeyer-
Villiger type reaction, which produces an intermediate anhydnde. Incorporation
of the second oxygen atom into the anhydride leads to the formation of the desired
product.
H
LIGAND
Coo
02
H20
[00C
(Fe)
'Fe..]
(Fe) HJI"
(Fe)
SUBSTRATEH
/ 0-0
H
(Fe)
PRODUCT
(Fe3)
ool
00C
o 0Fe
(Fe)
(Fe)
Figure 11.4 Proposed mechanism for 3,4-PCD.
Although initial studies proposed the existence of a dioxetane instead of an
anhydride intermediate, experiments using 1802 pyrogallol, and catechol 1,2-
13
dioxygenase (isolated from Pseudomonas arvilla)
15
provided evidence that is
inconsistent with this pathway.
As in the case of P450, crystallographic data obtained from 3,4-PCD has
revealed the existence of other residues in the active site (Figure 11.5) that could be
involved in catalysis.'° It has been proposed that the residue Arg-457 (labeled as
M457) could serve as a proton relay system to remove the substrate hydroxyl
protons of the active site. Arg-457 fonns a hydrogen bond with Gln-477 (labeled
as M477) which is part of a hydrogen bond network that reaches solvent
molecules. Therefore, an alternative path could involve stabilization of
developing carbanions by the positively charged Arg-457 side chain.
Figure 11.5 Active site in 3,4-PCD.
14
Acquisition of the first three dimensional structure of an extradiol ringcleavage type dioxygenase was not possible until
recently.'618
2,3
Dihydroxybiphenyl dioxygenase (BphC) is an octameric enzyme that catalyses the
degradation of polychiorinated 2,3 dihydroxyphenyls in Pseudomonas sp. Crystal
structures of this enzyme revealed that the active site is positioned so that catalytic
residues cannot be exposed to the surface of the molecule, suggesting that the
substrate needs to go through an entrance route into the active site. Prior to
substrate intake the iron ion possesses a square-pyramidal coordination geometry.
Residues His-209, Glu-260 and two water molecules occupy the equatorial
positions, while residue His-145 occupies the axial site (Figure 11.6). Three-
dimensional structures of substrate-bound enzyme show that after substrate
binding the geometry of the metal complex changes to a distorted tngonal
bipyramid.
2.1
A
Water iWater 2
2.5 A
2.3
2.1A
His-14
GIu-260
I
2.2 A
Figure 11.6 Active site of BphC.
His-209
15
It was later noted that prior to crystallization, ferrous substrate-bound
complexes oxidize easily to the femc state. Since the crystallographic data
corresponds to a ferric ion, it can only be speculated that a substrate bound ferrous
ion acquires a distorted square bipyramidal geometry. Other residues identified as
His-194 and Tyr-249 are thought to be crucial for catalysis.
Two possible paths for the mechanism of the reaction catalyzed by
dioxygenases have been proposed.19 Prior to the mechanisitic studies using 2,3
dihydroxyphenylpropionate 1,2 dioxygenase (MhpB) (isolated from Escherichia
coli) and labeled molecular oxygen, labeling patterns (Figure 11.7) were possible
for the intermediates of each of the two paths:
A = CH2CH2COO-
0
}
R
0
R'-
,9_o-
*'os
I
0-..
0
I
Fe(It)
Fe(II)
I
Fe(Il)
1H0/
)I:;o'
A
OHR
e(II)
H20
Figure 11.7 Proposed mechanisms for the reaction catalyzed by MhpB.
The formation of product via a lactone intermediate would be
accompanied by loss of label at the Cl position of the substrate. This loss arises
from exchange of the metal bound hydroxyl group with water. The latter is in
16
accordance with the labeling patterns observed, therefore, it has been suggested
that a lactone species is formed, most likely, through an acid catalyzed Criegee
rearrangement (showed by the bold arrows in Figure 11.7). It has also been
observed that incubation of MhpB with a saturated seven member lactone
analogue leads to time dependent production of the desired hydroxy acid (Figure
coo-
coo
H20
MhpB
I
OH
'Fe
0-
Figure 11.8 MhpB catalysis using a seven member lactone analogue.
Intradiol and extradiol dioxygenases are not the only types of
dioxygenases known. Lipooxygeases, a-ketoglutarate linked dioxygenase, as well
as dioxygenases that hydroxylate aromatic substrates in two adjacent positions
with loss of aromaticity and no ring cleavage, have also been studied.1
Dihydroxyacetanilide Epoxidases I and 11 (referred to as DHAE I and
DHAE H) have been the focus of Gould and coworkers' studies for a number of
years. They represent one of the first examples where two enzymes isolated from
different organisms produce enantiomeric compounds:2°
17
0
O
NH2
___ 0
0
NHAc
___
DHAEI
DHAEII
0
OH
MM14201
0
O
OH
NHAc
OH
oj
NHAc
O
)LNHAc
OH
LL-1 0037cz
2,5 clihydroxyacetanitide
Figure 11.9 Epoxidations catalyzed by DHAE I and DHAE II.
Antibiotic LL-C10037a, from Streptomyces LL-C10037a, was first
isolated in 1984 by Lee and coworkers, however, its structure was later revised on
the basis of X-ray diffraction and excitation circular dichroism analyses.2' A
closely related molecule with an oxirane functionality containing the opposite
stereochemistry, MM 14201 from Streptomyces MPP-305 1, was also isolated and
characterized by Box and coworkers at Beecham
Pharmaceuticals.22
The information presented below summarizes advances made towards the
understanding of the intriguing mechanism of action of the two oxygenases that
introduce the epoxide functionality into these molecules. These enzymes possess
characteristics that have not been previously observed and represent one of the few
cases where the mechanism of action is likely to involve a dioxetane intermediate.
DHAE I is a pentamer or a hexamer with a molecular weight of 117± 10
lcD. Each subunit possesses a molecular weight of 22.3 kD.23 This enzyme
follows classical Michaelis-Menten kinetics with an apparent Km of 10.7 ± 1.8 M
and an apparent V
of 0.0043 ± 0.0004 xmol/min; substrate inhibition is
observed at approximately 150 pM. Addition of NADH, NADPH, NAD,
NADP, FAD, FMN, EDTA, CN and CO have no effect on the enzymatic
activity. This indicates that no additional cofactor is required and that the enzyme
is not likely to be a P450. However, the enzyme does require a metal ion and a
sulfhydryl group. Addition of 1,10 phenanthroline (0.2 mM) and PCM1BA (0.5
mM) are detrimental to the enzyme. The optimal pH for enzymatic activity lies at
6.5. Prior to the elucidation of the mechanism for the epoxidation step, the
following biosynthetic scheme had been proposed and confirmed for the
production of the antibiotic LL-C 10037cc24
D-erythrose
OH
shikimic acid
OH 0
3-hydroxyanthranilic acid
OH
COOH
1.LNH2
HO&JLH
OH
HO?OH
-COOH
OH
0
)i
O
\OH
OH
NHAc
NH2
NH2
O
ii
Reduction
OH
OH
,J&NHAc
NHAc
DHAE
0
OH
LL-1 0037cL
Figure 11.10 Biosynthesis of LL-C10037a.
The epoxidase DHAE II is a dimer with a total molecular weight of 33 ±2
kD and a subunit molecular weight of 16 kD. Its optimal pH has been found to be
5.5. Like in the case of DHAE I, DHAE II does not require a cofactor; however,
the addition of CO does result in loss of enzymatic activity.23 DHAE II follows
19
classical Michaelis-Menten kinetics with substrate inhibition at around 100 pM.
The value of the apparent Km is 14.9 ± 3.2 p.M and the apparent
Vmax
is 0.0058 ±
0.0004 p.mo]Jmin. Studies on DHAE II have been limited by the instability of the
apoenzyme. As a result, more information needs to be obtained to gain
understanding of the similarities between DHAE I and II. It is unknown whether
the epoxide final product IMIlvfl420l is derived from shikimic acid, sinceno
biosynthetic analyses have been undertaken. However, the following path has
been proposed for its biosynthesis:
o
I
OH
NHAc
NHAc
DHAE U
0
0
0
0
OH
-kyNHAC
Reduction
OH
OH
MM1 4201
Figure 11.11 Proposed path for the biosynthesis of 1v11v114201.
Interestingly, the mechanism of the enzymatic epoxidation by DHAE I and
DHAE 11 has been proposed to resemble the mechanism for the regiospecific
oxygenation of vitamin K hydroquinone (vitamin KH2) proposed by Dowd and
coworkers in 1993.25 Vitamin K is an important cofactor used by carboxylase in
the process of blood clotting. As shown below (Figure 11.12) the epoxidation of
vitamin KH2 is coupled to a carboxylation step where glutamyl residues are
converted to y-carboxyglutarnyl residues (no metal ion is required):2527
20
0ii
H
J&
N
OH
I
N
H
1
/-GIuvitamin KH2
coo-
V
1802
OH
co2 A Carboxylase
0
N
vitamin
180
N
H
7'-coo-
COO-
Figure 11.12 Formation of vitamin K oxide.
Labeling studies have indicated that two oxygen atoms are incorporated
into vitamin K oxide. Analyses using the model compound naphthoxide (Figure
ll.13b) support the existence of a dioxetane intermediate which is thought to lead
to the formation of a strong base that can produce the glutamyl anion needed for
carboxylation28
(see Figure ll.13a). Both carboxylation and epoxidation are
inhibited by glutathione peroxidase which reduces hydrogen peroxide and organic
peroxides.26
21
a)
peroxide
0-
0-
OH
0-
(LR
OH
Lr
OH
vitamin KH2
0
-O
O-.11
0
-strong baseHO-
R
0
dioxetane
dialdoxide
-strong base-
b)
0-
0
0
C(7
naphthoxide
cis
cis
keto epoxy alcohol
relative stereochemistry obtained
from crystal structure29
Figure 11.13 Proposed mechanism of epoxidation of vitamin KH2.
Although labeling studies have only been done with DHAE I the
mechanism shown in Figure 11.13 has been applied to both DHAE epoxidases.
Both oxygen atoms are incorporated into the epoxyquinone, however immediate
reduction of the epoxyquinone to the semiquinone is required to avoid complete
loss of label at the C4 position.3° Incorporation of label in the oxirane ring was
quantitative, while incorporation of labeled oxygen at the C4 position was
22
reported to be 20 %. Figure 11.14 shows a mechanism that agrees with the
observed incorporations.
a)
(.\
'H B
L0
,LNHAc
0
)L,,.NHAC
[
I
0
)LNHAc
-
)NHAc
I
U
OH
OH
OH
o
°
1
cNHAd1
I
0 OH
j
O=O-ENZ
°"
)9 "Y"
0
HINHAc
o:y
0
b)
O
O
OP
HOL
2-acetamido-5,6-epoxy-1 ,4benzoquinone
dehydrogenase
NHAc
/OH
HO-0
NADPH
NADP
II
N HAc
OH
LL-10037a
OP
OP
spontaneous
(pH = 7)
0
HO0 glucose-6-phosphate
dehydrogenase
HOL\
HOOH
Figure 11.14 a) Proposed mechanism for the epoxidation catalyzed by DHAE. b)
Subsequent reduction to trap the label at C4.
The enzymatic activity of DHAE can be regenerated from the apoenzyme
by the addition of Mn(11) and Ni(11) in the case of DHAE I, and FeW), Cu(I),
Cu(11) in the case of DHAE 11. Enhanced activity is observed with both enzymes
after the addition of Co(11). The flexibility of the DHAE enzymes has been further
explored by means of inhibition studies.3° Assays using 2-,3- and 4-
23
acetamidophenol revealed that monohydroxylated acetanilides are weak
competitive inhibitors. However, modifications in the N-acetyl side chains have a
much stronger effect on the enzymatic activity.
K1
values as low as 2±28 pM for
DHAE II and 32±2 iM for DHAE I are observed when p-nitrobenzoyl replaces
the N-acetyl side chain.
Enzymes that require Co(II) for enzymatic activity are rare; therefore, the
role of the metal used by these enzymes is a puzzle. Whether the mechanism
proceeds via a two or one electron system still needs to be determined. Most of
the information collected has revealed interesting properties about DHAB I,
however, it is possible that DHAE II catalyzes the epoxidation by different means.
The inhibition of the latter by CO and the regeneration of activity of the
apoenzyme by ferrous ions could suggest that this enzyme is an internal
monooxygenase (it has not been fully established that DHAE II incorporates both
oxygen atoms into the substrate). Therefore, the analyses presented in the
following discussions will focus on attempting to gain more understanding on the
enzymatic activity of DHAE II, specifically, binding and activation of the
aromatic ring. Deuterium exchange analyses as well as kinetic studies are
described next.
24
CHAFFER III. RESULTS AND DISCUSSION
3.1 DEUTERIUM EXCHANGE ANALYSIS
Deutenumhydrogen exchange studies have been -a useful tool for the
study of enzyme catalysis. One of the most important applications of this
technique deals with the analyses of peptide and protein dynamics and
structures.31
The rates at which specific amide hydrogens exchange with
deuterium depend on solvent accessibility and the formation of specific hydrogen-
bonding patterns to the aniide hydrogen. Therefore, amide hydrogen exchanges
can be used to analyze the inherent stability of the folded polypeptide structure
and the constraints imposed by complexation with ligands. Early studies led by
Kaegi32
focused on the exchange behavior of the coenzyme (NAD, NADH) and
coenzyme-fragment complexes of yeast alcohol dehydrogenase and porcine heart
lactate dehydrogenase. The measurements that described the exchange behavior
relied on the interpretation of infrared spectra. In 1997 Tang and coworkers33
reported a similar study using dihydropicolinate reductase, an enzyme in the
succinylase pathway of bacterial L-lysine biosynthesis in
Escherichia coli.
In this
case, the exchange behavior was analyzed by a combination of mass spectrometric
methods, protease catalyzed protein hydrolysis, and HPLC separation at pH
values where exchange was significantly slowed. These developments have
25
simplified the analysis of conformational changes using hydrogen-deuterium
exchange.
OHO OH
CH3
HO
0
1
D20, H+
OHO
OHO OH
OH
H
D
I
0
0
CH3
DHQ
2b
2a
OH 0
from
CH3
0
OH 0
OH
HO_IIEXII5.H
from
OH
HOTI5cH3
NAD, D
H0
3b
3a
DAL
OH 0
OH
D11-CH3
DO
4
Scheme 1
Although deuterium exchange studies are widely used for peptide
dynamics, in 1988 Anderson, Scott, and coworkers applied this technique to the
26
study of the enzymatic removal of phenolic hydroxyl groups in the biosynthesis of
polyketide derived natural products (Scheme
The anthraquinone emodin 1 was incubated in a cell free system from
Pyrenochaeta terrestris
containing 50 % D20 buffer. Deoxygenation of 1 lead to
the formation of chrysophanol 4 which was deuteriated at positions C6, C5 and
C7 (Scheme 1). Deuteriated NAD2H mediates the exchange at C6. It is believed
that this deuteriated cofactor forms under the experimental conditions.
Incorporation was highest in the presence of active enzyme, therefore, it has been
proposed that ketotautomers 2a and 2b are stabilized in the active site.
As shown above, deuterium exchange studies can provide important
information about the mechanism of an enzymatic reaction by analyzing the
response of the enzyme to substrate or cofactor binding, or by the behavior of the
substrate as it reacts in the active site. In the case of DHAE II, the results obtained
from the exchange of the substrate (2,5 dihyclroxyacetanilide) with D20 were
expected to help answer standing questions in the proposed mechanism of
epoxidation.
The mechanism of epoxidation catalyzed by DHAE proposed by Gould
and coworkers4° relies on a series of nucleophilic attacks. These two-electron
processes start with the selective removal of one of the hydroxyl protons located
at carbons C2 and CS of the substrate 5 by a base in the active site. The removal
of either of these two protons can lead to the formation of an organo-peroxide 6
which acts as a nucleophile to form the dioxetane intermediate 7. Further
27
nucleophilic attack of the aromatic ring to one of the oxygens in the dioxetane ring
results in the formation of intermediate 8 which dehydrates to form the desired
product 9 (Scheme 2).°
,,,_cIIIJHAc
cjNHAc
OH
NHAc
I
0-
-O-
OH
6
OH
OH
0
I-
Oos*S'
NHAc
OH
9
I
-o
0
OH
NHA C
8
7
5
0
NHAc
NHAc
of;
- H20
Scheme 2
Labeling studies using
1802
have suggested that the hydroxyl group at the
C2 position is removed preferentially. However, it is not clear yet whether the
reaction is in fact a two or a one-electron process. The deuterium exchange
analysis was designed with the main objective of addressing this question.
If the reaction is an acid-base catalyzed process, in the absence of
molecular oxygen, acid-base interactions between residues in the active site and
one of the hydroxyl groups on the substrate would generate base lOa or lOb
(Scheme 3)3536 Exposure of these intermediates to D20 could lead to the
formation of the deuteriated products 12a,b which could not only help to confirm
which form is produced preferentially (by path A or B), but would also provide
evidence of the nucleophilic nature of the aromatic ring.
0
Q)LNHAC
Path A
0
H
DNHAC
D)JNHAc
D20
B:
OH
OH
lOa
ha
OH
OH
I 2a
3Lj,NHAC
c
-a
J1NHAc
s/B.
DO
Path B
OH
L,_NHAc
D4Y
H
0
llb
lOb
D>
OH
1 2b
Scheme 3
3.1.1
Control Experiments
3.1.1.1
T1 Measurements.
The results obtained from control studies and other deutenum exchange
analyses were evaluated using 1H and 2 NMR spectroscopic methods.
Incorporation of deuterium in the product was to be quantified directly from the
relative peak areas of the hydrogens in the spectra.
29
I
H
OH
'H N1\'IR shift
(acetone-d6)
T, value
Standard deviation
H
H-3
6.72
7.04 s
0.02
11-4
6.52
7.49 s
0.05
H-6
7.03
6.04 s
0.04
T1
6 H
OH
I
Table III.!
H
HtNCH3
I
values (in seconds) for hydrogens 3, 4 and 6 of DHA.
Reliable integration of the peaks was achieved by setting the relaxation
delay at 35 seconds, this value is approximately 5 times the T1 of the hydrogens in
the molecule.37 The spin-lattice relaxation times for the aromatic hydrogens of
2,5-dihydroxy-acetanilide (DHIA) are shown in Table ifi. 1.
3.1.1.2 Deuterium-hydrogen exchange of DHA at different pHs.
Exchangeability of the aromatic hydrogens of DHA in D20 was
investigated at basic, neutral, and acidic conditions. The reactions were carried
out under Ar for 1 hr at 25 °C. In addition, the substrate was exposed to D20 in a
buffered solution at pH 6.5 (with a final concentration of 0.1 M KH2PO4) to
observe if exchange occurred at the pH ideal for DHAE II activity.
30
pH
acid/base source site of exchange
time
>11
(10%)NaOD
---
lhr.
9
(2 %) NaOD
---
1 hr.
7
lhr.
6.5 (O.1M KH2PO4)
land2hrs.
4-5
(2%)D2SO4
---
lhr.
1
(10%)D2504
20%atC4
1-hr.
Table 111.2: Exchange of DHA in D20 at different pHs.
These control exchange experiments were carried out with two main
objectives:
a) Establish at what pH the aromatic hydrogens of DHA exchange in the absence
of enzyme catalysis.
b) Any exchange should be quantified and compared with the values obtained
from experiments carried out in the presence of enzyme.
The results obtained from this study are summarized in Table 111.2.
The formation of the deuteriated products 12a,b is anticipated to be a base
catalyzed reaction mediated by the enzyme (Scheme 3). Therefore, it was
expected that exchange of the substrate in D20 under highly basic conditions
31
would result in partial incorporation of deuterium at the activated carbons C3
and/or C4. However, only intact starting material was recovered from the basic
mixture. In addition, no exchange was observed in neutral and buffered
conditions. Prior exchange
studies38
earned out at high temperatures and long
reaction times at neutral and acidic pHs have shown that exchange is observed at
C4 and C6 only under harsh conditions (3M DC1, 80°C, 0.5 hrs.). In accordance
with these results, incorporation at C4 was observed only in highly acidic media.
It is possible that longer reaction times and higher temperatures could also give
exchange products under basic conditions. However, under these circumstances
high levels of oxidation of DHA to the corresponding quinone would take place.
As shown above, exchange of the aromatic hydrogens in DHA is slow. If
exchange occurs in the presence of enzyme, then that exchange can be attributed
to enzymatic activation of the ring. The chemical reactivity of the substrate shows
that C4 is more nucleophilic than C3.
3.1.1.3 Deuterium-hydrogen exchange
of DHA
in the presence of DHAE II.
Analysis of the exchangeability of DHA in the presence of DHAE II was
carried out in two separate systems. In the first system (A) compound 5 was
allowed to exchange for a 1 hr period in a buffered solution (pH 6.5) containing 78
% DO. The second system (B) contained the deuteriated compound 13(91 %-D
at C6, 64 %-D at C4, and 99 %-D at the acetyl group), which was allowed to
exchange for 1 hr in a buffered solution prepared in H20.
32
DHAE II
Path
D20
OH
DNHAc
H20
B:
OH
rYi NHAc
DHAE I!
Path A
OH
12a
4i.Ji6
OH
I
OH
2L
1
NHCOCD3
13lJ1Q
D
Path B
DO
______
DHAEII
D4r
OH
12b
H.kNHCOCD3
DD
OH
14a
OH
D
HLNHcoco3
OFJ
B:
OH
BJ
Path B
HO
DHAEII
OH
14b
Scheme 4
Systems A and B were constructed with the objective of monitoring the
exchange of the substrate in the presence of enzyme from two different directions.
The results from one system should be comparable with the other, and in
combination should provide reliable exchange values. Indication that exchange
occurred in one system and not in the other could help to identify any
inconsistencies between the two experiments.
After carrying out the exchange for 1 hr under Ar at room temperature the
organic material was extracted with EtOAc. Enzyme mixture (purified as
specified in the experimental section) was added to the system to initiate exchange
and after 30 mm to regenerate any enzyme activity lost. The identity of the
material recovered from each system was evaluated using 'H and 2H NMR
spectroscopy (Figure ifi. 1).
OH
H3
0
H6
1
OH
H4
I
9.0
8.5
8.0
7.0
7.5
ii
60
6.5
5.5
5.0
4.5
4.0
3.5
3.0
2.5
pzr
4.0
3.5
3.0
2.5
ppzr
H3
OH
H4
H6
9.0
8.5
8.0
7.5
/
7.0
6.5
6.0
5.5
5.0
Figure 111.1 1H NMR of material used in the exchange studies
4.5
34
Among the impurities found in each extraction lie protein residues and
buffer components, like glycerol, which are contained in the enzyme mixture.
Careful analyses of the deuterium and hydrogen spectra revealed that both 5 and
13 were recovered unchanged. The presence of a broad peak at 7.5 ppm in the
of the recovered 13 suggests that the mixture could contain traces of the
corresponding quinone. Comparison of the 1I4R spectrum of the mixture with
a spectrum obtained from unfed 13 revealed that oxidation occurred prior to the
addition of this compound to system B.
C
[iii
9
8
7
5
4
3 pm
Figure 111.2 Spectra acquired after enzymatic exchange. A) 2H NMR spectrum of compound 5. B) 1H NIMR spectrum of compound 5.
C) 2H NIMR spectrum of compound 13. D) 'H NtvIR spectrum of compound 13.
36
C-H
C3
Before Exchange
0.9959
After Exchange
1.0289
OH
C4
0.9922
1.0239
H1Q.CH3
6H
C6
0.9970
1.0442
OH
Table 111.3 Integration values measured before and after exchange relative to the
methyl group as 3.
The lack of formation of exchanged products could be justified if the
enzyme activity was lost immediately after its addition to the system. Therefore, a
second study was designed with the objective to determine whether the enzymatic
activity was retained for the half-hour period needed prior to the addition of more
enzyme mixture. Initial attempts focused in running a set of systems A' and B'
containing molecular oxygen parallel to a set of systems A and B run under Ar. If
the enzyme activity was retained during the two 30 mm periods, then partial
conversion of 5 to the epoxyquinone 9 should occur in system A' and conversion
of 13 to the corresponding epoxyquinone in B'. However, it was not possible to
identify any signals corresponding to the enzymatic products. This was due to
two main factors: a) overlapping signals arising from impurities in the system b)
the formation of only traces of epoxyquinone which gave weak signals with
questionable splitting patterns.
37
Successful product identification was possible by modifying a system
developed by M. Kirchmeier.4° In this case the enzymatic epoxidation was
carried out in a buffered solution (pH = 6.5) containing 78 % D20 for a period of
18 hrs. using increased concentrations of DHA. The levels of product formation
were monitored by HPLC.
material added
time
(miii)
0
%
Product
conversion
(mg)
3.00%
0.21
% glycerol, pH = 6.5) in
12 mL (50 mlvi KH2PO4, 5
D20
1 mL 10mM CoC12. 6 D20
4mL9.1 iTIMDHA
2 mL DHAE II (specific activity
8.0 imoI1(min mg))
---
15
30
2 mL 9.1 mM DHA
7.18
%
0.43
120
1 mL9.1 mMDHA, 1 mLDHAEII
11.34%
1.12
180
2.5mL9.1mIvIDHA
13.12%
1.51
260
4 mL DHAE II
15.46
%
2.41
360
21.82%
3.41
1080
57.52
%
9.00
Table 111.4 Values for the enzymatic conversion of 5 to 9 in 78 % D20.
Table ffl.4 shows the values obtained from this study. Epoxyquinone 9
was recovered in a final yield of 57 %, and no deuterium was incorporated during
the enzymatic epoxidation. These results confirm that in the absence of molecular
oxygen, active DHAE II does not catalyze hydrogen-deuterium exchange of the
substrate at pH 6.5 during the period of 1 hr.
Several hypotheses can help to explain why exchange of the substrate (5,
13) is not observed in the presence of active DHAE II. The simplest explanation
is that no solvent molecules can access the active site, therefore, even if carbanions
lOa,b are formed, the substrate will not be able to exchange.
However, if
molecular oxygen can access the active site it is likely that water molecules are
able to do so as well.
Alternatively, both protonation and deprotonation could occur
stereospecifically, leading to the recovery of unreacted substrate. Moreover, as in
the case of 3,4-PCD, it is also possible that residues in the active site of DHAE II
could help to stabilize the developing carbanions. It could be suggested that
conformational changes triggered by, for example, oxygen binding could free the
substrate from these residues to form the desired oxidized product. However, in
the absence of molecular oxygen the stabilizing residues could be constantly
exposed to the substrate.
39
A
<
OH/
NHAc
OH
B
H
(LNHAc
2<
Scheme 5
It should not be overlooked that the stabilization shown in Scheme 5
explains the lack of exchange of 5 and 13 through path A. If the amino residue is
a hydrogen donor and path B is part of the active mechanism then, contrary to
what has been observed experimentally, hydrogen would be incorporated at C4 of
compound 13.
The possibility that the bound substrate is not basic enough to accept a
proton/deuterium from the water should not be dismissed. Tn this case, the
substrate is unlikely to be nucleophilic enough to react with molecular oxygen.
Therefore, it would be appropriate to further investigate the role that the metal
plays in catalysis. As noted in the introduction, with the exception of VKH2
oxidase, most dioxygenases have a metal that serves as a spin flip catalyst to
convert triplet to singlet oxygen, or as a 1-electron donor. More information
about the metal complex could be gained by titrating DHAE using redox indicator
dyes of various potentials. This technique could be used to quantify the number
40
of reducing equivalents needed per metal center of DHAE I and II.
Massey39
and
coworkers used this technique to compare the redox potentials of the FAD and
Fe/S centers found in xanthine oxidase and xanthine dehydrogenase, which are
two forms of the same enzyme. In addition, this approach could help to determine
differences between the two DHAE enzymes that have not been considered
before.
41
3.2 UNEXPECTED RESISTANCE OF 2,5-DIMETHOXYANILIDES TO
ELECTROPHILIC CYCLIZATION.
The search for alternative substrates or inhibitors of DHAE I and II was
initiated by Gould and coworkers a few years
ago.4°
The main objective of this
study was to identify important features of the active site of both enzymes and
find information that could help to explain how each one controls the regio- and
stereochemistry of the epoxidation that leads to the selective formation of one of
the enantiomers of the epoxyquinone. It has been proposed that this selectivity
can be achieved either by the enzyme's manipulation of the orientation of the
planar 2,5-dihydroxyacetanilide, or of a putative oxygen-metal complex that
might be involved in the reaction mechanism. In previous work,41 compounds 1
through 5 had been tested as inhibitors. The analysis of the corresponding
inhibition constants helped to determine the importance of the different functional
groups around the aromatic ring of 2,5-dihydroxyacetanilide.
42
OH
NO (NO
H
çi
CH3
1
H
H
CH3
OH3
OH2
3
-O_
Scheme 6
It was observed that mono-hydroxylated acetanilides (1
3) only poorly
inhibit substrate binding. It has been suggested that in the presence of these
compounds interactions between active site residues and both hydroxyl groups
of the substrate (e.g. hydrogen bonding) help to stabilize its binding, leading to
high levels of epoxide formation. On the other hand, substitution of the N-acetyl
group by benzoyl (4) or p-nitrobenzoyl (5) side chains provided compounds that
showed strong inhibition of substrate epoxidation (Table 111.5).
43
Analog
(DHAE-I)
Inhibition
Type
1
Competitive
Competitive
No inhibition
Competitive
Competitive
2
3
4
5
K
Km
(pMJmin)
0.0046 ± 0.0004
0.0042 ± 0.0004
0.0043 ± 0.0004
0.0080 ± 0.0023
0.0092 ± 0.0023
(pM)
11.8 ± 1.8
10.0 ± 1.8
10.7 ± 1.8
QiM)
103 ± 8326
1348 ± 1854
24± 13
22± 10
35 ± 13
32±2
14.86 ±3.2
18.06 ± 3.2
15.94 ± 3.2
1208±2059
149±47
106±47
27±6.4
2±28
(DHAE-II)
Competitive
Competitive
Competitive
Competitive
Competitive
1
2
3
4
5
Table 111.5
40
through
0.0058±0.0004
0.0062 ± 0.0004
0.0058 ± 0.0004
0.022 ± 0.011
0.01 ± 0.011
505 ± 158
979 ± 544
Results obtained from the inhibition studies using anilides 1
In addition to the inhibition studies, compounds 1 through 3 were tested
as alternative substrates using the enzyme DHAE I.
No conversion was
observed in a period of time 10 times longer that what is needed for high
conversion levels in the presence of DHA. It was concluded that, although these
compounds are weak competitive inhibitors, they do not meet the structural
requirements needed for stable enzyme binding and, consequently, do not
undergo successful enzymatic oxidation.
In general, the study summarized above indicated two important factors:
a) The presence of both hydroxyl groups, at positions C2 and CS respectively,
is necessary for the formation of a stable enzyme-substrate complex.
b) Modifications on the N-acetyl side chain can result in the production of a
tight enzyme-substrate complex, where the enzyme looses its catalytic
ability.
The results presented above provided information, regarding the
importance of the functionality around the ring. The data suggests a degree of
flexibility in the identity of the N-acetyl substituent. To further investigate its
importance, a study using 1,4-dihydroxybenzene (6) was undertaken to learn if
the amide functionality is required for inhibition.
approaches
to dihydroquinolinone 7 are described.
In addition, different
In compound 7 the
orientation of the N-side chain is fixed using the aromatic ring as an anchor.
OH
OH
Scheme 7
3.2.1 Inhibitions Studies Using 1,4-Dihydroxybenzene and DHAE II.
Initial attempts to measure the binding constant
(Km) of the substrate
(DHA) following the procedure reported by Kirchmeier gave results that were
not in accordance with classical Michaelis-Menten kinetics. It was found that in
this procedure the stock solution of substrate (2 mM) was prepared using excess
of sodium dithionite to avoid chemical substrate oxidation to quinone which
could inhibit the enzyme. Therefore, the use of different volumes of the DIIA
stock solution signified the use of different volumes of the dissolved sodium
dithionite.
Attempts to keep the concentration of dithionite constant in the
system did not result in classical Michaelis-Menten behavior. This suggested
that if sodium dithionite was responsible for this result then it was part of a
complex reaction that is not proportional to its concentration. The reaction
could involve two processes:
1)
It was possible that this reducing agent was reacting with the cobalt and
decreasing the amount of metal available to activate the enzyme.
2) The sodium dithionite could be inactivating the enzyme.
The first probability was studied by removing the cobalt from the assay
conditions. It was found that in the absence of cobalt the enzyme was inactive,
therefore, no conclusion could be drawn in this case. To address the second
probability a stock solution of DHA was prepared lacking sodium dithionite.
Substrate oxidation could be avoided by carefully sparging the solution with Ar
and storing it at 78 °C.
After preparing this solution the effect of the
concentration of sodium dithionite on product formation was monitored. The
assays contained a constant concentration of DHA and varying concentrations of
sodium dithionite. The reaction was catalyzed by DHAE II and run for 6 mm at
30 °C (Figure 111.3).
Figure 111.3 Effect of the concentration of sodium hydrosulfite on percentage
conversion.
The results of kinetic studies performed in the absence of sodium
dithionite showed that under these conditions DHAE II follows MichaelisMenten kinetics. It was not possible to determine the side reactions that sodium
dithionite was initiating in the system, however loss of enzyme activity in its
presence has been previously
documented.41
In these cases, it has been
suggested that dithionite can reduce the disulfide bonds of the enzyme and so
affect its catalytic ability. Hydrosulfite has also been used to investigate the
redox potential of metal-containing enzymes. Since the oxidation state of the
metal is changed during this process the enzyme's catalytic ability is affected.42
In any event, because chemical substrate oxidation was most likely between
running the assay and HPLC analysis, dithionite was added to the terminating
solution rather than to the substrate.
47
5OuM
0.08
lOOuM
.5662x- 0.0133
= 0.9701
150 uM
x
0
contro'
y=
c3332x - 0.0081
y=
c1619x - 0.0023
0.04
=0.9735
A' = 9759
0.02
E
.0393x + 0.0027
A' =
-0 04
-02
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.9773
0 6
1/Es] (huM)
Figure 111.4 Results obtained from the inhibition study using 1 ,4-dihydroxybenzene. Protein concentration of the enzyme mixture: 1.45 tg/mL; enzyme
used in the each assay: 30 jiL. Control: Vmax (tMJmin) = 0.385 + 0.021; Km
(KM) = 15.57 + 1.81.
Once it was possible to measure the K of the substrate (DHA), the
compound 1,4-dihydroxybenzene was tested for inhibition. Figure ffl.4 shows
the results obtained using three different concentrations of inhibitor (50, 100,
and 150 jtM). It is important to mention that this compound is not an alternative
substrate at the conditions used for the inhibition studies; no epoxyquinone
product was observed.
These results show that 1 ,4-dihydroxybenzene is at best a weak inhibitor
(K = 146 tM). It is likely that, as predicted, the N-acetyl side chain group is
crucial for the formation of a stable substrate-enzyme complex.
3.2.2 Studies Towards the Synthesis of Dihydroquinoline 7.
The synthesis of compound 7 was undertaken with the main objective of
obtaining more information about the residues in the active site of DHAE II that
are responsible for substrate binding. In this compound the amide carbonyl (C9)
has a fixed orientation. The results obtained from this study could help to
determine if interactions between the residues in the active site and the molecule
are altered by the orientation of this functionality. In the approaches described
next, the formation of the bond between C6-C7 represented the key step of the
reaction.
OH
Initial attempts towards the formation of this crucial C-C bond focused
on the use of palladium chemistry.43"
The retrosynthetic analysis (Scheme 8)
shows the fragments that could be used to achieve the coupling.
OH H
CH3O
0
H
1NC(CH3)3
7
I
OH
CH3O
7
10
Scheme S
As shown in Scheme 9, the synthesis of fragment 10 relied on the orthodirecting effects of the amide carbonyl for selective
iodination45
at position C6.
The commercially availaMe dimethoxyaniline 8 was protected to give the
pivalated anilide 9 which was treated with 2 equivalents of BuLi and iodine to
give the desired intermediate 10 in 75 % yield. Synthesis of the precursor 12
was
straightforward.45
The allyl alcohol 11 was treated with TBDPSCI to give
the protected alcohol intermediate 12. In this case the use of TBDPSC1 was
important for the purification of 12, other protecting groups gave volatile oils
which were easily lost during solvent evaporation.
CH3O
CR3O
CH3O
NC(CH3)3
1 eq. PivCl
cNH2
0
quantitative
CH30
CH3O
(L1..C(CH3)3
75 %
CH3O
9
8
BuLi, 0 C, 12
10
TBDPSCI
OTBDPS
quantitative
12
11
Scheme 9
The procedure used for the Suzuki coupling relied on in situ formation of
the hydroborated compound 13. Attempts towards the coupling of fragments 10
and 13 were unsuccessful and gave principally recovered starting material and
decomposed fragments that are believed to be remaining residues of 9BBN
(Scheme 10).
Reactions using different Pd catalyst precursors (Pd(Ph3)4,
PdCl2dppf) were also run, however starting material and reduced allyl alcohol
were recovered from the mixture. Longer reaction times did not have an effect
on the outcome of the Pd coupling
CH3O
NyC(CH3)3
.OTBDPS
12
9BBN
KB-.OTBDPS
CH3O
13
OTBPDS
$
OH
CH3O
H
NC(CH3)3
-- - -
OH7
r°
CH3O
0
Scheme 10
A model study using the conditions shown above was done to test
whether the methoxy group at position C5 was hindering the oxidative addition
of the metal to the iodo intermediate 10. However, the use of iodobenzene
instead of 10 gave principally protected propyl alcohol and recovered starting
material. This was unexpected because as the reaction proceeded a color change
from brown to orange to brown was observed. This change in colors was
thought to indicate that the metal had successfully been reduced to Pd(0) and
undergone oxidative addition. The original procedure for the conditions of the
Pd coupling was based on the coupling of hydroborated intermediates with
51
organotriflates, it is possible that substitution of the triflate group with the iodo
group could alter the reactivity of the intermediates formed during catalysis.
Formation of the C6
reagents.
C7 bond was also attempted using cuprate
In this case compound 9 was reacted with BuLi to form the
intermediate 14. Addition of CuCN and ethyl acrylate 15 was expected to give
the coupled product
16.46
CH3O
CH30
CH3O
NC(cH3)3
1) CUCN, -78°C
BuLi, 0
75%
CH3O
9
Li
I
CH3O
14
H
)..NyC(CH3)3
0
2)
Et
ok
'-..OEt
CH3O
16
15
Ii
0
Scheme 11
Formation of 14 gave an opaque yellow mixture.
Addition of this
compound to a solution of CuCN in ethyl ether gave light brown solid
aggregates. In order to form the higher order organocuprate, the solution was
warmed to 0 °C to give an opaque beige solution.
If this mixture was
maintained at this temperature for more than a few minutes a black residue
would start accumulating in the flask walls which indicated that decomposition
of the active intermediate was taking place.
No coupling products were
observed after the addition of ethyl acrylate. The major compound found in the
reaction mixture was recovered starting material 9. Changes in temperature and
52
reaction times also resulted in recovery of the protected dimethoxyaniline 9.
Once again it was suspected that steric hindrance caused by the pivaloyl and
methoxy group at position C5 could be preventing the organocuprate from
reacting with the ethyl acrylate. It was believed that replacement of the ethyl
acrylate by a smaller more reactive compound like methyl iodide could help to
solve this problem. However, reaction with methyl iodide did not take place,
and once more, starting material was recovered from the mixture. Aggregates
formed in THF were found to be almost solid, if the reaction was run in ether the
aggregates were easily broken indicating that in this solvent the interactions
between the salts (lithium and copper) and the organic material were weakened.
In both cases no coupling product was observed.
A third and final approach focused on closing the ring using an
intramolecular reaction.
If steric hindrance was in fact the problem, then
approaching the two reacting sites could help to achieve product formation. The
intramolecular cyclization was envisioned in two different ways:
a) by means of a Friedel Crafts reaction
b) by promoting an aza-Claisen reaction
The synthesis of a derivative of 7 was reported over 30 years
ago.47
In
this case the dihydrocarbostyril 17 was produced (30 %) by an intramolecular
Friedel Crafts reaction.
53
CH3OO
OCH3
CH3O
Br
17
7a
Scheme 12
The procedure involved making a 1:1 mixture of 17 and ZnC12INaC1
which was heated up to 150 °C and left at this temperature for 1 hr. The same
approach was taken to the synthesis of 748
CH3O
CH3O
CH3O
propionyl chloride
cNH2
quantitative
Br
CH3O
CH3O
8
18
CH3O
19
Scheme 13
If the reaction was heated to 120 °C the major compound identified was
a derivative of 18 where the bromo functionality was replaced with a chlorine
group. Since this compound could also form the desired product 19 the reaction
was heated to 150 °C. However, in this case deprotection of the methoxy groups
to give the hydroxyl derivatives; again no cyclization was observed. At least
five major products were obtained from this reaction. As expected, deprotection
and decomposition occurred more easily if ZnC12 was replaced with AlCl3 (even
at 120 °C).
54
A final attempt to use this methodology consisted in bringing 18 into
solution and using a Lewis acid that would strongly bind the bromine group.
The Lewis acid selected for this purpose was AgOCOCF3.49 However, although
the desired product was not isolated, an unexpected compound was formed in
this reaction (Scheme 13).
CH3O
CH3O
H
H
cI1NTO
AgOCOCF3
83%
CH3O
CH3O
Br
18
20
CF3
Scheme 13
If the same reaction is carried out in benzene, starting material is
recovered from the mixture. The formation of compound 20 suggests that it is
possible that the conformation of the molecule does not allow the reactive side
chain to approach the ring. Since the aromatic ring is electron rich it represents a
much better nucleophile than the trifluoroacetic acid anion. It could be proposed
that the primary carbocation formed in this reaction is being stabilized by the
amide nitrogen:
Ag.
Br
CH3O
H
CH3O
H
Ag
-Br
or
CH3O
CH3O
55
The formation of compound 20 reaches highest conversion in
4-5
days.
If the reaction is left for longer reaction times, then deprotection of the methoxy
groups starts to take place.
The aza-Claisen
cyclization5°
was the next attempted. Scheme 14 shows
how cyclization leads to the desired bicyclic core.
CH3O
CH3O
H
0
CH3O
I1
N0H
CH3O
H
N0H
H
r10
CH3O
CH3O
CH3O
CH3O
Scheme 14
As shown in Scheme
15,
the unsaturated amide 21 needed for this
reaction was synthesized by treatment of compound 18 with base in THF at 0
°C. Although 21 is formed as the major product (88 %), the 13-lactam 22 was
also observed (10 %). The formation of this minor product shows that the amide
bond can rotate to allow interactions between the nitrogen and the 3-carbon. In
addition, its formation supports a
3-lactam intermediate as origin of
trifluoroacetate 20.
CH3O
CH3O
CH3O
Ii10
CH3O
Br
18
NaH, 0°C
CH3O
CH30
21
Scheme 15
22
56
Cyclization was expected to take place at 100 or 140 °C in the presence
of a Lewis acid. The reaction was carried out in a sealed NMR tube and
monitored periodically for hints that could indicate that cyclization was taking
place. A reaction carried out in benzene using
ZnCl2
as the acid showed that in
a period of 48 hrs at 100 °C the starting material remains intact. Heating
compound 21 to 100 °C in toluene using p-toluenesulfonic acid as the Lewis
acid also gave only starting material. If the temperature was raised to 140 °C,
deprotection of the methoxy groups would lead to decomposition of 21.
Based on these results, failure to construct the C6-C7 bond was
originally attributed to
steric effects
conformation of the amide side chain.
and the potentially unfavorable
However, intrigued by the outcome an
electron density map and the HOMO electron distribution of 21 were calculated
using Semi Empirical methods (AM1
model).51
The results show that the
electron density is centered around C4, suggesting that this atom may be the
most nucleophilic carbon in the ring (Figure 111.5).
A.
B.
C.
MeO
MeO
21
Figure 111.5 A. Electrostatic potential Map of amide 21. B. HOMO electron
distribution of amide 21. C. HOMO-1 electron density of amide 21.
A thorough literature search led to a study reported by Nakagawa and
coworkers52
in which target 7 is synthesized using a strongly electrophilic C7
and highly acidic conditions to force the desired ring closing reaction (Scheme
16). Future studies could utilize this route to obtain 7 for enzyme assays.
CH3O
CH3O
OR
CH3O
Ic
ROCH=CHCOCI
I
LO
NI-1
CH3O
CH3O
CH3O
24
23a R=CH2CH3
23b R=CH2CH(CH3)
8
I (CH3O)2CHCH2CO2CH3
1. H2, Raney-Ni
(74 %)
2. 47 % HBr
(81 %)
25%
LH3
áL
I
CH3O
OH
OCH3
CH3O
H2SO4
4
N
H
0
73%
OH7
25
Scheme 16
The information that could be acquired using 7 as an alternative substrate
or inhibitor of the DHAEs could be further complemented by the synthesis of
inhibitors containing a diazetane or dioxetane moiety. Although syntheses of
species of this type represent a challenge, the results could be of great relevance
to support the mechanism of epoxidation that has been proposed for these
enzymes.
59
3.3 REFERENCES
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I
62
46. M. Schiosser. Organometalics in Synthesis: A
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I
63
CHAPTER IV. EXPERIMENTAL SECTION
General.
Starting materials were obtained from common commercial
suppliers and used without further purification.
Unless otherwise stated,
concentration under reduced pressure refers to a rotatory evaporator at water
aspirator pressure.
Proton, carbon, and deuterium nuclear magnetic
resonance (NMR) spectra were acquired using a Bruker AM-400 or Bruker
AC-300 spectrometer. Infrared (IR) spectra were obtained using a Nicolet
5DXB Ff-IR spectrometer.
Melting points were measured using a Buchi
melting point apparatus.
T1
measurements. The T1 measurements of the aromatic hydrogens of DHA
were calculated in acetone-d6 using a Bruker AM400 spectrometer. A total of
9 points were used for every logarithmic curve (results are listed in Table
ffl.1).
64
Logarithmic curve iorH-4
Logarithmic curve forH4
10.00_i
10.00
s.00l
5.00
4
0.001
0.00
.E
-5.00
-5,00
1
-10.00
-10.00
0.00
5.00
10.00
15.00
000
20.00
5.00
10.00
15.00
20.00
seconds
sec onds
Logaritlinic curve for H-7
8.00
>.
6.001
4.001
ci
2.001
0.001
-2.001
:8 '
-8.00 t
-10.00
I
0.00
5.00
10.00
15.00
20.00
seconds
Figure IV.1 Logarithmic curves obtained for each aromatic hydrogen in
DHA.
Deuterium exchange of DHA in D20 at different pHs. The system shown
in Figure IV.2 was used for all exchange studies. The two-flask system was
used to maintain an Ar atmosphere while keeping the concentration of DHA
constant. For every control experiment flask A and B were charged with 10
mL of D20 (99 %-D). The pH of the solution was adjusted using NaOD
(99%-D) Or D2SO4 (99%-D). After sparging with Ar for approximately 10
mintues, DHA (1.3 mg, 7.78 x
102 mmol)
and sodium dithionite (2 mg, 1.14
x 102 mmol) were dissolved in flask A and exchanged for lhr. This was
followed by extraction of the organic material using EtOAc (5 X 2 mL). The
combined organic fractions were dried over Na2SO4 and concentrated in
65
vacuo. The beige crystals were dissolved in acetone to acquire the 2H NMR
spectra, and in
acetone-d6
of the acquisition for the 1H NIvIR spectra.
line
DHA
D20
acid or b
Hask A
Flask B
Figure IV.2 Two-flask system used for deuterium exchange studies with
constant DHA concentration.
Deuterium-hydrogen exchange of DIIA in the presence of DHAE II. Flask
A (Figure P1.2) was charged with 50 mL of a D20 buffered solution
containing 0.1 M
KH2PO4 (pH = 6.5),
and 6 mL of 0.2 mM CoC12.6H20 (in
D20) to give a clear light pink mixture. After adding 20 mL of D20 to flask 13
the system was sparged with Ar for
10-15
mm. DHA (1.3 mg, 7.78 x 102
mmol) was added to flask A and the solution was stirred until the colorless
crystals had fully dissolved. The reaction was started with the addition of 4
mL of DHAE II to flask A, no visual change was observed. Light stirring was
only allowed periodically to avoid denaturing of the enzyme. The specific
activity of DHAE II prior to its addition to the system was 15 tmo1/(min x mg
66
prot.). After 30 mm. flask A was charged with 4 mL of DHAE 11, which had
been carefully stored at 0 °C, this was done to regenerate the enzyme activity
lost during the first 30 mm. of exchange. No visual change was observed.
The organic material was extracted with EtOAc (4 X 8 mL) after one hour.
The organic fractions were dried over Na2SO4 and concentrated in vacuo.
Incorporation of deuterium was evaluated through 2H NIMIR spectra in acetone
and by comparison of the integrals measured for the peaks of the aromatic
hydrogens of the recovered and unfed DHA (Table 111.3).
Deuterium-hydrogen
exchange
of
[4,6,2',2',2'-2H5]-2,5-
dihydroxyacetaniide in the presence of DHAE II. The procedure described
above
was
followed,
replacing
DHA
for
[4,6,2',2',2'-2H5]-2,5-
dihydroxyacetanilide (colorless crystals) and D20 for
1120.
The substrate
consisted of 91 % D at C6 and 66 % D at C4 (determined by 1HNIMR). No
color change was observed after the addition of DHAE 11. The specific
activity of the enzyme prior to its addition to flask A was 12 J.Lmol/(min x mg
protein). Isolation of the acetanilide was performed as described above. The
organic material was analyzed by 111 NMR in acetone-d6.
Enzymatic synthesis of 2-acetamido-5,6-epoxyquinone in 78 % D20.4° A
50 mL Erlenmeyer was charged with 12 mL of D20 buffer containing 50
67
mM KH2PO4, 10 % glycerol (pH = 6.5), and 6 mL of 0.2 mM CoC12.6H20
(in D20). A 9.1 mM solution of DHA was prepared. In a period of 6 hrs a
total of 9.5 mL of DHA solution (0.086 mmol) and 7 mL of DHAE II
(specific acitivy = 8 p.mole/(min x mg protein)) were added periodically to
the clear light pink solution (final concentration of DHA = 3.4 mM). The
reaction was allowed to proceed for a total of 18 hrs in a Lab-Line Incubator
Shaker set at 120 rpm and 30 °C. The organic material was extracted with
EtOAc (3 x 20 mL) and purified with a YMC-Pack (250 x 10 mm L.D., S-
5tm, 120 A). Purification was achieved using a Waters HPLC (model M6000A) set at 311 nm and a solvent system of 85 % H20/ 15 % CH3CN. The
desired epoxyquinone had a retention time of 16 mm at a flow rate of 3
mljmin.
The fractions were combined, extracted with EtOAc, and
concentrated in vacuo. The epoxyquinone was obtained as yellow crystals.
This compound decomposes easily and should be treated with care.
Streptomyces MPP 3051. The process shown below represents a general
overview of the steps required for a 340-fold purification of DHAE II (Figure
IV.3).
Bacterial Cells
Sonication
Cell Free Extract
Anion Exchange column (DE-52)
Immobilized Metal Affinity Chromatography
Hydrophobic Interaction Chromatography
Partially pure DHAE Il
Figure IV.3 Purifications scheme of DHAE H.
The level of purification gained in each step is described below.
Volume
(mL)
Protein mass
Specific activity
(jimol/min mg prot.)
Fold purfication
(mg)
CFE
196
332
0.042
0
DE-52
132
72
0.203
5
IMAC
50
4
0.421
10
C-5 HIC
30
0.04
14.31
340
Purification step
Table IV.1* summarizes the level of purification gained in each step. The
fold purification was calculated by dividing the specific activity obtained
from the purification step by the specific activity found in the CFE.
*Based on a total cell mass of 25-36 g.
Growth media.4° Seed Broth: The spores where kept at 4°C in sterile soil.
A volume of 50 mL of seed medium (3 % glucose, 1 % bacteriological
69
peptone (Oxoid. Ltd.), 0.2 % KH2PO4.3 H20, 0.1 % NaC1, pH = 7) was
prepared in 250 mL Erlenmeyer flasks. After autoclaving for 15 mm at 256
&F the medium was inoculated and incubated for 72 hrs. at 28 °C (240 rpm)
in a Lab. Line Incubator Shaker. Production Broth: A volume of 200 mL of
production medium (1 % glucose, 1 % NaNO3, 0.1 % Pharmamedia (cotton
seed flour), 0.5 % CaCO3, pH = 7) was prepared in a 2 L Erlenmeyer flask.
Incubation with 5 % (V/V) seed broth was performed after autoclaving the
production broth for 20 mm at 256 °F and cooling it down. The flasks were
incubated for 24 hrs. at 28 °C (240 rpm) in a Lab. Line Incubator Shaker. Up
to 7 Erlenmeyer flasks were generally prepared.
Preparation of CFE. The bacterial cells were filtered though a 30 j.tm nylon
mesh and washed twice with 200 mL of H20, followed by 200 mL of 1.0 M
KCIand 200 mL of 0.8 M NaC1. A mass of 25-36 g of wet cells was isolated
from each growth. For cell rupture the cell mass was dissolved in 3 times its
volume of sonication buffer (50 mM KH2PO4.3 1120, 10 % glycerol, 0.1 mM
EDTA, 5 % PVP-10, pH = 7). Prior to sonication 9 mg of XAD-4 resin and 9
mg of PVPP were added to the mixture. The cells were somcated using a Heat
Systems-Ultrasonic sonicator (model W-225 R).
After sonicating for 20
seconds at 90 % duty (full power) the mixture was centrifuged in a JEC B-20A
refrigerated centrifuged at 4 °C for 10 mm at 14 K. This process was repeated
70
3
4 times to give 4 different supernatants that were combined for further
purification. Aliquots (300 ML) from each purification step were stored at 78
oc.
Diethylaminoethyl (52)-cellulose anion exchange column.
Resin
preparation: A total of 87 g of pre-swollen, micro granular anion exchange
resin DEAE-52 (Whatman) were initially rinsed twice with water. This was
followed by one wash with Buffer 1(50 mM KH2PO4, 10 % glycerol, 1.0 M
KC1, pH = 6.5) and two washes with Buffer 11(50 mM KH2PO4, 10 %
glycerol, 1.0 M KCI, pH = 6.5). The resin was poured in a 75 x 6 cm Econo-
column and left to settle overnight at 4 °C. Sample loading: After removal
of the excess buffer the CFE was loaded on the column and eluted at a rate of
0.65 mJJmin (fraction 1).
The following four solutions were used for
purification: a) 125 mL Buffer II, b) 125 mL of Salt Solution I (Buffer II
with 50 mM KC1), c) 125 mL Salt Solution II (Buffer II with 100 mM KC1),
d) 200 mL Salt Solution ifi (Buffer II with 200 mM KCI). Active fractions
eluted with Salt Solution ifi and were collected in volumes of 33 mL
(fractions 5a-g). The first four 33 mL fractions were carried on to the next
purification step.
71
DE-52 column
200
2.00
150
1.50
100
1.00
50
0.50
0
ooo
protein elution
1
2
(ug/mL)
3 4 5a Sb Sc 5d 5e 5f 5g
sific acthMy
(umol/mg mm)
fraction
j
Figure IV.4 Results observed after purification of the extract with a DE-52
column.
Concentration using an Amicon 'Ultracentrifuge (8200). A YM1O, 62
mm, 10K membrane was rinsed with water for 5 mm using a He pressure of
55 psi. The active fractions were added from the DE-52 column to the
centrifuge and concentrated to
psi.
1/4
of its original volume at a pressure of 60-65
The specific activity remained unchanged during this step if this
pressure was not exceeded.
The membranes were stored in a solution
containing 10 % ethanol.
Immobilized metal affinity chromatography. A volume of 36 mL of a
chelating sepharose resin (Fast Flow) was added to a 2.5 x 20 cm Econocolumn. The column was equilibrated with 100 mL of Buffer ifi (100 mM
KH2PO4, 10 % glycerol, 50 mM NaCI, pH = 6.5) at a flow rate of 2.5
72
mllmin using a Waters 650E FPLC system. A solution of CuSO4 (Smg/mL)
was loaded on the column and the excess copper was removed by eluting the
column with 500 mL of Buffer IV (100 mM KH2PO4, 10 % glycerol, 50 mM
NaCI, 83.3 mM imidazole, pH = 6.5). The column was rinsed with an
additional 50-70 mL of Buffer Ill followed by the addition of the
concentrated active fraction. After loading the sample additional Buffer ifi
was added to create a void volume of approximately
1%
of the resin height.
The following gradient was created to elute the active fractions:
A) 0 50 mm: 100 % Buffer 111/0 % Buffer IV (linear gradient)
B) 50 80 mm: 85 % Buffer ff1 15 % Buffer IV (linear gradient)
C) 80 - 260 mm: 85 % Buffer 111/15 % Buffer IV (isocratic)
D) 260
290 mm: 0 % Buffer ff/ 100 % Buffer IV (linear gradient)
Fractions were collected every 5 mm. Active enzyme was collected after the
first 100 mm.
73
IMAC column
180
160
9.00
140
120
100
7.00
80
60
40
20
0
4.00
8.00
6.00
5.00
3.00
2.00
1.00
iiiiii'
LIII!IJI
ii
0.00 s protein elution
(umL)
I
fraction
--specific actiMty
(umoVmg mm)
Figure IV..5 Results observed after purification using immobilized metal
affinity chromatography.
Pentyl Hydrophobic Interaction Chromatography. A 2.5 x 20 Bio-Rad Econocolumn was loaded with short extended unit C-5 agarose until a resin height of 10
cm was reached. The column was rinsed with 200 mL of MifliQ water followed
by 100 mL of 1 N NaOH. After a second rinse with MilliQ water (200 mL) the
column was equilibrated with over 200 mL of Buffer V (100 mM KH2PO4, 5 %
glycerol, 1.2 M
(N}L)2SO4,
pH = 6.5). The 4 7 most active fractions collected
from the previous purification step were combined and carefully brought to 1.2 M
(NH4)2SO4
using a stock solution of 3.75 M. The following gradient was used for
the isolation of active DHAE II: (Buffer VI = 100 mM KH2PO4, 5 % glycerol, pH
= 6.5)
A) 0 30 mm: 50 % Buffer V/ 50 % Buffer VI (linear gradient)
B) 30 70 mm: 50 % Buffer V/ 50 % Buffer VI (isocratic)
74
C) 70
D) 150
150 mm: 40 % Buffer V/ 60 % Buffer VI (linear gradient)
190 mm: 40 % Buffer V/ 6 0 % Buffer VI (isocratic)
E) 190 230 mm: 30 % Buffer V/ 70 % Buffer VI (linear gradient)
F) 230 270 mm: 30 % Buffer VI 70 % Buffer VI (isocratic)
G) 270 310 mm: 0 % Buffer V/ 100 % Buffer VI (linear gradient)
Samples were collected every 3 mm at a flow rate of 1.5 mlJmin.
The active fractions collected from this colunm are more diluted than the
fractions collected from the LL- 10037 organism.
Dialysis.
The samples obtained from the }IIC column were dialyzed to
remove the ammonium sulfate which interferes with protein purification.
Aliquots of the active fractions were placed in eppendorf tubes and covered
with wet dialysis tubing (Pierce Snake Skin, pleated, 7,000 MWCO, 22 mm x
35 feet dry diameter). The samples were left overnight face down in a
container with MI1IiQ water.
Bradford assay. A BSA standard (from Sigma or Bio-Rad) was diluted to a
final concentration of 1.4 mg/mL and stored in 1.5 mL eppendorf tubes at
20° C. One aliquot of the stock was further diluted (lOX) to create a standard
curve using the concentrations shown in Table 11.6.
The samples to be
quantified were prepared by adding X pL of sample, (800-X) pL of water, and
75
200 pL
of Bio-Rad dye. The standard curve was constructed plotting protein
concentration versus absorbance at
595
nm, this wavelength was used to read
the absorbance of the active samples.
Final concentration
tg/mL
Dilute BSA
dd H20
Bio-Rad coomassie blue dye
1.4
10
790
200
2.8
20
780
200
4.8
35
765
200
7.0
50
750
200
9.8
70
730
200
blank
0
800
200
Table IV.2 Standard used for protein quantification.
Standard enzyme activity assay. Identification of substrate and product was
achieved using a Waters 600E HPLC instrument (UIK-6 injector) with a
Waters 996 photodiodarray detector.
Successful compound separation was
possible using a C18 column (Econosphere,
5
mm, 250 X 4.6 mm) eluted with
a solvent system of 85 % MilhiQ water! 15 % CH3CN. A typical assay
mixture contained:
160 pL
MilliQ water, 100 pL of 10 mM CoC12 solution, 50
pL of I M KH2PO4, 65 tL of
with the addition of 25
enzyme solution. The reaction was initiated
p1. of 2 mM
DHA and was incubated at 30°C for
2-5
76
mm. After this period 100 pL of terminating solution (CH3CNIH2O/TFA:
66/27/7) were added to the mixture. The eppendorf tubes would then be
centrifuged for 3 mm at 13 K using a Biofuge centrifuge. This was followed
by analysis by HPLC using a wavelength of 254 nm and 100 pL injections.
The observed retention time for DHA was 4.3 mm, for the epoxyquinone 6.5
mm, and for the quinone 8 10 mm.
2,5-dihydroxyacetanilide (5). Grey powder with a m.p. of 165-168 °C. 'H
NMR (acetone-d6) 6Li 9.10 (1H, bs, exch D20), 8.57 (1H, s, exch D20), 7.89
(1H, s, exch D20), 7.03 (1H, d, J = 2.6 Hz), 6.72 (1H, d, J = 8.7 Hz), 6.52
(1H, dd, J = 8.6, 2.7 Hz), 2.18 (3H, s);
13C Nv1R (acetone-d6) &I1 170.9,
151.2, 141.9, 127.9, 118.8, 112.9, 109.3, 29.8.
2-acetamido-5,6-epoxyquinone (9):. Yellow crystals with a m.p. of 135137 °C. 'H NMR (acetone-d6) 8LII 7.89 (1H, bs, exch D20), 7.51 (1H, J = 2.2
liz), 3.91 (1H, d, J = 3.7 Hz), 3.83 (1H, dd, J = 3.7, 2.2 Hz), 2.22 (3H, s).
2-Acetamido-1,4-benzoquinone. Orange crystals with a m.p. of 148-149
°C. 'H NIIYIR (acetone-d6) L1 8.85 (1H, bs, exch D2O), 7.42 (1H, s, exch
D20), 7.89 (1H, d, J = 2.9 Hz), 6.78 (111, d, J = 10.3 Hz), 6.64 (1H, dd, J =
77
10.22, 2.2 Hz), 2.28 (3H, s); '3C NMR (acetone-d6) L1 189.2, 183.4, 171.6,
140.2, 118.2, 134.7, 114.7, 24.5.
Sodium dithionite inhibition. The assays were prepared by adding the
following components into a 1.5 mL Eppendorf tube:
Final Concentration
Volume
H20
10 mM CoCl2.6H20
20OXjtL
2 mM
1MKH2PO4
100 pL
50 p1.
0.5 mM DHA
20 j.tL
100mM
20 pM
11.5niMNa2S2O4
XpL
(0-2mM)
X= 0, 10, 20, 30, 40, 50, 60,70 jiL
The stock solution of DHA (0.5 niM) contained 11.5 mM sodium dithionite.
Table IV.3 Conditions used for assessing dithionite inhibition.
The reaction was initiated by adding 30 j.tL of DHAE II and was carried out
for 6 mm at 30°C. Terminating solution was added to stop the reaction (100
Inhibition studies using 1,4-dthydroxybenzene. Four sets of assays were
run using X = 0, 50, 100, 150 .LL. The reaction proceeded for 4.5 to 6 mm at
30 °C. Each assay was run in duplicate an in each case the reaction was
stopped with terminating solution (CH3CN/H20/TFA, 66/27/7; 100 p1.) which
78
contained sodium dithionite
(4.5
mM) to avoid any oxidation of the substrate
that could occur after the enzymatic reaction has finished (Table IV.4).
H20
200
200
200
200 -
200
(X+
(X+
(X+
(X+
(X+
(X+
(X+
Y)
Y)
Y)
Y)
Y)
Y)
Y)
200
200 -
1MKII2PO4
50
50
50
50
50
50
10mM
100
100
100
100
100
100
50
100
DHAEII
30
3030
30
30
30
30
30
Inhibitor
X
X
X
X
X
X
X
7
9
13
15
17
20
34
CoC12.6H20
(0.5 mM)
DHA=Y
(0.5 mM)
I
*Al1 values represent a volume in pL
Table IV.4 Assay conditions for inhibition studies.
The ideal HPLC solvent
(85
% water!
15
% acetonitrile) did not give
separation of the inhibitor and substrate peaks. Use of other solvent systems
was unsuccessful. In order to evaluate the product formation the values of
substrate conversion were obtained based on peak integrations calculated at
221 nm. At this wavelength (isosbestic point of the substrate and inhibitor
chromophores) it was possible to calculate the formation of product by
correcting the integrations of the epoxide peaks using the molar absorbtivity
of the epoxide at 221 nm. The equation shown below was used for the final
calculation of percentage conversion:
prnol Epoxyquinone = (initial prnol (DRA + inhibitor)) x {Integration
Epoxyquinone peak/(integration Epoxiquinone peak + integration inhibitor and
DHA peak)}
79
N-pivaloyl-2,5-dimethoxyaniline 9. Dimethoxyaniline (1.1 3g, 7.4 mmol)
was dissolved in 170 mL of dichioromethane at room temperature. A two
phase system was formed by combining the mixture with a saturated solution
of sodium carbonate (150 mL). Pivaloyl chloride (1.82 mL, 14.8 mmol) was
added while stirring to give a cloudy mixture. After stirring the solution
overnight, the organic layer was extracted, washed with water, and dried
over Na2SO4. The solvent was removed in vacuo to give compound 9 (3.4 g)
in quantitative yield as colorless crystals: m.p. 5 1-53 °C
d6)
;
1H NIVIR (acetone-
8.2 (1H, d, J = 2.9 Hz), 6.8 (1H, d, J = 8.9 Hz), 6.6 (1H, dd, J = 8.9, 2.9
Hz), 4.9 (1H, bs, exch D20), 3.9 (3H, s); 3.7 (3H, s), 1.3 (9H, s); 13C NIVIR
(acetone-d6) ö 177, 154, 142, 128, 111, 109, 105, 56.3, 55.7, 40, 27;
IR(KBr) Umax 3433, 1690, 618,
cm1.
N-pivalyl-6-iodo-2,5-dimethoxyaniline
10.
N-pivaloyl-2,5-dimethoxy-
aniline (1.25 g, 52.7 mmol) was dissolved in 15 mL of dry THF. The
solution was cooled under Ar to 0 °C to give a cloudy white mixture.
Dropwise addition of n-BuLi (1.6 M, 6.6 mL) to the flask produced a yellow
opaque solution which was stirred for 2 hrs. This was followed by the
addition of iodine (2.68 g, 0.106 mol) which had been dissolved in 4 mL of
THF. The red-brown solution was stirred for 1 hr at 0 °C and for 2 hrs at
80
room temperature. To quench the reaction the mixture was diluted with ethyl
ether, ice, and water. The organic material was washed with brine and a
solution of sodium dithionite, and dried over MgSO4. The solvent was
removed
and the desired product compound 10 (1.44 g, 75 %) was
in vacuo
obtained after purification by chromatography as dark brown crystals: m.p.
90-91 °C, 111 NMR (acetone-d6)
7.1 (1H, bs, exch D20), 6.9 (1H, d, J =
8.8 Hz), 6.3 (1H, d, J = 8.9 Hz), 3.85 (3H, s); 3.80 (3H, s), 1.4 (9H, s); 13C
NMIR (acetone-d6)
27.7, 27.2; IR(KBr)
130, 93, 153, 110, 112, 150, 177, 57.2, 56.9, 39.5, 31,
Umax
3313, 1675.7 cm1.
Protection of ally! alcohol 11. The allyl alcohol 11 (0.68 mL, 10 mmol)
was dissolved in 100 mL of dichloromethane. The solution was treated with
TBDPSCI (1.66 g, 11 mmol), TEA (1.67 mL, 12 mmol), and DMAP (0.05 g,
0.4 mmol) at room temperature. After the reaction was run under Ar for 15
hrs saturated ammonium chloride and water was added to the reaction
mixture. The organic layer was extracted and purified
in vacuo
to give a
yellow oil 12 as the only product: H NMR (CDC13) ö 7.7 (4H, m), 7.5 (6H,
m), 6.0 (1H, m), 5.5 (1H, dd), 5.2 (1H, dd), 4.3(IH, m), 1.2 (9H, s); 13C
NMR (CDCI3)
19.2.
137, 135.5, 134.7, 136.7, 129.6, 127.6, 113.8, 64.6, 26.8,
81
Palladium coupling. A flask equipped with a reflux condenser was charged
with 9-BBN (2.2 mL, 1.1 mmol) and compound 12 (0.79 g, 2.2 mmol) at 0
°C. The mixture was slowly warmed up to room temperature and stirred for
&hrs. Additional TF]F (3mL) was added to the white mixture followed by
K3PO4 (0.4g, 3.3 mmol), catalyst precursor (5.5
mol), and aryl or alkyl
halide (2.2 mmol). The solution was refluxed for 16-36 hrs. To quench the
reaction the mixture was diluted with hexane at room temperature. Residual
borane was oxidized with 3 M NaOH (0.5 mL) and 30 % H202 (0.5 mL) for
1 hr. The product was extracted, washed with brine and dried over MgSO4.
No coupled products were observed.
Coupling using organocuprates. CuCN (6.6 mg, 0.74 mmol) was diluted
in 1 mL of dry ethyl ether. A 0.1 M solution of aryl lithium 14 (6.3 mL, 1.44
mmol) was added dropwise to the reaction mixture and the solution was
stirred at 78 °C for 5 to 10 mm to give yellow gelatin aggregates. The
reaction mixture was warmed to 0 °C for 5 mm to produce a beige mixture
which easily decomposed if the reaction was left for longer reaction times at
this temperature.
The mixture was brought to 78 °C followed by the
addition of freshly distilled ethyl acrylate (54 pL, 0.5 mmol). The reaction
was left at this temperature for 1 - 24 hrs.
Attempts to increase the
82
temperature to make the organocuprate more reactive resulted in
decomposition. No coupling was achieved and anilide 9 was recovered.
-bromo 2,5-dimethoxypropylanilide (18)
Dimethoxyaniline (1.98 g, 13 mmol) was dissolved in
20 mL of
dichioromethane. The solution was heated to reflux and a solution of
bromo propionyl chloride (87
f3-
L, 13 mmol) in dichioromethane was added
dropwise. The reaction was quenched with brine after 1 hr. The organic
layer was washed with water and dried over MgSO4. The solvent was
removed in vacuo to give the desired product as colorless crystals (5.0 g, 96
%): m.p. 81-83 °C, 'H NMR (acetone-d6)
8.7 (1H, bs, exch D20), 8.1 (1H,
d, J = 3.0 Hz), 6.9 (IH, d, J = 8.9 Hz), 6.5 (111, dd, J = 8.9, 3.0 Hz), 3.8 (3H,
s); 3.75 (2H, t, J = 6.5 Hz), 3.72 (3H, s), 3.1 (2H, t, J = 6.5 Hz); 13C NIvIR
(acetone-d6)
28.6; IR(KBr)
168.9, 154.6, 143.6, 129.6, 112, 108, 107.8, 56.6, 55.8, 40.8,
3377, 1681, 655, 527 cm1.
Friedel Crafts reaction at high temperatures. A 1:1 mixture of anilide 18
(1.0 g, 3.48 mmol) and
ZnC12 (0.26
g, 3.48 mmol) or A1C13INaC1 (0.46 g, 3.48
mmol/ 0.12 g, 3.48 mmol) was heated to 120
150 °C. After 30 60 mm the
reaction was cooled to room temperature and the organic material was
extracted with ethyl acetate to give a complex mixture of products.
83
Friedel Crafts reaction at low temperatures. A solution of anilide 18 (1.3
g, 4.5 mmol) and AgOCOCF3 (1.0 g, 4.5 mmol) was prepared in dry
CH3CN (20 mL). The flask was covered with aluminum foil for 3-4 days at
room
temperature.
The
organic
material
was
extracted
using
dichioromethane and dried over MgSO4. Purification by semi preparative
HPLC using a reverse phase HPLC column (YMC C-18-HQ, 10 x 200 mm)
and a 80:20 solvent system of hexanes to ethyl acetate provided ester 20 light
yellow crystals (1.2 g, 83 %): 'H NMR (acetone-d6)
8.7 (1H, bs, exch
D20), 8.0 (1H, d, J = 2.9 Hz), 6.9 (1H, d, J = 8.9 Hz), 6.6 (1H, dd, J = 8.9,
2.9 Hz), 4.75 (2H, t, J = 6.0 Hz), 3.8 (3H, s); 3.72 (3H, s), 3.0 (2H, t, J = 6.0
Hz); 13C NMR (acetone-d6)
168, 157.7 (q, J = 41.8 Hz), 154.3, 143.6,
129.7, 115 (q, J = 284.8 Hz), 112, 108, 107.7, 64.5, 58, 55.7, 36. IR(KBr)
Umax
3412, 3335, 1788, 1692, 800
cm1.
Vinyl anilide 21. A suspension of NaH (60 mg, 1.5 mmol) in THF was
added slowly to a solution of compound 18 (157 mg, 0.55 mmol) in THF (10
mL) at 0 °C. The reaction was left overnight and quenched with saturated
ammonium
chloride.
The
organic
dichloromethane and washed with water.
material
was
extracted
with
The mixture was dried over
MgSO4 and the solvent was removed in vacuo. Purification was done by
semi preparative HPLC using a reverse phase HPLC column (YMC C-18HQ, 10 x 200 mm) and a 1:1 solvent system of ethyl acetate and hexanes to
84
give compound 21 (0.1 g, 87 %) and lactam 22 (10 %) as colorless crystals:
'H NMR (acetone-d6) 6 8.68 (1H, bs, exch D20), 8.18 (1H, d, J = 2.2 Hz),
6.9 (1H, d, J = 8.8 Hz), 6.6 (1H, dd, J = 8.2, 2.3 Hz), 6.4 (1H, dd, J = 10.3,
17.1 Hz), 6.34 (1H, dd, J = 1.48, 17.1 Hz), 5.71 (1H, dd, J = 1.48, 10.3 Hz),
3.8 (3H, s); 3.75 (3H, s); 13C NMR (acetone-d6) 6 183, 164, 155, 144, 133,
130, 127, 112, 108.6, 57, 56. IR(KBr) l)max 3234, 1660, 1630, 1461cm1. 22
1H NIMR (acetone-d6) 6 7.62 (1H, d, J = 2.9 Hz), 6.95 (1H, d, J = 8.9 Hz),
6.61 (1H, dd, J = 8.9, 2.9 Hz), 3.92 (2H, t, J = 4.5 Hz), 3.79 (3H, s); 3.72
(3H, s), 3.05 (2H, d, J = 4.5 Hz); 13C NMR (acetone-d6) 6 166.8, 154.9,
145.1, 129.5, 117.1, 110.9, 108.3, 57.8, 56.2, 44.1, 39.3. IR(KBr)
2939, 2841, 1732,
1222 cm1; HRFABMS calcd for
207.08954, found207.08970.
C11H13NO3
Umax
(M)
Acid catalyzed Aza-Claisen reaction. A solution of p-toluenesulfonic acid
(1.7 mg, 8 tmo1) and anilide 21 (3.8 mg, 18 mol) in toluene (100 %-D) was
prepared in an NIVIR tube and sealed in vacuo. The reaction was initially
monitored at 100 °C for a period of 18 hrs. Increasing the temperature to 140
°C led to decomposition.
STUDIES ON NITROGEN CONTAINING SECONDARY METABOLITES
FROM TERRESTRIAL AND MARINE ORIGIN: PART I. ENZYMATIC
EPDXIDATION OF 2,5-DIHYDROXYACETANILIDE IN
STREPTOMYCES SP. PART II. SYNTHESIS OF MARINE SPONGE
ALKALOIDS: SLAGENINS, AXINOHYDANTOINS, AND STUDIES
TOWARDS THE PYRROLOPIPERAZINE SYSTEM IN THE
AGELASTATIN CORE
PART II. SYNTHESIS OF MARINE SPONGE ALKALOIDS:
SLAGENINS, AXINOHYDANTOINS, AND STUDIES TOWARDS THE
PYRROLOPIPERAZINE SYSTEM IN THE AGELASTATIN CORE
SYNTHESIS OF MARINE SPONGE ALKALOIDS: SLAGENINS,
AXINOHYDANTOINS, AND STUDIES TOWARDS THE
PYRROLOPIPERAZINE SYSTEM IN THE AGELASTATIN CORE
CHAPTER V. INTRODUCTION
Marine natural products have been the subject of investigation for over
30 years. Marine alkaloids can be classified into guanidine, indole, pyrrole and
-carboline metabolites. Other groups include nitrogen containing polyketides,
peptides and miscellaneous alkaloids. Many of the nitrogen containing
metabolites are thought to originate from amino acids. However, in some cases
these units may be rearranged and derivatized to a degree where there is little
resemblance to the original building block.1
Of the more than 11 000 marine
natural products that have been characterized, those isolated from marine
sponges account for over 40 %2 The variety of structural features found in
sponge metabolites arises most likely from the fact that many of these
compounds may be biosynthesized by bacteria or microalgae associated with the
sponge.
One structural class exclusive to these primitive multicellular animals are
the pyrrole-imidazole alkaloids. Within this group of natural products lies a
structurally diverse and pharmacologically interesting class of
C1 1N41C1 1N5
secondary metabolites primarily isolated from sponges of the genera Agelas,
Hymeniacidon, and Phakellia. These pyrrole-imidazole metabolites, referred to
as the oroidin family of alkaloids, generally possess a functionalized or
unfunctionalized 3-carbon alkyl chain, a brominated or nonbrominated pyrrole
carboxamide unit, and a 2-aminoimidazole or glycocyamidine appendage.
Alkaloids containing an imidazolone or hydantoin nucleus are less commonly
found. Representative examples include the slagenins, the agelastatins and the
axinohydantoins.3
Slagenins A, B and C were isolated in 1999 by Kobayashi and coworkers
from extracts of the Okinawan marine sponge
Agelas nakamurai.4
These
marine alkaloids represent the first natural products with a tetrahydrofuro[2,3-
d]imidazolidin-2-one moiety (Scheme 1). Their absolute stereochemistry is
currently unknown. However, their relative stereochemistry was determined on
the basis of NOESY correlations.
Br
o
H
H
Ii
0
0
II
0
Slagenin C
Stagenrn A R = H
{[oJD27 +110 (C 1.2, MeOH)}
Slagenin B A = OH3
([aID26 +33° (C 0.2, MeOH)}
([aID25
HH
15:
H1
Br
.350 (c 0.2, MeOH)}
H
HH,N0
a H14
ZOH
Relative stereochemistry
in Slagenin A
Relative stereochemistry
in Slagenin C
Scheme 1
Slagenins B and C exhibit in vitro cytotoxicity against murine leukemia L1210
cells (IC50 = 7.5 and 7.0 pg/rnL respectively), whereas slagenin A does not show
such activity.
(Z)-Debromohymenialdisine was isolated by Sharma and coworkers in
1980 from the Caribbean sponge Phakellia fiabellata.5 This was followed by
the isolation of the corresponding 2-bromo derivative, (Z)-hymenialdisine, in
1982 by Kitagawa and coworkers from the sponges Axinella verrucosa and
Acanthella aurantiaca (Scheme 2).6 The structure of (Z)-hymenialdisine was
determined by X-ray crystallography, and its elucidation helped confirm the
structure of the 'yellow compound' (Z)-debromohymenialdisine. Several years
after the first isolation of (Z)-debromohymenialdisine, Proktsch and coworkers
isolated the dibrominated derivative, (Z)-3-bromohymenialdisine, from the
Indonesian sponge Axinella
bromohymenialdisine,8
Hymeniacidon
sp.9
carteri.7
Its corresponing E isomer, (E)-3-
was isolated in 1998 from an Okinawan sponge
Assignment of the C7-C8 geometry was deduced from the
1H and '3C chemical shifts of C-6. The C6 methylene protons of the (7E) isomer
were found to be further upfield than previously observed for the compound
possessing a (7Z)-geometry.
(Z)-3-Bromohymenialdisine exhibits cytotoxic
activity against the mouse lymphoma cell line L5178
(ED50
= 3.9 .tg/mL).
(E)-
3-bromohymenialdisine exhibits inhibitory activity against c-erbB-2 kinase (IC50
= 8.5 pg/mL) and cyclin-dependent kinase 4 (1050 =32 .g/mL). It was not until
1996 that the (E)-isomer of hymenialdisine was first isolated by Faulkner and
coworkers from the conmion shallow-water sponge Stylotella aurantium.10
(Z)-Debromohymenialdisine A = R' = H
(Z)-Hymenialdisine R = H, R' = Br
(Z)-3-Bromohymenialdisine R = A = Br
NH2
R'
(E)-Debromohymenialdisine R = R' = H
(E)-Hymenialdisjne A = H, R' = Br
(E)-3-Bromohymeniakhsine A = R' = Br
Scheme 2
In 1989 Pettit and coworkers isolated (E)-axinohydantoin from the
sponge Axinella
possess
sp.11
This close relative of hymenialdisine was found to
a hydantoin-lactam moiety in place
of the more common
glycocyan-iidine unit. As in the case of the hymenialdisines, an E configuration
for
the
hydantoin-lactam
C7-C8
double
bond
was
established
by
crystallographic methods. In addition, an angle of 36° was observed between
,j1
the two nearly planar five member rings, compared to the 43.8° angle found in
hymenialdisine.
In
1997
the related marine metabolites debromo-Z-
axinohydantoin and Z-axinohydantoin were isolated from the sponge Stylotella
aurantium and found to be inhibitors of PKC with IC50 values of 9.0 and 22 j.iM,
respectively.'2
Both alkaloids were later found in extracts of the sponge
Hymeniacidon sp. and given the names of spongiacidins D and
C.9
As seen in
the hymenialdisine family, these new hydantoin derivatives differ primarily in
the configuration around the C7-C8 bond and by the number of bromine atoms
present on the pyrrole group.
(E)-Axinohydantoin
(2)-Debromoaxinohydantoin R = H
(2)-Axinohydantoin R = Br
Scheme 3
Recent reports centering on (Z)-debromohymeni aldi sine (DBH) and
(Z)-hymenialdisine have appeared describing their role as modulators of protein.
kinase C and the proinflanimatory transcription factor, nuclear factor iB
()13
apoptosis,
NFiB regulates a number of genes critical for the control of
viral
replication,
tumorogenesis,
inflammation,
and various
autoimmune diseases. In particular, NFxB is thought to be involved in the
regulation of the inflammatory enzyme cyclooxygenase (COX-il), which plays
an important role in the mediation of inflammation associated with
arthritis.131
DBH has also been shown to slow joint deterioration and cartilage degradation
in animal models and, therefore, it is currently considered a potential drug
candidate for the treatment of osteoarthritis.'4
H2N
HN>O
Brfl'
0
Hymenlaldisine
Figure V.1 CDK2-hymenialdisine complex crystal structure (structure skeleton
in black).
93
In a recent inhibition study hymenialdisine strongly inhibited the kinases
GSK-3 and CDK1
(IC50
values of 10 and 35 nM respectively) and a crystal
structure of hymenialdisine bound to CDK2 (1050 value of 4OnM) was
determined (Figure
V.1).'5
It appears that the presence of the a-bromo
substituent in hymenialdisine may contribute significantly to the binding affinity
and specificity of its inhibition properties through interactions with the side
chains of the hydrophobic residues in CDK2.
The hymenialdisine-CDK2
complex represents the first example where the interactions between a pynoleimidazole alkaloid and its target could be identified.
In 1993 extracts of the sponge Agelas dendromorpha were shown to
contain two novel pyrroloaminopropylimidazoles, (-)-agelastatins A and B,
which possessed an unusual
C11
framework (Scheme 4)16
Initial
characterization studies were hindered by the unsuccessful separation of the two
alkaloids.
Exhaustive methylation of the agelastatin mixture yielded
tnmethylated derivatives, which were purified and used for characterization
purposes. This information was complemented by results obtained through a
series of degradation and derivatization
studies.'7
The 4,5-cis and 7,8-cis
fusions in the tetracyclic core were determined on the basis of NOE correlations
between the C5-OCH3 and H4 and between H7 and H8 of the methylated
derivative of agelastatin A. In addition, the 4,8-trans fusion was deduced based
on the small coupling observed between H4 and H8 (<0.5 Hz).
The absolute
stereochemistry of agelastatin A was determined to be 8S, 7S, 5S, 4R on the
basis of CD spectral data of a dibenzoyl derivative.
Shortly thereafter,
agelastatins C and D were isolated from the Indian Ocean sponge
sp.18
Cymbastela
The CD spectra for agelastatins C and D were essentially superimposable
with the CD spectrum of agelastatin A, indicating that the absolute configuration
of the agelastatins was identical. Agelastatin A exhibits strong cytotoxicity
against L1210 murine
(IC50
= 0.033 .tg/mL) and KB
(1050
= 0.075 jg/mL)
tumor cell lines.16
Me
HQ
Br
65
X_8NHHH
Agelastatin A, X = H
Agelastatin B, X = Br
Agelastatin C, R1 = Me, R2 = OH
Agelastatin D, R1 = R2 = H
Scheme 4
A few additional representative examples of oroidin alkaloids are
presented in Scheme 5.
These metabolites range from highly functionlized
linear systems, like the dispacamides and tauroacidins, to complex polycycles,
like phakellin and dibromoagelaspongin.
Unique dimeric species like the
axinellamides and sceptrin have also been isolated.3
95
NL
R1)y
H0
Br
NH
R2
R3 HN
NH
0
H
DispacamideAR1 =R2=Br, R3=H
Dispacamide B
Dispacamide C
Dispacamide D
Keramadine
= H, A2 = Br
= Br, A3 = OH
R1 = H, R2 = Br, R3 = OH
R1 = R3
A1 = A2
RN
Midpacamide R = Br
Debromomidpacamide R = H
OHHN-k+
HN
0
J1t°
Me 0
NH
Br
H
j)NH2
iI/ki
Br(N>
Br
Tauroacdin A R Br
Tauroacidin B R = H
0
NHH
Me 0
H3CO3S
Mauritamide A
OH
N4-.
N
0
H2N'
I
Br\/
0
Br"Br
I.,'
Phakellastatin
Phakellin
Dibromoagelaspongin
NBr
I
I-I
Rod
_-
I
.t%\
0
Br
Br
NH
Br
NH2
Sceptrin
Axinellamides
Scheme 5
Due to the similarities in their structural motif, it has been proposed that
most of the pyrrole-imidazole alkaloids may arise from the common linear
precursor,
oroidin,
or
the
related
sponge
metabolite
3-amino-1-(2-
aminoimidazolyl)-prop-1 -ene.3'19'20
H2N<jUrBr
Oroidin
3-amino-i -(2-am inoimidazolyl)-prop-i -ene
The work presented herein focuses on applying this hypothetical
approach
to
the
total
synthesis
of slagenins
A, B
and
C,
(Z)-
debromoaxinohydantoin and derivatives, and to studies towards the construction
of the pyrroloketopiperazine ring system in the agelastatin core (Scheme 5). Up
to this point, studies of this type have been hindered by the limited number of
methods available for the synthesis of substituted imidazolones. Included with
the axinohydantoin work, is the development of a practical route to the related
alkaloid (Z)-debromohymenialdisine.
97
o=)
0
R1
HQJ
Br H
NH
<LCH3
.NH
/N
-
N
0
Agelastatins
Z-Debromoaxinohydantoin
Slagenin C
Scheme 5
Our syntheses are noteworthy in that they are direct and do not require
the use of protecting groups.
In addition, access to synthetic samples of these
targets and their precursors will allow further investigation into the biomedical
properties associated with these alkaloids.
CHAPTER VI. RESULTS AND DISCUSSION
6.1. FIRST TOTAL SYNTHESIS OF (±) SLAGENINS A, B, AND C
Slagenins A (1), B (2), and C (3) are noteworthy in that they possess a
unique highly functionalyzed tetrahydrofuro[2,3-d}imidazolidin-2-one moiety.
HQR
N:
OCH3
o=< T'>11
0
NO
HN
H
o=<
N
HH
0
'"\
/9
HN'(
1R H
2R=CH3
B!
Br
Scheme 7
No prior synthesis of slagenins has appeared in the literature, and only
one
report
describes
the
preparation
of
heterocycles
containing
a
glycofurano[2,1-d}imidazolidin-2-one skeleton from a direct reaction of 2amino-2-deoxysugars with aryl isocyanates.21 Formation of the tetrahydrofuran
ring has been proposed to proceed via a monocyclic 5-hydroxyimidazolidine
intermediate (Scheme 8).
Ar\
/,P
N-
I
N-H
HO'
LOH
LOH
NHCONHAr
kOH
R
Scheme 8
Our approach to slagenins A (1), B (2), and C (3) centers on the
introduction of a f-hydroxy substituent in imidazolone 4 followed by its
oxidative cyclization to the desired core (Scheme 9). To our knowledge the
biosynthetic pathway to slagenins remains unknown; however, the amino acid
ornithine could be envisaged as a possible intermediate.
H
Q<
Slagenins
NHR
==
H2N-..NH2
CO2H
H
Scheme 9
Available methods for the synthesis of substituted imidazolones are
limited (Scheme 10).
Simple 4-alkyl- and 4-aryl-2-imidazolones have been
synthesized by initial condensation of a-aminocarbonyl compounds with
potassium cyanate followed by hydrolysis and decarboxylation to give the
monosubstituted products
(A).22
Alternatively, 4-aryl-2-imidazolones may also
100
be produced by condensation of cc-bromo ketones with urea
of N-arylphenylacylamine oximes with aryl isocyanates
00
(A)
R)LO
or by reaction
(C).24
R
R
KOCN
(B),23
1i>=b
0--N
NH2
0
R=CH3
N
H
H
A = CH2CH3
R = Ph
0
0
(B)
(C)
)LBr
Ph
NOH
H2NANH
Ph
N
H
0
OANHRI
H
Ar-R
RNC0
I
HN
A'
ArL>0
Arj
R'
TsOH
N
N
R
Scheme 10
Our initial investigations focused on developing an alternative method
for the direct construction of the imidazolone ring starting from available amino
acids. It has been shown that in situ condensation of a-amino aldehydes with
thiocyanate leads to the formation of 2-mercaptoimidazoles in good yields.25
This report prompted us to examine a similar set of conditions for the synthesis
of 2-imidazolones by condensation of a-amino aldehydes with potassium
101
Our approach began with Fisher esterification of commercially
cyanate.
available ornithine to afford methyl ester 6.26
Reduction under Akabori
conditions,25 followed by in situ condensation of the corresponding a-amino
aldehyde with potassium cyanate produced imidazolone
5
in an overall yield of
60 % after crystallization. Dimer 7 was obtained as a minor byproduct. Excess
heating, or addition of hydrochloric acid during workup, may lead to higher
levels of dimerization.
Multigram quantities of imidazolone
5
can be
conveniently prepared by this method (Scheme 11).
H
N
>=o
H2N.-NH
CO2Me
6
1. Na(Hg)
N
H
H
N
2. 1.1 eq
KOCN
H
5
7 (5-10%)
Scheme 11
Our strategy for the functionalization of the propylamine side chain in
imidazolone 5 was based on the relevant transformation of 2-aminoimidazole 5b
to dialkoxyimidazolines with NCS and alcohols. In this case, the stability of 2aminoimidazoline adducts allows for their isolation.27
102
OMe
H2N)NH2
H
H2N_<J7H2
NCS
MeOH
5b
HOMe
I
xylene
reflux
H2N-_j'
NH2
Scheme 12
Few examples of related imidazolidinones have been reported in the
literature.
For example, 4,5-dihydroxyimidazolidinones are formed upon
addition of glyoxal to urea under basic
conditions.28
The dialkoxyadduct 4,5-
dimethoxy-2-imidazolidinone-4-carboxylate has been prepared as a racemate by
anodic dimethoxylation of the double bond.29
R
HO
>=
HO"1
CH3O2C
H
MeOI1,>0
MeO
R
R=H or OH3
Based on this information, we envisioned functionalization of the
propylamine side chain of imidazolone 5 via in situ elimination of its
corresponding adduct followed by isomerization to the desired olefin 8 (Scheme
1
3)27
103
H
H
- -
-ø
H
N
H
5R=H
H
[
8R=H
Scheme 13
It was found, however, that bromination of 5 under mild and strong
acidic conditions and varying reaction times resulted in the formation of a
complex mixture of dimeric species, with dimer 9 formed as one of the
components. This complex mixture may arise from the nonselective oxidation
of monomers and dimers. Bromination followed by base catalyzed elimination
was attempted using the acylated imidazolone 10. In this case the a-substituted
carboxamide 11 was isolated in good yield (85 %) with no trace of the dialkoxy
adduct (Scheme 14).
Br2
5
TFA
H2
'.'J ,,.,/
0H
H
H
Br2
MeOH
H
10
Br
5 eq KOtBu
OMe
NJvN)LçBr
I
N
H
Br
(85
Scheme 14
104
Pyrrole-imidazolone 11 was found to be stable under basic conditions
(Et3N, KOtBu) at room temperature. Increasing temperatures (e.g. refluxing in
pyridine) gave only partial decomposition of the starting material. Elimination
of the alpha substituent was attempted by heating 11 to high temperatures.
Unfortunately, under these conditions (trimethylbenzene, reflux), the desired
olefin was found to decompose prior to the consumption of all starting material.
We then proceeded to attempt isolation of the dialkoxy adduct of 5 using
the reported conditions for the synthesis of 2-aminoimidazolines.27 However, it
was found that treatment of imidazolone 5 with NCS in methanol afforded only
the c-methoxy derivative 12. This compound was isolated as the free base in
good yield after purification of the reaction mixture by silica gel column
chromatography using a 6:4 CH2C12/MeOH(NH3) solvent system. Unlike 2aminodialkoxyimidazolines, derivative 12 was prone to further oxidation to the
corresponding a-ketone 13 (Scheme 15).
OMe
H
NOS
N
MeOH
N
H
2 NCS
__
2
H
5
MeOH
12 (70%)
H
N
0
NH2
N
H
13
Scheme 15
The difference in reactivity of 2-aminoimidazoles and imidazolones can
be explained by the difference in basicity between the two functionalities. In the
105
oxidative addition of alcohols to propylamine 5b, protonation of the guanidine
moiety is believed to help stabilize the 2-aminodialkoxyimidazoline product
(Scheme 16).
HoMel
o=
H
<N(R
I
N-i
1
12
N
I
HOMe]
[
j
H2N
HOMe
Scheme 16
In contrast to 2-aminodialkoxyimidazolines, thermal elimination of
methanol from 12 using xylene led primarily to decomposition. Dione 13 was
isolated in 25 % yield. Interestingly, treatment of a-methoxy derivative 12 with
trifluoroacetic acid gave the desired olefin 8 in moderate yields (35 %) with
dimer 9 formed as a byproduct (33 %). Dilution of the reaction (0.02 M vs. 0.15
M) gave no significant increase in the yield.
106
H
OMe
xytene
I
H
reflux
N
H
N
H
12
13b
jTFA
ojy
[H
o(jNH2 +9
ii
NH21
j
8
Scheme 17
Direct introduction of a hydroxyl group to the n-position in 8 (Scheme
17) using mildly acidic aqueous solutions proved to be problematic.
For
example, treatment of olefin 8 with aqueous acetic acid resulted in oxidation of
the imidazolone ring to give predominantly 13b over the desired beta
functionalized imidazolone.
However, reports of allylic amides undergoing
cyclization to oxazolines with sulfuric acid suggested an alternative
approach.3°
With this in mind, amide 14 was produced by acylation of olefin 8 with 4bromo-2-(tnchloroacetyl)pyrrole in good yield. Cyclization of carboxamide 14
to the oxazoline 15 was achieved with concentrated methanesulfonic acid in
nearly quantitative yield. A suspension of oxazoline 15 in aqueous HCI (5 %)
was stirred at 75 °C until starting material was detected by TLC. Evaporation of
the solvent yielded the corresponding ester 16 as the HCI salt.
Careful
107
neutralization of an aqueous solution of ester 16 with iN KOH facilitated acyl
transfer to produce the target -hydroxy amide 4 in good yield.
H
H
N
DMF
0
H
N
H
o=K
N
H
N
ci3c)Li:.l
8
Br
14 (90%)
Br
IMeSO3H
H
N-.-NH
0
KN
5 % HCI (aq)
o=<
0
jç'N
N
H
H
16
H
N
NH
15
(90%) B/
Br
!1N KOH
NH
H
N
H
4
Br
(85%)
Scheme 18
Oxidative cyclization of alcohol 4 with NCS in methanol at room
temperature produced a 1:1 mixture of slagenins B (2) and C (3) in yield of 90
% yield (Scheme 19). Purification by chromatography provided pure samples of
each synthetic alkaloid. Heating of a 1:1 mixture of slagenins 2 and 3 in the
presence of aqueous acid produced slagenin A (1) as the sole product. The acid
catalyzed conversion of slagenins B and C to A is thought to proceed via
FDI
elimination of methanol followed by isomerization at C15 (PATH A, Scheme
20).17
H
o=<fr
Br
H
4
MeOH
NCS, ii
90%
N:QCH3
H
OCH3
H
N
0
0
O
HH
HH
Slagenin B (2)
11
:
N
Slagenin C (3)
B!
Br
H20, H
80°C
HQH
°=THN
f-NH
Slagenin A (1)
Br
Scheme 19
Alternatively, treatment of alcohol 4 with NCS in a water:THF (1:5)
solution followed by work up with methanol at room temperature gave a mixture
of slagenins A (1), B (2), and C (3). The ratio of products was found to be 1:0.5:0.5 (1:2:3). It is currently unclear why the endo diastereomer of slagenin
109
A was not isolated in this case
However, the ratio of products observed
suggests that this diastereomer (endo 1) may rapidly react with methanol during
workup to form slagenins B and C (2, 3). This reactivity could be attributed to
the possible existence of an opened ketone intermediate of endo 1, as shown in
PATH B, Scheme 20. To our knowledge, the endo diastereomer of 1 has not
been found in nature.
PATH A
N
N
0=<
N:O
HH
O=<
N
NHR
HH
0
NHR
0=<
PATH B
N
HO
NHR
[:>]
slagenins B and C
4
endo 1
0
0
OH
ON(7NR ONIIHNR
NH2
ON+NR
Scheme 20
It should be noted that the facile interconversion of slagenins B and C to
A in aqueous acid, and the isolation of slagenins B and C during workup after
intramolecular oxidative addition of alcohol 4 in aqueous media suggest that
slagenins B and C are likely to be isolation artifacts and not natural products.
110
The relative stereochemistry of alkaloids 1, 2, and 3 was confirmed by
DPFGSE 1D-NOE experiments (Figures VI.1
VI.3). NOE correlations
between H15 and the hydroxyl or methoxy hydrogens were observed in
slagenins A (1) and B (2) respectively upon selective excitation of H15. An
additional correlation between H15 and H9 was observed in slagenin C (3).
These results are in agreement with the NOE correlations reported for the
natural products.4
'H'
11
10
9
8
7
6
''I
I,'
5
4
3
I
2
H15
NH14
OH11
Figure VI.1. DPFGSE 1D NOF correlations observed upon selective excitation of H15 in slagenin A (1).
ppm
11
10
9
8
7
5
6
.5
H.
Figure VI.2. DPFGSE 1D NOE correlations observed upon selective excitation ofHl 5 in slagenin B (2).
ppm
........................
11
10
9
7
8
6
5
4
3
2
[1
INH 14
H9
CH316
Figure VI.3. DPFGSE 1D NOE correlations observed upon selective excitation of H15 in slagenin C (3).
ppm
114
In summary, the first synthesis of slagenins A (1), B (2), and C (3) was
accomplished from commercially available L-ornithine. Activation of the beta
site for the key introduction of the hydroxy substituent was accomplished via
intramolecular cyclization of olefin 14 to the corresponding oxazoline. The
synthetic scheme incorporates steps that are conceivably biomimetic in nature
and shows that slagenins B and C are likely to be isolation artifacts. In addition,
it provides access to previously unavailable 4-subsituted imidazolones which
may be used for the synthesis of related alkaloids.
115
6.2. TOTAL SYNTHESIS OF (Z)-DEBROMOAXINOHYDANTOIN AND
RELATED ALKALAOIDS.
A wide range of tricyclic oroidin derived metabolites have been isolated
from marine sponges. The axinohydantoins are a small subgroup of structures
that differ from the rest by the presence of a hydantoin-lactam moiety. Thus far,
three
members
of
this
class
have
been
recently
isolated:
(Z)-
debromoaxinohydantoin (17), (Z)-axinohydantoion (18) and (E)-axinohyantoin
(19).
0
HNO
xfTh)
0
17 X=H
19
18 X=Br
Scheme 21
A biogenetic path for the axinohydantoins has not yet been proposed.
However, in analogy to the hymenialdisines, a linear precursor is a likely
intermediate.5'6
No synthetic work has been reported on these metabolites, and.
further investigation of their biomedical potential has been hindered by the
limited resources.
116
Currently available methods for the synthesis of simple unsaturated
hydantoins include the condensation of ureas with cyanides or esters,31 as well
as the condensation of (vinylimino)phosphoranes with aryl or alkyl isocyanates
(Scheme 22).32 However, a route for the direct access to unsaturated hydantoins
from imidazolones remains undeveloped.
PhNCO
PhMe
Ph
0
HNANH2
0
1.NaOMe, MeOH
2.HCI,water,MeOH
NCL1
NH
HNyLL)
0
Ph
Me
Me
Ac20
Scheme 22
During the course of our work on the axinohydantoin core, reports
centering on the biomedical potential of DBH and hymenialdisine appeared in
the literature. Although syntheses for DBH had been reported by our
group33a
and Annoura et al,33b ongoing investigations of this alkaloid as a potential
candidate for the treatment of osteoarthritis had reached the point where a
117
practical
synthesis of DBH was needed to obtain enough material for
preclinical/clinical trials. In response to the interest expressed by the
pharmaceutical sector, we chose to modify our original route to Z-DBH (20) to
meet large-scale demand. In this case, a straightforward oxidation of hymenin
20 to the glycocyamidine functionality would significantly improve the existing
route (Scheme 23). We recognized that a transformation of this type could also
be used for the synthesis of the hydantoin-lactam moiety in the axinohydantoins
starting from a related imidazolone derivative of 21. Therefore, we began our
study on the practical synthesis of Z-DBH (20).
H2N
H2N
HO
_N
H/)
_N
Br
Br
NH
NH
0
0
(±)Hymenin (21)
Z-DBH (20)
Scheme 23
118
6.2.1 Practical Synthesis of (Z)-Debromohymenialdisine.
In our original route to Z-DBH, the intermediate hymenin (21) is derived
from a key coupling reaction of the azepinone 22 and 2-aminoimidazole,
followed by a stepwise oxidation of hymenin (21) to form the cL,13unsaturated
amide system (Scheme 24).
H.,N
Br
20
B
21
Scheme 24
Synthesis of hymenin 21 began by conversion of the commercially
available bromoethyl dioxolane 23 to the known amine
Coupling of amine
25 with 4,5-dibromopyrrole trichioromethyl ketone followed by deprotection of
carboxamide 26 gave the desired aldehyde 27.
proved to be difficult,
methanesulfonic
acid
Cyclization of the aldehyde
and was only achieved using concentrated
and
long
reaction
times.
Regioselective
heterodimerization of pyrroloazepine 22 with 2-aminoimidazole was achieved in
methanesulfonic acid via an azafulvonium ion. Purification by chromatography
provided hymenin (21) in good yield (Scheme 25).
119
1. Ph3P, THF
2.H20
NaN3, H20
\...o
çNNH2
57%
75%
23
25
24
Br
CHCN
90%
Br,;,>Cc13
Br
H
0
N
/N4B
Br
H
refiux
91%
27
CH3SO3H
TsOH
H2Olacetone
26
80 %
via
H
Br
L-\
Br(Il
)
1rNH
0
22
IN
(±) Hymenin (21)
[Brç
MeSO3H
7d,RT
65%
Scheme 25
With the tricyclic
core in
hand, functionalization
of the
2-
aminoimidazole appendage was achieved by bromination of hymenin to give
bromohymenin 28. Hydrolysis gave derivative 29 as a mixture of diastereomers
as well as the natural product stevensine
as a reaction byproduct. Unsaturation
was achieved with a one-pot protodebromination-oxidation reaction with
catalytic HEr in methanesulfonic acid at 90 °C.
(Z)-DBH (20) and
120
hymenialdisine were isolated from a complex mixture of products, which also
yielded over-oxidized derivatives (Scheme 26).
H2N
H2N
Br2, TFA
B
Hymenin (21)
%
HBr
\
BriJ')
N
HOAc/H20
ref lux
_______
72 %
Br
HO
Br(
)_NH
N
II
0
0
29
28
90°C cat. HBr
CH3SO3H
+
Hymenialdisine X = Br (32 %)
DBH(20)X=H(15%)
Scheme 26
Initial modifications centered on the synthesis of linear carboxamide 26.
In this case, distillation of dioxolanes 23 and 24 was circumvented by replacing
amine 24 with commercially available diethoxypropylamine. Acylation of this
amine using standard coupling
conditions33
gave carboxamide 30 in good yield
(90 %) (Scheme 27). Next, attention was shifted to the synthesis of the pyrrole-
azepine 22 and its heterodimerization with 2-aminoimidazole, which initially
required a total of 14 days. It was found that treatment of carboxaniide 30 with
121
methanesulfonic acid at 45 °C provided the desired azepinone in comparable
yield in a total of 4 days. Furthermore, addition of 2-aminoimidazole directly to
the methanesulfonic acid mixture gave hymenin in an overall yield of 65 % from
carboxamide 30 (as opposed to an overall yield of 47 % starting from 26 using
the unmodified sequence). This modification circumvents the deprotection and
isolation of the corresponding aldehyde as well as the isolation of azepine 22.
Br
90 %
10
30
10
CH3SO3H
3-4d,45°C
H
[Bç]
N
(±)Hymenin(7)
3-4 d
45°C,
65%
Scheme 27
At
this
point,
a
method
for
installing
the
a,f-unsaturated
aminoimidazolidinone functionality of (Z)-DBH was investigated.
In the
original route, a protodebromination/oxidation step was used to introduce the abromine in hymenialdisine, which could not be successfully introduced by direct
bromination of DBH (compound 31 was isolated instead).33
122
H2N
Br2
Z-DBH(20)
>O
H
Br
NBS
position more reactive
0
31
Since selective introduction of an a-bromine substituent was not
necessary for a practical synthesis of DBH, we chose to include a hydrogenation
step for the removal of the bromine atoms with the hope that a direct oxidation
of a debrominated intermediate would cleanly provide the unsaturated
functionality. Hydrogenation of compound 29 with Pd/C gave the debrominated
glycocyamidine 32 in good yield.
Oxidation of 32 with bromine in
methanesulfonic acid at 90 °C provided alkaloid 20 in an improved yield of 40
%. The C9-substituted ether 33 was also isolated as a minor byproduct (Scheme
28).
H2NN
Br2,
H2 Pd/C
CH3SO3H
20
(40%)
29
85%
4.
90°C
/
0
32
33
Scheme 28
123
Substitution at C-9 in product 33 is thought to occur upon work up with
methanol. The time dependent elimination of methanol was studied by treating
a sample of ether 33 in DMSO-d6 with a minimum amount of methanesulfonic
acid. The NN'IR tube was heated to 60 °C, and monitored over time. Figure
VI.4 shows the time dependent formation of the unsaturated product 34.
124
H2NN
H2N
HJ,O
HF!J)°°O
OMe
MeSO3H
DMSO-d6
60°C
t=0
t
40 hrs
13
12
11
10
9
8
7
6
5
4
3
ppm
Figure VI.4 Time dependent elimination of compound 33 to 34 at 60°C.
The Z geometry at dO-Cu in 34 was elucidated through DPFGSE 1D
NOE and NOESY experiments. In the former, a correlation between H3 and the
glycocyamidine NH was observed upon irradiation at H3. Comparison of the
NOE spectra with a slice through H3 from a 2D gradient NOESY experiment
further corroborated this result (Figure VI.5).
125
K2N 14
2
H3
.H2H9
DPFGSE 1D NOE results
H7
H8
H15
H13
11
10
9
It
7
8
glycocyamidine NH
irradiate
'I'
glycocyamidine NH
H2
'Ii
irradiate
H8
H7
H9
irradiate
H9
Slice from 2D gradient NOESY
H8
glycocyamidine Nil
'V
slice through
Figure VI.5 Determination of the Z geometry of compound 34 by NMR
spectroscopy (in DMSO-d6).
126
The moderate yield of DBH obtained from the oxidation of compound
32 with bromine was partially attributed to the competition between bromination
of the pyrrole group and the glycocyamidine appendage. To circumvent this
problem we chose to perform a double oxidation of hymenin (21) to the sponge
metabolite (Z)-3-bromohymenialdisine (35). This transformation was achieved
successfully by using two equivalents of bromine in an acetic acid/sodium
acetate system. Alkaloid 35 was obtained in good yield after crystallization.
Spectral data of synthetic (Z)-3-bromohymenialdisine 35 were in satisfactory
agreement with those reported for the natural material. Debromination by
hydrogenation gave (Z)-DBH (20) in good yield after crystallization.
Br
CH3SO3H, 45°C,
4 d then 2-Al,
45°C,4d,
10
[Br>iNH]
60%
30
2 eq. Br2, 23°C,
HOAc/NaOAc,
85%
H
/1
H
(Z)-3-Bromohymenialdisine (35) R = Br i H2, 10% Pd/C,
DBH (20) R = H
HN
Br
NaOAc, 75%
Scheme 29
(j-Hymenin (21)
127
to produce (Z)-DBH
Efforts
didebromohymenin33
(20)
via
the
oxidation
of 2,3-
using Cu(OAc)2 or via base-catalyzed air oxidations
produced (Z)-DBH in a less efficient manner.
No evidence of the less
thermodynamically stable E isomer of Z-3-bromohymenialdisine (35) was
observed upon oxidation of hymenin (21). In accordance with earlier studies on
protonated and neutral glycocyamidines, comparison of 13C chemical shift
values of Z-3-bromohymenialdisine (35) for the free base and HCI salt revealed
a significant upfield shift upon protonation of the guanidine and imidazolidinone
(C=O) carbons.
M2Il 14
HN /O
free base
HCI salt
C12 176.7 ppm
C14 166.7 ppm
163.2 ppm
154.1 ppm
Br
Br
/
ic
8
2
N 5
NH
(Z)-3-Bromohymenialdisine (35)
In summary, a practical synthesis of Z-DBH (20) has been developed,
and the desired product can be synthesized in four steps from the commercially
available 3-diethoxypropylamine in an overall yield of 34 %. Noteworthy is the
fact that this route does not require the use of protecting groups for any of the
five nitrogen functionalities and no chromatographic techniques are needed for
purification. In addition, suitable conditions for a one-pot double oxidation of
128
hymenin (21) were successfully developed and could be used for an easy access
to the axinohydantoins. Table VI.1 summarizes some of the key aspects of this
practical route.
These results show that the synthesis should be directly
amenable to scale-up processes.
Purification
Longest reaction time
Scale
Original Route
Modified Route
4 chromatographies
2 distillations
No chromatography
No distillation
14d
6-8d
1 g Hymenin
60 g Hymenin
8
3
2%
34
Purified intermediates
Overall yield
Table VI.1 Improvements achieved for a practical synthesis of Z-DBH (20).
6.2.2 Total Synthesis of (Z)-Debromoaxinohydantoin (17).
Based on the structural similarity between the axinohydantoins and the
hymenialdisines, we initially considered a direct transformation of a 2aminoimidazole to a 2-imidazolone unit.
129
R
RH
N
1:1
,>NH
hydantoin
>=o
'11-
unit
N
H
A report by Cariello and coworkers described the conversion of the 2aminoimidazole units
in derivatives of the marine sponge metabolite
pseudozoanthoxanthin to the corresponding imidazolone functionality by
hydrolysis or
diazotization.34
However, use of these
methods35
conversion of the known 2-aminoimidazole derivatives 36 and
37,27
for the
to the
corresponding imidazolones was met with limited success. Heating of a solution
of propylamine 36 in concentrated hydrochloric acid in a sealed tube at 200 °C
led to decomposition of 36 and partial recovery of starting material (Scheme
31).
Several conditions for the diazotization of carboxamide 37 were
investigated. A trace of the desired imidazolone was formed as part of a
complex mixture of products using a 1:1 solution of iN HC1: AcOH as solvent.
130
H2N
HO
l-\
3N HC
N
NaNO3
NaC
H2N
H2N
N
TNNH
NNH
'
H3O
heat
H2Nj'NH2
H
-,--'
N
H2N
36
J
N
37
0H
Br
Scheme 31
Based on this outcome, we envisaged the synthesis of the tricyclic core
of hymenin derivative 38 to take place via a key cyclization of the linear amethoxyimidazolone 11 (Scheme 32). This approach complements our studies
on beta functionalization previously reported for the synthesis of slagenins. In
addtion, transformation of the imidazolone to the hydantoin lactam moiety could
be achieved with the conditions of a one-pot double oxidation developed for the
synthesis of Z-DBH from hymenin (21).
131
0
X.
HN
OMe
H
N
NR
o
N
H
axinohydantoins
----
11
38aX=H, X=Bi
38b X = X' = H
38cX=X'=Br
Scheme 32
Carboxamides ha-c were conveniently synthesized by acylation of aether 12 with 4,5-dibromo, 4-bromo- and debromo-pyrrol-2-yl tnchoromethyl
ketone (Scheme 33).
OMe
H
N
H
I
1 6h
o=(
H
OMe
DMF, rt
0
12
H
N
H
cI3c,'
Br
R'
ha R = R = H
lib R= H, A' = Br
lic R =
= Br
80%
Scheme 33
Carboxamides Ha-c were found to be stable under neutral and basic
conditions. Attempts to force cyclization using high temperatures and excess
base only led to partial decomposition of the starting material.
Therefore, an
acid catalyzed cyclization was investigated (Scheme 34). Treatment of lie with
trifluoroacetic acid at room temperature produced only a trace of the desired
132
hymenin derivative 38c (X = X' = Br). Interestingly, the major product of this
reaction was found to be diaza-spiro[4.5]dec-3-ene-2,6-dione 39.
lic
TFA
+ 38c
10%
39
70%
Scheme 34
The planar structure of the unusual polycyclic arrangement in 39 was
established primarily by 2D spectroscopic methods. Figure VI.6 shows the 1Hl3
HIvIBC spectrum of 39, key correlations between the methine proton at the
alpha site, the hydrolyzed pynole ring and the imidazole functionality (outlined)
support the diaza-spiro[4.5}dec-3-ene-2,6-dione framework.
H
133
!-7
N'
0
-24
/3NH
r
HN
7
6
pm
40
60
80
100
120
140
160
10
9
8
5
4
3
2
ppm
Figure VI.6 'H-13C HMBC correlations observed for compound 39.
The relative stereochemistry assigned to 39 is proposed to be as shown in
Figure VI.7 on the basis of DPFGSE 1D NOE experiments. Selective excitation
of Hi gave NOE enhancement at 112 and H3 (Figure VI.7 A) suggesting that the
imidazolone ring lies on the same face as Hi, most likely in a pseudo-equatorial
positon. Similarly, 113 was found to show NOE to Hi (Figure VI.7 B).
134
HH
selectively excited
B
selectively excited
A
A
II
selectively
excited
1
H3
H2
HI
/
VM
selectively
excited
B
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
4.5
5.0
4.0
3.5
3.0
A
2.5
2.0 Pp
Figure VI.7: DPFGSE 1D NOE correlations observed for compound 39.
Formation of this unexpected heterocycle is thought to proceed via the
path shown below (Scheme 35).
Br
lic
Br
0
39
- - - -
o==z(jj
N
H
Scheme 35
135
Based on the proposed path to 39, we proceeded to determine if the absence of
the 4-bromine substituent in lib could help circumvent the mode of cyclization
leading to the formation of 39 under similar conditions as to provide higher
yields of the desired hymenin derivative 38a (X = H, X' = Br). In this case,
evaporation of the solvent followed by trituration of the mixture with methanol
gave the major product of the reaction as a precipitate. Following filtration,
purification by chromatography yielded hymenin derivative 38a in an improved
yield of 30 %. Analysis of the major product by HMBC, COSY and 1H-
15N'C NIVIR experiments and mass spectroscopy revealed that this
compound is in full agreement with the unexpected product 40b. Oxazoline 15
was observed as a minor byproduct (7 %).
Similarly, cyclization of the
debrominated carboxamide ha gave the corresponding derivatives 38a (X = X'
= H) and 40b in comparable yields (Scheme 36).
0
HN
llaR=H
TFA
A
llbR=Br
+
3
rt,1
NH
N
0
38a R = H
38b R = Br
40a R = H
40b R Br
25 -35 %
65-70 %
Scheme 36
II
The 'H-'5N HMBC correlations supporting the connectivity assigned to
heterocycles 40b (R = Br) and 38b (R = Br) are shown in Figures VI.8 and VI.9.
In
38b,
the existence of an azepine backbone (7 member ring) is supported by
the correlations observed between the ct-methine proton and the imidazolone
nitrogens (Figure VI.8). Similarly, the a-methine proton in 40b was found to
show correlations to the imidazolone and pyrrole nitrogens supporting the
pyrrolophane system (Figure VI.9). In addition, cyclization of lib at C3 to give
38b
is accompanied by a downfield shift of this carbon from 112.2 to 123.4
ppm.
On the other hand, cyclization of
lib to 40b
is accompanied by a
downfield shift at C2 and C5 from 127.8 and 121.9 to 133.2 and 126.5
respectively. No significant chemical shift change was observed in this case for
C3.
(2,5)-Pyrrolophanes with the same ring dimension as 40 have been
synthesized in low yield from the photochemical reaction of bicyclic azirines
with electron-poor
alkynes.36
H4
14
13
12
11
10
9
9
7
6
5
4
3
2
lppm
137
Figure VI.8: H-'5N HMIBC correlations observed for 38b.
12
11
10
9
8
6
7
5
3
4
ppm
Figure VI.9: 1H-'5N HMBC correlations observed for 40b.
The unexpected formation of compounds 39 and 40 prompted us to take
a look at the electron distribution in carboxamides lib and lic to determine if
the mode of cyclization observed was a result of an inherent molecular property
(Table VI.2).
A.
C2
C4
C7
C9
Cli
C12
N8
06
Br15
Br 13
R=Br
R=H
0.29
0.3
-0.16
-0.11
H
10
N
H
0.13
-0.24
0.14
0.11
11
Br
138
B.
R=Br
R=H
C2
C4
C7
C9
-0.41718
-0.40 18
0.57 122
0.57 60
01 H
N
-0.16588
.0.1
-0.09172
-0.
Cli
-0.05192
-t12 53\ / 12
C12
-0.06446
-0.0285
N8
06
-0.00859
-0.55254
-0.17360
Br15
Br13
-0.06983
0.00175
8
-0.57160
41
A 15
Br
13
-0.09558
Table VI.2 A. Electrondensity distribution observed for carboxamides lib
and lie as calculated using AM1 Semi Empirical calculations (no solvent
effects). B. Ab initio electrondensity distribution on model compounds 41
(solvent = water).
Based on the values obtained for C7 and C9 in carboxamides lib and
lie, a difference in electron density is not likely responsible for the mode of
cyclization observed in these compounds.
With 38b in hand we proceeded to perform a double oxidation using the
conditions developed for the practical synthesis of (Z)-DBH (20). Oxidation of
38b with two equivalents of bromine was found to give a mixture of partially
oxidized structures 42a (35 %), 42b (20 %), with axinohydantoins Z-44 and K-
44 in only trace amounts.
Upon addition of three equivalents of bromine the
desired products Z- and E-44 were isolated after chromatography in 45 % and
30% yields, respectively (Scheme 37).
139
0
HN
2 eq Br2
38b
10 eq NaOAc
AcOH, ii, 18 h
H
NH
R>
Br(
Br__jr\)
Bri
-NH
ii
42a R = H
42b R = Br
o
0
Z-44
E-44
3 eq Br2
38b
Z-44
+
E-44
10 eq NaOAc
AcOH, rt, 18 h
Scheme 37
Based on this outcome it can be suggested that compounds 42a and 42b
may be possible intermediates of hydantoins 29 and 30 (Scheme 38).
42
-----
H2- - - -
Scheme 38
In
analogy
to
our
previous
work,
hydrogenation
of
Z-3-
bromoaxinohydantoin (44) with Pd/C gave the natural product 17 in good yield
after crystallization from methanol (Scheme 39).
140
10%Pd(C),H2
Z-44
3 eq NaOAc
MeOH,rt,3h
j
80%
)_.NH
0
17
Scheme 39
Interestingly, hydrogenation of the corresponding E-isomer 44 under
similar conditions gave derivatives 45a and 45b as a 1:1.3 mixture. Attempts to
drive debromination to completion only led to competing saturation of the ClO-
Cli bond, and compounds 46a and 46b were isolated in a 4:1 ratio. It is
believed that steric interactions between the E-hydantoin appendage and the
catalyst hinder complete removal of the 5-bromine prior to over-reduction.38
0
0
O(NH
HN.4r
/
N
NH
10% Pd(C), H2
10% Pd(C), H2
E-44
-4
0KNH
A
3 eq NaOAc
MeOH, rt, 8 h
3 eq NaOAc
MeOH, rt, 3 h
4:1 (82%)
1:1.3(85%)
/
N
0
NH
0
45aR=H
46
45b R = Br
Scheme 40
Alternatively, synthesis of hymenin derivative 38c can also be achieved
in 37 % yield via a key coupling reaction between azepinone 22 and imidizolone
in TFA at room temperature (Scheme 41).
Imidazolone is a commercially
141
available substance, however, we found that reduction of glycine ethyl ester
under Akabori
conditions25
followed by condensation of the aldehyde with
potassium cyanate gave the desired product in good yields (70 %) after
crystallization. In contrast to the coupling of 22 with 2-aminoimidazole, the use
of methanesulfonic acid led to decomposition of imidazolone. In this case the
oxidized protodebrominated product 42c was isolated from the complex
mixture. Although the yields of 38c are moderate when using TFA, unreacted
starting material can be recovered from the mixture and resubjected to coupling
conditions.
0
HN
Br
H
MeSO3H
p
I
N
H
NH
Br__c)
0
N
H
55°C
lOd
Br
35%
22
42c
TFA
2 eq Br2
Z-44+
38c
E-44
10 eq
AcOH, ii, 18 h
Scheme 41
As shown in Figure VI.10, E-3-Bromoaxinohydantoin 44 was found to
undergo slow isomerization to the corresponding Z isomer.
142
13
12
Figure VI.1O
11
10
9
8
7
6
5
4
3
2
i
ppm
Isomerization of E-3-bromoaxinohydantoin 44 at room
temperature. I = 6d, 30 % isomerization in DMSO-d6.
The heats of formation of the Z and E isomers of 44 were calculated
using Semi-Empirical calculations.
The Z isomer was found
to be
approximately 6 kcal/mol lower in energy than the corresponding E isomer
(Figure VI.1 1).
An energy difference of 5-6 kcal/mol was also observed
between Z and E isomers of the debrominated and monobrominated
axinohydantoins 17 and 19. This outcome suggests that the presence of bromine
143
atoms on the pyrrole group is not a predominant factor in determining the
energy difference between the Z and E isomers.
This may be due to the
geometry of the molecule, where a dihedral angle exists between the pyrrole
plane and the 5-member ring plane containing the hydantoin moiety.
0
HL>°
Br
Br
/
NH
N
B4>
0
-2U.U20 kcal/mol
- 4./U KcaI/moi
Figure VI.11 Heats of formation of Z and E Debromoaxinohydantoin
calculated using AM1 Semi Empirical methods
In a recent report published by Kobayashi and coworkers, isomerization
of the E-isomer of the hymenialdisines to the corresponding Z-isomer is
proposed to take place via a zwitterion intermediate.8
NH2
0
H2N
NH2
N
LNH
HNç>O
NH
o
v-NH
0
0
0
z
E
Based on our findings, it could be suggested that formation of this
intermediate may be facilitated by the unique electronic properties of the
glycocyamidine unit.27b
Therefore, in contrast to the axinohydantoin case,
absence of the E-isomer of 35 in the double oxidation of hymenin (21) may be
due to its ready isomerization to Z-35.
In
summary,
we
have
achieved
the
first
synthesis
debromoaxinohydantoin via a key oxidation of imidazolones to
hydantoins.
a-f
of Z-
unsaturated
Hymenin derivatives can be synthesized via cyclization of
a-
methoxy imidazolones or via coupling of azafulvene azepines and imidazolone.
Our route is noteworthy in that it provides direct access to a number of
previously unprecedented axinohydantoin derivatives and their corresponding Eand Z- isomers without the use of protecting groups in the nitrogens.
145
Based on our findings, it could be suggested that formation of this
intermediate may be facilitated by the unique electronic properties of the
glycocyamidine unit.27b
Therefore, in contrast to the axinohydantoin case,
absence of the E-isomer of 35 in the double oxidation of hymenin (21) may be
due to its ready isomerization to
In
summary,
we
Z-35.
have
achieved
the
first
synthesis
of
Z-
debromoaxinohydantoin via a key oxidation of imidazolones to a-13 unsaturated
hydantoins.
Rymenin derivatives can be synthesized via cyclization of c-
methoxy imidazolones or via coupling of azafulvene azepines and imidazolone.
Our route is noteworthy in that it provides direct access to a number of
previously unprecedented axinohydantoin derivatives and their corresponding Eand Z- isomers without the use of protecting groups in the nitrogens.
146
6.3. STUDIES TOWARDS THE PYRROLOPIPERAZINE SYSTEM IN
THE AGELASTATIN CORE AND RELATED FINDINGS
Of the many pyrroloaminopropylimidazoles that have been described
from sponges, the highly fused tetracyclic pyrrole skeleton containing a
pyrroloketopiperazine system is unique to agelastatins A (47), B (48), C (49),
andD (50).16
A1
Me
HO
_NH
HQ.1,j
Br
LNFt
47 X = H
48 X = Br
49 R1 = Me, R2 = OH
50 R1 = R2 = H
This unusual heterocyclic array has made the agelastatins an attractive
target for total synthesis. Thus far, synthetic efforts have centered on the total
synthesis of agelastatin A (47)39
of bromoagelastatin D
removal
51
Our synthetic strategy relies on the synthesis
as a precursor of agelastatin D
of the beta bromine
substituent of
protodebromination reaction (Scheme 4
51
(50),
with selective
taking place via a
147
Hc
0
50
0
51
Scheme 41
We envisioned the tetracyclic core of imidazolidinone 51 to be derived
from the key intermediate pyrroloketopiperazine 52. Oxidation of this precursor
followed by an acid-catalyzed C-ring annulation would furnish the tetracyclic
core. The desired heterocycle 51 would be obtained by final functionalization at
C5 (Scheme 42).'
H
N0
H
Br
[0]
- - - -
Br
"NH
Br_J1NH
0
0
52
H20
51
-
Br H
\
>çSNH
BrNH
0
Scheme 42
Recently, Taglialatela-Scafati et al. reported the isolation of cyclooroidin
53 from the sponge
This finding is suggestive of the
Agelas oroides.4°
intermediacy of a pyrroloketopiperazine precursor in the biosynthesis of the
agelastatins.
H
N
NH2
BrJT
53
The pyrrolopyrazine (N-C) and pyrrolopyridine (C-C) connections are
common structural motifs within the polycyclic framework of a variety of
secondary metabolites possessing a bromopyrrole moiety other than the
agelastatins. Examples of such compounds include phakellin,41a
isophe11in4lb
and longamide.41'
NN
N
H
Br
PhakeHin
Br
HOH
H2N-4
N\
H2N,
0
0
N
Br
A
Br
Br
Isophakellin
L.NH
0
Longamide
The work presented in this section begins with the description of
preliminary results on the synthesis of a pyrrolopyrazine (N-C) unit by
149
controlled cyclizations of 2-pyrrolecarboxamidoacetals (Scheme 43). This is
followed by analysis of the preferred mode of cyclization of 13-activated
imidazolones bearing an a,f3-unsaturated or 13-halogen functionality.
Br
OR
Br)j(NS*..YLOR
..
H0
Longamide
H
52
H
Br
Scheme 43
6.3.1
Controlling Cyclizations of 2-Pyrrolecarboxainidoacetals. Facile
Solvation of -Amido Aldehydes and Revised Structure of Synthetic
Homolongamide.
Early work by Johnson and coworkers described the acid-mediated
cyclization of 2-indolecarboxamidoacetals, where N and C act as competing
nucleophiles that lead to a mixture of pyrazinoindole and pyridindole products.
For example, upon treatment of the acetal with HC1/EtOH and H2SOilEt2O
conditions a 8:2 mixture of the (N-C) and (C-C) cyclized products was observed
(Scheme 44). 42
150
O1Et
H
LNH
Scheme 44
This outcome prompted us to investigate the controlled cyclizations of 2-
pyrrolecarboxamidoacetals 54-56 for the selective synthesis of fused bicyclic
pyrrolopyrazine (N-C) and pyrroloazepine (C-C) derivatives. We found that
preparation of the desired carboxamidoacetals was achieved in good yields by
acylation
of
the
corresponding
amines
with
4,5-dibromopyrrol-2-yl
tnchoromethyl ketone (Scheme 45)43
Br
OR
CH3CN
H2N-L.
Br(13
Br?YNYOR
54 n=1; A = -CH2CH255 n=2; R = -CH2CH256 n=3; A = -CH2CH3
Scheme 45
In the case of acetal 55, standard deprotection conditions (pTsOH,
acetonefH2O, reflux, 12h) gave aldehyde 27 as the sole product. However, no
trace of the corresponding aldehydes was observed upon exposure of acetals 54
151
and 56 to the same reaction conditions. Alternatively, acetal 54 was found to
cyclize in good yield to longamide (57), a marine sponge metabolite isolated by
Taglialatela-Scafati and coworkers from the Caribbean sponge Agelas
ion gissima.4c Similarly, N-C cyclization of acetal 56 was favored under these
conditions, and tricyclic pyrrole 58 was isolated in good yield. In contrast, upon
treatment of 54 and 55 with methanesulfonic acid at 45 °C, (C-C) cyclization
predominated to give pyrrolopyiridine 59 and, the previously described,
pyrroloazepine 22 respectively.
However, a complex mixture of products
resulted when acetal 56 was subjected to the same strongly acidic conditions
(Scheme 46).
0
0
Br_IT5H
TsOH
70%
H
N
Br\I
CH3SO3H
54
p
80%
NH
Br
longamide 57
59
0
T
NH
Br4
Br
Br
H
60
0
-
II
Br
N
Br
56
0
jj
TsOH
90%
CH3SO3H
55
27
H°
80%
22
Scheme 46
152
Treatment of longamide (57) with methanesulfonic acid led to formation of the
dehydration product pyrrolopyrazine 60 in nearly quantitative yield (Scheme
46).
The fused tricyclic structure 58 was determined on the basis of 1H-15N
HMIBC correlations. Figure VI.12 shows the key 'H-'5N correlations that
support the polycylic arrangement. Similarly, a series of 2D NMR experiments,
including, 1H-13C HSQC, 1H-'3C HIvIBC, 'H-'5N HMBC, helped to corroborate
carbinol 57 as the correct structure.
As shown in Figure VI.13, the C-N
connection in longamide (57) is supported by a 1H-'5N correlation between the
carbinol methine proton and the pyrrole nitrogen.
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4,5
4.0
3.5
3.0
2.5
2.0
1.5 ppiu
Figure VI.12 1H-'5N HIVIBC correlations observed for compound 58.
153
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
ppnt
Figure VI.13 1H-15N HN'lBC correlations observed for compound 57.
The heats of formation of all possible isomeric N-C and C-C cyclized
structures for the three acetals were calculated using Semi Empirical methods
(Figure VL14). In all cases, compounds with a C-C connection were found to
be the thermodynamic products over their corresponding N-C isomers by 10-18
kcal/mol.
154
Heat of formation
(kcal/mol)
-1 9.064
41 .252
-22.721
47.630
FgureVl.14
products."
B
OH
Br__JJH
Br
OH
Br
N
H
Heat of formation
(kcal/mol)
H
-29.581
0
Br_è]{
26.160
BriiiiH BrIIH
-36.007
BrH Br-H
29.605
Calculated heats of formation for isomeric N-C and C-C
It could be concluded from the experimental results that the ring size of
the product appears to be an important factor in the formation of the N-C bond.
While cyclization to form the pyrrolopyrazine longamide 57 occurs readily, the
corresponding seven membered ring closure is not observed. In related studies
involving the cyclization of amines with aldehydes and ketones to 7
9 member
rings, it was found that ring opening is favored under conditions that permit
equilibration between opened and closed forms.
During the progress of this work a communication by Al Mourabit and
coworkers reported a route identical to ours for the synthesis of longamide
5746
155
In addition, homolongamide
61,
a derivative of 57, was reported to arise by
intramolecular cyclization of aldehyde 27 in methanol at room temperature
(Scheme 47).
0
0
0
N)LN)
CH3OH
H
Br__T'H
Br
HO
Br
27
61
Scheme 47
Previous studies by our group revealed that aldehydes of this type are
readily solvated in protic
solvents.47
Close examination of the reported proton
chemical shifts for the carbinol hydrogens in 57 and homolongamide
61
revealed a difference of over 1 ppm. This, in conjunction with the splitting
pattern observed for this hydrogen in
61
(t vs. dd in 57), led us to believe that
the product from the reaction of 27 in methanol was in better agreement with an
acyclic structure. To further corroborate this conclusion, solvation of aldehyde
27 was monitored at room temperature in CD3OD. In this study it was found
that hemiacetal 27a formed readily upon dissolution (- 2 minutes) (Figure
VI.15).
In addition, an N-methylated derivative of 28, synthesized in
quantitative yield by treatment of the corresponding acetal with diazomethane
followed by acetal hydrolysis, was subjected to the same analysis (Figure
VI. 16).
0cD3
Ha9H
ON)Br
D3CO
H
D3CO>N
N
H
27b
27a
I
4.5
Br
Br
Br
5.0
H
Ha
4.0
I--.I-
3.5
3.0
2.5
2.0
Figure VI.15 Time dependent formation of hemiacetal 27a and acetal 27b in CD3OD at room
temperature as monitored by 'H NMR spectroscopy.
0
H
Me
00O3
Me
HaOH
0
Me
DC0
28b
28a
Br
Br
Br
t=Omin
IL
[ii3128a
III
II
.JL.
j
,.
t=llOmin j
III
I
a
t=24hrs
5.0
4.0
4.5
3.5
3.0
2.0
.2.5
28 b
4.5
4.0
3.5
3.0
2.5
Figure VI.16 Time dependent formation of hemiacetal 28a and acetal 28b in CD3OD at room
temperature as monitored by 1H NIIVIR spectroscopy.
2.0
158
The results obtained from the
NirviR
study confirmed that the
spectroscopic data for structure 61 is in better agreement with the hemiacetal of
aldehyde 27. This conclusion is supported by WVIBC correlations observed
between the methine hydrogen Ha and the deuteriated-methoxymethyl carbons
of the acetal moiety (Scheme 48).
Bryy0
Br
0
R
HO Ha
H
H
Br
0
reported (CD3OD): Ha = 64.57 (1 H, t, J = 5 Hz)
CD3OD
Br
Br(NO3 CD3OD
R
0
OH
Br
Br(NO3
R
CD3O
HajHMBC
R = H, Ha =64.42 (1 H, t, J = 5.8 Hz)
A = Me, Ha = 64.45 (1 H, t, J = 5.7 Hz)
A = H, Ha = 64.59 (1 H, t, J = 5.2 Hz)
R = Me, Ha = 64.57 (1 H, t, J = 5.2 Hz)
Br
0
HOHa
BrJNH
1H NMR (CD300): Ha = 65.76 (1 H, dd, J = 1.4, 2.9 Hz)
Scheme 48
In summary, it was found that altering the acidity of the reaction
conditions in the cyclization of pyrrolocarboxamidoacetals can lead to the
159
construction of two types of ring systems. In addition, cyclization was found to
be sensitive to ring size of the resulting product. Finally, on the basis of NIvIR
experiments, the reported structure for homolongamide 61 was found to be in
better agreement with hemiacetal 27a.
6.3.2
Intramolecular Cyclization of n-Activated Imidazolones
Weinreb's approach to the A-B ring system in agelastatin A relies on an
intramolecular Michael addition of the pyrrole nitrogen of compound 62 to give
a-amino ketone 63 (Scheme 49). Treatment of 63 with NBS converted the TMS
group to the desired bromine substituent. Addition of methyl isocyanate to the
resulting product accomplished the final D-ring annulation, affording racemic
agelastatin A (47).
TMS H
TMS
NHBoc Cs2CO3
NH
0
MeOH
61 %
NHBoc 1. NBS
B
NH
2.TMSI
47
3. MeNCO
0
63
62
Scheme 49
Our work on the synthesis of slagenins had shown that
beta-
functionalization of linear imidazolone 14 to build the tetrahydrofuro[2,3-
160
djimidazolidin-2-one moiety could be achieved via an intramolecular cyclization
of olefin 14 to oxazoline 15 in methanesulfonic acid.
H
MeSO3H
(90 %)
H
14R=H
Br
Br
slagenins
Scheme 50
Therefore, in contrast to Weinreb's approach, we envisioned the
functionalized pyrroloketopiperazine 51 to arise by a controlled cyclization of
activated linear imidazolone 14b (Scheme 51).
Br
R
H
14b(R=Br)
/
=<N J
L
H
NO
H
51
Scheme 51
However, initial results showed that formation of oxazoline 15b
prevailed under mildly acidic conditions. In this case, refluxing temperatures
were found to be necessary for cydlization to take place and product 15b was
161
isolated only in poor yields.
Similarly, palladium mediated intramolecular
amination of 14b using reported stoichiometric
products.
conditions48
favored oxazoline
In both cases, the low yield of isolated product was attributed to
concomitant hydrolysis of the oxazoline
ring49
carboxylic group (Scheme 52).
H
N
(
14b
________
N
H
and cleavage of the pyrrole
)N
15b
jNH
+
14b
35%
10-15%
A. TsOH, THF or CH2Cl2, sealed tube
B. 1- 3 equiv. PdCl2, Et3N, MeCN or THF, reflux
Scheme 52
Protection of the amide functionality with a non-sterically encumbered
group like SEMC15° was considered, in hopes that a protected aniide would
disfavor cyclization of 14b to the oxazoline ring. However, the preparation of
SEM protected precursors was hindered by the facile cleavage of this group
under the conditions needed for the synthesis and purification of imidazolones.
In light of this outcome, protection of the amide carbonyl oxygen of 14b as an
imino
ether
group
was
investigated
tetrafluoroborate (Meerwein's reagent).
instead
using
triethyloxonium
Interestingly, when olefin 14b was
treated with Meerwein's reagent under standard conditions5' and conditions
known to favor 0-alkylation in
DMF,52
alkylation of the pyrrole nitrogen
162
predominated. N-alkylation may proceed via an alkyl transfer process from the
imidate ether to the pyrrole group (Scheme 53).
Et3O BF4
14b
OH 1
RLN..Br
0
H
H
Br
64
Br
55%
Scheme 53
Our next approach to the pyrrolopyrazine connection centered around
the installation of a functional group at the beta site of the propylamine chain
which could be displaced in an
SN2
fashion by the pyrrole nitrogen.
The
synthesis of f3-chloro imidazolone 66 was investigated for this purpose.
Formation of 65 was achieved by reaction of olefin 8 in TFA with dilute HCI at
room temperature for 24 hrs. Acylation with 4,5-dibromo-2-(trichloroacetyl)pyrrole under standard conditions gave the desired product 66 in good yield.
163
H
N
H
<NJNH
N
H
TEA
J('NH2
O=::<
2%HCI
N
H
8
o
),
65
H
'DMF
j88%
c13c
\\.i(
Br
H
N
o=<
N
H
Br
66
Scheme 54
To our disappointment, treatment of this compound with acidic media
(TFA, MeSO3H) was found to give the corresponding oxazoline as the only
product.
Similarly, base catalyzed cyclization (Et3N, DBU, KOtBu) favored
oxazoline formation. In both cases, no trace of the desired piperazine was
observed (Scheme 55).
66
A. TEA, MeSO3H, (90 %)
B. Et3N, KOtBu, OBU, 50 °C, DMF, (40-50 %)
Scheme 55
The time dependent formation of oxazoline 15b from f-ch1oro
imidazolone 66 in CD3OD-d4 using K2CO3 is depicted in Figure VI.16.
T= 4h
Oxazoline
Oxazoline
Oxazoline
/
T
= 16 h
T
= 32 h
9.5
9.0
8.5
8,0
7.5
7.0
6.5
1N
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
ppm
Figure VI.17 Time dependant cyclization of t3-chloro amide 66 with K2CO3 at 50 °C to oxazoline 15b as
monitored by 1H NMR spectroscopy.
165
Elimination of the -chloro substituent was observed upon heating 66 in
MeOH. Interestingly, elimination of 66 in DMSO resulted in oxidation of the
imidazolone unit to give compound 67, which was isolated as part of a complex
mixture of products (Scheme 56). The chemical shifts observed in the 1H and
'3c
NMR spectra of 67 are consistent with the values expected for a hydantoin
unit and aj3-functionality.
MeOH
66
H
N
+
o=x:\
55°C
N
H
14b
Br
66
25%
52%
'DMSO
j 45°C
0H
<NN)Br
H
67
+ complex mixture
Br
43%
Scheme 56
In a final attempt to achieve nucleophilic addition of the pyrrole nitrogen
to an activated n-position, the synthesis of a n-halo ketone derivative of 14b was
studied. As previously described, ketone 13 can be readily synthesized from 12.
In contrast, a similar reaction of c-methoxy imidazolone 11 leads to an
intramolecular cyclization of the amide nitrogen yielding a bicyclic intermediate
as white crystals. This unstable product was found to react upon standing in
DMSO-d6 to give tetracyclic compound 68 (Scheme 57).
A cyclization
166
involving hydrolysis of the 4-bromine substituent of the pyrrole group has been
observed in our approach to the axinohydantoins (section 6.2.2 of this chapter).
0
OMe
oj'2
H
II
NCS
MeOH
13
N
H
OMe
OMe
H
0
H
Njt_Br NCSMeOH
N
H
11
H
N
0t
N
O
NI
Br
'.SO-d6
Scheme 57
The polycyclic framework of the unique imidazoline 68 was established
on the basis of 2D NMR experiments. Figure VI.18 shows key 1H-13C HMBC
correlations between the methine proton of the imidazoline ring and carbons
around each ring of the polycyclic system.
167
OMe
ppm
30
4°
so
60
70
80
90
100
110
120
130
140
i-so
160
170
10
9
8
7
6
5
4
3
2
ppm
Figure VI.18 'H-13C HMIBC spectrum of tetracyclic compound 68.
Synthesis of 3-bromo ketone 69 was achieved by addition of bromine to
ketone 13 in methanesulfonic acid. The desired product was isolated in good
yield after crystallization from methanol. However, acylation of 69 with 4,5dibromo-2-(trichloroacetyl)-pyrrole was hindered by the strong propensity of the
free amine to close to the corresponding aziridine 70 under mildly basic
conditions. Addition of excess NaOAc to a solution of 69 in DMF at 70°C gave
oxazoline 71 in low yield as the only coupled product (15 %). Similarly,
treatment of ketone 72 with bromine also gave oxazoline 71 as the major
product (Scheme 58).
0
0
H
Br2
13
0
MeSO3H
H
fNH2
II
o=<j94H
A
H 69 MeSO3H
H
70
+ 71
15%
70%
j85%
H
N
H
N
H
II
Br
72
Br
o=<
'
MeSO3H
Br2
N
H
r
65%
Br
A. 5 eq. 4,5-dibromo-2-(trichloroacetyl)-pyrrole, DMF, 10 eq.NaOAc, 60 °C
B. 4,5-dibromo-2-(trichloroacetyl)-pyrrole, DMF, 90 °C
Scheme 58
From the results presented above, it can be suggested that cyclization of
n-activated linear imidazolones via an
SN2
path favors formation of the
oxazoline over the pyrazine connection. Products containing a C-C connection
were not observed under these conditions. In Weinreb' s synthesis of agelastatin
A, nucleophilic addition of the pyrrole nitrogen in precursor 56 is believed to be
favored by two primary factors, the first one being the geometry of the system.
In this case, the double bond is locked in an E-geometry in close reach to the
pyrrole nitrogen functionality, where orbital alignment for the Michael addition
appears to be optimal.
Secondly, the formation of oxazoline byproducts is
possibly reversible and could lead to regeneration of the c-unsaturated
functionality.
'p
TMS
TMS H
NHBoc Cs2CO3
MeOH
61 %
0
0
62
63
Scheme 59
Based on these observations and the results obtained from our
preliminary
studies
on
the
N-C
and
C-C
cyclizations
of
pyrrolocarboxamidoacetals, we believe that a linear imidazolone bearing a
keto group may be an ideal substrate for the synthesis of a pyrazinone ring.
0
H
H
H
J'NH2
H
Br
H
74
73
In preliminary studies, the synthesis of precursor 73 was attempted by
oxidation of the corresponding n-alcohol. However, reagents like Dess-Martin
periodinane,53' PDC, and Jones'
reagent53"
gave a complex mixture of products
170
where functionalization of the imidazolone ring appeared to predominate.
Similarly, a number of conditions using a sulfur trioxide/pyridine complex54 and
triethylamine in DMSO gave decomposition with partial recovery of the starting
material.55
Finally, the synthesis of 74 was attempted via elimination of the o-
substituent in compound 75 (Scheme 60). However, treatment of this
imidazolone
with
diastereomeric dimers.
provided a complex
conditions
acidic
mixture of
Attempts to install an a-substituent in ester 76 to
perform a similar elimination, while avoiding dimerization, led to cleavage of
the pyrrole ester and formation of hydantoin 77.
OMe
8
F
H
40%
H
H2
I-i
NCS
MeOH
Br
77 60%
76 HCI
Scheme 60
In conclusion, methods for the synthesis of f-halogenated imidazolones
were described and their preferred mode of cyclization was studied. The results
show that acid and base mediated
SN2
type intramolecular cyclizations favor
171
formation of an oxazoline connection. However, based on our preliminary
studies on the ring forming reactions of pyrrolocarboxamidoacetals, we propose
that N-C connectivity could be achieved by cyclization from a 3-keto
carboxamide 73. Elimination of the resulting carbinol hydroxyl group with
methanesulfonic acid, as previously described for longamide 57, could give the
desired unsaturated pyrrolopyrazine (Scheme 61).
H
Br
H
73
Br"
--
-----
H
0
Scheme 61
51
172
6.4. REFERENCES
a) Chistophersen, C. in "The Alkaloids: Chemistry and Pharmacology",
A. Brossi Ed.; Academic Press: New York 1985, Vol. 24, Chapter 2. b)
Kobayashi, J.; Ishibashi, M.
in "The Alkaloids: Chemistry and
Pharmacology," A. Brossi; Cordell, G. A. Ed., Academic Press: New York
1992, Vol. 41, Chapter 2.
2.
Faulkner, D. J. Nat. Prod. Rep. 2001, 18, 1.
3.
Lindel, T.; Hoffmann, H.; Hochgurtel, M.
in "Bioorganic Chemitry:
Highlights and New Aspects," Diederichsen, U.; Lindhorst, T. K.;
Westermann, B.; Wessjohann, L. A. Ed., Wiley-VCH, Germany 1999,
Chapter 1.
4.
Tsuda, M.; Uemoto, H.; Kobayashi, J. Tetrahedron Lett. 1999, 40, 5709.
5.
Sharma, U. M.; Buyer, J. S.; Pomerantz, M. W. J. Chem. Soc. Chem.
Comm. 1980,435.
6.
Cimino, U.; De Rosa, S.; De Stefano, S.; Mazzarella, L.; Puliti, R.;
Sodano, G. Tetrahedron Lett. 1982, 23, 767.
7.
Supriyono, A.; Wray, S. V.; Witte, L.; Muller, W. E. G.; van Soest, R.;
Sumaryono, W.; Proksch P. Z. J'Jaturforsch. 1995, 50c, 669.
8.
This alkaloid was named spongiacidin A by Kobayashi and coworkers.
However, the name (E)-3-bromohymenialdisine is used in this manuscript
for simplicity.
9.
Inaba, K.; Sato, H.; Tsuda, M.; Kobayashi, J. J. Nat. Prod. 1998, 61, 693.
10.
Williams, D. H.; Faulkner, D. J. Nat. Prod. Lett. 1996, 9, 57.
11.
Pettit, G. R.; Herald, Ch. L.; Leet, J. E.; Gupta, R.; Schaufelberger, D. E.;
Bates, R. B.; Clewlow, P. J.; Doubek, D. L.; Manfredi, K. P.; RUtzler, K.;
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Chem. 1990, 68, 1621.
12.
Patil, A. D.; Freyer, A. J.; Kiflmer, L.; Hofmann, G.; Johnson, R. K. Nat.
Prod. Lett. 1997,9, 201.
173
13.
a) Breton, J. J.; Chabot-Fletcher, M. J. J. Phar,nacol. Exp. Ther. 1997,
282, 459. b) Roshak, A.; Jackson, J. R.; Chabot-Fletcher, M.; Marshall, L.
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SmithKline Beecham Corporation, Medicament, U.S. Patent 5,565,448,
1996.
14.
a) OsteoArthritis Sciences, Inc.
The Reagents of the University of
California. Use of Debromohymenialdisine for Treating Osteoarthritis. U.
S. Patent 5,591,740, 1997. b) Vaslos, G.; DiBenedetto, P.; IL-i-Induced
Gene Expression In Chondrocytes in Vitro. Presented at The American
College of Rheumatology, 63d Annual Scientific Meeting, Boston, MA,
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McPherson, J.; Oldham, C.; Palmer, J.; Tubo, R.; Wickham, A.; Vasios,
G.; Z-Debromohymenialdisine Shows Disease-Modifying Activity in
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15.
Meijer, L.; Thunnissen, A-MWH; White, A. W.; Gamier, M.; Nikolic, M.;
Tsai, L-H; Walter, J.; Cleverley, K. E.; Salinas, P. C.; Wu, Y-Z.; Biernat,
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D'Ambrosio, M.; Guerriero, A., Debitus, C., Ribes, 0.; Pusset, J.; Leroy,
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33, 4491. b) Jaafar, I.; Francis, G.; Danion-Bougot, R.; Danion, D.
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Chavignon, 0.; Teulade, J. C.; Roche, D.; Madesclaire, M.; Blache, Y.;
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35.
Godovikova, T. I., Rakitin, Khmel'nitskii, L.I.
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36.
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Stien, D.; Anderson, G. T.; Chase, Ch. E., Koh, Y-h.; Weinreb, S. M. J.
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a) Cook, G. A. Enamines: Synthesis, Structure and Reactions, 2' ed.;
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176
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a) Robins, M. J.; Samano, V. J. Org. Chem. 1990,
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1966, 2, 51.
55,
5180. b) Corey, E.
177
CHAPTER VII. EXPERIMENTAL SECTION
General. Starting materials were obtained from common commercial suppliers
and used without further purification. Unless otherwise stated, concentration
under reduced pressure refers to a rotatory evaporator at water aspirator pressure.
One and 2D nuclear magnetic resonance (NMR) spectra were acquired using a
Bruker AM-400, Bruker AC-300 or a Bruker DRX 600 spectrometer.
Exchangeable protons were identified by acquisition of a 1H spectrum after
addition of a drop of D20 to the NIVIR tube containing a solution of the sample
in DMSO-d6. For 13C spectra acquired using D20 as a solvent methanol was
used as a standard. Infrared (IR) spectra were obtained using a Nicolet 5DXB
FT-JR spectrometer.
Low-resolution FAB mass spectra (LRMS) and high-
resolution FBA mass spectra (FIRMS) were obtained on a Kratos MS 50 TC.
H2N-NJ
I>=o
H
1. Na(Hg)
H2N
CO2Me
6
2.1.leq
KOCN
0<
H
H
5
(3-Aminopropyl)-1H-imidazolidin-2-one (5).
7 (5-10%)
A solution of L-ornithine
methyl ester dihydrochioride (22 g, 0.1 mol) in 300 mL H20 was prepared and
cooled between 0 and 5 °C. The pH was adjusted between 1.2 and 1.5 by
addition of a 15 % solution of HCI. Over the course of 1 hr. 5 % Na(Hg) (545 g,
178
1.1 mol) was added in I g pieces maintaining the pH and temperature constant.
After bubbling ceased the solution was decanted, potassium cyanate was added
(9.4 g, 0.11 mol), bringing the pH to 3.4, and the solution was refluxed for 3 hrs.
The solvent was removed in vacuo and residual water was azeotroped with
ethanol (100 %) to give a light yellow residue. Ethanol was added to the residue
and NaCI was filtered off. Addition of a solution of ethanolldichloromethane
(2/1) resulted in precipitation of inorganic impurities which were filtered off.
Evaporation of the solvent gave a precipitate which was washed with a
minimum amount of ethanol to provide the desired product as a colorless solid
(17.7 g, 40 %). Chromatography of the ethanol wash (CH2C12/MeOH(NIH3),
6:4) provided an additional 20 % of the product 5 and 5 % of a dimenc
byproduct 7: 5 HCl 'H NMR (DMSO-d6) ö 1.77 (q, 2H, J = 7.4 Hz), 2.31 (t,
2H, J = 7.4 Hz), 2.70 (t, 2H, J = 7.4 Hz), 6.00 (s, 1H), 8.25 (bs, 3H, xch D20),
9.49 (bs, 1H, xch D20), 9.83 (bs, 1H, xch D20); '3C NIVIR (DMSO-d6)
55.8, 38.3, 104.7, 121.1, 155.3; IR(KBr)
)max
22.4,
3194, 3044, 1669 cm1; HRMS,
calcd for C6H12N30 (M + 1)142.09804, found 142.09777. 72 HC1 'H NMR
(DMSO-d6) 8 1.40-1.68 (m, 6H), 2.32-2.34 (m, 2H), 2.65-2.68 (m, 4H), 3.43 (d,
1H, J = 8.7 Hz), 4.12 (d, 1H, J = 8.7 Hz), 5.80 (bs, 6H, xch D20), 6.47 (bs, 1H,
xch D20), 6.58 (bs, 1H, xch D20), 9.80 (bs, 2H, xch D20); '3C NMR (DMSOd6) 621.2, 25.8, 29.3, 32.1, 39.4, 40.2, 53.3, 57.7, 1116.0, 120.0, 163.1; IR(KBr)
i
3294, 1651, 1625 cm1; HRMS, calcd for C,2H22N602 (M + 1) 282.18042,
found 282.18096.
179
H
H
N
DMF
0
H
H
5
N
Br
H
CI
10
Br
4,5-Dibromo-1H-pyrrole-2-carboxylic
acid
[3-(2-oxo-2,3-dihydro-1H-
imidazol-4-yl)-propyl]-amide (10). To a solution of (3-Aminopropyl)-1Himidazolidin-2-one free base 5 (0.35 g, 2.5 mmol) in 2 mL of DMF was added
4,5-dibromopyrrol-2-yl tnchoromethyl ketone (1.2 g, 3.1 mmol). The reaction
was stirred at room temperature under Ar for 16 h. Ether was added to the
reaction mixture (100 mL x 3) and decanted and the resulting residue was
triturated with methanol. The product was filtered and recrystallized from a 1:1
mixture of methanol and water to afford the desire coupled product as a
colorless solid (0.88 g, 90 %): 5 1H NMR (DMSO-d6) 6 1.60-1.63 (m, 2H), 2.22
(t, 2H, J = 7.3 Hz), 3.15 (t, 2H, J = 6.2 Hz), 5.95 (s, 1H), 6.90 (s, 1H), 8.10 (t,
1H, xch D20, J = 5.5 Hz), 9.38 (s, 1H, xch D20), 8.70 (s, 1H, xch D20), 12.64
(bs, 111, xch D20); 13C NN'W (DMSO-d6) 622.6, 27.6, 37.9, 97.7, 103.7, 104.4,
112.4, 121.3, 128.2, 154.9, 158.9; IR(KBr)
1;
Umax
3245, 3141, 1687, 1622 cm
HRMS, calcd for C11H13N4O279Br2 (M + 1) 390.94052, found 390.94126.
H
H
OMe
2
MeOH
o:jc1NCS
H
N
H
5
12
(1-Methoxy-3-aminopropyl)-1H-imidazolidin-2-one (12).
A solution of
imidazolone 5 (5 g, 29 mmol) in 125 mL CH3OH was prepared and NCS (3.9 g,
29 nm-iol) was added at room temperature. The reaction was stirred for 3 hrs
and then
loaded directly onto a silica gel column for purification
(CH2C12/MeOH(NH3), 6:4).
Attempts to remove the solvent under reduced
pressure prior to purification lead the formation of a complex mixture of dimeric
structures with none of the desired product remaining.
Direct purification
yielded the desired product as a light yellow oil (3.4 g, 70 %): 12 'H NMR
(DMSO-d6) ö 1.77 (q, 2H, J = 7.4 Hz), 2.31 (t, 211, J = 7.4 Hz), 2.70 (t, 2H, J =
7.4 Hz), 6.00 (s, 1H), 8.25 (bs, 3H, xch D20), 9.49 (bs, 1H, xch D20), 9.83 (bs,
111, xch D20); 13C NTVIR (DMSO-d6)
IR(KBr) u
22.4, 55.8, 38.3, 104.7, 121.1, 155.3;
3194, 3044, 1669 cm1; HRMS, calcd for C6H12N30 (M + 1)
142.09804, found 142.09777.
1131
N'NH NCS
2 _____
H
N
H
MeOH
5
H
0
N
H
13
(1-Oxo-3-aminopropyl)-1H-imidazolidin-2-one
(13).
A
solution
of
imidazolone 5 (2.5 g, 14 mmol) in 75 mL CH3OH was prepared and NCS (3.9 g,
29 mmol) was added at room temperature. The reaction was stirred for 1 hr.
Crystallization of the product was achieved upon concentration of the reaction at
room temperature to give the desired product as colorless crystals (1.9 g, 70 %):
13 H NMR (DMSO-d6) 62.79-3.14 (m, 4H), 7.67 (s, 1H), 7.71 (bs, 3H, xch
D20), 10.61 (bs, 1H, xch D20), 10.92 (bs, 1H, xch D20); 13C NIVIR (DMSO-d6)
633.8, 34.7, 122.2, 123.2, 154.5, 185.5; IR(KBr)
1;
Umax
3176, 1781, 1651 cm
HRMS, calcd for C6H9N302 (M + 1) 155.06944, found 155.06948.
H
OMe
I
=<NJLNH
N
H
12
xylene
reflux
H
N0
H
5-(3-Amino-propyl)-infidazolidine-2,4-dione
13b
(13b).
A
solution
of
imidazolone 12 (0.5 g, 2.9 mmol) in 10 mL CH3OH was prepared and NCS
(0.39 g, 2.9 mmol) was added at room temperature. The reaction was stirred for
1 hr, xylene was added and the reaction was refluxed in xylene for 30 mm.
Purification by chromatography (CH2C12IMeOH(NH3), 1:1) of a dark residue
gave the oxidized product 13 as a yellow oil (0.12 g, 25 %): 13 1H NtVIR
182
(CD3OD-d4) ö 1.69-1.92 (m, 4H), 2.95 (t, 2H, J = 6.7 Hz), 4.14 (t, 1H, J = 4.9
Hz);
13c
NMZR (CD3OD-d4) ö 23.9, 29.5, 40.6, 59.2, 160.0, 177.8; IR(KBr)
1.)max
3210, 1675, 1596 cm'; HRMS, calcd for C61111N302 (M) 157.08513, found
157.08533.
H2N
NNH
H
OMe
N
H
12
TEA
H
N
H
O<f'NH2
N
H
NH2
+
N
H
8
jj
9
3-Amino-1-(imidazolidin-2-one-4-yl)prop-1-ene (8). A solution of a-methoxy
imidazolone 12 (1 g, 5.9 mmol) in 23 mL TFA was prepared and stirred
overnight at room temperature. The solvent was evaporated without heat under
reduced pressure and the resulting residue was loaded to a silica gel column for
purification (CH2Cl2IMeOH(NH3), 7:3) to give the desired product 8 as a light
yellow oil (0.37 g, 35 %) and dimeric byproduct 9 (0.26 g, 33 %): 8 'H NIVIR
(DMSO-d6)
3.46 (t, 2H, J = 5.8 Hz), 5.71 (dt, 1H, J = 15.8, 5.8 Hz), 6.25 (d,
1H, J = 15.8 Hz), 6.49 (s, 1H), 7.99 (bs, 3H, xch D20), 10.04 (bs, 1H, xch D20),
10.49 (bs, 1H, xch D20); 13C NIVIR (DMSO-d6) ö41.4, 111.3, 116.8, 121.2,
124.3, 155.6; IR(KBr) u,, 3143, 3021, 1665 cm'; HRMS, calcd for C6H9N30
(M + 1)139.07456, found 139.0748 1. 9 'H NIvIR (D20)
1.61-1.79 (m, 1H),
1.81-1.87 (m, 1H), 1.88-2.01 (m, 1H), 2.35-2.39 (m, 1H), 2.50-2.62 (m, 2H),
3.89 (t, 1H, J = 5.4 Hz), 5.86 (dt, 1H, J = 15.9, 6.1 Hz), 6.26 (s, 1H), 6.26 (d,
183
1H, J = 15.9 HZ); '3C NIVIR (D20)
30.0, 33.8, 38.7, 42.7, 106.6, 115.5, 119.4,
120.2, 124.0, 127.8, 155.4, 155.5; IR(KBr)
1;
u1
3183, 3022, 1671, 1684 cm
HRMS, calcd for C,2H19N602 (M + 1) 279.15631, found 279.15695.
NNH
H
H
N
H
8
N
DMF
0
H
)LN)
H
Ci
Br
14
Br
4-Bromo-1H-pyrrole-2-carboxylic acid [3-(2-oxo-2,3-dihydro-1H-imidazol4-yl)-allyI]-amide (14).
To a solution of 3-Amino-1.-(imidazolidin-2-one-4-
yl)prop-1-ene (8) free base (0.35 g, 2.5 mmol) in 2 mL of DMF was added 5-
bromopyrrol-2-yl trichoromethyl ketone (0.8 g, 3.0 mmol). The reaction was
stined at room temperature under Ar for 16 h. Ether was added to the reaction
mixture (100 mL x 3) and decanted and the resulting residue was purified by
chromatography (CH2Cl2:MeOH, 9:1) to give the coupled product 14 as
colorless crystals (0.7 g, 90 %): 14 'H NMR (DMSO-d6)
3.90 (t, 2H, J
5.2
Hz), 5.77 (dt, 1H, J = 5.2, 15.9 Hz), 6.01 (d, 1H, J = 15.9 Hz), 6.04 (s, 1H), 6.34
(s, 1H), 6.88 (s, 1H), 8.28 (t, 1H, xch D20, J = 5.6 Hz), 9.81 (s, 1H, xch D20),
10.17 (s, IH, xch D20), 11.79 (bs, 1H, xch D20); '3C NMR (DMSO-d6)
40.90,
95.8, 109.2, 112.4, 119.4, 121.9, 122.1, 122.8, 127.7, 155.7, 160.2; IR(KBr) u
3286, 3145, 1684, 1615 cm1;HRMS, calcd for C,,H,2N4O279Br (M + 1)
311.0136, found 311.01419.
°H
H
H
N
MeSO3H
O(
j(N
N
H
sNH
Br
H
14
15
Br
4-[2-(4-Bromo-1H-pyrrole-2-carbonyl)-4,5-dihydro-oxazol-5-ylmethyl]-1,3-
dihydro-imidazol-2-one (15). A solution of olefin 14 (0.3 g, 0.96 mmol) in 3
mL CH3SO3H was prepared and stined at room temperature overnight. Ether
was added (3 X 100 mL) to remove the methansulfonic acid and the residue was
purified by chromatography (CH2C12IMeOH 8:2) to give the oxazoline 15 as
colorless crystals (0.27 g, 90 % yield): 15 1H NMR (DMSO-d6) 6 2.48
2.61
(m, 2H), 3.56 (dd, 1H, J = 6.8, 14.5 Hz), 3.93 (dd, 1H, J = 9.4, 14.5 Hz), 4.73 -
4.81 (m, 1H), 6.06 (s, 1H), 6.57 (s, 1H), 7.02 (s, 1H), 9.49 (s, 1H, exch D20),
9.71 (s, 1H, exch D20), 11.97 (bs, 1H, exch D20); 13C NMR (DMSO-d6) 6 31.8,
59.3, 78.3, 96.2, 106.6, 114.0, 117.9, 121.6, 122.8, 155.7, 156.8; IR(KBr) u
3143, 1683, 1665 cm1; HRMS, calcd for C11H11O2N479Br (M + 1) 310.00661,
found 3 10.00654.
H
N
2
o=<
5%HCI(aq)
N
H
H
4-Bromo-1H-pyrrole-2-carboxylic
acid
1-aminomethyl-2-(2-oxo-2,3-
dihydro-1H-imidazol-4-yI)-ethyl ester (16). A suspension of oxazoline 15
(0.150 g, 0.48 mmol) in 15 mL of aqueous HC1 (5 %) was stirred at 75 °C until
all oxazoline had reacted. The solvent was evaporated to give ester 17 as a light
tan solid in quantitative yield: 16 1H NMR (DMSO-d6) 62.66 (d, 2H, J = 5.8
Hz), 3.00
3.08 (m, 2H), 5.15 (d, 1H, J = 5.1 Hz), 6.05 (s, 1H), 6.94 (s, 111),
7.21 (s, 1H), 8.12 (bs, 3H, exch D20), 9.55 (s, 1H, exch D20), 9.83 (bs, 111,
exch D20), 12.33 (bs, 1H, exch D20); 13C NMR (DMSO-d6) 628.6, 41.0, 49.4,
70.1, 96.8, 107.3, 117.2, 117.9, 123.3, 124.7, 155.4, 159.3; IR(KBr) u
3220,
3105, 1713, 1665 cm1; HRMS, calcd for C11H14O3N479Br (M + 1) 329.02542,
found 329.02493.
H
1NKOH
16
N----,-.
O=:<
N
H
,IIINH
ti
I
OH
Br
4
Br
4-Bromo-1H-pyrrole-2-carboxylic acid
[2-hydroxy-3-(2-oxo-2,3-dihydro-
1H-imidazol-4-yI)-propyl]-amide (4). A solution of ester 16 (0.100 g, 0.30
mmol) in 15 mL of water was neutralized with 1 N KOH. Neutralization is
accompanied by a change in color from light purple to light brown. The solvent
was evaporated and the desired alcohol was extracted three times with ethanol
from the inorganic material. Evaporation of the solvent gave the alcohol as
colorless crystals (85 mg, 85 % from oxazoline 15): 4 H NMR (DMSO-d6)
2.23 2.39 (m, 2H), 3.08-3.30 (m, 2H), 3.70-3.75 (m, 1H), 4.90 (d, 1H, J =
5.2 Hz, exch with D20), 6.00(s, 1H), 6.88 (s, 1ff), 6.97 (s, 1H), 8.05 (t, 1H, J =
5.6 Hz, exch D20), 9.40 (s, 111, exch D20), 9.59 (bs, 1H, exch D20), 11.81 (bs,
1H, exch D20); 13C NIvIR (DMSO-d6) 6 32.0, 45.5, 69.11, 95.8, 105.8, 112.55,
119.8, 122.0, 127.7, 155.6, 160.7; IR(KBr)
Omax
3176, 1679 cm'; KRMS, calcd
for C11H14O3N479Br (M + 1) 329.0243 1, found 329.02493.
187
H
H
MeOH
NCS, rt
H
11
Br
4
H
N
o==(
N
H
Br
Slagenins B (2) and C (3). To a solution of alcohol 4 (0.075 g, 0.23 mmol) in
10 mL of methanol was added NCS (0.030 g, 0.23 mmol) and the reaction was
stirred at room temperature for 1 hr. The reaction produced cleanly a 1:1 mixture
of slagenins B and C. The mixture of diastereomers was subjected to
chromatography (CH2C12IMeOH (NH3), 9.5:0.5) to give pure slagenin B (2) (35
mg, 42 %) and slagenin C (3) (36 mg, 43%) as colorless crystals. Slagenin B:
1H NMIR (DMSO-d6) 6 1.76 (t, 1H, J = 11.5), 2.14 (dd, 1H, J = 3.9, 11.5), 3.13
(s, 3H), 3.40 (m, 2H), 4.00 (m, 111), 5.17 (s, 1H), 6.87 (s, 111), 6.96 (s, 1H), 7.46
(s, 1H, exch D20), 7.51 (s, IH, exch D20), 8.22 (t, 1H, J = 5.7 Hz, exch D20),
11.81 (bs, 1H, exch D20); '3C NIVIR (DMSO-d6) 641.3, 41.4, 50.40, 76.0, 88.4,
94.9, 97.9, 111.6, 121.3, 126.6, 159.7, 159.8; IR(KBr)
Umax
3435, 1695, 1635,
1205 cm1; HRFABMS C12H16O4N479Br (M + 1) 359.0352. Slagenin C: 'H
NMR (DMSO-d6) 6 1.89 (dd, 111, J = 6.5, 12.9), 2.27 (dd, 1H, J = 6.7, 12.9),
3.11 (s, 3H), 3.40 (m, 2H), 4.13 (m, 1H), 5.00 (s, 1H), 6.86 (s, 1H), 6.96 (s, 1H),
7.65 (s, 1H, exch D20), 7.69 (s, 1H, exch D20), 8.15 (t, 111, J = 5.5 Hz, exch
D20), 11.80 (bs, 1H, exch D20); 13C NMR (DMSO-d6) 840.9,42.8,49.8,76.0,
89.4, 94.9, 97.2, 111.7, 121.2, 126.7, 159.4, 159.7; IR(KBr) D
3435, 1695,
1635, 1205 cn-f'; HRFABMS C12H16O4N479Br (M + 1) 359.0355.
H OCH3
HH
NH
2
r
Br
H2O,H
80°C
I I
H
N
o=(
N
H
Slagenin A (1). A 1:1 mixture of 2 and 3 was heated to 80 °C in 2 % HC1 for
3.5 hrs to give slagenin A (1) as colorless crystals (60 mg, 85 %):
1H NIVIR
(DMSO-d6) 6 1.74 (t, ff1, J = 11.6), 2.07 (dd, 1H, J = 3.6, 11.6), 3.34 (m, 1H),
3.38 (m, 1H), 4.00 (m, 1H), 4.94 (s, 1H), 6.23 (s, 1H), 6.87 (s, 1H), 6.96 (s, 1H),
7.25 (s, 1H, exch D20), 7.28 (s, 1H, exch D20), 8.19 (t, 1H, J = 5.6 Hz, exch
D20), 11.76 (bs, 1H, exch D20); 13C NIVIR (DMSO-d6) 641.6, 43.1, 76.2, 91.9,
93.3, 94.9, 121.3, 126.7, 159.7, 159.7; IR(KBr) t
HRFABMS C12H13O4N479Br (M + 1) 345.0206.
3430, 1685
cm1;
HQR
Oz<J
H
H
)i..NN
THF:H20 1:5
1 R=H
NCS,
2 R = CH3
Br
H
4
H
N
o=<
N
H
Alternative Oxidative Cydization to Slagenins A (1), B (2) and C (3): To a
solution of alcohol 4 (0.075 g, 0.23 mmol) in 10 mL of TBF:H20 (5:1) was
added NCS (0.030 g, 0.23 mmol). The reaction was stined at room temperature
for 1.5 hrs. Two compounds were spotted by TLC in a 1:1 ratio, however, upon
treatment of the mixture with methanol one of the two compounds reacted
rapidly to give a mixture of slagenins B (2) and C (3).
Separation by
chromatography (CH2Cl2IMeOH (NH3), 9.5:0.5) gave slagenin A (34 mg, 43%)
and a 1:1 mixture of slagenins B and C as colorless crystals (33 mg, 45 %).
190
H2N
H2N
N
H
N
Br(
H
2eq. Br2
)_NH
F:i
NaOAc,
AcOH
Br?iJ'')
35
21
(Z)-3-Bromohymenialdisine
(35).
To
a
stirred
solution
of
(±)-
hymeninCH3SO3H (21) (7.5 g, 15 mmol) and sodium acetate (6.5 g, 75 mmol)
in 240 mL acetic acid was added bromine (1.6 mL, 30 mmol) at room
temperature. The reaction was stirred for 1 h, concentrated, and azeotroped with
hot ethanol, decolorized with activated charcoal, and filtered. Concentration of
the filtrate afforded a solid which was rinsed with a minimum amount of water
to remove any remaining inorganic salts. The resulting material was dried under
vacuum to afford (Z)-3-Bromohymenialdisine as a colorless solid (5.13 g, 85
%): 35 H NMR (DMSO-d6)
3.15 (bs, 4H), 6.58 (bs, 1H, xch D20), 7.88 (bs,
2H, xch D20), 8.80 (bs, 1H, xch D20), 13.09 (bs, 1H, xch D20); 13C NMR
(DMSO-d6) S 33.7, 38.6, 98.1, 106.4, 114.1, 123.3, 126.2, 130.2, 163.8, 166.9,
176.7; 35HCl 'H NMR (DMSO-d6) 5 3.25 (bs, 4H), 8.07 (bs, 1H, xch D20),
8.50 (bs, 1H, xch D20), 9.30 (bs, 2H, xch D20), 10.92 (bs, IH, xch D20), 13.4
(bs, 1H, xch D20); '3C NMR (DMSO-d6) 5 35.2, 38.6, 98.5, 107.4, 120.7, 123.7,
125.9, 127.3, 154.1, 162.5, 163.3; IR(KBr)
Umax
3273, 1749, 1706, 1641, 1282
cm'; LIHRMS, calcd for C,1H,0N5O279Br2 (M + 1) 401.9202, found 401.9201.
191
H2N
N
HrL>°
Pd/C
20
35
(Z)-Debromohymenialdisine
(20).
A
mixture
of
(Z)-3-
bromohymenialdisineHCI (35) (3.2 g, 8.0 mmol) in 200 mL methanol
containing sodium acetate (6.5 g, 75 mmol) in 240 mL acetic acid was added
bromine (3.2 g, 40 mmol) and 10 % Pd/C (1.0 g) was placed under a balloon of
hydrogen. After 10 hrs the reaction mixture was filtered through celite and the
resulting filtrate was concentrated and azeotroped with ethanol several times to
afford a yellow solid. The solid was dissolved in hot methanol, decolonzed with
activated charcoal, and filtered.
Upon concentration of the filtrate, (Z)-
DBIfMeOH (20) crystallized from solution as light yellow crystals (1.6 g, 74
%): 20HCl 1H NMR (DMSO-d6)
3.30 (m, 4H), 6.60 (t, 1H, J = 2.6 Hz), 7.10
(t, 1H, J = 2.6 Hz), 8.08 (t, 1H, J = 4.4 Hz), 8.80 (bs, 1H, xch D20), 9.20 (bs,
1H, xch D20), 11.30 (bs, 1H, xch D20), 12.14 (bs, 111, xch D20); 13C NMR
(DMSO-d6) 631.4, 39.2, 109.6, 119.6, 120.0, 122.8, 126.8, 130.2, 154.2, 162.9,
163.3; IR(KBr)
C11H13N502
Umax
3288, 2473, 1614, 1415, 1123 cm1; LIHRMS, calcd for
(M + 1) 246.0991, found 246.0986.
192
H
O=<NJNH2
N
H
0
OMe
H
DMF
0
H
H
5
CI3C
Br
4,5-Dibromo-1H-pyrrole-2-carboxylic
llaR=R=H
llbR=H,R=Br
llcR=R=Br
acid
acid
propyl]-amide (1 ib).
R
[3-methoxy-3-(2-oxo-2,3-
dihydro- 1H-imidazol-4-yl)-propyl]-amide (1 ic).
carboxylic
H
I
4-Bromo-1H-pyrrole-2-
[3-methoxy-3-(2-oxo-2,3-dihydro-1H-imidazol-4-yI)-
1H-pyrrole-2-carboxylic acid [3-methoxy-3-(2-oxo-
2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (ha). Representative example:
To a solution of imidazolone 6 (0.10 g, 0.58 mmol) in 2 mL of DMF was added
4,5-dibromopyrrol-2-yl trichoromethyl ketone (0.28 g, 0.76 mmol).
The
reaction was stirred at room temperature under Ar for 16 h. Ether was added to
the reaction mixture (100 mL x 3) and decanted and the resulting residue was
triturated with methanol. The resulting precipitate was filtered and washed with
1:1 mixture of methanol and water to afford the coupled product 7 as a colorless
solid (0.20 g, 80 %): lic 'H NMR (DMSO-d6)
1.74-2.00 (m, 2H), 3.08 (s,
3H), 3.13-3.18 (m, 2H), 3.88 (t, 1H, J= 6.7 Hz), 6.27 (s, 1H), 6.92 (s, 111), 8.07
(t, 1H, xch D20, J = 5.4 Hz), 9.62 (s, 1H, xch D20), 9.92 (s, 1H, xch D20), 12.6
(bs, 1H, xch D20); '3C NMR (DMSO-d6) 6 34.3, 36.4, 55.9, 73.4, 98.5, 105.4,
108.4, 113.3, 121.5, 129.2, 155.9, 159.8; IR(KBr)
Umax
3300, 3149, 1696, 1627,
1571, 1526 cm'; HRMS, calcd for C12H15N40379Br2 (M + 1) 419.94384, found
419.94326. lib 'H NMR (DMSO-d6) 6 1.73-1.98 (m, 2H), 3.09 (s, 3H), 3.16-
193
3.19 (m, 2H), 3.86 (t, 1H, J = 6.6 Hz), 6.26 (s, 1H), 6.81 (s, 1H), 6.94 (s, 1H),
8.03 (t, 1H, xch D20, J = 5.0 Hz), 9.62 (s, IH, xch D20), 9.89 (s, 1H, xch D20),
11.77 (bs, 1H, xch D20); 13C NMR (DMSO-d6) 634.4, 36.4, 55.9, 95.7, 108.4,
112.2, 121.6, 121.9, 127.8, 156.0, 160.5; IR(KBr)
Umax
3162, 3042, 1672, 1632,
1585, 1526 cm'; HRMS, calcd for C12H15N4O379Br (M + 1) 342.03317, found
342.03275. ha 1H NMR (DM80-cl6) 6 1.73-1.99 (m, 2H), 3.09 (s, 3H), 3.183.20 (m, 2H), 3.85 (t, 1H, J = 7.0 Hz), 6.05 (s, 1H), 6.26 (s, 1H), 6.71 (s, 1H),
6.81 (s, IH), 7.92 (t, 1H, xch D20, J = 5.1 Hz), 9.63 (s, IIH, xch D20), 9.90 (s,
1H, xch D20), 11.36 (bs, 1H, xch D20); 13C NMR (DMSO-d6) 634.5, 36.3,
55.9, 73.5, 108.3, 109.4, 110.6, 121.7, 122.0, 127.1, 156.0, 161.6; IR(KBr) Umax
3195, 1670, 1628 cm1; HRMS, calcd for C12H16N403 (M + 1) 264.12224,
found 264.1225 1.
H
OMe
=<j)
i
H
0
NBr TFA
(
Br
lic
OH
H
H
H
HN-4
Br
39
3-Bromo-iO-(2-oxo-2,3-dihydro- 1H-imidazol-4-yl)-i,7-diaza-spiro[4.5}dec-
3-ene-2,6-dione (39). A solution of carboxamide lie (0.10 g, 0.24 mmol) in 4
mL of CF3COOH was stirred at room temperature for id. The reaction mixture
was concentrated at reduced pressure. Trituration of the resulting residue gave a
precipitate which was filtered to afford the cyclic product 8 as tan crystals (60
194
mg, 70 %): 'H NMR (DMSO-d6)
1.88-1.92 (m, 1H), 2.26-2.45 (m, 1H), 3.07-
3.19 (m, 2H), 7.54 (s, 1H), 8.03 (s, 1H, xch D20), 8.46 (s, 1H, xch D20), 9.52
(s, 1H, xch D20), 9.62 (s, 111, xch D20); 13C NMR (DMSO-d6) ö 25.2, 39.0,
40.1, 70.4, 106.7, 120.0, 120.3, 145.6, 155.2, 166.9, 168.8; IR(KBr)
3073,
1693,
1674,
1661,
1488,
Umax
3217,
1343 cm'; HRMS, calcd for
1462,
CH12N4O379Br (M + 1) 327.00934, found 327.00928.
0
OMe
H
N
0
L,NH
__
TFA
o=<ll
N
H
H
N
HN
llbR=Br
llcR=H
N
H
=<
x:i-NH
0
38aR=H
38bR=Br
4OaR=H
4ObR=Br
3-Bromo-4-(2-oxo-2,3-dihydro- 1H-imidazol-4-yl)-4,5,6,7-tetrahydro-1H-
pyrrolo[2,3-c}azepin-8-one (38b); 4-(2-oxo-2,3-dihydro-1H-imidazol-4-yl)4,5,6,7-tetrahydro- 1H-pyrrolo[2,3-c]azepin-8-one (38a); 8-Bromo-6-(2-oxo2,3-dihydro-1H-imidazol-4-yI)-3,1O-diaza-bicyclo[5.2.1}deca-1(9),7-dien-2-
one
(40b).
6-(2-oxo-2,3-dihydro-1H-imidazol-4-yl)-3,1O-diaza-bicyclo-
[5.2.1]deca-1 (9),7-dien-2-one (40a).
Representative example: A solution of
carboxamide lib (0.10 g, 0.29 mmol) in 4 mL of CF3COOH was stirred at room
temperature for id. The reaction mixture was concentrated at reduced pressure,
and the resulting residue was triturated with methanol. The precipitate was
filtered to afford 40b (32 mg, 35 %) as tan crystals: (40b) 'H NIMR (DMSO-d6)
195
6 2.05-2.29 (m, 2H), 3.01-3.25 (m, 2H), 3.86 (bs, 1H), 6.04 (s, 1H), 6.77 (s, 1H),
8.06 (s, 1H, xch D20), 9.63 (s, 1H, xch D20), 9.99 (s, 1H, xch D20), 11.40 (s,
1H, xch D20); 13C NMR (DMSO-d6) 632.8, 32.8, 37.8, 95.3, 105.7, 112.4,
122.3, 126.5, 133.2, 155.6, 160.6; IR(KBr)
Umax
3420, 3230, 1685, 1532, 1461,
1325 cm'; LRMS, ln/z 311.1 (M + 1), 313.1 {i:1]; HRMS, calcd for
C,,H12N4O379Br (M
+ 1) 311.01381, found 311.1436.
The filtrate was
chromatographed (9:1 CH2C12:MeOH) to afford azepinone 38b (0.03 g, 35 %)
and oxazoline 15 (6 mg, 7 %) as colorless crystals: 38b 'H NMR (DMSO-d6)
6 1.80-1.90 (m, 111), 2.19-2.26 (m, 1H), 3.04-3.16 (m, 2H), 3.82 (bs, 1H), 5.38
(s, 1H), 7.01 (d, 1H, J = 3.13 Hz), 7.75 (bs, 1H, xch D20), 9.40 (s, 1H, xch
D20), 9.85 (s, 1H, xch D20), 11.6 (s, 1H, xch D20); 13C NIVIR (DMSO-d6)
6 31.5, 34.8, 37.3, 98.9, 107.4, 122.6, 123.4, 124.3, 125.1, 155.7, 162.7;
IR(KBr)
Umax
3273, 3209, 1697, 1635, 1481, 1441 cm'; HRMS, calcd for
C11H,2N4O379Br (M + 1) 311.01391, found 311.01436. (40a) 'H NIVIR (DMSOd6)
6 1.97-2. 14 (bm, 2H), 3.11-3.34 (bm, 2H), 3.73 (bs, 1H), 5.91 (s, 1H), 5.98
(s, 1H), 6.63 (s, 1H), 7.86 (s, 1H, xch D20), 9.49 (s, 1H, xch D20), 9.90 (s, 1H,
xch D20), 11.08 (s, 1H, xch D20); '3C NMR (DMSO-d6) 633.8, 33.8, 37.8,
104.9, 106.8, 110.8, 124.3, 126.1, 137.4, 155.7, 161.6; IR(KBr)
1682, 1541 cm'; LRMS,
C,,H,2N403
Umax
3245,
m/z 232.1 (M). 38a 'H NMR (DMSO-
d6) 6 1.91-1.98 (m, 1H), 2.00-2.13 (m, 1H), 3.04-3.18 (m, 2H), 3.86 (t, 1H, J =
6.1 Hz), 5.70 (s, 1H), 5.91 (t, 1H, J= 2.55 Hz), 6.79 (t, 1H, J 2.55 Hz), 7.58 (t,
196
1H, xch D20, J= 4.6 Hz), 9.40 (s, 1H, xch D20), 9.77 (s, 1H, xch D20), 11.09
(s, 1H, xch D20); 13C NMR (DMSO-d6)
33.4, 36.4, 39.2, 105.7, 111.2, 122.2,
123.4, 126.1, 126.7, 155.7, 164.1 ; IR(KBr)
Umax
3263, 1686, 1643 cm'; HRMS,
calcd for C11H13N403 (M + 1) 233.10385, found 233.10386.
0
0
HN4
HN4
L,NH
[,,NH
Br
Br
2eq Br2
NaOAc
AcOH
0
38b
NH
0
42aR=H
42b R = Br
3-Bromo-4-(2-oxo-2,3-dihydro-1H-imidazol-4-yl)-6,7-dihydro-1H-
pyrrolo[2,3-c]azepin-8-one
(42a);
2,3-Dibromo-4-(2-oxo-2,3-dihydro-1H-
imidazol-4-yl)-6,7-dihydro-1H-pyrrolo[2,3-c]azepin-8-one
(42b).
To
a
solution of azepinone 38b (65 mg, 0.21 mmol) in 15 mL of acetic acid was
added sodium acetate (172 mg, 2.1 mmol) and bromine (22 pL, 0.42 mmol).
The reaction was stirred at room temperature for 18 h. The reaction was
concentrated and dried under vacuum. The resulting residue was purified by
chromatography (9:1 CH2C12:MeOH) to afford 42a (20 mg, 35 %), 48b (15 mg,
20 %), (Z)-3-bromo axinohydantoin (Z-44) (4 mg, 5 %), and (E)-3bromoaxinohydantoin (Z-44) (4 mg, 4 %) as a colorless crystals: 42a 1H NMR
(CD3OD-d4) ö 3.54 (d, 2H, J = 7.1 Hz), 6.11 (t, 1H, J = 7.1 Hz), 6.33 (s, 1H),
197
7.14 (s, 1H); 13C NMR (CD3OD) ö 38.2, 96.2, 109.0, 121.9, 122.2, 122.4, 123.8,
126.9, 128.7, 155.2, 164.3; IR(KBr)
Umax
3404, 3170, 1717, 1653, 1541, 1457
cm1; HRMS, calcd for C11H10N4O279Br (M +1) 308.99087, found 308.99074.
42b
1HNMR (CD3OD) (S 3.52 (d, 2H, J_ 7.2 Hz), 6.11 (t, 1H, J= 7.2 Hz), 6.31
(s, 1H); 13C NMR (CD3OD) (S 38.0, 98.8, 108.4, 108.9, 121.9, 122.9, 123.2,
128.1, 128.5, 155.6, 163.5; IR(KBr)
Umax
3415, 3216, 1683, 1635, 1558, 1457
cm1; calcd for C11H9N4O279Br2 (M+1) 386.90150, found 386.90143.
H0
NH
HN
HN
Br
Br
3 eq Br2
/
I
NH
NaOAc
AcOH
Br
iflrNH
0
38b
Z-44
(Z)-3-Bromo axinohydantoin
(Z-44);
E-44
(E)-3-Bromo axinohydantoin
(E-44).
To a solution of azepinone 38b (65 mg, 0.21 mmol) in 15 mL of acetic acid was
added sodium acetate (172 mg, 2.1 mmol) and bromine (32 j.tL, 0.63 mmol).
The reaction was stirred at room temperature for 16 h. The reaction was
concentrated and dried under vacuum. The resulting residue was purified by
chromatography (9:1 CH2C12:MeOH) to afford (Z)-3-bromo axinohydantoin (Z44)
(36 mg, 45 %) and (E)-3-bromo axinohydantoin (E-44) (25 mg, 30 %) as a
colorless crystals.
Z-44
1H NMR (DMSO-d6) (S 3.12-3.18 (m, 4H), 7.88 (t, 1H,
xch D20, J= 4.7 Hz), 9.53 (s, 1H, xch D20), 11.01 (s, 1H, xch D20), 13.04 (bs,
1H, xch D20); 13C NIvIR (DMSO-d6) 535.8, 40.2, 99.8, 107.5, 118.1, 122.8,
126.9, 127.5, 154.9, 164.6, 165.3; IR(KBr)
Umax
3414, 3151, 1754, 1721, 1614,
1462, 1383, 1366 cm1; HRMS, calcd for C11H9N4O379Br2 (M + 1) 402.90485,
found 402.90414; E-44 1H NMIR (DMSO-d6) 52.49-2.77 (m, 2H) 3.12-3.18 (m,
2H), 7.88 (t, 1H, xch D20, J = 4.8 IE{z), 10.08 (s, 1H, xch D20), 10.90 (s, ff1,
xch D20), 12.90 (bs, 1H, xch D20); 13C NIvIR (DMSO-d6) S 38.3, 39.2, 102.9,
106.0, 115.9, 122.0, 126.9, 128.9, 155.2, 162.9, 165.2; IR (film) 3196, 1733,
1717, 1652, 1472, 1419, 1394 cm1; HRMS, calcd for C11H9N4O379Br2 (M + 1)
402.90507, found 402.90414.
0
NH
H2 Pd/C
NH
yNH
0
0
Z44
17
(Z)-Debromoaxinohydantoin (17). A solution of (Z)-3-bromo axinohydantoin
144
(R1
= R2 = Br) (25 mg, 0.06 mmol), sodium acetate (16 mg, 0.18 mmol)
and 10 % Pd/C (6.0 mg) in 20 mL of methanol was stirred under 1 atm of
hydrogen at room temperature for 3 h. The reaction mixture was concentrated
under reduced pressure and dried under vacuum. Crystallization from MeOH
and water afforded the product 17 (11 mg, 80 %) as a light yellow solid: 1H
NIVIR (DMSO-d6) 8 3.14-3.24 (m, 4H), 6.53 (bs, 1H), 6.97 (bs, 1H), 7.88 (bs,
199
1H, xch D20), 9.42 (s, 1H, xch D20), 11.03 (s, 1H, xch D20), 11.78 (bs, 1H,
xch D20); 3C NMIR (DMSO-d6)
30.7, 40.1, 109.9, 121.6, 122.1, 122.6, 122.7,
125.5, 154.7, 163.0, 165.8; IR(film) Umax 3425, 1686, 1633, 1212, 1042
cm1;
HRMS, calcd for C11H10N403 (M) 246.0729, found 246.0753.
110
H2PdIC
I::,
3h
N
E-44
NH
45a R = H
45b B = Br
(E)-4-Bromo-axinohydantoin (45b), (E)-debromoaxinohydantoin (45a). A
solution of E-44 (30 mg, 0.07 mmol), sodium acetate (20 mg, 0.21 mmol) and
10 % PdJC (8.0 mg) in 20 mL of methanol was stirred under 1 atm of hydrogen
at room temperature for 3 h. The reaction mixture was concentrated under
reduced pressure and dried under vacuum. Purification by HPLC using a YMC
ODS-AQ column (250x10 nm. 5pm) (1:1 H20:MeOH) afforded product 45b (8
mg, 45 %) and 45a (7 mg, 40 %) as light yellow solids: 45b 'H NMR (DMSOd6)
2.49-2.57 (m, 2H), 3.13-3.17 (m, 2H), 7.00 (s, 1H), 7.81 (t, 1H, J = 5.2
Hz), 10.01 (bs, xch D20), 10.81 (bs, 1H, xch D20), 12.01 (s, 1H, xch D20); 13C
NMR (DMSO-d6); 13C NMR (DMSO-d6) ö 38.2, 39.4, 100.3, 116.5, 120.8,
122.2, 125.7, 128.3, 155.2, 162.9, 166.0; HRMS, calcd for C11H10N4O379Br
200
(M) 323.8145, found 323.8137. 45a 'H NIMIR (DMSO-d6) 62.65-2.69 (m, 2H),
3.14-3.20 (m, 2H), 6.68 (t, IH, J = 2.57 Hz), 6.81 (t, 1H, J = 2.57 Hz), 7.79 (t,
1H, xch D20, J = 4.9 Hz), 9.74 (bs, 1H, xch D20), 10.82 (s, 1H, xch D20),
11.55 (s, 1H, xch D20); 13C NIvIR (DM80-cl6) 637.7, 39.7, 113.4, 120.8, 120.8,
123.0, 125.6, 125.8, 154.8, 163.7, 165.1; HRMS, calcd for
C11H10N403 (Mt)
HRMS, calcd for C11H,0N403 (M) 246.0731, found 246.0744.
h
iLo
O(H
o
H2 Pd/C
NH
8h
i:i
i::i
)T'
0
0
E-44
46
S-(8-Oxo-1,4,5,6,7,8-hexahydro-pyrrolo[2,3-c]azepjn-4-yI)-imidazoljdjne-
2,4-dione 12. A solution of (E)-3-bromo axinohydantoin E-44 (25 mg, 0.06
mmol), sodium acetate (16 mg, 0.18 mmol) and 10 % Pd/C (6.0 mg) in 20 mL of
methanol was stirred under 1 atm of hydrogen at room temperature for 8 h to
yield a 4:1 mixture of diastereomers. The reaction mixture was concentrated
under reduced pressure
and dried
under vacuum.
Purification by
chromatography (9.5:0.5 CH2C12:MeOH) afforded 46a (3 mg, 16 %) and 46b
(10 mg
,
66 %) as a colorless solids. 46a: 1H NMR (CD3OD) 6 1.80-2.11 (m,
2H), 3.21-3.47 (m, 2H), 3.52-3.63 (m, 1ff), 4.66 (d, 1H, J = 3.13 Hz), 6.30 (d,
201
1H, J = 2.74 Hz), 6.99 (d, 1H, J = 2.74 Hz), 'H NIvIR (DMSO-d6) 6 1.62-1.85
(m, 211), 3.04, 3.29 (m, 211), 4.53 (s, 111), 6.18 (s, 1H), 6.83 (s, 111), 7.60 (bs,
111, xch D20), 7.88 (bs, 1H, xch D20), 10.61 (bs, 1H, xch D20), 11.15 (bs, 1H,
xch D20) ; '3C NMR (DMSO-d6) 6 29.9, 40.0, 41.1, 63.9, 110.5, 123.6, 124.8,
126.2, 159.4, 165.6, 176.7; JR (film) 3111, 1734, 1713, 1631 cm1; HRMS, calcd
46b: 'H NMR
for C11H,3N403 (M + 1) 249.09903, found 249.09877.
(CD3OD) 6 1.94-2.33 (m, 2H), 3.22-3.46 (m, 2H), 3.63-3.71 (m, 1H), 4.33 (d,
1H, J = 2.35 Hz), 6.07 (d, 1H, J = 2.54 Hz), 6.91 (d, IH, J = 2.54 Hz), 'H NMR
(DMSO-d6) 6 1.79-2.11 (m, 2H), 3.07, 3.30 (m, 211), 4.21 (s, 1H), 5.84 (s, 1H),
6.79 (s, 1H), 7.64 (bs, 1H, xch D20), 7.89 (bs, 1H, xch D20), 10.64 (bs, 1H, xch
D20), 11.14 (bs, 1H, xch D20); '3C NIvJIR (DMSO-d6) 33.4, 40.2, 41.3, 63.3,
109.0, 122.3, 123.7, 124.4, 158.4, 164.4, 176.6; JR (film) 3196, 1733, 1717,
cm'; HRMS, calcd for
1652
C,,H,3N403
(M + 1) 249.09903, found
249.09877.
0
HN
Br
p-
H
N
,':.
Br
-\II
MeSO3H
I)
N"-NH
H
ii
0
N
H
55°C
Br
-I
22
42c
2-Bromo-4-(2-oxo-2,3-dihydro-1H-imidazol-4-yl)-6,7-dihydro-1H-
pyrrolo[2,3-c}azepin-8-one (42c). To a solution of azepinone 22 (0.10 g, 0.33
202
mmol) in 2 mL of MeSO3H was added imidazolone (32 mg, 0.40 mmol). The
reaction was stined at 55 °C for 4d. Ether was added to the reaction mixture
(100 mL x 3) and decanted.
The resulting residue was purified by
chromatography (9:1 CH2C12:MeOH) to afford the protodebrominated product
42c as colorless crystals (35 mg, 35 %): 42c 1H NIvIR (DMSO-d6) 6 3.33 (t, 2H,
J = 6.65 Hz), 5.99 (s, 1H, J = 6.65 Hz), 6.34 (s, 1H), 6.40 (s, 1H), 6.81 (s, 1H),
7.85 (t, 1H, xch D20, J = 4.8 Hz), 10.02 (s, IH, xch D20), 10.23 (s, 111, xch
D20), 12.63 (bs, 111, xch D20); 13C NMR (DMSO-d6) 638.4, 104.7, 109.5,
110.9, 118.4, 121.9, 123.8, 128.7, 128.9, 155.4, 163.3; TR(KBr) Umax 3168, 3025,
1684, 1619, 1484, 1456 cm1; HRMS, calcd for C11H9N4O279Br (M) 307.99038,
found 307.99089.
0
HN
Br
-
BrjI'
NH
TFA
+
Br
38c
2,3-Dibromo-4-(2-oxo-2,3-dihydro-1H-imidazol-4-yl)-4,5,6,7-tetrahydro-
lJi-pyrrolo[2,3-c]azepin-8-one (38c). To a solution of azepinone 22 (0.10 g,
0.33 mmol) in 2 mL of CF300H was added imidazolone (32 mg, 0.40 mmol).
The reaction was stirred at room temperature for 6d. Evaporation of the solvent
followed by purification of the residue by chromatography (9:1; CH2Cl2:MeOH)
203
affored 38c (39 mg, 37 %) as colorless crystals: 'H NIVIR (DMSO-d6)
1.80-
1.86 (m, 1H), 2.14-2.18 (m, 1H), 3.06-3.11 (m, 2H), 3.82 (t, 1H, J = 3.7 Hz),
5.40 (s, IH), 7.82 (bs, 1H, xch D20), 9.43 (s, 1H, xch D20), 9.85 (s, 1H, xch
D20), 12.47 (s, 1H, xch D20); 13C NMR (DMSO-d6)
31.7, 35.7, 37.2, 101.7,
106.7, 107.4, 124.6, 124.9, 125.9, 155.6, 162.0; IR(KBr)
Umax
3397, 3198, 1683,
1635, 1476, 1436 cm1; FIRMS, calcd for C11H11N4O279Br2 (M + 1) 388.92487,
found 388.92531.
0
Br
Br(Q70%
H0
TsOH
NH
Br
Br
OH
Iongamide57
54
Longamide
(57).
A
mixture
of
4,5-dibromo-N-[2-(1,3-dioxolan-
2y1)methyl]pyrrole-2-carboxaniide 54 (0.5 g, 1.4 mmol) and p-toluenesulfonic
acid monohydrate (0.13 g, 0.7 mmol) in 12 mL of acetone/water (1:1) was
refluxed for 16 h. After cooling the reaction mixture was extracted with ethyl
acetate (3 x 10 mL) followed by neutralization with a saturated solution of
sodium carbonate (20 mL).
The organic layer was dried over MgSO4 and
filtered. Removal of the solvent in vacuo provided 57 as white crystals (0.3 g,
70 %): 1H NMR (CD3OD)
3.59 (dd, IH, J = 1.4, 13.8 Hz), 3.81 (dd, 1H J
2.9, 13.8 Hz), 5.76 (dd, 1H, J = 1.4, 2.9 Hz), 6.94 (s, 1H); '3C NIVIR (CD3OD) 3
46.9, 74.2, 100.8, 106.7, 114.7, 126.5, 158.1. 1H NItvIR (DMSO-d6) ö 3.37 (dd,
204
1H, J = 5.5, 13.7 Hz), 3.7 (dd, 1H, J = 2.4, 13.7 Hz), 5.6 (d, 1H, J = 6.5 Hz),
6.97 (d, 1H, J = 6.7 Hz, exch. D20), 7.87 (d, 1H, J = 5.5 Hz, exch. D20); IR
(film) 3194, 3067, 1667, 1425, 1084 cm; HRMS calcd for C7H679Br2N2O2 (M)
307.87960, found 307.87937.
Br
0
oj
Br1
H
TsOH
75%
B(
\
Br
56
58
2,3-Dibromo-4a,5,6,7-tetrahydro-dipyrrolo[1,2-a ; 1 ',2'-cjimidazol-9-one
(58). A mixture of 4,5-Dibromo-N-(4,4-diethoxybutyl)pyrrole-2-carboxamide
56 (17.0 g, 41 mmol) with p-toluenesulfonic acid (4.0 g, 21 mmol) in (1:1)
acetone-water (300 mL) was refluxed for 1 8h. After cooling a white precipitate
was collected to give the desired product as colorless crystals (5.2 g, 40 %). The
rest of the organic material was extracted with dichioromethane (3 x 200 mL)
and neutralized with a saturated solution of sodium carbonate. The organic
layer was dried over MgSO4 and filtered. Removal of the solvent in vacuo and
purification by chromatography (MeOH(NH3):CH2C12, 5:95) gave an additional
20 % (3.6 g) of the desired product: 58 1H NMR (CDC13)
1.62 (dtt, 1H, J
8.39, 12.12, 15.0), 2.25 (m, 2H), 2.53 (m, 1H), 3.32 (ddd, 1H, J = 3.89, 8.39,
12.12), 3.76 (dt, 1H, J = 8.13, 11.37 Hz), 5.48 (dd, 1H, J = 5.8, 8.21 Hz);
I3C
NMR (CDCI3) ö 26.3, 30.1, 42.9, 76.4, 101.1, 104.0, 108.8, 128.9, 163.0.
1H
205
NMR (DMSO-d6) 6 1.56 (m, 1H), 2.19 (m, 2H), 2.41 (m, 1H), 3.57 (dt, 1H, J
8.15, 9.9 Hz), 5.76 (dd, lB. J = 5.9, 8.2 Hz); IR (film) 1701, 1358, 1323
cni1;
HRMS calcd for C9H879Br2N2O (M) 3 17.90034, found 3 17.90026.
Br
0
O-\
Br_______
CH3SO3H
80%
H
H
II
,NNH
Brj1J
Br
54
59
2,3-Dibromo-5,6-dihydro-1H-pyrroIo[2,3-c]azepjn7-one. A solution of 4,5dibromo-N- [2-( 1 ,3-dioxolan-2y1)methyl]pyrrole-2-carboxamide
(0.1
g,
0.3
mmol) in methanesulfonic acid (1 mL) was stirred at 45 °C for 3d. The reaction
mixture was diluted with ethyl acetate and neutralized with a saturated solution
of sodium carbonate. The organic layer was dried over MgSO4 and filtered.
Removal of the solvent in vacuo followed by purification by chromatography
(100 % ethyl acetate) gave the desired cyclic product 59 as colorless crystals (70
mg, 80 %): 'H NMR (DMSO-d6) 66.36 (d, 1H, J = 6.9 Hz), 7.00 (t, 1H, J = 6.9
Hz), 11.18 (bs, 1H, exch. D20), 13.26 (bs, 1H, exch. D20); 13C NIVIR (DMSOd6)
6 92.5, 97.7, 112.1, 124.4, 126.8, 130.2, 153.6; IR (film) 3131, 2988, 1677,
1658, 1402, 773 cm1; HRMS calcd for C7H579Br2N2O (M}fl 291.87502, found
291.87481.
206
0
0
CH3SO3H
Br_-<Iiiui
99 %
I)NH
Br,!)
B!
longamide 57
60
Dehydrologamide (60). A solution of longamide (57) (0.1 g, 0;32 mmol) in
methansulfonic acid (1 mL) was stirred at 45 °C for 3d. The organic material
was extracted with ethyl acetate and neutralized with a saturated solution of
sodium carbonate (10 mL).
The organic layer was dried over MgSO4 and
filtered. Removal of the solvent in vacuo gave the desired product as pale pink
crystals in quantitative yield: 60 'H NMR (DMSO-d6) 8 6.73 (d, 1H, J
Hz), 7.19 (s, 1H), 7.26 (d, 1H, J= 5.7 Hz), 10.5 (bs, 1H, xch.
D20);
5.7
'3C NMR
(DMSO-d6) 6 102.5, 103.6, 106.5, 111.8, 117.4, 126.4, 154.9; IR (film) 2985,
1681 cm'; HRMS calcd for C7H579Br2N2O (M) 290.73598, found 290.73486.
BrN3 Bry0
Br
Br
MeO
55
H
28
4,5-Dibromo-N-methyl-(3-oxopropyl)pyrrole-2-carboxamide 28. Acetal 55
(0.2 g, 0.3 mmol) was dissolved in 10 mL of dichioromethane and treated with
excess diazomethane. After 30 mm evaporation of the solvent and excess of
diazomethane with nitrogen gave the desired N-methyl dioxolane quantitatively
as white crystals. 'H NMR ((CD3)2C0) 6 1.90 (m, 2H), 3.47 (m, 2H), 3.84 (m,
207
2H), 3.95 (m, 211), 3.97 (s, 311), 4.91 (t, 1H, J = 4.65 Hz), 6.86 (s, 1H), 7.37 (bs,
1H, exch. D20); 13C NMR ((CD3)2C0) 6 34.4, 35.5, 35.9, 65.6, 98.1, 103.7,
111.1, 114.4, 129.6, 160.9. A mixture of the N-methyl acetal (0.2 g, 0.3 mmol)
and p-toluenesulfonic acid monohydrate (28 mg, 0.15 mmol) was dissolved in
(1:1) acetone:water (20 mL) and refluxed for 16 hrs. The organic material was
extracted with dichioromethane (3 x 10 mL) and neutralized with a saturated
solution of sodium carbonate. The organic layer was dried over MgSO4 and
filtered. Removal of the solvent in vacuo gave the desired N-methyl aldehyde
28 quantitatively as an opaque oil. 28 111
NIvIR
((CD3)2C0) 6 2.73 (dt, 2H, J =
1.4, 6.2 Hz), 3.65 (q, 2H, J = 6.1 Hz), 3.95 (s, 311), 6.87 (s, 1H), 7.54 (bs, 1H,
exch. D20), 9.77 (d, 111, J = 1.4 Hz); 13C NIvIR
((CD3)2C0)
6 34.1, 36.1, 44.5,
98.2, 111.5, 114.7, 129.1, 161.2, 202.0.
Solvation of aldehyde 27. Approximately 5 mg of aldehyde 27 were placed in
a 5 mm NIvIR tube and dissolved with approximately 0.6 mL of CD3OD.
1H
NMR spectra were acquired periodically.
Solvation of methyl aldehyde 28. Approximately 10 mg of aldehyde 28 were
placed in a 5 mm NIMR tube and dissolved in approximately 0.75 mL of
CD3OD. 1H NMR spectra were acquired periodically.
0
H
H
+
Et30 BF4
H
N
o=KI
H
Br
14b
N
H
64
Br
4,5-Dibromo-1-ethyl-1H-pyrrole-2-carboxylic acid [3-(2-oxo-2,3-dihydro1H-imidazol-4-yl)-allyl]-amide 64. To solution of 14b (0.075 g, 0.19 mmol) in
dry DMF (2 mL) was added KOtBu (26 mg, 0.23 mmol) and triethyloxonium
tetrafluoroborate (0.42 mmol, 423 j.tL (of 1M solution in CH2Cl2)). The solution
was stirred at room temperature under argon for 3.5 hrs. Ethyl ether was added
and decanted (3 X 20 mL) to remove excess DMF. The resulting residue was
purified by chromatography (9:1 CH2C12:MeOH) to afford the 64 as a colorless
solid (44mg, 55 %): 1H NIVIR (DMSO-d6) 6 1.20 (t, 3H, J = 6.9 Hz), 3.86 (t,
2H, J = 5.0 Hz), 4.43 (q, 2H, J = 6.9 Hz), 5.74 (dt, 1H, J
5.6, 15.9 Hz), 6.00
(d, 111, J = 15.9 Hz), 6.34 (s, 1H), 7.04 (s, 1H), 8.39 (t, 1H, J = 5.9 Hz), 9.83
(bs, 1H, xch D20), 10.17 (bs, 1H, xch D20); 3C NIVIR (DMSO-d6) 6 15.4, 40.8,
43.4, 98.2, 108.5, 109.9, 114.9, 118.7, 122.7, 127.2, 122.8, 155.1, 161.2;
IR(KBr) u
[1:2:1].
3195, 1691, 1635, cm1; LRMS, m/z 418.9 (M4+1), 416.9, 420.9
209
H
NJ'-NH
N
H
H
<NJ(NH
TFA
2%HCI
75%
8
H
65
4-(3-Amino-2-chloro-propyl)-1,3-dihydro-imidazol-2-one (65). To a solution
of olefin 8 HC1 (0.2 g, 1.3 mmol) in 12 mL of TFA was added HCI(2%) and the
reaction was stirred overnight at room temperature. The solvent was evaporated
without heat under reduced pressure and the resulting residue was loaded to a
silica gel column for purification (CH2C12IMeOH(NH3), 8:2) to give the desired
product 65 as a light yellow oil (0.15 g, 75 %): 1H NIvIR (DMSO-d6) ö 2.662.85 (m, 2H), 3.02 (dd, 1H, J = 10.1, 13.7 Hz), 3.24 (dd, 1H, J = 3.3, 13.7 Hz),
4.30-4.48 (m, 2H), 6.11 (s, 111), 8.14 (bs, 3H, xch D20), 9.61 (bs, 1H, xch D20),
9.80 (bs, 1H, xch D20); 13C NMR (DMSO-d6)
32.8, 39.8, 45.0, 58.3, 107.5,
117.5, 155.5; HRMS, calcd for C6H11N3O35C1 (M + 1) 176.05906, found
176.05922.
H
H
OjH'J
H
0
65
N
DMF
NH2
H
)L1.._Br
H
Br
66
Br
4,5-Dibromo-1H-pyrrole-2-carboxylic acid [2-chloro-3-(2-oxo-2,3-dihydro1H-imidazol-4-yl)-propyl]-amide 66. To a solution of (3-Aminopropyl)-1Himidazolidin-2-one free base 65 (0.1 g, 0.56 mmol) in 2 mL of DIvIF was added
210
4,5-dibromopyrrol-2-yl trichoromethyl ketone (0.25 g, 0.68 mmol).
The
reaction was stirred at room temperature under Ar for 16 h. Ether was added to
the reaction mixture (100 mL x 3) and decanted. The resulting residue was
purified by chromatography (CH2CJ2/MeOH; 9:1) to give 66 as colorless
crystals (0.21 g, 88%). 66 1H NIVIR (DMSO-d6)
2.56-262 (m, 1H), 2.71-2.81
(m, 1H), 3.3 8-3.56 (m, 2H), 4.22-4.28 (m, 1H), 6.11 (s, 1H), 6.96 (d, 1H, J = 2.7
Hz), 8.39 (t, 1H, xch D20, J = 5.9 Hz), 9.57 (bs, 111, xch D20), 9.81 (bs, 1H,
xch D20), 12.73 (bs, 1H, xch D20);
13C NIvIR
(DMSO.-d6)
32.9, 45.4, 60.7,
98.8, 105.8, 107.1, 113.9, 118.4, 128.6, 155.6, 1591.9; IR(KBr) u
1627 cm1; HRMS, calcd for
C11H12N4O279Br2
3109, 1678,
(M + 1) 424.90155, found
424.90193.
0H
H
H
DMSO
H
Br
66
=<NN)LBr
45°C
Br
67
4,5-Dibromo-1H-pyrrole-2-carboxylic acid [3-(2,5-dioxo-imidazolidin-4-yl)allyl]-amide. A solution of 66 (0.075 g, 0.17 mmol) in lmL DMSO was stirred
at 45 °C for 45 hrs. Ethyl ether was added to the reaction mixture (25 mL x 3)
and decanted.
The resulting residue was purified by chromatography
(CH2C12IMeOH; 8:2) and 67 was obtained as a colorless crystal (30mg, 43 %).
67 'H NMR (DMSO-d6)
3.84 (bd, 2H, J = 5.5 Hz), 4.61 (d, 1H, J = 5.5 Hz),
5.54 (dd, 1H, J = 5.5, 15.7 Hz), 5.78 (dt, 1H, J = 5.5, 15.7 Hz), 6.93 (d, 1H, J =
211
2.5, 15.9 Hz), 8.11 (bs, 1H, xch D20), 8.35 (t, 1H, xch D20, J = 5.7 Hz), 10.65
(bs, 1H, xch D20), 12.67 (bs, 1H, xch D20); '3C NIVIR (DMSO-d6) ö 49.5, 60.0,
98.7, 105.5, 113.5, 125.7, 128.8, 131.5, 158.0, 159.9, 174.8; 1R(KBr) u
3245,
1681, 1629cm'; LRMS,m/z406.9(M-i-1),408.9,404.9[1:2:1].
OMe
11
NOS
MeOH
I
DMSO-d6
I H
N
I
MeO
NH
B(L<I
L
BrJ
Imidazoline 68. A solution of imidazolone 11 (50 mg, 0.12 mmol
CH3OH was prepared and NCS (15 mg, 0.12 mmol) was added at room
temperature. The reaction was stirred for 1 hr. An intermediate proposed to be
the bicyclic
adduct in brackets was filtered as
a
colorless crystal.
Approximately 6 hrs after dilution in DMSO the tetracyclic imidazoline 68 had
been produced quantitatively (28 mg, 71 %):
Bicyclic adduct: 1H NIVIR
(DMSO-d6) 6 1.80-1.93 (m, 2H), 3.20 (s, 3H), 3.29 (s, 3H), 3.60-3.69 (m, 2H),
3.97 (t, 1H, J 4.7 Hz), 4.90 (s, 1H), 6.75 (s, 1H), 6.85 (bs, 111, xch D20), 7.56
(bs, 1H, xch D20), 12.69 (bs, 1H, xch D20); LRMS, mlz 450.9 (M + 1), 448.9,
452.9 1:2:1]. 68 1H NMR (DMSO-d6) 6 2.01-2.39 (m, 2H), 3.10-3.50 (m, 2H),
3.28 (s, 3H), 3.99 (q, 1H, J= 7.8, 10.4 Hz), 4.49 (s, 1H), 7.10 (bs, IH, xch D20),
7.58 (s, 1H), 7.78 (bs, 1H, xch D20), 9.41 (bs, 1H, xch D20); 13C NMR
212
(DMSO-d6) ö 29.5, 40.2, 57.4, 58.0, 81.0, 83.0, 120.0, 146.8, 160.9, 168.5,
169.4; HRMS, calcd for C13H13N4O479Br (M) 355.91456, found 355.91398.
0
HJ
N
N
H
H
NH2
Br2
MeSO3H
13
0
0
N'---O=<jj
H
II
NH2
Br
H 69 'MeSO3H
II
NH
0<jj
H
70
4-(3-Amino-2-bromo-propionyl)-1,3-dihydro-imidazol-2-one (69). 4-(Aziri-
dine-2-carbonyl)-1,3-dihydro-imidazol-2-one (70). A solution of ketone 13
(0.1 g, 0.64 mmol) in 1 mL CH3SO3H was prepared and Br2 (33 p.L, 0.64 mmol)
was added at room temperature. The reaction was stirred for 16 hrs and ethyl
ether was added to the reaction mixture (15 mL x 3) and decanted. Addition of
methanol to. the resulting residue gave
-bromo ketone 69 as tanned crystals
(0.17 g, 85 %). A DMSO solution of this material was loaded onto a silica gel
column (CH2C12/MeOH(NH3), 6:4) and aziridine 70 was isolated. 69 1H NMR
(DMSO-d6) ö 2.37 (s, 3H), 3.35 (bs, 1H), 3.49 (bs, 1H), 5.34 (t, 1H, J = 6.5 Hz),
7.94 (s, 111), 8.11 (bs, 3H, xch D20), 10.84 (bs, 111, xch D20), 11.21 (bs, 1H,
xch D20); '3C NMR (DMSO-d6) ö41.9, 42.3, 121.4, 125.5, 154.9, 179.8;
IR(KBr) Dmax 3301, 1695, 1656, cm1; HRMS, calcd for C6H9N3O279Br (M + 1)
233.9878 1, found 233.98769. 70 }I NMR (DMSO-d6)
1.59 (bs, 1H, xch D20),
1.73 (dd, 2H, J= 2.5, 5.5 Hz), 1.76 (t, 1H, J= 2.5 Hz), 3.15 (dd, 1H, J= 2.5, 5.5
Hz), 7.93 (s, 1H), 10.62 (bs, 111, xch D20), 10.94 (bs, 1H, xch D20); '3C NMR
213
28.8, 31.5, 123.5, 124.0, 154.8, 185.6; IR(KBr) Dmax 3274, 1655,
(DMSO-d6)
1613, cm1; HRMS, calcd for C6H7N302 LRMS, m/z 450.9 (M + 1), 448.9,
452.9 [1:2:1].
0
0
0
NJNH2
H
II
0
13
H
DMF
N
H
H
)L'..._Br
72
Br
C'
Br
4,5-Dibromo4H-pyrrole-2-carboxylic acid [3-oxo-3-(2-oxo-2,3-dihydro-1Himidazol-4-yI)-propyl]-amide (72). To a solution of the free base of ketone 13
(0.1
g, 0.64 mmol) in 2 mL of DIvIF was added 4,5-dibromopyrrol-2-yl
trichoromethyl ketone (0.28 g, 0.77 mmol). The reaction was stirred at 90 °C
under Ar for 3 h. Ether was added to the reaction mixture (20 mL x 3) and
decanted and the resulting residue was tnturated with methanol to give product
72 as tanned crystals (0.22 g, 85%). 72 1H NMIR (DMSO-d6)
2.78 (t, 2H, J =
6.8 Hz), 3.43 (q, 2H, J = 6.8 Hz), 6.85 (s, 1H), 7.56 (s, 1H), 8.16 (t, 2H, xch
D20, J = 5.6 Hz), 10.49 (bs, 1H, xch D20), 10.76 (bs, 1H, xch D20), 12.65 (bs,
IH, xch D20); 13C NIVIR (DMSO-d6)
35.9, 37.2, 98.6, 105.4, 113.4, 122.1,
124.3, 129.0, 154.9, 159.7, 187.0; IR(KBr)
1;
Umax
3206, 1691, 1675, 1663 cm
HRMS, calcd for C11H11N40379Br2 (M + 1) 404.9 1979, found 404.91885.
214
0
H
Br
Br2
p
MeSO3H
Br
Br
H
4-[2-(4,5-Dibromo-IH-pyrrol-2-yI)-4,5-dihydro-oxazole-5-carbonyl}-1,3dihydro-imidazol-2-one (71). A solution of ketone 72 (0.15 g, 0.37 mmol) in 1
mL CFI3SO3H was prepared and
Br2
(20 pL, 0.37 mmol) was added at room
temperature. The reaction was stirred for 16 his and ethyl ether was added to
the reaction mixture (15 mL x 3) and decanted. The residue was diluted in a
minimum amount of DMSO and purified by chromatography column
(CH2Cl2/MeOH, 8:2) to give 71 as tanned crystals (95 mg, 65 %). 71 'H NMR
(DMSO-d6)
3.88 (dd, 1H, J = 7.1, 14.6 Hz), 4.20 (dd, 1H, J = 10.6, 14.6 Hz),
5.59 (dd, 1H, J = 7.1, 10.6 Hz), 6.78 (s, 1H), 7.83 (s, 1H), 10.69 (bs, 1H, xch
D20), 11.04 (bs, 1H, xch D20), 13.01 (bs, 1H, xch D20); '3C NMR (DMSO-.d6)
59.4, 78.0, 99.5, 106.7, 115.7, 122.1, 122.2, 124.8, 154.8, 156.4, 184.3;
IR(KBr)
Umax
3278, 1685, 1668, 1657 cm1; HRMS, calcd for
(M + 1)402.90414, found 402.90423.
C11H9N4O379Br2
215
OMe
H
H
NCS
MeOH
I
OKj
H
H
6Me
75
4-(3-Amino-1,2-dimethoxy-propyl)-1,3-dihydro-imidazol-2-one (75). To a
solution of olefin 8 (0.1 g, 0.7 mmol) in 20 mL of methanol was added NCS
(0.09 g, 0.7 mmol). The reaction mixture was stirred at room temperature for 3
hrs. The solution was directly loaded to a silica gel column for purification
(CH2Cl2/MeOH(N}13), 7:3) to give the desired product 75 as a light yellow oil
(57 mg, 40 %) (2.5:1 inseparable mixture of diastereomers).
75 'H NIVIR
2.49-2.72 (m, 1H), 3.12 (s, 3H), 3.22 (s, 3H), 3.15-3.48 (m. 2H),
(DMSO-d6)
3.88 (dd, 111, J = 6.7 Hz), 6.28 (s, 1H), 9.69 (bs, 3H, xch D20), 9.99 (bs, 1H,
xch D20); 13C NMIR (DMSO-d6)
41.1, 56.6, 59.4, 77.6, 83.2, 109.3, 118.8,
156.9; H NIvIIR (DMSO-d6) 2.83-2.88 (m, 1H), 3.13 (s, 3H), 3.40 (s, 3H), 3.153.48 (m. 2H), 3.91 (dd, 1H, J = 9.0 Hz), 6.28 (s, 1H), 9.69 (bs, 3H, xch D20),
9.99 (bs, 111, xch D20); 13C NMR (DMSO-d6)
109.5, 119.4,
C8H,6N303
41.1, 56.6, 58.8, 75.8, 81.3,
155.8; IR(KBr) u, 3089, 1668 cm1; HRMS, calcd for
(M + 1) 202.11917, found 202.11927.
216
H2
p
o=<Lro
MeOH
NH2
77
76HC1
5-(3-Amino-propylidene)-imidazolidine.2,4-dione (77).
To a solution of the hydrochloric acid of ester 76 (0.075 g, 0.17 mmol) in 20 mL
of methanol was added NCS (22 mg, 0.17 mmol). The reaction mixture was
stirred at room temperature for 3 hrs. The solution was directly loaded to a silica
gel column for purification
(CH2Cl2/MeOH(NH3), 1:1)
to give the desired
product 77 as a light yellow oil (15 mg, 60 %). 75 'H NIN'IR (CD3OD-d4)
2.56
(dt, 2H, J = 7.0, 7.8 Hz), 3.10 (t, 2H, J = 7.0 Hz), 5.63 (t, 1H, J = 7.8 Hz);
NMR (CD3OD-d4)
24.7, 38.6, 113.5, 133.1, 155.8, 165.0; IR(KBr) U)( 3196,
1688, 1624 cm1;}iRMS, calcd for
155.92547.
'3C
C6H,0N302
(M + 1)155.92581, found
217
CHAPTER VIII. GENERAL CONCLUSIONS
The research described in this dissertation presented results on two studies
of nitrogen containing metabolites from terrestrial and marine origin.
Part I of the thesis focused on gaining understanding on the mechanism
of epoxidation of 2,5-dihydroxyacetanilide (5) by unusual oxidases isolated
from
Streptomyces
sp. In this case, a deuterium exchange analysis of 5 in the
absence and presence of DHAE II was performed. The results suggest that
stereospecific protonation and deprotonation of the substrate could have lead to
the recovery of unreacted substrate. The possibility that a metal complex may
play a crucial role in catalyzing the reaction of 5 with molecular oxygen could
also explain the outcome of this study. In addition, inhibition studies using 1,4-
dihydroxybenzene indicated that the N-acetyl side chain group of the substrate
may be crucial for the formation of a stable substrate-enzyme complex. Efforts
to synthesize dihydroquinoline 7 by formation of the C6-C7 bond using
palladium couplings, organocuprates, Lewis acid catalysts, and aza-Claisen
reactions were hindered by a combination of steric effects and an unfavorable
conformation of the amide chain. Semi Empirical calculations of the electron
distribution in amide 21 revealed that the electron density is centered around C4,
suggesting that this is the most nucleophilic carbon in the ring.
Part II of the thesis described the development of methods for the
synthesis of alkaloids of the oroidin family of marine sponge metabolites. Our
218
approach to this family of alkaloids relies on the hypothesis that members of this
class could be biogenetically derived from related linear precursors. For the
synthesis of slagenins, 13-functionalization of olefin 14 was performed using an
acid catalyzed intramolecular cyclization to the corresponding oxazoline 15.
Oxidative addition of the hydroxyl group of the hydrolyzed product 16 to the
imidazolone ring in the presence of methanol led to the synthesis of slagenins B
(2) and C (3). Heating 2 and 3 in aqueous acid gave slagenin A (1) as the sole
product. The facile conversion of slagenins B and C to slagenin A under aqueous
acid, in addition of the isolation of slagenins B and C upon workup with methanol
of an intramolecular oxidative addition of alcohol 4 in aqueous media, suggests
that slagenins B and C may be artifacts rather than natural products.
The
synthesis of (Z)-debromoaxinohydantoin (17) and derivatives was accomplished
by intramolecular cyclization of a-methoxy imidazolone lib under acidic
conditions followed by a double oxidation reaction to furnish the hydantoinlactam functionality. In addition a practical synthesis of the related alkaloid (Z)debromohymenialdisine (20) was accomplished using a similar double oxidation
reaction to furnish the glycocyamidine appendage.
This reaction played an
important role in improving the overall yield of the synthesis from a - 3 % to 34,
%.
Studies towards the synthesis of the pyrrolopiperazine ring system in the
agelastatins showed that SN2 type intramolecular cyclizations of imidazolones
bearing an ct-13 unsaturated system or a (3-halogen substituent catalyzed by acid or
base favor formation of an oxazoline ring system. However, it was found that
219
intramolecular cyclizations of simple pyrrolocarboxamidoacetals using mild
aqueous acid favored formation of C-N cyclyzed products including longanilde, a
simple pyrrolopiperazine alkaloid, while cyclization of these substrates with a
strong acid like methanesulfonic acid provided the more thermodynamically
stable pyrrolopyridine system. On the basis of these preliminary studies, -ketone
73 is proposed to be an appropriate substrate for the formation of the
pyrrolopiperazine system in the agelastatins.
The syntheses presented in Part II of the dissertation are noteworthy in that
they do not require the use of protecting groups on any of the nitrogens. In
addition, the routes are direct and provide access to a series of previously
unavailable derivatives which could be used for structural activity relation studies.
220
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228
APPENDIX: 1H AND '3C NMR SPECTRA
Q<jNH2
5HCI
H
in DMSO-d6
I-.---,---'---
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
H
Q<)'NH2
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
H
N
>=o
N
H
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
N
i
NH2
H
.1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
5
4
3
2
1
ppm
H
OMe
MLI_
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
o=<c' NH2
13b in CD1OD-d4
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
QNH2
13b in CD3OD-d4
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
NH2
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
..,...,,,
I
I
El
I
1
...,...,,
...,,,..
I
11
01
6
......,,
.........
I
I
I
8
L
9
I
.,,,,.,,,i.,,,,,,, ..,..,,,,
.1
uxdd
H.
H2
'J
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
0
H
N
o=(
N
H
13
12
III
11
10
9
CH3SO3H
m
I
8
7
6
5
4
3
2
1
ppnt
J
H
N
o=<
0
j'NH2
N
H
CH3SO3H
13 in DMSO-d6
i
190
180
170
160
150
140
130
120
r''
110
ii ""i"
100
90
80
70
60
50
40
30
20
10 ppm
H
N
H
13
12
LU
Br
0H
Br
H
)
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
I
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
j'
H
1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
NH2
NH
tX.)
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
NH2
16'HCI
Br
in DMSO-d6
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
H
N---.
o=<
OH
N
H
13
12
11
10
9
8
7
iL
I
I'
Br
6
5
4
3
2
1
ppm
H
N--O=<
I'
N
H
I
OH
Br
1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
OH
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
H OH
ONtNH
t.)
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
H OMe
o=<Uu
190
180
170
160
150
140
130
120
110
1
100
90
80
70
60
50
40
30
20
10 ppm
HOMe
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
OMe
ii
UII\
N
H
190
180
170
160
150
140
130
120
110
100
0
HN
/9
/ NH
90
80
70
60
50
40
30
20
10 ppm
HOMe
N
13
12
11
10
9
8
7
6
H
5
in DMSO-d6
4
3
2
1
ppm
OMe
N2H
0
I
inDMSO-d6
o=Kj
N
H
lib
Br
8o17O16O15O140130120hb0b0090807060 50403020
10 ppm
00
N
wdd
.1
I
I
I ...,,,,,, I ...,,,,,.I,,,,,,,,, I
J9
H
9POSNU UT
9
I
L
8
6
01
,,,,,,.,, I .........
I
TI
I
T
I
LI
I
H
314
eo
OMe
H
o=<
I
(I
H
190
180
170
16Q
150
140
130
120
110
100
in DMSO-do
90
lic
80
Br
70
60
50
40
30
20
10 ppm
rei
in DMSO-d6
39
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
in DMSO-d6
HN
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
.1
10 ppm
0
HNBr
13
12
11
10
9
8
7
6
5
4NH
4
in DMSO-d6
3
2
1
ppm
0
in DMSO-d6
H&1)
1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
ppm
in DMSO-d6
Br
40b
.................................................. .-.-.--.-,-.-1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
b..)
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
0
Br
190
180
170
160
150
140
130
120
110
100
90
HNyNH
80
70
60
50
40
30
20
10 ppm
0
HN-q
---l-
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
I..)
'I,,
190
180
170
160
150
140
130
120
110
100
90
I''''I'
70
80
II
60
50
40
30
20
''1
10 ppm
t'..)
NH
in DMSO-d6
HN
Br
Z-44
I
13
12
11
10
I
9
8
7
6
5
4
3
2
1
ppm
NH
in DMSO-d6
Br_-?Tj)
Z-44
190
180
170
160
150
140
130
120
110
100
90
80
70
60
40
30
20
iO ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
10
N-
in DMSO-do
H
Br
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20 ppm
0
HI
in DMSO-d6
P.
/
N
13
12
11
10
9
8
7
6
5
NH
4
3
2
1
ppm
0%_-NH
HL>°
in DMSO-d6
NH
0
17
,-
190
180
170
160
150
140
130
120
110
100
_'___'___'___I,,,,,,
90
80
70
60
50
40
30
20 ppm
uidd
£
S
9
L
.............................................
8
..
6
I
OT
IT
T
£1
I
11
HN\Q
110
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
1
10 ppm
00
N ii..
uidd
I.,
.1 . ..i....i
9
L
8
6
01
11
1
El
JLi
HNQ
:jo
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
ijo
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
H0
o
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
in DMSO-d6
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
13
12
1 LO'
2
1
ppm
00
UI
0
HN
-
NH
inDMSO-d6
Br
42c
I
.1 ,....,..
Z
I
I
S
9
L
8
6
I
I
01
I
11
1
£1
I
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
III
20
10 ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
©
II
i,,,.,,,,,I,,,.'',,,I.,',,,,, I
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
t.)
M
t'.)
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
H
H
Br
66
in rM()-tL
1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
H
<NN)L/Br
0
N
Br
67
in DMSO-d6
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
L)
0H
ii
190
180
170
160
150
140
130
120
110
100
N)LqBr
Br
0
90
80
70
60
50
40
30
20
10 ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
cit
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
0
3
2
1
ppm
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
0
H
in DMSO-d6
5
4
3
2
1
ppm
0
I-i
II
Li.)
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
-
0
0
H
190
180
170
160
150
140
130
120
110
100
M
-
90
80
70
60
50
40
30
20
1
10 ppm
13
12
11
10
9
8
7
6
5
4
3
2
1
ppm
'1
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10 ppm
C