The Pennsylvania State University
The Graduate School
Department of Chemistry
PART I. STUDIES TOWARD THE SYNTHESIS OF DIAZONAMIDE A. PART
II. A PROPOSAL FOR THE MECHANISM-OF-ACTION OF
DIAZOPARAQUINONE NATURAL PRODUCTS AND STUDIES TOWARD THE
SYNTHESIS OF KINMYCIN F.
A Thesis in
Chemistry
by
Kyle Joseph Eastman
© 2006 Kyle Joseph Eastman
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
December 2006
The thesis of Kyle Joseph Eastman was reviewed and approved* by the following:
Ken S. Feldman Professor of Chemistry Thesis Advisor Chair of Committee
Raymond L. Funk Professor of Chemistry
Christopher J. Falzone Senior Lecturer
J. Martin Bollinger, Jr. Associate Professor of Biochemistry & Molecular Biology and Chemistry
Ayusman Sen Professor of Chemistry Head of the Department of Chemistry
*Signatures are on file in the Graduate School
iii
ABSTRACT
The synthesis of an indole salicylate with the required axial chirality for diazonamide A are reported. Atropselectivity in this biaryl system are secured by a proximal sp3 stereogenic center.
A model system for a novel photochemically induced cyclization to of a
benzotriazole alkene to give a C(2) disubstituted indolenine is developed. Extension of this model system to an approach toward the synthesis of diazonamide A is described.
The putative reductive activation chemistry of the diazoparaquinone natural products was modeled with Bu3SnH and prekinamycin dimethyl ether along with prekinamycin itself. Reactions in a various combinations of aromatic solvents, with and without the nucleophile benzylmercaptan present, led to isolation of both radical trapping arene adducts and nucleophile capture benzyl thioether products. Based on these product distribution studies, the intermediacy of first, a cyclopentenyl radical, and subsequently, an orthoquinonemethide electrophile, is proposed.
Lastly, the preparation of a Nazarov cyclization precursor and attempted cyclizations aimed at securing the benzo[b]fluorenone core of kinamycin F is detailed. iv
TABLE OF CONTENTS
LIST OF FIGURES…………………………………………………………...….vii
LIST OF TABLES………………………………………………………………...xi
AKNOWLEDGEMENTS……………………………………………….….……xii
Chapter 1 Diazonamide A: Background and Significance…………………….….1
1.1 Isolation, Biological Activity and Structural Assignement…………...1
1.2 Approaches Toward Initially Assigned Diazonamide A……………...3
1.2.1 Nicolaou’s Horner-Wadsworth-Emmons Cyclization
Strategy…………………………………………………………...3
1.2.2 Nicolaou’s Heteropinacol Coupling Strategy……….……....5
1.2.3 Wipf’s Modified Chan Rearrangement……….………...…..7
1.2.4 Vedejes’ Imino Dieckmann Cyclization Strategy……...... 9
1.2.5 Wood’s Cyclopropanation Ring-Opening Strategy………..11
1.2.6 Magnus’ Photo-Ffies Strategy………………………….….12
1.2.7 Feldman’s Negishi Coupling/Lock Atropisomer Strategy...13
1.2.8 Harran’s Completion of Nominal 1a/Structural
Reassignment…………………………………………………….14
v
1.3 Approaches Toward Re-Assigned Diazonamide A………………….20
1.3.1 Nicolaou’s First Total Synthesis of Diazonamide A………20
1.3.2 Nicolaou’s Second Total Synthesis of Diazonamide A…...24
1.3.3 Harran’s Total Synthesis of Diazonamide A………………29
1.3.4 Other Approaches to the Revised Diazonamide A………...32
Chapter 2 Studies Toward Diazonamide A…………………………………..….37
2.1 Diazonamide-Related Biaryls with Defined Axial Chirality…….…..37
2.2 Benzotriazole Alkene Photoisomerization Approach
to DiazonamideA…………………………………………………….…..42
Chapter 3 Diazoparaquinone Natural Products: Background
and Significance………………………………………………….………………53
3.1 Isolation, History and Related Compounds……….….……….…...... 53
3.2 Biological Activity…………………………………….…………..….57
3.3 Synthetic Efforts Toward Diazoparaquinone Natural Products…...... 58
3.3.1 Hauser’s Synthesis of the Structure Proposed
for Prekinamcin……………………………………………….…..59
3.3.2 Gould’s Synthesis of Kinobscurinone (Extension
to Stealthin C)...... 60
3.3.3 Sniekus’ Synthesis of Kinobscurinone…………………..…62
3.3.4 Jones’ Synthesis of the Benzo[b]fluorenone Core
Structure………………………………………………………..…63 vi
3.3.5 Mal’s Synthesis of Benzo[b]flourenones………………….65
3.3.6 Biradical Cyclization Approaches to Benzo[b]flurenes…...65
3.3.6.1 Echavarren’s Arylalkyne-allene Cycloaddition….65
3.3.6.2 Dominguez’s Benzotriyne/Benzodiyne
Cycloaddition…………………………………………….66
3.3.6.3 Echavarren’s Diaryldiynone Cycloaddition……...66
3.3.7 Kamikawa’s Approach Toward O4,9-Dimethylstealthins
A and D…………………………………………………………..68
3.3.8 Jebaratnam’s Synthesis of Prekinamycin Analogues………70
3.3.9 Ishikawa’s Approach to Oxygenated Kinamycin
Analogues………………………………………………………..72
3.3.10 Dmitrienko’s Approach to Prekinamycin………………...75
3.4 Speculation into Diazoparaquinone Natural Product
Mechanism of Action…………………………………………………….76
3.4.1 Jebaratnam’s Oxidative Activation Proposal for
DNA Cleavage………………………………………………...…76
3.4.2 Dmitrienko’s Proposal For DNA Damage:
Diazo Electrophilicity…………………………………………....77
3.4.3 Formulating a New Mechanism of Action Hypothesis……79
Chapter 4 Efforts to Elucidate the Mechanism of Action of the
Diazoparaquinone Family of Natural Products………..………………....……84
vii
4.1 Initial Investigations with Prekinamycin and Derivatives with Bu3SnH……………………………………………………………..84
4.2 Investigations of the Reactive Intermediate Preceding the
Trapping Event…………………………………………………………105
Chapter 5 Efforts Toward the Total Synthesis of Kinmycin F…………………105
5.1 Construction of the Highly Oxygenated D-Ring……………………105
5.2 Functionalization of the AB Ring System………………………….108
5.3 Preparing the Nazarov Cyclization Precursor………………………109
5.4 Nazarov Cyclization Attempts……………………………………...114
Chapter 6 Experimentals………………………………………………………..116
6.1 Diazonamide A Studies.…………………………………………….116
6.2 Diazoparaquinone Mechanism of Action Studies………….……….150
6.3 Kinamycin F Studies………………………………………………..176
Bibliography………………………………………………………….…………191 viii
LIST OF FIGURES
Figure 1.1 Diazonamide A structure: initial and revised…………..………….....1
Figure 1.2 Nicolaou’s Horner-Wadsworth-Emmons cyclization………………..4
Figure 1.3 Nicolaou’s heteropinacol/macrolactamization strategy……………...6
Figure 1.4 Wipf’s advanced benzofuranone……………………………………..7
Figure 1.5 Wipf’s modified Chan rearrangement………………………………..8
Figure 1.6 Vedejes’ Imino-Dieckmann cyclization approach…………………...9
Figure 1.7 Wood’s cyclopropanation ring-opening strategy…………………….11
Figure 1.8 Magnus’ photo-Fries rearrangement…………………………………12
Figure 1.9 Feldman’s Negishi coupling/bridge-locked atropisomer strategy…....14
Figure 1.10 Harran’s early stage development of nominal diazonamide A…………………………………………………………………...16
ix
Figure 1.11 Harran’s completion of nominal diazonamide A…………………...18
Figure 1.12 Nicolaou’s early stage progress toward reassigned diazonamide A…………………………………………………………………...21
Figure 1.13 Nicolaou’s completion of the first total synthesis of diazonamide A………………………………………………………………..…23
Figure 1.14 Nicolaou’s building block syntheses for the second total synthesis of diazonamide A……………….……………………………………..25
Figure 1.15 Nicolaou’s heteropinacol coupling to fashion the lower macrocycle……………………………………………………………26
Figure 1.16 Completion of Nicolaou’s second total synthesis of diazonamide A……………………………………………………………….….28
Figure 1.17 Harran’s complete synthesis of diazonamide A……………………31
Figure 1.18 Wood’s cyclopropanation model studies toward diazonamide A……………………………………………………………….….33
Figure 1.19 Vedejes hemiaminal synthesis……………………………………..34 x
Figure 1.20 Completion of Vedejes’s heteroaromatic biarylmacrocycle………..35
Figure 2.1 Retrosynthetic plan…………………………………………………..38
Figure 2.2 Preparation of coupling partner 133…………………………………39
Figure 2.3 Preparation of coupling partner 139…………………………….……39
Figure 2.4 Completion of target 124……………………………………………..41
Figure 2.5 Retrosynthesis of benzotriazole alkene strategy……………………...43
Figure 2.6 Model benzotriazole alkene photochemical rearrangement………….44
Figure 2.7 Initial stages of our synthetic effort toward diazonamide A…………46
Figure 2.8 Dead end to macrolactam dimer……………………………………...48
Figure 2.9 Other substrates for attempted macrolactamization………………….51
Figure 3.1 Originally proposed structure for kinamycins A-D…………………..53
xi
Figure 3.2 Various historical depictions of prekinamycin……………………….54
Figure 3.3 Inclusive representation of known diazoparaquinone natural products…………………………………………………………………..55
Figure 3.4 Benzo[b]fluorene natural products from S. murayamaensis…………56
Figure 3.5 Diazo-containing natural products lacking the paraquinone moiety………………………………………………………………57
Figure 3.6 Hauser’s synthesis of prekinamycin…………………….……………59
Figure 3.7 Gould’s synthesis of kinobscurinone…………………………………60
Figure 3.8 Gould’s revised route to 211 and divergence from 232 to 233………61
Figure 3.9 Snieckus’s synthesis of kinobscurinone…………………………..….63
Figure 3.10 Jone’s synthesis of benzo[b]fluorenone 232………………………..64
Figure 3.11 Mal’s preparation of benzo[b]fluorenes…………………………….65
Figure 3.12 Echavarren’s aryl-alkyne-allene approach to kinamycin core……...66 xii
Figure 3.13 Dominguez’s benzotriyne and benzodiyne approach to kinamycin core…………………………………………………………………...67
Figure 3.14 Echavarren’s closure to benzo[b]- and benzo[a]fluorenones, and mechanistic speculation………………………………………………………….68
Figure 3.15 Kamikawa’s synthesis of O4, 9-Dimethylstealthins A and C………..69
Figure 3.16 Jebaratnam’s synthesis of kinamycin core analogues………………72
Figure 3.17 Ishikawa’s synthesis of highly oxygenated kinamycin
analogue 299………………………………………………………………..……73
Figure 3.18 Ishikawa’s synthesis of oxygenated tetracycle 300…………………74
Figure 3.19 Dmitrienko’s synthesis of isoprekinamycin analogue 305………….75
Figure 3.20 Jebaratnam’s hypothesis for kinamycin mode of action……………76
Figure 3.21 Dmitrienko’s hypothesis and relevant IR frequencies………………78
Figure 3.22 Mitomycin single electron reduction………………………………..80 xiii
Figure 3.23 Postulated mechanism for the formation of a C11 sp2 from
diazoparaquinones via single electron reduction………………...…….………...81
Figure 3.24 Neocarzinostatin chromophore derived sp2 radical and its fate…….81
Figure 3.25 Speculative pathways by which radical 314 might lead
to DNA damage………………………………………………………………….83
Figure 4.1 Model compounds for mechanism of action investigations………….85
Figure 4.2 The plan to probe the formal single electron reduction chemistry of the diazoparaquinone models………………………...………………………………86
Figure 4.3 Preliminary findings upon treatment of 188 with Bu3SnH/AIBN……87
Figure 4.4 Extension of Bu3SnH/AIBN chemistry to prekinamycin
derivatives 323/324……………………………..………………………………..88
Figure 4.5 Hammett Study of the reaction between 324 and arene solvents under
radical generating conditions…………………………………………………….91
xiv
Figure 4.6 A mechanistic proposal for the formation of observed products
333/336 from the diazoparaquinone 324………………………………………...93
Figure 4.7 Plausible routes to the C11-C11’ dimer 345…………………………95
Figure 4.8 Successful trapping of radical intermediate 339 with Ph2Se2………..96
Figure 4.9 Yield and ratio of aromatic trapping products
333/336c upon increasing [Bu3SnH]…………………………………...... …....97
Figure 4.10 Yield and ratio of aromatic trapping products
333/336f upon increasing [Bu3SnH]………………...... ……99
Figure 4.11 Thiol addition to reactive orthoquinonemethide
intermediate 342…………………………………………………………….….101
Figure 4.12 Deuterium labeling experiments with
diazoparaquinone 324……………………………………………………….…103
Figure 5.1 Retrosynthetic analysis of kinamycin F…………………………….106
Figure 5.2 Hudlicky’s approach to (+)-pinitol via enzymatic
Dihydroxylation………………………………………………………………..107 xv
Figure 5.3 Synthetic route to highly oxygenated kinamycin F D-ring…………108
Figure 5.4 Synthesis of juglone derivative 369 as per Brimble’s work……… 109
Figure 5.5 Attempted stannylation of alkenyl bromide 365……………………110
Figure 5.6 Attempted functionalization of alkenyl bromide 364……………….111
Figure 5.7 Preparation of stanyl-juglone derivative 374……………………….112
Figure 5.8 Attempted carbonylative coupling………………………………….112
Figure 5.9 Preparation of Wienreb amide 380………………………………….113
Figure 5.10 Formation of Nazarov precursor 381 and desired cyclization product……………………………………………………….……..113
Figure 5.11 Manipulations of 381 and down stream cyclization attempts……..115
xvi
LIST OF TABLES
Table 3.1 Kinamycin A-D activities against a section G+ and G- bacteria…...57
Table 3.2 Brief survey of lomaiviticins A’s anti-tumor activity………………58
Table 4.1 Control experiments with the dimethyl ether 324…………………..89
Table 4.2 Relative reactivity of electron rich and electron poor arene solvents with the reactive intermediate generated form 324 under reducing conditions Bu3SnH/AIBN……………………………...………………………90 xvii
AKNOWLEDGEMENTS
My first debt of gratitude is extended to my Lord and Savior Jesus Christ, to whom I owe everything that I am or ever will be. I am thankful to have been blessed with a cognizance of my surroundings and the privilege to investigate the creation at the deepest level. I am thankful for the work He has done in my life over the past six years.
Beginning as a naïve, newly married, first year graduate student, I had no concept of family, only a firm focus on hard work. I am grateful for the people he put in my life that taught me so much about balancing my commitment to Him, my family, my church and finally my career.
My thanks also go out to my beautiful and magnificently patient wife
Jennifer. Any other woman would have run away from the workaholic, impatient and spiritually aloof young man that I was six years ago. I thank you from the bottom of my heart for being there for me everyday throughout our journey through this very challenging period in our lives. Thank you for your patience, encouragement and prayer.
Without every bit of energy you poured into me, I don’t think I’d be writing these words today. Thank you to my son Joshua for giving me a reason to smile throughout the preparation of this thesis.
I would also like to thank my parents, George and Kathy and my in-laws
Emil and Barbara Pauli for their encouraging words, persistent prayers and advice over the past six years. I am grateful for the time we’ve been able to spend together throughout the many needed respites from the pursuit of my Ph.D.
I would like extend special thanks to my second family for the past four years, the State College Christian Church body, from whom I’ve learned a great deal xviii about myself and how to be a better man. Specifically, I would like to thank Vince and
Beth Smith (Melvin and Kathy too) for being an incredible example for me and Jen. I thank you for the many hours of fellowship and mentorship. The time and energy you spent on Jen and myself will have an effect on generations to come I am sure. If the only thing I took away from my time in State College was having had the opportunity to get to know you and gain your friendship, it would have been enough!
Thank you to my advisor Ken Feldman for letting me slip into the 1.0 positions you had available in 2000 when I arrived. There has never been a moment of regret for joining your research group. I appreciate all of your efforts to make me a better chemist, from guiding me in making good decisions in the lab and thinking critically about problems to your painstaking insistence on the highest quality in the written presentation of my work. I have learned a lot in the lab, in group meetings and at lunch conversing with you. I will especially miss our interesting lunchtime conversations.
Thanks also goes to my committee members, both past and present, Professor Ray Funk,
Professor Blake Peterson, Professor Tim Glass, Professor James Runt, Professor Martin
Bollinger, and Dr. Christopher Falzone for their time and commitment.
Last but certainly not least, I would like to thank those from my past who directed my current career path. Thanks to Bob Raymond who would not let me ditch honors chemistry for that easy bio class. Thanks to Peter and Rosalee Fraser for your encouragement to study organic chemistry and to attend graduate school. You played an instrumental role in getting me to where I am today, for that and for our friendship I am grateful. xix
This thesis is dedicated to my loving wife Jen. Without your love and support, this
accomplishment would not have been possible.
Chapter 1
Diazonamide A: Background and Significance
1.1 Isolation, Biological Activity, and Structural Assignemnt
Many would argue that the role of chemical synthesis as a tool for the structural elucidation of natural products has in large part been replaced by X-ray crystallographic and NMR spectroscopic techniques. Leaving synthesis indispensable only as a means to confirm absolute or relative stereochemical assignments particularly in cases where X-ray data is unattainable. However, in first part the 21st century we are often reminded of the
essential role of chemical synthesis, as this age old craft continues to provide the decisive
evidence in one structural revision after another. In 1991, Fenical and Clardy disclosed
the structure of two unique toxins, diazonamide A and B (1a and 1b, Figure 1.1), both
secondary metabolites isolated from the colonial ascidian Diazona angulata (initially
incorrectly identified as Diazona chinesis).1 The potent in vitro activity of 1a against
HCT-116 human colon carcinoma and B-16 murine melanoma cancer cell lines (IC50 values below 15 ng/mL) coupled with the strikingly rigid and unique structure of diazonamide A attracted much attention from the synthesis community. The X-ray crystal data accompanying diazonamide’s literature debut appeared to solidify its structural assignment and opened the door to pursue synthesis of this attractive target. In the ensuing decade, twenty-five papers addressing various approaches to the presumed diazonamide A structure were published.2-26
.
2
initial assignments iPr
(-)-diazonamide A 1a: X = H, R = NH2 O
(-)-diazonamide B 1b: X = Br, R = H Br HN H HN RHN N N N O O A N 2 O O N 2 O Cl B Cl O O Cl Cl E F 10 10 C NH NH 11 11 17 X O D Br 7 O O OH OH 2 1 H HN N N HO 2 O O N O Cl O Cl 10 NH 11 O NH diazonamide A 3 revised structure
Figure 1.1 Diazonamide structure: initial and revised assignments
Efforts toward elucidating the structure of the major isolate, diazonamide B (1b,
Figure 1.1), culminated in a skeletal assignment as determined by X-ray diffraction measurements from a crystal of the derived p-bromobenzamide 2. It was initially thought that the acylation of 1b resulted in a dehydration of the C(11) hemiacetal. Thus, the elucidation of structural information by extrapolation from 2 to 1b did not permit assignment of stereochemistry at C(11), and this point remained an ambiguity. The core of the minor metabolite (1a) was subsequently assigned by analogy, and additional spectral data supported the presence of a valine residue (of undetermined configuration) which was assigned at the C(2)-amine. In the crystal clear vision of hindsight, as pointed out by Harran,27 spectral evidence actually argued against this assignment of the valine
3 residue. The synthesis of this well recognized target (1a) was finally achieved by Harran and co-workers,27,28 but their exuberance turned to puzzlement upon their discovery that
the material they had synthesized did not match the authentic natural material, and
subsequent studies showed the actual structure of diazoanmide A to be that shown in 3
(Figure 1.1).
1.2 Approaches Toward the Structure Initially Assigned to Diazonamide A
1.2.1 Nicolaou’s Horner-Wadsworth-Emmons Cyclization Strategy
Perhaps one of the most difficult hurdles facing the investigator poised to undertake the challenge presented by 1a is the stereochemistry at C(10). This issue was addressed in an early model system by Nicolaou (Figure 1.2).9
4
a) mCPBA OTBS OTBS OCN b) KOtBu d) LiHMDS, O2 Br c)TBSCl, imid. e) SnCl2, HCl O 49 % NC O O Br 94 % Br 456
h) 1.2 EQ bis-(pinacolato) OTBS OTBS f) DIBAL-H diboron 15 mol % [Pd(dppf)Cl ] g) NaH, MeI 2 MeO O B O MeO 84 % O 50 % O 78Br O (MeO)2P N i) 30 mol % [Pd(dppf)Cl ] O 2 3.0 EQ K2CO3 and 9 NMe Br 60 %
9 j) aq. HF O P(OMe)2 N k) Dess-Martin [O] TBSO N O 85% O
O NMe l) 2.0 EQ NaH MeO 11 MeO O NMe THF, 0 oC, 1h 11 25 % 10
Figure 1.2 Nicolaou’s Horner-Wadsworth-Emmons cyclization approach. m-CPBA epoxidation of alkene 4, was followed by potassium t-butoxide mediated regioselective nucleophilic 5-exo-tet cyclization by the nitrile enolate, expelling the primary alcohol which is protected with TBSCl to furnish 5. Next, 5 was oxidized to the benzofuranone 6 via treatment with LHMDS followed by exposure to O2 and SnCl2. The lactone functionality was partially reduced to the lactol and subsequent treatment with
NaH and MeI afforded the acetal 7 in 79% from 5. While modifications of 7 into an intramolecular olefin metathesis precursor met little resistance, unfavorable sterics thwarted all attempts at olefin metathesis. Fortunately, only minor modifications were needed to achieve formation of the desired olefin 11. Pursuant of this revised approach, aryl bromide 7 was converted into the boronate 8, followed by Suzuki coupling to
5 phosphonate 9 employing the method developed by Ishiyama,29 to generate phosphonate
10. TBAF-mediated removal of the TBS protecting group followed by Dess-Martin oxidation of the resulting alcohol and finally treatment with NaH effected the HWE
olefination to give the desired macrocycle 11 in 25% as a single alkene isomer and a single epimer at C(11). It is interesting to note that only one of the diastereomers (at
C(11)) participates in the cyclization. This observation speaks to the fact that the success
or failure of the macrocyclization depends directly on the stereochemical features of the
chain, an ominous point that has implications for our own efforts toward the macrolactam
core of diazonamide A.
1.2.2 Nicolaou’s Heteropinacol Coupling/Macrolactamization Strategy
In a subsequent approach, Nicolaou was able to employ a novel macrocyclization
strategy, incorporating a heteropinacol coupling, en route to the core bicyclic system of
the originally proposed diazomanide A.30 To avoid the problems observed in the earlier
route arising from the stereochemistry at C(11), the aldehyde-oxime 12 (Figure 1.3) was
6
FmocHN OMe O O N NH N N a) 9.0 equiv SmI O O 2 N N Cbz 36 EQ HMPA Cbz HO O O THF, 25 oC, 1h NMOM O b) EDC, HOBt O NMOM MeO CO Me MeO 2 CO2Me FmocHN 12 13 14 O OH 45 % FmocHN d) aq. HF O e) Dess-Martin [O] c) Dess Martin [O] N f) NaClO2 N N g) Et NH d) POCl3 Cbz O 2 O 56 % 61 % O NMOM MeO CO2Me 15
H2N HO2C HN N h) HATU, CbzHN N CbzHN N N O collidine O O O 5-10% O
O NMOM O NMOM MeO CO Me CO Me 2 MeO 2 16 17
Figure 1.3 Nicolaou’s heteropinacol /macrolactamization strategy.
prepared. Treatment of 12 with a premixed complex of 9 equivalents of freshly prepared
SmI2 and 36 equiv of HMPA generated an intermediary amino alcohol (not shown) that
was subsequently coupled with valine residue 13 to generate alcohol 14 in 45% yield.
The subsequent oxazole formation involved a great deal of effort to execute, as all
literature precedented oxazole formation methods proved to have little utility in this
system for one reason or another. A simple solution was reached by following a two step
procedure: Dess-Martin oxidation followed by a novel pyridine buffered POCl3 cyclodehydration afforded oxazole 15 in 56% yield. The next four steps, HF-mediated acetonide cleavage, two step oxidation of the resultant alcohol, and finally Et2NH-
7 induced removal of the Fmoc protecting group, afforded amino acid 16 in 61% overall yield. Unfortunately, with only a single bond forming event in the way of reaching the core of diazonamide A, many standard macrolactamization attempts failed to produce anything apart from dimer- or trimerized products. Modest (5-10%) success was finally achieved with slow addition of 16 to HATU and collidine in a 1.0 X 10-4 M solution of
DMF:CH2Cl2 (9:1).
1.2.3 Wipf’s Modified Chan Rearrangement
Wipf and co-workers entered the diazonamide area with a disclosure in 199826 on
the synthesis of the benzofuranone-indoloxazole fragment 19 (Figure 1.4), which was
OBDPS Ph NH 2 O N O O 12 steps
N H N OEt 18 19 O
Figure 1.4 Wipf’s advanced benzofuranone.
followed shortly by a report on the utilization of a modified Chan rearrangement toward
polyoxazole synthesis.7 After a series of failed exploratory experiments focused on
employing the Chan rearrangement in the model benzyl pivaloate, attention was focused
on Hamada’s work on N→C acyl migrations of acyclic imides.31 With initial results
showing great promise, the strategy was extended to a more representative model (20,
Figure 1.5) for the planned synthesis of 1a, containing the indole nucleus and a more sterically demanding quaternary center. Thus, 20 was prepared in 7 steps from indole starting
8
Ph Ph Ph Ph Me Me O O a) LDA, THF N o N Boc -78 C, 30 min Boc N N O O
N N OEt OEt O O 20 21
Chan 78 % Rearrangement Me CbzHN O Me NHBoc O b) HCl Ph N c) Cbz-Valine Ph Ph d) TsOH Me N Ph O Me N O 41 %
N N OEt O OEt O 23 22
Figure 1.5 Wipf’s modified Chan rearrangement strategy.
material. The clever application of the LDA initiated Chan rearrangement of 20 resulted
in the clean formation of amino ketone 22 in 78% yield. Following the Chan rearrangement, 22 was efficiently converted to the requisite oxazole 23 in a three-step sequence, thus forming the contiguous A, B, C, D ring system of 1a.
9 1.2.4 Vedejs’s Imino Dieckmann Cyclization Strategy
In the approach of Vedejs and co-workers, the same region of the molecule was envisaged as a key point of entry to the challenging macrocycle, a line of attack that relied on a Dieckmann-type cyclization reaction to secure the lower portion of 1a
(32→33, Figure 1.6).5 Preparation of the cyclization precursor 32 began with the 7 step
preparation of benzofuran 24. The benzofuran was then oxidized upon treatment
a) MeOCOCl, Et N Br Br 3 97 % O CH3CO3H Br MeO OPMB OPMB b) MeOCOCl, DMAP 66 % OPMB O 89 % O O O O 24 25 26
d) MsCl, Et N f) NaH, 0 oC 3 O O Br c) DIBAL-H e) MsOH RO Br g) nBuLi, -78 oC MeO OPMB O 87 % 89 % h) B(OiPr)3 OH O O + 28 R = Me NaOH i) H3O 27 29 R = H 94 % j) TMSCHN2 77 %
Me N O O MeO B(OH)2 O TfO NBoc O 30
31 k) 1.5 EQ 31 66 % 10 mol % Pd(dppf)Cl2] 3.0 EQ Cs2CO3
N Me N O MeO l) 3.0 EQ LDA O O O THF, -23 oC, 5 min
O O NBoc 57 % O O 16 18 NBoc 33 32
Figure 1.6 Vedejs’ Imino-Dieckmann cyclization approach.
10 with peracid to give the oxindole 25, which was subsequently C-acylated using the method developed by Black32 to install the quaternary C10 (1a numbering) carbon.
Chemoselective reduction of 26 to the lactol 27, followed by mesylation and subsequent treatment with methanesulfonic acid resulted in the formation of stable acetal 28.
Saponification of 28, followed by temporary protection of the resulting acid 29 by
treatment with NaH, permitted lithium halogen exchange with n-BuLi and a subsequent
quench with triisopropyl boronate resulting in the boronic acid (not shown). The presence
of the carboxylic acid resulted in protodeboronation after only a few hours at room
temperature, and thus necessitated immediate reintroduction of the methyl ester with
TMSCHN2 to give the more stable boronic acid 30 in good yield. With the coupling
partners 30 and 31 in hand, attention was directed toward the Suzuki coupling. Whereas
studies in a model system gave good results14 the desired product bi-aryl 32 was formed
in yields that were quite low and in rates that were unacceptably slow (days).
Fortunately, employment of Cs2CO3 in place of K3PO4 in the coupling reaction greatly
increased the rate and yield of the reaction to give 32 in 66% in just 15 h. It should be noted at this point that spectroscopic studies showed the presence of two atropisomers that interconvert at room temperature, with an experimentally determined value for the barrier of rotation around the C(16)-C(18) biaryl bond of ΔG≠ = ca. 15.5 kcalmol-1. This point is interesting considering the lack of interconversion exhibited by 10 of an earlier synthesis discussed (Figure 2). Finally, treatment of 32 with LDA led cleanly to the
Imino-Dieckmann-cyclization product 33 in 57% yield as a single atropisomer, possessing the desire stereochemistry as confirmed by X-ray crystallography.
11
1.2.5 Wood’s Cyclopropanation Ring-Opening Strategy
The work from Nicolaou and Vedejs highlighted above relies on enolate chemistry to quaternarize C(10). In a clever application of cyclopropane ring opening,
Wood modeled a new and interesting approach to C(10) (Figure 1.7).10
b) 3.0 EQ LiOH O N 2 a) 1.3 mol % [Rh2(cap)4] THF/MeOH o O o O CH2Cl2, 40 C, 4 h 0 C, 30 min O Me Me OMe 89 % OMe O O 33 34
O O MeO2C Me OMe Me 76 % 10 O OMe O OMe O 35 36
Figure 1.7 Wood’s cyclopropanation ring-opening strategy.
Wood and co-workers focused their attention on the α-diazoester 33 (Figure 1.7),
available in 11 steps from commercially available hydroxycinnamate. Decomposition of
33 with dirohdium(II)caprolactamate gave rise to the desired cyclopropane 34 in excellent yield. With 34 in hand, attention was focused on the development of suitable conditions for cyclopropane ring opening. Gratifyingly, treatment of 34 with 3 equiv of
LiOMe lead directly to orthoester 36. This clever cascade reaction, which reveals the desired quaternary center, likely proceeds through initial formation of the methyl ester with simultaneous expulsion of the phenoxide (cf. 35), followed by cyclopropane ring opening. While this approach lengthens the synthesis to the quaternary center by a few
12 steps when compared to other approaches discussed herein, the use of transition metal catalyzed cyclopropanation reaction opens the door to the preparation of key precursors in optically active form. Wood was able to demonstrate the feasibility of this notion in preliminary experiments using Doyle’s catalyst33 to prepare optically active cyclopropane
34 in 45% ee.
1.2.6 Magnus’ Photo-Fries Strategy
A significant departure from macrocyclization strategies that rely on carbanionic
intermediates is embodied in the innovative and extremely efficient approach developed by Magnus. In a 2001 disclosure, Magnus demonstrated the application of a photo-Fries rearrangement of 39 (Figure 1.8) to construct the diazonamide macrocycle. The protected macrolactonization precursor 38 was efficiently prepared in 6 steps from the substituted indole 37 in 50% yield. Palladium catalyzed hydrogenolysis of the benzyl protecting groups of 38,
HN N a) H ,Pd/C N 2 O 6 steps NH O O 98 % CO2Bn 50 % b) DMAP, EDCI OBn BnO NCO2Me OMe 66 % 37 38
HN N HN N N O N O hν O O O O O O 76 % O NCO Me 2 HO NCO2Me OMe OMe 39 40
Figure1.8 Magnus’ photo-Fries rearrangement strategy.
13 followed by the Keck34 modification of the Stelich esterification35 procedure afforded
macrolactone 39 in 65% yield as a 1.5:1 mixture of atropisomers. Photolysis of a
solution of 39 in benzene resulted in the rearranged product 40 as a 2:1 mixture of
atropisomers in 76% yield.
1.2.7 Feldman’s Negishi Coupling/Bridge-Locked-Atropisomer Strategy
Yet another interesting and novel approach to diazonamide was added to the mix
in an account that literally straddled Harran’s disclosure, as Feldman and co-workers
detailed efforts toward 1a that relied on the stereogenicity of a proximal center to control atropselectivity in an intramolecular cyclization.36 This approach began by employing
the coupling procedure of Knochel37as modified by Fu38 to join iodoarene 41 (Figure
1.9), available in four steps from o-iodophenol, and the 4-iodoindole 42, available in five
steps from gramine, resulting in biaryl 43. A small survey of different palladium/ligand
protocols with 41 revealed that the Pd(t-Bu3P)2-based reaction provided the best yields.
Elaboration of the coupled product required the introduction of the tryptophan
functionality at C(3) of indole 43. To that end, Horner-Wadsworth-Emmons extension of
the aldehyde 43 with phosphonate 44 provided a 10:1 mixture of Z/E alkene 45. The use
39 of Schmidt’s protocol (DBU, CH2Cl2) was reported as a critical point for the
achievement of high yields and acceptable Z/E ratios. Asymmetric hydrogenation of 45
mediated by (S,S)-DuPHOS ligated rhodium affording a ca. 1.4:1 mixture of
14
a) i-PrMgCl O O b) ZnBr BnO OTMSE CO2TMSE 2 BnO OTMSE c) Pd(t-Bu3P)2, 42 d) DBU, 44
OBn 87 % 70 % CBzN CBzN I CHO CO2PMB 41 I CHO 43 O BOCHN (MeO) OP 45 2 OPMB N CBz NHBOC 42 44
O O HO OTMSE e) Rh((S,S) - DuPHOS)/H2 O O OTMSE f) Pd(CaCO3)/H2 g) DCC, DMAP H 82 % HN DMAP HCl CO H HN 2 91 % NHBOC H BOCHN 47 46
Figure 1.9 Feldman’s Negishi coupling/bridge-locked atropisomer strategy. atropisomers was followed by palladium-catalyzed hydrogenolysis of the benzyl- protecting groups to furnish the phenoxy acid 46 in 82% yield. Finally, 46 was cyclized using the Keck34 modification of the Stelich esterification35 procedure to provide the
desired lactone 47 in excellent yield. It is interesting to note that this cyclization
represents a preferential closure of one, of a mixture of two, freely rotating diastereomers
(avoiding a steric clash of the NHBOC and the phenyl ring during closure). These results
stand as a proof of concept for the thesis that the C(16)-C(18) (diazonamide numbering)
can be set under the influence of a proximal sp3 stereogenic center, a point that will be
addressed later in the current studies toward 3.
1.2.8 Harran’s Strategy for the Completion of Nominal 1a/Structural Reassignment
In the approach to 1a taken by Harran and co-workers, the challenge of
controlling atropselectivity is avoided. Instead, focus is directed at a triaryl acetaldehyde
15 epicenter as a means to form the macrocycle. In this well-designed and imaginative strategy, the core macrocycle is assembled in a very efficient manner through the union of several advanced, highly functionalized fragments to give rise to an adduct poised for the key bond forming reaction between rings E and F (Figure 1.1). This strategy proved fruitful, culminating in the first total synthesis of nominal diazonamide A (1a).
Construction of the upper peptidic portion of 1a began by treating dibutylzirconocene40 with α-chloro styrene 49 (Figure 1.10). The vinyl ZrIV species presumably generated in
situ undergoes a subsequent palladium catalyzed cross-coupling reaction with bromide 48
to generate 1,1-disubstituted ethylene 50. Harran notes that this variant of Takahashi’s
modification41 of a Negishi coupling neither requires nor benefits from a Zr-to-Zn
transmetallation prior to addition of the electrophile. Following the removal of the N-
BOC- and O-PMB protecting groups from 50, the iodotyrosine derivative 51 was coupled
to deprotected 50 to give 52 in excellent yield. Upon exposure of 52 to catalytic Pd0 in the presence of Ag3PO4, the putative pre-organization of the free phenol aids endocyclization to form triarylethylene 53, a substrate containing the content of the
16 Cl b) BBr3 c) O I PMBO 49 HO BocHN N 51 CN BOCHN BOCHN N a) Negishi Coupling 48 and 49 OBn CN O O 85 % 89 % Br 48 50 PMBO
d) 3 mol % [Pd2(dba)3] 6 mol % 2-(di-tert-butyl N HN phosphanyl)biphenyl N CN HN BOCHN O Ag3PO4 CN O BOCHN O O 82 %
I HO HO OBn OBn 52 53
OTf e) Br O OO f) 1.2 EQ Os N PrHN NHPr h) p-TsOH HN O HN N CN CN CBzHN O 54 BocHN O i) N-(Benzyloxy- O O OH carbonyloxy)- g) H2S (g) succinimide
67 % OH O 54 % OBn O O d.r. 93:7 OBn 55 Br 56 Br
Figure 1.10 Harran’s early stage development of nominal 1a. diazonamide core, needing only to be oxidatively restructured to reveal this feature.
After derivatization of phenol 53 to the corresponding 2-bromoethyl ether, the alkene was treated with the dihydroxylation reagent 54 followed by hydrogen sulfide to afford the glycol 55 in 67% overall yield in a 93:7 diastereomeric ratio favoring the desired facial selectivity. Interestingly, the use of this particular osmium reagent was necessary to overcome the intrinsic preference of 53 to undergo dihydroxylation from the opposite face of the olefin. Next, pinacol rearrangement of diol 55 under acidic conditions
17 furnished the triaryl acetaldehyde 56 as a single stereoisomer in good yield after amine protection.
Conversion of the nitrile 56 into the desired substituted oxazole along with the required functionalization of the core was completed over ten steps in 8.4% overall yield to furnish aryl bromide 57 (Figure 1.11). Included in this transformation was an interesting capitalization of an internal alcoholic nucleophile (reduced aldehyde) to generate a six member lactone upon treatment of the nitrile with p-TsOH, providing the platform needed to append a tryptamine fragment and elaborate the second oxazole ring.
Aryl bromide 57 was the foundation for construction of the second macrocyclization precursor, which eventually underwent successful cyclization to 58 utilizing conditions discovered by Witkop.42 A plausible mechanistic description of this transformation is an
intramolecular photoinduced electron transfer from the indole chromophore to the
adjacent bromoarene followed by a biradical collapse to form the pivitol C(16)-C(18)
bond. The brilliance in design of this approach is marked by the formation of 58 as a
single atropisomer, strategically driven by the asymmetry incorporated into the
cyclization precursor. Chlorination of 58 at C(27) and C(25) followed by palladium
catalyzed hydrogenolysis gave the free amine which was reacted with CBz-L-Val-OH to
give 59 in 55% yield. Acetyl hemiacetal 59 was deacylated under stannoxane catalysis
to give the free acetal, and final deprotection afforded 1a – the structure originally
proposed for (-)-diazonamide A.
18
a) hν, (300nm) N N HN HN N 2 EQ LiOAc N 27 CbzHN O CbzHN O O NH 3 EQ O O 10 steps O epichlorohydrin 56 AcO AcO 18 8.4 % 40 % 25 16 Br O O OBn NH OBn 57 58
b) NCS 55 % c) H2/Pd/C d) Cbz-L-Val-OH
HN N HN N H N Cl e) [(Bu2Sn(O)Cl)2] H N Cl N O N O H2N O O CbzHN O O f) H2/Pd/C O O HO AcO 80 % Cl Cl
O O OH NH OH NH 1a 59
Figure 1.11 Harran’s completion of nominal 1a.
Remarkably, the material prepared by Harran and co-workers was clearly different from a sample of natural (-)-diazonamide A, particularly by qualitative measures such as instability to handling and its mobility on a silica gel thin-layer chromatography plate. The former issue was reportedly the most troubling for obvious reasons. Mass spectrometry indicated that late stage synthesis intermediates began to decompose via net
C(10) deformylation almost immediately following hemiacetal deprotection. The process appears to complete upon HPLC purification of 1a under identical conditions used to isolate natural (-)-diazonamide A. While difficulty of obtaining pure 1a hampered exhaustive characterization, Harran reasoned that a correct structure of diazonamide A would need to reconcile the observed exact mass of 765.1998 amu with a closely related
19 polycyclic framework. Careful scrutiny of the original characterization data was critical, and uncovered the fact that hydrolysis of diazonamide A had failed to expel valine as expected. So, it became apparent that the first point of rectification was to reassign the appendage at C(2). Careful examination of the isolate’s NMR data revealed that the C(2) side chain contained a hydroxy group rather than the originally assigned amino group.
This NH2→OH revision required a compensatory permutation be made elsewhere in the molecule to account for the one Dalton increase in molecular mass.
In a fantastic bit of investigative work, Harran disclosed the following: “the exact mass of diazonamide B is 743.0340 amu. However, the structure proposed for this
+ material (1b) has the formula C35H26N5O6Cl2Br and an [[MH ]-H2O]] ion has the
+ calculated mass of 744.0416 amu. The formula C35H25N6O4Cl2Br [[MH ] = 743.0576] is more consistent with the observed mass (Δ= 2.4 ppm) and this suggests that a protonated nitrogen atom in diazonamide B has been mistaken for oxygen in 3.”27 This
point can be reconciled with the crystallographic data if a protonated nitrogen atom in
diazonamide B was mistaken for an oxygen atom. Careful analysis of the X-ray data is
revealing: the observed C(7)-O(2) (Figure 1.1) bond length of 1.371 Å does not differ
significantly from common aryl C-O bond lengths. On the other hand, the length of the
C(17)-O(3) bond (1.433 Å) is longer than the maximum value (1.409 Å) observed for
similar bonds of this type. The greater thermal motion associated with the O(3) atom is
suggestive of an element with fewer electrons and a larger covalent radii. Based on the
preceding findings and on a 1H-15N NMR correlation experiment, Harran concluded that the structure of the diazonamides was more likely the C(11) diaryl N,O-acetal.
Consequently, structure 3 was proposed as a more consistent structure given the available
20 data. One feature, the absolute configuration of the α-hydroxy acid side chain at C2, remained a mystery awaiting total synthesis to be confirmed.
1.3 Approaches Toward Re-assigned Diazonamide A
1.3.1 Nicolaou’s First Total Synthesis of Diazonamide A
The structural reassignment of diazonamide by Harran sent a shockwave of reorientation through the synthesis community. Those who had entertained the thought of wrestling with 1a and had suspended embarkation for one reason or another were perhaps grateful for their decision. On the other hand, all those committed to the pursuit of 1a were forced to implement major revisions or revise entire routes. Twenty three papers30,36,43-63 addressing the biology of 3, or synthesis approaches toward 3, have
appeared to date. The achievement of preparing 3 in an approach that, not surprisingly,
included the development of new synthetic technologies and strategies was revealed by
K. C. Nicolaou in the first publication following Harran’s disclosure.61
With all building blocks (60, 62, 64, 69, and 74) readily accessible from
commercially available starting materials utilizing literature procedures, a synopsis of
Nicolaou’s synthesis begins with oxazole 60 (Figure 1.12). After repeated failed attempts
to utilize the oxazole ester derivative 60 in the intended coupling reaction, it was reduced
with lithium borohydride and the resultant primary alcohol was protected as the benzyl
21 c) n-BuLi, O CO Me BOCHN 2 a) LiBH 4 CbzHN BOCHN b) NaHMDS, BOCHN N OBn O N BnBr 62 MOM Br O O N 60 % O N 73 % HO CO Me OBn N Br OH 602 61 63O MOM 64
H2N BOCHN MeO2C MeO2C e) BOC O (separation N OBn 2 N OBn CbzHN of C10 epimers) CbzHN d) p-TsOH O O Cl(CH2)2Cl f) MOMCl, K2CO3 10 10 33 % 61 % (31 %) N Br N Br OH O H MOMO O MOM 65 66
g) LiOH h) TFA CbzHN HN CbzHN HN N N i) HATU OBn OH collidine O O j) BCl3; NaOH O O 35 % 61 % N Br N Br MOM H MOMO O HO O 67 68
Figure 1.12 Nicolaou’s early stage progress toward 3. ether to give 61 in good yield. Treatment of 61 with two equivalents of n-BuLi resulted in formation of the dianion which was subsequently treated with isatin derivative 62 to afford the free alcohol 63 in 73% yield. The key step of establishing the appropriately elaborated quaternary center was effected by refluxing 63 with tyrosine derivative 64 in dichloroethane in the presence of p-TsOH to give 65, which had lost the acid sensitive
BOC and MOM protecting groups, in a mere 33% yield. Reprotection of the primary amine with BOC2O lead to two C(10) epimers that were conveniently separated via
chromatography. This separation was followed by MOM protection of the desired
isomer’s phenolic hydroxy group and the oxindole amide with treatment by MOMCl and
22
K2CO3 to afford 66 in 61% over two steps including both isomers (31% considering only usable material). Methyl ester 66 was then subjected to LiOH-mediated hydrolysis, followed by TFA deprotection of the amine, and, finally, subjection of the desired amino acid to HATU coupling conditions to achieve the coveted macrolactam 67 in 33% yield over three steps (36% for macrolactamization). It’s noteworthy to mention the fact that even at very high dilution, only one of the C(10) epimers of the deprotected 66 cyclized to macrolactam 67. Gratifying to Nicolaou and co-workers, the epimer that cyclized possessed the desired stereochemistry at C10. The next step in the sequence required the challenging orthogonal deprotection of a benzyl group in the presence of a Cbz protecting group. The selective deprotection was accomplished by the use of BCl3 followed by
treatment with NaOH to afford the desired debenzylated primary alcohol 68, also missing
the two MOM protecting groups, in 61% yield.
With 68 (Figure 1.13) in hand, elaboration continued with BOC protection of the phenol, followed by a two step oxidation, first with IBX and then with NaClO2, to furnish
the carboxylic acid which was coupled to the tryptamine derivative 69 to give the oxazole
precursor 70 in 41% yield over four steps. The keto amide 70 was then transformed to
the corresponding oxazole system with a novel modification of the Gabriel-Robinson
cyclodehydration employing POCl3 and pyridine. The ensuing macrocyclization under
radical conditions (PhSnH/AIBN) generated 71 in poor yield (10%).
23
a) BOC2O e) POCl3, py b) IBX CbzHN HN H CbzHN HN N f) hν, LiOAc or N OH c) NaClO2 N O Ph SnH O d) 69, EDC, HOBt O 3 O O O 16 % or 5 % 41 % E NH
O N Br D N Br NH2 H H BOCO O HO O TFA 70 68 69 N H
CbzHN HN CbzHN HN N N N N Cl O g) NCS O O h) TFA O O O Cl 52 % N NH N NH H H BOCO O HO O 71 72 i) DIBAL-H OH 56 % HO 74 H O N HN CbzHN HN HO N N N Cl j) H , Pd(OH) /C N Cl O O 2 2 O O k) 74, EDC, HOBt O O Cl O Cl 82 % N NH N NH O H O H 3 73
Figure 1.13 Nicolaou’s completion of the first total synthesis of diazonamide A.
The yield of the 70→71 conversion was improved to 30% by employing the Witkop- type42 conditions demonstrated as useful in a similar system by Harran.28 Interestingly,
in contrast to the Ph3SnH conditions (in which most of the of the starting material was
debrominated), the bulk of the remaining material from the photo-induced cyclization
remained unchanged and could be productively recycled. This observation supports a
mechanism based on intramolecular photo-induced electron transfer from the D-ring
indole chromophore to the adjacent benzenoid E-ring, rather than some exclusively
radical-based event during which the halogen would likely be removed.
24 With the successful establishment of upper and lower macrocyclic rings of diazonamide A, only a few manipulations remained to complete the synthesis. First, the two required chlorine atoms were installed by reacting 71 with NCS, which was followed by removal of the BOC protecting group to provide phenol 72 in 52% yield over two steps. Next, DIBAL-H reduction of 72 afforded the desired aminal in 56% overall yield.
Finally, hydrogenolysis of the Cbz group with Pearlman’s catalyst followed by installation of the necessary peptide chain using isovaleric acid (74) in the presence of
EDC and HOBt afforded 3 in 0.00015% yield over 21 steps from known starting materials.
1.3.2 Nicolaou’s Second Total Synthesis of Diazonamide A
The most interesting feature of the second total synthesis of diazonamide A59 is
that it comes from the laboratories of K. C. Nicolaou, representing a second entirely distinct route from this group. The critical difference from the previous strategy61 is the reversed order for the construction of the two macrocyclic domains of the natural product. In the present approach, employing the heteropinacol strategy developed for the construction of nominal diazonamide A (1a),50 the lower heterocyclic core was
constructed first, followed by the upper 12-membered AG macrolactam system.
The first set of tasks en route to the second synthesis of 3 was to prepare oxazole
76 and the functionalized EFG-ring system 84. The appropriately functionalized oxazole
(76, Figure 1.14) was prepared from indole derivative 75 (available in 6 steps from 4- bromoindole) through MOM protection followed by reaction with BPD and palladium to give the desired boronate ester in good yield. Progress toward tetracycle 84 began with
25 the protection of commercially available tyrosine methyl ester 77 to give phenol 78
OTBS OTBS
a) NaH, MOMCl O Br O N B O O N b) BPD, [Pd(dppf)Cl2] 80 %
N N H O O MOM 75B B 76 O O BPD
CO2Me CO2Me CO2Me CO2Me NHCbz e) SOCl2 NHCbz NH2 NHCbz c) CbzCl d) TiCl4, 79 f) NaCNBH3 94 % 58 % OH 76 %
O HO HO OH OH O N O N 77 78 80 H Br 81 H Br O N 79 H Br g) LiBH4 h) 2,2-DMP, 93 % p-TsOH
O O O k) TBSCl, imid l) LiOH N N i) TMSCl, Et N N Cbz m) BnBr, KF alumina Cbz 3 Cbz n) 9-BBN j) Yb(OTf)3, HCHO OTBS OH 57 % 70 %
BnO HO HO N O N O N 84 Bn Br 83 H Br 82 H Br
Figure 1.14 Nicolaou’s building block syntheses for the second total synthesis of 3.
without difficulty. Next, in a familiar reaction, TiCl4 mediated union of 78 and 79
generated 80, embodying the majority of the required functionality of the EFG target, in a
respectable 58% yield. Toward quaternarizing C(10) (diazonamide numbering), the
tertiary alcohol of 80 was exchanged for a hydrogen via an intermediate chloride upon
successive treatment by SOCl2 and NaCNBH3 to give oxindole 81. The tyrosine portion
of the molecule was protected as the acetonide via initial reduction of the methyl ester
followed by p-TsOH catalyzed reaction with 2,2-dimethoxypropane to give 82 in
26 excellent yield. The required hydroxy methyl group at C(10) was then installed using
Padwa’s two step protocol,64 to give 83 as an equimolar mixture of C(10) stereoisomers
in 70% overall yield. Finally, a four step functional group manipulation sequence was all
that remained to set up the Suzuki precursor. Silicon protection of the primary alcohol
with TBSCl and imidazole also resulted in protection of the phenol. Removal of the
undesired phenol silicon protecting group with LiOH was followed by benzyl protection
of the phenol and the oxindole nitrogen. A final unfortunate but necessary reduction of
the carbonyl portion of the lactam was accomplished with excess 9-BBN to give 84 in
very good 57% overall yield.
A palladium mediated Suzuki reaction united 76 and 84 (Figure 1.15) to give
biaryl compound 85 in very good yield. Next, a dual tandem deprotection/oxidation
sequence was effected by treating 85 with TBAF followed by SO3•Py to give the
O O OTBS TBDPSO N N O N Cbz O Cbz TBSO B O N a) [Pd(dppf)Cl2] O OTBS 78 % N NMOM BnO Bn BnO N N MOM 84 Bn Br 76 85 b) TBAF 78 % c) SO3 py d) MeONH2 HCl
FmocHN O OMe N O e) SmI2, DMA NH N N O f) FmocValOH, Cbz O N N EDC, HOBt O Cbz HO O 45-50 % N NMOM BnO Bn N NMOM BnO Bn 87 86
Figure 1.15 Nicolaou’s heteropinacol coupling to fashion the lower macrocycle.
27 dialdehyde (not shown). The more reactive aldehyde was selectively converted to the oxime upon stirring with MeONH2•HCl in DMSO to give the coupling precursor 86.
With 86 in hand, investigations into the heteropinacol cyclization began. It did not take
much investigation to discover that similar chemistry developed for the synthesis of
nominal diazonamide (see Figure 1.3) was sufficient in this case with only slight
modification. As such, treatment of 86 with a premixed complex of SmI2 (9 equiv) and
DMA (36 equiv) followed by (post work-up) immediate EDC/HOBt peptide coupling
with Fmoc protected L-valine gave rise to 87 in a reproducible 45-50% yield.
Elaboration of the upper oxazole ring, closure to the macrolactam, and appending
a few key functionalities was all that remained to complete the second synthesis of
diazonamide A. To that end, the hindered alcohol of 87 (Figure 1.16) was oxidized using
TPAP, and the resulting ketone was subjected to a Robinson-Gabriel cyclodehydration
with a mixture of POCl3 and pyridine (1:2) to generate the bis-oxazole compound 88 in a
low yield (22%). The low yield is perhaps a consequence of the high strain and steric
hindrance involved in the closure at hand, and the fact that other conditions failed
completely in this system speaks to the utility of the current recipe for other sterically or
highly strained systems.
With the completed heterocyclic core 88 (Figure 1.16) in hand, attention was
focused on the macrolactamization. Amino acid 89 was readily obtained from an acid
mediated acetonide opening, followed by a two step oxidation of the liberated primary
alcohol to the acid and final Fmoc removal in 83% yield. Macrolactamization proved to
be a formidable challenge, as an almost exhaustive survey of peptide coupling reagents
failed to close the desired macrolactam. Eventually, it was discovered that HATU and
28 -4 collidine at 1.0 x 10 M in DMF/CH2Cl2 (1:2) converted the amino acid to the desired macrolactam 90 in a scarce 10-15.
FmocHN FmocHN O O NH a) TPAP O N N N b) POCl3, py N O N Cbz HO Cbz O 22 % O
N NMOM N NMOM BnO Bn BnO Bn 87 88
c) aq. HF d) IBX H2N HO2C HN e) NaClO CbzHN N 2 N N f) Et NH O 2 CbzHN O N g) HATU, collidine O O O 83 % 15 % 11 N NMOM N NMOM Bn BnO Bn BnO 90 89
HN HN CbzHN N CbzHN N N h) H2, Pd(OH)2/C O j) NCS O N Cl i) CbzCl, NaHCO O k) BCl ; NaOH O 3 O 3 O Cl 35 % 75 % N NMOM N NH O H O H CbzO HO 91 92
OH HO 74 O HN H HN CbzHN N N N m) H2, Pd(OH)2/C N O N Cl HO O Cl l) DIBAL-H O n) 74, EDC, HOBt O O Cl O O Cl 56 % 79 % NH N NH N O H O H 93 3
Figure 1.16 Completion of Nicolaou’s second total synthesis of diazonamide A.
Next, in what was nothing short of a miraculous observation, sequential removal of the phenolic benzyl ether with Pearlman’s catalyst followed by Cbz protection not only afforded the desired benzyl protection, but the indoline ring had been oxidized to an
29 oxindole as shown in 91 with an overall conversation of 35%. Mechanistic investigations into this curious event are reportedly underway. Following this fortuitous oxidation, chlorine atoms were regioselectively installed with NCS. The MOM and phenolic Cbz protecting groups were removed with BCl3 followed by NaOH to give the dichlorinated macrocycle 92 in 75% yield. Finally, the same three step sequence employed for the first reported synthesis gave 3 in 44% from 92, thus completing a second independent synthesis of 3 in 40 steps and 0.00011% overall yield.
1.3.3 Harran’s Total Synthesis of Diazonamide A
In 2003 Harran and co-workers published their own route to the real diazonamide
A. While the route to follow differs significantly from their approach to nominal diazonamide A (1a), many lessons learned in the pursuit of the former target proved invaluable for the expeditious completion of 3. Armed with the knowledge that the halogenation and N(2) acyl substitution can occur on a late stage diazonamide core, and the fact that the desired atropisomer C(16)-C(18) can be accessed by forming the D/E biaryl bond intramolecularly within the context of a pre-existing, correctly configured
A/F macrolactam, shaped the design of Harran’s very concise route.
Beginning with condensation of racemic 7-bromotryptophan methyl ester (94,
Figure 1.17) with the acid chloride derived from Cbz-L-Val-OH, 95 was formed in 76% yield. Oxidation (DDQ) and cyclodehydration employing HBr and AcOH produced the coupling partner 96 as the hydrobromide salt in 85% yield. Next, TBTU coupling between salt 96 and the L-tyrosine derived sulfonamide 97 generated the adduct 98 containing all atoms needed for the final aminal 99. In amazingly short order, Harran and
30 co-workers converted 98 to N,O-acetal 99 directly and in 20-25% yield by treatment of the former species with a cold trifluoroethanol solution of PhI(OAc)2. In addition, 99’s
C(10) diastereomer (7-8%) was formed, along with some epimeric spiroindenones (10-
15%). While the yield of this reaction seems at first glance rather poor, its efficiency
(formal direct oxidative cycloaddition) greatly outweighs the loss of 75% of the starting
material. These few steps rapidly moved synthesis
31 a) Cbz-L-Val-OH,
HCl H2N Cl CO2Me HBr H2N N b) DDQ MeO C N CO2Me 2 CbzHN c) HBr in AcOH O O N 76 % N 85 % H H 94Br 95Br 96 N H Br
O
ArO2SHN N N e) PhI(OAc)2 HN N d) TBTU, (i-Pr) NEt, 97 H CO2Me LiOAc ArO2SHN 2 O O O CO Me 91 % 20 -25 % 2 99 HO 98 O N N Br ArO2SHN H O H OH Br
H2N 97 NH HO HCl OAc H 101 OH N
f) PhSH, Na2CO3 g) Teoc-Cl, K CO HN i) TBTU, (i-Pr)2NEt, 101 2 3 TeocHN N h) LiOH j) Ac2O, py HN O N O CO2H TeocHN NH 79% 86 % O O 100 102 O N Br O H N Br O H OH k) DDQ HO l) PPh3, Et3N 74 O m) LiOH/CH3CN p) differential acylation hν (300 nm) q) perchloro-2,4-cyclo n) 4-nitrophenyltriflate hexadiene-1-one H K CO r) (Me N) SSiMe N HN 2 3 TeocHN HN 2 3 3 HO N o) Pd(OH) /C, H N s) DEPC, NMM, 74 N Cl 2 2 N O O O O O 28 % O 33 % O Cl
NH N NH N O H O H 103 3
Figure 1.17 Harran’s complete synthesis of diazonamide A. efforts to late stage manipulations. Three straightforward functional group conversions on 99 (sulfonamide cleavage, Teoc protection and acid hydrolysis) prepared coupling partner 100 in 79% yield. Next, TBTU mediated coupling of 100 and 101 followed by acylation of the phenolic product gave the oxazole precursor 102 in excellent yield. Two step oxidation/cyclodehydration of 102 provided the bis-oxazole (not shown), which was
32 dissolved in aqueous CH3CN containing LiOH and subsequently irradiated at 300 nm to
effect biaryl coupling. The presence of the phenoxy group may seem a bit superfluous,
but additional electron density in the indole subunit reportedly benefited the process to an
extent worthy of spending two extra steps to remove it! As such, conversion of the
phenol to the triflate and subsequent reductive removal affords 103 in 28% yield over
five steps. End game manipulations commenced with differential acylation of the
remaining basic sites, careful regioselective chlorination with perchloro-2,4-
cyclohexadien-1-one, and Teoc deprotection to afford desbromo-diazonamide B (not
shown). Phosphoryl cyanide-mediated condensation of the resultant N(2) amine with the
commercially available 74 delivered (-)-diazonamide A (3) in 33% over the final four
steps. In an exquisite display of effective design and rapid implementation, Harran and
co-workers have demonstrated a concise route to 3 over 19 operations (9 steps – longest
linear sequence) in 0.009% overall yield.
1.3.4 Other approaches to the revised diazonamide A
Several other groups have been able to take the lessons learned from their own
approach to nominal diazonamide and apply them, in some cases, with the same or a
similar key step as in the old route, to an approach toward the revised structure of
diazonamide. Wood and co-workers have published model studies toward the
implementation of their cyclopropanation-ring opening strategy to 3.57 Based on their
original strategy10 and in accord with the revised structure, Wood and co-workers
33
H RHN HN N COOR' N N RHN HN O O O O O O NH N Br N2 H NO N Br 2 O H OH 104 105
a) TBSOTf, N Et3N 2 b) Rh2(OAc)4 O OTBS OTBS 54 % N N O O H H (2 steps) 106 107 N 110 108 H
O N O H TBS 109
Figure 1.18 Wood’s cyclopropanation model studies toward 3.
developed an approach that relies on the conversion of peptide 105 (Figure 1.18) to
indole precursor 104, which could serve as a substrate for tandem cyclopropanation-ring
opening to deliver quaternary C10. In model studies, 3-methyl oxindole (106) was
converted to silylenolether 107, which underwent Rh(II)-promoted coupling with 108 to
give oxindole 110 in good yield. This transformation likely proceeds through a Rh- promoted cyclopropanation followed by ring opening and finally silyl migration (e.g.
109).
In a 2004 disclosure,54 Vedejs highlighted his groups’ efforts toward the biaryl
macrocycle/hemiaminal core. Curiously enough, Vedejs had originally targeted a
bicyclic acetal, as in 2, based on the conjecture that the closed acetal form may be the
biologically active form of diazonamide.5 Consequently, the revised structure of
34 diazonamide A appears to fit well with their original approach, assuming of course, that the bicyclic N,O-acetal would serve in the same beneficial role as did the bicyclic acetal in the old route. To explore this hypothesis, oxindole 111 was prepared in seven steps from
OBn OBn OBn
a) ClCO2allyl, CO Me NaH, DMAP CO2Me b) NaBH4 CO2Me PMBO 2 PMBO PMBO O 99 % O HO N N N H O Br O Br Br O O 111 112 113
e) NaH f) n-BuLi CO2H CO2Me c) Ms2O, Et3N g) ClSnMe3 d) NaOH BnO h) TMSCHN2 BnO N O N O R 93 % Br 67 % SnMe3 O O (3 steps) Pd(PPh ) , 3 4 115 R = CO2allyl 114 1,3-diMethyl- 116 R = H barbituic acid quant.
Figure 1.19 Vedejs’s synthesis of hemiaminal.
N-protected bromosiatin. Acylation of 111 with allyl chloroformate and NaH in the presence of DMAP afforded protected oxindole 112 in nearly quantitative yield. Next, reduction of the amide to the hemiaminal with NaBH4, followed by closure to the
tetracyclic aminal and hydrolysis of the methyl ester proceeded in 93% yield over three steps. Temporary protection of the acid by treatment with NaH, followed by lithium halogen exchange with n-BuLi, an subsequent carbanion quench with trimethyltin
chloride and finally reinstallation of the methyl ester with TMSCHN2 produced stannane
115 in 67% yield over four steps. Deprotection of the allyl carbamate with Pd(PPh3)4 and
1,3-dimethylbarbituic acid gave coupling partner 116 in excellent yield.
35 Initial investigations into the cross coupling of 116 with an appropriately substituted oxazoyl indole triflate met absolutely no success. The prospect of employing stoichiometric palladium was investigated, and after much experimentation, it was discovered that heating the oxazoyl indole triflate (not shown) with Pd(PPh3)4 and benzyltrimethylammonium chloride (chloride source added to improve product stability) at 70 oC in THF provides the easily isolated crystalline palladium complex 117 (Figure
1.20) in 94% after chromatography. Simply heating 116 and 117 in benzene produced
the C16-
N N Me Me a) 116 b) H C=O, MeO2C O O 2 c) LDA (2.5 equiv) AcOH, MeOH NBOC 37 % Cl(Ph P) Pd NBOC 49 % N 3 2 BnO O MOM
117 118
AcHN N N d) KHMDS O O e) 120 O O f) Ac2O NBOC NBOC N N 14 % BnO O MOM BnO O MOM O 121 119 BuOC6H4 P NH O 2 g) POCl , BuOC H 80 % 3 6 4 pyr 120
N N O O
NBOC N BnO O H 122
Figure 1.20 Completion of Vedejs’ heteroaromatic biarylmacrocycle.
C18 (diazonamide numbering) bi-aryl adduct in 54% yield. Subsequent MOM protection of the aminal nitrogen with H2C=O and AcOH in MeOH gave rise to the
36 macrocyclization precursor 118 as a 2:1 mixture of atropisomers in 49% yield over two steps. Treatment of 118 with 2.5 equiv of LDA resulted in macrocyclization to the versatile ketone 119 in 37% yield, presumably through closure of the minor atropisomer
(which does not readily equilibrate at reaction temperatures: -30 oC/-78 oC). Fortunately, in the process of recovering starting material, the atropisomers re-equilibrated and could be re-submitted to the macrocyclization. Preliminary investigations with the diarylphosphinyl hydroxylamine reagent 120 have produced amino ketone 121 in 14%
yield. In a pleasing final result, it was found that treatment of amino ketone 121 with
POCl3 and pyridine smoothly effects dehydration to the oxazole. Investigations toward
an enantioselective route to 122, and further elaboration of 121, are reportedly underway.
Moody is the most recent contributor to the diazonamide literature with a report on studies involving the indole bis-oxazole fragment of diazonamide,44,46 and his most
recent disclosure on biomimetic approaches to diazaonamide A.43 While this work is certainly noteworthy, it fails to add novelty in the context of this exhaustive coverage of the diazonamide literature, and further examples will be forgone.
An interesting story has unfolded over the past fifteen years as the pursuit and eventual synthesis of 1a and the subsequent structural reassignment to 3 reminded the entire scientific community once again of the power and utility of synthesis. The great deal of effort that has gone into the investigation of 1a and 3 has resulted in many lessons learned and has made a noteworthy mark in the history of synthesis, with quite possibly much more to come.
Chapter 2
Studies Toward Diazonamide A
With the backdrop of the current state of affairs in the diazonamide literature
having been set in the previous chapter, herein lie our own multifaceted efforts toward
the revised structure of diazonamide A. The first approach addresses the control of
atropselectivity in the early stages of the synthetic endeavor via influence of a proximal
sp3 stereogenic center. A second approach, supported by model studies, relies on a
photochemically induced rearrangement of a benzotriazole alkene to a 2,2-disubstituted
inolinine, efficiently installing quaternary C(10).
2.1 Construction of Diazonamide-Related Biaryls with Defined Axial Chirality
The control of biaryl atropselectivity persists as an enduring challenge in the
design and implementation of synthetic strategies of structurally and functionally complex natural products presumably biosynthesized via oxidative coupling of arene rings.65 One prominent target in this context is diazonamide A. While the approach of
Feldman and co-workers to 1a was detailed in the previous chapter, herein lie our
redirected efforts toward the revised structure 3 (Figure 2.1) (change in structural
orientation made for clarity and consistency for the following section alone).
Our initial efforts toward 3 relied on two key strategic elements: 1) late stage
biomimetic transannular oxidative cyclization of a macrolactam precursor 123 to set the
38 PO H O H O [O] H O
HN 10 N HN N O O 30 NH H NH H 16 O OH O OH 18 O N O N HN H HN H 24 25 N N Cl 27 Cl 123 Cl 3 diazonamide A Cl
N CO2CH3 O O O
NBOC2 BOCN H 124
Figure 2.1 Retrosynthetic plan for the synthesis of 3.
stereochemistry at both C(10) and the C(24)/C(26) bond and 2) early installation of the
challenging C(16)/C(18) axial chirality via a conformational preference dictated by an sp3 center at C(27) (124, Figure 2.1). Molecular mechanics (MM) calculations indicate a
C(10)/C(30) distance of a mere 3.51Å in the low energy conformer of a benzofuran analog of 123, and we hoped that a similar surmountable distance in 123 will permit oxidative coupling to proceed with ease. This approach would, of course, depend on the successful implementation and maintenance of the correct atropisomer early in the synthetic effort.
The first step in pursuit of this effort was the successful construction of the coupling partners 133 (Figure 2.2) and 139 (Figure 2.3). To that end, indoline (125) was
BOC protected in quantitative yield to give N-BOC-indoline (126). Sequential carbamate directed ortholithiation at C(7), followed by iodine quench provided the N-protected-7- iodoindoline 127 in 69% yield. TFA removal of the BOC protecting group to give indoline 128, followed by exposure to salcomine/O2 oxidation conditions, transformed
128 to the known 7-iodoindole66 (129) in excellent overall yield.
39
BOC2O, s-BuLi, I2, TFA, THF TMEDA, Et2O CH2Cl2 N N N N quant. 60 % 93 % H H BOC BOC 125 126I 127I 128
O O H O salcomine, MnO2, NaCN, MeOH O2, MeOH POCl3, DMF N N N 87 % H 97 % H quant. H I I 129 I 130 131
O O ClOC OBn LHMDS, 132 OBn N 84 % 132 I O 133
Figure 2.2 Preparation of coupling partner 133.
Vilsmeier oxidation to the aldehyde 130 proceeded without incident. Subsequent manganese(IV)oxide oxidation of aldehyde 130 in methanol afforded methyl ester 131 in quantitative yield. Next, indole 131 was deprotonated with LHMDS and the resulting anion was reacted with acid chloride 132 to provide coupling partner 133 in 84% yield.
N N I N BuLi; t-BuLi; TIPSCl I2 TBAF N N N 92 % 82 % 60 % 134H 135TIPS 136 TIPS
O O N I I H I H HMT, BOC2O, propionic acid DMAP N N N 92 % 86 % 137H 138H 139 BOC
Figure 2.3 Preparation of coupling partner 139.
Our attention was then drawn to the synthesis of the known 4-iodoindole-3-
carboxaldehyde (138, Figure 2.3).67 Pursuant to this goal, C2 of gramine (134) was
40 sterically obstructed with installation of the bulky TIPS protecting group on nitrogen to give 135 in excellent yield. Next, lithiation at C4, followed by an iodine quench, afforded 4-iodogramine (136) in 82% yield. Straight forward TBAF de-silylation of 136 produced 137, which underwent subsequent conversion to the C2 aldehyde 138 in excellent yield using the hexamethylenetriazine (HMT) method developed by Snyder.68
Final BOC protection of 138 cleanly produced the second coupling partner 139 in 86% yield.
With both 133 and 139 in hand, efforts were directed toward the Negishi coupling69 to secure the pivotal C(16)/C(18) biaryl bond (Figure 2.4). Exposure of the
derived zincate, prepared by treatment 133 with i-PrMgCl at low temperatures followed
by addition of ZnBr2 and warming to room temperature, to 4-iodoindole 139 under
palladium catalysis resulted in the formation of the desired 4,7’-bis indole product 140 in
good yield. Through the course of investigation of the Negishi coupling, optimum yields
were achieved with the use of P(2-furyl)3 as the stabilizing ligand, in contrast to the P(t-
Bu)3 ligand favored in previous studies with the salicylate-derived zinc reagent 41 (see
Figure 1.9). Hindered aldehyde 140 was then subjected to DBU assisted Horner-
Wadsworth-Emmons olefination with known phosphonate 14270 to give Z-alkene 141 in
76% yield. For the installation of the key C(27) stereogenic center, alkene 141
underwent rhodium(I)-mediated asymmetric hydrogenation with (R,R)-DuPHOS as a
source of chirality to provide the desired tryptophan derivative in good yield.
41
O O i-PrMgCl; ZnBr2; Pd2(dba)3/P(2-furyl)3 BnO DBU, BnO 139 CO CH CO2CH3 142 N 2 3 OBn O N O N 73 % 76 % I O BOCN BOCN 133 CHO 140 141 CO Bn BOCHN 2 PO(OCH3)2
BOCHN CO2Bn 142 1) Rh[(R,R)-DuPhos] H2 2) BOC O, DMAP HO 2 DCC,DMAP, CO2CH3 3) Pd/C, H2 O N DMAP HCl N CO2CH3 O O O 44 % 65 % NBOC2 BOCN BOCN H 143 H 124 CO2H BOC2N
Figure 2.4 Completion of target 124.
Protection of the NHBOC functionality with a second BOC group, followed by palladium catalyzed hydrogenolysis of the benzyl protecting groups gave the free phenoxy acid 143 in 44% over three steps. The use of the (R,R)-DuPHOS reagent precipitated out of necessity for the (R)-configuration at C27 to lock the biaryl linkage in the desired (R)- axial chirality. As was the case with 46, biaryl 143 existed as a 1.5:1 mixture of diastereomers.
Finally, the phenoxy acid 143 was lactonized employing the Steglich esterification conditions as modified by Keck, to give the cyclized product 124 in good yield. Single crystal X-ray analysis provided evidence in support the gross structure and stereochemistry shown. As was observed in the case of 46, the presumably equilibrating mixture of diasteriomers 143 seem to have selectively cyclized through only one atropisomer, this time with a proton rather than the NBOC2 group directed toward the
42 adjacent C2 indole hydrogen. This bias was achieved through the steric bulk built into the system with the addition of a second BOC on the tryptophan nitrogen. In an earlier experiment with the mono-BOC compound, cyclization occurred to give a 1:1 mixture of isomers. A disappointing finding was uncovered when exposure of 124 to chiral HPLC analysis using a ss-Welk-01 column and eluting with 95:5 hexane/2-propanol revealed
(indirectly) the enantioselectivity of the DuPHOS-mediated hydrogenation. The fact the lactone 124 exists as a 75:25 mixture of enantiomers suggests that further pursuit of this approach would require addressing the enantioselectivity of the asymmetric hydrogenation. However, at this point in our studies, the approach at hand was set aside to enable pursuit of a more promising and potentially asymmetric approach to diazonamide A.
2.2 Benzotriazole Alkene Photoisomerization Approach to Diazonamide A
Examination of the central stereogenic center (C(10)) of target 3 reveals a sterically congested environment; consequently, a sound approach to generate this center, especially in the late stages of a synthetic endeavor, should minimize steric interactions.
Such an approach was envisioned in the context of the photochemically induced expulsion of nitrogen from an appropriately substituted benzotriazole alkene 146 (Figure
2.5) followed by closure to a useful indolenine 144 through the incipient diradical 145, a
43
CO2Et CO2Et O N N N N TIPSO TIPSO O N O O N O O O NH NH NH O TIPSO
NBOC2 NBOC2 NBOC2 144 145
O O O O N (EtO) P 2 O N O O NH N N O N N O NH TIPSO N O TIPSO N NBOC2 NBOC2 148 147 146
OH OTIPS ZnBr I O H N NH2 CbzHN O HCl H2N O O HO C O BnO C 2 2 149 150 151 152 O NH2 NBOC2 $$
Figure 2.5 Retrosynthesis of benzotriazole alkene strategy. precedented process71,72 embodying both a low energy of activation and minimal influence by steric hindrance upon diyl closure. Benzitriazole alkene 146 was envisioned to arise from phosphonate 147 and ketone 148, which could come from the union of 149
and 150, derivatives of the commercially available starting materials 151 and 152,
respectively.
Prior to investigating this transformation in the context of the real system (vide supra), we decided to test the feasibility through a model study. Progress toward this
44 goal began with the TIPS protection of commercially available 2-hydroxy-5-
1) IBC, NMM, NH3 O Cl O 2) ethyl glyoxalate/ H O (EtO) P(O)H, P 2 O refluxing acetone N Na metal BOCHN O N OH 3) Ac2O, pyr N BOCHN O O N N 72 % N O 70 % N 153 154 O 155 156
H O LHMDS; OH O TIPSO O TIPSO H N TIPSCl, TMSC(Li)N2 ZnBr2; BOCHN O 154 H imid H THF O 94 % 90 % 84 % OTIPS 157 158 159 160
N O N O BOCHN BOCHN n-BuLi, DMF, K2CO3 O O Dess-Martin O O 156 R 84 % 67 % O 74 %
OTIPS OTIPS SeO 2 161 R = H 163 60 % 162 R = OH
O O N N O BOCHN O BOCHN O N O O H O hν, 300 nm BOCHN N N O N N 71 % N OTIPS OTIPS OTIPS 164 165 166
Figure 2.6 Model benzotriazole alkene photo-rearrangement. methylbenzaldehyde (157, Figure 2.6) to give aldehyde 158 without incident. Exposure of 158 to Colvin’s homologation conditions73 gave rise to acetylene 159 in excellent
yield. Next, the in situ generated zincate derivative of 159 was combined with the valine derivative 154, formed in three straightforward steps from commercially available BOC- valine, to give the coupled acetylene 160 in very good yield. Whereas the acetate may not be the most intuitive substitution partner, initial explorations with the unstable
45 chlorine (vs. the acetate) derivative afforded no alkyne addition products. Potassium carbonate initiated 5-exo-dig cyclization of alkyne 160, followed by rearrangement of the resultant five membered heterocycle to the aromatic oxazole afforded 161 in 84% yield.
Treatment of 161 with selenium dioxide and crushed activated molecular sieves in refluxing 1,4-dioxane incompletely oxidized the doubly activated (e.g., di-aryl) benzylic methylene to a mixture of the alcohol (162) and the ketone (163). Efforts to drive this reaction to completion were unsuccessful; therefore a two step protocol is employed. The crude alcohol/ketone mixture was immediately submitted to Dess-Martin periodane oxidation conditions to furnish ketone 163 in an acceptable 49% yield over two steps after purification.
With ketone 163 in hand, attempts to prepare the desired benzotriazole alkene
164 began. Initial efforts focused on Peterson olefination74 with the known 1-
(trimethylsilyl)-1H-benzotriazole (not shown), which provided only trace amounts of the desired alkene. Fortunately, Horner-Wadsworth-Emmons olefination with phosphonate
156, available in one step from commercially available 1-(chloro)-1H-benzotriazole
(155), after n-BuLi deprotonation and condensation with 163, proceeds in very good yield to give the alkene 164. Much to our delight, the key cyclization required very little exploration. Our first experiment was performed in benzene and resulted in complete decomposition of the starting material. In the second experiment attempted, cyclohexane was the chosen solvent, and resulted in 29% yield of 166. Changing solvent from
cyclohexane to acetonitrile doubled the yield to 61% with decomposition accounting for
the remainder of the material. While there are obvious rigidity differences between 164
46 and 146, effectiveness of this strategy in a system of comparable sterics and electronics armed us with the confidence needed to pursue this line of attack on the real system.
1) CbzCl, K2CO3 2) ethyl glyoxalate/ H O refluxing acetone N 3) Ac O, pyr CbzHN O NH2 2 HCl H2N O O O 79 % 152 150 O
NH4OH, I2, BOC2O, NaOH, BnBr, NaHCO3, 95 % EtOH NH2 H O, dioxane NHBOC NH2 2 DMF CO H CO H CO H 97% HO 2 92 % HO 2 75 % HO 2 I I 151 167 168
BOC O, NHBOC TIPSCl, NHBOC 2 NBOC2 imid, DMF DMAP, toluene CO2Bn CO2Bn CO2Bn HO quant TIPSO 79 % TIPSO I I I 169 170 171 O H N ethynylMgBr, CbzHN O ZnBr2, Pd(PPh3)4, LHMDS, ZnBr2; O NBOC2 DMF 150, THF CO2Bn 66 % TIPSO 50 % TIPSO (76 % based on 172 recovered S.M.) 173 H BnO2C NBOC2
Figure 2.7 Initial stages of our synthetic effort to 3.
Implementation of this strategy (vide supra) first required the construction of macrolactam 148. With this goal in mind we began with 3-iodo-L-tyrosine. While this compound is available commercially, at the time of this work it was only available in small quantities at great expense. Fortunately, we were able to prepare tens of grams of
3-iodo-L-tyrosine ((167), Figure 2.7) with little effort and to greatly reduced cost, by simple iodination of L-tyrosine (151) in nearly quantitative yield. Protection of the amino functionality of 167 with BOC, followed by benzylester formation and finally
47 silylation of the phenol afforded the seemingly completely protected iodo-tyrosine derivative 170 in 69% over three steps. Early efforts carried the mono-BOC protected derivative through to the coupling reaction with the valine derivative, but unforeseen difficulties required the installation of the second BOC to fully protect the tyrosine functionality. Initial attempts at forming di-BOC protected 171, employed a standard di-
BOC protection protocol, i.e. addition of BOC2O to a stirring solution of 171 and DMAP
in CH3CN. Unfortunately, while effectively di-BOC protecting 171, these conditions
also rapidly removed the TIPS protecting group. Reasoning a less polar solvent would
not stabilize a siliconate ion intermediate in this deprotection, we tried the experiment in
the less polar solvent toluene. We were pleased to find that gently heating (40 oC) a
solution of 170, BOC2O and DMAP in toluene smoothly installed the second BOC
protecting group while preserving the phenolic TIPS protecting group.
With the aryl iodide 171 in hand, efforts toward elaborating the oxazole ring ensued. Initial advances were made by standard Sonogashira coupling of 171 with TMS- acetylene. However, difficulties in selectively removing the TMS group lead us to an alternative approach. A priori, the simplest line of attack would have been to simply couple 171 with an unprotected acetylene source. To accomplish this goal, preparation of the acetylene zincate (in situ) from the commercially available Grignard reagent, followed by treatment with Pd(PPh3)4 and aryl iodide 171 generated the desired alkyne
172 in an acceptable 66% yield. The next challenge was to couple the alkyne with valine
derivative 150, available in 79% over three steps from commercially available L-
valinamide•HCl. As previously mentioned, our first pass through this scheme carried the
mono-BOC alkyne (not shown) into the coupling with 150. With the precedent of the
48 coupling between alkyne 159 and valine derivative 154 in our model system, we did not anticipate any difficulty in this transformation with a mono-BOC protected alkyne. In practice, however, we found that preparation of the acetylide ion in the presence of the acidic carbamate NH of the mono-BOC compound was a futile effort. Presumably, the formed acetylide ion rapidly deprotonates the carbamate nitrogen, rendering the alkyne useless. Attempts to form the dianion, and subsequently couple the alkynyl zincate with the L-valine derivative 150, failed. These findings brought us to the di-BOC alkyne 172 currently at hand. Fortunately, di-BOC protection proved to be the key adjustment needed to successfully couple the in situ prepared zincate of 172 to valine derivative 150 to generate oxazole precursor 173 in 50% yield, 76% based on recovered starting material.
O O
N O N O
Cs CO , DMF Pd/C, H , THF 2 3 CbzHN O 2 H N O 173 2 93 % TIPSO 98 % TIPSO
174 12-94 % 175 BnO2CNBOC2 HO2CNBOC2 coupling conditions O coupling conditions
N O O O BOC2N O N O H TIPSO N
HN O O OTIPS H OTIPS O N NBOC2 BOC N O 2 O N 176 148 O
Figure 2.8 Dead end to macrolactam dimer.
49 Following the precedent from our model system (Figure 2.6), treatment of alkyne
173 (Figure 2.8) with K2CO3 gave the oxazole 174 in yields ranging from 39% to 54%.
However, simply substituting Cs2CO3 for K2CO3 in DMF increased the yield of 174 to
93%. With a reliable route to the protected amino acid 174, our attention was drawn to
macrolactamization. Toward this goal, the amino acid moiety of 174 was unmasked by
palladium-mediated hydrogenolysis to give 175 in nearly quantitative yield. At the time
this work began, the difficulties encountered within the context of the various efforts
toward diazonamide synthesis, had not been published. Still, we anticipated some level
of difficulty in closing the 12-membered lactam. Initial experiments with the powerful
benzotriazole coupling agent PyBOP employing a myriad of bases gave no cyclized
product. Taking a step back to the more conventional dicyclohexyl carbodiimide (DCC)
with DMAP and DMAP•HCl resulted in no cyclized product. However,
ethyldimethylaminopropylcarbodiimide (EDC) with 1-hydroxy-7-azabenzotriazole
(HOAt) gave the first taste of success, affording what appeared in all respects to be the
cyclized product 148 in 15% yield. Failure to improve this yield with carbodiimide based
methods lead us to investigate the phosphonate coupling agents. Attempted cyclization
with diphenylphosphoryl azide (DPPA) and Et3N gave no detectable cyclized product. pentafluorophenyl diphenylphosphinate (FDPP), on the other hand, gave the best results to this point in converting the amino acid to the presumed macrolactam 148 in 20% yield.
The very next experiment with diethylcyanophosphonate (DEPC) doubled the yield to
42%. This increase in yield gave us comfort that the macrolactamization would not be an impassible bottleneck in the synthetic effort to 3. Thus, we move onto the next set of
50 tasks: oxidation of C(10) (diazonamide numbering), followed by preparation of the benzotriazole alkene, and finally photochemical cyclization to the quaternary C(10).
Just as efforts toward the benzotriazole alkene got underway, Nicolaou published his second synthetic route48 to 3, which contained a similar macrolactamization to ours.
Employment of his cyclization conditions: HATU, 2,4,6-collidine in 2:1 CH2Cl2/DMF solution dramatically increased our yields to the high 80’s and low 90’s (depending on protecting groups present). The prospect for carrying enough material through the macrolactamization to late stage synthetic efforts looked very promising. During efforts to bring tens of grams through the macrolactamization, it was discovered that the mass spectrum obtained from the presumed 148 had a peak at M+H and then another peculiar peak at M+(H/2), it was doubly charged! A sample of assumed 148 was subsequently reexamined via mass spectrometry paying particular attention to the region beyond 1000 amu. Much to our discouragement, a peak equal to (Mx2)+H appeared quite prominently. We had been unknowingly been working with the dimer 176 not the macrolactam 148.
The revelation that we had the dimer 176 in hand in such good yield is not surprising in hindsight, especially in light of similar difficulties encountered by Nicolaou and co-workers.30,48-53 With the number of unsuccessful macrolactamization experiments run with C(10) methylene 175, even at 0.5 mM concentration, we had little hope of gaining much out of the use this particular compound. We did, however, make several attempts to resurrect the route through 175 by simulating high dilution conditions. Initial attempts to accomplish this goal focused on solid phase coupling agents such as polymer- bound EDC. Unfortunately, efforts to simulate high dilution conditions with polymer
51 supported coupling agents failed to provide any macrolactam. As a second order approach to this strategy, we appended the macrolactam to a Merifield resin as a means to simulate high dilution conditions, but cyclization did not occur after amino acid deprotection and subjection to coupling conditions.
Having exhausted attempts with 175, we decided to functionalize C(10) as a means to effectively remove a degree(s) of rotation in this large amino acid, and to possibly invoke a Thorpe-Ingold type effect to bring the two distal ends closer in proximity. The first C(10)
O O O
N O N O N O OTBS O O O O O H2N H2N H2N O TIPSO TIPSO TIPSO
177 178 179 HO2CNBOC2 HO2CNBOC2 HO2CNBOC2
O O O
N O N O N O N O N O O H2N H2N H2N TIPSO N N TIPSO HO2C NH O NBOC2 180 181 182 HO2CNBOC2 HO2CNBOC2
Figure 2.9 Other substrates used in attempted macrolactamizations. functionalized adduct to be subjected to coupling conditions was the C(10) OTBS ether
177. However, only 26% of the dimer was recovered from this experiment. Ketone 178 and dimethyl acetal 179 both shared the same fate, and only oligomeric products were suggested by TLC analysis. Unfortunately, independent treatment of amino acids 180,
52 181, and 182 with our best coupling conditions (HATU, collidine) produced no detectable cyclized products; presumably only oligomeric products were formed.
While much progress was made toward 3 and many synthetic challenges were overcome, the prospect of surmounting the very difficult feat of forming the 12- membered macrolactam was not very promising. In hindsight, perhaps a better line of attack would have been to form a larger macrocycle and in an energetically favorable process, close the macrocycle down to the 12-membered lactam. While attention was not directed completely away from our goal of preparing 3, at this point, a majority of our efforts were directed toward a new and exciting challenge before us, probing the mechanistic pathway by which diazoparaquinone natural products might exert their profound cytotoxicity.
53
Chapter 3
Diazoparaquinone Natural Products: Background and Significance
3.1 Isolation, History and Related Compounds
The diazoparaquinone family of natural products have been known since their first observation in 1970 by Omura and co-workers,75-78 who isolated “an orange
crystalline antibiotic,” kinamycins A-D (183-186, Figure 3.1), from the culture broth of
Streptomyces murayamaensis sp. nov. Hata et Ohtani. The skeletal structure of these kinamycins was originally designated as N-cyanobenzo[b]carbazoles (shown in Figure
3.1), based on the misinterpretation of an X-ray structure.
3 R4O OR O CH3 OR2 N OR1 OH O CN
183 kinamycin A R1 = Ac, R2 = Ac, R3 = Ac, R4 = H 184 kinamycin B R1 = H, R2 = Ac, R3 = H, R4 = H 185 kinamycin C R1 = Ac, R2 = H, R3 = Ac, R4 = Ac 186 kinamycin D R1 = Ac, R2 = H, R3 = Ac, R4 = H
Figure 3.1 Originally proposed structure for kinamycins A-D.
This structural assignment was accepted as fact for all synthetic and biological
investigations for nearly a quarter century. However, in 1994, following Echavarren’s
synthesis of the N-cyanobenzo[b]carbazole prekinamycin79 (187, Figure 3.2) a compound
whose spectral data did not match those of the natural product, Gould80 and Dmitrienko81 published successive communications revising the skeleton from an N- cyanobenzo[b]carbazole (187) to a N-diazobenzo[b]fluorene (188, Figure 3.2). The story
54
HO HO O HO O O
N OH O OH O CN OH O N2 N2 187 originally porposed 188 prekinamycin 189 isoprekinamycn: original structure of prekinamycin revised structure isolate identified as prekinamycin Echavarren 1994 Hauser 1996
Figure 3.2 Various historical depictions of prekinamycin. was not complete however, as a second structural revision was looming on the horizon.
In 1996, Hauser prepared prekinamycin (188, Figure 3.2),82 but direct comparison to
“prekinamycin” isolated from S. murayamaensis revealed that the two compounds were different, but the synthesized material was identical with a previously uncharacterized isolated compound from this microorganism. Simultaneously, Gould and co-workers83 were in the midst of an investigation that led to the same conclusion (vide supra), and to the discovery of a sample of true prekinamycin 188 in extracts of S. murayamaensis mutant MC2.
The true skeletal arrangement of the “original presumed prekinamycin” has recently been determined to be 189 (Figure 3.2) in a cooperative effort from Gould and
Demitrienko.84 It has been coined isoprekinamycin and its structure corresponds to a diazobenzo[a]fluorene vs. the more prevalent diazobenzo[b]fluorene. While the structure of isoprekinamycin still remains to be rigorously established by total synthesis, 30 years of well-deserved attention appears to have solved the structural mysteries within the diazoparaquinone natural products.
Over the past 35 years, the diazoparaquinone family of natural products has grown to include: kinamycins 190-206 (Figure 3.3), all varying as per the highly
oxygenated D-ring functionality or oxidation level, the biosynthetic precursor
55 prekinamycin (188),83,85,86 and lastly, the dimers lomaiviticin A (207) and B was recently added to the list by He and co-workers.87
3 R4O OR O 3 4 CH3 5 D 2 OR2 ABC 1 1 11 OR HO 9 10 O OH O N2
190 kinamycin A R1 = Ac, R2 = Ac, R3 = Ac, R4 = H 1 2 3 4 191 kinamycin B R = H, R = Ac, R = H, R = H OH O N2 1 2 3 4 192 kinamycin C R = Ac, R = H, R = Ac, R = Ac 188 prekinamycin 193 kinamycin D R1 = Ac, R2 = H, R3 = Ac, R4 = H 194 kinamycin E R1 = H, R2 = H, R3 = H, R4 = Ac OH 195 kinamycin F R1 = H, R2 = H, R3 = H, R4 = H H3CO 196 kinamycin G R1 = Ac, R2 = Ac, R3 = COi-Pr, R4 = Ac 2 197 kinamycin H R1 = Ac, R2 = Ac, R3 = H, R4 = COi-Pr O OH O O O 198 kinamycin I R1 = Ac, R2 = COi-Pr, R3 = H, R4 = COi-Pr 199 kinamycin J R1 = Ac, R2 = Ac, R3 = Ac, R4 = Ac 200 FL-120 A R1 = H, R2 = Ac, R3 = COi-Pr, R4 = O 201 FL-120 C R1= H, R2 = Ac, R3 = H, R4 = COi-Pr OH O N2 202 FL-120 C' R1= H, R2 = Ac, R3 = H, R4 = COEt O 203 FL-120 D' R1= H, R2 = H, R3 = H, R4 = COi-Pr 207 lomaiviticin A OH 204 FL-120 B R1= H, R2+ R3 = 2,3-oxirane, R4 = Ac N(CH3)2 205 FL-120 B' R1= H, R2+ R3 = 2,3-oxirane, R4 = COi-Pr 206 keto- R1= H, R2+ R3 = 2,3-oxirane, R4 = 1-C=O anhydro-kinamycin
Figure 3.3 Inclusive representation of known diazoparaquinone natural products.
In addition to the isolation of the diazoparaquinone natural products (vide supra), many naturally occurring benzo[b]fluorenes have been isolated from S. murayamaensis, several of which play a key role in the biosynthesis of the kinamycins. Figure 3.4 presents several of these compounds: kinobscurinone (211),88 kinafuorenone (212),89 stealthin A
(213),90 stealthin B (214),90 stealthin C (215),91 seongomycin (216),92 and cysfluorentin
(217) 90.
56 HO HO HO O OH OH R
OH O O MeO OH O OH O NH2 211 kinobscurinone 212 kinafluoenone 213 R = CH2OH stealthin A 214 R = CHO stealthin B 215 R = CH3 stealthin C HO OH OMe O OH
O S NHAc OH O S N HOOC MeO OH O H 216 seongomycin OH 217 cysfluorentin
Figure 3.4 Benzo[b]fluorene natural products isolated from S. murayamaensis.
The rarity of the diazo functionality outside of the diazoparaquinone family of
natural products is exemplified by the fact that, to the best of our knowledge, the current body of literature harbors only five other diazo natural products (189, 218-221, Figure
3.5).84,93-97 While none of these compounds shares the diazoparaquinone functionality
with the kinamycins and lomaiviticins, they do posses antitumor and/or antibiotic
activity, presumably through chemistry involving the reactive N2 moiety.
57
O HO O O O N2 N2 X OH OH O O N O NH2 N2 H O 219 X = O azaserine 189 isoprekinamycin 218 lagunamycin 220 X = CH2 6-diazo- 5-oxo-L-norleucine
OH O
221 SF2415A2 + 4 similar O congeners with further O functionalization at the epoxide and side chain N2 O
Figure 3.5 Diazo-containing natural products lacking the paraquinone moiety.
3.2 Biological Activity
Kinamycins have strong activity against Gram-positive, and to a lesser extent,
Gram negative bacteria (Table 3.1).75 Kinamycina B (191) and D (193) are more
Table 3.1 Kinamycin A-D activities against a section G+ and G- bacteria.
Minimum Inhibitory Concentration (μg/mL) Test organism A - 190 B - 191 C - 192 D - 193 Bacillus subtilis PCI-219 0.024 0.012 0.19 0.012 Bacillus anthrasis 0.19 0.012 0.19 0.024 Staphylococcus aureus FDA 209P 0.78 0.012 0.78 0.024 Staphylococcus albus 0.024 0.012 0.39 0.024 Mycobacterium 607 25 6.25 6.25 6.25 Escherichia coli NIHJ > 100 3.12 > 100 12.5 Klebsiella pneumonia >100 12.5 > 100 25 Pseudomonas aeruginosa P-1 > 100 > 100 > 100 > 100 Salmonella typhosa 901W > 100 6.25 > 100 12.5
58 active than A (190) and C (192). This evidence suggests a structure-activity relationship; as the number of acetates on the D-ring decreases the anti-microbial activity increases.
No biological activity studies have been performed for kinamycins E (194) and F (195).
Kinamycins are also reported to have weak antitumor activity against Ehrlich ascites carcinoma cells at 0.1 mg/kg (kinamycin C), and sarcoma-180, with an acute intravenous
76 toxicity (LD50) (190-193) in mice of 30-40 mg/kg. The dimeric diazobenzo[b]fluorene
glycoside lomaiviticin A (207) is reportedly active against Gram-positive bacteria as well
as variety of tumor cell lines (Table 3.2).87 Additionally, as an
Table 3.2 Brief survey of lomaiviticin A’s anti-tumor activity.
cell line Minimum Inhibitory Concentration (μg/mL) leukimia HL60 0.0009 lung A549 0.0048 brain T47D 0.025 colon HCT15 0.0082 ovarian A2780DDP 0.0065
allusion of things to come, in an “on-going study,” 207 reportedly cleaved double-
stranded DNA under reducing conditions. The intriguing diazoparaquinone functionality as well as the noteworthy biological activity has certainly warranted the synthesis pursuit and further biological study of the kinamycins.
3.3 Synthesis Efforts Toward Diazoparaquinone Natural Products
A great deal of synthesis effort has been exerted toward synthesis of the kinamycins since their isolation in 1970. Obviously, many of the earliest routes were
59 directed toward the synthesis of the N-cyanobenzo[b]carbazole moiety.79,98-103 While
these early efforts played a key role, along with some insightful analysis of original data,
in correctly identifying the kinamycins as diazobenzo[b]fluorenes, these efforts will not
be covered in any further detail. Since the structural revisions of the mid-nineties, there
have been many reports toward both the biosynthetic precursors (vide infra) and towards
the revised diazo structure of the kinamycins, many of which targeted or passed through
the benzo[b]fluorene core structure.
3.3.1 Hauser’s Synthesis of the Structure Proposed for Prekinamycin
The first total synthesis of prekinamycin was completed in 1996 by Hauser and
Zhou.82 The route began with an intramolecular Friedel-Crafts rearrangement
o 104 (NaCl/AlCl3, 180 C) of the previously known dihydrocoumarin 222 (Figure 3.6), followed by methylation with dimethylsulfate to give indanone 223 in 60% yield over two steps. Next, 223 was converted to the silyl enol ether (not shown) and subsequently treated with stoichiometric Pd(OAc)2 to furnish the unstable indenone 224.
a) NaCl, AlCl3 O OMe OMe SO Ph b) Me2SO4, c) TMSOTf, Et3N 2 O K2CO3 d) Pd(OAc)2 O 60 % 75 % O O OMe O 222 223 224 225 f) BBr MeO 3 HO HO g) H2NNH2 O e) LiOt-Bu h) Ag2CO3/Celite 73 % 38 %
MeO OH O OH O N2 226 188
Figure 3.6 Hauser’s synthesis of prekinamycin.
60 Immediate condensation of the anion of the commercially available phthalide sulfone 225 with indenone 224 gave the benzo[b]fluorene ketone 226 in 73% yield. Treatment of 226 with BBr3 gave the tetraol (not shown), which was sequentially reacted with anhydrous
hydrazine and Fetizon’s reagent to afford 188 in 38% yield over the final three steps.
3.3.2 Gould’s Synthesis of Kinobscurinone (Improvements and Extension to the
Synthesis of Stealthin C)
In the first synthesis approach to kinobscurinone (211, Figure 3.7), Gould and co-
workers envisioned fashioning the ABD ring through the coupling of a cyanophthalide
230 with an appropriately substituted cinnamate 229. The first step in realizing this
strategy was the global aromatic bromination of 2,5-dimethylphenol (227) to give the
perbrominated intermediate (not shown) which was immediately treated with hydroiodoic
b) K2S2O8,CuSO4,py; malonic acid, py; CN a) Br2, AlCl3; HI; OMe OH OMe c) LDA, O Me2SO4, NaOH CH2N2 2 O 70 % 35 % 68 % Br O Br MeO O 227 228 229OMe 230
MeO d) K2S2O4; MeO HO O MeO O Me2SO4/K2CO3 D e) t-BuLi f) BBr3, air A B Br 40 % 89 % CO2Me MeO O MeO OMe O HO O O 231 232 211 kinobscurinone
Figure 3.7 Gould’s synthesis of kinobscurinone. acid to give the monobromide (not shown). Methylation of the bromophenol with dimethyl sulfate then generated methyl ether 228 in 70% over three steps. After a great deal of effort, it was found that treatment of 228 with K2S2O8/CuSO4 in the presence of
61 pyridine gave the desired aldehyde (not shown). Knoevenagel condensation of the aldehyde with malonic acid gave the cinnamic acid, which was converted to the methyl ester by treatment with diazomethane to give 229 in 35% over two steps. Annulation to form the ABD ring system transpired upon treatment of cinnamate 229 with the lithium anion of 230 to furnish cleanly naphthoquinone 231 in 68% overall yield. Quinone 231 was reduced and methylated in situ (Na2S2O4/dimethylsulfate/K2CO3) to give the
dimethyl ether, which was treated with t-BuLi to effect lithium halogen exchange and
subsequent cyclization to the fluorenone 232 in good yield. Final deprotection of the
tetramethyl ether 232 with BBr3 gave the target 211 in 89% yield. While effective in
reaching the goal of fashioning 211, this route suffered from several mass-limiting steps.
The problems were addressed and subsequently corrected in a revised route to
kinobscurinone, Figure 3.8.
b) malonic acid, py, CN piperdine; OMe OMe OMe c) LDA, O a) K2S2O8, CuSO4 SOCl2, CH2N2 2 O 70 % CHO 78 % O MeO O 233 234 235OMe 230
MeO MeO e) PPA MeO O MeO MeO d) Na2S2O4; f) Me2SO4, Me2SO4/K2CO3 K2CO3 62 % 72 % CO2Me CO2Me MeO O MeO OMe MeO OMe O 236 237 h) NH2OH HCl 232 g) BBr3 99 % 80 %
HO MeO HO OH MeO O i) BBr3 j) Na2S2O4 80 % OH O NH2 MeO OMe NOH HO O O 215 stealthin C 238 211 kinobscurinone
Figure 3.8 Gould’s revised route to 211 and divergence from 232 to stealthin C.
62 Gould’s second route to kinobscurinone98 began with the oxidation of 233 (Figure
3.8) to afford aldehyde 234 in good yield. As in the previous route, Knoevenagel
condensation of the aldehyde 234 with malonic acid gave the cinnamic acid, which was
converted to the methyl ester by treatment with thionyl chloride followed by
diazomethane to give 235 in 78% over two steps. Again, reaction of cinnamate 235 with the lithium anion of 230 cleanly furnished the annulated naphthoquinone 236, which was subsequently reduced and methylated to give the benzofluorene precursor 237 in 62%. In
the crowning achievement of this approach, treatment of 237 with polyophosphoric acid,
followed by methylation or the resultant phenol furnished the ABCD ring system of 232
in 72% yield. Simple demethylation with BBr3 gave rise to kinobscurinone 211 in 80%
yield. In a later disclosure,91 Gould and co-workers capitalized on the versatility of
ketone 232 by treating it with hydroxylamine hydrochloride to give oxime 238 in nearly quantitative yield. Finally, demethylation of 238 with BBr3 followed by oxidation of the
resultant dihydroquinone with dithionite provided 215 in very good yield. In addition to
the synthesis of stealthin C, this report demonstrated the role of 215 in the kinamycin
biosynthesis.
3.3.3 Snieckus’ Synthesis of Kinobscurinone
Snieckus and co-workers formulated an approach to 211 based on a key remote-
carbamoyl migration reaction.105 En route to the pivotal intermediate 242 (Figure 3.9),
the ABD ring system 241 was prepared in excellent yield by the cross coupling of
bromonaphthalene 239 with boronic acid 240. Protection of the most reactive metalation site of 241 via deprotonation with s-BuLi followed by a silyl chloride quench of the
63 resulting aryl anion lead to silylamide 242 in nearly quantitative yield. Next, in the key transformation, treatment of 242 with LDA effected the critical O→C ring-to-ring carbamoyl transfer in good yield. Methylation of the resulting phenol with NaH and MeI cleanly gave 243 in 61% over two steps. Exposure of 243 to excess LDA effected a second remote-metalation-cyclization to furnish fluorenone 244 in 78% yield based on recovered starting material. The synthesis was completed by treatment of 244 with TFA which gave rise to kinobscurinone (211) in quantitative yield.
a)
B b) s-BuLi, OMe OMe OMe O OCONEt2 C TMEDA Br 240 3 c) Me3SiCl SiMe AB 3 O O O O Pd(PPH3)4, K3PO4 97 % toluene/ EtOH/H O MeO OMe 2 MeO OMe NEt2 MeO OMe NEt2 239 95 % 241 242
SiMe3 MeO MeO SiMe3 HO d) LDA OMe MeO O e) NaH, MeI, DMF f) LDA g) TFA, air
61 % NEt2 78 % quant. O MeO OMe O MeO OMe O HO O 243 244 211 kinobscurinone
Figure 3.9 Snieckus’ synthesis of kinobscurinone.
3.3.4 Jones’s Synthesis of the Benzo[b]fluorenone Core Structure
Taking a different approach, Qabaja and Jones utilized a Pd-mediated closure of aryl iodide 249 (Figure 3.10) to secure the C ring of benzo[b]fluorenone skeleton.106
Progress toward benzo[b]fluorenone 232 (Figure 3.10) began with the preparation of iodoaldehyde 246. To that end, dimethylanisole (245) was regioselectively brominated
64 with NBS, followed by lithium halogen exchange (n-BuLi) and subsequent iodine quench of the intermediate aryl anion to generate the iodoanisole (not shown). The aryl iodide was then subjected to Swern oxidation conditions which gave rise to the aldehyde 246 in
30% yield over three steps. Attention then turned toward the complementary coupling partner 248. Reduction of methyl juglone (247) with sodium dithionite followed by methylation of the resultant dihydroquinone with dimethylsulfite gave the fully protected dihydroquinone, which was brominated with Br2, and reprotected to afford aryl bromide
248, in 84% yield over two steps. Aldehyde 246 was subjected to 1,2-addition of the
lithioarene derived from aryl bromide 248 effectively uniting all atoms needed for the
palladium mediated coupling. The resulting benzylic alcohol was oxidized with PCC to
give ketone 249 in 80% yield over two steps.
a) NBS, K2CO3 d) Na2S2O4, OMe OMe O OMe b) n-BuLi, I2 Me2SO4 c) COCl2, DMF I e) Br2, Me2SO4 30 % 84 % OHC Br MeO OMe MeO O 245 246248 247
f) t-BuLi MeO g) PCC OMe OMe MeO h) PdCl2(PPh)3, 80 % I NaOAc, DMA
53 % MeO OMe O MeO OMe O 249 232
Figure 3.10 Jones’ synthesis of benzo[b]fluorenone 232.
Palladium mediated Heck cyclization was all that remained to secure the target. After an exhaustive survey of palladium mediated coupling conditions, Jones and Qabaja found that microwave-assisted closure catalyzed by PdCl2(PPh)3/NaOAc in DMA gave the best
results furnishing, 232 in 53% in 1 min.
65 3.3.5 Mal’s Synthesis of Benzo[b]fluorenones
At the same time that Jones’ work appeared in the literature, a disclosure by Mal and co-workers revealed their approach to benzo[b]fluorenones.107,108 Mal’s efforts
focused on overcoming the challenge of obtaining unstable indenones like the one used
by Hauser (224, Figure 3.6). Pursuant of this goal, flash vacuum pyrolysis (FVP) of adduct 250 (Figure 3.11)
HO SO2Ph a) FVP b) t-BuLi O 95 % 73 % O O O OH O 250 251 252 253
Figure 3.11 Mal’s preparation of benzo[b]fluorenones.
(450 oC/0.5 mmHg) furnished the indenone 251 in 95% yield after a quick purification on
silica gel. In an event that preceded Hauser’s route to prekinamycin (Figure 3.6), Mal
employed Hauser’s annulation strategy109 by exposing indenone 251 to the anion of the
sulfone 252 to secure the annulated tetracycle 253 in 73% yield.
3.3.6 Biradical Cyclization Approaches to Benzo[b]fluorenes
3.3.6.1 Echavarren’s Arylalkyne-Allene Cycloaddition
As part of an ongoing program of synthesis of kinamycin and kinamycin related
compounds, Echavarren and co-workers investigated the novel application of
Schmittel’s110 [4+2] cycloaddition to fashion the core of the benzo[b]fluorene antibiotics.
The key step in this transformation involved heating alkynyl-allene 254 (Figure 3.12) to
66 105 oC in toluene to effect the Schmittel cycloaddition and secure the tetracyclic 255 in
38% overall yield.
Me Si OMe MeO 3 Me3Si toluene, 105 oC 38 % MeO • Ph2OP H OMe POPh2 254 255
Figure 3.12 Echavarren’s aryl-alkyne-allene approach to kinamycin core.
3.3.6.2 Dominguez Benzotriyne/benzodiyne Cycloaddition
Dominguez and co-workers were able to demonstrate a similar cyclization to the
Echavarren work, employing benzotriynes and benzodiynes, in their own effort toward the core of the kinamycins.111 Dominguez found that after heating benzotriyne 256
(Figure 3.13) in benzene for several days, the starting material was totally unaffected.
However, moving to the higher boiling toluene and stirring benzotriyne 256 at 100 oC for
10 h resulted in smooth conversion to benzo[b]fluorene 257 in very good yield. This approach was extended to the benzodiyne 258. Under considerably more vigorous conditions (toluene, sealed tube, 170 oC, 20 h), 258 was cyclized in near quantitative yield to a mixture of 259 and 260.
67
OAc
toluene, 100 oC 80 % OAc SiMe3
256 257 SiMe3
OH Ph
toluene, 170 oC Ph 80 % Ph OH OH 258 259 (70 %) 260 (26 %)
Figure 3.13 Dominguez’s benzotriyne and benzodiyne approach to kinamycin core.
3.3.6.3 Echavarren’s Diaryldiynone Cycloaddition to Benzo [b]- and
Benzo[a]fluorenes
In synthesis investigations following their alkynyl-allene cycloaddition to benzo[b]fluorenes, Echavarren and co-workers exposed simple diaryldiynones to heat, and found that not only were the targeted benzo[b]fluorenes formed, but the synthetically useful benzo[a]fluorenes were formed in nearly equal portion. Refluxing variably substituted diaryldiynones such as 261 (Figure 3.14) in 1,2-dichlorobenzene at 180 oC
initiated the anticipated cyclization to benzo[b]fluorene 262 as well as the surprising
benzo[a]fluorene 263.
The presence of 263 in the product mixture unveiled a new and unexpected
rearrangement. Ring closure of a radical of type 266 (Figure 3.14) would lead to
intermediate 267, which, by formal [1,5]-hydrogen shift, could give benzo[b]fluorenone
262 directly. The Strained allene 267 could conceivably rearrange to carbene 268, which
could rearrange further to give the strained allene 269, an adduct needing only to undergo
68 a formal [1,5]-hydrogen shift to give observed product 263. Alternatively, the strained allene 267 could undergo ring opening to the ten member ring 270, which could electrocyclize to 269.
1,2-dichloro- SiMe3 Me Si 3 benzene SiMe3 180 oC
MeO O O O MeO MeO 261 262 (25 %) 263 (25 %)
Me3Si SiMe3 SiMe3
MeO MeO MeO O 264 265 O 266 O
Me3Si H SiMe3
262
MeO O MeO 267O 270
SiMe SiMe H 3 H 3
263
MeO MeO O O 268 269
Figure 3.14 Echavarren’s closure to benzo[b]- and benzo[a]fluorenones, and mechanistic speculation.
3.3.7 Kamikawa’s Approach Toward O4, 9-Dimethylstealthins A and C
Kamikawa and Koyama’s approach to O4, 9-dimethylstealthins A and C relied on
the Suziki coupling between boronic acid 271 (Figure 3.15), prepared from the aryl
bromide 239 in a single step, and aryl bromide 272 readily available from 3,5-dimethyl
69 b) NBS c) 2-nitropropane, NaOEt OMe OMe a) n-BuLi, OMe d) n-BuLi, OMe Br B(OMe) B(OH)2 2 Br BrCF2CF2CBr 82 % 33 % OHC MeO OMe MeO OMe 239 271 272 245
e) Pd(PPh3)4 99 %
MeO f) H2O2 MeO OMe MeO 4 g) (COCl)2, TiCl 4 2 OHC 72 % 9 MeO OMe MeO OMe O h) BnONH HCl, 273 2 232 k) NBS; CaCO NaOAc 3 l) Ac O i) CAN 20 % 2 m) BnONH HCl, 59 % 2 NaOAc; CAN MeO MeO O O CH2OAc
MeO O NOBn MeO O NOBn 274 276
j) Zn, AcOH n) Zn, AcOH 80 % 28 %
MeO MeO OH OH CH2OAc
MeO O NH2 MeO O NH2 275 277
Figure 3.15 Kamikawa’s synthesis of O4, 9-Dimethylstealthins A and C.
anisole in three steps to arrive at biaryl 273 in 99% yield. The key Friedel-Crafts
precursor (not shown) was prepared by conversion of 273 to the acid with H2O2 and then the acid chloride, which was subsequently cyclized upon exposure to TiCl4 to give the
versatile benzo[b]fluorenone 232 in 72% yield. At this point (just as in Gould’s stealthin
70 synthesis, Figure 3.8), the route diverges to independently functionalize 232 for both 275 and 277 synthesis. The former species was synthesized by conversion of ketone 232 to
O-benzyl ether oxime (not shown) followed by CAN oxidation to produce quinone 274 in
59% over two steps. Finally, zinc promoted de-benzylation of 274 gave rise to O4,O9- dimethylstealthin C (275) in 80% yield. As for O4,O9-dimethylstealthin A (277), the
appropriate C(2) appendage had to be installed prior to oxime formation. To that end,
232 was brominated under radical conditions at the C(2) methyl. Hydrolysis of the
benzylic bromide and acylation of the resultant alcohol produced the desired acetate
functionality. Ensuing O-benzyl ether oxime formation followed by CAN oxidation
furnished quinone 276 in 20% yield over five steps. Finally, zinc promoted
debenzylation resulted in a low yield conversion from 276 to 277. Unfortunately, all
efforts to demethylate 275 and 277 met with failure.
3.3.8 Jebaratnam’s Syntheses of Prekinamycin Analogues
In the late 1990’s, Jebaratnam and co-workers were focused on developing a unifying strategic approach to kinamycin analogues, one that allowed the construction of many compounds from a single synthesis route.112 With interest in both open and closed
ring (C ring) forms of the prekinamycin analogues, the primary disconnection was easily
determined to be the phenyl-phenyl bond of ring C. Additionally, due to the reactivity of
the diazo moiety, this functionality would be introduced in a final synthesis step. The
simplest closed chain analogue prepared was diazoparaquinone 282 (Figure 3.16).
Jabaratnam and co-workers employed an anionic coupling of 278 with acetanthranil
(279) to form ketone 280 followed by the use of the Pschorr reaction to close the C-ring.
71 The preparation of 282 began with the regioselective bromination of 1,4- dimethoxynapthalene (278) with bromine, followed by lithiation of the resultant aryl bromide with t-BuLi. The lithiate was quenched via reverse addition to a solution of 279 to give ketone 280 in 78% yield overall. Potassium hydroxide mediated hydrolysis of the acyl group revealed the amine, making way for ring closure via the hydroquinone catalyzed Pschorr reaction with isoamylnitrile in AcOH to furnish the cyclized tetracycle
281 in 42% after BBr3-mediated demethylation. Finally, preparation of the hydrazone of
282 by exposure of 281 to hydrazine, followed by oxidation of the dihydroquinone using
Fetizon’s reagent gave the target 282 in an acceptable 50% yield.
A second synthesis of the slightly modified kinamycin core 286 was completed utilizing a parallel strategy (vide supra). Beginning with the known phenol 283, containing the requisite A-ring oxygen, methylation with dimethylsulfate followed by lithiation and condensation by reverse addition to acetanthranil (279) gave amide 284 in
50% yield over two steps. Again, hydrolysis was followed by diazotization with isoamyl nitrile in AcOH and subsequent reductive cyclization catalyzed by hydroquinone to afford tetracycle 285 in 72% yield. Final de-methylation with BBr3, hydrazone formation
with anhydrous hydrazine and Ag2CO3 oxidation gave the target 286 in 70% yield over the final three steps.
72 c) KOH d) Isoamylnitrile, a) Br2 AcOH; OMe OMe OH O b) t-BuLi, hydroquinone f) NH2NH2; 279 e) BBr3 Ag2CO3 78 % 42 % 50 %
OMe OMe O NHAc OH O O N2 278 280 281 282 O
O
N 279 c) KOH g) (MeO)2SO4, d) Isoamylnitrile, NaOH AcOH; k) BBr3 OMe OMe OMe O h) t-BuLi, hydroquinone l) NH2NH2; 279 e) BBr3 Ag2CO3 50 % 72 % 70 % Br OMe OH OMe OMe O NHAc OMe OMe O OH O N2 283 284 285 286
Figure 3.16 Jebaratnam’s synthesis of kinamycin core analogues.
3.3.9 Ishikawa’s Approach to Highly Oxygenated Kinamycin Analogues
Quite surprisingly, only recently has the synthesis community embarked on the preparation of highly oxygenated (D-ring) kinamycin analogues. First on the scene was
Ishikawa and co-workers with their approach to the tricyclic racemate 295 (Figure 3.17), embodying the correct relative configurations at C1-C4 on the D-ring.113,114 In a later
disclosure, Ishikawa and co-workers reported the synthesis of the tetracycle 300 (Figure
3.18), with the intact kinamycin benzo[b]fluorene skeleton.
Synthesis efforts toward the highly oxygenated tricycle 295 started with oxidation
of 4-benzyloxy-1-indanone (not shown) using Saegusa’s method (treatment of a silyl enol
115 ether with Pd(OAc)2)-p-benzoquinone) to give indenone 287 in good yield. Next,
Diels-Alder reaction of 287 and Danishefsky’s diene 288 in refluxing benzene furnished
73 the endo cyclization product. Desilylation of the Diels-Alder product under acidic
(camphor sulfonic acid) conditions gave the respective enone, which was easily oxidized at the
a) benzene, O O OBn HO OTMS reflux; CSA BnO c) TMSOTf, Et3N BnO H H b) O2, KF d) OsO4, py 9 Me 63 % 57 % OMe OH OH O O O 287 288 289 290
OTBS k) NaH, S C, MeI e) DIBAL TBSO OTBS h) p-TsOH, 292 TBSO 2 BnO BnO o f) TBSCl, Et3N H i) TBAF H l) 300 C, g) OsO , py 20 mmHg 4 OH j) PDC O 51 % 36 % OH OH 20 % OH O OTBS O 291 O O 293
292
TBSO OTBS TBSO OTBS BnO m) NH NH BnO 2 2 4 3 n) Ag2CO3 2 O 1 O O 63 % O O N2 294 295
Figure 3.17 Ishikawa’s synthesis of highly oxygenated kinamycin analogue 295.
doubly activated C(9) position with molecular oxygen in the presence of catalytic
potassium fluoride to give 289 in 63% yield. Hydroxyenone 289 was then converted to
the corresponding silyl dienol ether followed by osmium tetroxide oxidation to afford a
mixture of α-hydroxy ketones 290 in a 5:1 ratio (α:β). DIBAL reduction of 290 followed
by crystallization resulted in the corresponding tetraol with the correct relative
stereochemistry for the kinamycins, which upon persilylation and osmylation gave 291
exclusively. Selective ketalization of triol 291 followed by desilylation of the southern
TBS protecting group with TBAF gave the corresponding diol as a single isomer whose
74 relative stereochemistry was ascertained by single crystal X-ray diffraction. PDC oxidation of the structurally secure diol gave the α-hydroxyketone 293 in 20% yield over three steps. Conversion of 293 to the corresponding xanthate, followed by pyrolysis under vacuum gave enone 294 in 51% yield. Finally, standard hydrazone formation with anhydrous hydrazine followed by oxidation with Ag2CO3 and catalytic KOH gave the
target 295 in 63% yield.
As mentioned above, in a continued effort toward the synthesis of kinamycin
analogues, Ishikawa subsequently published a route to the tetracyclic 300 (Figure 3.18).
While the key structure of their synthesis approach (ketone 297) had been prepared
a) n-BuLi, DMF b) malonic acid OMe OMe OMe c) Pd/C, H2 Br d) P2O5, MeSO3H e) IBX, DMSO 57 % 72 % OMe OMe OMe OMe O OMe OMe O 296 297 298 O O MeO MeO f) ZnCl2, 288 H H g) CSA h) KF, O2
H 48 % over 3 steps OH OMe OMe O OMe OMe O 299 300
Figure 3.18 Ishikawa’s synthesis of oxygenated tetracycle 300. previously by Rapoport,116 Ishikawa and co-workers designed a new route starting with
bromonaphthalene 296. Successive treatment of 296 with n-butyllithium and DMF gave
the corresponding aldehyde. A Knoevenagel reaction of this aldehyde with malonic acid
under sonication gave the α,β-usaturated carboxylic acid, which was hydrogenated with
Pd/C. The naphthalenepropionic acid (not shown) was then subjected to modified
Friedel-Crafts conditions (P2O5/MeSO3H) to effect cyclization to the Rapoport ketone
75 297 in 57% over four steps. IBX oxidation of indanone 297 furnished indenone 298 in
72% yield, making way for the familiar Diels-Alder cyclization. As such, treatment of indenone 298 with zinc chloride and diene 288 followed by acid hydrolysis led to the tetracyclic enone 299. Finally, facile oxidation with catalytic KF and air led to the targeted oxygenated tetracycle 300 in 48% yield over the final three steps.
3.3.10 Dmitrienko’s Approach to Prekinamycin
In the midst of an effort to study the potential mechanism of antibacterial and antitumor activity of the kinamycin family of natural products, Dmitrienko and co- workers prepared the benzo[a]fluorene 305 (Figure 3.19).117 The synthesis of the target
305, having the same core as isoprekinamycin (189, Figure 3.2), began with a modified
a) n-BuLi, B(OMe)3 302 b) Pd(PPh3)4, CO2Et c) NBS Na2CO3 d) CH3SO3H 83 % 81 % MeO CO2Et MeO 301 303 Br 302
e) Pd2(dba)3, BINAP, O t-BuOK, BnBNH2 O f) Pd/C, H2, AcOH g) NaNO2, HCl; NaHCO3 30 % MeO O Br N2 304 305
Figure 3.19 Dmitrienko’s synthesis of isoprekinamycin analogue 305.
Suzuki reaction between the boronic ester of 302, prepared in situ, with naphthalene derivative 301 to give biaryl 303 in excellent yield. Regioselective bromination, of 303 followed by intramolecular Friedel-Crafts cyclization gave benzo[a]fluorenone 304 in
76 81% yield over two steps. Palladium-catalyzed amination of 304 with benzylamine,118 followed by hydrogenolysis and finally concomitant demethylation and diazotization with HNO2 gave the target 305 in 30% yield over the final three steps.
3.4 Speculation About the Diazoparaquinone Natural Products Mechanism-of-
Action
3.4.1 Jebaratnam’s Oxidative Activation Proposal for DNA Cleavage
The profound cytotoxic activity of the diazoparaquinone-containing natural
products has stimulated the development of some thoughtful hypotheses governing the
possible mechanism-of-action of these structurally unique agents. In the midst of a
program to develop new reagents for DNA cleavage and looking primarily at highly
unstable and caustically activated diazonium compounds,119 Jebaratnam and co-workers
were intrigued by the structural similarity of diazonium compounds and
diazo[b]fluorenes (≈ deprotonated diazonium analogues). Ensuing investigations lead to
the observation that model compound diazofluorene 306 (Figure 3.20) nicks plasmid
120 DNA upon exposure to oxidizing conditions (Cu(OAc)2).
oxidation (Cu(OAc)2) DNA cleavage -N2
N2 AcO 306 307
Figure 3.20 Jebaratnam’s hypothesis for kinamycin mode of action.
Jebaratnam and co-workers speculated that oxidation of diazo 306 (via metal ion)
followed by loss of nitrogen and nucleophile (acetate) trapping of the resultant
77 carbocation leads to radical 307 which by itself, or a peroxy radical derived there from, induced DNA damage via known oxygen-mediated pathways.121-123 Jebaratnam then suggested similar chemistry may occur with the kinamycins, perhaps unaided, with the quinone moiety serving as an internal oxidant. While an intriguing idea at the time, this proposal is not consistent with the more recent observation of He and co-workers that lomaivaticin (207) is activated under reducing conditions.
3.4.2 Dmetrienko’s Proposal For DNA Damage Via Kinamycins Based On Diazo
Group Electrophilicity
Seven years after Jebaratnam’s proposal, Dmitrienko made a case for an electrophilic intermediate based on the facile alkylation of isoprekinamycin with β- naphthol as compared to the deshydroxyl prekinamycin analogue 310 (Figure 3.21).117
o When exposed to β-naphthol in the presence of Cs2CO3 at 0 C, diazobenzo[a]fluorene
189, shown with its suggestive resonance form 189a, undergoes smooth conversion to a
mixture of the azo addition product 308 and the hydrodediazonization product 309 in just
9 h. Conversely, when the deshydroxyl diazobenzo[a]fluorene 310 is exposed to β-
o naphthol in the presence of Cs2CO3 at 0 C there is negligible change to the starting
material after 17 h. From these observations, Dmitrienko suggested that enhanced
reactivity of the diazo group in the kinamycin natural products may play a role in their
bio-activity. Due to the observed difference in the IR frequencies of the H-bond capable
189 and the H-bond incapable 310, Dmitrienko hypothesized that the kinamycin natural products, exemplified by kinamycin F (195), are rendered more diazonium like (note IR
78 frequencies) and thus more electrophilic, by virtue of the intramolecular H-bonding present (195a).
Unfortunately, this hypothesis does not take into full consideration the relative N2 stretching frequencies of the kinamycin natural products. The H-bond capable kinamycin
-1 F has an N2 IR frequency of 2120 cm , a value not too disparate from that expected for a
diazonium like stretch. Hydrogen bonging incapable kinamycin J on the other hand has
an IR frequency of 2150 cm-1, a higher and more diazonium like stretch! The observation
of a higher IR frequency (N2) for the H-bond incapable species does not support
O HO O HO O HO O HO β-naphthol Cs2CO3
o OH OH OH IR (N2) = O 0 C, 9 h HO HO O -1 O N N OH H N2 2162 cm H N2 189 189a 308 (29 %) 309 (61 %)
O H β-naphthol No opportunity Cs2CO3 for intramolecular negligible conversion from SM IR (N ) = H-bonding 0 oC, 17 h H 2 O 2105 cm-1 N2 310
OH HO HO OH AcO OAc O CH3 O CH3 O CH3 OH OH OAc OH IR (N2) = OH OAc -1 N2 2120 cm OH O O O N2 OAc O N2 H -1 195 kinamycin F 195a 195 kinamycin J IR (N2) = 2150 cm
Figure 3.21 Dmitrienko’s hypothesis and relevant IR frequencies.
Dmitrienko’s hypothesis, at least in the context of diazoparaquinone natural products.
79 3.4.3 Formulating a New Hypothesis of the Diazoparaquinone Family of Natural
Products.
A significant observation with regard to diazoparaquinone natural product mechanism-of-action was made by He and co-workers in a report summarizing their discovery and biological assays of the novel dimeric diazo[b]fluorene glycosides lomaiviticins A (207) and B.87 Therein, He briefly notes, “An ongoing study showed that lomaiviticin A cleaved double stranded DNA under reducing conditions”. While
Dmitrienko was aware of this work when formulating his hypothesis, Jebaratnam’s work preceded this observation of reductive activation. It is not clear at this time how reductive activation might be involved in either previously proposed mechanistic hypothesis, especially in light of the fact that neither study included models containing the diazoparaquinone functionality. Therefore, it seems that the door is open for further consideration of alternative modes through which the diazoparaquinone natural products might elicit their well precedented cytotoxicity.
Synthesis efforts toward, and biological studies of, the mitomycins have lead to the general understanding that a lone pair of electrons adjacent to a paraquinone moiety is deactivated via vinylogous resonance (i.e., 311). However, upon single electron reduction of the paraquinone functionality, the nitrogen lone pair is freed to participate in further chemistry (Figure 3.22).124,125
O OCONH2 O (H) OCONH2 - H2N OMe e H2N OMe + N NH (H ) N NH O O 311 mitomycin C 312
Figure 3.22 Mitomycin single electron reduction.
80 Applying this model of activation to the diazoparaquinones leads to a postulate involving the formation of the 3-electron system 313 (Figure 3.23) by single electron reduction of 195 (a representative example). Illustration of 313a, an equivalent resonance form of 313, reveals a potential role for the diazo unit. Specifically, β-
2 elimination of N2 to give a reactive intermediate, the sp radical 314. The likelihood of
this speculative process of diazoparaquinone activation could very well depend on the
trade-off between the energetic penalty incurred with pyramidalization of C(11) required
for cloud/"*C-N overlap and the energy gained as a result of N2 expulsion. Predicting a thermodynamic vs. kinetic process is speculative at best, but if thermodynamics prevails and the energy gain outweighs the cost of pyramidalization, the above described process represents a formal addition of an electron to the diazoparaquinone function to form a carbon centered radical at C(11). A significant consequence of this elimination reaction is realized when considering the obligatory change in occupancy for this radical from a -
orbital to an sp2 orbital. Perhaps a single electron reduction process of diazoparaquinone
natural products, as so described, was engineered to serve as an efficient means to
convert biologically accessible single electron (radical?) reductants to more reactive sp2 carbon radicals.
81
HO OH HO OH HO OH O - (H)-O CH3 (H) O CH3 CH3 1 e- OH OH OH OH (H+) OH OH 11 N N OH O OH O N O O N N H N 195 kinamycin F 313 313a OH - HO (H) O CH3 - N2 OH OH OH O 314
Figure 3.23 Postulated mechanism for the formation of a C(11) sp2 radical from diazoparaquinones via single electron reduction.
The biologically relevant chemistry of sp2 radicals with DNA in general,122 and
cyclopentenyl sp2 radicals in particular,126-128 has been well documented. An example is
illustrated in Figure 3.24, where, based on the observation of Saito and co-workers,
neocarzinostatin chromophore (315) undergoes thiol addition at C(12) to give an
intermediate cumulene (not shown) which spontaneously forms the coresponding
diradical at C(2) and C(6). The C(2) radical cyclizes into the phenol leaving the C(6) sp2 radical (316) to elicit its damaging role; effecting single strand scission of doubly stranded DNA. With this knowledge of the fate of radical 316, the observation that the lomaiviticins doubly nick DNA could possibly be rationalized by invoking two DNA damaging events via a radical like (314), formed independently in each half.
OH OH O O O ArCO 12 2 2 O O S O ds DNA HS OH single strand : double strand scission > 100:1 (carb)O 6 O ArCO2
315 neocarzinostatin (carb)O chromophore 316
Figure 3.24 Neocarzinostatin chromophore-derived sp2 radical and its fate.
82 In contrast to the neocarzinostatin chromophore chemistry 316 + DNA-H, diazoparaquinones have inherent within their core structure the potential to go beyond simple hydrogen atom abstraction/radical reactions (i.e. path a, Figure 3.25) and become much more potent DNA damaging agents. One example envisioned (path b) is the rebound addition of DNA• with 317 to form the covalent adduct 318. An even more interesting possibility arises (path c) from the species resulting from hydrogen atom abstraction (317). Close inspection of 317 reveals the presence of a very reactive moiety; a hydroxymethyacylfulvene subunit, the key pharmacophore implicated in the potent cytotoxicity of a family of promising anticancer agents (cf. hydroxymethylacylfulvene
321).129-131 By analogy with the chemistry known for 321, base alkylation by the
electrophilic orthoquinonemethide 317, as illustrated by the postulated conjugate addition
of guanosine N(7) with C(11),125 provides an independent and orthogonal route to
introduce damage to DNA (path c, 317a→320). N(7)-alkylated guanosines are reported
to undergo spontaneous depurination to afford an abasic site.126,132-135 The role
cyclopentenyl radical 314 and/or orthoquinone methide 317 play in the context of
biological activity of the diazoparaquinone family of natural products remains a matter of
speculation, but it is certainly possible that this intriguing functionality was designed to
perform as an inducible (via single electron reduction) source of these two reactive
intermediates. Notably, the isolation of the shunt metabolite seongomycin (323) from S.
muramaensis serves an possible product of the transformation of 314→317 (with an
aromatic prekinamycin D ring), followed by trapping of 317 by N-acetylcysteine and
finally reoxidation of the corresponding hydroquinone.89,92
83 OH HO OH HO OH HO - - (H)-O (H) O CH3 (H) O CH3 CH3 path b DNA-H OH OH OH DNA OH OH OH H H DNA OH O OH O path a O OH O 314 3172 318
strand scission lesion HO HO OH H - O N OH (H) O CH3 NH2 OH N N 319 OH N OH O S HO O H O OPO(O-)ODNA 323 CO H H 317a path c AcHN 2 OPO(O-)ODNA OH HO CH3 HO HO (H)-O OH OH depurination, Nu nucleophiles H O strand scission = thiols, DNA, H O N 2 O- NH HO O OH Nu O H N N NH2 321 322 O 320 DNAO(O-)OPO OPO(O-)ODNA
Figure 3.25 Speculative pathways by which radical 314 might lead to DNA damage.
It is obvious that the intriguing diazoparaquinone moiety of the kinamycin family of natural products has attracted much well deserved attention, yet the biologically relevant mode through which these molecules operate remains unclear. The following chapter will describe our efforts to uncover the mechanism of action of these unique and
potent natural products.
Chapter 4
Efforts to Elucidate the Mechanism-of-Action of the Diazoparaquinone Family of Natural Products
4.1 Initial Investigations with Prekinamycin and Derivatives Thereof and Bu3SnH
Working with the hypothesis formulated in the previous chapter, we began our investigation with prekinamycin (188) and derivatives thereof and Bu3SnH as a model
single electron reductant. Whereas our experiments are performed outside of a biological
milieu, limiting our conclusions to be drawn by analogy only, they will serve to reveal
the intrinsic chemistry of the diazoparaquinone moiety under reducing conditions.
Insight into on the biological chemistry of the kinamycins or lomaiviticins will have to
await the results of further experimentation in a biological system. In the account of our
efforts to follow, convincing evidence is presented that suggests a mechanistic
progression similar to that presented in Figures 3.23 and 3.25; formation of an sp2 radical
314 from diazoparaquinones under single electron reduction and subsequent conversion to a well recognized electrophilic moiety, 317, by H-atom abstraction.
Prekinamycin (188), embodying the diazoparaquinone functional moiety we are interested in studying and having been previously synthesized (Hauser, Figure 3.5)82 made it an obvious choice for out mechanistic investigations. Unfortunately, the biological profile of prekinamycin has yet to be published. As a result, the validity of using aromatic D-ring species as a model for the highly D-ring oxygenated cytotoxic kinamycins is open to question. Efforts to determine the biological activity of prekinamycin and its derivatives are currently underway in our laboratory. In addition to
85 the natural product 188 itself, both the diacetate (323, Figure 4.1) and the dimethyl ether
(324) were prepared in an effort to probe the necessity of activation via H-bonding to the
C10 carbonyl as suggested by Dmitrienko, and to provide model compounds whose solubility and chromatography behavior would make experimentation easier and more efficient. Both 323 and 324 were prepared as per Hauser’s 1996 disclosure on these compounds.82
OH OAc OMe O O O
HO O N2 OAc O N2 OMe O N2 188 prekinamycin 323 324
Figure 4.1 Model compounds for mechanism of action investigations.
Our initial experiments were designed to probe the hypothesis that the formal
+ addition of H2 (as Bu3SnH, H ) to a model diazoparaquinone would lead to an isolable
orthoquinonemethide (325, Figure 4.2). Aware of a report by Kim that diazoketone 326
reacts with the H• delivering agent Bu3SnH to give the formal Sn-H addition product 327
provided encouragement for a similar transformation, 188→325.
86
OR 1 OR O R O 2 x H
H RO O N2 OR O 188, 323, or 324 325 R = H, Ac, or Me 1 R = Bu3Sn- or H-
RO OH M O
O O Bu SnO O OH Bu3Sn-H 3 4 OEt O AIBN OEt H N2 80 oC H OH O 326 327 PhH M = various metals 328 R = H monofulvenone A 329 R = Ac monofulveone B
Figure 4.2 The plan to probe the formal single electron reduction chemistry of the diazoparaquinone models.
On the other hand, concern regarding the stability/reactivity of the orthoquinonemethide
325 weighed heavily, especially in light of the disclosure that the related monofulveneones A (328) and B (329) decomposed within minutes in the presence of dilute acid.136 If the protonated version of 328/329 does not survive in even slightly
acidic conditions, i.e. acidic workup, the question is, will the aromatic D-ring model
(325), lacking the potentially activating C(4) carbonyl of 328/329 survive isolation attempts?
o Treatment of 188 with Bu3SnH and AIBN in d6-benzene at 80 C did not lead to
the recovery of any detectable product that possessed the orthoquinonemethide moiety of
325. Interestingly, the structure of the single major product that was recovered from this experiment presented a bit of a challenge to decipher. All of the peaks of the ABCD ring system were clearly present in the 1H NMR spectrum, but the identity of the substituent at
C(11) was a mystery. Surprisingly, after further spectroscopic analysis, it became clear
87 that a deuterobenzene solvent molecule was attached to this reactive center, 330 (Figure
4.3). With the relatively unreactive benzene solvent having participated in the reaction to form
OH OH OH O 1.1 eq Bu3SnH D 1.1 eq AIBN ABC D 80 oC HO O D HO O N2 d6-benzene D 188 59 % 330 D D
Figure 4.3 Preliminary findings upon treatment of 188 with Bu3SnH/AIBN.
330, it was clear a highly reactive intermediate was in play. At this point none of the
obvious candidates i.e.: carbene, carbocation (electrophilic orthoquinonemethide
equivalent), or radical could be excluded as possible candidates. Three short term goals
were set: a) determine if this reactivity profile extends to the prekinamycin derivatives
323 and 324, b) determine if C6H6 would participate similar to C6D6, and c) run a series
of control experiments to try and narrow the focus of the possible reactive
intermediate(s).
To accomplish our first and second goals, 188 (Figure 4.4) was subjected to a
o slow addition of a solution of AIBN in C6H6 in the presence of Bu3SnH at 80 C followed
by concentration and immediate chromatography on silica gel to give the benzene trapped
adduct 331 in 59% yield.
88 OAc OAc OAc OH OAc O 1) Bu3SnH, AIBN, 80 oC, benzene Ac2O, pyr 50 % 99 % OAc O OAc O OAc O N2 323 332 334 Ac2O, pyr 52 % OH OH OH O 1) Bu3SnH, AIBN, 80 oC, benzene
59 % MeI, HO O K CO HO O N2 2 3 188 331 90 %
OMe OMe OMe OH OMe O 1) Bu3SnH, AIBN, o 80 C, benzene MeI, K2CO3
80 % 91 % OMe O OMe O OMe O N2 324 333 335
Figure 4.4 Extension of Bu3SnH/AIBN chemistry to prekinamycin derivatives 323/324.
Next, di-acetate 323 was subjected to the exact same conditions (Bu3Sn/AIBN in benzene
at 80 oC) to give the benzene trapped adduct 332 in 50% yield. Finally, dimethyl ether
324 was also subjected to the aforementioned conditions to give the corresponding trapped adduct 333 in 80% yield. The increase in yield to 333 in the later case is most
likely due to the increased solubility of the starting material 324 in benzene. In a final set of correlation experiments, 331 and 332 were acylated in the presence of Ac2O and pyridine to give the peracylated adduct 334. Next, 331 and 333 were methylated with
MeI in the presence of K2CO3 to give the trimethyl ether 335. With the correlation
experiments lending confidence to the speculation that similar chemistry is occurring
with all three substrates (188, 323, 324), and with the highest yield coming from the most
soluble 324, subsequent experiments were run with substrate 324. Despite significant
89 efforts to modify the reaction parameters, workup and chromatographic techniques, no tin
1 bearing species (cf. 325 with R = Bu3Sn, as in 327) could be isolated. Attempts to
1 prepare a more stable version of 325 (R = SnPh3 or Si(TMS)3) with Ph3SnH or TMS3SiH
also failed. An additional lesson taken from these experiments is the apparent irrelevance
of intramolecular hydrogen bonding to activate the diazoparaquinone, as demonstrated by
the high reactivity of the H-bond incapable species 323 and 324 under our radical
generating conditions.
To accomplish our third short term goal, control experiments as indicated in Table
4.1 were run. Valuable information taken from these experiments included a) the fact
that these reactions demand the presence of all radical generating ingredients, and b) a
purely thermally or photochemically generated species could be dismissed.
Table 4.1 Control experiments with the dimethyl ether 324.
entry light heat Bu3SnH AIBN Result 1 X X X X 80% 333 2 X X X 79% 333 3 X X X recovered 324 4 X X X recovered 324 5 X X X recovered 324
4.2 Investigations into the Reactive Intermediate Preceding the Trapping Event
Identifying the reactive intermediate generated upon reduction of 324 that participates in C-C bond formation with benzene became the focus of the next series of experiments. A priori, carbene formation at C(11) seemed the least likely pathway given the types of products formed and the results of the control experiments. Therefore, distinguishing between the two possibilities, C(11) radical or C(11) electrophile (i.e.,
90 orthoquinonemethide ≈ secondary carbocation), became the next challenge. The use of aryl substituent effects in the context of a Hammett-type study proved to be a useful tool in this context. As such, the prekinamycin dimethyl ether 324 was treated with the standard recipe of Bu3SnH and AIBN in a brief survey of equimolar mixtures of benzene and other substituted aromatic solvents as described in Table 4.2. Absolute chemical yields of the benzene trapped adduct 333 and the substituted arene adducts 336 were noted, as were the ortho:meta:para ratios (or the 2,4-:2,6-:3,5- ratios). Standard
Hammett (para) substituent constants " were used to analyze this data, which required
normalization of the raw data.137
Table 4.2 Relative reactivity of electron rich and electron poor arene solvents with the reactive intermediate generated form 324 under reducing conditions, Bu3SnH/AIBN.
OMe OMe OMe OH OH O 1) Bu3SnH, AIBN, 80 oC, benzene S
PhH + Ar-H N 1:1 molar N OMe O OMe O 337 OMe O 2 mixture R1 324 333 336 2) SiO 2 R
a,b Entry R, R1 % yield rel. rate o : m : p 337 o : m : p 333 + 336a rel. rateb,d
A H, CH3 43±3 2.2 ± 0.1 62 : 23 : 15 2.1 60 : 23 : 17 B H, Cl 64±6 1.5 ± 0.2 48 : 32 : 20 0.9 43 : 33 : 19 C H, CN 67±5 2.1 ± 0.3 43 : 25 : 32 2.0 56 : 18 : 26
D H, OCH3 68±11 3.1 ± 0.7 72 : 16 : 12 2.2 63 : 14 : 23 c E CH3, CH3 52±11 4.0 ± 0.9 50 : 50 : 0 c F OCH3, OCH3 75±10 4.2 ± 1.5 31 : 69 : 0 G CN, CN 69±7 6.1 ± 0.5 24 : 76 : 0c aQuintuple measurements with standard deviation. bRate relative to benzene. cRatio of 2,4-:2,6-:3,5- adducts. dData from ref. 150. eExtrapolated from the results of 1:9 and 1:13 ratios of 1,3-dicyanobenzene to benzene solvent due to limited solubility.
91
0.8
d 0.6 c
ρ = −2.3
log(k/ko) 0.4 a ρ = 0.5 b 0.2
0 -0.5 0 0.5 1.0 1.5 σρ
Figure 4.5 Hammett Study of the reaction between 324 and arene solvents under radical generating conditions.
The deviation from linearity in the plot of entries a-d from Table 4.2 vs. "
(depicted in Figure 4.5) does not lend support to the intermediary of a C(11)
carbocationic species en route to 333 and 336. The fact that any non-hydrogen
substituent on the arene co-solvent accelerated formation of the corresponding trapped
adduct is apparently reinforced by entries e-g of Table 4.2, where the values for the 1,3-
dimethyl, 1,3-dimethoxy, and 1,3-dicyanobenzene are roughly twice the values corresponding to the respective monosubstituted species. The observation that the electron deficient 1,3-dicyanobenzene is the most reactive arene solvent examined, coupled with the observation that the second most reactive arene solvent was the electron rich 1,3-dimethoxybenzene, argues convincingly against a carbocationic or electrophilic orthoquinonemethide intermediate. However, these experiments alone do not permit the
92 exclusion of such a species from further consideration if it were perhaps a minor contributor with the electron rich arenes (vide infra).
By process of elimination, the mechanism for the conversion of 324 in to
333/336, or by comparison the conversion of 188 into 325, appears to proceed through an sp2 radical adding to the arene solvent,138-148 Figure 4.6. Specifically, the data presented
in Table 4.2 are consistent with a mechanistic sequence in which 324 is reduced to
2 semiquinone 338 and later sp radical 339 upon loss of N2, a species that adds directly to
the arene ring solvent to afford the transient cyclohexadienyl radical 340. With
149 knowledge of the work by Beckwith et. al. in the area of Bu3SnH-mediated aromatic
substitution, the fact that under globally reducing conditions, the radical intermediate
following arene addition is oxidized (presumably by AIBN, note stoichiometric
requirement) to afford the aryl ring of 341 is not a surprise. Finally, workup via SiO2 provides the observed product(s) 333/336.
93
OMe OMe OMe Bu SnO O Bu3SnO 3 -N2 Bu3Sn
N N OMe O OMe O N OMe O 324N 338N 338a N
OMe OMe OMe Bu3SnO 1.1 eq Bu3SnO Bu3SnO Bu3Sn-H
OMe O OMe O H OMe O H 339 342 342a
R R1
OMe OMe Bu3SnO Bu3SnO
H H H OMe O OMe O R1 R1 340 343 R R AIBN
OMe OMe OR OR air oxidation H
OMe O OMe OH R1 R1 344 R R 341 R = Bu3Sn SiO2 333/336 R = H
Figure 4.6 A mechanistic proposal for the formation of observed products 333/336 from the diazoparaquinone 324.
If the orthoquinonemethide 342 is indeed formed from 339 by hydrogen atom abstraction from Bu3SnH, the data acquired to this point does not permit a conclusion
about its role in adduct formation. It is interesting to point out that the chemical yields of
the arene trapped adducts ranges from 50-80%, with the fate of the remainder of the
94 material unknown. Some of the missing material could be accounted for by the diversion of 339 into the orthoquinonemethide 342, which does not appear to participate in direct adduct formation with the electron poor arenes (vide infra). On the other hand, the possibility that a minor role is played by the orthoquinonemethide in a Friedel-Crafts type alkylation with electron rich arenes to give the reduced hydroquinone 344 can not be ruled out. Exposure of 344 to air and SiO2 upon workup could then give the observed
product 333.
The speculative conclusion that radical addition chemistry prevails over
orthoquinonemethide chemistry only pertains to reactions with an aromatic solvent
nucleophile. Extrapolating these data to a biological setting, where much more
nucleophilic participants for orthoquinonemethide addition are present, does not seem
warranted. Fortunately, we were able to design a series of experiments that provide some
enlightenment regarding more biologically relevant nucleophiles (vide infra). Further
evidence in support of the role of a key radical intermediate (339) in adduct formation
lies in the correlation between the relative rates and ortho:meta:para ratios between our
data and the data from thiazole-derived radical 337 addition to substituted arenes as
recorded by Dou150 (see Table 4.2). Additionally, small and variable quantities of the
C(11) dimer 345 (Figure 4.7) were isolated from several experiments. It would be
difficult to rationalize the formation of 345 in the absence of the radical intermediate 339
even if its exact role in dimer formation (342, 339 + 342→345, or 342 +
342→346→345) is not clear.
95
OH OMe OMe OMe OH O Bu3SnH, AIBN, 80 oC OMe 11 O O 11' OMe ArH solvent
OMe O N2 OMe O 324 333 MeO HO 50 - 80 % 345 OMe Bu SnH, 5 - 15 % 3 -N2 Bu3SnO AIBN 2x
OMe O 339
H O OMe +H 339
OSnBu3 OMe O SnBu3 342 OMe MeO hetero Diels Alder O MeO O dimerization (342) H H
Bu SnO 3 OMe 346
Figure 4.7 Plausible routes to the C11-C11’ dimer 345.
Several additional experiments were run with the aim of trapping radical 339. To
our delight, standard reaction of 339 with Bu3SnH and AIBN in the presence of Ph2Se2 led to the formation of the C(11) selenium trapped adduct 347 (Figure 4.8) along with the normal benzene addition product 333. Unfortunately, all attempts to effect the tin hydride-mediated reduction of 324 in non-aromatic solvents met with failure, as reaction in either THF, 1,4-dioxane, CCl4, CH3CN, EtOH or Cl3CH returned only starting
material. Perhaps these observations point to a key role played by the aromatic solvent in
either promoting the formation of, or even stabilizing, the sp2 radical by -complexation.
96
Bu3SnH, AIBN, OMe OMe OMe OH OH O benzene, Ph2Se2 80 oC
OMe O SePh OMe O N2 OMe O 324 333 35 % 347 21 %
Figure 4.8 Successful trapping of radical intermediate 339 with Ph2Se2.
With confidence in the presence of the radical intermediate 339, our attention was
directed toward probing the questionable presence of the orthoquinonemethide 342, and
if present, elucidating its fate. The presence/fate of 342 was explored through a series of
experiments featuring either a) variation of the Bu3SnH/substrate ratio, or, separately, b)
examination of the product distribution when nucleophiles that are better than arene rings
are present. If the mechanistic model in Figure 4.6 is in operation, then diverting radical
339 away from the arene trapped adduct 340, to the orthoquinonemethide 342 should be
favored with a higher concentration of tin. In an experiment with an electron poor
solvent, it is presumed that any orthoquinonemethide (342) that is formed would not lead to adduct formation. Consequently, the overall yield of 333/336, which reflects passage only through 339, should decrease as [Bu3SnH] increases. This hypothesis was tested by
exposing 324 to AIBN with varying tin concentrations in 1:1 molar mixture of benzene
and cyanobenzene at 80 oC, Figure 4.9. Gratifyingly, the data obtained from these studies
lends support to our hypothesis, as the overall yield of arene trapped adducts decreases
linearly with increasing [Bu3SnH] over the range of 1-12 equivalents of Bu3SnH.
Additionally, there is an internal verification of the expectation that the arene adducts are
formed only from radical 339 and not orthoquinonemethide 342 for the electron poor
arene (cyanobenzene). Evaluation of the 333/336c ratio as a function of [Bu3SnH]
97 reveals no change within experimental error, thus providing no indication that any new mechanistic pathway is in operation.
OMe OMe OMe OH OH O varying SnBu3H AIBN, 80 oC 1:1 molar mix of N OMe O OMe O CN OMe O 2 and 324 333 336c CN
333 + 336c 333 + 336c % yield ratio 80 4
3.5 60
3 40
2.5
20 2
0 1.5 036912 equivalents of tributyltin hydride
333 + 336c 333 + 336c % yield ratio
Figure 4.9 Yield and ratio of aromatic trapping products 333/336c upon increasing [Bu3SnH]. All data points are averaged from pentuplicate measurements.
On the other hand, there is a obvious deviation in product distribution when an
electron rich aromatic solvent, 1,3-dimethoxy benzene, is used in place of cyanobenzene,
Figure 4.10. In experiments run with a 1:1 molar mixture of benzene and 1,3-
98 dimethoxybenzene, the overall yield of arene adducts 333 + 336f decreases in a nearly linear fashion at lower Bu3SnH concentrations, but the trend in reversed, and the overall
yield of the arene trapped adducts actually increases, at the highest tin concentration
examined. The question here is: could the orthoquinonemethide 342, in the presence of a
more nucleophilic solvent, be participating in adduct formation to an observable degree
(339→342→344→336f, c.f., Figure 4.6)? Evidence that supports this hypothesis can be
found by close inspection of the ratio of arene trapped adducts 333/336f as a function of
[Bu3SnH]. In contrast to the electron poor arene case, the ratio favoring the electron rich
adduct 336f increases at high tin hydride concentrations! These observations are
consistent with the divergence of radical 339, to some extent, down the path of
orthoquinonemethide 342 when favorable conditions (electron rich/more nucleophilic
arene ring) are present. Consequently, it seems that both reactive intermediates, the sp2 radical 339 and the orthoquinonemethide 342, are generated sequentially upon formal single electron reduction of diazoparaquinone 324, and both species can be trapped under optimal conditions.
99 OMe OMe OMe OH OH O varying SnBu3H AIBN, 80 oC 1:1 molar mix of OMe N OMe O OMe O OMe O 2 and 324MeO OMe 333 336f OMe
333 + 336f 333 + 336f % yield ratio 90 15
80 12
70
9 60
6 50
3 40
30 1 0 36912 equivalents of tributyltin hydride
333 + 336f 333 + 336f % yield ratio
Figure 4.10 Yield and ratio of aromatic trapping products 333/336f upon increasing [Bu3SnH]. All data points are averaged from pentuplicate measurements.
In a final series of experiments we hoped to demonstrate more definitively the
role of the orthoquinonemethide 342 in the mechanism at hand, i.e. probe its reactivity in
the presence of a species with greater nucleophilicity than electron rich arene rings. As
alluded to earlier, the experiments to follow begin to speak more relevantly to the
chemistry of diazoparaquinones in a biological setting, where nucleophiles such as thiols
100 and amines (c.f. Figure 3.25) might play a role in adduct formation. Inclusion of benzylmercaptan in the reaction mixture served as a means to evaluate this hypothesis.
The results from simply exposing a solution of diazoparaquinone 324 (Figure 4.11) to either 1.1 or 12 equivalents of Bu3SnH and 10 equivalents of benzylmercaptan in benzene
at reflux with a slow addition of a solution of 1.1 equivalents of AIBN in benzene speak
directly to this point. Specifically, the products of direct radical 339 addition to benzene,
333 and 349, are formed in a combined yield of 37% with 1.1 equivalents of tin hydride
and in an combined yield of 33% with 12 equivalents of tin hydride. The idea that 348
precedes 349 is supported by the independent synthesis of 349 from 333 and benzyl
mercaptan. The fate of the other 2/3 of the starting material still remains in question. We
were delighted to find that roughly half of the starting material had been converted to the
C(11) benzylmercaptan addition product 350, retaining the C(11) hydrogen. This
hydroquinone was relatively sensitive to air oxidation to the quinone (thus losing the
C(11) hydrogen), but careful handling under an N2 atmosphere during isolation and characterization reduced this undesired side reaction to nearly undetectable levels. The most plausible rationale for the observation of the benzylmercaptan trapped adduct 350
cites the orthoquinonemethide 342 as an intermediate, where now the greater
nucleophilicity of a thiol vis a’ vis an arene solvent serves to effectively trap this reactive
species. There was no evidence for the formation of the dimer 345 throughout this series
of experiments. In a revealing control experiment, refluxing a solution of
diazoparaquinone 324 with 100 equivalents of benzylmercaptan in refluxing benzene lead
to no detectable reaction, and 324 was recovered from the reaction unchanged.
101
MeO O Bu3SnH 1.1 eq AIBN
10 eq PhCH2SH o MeO O N2 PhH, 80 C 324 MeO Bu3SnO MeO MeO Bu3SnO Bu3SnO PhH Bu3SnH
MeO O H 348 MeO O MeO O 339 342 PhCH2SH SiO2 SiO2 PhCH2SH
MeO MeO HO HO MeO HO
MeO OH S MeO O H S 349 Bn 333 MeO OH Bn 350 PhCH2SH 90 %
eq of tin total yield 349 333 350
1.1 83 % 22 % 15 % 45 % 12 90 % 23 % 10 % 57 %
Figure 4.11 Thiol addition to reactive orthoquinonemethide intermediate 342.
Evidence presented herein suggests that the incorporation of radical generating
(reducing) conditions are an obligatory event for diazoparaquinone activation.
Additionally, the lack of direct nucleophilic addition of benzylmercaptan to the diazo moiety of 324 certainly does not lend support to Dmitrienko’s hypothesis (Figure 3.21), albeit the “required” H-bond activation in 189a is not present in 324. In an effort to examine this point further, the H-bond capable species prekinamycin (188) was subjected to an identical control experiment as described above with 324 (i.e. exposure to 100
102 equivalents of benzylmercaptan in refluxing benzene). This trial met the same fate; complete recovery of starting material (188) and no evidence for any chemical reaction.
The fact that the benzylmercaptan addition product 350 could be isolated with the
C(11) hydrogen intact, led to the obvious labeling experiments with Bu3SnD, Figure 4.12.
Surprisingly, when diazoparaquinone 324 was combined with 12 equivalents of Bu3SnD,
AIBN and 10 equivalents of benzylmercaptan in refluxing benzene, the expected C(11) thio-trapped adduct 352 was isolated in good yield, but analysis of the 1H NMR spectrum
revealed only 29% deuterium incorporation at C(11). The curiously high proton
incorporation in 352 was explained, in part, by rerunning the experiment with PhCH2SD
as the deuterium source. In this case, the hydroquinone 352 was found to contain 45%
deuterium incorporation at C(11). Control experiments involving heating a mixture of
either Bu3SnD/PCH2SH or, independently, Bu3SnH/PhSH2SD in benzene with AIBN
present did not lead to any detectable level of crossover. Finally, it was comforting to find that in the presence of both Bu3SnD and PhSH2SD with AIBN in refluxing benzene,
the deuterium incorporation of adduct 352 (76%) roughly matched the sum of the
deuterium incorporations of the two single-deuterium source experiments. The source of
the remaining 25% of proton incorporation remains a mystery, as no further labeling
studies were examined, although PhCH2SH is a likely candidate.
103
OMe OMe OMe HO O 12 eq Bu3SnD Bu3SnO 1.1 eq AIBN PhCH2SH
PhH, 80 oC D N 10 eq PhCH2SH D S OMe O 2 OMe O OMe OH Bn 324 351 352 29 % D
OMe OMe HO 12 eq Bu3SnH Bu3SnO 1.1 eq AIBN PhCH2SD 324 PhH, 80 oC D 10 eq PhCH2SD D S OMe O OMe OH Bn 351 352 45 % D
OMe OMe HO 12 eq Bu3SnD Bu3SnO 1.1 eq AIBN PhCH2SD 324 PhH, 80 oC D 10 eq PhCH2SD D S OMe O OMe OH Bn 351 352 76 % D
Figure 4.12 Deuterium labeling experiments with diazoparaquinone 324.
In conclusion, the results presented herein serve as a solid foundation on which to construct a reliable model for the chemistry that takes place upon single electron reduction (i.e., Bu3SnH/AIBN) of a diazoparaquinone-containing species such as
prekinamycin (188). Evidence in support of a mechanistic sequence involving an initial
formation of a C(11) sp2 radical, and a subsequent electrophilic orthoquinonemethide, has
been acquired. The radical intermediate appears to have sufficient reactivity to trap both
electron rich and electron poor arenes, whereas the downstream orthoquinonemethide
intermediate reacts only with electron rich arenes and nucleophiles such as an alkyl thiol.
104 Extension of these results to the larger question of kinamycin/lomaiviticin mechanism-of- action remains a challenge to be met, but at the very least, these results shed light on the intrinsic chemistry available to diazoparaquinone-containing species under reducing conditions, and they hint at a likely mechanistic picture within a biological setting.
Chapter 5
Efforts Toward the Total Synthesis of Kinamycin F
5.1 Construction of the Highly Oxygenated D-Ring
With a convincing model for the mechanism-of-action of the diazoparaquinone natural products having been established, our efforts turned toward kinamycin F (195) synthesis in the hope of providing enough material to test its behavior in our model system as well as in a biological setting. Kinamycin F was chosen as a synthesis target because to date, no publications have appeared detailing results on its biological activity, and for the obvious reason of avoiding the necessity of differentially acylating the D-ring tetraol. We envisioned that the fully elaborated 195 (Figure 5.1) could come from a novel Nazarov type cyclization of the ABD ring system 354 followed by oxidation to the
requisite unsaturation at the C/D ring fusion, global deprotection, hydrazone formation and oxidation to the diazoquinone. While the last series of steps is well precedented as demonstrated in Chapter 3, the Nazarov cyclization represents a new approach the closure of the C ring of the benzo[b]fluorenone core. We hoped we could gain access to
354 from some type of carbonylation chemistry or coupling between the juglone derivative 355 and the vaguely defined alkene 356. Both 355 and 356 were expected to be easily accessible from commercially available 5-hydroxy-1,4-naphthoquinone (357) and cis-(1S,2S)-3-bromo-3,5-cyclohexadiene-1,2-diol (358), respectively.
106
OH HO PO OP O OP D OH OP ABC OH OP N OH O 2 OP OP O 195 kinamycin F 353
OMe OP OMe OP OP OP
Br X OP OP OBn OMe O(H) OP OBn OMe OP 354 355 356
O
Br OH OH OH O 357 358
Figure 5.1 Retrosynthetic analysis of kinamycin F.
The construction of the highly oxygenated D-ring of kinamycin F was conceived from the work by Hudlicky and co-workers in the area of enzymatic dihydroxylation of aromatic compounds, specifically, their efforts involving the synthesis of poly- hyroxylated carbohydrate natural products (Figure 5.2).151-153 It has been shown that P.
pudita possesses an enzyme that efficiently catalyzes an enantiospecific dihydroxylation
of specific aromatic compounds to give cis-cyclohexadiene-diols (c.f, 360), which, despite their inherent instability, serve as versatile building blocks for highly oxygenated six member rings such as (+)-pinitol (361).154 Fortunately, at this time, some of these
107 diols are commercially available. Thus, elaboration to the fully functionalized D-ring of kinamycin F began with the commercially available diol 360.
OH P. putida F39/D MeO OH Br OH Br HO OH OH OH 359 358 360 (+)-pinitol
Figure 5.2 Hudlicky’s approach to (+)-pinitol via enzymatic dihydroxylation.
Our approach toward kinamycin F began with acetonide protection of the commercially available cis-(1S,2S)-3-bromo-3,5-cyclohexadiene-1,2-diol (358, Figure
5.3) with 2,2-dimethoxy propane, followed by regio- and diastereoselective epoxidation of the less substituted alkene to give the epoxide 361 in good yield. Next, a two step sequence of epoxide ring opening with 10% potassium hydroxide in DMSO at 140 oC
followed by acetonide deprotection under acidic conditions to afforded tetraol 362 in
80% over two steps. Notably, tetraol 362 contains all of the oxygen atoms needed in the
D-ring with the correct stereochemistry. Three of the four hydroxyls were then protected
with TBSCl and imidazole as per the method of Banwell and McRae to give 3,4,6-
protected alcohol 363 in very good yield.155 Interestingly, if the silylating reagent is
switched to TBS-OTf with 2,6-lutidine, the selectivity changes to afford the 3,4,5-
protected alcohol (not shown). Dess-Martin oxidation of the secondary alcohol gave rise
to ketone 364 in 69% yield. Notably, if the TBS-protected product is taken on crude to
the Dess-Martin oxidation the yield over two steps is 79%, representing an 89% yield per
step, a significant and worthwhile modification. Felkin-Ahn directed methylation of
ketone 364 with methyl lithium gave the tertiary alcohol 365 exclusively. Lastly,
108 protection of the tertiary alcohol with TMSOTf gave in good yield the fully protected cyclohexene 366 containing all of the appropriate functionality and stereochemistry of the D-ring of kinamycin.
O o 1) (CH3)2C(OCH3)2, p-TsOH 1) KOH, DMSO , 140 C o 2) MCPBA 2) AcOH, H2O, THF, 60 C Br Br OH 73 % O 80 % OH O
358 361
OH TBS-Cl, OTBS OTBS OH imid. OTBS DMP oxidation OTBS 81 % 69 % Br OH Br OH Br O OH OTBS OTBS 362 363 364
OTBS OTBS TMS-OTf OTBS MeLi 2,6-lutidine OTBS 97 % 76 % Br OH Br OTMS OTBS OTBS 365 366
Figure 5.3 Synthetic route to highly oxygenated kinamycin F D-ring.
5.2 Functionalization of the AB Ring System.
The AB ring system of kinamycin F arises through only minor modification of juglone (357). Juglone (357, Figure 5.4) was regioselectively brominated with treatment of Br2 followed by AcOH workup and recrystallization to afford 2-bromojuglone (366) in
acceptable yield. Subsequent phenolic benzylation with benzyl bromide and silver oxide
gave the benzyl protected bromo-juglone 367 in good yield. Reduction of the quinone
moiety of 367 with sodium dithionite gave the dihydroquinone 368, which was
109 immediately methylated by treatment with dimethyl sulfate to give the known juglone derivative 355 in 54% yield over the final two steps.156
O O O Br2, CHCl3, BnBr, Ag2O, AcOH CH2Cl2 50 % 79 % Br Br OH O OH O BnO O 357 367 368
OH OMe Na2S2O4, Me2SO4, Et2O, CH2Cl2 K2CO3, acetone 57 % 96 % Br Br BnO OH BnO OMe 369 355
Figure 5.4 Synthesis of juglone derivative 369 as per Brimble’s work.156
5.3 Preparing the Nazarov Cyclization Precursor.
At this point, it was anticipated that a palladium mediated carbonylative coupling
between 365 and 369, or perhaps independent carbonylation of one of these two coupling
partners (365/369) followed by a subsequent coupling event, would secure the pseudo di-
vinyl ketone (c.f. 354) required for the Nazarov cyclization. Initial efforts in this
direction focused on preparing the stannane of 355. To that end, treatment of alkenyl
bromide 365 (Figure 5.4) with n-BuLi in THF at -78 oC followed by trimethyltin chloride
addition gave a crude product that appeared to be the desired stannane 370. However,
after silica gel chromatography, the tertiary alcohol peak was observed in the 1H NMR and nine protons were missing from the area around 0.1 ppm. This observation suggested, and it was later confirmed, that the alkenyl bromide underwent the anticipated lithium halogen exchange and then suffered an unanticipated intermolecular silicon
110 transfer of the TMS to the lithiate with a final stannylation of the resulting alkoxide upon the Me3SnCl quench. Silica gel chromatography presumably removed the tin from the
tertiary alcohol to give the observed product 371 in near quantitative yield.
OTBS OTBS OTBS n-BuLi; OTBS OTBS OTBS Me3SnCl Me Sn TMS Br OTMS 3 OTMS OH OTBS OTBS OTBS 370 371 365
Figure 5.5 Attempted stannylation of alkenyl bromide 365.
Unfortunately, all attempts to functionalize 365 by metallation with an akyl
lithium led to the formation of 371 (Figure 5.5), regardless of the electrophile. Since the
alkenyl lithium appeared to have a singular and undesirable fate, efforts were directed
toward preparing the alkenyl Grignard in anticipation that the Grignard reagent would not
undergo similar silicon transfer. Unfortunately, all attempts to prepare the Grignard
reagent of 365 with magnesium metal or i-PrMgCl were unsuccessful, resulting only in
the recovery of unchanged starting material. In a final series of experiments directed
toward functionalizing alkene 365, we turned to attempts at direct palladium mediated
carbonylation. Again, the only result to come from these experiments was consistent
recovery of unchanged starting material. With the fate of the alkenyl lithiate apparently
sealed, and an inability to otherwise functionalize the alkenyl bromide, we turned to
operations on the tertiary alcohol 364, where the labile silicon is absent, and unavailable
to silylate the metallated alkene.
Treatment of the alkenyl bromide 364 (Figure 5.6) with two equivalents of t-BuLi
in THF followed by a addition of DMF led not to the desired aldehyde 372, instead the
111 undesired protonated product 373 was formed. Substitution of n-BuLi for t-BuLi gave the same result, as did the similar reaction conditions in Et2O or attempts to trap other
electrophiles
OTBS OTBS OTBS OTBS OTBS OTBS alkyl lithium H H Br OH OH OH OTBS O OTBS OTBS 372 373 364
Figure 5.6 Attempted functionalization of alkenyl bromide 364.
such as CO2 and benzaldehyde. Apparently the rate of proton transfer from the hydroxyl to the initially formed alkenyl lithiate is faster than the rate of deprotonation (dianion formation) of the alcohol. While this undesired proton transfer could have possibly been overcome by first deprotonating the alcohol with NaH and then performing the lithium halogen exchange, results from a different series of experiments (Figure 5.9) led to a shift in direction and rendered this potential solution untested.
While trying to functionalize the tertiary alcohol 364, we were making progress on another front; stannylating the complementary coupling partner 355. Gratifyingly, we found that treatment of aryl-bromide 355 (Figure 5.7) with t-BuLi gave a thermally unstable lithiate that, when prepared and quenched with an appropriate electrophile at very low temperatures, gave the substitution product in good yield. Specifically,
o treatment of 355 with t-BuLi at -110 C with subsequent Me3SnCl addition led to the
aryl-stannane 374 in good yield. This result taught us a great deal about the reactivity of
355, rendering this compound a versatile coupling partner in a later approach.
112 OMe OMe n-BuLi, Me3SnCl, THF, -110 oC 86 % Br SnMe3 BnO OMe BnO OMe 355 374
Figure 5.7 Preparation of stannyl-juglone derivative 374.
With stannane 374 in hand, a survey of carbonylative coupling conditions with
alkenyl bromide 365 ensued (Figure 5.8). A wide array of experimental techniques
OMe OTBS OMe OTBS OTBS OTBS Pd, CO
SnMe3 Br OTMS OTMS BnO OMe OTBS BnO OMe O OTBS 374 365 375
Figure 5.8 Attempted carbonylative coupling. including high and low pressure carbon monoxide, heat, different solvents and different palladium sources were tried to no avail. The desired ketone 375 was not detected in any of the coupling attempts. In fact, the alkenyl bromide could often be recovered unchanged from the reaction conditions. At this point, the steric bulk of the flanking
OTBS group was of concern.
It was assumed that removal of the sterically cumbersome OTBS might provide better access to the alkenyl bromide (make the corresponding lithiate a better nucleophile) and perhaps facilitate the attachment of the AB ring system in the context of nucleophilic acyl addition or substitution chemistry. To that end, persilylated 364 (Figure
5.9) was treated with 3.1 equivalents of TBAF in THF to afford the desired tetraol 376 in quantitative yield. Subsequent protection of 376 upon treatment with sodium hydride and benzyl bromide gave the tetrabenzylether 377 in 75% yield. We were very pleased to
113 find that treatment of the less sterically hindered alkenyl bromide 377 with t-BuLi at -78
o C followed by an immediate CO2 quench gave rise to the desired carboxylic acid 378 in
quantitative yield. Treatment of the acid 378 with N,O-dimethylamine•HCl (379),
ethyldimethylaminopropylcarbodiimide, and DMAP gave rise to Weinreb amide 380 in
good yield.
OTBS OH OBn NaH, BnBr, OTBS TBAF, THF OH DMF OBn quant. 75 % Br OH Br OH Br OBn OTBS OH OBn 364 376 377
OBn OBn t-BuLi,CO2, 379, EDC, o OBn OBn THF, -78 C DMAP, CH2Cl2 quant. HO N OBn 60 % O OBn O OBn O OBn 378 380 N H O HCl 379
Figure 5.9 Preparation of Weinreb amide 380.
With the Weinreb amide 380 in hand, we turned to coupling this D-ring fragment
with the AB ring fragment 369. As expected, treatment of 369 (Figure 5.10) with t-BuLi
in THF at -110 oC immediately followed by the addition of a solution of 380 in THF
smoothly effected the formation of the desired ketone 381 in very good yield.
BnO OBn OMe OMe OBn MeO t-BuLi; 380 Nazarov OBn THF conditions OBn 81 % Br OBn OBn BnO OMe BnO OMe O OBn BnO OMe O 369 381 382
Figure 5 10 Formation of Nazarov precursor 381 and the elusive cyclization product.
114 5.4 Nazarov Cyclization Attempts
With an efficient route to 381, experimentation was directed toward the key
Nazarov cyclization. Unfortunately, after more than 45 independent experiments using various energy sources such as heat, microwave and ultraviolet light in a number of different polar and non-polar solvents, and varying the activator from strong protic acids to weak chelating Lewis acids, none of the desired cyclized product 382 was observed.
Instead, it was often the case that no chemical reaction occurred or benzyl protecting groups were removed, especially under strong Lewis and protic acid conditions.
With little hope of cyclizing 381, the next obvious perturbation was to change the electronics of the starting material. To that end, in two independent experiments, we hope to reveal the quinone moiety of 381 and reduce the ketone of 381 to the corresponding alcohol. Specifically, treatment of 381 with cerium(IV)ammoniumnitrate should oxidatively removed the methyl protecting groups to afford the quinone 383
(Figure 5 11). Additionally, treatment of 381 with NaBH4 in MeOH is expected to
cleanly furnished the desired alcohol 384 as a mixture of diasteriomers. Once in hand, a
series of similar cyclization conditions to those employed with 381will be used to try and
effect cyclization of 383 to tetracycle 385. Additionally, attempts will be made at
dehydratetive cyclization of 384 to tetracycle 386.
115
BnO OBn O OBn O OBn Nazarov OBn OBn OBn BnO O O OMe OBn CAN BnO O O OBn 385 OBn 383
BnO OBn NaBH4 OBn OMe OBn MeO BnO OMe O OBn OBn dehydration OBn 381 OBn OBn BnO OMe OH OBn BnO OMe 384 386
Figure 5.11 Manipulations of 381 and down stream cyclization attempts.
Though the construction of the Nazarov cyclization precursor 381 was accomplished, the substrate appears to be resistant to cyclization under a large battery of conditions. Hope is certainly not lost though, as two additional substrates should soon be available to subject to cyclization conditions.
116
Chapter 6
Experimentals
6.1 Diazonamide A Studies
O O
N O I O
133
Methyl-1-(2-Benzyloxy-benzoyl)-7-iodo-1H-indole-3-carboxylate (133).
Phosphorus trichloride (1.61 mL, 17.3 mmol) was added dropwise to DMF (50 mL) at 0
ÿC. A solution of 7-iodoindole (129) (2.21 g, 9.09 mmol) in 50 mL of DMF was then
added at 0 ÿC. The reaction mixture was allowed to return to r.t. and the thick yellow
slurry was stirred overnight. A mixture of ice and water was added to the reaction
mixture which discharged the yellow color. Ice cold 3M NaOH was added to the
colorless solution affording an orange tinted solution. This solution was extracted 3x
with 95:5 CH2Cl2/MeOH (3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The orange solid obtained
was suspended in H2O and vigorously stirred for 1 h. Filtration and lyopholyzation
afforded 7-iodoindole-3-carboxaldehyde (130) as an orange tinted solid (8.85 g, 97%).
Sodium cyanide (1.31 g, 26.8 mmol) was added to a solution of 3-
carboxaldehyde-7-iodoindole (1.45 g, 5.35 mmol) in 50 mL of MeOH. After 10 min of
117 stirring, manganese dioxide (6.05 g, 69.7 mmol) was added in small portions. After stirring at room temperature for 20 h, the mixture was filtered through a pad of Celite, which was then rinsed with CH3OH and the combine filtrate was concentrated in vacuo.
The resulting pinkish purple solid was subjected to flash chromatography on silica gel
(hexanes/EtOAc, 75:25) to furnish methyl 7-iodoindole-3-carboxylate as a pink solid
(1.29 g, 80%). The spectral data matched that reported for 131 (prepared by a different method).157
Lithium hexamethyldisilazide (4.15 mL of a 1M solution in THF, 4.15 mmol) was
added to a stirring solution of the methyl ester from above (131) in 8 mL of THF (1.14 g,
3.78 mmol). After 1h, this solution was cooled in an ice bath and a solution of 2- benzyloxy benzoyl chloride (132) (4.00mL, 1.04M solution in CH2Cl2, 4.16 mmol) was
added. After returning to room temperature and stirring overnight, the reaction mixture
was diluted with CH2Cl2 and washed with H2O. The aqueous layer was extracted with
CH2Cl2 (3 x 20 mL). The combined organic extracts were washed with H2O and then
brine, dried over Na2SO4, filtered and concentrated in vacuo. The resulting semisolid
was purified by flash chromatography on silica gel (hexanes/EtOAc, 80:20) to give
methyl ester 133 as a yellow tinted white solid (1.76 g, 91%). mp 99-100 oC; IR (thin
-1 1 film) 1711cm ; H NMR (400 MHz, CDCl3) δ 8.20 (dd, J = 7.9. 1.1 Hz, 1H), 7.91 (dd, J
= 7.7, 1.0 Hz, 1H), 7.84 (s, 1H), 7.69 (dd, J = 7.6, 1.8 Hz, 1H), 7.56 (ddd, J = 9.4, 7.5, 1.8
13 Hz, 1H), 7.3 – 7.0 (m, 8H), 5.06 (s, 2H), 3.86 (s, 3H); C NMR (100 MHz, CDCl3) δ
164.4, 164.2, 158.7, 138.3, 137.8, 135.9, 135.1, 135.0, 132.3, 131.1, 128.5, 128.0, 126.9,
125.9, 123.2, 121.8, 121.4, 113.8, 111.6, 78.7, 70.6, 51.7; APCIMS m/z relative intensity
118
512 (M+H 15 ). Anal. Calcd for C24H18INO4: C, 56.38; H, 3.55; N, 2.74. Found: C,
56.45; H, 3.48; N, 2.71.
BnO CO2CH3 O N
BOCN CHO
140
1-tert-butyl-3’-Methyl-1'-(2-Benzyloxy-benzoyl)-3-formyl-1'H-
[4,7']biindolyl-1,3'-N-icarboxylate (140). Isopropylmagnesium chloride (0.52 mL mol of a 2M solution in THF, 1.04 mmol) was added to a solution of 133 (532 mg, 1.04 mmol) in THF (2.00 mL) at – 45 ÿC affording a dark amber solution. This solution was
stirred at -45 ÿC for 30 min. After cooling to -78 ÿC, a solution of ZnBr2 (235 mg, 1.04
mmol) in THF (2.00 mL) was added dropwise to the Grignard mixture affording a
precipitate. The temperature of the reaction was raised to ambient, and dioxane (5.0 mL)
was added, leading to a yellow/orange slurry. The mixture was stirred at room
temperature for 2 h and then filtered using an air-free funnel to furnish a yellow solution
of the zinc reagent, which was concentrated to about half the original volume under high
vacuum. Tris(dibenzylideneacetone)dipalladium (38 mg, 5% vs. 133) and tri-2-
furylphosphine (38 mg, 20 mol% vs. 133) were dissolved in 0.75 mL of THF and stirred
for 10 minutes (wine red to dark green color change). After the green color was obtained,
139 (303 mg, 0.817 mmol) was added as a solid. After an additional 5 minutes of
stirring, the zincate solution was added via cannula and the reaction mixture was stirred
at room temperature overnight. At this time, the mixture was diluted with an equal
119 volume of EtOAc and poured into 15 mL of a 1:1 mixture of aqueous saturatedurated
NH4Cl/NaCl. The aqueous phase was extracted with EtOAc (3 x 10 mL). The combined
organic layers were washed with brine, dried over Na2SO4, filtered and concentrated.
The yellow solid obtained was purified by flash chromatography on silica gel
(hexanes/EtOAc, 80:20) to afford the bisindole product 140 as a white solid (373 mg,
73%). mp 138-139 oC; IR (thin film) 1748, 1712, 1676 cm-1; 1H NMR (360 MHz,
CDCl3) δ 9.23 (s,1H), 8.26 (dd, J = 8.0, 1.2 Hz, 1H), 8.21 (s, 1H), 8.16 (dd, J = 8.5, 0.7
Hz, 1H), 7.91 (s, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.29 (dd, J = 7.4, 1.2 Hz, 1H), 7.3-7.2 (m,
5H), 7.1-6.9 (m, 4H), 6.74 (m, 2H), 4.87 (d, J = 12.0 Hz, 1H), 4.83 (d, J = 12.1 Hz, 1H),
13 3.91 (s, 3H), 1.66 (s, 9H); C NMR (90 MHz, CDCl3) δ 186.8, 165.9, 164.4, 156.1,
148.8, 136.4, 135.6, 135.0, 134.0, 133.8, 133.5, 131.3, 130.9, 129.5, 128.4, 128.37, 128.0,
127.8, 127.0, 125.1, 125.0, 124.7, 124.5, 123.0, 121.6, 121.4, 120.8, 114.4, 112.6, 112.2,
85.4, 70.0, 51.4, 28.0; APCIMS m/z relative intensity 651 (M+Na 100). Anal. Calcd for
C38H32N2O7: C, 72.60; H, 5.13; N, 4.46. Found: C, 72.42; H, 5.12; N, 4.50.
O O O P O O O N H O
142
Benzyl-tert-Butoxycarbonylamino-(dimethoxy-phosphoryl)-acetate (142).
Dicyclohexylcarbodiimide (89.0 mg, 4.31 mmol) was added to a solution of N-BOC-2-
(dimethylposphonato)glycine70 (1.11 g, 3.92 mmol) and benzyl alcohol (0.51 mL, 4.93 mmol) in 25 mL of CH2Cl2 at 0 ÿC. The reaction was slowly warmed to room
120 temperature and stirred at room temperature overnight. The reaction mixture was filtered and concentrated in vacuo. The residue was dissolved in EtOAc, filtered again, and the remaining solution was washed sequentially with citric acid (2 x 50 mL), saturatedurated
NaHCO3, water and finally brine. The resulting solution was dried over Na2SO4 and concentrated in vacuo. The oil obtained was purified by flash chromatography
(hexanes/EtOAc, 60:40). The phosphonate was obtained as a colorless oil (1.15 g, 78%).
Spectral data matched that of known material. IR(thin film): 1735, 1708 cm-1; 1H NMR
(300 MHz, CDCl3) 7.35 (m, 5H); 5.39 (br s, 1H); 5.29 (dd, J = 28.9, 12.2 Hz, 2H); 4.95
(dd, J = 23.0, 9.0 Hz, 1H); 3.74 (d, J = 8.6 Hz, 3H); 3.69 (d, J = 8.6 Hz, 3H); 1.42 (s,
9H).
BnO CO2CH3 O N
BOCN
CO Bn BOCHN 2
141
1-tert-butyl-3’-methyl-1'-(2-Benzyloxy-benzoyl)-3-(2-benzyloxycarbonyl-2-
tert-butoxycarbonylamino-vinyl)-1'H-[4,7']biindolyl-1,3'-dicarboxylate (141). 1,8-
Diazobicyclo[5.4.0]undec-7-ene (0.80 mL, 0.58 mmol) was added to a solution of 140
(230 mg, 0.62 mmol) in 2.5 mL of CH2Cl2. After ten min, a solution of 140 (66.4 mg,
0.106 mmol) in 3.0 mL of CH2Cl2 was added, resulting in a yellow solution. The mixture
was stirred at room temperature for 48 h. At this point the reaction solution was diluted
121 with an equal volume of EtOAc and poured into ice-cooled saturatedurated aqueous
NH4Cl. The aqueous layer was extracted with EtOAc (3 x 10 mL). The combined
organic layers were washed with water and brine, dried over Na2SO4, filtered and
concentrated. The resulting yellow oil was purified by flash chromatography on silica gel
(hexanes/EtOAc, 80:20). The alkene was obtained as a white solid (70 mg, 76%). mp
94-102 oC (dec); IR (thin film): 1737, 1732, 1720, 1714, 1698 cm-1; 1H NMR (300 MHz,
CDCl3) δ 8.18 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.97 (s, 1H), 7.79 (s, 1H),
7.7-7.0 (m, 10H), 6.80 (d, J = 7.1 Hz, 1H), 6.69 (m, 2H), 6.61 (t, J = 7.5 Hz, 1H), 6.56 (br
s, 1H), 6.11 (br s, 1H), 5.02 (d, J = 12.4 Hz, 1H), 4.93 (d, J = 12.4 Hz, 1H), 4.75 (d, J =
11.8 Hz, 1H), 4.68 (d, J = 11.9 Hz, 1H), 3.89 (s, 3H), 1.60 (s, 9H), 1.45 (s, 9H); 13C
NMR (75 MHz, CDCl3) δ 166.8, 164.8, 164.6, 156.7, 153.8, 149.2, 135.94, 135.92,
134.7, 134.4, 133.9, 133.7, 131.0, 129.3, 128.6, 128.5, 128.3, 128.2, 128.19, 128.12,
127.9, 127.4, 127.0, 126.3, 125.4, 125.3, 125.2, 124.2, 124.15, 123.8, 122.7, 121.6, 120.8,
114.5, 114.2, 112.7, 111.9, 84.5, 80.7, 69.9, 66.8, 51.5, 28.4, 28.2; APCIMS m/z relative intensity 898 (M+Na 100). Anal. Calcd for C52H49N3O10: C, 71.30; H, 5.64: N, 4.80.
Found: C, 71.00; H, 5.67; N 4.87.
122
HO CO2CH3 O N
BOCN H CO2H BOC2N
143
1-tert-butyl-3’-methyl-3-(2-di-tert-Butoxycarbonylamino-2-carboxy-ethyl)-
1'-(2-hydroxy-benzoyl)-1'H-[4,7']biindolyl-1,3'-dicarboxylate (143). In a nitrogen
atmosphere glovebox, Rh((S,S)-DuPHOS)(COD)OTf (3.0 mg, 5 mol%) was added to a
solution of 141 (70.0 mg, 0.080 mmol) in 5.0 ml of anhydrous MeOH. The solution was
placed in a Parr Hydrogenator and sealed while still in the glovebox. Outside of the glove box, the Hydrogenator was charged with 200 psi of H2. After stirring overnight
under these conditions, the reaction solution was concentrated and the residue was purified by flash chromatography on silica gel (hexanes/EtOAc, 75:25) to afford the tryptophan derivative as a white solid (61.7 mg, 88%). 1H NMR analysis indicated the
presence of two compounds in approximately 1.5:1 ratio. mp 90-100 oC (dec); IR( KBr
-1 1 cell,CCl4): 1758, 1737, 1720, 1701 cm ; H NMR (300 MHz, CDCl3, mixture of
diastereomers (M = major, m = minor where assignable)): 8.20 (d, J = 7.7 Hz, 1H); 8.10
(t, J = 7.3 Hz, 1H); 7.87(M) and 7.84(m) (s, 1H); 7.0-7.14 (m, 16H); 6.80-6.92 (m, 2H);
6.61-6.68 (m, 1H); 4.82-5.05 (m, 5H); 3.67(M), 3.81(M), 4.2(m) and 4.5(m) (br m, 1H);
3.92(M) and 3.91(m) (s, 3H); 2.7-2.93 (m, 1H); 2.38-2.47 (m, 1H); 1.61 (s, 9H); 1.12-
13 1.34 (m, 9H); C NMR (75 MHz, CDCl3): 172.3, 171.8, 165.8, 164.7, 156.7, 156.6,
123 155.3, 149.5, 136.7, 136.2, 135.9, 135.8, 135.74, 135.0, 134.27, 134.17, 134.0, 133.9,
133.8, 133.75, 131.9, 131.6, 129.3, 129.2, 128.9, 128.8, 128.63, 128.61, 128.59, 128.5,
128.4, 128.25, 128.21, 128.1, 128.0, 127.26, 127.1, 126.6, 125.6, 124.7, 124.3, 124.0,
123.9, 123.88, 122.9, 122.6, 121.3, 121.1, 121.0, 116.4, 116.2, 114.3, 114.25, 112.9,
112.3, 122.2, 83.5, 79.7, 79.5, 70.3, 70.2, 66.8, 66.7, 53.5, 53.3, 51.6, 51.56, 29.2, 28.4,
28.38, 28.32; APCIMS m/z relative intensity 878 (M+H 45). Anal. Calcd for
C52H51N3O10: C, 71.14; H, 5.85: N, 4.79. Found: C,70.84; H,5.83; N,4.80.
Di-tert-butyl dicarbonate (0.080 g, 0.37 mmol) was added in 0.02 g aliquots to a
stirring, refluxing solution of the tryptophan derivative from above (47.5 mg 0.054
mmol) and DMAP (1.0 mg, 0.008 mmol) in 0.4 mL of THF over a period of 72 h. At
that time, the reaction solution was concentrated in vacuo. The resulting orange residue
was purified by fash chromatography on silica gel (hexanes/EtOAc, 80:20) to furnish the
di-Boc protected tryptophan derivative as a white solid (29.3 mg, 55%). mp 80-90 oC
-1 1 (dec); IR(thin film): 1734, 1710, 1695 cm ; H NMR (300 MHz, CDCl3, mixture of diastereomers (M = major, m = minor where assignable)): 8.18 (dd, J = 7.7, 1.5 Hz,
1H); 8.08 (d, J = 8.1, 1H); 7.89 (s, 1H); 7.02- 7.41 (m, 16H); 6.70-6.80 (m, 3H); 4.81-
5.12 (m, 4H); 4.55 (dd, J = 11.1, 4.1 Hz, 1H); 3.87(M) and 3.89(m) (s, 3H); 2.86 (dd, J =
15.0, 11.2 Hz, 1H); 2.63 (dd, J = 15.0, 4.2 Hz, 1H); 1.61(M) and 1.63(m) (s, 9H);
13 1.20(M) and 1.37(m) (s, 18H); C NMR (75 MHz, CDCl3): 169.6, 165.9, 164.7, 156.5,
152.2, 149.6, 136.2, 136.1, 136.0, 135.1, 134.5, 134.0, 133.7, 131.3, 129.2, 128.64,
128.60, 128.56, 128.52, 128.48, 128.40, 128.2, 128.1, 128.0, 127.6, 127.3, 127.0, 125.8,
124.2, 124.1, 124.0, 123.2, 121.4, 120.9, 118.0, 116.6, 114.2, 112.9, 112.6, 112.2, 111.9,
83.4, 83.0, 82.6, 70.2, 70.0, 66.8, 66.6, 57.6, 51.5, 28.4, 28.0, 27.8, 26.3; APCIMS m/z
124 relative intensity 996 (M+NH4 45). Anal. Calcd for C57H59N3O12: C, 69.99; H, 6.08: N,
4.30. Found: C, 69.85; H, 6.08; N,4.34.
Palladium (10% on carbon, 3.0 mg) was added to the di-Boc protected tryptophan derivative from above (10.0 mg, 0.01 mmol) in 1.0 mL of THF. The reaction mixture was frozen (liquid N2) under argon, evacuated, thawed, immediately charged with
hydrogen and stirred overnight at room temperature. The reaction mixture was diluted
with CH2Cl2, filtered through a pad of Celite with several CH2Cl2 rinses, and the filtrate was concentrated. The resulting white solid was purified by flash chromatography on silica gel (hexanes/EtOAc/AcOH, 65:30:5). The phenolic acid 143 was obtained as a white solid (7.2 mg, 90%). mp 120-140 oC (dec); IR(thin film): br 3133, 1738, 1732,
-1 1 1722, 1715 cm ; H NMR ( 400 MHz, CDCl3, mixture of diastereomers (M = major, m =
minor where assignable)) δ 9.59 (br s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 8.9 Hz,
1H), 8.00 (s, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 6.2 Hz, 1H), 7.4-7.3 (m, 2H), 7.26
(s, 1H), 7.1-7.0 (m, 2H), 6.93 (d, J = 7.3 Hz, 1H), 6.8-6.6 (m, 2H), 4.43(M) and 5.03(m)
((M) dd, J = 10.2, 1.9 Hz, 1H), 4.10(M) and 4.09(m) (s, 3H), 2.77(M) and 3.01(m) ((M) dd, J = 14.3, 11.4 Hz, 1H), 2.26(M) and 2.63(m) ((M) dd, J = 14.0, 2.4 Hz, 1H), 1.63(M)
13 and 1.65(m) (s, 9H), 1.23(M) and 1.39(m) (s, 18H); C NMR (100 MHz, CDCl3) δ
175.2. 171.6, 165.0, 162.6, 152.0, 149.6, 137.0, 136.5, 135.9, 134.6, 132.6, 131.4, 128.5,
127.9, 127.8, 127.1, 126.1, 125.1, 124.7, 124.4, 122.2, 119.7, 118.6, 116.5, 115.1, 115.0,
112.7, 83.9, 83.5, 83.2, 57.5, 52.0, 28.65, 28.6, 28.3, 28.1, 26.4; APCIMS m/z relative intensity 698 (MH-BOC 5). Anal. Calcd for C43H47N3O12: C, 64.73; H, 5.94: N, 5.27.
Found: C, 64.39; H, 5.92; N, 5.22.
125
N CO2CH3 O O O
NBOC2 BOCN H
124
Lactone 124. A solution of phenolic acid 143 (17.9 mg, 0.022 mmol) in 0.75 mL
of THF was added via syringe pump over a period of 20 h to a stirring mixture of
dicyclohexylcarbodiimide (0.010 g, 0.047 mmol), DMAP*HCl (0.008 g, 0.05 mmol), and
DMAP (0.009 g, 0.07 mmol) in 0.75 mL of THF. When the addition was complete, the
reaction was allowed to stir for an additional 1 h, the reaction was filtered and the filtrate
was concentrated in vacuo. The resulting residue was purified by flash chromatography
on silica gel (hexanes/EtOAc, 85:15). Lactone 5 was obtained as a white solid (11.4 mg,
65%). mp 192-194 oC; IR(thin film) 1734, 1719, 1711, 1701 cm-1; 1H NMR (400 MHz,
CDCl3) δ 8.28 (dd, J = 8.0, 1.2 Hz, 1H), 8.19 (d, J = 7.3 Hz, 1H), 7.68 (d, J = 8.3 Hz,
1H), 7.5-7.0 (m, 9H), 5.38 (dd, J = 11.7, 2.2 Hz, 1H), 3.82 (s, 3H), 2.72 (dd, J = 15.9,
11.9 Hz, 1H), 2.03 (d(apparent)t, J = 15.8, 2.2 Hz, 1H), 1.65 (s, 9H), 1.4 (s, 18H); 13C
NMR (100 MHz, CDCl3) δ 166.9, 164.7, 164,6. 152.0, 149.9, 148.2, 135.2, 134.6, 134.4,
133.8, 132.9, 129.9 (2 overlapping signals), 129.6, 129.0, 128.6, 127.8, 126.7, 126.0,
124.8, 124.5, 123.0, 121.9, 121.4, 118.6, 114.6, 113.3, 84.0, 83.4, 58.8, 51.3, 28.4, 28.1,
26.7; ESMS m/z relative intensity 802 (M+Na 100). Anal. Calcd for C43H45N3O11: C,
66.23; H, 5.82: N, 5.39. Found: C, 66.35; H, 6.09; N, 5.31.
126
H O N BocHN O O O
O
154
Ethyl acetoxy-(2-tert-butoxycarbonylamino-3-methyl-butyrylamino)-acetate
(154). Isobutyl chloroformate (2.98 mL, 23.0 mmol) was added to a stirring solution of
N-BOC-L-valine (5.0 g, 23 mmol) and N-methyl morpholine (2.53 mL, 23.0 mmol) in
500 mL of DME at -10 oC. The solution became turbid after approximately 10 min of stirring. Next, gaseous ammonia was bubbled through the reaction mixture for 15 min at
o -10 C, and for an additional 1 h at room temperature. The reaction was diluted with H2O
(200 mL) and the mixture was extracted with CHCl3 (3 x 50 mL). The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to afford the desired
amide as a white fluffy solid (4.97 g, 100%). mp 220 oC (dec); ESMS m/z relative
intensity 217 (M+H 40); TOFHRMS (+ESI) Calcd for C10H20O3N2: 217.1542 , Found:
217.1546.
A 1:1 solution of ethyl glyoxalate/toluene (5.7 mL, 1.3 mmol of ethyl glyoxalate)
was added to a stirring solution of N-BOC-L-valinamide (2.5 g, 12 mmol) in 40 mL of
acetone. The solution was heated to reflux and allowed to stir at reflux for 12 h. The reaction was then diluted with H2O (50 mL) and the mixture was extracted with CH2Cl2
(6 x 20 mL). The organic extracts were dried over Na2SO4, filtered and concentrated in
vacuo to afford 3.7 g of a crude tan solid. This tan solid was triturated with vigorous
127 stirring in pentane (250 mL) overnight. Filtration of the mixture afforded the desired product as a white solid (3.1 g, 84%) of sufficient purity to take onto the next step. ESMS m/z relative intensity 219 (M+H 70); TOFHRMS (+ESI) Calcd for C14H26O6N2:
319.1859 , Found: 319.1863.
Acetic anhydride (4.0 mL, 42 mmol) was added to a stirring solution of the α- hydroxy ester (1.0 g, 3.1 mmol) from above in CH2Cl2 (40 mL) at room temperature.
The light yellow solution was allowed to stir at ambient temperature for 12 h. The
reaction solution was then concentrated in vacuo to afford the crude product as a yellow
sticky solid (>1.0 g). This sticky solid was triturated in pentane with very vigorous
stirring for 24 h. The trituration slurry was filtered and rinsed with pentane to afford the
desired product as an off-white solid (0.95 g, 84%) as a mixture of diastereomers.
Instability of this material prevented further purification. mp 210 oC (dec); IR(thin film)
-1 1 3322, 2972, 2359, 1745, 1667 cm ; H NMR (300 MHz, CDCl3) δ 7.67 (t, J = 8.5 Hz,
1H), 6.39 (d, J = 9.0 Hz, 1H), 5.25 (t, J = 8.5 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 2.19 (bs,
1H), 2.2-2.1 (m, 1H), 2.10 (t, J = 7.2 Hz, 3H), 1.44 (s, 9H), 1.28 (t, J = 7.1 Hz, 3H), 0.99
13 (d, J = 6.7 Hz, 3H) , 0.93 (d, J = 6.7 Hz, 3H); C NMR (75 MHz, CDCl3) δ 172.1, 170.3,
166.6, 155.9, 80.1, 72.3, 62.6, 59.6, 31.2, 28.4, 20.6, 19.2, 17.6, 14.0; APCI MS m/z
relative intensity 361 (M+H 100); TOFHRMS (+APCI) Calcd for C15H29O7N2: 361.1969,
Found: 361.1978.
128
O O P O N N N
156
Diethyl-benzotriazol-1-ylmethyl-phosphonate (156). A flame dried Schlenk
flask fitted with a reflux condenser was charged with finely sliced sodium metal and a
Teflon coated stir bar. The flask was the evacuated and filled with nitrogen. THF (10
mL) and diethyl phosphite (0.26 mL 2.0 mmol) were added to the flask and stirring
commenced and continued until all the sodium had been consumed. At this point, 1-
(chloromethyl)-1H-benzotriazole (0.34 g, 2.0 mmol) was added as a solid, and the
reaction mixture was heated to 60 oC and allowed to stir at this temperature for 12 h. The
reaction mixture was then cooled to room temperature, diluted with H2O (25 mL) and
extracted with CH2Cl2 (4 x 15 mL). The combined organic extracts were dried over
Na2SO4, filtered and concentrated to afford 400 mg of a crude oil. This oil was purified
by flash chromatography on silica gel (Et2O). The phosphonate 156 was obtained as a clear colorless oil (0.46 g, 72%). Spectral data matched that of the known compound.158
129 TIPSO O
H
158
5-Methyl-2-(triisopropylsilanyloxy)benzaldehyde (158). Imidazole (2.50 g,
36.7 mmol) was added in one solid portion to a stirring solution of 2-hydroxy-5-methyl
benzaldehyde (157) (2.50 g, 18.4 mmol) in DMF (20 mL) at room temperature. Next,
TIPSCl (5.90 mL, 27.6 mmol) was added to the stirring yellow solution, which immediately resulted in the dissipation of the yellow color to give a clear and colorless
solution. The reaction solution was allowed to stir at room temperature for 2 h, after
which time the reaction mixture was diluted with H2O (100 mL) and extracted with Et2O
(3 x 25 mL). The combined organic extracts were washed with H2O (3 x 15 mL). The
organic layer was then washed with brine, dried over Na2SO4, filtered and concentrated
in vacuo to afford 7.0 g of the crude product as a colorless oil. This oil was purified by
flash chromatography on silica gel (hexanes/EtOAc, 95/5). The aldehyde 158 was obtained as a clear colorless oil (4.99 g, 93%). IR(thin film) 2946, 2868, 1682 cm-1; 1H
NMR (300 MHz, CDCl3) δ 10.54 (s, 1H), 7.60 (s, 1H), 7.24 (d, J = 8.8 Hz, 1H), 6.81 (d,
J = 8.6 Hz, 1H), 2.28 (s, 3H), 1.39-1.28 (m, 3H), 1.12 (d, J = 7.2 Hz, 18H); 13C NMR (75
MHz, CDCl3) δ 190.1, 157.4, 136.5, 130.5, 128.1, 126.4, 119.6, 20.3, 18.0, 13.2; APCI
MS m/z relative intensity 293 (M+H 100); TOFHRMS (+APCI) Calcd for C17H29O2Si:
293.1931, Found: 293.1927.
130
TIPSO H
159
(2-Ethynyl-4-methyl-phenoxy)-triisopropylsilane (159). A solution of n-BuLi
(2.6 mL, 2.5 M/hexanes, 6.4 mmol) was added to a stirring solution of
(Trimethylsilyl)diazomethane (3.2 mL, 2.0 M/hexanes, 6.4 mmol) in 25 mL of THF at -
78 oC. The ensuing yellow solution was stirred at -78 oC for 30 min. Next, a solution of
158 in 5 mL of THF was added slowly to the stirring anionic solution (followed by 2 mL of a THF rinse). The resulting solution was stirred at -78 oC for 1 h. At this time, the
reaction mixture was added to aqueous NH4Cl and H2O. The mixture was diluted with
Et2O and the layers were separated. The aqueous layer was extracted with Et2O (4 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to afford 1.5 g of an orange oil. This oil was purified by flash chromatography on silica gel, eluting with hexanes. The acetylene 159 was obtained as a clear colorless oil (1.38 g, 90%). IR(thin film) 3314, 2945, 2867, 2360, 1496 cm-1; 1H
NMR (300 MHz, CDCl3) δ 7.21 (s, 1H), 6.96, (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.3 Hz,
1H), 3.13 (s, 1H), 2.22 (s, 3H), 1.35-1.23 (m, 1H), 1.11 (d, J = 7.0 Hz, 18H); 13C NMR
(75 MHz, CDCl3) δ 155.6, 134.4, 130.8, 130.1, 119.1, 114.1, 81.4, 80.7, 20.5, 18.2, 13.4;
APCI MS m/z relative intensity 289 (M+H 40); TOFHRMS (+APCI) Calcd for
C18H29OSi: 289.1982, Found: 289.1957.
131
H O N BOCHN O O
OTIPS
160
Ethyl 2-(2-tert-Butoxycarbonylamino-3-methyl-butyrylamino)-4-(5-methyl-
2-triisopropyl-silanyloxy-phenyl)-but-3-ynoate (160). A solution of n-BuLi (1.6 mL,
2.4 M/hexanes, 3.8 mmol) was added to a stirring solution of alkyne 159 (1.19 g, 4.12
mmol) in THF (3.5 mL) at -78 oC. The solution changed color from yellow to light
orange after 30 min of stirring at -78 oC. At this point, a solution of zinc bromide (1.03 g,
4.6 mmol) in THF (2.0 mL) was added to the acetylide solution via cannula. The reaction mixture was warmed to 0 oC and allowed to stir at this temperature for 30 min.
The solution was then cooled back down to -78 oC. A solution of valine derivative 154
o o in Et2O (10 mL, followed by a 5 mL Et2O rinse) at 0 C was added to the -78 C zincate
solution via cannula. The solution was allowed to stir at -78 oC for 10 min before it was
warmed to 0 oC and stirred at this temperature for 30 min. Finally, the reaction combined
with saturated aqueous NaHCO3, followed by extraction with CH2Cl2 (4 x 15 mL). The
combined organic extracts were dried over Na2SO4, filtered and concentrated to afford
2.0 g of a crude oil. The amber colored oil was purified by flash chromatography on silica gel (hexanes/EtOAc, 70:30). The coupled product 160 was obtained as a clear
132 colorless oil (1.35 g, 84%). IR(thin film) 3310, 2964, 2868, 1755, 1703, 1680, 1665 cm-
1 1 ; H NMR (300 MHz, CDCl3) δ 7.15 (s, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 8.4
Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 5.53 (d, J = 7.4 Hz, 1H), 5.20 (d, J = 8.8 Hz, 1H), 4.30-
4.15 (m, 2H), 4.14-4.05 (bm, 1H), 2.22 (s, 3H), 2.2-2.1 (m, 1H), 1.44 (s, 9H), 1.35-1.20
(m, 6H) 1.11 (d, J = 7.7 Hz, 18H), 0.98 (d, J = 6.7 Hz, 3H) , 0.95 (d, J = 6.7 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 171.1, 167.6, 155.8, 155.1, 134.3, 130.9, 130.0, 118.8, 113.5,
85.0, 82.5, 79.8, 62.4, 59.4, 45.2, 31.5, 31.0, 28.4, 19.3, 18.0, 14.0, 13.1; APCI MS m/z
relative intensity 589 (M+H 100); TOFHRMS (+APCI) Calcd for C32H53N2O6Si:
589.3664, Found: 589.3621.
N O BOCHN O O
OTIPS
161
Ethyl 2-(1-tert-Butoxycarbonylamino-2-methyl-propyl)-5-(5-methyl-2-
triisopropyl-silanyloxy-benzyl)-oxazole-4-carboxylate (161). Alkyne 160 (1.43 g, 2.42
mmol), and K2CO3 (0.67 g, 4.8 mmol) were combined in a round bottom flask,
evacuated, charged with nitrogen and submerged into a 0 oC ice water bath. Next, DMF
(10 mL) was syringed into the flask, resulting in immediate dissolution of the alkyne, followed by a rapid color change from yellow to peach. The K2CO3 suspension was
stirred in the cold water bath for 1 h. After this time, the reaction mixture was treated
with the addition of aqueous NH4Cl. The mixture was diluted with H2O and Et2O. The
133 layers were separated and the aqueous layer was extracted with Et2O (4 x 10 mL). The
combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to
afford 1.6 g of the crude product. The crude yellow oil was purified by flash
chromatography on silica gel (hexanes/Et2O, 70:30). The cyclized product 161 was
obtained as a clear colorless oil (1.19 g, 84%). IR(thin film) 3353, 2964, 2945, 2868,
-1 1 1738, 1715 cm ; H NMR (300 MHz, CDCl3) δ 6.89 (d, J = 8.2 Hz, 1H), 6.73 (s, 1H),
6.72 (d, J = 8.3 Hz, 1H), 5.31 (d, J = 9.3 Hz, 1H), 4.72 (dd, J = 9.7, 5.6 Hz, 1H), 4.41 (q,
J = 7.2 Hz, 2H), 4.40 (s, 2H), 2.19 (s, 3H), 2.15-2.07 (m, 1H), 1.42 (s, 9H), 1.42-1.25 (m,
6H) 1.11 (d, J = 7.2 Hz, 18H), 0.87 (d, J = 6.7 Hz, 3H) , 0.85 (d, J = 6.7 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 162.5, 162.3, 158.4, 155.4, 151.5, 130.3, 130.1, 128.4, 128.1,
125.9, 117.8, 79.8, 65.9, 61.1, 54.1, 33.2, 28.4, 26.7, 20.6, 18.2, 15.4, 14.4, 13.2; APCI
MS m/z relative intensity 589 (M+H 100); TOFHRMS (+APCI) Calcd for
C32H53N2O6Si: 589.3667, Found: 589.3629.
N O BocHN O O OH
OTIPS
162
Ethyl 2-(1-tert-Butoxycarbonylamino-2-methyl-propyl)-5-[hydroxy-(5-
methyl-2-triisopropylsilanyloxy-phenyl)-methyl]-oxazole-4-carboxylate (162). A
flame dried round bottom flask fitted with a reflux condenser was charged with selenium
dioxide (0.64 g, 5.8 mmol), and 3Å molecular sieves (0.5 g). Next, a solution of the
134 oxazole 161 (1.13 g, 1.92 mmol) in 1,4-dioxane (60 mL, followed by a 40 mL dioxane rinse) was added via cannula to the mixture. The resulting suspension was heated to 100
oC and allowed to stir at this temperature overnight. At this point, an additional 1.5 equiv of selenium dioxide (0.32 g, 2.9 mmol) was added to the dark brown mixture and stirring continued for an additional 24 h. The reaction solution was then cooled to room temperature, filtered through a pad of Celite, with an EtOAc rinse, and concentrated in vacuo to afford 1.3 g of an orange oil consisting of the alcohol (162) and a small amount
(20%) of the ketone (163) product. The crude oil was purified by flash chromatography on silica gel (hexanes/Et2O, 70:30). The alcohol was obtained as a clear colorless oil
(0.70 g, 60%) as a mixture of diastereomers. After determining the product mixture
contained the ketone 163 as a side product, material from this (SeO2) oxidation were
taken on crude, after filtration and concentration, to the Dess-Martin oxidation to
completely oxidize all material to the ketone. IR(thin film) 3370, 2965, 2868, 1719, 1701
-1 1 cm ; H NMR (360 MHz, CDCl3) δ 7.19 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H),
6.79 (d, J = 8.0 Hz, 1H), 6.52, (bs, 1H), 5.29 (d, J = 9.3 Hz, 1H), 5.22 (d, J = 9.3 Hz, 1H)
4.93 (d, J = 8.1 Hz, 1H), 4.67 (bm, 1H), 4.41 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 2.06-1.95
(m, 1H), 1.37 (s, 9H), 1.35-1.39 (q, J = 6.8 Hz, 3H) 1.22-1.31 (m, 3H) 1.05 (d, J = 7.3
13 Hz, 18H), 0.81 (d, J = 6.7 Hz, 6H); C NMR (90 MHz, CDCl3) δ 163.0, 162.2, 160.3,
155.8, 150.7, 130.2, 130.1, 129.5, 128.6, 128.0, 117.6, 79.7, 63.4, 63.3, 61.9, 54.0, 33.0,
28.2, 20.6, 18.6, 17.9, 14.3, 13.0; APCI MS m/z relative intensity 605 (M+H 100).
135
N O BocHN O O O
OTIPS
163
Ethyl 2-(1-tert-Butoxycarbonylamino-2-methyl-propyl)-5-(5-methyl-2-
triisopropyl-silanyloxy-benzoyl)-oxazole-4-carboxylate (163). Dess-Martin periodane
(0.268 g, 0.634 mmol) was added to a stirring solution of alcohol from above (0.174 g,
0.288 mmol) in CH2Cl2 (3.0 mL). The solution became cloudy over 30 min of stirring.
The reaction solution was diluted with CH2Cl2 and washed with saturated aqueous
NaHCO3. The aqueous layer was extracted with CH2Cl2 (1 x 10 mL) and the combined
organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in
vacuo to afford 0.23 g of a crude yellow semisolid. This crude solid was purified by
flash chromatography on silica gel (hexanes/EtOAc, 70:30). Ketone 163 was obtained as
a colorless foamy solid (0.117 g, 67%). IR(thin film) 3359, 2964, 2868, 1742, 1718 cm-1;
1 H NMR (300 MHz, CDCl3) δ 7.30 (s, 1H), 7.21 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 8.3 Hz,
1H), 5.35 (d, J = 9.5 Hz, 1H), 4.82 (dd, J = 9.5, 5.4 Hz, 1H), 4.10 (q, J = 7.1 Hz, 2H),
2.31 (s, 3H), 2.28-2.19 (m, 1H), 1.44 (s, 9H), 1.15-1.08 (m, 6H), 0.97 (d, J = 6.9 Hz,
13 18H), 0.95 (d, J = 6.7 Hz, 6H); C NMR (75 MHz, CDCl3) δ 183.1, 164.7, 160.6, 155.2,
153.0, 150.3, 134.4, 134.0, 130.9, 130.5, 129.5, 119.2, 79.9, 61.7, 54.2, 33.1, 28.3, 20.3,
136 18.9, 17.7, 13.7, 13.0; APCI MS m/z relative intensity 603 (M+H 100); TOFHRMS
(+APCI) Calcd for C32H51N2O7Si: 603.3460, Found: 603.3432.
O N BOCHN O O H N N N OTIPS
164
Ethyl 5-[2-Benzotriazol-1-yl-1-(5-methyl-2-triisopropylsilanyloxy-phenyl)-
vinyl]-2-(1-tert-butoxycarbonylamino-2-methyl-propyl)-oxazole-4-carboxylate (164).
A solution of n-BuLi (0.2 mL, 2.5 M/hexanes, 0.5 mmol) was added to a stirring solution
of phosphonate 156 (0.164 g, 0.613 mmol) in THF (1 mL) at -78 oC, immediately
producing an orange colored solution. The orange solution was stirred at -78 oC for 30 min. At this time, a solution of ketone 163 (0.147g, 0.245 mmol) in THF (1.0 mL, followed by 2 x 0.5 mL of a THF rinse) was added to the anionic solution at -78 oC via cannula. The solution immediately darkened to an amber color. The solution was allowed to stir for 5 min at -78 oC, and then it was slowly warmed to r.t over the period of
1 h and stirred for an additional 3 h. The reaction mixture was treated with the saturated
aqueous NH4Cl, water and Et2O. The layers were separated and the aqueous layer was
extracted with Et2O (3 x 10 mL). The combined organic layers were washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo to afford 0.15 g crude sticky solid.
The crude solid was purified by flash chromatography on silica gel (hexanes/Et2O,
70:30). A mixture of alkene isomers of the benzotriazole alkene 164 was obtained as a
137 colorless solid (0.124 g, 71%). The following data represents only one of two possible
-1 1 isomers: IR(thin film) 3436, 3333, 2965, 2868, 1720 cm ; H NMR (300 MHz, CDCl3)
δ 8.05 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.37 (t, J = 8.2 Hz, 1H),
7.28 (d, J = 7.9 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 6.82 (d, J = 8.2 Hz, 1H),
5.24 (d, J = 9.3 Hz, 1H), 4.68 (dd, J = 9.4, 5.2 Hz, 1H), 3.93 (q, J = 7.1 Hz, 2H), 2.29 (s,
3H), 1.88 (bs, 1H), 1.42 (s, 9H), 1.12-1.25 (m, 1H), 0.94-1.02 (m, 21H), 0.81 (d, J = 6.8
13 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H); C NMR (75 MHz, CDCl3) δ 163.4, 160.9, 155.4,
151.9, 151.6, 145.5, 132.1, 130.9, 130.4, 130.3, 128.5, 125.6, 126.6, 124.7, 120.8, 120.4,
119.2, 109.6, 79.9, 61.1, 54.1, 33.2, 28.4, 20.6, 18.6, 18.0, 17.5, 14.0, 13.3; APCIMS m/z
relative intensity 740 (M+Na 100).
O N O
BOCHN O
N OTIPS
166
Ethyl 2-(1-tert-Butoxycarbonylamino-2-methyl-propyl)-5-[3-(5-methyl-2-
triisopropyl-silanyloxy-phenyl)-3H-indol-3-yl]-oxazole-4-carboxylate (166).
Benzotriazole alkene 164 (0.223 g, 0.311 mmol) was dissolved in acetonitrile (50 mL) in
a quartz reaction vessel and the clear colorless solution was purged with N2 for 30 min.
The resulting deoxygenated solution was irradiated in a Rayonet photochemical reactor at
300 nm for 20 h. The amber colored solution reaction was concentrated in vacuo to
138 afford a dark semi-solid. The crude solid was purified by flash chromatography on silica gel (hexanes/Et2O, 70:30). Indolenine 166 (0.124 g, 71%) was isolated as a colorless
semisolid as a mixture of C10 (diazonamide numbering) epimers. IR(thin film) 3356,
-1 1 2966, 2868, 1720 cm ; H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.30 (t, J = 7.6 Hz,
1H) 7.16 (t, J = 7.2 Hz, 1H), 7.01 (m, 3H) 6.85 (d, J = 8.0 Hz, 1H), 6.67 (s, 0.5H), 6.66
(s, 0.5H), 5.28 (bs, 1H), 4.83 (q, J = 8.1 Hz, 2H), 4.91-3.09 (m, 1H), 2.31-2.20 (m,1H),
2.19 (s, 1.5H), 2.18 (s, 1.5H), 1.44 (s, 9H), 1.44-1.41 (m, 3H), 1.23-1.20 (m, 3H), 1.20-
13 1.03 (m, 18H), 1.05-0.91 (m, 6H); C NMR (100 MHz, CDCl3) δ 166.1, 166.0, 162.3,
162.1, 155.5, 151.7, 151.6, 151.5, 132.3, 130.5, 130.0, 129.9, 129.8, 129.1, 127.2, 125.9,
120.8, 120.6, 80.0, 61.5, 59.0, 58.7, 54.5, 54.4, 33.4, 31.4, 28.5, 23.4, 23.3, 21.3, 21.0,
19.0, 18.6, 18.5, 18.4, 18.3, 18.2, 18.1, 14.6, 14.5, 14.1, 13.7; APCIMS m/z relative
intensity 690 (M+H 100); TOFHRMS (+APCI) Calcd for C39H58N5O6Si: 690.3932,
Found: 690.3877
NH2 CO H HO 2 I
167
3-Iodo-L-tyrosine (167). A solution of iodine (4.2 g, 17 mmol) in 95% EtOH
(45 mL) was added to a stirring solution of L-tyrosine (3.0 g, 17 mmol) in concentrated
o NH4OH (300 mL) at 0 C via addition funnel over the period of 1h. Upon complete
addition, the reaction solution was concentrated to dryness. The resultant peach colored
solids were suspended in acetone at 0 oC and vigorously stirred for 2 h. The solids were
139 then filtered with an acetone rinse and then dried in vacuo to afford 2.41 g of the desired
3-iodo-L-tyrosine as a white solid. Spectral data for this compound matched those of a sample purchased from Acros.
NHBOC
CO H HO 2 I
168
2-tert-Butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionic acid
(168). BOC2O (0.78 g, 3.6 mmol) was added to a stirring solution of 168 in dioxane/H2O
(1:1, 8 mL) at 0 oC. The solution was allowed to stir 0 oC for 2 h, at which time the
reaction was warmed to room temperature and stirred for an additional 1 h. The reaction
solution was diluted with EtOAC and the layers were separated. The aqueous layer was
extracted with EtOAc (2 x 20 mL). The basic aqueous layer was then carefully acidified
with 4 M NaHSO4 which resulted in precipitation of the acid. The slightly acidic mixture
was extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed
with brine, dried over Na2SO4, filtered and concentrated to afford the desired product as a
white foamy solid with spectral data matching thosee of the commercially available
material.
140 NHBoc
CO Bn HO 2 I
169
Benzyl 2-tert-Butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionate
(169). Benzyl bromide (0.69 mL, 5.9 mmol) was added via syringe to a stirring
suspension of acid 169 (1.90 g, 4.66 mmol) and NaHCO3 (0.50 g, 5.9 mmol) in DMF (20
mL) at 0 oC. The resultant solution was stirred at 0 oC for 2 h, warmed to room
temperature and then allowed to stir at this temperature for 12 h. The reaction mixture
was then poured onto ice cold water. The mixture was extracted with EtOAc (3 x 20
mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated to afford a crude colorless oil. The crude oil was purified by flash chromatography on silica gel (hexanes/EtOAc, 70:30). The benzyl ester product (169) was obtained as a colorless oil (1.74 g, 75%). IR(thin film) 3355, 2977, 1735, 1685 cm-1;
1 H NMR (300 MHz, CDCl3) δ 7.42 (m, 5H), 6.87 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 8.3 Hz,
1H), 6.11 (s, 1H), 5.20 (d, J = 12 Hz, 1H), 5.15 (obscured peak, 1H), 5.11 (d, J = 12 Hz,
1H), 4.58 (dd, J = 13.7, 6.2, Hz, 1H), 3.03 (dd, J = 14 Hz, 6.0 Hz, 1H), 2.95 (dd, J = 14.0,
13 6.0 Hz, 1H), 1.46 (s, 9H); C NMR (75 MHz, CDCl3) δ 171.5, 155.1, 154.2, 139.0,
134.9, 130.8, 129.6, 128.6, 128.5, 128.4, 114.9, 85.2, 80.2, 67.2, 54.4, 36.8, 28.3;
APCIMS m/z relative intensity 398 (M-BOC 50); TOFHRMS (+APCI) Calcd for
C21H25NO5I: 498.0772, Found: 498.0784.
141 NHBOC
CO Bn TIPSO 2 I
170
Benzyl-2-tert-Butoxycarbonylamino-3-(3-iodo-4-triisopropylsilanyloxy- phenyl)-propionate (170). Triisopropylsilyl chloride (1.30 mL, 6.13 mmol) was added
to a stirring solution of imidazole (1.2 g, 18 mmol) and phenol 169 in DMF (10 mL) at
room temperature. The clear colorless solution was allowed to stir at this temperature for
2 h. The solution was then diluted with Et2O and H2O. The layers were separated and
the aqueous layer was extracted with Et2O (4 x 10 mL). The combined organic extracts were washed with H2O and then brine, dried over Na2SO4, filtered and concentrated in
vacuo to afford 3 g of a colorless oil. The crude oil was purified by flash
chromatography on silica gel (hexanes/Et2O, 70:30). The TIPS protected phenol 170 was
obtained as a clear colorless oil (2.77 g, 100%). IR(thin film) 3375, 2944, 2866, 1736,
-1 1 1715 cm ; H NMR (300 MHz, CDCl3) δ 7.50 (s, 1H), 7.38-7.28 (m, 5H), 6.83 (d, J =
8.4 Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 5.15 (d, J = 12.3 Hz, 1H), 5.09 (d, J = 12.2 Hz,
1H), 5.03 (d, J = 8.0 Hz, 1H), 4.55 (dd, J = 13.7, 5.9 Hz, 1H), 3.01 (dd, J = 13.8, 5.7 Hz,
1H), 2.91 (dd, J = 13.9, 6.0 Hz, 1H), 1.42, (s, 9H), 1.41-1.25 (m, 3 H), 1.12 (d, J = 7.0
13 Hz, 18H); C NMR (75 MHz, CDCl3) δ 171.7, 154.9, 154.4, 140.1, 134.9, 130.2, 129.9,
128.6, 128.5, 128.4, 117.7, 90.2, 79.9, 67.1, 54.4, 36.7, 28.2, 18.0, 12.8; APCIMS m/z
relative intensity 676 (M+Na 100); TOFHRMS (+APCI) Calcd for C30H44INO5Si:
676.1931, Found: 676.1948.
142
NBOC2 CO Bn TIPSO 2 I
171
Benzyl-2-di-tert-Butoxycarbonylamino-3-(3-iodo-4-triisopropylsilanyloxy-
phenyl)-propionate (171). A mixture of mono-BOC 170 (4.98 g, 7.63 mmol), BOC2O
(4.17 g, 19.1 mmol) and DMAP (0.93 g. 0.76 mmol) was dissolved in toluene and
warmed to 60 oC. The clear colorless solution was allowed to stir at this temperature for
12 h. The reaction mixture was cooled to room temperature and diluted with Et2O.
Imidazole (1.03 g, 15.3 mmol) was added to the solution and it was stirred for 30 min
(sufficient time for imidazole to react with excess BOC2O). The mixture was then cooled
with the addition of ice and was washed with ice cold 1M HCl (1 x 50 mL), followed by
successive washings with cold 0.1M HCl (2 x 100 mL). The combined aqueous layers
were extracted with Et2O (3 x 30 mL). The combined organic layers were washed with
brine and then saturated aqueous NaHCO3, dried over Na2SO4, filtered and concentrated
in vacuo to afford a yellow tinted oil. The crude oil was purified by flash
chromatography on silica gel (hexanes/Et2O, 70:30). The di-BOC protected 171 was
obtained as a clear colorless oil (4.55 g, 79%). IR(thin film) 2945, 2867, 2360, 1747,
-1 1 1698 7.56 cm ; H NMR (300 MHz, CDCl3) δ 7.55 (s, 1H), 7.33 (bs, 5H), 7.00 (d, J =
8.1 Hz, 1H), 6.71 (d, 8.2 Hz, 1H), 5.22 (d, J = 12.4 Hz, 1H), 5.16 (d, J = 12.3 Hz, 1H),
5.13 (obscured peak, 1H), 3.33 (dd, J = 14.2, 5.1 Hz, 1H), 3.14 (dd, J = 14.2, 10.6 Hz,
1H), 1.38 (s, 9H), 1.38-1.31 (obscured peak, 3H), 1.12 (d, J = 7.2 Hz, 18H); 13C NMR
(75 MHz, CDCl3) δ 170.2, 154.0, 151.7, 140.2, 135.4, 131.4, 130.2, 128.4, 128.0, 128.0,
143 117.4, 90.0, 82.9, 66.8, 59.1, 34.3, 27.7, 18.0, 13.0; APCIMS m/z relative intensity 754
(M+H 100).
NBOC2 CO Bn TIPSO 2
H
172
Benzyl 2-di-tert-Butoxycarbonylamino-3-(3-ethynyl-4-triisopropylsilanyloxy-
phenyl)-propionate (172). A solution of ethynyl magnesium bromide (29.6 mL, 0.5
M/THF, 14.8 mmol) was added to a stirring solution of zinc bromide (3.4 g, 15 mmol) in
DMF (20 mL) at room temperature and the suspension was allowed to stir for 30 min.
Next, a solution of aryl iodide (171) (4.45 g, 5.90 mmol) in DMF (20 mL) was added to
the zincate suspension, immediately followed by the addition of Pd(PPh3)4 (0.35 g, 0.30
mmol). The darkening mixture was heated to 60 oC and allowed to stir at this
temperature for 1.5 h. After this time, the mixture was promptly cooled to 0 oC and washed with ice cold 1M HCl. The mixture was extracted with Et2O (3 x 40 mL). The combined organic extracts were washed with water and then brine, dried over Na2SO4, filtered and concentrated in vacuo to afford a dark amber oil. The crude oil was purified by flash chromatography on silica gel (hexanes/Et2O, 90:10). Alkyne 172 was obtained
as an amber oil (2.53 g, 66%). IR(thin film) 3313, 2945, 2867, 1747, 1698 cm-1; 1H NMR
(300 MHz, CDCl3) δ 7.33 (bs, 5H), 7.21 (s, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.73 (d, J = 8.4
Hz, 1H), 5.21 (d, J = 12.5 Hz, 1H), 5.15 (dd, J = 12.6, 5.1 Hz, 1H), 3.34 (dd, J = 14.3, 5.2
144 Hz, 1H), 3.15 (dd, J = 14.5, 10.2 Hz, 1H), 3.14 (s, 1H), 1.38 (s, 18H), 1.36-1.26 (m, 3H),
13 1.10 (d, J = 7.2 Hz, 18H); C NMR (75 MHz, CDCl3) δ 170.2, 156.1, 151.8, 135.5,
134.8, 130.8, 129.6, 128.4, 128.1, 127.9, 118.7, 114.0, 82.9, 80.9, 80.6, 66.8, 59.1, 34.6,
27.7, 17.9, 12.8; APCIMS m/z relative intensity 652 (M+H 100).
O H N CbzHN O O
TIPSO
BnO2C NBOC2
173
Ethyl 2-(2-Benzyloxycarbonylamino-3-methyl-butyrylamino)-4-[5-(2-
benzyloxycarbonyl-2-di-tert-butoxycarbonylamino-ethyl)-2-triisopropylsilanyloxy-
phenyl]-but-3-ynate (173). A solution of lithium bis(trimethylsilyl)amide (18.4 mL, 1
M/hexanes, 18.4 mmol) was added to a stirring solution of alkyne 172 (12.0 g, 18.4
mmol) in THF (100 mL) at -78 oC. The clear colorless solution was allowed to stir at this temperature for 30 min. Next, a solution of ZnBr2 (4.14 g, 18.4 mmol) in 25 mL THF was added to the acetylide solution via cannula. Stirring continued as the temperature was allowed to slowly rise to 0 oC over 30 min. The reaction mixture was cooled back
down to -78 oC and a solution of valine derivative 150 (6.60 g, 16.8 mmol) in THF (30
mL) was added via cannula. The reaction mixture was allowed to slowly warm to room temperature over the period of 1 h, and stirring continued at this temperature for 2 h. The
145 mixture was then poured onto saturated aqueous NH4Cl and diluted with Et2O. The
layers were separated and the aqueous layer was extracted with Et2O (3 x 20 mL). The
combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to
afford a crude sticky solid. The crude solid was purified by flash chromatography on
silica gel (hexanes/Et2O, 70:30). A significant amount of starting alkyne 172 (4.0 g,
33%) was recovered from purification. The desired coupled alkyne 173 was obtained as
a colorless sticky solid (8.91 g, 50%, 76% based on recovery of starting material, 1:1
mixture of diastereomers). IR(thin film) 3332, 2971, 2868, 1744, 1698, 1672 cm-1; 1H
NMR (300 MHz, CDCl3) δ 7.32-7.25 (m, 10H), 7.15 (s, 1H), 7.0 (d, J = 8.5 Hz, 1H), 6.70
(d, J = 8.4 Hz, 1H), 5.63 (d, J = 8.7 Hz, 1H), 5.53 (d, J = 7.3 Hz, 1H), 5.20-5.10 (m, 5H),
4.27-4.19 (m, 3H), 3.33 (dd, J = 14.4, 5.4 Hz, 1H), 3.10 (dd, J = 14.3, 10.1 Hz, 1H), 2.15
(septet, J = 6.5 Hz, 1H), 1.38 (s, 18H), 1.34-1.21 (m, 6H), 1.09 (d, J = 7.2 Hz, 18H), 1.01
13 (d, J = 6.9 Hz, 3H), 0.96 (d, 7.0 Hz, 3H); C NMR (75 MHz, CDCl3) δ 170.8, 170.5,
169.9, 167.1, 156.0, 155.7, 151.6, 151.6, 136.0, 135.3, 134.6, 134.4, 131.0, 129.6, 128.3,
128.25, 128.2, 128.1, 128.0, 127.9, 127.8, 127.8, 127.75, 127..7, 118.5, 113, 84.7, 82.7,
82.1, 66.7, 66.6, 62.1, 58.9, 44.9, 34.4, 31.5, 27.7, 27.6, 27.5, 27.4, 17.7, 13.7, 13.6, 12.6;
APCIMS m/z relative intensity 986 (M+H 100).
146
O
N O
CbzHN O TIPSO
BnO2CNBOC2
174
Ethyl 2-(1-Benzyloxycarbonylamino-2-methyl-propyl)-5-[5-(2-
benzyloxycarbonyl-2-di-tert-butoxycarbonylamino-ethyl)-2-triisopropylsilanyloxy-
benzyl]-oxazole-4-carboxlyate (174). Solid Cs2CO3 (0.74 g, 2.3 mmol) was added in
one solid portion to a stirring solution of di-substituted alkyne 173 (1.12 g, 1.14 mmol) in
DMF (50 mL) at 0 oC. The solution immediately turned from clear and colorless to a red
mixture. The red mixture was allowed to stir at 0 oC for 2 h, at which time saturated
aqueous NH4Cl was added. The resultant yellow mixture was diluted with Et2O. The layers were separated and the aqueous layer was extracted with Et2O (3 x 20 mL). The
combined organic layers were washed with water (3 x 50 mL) and then brine, dried over
Na2SO4, filtered and concentrated in vacuo to afford a crude yellow oil. This crude oil was purified by flash chromatography on silica gel (hexanes/Et2O, 50:50). The desired
oxazole 174 was obtained as a colorless sticky solid (0.97 g, 86%). IR(thin film) 3352,
-1 1 2964, 2868, 2359, 1737, 1714, 1698 cm ; H NMR (300 MHz, CDCl3) δ 7.08 (bs, 10H),
6.92 (d, J = 8.2 Hz, 1H), 6.81 (s, 1H), 6.72 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 9.3 Hz, 1H),
5.17-5.03 (m, 4H), 4.80 (dd, J = 8.9, 6.3 Hz, 2H), 4.41 (d, J = 6.8 Hz, 1H), 4.38 (d, J =
6.8 Hz, 1H), 4.38 (obscured peak, 1H), 3.33, (dd, J = 14.2, 4.8 Hz, 1H), 3.10 (dd, J =
147 14.1, 10.2 Hz, 1H), 2.15 (septet, J = 6.3 Hz, 1H), 1.35 (s, 18H), 1.34 (obscured peak,
3H), 1.33 (obscured peak, 3H), 1.10 (d, J = 6.1 Hz, 18H), 0.88 (m, 6H); 13C NMR (75
MHz, CDCl3) δ 170.0, 161.8, 161.7, 157.5, 155.7, 152.1, 151.5, 136.1, 135.5, 130.6,
129.5, 128.7, 128.2, 128.1, 127.9, 127.8, 127.7, 127.7, 125.6, 117.3, 82.5, 66.6, 60.7,
59.0, 54.5, 34.8, 32.5, 27.5, 26.5, 18.5, 17.8, 14.1, 12.8; APCIMS m/z relative intensity
986 (M+H 100).
O
N O
O H2N TIPSO
HO2CNBOC2
175
Ethyl 2-(1-Amino-2-methyl-propyl)-5-[5-(2-tert-butoxycarbonylamino-2-
carboxy-ethyl)-2-triisopropylsilanyloxy-benzyl]-oxazole-4-carboxyate (175). A
mixture of the protected amino acid 174 (1.65 g, 1.67 mmol) and 10% Pd/C (1.65 g) in
THF (50 mL) was carried through a freeze-pump-thaw cycle with a final charge of H2 gas. The H2 blanketed, black mixture was stirred at room temperature for 2 h. The
mixture was then filtered through a pad of Celite with an EtOAc rinse. The filtrate was
concentrated in vacuo to afford the desired product 175 as a white sticky solid (1.25 g,
98%). IR(thin film) 3305, 2964, 2868, 2359, 1730, 1716, 1699 cm-1; 1H NMR (300
MHz, CDCl3) δ 6.86 (d, J = 8.1 Hz, 1H), 6.71 (s, 1H), 6.63 (d, J = 8.2 Hz, 1H), 4.79 (app t, J = 7.2 Hz, 1H), 4.31 (obscured peak, 1H), 4.30 (q, 7.1 Hz, 2H), 3.92 (d, J = 6.4 Hz,
1H), 3.20 (dd, J = 13.9, 7.6 Hz, 1H), 2.83 (dd, J = 13.8, 8.1 Hz, 1H), 2.07 (septet, J = 6.6
148 Hz, 1H), 1.35-1.16 (m, 6H), 1.31 (s, 18H), 1.03 (d, J = 7.3 Hz, 18H), 0.89 (d, J = 6.7 Hz,
13 3H), 0.80 (d, J = 6.7 Hz, 3H); C NMR (75 MHz, CDCl3) δ 174.1, 162.9, 162.0, 157.8,
151.9, 133.32, 133.31, 131.0, 128.7, 128.3, 125.4, 117.3, 82.5, 60.9, 60.5, 55.0, 36.0,
32.4, 27.8, 18.6, 18.2, 18.0, 14.3, 13.0; APCIMS m/z relative intensity 762 (M+H 100);
TOFHRMS (+APCI) Calcd for C39H63N3O10Si: 762.4355, Found: 762.4320.
O
N O O BOC N 2 N O H TIPSO
OTIPS H O N NBOC2 O O N O
176
Ethyl 2-(1-Amino-2-methyl-propyl)-5-[5-(2-tert-butoxycarbonylamino-2-
carboxy-ethyl)-2-triisopropylsilanyloxy-benzyl]-oxazole-4-carboxylate Dimer (176).
(EDC coupling): EDC•HCl (0.014 g, 0.070 mmol), Et3N (0.01 mL, 0.07 mmol) and
HOAt (0.01 g, 0.07 mmol) were sequentially added to stirring solution of amino acid 175
(0.049 g, 0.070 mmol) in DMF (50 mL) at room temperature. The resultant yellow
solution was stirred at room temperature for three days. The solution was then diluted
with Et2O and H2O. The layers were separated and the aqueous layer was extracted with
Et2O (3 x 10 mL). The combined organic extracts were washed with water (3 x 30 mL)
and then brine, dried over Na2SO4, filtered and concentrated in vacuo to afford 0.04 g
149 crude solid. This crude solid was purified by flash chromatography on silica gel
(hexanes/EtOAc, 70:30). The dimer 176, the only recognizable product obtained, was recovered as a colorless solid (0.07 g, 15%), mp 250 oC (dec).
(DEPC coupling): DEPC (0.80 mL, 5.3 mmol) and diisopropylethyl amine (1.84
mL, 10.6 mmol) were sequentially added to stirring solution of amino acid 175 (0.81 g,
1.1 mmol) in CH3CN (50 mL) at room temperature. The resultant clear colorless solution
was stirred at room temperature for two days, and then concentrated in vacuo to afford a
yellow residue. This residue was diluted with Et2O and saturated aqueous NH4Cl. The
layers were separated and the aqueous layer was extracted with Et2O (2 x 30 mL). The
combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to afford 1.5 g of a crude amber colored oil. This crude oil was purified by flash chromatography on silica gel (hexanes/EtOAc, 70:30). Dimer 176 was
recovered as a colorless solid (0.334 g, 42%).
(HATU coupling): HATU (1.37 g, 3.61 mmol) and 2,4,6-chollidine (1.44 mL,
10.8 mmol) were sequentially added to stirring solution of amino acid 179 (1.25 g, 1.64
mmol) in DMF/CH2Cl2 (600 mL, 2:1) at room temperature. The clear colorless solution
was stirred at room temperature for 24 h. The solution was then concentrated in vacuo to
afford a yellow orange residue. This residue was taken up in EtOAc and washed
successively with NaHCO3 (2 x 30 mL) and then brine, dried over Na2SO4, filtered, and
concentrated in vacuo to afford a crude residue. The crude material was purified by flash
chromatography on silica gel (hexanes/EtOAc, 70:30). The dimer 176 was recovered as
a colorless solid (0.150 g, 12%). IR (thin film) 3384, 2978, 2361, 1737, 1713 cm-1; 1H
NMR (300 MHz, CDCl3) δ 6.93 (d, J = 8.3 Hz, 2H), 6.72 (s, 2H), 6.69 (d, J = 8.3 Hz,
150 2H), 6.40 (d, J = 8.4 Hz, 2H), 4.93 (app t, J = 7.9 Hz, 2H), 4.76 (dd, J = 8.5, 6.1 Hz, 2H),
4.53 (d, J = 16.8 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.20 (d, J = 16.7 Hz, 2H), 3.33 (dd, J
= 14.5, 5.8 Hz, 2H), 3.05 (dd, J = 14.4, 9.1 Hz, 2H), 2.14 (septet, J = 7.0 Hz, 2H), 1.39 (s,
36H), 1.33 (t, J = 7.1 Hz, 6H), 1.29 (septet, J = 7.2 Hz, 6H), 1.11 (d, J = 7.4 Hz, 36H),
13 0.82 (d, J = 6.6 Hz, 6H), 0.77 (d, J = 6.6 Hz, 6H); C NMR (75 MHz, CDCl3) δ 169.2,
162.3, 161.9, 157.0, 152.2, 152.0, 130.4, 129.8, 129.4, 128.7, 126.4, 117.4, 81.1, 60.9,
60.7, 53.0, 34.7, 32.7, 27.9, 18.8, 18.6, 18.0, 14.3, 12.9; APCIMS m/z relative intensity
1537 (M+Na 100).
6.2 Diazoparaquinone Mechanism-of-Action Studies
General Procedure 1. Phenylation of Prekinamycin and Derivatives. AIBN
(1.1 equiv) in benzene (0.06 M) was added via syringe pump addition over a period of 1 h to a stirring solution of diazoquinones 188, 323 or 324 (1 equiv) and Bu3SnH (1.1
equiv) in benzene (0.06 M) at 80 oC. When the addition was complete, the reaction
solution was allowed to cool to room temperature. After reaching room temperature the
reaction mixture was concentrated in vacuo. The resulting residue was purified by flash
chromatography on silica gel using the specified eluent.
General Procedure 2. Aromatic Solvent Competition Experiments. AIBN
(1.1 equiv) in an equimolar solution of benzene and the appropriate aromatic solvent
(0.06 M) was added via syringe pump addition over a period of 1 h to a stirring solution
of diazoquinone 324 (1 equiv) and Bu3SnH (1.1 equiv) also in an equimolar solution of
151 benzene and the appropriate aromatic solvent (0.06 M) at 80 oC. When the addition was
complete, the reaction solution was allowed to cool to room temperature. After reaching
room temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica
gel column and purified eluting with CH2Cl2 with an increasing percentage of EtOAc
from 0% to 10%. Purification furnished the clean benzene trapped product and in most
cases a mixture of o,m,p or the 2,4,5 isomers of the given substituted aromatic trapped
product. Relative rates were quantified by product mass comparison of the substituted
aromatic adducts vs. the benzene addition product. Isolation of analytical samples of
pure o, m or p (or the 2, 4 or 5 isomers) from chromatography permitted 1H NMR identification. In cases where isomer separation was not achieved, the ratios were determined by inspection of 1H NMR spectra of the mixtures.
General Procedure 3. Aromatic Solvent Competition Eeperiments. AIBN
(1.1 equiv) in an equimolar solution of benzene and the appropriate aromatic solvent
(0.06 M) was added via syringe pump addition over a period of 1 h to a stirring solution
of diazoquinone 324 (1 equiv) and Bu3SnH (1.1 equiv) also in an equimolar solution of
benzene and the appropriate solvent (0.06 M) at 80 oC. When the addition was complete,
the reaction solution was allowed to cool to room temperature. After reaching room
temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica gel
column. Purification eluting with CH2Cl2 to remove tin residues, followed by an increase
in polarity to 10% acetone in CH2Cl2 afforded a mixture of the benzene and substituted
aromatic trapped products. Relative rates were quantified by 1H NMR integration of the
substituted aromatic adducts vs. the benzene addition product. Ratio determination was
152 made through similar comparison of the pure o,m,p (or the 2,4,5) isomer(s) 1H NMR
spectra to that of the mixture.
General Procedure 4. Aromatic Solvent Competition Experiments with
Varying Equivalents of Tin. General Proceeures 2 and 3 were both used varying only
in the equivalents of Bu3SnH.
O
O O
O O N2
O
323
Acetic acid 9-acetoxy-11-diazo-2-methyl-5,10-dioxo-10,11-dihydro-5H-
benzo[b]fluoren-4-yl ester (323). Pyridine (3.6 mL, 28 mmol) and Ac2O (3.6 mL, 44
mmol) were sequentially added to a mixture of 188 (150 mg, 0.471 mmol) and DMAP
(6.0 mg, 0.050 mmol) in CH2Cl2 (15.0 mL) at room temperature. The dark purple
mixture was stirred at room temperature for 2 days, turning to a dark red color. At this
time, the reaction solution was diluted with saturated aqueous NaHCO3 (50 mL) and
CH2Cl2 (30 mL). The layers were separated and the aqueous layer was extracted with
CH2Cl2 (3 x 10 mL). The combined organic layers were washed with water and brine,
dried over Na2SO4, filtered and concentrated. The resulting dark red solid was purified
by flash chromatography on silica gel eluting with CH2Cl2/EtOAc (99:1) to yield the di- acetate (323) as a red solid (16 mg, 85%). mp 205 oC (dec); IR (neat): 3365, 2924, 2102,
153
-1 1 1766 cm ; H NMR (300 MHz, CDCl3) δ 8.17 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 7.9 Hz,
1H), 7.30 (d, J = 7.9 Hz, 1H), 7.20 (s, 1H), 6.89 (s, 1H), 2.57 (s, 3H), 2.47 (s, 3H), 2.46
13 (s, 3H); C NMR (75 MHz, CDCl3) δ 180.0, 177.6, 171.0, 170.0, 150.0, 147.6, 139.7,
137.7, 137.2, 136.7, 136.4, 135.2, 129.0, 126.4, 126.0, 124.3, 122.3, 121.9, 116.9, 22.1,
21.9, 21.6; ESI m/z relative intensity 425 (M+Na 72); TOFHRMS (+ESI) Calcd for
C22H15N2O6Na: 425.0750, Found 425.0738.
HO OH
OH O
331
4,5,9-Trihydroxy-2-Methyl-11-Phenyl-Benzo[b]fluoren-10-one (331).
Following General Procedure 1, prekinamycin 188 (18 mg, 0.057 mmol) was converted
into benzo[b]fluorenone 331 (12 mg, 59%). mp 260 oC (dec); IR (neat): 3409, 1585 cm-1;
Presumably, due to the partial free radical nature of 331 analogous to kinobscurinone,1
331 appears to be “NMR silent” exhibiting neither a 1H- nor a 13C NMR signature; ESI
m/z relative intensity 391(M+Na 100); TOFHRMS (+ESI) Calcd for C24H16O4Na:
391.0946, Found 391.0947.
154 O
O OH
O O
O
332
Acetic Acid 9-Acetoxy-5-Hydroxy-2-Methyl-10-oxo-11-Phenyl-10H-
Benzo[b]fluoren-4-yl ester (332). Following General Procedure 1, diazoquinone 323
(18 mg, 0.044 mmol) was converted into benzo[b]fluorenone 332 (9.8 mg, 50%). mp
o -1 1 200 C (dec); IR (neat): 3330, 1789, 1766 cm ; H NMR (400 MHz, CDCl3) δ 9.39 (s,
1H), 7.88 (d, J = 7.9 Hz, 1H), 7.56 (t, J = 8.0 Hz, 2H), 7.55 (d, J = 7.7 Hz, 1H), 7.50-
7.41 (m, 3H), 7.06 (d, J = 8.0 Hz, 1H), 7.06 (s, 1H), 7.05 (s, 1H), 2.51 (s, 3H), 2.36 (s,
13 3H), 2.32 (s, 3H), C NMR (100 MHz, CDCl3) δ 180.2, 170.2, 166.9, 151.3, 149.9,
147.9, 144.4, 143.3, 138.5, 135.7, 133.9, 133.8, 129.7, 128.8, 128.7, 128.2, 126.5, 125.4,
123.3, 123.2, 123.0, 122.6, 114.6, 21.7, 21.6, 21.3; ESI m/z relative intensity 475 (M+Na
100); TOFHRMS (+ESI) Calcd for C28H20O6Na: 475.1158, Found 475.1142.
155
O OH
O O
333
5-Hydroxy-4,9-dimethoxy-2-methyl-11-phenyl-benzo[b]fluoren-10-one (333).
Following General Procedure 1, diazoparaquinone 324 (11 mg, 0.032 mmol) was converted into benzo[b]fluorenone 333 (10 mg, 79%). mp 220 oC (dec); IR (neat): 3181,
-1 1 1633 cm ; H NMR (400 MHz, CDCl3) δ 10.76 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.56
(d, J = 6.8 Hz, 2H), 7.48 (t, J = 8.2 Hz, 1H), 7.45 (t, J = 7.1 Hz, 2H), 7.39 (d, J = 7.2 Hz,
1H), 7.00 (d, J = 8.4 Hz, 1H), 6.81 (s, 1H), 6.67 (s, 1H), 4.09 (s, 3H), 3.90 (s, 3H), 2.32
13 (s, 3H); C NMR (100 MHz, CDCl3) δ 182.0, 161.8, 151.9, 151.1, 146.6, 144.3, 138.7,
136.9, 135.0, 134.6, 130.0, 129.4, 128.4, 128.2, 128.1, 120.0, 119.2, 117.8, 115.0, 114.9,
115.2, 56.9, 56.6, 22.2; ESI m/z relative intensity 497 (M+H 50); TOFHRMS (+ESI)
Calcd for C26H21O4: 397.1440, Found 397.1443.
156 O O O O
O O
O
334
Acetic Acid 4,5-Diacetoxy-2-Methyl-10-oxo-11-Phenyl-10H-benzo[b]fluoren-
9-yl Ester (334). From 331: Pyridine (0.26 mL, 3.1 mmol) and Ac2O (0.31 mL, 3.1
mmol) were sequentially added to a mixture of 331 (11.0 mg, 0.031 mmol) and DMAP
(1.0 mg, 0.0080 mmol) in CH2Cl2 (1.0 mL) at room temperature. The dark purple
mixture was stirred at room temperature for 30 min, turning to a dark red/orange color.
At this time, the reaction solution was diluted with saturated aqueous NaHCO3 (10 mL) and CH2Cl2 (10 mL). The layers were separated and the aqueous layer was extracted
with CH2Cl2 (3x 10 mL). The combined organic layers were washed with water and
brine, dried over Na2SO4, filtered and concentrated. The resulting dark red solid was
purified by flash chromatography on silica gel (CH2Cl2) to yield the tri-acetate as a bright
orange solid (8.1 mg, 52%).
From 332: Pyridine (0.070 mL, 0.90 mmol) and Ac2O (0.85 mL, 0.90 mmol)
were sequentially added to a mixture of 332 (4.0 mg, 0.90 mmol) and DMAP (1.0 mg,
0.0080 mmol) in CH2Cl2 (1.0 mL) at room temperature. The dark red mixture was stirred at room temperature for 30 min, changing to a dark red/orange color. At this time, the reaction solution was diluted with saturated aqueous NaHCO3 (10 mL) and CH2Cl2 (10
mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (3 x 10
157 mL). The combined organic layers were washed with water and brine, dried over
Na2SO4, filtered and concentrated. The resulting dark red solid was purified by flash
chromatography on silica gel (CH2Cl2). The tri-acetate was obtained as a orange solid
(4.4 mg, 99%). mp 195 oC (dec); IR (neat): 1769, 1635 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.59 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 7.3 Hz, 1H), 7.52-7.47 (m, 4H), 7.39 (d, J
= 8.3 Hz, 1H), 7.01 (d, J = 7.7 Hz, 1H), 6.99 (s, 1H), 6.80 (s, 1H), 2.53 (s, 3H), 2.43 (s,
13 3H), 2.31 (s, 6H); C NMR (125 MHz, CDCl3) δ 179.4, 169.6, 168.7, 167.6, 154.6,
151.4, 145.9, 145.2, 142.0, 141.1, 136.5, 133.8, 132.8, 129.4, 129.3, 129.3, 128.9, 128.0,
125.9, 125.3, 124.8, 124.2, 123.7, 121.8, 21.3, 21.2, 21.1, 20.9; ESI m/z relative intensity
517 (M+Na 100); TOFHRMS (+ESI) Calcd for C30H22O7Na: 517.1263, Found 517.1245.
O O
O O
335
4,5,9-Trimethoxy-2-Methyl-11-Phenyl-Benzo[b]fluoren-10-one (335). From
331: Methyl iodide (0.042 mL, 0.68 mmol) was added to a mixture of 331 (25.0 mg,
0.068 mmol) and K2CO3 (93.0 mg, 0.068 mmol) in DMF (2.0 mL) at room temperature.
The dark purple mixture was stirred at room temperature for 12 h, turning to a dark red color. The reaction solution was then diluted with saturated aqueous NH4Cl (10 mL) and
Et2O (10 mL). The layers were separated and the aqueous layer was extracted with Et2O
158 (3 x 10 mL). The combined organic layers were washed with water and brine, dried over
Na2SO4, filtered and concentrated. The resulting light red solid was purified by flash
chromatography on silica gel (CH2Cl2). The tri-methylether was obtained as a bright red
solid (11.5 mg, 41%).
From 333: Methyl iodide (0.031 mL, 0.50 mmol) was added to a mixture of 333
(20 mg, 0.050 mmol) and K2CO3 (69 mg, 0.050 mmol) in DMF (1.0 mL) at room
temperature. The dark red mixture was stirred at room temperature for 12 h, turning to a light red color. The reaction mixture was then diluted with saturated aqueous NH4Cl (10
mL) and Et2O (10 mL). The layers were separated and the aqueous layer was extracted
with Et2O (3X 10 mL). The combined organic layers were washed with water and brine, dried over Na2SO4, filtered and concentrated. The resulting light red solid was purified
by flash chromatography on silica gel (CH2Cl2). The tri-methylether was obtained as a
bright red solid (12 mg, 59%). mp 210 oC (dec); IR (neat): 1643 cm-1; 1H NMR (300
MHz, CDCl3) δ 7.60-7.50 (m, 2H), 7.50-7.40 (m, 5H), 6.96 (dd, J = 6.7, 2.7 Hz, 1H),
6.76 (s, 1H), 6.59 (s, 1H), 4.02 (s, 3H), 3.96 (s, 3H), 3.89 (s, 3H), 2.32 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ 181.5, 161.6, 155.2, 153.0, 151.7, 145.3, 140.3, 138.7, 134.3, 134.2,
130.1, 129.2, 128.3, 127.8, 126.8, 121.4, 118.3, 118.2, 117.4, 113.9, 113.6, 63.9, 56.1,
55.9, 21.7; ESI m/z relative intensity 411 (M+H 100); TOFHRMS (+ESI) Calcd for
C27H22O4: 411.1596, Found 411.1596.
159
O OH
O O
336a
5-Hydroxy-4,9-Dimethoxy-2-Methyl-11-p-Tolyl-Benzo[b]fluoren-10-one
(336a). Following General Procedure 2, diazoparaquinone 324 (20 mg, 0.060 mmol) was converted into a 2.2:1 mixture of benzo[b]fluorenone 336a (o,m,p = 62:23:15) (7.4 mg,
30%) and benzo[b]fluorenone 333 (3.6 mg, 15%). (o,m,p mixture) IR (neat): 3190, 1633
-1 1 cm ; (o-isomer) H NMR (500 MHz, CDCl3) δ 10.64 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H),
7.50 (t, J = 8.1 Hz, 1H), 7.36-7.22 (m, 3H), 7.19 (d, J = 7.3 Hz, 1H), 7.01 (d, J = 8.3 Hz,
1H), 6.69 (s, 1H), 6.54 (s, 1H), 4.11 (s, 3H), 3.89 (s, 3H), 2.31 (s, 3H), 2.16 (s, 3H);
1 (o,m,p mixture) H NMR (500 MHz, CDCl3) δ 10.76 (s, 0.4H, m,p), 10.68 (s, 0.6H, o)
7.60 (d, J = 7.7 Hz, 0.5H), 7.59 (d, J = 7.4 Hz, 0.5H), 7.51-7.47 (m, 1H), 7.36-7.33 (m,
1H), 7.30-7.22 (m, 2H), 7.19 (d, J = 6.8 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.85 (s,
0.25H), 6.79 (s, 0.25H), 6.69 (s, 1H), 6.54 (s, .5H), 4.11 (s, 1.8H, o), 4.10 (s, 1.2H, m,p),
3.90 (s, 1.2H, m,p) 3.89 (s, 1.8H, o), 2.35 (s, 1.8H, m,p), 2.31 (s, 1.8H, o), 2.16 (s, 3H);
13 (o,m,p mixture) C NMR (125 MHz, CDCl3) δ 181.5, 161.3, 151.5, 150.6, 145.9, 144.2,
138.5, 136.7, 136.5, 135.1, 134.0, 129.9, 129.5, 128.5, 127.6, 126.6, 125.4, 121.3, 119.4,
118.9, 118.6, 117.4, 114.6, 114.4, 111.1, 56.53, 56.5, 56.5, 21.8, 21.7, 19.9; ESI m/z
160 relative intensity 411 (M+H 30); TOFHRMS (+ESI) Calcd for C27H22O4: 411.1596,
Found 411.1611.
O OH
O O Cl
336b
11-(4-Chloro-Phenyl)-5-Hydroxy-4,9-Dimethoxy-2-Methyl-Benzo[b]fluoren-
10-one (336b). Following General Procedure 2, diazoparaquinone 324 (20 mg, 0.060
mmol) was converted into a 1.5:1 mixture of benzo[b]fluorenone 336b (o,m,p =
48:32:20) (9.5 mg, 37%) and benzo[b]fluorenone 333 (5.0 mg, 21%). 336b isomers were
separated via preparative TLC (20% EtOAc in benzene). (o-isomer) mp 280 oC (dec); (o-
-1 1 isomer) IR (neat): 3178, 1630 cm ; (o-isomer) H NMR (500 MHz, CDCl3) δ 10.76 (s,
1H), 7.60 (d, J = 7.8 Hz, 1H), 7.50 (m, 2H), 7.33 (m, 3H), 7.02 (d, J = 8.2 Hz, 1H), 6.69
(s, 1H), 6.58 (s, 1H), 4.11 (s, 3H), 3.90 (s, 3H), 2.33 (s, 3H); (m-isomer) mp 260 oC (dec);
-1 1 (m-isomer) IR (neat): 3378, 1630 cm ; (m-isomer) H NMR (500 MHz, CDCl3) δ 10.82
(s, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.51 (m, 2H), 7.44 (d, J = 6.8 Hz, 1H), 7.38 (t, J = 7.8
Hz, 1H), 7.37 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 6.75 (s, 1H), 6.71 (s, 1H), 4.12 (s, 3H),
3.91 (s, 3H), 2.36 (s, 3H); (p-isomer) mp 240 oC (dec); (p-isomer) IR (neat): 3166, 1631
-1 1 cm ; (p-isomer) H NMR (500 MHz, CDCl3) δ 10.81 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H),
7.52 (d, J = 8.6 Hz, 2H), 7.50 (t, J = 8.2 Hz, 1H), 7.42 (d, 8.6 Hz, 2H), 7.30 (d, J = 8.4
161 Hz, 1H), 6.79 (s, 1H), 6.71 (s, 1H), 4.12 (s, 3H), 3.92 (s, 3H), 2.35 (s, 3H); (o,m,p-
13 mixture) C NMR (125 MHz, CDCl3) δ 181.6, 181.5, 181.3, 161.3, 151.5, 151.4, 151.3,
151.1, 144.5, 143.5, 143.3, 142.3, 138.5, 136.6, 136.5, 136.4, 134.1, 133.8, 133.7, 133.2,
131.1, 130.7, 130.3, 129.5, 129.2, 129.1, 128.9, 128.3, 128.1, 127.9, 126.5, 121.1, 119.6,
119.5, 119.3, 118.5, 118.4, 118.3, 117.5, 114.7, 114.4, 114.2, 111.2, 111.1, 56.6, 56.5,
56.2, 56.1, 21.8, 21.7; ESI m/z relative intensity 431 (M+H 100); TOFHRMS (+ESI)
Calcd for C26H20O4Cl: 431.1050, Found 431.1060.
O OH
O O CN
336c
4-(5-Hydroxy-4,9-Dimethoxy-2-Methyl-10-oxo-10H-Benzo[b]fluoren-11-yl)-
Benzonitrile (336c). Following General Procedure 2, diazoparaquinone 324 (20 mg,
0.060 mmol) was converted into a 2.2:1 mixture of benzo[b]fluorenone 336c (o,m,p =
43:25:32) (13 mg, 51%) and benzo[b]fluorenone 333 (5.6 mg, 23%). 336c isomers were separated via preparative TLC (20% EtOAc in benzene). (o-isomer) mp 265 oC (dec); (o-
-1 1 isomer) IR (neat): 3394, 2232, 1631 cm ; (o-isomer) H NMR (500 MHz, CDCl3)
δ 10.87 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.61 (d, J = 7.9 Hz,
1H), 7.52 (t, J = 8.2 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.03 (d, J
= 8.2 Hz, 1H), 6.71 (s, 1H), 6.58 (s, 1H), 4.13 (s, 3H), 3.92 (s, 3H), 2.34 (s, 3H); (m-
162 isomer) mp 265 oC (dec); (m-isomer) IR (neat): 3412, 2216, 1633 cm-1; (m-isomer) 1H
NMR (500 MHz, CDCl3) δ 10.88 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.67 (d,
J = 7.7 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H) 7.47 (t, J = 8.1 Hz,
1H), 7.04 (d, J = 7.7 Hz, 1H), 6.72 (s, 1H), 6.70 (s, 1H), 4.13 (s, 3H), 3.93 (s, 3H), 2.37
(s, 3H); (p-isomer) mp 265 oC (dec); (p-isomer) IR (neat): 3412, 2223, 1631 cm-1; (p-
1 isomer) H NMR (500 MHz, CDCl3) δ 10.92 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.66 (d, J
= 8.5 Hz, 2H), 7.61 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 8.2 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H),
6.72 (s, 1H), 6.71 (s, 1H), 4.13 (s, 3H), 3.92 (s, 3H), 2.36 (s, 3H); (o,m,p-mixture) 13C
NMR (125 MHz, CDCl3) δ 181.7, 181.6, 181.4, 161.5, 161.4, 152.4, 152.1, 151.6, 151.5,
143.1, 143.0, 142.9, 139.9, 139.6, 138.7, 138.6, 138.5, 136.4, 136.3, 136.2, 134.4, 133.0,
132.9, 132.3, 131.7, 131.5, 130.8, 130.4, 130.0, 129.7, 128.8, 127.9, 121.0, 119.5, 118.3,
118.1, 118.0, 117.9, 117.8, 115.2, 114.4, 114.3, 114.2, 113.1, 112.1, 111.4, 111.3, 111.3,
56.6, 56.5, 56.3, 56.3, 56.2, 21.8; ESI m/z relative intensity 444 (M+Na 100); TOFHRMS
(+ESI) Calcd for C27H19NO4Na: 444.1212, Found 444.1215.
163
O OH
O O O
336d
5-Hydroxy-4,9-Dimethoxy-11-(4-Methoxy-Phenyl)-2-Methyl-
Benzo[b]fluoren-10-one (336d). Following General Procedure 2, diazoparaquinone 324
(20 mg, 0.060 mmol) was converted into a 3.2:1 mixture of benzo[b]fluorenone 336d
(o,m,p = 76:16:12) (15 mg, 57%) and benzo[b]fluorenone 333 (4.3 mg, 18%). (o-isomer)
mp 210 oC (dec); (o-isomer) IR (neat): 3182, 1632 cm-1; (o-isomer) 1H NMR (500 MHz,
CDCl3) δ 10.70 (s, 1H), 7.58, (d, J = 7.9 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.36 (t, J = 7.6
Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 7.0
(d, J = 8.3 Hz, 1H), 6.66 (s, 1H), 6.65 (s, 1H), 4.10 (s, 3H), 3.89 (s, 3H), 3.73 (s, 3H),
13 2.32 (s, 3H); (o-isomer) C NMR (125 MHz, CDCl3) δ 181.3, 161.2, 157.4, 151.4, 150.4,
144.0, 142.8, 138.1, 136.5, 133.8, 130.7, 130.0, 129.2, 124.3, 121.6, 120.4, 119.4, 118.7,
117.3, 114.7, 114.6, 111.3, 110.9, 56.5, 56.1, 55.7, 21.8; (m,p-isomer) IR (neat): 3178,
-1 1 2216, 1622 cm ; (m,p-isomer) H NMR (500 MHz, CDCl3) δ 10.78 (s, 0.43H, p), 10.76
(s, 0.57H, m), 7.59 (d, J = 7.3 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H),
7.37 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 7.6 Hz, 0.5H), 7.06-6.98 (m, 2H), 6.93 (d, J = 8.6
Hz, 0.5H), 6.88 (s, 0.5H), 6.82 (s, 0.5H), 6.69 (s, 1H), 4.10 (s, 3H), 3.91 (s, 3H), 3.88 (s,
1.5H), 3.88 (s, 1.5H), 2.35 (s, 1.5H), 2.34 (s, 1.5H); (p-isomer) IR (neat): 3412, 2223,
-1 13 1631 cm ; (m,p-isomer) C NMR (125 MHz, CDCl3) δ 181.6, 181.3, 161.4, 161.2,
164 159.6, 159.2, 157.4, 151.4, 150.8, 150.4, 150.2, 149.4, 146.2, 145.8, 144.0, 142.8, 138.6,
138.2, 136.5, 136.4, 136.2, 136.1, 134.0, 133.8, 133.7, 131.2, 130.7, 130.0, 129.2, 128.9,
128.7, 128.3, 126.6, 124.3, 122.1, 122.0, 121.7, 121.6, 120.4, 119.8, 119.4, 118.9, 118.8,
118.7, 117.4, 117.3, 114.8, 114.7, 114.6, 114.4, 113.7, 113.6, 113.2, 111.3, 111.1, 110.9,
110.5, 56.5, 56.4, 56.1, 56.0, 55.7, 55.3, 55.2, 21.8, 21.7; ESI m/z relative intensity 449
(M+Na 85); TOFHRMS (+ESI) Calcd for C27H22O5Na: 449.1365, Found 449.1346.
O O OH OH
O O O O
336e
11-(3,5-Dimethyl-phenyl)-5-Hydroxy-4,9-Dimethoxy-2-Methyl-
Benzo[b]fluoren-10-one (336e). Following General Procedure 2, diazoparaquinone 324
(20 mg, 0.060 mmol) was converted into a 3.14:1 mixture of benzo[b]fluorenone 336e
(2:4:5 = 50:50:0) (12 mg, 44%) and benzo[b]fluorenone 333 (3.4 mg, 14%). 336e
isomers were separated via preparative TLC (1% EtOAc in CH2Cl2). (4-isomer) mp 220
o -1 1 C (dec); (4-isomer) IR (neat): 3195, 1633 cm ; (4-isomer) H NMR (500 MHz, CDCl3)
δ 10.67 (s, 1H), 7.59 (d, J = 7.3 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.12 (s, 1H), 7.09 (d, J
= 7.7 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.68 (s, 1H), 6.58 (s,
1H), 4.11 (s, 3H), 3.89 (s, 3H), 2.37 (s, 3H), 2.30 (s, 3H), 2.13 (s, 3H); (4-isomer) 13C
NMR (125 MHz, CDCl3) δ 181.5, 161.3, 151.4, 150.4, 146.1, 144.3, 138.4, 137.1, 136.5,
165 136.5, 133.9, 131.9, 130.8, 129.9, 128.4, 126.1, 121.4, 119.4, 118.6, 117.3, 114.5, 114.4,
111.1, 56.5, 56.1, 21.7, 21.3, 19.8; (2-isomer) mp 250 oC (dec); (2-isomer) IR (neat):
-1 1 3194, 1633 cm ; (2-isomer) H NMR (500 MHz, CDCl3) δ 10.64 (s, 1H), 7.60 (d, J = 7.5
Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.18 (t, J = 8.1 Hz, 1H), 7.10 (d, J = 7.6 Hz, 2H), 7.01
(d, J = 8.1 Hz, 1H), 6.69 (s, 1H), 6.45 (s, 1H), 4.12 (s, 3H), 3.89 (s, 3H), 2.30 (s, 3H),
13 2.04 (s, 6H); (2-isomer) C NMR (125 MHz, CDCl3) δ 181.5, 161.3, 151.5, 150.3,
145.9, 143.3, 138.6, 136.6, 135.9, 134.8, 134.0, 130.8, 130.0, 127.1, 127.0, 121.1, 119.5,
118.0, 117.3, 114.5, 111.2, 56.4, 56.1, 20.7 20.1; ESI m/z relative intensity 425 (M+H
50); TOFHRMS (+ESI) Calcd for C28H25O4: 425.1753, Found 425.1740.
O O OH OH
O O O O O O O
O
336f
11-(3,5-Dimethoxy-Phenyl)-5-Hydroxy-4,9-Dimethoxy-2-Methyl-
Benzo[b]fluoren-10-one (336f). Following General Procedure 2, diazoparaquinone 324
(20 mg, 0.060 mmol) was converted into a 4.2:1 mixture of benzo[b]fluorenone 336f
(2:4:5 = 31:69:0) (16 mg, 59%) and benzo[b]fluorenone 333 (3.4 mg, 14%). (4-isomer) mp 190 oC (dec); (4-isomer) IR (neat): 3195, 1633 cm-1; (4-isomer) 1H NMR (500 MHz,
CDCl3) δ 10.68 (s, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.46 (t, J = 8.2 Hz, 1H), 7.31 (d, J =
8.9 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.70 (s, 1H), 6.65 (s, 1H), 6.59 (m, 2H), 4.08 (s,
166 3H), 3.89 (s, 3H), 3.87 (s, 3H), 3.72 (s, 3H), 2.32 (s, 3H); (4-isomer) 13C NMR (125
MHz, CDCl3) δ 181.3, 161.2, 160.9, 158.9, 151.4, 150.0, 144.1, 142.8, 138.0, 136.5,
133.6, 131.5, 128.3, 121.8, 119.5, 118.8, 117.2, 116.8, 114.7, 114.5, 110.9, 104.5, 99.0,
56.5, 56.1, 55.6, 55.4, 21.8; (2-isomer) mp 180 oC (dec); (2-isomer) IR (neat): 3190,
-1 1 1633 cm ; (2-isomer) H NMR (500 MHz, CDCl3) δ 10.80 (s, 1H), 7.58 (d, J = 8.1 Hz,
1H), 7.47 (t, J = 8.1 Hz, 1H), 7.30 (t, J = 8.3 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 6.65 (d, J
= 8.5 Hz, 2H) 6.64 (s, 1H), 6.57 (s, 1H), 4.08 (s, 3H), 3.89 (s, 3H), 3.68 (s, 6H), 2.30 (s,
13 3H); (2-isomer) C NMR (125 MHz, CDCl3) δ 181.1, 161.1, 158.5, 151.5, 150.0. 144.0,
130.4, 138.1, 136.7, 136.3, 133.6, 129.1, 121.6, 119.5, 118.5, 117.2, 114.9, 114.5, 113.7,
113.3, 110.9, 110.6, 104.3, 56.2, 56.1, 56.0, 21.8; ESI m/z relative intensity 457 (M+H
30); TOFHRMS (+ESI) Calcd for C28H25O6: 457.1651, Found 457.1653.
O O OH OH
CN CN O O O O NC
CN
336g
5-(5-Hydroxy-4,9-Dimethoxy-2-Methyl-10-oxo-10H-Benzo[b]fluoren-11-yl)- isophthalonitrile (336g). Due to the insolubility of 1,3-dicyanobenzene in benzene at 80
oC, the following experiment was run at a 13:1 ratio of benzene : 1,3-dicyanobenzene.
Solid AIBN (11 mg, 0.066 mmol) was added portionwise over a period of 1 h to a stirring solution of diazoparaquinone 324 (20 mg, 0.060 mmol), 1,3-dicyanobenzene (0.11 g, .86
167 o mmol) and Bu3SnH (0.018 mL, 0.066 mmol) in benzene (1.0 mL, 11 mmol) at 80 C.
When the addition was complete, the reaction solution was allowed to cool to room
temperature. After reaching room temperature the reaction mixture was diluted with
CH2Cl2 and purified with flash column chromatography, eluting with an increasing
percentage of EtOAc from 0% to 10% in CH2Cl2. Purification furnished a 1:2.2 mixture
of benzo[b]fluorenone 336g (2:4:5 = 24:76:0) (6.5 mg, 24%) and benzo[b]fluorenone 333
(12 mg, 52%). The ratio of 336g:333, ratioed up to equimolar amounts of benzene and
1,3-dicyanobenzene, is calculated to be 6.0:1. 336g isomers were separated via
preparative TLC (20% EtOAc in benzene). (4-isomer) mp 270 oC (dec); (4-isomer) IR
-1 1 (neat): 3213, 2526, 1633 cm ; (4-isomer) H NMR (500 MHz, CDCl3) δ 10.97 (s, 1H),
8.04 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H),
7.53 (t, J = 8.1 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H), 6.72 (s, 1H), 6.54 (s, 1H), 4.13 (s, 3H),
13 3.93 (s, 3H), 2.35 (s, 3H); (4-isomer) C NMR (125 MHz, CDCl3) δ 181.4, 161.5,
153.7, 151.7, 144.6, 141.8, 138.9, 137.4, 136.2, 136.2, 135.3, 134.8, 131.4, 131.3, 120.58,
119.6, 118.1, 117.2, 117.1, 116.2, 115.6, 115.0, 114.0, 112.3, 111.5, 56.6, 56.3, 21.8; (2-
isomer) mp 290 oC (dec); (2-isomer) IR (neat): 3213, 2238, 1631 cm-1; (2-isomer) 1H
NMR (500 MHz, CDCl3) δ 10.99 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.2 Hz,
1H), 7.60 (t, J = 7.9 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H) 6.72 (s,
1H), 6.48 (s, 1H), 4.13 (s, 3H), 3.93 (s, 3H), 2.34 (s, 3H); (2-isomer) 13C NMR (125
MHz, CDCl3) δ 161.5, 153.9, 151.7, 144.4, 141.5, 138.8, 136.5, 136.3, 135.0, 134.8,
132.2, 131.9, 128.4, 120.5, 119.6, 118.2, 117.1, 116.4, 115.5, 115.0, 114.1, 111.6, 56.6,
168 56.4, 21.9; ESI m/z relative intensity 469 (M+Na 100); TOFHRMS (+ESI) Calcd for
C28H18N2O4Na: 469.1164, Found 469.1156.
O OH
O O
O O
OH O
345
C(11) dimer of 5-Hydroxy-4,9-dimethoxy-2-methyl-benzo[b]fluoren-10-one
(345).
Following General Procedure 2, in many of the solvent competition experiments
aimed at converting diazoparaquinone (324) (20 mg, 0.06 mmol) to the aromatic solvent trapped adducts (333 + 336a-g), the dimeric adduct (345) was isolated as a minor product
(0-3.3 mg, 0-15%). mp 210 oC (dec); IR (neat): 3414, 2921, 1626 cm-1; 1H NMR (300
MHz, CDCl3) δ 10.64 (s, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.47 (t, J = 8.17 Hz, 1H), 6.96 (d,
J = 7.7 Hz, 1H), 6.62 (d, J = 8.1 Hz, 1H), 4.10 (s, 3H), 3.83 (s, 3H), 2.23 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 180.8, 161.2, 151.6, 150.2, 142.7, 141.2, 138.3, 136.7, 133.8,
131.1, 121.3, 120.2, 118.3, 117.3, 115.0, 114.4, 111.1, 56.5, 56.1, 21.8; ESI m/z relative
intensity 639 (M+H 100); TOFHRMS (+ESI) Calcd for C40H31O8: 639.2019, Found
639.2047.
169
O OH
O O Se
347
5-Hydroxy-4,9-dimethoxy-2-methyl-11-phenylselanyl-benzo[b]fluoren-10-one
(347). A Solution of AIBN (11 mg, 0.067 mmol) in benzene (1mL) was added over the
period of 1 h to a stirring solution of diazoparaquinone (324) (20 mg, 0.06 mmol),
o Bu3SnH (0.018 mL, 0.067 mmol), and diphenyl diselenide (0.11 g, 0.36 mmol) at 80 C.
The light red mixture turned to a dark red/purple color upon addition of AIBN. When the
addition was complete, the reaction solution was allowed to cool to room temperature.
After reaching room temperature the reaction mixture was diluted with CH2Cl2 and
poured onto a AgNO3 impregnated silica gel column (silica gel column chromatography
alone was insufficient for separation) and purified eluting with CH2Cl2 with an increasing
percentage of EtOAc from 0% to 10%. Purification furnished the benzene trapped adduct
(333) (5.0 mg, 21%), and the selenium trapped adduct (347) (10 mg, 35%). mp 162 oC
-1 1 (dec); IR (neat): 3414, 2919, 1631, 1607, 1583 cm ; H NMR (500 MHz, CDCl3) δ 10.42
(s, 1H), 7.80 (d, J = 7.0 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.52 (t, J = 8.1 Hz, 1H), 7.44
(t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.1 Hz, 2H), 7.06 (d, J = 8.2 Hz, 1H), 6.53 (s, 1H), 5.75 (s,
170
13 1H), 4.03 (s, 3H), 4.02 (s, 3H), 1.96 (s, 3H); C NMR (125 MHz, CDCl3) δ 180.6, 161.3,
151.1, 147.6, 143.8, 142.4, 137.4, 136.4, 136.3, 133.7, 131.7, 129.1, 128.9, 128.7, 121.3,
120.1, 117.2, 113.6, 113.5, 113.4, 110.9, 56.4, 56.1, 21.7; ESI m/z relative intensity 477
(M+H 100); TOFHRMS (+ESI) Calcd for C26H21O4Se: 477.0605, Found 477.0615.
O O OH OH
H S O OH O OH S
349 350
11-Benzylsulfanyl-4,9-dimethoxy-2-methyl-11-phenyl-benzo[b]fluorene-5,10-
diol (349), and 11-Benzylsulfanyl-4,9-dimethoxy-2-methyl-11H-benzo[b]fluorene-
5,10-diol (350). A Solution of AIBN (11 mg, 0.067 mmol) in benzene (1mL) was added over the period of 1 h to a stirring solution of diazoparaquinone (324) (20 mg, 0.06 mmol), Bu3SnH (0.018 mL, 0.067 mmol, 1.1 equivalents) (or 12 equivalents), and benzyl
mercaptan (0.077 mL, 0.60 mmol) at 80 oC. The light red mixture turned to a dark red
color upon addition of AIBN. When the addition was complete, the reaction solution was
allowed to cool to room temperature. After reaching room temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica gel column and purified by
eluting with hexanes/CH2Cl2 (1:1) followed by CH2Cl2 with an increasing percentage of
EtOAc from 0% to 5%. Oxidation of the air sensitive dihydroquinone (350) was
minimized during chromatography by employing N2 purged solvents and flashing with
171 low pressure N2 gas. Purification furnished the benzene trapped adduct (333) (3.5 mg,
15%), the benzene/benzyl mercaptan trapped adduct (349) (6.7 mg, 22%), mp 260 oC
-1 1 (dec); IR (neat): 3378, 3307, 2922, 1644, 1610 cm ; H NMR (500 MHz, CDCl3) δ 9.85
(s, 1H), 9.12 (s, 1H) 7.95 (d, J = 8.5 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.33 (t, J = 7.8 Hz,
1H), 7.27-7.19 (m, 3H), 7.04-6.94 (m, 5H), 6.91 (s, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.65 (s,
1H), 4.16 (s, 3H), 3.96 (s, 3H), 3.25 (d, J = 12.6 Hz, 1H), 3.14 (d, J = 12.6 Hz, 1H), 2.30
13 (s, 3H); C NMR (125 MHz, CDCl3) δ 156.5, 152.6, 151.0, 143.5, 141.4, 139.8, 138.9,
137.6, 128.8, 128.3, 128.1, 127.7, 126.9, 126.8, 126.2, 126.0, 125.0, 123.8, 120.4, 120.2,
116.7, 115.8, 111.3, 104.9, 64.0, 56.5, 55.8, 34.4, 29.7; ESI m/z relative intensity 543
(M+Na 40); TOFHRMS (+ESI) Calcd for C33H28O4SNa: 543.1606, Found 543.1647, and the benzyl mercpatan trapped adduct (350) (12.5 mg, 46%). mp 160 oC (dec); IR (neat):
-1 1 3389, 3311, 2921, 1611, 1580 cm ; H NMR (500 MHz, CDCl3) δ 9.64 (s, 1H), 9.26 (s,
1H), 7.86 (d, J = 8.5 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.03-6.89 (m, 6H), 6.76 (d, J = 7.6
Hz, 1H), 6.62 (s, 1H), 5.05 (s, 1H), 4.02 (s, 3H), 3.99 (s, 3H), 3.40 (d, J = 12.9 Hz, 1H),
13 3.22 (d, J = 12.9 Hz, 1H), 2.32 (s, 3H); C NMR (125 MHz, CDCl3) δ 156.3, 151.3,
147.4, 143.5, 139.3, 139.1, 138.4, 128.9, 128.0, 127.9, 126.3, 125.1, 124.8, 122.6, 120.8,
120.3, 116.8, 115.3, 111.3, 104.9, 56.5, 56.0, 48.0, 33.5, 29.7; ESI m/z relative intensity
467 (M+Na 95); TOFHRMS (+ESI) Calcd for C27H24O4SNa: 467.1293, Found
467.1301.
Control experiment: Benzyl mercaptan addition to diazoparaquinone (324)
with no radical ingredients. Benzyl mercaptan (0.019 mL, 0.15 mmol) was added to a
172 stirring solution of diazoparaquinone 324 (5.0 mg, 0.015 mmol) at 80 oC. The light red
mixture was stirred at 80 oC for 1 h. Following 1 h of stirring, the reaction solution was
allowed to cool to room temperature. After reaching room temperature the reaction
mixture was diluted with CH2Cl2 and poured onto a silica gel column and purified by
eluting with hexanes/CH2Cl2 (1:1), followed by CH2Cl2 with an increasing percentage of
EtOAc from 0% to 5%. Purification provided diazoparaquinone 324 (4.7 mg, 94%)
unchanged.
Control experiment: Benzyl mercaptan addition to prekinamycin (188) with
no radical ingredients. Benzyl mercaptan (0.015 mL, 0.12 mmol) was added to a
stirring solution of prekinamycin (188) (3.7 mg, 0.012 mmol) at 80 oC. The dark
purple/brown mixture was stirred at 80 oC for 1 h. Following 1 h of stirring, the reaction
solution was allowed to cool to room temperature. After reaching room temperature the
reaction mixture was diluted with CH2Cl2 and poured onto a silica gel column and
purified by eluting with hexanes/CH2Cl2 (1:1), followed by CH2Cl2 with an increasing
percentage of EtOAc from 0% to 5%. Purification provided prekinamycin (188) (3.7 mg,
100%) unchanged.
11-Benzylsulfanyl-4,9-dimethoxy-2-methyl-11-phenyl-benzo[b]fluorene-5,10-
diol (349) from benzene trapped adduct (333). Benzyl mercaptan (0.10 mL, 0.80
mmol) was added to a stirring solution of benzene trapped adduct 333 (3.7 mg, 0.0080
mmol) at 80 oC. The bright red solution turned to a dark red solution over the 2 h of
stirred at 80 oC. Upon consumption of the starting material by TLC, the reaction solution
173 was allowed to cool to room temperature. After reaching room temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica gel column and purified by
eluting with hexanes/CH2Cl2 (1:1), followed by CH2Cl2 with an increasing percentage of
EtOAc from 0% to 5%. Purification afforded benzyl mercaptan addition product 349
(3.7 mg, 90%).
Deuterium Labeling Experiment: PhCH2SD/Bu3SnH/AIBN: A Solution of
AIBN (11 mg, 0.067 mmol) in benzene (1mL) was added over the period of 1 h to a
stirring solution of diazoparaquinone 324 (20 mg, 0.060 mmol), Bu3SnH (0.018 mL,
0.067 mmol), and S-deutero-benzyl mercaptan (0.08 mL, 0.6 mmol) at 80 oC. The light
red mixture turned to a dark red color upon addition of AIBN. When the addition was
complete, the reaction solution was allowed to cool to room temperature. After reaching
room temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica
gel column and purified by eluting with hexanes/CH2Cl2 (1:1), followed by CH2Cl2 with
an increasing percentage of EtOAc from 0% to 5%. Purification furnished benzyl
mercaptan trapped adduct 352 (33% by 1H NMR) with 46% Deuterium incorporation at
1 C11. H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 9.26 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H),
7.26 (t, J = 7.8 Hz, 1H), 7.03-6.89 (m, 6H), 6.76 (d, J = 7.6 Hz, 1H), 6.62 (s, 1H), 5.05 (s,
0.54H), 4.02 (s, 3H), 3.99 (s, 3H), 3.40 (d, J = 12.9 Hz, 1H), 3.22 (d, J = 12.9 Hz, 1H),
2.32 (s, 3H).
Deuterium Labeling Experiment: PhCH2SH/Bu3SnD/AIBN: A Solution of
AIBN (11 mg, 0.067 mmol) in benzene (1mL) was added over the period of 1 h to a
174 stirring solution of diazoparaquinone 324 (20 mg, 0.060 mmol), Bu3SnD (0.018 mL,
0.067 mmol), and benzyl mercaptan (0.08 mL, 0.6 mmol) at 80 oC. The light red mixture
turned to a dark brown color upon addition of AIBN. When the addition was complete,
the reaction solution was allowed to cool to room temperature. After reaching room temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica gel
column and purified by eluting with hexanes/CH2Cl2 (1:1), followed by CH2Cl2 with an increasing percentage of EtOAc from 0% to 5%. Purification furnished benzyl mercaptan trapped adduct 352 (40% by 1H NMR) with 27% Deuterium incorporation at
1 C11. H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 9.26 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H),
7.26 (t, J = 7.8 Hz, 1H), 7.03-6.893 (m, 6H), 6.76 (d, J = 7.6 Hz, 1H), 6.62 (s, 1H), 5.05
(s, 0.73H), 4.02 (s, 3H), 3.99 (s, 3H), 3.40 (d, J = 12.9 Hz, 1H), 3.22 (d, J = 12.9 Hz, 1H),
2.32 (s, 3H).
Deuterium Labeling Experiment: PhCH2SD/Bu3SnD/AIBN: A Solution of
AIBN (11 mg, 0.067 mmol) in benzene (1mL) was added over the period of 1 h to a
stirring solution of diazoparaquinone 324 (20 mg, 0.060 mmol), Bu3SnD (0.018 mL,
0.067 mmol), and S-deutero-benzyl mercaptan (0.08 mL, 0.60 mmol) at 80 oC. The light
red mixture turned to a dark brown color upon addition of AIBN. When the addition was complete, the reaction solution was allowed to cool to room temperature. After reaching room temperature the reaction mixture was diluted with CH2Cl2 and poured onto a silica
gel column and purified by eluting with hexanes/CH2Cl2 (1:1) followed CH2Cl2 with an increasing percentage of EtOAc from 0% to 5%. Purification furnished benzyl mercaptan trapped adduct 352 (46% by 1H NMR) with 76% Deuterium incorporation at
175
1 C11. H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 9.26 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H),
7.26 (t, J = 7.8 Hz, 1H), 7.03-6.89 (m, 6H), 6.76 (d, J = 7.6 Hz, 1H), 6.62 (s, 1H), 5.05 (s,
0.24H), 4.02 (s, 3H), 3.99 (s, 3H), 3.40 (d, J = 12.9 Hz, 1H), 3.22 (d, J = 12.9 Hz, 1H),
2.32 (s, 3H).
Deuterium-Crossover Control Experiment: PhCH2SD/Bu3SnH/AIBN: AIBN
(12 mg, 0.073 mmol), S-deutero-benzyl mercaptan (0.010 mL, 0.073 mmol), and Bu3SnH
1 (0.020 mL, 0.073 mmol) were dissolved in d6-benzene (1mL) and inspected by H NMR, and it was noted that a triplet at 1.43 ppm(PhCH2SH) was absent. The reaction solution
was then heated at 80 oC for 1 h. After 1 h at 80 oC close inspection of the 1H NMR revealed a triplet at 1.43 (PhCH2SH) still remained absent.
Deuterium-Crossover Control Experiment: Bu3SnD/PhCH2SH/AIBN: AIBN
(12 mg, 0.073 mmol), Bu3SnD (0.020 mL, 0.073 mmol), and benzyl mercaptan (0.010
1 mL, 0.073 mmol) were dissolved in d6-benzene (1mL) and inspected by H NMR, and
the presence of a triplet at 1.43 ppm (1H) (PhCH2SH) was noted. The reaction solution
was then heated at 80 oC for 1 h. After 1 h at 80 oC close inspection of the 1H NMR revealed the triplet at 1.43 (PhCH2SH) remained and had not diminished in intensity.
176 6.3 Kinamycin F Synthetic Studies
O
Br O O
361
(3aS, 4R, 5R, 7aS)-7-Bromo-3a, 4, 5, 7a-tetrahydro-2, 2-dimethyl-4, 5-epoxy-
1, 3-benzodioxole (361). A catalytic amount of p-toluenesulfonic acid (50 mg, 0.26
mmol) was added to a solution of (1S-cis)-3-bromo-3,5-cyclohexadiene-1,2-diol (358)
(4.85 g, 25.4 mmol), and dimethoxypropane (4.7 mL, 38 mmol) in acetone (50 mL) and
the reaction mixture was stirred at room temperature under nitrogen. After 3 hours, 15%
aqueous NaOH (5 mL) and brine (15 mL) were added followed by ether (50 mL), and the
solution was stirred for 15 minutes. The reaction mixture was poured onto a brine
solution and extracted with ether (2 x 40 mL). The organic layers were combined and
dried over Na2SO4, filtered and concentrated to give 5.53 g (94%) of the desired
acetonide as a light yellow oil. This compound was used without further purification.
m-Chloroperbenzoic acid (6.25 g, 36.2 mmol) was added in 5 portions over 1 hour
to a suspension of the above acetonide (5.53 g, 23.9 mmol) and sodium bicarbonate
(6.04 g, 71.9 mmol) in CH2Cl2 (100 mL). After 4 hours, the reaction mixture was filtered
with an ether rinse (150 mL). The filtrate was then washed with saturated aqueous
NaHSO3 (50 mL), saturated aqueous NaHCO3 (3 x 50 mL, or until the pH of the water
layer was no longer acidic), and then with brine. The organic layer was dried over
Na2SO4, filtered and concentrated. The resulting white solid was purified via SiO2
177 chromatography using 10% ether in hexanes to give the epoxide 361 as a white solid
(4.48 g, 76%). Analytical data matches those previously reported.155
OH OH
Br OH OH
361
(1S, 2R, 3S, 4S)-5-Bromo-5-cyclohexene-1, 2, 3, 4-tetrol (361). A solution of epoxide 362 (2.21 g, 8.94 mmol) in DMSO (23 mL) was cooled to 0 oC, and a 10%
aqueous solution of KOH (23 mL) was added, resulting in a darkening of the solution.
The solution was warmed to room temperature after 15 minutes at 0 oC, and then heated
to reflux. After 3 hours of stirring at reflux, the solution was cooled to room temperature
and extracted with ethyl acetate (4 x 30 mL). The organic layer was washed with brine (2
x 30 mL) and then dried over Na2SO4, filtered and concentrated to give the diol as a
white solid (2.10 g, 88%). Analytical data matches those previously reported.155
A solution of the above diol (898 mg, 3.39 mmol) in AcOH:H2O:THF (2:1:1) was
heated to 60 oC for 18 h. The reaction mixture was cooled to room temperature and then
concentrated to give 361 as a white solid (687 mg, 89%). Analytical data matches those
previously reported.155
178
OTBS OTBS
Br OH OTBS
362
(1S, 2S, 5S, 6R)-3-Bromo-2, 5, 6-tris-(tert-butyldimethylsiloxy)-3-cyclohexen-
1-ol (362). t-Butyldimethylsilyl chloride (4.59 g, 30.5 mmol) was added to a solution of tetrol 361 (1.71 g, 7.60 mmol), and imidazole (4.15 g, 61.0 mmol) in anhydrous DMF (8 mL) under nitrogen. The solution was heated to 50 oC. After 24 h, the reaction solution was cooled to room temperature. The solution was diluted with water (50 mL) and extracted with ether (2 x 30 mL). The organic layer was then washed with brine, dried over Na2SO4, filtered and concentrated to a brown oil. This material was purified by silica gel chromatography, eluting with 15% toluene in petroleum ether, to afford 362
(2.69 g, 60%) as a colorless oil. Analytical data matches those previously reported.155
OTBS OTBS
Br O OTBS
363
(2S, 5S, 6R)-3-Bromo-2, 5, 6- tris-(tert-butyl-dimethylsiloxy)-cyclohex-3-enone
(363). Dess-Martin periodinane (3.61 g, 8.51 mmol) was added to a solution of alcohol
362 (2.41 g, 4.24 mmol) in CH2Cl2 (21 mL) under nitrogen. The reaction was stirred at
room temperature under nitrogen for 3 h. After this time, a 20% aqueous solution of
179 sodium thiosulfate (Na2S2O3) (10 mL) and saturated aqueous NaHCO3 (20 mL) was
SLOWLY added to the milky reaction suspension and stiring continued until the solids dissolved. The solution was then extracted with Et2O (2 x 40 mL), dried over Na2SO4, filtered and concentrated in vacuo to furnish a yellow solid. The crude product was purified by silica gel chromatography, eluting with 10% toluene in petroleum ether, to give the ketone 363 as a colorless oil (1.69 g, 70%). IR (neat) 1747, 1640 cm-1; 1H NMR
(300 MHz, CDCl3) δ 6.12 (d, J = 2.62 Hz, 1H), 4.61 (d, J = 6.8 Hz, 1H), 4.41 (s, 1H),
4.17 (dd, J = 6.8, 2.5 Hz, 1H), 0.93 (s, 9H), 0.92 (s, 9H), 0.91 (s, 9H), 0.21 (s, 3H), 0.11
13 (m, 9H), 0.07 (s, 3H), 0.06 (s, 3H); C NMR (75 MHz, CDCl3) δ 201.3, 135.5, 123.5,
77.9, 76.9, 74.3, 25.9, 25.9, 25.7, 18.3, 18.2, 18.1, -4.3, -4.5, -4.6, -4.8, -4.9 (2C’s); MS
+ APCI m/z (relative intensity) 565 (M+H, 80%); Anal. calcd. for C24H49BrO4Si3: C,
50.95; H, 8.73; Br, 14.12; Found: C, 50.76; H, 8.50; Br, 14.08.
OTBS OTBS
Br OH OTBS
364
(1S, 2S, 5S, 6R)-3-Bromo-2, 5, 6-tris-(tert-butyldimethylsiloxy)-1-methyl- cyclohex-3-ol (364). A solution of methyl lithium (2.3 mL, 1.5 M in ether, 3.5 mmol) was slowly added to a cooled (-30 oC) solution of the ketone 363 (0.961 g, 1.70 mmol) in
ether (35 mL) under nitrogen. After 30 minutes, no starting material remained by TLC. A saturated aqueous solution of NH4Cl (40 mL) was added to the reaction mixture, and it was extracted with Et2O (2 x 40 mL). The combined organic layers were dried over
180
Na2SO4, filtered and concentrated. The crude reaction product was purified by silica gel chromatography eluting with 15% toluene in petroleum ether to afford alcohol 364 (0.80
-1 1 g, 81%) as a white semisolid. IR (neat) 3564, 1646 cm ; H NMR (300 MHz, CDCl3) δ
6.01 (d, J = 3.2 Hz, 1H), 4.02 (s, 1H), 3.93 (dd, J = 6.5, 3.2 Hz, 1H), 3.73 (d, J = 6.4 Hz,
1H), 2.64 (s, 1H), 1.13 (s, 3H), 0.95 (s, 9H), 0.92 (s, 9H), 0.91 (s, 9H), 0.26 (s, 3H), 0.18
13 (s, 3H), 0.12 (s, 3H), 0.11 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H); C NMR (75 MHz, CDCl3)
δ 133.6, 124.2, 79.8, 76.0, 74.4, 74.2, 26.1 (2C’s), 26.0, 19.1, 18.5, 18.3, 18.2, -3.4, -3.5,
-3.8, -4.1, -4.1, -4.4; MS ESI- m/z (relative intensity) 579.1 (M-H, 100%); HRMS calcd. for C25H53BrO4Si3Na: 603.2333; Found: 603.2328.
OTBS OTBS
Br OTMS OTBS
365
1(3S, 4R, 5S, 6S)-1-Bromo-3, 4, 6-tris-(tert-butyldimethylsiloxy)-5-methyl-5- trimethylsiloxy-cyclohexene (365). Trimethylsilyl triflate (0.65 mL, 3.6 mmol) was added to a cooled (0 oC) solution of alcohol 364 (0.80 g, 1.4 mmol) and distilled 2,6-
lutidine (0.74 mL, 6.4 mmol) in CH2Cl2 (14 mL). The solution was slowly warmed to
room temperature and stirred overnight. The reaction mixture was poured into a saturated
aqueous solution of NH4Cl (30 mL) and was extracted with Et2O (2 x 30 mL). The
organic layer was dried over Na2SO4, filtered and concentrated. The crude product was
purified via silica gel chromatography, eluting with hexanes, to give 365 as a white solid
o -1 1 (0.73 g, 82%). m.p. 79–81 C; IR (neat) 1652 cm ; H NMR (300 MHz, CDCl3) 5.97 (m,
181 1H), 4.16 (s, 1H), 3.88 (m, 2H), 1.22 (s, 3H), 0.94 (s, 9H), 0.90 (s, 9H), 0.89 (s, 9H), 0.21
13 (s, 3H), 0.16 (s, 3H), 0.15 (m, 9H), 0.09 (m, 12H); C NMR (CDCl3, 75 MHz) δ 132.7,
125.5, 78.3 (2C’s), 77.2, 75.6, 29.7, 26.2 (2C’s), 26.1, 18.6, 18.2, 18.1, 3.0, -3.2 (2C’s), -
+ 3.7 (2C’s), -3.8 (2C’s); MS APCI m/z (relative intensity) 670 (M+NH4Cl, 100%);
HRMS calcd. for C28H62BrO4Si4: 653.2909; Found: 653.2903.
O
Br OH O
366
2-Bromo-8-hydroxy-1, 4-naphthoquinone (366). A solution of 5-hydroxy-1,4-
o naphthoquinone (357) (9.4 g, 54 mmol) in CHCl3 (100 mL) was cooled to 0 C under
nitrogen. A solution of bromine (3.05 mL, 59.4 mmol) in CHCCl3 (200 mL) was added
slowly. The dark orange solution was stirred at 0 oC for 8 h. The dark solution was
concentrated to give a dark solid. 10% aqueous AcOH (30 mL) was added and the
suspension was concentrated to a moist residue. This residue was resuspended in EtOH
(400 mL) and heated to reflux in a hot (100oC) water bath. After 1 h, the dark brown
suspension was cooled to room temperature and then placed in a freezer overnight (-20
C). After cooling in the freezer overnight, the reaction solution was filtered and rinsed with ice cold CHCl3 to give 10 g of a 20:1 mixture of the 2-bromo-:3-bromojuglone. This
solid was recrystallized from a minimal amount of refluxing EtOH to afford 2-
bromojuglone (366) as an orange solid (6.5 g, 50%). Analytical data matches those
previously reported.156
182 O
Br BnO O
367
18-Benzyloxy-2-bromo-1, 4-naphthoquinone (367). Silver oxide (13.2 g, 57.1
mmol) was added to a solution of 2-bromojuglone (366) (3.61 g, 14.3 mmol) and benzyl
bromide (3.4 mL, 28 mmol) in CH2Cl2 (36 mL) under nitrogen. The resulting suspension
was stirred at room temperature. After 3 hours, the suspension was filtered through Celite
with a CH2Cl2 rinse (50 mL) and the filtrate was concentrated to give an orange solid.
This orange solid was purified by silica gel chromatography, eluting with hexanes/Et2O
(80:20), to afford 367 as an orange solid (3.87 g, 79%). Analytical data matches those
previously reported.156
OH
Br BnO OH
368
8-Benzyloxy-2-bromonaphthalene-1,4-diol (368). A solution of sodium
dithionite (Na2S2O4) (4.04 g, 23.3 mmol) in 38 mL of water was added to a vigorously
stirring solution of bromoquinone 367 (1.14 g, 3.32 mmol) in a 3:1 mixture of Et2O and
CH2Cl2 (36 mL). The suspension was vigorously stirred for 10 minutes. The organic layer
was then separated, dried over Na2SO4, filtered and concentrated to afford 0.650 g (57%)
of crude hydroquinone 368. Analytical data matches those previously reported.156
183 OMe
Br BnO OMe
369
8-Benzyloxy-2-bromo-1,4-dimethoxy-naphthalene (369). The crude
hydroquinone 368 (0.50 g, 1.5 mmol) was dissolved in acetone (10 mL) and the resultant
solution was cannulated into a stirring suspension of K2CO3 (2.4 g, 17 mmol) in acetone
(25 mL) at room temperature. Dimethyl sulfate (0.96 mL, 10 mmol) was then added to
the mixture via syringe. The mixture was heated to reflux and allowed to stir at reflux for
48 h. The mixture was then filtered through a pad of Celite with an acetone rinse and the
filtrate was concentrated in vacuo to afford the crude product as a red oil. This oil was
dissolved in a solution of Et2O (5 mL) and triethylamine (0.5 mL) and stirred at room temperature for 30 min. After this time, the solution was washed successively with HCl
(1M, 2 x 5 mL), H2O, and brine. The organic layer was dried over Na2SO4, filtered and
concentrated to afford 0.5 g of the crude product as a red colored oil. This oil was
purified on silica gel chromatography (hexanes:EtOAc, 6:1) to afford the desired product
369 as a white solid (0.52 g, 96%). Analytical data matches those previously reported.156
184 OMe
SnMe3 BnO OMe
374
(8-Benzyloxy-1,4-dimethoxy-naphthalen-2-yl)-trimethylstannane (374). n-
Butyllithium (0.20 mL, 2.5 M/hexanes, 0.50 mmol) was added to a stirring solution of
aryl bromide 355 (0.075 g, 0.20 mmol) in THF (3 mL) at -120 oC. The cold solution was
o stirred at -120 C for 30 seconds before a solution of Me3SnCl (0.12 g, 0.60 mmol) in
THF (1 mL) was slowly added via syringe. The reaction solution was stirred for an
o additional 20 min at -120 C before it was poured onto saturated aqueous NH4Cl and
diluted with Et2O and H2O. The layers were separated and the aqueous layer was
extracted with Et2O (3 x 5 mL). The combined organic extracts were dried over Na2SO4, filtered and concentrated to afford 0.10 g of a crude oil. The crude product was purified by silica gel chromatography, eluting with hexanes/Et2O (80:20), to give the desired
1 stannane 374 as a clear colorless oil (0.080 g, 86 %). H NMR (300 MHz, CDCl3) δ 7.94
(d, J = 7.9 Hz, 1H), 7.59 (d, J = 7.3 Hz, 2H), 7.45-7.35 (m, 4H), 7.01 (d, J = 7.6 Hz, 1H),
6.86 (s, 1H), 5.18 (s, 2H), 4.02 (s, 3H), 3.15 (s, 3H), 0.41 (s, 9H); ESI m/z relative
intensity 459 (M+H 100).
185 OH OH
Br OH OH
376
6-Bromo-2-methyl-cyclohex-5-ene-1,2,3,4-tetraol (376). Tetrabutylammonium
fluoride (21 mL, 1 M/THF, 21 mmol) was slowly added via syringe to a stirring solution
of TBS protected alcohol 364 (3.45 g, 5.94 mmol) in THF (25 mL) at 0 oC, resulting in a
dark brown solution. The reaction mixture was allowed to stir at 0 oC for 1 h, after which
time it was warmed to room temperature and stirred for an additional 1 h. The solution
was then concentrated in vacuo to give a brown oil. The dark oil was dissolved in H2O and extracted with hexanes (3 x 20 mL). The aqueous layer was concentrated to a dark colored oil and purified by filtration through a silica gel plug with a 10% MeOH in
EtOAc rinse to afford the desired tetraol as a sticky amber colored residue (1.1 g, 78%).
1H NMR (300 MHz, MeOD) δ 6.04 (d, J = 3.3 Hz, 1H), 3.86 (s, 1H), 3.82 (dd, J = 7.4,
3.4 Hz, 1H), 3.62 (d, J = 7.3 Hz, 1H), 1.11 (s, 3H); ESI m/z relative intensity 239 (M+H
80).
186 OBn OBn
Br OBn OBn
377
6-Bromo-2-methyl-cyclohex-5-ene-1,2,3,4-tetrabenzyl ether (377). Sodium
hydride (0.432 g, 10.8 mmol) was added to a stirring solution of tetraol 376 (0.160 g,
0.675 mmol) and benzyl bromide (1.29 mL, 10.8 mmol) in DMF (10 mL) at 0 oC under
N2. The mixture was warmed to room temperature and stirred for 12 hours. Excess sodium hydride was then destroyed by a SLOW addition of ice chips. Upon consumption of the excess NaH, aqueous saturated NH4Cl was added, and the mixture was diluted with
Et2O. The layers were separated and the aqueous layer was extracted with Et2O (3 x 15 mL). The combined organic extracts were washed successively with H2O and brine,
dried over Na2SO4, filtered and concentrated in vacuo to afford 7.3 g of a crude oil. This
oil was purified by silica gel chromatography, eluting with 5% Et2O in hexanes, to give
the desired tetrabenzyl ether 377 as a clear colorless oil (0.30 g, 74%). IR (neat): 3029,
-1 1 2865 cm ; H NMR (300 MHz, CDCl3) δ 7.38-7.21 (m, 20H), 6.21 (d, J = 2.9 Hz, 1H),
4.95-4.55 (m, 8H), 4,20 (d, J = 7.3 Hz, 1H), 4.13 (s, 1H), 3.95 (dd, J = 7.3, 2.9 Hz, 1H),
13 1.34 (s, 3H); C NMR (75 MHz, CDCl3) δ 139.2, 139.0, 138.2, 137.8, 131.6, 128.3,
128.15 (2 C’s), 128.12, 127.9, 127.8, 127.7, 127.6, 127.5, 127.3, 127.1, 127.0, 122.8,
84.5, 81.4, 80.3, 80.1, 75.1, 74.1, 71.9, 64.3, 15.9; ESI m/z relative intensity 621 (M+Na
100); TOFHRMS (+ESI) Calcd for C35H35BrO4Na: 621.1616, Found 621.1642.
187 OBn OBn
HO OBn O OBn
378
3,4,5,6-Tetrakis-benzyloxy-5-methyl-cyclohex-1-enecarboxylic acid (378).
t-Butyllithium (1.31 mL, 1.7 M/pentane, 2.23 mmol) was slowly added via syringe to a
o stirring solution of alkenyl bromide 377 (0.89 g, 1.5 mmol) in Et2O (20 mL) at -78 C.
The resulting solution was stirred at this temperature for 15 min. The reaction mixture
was then purged with gaseous CO2 (sublimed dry ice introduced through a CaCl2 drying
tube and purging needle). The CO2 purge continued as the reaction was brought to room
temperature over the period of 1 h. Saturated aqueous NH4Cl was then added to the
milky white mixture and the layers were separated. The aqueous layer was extracted with Et2O (3 x 15 mL). The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to afford the desired acid as a yellow tinted oil (0.75 g, 90%).
-1 1 IR (neat): 3030, 2867, 1693 cm ; H NMR (300 MHz, CDCl3) δ 9.80 (bs, 1H), 7.35-7.18
(m, 20H), 7.06 (d, J = 2.5 Hz, 1H), 5.0-4.57 (m, 8H), 4.53 (s, 1H), 4.39 (d, J = 7.7 Hz,
13 1H), 4.13 (dd, J = 7.7, 2.5 Hz, 1H), 1.25 (s, 3H); C NMR (75 MHz, CDCl3) δ 171.1,
141.6, 139.3, 138.9, 138.7, 137.9, 131.1, 128.4, 128.3, 128.1, 128.0, 127.8, 127.7, 127.65,
127.56, 127.3, 127.2, 127.0, 126.9, 81.7, 79.9, 79.3, 76.2, 75.2, 73.9, 72.3, 64.4, 15.3;
ESI m/z relative intensity 587 (M+Na 100); TOFHRMS (+ESI) Calcd for C36H36O6Na:
587.2410, Found 587.2444.
188 OBn OBn
N O OBn O OBn
380
3,4,5,6-Tetrakis-benzyloxy-5-methyl-cyclohex-1-enecarboxylic acid Methoxy-
methylamide (380). Ethyldimethylamniopropylcarbodiimide•HCl (0.058 g, 0.30 mmol)
was added in five equal portions over the period of 2 h to a stirring solution of acid 378
(0.129 g, 0.212 mmol), DMAP (0.054 g, 0.45 mmol), and N,O-
dimethylhydroxylamine•HCl (0.023 g, 0.23 mmol) in CH2Cl2 (2 mL) at room
temperature. After stirring for 12 h at room temperature, the reaction mixture was poured
onto saturated aqueous NH4Cl. The layers were separated and the aqueous layer was
extracted with CH2Cl2 (3 x 10 mL). The combined organic extracts were dried over
Na2SO4, filtered and concentrated to afford 0.13 g of a crude oil. The crude material was
purified by flash chromatography, eluting with hexanes/Et2O (30:70), to give the desired
Weinreb amide 380 as a colorless oil (0.080g, 65%). IR (neat): 3028, 2933, 1633 cm-1;
1 H NMR (300 MHz, CDCl3) δ 7.36-7.21 (m, 20H), 6.21 (d, J = 2.6 Hz, 1H), 4.95 (d, J =
11.2 Hz, 1H), 4.83 (d, J = 11.2 Hz, 1H), 4.58-4.78 (m, 6H), 4.53 (s, 1H), 4.31 (d, J = 7.4
Hz, 1H), 4.09 (dd, J = 7.4, 2.8 Hz, 1H), 3.54 (s, 3H), 3.21 (s, 3H), 1.32 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ 169.6, 139.5, 139.0, 138.5, 138.3, 133.6, 131.4, 128.3, 128.1, 128.05,
128.03, 127.8, 127.7, 127.6, 127.56, 127.4, 127.3, 126.9, 126.88, 81.6, 79.5, 79.2, 77.9,
75.0, 73.8, 71.9, 64.3, 61.1, 33.8, 15.4; ESI m/z relative intensity 630 (M+Na 100);
TOFHRMS (+ESI) Calcd for C38H42NO6: 608.3012, Found 608.3051.
189 OMe OBn OBn
OBn BnO OMe O OBn
381
(8-Benzyloxy-1,4-dimethoxy-naphthalen-2-yl)-(3,4,5,6-tetrakis-benzyloxy-5-
methyl-cyclohex-1-enyl)methanone (381). n-Butyllithium was slowly added via syringe to a stirring solution of juglone derivative 369 (0.059 g, 0.16 mmol) in THF (1 mL) at -78 oC. Within 5 min, a solution of the Weinreb amide 380 (0.080 g, 0.13 mmol)
in THF (1 mL) was slowly added via syringe to this cold solution. The reaction mixture
was allowed to stir at -78 oC for 2 h. At this time, the reaction solution was combined
with saturated aqueous NH4Cl, diluted with Et2O, and the layers were separated. The
aqueous layer was extracted with Et2O (3 x 5 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated to afford 0.140 g of a
crude residue. The crude product was purified by silica gel chromatography, eluting with
hexanes/Et2O (70:30), to afford the desired product as a yellow sticky solid (0.089 g,
-1 1 66%). IR (neat): 3029, 2934, 1660 cm ; H NMR (300 MHz, CDCl3) δ 7.93 (d, J = 8.3
Hz, 1H), 7.55-7.0 (m, 27 H), 6.56 (s, 1H), 6.43 (d, J = 2.5 Hz, 1H), 5.18 (bs, 2H), 5.06-
4.5 (m, 8H), 4.55 (s, 1H), 4.45 (d, J = 7.6 Hz, 1H), 4.15 (dd, J = 7.7, 2.7 Hz, 1H), 3.86 (s,
13 3H), 3.60 (s, 3H), 1.33 (s, 3H); C NMR (75 MHz, CDCl3) δ 197.2, 155.6, 151.3, 148.1,
142.6, 139.9, 139.5, 139.19, 139.11, 138.1, 136.7, 129.9, 129.0, 128.4, 128.1 (2 C’s),
128.09 (2 C’s), 128.0, 127.8 (2 C’s), 127.7, 127.6, 127.5, 127.4, 127.2, 127.1, 127.0 (2
C’s), 126.9, 120.7, 115.3, 109.3, 103.1, 81.9, 80.2, 80.1, 75.2 (2 C’s), 73.8, 72.4, 71.4,
190 64.12, 64.07, 55.8, 15.2; ESI m/z relative intensity 663 (M+Na 100); TOFHRMS (+ESI)
Calcd for C55H53NO8: 841.3740, Found 841.3782.
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VITA
Kyle Joseph Eastman
EDUCATION Ph.D. candidate, Synthetic Organic Chemistry, Pennsylvania State University, University Park, PA, 16803 Adviser: Professor K. S. Feldman
B.S., Chemistry, Messiah College, Grantham, PA, 2000 Adviser: Professor D. Foster
HONORS Dalalian Fellowship, 2003 and 2005
Who’s Who Among America’s Colleges and Universities, 2000
Sigma Zeta National Honor Society for Math and Science Member, 1998-2000, President, 2000
PUBLICATIONS
Feldman, K. S.; Eastman, K. J. Studies on the Mechanism-of- Action of the Diazoparaquinone Family of Natural Products: Evidence for both sp2 Radical and Orthoquinonemethide Intermediates. Under Review.
Feldman, K. S.; Eastman, K. J. “A Proposal for the Mechanism-of- Action of Diazoparaquinone Natural Products.” J. Am. Chem. Soc. 2005, 127, 15344-15345.
Feldman, K. S.; Eastman, K. J.; Lessene, G. “Diazonamide Synthesis Studies: Use of Negishi Coupling to Fashion Diazonamide-Related Biaryls with Defined Axial Chirality.” Org. Let. 2002, 4, 3525-3528.