Synthesis of
7-N-Acetyl-Demethyl Lavendamycin t-Butyl Ester
Senior Honors Thesis
by
Brian M. Mathes
Thesis Advisor
Ball State University
May 2nd, 1994
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Abstract
The purpose of this thesis is to document the research being done on new possiblities for
cancer fighting drugs. Lavendamycin and its analogs have proven to be promising compounds in
this battle. The synthesis of Lavendamycin has gone along many pathways but our research uses
the Pictet-Spengler Condensation to achieve the molecule. In this research, the t-butyl ester analog
of lavendamycin is synthesized in the hopes that it can be used in fighting cancer. In addition, it is
possible for this analog to be used as an intermediate to other, possibily more effective
compounds.
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Table of Contents
Lavendamycin
1. Background
II. Biological Activity
Results and Discussion
1. Objective
II. Synthesis ofL-Tryptophan t-Butyl Ester
ID. Synthesis of 7-N-Acetyl-2-Formylquinoline-5,8-Dione
IV. Synthesis of7-N-Acetyl-Demethyl Lavendamycin t-Butyl Ester
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Experimental
1. General Information
II. Procedure
A. Preparation ofL-Tryptophan t-Butyl Ester
B. Preparation of7-N-Acetyl-2-Formylquinoline-5,8-Dione
C. Preparation of7-N-Acetyl-Demethyl Lavendamycin t-Butyl Ester
ID. Spectra and Chromatographs
References
Acknowledgements
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Lavendamycin
I. Historical
Lavendamycin (8) was first isolated from a Streptomyces Lavendulae bacteria fermentation
broth at Bristol Laboratories by Doyle and associates.! The lavendamycin was red solid with very
limited solubility in organic solvents. This fact hindered crystal growth for x-ray crystallography.
The structure was eventually determined by NMR, Mass Spectroscopy and IR techniques. The
structure bore a striking resemblance to the antibiotic streptogrin studied by Gould and it was
thought that this new lavendamycin compound might be an intermediate in the natural synthesis of
streptogrin .2
(9)
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This relationship sparked an explosion of research in the total synthesis of lavendamycin.
Kende and his associates were the first to publish a total synthesis of the lavendamycin methyl
ester (9).3 His method involved using the Friedlander condensation to make the quinoline moiety,
followed by a Bischler-Napieralski cyclodehydration with B-Methyl Tryptophan to form (9).
Hibano and his coworkers published a method using the Pictet-Spengler condensation the
form (9). The group first condensated B-methyl tryptophan methyl ester with a quinoline analog
and then added the necessary dione functional groups later. 4 Hibano also was the first to
5
synthesize the demethyl analog using tryptophan methyl ester instead of the B-methyl analog.
Work on lavendamycin was continued by Rao and his associates. The group used the
Bischler-Napieralski cyclodehydration to form (9) but used a different quinoline moiety to achieve
the moleculeS. Boger and his associates were also doing work at about the same time9 . The
uniqueness of their work comes in that they used a palladium reaction to close the pyridine ring on
the B-carboline.
Behforouz and his coworkers at Ball State decided to go the Pictet-Spengler route to the
methyl ester synthesis. The uniqueness of their research was the formation of the quinoline dione.
Instead of using the Friedlander condensation to form the quinoline analog, they use a Diels-Alder
type condensation to form the quinoline group6,7. Another part of the synthesis that is unique to
the Behforouz group is that the quinoline analog is complete before condensation. Therefore no
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further transformations are necessary to complete (9).
Recent developments in the synthesis of (9) have been published by Rocca and associates.
They appear to be the fIrst group to accomplish the total synthesis without the use of either the
Pictet-Spengler or Bischler-Napieralski reactions. The group uses a variety of cross-coupling
reaction starting with benzene and pyridine reagents8 .
n.
Biological Activity
In addition to the possibility of being an intermediate to streptonigrin, lavendamycin had the
same antimicrobial inhibition patterns as streptonigrin but was less potent. Streptonigrin is a very
active antitumor agent but is also a very potent bone marrow inhibitor lO• This fact stops the
streptonigrin from being used clinically. It was hoped that studying the structure differences the
mechanism that kills the tumors may be discovered.
It is believed that both lavendamycin and streptonigrin both work by cleaving DNA within
the cell. This cleavage probably occurs by reduction of the strand by a quinoline radical11. This
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cleavage is aided by metal cations and inhibited by the presence of chelating agents.
An interesting discovery is that biological studies done with partial structures do not exhibit
the same kind of results that studies done on the whole structure of lavendamycin. This points to
that fact that the whole skeleton is necessary for the cleavage of the DNAll.
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Results and Discussion
The objective of this research is to synthesize a new analog of lavendamycin,
7-N-acetyl-demethyl-Iavendamycin t-butyl ester (1), which may prove more biologically active or
more soluble in fats or water than previous analogs. Screening done in the past on lavendamycin
methyl ester (9) and 7-N-acetyl-Iavendamycin methyl ester (3) have shown that the latter
compound can selectively eradicate rask transformed rat kidney cells while the former showed no
such activityl3. One of the limitations of (3) was the lack of solubility in solvents generally used in
the pharmaceutical industry.
In addition to the possibility of increasing solubility, the t-butyl ester's unique ability as a
leaving group may allow the usage of (1) as an intermediate compound in further synthesis. The
replacement of the t-butyl group by sugar group is definitely a possibility for further study.
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Synthesis of L-Tryptophan t-Butyl Ester (4)
Compound (4) was synthesized by the esterification ofL-Tryptophan amino acid (5) with
isobutene (6) in the presence of sulfuric acid. (5) dissolved in l,4-dioxane and acid was allowed
to react with the isobutene gas for 36 hours at room temperature and produced (4), a brown solid,
in 37% yield after purification by flash chromatography. The structure of (4) was confirmed by
IH NMR and FAB-MS.
(4)
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Synthesis of 7-N-Acetyl-2-Formylquinoline-5,8-Dione (7)
Compound (7) was synthesized by the oxidation of
7-N-acetyl-2-methylquinoline-5,8-dione (2) in 1,4 dioxane, containing a small amount of water,
by selenium dioxide. The solution was allowed to reflux under argon for 25 hours producing (7),
an orange brown solid, at 71.3% yield after purification. Purification was achieved by dissolving
in chloroform and filtering. Structure confirmation of (7) was achieved by using 1H NMR.
(7)
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Synthesis of 7-N-Acetyl-Demethyl-Lavendamycin t-Butyl Ester (1)
Compound (1) was synthesized using the Pictet-Spengler condensation of
7-N-Acetyl-2-formylquinoline-5,8- dione (7) with L-Tryptophan t-Butyl Ester (4). The reaction
was done in hot xylene (140 C) under argon for 20 hrs. Mter the filtration of precipitates
(impurities) and purification by crystallization and flash chromatography, compound (1) obtained
in 27.4% yield. The structure of compound (1) was confirmed by IH NMR, HRMS and ElMS.
(7)
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Experimental
I. General Information
Rea~ents:
L-Tryptophan, Isobutene and selenium dioxide (99.9%) was purchased from the Aldrich
Chemical Company.
7-N-Acetyl-2-Methylquinoline-5,8-Dione was previously prepared by Christine Stoll and
other members of the Behforouz group according to the procedure of Behforouz.
Solvents:
1,4 dioxane and xylenes were previously dried and distilled before use.
All other solvents used were not distilled and were reagent grade.
Meltin~
Points:
Were obtained using a Thomas-Hoover Capillary melting point apparatus.
Nuclear Ma~netic Spectra:
H NMR were obtained using a Varian Gemini-200 spectrometer in deuterated
chloroform using the residual chloroform (7.24 ppm) as the internal standards.
Hi~h
Resolution Mass Spectra:
HRMS were obtained at Eli Lilly Research Laboratories using electron impact or F AB
ionization.
Mass Spectra:
ElMS were obtained at Ball State University using an Extrel ELQ 400 quadrupole
instrument set up for electron impact ionization.
Thin Layer Chromatography:
Performed using Eastman Silica gel sheets without indicator.
Elution Chromatography:
Performed using J.T. Baker silica gel using a flash chromatography procedure
published by Still.
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II. PROCEDURES
Preparation ofL-Tryptophan t-Butyl Ester (4)
In a 25 ml round bottom flask, equipped with a magnetic stir bar, was placed 15 ml of 1,4
dioxane and 1.5 ml of sulfuric acid. Into this solution, L-tryptophan (1.3093 g., 0.006418 mol)
was dissolved. After all of the compound was dissolved, a cold-finger apparatus, cooled with
acetone/dry ice, was attached to the round bottom. Isobutene was leaked into the cold-finger and
the condensate was allowed to drip into the round bottom. The isobutene was added in excess
over a 36 hour period while the reaction was being monitored by TLC. After the reaction appeared
to stop progressing, the solution was added to a cold mixture of 100 ml Ethyl Acetate, 100 ml
water and 15 ml of 1M NaOH. The pH was adjusted to 9.2 with NaOH and extracted 3 times with
ethyl acetate. The solution was dried with magnesium sulfate and rotovaped resulting in a thick
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brown liquid. From the TLCs there appeared to be some impurities so the liquid was redissolved
in chloroform and run through a flash column using a 96:4 mixture of chloroform and methanol.
The fractions were concentrated and vacuum pumped, resulting in a sticky solid. The product
weighed .6095g (36.52%): MP 65-67 C.
Preparation of 7 -N-Acetyl-2-Formylquinoline-5,8-Dione (7)
This procedure is similar to that reported by Stoll for the preparation of the same, using the
precursor made by Christine Stoll 14.
In a 25 ml round bottom flask, equipped with a magnetic stir bar, reflux condenser and
argon balloon, was placed 10 ml of 1,4 dioxane and 7-N-Acetyl-2-Methylquinoline-5,8-Dione
(.4722g., .00205 mol). Into this solution, .3004g of selenium dioxide and .3 ml of water was
added and the solution was allowed to reflux. The reaction was monitored by TLC and allowed to
reflux for 25 hrs. Upon completion, the reaction solution was cooled and filtered to remove the
selenium. The filtrate was rotovaped to dryness and redissolved in chloroform. The solution was
again filtered to remove the suspended solid. The filtrate was again rotovaped to dryness, resulting
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in a pure product. The product weighed .3565g (71.3%)
Preparation of7-N-Acetyl-Demethyl Lavendamycin t-Butyl Ester (1)
The procedure for the synthesis of this compound was similar to that reported by Gu for
the preparation of7-N-Acetyl Lavendamycin Methyl Ester 15 .
In a 50 ml round bottom flask, equipped with a reflux condenser, argon balloon, and
magnetic stir bar, the 7-N-Acetyl-2-Formylquinoline-5,8-Dione (.0637g., .0002611 mol),
L-Tryptophan t-Butyl Ester (.0672g., .0002585 mol) and 40 ml of xylene were slowly heated to
reflux over a period of three hours. The reaction was monitored by TLC and was allowed to reflux
for 18 additional hrs. The solution was cooled and filtered to remove a brown ppt (impurities).
The solution was rotovaped to dryness and redissolved in chloroform. After trying a variety of
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solvents it was found that by adding hexane to the chloroform, the lavendamycin and excess
aldehyde would precipitate out. This was done 3 times and each time the ppt was collected. The
ppt was redissolved in chloroform and a small amount of hexane was added. The solution was
refrigerated overnight and most of the excess aldehyde precipitated. The solution was allowed to
air evaporate to about 3 ml of solution when the lavendamycin precipitated. The ppt was filtered
and collected. The orange crystals collected weighed .02808g (22.5%). The remaining solution
still contained lavendamycin so a silica gel column was set-up using 100% Chloroform as the
mobile phase. There was some removal of the t-butyl group but the fractions were pure. The
remaining lavendamycin weighed .00613 g. (4.91 %,27.41 % Total) MP >300 C.
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Acknowledgements
I would like to give my greatest thanks to Dr. Mohammad Behforouz for giving me the
opportunity to work on this project. Dr. Behforouz gave me advice that not only helped to
complete this project but will help me succeed in life. Without him as a role model, I would
probably not be going on to graduate school.
I would also like to thank Wen eai. Her experience and constant advice made the project a
realleaming experience. In addition, her motherly attitude to myself and other students will
always be appreciated.
Thanks also goes out to Dr. Robert Morris for his advice and technical support, especially
in the Mass Spectrum area. I also wish to thank Eli Lilly and Co. for their technical support.
Finally I want to thank Christine Krumrich who has been working on similar projects for
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her advice and friendship throughout our projects.
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lli. Spectra and Chromatographs
1. NMR ofL-Tryprophan t-Butyl Ester
2. MS ofL-Tryprophan t-Butyl Ester
3. NMR of Quinoline Dione
4. NMR of7-N-Acetyl-Demethyl Lavendamycin t-Butyl Ester
5. MS of7-N-Acetyl-Demethyl Lavendamycin t-Butyl Ester
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References
1. Doyle, T.W.; Balitz, D.M.; Grulich, R.E. and Nettleton, D.E. Tetrahedron Lett. 1981,
22,4595
2. Kende, A. S.; Ebetino, F. H.; Tetrahedron Left. 1984, 25, 923.
3. Kende, A.S.; Ebetino, F H.; Tetrahedron Lett. 1984, 25, 923.
4. Hibino, S.; Okazaki, M.; Ichikawa, M.; Sato,K.; Ishizu, T.; Heterocycles, 1985, 23,
261.
5. Rao, A.; Chavan, S.; Sivadasan, L.; Tetrahedron, 1986,42, 5065.
6. Behforouz, M.; "Synthesis and Study of ras Specific Antitumor Drugs" Grant
Proposal, NIH, 1990.
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.
7. Gu, Z.; unpublished results, 1989 .
B. Rocca, F.; Marsais, F.; Godard, A.; Queguiner, G.; Tetrahedron Lett, 1993, 18,
2937.
9. Boger, D.; Duff, S.; Panek, J.; Yasuda, M.; J. Org. Chem., 1985,50,5790.
lO. Gould, S.; Chang, C.; J. Am. Chem. Soc., '-977, 99, 5496
11. Lown, J.; Mol. Cell. Biochem., 1983, 55, 17.
12. Behforouz, M.; Merriman,R.; Unpublished results, 1992.
13. Behforouz, M.; Merriman, R.; Unpublished results, 1992.
14. Stoll, C.; Unpublished resufts from lab book, 1994.
15. Gu, Z.; Unpublished results, 1989.
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